Halomethane hepatotoxicity: induction of lipid peroxidation and inactivation of cytochrome P-450 in rat liver microsomes under low oxygen partial pressures

Halomethane-induced lipid peroxidation and inactivation of cytochrome P-450 were studied in liver microsomes from phenobarbital-pretreated rats in the presence of NADPH at steady-state O2 partial pressures (PO2). As indicated by the formation of thiobarbituric acid-reactive material and the stimulation of O2 uptake, significant lipid peroxidation was induced by those halomethanes containing more than two Cl, Br, or I atoms. Lipid peroxidation decisively depended on the PO2 present, showing distinct maxima at PO2 between 1 and 10 mm Hg. Those halomethanes inducing lipid peroxidation also led to inactivation of microsomal cytochrome P-450, as indicated by a loss of cytochrome P-450 detectable as ferrous CO complex and an equimolar loss of microsomal heme. Under anaerobic conditions inactivation of cytochrome P-450 presumably resulted solely from an attack of halomethane radicals on its heme moiety. Under aerobic conditions lipid peroxidation made an additional contribution to the inactivation of cytochrome P-450. These results suggest that the reductive activation to free radicals, catalyzed by cytochrome P-450, and thus the induction of lipid peroxidation at low but physiological PO2 are characteristic not only of CCl4 but also of other polyhalogenated methanes, especially CBrCl3, CBr4, CHI3, CHBr3, and CHBr2Cl.     

Carbon tetrachloride-induced loss of microsomal glucose 6-phosphatase and cytochrome P-450 in vitro

Rat liver microsomes were incubated in the presence of NADPH and CCl4 under various conditions, and losses of glucose 6-phosphatase (G-6-Pase) and cytochrome P-450 were examined in terms of lipid peroxidation and CCl4 metabolism. Loss of G-6-Pase activity correlated well with the enhancement of lipid peroxidation. Loss of cytochrome P-450 was also dependent on the lipid peroxidation, under aerobic conditions. However, the cytochrome was destroyed under anaerobic conditions in which lipid peroxidation and loss of G-6-Pase were greatly suppressed. This anaerobic loss of cytochrome P-450 may be linked with the metabolism of CCl4 by this hemoprotein, as evidenced by the observation that CCl4 metabolism occurred only under anaerobic conditions and was inhibited by carbon monoxide accompanied by the suppression of the loss of cytochrome P-450.     

Early destruction of cytochrome P-450 in testis of carbon tetrachloride poisoned rats

There is 20--36 percent decrease in the microsomal cytochrome P-450 (P450) content of the testes 3 or 6 h after CCl4 administration to Sprague--Dawley male rats. Irreversible binding of CCl4 metabolites to testicular microsomal lipids is observed as early as 3 h while CCl4 induced lipid peroxidation does not occur within the first 6 h of poisoning. Results suggest that reactive metabolites rather than lipid peroxidation is involved in P-450 destruction in the testes.     

Effect of linoleic acid hydroperoxide on liver microsomal enzymes in vitro.

Rat liver microsomes incubated with linoleic acid hydroperoxide (LAHPO) lost cytochrome P-450 specifically among the enzymes of microsomal electron transport systems. The loss of cytochrome P-450 content and glucose-6-phosphatase activity by LAHPO was accompanied by an increase in malondialdehyde (MDA) production. Turbidity of microsomal suspensions was decreased with increasing MDA production, but not proportionately. Diethyldithiocarbamate (DTC), N,N'-diphenyl-p-phenylenediamine and alpha-tocopherol inhibited almost completely the LAHPO-induced MDA production of microsomes, however no perfect protection against the loss of cytochrome P-450 content and glucose-6-phosphatase activity was observed. The decrease of microsomal turbidity by LAHPO was little affected in the presence of DTC. Purified cytochrome P-450 was destroyed by LAHPO, with minimal protection by the compounds described above. These results suggest the possibility that the loss of microsomal enzyme activities during lipid peroxidation may be attributed largely to a direct attack on enzyme proteins by lipid peroxides rather than indirectly to a structural damage of microsomal membranes resulting from peroxidative breakdown of membrane lipids.     

