Synthesis of a nonacosapeptide (beta-fragment) corresponding to the N-terminal sequence 1-29 of human liver metallothionein II and its heavy metal-binding properties.

A nonacosapeptide (beta-fragment) corresponding to the N-terminal sequence 1-29 of human liver metallothionein II was synthesized by the fragment condensation method. The Cd-binding ability of the beta-fragment was much stronger than that of cysteine as thionein and synthetic alpha-fragment corresponding to the C-terminal sequence 30-61 of human liver metallothionein II. Both the alpha- and beta-fragments bound preferentially to Cu ions rather than Cd ions.

Induction of zinc-thionein by estradiol and protective effects on inorganic mercury-induced renal toxicity.

The castrated or unoperated male rats received an intravenous injection of HgCl2 at a dose of 0.7 mg/kg of body weight (b.w.) after pretreatment with 30% ethanol or estradiol dissolved in 30% ethanol at a dose of 0.5 mg/kg b.w. subcutaneously twice a day for six consecutive days. Renal total protein, gamma-GTP and K excretion in the rats treated with Hg and estradiol were significantly lower than the corresponding values in the rats treated with Hg alone, suggesting that pretreatment with estradiol ameliorates the renal toxicity of Hg in male rats. Pretreatment with estradiol significantly increased Hg and Hg-thionein(Hg-MT) concentrations in the renal cortex of the animals treated with Hg, though in the liver this agent did increase the Hg-MT without elevation of Hg concentration. Treatment with estradiol alone (0.5 mg/kg, s.c., twice a day, for six consecutive days) significantly increased the zinc-thionein (Zn-MT) concentration in the kidney and liver. Simultaneous treatment with 10(-5) M estradiol and Hg in human amniotic-fluid cells caused a significant increase in the uptake of Hg and the synthesis of Hg-MT, suggesting that estradiol may directly stimulate an accumulation of Hg into the cells and the synthesis of Hg-MT. Together, all of the above findings suggest that pretreatment with estradiol may increase the uptake of Hg, which in turn leads to the increase in the Hg-MT concentration. The induction of Zn-MT by pretreatment with estradiol may account for the protective effect of estradiol on Hg-induced renal toxicity.

Stimulation of cadmium uptake by estradiol in the kidney of male rats treated with cadmium.

The present study was carried out to analyze the sex differences in the retention of Cd in rats treated with a small amount of Cd, and its mechanisms. Cd and Zn concentrations in the kidney and liver of female rats treated with 28 nmol Cd or 1 nmole Zn were significantly higher than those in male rats. Pretreatment with estradiol (1.8 mumol/kg of b.w., twice a day, 6 consecutive days) increased the Cd and Zn concentrations in the kidney of male rats treated with Cd or Zn. Incubation of MDCK cells with 10(-5) M estradiol, 10(-5) M stilboestrol and 10(-5) M progesterone caused a significant increase in Cd uptake. These results suggest that endogenous female sex hormones may play a role in a higher concentration of Cd and Zn in the kidney of female rats than that in male rats. The basal level of metallothionein (MT) in the liver and kidney of control female rats was within the same range as that in the control male rats. Cd and Zn accumulations caused by pretreatment with estradiol in the kidney of male rats treated with Cd or Zn were so low (Cd: 38 ppb, Zn: 1.0 ppb) as to be probably unable to induce the synthesis of MT. An increase in the concentration of Cd in the cultured renal cells occurred 1 hr after treatment with estradiol and Cd. Pretreatment with estradiol alone also resulted in a modification of the concentration of Na and K, which cannot be bound to MT. Together, all of the above findings suggest that estradiol directly increases the accumulation of Cd into the renal cells without inducing the synthesis of MT.

Effects of cadmium and zinc on tissue levels of metallothionein.

