Binding sites for the (Hg-Se) complex on selenoprotein P.

The mechanism underlying the interaction between mercury (Hg), selenium (Se) and selenoprotein P (Sel P) in the bloodstream has been explained by the formation of the [(Hg-Se)n]m-Sel P complex. In the present study, the binding sites for the (Hg-Se)n complex on Sel P were studied by competitive assay of the binding of the (Hg-Se)n complex to Sel P with polymeric and monomeric amino acids with simultaneous detection of the Hg, Se of selenite origin and Se of Sel P origin by the high performance liquid chromatography-inductively coupled argon plasma-mass spectrometry method. The specific binding of the (Hg-Se) complex but not Hg2+ or selenide to Sel P was explained by the unique binding sites consisting of the cationic and anionic ends such as imidazolyl and selenol groups on Sel P, respectively. The number, n, in the (Hg-Se)n complex was estimated to be approx. 100, while the number, m, in the [(Hg-Se)n]m-Sel P complex was estimated to be 35. The formation of the unit complex (Hg-Se)100, followed by its binding to Sel P at up to the 35 binding sites on Sel P was suggested.

Expression of glutathione S-transferase P-form in primary cultured rat liver parenchymal cells by coplanar polychlorinated biphenyl congeners is suppressed by protein kinase inhibitors and dexamethasone.

Glutathione S-transferase P-form (GST-P, EC 2.5.1.18) mRNA was expressed by epidermal growth factor as well as by 3,4,5,3',4'-penta-chlorinated biphenyl (PenCB) in primary cultured rat liver parenchymal cells. The expression of GST-P was suppressed by inhibitors of protein kinase C and dexamethasone, an antagonist of AP-1 transcription factor activity, whereas expression of cytochrome P450IA2 by PenCB was not affected by these reagents. The AP-1 related transcription factor may be essential for the expression of GST-P by PenCB as also may be a protein kinase C type enzyme.

Induction of metallothionein in liver and changes of essential metal levels in selected tissues by three dextran derivatives.

Three dextran derivatives (dextran sulfate, DEAE-dextran and Dextran T500) were injected intraperitoneally into mice to study the induction of zinc-thionein in the liver, and the changes of essential metal levels in the liver, kidneys and spleen. The former was investigated with a high performance liquid chromatograph equipped with an atomic absorption spectrophotometer, and the latter with an inductively coupled plasma-atomic emission spectrometer. Dextran sulfate and DEAE-dextran induced a large amount of hepatic zinc-thionein dose-dependently, while Dextran T500 did not induce metallothionein substantially at the doses studied 20, 40 and 60 mg/kg body wt). The injection of dextran sulfate resulted in a significant increase of spleen weight, with a transitory increase of calcium concentration in the three tissues and a significant decrease of iron level in the spleen. In accord with the induction of zinc-thionein by dextran sulfate and DEAE-dextran, zinc concentration in the liver also increased transitionally after both injections and the time-course of the hepatic zinc level in dextran sulfate showed a close resemblance to that of the calcium one.

Excretion process of copper from copper pre-loaded rat liver parenchymal cells.

Excretion of copper from copper pre-loaded rat liver parenchymal cells was investigated. The copper pre-loaded cells were isolated from the liver of copper-administered rats and were maintained for 4 days in modified Williams medium E; sodium pyruvate was increased while calcium was reduced in concentration, and 10% fetal bovine serum and insulin were added. For the first 30 hr after plating, loss of copper was slow, with a half-life of more than 100 hr: subsequently the rate of loss was faster, with a half-life of 51 +/- 9 hr. Copper pre-loaded in the cells was mostly bound to metallothionein and the amount of copper-metallothionein decreased with time of culture. Furthermore, the relative ratio of the two iso-metallothioneins changed with length of culture.

Cadmium inhibits protein secretion from cultured rat liver parenchymal cells.

Depressive action of cadmium (Cd) to the protein secretion from liver was studied in vitro using primary cultures of rat liver parenchymal cells. The cells were cultured in the presence of 0.1-5 microM cadmium chloride. Over this concentration range Cd had no effect on protein synthesis, but caused a dose-dependent inhibition of protein secretion. Although the metallothionein level was increased more than 4-fold by incubation with 10(-6) M dexamethasone, the depressive action of Cd was not prevented by the induction of metallothionein.

Accumulation of newly synthesized serum proteins by cadmium in cultured rat liver parenchymal cells.

