Preferential DNA damage in the p53 gene by benzo[a]pyrene metabolites in cytochrome P4501A1-expressing xeroderma pigmentosum group A cells.
Gene-specific DNA damage levels were determined by quantitative polymerase chain reaction (QPCR) after treating cytochrome P450 (CYP) 1A1-expressing xeroderma pigmentosum fibroblasts with [3H]benzo[a]pyrene-trans-7,8-dihydrodiol ([3H]BPD) or [3H]benzo[a]pyrene-trans-7,8-dihydrodiol-9,10-epoxide ([3H]BPDE). DNA damage in the p53 gene (which is transcriptionally active) and the beta-globin gene (which is transcriptionally inactive) was measured in cells treated with [3H](+/-)-anti-BPDE, [3H](+/-)-BPD, and [3H](-)-BPD. DNA adduct formation in the genome overall was determined by measuring the incorporation of 3H into DNA. DNA damage in a p53 gene fragment (exons 8-9, 445 bp) was readily detected 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 more damage in the p53 gene than BPDE treatment did. The lesion frequencies in the p53 and beta-globin genes in purified DNA treated with BPDE in vitro were the same, indicating that there was no sequence-specific basis for preferential lesion formation in the p53 gene in treated cells. DNA damage in both the p53 and beta-globin genes showed a dose response to [3H](-)-BPD. The frequency of BPD-induced lesions in the p53 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 bulk DNA. The data indicate that the distribution of BPDE-induced DNA lesions is dramatically nonrandom and suggest that the nonrandomness is governed by DNA sequence composition, chromatin structure, and dose rate.
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Center for Molecular Medicine and Genetics, Wayne State University, Detroit, Michigan 48201, USA.
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D001483:Base Sequence; D001564:Benzo(a)pyrene / Q000031:analogs & derivatives; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D018736:DNA Adducts / Q000378:metabolism*; D004249:DNA Damage; D017931:DNA Primers / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D015870:Gene Expression; D016158:Genes, p53; D005914:Globins / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods; D014158:Transcription, Genetic; D014983:Xeroderma Pigmentosum / Q000378:metabolism
D018736:DNA Adducts; D017931:DNA Primers; D001564:Benzo(a)pyrene; D005914:Globins; D003577:Cytochrome P-450 Enzyme System