Displacement of zinc and copper from copper-induced metallothionein by cadmium and by mercury: in vivo and ex vivo studies

The in vitro affinity of metals for metallothionein (MT) is Zn less than Cd less than Cu less than Hg. In a previous study Cd(II) and Hg(II) displaced Zn(II) from rat hepatic Zn7-MT in vivo and ex vivo (Day et al., 1984, Chem. Biol. Interact. 50, 159-174). The ability of Cd(II) or Hg(II) to displace Zn(II) and/or Cu(II) from metallothionein in copper-preinduced rat liver (Zn, Cu-MT) was assessed. Cd(II) and Hg(II) can displace zinc from (Zn, Cu)-MT both in vivo and ex vivo. The in vitro displacement of copper from MT by Hg(II) was not confirmed in vivo and ex vivo. Cd(II) treatment did not alter copper levels in (Zn, Cu)-MT, as expected. Hg(II) treatment, however, did not decrease copper levels in MT, but rather increased them. The sum of the copper increase and mercury incorporation into MT matched the zinc decrease under in vivo conditions and actually exceeded the zinc decrease under ex vivo conditions. Short-term exposure of rat liver to exogenous metals can result in incorporation of these metals into MT by displacement of zinc from pre-existing MT. Displacement of copper from pre-existing MT by mercury, as predicted by in vitro experiments, was not confirmed under the conditions of our in vivo and ex vivo experiments. This result is explainable based on the differing affinities and/or preferences of the two metal clusters in MT.     

Radioimmunoassay of rat liver metallothionein

A simple, inexpensive and convenient radioimmunoassay for rat liver metallothionein has been developed. The double-antibody assay involves the labeling of homogeneous, rat liver zinc thionein with trace amounts of ¹⁰⁹Cd(II) to a specific activity of 1–2 × 10⁶ cpm/μg protein; the binding of this antigen by rabbit anti-rat liver metallothionein antiserum; the displacement of this antigen by unlabeled zinc thionein or cadmium, zinc-thionein; the precipitation of the rabbit antibody-rat antigen complex by goat anti-rabbit IgG immunoglobulins; and the binding of this precipitate to cellulose acetate filters. The radioimmunoassay is useful in the range of concentration of metallothionein of 10–500 ng protein. The assay is accurate as compared with a previous technique of quantitating metallothionein in extracts of rat liver. A radial immunodiffusion assay for metallothionein is also described.     

Formation and characterization of aurothioneins: Au,Zn,Cd-thionein, Au,Cd-thionein, and (thiomalato-Au)chi-thionein

Three gold-containing thioneins (Au,Zn,Cd-Th, Au,Cd-Th, and (TmSAu)chi Th, where Th = thionein and TmS = thiomalate) have been prepared by the reactions of horse kidney Zn,Cd-thionein with gold thiomalate (AuSTm). When thionein was present in excess, the thiomalate ligand was displaced and the protein chelated the gold in a bidentate fashion. Primarily zinc but also some cadmium was displaced to form Au,Zn,Cd-Th or Au,Cd-Th. Excess AuSTm reacted to form (TmSAu)chi-thionein with monodentate coordination of the protein to each bound gold, retention of the thiomalate, loss of zinc and cadmium, and an increase in the Stokes radius of the product. EXAFS/XANES studies of Au,Zn,Cd-Th and (TmSAu)chi Th established that the oxidation states and coordination environments of gold were Au(I)S2 and that the gold-sulfur bond distances were 229 and 230 pm, respectively. Radioimmunoassay established that the aurothioneins retained their antigenicity to native metallothionein antibodies. Metal exchange reactions with gold were complete within 5-10 min when Zincon or 4-(2-pyridylazo) resorcinol was used to monitor Cd2+ and Zn2+ displacement.     

The binding of gold(I) to metallothionein

The binding of gold(I) to metallothionein, MT, has been unambiguously established by the reaction of Na₂AuTM with purified horse kidney MT. Zinc was displaced more readily than cadmium although the latter could be displaced using large Au/Cd ratios. The metal exchange reactions were complete within 2 hr of mixing. Further evidence that such reactions might be physiologically significant were obtained by studying in vitro metal displacements in the liver cytosol of in vivo metal treated rats: When Na₂AuTM was added to the cytosol of rats administered CdCl₂ in vivo, zinc, copper and cadmium were displaced in 2/1/1 ratios from the metallothionein fraction. The zinc and cadmium displacement provide direct evidence that the gold was binding to MT. Addition of Cd⁺² to liver cytosol of gold-treated rats resulted in displacement of copper and zinc, but not gold, from the MT fractions. When liver MT is prepared from rats exposed to Au or Cd, the Cd/protein ratio increased during the preparation, but the Au/protein ratio decreased. The Mt-bound metals account for 95% of the cytosolic Cd but only 15%–30% of the cytosolic gold in these studies. Thus, the nonspecific binding of gold to MT in vivo should be considered as one aspect in its equilibration among protein binding sites, which include, inter alia, metallothionein. Gold was found to coelute with zinc and cadmium in the MT fraction of rat kidney cytosol, when both Cd and Na₂AuTM were administered to the rats. The possible significance of gold binding to MT in the treatment of rheumatoid arthritis-chrysotherapy-is briefly discussed.     

