Interaction of platinum with metallothionein-like ligands in the rat kidney after administration of cis-dichlorodiammine platinum II

After the administration of the anticancer drug cis-dichlorodiammine platinum II (cisplatin) to male rats, the Pt in the soluble fraction of the kidney is isolated, by gel filtration, in association with a high molecular weight component and a low molecular weight fraction. At 24 h, Pt is also recovered in a metallothionein-like fraction which elutes from Sephadex G-50 with a lower apparent molecular weight than endogenous (Cu, Zn)-thionein or Cd-thionein isolated from the kidneys of Cd2+-treated rats. None of these low molecular weight metal-binding fractions binds to Octyl Sepharose CL-4B. On DE-52 ion exchange chromatography, Cd-thionein is resolved into two isometallothioneins whereas the low molecular weight Pt-binding fraction is only partially purified and contains at least six components which elute at higher gradient concentrations than metallothionein. Pretreatment with Cd2+ which stimulates the synthesis of renal and hepatic metallothionein has no effect on the uptake and subcellular distribution of Pt in the liver and kidneys. Cisplatin treatment reduces the concentration of Cu and Zn in the renal metallothionein and other soluble protein fractions in the kidney. When administered to Cd2+-pretreated rats, cisplatin promotes the loss of Zn from the soluble protein fractions but causes the redistribution of Cd from the metallothionein to the high molecular weight fraction and fails to inhibit the Cd2+-induced accumulation of Cu in the kidneys and the binding of Cu to the soluble protein fractions. It is suggested that metallothionein probably does not have a significant role in the renal metabolism of Pt following the administration of cisplatin to rats.     

Turnover of metallothioneins in rat liver.

Two electrophoretically distinguishable metallothioneins were isolated from the livers of Cd2+-treated rats and had thiol group/metal ratios of 3:1, a total metal content, in each of these proteins, of 3.6 atoms of Cd2+ + 2.4 atoms of Zn2+/molecule and 4.2 atoms of Cd2+ + 2.8 atoms of Zn2+/molecule and respective apoprotein mol.wts. of 5844 and 6251. Studies with 1 h pulse labels of [3H]cysteine, given after a single injection of ZnCl2 or CdCl2, showed that these metals stimulated radioactive isotope incorporation into the metallothioneins over the control value by 10- and 15-fold respectively. This stimulation was maximal at 4 h after a single CdCl2 injection and decreased to control values by 16 h, suggesting that either a translational event is responding to free intracellular Cd2+ or a short-lived mRNA is being produced or stabilized in response to the metal treatment. In rats chronically exposed to CdCl2, the metallothioneins increased to 0.2% of the liver wet weight from a control value of 2--4 mumol/kg of liver, with a maximum rate of accumulation of 2--3 mumol/h per kg of liver. The turnover of these proteins in control animals was 0.3--0.6 mumoles/h per kg of liver, measured by the rate of disappearance of 203Hg2+, which binds irreversibly to the metallothioneins. Pretreatment with CdCl2 completely stopped the rapid 203Hg turnover observed in untreated animals. Unlike CdCl2, treatment with ZnCl2 increased the concentration of metallothioneins to a new steady-state pool, 11 mumole/kg of liver, after 10 h. The increase in the zinc-thionein pool by exposure to ZnCl2 in vivo was determined to be primarily due to a stimulation of metallothionein biosynthesis.     

Concentrations of cadmium,zinc, copper,iron, and metallothionein in liver and kidney of nonhuman primates

To evaluate the species specificity of Cd accumulation and the relationship of Cd with other essential metals and metallothionein (MT), the concentrations of Cd, Zn, Cu, and Fe in the liver and kidney and the MT concentrations in the soluble fractions of the liver and kidney were determined in Cd-uncontaminated nonhuman primates (11 species, 26 individuals) kept in a zoo and two wild-caught Japanese macaques. The compositions of metal-binding proteins in the soluble fractions were also investigated by high-performance liquid chromatography (HPLC). The hepatic Cd concentration was 0.03–14.0 μg/g and the renal Cd concentration was 0.35–99.0 μg/g, both varying greatly and being higher in nonhuman primates, which were more closely related to man. The hepatic Zn concentration was 24.0–176 μg/g and the renal Zn concentration was 13.5–138 μg/g, showing 7- to 10-fold differences, and a correlation (r=0.558, p<0.01) was found between renal Zn and renal Cd concentrations. It was proved that in the liver, MT is more closely correlated with Zn (r=0.795, p<0.001) than with Cd (r=0.492, p<0.01) and that in the kidney MT is correlated with both Cd (r=0.784, p<0.001) and Zn (r=0.742, p<0.001). HPLC analysis of metals bound to MT-like protein in chimpanzees, de Brazza’s monkeys, and Bolivian squirrel monkeys showed that more than 90% of Cd in both the liver and kidney, approx 40% of Zn in liver and 28–69% of Zn in kidney were bound to MT-like protein. The higher percentage Zn was bound to high-molecular protein.     

