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.     

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.     

Metals in squid,Loligo forbesi,adults, eggs and hatchlings. No evidence for a role for Cu- or Zn-metallothionein

An adult squid Loligo forbesi had the following metals in its liver/digestive gland: Mn, Fe, Ni, Zn, Cu, As, Cd, Ba and Pb in the range of 1-110 ppm wet wt. Adult mantle muscle, adult eyes, eggs and hatchlings contained a lesser number of these metals at concentrations above 1 ppm. Chromatographic analysis of non-heat-treated cytosols (in the presence of 5 mM 2-mercaptoethanol) gave no evidence for the presence of copper- or zinc-containing fractions with the molecular weights of mollusc metallothioneins in any of the above tissues. Copper and Zn were bound to either the particulate fraction or to very low molecular weight species.     

Immunological properties of low molecular-weight proteins binding heavy metals in rat kidney and liver

Two homogenous fractions of hepatic metallothioneins ((Cd,Zn) MT-1 and (Cd,Zn) MT-2) and renal metal binding proteins ((Bi,Cu) BP-1 and (Bi,Cu) BP-2) were isolated from rats exposed to heavy metals and specific antisera to them were produced in rabbits.These antisera were tested by immunodiffusion and immunoelectrophoresis for their ability to bind different fractions of hepatic Cd,Zn -metallothionein and renal (Bi,Cu)-, (Hg,Cu)- and (Cd,Cu)-binding proteins. It was found that anti (Bi,Cu) BP antisera did not cross-react with hepatic (Cd,Zn) MT-1 and (Cd,Zn) MT-2. Strong immunological cross-reactions were detected between anti (Bi,Cu) BP antisera and individual forms of (Cd,Cu)-, (Hg,Cu)- and (Bi,Cu)-binding proteins isolated from rat kidneys.     

Multiple forms of metallothionein from the digestive gland of naturally occurring and cadmium-exposed mussels, Mytilus galloprovincialis

Polymorphism of metallothioneins in the digestive gland of naturally occurring (control) and experimentally Cd-exposed mussels Mytilus galloprovincialis (200 μg Cd l^–1; 14 days) was studied by applying the conventional methods of Sephadex column liquid chromatography (G-75 and DEAE A-25), and by an electrochemical method (DPASV) for determination of Cd, Zn and Cu concentrations in chromatographic fractions. In both control and Cd-exposed mussels, two distinct molecular mass components of the metallothioneins, monomeric (MT-10) and dimeric (MT-20), were resolved by Sephadex G-75 gel filtration chromatography. In control mussels, the MT-10 component was predominantly expressed as containing markedly higher constitutive levels of Zn (100×) and Cu (10×) than of Cd. Each of these two molecular mass components was further resolved into seven metal-rich peaks by anion-exchange chromatography. In Cd-exposed mussels the larger proportion of Cd was bound to the MT-20 than to the MT-10 component, suggesting that the dimeric component may be considered as a primarily inducible metallothionein. The elution positions of metal-binding maxima of Cd-exposed and control mussels on the respective DEAE chromatographic profiles were comparable. A great similarity in elution positions of Cd maxima between the composite and single-specimen samples was also observed. Our study confirms a high multiplicity of MT forms in mussels from the Mytilus genus not only under the laboratory high-level metal exposure conditions, but also at a natural seawater metal exposure level. The ecotoxicological significance of dimeric and monomeric MT forms, as well as their possible application in the biomonitoring of seawater for trace metals, has been considered. Electronic Publication     

Cadmium bioaccumulation and detoxification in the gill and digestive gland of the Antarctic bivalve Laternula elliptica

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

Effects of dexamethasone on metallothionein induction by Zn, Cu, and Cd in Chang liver cells

Metallothioneins (MTs) were induced in Chang liver cells by the metals, Zn, Cu and Cd, and the glucocorticoid hormone, dexamethasone. When 116 microM Zn, 32 microM Cu and 18 microM Cd, and 10(-7) M dexamethasone, respectively, were administered for 9 h, MTs induced by each inducer in the cells reached maximum levels. The maximum accumulation of MT level induced by dexamethasone was the lowest of the four inducers investigated; the levels induced by Zn, Cu and Cd were 4.7, 1.2 and 1.5 times of that induced by dexamethasone. When dexamethasone was added to the cells together with the heavy metals (Zn, Cu and Cd), dexamethasone had an additive effect on the maximum MT accumulations induced by heavy metals as compared to when induction was conducted using one of heavy metals alone or by dexamethasone alone. However, dexamethasone did almost not effect the metal accumulations in the cells, although the maximum MT levels induced by heavy metal increased by dexamethasone. These results suggest that the process of MT induction by heavy metals and that by dexamethasone are independent of one another. When dexamethasone was added to the cells together with a high concentration of Cu (32 microM) induced the maximum MT accumulation, Cu transport into the cells decreased by 20-40% of that into non-treated cells, which was statistically significant.     

