The modulation by metallothionein of cadmium-induced cytotoxicity in primary hepatocyte cultures

The cytotoxicity of CdCl2 and 2 isoforms of hepatic cadmium-metallothionein (CdMT I and II), was investigated using primary cultures of rat hepatocytes. The cell cultures were exposed to cadmium as CdCl2 or as either isoform of CdMT for a 20-h period at concentrations ranging from 50 to 500 ng Cd X ml-1. Cytotoxicity was assessed by determining the amount of lactic dehydrogenase released from the cells into the incubation medium and the incorporation of [3H] arginine into cell protein. The uptake of Cd by the cells was also measured. Cadmium chloride and both isoforms of CdMT were found to be toxic to hepatocytes although partial protection was afforded by the binding of cadmium to metallothionein (MT). At the higher exposure concentrations and in accordance with the toxicity data, the cells exposed to CdCl2 were found to accumulate more cadmium than those exposed to CdMT. The distribution of cadmium in the culture medium was examined using Sephadex G-75 chromatography. The speciation of cadmium is discussed in relation to its cytotoxicity.     

Glutathione protection against hydrogen peroxide, tert-butyl hydroperoxide and diamide cytotoxicity in rat hepatoma-derived Fa32 cells

1. Several ozonides, peroxides and aldehydes are formed during ozone therapy, recently introduced in medicine. tert-Butyl hydroperoxide (t-BHP), H2O2 and diamide were investigated as model substrate in rat hepatoma-derived Fa32 cells. 2. The cytotoxicity was measured by the neutral red uptake inhibition assay after 1 h or 24 h treatment. The relative toxicities were quantified by the determination of the NI50. This is the concentration of test compound required to induce an inhibition of 50% in neutral red uptake as compared to the control cells. All test chemicals were more toxic after 24 h than after 1 h. 3. The influence of the glutathione (GSH) alteration on the cytotoxicity was measured by treating the cells with 2-oxo-4-thiazolidine carboxylic acid (OTC) or L-buthionine sulfoximine (BSO). OTC increased the endogenous GSH content in the cells. BSO pretreatment strongly decreased the NI50 of the three chemicals. OTC pretreatment increased the NI50 of H2O2 but not of t-BHP and diamide. This can be explained by the strong GSH-depletion after 1 h by t-BHP and diamide, which contrasted with a weak GSH-depletion by H2O2 after the same time period. 4. The three test chemicals increased the endogenous GSH content after 24 h. t-BHP and H2O2, but not diamide, increased the total GSH transferase (GST) activity. Several alterations of the GST subunits were observed. Most striking was the increase of class alpha GST subunits, also for diamide. 5. Since H2O2 and t-BHP are ozone metabolites thought to be responsible for the therapeutic effects of well-dosed ozone, the results show that Fa32 cells can be used as a valuable alternative model system for studying the effects encountered in human ozone therapy.     

Hepatoprotective constituents in plants. 14. Effects of soyasapogenol B,sophoradiol, and their glucuronides on the cytotoxicity of tert-butyl hydroperoxide to HepG2 cells

The effects of soyasapogenol B, sophoradiol, their glucuronides, and glycyrrhizin on the hepatotoxicity of tert-butyl hydroperoxide (t-BuOOH) in a human-liver-derived cell line (HepG2 cells) were investigated. Glycyrrhizin showed significant dose-dependent protective effects against the cytotoxicity of t-BuOOH. Among soyasapogenol B and its glucuronides, the monoglucuronide showed the most potent hepatoprotective activity, followed by soyasapogenol B itself. Soyasaponin III was weakly protective, while soyasaponin I increased the toxicity of t-BuOOH. Among sophoradiol and its glucuronides, sophoradiol itself showed the most potent hepatoprotective activity, which was equal to glycyrrhizin, while the monoglucuronide and kaikasaponin III showed an increase in cytotoxicity. These results were considerably different from those reported previously on the protective effects of these compounds using primary cultures of immunologically injured rat liver cells. Consequently, the hepatoprotective action of the triterpene derivatives investigated would be different in HepG2 cells and in rat primary hepatocyte cultures.     

