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Peroxyl Radical- and Cytochrome P-450-dependent Metabolic Activation of (+)-7,8-Dihydroxy-7,8-dihydrobenzo(a)pyrene in Mouse Skin in Vitro and in Vivo¹

Department of Chemistry, Wayne State University, Detroit, Michigan 48202

The role of peroxyl radicals and cytochrome P-450 in the metabolic activation of the (+)-enantiomer of 7,8-dihydroxy-7,8-dihydrobenzo-(a)pyrene [(+)-BP-7,8-diol] was investigated in the epidermis of CD-1 mice. In skin homogenates from untreated or acetone-pretreated animals [7-¹⁴C]-(+)-BP-7,8-diol (20 µM) was metabolized primarily to 7,8ß-dihydroxy-9ß,10ß-epoxy-7,8,9,10-tetrahydroBP [(-)-anti-BPDE] as detected by high performance liquid chromatography of the stable tetraol hydrolysis products. The amounts of anti-BPDE-tetraols increased with the length of time of incubation (0–90 min). Only small amounts of 7ß,8-dihydroxy-9ß,10ß-epoxy-7,8,9,10-tetrahydroBP [(+)-syn-BPDE]-tetraols were detected. Epoxidation was not dependent upon NADPH. The addition of butylated hydroxyanisole (BHA, a free radical scavenger) decreased the formation of both anti- and syn-BPDE-tetraols (I₅₀ < 1 µM). In epidermal homogenates from animals pretreated with ß-naphthoflavone (ß-NF, an inducer of cytochrome P-450c), (+)-BP-7,8-diol was metabolized almost exclusively to (+)-syn-BPDE. The amounts of syn-BPDE-tetraols also increased with time of incubation with only small amounts of anti-BPDE-tetraols being detected. Epoxidation was NADPH-dependent and was not inhibited by the addition of BHA. The addition of -naphthoflavone (an inhibitor of cytochrome P-450) inhibited syn-BPDE-tetraol formation (I₅₀ 2.5 µM). The DNA adducts formed in mouse epidermis after topical application of [1,3-³H]-(+)-BP-7,8-diol (200 nmol/mouse, 50 µCi/mouse) were analyzed by high performance liquid chromatography. The hydrocarbon-modified deoxyribonucleosides were identified by comparison with standards of (+)-syn-BPDE-dGuo and (-)-anti-BPDE-dGuo. In animals that received no pretreatment, similar amounts of (-)-anti-BPDE-dGuo and (+)-syn-BPDE-dGuo were formed after 3 h of exposure to (+)-BP-7,8-diol, whereas in ß-NF-pretreated animals larger proportions of (+)-syn-BPDE-dGuo were formed. Analysis of the tetraol hydrolysis products and DNA adducts formed from (+)-BP-7,8-diol in mouse skin demonstrates that two independent pathways of metabolic activation occur in vivo: in control animals peroxyl radical-mediated pathways are important contributors to metabolic activation, whereas in ß-NF-pretreated animals cytochrome P-450 is the major oxidizing agent. These results provide the first evidence that peroxyl radicals play a role in xenobiotic metabolism in vivo.

¹ This work was supported by Research Grants BC-244j from the American Cancer Society and CA47479 from the National Cancer Institute, and Training Grant T32-CA-09531 from the National Cancer Institute.

² Recipient of American Cancer Society Faculty Research Award FRA 243. To whom requests for reprints should be addressed.

Received 6/13/88. Revised 12/ 1/88. Accepted 1/ 4/89.

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