ascorbic-acid and bromobenzene

In the course of screening for hepatoprotective agents from natural products, the effects of the methanol extract (ME) of the rhizome of Alisma orientale (Alismataceae) and its major component, alisol B 23-acetate (ALB) on hepatic lipid peroxidation and drug-metabolizing enzymes were evaluated in rats intoxicated with bromobenzene (BB). Pretreatment with ME and ALB had no effect on hepatic antioxidant enzymes such as glutathione reductase and a-glutamylcysteine synthetase. ME and ALB had also no effect on the reduction in glutathione content caused by BB. In contrast, ME recovered the BB-induced decrease in epoxide hydrolase and glutathione S-transferase, enzymes that remove toxic epoxides. ME also attenuated the BB-induced increase in aminopyrine N-demethylase and aniline hydroxylase, enzymes that produce toxic intermediates. This effect was greater than that seen with ascorbic acid, which was used as a positive control. ALB had similar effects on the activities of antioxidant enzymes to ME, and may be partly responsible for the effects of ME.

Topics: Alisma; Animals; Ascorbic Acid; Bromobenzenes; Cholestenones; Diterpenes; Epoxide Hydrolases; Glutathione; Glutathione Transferase; Liver; Male; Plant Extracts; Rats; Rats, Sprague-Dawley

Water-soluble vitamin E (Trolox C), ascorbic acid and catalase were shown in our previous study to protect isolated rat hepatocytes against bromobenzene-induced toxicity.. In order to study the mechanisms of this protection and the pathogenesis of bromobenzene-induced hepatocellular injury, a fluorometric assay for the investigation of intracellular oxidation, indicated by conversion of dichlorofluorescein diacetate to dichlorofluorescein, was used. Single-strand DNA breakage was also evaluated in Hep G2 cells by a radio-labelling method.. Bromobenzene (2.4 and 4.8 mM) induced a significant increase in dichlorofluorescein fluorescence intensity compared to the controls. Trolox C, ascorbic acid or catalase significantly inhibited bromobenzene-induced enhancement of fluorescence intensity (p<0.05-0.001), as well as reduced auto-intracellular oxidation in untreated Hep G2 cells. Hydrogen peroxide (H2O2) evoked a dose-dependent increase in dichlorofluorescein fluorescence intensity in Hep G2 cells, and the effect was completely blocked by Trolox C (2.0 mM) and catalase (4800 unit/ml). Bromobenzene caused significant single-strand DNA breakage in Hep G2 cells during 2 h suspension incubation and 24 h primary incubation. H2O2 (400 microM) led to marked single-strand DNA breakage in 20 min, and the effect was attenuated by Trolox C.. Metabolism of bromobenzene in Hep G2 cells induces production of H2O2, indicated by enhancement of dichlorofluorescein fluorescence intensity, or other free radicals, which leads to single-strand DNA breakage in the cells. Vitamins E and C and catalase display strong intracellular antioxidative effects. Vitamin E could partially inhibit H2O2-induced single-strand DNA breakage in the cells.

Topics: Antioxidants; Ascorbic Acid; Bromobenzenes; Catalase; Cells, Cultured; Chromans; DNA Damage; DNA, Single-Stranded; Dose-Response Relationship, Drug; Fluoresceins; Fluorescence; Humans; Hydrogen Peroxide; Intracellular Fluid; Liver; Oxidants; Oxidation-Reduction; Vitamin E

The protective effects of trolox C (water-soluble vitamin E), vitamin C, and catalase on bromobenzene (BB)-induced toxicity to isolated rat hepatocytes were evaluated. The glutathione (GSH) content of the hepatocytes exposed to BB was measured.. BB caused acute damage to the cells during 2 h of incubation (short) when BB was added directly to the culture wells, whereas a late-occurring and time-dependent increase in lactate dehydrogenase (LDH) leakage rate was observed during 24 h of incubation (long) when BB was dissolved in a different way. Incubation of the cells with trolox C (0.5-2.0 mM) prevented the hepatocellular damage induced by BB at 2.4 mM during the long-term incubation. Vitamin C (0.1-1.0 mM) had a protective effect on BB-induced toxicity during both the short- (BB, 1.6 mM) and the long- (BB, 2.4 mM) term incubations. Catalase (3200 U/ml) also showed a beneficial effect on the cells during the short-term BB exposure. Trolox C (2.0 mM) and vitamin C (0.5 mM) restored BB-induced GSH depletion in the cells.. BB induced two patterns of LDH leakage from isolated hepatocytes on the basis of different ways of BB exposure and incubation periods. Trolox C, vitamin C, and catalase exerted protective effects on BB-induced toxicity during short- or/and long-term incubations. The effects were concentration-dependent. Restoration of GSH content in BB-exposed hepatocytes suggests that trolox C and vitamin C could reduce GSH consumption during BB metabolism and exert an antioxidant effect.

