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.     

Metal-binding proteins of the Syrian hamster ovaries: apparent deficiency of metallothionein

A deficiency of metallothionein, a high-affinity metal-binding protein thought to detoxify cadmium, has been observed in rat and mouse testes, tissues that are highly susceptible to the necrotizing and carcinogenic effects of cadmium. Like the testes, the ovaries undergo a hemorrhagic necrosis when exposed to cadmium, and female Syrian hamsters have recently been shown to be highly susceptible to cadmium. However, the nature of cadmium-binding proteins in the ovary is unknown; thus, this study was undertaken to define the nature of any such proteins in the Syrian hamster ovary. A low molecular weight (Mr) zinc- and cadmium-binding protein was detected in cytosol derived from the ovaries after gel filtration that eluted with a relative elution volume similar to authentic metallothionein. This protein was extractable by heat-treatment and sequential acetone precipitation. When such extracts were further purified with a reverse phase high performance liquid chromatography (HPLC) technique developed for the isolation of metallothionein isoforms, two forms were separated. However, neither of these could be classified as metallothionein on the basis of amino acid composition, since both were particularly low in cysteine, a very common amino acid in metallothionein. The ovarian protein also contained significant amounts of aromatic amino acids, unlike metallothionein--which is devoid of aromatics, and contained much more glutamate than metallothionein. Hamsters were also made resistant to cadmium-induced ovarian necrosis by zinc treatment. Such zinc treatment, however, did not alter levels of this protein, yet caused a marked induction of hepatic metallothionein. Likewise, cadmium treatment did not increase the levels of the ovarian metal-binding protein yet markedly induced hepatic metallothionein.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of superoxide dismutase deficiency on cadmium stress

Saccharomyces cerevisiae mutant strains deficient in superoxide dismutase (Sod), an antioxidant enzyme, were used to analyze cadmium absorption and the oxidation produced by it. Cells lacking the cytosolic Sod1 removed twice as much cadmium as the control strain, while those deficient in the mitochondrial Sod2 exhibited poor metal absorption. Interestingly, the sod1 mutant did not become more oxidized after exposure to cadmium, as opposed to the control strain. We observed that the deficiency of Sod1 increases the expression of both Cup1 (a metallothionein) and Ycf1 (a vacuolar glutathione S-conjugate pump), proteins involved with protection against cadmium. Furthermore, when sod1 cells were exposed to cadmium, the ratio glutathione oxidized/glutathione reduced did not increase as expected. We propose that a high level of metallothionein expression would relieve glutathione under cadmium stress, while an increased level of Ycf1 expression would favor compartmentalization of this metal into the vacuole. Both conditions would reduce the level of glutathione-cadmium complex in cytosol, contributing to the high capacity of absorbing cadmium by the sod1 strain. Previous results showed that the glutathione-cadmium complex regulates cadmium uptake. These results indicate that, even indirectly, metallothionein also regulates cadmium transport.     

Age-dependent variation for inducibility of metallothionein genes in mouse liver by cadmium

Cadmium is a toxic metal that induces the expression of metallothionein genes in many tissues and that binds avidly to metallothionein, a soluble transition metal binding protein. The present study examined the temporal pattern and magnitude of accumulation of metallothionein mRNA in liver of C57BL/6J mice of various ages treated with cadmium. In adult female mice, accumulation was dependent on the dosage level of cadmium and related to the concentration of this metal in liver. The accumulation of metallothionein mRNA in liver depended on age at exposure to cadmium. Intraperitoneal administration of 2 mg of cadmium per kg provoked small increases (two- to threefold) in levels of metallothionein mRNA in livers of 7- and 14-day-old mice. In contrast, cadmium treatment of 28- and 56-day-old mice resulted in 12- to 19-fold increases in levels of metallothionein mRNA in liver with maximum increases occurring 3 to 4 hr after treatment. Because similar patterns for the accumulation of cadmium of liver were found in 7-, 28-, and 56-day-old mice, observed age-dependent differences in induction of metallothionein mRNA in liver were probably not due to differences in the accumulation of cadmium in this organ. Taken together, these data suggest that tissue-specific factors controlling the expression of metallothionein genes may account for developmental variation in the inducibility of these genes by cadmium. Ontogenic variation in accumulation of metallothionein mRNA after cadmium treatment may be a factor in developmental variation in the acute lethality of cadmium in C57BL/6J mice.     

