Early effects of surgery on zinc and metallothionein levels in female rats

Time-response effects of experimental surgery on zinc (Zn) and metallothionein (MT) homeostasis were investigated in female rats up to 24 h. Hepatic Zn content increased at 20 and 24 h postsurgery, whereas serum Zn levels decreased. Hepatic MT increased significantly by 9 h postsurgery and peaked at up to twofold of control at 12 h after surgery. Following the peak at 12 h, hepatic MT content decreased with time but did not reach control levels at the end of this study. When MT isoforms were evaluated, MT-II levels were elevated to the highest extent by 12 h after surgery, whereas MT-I levels started to decrease after 3 h postsurgery but then increased by 20 h. The early increases in MT content are probably mediated by nonmetallic mediators released during the postsurgical inflammatory process, favoring the plasma/tissue mobilization of Zn. This process might be part of the overall mechanisms occurring in the inflammation.     

alpha-Tocopherol protects against alpha-naphthylisothiocyanate-induced hepatotoxicity in rats less effectively than melatonin

The protective effect of alpha-tocopherol (alpha-Toc), which exerts antioxidant and anti-inflammatory actions, against alpha-naphthylisothiocyanate (ANIT)-induced hepatotoxicity in rats was compared with that of melatonin because orally administered melatonin is known to protect against ANIT-induced hepatotoxicity in rats through its antioxidant and anti-inflammatory actions. Rats intoxicated once with ANIT (75 mg/kg, intraperitoneal (i.p.)) showed liver cell damage and biliary cell damage with cholestasis at 24 h, but not 12 h, after intoxication. ANIT-intoxicated rats received alpha-Toc (100 or 250 mg/kg) or melatonin (100 mg/kg) orally at 12 h after intoxication. The alpha-Toc administration protected against liver cell damage in ANIT-intoxicated rats, while the melatonin administration protected against both liver cell damage and biliary cell damage with cholestasis. ANIT-intoxicated rats had increased hepatic lipid peroxide concentration and myeloperoxidase activity at 12 and 24 h after intoxication. ANIT-intoxicated rats also had increased serum alpha-Toc and non-esterified fatty acid (NEFA) concentrations at 12 and 24 h after intoxication and increased serum triglyceride and total cholesterol concentrations at 24h. The administration of alpha-Toc to ANIT-intoxicated rats increased the hepatic alpha-Toc concentration with further increase in the serum alpha-Toc concentration and attenuated the increased hepatic lipid peroxide concentration and myeloperoxidase activity and serum NEFA concentration at 24 h after intoxication. The melatonin administration did not affect the hepatic alpha-Toc concentration but attenuated the increased hepatic lipid peroxide concentration and myeloperoxidase activity and serum alpha-Toc, NEFA, triglyceride, and total cholesterol concentrations at 24 h after ANIT intoxication. These results indicate that orally administered alpha-Toc protects against ANIT-induced hepatotoxicity in rats possibly through its antioxidant and anti-inflammatory actions less effectively than orally administered melatonin.     

Changes in plasma zinc,copper, iron,and hepatic metallothionein in adjuvant-induced arthritis treated with cyclosporin

The early changes in hepatic metallothionein (MT) and plasma zinc (Zn), copper (Cu), and iron (Fe) were investigated during the induction of adjuvant (AJ) arthritis in rats in conjunction with cyclosporin (CSA) treatment. Plasma Zn decreased after AJ injection (60% of control values at 8 h), and this was associated with a 4.5-fold increase in hepatic MT at 8 h. Plasma Zn was lowest at 16 h (40% of control), whereas hepatic MT concentrations increased to a maximum of 20-fold at 16 h. Changes in plasma Fe paralleled those of Zn, whereas plasma Cu levels were increased. Plasma metal and hepatic MT concentrations returned toward normal from d 1–7. At d 14, when marked paw swelling was apparent, hepatic MT and plasma Cu were again increased and plasma Zn decreased. Administration of CsA decreased MT induction in rats injected with AJ and also caused a marked recovery in plasma Zn and Fe levels. These changes were small but significant even in the early stages (up to 24 h) after AJ injection and were followed by a sustained improvement in all parameters, corresponding to the nonappearance of clinical arthropathy in CsA-treated rats. TNF-α and IL-6 production by peritoneal macrophages isolated from AJ-injected rats was significantly decreased by CsA treatment at d 7 and 14. The inhibition of hepatic MT induction during acute and chronic inflammation by cyclosporin emphasizes the role of the immune system in altered metal homeostasis in inflammation.     

