A comparison of metal-binding proteins and cadmium metabolism in the marine molluscs Littorina littorea (gastropoda), Mytilus edulis and Macoma balthica (bivalvia)

Studies on the processes of Cd accumulation, turnover and storage in marine molluscs have revealed important qualitative and quantitative differences between species. In Littorina littorea collected from uncontaminated sites, Cd is predominantly bound to metallothionein (MT)-like proteins. Exposure to Cd, in the laboratory and field, results in an increase in Cd bound to these proteins, together with limited increases in Cd in the high molecular weight (HMW) pool (the latter partly a result of haemocyanin-mediated transport of Cd in the haemolymph). Polarographic determination of heat-stable MT-like compounds in L. littorea indicates there is little overall increase in the amount of protein produced in response to Cd exposure, suggesting that much of the newly incorporated metal may be bound to previously synthesised material. Relatively high levels of MT-like proteins, notably in the digestive gland (the major Cd-storage organ), appear to be an inherent feature of this species even in uncontaminated areas.MT-like proteins also sequester much of the Cd in exposed mussels Mytilus edulis, though, unlike Littorina, protein induction can be quantitatively related to exposure. In contrast, tellinid clams, Macoma balthica, fail to produce MT-like compounds, even in highly contaminated conditions, and Cd is mainly bound to HMW proteins.These variations in metal-binding behaviour probably explain the large interspecific differences in experimentally-determined Cd uptake rates and may account for differences in bioaccumulation potential observed in the field. Despite such variability, all three species studied appear to tolerate an influx of Cd provided the normal partitioning of metal is not irreversibly disturbed. Maintaining intracellular homeostasis, albeit at elevated Cd concentrations, probably compensates for the inability of these animals to regulate Cd uptake and for their extremely slow rates of Cd excretion.     

Metallothionein in liver of eels Anguilla anguilla from the Thames Estuary: an indicator of environmental quality?

The purpose of this study was to examine the role of the cysteine-rich protein metallothionein (MT) in the detoxification and metabolism of metals in livers of eels Anguilla anguilla from the Thames Estuary, and to assess the value of MT measurements in environmental monitoring. Hepatic MT levels and associated metal concentrations were determined in A. anguilla collected on three occasions during 1998 at sites along the tidal Thames (from freshwater to the sea) and also from a reference site (Tamar Estuary) in southwest England. MT was present at basal levels of 2 mg g(-1) dry weight (dw), predominantly in association with the essential metals Cu and Zn. MT concentrations were variable within each eel 'population' but were generally highest (up to 11 mg g(-1) in individual eels) at the more contaminated upper- and mid-estuarine sites in the Thames (e.g. Brentford and Kew). Lowest MT levels were those in eels from Blythe Sands at the mouth of the estuary. Season, sex, reproductive status and salinity may have influenced MT levels to some extent, although metals were the most significant factor. Hepatic MT concentrations were highly correlated with associated metal burdens (Zn, Cu, Ag and Cd),--notably reflecting Cu and Ag enrichment in the upper- and mid-estuary. With the exception of Zn, metals in the supernatant fraction of eel livers were primarily associated with MT. Proportions of Cu, Ag and Cd bound to MT increased as a function of accumulated metal burdens, with no indication of saturation. Thus, despite causing induction of MT, excess bioavailable Cu, Ag and Cd appear to be successfully detoxified in eels over the range of environmental contamination encountered along the Thames Estuary. Paradoxically, it may be argued from conventional doctrine that the presence of raised levels of MT, at upstream sites, constitutes a response to contamination and, by definition, signals that the fish are affected by metals; however, obvious signs of deleterious effects were not detected. Superficially, from liver MT data, it would seem that eel populations have become biochemically adapted to metal contamination. The approach tested here has proved viable in helping to interpret trends in bioavailability and the toxicological significance of accumulated metal burdens in Thames eels. A. anguilla is a common inhabitant of estuarine and fresh waters throughout Europe, and related species occur world wide. Therefore, determination of hepatic MT (and associated metals) in eels could be useful for monitoring responses to metal exposure and environmental quality on a much broader basis. Ideally these determinations should be performed in tandem with other sublethal-effects measurements to maximise information.     

