Pirrone, C., F. Rossi, et al. (2018). "Evaluation of biomarkers in Mytilus galloprovincialis as an integrated measure of biofilm-membrane bioreactor (BF-MBR) system efficiency in mitigating the impact of oily wastewater discharge to marine environment: a microcosm approach." Aquat Toxicol 198: 49-62.

 

Rocha, T. L., E. Bilbao, et al. (2018). "Changes in metallothionein transcription levels in the mussel Mytilus galloprovincialis exposed to CdTe quantum dots." Ecotoxicology 27(4): 402-410.

                Quantum dots (QDs) are a class of engineered nanoparticles (ENPs) with several biomedical, industrial and commercial applications. However, their metabolism and detoxification process in aquatic invertebrates and environmental health hazards remain unclear. This study investigate the transcriptional changes of metallothioneins (MTs) isoforms (mt10IIIa and mt20IV) induced by CdTe QDs, in comparison with its dissolved counterpart, in the marine mussel Mytilus galloprovincialis. Mussels were exposed to CdTe QDs and to the same Cd concentration (10 microg Cd L(-1)) of dissolved Cd for 14 days and mt transcription levels were measured by real time quantitative PCR (qPCR). Tissue specific mt transcription patterns were observed in mussels exposed to both Cd forms, wherein the gills were a more sensitive organ compared to the digestive gland. No significant changes were observed in mt10IIIa transcription levels in mussels exposed to both Cd forms. In contrast, transcription of mt20IV was tissue and exposure time dependent, with higher mt20IV mRNA levels in mussels exposed to QDs and dissolved Cd when compared to unexposed mussels. Multivariate analysis indicates particle-specific effects after 14 days of exposure and a dual role of MTs in the QD metabolism and in the protection against oxidative stress in mussels exposed to Cd-based ENPs.

 

Nardi, A., M. Benedetti, et al. (2018). "Oxidative and interactive challenge of cadmium and ocean acidification on the smooth scallop Flexopecten glaber." Aquat Toxicol 196: 53-60.

                Ocean acidification (OA) may affect sensitivity of marine organisms to metal pollution modulating chemical bioavailability, bioaccumulation and biological responsiveness of several cellular pathways. In this study, the smooth scallop Flexopecten glaber was exposed to various combinations of reduced pH (pH/pCO2 7.4/ approximately 3000muatm) and Cd (20mug/L). The analyses on cadmium uptake were integrated with those of a wide battery of biomarkers including metallothioneins, single antioxidant defenses and total oxyradical scavenging capacity in digestive gland and gills, lysosomal membrane stability and onset of genotoxic damage in haemocytes. Reduced pH slightly increased concentration of Cd in scallop tissues, but no effects were measured in terms of metallothioneins. Induction of some antioxidants by Cd and/or low pH in the digestive gland was not reflected in variations of the total oxyradical scavenging capacity, while the investigated stressors caused a certain inhibition of antioxidants and reduction of the scavenging capacity toward peroxyl radical in the gills. Lysosomal membrane stability and onset of genotoxic damages showed high sensitivity with possible synergistic effects of the investigated factors. The overall results suggest that indirect effects of ocean acidification on metal accumulation and toxicity are tissue-specific and modulate oxidative balance through different mechanisms.

 

Sillero-Rios, J., A. Sureda, et al. (2018). "Biomarkers of physiological responses of Octopus vulgaris to different coastal environments in the western Mediterranean Sea." Mar Pollut Bull 128: 240-247.

                The increase of pollutants in coastal seawater could produce several harmful biological effects on marine organisms related to the production of reactive oxygen species (ROS) causing cellular and tissue damages through oxidative stress mechanisms. Common octopuses (Octopus vulgaris) inhabiting coastal areas under high anthropogenic activity of Mallorca (W-Mediterranean Sea) have the ability to control oxidative damage by triggering antioxidant enzyme responses. Analyzing the digestive glands, octopuses from human-altered coastal areas showed higher activity of superoxide dismutase (SOD), catalase (CAT) and glutathione S-transferase (GST) compared to octopuses from non-influenced coastal waters (i.e. marine reserve area). Higher metallothionein (MT) concentrations and lack of malondialdehyde (MDA) variations also reflect adaptations of O. vulgaris to polluted areas. This is the first study assessing the levels of the oxidative stress biomarkers on O. vulgaris in the Mediterranean Sea, revealing their usefulness to assess diverse environmental pollution effects on this relevant ecological and commercial species.

 

Sureda, A., X. Capo, et al. (2018). "Acute exposure to sunscreen containing titanium induces an adaptive response and oxidative stress in Mytillus galloprovincialis." Ecotoxicol Environ Saf 149: 58-63.

                The use of sunscreens to protect against ultraviolet radiation exposure progressively increases as result of a greater awareness of the people and the greater arrival of tourists. The components of these creams can end up in the waters affecting coastal species. Mediterranean mussels (Mytillus galloprovincialis) were subjected to an acute exposure of a sunscreen with TiO2 in their composition during 24h. The low and medium concentrations used in the assays contained a concentration of TiO2 in the range of values found in coastal waters of the Balearic Islands. Titanium and metallothionein concentrations were progressively increasing in gills with the sunscreen concentration in a dose-dependent manner. The activities of the antioxidant enzymes and the detoxification glutathione s-transferase evidenced a hormetic shape response with increased activities at lower sunscreen concentrations, a response that was abolished at the highest concentration. In accordance with these enzyme activities, the levels of malondialdehyde, as a marker of lipid peroxidation, were significantly elevated by the higher sunscreen concentrations. Acetylcholinesterase activity maintained control activities except for the highest sunscreen concentration, where a significant decrease was evidenced. In conclusion, the treatment of mussels with a sunscreen containing TiO2 in the range of Balearic coastal waters induces an adaptive response that is overcome by the highest concentration. Follow-up biomonitoring studies are necessary to control the concentration of sunscreen compound in coastal waters such as titanium since they can induce oxidative stress to affected organisms.

 

Rabei, A., A. Hichami, et al. (2018). "Fatty acid composition, enzyme activities and metallothioneins in Donax trunculus (Mollusca, Bivalvia) from polluted and reference sites in the Gulf of Annaba (Algeria): Pattern of recovery during transplantation." Environ Pollut 237: 900-907.

                The gulf of Annaba, the most important touristic and economic coastal zone located in Northeast Algeria, is contaminated by several pollutants from urban, agricultural, harbor and industrial activities. Elevated levels of heavy metals were detected in a locally prevalent edible mollusk Donax trunculus (Bivalvia, Donacidae) widely used as a sentinel species for the assessment of marine pollution. The present work aims to measure the difference between two localities, one being full of different pollutants (Sidi Salem) and the other being relatively clean (El Battah) and to evaluate the ability of D. trunculus to overcome the environmental stress during a transplantation experiment by a determination of fatty acid profile, the enzymes activities and the level of metallothioneins (MTs), a biomarker of metallic contamination. Adults of D. trunculus were collected at Sidi Salem (contaminated site) and transplanted into El Battah (reference site) for 21 days in cages (60x60x60cm with a 2mm mesh). Biochemical analyzes were conducted at different times (0, 7, 14 and 21 days). At 0-day experiment: the rate of the fatty acids, the enzymes activities and MT levels at the site of Sidi Salem (polluted site) were significantly different from those of El Battah. During the transplantation a gradual restoration of fatty acids rates, enzymes activities and MT levels was observed. At the end of the period of transplantation, the values are comparable to those of El Battah. A two-way ANOVA (time, site) on data revealed significant effects of time and site. Overally, D. trunculus is able to induce its detoxification system and to restore relatively rapidly the status of individuals from the reference site (El Battah).

 

Sforzini, S., C. Oliveri, et al. (2018). "Application of a new targeted low density microarray and conventional biomarkers to evaluate the health status of marine mussels: A field study in Sardinian coast, Italy." Sci Total Environ 628-629: 319-328.

                In the present study, we investigated the health status of marine mussels (Mytilus galloprovincialis) caged and deployed at three different sites on the Sardinian coastline characterized by different levels of contamination: Fornelli (F, the reference site), Cala Real (CR), and Porto Torres (PT). A new low density oligonucleotide microarray was used to investigate global gene expression in the digestive gland of mussels. Target genes were selected to cover most of the biological processes involved in the stress response in bivalve mollusks (e.g. DNA metabolism, translation, immune response, cytoskeleton organization). A battery of classical biomarkers was also employed to complement the gene expression analyses. Chemical analysis revealed higher loads of heavy metals (Pb and Cu) and total polycyclic aromatic hydrocarbons (PAHs) at PT compared to the other sites. In mussels deployed at CR, functional genomics analysis of the microarray data rendered 78 differentially expressed genes (DEGs) involved in 11 biological processes. Animals exposed at PT had 105 DEGs that were characterized by the regulation of 14 biological processes, including mitochondrial activity, adhesion to substrate, DNA metabolism, translation, metal resistance, and cytoskeleton organization. Biomarker data (lysosomal membrane stability, lysosomal/cytoplasm volume ratio, lipofuscin accumulation, metallothionein content, micronucleus frequency, and cytoskeleton alteration) were in trend with transcriptomic output. Biomarker data were integrated using the Mussel Expert System (MES), allowing defining the area in which the presence of chemicals is toxic for mussels. Our study provides the opportunity to adopt a new approach of integrating transcriptomic (microarray) results with classical biomarkers to assess the impact of pollutants on marine mussels in biomonitoring programs.

 

Pilote, M., C. Andre, et al. (2018). "Metal bioaccumulation and biomarkers of effects in caged mussels exposed in the Athabasca oil sands area." Sci Total Environ 610-611: 377-390.

                The Athabasca oil sands deposit is the world's largest known reservoir of crude bitumen and the third-largest proven crude oil reserve. Mining activity is known to release contaminants, including metals, and to potentially impact the aquatic environment. The purpose of this study was to determine the impacts of oil sands mining on water quality and metal bioaccumulation in mussels from the Fort McMurray area in northern Alberta, Canada. The study presents two consecutive years of contrasting mussel exposure conditions (low and high flows). Native freshwater mussels (Pyganodon grandis) were placed in cages and exposed in situ in the Athabasca River for four weeks. Metals and inorganic elements were then analyzed in water and in mussel gills and digestive glands to evaluate bioaccumulation, estimate the bioconcentration factor (BCF), and determine the effects of exposure by measuring stress biomarkers. This study shows a potential environmental risk to aquatic life from metal exposure associated with oil sands development along with the release of wastewater from a municipal treatment plant nearby. Increased bioaccumulation of Be, V, Ni and Pb was observed in mussel digestive glands in the Steepbank River, which flows directly through the oil sands mining area. Increased bioaccumulation of Al, V, Cr, Co, Ni, Mo and Ni was also observed in mussel gills from the Steepbank River. These metals are naturally present in oil sands and generally concentrate and increase with the extraction process. The results also showed different pathways of exposure (particulate or dissolved forms) for V and Ni resulting from different river water flows, distribution coefficient (Kd) and BCF. Increasing metal exposure downstream of the oil sands mining area had an impact on metallothionein and lipid peroxidation in mussels, posing a potential environmental risk to aquatic life. These results confirm the bioavailability of some metals in mussel tissues associated with detoxification of metals (metallothionein levels), and oxidative stress in mussels located downstream of the oil sands mining area. These results highlight a potential ecotoxicological risk to biota and to the aquatic environment downstream of the oil sands mining area, even at low metal exposure levels.

 

Chiesa, S., P. Chainho, et al. (2018). "Metals and As content in sediments and Manila clam Ruditapes philippinarum in the Tagus estuary (Portugal): Impacts and risk for human consumption." Mar Pollut Bull 126: 281-292.

                The Manila clam is emerging as a relevant species for the Portuguese market. The present work was conducted in the Tagus estuary to evaluate 1) the metals and As content in the sediments of the Tagus estuary, especially on those areas subjected to Manila clam harvesting 2) the metals and As content in clams, and the risk associated with their consumption 3) the physiological and biochemical responses of the clam to metals and As contamination, and its possible role as a pollution bioindicator in the estuarine environment. The most contaminated sediments were identified nearby industrial areas, nevertheless clams collected in low contaminated areas showed high metals and As concentrations. The condition index, glycogen content, membrane oxidative damage, biotransformation enzymes and metallothioneins showed consistent responses to metals and As content in clams. Results emphasize the need for the development of a management plan for the species exploitation in the Tagus estuary.

 

Liu, Q., F. Wang, et al. (2018). "Assessment of metal contamination in estuarine surface sediments from Dongying City, China: Use of a modified ecological risk index." Mar Pollut Bull 126: 293-303.

                Surface sediments and clam Meretrix meretrix were collected from a northern estuarine region in Dongying City, China. Sediments were analysed for heavy metals (Hg, As, Cd, Cr, Cu, Pb, and Zn) and the clams were tested for metallothioneins (MTs) and lysosomal membrane stability (LMS). The heavy metal total concentrations decreased in the order of Cr>Zn>Cu>Pb>As>Cd>Hg. The results of Bureau Communautaire de Reference (BCR) sequential extraction of heavy metals showed that the geochemical speciation of all heavy metals was dominated by residual fraction. According to the responses of biomarkers in M. meretrix, the modified potential ecological risk index (PERI-B) can more accurately reflect heavy metals pollution. PERI-B showed all sediment samples have low or moderate risk, except at site S10 (considerable risk), and the main contribution of ecological risk heavy metals were Cd and Hg.

 

Yin, Q. and W. X. Wang (2018). "Uniquely high turnover of nickel in contaminated oysters Crassostrea hongkongensis: Biokinetics and subcellular distribution." Aquat Toxicol 194: 159-166.

                Despite the environmental concerns regarding nickel (Ni) especially in China, it has received little attention in aquatic animals due to its comparatively weak toxicity. In the present study, we explored the bioaccumulation, biokinetics, and subcellular distribution of Ni in an estuarine oyster Crassostrea hongkongensis. We demonstrated that Ni represented a new pattern of bioaccumulation in oysters characterized by rapid elimination and low dissolved uptake. The waterborne uptake rate constant and dietary assimilation efficiency were 0.036L/g/h and 28%, respectively, and dissolved uptake was the predominant exposure route. The efflux rate constant was positively related to tissue Ni concentration, with the highest efflux of 0.155d(-1). Such high elimination resulted in a high Ni turnover and steady-state condition reached rapidly, as shown with a 4-week waterborne exposure experiment at different Ni concentrations. Ni in oysters was mainly sequestered in metallothionein-like protein (MTLP), metal-rich granule, and cellular debris. MTLP was the most important binding fraction during accumulation and depuration, and played a dynamic role leading to rapid Ni elimination. Pre-exposure to Ni significantly reduced the dissolved uptake, probably accompanied by depressed filtration activity. Overall, the high turnover and regulation of Ni in oysters were achieved by enhanced efflux, suppressed uptake, and sequestration of most Ni into the detoxified pool.

 

Falfushynska, H. I., L. L. Gnatyshyna, et al. (2018). "Detoxification and cellular stress responses of unionid mussels Unio tumidus from two cooling ponds to combined nano-ZnO and temperature stress." Chemosphere 193: 1127-1142.

                Bivalve mollusks from the cooling reservoirs of fuel power plants (PP) are acclimated to the chronic heating and chemical pollution. We investigated stress responses of the mussels from these ponds to determine their tolerance to novel environmental pollutant, zinc oxide nanoparticles (nZnO). Male Unio tumidus from the reservoirs of Dobrotvir and Burschtyn PPs (DPP and BPP), Ukraine were exposed for 14 days to nZnO (3.1 muM), Zn(2+) (3.1 muM) at 18 degrees C, elevated temperature (T, 25 degrees C), or nZnO at 25 degrees C (nZnO + T). Control groups were held at 18 degrees C. Zn-containing exposures resulted in the elevated concentrations of total and Zn-bound metallothionein (MT and Zn-MT) in the digestive gland, an increase in the levels of non-metalated MT (up to 5 times) and alkali-labile phosphates and lysosomal membrane destabilization in hemocytes. A common signature of nZnO exposures was modulation of the multixenobiotic-resistance protein activity (a decrease in the digestive gland and increase in the gills). The origin of population strongly affected the cellular stress responses of mussels. DPP-mussels showed depletion of caspase-3 in the digestive gland and up-regulation of HSP70, HSP72 and HSP60 levels in the gill during most exposures, whereas in the BPP-mussels caspase-3 was up-regulated and HSPs either downregulated or maintained stable. BPP-mussels were less adapted to heating shown by a glutathione depletion at elevated temperature (25 degrees C). Comparison with the earlier studies on mussels from pristine habitats show that an integrative 'eco-exposome'-based approach is useful for the forecast of the biological responses to novel adverse effects on aquatic organisms.

 

Aouini, F., C. Trombini, et al. (2018). "Assessing lead toxicity in the clam Ruditapes philippinarum: Bioaccumulation and biochemical responses." Ecotoxicol Environ Saf 158: 193-203.

                Lead (Pb) is a non-essential metal. Its occurrence in the environment is related principally to anthropogenic contamination. Pb is toxic to aquatic organisms and can provoke damage to membranes and inhibit the activity of essential enzymes. The filter-feeding, Manila clam Ruditapes philippinarum is widely used as a biomonitor organism to assess metal toxicity. Among biomarkers related to the Pb toxicity, the enzymatic activity of delta-aminolevulinic acid dehydratase (delta-ALAD) has been adopted as a specific tool. Metallothionein (MT), lipid peroxidation (LPO) and antioxidant enzymes activities, such as catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR) and glutathione S-transferase (GST) and superoxidase dismutase (SOD) have also been employed to assess the toxic effect of metals. Two target tissues, the gills and the digestive gland, were selected to examine biomarker responses. In order to assess the effects of Pb accumulation and the mechanisms involved in the recovery from it, clams were exposed at two Pb levels (10 and 100microg/L) for 7 days and were later maintained in clean water for 7 days as a depuration period. Pb accumulation was dependent on the exposure concentration and higher Pb levels were observed in the gills compared to the digestive gland. Inhibition of delta-ALAD, GST and SOD and the induction of MT and LPO over the exposure period were observed in the gills and the digestive gland of R. philippinarum. The depuration period showed a continuous inhibition of the delta-ALAD activity and induction of MT and LPO in both tissues. These results demonstrate that lead induced an exposure effect and the 7 days of depuration were not sufficient to recover the basal health status of the clams.

 

Chen, L., H. Wu, et al. (2018). "The role of GST omega in metabolism and detoxification of arsenic in clam Ruditapes philippinarum." Aquat Toxicol 204: 9-18.

                The major hazard of arsenic in living organisms is increasingly being recognized. Marine mollusks are apt to accumulate high levels of arsenic, but knowledge related to arsenic detoxification in marine mollusks is still less than sufficient. In this study, arsenic bioaccumulation as well as the role of glutathione S-transferase omega (GSTOmega) in the process of detoxification were investigated in the Ruditapes philippinarum clam after waterborne exposure to As(III) or As(V) for 30 days. The results showed that the gills accumulated significantly higher arsenic levels than the digestive glands. Arsenobetaine (AsB) and dimethylarsenate (DMA) accounted for most of the arsenic found, and monomethylarsonate (MMA) can be quickly metabolized. A subcellular distribution analysis showed that most arsenic was in biologically detoxified metal fractions (including metal-rich granules and metallothionein-like proteins), indicating their important roles in protecting cells from arsenic toxicity. The relative mRNA expressions of two genes encoding GSTOmega were up-regulated after arsenic exposure, and the transcriptional responses were more sensitive to As(III) than As(V). The recombinant GSTOmegas exhibited high activities at optimal conditions, especially at 37 degrees C and pH 4-5, with an As(V) concentration of 60 mM. Furthermore, the genes encoding GSTOmega significantly enhance the arsenite tolerance but not the arsenate tolerance of E. coli AW3110 (DE3) (DeltaarsRBC). It can be deduced from these results that GSTOmegas play an important role in arsenic detoxification in R. philippinarum.

 

Dallas, L. J., A. Turner, et al. (2018). "An integrated approach to assess the impacts of zinc pyrithione at different levels of biological organization in marine mussels." Chemosphere 196: 531-539.

                The mechanisms of sublethal toxicity of the antifouling biocide, zinc pyrithione (ZnPT), have not been well-studied. This investigation demonstrates that 14-d sublethal exposure to ZnPT (0.2 or 2muM, alongside inorganic Zn and sea water controls) is genotoxic to mussel haemocytes but suggests that this is not caused by oxidative DNA damage as no significant induction of oxidised purines was detected by Fpg-modified comet assay. More ecologically relevant endpoints, including decreased clearance rate (CR), cessation of attachment and decreased tolerance of stress on stress (SoS), also showed significant response to ZnPT exposure. Our integrated approach was underpinned by molecular analyses (qRT-PCR of stress-related genes, 2D gel electrophoresis of proteins) that indicated ZnPT causes a decrease in phosphoenolpyruvate carboxykinase (PEPCK) expression in mussel digestive glands, and that metallothionein genes are upregulated; PEPCK downregulation suggests that altered energy metabolism may also be related to the effects of ZnPT. Significant relationships were found between % tail DNA (comet assay) and all higher level responses (CR, attachment, SoS) in addition to PEPCK expression. Principal component analyses suggested that expression of selected genes described more variability within groups whereas % tail DNA reflected different ZnPT concentrations.

 

Hsu, W. H., Y. C. Lin, et al. (2018). "The neuronal protection of a zinc-binding protein isolated from oyster." Food Chem Toxicol 114: 61-68.

                Mitochondrial function is applied as oxidative stress and neuronal damage index. In this study, d-galactose was used to induce free radicals production and neuronal damage in HN-h cells, and the effect of novel 43kDa protein isolated from oyster on anti-mitochondrial dysfunction and zinc-binding ability were evaluated. Crystal violet stain results indicated zinc-binding protein of oyster (ZPO) attenuated neuronal cell death induced by 100mM of d-galactose on HN-h cells in a dose-dependent manner. ZPO alleviated mitochondrial inactivation, mitochondrial membrane potential decreasing, oxidative stress, and fusion/fission state in non-cytotoxic concentration of d-galactose (50mM)-treated HN-h cells. ZPO treatment recovered metallathionein-3 (MT-3) decrease and inhibited beta- and gamma-secretase as well as amyloid beta (Abeta) accumulation in HN-h cells caused by d-galactose induction. These results suggest ZPO could avoid oxidative stress and is a functional protein for zinc concentration maintainability, which has potential for development of functional foods for neuronal protection.

 

El Haimeur, B., M. Bouhallaoui, et al. (2017). "Use of biomarkers to evaluate the effects of environmental stressors on Mytilus galloprovincialis sampled along the Moroccan coasts: Integrating biological and chemical data." Mar Environ Res 130: 60-68.

                A biomonitoring study using wild Mytilus galloprovincialis mussels sampled from six sites along the Moroccan coasts evaluated whether select biomarkers are suitable for identifying and quantifying pollution-induced stress syndrome in mussels. Lysosomal membrane stability was confirmed to be a highly sensitive biological parameter, and acetylcholinesterase activity was found a suitable biomarker of neurotoxicity. Metallothionein concentrations were in line with heavy metal concentrations detected in mussel tissues. However, malondialdehyde was not sensitive, suggesting the need for alternative biomarkers of oxidative stress. Three different approaches were used for biomarker and chemical data integration. The Integrated Biomarker Response (IBR) was suitable for classifying the stress response but did not allow to evaluate the level of stress in the organisms. The Mussel Expert System (MES) was suitable for ranking the biological effects of pollutants, also providing an indication of the evolution of the stress syndrome in the animals. Finally, the use of Principal Component Analysis (PCA) provided indication of the inorganic chemicals contributing to the detrimental biological effects.

 

Pedrini-Martha, V., R. Schnegg, et al. (2017). "The physiological role and toxicological significance of the non-metal-selective cadmium/copper-metallothionein isoform differ between embryonic and adult helicid snails." Comp Biochem Physiol C Toxicol Pharmacol 199: 38-47.

                Metal regulation is essential for terrestrial gastropods to survive. In helicid snails, two metal-selective metallothionein (MT) isoforms with different functions are expressed. A cadmium-selective isoform (CdMT) plays a major role in Cd(2+) detoxification and stress response, whereas a copper-selective MT (CuMT) is involved in Cu homeostasis and hemocyanin synthesis. A third, non-metal-selective isoform, called Cd/CuMT, was first characterized in Cantareus aspersus. The aim of this study was to quantify the transcriptional activity of all three MT genes in unexposed and metal-exposed (Cd, Cu) embryonic Roman snails. In addition, the complete Cd/CuMT mRNA of the Roman snail (Helix pomatia) was characterized, and its expression quantified in unexposed and Cd-treated adult individuals. In embryos of Helix pomatia, the Cd/CuMT gene was induced upon Cu exposure. Its transcription levels were many times higher than that of the other two MT genes, and also exceeded by far the Cd/CuMT mRNA concentrations of adult snails. In the hepatopancreas of adult Roman snails, no Cd/CuMT could be detected at the protein level, irrespective of whether the snails had been exposed to Cd or not. This contrasts with the situation in the near relative, Cantareus aspersus. It appeared that the 3'-UTR of the Cd/CuMT mRNA differed largely between Cantareus aspersus and Helix pomatia, being larger in the latter species, with a number of putative binding sites for proteins and miRNAs known to inhibit mRNA translation. We suggest this as a possible mechanism responsible for the lack of Cd/CuMT protein expression in adult Roman snails.

 

Gonzalez-Fernandez, C., M. Albentosa, et al. (2017). "Interactive effects of nutrition, reproductive state and pollution on molecular stress responses of mussels, Mytilus galloprovincialis Lamarck, 1819." Mar Environ Res 131: 103-115.

                Marine bivalves including mussels Mytilus galloprovincialis are commonly used as sentinels for pollution monitoring and ecosystem health assessment in the coastal zones. Use of biomarkers to assess the pollution effects assumes that the effects of pollutants on the biomarkers exceed the natural background variability; yet this assumption has rarely been tested. We exposed mussels at different reproductive stages and nutritive states to two concentrations of a polycyclic aromatic hydrocarbon (fluoranthene, 3 and 60 mug L(-1)) for three weeks. Expression levels of the molecular biomarkers related to the detoxification and general stress response [cytochrome P450 oxidase (CYP450), glutathione S-transferases (GST-alpha; GST-S1; GST-S2), the multixenobiotic resistance protein P-glycoprotein (PgP), metallothioneins (MT10 and MT20), heat shock proteins (HSP22, HSP70-2; HSP70-3; HSP70-4), as well as mRNA expression of two reproduction-related genes, vitellogenin (Vitel) and vitelline coat lysin M7 (VCLM7)] were measured. The mussels' nutrition and reproductive state affected the baseline mRNA levels of molecular biomarkers and modulated the transcriptional responses of biomarker genes to the pollutant exposure. Thus, mussel physiological state could act as a confounding factor in the evaluation of the response of pollution through molecular biomarkers. The biomarker baseline levels must be determined across a range of physiological states to enable the use of biomarkers in monitoring programs.

 

Capolupo, M., S. Franzellitti, et al. (2017). "A comprehensive evaluation of the environmental quality of a coastal lagoon (Ravenna, Italy): Integrating chemical and physiological analyses in mussels as a biomonitoring strategy." Sci Total Environ 598: 146-159.

                This study aimed at evaluating the environmental quality of a coastal lagoon (Pialassa Piomboni, NW-Adriatic, Italy) by combining analyses of biomarkers of environmental stress and bioaccumulation of contaminants in marine mussels (Mytilus galloprovincialis) transplanted for 28days to six selected sites. Assessed biomarkers encompassed lysosomal endpoints, oxidative stress and detoxification parameters, specific responses to metals, neuro- and genotoxic substances; chemical analyses focused on PAHs, metals, pesticide and pharmaceuticals. Results showed up to a 67-fold bioaccumulation of 4- to 6-ring PAHs, including pyrene, fluoranthene, chrysene and benzo(ghi)perylene in transplanted mussels compared to reference conditions (T0). A 10-fold increase of Fe, Cr and Mn was observed, while pesticides and pharmaceuticals were not or slightly detected. The onset of a significant (p<0.05) general stress syndrome occurred in exposed mussels, as outlined by a 50-57.7% decrease in haemocytes lysosomal membrane stability and an increased lysosomal volume (22.6-26.9%) and neutral lipid storage (18.9-48.8%) observed in digestive gland. Data also revealed a diffuse lipofuscin accumulation (86.5-139.3%; p<0.05) in digestive gland, occasionally associated to a catalase activity inhibition in gill, indicating an increased vulnerability toward pro-oxidant factors. Higher levels of primary DNA damage (258%; p<0.05) and PAH accumulation were found in mussels exposed along the eastern shoreline, hosting a petrochemical settlement. Bioaccumulated metals showed a positive correlation with increased metallothionein content (85-208%; p<0.05) observed in mussels from most sites. Overall, the use of physiological and chemical analyses detected chronic alterations of the mussel health status induced by specific toxicological pathways, proving a suitable approach in the framework of biomonitoring programs of coastal lagoons.

 

Hawkins, C. A. and I. M. Sokolova (2017). "Effects of elevated CO2 levels on subcellular distribution of trace metals (Cd and Cu) in marine bivalves." Aquat Toxicol 192: 251-264.

                Hypercapnia (elevated CO2 levels) and pollution with trace metals such as Cu and Cd are common stressors in estuarine habitats that can negatively affect physiology and health of marine organisms. Hypercapnia can modulate toxicity of trace metals including Cu and Cd; however, the physiological and cellular mechanisms of the metal-CO2 interactions are not well understood. We investigated the effects of elevated PCO2 ( approximately 800 and 2000muatm) and metal exposure (50mugl(-1) of Cu or Cd) on subcellular distribution of metals in two common species of marine bivalves, Eastern oysters Crassostrea virginica and hard shell clams Mercenaria mercenaria. Oysters accumulated higher burdens of Cu and Cd in the gill tissues compared to clams. In both studied species, Cu was predominantly associated with the metabolically active cell compartments (mitochondria, lysosomes, microsomes and cytosolic enzymes), with a modest fraction sequestered by metallothioneins ( approximately 30%) and the insoluble metal-containing granules (MCG) ( approximately 15-20%). Unlike Cu, Cd was largely sequestered by metallothioneins ( approximately 60-70%), with a relatively small fraction associated with the organelles and the cytosolic enzymes. Mitochondria were the main intracellular target for trace metals accumulating higher concentrations of Cd (and in the case of oysters - of Cu) than other organelles or cytosolic enzymes. Cu accumulation in the metabolically active cellular compartments was independent of the CO2 levels, while Cd content of the organelles and cytosolic enzymes increased at elevated PCO2 in both studied species indicating that hypercapnia may enhance cellular toxicity of Cd in bivalves. Hypercapnia suppressed the sequestration capacity of metallothioneins for Cu and Cd in oysters but increased Cu and Cd load in clam metallothioneins. Thus, metal-induced metabolic injury in oysters may be exaggerated by hypercapnia which enhances metal accumulation in the potentially sensitive intracellular fractions and suppresses the metal detoxification capacity. In contrast, clams appear to be more resistant to the combined effects of hypercapnia and metal exposure reflecting more efficient and robust detoxification mechanisms of this species.

 

Kournoutou, G. G., P. C. Giannopoulou, et al. (2017). "Oxidative damage of 18S and 5S ribosomal RNA in digestive gland of mussels exposed to trace metals." Aquat Toxicol 192: 136-147.

                Numerous studies have shown the ability of trace metals to accumulate in marine organisms and cause oxidative stress that leads to perturbations in many important intracellular processes, including protein synthesis. This study is mainly focused on the exploration of structural changes, like base modifications, scissions, and conformational changes, caused in 18S and 5S ribosomal RNA (rRNA) isolated from the mussel Mytilus galloprovincialis exposed to 40mug/L Cu, 30mug/L Hg, or 100mug/L Cd, for 5 or 15days. 18S rRNA and 5S rRNA are components of the small and large ribosomal subunit, respectively, found in complex with ribosomal proteins, translation factors and other auxiliary components (metal ions, toxins etc). 18S rRNA plays crucial roles in all stages of protein synthesis, while 5S rRNA serves as a master signal transducer between several functional regions of 28S rRNA. Therefore, structural changes in these ribosomal constituents could affect the basic functions of ribosomes and hence the normal metabolism of cells. Especially, 18S rRNA along with ribosomal proteins forms the decoding centre that ensures the correct codon-anticodon pairing. As exemplified by ELISA, primer extension analysis and DMS footprinting analysis, each metal caused oxidative damage to rRNA, depending on the nature of metal ion and the duration of exposure. Interestingly, exposure of mussels to Cu or Hg caused structural alterations in 5S rRNA, localized in paired regions and within loops A, B, C, and E, leading to a continuous progressive loss of the 5S RNA structural integrity. In contrast, structural impairments of 5S rRNA in mussels exposed to Cd were accumulating for the initial 5days, and then progressively decreased to almost the normal level by day 15, probably due to the parallel elevation of metallothionein content that depletes the pools of free Cd. Regions of interest in 18S rRNA, such as the decoding centre, sites implicated in the binding of tRNAs (A- and P-sites) or translation factors, and areas related to translation fidelity, were found to undergo significant metal-induced conformational alterations, leading either to loosening of their structure or to more compact folding. These modifications were associated with parallel alterations in the translation process at multiple levels, a fact suggesting that structural perturbations in ribosomes, caused by metals, pose significant hurdles in translational efficiency and fidelity.

 

Zhang, B., C. D. Fang, et al. (2017). "Depuration of Cadmium from Blue Mussel (Mytilus edulis) by Protein Hydrolysate-Fe(2+) Complex: The Role of Metallothionein." J Food Sci 82(11): 2767-2773.

                Metallothionein (MT) plays a major role in the detoxification of toxic metal ions in mussel. This study served to investigate the effects of prepared protein hydrolysate-Fe(2+) (PH-Fe(2+) ) on depuration of cadmium salt (Cd(2+) ) from blue mussel (Mytilus edulis). The results indicated that Cd(2+) concentrations in control ranged from 46.1 to 47.1 mug/g during 15 d of depuration. While, 40 mg/L PH-Fe(2+) feed mussels exhibited obvious changes in Cd(2+) concentration variables, which decreased by 22.8 mug/g after 15 d of depuration, making them significantly lower than the values of the control. Our assumption was that Cd(2+) could be dissociated effectively from the complex of MT-Cd(2+) in mussels affected by the incorporation of PH-Fe(2+) during the feeding period. Further results of homology modeling and molecular dynamics (MD) confirmed that the combined power between MT and Cd(2+) weakened significantly by PH-Fe(2+) . This condition affected the charge density and/or the loop flexibility of MT and decreased the interaction energy within MT-Cd(2+) complex and resulted in the release of Cd(2+) from the complex, thereby exhibiting excretion detoxification. Finally, by comparing the experimental results to MD results, significant positive correlations were observed between PH-Fe(2+) and the depuration of Cd(2+) from MT-Cd(2+) complex. Overall, the findings of this study may help better understand the depuration mechanisms of Cd(2+) combined with MT, and the PH-Fe(2+) can be recommended as a depuration agent to decrease Cd(2+) concentrations in mussels. PRACTICAL APPLICATION: Metallothionein (MT) is a low-molecular-weight protein with high metal-ion affinity. If the intracellular concentrations of metals are too high or if toxic metals are present within the cell, then the synthesis of MTs is induced and generated. In our previous work, it was found that the prepared hydrolysate-metal element complex showed obvious depuration activity of heavy metals (Cd(2+) ) from blue mussel (Mytilus edulis). This study provided further the depuration mechanisms of Cd(2+) from mussel (M. edulis), in particular to the role of MT and its chelate during the depuration process.

 

Gueguen, Y., S. Denis, et al. (2017). "Response of the pearl oyster Pinctada margaritifera to cadmium and chromium: Identification of molecular biomarkers." Mar Pollut Bull 118(1-2): 420-426.

                This study was designed to identify in the pearl oyster Pinctada margaritifera, used as a bio-accumulator, molecular biomarkers for the presence of heavy metals in the lagoon environment. Pearl oysters were exposed to 2 concentrations (1 and 10mugL(-1)) of cadmium (Cd) and chromium (Cr) compared to a control. Twelve target genes encoding proteins potentially involved in the response to heavy metal contamination with antioxidant, detoxification or apoptosis activities were selected. P. margaritifera accumulated Cd but not Cr, and mortality was related to the amount of Cd accumulated in tissues. In response to Cd-Cr contamination, metallothionein (MT) was significantly up-regulated by Cd-Cr at both concentrations, while 7 others (SOD, CAT, GPX, GSTO, GSTM, CASP, MDR) were down-regulated. Based on the development of these molecular tools, we propose that the pearl oyster, P. margaritifera, could be used as a sentinel species for heavy metal contamination in the lagoons of tropical ecosystems.

 

Peric, L., V. Nerlovic, et al. (2017). "Variations of biomarkers response in mussels Mytilus galloprovincialis to low, moderate and high concentrations of organic chemicals and metals." Chemosphere 174: 554-562.

                The changes of acetylcholinesterase activity (AChE), metallothioneins content (MTs), catalase activity (CAT) and lipid peroxidation (LPO) were assessed after 4 days exposure of mussels Mytilus galloprovincialis to a wide range of sublethal concentrations of chlorpyrifos (CHP, 0.03-100 mug/L), benzo(a)pyrene (B(a)P, 0.01-100 mug/L), cadmium (Cd, 0.2-200 mug/L) and copper (Cu, 0.2-100 mug/L). The activity of AChE in the gills decreased after exposure to CHP and Cu, whereas no change of activity was detected after exposure to B(a)P and Cd. Both induction and decrease of MTs content in digestive gland occurred after exposure to CHP and B(a)P, while a marked increase was evident at highest exposure concentrations of Cd. The content of MTs progressively decreased of MTs with increasing concentration of Cu. CAT activity and LPO in the gills did not change after exposure to any of the chemicals. The results demonstrate different response profile in relation to the type of chemical compound, and highlight the potential implications for evaluation of biological effect of contaminants in marine environment. Furthermore, the AChE activity in the gills and MTs content in the digestive gland could be modulated by CHP and Cu at environmentally relevant concentrations indicating the potential risks of short-term transient mussels exposure that may occur due to run-off from land or accidental releases.

 

Reategui-Zirena, E. G., A. D. French, et al. (2017). "Cadmium Compartmentalization in the Pulmonate Snail Lymnaea stagnalis: Improving Our Understanding of Exposure." Arch Environ Contam Toxicol 72(4): 575-585.

                In ecotoxicology, analytical compartmentalization analysis can be used to better understand metal sequestration and detoxification. Metals are typically found in two main compartments, biologically detoxified metal (BDM) and metal sensitive fractions (MSF). The purpose of this study was to analyze the subcellular distribution of cadmium (Cd) in Lymnaea stagnalis. Adult snails were exposed to three concentrations of Cd for 56 days as part of a global ring test for L. stagnalis. At the end of the 56-day exposure, organisms were separated in two sections (viscera and foot). Each section was subsequently divided by differential centrifugation into five total fractions including (metal rich granules, debris, Organelles, heat denatured proteins, and heat stable proteins) followed by Cd analysis. The concentration in each compartment, BDM, MSF, and bioconcentration factors were estimated as well. There was significantly higher bioconcentration of Cd in the viscera section compared with the foot. Cadmium accumulation in all five fractions also increased with increasing exposure concentrations. Cadmium accumulated the most in the heat denatured protein fraction (enzymes) and accumulated the least in the heat stable protein fraction (metallothionein-like proteins). The MSF compartment (~65%) was in higher proportion than the BDM (~30%), but only in the lowest Cd exposure concentration was there a significant difference between these compartments. The results indicated that, in general, there was more Cd accumulated in the metal sensitive fractions, and that the detoxification mechanisms were not efficient enough to avoid toxicity at the two highest concentrations. This study provides evidence that improves our understanding of Cd tissue distribution in freshwater gastropods.

 

Balbi, T., R. Fabbri, et al. (2017). "Seasonal variability of different biomarkers in mussels (Mytilus galloprovincialis) farmed at different sites of the Gulf of La Spezia, Ligurian sea, Italy." Mar Pollut Bull 116(1-2): 348-356.

                Mussels (Mytilus spp.) are worldwide utilized in marine biomonitoring by a multi-biomarker approach. However, for a correct interpretation of different biomarker responses, information is needed on their natural seasonal variability due to environmental/physiological factors. In this work, the seasonal variations of different biomarkers were investigated in M. galloprovincialis from 4 different sites from the gulf of La Spezia (Ligurian sea, Italy), an intensive rearing area in the north-western Mediterranean near La Spezia harbor, an important commercial and touristic port. Lysosomal membrane stability-LMS, stress on stress-SoS, phagocytosis, tissue metallothionein-MT content, oxidative stress related enzyme activities (GST, catalase), and nitric oxide (NO) production were evaluated. The results underline the importance of LMS and SoS as core descriptors of the mussel health status in relation to seasonal variations in temperature and reproduction. These data represent the baseline information for ongoing biomonitoring studies related to dredging activities in this area.

 

Jimeno-Romero, A., E. Bilbao, et al. (2017). "Digestive cell lysosomes as main targets for Ag accumulation and toxicity in marine mussels, Mytilus galloprovincialis, exposed to maltose-stabilised Ag nanoparticles of different sizes." Nanotoxicology 11(2): 168-183.

                Bioavailability and toxicity of maltose-stabilised AgNPs of different sizes (20, 40 and 100 nm) in mussels were compared with bulk and aqueous forms of the metal through a two-tier experimental approach. In the first tier, mussels were exposed for 3 d to a range of concentrations (0.75, 75, 750 mug Ag/l) in the form of Ag20-Mal, Ag40-Mal, Ag100-Mal, bulk Ag and aqueous Ag (as AgNO3), as well as to the concentrations of maltose used in the formulation of NPs. Mortality, bioaccumulation, tissue and cell distribution and lysosomal responses were investigated. In the second tier, mussels were exposed for 21 d to Ag20-Mal, Ag100-Mal, bulk Ag and aqueous Ag at the lowest effective concentration selected after Tier 1 (0.75 mug Ag/l), biomarkers and toxicopathic effects were investigated. Aqueous Ag was lethal within 3 d at 75 mug Ag/l; Ag NPs or bulk Ag did not produce significant mortality at 750 mug Ag/l. Ag accumulation was limited and metallothionein gene transcription was not regulated although metal accumulation occurred in digestive, brown and stomach epithelial cells and in gut lumen after exposure to AgNPs and aqueous Ag starting at low concentrations after 1 d. Electrondense particles (<10 nm) in lysosomes and residual bodies after exposure to AgNPs contained Ag and S (X-ray). Intralysosomal metal accumulation and lysosomal membrane destabilisation were enhanced after exposure to all the forms of Ag and more marked after exposure to Ag20-Mal than to larger NPs. 21 d exposure to AgNPs provoked digestive cell loss and loss of digestive gland integrity, resulting in atrophy-necrosis in digestive alveoli and oedema/hyperplasia in gills (Ag NP), vacuolisation in digestive cells (aqueous Ag) and haemocyte infiltration of connective tissue (all treatments). Intralysosomal metal accumulation, lysosomal responses and toxicopathic effects are enhanced at decreasing sizes and appear to be caused by Ag(+ )ions released from NPs, although the metal was not substantially accumulated.

 

Zimmermann, S., N. Ruchter, et al. (2017). "Nanoparticulate versus ionic silver: Behavior in the tank water, bioaccumulation, elimination and subcellular distribution in the freshwater mussel Dreissena polymorpha." Environ Pollut 222: 251-260.

                Zebra mussels (Dreissena polymorpha) were exposed to polyvinylpyrrolidone (PVP)-coated silver nanoparticles (AgNP; hydrodynamic diameter 80 nm; solid diameter 50 nm) to investigate the behavior of Ag in the tank water with respect to its uptake, bioaccumulation, elimination and subcellular distribution in the mussel soft tissue. Parallel experiments were performed with ionic Ag (AgNO3) to unravel possible differences between the metal forms. The recovery of the applied Ag concentration (500 mug/L) in the tank water was clearly affected by the metal source (AgNP < AgNO3) and water type (reconstituted water < tap water). Filtration (<0.45 mum) of water samples showed different effects on the quantified metal concentration depending on the water type and Ag form. Ag accumulation in the mussel soft tissue was neither influenced by the metal source nor by the water type. Ag concentrations in the mussel soft tissue did not decrease during 14 days of depuration. For both metal forms the Ag distribution within different subcellular fractions, i.e. metal-rich granules (MRG), cellular debris, organelles, heat-sensitive proteins (HSP) and metallothionein-like proteins (MTLP), revealed time-dependent changes which can be referred to intracellular Ag translocation processes. The results provide clear evidence for the uptake of Ag by the mussel soft tissue in nanoparticulate as well as in ionic form. Thus, zebra mussels could be used as effective accumulation indicators for environmental monitoring of both Ag forms.

 

Boulajfene, W., E. Strogyloudi, et al. (2017). "Bio-monitoring of metal impact on metallothioneins levels in the gastropod Phorcus turbinatus (Born, 1778) in the northeastern and the eastern coasts of Tunisia." Mar Pollut Bull 120(1-2): 274-285.

                This work is an evaluation of metal contamination degree in Phorcus turbinatus and a monitoring of metals impact on metallothioneins functioning. The gastropod was sampled from six stations along the northeastern and eastern coasts of Tunisia during four seasons (2014-2015). Our results suggested that sedimentological metallic contents (Copper, Zinc and Cadmium) vary significantly depending on stations and not significantly according to seasons except for copper. Nevertheless, differences were no significant between stations and significant between seasons as for ambient factors, metallic intrinsic contents and metallothioneins rates. The monodonta tissue seems to be enriched in the four analyzed metals (Cu, Zn, Cd and Hg) and this species seems to have an ability to accumulate metals. Metal effect on the protein induction may be linked to physicochemical factors (temperature, O2 and Cu contents in sediment). In fact, metallothioneins levels were positively correlated to the dissolved oxygen rates and negatively linked to temperature.

 

Niederwanger, M., S. Calatayud, et al. (2017). "Biomphalaria glabrata Metallothionein: Lacking Metal Specificity of the Protein and Missing Gene Upregulation Suggest Metal Sequestration by Exchange Instead of through Selective Binding." Int J Mol Sci 18(7).

                The wild-type metallothionein (MT) of the freshwater snail Biomphalaria glabrata and a natural allelic mutant of it in which a lysine residue was replaced by an asparagine residue, were recombinantly expressed and analyzed for their metal-binding features with respect to Cd(2+), Zn(2+) and Cu(+), applying spectroscopic and mass-spectrometric methods. In addition, the upregulation of the Biomphalaria glabrataMT gene was assessed by quantitative real-time detection PCR. The two recombinant proteins revealed to be very similar in most of their metal binding features. They lacked a clear metal-binding preference for any of the three metal ions assayed-which, to this degree, is clearly unprecedented in the world of Gastropoda MTs. There were, however, slight differences in copper-binding abilities between the two allelic variants. Overall, the missing metal specificity of the two recombinant MTs goes hand in hand with lacking upregulation of the respective MT gene. This suggests that in vivo, the Biomphalaria glabrata MT may be more important for metal replacement reactions through a constitutively abundant form, rather than for metal sequestration by high binding specificity. There are indications that the MT of Biomphalaria glabrata may share its unspecific features with MTs from other freshwater snails of the Hygrophila family.

 

Palacios, O., E. Jimenez-Marti, et al. (2017). "Analysis of Metal-Binding Features of the Wild Type and Two Domain-Truncated Mutant Variants of Littorina littorea Metallothionein Reveals Its Cd-Specific Character." Int J Mol Sci 18(7).

                After the resolution of the 3D structure of the Cd(9)-aggregate of the Littorina littorea metallothionein (MT), we report here a detailed analysis of the metal binding capabilities of the wild type MT, LlwtMT, and of two truncated mutants lacking either the N-terminal domain, Lltr2MT, or both the N-terminal domain, plus four extra flanking residues (SSVF), Lltr1MT. The recombinant synthesis and in vitro studies of these three proteins revealed that LlwtMT forms unique M(9)-LlwtMT complexes with Zn(II) and Cd(II), while yielding a complex mixture of heteronuclear Zn,Cu-LlwtMT species with Cu(I). As expected, the truncated mutants gave rise to unique M(6)-LltrMT complexes and Zn,Cu-LltrMT mixtures of lower stoichiometry with respect to LlwtMT, with the SSVF fragment having an influence on their metal binding performance. Our results also revealed a major specificity, and therefore a better metal-coordinating performance of the three proteins for Cd(II) than for Zn(II), although the analysis of the Zn(II)/Cd(II) displacement reaction clearly demonstrates a lack of any type of cooperativity in Cd(II) binding. Contrarily, the analysis of their Cu(I) binding abilities revealed that every LlMT domain is prone to build Cu(4)-aggregates, the whole MT working by modules analogously to, as previously described, certain fungal MTs, like those of C. neoformans and T. mesenterica. It is concluded that the Littorina littorea MT is a Cd-specific protein that (beyond its extended binding capacity through an additional Cd-binding domain) confers to Littorina littorea a particular adaptive advantage in its changeable marine habitat.

 

Oaten, J. F. P., M. D. Hudson, et al. (2017). "Seasonal effects to metallothionein responses to metal exposure in a naturalised population of Ruditapes philippinarum in a semi-enclosed estuarine environment." Sci Total Environ 575: 1279-1290.

                The Manila clam (Ruditapes philippinarum), an invasive species in Northern Europe, can be used as a bioindicator of metal pollution. Seasonal effects on metallothionein (MT) production have not been considered in this species at the northernmost extent of its European distribution. This study assesses the annual seasonal effects on MT and metal concentrations in R. philippinarum from Poole Harbour, UK. R. philippinarum were collected in winter, spring, summer, and autumn throughout 2015, and MT and metal concentrations, as well as biotic and abiotic variables, were quantified. During winter, linear regression analysis showed significant positive relationships between tissue metal and MT concentrations. However, during spring and summer, these relationships were mostly insignificant. MT concentrations during spring had significant positive relationships with tissue and whole weight. Significant positive relationships were also observed between MT and condition index, during summer. During spring and summer, biotic factors seem to override the role of MT as a detoxification mechanism for metal exposure in this species. This is probably due to an increase in MT concentration in spring caused by gametogenesis, associated with increased tissue weight as the gonads expand. A depletion of energy resources, or physical stressors such as heat, may be attributed to the reduced MT production in clams of poor body condition in summer. The evidence from this study suggests that MT may only be a useful biomarker of metal pollution during winter in R. philippinarum in the UK. This verifies the natural variability of MT in this species at high latitudes, and highlights the potential and limits to a widely available bioindicator of metal pollution.

 

Martinez-Paz, P., M. Morales, et al. (2017). "Cadmium in vivo exposure alters stress response and endocrine-related genes in the freshwater snail Physa acuta. New biomarker genes in a new model organism." Environ Pollut 220(Pt B): 1488-1497.

                The freshwater snail Physa acuta is a sensitive organism to xenobiotics that is appropriate for toxicity testing. Cadmium (Cd) is a heavy metal with known toxic effects on several organisms, which include endocrine disruption and activation of the cellular stress responses. There is scarce genomic information on P. acuta; hence, in this work, we identify several genes related to the hormonal system, the stress response and the detoxification system to evaluate the effects of Cd. The transcriptional activity of the endocrine-related genes oestrogen receptor (ER), oestrogen-related receptor (ERR), and retinoid X receptor (RXR), the heat shock proteins genes hsp70 and hsp90 and a metallothionein (MT) gene was analysed in P. acuta exposed to Cd. In addition, the hsp70 and hsp90 genes were also evaluated after heat shock treatment. Real-time reverse transcriptase-polymerase chain reaction (qRT-PCR) analysis showed that Cd presence induced a significant increase in the mRNA levels of ER, ERR and RXR, suggesting a putative mode of action that could explain the endocrine disruptor activity of this heavy metal at the molecular level on Gastropoda. Moreover, the hsp70 gene was upregulated after 24-h Cd treatment, but the hsp90 gene expression was not affected. In contrast, the hsp70 and hsp90 genes were strongly upregulated during heat shock response. Finally, the MT gene expression showed a non-significant variability after Cd exposure. In conclusion, this study provides, for the first time, information about the effects of Cd on the endocrine system of Gastropoda at the molecular level and offers new putative biomarker genes that could be useful in ecotoxicological studies, risk assessment and bioremediation.

 

Ozkan, D., M. Dagdeviren, et al. (2017). "Multi-Biomarker Responses After Exposure to Pollution in the Mediterranean Mussels (Mytilus galloprovincialis L.) in the Aegean Coast of Turkey." Bull Environ Contam Toxicol 98(1): 46-52.

                In this study, sublethal effects on the Mediterranean mussels (Mytilus galloprovincialis L.) collected from the Aegean coast of Turkey were determined. Enzymes such as glutathione-S-transferase (GST), superoxide dismutase (SOD), catalase (CAT), and acetylcholinesterase (AChE), metallothionein (MT) mRNA expressions, thiobarbituric acid reactive substances (TBARS) contents, determination of 14 heavy metals and micronucleus frequency were selected as multibiomarkers. Results show that heavy metals and an increase in the level of MT gene expression have been determined in tissues of mussels collected from all stations. The GST, SOD and CAT enzymes were increased in mussels of Aliaga and Old Foca, compared to the mussels of Urla, while it was showed inhibition at AChE levels. Extensive LP is determined on mussels of Aliaga. It was determined that mussels in Aliaga region have exposed more oxidative stress than Old Foca and Urla. These biomarkers were carried out for the first time in these stations to assess environmental quality.

 

Nardi, A., L. F. Mincarelli, et al. (2017). "Indirect effects of climate changes on cadmium bioavailability and biological effects in the Mediterranean mussel Mytilus galloprovincialis." Chemosphere 169: 493-502.

                Despite the great interest in the consequences of climate change on the physiological functioning of marine organisms, indirect and interactive effects of rising temperature and pCO2 on bioaccumulation and responsiveness to environmental pollutants are still poorly explored, particularly in terms of cellular mechanisms. According to future projections of temperature and pH/pCO2, this study investigated the main cellular pathways involved in metal detoxification and oxidative homeostasis in Mediterranean mussels, Mytilus galloprovincialis, exposed for 4 weeks to various combinations of two levels of pH/pCO2 (8.2/ approximately 400 muatm and 7.4/ approximately 3000 muatm), temperature (20 and 25 degrees C), and cadmium addition (0 and 20 mug/L). Bioaccumulation was increased in metal exposed organisms but it was not further modulated by different temperature and pH/pCO2 combinations. However, interactions between temperature, pH and cadmium had significant effects on induction of metallothioneins, responses of the antioxidant system and the onset of oxidative damages, which was tissue dependent. Multiple stressors increased metallothioneins concentrations in the digestive gland revealing different oxidative effects: while temperature and cadmium enhanced glutathione-dependent antioxidant protection and capability to neutralize peroxyl radicals, the metal increased the accumulation of lipid peroxidation products under acidified conditions. Gills did not reveal specific effects for different combinations of factors, but a general stress condition was observed in this tissue after various treatments. Significant variations of immune system were mainly caused by increased temperature and low pH, while co-exposure to acidification and cadmium enhanced metal genotoxicity and the onset of permanent DNA damage in haemocytes. Elaboration of the whole biomarker data in a cellular hazard index, corroborated the synergistic effects of temperature and acidification which increased the toxicological effects of cadmium. The overall results confirmed that climate change could influence ecotoxicological effects of environmental contaminants, highlighting the importance of a better knowledge of cellular mechanisms to understand and predict responsiveness of marine organisms to such multiple stressors.

 

Giannetto, A., M. Maisano, et al. (2017). "Effects of Oxygen Availability on Oxidative Stress Biomarkers in the Mediterranean Mussel Mytilus galloprovincialis." Mar Biotechnol (NY) 19(6): 614-626.

                In aquatic environments, hypoxia and oxygen-deficient areas are increasing worldwide. Transitions in oxygen levels can influence the production of reactive oxygen species (ROS), eventually leading to oxidative stress. The transcriptional response of oxidative stress biomarkers was evaluated by qPCR in gill tissue from Mytilus galloprovincialis experimentally subjected to 48-h air exposure followed by 48-h re-oxygenation, as compared to normoxic control mussels. Superoxide dismutases (CuZnsod and Mnsod), catalase (cat), and glutathione S-transferase (gst) were over-expressed early after 8-h air exposure and returned to normoxic levels during re-oxygenation. Moreover, the mRNAs and protein expression patterns of heat shock proteins (HSP70 and HSP90) and metallothioneins (MT-10 and MT-20) were modulated by oxygen availability with increased levels during re-oxygenation suggesting the participation of these cytoprotective mechanisms in the physiological oxidative stress response when oxygen concentration was restored. Overall, the observed modulation of the oxidative stress-related and general stress genes indicates that M. galloprovincialis responds to changes in oxygen availability enhancing the antioxidant potential under low oxygen conditions for dealing with the oxidative burst during future re-oxygenation. The present investigation brings further insights in understanding how intertidal molluscs cope with short-term oxygen variations and gives useful biomarkers for environmental monitoring of hypoxic areas that are predicted to occur in the next future.

 

Benito, D., M. Niederwanger, et al. (2017). "Successive Onset of Molecular, Cellular and Tissue-Specific Responses in Midgut Gland of Littorina littorea Exposed to Sub-Lethal Cadmium Concentrations." Int J Mol Sci 18(8).

                Cadmium (Cd) is one of the most harmful metals, being toxic to most animal species, including marine invertebrates. Among marine gastropods, the periwinkle (Littorina littorea) in particular can accumulate high amounts of Cd in its midgut gland. In this organ, the metal can elicit extensive cytological and tissue-specific alterations that may reach, depending on the intensity of Cd exposure, from reversible lesions to pathological cellular disruptions. At the same time, Littorina littorea expresses a Cd-specific metallothionein (MT) that, due to its molecular features, expectedly exerts a protective function against the adverse intracellular effects of this metal. The aim of the present study was, therefore, to assess the time course of MT induction in the periwinkle's midgut gland on the one hand, and cellular and tissue-specific alterations in the digestive organ complex (midgut gland and digestive tract) on the other, upon exposure to sub-lethal Cd concentrations (0.25 and 1 mg Cd/L) over 21 days. Depending on the Cd concentrations applied, the beginning of alterations of the assessed parameters followed distinct concentration-dependent and time-dependent patterns, where the timeframe for the onset of the different response reactions became narrower at higher Cd concentrations compared to lower exposure concentrations.

 

Niederwanger, M., M. Dvorak, et al. (2017). "Challenging the Metallothionein (MT) Gene of Biomphalaria glabrata: Unexpected Response Patterns Due to Cadmium Exposure and Temperature Stress." Int J Mol Sci 18(8).

                Metallothioneins (MTs) are low-molecular-mass, cysteine-rich, metal binding proteins. In most animal species, they are involved in metal homeostasis and detoxification, and provide protection from oxidative stress. Gastropod MTs are highly diversified, exhibiting unique features and adaptations like metal specificity and multiplications of their metal binding domains. Here, we show that the MT gene of Biomphalaria glabrata, one of the largest MT genes identified so far, is composed in a unique way. The encoding for an MT protein has a three-domain structure and a C-terminal, Cys-rich extension. Using a bioinformatic approach involving structural and in silico analysis of putative transcription factor binding sites (TFBs), we found that this MT gene consists of five exons and four introns. It exhibits a regulatory promoter region containing three metal-responsive elements (MREs) and several TFBs with putative involvement in environmental stress response, and regulation of gene expression. Quantitative real-time polymerase chain reaction (qRT-PCR) data indicate that the MT gene is not inducible by cadmium (Cd) nor by temperature challenges (heat and cold), despite significant Cd uptake within the midgut gland and the high Cd tolerance of metal-exposed snails.

 

Guo, R., L. Pan, et al. (2017). "A multi-biomarker approach in scallop Chlamys farreri to assess the impact of contaminants in Qingdao coastal area of China." Ecotoxicol Environ Saf 142: 399-409.

                A multi-biomarker approach was carried out to classify the environmental quality and the adverse effects of contaminants on scallop Chlamys farreri. The scallops were collected from three sampling stations in Qingdao coastal area of China in March, May, August and October of 2015. A suite of environmental factors and biomarkers, including temperature, salinity, pH, the concentrations of polycyclic aromatic hydrocarbons (PAHs), tetrabromobisphenol A (TBBPA) and metals (Cr, Mn, Cu, Zn, Cd, Pb, As) in seawater and soft tissue, mRNA expression of aryl hydrocarbon receptor (AhR) and P-glycoprotein (P-gp), 7-ethoxyresorufin O-deethylase (EROD), glutathione-S-transferase (GST), uridine-diphosphate-glucuronyl-transferase (UGT), sulfotransferase (SULT), metallothionein (MT), Superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT), lipid peroxidation (LPO) and protein carbonyl (PC) contents and DNA strand breaks, were measured in the gill and digestive gland. The results showed that S2 was the most polluted while S1 was identified the least polluted. Despite the differentiation of pollution levels and environmental parameters the selected biomarkers responded efficiently to contaminants. Principal component analysis (PCA) revealed that EROD for PAHs, AhR for TBBPA, MT for Cr, Pb and Mn, LPO and PC for Zn were the effective biomarkers respectively. This study demonstrated that the application of multi-biomarker approach in conjunction with the traditional analysis of environmental parameters and contaminants provided valuable information in environmental risk assessment.

 

Hanana, H., P. Turcotte, et al. (2017). "Comparative study of the effects of gadolinium chloride and gadolinium - based magnetic resonance imaging contrast agent on freshwater mussel, Dreissena polymorpha." Chemosphere 181: 197-207.

                Gadolinium (Gd), a metal of the lanthanide series used in various industrial and medical purposes is released into the aquatic environment. However, there are few aquatic toxicological studies addressing environmental effects of Gd which remains unknown in aquatic animals. Therefore, this study aimed to compare the effects of GdCl3 and a gadolinium-based MRI contrast agent (Omniscan), in zebra mussels after 28 days through a multibiomarker approach. Data revealed that after GdCl3 exposure, the mRNA level of metallothionein (MT) was modulated, those of cytochrome c oxidase (CO1) and superoxide dismutase (SOD) were increased, while gene expressions of catalase (CAT) and glutathione-S-transferase (GST) were downregulated. Furthermore, neither lipoperoxidation (LPO) nor genotoxicity were detected but only a decrease in the cyclooxygenase (COX) activity was observed. In addition, a significant correlation was found between biomarkers and bioaccumulated Gd, suggesting that mitochondrial and anti-inflammatory pathways were triggered with GdCl3. By opposition, the contrasting agent formulation induced downregulation of SOD, CAT, GST and CO1, a decrease in the level of LPO and an increase in the GST and COX activities. This suggests that the chelated form of Gd did not promote reactive oxygen species (ROS) production and exhibits antioxidant and proinflammatory effects in mussels. Therefore, this study revealed that ionic and the chelated form of Gd influence different cellular pathways to initiate cellular changes.

 

Boukadida, K., J. Cachot, et al. (2017). "Early and efficient induction of antioxidant defense system in Mytilus galloprovincialis embryos exposed to metals and heat stress." Ecotoxicol Environ Saf 138: 105-112.

                The present study aims to elucidate the stress response of early life stages of Mytilus galloprovincialis to the combine effects of selected metals and elevated temperature. For this purpose, we investigated the response of a large panel of oxidative stress markers such as catalase (CAT), superoxide dismutase (SOD), glutathione-S-transferase (GST) activities and lipid peroxidation (thiobarbituric acid reactive substrates (TBARS) concentration) and metallothionein accumulation (MT) as well as selected gene transcription level and metal accumulation in mussels larvae exposed to a sub-lethal concentration of Cu (9.54microg/L), Ag (2.55microg/L) and mixture of the two metals (Cu (6.67microg/L)+Ag (1.47microg/L)) along with a temperature gradient (18, 20 and 22 degrees C) for 48h. Cu and Ag applied as single or mixture were differentially accumulated in mussel larvae according to the exposure temperature. Sod, cat, gst and mt-10 gene transcription levels showed an important increase in larvae exposed to Cu, Ag or to the mix compared to the control condition at 18 degrees C. The same pattern but with higher induction levels was recorded in larvae co-exposed to metals at 20 degrees C. At 22 degrees C, a significant decrease in mRNA abundance of cat, gst and sod and a significant up-regulation of mts targets (mt10 and mt20) were observed. RESULTS: suggest that co-exposure to metals and moderate elevated temperature (20 and 22 degrees C) significantly increased the antioxidant enzyme activities of catalase (CAT), and glutathione-S-transferase (GST) and caused an increase of metal and metallothionein concentrations. In contrast, no significant change in lipid peroxidation products measured as TBARS content was observed indicating a protective response of anti-oxidative system. This study provides first evidences of the early and efficient protective response of antioxidant defense mechanisms in mussel's early life stages facing in multi stressors situations.

 

Moncaleano-Nino, A. M., S. A. Barrios-Latorre, et al. (2017). "Alterations of tissue metallothionein and vitellogenin concentrations in tropical cup oysters (Saccostrea sp.) following short-term (96h) exposure to cadmium." Aquat Toxicol 185: 160-170.

                Metallothioneins and vitellogenins are low molecular weight proteins that have been used widely in environmental monitoring as biomarkers of exposure and damage to metals and estrogenic compounds, respectively. In the present study, the responses of metallothionein and vitellogenin tissue concentrations were measured following acute (96h) aqueous exposures to cadmium in Saccostrea sp., a tropical cup oyster native to the Western Pacific Ocean that has recently established itself in the Caribbean Sea. Adult oysters (1.5-5.0cm shell length) collected from the municipal marina of Santa Marta, Colombia (Caribbean Sea) and acclimated for 5days in the laboratory, were exposed to Cd at five concentrations (0, 1, 10, 100 and 1000mug/L) and their tissues (gills, digestive gland and adductor muscle) were analyzed in pools of 5 individuals (3 replicates per concentration). Metallothioneins in digestive glands of oysters exposed to Cd concentrations>/=100mug/L showed a significant increase, from 8.0 to 14.8mug MT/mg total protein, whereas metallothionein concentrations in gills increased to lesser extent, and no differences were observed in adductor muscle. Metallothionein concentrations in digestive gland and gills correlated directly with whole soft tissue Cd concentrations (ranging from 2 to 297mug/g dw Cd). Vitellogenin in homogenates of oyster gonad tissue, after 96h of exposure to 1000mug/L Cd, were significantly lower (0.04mg P/g gonad) compared to control oysters (0.68mg P/g gonad), suggestive of an anti-estrogenic effect of Cd at high concentrations, whereas no significant changes in vitellogenin concentrations were observed at intermediate Cd exposure concentrations. This study confirms acute responses of metallothionein and vitellogenin concentrations in tissues of Saccostrea sp. exposed to high concentrations of cadmium (Cd>/=100mug/L, 96h). The present results are first step towards validating the use of these two proteins as biomarkers of metal exposure in this species.

 

Bighiu, M. A., E. Gorokhova, et al. (2017). "Metal contamination in harbours impacts life-history traits and metallothionein levels in snails." PLoS One 12(7): e0180157.

                Harbours with limited water exchange are hotspots of contaminant accumulation. Antifouling paints (AF) contribute to this accumulation by leaching biocides that may affect non-target species. In several leisure boat harbours and reference areas in the Baltic Sea, chronic exposure effects were evaluated using caging experiments with the snail Theodoxus fluviatilis. We analysed variations in ecologically relevant endpoints (mortality, growth and reproduction) in concert with variation in metallothionein-like proteins (MTLP) levels. The latter is a biomarker of exposure to metals, such as copper (Cu) and zinc (Zn), which are used in AF paints as active ingredient and stabilizer, respectively. In addition, environmental samples (water, sediment) were analysed for metal (Cu and Zn) and nutrient (total phosphorous and nitrogen) concentrations. All life-history endpoints were negatively affected by the exposure, with higher mortality, reduced growth and lower fecundity in the harbours compared to the reference sites. Metal concentrations were the key explanatory variables for all observed adverse effects, suggesting that metal-driven toxicity, which is likely to stem from AF paints, is a source of anthropogenic stress for biota in the harbours.

 

Kerkvliet, J., T. de Boer, et al. (2017). "Candidate genes for shell colour polymorphism in Cepaea nemoralis." PeerJ 5: e3715.

                The characteristic ground colour and banding patterns on shells of the land snail Cepaea nemoralis form a classic study system for genetics and adaptation as it varies widely between individuals. We use RNAseq analysis to identify candidate genes underlying this polymorphism. We sequenced cDNA from the foot and the mantle (the shell-producing tissue) of four individuals of two phenotypes and produced a de novo transcriptome of 147,397 contigs. Differential expression analysis identified a set of 1,961 transcripts that were upregulated in mantle tissue. Sequence variant analysis resulted in a set of 2,592 transcripts with single nucleotide polymorphisms (SNPs) that differed consistently between the phenotypes. Inspection of the overlap between the differential expression analysis and SNP analysis yielded a set of 197 candidate transcripts, of which 38 were annotated. Four of these transcripts are thought to be involved in production of the shell's nacreous layer. Comparison with morph-associated Restriction-site Associated DNA (RAD)-tags from a published study yielded eight transcripts that were annotated as metallothionein, a protein that is thought to inhibit the production of melanin in melanocytes. These results thus provide an excellent starting point for the elucidation of the genetic regulation of the Cepaea nemoralis shell colour polymorphism.

 

Falfushynska, H., L. Gnatyshyna, et al. (2016). "Interpopulational variability of molecular responses to ionizing radiation in freshwater bivalves Anodonta anatina (Unionidae)." Sci Total Environ 568: 444-456.

                Freshwater ecosystems are exposed to multiple anthropogenic stressors including chemical pollution and warming that can affect health of the resident organisms and their responses to novel challenges. We investigated the of in situ exposure history on molecular responses to a novel stressor, ionizing radiation, in unionid mollusks Anodonta anatina. Males from pristine (F-), agricultural (A-) sites and a cooling reservoir of a nuclear power plant (N-site) were exposed to acute low dose (2mGy) X-ray radiation followed by 14days of recovery (R-groups) or to control conditions (C-groups). Biomarkers of oxidative stress, geno-, cyto- and neurotoxicity were used to assess cellular injury and stress. Control group from the cooling reservoir (CN) had higher background levels of caspase-3 activity, metallothionein concentrations and nuclear lesions and lower levels of superoxide dismutase (SOD) and glutathione in the gills compared to other control groups (CF and CA). Irradiation induced cellular damage in mussels from all three sites including increased levels of nuclear lesions in hemocytes, depletion of caspase-3, suppression of superoxide dismutase and catalase activities, an increase of the lipid peroxidation and oxidized glutathione levels, as well as down-regulation of cholinesterase indicating neurotoxicity. The up-regulation of ethoxyresorufin-O-deethylase activity in the digestive gland and vitellogenin-like protein level in gonads were also found in radiation-exposed groups indicating feminization of males and disturbances of xenobiotic metabolism. The RA-group showed the greatest magnitude of radiation-induced stress responses compared to the other two groups. Overall, unionid mollusks, particularly those from a chronically polluted agricultural site, were highly sensitive to low-dose radiation (2mGy) indicating limitations of stress protection mechanisms to deal with multiple stressors.

 

Rodriguez-Iruretagoiena, A., A. Rementeria, et al. (2016). "Is there a direct relationship between stress biomarkers in oysters and the amount of metals in the sediments where they inhabit?" Mar Pollut Bull 111(1-2): 95-105.

                The effects exerted by metals in oysters are still a matter of debate and require more detailed studies. In this work we have investigated whether the health status of oysters are affected by the amount of metals present in the sediments of their habitat. Sediments and oysters were collected in the tidal part of the estuary of the Oka River (Basque Country), representative of other mesotidal, well mixed and short estuaries of the European Atlantic coast. The concentrations of 14 elements were determined in all the samples. Several biomarkers were also measured in the soft tissues of oysters. According to the concentrations found, the sediments were classified as non-toxic or slightly toxic. In good agreement, the histological alterations observed in oysters were not severe. Interestingly, in those sampling sites where the sediments showed relatively high metal concentrations, the metallic content in oysters was lower, and vice versa.

 

Benedetti, M., I. Lanzoni, et al. (2016). "Oxidative responsiveness to multiple stressors in the key Antarctic species, Adamussium colbecki: Interactions between temperature, acidification and cadmium exposure." Mar Environ Res 121: 20-30.

                High-latitude marine ecosystems are ranked to be among the most sensitive regions to climate change since highly stenothermal and specially adapted organisms might be seriously affected by global warming and ocean acidification. The present investigation was aimed to provide new insights on the sensitivity to such environmental stressors in the key Antarctic species, Adamussium colbecki, focussing also on their synergistic effects with cadmium exposure, naturally abundant in this area for upwelling phenomena. Scallops were exposed for 2 weeks to various combinations of Cd (0 and 40 mugL-1), pH (8.05 and 7.60) and temperature (-1 and +1 degrees C). Beside Cd bioaccumulation, a wide panel of early warning biomarkers were analysed in digestive glands and gills including levels of metallothioneins, individual antioxidants and total oxyradical scavenging capacity, onset of oxidative cell damage like lipid peroxidation, lysosomal stability, DNA integrity and peroxisomal proliferation. Results indicated reciprocal interactions between multiple stressors and their elaboration by a quantitative hazard model based on the relevance and magnitude of effects, highlighted a different sensitivity of analysed tissues. Due to cellular adaptations to high basal Cd content, digestive gland appeared more tolerant toward other prooxidant stressors, but sensitive to variations of the metal. On the other hand, gills were more affected by various combinations of stressors occurring at higher temperature.

 

Gagne, F., P. Turcotte, et al. (2016). "Elemental profiles of freshwater mussels treated with silver nanoparticles: A metallomic approach." Comp Biochem Physiol C Toxicol Pharmacol 188: 17-23.

                Nanoparticles released into the environment could pose a risk to resident organisms that feed on suspended particles in aquatic ecosystems. The purpose of this study was to examine the effects of silver nanoparticles (nanoAg) of different sizes in freshwater mussels using a multi-elemental (metallomic) approach in order to determine signature effects of nanoparticulate and ionic Ag. Mussels were exposed to three concentrations (0.8, 4 and 20mug/L) of 20-nm and 80-nm nanoAg and AgNO3 for 48h at 15 degrees C. After the exposure period, mussels were placed in clean, aerated water for a depuration step and analyzed for the following total elements in gill, digestive gland and gonad tissues: Al, Ag, As, Ba, Be, Ca, Cd, Co, Cr, Cu, Fe, K, Mg, Mn, Mo, Pb, Na, Ni, Se, Sr, Th, U, V and Zn. Metallothioneins (MT; digestive gland only) and lipid peroxidation (LPO) were also determined in gills, digestive glands and gonads. The 20-nm-diameter nanoAg was detected in all three tissues at 20mug/L, while the 80-nm nanoAg was detected more strongly in the digestive gland. Ionic Ag was found at higher levels in gills than in other tissues. Correlation analysis revealed that gonad Ag levels were significantly correlated with Al (r=0.28), V (r=0.28), Cr (r=0.31), Co (r=0.32), Se (r=0.34) and MT levels (r=0.28). Indeed, the MT levels in the digestive gland were significantly increased by 20-nm nanoAg (20mug/L) and 80-nm nanoAg (4mug/L) and AgNO3 (<0.8mug/L). LPO was observed in gills, digestive glands and even gonads for all Ag forms. Discriminant function analysis revealed that all forms of Ag differed from each other and from unexposed mussels, where ionic Ag was more closely related to the 80-nm-diameter nanoAg. Factorial analysis revealed that Ba, Ca, Co, Mn, Sr, U and Zn had consistently high factorial weights in all tissues; that explained 80% of the total variance. Moreover, the following elements showed strong correlations (r>0.7) with each other: Sr, Ba, Zn, Ca, Mg Cr, Mn and U. Comparisons of these elements with other elements showing low or no correlations (e.g., transition elements) revealed that these elements had significantly lower standard reduction potential and electronegativity, suggesting that stronger reducing elements were most influenced by the oxidizing effects of nanoAg and ionic Ag in tissues. Indeed, tissues with oxidative stress (LPO) had decreased levels for most of these reducing elements. We conclude that exposure to Ag nanoparticles produces a characteristic change in the elemental composition of gills, digestive gland and gonad tissues in freshwater mussels. Elements most responsive to oxidative stress were more influenced by both nanoAg and ionic Ag. Sr and Ba were readily decreased by Ag and appeared to respond more sensitively to nanoAg than to ionic Ag. The metallomic approach could contribute in the understanding of fundamental mode of action of nanoparticles in mussels.

 

Jenny, M. J., S. L. Payton, et al. (2016). "Phylogenetic Analysis of Molluscan Metallothioneins: Evolutionary Insight from Crassostrea virginica." J Mol Evol 83(3-4): 110-125.

                Mechanisms by which organisms genetically adapt to environmental conditions are of fundamental importance to studies of evolutionary biology and environmental physiology. Natural selection acts on existing genetic variation leading to adaptation through selection of new mutations that confer beneficial advantages to populations. The American oyster, Crassostrea virginica, is an excellent model to investigate interactions between environmental and ecological factors as driving forces for natural selection. A great example of this is represented by the diversity of C. virginica metallothioneins (CvMT), metal-binding proteins involved in homeostasis and tolerance, that have resulted from a series of duplication events to produce the greatest structural diversity of MT proteins found in a single species. We present phylogenetic evidence of two distinct ancestral beta-domain MTs that gave rise to a variety of betabeta and alphabeta CvMT proteins, as well as CvMT-II proteins consisting solely of one to four alpha-domains. Furthermore, we annotate the complete locus containing the paralogous CvMT-I, -II, and -IV genes, providing supporting evidence of a hypothesized series of exon and gene duplication events that gave rise to the various CvMT-I and -II isoforms. We also highlight unique MT expression profiles from four separate C. virginica populations to demonstrate differences in gene diversity and copy number which appear to be enriched in southeastern U.S. oyster populations. These observations contribute to a better understanding of the molecular mechanisms leading to adaptation in organisms that experience substantial environmental stress, with a specific focus on evolutionary adaptations of gene structure.

 

Wang, Z., Y. Shao, et al. (2016). "RNA-seq analysis revealed ROS-mediated related genes involved in cadmium detoxification in the razor clam Sinonovacula constricta." Fish Shellfish Immunol 57: 350-361.

                The razor clam Sinonovacula constricta is a eukaryotic benthic intertidal bivalve species that is tolerant to different heavy metals, such as cadmium ion (Cd(2+)). However, the mechanism by which S. constricta responds to Cd(2+)-induced stress remains unclear. In this study, eight transcriptome libraries were constructed and sequenced using razor clams exposed to Cd(2+) for 12 and 48 h. A total of 18,330 unigenes with an average length of 500 bp were annotated. Among these 18,330 unigenes, 582 and 649 displayed differential expression profiles at 12 and 48 h in the gill, respectively. The corresponding differential unigenes in the hepatopancreas were 1056 and 382. Gene Ontology annotation revealed that these unigenes were highly pronounced in metabolic process, cellular process, binding, and catalytic activity. Notably, ROS production-related genes, such as heat shock proteins 32, metallothionein, and glutathione, were synchronously enriched in all experimental samples with induced expression profiles, which was also validated by qPCR. Our results highlighted the relation between immune regulation and Cd(2+)-induced stress in razor clam and provided new insights into the molecular mechanisms of heavy toxicology.

 

Oliveira, L. F., M. T. Cabral, et al. (2016). "Metals bioaccumulation and biomarkers responses in the Neotropical freshwater clam Anodontites trapesialis: Implications for monitoring coal mining areas." Sci Total Environ 571: 983-91.

                As one of the most impactful industries, coal mining can promote several alterations at surrounding environment. In surface water, elevated concentrations of metals like Mn, Zn, Fe and Al are often observed. Thus, the aim of this study was to investigate the bioaccumulation and the sub-lethal effects of these metals on various organs of the Neotropical bivalve Anodontites trapesialis confined along a stream located near a coal mine, in order to assess a set of biomarkers that could be used for effectively monitoring coal mining areas. Clams were caged, for 96h, at two sites located upstream (Up1 and Up2) and two sites downstream (Dw1 and Dw2) from the mine. Metals bioaccumulation was determined in gills, mantle, digestive gland, muscle and hemolymph and the following biomarkers were measured in A. trapesialis tissues: total antioxidant capacity against peroxyl radicals, metallothionein content, lipid peroxidation (LPO), proteins carbonylation, glutathione S-transferase activity, superoxide dismutase activity and acetylcholinesterase (AChE) activity. The results showed that Al and Fe bioaccumulation in the gills and hemolymph, Al bioaccumulation in the mantle and muscle, increased LPO in the gills (Dw1 and Dw2) and mantle (Dw1), as well as reduced AChE activity in the muscle (Dw1 and Dw2) should be considered effective biomarkers for monitoring coal mining areas. A. trapesialis proved to be an efficient biological model, considering that biomarkers responses were observed in the clams after only 96h of confinement at Dw sites, accordingly this species could be a good candidate for monitoring Neotropical freshwaters.

 

Lee, S. Y. and Y. K. Nam (2016). "Transcriptional responses of metallothionein gene to different stress factors in Pacific abalone (Haliotis discus hannai)." Fish Shellfish Immunol 58: 530-541.

                A novel metallothionein (MT) gene from the Pacific abalone H. discus hannai was characterized and its mRNA expression patterns (tissue distribution, developmental expression and differential expression in responsive to various in vivo stimulatory treatments) were examined. Abalone MT shares conserved structural features with previously known gastropod orthologs at both genomic (i.e., tripartite organization) and amino acid (conserved Cys motifs) levels. The 5'-flanking regulatory region of abalone MT gene displayed various transcription factor binding motifs particularly including ones related with metal regulation and stress/immune responses. Tissue distribution and basal expression patterns of MT mRNAs indicated a potential association between ovarian MT expression and sexual maturation. Developmental expression pattern suggested the maternal contribution of MT mRNAs to embryonic and early larval developments. Abalone MT mRNAs could be significantly induced by various heavy metals in different tissues (gill, hepatopancreas, muscle and hemocyte) in a tissue- and/or metal-dependent fashion. In addition, the abalone MT gene was highly modulated in responsive to other non-metal, stimulatory treatments such as immune challenge (LPS, polyI:C and bacterial injections), hypoxia (decrease from normoxia 8 ppm-2 ppm), thermal elevation (increase from 20 degrees C to 30 degrees C), and xenobiotic exposure (250 ppb of 17alpha-ethynylestradiol and 0.25 ppb of 2,3,7,8-tetrachlorodibenzodioxin) where differential expression patterns were toward either up- or down-regulation depending on types of stimulations and tissues examined. Taken together, our results highlight that MT is a multifunctional effector playing in wide criteria of cellular pathways especially associated with development and stress responses in this abalone species.

 

Liu, X. and W. X. Wang (2016). "Antioxidant and detoxification responses of oysters Crassostrea hongkongensis in a multimetal-contaminated estuary." Environ Toxicol Chem 35(11): 2798-2805.

                The contaminated oysters discovered in the Pearl River Estuary (Guangdong province, China) contained high levels of metals in their tissues, especially Cu and Zn, indicating that this large and densely urbanized estuary in Southern China suffers from serious metal pollution. The present study aimed to investigate the impacts of multimetal pollution in the Pearl River Estuary on oyster antioxidant and detoxification systems. The responses of various biochemical biomarkers in the ecologically important oyster Crassostrea hongkongensis collected from 7 sites in the Pearl River Estuary were quantified. Significant correlations were demonstrated between the accumulation of Cu and Zn and oxidative stress (lipid peroxidation) and oxidative stress defenses (catalase, glutathione peroxidase) in the oyster gills. Significant correlations between the accumulation of Cd and Cu and detoxification (glutathione and glutathione transferase) in the gills were also documented. Interestingly, metallothionein concentrations were positively correlated with Cd, but negatively correlated with Cu, Ni, and Zn concentrations in the gills. These measurements indicated that Cu in the Pearl River Estuary induced various biochemical responses in the oysters and influenced the susceptibility of oysters to environmental stress. The present study has provided the first evidence of antioxidant and detoxification responses in native contaminated oysters from a field environment seriously contaminated by metals. Coupling biomarkers with tissue metal concentration measurements was a promising approach to identify the metals causing biological impacts in a multimetal-contaminated estuary. Environ Toxicol Chem 2016;35:2798-2805. (c) 2016 SETAC.

 

Leomanni, A., T. Schettino, et al. (2016). "Mercury induced haemocyte alterations in the terrestrial snail Cantareus apertus as novel biomarker." Comp Biochem Physiol C Toxicol Pharmacol 183-184: 20-7.

                The aim of the present work was to study the response of a suite of cellular and biochemical markers in the terrestrial snail Cantareus apertus exposed to mercury in view of future use as sensitive tool suitable for mercury polluted soil monitoring and assessment. Besides standardized biomarkers (metallothionein, acetylcholinesterase, and lysosomal membrane stability) novel cellular biomarkers on haemolymph cells were analyzed, including changes in the spread cells/round cells ratio and haemocyte morphometric alterations. The animals were exposed for 14 days to Lactuca sativa soaked for 1h in HgCl2 solutions (0.5 e 1 muM). The temporal dynamics of the responses were assessed by measurements at 3, 7 and 14 days. Following exposure to HgCl2 a significant alteration in the relative frequencies of round cells and spread cells was evident, with a time and dose-dependent increase of the frequencies of round cells with respect to spread cells. These changes were accompanied by cellular morphometric alterations. Concomitantly, a high correspondence between these cellular responses and metallothionein tissutal concentration, lysosomal membrane stability and inhibition of AChE was evident. The study highlights the usefulness of the terrestrial snail C. apertus as bioindicator organism for mercury pollution biomonitoring and, in particular, the use of haemocyte alterations as a suitable biomarker of pollutant effect to be included in a multibiomarker strategy.

 

Le, T. T. Y., S. Zimmermann, et al. (2016). "How does the metallothionein induction in bivalves meet the criteria for biomarkers of metal exposure?" Environ Pollut 212: 257-268.

                Metallothionein (MT) concentrations in the whole soft tissue or in a particular tissue of bivalves have widely been used in ecotoxicological studies and biomonitoring programmes. This approach is based on the reported results on the enhancement of MT induction in bivalves in response to metal exposure. The validity of using MT induction as a biomarker is briefly assessed in the present study. The sensitivity of MT induction in these organisms is questionable due to the high basal MT level as well as the high natural variability related to the effects of a number of biotic and abiotic factors, which are not well described yet. Moreover, the relationship between exposure to metals, the toxic effects of that exposure, and the appearance of MT in soft tissue, is not well characterized. A variety of factors may influence the appearance and distribution of MT: 1) the uneven distribution of metals in particular portions of the soft tissue and in particular subcellular compartments; 2) pre-exposure to metals, perhaps at non-toxic levels; 3) metal-metal competition and metal-protein interactions; and 4) tissue-specific induction, functions, and isoforms of MT. Therefore, attention is required when using MT induction in bivalves for assessment of metal exposure or consequent toxic effects. The MT concentration can be a reliable indicator only when it is considered in relation with metal uptake kinetics and subcellular partitioning while specifying the isoform of MT synthesised and considering various confounding factors. The kinetic turnover of MT may provide useful information on metal exposure and biological effects since it covers both the synthesis and breakdown of MT as well as the chemical species of metals accumulated and MT.

 

Marques, A., D. Pilo, et al. (2016). "Propensity to metal accumulation and oxidative stress responses of two benthic species (Cerastoderma edule and Nephtys hombergii): are tolerance processes limiting their responsiveness?" Ecotoxicology 25(4): 664-76.

                The chronic exposure of benthic organisms to metals in sediments can lead to the development of tolerance mechanisms, thus diminishing their responsiveness. This study aims to evaluate the accumulation profiles of V, Cr, Co, Ni, As, Cd, Pb and Hg and antioxidant system responses of two benthic organisms (Cerastoderma edule, Bivalvia; Nephtys hombergii, Polychaeta). This approach will provide clarifications about the ability of each species to signalise metal contamination. Organisms of both species were collected at the Tagus estuary, in two sites with distinct contamination degrees (ALC, slightly contaminated; BAR, highly contaminated). Accordingly, C. edule accumulated higher concentrations of As, Pb and Hg at BAR compared to ALC. However, antioxidant responses of C. edule were almost unaltered at BAR and no peroxidative damage occurred, suggesting adjustment mechanisms to the presence of metals. In contrast, N. hombergii showed a minor propensity to metal accumulation, only signalising spatial differences for As and Pb and accumulating lower concentrations of metals than C. edule. The differences in metal accumulation observed between species might be due to their distinctive foraging behaviour and/or the ability of N. hombergii to minimise the metal uptake. Despite that, the accumulation of As and Pb was on the basis of the polychaete antioxidant defences inhibition at BAR, including CAT, SOD, GR and GPx. The integrated biomarker response index (IBRv2) confirmed that N. hombergii was more affected by metal exposure than C. edule. In the light of current findings, in field-based studies, the information of C. edule as a bioindicator should be complemented by that provided by another benthic species, since tolerance mechanisms to metals can hinder a correct diagnosis of sediment contamination and of the system's health. Overall, the present study contributed to improve the lack of fundamental knowledge of two widespread and common estuarine species, providing insights of the metal accumulation profiles under a scenario of chronic contamination. Finally, this work provided useful information that can be applied in the interpretation of future environmental monitoring studies.

 

Velez, C., R. Freitas, et al. (2016). "Clams sensitivity towards As and Hg: A comprehensive assessment of native and exotic species." Ecotoxicol Environ Saf 125: 43-54.

                To assess the environmental impact of As and Hg, bioindicator organisms such as bivalves have been used. Nevertheless, few studies have assessed the impacts of As and Hg in Ruditapes decussatus and Ruditapes philippinarum, which are native and exotic species in Europe, respectively. The main goal of the present study was to assess elements' partitioning and detoxification strategies of R. decussatus and R. philippinarum. Both clams showed a higher capacity to bioconcentrate Hg (BCF 2.29-7.49), when compared to As (0.59-1.09). Furthermore, As accumulation in both species was similar in the soluble and insoluble fractions, while in both species the majority of Hg was found in the insoluble fraction. Clams exposed to As showed different detoxification strategies, since R. decussatus had higher ability to enhance antioxidant enzymes and metallothioneins in order to reduce toxicity, and R.philippinarum increased glutathione S-transferase Omega activity, that catalyzes monomethyl arsenate reduction, the rate-limiting reaction in arsenic biotransformation. When exposed to Hg, R. decussatus presented, higher synthesis of antioxidant enzymes and lower LPO, being able to better tolerate Hg than the exotic species R. philippinarum. Thus under relevant levels of As and Hg contamination our work evidenced the higher ability of R. decussatus to survive and inhabit coastal environments not heavily contaminated by Hg and As.

 

Rocha, T. L., T. Gomes, et al. (2016). "Subcellular partitioning kinetics, metallothionein response and oxidative damage in the marine mussel Mytilus galloprovincialis exposed to cadmium-based quantum dots." Sci Total Environ 554-555: 130-41.

                The environmental health impact of metal-based nanomaterials is of emerging concern, but their metabolism and detoxification pathways in marine bioindicator species remain unclear. This study investigated the role of subcellular partitioning kinetics, metallothioneins (MTs) response and oxidative damage (lipid peroxidation - LPO) in the marine mussel Mytilus galloprovincialis exposed to CdTe quantum dots (QDs) in comparison with its dissolved counterpart. Mussels were exposed to QDs and dissolved Cd for 21 days at 10 mug Cd L(-1) followed by a 50 days depuration. Higher Cd concentrations were detected in fractions containing mitochondria, nucleus and lysosomes, suggesting potential subcellular targets of QDs toxicity in mussel tissues. Tissue specific metabolism patterns were observed in mussels exposed to both Cd forms. Although MT levels were directly associated with Cd in both forms, QDs subcellular partitioning is linked to biologically active metal (BAM), but no increase in LPO occurred, while in the case of dissolved Cd levels are in the biologically detoxified metal (BDM) form, indicating nano-specific effects. Mussel gills showed lower detoxification capability of QDs, while the digestive gland is the major tissue for storage and detoxification of both Cd forms. Both mussel tissues were unable to completely eliminate the Cd accumulated in the QDs form (estimated half-life time>50 days), highlighting the potential source of Cd and QDs toxicity for human and environmental health. Results indicate tissue specific metabolism patterns and nano-specific effects in marine mussel exposed to QDs.

 

Okay, O. S., M. Ozmen, et al. (2016). "Heavy metal pollution in sediments and mussels: assessment by using pollution indices and metallothionein levels." Environ Monit Assess 188(6): 352.

                In the present work, the concentration of eight metals (Cd, Cr, Cu, Fe, Mn, Ni, Pb, Zn) was determined in the sediments and transplanted and native mussels (Mytilus galloprovincialis). The study was conducted in Turkish marinas, shipyards, and shipbreaking yards. The effect of metal pollution was evaluated by determining the levels of metallothionein (MT) in the mussels. The extent of contamination for each single metal was assessed by using the geoaccumulation index (I geo) and enrichment factor (EF). Whereas, to evaluate the overall metal pollution and effect, the pollution load index (PLI), modified contamination degree (mC d), potential toxicity response index (RI), mean effects range median (ERM) quotient (m-ERM-Q), and mean PEL quotient (m-PEL-Q) were calculated. The influence of different background values on the calculations was discussed. The results indicated a significant metal pollution caused by Cu, Pb, and Zn especially in shipyard and shipbreaking sites. Higher concentrations of MT were observed in the ship/breaking yard samples after the transplantation.

 

Acs, A., A. Vehovszky, et al. (2016). "Seasonal and size-related variation of subcellular biomarkers in quagga mussels (Dreissena bugensis) inhabiting sites affected by moderate contamination with complex mixtures of pollutants." Environ Monit Assess 188(7): 426.

                The size-related differences in subcellular biomarker responses were assessed in Dreissena bugensis mussels inhabiting harbours moderately affected by pollution with complex mixtures of heavy metals and polycyclic aromatic hydrocarbons (PAHs). Adult D. bugensis samples were collected from three harbours of Lake Balaton (Hungary) characterized by moderate shipping activity, and as reference site, from a highly protected remote area of the lake. Biomarkers of exposure (metallothioneins (MTs), ethoxyresorufin-o-deethylase (EROD)), oxidative stress (lipid peroxidation (LPO), DNA strand breaks (DNAsb)) and possible endocrine disruption (vitellogenin-like proteins (VTG)) were analysed in whole-tissue homogenates of differently sized groups of mussels in relation to environmental parameters and priority pollutants (heavy metals and polycyclic aromatic hydrocarbons). Integrated biomarker response (IBR) indices were calculated for biomarker responses gained through in situ measurements to signalize critical sites and to better distinguish natural tendencies from biological effects of contaminants. Biomarker responses showed close positive correlation in case of MT, EROD, LPO, and DNAsb and negative correlation with VTG levels with mussel shell length in autumn, when higher levels of biomarkers appeared, possibly due to natural lifecycle changes of animals.

 

de Oliveira, L. F., C. Santos, et al. (2016). "Biomarkers in the freshwater bivalve Corbicula fluminea confined downstream a domestic landfill leachate discharge." Environ Sci Pollut Res Int 23(14): 13931-42.

                Landfills represent a severe environmental problem mainly due to the generation of leachates, and this study aimed to evaluate sublethal effects of a domestic landfill leachate in the freshwater bivalve Corbicula fluminea. Clams were submitted to in situ tests along a stream, at three sites, representing increasing distances from the leachate discharge (Pq1, Pq2, and Pq3), for 1, 5, and 15 days. The following biomarkers were analyzed in the gills and digestive glands: 7-ethoxyresorufin-O-deethylase (EROD) and glutathione S-transferase (GST) activities, multixenobiotic resistance mechanism (MXR), total antioxidant capacity (TAC), reactive oxygen species (ROS), and lipid peroxidation (LPO). Metallothionein (MT) content was determined in the gills and DNA damage in hemocytes. The mortality rate of animals during in situ tests was reduced as the distance from the leachate discharge source increased. On the other hand, biomarker results showed sublethal effects on C. fluminea confined at all sites of PqS. GST, TAC, ROS, and DNA damage were the most significant biomarkers for this species and should be considered for future monitoring and assessment of freshwater environments located in landfill areas.

 

Lavradas, R. T., R. C. Rocha, et al. (2016). "Differential metallothionein, reduced glutathione and metal levels in Perna perna mussels in two environmentally impacted tropical bays in southeastern Brazil." Ecotoxicol Environ Saf 129: 75-84.

                Mussel farming is an important economic activity in Brazil, and these organisms are consumed by the majority of the population in most coastal zones in the country. However, despite the increasing pollution of aquatic ecosystems in Brazil, little is known about the biochemical activity in mussels in response to metal exposure. In this context, the aim of the present study was to investigate metal and metalloid exposure effects in Perna perna mussels, by determining metal levels, the induction of metallothionein (MT) synthesis, and oxidative stress, in the form of reduced glutathione (GSH) in 3 contaminated areas from the Guanabara Bay in comparison to a reference site, Ilha Grande Bay, both in summer and winter. Metal and metalloid concentrations were also compared to Brazilian and international guidelines, to verify potential health risks to human consumers. Mussels from all sampling sites were shown to be improper for human consumption due to metal contamination, including Ilha Grande Bay, which has previously been considered a reference site. Several statistically significant correlations and seasonal differences were observed between MT, GSH and metals and metalloids in both analyzed tissues. A Discriminant Canonical Analysis indicated that the digestive gland is a better bioindicator for environmental contamination by metals and metalloids in this species and offers further proof that MT variations observed are due to metal exposure and not oxidative stress, since GSH influence for both muscle tissue and the digestive glands was non-significant in this analysis. These results show that P. perna mussels are an adequate sentinel species for metal contamination with significant effects on oxidative stress and metal exposure biomarkers. To the best of our knowledge, this is the first study to report metals, metalloids, MT and GSH levels in the muscle tissue of this species.

 

Wang, C., J. Sheng, et al. (2016). "Molecular characterization and expression of metallothionein from freshwater pearl mussel, Hyriopsis schlegelii." Biosci Biotechnol Biochem 80(7): 1327-35.

                Two metallothionein genes (HsMT1 and HsMT2) were first identified and described from Hyriopsis schlegelii. The open reading frame of HsMT1 and HsMT2 were 216 and 222 bp, encoding a protein of 71 and 73 amino acid residues. The deduced amino acid sequences showed they contained parts of typical MT characteristics, apart from HsMT2 lacked Cys-Cys motifs. The phylogenetic tree showed HsMT1 shared a high similarity with that of other molluscs, but HsMT2 was split into a distinct group separated from known molluscan MTs. HsMT1 exhibited constitutive expression in all examined tissues and the highest expression occurred in hepatopancreas, however, nearly all HsMT2 was just detected in gonad. After Cd exposure, their mRNA levels presented similar expression patterns. The transgenic bacteria of HsMT1 showed higher tolerance than HsMT2 in Cd environment. It was implied that HsMT1 and HsMT2 were involved in metal response but HsMT2 might have other physiological functions.

 

Wang, Z., C. Feng, et al. (2016). "Subcellular partitioning profiles and metallothionein levels in indigenous clams Moerella iridescens from a metal-impacted coastal bay." Aquat Toxicol 176: 10-23.

                In this study, the effect of environmental metal exposure on the accumulation and subcellular distribution of metals in the digestive gland of clams with special emphasis on metallothioneins (MTs) was investigated. Specimens of indigenous Moerella iridescens were collected from different natural habitats in Maluan Bay (China), characterized by varying levels of metal contamination. The digestive glands were excised, homogenized and six subcellular fractions were separated by differential centrifugation procedures and analyzed for their Cu, Zn, Cd and Pb contents. MTs were quantified independently by spectrophotometric measurements of thiols. Site-specific differences were observed in total metal concentrations in the tissues, correlating well with variable environmental metal concentrations and reflecting the gradient trends in metal contamination. Concentrations of the non-essential Cd and Pb were more responsive to environmental exposure gradients than were tissue concentrations of the essential metals, Cu and Zn. Subcellular partitioning profiles for Cu, Zn and Cd were relatively similar, with the heat-stable protein (HSP) fraction as the dominant metal-binding compartment, whereas for Pb this fraction was much less important. The variations in proportions and concentrations of metals in this fraction along with the metal bioaccumulation gradients suggested that the induced MTs play an important role in metal homeostasis and detoxification for M. iridescens in the metal-contaminated bay. Nevertheless, progressive accumulation of non-essential metals (Cd, and especially Pb) resulting from "spillover" was observed in putative metal- sensitive (e.g., mitochondria and heat-denaturable protein (HDP)) or lysosome/microsome fractions, demonstrating that metal detoxification was incomplete and increased the toxicological risk to M. iridescens inhabiting the metal-impacted environments. Through multiple stepwise regression analysis, the induction of MTs was statistically correlated with the HSP concentrations of Cu, and to a lesser extent with Zn, and ultimately to the Cd concentrations, exhibiting significant dose-dependent relationships. Overall, these findings not only revealed the fates of accumulated metals, but scientifically favored an improved understanding of the detoxification at the subcellular level in response to metal accumulation, supporting the focus of metabolic availability assessment on the intracellular processes or events occurring within organisms.

 

Tsangaris, C., V. Moschino, et al. (2016). "Biochemical biomarker responses to pollution in selected sentinel organisms across the Eastern Mediterranean and the Black Sea." Environ Sci Pollut Res Int 23(2): 1789-804.

                Pollution effects were assessed by means of biochemical biomarkers (catalase, glutathione S-transferase and acetylcholinesterase activities, and metallothioneins content) in five species at selected coastal sites across the Eastern Mediterranean and the Black Sea. The mussel Mytilus galloprovincialis, a well-established sentinel species, was investigated in the Adriatic Sea, Aegean Sea, and Black Sea. The mussel Brachidontes pharaonis and the striped red mullet Mullus surmuletus were used in the Levantine Sea where M. galloprovincialis is not present. The white seabream Diplodus sargus sargus and the gastropod Rapana venosa were additionally sampled in the Adriatic and the Black Sea, respectively. Mussels showed catalase, glutathione S-transferase, and acetylcholinesterase responses to pollution in most geographical areas while the response of metallothioneins was restricted to a few sites. R. venosa showed marked responses of catalase and metallothioneins whereas both fish species did not generally exhibit variations in biomarker values among sites. The approach based on the reference deviation concept using the "Integrated Biological Responses version 2" index was useful for the interpretation of overall biomarker responses.

 

Liu, X. and W. X. Wang (2016). "Time changes in biomarker responses in two species of oyster transplanted into a metal contaminated estuary." Sci Total Environ 544: 281-90.

                The Jiulong Estuary in Southern China suffers from serious metal pollution, leading to the appearance of 'colored' oysters in this estuary. In this study, two species of oysters Crassostrea hongkongensis and Crassostrea angulata were transplanted to three sites in the Jiulong Estuary over a two-month period. The time-series changes of various biomarkers were measured, coupled with simultaneous quantification of metal bioaccumulation (Ag, Cd, Cr, Cu, Ni and Zn). Cu and Zn accumulation increased linearly and reached up to 2% and 1.5% dry tissue weight by the end of exposure. Negative correlations between the tissue Cu or Zn accumulation and catalase or superoxide dismutase activities strongly indicated that Cu and Zn in 'colored' oysters induced the adjustments of oyster antioxidant systems. Metallothionein (MT) detoxification was insufficient for sequestering all the absorbed metals and its concentrations in the oysters were suppressed following an initial increase, primarily due to the high metal accumulation in the tissues. Interestingly, gradual recoveries of lysosomal membrane stability after the initial strong inhibitions were observed in both oysters. We also documented an increasing 'watering' of oyster tissues presumably as a result of rupturing of tissue cells under metal stress. This study demonstrated the complexity of biomarker responses under field condition, therefore the time changes of biomarker responses to metals need to be considered in evaluating the biological impacts of metal pollution on estuarine organisms.

