Mutagenesis of the N-(deoxyguanosin-8-yl)-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine DNA adduct in mammalian cells - Sequence context effects

Site-specifically modified oligodeoxynucleotides were used to investigate t he mutagenic properties of a major cooked food mutagen-derived DNA adduct, N-(deoxyguano sin-8-yl)-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (dG -C8-PhIP). dG-C8-PhIP-modified oligodeoxynucleotides were prepared by react ing an oligodeoxynucleotide containing a single dG (5'-TCCTC-CTX (G) under bar CCTCTC, where X = C, A, G, or T) with N-acetoxy-PhIP. The unmodified an d dG-C8-PhIP-modified oligomers were inserted into single stranded phagemid vectors. These single-stranded vectors were transfected into simian kidney (COS-7) cells. The progeny plasmid obtained was used to transform Escheric hia coli DH10B, When dC was at the 5'-flanking position to dG-C8-PhIP, pref erential incorporation of dCMP, the correct base, was observed opposite the dG-C8-PhIP. Targeted G --> T transversions were detected, along with lesse r amounts of G --> A transitions and G --> C transversions. No mutations we re detected for the unmodified vector. The influence of sequence context on the dG-C8-PhIP mutation frequency and spectrum was also explored. When the dC 5'-flanking base was replaced by dT, dG or dG, the mutational spectra w ere similar to that observed with dC-flanking base. Higher mutational frequ encies (28-30%) were observed when dC or dG was 5' to dG-C8-PhIP, A lower m utational frequency (13%) was observed when dA was at the 5' to the lesion. Single-base deletions were detected only when dG or dT flanked the adduct, We conclude that dG-C8-PhIP is mutagenic, generating primarily G --> T tra nsversions in mammalian cells. The mutational frequency and specificity of dG-C8-PhIP vary depending on the neighboring sequence context.