Effects of Cadmium in Stylonychia lemnae, Stylonychia notophora and Oxytricha granulifera: Isolation of a Cadmium-Binding Protein

ABSTRACT The effects of cadmium on three ciliates are reported here. Cultures of Stylonychia lemnae, Stylonychia notophora and Oxytricha granulifera were treated with different doses of Cd according to tolerance. The two species of Stylonychia are very sensitive to the metal, white O. granulifera tolerates higher doses. Adding 50 μM of Cd to the medium did not damage cells. The accumulated metal is almost totally present in the particulate fraction after day 3. Two Cd-Zn linking fractions were separated from the soluble fraction of culture treated on day 1. The first protein linking 17 μg Cd/mg showed an ultraviolet absorption spectrum similar to that of Cd-thioneins. Preliminary amino acid analyses indicated that it contained 13% cysteine. The second protein, linking 60 μg Cd/mg, was a glycoprotein. Its ultraviolet absorption spectrum and amino acid analysis showed that this binding protein was far from being a metallothionein: its cysteine content was very low and aromatic and cyclic residues were present. This Cd-linking compound seems to be unique, since it was very different both from metallothioneins and chelatins isolated by other protozoa. The protective role of these chelating proteins is discussed.     

Relationship between metallothionein and metal contents in red-blooded and white-blooded Antarctic teleosts

Metal bioaccumulation and metallothionein were investigated in different organs of the red-blooded teleost, Trematomus bernacchii and the haemoglobinless Chionodraco hamatus. Specimens of the two Antarctic fish were sampled from Terra Nova Bay (Ross Sea), and their levels of Cd, Cu and Zn in homogenates and in soluble fractions of liver, muscle, gills, heart and plasma were determined. Dosages of metallothioneins (MTs) or MT-like proteins were assayed by the silver saturation method in soluble fractions of the same organs. In both T. bernacchii and C. hamatus the highest MT contents were found in liver. Hepatic MT and Cd, Cu and Zn concentrations correlated positively in T. bernacchii, whereas in C. hamatus hepatic MT showed a positive correlation only with Cd. Positive correlations were also found between Cd and MT in gills of the two species. A metal-binding protein containing a high percentage of cysteine from C. hamatus was purified and compared with the MT from T. bernacchii and mammals. Accepted: 27 November 1999     

The molecular characteristics of two metallothioneins in Ostrinia furnacalis and expression under conditions of heavy metal stress

Metallothioneins are ubiquitously-expressed metal-binding proteins. Despite their potential ecological relevance, no prior reports have identified any metallothioneins in Ostrinia furnacalis or other Lepidoptera species. A better understanding of the molecular characteristics and regulatory dynamics of metallothionein genes in O. furnacalis under heavy metal stress conditions would enable future studies of the roles played by these proteins in the context of heavy metal detoxification. Herein, we identified and characterized two metallothionein (OfMT) genes in O. furnacalis, including the 147 bp OfMT1 gene encoding a 48 amino acid protein containing 10 cysteine residues, and the 141 bp OfMT2 gene encoding a 46 amino acid protein containing 12 cysteine residues. The expression of OfMT2 was found to be related to Cu and Cd concentrations in a dose-dependent manner but was unaffected by Zn exposure. Overall, these results indicate that OfMT genes likely encode metal-binding proteins consistent with their potential role in the maintenance of heavy metal homeostasis.     

From heavy metal‐binders to biosensors: Ciliate metallothioneins discussed

Metallothioneins (MTs) are ubiquitous proteins with the capacity to bind heavy metal ions (mainly Cd, Zn or Cu), and they have been found in animals, plants, eukaryotic and prokaryotic micro‐organisms. We have carried out a comparative analysis of ciliate MTs (Tetrahymena species) to well‐known MTs from other organisms, discussing their exclusive features, such as the presence of aromatic amino acid residues and almost exclusive cysteine clusters (CCC) present in cadmium‐binding metallothioneins (CdMTs), higher heavy metal‐MT stoichiometry values, and a strictly conserved modular–submodular structure. Based on this last feature and an extensive gene duplication, we propose a possible model for the evolutionary history of T. thermophila MTs. We also suggest possible functions for these MTs from consideration of their differential gene expressions and discuss the potential use of these proteins and/or their gene promoters for designing molecular or whole‐cell biosensors for a fast detection of heavy metals in diverse polluted ecosystems.     