 

Baurand, P. E., R. Dallinger, et al. (2016). "Metallothionein gene expression in embryos of the terrestrial snail (Cantareus aspersus) exposed to cadmium and copper in the Bordeaux mixture." Environ Sci Pollut Res Int 23(4): 3068-72.

                The response specificity of three metallothionein (MT) genes (CdMT, CuMT and Cd/CuMT) was assessed after long-term exposure (20 days) of Cantareus aspersus eggs to cadmium (Cd) (2 to 6 mg/L) or to the fungicide Bordeaux mixture (BM) (2.5 and 7.5 g/L). MT gene expression measured by quantitative real-time PCR (qRT-PCR) revealed that in the unexposed embryos, the transcript levels of the three MT genes decreased significantly through embryonic development. However, the CdMT gene was strongly upregulated with increasing Cd exposure concentration, whereas the transcript levels of the other two genes increased less pronouncedly, but significantly above an exposure concentration of 4 mg Cd/L. Upon exposure to BM, all three MT genes were significantly upregulated above a BM concentration of 2.5 g/L. It is concluded that long-term Cd exposure in hatched snails induced patterns of MT gene expression that differed from those obtained after short-term exposure (24 h).

 

Baurand, P. E., R. Dallinger, et al. (2016). "Differential sensitivity of snail embryos to cadmium: relation to age and metallothionein gene expression." Environ Sci Pollut Res Int 23(4): 3062-7.

                The aim of this study was to determine whether cadmium (Cd) sensitivity of Cantareus aspersus embryos is age-dependent and influenced by metallothionein (MT) gene expression. Hatching success and the expression of three MT isoform genes (Ca-CdMT, Ca-CuMT and Ca-Cd/CuMT) were measured in embryos exposed to increasing Cd concentrations for 24 h starting on the sixth day of development. Isoform gene expression was quantified on days 7 and 12 after exposure. Results were compared to those of embryos exposed to the same conditions as above, but from the beginning of embryogenesis (day 0). Transcription of the Cd-specific MT gene (Ca-CdMT) was observed from the first day of development, whereas the two other genes did not respond to Cd at all. Overall, Cd sensitivity of embryos decreased with increasing age of development, as assessed by age-dependent increase of EC50 values for hatching rate, and increasing Cd threshold concentrations for Ca-CdMT expression.

 

Chalghmi, H., J. P. Bourdineaud, et al. (2016). "Transcriptomic, Biochemical, and Histopathological Responses of the Clam Ruditapes decussatus from a Metal-Contaminated Tunis Lagoon." Arch Environ Contam Toxicol 70(2): 241-56.

                This study was designed to investigate the molecular (transcriptional expression), biochemical (oxidative stress and neurotoxicity), and histopathological effects of metal contamination in the gill of clams (Ruditapes decussatus) sampled from the Tunis lagoon. The concentrations of five heavy metals (Cd, Pb, Hg, Cu, and Zn) in surface sediments and their accumulation in soft tissues of R. decussatus were evaluated in three sites (Z1, Z2, and Z3). A metal contamination state of Tunis lagoon sediments was noted with spatial variations with relatively high levels at Z2. Biomarker analyses showed an increase in glutathione S-transferase and catalase activities and lipid peroxidation levels and a decrease in acetylcholinesterase activity in the studied sites. Molecular investigation showed a significant overexpression of: cytochrome c oxidase subunit I, ribosomal RNA 16S, Cu/Zn superoxide dismutase, heat shock protein 70, and metallothioneins in the three sampling sites. Moreover, our data were correlated to severe and diverse histopathological alterations in the clam gills. The principal component analysis showed that the Z2 region is more affected by metal contamination than Z1 and Z3 regions. Current field results suggest the use of several combined biomarkers at different cell levels instead of individual ones in monitoring programs.

 

Giltrap, M., J. Ronan, et al. (2016). "Application of a weight of evidence approach utilising biological effects, histopathology and contaminant levels to assess the health and pollution status of Irish blue mussels (Mytilus edulis)." Mar Environ Res 122: 33-45.

                A weight of evidence (WOE) approach, integrating biological effects, mussel histopathology and tissue contaminant levels is proposed to evaluate mussel health and pollution status. Contaminant levels, histopathology and several biological effects (BEs) including Lysosomal membrane stability (LMS), acetylcholinesterase (AChe), metallothionein proteins (MT) and alkali labile phosphate (ALP), in Mytilus edulis are presented, improving the current knowledge base for these data. Potential links between histopathology, BEs and contaminants and ranking of sites are investigated with an integrated response (IR) indexing technique. Histopathological condition indices (Ih) in mussel digestive gland are used to calculate health indices. A spatial and temporal assessment is conducted at Irish coastal locations. Linear mixed effects modelling revealed effects of confounding factors such as reproductive condition on NRRT (gonad stage (p < 0.001)). Higher prevalence of inflammation, brown cells and epithelial thinning of the digestive gland was evident at Dublin and Wexford and this linked well with the Ih. Levels of contaminants were generally found to be low with few exceptions as were BE responses. Using the IR approach, Dublin was ranked as being most impacted while Shannon ranked the least impacted, this being consistent with the BE ranking.

 

Won, E. J., K. T. Kim, et al. (2016). "Target organs of the Manila clam Ruditapes philippinarum for studying metal accumulation and biomarkers in pollution monitoring: laboratory and in-situ transplantation experiments." Environ Monit Assess 188(8): 478.

                To characterize the target organs of the Manila clam Ruditapes philippinarum for use in environmental study, the accumulation of trace metals and three biomarkers was measured in different organs. Exposure with Cu and Pb carried out under laboratory conditions revealed a linear uptake of metals throughout the experimental period in each tissue. In particular, significant increase was observed in gills and mantle. The increase of intracellular reactive oxygen species showed the great potential of gills as a target tissue for both Cu and Pb exposure. The highest activity of glutathione S-transferase and their relative increase in activity were also observed in gills. Metallothionein-like protein levels, however, increased greatly in the digestive gland and mantle during Cu and Pb exposure, respectively, although all tissues, except the foot, showed significant changes after 24 h of metal exposure. In the field study, the highest concentration of metals was recorded in the gills and mantle, accounting for over 50 % of the total accumulated metal in all sites. Additionally, Cu and Pb increased significantly in these two organs, respectively. However, the order of accumulation rate in laboratory exposure was not concomitant with those of the lab-based study, suggesting that different routes of metal uptake and exposure duration induce distinct partitioning of metals and regulating system in R. philippinarum. These series of exposure studies demonstrated that gills, mantle, and digestive gland in R. philippinarum are potential target tissues in environmental monitoring study using metal concentrations and biomarkers.

 

Xu, L., X. Peng, et al. (2016). "Proteomic responses reveal the differential effects induced by cadmium in mussels Mytilus galloprovincialis at early life stages." Fish Shellfish Immunol 55: 510-5.

                Cadmium (Cd) has become an important metal contaminant and posed severe risk on the organisms in the coastal environments of the Bohai Sea. Marine mussel Mytilus galloprovincialis is widely distributed along the Bohai coast and consumed as seafood by local residents. Evidences indicate that the early stages of marine organisms are more sensitive to metal contaminants. In this study, we applied two-dimensional electrophoresis-based proteomics to characterize the biological effects of Cd (50 mug L(-1)) in the early life stages (D-shape larval and juvenile) of mussels. The different proteomic responses demonstrated the differential responsive mechanisms to Cd exposure in these two early life stages of mussels. In details, results indicated that Cd mainly induced immune and oxidative stresses in both D-shape larval and juvenile mussels via different pathways. In addition, the significant up-regulation of triosephosphate isomerase and metallothionein confirmed the enhanced energy demand and mobilized detoxification mechanism in D-shape larval mussels exposed to Cd. In juvenile mussels, Cd exposure also induced clear apoptosis. Overall, this work suggests that Cd is a potential immune toxicant to mussel M. galloprovincialis at early life stages.

 

Wlostowski, T., P. Kozlowski, et al. (2016). "Cadmium Accumulation and Pathological Alterations in the Midgut Gland of Terrestrial Snail Helix pomatia L. from a Zinc Smelter Area: Role of Soil pH." Bull Environ Contam Toxicol 96(4): 484-9.

                The purpose of this study was to determine whether cadmium (Cd) accumulation and toxicity in the midgut gland of Helix pomatia snails living in a Cd-contaminated area were related to soil pH. Toxic responses in the midgut gland (i.e., increased vacuolization and lipid peroxidation) occurred in H. pomatia snails exhibiting the highest Cd levels in the gland (265-274 microg/g dry wt) and living on acidic soil (pH 5.3-5.5), while no toxicity was observed in snails accumulating less Cd (90 microg/g) and ranging on neutral soil (pH 7.0), despite the fact that total soil Cd was similar in the two cases. The accumulation of Cd in the gland was directly related to the water extractable Cd in soil, which in turn correlated inversely with soil pH, indicating that this factor had a significant effect on tissue Cd. It appeared further that the occurrence of Cd toxicity was associated with low levels of metallothionein in the gland of snails ranging on acidic soil.

 

Pedrini-Martha, V., M. Niederwanger, et al. (2016). "Physiological, Diurnal and Stress-Related Variability of Cadmium-Metallothionein Gene Expression in Land Snails." PLoS One 11(3): e0150442.

                The terrestrial Roman snail Helix pomatia has successfully adapted to strongly fluctuating conditions in its natural soil habitat. Part of the snail's stress defense strategy is its ability to express Metallothioneins (MTs). These are multifunctional, cysteine-rich proteins that bind and inactivate transition metal ions (Cd(2+), Zn(2+), Cu(+)) with high affinity. In Helix pomatia a Cadmium (Cd)-selective, inducible Metallothionein Isoform (CdMT) is mainly involved in detoxification of this harmful metal. In addition, the snail CdMT has been shown to also respond to certain physiological stressors. The aim of the present study was to investigate the physiological and diurnal variability of CdMT gene expression in snails exposed to Cd and non-metallic stressors such as desiccation and oxygen depletion. CdMT gene expression was upregulated by Cd exposure and desiccation, whereas no significant impact on the expression of CdMT was measured due to oxygen depletion. Overall, Cd was clearly more effective as an inducer of the CdMT gene expression compared to the applied non-metallic stressors. In unexposed snails, diurnal rhythmicity of CdMT gene expression was observed with higher mRNA concentrations at night compared to daytime. This rhythmicity was severely disrupted in Cd-exposed snails which exhibited highest CdMT gene transcription rates in the morning. Apart from diurnal rhythmicity, feeding activity also had a strong impact on CdMT gene expression. Although underlying mechanisms are not completely understood, it is clear that factors increasing MT expression variability have to be considered when using MT mRNA quantification as a biomarker for environmental stressors.

 

Wong, Y. H., J. Sun, et al. (2015). "High-throughput transcriptome sequencing of the cold seep mussel Bathymodiolus platifrons." Sci Rep 5: 16597.

                Bathymodiolid mussels dominate hydrothermal vents, cold methane/sulfide-hydrocarbon seeps, and other sites of organic enrichment. Here, we aimed to explore the innate immune system and detoxification mechanism of the deep sea mussel Bathymodiolus platifrons collected from a methane seep in the South China Sea. We sequenced the transcriptome of the mussels' gill, foot and mantle tissues and generated a transcriptomic database containing 96,683 transcript sequences. Based on GO and KEGG annotations, we reported transcripts that were related to the innate immune system, heavy metal detoxification and sulfide metabolic genes. Our in-depth analysis on the isoforms of peptidoglycan recognition protein (PGRP) that have different cellular location and potentially differential selectivity towards peptidoglycan (PGN) from gram-positive and gram-negative bacteria were differentially expressed in different tissues. We also reported a potentially novel form of metallothionein and the production of phytochelatin in B. platifrons, which has not been reported in any of its coastal relative Mytilus mussel species. Overall, the present study provided new insights into heavy metal and sulfide metabolism in B. platifrons and can be served as the basis for future molecular studies on host-symbiont interactions in cold seep mussels.

 

Francois, G., D. Melanie, et al. (2015). "Effects of a municipal effluent on the freshwater mussel Elliptio complanata following challenge with Vibrio anguillarum." J Environ Sci (China) 37: 91-9.

                The purpose of this study was to examine the cumulative effects of exposure to a pathogenic bacteria and municipal effluent in the freshwater mussel Elliptio complanata. Mussels were exposed to increasing concentrations of an ozone-treated effluent at 15 degrees C for 7days. A sub-group of mussels was inoculated with Vibrio anguillarum and exposed to the same conditions as above. After the exposure period, mussels were collected to assess hemocyte count and viability, immunocompetence (phagocytosis and nitrite production), oxidative stress/inflammation (cyclooxygenase and lipid peroxidation) and oxygen radical/xenobiotic scavenging activity (metallothioniens, glutathione S-transferase). The results showed that mussels exposed to municipal effluent had increased hemocyte counts, phagocytosis, nitrites, lipid peroxidation and metallothioneins. In the inoculated mussels, the same responses were observed, in addition to cyclooxygenase and glutathione S-transferase activities. Multivariate analyses revealed that (1) the response pattern changed with effluent concentration, where increased responses observed at low effluent concentrations (>10%, V/V) were attenuated at higher effluent concentrations, (2) the effluent produced more pronounced changes in lipid peroxidation, metallothionein and hemocyte viability, and (3) the simultaneous presence of V. anguillarum led to more important changes in hemocyte count and viability and nitrite levels. In conclusion, the presence of V. anguillarum could alter the response of mussels to municipal effluent, which could lead to increased inflammation in mussels.

 

Karray, S., E. Tastard, et al. (2015). "Transcriptional response of stress-regulated genes to industrial effluent exposure in the cockle Cerastoderma glaucum." Environ Sci Pollut Res Int 22(22): 17303-16.

                This study assessed the responses of molecular biomarkers and heavy metal levels in Cerastoderma glaucum exposed for 1 week to two industrial effluents (1%) discharged into the Tunisian coastal area, F1 and F2, produced by different units of production of a phosphate treatment plant. A significant uptake of metals (Cd, Cu, Zn, and Ni) was observed in exposed cockles compared to controls, with an uptake higher for F1 than for F2. A decrease in LT50 (stress on stress test) was also observed after an exposure to the effluent F1. Treatments resulted in different patterns of messenger RNA (mRNA) expression of the different genes tested in this report. Gene transcription monitoring performed on seven genes potentially involved in the tolerance to metal exposure showed that for both exposures, mechanisms are rapidly and synchronically settled down to prevent damage to cellular components, by (1) handling and exporting out metal ions through the up-regulation of ATP-binding cassette xenobiotic transporter (ABCB1) and metallothionein (MT), (2) increasing the mRNA expression of antioxidant enzymes (catalase (CAT), superoxide dismutases, CuZnSOD and MnSOD), (3) protecting and/or repairing proteins through the expression of heat shock protein 70 (HSP70) mRNAs, and (4) increasing ATP production (through the up-regulation of cytochrome c oxidase 1 (CO1)) to provide energy for cells to tolerate stress exposure. The tools developed may be useful both for future control strategies and for the use of the cockle C. glaucum as a sentinel species.

 

Bebianno, M. J., C. G. Pereira, et al. (2015). "Integrated approach to assess ecosystem health in harbor areas." Sci Total Environ 514: 92-107.

                UNLABELLED: Harbors are critical environments with strategic economic importance but with potential environmental impact: health assessment criteria are a key issue. An ecosystem health status approach was carried out in Portimao harbor as a case-study. Priority and specific chemical levels in sediments along with their bioavailability in mussels, bioassays and a wide array of biomarkers were integrated in a biomarker index (IBR index) and the overall data in a weight of evidence (WOE) model. Metals, PAHs, PCBs and HCB were not particularly high compared with sediment guidelines and standards for dredging. Bioavailability was evident for Cd, Cu and Zn. Biomarkers proved more sensitive namely changes of antioxidant responses, metallothioneins and vittellogenin-like proteins. IBR index indicated that site 4 was the most impacted area. Assessment of the health status by WOE approach highlighted the importance of integrating sediment chemistry, bioaccumulation, biomarkers and bioassays and revealed that despite some disturbance in the harbor area, there was also an impact of urban effluents from upstream. CAPSULE ABSTRACT: Environmental quality assessment in harbors.

 

Falfushynska, H., L. Gnatyshyna, et al. (2015). "The effects of zinc nanooxide on cellular stress responses of the freshwater mussels Unio tumidus are modulated by elevated temperature and organic pollutants." Aquat Toxicol 162: 82-93.

                Nanoparticle toxicity is a growing concern in freshwater habitats. However, understanding of the nanoparticle effects on aquatic organisms is impeded by the lack of the studies of the nanoparticles effects in the environmentally relevant context of multiple stress exposures. Zinc oxide nanoparticles (n-ZnO) are widely used metal-based nanoparticles in electronics and personal care products that accumulate in aquatic environments from multiple non-point sources. In this study, we evaluated the effects of n-ZnO in a model organism, a mussel Unio tumidus, and the potential modulation of these effects by common co-occurring environmental stressors. Male U. tumidus were exposed for 14 days to n-ZnO (3.1 muM), Zn(2+) (3.1 muM), Ca-channel blocker nifedipine (Nfd 10 muM), combinations of n-ZnO and Nfd or n-ZnO and thiocarbamate fungicide Tattoo (Ta, 91 mug L(-1)) at 18 degrees C, and n-ZnO at 25 degrees C (n-ZnO+t degrees ). Total and metallothionein-bound Zn levels as well as levels of metallothioneins (MT), cellular stress responses and cytotoxicity biomarkers were assessed in the mussels. The key biomarkers that showed differential responses to different single and combined stressors in this study were activities of caspase-3 and lysosomal cathepsin D, as well as protein carbonyl content. At 18 degrees C, exposures to n-ZnO, organic pollutants and their combinations led to a prominent up-regulation of MT levels (by approximately 30%) and oxidative stress response including up-regulation of superoxide dismutase activity, an increase in oxyradical production, and a 2-3-fold decrease in the levels of protein carbonyls in all exposures except nZnO+Ta. Expos ure to n-ZnO in the absence of other stressors also led to a strong ( approximately 7-fold) elevation of cathepsin D activity. Cellular responses to Zn(2+) and n-ZnO were different indicating that n-ZnO was not due exclusively to Zn release. Ca-channel blocker Nfd affected intracellular Zn distribution (reflected in the prominent elevation of Zn-MT levels) and caused reductive stress indicated by elevated levels of reduced glutathione levels and an increase in lactate/pyruvate ratio (reflecting higher NADH/NAD ratio). Elevated temperature (25 degrees C) abolished most of the typical responses to n-ZnO and induced oxidative injury, DNA fragmentation and caspase-3 mediated apoptosis in n-ZnO-exposed mussels. DNA fragmentation was also induced by exposure to organic toxins (alone and in combination with n-ZnO) but not by n-ZnO alone. These data indicate that n-ZnO toxicity to freshwater organisms is modulated by organic pollutants and enhanced by elevated temperatures.

 

Mottier, A., A. Seguin, et al. (2015). "Effects of subchronic exposure to glyphosate in juvenile oysters (Crassostrea gigas): From molecular to individual levels." Mar Pollut Bull 95(2): 665-77.

                Glyphosate-based herbicides are extensively used and can be measured in aquatic ecosystems, including coastal waters. The effect of glyphosate on non-target organisms is an issue of worldwide concern. The aim of this study was to investigate the effects of subchronic exposure to glyphosate in juvenile oysters, Crassostrea gigas. Yearling oysters were exposed to three concentrations of glyphosate (0.1, 1 and 100mugL(-1)) for 56days. Various endpoints were studied, from the individual level (e.g., gametogenesis and tissue alterations) to the molecular level (mRNA quantification), including biochemical endpoints such as glutathione-S-transferase (GST) and catalase activities and malondialdehyde content. No mortality and growth occurred during the experiment, and individual biomarkers revealed only slight effects. The levels of gene expression significantly increased in oysters exposed to the highest glyphosate concentration (GST and metallothioneins) or to all concentrations (multi-xenobiotic resistance). These results suggested an activation of defence mechanisms at the molecular level.

 

Oaten, J. F., M. D. Hudson, et al. (2015). "Effects of organism preparation in metallothionein and metal analysis in marine invertebrates for biomonitoring marine pollution." Sci Total Environ 518-519: 238-47.

                Metallothionein (MT) is established as a potentially useful biomarker for monitoring aquatic pollution. This paper addresses widespread inconsistencies in storage conditions, tissue type selection and pre-treatment of samples before MT and metal analysis in biomarker studies. This variation hampers comparability and so the widespread implementation of this monitoring approach. Actively sampled Mytilus edulis in Southampton Water, UK were exposed to different storage temperatures, a variety of tissue types were analysed, and various pre-treatments of transportation on ice, transportation in seawater, depuration, and rapid dissection in the field were examined. Storage temperatures of -20 degrees C were found to be adequate for periods of at least ten weeks, as MT was not reduced by protein degradation compared with samples kept at -80 degrees C. Whole tissue and digestive gland concentrations of MT and metals were significantly positively correlated and directly relatable. MT in the digestive gland appeared to be more responsive to metals than in whole tissue, where it may be diluted, masking MT responses. However, longer study periods may suffer the effects of mass changes to the digestive gland, which alters MT concentration, and it may therefore be advisable to measure whole tissue. Depuration and transportation in seawater reduced both MT and metal concentrations in the digestive gland, and few correlations between MT and metals were identified for these treatments. It is therefore recommended that: i) samples are transported to the laboratory on ice and dissected as soon as possible thereafter, ii) depuration should not be used when examining MT response to metal exposure until further research clarifying its utility is reported, iii) either whole tissue or the digestive gland can be used to measure MT, though whole tissue may be preferable on long-term studies, and iv) organisms can be stored at -20 degrees C before analysis for up to ten weeks. These practices can be applied to future biomonitoring studies and will improve the comparability and repeatability of using MT as a biomarker.

 

Geng, N., C. Wang, et al. (2015). "Cadmium Accumulation and Metallothionein Response in the Freshwater Bivalve Corbicula fluminea Under Hydrodynamic Conditions." Biol Trace Elem Res 165(2): 222-32.

                Freshwater bivalves such as Corbicula fluminea (Muller) are useful biomonitors for cadmium pollution because they absorb heavy metals and accumulate them in their tissues. We exposed C. fluminea in the laboratory to natural and cadmium (Cd)-spiked sediments below flowing water in order to evaluate the organisms' Cd accumulation and metallothionein (MT) response under hydrodynamic conditions. The accumulation of Cd and the induction of MT in C. fluminea were determined at 0, 1, 3, 6, 10, 16, and 23 days. Hydrodynamic conditions, represented by a water flow rate of 14 or 3.2 cm/s, increased Cd accumulation in the visceral mass, gill, foot, and mantle of C. fluminea in the first 3 or 6 days in the natural sediment. Cd concentrations in the C. fluminea tissues kept increasing over time in the three treatments, and significant differences were observed in Cd accumulation after 6 (visceral mass), 10 (foot) and 16 (gill and mantle) days among the three groups. The MT concentrations were barely affected by hydrodynamic conditions and were significantly linearly related to the Cd concentration in the visceral mass in the natural sediment and binomially related to it in the Cd-spiked sediment. Hydrodynamic conditions enhanced the accumulation of Cd in the soft tissues of C. fluminea, especially in the Cd-spiked sediment, but stronger hydrodynamic forces did not increase Cd accumulation. MT may be considered an indicator for Cd accumulation in C. fluminea under hydrodynamic conditions, but only when the Cd concentrations in the tissue remain below the toxic threshold values.

 

Wan, R., F. Meng, et al. (2015). "Biochemical responses in the gills of Meretrix meretrix after exposure to treated municipal effluent." Ecotoxicol Environ Saf 111: 78-85.

                The biochemical effects in marine bivalves exposed to increasing concentrations of treated municipal effluent (TME), as discharged into receiving marine waters, are investigated. The effluent was collected from a municipal sewage treatment plant (STP) in Qingdao (China). Meretrix meretrix were exposed to effluent volume ratio (EVR, ratio of effluent volume accounted for tailwater seawater mixture) 0%, 1%, 5%, 10%, and 20% (v/v) TME for 15 days and the following biochemical responses in gills were measured: (1) the activity of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), and glutathione (GSH) content, and lipid peroxidation levels of malondialdehyde (MDA), as oxidative stress biomarkers; (2) the activity of 7-ethoxyresorufin O-deethylase (EROD) and gluthathione S-transferase (GST), as phase I and phase II conjugation enzymes, respectively; (3) acetylcholinesterase (AChE), as a biomarker of neurotoxicity, and (4) metallothioneins (MTs), as proteins strongly induced by heavy metals. Most of the biochemical indices present high and significant variation frequency (above 50%). There is enhancement in the antioxidant enzymes, EROD, GST, AChE, and MTs, as well as consumption of GSH. The current experimental results suggest that effluent with concentrations less than 20% (v/v) do not cause lipid peroxidation damage. This implies that the activated defense is sufficient to protect the bivalves' gill tissues from cytotoxicity produced by the effluent. Furthermore, GSH, GPx, MTs, and GR are suitable, and sufficiently sensitive, biomarkers to indicate the pollution levels in marine environments receiving such effluent.

 

Zhang, W., Z. Guo, et al. (2015). "Biotransformation and detoxification of inorganic arsenic in Bombay oyster Saccostrea cucullata." Aquat Toxicol 158: 33-40.

                Arsenic (As) exists as the toxic inorganic forms in marine water and sediment, while marine oysters usually accumulate high As contents mostly as the less toxic organic forms. It has not yet been clear that how As is biotransformed in marine oysters. This study therefore investigated the biotransformation and detoxification of two inorganic As forms (As(III) and As(V)) in Bombay oyster Saccostrea cucullata after waterborne exposures for 30 days. Seven treatments of dissolved As exposure (clean seawater, 1, 5, 20 mg/L As(III), and 1, 5, 20 mg/L As(V)) were performed. Body As concentration increased significantly after all As exposure treatments except 1mg/L As(V). Total As, As(III), and As(V) concentration were positive correlated with glutathione-S-transferases (GST) activities, suggesting GST might play an important role in the As biotransformation and detoxification process. Organic As species were predominant in control and the low As exposed oysters, whereas a large fraction of As was remained as the inorganic forms in the high As exposed oysters, suggesting As could be biotransformed efficiently in the oysters in clean or light contaminated environment. The results of As speciation demonstrated the As biotransformation in the oysters included As(V) reduction, methylation to monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA), and subsequent conversion to arsenobetaine (AsB). More As was distributed in the subcellular metallothionein-like proteins fraction (MTLP) functioning sequestration and detoxification in the inorganic As exposed oysters, suggesting it was also a strategy for oysters against As stress. In summary, this study elucidated that marine oysters had high ability to accumulate, biotransform, and detoxify inorganic As.

 

Kalman, J., E. Bonnail-Miguel, et al. (2015). "Toxicity and the fractional distribution of trace metals accumulated from contaminated sediments by the clam Scrobicularia plana exposed in the laboratory and the field." Sci Total Environ 506-507: 109-17.

                The relationship between the subcellular distribution of accumulated toxic metals into five operational fractions (subsequently combined into presumed detoxified and non-detoxified components) and toxicity in the clam Scrobicularia plana was investigated under different laboratory exposures. Clams were exposed to metal contaminated media (water and diet) and analysed for the partitioning of accumulated As, Cu and Zn into subcellular fractions. In general, metallothionein-like proteins, metal-rich granules and cellular debris in different proportions acted as main storage sites of accumulated metals in the clam soft tissues for these three metals. No significant differences were noted in the accumulation rates of As, Cu and Zn of groups of individuals with or without apparent signs of toxicity after up to 30 days of exposure to naturally contaminated sediment mixtures. There was, however, an increased proportional accumulation of Cu in the non-detoxified fraction with increased Cu accumulation rate in the clams, suggesting that the Cu uptake rate from contaminated sediments exceeded the combined rates of elimination and detoxification of Cu, with the subsequent likelihood for toxic effects in the clams.

 

Caro, A., G. Chereau, et al. (2015). "Contrasted responses of Ruditapes decussatus (filter and deposit feeding) and Loripes lacteus (symbiotic) exposed to polymetallic contamination (Port-Camargue, France)." Sci Total Environ 505: 526-34.

                The use of symbiotic bivalve species to assess the effect of anthropogenic metal pollution was rarely investigated whereas data on filter feeding bivalves are common. The aim of this study was the exposure of two bivalve species, Ruditapes decussatus and Loripes lacteus to polymetallic pollution gradient, originating from harbor activities (Port-Camargue, south of France). Both bivalves differ by their trophic status, filter and deposit feeder for Ruditapes and symbiotic for Loripes that underlies potential differences in metal sensibility. The bivalves were immerged in July (for Ruditapes during 2 and 8 days) and in August 2012 (for Loripes during 2, 6 and 8 days) in the water column of the harbor, at 3 stations according to pollution gradient. Metal concentrations (Cu, Mn, Zn) in the water column were quantified as dissolved metals (measured by ICP-MS) and as labile metals (measured by ICP-MS using DGT technique). For each exposure time, accumulation of metals in the soft tissue of bivalves ("bioaccumulation") was measured for both species. In addition, specific parameters, according to the trophic status of each bivalve, were investigated: filtering activity (specific clearance rate, SCR) for Ruditapes, and relative cell size (SSC) and genomic content (FL1) of bacterial symbionts hosted in the gills of Loripes. The SCR of Ruditapes drops from 100% (control) to 34.7% after 2 days of exposure in the less contaminated site (station 8). On the other hand, the relative cell size (SSC) and genomic content (FL1), measured by flow cytometry were not impacted by the pollution gradient. Bioaccumulation was compared for both species, showing a greater capability of Cu accumulation for Loripes without lethal effect. Mn, Fe and Zn were generally not accumulated by any of the species according to the pollution gradient. The trophic status of each species may greatly influence their respective responses to polymetallic pollution.

 

Fan, W., Z. Xu, et al. (2015). "Contrasting metal detoxification in polychaetes, bivalves and fish from a contaminated bay." Aquat Toxicol 159: 62-8.

                Jinzhou Bay in Bohai, Northern China, is historically contaminated with metals, but the organisms living in such contaminated environments are much less well studied. In this study, we contrasted the different subcellular and detoxification responses of polychaetes, bivalves and fish collected from different contaminated sites in Jinzhou Bay. In polychaete Neanthes japonica, metal-rich granule (MRG) was the main biologically detoxified metal compartment, and metallothionein-like protein (MTLP) detoxified a relatively smaller fraction of accumulated metals. The importance of MRG increased whereas that of MTLP decreased with increasing metal bioaccumulation. Detoxification in the two bivalves was similar to that in the polychaetes. However, the MRG appeared to play only a minor role in metal binding and detoxification in the gills and livers of fish, whereas MTLP was the dominating detoxification pool. Cellular debris was an important pool binding with metals in the three marine animals. Our study highlighted the contrasting cellular binding and detoxification among different marine organisms living in contaminated environments.

 

Le Pabic, C., C. Caplat, et al. (2015). "Trace metal concentrations in post-hatching cuttlefish Sepia officinalis and consequences of dissolved zinc exposure." Aquat Toxicol 159: 23-35.

                In this study, we investigated the changes of 13 trace metal and metalloid concentrations (i.e. Ag, As, Cd, Co, Cr, Cu, Fe, Mn, Ni, Pb, Se, V, Zn) and their subcellular fractionation in juvenile cuttlefish Sepia officinalis reared in controlled conditions between hatching and 2 months post-hatching. In parallel, metallothionein concentrations were determined. Our results highlighted contrasting changes of studied metals. Indeed, As and Fe concentrations measured in hatchlings suggested a maternal transfer of these elements in cuttlefish. The non-essential elements Ag and Cd presented the highest accumulation during our study, correlated with the digestive gland maturation. During the 6 first weeks of study, soluble fractions of most of essential trace metals (i.e. Co, Cr, Cu, Fe, Se, Zn) slowly increased consistently with the progressive needs of cuttlefish metabolism during this period. In order to determine for the first time in a cephalopod how metal concentrations and their subcellular distributions are impacted when the animals are trace metal-exposed, we studied previously described parameters in juveniles exposed to dissolved Zn at environmental (i.e. 50 mug l(-1)) and sublethal (i.e. 200 mug l(-1)) levels. Moreover, oxidative stress (i.e. glutathione S-transferase (GST), superoxide dismutase (SOD) and catalase activities, and lipid peroxidation (LPO)) was assessed in digestive gland and gills after 1 and 2 months exposures. Our results highlighted no or low ability of this stage of life to regulate dissolved Zn accumulation during the studied period, consistently with high sensitivity of this organism. Notably, Zn exposures caused a concentration-dependent Mn depletion in juvenile cuttlefish, and an increase of soluble fraction of Ag, Cd, Cu without accumulation modifications, suggesting substitution of these elements (i.e. Mn, Ag, Cd, Cu) by Zn. In parallel, metallothionein concentrations decreased in individuals most exposed to Zn. Finally, no perturbations in oxidative stress management were detected in gills, whereas modifications of GST, SOD and catalase activity levels were recorded in digestive gland, resulting in an increase of LPO content after a 6-week exposure to low Zn concentration. Altogether, these perturbations are consistent with previously described high sensitivity of juvenile cuttlefish towards Zn. Our results underlined the need to study deeply contamination impact on this animal at this stage of life.

 

Gil-Moreno, S., E. Jimenez-Marti, et al. (2015). "Does Variation of the Inter-Domain Linker Sequence Modulate the Metal Binding Behaviour of Helix pomatia Cd-Metallothionein?" Int J Mol Sci 17(1).

                Snail metallothioneins (MTs) constitute an ideal model to study structure/function relationships in these metal-binding polypeptides. Helix pomatia harbours three MT isoforms: the highly specific CdMT and CuMT, and an unspecific Cd/CuMT, which represent paralogous proteins with extremely different metal binding preferences while sharing high sequence similarity. Preceding work allowed assessing that, although, the Cys residues are responsible for metal ion coordination, metal specificity or preference is achieved by diversification of the amino acids interspersed between them. The metal-specific MT polypeptides fold into unique, energetically-optimized complexes of defined metal content, when binding their cognate metal ions, while they produce a mixture of complexes, none of them representing a clear energy minimum, with non-cognate metal ions. Another critical, and so far mostly unexplored, region is the stretch linking the individual MT domains, each of which represents an independent metal cluster. In this work, we have designed and analyzed two HpCdMT constructs with substituted linker segments, and determined their coordination behavior when exposed to both cognate and non-cognate metal ions. Results unequivocally show that neither length nor composition of the inter-domain linker alter the features of the Zn(II)- and Cd(II)-complexes, but surprisingly that they influence their ability to bind Cu(I), the non-cognate metal ion.

 

Ivanina, A. V., C. Hawkins, et al. (2015). "Effects of environmental hypercapnia and metal (Cd and Cu) exposure on acid-base and metal homeostasis of marine bivalves." Comp Biochem Physiol C Toxicol Pharmacol 174-175: 1-12.

                Elevated CO2 levels reduce seawater pH and may affect bioavailability of trace metals in estuaries. We studied the interactive effects of common metal pollutants (50 mug l(-1) Cd or Cu) and PCO2 (~395, 800 and 2000 muatm) on metal levels, intracellular pH, expression of metal binding proteins and stress biomarkers in estuarine bivalves Crassostrea virginica (oysters) and Mercenaria mercenaria (hard clams). Cd (but not Cu or hypercapnia) exposure affected the acid-base balance of hemocytes resulting in elevated intracellular pH. Cd and Cu exposure led to the increase in the tissue metal burdens, and metal accumulation was reduced by elevated PCO2 in the mantle but not hemocytes. No change was found in the intracellular free Cd(2+), Cu(2+) or Fe(2+) during Cu or Cd exposure indicating that these metals are bound to intracellular ligands. Free Zn(2+) content in oyster hemocytes was suppressed by Cd and Cu exposure and below the detection limits in clam hemocytes, which went hand-in-hand with the elevated mRNA expression of metallothioneins and ferritin in Cd- and Cu-exposed bivalves, enhanced by hypercapnia. The metal-binding and antioxidant mechanisms of oysters and clams were sufficient to effectively maintain intracellular redox status, even though metal exposure combined with moderate hypercapnia (~800 muatm PCO2) led to the elevated production of reactive oxygen species in hemocytes. Overall, while hypercapnia modulates metal accumulation, binding capacity and oxidative stress in estuarine bivalves, the physiological effects of elevated CO2 are mild compared to the effects of other common stressors.

 

Santovito, G., F. Boldrin, et al. (2015). "Metal and metallothionein distribution in different tissues of the Mediterranean clam Venerupis philippinarum during copper treatment and detoxification." Comp Biochem Physiol C Toxicol Pharmacol 174-175: 46-53.

                Filter feeding animals can accumulate large amount of contaminants in their body through particles filtered from seawater. In particular, copper is interesting since it plays important roles as co-factor of numerous proteins but its toxicity is well established, also because it can readily generate free radicals or oxidize cellular components through their redox activity. Its availability is tightly regulated within cells: it is immediately transferred to metallothionein (MT) that in turn provides efficient and specific mechanisms for its intracellular storage and transport. The aim of this study was to evaluate the acute effect of sublethal copper concentrations in Venerupis philippinarum, by studying the kinetics of copper, zinc (for its interactions at the sites of intake or elimination with the accumulation of other essential and not essential trace metals) and metallothionein accumulation under laboratory conditions. The time-course of metal accumulation/elimination is similar in digestive gland and gills and importantly it is dose-dependent. Both copper and zinc increase slowly within cells, reaching a maximum concentration at the end of the exposure period. During the detoxification period, the metal levels in digestive gland and gills rapidly decrease, with different kinetics in the two tissues. Positive correlations between metallothionein accumulation and copper or zinc concentrations have been verified in both treated groups. The obtained data demonstrated the involvement of MTs in detoxification strategies after a recovery period in clean seawater.

 

Jin, Y. and W. X. Wang (2015). "Biokinetics and metallothionein-like proteins response in oysters facing metal challenges in an estuary." Environ Toxicol Chem 34(8): 1818-25.

                The discovery of colored oysters in an estuary in Southern China raised questions of how these oysters could survive in such an unpredictable estuary. In the present study, the authors conducted a transplant experiment using oysters Crassostrea hongkongensis in the estuary to quantify the changes of metal biokinetics (Cd and Zn) and the responses of metallothionein-like proteins. Oysters in the transplantation experiment accumulated extremely high concentrations of Cd, Cu, Cr, Ni, and Zn. The present study provided the field evidence of the time-course coupling relationship between Cd and Zn bioaccumulation. Over the 2-mo transplantation, the uptake rate constants of Cd and Zn varied greatly during the early stage of exposure but subsequently became comparable among the different locations. The dietary Zn assimilation remained relatively constant, whereas the Cd assimilation increased gradually with increasing period of exposure. No notable difference was seen in dietary metal assimilation among the different locations. In contrast, the efflux rates quantified by the end of transplantation were 26% to 42% higher for Cd and 12% to 37% higher for Zn than that in the unexposed oysters. The authors further demonstrated that the incoming Cu and Zn were not stored in metallothionein-like pools and that metallothionein-like proteins synthesis and breakdown were little affected by metal exposure. Overall, the authors' transplantation experiments suggested that oysters displayed a rather weak ability to modify their biokinetics and metallothionein turnover under metal exposure.

 

Chandurvelan, R., I. D. Marsden, et al. (2015). "Assessment of a mussel as a metal bioindicator of coastal contamination: relationships between metal bioaccumulation and multiple biomarker responses." Sci Total Environ 511: 663-75.

                This is the first study to use a multiple biomarker approach on the green-lipped mussel, Perna canaliculus to test its feasibility as a bioindicator of coastal metal contamination in New Zealand (NZ). Mussels were collected from six low intertidal sites varying in terms of anthropogenic impacts, within two regions (West Coast and Nelson) of the South Island of NZ. Trace elements, including arsenic (As), cadmium (Cd), copper (Cu), lead (Pb), nickel (Ni), and zinc (Zn), were measured in the gills, digestive gland, foot and mantle, and in the surface sediments from where mussels were collected. Metal levels in the sediment were relatively low and there was only one site (Mapua, Nelson) where a metal (Ni) exceeded the Australian and New Zealand Interim Sediment Quality Guideline values. Metal levels in the digestive gland were generally higher than those from the other tissues. A variety of biomarkers were assessed to ascertain mussel health. Clearance rate, a physiological endpoint, correlated with metal level in the tissues, and along with scope for growth, was reduced in the most contaminated site. Metallothionein-like protein content and catalase activity in the digestive gland, and catalase activity and lipid peroxidation in the gill, were also correlated to metal accumulation. Although there were few regional differences, the sampling sites were clearly distinguishable based on the metal contamination profiles and biomarker responses. P. canaliculus appears to be a useful bioindicator species for coastal habitats subject to metal contamination. In this study tissue and whole organism responses provided insight into the biological stress responses of mussels to metal contaminants, indicating that such measurements could be a useful addition to biomonitoring programmes in NZ.

 

Chatel, A., V. Faucet-Marquis, et al. (2015). "Genotoxicity and activation of cellular defenses in transplanted zebra mussels Dreissena polymorpha along the Seine river." Ecotoxicol Environ Saf 114: 241-9.

                The aim of the present study was to confirm the relevance of studying DNA adduct formation in a field study. In that context, freshwater mussels Dreissena polymorpha, collected in a reference station, were transplanted in different sites with a pollution gradient. After one and two months, mussels were collected and DNA adduct formation was analyzed using the (32)P post labelling technique on both gills and digestive glands. In addition, the expression of genes involved in the detoxification system (catalase (CAT), superoxide dismutase (SOD), glutathione S-transferase (GST), HSP70, aryl hydrocarbon receptor (AHR), P glycoprotein (PgP), metallothionein (MT)) was assessed by RT-PCR. DNA adducts were observed at amount comparable to data from literature. Increase of DNA adducts after two months of transplantation could be correlated with strong modulation of gene expression implicated in detoxification processes. Indeed, PgP and HSP70 gene expressions were similarly induced in gills and digestive glands while SOD and CAT expressions were down regulated in both tissues. AHR, GST and MT genes were differently regulated depending upon the tissue studied and the level of contamination in the different sites. We demonstrated that mussels transplanted in the different stations with pollution gradient were able to biotransform PAHs, assessed by DNA adduct formation and the high decrease of detoxification genes. Specific DNA adducts pattern obtained after one and two month mussel transplantations demonstrated the relevance of DNA adduct as biomarker of environmental pollution.

 

Macias-Mayorga, D., I. Laiz, et al. (2015). "Is oxidative stress related to cadmium accumulation in the Mollusc Crassostrea angulata?" Aquat Toxicol 161: 231-41.

                The kinetics of cadmium (Cd) accumulation in the gills and digestive gland of Crassotrea angulata at three concentrations of cadmium (0.088 muM, 0.44 muM and 2.22 muM) was monitored for 28 days. The relationship between accumulation and toxicity was studied using metallothionein-like protein (MTLP) concentration and reduced glutathione levels (GSH) as biochemical endpoints. The activity of enzymes which form part of the antioxidant defense system, in particular glutathione reductase (GR), total glutathione peroxidase (GPx), superoxide dismutase (SOD) and catalase (CAT), as enzymatic endpoints, was also assessed. A first order kinetic model demonstrated that the accumulation process does not take place linearly, as the Cd concentration in gills and digestive gland tended toward a stationary state. Metallothionein-like protein is clearly induced by Cd accumulation; however, at high Cd concentrations the detoxification mechanism of this protein is affected. High Cd concentrations (2.22 muM) lead to a decrease in GSH levels, and also inhibit antioxidant enzyme activities, demonstrating the adverse effect of this metal on the antioxidant balance system.

 

Baurand, P. E., V. Pedrini-Martha, et al. (2015). "Differential expression of metallothionein isoforms in terrestrial snail embryos reflects early life stage adaptation to metal stress." PLoS One 10(2): e0116004.

                The aim of this study was to analyze the expression of three metallothionein (MT) isoform genes (CdMT, CuMT and Cd/CuMT), already known from adults, in the Early Life Stage (ELS) of Cantareus aspersus. This was accomplished by detection of the MT isoform-specific transcription adopting Polymerase Chain Reaction (PCR) amplification and quantitative Real Time (qRT)-PCR of the three MT genes. Freshly laid eggs were kept for 24 hours under control conditions or exposed to three cadmium (Cd) solutions of increasing concentration (5, 10, and 15 mg Cd/L). The transcription of the three MT isoform genes was detected via PCR in 1, 6 and 12-day-old control or Cd-exposed embryos. Moreover, the transcription of this isoform genes during development was followed by qRT-PCR in 6 and 12-day-old embryos. Our results showed that the CdMT and Cd/CuMT genes, but not the CuMT gene, are expressed in embryos at the first day of development. The transcription of the 3 MT genes in control embryos increased with development time, suggesting that the capacities of metal regulation and detoxification may have gradually increased throughout embryogenesis. However in control embryos, the most highly expressed MT gene was that of the Cd/CuMT isoform, whose transcription levels greatly exceeded those of the other two MT genes. This contrasts with the minor significance of this gene in adult snails and suggests that in embryos, this isoform may play a comparatively more important role in metal physiology compared to adult individuals. This function in adult snails appears not to be related to Cd detoxification. Instead, snail embryos responded to Cd exposure by over-expression of the CdMT gene in a concentration-dependent manner, whereas the expression of the Cd/CuMT gene remained unaffected. Moreover, our study demonstrates the ability of snail embryos to respond very early to Cd exposure by up-regulation of the CdMT gene.

 

Li, Y., H. Yang, et al. (2015). "Cadmium accumulation and metallothionein biosynthesis in cadmium-treated freshwater mussel Anodonta woodiana." PLoS One 10(2): e0117037.

                This study investigated the distribution of cadmium (Cd) and the protein level of metallothionein (MT) and examined the relationship of Cd accumulation and the MT concentration in different tissues of freshwater mussel Anodonta woodiana following Cd treatment. The mussels were exposed to Cd (4.21, 8.43, 16.86, 33.72 and 67.45 mg L-1) for 24, 48, 72 and 96 h, respectively. After Cd treatment, the gills, mantle, foot, visceral mass and digestive gland tissues were collected for analysis. We found that, in the controls, Cd distributed in all tissues in the concentration order of gills>mantle>foot>visceral mass>digestive gland. Upon Cd treatment, Cd concentration significantly increased in all tissues. The highest Cd accumulation was found in the digestive gland, which was 0.142 mg g-1 (P<0.05). MT levels in the gills and mantle of the mussels increased significantly (P<0.05), which were in positive correlation with Cd accumulation in the tissues (P<0.05). In conclusion, our results demonstrated a correlation between Cd accumulation and MT up-regulation in gills and mantle of the mussels after Cd treatment. It is suggested that the protein level of MT in gills and mantle of Anodonta woodiana is a good biomarker for Cd contamination.

 

Sone, T., Y. Haraguchi, et al. (2015). "Structural characterization reveals the keratinolytic activity of an arthrobacter nicotinovorans protease." Protein Pept Lett 22(1): 63-72.

                Elevated cadmium (Cd) concentrations in fishery byproducts are an environmental concern, that might be reduced by enzymatic removal and adsorption of the contaminants during recycling the byproducts as animal food. We cloned the gene for Arthrobacter nicotinovorans serine protease (ANISEP), which was isolated from the hepatopancreas of the Japanese scallop (Patiopecten yessoensis) and has been found to be an effective enzyme for Cd(II) removal. The gene is 993 bp in length and encodes 330 amino acids, including the pre (1-30) and pro (31-111) sequences. The catalytic triad consists of His, Asp, and Ser. Sequence similarities indicate that ANISEP is a extracellular serine protease. X-ray crystallography revealed structural similarities between ANISEP and the trypsin-like serine protease NAALP from Nesterenkonia sp. Site-directed mutagenesis identified Ser171 as catalytic residue. The keratinolytic activity of ANISEP was 10-fold greater than that of trypsin. ANISEP digested Cd(II)-bound recombinant metallothionein MT-10a from Laternula elliptica, but did not release Cd. These results further suggest ANISEP is a trypsin-like serine protease that can release Cd from the Japanese scallop hepatopancreas because of its strong keratinolytic activity.

 

Balbi, T., A. Smerilli, et al. (2014). "Co-exposure to n-TiO2 and Cd2+ results in interactive effects on biomarker responses but not in increased toxicity in the marine bivalve M. galloprovincialis." Sci Total Environ 493: 355-64.

                The increasing production of nanoparticles (NPs) will lead to their release into the aquatic environment, where they could modify the bioavailability/bioconcentration and consequent biological impact of other contaminants. Interactive effects of n-TiO2, one of the most widespread NP type, and Cd(2+), a common heavy metal pollutant, have been described in freshwater species, whereas no information is available in marine organisms. In this work, the effects of co-exposure to n-TiO2 and Cd(2+) were investigated in the marine bivalve Mytilus galloprovincialis. Experimental conditions (100 mug/L, 96 h), were chosen in order to induce early but measurable stress responses (biomarkers) without toxicity. Several biomarkers, from molecular to tissue level, were measured in hemolymph and digestive gland; the effects on embryo development were also evaluated. In hemolymph, Cd(2+) abolished the increase in immune parameters induced by n-TiO2 (NO production and lysozyme activity). In the digestive gland, distinct interactive effects of n-TiO2 and Cd(2+) were observed on different lysosomal biomarkers (lysosomal membrane stability, lipid accumulation and lysosome/cytoplasm volume ratio) and transcription of the immune genes lysozyme and toll-like receptor (TLR). However, n-TiO2 did not affect specific metal-induced responses (metallothionein induction) and tissue metal accumulation. Cd(2+) alone, but not in combination with n-TiO2, affected embryo development. The interactive effects observed on different biomarkers were not apparently due to differences in bioavailability/bioaccumulation of Cd(2+) in the presence of n-TiO2 agglomerates; these effects may result from interactions of either contaminant with both common and distinct targets/mechanisms of action at different levels of biological organization. Overall, the results indicate that co-exposure to n-TiO2 and Cd(2+) did not result in increased adverse effects in M. galloprovincialis. These data underline the need for further knowledge on the potential interactions of NPs with existing contaminants in marine organisms.

 

Arini, A., G. Daffe, et al. (2014). "Detoxification and recovery capacities of Corbicula fluminea after an industrial metal contamination (Cd and Zn): a one-year depuration experiment." Environ Pollut 192: 74-82.

                This study aimed to assess the recovery capacity of the freshwater bivalve Corbicula fluminea subjected to industrial metal discharges (Cd, Zn). After a 24-day exposure in a metal-contaminated river, bivalves were transferred and maintained in the laboratory for one year under metal-free conditions. Metal accumulation, metallothionein production and genetic expressions of genes involved in metal stress were studied. Results demonstrated the high persistence of Cd in tissues (only 73% eliminated after 365 days) whereas Zn was rapidly depurated. The Cd half-life was estimated around 240 days. Metallothioneins were strongly induced within the 28 first days of decontamination, then decreased by 45% after 365 days. The metal exposure of bivalves led to a significant gene induction. After 28 days, most of the genes were no longer overexpressed, suggesting that the bivalves may withstand small amounts of non-essential metals in their tissues without showing signs of detrimental effects on the tested genes.

 

Giarratano, E., M. N. Gil, et al. (2014). "Biomarkers of environmental stress in gills of ribbed mussel Aulacomya atra atra (Nuevo Gulf, Northern Patagonia)." Ecotoxicol Environ Saf 107: 111-9.

                In this study, we assessed in gills of native ribbed mussels Aulacomya atra atra from three sites within Nuevo Gulf (Northern Patagonia) several biomarkers such as reactive oxygen species (ROS), lipid radicals (LR), malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST) and metallothionein (MT). Furthermore, concentrations of main trace metals (Fe, Al, Zn, Cu, Cd and Pb) were quantified in mussel tissue. Results showed significant induction of SOD, GST, MT and MDA, as well as, higher concentration of Fe, Al and Cd in winter than in summer. The high MDA content measured in mussels from Folias Wreck seemed to be caused by the very high levels of Fe that would come from the corrosion of the vessel. Mussels from the control site Punta Cuevas presented the lowest levels of Cd and the highest of Al in winter. Despite positive correlations were found between Al and GST and MT, no spatial differentiation was detected in those biomarkers. On the other hand, MT was only related to Al been most likely influenced by environmental variables than by the trace metals. It has to be highlighted that the relationship detected among water temperature, nutrients and antioxidant responses in gills is probably related to the fact that this tissue is in direct contact with water and it is sensitive to its fluctuations. Taking into account that mussel gill is a tissue actively proliferating and the first target of contaminants present in water, so that changes in its antioxidant system can provide an earlier warning signal than in other tissues.

 

Kournoutou, G. G., S. Pytharopoulou, et al. (2014). "Changes of polyamine pattern in digestive glands of mussel Mytilus galloprovincialis under exposure to cadmium." Comp Biochem Physiol C Toxicol Pharmacol 165: 1-8.

                Polyamines, in particular spermidine and spermine, have been identified as important antioxidants, highly induced by oxidative stress in a variety of organisms. However, little is known about changes in polyamine content of metal-stressed marine organisms. In the present study, mussels (Mytilus galloprovincialis) were experimentally exposed to 25 mug/L Cd(2+) or 100 mug/L Cd(2+) for up to 15 days. Cd(2+) was progressively accumulated in mussel tissues, leading to a characteristic oxidative-stress status. Free putrescine (PUT) production was noticeably induced in response to Cd(2+) at day 5 and then declined. In contrast, free spermidine (SPD) content was gradually reduced, whereas the concentration of free spermine (SPM) increased. In combination, these changes led to a 69% or 88% reduction in the ratio of (SPD+SPM)/PUT at day 5, dependent on the Cd(2+) concentration used, which subsequently followed an upward trend in values, albeit not reaching those of controls. Conjugated polyamines constantly increased, in particular conjugated spermidine and spermine, tagging along with metallothionein production. Acetylated polyamines showed a diverse profile of changes, but their content was generally kept at low levels throughout the exposure period. Collectively, our results suggest that certain polyamine compounds could play a significant role in the tolerance of mussels against Cd(2+)-mediated stress, and that the ratio (SPD+SPM)/PUT could be a good indicator of the metal-stress status.

 

Perez-Rafael, S., F. Monteiro, et al. (2014). "Cantareus aspersus metallothionein metal binding abilities: the unspecific CaCd/CuMT isoform provides hints about the metal preference determinants in metallothioneins." Biochim Biophys Acta 1844(9): 1694-707.

                In Proteomics, gene/protein families including both specialized and non-specialized paralogs are an invaluable tool to study the evolution of structure/function relationships in proteins. Metallothioneins (MTs) of the pulmonate gastropod molluscs (snails) offer one of the best materials to study the metal-binding specificity of proteins, because they consist of a polymorphic system that includes members with extremely distinct metal preferences but with a high protein sequence similarity. Cantareus aspersus was the first snail where three paralogous MTs were isolated: the highly specific cadmium (CaCdMT) and copper (CaCuMT) isoforms, and an unspecific CaCd/CuMT isoform, so called because it was natively isolated as a mixed Cd and Cu complex. In this work, we have thoroughly analyzed the Zn(2+)-, Cd(2+)- and Cu(+)-binding abilities of these three CaMTs by means of the spectroscopic and spectrometric characterization of the respective recombinant, as well as in vitro-substituted, metal-complexes. The comparison with the orthologous HpMTs and the study of the isoform-determinant residues allow correlating the protein sequence variability with the coordination capabilities of these MTs. Surprisingly, the CaCuMT isoform exhibits a stronger Cu-thionein character than the HpCuMT ortholog, and the CaCd/CuMT isoform could be defined as a non-optimized Cu-thionein, which has not attained any defined functional differentiation in the framework of the snail MT gene/protein family.

 

Gomes, T., C. G. Pereira, et al. (2014). "Effects of silver nanoparticles exposure in the mussel Mytilus galloprovincialis." Mar Environ Res 101: 208-214.

                Silver nanoparticles (Ag NPs) have emerged as one of the most commonly used NPs in a wide range of industrial and commercial applications. This has caused increasing concern about their fate in the environment as well as uptake and potential toxicity towards aquatic organisms. Accordingly, mussels Mytilus galloprovincialis were exposed to 10 mug L(-1) of Ag NPs and ionic silver (Ag+) for 15 days, and biomarkers of oxidative stress and metal accumulation were determined. Accumulation results show that both Ag NPs and Ag+ accumulated in both gills and digestive glands. Antioxidant enzymes (superoxide dismutase, catalase and glutathione peroxidase) were activated by Ag NPs and Ag+, showing different antioxidant patterns in both gills and digestive glands. Moreover, metallothionein was inducted in gills, directly related to Ag accumulation, while in the digestive glands only a small fraction of Ag seems to be associated with this protein. Lipid peroxidation was higher in gills exposed to Ag NPs, whereas in the digestive glands only Ag+ induced lipid peroxidation. Ag NPs and Ag+ cause oxidative stress with distinct modes of action and it's not clear if for Ag NPs the observed effects are attributed to free Ag+ ions associated with the nanoparticle effect.

 

Wang, L. and W. X. Wang (2014). "Depuration of metals by the green-colored oyster Crassostrea sikamea." Environ Toxicol Chem 33(10): 2379-85.

                The accumulation of metals (especially copper) in oysters has led to green-color now being found in Chinese estuaries. In the present study, the authors quantified the depuration of 8 metals (Ag, Cd, Co, Cr, Cu, Ni, Pb, and Zn) in green-colored oysters (Crassostrea sikamea) collected from an estuary that is heavily contaminated by metals as a result of industrial effluent releases. The oysters were depurated under laboratory conditions for 4 mo; the accumulated concentrations and the subcellular distribution of metals were measured at different time intervals. Results showed that the green color of oysters faded to light yellow (nearly normal) after 4 mo of depuration. Depuration of metals could be described by a first-order kinetic process. The calculated overall depuration rate constants of metals were in the range of 0.008 d(-1) to 0.024 d(-1) , with a biological retention half-life of 30 d to 70 d. The depuration rates of green-colored contaminated oysters were significantly higher for Cd, Cu, Cr, and Ni than the rates of oysters from a less contaminated site, whereas the depuration rates of Ag, Co, Pb, and Zn were comparable between the 2 populations. When corrected for the change of oyster tissue weight, the actual efflux rate constants of the metals (0.0708-0.1014 d(-1) ) were much higher than the overall depuration rate constants. Cellular debris and metallothionein-like proteins were the important fractions binding with the metals in the oysters. Significant changes in metal subcellular distribution were observed during the 4-mo depuration for Ag, Cd, Cu, and Zn. Metallothionein-like protein became more important in sequestering the metals during the depuration period, with a concomitant decrease in metals associated with the cellular debris fraction.

 

Jarque, S., E. Prats, et al. (2014). "Seasonal variations of gene expression biomarkers in Mytilus galloprovincialis cultured populations: temperature, oxidative stress and reproductive cycle as major modulators." Sci Total Environ 499: 363-72.

                The blue mussel Mytilus galloprovincialis has been used as monitoring organism in many biomonitoring programs because of its broad distribution in South European sea waters and its physiological characteristics. Different pollution-stress biomarkers, including gene expression biomarkers, have been developed to determine its physiological response to the presence of different pollutants. However, the existing information about basal expression profiles is very limited, as very few biomarker-based studies were designed to reflect the natural seasonal variations. In the present study, we analyzed the natural expression patterns of several genes commonly used in biomonitoring, namely ferritin, metallothionein, cytochrome P450, glutathione S-transferase, heat shock protein and the kinase responsive to stress KRS, during an annual life cycle. Analysis of mantle-gonad samples of cultured populations of M. galloprovincialis from the Delta del Ebro (North East Spain) showed natural seasonal variability of these biomarkers, pointing to temperature and oxidative stress as major abiotic modulators. In turn, the reproductive cycle, a process that can be tracked by VCLM7 expression, and known to be influenced by temperature, seems to be the major biotic factor involved in seasonality. Our results illustrate the influence of environmental factors in the physiology of mussels through their annual cycle, a crucial information for the correct interpretation of responses under stress conditions.

 

Chatel, A., V. Faucet-Marquis, et al. (2014). "DNA adduct formation and induction of detoxification mechanisms in Dreissena polymorpha exposed to nitro-PAHs." Mutagenesis 29(6): 457-65.

                Derived polycyclic aromatic hydrocarbons (PAHs) such as nitro-PAHs are present in the environment and are known to be much more toxic than PAHs compounds. However, very few studies have analysed their effects on the aquatic environment and none have investigated the freshwater environment. In the present study, we determined whether 1-nitropyrene (1-NP), a model of nitro-PAHs, can induce DNA adducts in gills and digestive glands of the freshwater mussel Dreissena polymorpha. Two concentrations of 1-NP (50 and 500 muM) were tested. In addition, in order to understand the metabolic pathways involved in 1-NP genotoxicity, mRNA expression of genes implicated in biotransformation mechanisms was assessed by quantitative reverse transcription-PCR. Results showed the presence of DNA adducts in both gills and digestive glands, with highest levels obtained after 5 days of exposure to 500 muM. Metallothionein mRNA levels were enhanced in digestive glands exposed to 50 muM. Surprisingly, at the higher concentration (500 muM), aryl hydrocarbon receptor and HSP70 genes were only up-regulated in digestive glands while PgP mRNA levels were increased in both tissues. Results suggested a cytotoxic and genotoxic effect of 1-NP. Mussels seemed to be able to partially detoxify this compound, in view of the low amount of DNA adducts observed after 5 days exposure to 50 muM. For the first time, 1-NP biotransformation and detoxification systems have been characterised in D. polymorpha.

 

Marasinghe Wadige, C. P., A. M. Taylor, et al. (2014). "Bioavailability and toxicity of zinc from contaminated freshwater sediments: linking exposure-dose-response relationships of the freshwater bivalve Hyridella australis to zinc-spiked sediments." Aquat Toxicol 156: 179-90.

                To evaluate the use of the freshwater bivalve Hyridella australis as a potential biomonitor for zinc contamination in freshwater sediments, the bioavailability and toxicity of zinc contaminated sediments (low 44 +/- 5, medium 526 +/- 41, high 961 +/- 38 mug/g dry mass) were investigated in laboratory microcosms for 28 days by examining H. australis exposure-dose-response relationships. Zinc concentrations in sediments and surface waters were measured as zinc exposure. Zinc in whole organism soft body tissues and five individual tissues were measured as organism zinc dose. Sub-cellular localisation of zinc in hepatopancreas tissues was investigated to further understand the zinc handling strategies and tolerance of H. australis. Total antioxidant capacity, lipid peroxidation and lysosomal membrane stability were measured in hepatopancreas tissues as zinc induced biomarker responses. Accumulated zinc concentrations in whole body tissues of H. australis reflected the zinc exposure and exhibited exposure dependent zinc accumulation at day 28. Gills accumulated significantly higher zinc concentrations than other tissues, however, no significant differences in zinc accumulation between treatments were detected for any of the individual tissues analysed. Analysis of individual tissue zinc concentrations, therefore, may not offer any advantages for monitoring bioavailable zinc in freshwater environments with this organism. Relationships between tissue zinc and calcium concentration suggest accumulation of zinc by H. australis may have occurred as an analogue of calcium which is a major constituent in shell and granules of unionid bivalves. A high percentage of accumulated zinc in the hepatopancreas tissues was detoxified and stored in metallothionein like proteins and metal rich granules. Of the zinc accumulated in the biologically active metal pool, 59-70% was stored in the lysosome+microsome fraction. At the concentrations tested, increasing zinc exposure resulted in decreasing total antioxidant capacity and measurable increases in the sublethal effects, lipid peroxidation and lysosomal membrane destabilisation, were observed. Based on exposure-dose analysis, H. australis partially regulates zinc uptake and weakly exhibits bioavailability of zinc in freshwater environments, however, exposure-response analysis shows zinc induced toxicological effects, suggesting the potential of this organism as a biomonitor for zinc in heavily contaminated freshwater environments.

 

Taylor, A. M. and W. A. Maher (2014). "Exposure-dose-response of Tellina deltoidalis to contaminated estuarine sediments 3. Selenium spiked sediments." Comp Biochem Physiol C Toxicol Pharmacol 166: 34-43.

                The metalloid selenium is an essential element which at slightly elevated concentrations is toxic and mutagenic. In Australia the burning of coal for power generation releases selenium into estuarine environments where it accumulates in sediments. The relationship between selenium exposure, dose and response was investigated in the deposit feeding, benthic, marine bivalve Tellina deltoidalis. Bivalves were exposed in microcosms for 28 days to individual selenium spiked sediments, 0, 5 and 20 mug/g dry mass. T. deltoidalis accumulated selenium from spiked sediment but not in proportion to the sediment selenium concentrations. The majority of recovered subcellular selenium was associated with the nuclei and cellular debris fraction, probably as protein bound selenium associated with plasma and selenium bound directly to cell walls. Selenium exposed organisms had increased biologically detoxified selenium burdens which were associated with both granule and metallothionein like protein fractions, indicating selenium detoxification. Half of the biologically active selenium was associated with the mitochondrial fraction with up to 4 fold increases in selenium in exposed organisms. Selenium exposed T. deltoidalis had significantly reduced GSH:GSSG ratios indicating a build-up of oxidised glutathione. Total antioxidant capacity of selenium exposed T. deltoidalis was significantly reduced which corresponded with increased lipid peroxidation, lysosomal destabilisation and micronuclei frequency. Clear exposure-dose-response relationships have been demonstrated for T. deltoidalis exposed to selenium spiked sediments, supporting its suitability for use in selenium toxicity tests using sub-lethal endpoints.

 

Wlostowski, T., P. Kozlowski, et al. (2014). "Accumulation of cadmium in and its effect on the midgut gland of terrestrial snail Helix pomatia L. from urban areas in Poland." Bull Environ Contam Toxicol 93(5): 526-31.

                The objectives of this study were (1) to determine cadmium (Cd) accumulation in the midgut gland of a land snail Helix pomatia L. inhabiting residential areas of the 14 largest cities in Poland, and (2) to examine whether the accumulated Cd exerted any toxic effects. The average accumulation of Cd in the midgut gland of snails, weighing 16-18 g, ranged from 7.00 to 87.3 microg/g dry weight (0.06-0.77 micromol/g) and differed significantly among animals from the various urban areas. This difference in Cd accumulation was not related to city population, but was associated with the topsoil Cd (R(2) = 0.868, p < 0.0001). The tissue Cd was not found to produce toxicity (histopathology, programmed cell death, lipofuscin formation or lipid peroxidation), probably due to the induction of sufficiently high quantities of metallothionein and glutathione, well-known protective molecules.

 

Zhou, Q., Y. Zhang, et al. (2014). "Toxicological responses of the hard clam Meretrix meretrix exposed to excess dissolved iron or challenged by Vibrio parahaemolyticus." Aquat Toxicol 156: 240-7.

                The responses of genes encoding defense components such as ferritin, the lipopolysaccharide-induced tumor necrosis factor-alpha factor (LITAF), the inhibitor of nuclear factor-kappaB (IkappaB), metallothionein, and glutathione peroxidase were assessed at the transcriptional level in order to investigate the toxicological and immune mechanism of the hard clam Meretrix meretrix (HCMM) following challenge with iron or a bacterium (Vibrio parahaemolyticus). Fe dissolved in natural seawater led to an increase of Fe content in both the hepatopancreas and gill tissue of HCMM between 4 and 15 days of exposure. The ferritin gene responded both transcriptionally as indicated by real-time quantitative PCR and translationally as shown by western blotting results to iron exposure and both transcriptional and translational ferritin expression in the hepatopancreas had a positive correlation with the concentration of dissolved iron in seawater. Both iron and V. parahaemolyticus exposure triggered immune responses with similar trends in clam tissues. There was a significant post-challenge mRNA expression of LITAF and IkappaB at 3h, ferritin at 24h, and metallothionein and glutathione peroxidase at 48h. This behavior might be linked to their specific functions in physiological processes. These results suggested that similar signaling pathways were triggered during both iron and V. parahaemolyticus challenges. Here, we indicated that the ferritin of Meretrix meretrix was an intermediate in the pathway of iron homeostasis and in its innate immune defense mechanism.

 

Kamel, N., T. Burgeot, et al. (2014). "Effects of increasing temperatures on biomarker responses and accumulation of hazardous substances in rope mussels (Mytilus galloprovincialis) from Bizerte lagoon." Environ Sci Pollut Res Int 21(9): 6108-23.

                This study examined the influence of increasing temperatures in spring and summer on biochemical biomarkers in Mytilus galloprovincialis mussels sampled from Bizerte lagoon (northern Tunisia). Spatial and seasonal variations in a battery of seven biomarkers were analyzed in relation to environmental parameters (temperature, salinity, and pH), physiological status (condition and gonad indexes), stress on stress (SoS), and chemical contaminant levels (heavy metals, polycyclic aromatic hydrocarbons (PAHs), and PCBs) in digestive glands. Integrated biological response (IBR) was calculated using seven biomarkers (acetylcholinesterase (AChE), benzo[a]pyrene hydroxylase (BPH), multixenobiotic resistance (MXR), glutathione S-transferase (GST), catalase (CAT), malondialdehyde (MDA), and metallothioneins (MT). Seasonal variations in biological response were determined during a critical period between spring and summer at two sites, where chemical contamination varies by a factor of 2 for heavy metals and a factor 2.5 for PAHs. The analysis of a battery of biomarkers was combined with the measurement of physiological parameters at both sites, in order to quantify a maximum range of metabolic regulation with a temperature increase of 11 degrees C between May and August. According to our results, the MT, MDA, CAT, and AChE biomarkers showed the highest amplitude during the 11 degrees C rise, while the BPH, GST, and MXR biomarkers showed the lowest amplitude. Metabolic amplitude measured with the IBR at Menzel Abdelrahmen-the most severely contaminated station-revealed the highest metabolic stress in Bizerte lagoon in August, when temperatures were highest 29.1 degrees C. This high metabolic rate was quantified for each biomarker in the North African lagoon area and confirmed in August, when the highest IBR index values were obtained at the least contaminated site 2 (IBR = 9.6) and the most contaminated site 1 (IBR = 19.6). The combined effects of chemical contamination and increased salinity and temperatures in summer appear to induce a highest metabolic adaptation response and can therefore be used to determine thresholds of effectiveness and facilitate the interpretation of monitoring biomarkers. This approach, applied during substantial temperature increases at two sites with differing chemical contamination, is a first step toward determining an environmental assessment criteria (EAC) threshold in a North African lagoon.

 

Oliveira, L. F., S. M. Silva, et al. (2014). "Assessment of domestic landfill leachate toxicity to the Asian clam Corbicula fluminea via biomarkers." Ecotoxicol Environ Saf 103: 17-23.

                In order to evaluate the effects of domestic landfill leachate to bivalves Corbicula fluminea, clams were exposed to different leachate concentrations (v/v): 2, 3, 6 and 10 percent, corresponding to dilutions observed along a stream that receives this effluent, or only to clean water for comparisons. After 5 and 15 days of exposure the activity of the biotransformation enzymes 7-ethoxyresorufin-O-deethylase (EROD) and glutathione S-transferase (GST), the multixenobiotic resistance mechanism (MXR) and lipid peroxidation (LPO) in gills and digestive gland and metallothionein (MT) content in gills were evaluated. Differences in biomarkers responses were observed between gills and digestive gland, except for MXR that decreased in both tissues of clams exposed to 6 percent for 5 days. EROD activity in gills was reduced in all leachate concentrations after 5 days and only in 2 percent after 15 days exposure, while an EROD increase was observed in digestive gland after 15 days exposure to 6 percent. GST activity increased only in the gills of clams exposed to 10 percent for 5 days. LPO varied between tissues and different conditions. A significant increase in LPO was observed in the gills, after 5 days exposure to 2 and 6 percent, and in digestive gland after 5 and 15 days exposure to 2 and 3 percent. MT content in the gills increased after 15 days exposure to 2 percent. In conclusion, different leachate concentrations tested here caused biochemical changes in C. fluminea, but due to the observed variability in biomarkers responses among leachate concentrations, it was difficult to determine patterns or thresholds concentrations.

 

Aly, W., I. D. Williams, et al. (2014). "Limitations of metallothioneins in common cockles (Cerastoderma edule) and sponges (Haliclona oculata) as biomarkers of metal contamination in a semi-enclosed coastal area." Sci Total Environ 473-474: 391-7.

                Poole Harbour is typical of many heavily anthropologically impacted semi-enclosed estuarine coastal areas under pressure from metal pollution across the world. This study examined the physiological significance of metal burdens within that sensitive area, and assessed the potential use of metallothionein (MT) concentrations in two organisms: the common cockle (Cerastoderma edule) and the Mermaid's glove sponge (Haliclona oculata) for mapping the spatial extent of the biological response to metal contamination. A spectrophotometric method was applied for detection of MT in the bivalve, and for the first time to detect MT in sponges. The results show that while some metal concentrations in cockle and sponge tissues and in their surrounding environment (water and sediment) could be related to sources of metal contamination, MT values in the soft tissue of cockles and whole tissue of sponges are not. No relation could be found between MT in both cockles and sponges, and any of the tested metals (As, Cd, Cr, Cu, Pb, Hg, Ni, Ag, Sn, Zn). Furthermore, some of the lowest MT concentrations were detected in heavily polluted areas, indicating that MT concentrations in tested organisms are not exclusively associated with metal concentrations, as other environmental factors could affect induction of this protein. Organisms probably have a high tolerance to metal contamination and chronic exposure to a high level of contamination resulted in developing a variety of detoxification mechanisms. Results indicate that further study of metal stress in this type of ecosystem may need to examine other indicator species and/or apply a different biomonitoring technique.

 

Attig, H., N. Kamel, et al. (2014). "Effects of thermal stress and nickel exposure on biomarkers responses in Mytilus galloprovincialis (Lam)." Mar Environ Res 94: 65-71.

                The present work aimed to assess the Mytilus galloprovincialis digestive gland biomarkers responses to nickel (Ni) exposure along with a heat stress gradient. Mussels were exposed to a sublethal dose of nickel (13 muM) along with a temperature gradient (18 degrees C, 20 degrees C, 22 degrees C, 24 degrees C and 26 degrees C) for 4 days. Metallothionein (MTs) content was assessed as specific response to metals. Catalase (CAT), glutathione S-transferase (GST) activities and malondialdehyde (MDA) were measured as biomarkers of oxidative stress and lipid peroxidation. The cholinergic system was monitored using the acetylcholinesterase activity (AChE). Moreover, Ni uptakes along with the exposure temperatures were assessed. A correlation matrix (CM) between the investigated biomarkers and the exposure temperatures and a Principal Component Analysis (PCA) were achieved. Our data showed a negative effect of temperature increase on mussel's antioxidant and detoxification response to Ni exposure being more pronounced in animals exposed to the 24 degrees C and 26 degrees C.

 

Banni, M., A. Hajer, et al. (2014). "Transcriptional expression levels and biochemical markers of oxidative stress in Mytilus galloprovincialis exposed to nickel and heat stress." Comp Biochem Physiol C Toxicol Pharmacol 160: 23-9.

                The present study aims to evaluate transcriptional expression levels and biochemical markers of oxidative stress responses to nickel (Ni) exposure along with heat stress gradient in a mussel (Mytilus galloprovincialis). For this purpose, we investigated the response of oxidative stress markers, metallothionein accumulation and gene expression in digestive gland of mussels exposed to a sublethal concentration of Ni (2.5muM) along with a temperature gradient (18 degrees C, 22 degrees C, and 26 degrees C) for 24h and 72h. Ni digestive gland uptake was evaluated after the exposure periods. Co-exposure to Ni and higher temperature (26 degrees C) for 72h significantly decreased the antioxidant enzyme activities termed as catalase (CAT), superoxide dismutase (SOD) and glutathione-S-transferase (GST) and caused a pronounced increase of lipofuscin and neutral lipid (NL) accumulation. Ni-uptake was different with respect to the exposure periods and temperatures in Ni-exposed mussels. Sod, cat, gst, mt-10 and mt20 gene expression levels showed a substantial increased pattern in animals exposed for one day to heat stress compared to the control condition (18 degrees C). The same pattern but with highest level was registered in animals co-exposed to Ni and temperatures within one day. Three days exposure to 18 degrees C, 22 degrees C and 26 degrees C, resulted in a significant decrease in mRNA abundance of cat, gst and sod and a significant down-regulation of mts targets (22 degrees C and 26 degrees C). Our data provide new insights into the importance of the early protective response of oxidative stress related-gene expression and regulation in mussels challenging heat stress and sublethal Ni concentration.

 

Liu, F. and W. X. Wang (2014). "Differential influences of Cu and Zn chronic exposure on Cd and Hg bioaccumulation in an estuarine oyster." Aquat Toxicol 148: 204-10.

                In this study, the effects of Cu and Zn exposure, alone and in combination, on the bioaccumulation of Cd and Hg were investigated in an estuarine oyster Crassostrea hongkongensis under different salinity gradients. We showed that Zn, but not Cu, exposure significantly enhanced the Cd bioaccumulation. In contrast, both Cu and Zn exposure significantly enhanced the Hg bioaccumulation. Combined exposure and salinity did not affect the metal interactions in oysters. The increased tissue concentrations of Cd or Hg were associated with their increased storage in inducible metal-binding ligands (e.g. metallothionein-like proteins, MTLP) by Cu/Zn exposure. The differential roles of Cu and Zn exposure in Cd and Hg bioaccumulation resulted from their contrasting ligand induction and affinities. Analysis of field collected oysters indicated that Cu/Zn exposure was a significant contributor to tissue concentrations of Cd, Cu and Hg. Overall, biochemical/physiological changes of the animals chronically exposed to metal stressors played a key role in affecting tissue concentrations of other metals. One metal's ability to enhance the bioaccumulation of other metals depended upon the relative affinities of the metals for MTLP.

 

Luo, L., C. Ke, et al. (2014). "Metal accumulation and differentially expressed proteins in gill of oyster (Crassostrea hongkongensis) exposed to long-term heavy metal-contaminated estuary." Fish Shellfish Immunol 38(2): 318-29.

                Bio-accumulation and bio-transmission of toxic metals and the toxicological responses of organisms exposed to toxic metals have been focused, due to heavy metal contaminations have critically threatened the ecosystem and food security. However, still few investigations focused on the responses of certain organisms exposed to the long term and severe heavy metal contamination in specific environments. In present investigation, the Hong Kong oyster, Crassostrea hongkongensis were obtained from 3 sites which were contaminated by different concentrations of heavy metals (such as zinc, copper, manganese and lead etc.), respectively. Heavy metal concentrations in the sea water samples collected from the 3 sites and the dissected tissues of the oysters with blue visceral mass were determinated to estimate the metal contamination levels in environments and the bio-accumulation ratios of the heavy metals in the different tissues of oysters. Moreover, Proteomic methods were employed to analyze the differentially expressed proteins in the gills of oysters exposed to long-term heavy metal contaminations. Results indicated that the Jiulong River estuary has been severely contaminated by Cu, Zn and slightly with Cr, Ni, Mn, etc, moreover, Zn and Cu were the major metals accumulated by oysters to phenomenally high concentrations (more than 3.0% of Zn and about 2.0% of Cu against what the dry weight of tissues were accumulated), and Cr, Ni, Mn, etc were also significantly accumulated. The differentially expressed proteins in the gills of oysters exposed to heavy metals participate in several cell processes, such as metal binding, transporting and saving, oxidative-reduction balance maintaining, stress response, signal transduction, etc. Significantly up-regulated expression (about 10 folds) of an important metal binding protein, metallothionein (MT) and granular cells was observed in the gills of oysters exposed to long-term and severely heavy-metal-contaminated estuary, it suggested that binding toxic metals with metallothionein-like proteins (MTLP) and storing toxic metals in metal-rich granules (MRG) with insoluble forms were the important strategies of oyster to detoxify the toxic metals and adapt to the high level of metal-contaminated environment. Most of the stress and immunity responsive proteins, such as heat shock proteins (HSP), extracellular superoxide dismutase (ECSOD) and cavortin, and the cellular redox reaction relative proteins such as 20G-Fe (II) oxygenase family oxidoreductase, aldehyde dehydrogenase and retinal dehydrogenase 2, were detected significantly down-regulated in the gills of oysters exposed to long term heavy metal contaminated environments, it indicated that long term exposure different from emergent exposure to heavy metal contamination may significantly suppress the stress and immunity response system of oysters. Moreover, Formin homology 2 domain containing protein (FH2). The only protein domain to directly nucleate actin monomers into unbranched filament polymers, by which will subsequently control gene expression and chromatin remodelling complexes, was also detected greatly up-regulated in the gills of oysters exposed to long-term heavy metal contaminations. It indicated that nuclear activity regulation may also be important for oyster to adapt to the long-term heavy-metal-contaminated environment.

 

Scudiero, R., P. Creti, et al. (2014). "Evaluation of cadmium, lead and metallothionein contents in the tissues of mussels (Mytilus galloprovincialis) from the Campania coast (Italy): levels and seasonal trends." C R Biol 337(7-8): 451-8.

                The biological effect of seasonality on cadmium, lead and metallothionein contents was assessed in mussels Mytilus galloprovincialis from natural banks located along the coastline of the Gulf of Naples (Campania, Italy). Heavy metals and metallothionein concentrations were measured in digestive and reproductive glands. The results showed a clear correlation between metallothionein content and the reproductive gland status determined during the seasons; on the contrary, no correlation was found between metallothionein and metal contents. Data allow us to hypothesize that metallothionein functions go beyond metal detoxification, thus opening new scenarios for these proteins in invertebrates. The effect of seasons on metals concentration in mussel tissues showed similar seasonal patterns between the sites, regardless of their anthropogenic impacts. Cadmium content was not strictly related to seasonal periods, whereas lead content was significantly lower in summer. The results also indicate that the metal contents in mussels from the Gulf of Naples do not represent a risk to human health, even in the period of their maximum accumulation, and that the relaying of mussels before marketing could improve the animal stress conditions, but having a slight effect on metal excretion.

 

Marasinghe Wadige, C. P., W. A. Maher, et al. (2014). "Exposure-dose-response relationships of the freshwater bivalve Hyridella australis to cadmium spiked sediments." Aquat Toxicol 152: 361-71.

                To understand how benthic biota may respond to the additive or antagonistic effects of metal mixtures in the environment it is first necessary to examine their responses to the individual metals. In this context, laboratory controlled single metal-spiked sediment toxicity tests are useful to assess this. The exposure-dose-response relationships of Hyridella australis to cadmium-spiked sediments were, therefore, investigated in laboratory microcosms. H. australis was exposed to individual cadmium spiked sediments (<0.05 (control), 4+/-0.3 (low) and 15+/-1 (high) mug/g dry mass) for 28 days. Dose was measured as cadmium accumulation in whole soft body and individual tissues at weekly intervals over the exposure period. Dose was further examined as sub-cellular localisation of cadmium in hepatopancreas tissues. The biological responses in terms of enzymatic and cellular biomarkers were measured in hepatopancreas tissues at day 28. H. australis accumulated cadmium from spiked sediments with an 8-fold (low exposure organisms) and 16-fold (high exposure organisms) increase at day 28 compared to control organisms. The accumulated tissue cadmium concentrations reflected the sediment cadmium exposure at day 28. Cadmium accumulation in high exposure organisms was inversely related to the tissue calcium concentrations. Gills of H. australis showed significantly higher cadmium accumulation than the other tissues. Accumulated cadmium in biologically active and biologically detoxified metal pools was not significantly different in cadmium exposed organisms, which suggests that H. australis has some tolerance to cadmium. The metallothionein like protein fraction played an important role in the sequestration and detoxification of cadmium and the amount sequestered in this fraction increased with increased cadmium exposure. The highest percentage of biologically active cadmium was associated with the lysosome+microsome and mitochondrial fractions. Cadmium concentrations in these two fractions of cadmium exposed organisms were significantly higher with respect to controls. Total antioxidant capacity decreased with increased cadmium exposure and tissue dose. Lipid peroxidation increased and lysosomal membrane stability decreased significantly with increased cadmium exposure and tissue dose. Based on exposure-dose-response analysis in this study, H. australis would be a suitable organism for assessing cadmium sediment exposure and toxicity.

 

Taylor, A. M. and W. A. Maher (2014). "Exposure-dose-response of Tellina deltoidalis to metal contaminated estuarine sediments 2. Lead spiked sediments." Comp Biochem Physiol C Toxicol Pharmacol 159: 52-61.

                Lead accumulation in estuarine sediments, as a result of activities such as mining and ore smelting, and through urban runoff is a continuing problem in the increasingly developed world. Marine organisms accumulate lead, which is known to be highly toxic to biological processes and to degrade organism and ecosystem health. Here the relationship between lead exposure, dose and response was investigated in the sediment dwelling, deposit feeding, marine bivalve Tellina deltoidalis. Bivalves were exposed in the laboratory to individual lead spiked sediments at < 0.01, 100 and 300 mug/g dry mass, for 28 days and accumulated total tissue lead concentrations of 4, 96 and 430 mug/g, respectively. Subcellular fractionation indicated that around 70% of the total accumulated tissue lead was detoxified, three quarters of the detoxified lead fraction was converted into metal rich granules, with the remainder in the metallothionein like protein fraction. The majority of biologically active lead was associated with the mitochondrial fraction with up to a 128 fold increase in lead burden in exposed organisms compared to controls. This indicates lead detoxification was occurring but the organism was unable to prevent lead interacting with sensitive organelles. With increased lead exposure T. deltoidalis showed a suppression in glutathione peroxidase activity, total glutathione concentration and reduced GSH:GSSG ratios, however, these differences were not significant. Lead exposed T. deltoidalis had a significantly reduced total antioxidant capacity which corresponded with increased lipid peroxidation, lysosomal destabilisation and micronuclei frequency. The exposure-dose-response relationships demonstrated for lead exposed T. deltoidalis supports its potential for the development of sublethal endpoints in lead toxicity assessment.

 

Weng, N. and W. X. Wang (2014). "Improved tolerance of metals in contaminated oyster larvae." Aquat Toxicol 146: 61-9.

                Environmental stress experienced by parents may make a significant difference in the response of their offspring. However, relevant studies on marine bivalves are very limited especially for the field populations. In the present study, we examined the relative metal tolerance of offspring produced by four natural populations of oyster Crassostrea sikamea that were contaminated by metals to different degrees. We demonstrated that the resistance of oyster offspring to copper and zinc was correlated with the level of metal pollution experienced by the parent oysters. Specifically, the oyster embryo and larvae produced by adult oysters from contaminated sites had a much higher tolerance to metal stress than those from the reference sites. Furthermore, tissue concentration-dependent maternal transfer of Cu and Zn was found in this study, and the metallothionein concentrations in eggs were positively related to the total concentrations of maternally transferred Cu and Zn. Thus, the maternally transferred metals inducing high level of MT synthesis in eggs was one of the possible mechanisms responsible for the enhanced metal tolerance of oyster embryos and larvae from heavily contaminated sites. We concluded that environmental exposure history of adult oysters significantly influenced the ability of their offspring to cope with metal stress. Our findings offered the field evidence of the possible transfer of metal tolerance from adults to offspring in marine bivalves.

 

Strizak, Z., D. Ivankovic, et al. (2014). "Characterization of the cytosolic distribution of priority pollutant metals and metalloids in the digestive gland cytosol of marine mussels: seasonal and spatial variability." Sci Total Environ 470-471: 159-70.

                Cytosolic profiles of several priority pollutant metals (Cu, Cd, Zn, Pb) and metalloid As were analyzed in the digestive gland of the mussel (Mytilus galloprovincialis) sampled at locations with different environmental pollution levels along the Croatian coast in the spring and summer season. Size-exclusion chromatography (SEC) connected to inductively coupled plasma mass spectrometry (ICP-MS) was used to determine selected elements bound to cytosolic biomolecules separated based on their molecular size. Copper, cadmium and zinc eluted mostly associated with high molecular weight (HMW) and medium molecular weight (MMW) biomolecules, but with a more prominent elution in the MMW peak at polluted locations which were probably associated with the 20 kDa metallothionein (MT). Elution of all three metals within this peak was also strongly correlated with cytosolic Cd as strong inducer of MT. Lead mostly eluted in HMW biomolecule range, but in elevated cytosolic Pb concentrations, significant amount eluted in low molecular weight (LMW) biomolecules. Arsenic, on the other hand eluted almost completely in LMW range, but we could not distinguish specific molecular weight biomolecules which would be predominant in detoxification mechanism. Seasonal variability in element abundance within specific peaks was present, although not in the same extent, for all elements and locations, especially for As. The results confirm the suitability of the distribution of selected metals/metalloids among different cytosolic ligands as potential indicator for metal exposure. Obtained findings can also serve as guidelines for further separation and characterization of specific cytosolic metal-binding biomolecules.

 

Gagnon, C., P. Turcotte, et al. (2014). "Impacts of municipal wastewater oxidative treatments: changes in metal physical speciation and bioavailability." Chemosphere 97: 86-91.

                The environmental repercussions of the discharge of disinfected effluents are still poorly understood. This study assessed the impact of ozonation and UV oxidative treatment processes on metal forms - particulate, colloidal and permeable fractions - and bioavailability in disinfected wastewaters. In addition to wastewater analyses, mussels were placed in continuous flow-through aquaria and exposed for 4wk to wastewater, then metals in their tissues were analysed in parallel with exposure biomarkers. Metal size distribution was affected by oxidative processes; results showed that ozonation treatment generally increases the permeable fraction of some metals, particularly Cd and Cu, in treated waters, whereas UV treatment fosters the formation of permeable Zn. Ozone treatment of wastewater generally increased the bioavailability of specific metals. Metal bioaccumulation was in most cases significantly higher in mussels exposed to ozone-treated effluent compared to the UV treatment: 58%, 32%, 42% and 47% higher, respectively, for Ag, Cd, Cr and Cu. Physical metal speciation in these wastewaters comparatively measured the permeable fraction of metals to relate them to the bioaccumulation results for the exposed mussels. The levels of lipid peroxidation were significantly increased in gills but not in the digestive gland. The levels of metallothionein in the digestive gland were also significantly reduced suggest decreased input of particulate metals. Results of bioaccumulation in mussels suggested that metal bioavailability can be modified by the different oxidative processes. Despite this disadvantage, ozonation still represents a great choice of treatment considering the overall environmental benefits.

 

Falfushynska, H., L. Gnatyshyna, et al. (2014). "Habitat pollution and thermal regime modify molecular stress responses to elevated temperature in freshwater mussels (Anodonta anatina: Unionidae)." Sci Total Environ 500-501: 339-50.

                Elevated temperature and pollution are common stressors in freshwater ecosystems. We study cellular stress response to acute warming in Anodonta anatina (Unionidae) from sites with different thermal regimes and pollution levels: a pristine area and an agriculturally polluted site with normal temperature regimes (F and A, respectively) and a polluted site with elevated temperature (N) from the cooling pond of an electrical power plant. Animals were exposed to different temperatures for 14 days and stress response markers were measured in gills, digestive gland and hemocytes. Mussels from site N and A had elevated background levels of lactate dehydrogenase activity indicating higher reliance on anaerobic metabolism for ATP production and/or redox maintenance. Exposure to 25 degrees C and 30 degrees C induced oxidative stress (indicated by elevated levels of lipid peroxidation products) in digestive gland and gills of mussels from A and F sites, while in mussels from N sites elevated oxidative stress was only apparent at 30 degrees C. Temperature-induced changes in levels of antioxidants (superoxide dismutase, metallothioneins and glutathione) were tissue- and population-specific. Acute warming led to destabilization of lysosomal membranes and increased frequencies of nuclear lesions in mussels from F and A sites but not in their counterparts from N site. Elevated temperature led to an increase in the frequency of micronuclei in hemocytes in mussels from F and A sites at 25 degrees C and 30 degrees C and in mussels from N site at 30 degrees C. The mussels from N site also demonstrated better survival at elevated temperature (30 degrees C) than their counterparts from the F and A sites. Taken together, these data indicate that long-term acclimation and/or adaptation of A. anatina to elevated temperatures result in increased thermotolerance and alleviate stress response to moderate temperature rise. In contrast, extreme warming (30 degrees C) is harmful to mussels from all populations indicating limit to this induced thermotolerance.

 

Freitas, R., R. Martins, et al. (2014). "Venerupis decussata under environmentally relevant lead concentrations: Bioconcentration, tolerance, and biochemical alterations." Environ Toxicol Chem 33(12): 2786-94.

                The edible clam Venerupis decussata is widely distributed in European aquatic systems, some of which are under strong anthropogenic pressure, which can contribute to trophic transfer of xenobiotics to humans. Accordingly, the present study focused on the tolerance, bioconcentration, and biochemical responses of V. decussata after exposure to ecologically relevant concentrations of lead. Health risks to humans after consumption of clams was also explored. An acute toxicity assay (96 h) was conducted with wild clams, using Pb exposure concentrations ranging from 0 mg L(-1) to 1.80 mg L(-1). Lethality, bioconcentration factor (BCF), intracellular partitioning, and a relevant set of biomarkers were used as endpoints. Clams, interstitial water, water column, and sediment samples were collected to analyze Pb concentration. The Pb concentration in wild clams was below international consumption guidelines. Under laboratory conditions, clams revealed high sensitivity to Pb (median lethal concentration of 0.65 mg L(-1)), with a high bioconcentration ability (bioconcentration factor > 1) during exposure. The intracellular partitioning data showed that most of the Pb had accumulated in the insoluble fraction (>80%). Several significant biochemical changes were observed, namely on catalase and glutathione-S-tranferase activities and metalothionein content. Overall, it was demonstrated that the European clam has a reduced tolerance to Pb, compared with other bivalves. However, consumption of clams from the Ria de Aveiro lagoon (Portugal) does not raise public health concerns in terms of Pb.

 

Arini, A., G. Daffe, et al. (2014). "What are the outcomes of an industrial remediation on a metal-impacted hydrosystem? A 2-year field biomonitoring of the filter-feeding bivalve Corbicula fluminea." Chemosphere 108: 214-24.

                The Riou-Mort watershed (southwest France) exhibits high metal contaminations (Cd and Zn) related to an important mining past. In this context, a remediation process has been implemented in 2007 to reduce the watershed contamination. The aim of this study was to assess the early effectiveness of the remediation process on the hydrosystem contamination state. A biomonitoring was realized over two years (2008-2010) with the filter-feeding bivalve Corbicula fluminea, exposed along a contamination gradient. Several biological parameters were monitored: (1) Cd and Zn bioaccumulation, (2) Metallothionein (MT) production as detoxification mechanism, (3) differential gene expression (cat, sod, gst, 12S, cox1, mt). The physicochemical data highlighted strong metal contamination persistence in the river water and failed to demonstrate a significant decrease of metal contamination during the 2-year monitoring. The bioaccumulation results confirmed the persistence of a water contamination despite remediation works, with maximum values measured downstream from the industrial site (Joany). The bioaccumulation increased in 2010, reaching 69.3+/-5.3 mug Cd g(-1) DW at Joany in July 2010, whereas it did not exceed 1.4+/-0.2 mug Cd g(-1) DW at the reference site throughout the biomonitoring. MT concentrations were closely related to the contamination gradient, especially at Joany, demonstrating their strong involvement in the detoxification processes. The mt gene induction was strongly correlated to the MT and metal concentrations. The gene inductions of cat, sod, gst and 12S were correlated to both the metal concentrations and the seasonal variations, especially temperatures. This suggests that environmental factors require serious consideration for the interpretation of bioaccumulation kinetics and thus for the assessment of the remediation effectiveness. Consequently, the whole results did not yet highlight strong beneficial effects of remediation work on the hydrosystem contamination state. First benefits of that process should be progressively felt, once the remediation achieved, and should grandly accelerate the decontamination process of the contaminated area.

 

D'Agata, A., S. Fasulo, et al. (2014). "Enhanced toxicity of 'bulk' titanium dioxide compared to 'fresh' and 'aged' nano-TiO2 in marine mussels (Mytilus galloprovincialis)." Nanotoxicology 8(5): 549-58.

                Marine bivalves (Mytilus galloprovincialis) were exposed to titanium dioxide (10 mg L(-1)) either as engineered nanoparticles (nTiO2; fresh, or aged under simulated sunlight for 7 days) or the bulk equivalent. Inductively coupled plasma-optical emission spectrometry analyses of mussel tissues showed higher Ti accumulation (>10-fold) in the digestive gland compared to gills. Nano-sized TiO2 showed greater accumulation than bulk, irrespective of ageing, particularly in digestive gland (>sixfold higher). Despite this, transcriptional expression of metallothionein genes, histology and histochemical analysis suggested that the bulk material was more toxic. Haemocytes showed significantly enhanced DNA damage, determined by the modified comet assay, for all treatments compared to the control, but no significant differences between the treatments. Our integrated study suggests that for this ecologically relevant organism photocatalytic ageing of nTiO2 does not significantly alter toxicity, and that bulk TiO2 may be less ecotoxicologically inert than previously assumed.

 

Leung, P. T., T. J. Park, et al. (2014). "Isoform-specific responses of metallothioneins in a marine pollution biomonitor, the green-lipped mussel Perna viridis, towards different stress stimulations." Proteomics 14(15): 1796-807.

                Metallothioneins (MTs) are commonly used as biomarker for metal pollution assessment in marine ecosystems. Using integrated genomic and proteomic analyses, this study characterized two types of MT isoform in the digestive gland of a common biomonitor, the green-lipped mussel Perna viridis, towards the challenges of a metal (cadmium; Cd) and a non-metal oxidant (hydrogen peroxide; H2 O2 ) respectively. The two isoforms differed in their deduced protein sequences, with 73 amino acids for MT10-I and 72 for MT10-II (a novel type), but both consisted of a high percentage (27.4 to 29.2%) of cysteine. Two-dimensional gel and Western blot showed that the MT proteins were present in multiple isoform spots, and they were further validated to be MT10-I and MT10-II using MS analysis coupled with unrestricted modifications searching. Expression of mRNA revealed that MT10-I responded promptly to Cd but had a lagged induction to H2 O2 treatments, while MT10-II was exclusively induced by Cd treatment over the course of exposure. Expression of the MT proteins also showed a delayed response to H2 O2 , compared to Cd treatments. This study uncovered the potential different functional roles of various MTs isoforms in P. viridis and thus advances the resolution of using MTs as biomarkers in future applications.

 

Palacios, O., S. Perez-Rafael, et al. (2014). "Cognate and noncognate metal ion coordination in metal-specific metallothioneins: the Helix pomatia system as a model." J Biol Inorg Chem 19(6): 923-35.

                The Helix pomatia metallothionein (MT) system, namely, its two highly specific forms, HpCdMT and HpCuMT, has offered once again an optimum model to study metal-protein specificity. The present work investigates the most unexplored aspect of the coordination behavior of MT polypeptides with respect to either cognate or noncognate metal ions, as opposed to the standard studies of cognate metal ion coordination. To this end, we analyzed the in vivo synthesis of the corresponding complexes with their noncognate metals, and we performed a detailed spectroscopic and spectrometric study of the Zn(2+)/Cd(2+) and Zn(2+)/Cu(+) in vitro replacement reactions on the initial Zn-HpMT species. An HpCuMTAla site-directed mutant, exhibiting differential Cu(+)-binding abilities in vivo, was also included in this study. We demonstrate that when an MT binds its cognate metal, it yields well-folded complexes of limited stoichiometry, representative of minimal-energy conformations. In contrast, the incorporation of noncognate metal ions is better attributed to an unspecific reaction of cysteinic thiolate groups with metal ions, which is dependent on their concentration in the surrounding milieu, where no minimal-energy structure is reached, and otherwise, the MT peptide acts as a multidentate ligand that will bind metal ions until its capacity has been saturated. Additionally, we suggest that previous binding of an MT polypeptide with its noncognate metal ion (e.g., binding of Zn(2+) to the HpCuMT isoform) may preclude the correct folding of the complex with its cognate metal ion.

 

Gillis, P. L., S. K. Higgins, et al. (2014). "Evidence of oxidative stress in wild freshwater mussels (Lasmigona costata) exposed to urban-derived contaminants." Ecotoxicol Environ Saf 102: 62-9.

                The physiological effect of complex mixtures of anthropogenic contaminants on aquatic organisms is not well understood. This study employed a suite of sub-cellular biomarkers and general health measurements to assess the effect of urban-derived contaminants on wild freshwater mussels. Adult Lasmigona costata were collected from four sites in the Grand River (ON, Canada) that receive incremental amounts of municipal wastewater effluents and road runoff. Biomarkers of metal exposure, oxidative stress, and general health were examined in the gills of wild mussels. Concentrations of nine metals as well as the metal-binding protein, metallothionein (MT), were significantly higher (p<0.05) in mussels living downstream of the urban area. For example the concentrations of Pb, Cr and Zn were five-fold, and Ag more than 20 fold higher in mussels collected downstream of 11 municipal wastewater treatment plants and four cities compared to levels in upstream mussels. Downstream mussels showed evidence of oxidative stress, such that lipid peroxidation (LPO) (as thiobarbiturate reactive substances) was significantly elevated and the antioxidant capacity against peroxyl radicals (ACAP) was significantly decreased (p<0.01) in downstream mussels compared to upstream mussels. Regarding general health indicators, although gill lipid concentrations were similar across sites, protein concentration was significantly (p<0.001) higher in mussels collected from the upstream reference site compared to all downstream sites. The trends observed indicate that there are physiological effects in mussels associated with chronic exposure to complex urban inputs and that some biomarkers respond to municipal wastewater effluent and road runoff exposure in a cumulative manner. The observed oxidative stress response (ACAP) along with the elevation in MT, suggest that even though the defense mechanisms in the chronically exposed mussels have been activated, there is still an excess of reactive oxygen species that result in oxidative damage. The physiological effects of exposure reported in this study correspond with previously reported whole-organism impacts and declines in freshwater mussel populations in the urban-impacted region of this watershed.

 

Gotze, S., O. B. Matoo, et al. (2014). "Interactive effects of CO(2) and trace metals on the proteasome activity and cellular stress response of marine bivalves Crassostrea virginica and Mercenaria mercenaria." Aquat Toxicol 149: 65-82.

                Increased anthropogenic emission of CO2 changes the carbonate chemistry and decreases the pH of the ocean. This can affect the speciation and the bioavailability of metals in polluted habitats such as estuaries. However, the effects of acidification on metal accumulation and stress response in estuarine organisms including bivalves are poorly understood. We studied the interactive effects of CO2 and two common metal pollutants, copper (Cu) and cadmium (Cd), on metal accumulation, intracellular ATP/ubiquitin-dependent protein degradation, stress response and energy metabolism in two common estuarine bivalves-Crassostrea virginica (eastern oyster) and Mercenaria mercenaria (hard shell clam). Bivalves were exposed for 4-5 weeks to clean seawater (control) and to either 50 mug L(-1) Cu or 50 mug L(-1) Cd at one of three partial pressures of CO2 ( [Formula: see text] approximately 395, approximately 800 and approximately 1500 muatm) representative of the present-day conditions and projections of the Intergovernmental Panel for Climate Change (IPCC) for the years 2100 and 2250, respectively. Clams accumulated lower metal burdens than oysters, and elevated [Formula: see text] enhanced the Cd and Cu accumulation in mantle tissues in both species. Higher Cd and Cu burdens were associated with elevated mRNA expression of metal binding proteins metallothionein and ferritin. In the absence of added metals, proteasome activities of clams and oysters were robust to elevated [Formula: see text] , but [Formula: see text] modulated the proteasome response to metals. Cd exposure stimulated the chymotrypsin-like activity of the oyster proteasome at all CO2 levels. In contrast, trypsin- and caspase-like activities of the oyster proteasome were slightly inhibited by Cd exposure in normocapnia but this inhibition was reversed at elevated [Formula: see text] . Cu exposure inhibited the chymotrypsin-like activity of the oyster proteasome regardless of the exposure [Formula: see text] . The effects of metal exposure on the proteasome activity were less pronounced in clams, likely due to the lower metal accumulation. However, the general trends (i.e. an increase during Cd exposure, inhibition during exposure to Cu, and overall stimulatory effects of elevated [Formula: see text] ) were similar to those found in oysters. Levels of mRNA for ubiquitin and tumor suppressor p53 were suppressed by metal exposures in normocapnia in both species but this effect was alleviated or reversed at elevated [Formula: see text] . Cellular energy status of oysters was maintained at all metal and CO2 exposures, while in clams the simultaneous exposure to Cu and moderate hypercapnia ( approximately 800 muatm [Formula: see text] ) led to a decline in glycogen, ATP and ADP levels and an increase in AMP indicating energy deficiency. These data suggest that environmental CO2 levels can modulate accumulation and physiological effects of metals in bivalves in a species-specific manner which can affect their fitness and survival during the global change in estuaries.

