Oxidation of (+)-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene by mouse keratinocytes: evidence for peroxyl radical- and monoxygenase-dependent metabolism

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Abstract
The role of prostaglandin H (PGH) synthase and peroxyl radicals as well as cytochrome P-450 in the metabolism of 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (BP-7,8-diol) was examined in fresh skin keratinocytes isolated from hairless mice. Labeled (+)-BP-7,8-diol was oxidized after incubation with the keratinocytes to syn - and anti -diolepoxides in greater than a 4:1 ratio as estimated by h.p.l.c. analysis of the stable hydrolysis products. Formation of diolepoxides was dependent on cell number and the concentration of BP-7,8-diol. Incubation in the presence of the PGH synthase substrate, 20:4 or the inhibitor, indomethacin did not alter the total formation or the ratio of diolepoxides. However, the addition of butylated hydroxyanisole (1 μm) an inhibitor of peroxyl radical dependent-metabolism significantly inhibited diol-epoxide formation. The time course for the formation of the anti -diolepoxide and lipid peroxidation, measured as malon-dialdehyde was determined. The results suggest an excellent correlation between peroxyl radical and diolepoxide formation. Pretreatment of mice with the cytochrome P-450 in-ducer, β-naphthoflavone greatly altered the metabolism of (+)-BP-7,8-diol by keratinocytes. The major metabolite was the syn -diolepoxide with significant formation of two unknown metabolites. Pretreatment of mice with BP-7,8-diol did not induce aryl hydrocarbon hydroxylase activity but did increase the yield of syn -diolepoxide formed from labeled (+)-BP-7,8-diol by 1.5-fold. Our results suggest that peroxyl radical-mediated metabolism is primarily responsible for the oxidation of (+)-BP-7, 8-diol in control animals while the cytochrome P-450 system is primarily responsible for oxidation in animals pretreated with inducers.

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