Effects of cadmium on secretion of the two major serum proteins, albumin and transferrin were studied in primary cultured rat liver parenchymal cells. Exposure to 7 microM cadmium for 8 hr did not inhibit synthesis of both albumin and transferrin whereas secretions of the two proteins were effectively depressed. In the cells exposed to cadmium serum type albumin and proalbumin were accumulated. Cadmium may affect intracellular processing of secretory proteins and also retard the discharge of these proteins resulting in inhibition of protein secretion from the liver parenchymal cells.

Pregnancy-associated changes in renal metallothionein concentration and plasma distributions of metals.

Pregnancy-associated changes in concentrations and distributions of selected essential elements were examined in the blood and tissues of rats. Concentrations of copper (Cu) and zinc (Zn) in the kidneys of dams significantly decreased with gestational age and recovered after delivery. Distribution profiles of multi-elements in the supernatant of the kidneys indicated that Cu and Zn bound to metallothionein decreased with gestational age without affecting their distributions to other components. Although concentrations of Cu and Zn in the liver did not show significant changes during gestational period, Zn bound to metallothionein decreased with gestational age. Plasma concentrations of Cu, iron, phosphorus, sulfur, Zn and other elements were altered by the physiological change, some of those chemical forms being assigned.

Studies of cadmium uptake and metabolism by the kidney.

Our investigation was centered on a possible relationship between the toxicity of cadmium and changes in its chemical forms in tissues. Two models have been studied: one is the renal damage induced by a single injection of cadmium-containing metallothionein and the other is the renal damage induced by repeated injections of cadmium salt. Parenteral loading of cadmium-containing metallothionein caused acute and transitory necrotic damage of renal tubular lining cells. This was explained by the selective and rapid uptake of metallothionein at the proximal tubules and degradation of the protein, resulting in liberation of cadmium ions. Cadmium ions were injected repeatedly into rats, and the changes in the chemical forms of cadmium, zinc and copper in the liver and kidneys were correlated with the histological observations. The transitory necrotic damage of the proximal tubules caused during the repeated injections of cadmium was accompanied with a rapid decrease of the copper content in the kidney metallothionein. Further loading of cadmium ions induced increases in the amounts of cadmium not bound to metallothionein and its oxidation products as well as an increase of the Cd/Zn ratio in metallothionein. With these changes in the chemical forms of cadmium, persistent damage of the kidneys occurred. The transitory renal damage caused both by a single injection of cadmium-containing metallothionein and by repeated injections of cadmium salt can be explained by a limit of the native biosynthetic capacity of metallothionein in the kidney, while the persistent damage appears to be due to a limit of the induced capacity.

Exposure, metabolism, and toxicity of rare earths and related compounds.

For the past three decades, most attention in heavy metal toxicology has been paid to cadmium, mercury, lead, chromium, nickel, vanadium, and tin because these metals widely polluted the environment. However, with the development of new materials in the last decade, the need for toxicological studies on those new materials has been increasing. A group of rare earths (RE) is a good example. Although some RE have been used for superconductors, plastic magnets, and ceramics, few toxicological data are available compared to other heavy metals described above. Because chemical properties of RE are very similar, it is plausible that their binding affinities to biomolecules, metabolism, and toxicity in the living system are also very similar. In this report, we present an overview of the metabolism and health hazards of RE and related compounds, including our recent studies.

Essential metal contents and metallothionein-like protein in testes of mice after cadmium administration.

To study the testicular injury by Cd from the viewpoint of essential element levels, male mice were injected with Cd subcutaneously at a dose of 30 mumol/kg body weight singly and the concentrations of 8 elements (Na, Mg, P, K, Ca, Fe, Zn, and Cd) in the testis were measured for 6 weeks. The most pronounced alteration was an increase of Ca content with time after the injection; the experimental group had 270 times more Ca than the control despite a decrease of the testis weight to one-third of the control at 4 weeks. K content decreased markedly and Fe remained at an elevated level throughout the experimental period. Decreased Mg, P, and Zn levels started to be restored after 2 weeks. Metallothionein (MT)-like Zn-binding proteins were studied in the cytosol fraction with a high performance liquid chromatograph-atomic absorption spectrophotometer. MT-like proteins were observed at a higher concentration in the control than in the Cd-injected group. No indications of new induction of MTs or MT-like proteins could be detected.

