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 init iation, yet neither a dependence upon bioactivation nor the critical e nzymes have been demonstrated. Using rat skin fibroblasts, protein oxi dation (carbonyl formation) and content of prostaglandin H synthase (P HS) and cytochrome P4501A1 (CYP1A1) protein were determined by Western blot/immunodetection with enhanced chemiluminescence. DNA oxidation a s 8-hydroxy-2'-deoxyguanosine formation was quantified using high-perf ormance liquid chromatography with electrochemical detection. Fibrobla st CYP1A1 activity assessed as ethoxyresorufin-O-deethylase was not de tectable, and even CYP1A1 protein was measurable only after induction with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). However, TCDD additio nally induced prostaglandin H synthase (PHS), which also was detectabl e constitutively. B[a]P 10 mu M initiated the oxidation of DNA and pro tein, and the formation of micronuclei, all of which were enhanced ove r 2-fold by the dual CYP1A1/PHS inducer TCDD 10 nM, as well as by othe r PHS inducers, 12-O-tetradecanoylphorbol-13-acetate 1 mu M and interl eukin-1 alpha 0.625 or 1.25 ng/ml, that do not induce CYP1A1 (p <.05). Conversely, B[a]P target oxidation and micronucleus formation were ab olished by 1-aminobenzotriazole 1mM (p <.05), which was a potent inhib itor of both peroxidases and P450. These results provide the first dir ect evidence that B[a]P-initiated micronucleus formation, like carcino genic initiation, requires enzymatic bioactivation, and that peroxidas e-dependent, reactive oxygen species-mediated oxidation of DNA, and po ssibly protein, constitutes a molecular mechanism of initiation in uni nduced cells. Induction of either CYP1A1 or peroxidases such as PHS su bstantially enhances this genotoxic initiation, which may reflect canc er risk. (C) 1997 Elsevier Science Inc.