Role of cytochrome P450 in the metabolism and toxicity of hydroperoxides in isolated rat hepatocytes.

The contributions of cytochromes P450 (P450) to the metabolism and toxicity of hydroperoxides in freshly isolated rat hepatocytes were investigated utilizing 2,6-di-tert-butyl-4-hydroperoxy-4-methyl-2,5-cyclohexadienone (BHTOOH). This hydroperoxide was rapidly degraded in cell suspensions, and the products were identical to those determined previously with subcellular preparations of ferric P450. With 250 microM BHTOOH, the ratio of glutathione peroxidase-mediated:P450-mediated metabolism was estimated to be about 3:1. Surprisingly, BHTOOH was found to be a more potent cytotoxin than cumyl hydroperoxide (CuOOH), despite the fact that it caused substantially less lipid peroxidation than the latter. P450 inhibition enhanced the toxicity of BHTOOH, but lowered the toxicity of CuOOH. These data demonstrate that intracellular ferric P450 can compete with glutathione peroxidase to reduce hydroperoxides by 1- and 2-electron processes. If the alkoxy radical from homolytic cleavage of the O-O bond can undergo facile intramolecular reactions to nontoxic products, as with BHTOOH, the role of P450 is detoxification. On the other hand, if the alkoxy radical preferentially attacks membrane lipids, as with CuOOH, P450 contributes to lipid peroxidation and toxicity. It was determined that the levels of glutathione, protein thiols, and ATP decreased in parallel with BHTOOH-induced cell death, but no conclusions are possible concerning mechanisms underlying the relatively potent toxicity of BHTOOH. Toxicity may be related to the high lipophilicity of this hydroperoxide which, presumably, facilitates its passage into cells and distribution to various intracellular sites. BHTOOH appears to be an excellent model compound for investigating mechanisms of hydroperoxide-mediated cytotoxicity which do not involve lipid peroxidation.