Protective effect of Ninjin-yoei-to on damage to isolated hepatocytes following transient exposure to tert-butyl hydroperoxide

To establish a simple screening system for estimating efficacy of an agent for an oxidative-related lesion, we investigated the damage in isolated rat hepatocytes exposed to 75 microM tert-butyl hydroperoxide (t-BuOOH) and then subsequently incubated the cells in fresh medium. By electron spin resonance spectroscopy analysis using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), DMPO adducts of tert-butoxyl radicals and carbon center radicals were detected during the t-BuOOH exposure, and DMPO-OH formation was detected after t-BuOOH removal. In t-BuOOH-exposed cells, the level of phosphatidylcholine hydroperoxide (PCOOH), a peroxidative product of biomembranes in the hepatocytes, and the leakage of enzymes into the culture medium were significantly increased. An increase in acid phosphatase (AP) activity representing lysosome destabilization preceded the aspartate oxoglutarate aminotransferase (AST), alanine oxoglutarate aminotransferase (ALT) and lactate dehydrogenase (LDH) leakage. Ninjin-yoei-to added to the culture medium following the t-BuOOH exposure significantly inhibited the PCOOH formation and the leakage of AP, AST, ALT and LDH, concentration-dependently. Ninjin-yoei-to at 1 mg/ml in culture medium completely diminished these increases in enzyme activities down to the background levels found in control experiments and this reduction was greater than the most effective alpha-tocopherol concentration of 20 micromol/ml. Considering all of these results, it is likely Ninjin-yoei-to may exert its protective effect by antioxidative action and membrane stabilization.     

The effect of aging on the response of isolated hepatocytes to hydrogen peroxide and tert-butyl hydroperoxide

Aging is associated with increased susceptibility to oxidative stress. To study this in the liver and to elucidate underlying mechanisms, hepatocytes from young (4–6 months) and old (24–26 months) rats were exposed to two oxidants, hydrogen peroxide and tert-butyl hydroperoxide. ATP content and mitochondrial activity were lower in old hepatocytes and decreased further with oxidative stress. Expression of Cu/Zn superoxide dismutase, Mn superoxide dismutase and catalase was not substantially influenced by oxidative stress in young and old hepatocytes, whereas glutathione peroxidase 1 expression was markedly increased only in young hepatocytes. Oxidative stress in young hepatocytes led to increased expression of apoE and movement of apoE to the early endosomes. In old hepatocytes, oxidative stress did not increase apoE expression and apoE was co-localized with early endosomes under control conditions. The results show that old age is associated with impaired hepatocyte responses of mitochondria, ATP, glutathione peroxidase 1 and apoE to oxidative stress.     

Metabolism of pyridine nucleotides in cultured rat hepatocytes intoxicated with tert-butyl hydroperoxide.

The alterations in the metabolism of pyridine nucleotides, as well as the role such changes play in the genesis of lethal cell injury, were explored in cultured rat hepatocytes intoxicated with tert-butyl hydroperoxide (TBHP). The loss of NADPH, NADH, and NAD equalled the increase in NADP, with little if any change in the total content of pyridine nucleotides. Identical alterations occurred in the presence of N,N'-diphenyl-p-phenylenediamine, an antioxidant that prevented the death of the cells. Inhibition of glutathione reductase by 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) reduced the extent of the increase in NADP and the decrease in NADPH. At the same time, BCNU increased the cell killing. Depletion of ATP with oligomycin reduced the loss of NAD and the accumulation of NADP. Treatment of the hepatocytes with the poly(ADP-ribose) polymerase inhibitor 3-aminobenzamide had no effect on the depletion of NAD. Thus, all of the alterations in pyridine nucleotides that accompany the exposure of cultured hepatocytes to TBHP can be dissociated from the development of lethal cell injury. The changes do suggest, however, a rapid interconversion of the respective species. The initial response reflects activation of glutathione reductase with the consequent oxidation of NADPH to NADP. The conversion of NADH to NAD and then NAD to NADP, the latter by nicotinamide adenine dinucleotide kinase, can account for the increase in NADP over the resulting from the oxidation of NADPH by glutathione reductase. Finally, there was no evidence in cultured hepatocytes treated with TBHP for changes in NAD that reflect the activation of poly(ADP-ribose) polymerase.     

Effects of vitamin E on the killing of cultured hepatocytes by tert-butyl hydroperoxide.

The disposition of vitamin E was examined in cultured rat hepatocytes intoxicated with tert-butyl hydroperoxide (TBHP). Culturing of the cells overnight (18-20 hr) with approximately 60 nM alpha-tocopherol (alpha-T) equivalents [Williams' E medium, 18 nM tocopherol phosphate (alpha-TP), 9% fetal calf serum, 43 nM alpha-T] resulted in a content of alpha-T that was 16% of the concentration of vitamin E measured in freshly isolated hepatocytes. Supplementation of the medium with 1 microM alpha-TP maintained the alpha-T concentration of the cultured cells at the level of freshly isolated hepatocytes. Supplemented hepatocytes exposed to TBHP showed decreased lipid peroxidation and delayed cell killing, compared with hepatocytes not cultured overnight with alpha-TP. Killing of the supplemented cells by TBHP was accompanied by a loss of alpha-T. Pretreatment of supplemented hepatocytes with the iron chelator deferoxamine prevented much of the loss of alpha-T. At the same time, deferoxamine inhibited both the lipid peroxidation and cell killing. The antioxidant N,N'-diphenyl-1,4-phenylenediamine reduced the loss of alpha-T and significantly decreased lipid peroxidation. In the presence of N,N'-diphenyl-1,4-phenylenediamine, cell killing was delayed by 15 min and reduced in extent. Overnight supplementation of hepatocytes with nonesterified alpha-T, or vitamin E esters other than alpha-TP, similarly rendered the cells less sensitive to TBHP. The nonesterified alpha-T produced a higher cell-associated vitamin E concentration than did the esters; however, nonesterified alpha-T did not result in greater protection against TBHP. These data indicate that the mechanisms of the cell killing by TBHP are the same in cultured hepatocytes that contain low or physiological concentrations of vitamin E.     

Strain differences in susceptibility of mice to cadmium-induced metallothionein

Strain differences in inducible biosynthesis of metallothionein of liver among five closed colonies of mice (DDY-Saitama, DDY (N), DDN-Saitama, STD-DDY and SWR) receiving repeated injections of cadmium have been demonstrated. No relationship could be observed between the degree of metallothionein induction and the percentage of testicular necrosis. The results of amino acid and metal content analyses of metallothionein did not differ significantly among the five closed colonies.     

In vivo metabolism of tert-butyl hydroperoxide to methyl radicals. EPR spin-trapping and DNA methylation studies

Metabolic activation of peroxides and hydroperoxides to free radicals is associated with the tumor promoting activity of these compounds. tert-Butyl hydroperoxide (t-BOOH) metabolism has been extensively studied as a model of peroxide biotransformation. In vivo studies are limited, and the hemoglobin-thiyl radical was the only species thus far identified in the blood of treated rats. Here we further examine t-BOOH metabolism in vivo with regard to free radical and DNA adduct production. Spin-trapping experiments with phenyl-N-tert-butylnitrone (PBN) led to the detection of EPR signals in the blood, bile, and organic extracts of the liver and stomach of rats treated with t-BOOH. Analysis of these signals demonstrated that t-BOOH metabolism in vivo produces alkyl radicals, detected in the bile and organic extracts of liver and stomach, in addition to the previously identified hemoglobin-thiyl radical. To characterize the produced alkyl radicals, experiments were performed with (13)C-labeled t-BOOH and two spin traps, PBN and alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN). The latter was used because the EPR signals obtained with PBN were too weak to be unambiguous. Nevertheless, the EPR signals present in the bile of animals treated with (13)C-labeled t-BOOH and PBN or POBN were consistent with adducts of (13)C-labeled methyl radical and an unidentified alkyl radical. The latter is probably derived from lipids oxidized by the metabolically produced primary radicals, methyl and its precursor, tert-butoxyl. The presence of 8-methylguanine and 7-methylguanine in hydrolysates of DNA from liver and stomach of rats treated with t-BOOH was also examined. 8-Methylguanine, a typical product of methyl radical attack on DNA, was detectable in both the liver and stomach of treated rats. The results may be relevant to the understanding of the genotoxic properties of other peroxides, particularly of cumene hydroperoxide.     

Cleavage of supercoiled DNA by horseradish peroxidase plus tert-butyl hydroperoxide is not due to tert-butylperoxyl radicals

Two supercoiled (SC), double-stranded DNAs, pBR 322 and pUC 19, have been subjected to oxidative stress using horseradish peroxidase (HRP) and HRP + tert-butyl hydroperoxide (BOOH). HRP alone causes single-strand cleavage of these SC DNAs. Strand cleavage is enhanced substantially in the presence of commercial BOOH (which contains H(2)O(2)) but is, at best, only very slightly enhanced in the presence of pure BOOH. In the HRP/pure BOOH system, the DNA single-strand-scission which does occur appears to be due to a direct action of oxidized HRP. It is not due to tert-butylperoxyl radicals because strand-scision is not even retarded by 10 mM Trolox, an outstanding water-soluble trap for peroxyl radicals. The present results are congruent with our earlier conclusion [Paul, T., et al. (2000) Biochemistry 39, 4129] that neutral alkylperoxyl radicals produce little or no direct single-strand-scission in SC DNAs.     