Effect of cadmium on essential metal concentrations in testis, liver and kidney of five inbred strains of mice.

To compare the organ injuries by cadmium administration among inbred strains of mice, CdCl2 was injected subcutaneously into C3H/He, BALB/c, DBA/2, CBA/J and C57BL/6 strains at a dose of 30 mumol/kg body weight, and the changes of 6 essential metal [sodium (Na), magnesium (Mg), potassium (K), calcium (Ca), iron (Fe) and zinc (Zn)] concentrations were determined at 6, 24 and 48 h. Testicular Cd concentrations at 6 h in DBA/2 and CBA/J were significantly higher than those in the other 3 strains. Marked alterations in testicular metal concentrations (increases in Na, Ca and Fe and decreases in K and Mg) were observed in the 2 strains. At 48 h, hepatic Cd concentration in BALB/c was decreased from the higher level at 24 h and an abrupt increase in renal Cd concentration was detected. Changes in hepatic essential metal concentrations at 48 h in BALB/c were in the same direction as those in the testis of DBA/2 and CBA/J. As a whole, C3H/He, BALB/c and C57BL/6 strains showed more remarkable alterations in hepatic essential metal concentrations, except Zn level, than DBA/2 and CBA/J which showed larger increases in Zn concentration (probably due to larger metallothionein induction). It was suggested from the data that C3H/He, BALB/c and C57BL/6 were strains sensitive to the hepatotoxicity of Cd and that DBA/2 and CBA/J were susceptible to the testicular toxicity of Cd.

Effect of intratracheal instillation of cadmium chloride on phospholipids in alveolar wash fluid.

The effect of cadmium chloride on phospholipids in the alveolar wash fluid was investigated by instilling the chemical (2.5 micrograms Cd/0.4 ml saline per rat) into rat trachea. Two days later the phospholipid content of the wash fluid increased markedly in the cadmium-treated group to about 2.4 times that in the control (saline-treated) group. The relative amount of phosphatidylcholine and the fatty acid composition of phosphatidylcholine were not altered by the cadmium treatment, and retained a composition typical of lung surfactant. These findings suggest that cadmium induces a marked increase in surfactant phospholipids in the lung at a very low dose.

Selenium distribution and metabolic profile in relation to nutritional selenium status in rats.

The disposition of selenium (Se) was investigated in Wistar rats of various Se status after an intravenous injection of 82Se-selenite. Various fractions of plasma, urine, and cytosols from liver and kidney were separated by high performance liquid chromatography (HPLC), coupled with an inductively coupled argon plasma-mass spectrometry (ICP-MS). The technique allowed simultaneous differentiation of the fate of injected and endogenous Se, and if it was influenced by the previous Se burden in the tissues. A broad Se-peak from plasma was resolved in two fractions by assessing the m/z 82/78 ratios. Urinary profiles indicated that the metabolism of Se was dose-dependent; monomethylselenol being the primary metabolite of Se in untreated animals, whereas noticeable amount of trimethylselenonium ion was detected after the injection of 82Se. Liver and kidney cytosols contained complex Se-enriched fractions, a positive identification of which was not done in this study. In most cases, the enrichment of tissue fractions with the stable isotope was altered by the dietary Se levels, the isotope nevertheless was exchanged with the endogenous Se in various macromolecules to a varying degree.

Mechanisms of selective copper removal by tetrathiomolybdate from metallothionein in LEC rats.

Copper (Cu) was selectively removed from metallothionein (MT) in the liver of LEC rats (Long-Evans rats with a cinnamon-like coat color) in vivo and in vitro by tetrathiomolybdate (TTM). Female LEC rats were injected intraperitoneally with TTM at a dose of 10 mg/kg body weight for 8 consecutive days. More than 2/3 of the Cu accumulating in the liver was removed by TTM treatment 24 h after the last injection. Although most Cu was bound to MT in the soluble fraction before TTM treatment, the Cu remaining in the liver was present almost exclusively in the non-soluble fraction together with molybdenum (Mo). Cu,Zn,Cd-MT was separated from the liver of LEC rats that had been injected with cadmium (Cd) and reacted with TTM at mol ratios of 0, 0.25, 0.50, 1.0, 2.0 and 4.0 to Cu bound to MT for 10 min at 37 degrees C. When TTM was added at a mol ratio of less than 1.0, a Cu,Zn,Cd-MT/TTM complex was detected, while addition of TTM at a mol ratio of greater than 1.0 selectively removed Cu from MT and produced a Cu/TTM complex via liberation of Zn,Cd-MT from the Cu,Zn,Cd-MT/TTM complex. Excessive TTM appeared to facilitate polymerization of the Cu/TTM complex to insoluble polymers. The dose-related formation of differing MT/TTM complexes explains the findings observed in vivo.

