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.     

Metallothionein synthesis in foetal, neonatal and maternal rat liver

The synthesis of hepatic metallothionein relative to other cytosol proteins was measured by [35S]cysteine incorporation in foetal, neonatal and pregnant rats. The relative rate of hepatic metallothionein synthesis reached a maximum in foetal liver on days 18-21 of gestation. Metallothionein synthesis then declined until weaning, when adult levels were established. The rate of metallothionein synthesis was greater in pregnant rats at term than in nulliparous rats. To determine if circulating inducing agents could play a role in the regulation of metallothionein synthesis in foetal liver we treated pregnant rats with inducers at a time prior to the normal rise in foetal liver metallothionein synthesis. Injections of copper, cadmium or hydrocortisone to 17-day-pregnant dams failed to induce foetal metallothionein synthesis. In contrast, zinc injection to the dam was an effective inducer in the foetuses. Maternal laparotomy (performed to expose the foetus for direct injection of inducers) induced foetal metallothionein synthesis. Metallothionein synthesis in the livers of 17-day-gestation dams was induced by all metal injections and laparotomy but, surprisingly, not by hydrocortisone injection. Maternal adrenalectomy did not influence the subsequent normal elevation in foetal or maternal metallothionein synthesis. These results, in conjunction with previous reports, suggest that mobilization of zinc in serum during late gestation may regulate foetal and maternal changes in metallothionein synthesis.     

Intestinal metallothionein and the mutual antagonism between copper and zinc in the rat.

A study has been made of the mechanism of the mutual antagonism between copper and zinc in rats. Dietary zinc concentrations of up to 450 mg/kg had no effect on intestinal 64Cu absorption but 900 mg/kg caused a 40% reduction. This was associated with an increase in the mucosal uptake of 64Cu in the small intestine. This occurred mainly in the form of metallothionein and it appeared that copper displaced zinc from the protein after its synthesis had been induced by zinc. Ths intestinal absorption of 65Zn was decreased by 20% when the dietary copper intake was increased from 3 to 24 mg/kg. Further increases in copper intake to 300 mg/kg did not cause any additional decrease in 65Zn absorption or any change in the association of intestinal 65Zn with metallothionein. Concentrations of this protein in the intestinal mucosa were not influenced by dietary copper intake.     

Regulation of liver metallothionein and plasma zinc by the glucocorticoid dexamethasone.

Administration of the glucocorticoid dexamethasone to adrenalectomized rats significantly decreased the serum zinc concentration within 14 hr. Dexamethasone did not detectably alter the liver zinc content, but markedly increased the proportion of zinc associated with liver metallothionein. The rate of incorporation of ³⁵S-cystine into this protein was stimulated to a maximal extent 7 hr after administration of the glucocorticoid. Poly(A)⁺ mRNA from liver polysomes was isolated and translated in a cell-free protein synthesizing system. Nearly twice as much polysomal metallothionein mRNA was found 7 hr following treatment with dexamethasone. These results suggest that glucocorticoids can regulate the plasma zinc concentration by a process that is related to the biosynthesis of the hepatic zinc-binding protein, metallothionein.     

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.     

Zinc accumulation and metabolism in primary cultures of adult rat liver cells. Regulation by glucocorticoids

Adult rat liver parenchymal cells were isolated by the collagenase perfusion technique and cultured as a monolayer for up to 20 h. The quantity of zinc accumulated from the extracellular environment was significantly increased by adding physiological concentrations of certain glucocorticoids to the medium. The degree of stimulation was directly related to glucocorticosteroids potency. Sex steroids, certain peptide hormones and prostaglandins E₂ and F_2α did not influence zinc accumulation.Control cells exhibited a decline of zinc accumulation after 4 h in culture although uptake processes were still operative. When dexamethasone, the most potent glucocorticoid used, was present in the medium the cells accumulated zinc at a linear rate greater than that seen in control cells, for at least 20 h. The dexamethasone-induced stimulation of zinc accumulation was relatively specific since ⁴⁵Ca, ¹⁴C-labelled amino acids and [³⁵S]cystine accumulation was not influenced by the hormone. A lag of 4 h was observed before an effect of dexamethasone on zinc accumulation could be detected. Moreover, the hormone-stimulated phase of accumulation was blocked when the cells were simultaneously incubated with either actinomycin D or cycloheximide. The additional complement of zinc accumulated by the dexamethasone-treated cells was localized in the cytosol fraction. Gel filtration and ion-exchange chromatorgraphy confirmed that this additional cytosol zinc was bound to metallothionein. [³⁵S]Cystine was incorporated into metallothionein in hormone-treated cells indicating that the protein was synthesized de novo during periods of enhanced zinc accumulation.     

