Cadmium: tissue distribution and binding protein induction in the painted turtle, Chrysemys picta

The freshwater painted turtle, Chrysemys picta, was used to investigate (a) the distribution of an injected dose of ¹⁰⁹Cd in tissues over a period of 192 h (8 days) and (b) the effect of non-isotopic cadmium injection on tissue metal-binding protein levels. Cadmium is cleared from the blood with 9% remaining in the circulation at 192 h. ¹⁰⁹Cd is found in all tissues, but is accumulated preferentially in liver, kidney, pancreas, and gastrointestinal tract. The liver is the primary site of Cd accumulation, accounting for 46.4% of the injected dose by 192 h and the highest Cd concentration (cpm/mg tissue). Steroidogenic tissues and the oviduct accumulate significant amounts of ¹⁰⁹Cd and the isotope is present in yolk. An increase in tissue metal-binding protein level after non-isotopic CdCl₂ injection is consistent with ¹⁰⁹Cd distribution, in that metal-binding protein concentration after CdCl₂ injection is highest in liver, followed by pancreas and kidney with low, but with significant levels of cadmium-binding protein in gonads and steroid target organs. We conclude that the liver is the major site of storage after a single injection of isotopic cadmium and induction of a metal-binding protein may be an adaptive response to exposure to cadmium.     

Studies on the role of iron in the reversal of cadmium toxicity in chicks

Studies were conducted to determine the effect of dietary iron (Fe) levels ranging from a deficiency to an excess on the toxicity of cadmium (Cd) in chicks. In Fe-deficient animals, cadmium was found to be more toxic than in Fe supplemented animals as measured by growth. The liver Cd burdens were increased significantly in the presence of dietary Fe supplementation, and there was a significant Cd−Fe interaction in the Cd concentration of the kidney, indicating that iron deficiency increased the concentration of Cd in the kidneys of those chicks receiving this element. Cd tended to reduce the Fe concentration in both the liver and kidney. The absorption of Cd as measured by the amount of¹⁰⁹Cd that disappeared from an isolated duodenal segment in one h was not affected by the Fe content of the diet, but the amount of isotope appearing in the liver compared to the amount present in the blood was increased in the Fe supplemented chicks. Separation of the Cd binding ligands by column chromatography revealed that more of the Cd in the liver, but not the kidney, was associated with ligands which eluted in a column volume that contained metallothionein in those chicks receiving Fe than in the livers from Fe deficient animals. The inverse relationship between the amount of Cd bound to the metallothionein containing fraction and toxicity may be related causally. Paper No. 10538 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC 27695-7601. The use of trade names in this publication does not imply endorsement by the NC Agricultural Research Service of the products named nor criticism of similar ones not mentioned.     

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.     

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.     

Correlation between metallothionein and energy metabolism in sea bass,Dicentrarchus labrax,exposed to cadmium

Specimens of sea bass (Dicentrarchus labrax) were exposed to two different cadmium concentrations (0.5 and 5 μg Cd²⁺/ml seawater) for a period of 7 days. Cadmium accumulated in the tissues of D. labrax in the following order: kidney > liver > gills at both concentrations. Accumulation patterns in fish exposed to 0.5 μg Cd²⁺/ml seawater were different with respect to 5.0 μg Cd²⁺/ml seawater. At both Cd concentrations a similar stress situation occurred during the first 4 hr as noted by the depletion of glycogen stores and the increase in free glucose in the muscle; metallothionein was induced in the liver, but failed to bind all the cytosolic Cd, which was in part bound to high-molecular-weight ligands. Fish recovered from this initial stress situation within 24 hr as indicated by the increase in glycogen and the decrease of glucose. Long-term effects were clearly dependent upon metal concentration: at lower Cd exposure, metallothionein induction increased linearly with time and counteracted the toxic effect of the metal; on the other hand, when fish were exposed to 5.0 μg Cd²⁺/ml seawater a clear stress occurred at the end of the exposure, as indicated by the notable decrease of glycogen stores, the increase of free glucose, the decrease of AEC in the muscle and the increase of Cd bound to high-molecular-weight ligands in the liver.     

Metallothionein as Potential Biomarker of Cadmium Exposure in Persian Sturgeon (<Emphasis Type="Italic">Acipenser persicus</Emphasis>)

Metallothionein (MT) concentration in gills, liver, and kidney tissues of Persian sturgeon (Acipenser persicus) were determined following exposure to sublethal levels of waterborne cadmium (Cd) (50, 400, and 1,000 μg l⁻¹) after 1, 2, 4, and 14 days. The increases of MT from background levels were 4.6-, 3-, and 2.8-fold for kidney, liver, and gills, respectively. The results showed that MT level change in the kidney is time and concentration dependent. Also, cortisol measurement revealed elevation at the day 1 of exposure and followed by MT increase in the liver. Cd concentrations in the cytosol of experimental tissues were measured, and the results indicated that Cd levels in the cytosol of liver, kidney, and gills increased 240.71-, 32.05-, and 40.16-fold, respectively, 14 days after exposure to 1,000 μg l⁻¹ Cd. The accumulation of Cd in cytosol of tissues is in the order of liver > gills > kidney. Pearson correlation coefficients showed that the MT content in kidney is correlated with Cd concentration, the value of which is more than in liver and gills. Thus, kidney can be considered as a tissue indicator in A. persicus for waterborne Cd contamination.     

Radioimmunoassay of rat liver metallothionein

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

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.     

Mercury,cadmium, zinc and copper concentrations and stable isotope ratios of carbon and nitrogen in tiger sharks (Galeocerdo cuvier) culled off Ishigaki Island,Japan

We analyzed Hg, Cd, Zn and Cu in the liver as well as Hg and stable isotope ratios of carbon and nitrogen (δ¹³C and δ¹⁵N) in muscle from tiger sharks (Galeocerdo cuvier) in Japan. The Hg concentration in the muscle increased slightly and proportionally with increases in body length, but the Hg concentration in the liver increased markedly after maturation (exceeding 2.7 m precaudal length). The Hg concentration in the liver of mature shark was higher than that in the muscle. The Cd concentration in the liver increased with increases in body length. On the other hand, the Zn and Cu concentrations in the liver decreased during the growth stage, but thereafter increased with increased Cd burden due to growth. The marked increase in hepatic Hg in mature sharks may be explained by the continuous intake of Hg via food, slower growth and Hg–Se complex formation. High concentrations of Zn and Cu in the liver of immature sharks and concomitant increases in Zn and Cu with the Cd burden in the liver of mature sharks may be explained by the physiological requirements of Zn and Cu during the growth stage, the induction of metallothionein synthesis due to the Cd burden and the subsequent binding of these metals to metallothionein. The δ¹⁵N and δ¹³C values decreased with increases in body length, suggesting a shift from coastal feeding to pelagic feeding with shark growth. The Hg and Cd concentrations tended to be negatively correlated with the δ¹⁵N and δ¹³C values as a result of the increase in Hg and Cd accumulation due to the growth and the decreases in δ¹⁵N and δ¹³C values due to the sift of feeding area.     

Metallothionein mRNA expression in fetal mouse organs

The regulation of metallothionein biosynthesis in mammalian development was investigated by examining organs of 17-day fetal mice for biologically active metallothionein mRNA. Metallothionein was identified in cell-free translation products by migration in polyacrylamide gels and its characteristic elution on Sephadex G-50 columns. Metallothionein constitutes ～7.5% of [³⁵S]cysteine incorporated into polypeptides directed by mRNA from fetal liver, but it is not detectable in mRNA-directed products of fetal kidney, small bowel, heart, or adult liver. Consistent with a fetal-specific role, hepatic metallothionein mRNA content decreases abruptly in newborn mice, becoming undetectable within 12 days.     

Kinetic characterization of Zinc transport process and its inhibition by Cadmium in isolated rat renal basolateral membrane vesicles: In vitro and In vivo studies

We firstly characterized zinc uptake phenomenon across basolateral membrane vesicles (BLMVs) isolated from normal rat kidney. The process was found to be time, temperature, and substrate concentration dependent, and displayed saturability. Zn²⁺ uptake was competitively inhibited in the presence of 2 mM Cd with Ki of 3.9 mM. Zinc uptake was also inhibited in the presence of sulfhydryl reacting compound suggesting involvement of {–}SH groups in the transport process. Further, to elucidate the effect of in vivo Cd on zinc transport in BLMVs, Cd nephrotoxicity was induced by subcutaneous administration of CdCl₂ at dose of 0.6 mg/kg/d for 5 days in a week for 12 weeks. An indolent renal failure developed in Cd exposed rats was accompanied with a significantly high urinary excretion of Cd²⁺, Zn²⁺ and proteins. The histopathology and electron microscopy of kidneys of Cd exposed rats documented changes of proximal tubular degeneration. Notably, Cd content in renal cortex of Cd exposed rats was 215 μg/g tissue that was higher than the critical concentration of Cd in kidneys which was associated with significantly higher Zn and metallothionein (MT) contents. Zinc uptake in BLMVs isolated from kidneys of Cd exposed rats was significantly reduced. Further, kinetic studies revealed that decrease in zinc uptake synchronized with decrease in maximal velocity (V_max) and increase in affinity constant which is suggestive of decreased number of active zinc transporters. Furthermore, conformational modulation of Zn transporter in BLM was further supported by observed variation in transition temperature for zinc transport in BLMVs isolated from Cd-exposed kidney.     

Relative cadmium-binding capacity of metallothionein and other cytosolic fractions in various tissues of the rat.

The Cd-binding capacity of soluble proteins in 10 tissues of normal rats not excessively exposed to heavy metals was measured by saturation of freshly isolated cytosol with 109CdCl2 in vitro followed by Sephadex G-75 chromatography. The Cd-binding capacity of a 10,000 molecular weight Cd-binding peak (10,000 MW Cd-BP), which had a high affinity for Cd and was probably metallothionein, was the highest in kidney (78nmol Cd/g fresh tissue), followed by testis (63 nmol/g), liver (38 nmol/g) and then by brain (14 nmol/g). The amount of the Cd-BP in these tissues (assuming that it was metallothionein and bound 9 mol Cd/10,000g) was calculated to be 87, 70, 42 and 16 mg/kg fresh tissue in kidney, testis, liver and brain, respectively, or in the order of 10(-5) to 10(-6) mol/kg tissue. A significant amount of the 10,000 MW Cd-BP was also found in small intestine. It was present in rather small amounts in heart and lung, and possibly in spleen and skeletal muscle as well. In contrast, the protein was not detectable by this technique in plasma. The results suggest that metallothionein is a rather ubiquitous, intracellular protein in tissues of normal animals and may have other biological functions, besides its possible fortuitous role in heavy metal detoxification. A 30,000 molecular weight Cd-binding peak (30,000 MW Cd-BP) having a very high affinity Cd, apparently higher than that of the 10,000 MW Cd-BP, was found only in testes, among the 10 tissues examined. Its estimated Cd-binding capacity was 51 nmol Cd/g of testis, slightly less than that of metallothionein in testis. These findings support the hypothesis that the 30,000 MW Cd-BP is a plausible target of Cd in Cd-induced testicular injury, and suggest a basis for the peculiar sensitivity of the rat testis to Cd.     

Effects of treated sewage on the availability of cadmium to coho salmon

Abstract

Juvenile coho salmon exposed for 30 days to dilutions of sewage treatment plant effluent (STPE) with approximately 2 μg Cd L^?1 showed increased Cd in liver, gill, and kidney tissues. In the liver the increase was unrelated to concentration of STPE, whereas in the gills some treatment effects were apparent in the lower dilutions. In the kidneys, Cd concentration varied inversely with percentage of STPE and levels of total alkalinity as well as total organic carbon in test solutions. It would appear that the ligands in STPE complex Cd and thereby reduce the amount available to bioconcentrate. Neither the average concentration of total Cd nor that of Chelex-labile Cd in the test solutions was indicative of Cd bioavailability in this work.     

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.     

Development of a stable isotope approach for the inductively coupled plasma-mass spectrometry determination of oxidized metallothionein in biological materials

The use of isotope dilution analysis (IDA) with inductively coupled plasma-mass spectrometry (ICP-MS) for the determination of oxidized metallothionein (MT) by a Cd-saturation method is investigated. The method developed here is a modification of an earlier methodology which used a radioactive Cd isotope ((109)Cd). While retaining the many advantages of this previous approach, the procedure presented here uses stable isotope ratio measurements ((114)Cd/(111)Cd) for the determination of MT. Experimental parameters governing the instrumental precision and accuracy for isotope ratio measurements of Cd by ICP-MS were characterized. Systematic errors, including mass bias, detector dead time, and spectroscopic interferences, could be easily corrected. The isotope dilution ICP-MS method was validated by the determination of very low levels of cadmium in biological certified reference materials (NIST SRM 2670 freeze-dried urine, IAEA H-8 horse kidney, and BCR TP-25 lichens). Finally, the IDA procedure was evaluated for the determination of oxidized MT by a Cd-saturation method previously developed using radioactive (109)Cd. The final procedure was applied to the quantification of MT in Long-Evans Cinnamon rat liver cytosol samples and the results were compared with data obtained for the same samples using the reference (109)Cd methodology. A good agreement between the analytical values obtained by both methods was observed.     

Cd-metallothionein in liver and kidney of goldfish (Carassius auratus): effects of temperature and salinity.

Treatment of goldfish with Cd, by intraperitoneal injection, resulted in Cd-metallothionein (Cd-MT) synthesis mainly in liver and kidney. The relative amount of Cd sequestered by liver metallothionein was always greater in fish maintained at 20 degrees C compared to those reared at 10 degrees C, indicating a temperature dependence of metallothionein biosynthesis; in the kidney this dependence was not so clearly evident. Changes in MT levels induced by adapting fish to different salinities did not correlate with the salinity change.     

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.     

Effect of Selenium Pre-treatment on Antioxidative Enzymes and Lipid Peroxidation in Cd-exposed Suckling Rats

Since there are no data about the protective role of selenium (Se) against cadmium (Cd)-induced oxidative damage in early life, we studied the effect of Se supplementation on antioxidative enzyme activity and lipid peroxidation (through thiobarbituric acid reactive substances; TBARS) in suckling Wistar rats exposed to Cd. Treated animals received either Se alone for 9 days (8 μmol, i.e., 0.6 mg Se as Na₂SeO₃ kg⁻¹ b.w., daily, orally; Se group), Cd alone for 5 days (8 μmol, i.e., 0.9 mg Cd as CdCl₂ kg⁻¹ b.w., daily, orally; Cd group), or pre-treatment with Se for 4 days and then co-treatment with Cd for the following 5 days (Se + Cd group). Our results showed that selenium supplementation, with and without Cd, increased SOD activity in the brain and kidney, but not in the liver and GSH-Px activity across all tissues compared to control rats receiving distilled water. Relative to the Cd group, Se + Cd group had higher kidney and brain SOD and GSH-Px activity (but not the liver), while in the liver caused increased and in the brain decreased TBARS level. These results suggest that Se stimulates antioxidative enzymes in immature kidney and brain of Cd-exposed rats and could protect against oxidative damage.     

Cadmium,zinc, and copper metabolism in the mottled mouse,an animal model for menkes' kinky hair syndrome

Studies of uptake and release of ⁶⁴Cu, ¹⁰⁹Cd, and ⁶⁵Zn in suckling C57BL/6J male mice revealed kinetics and distributions that differed for each metal both within and among the organs analyzed, suggesting distinct, albeit overlapping, mechanisms for transport and binding of each metal. In mutants, there were tissue-specific increases in copper-binding capacity. In hemizygotes (Mo^blo/y) accumulation of ⁶⁴Cu was increased in kidney, lung, and duodenum. In heterozygotes (Mo^blo/+), ⁶⁴Cu content was increased in kidney, with a smaller increase in lung, and no change in duodenal Cu. Decreased ⁶⁴Cu accumulation was seen in liver in both hemi- and heterozygotes. In contrast, ⁶⁴Zn and ¹⁰⁹Cd accumulation in organs of heterozygote mice was not significantly distinguishable from normal. In skin and connective tissues there is excessive accumulation of ⁶⁴Cu in Mo^blo/+ and Mo^blo/y, no abnormality in heterozygote ⁶⁵Zn accumulation, but a clear decrease in heterozygote ¹⁰⁹Cd content. In both mutant kidney and liver, there was an aberrant subcellular distribution of ⁶⁴Cu, with the major fraction of sequestered ⁶⁴Cu in the cytosol. Our studies establish that in spite of the ubiquity of metallothioneins and the structural similarities of those that have been characterized, there is specificity and functional heterogeneity in metal binding among tissues. The aggregate data suggest that there are unique regulatory mechanisms for the metabolism