metallothionein and Carbon-Tetrachloride-Poisoning

Many Chinese medicines have the potential to be hepatoprotective and therefore can be used to treat acute and chronic liver diseases. The challenge is to identify the molecular target for their protective mechanism. This study investigated the induction of nuclear factor-erythroid 2(NF-E2)-related factor 2 (Nrf2) antioxidant genes and metallothionein as a common mechanism of hepatoprotective effects of Chinese medicines such as Piper puberulum. Mice were pretreated with Piper puberulum extract (PPE, 500 mg/kg, po) or vehicles for seven days, followed by intoxication with CCl 4 (25 μl/kg, ip for 16 h), D-galactosamine (800 mg/kg, ip for 8 h), or acetaminophen (400 mg/kg, ip for 8 h). Hepatotoprotection was evaluated by serum enzyme activities and histopathology. To determine the mechanism of protection, mice were given PPE (250-1000 mg/kg, po for seven days) and livers were collected to quantify the expression of Nrf2-targeted genes and metallothionein. Nrf2-null mice were also used to determine the role of Nrf2 in PPE-mediated hepatoprotection.PPE pretreatment protected against the hepatotoxicity produced by CCl 4, D-galactosamine, and acetaminophen, as evidenced by decreased serum enzyme activities and ameliorated liver lesions. PPE treatment increased the expression of hepatic Nrf2, NAD(P)H:quinone oxidoreductase1 (Nqo1), heme oxygenase-1 (Ho-1), glutamate-cysteine ligases (Gclc), and metallothionein (MT), at both transcripts and protein levels. PPE protected wild-type mice from CCl 4 and acetaminophen hepatotoxicity, but not Nrf2-null mice, fortifying the Nrf2-dependent protection. In conclusion, induction of the Nrf2 antioxidant pathways and metallothionein appears to be a common mechanism for hepatoprotective herbs such as PPE.

Topics: Acetaminophen; Animals; Antioxidants; Carbon Tetrachloride Poisoning; Chemical and Drug Induced Liver Injury; Galactosamine; Glutamate-Cysteine Ligase; Heme Oxygenase-1; Liver; Male; Metallothionein; Mice; Mice, Inbred Strains; Molecular Targeted Therapy; NAD(P)H Dehydrogenase (Quinone); NADP; NF-E2-Related Factor 2; Oxidative Stress; Phytotherapy; Piper; Plant Extracts

The aim of this study was to investigate the hepatoprotective effects of anthocyanidin delphinidin in carbon tetrachloride (CCl(4))-induced liver fibrosis in mice. Male Balb/C mice were treated with CCl(4) dissolved in olive oil (20%, v/v, 2mL/kg) intraperitoneally (i.p.), twice a week for 7 weeks. Delphinidin was administered i.p. once daily for next 2 weeks, in doses of 10 and 25mg/kg of body weight. The CCl(4) control group has been observed for spontaneous reversion of fibrosis. CCl(4)-administration induced an elevation in serum transaminase and alkaline phosphatase levels and increased oxidative stress in the liver. Delphinidin has successfully attenuated oxidative stress, increased matrix metalloproteinase-9 and metallothionein I/II expression and restored hepatic architecture. Furthermore, the overexpression of tumor necrosis factor-alpha and transforming growth factor-beta1 has been withdrawn by delphinidin. Concomitantly, the expression of alpha-smooth muscle actin indicated returning of hepatic stellate cells (HSC) into inactive state. Our results suggest the therapeutic effects of delphinidin in CCl(4)-induced liver fibrosis by promoting extracellular matrix degradation, HSC inactivation and down-regulation of fibrogenic stimuli, with strong enhancement of hepatic regenerative capability.

