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.

Authors: Shen, Yu-Min; Troxel, Andrea B; Vedantam, Srilakshmi; Penning, Trevor M; Field, Jeffrey

Published In Chem Res Toxicol, (2006 Nov)

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.

PubMed ID: 17112231

MeSH Terms: 7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide/toxicity*; Genes, Reporter/drug effects; Humans; Molecular Structure; Mutagenicity Tests; Mutagens/toxicity; Mutation*; Polycyclic Aromatic Hydrocarbons/metabolism; Polycyclic Aromatic Hydrocarbons/toxicity*; Quinones/metabolism; Quinones/toxicity*; Reactive Oxygen Species/chemistry; Reactive Oxygen Species/toxicity*; Saccharomyces cerevisiae; Tumor Suppressor Protein p53/drug effects; Tumor Suppressor Protein p53/genetics*