Although the induced synthesis of metallothionein (MT) after exposure to certain metals has been known for some time, there is little information on the quantitation of MT in various tissues. In this study, tissue MT concentrations were measured by a modified Cd-saturation method in tissues of adult male rats after injection of different metal salts. There were differences in tissue levels of MT, depending on the injected metals. Of all the metals studied, Cd2+ was the most effective element in increasing MT concentrations in liver, kidney, pancreas and small intestine. The highest increase in tissue MT concentration after CdCl2 injection was found in the liver, while the pancreas contained the highest MT level after ZnSO4 injection. Co and Ni salts increased MT levels in both liver and kidney, while Mn and Ca increased MT levels only in liver. A direct correlation between tissue MT levels and Cd or Zn concentration was observed in most of the tissues after injection of CdCl2 or ZnSO4. Although there was no positive relationship between tissue levels of MT and tissue Mn and Ni concentrations, the increase in hepatic Zn after injection of these metals was related to hepatic MT levels. The tissue distribution of injected Cd2+ in control adult rats and Zn-deficient rats was similar. However, there was no increase in pancreatic MT levels in Zn-deficient rats after injection of CdCl2. The high concentration of MT in pancreas after ZnSO4 injection in adult rats and the inability of the pancreas to synthesize MT in Zn-deficient rats suggest that the induction of pancreatic MT synthesis is sensitive to Zn status.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding of retinol-binding protein obtained from human urine with vitamin A derivatives and terpenoids.

Retinol-binding protein(RBP) was purified from fresh urine of patients suffering from Itai-Itai disease. The purified preparation contained two types of apo-RBP(Apo I and II) in equal amounts as major components (about 85% of the total RBP). The corresponding two retinol-binding forms (Holo I and II) were present as minor components (about 15% of the total)...

The induced synthesis of metallothionein in various tissues of rats in response to metals. II. Influence of zinc status and specific effect on pancreatic metallothionein.

Metallothionein (MT) of various tissues contains bound zinc (Zn) and any change in Zn status can alter its synthesis and tissue deposition. The changes in MT levels and its inducibility in Zn-injected and Zn-deficient (Zn-D) rats were studied. MT levels in 11 tissues (brain, lung, heart, liver, kidney, stomach, small intestine, pancreas, spleen, testes and muscle) of control and rats injected with different doses of ZnSO4 (20 mg Zn/kg for 2, 4 or 7 times) were measured by the cadmium-hemolysate (Cd-hem) method. A dose dependent increase in MT levels was observed only in the pancreas, liver, small intestine and kidney after ZnSO4 injection--the highest level being in the pancreas. A positive correlation was found between Zn and MT concentrations and also the relative inducibility of MT was similar in these 4 tissues (slopes of regression equations were 12.6--15.5). In order to study the effect of Zn-D in MT induction, rats were fed a diet containing 1 ppm Zn for 18 days and CdCl2 (1 mg Cd/kg) was injected subcutaneously 3 times at 48-h intervals to control and Zn-D rats. Although the tissue distribution of Cd was similar in both the groups, MT concentrations in pancreas and kidney were significantly decreased in Zn-D. The plasma and tissue levels of Zn were also decreased in Zn-D rats injected with CdCl2. The decrease in both Zn and MT levels was more prominent in pancreas than other organs of Zn-D rats. The results suggest that of all the organs studied, the induction of pancreatic MT is sensitive to Zn status and Zn may be a primary inducer of MT.

The induced synthesis of metallothionein in various tissues of rat in response to metals. I. Effect of repeated injection of cadmium salts.

Even though the induce synthesis of metallothionein (MT) after exposure to metals has been known for some time, there is little data on the quantitation of MT in various tissues. In this study we have measured MT levels in eleven different tissues of rat after injection of CdCl2 and also compared the relative MT inducibility of these tissues. Over a period of 16 days, rats were injected subcutaneously once every second day with either saline or 3 different doses - 0.8, 1.5 or 3 mg (7.2, 13.4 or 26.8 mu mol) Cd/kg of CdCl2 and sacrificed 48 h after the last injection. Cadmium and MT were determined in brain, lung, heart, liver, kidney, stomach, small intestine, pancreas, spleen, testes and muscle. A dose dependent increase in MT accumulation was observed in a number of tissues after CdCl2 injection, the highest amount being in liver, kidney and pancreas. Further analysis of the data showed a positive correlation between Cd and MT concentrations in 8 tissues. The relative MT inducibility in liver, pancreas, heart and stomach in response to Cd injection was identical.

The interaction of zinc and cadmium in the synthesis of hepatic metallothionein in rats.

The interaction of injected zinc salts (Zn) and cadmium salts (Cd) with regard to the synthesis of metallothionein (MT) in adult rat liver was investigated. Male rats received an i.p. injection of Zn (20 mg/kg) or Cd (0.6 mg/kg) with or without pretreatment with Zn (20 mg/kg 16 h prior to the second injection). It was found that both metals, when administered singly, induced the synthesis of significant levels of hepatic MT, but that, when the Cd injection followed the Zn injection, synthesis of MT was not additive. When Zn pretreatment was followed by a second Zn injection, MT accumulation was additive (approx. 2-fold of that observed after a single Zn injection). Also, a highly significant positive correlation, (r = 0.97, P less than 0.01) was noted between hepatic Zn concentration and hepatic MT concentration, a relationship which was independent of the mode of MT induction. The results of the investigation indicate that: (1) in the presence of pre-existing hepatic Zn--MT, the ability of Cd to induce new MT synthesis is greatly reduced; rather, Cd is sequestered by the pre-existing MT; and (2) Zn may play a major role in the induction of MT synthesis both after Zn administration and after Cd administration.