 

Goswami, P., G. Hariharan, et al. (2014). "An integrated use of multiple biomarkers to investigate the individual and combined effect of copper and cadmium on the marine green mussel (Perna viridis)." J Environ Sci Health A Tox Hazard Subst Environ Eng 49(13): 1564-77.

                The present study documents individual and combined sub-lethal effect of one redox active (copper) and one non-redox active (cadmium) metal on green mussel (Perna viridis). The mussels were exposed to 60 mug L(-1) of Cu and 150 mug L(-1) of Cd (individually and in combination) for 21 days. Histopathological and ultrastructural studies revealed significant metal induced alterations such as vacuolization, fusion of gill lamellae, enhance mucous deposition, hyperplasia and necrosis in gills. Antioxidant enzyme assays revealed significant increase in superoxide dismutase (SOD), glutathione S-transferase (GST) and glutathione peroxidase (GPx) activity. Similarly, single exposure to Cd and Cu caused significant induction in Malate dehydrogenase (MDH) activity. However, combined Cu+Cd exposure modulated suppression in MDH activity. Unlike MDH, Cu and Cd individual exposure resulted in a decrease in esterase (EST) activity, but their combined exposure caused an induction. Non-enzymatic biomarkers such as lipid peroxidation (LPO) and metallothionein (MT) levels showed no significant change in response to Cu exposure, whereas, individual Cd exposure or Cd exposure in combination with Cu caused significant changes in their levels. Comet assay revealed a significant increase in DNA damage upon metal exposure. These results indicate that Cu (redox active) and Cd (non-redox active) can induce measurable physiological, biochemical as well as genotoxic perturbations in mussels even at sub-lethal concentrations. A monitoring programme based on the biomarkers discussed here would be useful to study the effect of metal pollutants reaching the coastal waters.

 

Chandurvelan, R., I. D. Marsden, et al. (2013). "Biochemical biomarker responses of green-lipped mussel, Perna canaliculus, to acute and subchronic waterborne cadmium toxicity." Aquat Toxicol 140-141: 303-13.

                The biochemical responses of the green-lipped mussel, Perna canaliculus, to waterborne cadmium (Cd) were investigated in order to delineate toxic mechanisms, and the impacts of exposure dose and duration, of this important toxicant in a potential sentinel species. Mussels were exposed for either 96 h (acute: 0, 2000, 4000 mugL(-1) Cd) or for 28 d (subchronic: 0, 200, 2000 mugL(-1) Cd), and the digestive gland, gill and haemolymph were examined for impacts. Biochemical responses measured included those associated with metal detoxification (metallothionein-like protein; MTLP), oxidative stress (catalase, lipid peroxidation), cellular homeostasis (alkaline phosphatase, Na(+), K(+)-ATPase; NKA), and energy utilisation (glycogen, haemolymph protein). Following acute exposure, digestive gland glycogen and gill NKA activity were significantly altered by Cd exposure relative to levels in mussels exposed to Cd-free seawater. Subchronic Cd exposure resulted in a significant increase in MTLP levels in both the gill and the digestive gland. This increase was correlated strongly with the levels of Cd accumulation measured in these tissues (R=0.957 for gill, 0.964 for digestive gland). Catalase activity followed a similar pattern, although the correlation with tissue Cd accumulation was not as strong (R=0.907 for gill, 0.708 for digestive gland) as that for MTLP. Lipid peroxidation increased in the digestive gland at Days 7 and 14 at both subchronic Cd levels tested, but this effect had largely dissipated by Days 21 and 28 (with the exception of the 2,000 mugL(-1) group at Day 28). Alkaline phosphatase activity decreased significantly with Cd exposure in both tissues. This effect was observed at both tested concentrations in the gill, but only at the highest concentration for digestive gland. A decrease in digestive gland glycogen levels was observed in Cd-exposed mussels (Days 14 and 21 at 2,000 mugL(-1)), while haemolymph protein levels increased as a result of subchronic Cd exposure. These findings indicated that biochemical responses in Cd-exposed mussels were tissue-specific, dose- and time-dependent, with duration of exposure being the predominant effect. This study shows that biochemical changes in Cd-exposed green-lipped mussels can be linked to tissue metal accumulation and are consistent with previously reported physiological effects. It also suggests that green-lipped mussels are amenable to a multiple biomarker approach and may be of use as a bioindicator species for monitoring coastal metal pollution.

 

Ivanina, A. V., E. Beniash, et al. (2013). "Short-term acute hypercapnia affects cellular responses to trace metals in the hard clams Mercenaria mercenaria." Aquat Toxicol 140-141: 123-33.

                Estuarine and coastal habitats experience large fluctuations of environmental factors such as temperature, salinity, partial pressure of CO2 ( [Formula: see text] ) and pH; they also serve as the natural sinks for trace metals. Benthic filter-feeding organisms such as bivalves are exposed to the elevated concentrations of metals in estuarine water and sediments that can strongly affect their physiology. The effects of metals on estuarine organisms may be exacerbated by other environmental factors. Thus, a decrease in pH caused by high [Formula: see text] (hypercapnia) can modulate the effects of trace metals by affecting metal bioavailability, accumulation or binding. To better understand the cellular mechanisms of interactions between [Formula: see text] and trace metals in marine bivalves, we exposed isolated mantle cells of the hard clams (Mercenaria mercenaria) to different levels of [Formula: see text] (0.05, 1.52 and 3.01 kPa) and two major trace metal pollutants - cadmium (Cd) and copper (Cu). Elevated [Formula: see text] resulted in a decrease in intracellular pH (pHi) of the isolated mantle cells from 7.8 to 7.4. Elevated [Formula: see text] significantly but differently affected the trace metal accumulation by the cells. Cd uptake was suppressed at elevated [Formula: see text] levels while Cu accumulation has greatly accelerated under hypercapnic conditions. Interestingly, at higher extracellular Cd levels, labile intracellular Cd(2+) concentration remained the same, while intracellular levels of free Zn(2+) increased suggesting that Cd(2+) substitutes bound Zn(2+) in these cells. In contrast, Cu exposure did not affect intracellular Zn(2+) but led to a profound increase in the intracellular levels of labile Cu(2+) and Fe(2+). An increase in the extracellular concentrations of Cd and Cu led to the elevated production of reactive oxygen species under the normocapnic conditions (0.05 kPa [Formula: see text] ); surprisingly, this effect was mitigated in hypercapnia (1.52 and 3.01 kPa). Overall, our data reveal complex and metal-specific interactions between the cellular effects of trace metals and [Formula: see text] in clams and indicate that variations in environmental [Formula: see text] may modulate the biological effects of trace metals in marine organisms.

 

Pytharopoulou, S., G. G. Kournoutou, et al. (2013). "Cadmium versus copper toxicity: insights from an integrated dissection of protein synthesis pathway in the digestive glands of mussel Mytilus galloprovincialis." J Hazard Mater 260: 263-71.

                The main purpose of this study was to investigate the impact of metal-mediated stress on the protein-synthesis pathway in mussels. To this end, mussels (Mytilus galloprovincialis) underwent a 15 days exposure to 100 mug/L Cu(2+) or Cd(2+). Both metals, in particular Cd(2+), accumulated in mussel digestive glands and generated a specific status of oxidative-stress. Exposure of mussels to each metal resulted in 40% decrease of the tRNA-aminoacylation efficiency, at the end of exposure. Cu(2+) also caused a progressive loss in the capability of 40S-ribosomal subunits to form 48S pre-initiation complex, which reached 34% of the control at the end of exposure. Other steps of translation underwent less pronounced, but measurable damages. Mussels exposed to Cd(2+) for 5 days presented a similar pattern of translational dysfunctions in digestive glands, but during the following days of exposure the ribosomal efficiency was gradually restored. Meanwhile, metallothionein levels significantly increased, suggesting that upon Cd(2+)-mediated stress the protein-synthesizing activity was reorganized both quantitatively and qualitatively. Conclusively, Cd(2+) and Cu(2+) affect translation at several levels. However, the pattern of translational responses differs, largely depending on the capability of each metal to affect cytotoxic pathways in the tissues, such as induction of antioxidant defense and specific repair mechanisms.

 

Yang, C., L. Wang, et al. (2013). "The polymorphism in the promoter region of metallothionein 1 is associated with heat tolerance of scallop Argopecten irradians." Gene 526(2): 429-36.

                Metallothioneins (MTs), a superfamily of cysteine-rich proteins, perform multiple functions, such as maintaining homeostasis of essential metals, detoxification of toxic metals and scavenging of oxyradicals. In this study, the promoter region of a metallothionein (MT) gene from Bay scallop Argopecten irradians (designed as AiMT1) was cloned by the technique of genomic DNA walking, and the polymorphisms in this region were screened to find their association with susceptibility or tolerance to high temperature stress. One insert-deletion (ins-del) polymorphism and sixteen single nucleotide polymorphisms (SNPs) were identified in the amplified promoter region. Two SNPs, -375 T-C and -337 A-C, were selected to analyze their distribution in the two Bay scallop populations collected from southern and northern China coast, which were identified as heat resistant and heat susceptible stocks, respectively. There were three genotypes, T/T, T/C and C/C, at locus -375, and their frequencies were 25%, 61.1% and 13.9% in the heat susceptible stock, while 34.2%, 42.1% and 23.7% in the resistant stock, respectively. There was no significant difference in the frequency distribution of different genotypes between the two stocks (P>0.05). In contrast, at locus -337, three genotypes A/A, A/C and C/C were revealed with the frequencies of 11.6%, 34.9% and 53.5% in the heat susceptible stock, while 45.7%, 32.6% and 21.7% in the heat resistant stock, respectively. The frequency of C/C genotype in the heat susceptible stock was significantly higher (P<0.01) than that in the heat resistant stock, while the frequency of A/A in the heat resistant stock was significantly higher (P<0.01) than that in the heat susceptible stock. Furthermore, the expression of AiMT1 mRNA in scallops with C/C genotype was significantly higher than that with A/A genotype (P<0.05) after an acute heat treatment at 28 degrees C for 120min. These results implied that the polymorphism at locus -337 of AiMT1 was associated with the susceptibility/tolerance of scallops to heat stress, and the -337 A/A genotype could be a potential marker available in future selection of Bay scallop with heat tolerance.

 

Guo, F., J. Yao, et al. (2013). "Bioavailability of purified subcellular metals to a marine fish." Environ Toxicol Chem 32(9): 2109-16.

                In the present study, the authors used a supply of naturally contaminated oysters to investigate how the subcellular metal distribution and the metal burden in prey affected the transfer of metals to a marine fish, the grunt Terapon jarbua. The oysters, Crassostrea hongkongensis, each with different contamination histories, were collected and separated into 3 subcellular fractions: 1) metal-rich granules, 2) cellular debris, and 3) a combined fraction of organelles, heat-denatured proteins, and metallothionein-like proteins, defined as the trophically available metal (TAM). These purified fractions showed a wide range of metal concentrations and were fed to the fish for a period of 7 d at a daily comparable feeding rate of 3% of fish body weight. After 7 d exposure, the newly absorbed metals were mainly distributed in the intestine and liver, indicating a significant tissue-specific trophic transfer, especially for Cd and Cu. The trophic transfer factors (TTFs) showed a sequence of cellular debris >TAM > metal-rich granules, suggesting the impact of subcellular distribution in prey on metal bioavailability. However, significant inverse relationships between the TTFs and the metal concentrations in diets were also found in the present study, especially for Cd and Zn. The subcellular metal compartmentalization might be less important than the metal concentration in prey influencing the trophic transfer. The authors' results have important implications for bioavailability and environmental assessment of dietary metals.

 

Abdel-Halim, K. Y., A. M. Abo El-Saad, et al. (2013). "Oxidative stress on land snail Helix aspersa as a sentinel organism for ecotoxicological effects of urban pollution with heavy metals." Chemosphere 93(6): 1131-8.

                The oxidative stress in the digestive gland of the land snail Helix aspersa was considered as a bioindicator for atmospheric pollution with heavy metals from several industries and vehicular traffic in Kafr El-Zayat city. Regional means of heavy metals concentration of all sites were 0.71, 7.09, 0.71, 2.68, 41.44 and 18.01 mg kg(-1) wet mass for Cd, Mn, Ni, Pb, Zn and Cu, respectively. In addition, the highest values of Cd concentrations were found 1.22 and 1.73 mg kg(-1) wet mass in S1 (Potato International Center) and S4 (The Nile bank), respectively. Lactate dehydrogenase (D-LDH(and recorded lipid peroxidation (LPO) levels were significantly high in S1 and S2 (Traffic station). On the other hand, the highest activity of catalase (CAT) was found in S2 (194.04% of control), while the activity of glutathione peroxidase (GPx) reached the highest significant value in S1. As a matter of fact, glutathione-S-transferase (GST) and glutathione reductase (GR) activities were significantly higher in polluted sites than in reference zone. In contrast, the glutathione (GSH) concentration of exposed animals showed significant decrease in all sites, with the lowest value in S1 (57.61% of control). However, metallothioneins concentration (MT) showed no significant difference in all sites except in S1 which accounted for 127.81% of control. Therefore, the overall results of this study showed the importance of H. aspersa as a sentinel organism for biomonitoring the biologic impact of atmospheric pollution in urban areas.

 

Cooper, S., E. Bonneris, et al. (2013). "Influence of a step-change in metal exposure (Cd, Cu, Zn) on metal accumulation and subcellular partitioning in a freshwater bivalve, Pyganodon grandis: a long-term transplantation experiment between lakes with contrasting ambient metal levels." Aquat Toxicol 132-133: 73-83.

                The objective of the present field experiment was to identify detoxification responses in the gills and digestive gland of a freshwater unionid bivalve, Pyganodon grandis, subjected to a step-change in metal exposure. Adult bivalves were transferred from a reference site (Lake Opasatica) and a metal-contaminated lake (Lake Heva) to a second contaminated lake (Lake Vaudray) in northwestern Quebec, Canada. Changes in organ metal concentrations, in the subcellular distribution of metals and in metallothionein concentrations were followed over time (t=0, 132, (400) and 860 days). At each collection time and for each bivalve, the gills and digestive gland were excised and gently homogenized; six sub-cellular fractions were separated by differential centrifugation and analyzed for their Cd, Cu and Zn content, and metallothionein was quantified independently. Metal detoxification strategies were shown to differ between target organs: in the gills, incoming metals were sequestered largely in the granules, whereas in the digestive gland the same metals primarily accumulated in the cytosol, in the metallothionein-like protein fraction. These metal-handling strategies, as employed by the metal-naive bivalves originating in the reference lake, closely resemble those identified in free-living P. grandis chronically exposed in the metal-contaminated lake, suggesting that the ability to handle incoming metals (Cd in particular) is inherent in P. grandis and is not a trait acquired after long-term adaptation of the bivalve to metal-contaminated environments. The bivalves transplanted from both Lakes Opasatica and Heva were able to tolerate their new surroundings during the first 400 days of the transplant experiment, as indicated by the absence of mortality and the presence of gravid animals. Over the final 460 days, mortality remained low for the bivalves transplanted from the reference lake (20%) but reached 100% in the transplanted group from the contaminated lake. It would seem that the Lake Heva bivalves were compromised by their initial exposure to metals in their home lake and that the added stress of being transplanted to and caged in a lake with comparable or slightly higher concentrations of metals was sufficient to cause mortality.

 

Yu, X. J., K. Pan, et al. (2013). "Spatial variation and subcellular binding of metals in oysters from a large estuary in China." Mar Pollut Bull 70(1-2): 274-80.

                Pearl River Estuary (PRE) is the largest estuary in Southern China and there has been an increasing concern of metal pollution due to regional industrialization. In this study, we investigated the spatial variation of metal pollution (Ag, As, Cd, Cu, Pb, and Zn) as well as their subcellular handling in the oyster Crassostrea hongkongensis. Hot spots of metal contamination in the oysters were found in different sites, suggesting that there were different sources of metals in the estuary associated with industrial activity. Metals differed in their subcellular bindings in the oysters from different locations. Metal distribution in the biologically detoxified fraction decreased for Cu but increased for Zn with increasing contamination in the oysters. For Zn, there was a significant difference in its two detoxification pools (metal-rich granules and metallothionein-like proteins) in response to Zn contamination. The high Cd concentrations in oysters may carry a high Cd hazard to the consumers.

 

Guo, F., R. Tu, et al. (2013). "Different responses of abalone Haliotis discus hannai to waterborne and dietary-borne copper and zincexposure." Ecotoxicol Environ Saf 91: 10-7.

                To investigate the potential influence of the contamination of copper (Cu) or zinc (Zn), the abalone Haliotis discus hannai was exposed to waterborne or macroalgae-borne Cu or Zn over a period of 8weeks. Both Cu and Zn were effectively accumulated by the abalones from water or macroalgae, but their concentration factors and trophic transfer factors were low due to the regulation of Cu and Zn accumulation. Following waterborne or dietary exposure, the abalones exhibited different accumulation patterns of Cu and Zn. The tissue Zn burden decreased quickly after the initial accumulation, and the incoming Zn was mainly deposited in the viscera. In contrast, the tissue Cu burden increased rapidly and gradually reached a steady state. The abalone muscle exhibited a comparable storage capacity of Cu as the viscera and the accumulated Cu in muscle was mainly derived from the dissolved phase instead of trophic transfer. The feeding and growth of the abalone were not influenced in all the exposure regimes. Moreover, the significant induction of metallothionein indicated that the bioaccumulated metals were actively detoxified. In the metal-exposed abalones, more Cu was distributed into the biologically detoxified fractions (metallothionein-like protein and/or metal-rich granule), whereas no significant subcellular redistribution of Zn was observed. Our study suggested that the abalone may have high endurance to the contamination of Cuor Zn.

 

Sheir, S. K., R. D. Handy, et al. (2013). "Effect of pollution history on immunological responses and organ histology in the marine mussel Mytilus edulis exposed to cadmium." Arch Environ Contam Toxicol 64(4): 701-16.

                The effect of previous toxicant exposure (i.e., exposure history) on an organism's response to re-exposure to the toxicant is of considerable interest. The marine mussel Mytilus edulis was collected from reference and polluted sites in southwest England, and groups of mussels from each site were exposed to 20 mug/L CdCl2 for 0, 1, 4, and 8 days and compared with unexposed controls. End points evaluated were tissue metal and electrolyte concentrations, haemolymph chemistry, haemocyte characteristics [counts, neutral red uptake (NRU), and phagocytosis], histology, and expression of metallothionein gene (mt10) expression in digestive glands. Field-collected animals differed by collection site for some end points at time zero, at which time tissue Fe and Pb concentrations were greater and NRU and condition index lower in mussels from the polluted site. Subsequent exposure to cadmium (Cd) in the laboratory caused Cd accumulation mainly in digestive gland, but there were no site-specific effects on tissue trace-metal concentrations. NRU, phagocytosis, and haemolymph Na(+) and K(+) concentrations differed among sites and Cd treatment, but there were no clear trends. Exposure to Cd resulted in lower Ca(2+) concentrations in gill, digestive gland, and haemolymph in animals from the polluted site compared with controls (Kruskal-Wallis, p </= 0.05). Lesions, including necrosis, inflammation, and neoplasia, were observed in animals from the polluted site, but the frequency of these lesions appeared to decrease unexpectedly after Cd exposure. Expression of mt10 increased 3-fold in Cd-exposed animals from the polluted site compared with all other groups (Kruskal-Wallis, p = 0.01). We conclude that Cd exposure affected some immune responses in M. edulis, but pre-exposure history influenced toxicological outcomes of Cd exposure in the laboratory.

 

Falfushynska, H. I., L. L. Gnatyshyna, et al. (2013). "Effect of in situ exposure history on the molecular responses of freshwater bivalve Anodonta anatina (Unionidae) to trace metals." Ecotoxicol Environ Saf 89: 73-83.

                The goal of the study was to assess the adequacy of molecular responses in mollusks in relation to their in situ exposure history. Freshwater male bivalve mollusks Anadonta anatina (Unionidae) from polluted (A) and unpolluted (F) sites were subjected to 14 days of exposure to copper (Cu(2+), 10 mug L(-1)), zinc (Zn(2+), 130 mug L(-1)) or cadmium (Cd(2+), 15 mug L(-1)). The comparison of two control groups showed that the specimens from site A had higher levels of Cu, Zn and Cd and metallothionein (measured both through metal (MT-Me), and protein (MT-SH) levels) in the tissues. Cytotoxicity (low lysosomal membrane stability), low glutathione level, high antioxidant and apoptotic enzymes activities, lipid and protein oxidative injury, depletion of ethoxyresorufin-O-deethylase (EROD) in digestive gland, high vitellogenin-like protein (Vtg-LP) concentration in gonads confirmed the effect of toxic environment on this group. Exposures provoked increased number of hemocytes with micronuclei (by 100-500%) and nuclear abnormalities (by 50-400%) (genotoxicity), elevation of caspase-3 (in 1.5-10 times) and/or Vtg-LP (by 70-310%) levels in all groups. However, the responses were strongly dependent on the origin of mussels. Exposed mussels from site F demonstrated typical for the effect of toxic metals elevation of MT-SH (by 100-380%) and MT-Me (up to seven times) levels and accumulation of metals (with a few exceptions) in the tissues. Conversely, in the mussels inhabiting site A, exposures caused the decrease of metal (by 37% for Cu, by 62% for Zn, by 50% for Cd), MT-SH (by 68% in ZnA group) and MT-Me (by 50-68%) levels. That was accompanied with increase of cytotoxicity and EROD activity (by 144-240%). High level of protein carbonyls was the distinguished feature of all groups from site A. Hence, despite high efficiency of metal detoxification and oxidative stress responses in the mussels, in the specimens from spontaneously polluted site they were impaired.

 

Gagne, F., P. Turcotte, et al. (2013). "The effects of zinc oxide nanoparticles on the metallome in freshwater mussels." Comp Biochem Physiol C Toxicol Pharmacol 158(1): 22-8.

                The use of zinc oxide nanoparticles (nanoZnO) as sunscreens has raised concerns about their safety and release in the aquatic environment through swimming activities and within municipally treated wastewaters. This study's purpose was to examine the effects of nanoZnO on the elemental composition (metallome) in exposed freshwater mussels, Elliptio complanata. Mussels were exposed for 21 days to an environmentally realistic (low) concentration (2 mug/L) of nanoZnO and zinc chloride. The mussels were also exposed to a physically and chemically treated municipal effluent (ME), both alone and in the presence of both forms of Zn. The metallome profile was characterized by the following 15 elements in gills, digestive gland and gonad tissues: Ag, Al, As, Cd, Co, Cr, Cu, Fe, Mn, Mo, Ni, Pb, Se, V and Zn. The levels of metallothioneins (MT) and lipid peroxidation (LPO) in the digestive gland were also measured as biomarkers of toxic effects. The data revealed that exposure to nanoZnO increased the total levels of Zn, MT and LPO in the digestive gland. Discriminate function analysis revealed that the digestive gland responded the most to exposure to either nanoZnO or Zn(2+). For nanoZnO, the observed changes in Al, As and Mo in the digestive gland offered the best discrimination from dissolved Zn(2+). Co-exposure of nanoZnO with the ME changed the metallome profile closer to dissolved Zn(2+), suggesting a common interaction site within the ME. This was observed in changes in Ni, Cu, Se and Zn in the digestive gland of exposed mussels. Canonical analysis of essential and non-essential elements revealed that exposure to nanoZnO increased the relationships between LPO and the sum of essential elements in the digestive gland. Conversely, exposure to dissolved Zn(2+) and the ME decreased the relationship between the sum of non-essential elements and LPO and MT. In conclusion, the use of a "metallomic" approach was used to discriminate changes following exposure to nanoZnO and dissolved Zn in freshwater mussels and provided insights into the interaction of forms of Zn in ME towards mussels.

 

Lemus, M., N. Rojas, et al. (2013). "[Metallothioneins in Perna viridis (Bivalvia: Mytilidae): seasonal variation and its relation to reproductive biology]." Rev Biol Trop 61(2): 701-9.

                Metallothionein is a cytosolic protein found in a variety of tissues and have been involved in the regulation of essential trace metals such as copper and zinc, and in the detoxification of essential and nonessential metals. With the aim to study their seasonal variation and their possible role in reproductive behavior, we evaluated metallothioneins (Mts) in Perna viridis, taken from Rio Caribe and Chacopata localities in the North coast of Sucre state, Venezuela. A total of 325 samples were obtained from February to December 2003. We determined the following biometric indices in bivalves: Condition Index (CI), meat yield (RC) and dry weight-length relationship (PSL). Besides, Mts in whole tissue were separated by molecular exclusion chromatography, Sephadex G-50 and quantified by saturation with cadmium. Our results showed that the biometric indices (RC and PSL) had seasonal variations between localities and maturity stages, with the exception of IC. No significant differences were found between sexes. Mts showed seasonal variations between localities, with the highest concentrations between February and March, and minimum ones between September and December, coinciding with the respectively high and low productivity periods in the area. The mussels from Rio Caribe had higher Mts concentration than those from Chacopata. Furthermore, immature mussels showed the highest Mts concentration while the lowest was found in spawned specimens. We found a significant negative relationship between Mts and CI. Our results demonstrated that MTs in Perna viridis are influenced by the condition index and reproductive status, as well as physico-chemical factors in the marine environment.

 

Taylor, D. A., E. L. Thompson, et al. (2013). "Differential effects of metal contamination on the transcript expression of immune- and stress-response genes in the Sydney Rock oyster, Saccostrea glomerata." Environ Pollut 178: 65-71.

                Environmental contamination by metals is a serious threat to the biological sustainability of coastal ecosystems. Our current understanding of the potential biological effects of metals in these ecosystems is limited. This study tested the transcriptional expression of immune- and stress-response genes in Sydney Rock oysters (Saccostrea glomerata). Oysters were exposed to four metals (cadmium, copper, lead and zinc) commonly associated with anthropogenic pollution in coastal waterways. Seven target genes (superoxide dismutase, ferritin, ficolin, defensin, HSP70, HSP90 and metallothionein) were selected. Quantitative (real-time) PCR analyses of the transcript expression of these genes showed that each of the different metals elicited unique transcriptional profiles. Significant changes in transcription were found for 18 of the 28 combinations tested (4 metals x 7 genes). Of these, 16 reflected down-regulation of gene transcription. HSP90 was the only gene significantly up-regulated by metal contamination (cadmium and zinc only), while defensin expression was significantly down-regulated by exposure to all four metals. This inhibition could have a significant negative effect on the oyster immune system, promoting susceptibility to opportunistic infections and disease.

 

Guo, F., Y. Yang, et al. (2013). "Metal bioavailability from different natural prey to a marine predator Nassarius siquijorensis." Aquat Toxicol 126: 266-73.

                Gastropods are often the top predators in marine benthic environments, and trophic transfer is the predominant route by which metals are accumulated in these predators. In the present study, the potential influences of prey composition on the trophic transfer, accumulation, subcellular distribution and metallothionein induction of six metals (Ag, As, Cd, Cu, Pb and Zn) in a predator Nassarius siquijorensis were investigated. The snails were fed venerid clams Ruditapes philippinarum, mussels Perna viridis, oysters Crassostrea angulata or barnacles Fistulobalanus albicostatus, each differing greatly in their metal accumulation and handling patterns. N. siquijorensis showed prey-specific bioaccumulation and trophic transfer of the six metals. In general, the body burdens of metals in the viscera and muscles of N. siquijorensis increased with increasing exposure period and metal concentration in the four prey. The calculated trophic transfer factors (TTFs) of the metals in different prey varied and were the highest for clams and mussels prey, indicating that metal bioavailability from these prey was higher than that from barnacles and oysters. All the studied metals except Pb were enriched during transfer to the snails. The subcellular metal distribution in the viscera was affected by prey composition. Exposure to the four natural prey induced MTs, which may be used as a better biomarker for muscle than for viscera for metal stress. Our results imply that metals from different natural prey have different bioavailability and may help better understand the trophic transfer of metals in marine benthic food chain.

 

Carreira, S., P. M. Costa, et al. (2013). "Ecotoxicological heterogeneity in transitional coastal habitats assessed through the integration of biomarkers and sediment-contamination profiles: a case study using a commercial clam." Arch Environ Contam Toxicol 64(1): 97-109.

                Transitional waterbodies, such as estuaries, are highly diversified environments with respect to ecology, geophysics, and nature of anthropogenic impacts. This spatial heterogeneity may pose important constraints when developing monitoring programmes for aquatic pollution. The present study compared three distinct coastal ecosystems located in Southern Portugal (subjected to different anthropogenic stressors), namely, two estuaries and a coastal lagoon, through the characterisation of sediment contamination and a biomarker approach to an important commercial clam (Ruditapes decussatus) obtained from local fishing grounds. The results showed high heterogeneity of sediment contamination for both estuaries and a marked distinction between industrially and agriculturally influenced areas as well as between natural and artificialized sites. Hydrodynamics and oceanic influence (in essence dictating sediment type) play a major role in environmental quality. Environmental heterogeneity constituted an important confounding factor for biomarker analysis in the clams' digestive glands since the animals appeared to respond to their immediate surroundings' characteristics rather than the geographical area where they were collected from, despite the relative distance to pollution hot spots. Still, oxidative stress biomarkers (lipid peroxidation and catalase activity) could correlate with each other and to both organic and metallic contamination, whereas metallothionein-like protein induction failed to correlate to any class of sediment toxicants (albeit metals being the most representative pollutants) and appeared to be strongly affected (unlike the previous) by clam size and probably other unknown internal and external variables, among which contaminant interactions should play a major role.

 

Fang, Y., H. Yang, et al. (2013). "Transcriptional response of lysozyme, metallothionein, and superoxide dismutase to combined exposure to heavy metals and bacteria in Mactra veneriformis." Comp Biochem Physiol C Toxicol Pharmacol 157(1): 54-62.

                The response of the defense components lysozyme (LYZ), metallothionein (MT), and superoxide dismutase (SOD) to combined exposure to heavy metals and bacteria was assessed at transcriptional level in the surf clam Mactra veneriformis. First, the full-length LYZ cDNA containing 808 nucleotides and encoding 194 deduced amino acids was identified from the clam. Multiple alignments revealed that MvLYZ had a high identity with invertebrate-type LYZs from other mollusks. Next, clams were exposed to Vibrio parahaemolyticus and a mixture of cadmium and mercury, alone or in combination, for 7 days. Cumulative mortality of clams and mRNA expressions of the three defense components were analyzed. The highest cumulative mortality took place in the combined treatment on day 7. The expression of the three genes was up-regulated in response to treatments compared to the control with different response times and transcriptional levels; the response to combined exposure occurred earlier than to single exposure. Among the experimental groups, MvLYZ expression and MvSOD expression peaked in the combined treatment on day 3, whereas MvMT expression peaked in heavy metals treatment on day 5. Furthermore, interactive effects of heavy metals and Vibrio on transcriptional response changed over the exposure time. Therefore, transcriptional regulation of the three genes under combined exposure was more complex than under single exposure.

 

Buffet, P. E., M. Richard, et al. (2013). "A mesocosm study of fate and effects of CuO nanoparticles on endobenthic species (Scrobicularia plana, Hediste diversicolor)." Environ Sci Technol 47(3): 1620-8.

                The fate and effects of CuO nanoparticles (CuO NPs) were examined in endobenthic species (Scrobicularia plana , Hediste diversicolor), under environmentally realistic conditions in outdoor mesocosms (exposure to Cu at 10 mug L(-1) in particulate (CuO NPs) or soluble salt (CuNO(3)) forms) for 21 days. Labile Cu was determined in water and sediment by using diffusive gradient in thin films. No labile Cu being detected from CuO NPs; the observed effects in invertebrates exposed to CuO NPs were mainly attributed to the toxicity of nanoparticulate rather than dissolved Cu toxicity. Bioaccumulation of CuO NPs was observed in both species. Biomarkers were examined at different levels of biological organization: biochemical markers of defense and damage, biomarkers of genotoxicity (comet assay), and behavioral biomarkers (feeding and burrowing). Behavioral biomarkers, antioxidant defenses (catalase, glutathion S-transferase, metallothionein), and genotoxicity are the most sensitive tools to highlight the effect of soluble or nanoparticulate metal forms. Concerning other biomarkers of defense (superoxide dismutase, lactate dehydrogenase, laccase) and damage (thiobarbituric acid reactive substances, acetylcholinesterase, acid phosphatase), no significant effects were detected. This experiment shows the suitability of mesocosms for studying the environmental effects of nanoparticles.

 

Liu, F. and W. X. Wang (2013). "Facilitated bioaccumulation of cadmium and copper in the oyster Crassostrea hongkongensis solely exposed to zinc." Environ Sci Technol 47(3): 1670-7.

                Exposure to one metal might have significant effects on the bioaccumulation of other metals. In the present study, we examined the possible effects of Zn exposure on the bioaccumulation of Cd and Cu in three populations of the oyster Crassostrea hongkongensis. We found that Zn exposure significantly enhanced the tissue concentrations of Cd and Cu in all populations, and the tissue concentrations of Cd and Cu were highly and positively related to the tissue Zn concentration. Furthermore, the enhanced bioaccumulation of Cd and Cu resulted mainly from their increasing accumulation and distribution in two subcellular fractions (i.e., metallothionein-like proteins and metal-rich granules). Tissue concentrations of Cd and Cu in the natural Zn-contaminated oysters also covaried with tissue Zn concentration, and prediction analyses revealed that Zn exposure was a significant contributor to tissue Cd and Cu concentrations. Therefore, we concluded that the increased Zn bioavailability in ambient waters not only increased the tissue Zn concentration but also enhanced the overall bioaccumulation of Cd and Cu. This study strongly demonstrates that contamination of metals in oysters may result from concurrent exposure to other metals. Thus, environmental managers should consider the possible exposure to other metals such as Zn in order to interpret/predict the tissue concentrations of toxic metals in oysters.

 

Cravo, A., B. Lopes, et al. (2013). "Spatial and seasonal biomarker responses in the clam Ruditapes decussatus." Biomarkers 18(1): 30-43.

                The clam Ruditapes decussatus is an important resource to preserve in coastal lagoon systems around the Mediterranean including the South Portugal. To assess spatial and temporal biomarker responses to contamination in the species, a multibiomarker approach was conducted using antioxidant enzymes, MFO system phase I and II; acetylcholinesterase, metallothionein (MT), delta-aminolevulinic acid dehydratase and lipid peroxidation (LPO). The condition index (CI), metals and polycyclic aromatic hydrocarbons (PAHs) were also determined. The levels of contaminants were not particularly high and the antioxidant enzymes, acetylcholinesterase (AChE), MT in the digestive gland, and delta-aminolevulinic acid dehydratase (ALAD) do not provide a suitable seasonal and spatial discrimination reversely to that regarding CYP450, glutathione-S-transferase (GST), MT in the gills, and LPO in both tissues. However, even those could vary with natural variables that may act as confounding factors. Thus, seasonal variability and natural range of biomarker responses must be carefully and accurately taken into account in ecotoxicological approaches of environmental quality assessment programmes.

 

Rodrigo, A. P., P. M. Costa, et al. (2013). "Integration of sediment contamination with multi-biomarker responses in a novel potential bioindicator (Sepia officinalis) for risk assessment in impacted estuaries." Ecotoxicology 22(10): 1538-54.

                For the purpose of biomonitoring, species that combine ecological and commercial importance may provide a link between ecological and human health risk. The common cuttlefish, Sepia officinalis, holds both characteristics in south-western Europe, albeit remaining unsurveyed in ecotoxicological studies. Cuttlefish collected from an impacted estuary in SW Portugal and a reference location off the coast were analysed for a battery of biomarker responses in the digestive gland and gills. The contrast to reference animals revealed that biomarker responses, especially those related to oxidative stress, were consistent with sediment contamination by PAHs, even in a situation that combines complex toxicant mixtures, moderate levels of contamination and high ecotoxicological diversity. However, environmental parameters related to the differences between shore and estuarine habitats should not be overruled. Also, digestive gland metallothionein retained significant specificity to metals even though previous studies in the area with clams and fish failed to trigger a conclusive response. The highest net differences in biomarker responses were detected in the gills, likely indicating higher sensitivity to environmental stressors. Still, the digestive gland responses were overall the most consistent with sediment contamination and effectively differentiated between estuarine industrial- and rural-impacted sites. The results indicate that S. officinalis may be a candidate to meet the European Union's requirements for efficient biomonitoring programmes, with the additional importance of being cosmopolitan, abundant, commercially valuable and combining the molluscan biology that has been granting bivalves their high value for biomonitoring with foraging behaviour, thus better able to reflect anthropogenic stressors impacting a wider area than sedentary organisms. Nevertheless, further investigations in unpolluted sites are needed to better evaluate the background levels of biomarker responses in the species.

 

Moschovaki-Filippidou, F., A. Itziou, et al. (2013). "Effect of starvation and hibernation on the values of five biomarkers of general and specific stress using the land snail Eobania vermiculata." Comp Biochem Physiol B Biochem Mol Biol 165(4): 236-42.

                Values of five biomarkers related to cell stress or pollution were evaluated in tissues of the land snail Eobania vermiculata under starvation or hibernation conditions. The biomarkers applied were lysosomal membrane stability in digestive gland cells (LMS) or in haemocytes (neutral red retention assay (NRR)), acetylcholinesterase activity (AchE; EC 3.1.1.7), metallothionein content (MTs), and cyclic AMP content (cAMP). Three groups of snails were studied that were kept under starvation, hibernation and normal conditions, respectively. The results indicated statistically lower values of LMS and NRR in snails kept under starvation or hibernation compared to control ones. Higher values of AChE activity were measured in snails under hibernation compared to controls. MT contents were statistically higher in snails under starvation compared to controls. Measurement of cAMP contents showed no significant differences among the tested groups. The values of the first four biomarkers may be affected by factors other than pollution, such as starvation or hibernation. Therefore, these factors should be taken into consideration when biomonitoring studies are performed in time intervals of hibernation or starvation.

 

Falfushynska, H. I., L. L. Gnatyshyna, et al. (2013). "In situ exposure history modulates the molecular responses to carbamate fungicide Tattoo in bivalve mollusk." Ecotoxicology 22(3): 433-45.

                The aim of the present study was the investigation of the effect of in situ exposure history on the responses of freshwater mussels to thiocarbamate fungicide. Male bivalve mollusks Anodonta anatina (Unionidae) from polluted (A) and unpolluted (F) sites were subjected to 14 days of exposure to fungicide Tattoo (mixture of propamocarb and mancozeb, 91 mug L(-1)). When unexposed mussels were compared, chronic effect of toxic environment in site A was confirmed by oxidative stress indices (high levels of superoxide dismutase and catalase activities, lipid peroxidation, protein carbonyls and oxyradical production, low level of total glutathione (GSH)), genotoxicity (high levels of DNA-strand breaks and caspase-3 activity in digestive gland), and cytotoxicity (low lysosomal membrane stability in hemocytes), elevated vitellogenin-like proteins (Vtg-LP) concentration in gonads, high levels of Cu, Zn, Cd, metallothionein (MT)-bound metals (MT-Me) and MT-related thiol (MT-SH), and low ethoxyresorufin-O-deethylase (EROD) activity in digestive gland. The major differences in the responses of the two exposed groups were related to antioxidant defense and MT: in the group A, prominent oxidative stress response with the participation of MT-SH and GSH in the gills, EROD activation, but decrease of MT-Me level was shown, whereas in group F exposure provoked the elevation of MT-Me, caspase-3 and Vtg-LP values. Carbamate did not cause cholinesterase depletion and cytotoxicity. However, genotoxic and pro-oxidant effects (increased levels of hemocytes with micronuclei and nuclear abnormalities, DNA-strand breaks and oxyradical in digestive gland), were common responses for both the exposed groups.

 

Gagne, F., J. Auclair, et al. (2013). "Bioavailability and immunotoxicity of silver nanoparticles to the freshwater mussel Elliptio complanata." J Toxicol Environ Health A 76(13): 767-77.

                The purpose of this study was to examine the effects of Ag nanoparticles (nAg) of two different sizes (20 and 80 nm) and Ag(+) on the immune system of the freshwater mussel Elliptio complanata. Mussels were exposed to increasing concentrations of nAg and dissolved Ag (AgNO3) for 48 h at 15 degrees C and concentration of 0, 0.8, 4, or 20 mug/L. Immunocompetence was determined by hemocyte viability, phagocytosis, and cell cytotoxicity. Ag tissue loadings and levels of metallothioneins (MT), lipid peroxidation (LPO), and labile zinc (Zn) were also determined. Results revealed first that 20- and 80-nm nAg readily formed aggregates in freshwater. Ag was detected in soft tissues with each form of Ag with bioconcentration factors of 20, 9, and 7 for Ag(+), 20-nm nAg, and 80-nm nAg, respectively. Significant induction in phagocytosis and decreased cell cytotoxicity were observed. All forms of Ag were able to induce LPO in gills and digestive glands at concentrations below those from the initial fraction of dissolved Ag. The effects of nAg on MT levels in mussels were not discernible from those of dissolved Ag, but the 80-nm was 25-fold more potent than 20-nm nAg in inducing MT. Multivariate analysis revealed that the global responses of the 20- and 80-nm nAg were generally similar to those of dissolved Ag. Data also demonstrated that nAg are bioavailable for mussels where the immune system is a target during early exposure to nanoparticles.

 

Galitsopoulou, A., D. Georgantelis, et al. (2013). "Effect of thermal processing and canning on cadmium and lead levels in California market squid: the role of metallothioneins." Food Addit Contam Part A Chem Anal Control Expo Risk Assess 30(11): 1900-8.

                The effects of two common seafood preparation practices (roasting and industrial canning) on the heavy metal content--cadmium (Cd) and lead (Pb)--of various tissues of California market squid were studied. Emphasis was placed on the role of metallothioneins (MT) in Cd and Pb behaviour during processing. Cd and Pb analysis was conducted by a Zeeman GTA-AAS atomic absorption spectrometry system; MT analysis was performed by a mercury saturation assay. Results showed that Cd levels in the mantle and whole squid were considerably affected by both processing practices, reaching a 240% increase in mantle and a 40% increase in whole squid. Interestingly, Cd behaviour was associated with MT changes during squid processing. On the other hand, Pb content was not affected from either processing or associated with MT content in the raw or processed squid. Therefore, processing operations may affect Cd and Pb content differently due to the specific metal bioaccumulation and chemical features of each heavy metal type.

 

Ciacci, C., C. Barmo, et al. (2012). "Effects of sublethal, environmentally relevant concentrations of hexavalent chromium in the gills of Mytilus galloprovincialis." Aquat Toxicol 120-121: 109-18.

                Hexavalent chromium Cr(VI) is an important contaminant released from both domestic and industrial effluents, and represents the predominant chemical form of the metal in aquatic ecosystems. In the marine bivalve Mytilus galloprovincialis exposure to non-toxic, environmentally relevant concentrations of Cr(VI) was shown to modulate functional parameters and gene expression in both the digestive gland and hemocytes. In this work, the effects of exposure to Cr(VI) (0.1-1-10 mug L(-1) animal(-1) for 96 h) in mussel gills were investigated. Gill morphology and immunolocalization of GSH-transferase (GST), of components involved in cholinergic (AChE and ChAT), adrenergic (TH) and serotoninergic (5-HT(3) receptor) systems, regulating gill motility, were evaluated. Total glutathione content, activities of GSH-related enzymes (glutathione reductase - GSR, GST), of catalase, and of key glycolytic enzymes (phosphofructokinase - PFK and pyruvate kinase - PK) were determined. Moreover, mRNA expression of selected Mytilus genes (GST-pi, metallothionein isoforms MT10 and MT20, HSP70 and 5-HT receptor) was assessed by RT-q-PCR. Cr(VI) exposure induced progressive changes in gill morphology and in immunoreactivity to components involved in neurotransmission that were particularly evident at the highest concentration tested, and associated with large metal accumulation. Cr(VI) increased the activities of GST and GSR, and total glutathione content to a different extent at different metal concentrations, this suggesting Cr(VI) detoxication/reduction at the site of metal entry. Cr(VI) exposure also increased the activity of glycolytic enzymes, indicating modulation of carbohydrate metabolism. Significant changes in transcription of different genes were observed. In particular, the mRNA level for the 5-HTR was increased, whereas both decreases and increases were observed for GST-pi, MT10, MT20 and HSP70 mRNAs, showing sex- and concentration-related differences. The results demonstrate that Cr(VI) significantly affected functional and molecular parameters in mussel gills, and indicate that this tissue represents the major target of exposure to environmentally relevant concentrations of the metal.

 

Mottin, E., C. Caplat, et al. (2012). "Effect of zinc sacrificial anode degradation on the defence system of the Pacific oyster, Crassostrea gigas: chronic and acute exposures." Mar Pollut Bull 64(9): 1911-20.

                Two types of exposures were performed to assess the effects of zinc released from sacrificial anode degradation: a chronic exposure, in which oysters were exposed to 0.53+/-0.04 mg Zn L(-1) for 10 weeks, and an acute exposure, where oysters were exposed to 10.2+/-1.2 mg Zn L(-1) for 1 week. At the end of the acute exposure experiment, 81.8% mortality was recorded. In contrast, no mortality was detected after 10 weeks exposure. Moreover, all of the immune system biomarkers studied, except the number of circulating haemocytes, were stimulated by a moderate level of zinc and inhibited by a high level. Our exposure conditions did not induce SOD or MXR mRNA expression in gills and digestive gland. However, an increase of MT mRNA is observed in these tissues. The results indicate that oysters are sensitive to acute zinc toxicity but are only moderately affected by a mild zinc concentration.

 

Pan, J. F., P. E. Buffet, et al. (2012). "Size dependent bioaccumulation and ecotoxicity of gold nanoparticles in an endobenthic invertebrate: the Tellinid clam Scrobicularia plana." Environ Pollut 168: 37-43.

                Gold nanoparticles (AuNPs) have important technological applications resulting in an increased potential for release to the environment, and a greater possibility of toxicological effects. The marine bivalve Scrobicularia plana was exposed to AuNPs of size 5, 15 and 40 nm during a 16 d laboratory exposure at 100 mug Au L(-1). After exposure to AuNPs forming aggregates (>700 nm), the clams accumulated Au in their soft tissues. Biochemical (biomarkers) and behavioral (burrowing and feeding) responses were investigated. Au NPs were responsible of metallothionein induction (5, 40 nm), increased activities of catalase (15, 40 nm) and superoxide dismutase (40 nm) and of glutathione S-transferase by the three sizes of AuNPs indicating defense against oxidative stress. Exposure to AuNPs impaired burrowing behavior. However, it must be underlined that these effects were observed at a dose much higher than expected in the environment.

 

Schmidt, W., T. O'Shea, et al. (2012). "The effect of shore location on biomarker expression in wild Mytilus spp. and its comparison with long line cultivated mussels." Mar Environ Res 80: 70-6.

                Biomarkers are a common tool in the assessment of potential effects of contaminants in aquatic organisms. In order to identify the effects of anthropogenic pollution it is essential to identify background levels and to know the range of natural variability in the biomarker response. In this study, we examined various biomarkers of stress (glutathione S-transferase and metallothionein), damage (lipid peroxidation (LPO) and DNA damage (DNA)) and reproduction (vitellin-like proteins) in marine mussels (Mytilus spp.) from four locations along a vertical transect from high to low shore and compared them with cultivated long line mussels. High shore and cultivated mussels showed significantly higher LPO and DNA damage expression than the low shore mussels indicating a level of oxidative stress resulting from mussel location. Significant effects in physiological endpoints were also found. This study highlights the need to consider the diversity of natural environmental stress factors when using biomarkers in environmental assessment.

 

Buffet, P. E., C. Amiard-Triquet, et al. (2012). "Fate of isotopically labeled zinc oxide nanoparticles in sediment and effects on two endobenthic species, the clam Scrobicularia plana and the ragworm Hediste diversicolor." Ecotoxicol Environ Saf 84: 191-8.

                Although it is reported that metal and metal oxide nanoparticles, which are among the most rapidly commercialized materials, can cause toxicity to organisms, their fate in the environment and toxicity to marine organisms are not well understood. In this study, we used a stable isotope labelling approach to trace the fate of nanoparticles (NPs) in sediments and also investigated bio-uptake in two estuarine intra-sedimentary invertebrates Scrobicularia plana and Nereis diversicolor. We selected exposure to 3 mg kg(-1) sediment ZnO NPs since this level is a realistic prediction of the environmental concentration in sediments. 67ZnO NPs (DLS: 21-34 nm, positively charged: 31.3 mV) suspensions were synthesised in diethylene glycol (DEG). We explored the fate of 67ZnO NPs in sediment, 67Zn bioaccumulation and the biochemical (biomarkers of defence and damage) and behavioural (burrowing kinetics and feeding rates) biomarkers in both species to 67ZnO NPs and DEG on its own during a 16 d laboratory exposure. After exposure, 67Zn concentrations in sediment showed higher levels in the upper section (1cm: 2.59 mg kg(-1)) decreasing progressively (2 cm: 1.63 mg kg(-1), 3 cm: 0.90 mg kg(-1), 4 cm: 0.67 mg kg(-1)) to a minimum value at the bottom (5 cm: 0.31 mg kg(-1)). 67Zn bioaccumulation was observed in both organisms exposed to 67ZnO NPs in DEG but no major inter-species differences were found. At the biochemical level, 67ZnO NPs exposure significantly induced increased glutathione-S-transferase activity in worms and catalase activity in clams whereas superoxide dismutase activity and thiobarbituric acid reactive substance levels were not affected in any species. Exposure to DEG on its own leads to a significant increase of metallothionein-like protein levels in clams compared with those exposed to 67ZnO NPs or controls. Burrowing behaviour as well as feeding rate were significantly impaired in both species exposed to 67ZnO NPs. Concerning exposure to DEG on its own, burrowing behaviour impairments were also shown in both species and feeding rate was impaired in bivalves. At environmentally realistic concentration of 67ZnO NPs in sediment, there is no strong evidence for a severe nanoparticle effect since most effects were also observed in the presence of DEG alone.

 

Cong, M., H. Wu, et al. (2012). "Effects of heavy metals on the expression of a zinc-inducible metallothionein-III gene and antioxidant enzyme activities in Crassostrea gigas." Ecotoxicology 21(7): 1928-36.

                Sequestration by metallothioneins and antioxidant defense are two kinds of important defense mechanisms employed by mollusks to minimize adverse effects caused by heavy metal contaminants in marine environment. In the present study, a novel metallothionein gene, CgMT-III, was cloned from Crassostrea gigas, consisting of eighteen conserved cysteine residues and encoding a MT III-like protein with two tandem beta domains. The expression level of CgMT-III transcript induced by zinc was much higher than that induced by cadmium exposure. It suggested that CgMT-III was perhaps mainly involved in homeostatic control of zinc metabolism, which was distinct from previously identified MTs in C. gigas. Among the tested antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), SOD and GPx showed varying up-regulations in a tissue-specific manner, while CAT activities were inhibited in both gill and hepatopancreas from C. gigas exposed to heavy metals. It can be inferred that CgMT-III was mainly involved in zinc homeostasis, and CgMT-III gene together with CAT enzyme could be potential biomarkers to indicate heavy metal, especially zinc pollution in marine organisms.

 

Figueira, E., D. Branco, et al. (2012). "Are metallothioneins equally good biomarkers of metal and oxidative stress?" Ecotoxicol Environ Saf 84: 185-90.

                Several researchers investigated the induction of metallothioneins (MTs) in the presence of metals, namely Cadmium (Cd). Fewer studies observed the induction of MTs due to oxidizing agents, and literature comparing the sensitivity of MTs to different stressors is even more scarce or even nonexistent. The role of MTs in metal and oxidative stress and thus their use as a stress biomarker, remains to be clearly elucidated. To better understand the role of MTs as a biomarker in Cerastoderma edule, a bivalve widely used as bioindicator, a laboratory assay was conducted aiming to assess the sensitivity of MTs to metal and oxidative stressors. For this purpose, Cd was used to induce metal stress, whereas hydrogen peroxide (H2O2), being an oxidizing compound, was used to impose oxidative stress. Results showed that induction of MTs occurred at very different levels in metal and oxidative stress. In the presence of the oxidizing agent (H2O2), MTs only increased significantly when the degree of oxidative stress was very high, and mortality rates were higher than 50 percent. On the contrary, C. edule survived to all Cd concentrations used and significant MTs increases, compared to the control, were observed in all Cd exposures. The present work also revealed that the number of ions and the metal bound to MTs varied with the exposure conditions. In the absence of disturbance, MTs bound most (60-70 percent) of the essential metals (Zn and Cu) in solution. In stressful situations, such as the exposure to Cd and H2O2, MTs did not bind to Cu and bound less to Zn. When organisms were exposed to Cd, the total number of ions bound per MT molecule did not change, compared to control. However the sort of ions bound per MT molecule differed; part of the Zn and all Cu ions where displaced by Cd ions. For organisms exposed to H2O2, each MT molecule bound less than half of the ions compared to control and Cd conditions, which indicates a partial oxidation of thiol groups in the cysteine residues through ROS scavenging. The present results suggest that MTs are excellent markers of metal stress, but not of oxidative stress.

 

Franzellitti, S., A. Viarengo, et al. (2012). "Molecular and cellular effects induced by hexavalent chromium in Mediterranean mussels." Aquat Toxicol 124-125: 125-32.

                The present study evaluated the effects of Cr(VI) in digestive gland of the Mediterranean mussel (Mytilus galloprovincialis) exposed for 1 week to the metal at 1, 10, and 50 ng/L. Tissue accumulation of Cr and lysosomal biomarkers were measured. Moreover, a low-density DNA microarray was used to identify early molecular markers of metal exposure. A concentration-dependent increase in tissue Cr concentrations was observed in both digestive gland and remaining soft tissues. A reduction of lysosomal membrane stability was detected in digestive gland at 10 and 50 ng/L of Cr(VI), indicating a loss of cell functional integrity. The expression of mRNAs encoding 13 genes involved in metal resistance (mt10, mt20), molecular chaperoning (hsp70), immune response (mytlB, mytcA and lys), transcriptional (histones h1, h2-a and h4), and antioxidant/detoxification (cat, mrp2, mvp) processes were significantly altered already at the lowest Cr(VI) concentration, where the effects at the histological level were nonsignificant. Altogether, data point out that depending on the exposure concentration Cr(VI) may cause or not oxidative stress altering the efficiency of the antioxidant system in counteracting the effects of Cr as a redox-active metal. Moreover, changes of mRNA expression profiles induced by Cr(VI) concentrations as low as 1-50 ng/L were related to altered immunomodulation, DNA stability, and stress response pathways previously proven to be affected by the metal. The molecular targets presently identified may drive the development of new biomarkers for Cr exposure or help their interpretation.

 

Tsarpali, V. and S. Dailianis (2012). "Investigation of landfill leachate toxic potency: an integrated approach with the use of stress indices in tissues of mussels." Aquat Toxicol 124-125: 58-65.

                The present study investigates the harmful impacts of landfill leachate release and/or disposal into the marine environment, as well as its ability to induce lethal and pre-pathological alterations in marine organisms, such as the mussel Mytilus galloprovincialis. In specific, mortality test (96 h), performed first in order to estimate leachate lethal endpoints, showed increased levels of mussel mortality after exposure to leachate higher than 0.5%, v/v (96 h LC(50)=0.526%, v/v), while the exposure to 0.01 and 0.1% (v/v) of leachate showed negligible levels of mortality (96 h LC(10)=0.167%, v/v). Furthermore, the estimation of lysosomal membrane integrity in hemocytes of exposed mussels (Neutral Red Retention Time assay) showed increased levels of lysosomal destabilization in cells of mussels exposed to sub-lethal concentrations of leachate (0.01, 0.1 and 0.5%, v/v) for 4 days. In order to exclude parameters, such as mussel mortality and cell death, which could interfere with the obtained results, leachate at final concentrations of 0.01 and 0.1% (v/v) were finally used for the estimation of a battery of stress indices in target tissues of mussels, such as hemolymph, gills and digestive gland. According to the results, leachate-exposed mussels showed a significant inhibition of acetylcholinesterase activity, increased levels of nuclear abnormalities, as well as increased levels of metallothionein, superoxide anion and lipid peroxides (in terms of malondialdehyde equivalents) in each tissue tested. The results of the present study clearly indicate leachate-induced lethal effects as well as the ability of leachate to induce disturbances on different levels of organism function before mortality occurs.

 

Lavilla, I., M. Costas, et al. (2012). "Simplified and miniaturized procedure based on ultrasound-assisted cytosol preparation for the determination of Cd and Cu bound to metallothioneins in mussel tissue by ICP-MS." Talanta 93: 111-6.

                A simplified and miniaturized procedure for the determination of Cd and Cu bound to metallothioneins (MTs) by ICP-MS in mussel tissue has been developed. Cytosol preparation was based on the indirect sonication of slurries containing the lyophilized sample dispersed in 1 mL of extractant by means of a sonoreactor Cup-Horn. Rabbit liver MTs (Apo-MT-I, Apo-MT-II and Cd(7)-MT-II) and a conventional cytosol preparation procedure were used for validation purposes. The usual heating step and additional centrifugations of the conventional procedure for cytosol preparation can be omitted when using ultrasound treatment. The possible effect of denaturation on MTs and its effect on the metal bound to MTs were evaluated. Variables influencing the ultrasound-assisted cytosol preparation procedure were carefully optimized for simultaneous determination of both metals. Chromatographic conditions to separate the MT fraction from other proteins present in cytosols were also studied. Six samples can be processed within 3 min of sonication. An acid ultrasound-assisted extraction procedure with diluted acid was also proposed for determining total Cd and Cu. Finally, Cd and Cu bound to MTs as well as total Cd and Cu were determined in mussels from Pontevedra and Ares-Betanzos coastal inlets (Galicia, Spain).

 

David, E., A. Tanguy, et al. (2012). "Responses of Pacific oyster Crassostrea gigas populations to abiotic stress in environmentally contrasted estuaries along the Atlantic coast of France." Aquat Toxicol 109: 70-9.

                Genetic and ecophysiological responses of oyster, Crassostrea gigas, populations to environmental stress were investigated in three highly contaminated French estuaries (the Gironde, Loire and Vilaine) and compared to a control, the Belon estuary. A strong response in both metallothionein CgMT4 mRNA expression, as determined by semi-quantitative RT-PCR, and amount of protein, as determined by ELISA tests, was demonstrated in estuarine populations subjected to differential contamination, with an inhibition in the area most highly contaminated with metals. In these same estuarine populations, we found polymorphisms of the metallothionein CgMT4 gene and three other genes (glutamine synthetase--GS, delta-9 desaturase--D9 and phosphoglucomutase--PGM) involved in stress response of C. gigas. We showed that genetic differentiation was observed for MT4 and PGM genes in the Gironde estuary which is highly contaminated with metals. A strong seasonal effect was observed. Phenotype-genotype coupling revealed that one particular MT4 allele and one PGM allele seemed to be associated with metal sensitivity expressed as lower detoxification efficiency and higher metal bioaccumulation. The MT4 gene is a good physiological and genetic marker of stress response and susceptibility.

 

Perez-Rafael, S., A. Mezger, et al. (2012). "The metal binding abilities of Megathura crenulata metallothionein (McMT) in the frame of gastropoda MTs." J Inorg Biochem 108: 84-90.

                Metallothioneins (MTs) are proteins that play a major role in metal homeostasis and/or detoxification in all kind of organisms. The MT gene/protein system of gastropod molluscs provides an invaluable model to study the diversification mechanisms that have enabled MTs to achieve metal-binding specificity through evolution. Most pulmonate gastropods, particularly terrestrial snails, harbor three paralogous isogenes encoding three MT isoforms with different metal binding preferences: the highly specific CdMT and CuMT isoforms, for cadmium and copper respectively, and the unspecific Cd/CuMT isoform. Megathura crenulata is a non-pulmonate gastropod in which only one MT isogene has so far been reported. In order to elucidate the metal binding character of the corresponding peptide (McMT), it has been recombinantly synthesized in the presence of Cd(2+), Zn(2+) or Cu(2+), and the corresponding metal complexes have been analyzed using electrospray mass spectrometry, and CD and UV-visible spectroscopy. The metal-binding traits exhibited by McMT revealed that it is an unspecific MT, similarly to the pulmonate Cd/CuMT isoforms. This is in full concordance with the protein sequence distance analysis in relation to other gastropod MTs.

 

Taylor, A. M. and W. A. Maher (2012). "Exposure-dose-response of Anadara trapezia to metal contaminated estuarine sediments. 1. Cadmium spiked sediments." Aquat Toxicol 109: 234-42.

                The relationship between cadmium exposure dose and response was investigated in Anadara trapezia exposed to cadmium spiked sediment (10 mug/g and 50 mug/g dry mass) for 56 days. A. trapezia reached an equilibrium cadmium tissue concentration (13 mug/g and 25 mug/g, respectively) by day 42. Gills accumulated significantly more cadmium than the hepatopancreas and haemolymph. After 56 days exposure between 46 and 73% of accumulated gill and hepatopancreas cadmium was detoxified and in the metallothionein like protein fraction. Approximately half of the biologically active cadmium in both tissues was in the mitochondrial fraction which has the potential to cause dysfunction in mitochondrial activity. Cadmium exposed A. trapezia generally had reduced GPx activity with an associated increase in total glutathione concentrations and reduced GSH:GSSG ratios due to a build up of oxidised glutathione. The changes in the glutathione pathway were reflected in the total antioxidant capacity of cadmium exposed A. trapezia which were significantly reduced compared to control organisms. There was a trend of increased lipid peroxidation with increased cadmium exposure but this was not significant. Increased cadmium exposure resulted in significant lysosomal destabilisation and increased frequency of micronuclei. The significant exposure-dose-response relationship for A. trapezia exposed to cadmium enriched sediments indicates that elevated sediment cadmium concentrations have the potential to lead to increased biologically active cadmium burdens and impairment of individual A. trapezia at cellular and subcellular levels.

 

Fernandez, B., J. A. Campillo, et al. (2012). "Assessment of the mechanisms of detoxification of chemical compounds and antioxidant enzymes in the digestive gland of mussels, Mytilus galloprovincialis, from Mediterranean coastal sites." Chemosphere 87(11): 1235-45.

                In this study the effects of the main marine pollutants (metals, PAHs, PCBs and DDTs) were assessed in native mussels from the Mediterranean coast of Spain. For this purpose several biomarkers such as benzo[a]pyrene hydroxylase (BPH), DT-diaphorase (DTD), glutathione S-transferase (GST), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidases (GPs), glutathione reductase (GR), metallothionein (MT) and lipid peroxidation (LPO) were measured in the digestive gland. Results showed increased LPO levels in mussels which accumulated high loads of organic compounds and arsenic in their tissues. BPH levels correlated to the concentrations of organic compounds in mussel tissues, though the range of BPH response was low in relation to the high gradient of accumulation of organic pollutants. Increased BPH levels, concomitant to low DTD and GST activities, were detected in mussels which presented high levels of organic pollutants in their tissues. This suggests that signs of LPO present in these organisms are related to the imbalance between phase I and phase II biotransformation processes. Furthermore, the increased levels of MT and CAT detected in mussels which showed high levels of Cd in their tissues appear to reflect a coordinated response which protects against the toxicity of this metal. The application of these biomarkers in environmental assessment is discussed.

 

Perrigault, M. and B. Allam (2012). "Differential immune response in the hard clam (mercenaria mercenaria) against bacteria and the protistan pathogen QPX (quahog parasite unknown)." Fish Shellfish Immunol 32(6): 1124-34.

                The immune response of the hard clam (quahog) Mercenaria mercenaria following challenge with live bacteria (Vibrio alginolyticus) and the protist QPX (Quahog Parasite Unknown) was investigated. The study also compared immune responses following QPX challenge in two different hard clam broodstocks exhibiting different degrees of susceptibility toward this parasite. Different immune and stress-related cellular and humoral factors were assessed including general hemocyte parameters (total and differential hemocyte counts, percentage of dead cells, reactive oxygen production, phagocytosis), parameters geared toward QPX (anti-QPX activity in plasma and hemocyte resistance to the cytotoxicity of QPX extracellular products). Two genes (ferritin and metallothionein) previously shown to be modulated following QPX exposure were molecularly characterized by rapid amplification of cDNA ends (RACE) and their transcription levels were determined in resistant and susceptible clams in response to QPX and bacterial challenge. Results indicated that both V. alginolyticus and QPX challenge triggered significant immune responses in clams with similar trends for most measured parameters. However, specific responses were observed for anti-QPX activity in plasma and hemocyte resistance to QPX products as well as ferritin and metallothionein expression according to each inoculum. Similarly, different response patterns were detected following QPX challenge in susceptible and resistant clam stocks. Resistant clams were able to elicit effective response against the parasite leading to the elimination of QPX and the restoration of constitutive immune status whereas QPX-susceptible clams triggered a strong immune modulation characterized by an acute phase response and associated acute phase protein but appeared to be less active in eliminating the parasite. These results suggest that different signaling pathways are triggered during V. alginolyticus and QPX challenge. Moreover, differences in the immune response toward QPX might be linked to the susceptibility or resistance of different clam stocks to the infection by this parasite.

 

Taylor, A. M. and W. A. Maher (2012). "Exposure-dose-response of Anadara trapezia to metal contaminated estuarine sediments. 2. Lead spiked sediments." Aquat Toxicol 116-117: 79-89.

                The composition of near shore marine environments is increasingly being altered by contaminants from human activities. The ability of lead, which has no known biological function, to mimic biologically essential metals makes it one of the most toxic to marine biota. The relationship between lead exposure, dose and response was investigated in Anadara trapezia exposed for 56 days to lead spiked sediment (100 and 300 mug/g dry mass). Lead tissue concentrations of the 300 mug/g exposed A. trapezia doubled in the last 2 weeks of exposure with final lead tissue concentrations of exposed organisms of 1 and 12 mug/g, respectively. Tissue lead accumulation of exposed organisms followed the pattern haemolymph>gill>hepatopancreas during much of the 56 day exposure. Between 30 and 69% of accumulated lead in the gill and hepatopancreas was detoxified and fairly evenly distributed between the metal rich granule and the metallothionein like protein fractions. Approximately half of the biologically active lead in both tissues was in the mitochondrial fraction which showed increased cytochrome c oxidase activity in lead exposed organisms. There was a reduction in GPx activity, an associated increase in total glutathione concentrations and reduced GSH:GSSG ratios due to a build up of oxidised glutathione. These changes in the glutathione pathway were reflected in the total antioxidant capacity of lead exposed A. trapezia which were significantly reduced compared to control organisms. Increased lead exposure significantly increased lipid peroxidation, lysosomal destabilisation and frequency of micronuclei. A significant exposure-dose-response relationship for A. trapezia exposed to lead enriched sediments indicates that elevated sediment lead concentrations have the potential to increase biologically active lead burdens and impair the antioxidant reduction capacity leading to a series of associated effects from lipid peroxidation to cellular perturbation and genotoxic damage.

 

Itziou, A. and V. K. Dimitriadis (2012). "Effects of organic pollutants on Eobania vermiculata measured with five biomarkers." Ecotoxicology 21(5): 1484-94.

                In the present study, the effect of organic pollution on land snails Eobania vermiculata was investigated. Five pollution biomarkers (neutral red retention assay, morphometry of lysosomes and neutral lipids, acetylcholinesterase activity and metallothioneins content), were applied on tissues of the land snails. The results showed intense differentiations between the snails treated with organic pollutants and the control ones, as indicated by the results obtained. Statistically significant correlations among the results obtained emphasize the usefulness of these biomarkers.

 

Xue, M. and C. H. Ke (2012). "[Cadmium bioaccumulation and its toxicity in Babylonia areolata under different nutritional status]." Ying Yong Sheng Tai Xue Bao 23(7): 1965-72.

                An indoor exposure experiment with juvenile Babylonia areolata was conducted to study its survival, growth, cadmium (Cd) accumulation, metallothionein (MT) induction, and glycogen content as well as the DNA integrity of hepatopancreas tissue. The juveniles were starved or fed with mussel (Perna viridis) or clamworm (Perinereis aibuhitensis), and exposed to 50 microg x L(-1) of Cd2+ for 10 weeks. Prolonged starvation and simultaneous exposure to Cd reduced the survival rate of B. areolata, and its glycogen was mobilized in great extent. Feeding with P. viridis or P. aibuhitensis helped the B. areolata to combat Cd toxicity and lessen mortality. After exposed to Cd, the damage of the DNA integrity of hepatopancreas tissue for the B. areolata fed with P. viridis or P. aibuhitensis could be recovered with time, but not for the starved B. areolata. Prolonged starvation caused tissue atrophy and led to Cd accumulation and MT increase, while feeding with P. viridis or P. aibuhitensis increased the B. areolata mass and lowered the Cd accumulation and MT level because of the tissue dilution effect. The B. areolata fed with P. viridis had better growth and lower Cd content than that fed with P. aibuhitensis. This study indicated that starvation intensified the toxicity of Cd to B. areolata, while prey type had significant effects on the growth rate of the B. areolata and indirectly affected its Cd accumulation, MT induction, and glycogen consumption. It was suggested that when using gastropods such as B. areolata as the indicator species to monitor marine environmental pollution, it would be necessary to consider the effects of habitat ecological data including food richness and prey type. Moreover, in the high-density cultivation of B. areolata in factory, rational feeding and periodic measurement of Cd concentration in seawater should be made.

 

Fal'fushins'ka, H. I., L. L. Hnatyshyna, et al. (2012). "[Population-related peculiarities of molecular stress-responsive systems of bivalve mollusk under the effect of tetrazine pesticide]." Ukr Biokhim Zh (1999) 84(1): 90-7.

                Tetrazine pesticides are widely used for the treatment of crops in most EU countries and USA. However, data about the effect of environmentally realistic concentrations of biocides on the molecular stress response system in non-target organisms are absent. The aim of our study was the comparison of adaptive capability of bivalve mollusk Anodonta cygnea from two populations under the effects of commercial pesticide Apollo in terms of biochemical parameters of the digestive gland. The differences between parameters of oxidative stress and glutathione transferase activity in specimens of control groups from clean (I group) and polluted (B group) areas have been shown. Under the effect of Apollo, the level of protein carbonyls and microsomal oxidation processes increased, and the level of metallothioneins and oxyradical formation decreased in the specimens from both populations. However, the treatment provoked the activation of antioxidant processes in the I group and their inhibition in B group. Potentially the injury of cellular thiols, glutathione and metallothioneins, seems to be key point of tetrazine pesticides toxicity.

 

Freitas, R., E. Costa, et al. (2012). "Looking for suitable biomarkers in benthic macroinvertebrates inhabiting coastal areas with low metal contamination: comparison between the bivalve Cerastoderma edule and the Polychaete Diopatra neapolitana." Ecotoxicol Environ Saf 75(1): 109-18.

                Metals accumulated in marine sediments are often a threat to benthic communities. With the recognized importance and wide use of stress biochemical responses as indicators of metal contamination it becomes essential to compare these markers between different species and verify their ubiquity and accuracy. Using wild Diopatra neapolitana and Cerastoderma edule, collected at several areas differing in metal contamination, this study aimed to assess the use of these two macrobenthic species as sentinel organisms and to determine the applicability of currently used biomarkers in benthic species exposed to a range of low metal and As concentrations. Total metal accumulation and intracellular partitioning was analyzed and metal-induced alterations were assessed through the analysis of several biochemical parameters in both organisms, including stress-induced reactive oxygen species (ROS), lipid peroxidation and protein content, the activity of antioxidant enzymes catalase (CAT), superoxide dismutase (SOD) and of glutathione S-transferase (GST). Metal chelation by Metallothioneins (MTs) was also determined. Results revealed that D. neapolitana accumulated higher levels of metals when compared to C. edule, independently of the sediment concentration. Results also showed strong species-specific responses to metals and differences in the ability to sequester metals. Overall, C. edule showed to be more efficient metal chelator and precipitatior than D. neapolitana, which was less tolerant and presented oxidative stress. MTs proved to be a good predictor of metal accumulation in both species, even under low metal exposures. On the other hand, lipid peroxidation was a good indicator of oxidative damage, only observed in D. neapolitana, which was a result of higher metal retention in the soluble fraction.

 

Paul-Pont, I., P. Gonzalez, et al. (2012). "Cloning, characterization and gene expression of a metallothionein isoform in the edible cockle Cerastoderma edule after cadmium or mercury exposure." Ecotoxicol Environ Saf 75(1): 119-26.

                Metallothionein (MT) genes encode crucial metal-binding proteins ubiquitously expressed in living organisms and which play important roles in homeostasis of essential metals and detoxification processes. Here, the molecular organization of the first metallothionein gene of the edible cockle Cerastoderma edule and its expression after cadmium (Cd) or mercury (Hg) exposures were determined. The resulting sequence (Cemt1) exhibits unusual features. The full length cDNA encodes a protein of 73 amino acids with nine classical Cys-X((1-3))-Cys motifs, but also one Cys-Cys not generally found in molluscan MT. Moreover, characterization of the molecular organization of the Cemt1 gene revealed two different alleles (A1 and A2) with length differences due to large deletion events in their intronic sequences involving direct Short Interspersed repeated Elements (SINE), while their exonic sequences were identical. To our knowledge, such large excision mechanisms have never before been reported in a bivalve gene sequence. After 10 days of Cd exposure at environmentally relevant doses, quantitative real-time PCR revealed a strong induction of Cemt1 in gills of C. edule. Surprisingly, neither induction of the Cemt1 gene nor of MT protein was shown after Hg exposure, despite the fact that this organism is able to bioaccumulate a high amount of this trace metal which is theoretically one of the most powerful inducers of MT biosynthesis.

 

An, L. H., B. H. Zheng, et al. (2012). "Biomarker responses and genotoxicity in the mud snail (Bullacta exarata) as indicators of coastal contamination." Mar Pollut Bull 64(2): 303-9.

                In the present study different biomarker responses and genotoxicity were determined in wild mud snails (Bullacta exarata) collected from 3 sampling sites in Bohai Bay in northeastern China, which is a region that is under considerable anthropogenic influence. Significant spatial variability of superoxide dismutase (SOD) and metallothionein (MT)-like proteins were recorded, while glutathione transferase (GST), catalase (CAT), and vitellin-like proteins (Vn's) were not observed. Furthermore, genomic DNA that was amplified with 4 fluorescence-labeled primer pairs showed variable genetic distances among the 3 wild mud snail populations found in Bohai Bay, which may be correlated with differences in the types of environmental genotoxicants, such as heavy metals and polycyclic aromatic hydrocarbons. This multi-biomarker approach provided an improved understanding of the potential toxicological impact of contaminated sediments on benthic organisms.

 

David, E., A. Tanguy, et al. (2012). "Characterisation and genetic polymorphism of metallothionein gene CgMT4 in experimental families of Pacific oyster Crassostrea gigas displaying summer mortality." Biomarkers 17(1): 85-95.

                Summer mortality events have been observed in Pacific oyster Crassostrea gigas for several decades. This paper examines the selective pressure exerted by summer mortality on the polymorphism of a newly identified oyster metallothionein gene. CgMT4 cDNA and genomic sequences were obtained. CgMT4 was studied in two generations of oysters reared in three sites on the French Atlantic coast, using single strand conformation polymorphism analysis. Four alleles were detected. Individuals carrying genotype MT4-CD seem to have higher susceptibility to summer risk conditions. The MT4 gene could be a potential new genetic marker for susceptibility; further validation studies are recommended.

 

Devos, A., C. Voiseux, et al. (2012). "Effect of chronic exposure to zinc in young spats of the Pacific oyster (Crassostrea gigas)." Environ Toxicol Chem 31(12): 2841-7.

                The marine coastal environment is exposed to a mixture of environmental pollutants of anthropogenic origin, resulting in chronic low concentrations of contaminants. As a consequence, most coastal marine species are exposed to low doses of such pollutants during their entire life. Many marine species live for years in their natural environment, whereas they do not under laboratory exposure conditions. Using early stages of development in laboratory work allows animals to be chronically exposed from an early age over a reasonable experiment period. In the present study, the authors investigated the effect of chronic exposure to zinc in spats of the Pacific oyster (Crassostrea gigas), from metamorphosis up to 10 weeks. The authors investigated integrated biological endpoints that would account for the apparent general health of the animals as well as molecular markers showing more subtle effects that could potentially go unnoticed at a biologically integrated level. The authors measured in parallel both growth and the transcriptional level of target stress genes. Growth was monitored by image analysis of large samples to avoid high variability and ensure statistical robustness. A dose-response relationship was derived from growth data, yielding a median effective concentration (EC50) of 7.55 microM. Stress genes selected on the basis of available RNA sequences in C. gigas included genes involved in chaperone proteins, oxidative stress, detoxification, and cell cycle regulation. Out of nine stress target genes, only metallothionein displayed overexpression in response to high levels of zinc.

 

Itziou, A. and V. K. Dimitriadis (2012). "Multiple pollution biomarker application on tissues of Eobania vermiculata during two periods characterized by augmented and reduced snail activity." Ecotoxicol Environ Saf 86: 13-22.

                In the present study a package of biomarkers was applied on land snails E. vermiculata collected from polluted areas, as well as from an unpolluted reference one. Snail collection was performed during two different sampling periods characterized by reduced and augmented organism activity, October and May, respectively. The biomarkers applied were lysosomal membrane stability on digestive cells (LMS), neutral red lysosomal retention assay on haemocytes (NRR), morphometric changes of the lysosomal system (VDL, NDL), morphometric alterations of the neutral lipids (VDLP, NDLP), acetylcholinesterase activity on digestive gland and hemolymph (AChE), metallothionein content on digestive gland (MTs) and cyclic AMP content on digestive gland (cAMP). The results revealed significant differences in biomarker values between the two sampling periods. Significant differences were also detected among the sampling groups. The fluctuation of the parameters applied indicated that spring is a more suitable period for sampling conduction compared to autumn and that biomonitoring studies should be performed with special attention during the last mentioned period.

 

Moschino, V., E. Delaney, et al. (2012). "Assessing the significance of Ruditapes philippinarum as a sentinel for sediment pollution: bioaccumulation and biomarker responses." Environ Pollut 171: 52-60.

                The present study assessed whether the bivalve Ruditapes philippinarum may be appropriately deployed as a bioindicator in monitoring transitional environments, in terms of bioaccumulation potential and biomarker responses. The concentrations of trace metals, PAHs and PCBs were determined in sediments and clam tissue, and biomarkers were estimated at various levels of biological complexity (i.e. metallothioneins, lipofuscins, survival-in-air and reburrowing behaviour). Sediments and clams were collected biannually in 2004 and 2005 at eight sites within Venice lagoon, which were influenced differently by natural and anthropogenic impacts. Results highlighted that the broad variations of pollutant concentrations in sediments were not consistent either with the body residuals or with the biomarker responses. Consequently, on the basis of the observed weak responsiveness and sensitivity to anthropogenic stressors we suggest a more cautious use for R. philippinarum as sentinel organism, at least in estuarine sediments in the north Adriatic transitional areas.

 

Strogyloudi, E., M. O. Angelidis, et al. (2012). "Metal concentrations and metallothionein levels in Mytilus galloprovincialis from Elefsis bay (Saronikos gulf, Greece)." Environ Monit Assess 184(12): 7189-205.

                Spatial and temporal variability of Cd, Cu, Cr, Ni, Zn, Fe and Mn and metallothionein (MT) concentrations were determined in mussels Mytilus galloprovincialis from Elefsis bay (Saronikos gulf, Greece). Higher concentrations of both metal concentrations and MTs were recorded in mussels inhabiting industrial locations (steelworks and shipyard), indicating a markedly higher metal bioavailability. However at these sites, located at the eastern part of the bay, mussel metal concentrations were not always correlated with both seawater metal concentrations and MTs possibly due to different time scales of integration of the metal sources into mussels and/or the participation of other metal regulatory mechanisms except MT induction. The pattern of the temporal variation of mussel metal concentrations and the MT levels was similar among stations with higher values during the winter-spring season and lower during the summer-autumn period. The inverse relationship of flesh condition index with mussel metal concentrations was attributed to the influence of mussel annual reproductive cycle.

 

Gomes, T., C. G. Pereira, et al. (2012). "Accumulation and toxicity of copper oxide nanoparticles in the digestive gland of Mytilus galloprovincialis." Aquat Toxicol 118-119: 72-9.

                Given the wide use of CuO nanoparticles in various industrial and commercial applications they will inevitably end up in the aquatic environment. However, little information exists on their biological effects in bivalve species. Accordingly, mussels Mytilus galloprovincialis were exposed to 10 mug Cu L(-1) as CuO nanoparticles and Cu(2+) for 15 days, and biomarkers of oxidative stress (superoxide dismutase, catalase and glutathione peroxidase), damage (lipid peroxidation) and metal exposure (metallothionein) were determined along with Cu accumulation in the digestive glands of mussels. Cu was linearly accumulated with time of exposure in mussels exposed to CuO nanoparticles, while in those exposed to Cu(2+) elimination was significant by day 15. Both forms of Cu cause oxidative stress with distinct modes of action. Exposure to CuO nanoparticles induces lower SOD activity in digestive glands compared to those exposed to Cu(2+), while CAT was only activated after 7 days of exposure to nano and ionic Cu, with contradictory effects after 15 days of exposure and GPX activities were similar. Lipid peroxidation levels increased in both Cu forms despite different antioxidant efficiency. Moreover, a linear induction of metallothionein was detected with time in mussels exposed to CuO nanoparticles, directly related to Cu accumulation, whereas in those exposed to Cu(2+) metallothionein was only induced after 15 days of exposure. Since only a small fraction of soluble Cu fraction was released from CuO nanoparticles, the observed effects seem to be related to the nano form of Cu, with aggregation as a key factor. Overall, our results show that the digestive gland is susceptible to CuO nanoparticles related oxidative stress, and is also the main tissue for their accumulation.

 

Falfushynska, H., L. Gnatyshyna, et al. (2012). "Evaluation of biotargeting and ecotoxicity of Co(2)(+)-containing nanoscale polymeric complex by applying multi-marker approach in bivalve mollusk Anodonta cygnea." Chemosphere 88(8): 925-36.

                Cobalt (Co(2+)) is present in many nanoscaled materials created for various applications. The key goal of our study was to develop sensitive approaches for assessing the bio-risks associated with using novel Co(2+)-containing nanoscaled polymeric complex (Co-NC). Freshwater bivalve mollusk Anodonta cygnea (Unionidae) was subjected to 14 d action of the developed Co-NC, as well as of Co(2+) applied in the corresponding concentration (50 mug L(-1)) or polymeric substance (PS). All experimental groups under study have demonstrated signs of toxic targeting, notably changes in DNA characteristics, oxidative stress (with particularities in each exposed group) and activation of anaerobiosis (Co(2+) and Co-NC). However, the group exposed to Co-NC showed some advantages that can be related to the activation of metallothionein (MT) function (increase in the level of MT-related SH-groups (MT-SH)): low level of oxyradical formation, no increase in protein carbonylation and vitellogenin-like proteins concentration unlike in Co(2+) and PS exposed groups. On the other hand, Co(2+) increased metal (Co, Cu, Zn and Cd) binding to MT (MT-Me) without changes in MT-SH level jointly with activation of oxyradical formation and apoptosis and decreasing of lysosomal membrane stability. PS per se initiated unbalanced changes in activities of the biotransformation enzymes ethoxyresorufin-O-deethylase and glutathione-S-transferase. Thus, Co(2+) complexing with the developed PS prevented bio-toxic effects of free Co(2+) ions and PS per se, at least in the studied hydrobiont. The MT-SH was the main distinguishing index of Co-NC group selected by classification and regression tree analysis.

 

Fang, Y., H. Yang, et al. (2012). "Tissue-specific response of metallothionein and superoxide dismutase in the clam Mactra veneriformis under sublethal mercury exposure." Ecotoxicology 21(6): 1593-602.

                To identify the relationship between mercury (Hg) and stress responses in the clam Mactra veneriformis, metallothionein (MT) and superoxide dismutase (SOD) mRNA expression in the digestive gland, gill, and mantle as well as MT protein content and SOD activity in the digestive gland were examined under sublethal Hg exposure at doses of 10, 20, and 40 mug/L for 21 days. The ranking of the tissues in decreasing order of their basal MvMT and MvSOD mRNA expression is as follows: digestive gland > mantle > gill > adductor muscle > foot and digestive gland > mantle > gill > foot > adductor muscle, respectively. Hg exposure significantly elevated MvMT and MvSOD mRNA transcripts in the digestive gland, gill, and mantle in a tissue-specific way. In the digestive gland, a dose-dependent increase of MvMT and MvSOD mRNA expression, stimulation of MT protein, and alteration of SOD activity were observed under Hg stress. MT protein responded later than MT mRNA to Hg exposure and no clear relationship was found between them, indicating the occurrence of posttranscriptional events. All of the results suggest that MT and SOD cooperate in resisting Hg toxicity and maintaining cellular metabolic homeostasis in M. veneriformis. MT and SOD mRNA expressions have great potential as biomarkers of Hg pollution in the aquatic environment for the studied species.

 

Figueira, E., P. Cardoso, et al. (2012). "Ruditapes decussatus and Ruditapes philippinarum exposed to cadmium: toxicological effects and bioaccumulation patterns." Comp Biochem Physiol C Toxicol Pharmacol 156(2): 80-6.

                Since differences in metal accumulation may exist between bivalve species, the aim of this study was to assess the impact of cadmium (Cd) on Ruditapes decussatus and Ruditapes philippinarum. For this, the Cd accumulation, mortality rates and biochemical responses were analysed in the two species after 5days of exposure, under laboratory-controlled conditions. The concentration of Cd that caused 50% of mortality on clams was two-times higher in R. decussatus than in R. philippinarum. For both species, higher percentage (84.5-98.2%) of the Cd was in the insoluble fraction, but the Cd concentration in solution was 3 to 8 times higher in R. decussatus. Nevertheless, R. philippinarum presented higher oxidative stress and higher CAT activity. The paradox observed between the two clams can be explained by the higher capacity of R. decussatus to increase the expression of MTs when exposed to Cd.

 

Kamel, N., J. Jebali, et al. (2012). "Biochemical responses and metals levels in Ruditapes decussatus after exposure to treated municipal effluents." Ecotoxicol Environ Saf 82: 40-6.

                This study assessed the responses of biochemical biomarkers and metals levels in Ruditapes decussatus exposed to the increasing concentrations of treated municipal effluents (TME) discharged into the Tunisian coastal area. Clams were exposed to 0%, 1%, 3% and 10% for 7 and 14 day and the following biochemical responses were measured: (1) catalase activity and lipid peroxidation levels (TBARS) as oxidative stress biomarkers, (2) gluthathione S-transferase (GST) activity as a phase II conjugation enzyme; (3) cholinesterase activity (ChE) as biomarker of neurotoxicity, and (4) metallothioneins as a proteins highly induced by heavy metals. A significant uptake of Cu, Cd and Zn in digestive gland and serious biochemical alterations were observed. Thus, exposure of clams to croissant concentration of TME have the potential to increase the oxidative stress biomarkers (TBARS, CAT activity) and MT levels; and decrease ChE activity in both gills and digestive gland. Current experimental results suggest that CAT, GST, ChE activities and MT and TBARs levels in gills and digestive gland of clam R. decussatus are sensitive and suitable responses for assessing the effects of anthropogenic contaminants on the aquatic ecosystems, particularly effluent complex mixtures.

 

Cravo, A., C. Pereira, et al. (2012). "A multibiomarker approach in the clam Ruditapes decussatus to assess the impact of pollution in the Ria Formosa lagoon, South Coast of Portugal." Mar Environ Res 75: 23-34.

                The Ria Formosa lagoon is an ecosystem whose water quality reflects the anthropogenic influence upon the surrounding areas. In this lagoon, the clam Ruditapes decussatus has a great economical importance and has been widely used as a biomonitor. A multibiomarker approach (delta-aminolevulinic acid dehydratase, metallothionein, lipid peroxidation, acetylcholinesterase, alkali-labile phosphates, DNA damage) was applied to assess the environmental quality of this ecosystem and the accumulation of contaminants and their potential adverse effects on clams. Clams were sampled in different shellfish beds in the period between July 2007 and December 2008 and abiotic parameters (temperature, salinity, pH and dissolved oxygen of seawater and organic matter in the sediment), condition index, metals (Cd, Cu, Zn, Ni, Pb), TBTs and PAHs concentrations were measured in clam tissues. Data was integrated using Principal Component Analyses and biomarker indices: IBR (Integrated Biomarker Response) and HSI (Health Status Index). This multibiomarker approach enabled discrimination of a time and space trend between sites with different degrees of anthropogenic contamination, identifying one of them (site 2) as the most stressful and summer months as the most critical period for clams due to an increase of environmental stress (anthropogenic pressure along with extreme environmental conditions, e.g. temperature, dissolved oxygen, organic matter in the sediments, etc). The selected biomarkers provided an integrated response to assess the environmental quality of the system, proving to be a useful approach when complex mixtures of contaminants occur.

 

Mao, H., D. H. Wang, et al. (2012). "The involvement of metallothionein in the development of aquatic invertebrate." Aquat Toxicol 110-111: 208-13.

                The many documents on metallothioneins (MTs) in aquatic organisms focus especially on their use as biomarkers in environmental monitoring programs, but there are a few papers that summarize the physiological role of MTs in aquatic organisms especially in their development. The multifaceted role of MTs include involvement in homeostasis, protection against heavy metals and oxidant damage, metabolic regulation, sequestration and/or redox control. MTs could be induced by heavy metals which are able to hinder gametogenesis, suppress embryogenesis, and hamper development. Here we pay more attention on the non-essential metal cadmium, which is the most studied heavy metal regarding MTs, and its effects on the development of aquatic invertebrates. In this paper, we have collected published information on MTs in aquatic organisms - mollusks, crustaceans, etc., and summarize its functions in aquatic invertebrates, especially those related to their development.

 

Palais, F., O. Dedourge-Geffard, et al. (2012). "One-year monitoring of core biomarker and digestive enzyme responses in transplanted zebra mussels (Dreissena polymorpha)." Ecotoxicology 21(3): 888-905.

                A 12-month active biomonitoring study was performed in 2008-2009 on the Vesle river basin (Champagne-Ardenne, France) using the freshwater mussel Dreissena polymorpha as a sentinel species; allochthonous mussels originating from a reference site (Commercy) were exposed at four sites (Bouy, Sept-Saulx, Fismes, Ardre) within the Vesle river basin. Selected core biomarkers (acetylcholinesterase (AChE) activity, glutathione-S transferase (GST) activity, metallothionein concentration), along with digestive enzyme activities (amylase, endocellulase) and energy reserve concentrations (glycogen, lipids), were monitored throughout the study in exposed mussels. At the Fismes and Ardre sites (downstream basin), metallic and organic contamination levels were low but still high enough to elicit AChE and GST activity induction in exposed mussels (chemical stress); besides, chemical pollutants had no apparent deleterious effects on mussel condition. At the Bouy and Sept-Saulx sites (upstream basin), mussels obviously suffered from adverse food conditions which seriously impaired individual physiological state and survival (nutritional stress); food scarcity had however no apparent effects on core biomarker responses. Digestive enzyme activities responded to both chemical and nutritional stresses, the increase in energy outputs (general adaptation syndrome-downstream sites) or the decrease in energy inputs (food scarcity-upstream sites) leading to mid- or long-term induction of digestive carbohydrase activities in exposed mussels (energy optimizing strategy). Complex regulation patterns of these activities require nevertheless the use of a multi-marker approach to allow data interpretation. Besides, their sensitivity to natural confounding environmental factors remains to be precised.

 

Fal'fushins'ka, G. I., L. L. Gnatyshina, et al. (2011). "[Evaluation of biological effects of cobalt-nanocomposites with the use of biochemical markers of bivalve mollusk Anodonta cygnea]." Ukr Biokhim Zh (1999) 83(5): 40-7.

                Intensive implementation of nanomaterials requires development of novel methods for evaluation of their potential ecotoxicity. The aim of our study was to identify specific characteristics of the effect of cobalt-nanocomposite (Co-NC) on the molecular stress-responsive system in the digestive gland of bivalve mollusk Anodonta cygnea. Nanocomposite was synthesized by mixing alcohol solution of copolymer N-vinylpirrolidone, 5-(tret-butylperoxy)-5-methyl-1-hexene-3-yne and dimethylaminoethylmetacrylate and cobalt (II) chloride. After 14 days of the mollusk exposure in the presence of Co-NC, CoCl, or corresponding polymer substance it was shown that the Co-NC, in contrast to other agents, does not cause an oxidative stress due to the superoxide dismutase activity, metallotioneins (MTs) level, glutathione redox index and oxyradical production. Multivariate analysis confirmed specific features of the Co-NC's effect related to an enhanced expression of MTs, while CoCl2 activated lactate dehydrogenate and oxyradical production, and polymer substance enhanced glutathione transferase activity.

 

Ng, T. Y., N. M. Pais, et al. (2011). "Mechanisms of waterborne Cu toxicity to the pond snail Lymnaea stagnalis: physiology and Cu bioavailability." Ecotoxicol Environ Saf 74(6): 1471-9.

                We examined the mechanisms of toxicity of waterborne Cu to the freshwater pond snail Lymnaea stagnalis. The snail is one of the most sensitive species to acute Cu exposure (96 h LC(50), LC(20): 24.9, 18.0 mugl(-1)); they are not protected by the water quality criteria of the US EPA. Tissue Na and Ca were also reduced by Cu in the acute exposure. In contrast, during 28 d chronic exposures to Cu in the presence of food, which resulted in higher DOC concentrations, there was no significant mortality but an inhibition of growth, which may reflect a re-allocation of resources to detoxification. Cu detoxification was evidenced in chronic exposure by increases in metallothionein-like protein concentrations and Cu binding to metal-rich granules, decreases in thiobarbituric acid-reactive substances, and changes in the subcellular distribution in the soft tissues. Our results demonstrated that apart from external Cu bioavailability, compartmentalization of metals within the cells can alter toxicity of Cu to the snails.

 

Pytharopoulou, S., K. Grintzalis, et al. (2011). "Translational responses and oxidative stress of mussels experimentally exposed to Hg, Cu and Cd: one pattern does not fit at all." Aquat Toxicol 105(1-2): 157-65.

                Certain metals, like Hg, Cu and Cd, are capable of down-regulating protein synthesis in several marine organisms, including Mytilus galloprovincialis. Nevertheless, due to the complexity of the environmental stress, it is difficult to evaluate the influence of individual metals on protein synthesis via field studies. To bypass this difficulty, experimental studies were carried out on M. galloprovincialis exposed in aquarium for 15 days to one of three selected metal salts, HgCl(2), CuCl(2) and CdCl(2). Polysome profile was determined in digestive gland extracts of the exposed mussels as a way of measuring the functional status of ribosomes, superoxide radical production and lipid peroxidation as indicators of oxidative stress, metallothionein content as a metal detoxification index, and superoxide dismutase activity as a free radicals-scavenging index. Exposure of mussels to Hg(2+) or Cu(2+) resulted in a concentration- and time-dependent decrease in the polysome content of digestive gland cells, which at 15th day of exposure and at the highest metal concentrations tested, was 32% and 19% of the control, respectively. Both metals, at the concentrations used (<40 mug/L), did not significantly influence the oxidative stress biomarkers. By contrast, Cd(2+) treatment significantly induced superoxide radical production and lipid peroxidation in digestive gland cells, hinting that mussels suffered from oxidative stress. Polysome levels in Cd(2+)-exposed mussels were initially decreased by day 5 in digestive gland cells and then elevated to reach nearly the control levels by 15 days of exposure. Elevated protein synthesis was associated with significantly increased production of metallothioneins, whereas such increase was not recorded in Hg(2+)- or Cu(2+)-exposed mussels. Interestingly, the ribosome efficiency at initiating protein synthesis followed a similar pattern of polysome alterations, a fact suggesting that regulation of protein synthesis mainly occurred at the initiation phase of translation. Overall, these results suggest that the effect of each metal on protein synthesis is idiosyncratic and depends on its ability to induce specific cellular defense mechanisms against oxidative stress.

 

Tankoua, O. F., P. E. Buffet, et al. (2011). "Potential influence of confounding factors (size, salinity) on biomarkers in the sentinel species Scrobicularia plana used in programmes monitoring estuarine quality." Environ Sci Pollut Res Int 18(8): 1253-63.

                INTRODUCTION: To use biomarkers in monitoring programmes, potential confounding factors must be considered. In the clam Scrobicularia plana, the influence of size and salinity on biomarkers at different levels of biological organisation has been examined. MATERIAL AND METHODS: Biochemical (glutathione-S-transferase, lactate dehydrogenase, acetylcholinesterase, digestive enzymes, metallothionein), physiological (energy reserves) and behavioural (burrowing) responses were compared (a) in specimens of different sizes from the Loire estuary; (b) in specimens from the Belon estuary at two sites with salinities of 30.1 or 11.5. RESULTS: Amongst the biomarkers able to reveal pollution effects, several are influenced by the size of the clams (Ag, Cu, Ni and glycogen concentrations, GST and AChE activities, condition indices). Salinity differences induced variations of the same order of magnitude (GST, AChE) or even higher (lactate dehydrogenase, digestive enzymes in the crystalline style) than contamination-induced variations. In burrowing tests, the number of burrowed specimens was similar at both salinities after an experiment time <3 h. CONCLUSIONS: Size is a factor necessarily but easily controlled. Because the weight may be different in clams of identical size, correction factors may be used to minimise the influence of weight changes on biomarkers. A correction factor taking into account salinity levels can also be used. The protein concentrations in the clams did not differ with salinity, a very favourable outcome since all enzyme activities are classically expressed by reference to total protein concentrations. For burrowing tests, the number of burrowed specimens at a particular time is an endpoint that is preferable to measures of burrowing speed.

 

Contardo-Jara, V., C. Lorenz, et al. (2011). "Exposure to human pharmaceuticals Carbamazepine, Ibuprofen and Bezafibrate causes molecular effects in Dreissena polymorpha." Aquat Toxicol 105(3-4): 428-37.

                Carbamazepine (CBZ), Ibuprofen (IBU) and Bezafibrate (BEZ) were tested for their potential to bioaccumulate and provoke molecular changes in the non-target organism Dreissena polymorpha. mRNA changes of enzymes and other proteins involved in the prevention from protein damage (heat shock protein 70, hsp70) and oxidative stress (superoxide dismutase, SOD; catalase, CAT; metallothionein, MT), biotransformation (pi-class glutathione S-transferase, piGST; aryl hydrocarbon receptor, AH-R), elimination (P-glycoprotein, P-gp) and reversible protein posttranslational modification (protein phosphatase 2A, PP2A) served as molecular biomarkers. Mussels were exposed in a flow-through system to increasing concentrations of the three substances (1, 10, 100 and 1000 nM). The two lower concentrations correspond to environmentally relevant concentrations detected in surface and effluent waters, respectively. Measuring tissue concentration after one, four and seven days the uptake of CBZ and IBU by the mussels could be evidenced, whereas no accumulation data could be achieved for BEZ. The bioconcentration factor was highest for mussels exposed to the lowest CBZ and IBU concentrations, with 90 and 460-fold higher tissue concentration, respectively, after seven days. CBZ was the only substance tested which caused a significant increase in gill mRNA level of hsp70 after only one day exposure, evidencing the potential of CBZ to immediately provoke a stress condition and assumingly protein damage in gills. After longer exposure, mussels displayed down-regulated mRNA levels of hsp70 and SOD in gills, as well as of MT and P-gp in the digestive gland, hinting on an inhibitory character of CBZ. In IBU exposed mussels increased oxidant stress conditions were evidenced by induced mRNA levels in the digestive gland of CAT and MT, as well as SOD after one and four days, respectively. A concentration as found at sewage treatment plant effluents provoked an increase in transcript levels of piGST, suggesting enhanced need for biotransformation of IBU or by-products derived from oxidative stress. Also exposure to an environmentally relevant BEZ concentration provoked an immediate increase in piGST transcript level in the digestive gland followed by up-regulated hsp70 after four and seven days evidencing a chronic stress condition for the mussels.

 

Hockner, M., R. Dallinger, et al. (2011). "Nematode and snail metallothioneins." J Biol Inorg Chem 16(7): 1057-65.

                Metallobiologists have, at large, neglected soil dwelling invertebrates; exceptions are the nematode (Caenorhabditis elegans) and snails (Helix pomatia and Cantareus aspersus). This review aims to compare and contrast the molecular, protein and cellular mechanisms of the multifunctional nematode and snail metallothioneins (MTs). The C. elegans genome contains two MT genes, mtl-1, which is constitutively expressed in the pharynx and likely to act as an essential and/or toxic metal sensor, and mtl-2, which plays a negligible role under normal physiological conditions but is strongly induced (as mtl-1) in intestinal cells upon metal exposure. It has been possible to follow the intricate phenotypic responses upon the knockdown/knockout of single and multiple MT isoforms and we have started to decipher the multifunctional role of C. elegans MTs. The snails have contributed to our understanding regarding MT evolution and diversity, structure and metal-specific functionality. The H. pomatia and C. aspersus genomes contain at least three MT isoform genes. CdMT is responsible for cadmium detoxification, CuMT is involved in copper homeostasis and Cd/CuMT is a putative ancestral MT possibly only of minor importance in metal metabolism. Further investigations of nematode, snail and other invertebrate MTs will allow the development of alternative biomarker approaches and lead to an improved understanding of metallobiology, protein evolution and toxicogenomics.

 

Liu, F. and W. X. Wang (2011). "Metallothionein-like proteins turnover, Cd and Zn biokinetics in the dietary Cd-exposed scallop Chlamys nobilis." Aquat Toxicol 105(3-4): 361-8.

                In this study, we tested whether metallothionein-like proteins (MTLPs) affected the biokinetics of Cd and Zn in the scallops Chlamys nobilis following dietary Cd exposure. The scallops were fed Cd-contaminated diatoms for 8 or 40 days, and then the tissue Cd concentrations, MTLP turnover and their standing stocks, Cd and Zn kinetics were monitored. After 8 days of dietary Cd exposure (at 98 or 196 mug Cdg(-1)), their Cd levels were increased by 7.4-12.5 times, newly synthesized MLTPs by 1.7-2.1 times, and their MLTP stocks doubled. However, after 40 days of dietary Cd exposure (at 58 or 115 mug Cdg(-1)), MTLP synthesis and MTLP stocks did not change despite the fact that Cd bioaccumulation increased by 2.7-4.2 times. MTLPs played little role in the overall Cd and Zn uptake from either food or water, since enhanced MTLP induction did not improve the sequestration of newly incoming Cd or Zn. As MTLP-metal complexes degraded, the released Cd was immediately sequestered by newly synthesized MTLPs while most Zn was depurated. This may explain why scallops eliminated Cd more slowly than Zn. Induced proteins were shown to play a minor role in the detoxification of dietary Cd in C. nobilis, but the species can modify its Cd assimilation to reduce the impact of high dietary Cd levels. MTLPs are probably unsuitable as biomarkers for environmental monitoring involving C. nobilis.