Induction of reactivation of herpes simplex virus in murine sensory ganglia in vivo by cadmium.

Herpes simplex viruses maintained in a latent state in sensory neurons in mice do not reactivate spontaneously, and therefore the factors or procedures which cause the virus to reactivate serve as a clue to the mechanisms by which the virus is maintained in a latent state. We report that cadmium sulfate induces latent virus to reactivate in 75 to 100% of mice tested. The following specific findings are reported. (i) The highest frequency of induction was observed after two to four daily administrations of 100 micrograms of cadmium sulfate. (ii) Zinc, copper, manganese, or nickel sulfate administered in equimolar amounts under the same regimen did not induce viral reactivation; however, zinc sulfate in molar ratios 25-fold greater than those of cadmium induced viral replication in 2 of 16 ganglia tested. (iii) Administration of zinc, nickel, or manganese prior to the cadmium sulfate reduced the incidence of ganglia containing infectious virus. (iv) Administration of cadmium daily during the first week after infection and at 2-day intervals to 13 days after infection resulted in the recovery from ganglia of infectious virus in titers 10- to 100-fold higher than those obtained from animals given saline. Moreover, infectious virus was recovered as late as 11 days after infection compared with 6 days in mice administered saline. (v) Administration of cadmium immediately after infection or repeatedly after establishment of latency did not exhaust the latent virus harbored by sensory neurons, inasmuch as the fraction of ganglia of mice administered cadmium and yielding infectious virus was similar to that observed in mice treated with saline. We conclude that induction of cadmium tolerance precludes reactivation of latent virus. If the induction of metallothionein genes was the sole factor required to cause reactivation of latent virus, it would have been expected that all metals which induce metallothioneins would also induce reactivation, which was not observed. The results therefore raise the possibility that in addition to inducing the metallothionein genes, cadmium inactivates the factors which maintain the virus in latent state.     

Implication of distinct proteins in cadmium uptake and transport by intestinal cells HT-29

The mechanisms of intestinal absorption have not been clearly elucidated for cadmium, a toxic metal. In this work, we show the implication of distinct proteins in cadmium transport, and the transport step where these proteins are involved. We first validated the HT-29 model by evaluating nontoxic doses of cadmium (ranging from 1 to 20 μmol/L), and by quantifying metal uptake and transepithelial transport. The time-course of 1 μmol/L cadmium uptake at pH 7.5 showed three steps: a rapid one during the first 4 min, probably due to cadmium binding to the membrane; a slower one, characterized by K _m of 1.65±0.54 μmol/L and V _max of 3.9±0.3 pmol/min per mg protein; and a third, corresponding to slow accumulation that was not equilibrated even after 48 h of cadmium exposure. Intracellular metallothionein content following 1 or 5 μmol/L cadmium exposure showed a significant increase after 6 h of exposure, and was not equilibrated even after 72 h, allowing cadmium accumulation. After 24 h of exposure, metallothionein content was 5-fold, 14-fold, 26-fold, and 50-fold, respectively, for cells grown in the presence of 1, 5, 10, and 20 μmol/L cadmium, compared to control cells. The second step of uptake, characterized by carrier-mediated transport, was markedly increased at pH 5.5, compared to pH 7.5, and strongly inhibited by the metabolic inhibitor dinitrophenol. Moreover Nramp2 transporter cDNA was present in HT-29 cells. These data suggest the involvement of a proton-coupled transporter, which may be the divalent cation transporter Nramp2 (natural resistance-associated macrophage protein 2). Cadmium uptake was also inhibited by copper, zinc, and para-chloromercuribenzenesulfonate (pCMBS), but not by verapamil or ouabain. Taken together, our results indicate that cadmium could enter HT-29 cell by Nramp2 proton-coupled active transport and by diffusion, and accumulates in the cell as long as it binds to metallothionein. Cadmium toxicity could depend partly on the activity of Nramp2, and partly on metallothionein content. This revised version was published online in July 2006 with corrections to the Cover Date.     

Responses of liver enzymes to cadmium administration in the goldfish (Carassius auratus) at different times of the year

1. The activity of goldfish liver ornithine decarboxylase increased upon cadmium chloride injection in April and November (water temperature 16 ± 1°C) and did not change significantly in June and July (water temperature 20 ± 1°C and 22 ± 1°C, respectively).2. Adenosylmethionine decarboxylase exhibited similar patterns.3. Tyrosine aminotransferase increased significantly in April, but this was not confirmed the following year.4. Liver metal concentration always increased after the injection and hepatic metallothionein was induced at all times independently of the responses of the three enzymes.     

