Peroxidase-dependent bioactivation and oxidation of DNA and protein in benzo[a]pyrene-initiated micronucleus formation.
Micronucleus formation initiated by benzo[a]pyrene (B[a]P) and related xenobiotics is widely believed to reflect potential carcinogenic initiation, yet neither a dependence upon bioactivation nor the critical enzymes have been demonstrated. Using rat skin fibroblasts, protein oxidation (carbonyl formation) and content of prostaglandin H synthase (PHS) and cytochrome P4501A1 (CYP1A1) protein were determined by Western blot/immunodetection with enhanced chemiluminescence. DNA oxidation as 8-hydroxy-2'-deoxyguanosine formation was quantified using high-performance liquid chromatography with electrochemical detection. Fibroblast CYP1A1 activity assessed as ethoxyresorufin-O-deethylase was not detectable, and even CYP1A1 protein was measurable only after induction with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). However, TCDD additionally induced prostaglandin H synthase (PHS), which also was detectable constitutively. B[a]P 10 microM initiated the oxidation of DNA and protein, and the formation of micronuclei, all of which were enhanced over 2-fold by the dual CYP1A1/PHS inducer TCDD 10 nM, as well as by other PHS inducers, 12-O-tetradecanoylphorbol-13-acetate 1 microM and interleukin-1alpha 0.625 or 1.25 ng/ml, that do not induce CYP1A1 (p < .05). Conversely, B[a]P target oxidation and micronucleus formation were abolished by 1-aminobenzotriazole 1 mM (p < .05), which was a potent inhibitor of both peroxidases and P450. These results provide the first direct evidence that B[a]P-initiated micronucleus formation, like carcinogenic initiation, requires enzymatic bioactivation, and that peroxidase-dependent, reactive oxygen species-mediated oxidation of DNA, and possibly protein, constitutes a molecular mechanism of initiation in uninduced cells. Induction of either CYP1A1 or peroxidases such as PHS substantially enhances this genotoxic initiation, which may reflect cancer risk.
Kim
P M
PM
Faculty of Pharmacy, University of Toronto, Ontario, Canada.
DeBoni
U
U
Wells
P G
PG
D000818:Animals; D001564:Benzo(a)pyrene / Q000494:pharmacology*; D002467:Cell Nucleus / Q000502:physiology*; D002851:Chromatography, High Pressure Liquid; D003571:Cytochalasin B / Q000494:pharmacology; D019363:Cytochrome P-450 CYP1A1 / Q000378:metabolism; D065607:Cytochrome P-450 Enzyme Inhibitors; D004247:DNA / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005347:Fibroblasts / Q000378:metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008862:Microsomes, Liver / Q000378:metabolism; D010084:Oxidation-Reduction; D009195:Peroxidase / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000494:pharmacology*; D000072317:Polychlorinated Dibenzodioxins / Q000494:pharmacology; D011506:Proteins / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar
D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors; D000072317:Polychlorinated Dibenzodioxins; D011506:Proteins; D001564:Benzo(a)pyrene; D003571:Cytochalasin B; D004247:DNA; D009195:Peroxidase; D019363:Cytochrome P-450 CYP1A1