 

Gomes, T., J. P. Pinheiro, et al. (2011). "Effects of copper nanoparticles exposure in the mussel Mytilus galloprovincialis." Environ Sci Technol 45(21): 9356-62.

                CuO NPs are widely used in various industrial and commercial applications. However, little is known about their potential toxicity or fate in the environment. In this study the effects of copper nanoparticles were investigated in the gills of mussels Mytilus galloprovincialis, comparative to Cu(2+). Mussels were exposed to 10 mug Cu.L(-1) of CuO NPs and Cu(2+) for 15 days, and biomarkers of oxidative stress, metal exposure and neurotoxicity evaluated. Results show that mussels accumulated copper in gills and responded differently to CuO NPs and Cu(2+), suggesting distinct modes of action. CuO NPs induced oxidative stress in mussels by overwhelming gills antioxidant defense system, while for Cu(2+) enzymatic activities remained unchanged or increased. CuO NPs and Cu(2+) originated lipid peroxidation in mussels despite different antioxidant efficiency. Moreover, an induction of MT was detected throughout the exposure in mussels exposed to nano and ionic Cu, more evident in CuO NPs exposure. Neurotoxic effects reflected as AChE inhibition were only detected at the end of the exposure period for both forms of copper. In overall, these findings show that filter-feeding organisms are significant targets for nanoparticle exposure and need to be included when evaluating the overall toxicological impact of nanoparticles in the aquatic environment.

 

Bigot, A., L. Minguez, et al. (2011). "Early defense responses in the freshwater bivalve Corbicula fluminea exposed to copper and cadmium: Transcriptional and histochemical studies." Environ Toxicol 26(6): 623-32.

                The aim of the present study was to measure the early effects of copper (10 and 50 mug L(-1)), cadmium (2, 10, and 50 mug L(-1)) and mixtures of these metals in the freshwater bivalve Corbicula fluminea exposed for 12 h in laboratory. Transcription levels of superoxide dismutase (SOD), catalase (CAT), selenium-dependent glutathione peroxidase (Se-GPx), pi-class glutathione S-transferase (pi-GST), metallothionein (MT) in digestive gland and gills, and response of lysosomal system and neutral lipids in digestive gland were determined after the exposure period. Results showed that lysosomal system, neutral lipids content, and mRNA levels were modified, suggesting their early response against oxidative stress and their important role in cell integrity. The integrated biomarker response was calculated and showed that the effects of the combinations of Cu and Cd on the biomarker responses are additive. MT and pi-GST mRNA expression correspond to the largest ranges of response. As efficient biomarkers should have an early warning capacity, SOD, CAT, Se-GPx, pi-GST, MT transcripts levels, lysosomal system, and neutral lipids could be used as biomarkers of metal contamination in the aquatic environment.

 

Gomiero, A., L. Da Ros, et al. (2011). "Integrated use of biomarkers in the mussel Mytilus galloprovincialis for assessing off-shore gas platforms in the Adriatic Sea: results of a two-year biomonitoring program." Mar Pollut Bull 62(11): 2483-95.

                Despite a large number of gas platforms existing in the Adriatic Sea, which is a semi-enclosed basin characterized by a slow turnover rate and increasing industrial as well as other anthropogenic activities, the effects of these structures on the aquatic ecosystem require further investigation. Since 1998, multidisciplinary studies have been performed by CNR-ISMAR to comply with legislation and to support the development of protocols for the monitoring of offshore activities in the Adriatic Sea. The present study was developed to implement a biomonitoring plan to assess the ecotoxicological effects of the extraction activities of an off-shore gas platform. Biomarkers were evaluated in mussels collected from the platform in relation to physiological stress, DNA damage, cellular damage, oxidative stress and exposure effects. Organic contaminants and trace element bioaccumulation were also assessed in the soft body of the mussels to correlate bioaccumulation of pollutants with biomarker responses. The results indicate an absence of platform-related environmental stress.

 

Serafim, A., B. Lopes, et al. (2011). "A multi-biomarker approach in cross-transplanted mussels Mytilus galloprovincialis." Ecotoxicology 20(8): 1959-74.

                The present work integrates the active biomonitoring (ABM) concept in mussels Mytilus galloprovincialis from the South coast of Portugal transplanted during 28 days between two sites with different sources of contamination, and vice versa, in order to assess biological effects in these mussels. For that purpose a multibiomarker approach was used. The suit of biomarkers indicative of metal contamination were metallothioneins (MT) and the enzyme delta-aminolevulinic acid dehydratase (ALAD), for organic contamination mixed function oxidase system (MFO), glutathione-S-transferase (GST) and acetylcholinesterase (AChE), as oxidative stress biomarkers superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and lipid peroxidation (LPO). These biomarkers were used to determine an index to evaluate the stress levels in these two sites. Site A is strongly influenced by metallic contamination, with higher Cu, Cr and Pb in M. galloprovincialis, as well as higher MT levels, antioxidant enzymes activities and LPO concentrations, and lower ALAD activity. In site B organic compounds (PAHs) are prevalent and native mussels show higher activities of the MFO system components and GST. Transplanted mussels had significant alterations in some biomarkers that reflect the type of contaminants present in each site, which demonstrates the primary role of the environment in determining the physiological characteristics of resident mussels. Therefore the application of ABM using a battery of biomarkers turns out to be a useful approach in sites where usually complex mixtures of contaminants occurs. In this study the biomarkers that better differentiate the impact of different contaminants at each site were MT, CYP450, SOD and CAT.

 

Barmo, C., C. Ciacci, et al. (2011). "Pleiotropic effects of hexavalent chromium (CrVI) in Mytilus galloprovincialis digestive gland." Chemosphere 83(8): 1087-95.

                Hexavalent Chromium Cr(VI) is an important contaminant considered as a model oxidative toxicant released from both domestic and industrial effluents, and represents the predominant chemical form of the metal in aquatic ecosystems. On the other hand, in mammals the reduced form Cr(III) is considered an essential microelement, involved in regulation of lipid and carbohydrate metabolism; moreover, recent evidence suggests that Cr may have endocrine effects. In this work, the effects of Cr(VI) were investigated in the digestive gland of the marine bivalve Mytilus galloprovincialis. Mussels were exposed to 0.1-1-10-100 mug Cr(VI) L(-1) animal(-1) for 96 h. At 100 mug L(-1), a large increase in total Cr tissue content was observed; in these conditions, the lysosomal membranes were completely destabilized, whereas other lysosomal biomarkers (neutral lipids-NL and lipofuscin-LF), as well as different enzyme activities and gene expression were unaffected, this indicating severe stress conditions in the tissue. On the other hand, at lower concentrations, changes in other histochemical, biochemical and molecular endpoints were observed. In particular, at both 1 and 10 mug L(-1), lysosomal destabilization was associated with significant NL and LF accumulation; however, no changes in catalase and GSH transferase (GST) activities were observed. At the same concentrations, GSSG reductase (GSR) activity was significantly increased, this probably reflecting the recycling of GSSG produced in the GSH-mediated intracellular reduction of Cr(VI). Increased activities of the key glycolytic enzymes PFK (phosphofructokinase) and PK (pyruvate kinase) were also observed, indicating that Cr(VI) could affect carbohydrate metabolism. Cr(VI) induced downregulation or no effects on the expression of metallothioneins MT10 and MT20, except for an increase in MT20 transcription in males. Moreover, significant up-regulation of the Mytilus estrogen receptor MeER2 and serotonin receptor (5-HTR) were observed in both sexes. The results demonstrate that exposure to Cr(VI) in the low ppb range did not result in strong toxicity or oxidative stress conditions in mussel digestive gland. On the other hand, our data support the hypothesis that low concentrations of the metal can exert pleiotropic effects on mussel physiology, from modulation of lipid and carbohydrate metabolism, to effects on the expression of estrogen-responsive genes.

 

Strady, E., J. Schafer, et al. (2011). "Tracing cadmium contamination kinetics and pathways in oysters (Crassostrea gigas) by multiple stable Cd isotope spike experiments." Ecotoxicol Environ Saf 74(4): 600-6.

                Laboratory experiments using stable Cd isotopes ((110)Cd and (112)Cd) were conducted to separately and simultaneously characterize Cd accumulation in different tissues of Pacific oysters (Crassostrea gigas) via the (i) trophic and (ii) direct pathways. For this, we exposed juvenile oysters to (110)Cd-spiked seawater ((110)Cd: 2 mug l(-1); constant level) and (112)Cd-spiked food (Thalassiossera weissflogii, (112)Cd: 2 mug l(-1) in 35x10(3) cells/oyster/L) in four experimental treatment groups, each containing 6 oysters, for 21 days with constant trophic feeding. These Cd contamination levels were approximately 10 times lower than those typically used in experimental accumulation studies. Three oysters per treatment group were dissected every 7 days with separate sampling of the gills, digestive gland and the rest of the body. Metallothioneins were analysed in the digestive gland and gills. Cadmium concentrations and isotope ratios were measured in water (daily) and tissues (weekly) by GF-AAS and ICP-MS. The observed time-dependant evolution in Cd concentrations and (110)Cd/(114)Cd and (112)Cd/(114)Cd isotope ratios clearly revealed the bio-accumulation short-term kinetics and pathways of Cd contamination in the different tissues. Under the experimental conditions, significantly changed isotope ratios in gills and the digestive gland of oysters suggested rapid and efficient contamination by (110)Cd derived from direct exposure followed by internal Cd transfer between organs. Trophic contamination became measurable after 14 days of exposure corresponding to a trophic transfer rate of 1%. Constant metallothionein levels during the experiment suggested that the initially present metallothionein levels were sufficient to deal with the experimental Cd exposure.

 

Aceto, S., G. Formisano, et al. (2011). "The metallothionein genes of Mytilus galloprovincialis: genomic organization, tissue expression and evolution." Mar Genomics 4(1): 61-8.

                Recently, increasing interest has been directed to the study of metallothioneins (MTs), which are small proteins that are able to bind metal ions. The induction of MT synthesis after exposure to metal or other environmental contaminants in a large number of aquatic invertebrates makes these proteins good biomarkers in water monitoring programs. Within bivalves, the species Mytilus galloprovincialis and Mytilus edulis represent model organisms for these types of studies, as well as for molecular studies regarding the expression and characterization of MT encoding genes. In the present paper, we focused on the genomic characterization, evolutionary, and tissue-expression analyses of the MT-10, MT-10 Intronless, and MT-20 genes in M. galloprovincialis. The comparison of the genomic sequences showed the presence of long nucleotide stretches within the introns of the MT genes that are conserved between M. galloprovincialis and M. edulis. These non-coding conserved sequences may contain regulatory motifs. Real-Time RT-PCR experiments revealed that, at the basal conditions, the MT-10 and MT-10 Intronless genes are expressed at levels considerably higher than the MT-20 gene, mainly in the digestive gland and gill tissue. The strong induction of the MT-20 gene expression detected in a field-collected sample is associated with the up-regulation of both the MT-10 and MT-10 Intronless genes. Evolutionary analysis revealed signals of localized positive selection that, together with the tissue-expression data, support a possible functional diversification between the MTs encoded by the MT-10 and MT-10 Intronless genes.

 

Andreani, G., E. Carpene, et al. (2011). "Metallothionein cDNA cloning, metallothionein expression and heavy metals in Scapharca inaequivalvis along the Northern Adriatic coast of Italy." Ecotoxicol Environ Saf 74(3): 366-72.

                The aims of this work were: (1) identification of the metallothionein (MT) gene coding sequence in order to prepare an MT probe in Scapharca inaequivalvis and (2) quantification of Cd, Zn, Cu, MT and MTmRNA expression in tissues of molluscs from three areas along the Northern Adriatic coast of Italy. By RT-PCR we cloned the MTcDNA of S. inaequivalvis using the RNA extracted from hepatopancreas of specimens exposed to Cd. The 61 amino acids sequence of MT was deduced and was 70% identical to S. brughtonii MT. Cd concentration in molluscs from the wild was significantly higher in gills from specimens sampled near Ravenna. Zn concentration in the same tissue was significantly higher in Ravenna with respect to Porto Garibaldi while no difference with respect to Cesenatico was detected. Cu levels showed significant differences among sites in gills and mantle whereas values in the hepatopancreas were similar in all sites. The low MT levels were indicative of a low metal exposure; few differences were found in MTmRNA concentrations, which resulted significantly higher in hepatopancreas of molluscs from Porto Garibaldi.

 

Liu, F. and W. X. Wang (2011). "Differential roles of metallothionein-like proteins in cadmium uptake and elimination by the scallop Chlamys nobilis." Environ Toxicol Chem 30(3): 738-46.

                Metallothionein-like proteins (MTLPs) are important inducible ligands for Cd detoxification, but whether MTLPs are involved in Cd bioaccumulation remains unclear. The present study investigated the potential interrelationships between MTLP turnover and Cd biokinetics in the scallop Chlamys nobilis with or without dissolved Cd pre-exposure. After Cd pre-exposure, increased synthesis rate and standing stock concentration of MTLPs as well as increased dissolved Cd uptake rate, were found in C. nobilis. However, the level of newly sequestrated Cd by MTLPs in the pre-exposed scallops was similar to that of the controls, indicating that MTLPs had no effect on Cd uptake from the dissolved phase. Instead, Cd uptake rate was positively related to the Cd binding rate of cellular debris and organelles. In addition, no significant difference was observed in the Cd dietary assimilation efficiency of the scallops, showing that MTLPs played a minimal role in Cd uptake from the dietary phase. Conversely, the Cd efflux rate constant decreased by 68 to 75% in the pre-exposed scallops in comparison with that in the control. Correspondingly, progressive elevations of MTLP-bound Cd and standing stock MTLP concentration were observed in the pre-exposed individuals during the Cd elimination period. In the pre-exposed scallops, the faster de novo MTLP synthesis could remobilize and recycle more Cd from other intracellular pools, effectively reducing the Cd elimination rate. The present study thus showed the differential roles of MTLPs in Cd uptake and elimination in the bivalves.

 

Buffet, P. E., O. F. Tankoua, et al. (2011). "Behavioural and biochemical responses of two marine invertebrates Scrobicularia plana and Hediste diversicolor to copper oxide nanoparticles." Chemosphere 84(1): 166-74.

                Engineered nano-sized Cu oxide particles are extensively used in diverse applications. Because aquatic environments are the ultimate "sink" for all contaminants, it is expected that nanoparticles (NP) will follow the same fate. In this study, two marine invertebrates Scrobicularia plana and Hediste diversicolor were chosen as ecotoxicological models. The aim was to evaluate behavioural (burrowing kinetics, feeding rate) and biochemical (biomarkers) responses of S. plana and H. diversicolor exposed in the laboratory to Cu (10 mug L(-1)) added in natural seawater either in the form of engineered nanoparticles (NPs) of CuO or as dissolved Cu in 2% HNO(3). Exposure was characterized by considering (i) the physico-chemical fate of NP (ii) the fraction of labile Cu in experimental media and (iii) Cu bioaccumulation. Results showed high aggregation of CuO NPs in seawater and no additional bioavailable Cu concentrations. Behavioural impairments were observed in S. plana exposed to CuO NPs or soluble Cu whereas in H. diversicolor, only the exposure to soluble Cu led to a burrowing decrease. No obvious neurotoxicity effects were revealed since in both species, no changes in cholinesterasic activity occurred in response to both forms of Cu exposure. Biomarkers of oxidative-stress catalase and glutathione-S-transferase were enhanced in both species whereas superoxide dismutase was increased only in S. plana exposed to CuO NPs. Metallothionein-like protein was increased in bivalves exposed to both forms of Cu. Since, no detectable release of soluble Cu from CuO NPs occurred during the time of experiment, ecotoxicity effects seem to be related to CuO NPs themselves.

 

Maria, V. L. and M. J. Bebianno (2011). "Antioxidant and lipid peroxidation responses in Mytilus galloprovincialis exposed to mixtures of benzo(a)pyrene and copper." Comp Biochem Physiol C Toxicol Pharmacol 154(1): 56-63.

                This study aimed to assess the antioxidant system potential and lipid peroxidative effects, in the gill and digestive gland of Mytilus galloprovincialis exposed to individual and binary mixtures of benzo(a)pyrene (BaP) and Cu for 7 days. Data demonstrated that in mussels exposed to BaP antioxidant enzymes (catalase--CAT, total glutathione peroxidase--tGPx, glutathione S-transferase--GST and glutathione reductase--GR) and lipid peroxidation (LPO) increased in the gill. On the contrary, in the digestive gland inhibitory antioxidant effects (superoxide dismutase-SOD, GR, metallothioneins-MT) and no changes in LPO levels were detected. Cu was also a potent oxidant agent since MT and LPO levels increased in mussel gill, despite no LPO effect in the digestive gland. For both single contaminants the organ specificity and distinct physiologic/metabolism roles were evident in terms of antioxidant capacity. Gill SOD inhibition, MT and GST unchanged was a result of "simple independent action" of exposure to BaP and Cu. "Interactions" in the binary mixtures, led to absence of changes in LPO effects. In the digestive gland, BaP and Cu interactions were also responsible for the GST and LPO enhancement (antagonistic effects). The current findings demonstrate the differences in antioxidant responses where the organ dependency highlights each contaminant particular mode of action. Generally, in the gill "non-interactive" effects occurred with the lowest Cu concentration while "interactions" exist for the mixture with the highest Cu concentrations. In the digestive gland, "interactions" and "no interaction" effects occurred in all the binary mixtures. Complex contaminant mixtures interact differently based on target tissue which may lead to an imbalance in the mussels health status.

 

Strady, E., G. Blanc, et al. (2011). "Roles of regional hydrodynamic and trophic contamination in cadmium bioaccumulation by Pacific oysters in the Marennes-Oleron Bay (France)." Chemosphere 84(1): 80-90.

                The Marennes-Oleron Bay, hosting the largest oyster production in France, is influenced by the historic polymetallic pollution of the Gironde Estuary. Despite management efforts and decreasing emissions in the Gironde watershed, cadmium levels in oysters from the bay are close to the consumption limit (5 mug g(-1) dw, EC). From mid April to mid July 2009, we investigated the role of tidal resuspension and regional hydrodynamics on Cd speciation (seawater, SPM, phytoplankton, sediment, microphytobenthos) and bioaccumulation in 18 month-old oysters (gills, digestive glands, rests of tissues) reared under natural conditions (i) at approximately 60 cm above the sediment and (ii) on the sediment surface. Dissolved and particulate Cd concentrations in surface and bottom waters were similar and constant over tidal cycle suggesting the absence of Cd release during sediment resuspension. Temporal dissolved and particulate Cd concentrations were closely related to Gironde Estuary water discharges, showing increasing concentrations during flood situations and decreasing concentrations afterwards. Cd depletion in the water column was associated with increasing Cd in the [20-100 mum] plankton fraction, suggesting Cd bioaccumulation. After 3 months, enrichment factors of Cd in tissues of oysters exposed in the water column and directly on the sediment were respectively 3.0 and 2.2 in gills, 4.7 and 3.2 in digestive glands and 4.9 and 3.4 in remaining tissues. Increasing Cd bioaccumulation in gills, digestive glands and remaining tissues can be related to elevated dissolved Cd in the bay, suggesting gill contamination via the direct pathway and subsequent internal redistribution of Cd to other organs and tissues. Elevated Cd contents in oysters reared on tables could be attributed to different trophic Cd transfer (phytoplankton versus microphytobenthos) or to different oyster metabolisms between the rearing conditions as suggested by metallothionein concentrations.

 

Farcy, E., C. Voiseux, et al. (2011). "Effect of ionizing radiation on the transcription levels of cell stress marker genes in the Pacific oyster Crassostrea gigas." Radiat Res 176(1): 38-48.

                In the North-Cotentin (Normandy, France), the marine environment is chronically exposed to liquid releases from the La Hague nuclear fuel recycling plant (Areva NC), resulting in a small increase in radioactivity compared to natural background. The transcriptional expression levels of stress genes were investigated in oysters exposed to ionizing radiation. Adult oysters were kept for 6 weeks in (60)Co-labeled seawater (400 Bq liter(-1)), resulting in a total dose of 6.2 mGy. Transcriptional expression of target genes was monitored by reverse-transcription quantitative polymerase chain reaction. Nine genes were selected for their sensitivity to ionizing radiation based on the literature and available DNA sequences. They included genes encoding chaperone proteins and genes involved in oxidative stress regulation, cell detoxification and cell cycle regulation. Of the nine genes of interest, metallothionein (MT) and multi-drug resistance (MDR) displayed significant overexpression in response to chronic exposure to an internal low dose. For comparison, oysters were acutely exposed to an external high dose for 100 min, resulting in 20 Gy, and the same target gene expression analysis was carried out. As in the case of chronic exposure to the low dose, MT and MDR displayed significant increases. The results suggest that the transcriptional expression levels of cell stress genes may be used as a biosensor of exposure of oysters to ionizing radiation, with a particular focus on the MT and MDR genes. However, the upregulation of these potential players in the cellular response to radiation-induced stress was not correlated with mortality or apparent morbidity. The possible role of these stress genes in the resistance of oysters to ionizing radiation is discussed.

 

Quinn, B., W. Schmidt, et al. (2011). "Effects of the pharmaceuticals gemfibrozil and diclofenac on biomarker expression in the zebra mussel (Dreissena polymorpha) and their comparison with standardised toxicity tests." Chemosphere 84(5): 657-63.

                Pharmaceuticals, including the lipid regulator gemfibrozil and the non-steroidal anti-inflammatory drug diclofenac have been identified in waste water treatment plant effluents and receiving waters throughout the western world. The acute and chronic toxicity of these compounds was assessed for three freshwater species (Daphnia magna, Pseudokirchneriella subcapitata, Lemna minor) using standardised toxicity tests with toxicity found in the non-environmentally relevant mid mg L(-1) concentration range. For the acute endpoints (IC(50) and EC(50)) gemfibrozil showed higher toxicity ranging from 29 to 59 mg L(-1) (diclofenac 47-67 mg L(-1)), while diclofenac was more toxic for the chronic D. magna 21 d endpoints ranging from 10 to 56 mg L(-1) (gemfibrozil 32-100 mg L(-1)). These results were compared with the expression of several biomarkers in the zebra mussel (Dreissena polymorpha) 24 and 96 h after exposure by injection to concentrations of 21 and 21,000 mug L(-1) corresponding to nominal concentrations of 1 and 1000 mug L(-1). Exposure to gemfibrozil and diclofenac at both concentrations significantly increased the level of lipid peroxidation, a biomarker of damage. At the elevated nominal concentration of 1000 mug L(-1) the biomarkers of defence glutathione transferase and metallothionein were significantly elevated for gemfibrozil and diclofenac respectively, as was DNA damage after 96 h exposure to gemfibrozil. No evidence of endocrine disruption was observed using the alkali-labile phosphate technique. Results from this suite of biomarkers indicate these compounds can cause significant stress at environmentally relevant concentrations acting primarily through oxidation pathways with significant destabilization of the lysosomal membrane and that biomarker expression is a more sensitive endpoint than standardised toxicity tests.

 

Schmidt, W., K. O'Rourke, et al. (2011). "Effects of the pharmaceuticals gemfibrozil and diclofenac on the marine mussel (Mytilus spp.) and their comparison with standardized toxicity tests." Mar Pollut Bull 62(7): 1389-95.

                Human pharmaceuticals, like the lipid lowering agent gemfibrozil and the non-steroidal anti-inflammatory drug diclofenac are causing environmental concern. In this study, the marine mussel (Mytilus spp.) was exposed by injection to environmentally relevant and elevated (1 mug/L and 1000 mug/L) concentrations of both compounds and biomarker expression was observed. Gemfibrozil exposure induced biomarkers of stress (glutathione S-transferase and metallothionein) at both concentrations 24h and 96 h after exposure, respectively. Biomarkers of damage (lipid peroxidation (LPO) and DNA damage) were significantly affected, as well as the biomarker for reproduction, alkali-labile phosphate assay, indicating the potential oxidative stress and endocrine disrupting effect of gemfibrozil. Diclofenac significantly induced LPO after 96 h indicating tissue damage. Additionally standard toxicity tests using the marine species Vibrio fischeri, Skeletonema costatum and Tisbe battagliai showed differences in sensitivity to both drugs in the mg/L range. Results indicate a suite of tests should be used to give accurate information for regulation.

 

Palacios, O., A. Pagani, et al. (2011). "Shaping mechanisms of metal specificity in a family of metazoan metallothioneins: evolutionary differentiation of mollusc metallothioneins." BMC Biol 9: 4.

                BACKGROUND: The degree of metal binding specificity in metalloproteins such as metallothioneins (MTs) can be crucial for their functional accuracy. Unlike most other animal species, pulmonate molluscs possess homometallic MT isoforms loaded with Cu(+) or Cd(2+). They have, so far, been obtained as native metal-MT complexes from snail tissues, where they are involved in the metabolism of the metal ion species bound to the respective isoform. However, it has not as yet been discerned if their specific metal occupation is the result of a rigid control of metal availability, or isoform expression programming in the hosting tissues or of structural differences of the respective peptides determining the coordinative options for the different metal ions. In this study, the Roman snail (Helix pomatia) Cu-loaded and Cd-loaded isoforms (HpCuMT and HpCdMT) were used as model molecules in order to elucidate the biochemical and evolutionary mechanisms permitting pulmonate MTs to achieve specificity for their cognate metal ion. RESULTS: HpCuMT and HpCdMT were recombinantly synthesized in the presence of Cd(2+), Zn(2+) or Cu(2+) and corresponding metal complexes analysed by electrospray mass spectrometry and circular dichroism (CD) and ultra violet-visible (UV-Vis) spectrophotometry. Both MT isoforms were only able to form unique, homometallic and stable complexes (Cd(6)-HpCdMT and Cu(12)-HpCuMT) with their cognate metal ions. Yeast complementation assays demonstrated that the two isoforms assumed metal-specific functions, in agreement with their binding preferences, in heterologous eukaryotic environments. In the snail organism, the functional metal specificity of HpCdMT and HpCuMT was contributed by metal-specific transcription programming and cell-specific expression. Sequence elucidation and phylogenetic analysis of MT isoforms from a number of snail species revealed that they possess an unspecific and two metal-specific MT isoforms, whose metal specificity was achieved exclusively by evolutionary modulation of non-cysteine amino acid positions. CONCLUSION: The Roman snail HpCdMT and HpCuMT isoforms can thus be regarded as prototypes of isoform families that evolved genuine metal-specificity within pulmonate molluscs. Diversification into these isoforms may have been initiated by gene duplication, followed by speciation and selection towards opposite needs for protecting copper-dominated metabolic pathways from nonessential cadmium. The mechanisms enabling these proteins to be metal-specific could also be relevant for other metalloproteins.

 

Perez-Rafael, S., S. Atrian, et al. (2011). "Differential ESI-MS behaviour of highly similar metallothioneins." Talanta 83(3): 1057-61.

                ESI-MS can only be accepted as a quantification method when using standards with a high resemblance to the analyte(s). Unfortunately, this is usually not applicable to metallothioneins (MTs), a superfamily of singular metal-binding cysteine-rich proteins, present in all living organisms, since the absence of suitable reference material due to the high diversity among metal-MT species precludes their quantification by molecular mass spectrometry. Even thus, it is widely assumed that the intensities of the ESI-MS peaks of similar species are directly correlated with their relative concentration in the sample, and this has been extended to the determination of different MT proteins coexisting in a sample. Practically all organisms contain several MT isoforms, some of them exhibiting highly similar sequences, with conserved coordinating Cys residues. For the current analysis, we used as a model system the MT isoforms of two terrestrial snails (Helix pomatia and Cornu aspersum). Hence, distinct samples were prepared by mixing, at different molar ratios, the recombinant HpCuMT and HpCdMT isoforms from H. pomatia, or the recombinant CaCuMT, CaCdMT and CaCdCuMT isoforms from C. aspersum, and they were analyzed by ESI-MS both at neutral pH (for Zn-loaded MT forms) and at acidic pH (for the corresponding apo-forms). The results here presented reveal that the ESI-MS peak intensity of a single MT species strongly depends on its sensitivity to be ionized, and thus, on the presence or absence of metal ions bound. Furthermore, our data demonstrate that very similar MT isoforms of the same organism with similar pI (ranging from 7.9 to 8.3) can show a clear different sensitivity to ES ionization, something that cannot be readily predicted only by consideration of their amino acid content. In conclusion, even in this optimum case, deductions about quantity features of MT samples drawn from ESI-MS measurements should be carefully considered.

 

Martins, I., R. Bettencourt, et al. (2011). "The influence of nutritional conditions on metal uptake by the mixotrophic dual symbiosis harboring vent mussel Bathymodiolus azoricus." Comp Biochem Physiol C Toxicol Pharmacol 153(1): 40-52.

                The vent mussel Bathymodiolus azoricus, host thioautotrophic and methanotrophic bacteria, in their gills and complementary, is able to digest suspended organic matter. But the involvement of nutritional status in metal uptake and storage remains unclear. The influence of B. azoricus physiological condition on its response to the exposure of a mixture of metals in solution is addressed. Mussels from the Menez Gwen field were exposed to 50 mugL(-1) Cd, plus 25 mugL(-1) Cu and 100 mugL(-1) Zn for 24 days. Four conditions were tested: (i) mussels harboring both bacteria but not feed, (ii) harboring only methanotrophic bacteria, (iii) without bacteria but fed during exposure and (iv) without bacteria during starvation. Unexposed mussels under the same conditions were used as controls. Eventual seasonal variations were assessed. Metal levels were quantified in subcellular fractions in gills and digestive gland. Metallothionein levels and condition indices were also quantified. Gill sections were used for fluorescence in situ hybridization (FISH) to assess the temporal distribution of symbiotic associations. Starvation damages metal homeostasis mechanisms and increase the intracellular Zn and MT levels function. There is a clear metallic competition for soluble and insoluble intracellular ligands at each condition. Seasonal variations were observed at metal uptake and storage.

 

Navarro, A., M. Faria, et al. (2011). "Transcriptional response of stress genes to metal exposure in zebra mussel larvae and adults." Environ Pollut 159(1): 100-107.

                Development of stress markers for the invader freshwater zebra mussel (Dreissena polymorpha) is of great interest for both conservation and biomonitoring purposes. Gene expression profiles of several putative or already established gene expression stress markers (Metallothionein, Superoxide dismutase, Catalase, Glutathione S transferase, Glutathione peroxidase, Cytochrome c oxidase, the multixenobiotic resistance P-gp1, and heat shock proteins HSP70 and HSP90) were analyzed by quantitative Real-Time PCR in adults and pediveliger larvae after exposure to metals (Hg, Cu, Cd). A defined pattern of coordinated responses to metal exposure and, presumably, to oxidative stress was observed in gills and digestive gland from adults. A similar, albeit partial response was observed in larvae, indicating an early development of stress-related gene responses in zebra mussel. The tools developed in this study may be useful both for future control strategies and for the use of zebra mussel as sentinel species in water courses with stable populations.

 

Itziou, A. and V. K. Dimitriadis (2011). "Introduction of the land snail Eobania vermiculata as a bioindicator organism of terrestrial pollution using a battery of biomarkers." Sci Total Environ 409(6): 1181-92.

                The present study aimed to enrich the group of sentinel organisms of terrestrial pollution biomonitoring, by investigating the efficacy of the land snail Eobania vermiculata. For this reason, a package of biomarkers was performed on land snails E. vermiculata collected from polluted areas in the field or treated with heavy metals in the laboratory. The biomarkers used were neutral red lysosomal retention assay of the haemocytes, acetylcholinesterase activity in the digestive gland and the haemolymph, and metallothionein content of the digestive gland. Moreover, the morphometric changes in the lysosomal system and the morphometric alterations of the neutral lipids were also investigated. In addition, the content of cadmium, lead and copper was evaluated in the digestive gland of the snails. The results revealed appreciable alterations in the biomarker values both in field- and laboratory-conditions, accompanied by significant correlations among the biomarkers. Therefore, this exploratory study suggests the utility of E. vermiculata as a sentinel organism for biomonitoring the biologic impact of terrestrial pollution, and supports the package's efficacy of the selected biomarkers.

 

Sureda, A., A. Box, et al. (2011). "Biochemical responses of Mytilus galloprovincialis as biomarkers of acute environmental pollution caused by the Don Pedro oil spill (Eivissa Island, Spain)." Aquat Toxicol 101(3-4): 540-9.

                In the present work, the potential use of several antioxidant and detoxification biomarkers in the digestive gland of wild mussels (Mytilus galloprovincialis) for biomonitoring the marine pollution induced by the Don Pedro oil spill has been investigated. Two locations from the East to South-East of Eivissa (Ibiza) and Formentera islands were selected, one extensively affected by the oil spill and the other one not affected and considered as the control area. Mussels were sampled one, two and six months after the Don Pedro accident. Polycyclic aromatic hydrocarbon (PAH) levels were significantly increased in the soft tissues of mussels in the affected area one month after the disaster, returning to normal values after six months. Markers of oxidative damage in lipids--malondialdehyde, and in proteins--carbonyl derivates, and antioxidant enzyme--catalase, superoxide dismutase and glutathione peroxidase, activities significantly increased as result of the spill oil after one month, returning to basal values at two month sampling time. Glutathione/glutathione disulfide ratio (GSH/GSSG), as a marker of the redox status, was reduced after one and two months indicating a more oxidized situation. Markers of detoxification--glutathione-S-transferase and cytochrome P4501A activities and metallothionein gene expression--were significantly increased by the oil spill one month after the accident, returning to the basal values at two month sampling time. In conclusion, the Don Pedro accident induced a transient situation of PAHs pollution resulting in enhanced antioxidant and detoxification defense systems in the wild mussel M. galloprovincialis returning to normal levels six months from the spill. The selected biomarkers are a useful tool for biomonitoring the response to acute exposure to pollutants in marine mussels.

 

Hockner, M., K. Stefanon, et al. (2011). "Physiological relevance and contribution to metal balance of specific and non-specific Metallothionein isoforms in the garden snail, Cantareus aspersus." Biometals 24(6): 1079-92.

                Variable environmental availability of metal ions represents a constant challenge for most organisms, so that during evolution, they have optimised physiological and molecular mechanisms to cope with this particular requirement. Metallothioneins (MTs) are proteins that play a major role in metal homeostasis and as a reservoir. The MT gene/protein systems of terrestrial helicid snails are an invaluable model for the study of metal-binding features and MT isoform-specific functionality of these proteins. In the present study, we characterised three paralogous MT isogenes and their expressed products in the escargot (Cantareus aspersus). The metal-dependent transcriptional activation of the three isogenes was assessed using quantitative Real Time PCR. The metal-binding capacities of the three isoforms were studied by characterising the purified native complexes. All the data were analysed in relation to the trace element status of the animals after metal feeding. Two of the three C. aspersus MT (CaMT) isoforms appeared to be metal-specific, (CaCdMT and CaCuMT, for cadmium and copper respectively). A third isoform (CaCd/CuMT) was non-specific, since it was natively recovered as a mixed Cd/Cu complex. A specific role in Cd detoxification for CaCdMT was revealed, with a 80-90% contribution to the Cd balance in snails exposed to this metal. Conclusive data were also obtained for the CaCuMT isoform, which is involved in Cu homeostasis, sharing about 30-50% of the Cu balance of C. aspersus. No apparent metal-related physiological function was found for the third isoform (CaCd/CuMT), so its contribution to the metal balance of the escargot may be, if at all, of only marginal significance, but may enclose a major interest in evolutionary studies.

 

Wang, L., L. Pan, et al. (2011). "Biomarkers and bioaccumulation of clam Ruditapes philippinarum in response to combined cadmium and benzo[alpha]pyrene exposure." Food Chem Toxicol 49(12): 3407-17.

                Biochemical and molecular biomarkers (the contents of metallothionein (MT), glutathione (GSH), the activities of aryl hydrocarbon hydroxylase (AHH), glutathione S-transferase (GST) and superoxide dismutase (SOD) and the mRNA expressions of GST-pi and Cu, Zn-SOD) were evaluated in clams Ruditapes philippinarum exposed to cadmium (Cd, 15 mug/L) and benzo[alpha]pyrene (BaP, 0.01 mug/L) individually and in combination (15 mug/L Cd+0.01 mug/L BaP) for 21 days. The accumulation of Cd, BaP and the biomarkers measured in the gills and digestive glands of the clam showed significant increase in combination treatment and it was significantly higher than the Cd or BaP treatment (P>0.05). The contents of MT increased in Cd and Cd+BaP treatment, while AHH activities were increased in Bap and Cd+BaP treatment (P>0.05). GSH levels enhanced in Cd group and declined significantly in Cd+BaP treatment (P>0.05). The activities of GST, SOD, and mRNA expressions of GST-pi, Cu, Zn-SOD increased remarkably in the clams exposed to combined pollutants. In this study, a significant interaction was observed for Cd and BaP accumulation in the clam and the current findings demonstrate the differences in antioxidant response of the biomarkers in clam to single contaminant and the mixtures.

 

Baraj, B., F. Niencheski, et al. (2011). "Assessing the effects of Cu, Cd, and exposure period on metallothionein production in gills of the Brazilian brown mussel Perna perna by using factorial design." Environ Monit Assess 179(1-4): 155-62.

                Factorial design plan was experimented in Perna perna in order to find out the contribution of day exposure, Cd and Cu concentrations in water, and their interactions on metallothioneins (MT), Cd, and Cu (the dependent measured variables) in the gills. The picture obtained is more adequate than by studying the factor effect separately. Compared with the control group, the MT concentration after 22 days exposure period in the mixture of 100 mug/mL Cu and Cd is increased almost two times, showing that P. perna might be used as a biomonitor. Cd showed stronger effect than Cu on MT inducing.

 

Wang, W. X., Y. Yang, et al. (2011). "Copper and zinc contamination in oysters: subcellular distribution and detoxification." Environ Toxicol Chem 30(8): 1767-74.

                Metal pollution levels in estuarine and coastal environments have been widely reported, but few documented reports exist of severe contamination in specific environments. Here, we report on a metal-contaminated estuary in Fujian Province, China, in which blue oysters (Crassostrea hongkongensis) and green oysters (Crassostrea angulata) were discovered to be contaminated with Cu and other metals. Extraordinarily high metal concentrations were found in the oysters collected from the estuary. Comparison with historical data suggests that the estuary has recently been contaminated with Cr, Cu, Ni, and Zn. Metal concentrations in blue oysters were as high as 1.4 and 2.4% of whole-body tissue dry wt for Cu and Zn, respectively. Cellular debris was the main subcellular fraction binding the metals, but metal-rich granules were important for Cr, Ni, and Pb. With increasing Cu accumulation, its partitioning into the cytosolic proteins decreased. In contrast, metallothionein-like proteins increased their importance in binding with Zn as tissue concentrations of Zn increased. In the most severely contaminated oysters, only a negligible fraction of their Cu and Zn was bound with the metal-sensitive fraction, which may explain the survival of oysters in such contaminated environments.

 

Won, E. J., S. Hong, et al. (2011). "Evaluation of the potential impact of polluted sediments using Manila clam Ruditapes philippinarum: bioaccumulation and biomarker responses." Environ Sci Pollut Res Int 19(7): 2570-80.

                An assessment was made to monitor the short-term impact of heavily polluted sediments that may move out from the brackish man-made Lake Shihwa outside of the sea dike due to operations of a tidal power plant. Here, we exposed the Manila clam Ruditapes philippinarum collected from the western coast of Korea to natural sediment under lab condition for 96 h. Sediments were collected from Lake Shihwa and outside of the sea dike representing polluted and reference conditions, respectively. The results of chemical analysis revealed that the concentrations of nonylphenol and heavy metals in water and sediment from the inner region of Lake Shihwa were significantly higher than those of reference sediments. After 48 and 96 h of exposure, 30 specimens of clams were sampled from each experimental condition, and concentrations of nonylphenol and metals were measured in clams, water, and sediments. Several biomarkers, including concentrations of metallothionein-like proteins, and activities of the antioxidant enzymes glutathione S-transferase and catalase were determined in clams to characterize the effects of polluted sediments to clams. After 96 h of exposure, R. philippinarum assimilated nonylphenol up to 71 times compared to initial concentrations. However, there was no apparent uptake of heavy metals into the clams. Additionally, antioxidant enzymes exhibited higher activities in clams exposed to the polluted sediment. The results of the present study with physiological responses in R. philippinarum suggest that sediment transportation caused by the operation of a tidal power plant in Lake Shihwa will have striking effects on benthic organisms in the adjacent coastal area.

 

Da Ros, L., V. Moschino, et al. (2011). "An ecotoxicological approach for the Boka Kotorska Bay (south-eastern Adriatic Sea): first evaluation of lysosomal responses and metallothionein induction in mussels." Mar Pollut Bull 63(5-12): 326-33.

                Mytilus galloprovincialis was used as a biomonitoring organism in Boka Kotorska Bay, a coastal transitional ecosystem in south-western Montenegro. Native mussels were collected in June 2008 at four sites thought to be differently impacted. Biological effects were investigated analysing both generic and specific biomarkers at cellular level (metallothionein content, lysosomal membrane stability, lipofuscin and neutral lipid accumulation, lysosomal structural changes). Trace element levels in mussels were quite low, only Cu and Zn exhibiting slight increases in the two sampling sites of Tivat Bay, the first one in front of a small working dockyard and the other in the water area of a former Naval dockyard. Mussels collected from these sites exhibited the highest values in neutral lipid and lysosomal volume density and the lowest neutral red retention times. Metallothionein content was always low, reflecting the minor body burden of the trace elements and suggesting a non-relevant environmental induction.

 

Serafim, A. and M. J. Bebianno (2010). "Effect of a polymetallic mixture on metal accumulation and metallothionein response in the clam Ruditapes decussatus." Aquat Toxicol 99(3): 370-8.

                The toxic effects of metals are related to changes in natural physiological and biochemical processes in organisms, which are not easily detected. Marine invertebrates developed strategies to survive in the presence of toxic metal levels, by the removal of these compounds from the soluble fraction of the cell through different processes, including the synthesis of metallothionein (MT). To better understand the effect of Cd, Cu and Zn in MT response it is important to consider that in their natural environment these metals are normally mixed. The exposure to a metal mixture result in toxicological interactions that produce different biological responses when compared to the effects of a single metal exposure. The aim of this study was to assess metal accumulation and MT response in different tissues of the clam Ruditapes decussatus exposed to a mixture of sublethal Cd, Cu and Zn concentrations. Kinetic models were applied to determine metal uptake and loss to better predict and understand the detoxification mechanisms in this species. The kinetic model showed that the effect of this polymetallic exposure compared to a single exposure to these metals caused different metal uptake and loss rates in several tissues. Moreover, the exposure of R. decussatus to a polymetallic mixture caused an increase in MT induction compared to a single metal exposure. This is probably related to the interaction between the different metals and their different affinity to this protein. Metal interactions within aquatic organisms are very important in MT synthesis and the effect of polymetallic mixtures in the environment must be taken into account in field studies.

 

Caricato, R., M. G. Lionetto, et al. (2010). "Carbonic anhydrase activity in Mytilus galloprovincialis digestive gland: sensitivity to heavy metal exposure." Comp Biochem Physiol C Toxicol Pharmacol 152(3): 241-7.

                Heavy metals are known to in vitro inhibit carbonic anhydrase (CA) activity in a variety of organisms; however, little is known about their in vivo effects on the activity and the expression of this metalloenzyme. The aim of this work was to investigate the in vitro and in vivo sensitivity to cadmium of CA in the digestive gland of Mytilus galloprovincialis. CA activity and protein expression (apparent molecular mass of about 28 kDa) were demonstrated in mussel digestive gland for the first time. CA activity showed week sensitivity to in vitro cadmium exposure, while it was significantly increased (about 40%) following two weeks in vivo exposure. In parallel, CA protein expression appeared significantly enhanced as demonstrated by Western blotting. Laboratory experimental results were confirmed by a field experiment. Mussels exposed for 30 days to an impacted site showed a significant increase of the CA activity and protein expression with respect to animals exposed to the control site in parallel to the increase of the metallothionein tissutal concentration. In conclusion in the present work for the first time CA activity and protein expression have been demonstrated to be enhanced by the exposure to the trace element cadmium in animals.

 

Huang, X., F. Guo, et al. (2010). "Responses of abalone Haliotis diversicolor to sublethal exposure of waterborne and dietary silver and cadmium." Ecotoxicol Environ Saf 73(6): 1130-7.

                In this study, we examined the chronic waterborne and dietary exposure of silver (Ag) and cadmium (Cd) to the abalone Haliotis diversicolor using various endpoints such as growth and feeding rates, metal body burden, subcellular distribution, and metallothionein (MT) concentration over a period of 7 weeks of exposure. The growth and feeding rates of abalones were inhibited during the early stage of exposure to different extents, but then recovered to nearly the control levels. A large portion of Ag was redistributed to organelles and metal-rich granules from the cellular debris fraction, whereas cellular debris and metallothionein-like protein were the dominant pools for the storage of Cd, which remained comparable during the exposure period. The MT concentrations were significantly elevated (in a dose-dependent manner) within the first 2 weeks of exposure, after which the MT concentrations started to decrease. All these results implied that abalones respond rapidly to metal exposure, but apparently developed subsequent acclimation.

 

Guidi, P., G. Frenzilli, et al. (2010). "Antioxidant, genotoxic and lysosomal biomarkers in the freshwater bivalve (Unio pictorum) transplanted in a metal polluted river basin." Aquat Toxicol 100(1): 75-83.

                The freshwater painter's mussel (Unio pictorum) was used as sentinel species to assess the chemical disturbance in an Italian river (the river Cecina) characterized by elevated levels of trace metals of both natural and anthropogenic origin. Organisms were transplanted for 4 weeks in different locations of the river basin and the bioaccumulation of metals was integrated with a wide battery of biomarkers consisting of oxidative, genotoxic and lysosomal responses. Such parameters included the levels of individual antioxidants (catalase, glutathione-S-transferases, glutathione reductase, Se-dependent and Se-independent glutathione peroxidases, total glutathione), the total oxyradical scavenging capacity (TOSC), metallothionein-like proteins, the assessment of DNA integrity, chromosomal damages and lysosomal membrane stability. Elevated levels of several metals were measured in sediments, but the relatively low tissue concentrations suggested a moderate bioaccumulation, possibly due to a high excretion efficiency, of U. pictorum and/or to a limited bioavailability of these elements, partly deriving from erosion of bedrocks. Among antioxidant responses, those based on glutathione metabolism and the activity of catalase were mostly affected in bivalves showing a significant accumulation of arsenic, mercury and/or nickel. In these specimens, the content of glutathione and the activities of glutathione reductase and glutathione peroxidases (H2O2) were respectively 9-, 6- and 4-fold lower than in controls, while a 3-fold increase was observed for catalase. Despite some differences in the response of individual antioxidants, a significant reduction of the capability to neutralize peroxyl radicals was observed in bivalves caged in all the impacted sites of the river basin; these organisms also exhibited a significant impairment at the DNA, chromosomal and lysosomal levels. Considering the mild contamination gradient in the investigated area, the overall results suggested that some oxidative biomarkers, as well as those evaluating chromosomal and cell damages, are highly sensitive and could be profitably applied to caged painter's mussels for environmental quality assessment in freshwater.

 

Attig, H., A. Dagnino, et al. (2010). "Uptake and biochemical responses of mussels Mytilus galloprovincialis exposed to sublethal nickel concentrations." Ecotoxicol Environ Saf 73(7): 1712-9.

                In the present study, mussel (Mytilus galloprovincialis) digestive gland oxidative stress biomarkers and detoxification responses to acute exposure to nickel (Ni) were investigated. Mussels were exposed to two sublethal concentrations of Ni (135 mug/L per animal (2.5 muM) and 770 mug/L per animal (13 muM)) for 24, 48, 72, 96 h and 8 days. Following biological responses were measured: (1) glutathione S-transferase (GST) activity as a phase II conjugation enzyme, (2) catalase activity as antioxidant response, (3) malondialdehyde accumulation (MDA) as lipid peroxydation marker and metallothionein as specific response to metals exposure. The cholinergic system was evaluated using the acetylcholinesterase activity (AChE). Moreover, Ni uptakes during the exposure periods were assessed and the uptake rate constant determined. A correlation matrix (CM) between the investigated biomarkers and a principal component analysis (PCA) were achieved for the two tested concentrations. The Ni-uptake constant was higher in animals exposed to the lowest concentration. The CM and the PCA showed a time-dependent effect of the Ni exposure on the investigated biomarkers being more pronounced in animals exposed to the highest Ni concentration. While AChE showed a significant increase after 48 h and a further return to control values in the lowest concentration, it was drastically maintained inhibited in the highest concentration. Our data provided clues about the occurrence of different toxicokinetics and toxicodynamics of two Ni sublethal concentrations in an ecologically relevant organism.

 

Boldina-Cosqueric, I., J. C. Amiard, et al. (2010). "Biochemical, physiological and behavioural markers in the endobenthic bivalve Scrobicularia plana as tools for the assessment of estuarine sediment quality." Ecotoxicol Environ Saf 73(7): 1733-41.

                The aim of this study was to link the responses at different levels of biological organisation of the endobenthic bivalve Scrobicularia plana differentially exposed to anthropogenic pressure. Clams were collected in April 2008 from three estuaries along a pollution gradient (Goyen < Loire < Seine). Biomarkers of defence (metallothionein concentration and glutathione-S-transferase activity) were activated in the Loire and the Seine. Biomarkers of damage revealed neurotoxicity (decreased AChE activity) and impairment of digestive enzyme activities (cellulase or amylase) in these estuaries. The highest lactate dehydrogenase activity was registered in the Loire estuary, in parallel with enhanced levels of vanadium (a metal present in petroleum), likely as a consequence of a small oil spill that occurred one month before the sampling collection. Physiological biomarkers (energy reserves as glycogen, lipids and proteins, condition and gonado-somatic indices) showed a few intersite differences. However, the median size was significantly lower in clams exposed to direct (chemicals) or indirect (available food) effects in the most contaminated site. Burrowing behaviour was disturbed in clams from both of the Loire and Seine estuaries, a response probably due to physiological impairment rather than to avoidance of contaminated sediment. The activation of defence mechanisms towards metals (metallothionein) and other classes of contaminants (the biotransformation enzyme glutathione-S-transferase) do not ensure a total protection since a number of impairments were observed at the infra-organismal (AChE and digestive enzyme activities) and individual (burrowing behaviour) levels in relation to the degree of anthropogenic pressure. However, even in the most contaminated estuary (Seine), historical records do not show a consistent decrease of S. plana populations.

 

Wang, Q., X. Wang, et al. (2010). "Analysis of metallotionein expression and antioxidant enzyme activities in Meretrix meretrix larvae under sublethal cadmium exposure." Aquat Toxicol 100(4): 321-8.

                To investigate the possible role of metallothioneins (MTs) and antioxidant enzymes in cadmium (Cd) tolerance in Meretrix meretrix larvae, a new MT (designated MmMT) gene was identified and cloned from M. meretrix. The full-length cDNA of MmMT consisted of an open reading frame (ORF) of 231bp encoding a protein of 76 amino acids, with 21 cysteine residues and a conserved structural pattern Cys-x-Cys-x(3)-Cys-Tyr-Gly-x(3)-Cys-x-Cys-x(3)-Cys-x-Cys-Lys at the C-terminus. The deduced amino acid sequence of MmMT showed about 57-84% identity with previously published MT sequences of mussels and oysters. Real-time PCR was used to analyze the expression level of MmMT mRNA at different M. meretrix larval stages under Cd exposure (25mugL(-)(1)). Results showed that Cd could induce the expression of MmMT mRNA in the larvae, and the expression level increased 5.04-fold and 3.99-fold in D-shaped larvae and pediveligers, respectively. Immunolocalization of MmMT in the stressed larvae revealed that MmMT was synthesized in almost all of the soft parts at the trochophore and postlarva stages, whereas it was only synthesized in the velum and epidermis at the D-shaped larva and pediveliger stages. The activities of three antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx), also were measured in larvae at different developmental stages. Increased enzymatic activities were detected mainly in D-shaped larvae and pediveligers under Cd stress, suggesting that these enzymes respond synchronously with MT. Our results indicate that MmMT and antioxidant enzymes played important roles in counteracting Cd stress in M. meretrix larvae.

 

Falfushynska, H. I., L. L. Gnatyshyna, et al. (2010). "Vulnerability of biomarkers in the indigenous mollusk Anodonta cygnea to spontaneous pollution in a transition country." Chemosphere 81(10): 1342-51.

                The aim of this study was to estimate the sensitivity of biomarkers of stress and exposure in the bivalve mollusk Anodonta cygnea to spontaneous anthropogenic activities typical for the Western Ukraine. Three sites were examined during spring, summer and autumn: an agricultural site (A); the cooling pond of nuclear power plant (N) and a forestry close to the municipal water inlet (F). Common temporal changes of a battery of biochemical markers in the gills and hemolymph and morphological characteristics were shown by discriminant functional analysis. Classification trees built on the basis of the screened biomarkers demonstrated persistent peculiarities at each site: genotoxicity (nuclear abnormalities) at site A and endocrine disruption (high levels of vitellogenin-like proteins (Vtg-LP) in hemolymph) at site F. Interim local effects were best characterized by metallothionein (MT) concentrations, lipid peroxidation (LPO), activities of glutathione S-transferase and lactate dehydrogenase, and the conditional index of the gills. In autumn, the mollusks from the three sites revealed the highest differences in pollution status: the activation of antioxidant defense and cholinesterase were typical for site A, highest levels of MT related to high levels of Cu and Cd in the water at site B, and a steep increase in the level of Vtg-LP and the decrease of lysosomal membrane stability were recorded at the site selected as reference (F). The biomarker alterations recorded at site F were later related to an emergency event at the municipal dump located nearby. Thus, our case study demonstrated the reliability of using biomarkers of exposure to assess both long-term and accidental environmental pollution loads.

 

Faria, M., M. A. Lopez, et al. (2010). "Are native naiads more tolerant to pollution than exotic freshwater bivalve species? An hypothesis tested using physiological responses of three species transplanted to mercury contaminated sites in the Ebro River (NE, Spain)." Chemosphere 81(10): 1218-26.

                In the lower Ebro River exist the paradoxical convergence of relatively well preserved river dynamics with the historical presence of a chloralkali plant with a long history of mercury discharges and the recent invasion of foreign bivalves species. Here we performed a comparative study on two alien bivalves, the Zebra mussel and the Asian clam (Dreissena polymorpha and Corbicula fluminea), and one protected species of naiads (Psilunio littoralis), which is the most common species of the freshwater mussel assemblages in this river. Individuals of the three species were transplanted to three sites that included a clean unpolluted upstream site, a contaminated location next to the mercury source and a downstream one. The study focused on digestive gland antioxidant and oxidative stress responses such as antioxidant enzymes, glutathione S transferase, glutathione levels, metallothionein proteins, DNA strand breaks and lipid peroxidation levels. Results evidenced interspecies differences on accumulation levels of mercury, antioxidant defensive systems and oxidative tissue damage. The naiad species, despite of accumulating more mercury showed the greatest antioxidant defensive potential, which was characterized by having high constitutive activities of glutathione S transferase and inducible activities and levels of key antioxidant enzymes and glutathione. Exposed individuals of C. fluminea had moderate levels of metal accumulation, the highest activities of antioxidant enzymes but also high levels of lipid peroxidation. D. polymorpha mussels showed the lowest levels of mercury but the lowest antioxidant responses and consequently the highest levels of oxidative injuries in the DNA and of mortality. Our results support the hypothesis that naiad species might be more tolerant to pollution than exotic species.

 

Lobo, J., P. M. Costa, et al. (2010). "Evaluation of the potential of the common cockle (Cerastoderma edule L.) for the ecological risk assessment of estuarine sediments: bioaccumulation and biomarkers." Ecotoxicology 19(8): 1496-512.

                Common cockles (Cerastoderma edule, L. 1758, Bivalvia: Cardiidae) were subjected to a laboratory assay with sediments collected from distinct sites of the Sado Estuary (Portugal). Cockles were obtained from a mariculture site of the Sado Estuary and exposed through 28-day, semi-static, assays to sediments collected from three sites of the estuary. Sediments from these sites revealed different physico-chemical properties and levels of metals and organic contaminants, ranging from unimpacted (the reference site) to moderately impacted, when compared to available sediment quality guidelines. Cockles were surveyed for bioaccumulation of trace elements (Ni, Cu, Zn, As, Cd and Pb) and organic contaminants (PAHs, PCBs and DDTs). Two sets of potential biomarkers were employed to assess toxicity: whole-body metallothionein (MT) induction and digestive gland histopathology. The bioaccumulation factor and the biota-to-soil accumulation factor were estimated as ecological indices of exposure to metals and organic compounds. From the results it is inferred that C. edule responds to sediment-bound contamination and might, therefore, be suitable for biomonitoring. The species was found capable to regulate and eliminate both types of contaminants. Still, the sediment contamination levels do not account for all the variation in bioaccumulation and MT levels, which may result from the moderate metal concentrations found in sediments, the species' intrinsic resistance to pollution and from yet unexplained xenobiotic interaction effects.

 

Paul-Pont, I., X. de Montaudouin, et al. (2010). "Interactive effects of metal contamination and pathogenic organisms on the introduced marine bivalve Ruditapes philippinarum in European populations." Environ Pollut 158(11): 3401-10.

                In natural environment, marine organisms are concomitantly exposed to pollutants and multiple disease agents resulting in detrimental interactions. The present study evaluated interactive effects of metal contamination (cadmium) and pathogenic organisms (trematode parasites Himasthla elongata and pathogenic bacteria Vibrio tapetis) singularly and in combination on the bivalve Ruditapes philippinarum, an introduced species to Europe, under laboratory controlled conditions. After 7 days, metal bioaccumulation and pathogen load were analyzed as well as metallothionein (MT) response and hemocyte concentrations and activities. Results showed that infection by opportunistic pathogens affects metal accumulation, leading to maximal Cd accumulation in co-infected clams. Among stressors only V. tapetis induced significant effects on immune parameters whereas a particular interaction "trematode-bacteria" was shown on MT responses. Despite low trematode infection in agreement with the resistant status of R. philippinarum to these macroparasites, significant interaction with bacteria and metal occurred. Such results highlight the necessity of taking pathogens into account in ecotoxicological studies.

 

Paul-Pont, I., P. Gonzalez, et al. (2010). "Short-term metallothionein inductions in the edible cockle Cerastoderma edule after cadmium or mercury exposure: discrepancy between mRNA and protein responses." Aquat Toxicol 97(3): 260-7.

                Metallothioneins (MT) are essential metal binding proteins involved in metal homeostasis and detoxification in living organisms. Numerous studies have focused on MT response to metal exposure and showed an important variability according to species, metal, concentration and time of exposure. In this study, the expression of one isoform of MT gene (Cemt1) and associated MT protein synthesis were determined after 1, 3, 9, 24, 72 and 168h of cadmium (Cd) or mercury (Hg) exposures in gills of the cockle Cerastoderma edule. This experiment, carried out in laboratory conditions, revealed that in Cd-exposed cockles, induction of Cemt1 is time-dependent following a "pulse-scheme" with significant upregulation at 24h and 168h intersected by time point (72h) with significant downregulation. MT protein concentration increases with time in gills of exposed cockles in relation with the progressive accumulation of Cd in soluble fraction. On contrary, Hg exposure does not lead to any induction of Cemt1 mRNA expression or MT protein synthesis compared to control, despite a higher accumulation of this metal in gills of cockles compared to Cd. The localization of Hg (85-90%) is in insoluble fraction, whereas MT was located in the cytoplasm of cells. This gives us a first clue to understand the inability of Hg to activate MT synthesis. However, other biochemical processes probably occur in gills of C. edule since the remaining soluble fraction of Hg exceeds MT sequestration ability. Finally, since one of the first main targets of metal toxicity in cells was the mitochondria, some genes involved in mitochondria metabolism were also analyzed in order to assess potential differences in cellular damages between two metal exposures. Indeed, until T(168), no impact on mitochondrial genes was shown following Hg exposure, despite the complete lack of MT response. This result indicated the presence of other effective cellular ligands which sequester the cytosolic fraction of this metal and consequently inhibit metal reactivity. Such competition mechanisms with other cytosolic ligands more sensitive to Hg were particularly argued in the discussion.

 

Dragun, Z., M. Erk, et al. (2010). "Assessment of low-level metal contamination using the Mediterranean mussel gills as the indicator tissue." Environ Sci Pollut Res Int 17(4): 977-86.

                PURPOSE: The aim of this study was to compare the level of metal contamination in two bays in the middle part of the Eastern Adriatic coastal zone in Croatia using the gills of mussels Mytilus galloprovincialis as indicator tissue. Despite the existing sources of contamination, previous studies with caged mussels only indicated moderate metal contamination of the Kastela Bay, contrary to the Trogir Bay in which marina and shipyard present a probable source of Cu- and Zn-contamination. METHODS: The measurements of metallothioneins (MTs) and metals that induce MT synthesis (Cu, Zn, and Cd) were performed in the heat-treated gill cytosol and total proteins (TPs) in the untreated gill cytosol. MTs were determined by differential pulse voltammetry, Cu and Zn by flame atomic absorption spectrometry (AAS), Cd by graphite furnace AAS, and TPs by Bradford spectrophotometric procedure. RESULTS: The results collected in four sampling campaigns (autumn periods from 2001 to 2004) indicated that MT levels in mussel gills (expressed on dry mass basis 2.3+/-0.3 mg g(-1)) were comparable with basal levels reported in the literature (2.5+/-0.8 mg g(-1)). Observed interindividual, temporal, and spatial MT variability could be associated with different confounding factors, such as the time of sampling, total protein concentration, and mussel size rather than cytosolic levels of Cu and Zn. Metal levels, expressed on wet mass basis, in the heat-treated gill cytosol ranged from 1.33 to 11.31 microg g(-1) for Zn, from 0.72 to 2.96 microg g(-1) for Cu, and from 0.036 to 0.100 microg g(-1) for Cd. The highest Zn level was measured at Vranjic (Kastela Bay)-the site influenced by untreated domestic wastewater, while somewhat increased Zn and the highest Cu levels were found at marina and shipyard locations (Trogir Bay). The highest Cd level was measured at Inavinil (Kastela Bay). CONCLUSIONS: The observed association of gill MT levels with several biotic and abiotic factors limits its use as the biomarker of low-level metal exposure. Therefore, the use of the metal concentrations in the heat-treated gill cytosol of Mediterranean mussels should be considered for the assessment of the low-level metal contamination of coastal marine areas.

 

Paul-Pont, I., X. de Montaudouin, et al. (2010). "How life history contributes to stress response in the Manila clam Ruditapes philippinarum." Environ Sci Pollut Res Int 17(4): 987-98.

                INTRODUCTION: Within the last decade, numerous studies have investigated the role of environmental history on tolerance to stress of many organisms. This study aims to assess if Manila clams Ruditapes philippinarum may react differently to cadmium exposure and trematode parasite infection (Himasthla elongata) depending on their origin and environmental history in Arcachon Bay (France). MATERIALS AND METHODS: Clams were exposed to Cd (15 microg L(-1)) and parasites (25 cercariae per clam), alone or in combination, at 15 degrees C under controlled laboratory conditions for 7 days. Metal accumulation and success of parasite infestation were examined, also physiological parameters such as metallothionein response and hemocyte counts and activities (phagocytosis, oxidative burst, viability, and adhesion). RESULTS AND DISCUSSION: Sensitivity of Manila clams to both stressors differed from one site to another, suggesting local adaptation of populations. Clams from the more parasitized site presented better resistance to trematodes than the others in terms of first line defense, i.e., avoidance of infection. On the other hand, clams that adapted to chronic Cd contamination showed better detoxification mechanisms, both in a faster transfer of metal from gills to visceral mass and in a higher metallothionein baseline, than clams which had never experienced Cd contamination. Finally, hemocyte concentration and viability differed between clam origin site, highlighting the fact that populations living in different environments may adapt their physiological and biochemical responses to environmental stressors. CONCLUSION: It is therefore important to be cautious when extrapolating results from field studies of one species and one site, if the life history of the organisms is not taken into account.

 

Van den Broeck, H., H. De Wolf, et al. (2010). "Effect of metal accumulation on metallothionein level and condition of the periwinkle Littorina littorea along the Scheldt estuary (the Netherlands)." Environ Pollut 158(5): 1791-9.

                Metal (i.e. Ag, As, Ca, Cd, Co, Cu, Mn, Pb and Zn) and metallothionein (MT) concentrations in the soft tissue of Littorina littorea were measured along the heavily polluted Western Scheldt (WS) and relatively clean Eastern Scheldt (ES) estuary. Along the WS metal and MT levels in periwinkles reflected the known downstream decreasing pollution gradient. Surprisingly in ES animals As, Mn and Zn concentrations decreased from east to west reflecting past pollution. Compared to the WS metal concentrations of ES periwinkles were significantly lower and both estuaries were maximally discriminated from each other based on their Cd soft tissue concentration using a canonical discriminant analysis. Furthermore, no overall difference was found in MT levels among animals from both estuaries. Using previously obtained condition data (i.e. dry/wet weight ratio and lipid content) the relation between soft tissue metal concentration (i.e. Cd, Cu and Zn) and fitness indicators (i.e. MT and condition data) was examined using a canonical correlation analysis. Periwinkles with a high metal load (i.e. Cd and Zn) also had high MT levels but were in a relatively poor condition.

 

Contardo-Jara, V., S. Pflugmacher, et al. (2010). "The beta-receptor blocker metoprolol alters detoxification processes in the non-target organism Dreissena polymorpha." Environ Pollut 158(6): 2059-66.

                Due to increasing amounts of pharmaceutically active compounds (PhACs) in the aquatic environment, their largely unknown effects to non-target organisms need to be assessed. This study examined physiological changes in the freshwater mussel Dreissena polymorpha exposed to increasing concentrations (0.534, 5.34, 53.4 and 534 microg L(-1)) of the beta-blocker metoprolol in a flow-through system for seven days. The two lower concentrations represent the environmentally relevant range. Surprisingly, metallothionein mRNA was immediately up-regulated in all treatments. For the two higher concentrations mRNA up-regulation in gills was found for P-glycoprotein after one day, and after four days for pi class glutathione S-transferase, demonstrating elimination and biotransformation processes, respectively. Additionally, catalase and superoxide dismutase were up-regulated in the digestive gland indicating oxidative stress. In all treated mussels a significant up-regulation of heat shock protein mRNA was observed in gills after four days, which suggests protein damage and the requirement for repair processes. Metoprolol was 20-fold bioaccumulated for environmentally relevant concentrations.

 

Franzellitti, S., S. Buratti, et al. (2010). "Exposure of mussels to a polluted environment: insights into the stress syndrome development." Comp Biochem Physiol C Toxicol Pharmacol 152(1): 24-33.

                Coastal environments are often subjected to contamination, whose biological impact is profitably evaluated through sentinel organisms and biomarkers. mRNA profiling was also proposed as a potential biomarker, whose relevance is still under discussion. Indeed, correlation between molecular and cell-organism responses need further investigations, especially under field conditions. In this study, we followed the development of physiological alterations in Mytilus galloprovincialis transplanted into a polluted coastal lagoon for 2, 4, 7, 14 and 30 days. Three consolidated biomarkers were measured, i.e. lysosomal membrane stability, lipofuscin and metallothionein contents. In parallel, the expressions of stress-related genes encoding metallothioneins (mt10 and mt20), 70-kDa heat shock proteins (MgHSC70 and MgHSP70), and Multi Xenobiotic Resistance-related transporters (MgPgp, MgMrp2, and MgMvp) were analyzed, to have a greater insight into the time-related evolution of the response. Significant (p<0.05) biomarker responses were induced after 7 days of exposure and further increased with time, whereas gene expression profiles were dramatically altered 2 days after transplanting. Biomarkers and gene expression profiles indicated a stress syndrome development in mussels, although with different temporal patterns. Their combined application provided insights into the molecular and cellular basis of mussel responses to challenging environments, and may have far-reaching implications for monitoring environmental health.

 

Company, R., A. Serafim, et al. (2010). "Sub-lethal effects of cadmium on the antioxidant defence system of the hydrothermal vent mussel Bathymodiolus azoricus." Ecotoxicol Environ Saf 73(5): 788-95.

                The mussel Bathymodiolus azoricus is one of the most abundant species in the Mid-Atlantic Ridge hydrothermal vents and is continually exposed to the high-temperature venting fluids containing high metal concentrations and enriched in sulphides and methane, which constitute a potential toxic environment for marine species. The aim of this study was to assess the effects of a sub-lethal Cd concentration on the antioxidant defence system of this mussel. B. azoricus were collected at Menez Gwen vent site (37 degrees 51'N, 32 degrees 31'W) and exposed to Cd (50 microg l(-1)) during 24 days, followed by a depuration period of six days. A battery of stress related biomarkers including antioxidant enzymes (superoxide dismutase-SOD, catalase-CAT; glutathione peroxidases-GPx), metallothioneins (MT), lipid peroxidation (LPO) and total oxyradical scavenging capacity (TOSC) were measured in the gills and mantle of B. azoricus. Cd was accumulated linearly during the exposure period in both tissues and no significant elimination occurred after the 6 days of depuration. Antioxidant enzymes activities were significantly higher in the gills. Cyt-SOD, T-GPx and Se-GPx were induced during the experiment but this was also observed in control organisms. Mit-SOD and CAT activities remained relatively unchanged. MT levels increased linearly in the gills of exposed mussels in the first 18 days of exposure. No significant differences were observed between LPO levels of control and exposed mussels. TOSC levels remained unchanged in control and exposed mussels. This suggests that although Cd is being accumulated in the tissues of exposed mussels, MT defence system is enough to detoxify the effect of Cd accumulated in the tissues. Furthermore, other factors besides the presence of Cd are influencing the antioxidant defence system in B. azoricus.

 

Hodl, E., E. Felder, et al. (2010). "Cadmium stress stimulates tissue turnover in Helix pomatia: increasing cell proliferation from metal tolerance to exhaustion in molluscan midgut gland." Cell Tissue Res 341(1): 159-71.

                In terrestrial pulmonate snails, cadmium (Cd) uptake leads to the induction of a Cd-specific metallothionein isoform (Cd-MT) that protects against adverse interactions of this toxic metal ion. Increasing concentrations of Cd cause increased individual mortality possibly linked to pathological alterations in the snail midgut gland. Histological, immuno-histochemical, and electron-microscopic methods in combination with tissue metal analyses and quantification of MT induction parameters were applied to the midgut gland of Cd-exposed Roman snails (Helix pomatia). Conspicuous concentration-dependent alterations occurred in this organ, including the metal-induced increase of Cd-MT concentration and manifestation of Cd-MT mRNA precipitations in all midgut gland cell types. The most evident alteration was an increase of cellular turnover reflected by enhanced cell proliferation. Intensified vesiculation of endoplasmic reticulum was noted in basophilic cells and an increasing formation of lipofuscin granules in excretory cells. At the highest Cd concentrations, mitochondrial membranes were disrupted in basophilic cells, and lipofuscin granules were released from excretory cells into the midgut gland tubular system. Some of these alterations (e.g., increased cell proliferation rate, vesiculation of endoplasmic reticulum) detected at low Cd concentrations were interpreted as adaptive response processes enhancing the tolerance of exposed individuals to metal stress. Cellular alterations at higher Cd concentrations (e.g., mitochondrial structural damage) clearly represented ongoing irreversible cellular disruption. Combined evaluation of cellular biomarkers and MT saturation levels indicated that the transition from stress resistance to depletion of resistance capacity occurred above a threshold of 0.8 micromol Cd/g dry weight in the midgut gland of H. pomatia. At these Cd concentrations, Cd-MT was saturated with Cd(2+) ions, whereas at the cellular level, structural alterations turned into pathological deterioration.

 

Urena, R., M. Joao Bebianno, et al. (2010). "Metallothionein in the freshwater gastropod Melanopsis dufouri chronically exposed to cadmium: a methodological approach." Ecotoxicol Environ Saf 73(5): 779-87.

                Previous studies have demonstrated that the use of differential pulse polarography (DPP) for metallothionein (MT) determination in marine gastropod tissues, particularly the digestive gland, requires taking into account the presence of heat-stable high molecular weight compounds that exhibit polarographic signal. In the present paper, similar compounds were identified in tissues from the freshwater snail Melanopsis dufouri which also interfere with MT determination by DPP and, due to their silver binding capacity, also interfere in the silver assay for MT quantification. Ultrafiltration seems to be effective in removing these high molecular weight compounds from heat-denatured homogenate supernatant allowing direct MT quantification by DPP. A fully validated procedure for metallothionein determination in M. dufouri is described. In spite of a considerable accumulation of cadmium in the visceral complex of M. dufouri following exposure to 100 microg CdL(-1) for 8 weeks (up to 37 microgg(-1)) only a small increase in MT concentration was found.

 

Gourgou, E., I. K. Aggeli, et al. (2010). "Hyperthermia-induced Hsp70 and MT20 transcriptional upregulation are mediated by p38-MAPK and JNKs in Mytilus galloprovincialis (Lamarck); a pro-survival response." J Exp Biol 213(2): 347-57.

                In the present study we investigated the signal transduction cascades triggered by acute thermal stress in Mytilus galloprovincialis gills. This particular species has been reported to exhibit a significant tolerance to high temperatures; thus, it was intriguing to examine the molecular mechanisms responsible for this extraordinary trait. In particular, exposure to 30 degrees C was found to cause a significant and sustained stimulation of p38-MAPK phosphorylation while the activation profile of JNKs was transient and relatively moderate. We also observed that hyperthermia induced apoptosis as a delayed response, with both MAPK subfamilies rapidly translocating to the nucleus. The phosphorylation of cJun, ATF2 and NFkappaB was detected next. Using selective inhibitors, phosphorylation of these transcription factors was established to be dependent on p38-MAPK or JNKs. Subsequently, potential changes in gene expression were assessed. In this context, hyperthermia resulted in the transcriptional upregulation of Hsp70 and MT20 genes with a widely known salutary effect, preserving mussel fitness and performance under adverse environmental conditions. Interestingly, p38-MAPK and JNKs were found to mediate the hyperthermia-induced Hsp70 and MT20 upregulation as well as the delayed induction of apoptosis under the interventions studied. Overall this is, to our knowledge, the first time that an insight into the compensatory survival ;programme' initiated in Mytilus galloprovincialis gills, contributing to this organism's exceptional tolerance to thermal stress, has been gained. In particular, we provide evidence demonstrating the principal role of p38-MAPK and JNKs in transducing the stress signal via mobilization of specific transcription factors and the transcriptional upregulation of cytoprotective genes.

 

Faria, M., D. Huertas, et al. (2010). "Contaminant accumulation and multi-biomarker responses in field collected zebra mussels (Dreissena polymorpha) and crayfish (Procambarus clarkii), to evaluate toxicological effects of industrial hazardous dumps in the Ebro river (NE Spain)." Chemosphere 78(3): 232-40.

                Large amounts of industrial waste containing high concentrations of mercury, cadmium and organochlorine residues were dumped in a reservoir adjacent to a chlorine-alkali plant in the village of Flix(Catalonia, Spain), situated at the shore of the lower Ebro river. Effects of these contaminants to aquatic river invertebrates were assessed by integrating analyses of metals and organochlorine residues in field collected zebra mussels and crayfish with a wide range of biomarkers. Biological responses included levels of metallothioneins, activities of ethoxyresorufin-O-deethylase, oxidative stress biomarkers (glutathione content, enzymatic activities of superoxide dismutase, catalase, glutathione s-transferase, glutathione peroxidise and glutathione reductase), levels of lipid peroxidation and of DNA strand breaks. The results obtained evidenced similar response patterns in mussels and crayfish with increasing toxic stress levels from upper parts of the river towards the meander located immediately downstream from the most polluted site, close to the waste dumps. The aforementioned stress levels could be related with concentrations of mercury, cadmium, hexachlorobenzene, polychlorobiphenyls and dichlorodiphenyltrichloroethanes from 4- to 195-fold greater than local background levels. The response of biomarkers to these pollutant concentrations differences was reflected in high activities and levels of antioxidant enzymes, metallothioneins, lipid peroxidation and DNA strand breaks and decreased levels of glutathione.

 

Masson, S., Y. Couillard, et al. (2010). "Responses of two sentinel species (Hexagenia limbata--mayfly; Pyganodon grandis--bivalve) along spatial cadmium gradients in lakes and rivers in northwestern Quebec." J Environ Monit 12(1): 143-58.

                Specimens of the mayfly larva Hexagenia limbata and of the floater mussel Pyganodon grandis were sampled in rivers and lakes contaminated by trace metals in the Abitibi-James Bay region in northwestern Quebec. Water samples were collected at each sampling site with in situ diffusion samplers and analyzed for major cations, anions and trace metals (Cd, Cu, Mn, Zn). Surficial sediment samples were also collected at each site and analyzed for Cd, Cu and Zn. In response to Cd contamination at river and lake sites, both sentinel organisms accumulated the metal and synthesized metallothionein (MT), a metal-binding protein synthesized by organisms as a defence mechanism against excess metals in the surrounding media. At the river sites, H. limbata unexpectedly maintained much higher concentrations of MT per unit of accumulated Cd than at the lake sites; this difference between lentic and lotic environments may reflect the response of the species to the more stressful hydrodynamic conditions that prevail in a river. The accumulation of Cd in the mayflies at lake and river sites decreased as a function of the ambient manganese concentration. We hypothesize that dissolved Mn protects against Cd bioaccumulation in H. limbata. The present results support the contention that one cannot extrapolate conclusions drawn from the use of a single sentinel species to a larger set of freshwater invertebrates--both the mayfly and the bivalve are promising biomonitors.

 

Bigot, A., P. Vasseur, et al. (2010). "SOD and CAT cDNA cloning, and expression pattern of detoxification genes in the freshwater bivalve Unio tumidus transplanted into the Moselle river." Ecotoxicology 19(2): 369-76.

                The cDNA sequences encoding manganese superoxide dismutase (Mn-SOD) and catalase (CAT) were isolated in the freshwater bivalve Unio tumidus by reverse-transcription polymerase chain reaction (RT-PCR) using degenerate primers. Quantitative real-time PCR approach was used to evaluate the mRNA expression patterns of SOD, CAT, selenium-dependent glutathione peroxidase (Se-GPx), pi class glutathione S-transferase (pi-GST) and metallothionein (MT), in the digestive gland of Unio tumidus transplanted from a control site to four stations in the Moselle River (M1-M4), for periods of 8 and 21 days. These sites were chosen upstream and downstream of populated areas. Chemical analysis performed on sediments from the Moselle river sites did not show high levels of pollutants. Decrease of SOD, CAT, Se-GPx and MT mRNA levels were observed at M3 site after a 21-day exposure compared to control site. These results suggest inefficiency of antioxidant systems affected by cytotoxic mechanisms and confirm an environmental perturbation. Organisms transplanted at M4 site showed a strong increase of biomarkers transcription levels after 21 days of exposure. These inductions could correspond to an adaptive response to an altered environment. Our results showed that biological approaches using multibiomarkers appear as essential tools complementary to measurement of contaminants, to detect environmental degradations.

 

Tsangaris, C., E. Cotou, et al. (2010). "Assessment of contaminant impacts in a semi-enclosed estuary (Amvrakikos Gulf, NW Greece): bioenergetics and biochemical biomarkers in mussels." Environ Monit Assess 161(1-4): 259-69.

                A combination of bioenergetics and biochemical biomarkers in mussels was applied to assess possible pollution impacts in a protected semi-enclosed estuary (Amvrakikos Gulf, NW Greece) that receives pesticide discharges through riverine transport. Scope for growth, a physiological condition index representing the energy budget of the organism, was applied to detect general stress effects on the health status of mussels. The low energy budgets of mussels revealed stress conditions and provided early warning signals of possible consequences at higher levels of biological organization. Biochemical markers of exposure confirmed a risk of pesticide contamination. Decreased acetylcholinesterase activities indicated exposure to organophosphate and carbamate pesticides. Responses of the antioxidant enzyme glutathione peroxidase suggested the presence of contaminants capable of reactive oxygen species production that could be related to organochlorine pesticide contamination in the area. On the other hand, metallothionein levels implied low metal contamination.

 

Fernandez, B., J. A. Campillo, et al. (2010). "Antioxidant responses in gills of mussel (Mytilus galloprovincialis) as biomarkers of environmental stress along the Spanish Mediterranean coast." Aquat Toxicol 99(2): 186-97.

                Antioxidant response was used to assess the effects of the main pollutants in wild mussels (Mytilus galloprovincialis) along the Mediterranean coast of Spain. Antioxidant enzyme activities - those of catalase, superoxide dismutase, glutathione peroxidases, glutathione reductase, glutathione S-transferase and DT-diaphorase - as well as lipid peroxidation and metallothionein concentrations were measured in gills of mussels from 16 selected sites. Furthermore, concentrations of the main contaminants (Hg, Pb, Cd, Cu, Zn, As, PAH, PCB, and DDT) were quantified in mussel tissue, and environmental parameters were measured in water samples collected at each site. Results showed that the glutathione-dependent antioxidant enzymes offered an increased and coordinated response against metal (Hg, Pb and Cd) contamination. These enzymatic activities correlated positively to temperature, suggesting the influence of this environmental parameter on antioxidant responses in gill tissues. Furthermore, although temperature did not reach stressful levels in the study area, it seemed to add a synergistic effect to that produced by metals to induce antioxidant enzymes in the most metal-polluted sites. Catalase activity appeared to be involved in a different antioxidant pathway, more related to organic pollutant bioaccumulation, offering an efficient protection mechanism against reactive oxygen species generation due both to organic exposure and high physiological activity, reflected by high condition indices. In general terms, increased levels of antioxidant enzymes at some sites suffering from metal and organic pollution indicated a situation of oxidative stress that nevertheless did not appear to be harmful, since lipid peroxidation levels showed no peroxidative damage in gill tissues of mussels collected from even the most heavily polluted sites. On the other hand, metallothionein and DT-diaphorase did not reflect pollutant exposure and seemed to be more influenced by environmental variables than by the pollutants.

 

de Montaudouin, X., I. Paul-Pont, et al. (2010). "Bivalve population health: multistress to identify hot spots." Mar Pollut Bull 60(8): 1307-18.

                This study investigated some stress (metals, parasites) and response (immunity, metallothionein) factors in two cockle and two Manila clam populations. Data from eight seasons were averaged to obtain global baseline values. Stress/response characteristics of each population were compared to population health status that was determined through population dynamics parameters. Four different scenarios were discussed: (1) a lightly stressed cockle population with correct population health but with a risk of deterioration (hot spot); (2) a lightly stressed introduced cockle population threatened of extinction. In this case ecological factors were suspected; (3) a moderately stressed clam population with moderate adaptative response. The population was sustainable but the level of stress should not increase (hotspot); and (4) a stressed clam population and unfavourable ecological conditions preventing clam settlement. This monitoring highlighted that the discrepancy between population health and stress levels could be due to insufficient response by bivalves and/or by unfavourable ecological factors.

 

Escobedo-Fregoso, C., L. C. Mendez-Rodriguez, et al. (2010). "Assessment of metallothioneins in tissues of the clam Megapitaria squalida as biomarkers for environmental cadmium pollution from areas enriched in phosphorite." Arch Environ Contam Toxicol 59(2): 255-63.

                The aim of this study was to evaluate the use of metallothionein (MT) concentrations in tissues of the clam Megapitaria squalida as biomarkers of environmental cadmium (Cd) pollution from phosphorite enrichments in the marine environment, which resulted from mining activities in La Paz Bay, Baja California Sur, Mexico. Cd and MT were quantified in gills, digestive gland, and kidney of clams exposed to 0.2 or 0.5 mg Cd l(-1) for 10, 20, or 30 days. In addition, clams from four strategically selected natural sites of La Paz Bay were collected for analysis. In tissues of bioassayed and untreated clams, the gradient of Cd concentrations was digestive gland>>gills>kidney, whereas that of MT was digestive gland>gills>kidney. Digestive gland of the clams exposed to 0.5 mg Cd l(-1) for 30 days showed the highest concentrations of Cd (16.3+/-3.9 microg Cd g(-1)). The highest statistically significant MT concentrations were found in digestive gland at 10 days of exposure to Cd. In the untreated clams, one of the highest Cd concentrations, but not MT levels, was found in digestive glands of the organisms collected from the area close to phosphorite mining activities. For environmental monitoring, MT levels in digestive gland can be used as a first approximation of the presence of high levels of divalent metals in the environment. However, in this study, MT levels did not correlate with high Cd levels in clams that had been collected from areas associated with phosphorite enrichment.

 

Raimundo, J., P. M. Costa, et al. (2010). "Metallothioneins and trace elements in digestive gland, gills, kidney and gonads of Octopus vulgaris." Comp Biochem Physiol C Toxicol Pharmacol 152(2): 139-46.

                Metallothionein-like proteins (MT) and V, Cr, Co, Ni, Zn, Cu, As and Cd were determined in digestive gland, gills, kidney and gonads of Octopus vulgaris, from the Portuguese coast. To our knowledge these are the first data on MT in octopus. High concentrations (microgg(-1), dry mass) of Zn (48050) and Cd (555) were found in digestive gland, and MT reached levels one order of magnitude above the ones registered in wild bivalves. Significantly higher levels of MT in digestive gland and gills of specimens from A and B were in line with elevated Cd concentrations. Principal component analyses (PCA) point to MT-Cd and MT-Cr associations in digestive gland and gills. Despite the high levels of Zn in specimens from B, association with Zn was not obtained. Due to the affinity of MT to various elements, it should not be excluded the possibility of Cd replacing Zn in Zn-MT. Kidney presented higher levels of Cd, Co, Ni and As than gills and gonads, and in the case of As surpassing the levels in digestive gland, but PCA showed no relation with MT. Likewise the MT levels in gonads had no correspondence to the metal concentration variation.

 

Bernal-Hernandez, Y. Y., I. M. Medina-Diaz, et al. (2010). "Acetylcholinesterase and metallothionein in oysters (Crassostrea corteziensis) from a subtropical Mexican Pacific estuary." Ecotoxicology 19(4): 819-25.

                Substantial efforts have been devoted to developing and applying biomarkers for ecological risk assessment. Bivalve mollusks, such as mussels and oysters, are commonly used in environmental monitoring programs because of their wide geographical distribution, great sensitivity to environmental pollutants, and ability to accumulate anthropogenically derived chemicals at a high rate. Acetylcholinesterase (AChE) activity and metallothionein (MT's) content are representative specific biomarkers that indicate the presence of anticholinesterasic compounds (like organophosphorus and carbamate pesticides) and metals, respectively. The aim of this study was to evaluate AChE activity and MT's content in Crassostrea corteziensis from Boca de Camichin estuary. The results obtained here showed that AChE activity was 65% lower in oysters from Boca de Camichin than in control organisms. In contrast, MT's content in collected organisms was not statistically different from that in control organisms. AChE activity and MT's content in oysters could be used as early biomarkers of effects and exposure to pesticides and heavy metals, respectively, in aquatic environments.

 

Fang, Y., H. Yang, et al. (2010). "Metallothionein and superoxide dismutase responses to sublethal cadmium exposure in the clam Mactra veneriformis." Comp Biochem Physiol C Toxicol Pharmacol 151(3): 325-33.

                The objective of this study was to identify the relationship between cadmium (Cd) and stress responses in the clam Mactra veneriformis. Metallothionein (MT) and Cu-Zn superoxide dismutase (SOD) cDNAs from the clam were isolated and characterized. The full-length cDNA of MvMT and MvSOD contained 830 and 689 nucleotides encoding 59 and 159 amino acids, respectively. Multiple alignments indicated that deduced amino acid sequences of MvMT and MvSOD shared high homology with MT and SOD sequences of other mollusks. Clams were exposed to 0, 50, 100, and 200microg/L Cd for 21days. The mRNA transcripts of the two genes, MT protein content and SOD activity in the digestive gland were examined. Cd treatment significantly elevated MvMT and MvSOD mRNA expression in a dose-dependent manner and MT protein level in a dose- and time-dependent manner. SOD activity significantly increased at the start of Cd exposure, then decreased and finally returned to the normal level. These results indicate that MT and SOD play an important role in maintaining cellular metabolism homeostasis and protecting M. veneriformis from Cd toxicity. MT and SOD could be used as biomarkers of Cd pollution in aquatic environment for the studied species.

 

Ivankovic, D., J. Pavicic, et al. (2010). "Inducibility of metallothionein biosynthesis in the whole soft tissue of zebra mussels Dreissena polymorpha exposed to cadmium, copper, and pentachlorophenol." Environ Toxicol 25(2): 198-211.

                Freshwater mussels Dreissena polymorpha (Pallas, 1771) were exposed to the elevated concentrations of Cd (10, 50, 100, and 500 microg/L), Cu (10, 30, 50, and 80 microg/L), and an organochlorinated pesticide, pentachlorophenol (PCP) (1, 10, and 100 microg/L). Induced synthesis of biomarker metallothionein (MT) and changes in concentrations of cytosolic Cd, Cu, and Zn in the whole soft tissue of mussels were monitored after a 7-day laboratory exposure to the contaminants. A clear dose-dependent elevation in the MT concentration was observed after exposure to Cd at doses of 10-100 microg/L, and this increase of MT content was accompanied with a linear increase of cytosolic Cd. Cd concentration of 500 microg/L caused no additional increase of MT and Cd in mussel cytosol, suggesting possible toxic effects due to exceeding cellular inducible/defense capacity. Cu exposure resulted with variable changes in MT concentrations, with no clear linear relationship between MT and Cu concentrations in water, although a progressive dose-dependent accumulation of Cu in the soluble fraction of mussel tissues was recorded. A decrease of cytosolic Zn was evident at higher exposure concentrations of both metals used. PCP in concentrations applied was unable to induce MT synthesis, but the higher concentrations of PCP influenced the cytosolic metal concentrations. In conclusion, the results obtained confirm the specificity of MT induction in D. polymorpha as an biological response on metal stimulation, especially by cadmium, being more closely correlated to MT than copper within the ecologically relevant concentration range. The strong induction potential of cadmium as well as an absence of MT induction following exposure to PCP as an organic chemical contaminant are supporting evidences for usage of zebra mussel MT as a specific biomarker of Cd exposure in biomonitoring programs.

 

Paul-Pont, I., P. Gonzalez, et al. (2010). "Interactive effects of metal contamination and pathogenic organisms on the marine bivalve Cerastoderma edule." Mar Pollut Bull 60(4): 515-25.

                The present study evaluated the interactive effects of cadmium contamination and pathogenic organisms (trematodes Himasthla elongata and bacteria Vibrio tapetis) singularly and in combination during 7 days on the bivalve Cerastoderma edule. Some defense-related activities were analyzed such as genetic expression, metallothionein and immune responses. Trematode metacercarial infection, similar whatever the treatment, induced the strongest responses of immune parameters. Particularly, the interaction between cadmium and parasite exposures induced unusual responses on gene expression and immune responses. No effect of bacterial challenge appeared on bivalve responses, nevertheless a strong mortality of V. tapetis infected cockles occurred between 7 and 14 days. Cadmium bioaccumulation was significantly modulated by both pathogenic organisms. Furthermore, an antagonistic effect of trematodes and bacteria was shown on metal bioaccumulation of co-infected cockles. These results highlighted the importance of considering the multiplicity of perturbation sources in coastal ecosystems to assess the health status of organisms.

 

Tsangaris, C., K. Kormas, et al. (2010). "Multiple biomarkers of pollution effects in caged mussels on the Greek coastline." Comp Biochem Physiol C Toxicol Pharmacol 151(3): 369-78.

                A suite of biomarkers was measured in caged mussels at areas impacted by different anthropogenic activities along the Greek coastline to assess biological effects of environmental pollution. Mussels were caged at coastal sites in the vicinity of major cities, in areas influenced by major industries, agricultural practices and in islands away from known sources of pollution. Biomarkers indicative of neurotoxicity (acetylcholinesterase, AchE), oxidative stress (catalase, CAT), phase II biotransformation of xenobiotics (glutathione S-transferase, GST), metal exposure (metallothioneins, MTs) and protein synthesis (RNA:DNA ratio) were measured to assess effects of various types of pollutants. AchE activity proved to be the most responsive biomarker with decreased values at sites influenced by agricultural, urban and industrial activities. Decreased CAT and GST activities and increased MTs levels were recorded at a number of anthropogenic-impacted sites. RNA:DNA ratio showed a biphasic response as both high and low values were found at impacted sites. Principal component analysis clearly distinguished sites receiving pollution inputs from non-polluted sites. The combination of the selected biomarkers used in caged mussels resulted useful in the assessment of the effects of environmental pollution.

 

Long, A., C. Li, et al. (2010). "Short-term metal accumulation and MTLP induction in the digestive glands of Perna virdis exposed to Zn and Cd." J Environ Sci (China) 22(7): 975-81.

                Time-dependent Zn and Cd accumulation and metallothionein like protein (MTLP) induction in the digestive glands of mussels, Perna virdis, were measured under different exposure conditions. The initial uptake rate at start of chase (rho0) and mean residence time (tau) were calculated to determine the physiological response of organisms and their potential detoxification mechanisms. It was found that in digestive glands, Zn had obviously higher rho0 and shorter mean residence time than Cd, indicating that these two metals had different accumulation dynamics even though they were very close in the periodic element table. MTLP levels in digestive glands varied from 0.51 to 1.05 microg/g ww (wet weight). The MTLP level increased continuously when mussels were exposed to low and middle levels of Zn and Cd media, and reached maximal levels at day 4, then decreased when they were exposed to high level Zn and Cd solutions. With regard to the fraction of Zn and Cd accumulated in the digestive glands, the ratios of soluble metal to total metal decreased continuously after exposure in low and middle levels of Zn and Cd media, and decreased continuously in the first 4 days and then to level off when mussels were exposed to media with high concentration of Zn and Cd. Results suggested that both MTLP induction and metal insolubilization were detoxification processes in digestive glands of mussels.

 

Ringwood, A. H., M. McCarthy, et al. (2010). "The effects of silver nanoparticles on oyster embryos." Mar Environ Res 69 Suppl: S49-51.

                Nanoparticles may be introduced into aquatic environments during production processes and also as a result of release following their use in various commercial formulations and biologic applications. Filter-feeding bivalve mollusks such as oysters are valuable model species for characterizing nanoparticle bioavailability and interactions with basic cellular processes. The adults release their gametes into the environment, so their embryos and larvae are also likely targets of nanoparticles. The purpose of these studies was to characterize the toxicity of metal nanoparticles on embryonic development of oysters, Crassostrea virginica and to compare the relative sensitivity of embryos to adults. Newly-fertilized oyster embryos were exposed to silver nanoparticles (AgNP) and then the percent normal development after 48h was assessed. Studies were conducted with adult oysters in which they were also exposed to AgNP for 48h, and the effects on lysosomal destabilization were determined. The expression of metallothionein (MT) gene expression was also assessed in both embryos and adults. Adverse effects on embryonic development were observed at concentrations similar to those that caused both statistically and biologically significant effects on lysosomal destabilization of adults. Significant increases in MT mRNA levels were observed in both embryos and adult oysters, and MT levels were highly induced in embryos. While we do not know whether the toxicity and gene expression responses observed in this study were due to the nanoparticles themselves or the Ag ions that dissociated from the nanoparticles, these kinds of basic studies are essential for addressing the potential impacts of nanoengineered particles on fundamental cellular processes as well as aquatic organisms.

 

Maltez, H. F., M. Villanueva Tagle, et al. (2009). "Metal-metallothioneins like proteins investigation by heteroatom-tagged proteomics in two different snails as possible sentinel organisms of metal contamination in freshwater ecosystems." Anal Chim Acta 650(2): 234-40.

                Metal speciation analysis in MLPs was carried out in two snails, Marisa cornuarietis and Pomacea bridgesi, in order to investigate them as possible sentinel organisms of heavy metal contamination. To carry out this study snails born in a non-contaminated environment were divided into two groups: a control group and a contaminated one with cadmium administered for 40 days. Subsequently, we investigated the speciation of the induced MLPs in exposed animals in relation to controls. In order to obtain the MLP fraction, cytosols from both snail species where subjected to size-exclusion fractionation, monitoring on-line the metal signal (Cd, Cu and Zn) by ICP-MS while protein elution was followed by on-line UV detection. MLP fraction was then separated by anion-exchange (AE)-FPLC using optimal chromatographic conditions for the separation of the different MLP isoforms in both snail species. Specific detection of separated metalloforms was carried out again by the hyphenation of the AE chromatographic system with ICP-MS. The determination of the amount of metal bound to MLPs was carried out by post-column isotope dilution analysis ICP-MS, finding that the snail M. cornuarietis accumulated higher concentrations of cadmium than P. bridgesi. Thus this first snail could therefore be a better candidate sentinel organism of pollution in natural waters. Identification and characterization of the isoforms separated in M. cornuarietis was carried out for the entire or intact isoforms by MALDI-TOF and then conventional triptic digestion was also carried out to identify the nature of the formed peptides. The presence identification of a MLP isoform of relatively low molecular weight in M. cornuarietis is reported.

 

Cravo, A., B. Lopes, et al. (2009). "A multibiomarker approach in Mytilus galloprovincialis to assess environmental quality." J Environ Monit 11(9): 1673-86.

                A multibiomarker approach was carried out for the first time in the South Portuguese Coast using Mytilus galloprovincialis, to assess environmental quality, establish if there are adverse biological responses associated to different sources of anthropogenic contamination and to determine spatial and seasonal trends. For this purpose the battery of biomarkers selected was: superoxide dismutase (SOD), catalase (CAT), glutathione peroxidases (GPx total and Se dependent), Cytochrome P450 component system, Glutathione-S-transferase (GST) and acetylcholinesterase (AChE), metallothionein (MT) and lead-delta-aminolevulinic acid dehydratase (ALAD), lipid peroxidation (LPO) and Condition Index (CI) along with the determination of PAHs and metals (Cd, Cu, Ni, Pb and Zn). Results show that despite the levels of both organic and metallic contaminants in these eight spots in the South Coast of Portugal not being particularly high compared with other contaminated/polluted sites worldwide, the selected battery of biomarkers responded efficiently to the environmental changes and allowed an environmental assessment between seasons and sites. Different spatial and seasonal responses were evident along the South Coast of Portugal, meaning that the contamination is not homogeneous. This does not only reflect different competition, origin and intensity of contamination, but also different environmental conditions (e.g. temperature, salinity). Along the South Portuguese Coast site 8 was the most contaminated, while site 2 was considered the least contaminated. Despite environmental factors possibly causing difficulties in the general interpretation of biomarker data, those that better responded to environmental contamination were CYP450, SOD-mit and T-GPx for the summation SigmaPAHs, MT (digestive gland) for metals (especially Cu), ALAD for Pb and LPO for both organic and metallic contamination. These biomarkers were also positively correlated with temperature in summer, revealing this as a more stressful/critical season. In future environmental contamination assessments there is no need to analyse the components b5, P418, NADH and NADPH of phase I MFO system, and MT in the gills, since their responses are not evident.

 

Gagne, F., C. Blaise, et al. (2009). "Impacts of pollution in feral Mya arenaria populations: the effects of clam bed distance from the shore." Sci Total Environ 407(22): 5844-54.

                This study examined the relationships between population characteristics and the expression of physiological biomarkers of stress in an intertidal clam population under pollution at sites differing in thermal history and coastline distance. The clam population metrics were age distribution, growth, condition factor, distance of the clam beds from the shore, and gonad development. Physiological biomarkers comprised biomarkers of defence such as superoxide dismutase, labile IIb metals in tissues, redox status of metallothioneins and glutathione S-transferase, of tissue damage such as lipid peroxidation and DNA strand breaks, of reproduction as determined by vitellogenin-like proteins and gonadosomatic index and immunocompetence such as phagocytosis and hemocyte viability. Age-related pigments were also examined to compare the physiological age of the clams with their chronological age. The results showed that all the above biomarkers were significantly affected at one of the two polluted sites at least. Distance from the shore was significantly correlated with most (81%) of the biomarkers examined. Clams collected at one polluted site were physiologically older than clams from the corresponding reference site. Canonical and adaptive regression (artificial neural networks) analyses found that the biomarkers measured in this study were able to predict the ecologically relevant endpoints. Biomarkers implicated in defense mechanisms, tissue damage and age-related pigments were most closely related to the clam population characteristics. Sensitivity analysis of the learning algorithm found that the following physiological and biochemical markers were the most predictive, in decreasing order, of clam population characteristics: glutathione S-transferase, phagocytosis, age pigments, lipid peroxidation in the gills, labile IIb metals and total MT levels. These biomarkers were affected by the distance of the clam beds from the shore, site quality (pollution) and reproduction activity.

 

Ladhar-Chaabouni, R., R. Mokdad-Gargouri, et al. (2009). "Cloning and characterization of cDNA probes for the analysis of metallothionein gene expression in the Mediterranean bivalves: Ruditapes decussatus and Cerastoderma glaucum." Mol Biol Rep 36(5): 1007-14.

                cDNA probes have been developed for subsequent use in monitoring the cadmium exposure of the clam Ruditapes decussatus and the cockle Cerastoderma glaucum using metallothionein (MT) gene expression in different tissues of these species. Two partial MT cDNAs were isolated from Ruditapes decussatus and Cerastoderma glaucum. The identification of the nucleotide sequences showed that the cDNAs consist of 480 bp coding 72 amino acid proteins containing 21 cysteine residues organized in Cys-X-Cys motifs as classically described for MTs. The induction of MT gene expression in CdCl(2) treated bivalves was confirmed by dot blot analysis and suggests a potential specific tissue expression rate.

 

Serafim, A. and M. J. Bebianno (2009). "Metallothionein role in the kinetic model of copper accumulation and elimination in the clam Ruditapes decussatus." Environ Res 109(4): 390-9.

                In order to clarify the role of metallothioneins (MT) in copper (Cu) toxicity, this work aimed to assess the involvement of this protein in the accumulation and elimination strategies of Cu in the clam Ruditapes decussatus exposed to two sublethal concentrations (25 and 50 microgCul(-1)). The behaviour of MT in three different tissues of clams during the accumulation and depuration processes was also followed by gel-filtration chromatography to assess if Cu was bound to MT or to other cytosolic components. The 96 h LC50 for water-borne copper was 715 microgL(-1) in R. decussatus. The Cu accumulation pattern was dependent on Cu exposure concentrations. In clams exposed to 25 microgl(-1), total Cu accumulation in the three tissues increased linearly during the exposure period, while in those exposed to 50 microgl(-1) it followed the first order kinetic model. The greatest amount of Cu accumulated in all tissues is associated to the low molecular weight cytosolic fraction (>50%). The chromatographic assay indicated that Cu in the cytosolic fraction is bound to MT and MT levels increase with the increase of Cu exposure confirming the binding affinity of Cu to MT in all tissues. However, a smaller percentage of Cu seems to be bond to other ligands, such as GSH. Copper was exponentially eliminated (only studied in clams exposed to 25 microgl(-1)) and the estimated half-life was tissue dependent (9, 5 and 14 days for the gills, digestive gland and remaining tissues, respectively). Copper bound to the thermostable compounds was eliminated more quickly (t(1/2)=4-7 days) in all tissues than those bound to the thermolabile compounds (t(1/2)=7-18 days). Interestingly, MT is rapidly degraded (t(1/2)=7 and 18 days), suggesting that this protein is actively involved in the elimination of this metal, through the Cu-MT complex since MT and Cu are turning over simultaneously. Therefore, when Cu exposure is low, the clam can cope efficiently with the excess of Cu levels by increasing MT induction as well as rapidly eliminating this metal via the MT-Cu complex. Copper toxicity in the clam R. decussatus is associated to the limited capacity of MT induction at higher and environmental unrealistic Cu exposures especially in the gills and remaining tissues.