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.     

Increased urinary excretion of zinc and copper by mercuric chloride injection in rats

The effects of HgCl₂ on urinary excretion of Zn, Cu and metallothionein at different time intervals were observed in male Wistar rats. The rats were given a daily intraperitoneal injection of²⁰³HgCl₂ (0.5 or 1.0 mg Hg kg^–1) for 2 days.²⁰³Hg, Zn, Cu and metallothionein in urine, kidney and liver were analyzed. Significant increases in urinary Zn and Cu concentrations were found in HgCl₂-dosed groups. Elevated urinary Zn and Cu concentrations were accompanied by an increased metallothionein excretion in urine at different time periods. Zn concentration in urine remained elevated during the entire observation period of 7 days. There were also increased concentrations of Cu and Zn in the renal cortex in one of the two exposed groups. The results indicate that urinary Cu and Zn are related to the manifestation of renal toxicity and/or the synthesis of metallothionein in kidney induced by mercury.     

Chronic Cadmium Exposure Lead to Inhibition of Serum and Hepatic Alkaline Phosphatase Activity in Wistar Rats

Alkaline phosphatase (ALP) activity in the serum and liver from rats administered with cadmium (Cd) in drinking water was studied. After metal administration, Cd showed a time‐dependent accumulation in the liver, meanwhile metallothionein had a maximum increase at 1 month, remaining in this level until the end of the study. On the other hand, serum and liver ALP activity was decreased after 3 months exposure. To determine if Cd produced an inhibition on enzyme, apo‐ALP prepared from both nonexposed and exposed rats was reactivated with Zn, showing 60% more activity as compared with the enzyme isolated from nonexposed rats. In vitro assays showed that Cd‐ALP was partially reactivated with Zn; however, in the presence of cadmium, Zn‐ALP was completely inhibited. Kinetic studies indicate a noncompetitive inhibition by Cd; these results suggest that Cd can substitute Zn, and/or Cd can interact with nucleophilic ligands essential for the enzymatic activity.     

Identification of metallothionein in parenchymal and non-parenchymal liver cells of the adult rat.

Parenchymal and non-parenchymal cells were isolated from the livers of control, starved, Zn2+-injected and Cd2+-injected rats. Parenchymal cells were prepared by differential centrifugation after perfusion of the liver with collagenase. Non-parenchymal cells were separated from parenchymal cells by unit-gravity sedimentation and differential centrifugation. Yields of 2 x 10(8) non-parenchymal cells with greater than 95% viability and less than 0.2% contamination with parenchymal cells were obtained without exposing cells to Pronase. Metallothioneins-I and -II were identified in parenchymal cells and non-parenchymal cells from Zn2+-treated rats. The metallothionein contents of parenchymal cells, non-parenchymal cells and intact liver were quantified by a competitive 203Hg-binding assay. Administration of heavy-metal salts significantly increased the metallothionein content of both cell populations, although the concentration of the protein was approx. 2.5-fold greater in parenchymal cells than in non-parenchymal cells. Overnight starvation increased the metallothionein content of parenchymal cells without altering that of non-parenchymal cells. The potential significance of this differential response by different liver cell types with regard to the influence of Zn2+ on stress-mediated alterations in hepatic metabolism is discussed.     

The biosynthesis of metallothionein in rat liver and kidney after administration of cadmium

The biosynthesis of the cadmium-binding protein, metallothionein, was studied in rat liver and kidney after injection of cadmium chloride. A simplified procedure for the isolation of metallothionein from liver and kidney tissues was devised. It was found that the concentration of a subcutaneously injected dose of 30 μmoles of ¹⁰⁹CdCl₂/kg in the liver reached the maximum within 36 h. Thereafter, a slow decrease in the concentration of the isotope was noted during the 3 week period. In the kidney, the isotope was taken up in two phases. During the first phase the uptake was faster and lasted for about 4 days. The second phase of ¹⁰⁹Cd accumulation showed a slower increase in the concentration of the isotope. In both liver and kidney tissues 75–80% of the ¹⁰⁹Cd was associated with metallothionein. Amino acid incorporation studies revealed that active biosynthesis of metallothionein took place in the kidney as well as in the liver of cadmium-exposed rats. The turnover of ³⁵S-labeled metallothionein was also investigated and the half-lives of the hepatic and the renal metallothionein were found to be 2.8 and 5 days, respectively.     

delta-Aminolevulinic acid dehydratase: effects of succinylacetone in rat liver and kidney in an in vivo model of the renal Fanconi syndrome.

