Changes in metallothionein concentrations in response to variation in natural factors (salinity, sex, weight) and metal contamination in crabs from a metal-rich estuary

Intermoult male and female crabs Pachygrapsus marmoratus and Carcinus maenas were sampled from three sites between the mouth and 25 km upstream in the Gironde, the most Cd-contaminated estuary in France, in order to study the relative importance of natural factors (salinity, sex, weight) and accumulated metal concentrations on metallothionein (MT) concentrations. In the two species studied, higher metal, total protein and MT concentrations were observed in the hepatopancreas than in the gills. In P. marmoratus, MT concentrations were mainly related to changes in the natural factors even if MT and Zn concentrations were positively correlated in the hepatopancreas whereas in C. maenas, the main relationships were with accumulated metal levels. In the case of the natural factors, the most important ones were weight in gills of both crab species, and salinity changes in both hepatopancreas and gills of P. marmoratus. Cd and Cu concentrations in both organs of the two species were inversely related to salinity. The same observation was found for Zn concentrations in C. maenas but not in P. marmoratus. In the hepatopancreas of both species, the highest total protein concentrations were found in crabs from the site with the highest salinity, whereas there were no such differences in the gills. It seems that changes in MT concentrations are linked more to changes in general protein metabolism than to changes in metal accumulation. Thus it was important to examine the storage of metals in other tissue compartments, particularly the insoluble fraction which includes mineral granules which is known to also contribute to trace metal detoxification in invertebrates. In the gills of the crabs, Zn was present mainly in the insoluble fraction, whereas Cd was nearly equally distributed between soluble and insoluble fractions. In contrast, Cu in the gills and all three metals in the hepatopancreas of both species were mainly cytosolic, but this does not necessarily imply a predominant role for MT since the cytosolic fraction also includes other macromolecules which may be the target binding site for accumulated trace metals.     

Metal accumulation and metallothionein in brown trout, Salmo trutta, from two Norwegian rivers differently contaminated with Cd, Cu and Zn

In this work we have studied the accumulation of heavy metals in two brown trout (Salmo trutta) populations in their natural environment and the participation of metal binding to metallothionein (MT) in this process. Cd, Cu and Zn concentrations, total MT (including Cu MT) and Cd/Zn MT were measured in the gills, liver and kidney of trout inhabiting two rivers, one Cu-contaminated and the other Cd/Zn-contaminated, located at Røros, Central Norway. In both populations, high levels of Cu were found in the liver, whereas Cd was accumulated in liver and particularly in the kidney. The proportions of Cd/Zn MT and Cu MT in liver and kidney, but not in gills, reflected the accumulated and the environmental concentrations of these metals. The total Cu MT concentrations in the investigated tissues, however, were highest in trout from the river with the lowest ambient Cu concentration. It is suggested that MTs are of less importance in Cu-acclimated trout. The data also suggest that acclimation to a Cu-rich environment involves reduced Cu accumulation or increased Cu elimination. In trout from the Cd-rich environment, this metal was mainly bound to MT, whereas in trout from the Cu-rich environment Cd was also associated with non-MT proteins. These findings emphasize the importance to determine both Cd/Zn MT and Cu MT levels, when the participation of this protein in metal handling in trout tissues is investigated.     

Dissolved and particulate trace metal geochemistry in the scheldt estuary,S. W. Netherlands (water column and sediments)