 

Pan, K. and W. X. Wang (2009). "Biodynamics to explain the difference of copper body concentrations in five marine bivalve species." Environ Sci Technol 43(6): 2137-43.

                Copper is an essential metal for organisms but can be toxic when high intake occurs. The interspecific and intraspecific differences in concentrations of Cu in the tissues of different bivalve species have been recognized but the underlying mechanisms leading to these differences remain speculative. In this study, the biokinetics of Cu, using 67Cu as a radioactive tracer, was investigated in five species of marine bivalves, including scallops Chlamys nobilis, clams Ruditapes philippinarum, green mussels Perna viridis, black mussels Septifer virgatus, and oysters Saccostrea cucullata. All species have contrasting Cu concentrations in their bodies. Our results demonstrated the clear importance of biokinetics in controlling Cu concentrations in the bodies of bivalves. Assimilation efficiency (AE), feeding activity (filtration rate), and efflux rate all contributed to the observed interspecies differences. The AE and efflux rate were positively correlated with the respective Cu body burden, indicating their important roles in Cu concentrations in the body. The efflux rate also explained the intraspecies difference in Cu bioaccumulation. The subcellular distribution of Cu varied among different species of bivalves. There was a positive relationship between the newly accumulated Cu in the metallothionein-like protein (MTLP) fraction and the efflux rate, suggesting that MTLP may be responsible for Cu elimination and may control Cu regulation in bivalves. Calculations showed that the Cu partitioning coefficient and ingestion are important in determining the relative contribution of waterborne and dietborne exposure pathways to overall Cu accumulation. However, the dietary pathway was the most important source of Cu in bivalves under most circumstances.

 

Voets, J., E. S. Redeker, et al. (2009). "Differences in metal sequestration between zebra mussels from clean and polluted field locations." Aquat Toxicol 93(1): 53-60.

                Organisms are able to detoxify accumulated metals by, e.g. binding them to metallothionein (MT) and/or sequestering them in metal-rich granules (MRG). The different factors involved in determining the capacity or efficiency with which metals are detoxified are not yet known. In this work we studied how the sub-cellular distribution pattern of cadmium, copper and zinc in whole tissue of zebra mussels from clean and polluted surface waters is influenced by the total accumulated metal concentration and by its physiological condition. Additionally we measured the metallothionein concentration in the mussel tissue. Metal concentration increased gradually in the metal-sensitive and detoxified sub-cellular fractions with increasing whole tissue concentrations. However, metal concentrations in the sensitive fractions did not increase to the same extent as metal concentrations in whole tissues. In more polluted mussels the contribution of MRG and MT became more important. Nevertheless, metal detoxification was not sufficient to prevent metal binding to heat-sensitive low molecular weight proteins (HDP fraction). Finally we found an indication that metal detoxification was influenced by the condition of the zebra mussels. MT content could be explained for up to 83% by variations in Zn concentration and physiological condition of the mussels.

 

Egg, M., M. Hockner, et al. (2009). "Structural and bioinformatic analysis of the Roman snail Cd-Metallothionein gene uncovers molecular adaptation towards plasticity in coping with multifarious environmental stress." Mol Ecol 18(11): 2426-43.

                Metallothioneins (MTs) are a family of multifunctional proteins involved, among others, in stress response. The Cadmium (Cd)-MT gene of the Roman snail (Helix pomatia), for example, encodes for a protein induced upon cadmium exposure. While our previous studies have demonstrated that the expressed Cd-MT isoform of Roman snails assists detoxification of cadmium, the present work focuses on the potential plasticity of this gene in response to a variety of environmental stressors playing a crucial role in the specific ecological niche of H. pomatia. Our hypothesis is based on a bioinformatic approach involving gene sequencing, structural and in silico analysis of transcription factor binding sites (TFBs), and a comparison of these features with other MT genes. Our results show that the Roman snail's Cd-MT gene not only is the largest known MT gene, but also contains--apart from the regulatory promoter region--several intronic repeat cassettes of putative TFBs suggested to be involved in environmental stress response, immune competence, and regulation of gene expression. Moreover, intronic scaffold/matrix attachment regions (S/MARs) and stress-induced duplex destabilization sites confer a high potential for epigenetic gene regulation. This suggested regulatory plasticity is also supported by physiological data showing that Cd-MT in Roman snails can be induced differentially not only after cadmium exposure, but also in response to nonmetallic environmental stressors. It is concluded that structural analysis combined with bioinformatic screening may constitute valuable tools for predicting the potential for plasticity and niche-specific adaptation of stress-responsive genes in populations living under rapidly changing environmental conditions.

 

Falfushynska, H. I., L. Delahaut, et al. (2009). "Multi-biomarkers approach in different organs of Anodonta cygnea from the Dnister Basin (Ukraine)." Arch Environ Contam Toxicol 57(1): 86-95.

                The aim of this study was to compare environmental quality in two sites in western Ukraine-rural (R) and urbanized (U)-with the usage of the resident bivalve mollusk Anodonta cygnea. The study was realized during three seasons. The metal uptake and a set of biochemical markers were determined. For each season, Cd and metallothioneins (MTs) contents in the digestive gland and gills of the mollusc were higher at the U site, reflecting its chronic pollution. The oxidative stress in the mollusk was observed at the U site during spring and at the R site during summer and autumn according to the differences in Mn-superoxide dismutase and catalase activities, O (2) (*-) production, lipid peroxidation, and glutathione levels. The elevated vitellogenin-like protein levels in the hemolymph and the ethoxyresorufin-O-deethylase activity in the digestive gland in summer-autumn suggested pollutions by organic substances at the R site. The acetylcholinesterase activity was similar in both groups. The centroid grouping analysis of biomarkers and morphological and water indexes demonstrated the clear differentiation of general response in each group in spring and, at the R site, in summer and autumn but its similarity at the U site in summer and autumn.

 

Farcy, E., C. Voiseux, et al. (2009). "Transcriptional expression levels of cell stress marker genes in the Pacific oyster Crassostrea gigas exposed to acute thermal stress." Cell Stress Chaperones 14(4): 371-80.

                During the annual cycle, oysters are exposed to seasonal slow changes in temperature, but during emersion at low tide on sunny summer days, their internal temperature may rise rapidly, resulting in acute heat stress. We experimentally exposed oysters to a 1-h acute thermal stress and investigated the transcriptional expression level of some genes involved in cell stress defence mechanisms, including chaperone proteins (heat shock proteins Hsp70, Hsp72 and Hsp90 (HSP)), regulation of oxidative stress (Cu-Zn superoxide dismutase, metallothionein (MT)), cell detoxification (glutathione S-transferase sigma, cytochrome P450 and multidrug resistance (MDR1)) and regulation of the cell cycle (p53). Gene mRNA levels were quantified by reverse transcription-quantitative polymerase chain reaction and expressed as their ratio to actin mRNA, used as a reference. Of the nine genes studied, HSP, MT and MDR1 mRNA levels increased in response to thermal stress. We compared the responses of oysters exposed to acute heat shock in summer and winter and observed differences in terms of magnitude and kinetics. A larger increase was observed in September, with recovery within 48 h, whereas in March, the increase was smaller and lasted more than 2 days. The results were also compared with data obtained from the natural environment. Though the functional molecule is the protein and information at the mRNA level only has limitations, the potential use of mRNAs coding for cell stress defence proteins as early sensitive biomarkers is discussed.

 

Hoang, T. C. and G. M. Rand (2009). "Exposure routes of copper: short term effects on survival, weight, and uptake in Florida apple snails (Pomacea paludosa)." Chemosphere 76(3): 407-14.

                The uptake and effects (survival, weight) of copper (Cu) on Florida apple snails (Pomacea paludosa) via exposures to copper-enriched agricultural soil-water and water-only treatments were investigated. Soils were collected from citrus sites in south Florida and flooded with laboratory freshwater for 14d. Neonate apple snails (96-h-old) were then exposed to either Cu from a soil-overlying water (i.e., flooded agricultural soils) treatment or overlying water-only (i.e., equilibrated overlying water produced from 14d flooding of agriculture soils) treatment for 14d under standard laboratory conditions. Survival, weight (dry, wet), and whole body Cu uptake were measured. Copper exposure via soil-water exposures resulted in higher mortality and whole body Cu uptake than water-only exposures, indicating Cu uptake from soils. However, snail wet and dry weights were higher in soil-water treatments than in water-only treatments. Micronutrients from soils may be consumed by snails increasing weights. Survival, apple snail dry weight, and whole body Cu concentrations were significantly correlated with soil and water Cu concentrations in soil-water treatments. Survival was significantly correlated with the concentration of Cu(CO3)2(2-) in water-only treatments. This suggests that Cu(CO3)2(2-) is toxic to apple snails. Whole body Cu concentrations were higher in surviving snails than dead snails, suggesting that apple snails have the ability to detoxify accumulated Cu (e.g., through metallothionein induction, granules).

 

Santiago-Rivas, S., A. Moreda-Pineiro, et al. (2009). "Characterization of raft mussels according to total trace elements and trace elements bound to metallothionein-like proteins." J Environ Monit 11(7): 1389-96.

                In the current work, samples of Mytilus galloprovincialis collected in different sites from Ria de Arousa estuary (Galicia, north-western Spain) were analysed for total Al, As, Ba, Cd, Cr, Cu, Fe, Mn, Ni, Pb and Zn, and for Ba, Cu, Mn and Zn bound to metallothionein-like proteins isoform I (MLP-I) contents. Inductively coupled plasma-optical emission spectrometry (ICP-OES) was used to assess total metal contents in raft mussels, while high performance liquid chromatography (HPLC) with an on-line metal detection with ICP-OES was used to measure metals bound to MLP-I. A microwave assisted acid digestion procedure was used as a sample pre-treatment for total metal contents, while a cytosolic preparation method based on a blending procedure with TRIS-HCl (pH 7.4) as an extracting solution was used to isolate MLP-I. Concentrations of total metals and metals bound to MLP-I were used as discriminating factors to establish different regions in the Ria de Arousa estuary. Principal component analysis (PCA) and cluster analysis (CA) were used as unsupervised pattern recognition procedures, and the half-range central value transformation was used as a data pre-treatment to homogenize data sets. Results have revealed a separation of raft mussels in good agreement with water circulation pattern and oceanographic processes in the estuary only when concentrations of metals to bound MLP-I are used as discriminating factors. Otherwise, raft mussels are classified as samples harvested in the inner or outer sides of the Ria.

 

Smaoui-Damak, W., B. Berthet, et al. (2009). "In situ potential use of metallothionein as a biomarker of cadmium contamination in Ruditapes decussatus." Ecotoxicol Environ Saf 72(5): 1489-98.

                Cadmium (Cd) and metallothionein (MT) concentrations were analysed in the gills and digestive gland of clams Ruditapes decussatus collected from two sites of the Gulf of Gabes (Tunisia) which differ by their degree of pollution. The effect of biotic (maturity stages, protein, sex and body mass) and abiotic factors (temperature, salinity, site and season) on Cd and MT concentrations showed that in the reference site "Bordj d'Ungha" physiological changes caused by gamete development and maturity contributed more to changes in MT concentrations than the bioavailable Cd concentrations; whereas, in the contaminated site "El Hofra", MT induction responded to raised Cd bioavailabilities rather than to physiological changes. With a view to using MTs as metal exposure biomarker in monitoring program, our results show that the analysis in the digestive gland seem to be more relevant than in gills, and that males appear as the most promising candidates compared to females in the determination of this biomarker.

 

Wang, L., L. Song, et al. (2009). "Alteration of metallothionein mRNA in bay scallop Argopecten irradians under cadmium exposure and bacteria challenge." Comp Biochem Physiol C Toxicol Pharmacol 149(1): 50-7.

                Metallothionein (MT) is a superfamily of cysteine-rich proteins contributing to metal metabolism, detoxification of heavy metals, and immune response such as protecting against ionizing radiation and antioxidant defense. A metallothionein (designated AiMT2) gene was identified and cloned from bay scallop, Argopecten irradians. The full length cDNA of AiMT2 consisted of an open reading frame (ORF) of 333 bp encoding a protein of 110 amino acids, with nine characteristic Cys-X-Cys, five Cys-X-X-Cys, five Cys-X-X-X-Cys and two Cys-Cys motif arrangements and a conserved structural pattern Cys-x-Cys-x(3)-Cys-Tyr-x(3)-Cys-x-Cys-x(3)-Cys-x-Cys-Arg at the C-terminus. The cloned AiMT showed about 50% identity in the deduced amino acid sequence with previously published MT sequences of mussels and oysters. The conserved structural pattern and the close phylogenetic relationship of AiMT2 shared with MTs from other mollusc especially bivalves indicated that AiMT2 was a new member of molluscan MT family. The mRNA transcripts in hemolymph of AiMT2 under cadmium (Cd) exposure and bacteria challenge were examined by real-time RT-PCR. The mRNA expression of AiMT2 was up-regulated to 3.99-fold at 2 h after Listonella anguillarum challenge, and increased drastically to 66.12-fold and 126.96-fold at 16 and 32 h post-challenge respectively. Cadmium ion exposure could induce the expression of AiMT2, and the expression level increased 2.56-fold and 6.91-fold in hemolymph respectively after a 10-day exposure of 100 microg L(- 1) and 200 microg L(- 1) CdCl(2). The sensitivity of AiMT2 to bacteria challenge and cadmium stress indicated it was a new Cd-dependent MT in bay scallop and also regulated by an immune challenge. The changes in the expression of AiMT2 could be used as an indicator of exposure to metals in pollution monitoring programs and oxidative stress, and bay scallop as a potential sentinel organism for the cadmium contamination in aquatic environment.

 

Ivanina, A. V., C. Taylor, et al. (2009). "Effects of elevated temperature and cadmium exposure on stress protein response in eastern oysters Crassostrea virginica (Gmelin)." Aquat Toxicol 91(3): 245-54.

                Stress proteins such as heat shock proteins (HSPs) and metallothioneins (MTs) play a key role in cellular protection against environmental stress. Marine ectotherms such as eastern oysters Crassostrea virginica are commonly exposed to multiple stressors including temperature and pollution by metals such as cadmium (Cd) in estuaries and coastal zones; however, the combined effects of these stressors on their cellular protection mechanisms are poorly understood. We acclimated C. virginica from populations adapted to different thermal regimes (Washington, North Carolina and Texas) at a common temperature of 12 degrees C, and analyzed their expression of MTs and HSPs (cytosolic HSP69, HSC72-77, HSP90 and mitochondrial HSP60) in response to the combined acute temperature stress and long-term Cd exposure. Overall, HSP and MT induction patterns were similar in oysters from the three studied geographically distant populations. HSP69 and MTs were significantly up-regulated by Cd and temperature stress implying their important role in cellular stress protection. In contrast, HSC72-77, HSP60 and HSP90 were not consistently induced by either acute heat or Cd exposure. The induction temperature for MTs was higher than for HSP69 (>28 degrees C vs. 20 degrees C, respectively), and MTs were more strongly induced by Cd than by temperature stress (to up to 38-94-fold compared by 3.5-7.5-fold, respectively) consistent with their predominant role in metal detoxification. Notably, heat stress did not result in an additional increase in metallothionein expression in Cd-exposed oysters suggesting a capacity limitation during the combined exposure to Cd and temperature stress. Levels of HSP69 and in some cases, HSC72-77 and HSP90 were lower in Cd-exposed oysters as compared to their control counterparts during heat stress indicating that simultaneous exposure to these two stressors may have partially suppressed the cytoprotective upregulation of molecular chaperones. These limitations of stress protein response may contribute to the reduced thermotolerance of oysters from metal-polluted environments.

 

Bigot, A., P. Doyen, et al. (2009). "Metallothionein coding sequence identification and seasonal mRNA expression of detoxification genes in the bivalve Corbicula fluminea." Ecotoxicol Environ Saf 72(2): 382-7.

                The aim of this study was to identify a metallothionein (MT) coding sequence from the freshwater bivalve Corbicula fluminea and to measure the seasonal transcriptional pattern of MT in parallel with several detoxification genes: superoxide dismutase (SOD), catalase (CAT), glutathione S-transferases (GST) and glutathione peroxidases (GPx), in the digestive gland and the gills of this bivalve during a 1-year period. We identified a C. fluminea MT complete cDNA sequence using RT-PCR and RACE-PCR. The amino acid sequence deduced from the coding sequence encodes for a protein of 73 amino acids containing 21 cysteine residues. This protein exhibits high identities and similarities with the MT sequences of numerous bivalves. MT, SOD, CAT, pi-GST and Se-GPx expression patterns did not exhibit major seasonal variations. A slight increase of MT was observed in July. Therefore, the mRNA expression of these five genes could be used as biomarkers for monitoring studies.

 

Digilio, G., C. Bracco, et al. (2009). "The cadmium binding domains in the metallothionein isoform Cd(7)-MT10 from Mytilus galloprovincialis revealed by NMR spectroscopy." J Biol Inorg Chem 14(2): 167-78.

                The metal-thiolate connectivity of recombinant Cd(7)-MT10 metallothionein from the sea mussel Mytilus galloprovincialis has been investigated for the first time by means of multinuclear, multidimensional NMR spectroscopy. The internal backbone dynamics of the protein have been assessed by the analysis of (15)N T (1) and T (2) relaxation times and steady state {(1)H}-(15)N heteronuclear NOEs. The (113)Cd NMR spectrum of mussel MT10 shows unique features, with a remarkably wide dispersion (210 ppm) of (113)Cd NMR signals. The complete assignment of cysteine Halpha and Hbeta proton resonances and the analysis of 2D (113)Cd-(113)Cd COSY and (1)H-(113)Cd HMQC type spectra allowed us to identify a four metal-thiolate cluster (alpha-domain) and a three metal-thiolate cluster (beta-domain), located at the N-terminal and the C-terminal, respectively. With respect to vertebrate MTs, the mussel MT10 displays an inversion of the alpha and beta domains inside the chain, similar to what observed in the echinoderm MT-A. Moreover, unlike the MTs characterized so far, the alpha-domain of mussel Cd(7)-MT10 is of the form M(4)S(12) instead of M(4)S(11), and has a novel topology. The beta-domain has a metal-thiolate binding pattern similar to other vertebrate MTs, but it is conformationally more rigid. This feature is quite unusual for MTs, in which the beta-domain displays a more disordered conformation than the alpha-domain. It is concluded that in mussel Cd(7)-MT10, the spacing of cysteine residues and the plasticity of the protein backbone (due to the high number of glycine residues) increase the adaptability of the protein backbone towards enfolding around the metal-thiolate clusters, resulting in minimal alterations of the ideal tetrahedral geometry around the metal centres.

 

Meistertzheim, A. L., M. Lejart, et al. (2009). "Sex-, gametogenesis, and tidal height-related differences in levels of HSP70 and metallothioneins in the Pacific oyster Crassostrea gigas." Comp Biochem Physiol A Mol Integr Physiol 152(2): 234-9.

                Pacific oysters, Crassostrea gigas, living at a range of tidal heights, routinely encounter large fluctuations in temperature. We demonstrate that levels of heat shock proteins (HSP) and other stress proteins (metallothioneins, MTs) quantified by ELISA, remained similar in gills, mantle and digestive gland between oysters inhabiting low and high tidal heights. In contrast, endogenous HSPs and MTs levels in gonad changed significantly during gametogenesis. In female gonads, the constitutive form of HSP70 and the MTs increased from immature (or resting) to mature stages (about more than 3-fold) and decreased after spawning. In male gonads, the same expression patterns were observed, whereas proteins levels decreased once fully mature. Females presented higher concentration of HSP70 and MTs than males during the spawning period. No significant difference in HSPs and MTs patterns was found among oysters sampled at low and high tidal heights. We hypothesize that the high level of stress proteins in eggs may increase survival of oyster progeny.

 

Yap, C. K., A. Noorhaidah, et al. (2009). "Telescopium telescopium as potential biomonitors of Cu, Zn, and Pb for the tropical intertidal area." Ecotoxicol Environ Saf 72(2): 496-506.

                The distributions of Cu, Zn, and Pb concentrations in the selected soft tissues (foot, cephalic tentacle, mantle, muscle, gill, digestive caecum, and remaining soft tissues) and shells of the mud-flat snail Telescopium telescopium were determined in snails from eight geographical sites in the south-western intertidal area of Peninsular Malaysia. Generally, the digestive caecum compared with other selected soft tissues, accumulated higher concentration of Zn (214.35+/-14.56 microg/g dry weight), indicating that the digestive caecum has higher affinity for the essential Zn to bind to metallothionein. The shell demonstrated higher concentrations of Pb (41.23+/-1.20 microg/g dry weight) when compared to the selected soft tissues except gill from Kuala Sg. Ayam (95.76+/-5.32 microg/g dry weight). The use of different soft tissues also can solve the problem of defecation to reduce error in interpreting the bioavailability of heavy metals in the intertidal area.

 

Hockner, M., K. Stefanon, et al. (2009). "Coping with cadmium exposure in various ways: the two helicid snails Helix pomatia and Cantareus aspersus share the metal transcription factor-2, but differ in promoter organization and transcription of their Cd-metallothionein genes." J Exp Zool A Ecol Genet Physiol 311(10): 776-87.

                Gastropods are able to withstand fluctuating availabilities of nonessential trace elements such as cadmium by induction of Cd-specific metallothionein isoform (Cd-MT) expression. As in other species, the induction mechanism involves the binding of metal-regulatory transcription factors (MTF-1 or MTF-2) to metal responsive elements (MREs) in the MT promoter regions. Cd-dependent transcription of Cd-MT genes was assessed by quantitative real time PCR in two helicid gastropods, Helix pomatia and Cantareus aspersus, over a period of eight days. The promoter regions of the Cd-MT genes of the two species were sequenced and compared regarding the position of MREs and other relevant potential transcription factor binding sites (TFBs). Cd-MT gene transcription is induced after Cd exposure in Helix pomatia and Cantareus aspersus, showing a transient peak in Helix pomatia, contrasting with a persistent induction rate in Cantareus aspersus. Since the existence of MTF-2 was verified in both species, differing transcription patterns of Cd-MT genes must be due to functional differences in their metal-responsive promoter regions. Both promoters contain a proximal cluster of three MREs overlapping with TFBs for the transcriptional regulator Sp1. In contrast to Cantareus aspersus, however, the Cd-MT gene of Helix pomatia hosts an additional distal MRE overlapping with a Sp1 binding site and a CACCC box. Inhibitory effects of MRE overlapping Sp1 binding sites were observed in other MT genes. We therefore suggest that transient Cd-MT transcription upon Cd(2+) exposure in Helix pomatia may be the result of an inhibitory action of the distal MRE cluster.

 

Costa, P. M., H. M. Santos, et al. (2009). "Toxicokinetics of waterborne trivalent arsenic in the freshwater bivalve Corbicula fluminea." Arch Environ Contam Toxicol 57(2): 338-47.

                Arsenite (As(III)) uptake and elimination kinetics were studied in a freshwater bivalve, Corbicula fluminea, exposed to several nominal concentrations of As(III) (0, 100, 300, 500, and 1000 microg L(-1)) in a static 28-day assay, followed by a depuration stage of 14 days. At the end of each sampling time (days 0, 7, 28, and 42) whole-body portions were surveyed for total As concentrations and, complimentarily, surveyed for whole-body metallothionein (MT) induction to assess its role as a defense mechanism against exposure to As(III). Histochemical evaluation of the digestive gland was performed to verify As deposition and elimination in the tissue. Results show a significant increase in whole-body total As after 28 days of exposure for all treatments, followed by a decrease at the end of the depuration phase. Biodynamic kinetic models for As uptake and elimination were obtained from bioaccumulation data during the exposure phase, for all As treatments, by estimating uptake and elimination rate constants. Bioconcentration factors (BCFs) were estimated by the ratio of these constants. Results revealed that exposure to higher concentrations of As(III) causes a decrease in BCFs, suggesting that C. fluminea triggers effective regulatory mechanisms when exposed to higher concentrations of the metalloid. Significant induction of MT was detected during the exposure phase, followed by a decrease in MT concentration to control levels after depuration for all treatments. No significant differences in MT concentrations were observed between treatments. This finding may confirm the role of MT as part of the As regulation process, but its independence relative to concentrations of As(III) in water suggests that MT induction is not dose dependent. The histochemical evaluation provided clear evidence that As was effectively accumulated in the digestive gland during exposure and eliminated during depuration. The present work demonstrated that C. fluminea is capable of regulating As, even at exposures as high as 1000 microg L(-1) of waterborne As(III).

 

Pellerin, J. and J. C. Amiard (2009). "Comparison of bioaccumulation of metals and induction of metallothioneins in two marine bivalves (Mytilus edulis and Mya arenaria)." Comp Biochem Physiol C Toxicol Pharmacol 150(2): 186-95.

                The St. Lawrence maritime estuary (Quebec, Canada) is subjected to mixed inputs of pollutants and the study of the induction of metallothionein in species of economic and ecologic importance such as Mytilus edulis and Mya arenaria was pertinent to assess the consequences of pollution in this northern estuary. Bivalves from an area devoid of anthropogenic influences but characterized by background metal contamination (Franquelin) were actively transplanted within this location and in a site contaminated by urban, industrial and endogenous pollutants, Baie-Comeau (Baie-des-Anglais). Spatial differences in metal concentrations were shown between sites. Cu and Zn concentrations were higher in mussels from Baie-des-Anglais at the beginning of the transfer and after 1 and 2 months. In clams, Zn concentrations were significantly higher in gills and digestive gland tissues for organisms transplanted in Baie-des-Anglais thus showing that spatio-temporal variations of metal concentrations were different between the two species studied. Mussels and clams partitioning of metals were shown to be different depending of the species, metal and/or tissue studied. In mussels, Cd and Cu concentrations decreased in both organs and both groups after the 3-month transfer in the polluted site. In mussels, total metal and metallothionein (MT) concentrations were positively correlated in digestive gland while in clams a positive correlation was only observed in gills.

 

Peyrot, C., C. Gagnon, et al. (2009). "Effects of cadmium telluride quantum dots on cadmium bioaccumulation and metallothionein production to the freshwater mussel, Elliptio complanata." Comp Biochem Physiol C Toxicol Pharmacol 150(2): 246-51.

                Nanotechnology has gained increasing commercial attention over recent years and its use has raised concerns about its potential release in the environment. The purpose of this study was to determine the size distribution of CdTe in freshwater, bioavailability and potential toxic effects of cadmium telluride quantum dots (CdTe QD) to the freshwater mussel Elliptio complanata. Mussels were exposed to increasing concentrations (0 to 8 mg Cd L(-1)) of CdTe and 0.5 mg/L CdSO4 for 24 h at 15 degrees C to examine the initial uptake and toxic effects of Cd from CdTe QDs and dissolved CdSO4. After the exposure period, Cd bioaccumulation in the gills, digestive gland and gonad tissues and metallothionein (MT) levels were determined. The results revealed that about 80% of Cd was retained by a 450 nm pore filter (aggregates) and that 14% of the Cd was in the dissolved phase (i.e., eluted through a 1 kDa ultrafiltration membrane) which suggested that uncoated CdTe QDs were not stable in freshwater. In mussels, Cd was accumulated principally by the gills and digestive gland and the bioaccumulation factors of Cd from CdTe were similar to that of dissolved Cd. Indeed, tissue-levels of Cd were below the proportion of dissolved Cd from CdTe which suggests that Cd rather comes from the dissociation of Cd from the ingested QDs than from the internalization of the QDs in mussel tissues. The levels of MT were induced in both the digestive gland and gonad but were readily decreased in the gills by both CdTe and CdSO4. The observed decrease in the metallic form of MT might result from the oxidative stress by CdTe and dissolved Cd. In conclusion, uncoated CdTe QD in freshwater leads to aggregates and a dissolved component of Cd where the latter explained the contribution of the observed accumulation pattern in mussel tissues and effects on MT levels in mussels.

 

Faria, M., L. Carrasco, et al. (2009). "Multi-biomarker responses in the freshwater mussel Dreissena polymorpha exposed to polychlorobiphenyls and metals." Comp Biochem Physiol C Toxicol Pharmacol 149(3): 281-8.

                Contaminant related changes in behavioral, phase I and II metabolizing enzymes and pro-oxidant/antioxidant processes in the freshwater mussels Dreissena polymorpha exposed to metals and PCBs were assessed. Behavioral and biochemical responses including filtering rates, key phase I, II and antioxidant enzymes and levels of metallothioneins, glutathione, lipid peroxidation and DNA strand breaks were determined in digestive glands of mussels after being exposed to sublethal levels of mercury chloride, methyl mercury, cadmium and Aroclor 1260 during 5 days. In 7 out of 12 responses analyzed, mussels showed significant differences across treatments. Unusual properties of measured ethoxyresorufin-O-deethylase (EROD) activities indicated that mussels lack an inducible CYP1A enzymatic activity. Despite of using similar exposure levels, inorganic and organic mercury showed different biomarker patterns of response with methyl mercury being more bio-available and unable to induce metallothionein proteins. Mussels exposed to Cd presented higher levels of metallothioneins and an enhanced metabolism of glutathione, whereas those exposed to Aroclor showed their antioxidant glutathione peroxidase related enzyme activities inhibited. Although there was evidence for increased lipid peroxidation under exposure to inorganic and organic mercury, only mussels exposed to Aroclor had significant greater levels than those in controls.

 

Sole, M., J. Kopecka-Pilarczyk, et al. (2009). "Pollution biomarkers in two estuarine invertebrates, Nereis diversicolor and Scrobicularia plana, from a Marsh ecosystem in SW Spain." Environ Int 35(3): 523-31.

                The polychaete worm Nereis diversicolor and the clam Scrobicularia plana were collected from several sites, affected by different types of contamination, in a littoral enclosure in the SW Spain (Cano Sancti-Petri and Rio San Pedro). N. diversicolor was present in 6 sampling sites whereas S. plana in 4 of them. The aim of our study was to relate several pollution biomarkers to chemical sources (metals and organic pollutants e.g. PCB, PAH) in these species, thereby confirming their adequacy as sentinels for this habitat. The biomarkers surveyed in the two invertebrates were the activities of the antioxidant enzyme catalase (CAT), the phase II detoxifying enzyme glutathione S-transferase (GST) and the neurotoxicity marker acetylcholinesterase (AChE). Metallothionein (MT) levels were measured as a biomarker of exposure to metals. The results suggested a different response in the two sediment-dwelling organisms, the sediment-eating polychaete and the water-filtering clam, probably as a consequence of different contamination exposures. The results also suggested that samples from the "Cano Sancti-Petri" were exposed to biologically active compounds that altered some of their biochemical responses. Of all the biomarkers tested, AChE was the most sensitive one and N. diversicolor the potentially most robust sentinel in this ecosystem. In this low to moderately polluted environment, the biochemical approach better reflected temporal trends than site-related differences although it was also able to detect punctual chemical insults.

 

Mishchuk, O. V. and O. B. Stoliar (2008). "[The effect of pesticide acetamiprid on biochemical markers in tissues of fresh water bivalve mussels Anodonta cygnea L. (Unionidae)]." Ukr Biokhim Zh (1999) 80(5): 117-24.

                The effects of ecological real concentration of pesticide "Mospilan" (active substance - acetamiprid) on the status of oxidative stress biomarkers, neurotoxicity and metallothioneins' (MTs) characteristics of freshwater bivalve mussels Anodonta cygnea were investigated. The prooxidative changes (decrease of superoxide dismutase activity, reduced glutathione content, increase of content of lipid and proteins peroxidation products) and decrease of the MTs content in gills and especially in digestive gland in the experimental group were reflected, whereas acetylcholinesterase activity was not changed. The effect of acetamiprid induces the appearance of additional chromatographic MTs form.

 

Bocchetti, R., D. Fattorini, et al. (2008). "Contaminant accumulation and biomarker responses in caged mussels, Mytilus galloprovincialis, to evaluate bioavailability and toxicological effects of remobilized chemicals during dredging and disposal operations in harbour areas." Aquat Toxicol 89(4): 257-66.

                Remobilization of chemicals from contaminated sediments is a major risk associated with dredging and disposal operations in harbour areas. In this work caged mussels, Mytilus galloprovincialis, were chosen as bioindicator organisms to reveal the impact and recovery of organisms from these activities in the harbour of Piombino (Tuscany, Italy) where approximately 100,000 m(3) of sediments were removed and disposed in a local confined disposal facility (CDF). Organisms were deployed before, during and after the end of operations, selecting sites differently impacted by these activities. Temporal changes in environmental bioavailability and biological effects of pollutants were assessed by integrating analyses of trace metals and polycyclic aromatic hydrocarbons (PAHs) accumulated in tissues of caged mussels with a wide array of biomarkers reflecting exposure to specific classes of pollutants and different levels of cellular unbalance or toxicity. Such biological responses included levels of metallothioneins, activity of acyl CoA oxidase (AOX) as a marker of peroxisome proliferation, oxidative stress biomarkers (content of glutathione, enzymatic activities of catalase, glutathione S-transferases, glutathione reductase, glutathione peroxidases), total oxyradical scavenging capacity (TOSC) toward peroxyl and hydroxyl radicals, lysosomal membrane stability and genotoxic effects measured as DNA strand breaks and frequency of micronuclei. Obtained results indicated that a general disturbance was already present in the whole harbour area and especially in the inner site before the beginning of operations, when caged mussels exhibited a significant accumulation of PAHs and Pb, lower TOSC values and higher levels of both lysosomal and genotoxic damages. Bioavailability of trace metals and PAHs markedly increased during dredging activities with values up to 40 microg/g for Pb and up to 2200 ng/g for PAHs in tissues of caged mussels, a significant inhibition of antioxidant efficiency and increase of oxidative damages. While bioavailability of trace metals returned to the pre-dredging values after the end of operations, the accumulation of PAHs, oxidative effects and genotoxic damages remained elevated in mussels caged in the inner area and in front of CDF. Overall this study confirmed the utility of caged mussels to assess the remobilization of chemicals from dredged sediments and the onset of potentially harmful biological effects.

 

Hamer, B., Z. Jaksic, et al. (2008). "Effect of hypoosmotic stress by low salinity acclimation of Mediterranean mussels Mytilus galloprovincialis on biological parameters used for pollution assessment." Aquat Toxicol 89(3): 137-51.

                In the present study, we investigated the progressive acclimation of the mussel Mytilus galloprovincialis to different reduced seawater (SW) salinities and its effect on several biochemical markers and biotests. Mussels were purchased from a local mariculture facility during summer (SW temperature 27 degrees C, salinity 37.5 psu) and winter (13 degrees C, 37 psu) seasons, and transferred to the laboratory for acclimation to reduced SW salinities (37, 28, 18.5 and 11 psu). At the beginning and at the end of acclimation processes tests of mussel survival in air were provided. After 14 days of acclimation the DNA integrity, p38-MAPK activation, metallothionein induction, oxygen consumption rate, and condition index were measured. Survival in air (SOS test), as a physiological index of mussel's health and vitality, had significantly lower LT50 values (11 psu) in the summer than in the winter, and it seems to be negatively affected by acclimation in comparison to controls (37 psu and mariculture). Condition indexes (CIs) were not significantly different, but mussel's acclimation resulted in decline (i.e., a negative trend), especially of CI-2 and CI-3 calculated on the basis of mussel tissue weight and shell sizes. Oxygen consumption rate (VO2) of M. galloprovincialis acclimated to reduced salinities was a concentration-dependent process and increased considerably to about 51 and 65% in lower SW concentrations (28 and 18 psu) compared to control mussels (37 psu). DNA integrity, determined by Fast Micromethod, was negatively impacted by salinity acclimation and corresponding physiological stress as well. Some differences in 1D protein expression patterns between control groups and mussels acclimated to 28, 18.5 and 11 psu (SW) were established. Reduced SW salinities (18.5 and 11 psu) resulted in significantly higher p38-MAPK phosphorylation, whereas the SW salinity of 28 psu decreased p-p38 significantly compared to control (37 psu). The concentration of metallothioneins in mussels' gills was reduced at 28 and 18.5 psu, while it was significantly higher at 11 psu. Results indicated that SW salinity variation (i.e., hypoosmotic stress) in the marine environment can affect all investigated parameters. This investigation expands our understanding of multifactorial effects of the physical marine environment on the specificity of investigated biomarkers and biotests, providing insight into the acclimation, adaptive and stress response processes of mussels. Effects of environmental factors have to be considered in sampling strategies for monitoring programmes to prevent false interpretation of results.

 

Del Castillo, E. and W. E. Robinson (2008). "Nuclear and cytosolic distribution of metallothionein in the blue mussel Mytilus edulis L." Comp Biochem Physiol B Biochem Mol Biol 151(1): 46-51.

                Four tissues from the blue mussel, Mytilus edulis L., were examined for the presence of nuclear metallothionein (MT), and the nuclear:cytosolic (N:C) MT ratios and nuclear MT:DNA ratios investigated. Gill, digestive gland, gonad and posterior adductor muscle tissues were dissected, homogenized and subjected to differential centrifugation in order to isolate the nuclear and cytosolic fractions, which were then analyzed for MT and DNA. MT was present in all samples of the nuclear fractions from all four tissues. The nuclear MT concentration was either lower or the same as the cytosolic MT concentration from the same tissue. The mean N:C MT ratio of the digestive gland was significantly lower than that of the gill. The mean nuclear MT:DNA ratio of the digestive gland was significantly higher than that of the gill and posterior adductor muscle. In addition to being the first report of nuclear MT in bivalves, we showed that N:C MT ratios and nuclear MT:DNA ratios differ among tissues of the same organism. This raises important questions concerning the regulation of nuclear MT concentrations and the role of nuclear MT in metal regulation and DNA protection.

 

Metian, M., M. Warnau, et al. (2008). "Bioaccumulation and detoxification processes of Hg in the king scallop Pecten maximus: field and laboratory investigations." Aquat Toxicol 90(3): 204-13.

                Hg bioaccumulation was investigated in the king scallop Pecten maximus in the laboratory and in the field. In controlled conditions, scallops were exposed to (203)Hg through seawater, sediment and food in order to determine its uptake and depuration kinetics. In the field, Hg and metallothionein (MT) concentrations and the metal subcellular distribution were determined in scallops from two sites of the Bay of Seine (France) differently subjected to the Seine river inputs. While Hg concentrations in the whole soft parts and kidneys (viz. the highest accumulator organ) did not differ between scallops from both sites (74-156 ng g(-1)dry wt), they did for the digestive gland and the gills. According to the experimental results, a higher exposure to dissolved Hg might occur in the site close to the estuary whereas Hg would be mainly incorporated via the dietary pathway in the site away from the estuary. Within the cells of wild scallops, Hg was mainly associated to the cytosolic fraction in the digestive gland and gills (60-100%). However, the lack of relationship between Hg and MT levels suggests that Hg detoxification in P. maximus involves other, non-MT, soluble compounds. In kidneys, insoluble compounds played an important role in Hg sequestration. No effect of scallop age was observed neither on Hg and MT concentrations nor on the subcellular distribution of the metal. Finally, according to FAO/WHO recommendations (maximum weekly Hg intake), our results clearly indicate that the low Hg contents in the edible part of the king scallops from the Bay of Seine prevent any risk for human consumers.

 

Frank, S. N., C. Singer, et al. (2008). "Metallothionein (MT) response after chronic palladium exposure in the zebra mussel, Dreissena polymorpha." Environ Res 108(3): 309-14.

                The effects of different exposure concentrations of palladium (Pd) on relative metallothionein (MT) response and bioaccumulation were investigated in zebra mussels (Dreissena polymorpha). The mussels were exposed to 0.05, 5, 50, and 500 microg/L Pd2+ for 10 weeks under controlled temperature and fasting conditions. Relative MT contents were assessed by a modified Ag-saturation method, which allows to discriminate between MT bound to Pd (Pd-MT) and MT bound to unidentified metals (Ag-MT). Determination of metal contents resulted from atomic absorption spectrometry following a microwave digestion. For unexposed mussels and mussels exposed to 0.05 microg/L Pd no metal accumulation could be detected. All other exposure concentrations resulted in detectable Pd accumulation in mussels with final tissue concentrations of 96 microg/g (500 microg/L), 45 microg/g (50 microg/L), and 9 microg/g (5 microg/L). Compared with initial levels Pd-MT concentrations at the end of the exposure period were 600 (500 microg/L), 160 (50 microg/L), and 27 (5 microg/L) times higher. These results show that an increase in MTs in D. polymorpha already occurs at relatively low aqueous Pd concentrations indicating that there is the need for detoxification of Pd in the mussel. Furthermore, correlations between Ag-MT and Pd accumulation indicate that higher exposure concentrations are associated with adverse effects on the mussels. Thus, harmful effects of chronic Pd exposure of organisms even in lowest concentrations cannot be excluded in the environment.

 

Machreki-Ajmi, M. and A. Hamza-Chaffai (2008). "Assessment of sediment/water contamination by in vivo transplantation of the cockles Cerastoderma glaucum from a non contaminated to a contaminated area by cadmium." Ecotoxicology 17(8): 802-10.

                In this study the cockle Cerastoderma glaucum, a filter-feeding bivalve living in the upper layer of sediment was used to investigate the cadmium contamination at a heavily urbanised and industrial area, with a view to using them as an indicator of water/sediment contamination. To this end, cockles collected from indigenous population in a relatively uncontaminated site (Ras Ungha) were in vivo transplanted into sediment and water removed from cadmium contaminated site (El Hofra) for 45 days. The manipulative experiment was undertaken in order to examine the trace metal bioavailability in the contaminated area and to establish an analytical framework between the bioaccumulation of cadmium in the tissues and their biological effect in transplanted cockles. For this purpose, a range of sublethal stress biomarkers were selected on the basis of their potential to provide relevant information. Cadmium concentrations were determined in the sediment and in the soft tissue of the cockles from the two studied stations at time 0. Compared to the reference site, cadmium concentrations in the contaminated site were 53 higher in the sediment and 15 higher in the whole soft tissues. The variation of cadmium concentrations and biomarkers responses in transplanted cockles were determined as a function of exposure time. After 45 days' experience, cadmium concentrations increased by a factor of 5 compared with time 0. No significant change could be detected in controls. In the digestive gland of exposed cockles cadmium was mainly associated with the cytosolic fraction. The significant increase of cadmium concentration in the soluble fraction was followed by a significant increase in the concentration of the Sulphydryl-Containing Heat Stable Compounds (SCHSC) including metallothionein like proteins by approximately 86%. This is led to investigation into possible existence of an induction of MT-like proteins in relation to cadmium accumulated by exposed cockles. Transplanted cockles were also subjected to some stress effect consisting in significant inhibition of cholinesterase activity by 26.5% when compared with control cockles. Increased levels of malondialdehydes (MDA) following cadmium exposure have been also reported, suggesting that exposed cockles have been submitted to an oxidative stress probably due to the presence of high cadmium contamination in the sediment. Principal component analyses showed that cockles in vivo transplanted into cadmium contaminated sediment and water were standing out progressively from the control group as a function of exposure time. A clear separation of the transplanted cockles from their controls was observed after 45 days' experience. But, the transplanted cockles appeared not having yet reached the same characteristics as the resident cockles from the contaminated site.

 

Zaldibar, B., I. Cancio, et al. (2008). "Changes in cell-type composition in digestive gland of slugs and its influence in biomarkers following transplantation between a relatively unpolluted and a chronically metal-polluted site." Environ Pollut 156(2): 367-79.

                Changes in cell-type composition (CCTC) is a general phenomenon that takes place in the digestive gland epithelium of stressed molluscs. The aim of the present work was to determine whether CCTC is a reversible process in the digestive gland of sentinel slugs chronically exposed to metal pollution and how CCTC affects metal accumulation parameters and different cell and tissue biomarkers of exposure and effect. Slugs (Arion ater) from an abandoned zinc mine were transferred to a relatively unpolluted site and the other way around for 3, 10 and 28 d. The volume density of black silver deposits (Vv(BSD)) after autometallography, and metallothionein (MT) levels were used as biomarkers of exposure to metals and CCTC and lysosomal responses were selected as effect biomarkers. Results indicated that slugs were sensitive to recent metal pollution; however, slugs chronically exposed to metals presented some characteristic features and were less responsive to pollution cessation without signs of CCTC reversal.

 

Verlecar, X. N., K. B. Jena, et al. (2008). "Modulation of antioxidant defences in digestive gland of Perna viridis (L.), on mercury exposures." Chemosphere 71(10): 1977-85.

                Sub-lethal effects of mercury exposure (110th of LC(50), i.e. 0.045 mg l(-1)) for 5, 10 and 15 d was investigated on oxidative stress parameters and antioxidant defences in digestive gland of Perna viridis. In addition to this an in vitro effect of mercury single and supplemented with reduced glutathione on lipid peroxidation was studied. Increased lipid peroxidation (during first 10 days and also during in vitro exposures), protein carbonyl and hydrogen peroxides (from 5th till last day of exposure) indicate the resultant oxidative stress in the mercury exposed specimen. DNA damage (F-value) response although less distinct on 5th and 15th d, its low values on 10th d and significant correlation with hydrogen peroxide suggests the toxic role of free radicals towards DNA integrity. Superoxide dismutase, which remains low initially (5th d) and increases later suggests its immediate response against superoxide radical. Higher activities of catalase, glutathione peroxidase and glutathione reductase on 15th d and glutathione-S-tranferase from 10th d onwards suggests the adaptive behaviour of the tissue against oxyradicals. Increasing levels of non-enzymatic antioxidant molecules, such as reduced glutathione and ascorbic acid indicated its involvement in counteracting oxidative damage. Further role of reduced glutathione in reducing Hg toxicity is evident in in vitro experiments where lipid peroxidation remains low in mercury concentrations supplemented with reduced glutathione. The elevated levels of metallothionein from 5th to 10th d suggest involvement of this protein in detoxification of reactive oxygen species and toxic metal. The above results suggest that both enzymatic and non-enzymatic antioxidants play an important role in protecting cell against Hg toxicity, which can be used as a biomarker of metal contamination in aquatic environment.

 

Fasulo, S., A. Mauceri, et al. (2008). "Expression of metallothionein mRNAs by in situ hybridization in the gills of Mytilus galloprovincialis, from natural polluted environments." Aquat Toxicol 88(1): 62-8.

                Metallothioneins (MTs), metal-inducible proteins, are crucial proteins for the regulation of essential metals, and are transcriptionally induced in all organisms by certain heavy metals, oxidative stress and inflammation. The gills represent an organ of uptake and loss of metals in which different mechanisms are present controlling the functions directly involved in the maintenance of homeostasis. In this study, the morphological and histomorphological aspects of branchial epithelium in Mytilus galloprovincialis from polluted environment (Faro swamp, Messina, Italy) have been investigated. The reverse transcriptase-polymerase chain reaction (PCR) has been used to isolate complementary DNA of both MT isoforms present from RNA extracted from mussel gills. The respective mRNAs on histological sections have been visualized by in situ hybridization. These methods showed that MT-10 mRNA is expressed at the basal level. In contrast, the MT-20 expression level was very low under basal conditions, while its mRNA increased dramatically in individuals collected in Faro. The presence of acid mucocytes and MTs in the gills may be considered a further defensive mechanism also related to the significantly higher concentration of Cd, Pb and Cr found in gills of M. galloprovincialis from Faro than specimens from the reference site (Goro). The results obtained show that, in stressed mussels, the defensive processes increase to maintain the normal functions of the organs more exposed to the action of polluted substances.

 

Calisi, A., M. G. Lionetto, et al. (2008). "Morphometric alterations in Mytilus galloprovincialis granulocytes: a new biomarker." Environ Toxicol Chem 27(6): 1435-41.

                Bivalve molluscs, particularly marine mussels, are used widely as sentinel organisms in environmental quality monitoring and assessment. Biochemical and cellular responses to pollutant exposure (i.e., biomarkers) increasingly are investigated in mussel tissues and their measurements largely used in marine environmental monitoring. The aim of the present study was to investigate possible pollutant-induced morphometric alterations in Mytilus galloprovincialis granulocytes in view of future applications as a sensitive, simple, and quick biomarker for monitoring and assessment applications. Granulocyte morphometric alterations were determined by image analysis on Diff-Quick stained cells. For the first time, the rapid alcohol-fixed Diff-Quick stain protocol, utilized in clinical and veterinary applications for immediate interpretation of histological samples, was shown to be suitable for rapid cytological staining of M. galloprovincialis haemocytes. The present study was carried out in standardized laboratory conditions and further validated in field conditions. Results show consistent pollutant-induced enlargement of mussel granulocytes. This was verified by standardized biomarkers such as metallothionein concentrations in the digestive gland or lysosomal membrane stability in laboratory and field exposures. Results further suggest that the observed morphometric alterations can be used as a biomarker of chemical stress. Because no single biomarker generally is adequate for describing the complexity of effects induced by environmental pollutants on the organisms, we propose that morphometric alterations of granulocytes should be used in a biomarker battery in marine environmental monitoring programs such as Mussel Watch.

 

Orihuela, R., J. Domenech, et al. (2008). "The metal-binding features of the recombinant mussel Mytilus edulis MT-10-IV metallothionein." J Biol Inorg Chem 13(5): 801-12.

                In contrast with the paradigmatic mammalian metallothioneins (MTs), mollusc MT systems consist at least of a high-cadmium induced form, possibly involved in detoxification, and another isoform either constitutive or regulated by essential metals and probably associated with housekeeping metabolism. With the aim of providing a deeper characterization of the coordination features of a molluscan MT peptide of the latter kind, we have analyzed here the metal-binding abilities of the recombinant MeMT-10-IV isoform of Mytilus edulis (MeMT). Also, comparison with other MTs of this type has been undertaken. A synthetic complementary DNA was constructed, cloned and expressed into two Escherichia coli systems. Upon zinc coordination, MeMT folds in vivo into highly chiral and stable Zn(7) complexes, with an exceptional reluctance to fully substitute cadmium(II) and/or copper(I) for zinc(II). In vivo cadmium binding leads to homometallic Cd(7) complexes that structurally differ from any of the in vitro prepared Cd(7) complexes. Homometallic Cu-MeMT can only be obtained in vitro from Zn(7)-MeMT after a great molar excess of copper(I) has been added. In vivo, two different heterometallic Zn,Cu-MeMT complexes are recovered, which nicely correspond to two distinct stages of the in vitro zinc/copper replacement. These MeMT metal-binding features are consistent with a physiological role related to basal/housekeeping metal, mainly zinc, metabolism, and confirm the correspondence between the MeMT gene response pattern and the functional properties of the encoded protein.

 

Hispard, F., A. de Vaufleury, et al. (2008). "Effects of subchronic digestive exposure to organic or inorganic cadmium on biomarkers in rat tissues." Ecotoxicol Environ Saf 70(3): 490-8.

                In an experimental food chain, Wistar rats were fed cadmium (Cd) in an inorganic (CdCl(2)) or organic (mainly associated with metallothionein from Helix aspersa snail viscera) form. After 1 month of exposure to 100 microg inorganic Cd g(-1) in food, an induction of metallothionein was observed in all target tissues. In liver, glutathione peroxidase (GSH-Px) activity decreased and alanine aminotransferase (ALAT) activity increased, suggesting that Cd causes hepatotoxicity. However, lipid peroxidation as well as catalase and caspase 3 (a marker of apoptosis) activities were not modified. At a rather low exposure (2.5 microg Cd g(-1)), metallothionein level in the kidney was found to be the most sensitive biomarker of exposure for both Cd forms. In the small intestine of rats ingesting inorganic Cd, metallothionein expression was significantly higher than that observed for rats fed organic Cd. Present results allowed proposing a simple design to assess the effect of a chemical in a trophic transfer approach.

 

Hispard, F., D. Schuler, et al. (2008). "Metal distribution and metallothionein induction after cadmium exposure in the terrestrial snail Helix aspersa (Gastropoda, Pulmonata)." Environ Toxicol Chem 27(7): 1533-42.

                The aim of the present work was to study the effect of Cd2+ exposure on metallothionein (MT) induction and on the distribution of metals (Cd, Cu, and Zn) in the terrestrial pulmonate Helix aspersa. In particular, the soluble and nonsoluble pools of the accumulated metals and their tissue distribution in uncontaminated and contaminated edible snails were investigated after a two-week exposure to Cd2+. In the soluble cytosolic pool of the midgut gland of H. aspersa, three metal-specific putative MT isoforms were separated following a fractionation protocol with diethylaminoethyl cellulose, size-exclusion chromatography, ultrafiltration, and reversed-phase high-performance liquid chromatography (RP-HPLC). Interestingly, one of the above isoforms seems to bind both Cd and Cu, which may in addition mobilize, after induction by Cd2+, some of the intracellular Cu and, thus, perhaps increase the Cu pool in the cytosolic fraction. The cDNA and its translated amino acid sequence of a Cd2+-binding MT isoform from the snail midgut gland was characterized and attributed to one of the putative MT isoforms obtained by RP-HPLC. The amino acid sequence of this Cd-MT isoform of H. aspersa differed from similar sequences described in other terrestrial pulmonates, such as Helix pomatia or Arianta arbustorum, by only a few amino acids (n = 4 and 8, respectively). That the identified Cd-MT from H. aspersa is inducible by Cd2+ also was shown, chromatographic evidence aside, by a specific polymerase chain reaction protocol on a cDNA basis, which included a noninducible housekeeping gene as a control.

 

Company, R., A. Serafim, et al. (2008). "Antioxidant biochemical responses to long-term copper exposure in Bathymodiolus azoricus from Menez-Gwen hydrothermal vent." Sci Total Environ 389(2-3): 407-17.

                Copper (Cu) is essential to various physiological processes in marine organisms. However, at high concentrations this redox-active transition metal may enhance the formation of reactive oxygen species (ROS) and subsequently initiate oxidative damage. High concentrations of Cu may increase oxidative damage to lipids, proteins and DNA. Bathymodiolus azoricus is a Mytilid bivalve very common in hydrothermal environments near the Azores Triple Junction continuously exposed to high metal concentrations, including Cu, emanating from the vent fluids. The knowledge of antioxidant defence system and other stress related biomarkers in these organisms is still scarce. The aim of this work was to study the effect of Cu (25 microg l(-1); 24 days exposure; 6 days depuration) on the antioxidant stress biomarkers in the gills and mantle of B. azoricus. The expression of stress related biomarkers was tissue-dependent and results suggest that other factors than metal exposure may influence stress biomarkers, since little variation in antioxidant enzymes activities, MT concentrations, LPO and total oxyradical scavenging capacity (TOSC) occurred in both control and Cu-exposed mussels. Moreover, there is a general tendency for these parameters to increase with time, in both control and Cu-exposed mussels, suggesting that reactive oxygen species (ROS) formation is not metal dependent, and may be related with poor physiological conditions of the animals after long periods in adverse conditions compared to those in hydrothermal environments.

 

Machreki-Ajmi, M., I. Ketata, et al. (2008). "The effect of in situ cadmium contamination on some biomarkers in Cerastoderma glaucum." Ecotoxicology 17(1): 1-11.

                The present study was designed to detect in situ effects of cadmium on marine organisms Cerastoderma glaucum from the gulf of Gabes (Tunisia). Six sampling stations were chosen: one site, relatively far from known local source of pollution, was considered as uncontaminated site and five sites clearly exposed to anthropogenic impact. Metallothionein like protein (MTLP) and sub-cellular metal partitioning were measured in cockles (C. glaucum) gills and digestive gland. Various biomarkers were also measured, including malondialdehyde (MDA) in the digestive gland and acetylcholinesterase activity (AChE) in the remainder. The health status of the cockles was assessed by using the condition index (CI). Significant differences between sites were noted for Cd concentrations, (CI) and also for the three studied biomarkers. Significant higher biomarkers response was measured in cockles from stations located in the northern part of the gulf, which are exposed directly to industrial and urban effluents, whereas the response of most biomarkers was minimal at the reference station. Positive and significant correlations were observed between MTLP and Cd concentrations in the digestive gland and in the gills. However, it must be noted that these correlations were more significant in the digestive gland, suggesting that compared to the gills, the digestive gland of C. glaucum is more suitable for monitoring metal pollution. The subcellular distribution of Cd showed that the soluble fraction was the major compartment for Cd storage, a pattern which is due to the role of MTLP in Cd detoxication. But at the most contaminated site (EH), cadmium in the digestive gland was preferentially accumulated in the insoluble fraction (P1) suggesting that the MTLP capacity in binding metals was not sufficient to avoid the binding of Cd to the insoluble fraction. Furthermore, the MTLP concentrations in the cockles from this site are lower than expected. So in highly polluted sites, MTLP in C. glaucum should not be used as a useful biomarker for metal pollution. The lipid peroxidation as presented by malondialdehyde levels, and MT-like protein concentrations increased in cockles exposed to cadmium contamination. We can, therefore, hypothesize that Cd could induce MTLP synthesis and MDA increase. While AChE had distinct and specific pattern showing that cadmium is not the only factor of the inhibition of cholinesterase activity. There are other polluting inputs engendering this inhibition.

 

Gorbi, S., C. Virno Lamberti, et al. (2008). "An ecotoxicological protocol with caged mussels, Mytilus galloprovincialis, for monitoring the impact of an offshore platform in the Adriatic Sea." Mar Environ Res 65(1): 34-49.

                An ecotoxicological protocol with caged mussels, Mytilus galloprovincialis, was developed to evaluate the potential impact of an offshore gas platform in the central Adriatic Sea. Reference organisms were collected on a seasonal basis from an unpolluted site and transplanted for four weeks in both the sampling area and to the investigated platform. Chemical analyses of trace metals in mussel tissues were integrated with a multi-biomarker approach for the early detection of biological responses at several cellular targets. Induction of metallothioneins, peroxisomal proliferation and activity of acetylcholinesterase were measured as markers for specific classes of chemicals. Special attention was given to oxyradical metabolism and appearance of oxidative-mediated toxicity to reveal a more general onset of cellular disturbance. In addition to individual antioxidants (superoxide dismutase, catalase, glutathione S-transferases, glutathione reductase, Se-dependent and Se-independent glutathione peroxidases, and levels of total glutathione), the total oxyradical scavenging capacity (TOSC) allowed a quantification of the overall capability to neutralize specific forms of intracellular reactive oxygen species (ROS; i.e. peroxyl and hydroxyl radicals). Cellular damages were evaluated as lysosomal destabilization (membrane stability, accumulation of lipofuscin and neutral lipids), lipid peroxidation products (malondialdehyde) and DNA integrity (strand breaks and micronuclei); the air survival test was finally applied to evaluate the overall physiological condition of mussels. Concentration of trace metals (As, Ba, Cd, Cr, Cu, Fe, Hg, Mn, Ni, Pb, Zn) revealed only limited variations in transplanted mussels during various experimental periods and such changes appeared partly related to natural fluctuations. Among biological responses, variations of antioxidants and lysosomal stability were confirmed as sensitive early warning signals for biological disturbance of both natural and anthropogenic origin. The presented protocol with caged mussels allowed marked biological effects caused by the investigated platform to be excluded, and represented a useful approach that is easy to extend for monitoring the impact of offshore activities in the Adriatic sea.

 

Ivanina, A. V., A. S. Cherkasov, et al. (2008). "Effects of cadmium on cellular protein and glutathione synthesis and expression of stress proteins in eastern oysters, Crassostrea virginica Gmelin." J Exp Biol 211(Pt 4): 577-86.

                Cadmium (Cd) is an important toxicant in estuarine and coastal environments that can strongly affect energy balance of aquatic organisms by increasing the organism's basal energy demand and reducing its aerobic capacity. Mechanisms of cadmium-induced increase in basal metabolic costs are not well understood and may involve elevated detoxification costs due to the synthesis of cellular protective proteins and glutathione. We studied the short-term effects of cadmium exposure (4 h) on protein and glutathione (GSH) synthesis and expression of stress proteins (heat shock proteins HSP60, HSP70 and HSP90) and metallothioneins in isolated gill and hepatopancreas cells of the eastern oyster, Crassostrea virginica. Our study showed that exposure to cadmium resulted in a dose-dependent increase in the rate of protein synthesis in oyster cells, which reached 150% of the control at the highest tested Cd level (2000 micromol l(-1)). GSH synthesis was significantly inhibited by the highest Cd concentrations, especially in hepatopancreas, which resulted in a slight but significant decrease in the total GSH concentrations. Elevated protein synthesis was associated with the increased expression of metallothioneins and heat shock proteins. Interestingly, stress protein response differed considerably between gill and hepatopancreas cells. In hepatopancreas, expression of metallothionein mRNA (measured by real-time PCR) increased 2-8-fold in response to Cd exposure, whereas no significant increase in metallothionein expression was found in Cd-exposed gill cells. By contrast, HSP60 and HSP70 protein levels increased significantly in Cd-exposed gill cells (by 1.5-2-fold) but not in hepatopancreas. No change in HSP90 expression was detected in response to Cd exposure in oyster cells. These data indicate that metallothionein expression may provide sufficient protection against Cd-induced damage to intracellular proteins in hepatopancreas, alleviating the need for overexpression of molecular chaperones. By contrast, Cd detoxification mechanisms such as inducible metallothioneins and GSH appear to be insufficient to fully prevent protein damage in gill cells, thus necessitating induction of HSPs as a secondary line of cellular defense. Therefore, gills are likely to be among the most Cd-sensitive tissues in oysters, which may have important implications for impaired oxygen uptake contributing to energy misbalance and reduced aerobic scope in Cd-exposed oysters.

 

Martin-Diaz, M. L., J. Blasco, et al. (2008). "Field validation of a battery of biomarkers to assess sediment quality in Spanish ports." Environ Pollut 151(3): 631-40.

                Two marine invertebrates, the crab Carcinus maenas and the clam Ruditapes philippinarum, were used as bioindicator species to assess contamination when exposed in situ to sediment from different sites from four Spanish ports Cadiz (SW Spain), Huelva (SW Spain), Bilbao (NE Spain) and Pasajes (NE Spain). In an attempt to determine sediments toxicity, a combination of exposure biomarkers was analyzed in both species: metallothionein-like-proteins (MTLPs), ethoxyresorufin O-deethylase (EROD), glutathione S-transferase activity (GST), glutathione peroxidase (GPX) and glutathione reductase (GR). In parallel, physical and chemical characterization of the different sediments was performed and biological responses related to the contaminants. Significant induction of MTLPs was observed when organisms were exposed to metal contaminated sediments (port of Huelva), and EROD and GPX activities after exposure to sediments containing organic compounds (port of Bilbao and Pasajes). No significant interspecies differences were observed in biomarker responses except for the GST and GR.

 

Pan, K. and W. X. Wang (2008). "The subcellular fate of cadmium and zinc in the scallop Chlamys nobilis during waterborne and dietary metal exposure." Aquat Toxicol 90(4): 253-60.

                Subcellular metal distribution has received increasing attention in aquatic toxicology studies, but the relationship between metal distribution and metal biokinetics remains largely unexplored. A series of short-term experiments on different concentrations of dissolved and dietary metals and on metal elimination were conducted to investigate the dynamics of subcellular distribution of Cd and Zn in the scallop Chlamys nobilis, a bivalve species that is known to accumulate very high concentrations of Cd and Zn in its tissues. Our results showed that, in general, both Cd and Zn were sequestered in insoluble forms (organelles, metal-rich granules, and cellular debris). The main binding pool for the newly acquired metals was organelles for Cd and cellular debris for Zn. Metallothionein-like protein (MTLP) was the most important storage pool for Cd in the scallops. Storage in the non-toxic form both in organelles and MTLP instead of through exocytosis was the major detoxification strategy to control Cd and accounted for the low efflux rate of Cd from scallops. In contrast to Cd, the main binding pool for Zn was cellular debris. Significant changes were found in the scallops when they were challenged with different concentrations of metals in the aqueous and food phases. Such changes provide important information on how scallops handle metals when there is increasing metal uptake. The redistribution of Zn among each subcellular compartment was much faster than the redistribution of Cd, suggesting an effective regulation mechanism for Zn in scallops. Thus, knowing subcellular metal distribution helps in studying the toxicity of both waterborne and dietborne metals.

 

Romero-Ruiz, A., J. Alhama, et al. (2008). "New metallothionein assay in Scrobicularia plana: heating effect and correlation with other biomarkers." Environ Pollut 156(3): 1340-7.

                Metallothionein (MT) and other biomarker levels were measured in Scrobicularia plana clams to assess pollution of the Guadalquivir Estuary possibly affected by metals released from Aznalcollar pyrite mine in 1998. After optimizing reagent concentrations for monobromobimane derivatization, MT levels were quantified by reversed-phase high-performance liquid chromatography coupled to fluorescence detection (RP-HPLC-FD) in heated or unheated digestive gland extracts and compared to those obtained by differential pulse polarography (DPP). MT content assayed by RP-HPLC-FD in unheated samples was higher than that obtained by DPP and correlated better with metals and anti-oxidant activities. MT assay by RP-HPLC-FD in unheated extracts would be preferable for assessing metal pollution, due to its greater sensitivity and specificity. In addition to MT induction, glyoxalase II inhibition was well correlated with metal contents. Our results suggest that metals at the estuary do not originate from Aznalcollar spill, but from those carried along by the river and deposited at its concave bank.

 

Torreggiani, A., J. Domenech, et al. (2008). "Raman study of in vivo synthesized Zn(II)-metallothionein complexes: structural insight into metal clusters and protein folding." Biopolymers 89(12): 1114-24.

                Metallothioneins (MTs) are metal-chelating peptides that play an active role in zinc homeostasis. The participation of metal ligands other than cysteines and the presence of secondary structure elements in metal-MT complexes are fairly unknown, especially in nonvertebrate MTs. Here, four Zn(II) complexes of invertebrate MTs (mollusc, insect, nematode, and echinoderm) and the Zn(II)-MT complex of the mammalian MT1 isoform, heterologously synthesized in E. coli, were studied by analytic and spectroscopic techniques. By Raman and circular dichroism spectroscopy, new structural informations were obtained. The five analyzed MT isoforms consist largely of beta-turns with the near exclusion of alpha-helical segments. Raman spectroscopy was revealed as an useful tool, providing information about the state of the cysteine sulfur atoms (metal coordinated and oxidized), the participation of histidine in metal coordination, and the molecular environment of tyrosine residues. In all the five Zn(II)-MT studied samples, acid-labile sulfide anions were found as nonproteic ligands, since sulfide-containing and sulfide-devoid species coexisted in the corresponding preparations. Significantly, Raman bands useful as markers of sulfide bridging ligands were identified. Overall, this work illustrates how the combination of analytical and spectroscopic techniques can be a very informative approach for the analysis of in vivo-synthesized metal-MT complexes, providing new data on the metal binding behavior of MTs from the most diverse organisms.

 

Pytharopoulou, S., E. Sazakli, et al. (2008). "Translational responses of Mytilus galloprovincialis to environmental pollution: integrating the responses to oxidative stress and other biomarker responses into a general stress index." Aquat Toxicol 89(1): 18-27.

                Heavy metals are commonly associated with the generation of reactive oxygen species (ROS), which may cause oxidative damage to several cellular macromolecules and organelles. In an attempt to correlate biomarker responses to oxidative stress, caged mussels (Mytilus galloprovincialis) were exposed for 30 days in a relatively clean site and two areas (Stations 1 and 2) unevenly polluted by heavy metals in Gulf of Patras (Greece). Three periods of caging were: one in winter, the second in spring, and the third in autumn. Heavy metal content was determined in digestive glands of the exposed mussels as a measure of metal pollution, metallothionein content as an adaptive and detoxifying index, lysosomal membrane stability as a biomarker of general stress, superoxide radical production and lipid peroxidation as indicators of oxidative stress, and micronucleus frequency in gill cells as an index of chromosomal damage. Considering that protein-synthesizing machinery is one of the candidate targets for ROS, the in vivo activity of ribosomes in digestive glands was also tested. Compared with the reference samples, mussels transplanted to Station 1 showed increased levels of heavy metals and metallothionein in digestive glands, lower lysosomal membrane stability, higher values in oxidative stress indices, reduced activity of ribosomes, and increased chromosomal damage in gill cells. In addition, run-off ribosomes isolated from mussels transplanted to Station 1 were less efficient at initiating protein synthesis in a cell-free system than those from mussels in the reference site. Mussels transplanted to Station 2 exhibited similar but less pronounced responses. Statistical analysis revealed a strong positive correlation of ribosomal activity with lysosomal membrane stability, as well as a significant negative correlation with the oxidative stress indices, metallothionein content, micronucleus frequency, and the digestive gland content in Cr, Cu and Mn. Integration of all the measured biomarker responses into one general "stress index" demonstrated a clear distinction between the sampling sites, allowing classification along a pollution gradient (reference site<Station 2<Station 1), independently from the season. Moreover, this analysis allowed us to compare responses between sampling campaigns and showed that the biomarker responses show best integration in winter. It was also evident that climatic or metabolic changes could modulate bioavailability of pollutants and priming of cellular defence processes.

 

Lilja, K., A. Prevodnik, et al. (2008). "Regional differences in mRNA responses in blue mussels within the Baltic proper." Comp Biochem Physiol C Toxicol Pharmacol 148(2): 101-6.

                Mussels (Mytilus sp.) from two regions along the permanent salinity gradient within the Baltic proper were exposed to copper (35 ppb) or petrol (0.3 mL/L) for 10 days and analyzed for mRNA expressions in gill tissue. Expression of mRNAs for the heat shock proteins HSP70 and HSP90 was significantly induced by copper, but not by petrol. For the metallothioneins MT10 and MT20, regional differences in mRNA expressions could be seen. In mussels from the northern Baltic proper, MT20 expression increased 2.8 and 3.4 times, after exposure to copper and petrol, respectively. In contrast, no change could be seen in MT20 expression for mussels from the southern Baltic proper. MT10 showed a peculiar expression not previously described. For some mussels, no expression at all was detected, some showed a weak expression and for some individuals a strong expression could be seen. For the mussels from the southern Baltic proper, the number of individuals with a strong expression of MT10 increased from 1 out of 18 (control), to 7 and 8, after exposure to copper and petrol, respectively. The results clearly show that responses vary between different regions within the Baltic proper, which emphasises the importance to study interactions between contaminants, populations and regions.

 

Amiard, J. C., R. Journel, et al. (2008). "Influence of field and experimental exposure of mussels (Mytilus sp.) to nickel and vanadium on metallothionein concentration." Comp Biochem Physiol C Toxicol Pharmacol 147(3): 378-85.

                In mussels impacted by the V- and Ni-rich oil dispersed in the environment after the << Erika >> wreck, a significant and positive correlation was observed between metallothionein (MT) and V concentrations whereas no correlation was observed between MT and Ni concentrations. Mussels were exposed separately to V and Ni in the laboratory. The highest concentrations tested (> or =2000 ng V per individual; > or =50 ng Ni per individual) were responsible for V and Ni bioaccumulation. In the case of V, the metal was preferentially stored in the insoluble fraction of the soft tissues but with increasing body burden, the part of soluble V increased from 19 to 41%. Metallothionein was induced in mussels exposed to V but not in those exposed to Ni. In addition, in mussels originating from a shellfish farming area which showed abnormally high levels of V, depuration showed concomitant decreases of V and MT. It is the first report in the literature of a functional link between MT and V. When interpreting surveys of metal pollution based upon the use of MT, one must be aware that V may be responsible for MT synthesis, explaining likely high levels of this biomarker in the absence of metals known as MT inducers.

 

Buico, A., C. Cassino, et al. (2008). "Radical scavenging abilities of fish MT-A and mussel MT-10 metallothionein isoforms: An ESR study." J Inorg Biochem 102(4): 921-7.

                Metallothioneins (MTs) are cysteine-rich proteins involved in homeostasis of essential metals, detoxification of toxic metals and scavenging of free radicals. Scavenging of the stable 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical was measured by means of ESR spectroscopy for two recombinant MTs from aquatic species: MT-10 from the sea mussel Mytilus galloprovincialis, and MT-A from the fish Oncorhyncus mykiss. Both the zinc- and the cadmium-loaded forms (Zn(7)-MTs and Cd(7)-MTs) were analysed, using the commercial MT-II (Zn(7)-MT-II and Cd(7)-MT-II, respectively) from rabbit liver as a reference. A decrease in the scavenging ability was observed for all the three MTs passing from the Zn- to the Cd-loaded forms, because of the higher stability of the Cd-mercapto complex. The Zn(7)-MTs from aquatic species were more effective in scavenging DPPH signal than the rabbit Zn(7)-MT-II (2.8 and 4-folds, respectively). Similar results were obtained also for the Cd(7)-MTs, thus confirming the stronger antioxidant power of MTs from aquatic organisms compared with the rabbit MT-II. Moreover, mussel MT-10 was more active in DPPH scavenging than fish MT-A. When the complete release of metals from MTs was obtained by lowering the pH to 3 or, alternatively, by adding the chelating agent diethylenetriaminepentaacetic acid (DTPA), an increase in the scavenging ability of MTs was observed.

 

Choi, Y. K., P. G. Jo, et al. (2008). "Cadmium affects the expression of heat shock protein 90 and metallothionein mRNA in the Pacific oyster, Crassostrea gigas." Comp Biochem Physiol C Toxicol Pharmacol 147(3): 286-92.

                Cadmium (Cd) is a widespread nonessential heavy metal that enters the aquatic environment as a result of natural processes and human activities such as wastewater production, agriculture, and mining. To determine the effects of Cd on organisms, we investigated its time- and dose-related effects on mRNA levels of heat shock protein 90 (HSP90) and metallothionein (MT) in the gill and digestive gland and changes enzyme levels in the hemolymph of the Pacific oyster Crassostrea gigas. Full-length HSP90 cDNA was isolated from C. gigas by rapid amplification of cDNA end (RACE) techniques and found to contain 2154 nucleotides, including an open reading frame, and was predicted to encode a protein of 717 amino acids. BLAST analysis indicated that the HSP90 gene of C. gigas shared high homology with known HSP90 genes of other mollusks. The expression of HSP90 mRNA increased significantly with exposure to 0.01 ppm Cd for 11 days or 0.05 or 0.1 ppm Cd for 7 days. The expression of MT mRNA increased significantly with exposure to 0.01, 0.05, or 0.1 ppm Cd for 11 days. Glutamate oxaloacetate and glutamate pyruvate levels increased significantly with exposure to 0.05 or 0.1 ppm Cd for 7 days. These results indicate that HSP90 and MT play important roles in the physiological changes related to metabolism and cell protection that occur in Pacific oysters exposed to Cd.

 

Hispard, F., A. de Vaufleury, et al. (2008). "Comparison of transfer and effects of Cd on rats exposed in a short experimental snail-rat food chain or to CdCl2 dosed food." Environ Int 34(3): 381-9.

                Transfer and toxic effects of two cadmium (Cd) forms, inorganic (CdCl2 dosed rat food) or organic (contaminated snail-based rat food) were studied in Wistar rat. Cd concentrations in rat food were 0 and 2.5 microg Cd g(-1) for both inorganic and organic forms and a high concentration of 100 microg Cd g(-1) was also tested for the inorganic form. Rats were exposed for four weeks to contaminated food. Both forms of Cd were bioavailable to rats, with a percentage of transfer from food to rats of around 1% for all contaminated groups. Cd concentrations in rat tissues increased with increasing Cd concentrations in the food. Rats fed with organic form of Cd accumulated significantly more Cd in the main organ for Cd toxicity, the kidney, than those eating the inorganic form. Survival was not affected for any rat group but a decrease in growth and food consumption was observed for the inorganic form. As a defence system against Cd toxicity, rats increased their metallothionein (MT) synthesis at the highest Cd concentration in the target organs (kidney, liver and small intestine) and even did the same at low Cd concentrations (2.5 microg Cd g(-1)) in the kidney. At this low Cd concentration, MT induction was lower in the small intestine of rats ingesting organic Cd than those ingesting inorganic Cd. Bioavailability of organic and inorganic forms of Cd was similar, but subsequent Cd distribution within organs was different. This quantification of the trophic transfer of both inorganic and organic forms of a toxicant is a basis for a better assessment of the fate and effects of chemicals in food webs.

 

Vergani, L., M. Grattarola, et al. (2007). "Molecular characterization and function analysis of MT-10 and MT-20 metallothionein isoforms from Mytilus galloprovincialis." Arch Biochem Biophys 465(1): 247-53.

                Structure and function of molluscan metallothioneins (MTs) are still poorly understood. The sea mussel Mytilus galloprovincialis displays two MT isoforms which differ in both primary sequences and physiological functions. MT-10 is the constitutive isoform, whereas MT-20 is mainly induced by cadmium (Cd). Both MTs were produced as recombinant proteins and showed identical Cd content and similar Cd-binding properties. Conversely, circular dichroism disclosed marked differences in the secondary conformations of the two Cd(7)-MTs. The possible relapses of these structural differences on protein stability and function were assessed. MT-10 presented a higher thermal stability and a more compact structure than MT-20, as it was inferred by absorption and emission spectroscopy studies. Moreover, the kinetics of Cd-release clearly indicated that MT-10 is much more sensitive to oxidation than is MT-20. The observed differences between MT-10 and MT-20 are discussed in terms of the different physiological roles exerted by the two isoforms in mussel.

 

Donnini, F., E. Dinelli, et al. (2007). "A biological and geochemical integrated approach to assess the environmental quality of a coastal lagoon (Ravenna, Italy)." Environ Int 33(7): 919-28.

                The assessment of the environmental quality of coastal lagoons is rather difficult as their intrinsic high variability limits the power of analytical chemistry and ecotoxicological approaches. Aim of the present work was to integrate geochemical and biological data which were simultaneously collected from the Pialassa Baiona lagoon (Ravenna, Italy), and evaluate the usefulness of a biomonitoring strategy based on sentinel organisms and biomarkers. Geochemical analysis on suspended, surface, and subsurface sediments were performed at five different sites of the lagoon; moreover the heavy metal bioaccumulation and a battery of biomarkers was evaluated in Mytilus galloprovincialis transplanted in the same sites. Heavy metals appeared to be diffused throughout the lagoon reaching higher levels in the southernmost sites, and mainly concentrated in suspended sediments. Lysosome membrane stability, neutral lipid and lipofuscin accumulation, and metallothionein levels in mussels indicated the development of a stress syndrome in animals exposed to the polluted sites.

 

Santos, H. M., M. S. Diniz, et al. (2007). "Toxicological effects and bioaccumulation in the freshwater clam (Corbicula fluminea) following exposure to trivalent arsenic." Environ Toxicol 22(5): 502-9.

                Contamination of aquatic environments by arsenic is a serious worldwide problem. The main objective of this work was to evaluate the response of a freshwater clam (Corbicula fluminea) to arsenic (As III) exposure and infer its potential as a biological indicator of contamination. Metallothioneins (MTs) were used as indicators of metalloid toxicity in combination with an histological and histochemical evaluation. After a period of acclimatization in the laboratory, 50 C. fluminea (0.4 g +/- 0.1) were exposed to different nominal concentrations of arsenic (100, 300, 500, and 1000 microg L(-1)) for 7 days. The concentration of total As III in the water and in the tissues of the organisms was determined by atomic absorption spectrometry, and MTs were quantified through differential pulse polarography. Results suggest that the organisms exposed to the concentrations of 300 and 1000 microg As L(-1) accumulated the highest levels of As III in the tissues (17 +/- 9 and 15 +/- 3 microg g(-1) distilled water, respectively), which was confirmed through histochemical analysis. An apparent induction of MTs was also observed in the organisms exposed to As III, suggesting that C. fluminea possesses some capacity for arsenic regulation. The results suggest that the induction of MTs may be of high interest as a biomarker for arsenic contamination in aquatic environments, and confirms the potential of C. fluminea as a biological indicator.

 

Diniz, M. S., H. M. Santos, et al. (2007). "Metallothionein responses in the Asiatic clam (Corbicula fluminea) after exposure to trivalent arsenic." Biomarkers 12(6): 589-98.

                The main objective of this work was to evaluate arsenic effects on metallothionein (MT) induction by exposing a freshwater Asiatic clam (Corbicula fluminea) to different concentrations of this metalloid. The presence of MT-like proteins was detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis and compared with a standard rabbit MT. In addition, the polarographic response showed good correspondence between standard MT and MT-like curves from C. fluminea, allowing MT quantification. The results show that clams exposed to different concentrations of arsenic are able to induce significant levels of MTs. Although variability was found in MT induction, significant differences in MT levels were found after 28 days of exposure in all treatments in comparison with the controls, suggesting that exposure to arsenic induced MT-like proteins in C. fluminea.

 

Santiago-Rivas, S., A. Moreda-Pineiro, et al. (2007). "Pressurized liquid extraction-assisted mussel cytosol preparation for the determination of metals bound to metallothionein-like proteins." Anal Chim Acta 603(1): 36-43.

                The possibilities of pressurized liquid extraction (PLE) have been novelty tested to assist the cytosol preparation from wet mussel soft tissue before the determination of metals bound to metallothionein-like proteins (MLPs). Results obtained after PLE were compared with those obtained after a classical blending procedure for mussel cytosolic preparation. Isoforms MLP-1 (retention time of 4.1 min) and MLP-2 (retention time of 7.4 min) were separated by anion exchange high-performance liquid chromatography (HPLC) and the concentrations of Ba, Cu, Mn, Sr and Zn bound to MLP isoforms were directly measured by inductively coupled plasma-atomic emission spectrometry (ICP-OES) as a multi-element detector. The optimized PLE-assisted mussel cytosol preparation has consisted of one extraction cycle at room temperature and 1500 psi for 2 min. Since separation between the solid mussel residue and the extract (cytosol) is performed by the PLE system, the cytosol preparation method is faster than conventional cytosol preparation methods by cutting/blending using Ultraturrax or Stomacher devices.

 

Gagne, F., C. Andre, et al. (2007). "Toxicological effects of primary-treated urban wastewaters, before and after ozone treatment, on freshwater mussels (Elliptio complanata)." Comp Biochem Physiol C Toxicol Pharmacol 145(4): 542-52.

                This study examined the toxic potential of a primary-treated municipal effluent, before and after ozonation, in freshwater mussels. Animals were exposed to various concentrations (0, 1, 3, 10 and 20% v/v) of a primary-treated effluent and also after a treatment with ozone at 10 mg/L in continuous flow-through mode for seven weeks. A suite of biomarkers was used to assess the potential toxic effects of various contaminants typically present in municipal wastewaters: heavy metal metabolism (metallothioneins and labile zinc), cytochrome P4501A1 and 3A4, glutathione S-transferase activities (biotransformation of organic compounds), lipid peroxidation and xanthine oxidoreductase (oxygen radical scavenging), DNA damage, mitochondrial electron transport activity at various temperatures and gonad lipid levels (cellular energy allocation) and aspartate transcarbamoylase and dihydrofolate reductase (gonad activity). On the one hand, some biomarkers, including metallothioneins, labile zinc, glutathione S-transferase, cytochrome P4503A4 activity, dehydrofolate reductase and aspartate transcarbamoylase, were readily decreased. In contrast, these biomarkers, cytochrome P4501A1, gill lipid peroxidation, DNA strand breaks in gills and digestive gland, mitochondrial electron transport at high and low temperatures (temperature-dependent activity) and total gonad lipids, were readily increased. In general, ozone treatment reduced adverse effects by either decreasing the intensity of the toxic responses or increasing the threshold concentration. For gill lipid peroxidation, however, intensity was greater at a higher threshold concentration. Ozone treatment eliminated the temperature sensitivity of the mitochondrial electron transport system, indicating a loss of interaction between temperature and urban pollution in terms of energy expenditure in mussels. Ozone treatment could significantly decrease either the toxic potency or intensity of urban pollutants at the expense of increased oxidative stress in gills of freshwater mussels.

 

Serafim, A. and M. J. Bebianno (2007). "Kinetic model of cadmium accumulation and elimination and metallothionein response in Ruditapes decussatus." Environ Toxicol Chem 26(5): 960-9.

                The aim of the present study was to determine the response of metallothionein (MT) during Cd accumulation and elimination in different tissues of the estuarine bivalve Ruditapes decussatus exposed to two nominal Cd concentrations (4 and 40 microg/L) for 40 d, followed by a depuration period of 50 d. Cadmium was accumulated in all tissues of R. decussatus at both exposure concentrations, and the accumulation was tissue dependent. Use of the kinetic model showed that in the gills and remaining tissues, Cd was assimilated faster at the beginning of the exposure and decreased with time, possibly limited by the diffusion rate of this metal within the cell. In the digestive gland, however, the Cd was continuously accumulated. This could reflect that the Cd uptake rate is considerably higher than the loss rate and, therefore, that this tissue has a higher capacity to accumulate Cd compared to the other two tissues. Moreover, the application of this kinetic model in the different subcellular fractions showed that the bioconcentration factor was significantly higher in the low-molecular-weight fraction (where MT is found), suggesting that this fraction binds Cd faster, with a high uptake rate (K(u) = 32/d), and eliminates this metal more slowly (K(1) = 0.005/d). During the depuration phase, MT decreased simultaneously with Cd elimination in all tissues, although with a shorter half-life. In conclusion, the MT response prevented Cd in the tissues of R. decussatus from interfering in the normal clam metabolism; therefore, MT acts as a detoxification mechanism of Cd.

 

Farcy, E., C. Voiseux, et al. (2007). "Seasonal changes in mRNA encoding for cell stress markers in the oyster Crassostrea gigas exposed to radioactive discharges in their natural environment." Sci Total Environ 374(2-3): 328-41.

                The North Cotentin area (Normandy, France) hosts several nuclear facilities among which the AREVA reprocessing plant of La Hague is responsible for controlled discharges of liquid radioactive wastes into the marine environment. The resulting increase in radioactivity is very small compared to natural radioactivity. However, concerns about environment protection prompted the scientific community to focus on the effects of the chronic exposure to low concentrations of radionuclides in non-human biota. This study contributes to the evaluation of the possible impact of radioactive discharges on the oyster Crassostrea gigas in the field. Real-time polymerase chain reaction was used to quantify the expression levels of genes involved in cell stress in the oyster. They included members of the heat shock protein family (Hsp70, Hsc72, Hsp90), superoxide dismutase (SOD) and metallothionein (MT). Times series measurements were built from periodic samplings in the natural environment in order to characterize the natural variability as well as possible seasonal fluctuations. The genes studied exhibited a general seasonal expression pattern with a peak value in winter. The data inversely correlated with seawater temperature and the nature of the relationship between gene expression and temperature is discussed. In parallel, oysters were collected in four locations on the French shores, exposed or not to radioactive liquid wastes from the nuclear facilities hosted in the North Cotentin. The comparison of data obtained in the reference location on the Atlantic coast (not exposed) and data from oysters of the English Channel (exposed) gave no evidence for any statistical difference. However, because of the complexity of the natural environment, we cannot rule out the possibility that other parameters may have masked the impact of radioactive discharges. This dense set of data is a basis for the use of the expression levels of those genes as biomarkers to address the question of the possible effects of chronic exposure of the oyster to low concentrations of radionuclides in controlled laboratory experimental conditions.

 

Choi, H. J., J. Ji, et al. (2007). "Cadmium bioaccumulation and detoxification in the gill and digestive gland of the Antarctic bivalve Laternula elliptica." Comp Biochem Physiol C Toxicol Pharmacol 145(2): 227-35.

                Exposure to a sublethal concentration of cadmium (Cd; 50 microg L(-1)) resulted in significantly increased Cd concentrations in the gill and digestive gland of the Antarctic bivalve Laternula elliptica. Continuous accumulation of Cd in the two organs during the 14-day exposure period was associated with sequestration of Cd to both the soluble cytosolic and insoluble particulate cell fractions. However, the contribution of each cell fraction to Cd sequestration differed between the two organs; in the gill, a larger portion of Cd was associated with the insoluble fraction, while in the digestive gland, both the soluble and insoluble fractions sequestered similar amounts of Cd. Metal-binding components in the insoluble cell fraction were not identified in this study. On the other hand, a metallothionein-like protein (MTLP) was the major Cd-detoxifying component in the soluble cell fraction of the gill and digestive gland. The amount of MTLP increased linearly with exposure time and the amount of Cd accumulated in the tissue, which suggests a potential utility of MTLP as a biomarker for exposure to Cd and possibly other metals.

 

Park, H., I. Y. Ahn, et al. (2007). "Cloning, expression and characterization of metallothionein from the Antarctic clam Laternula elliptica." Protein Expr Purif 52(1): 82-8.

                The genes for two apparent subtypes of metallothionein (MT) isoform were isolated from the Antarctic clam Laternula elliptica. Determination of the nucleotide sequence showed that the gene consists of 222 bp that code a 73-amino acid protein. The comparison between MT cDNA sequences of L. elliptica and other bivalves showed strong homologies on positions of cysteine residues, which are important for their metal binding abilities. The gene for the MT was inserted into a pET vector and overexpressed as a carboxyl terminal extension of glutathionein-S-transferase (GST) in Escherichia coli. After the GST fusion proteins had been purified by glutathione-Sepharose affinity chromatography column and digested with enterokinase, the MT was purified with gel filtration and analyzed for its biochemical properties. Recombinant MTs were reconstituted with Cd, Cu, and Zn, and kinetic studies of the reactions with electrophilic disulphide, DTNB, were investigated to explore their metal binding ability. It is revealed that the Cd-MT and Zn-MT react with DTNB biphasically, and that Zn-MT reacts with DTNB more rapidly, and with a significantly greater pseudo-first-order rate constant. Cu-MT reacts monophasically and releases metal slowly from MT.

 

Cheung, M. S., L. Zhang, et al. (2007). "Transfer and efflux of cadmium and silver in marine snails and fish fed pre-exposed mussel prey." Environ Toxicol Chem 26(6): 1172-8.

                Subcellular metal distribution may play an important role in the bioaccumulation and trophic transfer of metals in marine food chains. In the present study, we preexposed the green mussel Perna viridis to Ag and Cd and quantified their trophic transfer efficiencies to two predators (whelks [Thais clavigera] and fish [Terapon jarbua]). For the mussels, more Ag was distributed in the metal-rich granule (MRG) fraction following Ag exposure, and more Cd was distributed in the metallothionein-like protein following Cd exposure. In addition, Cd was mainly bound with the proteins having a molecular size of approximately 20 kDa. After being fed with metal-exposed mussels, the assimilation efficiencies of Ag decreased significantly (from 77 to 29-60% in whelks and from 9 to 2% in fish) with an increasing percentage of Ag deposited in the MRG fraction of the prey. In contrast, the assimilation efficiencies of Cd remained comparable (81-85% in whelks and 6-8% in fish), because its partitioning in the soluble fraction of different treatments of the prey was similar. The efflux of Ag and Cd in the two predators was comparable after feeding on preexposed mussel prey. Our results imply that the subcellular distribution of metals in prey may affect the dietary assimilation of metals in predators, but such influence is clearly metal-specific. The present study may lead to a better understanding of metal trophic transfer in different marine food chains.

 

Banni, M., F. Dondero, et al. (2007). "Assessment of heavy metal contamination using real-time PCR analysis of mussel metallothionein mt10 and mt20 expression: a validation along the Tunisian coast." Biomarkers 12(4): 369-83.

                In mussel Mytilus galloprovincialis tissues, metallothionein belongs to two different gene classes, mt10 and mt20, showing differential expression at both basal conditions and under heavy metal challenge. In this study, a new more highly sensitive technique, expression analysis of mt10 and mt20 mRNA levels by quantitative reverse transcription polymerase chain reaction, was used to assess the effects of heavy metal contamination in the digestive glands of mussels caged along the Tunisian coast. To validate the new assay, total metallothionein protein, amount of heavy metals (zinc, copper, cadmium), and a biomarker of oxidative stress such as malondialdehyde content, were assessed in the same tissues. At the investigated sites, the molecular assay showed variations of mt20 relative gene expression levels within one or two orders of magnitude, with maximum values at two sites severely polluted with cadmium, Mahres (100-fold) and Menzel Jemile (165-fold). Changes in mt10 expression were recorded at all sites where copper had significantly accumulated, although fold induction levels were less pronounced than those of mt20. In this paper, gene expression data are discussed in relation to the studied biomarkers, demonstrating that the molecular technique based on the differential expression of mt10 and mt20 genes represents (i) a useful and robust tool for studying and monitoring heavy metal pollution under field conditions, and (ii) an improvement in the application of metallothionein as a biomarker of response to exposure to heavy metals in marine mussels.

 

Geffard, A., O. Geffard, et al. (2007). "Bioaccumulation of metals in sediment elutriates and their effects on growth, condition index, and metallothionein contents in oyster larvae." Arch Environ Contam Toxicol 53(1): 57-65.

                The bioavailability of Cd, Cu, Zn, and Pb from two metal-contaminated sediments (Bidassoa and Dunkerque) was studied using Crassostrea gigas larvae exposed to sediment elutriates. The metal contents within the sediments, the larvae and larval growth, the condition index, and the induction of metallothionein in the larvae were measured. The larval growth and condition index were only affected after exposure to the highest elutriates concentration (5 to 25%) from the most contaminated sediment (Dunkerque). Bioaccumulation of all metals was observed in larvae exposed to Dunkerque elutriatre; only Cu bioaccumulation was observed in the Bidassoa elutriate. The results from larvae exposed to both sediment elutriates show a strong correlation between bioaccumulated metal considered individually or in combination and the metallothionein level in larvae presenting no detrimental effect. On the other hand, in the case of larvae exposed to the highest Dunkerque elutriate concentration and showing the highest metal body burden, we observed a drop in the metallothionein level. These results indicate that metallothionein is a more sensitive indicator of heavy metal pollution than physiological endpoints taken into account in bioassays and could be proposed as an early biomarker of metal exposure in larvae. However, care must be taken with "fault control" due to the toxicological effect on larvae metabolism in the case of substantial contaminant exposure.

 

Martin-Diaz, M. L., J. Blasco, et al. (2007). "Biomarkers study for sediment quality assessment in spanish ports using the crab Carcinus maenas and the clam Ruditapes philippinarum." Arch Environ Contam Toxicol 53(1): 66-76.

                Intermolt crab Carcinus maenas and clam Ruditapes philippinarum were used to determine the toxicity of sediments collected in four Spanish ports (Cadiz, Huelva, Pasajes, and Bilbao) under laboratory conditions during 28 days. Sediment samples were analyzed to determine chemical concentration of metals (As, Cd, Cr, Cu, Fe, Hg, Mn, Ni, Pb, and Zn), polyaromatic hydrocarbons, polychlorinated biphenyls, grain size distribution, and organic matter content. Different biomarkers of exposure of early biological stress were determined after 28-day exposure in crabs and clams, in the hepatopancreas and in the digestive gland, respectively: metallothionein, ethoxyresorufin O-deethylase (EROD), glutathione peroxidase (GPX), glutathione S-transferase (GST), and glutathione reductase activities (GR). The battery of biomarkers tested resulted in showing and linking the bioavailability of various contaminants and sediment characteristics to the toxicity of the different sediments. Significant induction of MTs was observed when organisms were exposed to metal-contaminated sediments (port of Huelva), and induction of EROD and GPX activities after exposure to sediments containing organic compounds (port of Bilbao and Pasajes). Higher induction was shown in biomarkers tested in crabs; nevertheless, only interspecies significant differences were observed in the induction of GR and GST activities. The present work confirms the necessity of using species with different ecological lifestyles for sediment toxicity assessment and validates the use of this set of biomarkers as a potential tool in sediment toxicity assessment.

 

Zorita, I., I. Apraiz, et al. (2007). "Assessment of biological effects of environmental pollution along the NW Mediterranean Sea using mussels as sentinel organisms." Environ Pollut 148(1): 236-50.

                With the aim of assessing the biological effects of pollution along three gradients of pollution in the NW Mediterranean Sea, a biomonitoring survey was implemented using a battery of biomarkers (lysosomal membrane stability, lysosomal structural changes, metallothionein (MT) induction and peroxisome proliferation) in mussels over a period of two years as part of the EU-funded BEEP project. Mussels from the most impacted zones (Fos, Genova and Barcelona harbours) showed enlarged lysosomes accompanied by reduced labilisation period of lysosomal membranes, indicating disturbed health. MT levels did not reveal significant differences between stations and were significantly correlated with gonad index, suggesting that they were influenced by gamete development. Peroxisomal acyl-CoA oxidase (AOX) activity was significantly inhibited in polluted stations possibly due to interactions among mixtures of pollutants. In conclusion, the application of a battery of effect and exposure biomarkers provided relevant data for the assessment of biological effects of environmental pollution along the NW Mediterranean Sea.

 

Quiniou, F., G. Damiens, et al. (2007). "Marine water quality assessment using transplanted oyster larvae." Environ Int 33(1): 27-33.

                Active bio-monitoring in terms of biomarkers was attempted using Crassostrea gigas larvae produced in the laboratory and transplanted using special containers to two sites at the entrance (A) and inner part (P) of the harbour of Arcachon (French Atlantic Coast). The larvae were kept in the medium for 48 h. Their physiological status and their biomarker levels : acetylcholinesterase AChE, catalase CAT and glutathione S-transferase GST activities were determined together with metallothionein MT and Thiobarbituric Acid Reactive Substances TBARS concentrations. Copper and PAH (polycyclic aromatic hydrocarbon) concentrations were determined in the exposed larvae and in the sediments collected under the containers. Cadmium, lead and zinc could be also analyzed in the sediments. Toxicity tests demonstrate that the larvae are in better physiological conditions in A compared to P. Larvae transplanted in the inner harbour (P) present relatively high GST activity (869.1+/-39.3 nmol min(-1)mg protein(-1)), TBARS (2.74+/-0.19 nmol mg protein(-1)), compared to those exposed at the harbour entrance (A). Copper measured in the sediments (65+/-1 mg kg(-1) d.w.) collected under the cages at P is higher than at A. Larvae placed in A present higher total PAH concentrations compared to the inner part. The data tend to reveal a lower copper and higher PAH contamination in A than in P. Therefore larvae, developing in the natural medium, show different responses according to their immersion sites. These responses, obtained within 48 h, may be related to the chemical contamination of the environment and may be used for seawater quality assessment in future studies.

 

Baudrimont, M. and X. de Montaudouin (2007). "Evidence of an altered protective effect of metallothioneins after cadmium exposure in the digenean parasite-infected cockle (Cerastoderma edule)." Parasitology 134(Pt 2): 237-45.

                The aim of the present study was to analyse the relation between parasitism and subsequent metallothioneins (MT) in the case of metal contamination. Experimental exposure of parasitized and unparasitized cockles (Cerastoderma edule) to cadmium (Cd) was performed, with the cockle as first or second intermediate host of 2 digenean species. After 7 days of Cd exposure in microcosms, cockles infected as first intermediate host by Labratrema minimus exhibited metal concentrations in tissues double that in uninfected cockles. Jointly, MT concentrations of parasitized cockles were not modified in comparison with uninfected individuals in which concentrations were increased 4.3-fold compared with controls. In cockles experimentally infected as the second intermediate host by Himasthla elongata, cadmium concentrations significantly increased again in parasitized cockles compared with uninfected individuals in contaminated conditions. Simultaneously, MT concentrations in healthy cockles increased, whereas they significantly decreased in parasitized individuals. Therefore, the presence of digenean parasites in Cd-exposed cockles leads to a maintenance or a decrease in MT concentrations compared with healthy individuals, whereas Cd accumulation in tissues is significantly increased. These experiments indicate a significant alteration of the protective effect of metallothioneins towards metals which could consequently enhance cockle vulnerability. Moreover, these results highlight the limit of the use of MT as a biomarker of metal pollution in field monitoring if parasitism is not taken into account.

 

Serafim, A. and M. J. Bebianno (2007). "Involvement of metallothionein in Zn accumulation and elimination strategies in Ruditapes decussatus." Arch Environ Contam Toxicol 52(2): 189-99.

                The aim of this study was to identify the involvement of metallothionein (MT) in Zn accumulation and elimination strategies in different tissues (gills, digestive gland, and remaining tissues) of Ruditapes decussatus. Clams were exposed to two Zn concentrations (100 and 1000 microg/L) for 40 days, followed by a depuration period of 50 days. The accumulation and elimination processes were complemented by subcellular Zn distribution, to determine the affinity of this element for the different cellular partitions. Subsequently, the involvement of MT was followed. Zinc concentrations in the tissues of R. decussatus exposed to 100 microg/L was partially regulated, whereas in those exposed to 1000 microg/L, Zn was accumulated (exponentially and linearly) throughout the exposure period. The greatest amount of Zn is associated to the insoluble fraction; however, in the highest Zn exposure, approximately 30% is in the thermostable fraction, where MT occurs. Gel filtration chromatography confirmed that the Zn ions accumulated in this fraction were bound to MT. This protein is actively involved in the elimination of this metal, through the rapid degradation of the Zn-MT complex because MT and Zn are turning over simultaneously. Therefore, MT participates in Zn homeostasis in R. decussatus when Zn exposure is low. When Zn accumulation increases, the role of this protein changes from a constitutive function to the detoxification of the excess of Zn ions.

 

Canesi, L., C. Borghi, et al. (2007). "Effects of 17beta-estradiol on mussel digestive gland." Gen Comp Endocrinol 153(1-3): 40-6.

                In bivalve molluscs the digestive gland (hepatopancreas) plays a central role in metabolism. In this work, the effects of 17beta-estradiol (E(2)) on digestive gland were evaluated in Mytilus galloprovincialis. Mussels were injected into the adductor muscle sinus with different amounts of the hormone (5, 25 and 100pmol) and tissues were sampled 24h post-injection. Functional parameters (lysosomal membrane stability-LMS, lysosomal accumulation of neutral lipids-NL and of lipofuscin-LF), as well as the activity of the key glycolytic enzymes PFK (phosphofructokinase) and PK (pyruvate kinase), and of the antioxidant enzyme catalase were evaluated. Selected genes, whose expression can be modulated by estrogens in mammalian systems and whose sequences have been identified in Mytilus, were investigated as possible targets for the action of E(2). E(2) induced a concentration-dependent decrease in LMS; such an effect was accompanied by an increase in NL accumulation, whereas the level of lipofuscin showed a slight, although not significant decrease. E(2) exposure also led to a significant increase in the activity of PFK and catalase but not of PK. Moreover, E(2) induced significant changes in the pattern of gene expression at the lower concentrations tested (5 and 25pmol) as evaluated by quantitative RT-PCR. In particular, increased transcription of catalase, as well as of the metallothionein 20 (MT20) isoform were observed; on the other hand, a decreased transcription of the p53 gene was detected. The results demonstrate that in Mytilus the digestive gland represents a target for the action of E(2), and that the hormone can modulate the lysosomal function, as well as lipid and glucose metabolism. Moreover, these data suggest that E(2) may also alter oxidative stress conditions in this tissue, as indicated by the increased transcription of genes (metallothionein and catalase) that play a role in antioxidant defences. Overall, the results indicate that E(2) can modulate both functional parameters and gene expression in mussel hepatopancreas and underline the importance of investigating also non-reproductive effects of estrogenic compounds in bivalve molluscs.

 

Ng, T. Y., P. S. Rainbow, et al. (2007). "Metallothionein turnover, cytosolic distribution and the uptake of Cd by the green mussel Perna viridis." Aquat Toxicol 84(2): 153-61.

                We examined the relationship between Cd kinetics (uptake from solution and diet, and efflux), metallothionein turnover, and changes in the cytosolic distribution of accumulated Cd between protein fractions in the green mussel Perna viridis. We pre-exposed the mussels to 5, 20, 50 and 200 microg l(-1) of Cd for 1 week and determined the biokinetics of Cd uptake and efflux in the mussels. The dietary assimilation efficiency of Cd increased by 2 times following exposure to 20-200 microg l(-1) Cd, but the dissolved uptake rate was unchanged by pre-exposure to any Cd concentrations. The efflux rate of Cd was also similar among control and Cd pre-exposed mussels. The cytosolic distribution of Cd in the mussels that had been exposed to dissolved Cd, showed that besides metallothionein (7000 - 20,000 Da), high molecular weight proteins (>20,000 Da) were important for Cd binding and depuration. In general, the Cd pre-exposed mussels had higher metallothionein turnover with a higher metallothionein synthesis rate, but similar metallothionein breakdown rates as the control mussels. Metallothionein synthesis rate was correlated to the dietary assimilation of Cd, whereas metallothionein breakdown and Cd efflux rate were independent of each other. This study provides important new information for the role of metallothionein turnover on Cd kinetics in an aquatic invertebrate.

