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 vivo.
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 microM) was metabolized primarily to 7 alpha,8 beta-dihydroxy-9 beta,10 beta-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 7 beta, 8 alpha-dihydroxy-9 beta,10 beta-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 less than 1 microM). In epidermal homogenates from animals pretreated with beta-naphthoflavone (beta-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 alpha-naphthoflavone (an inhibitor of cytochrome P-450) inhibited syn-BPDE-tetraol formation (I50 approximately 2.5 microM). The DNA adducts formed in mouse epidermis after topical application of [1,3-3H]-(+)-BP-7,8-diol (200 nmol/mouse, 50 microCi/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 beta-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 beta-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.
Pruess-Schwartz
D
D
Department of Chemistry, Wayne State University, Detroit, Michigan 48202.
Nimesheim
A
A
Marnett
L J
LJ
D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide / Q000378:metabolism; D000818:Animals; D001571:Benzoflavones / Q000494:pharmacology; D001711:Biotransformation; D003577:Cytochrome P-450 Enzyme System / Q000502:physiology*; D004247:DNA / Q000378:metabolism; D018736:DNA Adducts; D004101:Dihydroxydihydrobenzopyrenes / Q000378:metabolism*; D004852:Epoxy Compounds / Q000378:metabolism; D005260:Female; D066298:In Vitro Techniques; D051379:Mice; D009249:NADP / Q000494:pharmacology; D010545:Peroxides; D012867:Skin / Q000378:metabolism*; D019324:beta-Naphthoflavone
D001571:Benzoflavones; D018736:DNA Adducts; D004101:Dihydroxydihydrobenzopyrenes; D004852:Epoxy Compounds; D010545:Peroxides; C043931:benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide-DNA; C017228:benzo(a)pyrene 7,8-dihydrodiol; C049375:perhydroxyl radical; D009249:NADP; D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide; C011512:alpha-naphthoflavone; D019324:beta-Naphthoflavone; D004247:DNA; D003577:Cytochrome P-450 Enzyme System