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 and 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 the 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 (±) antibenzo[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 μM 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 would be expected by a random distribution. The dependence of G to T transversions on redoxcycling 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 μM). 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-N 2 -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. KeywordsAldo-keto reductase; polycyclic aromatic hydrocarbon; ortho-quinones; reactive oxygen species Supporting Information Available: Tables of supplemental data S1-S6. These tables list the mutations observed for each PAH o-quinone, BPDE and MNNG in unmethylated or methylated p53 cDNA by codon number, the nucleotide change observed, the sequence context (5′ and 3′-codons), the type of mutation, and the amino acid substitution. This material is available free of charge via the internet at

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