Sepsis-associated cholestasis is critically dependent on P-selectin-dependent leukocyte recruitment in mice

Cholestasis is a major complication in sepsis although the underlying mechanisms remain elusive. The aim of this study was to evaluate the role of P-selectin and leukocyte recruitment in endotoxemia-associated cholestasis. C57BL/6 mice were challenged intraperitoneally with endotoxin (0.4 mg/kg), and 6 h later the common bile duct was cannulated for determination of bile flow and biliary excretion of bromosulfophthalein. Mice were pretreated with an anti-P-selectin antibody or an isotype-matched control antibody. Leukocyte infiltration was determined by measuring hepatic levels of myeloperoxidase. Tumor necrosis factor-alpha and CXC chemokines in the liver was determined by ELISA. Liver damage was monitored by measuring serum levels of alanine aminotransferase and aspartate aminotransferase. Apoptosis was quantified morphologically by nuclear condensation and fragmentation using Hoechst 33342 staining. Endotoxin induced a significant inflammatory response with increased TNF-alpha and CXC chemokine concentrations, leukocyte infiltration, liver enzyme release, and apoptotic cell death. This response was associated with pronounced cholestasis indicated by a >70% decrease of bile flow and biliary excretion of bromosulfophthalein. Immunoneutralization of P-selectin significantly attenuated endotoxin-induced leukocyte infiltration reflected by a >60% reduction of hepatic myeloperoxidase levels. Interference with P-selectin decreased endotoxin-mediated hepatocellular apoptosis and necrosis, but did not affect hepatic levels of tumor necrosis factor-alpha and CXC chemokines. Of interest, inhibition of P-selectin restored bile flow and biliary excretion of bromosulfophthalein to normal levels in endotoxin-challenged animals. Our study demonstrates for the first time that P-selectin-mediated recruitment of leukocytes, but not the local production of proinflammatory mediators, is the primary cause of cholestasis in septic liver injury.     

Activation of glycolysis by zinc is diminished in hepatocytes from metallothionein-null mice

The influence of hepatic metallothionein (MT) and zinc (Zn) on glycolysis was investigated in primary cultures of mouse hepatocytes prepared from MT-normal (+/+) and MT-null (−/−) mice. In MT +/+ mice, a close relationship was observed between the Zn concentration in the incubation medium (10–150 μM), increased MT levels in the cells, and increased glycolysis (accumulation of lactate + pyruvate) over 24 h, with significant effects seen at physiological levels of Zn (10–25 μM). Hepatocytes from MT −/− mice had significantly lower basal rates of glycolysis and demonstrated increased glycolysis only at Zn concentrations of 50 μM or greater. The lactate: pyruvate ratio was higher in the MT +/+ hepatocytes. The oxidation of endogenous fatty acid (accumulation of the ketone bodies, 3-hydroxybutyrate and acetoacetate) was initially greater in the MT +/+ hepatocytes, although only MT −/− hepatocytes showed increased ketone body production in response to Zn. The 3-hydroxybutyrate: acetoacetate ratio was higher in the MT +/+ hepatocytes and increased with increasing Zn concentrations. Intracellular Zn accumulation was 60% greater in the MT +/+ hepatocytes, with approximately 80% of the extra Zn associated with MT. The results implicate MT-associated Zn rather than increased intracellular Zn per se in the regulation of hepatic carbohydrate metabolism.     

Interaction of metallothionein and carbon tetrachloride on the protective effect of zinc on hepatotoxicity

To study the influence of hepatic metallothionein (MT) on the hepatotoxic response to carbon tetrachloride (CCl4), adult male rats were pretreated with a 10 mg X kg-1 dose of zinc (Zn) 24 h prior to CCl4 (i.p., l mL X kg-1) treatment. Zn pretreatment increased the hepatic MT concentrations markedly and reduced the magnitudes of the CCl4-induced reduction of cytochrome P450 concentration as well as elevation of serum alanine aminotransferase and aspartate aminotransferase activities when determined at 4 or 24 h following CCl4 treatment. Treatment of Zn-exposed animals with CCl4 also resulted in significant reduction of the concentrations of hepatic MT (as determined by the cadmium-saturation method) as well as cytosolic Zn. Sephadex G-75 chromatographic study of hepatic cytosols showed that MT-bound Zn was selectively depleted by CCl4 exposure. Moreover, it was demonstrated that CCl4, after metabolic activation, reduced the cadmium binding capacity of Zn-induced hepatic MT in vitro. To examine the possible protective effect of Zn independent of induction of MT synthesis, CCl4 was administered 2 h following Zn pretreatment and the hepatotoxic response was examined 4 h later. This study revealed limited protection by Zn prior to the induction of MT synthesis. These data further support a role of MT in the modulation of CCl4 hepatotoxicity.     

Effect of gamma irradiation on liver metallothionein synthesis and lipid peroxidation in rats.

Several studies have recently shown that metallothionein (MT), a protein characterized by a high thiol content and that binds Zn2+ and Cu+, might be involved in the protection against oxidative stress and can act as a free radical scavenger. Oxidative stresses, such as irradiation, increase lipid peroxidation (LP) and subsequent tissue damage through free radical production. The induction of hepatic MT synthesis by gamma-irradiation (20 Gy) at 8, 24, 30 and 48 hrs. post-irradiation in two different age groups of Sprague-Dawley rats (39-40 and 48-49 days old) was studied. LP measured by the thiobarbituric acid reactive substances (TBARS) assay and Cu and Zn levels in liver have also been determined. In the younger group, the gamma-irradiation induced hepatic MT synthesis and increased LP that peaked 24 hrs. after irradiation. During the first 30 hrs. post-irradiation, a positive and statistically significant correlation between hepatic MT content and LP level in liver was found. In the older group, liver MT synthesis was only increased 1.7-fold and LP levels were not altered at 24 hrs. post-irradiation compared with sham-irradiated rats.Therefore it appears that LP is not necessary for induction of MT synthesis by gamma-irradiation.     

Cell proliferation and hepatocarcinogenesis in rat initiated by diethylnitrosamine and promoted by phenobarbital: potential roles of early DNA damage and liver metallothionein expression