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.     

Zinc depletion suppresses tumor growth in mice

The hypothesis that in tumor-bearing animals an increase of host hepatic zinc metallothionein (Zn-MT) causes a restriction of zinc in the tumor tissue was studied. Three types of tumors were induced in laboratory mice by cell transplant. Tumor growth appears to be inhibited under zinc-deficient conditions, even in cases where zinc deficiency was started after tumor cell transplant. The survival times of tumor-bearing mice were prolonged by administration of cadmium chloride, which induces the synthesis of a combined zinc-cadmium metallothionein derivative in the host liver, but not in the tumor tissue, leading to an increase of hepatic zinc in the treated animals. The uptake of⁶⁵Zn by the liver of Cd-treated, tumor bearing mice was significantly higher than that of controls whereas uptake of⁶⁵Zn by tumor cells was significantly higher in controls than in the treated animals. These results suggest that restriction of zinc intake suppresses tumor growth.     

Glucocorticoid independent mediation of interleukin-1 induced changes in serum zinc and liver metallothionein levels

Interleukin-1 (IL-1) causes changes in zinc metabolism which have been attributed to mediation, at least in part, by glucocorticoids. However, IL-1 was found to actually lower serum corticosterone levels in rats. In addition, adrenalectomy only partially inhibited the ability of IL-1 to depress serum zinc levels and increase the amount of zinc associated with hepatic metallothionein. Furthermore, IL-1 increased total liver metallothionein protein to similar levels in both adrenalectomized and normal rats. Administering the synthetic glucocorticoid dexamethasone with IL-1 to adrenalectomized rats produced additive, but not synergistic effects on serum zinc and metallothionein concentrations. Studies with actinomycin D suggested that IL-1 induction of metallothionein might involve glucagon.     

Inhibition of zinc absorption by iron depends on their ratio

Previous studies upon zinc-iron interactions gave conflicting results that could come from differences in protocol design or in trace element status of subjects. The present work assessed the influence of zinc : iron ratio and iron deficiency upon zinc absorption. The digestive absorption of zinc sulphate (100 mol Zn/l) in presence of iron gluconate was studied in perfused jejunal loops (n = 6/group) of normal rats (range 0–1000 mol Fe/l) and iron deficient rats (200–750 mol Fe/l). In normal rats no significant iron inhibition on zinc absorption occurred at Fe:Zn ratio below 2:1. At higher ratios zinc uptake and net absorption decreased significantly (p<0.05). Between 2:1 and 5:1 a dose dependent inhibition of zinc absorption occurred and reached a plateau beyond this ratio. In iron deficient animals no changes in zinc uptake, mucosal retention and absorption compared to normal animals occurred at ratio 2:1. At higher ratios differences were observed at every zinc absorption step except for mucosal retention at 7.5:1 ratio.

Iron-zinc interactions depend on their ratio and on previous trace elements status of subjects. Due to the wide and unknown variations that were likely to occur between the subjects of previous human and experimental studies, these results could explain some of the discrepancies between their results.     

Cadmium accumulation and metabolism by rat liver parenchymal cells in primary monolayer culture

Primary cultures of adult rat liver parenchylmal cells, isolated by the collagenase perfusion technique and maintained as a monolayer,l were used to investigate the characteristics of hepatic cadmium accumulation and metabolism. Cadmium accumulation was found to be temperature- and concentration-dependent process that required sulfhydryl groups and was significantly stimulated by the addition of dexamethasome to the medium. Once taken up, cadmium was less available for exit-exchange processes than its biologically required congener, zinc. Moreover, cadmium influx enhanced zinc efflux. While most of the intracellular cadmium was located in the cytosol, its distribution within this fraction was altered with time. Initially the metal was bound to both high molecular weight species (>50 000) and metallothionein. As the incubation period increased, the cytosol concentration of cdmium and the percentage of this metal associated with metallothionein was likewise increased. [³H]Amino acid incorporation studies indicated that the accumulation of cadmium resulted in de novo synthesis of the 1 and 2 forms of metallothionein.     