Menadione and cumene hydroperoxide induced cytotoxicity in biliary epithelial cells isolated from rat liver

Biliary epithelial cells (BEC) and parenchymal cells isolated from normal rat liver were exposed in vitro to a number of toxic compounds. BEC were found to be highly sensitive to concentrations of menadione (100 microM) and cumene hydroperoxide (10 microM) that are usually not effective as toxic agents in hepatocytes under normoxic conditions. On the other hand, BEC were not affected by concentrations of carbon tetrachloride or 7-ethoxycoumarin that are known to exert toxic effects on hepatocytes. The development of both menadione- and cumene hydroperoxide-induced toxic injury in BEC followed a common and time-correlated pattern, and included a strong depletion of GSH, depletion of protein thiols and an increase in the extent of cell death. The damage induced by cumene hydroperoxide was found to be independent of lipid peroxidative processes and was prevented by a pre-incubation with desferrioxamine. The cytotoxicity of menadione was further exacerbated by dicoumarol but was not prevented by desferrioxamine or promethazine. The mechanisms underlying BEC injury and death induced by the quinone and by the organic hydroperoxide are discussed in relation to the known biochemical characteristics of BEC.     

Evidence for a role of tert-butyl hydroxylation in the induction of pneumotoxicity in mice by butylated hydroxytoluene

Previous studies have shown that BHT must be biotransformed, probably to a quinone methide, in order to cause pneumotoxicity in mice. When BHT is incubated with mouse hepatic or pulmonary microsomes, a major metabolite that is formed is the tert-butyl-hydroxylated derivative of BHT (BHT-BuOH). Herein we show that BHT-BuOH has a fourfold greater potency than BHT in increasing the lung wt/body wt ratio, decreases lung cytosolic Ca2+-dependent protease activity at 1/10 the dose required for BHT to do this, and causes pulmonary histopathology at 1/20 the dose of BHT. Lung damage occurs earlier and is repaired faster at lower concentrations of BHT-BuOH than of BHT, but the nature of the damage (type I cell death) and regenerative response (type II cell hyperplasia and differentiation) is apparently identical. Neither BHT-BuOH nor BHT cause damage to liver, kidney, or heart as assessed by light microscopy, so they are both specific pulmonary toxicants. We postulate that BHT-BuOH formation is an essential step in the conversion of BHT to the ultimate pneumotoxin, which might be the corresponding quinone methide.     

Use of transgenic mice to study voltage-dependent Ca2+ channels

During the past decade a great number of genes encoding high- and low-voltage-dependent Ca(2+) channels and their accessory subunits have been cloned. Studies of Ca(2+) channel structure-function relationships and channel regulation using cDNA expression in heterologous expression systems have revealed intricate details of subunit interaction, regulation of channels by protein kinase A (PKA) and protein kinase C (PKC), drug binding sites, mechanisms of drug action, the ion conduction pathway and other aspects of channel function. In recent years, however, we have arrived at the brink of an entirely new strategy to study Ca(2+) channels by overexpressing or knocking out genes encoding these channels in transgenic mice. In this article, various models of gene knockout or gene overexpression will be discussed. This new approach will reveal many secrets regarding Ca(2+) channel regulation and the control of Ca(2+)-dependent cellular processes.     

Subacute oral and dermal toxicity of tert-butyl hydroperoxide in Fischer F344/N rats and B6C3F1 mice