Cell proliferation plays an important role in multistage chemical carcinogenesis. Again, several reports demonstrated that upregulation of metallothionein (MT) expression is associated with increased cell proliferation that may contribute to the pathogenesis of preneoplastic phenotype to frank malignancy. In this study, we evaluated the roles of early DNA damage, altered expressions of liver MT and Ki-67 nuclear antigen, and altered hepatic levels of zinc (Zn) and copper (Cu) on cell proliferation and the progression of hepatocarcinogenesis through premalignant, late premalignant and malignant transformation phases in male Sprague-Dawley rats. We have further studied the association between MT expression and cell proliferation in hepatocarcinogenesis. There was substantial induction of DNA single-strand breaks (SSBs) (P < 0.001) and development of hepatocellular premalignant lesions along with significant decrease in hepatic levels of Zn and increase in Cu content following a single, necrogenic, intraperitoneal (i.p.) injection (200 mg/Kg body weight) of diethylnitrosamine (DEN) at week 4 of the experimental protocol. Moreover, DEN + phenobarbital (PB)-treatment significantly elevated MT-, Ki-67-, and BrdU-immunoexpressions along with their immunolabeling indices. Furthermore, positive correlations between MT- and Ki-67- labeling (P = 0.0006) at various time intervals, as well as, between MT immunoreactivity and 5’-bromo-2’-deoxyuridine-labeling index (BrdU-LI) (P = 0.0007) indicate that, MT expression might be associated with Ki-67 expression and cell proliferation thereby. The study suggests that DEN treatment may lead to alteration of Zn and Cu levels resulting in early DNA damage along with elevation of MT expression that may ultimately lead to hepatic cell proliferation. The results thus provide evidence in support of the role of MT as a potential positive regulator of cell growth during the early stages of hepatocellular transformation in rats.     

Zinc supplementation partially prevents renal pathological changes in diabetic rats

We have demonstrated that Zn supplementation mediated up-regulation of cardiac metallothionein (MT) as a potent antioxidant prevented the development of diabetic cardiomyopathy. The present study was undertaken to test whether induction of renal MT synthesis by Zn supplementation protects the kidney from diabetes-induced damage. Streptozotocin-induced diabetic rats were treated with and without Zn supplementation at 5 mg/kg in drinking water for 3 months. Diabetic renal damage was detected by examining renal pathological alterations and 24-h urinary protein levels. Three-month Zn supplementation immediately after the onset of diabetes, partially but significantly, prevented the kidney from diabetes-induced increases in 24-h urinary proteins and pathological alterations. Diabetes-induced renal oxidative damage, inflammation and up-regulated expression of profibrosis mediator connective tissue growth factor (CTGF) were also markedly attenuated by Zn supplementation, along with significant increases in Zn levels concomitant with MT expression in renal tubular cells. Direct exposure of renal tubular (HK11) cells to high levels of glucose (HG) induced CTGF up-regulation predominantly through ERK (extracellular signal-regulated kinase)1/2-dependent, and partially through p38 mitogen-activated protein kinase (MAPK)-dependent pathways. Pretreatment of HK11 cells with Zn or cadmium induced MT expression and also significantly suppressed HG-induced CTGF expression. These results provide the first evidence for Zn supplementation to attenuate diabetes-induced renal pathological changes, likely through prevention of hyperglycemia-induced CTGF expression by Zn-induced MT in renal tubular cells.     

Selenium, iron, copper, and zinc levels and copper-to-zinc ratios in serum of patients at different stages of viral hepatic diseases

Viral hepatic diseases, especially those induced by the hepatitis B virus, can progress into more serious pathological outcomes and eventually to hepatocellular carcinoma. A growing body of evidence indicates that many trace elements play important roles in a number of carcinogenic processes that proceed through various mechanisms. To examine the status of trace elements during the development of hepatic carcinoma, we determined the selenium, iron, copper, and zinc levels and copper-to-zinc ratios in the serum of patients at different stages of viral hepatic disease. We observed significant changes in the selenium, iron copper, and zinc levels in the serum of patients having hepatocellular carcinoma, relative to those of healthy controls (p<0.05). The mean serum copper level in patients with hepatocellular carcinoma was significantly higher than that of the control group. In contrast, the mean selenium, iron, and zinc levels in patients having hepatocellular carcinoma were significantly lower than those of the control group. In addition, the mean zinc level in the serum of patients with hepatic cirrhosis was significantly lower than that of the control group (p<0.05). Moreover, we found markedly elevated Cu: Zn ratios (p<0.05) in patients having hepatic cirrhosis or hepatocellular carcinoma. Our findings imply that the levels of some trace elements, such as selenium, iron, copper, and zinc, and Cu:Zn ratios, might serve as biomarkers for the increased severity of viral hepatic damage.     