The geochemistry of dissolved and particulate trace metals has been studied in the water column and the sediments of the Scheldt estuary between 1987 and 1990. A strong seasonal influence on the behaviour of dissolved Cd, Cu and Zn is observed, related to the redox conditions in the upper estuary and phytoplankton activity in the lower estuary (which are both seasonally dependent variables). The dissolved trace metal concentrations in the fresh water end-member are remarkably low during spring and summer, due to metal sulphide precipitation in the anoxic Scheldt river. However, the dissolved concentrations increase rapidly with increasing salinity, due to oxidation of metal sulphides that are present in the suspended matter, accompanied by (e.g. chloro-)complexation of the released metals. Readsorption of Cd and Zn occurs in the lower estuary during the spring phytoplankton bloom. During winter, when the Scheldt river is not completely anoxic, much higher dissolved trace metal concentrations are observed in the fresh water end-member since metal sulphide precipitation in the water column is precluded. Rapid trace metal removal is observed in the low salinity, high turbidity zone, due to adsorption onto suspended matter and freshly precipitated iron and manganese oxyhydroxides. Upon further mixing, desorption is apparent, due to a similar oxidation-complexation mechanism as observed during spring and summer. Pore water infusion may also contribute to the enrichment of dissolved Cd, Cu and Zn in the mid-estuarine region. The trace metal contents of the suspended matter and the sediments show a continuous decrease with increasing salinity. This behaviour is to a very large extent due to physical mixing of contaminated fluvial particulates and relatively unpolluted marine particulates. Desorption of Cd, Cu and Zn can be identified but is of minor importance compared to the conservative mixing process. The distribution of dissolved Cd, Cu and Zn in the pore waters of the mid-estuarine region reflects the impact of early diagenetic processes. Trace metal peaks are observed near the sediment-water interface, and at greater depth in the manganese and iron reduction zones. These peaks are attributed to oxidation of reduced trace metal compounds (e.g. sulphides) and reduction of the (iron and manganese) oxide carrier phases, respectively. At greater depth, the dissolved trace metal concentrations are much lower due to metal sulphide precipitation in the sulphate reduction zone. Analysis of a large sediment dataset indicates severe trace metal pollution of the Scheldt estuary at the end of the fifties. A major reduction of the pollution by As, Cr, Hg, Pb, and Zn has occurred in the seventies, and of Cd and Cu in the eighties. The Ni pollution has increased over the time period considered. In spite of this improvement, the present-day pollution status of the Scheldt estuary is still reason for concern.     

Metallothionein, zinc, and mercury levels in tissues of young rats exposed to zinc and subsequently to mercury

Several studies have described mercury toxicity and the role of metallothioneins (MT) in the detoxification and regulation of metal homeostasis. However, little data exist on this topic during the specific post-natal developmental phase in young mammals. This developmental phase is particularly important since young animals are more sensitive to toxicants than adults. The objective of this work was to investigate whether MT participates in the mechanism of protection conferred by zinc pre-treatment on the toxic effects induced by mercury in neonate rats. Pups were exposed to ZnCl(2) (5 doses of 27 mg/kg/day, s.c.) and subsequently to HgCl(2) (5 doses of 5 mg/kg/day, s.c.); metal (Zn and Hg) and MT contents were analyzed in the liver, kidney, and blood. MT was induced in the liver and kidney of pups of both Zn-sal and Zn-Hg groups, although the greatest increase was in neonates exposed to Zn only. A direct relationship exists between MT and metals for both hepatic and renal tissues, which indicates that the increase in metal levels occurs in parallel to the increase in MT content. Although the heat-treated cytosolic fraction is rich in MT and metals, higher Zn and Hg contents were detected in the insoluble fraction of all tissues. These results suggest that MT is, at least in part, responsible for preventing Hg accumulation in the liver and blood and decreasing renal toxicity.     

Heavy metal intracellular balance and relationship with metallothionein induction in the gills of carp

Determination of metal levels (Cu, Zn, Cd, Ag, Hg) in soluble and insoluble fractions of gill homogenates has been performed after 7 d exposure of carp (Cyprinus carpio) to moderate concentrations of Cd, Ag, and Hg in water. Metallothionein levels have been quantified by polarographic method before and after contamination and a subsequent decontamination phase (7 d). The influence of pretreatment by zinc (7 d) has also been evaluated. Metallothionein level variations have been interpreted as having regard to interrelated flows of metal between subcellular fractions. Special interest has been focused on heat-stable compound (HSC)-bound heavy metal flows within the cytosol, taking in account that MT is the major component of these ligands. Our data showed differences between the ability of metals to bind cytosolic ligands and HSCs, and their respective potency for MT induction in gill. Regardless of pretreatment, mercury gave the highest increase of gill MT, and after the decontamination MT level remained high compared to control. Cadmium and silver gave similar increases, but a significant difference with control appeared only after the decontamination step with silver, whereas 1 week of contamination was enough for cadmium. Our experimental conditions gave the following order of potency for MT induction in gill: Hg≫Cd>Ag>Zn.     