Topics: Analysis of Variance; Animals; Antioxidants; Ascorbic Acid; Bromobenzenes; Catalase; Cells, Cultured; Chromans; Female; Glutathione; L-Lactate Dehydrogenase; Liver; Rats; Rats, Sprague-Dawley; Vitamin E

The mechanisms of the liver damage produced by three glutathione (GSH) depleting agents, bromobenzene, allyl alcohol and diethylmaleate, was investigated. The change in the antioxidant systems represented by alpha-tocopherol (vitamin E) and ascorbic acid were studied under conditions of severe GSH depletion. With each toxin liver necrosis was accompanied by lipid peroxidation that developed only after severe depletion of GSH. The hepatic level of vitamin E was decreased whenever extensive lipid peroxidation developed. In the case of bromobenzene intoxication, vitamin E decreased before the onset of lipid peroxidation. Changes in levels of the ascorbic and dehydroascorbic acid indicated a redox cycling of vitamin C with the oxidative stress induced by all the three agents. Such a change of the redox state of vitamin C (increase of the oxidized over the reduced form) may be an index of oxidative stress preceding lipid peroxidation in the case of bromobenzene. In the other cases, such a change is likely to be a consequence of lipid peroxidation. Experiments carried out with vitamin E deficient or supplemented diets indicated that the pathological phenomena occurring as a consequence of GSH depletion depend on hepatic levels of vitamin E. In vitamin E deficient animals, lipid peroxidation and liver necrosis appeared earlier than in animals fed the control diet. Animals fed a vitamin E supplemented diet had an hepatic vitamin E level double that obtained with a commercial pellet diet. In such animals, bromobenzene and allyl alcohol had only limited toxicity and diethylmaleate none in spite of comparable hepatic GSH depletion. Thus, vitamin E may largely modulate the expression of the toxicity by GSH depleting agents.

Topics: 1-Propanol; Animals; Antioxidants; Ascorbic Acid; Bromobenzenes; Chromatography, High Pressure Liquid; Glutathione; Lipid Peroxidation; Liver; Male; Maleates; Malondialdehyde; Mice; Necrosis; Propanols; Time Factors; Vitamin E

Topics: Animals; Antioxidants; Ascorbic Acid; Bromobenzenes; Dehydroascorbic Acid; Glutathione; Liver; Male; Mice; Vitamin E

Bromobenzene undergoes metabolic activation via 2,3- and 3,4-epoxidation catalyzed by the hepatic cytochrome P-450 mixed-function oxidase system. Its reactive metabolites, especially bromobenzene 3,4-oxide, presumably lead to severe centrolobular necrosis. A study of relative rate of binding of 14C-bromobenzene metabolites to hepatic microsomal protein indicated a significant difference in the rate of binding of the bromobenzene 3,4-oxide compared to its positional isomer, bromobenzene 2,3-oxide. However, the rate of bromobenzene metabolism indicated no significant difference in the formation of products o-bromophenol and p-bromophenol. A search for protective agents revealed that 6,7-dimethyl-5,6,7,8-tetrahydropterine and ascorbyl palmitate were very effective in protecting against macromolecular adduct formation at a concentration of 1 mM-in fact, at least a twofold increase in protection compared to the known protective agents such as glutathione or cysteine. Furthermore, 6,7-dimethyl-5,6,7,8-tetrahydropterine and ascorbyl palmitate inhibited the metabolism of bromobenzene over 90% at a concentration of 2.5 mM.

Topics: Animals; Ascorbic Acid; Biotransformation; Bromobenzenes; Enzyme Induction; Male; Microsomes, Liver; Mixed Function Oxygenases; Oxidation-Reduction; Phenols; Protein Binding; Rats; Rats, Inbred Strains