Colchicine-induced elevation of tissue metallothionein contents is mediated by inflammation-independent serum factor.

Subcutaneous injection of colchicine caused dose-dependent and time-dependent induction of hepatic MT in mice. Other than colchicine, similar MT induction was observed in vincristine- or vinblastine-injected mice, but not in beta-lumicolchicine-injected mice. MT contents were also elevated in the kidney, spleen, lung and heart by colchicine injection. Isoforms of colchicine-induced MT in the liver were identified to be MT-I and II by immunoblot analysis. Unlike turpentine-induced MT synthesis, dexamethasone, an anti-inflammatory agent, could not block the MT-inducing activity of colchicine. Therefore, the MT-inducing activity of colchicine does not appear to be due to inflammation. Mouse serum, obtained at 4-24 h after colchicine treatment, stimulated MT induction in rat hepatoma H4IIEC3 cells. The MT-inducing activity in the serum from colchicine-treated mice was determined to be highest at 12 h after colchicine injection. The MT-inducing activity from sera of colchicine-treated mice was completely blocked by glucocorticoid antagonist, RU38486, similar to such activity in the serum from lipopolysaccharide-treated mice. The ability of sera to induce MT was abolished by heat treatment (56 degrees C, 30 min). The molecular weight of the MT-inducing factor estimated by gel filtration was approximately 20 000 Da. Thus, colchicine-induced stimulation of MT production is mediated by some humoral factor. The production of the MT-inducing factor was not blocked by dexamethasone. We conclude that the mediator is not an inflammatory cytokine or a glucocorticoid and suspect that the disruption of microtubule triggers production or release of such humoral mediator which stimulates MT induction.

Induced synthesis of metallothionein by ascorbic acid in mouse liver.

The concentration of metallothionein in mouse tissues after administration of ascorbic acid was determined by the Cd-hem method. The concentration of metallothionein in the liver was increased significantly after an intraperitoneal injection of L-ascorbic acid at the dose of 1000 mg/kg. A simultaneous injection of cycloheximide inhibited the increase of the metallothionein concentration. Mortality of mice injected with a lethal dose of cadmium was decreased significantly by the pre-injection of L-ascorbic acid. All of these results indicate that metallothionein is induced in the liver after the administration of ascorbic acid.

High concentration of pancreatic metallothionein in normal mice.

An elution profile of supernatant of normal mouse pancreas on a Sephadex G-75 column showed a high peak of zinc in the metallothionein fraction. Cd was added to the supernatant of the mouse pancreas, and 2 cadmium binding proteins were purified by gel filtration and DEAE chromatography. Absorption spectrum and amino acid compositions of the purified proteins agreed with those of MT-I and -II. The concentration of metallothionein in the mouse pancreas was high (227 +/- 11 micrograms/g on 36th day after the birth). The metallothionein concentration in the mouse pancreas was increased by injections of zinc, alloxan or streptozotocin. The results indicate that metallothionein is one of the main zinc-binding components in mouse pancreas.

Concentrations of metallothionein and metals in malignant and non-malignant tissues in human liver.

Concentration of metallothionein in the malignant and non-malignant tissues of the human liver was determined by the Cd-hem method. The concentration of metallothionein was high in the non-malignant tissue (471 +/- 306 micrograms/g). Metallothionein in the non-malignant tissue was mainly Zn-thionein and metallothionein-bound Zn was a major chemical form of Zn in the non-malignant tissue. The concentration of metallothionein in the malignant tissue was 75.6 +/- 79.6 micrograms/g, and significantly lower than that in the nonmalignant tissue. A strong, positive relationship was observed between the Zn and metallothionein concentrations, and the regression equation was MT (microgram/g) = -94.7 + 11.9 Zn (micrograms/g).

Protective effect of metallothionein to ras DNA damage induced by hydrogen peroxide and ferric ion-nitrilotriacetic acid.