Sources,distribution and temporal variability of trace metals in the Thames Estuary

Water and intertidal sediment samples were collected from 28 stations along the Thames Estuary, from the tidal limit to the outer estuary. Surveys were conducted in 1997, 1999 and 2001 to assess spatial distributions and short-term variability. Highest concentrations of trace metals in water coincided with high turbidity in the mid-estuarine region, although the particle-associated fraction varied from 22% (As) to 95% (Pb). Theoretical dilution line (TDL) plots showed that dissolved metals were largely derived from a combination of diffuse (sediment desorption) and localised point sources (outfalls, industry). Dissolved Cu and Zn both exceeded environmental quality standard (EQS) levels during the survey period. The majority of sediment metals showed common distributional patterns, with increasing concentrations upstream. This increase was greatest for pollutant metals: Ag, Cd, Cu, Hg, Pb and Zn. Partial extraction of sediment metals with 1 M HCl showed that >50% of Ag, Cd, Cu, Mn, Pb and Zn were potentially bioavailable and that the proportion of bioavailable sediment metals also increased upstream. The majority of sediment metal concentrations exceeded Interim Sediment Quality Guideline threshold effects levels (TELs) over much of the estuary. Sediment concentrations of Cu, Hg, Pb and Zn also exceeded probable effects levels (PELs) at many estuarine sites. Despite improvements in recent years, the Thames Estuary remains chronically contaminated with a range of metals.     

The Lusitanian toadfish as bioindicator of estuarine sediment metal burden: The influence of gender and reproductive metabolism

Early diagenetic processes and anthropogenic activities are responsible for metal enrichment in estuarine sediments. The Tagus estuary (Portugal) is no exception, and as a result of past and present pressures, surface sediment contamination is still an issue in some areas. Since such metal loads may be incorporated by benthic organisms, this study analyzed the accumulation of trace metals in the Lusitanian toadfish (Halobatrachus didactylus) in the Tagus estuary. In order to determine the role played by the seasonal reproductive cycle of the Lusitanian toadfish in the bioaccumulation process of trace metals in its tissues, the concentrations of Cd, Co, Cr, Cu, Ni, Pb and Zn were determined in the liver of male and female specimens captured during reproductive and non-reproductive periods. The results showed that metal accumulation in the liver was related simultaneously with gender and season, with females having higher levels of Cd, Cu and Zn during the reproductive period. The metabolic roles of Cu and Zn in embryonic development may explain such results, as both metals accumulated in the female liver to be transported to the gonads later on. Cd, on the other hand, does not have a metabolic role, and the higher concentrations of this metal found in spawning females could be related to the high affinity of Cd to vitellogenin, which is produced in the liver. To assess the species’ potential as an indicator of metal contamination, the concentrations of the seven elements were compared in the muscle tissue of adult, type I males (age ≥5), from two areas with distinct sediment metal loads. Non-essential metals in the muscle reflected the same differences between areas that were found in the sediment samples, evidencing H. didactylus as a potential indicator of those elements bioavailability from the sediment. The results showed that the muscle tissue of adult specimens of a relatively sedentary species such as H. didactylus is a useful indicator of long term accumulation of trace metals. On the contrary, liver concentrations of trace metals showed variation according to the reproductive status, which could lead to overestimate of the environmental status concerning trace metals bioavailability. Spawning season and liver tissue should thus be avoided in biomonitoring studies targeting this benthic fish.     