Variation in cadmium accumulation potential and tissue distribution of cadmium in tobacco

Variation in Cd accumulation between Nicotiana species but not varieties has been observed in seedlings grown in solution culture with moderate-to-low levels of Cd. Nicotiana tabacum has been characterized as a leaf and root accumulator while Nicotiana rustica is shown to be primarily a root accumulator, having about half the leaf Cd per gram dry weight of N. tabacum. This phenotype is retained in the mature N. rustica plant. To characterize these two species which differ in their modes of Cd accumulation, tissue Cd distribution, partitioning of metal in soluble and insoluble fractions and the contribution of soluble Cd-binding proteins (peptides) to total plant Cd was assessed using mature solution cultured plants. Metal accumulation was highest in the most mature leaves and in young roots. The preponderance of young roots in N. rustica may, in part, account for low leaf/high root Cd accumulation in this species. While Cd-binding peptides appear to be a principal form of Cd in leaves and roots of seedlings and these also occur in mature leaves, Cd is equally distributed between soluble (about 80% as Cd-binding peptide) and uncharacterized insoluble forms in mature plant roots.     

Interspecific comparison of Cd bioaccumulation in European Pectinidae (Chlamys varia and Pecten maximus)

The uptake and loss kinetics of Cd were determined in two species of scallops from the European coasts, the variegated scallop Chlamys varia and the king scallop Pecten maximus, following exposures via seawater, phytoplankton and sediment using highly sensitive radiotracer techniques (¹⁰⁹Cd). Results indicate that, for seawater and dietary pathways, C. varia displays higher bioaccumulation capacities in terms of uptake rate from water and fraction absorbed from ingested food (assimilation efficiency) than Pecten maximus. Regarding sediment exposure, P. maximus displayed low steady-state Cd transfer factor (TF_SS < 1); however, once incorporated, a very large part of Cd transferred from sediment (92%) was strongly retained within P. maximus tissues.Both species showed a high retention capacity for Cd (biological half-life, T_b1/2 > 4 months), suggesting efficient mechanisms of detoxification and storage in both species. The digestive gland was found to be the main storage organ of Cd in the two scallops regardless of the exposure pathway. However, Cd was stored differently within this organ according to the species considered: 40% of the total Cd was found in the soluble cellular fraction in C. varia whereas this soluble fraction reached 80% for P. maximus. This suggests that the two species displayed different Cd detoxification/storage mechanisms.Finally, the present study has determined the relative contribution of the different exposure pathways to global Cd bioaccumulation for the two scallop species. Results clearly show that for both species, food constitutes the major accumulation pathway, contributing for > 99% and 84% of the global Cd bioaccumulation in C. varia and P. maximus, respectively. This work confirms the previous assumption, derived from a bibliographic overview, that dietary pathway plays a prevalent role in metal bioaccumulation in Pectinidae.     

The transfer of cadmium, mercury, methylmercury, and zinc in an intertidal rocky shore food chain

