diethyl-maleate and Liver-Diseases

A gradual but extensive depletion of hepatic GSH has long been known to accompany development of galactosamine-induced hepatotoxicity in rats, and some protection from liver injury has been observed after administration of sulfhydryl-donating compounds. Although these observations support a key role for GSH in the underlying mechanism, the impact of GSH depletion and repletion on the hepatotoxic response to galactosamine is unclear. To investigate the role of GSH in galactosamine-induced liver injury, we examined the effect of modulating GSH content on galactosamine toxicity in rat primary hepatocyte cultures. Galactosamine (4 mM) cytotoxicity was assessed by release of lactate dehydrogenase into the culture medium, and hepatocellular GSH content was measured by HPLC with electrochemical detection. The data indicated that prior depletion of GSH with either diethyl maleate or buthionine sulfoximine significantly enhanced galactosamine toxicity; however, addition of GSH-ester or alternate sulfur nucleophiles at various times during the incubation did not abrogate toxicity. In contrast, co-addition of S-adenosylmethionine (SAMe) with galactosamine exerted a marked protective effect without significantly altering hepatocyte GSH content. These data suggest that GSH depletion is not directly involved in the sequelae for galactosamine-induced hepatotoxicity, and raise the possibility that SAMe may have hepatoprotective effects that are not dependent on its ability to enhance GSH synthesis.

Topics: Animals; Cells, Cultured; Galactosamine; Glutathione; Hepatocytes; L-Lactate Dehydrogenase; Liver Diseases; Male; Maleates; Rats; Rats, Long-Evans; S-Adenosylmethionine

We have shown that urinary taurine level may be used as a biomarker of pathological and biochemical lesions. Detection of changes in the urinary concentration of this low molecular weight metabolite indicates biochemical lesions which may also be associated with pathological damage. Hepatotoxic compounds such as CCl4, galactosamine and thioacetamide that cause hepatic necrosis and compounds such as hydrazine and ethionine that cause fatty liver all result in elevated urinary taurine levels in rats. However compounds which do not cause liver damage, such as cycloheximide, also raise urinary taurine levels. All of these substances are known to or are believed to inhibit protein synthesis. Conversely, compounds which increase protein synthesis, such as phenobarbital and clenbuterol, significantly decrease urinary taurine levels. Compounds which interfere with hepatic GSH synthesis will also change urinary taurine levels. Thus, depletion of GSH with diethyl maleate or phorone decreases urinary taurine whereas inhibition of GSH synthesis with compounds such as buthionine sulphoximine increases urinary taurine levels. In isolated hepatocytes in vitro, leakage of taurine occurs in response to cytotoxic compounds such as hydrazine and allyl alcohol. However, total taurine levels were increased by the hepatotoxicant CCl4. Taurine synthesis is decreased by depletion of GSH with allyl alcohol in isolated hepatocytes. Therefore taurine levels are an important potential biomarker for biochemical lesions induced by chemicals both in vivo and in vitro, in particular changes in protein and GSH synthesis.

Topics: Animals; Biomarkers; Carbon Tetrachloride; Carbon Tetrachloride Poisoning; Cells, Cultured; Clenbuterol; Galactosamine; Glutathione; Ketones; Liver; Liver Diseases; Male; Maleates; Necrosis; Phenobarbital; Rats; Rats, Sprague-Dawley; Taurine; Thioacetamide

Metallothionein (MT) is a sulfhydryl-rich protein whose levels are increased by administration of a variety of agents including metals, cytokines, and oxidative stress agents. Recent studies have suggested that MT is involved in protecting against various forms of oxidative stress, but little is known about the induction of MT by oxidative stress agents. Diethyl maleate (DEM) causes oxidative stress by depleting glutathione levels and is quite effective at increasing hepatic concentrations of MT. The purpose of the current study was to learn more about the relationship between induction of MT and oxidative stress by characterizing this increase in hepatic MT levels produced by DEM. Administration of DEM (3 to 9 mmol/kg, sc) increased hepatic MT concentration in mice as much as 37-fold to 213 micrograms MT/g liver, which is similar to the hepatic MT level seen after administration of other effective MT inducers, such as Cd. The maximal increase of hepatic MT took place 12 to 24 hr after administration of 5 mmol DEM/kg. This rise in MT was preceded by a 60% depletion of hepatic glutathione 3 hr after DEM and increases in both MT-I and MT-II mRNA, which reached a peak 6 to 9 hr after DEM. Administration of DEM (3-5 mmol/kg, sc) also increased MT levels in Sprague-Dawley rats. Pretreatment with DEM protected against Cd-induced hepatotoxicity in a fashion which suggested that a functional MT was being synthesized. In summary, DEM is a highly effective inducer of MT which increases MT at the mRNA level.