Age dependent changes in metallothionein and accumulation of cadmium in horses

1. Analysis of livers and kidneys from 28 horses for cadmium, zinc and metallothionein showed low cadmium content in liver. There was a gradual increase in cadmium content in kidney with age. 2. Metallothionein values varied with zinc content in the liver and with cadmium content in the kidney; copper values did not vary in either tissue. 3. Metallothionein was localized mainly in the cytoplasms in liver and kidney of horses by immunohistochemistry.     

Increased synthetic capacity for metallothionein in cultured liver cells following dimethyl sulfoxide pretreatment

Cultured TRL 1215 cells in log phase of growth were exposed to dimethyl sulfoxide (DMSO; 14-280 mM) followed 48 h later by cadmium (10 micron). Intracellular concentrations of metallothionein (MT) were measured 24 h after cadmium addition. Cadmium alone caused a 10-fold increase in the levels of MT, while DMSO alone had no effect on cellular MT levels. DMSO pretreatment followed by cadmium exposure, however, resulted in MT levels that were elevated by a factor of as much as 25-fold those observed in control cells. Concurrent treatment with the DNA synthesis inhibitor hydroxyurea (HU) eliminated the enhancing effect of DMSO pretreatment on cadmium induction of MT, indicating the requirement of DNA synthesis. An enhancement of the cellular accumulation of the metal ion did not account for the increased cadmium-induced MT synthesis in DMSO-pretreated cells as these cells did not show significantly increased uptake of cadmium during the initial period of exposure. DMSO pretreatment enhances cadmium induction of MT synthesis through a mechanism that appears to be dependent on the synthesis of DNA.     

Biological function of metallothionein. VI. Metabolic interaction of cadmium and zinc in rats

The accumulation and depletion of cadmium in liver and kidney metallothionein (MT) and the effects of dietary zinc deficiency on cadmium metabolism were studied in rats. The accumulation of cadmium in liver MT started to plateau after 80 days, but there was a linear accumulation of this element in kidney MT over the entire 300-day experiment. Cadmium in MT fractions was depleted very slowly when rats were changed to a diet without cadmium. The accumulation of cadmium in MT also caused zinc to accumulate in this protein, even in rats fed zinc-deficient diets. However, the reverse situation was found not to be true; zinc did not cause cadmium to accumulate in MT. Dietary zinc deficiency limited the binding of injected¹⁰⁹Cd to MT of liver, but not of kidneys or testes. However, zinc-deficient rats fed cadmium in their diets metabolized cadmium similarly to zinc-supplemented rats, suggesting that zinc deficiency does not limit the ability of cadmium to stimulate MT synthesis.     

Hepatic metallothionein production and resistance to heavy metals by rainbow trout (Salmo gairdneri)--I. Exposed to an artificial mixture of zinc, copper and cadmium

Rainbow trout developed elevated hepatic metallothionein concentrations after 4 weeks in a solution containing zinc, copper and cadmium in a fixed ratio of 400:20:1. Resistance to a combination of these metals increased in proportion to the concentration to which they were exposed for 4 weeks. The concentration of copper but not zinc or cadmium in the low molecular weight proteins separated by gel filtration was related to the concentrations of metallothionein present. The combined toxicity of the metals in the mixture was additive.     

The effect of cadmium on the survivability of colony-forming hematopoietic cells in mice exposed to x-ray irradiation]

It has been reported elsewhere that in addition to enhancing the expression of metallothionein genes, the previous injection of cadmium salts into sublethally X-irradiated mice increases by 10 times the number of endogenous spleen colonies. To understand the mechanism of the strong radioprotective cadmium effect donors and recipients were treated separately. It is shown that the survival of exogenous bone marrow colony-forming cells in lethally irradiated recipient remains at the control level independently of the donor cadmium treatment, whereas the injection of cadmium nitrate to recipient mice leads to the stimulation of colony formation by 1.7-1.8 times. The data allow to conclude that the cadmium effect on the survival of colony-forming hemopoietic murine cells after X-irradiation is not mediated by the enhanced expression of metallothionein genes.     