Induction and degradation of Zn-, Cu- and Cd-thionein in Chang liver cells

Human liver cells (Chang liver) were exposed to 5 micrograms Zn, 2.5 micrograms Cu or 1 microgram Cd/ml in cultured medium. These exogeneous heavy metals were accumulated by the cells and induced de novo synthesis of metallothionein after a 3-h incubation period. The production of Zn-, Cu- or Cd-thionein started in the cells with accumulation of 1 nmol Zn, 0.3 nmol Cu and 0.1 nmol Cd/mg cytosol protein and subsequently the amounts of metal-binding thioneins increased in agreement with the relative amount of metal accumulated in the cytosol over a 24-h period. When cells containing Zn- or Cu-thionein were placed in metal free medium, 70% or 25% of the zinc or copper bound to each original metallothionein was released after 3 h; bound metals decreased to 85% and 65% respectively after 24 h. The disappearance of metal from metallothionein correlated with increases of metal in the medium. On the other hand, 35S-counts incorporated into Zn- and Cu-thionein decreased only to 40% and 15% of the levels in the original metallothionein after 3 h; 35S-counts decreased to 65% and 45%, respectively, after 24 h, indicating that metals bound to metallothionein decreased more quickly than 35S-counts. These results suggest that metals were released from metallothionein and were excreted into the medium. However, 35S- and 109Cd-counts in Cd-thionein changed very little, if at all, in the cells even after a 24-h incubation period. Our data strongly suggest that Zn- and Cu-thionein are degraded in the cells, but that Cd-thionein remains longer than either Zn- or Cu-thionein. When cells containing Zn-thionein were incubated in metal-free medium, Zn-thionein was digested in the cells and peptide fragments ranging about 200-400 daltons were excreted from the cells.     

Effect of 6-mercaptopurine on 65Zn distribution in the pregnant rat

The effect of 6-mercaptopurine (6-MP) on the distribution of gavaged 65Zn in maternal and embryonic tissues of Sprague-Dawley rats was examined 24 hr after injection of the drug on day 13 of pregnancy. 6-MP injection resulted in a significantly higher retention of counts of 65Zn in maternal liver and lower counts in maternal plasma, uterus, placenta, and embryo than in controls. Compared to controls, gel chromatography of maternal liver from 6-MP injected dams showed higher counts associated with a protein peak of molecular weight 6,000-8,000, the approximate molecular weight of the zinc-binding protein metallothionein. These results support the idea that the zinc deficiency, which is observed in day 21 fetuses from dams injected with 6-MP during midgestation, may be the result of a drug-induced sequestering of zinc into maternal liver followed by a decrease in maternal plasma zinc and subsequent reduction in fetal zinc uptake. We suggest that this 6-MP-associated redistribution of zinc into maternal liver may be due to induction of maternal metallothionein synthesis by the drug.     

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.     

Induction of metallothionein synthesis in Menkes' and normal lymphoblastoid cells is controlled by the level of intracellular copper

A study was carried out on the uptake of copper, zinc, or cadmium ions and their induction of metallothionein synthesis in Menkes' and normal lymphoblastoid cells. The main difference between Menkes' and normal cells in the uptake of these metal ions was an increased uptake of copper ions in Menkes' cells at a low concentration of CuCl2 (2.1 microM). The CuCl2 concentration necessary to induce metallothionein synthesis in Menkes' cells was 50 microM, whereas that in normal cells was about 200 microM. The levels of zinc or cadmium ions needed to induce metallothionein in Menkes' cells were similar to those in normal cells. At least four isomers of metallothionein were induced by copper, zinc, and cadmium ions in both types of cells. Metallothionein synthesis in Menkes' and normal cells was induced when the amounts of intracellular copper reached a threshold level of approximately 0.2 nmol/10(6) cells, and the rate of metallothionein synthesis in these cells was increased as a function of the amounts of intracellular copper (0.2-1.7 nmol/10(6) cells). These results indicate that the induction of metallothionein synthesis in lymphoblastoid cells is controlled by the level of intracellular copper, suggesting that the major defect in Menkes' cells is not due to the abnormal regulation of metallothionein synthesis but to an alteration of the copper metabolism in cells by which the levels of intracellular copper become larger than those in normal cells and just lower than the threshold level for induction of metallothionein synthesis.     