Topics: Animals; Base Sequence; Blotting, Northern; Cadmium; Chemical and Drug Induced Liver Injury; Cytosol; Dose-Response Relationship, Drug; Glutathione; Immunoblotting; Liver; Liver Diseases; Male; Maleates; Metallothionein; Mice; Mice, Inbred Strains; Molecular Sequence Data; Rats; Rats, Inbred Strains; RNA, Messenger; Subcellular Fractions; Time Factors; Transcription, Genetic

Loxistatin is a possible therapeutic agent of muscular dystrophy. A single oral administration of loxistatin to male rats caused focal necrosis of the liver with inflammatory cell infiltration. The severity of the lesions was dose-dependent up to 200 mg/kg and also manifest by an increase in serum alanine aminotransferase and aspartate aminotransferase activities. Hepatic glutathione (GSH) levels decreased with a maximum 20% depletion within 5 hr after the oral administration of loxistatin. Pretreatment with diethyl maleate did not potentiate the loxistatin-induced hepatic injury. On the other hand, the hepatoprotective effect of cysteamine was observed when cysteamine was administered 24 hr before loxistatin dosing, but the effect was not observed when the antidote was administered concomitantly with loxistatin. Pretreatment of rats with phenobarbital or trans-stilbene oxide provided partial protection against the hepatotoxic effect of loxistatin. Pretreatment with SKF-525A resulted in increased hepatic injury, while pretreatment with piperonyl butoxide, cimetidine, or 3-methylcholanthrene had no effect on hepatic damage by loxistatin. Five hours after [14C]loxistatin administration to rats, the covalent binding of the radioactivity to proteins was greatest in the liver, followed by the kidney, then muscle and blood to a lesser extent. [14C]Loxistatin acid, the pharmacologically active form of loxistatin, irreversibly bound to rat liver microsomal proteins; more binding occurred when the NADPH-generating system was omitted and when the microsomes were boiled first. GSH did not alter the extent of irreversible binding, whereas N-ethylmaleimide decreased the binding of [14C]loxistatin acid to rat liver microsomal proteins by 75%. Unlike the rat, administration of loxistatin to hamsters caused neither hepatic injury nor hepatic GSH depletion even at a high dose (500 mg/kg). Both the distribution and covalent binding of radioactivity in the hamster liver were one-third of those in rats following [14C]loxistatin dosing. These results suggest that loxistatin causes species-specific hepatotoxicity and that, at least in part, some of the toxic effects of loxistatin are mediated by the nonenzymatic covalent binding of loxistatin acid to thiol residues on cellular macromolecules.

Topics: Alanine Transaminase; Animals; Aspartate Aminotransferases; Chemical and Drug Induced Liver Injury; Cricetinae; Cysteamine; Cysteine Proteinase Inhibitors; Drug Interactions; Glutathione; In Vitro Techniques; Leucine; Liver Diseases; Male; Maleates; Mesocricetus; Necrosis; Protein Binding; Rats; Rats, Inbred Strains; Species Specificity; Tissue Distribution

Recently, we have reported that 3,5-dialkyl substitution of paracetamol, in contrast to 3-monoalkyl substitution, prevented the paracetamol-induced toxicity in freshly isolated rat hepatocytes without having any effect on its cytochrome P-450 mediated bioactivation to reactive N-acetyl-p-benzoquinone imines (NAPQI). In the present study the mechanism of this prevention of toxicity, with special emphasis on oxidative stress, was studied in more detail in freshly isolated rat hepatocytes, using paracetamol, 3-methyl-, 3,5-dimethyl-paracetamol, synthetic NAPQI and 3,5-dimethyl-NAPQI. 3-Methyl-paracetamol was found to induce glutathione (GSH) depletion, lipid-peroxidation and cytotoxicity in hepatocytes to the same extent as paracetamol. 3,5-Dimethyl-paracetamol, however, even when added in a ten-fold higher concentration when compared to paracetamol, did not induce any of these effects. Similar differences of toxicity were observed between NAPQI and 3,5-dimethyl-NAPQI; 3,5-dimethyl-NAPQI, in contrast to NAPQI, did not reduce protein thiol levels, did not induce GSH depletion, lipid-peroxidation nor cytotoxicity. Only after artificial depletion of GSH levels in the hepatocytes by DEM or BCNU, 3,5-dimethyl-NAPQI was cytotoxic. This effect was accompanied by depletion of protein thiol levels, but not by lipid-peroxidation. Addition of the disulfide reducing agent, dithiothreitol, prevented the artificially created cytotoxicity of 3,5-dimethyl-NAPQI. It is concluded that prevention of paracetamol-induced toxicity by 3,5-dialkyl substitution is primarily due to prevention of irreversible GSH-depletion, presumably caused by the inability of 3,5-dialkyl-NAPQI to conjugate with thiols. As a result, the GSH-dependent cellular defense mechanism against potential oxidative cellular injury by 3,5-dialkyl-NAPQI is left unimpaired. Our observations indicate that a compound, not capable of covalent binding to thiol groups of proteins, can induce toxicity solely as a result of protein thiol oxidation without inducing lipid-peroxidation.