Inducibility and amounts of metallothionein as influenced by cadmium and zinc in the developing rat

Information on the accumulation and/or depletion of Zn in metallothionein (MT) of rat fetus, rat pup, and maternal rat liver at various ages was obtained with pregnant rats fed a basal casein diet or this diet plus either 100 ppm Zn or 50 ppm Cd. Rats fed each of the respective diets were sacrificed on 12, 16, and 20 d of gestation and 0, 7, 14, and 28 d post-partum. No Cd was detected in the placenta or fetal tissue and the Cd did not affect the accumulation of Zn in the fetal MT, but it did increase the Zn content in liver MT of the dams. Very little Zn in MT was found on day 12 of gestation, but Zn rapidly increased in MT to a maximum at time of birth. The accumulation of Zn in MT was independent of the diet for the fetuses, but the Zn accumulation in the dam and pup tissues was diet dependent. In order to study age-dependent difference in the inducibility of MT, newborn, 5-week-old, or 24-week-old rats were injected with zinc at the levels of 0, 3, 6, or 9 mg/kg and 5 h later injected with³⁵S-cystine. In rats sacrificed 1 h later, the amount of radioactivity in liver MT demonstrated that this protein in older animals was more readily induced by Zn than in younger animals.     

Effects of Kupffer cell blockade on the hepatic expression of metallothionein and heme oxygenase genes in endotoxemic rats with obstructive jaundice

AimsHeme oxygenase (HO) and metallothionein (MT) genes are rapidly upregulated in the liver by pro-inflammatory cytokines and/or endotoxin as protection against cellular stress and inflammation. Gadolinium chloride (GdCl₃)-induced Kupffer cell blockade has beneficial consequences in endotoxemia following bile duct ligation. Herein we further characterized the effects of Kupffer cell inhibition on the activation of the antioxidant defense system (HO and MT gene expressions, and antioxidant enzyme activities) in response to endotoxemia and obstructive jaundice.Main methodsThe isoform-specific expression of MT and HO genes was assessed (RT-PCR) in rat livers following 3-day bile duct ligation, 2-h lipopolysaccharide treatment (1 mg/kg) or their combination, with or without GdCl₃ pretreatment (10 mg/kg, 24 h before endotoxin). Lipid peroxidation, DNA damage and hepatic antioxidant enzyme activities were also assessed.Key findingsAll these challenges induced similar extents of DNA damage, whereas the lipid peroxidation increased only when endotoxemia was combined with biliary obstruction. The MT and HO mRNA levels displayed isoform-specific changes: those of MT-1 and HO-2 did not change appreciably, whereas those of MT-2 and HO-1 increased significantly in 2-h endotoxemia, with or without obstructive jaundice. Among the enzymes reflecting the endogenous protective mechanisms, the catalase and copper/zinc-superoxide dismutase levels decreased, while that of Mn-SOD slightly increased. Interestingly, GdCl₃ alone induced lipid peroxidation, DNA damage and MT-2 expression. In response to GdCl₃, HO-1 induction was significantly lower in each model.SignificanceDespite its moderate hepatocellular toxicity, the ameliorated stress-induced hepatic reactions provided by GdCl₃ may contribute to its protective effects.     

