PREFERENTIAL DNA-DAMAGE IN THE P53 GENE BY BENZO[A]PYRENE METABOLITESIN CYTOCHROME P4501A1-EXPRESSING XERODERMA-PIGMENTOSUM GROUP-A CELLS

Gene-specific DNA damage levels were determined by quantitative polyme rase chain reaction (QPCR) after treating cytochrome P450 (CYP) 1A1-ex pressing xeroderma pig mentosum fibroblasts with [H-3] benzo[a]pyrene- trans-7,8-dihydrodiol ([H-3]BPD) or ]benzo[a]pyrene-trans-7,8-dihydrod iol-9,10-epoxide ([H-3]BPDE). DNA damage in the p53 gene (which is tra nscriptionally active) and the beta-globin gene (which is transcriptio nally inactive) was measured in cells treated with [H-3](+/-)-anti-BPD E, [H-3](+/-)-BPD, and [H-3](-)-BPD. DNA adduct formation in the genom e overall was determined by measuring the incorporation of H-3 into DN A. DNA damage in a p53 gene fragment (exons 8-9, 445 bp) was readily d etected by QPCR. DNA damage was either not detected or much reduced in a similarly sized target in the beta-globin gene (exons 1-2, 551 bp). At equivalent levels of genomic DNA adducts, BPD treatment induced mo re damage in the p53 gene than BPDE treatment did. The lesion frequenc ies in the p53 and beta-globin genes in purified DNA treated with BPDE in vitro were the same, indicating that there was no sequence-specifi c basis for preferential lesion formation in the p53 gene in treated c ells. DNA damage in both the p53 and beta-globin genes showed a dose r esponse to [H-3](-)-BPD. The frequency of BPD-induced lesions in the p 53 gene was sixfold to sevenfold greater than in the beta-globin gene and 200- to 300-fold greater than in bulk DNA. The BPD-induced lesion frequency in the beta-globin gene was 30- to 50-fold greater than in b ulk DNA. The data indicate that the distribution of BPDE-induced DNA l esions is dramatically nonrandom and suggest that the nonrandomness is governed by DNA sequence composition, chromatin structure,and dose ra te. (C) 1996 Wiley-Liss, Inc.