We examined the transfer of cadmium (Cd), inorganic mercury [Hg(II)], methylmercury (MeHg), and zinc (Zn) in an intertidal rocky shore food chain, namely from marine phytoplankton to suspension-feeding rock oysters (Saccostrea cucullata) and finally to predatory whelks Thais clavigera. The uptake of metals from the dissolved phase was also concurrently quantified in the oysters and the whelks. Metal uptake by the oysters was not directly proportional, whereas metal uptake by the whelks was directly proportional to metal concentration in the water. The order of uptake was MeHg>Hg(II)>Zn>Cd, and was much higher in the oysters than in the whelks. The relative uptake of Zn and Cd was comparable between oysters and whelks, whereas MeHg and Hg(II) showed disproportionally higher uptake in oysters than in whelks as compared to Zn and Cd. The assimilation efficiencies (AEs) were in the order of MeHg>Zn>Cd=Hg(II) in oysters, whereas the AEs were highest for MeHg and comparable for Zn, Cd, and Hg(II) in the whelks. Pre-exposure of the oysters to different dissolved concentrations of Cd significantly elevated the AEs of Cd and Hg(II) but not of Zn, in association with the induction of metallothioneins in the oysters. The whelks significantly assimilated Cd and Zn from various prey (barnacles, oysters, mussels, and snails) with contrasting strageties of metal sequestration and storage. There was no significant relationship between the metal AE and the metal partitioning in the soluble fraction (including metallothionein-like proteins, heat stable protein, and organelles). The insoluble fraction of metals was also available for metal assimilation. Our calculations show that the dietary uptake of metals can be dominant in the overall bioaccumulation in the oysters and whelks, and the trophic transfer factor was >1 for all metals. Thus, the four metals have a high potential of being biomagnified in the intertidal rocky shore food chain. MeHg possessed the highest and Hg(II) and Cd the lowest potential of trophic transfer among the four metals considered.     

Importance of metallothioneins in the cadmium detoxification process in Daphnia magna

Good knowledge of the relationship between toxic metals and biological systems, particularly the sub-cellular fraction, could be a suitable early indicator of toxic effects. These effects and the sub-cellular behaviour of cadmium were studied with a widely used species in freshwater toxicity bioassays, Daphnia magna. In spite of this very commonplace usage in ecotoxicological studies, very few data are available on its toxicant metabolism and in particular metal homeostasis. Combining multi-tools analysis, a soluble protein was found: it is heat-stable, rich in sulfhydryl groups (differential pulse polarography), characterised by a molecular mass of approximately 6.5 kDa, with a G-75 chromatographic profile corresponding to the rabbit metallothioneins monomer, with few if any aromatic-containing amino acids, it binds metals (e.g. Cd, Cu), and its concentration increases with Cd exposure. This evidence led us to hypothesise that metallothioneins (MTs) are present in D. magna. Up to 75% of the Cd body burden with Cd exposure is bound to the MTs fraction. The increase in the Cd concentration in the surrounding medium and concomitantly in daphnids induces sub-cellular reorganisation of essential metals such as Cu and Zn. The rate of metals in the soluble cellular fraction and associated with MTs increases with the Cd body burden. Monitoring sub-cellular distribution of metals after exposure in the natural environment could be very useful for ecotoxicological assessment.     

Enrichment in Trace Metals (Al,Mn, Co,Cu, Mo,Cd, Fe,Zn, Pb and Hg) of Macro-Invertebrate Habitats at Hydrothermal Vents Along the Mid-Atlantic Ridge

The present study describes several features of the aquatic environment with the emphasis on the total vs. filter-passing fraction (FP) of heavy metals in microhabitats of two typical deep-sea vent organisms: the filter-feeder, symbiont-bearing Bathymodiolus and the grazer shrimps Rimicaris/Mirocaris from the Mid-Atlantic Ridge (MAR). The concentration of 10 trace elements: Al, Mn, Co, Cu, Mo, Cd, Fe, Zn, Pb and Hg was explored highlighting common and distinctive features among the five hydrothermal vent sites of the MAR: Menez Gwen, Lucky Strike, Rainbow, Saldanha, and Menez Hom that are all geo-chemically different when looking at the undiluted hydrothermal fluid composition. The drop off in the percentage of FP from total metal concentration in mussel and/or shrimp inhabited water samples (in mussel beds at Rainbow, for instance, FP fraction of Fe was below 23%, Zn 24 %, Al 65%, Cu 70%, and Mn 89%) as compared to non-inhabited areas (where 94% of the Fe, 90% of the Zn, 100% of the other metals was in the FP fraction) may indicate an influence of vent organisms on their habitat’s chemistry, which in turn may determine adaptational strategies to elevated levels of toxic heavy metals. Predominance of particulate fraction over the soluble metals, jointly with the morphological structure and elemental composition of typical particles in these vent habitats suggest a more limited metal bioavailability to vent organisms as previously thought. In addition, it is evoked that vent invertebrates may have developed highly efficient metal-handling strategies targeting particulate phase of various metals present in the mixing zones that enables their survival under these extreme conditions.     