Topics: Animals; Anthocyanins; Carbon Tetrachloride; Carbon Tetrachloride Poisoning; Down-Regulation; Drug-Related Side Effects and Adverse Reactions; Fibrosis; Hepatic Stellate Cells; Liver; Liver Cirrhosis; Male; Matrix Metalloproteinase 9; Metallothionein; Mice; Mice, Inbred BALB C; Olive Oil; Plant Oils; Transforming Growth Factor beta1; Tumor Necrosis Factor-alpha

Hepatic fibrosis is effusive wound healing process in which excessive connective tissue builds up in the liver. Because specific treatments to stop progressive fibrosis of the liver are not available, we have investigated the effects of luteolin on carbon tetrachloride (CCl(4))-induced hepatic fibrosis. Male Balb/C mice were treated with CCl(4) (0.4 ml/kg) intraperitoneally (i.p.), twice a week for 6 weeks. Luteolin was administered i.p. once daily for next 2 weeks, in doses of 10, 25, and 50 mg/kg of body weight. The CCl(4) control group has been observed for spontaneous reversion of fibrosis. CCl(4)-intoxication increased serum aminotransferase and alkaline phosphatase levels and disturbed hepatic antioxidative status. Most of these parameters were spontaneously normalized in the CCl(4) control group, although the progression of liver fibrosis was observed histologically. Luteolin treatment has increased hepatic matrix metalloproteinase-9 levels and metallothionein (MT) I/II expression, eliminated fibrinous deposits and restored architecture of the liver in a dose-dependent manner. Concomitantly, the expression of glial fibrillary acidic protein and alpha-smooth muscle actin indicated deactivation of hepatic stellate cells. Our results suggest the therapeutic effects of luteolin on CCl(4)-induced liver fibrosis by promoting extracellular matrix degradation in the fibrotic liver tissue and the strong enhancement of hepatic regenerative capability, with MTs as a critical mediator of liver regeneration.

Topics: Actins; Animals; Carbon Tetrachloride Poisoning; Chemical and Drug Induced Liver Injury; Copper; Expectorants; Glial Fibrillary Acidic Protein; Glutathione; Hydroxyproline; Immunohistochemistry; Liver; Liver Cirrhosis; Luteolin; Male; Matrix Metalloproteinases; Metallothionein; Mice; Mice, Inbred BALB C; Superoxide Dismutase; Vitamin A; Zinc

The aim of this study was to investigate the protective effect of luteolin on liver Ca, Mg, Zn, Cu, Fe, and Mn content in mice with carbon tetrachloride (CCl4)-induced hepatotoxicity. Additionally, liver metallothionein (MT) expression was studied. Luteolin was administered intraperitoneally (i.p.) as a single 5- or 50-mg/kg dose or once daily for two consecutive days, respectively. Two hours after the last injection, the mice were treated with CCl4 (20 mg/kg, i.p.). CCl4 injection reduced hepatic level of all metals except Ca, with an intense cytoplasmic staining pattern in hepatocytes located in periportal areas, indicating induction of MTs. Pretreatment with 50 mg/kg of luteolin for 2 days remarkably elevated metal content to control values (Mg and Cu) or even above them (Zn and Fe). Luteolin pretreatment increased pericentral MTs immunopositivity and histological architecture improvement in a time- and dose-dependent manner, being the most prominent in mice pretreated with 50 mg/kg for 2 days. The liver in this group showed pronounced MT expression in almost all hepatocytes throughout the liver parenchyma. In conclusion, these results suggest the protective effect of luteolin on CCl4-induced hepatotoxicity and an enhancement of hepatocyte proliferative capabilities.

Topics: Animals; Carbon Tetrachloride Poisoning; Copper; Immunohistochemistry; Iron; Liver; Luteolin; Magnesium; Male; Manganese; Metallothionein; Metals; Mice; Mice, Inbred BALB C; Zinc

Metallothioneins (MTs), are low molecular weight proteins, mainly implicated in metal ion detoxification. In the present study, we investigated the expression of hepatic MT in a rat model of injury and regeneration, induced by carbon tetrachloride (CCl(4)) administration. A single intraperitoneal injection of 1 ml CCl(4)/kg body weight was performed in male Wistar rats, killed at different time points post-administration. The enzymatic activities of aspartate and alanine aminotransferases in serum were determined, in addition to the liver histological findings, to estimate hepatotoxicity. The rate of tritiated thymidine incorporation into hepatic DNA, the enzymatic activity of thymidine kinase in liver tissue and the assessment of the mitotic index in hepatocytes, were used as indices of regeneration. MT was detected immunohistochemically in liver tissue sections. CCl(4) administration caused severe hepatic injury, followed by regeneration. MT expression became prominent as early as 12 h after the administration of CCl(4), in the nuclei of hepatocytes, while at 24 and 36 h intense cytoplasmic staining for MT appeared in the hepatocytes in the vicinity of necrotic areas. The peak of hepatocyte proliferative capacity, occurring at 48 h post-CCl(4) administration coincides with the maximum nuclear and cytoplasmic MT expression. At further time points MT expression presented a decreasing trend. Induction of MT expression was observed in the liver after a single administration of CCl(4), being more prominent at the time of maximum hepatocellular proliferation, participating actively in the replication of hepatocytes.