Cadmium removal from human plasma by Cibacron Blue F3GA and thionein incorporated into polymeric microspheres

Poly(2-hydroxyethylmethacrylate–ethyleneglycoldimethacrylate) [poly(HEMA–EGDMA)] microspheres carrying Cibacron Blue F3GA and/or thionein were prepared and used for the removal of cadmium ions Cd(II) from human plasma. The poly(HEMA–EGDMA) microspheres, in the size range of 150–200 μm in diameter, were produced by a modified suspension copolymerization of HEMA and EGDMA. The reactive triazinyl dye-ligand Cibacron Blue F3GA was then covalently incorporated into the microspheres. The maximum dye incorporation was 16.5 μmol/g. Then, thionein was bound onto the Cibacron Blue F3GA-incorporated microspheres under different conditions. The maximum amount of thionein bound was 14.3 mg/g. The maximum amounts of Cd(II) ions removed from human plasma by poly(HEMA–EGDMA)–Cibacron Blue F3GA and poly(HEMA–EGDMA)–Cibacron Blue F3GA–thionein were of 17.5 mg/g and 38.0 mg/g, respectively. Cd(II) ions could be repeatedly adsorbed and desorbed with both types of microspheres without significant loss in their adsorption capacity.     

Metal binding in metallothioneins: Competition for cadmium and zinc between chelex-100 and metal binding sites in metallothionein

A study of the use of the metal chelation properties of Chelex-100 in metal binding reactions of metallothionein (MT), is described. The stoichiometric ratios of bound metals in MT were determined at several stages during a titration in which the Zn(II) in Zn₇MT was displaced by Cd(II), by using Chelex-100 to sequester the free zinc. The stoichiometric ratios provide convincing supporting evidence that the complicated circular dichroism spectral properties observed during the titration arise because the incoming cadmium is distributed across both domains in the protein. It is shown that Chelex-100 does not sequester zinc or cadmium directly from the metallothionein binding sites. Use of Chelex-100 over the temperature range −20 to 65 °C is demonstrated. The chelation capacity of Chelex-100 (in terms of μ metal ion/mg resin) has been determined for a range of elements important in metal toxicology, including: cadmium (33 μ), zinc (22 μ), copper (19 μ), silver (38 μ), lead (40 μ) and mercury (40 μ).     

Postinductive actinomycin D effects on the concentrations of cadmium thionein, zinc thionein, and copper chelatin in rat liver

The time courses of induction in rat liver of copper chelatin by copper, cadmium thionein by cadmium, and zinc thionein by copper, cadmium, and zinc were monitorg metal were used in order to avoid toxic effects, being 5 mg zinc, 0.5 mg copper, and 0.25 mg cadmium per kg body weight. Peak times of induction and half times of decay observed were: copper chelatin (9 h, 8.6 h), cadmium thionein (18 h, 6.80 days), and zinc thionein (zinc rats, 18 h, 10.1 h; copper rats, 9 h, 18.2 h; cadmium rats, 24 h, 4.53 days). Administration of actinomycin D (1 mg per kg body weight) at the peak times of induction of the various proteins had no effect on the concentrations of chelatin or cadmium thionein observed up to 24 hours later, but in the case of zinc thionein, induced by zinc, copper, or cadmium, elevated concentrations were observed up to 23 h after administration of the drug. Such behavior is reminiscent of superinduction previously seen with other proteins and enzymes. We postulate that the intracellular concentration of free zinc in liver is of fundamental importance in the induction of zinc thionein, and this can be distributed by exogenous copper or cadmium resulting in the induction of synthesis of zinc thionein.     

Zinc-, copper- and cadmium-binding protein in Ehrlich ascites tumour cells.