Ruditapes decussatus and Ruditapes philippinarum exposed to cadmium: toxicological effects and bioaccumulation patterns

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

Metal binding of metallothioneins in human astrocytomas (U87 MG,IPDDC-2A)

Astroglia cells structurally and nutritionally support neurons in the central nervous system. They play an important role in guiding the construction of the nervous system and controlling the chemical and ionic environment of neurons. They also represent the major sites for accumulation and immobilisation of toxic metal ions most probably connected with metallothioneins. For this reason astroglia cells possess high cytosolic levels of metallothioneins I, II and III (MT-I,II,III). Our aim was to establish the inducibility and metal binding of MTs in two human astrocytoma cell lines, U87 MG (astrocytoma–glioblastoma, grade IV) and IPDDC-2A (astrocytoma, grade II), on exposure to cadmium chloride (1 μM). MTs were identified by molecular weight (size exclusion chromatography) and their metal content (Cd, Zn and Cu) to follow the interactions between metals. We showed that MTs are constitutively expressed in both human astrocytoma cell lines. In accordance with the higher malignancy grade of U87 MG, the amount of MTs was higher in U87 MG than in IPDDC-2A cells. After 24 hours of exposure to Cd their expression greatly increased in both cell lines and they were capable of immobilising almost all water soluble Cd. Induction of MTs in U87 MG cells was additionally followed up to 48 hours with exposure to different concentrations of CdCl₂ (1, 10 μM). Induction was a time dependent process throughout the period. Isoform III (identified by chromatographic separation of isoform III from I/II) was present at all exposure times, but only in traces with respect to the prevailing amounts of MT-I/II isoforms. So induction can be attributed to isoform I/II only.     

Heavy Metal Accumulation and Distribution in Organs of the Mussel Crenomytilus grayanus from Upwelling Areas of the Sea of Okhotsk and the Sea of Japan

The subcellular and cytosolic distribution of Zn, Fe, Ni, Cu, Mn, Cd, and Pb in the digestive gland and kidney of the mussel Crenomytilus grayanus from upwelling areas of the Sea of Okhotsk and the Sea of Japan was studied. Cd, Zn, Pb, and Ni have accumulated in the kidneys of mussels from these areas. When the concentrations of both essential and toxic metals in the mussel organs had increased significantly, their redistribution into the cytosol took place. Gel chromatography of cytosolic proteins in the kidneys of mussels sampled in the area with a strong stationary upwelling revealed metallothioneins. This is uncommon for bivalve mollusks from unpolluted areas. High molecular weight proteins act as preliminary ligands for Cd.     

Metallothioneins in liver of Rutilus rutilus exposed to Cu2+. Analysis by metal summation, SH determination and spectrofluorimetry

Metallothioneins (MTs) have important roles in the homeostasis of essential metals and in the detoxication of heavy metals. They also represent a potential indicator of aquatic contamination by metals. Routine methods are needed for MTs quantification in ecotoxicological studies. This paper investigates the possibility to use the spectrofluorescent properties of Cu-MTs for MTs quantification. Cu displacement of metals coordinated to MTs and spectrofluorimetric determination of the obtained Cu-MTs was tested with commercial MTs and Cu2+-induced MTs in roach liver (Rutilus rutilus). Results of this original and simple spectrofluorimetric quantification of MTs presented a good correlation with data obtained with SH quantification, but not with metal summation evaluation of MTs (analysis of Zn, Cu and Cd coordinated to MTs). The three methods showed an clear induction of MTs in roach liver after 7 days of Cu2+ exposure. After 14 days of contamination, a reduction of hepatic MTs content was observable and not correlated to liver recovery. Results show that this low cost spectrofluorimetric method is useful to quantify MTs.     

