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.     

A comparison of cadmium-induced metallothionein gene expression and Me2+ distribution in the tissues of cadmiumsensitive (rainbow trout; Salmo gairdneri) and tolerant (stone loach; Noemacheilus barbatulus) species of freshwater fish

1. The accumulation of cadmium in the liver, kidney and gills of rainbow trout and stone loach was measured during exposure of the fish to the metal at 3 smg/l in their aquarium water. The pattern of accumulation of the toxic metal in the individual organs was different between the two species.2. The tissue concentrations of metallothionein-specific mRNA and metallothionein protein were also determined in these organs from the same fish. In rainbow trout, the induction of metallothionein gene expression resulted in a gradual increase in metallothionein concentration in gill over the course of the experiment whereas increases in metallothionein in the liver and kidney were detected only at the later time points of analysis (beyond 19 weeks). By contrast, in the same tissues from stone loach, relatively minor changes were quantified in specific mRNA and metallothionein concentrations.3. Throughout the experimental period, tissue concentrations of zinc and copper were determined in the liver, kidney and gills of the rainbow trout and stone loach. Subtle decreases were observed in the zinc concentration of gills in rainbow trout and substantial increases were observed in the hepatic copper concentrations in both species at the later time points of analysis.4. The ability of cadmium to induce metallothionein gene expression and its subsequent ability to compete for the sequestration sites on the newly-synthesized protein is discussed with regard to the relative levels of cadmium, zinc and copper in the organs studied and differing regimes of cadmium administration.     

Isolation and characterization of metallothionein from the tortoise Clemmys mutica

A relatively high concentration of metallothionein was present in the liver of the control mature tortoise Clemmys mutica. The administrations of cadmium and copper to Clemmys mutica increased the metals in the liver with concomitant decrease of zinc, which was due to substitution of zinc in the native metallothionein with cadmium and copper. The Clemmys metallothionein was a mixture of three isoproteins which were abundant in half-cystine (34.2-34.6 mol%) and lysine (10.8-11.9 mol%), but lacked aromatic amino acids. One of the isoproteins, MT-I, contained one residue of histidine, which was an identical feature with frog and avian metallothioneins.     

Influence of dietary cadmium on the distribution of the essential metals copper,zinc and iron in tissues of the rat

The effect of long-term dietary cadmium treatment upon the distribution of the metals copper, iron and zinc has been compared in various organs of male and female rats. The renal accumulation of cadmium was similar in both sexes without a plateau being reached. In contrast, the hepatic accumulation of cadmium was higher in the female than in the male rat and a plateau was observed after 30–35 weeks of dietary cadmium treatment. Most of the cadmium which accumulated in these organs was recovered in the metallothionein fraction and the concentration of hepatic cadmiumthionein in the female rat was correspondingly higher than in the male rat. Accumulation of cadmium was associated with an increased zinc concentration in the liver and an increased copper concentration in the kidney; these increases were correlated with increases in liver and kidney metallothioneins induced by cadmium. Uptake of cadmium into organs other than liver and kidney occurred to a small extent but was not associated with changes in the concentration of copper and zinc. Cadmium also accumulated in the intestinal mucosa where it could be recovered in a fraction corresponding to metallothionein. A loss of iron from the liver and kidney was also observed following dietary cadmium treatment and involved mainly a loss of iron from ferritin.     

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.     

Heavy metals in relation to the biology of the mummichog, Fundulus heteroclitus

Heavy metal levels of cadmium, copper, mercury, manganese, and zinc were examined in the mummichog, Fundulus heteroditus from industrialized and non-industrialized environments. With one exception, the environment with the highest trace metal in its waters, had the fishes with the highest metal concentration. Except for mercury, the concentration factor varied inversely with the metal concentrations of the fish and water, suggesting a possible regulatory mechanism for metals in the tissues of mummichogs from environments with high metal concentrations. There was an inverse relationship between standard length and concentrations of zinc, manganese, copper and cadmium in whole male and female fishes. The viscera contained significantly greater concentrations of these metals than somatic muscle tissue. There were also significant differences between males and females with respect to whole-body zinc and copper concentrations, but no sex differences for manganese and cadmium.     

Change in ratio of the two hepatic isometallothioneins with development from prenatal to neonatal rats

Concentrations and contents of essential metals in the livers of prenatal and neonatal rats were determined simultaneously by inductively coupled plasma-atomic emission spectrometry. The concentrations and contents changed with development in a mode characteristic to the respective metals. Changes in the concentrations and contents of zinc and copper were correlated to those bound to metallothionein, the two metals being high in concentration during prenatal and neonatal periods. The relative ratio of the two isoforms of metallothionein changed with development, the form I being predominant in prenatal livers.     