Renal damage induced by cadmium-metallothionein: effects on biochemical indicators.

Time dependent changes in urinary biochemical indicators for renal tubular injury and dysfunction were determined in female Wistar rats after an intraperitoneal injection of cadmium-metallothionein (Cd-MT) (50, 150 or 300 micrograms Cd/kg body wt) to further characterize the tubular damage caused by Cd. The Cd-MT injection caused dose-dependent increases in urinary activities of the enzymes (alkaline phosphatase; gamma-glutamyl transpeptidase; lactate dehydrogenase, LDH; N-acetyl-beta-D-glucosaminidase) on day 1, which appeared to reflect the tubular injury. The rate of increase in LDH, a cytosolic enzyme, was the largest among those of the enzymes. This result coincided with the data reported for repeated administration of ionic Cd to rats, suggesting that the feature of tubular injury caused by an injection of Cd-MT is similar to that by chronic exposure to ionic Cd. Changes in urinary glucose and total protein, indicators of tubular dysfunction, and metals (Cd, zinc and copper) were accompanied with those in urinary enzymes. Hydrocarbons in breath of rats injected with Cd-MT at a dose of 300 micrograms Cd/kg body weight were also determined as an indicator of in vivo lipid peroxidation. The levels of ethane and propane were significantly increased at 12 h after injection, which suggests that lipid peroxidation is partly involved in the tubular damage reflected by the increases in urinary enzymes.

Cadmium found in non-soluble fraction of kidney homogenates and its relation to renal dysfunction after cadmium-cysteine administration.

The critical concentration of cadmium (Cd) in the kidney after acute administration of Cd-cysteine (Cys) is 10 micrograms/g wet wt in whole kidney and is equal to that following Cd-thionein (Th) injection. This fact suggests that the nephrotoxicity of Cd-Th may be due to the Cd ions liberated from Cd-Th. To clarify the mechanism of the nephrotoxicity, the subcellular distribution of Cd injected as Cd-Cys was determined between supernatant and sediment fractions. Renal Cd level observed at 4 h increased with dose, but the Cd concentration in the supernatant fraction was kept almost constant at higher doses. Renal dysfunction measured by urinary protein and glucose levels was seen at the higher doses. This suggested that Cd in the sediment fraction of the kidney homogenates may be the ultimate cause for the nephrotoxicity of Cd-Cys. The sedimental Cd was eliminated from the kidney by 24 h. Much of the Cd found in the supernatant fraction was bound to high-molecular-weight proteins (HMWP) at 4 h, and almost all Cd ions were found to be bound to metallothionein at 24 h. Therefore, the HMWP-bound Cd in the supernatant fraction also may be a cause for the renal dysfunction. It is concluded that in addition to the HMWP-bound Cd in the supernatant fraction, CD distribution into the sediment fraction should be considered as a factor in causing nephrotoxicity after Cd-Cys and possibly Cd-Th administration.

Protective effect of metallothionein on intracellular pH changes induced by cadmium.

In order to gain further insight into the protective mechanism of metallothionein (MT) against Cd cytotoxicity, the effects of in vivo Zn- or Cd-pretreatment on the cytotoxicity and alteration in cellular pH induced by Cd were examined in isolated rat hepatocytes and testicular Leydig cells. These pretreatments both induced the synthesis of MT in the hepatocytes, but not in the Leydig cells. Both pretreatments alleviated Cd cytotoxicity in the hepatocytes. Cd- or Zn-pretreatment was also effective in preventing Cd-induced cellular acidification in hepatocytes but neither pretreatment was effective in Leydig cells. In fact, Cd-pretreatment stimulated acidification in Leydig cells. Exposure in vitro of hepatocytes from untreated rats to probenecid, an inhibitor of HCO3-/Cl- exchange, also ameliorated Cd-induced cellular acidification, suggesting an involvement of HCO3-/Cl- exchange in the preventive action of MT against Cd-induced acidification. These results suggest that Cd cytotoxicity in various cells may be initiated by alterations in plasma membrane ion transport systems such as the HCO3-/Cl- exchange and consequential cellular acidification. Induction of MT, therefore, may prevent Cd cytotoxicity, at least in rat hepatocytes, by preventing an alteration in ion transport at the plasma membrane as well as by intracytoplasmic binding of the metal molecules.