Topics: Acetaminophen; Animals; Benzoquinones; Carmustine; Chemical and Drug Induced Liver Injury; Glutathione; Imines; Lipid Peroxides; Liver; Liver Diseases; Male; Maleates; Oxidation-Reduction; Quinones; Rats; Rats, Inbred Strains; Structure-Activity Relationship

The mechanisms of bromobenzene and iodobenzene hepatotoxicity in vivo were studied in mice. Both the intoxications caused a progressive decrease in hepatic glutathione content. In both instances liver necrosis was evident only when the hepatic glutathione depletion reached a threshold value (3.5-2.5 nmol/mg protein). The same threshold value was evident for the occurrence of lipid peroxidation. Similar results were obtained in a group of mice sacrificed 15-20 hours after the administration of diethylmaleate, a drug which is mainly conjugated with hepatic glutathione without previous metabolism. The correlation between lipid peroxidation and liver necrosis was much more significant than that between covalent binding and liver necrosis. This fact supports the view that lipid peroxidation is the major candidate for the liver cell death produced by bromobenzene intoxication. Moreover, a dissociation of liver necrosis from covalent binding was observed in experiments in which Trolox C (a lower homolog of vitamin E) was administered after bromobenzene poisoning. The treatment with Trolox C, in fact, almost completely prevented both liver necrosis and lipid peroxidation, while not changing at all the extent of the covalent binding of bromobenzene metabolites to liver protein.

Topics: Alanine Transaminase; Animals; Aspartate Aminotransferases; Bromobenzenes; Chemical and Drug Induced Liver Injury; Chromans; Glutathione; Iodobenzenes; Lipid Peroxides; Liver; Liver Diseases; Male; Maleates; Mice; Necrosis; Proteins; Rats; Rats, Inbred Strains

Cd has a strong affinity for sulfhydryl groups and is hepatotoxic. Thus, to further understand the mechanism of Cd-induced liver injury, the effect of increased and decreased hepatic glutathione (GSH) concentration on Cd-induced liver injury was examined. Liver GSH was lowered by pretreating rats with phorone (250 mg/kg, ip) or diethyl maleate (0.85 mg/kg, ip) 2 hr prior to challenge with various doses of Cd. Ten hours after Cd (1) 40-80% of the rats pretreated with phorone or diethyl maleate and challenged with 1.0-2.0 mg Cd/kg died whereas no mortality was observed in the control group; (2) plasma enzyme activities of alanine (ALT) and aspartate (AST) aminotransferase and sorbitol dehydrogenase (SDH) were markedly increased in phorone and diethyl maleate-pretreated rats challenged with Cd (0.7-2.0 mg/kg) versus control rats; and (3) moderate changes in liver histology were observed in corn oil pretreated and Cd challenged rats, while prior depletion of GSH potentiated histopathologic changes in liver produced by Cd alone. Another group of rats received cysteine (1.9 g/kg, po) 3 hr prior to injection of a lethal dose of Cd. Cysteine pretreatment increased liver GSH levels by 22% 3 hr after administration and attenuated Cd-induced liver injury as evidenced by marked decreases in plasma ALT, AST, and SDH activities. Pathological changes in liver were also reduced. These data indicate that liver reduced GSH concentration is important in modulating Cd-induced hepatotoxicity.

Topics: Alanine; Animals; Aspartic Acid; Cadmium Poisoning; Chemical and Drug Induced Liver Injury; Cysteine; Drug Synergism; Glutathione; Ketones; L-Iditol 2-Dehydrogenase; Liver; Liver Diseases; Male; Maleates; Rats; Rats, Inbred Strains; Transaminases; Triglycerides