Environmental and injected cadmium are sequestered by two major isoforms of basal copper, zinc-metallothionein in gibel (Carassius auratus langsdorfi) liver

Gibels were exposed to cadmium in their aquarium at a concentration of 10 micrograms Cd/l for up to 39 weeks. Distributions of cadmium, copper and zinc in the liver soluble fraction were determined along with sulfur by high performance liquid chromatography-inductively coupled argon plasma-atomic emission spectrometry. Cadmium was sequestered by the two major isoforms of gibel metallothionein as in the case of cadmium injected intraperitoneally into gibel. Several peaks with cadmium, copper, zinc and sulfur were observed other than the two major isoforms and their relative ratios were different between the control and cadmium-exposed fishes.     

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.     

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.     

Kidney and liver metallothioneins in rats after administration of an organic compound

Intubation of rats with alpha-mercapto-beta-(2-furyl)-acrylic acid (MFA) for 5 days at 50 mg/kg caused a 7-fold increase in kidney copper concentration, a 2-fold increase in kidney zinc concentration, and a 20% increase in liver zinc concentration. The proteins which bound the increased metals were purified and identified as metallothioneins by their amino acid compositions. Two isoforms were isolated from each organ. Renal thioneins appeared identical to counterpart hepatic apoproteins, but the former bound Cu and Zn in a 2:1 mole ratio and the latter bound only Zn. Kidney contained over 10 times more metallothionein per g of tissue than did liver. In rats previously administered MFA, injection of cadmium sulfate resulted in rapid displacement of liver metallothionein-bound Zn by Cd under conditions where minimal metallothionein was found in Cd-dosed animals not administered MFA. We conclude that MFA induces metallothionein biosynthesis in kidney and liver of normal rats; this is a novel effect for an organic compound.     

Rainbow trout metallothionein

Two forms of hepatic metallothionein were isolated and purified from rainbow trout injected intraperitoneally with cadmium chloride. Both forms showed similarities with mammalian metallothioneins, had a high cystein content (30 mol%), and were void of aromatic amino acids and histidine. The molecular weight was estimated to be about 6000 dalton for the apothioneins, and the thiol groups of the cysteine residues complexed with the heavy metals (Cd, Cu, Zn) in a SH/Me⁺⁺ ratio of about 2.4. The amount of copper in metallothionein from rainbow trout was very high, greater than the amount of cadmium and zinc after injections of 3 mg cadmium/kg body weight. The total metal content of cadmium, copper and zinc in metallothionein 1 and 2 were about 7 and 8 atoms per molecule respectively.     

Metallothionein accumulation may account for intracellular copper retention in Menkes' disease

Cultured lymphoblasts derived from infants with Menkes' disease exhibit the same increased avidity for copper as do fibroblasts and most extrahepatic tissues from these patients. The Menkes' cells preferentially take up not only copper but also, on exposure to elevated metal concentrations, the other metallothionein-binding metals, zinc and cadmium. Menkes' lymphoblasts contain larger amounts of metallothionein than normal cells following exposure to each of these metals; the amount bound to this protein quantitatively accounted for the total cellular increment in metal in Menkes' cells. Induction of metallothionein synthesis caused both normal and Menkes' cells to subsequently take up increased amounts of 67Cu. These observations suggest that an enhanced capacity of Menkes' cells to accumulate metallothionein may be responsible for their increased uptake and retention of copper.     

Dietary cadmium decreases lipid peroxidation in the liver and kidneys of the bank vole (Clethrionomys glareolus).

The effect of elevated levels of dietary cadmium on lipid peroxidation in the liver and kidneys of a small rodent, the bank vole, was determined in the present study. Males and females, aged 1 month, were given diets containing 0.40 and 80 mg Cd per kg; liver and kidneys were removed for TBA-RS as well as iron, copper, zinc, cadmium and metallothionein analyses at the end of 6 weeks. Dietary Cd significantly decreased the TBA-RS level in the liver and kidneys of both sexes; however, this effect appeared to be dose-dependent only for the male liver. The changes in hepatic and renal TBA-RS paralleled closely those of tissue iron. Copper concentration decreased significantly only in the male liver, while hepatic and renal zinc were not influenced by dietary Cd. The concentrations of Cd and metallothionein in the liver and kidneys increased significantly in a dose-dependent fashion. Regression analysis confirmed that TBA-RS in both organs correlated closely with iron. The data suggest that dietary Cd decreases hepatic and renal lipid peroxidation indirectly, through lowering the tissue iron concentration.     