Topics: Animals; Carbon Tetrachloride Poisoning; DNA; Immunohistochemistry; Injections, Intraperitoneal; Liver; Liver Regeneration; Male; Metallothionein; Rats; Rats, Wistar; Thymidine Kinase

Although recent studies have shown that various stress can induce metallothionein (MT) synthesis in animal tissues, the induction of MT synthesis by exposure to static magnetic fields (SMF) has not been reported. We measured MT levels in the liver, kidney and brain of mice exposed to SMF and also evaluated the effect of SMF exposure on the induction of hepatic MT by treatment with carbon tetrachloride (CCl4). The MT content in the liver was significantly increased by exposure to 4.7 T of SMF for 6, 24, or 48 h, whereas that in the kidney or brain was not changed compared to the control. The combination of CCl4 injection and SMF exposure induced elevation of the hepatic MT content exceeding that induced by either treatment alone. These results indicate that exposure to the strong SMF induces MT synthesis in the liver in mice and enhances the hepatic MT synthesis induced by CCl4 administration.

Topics: Animals; Brain Chemistry; Carbon Tetrachloride Poisoning; Electromagnetic Fields; Enzyme Induction; Kidney; Liver; Male; Metallothionein; Mice; Mice, Inbred BALB C; Rats

Inflammation, induced by turpentine (0.1 ml i.m.), protected against carbon tetrachloride (CCl4)-induced hepatotoxicity based on serum activities of sorbitol dehydrogenase. Inflammation was confirmed by elevated serum ceruloplasmin activities, and was associated with high hepatic levels of metallothionein, a zinc protein proposed to protect against CCl4-induced injury. Inflammation suppressed cytochrome P-450 activities, but this was not associated with protection against CCl4-promoted liver microsomal injury as assessed by glucose-6-phosphatase activity loss. Thus, protection against plasma membrane injury did not result primarily from depressed microsomal activation of CCl4. Each effect of inflammation reported here resembled effects of zinc injections. This similarity strengthens the hypothesis that metallothionein protects against CCl4-induced hepatic plasma membrane injury.

Topics: Animals; Carbon Tetrachloride Poisoning; Ceruloplasmin; Cytochrome P-450 Enzyme System; Glucose-6-Phosphatase; Inflammation; Injections, Intramuscular; L-Iditol 2-Dehydrogenase; Liver; Male; Metallothionein; Rats; Rats, Inbred Strains; Turpentine

A study has been made of factors which may influence the induction of metallothionein-I (MT-I) synthesis by the superoxide radical generating agent, paraquat (PQ). Hepatic concentrations of zinc (Zn) and MT-I increased in rats injected with PQ (40 mg/kg, s.c.) or fasting, but were greater in the former. Renal concentration of MT-I increased in fasted rats but not in PQ-treated rats. The data suggest that the increase in MT-I concentrations in PQ-treated rats is not caused by reduction in food intake. Administration of PQ increased hepatic concentrations of Zn, MT-I and thiobarbituric acid-reactive substances (TBA-RS), indicating the occurrence of lipid peroxidation. Treatment of rats with vitamin E (400 mg/kg, s.c.) on 4 successive days before injection of PQ prevented only the enhancement of lipid peroxidation. The data indicate that the induction of MT synthesis by PQ is not correlated with enhancement of lipid peroxidation. Similar results were obtained in the liver of rats subjected to the radical-generating conditions, such as fasting and exposure to carbon tetrachloride. Free radicals may induce MT synthesis by direct or indirect mechanisms.