In vivo and ex vivo displacement of zinc from metallothionein by cadmium and by mercury

Divalent cadmium and mercury ions are capable in vitro of displacement of zinc from metallothionein. This process has now been studied in vivo and ex vivo, using the isolated perfused rat liver system, in order to determine if this process can occur in the intact cell. Rats with normal and elevated (via preinduction with zinc) levels of hepatic zinc thionein were studied. Cd(II) completely displaces zinc from normal levels of metallothionein and on a one-to-one basis from elevated levels of metallothionein, both in vivo and ex vivo. Hg(II) displaces zinc from metallothionein (normal or elevated) rather poorly, as compared with Cd(II), in vivo, probably due to the kidneys preference for absorbing this metal. Ex vivo Hg(II) displaces zinc from metallothionein (normal or elevated) on a one-to-one basis, with considerably more mercury being incorporated into the protein than in vivo. The results of double-label ex vivo experiments using metal and [35S]cysteine (+/- cycloheximide) were consistent with the above experiments, indicating that de novo thionein synthesis was not required for short term incorporation of cadmium and mercury into metallothionein. These data are supportive of the hypothesis that cadmium and mercury incorporation into rat hepatic metallothionein during the first few hours after exposure to these metals can occur primarily by displacement of zinc from preexisting zinc thionein by a process which does not require new protein synthesis.     

Purification and immunological characterization of catfish (Heteropneustes fossilis) metallothionein

Catfish hepatic metallothionein was purified to homogeneity by Sephadex G-75 gel filtration, DEAE-Sephadex A-25 column chromatography and preparative polyacrylamide gel electrophoresis. Induction by cadmium and zinc, characteristic UV spectrum, cadmium binding property and its low MW established that it was a metallothionein. Antibody was raised in rabbit against catfish metallothionein. Catfish antimetallothionein cross-reacted with other fish metallothioneins but not with chicken or rodent metallothionein. Catfish metallothionein is more electronegative as compared to mouse, rat, chicken or hamster metallothionein. Catfish MT appeared to aggregate readily on storage and to be less electronegative.     

Characterization and use of the Drosophila metallothionein promoter in cultured Drosophila melanogaster cells.

The promoter from the metallothionein gene may be a useful conditional promoter for the construction of chimeric genes to be expressed in Drosophila cells in culture. To explore this possibility the responses of the endogenous metallothionein gene and an in vitro constructed chimeric gene containing the metallothionein promoter were examined. Copper and cadmium, when added to the growth medium of Drosophila Schneider's line 2 cells, can produce a 30-100 fold induction of metallothionein mRNA levels. The level of induction depends on the amount of copper or cadmium added to the medium and these mRNA levels remain high for at least four days. Copper is less toxic than cadmium and does not induce a typical heat-shock response in the cells. Finally, a chimeric gene containing the metallothionein promoter shows a similar induction when transformed into the cells.     

The effects of cadmium and copper on the renal uptake and metallothionein binding of gold in the rat and hamster

Rats and hamsters, (pre)-treated with copper and cadmium, were used to investigate whether species-differences in renal metallothionein synthesis in response to gold were determined by changes in the kidney concentrations of other metals. The effects of both dietary copper limitation and excess on the renal metabolism of gold also were studied in the rat. In this species, all of the pre-treatments affected the renal concentrations of total and metallothionein-bound copper, but none of them altered either the kidney uptake or thionein-binding of gold. Incorporation of zinc into the metallothionein, which accompanied the binding of gold in this fraction of the kidney, however, was influenced slightly by the pretreatments. In hamsters, pretreatment with cadmium, which increased the concentrations of total and thionein-bound zinc in the kidneys, also did not affect the renal uptake of gold, although it increased significantly the binding of gold to the metallothionein fraction of the renal cytosol. This increased binding of gold also was accompanied by further increases in the zinc and copper contents of the metallothionein; the contents of total and thionein-bound cadmium, however, remained essentially unchanged. Concentrations of copper and zinc in the hamster kidney were not affected significantly by subcutaneous administration of copper alone (five daily doses, each of 3.2 mg Cu/kg body wt.), but were increased when gold was given during the copper-treatment. The concentrations of gold, copper and zinc in the renal metallothionein fraction also were increased under these conditions. From these results it seems that kidney metallothionein synthesis in response to gold may be related to the changes in either the concentration or distribution of zinc, rather than copper.