The partition of copper and cadmium between different charge-forms of metallothionein in the digestive tubules of the crab, Cancer pagurus

In the presence of 2-mercaptoethanol DEAE cellulose chromatography of desalted hepatopancreas cytosols of crabs rich in both copper and cadmium yielded three peaks, probably all due to metallothioneins, with very different copper to cadmium ratios. Addition of a copper glycine complex to a desalted crab cytosol containing cadmium metallothionein gave a chromatogram similar to that obtained by in vivo administration of copper and cadmium. The chromatographic distribution of the metals between the different charge-forms in the distal and proximal portions of the tubules was basically similar although the concentrations of the metals were different.     

Effects of interaction between65Zn,cadmium, and copper in rats

Distribution and retention of zinc in the presence of cadmium and copper was studied in rats exposed repeatedly to these metals. The experiment was performed on white rats of the Wistar strain. The animals were divided into four groups/five rats each: 1)⁶⁵ZnCl₂; 2)⁶⁵ZnCl₂+CdCl₂; 3)⁶⁵ZnCl₂+CuCl₂; and 4) control group. Rats were administered sc every other day for two weeks:⁶⁵ZnCl₂−5 mg Zn/kg; CdCl₂−0,3 Cd/kg; and CuCl₂−2 mg Cu/kg. The zinc content was measured in rat tissues by γ-counting. Effect of Cd and Cu on subcellular distribution of zinc in the kidney and liver and on the level of metallothionein were also examined. Whole body retention of zinc under the influence of cadmium was lower than that observed in animals treated with zinc alone. However, copper increased twofold the whole body retention of zinc. Cadmium elevated the accumulation of zinc only in the kidneys nuclear fraction and liver soluble fraction. In the kidneys and liver, copper elevated the accumulation of zinc, in the nuclear, mitochondrial, and soluble fractions. The level of metallothionein-like proteins (MT) in the kidneys after a combined supply of zinc and copper was significantly increased with respect to the group of animals treated with zinc alone. These results indicated complex interactions between cadmium, copper, and zinc that can affect the metabolism of each of the metals.     

The metabolism of metals in rat placenta

The status and transfer of metals across the rat placenta were studied by subcellular and molecular fractionations of this organ at 2 and 24 h after iv injection of radiolabeled metals. The soluble and nuclear fractions showed higher contents of copper and zinc, whereas most of the nickel was associated with the soluble fraction. Cadmium was almost evenly distributed between the microsomal and nuclear fractions. Gel filtration of the soluble fractions showed nickel associated with an unknown low molecular weight form; zinc with high molecular weight proteins; copper with metallothionein, ceruloplasmin, and high molecular weight proteins; and cadmium with high molecular weight proteins and metallothionein.     

Cadmium, copper and zinc in tissues of deceased copper smelter workers

Workers at a copper and lead smelter in northern Sweden have a multifactorial exposure to a number of heavy metals. The concentrations of cadmium, copper and zinc in liver, lung, kidney and brain tissues have been determined by atomic absorption spectrometry in 32 deceased long-term exposed male lead smelter workers, and compared with those of 10 male controls. Furthermore, copper and zinc levels in hair and nails were determined by energy-dispersive X-ray fluorescence.

The highest cadmium concentrations among both workers and controls were observed in kidney, followed in order by liver, lung and brain. The levels in kidney, liver and lung were all significantly higher in the workers than in the controls (p < 0.03). Among the workers relatively strong positive correlations (p < 0.03) were observed between cadmium concentrations in liver and lung, liver and kidney, liver and brain, and lung and brain. In the exposed workers a positive correlation was observed between cadmium and zinc concentrations in the kidney (r_s = 0.38; p = 0.034). This is probably mainly due to the protein metallothionein, which is stored in the kidney, binding equimolar amounts of these two metals.

The highest concentrations of copper were found in hair and nails among both workers and controls, followed in order by liver, brain, kidney and lung. The tissue concentrations of copper in brain, lung and kidney were all significantly higher among the smelter workers than in the controls (p ≤ 0.036). Copper levels in lung and age at time of death were positively correlated among the exposed workers (r_s = 0.39; p = 0.029). In the same group, positive correlations between copper and zinc concentrations in kidney (r_s = 0.45; p = 0.009) and nails (r_s = 0.68; p < 0.001) were also observed, reflecting possible biological interactions between these two metals.

Among both workers and controls, the highest zinc concentrations were found in hair, followed in order by nails, liver, kidney, brain and lung. Significantly higher tissue concentrations among the workers as compared with the reference group were noted in kidney, liver and brain (p ≤ 0.033).

Neither copper nor zinc concentrations in hair and nails seemed to provide a useful measure of the trace element status of the smelter workers.