Localization effect on the metal biosorption capability of recombinant mammalian and fish metallothioneins in Escherichia coli

In this study, we examined the expression of mammalian and fish metallothioneins (MTs) in Escherichia coli as a strategy to enhance metal biosorption efficiency of bacterial biosorbents for lead (Pb), copper (Cu), cadmium (Cd), and zinc (Zn). In addition, MT proteins were expressed in either the cytoplasmic or periplasmic compartment of host cells to explore the localization effect on metal biosorption. The results showed that MT expression led to a significant increase (5-210%) in overall biosorption efficiency (eta(ads)), especially for biosorption of Cd. The MT-driven improvement in metal biosorption relied more on the increase in the biosorption rates (r(2), a kinetic property) than on the equilibrium biosorption capacities (q(max), a thermodynamic property), despite a 10-45% and 30-80% increase in q(max) of Cd and Zn, respectively. Periplasmic expression of MTs appeared to be more effective in facilitating the metal-binding ability than the cytoplasmlic MT expression. Notably, disparity of the impacts on biosorption ability was observed for the origin of MT proteins, as human MT (MT1A) was the most effective biosorption stimulator compared to MTs originating from mouse (MT1) and fish (OmMT). Moreover, the overall biosorption efficiency (eta(ads)) of the MT-expressing recombinant biosorbents was found to be adsorbate-dependent: the eta(ads) values decreased in the order of Cd > Cu > Zn > Pb.     

Stress response in Cu2+ and Cd2+ exposed oysters (Crassostrea gigas): an immunohistochemical approach

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

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.     

Evidence for copper homeostasis function of metallothionein (MT3) in the hyperaccumulator Thlaspi caerulescens

Metallothioneins chelate metals and consequently may be a control point of metal homeostasis. Homologous to type 3 metallothioneins, TcMT3 cDNA was identified in the Cd/Zn hyperaccumulator, Thlaspi caerulescens. TcMT3 amino acid sequence showed modifications in the Cys positions when compared with its Arabidopsis orthologue. A structural model established that the MT3 carboxyterminal domain is similar to the beta domain of animal metallothioneins and predicts a smaller cavity to chelate metals for A. thaliana than for T. caerulescens. Functional testing in yeast and Northern blot analysis added further evidence for adaptative variations of MT3 for the maintenance of Cu homeostasis in a metal hyperaccumulator.     

Cd, Cu, Zn, Se, and Metallothioneins in Two Amphibians, Necturus maculosus (Amphibia, Caudata) and Bufo bufo (Amphibia, Anura)

The accumulation of cadmium, its affinity for metallothioneins (MTs), and its relation to copper, zinc, and selenium were investigated in the experimental mudpuppy Necturus maculosus and the common toad Bufo bufo captured in nature. Specimens of N. maculosus were exposed to waterborne Cd (85???g/L) for up to 40?days. Exposure resulted in tissue-dependent accumulation of Cd in the order kidney, gills > intestine, liver, brain > pancreas, skin, spleen, and gonads. During the 40-day exposure, concentrations increased close to 1???g/g in kidneys and gills (0.64?C0.95 and 0.52?C0.76; n?=?4), whereas the levels stayed below 0.5 in liver (0.14?C0.29; n?=?4) and other organs. Cd exposure was accompanied by an increase of Zn and Cu in kidneys and Zn in skin, while a decrease of Cu was observed in muscles and skin. Cytosol metallothioneins (MTs) were detected as Cu,Zn?Cthioneins in liver and Zn,Cu?Cthioneins in gills and kidney, with the presence of Se in all cases. After exposure, Cd binding to MTs was clearly observed in cytosol of gills as Zn,Cu,Cd?Cthionein and in pellet extract of kidneys as Zn,Cu,Cd?Cthioneins. The results indicate low Cd storage in liver with almost undetectable Cd in liver MT fractions. In field trapped Bufo bufo (spring and autumn animals), Cd levels were followed in four organs and found to be in the order kidney > liver (0.56?C5.0???g/g >0.03?C0.72???g/g; n?=?11, spring and autumn animals), with no detectable Cd in muscle and skin. At the tissue level, high positive correlations between Cd, Cu, and Se were found in liver (all r?>?0.80; ???=?0.05, n?=?5), and between Cd and Se in kidney (r?=?0.76; n?=?5) of autumn animals, possibly connected with the storage of excess elements in biologically inert forms. In the liver of spring animals, having higher tissue level of Cd than autumn ones, part of the Cd was identified as Cu,Zn,Cd?Cthioneins with traces of Se. As both species are special in having liver Cu levels higher than Zn, the observed highly preferential Cd load in kidney seems reasonable. The relatively low Cd found in liver can be attributed to its excretion through bile and its inability to displace Cu from MTs. The associations of selenium observed with Cd and/or Cu (on the tissue and cell level) point to selenium involvement in the detoxification of excessive cadmium and copper through immobilization.     

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.     

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.