Succinylacetone (SA) is a known inhibitor of the heme biosynthetic pathway in liver. We have demonstrated previously the SA enhancement of delta-aminolevulinic acid dehydratase (ALAD) in renal tubules, while this enzyme is known to be impaired by SA in the liver. The present studies, based on in vivo treatment of animals with SA, show equivalent degree of inhibition of specific ALAD activity in liver and kidney. Both tissues evidenced an ability to restore enzyme activity with time, the recovery occurring much more slowly in kidney than in liver. The discrepant in vitro and in vivo effect of SA on renal ALAD may be due to differences between a direct inhibitor-enzyme interaction and inhibitor actions in the living cell, respectively. Persistent tissue levels of SA, consistent with demonstrated SA in plasma and urine, might account for continuing inhibition, with the greatest tissue accumulation in kidney where the substance must be cleared for excretion.     

Evidence for a synergistic interaction between cadmium and endotoxin toxicity and for nitric oxide and cadmium displacement of metals in the kidney.

This study was undertaken to examine changes in Zn and Cu homeostasis in the liver and kidney of rats caused by cadmium (Cd) or lipopolysaccharide (LPS) administration. Twenty-five male, 7- to 8-week-old Wistar rats were divided into five groups: saline only treatment, saline treatment and food deprivation, exposure to a single dose of Cd, exposure to LPS alone, and exposure to Cd + LPS. Changes in plasma nitrate concentrations and hepatic and renal Zn and Cu contents were measured together with urinary excretion rates for the metals and nitrate on 3 consecutive days: 24 h before treatment and 24 and 48 h after treatments. Cd exposure alone for 48 h caused a nearly 2-fold increase in plasma nitrate levels with no changes in urinary nitrate excretion whereas LPS treatment caused plasma nitrate levels to increase by 10-fold and urinary nitrate excretion to increase by 4-fold. Administration of LPS 24 h after Cd exposure caused a 10-fold increase in plasma nitrate concentrations and a 100-fold increase in urinary nitrate excretion compared to the rates prior to LPS administration. These results indicate a synergistic interaction between Cd and LPS toxicity. Cd exposure also caused a marked increase in hepatic Zn levels, but LPS did not cause any changes in hepatic Zn or Cu content. In sharp contrast, both Zn and Cu contents were decreased in the kidneys by 16 and 36% in animals exposed to Cd or LPS. A correlation analysis of measured variables reveals that renal Cu contents were inversely associated with plasma nitrate concentrations while urinary Cu excretion on day 3 showed a strong positive correlation with both urinary nitrate and Cd excretions on the same day. A linear regression analysis shows 20% of the variation in urinary Cu excretion was associated with urinary Cd excretion on the same day. It is concluded that reductions in renal Cu contents caused by Cd or LPS administration may be a result of Cd and NO displacement of Cu previously bound to metallothionein.     

Metal concentrations in the liver and kidney of aquatic mammals and penguins

We determined the hepatic and renal concentrations of Cd, Pb, Zn, Cu, and Fe in (1) marine mammals (three bottle-nosed dolphins, six California sea lions, and one sea otter), (2) freshwater and brackish-water mammals (one Oriental short-clawed otter and four European river otters), and (3) sea birds (three rock-hopper penguins, two king penguins, three Humboldt penguins, four Macaroni penguins, and four Magellanic penguins), all of which were kept in a zoo and an aquarium in Japan. We investigated the species-specificity of Cd accumulation in these aquatic animals. We also presented the basic data on metal concentrations. The concentrations of Cd in liver and kidney tended to be higher in marine mammals than in freshwater mammals. Many penguins, sea birds, showed high Cd concentrations. These results suggest that the habits of these animal species may be involved in accumulation of Cd. Pb concentrations were below the detection limit or low in both liver and kidney [not detected (ND)=0.132 μg/g and ND=0.183 μg/g, respectively]. The hepatic concentrations of Zn and Cu were high in young animals. In penguins, a positive correlation was found between the Zn and Cd concentrations in the liver and kidney and between the Cu and Cd concentrations in the liver. Individual variation was large in Fe concentration (48–3746 μg/g in the liver and 51–980 μg/g in the kidney).     