 

Li, C. D., W. Yan, et al. (2007). "[Metal accumulation and MTLP induction in the digestive glands of Perna viridis exposed to Cu]." Huan Jing Ke Xue 28(8): 1788-95.

                Mussels have been proposed as appropriate biomonitors of marine pollution, especially for monitoring metallic pollution based on variations of metallothionein as biomarkers. Under 2 exposure levels (12.7 microg/L, 63.5 microg/L), Cu accumulation and metallothionein-like protein (MTLP) induction by mussel (Perna viridis) digestive glands were investigated and simulated into dynamic models in the present work, and the soluble and total Cu burden of digestive glands were also determined. Calculated mean Cu uptake rates by mussel target organ were 2.045 and 7.028 microg x (g x d)(-1) respectively, and the theoretical equilibrium kinetic BCFs of Cu were 2074 and 1619 correspondingly. And within the exposure duration, different changing trends of ratio of soluble Cu to total Cu in digestive glands were observed in the two groups. The MTLP level of control samples was (0.551 +/- 0.037) mg/g, and the counterparts are 0.407 - 0.699 mg/g, 0.826 - 0.942 mg/g respectively when mussels were exposed to 12.7 microg/L and 63.5 microg/L Cu solutions. Statistically significant MTLP induction (p < 0.001) was observed under higher exposure level. MTLP contents in digestive glands increased with the exposure Cu concentration and body accumulation of metal. There is a significantly negative exponential rise relationship (p < 0.000 1) between MTLP and Cu concentrations accumulated in the digestive glands of mussels.

 

Ng, T. Y. and W. X. Wang (2007). "Interactions of silver, cadmium, and copper accumulation in green mussels (Perna viridis)." Environ Toxicol Chem 26(8): 1764-9.

                Metal interaction is vital for assessing the use of aquatic organisms in monitoring metal contamination. The present study examined the interactions between Ag and Cd accumulation and between Ag and Cu accumulation in the green mussel (Perna viridis). Accumulation of Ag and Cd in the whole tissue of green mussels exposed to 5 microg/L of Ag and 20 microg/L of Cd for two weeks was independent; however, interaction was observed at the subcellular level. Approximately 25% of Ag shifted from the insoluble fraction (IF) to the metallothionein-like protein in the presence of Cd, which probably resulted from the competition of Cd on IF in the Ag-Cd coexposure. On the other hand, coexposure of the mussels to Ag (5 microg/L) and Cu (30 microg/L) for two weeks increased the Ag and Cu concentrations in the tissue synergistically (two- to fivefold), but Ag and Cu subcellular distributions were similar in the coexposed and the singly exposed mussels. Exposure to Ag alone increased the dietary uptake of Ag by 30%, but the effect was reduced in the presence of Cd. No interaction, however, was observed between uptake rates of metals from the dissolved phase. To conclude, a significant interaction was observed for total Ag and Cu accumulation, but not for total Ag and Cd accumulation, in the mussels. Metal interaction is more likely to be observed at the subcellular level than at the whole-tissue level.

 

Zorita, I., E. Bilbao, et al. (2007). "Tissue- and cell-specific expression of metallothionein genes in cadmium- and copper-exposed mussels analyzed by in situ hybridization and RT-PCR." Toxicol Appl Pharmacol 220(2): 186-96.

                Metallothioneins (MTs) are metal-inducible proteins that can be used as biomarkers of metal exposure. In mussels two families of MT isoforms (MT10 and MT20) have been characterized. In this study, mussels (Mytilus galloprovincialis) were exposed to 200 ppb Cd and 40 ppb Cu for 2 and 9 days to characterize the tissue and isoform specificity of metal-induced MT expression. Non-radioactive in situ hybridization demonstrated that both MT isoforms were mainly transcribed in digestive tubule epithelial cells, especially in basophilic cells. Weaker MT expression was detected in non-ciliated duct cells, stomach and gill epithelial cells, haemocytes, adipogranular cells, spermatic follicles and oocytes. RT-PCR resulted in cloning of a novel M. galloprovincialis isoform homologous to recently cloned Mytilus edulis intron-less MT10B isoform. In gills, Cd only affected MT10 gene expression after 2 days of exposure while increases in MT protein levels occurred at day 9. In the digestive gland, a marked increase of both isoforms, but especially of MT20, was accompanied by increased levels of MT proteins and basophilic cell volume density (Vv(BAS)) after 2 and 9 days and of intralysosomal metal accumulation in digestive cells after 9 days. Conversely, although metal was accumulated in digestive cells lysosomes and the Vv(BAS) increased in Cu-exposed mussels, Cu exposure did not produce an increase of MT gene expression or MT protein levels. These data suggest that MTs are expressed in a tissue-, cell- and isoform-specific way in response to different metals.

 

Da Ros, L., V. Moschino, et al. (2007). "Lysosomal responses and metallothionein induction in the blue mussel Mytilus edulis from the south-west coast of Iceland." Environ Int 33(3): 362-9.

                It has recently been emphasized that high levels of inorganic and organic micropollutants (particularly organometals, POPs and PAHs) may be present in coastal waters at high latitudes, stressing the need to evaluate the effects of contaminants on marine organisms from sub-arctic zones. With this aim, specimens of the blue mussel Mytilus edulis were sampled in polluted and reference areas along the south-west coast of Iceland in July 2004. Samples were collected from the intertidal zone at three sites in Reykjavik harbour which are differently exposed to contaminants, and at three reference coastal sites, two located along the Reykjanes Peninsula and the third one on the northern part of Hvalfjordur fiord. Lipofuscin content, neutral lipid accumulation and lysosomal enlargement were evaluated in digestive cells from cryostat sections of the mussel hepatopancreas, and quantified by automated image analysis. Metallothionein induction was also determined in the same tissue. Results indicate that mussels from the inner part of Reykjavik harbour, which is the most sheltered and most influenced by extensive shipping traffic, were the worst affected, with the highest values in neutral lipids, lipofuscin and lysosomal swelling. At the other two harbour sites, mussels exhibited lower values, similar to those observed in organisms collected in Hvalfjordur fiord and in bay of Osar. Mussels from Kuagerdi had the lowest values.

 

Dailianis, S. and M. Kaloyianni (2007). "Role of cAMP in tissues of mussel Mytilus galloprovincialis as a potent biomarker of cadmium in marine environments." Arch Environ Contam Toxicol 52(3): 371-8.

                The present study investigated the signal transduction molecule cAMP as a biomarker of exposure to cadmium in mussels Mytilus galloprovincialis. Mussels were exposed to 10 and 100 microg/l cadmium for 3, 6, and 9 days, and cAMP content in three tissues-digestive gland, gills and mantle-gonad complex-was estimated. The results showed significantly increased levels of cAMP in all tissues at all time points tested. In support of our results, cAMP levels were positively correlated with the established metal biomarker, metallothionein. Therefore, we could suggest that mussels exposed to cadmium respond by increasing cAMP content in digestive gland, gills and mantle-gonad complex, thus indicating that cAMP could constitute a promising biomarker of exposure to cadmium.

 

Nesto, N., S. Romano, et al. (2007). "Bioaccumulation and biomarker responses of trace metals and micro-organic pollutants in mussels and fish from the Lagoon of Venice, Italy." Mar Pollut Bull 55(10-12): 469-84.

                The aims of this study were to evaluate levels and effects of trace metals (Cd, Cr, Cu, Fe, Mn, Pb, Zn), PAHs and PCBs in mussels and fish native to the Lagoon of Venice. To this end, bioaccumulation and various biomarkers of generic stress and exposure (malondialdehyde, MDA and metallothioneins, MT; ethoxyresorufin O-deethylase activity, EROD; fluorescent bile metabolites and somatic indices in fish; lysosomal membrane stability, LMS; lipofuscins, LF; neutral lipids, NL and survival-in-air, SOS in mussels) were measured in organisms seasonally collected from two differently influenced areas. In mussels, metal bioaccumulation levels at the two sites were low and fluctuated similarly, exhibiting decreasing levels in summer, like MDA and MT. Micro-organic pollutants and the probably related biological response of LMS did not show either significant site differences or seasonal trends. LF and NL revealed inconclusive patterns. The results of the survival-in-air test were quite erratic, showing that they were associated with the high variability of both natural and physiological parameters temperature, food, reproduction, and body reserve cycle. In fish, both inorganic and organic micropollutant levels were low, and differences were detected between sites only for PCBs. The pattern exhibited by micro-organic contaminants was clearly related to the reproductive cycle at both sites; a relationship with PCBs, EROD and MDA was established at the site where their levels were generally higher. Bile metabolites (3-OH benzo(a)pyrene, 1-OH pyrene) were associated with PAH contents.

 

Znidaric, M. T., I. Falnoga, et al. (2006). "Induction of metallothionein-like proteins by mercury and distribution of mercury and selenium in the cells of hepatopancreas and gill tissues in mussel Mytilus galloprovincialis." Biol Trace Elem Res 111(1-3): 121-35.

                The binding of mercury (Hg) to metallothioneins (MTs) and the relation between Hg and selenium in supernatants of hepatopancreas and gill tissues of the common mussel Mytilus galloprovincialis (Lamarck, 1819) was investigated. The mussels were exposed to different Hg concentrations in laboratory conditions: 2.5 microgHg/L, 4 d exposure (short term) and 60 microgHg/L, 33 d exposure (long term). In addition, the results were compared to those found for mussels from nature (polluted and unpolluted region). In control and short-term-exposed mussels, the level of Hg extraction (cytosol) from hepatopancreas and gill cells was very low with respect to the total Hg concentrations in the corresponding tissues, around 10% in control and around 20% after exposure. As expected, Hg exposure was followed by Se increase. For Se, the levels of extraction were higher, around 20% in control and up to 50% (heaptopacrease) of 70% (gills) after exposure. In order to study the distribution of Hg and Se in the cells of these organs, the total Hg and Se concentrations were analyzed in the subcellular fractions obtained after differential centrifugation. Although after exposure the concentrations of both element increased in all subcellular fractions, their percentages in particular fractions were lower or higher. In this study, the convincing binding of Hg to metallothionein-like proteins was perceived after long-term laboratory exposure (gills, heapatopancreas) and in wild mussels collected near industrial port (hepatopancreas). In latter case, we also detected the traces of Se bound to the MT fractions after size-exclusion chromatography.

 

Raftopoulou, E. K., S. Dailianis, et al. (2006). "Introduction of cAMP and establishment of neutral lipids alterations as pollution biomarkers using the mussel Mytilus galloprovincialis. Correlation with a battery of biomarkers." Sci Total Environ 368(2-3): 597-614.

                The results of the present study suggest that the signaling molecule cAMP could constitute a reliable biomarker of pollution monitoring. Increased levels of cAMP were observed in tissues of mussels Mytilus galloprovincialis exposed to PAHs, 2,4-dichlorophenoxyacetic acid and lindane for 15 days. cAMP levels were higher in mantle/gonad complex, in relation to other tissues studied (digestive gland and gills). In support of our suggestion, cAMP was significantly correlated with established biomarkers, such as lysosomal membrane stability, AChE activity and metallothionein content. In addition, our results support the establishment of morphometrical alterations of neutral lipids as biomarker of organic pollution, since high accumulation of neutral lipids in the digestive gland of mussels exposed to organic pollutants in relation to non-exposed mussels was noted. In addition, correlation analysis between cellular and biochemical biomarkers showed that the volume density of neutral lipids negatively correlated with both lysosomal membrane stability and AChE activity. Regarding micronucleus test, our results do not support its use as a biomarker of organic pollution, since no statistical differences were found between control and exposed mussels. On the other hand, significant correlations were observed among the battery of biomarkers, such as lysosomal membrane stability, AChE activity and metallothionein content after the mussels' exposure to the specific organic compounds, thus supporting the important role of these biomarkers as reliable indicators of organic pollution. In conclusion, our results support cAMP and morphometrical alterations of neutral lipids as biomarkers of environmental pollution.

 

Jenny, M. J., G. W. Warr, et al. (2006). "Regulation of metallothionein genes in the American oyster (Crassostrea virginica): ontogeny and differential expression in response to different stressors." Gene 379: 156-65.

                Metallothioneins (MTs) are typically low molecular weight (6-7 kDa), metal-binding proteins with characteristic repeating cysteine motifs (Cys-X-Cys or Cys-Xn-Cys) and a prolate ellipsoid shape containing single alpha- and beta-domains. While functionally diverse, they play important roles in metals homeostasis, detoxification and the stress response. The present study, combined with previous observations (e.g., Jenny et al., Eur. J. Biochem. 2005; 271:1702-1712) defines an unprecedented diversity of MT primary structure and domain organization in the American oyster, Crassostrea virginica. Two novel molluscan MT families are described. One of these (CvMT-III) is characterized by the presence of two beta-domains and the absence of alpha-domains. This family exhibits constitutive expression during larval development and is the dominant CvMT isoform expressed in larvae. CvMT-III displays low basal levels of expression in adult tissues and only moderate responsiveness to metal challenges in both larvae and adults. A second novel MT isoform (CvMT-IV) was isolated from hemocytes by subtractive hybridization techniques following a 4-hour immune challenge with heat-killed bacteria (Vibrio, Bacillus, Micrococcus spp. mixture). Based on conservation of the cysteine motifs, this isoform appears to be a sub-family related to the molluscan alphabeta-domain MTs. A series of amino acid substitutions has resulted in four additional cysteines which give rise to a Cys-Cys motif and three Cys-Cys-Cys motifs. Northern blot analyses demonstrate that CvMT-IV is down-regulated upon sterile wounding and immune challenge, displays moderate expression in larvae and adults and differential gene induction in response to metals exposure.

 

Linde, A. R. and E. Garcia-Vazquez (2006). "A simple assay to quantify metallothionein helps to learn about bioindicators and environmental health." Biochem Mol Biol Educ 34(5): 360-3.

                We present a laboratory practice aimed to clarify and discuss the concept of bioindicator molecules in environmental health. It has been implemented as a hands-on laboratory exercise in postgraduate courses in public health (in Brazil) and in a course entitled Biology Applied to Natural Resources Sustainability (in Spain) but can be useful for undergraduate studies. By simple, repeatable, and low cost spectrophotometry methodology, students detect minimal concentrations (nmol) of metallothionein in biological samples obtained from sites with various status of pollution. They can also expose experimental fish or mollusks to heavy metals in aquarium conditions and then detect metallothionein. This method is excellent for didactic purposes because it can be developed even in modest laboratories with basic equipment for molecular biology.

 

Pytharopoulou, S., E. C. Kouvela, et al. (2006). "Evaluation of the global protein synthesis in Mytilus galloprovincialis in marine pollution monitoring: seasonal variability and correlations with other biomarkers." Aquat Toxicol 80(1): 33-41.

                Protein synthesis down-regulation is a life-saving mechanism for many organisms exposed to xenobiotics that threaten normal life. The present study was designed to assess the spatial and seasonal variability of global protein synthesis, determined in the microsomal fraction of digestive glands from caged Mytilus galloprovincialis mussels exposed for 30 days in a relatively clean region and two unevenly polluted areas (Stations 1 and 2) along the Gulf of Patras (Greece). The in vivo activity of translating ribosomes was evaluated by analyzing the translating ribosomes, polysome content, which may serve as an indicator of the efficiency of the protein-synthesizing machinery. To correlate with classical biomonitoring strategies, various biomarkers were measured in digestive glands, including metallothionein content, heavy-metal content, and lysosomal membrane stability. In parallel, gill cells were examined for micronucleus frequency. Metal ion concentrations were also estimated in the surrounding waters as a measure of metal exposure. Substantially lower polysome content was recorded in caged mussels collected from Station 1, in particular during the winter and spring sampling. As verified by chemical analysis of the seawater and measurement of other biomarkers, Station 1 was more contaminated than Station 2. Polysome content was found negatively correlated with metallothionein levels, micronucleus frequency and cytosolic Cu and Hg in all seasons. In addition, negative correlations were obtained between polysome content and lysosomal membrane stability in winter and spring. A progressive increase in polysomes was observed from winter to autumn, in particular in samples from Station 1. A non-uniform trend was detected in 80S ribosomal monosomes, whereas the seasonal changes in ribosomal subunits were opposite to those found in polysome content. Comparisons between seasonal and local site-specific influences on polysome content provides evidence that winter and spring are the most appropriate sampling seasons for application of translation activity as a possible biomarker in biomonitoring studies.

 

Perceval, O., Y. Couillard, et al. (2006). "Long-term trends in accumulated metals (Cd, Cu and Zn) and metallothionein in bivalves from lakes within a smelter-impacted region." Sci Total Environ 369(1-3): 403-18.

                Temporal monitoring studies are needed to detect long-term trends in ecosystem health. In the present study, we tested metallothionein (MT) as a potential biomarker for long-term variations in trace metal levels in lakes subject to atmospheric metal inputs from a nearby copper smelter. Over a 13-year period, we estimated on several occasions ambient free Cd2+, Cu2+ and Zn2+ concentrations at the sediment-water interface in six lakes with contrasting metal levels, and measured metal and metallothionein concentrations in gills of bivalves (Pyganodon grandis) living in these lakes. All but one of the study lakes had comparable drainage ratios, so inter-lake differences in hydrological export of metals from contaminated watersheds to receiving waters were likely minimal. Declines in the metal emissions from the smelter (especially for Cd and Zn) during the 1980s led to appreciable decreases in both calculated free Cd2+ ion concentrations in the study lakes (-59+/-21% between 1989 and 1998) and accumulated Cd levels in their clam populations (-46+/-12% between 1989 and 2002). Taking all lakes into account, MT concentrations in bivalves have comparatively dropped by 44% (+/-10%) since 1989. In contrast to what we found for Cd, there were no significant reductions in the calculated free Cu2+ and Zn2+ concentrations in the various lakes during our study period (-2 and -10%, respectively, with 95% confidence intervals spanning zero). Overall, observed decreases in MT in bivalves over time were best correlated with similar decreases in both ambient and accumulated Cd levels (r = 0.77, P = 0.0003 and r = 0.79, P < 0.0001, respectively, both P-values corrected for temporal autocorrelation), suggesting that long-term trends in gill MT concentrations in P. grandis may be useful as predictors of temporal changes in environmental Cd contamination in lakes.

 

Girotti, S., L. Bolelli, et al. (2006). "Trace metals in arcid clam Scapharca inaequivalvis: effects of molluscan extracts on bioluminescent bacteria." Chemosphere 65(4): 627-33.

                The relationship between a supposed effect of molluscan extracts on bioluminescent bacteria and metal concentrations in the extracts was investigated. For this purpose a biotoxicological assay based on bioluminescent bacteria (BLB) and extracts from metal exposed molluscs, Scapharca inaequivalvis, was optimized to monitor Cd and Cu marine pollution. Cu and Cd concentrations increased in tissues of experimentally exposed molluscs. Molluscan extracts inhibited the bacterial luminescence, the inhibition decreasing as the time of mollusc exposure to metals increased, suggesting a reduction of the "bioactive" metals. In regard to the use of BLB test in environmental monitoring, the analysis of Cu, Cd, and metallothionein (MT) was first performed in tissues from molluscs collected in three different areas of Northern Adriatic Sea. Metal concentrations reached maximum values in the gills, while Cd was mostly bound to MT in the kidney. Significant differences in metals and MT concentrations were found depending on the sampling sites. The biotoxicological assay resulted slightly correlated with the biochemical parameters.

 

Marie, V., M. Baudrimont, et al. (2006). "Cadmium and zinc bioaccumulation and metallothionein response in two freshwater bivalves (Corbicula fluminea and Dreissena polymorpha) transplanted along a polymetallic gradient." Chemosphere 65(4): 609-17.

                This study was designed to compare the metallothionein (MT) response capacity of two freshwater bivalves, Corbicula fluminea and Dreissena polymorpha, along an environmental gradient of polymetallic pollution. The bivalves were transplanted into 4 stations in southwestern France, with a cadmium and zinc pollution gradient. MT and metal concentrations were measured in the soft bodies of the clams and mussels over 2.5 months. In the zebra mussels, variations in MT concentrations were strictly correlated to progressive Cd and Zn bioaccumulation. In contrast, the faster response in the clams appeared positively correlated to Cd accumulation only, with the activation of efficient detoxification mechanisms which limited Cd bioaccumulation and reduced Zn concentrations over time. Nevertheless, despite stronger metal accumulation factors in D. polymorpha, C. fluminea revealed higher sensitivity of MT response along the pollution gradient.

 

Bocchetti, R. and F. Regoli (2006). "Seasonal variability of oxidative biomarkers, lysosomal parameters, metallothioneins and peroxisomal enzymes in the Mediterranean mussel Mytilus galloprovincialis from Adriatic Sea." Chemosphere 65(6): 913-21.

                The Mediterranean mussel, Mytilus galloprovincialis, is a classical sentinel organism for monitoring the biological effects of contaminants through the use of molecular and cellular biomarkers. These biological responses can be modulated also by seasonal changes of both environmental and biological factors, potentially influencing responsiveness and sensitivity to pollutants. The aim of this study was to characterize in a reference mussel population from the Adriatic Sea, the natural fluctuations of several oxidative stress biomarkers widely used in ecotoxicological applications. Analyses of individual antioxidant defenses (catalase, glutathione S-transferases, glutathione reductase, glutathione peroxidases, levels of glutathione) were integrated with those of the total oxyradical scavenging capacity (TOSC-assay), which quantify the overall capability of a tissue to neutralize different forms of oxyradicals. Due to the close relationship between antioxidant efficiency and onset of various cellular alteration, the seasonal characterization was carried out also on lysosomal membrane stability, accumulation of malondialdehyde, neutral lipids and lipofuscin, levels of metallothioneins and activity of peroxisomal enzymes (acyl-CoA oxidase and d-aminoacid oxidase). Results indicated a significant seasonality for several biological responses; major variations occurred especially in spring and summer months suggesting the influence of natural factors, such as temperature, reproductive cycle and food availability. The observed seasonal oscillations revealed both similarities and differences with those reported for other Mediterranean mussel populations suggesting that opposite trends can occur when the same environmental prooxidant factors have a different regional influence.

 

Bourdineaud, J. P., M. Baudrimont, et al. (2006). "Challenging the model for induction of metallothionein gene expression." Biochimie 88(11): 1787-92.

                Metallothioneins (MTs) are low-molecular-weight, cysteine-rich metal-binding proteins found in a wide variety of organisms including bacteria, fungi and all eukaryotic plant and animal species. MTs bind essential and non-essential heavy metals. In mammalian cells MT genes are highly inducible by many heavy metals including Zn, Cd, Hg, and Cu. Aquatic systems are contaminated by different pollutants, including metals, as a result of man's activities. Bivalve molluscs are known to accumulate high concentrations of heavy metals in their tissue and are widely used as bioindicators for pollution in marine and freshwater environments, with MTs frequently used as a valuable marker of metal contamination. We here describe the MT isoform gene expression patterns of marine and freshwater molluscs and fish species after Cd or Zn contamination. Contamination was carried out at a river site polluted by a zinc ore extraction plant or in the laboratory at low, environmentally relevant metal concentrations. A comparison for each species based on the accumulated MT protein levels often shows discrepancies between gene expression and protein level. In addition, several differences observed in the pattern of MT gene expression between mollusc and mammalian species enable us to discuss and challenge a model for the induction of MT gene expression.

 

Bouskill, N. J., R. D. Handy, et al. (2006). "Differentiating copper and arsenic toxicity using biochemical biomarkers in Asellus aquaticus and Dreissena polymorpha." Ecotoxicol Environ Saf 65(3): 342-9.

                Biomarkers of metal exposure are well known, but how a suite of such biomarkers will respond if the metal is also an oxidizing agent or causes oxidative stress is unclear. This study compares the effects of copper and arsenic, two metals with different oxidizing potential, on freshwater invertebrates. Dreissena polymorpha and Asellus aquaticus were exposed to nominal concentrations of copper (100 microg/L) or arsenic (80 microg/L) over 7 days, and physiological stress was examined by measuring metallothionein (MT) induction, thiobarbituric acid reactive substances (TBARS), and Na(+)/K(+)-ATPase activity. Both species showed increased levels of MT during 7-day Cu exposure tests and transient changes in lipid peroxidation (TBARS) which decreased to control levels by day 7. Arsenic had no effect on TBARS and only a transitory effect on MT in D. polymorpha over 7 days, although it initially induced lipid peroxidation in A. aquaticus on day 3. No inhibition of the Na(+)/K(+)-ATPase enzyme was observed for exposed organisms, and baseline values reported here, for A. aquaticus, 1.1 micromol Pi/mg/h, and for D. polymorpha, 0.38 micromol Pi/mg/h, are probably the first reported for these species.

 

Podgurskaya, O. V. and V. Y. Kavun (2006). "Cadmium concentration and subcellular distribution in organs of the mussel Crenomytilus grayanus from upwelling regions of Okhotsk Sea and Sea of Japan." Arch Environ Contam Toxicol 51(4): 567-72.

                Cadmium (Cd) accumulation and subcellular distribution in the digestive gland and kidney of mussel Crenomytilus grayanus from naturally Cd elevated areas was studied. Mussels were collected from three sites of Okhotsk Sea and Sea of Japan: control area (site 1), seasonal upwelling region (site 2), and stationary upwelling region (site 3). Mussel from site 3 was shown to accumulate a significantly increased Cd concentration in the digestive gland and an extremely high Cd concentration (1780+/-732 microg g-1 dry weight) in the kidney. Cd was mainly sequestered into the kidney cytosol of the mussels from both upwelling regions and control sites (73% to 96%). However, digestive gland cytosol bound about 55% of Cd for the mussels from sites 1 and 2 and only 22.4% of Cd for the mussel from site 3. In the organs of the mussels from upwelling regions, the most of cytosolic Cd was associated with the protein fraction corresponding to molluscan metallothioneins. MTLP was isolated from kidney cytosol of the mussels from site 3 (powerful upwelling regions) by gel filtration chromatography, whereas in the organs studied of the mussels from other sites, MTLP content was below the detection limit.

 

Desouky, M. M. (2006). "Tissue distribution and subcellular localization of trace metals in the pond snail Lymnaea stagnalis with special reference to the role of lysosomal granules in metal sequestration." Aquat Toxicol 77(2): 143-52.

                The present study was undertaken to elucidate the cellular mechanisms, which govern metal sequestration and detoxification in gastropods. For this purpose the pond snail, Lymnaea stagnalis was exposed to environmentally relevant concentrations of three species of metals (Al, Zn and Cd) for 30 days and the localization and fate of these metals were followed in different tissues of the snails. The measurement of relative distribution of metals between tissues revealed that the digestive gland and kidney account for most of the accumulated metals. Al and Cd (non-essential metals) were redistributed to the digestive gland, possibly because of the presence of specific binding entities in the digestive glands of the herein species. This study focuses on the role of intracellular metal-containing granules on metal sequestration. Three main types of granules were identified in the digestive gland cells namely small, green and yellow granules. The morphological examination and the progressive accumulation of elements within these granules revealed that they are developmental stages with the yellow granule being the mature one. The total number of these granules was found to be significantly increased upon exposure of the snails to Al only. This increase may be a response to the large amount of Al that is accumulated through feeding route of this grazing snail. X-ray microanalysis (XRMA) revealed that metals were localized in all three types of digestive gland granules. The increased amount of ligands (P and S) in the granules may give evidence for their role in metal sequestration. Levels of Al and P were positively correlated in the digestive gland granules. It is possible that aluminium is bound to phosphorus to render it insoluble and so to both immobilize it within the lysosome and to be excreted in a highly insoluble form. On the other hand, both Zn and Cd induced marked upregulation of S in mature (yellow) granules by 26- and 11-folds, respectively. The lysosomal codeposition of S and either Cd or Zn in the lysosomal granules in addition to the increase in RER cisternae may indicate that the exposure to these metals could induce metallothionein synthesis in the cells. The microscopical examinations in the present study revealed that metal detoxification from the digestive gland cells may occur via faeces or via basal exocytosis towards hemocytes dispersed by the connective tissue in the visceral mass. In the kidney, one type of granules, the excretory concretions, was identified in the nephrocytes. The significant increase in the number of these concretions in the snail L. stagnalis upon exposure to metals may give further evidence for their role in metal excretion.

 

Baudrimont, M., X. de Montaudouin, et al. (2006). "Impact of digenean parasite infection on metallothionein synthesis by the cockle (Cerastoderma edule): a multivariate field monitoring." Mar Pollut Bull 52(5): 494-502.

                Metallothioneins (MT) are proteins that play an important role in metabolism of essential metals and detoxification of trace metals from living organisms. Their synthesis is induced by metal pollution but can also be exacerbated by other factors such as reproduction processes. In this context, we monitored MT concentrations in a cockle Cerastoderma edule (marine bivalve) population and highlighted the effect of a castrating digenean parasite, Labratrema minimus. In spent cockles, MT levels were low (ca. 5 nmol sites g(-1), fresh weight) but slightly higher in parasitized individuals. During gametogenesis, MT synthesis increased in all cockles, but concentrations were lower in parasitized individuals (18 against 27 nmol sites g(-1), fw in unparasitized cockles) in relation with gonad damage by parasites. Therefore, it is suggested that parasite infection in cockles can modulate MT synthesis that could consequently interfere with the response of these protective proteins in case of metal contamination.

 

Sarkar, A., D. Ray, et al. (2006). "Molecular Biomarkers: their significance and application in marine pollution monitoring." Ecotoxicology 15(4): 333-40.

                This paper presents an overview of the significance of the use of molecular biomarkers as diagnostic and prognostic tools for marine pollution monitoring. In order to assess the impact of highly persistent pollutants such as polychlorinated biphenyls (PCB), polychlorinated dibenzo-dioxins (PCDD), polychlorinated dibenzo-furans (PCDF), polynuclear aromatic hydrocarbons (PAH), tributyltin (TBT) and other toxic metals on the marine ecosystem a suite of biomarkers are being extensively used worldwide. Among the various types of biomarkers, the following have received special attention: cytochrome P4501A induction, DNA integrity, acetylcholinesterase activity and metallothionein induction. These biomarkers are being used to evaluate exposure of various species of sentinel marine organisms (e.g. mussels, clams, oysters, snails, fishes, etc.) to and the effect of various contaminants (organic xenobiotics and metals) using different molecular approaches [biochemical assays, enzyme linked immuno-sorbent assays (ELISA), spectrophotometric, fluorometric measurement, differential pulsed polarography, liquid chromatography, atomic absorption spectrometry]. The induction of the biotransformation enzyme, cytochrome P4501A in fishes (Callionymus lyra, Limanda limanda, Serranus sp., Mullus barbatus) and mussels (Dreissena polymorpha) by various xenobiotic contaminants such as PCBs, PAHs, PCDs is used as a biomarker of exposure to such organic pollutants. The induction of cytochrome P4501A is involved in chemical carcinogenesis through catalysis of the covalent bonding of organic contaminants to a DNA strand leading to formation of DNA adduct. Measurement of the induction of cytochrome P4501A in terms of EROD (7-ethoxy resorufin O-deethylase) activity is successfully used as a potential biomarker of exposure to xenobiotic contaminants in marine pollution monitoring. In order to assess the impact of neurotoxic compounds on marine environment the evaluation of acetylcholinesterase activity in marine organisms is used as a biomarker of exposure to neurotoxic agents such as organophosphorus, carbamate pesticides etc. Metallothioneins (MTs) are induced by toxic metals such as Cd, Hg, and Cu by chelation through cysteine residues and are used in both vertebrates and invertebrates as a biomarker of metal exposure. The measurement of the levels of DNA integrity in marine organisms such as Sea stars (Asterias rubens) from the North Sea and the marine snails (Planaxis sulcatus) from the Arabian Sea along the Goa coast exposed to environmental xenobiotic contaminants clearly indicated the extent and the nature of pollution at the sampling sites along coastal environment.

 

Dragun, Z., B. Raspor, et al. (2006). "The influence of the biometric parameters on metallothionein and metal level in the heat-treated cytosol of the whole soft tissue of transplanted mussels." Environ Monit Assess 114(1-3): 49-64.

                The influence of the biometric parameters (shell mass, whole soft tissue mass, condition index) on MT and metal levels in the heat-treated cytosol of the whole soft tissue of transplanted mussels was studied over the period of one year. The positive correlation of three metals (Cd, Fe, Zn) with the shell mass indicated to time-dependent increase of their contents. Strong correlation of Mn content with the whole soft tissue mass (r = 0.74, p < 0.0001), and almost identical changes of these two parameters over the year make Mn a good indicator of mussel's condition. As opposed to the other metals, Cu content does not exhibit connection with biometry. On the other hand, MTs are highly influenced by biometry. As much as 65% of their variability could be explained by the changes of the shell mass and the whole soft tissue mass. Consequently, it is difficult to distinguish if the obtained positive correlation between Cd and MTs (r = 0.48, p < 0.05) reflects MT induction by Cd, or Cd accumulation as a result of age-dependent increase of MTs. Due to the strong influence of the biometry on MT level, the whole soft tissue is not considered as the best choice for measuring MTs as a biomarker. Better option would be to isolate a specific tissue that shows indisputable connection between MT induction and metal accumulation.

 

Dondero, F., A. Dagnino, et al. (2006). "Assessing the occurrence of a stress syndrome in mussels (Mytilus edulis) using a combined biomarker/gene expression approach." Aquat Toxicol 78 Suppl 1: S13-24.

                A combination of biomarkers and gene expression analyses was used to investigate the occurrence of a stress syndrome in mussels (Mytilus edulis) caged along a copper pollution gradient in the Visnes fjord, Norway. The stress level in mussels, as calculated by a novel algorithm (the "Expert System") from a set of seven biomarkers, was compared with gene expression changes utilising a low-density oligonucleotide microarray, employing 24 different genes involved in both cellular homeostasis and stress-related responses. The biomarker battery included lysosomal membrane stability, lysosomal accumulation of neutral lipids and lipofuscin, lysosomal/cytoplasm volume ratio, Ca(2+)-ATPase and catalase activities, and total metallothionein content. Integration of the biomarkers into the Expert System ranked individuals sampled at site 2 as unstressed, mussels sampled at site 3 as being subject to low stress, and those from site 4, which is adjacent to what used to be a copper mine, as being highly stressed, with respect to specimens sampled from the reference site. Microarray analyses demonstrated that at the two innermost and mostly polluted sites, gene expression patterns where severely altered. In particular, some genes exhibited a linear activation response along the copper gradient, e.g. metallothioneins mt 20 and mt 10, and catalase. In addition, stress responsive kinase (krs), glutathione transferase (gst), major vault protein and histones (h1, h2a and h4) were significantly up-regulated at the innermost site. In conclusion, these results demonstrated that sites could be discriminated using both a physiological and a molecular approach. The development of a stress syndrome along the pollution gradient was evidenced with a novel mussel microarray, both in terms of numbers of regulated genes and level of gene response.

 

Dondero, F., L. Piacentini, et al. (2006). "Gene transcription profiling in pollutant exposed mussels (Mytilus spp.) using a new low-density oligonucleotide microarray." Gene 376(1): 24-36.

                In this study we describe the design and implementation of a novel low-density oligonucleotide microarray (the "Mytox-chip"). It consists of 24 mussel genes involving both normalizing elements and stress response related genes, each represented on the array with one or two different 50 mer oligonucleotide-probe reporters spotted in replicated samples on glass-activated slides. Target genes were selected on the basis of their potential involvement in mechanisms of pollutant and xenobiotic response. They are implicated in both basic and stress related cellular processes such as shock response, biotransformation and excretion, cell-cycle regulation, immune defense, drug metabolism, etc. The microarray was tested on mussels exposed to sublethal concentrations of mercury or a crude North Sea oil mixture. RNA samples were extracted from digestive glands of control and treated mussels for the synthesis of fluorescence labeled cDNAs to be used in dual color hybridizations. Transcription rates of two metallothionein iso-genes (mt10 and mt20), a p53-like gene and actin were quantitatively estimated also by real-time PCR to confirm microarray data. Significant alterations in the gene transcription patterns were seen in response to both treatments.

 

Hardivillier, Y., F. Denis, et al. (2006). "Metal influence on metallothionein synthesis in the hydrothermal vent mussel Bathymodiolus thermophilus." Comp Biochem Physiol C Toxicol Pharmacol 143(3): 321-32.

                The present study reports on the metallothionein expression in the hydrothermal vent mussel Bathymodiolus thermophilus. Metallothioneins (MT) are proteins involved in intracellular metal regulation and conserved throughout the animal kingdom. The hydrothermal vent environment presents peculiarities (high levels of sulfides and metals, low pH, anoxia) that may have driven associated species to develop original evolutionary ways to face these extreme living conditions. Mussels were exposed to different metal solutions at the atmospheric pressure. The MT mRNA levels and MT contents were measured in gills and mantles of each exposed mussel. The intracellular metal distribution was estimated in fractions obtained after the centrifugation of tissue homogenates. A few of the tested metals (Ag, Cu, Cd, Hg and Zn) were able to significantly induce MT mRNA levels. Silver was the only one that produced a significant increase of the MT protein level in both mantle and gills. The gills always presented higher MT protein levels than the mantle did, while their MT mRNA levels were similar. Our data show that MT mRNA and MT protein levels do not follow a clear relationship in the gills and mantle of B. thermophilus and we assume that a posttranscriptional control occurs in these mussels.

 

Leignel, V. and M. Laulier (2006). "Isolation and characterization of Mytilus edulis metallothionein genes." Comp Biochem Physiol C Toxicol Pharmacol 142(1-2): 12-8.

                Metallothioneins (MTs) are crucial proteins in all organisms for the regulation of essential metals and the detoxification of heavy metals. Many studies have estimated MT levels in mussel tissues to detect marine metal pollution. In this study, we investigated the MT gene structures of the forms present in Mytilus edulis (blue mussel). One MT-10 (2413 bp) gene and one MT-20 (1906 bp) gene were obtained. These MT genes contain three exons and two long introns. The splicing signals for MT-10 and MT-20 were GTA(T/A)GT-(C/T)AG. The structural organization (length of intron, splicing signals, AT content) of MT-10 and MT-20 is compared with other MT genes.

 

Cheung, M. S., E. M. Fok, et al. (2006). "Subcellular cadmium distribution, accumulation, and toxicity in a predatory gastropod, thais Clavigera, fed different prey." Environ Toxicol Chem 25(1): 174-81.

                Bioaccumulation and toxicity of Cd were investigated in a marine predatory whelk, Thais clavigera, after being fed with the rock oyster, Saccostrea cucullata, or the herbivorous snail, Monodonta labio, for up to four weeks. The oysters and snails had different subcellular Cd distributions and concentrations in their bodies given their different metal-handling strategies and were exposed to dissolved Cd for two weeks before being fed to the whelks. After four weeks of dietary exposure, the Cd body concentrations in T. clavigera increased from 3.1 microg/g to between 22.9 and 41.8 microg/g and to between 22.7 and 24.1 microg/g when they were fed with oyster and snail prey, respectively. An increasing proportion of Cd was found to be distributed in the metallothionein (MT)-like proteins and organelle fractions, whereas the relative distribution in the metal-rich granules fraction decreased when the whelks were fed Cd-exposed prey. At the highest Cd dosage, more Cd was distributed in the pool of metal-rich granules when the whelks were fed the oysters than when they were fed the snails. Among all the biomarkers measured (MT induction, condition index, lipid peroxidation, and total energy reserve including carbohydrate, lipid, and protein), only MT showed a significant difference from the control treatments, and MT was the most sensitive biomarker for dietary Cd exposure. No toxicity was found in the whelks fed different Cd-exposed prey as revealed by various biomarkers at the different biological levels. Our results imply that metal fractionation in prey can alter the subsequent subcellular metal distribution in predators and that dietary Cd toxicity to the whelks was low, even when the accumulated Cd body concentrations were high.

 

Funes, V., J. Alhama, et al. (2006). "Ecotoxicological effects of metal pollution in two mollusc species from the Spanish South Atlantic littoral." Environ Pollut 139(2): 214-23.

                Metal accumulation and some of their biochemical effects have been studied in oysters (Crassostrea angulata) and mussels (Mytilus galloprovincialis) of the South Atlantic Spanish littoral. Especial attention has been paid to antioxidant defences and oxidative damage to biomolecules. Deep differences in the response of oysters and mussels to metal pollution were found. Oysters, with the higher metal loads of both species, showed increased antioxidant defences, and less extensive oxidative damage. In contrast, mussels, which accumulated much lower metal concentrations, showed clear increases in oxidized biomolecules, in agreement with their low increases in the antioxidant defence mechanisms. Our results suggest that mussels are more sensitive and less well adapted to metal pollution, probably explaining their absence in the most contaminated studied site, Mazagon. We conclude that oysters can be used as more sensitive bioindicator of pollution in the South Spanish littoral, and as a suitable model to study the adaptation to metal pollution.

 

Regoli, F., S. Gorbi, et al. (2006). "Use of the land snail Helix aspersa as sentinel organism for monitoring ecotoxicologic effects of urban pollution: an integrated approach." Environ Health Perspect 114(1): 63-9.

                Atmospheric pollution from vehicular traffic is a matter of growing interest, often leading to temporary restrictions in urban areas. Although guidelines indicate limits for several parameters, the real toxicologic impacts remain largely unexplored in field conditions. In this study our aim was to validate an ecotoxicologic approach to evaluate both bioaccumulation and toxicologic effects caused by airborne pollutants. Specimens of the land snail Helix aspersa were caged in five sites in the urban area of Ancona, Italy. After 4 weeks, trace metals (cadmium, chromium, copper, iron, manganese, nickel, lead, and zinc) and polycyclic aromatic hydrocarbons (PAHs) were measured and these data integrated with the analyses of molecular and biochemical responses. Such biomarkers reflected the induction of detoxification pathways or the onset of cellular toxicity caused by pollutants. Biomarkers that correlated with contaminant accumulation included levels of metallothioneins, activity of biotransformation enzymes (ethoxyresorufin O-deethylase, ethoxycoumarin O-deethylase), and peroxisomal proliferation. More general responses were investigated as oxidative stress variations, including efficiency of antioxidant defenses (catalase, glutathione reductase, glutathione S-transferases, glutathione peroxidases, and total glutathione) and total oxyradical scavenging capacity toward peroxyl and hydroxyl radicals, onset of cellular damages (i.e., lysosomal destabilization), and loss of DNA integrity. Results revealed a marked accumulation of metals and PAHs in digestive tissues of organisms maintained in more traffic-congested sites. The contemporary appearance of several alterations confirmed the cellular reactivity of these chemicals with toxicologic effects of potential concern for human health. The overall results of this exploratory study suggest the utility of H. aspersa as a sentinel organism for biomonitoring the biologic impact of atmospheric pollution in urban areas. Key words: atmospheric pollutants, bioindicators, biomarkers, DNA integrity, lysosomes, metallothioneins, oxidative stress, peroxisomes, polycyclic aromatic hydrocarbons, trace metals.

 

Gagnon, C., F. Gagne, et al. (2006). "Exposure of caged mussels to metals in a primary-treated municipal wastewater plume." Chemosphere 62(6): 998-1010.

                The biological availability of metals in municipal wastewater effluents is strongly influenced by the physical and chemical conditions of both the effluent and the receiving water. Aquatic organisms are exposed to both dissolved and particulate (food ingestion) forms of these metals. In the present study, the distribution of metals in specific tissues was used to distinguish between exposure routes (i.e. dissolved vs. particulate phase) and to examine metal bioavailability in mussels exposed to municipal effluents. Caged Elliptio complanata mussels were deployed at sites located between 1.5 km upstream and 12 km downstream of a major effluent outfall in the St. Lawrence River. Metals in surface water samples were fractionated by filtration techniques to determine their dissolved, truly-dissolved (<10 kDa), total-particulate and acid-reactive-particulate forms. At the end of the exposure period (90 days), pooled mussel soft tissues (digestive gland, gills, gonad, foot and mantle) were analyzed for several metals. The results showed that gills and digestive gland were generally the most important target tissues for metal bioaccumulation, while gill/digestive gland metal ratios suggest that both exposure routes should be considered for mussels exposed to municipal effluents. We also found that Ag and Cd in the dispersion plume nearest the outfall, in contrast to other metals such as Cu and Zn, are more closely associated with colloids and were generally less bioavailable than at the reference site in the St. Lawrence River.

 

Marie, V., P. Gonzalez, et al. (2006). "Metallothionein gene expression and protein levels in triploid and diploid oysters Crassostrea gigas after exposure to cadmium and zinc." Environ Toxicol Chem 25(2): 412-8.

                Quantitative real-time polymerase chain reaction (PCR) was used to compare for the first time the differential expression of metallothionein (MT) isoform genes, together with biosynthesis of the total MT proteins, in the gills of triploid and diploid juvenile Pacific oyster Crassostrea gigas in response to cadmium (Cd) and zinc (Zn) exposure. Oysters were exposed to Cd (0.133 microM), Zn (15.3 microM), and Cd+Zn for 14 d. Results showed similar response capacities to metal exposures in the two populations. No significant difference was revealed in terms of MT gene expression, MT protein synthesis, and Cd accumulation. However, triploid oysters bioaccumulated Zn 30% less efficiently than diploid oysters. Among the three MT isoform genes, CgMT2 appeared to be more expressed than CgMT1, whereas CgMT3 appeared to be anecdotal (10(6) times lower than CgMT2). CgMT2 and CgMT1 gene expression levels were increased sevenfold in the presence of Cd, whereas Zn appeared to have no effect. A twofold increase in MT protein levels occurred in response to Cd exposure. Discrepancies between mRNA and protein levels suggest that in C. gigas MT are regulated at the transcriptional level, as well as at the translational level.

 

Grattarola, M., M. Carloni, et al. (2006). "Expression, purification and preliminary characterization of mussel (Mytilus galloprovincialis) metallothionein MT20." Mol Biol Rep 33(4): 265-72.

                Metallothioneins are rather ubiquitous metal-binding proteins induced by stressing or physiological stimuli. Two major metallothionein isoforms have been identified in mussel: MT10 and MT20. Nevertheless the high sequence homology, the two isoforms exhibit different expression and inducibility in vivo. We cloned and produced in Escherichia coli the MT20 isoform from Mytilus galloprovincilis. cDNA was subcloned into pGEX-6P.1 vector, in frame with a sequence encoding a glutathione-S-transferase (GST) tail. Recombinant protein was purified to electrophoretic homogeneity by affinity chromatography. After enzymatic cleavage of the GST tail the MT moiety was recovered with a final yield of about 5 mg of protein per litre of bacterial culture. The metal-binding ability of MT20 was assessed by absorption spectroscopy upon addition of cadmium equivalents and the metal release was checked as a function of the environment pH. Moreover the protein was analysed for the propensity to polymerization, typical of this class of protein, before and after exposure to reducing and alkylating agents.

 

Perceval, O., Y. Couillard, et al. (2006). "Linking changes in subcellular cadmium distribution to growth and mortality rates in transplanted freshwater bivalves (Pyganodon grandis)." Aquat Toxicol 79(1): 87-98.

                Relationships between Cd accumulation and subcellular distribution, and growth and mortality rates were examined in the freshwater bivalve Pyganodon grandis in a transplant experiment. Organisms were transferred from a clean lacustrine site to four lakes situated along a Cd concentration gradient in the mining region of Rouyn-Noranda. The bivalves were maintained in open enclosures placed in the bottom sediments of the littoral zone of all five lakes for 400 days. At the end of the experiment, metallothionein (MT) was measured in the bivalve gills with a Hg-saturation assay and Cd partitioning among the various cytosolic protein pools was determined by size-exclusion chromatography. Marked differences were observed among the five sites: the range in calculated free-cadmium ion concentrations in water overlying the sediments was 35-fold whereas Cd concentrations in the gill cytosol of the transplanted bivalves varied three-fold. In the transplanted bivalves, the distribution of gill Cd among the various cytosolic complexes also varied significantly among sites. For bivalves transplanted to the three most contaminated sites, Cd concentrations in the high molecular weight pool (HMW>25 kDa) were significantly higher than the baseline levels determined from bivalves caged at the reference site; a similar trend was seen for Cd concentrations in the metallothionein pool (Cd-MT). For bivalves transferred to two of the high contamination sites, proportionately less of the gill cytosolic Cd was sequestered (i.e. detoxified) by MT-like proteins. Reductions in survival were also observed at these two sites, and these elevated mortalities, in turn, were consistent with the absence of indigenous bivalve populations at these sites. This result is compatible with our recent work on P. grandis populations living in lakes of the Rouyn-Noranda area, in which we demonstrated that excessive accumulation of Cd in the HMW pool of the gill cytosol of the individual mollusks could be related to the impairment of population health status.

 

Marie, V., P. Gonzalez, et al. (2006). "Metallothionein response to cadmium and zinc exposures compared in two freshwater bivalves, Dreissena polymorpha and Corbicula fluminea." Biometals 19(4): 399-407.

                Metallothionein (MT) response to cadmium (Cd) and zinc (Zn) bioaccumulation after single or combined direct exposure was compared in two freshwater bivalves, Dreissena polymorpha (zebra mussel) and Corbicula fluminea (Asiatic clam). Bivalves were exposed to 0.133 microM Cd and/or 15.3 microM Zn, with metal and MT concentrations analysed in the whole soft body after 1, 3, 10 and 24 days of exposure and compared with controls. Results showed significant increase in MT concentrations in both species exposed to Cd and Cd+Zn with a higher accumulation of the protein compared to the control in D. polymorpha for nevertheless similar Cd levels accumulated with time. Exposure to Zn alone led to a significant increase in MT concentrations only in C. fluminea, whereas there was a lack of MT gene induction in the zebra mussels which was confirmed by MT mRNA quantification in gills (RT-PCR). Mussel mortality after 10 days of exposure to Zn and Cd + Zn is discussed with regard to detoxification mechanisms, which include metallothioneins.

 

Damiens, G., C. Mouneyrac, et al. (2006). "Metal bioaccumulation and metallothionein concentrations in larvae of Crassostrea gigas." Environ Pollut 140(3): 492-9.

                Larval stages of bivalve molluscs are highly sensitive to pollutants. Oysters from a hatchery from Normandy (English Channel) were induced to spawn, and fertilized eggs were exposed to copper or cadmium for 24 h. Metal accumulation (from 0.125 to 5 microg Cu L(-1) and from 25 to 200 microg Cd L(-1)) and MT concentrations were measured in larvae. Compared to controls, larvae accumulated copper and cadmium with an increase in MT concentrations particularly with cadmium (i.e. 130.96 ng Cu (mg protein)(-1) and 12.69 microg MT (mg protein)(-1) at 1 microg Cu L(-1) versus 23.19 ng Cu (mg protein)(-1) and 8.92 microg MT (mg protein)(-1) in control larvae; 334.3 ng Cd (mg protein)(-1) and 11.70 microg MT (mg protein)(-1) at 200 microg Cd L(-1) versus 0.87 ng Cd (mg protein)(-1) and 4.60 microg MT (mg protein)(-1) in control larvae). Larvae were also obtained from oysters of a clean area (Arcachon Bay) and a polluted zone (Bidassoa estuary) and exposed to copper in the laboratory, their MT concentration was measured as well as biomarkers of oxidative stress. Biomarker responses and sensitivity to copper for the larvae from Arcachon oysters were higher than for those from Bidassoa.

 

Barsiene, J., K. K. Lehtonen, et al. (2006). "Biomarker responses in flounder (Platichthys flesus) and mussel (Mytilus edulis) in the Klaipeda-Butinge area (Baltic Sea)." Mar Pollut Bull 53(8-9): 422-36.

                During the EU project BEEP a battery of biomarkers was applied in flounder (Platichthys flesus) and the blue mussel (Mytilus edulis) collected at three locations off the Lithuanian coast (Baltic Sea) in June and September 2001 and 2002. The elevated biomarker responses in specimens sampled in September 2001 were apparently related to the extensive dredging activities in the Klaipeda port area and subsequent dumping of contaminated sediments. High concentrations of organic pollutants (organochlorines and PBDEs) were also measured in the tissues of both indicator species. In addition, response levels of genotoxicity, cytotoxicity, immunotoxicity as well as concentrations of PAH metabolites in the bile of flounder showed elevations in 2002 after an oil spill in the Butinge oil terminal in November 2001. In flounder, biomarker measurements 10 months after the spill indicated recovery processes but in mussels a high level of genotoxicity could still be observed 22 months later. The present study illustrates the usefulness of the multi-biomarker approach in the detection of biological effects of pollution in this region of the Baltic Sea.

 

Kopecka, J., K. K. Lehtonen, et al. (2006). "Measurements of biomarker levels in flounder (Platichthys flesus) and blue mussel (Mytilus trossulus) from the Gulf of Gdansk (southern Baltic)." Mar Pollut Bull 53(8-9): 406-21.

                In the framework of the EU funded BEEP project a set of biomarkers, gross morphometric indices and tissue concentrations of selected organic pollutants were measured in flounder (Platichthys flesus) and mussels (Mytilus trossulus) collected twice a year (April and October) from three sites in the inner Gulf of Gdansk between autumn 2001 and spring 2003. In flounder, seasonal differences in most biomarkers were observed, but no correlations with tissue pollutant levels could be found. In mussels, highly variable levels in biomarker responses were seen, but no clear seasonal or spatial trends, directly related to tissue concentrations, could be established. The observed biomarkers distribution the study sites are probably mostly caused by interannual, seasonal and individual variability and, in case of flounder, possibly by exchange of stocks between the sampling sites.

 

Lehtonen, K. K., D. Schiedek, et al. (2006). "The BEEP project in the Baltic Sea: overview of results and outline for a regional biological effects monitoring strategy." Mar Pollut Bull 53(8-9): 523-37.

                Field studies in the framework of the EU funded BEEP project (Biological Effects of Environmental Pollution in Marine Coastal Ecosystems, 2001-2004) aimed at validating and intercalibrating a battery of biomarkers of contaminant exposure and effects in selected indicator species in the Mediterranean, the North Atlantic and the Baltic Seas. Major strategic goals of the BEEP project were the development of a sensitive and cost-efficient biological effects monitoring approach, delivery of information and advice to end-user groups, and the implementation of a network of biomarker researchers around Europe. Based on the main results obtained in the Baltic Sea component of the BEEP the present paper summarises and assesses the applicability of biomarkers for different regions and species in this sea area. Moreover, a general strategy and some practical considerations for the monitoring of biological effects in the Baltic Sea are outlined.

 

Schiedek, D., K. Broeg, et al. (2006). "Biomarker responses as indication of contaminant effects in blue mussel (Mytilus edulis) and female eelpout (Zoarces viviparus) from the southwestern Baltic Sea." Mar Pollut Bull 53(8-9): 387-405.

                During a field study performed in spring and autumn 2001 and 2002, blue mussels (Mytilus edulis) and female eelpout (Zoarces viviparus) were collected at three locations in the Wismar Bay (Baltic Sea), and several biomarkers of contaminant effects were analysed. Besides seasonal and inter-annual variations, biomarker signals were most pronounced at the location closest to Wismar Harbour (Wendorf) in both species. Lysosomal membrane stability (LMS) was lowest and acetylcholinesterase activity (AChE) was significantly reduced. Frequency of micronuclei (MN) was significantly higher (in blue mussels), indicating mutagenic effects. In eelpout elevated levels of DNA adducts, EROD induction and PAH-metabolites were measured. Metallothionein (MT), biomarker for trace metal exposure, showed a gradient only in spring. Organochlorine contaminant analyses (PCBs, DDTs) corresponded to the observed biomarker levels. The results obtained clearly demonstrate pollution effects in the southwestern Baltic Sea. Moreover, they show that a multibiomarker approach is also applicable in a brackish water environment.

 

Zorita, I., E. Strogyloudi, et al. (2005). "Application of two SH-based methods for metallothionein determination in mussels and intercalibration of the spectrophotometric method: laboratory and field studies in the Mediterranean Sea." Biomarkers 10(5): 342-59.

                Metallothionein (MT) induction is widely used as a biomarker of exposure to metals in mussels. The aims of the present work were first to compare the suitability of spectrophotometry and differential pulse polarography (DPP) for MT detection in mussels exposed to 200 ppb cadmium for 9 days in a laboratory experiment and in mussels sampled in different seasons from expected pollution gradients along the Mediterranean Sea; second, to intercalibrate the widely used spectrophotometric method using mussels from Saronikos Gulf. In the intercalibration of the spectrophotometric method, similar results (p>0.05) were obtained by two different research teams indicating a good reproducibility of the technique. However, polarographic and spectrophotometric methods gave significantly (p<0.05) different results in laboratory and field studies. In the laboratory experiment, MT values detected with DPP were nine times higher than with spectrophotometry. The results obtained by the two methods were significantly correlated. Both methods could discriminate between control and exposed mussels. In field studies, MT values obtained by DPP were 34-38-fold higher than with spectrophotometry, and MT concentrations measured by both methods were not correlated. This discrepancy could be due to several factors, including the low levels of bioavailable metals in the studied areas and the possibility that the different methods can measure MT isoforms differentially. Further work is needed to decipher the functions of MT isoforms in mussels. This information is relevant for the application of MT as a biomarker in biomonitoring programmes.

 

Legeay, A., M. Achard-Joris, et al. (2005). "Impact of cadmium contamination and oxygenation levels on biochemical responses in the Asiatic clam Corbicula fluminea." Aquat Toxicol 74(3): 242-53.

                The aim of this work was to evaluate the potential utility of several biochemical parameters as indicators of the toxic effects of cadmium in the freshwater clam Corbicula fluminea under two levels of oxygenation (normoxia 21 kPa and hypoxia 4 kPa). These variations in oxygenation are representative of the natural environments of bivalves living at the bottom of the water column, where hypoxic episodes may occur regularly. Cadmium accumulation, metallothionein synthesis, MXR protein induction, lipoperoxidation and antioxidant enzyme activities (catalase, glutathione reductase and total and selenium-dependent glutathione peroxidases) were assessed in the gills of C. fluminea in four experimental conditions: normoxia, hypoxia, normoxia with cadmium and hypoxia with cadmium ([Cd]=30 microg l(-1)) over a 14-day period. Behavioural reactions were also followed for the duration of the experiment by monitoring clam activity and valve movements. This study is a first report on biochemical responses under cadmium contamination and hypoxia and will enable us to determine better biomarkers for C. fluminea as they were measured simultaneously. In metal-exposed animals, we found an increasing accumulation of cadmium in the gills with time, and this was more severe in hypoxic conditions. Metallothionein synthesis occurred in contaminated clams and was precocious in hypoxic conditions. MXR protein induction appeared promising due to its quick and significant response to metal with a strong impact from hypoxic contamination. On the other hand, in our experimental conditions, antioxidant parameters did not show decisive responses to contamination and hypoxia, except glutathione peroxidases which decreased systematically with time in a cadmium-independent manner. Lipid peroxidation, expressed as malondialdehyde content, was not stimulated by normoxic contamination, as has been shown in other studies, but was stimulated under hypoxic cadmium contamination. Our study confirms the importance of a multi-biomarker approach in environmental studies as some are not appropriate to all organisms.

 

Ng, T. Y. and W. X. Wang (2005). "Dynamics of metal subcellular distribution and its relationship with metal uptake in marine mussels." Environ Toxicol Chem 24(9): 2365-72.

                We examined the dynamics of subcellular distribution of metals (Cd, Ag, and Zn) in the marine green mussel Perna viridis by partitioning the metals into the insoluble fraction (IF), heat-sensitive proteins (HSP), and metallothionein-like proteins (MTLP) during metal uptake and elimination. Variations in metal uptake and elimination then were correlated with the subcellular distributions of these metals. The IF and HSP were the first ligands to bind with the metals during the dissolved exposure, and more metals were found in the HSP when the metal influx rate was higher. However, to minimize toxicity, metals were redistributed from HSP to MTLP afterwards. The subcellular distribution of metals was dependent of the exposure route in the mussels. During dietary metal exposure, the metals attained equilibrium before they were assimilated and the metal assimilation efficiency was independent of the metal partitioning in different subcellular fractions. During the efflux, metals in the soluble fraction mediated depuration, whereas metals in the insoluble fraction acted as a final storage pool. Redistribution also may occur between the metal-sensitive and inactive pools without significant depuration as a secondary protective mechanism. We further demonstrated that the higher efflux rate of Ag and Cd was related to a higher partitioning in the MTLP and a lower partitioning in the IF. Our study shows that subcellular pools other than MTLP were involved in immediate metal handling in the bivalves. The wide dynamics of subcellular metal distribution suggests that the relevance of individual subcellular fractions is dependent on the exposure pathway.

 

Ng, T. Y. and W. X. Wang (2005). "Modeling of cadmium bioaccumulation in two populations of the green mussel Perna viridis." Environ Toxicol Chem 24(9): 2299-305.

                This study attempted to quantify differences in Cd biokinetics from two populations of green mussels (Perna viridis) from two sites (eastern and western) in Hong Kong with contrasting hydrological conditions. Body Cd concentrations were modeled using a simple biokinetic model coupled with measurements of dissolved Cd concentrations at each site. Mussels collected from the western site had three to six times higher Cd tissue concentration than the eastern population collected during two seasons (summer wet and winter dry), but the salinity was only lower in the western site during the summer. More Cd was distributed in the metallothionein-like and heat-sensitive proteins in the western population than the eastern population, and Cd predominantly was distributed in the insoluble fraction during summer. The Cd uptake rate constant from the dissolved phase was higher in the western population during summer due to a much lower salinity, but was comparable during winter. Dietary uptake of Cd was similar in both populations, and assimilation was lower from ingested radiolabeled seston than from diatoms. Efflux of Cd remained comparable between the two populations from two seasons (0.02-0.03 /d). Kinetic modeling demonstrates that the faster influx of Cd from aqueous phase caused the higher body Cd concentrations in the western population. The predicted Cd concentrations in mussels were comparable to those observed in the field. Our study highlights differences in Cd accumulation kinetics in different populations of mussels likely caused by the different physical environments.

 

Dallinger, R., M. Chabicovsky, et al. (2005). "Copper in Helix pomatia (Gastropoda) is regulated by one single cell type: differently responsive metal pools in rhogocytes." Am J Physiol Regul Integr Comp Physiol 289(4): R1185-95.

                Like all other animal species, terrestrial pulmonate snails require Cu as an essential trace element. On the other hand, elevated amounts of Cu can exert toxic effects on snails. The homeostatic regulation of Cu must therefore be a pivotal goal of terrestrial pulmonates to survive. Upon administration of Cu, snails accumulate the metal nearly equally in most of their organs. Quantitative studies in connection with HPLC and electrospray ionization mass spectrometry reveal that a certain fraction of Cu in snails is bound to a Cu-metallothionein (Cu-MT) isoform that occurs in most organs at constant concentrations, irrespective of whether the animals had been exposed to physiological or elevated amounts of Cu. In situ hybridization demonstrates that at the cellular level, the Cu-binding MT isoform is exclusively expressed in the so-called pore cells (or rhogocytes), which can be found in all major snail organs. The number of pore cells with Cu-MT mRNA reaction products remains unaffected by Cu exposure. Rhogocytes also are major storage sites of Cu in a granular form, the metal quickly entering the snail tissues upon elevated exposure. The number of rhogocytes with granular Cu precipitations strongly increases upon Cu administration via food. Thus, whereas Cu-MT in the rhogocytes represents a stable pool of Cu that apparently serves physiological tasks, the granular Cu precipitations form a second, quickly inducible, and more easily available pool of the metal that serves Cu regulation by responding to superphysiological metal exposure.

 

La Porte, P. F. (2005). "Mytilus trossulus hsp70 as a biomarker for arsenic exposure in the marine environment: laboratory and real-world results." Biomarkers 10(6): 417-28.

                The highly conserved heat shock protein 70 (hsp70) is induced by heat and chemical toxins, particularly heavy metals such as arsenic (As). The use of Mytilus trossulus (bay mussel) hsp70 as a 'screening' biomarker for marine heavy metals contamination was assessed. Some studies have found high hsp70 sensitivity to heavy metals, while others have found the opposite. Few studies have realistically used low heavy metals exposures, and fewer have used real-world contamination exposures. Clean sub-tidal mussels from the Puget Sound, Washington State (WA), USA, were acclimatized for 2 weeks and exposed for 24 h to As-spiked seawater (n=9) or to contaminated seawater from an arsenical pesticide plant in Tacoma, WA (n=10) followed by a Western blot for hsp70. Hsp70 inductions were insignificant at 10 microg l(-1) As(III), but were strong at 100 microg l(-1) (p<0.05) and 1000 microg l(-1) (p<0.01), with the induction threshold estimated at 30-50 microg l(-1) As(III). Hsp70 induction roughly correlated with arsenical toxicity, with As(III) > As(V) > (CH(3))(2)As(V). Altogether, the inter-individual variability of hsp70 levels tends to mask inductions at low As concentrations, making it a crude toxicity biomarker. In addressing this problem, the following options could prove promising: (1) pre- or post-stressing specimens for greater hsp70 sensitivity, (2) use of internal protein controls such as actin, (3) use of hsp70-reporter gene constructs, and (4) detection with hsp60, heme oxygenase-1, metallothionein, CYP450, MXR or GPx.

 

Bebianno, M. J., R. Company, et al. (2005). "Antioxidant systems and lipid peroxidation in Bathymodiolus azoricus from Mid-Atlantic Ridge hydrothermal vent fields." Aquat Toxicol 75(4): 354-73.

                Enzymatic defenses involved in protection from oxygen radical damage were determined in gills and mantle of Bathymodiolus azoricus collected from three contrasting Mid-Atlantic Ridge (MAR) hydrothermal vent fields (Menez-Gwen, Lucky Strike and Rainbow). The activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidases (GPx) (total and Se-dependent), and levels of total oxyradical scavenging capacity (TOSC), metallothioneins (MT) and lipid peroxidation (LPO) were determined in B. azoricus tissues and the impact of metal concentrations on these antioxidant systems and lipid peroxidation assessed. SOD, CAT, TOSC, MTs and LPO levels were higher in B. azoricus gills while glutathione peroxidases (total and Se-dependent) were higher in the mantle, and with the exception of CAT, were of the same order of magnitude as in other molluscs. TOSC levels from Menez-Gwen indicate that the vent environment at this site is less stressful and the formation of ROS in mussels is effectively counteracted by the antioxidant defense system. TOSC depletion indicates an elevated ROS production in molluscs at the other two vent sites. Cytosolic SOD, GPx and LPO were more relevant at Lucky Strike (Bairro Alto) where levels of essential (Cu and Zn) and toxic metals (Cd and Ag) were highest in the organisms. CAT activity and LPO were predominant at the Rainbow vent site, where an excess of Fe in mussel tissues and in vent fluids (the highest of all three vent sites) may have contributed to increased LPO. Therefore, three distinct pathways for antioxidant enzyme systems and LPO based on environmental metal speciation of MAR vent fields are proposed for Bathymodiolus gills. At Menez-Gwen, TOSC towards peroxyl and hydroxyl radicals and peroxynitrite are predominant, while at Lucky Strike cytosolic SOD activity and GPx are the main antioxidant mechanisms. Finally at Rainbow, catalase and lipid peroxidation are dominant, suggesting that resistance of mussels to metal toxicity at these vent fields decreases in the sequence Menez-Gwen > Lucky Strike and Rainbow.

 

David, E., A. Tanguy, et al. (2005). "Response of the Pacific oyster Crassostrea gigas to hypoxia exposure under experimental conditions." FEBS J 272(21): 5635-52.

                The molecular response to hypoxia stress in aquatic invertebrates remains relatively unknown. In this study, we investigated the response of the Pacific oyster Crassostrea gigas to hypoxia under experimental conditions and focused on the analysis of the differential expression patterns of specific genes associated with hypoxia response. A suppression subtractive hybridization method was used to identify specific hypoxia up- and downregulated genes, in gills, mantle and digestive gland, after 7-10 days and 24 days of exposure. This method revealed 616 different sequences corresponding to 12 major physiological functions. The expression of eight potentially regulated genes was analysed by RT-PCR in different tissues at different sampling times over the time course of hypoxia. These genes are implicated in different physiological pathways such as respiration (carbonic anhydrase), carbohydrate metabolism (glycogen phosphorylase), lipid metabolism (delta-9 desaturase), oxidative metabolism and the immune system (glutathione peroxidase), protein regulation (BTF3, transcription factor), nucleic acid regulation (myc homologue), metal sequestration (putative metallothionein) and stress response (heat shock protein 70). Stress proteins (metallothioneins and heat shock proteins) were also quantified. This study contributes to the characterization of many potential genetic markers that could be used in future environmental monitoring, and could lead to explore new mechanisms of stress tolerance in marine mollusc species.

 

Ivankovic, D., J. Pavicic, et al. (2005). "Evaluation of the Mytilus galloprovincialis Lam. digestive gland metallothionein as a biomarker in a long-term field study: seasonal and spatial variability." Mar Pollut Bull 50(11): 1303-13.

                A study was made of seasonal and spatial variability of metallothionein (MT) concentrations, determined spectrophotometrically in the soluble fraction of the digestive gland of mussels Mytilus galloprovincialis, collected between 1999 and 2001 from several coastal and estuarine locations along the central Eastern Adriatic coast (Croatia). The seasonal influence on the MT and metal concentrations (higher values in winter-spring season, than in summer-autumn season) is more pronounced than the local site-specific influence. Furthermore, within each season a significant site-specific dependence on the MT and trace metal variations can be detected. An inverse relationship of mussel condition index (CI) and temperature with MT and trace metals levels indicates the influence of food abundance and mussel annual reproductive cycle. Substantially higher concentrations of both MT and Cd were recorded in mussels inhabiting estuarine locations, possibly indicating a markedly higher Cd bioavailability at these locations. The positive correlations obtained between MT and Cd in all seasons except autumn support an argument for application of digestive gland MT as a biomarker of Cd exposure, providing evidence for assessing the most appropriate season for mussel sampling.

 

Leignel, V., Y. Hardivillier, et al. (2005). "Small metallothionein MT-10 genes in coastal and hydrothermal mussels." Mar Biotechnol (NY) 7(3): 236-44.

                Metallothioneins (MTs) are important proteins in the intracellular regulation of metals. In the Mytilidae family, which includes many economically important species, 2 major forms of MTs have been reported: MT-10 (10 kDa) and MT-20 (20 kDa). Many different MT-10 proteins have been isolated from the common species Mytilus edulis, which suggests that distinct MT-10 genes may occur in a single specimen. Some MT genes, involving 3 exons and 2 large introns, have been isolated in Mytilidae. Our aim was to determine whether intron-free forms of the MT-10 genes can exist, which could allow rapid transcription in response to exposure to metals. Our study focused on 2 species living under very different environmental conditions: Mytilus edulis (a coastal mussel) and Bathymodiolus thermophilus (a hydrothermal mussel). We report here the first description of small, intron-free MT-10 genes, possessing a correct open reading frame in these 2 species.

 

Bonneris, E., O. Perceval, et al. (2005). "Sub-cellular partitioning of Cd, Cu and Zn in tissues of indigenous unionid bivalves living along a metal exposure gradient and links to metal-induced effects." Environ Pollut 135(2): 195-208.

                We studied organ and sub-cellular distributions of several trace metals in a freshwater bivalve that has been proposed for use as a metal biomonitor. Specimens of Pyganodon grandis were collected from nine lakes located along a Cd, Cu and Zn concentration gradient (Rouyn-Noranda area, Quebec). Gills and digestive gland were isolated, homogenized and six sub-cellular fractions were separated by differential centrifugation and analysed for their Cd, Cu and Zn content. Metallothionein was quantified independently. Gill tissues contained abundant calcium concretions that accounted for over 60% of the total gill burden of each metal. Cadmium and Zn concentrations in this granule fraction reflected ambient metal concentrations. Metal concentrations in the digestive gland also responded to the metal contamination gradient, but to a lesser extent than the gills, reflecting the lower abundance of granules in the digestive gland. Metals (Cd, Cu) in this organ were present largely in the "heat-stable proteins" fraction, and metal concentrations in this fraction were strongly correlated with those of both metallothionein and, to a lesser extent, the "lysosomes+microsomes" and "mitochondria" fractions. In both organs, Cd concentrations in the "heat-denaturable protein" fraction remained low and constant, suggesting reasonably effective metal detoxification. Some evidence for oxidative stress was noted in the gills but not in the digestive gland. Overall, we conclude that in nature metals in P. grandis are bound differently in the gills and in the digestive gland and that metal detoxification in the former organ may be less effective than in the latter.

 

Leinio, S. and K. K. Lehtonen (2005). "Seasonal variability in biomarkers in the bivalves Mytilus edulis and Macoma balthica from the northern Baltic Sea." Comp Biochem Physiol C Toxicol Pharmacol 140(3-4): 408-21.

                Metallothionein level (MT), and acetylcholinesterase (AChE), catalase (CAT) and glutathione-S-transferase (GST) enzyme activities in the bivalves Mytilus edulis and Macoma balthica were investigated for seasonal variations from an inshore and an offshore site in the northern Baltic Sea. All the biomarkers showed variability, following mostly a similar pattern at both sites. Relationships between biomarkers and environmental factors and protein concentration and weight of target tissues were examined. In M. edulis, GST activity was related to Secchi depth, while in M. balthica a correlation with near-bottom oxygen saturation was observed. AChE activity correlated with the weight of the foot tissue of M. balthica. In both species, an integrated biomarker index indicated a stressed condition during the spring/early summer period. Strong seasonal variability in temperature and a concentrated period of food availability in spring-both governing the reproductive cycle of the bivalves-probably explains most of the observed natural variability in biomarkers in this sea area.

 

Campbell, P. G., A. Giguere, et al. (2005). "Cadmium-handling strategies in two chronically exposed indigenous freshwater organisms--the yellow perch (Perca flavescens) and the floater mollusc (Pyganodon grandis)." Aquat Toxicol 72(1-2): 83-97.

                Laboratory experiments on a variety of aquatic organisms suggest that metallothionein-like proteins (MT) play an important role in the regulation of essential metals, and in the sequestration and detoxification of non-essential metals (e.g., Cd). However, the importance of metallothionein production relative to alternative strategies of metal detoxification, and its effectiveness in metal detoxification, remain largely unexplored in field situations. In the present study we explored metal-handling strategies in an adult benthic bivalve (Pyganodon grandis) and in juvenile yellow perch (Perca flavescens), exposed to Cd in their natural habitat. The two biomonitor species were collected from lakes located along a Cd concentration gradient. Ambient dissolved Cd concentrations were determined by in situ dialysis as a measure of metal exposure. Sub-cellular Cd partitioning was determined in target tissues (bivalve gills and digestive gland; perch liver) by differential centrifugation, and metallothionein was measured independently by a mercury-saturation assay in the bivalve tissues. Malondialdehyde concentrations were measured as a potential indicator of oxidative stress. Ambient dissolved Cd concentrations ranged from 0.06 to 0.57 nM in the nine lakes from which bivalves were collected, and from < 0.3 to 6.7 nM in the eight lakes from which yellow perch were sampled. Bioaccumulated Cd also varied from lake to lake, more so for the bivalve than for the yellow perch; the [Cd]max/[Cd]min ratios for the various tissues decreased in the order: bivalve gill Cd 28 > bivalve digestive gland Cd 18 > perch hepatic Cd 14. In the two lakes that were common to both the bivalve and perch studies, i.e. lakes Opasatica and Vaudray, accumulated Cd concentrations were consistently higher in the bivalve than in the perch. Cadmium-handling strategies were similar in the bivalve digestive gland and perch liver, in that Cd was mainly associated with the heat-stable protein (HSP) fraction. Furthermore, in these organs the contributions from the "mitochondria" and "lysosomes + microsomes" fractions were consistently higher than in the gill tissue. In the bivalve gill, the HSP fraction could only account for a small proportion (10+/-3%) of the total Cd burden, and the metal was instead largely sequestered in calcium concretions (58+/-13%). Along the Cd-exposure gradient, Cd detoxification appeared to be reasonably effective in the bivalve gill and digestive gland, as judged from the protection of the heat-denaturable protein (HDP) fraction. However, in both organs Cd concentrations did increase in potentially metal-sensitive organelles (mitochondria), and malondialdehyde concentrations increased along the exposure gradient in the gills (but not in the digestive gland). Cadmium detoxification seemed less effective in juvenile yellow perch. As total hepatic Cd increased, Cd concentrations increased in all sub-cellular fractions, including the HDP fraction that was well protected in the bivalve. The relative proportions of Cd in the various fractions did not vary appreciably along the exposure gradient and there was no evidence of a threshold exposure concentration below which sensitive metal pools were protected.

 

Bonneris, E., A. Giguere, et al. (2005). "Sub-cellular partitioning of metals (Cd, Cu, Zn) in the gills of a freshwater bivalve, Pyganodon grandis: role of calcium concretions in metal sequestration." Aquat Toxicol 71(4): 319-34.

                Indigenous unionid molluscs, Pyganodon grandis, were collected from nine lakes in the Rouyn-Noranda area (Quebec, Canada) along a polymetallic concentration gradient (Cd, Cu, Zn). After excision, the gills were gently homogenised and the cellular compartments were separated by a differential centrifugation procedure that yielded the following particulate fractions: "nuclei + cellular debris", "mitochondria", "lysosomes + microsomes" and "granules". The supernatant remaining after the final ultracentrifugation step, i.e., the operationally-defined cytosol, was separated into a "heat-denaturable proteins" (HDP) fraction and a "heat-stable proteins" (HSP) fraction containing metallothionein (MT). The Cd, Cu and Zn content of each particulate and cytosolic fraction was determined and gill metallothionein was quantified independently by a mercury saturation assay. Cytosolic Cd concentrations were significantly related to the dissolved Cd concentrations at each site, but cytosolic Cu and Zn (essential metals) were not related to their respective ambient dissolved metal concentrations. Metallothionein concentrations increased along the metal contamination gradient and were related to cytosolic Cd (and Zn) in a concentration-dependent manner. However mass balance calculations showed that binding to metallothionein could only account for a small proportion of total gill metal ( approximately 10% Cd; approximately 3% Cu; approximately 1% Zn). Under these chronic exposure conditions, the three metals (Cd, Cu and Zn) were mainly located in calcium concretions present in the gills (respectively 58 +/- 13% of the total gill Cd, 64 +/- 6% of the total gill Cu and 73 +/- 6% of the total gill Zn). The overall contribution of granules to the total gill dry weight remained relatively constant among the different lakes, suggesting that lake-to-lake variations in granule synthesis were independent of the metal contamination gradient, i.e., these constituent elements of unionid gills act as non-inducible metal sinks at the cellular level. Metal concentrations increased proportionally in both the granules and the MT pool along the polymetallic gradient, suggesting a constant partitioning between these two compartments. Overall, despite an increase in Cd in the "mitochondria" fraction, metal sequestration mechanisms seem to be reasonably effective in detoxifying cadmium: in the cytosol, Cd concentrations in the potentially metal-sensitive HDP fraction remained relatively low and constant, even in specimens collected from the most contaminated lakes.

 

Chelomin, V. P., M. V. Zakhartsev, et al. (2005). "An in vitro study of the effect of reactive oxygen species on subcellular distribution of deposited cadmium in digestive gland of mussel Crenomytilus grayanus." Aquat Toxicol 73(2): 181-9.

                The study was performed to assess in vitro effects of reactive oxygen species (ROS, oxyradicals) on intracellular distribution of accumulated cadmium in digestive gland of the mussel Crenomytilus grayanus. In vitro induction of ROS (by Fe/ascorbate reaction) in tissue homogenates of Cd-accumulated mussels led to a significant increase in lipid peroxidation (as conjugated dienes and malondialdehyde) and also to decrease in reduced glutathione and Cd-binding protein contents. Also fraction of MT-like proteins (20-22 kDa) has been shifted to a higher molecular weight area (40-45 kDa), which indicates dimerization of the protein. The level of intracellular vesicle-stored cadmium (within membrane compartments like lysosomes) was decreased significantly in oxyradicals-exposed tissue crude homogenate of mussels in comparison with controls. Additionally, Cd distribution among three weight classes of cytosol proteins has been significantly changed after ROS exposure. Taken together the results, there is a clear indication that ROS induce an oxidative stress resulting in damaging of intracellular Cd-binding compartments that may trigger (or contribute) the toxicity of this metal. Thus, from our experimental results and reviewed information follows that under high "pressure" of heavy metals on marine environment the aquatic organisms can show higher sensitivity to normal variations of natural factors of the environment or even decrease the range of tolerance to their variations.

 

Geffard, A., C. Amiard-Triquet, et al. (2005). "Do seasonal changes affect metallothionein induction by metals in mussels, Mytilus edulis?" Ecotoxicol Environ Saf 61(2): 209-20.

                Mussels have been proposed as biomonitors of metal pollution based on the determination of metallothionein (MT) concentrations as a biomarker, but a comprehensive study taking into account both intersite and long-term temporal variations in MT and metal concentrations in different organs is lacking. Thus, the present study was designed to examine the concentrations of cytosolic and insoluble Cd, Cu, Zn, and MT in gills and digestive gland of mussels (Mytilus edulis) of homogeneous size and age obtained from aquaculture and kept on a reference site or translocated to a metal-rich site throughout their reproductive season (March-October 1997). Relatively significant binding of metals to the insoluble fraction was observed in both tissues. In the digestive gland, monthly MT concentrations were strongly correlated to cytosolic metal levels. Moreover, despite significant temporal variations, the grand mean MT concentration based on all individual determinations in the digestive gland (reference, n=54; transplants, n=50) was significantly higher in mussels from the metal-rich site. On the other hand, gill MT concentrations did not reflect metal contamination as reliably.

 

Moraga, D., A. L. Meistertzheim, et al. (2005). "Stress response in Cu2+ and Cd2+ exposed oysters (Crassostrea gigas): an immunohistochemical approach." Comp Biochem Physiol C Toxicol Pharmacol 141(2): 151-6.

                Localization of heat shock proteins (HSPs) and metallothioneins (MTs) was investigated in a marine bivalve (Crassostrea gigas) by immunohistochemical methods. Differential protein expression was demonstrated in digestive gland, gonad and gills, using a polyclonal antibody against C. gigas proteins. Application of this technique showed the cellular and tissue immunolabelling specificity of the two proteins. HSPs and MTs were localized in the epithelium of the digestive gland and gills in contact with the palleal compartment. For the first time, localization of MTs was observed in mature gametes of bivalve molluscs. Our results establish a basis for the use of immmunodetection techniques to study the tissue-specific localization of stress proteins in marine bivalves exposed to metal stress.

 

Raspor, B., Z. Dragun, et al. (2005). "Examining the suitability of mussel digestive gland to serve as a biomonitoring target organ." Arh Hig Rada Toksikol 56(2): 141-9.

                In coastal marine areas, the level of metallothioneins (MTs) is monitored as the specific response to trace heavy metals and is considered as a biomarker of metal exposure. Laboratory experiments with bivalves indicate that MT synthesis is induced by Cd, Cu, Hg, Ag and Zn ions. The results of our study, conducted with the mussel Mytilus galloprovincialis, deployed 12 months in the "hot spot" area of the Kastela Bay, Dalmatia, Croatia, are evaluated considering the recommendation of MED POL Programme for estimating exposure of mussels to trace heavy metals by means of MTs. Our data clearly indicate that the MT level in the digestive gland of the Mediterranean mussel depends on the biotic and abiotic parameters. Beside MT level, a number of parameters, like digestive gland mass, shell mass, cytosolic metal concentration is needed in order to define if MT level in the digestive gland of mussels is indicative as a biomarker of cadmium exposure.

 

Wang, W. X. and P. S. Rainbow (2005). "Influence of metal exposure history on trace metal uptake and accumulation by marine invertebrates." Ecotoxicol Environ Saf 61(2): 145-59.

                This paper reviews recent studies on the influence of trace metal exposure history on subsequent metal uptake in marine invertebrates, particularly bivalves and barnacles. Trace metal exposure may induce specific metal-binding ligands. Metallothionein-like proteins appear to play an important role in mediating metal uptake and hence accumulation, especially for Cd. Other ligands such as sulfide are important for Ag biokinetic changes in bivalves. The incorporation of trace metals into metal-rich granules appears to have little if any effect in modifying trace metal uptake. Furthermore, metals also interact strongly in their accumulation by marine animals. Generally, dissolved Hg uptake is reduced following exposure to other metals such as Ag, Cd, Cu, and Zn. Preexposure of marine mussels to Zn also causes concomitant changes in the uptake of other metals such as Cd sharing similar transport pathways. In general, barnacles are less responsive than mussels to trace metal exposure, presumably because of the dominance of detoxificatory pathways in which the accumulated metals are bound in granules (relatively biologically inactive pools). The tissue body burden and the detoxificatory fate of metals in animals seem to be more important in affecting metal accumulation than the nature of the exposure routes (aqueous vs. dietary) or of the exposure regimes. Trace metal accumulation may also be variable in different natural populations of bivalves as a result of different physicochemical environments and histories of exposure. We finally suggest a few areas for future research in this field.

 

Pampanin, D. M., I. Marangon, et al. (2005). "Stress biomarkers and alkali-labile phosphate level in mussels (Mytilus galloprovincialis) collected in the urban area of Venice (Venice Lagoon, Italy)." Environ Pollut 136(1): 103-7.

                In this study, a spatial and temporal survey at three sites located in the "canals" of the Venice historic centre (Italy) and at a reference site was undertaken to evaluate stress effects on mussels sampled in the Venice urban area, where raw sewage is discharged without treatment directly into the water. A battery of biomarkers (metallothionein, micronuclei, condition index and survival in air) was used to evaluate the stress condition of the animals. At the same time the alkali-labile phosphate assay (ALP) was performed in mussel' hemolymph with the aim to find an estrogenic effect biomarker in this mussel species. Biomarker results showed an impairment of the general health condition in the mussels coming from the urban area, in agreement with the chemical analysis. Significantly higher level of the ALP was found in male mussels sampled in April in the urban area, in comparison with the ones from the reference site (P<0.001). Finally, the PCA proved an easy and useful tool to summarize the obtained results, also able to classify the data to indicate a pollution gradient in the Venice urban area.

 

Dondero, F., L. Piacentini, et al. (2005). "Quantitative PCR analysis of two molluscan metallothionein genes unveils differential expression and regulation." Gene 345(2): 259-70.

                The mRNA levels of two components of the mussel (Mytilus galloprovincialis) metallothionein (MT) gene families, MT10 and MT20, were evaluated using real-time quantitative-PCR and Sybr Green I chemistry in animals exposed to heavy metals in vivo and in primary cell cultures. This method was highly specific in detecting the expression of the two genes over a widely dynamic range of starting DNA amounts, showing that the basal level of MT expression is mostly due to MT10 mRNA. Basal MT expression reflected the intracellular concentration of heavy metal as indicated by the use of the heavy metal chelator TPEN on primary cells. MT10 was observed to be inducible by Cd, Zn, and Cu ions, and to a lesser extent by Hg. By contrast, the MT20 expression level was very low under basal conditions, while its mRNA increased dramatically in response to Cd exposure, and to a lesser extend to Hg, leading to levels of expression similar to those of the MT10 gene. The essential metals Cu and Zn had a very small effect on the MT20 gene, whereas the concomitant exposure to Cu and H(2)O(2) produced a rapid rise of expression. In summary, data indicate that the MT isogenes are differentially regulated by heavy metals, while hydroxyl radicals may have a role in MT20 gene activation. Also, protein expression showed metal inducibility only after Cd exposure, suggesting the occurrence of posttranscriptional control mechanisms.

 

Shi, D. and W. X. Wang (2005). "Uptake of aqueous and dietary metals by mussel Perna viridis with different Cd exposure histories." Environ Sci Technol 39(23): 9363-9.

                The influences of different Cd pre-exposure regimes (route, concentration, and duration of Cd exposure) on the bioavailability of Cd, Ag, Hg, and Zn to the green mussels Perna viridis were quantified in this study. Following pre-exposing the mussels to Cd, we measured the mussel's tissue Cd concentration and clearance rate, as well as the metal dietary assimilation efficiency (AE) and the influx rate from the dissolved phase of the four studied metals. Differences in the route (aqueous and dietary pathways) and the history of pre-exposure (combined Cd concentration and duration) did not significantly affect the subsequent Cd dietary and aqueous uptake. The Cd dietary AEs increased following both the dissolved and dietary Cd pre-exposure. There was a significant correlation between the Cd AE and the accumulated Cd body concentration in the mussels. Dietary assimilation of Hg and Zn also increased slightly (but not significantly) after Cd pre-exposure, but the AEs of Ag remained constant. Except for the significant decrease in the dissolved uptake of Hg, Cd pre-exposure did not apparently affect the uptake of the other three metals from the solution. Metal-metal interactions are likely to be affected by the specificity of metallothionein induction. Our study demonstrated that the Cd body concentration as well as the environmental Cd concentration instead of the history of pre-exposure was more important in affecting the Cd accumulation in the mussels. Such factors need to be considered in interpreting metal body concentrations in biomonitors.

 

Pampanin, D. M., L. Camus, et al. (2005). "Susceptibility to oxidative stress of mussels (Mytilus galloprovincialis) in the Venice Lagoon (Italy)." Mar Pollut Bull 50(12): 1548-57.

                The aim of this study was to evaluate the susceptibility to pollutant mediated oxidative stress of the Mediterranean mussel Mytilus galloprovincialis in the Venice lagoon (Italy). In June 2003, mussels from a farm were transplanted to eight sites in the lagoon for five weeks. Oxidative stress responses were measured by: (i) total oxyradical scavenging capacity (TOSC) assay, for an overall evaluation of the oxidative stress response capability; (ii) catalase (CAT), as a key enzyme involved in the antioxidant defence system; (iii) malondialdehyde (MDA), as an indicator of lipid peroxidation, to evaluate an oxidative damage; (iv) metallothioneins (MTs), as they play a role in the antioxidant defence. The TOSC analysis revealed a reduced capability to eliminate: (i) peroxyl radical in mussels transplanted at Palude della Rosa, Valle Millecampi and Chioggia; (ii) hydroxyl radical at Campalto and Valle Millecampi; (iii) peroxynitrite at Valle Millecampi. Inhibition in CAT activity, observed in all the monitored sites, confirms the presence of an oxidative pressure in transplanted mussels. In addition, Pearson correlation analysis was performed in order to observe possible links between the various parameters. The PCA was a powerful tool to discriminate impacted sites, suggesting that the mussels transplanted throughout the Venice lagoon were subjected to different levels of oxidative pressure. Furthermore, it provided an easy and useful tool to summarize the obtained results.

 

Vergani, L., M. Grattarola, et al. (2005). "Fish and molluscan metallothioneins." FEBS J 272(23): 6014-23.

                Metallothioneins (MTs) are noncatalytic peptides involved in storage of essential ions, detoxification of nonessential metals, and scavenging of oxyradicals. They exhibit an unusual primary sequence and unique 3D arrangement. Whereas vertebrate MTs are characterized by the well-known dumbbell shape, with a beta domain that binds three bivalent metal ions and an alpha domain that binds four ions, molluscan MT structure is still poorly understood. For this reason we compared two MTs from aquatic organisms that differ markedly in primary structure: MT 10 from the invertebrate Mytilus galloprovincialis and MT A from Oncorhyncus mykiss. Both proteins were overexpressed in Escherichia coli as glutathione S-transferase fusion proteins, and the MT moiety was recovered after protease cleavage. The MTs were analyzed by gel electrophoresis and tested for their differential reactivity with alkylating and reducing agents. Although they show an identical cadmium content and a similar metal-binding ability, spectropolarimetric analysis disclosed significant differences in the Cd7-MT secondary conformation. These structural differences reflect the thermal stability and metal transport of the two proteins. When metal transfer from Cd7-MT to 4-(2-pyridylazo)resorcinol was measured, the mussel MT was more reactive than the fish protein. This confirms that the differences in the primary sequence of MT 10 give rise to peculiar secondary conformation, which in turn reflects its reactivity and stability. The functional differences between the two MTs are due to specific structural properties and may be related to the different lifestyles of the two organisms.

 

Bataille, C., G. Baldacchino, et al. (2005). "Effect of pressure on pulse radiolysis reduction of proteins." Biochim Biophys Acta 1724(3): 432-9.

                Pulse radiolysis experiments were performed on proteins under pressure. Whereas many spectroscopic techniques have shown protein modifications at different pressure ranges, the present measurements performed using the water radiolysis allowed to generate radical species and to study the mechanisms implied in their reactions with proteins. This work gives the first results obtained on the effects of pressure on the rate constants of the proteins reduction by the hydrated electron at pressures up to 100 MPa. The reaction with the hydrated electron was investigated on two classes of protein: the horse myoglobin and the mussel metallothioneins. We have successively studied the influence of the pH value of metmyoglobin solutions (pH 6, 7 and 8) and the influence of the metals nature (Zn,Cu,Cd) bound to metallothioneins. For both protein, whatever the experimental conditions, the pressure does not influence the value of the reduction rate constant in the investigated range (0.1-100 MPa).

 

Leung, K. M., R. E. Dewhurst, et al. (2005). "Metallothioneins and trace metals in the dogwhelk Nucella lapillus (L.) collected from Icelandic coasts." Mar Pollut Bull 51(8-12): 729-37.

                Different sizes of the dogwhelk Nucella lapillus were collected from eight locations along the southwest and north coasts of Iceland. Concentrations of total metallothioneins (MTs) and heavy metals (Cd, Cu, Cr, Mn, Ni, and Zn) were analysed using the silver saturation method and inductively coupled plasma-atomic emission spectrometry. The level of tributyltin (TBT) contamination was also assessed using imposex indices, the vas deferens stage index (VDSI) and relative penis size index (RPSI). Gufunes N. lapillus presented the highest values of VDSI (4.0) and RPSI (11.1), followed by Grenivik individuals (VDSI = 3.0; RPSI = 0.9), while the Strandakirkja population showed the lowest VDSI (0.3) and zero RPSI. At a standardised size (0.25 g dry soft-body weight), Grenivik N. lapillus exhibited significantly higher concentrations of all metals whereas overall metal concentrations were significantly lower in individuals from Strandakirkja and Garethskagi compared to other study sites. Partial correlation analyses with size correction indicated that MT concentrations were better correlated with Cd and Cu concentrations than with other metals. At the standard size, the pattern of MT concentrations in N. lapillus from different sites was, however, very different from those of metal profiles. Such discrepancies between the patterns of MT and metals in N. lapillus might be explained by the fact that MT induction could be influenced by various factors such as temperature, dietary metal intake, growth rate and co-existence of other MT-inducing chemicals.

 

Quinn, B., F. Gagne, et al. (2005). "Ecotoxicological effects of a semi-submerged municipal dump (Castle harbour, Bermuda) on the Calico scallop Argopecten gibbus." Mar Pollut Bull 51(5-7): 534-44.

                A biomarker study was undertaken using the Calico scallop Argopecten gibbus to assess the ecotoxicological effects of a semi-submerged municipal dump on the adjacent patch reef lagoon ecosystem (Castle harbour, Bermuda). Caged scallops were deployed in situ for 2 months at various distances from the dump (50 m, 900 m and 2.7 km) and at a reference site (14 km). A suite of biomarkers comprising metallothionein (MT), lipid peroxidation (LPO), vitellin-like proteins (Vn), glutathione S-transferase (GST), DNA strand breaks and condition factor (CF) were investigated in various tissues of the scallop (gill, digestive gland and gonad). Levels of heavy metals were also measured in the whole scallop soft tissue. While there was some variation in response between tissues, in general the results indicated that the dump was negatively impacting scallops deployed in the adjacent marine environment: elevated levels of MT, DNA strand breaks, Vn and GST and reduced condition factor were found for scallops deployed nearest to the dump and at the site 1.5 km from this point source of contamination (Tuckers town) in Castle harbour, with respect to the reference site, North Rock (although this exhibited some degree of metal contamination). The gills from scallops deployed at the dump site were the most responsive tissue, with the highest expression of MT, LPO and DNA damage. This study indicates the potential of the Calico scallop as a convenient bioindicator species in the marine tropical benthos.

 

Stolyar, O. B., R. L. Myhayliv, et al. (2005). "The concentration-specific response of metallothioneins in copper-loading freshwater bivalve Anodonta cygnea." Ukr Biokhim Zh (1999) 77(6): 68-72.

                Metallothioneins (MTs) from digestive gland of freshwater bivalve mollusc Anodonta cygnea, exposed to 10 or to 200 microg x l(-1) copper ions during 14 days, were resolved using subsequent gel-permeation and ion-exchange chromatography on MT-1 and MT-2. In both groups of treated molluscs the content of copper in MT-2 was elevated twice. The significant elevation of this isoform, which is minor in control animals, and the increasing of the zinc level in it were also observed after treatment with 10 microg copper x l(-1). It was accompanied with Cu,Zn-superoxide dismutase (SOD) activation. Under the treatment with the 200 microg copper x l(-1) the zinc in MT-2 was at the control level, the inhibition of Cu,Zn-SOD but activation of Mn-SOD was revealed. The properties of MT-1 in all cases were similar.

 

Piano, A., P. Valbonesi, et al. (2004). "Expression of cytoprotective proteins, heat shock protein 70 and metallothioneins, in tissues of Ostrea edulis exposed to heat and heavy metals." Cell Stress Chaperones 9(2): 134-42.

                Heat shock proteins (Hsps) are constitutively expressed in cells and involved in protein folding, assembly, degradation, intracellular localization, etc, acting as molecular chaperones. However, their overexpression represents a ubiquitous molecular mechanism to cope with stress. Hsps are classified into families, and among them the Hsp70 family appears to be the most evolutionary preserved and distributed in animals. In this study, the expression of Hsp70 and the related messenger ribonucleic acid (mRNA) has been studied in Ostrea edulis after exposure to heat and heavy metals; moreover, levels of metallothioneins (MTs), another class of stress-induced proteins, have contemporaneously been assessed in the same animals. Thermal stress caused the expression of a 69-kDa inducible isoform in gills of O edulis but not in the digestive gland. Northern dot blot analysis confirmed that the transcription of Hsp69-mRNA occurs within 3 hours of stress recovery after oyster exposure at 32 and 35 degrees C. Hsp69-mRNA transcripts were not present in the gills of animals exposed to 38 degrees C after 3 hours of poststress recovery, but they were detected after 24 hours. The expression of the 69-kDa protein in O edulis exposed to 38 degrees C was rather low or totally absent, suggesting that the biochemical machinery at the base of the heat shock response is compromised. Together with the expected increase in MT content, the oysters exposed to Cd showed a significant enhancement of Hsp70, although there was no clear appearance of Hsp69. Interestingly, the levels of MT were significantly increased in the tissues of individuals exposed to thermal stress. Unlike oysters, heat did not provoke the expression of inducible Hsp isoforms in Mytilus galloprovincialis, Tapes philippinarum, and Scapharca inaequivalvis, although it significantly enhanced the expression of constitutive proteins of the 70-kDa family. The expression of newly synthesized Hsp70 isoforms does not seem therefore a common feature in bivalves exposed to thermal stress.

 

Perceval, O., Y. Couillard, et al. (2004). "Metal-induced stress in bivalves living along a gradient of Cd contamination: relating sub-cellular metal distribution to population-level responses." Aquat Toxicol 69(4): 327-45.

                The use of biomarkers to assess the impacts of contaminants on aquatic ecosystems has noticeably increased over the past few years. Few of these studies, however, have contributed to the prediction of ecologically significant effects (i.e., at the population or community levels). The present field study was designed to evaluate the potential of metallothionein (MT) and sub-cellular metal partitioning measurements for predicting toxic effects at higher levels of the biological organization in freshwater bivalves (Pyganodon grandis) chronically exposed to Cd. For that purpose, we quantitatively sampled P. grandis populations in the littoral zone of nine lakes on the Precambrian Canadian Shield during two consecutive summers (1998 and 1999); lakes were characterized by contrasting Cd levels but similar trophic status. We tested relationships between the population status of P. grandis (i.e., growth parameters, density, biomass, secondary production, turnover ratio and cumulative fecundity) and (i) ambient Cd concentrations, (ii) sub-organismal responses (MT concentrations in the gill cytosol of individuals and Cd concentrations in three metal-ligand pools identified as M-HMW, the high molecular weight pool, M-MT, the metallothionein-like pool and M-LMW, the low molecular weight pool) and (iii) ecological confounding factors (food resources, presence of host fishes for the obligatory parasitic larval stage of P. grandis). Our results show that littoral density, live weight, dry viscera biomass, production and cumulative fecundity decreased with increasing concentrations of the free-cadmium ion in the environment (Pearson's r ranging from -0.63 to -0.78). On the other hand, theoretical maximum shell lengths (L( infinity )) in our populations were related to both the dissolved Ca concentration and food quality (sestonic C and N concentrations). Overall, Cd concentrations in the gill cytosolic HMW pool of the individual molluscs were the biomarker response that was most frequently and most strongly correlated with the population variables (Pearson's r ranging from -0.58 to -0.80). Our findings demonstrate, however, the difficulty of currently assigning to sub-cellular metal partitioning measurements (mainly Cd bound to the HMW fraction) any predictive role for population health, notably because of the influence of ecological confounding variables (e.g., the cumulative number of degree-days in the littoral zone, as is the case here). Metal contamination of our lakes has decreased markedly in the past 10 years and consequently we believe that the toxic effects of metals may have been replaced by some natural factors as the main agent for structuring the clam populations in these lakes.

 

Raspor, B., Z. Dragun, et al. (2004). "Is the digestive gland of Mytilus galloprovincialis a tissue of choice for estimating cadmium exposure by means of metallothioneins?" Sci Total Environ 333(1-3): 99-108.

                A study performed over 12 months with caged mussels Mytilus galloprovincialis in the coastal marine zone, which is under urban pressure, reveals a temporal variation of digestive gland mass, which causes "biological dilution" of cytosolic metallothionein (MT) and trace metal (Cd, Cu, Zn, Fe, Mn) concentrations. The dilution effect was corrected by expressing the cytosolic MT and metal concentrations as the tissue content. Consequently, the changes of the average digestive gland mass coincide with the changes of MT and trace metal contents. From February to June, MT contents are nearly twice and trace metal contents nearly three times higher than those of the other months. The period of increased average digestive gland mass, of MT and trace metal contents probably overlaps with the sexual maturation of mussels (gametogenesis) and enhanced food availability. Since natural factors contribute more to the MT content than the sublethal levels of Cd, the digestive gland of M. galloprovincialis is not considered as a tissue of choice for estimating Cd exposure by means of MTs.

 

Dragun, Z., M. Erk, et al. (2004). "Metal and metallothionein level in the heat-treated cytosol of gills of transplanted mussels Mytilus galloprovincialis Lmk." Environ Int 30(8): 1019-25.

                A stock of mussels Mytilus galloprovincialis was transplanted over 1 year to four sites in a semi-enclosed bay in Croatia which is under the influence of various sources of pollution. The positive correlation of metal (Cd, Zn, Cu, Mn, Fe) and metallothionein (MT) tissue contents based on the analysis of heat-treated cytosol of gills with shell mass, as an indicator of mussel age, indicated to accumulation of metals, as well as the increase of MTs with mussel age. The principal component analysis (PCA) revealed that 74% of total variance of obtained results could be explained through two principal components. The first principal component was highly correlated with MT, Cd and Zn indices (metal or MT content/shell mass), and the second one with Cu, Mn, and Fe indices, as well as the gill index. High correlation of MTs with Zn and Cd is consistent with their affinity for binding to MTs.

 

Hardivillier, Y., V. Leignel, et al. (2004). "Do organisms living around hydrothermal vent sites contain specific metallothioneins? The case of the genus Bathymodiolus (Bivalvia, Mytilidae)." Comp Biochem Physiol C Toxicol Pharmacol 139(1-3): 111-8.