Seaweed Alaria esculenta as a biomonitor species of metal contamination in Aughinish Bay (Ireland)

The concentrations of ten metals (As, Cd, Cu, Fe, Hg, K, Na, Pb, Sn and Zn) were monitored in coastal seawater and biomass of the seaweed Alaria esculenta from Aughinish Bay on the West coast of Ireland during March–June 2014 in order to study their temporal variations and assimilation efficiencies and to assess the ecological quality of these ecosystems. Seawater and A. esculenta showed significant temporal variations in their metal concentrations during March–June 2014 and A. esculenta accumulated more efficiently Fe and Zn, but showed low sensitivity to Na and K. On the other hand, A. esculenta showed no active detoxication mechanisms for Cd and Pb, but no saturation point was observed during this work to any metal. Considering metal concentrations bioaccumulated by A. esculenta, the Irish coast of Aughinish Bay was always ecologically classified as “Class I – Unpolluted” during March–June 2014. The significant correlations between seawater and A. esculenta obtained to all metals proved that this seaweed species: (i) is a suitable biomonitor of metal contamination in Irish coasts; (ii) it can be included in the European Environmental Specimen Banks and (iii) it can be used in European Real-Time Environmental Monitoring Networks.     

The Response of Metallothionein and Malondialdehyde after Exclusive and Combined Cd/Zn Exposure in the Crab Sinopotamon henanense

The purpose of this paper is to show the interactions of Cd and Zn in the freshwater crab Sinopotamon henanense through metallothionein (MT) and malondialdehyde (MDA) level measurements. Laboratory acclimated S.henanense were exposed to Cd (50 µg/L, 100 µg/L, 500 µg/L ), and Zn (100 µg/L, 1000 µg/L) alone and in combined treatments (100 µg/L Zn+50 µg/L Cd, 100 µg/L Zn+100 µg/L Cd, 100 µg/L Zn+500 µg/L Cd, 1000 µg/L Zn+50 µg/L Cd, 1000 µg/L Zn+100 µg/L Cd, 1000 µg/L Zn+500 µg/L Cd) for 7, 14, 21, 28, 35 days. The results demonstrated that the MDA contents increased with exposure time and dose and showed time- and dose-dependence in both gills and hepatopancreas of S.henanense after single Cd exposure, while the changes of MDA levels were not significant with single Zn exposure. The MDA levels decreased when the crabs were exposed to metal mixtures compared to Cd exposure alone, indicating that Zn mediated the cellular toxicity of Cd. MT contents increased after single Cd exposure and also showed a time- and dose-dependence, in a tissue-specific way. Zn showed a limited ability of MT induction both in gills and hepatopancreas of S.henanense. The MT contents represented not a simple addition of single metal exposures but were enhanced at a higher concentration of Zn combined with different Cd concentrations compared to single metal exposure. Whether MT can be used as a biomarker for complex field conditions need to be considered cautiously since different induction patterns of MT were found among single Zn, Cd and combined groups. It is suggested that several biomarkers together as a suite should be used in the monitoring of heavy metal pollution in the aquatic environment.     

黄河口盐地碱蓬湿地土壤-植物系统重金属污染评价

以黄河口盐地碱蓬湿地为例,评价了淹水和非淹水区湿地表层土壤As、Cd、Cu、Cr、Pb和Zn 6种重金属的污染程度及其在土壤-植物系统中的迁移、富集特征,分析了不同积水深度和土壤理化性质对研究区土壤重金属含量的影响.研究结果表明,与土壤或沉积物质量标准相比,黄河口盐地碱蓬湿地土壤受As和Cd污染最严重,而其它重金属污染较轻;非淹水土壤Cd、Cr和Zn含量高于淹水湿地,而As、Cu和Pb则较低;而且淹水土壤As含量随积水深度增加而呈下降趋势,但积水深度对其他重金属含量的影响不明显.相关性分析结果表明,按照受土壤关键影响因子的不同重金属(除As外)可以分为两类:第一类为Cd、Cr和Zn,这些重金属含量受土壤pH值和盐分影响较大,且相互间存在显著正相关关系,表明它们可能有相同的来源;第二类为Pb和Cu,它们受土壤pH值、盐分和有机质的影响,且Pb和Cu之间存在显著正相关关系.除Cr、Cu和Zn外,重金属在盐地碱蓬的根系内一般不发生显著富集,但绝大多数重金属都表现出地上部分的含量比根系更高的现象.     