Topics: Animals; Body Weight; Carbon Tetrachloride Poisoning; Fasting; Free Radicals; Kidney; Lipid Peroxides; Liver; Lung; Male; Metallothionein; Organ Size; Oxidation-Reduction; Paraquat; Rats; Rats, Inbred Strains; Stress, Physiological; Vitamin E; Zinc

Topics: Animals; Cadmium Poisoning; Carbon Tetrachloride Poisoning; Chemical and Drug Induced Liver Injury; In Vitro Techniques; Liver; Male; Metallothionein; Rats; Rats, Inbred Strains; Zinc

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.

Topics: Animals; Cadmium; Carbon Tetrachloride Poisoning; Chemical and Drug Induced Liver Injury; Cytosol; In Vitro Techniques; Liver Diseases; Male; Metallothionein; Protein Binding; Rats; Rats, Inbred Strains; Zinc

These studies were designed to investigate the effects of stress and of changes in zinc status on plasma and liver concentrations of metallothionein-I (MT-I) in rats and to assess the value of plasma MT-I assays in the diagnosis of zinc deficiency. No MT-I was detected by radioimmunoassay in the plasma or liver of rats made hypozincaemic by feeding diets with less than 1 or 3 mg Zn/kg. Injection of normal rats with endotoxin or CCl4 also decreased plasma zinc levels, but these treatments greatly increased MT-I concentrations in both liver and plasma. Moreover plasma MT-I levels in zinc-deprived rats given endotoxin were only slightly greater than those in untreated rats of normal zinc status. Neither plasma zinc nor MT-I levels were altered in starved rats despite increased levels of the protein in the liver, although a slight increase in plasma MT-I was found in rats pair-fed with zinc-deficient animals. It appears therefore that reduced plasma levels of both zinc and MT-I are indicative of a zinc deficiency state and that assay of plasma MT should be of value in the diagnosis of zinc deficiency.

Topics: Animals; Carbon Tetrachloride Poisoning; Endotoxins; Liver; Male; Metallothionein; Protein Deficiency; Rats; Starvation; Stress, Physiological; Zinc

In order to test the possibility of metallothionein (MT) transfer from liver to kidney, experimental hepatic disorders produced by hepatotoxins were examined to study the release of MT from liver. 109Cd exposed rats were treated with carbon tetrachloride (CCl4) and the distribution of cadmium (Cd) in the body was studied. Hepatic Cd was significantly decreased corresponding to the dose of CCl4. Cd in plasma, kidney, and urine was increased remarkably in contrast with the decrease of hepatic Cd. No remarkable changes in Cd of other tissues and feces were observed. These phenomena were produced by other hepatotoxins like galactosamine and ethionine, and long-term administration of Cd, too. In every case that plasma Cd increased markedly, plasma levels of glutamate oxaloacetate transaminase (GOT), glutamate pyruvate transaminase (GPT), and lactate dehydrogenase (LDH) rose simultaneously, and a significant, positive correlation between Cd concentration and each of enzyme activities in plasma was observed. Cd in hepatic supernatant of CCl4 treated rats was bound mostly to MT fraction, and in kidney, plasma or urine, Cd was also in the form of MT. These results suggest that hepatic MT can be released into blood in the same manner as hepatic enzymes and transported to kidney and urine in some types of hepatic disorders.

Topics: Animals; Cadmium; Cadmium Poisoning; Carbon Tetrachloride Poisoning; Dose-Response Relationship, Drug; Female; Kidney; Liver; Liver Diseases; Male; Metallothionein; Rats; Rats, Inbred Strains; Time Factors; Tissue Distribution

Topics: Alanine Transaminase; Animals; Aspartate Aminotransferases; Carbon Tetrachloride Poisoning; Chlorides; Glutathione; Liver; Metalloproteins; Metallothionein; Microsomes, Liver; Rats; Zinc; Zinc Compounds

Topics: Animals; Carbon Tetrachloride; Carbon Tetrachloride Poisoning; Chemical and Drug Induced Liver Injury; Cytochrome P-450 Enzyme System; Cytosol; Lipid Metabolism; Male; Metalloproteins; Metallothionein; Microsomes, Liver; Rats; Time Factors; Zinc