Inhibition of Ca2+ sequestration in foetal liver microsomes by carbon tetrachloride and bromotrichloromethane

The purpose of the present work is to establish to what extent the calcium uptake of foetal liver microsomes can be modified, as in the adult, by classical hepatotoxins. The administration of liver toxins (BrCCl3, CCl4) to the pregnant rat or their addition to foetal and maternal liver microsome preparations causes a decrease in the level of cytochrome P-450 and a drop in the calcium storage capacity of microsomes. Lipid peroxidation of membrane phospholipids is observed in the mother but not in the foetus. On the 20th day of gestation, the foetal liver shows cytochrome P-450 dependent metabolic activity and constitutes a good model illustrating the hypothesis of calcium pump inhibition by .CCl3 radicals without lipoperoxidation.     

Action of carbon tetrachloride in the reconstituted monooxygenase system using partially purified cytochrome P-450 and P-448

In the cytochrome P-450-reconstituted system, CCl4 stimulated NADPH-dependent lipid peroxidation of the system containing the P-450 form to a much greater extent than that of the system containing the P-448 form. When the P-450-reconstituted system was preincubated in the presence of both NADPH and CCl4, 7-ethoxycoumarin O-deethylase, aminopyrine N-demethylase and aniline hydroxylase activities were decreased by 40-60%, whereas, with P-448 form reconstituted system, no suppression was observed in these enzyme activities or in 7-ethoxyresorufin O-deethylase activity. These observations suggest that the P-450 form, but not the P-448 form, is active in metabolizing CCl4 to a reactive species that subsequently impairs the hemoprotein.     

Lipid peroxidation and irreversible cell damage: synergism between carbon tetrachloride and 1,2-dibromoethane in isolated rat hepatocytes

The combination of 1,2-dibromoethane (DBE) with carbon tetrachloride (CCl4) in the isolated rat hepatocyte model produces a significant potentiation of both lipid peroxidation and plasma membrane damage induced by the latter compound. The increase in malondialdehyde production precedes the hepatocyte damage, evaluated in terms both of lactate dehydrogenase leakage and trypan blue exclusion. When hepatocytes are isolated from vitamin E pretreated rats, both the prooxidant and the cytotoxic effects of CCl4 are prevented. Also the synergism between CCl4 and DBE on lipid peroxidation disappears completely while that on cell damage is strongly reduced. The increased lipid peroxidation appears to be one of the mechanisms of the observed synergism between CCl4 and DBE on hepatocyte damage. Regarding the antioxidant status of the hepatocyte challenged with CCl4 and DBE, an early and significant consumption of vitamin E is observed only in the presence of the mixture of these xenobiotics. Total nonprotein thiol content is not significantly modified by CCl4 poisoning while DBE, alone and in association with CCl4, markedly decreases it. Vitamin E supplementation does not prevent but moderately delays total nonprotein thiol depletion due to DBE or to the mixture. Finally, glutathione transferase activity is significantly reduced by CCl4 treatment and not by DBE, and vitamin E supplementation totally prevents such inhibition. The increased prooxidant effect of CCl4 plus DBE compared to CCl4 alone seems related to the shift in DBE metabolism consequent to the CCl4-dependent inactivation of glutathione transferase.     

Effects of carbon tetrachloride on the liver of chickens. Early biochemical and ultrastructural alterations in the absence of detectable lipid peroxidation

Administration of CCl4 i.p. to Leghorn chickens did not promote lipid peroxidation of liver microsomal lipids, as evidenced by either increased diene conjugation or by decreased arachidonic acid content. The hepatotoxin did not produce liver necrosis 24 h after dosing, but decreased the cytochrome P-450 content, and aminopyrine N-demethylase and glucose 6 phosphatase activities at 1, 3, 6 and 24 h. CCl4 administration produced dilation of the rough endoplasmic reticulum and detachment of ribosomes from their membranes. These observations suggest that lipid peroxidation is not the key event in the production of these biochemical and ultrastructural alterations, elicited by CCl4.