Interactions of cadmium with sulfhydryl-containing compounds in striped mullet (mugil cephalus L.)

Interactions between cadmium and sulfhydryl-containing compounds in striped mullet, Mugil cephalus, were examined. Exposure to 10 mg Cd/liter for up to 6 weeks caused an increase in the acid-soluble thiol (AST) content of the liver. Although the AST concentration was elevated at 1·4 weeks, increases in the glutathione and cysteine contents were not observed until after 4 weeks of exposure. Homogenates of livers were fractionated on a Sephadex G-75 column. A homogenate of livers of mullet injected with ¹⁰⁹Cd 2 days prior to sampling had an elution profile for thiols similar to controls. After 3 weeks exposure to 10 mg Cd/liter the 9400 MW thiol peak increased 3·6-fold and the peak with the same retention volume as glutathione increased 4·3-fold. Greater than 70% of the thiols in these two peaks were acid-soluble. In both experiments, almost all the cadmium was associated with the 9400 MW peak. The ratio in the 9400 MW peak declined from 14·1 (controls) to 3·2 after 3 weeks exposure. No cadmium was associated with glutathione in the bile.     

The impact of metals on the reproductive mechanisms of the ascidian Ciona intestinalis

Environmental pollution due to anthropogenic activities may exert an adverse impact on the reproductive mechanisms of animals. In particular, environmental chemicals introduced into seawater are able to disrupt the normal development and function of the reproductive system of marine animals. In this study, we have used the whole‐cell voltage clamp technique to examine the effects of four metals – lead, cadmium, mercury, and zinc – on reproductive mechanisms of the marine ascidian Ciona intestinalis (tunicates). In particular, we measured the effect of metals on plasma membrane electrical properties, the steady‐state conductance, the fertilization current in the mature oocyte, and larval development. Results show that oocyte voltage‐gated sodium currents are significantly reduced by all four metals, steady‐state conductance is affected only by zinc, and post‐fertilization contraction is inhibited only by lead. The fertilization current is suppressed in the presence of zinc and mercury. Embryo development up to larval stage is inhibited by zinc and mercury exposure with a reversible effect; however, a long‐term effect on larval morphology was observed. After exposure to cadmium, fertilization occurs but gives rise to abnormal larval development. These findings highlight the point that exposure to metals represents a significant risk factor for the physiology of reproduction of marine species and suggest a possible role of the C. intestinalis as a bioindicator for marine pollution monitoring.     

Metallothionein and mercury tolerance in the killifish, fundulus heteroclitus

Synthesis of metallothionein (MT) as a possible mechanism by which Fundulus heteroclitus adapts to heavy metal pollution has been investigated in relation to responses to methylmercuric chloride (meHg) and HgCl₂(Hg²⁺) at different life stages.Clutches of embryos from unpolluted sites exhibit teratogenic responses to a standard dose of meHg or of Hg²⁺ which range from little or no effect (tolerant) to severely affected or almost complete inhibition of development (susceptible). At the time of hatching, untreated embryos from tolerant clutches had twice as much of a protein which co-migrates in SDS-PAGE gels with Fundulus MT as do those of susceptible clutches. This difference could be a mechanism for conveying tolerance to Hg²⁺ and also to meHg if demethylation occurs. However, eggs at the time of deposition had virtually no MT, suggesting that the protein is synthesized too late to provide tolerance in the early stages of development which are critical in the etiology of malformations. Treatment of embryos with non-teratogenic doses of either meHg or Hg²⁺ did not increase the amount of MT-like protein. This may indicate that the thionein gene is already fully active, and would also explain observations that Hg²⁺ pretreatment of embryos does not enhance tolerance to Hg²⁺ in the larval stage.MT, isolated from livers of Hg²⁺- or Cu²⁺-dosed or control adults, is associated with Cu and Zn but not with Hg, although it can bind Hg²⁺in vitro. Fundulus, like other fish, methylates much (ca. 75 %) of its Hg burden, and meHg is considered by some not to react with MT. Fundulus liver has its Hg burden distributed between homogenate sediment (25 %), the cytosol (60 %) and the remainder in lipid. The liver responds to Hg²⁺ treatment by increasing lipid content. This may allow sequestration of some lipid-soluble meHg. Of the cytosolic Hg, most is associated with larger proteins and some (ca. 25 %) is dialyzable.These data suggest that metallothionein is not a significant factor in Hg tolerance in Fundulus adults, and has a questionable role in embryonic and larval tolerance to Hg²⁺ and none to the more toxic meHg.     