Metallothionein (MT) is a strong antioxidant, due to a large number of thiol groups in the MT molecule and MT has been found in the nucleus. To investigate whether MT can directly protect DNA from damage induced by hydroxyl radical, the effects of MTs on DNA strand scission due to incubation with ferric ion-nitrilotriacetic acid and H2O2 (Fe3+ -NTA/H2O2) were studied. The Fe3+-NTA/H2O2 resulted in a higher rate of deoxyribose degradation, compared to incubation of Fe3+/H2O2, presumably mediated by the formation of hydroxyl radicals (*OH). This degradation was inhibited by either Zn-MT or Cd-MT, but not by Zn2+ or Cd2+ at similar concentrations. The Fe3+ -NTA/H2O2 resulted in a concentration dependent of increase in DNA strand scission. Damage to the sugar-phosphodiester chain was predominant over chemical modifications of the base moieties. Incubation with either Zn-MT or Cd-MT inhibited DNA damage by approximately 50%. Preincubation of MT with EDTA and N-ethylmaleimide, to alkylate sulfhydryl groups of MT, resulted in MT that was no longer able to inhibit DNA damage. These results indicates that MT can protect DNA from hydroxyl radical attack and that the cysteine thiol groups of MT may be involved in its nuclear antioxidant properties.

Binding of Cd to metallothionein in the placenta of Cd-treated mouse.

The toxicological significance of the placental metallothionein (MT) was studied from the viewpoint of cadmium (Cd) intoxication. The Cd concentration was higher in the placenta than the kidneys until 8 hr after a single injection of 109CdCl2, but was very low after administration of 109Cd-MT. Compared with lower doses, fetal Cd began to increase at the dose of 2 mg/kg. Alkaline phosphatase activity in the placenta was significantly decreased, and dead fetuses appeared at this dose. The MT concentration was not increased by the Cd injection but the ratio of Cd/Zn in MT increased proportionally to the dose up to 2 mg/kg. MT was detected in the placenta during all stages of pregnancy, and its concentration was higher in the earlier stage. The metal bound to MT was mainly Zn, and very little Cu was bound. In the mice that became pregnant after 109Cd accumulation, redistribution of 109Cd was not observed. MT-I and -II in the placenta were identified by column chromatography and immunoblotting methods. These findings suggest that the placental MT might play a protective role against Cd toxicity by trapping of the metal.

Cosinor analysis of feed intake cycle of rats fed a zinc-deficient diet and the effect of zinc supplementation.

Rats fed a Zn-deficient diet develop a characteristic cyclic variation in feed intake (Mills et al., Am J Clin Nutr 22: 1240-1249 (1969). A preliminary analysis (Tamaki et al., Br J Nutr 73: 711-722 (1995)) of the cyclic variations was followed with a personal computer. Cosinor analysis revealed that the cyclic period of the feed intake of male rats was 3.5 +/- 0.05 d. The mesor, amplitude and acrophase value were 10.0 +/- 0.3 g/d, 4.4 +/- 0.2 g/d and 3.5 +/- 0.3 radian, respectively. The cycle of body-weight change of the Zn-deficient rats was well synchronized with that of feed intake. The parameters of the feed intake cycle had a high correlation to the corresponding parameters of body-weight change (mesor: r = 0.846; amplitude: r = 0.771; period: r = 0.925; acrophase: r = 0.452). With the supplementation of Zn (0.95-3.80 mg/kg of the Zn-deficient diet), cyclic variations in feed intake and body-weight change were also found. The mesor, amplitude and period of feed intake cycle were is good correlation with Zn intake (r = 0.856, p < 0.001, r = 0.804, p < 0.001 and r = 0.613, p < 0.01, respectively). The cycle of feed intake of the rats fed a Zn-free diet was simulated to be: mesor 9.7 +/- 0.1 g/d, amplitude 6.5 +/- 0.1 g/d and period 3.4 +/- 0.02 d. The concentration of Zn intake given the half-maximal value of the amplitude was assumed to be 56 +/- 1 microgram/d.

Induction of hepatic metallothionein by nonmetallic compounds associated with acute-phase response in inflammation.