The properties of Ehrlich ascites tumour cells exposed in vivo to cadmium were investigated as a function of the zinc status of the host animals. Tumour-cell growth was inhibited by cadmium in both zinc-sufficient and zinc-deficient animals. However, cells in zinc-sufficient tumours accumulate much less cadmium than those in deficient tumours. The subcellular distributions of cadmium and zinc do not depend on zinc status. Cadmium and zinc are bound to a low-molecular-weight protein with properties similar to metallothionein. Without exposure to cadmium, a zinc- and copper-binding protein is still present that behaves like a metallothionein. This protein can rapidly bind cadmium added to Ehrlich cells in vitro. It is shown that the zinc- and copper-binding protein contains free thiol groups. Ehrlich cells isolated from cadmium-treated animals are viable and show normal incorporation of uridine into RNA, but the cellular uptake of thymidine and its incorporation into DNA are inhibited.     

On the sensitivity of metallothioneins to oxidation during isolation.

It is demonstrated that the distribution of metals among the Sephadex G-75 fractions of rat liver and horse kidney supernatant is altered by exposure to oxidizing conditions. In particular, the metals bound to metallothionein are displaced into high-molecular-weight fractions and, to a lesser extent, into the low-molecular-weight forms, under aerobic conditions. In this process, metallothionein zinc is much more labile than cadmium. An appreciable proportion of the thionein is also found in the high-molecular-weight fractions and can be recovered along with the metals by treatment with mercaptoethanol. This result shows that the distributions obtained aerobically with large cadmium content in the high-molecular-weight fractions are an artefact due to metallothionein oxidation and suggests that 'spillage' of metals such as cadmium may be due in large part to oxidative processes rather than saturation effects. Evidence is presented that disulphide-bond formation occurs as thionein becomes bound in the high-molecular-weight region and that chemical reduction is necessary to restore its normal elution behaviour. Mercaptoethanol added to the homogenates maintains the reducing conditions normally found in the cellular milieu and prevents the oxidation of the metallothionein redistribution of the metals during isolation. Under these conditions the rat liver metallothionein isolated from animals exposed to chronic low concentrations of cadmium in drinking water contains appreciable quantities of copper as well as zinc and contains much of the zinc that is present in horse kidney supernatants. Metallothionein can also be extracted from a 40 000g pellet after sonication of the pellet. Thus careful analytical studies of the sites of cadmium deposition in rat liver indicate that greater than 95% is bound to metallothionein.     

Embryo-larval tolerance of Mytilus galloprovincialis,exposed to the elevated sea water metal concentrations—I. Toxic effects of Cd,Zn and Hg in relation to the metallothionein level

Developing eggs of Mytilus galloprovincialis were exposed to elevated concentrations of sea water cadmium, zinc and mercury, 48 hr post-fertilization, in order to study the toxic effects at the morphological and biochemical level comparatively. Decreasing order of metal toxicity (Hg > Zn > Cd) was based on the proportion of normally formed straight-hinge veliger larvae and upon the growth inhibition of the primary larval shell. Metal-induced proteins isolated from homogenate extract of the straight-hinge veliger larvae by Sephadex G-75 chromatography were considered to belong to the metallothionein class due to their high metal content as well as according to their chromatographic and spectral properties. The inverse relationship between relative toxicity level and amounts of total and induced metal bound to MT has been found to be described by the function of a potency type. Combined exposure to zinc and cadmium simultaneously, resulted in an antagonistic effect, showing a larger proportion of normally formed veliger larvae and a smaller growth inhibition in comparison with the sum of single metal effects. The induction of MT in larvae subjected to cadmium and zinc mixture was markedly larger than the addition of individual metal effects.     

Metabolism of parenterally administered zinc and cadmium in livers of newborn rats

The interaction of injected zinc and cadmium with metallothionein was investigated in newborn rats. Tissues of 5-day-old rats were removed 24 h after a single injection (Sc) of saline or zinc (20 mg/kg, body wt.) or cadmium (1 mg/kg, body wt.) with 2.5 μCi of ⁶⁵Zn or ¹⁰⁹Cd or 5 μCi of [³⁵S]cysteine. Injection of zinc resulted in a 75% increase in the hepatic zinc concentration with a concomitant elevation of metallothionein (P < 0.001), zinc in metallothionein increased by 45% (P < 0.05); [³⁵S]cysteine incorporation indicated the induced synthesis of metallothionein. Injection of cadmium did not alter either metallothionein or zinc levels in liver, but cadmium in cytosol was preferentially bound to metallothionein. Neither treatment altered hepatic copper metabolism and copper in metallothionein, nor renal zinc and metallothionein levels. These data indicate that zinc injection can elevate hepatic zinc levels and induce metallothionein synthesis in newborn rats despite high basal levels; cadmium injection does not induce metallothionein synthesis, though cadmium is avidly sequestered by pre-existing metallothionein. The differences in the induction of metallothionein by these divalent cations can be explained by the differences in their binding affinities for thiol groups in intracellular metallothionein.     

