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.     

Use of metallothionein in gills from oysters (Crassostrea gigas) as a biomarker: seasonal and intersite fluctuations

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.     

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.     

Comparison of metallothionein concentrations and tissue distribution of trace metals in crabs (Pachygrapsus marmoratus) from a metal-rich estuary,in and out of the reproductive season

Crabs, Pachygrapsus marmoratus, were sampled in June 1997 and February 1998 from two sites (at the mouth and 25 km upstream) in the metal-rich Gironde estuary, France. Gills and hepatopancreas were analysed for metal (Cd, Cu, Zn) and metallothionein (MT) contents, in order to examine the influence of both biological and environmental factors on the physico-chemical forms of detoxified metal storage in the crabs. The concentrations of MT and both cytosolic and insoluble metals were not greatly different between males and females, and the influence of organ weights was also minimal. Intersite differences were observed, probably resulting from the gradient of salinity in the estuary, which interacts with both the chemical speciation and bioavailability of metals, and the general protein metabolism of the crabs. Seasonal changes were also important, probably in interaction with the moult and reproductive cycles. In February, concentrations of insoluble metals were generally higher than in June, in both organs, suggesting that essential metals, particularly Zn, are stored during winter then remobilised during the breeding season. The natural variability in the concentrations of MT often concealed any relationship with accumulated metal concentrations. Thus MT in crabs cannot be considered as a useful biomarker of metal pollution.     

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.     

Nuclear and cytosolic distribution of metallothionein in the blue mussel Mytilus edulis L

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.     

Spatio-temporal variation in energy content of the periwinkle Littorina littorea, along a pollution gradient in the western Scheldt estuary, The Netherlands

Energy reserves, expressed as lipid, glycogen and protein fractions, were measured in the periwinkle L. littorea after 4 weeks of translocation from a relatively clean, marine (i.e. Westkapelle) to an intermediate (i.e. Ellewoutsdijk) and a highly heavy metal polluted, brackish site (i.e. Hansweert) in the Western Scheldt estuary (The Netherlands). Energy reserves of resident periwinkles from all three sites were assessed over time as well (i.e. 16/03/05, 23/03/05, 30/03/05, 13/04/05 and 11/05/05). None of the energy fractions reflected the upstream decreasing salinity and increasing heavy metal gradients. Glycogen and protein fractions decreased significantly in the translocated specimens, despite no differences were detected among the resident populations, suggesting adaptation and/or acclimation effects at the upstream parts of the estuary. The temporal data revealed a consistent post-winter, pre-spawning increase of glycogen and protein levels and a subsequent drop after spawning. Compared to glycogen and proteins, lipids follow a slightly different pattern, which may be explained by sex related and site specific interactions between biotic and abiotic factors.     

Metallothionein concentration in the mussel Mytilus galloprovincialis as a biomarker of response to metal contamination: validation in the field.

Mussels were translocated from a shell-fish breeding area (Sète, 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.     

Purification and characterization of metallothionein-like cadmium binding protein from Asian periwinkle Littorina brevicula

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.     

Metallothioneins and trace elements in digestive gland,gills, kidney and gonads of Octopus vulgaris

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 (µg g^− 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.     

Metallothionein concentration in the mussel Mytilus galloprovincialis as a biomarker of response to metal contamination: validation in the field

Mussels were translocated from a shell-fish breeding area (Sète, 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.     

Biomonitoring of trace metal contamination in mangrove-lined Brazilian coastal systems using the oyster Crassostrea rhizophorae: comparative study of regions affected by oil, salt pond and shrimp farming activities

The oyster Crassostrea rhizophorae has been used as a biomonitor of trace metal contamination in two Brazilian coastal systems. C. rhizophorae were collected in January 1998 from 15 stations (from 4 coastal inlets (including 1 estuary) and 1 coastal beach) near Macau, Rio Grande do Norte (RN), Brazil, a region affected by the activities of the oil industry and salt manufacture in coastal salt ponds; oysters were also collected in September 1999 from 8 stations in the Curimatau estuary (RN), an estuary becoming increasingly affected by shrimp farming activities. C. rhizophorae is a net accumulator of trace metals and can be used as a biomonitor, the accumulated soft tissue concentrations representing integrated records of bioavailable metal over the life of the oyster. At Macau, significant differences in oyster accumulated concentrations (and hence bioavailabilities to the oyster) of Fe, Zn, Cu, and Mn were found between stations; raised zinc availabilities at the coastal site are in close proximity to oil industry activities but the very high availabilities of Fe, Cu and Mn in the Rio dos Cavalos estuary originate from an unknown source. In the Curimatau estuary, bioavailabilities of Mn, Pb and Cd, but particularly of Cu and Zn, to the oysters are raised at the two most downstream sites, the only sites below the effluent of a large shrimp farming enterprise. The oysters also act as a local food source, and concentrations of Zn, Cu and Pb of some of the oysters are above typical public health recommended limits.     

Cadmium affects the expression of heat shock protein 90 and metallothionein mRNA in the Pacific oyster, Crassostrea gigas

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.     

Brown trout (Salmo trutta) metallothioneins as biomarkers for metal exposure in two Norwegian rivers

The potential use of the metal binding protein metallothionein (MT) as a biomarker for trace metal exposure has been evaluated in brown trout (Salmo trutta)     

Brown trout (Salmo trutta) metallothioneins as biomarkers for metal exposure in two Norwegian rivers

The potential use of the metal binding protein metallothionein (MT) as a biomarker for trace metal exposure has been evaluated in brown trout (Salmo trutta)     

Spatial and seasonal biomarker responses in the clam Ruditapes decussatus

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), δ-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 δ-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.     

Mechanisms of detoxication of cadmium by the oyster Crassostrea gigas (Mollusca, Bivalve. II. Intracellular sites of accumulation of the metal in absorbant and excretory organs

In oysters exposed to CdCl2 or under natural conditions (Gironde), concentrations of cadmium occur in absorbant and excretory organs, without cellular injury. The lysosomal system, involved in the storage of the metal, is implicated in detoxification processes.     

Trace metal transfer in a soil–plant–snail microcosm field experiment and biomarker responses in snails

A microcosm experiment was performed to investigate temporal (up to 16 weeks) and spatial variation in metal transfer in a soil–food (nettle)–snail (Cepaea nemoralis) food chain and biomarker responses in the digestive gland of the same snails. Adult snails were sampled from an uncontaminated site and transferred to five sites located 0.5, 2.5, 3, 5, and 10 km from a historically polluted point source. All sites were park areas where grasslands interfered with patches of deciduous forest. Soil physicochemical properties (pH, clay%, OC%) significantly explained the uptake of metals by nettle. Concentrations of metals in the digestive gland (DG) of snails were significantly related to those in nettle, but rarely to soil physicochemical properties. In general metal concentrations in the DG fluctuated while As, Ni, Pb and Zn showed a site dependent increase with time. Despite the long term exposure, biomarker concentrations (lipid, glycogen, proteins, glutathione-S-transferases), and shell morphology, were not related to DG metal concentrations. Our investigation emphasizes the need for controlled long-term studies on the transfer and effects of metals in food chains since short term studies might only show temporary physiological changes due to experimental acclimation.     

Cadmium Accumulation and Metallothionein Biosynthesis in Cadmium-Treated Freshwater Mussel Anodonta woodiana

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.