Induction of hepatic metallothionein (MT) synthesis by several nonmetallic compounds and its relationship to an acute-phase response in inflammation were studied in mice. Subcutaneous injections of menadione, paraquat, carbon tetrachloride (CCl4), and several organic solvents caused an increase of hepatic MT concentration. This MT contained only zinc. Menadione and n-hexane caused the greatest accumulation of hepatic MT among these nonmetallic compounds (about 13-fold). The concentration of Zn was significantly decreased in plasma in contrast to liver after an injection of these nonmetallic compounds. When 65ZnCl2 was injected iv after these injections, uptake of 65Zn to the liver was increased. This effect was not observed after treatment with cycloheximide. The association with inflammation of this induction of MT accumulation was examined by determination of acute-phase proteins. The concentration of fibrinogen in the plasma was significantly increased following injection of those nonmetallic compounds which caused marked hepatic MT accumulation. An injection of 1 N NaOH, 1 N HCl, turpentine oil, or endotoxin caused a significant increase in the plasma concentration of fibrinogen and in the hepatic MT concentration. Injections of n-hexane as well as turpentine oil significantly increased hepatic MT concentration and plasma concentration of fibrinogen and ceruloplasmin with time. The concentration of fibrinogen was significantly correlated (r = 0.789) with the concentration of hepatic MT. Neither adrenalectomy nor pretreatment with dexamethasone prevented hepatic MT accumulation caused by these compounds. These results indicate that induction of hepatic MT synthesis by these nonmetallic compounds is associated with an acute-phase response in inflammation and is independent of glucocorticoids.

Induction of metallothionein synthesis by menadione or carbon tetrachloride is independent of free radical production.

The relationship between induction of hepatic metallothionein (MT) synthesis and lipid peroxidation by free radical production following an injection of menadione or carbon tetrachloride (CCl4) in mice was studied. The hepatic concentration of MT was increased by menadione significantly at 25 mg/kg or higher. A significant increase in thiobarbituric acid (TBA) value, indicative of lipid peroxidation, was observed in the liver at menadione doses of 62.5 mg/kg or higher. Both the MT and the TBA value in the liver were significantly increased at the low dose of CCl4. The concentration of MT was increased significantly 4-8 hr after administrations of these compounds. The increase of TBA value over time was similar to that of MT concentration after administration of CCl4, but not after administration of menadione. The MT concentration in the menadione group was higher than that in the CCl4 group, and the TBA level in the menadione group was lower than that in the CCl4 group. Pretreatment with vitamin E caused a significant reduction in the TBA value, but did not affect the MT level in the liver. The concentration of MT did not significantly correlate with the TBA value in either the menadione or the CCl4 group. Pretreatment with phenobarbital, which promotes free radical production, did not influence induction of MT synthesis following an injection of menadione or CCl4. Neither L-buthionine sulfoximine nor 2-cyclo-hexen-1-one, which decreases hepatic glutathione, influenced the induction of MT by menadione. These data suggest that induction of MT synthesis by menadione or CCl4 is independent of free radical production in the liver.

Synthesis and degradation of erythrocyte metallothionein in cadmium-administered mice.

The origin of erythrocyte metallothionein (MT) in cadmium (Cd) administered mice and its fate were studied in connection with erythrocyte kinetics. It was shown that Cd-MT in the erythrocyte is synthesized in the precursor cells of the erythropoietic tissues, and Cd-MT containing erythrocytes come into the circulation after maturation. In regard to the degradation, it was found that Cd-MT is stable in the erythrocyte, but decomposed in the spleen or liver with the breakdown of erythrocyte when its life span is over.

Role of intestinal metallothionein in absorption and distribution of orally administered cadmium.

The effects of mucosal metallothionein (MT) preinduced by Zn on the intestinal absorption and tissue distribution of Cd were studied. 109CdCl2 was administered to control and Zn-pretreated rats. The total amount of Cd distributed to the liver and the kidney in the group pretreated with 100 mg/kg of Zn was about 70% that of the control group. In the control group, the Cd concentration in the intestinal mucosa reached a maximum 16-24 hr after its administration and then gradually decreased with time, unlike that in the liver and the kidney. The concentration of intestinal Cd in the pretreated group reached a maximum earlier than it did in the control group and most of the Cd was in the MT fraction. Pretreatment with Zn (100 mg/kg or higher, po) caused a reduction in the Cd concentration in the liver and an increase in the kidney. Pretreatment with Zn (5 X 10 mg/kg, sc) or Cd (5 mg/kg, po) also increased renal Cd concentration. This was effective at 24 hr but not at 0.5 hr after pretreatment. These effects of pretreatment with Zn (100 mg/kg, po) on tissue distribution of Cd were also observed after an intraintestinal injection of Cd but not after an iv injection. The results indicate that MT in intestinal mucosa plays a significant role not only in the absorption of Cd but also in its transport to the kidney.