Diatom assemblages from surface sediments of the Río de la Plata estuary,Uruguay

The Río de la Plata estuary (RdlP) exhibits environmental gradients associated with the freshwater input and oceanic water intrusion. The aim of this study was to assess diatom species distribution in surface sediment samples related to such environmental gradients. The internal section of RdlP was dominated by Aulacoseira spp., Eunotia spp., Staurosirella martyi, Actinocyclus normanii and Thalassiosira baltica, indicatives of low salinity levels and high trophic conditions, associated with the riverine and estuarine regimes. The external section was dominated by Coscinodiscus radiatus, Thalassiosira spp., Paralia sulcata, Cyclotella striata, among other marine taxa, indicatives of high salinity and low trophic conditions, associated with the influence of the Southwestern Atlantic Ocean. Furthermore, the intermediate section presents a mixture of both diatom groups representing mixing conditions. The observed diatom species groups capture fairly well the RdlP environmental variability and can be reliably used for paleoenvironmental studies in this and other similar estuarine systems.     

Assessment of potential biomarkers, metallothionein and vitellogenin mRNA expressions in various chemically exposed benthic Chironomus riparius larvae

The objective of this study was conducted to identify the possibility of using Chironomus metallothionein (MT) and vitellogenin (VTG) as biomarkers of stress caused by endocrinedisrupting chemicals (EDCs), heavy metals, herbicides and veterinary antibiotics. We characterized the MT and VTG cDNA in Chironomus riparius and evaluated their mRNA expression profiles following exposure to different environmental pollutants. The gene expression analysis showed that the MT mRNA levels increased significantly after long-term exposure to cadmium (Cd), copper (Cu), Lead (Pb), di(2-ethylhexyl) phthalate (DEHP), and 2,4-dichlorophenoxyacetic acid (2,4-D). Moreover, the VTG mRNA expression increased significantly in C. riparius larvae exposed to BPA, NP, DEHP, Cd, 2,4-D and fenbendazole. Evaluation of the long-term effects of environmental pollutants revealed up regulation of Chironomus MT mRNA in response to DEHP exposure among EDCs, and the level of the VTG mRNA was increased significantly following treatment with Cd and herbicide 2,4-D at all concentrations in a dose-dependent manner. These results indicate that VTG could be used as a potential biomarker of herbicide and Cd as well as EDCs, while MT was a potential biomarker of heavy metals such as Cd, Cu, and Pb in aquatic environments.     

Effects of water-borne copper on metallothionein and lipid peroxidation in the marine amphipod gammarus locusta

The aim of the current work was to determine over 10 days the effects of water-borne exposure of the marine amphipod Gammarus locusta to 4 μgCu l⁻¹ on the metallothionein (MT; measured by differential pulse polarography) protection system and lipid peroxidation (LP; thiobarbituric acid-reactive malondialdehyde equivalents) as a measure of oxidative damage. MT levels in exposed animals increased significantly at day 2 (36% > control; P<0.001) and remained high at days 6 and 10 (55 and 38%, respectively, P<0.001). The maximum level of MT at day 6 coincided with the highest Cu body-burden. LP increased within 1 day of exposure, indicative of Cu as an oxidative stressor. However, in contrast to MT, the highest LP level was seen at day 4 (68% > control, P<0.001) before returning to control values by day 6, indicating a protective role of MT against the pro-oxidant effects of Cu.     