Tert-butyl hydroperoxide (TBHP) is a catalyst frequently used in oxidation and sulfonation reactions in the plastics industry. Since the toxicological evaluation of TBHP remains unknown, the National Toxicology Program (NTP) designed studies to characterize and compare TBHP toxicity by the dermal and oral (gavage) routes in male and female Fischer 344 rats and B6C3F1 mice in 14-day exposures. Rats and mice were administered TBHP at 22, 44, 88, 176 or 352 mg/kg in 0.5% aqueous methylcellulose for the gavage studies. In the dermal studies, mice were administered the same doses as above, while rats were administered four doses (22, 44, 88, 176 mg/kg) in 50% aqueous acetone. Results from the gavage studies revealed treatment-related decreases in survival in male rats and body weights in both male and female rats in the 352 mg/kg group. Clinical signs included post-treatment lethargy, thinness, abnormal breathing, ruffled fur, and/or ataxia which occurred sporadically. The male mice showed a statistically significant decrease in body weight in the 44, 88, 176, and 352 mg/kg groups. The major target organs of toxicity were the forestomach in male and female rats and mice, and the esophagus in male and female rats and in male mice. In addition, there was an increase in the absolute and relative liver weight in female mice with hepatocellular hypertrophy in the top-dose group only. Results from spin trapping experiments revealed the presence of electron paramagnetic resonance signals from radical adducts in the blood and organic extracts of the liver and kidneys of rats treated by gavage with 176 mg/kg TBHP, suggesting the involvement of free- radical generation. The no observed adverse effect level (NOAEL) was considered to be 22 mg/kg in rats and male mice, and 44 mg/kg in female mice. In the dermal studies, there was no effect on survival, body weight, or organ weights in either rats or mice. TBHP administration at the site of application resulted in dermal irritation, hyperkeratosis, hyperplasia, and/or inflammation of the epidermis and inflammation of the dermis at 176 mg/kg and above in male and female rats. Dermal irritation at the site of application was noted in all the mice exposed to 352 mg/kg TBHP. Histopathological lesions in the epidermis and dermis were seen in the 88–352 mg/kg males and in the 176–352 mg/kg females. The NOAEL was found to be 88 mg/kg for male rats and female mice, and 44 mg/kg for female rats and male mice. In conclusion, these studies demonstrate that TBHP is metabolized to free radicals and is a contact irritant affecting skin by the dermal route of exposure, and forestomach and esophagus by oral administration. There was no evidence of systemic absorption by the dermal route of exposure based on lack of pathological findings (Supported by National Institute of Environmental Health Sciences Contract No. N01-ES-65406).     

Subacute oral and dermal toxicity of tert-butyl hydroperoxide in Fischer F344/N rats and B6C3F1 mice

Tert-butyl hydroperoxide (TBHP) is a catalyst frequently used in oxidation and sulfonation reactions in the plastics industry. Since the toxicological evaluation of TBHP remains unknown, the National Toxicology Program (NTP) designed studies to characterize and compare TBHP toxicity by the dermal and oral (gavage) routes in male and female Fischer 344 rats and B6C3F1 mice in 14-day exposures. Rats and mice were administered TBHP at 22, 44, 88, 176 or 352 mg/kg in 0.5% aqueous methylcellulose for the gavage studies. In the dermal studies, mice were administered the same doses as above, while rats were administered four doses (22, 44, 88, 176 mg/kg) in 50% aqueous acetone. Results from the gavage studies revealed treatment-related decreases in survival in male rats and body weights in both male and female rats in the 352 mg/kg group. Clinical signs included post-treatment lethargy, thinness, abnormal breathing, ruffled fur, and/or ataxia which occurred sporadically. The male mice showed a statistically significant decrease in body weight in the 44, 88, 176, and 352 mg/kg groups. The major target organs of toxicity were the forestomach in male and female rats and mice, and the esophagus in male and female rats and in male mice. In addition, there was an increase in the absolute and relative liver weight in female mice with hepatocellular hypertrophy in the top-dose group only. Results from spin trapping experiments revealed the presence of electron paramagnetic resonance signals from radical adducts in the blood and organic extracts of the liver and kidneys of rats treated by gavage with 176 mg/kg TBHP, suggesting the involvement of free- radical generation. The no observed adverse effect level (NOAEL) was considered to be 22 mg/kg in rats and male mice, and 44 mg/kg in female mice. In the dermal studies, there was no effect on survival, body weight, or organ weights in either rats or mice. TBHP administration at the site of application resulted in dermal irritation, hyperkeratosis, hyperplasia, and/or inflammation of the epidermis and inflammation of the dermis at 176 mg/kg and above in male and female rats. Dermal irritation at the site of application was noted in all the mice exposed to 352 mg/kg TBHP. Histopathological lesions in the epidermis and dermis were seen in the 88-352 mg/kg males and in the 176-352 mg/kg females. The NOAEL was found to be 88 mg/kg for male rats and female mice, and 44 mg/kg for female rats and male mice. In conclusion, these studies demonstrate that TBHP is metabolized to free radicals and is a contact irritant affecting skin by the dermal route of exposure, and forestomach and esophagus by oral administration. There was no evidence of systemic absorption by the dermal route of exposure based on lack of pathological findings (Supported by National Institute of Environmental Health Sciences Contract No. N01-ES-65406).     