Isolation of copper thionein from rat liver

The inducible Cu-binding protein from adult rat liver previously referred to as Cu-chelatin has been purified and shown to be Cu-thionein. The Cu-protein was purified to homogeneity by gel filtration and thiopropyl-Sepharose chromatography. The Cu-thionein exhibited an amino acid composition similar but not identical to that of the two forms of rat liver Cd,Zn-thionein. The polypeptide-chain molecular weight of Cu-thionein was indistinguishable from that of Cd,Zn-thionein. The identification of the Cu-protein as metallothionein was substantiated by the complete immunological cross-reactivity with antisera prepared against purified rat liver Cd,Zn-thionein. Purified Cu-thionein bound 9–11 g atoms of Cu per mole of protein in an electron paramagnetic resonance nondetectable form. The $\text{Cu}\text{Zn}$ ratio of the protein is about 100. Ion-exchange chromatography resolved the Cu-protein into three polymorphic forms which differed from the polymorphism of Cd,Zn-thionein.     

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 zinc status of rats on the synthesis and degradation of copper-induced metallothioneins.

Injection of Zn2+-adequate and Zn2+-deficient rats with Cu2+ stimulated the incorporation of l-[35S]cysteine into a low-molecular-weight Cu2+-binding protein in both liver and kidney. No significant incorporation of l-[4,5-3H]leucine into this protein occurred, confirming the previous claim that it was metallothionein and not some other leucine-rich protein. The half-life of the protein was found to be 16.9 +/- 1.0 (S.E.)h in the liver of Zn2+-adequate rats but only 12.3 +/- 0.5h in Zn2+-deficient animals. The degradation rate of the metallothionein was similar to the rate of disappearance of Cu2+ and Zn2+ from the protein, indicating that the release of mental from the protein and its catabolism occurred simultaneously. There was no significant difference in the half-lives of the hepatic or renal copper-thioneins in Zn2+-adequate rats.     

Circular dichroism of metallothioneins. A structural approach.

A comprehensive study on circular dichroism of metallothioneins containing Zn, Cd and Cu was carried out. The contributions of the metals, the sulphur and the polypeptide chain to the observed Cotton effects was shown. From the pH dependency of the extrinsic Cotton effects which are due to the metal-thiolate chromophore the stability of the metal clusters was found to decrease in the order Cu greater than Cd greater than Zn. The pH values corresponding to the dissociation of half of the bound metal ions are 0.44 for Cu-thionein, 3.05 for Cd-thionein and 4.6 for Zn-thionein. The extrinsic Cotton effects of Cd, Zn-thioneins of varying Cd to Zn ratio could be simulated using the difference circular dichroic spectra of Cd-thionein (bands at 227, 242.5 and 262 nm), Zn-thionein (bands at 225 and 244 nm) and the circular dichroic spectrum of cysteine-thionein (band at 200 nm, shoulder at 225 nm). Since during the dissociation of the metals the circular dichroic spectra exhibited changes only in amplitude and not in shape we can conclude that the dissociation of the metal ions involves the complete sequential degradation of metal clusters. In the near-ultraviolet region the metal-free proteins show only Cotton effects attributable to a disulphide chromophore. Thus Cotton bands are observed for cystine-thionein at 282.5 and 260 nm. From the intrinsic circular dichroism of Cd- and Zn-thionein (negative Cotton effect at 200 nm, shoulder at 225 nm) it follows that the protein conformation consists of less than 5% helical or pleated sheet structure and therefore has to be classified as unordered structure or "fixed" random coil     

Metal composition of human hepatic and renal metallothionein

This article is based on data on the levels of metals (Cd, Zn, Cu) and metallothionein (MT) determined radiochemically with²⁰³Hg in renal cortex and liver of 137 autopsy cases. From this number, for 23 cases, the gel filtration of the cytoplasmic fraction of the organs was performed. The molar content of metals in the MT fraction (Sephadex G-50) amounted to 46.9, 50.2, and 2.0% for Cd, Zn, and Cu in renal cortex, respectively, and to 8.3, 83.6, and 9.1% for Cd, Zn, and Cu in the liver, respectively. In parallel with the increase of Cd and MT in renal cortex, increasing saturation was found of the MT fraction by Cd, occurring at the expense of Zn and Cu. Equimolar amounts of Cd and Zn in the MT fraction are found at Cd level of 0.5 μmol Cd/g wet wt of renal cortex. In the liver, analogous dependency (elevation of %Zn, depression of %Cd and %Cu) were observed in relation to Zn and MT levels in this organ. The basic level of Zn (not bound with MT) was estimated at 0.5 μmol/g for both renal cortex and liver. A deficit of non-MT Zn in kidneys is proposed as an alternative mechanism of toxic Cd action.     