                The unusual characteristics of the hydrothermal vent environment (high pressure and metal concentrations, low pH, etc.) leads us to wonder how species living in this particular biotope have adjusted to these severe living conditions. To investigate the consequences of high metal concentrations, filter-feeding organisms are commonly used in ecotoxicological studies. Metallothioneins (MTs) are proteins conserved throughout the animal kingdom and involved in intracellular metal regulation. Therefore, we tried here to find out whether the metallothioneins of hydrothermal bivalves are different from those of coastal bivalves. The characterization of DNA sequences coding MTs from some of the most common hydrothermal bivalves, belonging to the genus Bathymodiolus (Mytilidae) was performed by reverse transcription polymerase chain reactions (RT-PCRs). The complementary DNAs (cDNAs) of MT-10 and MT-20 isoforms were obtained for the Atlantic and Pacific hydrothermal mussels (Bathymodiolus azoricus and Bathymodiolus thermophilus). The MT-10 transcripts were 222 nucleotides long and the MT-20 transcripts, 207 nucleotides. The polymorphism of the MT cDNAs in these two hydrothermal species is discussed. The comparison between metallothionein cDNA sequences of the Mytilus and the Bathymodiolus genera shows strong homologies among metallothioneins of coastal and hydrothermal mussels.

 

Dallinger, R., B. Lagg, et al. (2004). "Cd accumulation and Cd-metallothionein as a biomarker in Cepaea hortensis (Helicidae, Pulmonata) from laboratory exposure and metal-polluted habitats." Ecotoxicology 13(8): 757-72.

                Cepaea hortensis is a widespread terrestrial pulmonate, contributing significantly to element fluxes in soil ecosystems. Due to its capacity of accumulating certain trace elements in its tissues, Cepaea hortensis can serve as a biological indicator of metal accumulation in contaminated areas. In response to Cd exposure this species and related helicid pulmonates are also able to synthesize an inducible, Cd-binding metallothionein (MT) isoform specifically serving in binding and detoxification of this metal. As shown by field-collected garden-snails from a metal-contaminated site near a zinc smelting works in Avonmouth (UK) and an unpolluted reference site in Reutte (Tyrol, Austria), Cd and Cd MT concentrations in midgut gland of C. hortensis from these sites increased with rising Cd concentrations in the soil substrate from the same contaminated sites. By combining the results of these field data with laboratory experiments it appears that midgut gland Cd-MT of Cepaea hortensis seems to fulfil the criteria of a successful biomarker in many respects. First, the synthesis of the protein can rapidly be induced by Cd exposure. Second, the level of Cd MT induction in C. hortensis directly reflects the intensity of metal exposure. Third, the induced signal of increased Cd-MT concentration in C. hortensis is persistent over extended periods of time. Fourth, the Cd-MT signal in C. hortensis seems to be very specific for Cd exposure. Regression analyses demonstrate that tissue levels of Cd and Cd MT in C. hortensis depend on Cd concentrations in the substrate which is represented by either soil or plant material on which snails normally feed. In both cases the best fit for this dependence is exhibited by a semi-logarithmic relationship, with substrate (soil or plant feed) concentrations expressed on a logarithmic scale. It is concluded that C. hortensis and other related pulmonates can successfully be used either as biological indicators of Cd accumulation, or as key species in biomonitoring studies focusing on Cd-MT induction as a biomarker for Cd exposure.

 

Shi, D. and W. X. Wang (2004). "Modification of trace metal accumulation in the green mussel Perna viridis by exposure to Ag, Cu, and Zn." Environ Pollut 132(2): 265-77.

                To examine the Cd, Hg, Ag, and Zn accumulation in the green mussel Perna viridis affected by previous exposure to Cu, Ag, or Zn, the dietary metal assimilation efficiency (AE) and the uptake rate from the dissolved phase were quantified. The mussel's filtration rate, metallothionein (MT) concentration, and metal tissue burden as well as the metal subcellular partitioning were also determined to illustrate the potential mechanisms underlying the influences caused by one metal pre-exposure on the bioaccumulation of the other metals. The green mussels were pre-exposed to Cu, Ag, or Zn for different periods (1-5 weeks) and the bioaccumulation of Cd, Hg, Ag, and Zn were concurrently determined. Pre-exposure to the three metals did not result in any significant increase in MT concentration in the green mussels. Ag concentration in the insoluble fraction increased with increasing Ag exposure period and Ag ambient concentration. Our data indicated that Cd assimilation were not influenced by the mussel's pre-exposure to the three metals (Cu, Ag, and Zn), but its dissolved uptake was depressed by Ag and Zn exposure. Although Hg assimilation from food was not affected by the metal pre-exposure, its influx rate from solution was generally inhibited by the exposure to Cu, Ag, and Zn. Ag bioaccumulation was affected the most obviously, in which its AE increased with increasing Ag tissue concentration, and its dissolved uptake decreased with increasing tissue concentrations of Ag and Cu. As an essential metal, Zn bioaccumulation remained relatively stable following the metal pre-exposure, suggesting the regulatory ability of Zn uptake in the mussels. Zn AE was not affected by metal pre-exposure, but its dissolved uptake was depressed by Ag and Zn pre-exposure. All these results indicated that the influences of one metal pre-exposure on the bioaccumulation of other metals were metal-specific due to the differential binding and toxicity of metals to the mussels. Such factors should be considered in using metal concentrations in mussel's soft tissues to evaluate the metal pollution in coastal waters.

 

Gagne, F., C. Blaise, et al. (2004). "Endocrine disruption and health effects of caged mussels, Elliptio complanata, placed downstream from a primary-treated municipal effluent plume for 1 year." Comp Biochem Physiol C Toxicol Pharmacol 138(1): 33-44.

                Freshwater mussels, Elliptio complanata, were caged in special benthic pens and were immersed at one upstream (Ups) site and two downstream sites (8 and 11 km) of a primary-treated municipal effluent plume for 1 year. The levels of metallothionein-like proteins (MT), lipid peroxidation, protein-free DNA strands and glutathione S-transferase (GST) activity were assayed in digestive gland, gill and gonad tissues to evaluate biological effects and damage. The levels of monoamines (serotonin and dopamine) in nerve ganglia, ATP-dependent transport activity and monoamine oxidase (MAO) activity were also investigated in the homogenates, synaptosomes and mitochondria, respectively. Results showed that significant amounts of sediment accumulated in cages and 82% of mussels survived the yearlong exposure period at the downstream sites. MT-like proteins were induced in all tissues with the following response intensity: gill (3-fold), digestive gland (1.4-fold) and gonad tissues (1.3-fold). Lipid peroxidation decreased (2.5-fold) in digestive gland but increased in gill (1.6-fold) and in gonad tissues (1.5-fold). GST activity was readily increased in digestive gland (2.5-fold), suggesting the presence of organic contaminants in the plume. Levels of protein-free DNA strands did not vary significantly in digestive gland and gill tissues but were significantly reduced in gonad tissues (2.5-fold) relative to the upstream site. In visceral nerve ganglia, both serotonin and ATP-dependent serotonin transport decreased 1.7-fold with a 4-fold increase of 5-hydroxyindole acetate (5-HIAA, a serotonin metabolite) level relative to the upstream site. However, MAO activity was somewhat reduced at downstream sites (0.7- to 0.9-fold of the activity at the upstream site). Dopamine levels were found to be decreased (1.5-fold), but dopamine ATP-dependent transport activity was increased 1.8-fold, suggesting reduced dopaminergic activity. These results indicate that estrogenic chemicals are likely at play, and the increased dopamine and decreased serotonin ATP-dependent transport suggest that the municipal plume was serotonergic for mussels located at the downstream sites. Mussels exposed for 1 year display a complex but characteristic pattern of responses that could lead to harmful health effects including neuroendocrine disruption of reproduction.

 

Jenny, M. J., A. H. Ringwood, et al. (2004). "Diversity of metallothioneins in the American oyster, Crassostrea virginica, revealed by transcriptomic and proteomic approaches." Eur J Biochem 271(9): 1702-12.

                Metallothioneins are typically low relative molecular mass (6000-7000), sulfhydryl-rich metal-binding proteins with characteristic repeating cysteine motifs (Cys-X-Cys or Cys-X(n)-Cys) and a prolate ellipsoid shape containing single alpha- and beta-domains. While functionally diverse, they play important roles in the homeostasis, detoxification and stress response of metals. The originally reported metallothionein of the American oyster, Crassostrea virginica showed the canonical molluscan alphabeta-domain structure. Oyster metallothioneins have been characterized as cDNA and as expressed proteins, and here it is shown that the previously reported metallothionein is a prototypical member of a subfamily (designated as CvMT-I) of alphabeta-domain metallothioneins. A second extensive subfamily of oyster metallothioneins (designated as CvMT-II) has apparently arisen from (a) a stop mutation that truncates the protein after the alpha-domain, and (b) a subsequent series of duplication and recombination events that have led to the development of metallothionein isoforms containing one to four alpha-domains and that lack a beta-domain. Analysis of metallothioneins revealed that certain CvMT-I isoforms showed preferential association either with cadmium or with copper and zinc, even after exposure to cadmium. These data extend our knowledge of the evolutionary diversification of metallothioneins, and indicate differences in metal-binding preferences between isoforms within the same family.

 

Galloway, T. S., R. J. Brown, et al. (2004). "A multibiomarker approach to environmental assessment." Environ Sci Technol 38(6): 1723-31.

                Incorporation of ecologically relevant biomarkers into routine environmental management programs has been advocated as a pragmatic means of linking environmental degradation with its causes. Here, suites of biomarkers, devised to measure molecular damage, developmental abnormality and physiological impairment, were combined with chemical analysis to determine exposure to and the effects of pollution at sites within Southampton Water (UK). Test species included a filter feeder, a grazer, and an omnivore to determine the sensitivity of organisms occupying different trophic levels. Linear regression confirmed a significant association between incidence of intersex in Littorina littorea and tributyltin (TBT) concentrations (R2 = 0.954) and between PAH metabolites in Carcinus maenas urine and PAHs in sediments (R2 = 0.754). Principal component analysis revealed a gradient of detrimental impact to biota from the head to the mouth of the estuary, coincident with high sediment concentrations of heavy metals, PAHs, and biocides. Multidimensional scaling identified C. maenas as the organism most sensitive to contamination. Carboxylesterase activity, metallothionein and total haemolymph protein were the most discriminating biomarkers among sites. This holistic approach to environmental assessment is encouraged as it helps to identify the integrated impact of chemical contamination on organisms and to provide a realistic measure of environmental quality.

 

Chabicovsky, M., W. Klepal, et al. (2004). "Mechanisms of cadmium toxicity in terrestrial pulmonates: programmed cell death and metallothionein overload." Environ Toxicol Chem 23(3): 648-55.

                A sublethal dose of cadmium (Cd2+) administered via the diet during short-term exposure over 10 d induced programmed cell death in the hepatopancreas of the terrestrial pulmonate snail Helix pomatia. Condensed cell residues were predominantly phagocytosed by calcium cells, suggesting a specific function of these epithelial cells in metal detoxification or in clearing the organ of cellular debris from cell death. The considerable cell loss recorded by histological analysis was accompanied by enhanced cell proliferation. Intoxication with Cd was further associated with the pronounced abundance of residual bodies, predominantly recorded in excretory cells, and with pathological changes in the endoplasmic reticulum. During long-term Cd exposure, mortality increased with increasing Cd concentrations in the diet, as demonstrated by feeding experiments in the laboratory. Lethal effects of Cd appeared to be correlated with Cd overloading of the Cd-specific metallothionein isoform (Cd-MT), isolated and characterized previously from the animal's hepatopancreas. Stoichiometric analysis shows that the capacity of Cd-MT to bind six molar equivalents of Cd corresponds to a tissue Cd concentration of approximately 4 micromol/g dry weight. At this tissue concentration, all high-affinity metal-binding sites of Cd-MT are occupied by Cd2+. Cadmium exposure beyond this level gives rise to progressive destabilization of Cd-MT cluster structure in vitro, resulting in increasing proportions of weakly bound, or even unbound, Cd2+ ions. Our results suggest that in vivo, the observed overburdening of Cd-MT with Cd2+ reduces the viability of affected animals.

 

Domouhtsidou, G. P., S. Dailianis, et al. (2004). "Lysosomal membrane stability and metallothionein content in Mytilus galloprovincialis (L.), as biomarkers. Combination with trace metal concentrations." Mar Pollut Bull 48(5-6): 572-86.

                The simultaneous study of two biomarkers, the lysosomal membrane stability (LMS) of the digestive gland and the metallothionein (MT) content of the digestive gland, the gills and the mantle/gonad complex of the mussel Mytilus galloprovincialis was examined in an enclosed estuarine system in June and October 2001. Mussels were collected along the Gulf of Thermaikos (northern Greece) from stations displaying a pollution gradient, while Olympiada in the Gulf of Strymonikos was used as a reference station. In addition, the heavy metal (Cd, Pb, Cu and Zn) content, using atomic absorption spectrophotometry (AAS), were evaluated in the digestive gland, the gills and the mantle/gonad complex of mussels collected from the same sites and seasons. LMS values were significantly greater, and the MT content of the studied tissues were significantly less in mussels collected from the reference station compared to mussels from more polluted stations located in the Gulf of Thermaikos. Significant correlation was observed between the MT content of the gills and the mantle/gonad complex with the LMS values.

 

Geret, F. and M. J. Bebianno (2004). "Does zinc produce reactive oxygen species in Ruditapes decussatus?" Ecotoxicol Environ Saf 57(3): 399-409.

                Zinc is an essential trace element with many enzymatic functions that include antioxidant properties. To investigate whether an excess of Zn in the cells produces cytotoxicity or tissue damage or an imbalance in the antioxidant systems, marine clams (Ruditapes decussatus) were exposed to two sublethal Zn concentrations (100 and 1000 microg L(-1)) for 28 days. The effects of Zn on the activities of protective antioxidant enzymes (superoxide dismutase, catalase, and glutathione peroxidase, both total and selenium-dependent), lipid peroxidation, and metallothionein induction were followed in the gills and digestive gland of these clams. The results indicate that the effect of Zn exposure in this clam species depends not only on the tissue but also on the Zn concentration present. In the gills, catalase activity was enhanced by Zn exposure, whereas total glutathione peroxidase activity was inhibited. Lipid peroxidation occurred only in the clams exposed to the highest Zn concentration. In the digestive gland, the impact of Zn exposure on metabolic activity was less evident than in the gills. The most evident effect in both tissues was the enhancement of catalase activity by Zn exposure. Catalase and total glutathione peroxidase activities as well as lipid peroxidation are promising biomarkers to assess the effects of Zn in the gills of R. decussatus.

 

Bebianno, M. J., F. Geret, et al. (2004). "Biomarkers in Ruditapes decussatus: a potential bioindicator species." Biomarkers 9(4-5): 305-30.

                The clam Ruditapes decussatus is distributed worldwide and due to its ecological and economical interest has been proposed as a bioindicator in areas where mussels are not available. The accumulation of several anthropogenic compounds in their tissues suggests that they possess mechanisms that allow them to cope with the toxic effects of these contaminants. Besides pollutant uptake, the use of biomarkers is pointed out in this paper since it is a promising approach to monitor the effect of these contaminants in the marine environment. Biomarkers complement the information of the direct chemical characterization of different types of contaminants. Therefore, the aim of this paper is to review the role of several biomarkers: (metallothioneins (MT), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidases (GPx) (total and selenium-dependent), lipid peroxidation (measured as MDA, one of the final products of lipid peroxidation), glutathione S-transferase (GST) and acetylcholinesterase (AChE), measured in different tissues of the clam R. decussatus, in laboratory conditions and under various environmental stresses, in two ecosystems (Ria Formosa lagoon- Portugal) and Bizerta lagoon (Tunisia) in a perspective of a multibiomarker approach to assess environmental changes. Experiment and field studies are in good agreement since MT levels, especially in the gills, the first target tissue of these contaminants, can be used as biomarker of exposure to Cd. GPx and MDA may also be determined in this respect. AChE activity is inhibited by pesticide and, to a less extent, by metal exposure in the gills and whole soft body of clams. However, the induction of GST isoforms experimentally demonstrated is not observed in the field because only global GST activity was determined. The whole set of results opens new research perspectives for the use of this species to assess the effect of mixtures of pollutants in the aquatic environment.

 

Siboni, N., M. Fine, et al. (2004). "Coastal coal pollution increases Cd concentrations in the predatory gastropod Hexaplex trunculus and is detrimental to its health." Mar Pollut Bull 49(1-2): 111-8.

                Parameters of environmental health, including paracellular permeability of external epithelia, functional state of lysosomes and the level of metallothioneins (MTs), were examined using fluorescent markers and vital microfluorometry in different tissues of the marine gastropod, Hexaplex trunculus, from a coal-polluted and coal-free site. Vital microfluorometrical examinations exhibited enhanced paracellular permeability of external epithelia to the anionic marker, fluorescein (FLU), lower lysosomal accumulation of neutral red (NR) as well as higher levels of MTs, when compared with epithelia of gastropods from the coal-free site. Those differences were particularly marked in the foot epithelium, which is in direct contact with the substrate. In addition, cadmium was measured by ICP-AES in the hepatopancreas of gastropods sampled from the coal-polluted site and two coal-free sites. Significantly higher levels of Cd were found in gastropod hepatopancreas from the coal-polluted site. In addition, two months feeding experiments conducted in aquaria containing: (a) coal pieces covered by barnacles; (b) natural rocks covered by barnacles; and (c) natural rocks with barnacles + bare coal pieces, demonstrated significant increase of Cd concentration in the hepatopancreas of the gastropods exposed to coal. We suggest that coal in the marine environment has detrimental effects on marine gastropods, both directly through contact with the organisms and indirectly through the food web.

 

Shi, D. and W. X. Wang (2004). "Understanding the differences in Cd and Zn bioaccumulation and subcellular storage among different populations of marine clams." Environ Sci Technol 38(2): 449-56.

                The marine clams Mactra veneriformis were collected from three different locations in a contaminated bay in Northern China. Another species of clams Ruditapes philippinarum was collected from the same contaminated bay as well as from a relatively clean site in Hong Kong. The indices of Cd and Zn bioaccumulation (assimilation efficiency, dissolved uptake rate, and efflux rate), tissue concentration, subcellular distribution, metallothionein (MT) content, and clearance rate of the clams were subsequently quantified in these populations in the laboratory. In the two species of clams, the population with a higher Cd tissue concentration assimilated Cd and Zn more efficiently, in correlation with an increase in the Cd associated with the metallothionein-like protein (MTLP) fraction. The subcellular partitioning of Zn was similar among the different populations. The dissolved uptake rates of Cd and Zn were not influenced by the different tissue concentrations of metals in the clams. However, the clam R. philippinarum from the contaminated site reduced their Zn uptake rate constants in response to increasing Zn concentration in the water. Differences in Cd and Zn tissue concentrations had little influence on the metal efflux rate constant and the clams' clearance rate. Our results indicate that the higher Cd and Zn tissue concentrations observed in these two species may be partially caused by the high levels of metal assimilation. Populations living in contaminated environments may be able to modify their physiological and biochemical responses to metal stress, which can subsequently alter trace metal bioaccumulation to aquatic animals. The relative significance of dietary uptake and the potential trophic transfer of metals in the contaminated areas may be substantially different from those in the clean environments.

 

Brown, R. J., T. S. Galloway, et al. (2004). "Differential sensitivity of three marine invertebrates to copper assessed using multiple biomarkers." Aquat Toxicol 66(3): 267-78.

                Understanding how biomarkers relate to each other on exposure to particular contaminants in different species is key to their widespread application in environmental management. However, few studies have systematically used multiple biomarkers in more than a single species to determine the variability of sublethal effects of a particular contaminant. In this study, three marine invertebrates, the shore crab Carcinus maenas, the common limpet Patella vulgata and the blue mussel Mytilus edulis, were exposed over 7 days in the laboratory to environmentally realistic concentrations of the priority pollutant copper. A combination of molecular, cellular and physiological biomarkers was measured in each organism to detect the toxic effects of copper. Biomarkers included lysosomal stability (neutral red retention), neurotoxicity (acetylcholinesterase activity), metabolic impairment (total haemolymph protein), physiological status (heart rate) and induction of protective metallothionein proteins. P. vulgata was the most sensitive to copper with significant effects measured in all biomarkers at concentrations of 6.1 microg Cu l(-1). In C. maenas, cellular and neurotoxic endpoints were affected significantly only at 68.1 microg Cu l(-1). Exposure to copper also induced metallothionein production in crabs. Over a 7-day exposure period, M. edulis was the most tolerant species to copper with significant effects being observed at the cellular level only at 68.1 microg Cu l(-1) . In all three species, cellular and neurotoxic pathways were more sensitive to disruption than physiological processes (protein and heart rate). Results illustrate how a suite of biomarkers applied to different sentinel species can provide a 'diagnosis of stress', whereby, effects at the molecular level can be used to interpret the level of physiological impairment of the organism.

 

Ciocan, C. M. and J. M. Rotchell (2004). "Cadmium induction of metallothionein isoforms in juvenile and adult mussel (Mytilus edulis)." Environ Sci Technol 38(4): 1073-8.

                Isoforms of metallothionein in the digestive gland of control and experimentally Cd-exposed mussels (Mytilus edulis) (200 microg L(-1) Cd2+ and 400 microg L(-1) Cd2+; 20 days) were studied using the reverse transcriptase-polymerase chain reaction. In addition, glutathione S-transferase (GSTpi) primers were designed to evaluate the reduction in the antioxidant defense systems (glutathione) accompanying the aging process in the same organisms. Following experimental exposure, an accumulation of Cd was observed in the digestive gland of exposed mussels, both adults and juveniles, up to 500 times higher than in the control. An induction of the dimeric form MT20 II was detected in 400 microg L(-1) exposed mussels, as well as a visible inhibition of the monomeric form MT10 IV. After 20 days of exposure juveniles expressed increased GSTpi compared with adults. Results reveal individual variation of both metallothioneins and GSTpi expression among control and Cd2+-exposed mussels of different ages. The ecotoxicological significance of MT utilization in biomonitoring of seawater for trace metals has been considered in light of these results.

 

Kalpaxis, D. L., C. Theos, et al. (2004). "Biomonitoring of Gulf of Patras, N. Peloponnesus, Greece. Application of a biomarker suite including evaluation of translation efficiency in Mytilus galloprovincialis cells." Environ Res 94(2): 211-20.

                Specimens of Mytilus galloprovincialis were placed in bow nets and immersed at 3-10 m depth in a clean coastal region (reference area), Itea, and two marine stations along Gulf of Patras, N. Peloponnesus, Greece. One site is near the estuaries of the Glafkos River, which are influenced by local industrial and urban sources (Station 1); the second site, Agios Vasilios, has no evident organic pollution but is enriched in metals, particularly zinc (Station 2). One month after immersion, digestive glands were removed from the mussels and tested for lysosomal membrane stability, metallothionein content, and translational efficiency of ribosomes. In addition, gill cells were isolated and their micronuclei content was determined. Compared with the reference samples, mussels transplanted to Gulf of Patras showed a significant increased lysosomal membrane permeability and metallothionein content, reduced polysome levels, and increased chromosomal damage in relation to the contamination burden of each sampling area. Also, runoff ribosomes from mussels transplanted to Gulf of Patras (that is, ribosomes stripped of endogenous messengers and peptidyl- or/and aminoacyl-tRNAs) were less efficient at initiating protein synthesis in an in vitro-translation system than those prepared from reference samples. The whole set of data suggests that the degree of Gulf of Patras pollution differs between different sites and depends on the proximity of urban sewage and industrial outfalls. In addition, our results emphasize the importance of protein synthesis regulation as a component of the cellular stress response.

 

Lecoeur, S., B. Videmann, et al. (2004). "Evaluation of metallothionein as a biomarker of single and combined Cd/Cu exposure in Dreissena polymorpha." Environ Res 94(2): 184-91.

                The effects of metal mixture (Cd+Cu) versus single-metal exposure on total MT response and bioaccumulation were investigated in the freshwater bivalve Dreissena polymorpha. A two-month exposure period, including two levels of contamination, was chosen for each of the two metals: 5, 10 microg/L for Cu, and 2, 20 microg/L for Cd, with mixtures of, respectively, 5 microg/L Cu+2 microg/L Cd, 5 microg/L Cu+20 microg/L Cd, 10 microg/L Cu+2 microg/L Cd, and 10 microg/L Cu+20 microg/L Cd. Total MT contents were assessed by an Ag-saturation method, and metals contents were determined by atomic absorption spectrometry. Results at the whole-organism level showed a significant and early increase of total MT biosynthesis after exposure to Cd. This increase was significantly correlated with Cd bioaccumulation. By contrast, Cu did not modify total MT response, and mussels limited Cu bioaccumulation. The mixture either did not influence or only weakly influenced metal accumulation and MT response to Cu and Cd after long-term exposure. Our results suggest that the form of MT existing in D. polymorpha was not Cu-inducible. This could limit the use of MT in D. polymorpha as a biomarker of heavy metal pollution in freshwater ecosystems.

 

Arai, T., T. Ikemoto, et al. (2004). "Chemical forms of mercury and cadmium accumulated in marine mammals and seabirds as determined by XAFS analysis." Environ Sci Technol 38(24): 6468-74.

                Marine mammals and seabirds tend to exhibit high accumulations of mercury, cadmium, and selenium in their livers and kidneys. In this study, chemical forms of mercury, cadmium, and selenium accumulated in the livers and kidneys of northern fur seal (Callorhinus ursinus), Risso's dolphin (Grampus griseus), and black-footed albatross (Diomedea nigripes) were studied by extended X-ray absorption fine structure (EXAFS) spectroscopy to reveal the detoxification mechanisms of these metals. It was found that mercury and selenium exist in the form of HgSe in the liver of northern fur seal. Mercury levels were found to be higher than those of Se, based on their molar ratio, in black-footed albatross. XAFS analysis disclosed an existence of chalcogenide containing both Hg-Se and the Hg-S bonds, suggesting the existence of a solid solution Hg(Se, S) as granules in black-footed albatross. In contrast, Cd concentrations in the kidney were higher than those in the liver for northern fur seal, black-footed albatross, and Risso's dolphin. It was found that Cd was bound to sulfur, which was probably derived from the metallothionein. The Cd-O bond was observed in the tissues of northern fur seal.

 

Smaoui-Damak, W., A. Hamza-Chaffai, et al. (2004). "Variation of metallothioneins in gills of the clam Ruditapes decussatus from the Gulf of Gabes (Tunisia)." Comp Biochem Physiol C Toxicol Pharmacol 139(4): 181-8.

                Cadmium (Cd) and metallothionein (MT) concentrations were determined in the subcellular fractions of the gills of the clam Ruditapes decussatus. Clams were collected monthly during the period of sexual development (from June to August 2001) from two different sites situated in the Gulf of Gabes (Tunisia). These sites differ by their degree of metal contamination. Differences in condition index (CI) were observed in June and August between clams from both sites. In clams from both sites, cadmium was approximately equally distributed between the soluble (S1) and insoluble (C1) fractions. MT levels varied according to month but not sex. Site and body mass of clams seem to be important factors to explain the variation of MT levels compared to cadmium.

 

Garcia-Luque, E., T. A. DelValls, et al. (2004). "Simulating a heavy metal spill under estuarine conditions: effects on the clam Scrobicularia plana." Mar Environ Res 58(2-5): 671-4.

                We describe the effect of heavy metals Zn, Cd, Pb and Cu on the induction of methallothioneins on the clam Scrobicularia plana along a salinity gradient simulated under laboratory conditions. The clams were exposed to constant heavy metal concentrations in a dynamic estuary simulator during a 15-day assay to investigate possible induction of metal-binding proteins in them. The concentration of heavy metals in water was analysed. Clams were analysed for methallothionein concentrations. The speciation of Zn, Cd, Pb and Cu along the salinity gradient was modelled. Zn showed the highest concentrations and its prevalent species was the free ion. Intersite differences have been observed in methallothionein concentration and related to the salinity gradient. It seems that synthesis of methallothioneins is the result of physiological forces acting in concert with the changes in the chemical speciation of metals, owing to the trace metals uptake is controlled by means of an interaction of physiology and physicochemistry.

 

Perez, E., J. Blasco, et al. (2004). "Biomarker responses to pollution in two invertebrate species: Scrobicularia plana and Nereis diversicolor from the Cadiz bay (SW Spain)." Mar Environ Res 58(2-5): 275-9.

                The clam Scrobicularia plana and the polychaete worm Nereis diversicolor were collected in several sites from a littoral enclosure in SW Spain. The aim of our study was to relate various biomarker responses in these species to a pollution gradient caused by untreated domestic discharges and to verify the adequacy of the selected species as sentinels in this habitat. The biomarkers selected were the antioxidant enzymes catalase (CAT), glutathione peroxidase (GPX) and DT-diaphorase (DT-D). In addition, the activities of cytochrome P450-dependent ethoxyresorufin O-deethylase (EROD) activity, the phase II detoxifying enzyme glutathione S-transferase (GST) and the neurotoxicity marker acetylcholinesterase (AChE) were measured. Metallothionein levels were selected as biomarkers of heavy metals exposure in both species. The results suggest a different response in the water filtering organism (clam) and the sediment eater (polychaete), probably as a consequent of different pollution exposure and that samples from the "Cano Sancti-Petri" were exposed to biologically active compounds that altered some of their biochemical responses. AChE was the most sensitive biomarker in both species and N. diversicolor proved to be a more robust sentinel in this ecosystem.

 

Riba, I., M. C. Casado-Martinez, et al. (2004). "Bioavailability of heavy metals bound to sediments affected by a mining spill using Solea senegalensis and Scrobicularia plana." Mar Environ Res 58(2-5): 395-9.

                After the Aznalcollar mining spill (April, 1998) different ecosystems along the Guadiamar river and the Guadalquivir estuary were impacted by high concentrations of metals. The concentration of metals Zn, Cd, Pb, Cu and As has been detected as a good tracer of the pathway of the toxic spill. Different individuals of the fish Solea senegalensis and the clam Scrobicularia plana were exposed to contaminated sediments after the accidental spill in the south-west Spain. The induction of metallothioneins in fish and the bioaccumulation of metals in clams were determined in different tissues to determine the biological adverse effect associated with the heavy metals from the spill. A relationship between the enrichment of metals in the tissues and the induction of metallothioneins was determined for those assays performed using contaminated sediments. The bioaccumulation of metals indicates that the risk associated with the spill should be continuously evaluated to establish the sublethal effects related to the accident.

 

Dallinger, R., M. Chabicovsky, et al. (2004). "Isoform-specific quantification of metallothionein in the terrestrial gastropod Helix pomatia. I. Molecular, biochemical, and methodical background." Environ Toxicol Chem 23(4): 890-901.

                The biomarker concept is an important approach of modern ecotoxicology based on the detection and quantification of molecular, biochemical, cellular, or physiological alterations in organisms as the result of exposure to toxic compounds. In the present study, the biomarker potential of two metallothionein (MT) isoforms from hepatopancreas and mantle tissue of the Roman snail, Helix pomatia L., is evaluated. This species possesses two MT isoforms with specific functions: The Cd-specific isoform (Cd-MT) is predominantly induced and expressed in the hepatopancreas upon Cd exposure. It binds and inactivates this potentially toxic trace element. The second isoform is Cu-specific (Cu-MT), primarily expressed in the mantle tissue and responsible for the homeostatic regulation of Cu. The differential expression and metal-specificity of the two isoforms make them potential candidates for a dual MT biomarker system based on an isoform-specific approach. This study shows that Cd-MT and Cu-MT of H. pomatia can reliably be quantified by a differential metal saturation approach that could be used for environmental biomarker studies. The approach works by virtue of the following preconditions: The two isoforms differ from each other with respect to their molecular and biochemical features, they show metal-specific binding preferences, they are organ-specific, they exhibit distinct induction patterns in response to the metals to which they bind, and their biological and functional background is thoroughly known.

 

Dallinger, R., M. Chabicovsky, et al. (2004). "Isoform-specific quantification of metallothionein in the terrestrial gastropod Helix pomatia. II. A differential biomarker approach under laboratory and field conditions." Environ Toxicol Chem 23(4): 902-10.

                The two function-specific metallothionein (MT) isoforms characterized from the midgut gland and mantle tissue of Helix pomatia differ substantially in their metal-binding preferences, as well as molecular and biochemical features. These differences make them potential candidates for biomarker studies based on a differential, isoform-specific approach. To prove this hypothesis, induction experiments with two metals (Cd and Cu) that are normally bound by the two isoforms were compared with a range of organic chemicals and physical stressors under laboratory conditions to test the responsiveness of the two isoforms to the stressors applied. In addition, field studies were conducted with Roman snails and substrate samples collected from different metal-contaminated sites in Austria to test the suitability of the two isoforms as biomarkers under field conditions. The results of these combined laboratory and field studies confirmed the validity of the biomarker approach with the two metal- and tissue-specific isoforms. It is demonstrated that the Cd-binding MT specifically and exclusively responds to Cd exposure by increasing concentrations, whereas the Cu-binding MT isoform decreases in its concentration upon exposure to physical stress (X-ray irradiation and cold). This suggests researchers should adopt, under certain preconditions, a dual biomarker approach by combining the simultaneous quantification of Cd-MT concentrations in the midgut gland as a biomarker for Cd pollution and of Cu-MT concentration in the mantle as a biomarker for the impairment of snails by additional physical stressors.

 

Corporeau, C. and M. Auffret (2003). "In situ hybridisation for flow cytometry: a molecular method for monitoring stress-gene expression in hemolymph cells of oysters." Aquat Toxicol 64(4): 427-35.

                In the present study, we developed a molecular method for flow cytometry to detect the effects of environmental factors on the stress-response in immunocompetent cells of the European flat oyster, Ostrea edulis. Stress-generating conditions were applied to individuals acclimated in the laboratory to environmental salinity and temperature. Oligonucleotidic probes were applied to quantify the expression of HSP/C70 or metallothionein genes. After a heat-stress, a response was reflected in hemocytes by an increased amount of HSP/C70 mRNA and quantitative changes in HSP/C70 protein expression. The technique of in situ hybridisation described here also allowed to quantify the expression of metallothionein mRNA in oysters exposed to a heavy metal-exposure. The detection of stress protein markers by using such quantitative methods could be applied in contamination studies in oysters and other bivalves where monitoring the status of hemolymph cells is required.

 

Lehtonen, K. K. and S. Leinio (2003). "Effects of exposure to copper and malathion on metallothionein levels and acetylcholinesterase activity of the mussel Mytilus edulis and the clam Macoma balthica from the northern Baltic Sea." Bull Environ Contam Toxicol 71(3): 489-96.

               

Geret, F., A. Serafim, et al. (2003). "Antioxidant enzyme activities, metallothioneins and lipid peroxidation as biomarkers in Ruditapes decussatus?" Ecotoxicology 12(5): 417-26.

                Antioxidant enzymes (superoxide dismutase (SOD), catalase (CAT), glutathione peroxidases (GPX)) were studied in the gills and digestive gland of clams Ruditapes decussatus from different sites of the Ria Formosa (south coast of Portugal) in order to validate these enzymes as biomarkers of metal exposure. Two other potential biomarkers, lipid peroxidation expressed as malondialdehyde (MDA), and metallothionein (MT) levels were also measured. This study demonstrates that mitochondrial and cytosolic SOD activity changed while CAT activity (cytosolic and mitochondrial) was higher in the gills of clams from the three sites affected by different sources of contamination. GPXs (total and Se-dependent) were inhibited in the gills of clams from sites A (directly influenced by sewage industrial wastes) and B (directly influenced by harbor facilities). MT and MDA levels were higher in the gills of clams from these two sites and in the digestive gland of clams from the third site (C, main navigation channel and exchange with Atlantic ocean). These results suggest that CAT and GPX activities and MDA levels especially in the gills of clams can be used as biomarkers for monitoring environmental pollution.

 

Romeo, M., Y. Mourgaud, et al. (2003). "Multimarker approach in transplanted mussels for evaluating water quality in Charentes, France, coast areas exposed to different anthropogenic conditions." Environ Toxicol 18(5): 295-305.

                An active biomonitoring experiment was performed using mussels collected at a clean site, Fier d'Ars, and transplanted to two locations, outside the harbor of La Rochelle and in the Baie de L'Aiguillon along the coast of Charentes (French Atlantic coast) beginning in April for several months. Mussels were collected in June and October. The cadmium, copper, and zinc concentrations of all resident and transplanted mussel samples and the polycyclic aromatic hydrocarbon and polychlorinated biphenyl concentrations in some mussel samples and in the sediment samples were determined. Mussel response was evaluated for several biochemical biomarkers: concentrations of metallothionein, activities of glutathione S-transferase and acetylcholinesterase (AChE) and levels of thiobarbituric reactive substance (TBARS). The physiological status of the animals was assessed using the condition index. A principal component analysis performed with the chemical and biochemical results of the evaluations of the resident and transplanted mussels collected in June allowed them to be separated into three groups: resident mussels from la Rochelle with high metal and TBARS levels, resident mussels from Baie de L'Aiguillon with a very high condition index, and resident mussels from Fier d'Ars and transplanted mussels at La Rochelle and Baie de L'Aiguillon with low TBARS and AChE activities. Strong seasonal variation from June to October of all parameters was noted. Mussels transplanted to La Rochelle appeared to be the most "polluted" in their pollutant concentrations and biochemical responses; moreover, the La Rochelle site had the highest concentration of organics in sediments of all the sites. The choice of Fier d'Ars as a reference site may be questionable because some of the biomarker responses of the mussels were higher than expected there, although these pollutants in mussels and sediment were present at the lowest concentrations measured.

 

Chavez-Crooker, P., P. Pozo, et al. (2003). "Cellular localization of calcium, heavy metals, and metallothionein in lobster (Homarus americanus) hepatopancreas." Comp Biochem Physiol C Toxicol Pharmacol 136(3): 213-24.

                This investigation combines confocal microscopy with the cation-specific fluorescent dyes Fluo-3 and BTC-5N to localize calcium and heavy metals along the length of intact lobster (Homarus americanus) hepatopancreatic tubules and isolated cells. A metallothionein-specific antibody, developed in mollusks with cross-reactivity in crustaceans, showed the tissue-specific occurrence of this metal-binding protein in several organ systems in lobster and in single cell types isolated from lobster hepatopancreas. Individual lobster hepatopancreatic epithelial cell types were separated into pure single cell type suspensions for confocal and antibody experiments. Intact hepatopancreatic tubules showed high concentrations of both calcium and heavy metals at the distal tips of tubules where mitotic stem cells (E-cells) are localized. In addition, a concentrated distribution of calcium signal within isolated single premolt E-cells in solution was disclosed that might suggest an endoplasmic reticulum compartmentation of this cation within these stem cells. Both E- and R-cells showed significantly (P < 0.05) greater intracellular calcium concentrations in premolt than intermolt, suggesting the accumulation of this cation in these cells prior to the molt. Antibody studies with lobster tissues indicated that the hepatopancreas possessed 5-10 times the metallothionein concentration as other lobster organ systems and that isolated E-cells from the hepatopancreas displayed more than twice the binding protein concentrations of other cells of this organ or those of blood cells. These results suggest that crustacean hepatopancreatic stem cells (E-cells) and R-cells play significant roles in calcium and heavy metal homeostasis in this tissue. Interactions between the four hepatopancreatic cell types in this regulatory activity remain to be elucidated.

 

Leung, K. M., H. Ibrahim, et al. (2003). "Concentrations of metallothionein-like proteins and heavy metals in the freshwater snail Lymnaea stagnalis exposed to different levels of waterborne cadmium." Bull Environ Contam Toxicol 71(5): 1084-90.

               

Soazig, L. and L. Marc (2003). "Potential use of the levels of the mRNA of a specific metallothionein isoform (MT-20) in mussel (Mytilus edulis) as a biomarker of cadmium contamination." Mar Pollut Bull 46(11): 1450-5.

                Metallothionein (MT) is a metal-inducible protein and a potential biomarker of metal pollution. Mussel MTs are characterized by having both monomeric (MT-10) and dimeric (MT-20) isoforms. We have carried out a series of experiments in which we studied mRNA levels of both MTs in Mytilus edulis after a brief exposure (6-72 h) to cadmium, zinc and copper. The induction of MT-10 and MT-20 mRNA in the heavy metal-treated mussels was detected by Northern blot analysis. The exposure of mussels to copper (40 microgl(-1)), cadmium (200 microgl(-1)) or zinc (1,000 microgl(-1)) resulted in different MT-gene induction patterns. In the cadmium-intoxicated mussels levels of both MT-10 and MT-20 mRNA were increased. In the zinc-treated mussels, a rapid response of the MT-10 gene was detected within 6 h. Induction of MT-20 transcription was specific for cadmium, and can be envisaged as a potential biomarker of cadmium contamination. Copper had no effect on mRNA levels. We suggest the possibility that there is a hitherto-unknown copper-specific isoform.

 

Chevre, N., F. Gagne, et al. (2003). "Development of a biomarker-based index for assessing the ecotoxic potential of aquatic sites." Biomarkers 8(3-4): 287-98.

                The use of biochemical or physiological measurements as indicators of ecotoxicity is under constant development and has the advantage of delineating effects before the appearance of disease. However, these biomarkers are often part of a battery of tests, and it is difficult to integrate them together to gain an overall view of an organism's health. The aim of this study was to develop an index that could integrate the data derived from a battery of biomarkers for application to both spatial and temporal studies. Mya arenaria clams were collected at different sites along the Saguenay Fjord (Quebec, Canada). Six biomarkers were measured: metallothioneins, DNA strand breakage, lipid peroxidation, vitellin-like proteins, phagocytosis, and non-specific esterase activity in haemocytes. A biomarker index was obtained by summing the biomarker values expressed in term of classes. Classes were determined by a distribution-free approach derived from the theory of rough sets. The results of the spatial study show that the index values discriminated well between contaminated and uncontaminated sites. The highly polluted sites had the highest index values (18 compared with a reference value of 14). In the temporal study, the index was also able to highlight possible contamination-induced alterations, even though the interpretation of temporal variation is complicated by natural variations occurring throughout the year. A control chart approach is proposed for determining contaminated sites in both spatial and temporal surveys.

 

Simes, D. C., M. J. Bebianno, et al. (2003). "Isolation and characterisation of metallothionein from the clam Ruditapes decussatus." Aquat Toxicol 63(3): 307-18.

                Metallothioneins (MT) were obtained after purification from metal-exposed clams (Ruditapes decussatus) using gel-permeation and ion-exchange chromatography. Four cadmium-metallothioneins (CdMTs) were resolved by ion-exchange chromatography and they all had similar molecular weights, high cadmium content and an absorption spectra indicative of the presence of characteristic Cd-S aggregates. The NH(2)-terminal sequence suggests the presence of at least two class I clam MT isoforms. For the other two putative clam CdMTs isolated, the results of the amino acid determination were inconclusive. One was slightly contaminated and the other one had a blocked NH(2)-terminal. These clam metalothioneins contain glycine, which seems to be a common feature of molluscan MT family and exhibited more similarity to oysters than to mussels. Further investigation on the inducibility of these isoforms will be necessary if clams are to be used as biomarkers of metal exposure.

 

Han, S. J., J. S. Park, et al. (2003). "Accumulation and elimination of cadmium and zinc in the Asian periwinkle Littorina brevicula." J Environ Sci Health A Tox Hazard Subst Environ Eng 38(5): 965-74.

                To elucidate the differences between the detoxification mechanisms of essential metal (Zn) and non-essential metal (Cd) in Littorina brevicula that is highly resistant to a wide range of heavy metal concentrations, Asian periwinkles were exposed to Cd (400 microg/L), Zn (3000 microg/L) and a mixture of both metals. We examined metal accumulation, elimination and subcellular distribution for binding to proteins. The metal concentration in L. brevicula increased gradually with exposure time (up to 70 days), following which accumulated levels reached saturation point. The accumulated Zn content was increased in the presence of Cd, while Cd uptake was decreased when Zn was present. During the depuration period (42 days), Cd was not removed from periwinkles, while Zn was eliminated in a rate of 2.19 microg Zn g(-1) day(-1). This elimination rate was particularly high on exposure to the metal mixture (3.80 microg Zn g(-1) day(-1)). Subcellular distribution studies on Cd and Zn revealed that most Cd (80%) was bound to cytosolic ligand, while more than 75% Zn was distributed in the membrane (insoluble) fraction. An additional difference in sequestering of metal in the cytosol was noted between Cd and Zn; most Cd in the cytosol was bound to metallothionein-like cadmium binding protein, MBP-1 (9.8 kDa), while the profile for Zn distribution revealed the presence of four Zn-binding ligand peaks, specifically, HMW (60 kDa), MBP-1 (9.8 kDa), MBP-2 (5 kDa) and LMW (<1 kDa). Our data confirm that metallothionein-like cadmium binding protein, MBP-1, has the same affinity for Zn, while MBP-2 displays comparatively higher affinity for Zn than Cd.

 

Irato, P., G. Santovito, et al. (2003). "Metal accumulation and binding protein induction in Mytilus galloprovincialis, Scapharca inaequivalvis, and Tapes philippinarum from the Lagoon of Venice." Arch Environ Contam Toxicol 44(4): 476-84.

                Heavy metal pollution is known to be widespread in the sediments of the Lagoon of Venice. Therefore, physiological parameters influenced by this form of contamination were examined. The bivalve molluscs blue mussel (Mytilus galloprovincialis), ark clam (Scapharca inaequivalvis), and Japanese littleneck (Tapes philippinarum) were sampled in two sites (Marghera, Chioggia) every 3 months for 1 year. The digestive gland and gills of each bivalve were analyzed. The concentrations of essential and nonessential metals (including chromium, manganese, iron, cobalt, nickel, copper, zinc, and cadmium) were determined. Because glutathione and metallothioneins (MTs) are involved in metal homeostasis and detoxification, their concentrations were evaluated in relation to metal concentrations. Results show that (1) all three studied species accumulate metals to a considerable extent, with some species-specific differences between the digestive gland and gills; (2) gills are a good tissue to evaluate pollution by examining the MT content. In particular, the correlation between Zn and MT levels in the gills indicates that M. galloprovincialis and S. inaequivalvis are sentinel organisms and can be used specifically for Zn pollution; (3) T. philippinarum accumulates Cu in the digestive gland more readily than the other two bivalves and therefore has the highest MT.

 

Shi, D., G. Blackmore, et al. (2003). "Effects of aqueous and dietary preexposure and resulting body burden on silver biokinetics in the green mussel Perma viridis." Environ Sci Technol 37(5): 936-43.

                To determine whether preexposure of green mussel Perna viridis to Ag influenced metal uptake kinetics we compared various physiological indicators of metal uptake kinetics between the control mussels and mussels preexposed to Ag in both diet and water at different levels (up to 5 weeks). In all preexposed mussels, the assimilation of Ag increased by 1.1-3.0 times with increasing Ag body concentration (0.651-19.3 microg g(-1)) as compared with the controls (Ag body concentration of 0.311-0.479 microg g(-1)), whereas the efflux rate constants decreased by 45-88%. There was no significant increase in Ag associated with the metallothionein-like protein (MTLP) fraction following exposure of the mussels to Ag through either the dissolved or food phase. The clearance rates were little affected or depressed byAg preexposure, and the relationship between the Ag influx rate from the dissolved phase and the Ag preexposure was somewhat complicated. The influx rate decreased with increasing Ag body burden at <2.5 microg g(-1), above which it increased with increasing Ag body burden. Our results indicate that the mussels may modify physiological processes to ambient chronic Ag exposure, consequently accumulating more Ag. Ag body concentration in these mussels may therefore increase disproportionally in response to increasing Ag concentration in the ambient environments. Ag preexposure and resulting body burden should be considered carefully when interpreting the observed Ag concentration in biomonitoring animals to evaluate the Ag pollution in seawater.

 

Craig, S. and J. Overnell (2003). "Metals in squid, Loligo forbesi, adults, eggs and hatchlings. No evidence for a role for Cu- or Zn-metallothionein." Comp Biochem Physiol C Toxicol Pharmacol 134(3): 311-7.

                An adult squid Loligo forbesi had the following metals in its liver/digestive gland: Mn, Fe, Ni, Zn, Cu, As, Cd, Ba and Pb in the range of 1-110 ppm wet wt. Adult mantle muscle, adult eyes, eggs and hatchlings contained a lesser number of these metals at concentrations above 1 ppm. Chromatographic analysis of non-heat-treated cytosols (in the presence of 5 mM 2-mercaptoethanol) gave no evidence for the presence of copper- or zinc-containing fractions with the molecular weights of mollusc metallothioneins in any of the above tissues. Copper and Zn were bound to either the particulate fraction or to very low molecular weight species.

 

Giguere, A., Y. Couillard, et al. (2003). "Steady-state distribution of metals among metallothionein and other cytosolic ligands and links to cytotoxicity in bivalves living along a polymetallic gradient." Aquat Toxicol 64(2): 185-200.

                The present study was designed to assess the environmental effects of metals in a field setting. We explored exposure-->bioaccumulation-->effects relationships in freshwater molluscs exposed to metals in their natural habitat. Indigenous floater mussels (Pyganodon grandis) were collected from ten limnologically similar lakes located along a Cd, Cu and Zn gradient. Ambient free-metal ion concentrations were estimated as a measure of metal exposure. Metallothionein (MT) was measured in mussel gills and metal partitioning among the various cytosolic protein pools was determined by size exclusion chromatography. Various biomarkers were also measured, including malondialdehyde (MDA) concentrations in the gills and in the digestive gland, glutathione-peroxidase and glutathione-reductase activities in the digestive gland, and lipid concentrations in the gonad. Cadmium and MT concentrations in the gill cytosol increased along the contamination gradient, but Cu and Zn levels were independent of the ambient free-metal ion concentrations. The distribution of Cd among the various cytosolic complexes remained quite constant: 80% in the MT-like pool, 7% in the low molecular weight pool (LMW<1.8 kDa) and 13% in the high molecular weight pool (HMW>18 kDa). For these chronically exposed molluscs there was thus no threshold exposure concentration above which spillover of Cd occurred from the MT pool to other cytosolic ligands. However, the presence of Cd in the LMW and HMW fractions suggests that metal detoxification was imperfect, i.e. that P. grandis was subject to some Cd-related stress at low chronic exposure concentrations. Consistent with this suggestion, MDA concentrations, an indicator of oxidative stress, increased with gill cytosolic Cd. In the digestive gland, MDA concentrations were unrelated to any of the measured metals, but glutathione-peroxidase and glutathione-reductase activities increased with gill cytosolic copper. We speculate that cytosolic Cu catalyses the production of reactive oxygen species, to which the organism reacts by increasing activities of the two enzymes, thus preventing the accumulation of reactive oxygen species. Lipid concentrations in the gonad did not decrease with any of the measured toxicological parameters, suggesting that energy reserves for reproduction were not compromised in the metal-contaminated mussels. The results of the present study, where chronically exposed bivalves were collected from their natural habitat along a metal contamination gradient, contrast markedly with what would have been predicted on the basis of experimental metal exposures, and clearly demonstrate the need to study metal exposure-->bioaccumulation-->effects relationships in natural populations.

 

Chabicovsky, M., H. Niederstatter, et al. (2003). "Localization and quantification of Cd- and Cu-specific metallothionein isoform mRNA in cells and organs of the terrestrial gastropod Helix pomatia." Toxicol Appl Pharmacol 190(1): 25-36.

                A quantitative assay based on real-time detection polymerase chain reaction (rtdPCR) was applied to analyze basal and metal-induced mRNA levels of two metallothionein (MT) isoforms (Cd-MT and Cu-MT) in organs of the terrestrial gastropod Helix pomatia. The results show that specific Cd-MT mRNA levels increase with Cd tissue burden, identifying hepatopancreas and gut as the main organs of Cd accumulation and, accordingly, the predominant organs of Cd-MT mRNA expression. In situ hybridization localized this isoform in epithelial cells of hepatopancreas, gut, and kidney. In contrast to the observed Cd-dependent inducibility of the Cd-binding MT isoform, gene expression of the Cu-binding MT could not be induced by either Cd or Cu exposure. Only very low mRNA amounts of the Cu-MT isoform were found in snail hepatopancreas and kidney, whereas the mantle exhibited high basal mRNA levels of this isoform. In situ localization revealed that the Cu-MT gene expression was restricted to one cell type, the so-called rhogocytes, which are present to various extents in the different organs examined. These results suggest a metal-specific sharing of functions between the two MT isoforms. The Cd-MT isoform apparently plays a crucial role in Cd detoxification, as demonstrated by the inducibility of this isoform, as well as its specific localization in the main metabolic and Cd storing organs. The predominant presence of Cu-MT in rhogocytes of snail mantle strengthens the hypothesis that this isoform may regulate Cu availability in hemocyanin synthesis.

 

English, T. E. and K. B. Storey (2003). "Freezing and anoxia stresses induce expression of metallothionein in the foot muscle and hepatopancreas of the marine gastropod Littorina littorea." J Exp Biol 206(Pt 14): 2517-24.

                Differential screening of cDNA libraries constructed from the foot muscle of marine snails Littorina littorea revealed several cDNAs that are upregulated during anoxia or freezing exposures, environmental stresses that are naturally endured by this species. One full-length clone of 1196 nucleotides (GenBank accession number AY034179) hybridized with a 1200-nucleotide band on northern blots and encoded a 100-amino-acid protein that was identified as belonging to the metallothionein (MT) family. L. littorea MT shared 45% and 56% identity with the copper- and cadmium-binding MT isoforms, respectively, from another gastropod, Helix pomatia and 43-47% identity with marine bivalve MTs. The L. littorea sequence included the mollusc-specific C-terminal motif Cys-X-Cys-X(3)-Cys-Thr-Gly-X(3)-Cys-X-Cys-X(3)-Cys-X-Cys-Lys that identifies it as a family 2 (mollusc) MT. Northern blot analysis showed that L. littorea MT was upregulated in both foot muscle and hepatopancreas in response to both freezing and anoxia stresses; within 1 h of the beginning of the stress transcript levels rose 2.5- to sixfold of control levels, reaching maximal levels at 12 or 24 h. After 24 h recovery from either stress, transcript levels were reduced again in three cases but remained elevated in hepatopancreas from anoxia-treated snails. Upregulation of MT during environmental stress could serve one or more possible roles, including a function in antioxidant defense.

 

Gueguen, Y., J. P. Cadoret, et al. (2003). "Immune gene discovery by expressed sequence tags generated from hemocytes of the bacteria-challenged oyster, Crassostrea gigas." Gene 303: 139-45.

                An expressed sequence tag program was undertaken to isolate genes involved in defense mechanisms of the Pacific oyster, Crassostrea gigas. Putative function could be assigned to 54% of the 1142 sequenced cDNAs. We built a public database where all EST information are accessible through numerous search profiles (http://www.ifremer.fr/GigasBase). Based on sequence similarities we identified 20 genes that may be implicated in immune function. We investigated the expression of four of these genes during bacterial challenge of oysters. Three of them were induced in response to challenge lending support to their involvement in oyster immunity. Moreover, four other genes were highly homologous to components of the NF-kappa B signaling pathway which is involved in innate immune response in Drosophila and mammals. Altogether, our results open a new way to investigate the immune response in mollusks.

 

Bebianno, M. J., A. Cravo, et al. (2003). "Metallothionein concentrations in a population of Patella aspera: variation with size." Sci Total Environ 301(1-3): 151-61.

                The determination of metallothionein (MT) concentrations in species with different feeding habits is important from the ecotoxicological point of view because it provides a better understanding of the role of these proteins in metal uptake pathways. The main objective was study the variation of MT and metal (Cd, Cu and Zn) concentrations with size and weight in the limpets Patella aspera. In addition investigate the relationship between MT and metal concentrations in limpets from different metal load environments of the south coast of Portugal with the aim to use MT in P. aspera as a biomarker of metal exposure. MT concentrations in the whole soft tissues of P. aspera increased with size and weight while metals decrease with size and weight. MT concentrations showed no significant relationship with Cd or Cu concentrations in the limpets from the South Portuguese Coast. However, a negative exponential relationship detected between MT and Zn concentrations suggest that Zn bound to MT might be displaced by Cd or Cu ions. The ability of limpets to store both Cd and Cu bound to MT may be responsible for the tolerance of this species to contaminated environments. The relationship between MT concentrations and Zn indicates that MT seems to play a minor role in binding Zn in Patella species.

 

Bebianno, M. J. and M. A. Serafim (2003). "Variation of metal and metallothionein concentrations in a natural population of Ruditapes decussatus." Arch Environ Contam Toxicol 44(1): 53-66.

                The spatial and seasonal variation of total and subcellular distribution of Cd, Cu, and Zn was followed in different tissues (gills, digestive gland, and remaining tissues) of the clam Ruditapes decussatus collected along a metal contamination gradient in the Ria Formosa lagoon (southern Portugal) and compared with metallothionein (MT) concentrations.Total metal concentrations decreased according to the sequence digestive gland > gills > remaining tissues for Cd, digestive gland approximately gills > remaining tissues for Cu and gills > digestive gland > remaining tissues for Zn. MT concentrations in these tissues decreased according to the same sequence observed for Cd. In all the tissues, the highest subcellular concentration was in the cytosol for Cd and Cu and in the pellet for Zn. Among the three metals, Cd concentrations showed the most evident spatial variation. In all tissues, total and subcellular Cd concentrations decreased from the inner parts of the lagoon toward the ocean. However, no significant spatial or seasonal variation occurred in clam tissues for the other two metals, though marginal elevated Cu concentrations were observed in the inner parts of the lagoon. Therefore, Cu subcellular distribution in clam tissues was not significantly altered by Cu changes in the lagoon and are the baseline levels for normal metabolism of this clam population. The fact that total Zn concentrations remained unchanged both spatial and seasonal suggested that these clams regulate Zn in their tissues. In the three tissues, MT bind most significantly to Cd and Cu, while Zn, although binding to MT, is preferably bound to other ligands. MT concentrations showed the same spatial and seasonal variation of Cd and were significantly related with total and heat-treated cytosolic Cd in all tissues. For Cu a significant relationship between MT and total or cytosolic Cu was only observed in the remaining tissues. No relationship was observed between MT and total or cytosolic Zn concentrations. Metals and MT concentrations increased with the increase in the condition index for the gills and the digestive gland and decreased from the remaining tissues.Cd concentrations in the gills increased only in the heat-treated cytosolic fraction while Zn in this fraction decreased. Thus Cd concentrations in this tissue displaced Zn from the MT-fraction, leading to a modification of the soluble/insoluble Zn ratio once total Zn concentrations remained unchanged. This modification reflects a perturbation in the normal metabolism in this tissue due to the excess of Cd present. With the exception of the gills, Zn subcellular distribution in the other two tissues was similar among sites and season. The model that describes the relationship between MT, metals, and weight in the gills, digestive gland and remaining tissues also indicates that Cd was the only metal that influence MT synthesis significantly in all the tissues. The induced and/or existent MT was sufficient to bind free Cd ions present in the cells, preventing any damage to cellular metabolism in this clam population. Therefore, MT in the gills and digestive gland of R. decussatus can be used as an early warning signal for Cd exposure and are a useful biomarker to assess the toxicological status of this population in the Ria Formosa lagoon.

 

Bragigand, V. and B. Berthet (2003). "Some methodological aspects of metallothionein evaluation." Comp Biochem Physiol A Mol Integr Physiol 134(1): 55-61.

                The specificity of the methods used for metallothioneins (MTs) determination in both field and laboratory studies is crucial to a relevant interpretation of the results. The technique applied in the present study is commonly used, but several potential problems may limit its validity: (i). the stability of the metal-SH links during heating and the possibility of metal exchanges between MTs and others compounds; (ii). the presence of heat-stable thiol compounds with high molecular weight or very low molecular weight, which may subsist in the supernatant and interfere during global analysis of MTs; (iii). the subsistence of metals not bound to MT in the supernatant after heating; and (iv). the impact of reducing agents such as mercaptoethanol on the metal-binding ligands. Our investigations were conducted mainly on oysters (Crassostrea gigas) and polychaetes (Hediste/Nereis diversicolor) sampled, respectively, in two French metal-rich sites, the Gironde estuary and Boulogne harbour. This study, which allowed us to clarify most of the potential problems noted above, indicates the importance of dealing with methodological difficulties.

 

Hamza-Chaffai, A., J. Pellerin, et al. (2003). "Health assessment of a marine bivalve Ruditapes decussatus from the Gulf of Gabes (Tunisia)." Environ Int 28(7): 609-17.

                Metallothioneins (MTs), malonedialdehyde (MDA), and glycogen concentrations were determined in order to assess the health status of Ruditapes decussatus exposed to in situ contamination, showing the effects of time, site, and metal contamination on these three biomarkers. Metallothionein was positively correlated with Cd and Zn. If introduced in a multiple model, the factors site and time were shown to decrease significantly MT concentrations. MDA was inversely correlated with size of the animals and was affected by the factor time. Glycogen was inversely correlated with zinc and was affected by both site and time.This study constitutes a field-based validation of a multiparametric approach using specific and nonspecific biomarkers.

 

Lieb, B. (2003). "A new metallothionein gene from the giant keyhole limpet Megathura crenulata." Comp Biochem Physiol C Toxicol Pharmacol 134(1): 131-7.

                Metallothioneins (MTs) are small soluble proteins ubiquitously expressed in animals and plants. Different isoforms are present in deuterostomes and protostomes. They do not differ greatly in primary structure, but are clearly distinguishable. Here, I present the gene and the complete cDNA of a novel MT from the mollusk Megathura crenulata. This protein is closely related to the Cu-inducible MTs of the vineyard snail Helix pomatia, but has also some minor sequence features typical of Cd-inducible isoforms of H. pomatia and other molluscs. Overall, the deduced primary structure is similar to the known molluscan MTs, but in addition possesses an insertion of 5 amino acids not found in any other molluscan MTs, protostomic or deuterostomic MTs. In addition, a pentapeptide insertion, characteristic of mammalian MT-3 is present but it lacks the functional tetrapeptide CPCP within the beta-region of those MT-3 proteins that are known to suppress neuronal growth processes. The M. crenulata MT is a novel form of MT in comparison to all other known MTs. Possible functional aspects for this new MT are discussed.

 

Romero-Ruiz, A., O. Amezcua, et al. (2003). "Oxidative stress biomarkers in bivalves transplanted to the Guadalquivir estuary after Aznalcollar spill." Environ Toxicol Chem 22(1): 92-100.

                Biological effects of metals were studied in clams (Scrobicularia plana) transplanted to Guadalquivir estuary (Spain) at several times after the spill of toxic metals from Aznalcollar pyrite mine (southwest Spain) (April 1998) using biochemical biomarkers responsive to reactive oxygen species. Significant As, Cd, Fe, Mn, Ni, and Pb uptake was found in clams living for seven months at the estuary (from July 1999). Increased activity of antioxidant (catalase, glucose-6-phosphate, and 6-phosphogluconate dehydrogenase) and glutathione-related (glutathione reductase and glyoxalase I and II) enzymes was also found after short exposures; the levels of malondialdehyde and metallothionein increased also, particularly with long exposures. Clams living four weeks at the estuary (from March 2000) but not at a reference site also accumulated metals. The higher malondialdehyde and lower reduced-glutathione levels and the more oxidized glutathione status confirmed the oxidative stress of clams living at the estuary, while no marked increase of antioxidant activities was found this time. Lower metal availability along the second transplant could explain the limited responses in this shorter experiment. Although the status of Guadalquivir estuary has recovered since Aznalcollar spill, continuous monitoring is needed to confirm its progress and to be alert to possible deterioration after heavy rains.

 

Ryu, S. K., J. S. Park, et al. (2003). "Purification and characterization of a copper-binding protein from Asian periwinkle Littorina brevicula." Comp Biochem Physiol C Toxicol Pharmacol 134(1): 101-7.

                The Asian periwinkle, Littorina brevicula, is highly resistant to a wide range of heavy metal concentrations and its metal-binding protein(s) are induced in the presence of cadmium (Cd) and zinc (Zn). In this study, we isolated and characterized a novel copper-binding protein (Cu-BP). Following purification by Sephacryl S-100 chromatography, Cu-BP contained an equal amount of Zn in non-exposed physiological conditions. However, Zn is replaced by Cu at the binding site upon addition of excess Cu (100 microM CuCl(2)) to the cytosol or after a long period (60 days) of exposure of the periwinkles to the metal ion (150 microg/l CuCl(2)). The ligand was further purified by DEAE-Sepharose anion-exchange chromatography and C(18) reverse-phase HPLC. The molecular weight of the purified protein was determined as 11.38 kDa by MALDI-TOF MS analyses. This Cu-BP is distinct from common mollusk metallothionein (MT) in that it contains significantly lower number of Cys (8 residues) and high levels of aromatic amino acids, Tyr and Phe. The protein additionally contains His and Met, which are absent in the MT-like Cd-BP of L. brevicula. The finding that Cu-BP in the Asian periwinkle is distinct from MT-like Cd-BP suggests that the timely expression of specific metal-binding proteins allows added protection against each heavy metal in severely polluted conditions.

 

Riveros, A., M. Zuniga, et al. (2003). "Copper metallothionein-like proteins as exposure biomarker in native and transplanted intertidal populations of the mussel Perumytilus purpuratus from San Jorge Bay, Antofagasta, Chile." Bull Environ Contam Toxicol 70(2): 233-41.

               

El Ghazi, I., S. Menge, et al. (2003). "Quantification of metallothionein-like proteins in the mussel Mytilus galloprovincialis using RP-HPLC fluorescence detection." Environ Sci Technol 37(24): 5739-44.

                A HPLC-fluorescence method, using the fluorophore SBD-F (ammonium-7-fluorobenz-2-oxa-1,3-diazole-4-sulfonate), was adapted for the quantification of metallothioneins and their isoforms from the Moroccan mussel Mytilus galloprovincialis. The method was first optimized using a rabbit liver metallothionein. The effects of EDTA, tris(2-carboxyethyl)phosphine, and SBD-F on the labeling efficiency were studied. The optimized method was then applied to evaluate the amount of metallothionein in the mussels either exposed to cadmium in the laboratory or collected from the Casablanca coast, Morocco. The concentrations of metallothioneins measured in the field samples describe the degree of contamination of the sites and are reflected by distinct isoform patterns.

 

Rebelo, M. F., W. C. Pfeiffer, et al. (2003). "Cloning and detection of metallothionein mRNA by RT-PCR in mangrove oysters (Crassostrea rhizophorae)." Aquat Toxicol 64(3): 359-62.

                A semi-quantitative RT-PCR protocol was developed to directly evaluate metallothionein (MT) mRNA expression in different tissues of mangrove oysters (Crassostrea rhizophorae), using beta-Actin (ACT) as a normalizing gene. Clones with high degree of identity from partial coding sequences were obtained for both MT and ACT. Although not statistically significant, high relative accumulation of MT mRNA was observed in the digestive gland (DGG), but not in the gills, from samples collected from both control and contaminated sites. Nevertheless, MT expression was not comparable to the high levels of metal in the contaminated oysters. Results indicate that the variation in relative MT mRNA levels from different samples of the same site could be due to multiple gene copies or different MT isoform induction.

 

Baudrimont, M., S. Andres, et al. (2003). "The key role of metallothioneins in the bivalve Corbicula fluminea during the depuration phase, after in situ exposure to Cd and Zn." Aquat Toxicol 63(2): 89-102.

                An experimental study of the role of metallothioneins (MTs) in Cd and Zn depuration processes in the freshwater bivalve Corbicula fluminea was conducted after in situ exposure on the river Lot (France). Specimens of adult C. fluminea were first transplanted from a lacustrine reference site to a polymetallic polluted station (Bouillac, (B)) for a 42-days' exposure period from September to November 1996. They were then depurated after transfer to the laboratory, and were sub-sampled periodically until May 1997. During the first phase, MT concentrations measured with the Mercury-Saturation Assay were induced for a factor of 3.5 compared with time 0, whereas metal uptake showed accumulation factors of 17 and 4 for Cd and Zn, respectively. During the depuration phase, Cd and Zn concentrations decreased by 18 and 70%, respectively, giving estimated biological half-lives of 500 and 40 days. During the same period, MT concentrations decreased by 37% after transfer under unpolluted conditions, especially between 0 and 3 days, suggesting that MTs play a predominant role in Cd depuration. The quantity of Cd sequestered by the MT fraction, after size-exclusion liquid chromatography, represents on average 40% of the total Cd bioaccumulated in the soft body of the molluscs, compared with only 4-9% for total accumulated Zn. This essential metal was principally bound to low molecular weight proteins, which represented 20% of total Zn. Furthermore, it was observed that MTs had a key role in Cd remanence in the bivalves, and it was also reported that other proteins or small peptides were involved in the depuration of Zn.

 

Chevre, N., F. Gagne, et al. (2003). "Application of rough sets analysis to identify polluted aquatic sites based on a battery of biomarkers: a comparison with classical methods." Chemosphere 51(1): 13-23.

                The evaluation of toxicological effects at the cellular and molecular levels in organisms are often used to determine sites subjected to contamination problems that pose a threat to the long-term survival of organisms. However, the integration of multiple measurements on the health status of organisms into a model for site discrimination is challenging. This study compares two discrimination methods which are based on rule inference: rough sets (RS) analysis and classification trees (CT) with classical multivariate discriminant analysis (DA). Site classification was attempted with six biomarkers of effects: metallothionein levels, lipid peroxidation, DNA damage, levels of lipophosphoproteins (i.e., vitellins), phagocytosis activity and haemocyte cell viability on clam (Mya arenaria) populations from the Saguenay River fjord (Quebec, Canada). Rule based methods have the advantage of complete independence from data distribution constraints in contrast to the classification methods from multivariate analysis that are more commonly used in ecotoxicology. Results show that RS and CT gave better classifications than DA because they do not require strong distributional assumptions. Moreover, RS provided classification rules that could identify the most important biomarker(s) for site discrimination. RS and CT were shown to be simple and efficient methods for classifying multivariable ecotoxicological data. This methodology would be especially useful when freedom from distributional assumptions is required.

 

Geffard, O., A. Geffard, et al. (2003). "Assessment of the bioavailability and toxicity of sediment-associated polycyclic aromatic hydrocarbons and heavy metals applied to Crassostrea gigas embryos and larvae." Mar Pollut Bull 46(4): 481-90.

                Sediments represent a vast sink for contaminants in aquatic systems, and may pose a threat to pelagic and benthic organisms. The objective of this research was to determine the bioavailability and toxicity of sediment-associated PAHs and heavy metals, using embryos and larvae of the oyster Crassostrea gigas, exposed to two sediment fractions: the whole sediment and the elutriate. The percentages of abnormal larvae, the contaminant accumulation and, (in the case of metal contamination), the induction of metallothionein in the larvae, were investigated. Sediment-associated PAHs and heavy metals were available for exposure, as indicated by their accumulation in C. gigas larvae and by the abnormalities induced during larval development. The critical body burden of PAHs (Fluo, Pyr, BaA, Triph, Chrys, BbF, BkF, BjK, BeP, BaP, Per, IP, BPer and the DahA) in the larvae was 0.3 micro g g(-1), above which abnormalities were observed. This value corresponds to concentrations observed for most vertebrate and invertebrate species. The bioavailability of PAHs is determined by their solubility; only the soluble fraction of PAHs is accumulated by the embryos. The bioavailability of metals for the larvae is substantiated by MT induction, correlated with cytosolic metal concentrations. MT induction provided a better early-warning response than the embryotoxicity test currently used for evaluating environmental contamination by metals. This study recommends choosing oyster embryos as a particularly sensitive tool for evaluating sediment quality.

 

Geret, F., A. Serafim, et al. (2002). "Response of antioxidant systems to copper in the gills of the clam Ruditapes decussatus." Mar Environ Res 54(3-5): 413-7.

                Copper (Cu) is an essential element for biological systems, however, when present in excess, is toxic. Metallothioneins can play an important role in Cu homeostasis and detoxification. Moreover, Cu can catalyse the production of toxic hydroxyl radicals that cause lipid peroxidation but defence systems in the cells can limit the oxidative damage. The present study was performed to investigate the effect of three Cu concentrations (0.5, 2.5 and 25 microg l(-1)) on the response of antioxidant enzyme activities (superoxide dismutase (SOD), catalase (CAT), selenium-dependent glutathion peroxidase and total glutathion peroxidase), total proteins, metallothioneins (MT), malondialdehyde (MDA) concentrations in the gills of the clam, Ruditapes decussatus. The activity of antioxidant enzymes and total proteins, MT and MDA concentrations were measured in the gills of the clams after 1, 3, 7, 14, 21 and 28 days of Cu exposure. Results indicate that Cu only induces an imbalance in the oxygen metabolism during the first week of Cu exposure due to a decrease in mitochondrial SOD and CAT, selenium-dependent and total glutathion peroxidase activities. Cu also causes lipid peroxidation, measured by the MDA formation, that was Cu dependent. In the gills of clams exposed to 25 microg Cu/l, the excess of Cu triggers the induction of MT synthesis after 3 days of exposure.

 

Mason, A. Z. and M. R. Borja (2002). "A study of Cu turnover in proteins of the visceral complex of Littorina littorea by stable isotopic analysis using coupled HPLC-ICP-MS." Mar Environ Res 54(3-5): 351-5.

                A two-dimensional HPLC system, tandemly coupled to an ICP-MS, has been used to study copper accumulation and turnover in the visceral complex cytosol of the gastropod, Littorina littorea. Animals were exposed for 8 weeks to NTA-buffered seawater containing stable isotopic 65Cu and then transferred to media containing stable isotopic 63Cu. The free ion activity of each isotope was maintained at 10(-11) M. Size exclusion (SE) HPLC showed Cu associated with haemocyanin (HC) and metallothionein-like (MT) proteins in two ligand pools with apparent molecular weights of >300 kDa and approximately 17 kDa, respectively. The MT pool was inducible by Cu, could assimilate the metal from both intrinsic and extrinsic sources and showed a higher rate of Cu accumulation and turnover than the HC pool. The induction of this pool also caused the sequestration and cytosolic redistribution of Zn, Cd, Pb, Mn and Co. Further fractionation of the MT pool by ion-exchange (IE) HPLC revealed that the Cu was associated with a single, major isoform of the protein that was Cu inducible and also bound trace quantities of Zn and Pb. A number of additional metal containing proteins were also resolved by IE. the most prominent of which also bound Pb, Mn and minor quantities of Zn. The significance of these findings in metal homeostasis and detoxification is discussed.

 

Moraga, D., E. Mdelgi-Lasram, et al. (2002). "Genetic responses to metal contamination in two clams: Ruditapes decussatus and Ruditapes philippinarum." Mar Environ Res 54(3-5): 521-5.

                Coastal ecosystems are subjected to a wide variety of disturbances, including those due to xenobiotics of agricultural and industrial origin. These pollutants as heavy metals can modify the genetic diversity of populations by favouring or counter-selecting certain alleles or genotypes by differential mortality. In the present study, two genetic markers (phosphoglucomutase and glucosephosphate isomerase) and a protein marker (metallothionein) were monitored in order to determine the impact of heavy metals in different clam populations. Analysis of the genetic structure of the clam populations examined reveals that those inhabiting environments contaminated by heavy metals exhibit a higher allelic diversity and possess alleles at PGM loci that could be selected by the presence of heavy metals. The evaluation of metallothionein levels using a specific polyclonal antibody developed in the Pacific oyster (Crassostrea gigas) demonstrated the existence of a relationship between metallothionein concentrations and the level of metal pollution for clam populations sampled from different sites. An inter-specific difference was also detected between Ruditapes decussatus and Ruditapes philippinarum living in sympatry at the same site, suggesting a differential response of these two species upon exposure to an identical heavy metal concentration.

 

Serafim, M. A., R. M. Company, et al. (2002). "Effect of temperature and size on metallothionein synthesis in the gill of Mytilus galloprovincialis exposed to cadmium." Mar Environ Res 54(3-5): 361-5.

                Although the degree of metal contamination is expected to be a primary determinant of metallothionein (MT) induction in marine mussels, at least at polluted sites, variability caused by temperature, and biotic factors such as size, may need to be considered when interpreting field data. To test the effects of these variables, mussels, Mytilus galloprovincialis, of different sizes (small: 3.5 +/- 0.5 cm and large: 5.2 +/- 0.7 cm) were exposed to Cd (100 microg l(-1)) at different water temperatures (5, 18 and 25 degrees C) for 34 days. Resultant Cd and MT concentrations in gills were shown to be size dependent and increased significantly with temperature. At the highest temperature tested (25 degrees C) there appears to be a disproportionate effect on Cd accumulation, which raises MT synthesis to exceptionally high levels. The effect of size on MT concentrations was also temperature-dependent: at 18 and 25 degrees C, large mussels exhibited higher MT levels than smaller individuals, whilst at 5 degrees C there were no significant differences between contaminated and control mussels, in either size-group, as a result of the reduced level of MT production at this temperature. When considering the use of MT levels in mussels as a biochemical indicator of metal contamination, the potential influence of factors such as size and temperature on MT-metal relationships needs to be considered. Samples should be of uniform size as far as possible, and collection should be limited to a fixed season (avoiding climatic extremes) to ensure that the effects of these factors on baseline levels of MT is minimised.

 

Zaroogian, G. and C. Norwood (2002). "Glutathione and metallothionein status in an acute response by Mercenaria mercenaria brown cells to copper in vivo." Ecotoxicol Environ Saf 53(2): 285-92.

                Red glands of Mercenaria mercenaria comprise brown cells that accumulate, detoxify, and excrete copper. Brown cell involvement in metal detoxification is due in part to endogenous glutathione (GSH) and metallothionein (MT). The intent of this study was to test the hypotheses that brown cell GSH functions in protection against Cu2+ toxicity, that brown cell GSH provides the initial defense against Cu2+ prior to brown cell MT induction, and that MT variants (MTI, MTII), if present are unequal in response to Cu2+. Brown cells were analyzed for GSH and MT after 0.25, 1, 2, 3, and 4 days of treatment of Mercenaria with 0.01 and 0.05 ppm Cu2+. Glutathione initiated the brown cell acute response (within the first day of treatment) to both the 0.01 and 0.05 ppm Cu2+ treatments. Metallothionein in brown cells increased to Day 4 during treatment with 0.01 ppm Cu2+, whereas MT concentration was greatest at Day 2 after which it decreased to Day 4 with treatment of 0.05 ppm Cu2+. The change in MTII relative to its control was greater than that of MTI in the brown cell acute response to 0.01 ppm Cu2+ and also for Days 0.25 to 2 in response to the 0.05 ppm Cu2+ treatment. At Days 3 and 4 with the 0.05 ppm Cu2+ the change in MTI/MTII ratio was due to a greater change in MTI than MTII relative to their respective controls. The variants of brown cell MT appear to respond differently to Cu2+ depending on the Cu2+ treatment concentration.

 

Mourgaud, Y., E. Martinez, et al. (2002). "Metallothionein concentration in the mussel Mytilus galloprovincialis as a biomarker of response to metal contamination: validation in the field." Biomarkers 7(6): 479-90.

                Mussels were translocated from a shell-fish breeding area (Sete, on the French Mediterranean coast) to sites exposed to trace element inputs in April 2000. They were recovered 3 months later. Whole soft tissues from all of the sites (n = 97) were analysed for arsenic, cadmium, chromium, copper, mercury, nickel, lead and zinc. Metallothioneins (MTs) were also measured in the digestive gland and in the remaining tissues (allowing calculation of whole soft tissue concentrations) at 22 of the 97 sites. MT concentrations in the digestive gland and the whole soft tissues were strongly correlated. The condition index varied with food availability at different sites. This did not influenced MT concentrations in the whole soft tissues, whereas the condition index was negatively correlated to trace element concentrations. A model is proposed to minimize this influence of condition. Metal concentrations adjusted using this model showed significant correlations with MT levels for those metals (cadmium, copper, nickel and zinc) that are known to bind to this protein, with the exception of mercury. Even in moderately contaminated sites, measurement of the MT level in the soft tissues of mussels was generally able to discriminate between different levels of contamination, allowing the use of a simplified procedure compared with dissection of the digestive gland. It is recommended to avoid translocation and sampling during the reproductive period, which is well documented for commercial species such as Mytilus sp.

 

Tanguy, A., I. Boutet, et al. (2002). "Polymorphism of metallothionein genes in the Pacific oyster Crassostrea gigas as a biomarker of response to metal exposure." Biomarkers 7(6): 439-50.

                Quantification of metallothioneins (MTs) is classically associated with a cellular response to heavy metal contamination and is used in the monitoring of disturbed ecosystems. Despite the characterization of several MT genes in marine bivalves, only a few genetic studies have used MT genes as potential biomarkers of pollution. The aim of this study was to assess whether MT gene polymorphism could be used to monitor exposure of the Pacific oyster Crassostrea gigas to heavy metals and to develop specific genetic markers for population genetic studies in relation to environmental stress. The polymorphism of two exons of the C. gigas MT gene CgMT1 were studied using polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) in both field populations exposed to various metals concentrations and in experimentally exposed populations. High frequencies of two SSCP types in exons 2 and 3 of the CgMT1 gene have found to be significantly associated with tolerance to metals in experimental and field oyster populations. The use of MT1 gene polymorphism in C. gigas as in the present study should therefore be of high ecological relevance. In conclusion, the analysis of the types in these two CgMT1 gene exons, which can confer a greater tolerance to heavy metals, can constitute a good biomarker of effect of the presence of heavy metals in ecosystems.

 

Geret, F., A. Serafim, et al. (2002). "Effect of cadmium on antioxidant enzyme activities and lipid peroxidation in the gills of the clam Ruditapes decussatus." Biomarkers 7(3): 242-56.

                Metals are known to influence the oxidative status of marine organisms, and antioxidant enzymes have been often proposed as biomarkers of effect. The clam Ruditapes decussatus is a well-known metal bioindicator. In this species cadmium (Cd) induces metallothionein (MT) synthesis only after 7 days of exposure. Before MT synthesis is induced, the other mechanisms capable of handling the excess of Cd are unknown. In order to identify some of these mechanisms, variations in antioxidant systems (superoxide dismutase, catalase, selenium-dependent glutathione peroxidase and non-selenium-dependent glutathione peroxidase), malondialdehyde (MDA) and MT were studied in the gills of R. decussatus exposed to different Cd concentrations (4, 40 and 100 gl-1) for 28 days. These parameters, together with total proteins and Cd concentrations, were measured in the gills of the clams over different periods of exposure. Results indicate that Cd accumulation increased linearly in the gills of R. decussatus with the increase in Cd concentration. This increase induces an imbalance in the oxygen metabolism during the first days of Cd exposure. An increase in cytosolic superoxide dismutase (SOD) activity and a decrease in mitochondrial SOD activity was observed at the same time as or after a decrease in cytosolic and mitochondrial catalase activity and of selenium-dependent and non-selenium-dependent glutathione peroxidase activity. After 14 days of exposure, Cd no longer affect these enzymes but there was elevation of other cellular activities, such as MDA and MT production. MT bound excess Cd present in the cell. These variations in these parameters suggest their potential use as biomarkers of effects such as oxidative stress resulting from Cd contamination in molluscs.

 

Boutet, I., A. Tanguy, et al. (2002). "Immunochemical quantification of metallothioneins in marine mollusks: characterization of a metal exposure bioindicator." Environ Toxicol Chem 21(5): 1009-14.

                A recombinant metallothionein CgMT1, from the Pacific oyster Crassostrea gigas, was synthesized and used as antigen in the development of antibodies and a specific enzyme-linked immunosorbent assay (ELISA). The ELISA showed that rabbit anti-CgMT1 IgG reacted with purified CgMT1 and MTs from other marine bivalves, indicating its suitability as a reagent to quantify MTs and for monitoring of metal contamination in field animals. Application of this assay to tissues excised from metal exposed C. gigas, showed that MT induction reached a saturation level in gills that was not observed in digestive gland. Quantification of MTs in gills and digestive gland of field-collected C. gigas showed that the MT level depended on the metal concentrations at the collection sites and may have been influenced by salinity variations at estuarine sites. Oysters from metal-contaminated estuarine sites showed lower MT concentrations than those from nonestuarine contaminated sites.

 

Geffard, A., J. C. Amiard, et al. (2002). "Use of metallothionein in gills from oysters (Crassostrea gigas) as a biomarker: seasonal and intersite fluctuations." Biomarkers 7(2): 123-37.

                The use of oyster gills for the analytical determination of metallothionein (MT) concentration as a biomarker of metal exposure was investigated. Temporal variations in MT and metal concentrations (which can interfere with inter-site differences) were examined over a 7 month period (from spring to autumn) in Japanese oysters from a clean site (Bay of Bourgneuf, France) and a metal-rich site (Gironde estuary, France) as well as in individuals translocated from the clean to the contaminated area. The ratio between the annual average of MT concentrations in specimens from the clean and the metal-rich sites was 1.3. During the last 3 months of the experiment, significant differences were no longer registered between transplants and residents from the Gironde estuary. Metals concentrations in oyster gills differed consistently between the clean and the metal-rich sites (annual average ratios of 1.5, 2.7 and 9.8, respectively, for zinc, copper and cadmium) and a fast increase in metal concentrations (over a few months) was observed in transplants, mainly for cadmium. MT and soluble metal concentrations were found to be positively and significantly correlated over the period of the study. This relationship is a positive argument for a possible use of gill MT concentration as a biomarker of metal pollution in contrast to previous findings on the digestive gland, there being a smaller amount of seasonal variability in the weight of oyster gills.

 

Desouky, M., R. Jugdaohsingh, et al. (2002). "Aluminum-dependent regulation of intracellular silicon in the aquatic invertebrate Lymnaea stagnalis." Proc Natl Acad Sci U S A 99(6): 3394-9.

                Silicon is essential for some plants, diatoms, and sponges but, in higher animals, its endogenous regulation has not been demonstrated. Silicate ions may be natural ligands for aluminum and here we show that, in the freshwater snail (Lymnaea stagnalis), intracellular silicon seems specifically up-regulated in response to sublethal aluminum exposure. X-ray microanalysis showed that exposure of snails to low levels of aluminum led to its accumulation in lysosomal granules, accompanied by marked up-regulation of silicon. Increased lysosomal levels of silicon were a specific response to aluminum because cadmium and zinc had no such effect. Furthermore, intra-lysosomal sulfur from metallothionein and other sulfur-containing ligands was increased after exposure to cadmium and zinc but not aluminum. To ensure that these findings indicated a specific in vivo response, and not ex vivo formation of hydroxy-aluminosilicates (HAS) from added aluminum (555 microg/liter) and water-borne silicon (43 microg/liter), two further studies were undertaken. In a ligand competition assay the lability of aluminum (527 microg/liter) was completely unaffected by the presence of silicon (46 microg/liter), suggesting the absence of HAS. In addition, exogenous silicon (6.5 mg/liter), added to the water column to promote formation of HAS, caused a decrease in lysosomal aluminum accumulation, showing that uptake of HAS would not explain the loading of aluminum into lysosomal granules. These findings, and arguments on the stability, lability, and kinetics of aluminum-silicate interactions, suggest that a silicon-specific mechanism exists for the in vivo detoxification of aluminum, which provides regulatory evidence of silicon in a multicellular organism.

 

Blackmore, G. and W. X. Wang (2002). "Uptake and efflux of Cd and Zn by the green mussel Perna viridis after metal preexposure." Environ Sci Technol 36(5): 989-95.

                Cadmium and zinc uptake from the dissolved phase, assimilation efficiency from the dietary phase, efflux rate constants, and body burden as well as clearance rate were measured in the green mussel Perna viridis with or without laboratory preexposure to Cd or Zn. Efflux rate constants and clearance rates were little affected by preexposure to either Cd or Zn. In contrast, the assimilation of Cd increased by 1.2-1.6 in mussels preexposed to Cd (subsequent Cd concentrations 10.2-25.9 microg(-1)) as compared to controls (0.19-0.39 microg g(-1)). This increase corresponded to an elevation in the proportion of Cd associated with the metallothionein-like proteins (MTLPs) in the mussels, suggesting that exposure to Cd and subsequent induction of MTLPs affected Cd accumulation. Exposure to Zn only resulted in elevated body concentrations following 7-d exposure to 250 microg L(-1), although Zn and Cd uptake from the dissolved phase were reduced by 24-47% by exposure to a lower concentration (100 microg L(-1)) for 7 and 21 d. Despite the lack of an increase in body Zn concentration, the subcellular distribution was altered such that the proportion of Zn associated with the metal-rich granules increased. This study indicates the importance of the subcellular distribution of metals in affecting the biokinetics and thus the toxic effects of metals on aquatic animals. Cd preexposure has potential effects on its influx from the dietary phase, e.g., increasing the importance of dietary uptake and further increasing the body burdens. In contrast, preexposure to Zn has a negative effect on Cd and Zn influx from the dissolved phase, suggesting the mechanism of Zn regulation but also potentially reducing Cd uptake and body concentrations over the long-term exposure. Such effects may have implications for biomonitoring studies involving a single species that modifies physiological processes affecting metal uptake (and hence bioavailability). Caution is needed in extrapolating data to species not capable of making such changes, particularly for Cd, which is not regulated and for which the effects of an elevated body burden are most obvious.

 

Ceratto, N., F. Dondero, et al. (2002). "Cloning and sequencing of a novel metallothionein gene in Mytilus galloprovincialis Lam." Comp Biochem Physiol C Toxicol Pharmacol 131(3): 217-22.

                Metallothionein (MT) is a ubiquitous, metal-inducible protein with an important role in the homeostasis and in the detoxification of heavy metals. This work reports the cloning and sequencing of a MT gene encoding a MT isoform (MT20-IIIa) in the mussel Mytilus galloprovincialis Lam, a lamellibranch mollusc known to accumulate and to detoxify large amounts of metal. The MT gene, lacking the 5' promoter region, is 1865 bp long and has a tripartite structure consisting of three exons and two introns. The putative open reading frame (ORF) encodes a polypeptide of 72 amino acids, which corresponds to the MT-I class, type 2 family (http://www.unizh.ch/~mtpage/classif.html). The structure of the gene and the putative MT20-III protein have been compared with those of other species. The putative biological significance of the differences at the amino acid level among the different MTs is discussed.

 

Rodriguez-Ortega, M. J., J. Alhama, et al. (2002). "Biochemical biomarkers of pollution in the clam Chamaelea gallina from south-Spanish littoral." Environ Toxicol Chem 21(3): 542-9.

                Fourteen biochemical pollution biomarkers were analyzed in the clam Chamaelea gallina sampled at seven South-Spanish littoral sites at different times in 1999. They included enzymes that regenerate reduced nicotinamide adenine dinucleotide phosphate (NADPH) or maintain the cytosol-reduced (glucose-6-P- and isocitrate dehydrogenases, glutathione reductase), that decompose reactive oxygen species (catalase, superoxide dismutase, glutathione peroxidase), or detoxify glutathione-reactive electrophiles (soluble and microsomal glutathione transferases, glyoxalases II and I). The levels of reduced glutathione, malondialdehyde, and metallothionein and the glutathione redox status were also determined. Clams from Huelva sites with higher contaminant loads and metallothionein levels showed higher antioxidant and glutathione-related activities and a decreased glutathione level, lower malondialdehyde content, and a less oxidized glutathione status. This suggests that clams chronically exposed to contaminants released by Huelva and Guadalquivir Estuaries are better protected from oxidative stress than reference animals. Most biomarkers showed six-month cyclicity with up to threefold amplitudes, further supporting the inverse relationship between antioxidant defenses and oxidative damages. Thus the lower antioxidant defenses in June fit to maximum oxidative damages, whereas the high antioxidant defenses found in March and September through October agree with lower lipid damages and less oxidized glutathione status. The effect of environmental or endogenous factors on this cyclic response is discussed.

 

Soto, M., B. Zaldibar, et al. (2002). "Subcellular distribution of cadmium and its cellular ligands in mussel digestive gland cells as revealed by combined autometallography and X-ray microprobe analysis." Histochem J 34(6-7): 273-80.

                Autometallography (AMG) and electron probe X-ray microanalysis (EPXMA) were applied in combination to determine the subcellular distribution of Cd and its subcellular ligands in the digestive gland cells of Cd-exposed mussels Mytilus galloprovincialis. Black silver deposits (BSD), which reveal the presence of metals when AMG is applied, were only localized in digestive cell lysosomes. Digestive cell cytoplasm and basophilic cells were devoid of BSD. EPXMA (static probe and X-ray mapping) indicated that Cd, S (possibly associated with metallothioneins or metallothionein-like proteins) and autometallographical Ag ions are co-localized within digestive cell lysosomes. In addition, Cd and S co-occur in the absence of Ag in the cytosol of digestive cells. AMG does not reveal the presence of the Cd 'pool' strongly bound to cytosolic Cd-metallothionein complexes; only 'free' Cd or Cd supposedly loosely bound to (semi)digested metallothionein within lysosomes was revealed. The levels of lysosomal Cd were indirectly quantified by stereology as the volume density of BSD (V(v)BSD). Significantly higher values were recorded in Cd-exposed mussels compared with controls at all exposure times. However, V(v)BSD values were lower at days 7 and 21 than at day 1. This relative decrease in V(v)BSD reflected another (and confounding) response elicited by Cd-exposure in the digestive epithelium: the volume density of basophilic cells (V(v)BAS) increased significantly as exposure progressed. Due to this cell-type replacement, the net accumulative capacity of the digestive epithelium decreases at long exposure times.

 

Geret, F. and R. P. Cosson (2002). "Induction of specific isoforms of metallothionein in mussel tissues after exposure to cadmium or mercury." Arch Environ Contam Toxicol 42(1): 36-42.

                The synthesis of metallothioneins has been established for Mytilus edulis exposed to cadmium and mercury. We checked if this induction resulted in the synthesis of tissue- or metal-specific isoforms in the gills, the mantle, and the digestive gland that could be used as tool for the characterization of undefined metallic contamination of aquatic ecosystems. An accumulation of metals was observed in the selected organs after 21 days of exposure. The levels of metallothioneins measured by using the polarographic method were significantly increased by cadmium and mercury in the gills (21 days). Size exclusion chromatography showed the presence of a monomer and a dimer of metallothionein of respective apparent molecular weight about 12 kDa and 20 kDa in all samples. They were resolved into five components by anion exchange chromatography in the gills of control or Hg-treated mussels, whereas a sixth isoform was isolated in the gills of cadmium-exposed mussels. In the mantle of mussels exposed or not, five isoforms were separated, and in the digestive gland of mussels exposed or not, six isoforms were separated. The occurrence of a specific cadmium-binding isoform in the gills has to be confirmed in cadmium-contaminated specimens collected in situ before its detection may be used as biomarker of cadmium contamination.

 

Chan, M. K., R. Othman, et al. (2002). "Induction of a putative metallothionein gene in the blood cockle, Anadara granosa, exposed to cadmium." Comp Biochem Physiol C Toxicol Pharmacol 131(2): 123-32.

                The relationship between a putative metallothionein gene (MT) and exposure to cadmium (Cd) in blood cockles (Anadara granosa) is reported. In a 96-h dose-response experiment, mortality of cockles was found to proportionately increase in the range of 0.2-5.0 mg/l Cd with a calculated LC(50) of 2.94 mg/l. Exposure to 0.25 mg/l Cd for 16 days caused significant increases (P<0.05) in Cd concentrations in whole tissues, gills and hepatopancreas, and the accumulation of Cd in these tissues increased with the duration of exposure. Two cDNA libraries constructed using the hepatopancreas from control and Cd-treated cockles gave titres of 5.62 x 10(5) and 1.94 x 10(5) pfu/microg vector, respectively. A putative MT gene, AnaMT, of 510 nucleotides in length, was isolated from the treated cDNA library using a heterologous probe MT20 from the blue mussel, Mytilus edulis. Northern analyses using AnaMT as a probe indicated low expression of the MT mRNA in control animals. In cockles treated with 0.25 mg/l Cd for 4 days, MT mRNA level increased to approximately 168%, but declined to 108% at day 8. After 12 and 16 days of Cd treatment, expression of the MT gene was 138% and 187%, respectively, compared to the controls. These observations suggest that induction of the MT gene by a sublethal dose of Cd is rapid, occurring within 4 days of treatment.

 

Bustamante, P., R. P. Cosson, et al. (2002). "Cadmium detoxification processes in the digestive gland of cephalopods in relation to accumulated cadmium concentrations." Mar Environ Res 53(3): 227-41.

                The high concentrations of cadmium recorded in the digestive gland of cephalopods from various temperate and subpolar waters suggest that these molluscs have developed efficient cadmium detoxification mechanisms. The subcellular distribution of cadmium in the digestive gland cells was investigated in seven cephalopod species from the Bay of Biscay (France) and the Faroe Islands. In most species, cadmium was mainly found in the cytosolic fraction of the digestive gland cells, reaching up to 86% of the total cadmium for the squid Loligo vulgaris from the Bay of Biscay. But species with the highest total level of cadmium showed a higher percentage of cadmium associated to insoluble compounds. The quantification of metallothioneins (MTs) by the polarographic method was performed in order to evaluate the involvement of these proteins in the detoxification of the high amounts of bioaccumulated cadmium. Metallothionein levels in cephalopods ranged form 742 +/- 270 to 3478 +/- 1572 microg/g wet weight. No relationship could be established between total cadmium, cytosolic cadmium and MT levels suggesting the occurrence of other Cd-binding ligands. Although these proteins have not been characterised, as cadmium in the digestive gland of cephalopods is mainly associated with soluble ligands, a high potential transfer to predators can be predicted.

 

Cavaletto, M., A. Ghezzi, et al. (2002). "Effect of hydrogen peroxide on antioxidant enzymes and metallothionein level in the digestive gland of Mytilus galloprovincialis. cavalett@unipmn.it." Comp Biochem Physiol C Toxicol Pharmacol 131(4): 447-55.

                The pro-oxidant effect of H2O2 at a concentration of 20 microM was examined in the digestive gland of Mytilus galloprovincialis, a bivalve mollusc frequently used in biomonitoring programs. The oxidative stress caused by H2O2 has been evaluated in terms of lipid peroxidation and lysosomal system alteration. Complex cellular antioxidant defence mechanisms of the mussel were investigated at the enzymatic and non-enzymatic level in order to explain their relative role in reducing the risk of oxidative injury. Metallothionein, glutathione, superoxide dismutase, catalase and glutathione peroxidase were assayed after 1, 4 and 7 days of exposure to H2O2. The metallothionein content showed an increase by 43% after 4 days of exposure, followed by a decrease back to control values at 7 days. Antioxidant enzyme activities followed a similar pattern with a moderate increase after 1 or 4 days of treatment and a return to control values at 7 days. All data indicate a 'transient' oxidative stress response, after which mussel cells restore the redox balance.

 

Gagne, F., C. Blaise, et al. (2002). "Alteration of the biochemical properties of female gonads and vitellins in the clam Mya arenaria at contaminated sites in the Saguenay Fjord." Mar Environ Res 53(3): 295-310.

                Vitellins (Vn) are the major egg yolk proteins that constitute an important energy reserve for mollusc embryos. The purpose of the present study was to examine whether the relative levels of sugars, lipids, phosphates, and labile IIb metals and calcium normally associated with Vn would differ in clam populations living at contaminated sites. Softshell clams (Mya arenaria) were collected at three sites in the area of the Saguenay Fjord: a marina, a municipal sewer outfall zone, and a reference site. The condition factor (weight:length ratio), metallothionein-like proteins, cytochrome P450 1A1 activity and DNA damage were all determined in the clam's digestive gland. Levels of total sugars, lipids, alkali-labile phosphates, proteins, and labile zinc and calcium were determined in female gonad homogenates and in purified Vn. The results show that clam gonads at the contaminated site by a marina displayed a lower index of gonad activity than the reference site. In addition, the condition factor was significantly lower at the marina site as compared to the reference site, with a concomitant increase in DNA damage and metallothionein (MT) induction. In fact, the condition factor was significantly correlated with DNA damage (R = -0.413, P = 0.045) and MT levels (R = -0.622, P = 0.03). Homogenates of female gonads were found to contain higher levels of labile IIb metals and calcium, with lower lipid content at the marina site compared to the reference site, and much higher levels of alkali-labile phosphates (ALP) and calcium at the municipal outfall site. Vn from the marina site were significantly higher in labile IIb metals but lipid content appeared to be somewhat lower than at the reference site. Vn from the municipal site were found to be highly phosphorylated, with higher levels of lipids and calcium. These results suggest that the chemical composition of the gonads and Vn are altered in contaminated sites.

 

Park, J. S., S. Chung, et al. (2002). "Purification and characterization of metallothionein-like cadmium binding protein from Asian periwinkle Littorina brevicula." Comp Biochem Physiol C Toxicol Pharmacol 131(4): 425-31.

                Although mussels and oysters in the ocean are known to act as bioconcentrators for contaminants such as heavy metals, their ability to survive in heavily polluted water is relatively limited. The Asian periwinkle, Littorina brevicula, is one species that can accumulate a variety of environmental heavy metals, and the expression of its metal binding protein (MBP) is induced by cadmium. To better characterize this protein and its detoxification mechanism against cadmium, the present work examined the induction of a cadmium binding protein (Cd-BP) in Littorina brevicula exposed to 400 microg/l CdCl(2) for 30 days. The induced Cd-BP was purified by chromatography from the supernatants of homogenized organs (digestive gland, gonad, gill and kidney). This Cd-BP was found to consist of 103 amino acids, was rich in Cys (21 residues), and partial C-terminal sequence obtained by MALDI-TOF MS analysis revealed a Cys-XXX-Cys motif, which resembles a typical feature of mollusc metallothionein (MT). The Cd-BP molecular weight of 9.8 kDa is a little larger than that of other MTs.

 

Blaise, C., F. Gagne, et al. (2002). "Molluscan shellfish biomarker study of the Quebec, Canada, Saguenay Fjord with the soft-shell clam, Mya arenaria." Environ Toxicol 17(3): 170-86.

                A spatial and temporal survey of six sites in the Saguenay Fjord and of one adjacent site in the St. Lawrence River estuary (Quebec, Canada) was undertaken to study the possible effects of anthropogenic contaminant input on soft-shell clam (Mya arenaria) populations. Bivalve sampling sites were selected because they reflected a range of areas representative of either no known (or apparent) pollution sources or of areas potentially influenced by different gradients and types of contamination sources. The most upstream site selected in the Saguenay Fjord, nearest to a highly populated and industrialized sector, and the most downstream site, near its mouth with the St. Lawrence River estuary, spanned a distance of some 70 km and encompassed the entire intertidal area suitable for Mya arenaria habitat. To measure effects in collected animals, we used a comprehensive battery of biomarkers composed of metallothionein-like proteins (MT), 7-ethoxyresorufin O-deethylase activity (EROD), DNA damage (DD), lipid peroxidation (LPO), vitellinlike proteins (Vn), phagocytosis (PHAG), nonspecific esterase (NspE) activity, and condition factor (weight-to-length ratio of clams). Vn, PHAG, DD, and NspE biomarkers were assayed in hemolymph (or hemocytes), whereas others (MT, EROD, LPO) were determined in the digestive gland. Whole-tissue metal content was also quantified in clams collected in the spatial survey. The spatial survey conducted in June 1997 showed significant effects at all sites, and principal component analysis indicated in addition that the more important responses were linked to the MT, LPO, and NspE biomarkers. Clams collected from sites closest to the upstream reaches of the fjord generally displayed higher levels of tissue metals (cadmium, manganese), as well as greater responses of NspE activity, MT, LPO, and PHAG. Animals collected from sites influenced by municipal wastewaters had higher levels of Vn, suggesting the presence of environmental estrogens. The results of the temporal survey (six monthly samplings of clams at three sites from May through October, 1997) showed that the bivalve reproductive cycle (vitellogenesis and spawning) can modulate the expression of several biomarkers. Vn levels, for example, were positively correlated with DD and EROD and negatively correlated with MT, suggesting that reproduction can influence the susceptibility of clams to some contaminants. Discrimination analysis over the 6 months of sampling revealed that the mean value of the discriminant function changed significantly over time, suggesting important changes in the relative contribution of each biomarker. In short, this study has provided evidence that clam populations in the Saguenay Fjord are impacted by multiple sources of contamination whose effects can be modulated by reproduction.

