title: p53 controls global nucleotide excision repair of low levels of structurally diverse benzo(g)chrysene-DNA adducts in human fibroblasts.
abstract: Benzo(g)chrysene is a widespread environmental contaminant and potent carcinogen. We have measured the formation and nucleotide excision repair of covalent DNA adducts formed by the DNA-reactive metabolite of this compound in human fibroblasts, in which expression of the p53 tumor suppressor gene could be controlled by a tetracycline-inducible promoter. Cells were exposed for 1 h to 0.01, 0.1, or 1.2 microM (+/-)-anti-benzo(g)chrysene diol-epoxide, and DNA adducts were assessed at various post-treatment times by subjecting isolated DNA to (32)P-postlabeling analysis. Four major DNA adducts were detected, corresponding to the reaction of either the (+)- or (-)-anti-benzo(g)chrysene diol-epoxide stereoisomer with adenine or guanine. Treatment with 1.2 microM resulted in a level of 1100 total adducts/10(8) nucleotides for both p53-proficient and -deficient cells; removal of adducts was not observed in either case. In cells treated with 0.1 microM, the maximum level of total adducts at 24 h was 150/10(8) nucleotides in p53-proficient cells and 210 adducts/10(8) nucleotides in p53-deficient cells. A concentration of 0.01 microM resulted in a maximum of 20 adducts/10(8) nucleotides in p53-proficient cells at 4 h, but 40 adducts/10(8) nucleotides persisted in p53-deficient cells at 24 h. Whereas there were clear differences in the time course of adduct levels in p53-proficient compared with p53-deficient cells treated with 0.1 microM or 0.01 microM, these levels did not decrease extensively over 3 days. This is likely because of the stabilization of the diol-epoxide in cells, and consequent exposure and formation of adducts for many hours after the initial treatment. Furthermore, despite minor quantitative differences, all 4 of the adducts behaved similarly with respect to the effect of p53 expression on their removal. p53 appears to minimize the appearance of benzo(g)chrysene adducts in human cells by up-regulating global nucleotide excision repair and reducing the maximum adduct levels achieved. The fact that this p53-dependent effect is noted at levels of DNA adducts that are commonly found in human tissues (i.e., <100 adducts/10(8) nucleotides) because of environmental factors such as smoking is particularly significant with respect to human carcinogenesis related to environmental exposure.
authors.affiliation: Department of Biological Sciences, Stanford University, 371 Serra Mall, Stanford, CA 94305-5020, USA.
authors.forename: Daniel R
authors.initials: DR
authors.lastname: Lloyd
authors.forename: Philip C
authors.initials: PC
authors.lastname: Hanawalt
mesh_terms: D002274:Carcinogens, Environmental / Q000378:metabolism* / Q000633:toxicity; D002478:Cells, Cultured; D002911:Chrysenes / Q000378:metabolism* / Q000633:toxicity; D004247:DNA / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D018736:DNA Adducts / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D004260:DNA Repair / Q000502:physiology*; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D006801:Humans; D010761:Phosphorus Radioisotopes; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism / Q000502:physiology*
chemical_list: D002274:Carcinogens, Environmental; D002911:Chrysenes; D018736:DNA Adducts; D010761:Phosphorus Radioisotopes; D016159:Tumor Suppressor Protein p53; C100422:benzo(g)chrysene; D004247:DNA