Acute exposure to formaldehyde induces hepatic metallothionein synthesis in mice

Humans risk inadvertent intraperitoneal or intravenous exposure to formaldehyde (HCHO), commonly used for disinfection of implanted or extracorporeal medical devices. Various chemical and physical stresses are known to induce hepatic metallothionein. This study examined the effect of acute parenteral administration of HCHO on induction of hepatic metallothionein synthesis. Adult male CF1 mice were administered HCHO ip and hepatic metallothionein was quantified by the cadmium-radioassay method. HCHO (50 mg/kg) increased hepatic metallothionein as early as 8 hr after dosing with maximal levels (27-fold increase) occurring at 72 hr. Metallothionein concentrations were elevated (15-fold) 24 hr after 50 or 100 mg HCHO/kg but not at lower dosages. Concomitant elevations in hepatic zinc and copper content were observed. No increases in metallothionein were observed in kidney, pancreas, or intestine 24 hr after HCHO administration (100 mg/kg, ip). Induction of metallothionein by HCHO may reflect direct de novo synthesis since the response was abolished by pretreatment with the RNA synthesis inhibitor, actinomycin D. HCHO induction of metallothionein also does not appear to be mediated by stress-induced release of corticosteroids or catecholamines from the adrenal since the response was unaltered in adrenalectomized mice. Interference with the glutathione (GSH)-dependent oxidation of HCHO by reducing hepatic GSH concentrations to 40% of control after a 2-hr pretreatment with phorone decreased the metallothionein induction response to HCHO by 33%. This result suggests that the induction may be partially due to a HCHO metabolite, e.g., formate. Confirmation of metallothionein synthesis was obtained following spectral and chromatographic analysis. Thus, HCHO and/or a metabolite produces a marked increase in hepatic metallothionein and alters hepatic zinc and copper homeostasis, all of which are transient responses. Although HCHO was only mildly hepatotoxic at the highest dose (as evidenced by an increase in plasma alanine aminotransferase activity), such changes in metallothionein synthesis and essential metal homeostasis may be part of a cellular repair mechanism operant after acute toxic cell injury.     

Flavonoids suppress the cytotoxicity of linoleic acid hydroperoxide toward PC12 cells

The suppressive effect of flavonoids on the cytotoxicity of linoleic acid hydroperoxide (LOOH) toward rat phenochromocytoma PC12 cells was examined. The extent of cytotoxicity was shown on the basis of % survival determined by the trypan blue exclusion test. On preincubation of cells with either 3-hydroxyflavone, quercetin, or luteolin prior to LOOH exposure, the cytotoxicity was considerably suppressed. In contrast, on coincubation of cells with either eriodictyol, quercetin, kaempherol, luteolin, or 3-hydroxyflavone and LOOH, it was markedly suppressed. Regardless of incubation conditions, quercetin, 3-hydroxyflavone, and luteolin were thus more effective as protective agents against the cytotoxicity than the other flavonoids. These flavonoids further showed a suppressive effect on coincubation rather than on preincubation. These results suggest that such flavonoids are beneficial for cells under oxidative stress.     

Antioxidant property of metallothionein in fasted mice

Metallothionein (MT) is a low-molecular-weight and sulfur-rich protein that is induced by not only heavy metals but also physiological stresses such as fasting and restraint stresses. Although MT plays a role as a radical scavenger and a regulator of metabolism of metals, the biological function of MT induced by fasting stress has not been elucidated. In this study, we investigated the antioxidative role of MT in fasted mice. In fasted mice, the lipid peroxidation level of the liver was elevated by 24-h fasting stress, and pre-induction of hepatic MT by Zn diminished hepatic lipid peroxidation. Although 24-h fasting stress induced MT synthesis in the liver, other antioxidants such as catalase, manganese-superoxide dismutase (Mn-SOD), and glutathione peroxidase (GSHPx) were not activated in the liver. Moreover, the hepatic MT level was still elevated by fasting stress after seven cycles of repetition of alternate fasting and feeding every 24 h, but the activities of catalase, Mn-SOD and GSHPx were unchanged. These results indicate that MT induced by fasting stress plays partly as an antioxidant.