Hepatic metallothionein synthesis in neonatal mottled-brindled mutant mice

Mottled-brindled mutant mice did not display the elevated hepatic metallothionein synthesis normally observed in 2- to 6-day-old wild-type mice. This difference between normal and mutant mice was not due to a decreased ability to synthesize metallothionein in the liver, since hepatic metallothionein synthesis was inducible in response to copper, cadmium, zinc, or hydrocortisone administration to neonatal mutant mice. Hydrocortisone treatment resulted in increased metallothionein synthesis in liver of mutant mice but had no ameliorative effect on the mottled-brindled disease.This work was supported by Contract DE AM03 76 SF00012 between the Department of Energy and the Regents of the University of California. JEP is the recipient of a National Research Service Award from the National Institutes of Health.     

Induction of metallothionein by zinc in lethal milk mutant mice

Adult lethal milk (lm/lm) mutant mice display increased induction of hepatic metallothionein synthesis compared to wild-type mice following the subcutaneous injection of 40 µmol ZnCl₂/kg mouse. At this zinc dose the rate of incorporation of |³⁵S| cysteine into hepatic metallothionein in adult (100-to 230-day-old) lm/lm mice was approximately 2.4-fold greater than the rate of incorporation of isotope in wild-type animals. At a higher zinc dose (160 µmol ZnCl₂/kg) the incorporation of |³⁵S| cysteine into hepatic metallothionein was similar in lm/lm and wild-type mice. The altered dose-response to zinc administration was not due to a change in hepatic zinc, copper, or manganese levels, to a difference in ⁶⁵Zn uptake, or to an alteration in ⁶⁵Zn bound to differential centrifugation fractions of adult lm/lm liver. ⁶⁵Zn bound to hepatic metallothionein was, however, increased in aging lm/lm mice with symptomatic skin lesions.     

Parenteral zinc and tissue metallothionein in normal and diabetic rats

The effect of parenteral zinc on tissue metallothionein (MT) was studied in normal and streptozotocin-induced diabetic rats. The accumulation of Zn-MT in liver and pancreas of normal and diabetic rats following the administration (ip) of various amounts of zinc was not different. Renal Zn-MT was higher in the diabetic group, and this was not changed by zinc injection. Although diabetic rats, relative to normal, possessed a markedly higher concentration of Cu-MT in kidney initially, this difference decreased considerably after zinc injection. The ratio of Cu-MT to cytosolic Cu in kidney was not affected by parenteral zinc and was highest in diabetic rats. Zinc injection markedly reduced food intake, water consumption, and urine output in both normal and diabetic rats. Blood glucose of diabetic rats also decreased 24 h after zinc administration. Our results indicate that relative to normal, MT and zinc metabolism are different in kidney, and to some extent liver, but not different in the pancreas of the chemically induced diabetic rat.     

Zinc suppression of free radicals induced in cultures of rat hepatocytes by iron, t-butyl hydroperoxide, and 3-methylindole

The effect of zinc on lipid peroxidation initiated by either ferric-nitrilotriacetate, t-butyl hydroperoxide, or 3-methylindole was studied using primary monolayer cultures of rat liver parenchymal cells. The malondialdehyde content of the cells and culture medium was used to estimate the extent of lipid peroxidation. As the zinc concentration of the culture medium was increased from 1 to 48 microM, peroxidation was diminished. Cellular zinc and metallothionein levels were proportionally increased by supplemental zinc. Zinc supplementation of the medium inhibited NADPH-cytochrome c reductase activity and stimulated glutathione peroxidase activity. The uptake of iron into the hepatocytes was significantly reduced as the level of zinc was raised, suggesting that zinc antagonizes uptake of chelated iron into isolated hepatocytes and in this way blocks iron-induced peroxidation. Furthermore, induction of metallothionein synthesis by zinc may contribute to the reduction in free radicals. Spectra from electron spin resonance studies, using phenylbutylnitrone as a spin-trapping reagent, demonstrated that free radical production was inversely related to the zinc concentration of the culture medium. Spin trap data suggest that metallothionein added to lysed cells in vitro decreases free radical production. Studies using the spin trap, 3,3,5,5-tetramethylpyrroline-N-oxide indicated that cumulatively the predominant radical present in the cultures was a phenyl radical with hydroperoxide or methylindole. Collectively, our data demonstrate that zinc inhibits free radical production and lipid peroxidation in cultured hepatocytes. The mode of action of zinc could occur via free radical scavenging by zinc-induced metallothionein and/or by processes related to cytochrome P-450 and glutathione peroxidase, since these were also found to be sensitive to zinc supplementation levels of the culture medium.