Dithiocarbamates and prevention of cadmium teratogenesis in the hamster

Certain dithiocarbamates (DTC) have been reported to protect against cadmium (Cd)-induced lethality and to decrease Cd body burden. The present study evaluated the influence of sodium N-benzyl-D-glucamine dithiocarbamate, sodium N-di(hydroxyethyl)amine dithiocarbamate, sodium 4-carboxyamidopiperidine-N-dithiocarbamate, and sodium N-methyl-D-glucamine dithiocarbamate on Cd-induced teratogenesis in the hamster. When given as a single ip injection at 2.2 mmol/kg 15 min prior to iv CdCl2 (2 mg/kg), all of the DTC afforded significant protection against Cd-induced developmental toxicity and reduced kidney [Cd] in the dam. Maternal liver [Cd] was reduced with the glucamine and dihydroxyethyl amine analogs, but treatment with the piperidine failed to influence hepatic [Cd]. Pretreatment of the dams with DTC 24 hr prior to Cd challenge failed to protect against Cd-induced embryotoxicity, and provided minimal, if any, reduction in renal or hepatic [Cd]. Pretreatment with the N-methyl-D-glucamine congener 24 hr prior to Cd exposure increased embryolethality. The dose-time relationships found here suggest that pharmacologically effective levels of these DTC decline within 24 hr of treatment and that induction of metallothionein does not play a major role in DTC antagonism of Cd poisoning.     

Molecular biology of copper. A circular dichroism study on copper complexes of thionein and penicillamine.

Chicken liver Cd, Zn-thionein (metallothionein) was isolated from Cd-pretreated chickens weighing 1 500 g. The native Cd, Zn-thionein contained 9 g-atoms of metals per 12 000 g of protein. Upon the addition of Cu(CH3CN)4ClO4, all Cd2 and Zn2 were successfully replaced. 15 g-atoms of Cu from the acetonitrile perchlorate complex were bound to the protein. Due to the absence of aromatic amino acid residues, thionein has unique ultraviolet and circular dichroism properties. The shoulder of the ultraviolet spectrum at 250 nm (A250 X A280(-1) = 23.9) was shifted to 275 nm (A250 X A280(-1) = 1.6). No significant absorption was detected in the visible region. Th conformational changes of the protein moiety were much more visible in the circular dichroism spectra. The titration with Cu(CH3CH)2 caused the appearence of three new Cotton effects: 257.5 nm (+), 350 nm (+) and 301 nm (-). The negative Cotton effect at 239 nm of the original metallothionein was completely levelled off. The binding strength of copper with thionein is extraordinarily high: it survives proton treatment up to pH 1.9. Displacement of the Cd2 by Cu employing Cd-thionein which was formed at pH 2.2 resulted in the same circular dichroism properties as observed for Cu-thionein. D-Penicillamine proved a suitable model for the metal-free thionein, since redox reactions and polymerization of the sterically hindered thiol residue are known to be slow. The correlation of the circular dichroism properties of either copper complex using thionein or D-penicillamine was surprisingly high. Circular dichroism measurements of Cu(I)-D-penicillamine revealed Cotton effects at 255 nm (+), 280 nm (+) and 355 nm (-). Upon examining the red-violet mixed Cu(-i)-cu(II)-D-penicillamine complex, Cotton bands in the visible region at 425 nm (-) and 495 nm (+) were seen. In many blue copper enzymes, the copper is assumed to be in the neighborhood of both cysteine and aromatic amino acid residues, which are known to play an important role in the electron transfer. This is not the case in the Cu-thionein, which would explain many different properties of this copper protein. It is very attractive to conclude that the sterically hindered SH-group of D-penicillamine reacts with excess copper in a specific way, similar to the Cu-thionein. This phenomenon could explain the considerable success of D-penicillamine in the treatment of Wilson's disease.     

An immunological study of a Pb-thionein like protein in rat liver

Administration of a sublethal dose of lead acetate to rats induced the simultaneous synthesis of a Pb-thionein like protein (Pb-BP) and Zn-thionein in the liver. To determine of the Pb-BP is a species of metallothionein, immunological properties of this protein were investigated. The results indicate that the Pb-BP is cross-reactive with the antibody against rat Zn-thionein II, strongly suggesting that the Pb-BP is a metallothionein.