Hepatic zinc response via metallothionein induction after tumor transplantation

Based on previous findings that liver zinc and metallothionein (MT) levels increase after tumor transplantation, zinc metabolism in tumor-bearing mice was studied to clarify the role of zinc-MT in host defense systems. Zinc in the hepatic cytosolic MT fraction did not increase in tumor-bearing mice fed a zinc-deficient diet, suggesting that dietary zinc is necessary for apo-MT induction in the liver after tumor transplantation and is then incorporated into the apo-MT. When (65)ZnCl(2) was intravenously injected, liver (65)Zn levels in the tumor-bearing mice were higher than those in control mice for 72 h after the injection. Pancreatic and blood (65)Zn levels in tumor-bearing mice were lower than those in controls for 24 h (pancreas) and 6 h (blood) after the injection. These findings indicate that the hepatic zinc response via MT induction influences zinc metabolism in the body after tumor transplantation. Moreover, (65)Zn uptake in the liver of MT-deficient tumor-bearing mice was lower than that in control tumor-bearing mice 1 h after injection. (65)Zn uptake in the tumor and blood (65)Zn levels in the MT-deficient tumor-bearing mice were higher than those in the control tumor-bearing mice. Tumor weight increased more in MT-deficient mice than in control mice. The formation of zinc-MT in the liver of tumor-bearing mice might decrease blood zinc availability for tumors and other tissues, such as the pancreas.     

Involvement of oxidative stress in the impairment in biliary secretory function induced by intraperitoneal administration of aluminum to rats

We have shown that aluminum (Al) induces cholestasis associated with multiple alterations in hepatocellular transporters involved in bile secretory function, like Mrp2. This work aims to investigate whether these harmful effects are mediated by the oxidative stress caused by the metal. For this purpose, the capability of the antioxidant agent, vitamin E, to counteract these alterations was studied in male Wistar rats. Aluminum hydroxide (or saline in controls) was administered ip (27 mg/kg body weight, three times a week, for 90 d). Vitamin E (600 mg/kg body weight) was coadministered, sc. Al increased lipid peroxidation (+50%) and decreased hepatic glutation levels (-43%) and the activity of glutation peroxidase (-50%) and catalase (-88%). Vitamin E counteracted these effects total or partially. Both plasma and hepatic Al levels reached at the end of the treatment were significantly reduced by vitamin E (-40% and -44%, respectively;p< 0.05). Al increased 4 times the hepatic apoptotic index, and this effect was fully counteracted by vitamin E. Bile flow was decreased in Al-treated rats (-37%) and restored to normality by vitamin E. The antioxidant normalized the hepatic handling of the Mrp2 substrates, rose bengal, and dinitrophenyl-S-glutathione, which was causally associated with restoration of Mrp2 expression. Our data indicate that oxidative stress has a crucial role in cholestasis, apoptotic/necrotic hepatocellular damage, and the impairment in liver transport function induced by Al and that vitamin E counteracts these harmful effects not only by preventing free-radical formation but also by favoring Al disposal.     

Laboratory assessment of hepatic injury in the woodchuck (Marmota monax)

Normal reference values for total serum protein, albumin, cholesterol, alanine aminotransferase (ALT), aspartate aminotransferase (AST), sorbitol dehydrogenase (SDH), gamma glutamyl transferase (GGT), alkaline phosphatase (AP), and total bilirubin were established in 48 clinically healthy woodchucks. To validate the use of these biochemical tests in the woodchuck for assessment of liver injury, carbon tetrachloride was administered to produce hepatocellular necrosis and the common bile duct was surgically occluded to produce cholestasis. Biochemical tests were performed prior to experimental treatment and thereafter in surviving woodchucks for a period of 6 weeks. There were marked increases in the serum activities of AST, ALT, and SDH following carbon tetrachloride administration and all 3 enzymes appeared to be useful markers of acute hepatocellular injury. The predominate biochemical abnormalities in woodchucks with bile duct obstruction were hyperbilirubinemia, hypercholesterolemia and increased serum AP and GGT activities. The increase of GGT occurred earlier following bile duct obstruction and the magnitude of increase was greater than that of AP, suggesting that GGT would be the preferred serum enzyme test in the woodchuck for assessment of cholestatic liver injury.     

Elevation of hepatic levels of metallothionein and zinc in mice bearing experimental tumors.