Temporal variations of metallothionein and metal concentrations in the digestive gland of oysters (Crassostrea gigas) from a clean and a metal-rich site

The concentrations of metallothionein (MT) in bivalves, a potential biomarker of metal pollution, are variable according to specific organs, the highest concentrations being encountered in the digestive glands of oysters. Thus, the present study has been focussed on this organ with a view to validate the use of MT as a biomarker in the field, the temporal changes of metal and metallothionein concentrations have been examined from March to October 1997 in the digestive gland of resident oysters from a clean site (Bay of Bourgneuf, France) and a metal-rich site, the Gironde estuary which has been shown as the most Cd-contaminated marine area in France but is also enriched with Cu and Zn. Moreover, oysters from the clean site have been translocated to the Gironde estuary over the same period. Taking into account all the samples collected over the 7 months of the study, MT concentrations in the digestive gland were positively correlated with weight whereas metal levels were negatively correlated with weight. However, considering monthly samples including specimens from both sites (resident or translocated oysters), a positive correlation was shown between MT and metal concentrations in autumn (September and October) but not in spring and summer. These findings limit the interest of using the digestive gland of oysters as the preferred tissue for the determination of MT concentration as a biomarker.The alternative use of gills should be considered.     

Temporal variations of metallothionein and metal concentrations in the digestive gland of oysters (Crassostrea gigas) from a clean and a metal-rich site

The concentrations of metallothionein (MT) in bivalves, a potential biomarker of metal pollution, are variable according to specific organs, the highest concentrations being encountered in the digestive glands of oysters. Thus, the present study has been focussed on this organ with a view to validate the use of MT as a biomarker in the field, the temporal changes of metal and metallothionein concentrations have been examined from March to October 1997 in the digestive gland of resident oysters from a clean site (Bay of Bourgneuf, France) and a metal-rich site, the Gironde estuary which has been shown as the most Cd-contaminated marine area in France but is also enriched with Cu and Zn. Moreover, oysters from the clean site have been translocated to the Gironde estuary over the same period. Taking into account all the samples collected over the 7 months of the study, MT concentrations in the digestive gland were positively correlated with weight whereas metal levels were negatively correlated with weight. However, considering monthly samples including specimens from both sites (resident or translocated oysters), a positive correlation was shown between MT and metal concentrations in autumn (September and October) but not in spring and summer. These findings limit the interest of using the digestive gland of oysters as the preferred tissue for the determination of MT concentration as a biomarker.The alternative use of gills should be considered.     

The influence of nutritional conditions on metal uptake by the mixotrophic dual symbiosis harboring vent mussel Bathymodiolus azoricus

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 μg L^− 1 Cd, plus 25 μg L^− 1 Cu and 100 μg L^− 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.     

Kinetics of metal elimination in oysters from a contaminated estuary

In oysters Crassostrea gigas translocated from a metal-enriched estuary (Gironde, France) to a comparatively clean site, the Bay of Bourgneuf (France), Cd, Cu and Zn concentrations were determined monthly in the whole soft tissues, or in different fractions (cytosolic or insoluble) of gills and digestive glands. In all cases, the concentrations of all of the three metals decreased logarithmically and half-lives were always shortest for Cd (86-251 days). After 4 months, the Cd concentration had become not significantly different from the threshold considered safe for human consumption (1 mg kg(-1) wet wt.). In the digestive gland, half-lives were similar in cytosolic and insoluble fractions. In contrast, in the gills, elimination patterns differed markedly between these fractions. The long half-lives calculated for divalent metals in the insoluble fraction of the gills (1505 and 3010 days for Zn and Cu, respectively) is possibly due to a fossilization of metals in intracellular membrane-bound inclusions as shown previously in Ostrea edulis. It is interesting to underline that elimination is fastest for cytosolic metals compared to the insoluble fraction.     

Variation of metallothioneins in gills of the clam Ruditapes decussatus from the Gulf of Gabès (Tunisia)

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 Gabès (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.     

The influence of starvation and copper exposure on the composition of the dorsal carapace and distribution of trace metals in the shore crab Carcinus maenas (L.)