Induction of heat shock protein (hsp)60 in Isochrysis galbana exposed to sublethal preparations of dispersant and Prudhoe Bay crude oil

Adaptation to sublethal exposure to crude oil by phytoplankton is poorly understood. Use of chemical dispersants for oil spill remediation increases petroleum hydrocarbon concentrations in water, while exposing marine organisms to potentially toxic concentrations of dispersant. Heat shock proteins (hsps) have been found to serve as an adaptive and protective mechanism against environmental stresses. The objective of this project was to examine the induction of hsps in Isochrysis galbana, a golden-brown algae, following exposure to the water-accommodated fraction (WAF) of Prudhoe Bay crude oil (PBCO) and PBCO chemically dispersed with Corexit 9527® (dispersed oil: DO). Initial experiments using ³⁵S-labeled amino acids and 2-dimensional electrophoresis with subsequent western blotting identified and confirmed hsp60, a member of the chaperonin family of stress proteins, as being efficiently induced by heat shock in this species. One-dimensional SDS PAGE and western blotting, with hsp60 antibodies and chemiluminesence detection, were used to quantitate hsp60 following exposure to a range of environmental temperatures and concentrations of WAF and DO preparations. I. galbana cultured in 22 parts per thousand (‰) salinity showed a statistically significant increase (p<0.05) in hsp60 after exposure to 25, 30, and 35°C for 1 h compared to controls (20°C), while 34‰ cultures showed no significant increase in hsp60 with increasing temperature. Cultures (22 and 34‰) exposed to varying doses of WAF and DO exhibited statistically significant (p<0.05) increases in levels of hsp60 in all cases. A dose-related response was observed in all exposures (r²≥0.90) with the exception of 34‰ WAF. In independent exposures, a dose-related hsp60 response to naphthalene, a relatively abundant water-soluble aromatic hydrocarbon, was observed in both 22 and 34‰ cultures. Results of this study are consistent with previous studies in other species documenting increases in hsp60 levels with exposure to xenobiotics. Further studies are investigating the protective function of hsp60 against the toxic effects of exposure to WAF and DO preparations.     

Quantification of cadmium-induced metallothionein in crustaceans by the silver-saturation method

Metallothionein (MT) has been proposed as a specific biochemical probe for metal exposure of aquatic organisms. It is recognized that its usefulness as a monitoring tool depends on the full understanding of its function and on the possibility of measuring its concentration in tissues. Therefore the study of MT in crustaceans is interesting from two different points of view: the need to understand mechanisms associated with the toxicology of metals, and the potential use of this protein for monitoring metal-contamined environments. Several methods have been developed for quantitative measurements of MT in biological samples. Immunochemical and electrochemical procedures are reliable techniques but they require sophisticated equipment and highly trained staff. In the present work, modifications to the silver-saturation method have been developed for the quantitative determination of MT in crustacean tissues, estimating silver concentrations by flame in an atomic absorption spectrophotometer. Cadmium and MT were measured both in Artemia and Procambarus clarkii after sublethal exposure to cadmium. An increase in MT content was recorded in crustacean tissues after 12 h of cadmium exposure. A high responsiveness was found in MT induction by cadmium in the two crustaceans and there was a clear relationship between cadmium concentration in water and MT levels in tissues. The silver-saturation assay may be a rapid assay technnique for biomonitoring purposes.     

Immunodetection of hepatic peroxisomal PMP70 as an indicator of peroxisomal proliferation in the mummichog, Fundulus heteroclitus.

Peroxisomes are important sites for beta-oxidative fatty acid metabolism and peroxidative detoxification. Agents causing peroxisomal proliferation have been associated with reproductive and developmental toxicity and hepatocarcinogenesis. Female mummichog (Fundulus heteroclitus) were exposed to waterborne 2,4-dichlorophenoxyacetic acid (2,4-D), a model peroxisome proliferator, at sublethal concentrations of 0.01, 0.10, and 1.00 ppm or dimethyl sulfoxide (DMSO) vehicle for 7, 14, or 21 days. A polyclonal antibody to rat PMP70 protein (70 kDa peroxisomal membrane protein, a major component of peroxisomes and member of the ABC transporter superfamily) was used for Western blotting after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to determine possible peroxisome proliferation. Significant increases of an approximately 70-kDa protein band recognized by anti-PMP70 were observed on all days, especially at the highest exposure concentration. The data suggest that immunoassay of PMP70 is a useful biomarker assay for peroxisome proliferation, and may be applicable to a wide range of species. The response also suggests that this assay could be used for measuring chronic exposures to environmental peroxisome proliferating agents.     