Extrachromosomal ribosomal RNA genes in Tetrahymena: structure and evolution

The macronuclear ribosomal RNA genes from a number of strains within several species of Tetrahymena have been characterized. Restriction enzyme analysis revealed that individual strains all contained entirely homogeneous populations of extrachromosomal palindromic ribosomal DNA, varying in molecular size from 12 × 10⁶ to 14 × 10⁶ in different strains. Considering that the evolutionary distance among some of the species is estimated to be of the order of 10⁶ years, the rDNA from all the species exhibited a strikingly high similarity in the localization of their restriction sites. Nevertheless, differences both inside and outside the gene region were clearly detectable, showing that the rDNA sequences have diverged in all species.Genetic polymorphism with respect to rDNA structure exists in Tetrahymena, but seems to be rare. In only two out of five species examined (T. borealis and T. pigmentosa) interbreeding strains differing in rDNA structure were found. While the differences detected in the T. borealis rDNA were confined to a small size difference located at the non-coding ends of the molecule, several differences were detected in the rDNA from the T. pigmentosa strains. One of the differences was shown to be due to the presence of an intervening sequence within the structural gene for 26 S rRNA in some of the strains. An intervening sequence of similar size located at the same position within the 26 S gene region was found by R-loop mapping in all strains of the species T. thermophila. Restriction enzyme analysis indicates that the rDNA from two other species contains a similar intervening sequence, and we therefore suggest that the size and localization of the intervening sequence is evolutionarily stable. The two intervening sequences examined so far, however, are not identical, as revealed by restriction enzyme mapping.     

Subcellular Distribution of Cadmium and Nickel in Chronically Exposed Wild Fish: Inferences Regarding Metal Detoxification Strategies and Implications for Setting Water Quality Guidelines for Dissolved Metals

The objective of this study was to investigate metal detoxification in chronically exposed juvenile yellow perch (YP: Perca flavescens) and to field test the commonly assumed threshold toxicity model. Fish were collected from lakes located along a cadmium (Cd) and nickel (Ni) concentration gradient. Ambient dissolved metal concentrations were measured to evaluate exposure and total hepatic metal concentrations were determined as a measure of metal bioaccumulation. Hepatic metal partitioning among potentially metal-sensitive fractions (heat-denatured proteins, organelles) and detoxified metal fractions (metallothionein) was determined after differential centrifugation of YP liver homogenates. Major proportions of hepatic Cd were found in the heat-stable cytosolic peptides and proteins fraction (HSP; including metallothioneins), whereas Ni was mainly found in the potentially metal-sensitive heat-denaturable proteins fraction (HDP). For these chronically exposed fish there was no threshold exposure concentration below which binding of Cd or Ni to the heat-denaturable protein fraction or the organelle fraction did not occur. Metal detoxification was clearly incomplete and P. flavescens was subject to some metal-related stress, as evidenced notably by endocrine perturbations. Similar subcellular partitioning results were obtained when juvenile yellow perch were transferred from a reference lake to a Cd-contaminated lake and Cd accumulation was followed over time; there was no accumulation threshold below which Cd binding to the putative metal-sensitive fractions (HDP and organelles) did not occur. The presence of Cd and Ni in these fractions, even for low exposure concentrations and low hepatic accumulation, contradicts the threshold toxicity model that underpins metal toxicology theory and that is implicitly used in setting water quality guidelines for metals. Chronically exposed YP appear to have settled for a tradeoff between the cost of turning on their detoxification apparatus at full capacity, to completely suppress metal binding to metal-sensitive sites, and the alternative cost of allowing some binding of inappropriate metals to metal-sensitive sites.     