 

Gagne, F., C. Blaise, et al. (2002). "Biomarker study of a municipal effluent dispersion plume in two species of freshwater mussels." Environ Toxicol 17(3): 149-59.

                The toxicological effects of a primary-treated municipal effluent plume were investigated in two species of freshwater mussels, Elliptio complanata and Dreissena polymorpha, exposed for 62 days at sites upstream and downstream of an effluent outfall in the St. Lawrence River (Quebec, Canada). Levels of metallothioneins (MT), cytochrome P4501A1 activity, DNA damage, total lipids, relative levels of vitellins, and phagocytic activity (in E. complanata hemocytes) were determined after the exposure period. A parallel analysis measured heavy metals and coprostanol in mussel tissues. The results show that significant levels of coprostanol and some metals (specifically, Cu, Hg, Sb, Se, and Zn) had accumulated in mussels caged 5 km downstream of the effluent plume. Mixed-function oxidase activity, MT in gills, total lipids, DNA damage (in D. polymorpha only), and total hemolymph bacteria (in E. complanata only) had increased in these mussels, while levels of total cadmium (Cd), MT in digestive glands or whole soft tissues, phagocytic activity, and DNA damage in the digestive gland (in E. complanata only) were diminished. The exposure of mussels to surface waters contaminated by a municipal effluent led to many stress responses, depending on both the tissues and the species being examined.

 

Perceval, O., B. Pinel-Alloul, et al. (2002). "Cadmium accumulation and metallothionein synthesis in freshwater bivalves (Pyganodon grandis): relative influence of the metal exposure gradient versus limnological variability." Environ Pollut 118(1): 5-17.

                The relative influence of limnological confounding factors on cadmium (Cd) bioaccumulation and metallothionein (MT) synthesis was quantified in natural populations of freshwater bivalves (Pyganodon grandis) living in lakes along a Cd concentration gradient. During the ice-free period, we measured 15 environmental variables in the water compartment and determined total concentrations of Cd and MT in the gills of bivalves at 37 littoral stations in 20 lakes distributed across the mining area of Rouyn-Noranda in northwestern Quebec. A multiple linear regression model including pH (+), dissolved Ca concentrations (-) and free Cd2+ concentrations at the sediment-water interface (+) explained 74% of the variability in Cd concentrations in the bivalve gills. Dissolved Ca (-) and free Cd2+ (+) together explained 62% of the variation in MT concentrations in the bivalve gills. Partial linear regression analyses indicated that the limnological factors' pure and shared effects together accounted for 48 and 45% of the total variation in Cd and MT concentrations in the gills, respectively. A lake selection procedure that could be applied in monitoring programs is proposed to minimise the relative influence of these confounding variables.

 

Cotou, E., C. Vagias, et al. (2001). "Metallothionein levels in the bivalves Callista chione and Venus verrucosa from two Mediterranean sites." Z Naturforsch C 56(9-10): 848-52.

                Metallothioneins levels (MTs) in the clams Callista chione and Venus verrucosa, collected from two coastal sites in Greece, were determined and quantified by SDS polyacrylamide gel electrophoresis (SDS-PAGE) and a spectrophotometric assay (Ellman's reaction). SDS-PAGE separation in the digestive gland, which represents the hepato-pancreas in clams, demonstrated the presence of MTs similar to mammalian MT (rabbit liver Cd, Zn-thionein). No other SH-containing proteins apart from the MTs were detected. MT levels quantified by the Ellman's reaction indicated seasonal variation for both species. The highest values were recorded in the spring and the lowest in the autumn. The seasonal variation and the differences in the MT levels of the two areas seem to be related to the reproductive cycle of the organisms as well as to abiotic factors of each area. Our results show that both C. chione and V. verrucosa have the potential to be used as biomarkers of metal pollution, provided that the influence of the external factors is safely quantified.

 

Tanguy, A., C. Mura, et al. (2001). "Cloning of a metallothionein gene and characterization of two other cDNA sequences in the Pacific oyster Crassostrea gigas (CgMT1)." Aquat Toxicol 55(1-2): 35-47.

                Metallothionein (MT) genes encode essential metal-binding proteins involved in metallic homeostasis and detoxification in living organisms. Here, we describe the structure of the first Pacific oyster Crassostrea gigas metallothionein (CgMT1) gene and the sequences of two other MT cDNA. The CgMT1 gene sequence contains three coding exons plus a 5' entirely non-coding exon, and the predicted protein contains 21 cysteine residues organized in Cys-X-Cys motifs as classically described for MTs. The three cDNA sequences present few substitutions in either coding sequence or UTRs. Induction of these MT-mRNA in heavy metal-treated oysters (i.e. cadmium) was confirmed by Northern blot analysis and RT-PCR and suggests a potential specific tissue expression rate. Southern blot analysis suggested the presence of multiple CgMT genes, and allowed the detection of restriction fragment length polymorphisms (RFLPs). Although the CgMT1 coding sequence showed 30-73% nucleotide identities with known sequences in other mollusks, it included the specific motif Cys-X-Cys-X(3)-Cys-Thr-Gly-X-X-X-Cys-X-Cys-X(5)-Cys-X-Cys-Lys found in Mollusk family 2. Marine bivalves are commonly used as pollution bioindicators, thus the development of genetic markers based on CgMT1 polymorphism will allow a monitoring of heavy metal exposure in anthropogenically disturbed ecosystems.

 

Serafim, M. A. and M. J. Bebianno (2001). "Variation of metallothionein and metal concentrations in the digestive gland of the clam Ruditapes decussatus: sex and seasonal effects." Environ Toxicol Chem 20(3): 544-52.

                Metallothionein (MT) and metal (Cd, Cu, and Zn) concentrations were determined in the digestive gland of male and female Ruditapes decussatus. Clams were collected monthly during the period of sexual development, from June to September, at two different sites of a coastal lagoon, the Ria Formosa Lagoon, on the south coast of Portugal. The MT concentrations were determined in the heat-treated cytosolic fraction of the digestive gland of both male and female clams. Total metal (Cd, Cu, and Zn) concentrations in the heat-treated cytosol of the digestive gland were also determined in both sexes. The MT and metal concentrations in the digestive gland were not sex dependent in this species. Therefore, these results suggest that random samples can be used to determine MT and metal concentrations in the digestive gland of R. decussatus. Seasonal and site-specific dependency, however, were detected for MT, Cd, and Cu concentrations. The maximum concentration levels of MT, Cd, and Cu were detected in July and August for both sexes in the same area. Zinc concentrations, however, did not show marked seasonal variations, indicating that this species is able to regulate zinc concentrations. The highest MT concentrations were significantly related to the highest metal concentrations.

 

Tanguy, A. and D. Moraga (2001). "Cloning and characterization of a gene coding for a novel metallothionein in the Pacific oyster Crassostrea gigas (CgMT2): a case of adaptive response to metal-induced stress?" Gene 273(1): 123-30.

                Cases of heavy metal resistance acquisition have already been demonstrated in eukaryotes, which involve metallothionein (MT) gene duplication or amplification mechanisms. We characterized in a marine bivalve, Crassostrea gigas, a gene coding for an unusual MT, which has never been described in other species. Our results illustrate a unique case of exon duplication and rearrangement in the MT gene family. The particular organization of the third exon of this gene allows the synthesis of a MT that presents a higher metal ion binding capacity compared to previously described MTs. The formation of a supplementary third structural beta-domain is proposed to explain results obtained in in vitro experiments. Differences in the metal responsive element (MRE) copy number and MRE core sequence observed in the promoter of CgMT2 also suggest differential regulation of CgMT2 transcription and possible implication in the detoxification processes.

 

Leung, K. M. and R. W. Furness (2001). "Metallothionein induction and condition index of dogwhelks Nucella lapillus (L.) exposed to cadmium and hydrogen peroxide." Chemosphere 44(3): 321-5.

                It has been suggested that metallothionein (MT) not only can regulate essential metals and detoxify toxic metals, but that MT can also play a significant role as an antioxidant and can be induced by oxidative stresses other than metals. This study is aimed at investigating the effect of hydrogen peroxide (H2O2), and the combined effect of H2O2 and cadmium (Cd) on MT induction and condition index (CI) in dogwhelks Nucella lapillus. Adult male dogwhelks (27 +/- 1 mm in shell length) were exposed for 20 days to (1) control (filtered natural seawater only); (2), 0.50 ppm Cd; (3) 2.0 ppm H2O2 + 0.50 ppm Cd; (4) 1.0 ppm H2O2 + 0.25 ppm Cd; (5) 2.0 ppm H2O2; (6) 1000 ppm H2O2 or (7) 1000 ppm H2O2 + 0.50 ppm Cd. The concentration of MT in the Leiblein gland of N. lapillus was quantified using the silver saturation method. MT or MT-like proteins in the animals were induced by Cd (0.5 ppm), H2O2 (2.0 ppm) or Cd + H2O2, indicating that MT in this gastropod species can be induced by either metal or oxidative stresses. Exposure to high H2O2 (1000 ppm) alone or combined with Cd, and exposure to Cd (0.50 ppm) or H2O2 (2.0 ppm), resulted in significant weight loss, indicated by a reduction of CI. However, CIs of groups (3) and (4) were similar to that of the control suggesting that Cd antagonistically reduces toxicity caused by H2O2 since Cd-induced MT may have a protective function against hydroxyl radicals.

 

Butler, R. A. and G. Roesijadi (2001). "Disruption of metallothionein expression with antisense oligonucleotides abolishes protection against cadmium cytotoxicity in molluscan hemocytes." Toxicol Sci 59(1): 101-7.

                The relationship between metallothionein (MT) induction and cytotoxicity was examined in isolated oyster hemocytes exposed in vitro to cadmium, copper, and zinc. In all cases MT induction increased to peak levels with increased metal dose, then declined with continued increases in dose. The effectiveness of these metals as inducers of MT was cadmium > copper > zinc, with cadmium the most effective inducer in magnitude of induction and sensitivity to dose. Cytotoxicity of the metals, based on decreases in lysosomal neutral red retention times, was copper > cadmium > zinc. Zinc was relatively nontoxic and a poor inducer of MT. Cadmium, which was intermediate in toxicity, required concentrations in excess of that causing peak induction of MT to have substantial effects on lysosomal membrane integrity. In contrast, copper was highly cytotoxic at concentrations below that which resulted in peak MT induction. This reversal in the rank order of cadmium and copper suggested that the toxicity of cadmium was tempered by the protective effects of MT. This was verified by disrupting MT expression with antisense phosphorothioate-substituted oligodeoxynucleotides. Administration of 2.5 microM CdCl(2), which induced MT but had no effect on lysosomal membrane stability when administered alone, reduced neutral red retention time to 41% of control levels when administered in the presence of antisense. The resulting toxicity was greater than that caused by a comparable concentration of copper. The findings represented evidence that expression of MT protects against cadmium toxicity in an estuarine mollusc.

 

Ettajani, H., B. Berthet, et al. (2001). "Determination of cadmium partitioning in microalgae and oysters: contribution to the assessment of trophic transfer." Arch Environ Contam Toxicol 40(2): 209-21.

                Alternative methodologies have been applied to the study of cadmium transfer in a food chain: water, microalgae (Skeletonema costatum and Tetraselmis suecica), oysters (Crassostrea gigas). The potential bioavailability of Cd in organisms was assessed through partitioning at the cell or tissue levels, and the predictive value of this method was evaluated by determining directly the metal transfer in an experimental food chain model. Cd concentrations were lower in S. costatum than T. suecica, in controls as well as in contaminated algae. In both algal species, Cd was firmly bound to the cell wall or had entered the cell. Cytosolic Cd was bound to intracellular ligands, the biochemical characteristics of which were not consistent with the hypothesis of detoxification via phytochelatins. In both algal species, Cd was predominantly present in the insoluble fraction, but at pHs such as those existing in the digestive tract of bivalves, it was easily extracted from the cells. Thus, exposure to Cd through phytoplanktonic food induced a significant uptake of this metal in soft tissues of bivalves. Due to the difference in Cd accumulation in algae, Cd doses associated with S. costatum were lower than those bound to T. suecica. Moreover, oysters retained a lower percentage of the metal associated with S. costatum compared to T. suecica (9 and 20%, respectively, after 21 days of exposure). Cd doses potentially available to oysters exposed directly in sea water were considerably higher, and direct uptake induced the highest levels of Cd incorporation but only 2% of dissolved Cd was actually retained by oysters over 21 days of exposure. In the soft tissues of oysters, Cd was distributed equally between soluble and insoluble fractions. Cytosolic Cd was present predominantly in the heat-stable fraction and mainly bound to compounds of molecular weight equal to 13.5 kDa. Moreover, a positive correlation was observed between metallothionein-like protein (MTLP) levels and gross concentrations of Cd in the soft tissues of oysters. These data are consistent with the hypothesis of an important role of metallothioneins in Cd metabolism in oysters and suggest a potential availability of MT-bound fraction of Cd to the consumers. These data are in agreement with the response of oysters exposed to Cd in the field.

 

Petrovic, S., B. Ozretic, et al. (2001). "Lysosomal membrane stability and metallothioneins in digestive gland of Mussels (Mytilus galloprovincialis Lam.) as biomarkers in a field study." Mar Pollut Bull 42(12): 1373-8.

                The lysosomal membrane destabilization and the metallothionein content in the digestive gland cells of mussels (Mytilus galloprovincialis Lam.), collected along the east coast of the North Adriatic (Istrian and Kvarnerine coast, Croatia), were examined over a period of four years (1996-1999). The lysosomal membrane stability, as a biomarker of general stress, showed that the membrane labilization period in mussels from polluted, urban- and industrial-related areas was significantly decreased (p < 0.05) when compared to mussels from control, clean seawater sites. In the harbour of Rijeka, the most contaminated site, the lysosomal membrane stability was reduced by more than 70% compared to the control. This method also proved to be a useful biomarker for detection of additional stress caused by short-term hypoxia that occurred once during this study inside the polluted and periodically quite eutrophic Pula Harbour. The concentration of metallothioneins in the mussel digestive gland, as a specific biomarker of exposure to heavy metals, did not reveal significant differences (p < 0.05) between sites covered by this study.

 

Dallinger, R., Y. Wang, et al. (2001). "Spectroscopic characterization of metallothionein from the terrestrial snail, Helix pomatia." Eur J Biochem 268(15): 4126-33.

                The Cd-sequestering metallothionein (MT) isoform isolated from the midgut gland of Roman snails exposed to Cd supplements in the feed was characterized by compositional and spectroscopic analysis. The preparations contained nearly 5 mol of Cd, small amounts of Cu and about 1 mol of Zn per chain mass of 6620 Da, in numerical agreement with the apoprotein's measured capacity of firmly binding a maximum of 6 equivalents of Cd per molecule. As with other Cd-containing MTs the occurrence of a prominent Cd-mercaptide-specific shoulder at 250 nm in its absorption spectrum showed that Cd is complexed in tetrahedral symmetry by the cysteine residues of the protein, and the multiphasic ellipticity profile in the CD spectrum revealed that these complexes are joined to form one or more oligonuclear Cd-mercapto clusters. Both spectral features vanished with the removal of the metal but were reconstituted to maximum amplitudes by readdition of Cd to the metal-free apoprotein, provided precautions were taken to prevent air oxidation of the latter. Quantitative analysis of snail MT reconstituted with Cd established that the 18 cysteine side chains bind the metal in a 3-to-1 ratio; spectroscopic studies on fractionally restored forms demonstrated that the six Cd ions were bound to the apoprotein molecule in succession in two sets of three Cd ions each. Thus, one can infer from the observed stoichiometry and the coordinating preferences of Cd that this gastropod MT, like the Cd-bearing MTs of marine crustaceans, harboured the metal in two separate cyclically constructed Cd3Cys9 clusters. The snail clusters differed, however, from other MTs in their response to acidification. Their protolytic dissociation proceeded through two separate protonation steps with the manifestation of spectroscopically distinguishable intermediate forms. Thus, this snail isoform displays in its metal composition and its chemical and spectroscopic features both similarities and differences to other animal kingdom MTs. Its properties suggest that it serves an important role in the protection of the terrestrial gastropod from Cd.

 

Leung, K. M. and R. W. Furness (2001). "Survival, growth, metallothionein and glycogen levels of Nucella lapillus (L.) exposed to subchronic cadmium stress: the influence of nutritional state and prey type." Mar Environ Res 52(2): 173-94.

                Dogwhelks Nucella lapillus feed mainly on mussels and barnacles, and may experience periods of starvation. We report effects of nutritional state and prey type on the survival, growth, cadmium (Cd) accumulation, metallothionein (MT) induction and glycogen stores in N. lapillus exposed to Cd in water. Adult dogwhelks, with similar shell length (30.0+/-1.5 mm), were either starved or fed to satiation with barnacles Semibalanus balanoides, mussels Mytilus edulis or Cd-dosed M. edulis, and kept in filtered natural seawater (< 0.01 microg Cd 1(-1)) or Cd-contaminated (400 microg Cd 1(-1)) seawater for 80 days. Mortality and individual growth rate were determined. Cd, MT and glycogen were measured in different tissues. Prolonged starvation and exposure to Cd significantly reduced the survivorship of N. lapillus, but feeding could help dogwhelks to combat Cd toxicity and minimise mortality. Extended starvation also caused tissue wastage, leading to higher concentrations of Cd and MT in tissues, whereas fed animals increased in weight and had lower Cd and MT concentrations because of the tissue dilution effect. Prey type significantly affected growth rate of dogwhelks and indirectly influenced Cd accumulation, MT induction and glycogen stores. Eating mussels promoted better growth and higher glycogen reserves than eating barnacles. Individual growth rate decreased with increasing Cd accumulation. Cd-exposed survivors grew faster and consumed more than control animals, implying that these survivors may have better fitness and greater tolerance to Cd toxicity. The use of growth, condition index, MT and glycogen as biomarkers of environmental pollution are discussed. These results indicate a need to incorporate biological data including growth (or at least condition index) and prey type into biomonitoring programmes to allow sound interpretation.

 

Hamza-Chaffai, A., J. C. Amiard, et al. (2000). "The potential use of metallothionein in the clam Ruditapes decussatus as a biomarker of in situ metal exposure." Comp Biochem Physiol C Toxicol Pharmacol 127(2): 185-97.

                This work aimed to validate the relationship between metallothioneins (MTs) and metals (Cd, Cu and Zn) in field conditions. Specimens of the marine bivalve Ruditapes decussatus (Linne, 1758) from Gargour were transferred in two sites: Gargour and Sidi Mansour, both situated along the south-eastern coast of Tunisia. The bivalves were removed from pairs of cages at day 0 (date of transplantation), day 62 and day 132. Metals (Cd, Cu and Zn) and MTs were determined in the subcellular fractions of the digestive gland. In Gargour, metal and MT levels increased significantly after 62 days of transplantation. However, they showed modest and non-significant variations in Sidi Mansour. Zn was mainly associated with the insoluble fraction, whereas Cd and Cu percentages in the soluble and the insoluble fractions were equivalent. Simple correlation analysis showed a positive and significant relationship between MTs and each metal. If all metals were taken together, multiple correlations showed that MTs were significantly correlated with Cd and Zn, with an important coefficient for Cd, but no significant relationship was observed for Cu. Gel filtration chromatography showed that in the heat stable fraction, the only cytosolic SH rich compounds have an apparent low molecular mass (about 15 kDa), which could correspond to metallothioneins. In the digestive gland of R. decussatus MTs responded to moderate increases of metal contamination, without interference with other factors, and could be a promising biochemical indicator of metal exposure.

 

Cosson, R. P. (2000). "Bivalve metallothionein as a biomarker of aquatic ecosystem pollution by trace metals: limits and perspectives." Cell Mol Biol (Noisy-le-grand) 46(2): 295-309.

                Owing to their induction by metals metallothioneins (MTs) have been proposed as biomarkers of the metallic contamination of the environment. On the other hand, bivalves are regarded as very convenient bioindicators of the aquatic ecosystems and an extensive literature has been dedicated to their response to metals. Among studies supporting the involvement of MTs in metal detoxification some discrepancy appears due to inter- and intra-specific variations, or to heterogeneous exposure conditions. A lesser number of papers are dealing with the use of metallothionein levels as biomarkers, and sometimes they evidence that natural factors influencing metallothionein synthesis have to be taken into account before final conclusions can been drawn. Moreover, there is still a large number of non-intercalibrated protocols used to quantify amounts of metallothioneins in organisms. As comparisons are necessary to assess the relative abundance of metallothioneins in a studied species, more work has to be completed before such comparisons could be validated. In the present paper we wish to establish the limits of the use of mollusc metallothioneins as a biomarker of aquatic ecosystem contamination by trace metals, using published and recent data as support for our conclusions and perspectives.

 

Dallinger, R., B. Berger, et al. (2000). "Metallothioneins in terrestrial invertebrates: structural aspects, biological significance and implications for their use as biomarkers." Cell Mol Biol (Noisy-le-grand) 46(2): 331-46.

                During the last few years the subject of metallothioneins (MTs) in terrestrial invertebrates has gained increasing attention. One reason for this may be that terrestrial invertebrates provide new insights into the biological diversity of MTs, with the potential of discovering alternative models of structural and functional relationships. Four groups of terrestrial invertebrates have been studied in detail, namely nematodes, insects, snails and earthworms, with the present article focusing on MTs from the latter two groups. Snails are interesting because they possess distinct MT isoforms involved in different metal-specific tasks. In the Roman snail (Helix pomatia), for example, one isoform is predominantly expressed in the midgut gland, accounting for the accumulation, binding and detoxification of cadmium. The second isoform, which is present in the snail's mantle, is substantially different regarding its primary structure. Furthermore, it binds nearly exclusively copper, and thus is probably involved in the homeostatic regulation of essential trace elements. Earthworm MTs merit our attention because of another peculiarity: they seem to be much more unstable than snail MTs, particularly under conventional conditions of preparation. The cDNA of the brandling worm (Eisenia foetida), for instance, codes for a putative MT, which is about twice the size of the actual protein. The isolated MT peptide binds four Cd2+ ions and represents a one-domain MT entity that is stable and functional in vitro. This strongly suggests that earthworm MTs are either posttranslationally modified, or subjected to enzymatic cleavage during preparation. Both snail and earthworm MTs are inducible by metal exposure, especially by cadmium, thus supporting the idea of using them as potential biomarkers for environmental metal pollution. Whilst snail MTs have already been tested in this respect with some success, the use of earthworm MTs as biomarkers still remains to be evaluated, especially in the light of the unknown significance of their posttranslational instability.

 

Erk, M. and B. Raspor (2000). "Advantages and disadvantages of voltammetric method in studying cadmium-metallothionein interactions." Cell Mol Biol (Noisy-le-grand) 46(2): 269-81.

                A sensitive and chemical species-selective technique of differential pulse anodic stripping voltammetry (DPASV) was applied in studying the cadmium-metallothionein (Cd-MT) interaction. The amperometric titrations of the purified MT20 and MT10 fractions, isolated by verified biochemical procedures from the digestive gland of cadmium-exposed mussels Mytilus galloprovincialis, with Cd2+ ions were performed in the buffered sodium chloride solution of 0.59 M ionic strength, pH 7.9 and 25 degrees C. Applying the DPASV method at various cadmium to metallothionein ratio several groups of chemical species were recorded. The data on the available ligand concentration to complex cadmium ions (CL), the apparent concentration stability constants (K,) of the respective complexes and the reliability of the determined complexing parameters are discussed. In quantifying the Cd-MT interaction the interference of dithiotreitol (DTT), which is used as the reducing agent in isolation and purification of MTs, is documented.

 

Isani, G., G. Andreani, et al. (2000). "Metallothioneins (MTs) in marine molluscs." Cell Mol Biol (Noisy-le-grand) 46(2): 311-30.

                The presence of MTs in marine molluscs was firstly hypothesized in oyster and in mussel during the seventies, however mussel's and oysters' MTs were completely purified and sequenced rather later. Already from the first studies it was evident that the purification of molluscan MTs was more difficult than in mammals. Mussel's MTs are characterized by the presence of a monomeric and a dimeric form. Several physiological and biochemical parameters can influence the concentration and the isolation of MT from molluscan tissues. Remarkable variations in MT isolation and quantification might depend on the purification and storage protocol. Because of possible artefacts due to the isolation procedure the establishment of a standard protocol for MT quantification in marine mollusc is still an important goal. In a few species the presence of very low molecular weight metal binding ligands has also been reported, in these cases it cannot be excluded that the native MT has been cleaved by the action of proteases. This review aims to report: 1) importance of a standard method for MT purification and quantification in molluscs; 2) distribution of MT among molluscan species; 3) data concerning oyster's and mussel's MTs which are the two more deeply investigated marine molluscs; 4) biotic and abiotic factors influencing MT concentration, and 5) biological role of MT and use of MT as a biochemical marker of heavy metal pollution.

 

Syring, R. A., T. Hoexum Brouwer, et al. (2000). "Cloning and sequencing of cDNAs encoding for a novel copper-specific metallothionein and two cadmium-inducible metallothioneins from the blue crab Callinectes sapidus." Comp Biochem Physiol C Toxicol Pharmacol 125(3): 325-32.

                Metallothioneins (MTs) are cysteine-rich metal-binding proteins found in micro-organisms, plants and all invertebrate and vertebrate animals. Unicellular eukaryotes such as yeast have a copper-MT whose synthesis is induced by a copper-activated transcription factor. Most higher organisms have two major cadmium/zinc MT isoforms, whose synthesis is controlled by a zinc-activated transcription factor. The blue crab, Callinectes sapidus, has two cadmium-inducible isoforms, CdMT-I and CdMT-II, and a third isoform, CuMT-II, which is induced by copper, but not by cadmium. The cDNA sequence of the copper-specific MT, along with those of the two CdMTs, was determined utilizing 3' and 5' rapid amplification of cDNA ends (RACE). CuMT-II cDNA encodes a 63 amino acid protein containing 21 cysteine residues. CdMT-I and CdMT-II cDNA encode a 58 and 57 amino acid protein, respectively, each with 18 cysteines. Molecular phylogeny analysis shows that the CdMT isoforms cluster with other crustacean CdMTs, whereas the copper-specific MT is more closely related to mollusk MTs. CuMT-II shows considerable homology to a copper-specific, non-cadmium inducible, MT from the snail, Helix pomatia. The presence of copper-specific MTs in mollusks and crustaceans, both of which are dependent on hemocyanin for oxygen transport, suggests that CuMT-II is involved in copper homeostasis associated with the synthesis and degradation of hemocyanin.

 

Viarengo, A., B. Burlando, et al. (2000). "Antioxidant role of metallothioneins: a comparative overview." Cell Mol Biol (Noisy-le-grand) 46(2): 407-17.

                Metallothioneins (MTs) are sulfhydryl-rich proteins binding essential and non-essential heavy metals. MTs display in vitro oxyradical scavenging capacity, suggesting that they may specifically neutralize hydroxyl radicals. Yet, this is probably an oversimplified view, as MTs represent a superfamily of widely differentiated metalloproteins. MT antioxidant properties mainly derive from sulfhydryl nucleophilicity, but also from metal complexation. Binding of transition metals displaying Fenton reactivity (Fe,Cu) can reduce oxidative stress, whereas their release exacerbates it. In vertebrates, MT gene promoters contain metal (MRE) and glucocorticoid response elements (GRE), Sp and AP sequences, but also antioxidant response elements (ARE). MT neosynthesis is induced by heavy metals, cytokines, hormones, but also by different oxidants and prooxidants. Accordingly, MT overexpression increases the resistance of tissues and cells to oxidative stress. As for invertebrates, data from the mussel show that MT can actually protect against oxidative stress, but is poorly inducible by oxidants. In yeast, there is a Cu(I)-MT that in contrast to mammalCu-MT exhibits antioxidant activity, possibly due to differences in metal binding domains. Finally, as the relevance of redox processes in cell signaling is becoming more and more evident, a search for MT effects on redox signaling could represent a turning point in the understanding of the functional role of these protein.

 

Werner, I. and D. E. Hinton (2000). "Spatial profiles of hsp70 proteins in Asian clam (Potamocorbula amurensis) in northern San Francisco Bay may be linked to natural rather than anthropogenic stressors." Mar Environ Res 50(1-5): 379-84.

                Multi-year investigations in northern San Francisco Bay by United States Geological Survey have linked reduced condition indices in populations of Asian clam (Potamocorbula amurensis) with elevated cadmium tissue concentrations. Our study seeks to determine whether levels of hsp70 proteins in P. amurensis can be correlated with these findings, and/or are related to histopathologic alterations and concentrations of metallothionein-like proteins. Here we present our results on stress proteins in clams collected monthly from four field stations between July 1996 and January 1998. In addition, animals were exposed in the laboratory to a range of salinities. Stress proteins were analyzed by Western blotting using monoclonal antibodies. Hsp70 protein levels in field-collected clams were significantly higher at the seaward (high salinity/low cadmium) stations (12.5, 8.1) than at the landward (low salinity/high cadmium) stations (6.1, 4.1). Laboratory studies showed that clams exposed to 0.1 ppt salinity had markedly lower hsp70 levels than clams exposed to higher salinities. In view of our previous laboratory studies showing that cadmium induces hsp70 in P. amurensis, our present results indicate that reduced hsp70 protein levels in field-collected clams may be linked to salinity effects rather than cadmium tissue concentrations.

 

Viarengo, A., M. Lafaurie, et al. (2000). "Critical evaluation of an intercalibration exercise undertaken in the framework of the MED POL biomonitoring program." Mar Environ Res 49(1): 1-18.

                The results of an intercalibration exercise among the laboratories participating in the MED POL program for monitoring biological effects of pollutants along the Mediterranean coasts are presented. Three established biomarkers, i.e. lysosomal membrane stability, metallothionein concentration and ethoxyresorufin-O-deethylase (EROD) activity, were intercalibrated. The stability of lysosomal membranes in mussels (Mytilus galloprovincialis Lam.) was assessed with a cytochemical method. The four participating laboratories were able to discriminate between control animals (membrane labilization times ranging from 21 to 35 min) and Cu-exposed animals (40 micrograms/l Cu for 3 days) (labilization times ranging from 4.5 to 7.4 min). The metallothionein concentration was evaluated in digestive gland homogenates of control mussels and of animals exposed to 200 micrograms/l Cd for 7 days. The eight participating laboratories were able to discriminate between controls and treated samples using a spectrophotometric method. The EROD activity was evaluated by 11 laboratories. All laboratories were able to discriminate between liver microsomal preparations obtained from control and from benzo-a-pyrene exposed fish (Dicentrarchus labrax), with values ranging from 0.5 to 15.88 pmol/min/mg protein in controls and from 5.41 to 165.13 pmol/min/mg protein in treated animals. Using S9 fractions, it was possible to correctly identify control and treated fish, with a variation similar to that found using microsomal fractions, albeit with an inevitable difference in specific activity. As a corollary, all laboratories involved produced comparable data and were able to identify pollutant-induced stress syndromes in sentinel organisms. Thus, intercalibration enables the use of biomarkers in large biomonitoring programs.

 

Ferrarello, C. N., M. R. Fernandez de la Campa, et al. (2000). "Speciation of metallothionein-like proteins of the mussel Mytilus edulis at basal levels by chromatographic separations coupled to quadrupole and double-focusing magnetic sector ICPMS." Anal Chem 72(24): 5874-80.

                Characterization and partial purification of metallothionein-like proteins (MLPs) of the mussel Mytilus edulis from natural populations of three coastal regions in Spain were performed. Size exclusion chromatography (SEC) with quadrupole (Q-ICPMS) or double-focusing inductively coupled plasma mass spectrometry (DF-ICPMS) detection was used first for speciation of cadmium in such natural samples and those of mussels exposed to 500 mg x L(-1) Cd in an aquarium tank. SEC results showed always a single Cd-MLP peak (MLP fraction). The contents in Cd, Cu, and Zn of this MLP fraction, of the high molecular weight protein pool (HMW), and of the whole cytosol were then measured by DF-ICPMS. Then, a given aliquot (50 microL) of MLPs with the highest values for UV molecular absorption at 254 nm (also the maximum sulfur and Cd, Cu, or Zn contents) was used to further fractionation. Fast protein liquid chromatography "on line" with Q-ICPMS was used for the purpose. Two Cd-MLP isoforms (MLP-1, MLP-2), with retention times (tR) of 15.7 and 16.0 min, were then detected in cytosols of the mussel samples of aquarium tank and also of the industrial area and Galicia coast. Conversely, wild coast mussels did not show any Cd-MLP signals at all. Analysis of essential elements copper and zinc in such cytosols by FPLC-Q-ICPMS revealed that these two metals were associated just to MLP-1. These results tend to indicate a different role for the two MLP isoforms detected in mussels (i.e., essential metals' homeostasis role seems to be tied to the MLP-1 isoform only). They illustrate the fact that trace metal speciation of unknown species in biological materials is becoming a challenge and points to the use of several complementary analytical techniques to obtain the required speciation information.

 

Zaroogian, G. and E. Jackim (2000). "In vivo metallothionein and glutathione status in an acute response to cadmium in Mercenaria mercenaria brown cells." Comp Biochem Physiol C Toxicol Pharmacol 127(3): 251-61.

                Brown cells that are found in the red glands of Mercenaria mercenaria accumulate, detoxify and excrete cadmium. Brown cell involvement in metal detoxification was due in part to endogenous glutathione (GSH) and protein sulfhydryl. Metallothionein (MT) and GSH have been shown to play an important role in metal detoxification in bivalve molluscs. This study showed that the protein sulfhydryl in brown cells of Mercenaria was in fact MT, that brown cell GSH functioned in acute protection against Cd2+ toxicity, that GSH provided the initial defense against Cd2+ toxicity prior to MT induction and that MT variants were unequal in response to Cd2+. During treatment of Mercenaria with 0.5 and 1.0 ppm Cd2+, brown cells were analyzed for MT by capillary electrophoresis and GSH colorimetrically after 0.25, 1, 2, 3, and 4 days. The data indicated that the cadmium-binding protein was MT with an apparent molecular weight of 9 kDa determined by gel filtration or 6 kDa as indicated by capillary electrophoresis. Glutathione appeared to prevail in the brown cell acute response to 0.5 ppm Cd2+, whereas MT appeared to prevail in the acute response to 1.0 ppm Cd2+. Capillary electrophoresis can be used to monitor and quantify MT and its variants in brown cells without need for prior separation of cytosolic components by chromatography. The change in MT-II was greater relative to the change in MT-I in the brown cell acute response to 0.5 ppm Cd2+, whereas the change in MT-1 was greater relative to the change in MT-II in the acute response to 1.0 ppm Cd2+. The variants of brown cell MT appeared to respond differentially to Cd2+ depending upon the Cd2+ treatment concentration.

 

Bose, R. and S. Bhattacharya (2000). "C-reactive protein in the hemolymph of Achatina fulica: interrelationship with sex steroids and metallothionein." Comp Biochem Physiol A Mol Integr Physiol 125(4): 485-95.

                C-reactive protein in Achatina fulica (ACRP) is a normal component of the hemolymph. Its concentration varied from 1mg/ml in the newly hatched male, 3-5 mg/ml in the most active hermaphrodite and 1.5-2.8 mg/ml in the sedentary female showing a direct relationship of the protein with the active phase of the animal. ACRP has a molecular mass of 400 kDa and showed high absorbance in the region of 200-230 nm. It has four subunits with relative molecular masses of 110, 90, 62 and 60 kDa, respectively. Interestingly, rat platelet aggregation in vitro was significantly enhanced by ACRP in presence of 10 microM ADP and 2 mM Ca(2+) suggesting a probable role of ACRP in the aggregation of amoebocytes during the formation of plug in injured tissue. Like other vertebrate CRPs, ACRP also acts as a scavenger of chromatin fragments as evidenced by its binding to poly-L-arginine. Among the sex steroids, 4-androstenedione induces ACRP synthesis in the newly hatched male reaching the level found in the most active hermaphrodite phase (4 mg/ml). A very high molar ratio (5) of mercury binding to ACRP confirmed its sequestration property of heavy metals as observed in vertebrates. The level of metallothionein (MT) in the hemolymph gradually increased from the male to the hermaphrodite to the female, a pattern distinctly different from that of the ACRP titer. Since both MT and ACRP can sequester inorganic mercury, the high level of MT compensates functionally for the low titer of ACRP in the sedentary female.

 

Khoo, H. W. and K. H. Patel (1999). "Metallothionein cDNA, promoter, and genomic sequences of the tropical green mussel, Perna viridis." J Exp Zool 284(4): 445-53.

                The primary structure of the cDNA and metallothionein (MT) genomic sequences of the tropical green mussel (Perna viridis) was determined. The complete cDNA sequences were obtained using degenerate primers designed from known metallothionein consensus amino acid sequences from the temperate species Mytilus edulis. The amino acid sequences of P. viridis metallothionein deduced from the coding region consisted of 72 amino acids with 21 cysteine residues and 9 Cys-X-Cys motifs corresponding to Type I MT class of other species. Two different genomic sequences coding for the same mRNA were obtained. Each putative gene contained a unique 5'UTR and two unique introns located at the same splice sites. The promoters for both genes were different in length and both contained metal responsive elements and active protein-binding sites. The structures of the genomic clones were compared with those of other species. J. Exp. Zool. 284:445-453, 1999.

 

Anderson, R. S., K. M. Patel, et al. (1999). "Oyster metallothionein as an oxyradical scavenger: implications for hemocyte defense responses." Dev Comp Immunol 23(6): 443-9.

                In order to better understand the interplay between metallothionein (MT) and reactive oxygen species (ROS) in oyster hemocytes, studies of the hydrogen peroxide (H2O2) scavenging properties of MT were carried out in a cell-free system. Mammalian MT is involved in protection against oxidative stress by virtue of its ability to scavenge free radicals; therefore, the H2O2 scavenging potentials of Crassostrea virginica and rabbit MTS were compared. Oyster and rabbit MTs showed similar dose-dependent suppression of H2O2-stimulated, luminol-augmented chemiluminescence (CL); the EC50 for CL (25 microM H2O2) was approximately 1.0 microM MT for both species. The interaction of ROS with MT in hemocytes could play a role in protection of the cells and surrounding tissues from oxidants associated with antimicrobial responses. Mobilization of bound zinc from MT by hemocyte-derived ROS may produce aberrant regulatory effects on various cellular processes. The data suggest that MT may be involved in immunoregulatory pathways in oyster hemocytes as a result of its ability to scavenge antimicrobial ROS.

 

Hamza-Chaffai, A., J. C. Amiard, et al. (1999). "Relationship between metallothioneins and metals in a natural population of the clam Ruditapes decussatus from Sfax coast: a non-linear model using Box-Cox transformation." Comp Biochem Physiol C Pharmacol Toxicol Endocrinol 123(2): 153-63.

                Cadmium, copper and zinc were determined concomitantly with metallothionein-like proteins (MTLPs) in the subcellular fractions of Ruditapes decussatus digestive gland. This study covered 4 months and aimed to evaluate the effect of metal pollution and other factors such as sex, size and reproductive state on MTLP levels. Copper concentrations did not vary with month, however Cd and Zn concentrations showed high levels during August. Organisms showing low cadmium concentrations presented the highest cadmium percentages in the soluble fraction (SF) containing MTLPs. However for high cadmium concentrations, the insoluble fraction (IF) was implicated in cadmium association. MTLP levels varied according to the month, the sex and the size of the organisms. A non-linear model based on the Box-Cox transformation, was proposed to describe a positive and a significant relationship between MTLPs and the studied metals. A model including sex and size showed that these two factors affected MTLP levels, but were less important than metals. Males of R. decussatus showed higher significant correlations between MTLP levels and cadmium than females. Moreover, the effect of size and reproductive state on MTLP levels was less perceptible in males than in females. As a result, MTLPs in males of R. decussatus could be proposed as suitable biomarker for detecting metal contamination.

 

Leung, K. M. and R. W. Furness (1999). "Induction of metallothionein in dogwhelk Nucella lapillus during and after exposure to cadmium." Ecotoxicol Environ Saf 43(2): 156-64.

                Induction of metallothionein (MT) was investigated in a common biomonitor, the dogwhelk Nucella lapillus (shell length: 27.7+/-1.4 mm; wet tissue weight: 667+/-196 mg), during and after exposure to cadmium (Cd) under controlled laboratory conditions (10+/-1 degrees C and 34+/-1 per thousand salinity). The dogwhelks were exposed to 500 microg Cd l-1 (2.2% of 96 h LC50) for 60 days and then placed into clean seawater for 110 days. MT concentration in whole animal increased during the exposure period, peaked at Day 70, and then declined gradually. Half-life of MT was ca. 40 days. MT concentration increased very significantly with increasing Cd concentration (r=0.74, n=24, P<0.001). Nevertheless, Cd concentration increased throughout the period of exposure and while in clean seawater, leveling off only after Day 120, indicating that Cd concentration could not be regulated by N. lapillus. Throughout the study, MT and Cd concentrations in gills, Leiblein gland, kidney, digestive gland, and gonad tissues increased gradually. Highest concentrations of MT and Cd were found in the Leiblein gland. Measurement of MT induction in the Leiblein gland of N. lapillus may therefore prove useful as a sublethal biological response to Cd contamination.

 

Serra, R., G. Isani, et al. (1999). "Seasonal dependence of cadmium accumulation and Cd-binding proteins in Mytilus galloprovincialis exposed to cadmium." Comp Biochem Physiol C Pharmacol Toxicol Endocrinol 123(2): 165-74.

                At different periods of the year specimens of Mytilus galloprovincialis were exposed to 0.5 microg Cd/ml seawater for 7 days. Concentrations of trace elements (Cd, Zn, Cu and Fe) and Cd-binding proteins in gills, viscera, muscle and mantle were determined after 1 weeks exposure. Cadmium accumulation was higher in May and June and was tissue dependent; it was highest in the gills and decreased in the order: gills > viscera > mantle and adductor muscle. Significant seasonal variations of zinc, copper and iron, were also found, in both exposed and control molluscs. The percentage of Cd distribution between cytosol and pellet changed during the year; a clear shift from the particulate fractions to the cytosolic fractions was measured during May and June, especially in the gills. Metallothionein (MT) was the main ligand responsible for Cd accumulation, and this protein reached a maximum between May and June. Inclusion of mercaptoethanol during the purification procedure was found to improve MT isolation by gel chromatography. In the absence of mercaptoethanol, MT showed polymerization patterns which were season dependent and temperature independent, whereas its concentration was increased in mussels kept at higher temperature.

 

Barsyte, D., K. N. White, et al. (1999). "Cloning and characterization of metallothionein cDNAs in the mussel Mytilus edulis L. digestive gland." Comp Biochem Physiol C Pharmacol Toxicol Endocrinol 122(2): 287-96.

                Metallothioneins are small metal-binding proteins found in all species of animals and are transcriptionally-induced by heavy metal ions, oxidative stresses, and inflammation. In the blue sea mussel, Mytilus edulis, several apparent subtypes of each isoform have been purified and biochemically sequenced. To determine whether the high number of metallothionein forms present in M. edulis were specific to the digestive gland, and to understand how these proteins evolved, we cloned five variants of metallothionein from M. edulis. MT10 and MT20 isoform fragments were amplified by PCR, and used as radiolabelled probes to screen digestive gland cDNA libraries. The MT10 transcripts were 321-353 nucleotides long and the MT20 transcripts, 513-555 nucleotides. Previously identified primary structures of MT10 subtypes were confirmed and, in addition, a novel subtype was identified. Expression of MT10 and MT20 isoforms shown by clonal representation and Northern blot analysis indicated that the MT10 message was more prevalent than the MT20 message. Only the MT20 II transcript could be identified among the MT20 clones. The high degree of untranslated region similarity between each isoform indicates that these additional forms are recent gene duplication events in the Mytilus lineage. Exposure of 0.4 mg l-1 of cadmium to the mussels resulted in a marked increase in both mRNAs suggesting that the MT20 isoform represents a primarily inducible metallothionein not highly expressed under basal conditions.

 

Viarengo, A., B. Burlando, et al. (1999). "Role of metallothionein against oxidative stress in the mussel Mytilus galloprovincialis." Am J Physiol 277(6): R1612-9.

                Metallothionein (MT) is a sulfhydryl-rich protein involved mainly in heavy metal homeostasis and detoxification. In this study, the use of the mussel as an experimental model allowed us to test MT antioxidant properties at the molecular, cellular, and organism level. MT induction was achieved by mussel exposure to Cd (200 microg/l) in aquaria for 7 days followed by detoxification in the sea for 28 days. Cd-preexposed and nonexposed mussels were then treated with Fe (300-600 microg/l) in aquaria for 3 days. Biochemical assays on digestive gland tissue showed that treatment with Fe led to a significant increase in oxyradical production and malondialdehyde level only in mussels not preexposed to Cd. The Cd-dependent resistance to oxidative stress was ascribed to MT induction, as Cd produced no significant variation of reduced glutathione and major antioxidant enzymes. Digital imaging of isolated digestive gland cells showed lower oxyradical rise and higher viability in cells from Cd-preexposed mussels after treatments with 0.5-5 mM H2O2. Analyses on whole organisms showed that anoxic survival was lowered in mussels that had been treated with Fe, but such an effect was less pronounced in Cd-preexposed mussels compared with nonpreexposed ones. In conclusion, data suggest an antioxidant role for MT, which seems to occur through oxyradical scavenging and is able to protect both isolated cells and the entire organism from oxidative stress.

 

Bolognesi, C., E. Landini, et al. (1999). "Genotoxicity biomarkers in the assessment of heavy metal effects in mussels: experimental studies." Environ Mol Mutagen 33(4): 287-92.

                Heavy metals are stable and persistent environmental contaminants. The range of metal concentrations is generally below acute thresholds in coastal areas, where recognition of chronic sublethal effects is more relevant. Evidence of long-term adverse effects, such as cancer, due to heavy metals in marine animals comes from a number of field and experimental studies. The mechanism of metal carcinogenicity remains largely unknown, although several lines of experimental evidence suggest that a genotoxic effect may be involved. The aim of our study was to evaluate the sensitivity of genotoxicity tests, alkaline elution and micronucleus test, as biomarkers for the detection of heavy metals in mussels as the sentinel species. Experimental studies were carried out on Mytilus galloprovincialis exposed in aquarium (5 days) to different concentrations of three selected metal salts, CuCl2 (5, 10, 20, 40, 80 micrograms/l/a), CdCl2 (1.84, 18.4, 184 micrograms/l/a), and HgCl2 (32 micrograms/l/a), and to a mixture of equimolar doses of the three metals to study the results of their joint action. Metallothionein quantitation was used as a marker of metal exposure. Lysosomal membrane stability was applied to evaluate the influence of physiological status on genotoxic damage. The ranking of genotoxic potential was in decreasing order: Hg > Cu > Cd. Cu and Hg caused an increase of DNA single-strand breaks and micronuclei frequency. Cd induced a statistical increase of DNA damage, but gave negative results with the micronucleus test. A relationship between genotoxic effects and metallothionein content was observed. Reduction in lysosomal membrane stability with the increasing concentration of heavy metals was also evident.

 

Engelken, J. and A. Hildebrandt (1999). "cDNA cloning and cadmium-induced expression of metallothionein mRNA in the zebra mussel Dreissena polymorpha." Biochem Cell Biol 77(3): 237-41.

                Using pooled degenerate oligonucleotides inferred from the N-terminal amino acid sequence of Dreissena polymorpha metallothionein and a Cys-X-Cys motif characteristic for known metallothioneins, a 150-bp metallothionein-specific reverse transcription PCR product was generated. The PCR product was used to screen a Dreissena polymorpha cDNA library, and a complete metallothionein cDNA sequence from Dreissena was identified. Four clones with the identical sequence were detected, supporting the idea of a single metallothionein gene in Dreissena. The sequence contains a 141-bp 5' untranslated region and a 572-bp 3' untranslated region with two polyadenylation signals. The coding region spans 219 bp. The deduced amino acid sequence shows 21 cysteine residues present in the metallothionein-typical motifs. Induction studies were performed with 50 microg Cd2+/L for up to 16 days. The exposed mussels show a sevenfold higher metallothionein mRNA level compared with uninduced control mussels.

 

Amiard-Triquet, C., F. Rainglet, et al. (1998). "Metallothioneins in Arctic bivalves." Ecotoxicol Environ Saf 41(1): 96-102.

                In the framework of an International Association for the Promotion of Cooperation with Scientists from the Independent States of the Former Soviet Union (INTAS) Project on biodiversity and adaptation strategies of Arctic coastal marine benthos, research was focused on the role of metallothioneins as a possible indicator of the effect on animals and availability of trace metals in the Arctic. Metallothioneins are low-molecular-weight, cysteine-rich proteins known to be induced by high environmental levels of trace metals. Specimens of Macoma balthica and Mytilus edulis were collected along several Arctic estuaries in the White and Pechora seas; whole tissues for M. balthica and the digestive gland and gills for M. edulis were dissected, frozen in liquid nitrogen, and lyophilized onboard. Metallothionein concentrations were determined by a polarographic assay. From the same stations organisms and sediments were also collected for metal analysis. The results revealed significant differences in metallothionein concentrations among the stations for M. balthica. Similar, although less marked, differences were also obtained in the organs of M. edulis. Data on metallothionein were compared with trace metal concentrations in both the organisms and sediments. Also, the relationship with abiotic factors (salinity) and biological variables (size of sampled organisms) was determined. In particular, biological variables seemed to influence metallothionein concentration in the organisms and their effect should be carefully considered for a correct assessment of differences between stations.

 

Hamza-Chaffai, A., M. Romeo, et al. (1998). "Effect of copper and lindane on some biomarkers measured in the clam Ruditapes decussatus." Bull Environ Contam Toxicol 61(3): 397-404.

               

Bebianno, M. J. and M. A. Serafim (1998). "Comparison of metallothionein induction in response to cadmium in the gills of the bivalve molluscs Mytilus galloprovincialis and Ruditapes decussatus." Sci Total Environ 214: 123-31.

                Metal binding characteristics and metallothionein induction differ markedly among the gills of the bivalve molluscs Mytilus galloprovincialis and Ruditapes decussatus exposed to sublethal cadmium concentrations (100 micrograms/l). The rates of cadmium uptake as well as the percentage of cadmium bound to the cytosol were significantly higher in the gills of M. galloprovincialis when compared with that of R. decussatus. Similarly metallothionein concentration detected in the gills of M. galloprovincialis increase fourfold while for the clam R. decussatus metallothionein concentrations increased only twofold.

 

Roesijadi, G., L. L. Brubacher, et al. (1997). "Metallothionein mRNA induction and generation of reactive oxygen species in molluscan hemocytes exposed to cadmium in vitro." Comp Biochem Physiol C Pharmacol Toxicol Endocrinol 118(2): 171-6.

                (1) Hemocytes of the oyster Crassostrea virginica were exposed to Cd in vitro to examine the concomitant induction of metallothionein (MT) mRNA and production of reactive oxygen species (ROS) during the oxidative burst response of phagocytic cells. (2) MT mRNA induction was concentration-dependent, exhibiting a threshold between 5 and 10 microM cdCl2, and an increase at higher Cd concentrations up to a maximum level of 36 microM cdCl2. Continued increases in the Cd exposure concentrations resulted in declines in the levels of MT mRna. (3) No significant effects of Cd on ROS production were observed at Cd concentrations up to 36 microM CdCl2. However, ROS production decreased in a concentration-dependent fashion at CdCl2 concentrations from 36 to 356 microM, the highest concentration tested. (4) At these higher concentrations of Cd, the concomitant decrease in MT mRNA inducibility and suppression of ROS production were probably manifestations of the general cytotoxicity of Cd. ROS suppression can result in reduced resistance to infectious agents, suggesting that Cd is immunotoxic to C. virginica.

 

Berger, B., R. Dallinger, et al. (1997). "Primary structure of a copper-binding metallothionein from mantle tissue of the terrestrial gastropod Helix pomatia L." Biochem J 328 ( Pt 1): 219-24.

                A novel copper-binding metallothionein (MT) has been purified from mantle tissue of the terrestrial snail Helix pomatia using gel-permeation chromatography, ion-exchange chromatography and reverse-phase HPLC. Copper was removed from the thionein by addition of ammonium tetrathiomolybdate. The resulting apothionein (molecular mass 6247 Da) was S-methylated and digested with trypsin, endoproteinase Arg-C and endoproteinase Lys-C. Amino acid sequences of the resulting peptides were determined by collision-induced dissociation tandem MS. The protein is acetylated at its N-terminus, and consists of 64 amino acids, 18 of which are cysteine residues. A comparison with the cadmium-binding MT isolated from the midgut gland of the same species shows an identical arrangement of the cysteines, but an unexpectedly high variability in the other amino acids. The two MT isoforms differ in total length and at 26 positions of their peptide chains. We suggest that the copper-binding MT isoform from the mantle of H. pomatia is responsible for regulatory functions in favour of copper, probably in connection with the metabolism of the copper-bearing protein, haemocyanin.

 

Dallinger, R., B. Berger, et al. (1997). "Metallothionein in snail Cd and Cu metabolism." Nature 388(6639): 237-8.

               

Alonso, J. I. and M. C. Martin-Mateo (1996). "Induction and characterization of metallothionein in different organs of Ostrea edulis L." Biol Trace Elem Res 53(1-3): 85-94.

                In this study, the induction of metallothionein (MT) in one species of oyster Ostrea edulis exposed to copper (Cu) and zinc (Zn) was examined. The quantity of total protein (mg protein/mg dry wt) in each sample and the measurement of MT were determined by the Lowry and silver (Ag)-saturation methods. Our results show that the gonads are the organs producing the greatest quantity of MT in the controls and in the groups induced by Cu and Zn, followed by the mantle, gut, and by muscle and plasma. Competition by Zn with respect to Cu for the production of MT has been established.

 

Roesijadi, G., K. M. Hansen, et al. (1996). "Cadmium-induced metallothionein expression during embryonic and early larval development of the mollusc Crassostrea virginica." Toxicol Appl Pharmacol 140(2): 356-63.

                Newly fertilized eggs of the oyster Crassostrea virginica were exposed to 0.2 microM Cd and sampled during the first 24 hr of embryonic and larval development for determination of the temporal patterns of total bioaccumulated and metallothionein (MT)-bound Cd concentrations and the concentrations of MT mRNA. In comparison with controls, exposure to this concentration of Cd resulted in delayed development to the D-stage veliger larval stage. Maternal MT mRNA, which was carried over into the egg, declined immediately after fertilization. In controls, levels of MT mRNA were stable after this initial decline, and little or no detectable Cd was bound to MTs. Accumulation of new MT mRNA was observed in the exposed group after 12 hr of exposure when increases due to induction by Cd had occurred. Total accumulated Cd concentrations, which ranged from 0.04 to 0.1 pmol Cd/microg DNA in unexposed individuals, increased to 13.5 pmol Cd/microg DNA after 24 hr in Cd-exposed individuals. There was an increase in MT-bound Cd in exposed individuals prior to MT induction, which may be explained in part as Cd binding to maternal MT. This was detected as binding to CvNAcMT, the expected N-acetylated form of the oyster MT. Amounts of Cd bound to this form of MT increased, thereafter, and was followed by increases in binding to CvMT, the unexpected, nonacetylated form of the oyster MT, which has been associated with Cd toxicity, possibly in relation to the effects of Cd on normal cotranslational processing.

 

Pedersen, S. N., K. L. Pedersen, et al. (1996). "Primary structures of decapod crustacean metallothioneins with special emphasis on freshwater and semi-terrestrial species." Biochem J 319 ( Pt 3): 999-1003.

                Cadmium injections induced only a single form of metallothionein (MT) in the midgut gland of Potamon potamios, whereas the same treatment induced two isoforms in Astacus astacus. The only difference between the two latter isoforms was that one had an extra N-terminal methionine residue. MT from P. potamios showed structural differences from other decapod crustacean MTs. It contained a Gly-Thr motif at positions 8 and 8a, which had previously been found only in certain vertebrate and molluscan MTs. Furthermore P. potamios MT contained two to three times as many glutamic acid residues as normally found in decapod crustacean MT. The primary structure of MT from the freshwater crayfish A. astacus showed a high degree of sequence identity with MT from other decapod crustaceans, especially the marine astacidean Homarus americanus, although two valine residues were unexpectedly found at positions 8 and 21, where lysine residues are normally found.

 

Milcent, M. C., F. Goudard, et al. (1996). "Identification of 137Cs-and 241Am-binding sites in the oyster Crassostrea gigas." Biochem Mol Biol Int 39(1): 137-48.

                The subcellular distribution and binding of 241Am and 137Cs in the visceral mass of the oyster Crassostrea gigas were investigated following exposure to sea water contaminated with these radionuclides. 241Am was predominantly sequestered by the lysosomal system. Approximately, 10% of 241Am was associated with soluble macromolecules. 241Am was bound to lipofuscin, ferritin and to unidentified ligands of 60 to 15 kdaltons mol. wt. No evidence was found for binding of 241Am to metallothionein synthesized de novo. In contrast, only small amounts of 137Cs were present in lysosomes and 137Cs was not associated with soluble cellular proteins. These results indicate that they enter complete separate metabolic pathways.

 

Dallinger, R. (1996). "Metallothionein research in terrestrial invertebrates: synopsis and perspectives." Comp Biochem Physiol C Pharmacol Toxicol Endocrinol 113(2): 125-33.

                While most of metallothionein research during the past years has been carried out on mammals or vertebrates, only relatively few studies have been directed towards invertebrates. Even fewer investigations have focussed on terrestrial invertebrates. The best studied metallothioneins and/or metallothionein genes among terrestrial invertebrates are those from an insect species (Drosophila melanogaster), a nematode (Caenorhabditis elegans) and some terrestrial gastropods (Helix pomatia, Arianta arbustorum). From these few examples it already appears that terrestrial invertebrate metallothioneins provide intriguing models to better understand the multiplicity of functions of these proteins and their evolution within the animal kingdom. Like in mammals, metallothioneins in terrestrial invertebrates seem to perform different functions simultaneously. This is exemplified by terrestrial gastropods, which are able to accumulate different metals in different tissues, in which metal-specific metallothionein isoforms or conformation forms are expressed, allowing these organisms to detoxify more efficiently nonessential trace elements such as cadmium, and at the same time to maintain the homeostasis of essential trace elements such as copper. A major proportion of metallothionein research in terrestrial invertebrates addresses the ecophysiological and ecotoxicological significance of these proteins with regard to the increasing risk due to chemical pollution. One promising aspect in this concern is the potential utilization of metallothioneins as biomarkers for risk assessment in terrestrial environments.

 

Tessier, C. and J. S. Blais (1996). "Determination of cadmium-metallothioneins in zebra mussels exposed to subchronic concentrations of Cd2+." Ecotoxicol Environ Saf 33(3): 246-52.

                Metallothioneins have been detected and investigated in zebra mussels (Dreissena polymorpha) using high-performance liquid chromatography (size exclusion) coupled with microatomization-AAS or inductively coupled plasma mass spectrometry. The mussels were exposed to 0.2, 2, and 20 micrograms/liter Cd2+ (as CdCl2) for 1 month under controlled temperature and dietary conditions. Elevated (relative to control) concentrations of tissue Cd2+ were detected in all specimens exposed to 2 micrograms Cd/liter and more than 50% of the specimens exposed to 0.2 micrograms Cd/liter, demonstrating that Dreissena cannot regulate Cd2+ at trace exposure concentrations. In most specimens, at least 85% of the measured Cd2+ was bound to metallothioneins. After reduction and exposure to excess Cd2+, the metallothionein fraction of all extracts adsorbed similar quantities of Cd2+, indicating that the physiological concentration of metallothionein in the exposed specimens remained similar to the basal concentration in the control specimens. Thus, a short-term exposure to environmentally relevant concentrations of Cd2+ did not produce a genetic induction of metallothionein biosynthesis as generally observed in specimens exposed to higher concentrations of d10 metals.

 

Berger, B., P. E. Hunziker, et al. (1995). "Mass spectrometry and amino acid sequencing of two cadmium-binding metallothionein isoforms from the terrestrial gastropod Arianta arbustorum." Biochem J 311 ( Pt 3): 951-7.

                1. Two cadmium-binding metallothionein (Mt) isoforms, called Mta and Mtb, were isolated from terrestrial snails (Arianta arbustorum), using various chromatographic techniques, such as gel-permeation chromatography and reversed-phase HPLC. The purified proteins were S-methylated and cleaved by means of different enzymes (trypsin, endoproteinase Glu-C, and endoproteinase Asp-N). Amino acid sequences were determined by automated Edman degradation and collision-induced dissociation (CID) tandem MS. According to their primary structures, both isoforms should be attributed to class-I Mts. 2. The two forms are structurally identical, differing only by one amino acid exchange in position 60 of the peptide chain. Both isoproteins consist of 66 amino acids, 18 of which are cysteine residues. Most of the cysteine residues are arranged in seven Cys-Xaa-Cys motifs. Mta and Mtb possess an N-terminal acetylated-serine residue and contain a short N-terminal motif which shows a high degree of similarity with the N-termini of histones H4 and H2A. 3. A comparison of Mta and Mtb with other invertebrate Mts shows a very high degree of sequence similarity with a cadmium-binding Mt from Helix pomatia, a species that is closely related to Arianta arbustorum. Moreover, Mta and Mtb, as expected, also exhibit structural similarities with Mts from other molluscan species, such as mussels and oysters. It is suggested that Mta and Mtb represent two allelic isoforms, reflecting the genetic polymorphism of Mt in Arianta arbustorum.

 

Roesijadi, G., K. M. Hansen, et al. (1995). "Cadmium-induced expression of metallothionein and suppression of RNA to DNA ratios during molluscan development." Toxicol Appl Pharmacol 133(1): 130-8.

                One-week-old larvae of the mollusc Crassostrea virginica, an oyster, were exposed to Cd concentrations ranging from the control with no added Cd to 0.2 microM CdCl2 for 24 hr. Concentration-dependent increases in total cadmium accumulation, cytosolic and metallothionein-bound Cd concentrations, and levels of the MT mRNA were detectable over this range of exposure concentrations. Increases in these measures were apparent at the lowest exposure concentration of 0.005 microM, with dramatic increases observed between 0.04 and 0.2 microM Cd. Although the concentrations of MT-bound Cd increased, the fraction of total Cd that was bound to MTs declined in larvae treated with 0.04 and 0.2 microM Cd, presumably due to the inability of MT biosynthesis to keep up with the higher rates of Cd uptake at the higher concentrations. This decreased effectiveness of MTs in sequestering Cd in relation to the total Cd load and the associated increase in the nonthionein Cd fraction at these higher treatment conditions coincided with a decline in the RNA to DNA ratio and an increase in a nonacetylated variant of the MT. The former is indicative of an inhibition of larval metabolism and growth. The latter is an unexpected form for this MT and is believed to signify disruption of normal cotranslational modification of the N-terminal amino acid sequence.

 

Bebianno, M. J., M. A. Serafim, et al. (1994). "Involvement of metallothionein in cadmium accumulation and elimination in the clam Ruditapes decussata." Bull Environ Contam Toxicol 53(5): 726-32.

               

Roesijadi, G. (1994). "Metallothionein induction as a measure of response to metal exposure in aquatic animals." Environ Health Perspect 102 Suppl 12: 91-5.

                Metallothioneins (MTs) are considered central in the intracellular regulation of metals such as copper, zinc, and cadmium. Increased MT synthesis is associated with increased capacity for binding these metals and protection against metal toxicity. Recent advances in the biochemistry and molecular biology of MTs have facilitated research on MTs in aquatic species. For the bivalve mollusc Crassostrea virginica, a species frequently used in studies on the toxicology and environmental monitoring of metals, the primary structure for MT has been deduced from analysis of the proteins and cDNA. Procedures for analysis of MT synthesis and MT gene expression have been applied in studies of response to metal exposure. Induction of specific MT forms by Cd is concentration- and time-dependent. The levels of MT-bound metals exhibit a strong relationship with the cytosolic metal concentrations in a metal-exposed natural population of oysters. Ribonuclease protection assays using sequence-specific antisense RNA probes have shown that the MT mRNA structure in this natural population exhibits considerable individual variability in the 3'-untranslated region. Although yet to be substantiated, the possibility exists that the distribution of this variability may be related to the level of environmental metal contamination. One probe derived from the coding region is suitable for use in quantitative RPAs for oyster MT mRNAs.

 

Bebianno, M. J. and W. J. Langston (1993). "Turnover rate of metallothionein and cadmium in Mytilus edulis." Biometals 6(4): 239-44.

                The results demonstrate the first attempt to determine metallothionein turnover in the whole soft tissues of mussels Mytilus edulis exposed to cadmium. Half-lives for metallothionein and cadmium are 25 and 300 days, respectively. As metallothionein degrades the released cadmium induces further synthesis of the protein, to which the metal becomes resequestered. The slow metallothionein turnover rates (compared with mammals) and the lack of significant cadmium excretion testify to the relatively stable nature of the cadmium-metallothionein complex in these invertebrates and supports the view of a detoxifying role for metallothionein in the mussels.

 

Dallinger, R., B. Berger, et al. (1993). "Purification and primary structure of snail metallothionein. Similarity of the N-terminal sequence with histones H4 and H2A." Eur J Biochem 216(3): 739-46.

                A cadmium-binding metallothionein has been purified from metal-exposed Roman snails (Helix pomatia) using gel-permeation, ion-exchange and reverse-phase high-performance liquid chromatography. The S-methylated protein was digested with trypsin and the endoproteinases Asp-N, Glu-C and Arg-C. While most of the resulting peptides could be sequenced by Edman degradation, the intact protein, as well as the N-terminal peptide, proved to be blocked. Analysis by mass spectrometry showed that the N-terminal amino acid was an acetylated serine residue. Snail metallothionein, which is suggested to be involved in the detoxification of cadmium, contains 66 amino acid residues, 18 of which are cysteine residues arranged in seven Cys-Xaa-Cys motifs. The calculated molecular mass of the protein is 6.62 kDa. The primary structure of snail metallothionein reveals a clear relationship with molluscan and vertebrate metallothioneins, but lower similarity with metallothioneins of other invertebrate species. The N-terminal region of the isolated protein proved to be unique among the metallothionein sequences determined so far, showing high degrees of similarity with the N-terminal sequences of histones H2A and H4 which may be important for regulatory functions.

 

Unger, M. E. and G. Roesijadi (1993). "Sensitive assay for molluscan metallothionein induction based on ribonuclease protection and molecular titration of metallothionein and actin mRNAs." Mol Mar Biol Biotechnol 2(5): 319-24.

                We developed a sensitive assay for oyster metallothionein (MT) mRNA based on ribonuclease protection and molecular titration. Deletion constructs derived from previously described MT cDNA and newly constructed actin cDNA clones were used to create templates for MT and actin riboprobe synthesis, respectively. MT induction was assessed by normalizing MT expression to that of actin. MT and actin transcripts were quantified in gills of cadmium-exposed (50 micrograms/L-1 cadmium for 2 weeks) and control oysters. Assays for actin and MT were sensitive to 150 and 35 amol mRNA, respectively. Actin was not affected by cadmium exposure and was suitable for use as a normalization factor. For detection of MT mRNA of controls, it was necessary to increase the amount of total input RNA 7.5-fold over that of cadmium-exposed samples due to lower concentrations of target. MT mRNA was induced approximately 15-fold in the cadmium-exposed sample.

 

Mackay, E. A., J. Overnell, et al. (1993). "Complete amino acid sequences of five dimeric and four monomeric forms of metallothionein from the edible mussel Mytilus edulis." Eur J Biochem 218(1): 183-94.

                Cadmium-induced metallothioneins from the common sea mussel, Mytilus edulis, were shown to comprise of two groups of isoforms having apparent molecular masses of 10 kDa and 20 kDa. The 10-kDa group was resolved by anion-exchange chromatography into four fractions while the 20-kDa group was resolved into three fractions using this method. After metal removal and S-methylation of the cysteine residues using methyl-p-nitrobenzenesulphonate the complete amino acid sequences were determined. Five isoforms of the 20-kDa group were shown to possess monomeric units consisting of 71 amino acids. These proteins were distinct from the four 72-amino-acid proteins of the 10-kDa group. The FASTA algorithm has been used to compare the degree of similarity between the mussel metallothionein MT-10-IV isoform and other metallothioneins. The mussel MT-10-IV isoform exhibited substantial similarity to other molluscan metallothioneins. Moreover, the mussel metallothionein exhibited more similarity to vertebrate metallothioneins than to those of non-molluscan invertebrates, thus suggesting that the mussel metallothioneins are class I metallothioneins.

 

Pokora, Z., P. Szilman, et al. (1993). "[Histochemical estimation of zinc accumulation in hepato-pancreatic gland cells of the snail Lymnaea stagnalis (L.)after subacute poisoning with zinc acetate considering infection with Digenea larvae]." Wiad Parazytol 39(3): 225-32.

                Accumulation of zinc in epithelial cells of the digestive gland tubules of the fresh-water snail Lymnaea stagnalis after subacute intoxication with zinc acetate (strong increasing doses of the metal in short period of time) was histochemically examined using dithizone. Large zinc granules observed in medial and basal parts of calcium cells permitted to conclude that this metal is bound in cytoplasm mainly on granules built of calcium and magnesium phosphate. There were no smaller concentrations of zinc, that would be indicative for metal binding with cytoplasmatic metallothionein-like proteins. In calcium cells of the digestive gland tubules of snails infected with Digenea larvae accumulation of zinc was stronger, that would be an aspect of compensatory reaction.

 

Chou, C. L., R. D. Guy, et al. (1991). "Isolation and characterization of metal-binding proteins (metallothioneins) from lobster digestive gland (Homarus americanus)." Sci Total Environ 105: 41-59.

                Two metallothionein (low-molecular-weight, metal-binding proteins) preparations, MT-1 and MT-2, have been isolated from the digestive gland of American lobster (Homarus americanus) contaminated with Cd. MT-1 contains Cd- and Cu-binding proteins, whereas MT-2 is a reasonably pure Cd-binding protein. The properties of MT-1 and MT-2 with respect to amino acid and elemental compositions, heat stabilities, polarographic, high-performance liquid chromatography (HPLC), and isoelectric focussing behaviors are reported. Lobster metallothioneins share a number of similarities with mammalian metallothioneins with respect to the presence of Cd and Cu, apparent molecular weights, amino acid compositions, UV absorption spectra at various pH, and polarographic behavior, but differ substantially in their electrophoretic behavior.

 

Roesijadi, G., M. M. Vestling, et al. (1991). "Structure and time-dependent behavior of acetylated and non-acetylated forms of a molluscan metallothionein." Biochim Biophys Acta 1074(2): 230-6.

                Cadmium-induced metallothionein in a mollusc, the oyster Crassostrea virginica, occurs in both blocked and unblocked forms (Roesijadi, G., Kielland, S.L. and Klerks, P. (1989) Arch. Biochem. Biophys. 273, 403-413). The block, which is the sole difference in the structure of the two proteins, was identified as an acetyl group with use of tandem mass spectrometry. The blocked and unblocked proteins carried N-acetylserine and serine, respectively, at the N-terminus and were designated CvNAcMT and CvMT. Only CvNAcMT was detected under basal conditions. Both forms were induced by Cd. Pulse-labeling with [35S]cyteine at specified times during exposure showed that the rate of CvNAcMT synthesis in gills increased rapidly, initially exceeding that of CvMT, then declined to the rate attained by CvMT. Turnover rates for Cd-induced CvMT and CvNAcMT were similar to each other. They appeared to be faster when measured in the absence of Cd in the external medium (k = 0.18 and 0.16/day, respectively), than in its presence (k = 0.03 and 0.06/day, respectively).

 

den Besten, P. J., P. T. Bosma, et al. (1991). "Effects of cadmium on metal composition and adenylate energy charge in the sea star Asterias rubens L." Arch Environ Contam Toxicol 21(1): 112-7.

                Sea stars, Asterias rubens, were exposed to 200 micrograms Cd/L or fed with mussels which contained about 70 micrograms Cd/g dry wt. After 5 weeks, cadmium in the pyloric caeca of directly and indirectly exposed sea stars had reached levels of 12 and 9 micrograms Cd/g dry wt, respectively. For both types of exposure, a reduction of 30% of the zinc levels in the pyloric caeca was found, which was correlated with a comparable displacement of zinc from the metallothionein-like proteins. Copper levels were increased in the pyloric caeca of directly exposed sea stars. In gonads, stomachs, and body wall of directly exposed sea stars, cadmium concentrations were 4 to 9 times higher than those in animals fed with Cd-contaminated mussels. Cadmium exposure also affected metal composition in these tissues. The ovaries contained relatively large amounts of zinc. Gel filtration chromatography revealed that this zinc and the accumulated cadmium were distributed over a large range of high-molecular-weight proteins. Both direct and indirect cadmium exposure resulted in a small, but significant decrease of the adenylate energy charge (AEC) in the pyloric caeca. In the gonads, no effect of the cadmium exposure could be demonstrated on the AEC, but in the ovaries a reduction of the adenylate pool was found. In semi-field experiments, stars were exposed to 25 micrograms Cd/L or fed with mussels collected from the heavily polluted Dutch Western Scheldt. After 6 months of direct or indirect exposure, cadmium in the pyloric caeca had reached comparable levels of 8 and 7 micrograms/g dry wt, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

 

Unger, M. E., T. T. Chen, et al. (1991). "Primary structure of molluscan metallothioneins deduced from PCR-amplified cDNA and mass spectrometry of purified proteins." Biochim Biophys Acta 1074(3): 371-7.

                The primary structure of metallothioneins (MT) of a mollusc, the oyster Crassostrea virginica, was determined by molecular cloning and mass spectrometry of purified proteins. The cloning strategy included PCR amplification of the responsible cDNAs from total cDNA using completely degenerate oligonucleotides (derived from the N-terminal amino acid sequence) and oligo(dT)20 as primers. Primer extension off mRNA was used as an independent determination of the nucleotide sequence represented by the degenerate PCR primers. The deduced amino acid sequence was consistent with characteristics of class I MT. Twenty-one cysteine residues, were arranged in nine Cys-X-Cys motifs, five as Cys-Lys-Cys. A single Cys-X-X-Cys motif was also observed. Two MTs that differ only in the presence or absence of an N-acetyl group exist in this organism. Masses of tryptic peptides of purified MTs corresponded with those of peptides predicted from tryptic cleavages of the deduced amino acid sequence. Allowing for known N-terminal modifications, 96% of the deduced sequence was confirmed by mass spectrometry. Comparison (FASTA algorithm) of the primary structure of the oyster MTs with those of other species indicated a higher similarity with vertebrate MTs than with those of other invertebrates.

 

Roesijadi, G., S. Kielland, et al. (1989). "Purification and properties of novel molluscan metallothioneins." Arch Biochem Biophys 273(2): 403-13.

                Two low-molecular-mass cadmium-induced, cadmium-, zinc-binding proteins were purified from the oyster Crassostrea virginica using procedures that included acetone precipitation, Sephadex gel chromatography, and anion-exchange and reverse-phase high-performance liquid chromatography. Although they could be cleanly separated from each other, they exhibited similar molecular weights, metal and amino acid compositions, and electrophoretic behavior. These proteins were glycine-rich, in addition to being cysteine-rich, and lacked methionine, histidine, arginine, and the aromatic amino acids phenylalanine and tyrosine. Determination of the NH2-terminal amino acid sequence of these molecules showed that they were identical in primary structure in this region and differed only in that one had a blocked NH2-terminal. This provided an explanation for the isolation of two proteins with otherwise identical characteristics. Serine was the NH2-terminal amino acid. The sequence was most similar to that of vertebrate metallothioneins when compared with other proteins, which included metallothioneins from other invertebrate phyla. All cysteines in the first 27 residues of the oyster metallothionein aligned with those in the mammalian forms. On this basis, these proteins were classified as class I metallothioneins.

 

Dallinger, R., B. Berger, et al. (1989). "Purification of cadmium-binding proteins from related species of terrestrial Helicidae (Gastropoda, Mollusca): a comparative study." Mol Cell Biochem 85(2): 135-45.

                Three species of terrestrial Helicidae (Helix pomatia, Cepaea hortensis and Arianta arbustorum) were fed cadmium-rich diet in the laboratory. The snails accumulated high amounts of the metal in their hepatopancreas. Most cadmium and some zinc were found, after centrifugation, in the soluble fractions from which a cadmium-binding protein was isolated for each species by ion exchange and gel chromatography. The proteins contained different amounts of cadmium, but little or no zinc, and showed high absorption at 254 nm indicating the presence of cadmium-mercaptide bonds. After gel filtration, a molecular weight of 12,000 was found for cadmium-binding proteins from Helix pomatia and Arianta arbustorum, whereas a molecular weight of 10,000 was found for a cadmium-binding protein from Cepaea hortensis. SDS-polyacrylamide gel electrophoresis showed one single band for each protein from Helix pomatia and Arianta arbustorum and suggested a molecular weight of 11,000 for both species. Amino acid analysis revealed, for each protein, high amounts of cysteine (12-20%), glycine (15-19%), and serine (12-14%), and moderately elevated contents of lysine (9-13%) and alanine (4-8%), but no methionine and only traces, if any, of aromatic amino acids. The ratios of cadmium to cysteine wer 1:5, 1:10 and 1:3 in the proteins from Helix pomatia, Cepaea hortensis and Arianta arbustorum, respectively. Some features of the isolated proteins resembled mammalian metallothioneins. Most characteristics, however, differed from true metallothioneins and were similar to cadmium-binding proteins found in some marine molluscs.

 

Imber, B. E., J. A. Thompson, et al. (1987). "Metal-binding protein in the Pacific oyster, Crassostrea gigas: assessment of the protein as a biochemical environmental indicator." Bull Environ Contam Toxicol 38(4): 707-14.

               

Lobel, P. B. and J. F. Payne (1987). "The mercury-203 method for evaluating metallothioneins: interference by copper, mercury, oxygen, silver and selenium." Comp Biochem Physiol C 86(1): 37-9.

                Metallothioneins are low molecular weight proteins rich in sulfhydryl groups (cysteinyl) which readily bind various heavy metal cations, e.g. cadmium, copper, gold, mercury, silver and zinc. Mercury has a particular affinity for sulfhydryl groups and mercury-203 has been used as the basis of a rapid, sensitive, radiometric assay for metallothionein. The potential of 16 metals and oxygen for interfering with this test was examined. The mercury-203 test appears to be sensitive to the presence of copper, mercury, oxygen, selenium and silver.

 

Dohi, Y., K. Kosaka, et al. (1986). "Cadmium-binding proteins of three marine molluscs and characterization of two cadmium-binding glycoproteins from the hepatopancreas of a whelk, Buccinum tenuissimum." Environ Health Perspect 65: 49-55.

                The cadmium-binding proteins were shown to exist in the hepatopancreas of three molluscs, a whelk, Buccinum tenuissimum, a turbo, Batillus cornutus, and a squid, Todarodes pacificus. Cadmium was efficiently accumulated in nature to a mean concentration of 119, 33, and 50 micrograms/g wet tissue in the hepatopancreas of three species of molluscs, and 30%, 11%, and 43% of the element in each tissue of whelk, turbo, and squid was extracted to the soluble fraction, respectively. Separation of the soluble fraction by Sephadex G-75 in the presence of 2-mercaptoethanol revealed that cadmium was mainly bound to the protein fraction FII of molecular weight 10,000. Two cytoplasmic cadmium-binding glycoproteins from the hepatopancreas of Buccinum tenuissimum were purified to homogeneity by Sephadex G-75 gel filtration and double DEAE-Sephadex A-25 chromatographies in the presence of 2-mercaptoethanol. These two cadmium-binding glycoproteins, termed FIIA and FIIB, had molecular weights of 8000 and 13,000 and consisted of 52 and 94 amino acid residues, respectively. Three and two cysteine residues in FIIA and FIIB, respectively, were found and two more half-cystine were also detected in FIIB. The sugar contents of FIIA and FIIB were about 20.5% and 8.7% by weight, respectively, consisting of galactose, mannose, fucose, and amino sugar. Both showed strong metal-binding ability, especially for cadmium, copper, and mercury.

 

Fowler, B. A., D. W. Engel, et al. (1986). "Purification and characterization studies of cadmium-binding proteins from the American oyster, Crassostrea virginica." Environ Health Perspect 65: 63-9.

                The previously reported low molecular weight cadmium-binding protein (CdBP) from the American oyster, Crassostrea virginica, has been further purified and characterized by improved technical methods. The internal organ distribution of the protein within the oyster and effects of life cycle/season on CdBP production also have been evaluated. CdBP isolated by extended ion-exchange gradients or double ion-exchange chromatography followed by HPLC analysis possesses an electrophoretic Rf of about 0.7 and contains relatively little Zn, as previously reported. Cysteine, lysine, and glycine are the dominant amino acids. When ion-exchange columns are developed with NaCl gradients, the aromatic residues tryptophan, tyrosine, and phenylalanine are found to be present, but these may be largely removed depending upon whether the protein is denatured and carboxymethylated prior to analysis. The ultraviolet absorption spectrum of CdBP also was variable, with 250/280 nm ratios ranging from 17:1 immediately after ion-exchange chromatography to 2:1 following concentration procedures. Internal organ distribution studies showed that the visceral mass contained most of the Cd present with lesser amounts in the gills and mantle. In contrast with mammals, CdBP accounts for only about 30% of the total cell Cd burden in these tissues. Cu displacement of Cd from the protein is a particular problem during the summer spawning season and appears to stem from altered Cu metabolism during this period. Relative oyster dormancy during the winter also reduces CdBP production in response to Cd, and the protein is obtained most readily during the fall and spring.(ABSTRACT TRUNCATED AT 250 WORDS)

 

Fowler, B. A. and M. M. Megginson (1986). "Isolation and partial characterization of a high molecular weight Cd/Zn-binding protein from the kidney of the scallop Placopecten magellanicus: preliminary studies." Environ Health Perspect 65: 199-203.

                Exposure of the scallop Placopecten magellanicus to 20 ppb Cd2+ in seawater for 7 weeks results in a 7-fold increase in the kidney cytosol content of Cd and 5-fold increase in Zn. Sephadex G-75 column chromatography of the kidney cytosol showed that most of the Cd and Zn were bound to a protein complex with an estimated molecular mass of 45,000 daltons. Further purification of this complex by DEAE A-25 column chromatography disclosed the presence of five peaks with varying degrees of affinity for the ion-exchange resin. One of these peaks (III) was successfully rechromatographed by ion-exchange chromatography and further purified by HPLC using a gel-permeation column. The resultant protein peak which was resistant to disaggregation by 20 mM dithiothreitol gave a preliminary amino acid composition with cysteine, glycine, alanine, and lysine as the major amino acids. The aromatic amino acid phenylalanine was also present. The ultraviolet absorption spectrum gave a 250/280 nm ratio of 2.5:1. Metal analysis of the purified protein showed that it contained Cd, Zn, and Cu in ratios of 1:1:1. Results of these studies indicate that scallop kidney produces a protein complex which appears to share both similarities with mammalian metallothionein with respect to the presence of both Cd and Zn but different with respect to apparent size, amino acid composition, and ultraviolet absorption spectrum.

 

Frazier, J. M. (1986). "Cadmium-binding proteins in the mussel, Mytilus edulis." Environ Health Perspect 65: 39-43.

                Inducible cadmium-binding proteins (Cd-BP) in the mussel, Mytilus edulis, were resolved into two molecular weight components, designated Cd-BP10 and Cd-BP20, by gel-permeation chromatography on Sephadex G-75. Each of these two molecular weight components were further resolved into four subcomponents by DEAE-ion-exchange chromatography. All eight subcomponents bound cadmium and exhibited significant UV absorption at 254 nm and little absorption at 280 nm. Each subcomponent was purified and subjected to amino acid composition analysis. Two classes were identified, one having higher cysteine (23.9-26.6 mole-%) and lower glutamic acid contents compared to the other class (11.6-18.2 mole-% cysteine). All subcomponents have a relatively high glycine content (approximately 15 mole-%) relative to mammalian metallothioneins (approximately 8 mole-%). Although the Cd-BP20 have apparent molecular weights almost twice the Cd-BP10, the exact molecular relationship between these binding proteins is not known.

 

Hennig, H. F. (1986). "Metal-binding proteins as metal pollution indicators." Environ Health Perspect 65: 175-87.

                The fact that metal-binding proteins are a consequence of elevated metal concentration in organisms is well known. What has been overlooked is that the presence of these proteins provides a unique opportunity to reformulate the criteria of metal pollution. The detoxification effect of metal-binding proteins in animals from polluted areas has been cited, but there have been only very few studies relating metal-binding proteins to pollution. This lack is due partly to the design of most experiments, which were aimed at isolation of metal-binding proteins and hence were of too short duration to allow for correlation to adverse physiological effects on the organism. In this study metal-binding proteins were isolated and characterized from five different marine animals (rock lobster, Jasus lalandii; hermit crab, Diogenes brevirostris; sandshrimp, Palaemon pacificus; black mussel, Choromytilus meridionalis; and limpet, Patella granularis). These animals were kept under identical metal-enriched conditions, hence eliminating differences in method and seasons. The study animals belonged to different phyla; varied in size, mass, age, behavior, food requirements and life stages; and accumulated metals at different rates. It is possible to link unseasonal moulting in crustacea, a known physiological effect due to a metal-enriched environment, to the production of the metal-binding protein without evidence of obvious metal body burden. Thus a new concept of pollution is defined: the presence of metal-binding proteins confirms toxic metal pollution. This concept was then tested under field conditions in the whelk Bullia digitalis and in metal-enriched grass.

 

Nordberg, M., I. Nuottaniemi, et al. (1986). "Characterization studies on the cadmium-binding proteins from two species of New Zealand oysters." Environ Health Perspect 65: 57-62.

                Two different types of New Zealand oysters--Ostrea lutaria (OL) and Crassostrea glomerata (CG)--contained different concentrations of zinc, copper, and cadmium. OL oysters had 5.3 micrograms Cd/g, 3.4 micrograms Cu/g, 100 micrograms Zn/g; CG oysters had 1.4 micrograms Cd/g and 936 micrograms Zn/g. Both kinds of oysters were shown by gel filtration (G-75) to contain cadmium and zinc in fractions corresponding to a high molecular weight protein (corresponding to the size of albumin or larger) which was heat labile. OL oysters contained cadmium in fractions corresponding to a molecular weight of approximately 6500. The cadmium-binding protein in these fractions was heat-stable. This protein contained no detectable amounts of zinc and was not present in the CG oysters. Further purification by gel filtration (G-50) was performed to obtain a purer protein fraction. Isoelectric focusing of the protein obtained by G-50 filtration showed one main fraction of protein with a pI approximately 5.9 at approximately 13 degrees C. CG oysters contained cadmium and zinc in a polypeptide with low molecular weight (MW 1000). The cadmium-binding oyster proteins are minimally reactive in a competitive binding radioimmunoassay in comparison to the reactivity of a typical vertebrate metallothionein; the proteins may be metallothioneins, but, if so, they do not exhibit the principal determinants characteristic of vertebrate metallothioneins.

 

Stone, H. C., S. B. Wilson, et al. (1986). "Cadmium-binding proteins in the scallop Pecten maximus." Environ Health Perspect 65: 189-91.

                Scallops, Pecten maximus, accumulate cadmium naturally in the digestive gland to a level of approximately 100 ppm wet weight. Of this cadmium, 60% was soluble and was composed of three weight classes as judged by Sephadex G-100 chromatography. Of the soluble cadmium, 60% was in the 55,000 molecular weight range and 20% each in an excluded fraction and a 10,000 molecular weight fraction. The 55,000 molecular weight fraction, after further purification, showed a maximum cadmium concentration of 1.4% by weight. The cadmium was thiolate bound but not as strongly bound as in the case of metallothionein. The 10,000 molecular weight fraction was a metallothionein-like protein.

 

Evtushenko, Z. S., N. N. Belcheva, et al. (1986). "Cadmium accumulation in organs of the scallop Mizuhopecten yessoensis--II. Subcellular distribution of metals and metal-binding proteins." Comp Biochem Physiol C 83(2): 377-83.

                Cd accumulation in the hepatopancreas and gills of the scallop resulted in an increase of metal concentration in the cytoplasm, while in control animals the general amount of Cd in the hepatopancreas was concentrated in the microsomes and cytoplasm. Cd appears to be equally distributed among high molecular weight proteins and metallothioneins in control animals. It was shown that during the experiment the metal was bound mainly to high molecular weight proteins. After 30 days of exposure of scallops to flowing water Cd was redistributed to metallothionein (MT)-like proteins. Cd concentration in the lipids of the hepatopancreas of control and experimental scallops was equal.

 

Ray, S. (1986). "Bioaccumulation of cadmium in marine organisms." Experientia Suppl 50: 65-75.

                It has been established that, although Cd occurs in the marine environment in only trace concentrations, most marine organisms, especially molluscs and crustaceans, can accumulate it rapidly. Cadmium is not uniformly distributed in the body and selectively accumulates in specific organs like liver, kidney, gills, and exoskeleton. The concentrations in muscle tissues are several orders of magnitude lower. The disposition of Cd in the organisms in the laboratory studies generally parallels those in nature. A number of biotic factors like body size, maturity, sex, etc. influence bioaccumulation but extensive studies are still lacking. The chemical form of Cd in the environment is of prime importance in bioaccumulation by marine organisms. Salinity can affect the speciation of Cd, and bioaccumulation is affected by both temperature and salinity. The ultimate level of Cd in the organisms will depend not only on the biotic and abiotic factors but also on metabolism of the metal by the organisms. A few studies indicate depuration of Cd by some bivalves but other organisms show very effective retention of Cd. Metallothionein formation for detoxification and storage has been observed in a large variety of marine organisms. Recent reports indicate an alternate storage and excretion mechanism in the formation of membrane-limited vesicles or granules. There seems to be a common link between intracellular localisation of Cd in metal-binding proteins and Cd containing vesicles as detoxifying mechanisms in the marine organisms. Much of what is known about Cd bioaccumulation by marine organisms has come from laboratory studies and there are inherent dangers in trying to extrapolate the results to field situations. In spite of tremendous progress made over the years, the basic understanding of the bioaccumulation process is still very nebulous and will remain so until the uptake, storage, and elimination processes are fully understood.

 

Marafante, E., C. Nolan, et al. (1985). "Metabolism in the rat of cadmium biocomplexes from edible mussel exposed to 109CdCl2." Ecotoxicol Environ Saf 9(2): 209-15.

                The metabolism in the rat of 109Cd biocomplexes present in the tissues of the edible mussel Mytilus galloprovincialis, previously exposed to 109CdCl2, was studied. The tissue distribution and binding of 109Cd were compared to those caused by an equal dose of 109Cd as CdCl2 or rat liver Cd-metallothionein. Administration of mussel 109Cd to rats resulted in an initial accumulation of 109Cd in the kidneys due to the presence of 109Cd-metallothionein, which constituted 25% of the 109Cd in the tissues of the mussels. Other 109Cd biocomplexes present in the mussel tissues were metabolized in the rat in a way similar to that of inorganic cadmium, i.e., initial accumulation in the liver. These findings indicate that the ingestion of seafood rich in metallothionein may give rise to a faster increase of renal cadmium levels than the consumption of a similar amount of inorganic cadmium.

 

Frazier, J. M., S. S. George, et al. (1985). "Characterization of two molecular weight classes of cadmium binding proteins from the mussel, Mytilus edulis (L.)." Comp Biochem Physiol C 80(2): 257-62.

                Inducible cadmium binding proteins (Cd-BP) in the mussel, Mytilus edulis, were resolved into two molecular weight components by gel permeation chromatography on Sephadex G-75. Each of these two molecular weight components was further resolved into four subcomponents by DEAE ion exchange chromatography. All eight subcomponents bound cadmium and exhibited significant u.v. absorption at 254 and little absorption at 280 nm. Based on amino acid composition analysis two classes of proteins were identified, one having higher cysteine (approximately 25 mole %) and lower serine and glutamic acid contents compared to the other class.

 

Stone, H. and J. Overnell (1985). "Non-metallothionein cadmium binding proteins." Comp Biochem Physiol C 80(1): 9-14.

                In this review we discuss cadmium binding proteins which are similar to equine metallothionein but have significant differences and also binding proteins which appear to be totally unrelated.

 

Lobel, P. B. and J. F. Payne (1984). "An evaluation of mercury-203 for assessing the induction of metallothionein-like proteins in mussels exposed to cadmium." Bull Environ Contam Toxicol 33(2): 144-52.

               

Dohi, Y., K. Ohba, et al. (1983). "Purification and molecular properties of two cadmium-binding glycoproteins from the hepatopancreas of a whelk, Buccinum tenuissimum." Biochim Biophys Acta 745(1): 50-60.

Buccinum tenuissimum에 대한 읢¥ë¯¢¬ì¡×€ ê©÷€ìƒ‰ê©÷¡Æê©ø¨ù                Two cytoplasmic cadmium-binding glycoproteins from the hepatopancreas of a whelk, Buccinum tenuissimum, were purified to homogeneity by Sephadex G-75 gel filtration and two DEAE-Sephadex A-25 chromatographies in the presence of 2-mercaptoethanol. These two cadmium-binding glycoproteins, termed FIIA and FIIB, each showed a single band after electrophoresis on 10% polyacrylamide gel at pH 8.9. The molecular weight of FIIA was estimated as 8000 and that of FIIB as 13000 by SDS-polyacrylamide gel electrophoresis. Based on molecular weights of 6500 and 11900 for the protein moiety of FIIA and FIIB, respectively, the total number of amino acid residues was 52 in the former and 94 in the latter. Three and two cysteine residues in FIIA and FIIB, respectively, were titratable with p-chloromercuribenzoate. FIIB also contained two more half-cystine residues. The sugar contents of FIIA and FIIB were about 20.5% and 8.7% by weight, respectively, consisting of galactose, mannose, fucose and amino sugar. The purified glycoproteins FIIA and FIIB contained about 0.6% and 1.0% cadmium by weight, respectively, and both showed strong metal-binding capacity, especially for cadmium, copper and mercury. The apparent cadmium dissociation constants for FIIA and FIIB after treatment with 2-mercaptoethanol were 7.3 X 10(-6) and 9.1 X 10(-7) M, respectively. Cadmium contents at saturation were nearly 6 and 8 gatom per mole for FIIA and FIIB, respectively.

 

Siewicki, T. C., J. S. Sydlowski, et al. (1983). "The nature of cadmium binding in commercial eastern oysters (Crassostrea virginica)." Arch Environ Contam Toxicol 12(3): 299-304.

               

Bray, J. T., L. A. Webb, et al. (1983). "Multielement analysis of metal-binding proteins in cytosol fractions." Sci Total Environ 28: 367-74.

                The distribution of 24 elements among the cytosol protein fractions was determined for specimens of the bivalve mollusc Macoma balthica obtained from three estuarine locations subject to varying levels of metal pollution and on specimens of Rangia cuneata from three areas subject to varying degrees of thermal pollution. Of the 24 elements examined 15 occurred at levels above detection limits and in association with one or more of four distinct protein fractions. Levels of Ag and Cu associated with high molecular weight proteins and with "metallothionein-like" proteins permitted identification of those Macoma balthica specimens exposed to the greatest degree of metal stress.

 

Engel, D. W. (1983). "The intracellular partitioning of trace metals in marine shellfish." Sci Total Environ 28: 129-40.

                Marine organisms have evolved a number of metabolic strategies to deal with potentially toxic trace metals. To determine how "detoxification" mechanisms such as metal-binding proteins and concretions are involved in the intracellular partitioning of trace metals in marine shellfish, the oyster, Crassostrea virginica, and the blue crab, Callincetes sapidus, were exposed to controlled levels of trace metals. Oysters accumulated cadmium readily and produced specific low molecular weight, cadmium-binding proteins. Blue crabs produced a single cadmium-binding protein in the hepatopancreas and gills when exposed to cadmium through food or water, respectively. Concretions appear to be of limited importance in detoxification.

 

Sharma, R. P. (1983). "Ligands binding cadmium, zinc, and copper in a species of New Zealand oyster (Ostrea lutaria)." Bull Environ Contam Toxicol 30(4): 428-34.

               

George, S. G. (1983). "Heavy metal detoxication in the mussel Mytilus edulis-composition of Cd-containing kidney granules (tertiary lysosomes)." Comp Biochem Physiol C 76(1): 53-7.

                A new method for isolation and purification of lipofuschin granules (tertiary lysosomes) from mussel kidney is described. The results confirm previous ultrastructural evidence for the accumulation of Cd in these organelles. Granules from Cd-exposed animals contain higher concentrations of protein, Ca, Cd and S than those from control animals. Gel filtration experiments indicate that secondary lysosomes contain metallothionein whilst only low molecular weight Cd-complexes can be solubilised from tertiary lysosomes. Amino acid analysis of Cd-containing tertiary lysosomes show that they are not enriched in cysteine, indicating the absence of insoluble polymerised metallothionein.

 

Carpene, E. and G. Hakim (1981). "[Covalent chromatography of Mytilus edulis L. thioneine using thiosepharose 4B]." Boll Soc Ital Biol Sper 57(4): 421-7.

               

George, S. G., E. Carpene, et al. (1979). "Characterisation of cadmium-binding proteins from mussels, Mytilus edulis (L), exposed to cadmium." Biochim Biophys Acta 580(2): 225-33.

                Three Cd2+-binding proteins have been purified and partially characterised from the digestive gland of the bivalve mollusc, Mytilus edulis, after exposure to Cd2+. The major protein, which was judged to be pure on polyacrylamide gel electrophoresis, showed many of the characteristics of mammalian metallothionein; having a high -SH content, few aromatic amino acids and a high A250/A280 nm ratio which disappears on acidification. It also contains Zn and Cu, but differs in its higher apparent molecular weight of about 25 000 and high glycine content (12-19%). The two additional Cd2+-binding proteins had lower cysteine contents and different molar proportions of Cd2+, Zn2+ and Cu2+.

 

George, S. G. and B. J. Pirie (1979). "The occurrence of cadmium in sub-cellular particles in the kidney of the marine mussel, Mytilus edulis, exposed to cadmium. The use of electron microprobe analysis." Biochim Biophys Acta 580(2): 234-44.

                In mussels (Mytilus edulis) chronically exposed to cadmium, 85% of the Cd2+ was found to be associated with membrane-limited granular structures when elemental analyses were carried out on cryo-sectioned tissue by electron probe X-ray microanalysis. These granules also contained high concentrations of sulphur and phosphorus as well as other metalions, including Ca2+, iron and Zn2+. In contrast, after homogenisation and fractionation by differential centrifugation, the major proportion of the Cd2+ was found in the cytoplasmic fraction. However, many lysosomes were also ruptured by this treatment. Gel filtration chromatography of this fraction indicated the presence of a Cd2+-binding component of similar molecular weight to the metallothionein purified from the digestive gland of the same animals. It is therefore proposed that metallothionein may be associated with particulate structures which would thus reduce its cellular toxicity.

 

Olafson, R. W., R. G. Sim, et al. (1979). "Physiological and chemical characterization of invertebrate metallothionein-like proteins." Experientia Suppl 34: 197-204.

                Metallothionein-like proteins have been isolated from marine invertebrates. Three crustaceans, Scyllus serratus, Cancer magister and Acetes sibogae together with a mollusc, Cryptochiton stelleri, have been investigated. S. serratus hepatopancreas was shown to contain cadmium and zinc inducible metallothionein-like proteins with an amino acid analysis very similar to vertebrate metallothioneins. The molecular weight, ultra-violet spectrum and isoelectric points of S. serratus metallothioneins are also comparable to the vertebrate proteins, suggesting a fundamental biological function conserved throughout evolution.

 

Talbot, V. and R. J. Magee (1978). "Naturally-occurring heavy metal binding protein in invertebrates." Arch Environ Contam Toxicol 7(1): 73-81.

                Low molecular weight cadmium, copper, and zinc binding protein has been isolated from the mussel Mytilus edulis in the cadmium polluted areas of Corio Bay adjacent to Melbourne. Its molecular weight and other properties suggest that it belongs to the metallothionein family of metal binding proteins. Its synthesis may have been induced in the cyctoplasmic solutions of the cells as a regulatory mechanism to avoid intoxication by excess uptake of any of the metals.

 

Howard, A. G. and G. Nickless (1977). "Heavy metal complexation in polluted molluscs II. Oysters (Ostrea edulis and Crassostrea gigas)." Chem Biol Interact 17(3): 257-63.

                Cadmium, zinc and copper, accumulated from polluted habitats by the oysters Ostrea edulis and Crassostrea gagas, were studied. Three distinct low molecular weight (less than 3000 daltons) zinc complexes were separated from highly contaminated C. gigas, one of which may be a complex with the amino acid taurine. Only one of these zinc complexes was present in less contaminated specimens of O. edulis. On gel permeation chromatography of oyster extracts, copper was eluted together with amino acids (principally taurine) and the betaine homarine. No evidence of metallothionein type proteins was found.

 

Howard, A. G. and G. Nickless (1977). "Heavy metal complexation in polluted molluscs. I. Limpets (Patella vulgata and Patella intermedia)." Chem Biol Interact 16(1): 107-14.

                The accumulation of cadmium, zinc and copper by the marine gastropod molluscs Patella vulgata and Patella intermedia has been studied by gel permeation chromatography of water-soluble extracts of environmentally contaminated shellfish. A major proportion of the water-soluble cadmium and copper in these molluscs is associated with a protein of molecular weight 10 800 daltons. Evidence is presented supporting the similarity of this protein with mammalian metallothionein. This protein contained only a small proportion of the zinc found in the samples.

                 The large volumes of oily wastewater discharged to marine environment cause heavy impacts on the coastal marine ecosystem. The selection of an appropriate technology to reduce these impacts should be based on the respect of the discharge limits and on the effective assessment and monitoring of its effects on biological organism preservation. To this aim, we set up a controlled microcosm-scale system to compare the effects of a treated and untreated oily wastewater discharge in which the restore process is performed through a Membrane Bio-Reactor. The system is completed by other three microcosms to control and isolate any possible concurrent effect on the Mytilus galloprovincialis, used as sentinel organism. Mytilus galloprovincialis have been kept in all these microcosms, and then mRNA expression and morphology were evaluated on gills and digestive gland. The genes considered in this work are Heat Shock Protein 70 and Metallothionein 10, involved in response to physicochemical sublethal stressors, Superoxide dismutase 1, Catalase, and Cytochrome P450 involved in oxidative stress response. Our results evidenced a significant overexpression, both in gills and digestive gland, of HSP70 in samples maintained in the microcosm receiving the untreated effluent, and of MT10 in those animals kept in microcosm where the effluent was treated. Even though the mRNA modifications are considered "primary" and transient responses which do not always correspond to protein content, the study of these modifications can help to gain insights into the mechanisms of action of xenobiotic exposure. Morphological analysis suggested that, although different, depending on the microcosm, the most serious damages were found in the gill epithelium accompanied with severe haemocyte infiltration, whilst in digestive gland the tissue architecture alterations and the haemocyte infiltration were less pronounced. These observations suggest that the immune system was activated as a general response to stressful stimuli such as the presence of toxic compounds. Moreover, the results indicate that the treatment process is useful. In fact, samples derived from the microcosm receiving the treated effluent, even though presenting some signs of stress, seemed to partially recover the normal structure, although their mRNA expression indicated some cellular suffering.