Relationship between visible and heritable chromosome damage in trout cells and embryos

The disruption of chromosome segregation which is detected visually by the anaphase aberration (aa) test suggests that unequal amounts of DNA are distributed to daughter cells and possibly to subsequent cell generations. To investigate this possibility trout cell cultures and trout embryos (blastodisc) were exposed to benzo[a]pyrene (B[a]P) and the nitrosamide, MNNG, respectively. They were then examined by flow cytometry to determine if anaphase damage resulted in unequal DNA distribution to daughter cells. Both B[a]P and MNNG produced a significant increase (P < 0·01) in aa in cultured cells after 48 h exposure. These values returned to normal by 10 days in the absence of the genotoxic agents, except for the highest concentration (0·5 μg/ml MNNG), which showed only a 50% recovery by that time. Likewise, the coefficient of variation (CV) of nuclear DNA content of the cells also rose significantly after treatment and remained elevated for as long as 14 days following exposure. Experiments with rainbow trout embryos also showed a significant increase in both aa and CV following exposure to MNNG. Flow cytometric analysis of DNA content of trout cells and embryos following exposure to mutagens showed an unequal distribution of DNA that was transmissible through several cell generations. These findings indicate that visible anaphase aberrations could predict heritable genetic defects such as those associated with aneuploidy.     

Biology of three Malacostraca (Decapoda) in a Mediterranean lagoon with particular emphasis on the effect of rapid environmental changes on the activity (catchability) of the species

The activity of three Malacostraca (Decapoda), Crangon crangon, Carcinus aestuarii and Paleamon squilla, was studied following rapid environmental changes in a Mediterranean lagoon linked temporarily with the sea. Nekton was sampled every 2–3 days using fixed fyke nets. A canonical analysis and a stepwise multiple regression were computed to determine which factor(s) can predict or account for the catch of these three decapods. In spite of rapid environmental changes no exceptional mortality was observed in each of the three species. Temperature is the environmental factor with major impact on the activity of C. crangon. Activity of P. squilla is controlled by water depth. At a depth of less than 20 cm two factors, salinity and phase of the moon, have a significant influence on the activity of this species. At depths deeper than 20 cm the activity is inhibited. There is only a weak relationship between the catch of C. aestuarii and salinity. Additional data on the life cycle of these species are given. The limitations of a monthly sampling period are discussed and the need for more short-term, consecutive sampling periods is stressed in order to understand the dynamics of systems with rapid environmental fluctuations.     

海水盐度变化影响裂片石莼(Ulva fasciata)(Ulvales, Chlorophyta)幼体的生长和光合作用

Seawater salinity is greatly influenced by tide, evaporation and rain falls. In this study, we investigated the growth and photosynthetic responses of zygote-derivedUlva fasciata Delile germlings to sh...     

Natural modulation of hepatic metallothionein and cytochrome P4501A in flounder, Platichthys flesus L.

The use of biomarkers such as cytochrome P4501A (CYP1A) or metallothionein (MT) for pollution monitoring is based on the assumption that an increased activity or concentration is primarily caused by exposure to contaminants. Previous studies have shown that the response of biomarkers may be affected by factors such as season, temperature, gender, nutritional status or size. The objective of the present study was to identify natural factors that affect the hepatic activity of CYP1A and hepatic concentration of MT in flounder (Platichthys flesus L.). Thirty flounder were sampled at each of two sites in the Hvaler archipelago, southern Norway, monthly through one year. A set of variables were recorded for each sampling and individual including water temperature, salinity, external and internal lesions, intestinal content, sex, size and organ weights. Hepatic CYP1A activity (EROD), MT and metal concentrations were determined for each individual flounder. The influence of environmental and endogenous factors on the response of these two biomarkers was assessed in multiple regression models. For both biomarkers, 50–60% of the total variability could be explained from factors directly related to season, gender and maturation. Season contributed significantly in the model for EROD, as did external lesions. Size, condition and diet did not contribute greatly when the above factors were included. The results confirm previous findings that season, gender and maturation must be taken into account in biomarker monitoring, but also indicate that other factors such as external lesions should be considered.     