A tumor growth-dependent elevation in the hepatic levels of Zn and metallothionein (MT), without a change in the level of Cu, was found in mice and rats bearing solid tumors in the inguinal region. The levels of Zn and MT thus elevated gave a significant correlation (r = 0.95) between them. Nevertheless, when tumor-bearing mice and rats were fed a Zn-deficient diet, the hepatic levels of Zn and MT did not increase. In mice in which inflammation was induced at the same region, on the other hand, hepatic levels of Zn and MT increased transiently after the injection of turpentine or carrageenan even when they were fed the Zn-deficient diet. These results suggest that the elevation of MT and Zn levels can be a helpful marker for detecting malignancy.     

Induction of Metallothionein in Rat Liver by Zinc Exposure: A Dose and Time Dependent Study

Metallothioneins (MTs) are low molecular weight ubiquitous metalloproteins with high cysteine (thiol) content. The intracellular concentration of zinc (Zn) is tightly regulated and MT plays a crucial role in it. The present study investigates the relationship between the Zn status (as a function of Zn concentration and time) in the rat liver and the occurrence of hepatic MT. For dose dependent study, four experimental groups, one control and three receiving different levels of metal supplementation, were chosen [Group 1 control and Group 2, Group 3, Group 4 receiving subcutaneous dose of 10, 50 and 100 mg of Zn/kg body weight (in the form of ZnSO₄·7H₂O), respectively]. For the time dependent expression of MT, again four experimental groups, i.e. Group 5 control and Group 6, Group 7, Group 8 receiving 50 mg of Zn/kg body weight (in the form of ZnSO₄·7H₂O) subcutaneously and sacrificed at different time intervals after last injection i.e. 6, 18, 48 h, respectively were chosen. Isolation of MT was done by using combination of gel filtration and ion exchange chromatography while characterization of MT fraction was carried in the wavelength range 200–400 nm. Expression of MT was studied by using Western blot analysis. The results revealed that the MT expression increases with increasing the dose of Zn administered and maximum at 18 h after last Zn injection. Accumulation of MT with increase dose would help in maintaining the intracellular Zn concentration by its sequestration which further reduces the possibility of undesirable binding of Zn to other proteins significantly and maintains Zn homeostasis. The maximum expression of MT at 18 h is indicative of its half life.     

Induction of metallothionein in the livers of female Sprague-Dawley rats treated with 2,3,7 ,8-tetrachlorodibenzo-p-dioxin

Metallothioneins (MTs) are cysteine-rich metal-binding proteins that exert cytoprotective effects against metal toxicity and external stimuli including ionizing or ultraviolet B irradiation. Since 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is known to cause an exaggerated oxidative stress response in animals and in different organs, we have studied possible involvement of MT in the oxidative responses induced by TCDD. Female Sprague-Dawley (SD) rats (6-week old) were administered a single oral dose of TCDD that varied from 1.0 to 4.0 microg/kg body weight. The serum and tissues were collected 7 days after dosing. Indicators of oxidative damage were assessed. Significant increases in serum 8-hydroxydeoxyguanosine (8-OHdG) levels were observed in the rats dosed with 2.0 and 4.0 microg TCDD/kg bw. Only 4.0 microg TCDD/kg bw produced a decrease in reduced glutathione concentration in the liver. Immunohistochemical staining revealed a TCDD-induced increase in heme oxygenase-1 (HO-1) expression in the hepatic macrophages (Kupffer cells). Under these conditions, MT protein as well as the mRNAs of MT-I and MT-II, were dose-dependently induced in the liver by TCDD doses from 1.0 microg/kg bw. TCDD-induced MT was found to localize in the parenchymal cells of the liver. Serum concentrations of cytokines (TNF-alpha, IL-1beta and IL-6) were not affected by TCDD. The hepatic concentrations of Cu, Zn and Fe were all increased significantly by TCDD administration. Our results suggest that MT levels are increased in the liver upon exposure to TCDD, perhaps by TCDD-generated reactive oxygen species, and that it may play a protective role in TCDD-induced oxidative stress responses as an antioxidant.