Physiological levels of glutathione enhance Zn(II) binding by a Cys4 zinc finger

Using fluorescence and UV-vis spectroscopies and mass spectrometry, we demonstrated that the presence of physiological levels of reduced glutathione enhances the binding of Zn(II) to XPAzf, a Cys4 zinc finger peptide derived from the XPA protein, by means of formation of a ternary complex of a general formula ZnXPAzf[GSH]. Similar complexes were also indicated by ESI-MS for isostructural Co(II)- and Cd(II)-substituted XPAzf. The observed enhancement of the Zn(II) binding to XPAzf by a factor of 50 over the physiological range of GSH concentrations of 1-20 mM corresponds to a dissociation constant of GSH from the ZnXPAzf[GSH] complex of 0.05 μM. This effect may account for an apparent discrepancy between relatively low Zn(II) binding constants measured in vitro for many zinc fingers, and the requirement of tight Zn(II) binding enforced by intracellular zinc buffering by the thionein/metallothionein couple.     

Expression of the mouse metallothionein-I gene in Escherichia coli: increased tolerance to heavy metals

The cDNA of mouse metallothionein, a small metal-binding protein rich in cysteine, has been cloned downstream from a bacterial inducible promoter and expressed in Escherichia coli. Upon induction, E. coli harboring this cDNA clone contained a protein species readily labelled by [35S]cysteine in vivo and incorporated 10-times as much 109Cd from the medium than would otherwise be the case. We show that expression of metallothionein endows resistance in E. coli to heavy metal ions such as mercury, silver, copper, cadmium and zinc by sequestering rather than exclusion or conversion, common mechanisms of metal resistance in bacteria.     

Patterns of tolerance to heavy metals among methane-utilizing bacteria

The susceptibility of obligate methane-utilizing bacteria, including 14 reference strains and 175 environmental isolates, to five readily available heavy metal pollutants was determined. The chloride salts of Hg(II), Cd(II), Cu(II), Cr(III) and Zn(II) were tested in a system free from organic matter. Methane-utilizers appeared to have relatively discrete metal tolerance patterns with resistances to all metals varying with isolation site. Methanotrophs proved to be quite sensitive to mercury and cadmium but relatively resistant to copper, chromium and zinc     

Affinity labeling of phosphoenolpyruvate carboxykinase with 1, 5-I-AEDANS.

The concentrations of zinc thionein and cytosolic zinc in rat liver were examined in male rats five days after bilateral adrenalectomy. Zinc in metallothionein increased 10 fold, as compared with control animals. Cytosolic zinc increased 79% as compared with controls. 65% of this increase could be accounted for bound to metallothionein. Sham operated animals after five days showed a 4 fold increase in hepatic zinc thionein and a 23% increase in cytosolic zinc, 71% of this increase being bound to metallothionein. Adrenalectomized rats, maintained on daily injections of corticosterone (4mg/100g b.w.), exhibited the same levels of zinc thionein and cytosolic zinc as adrenalectomized rats receiving no treatment. Adrenalectomized rats, maintained on daily injections of aldosterone (5μg/100g b.w.), exhibited the same levels of zinc thionein as the sham operated rats, but the cytosolic zinc remained elevated at the level found in adrenalectomized rats receiving no treatment. These results indicate that there is adrenal involvement in the control of hepatic zinc and zinc thionein levels in the rat.     

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.     

Maleate induced change in the kidney binding of mercury in rats pretreated with cadmium

The kidney uptake of Hg2+ was increased by Cd2+-pretreatment when Hg2+ was given intraperitoneally but not subcutaneously. Subsequent s.c. administration of maleate increased Hg2+ release from the kidneys only if Hg2+ was given subcutaneously. Neither the effect of Cd2+, nor that of maleate, on the distribution of Hg2+ among the renal soluble protein fractions was affected by the route of Hg2+ administration. The protective effect of Cd2+-pretreatment against the nephrotoxic effect of Hg2+ was also independent of the route of Hg2+ administration. Maleate given in nephrotoxic doses removed Hg2+ and copper, but not Cd2+ from the renal metallothionein fraction. Mercury in the urine, however, was not complexed by proteins with the molecular weight of thionein, but was bound to high molecular weight proteins and diffusible molecules. These findings are discussed in relation to the role of metallothionein in the interaction between Cd2+ and Hg2+.