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.

Figure 1

Metabolic formation of diol epoxides and PAH o-quinones.

Figure 2

Comparison of the mutation frequencies of PAH o-quinones. Data is presented as percentage of red colonies vs. the treatment indicated. Incubation conditions are described in Materials and Methods.

Figure 3

Patterns of mutations induced in p53 by PAH o-quinones. Data are presented as % mutation for the given base change. The twelve possible changes are reduced to six when complimentary changes are plotted together since the original base lesion cannot be determined from the mutant sequence. For comparison, the base substitution data for lung cancer (2,086 total substitutions) and all other cancers excluding lung cancer (16,557 total substitutions) are shown.

Figure 4

Mutation spectra induced in p53 by PAH o-quinones. The occurrence of point mutations are plotted against codon number for each PAH o-quinone tested under redox cycling conditions. For comparison, spectra of single base substitutions for lung cancer (2,027 singlet mutations) and all cancer excluding lung cancer (16,117 singlet mutations) are shown. Data was extracted from the p53 IARC database R10 release (50).

Figure 5

Mutation frequency of (±)-anti-BPDE, on unmethylated vs. methylated DNA.

Figure 6

Mutational patterns of (±)-anti-BPDE, induced in unmethylated p53 and methylated p53.

Figure 7

Mutational spectra of (±)-anti-BPDE, induced in unmethylated p53 and methylated p53.