1. The influence of starvation and copper exposure on the composition of the carapace of the shore crab, Carcinus maenas has been investigated. The effects of the concentrations of selected trace metals in the carapace and midgut gland have also been examined.2. Differences in nutritional state did not affect the concentrations or relative proportions of the principle carapace components (calcium, chitin and protein), but starvation was associated with a reduction in carapace copper concentration. Copper concentration in the midgut gland increased in “starved” crabs, while midgut gland zinc and calcium concentrations remained unchanged.3. Starvation in combination with copper exposure (0.5 mg Cu/1) resulted in alterations in both carapace composition and trace metal loads. Carapace calcium concentrations were reduced significantly following copper exposure irrespective of the nutritional state of the crabs. However, the reduction was more marked in “fed” crabs than in “starved” animals.4. Copper-exposed crabs exhibited raised carapace copper concentrations while carapace zinc concentrations were depressed. Such changes were most pronounced in copper-exposed “starved” crabs.5. Calcium concentrations in the midgut glands of “starved” copper-exposed animals were significantly elevated compared with those in either control or “fed”, copper-exposed crabs.6. The findings are discussed with regard to their ecotoxicological significance.     

Influence of maternal mineral deficiency on the hepatic metallothionein and zinc in newborn rats

The effects of maternal Zn, Cu, or Fe deficiencies during late gestation on hepatic levels of metals and metallothionein (MT) and the binding of Zn and Cu to protein fractions were investigated in newborn rats. Timed pregnant rats were fed one of the following diets: Zn deficient (Zn-D), Cu-D, Fe-D, or control from day 12 of gestation until birth. The specific nutritional deficiency status of the dams was confirmed by low plasma levels of the deficient metal. Livers from pups were analyzed for MT, metal content, and metal-protein binding. Maternal Zn-D resulted in a greater than 50% reduction of hepatic MT levels in pups, whereas Cu-D and Fe-D had no significant effects. Pups in each deficient group showed a significant decrease in the hepatic levels of the respective metals. Fractionation of hepatic cytosols from the pups by Sephadex G-75 gel filtration showed that in both Fe-D and Cu-D pups the respective metals were depleted from the high molecular weight protein fraction, whereas in Zn-D pups the Zn was depleted mainly from the MT fraction (Ve/V0 approximately 2). Incorporation of [35S] cysteine into MT fractions was significantly lower in Zn-D pups as compared with control pups. These results indicate that there is a specific effect of the maternal Zn-D on the hepatic storage of Zn as MT in newborn rats.     

Alterations of tissue glutathione levels and metallothionein mRNA in rainbow trout during single and combined exposure to cadmium and zinc

The objective of this study was to assess the effects of Cd and Zn exposure of rainbow trout (Oncorhynchus mykiss) on (a) hepatic glutathione (GSH) levels; and (b) hepatic and branchial metallothionein (MT) mRNA expression. Juvenile rainbow trout were exposed to waterborne Cd (nominal concentrations: 1.5 or 10 microg Cd l(-1)), Zn (150 or 1000 microg Zn l(-1)) or Cd/Zn mixtures (1.5 microg Cd l(-1) with 200 microg Zn l(-1) or 10 microg Cd l(-1) with 1000 microg Zn l(-1)). After 14 and 28 days of treatment, hepatic concentrations of total glutathione, oxidized glutathione (GSSG) and cysteine were determined by means of fluorometric high performance liquid chromatography (HPLC). Branchial and hepatic expression of MT mRNA was measured by means of semi-quantitative RT-PCR. Exposure of trout to Zn did not result in significantly elevated tissue levels of Zn, whereas Cd accumulation factors changed significantly with time and concentration. Despite of the absence of Zn accumulation, hepatic GSH but not MT mRNA levels were significantly altered in Zn-exposed fish. Cd, on the contrary, affected mainly the MT response but not GSH. Also tissue specific differences in the regulation of the two thiol pools were expressed. The thiol response after exposure to metal mixtures could not be explained by simple addition of the effects of the individual metals. The results indicate that cellular thiol pools show different reaction patterns with respect to specific metals and metal mixtures. Under conditions of long-term, low dose metal exposure, the function of GSH appears to go beyond that of a transitory, first line defense.