Acute dietary pre-exposure and trace metal bioavailability to the barnacle Balanus amphitrite

Rates of uptake from solution and assimilation efficiencies of the trace metals Ag, Cd and Zn were investigated in the barnacle Balanus amphitrite after exposure in the laboratory for 19 days to low and high doses of added Ag and Cd in a diatom (Thalassiosira weissflogii) diet, the major route of metal uptake in barnacles. The hypothesis under test was that acute metal pre-exposure would affect the assimilation efficiency (AE) of that and other metals and their rate of uptake from solution. It was found that pre-exposure of the barnacles to atypically high dietary challenges of Cd and Ag did not cause changes in the rates of uptake of Cd, Ag or Zn from solution. Similarly, there was no clear consistent effect of dietary pre-exposure to Cd or Ag on the assimilation efficiency of Cd, Ag or Zn. The efflux rates of the metals were also comparable following the acute dietary exposure. Subcellular fractionation data indicated that the majority of the three metals were partitioned in the insoluble fraction, with very little in the soluble fraction consisting of metallothionein-like proteins and other (heat-sensitive) proteins. The lack of induction of increased Cd or Ag AE after pre-exposure in barnacles contrasts with results for mussels; this inconsistency is interpreted to result from differences in physiological accumulation patterns, the barnacles relying to an extreme extent on insoluble detoxification.     

Isolation and characterization of metallothionein from guinea pig liver

The amino acid composition, and the absorption, circular dichroism (CD) and magnetic circular dichroism spectra of a metalloprotein induced in the livers of guinea pigs by the injection of CdCl₂ are reported. The amino acid composition of this protein closely resembles that of rat liver metallothionein (MT). We show that this protein has spectroscopic properties that closely follow the behaviour previously reported for several other cadmium-containing metallothioneins in its spectral response to changes in pH, and to the addition of cadmium and copper(I). Dramatic changes are observed in the CD spectrum during the addition of copper(I); it is suggested that these changes are the result of the formation of a mixed Cu(I)/Cd(II) cluster that forms in the α domain once the β domain has been saturated with Cu(I). These results are of particular importance in the characterization of this protein as belonging to the metallothionein class of proteins, as spectral changes of this type are directly related to the displacement of Cd²⁺ and Zn²⁺ from the two, thiolatecluster binding sites that are amongst the unique properties of mammalian metallothioneins. It is demonstrated that the CD spectrum provides a sensitive indicator of the presence of these special metal binding sites by indicating changes in the binding geometry and stoichiometry in response to an incoming metal. These results indicate that the guinea pig liver metallothionein induced by injections of CdCl₂ uses the same α and β type of clusters for cadmium binding as rat liver Cd, Zn-MT, even though there are minor differences in the amino acid composition between the guinea pig and rat liver proteins.     

Metallothionein research in terrestrial invertebrates: Synopsis and perspectives

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.     

Updating rDNA Restriction Enzyme Maps of Tetrahymena Reveals Four New Intron-Containing Species1

The extrachromosomal rDNA molecules from a number of Tetrahymena strains wered racterized by restriction enzyme mapping using three different restriction enzymes combined with gel blotting and hybridization analysis. Strains from four out of six recently described species were found to contain an intron in the 26s rRNA coding region. The evolutionary relationship among the species of the T. pyriformis complex was examined on the basis of the rDNA maps with emphasis on similarities between two of the new species and the widely studied T. thermophila and T. pigmentosa. Examination of a large number of T. pigmentosa strains showed this species to exhibit an unusual polymorphism with respect to its rDNA. It is suggested that recombinational cross-over events play a role in the formation of new rDNA alleles in this species.     

Glutamate dehydrogenase from pumpkin cotyledons: characterization and isoenzymes

Glutamate dehydrogenase from pumpkin (Cucurbita moschata Pior. cultivar Dickinson Field) cotyledons was found in both soluble and particulate fractions with the bulk of the activity in the soluble fraction. Both enzymes used NAD(H) and NADP(H) but NAD(H) was favored. The enzymes were classified as glutamate-NAD oxidoreductase, deaminating (EC 1.4.1.3). Both enzymes were heat stable, had a pH optimum for reductive amination of 8.0, and were inhibited by high concentrations of NH₄⁺ or α-ketoglutarate. The soluble enzyme was more sensitive to NH₄⁺ inhibition and was activated by metal ions after ammonium sulfate fractionation while the solubilized particulate enzyme was not. Inhibition by ethylenediaminetetraacetate was restored by several divalent ions and inhibition by p-hydroxymercuribenzoate was reversed by glutathione. Particulate glutamate dehydrogenase showed a greater activity with NADP. The molecular weights of the enzymes are 250,000. Separation of the enzymes by disc gel electrophoresis showed that during germination the soluble isoenzymes increased from 1 to 7 in number, while only one particulate isoenzyme was found at any time. This particulate isoenzyme was identical with one of the soluble isoenzymes. A number of methods indicated that the soluble isoenzymes were not simply removed from the particulate fraction and that true isoenzymes were found.     

Characterization of arginase activity from mouse epidermis and its relation to ornithine decarboxylase induction by the tumor-promoting agent, 12-O-tetradecanoylphorbol-13-acetate

Arginase, which catalyzes the cleavage of l-arginine to urea and ornithine, was detected in both soluble and particulate fractions of mouse epidermis. In a typical experiment, about 75 and 25% of the total arginase activity was associated with the soluble (100 000 × g supernatant) and the washed particulate fraction, respectively. Both soluble and particulate enzymes required the presence of divalent Mn²⁺ for activity. Arginase activity was increased by about 50% in the particulate fraction, but not in the soluble fraction, by preheating the fractions at either 50 or 55°C in the presence of 15 mM MnCl₂. Enzyme activity in both fractions, in the absence of 15 mM MnCl₂, dropped precipitously during heating. A comparison of the nature of arginases in the soluble and particulate fractions revealed similar K_m values (13 mM) and pH optima (9.5) and identical heat denaturation curves. Application of 10 nmol of 12-O-tetradecanoylphorbol-13-acetate to mouse skin did not increase arginase activity in either fraction over a period of 24 h. In contrast, there was a large increase in ornithine decarboxylase activity in the soluble fraction 4.5 h after treatment. Mouse epidermal ornithine decarboxylase activity was much less than arginase activity and was predominantly localized in the soluble fraction. These results indicate that the normal level of arginase activity is not a limiting factor for the stimulation of polyamine biosynthesis by TPA. High arginase activity in mouse epidermis may play a role in providing ornithine for polyamine biosynthesis and in the production of glutamate and proline as well as in the production of keratinous proteins.     

Identification and Comparative Analysis of the Tegillarca granosa Haemocytes MicroRNA Transcriptome in Response to Cd Using a Deep Sequencing Approach

Background

MicroRNAs (miRNAs) are endogenous non-coding small RNAs (sRNAs) that can base pair with their target mRNAs, which represses their translation or induces their degradation in various biological processes. To identify miRNAs regulated by heavy metal stress, we constructed two sRNA libraries for the blood clam Tegillarca granosa: one for organisms exposed to toxic levels of cadmium (Cd) and one for a control group.

Results

Sequencing of the two libraries and subsequent analysis revealed 215 conserved and 39 new miRNAs. Most of the new miRNAs in T. granosa were up- or down-regulated in response to Cd exposure. There were significant differences in expression between the Cd and control groups for 16 miRNAs. Of these, five miRNAs were significantly up-regulated and 11 were significantly down-regulated in the Cd stress library. Potential targets were predicted for the 16 differential miRNAs in pre-miRNAs identified according to sequence homology. Some of the predicted miRNA targets are associated with regulation of the response to stress induced by heavy metals. Five differentially expressed miRNAs (Tgr-nmiR-8, Tgr-nmiR-21, Tgr-miR-2a, Tgr-miR-10a-5p, and Tgr-miR-184b) were validated by qRT-PCR.

Conclusion

Our study is the first large-scale identification of miRNAs in T. granosa haemocytes. Our findings suggest that some miRNAs and their target genes and pathways may play critical roles in the responses of this species to environmental heavy metal stresses.     

The potential use of metallothionein in the clam Ruditapes decussatus as a biomarker of in situ metal exposure

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 (Linné, 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.