title: Comparison of p53 mutations induced by PAH o-quinones with those caused by anti-benzo[a]pyrene diol epoxide in vitro: role of reactive oxygen and biological selection.
abstract: Polycyclic aromatic hydrocarbons (PAH) are one of the major carcinogens in tobacco smoke. They are metabolically activated through different routes to form either diol-epoxides, PAH o-quinones, or radical cations, each of which has been proposed to be an ultimate carcinogen. To study how PAH metabolites mutate p53, we used a yeast reporter gene assay based on p53 transcriptional activity. Colonies expressing wt p53 turn white (ADE +) and those expressing mutant p53 turn red (ADE -). We examined the mutagenicity of three o-quinones, benzo[a]pyrene-7,8-dione, benz[a]anthracene-3,4-dione, and dimethylbenz[a]anthracene-3,4-dione, and compared them with (+/-)-anti-benzo[a]pyrene diol epoxide ((+/-)-anti-BPDE) within the same system. The PAH o-quinones tested gave a dose-dependent increase in mutation frequency in the range of 0.160-0.375 microM quinone, provided redox-cycling conditions were used. The dominant mutations were G to T transversions (>42%), and the incidence of hotspot mutations in the DNA-binding domain was more than twice than that expected by a random distribution. The dependence of G to T transversions on redox cycling implicates 8-oxo-dGuo as the lesion responsible, which is produced under identical conditions (Chem. Res. Toxicol. (2005) 18, 1027). A dose-dependent mutation frequency was also observed with (+/-)-anti-BPDE but at micromolar concentrations (0-20 microM). The mutation pattern observed was G to C (63%) > G to A (18%) > G to T (15%) in umethylated p53 and was G to A (39%) > G to C (34%) > G to T (16%) in methylated p53. The preponderance of G mutations is consistent with the formation of anti-BPDE-N2-dGuo as the major adduct. The frequency of hotspots mutated by (+/-)-anti-BPDE was essentially random in umethylated and methylated p53, suggesting that 5'-CpG-3' islands did not direct mutations in the assay. These data suggest that smoking may cause mutations in p53 by formation of PAH o-quinones, which produce reactive oxygen species. The resultant 8-oxo-dGuo yields a pattern of mutations but not a spectrum consistent with that seen in lung cancer; we suggest that the emergence of the spectrum requires biological selection.
authors.affiliation: Department of Pharmacology, Center of Excellence in Environmental Toxicology and Center for Clinical Epidemiology and Biostatistics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6084, USA.
authors.forename: Yu-Min
authors.initials: YM
authors.lastname: Shen
authors.forename: Andrea B
authors.initials: AB
authors.lastname: Troxel
authors.forename: Srilakshmi
authors.initials: S
authors.lastname: Vedantam
authors.forename: Trevor M
authors.initials: TM
authors.lastname: Penning
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Field
mesh_terms: D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide / Q000633:toxicity*; D017930:Genes, Reporter / Q000187:drug effects; D006801:Humans; D015394:Molecular Structure; D009152:Mutagenicity Tests; D009153:Mutagens / Q000633:toxicity; D009154:Mutation; D011084:Polycyclic Aromatic Hydrocarbons / Q000378:metabolism / Q000633:toxicity*; D011809:Quinones / Q000378:metabolism / Q000633:toxicity*; D017382:Reactive Oxygen Species / Q000737:chemistry / Q000633:toxicity*; D012441:Saccharomyces cerevisiae; D016159:Tumor Suppressor Protein p53 / Q000187:drug effects / Q000235:genetics*
chemical_list: D009153:Mutagens; D011084:Polycyclic Aromatic Hydrocarbons; D011809:Quinones; D017382:Reactive Oxygen Species; D016159:Tumor Suppressor Protein p53; D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide