A Single Site-specific trans-Opened 7,8,9,10-Tetrahydrobenzo[a]pyrene 7,8-Diol 9,10-EpoxideN 2-Deoxyguanosine Adduct Induces Mutations at Multiple Sites in DNA

Site-specific mutagenicity of trans- opened adducts at the exocyclic N 2 -amino group of guanine by the (+)-(7 R, 8 S, 9 S ,10 R )- and (−)-(7 S, 8 R, 9 R ,10 S )-enantiomers of a benzo[ a ]pyrene 7,8-diol 9,10-epoxide (7-hydroxyl and epoxide oxygen are trans , BPDE-2) has been determined in Chinese hamster V79 cells and their repair-deficient counterpart, V-H1 cells. Four vectors containing single 10 S -BPDE-dG or 10 R -BPDE-dG adducts positioned at G 0 or G −1 in the analyzed 5′-ACTG 0 G −1 GA sequence of the non-transcribed strand were separately transfected into the cells. Mutations at each of the seven nucleotides were analyzed by a novel primer extension assay using a mixture of one dNTP complementary to the mutated nucleotide and three other ddNTPs and were optimized to quantify levels of a mutation as low as 1%. Only G → T mutations were detected at the adducted sites; the 10 S adduct derived from the highly carcinogenic (+)-diol epoxide was 40–50 and 75–140% more mutagenic than the 10 R adduct in V79 and V-H1 cells, respectively. Importantly, the 10 S adducts, but not the 10 R adducts, induced separate non-targeted mutations at sites 5′ to the G −1 and G 0 lesions (G 0 → T and C → T, respectively) in both cell lines. Neither the T 5′ to G 0 nor sites 3′ to the lesions showed mutations. Non-targeted mutations may enhance overall mutagenicity of the 10 S -BPDE-dG lesion and contribute to the much higher carcinogenicity and mutagenicity of (+)-BPDE-2 compared with its (−)-enantiomer. Our study reports a definitive demonstration of mutations distal to a site-specific polycyclic aromatic hydrocarbon adduct.