Cytochrome P450 dependent xenobiotic activation by physiological hydroperoxides in intact hepatocytes

Xenobiotic metabolic activation by intact hepatocytes was recently shown to be enhanced by the addition of nontoxic concentrations of t-butyl hydroperoxide and prevented by cytochrome P450 inhibitors. Furthermore, H2O2 (Km = 103 microM) was found to be highly effective in supporting the human microsomal CYP1A2 catalyzed metabolic activation of the heterocyclic aromatic amine 2-amino-3-methylimidazo (4,5-f) quinoline (IQ) to mutagenic metabolites and the DNA adduct formed was the same as that formed by the mixed-function oxidase catalyzed activation system. In the following, it is shown that the cytotoxicity of other xenobiotics including carcinogenic arylamines and their N-hydroxyarylamine metabolites were markedly enhanced by hydroperoxide addition but not in the presence of cytochrome P450 inhibitors. The CYP1A2 dependent O-demethylation of methoxyresorufin in 3-methylcholanthrene induced hepatocytes was also markedly enhanced when intracellular H2O2 was generated by the mitochondrial monoamine oxidase (MAO) substrates tyramine or kynurenamine. Linoleic acid hydroperoxide also dramatically enhanced the cytotoxicity of phenelzine towards isolated hepatocytes and the microsomal metabolism of phenelzine to form ethylbenzene. The P450 inhibitors phenylimidazole, benzylimidazole prevented the metabolic activation of phenelzine but not lipid peroxidation. These results suggest that linoleic acid hydroperoxide can activate hydrazines via a cytochrome P450 peroxidase catalyzed one electron oxidation to form highly cytotoxic reactive intermediates. Furthermore, increased hydrogen peroxide formation, e.g. as a result of oxidative stress, would also be expected to enhance the metabolic activation of carcinogenic arylamines via the peroxygenase function of CYP1A2.