Metallothionein-like proteins induced by cadmium stress in the scallop Mizuhopecten yessoensis

Organisms have evolved a cellular response called stress protein response that increases their tolerance in adverse environmental conditions. Well known stress proteins that bind essential and toxic metals are metallothionein (MT). The scallop Mizuhopecten yessoensis is the most interesting organism because it is able to accumulate toxic cadmium in its digestive gland. However, in the tissue of the digestive gland of Mizuhopecten yessoensis MT (metallothioneins) have not been found. Eastern scallops, Mizuhopecten yessoensis, were collected from two locations ?? one clean and one polluted site. The concentrations of cadmium (Cd), copper (Cu) and zinc (Zn) were measured in the digestive gland. There was a significant increase in Cd concentrations in this studied tissue. We found that in the presence of cadmium Mizuhopecten yessoensis can induce high molecular proteins. The results of experiments have shown that Cd-binding ligands have a number of properties similar to MT: acetone and temperature stability; the ability to bind some metals, including Cd, Cu and Zn. Protein chromatography (FPLC, Superosa 12) from the digestive gland of scallop M. yessoensis has shown that cadmium is associated with high molecular weight Cd-binding proteins (72 kDa and 43 kDa). The major cadmium-binding protein 72 kDa is glycoprotein. In experiments we have demonstrated that Cd-binding proteins can be induced when there is cadmium exposure. The results of this study strongly suggest that the far eastern scallop Mizuhopecten yessoensis has a unique and well-developed system for the detoxification of heavy metals and it allows for biochemical systems to be maintained in a relatively stable manner in the presence of heavy metals.     

盐度对不同卤虫品系存活和生殖性能的影响

作者采用中国渤海湾盐田固有的渤海湾孤雌生殖卤虫(Bohai Bay Artemia parthenogenetica,BHB)和分别源自美国旧金山湾盐田(SFB)与越南Vinh Chau 人工养殖(VSFB)的两性生殖卤虫(A. franciscana),研究盐度(分别为70, 125 和180)对上述品系卤虫存活和生殖力的影响。结果表明, 在实验盐度范围内, 所有品系卤虫存活率随盐度升高而显著降低, 但与BHB 卤虫相比, SFB 和VSFB 卤虫对高盐度的耐受力较强。盐度显著影响BHB 卤虫性成熟时间和产后代间隔时间, 并随盐度的升高而延长,但对SFB 和VSFB 卤虫没有显著影响。所有品系卤虫每窝产幼体或产卵数量均随盐度升高而有降低的趋势。较低盐度(70)下, BHB 卤虫以产幼体占绝对优势, VSFB 产幼体较多, SFB 产卵较多; 而在较高盐度(125 和180)下, VSFB 以产幼体占绝对优势, SFB 和BHB 也以产幼体居多。总之, 与SFB 和BHB 相比, VSFB 在高盐环境下存活率较高, 且以产幼体占绝对优势, 表明VSFB 卤虫更适应于高盐盐田环境,有利于该品系卤虫在盐场建立优势种群。     

Factors Affecting Long-term Trends in the Estuarine Abundance of Pogge (Agonus cataphractus )

Long-term data assembled from a power station on the Thames estuary were used to model fluctuations in the abundance of pogge (Agonus cataphractus) as a function of estuarine environmental, seasonal and prey availability variables using multiple regression. Temperature was the most important determinant of abundance, with seasonal variables and changes in the relative abundance of age-0 dab, Limanda limanda, possibly as prey, also holding important consequences for pogge abundance. Pogge abundance was also moderately influenced by: salinity, flow, salinity-flow interactions, changes in the relative abundance of Dover sole, Solea solea, and a trend variable. Regular patterns of seasonal (spring/winter) occurrences were associated with the use of warmer estuarine waters to optimize growth and reproductive potential and/or correlated with inshore spawning migrations and the availability of prey species. Both mechanisms suggest opportunistic use of estuarine habitats by pogge. Available temperature-abundance relationships further suggest a well-defined estuarine thermal niche, with an optimum at 9·5 °C. There remains a requirement for improved information on the biology of pogge and factors governing the population dynamics of this important estuarine species.     

Response of antioxidant systems to copper in the gills of the clam Ruditapes decussatus

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 μg 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 μg Cu/l, the excess of Cu triggers the induction of MT synthesis after 3 days of exposure.