Potential mechanism of cadmium-induced cytotoxicity in rat hepatocytes: inhibitory action of cadmium on mitochondrial respiratory activity.

The present study was designed to clarify the mechanism of cadmium (Cd)-induced toxicity in rat hepatocytes. Cd and Mg-ATP induced cellular acidification at concentrations lower than 25 microM. In Mg-ATP-treated cells, maximal acidification occurred within 2.5 min, with a subsequent return to control levels. In Cd-treated cells, maximal acidification (pH 6.76) occurred 10 min after exposure to the metal, then the cytoplasmic pH began to rise but did not return to normal. Cd eliminated the membrane potential of isolated mitochondria in media at both pH 6.5 and 7.4. This effect of Cd on membrane potential was approximately equivalent in both media when the metal concentration was 5 microM, but was more intense in the medium at pH 6.5 than in the medium at pH 7.4 at the metal concentration > 5 microM. Acidic medium alone had no effect on membrane potential. Mitochondrial uptake of Cd increased in a dose-dependent manner in media at both pH 7.4 and 6.5. The uptake of 5 microM Cd was significantly increased by acidic medium, however at Cd concentrations > 5 microM, there were no pH-dependent differences in Cd uptake. The incubation of hepatocytes in the medium at pH 6.0 and 6.5 resulted in 5 and 7.5% inhibition of mitochondrial respiration in intact cells, respectively. The presence of 10 microM Cd in the medium at pH 6.0 enhanced this inhibition. Mitochondrial respiration was inhibited to 60% of the control mitochondria at pH 7.4 when exposed to the medium at pH 6.5 without Cd and this inhibition was extended to 70% by the presence of 5 microM Cd. Cd in the medium at pH 7.4 had no deleterious effect on mitochondrial respiration at all concentrations examined. The results indicate that the respiratory activity of mitochondria is sensitive to the low pH rather than to Cd although the metal strongly inhibits the activity when in the medium at low pH. Therefore, it appears Cd-induced acidification plays an important role in the initiation of deteriorative processes in mitochondria.

Copper metabolism leading to and following acute hepatitis in LEC rats.

The accumulation process of copper (Cu) in the liver and the following metabolic disorder of Cu were examined in LEC rats, a mutant strain which accumulates Cu with age and shows spontaneous acute hepatitis and/or hepatoma. Cu concentration in the liver of female rats was approximately 220 micrograms/g liver at 2 weeks of age, decreased to 100 micrograms/g liver at 4-6 weeks, and then started to increase with age linearly to the highest concentration of 250 micrograms/g liver at 16 weeks. Although the Cu level expressed by concentration (microgram/g liver) decreased during weaning, it increased linearly with age when it was expressed by content (mg/liver), indicating a constant and preferential accumulation of Cu in the liver. Cu concentration stopped increasing at 16 weeks in the liver, followed by a sudden decrease to 1/2 the highest level. Biological markers (serum lactate dehydrogenase and glutamic-oxaloacetic transaminase activities) for liver damage started to increase, together with the appearance of signs of jaundice, when Cu attained the highest concentration. Distributions of Cu and zinc (Zn) in the supernatant fraction of the liver indicated that both metals were mostly distributed to metallothionein (MT) and, to a small extent, to superoxide dismutase on a gel filtration column throughout the course of the experiments. Serum Cu concentration started to increase in a form of ceruloplasmin, together with serum marker enzyme activities for liver damage. Cu concentration in the kidneys also started to increase after the increase of serum Cu. The results indicate that Cu accumulates in the form of MT in the liver of LEC rats to a maximum level of approximately 250 micrograms/g liver, and then decreases suddenly with the onset of acute hepatitis. The maximum level seems to be related to the capacity of MT synthesis, and acute hepatitis is assumed to occur when Cu accumulates beyond the capacity. Serum Cu started to increase, from the abnormally low level, when the metal accumulated beyond the capacity of MT synthesis in the liver, and it was partly reabsorbed by the kidneys and the rest was excreted into urine. Changes in iron and zinc levels were determined and discussed in relation to those of Cu.