Mutagenic specificity of 2-acetylaminonaphthalene-derived DNA adduct in mammalian cells

2-Acetylaminonaphthalene (2-AAN) has been recognized as a urinary bladder carcinogen in humans. The deacetylated form, 2-aminonaphthalene (2-AN), is metabolized in vivo and reacts primarily with guanine residues in DNA, resulting in the formation of dG-N2-aminonaphthalene (dG-N2-AN) adduct. Phosphoramidite chemical procedure has recently been established in our laboratory to prepare oligodeoxynucleotides containing a single dG-N2-acetylaminonaphthalene (dG-N2-AAN) adduct. Oligodeoxynucleotides (5′TCCTCCTNXCCTCTC, where X is dG or dG-N2-AAN and N is C, A, T or G) with different bases 5′ flanking to the lesion were prepared and were inserted into a single-strand shuttle vectors and used to establish the mutational frequency and specificity of dG-N2-AAN adduct in simian kidney cells. dG-N2-AAN adduct promoted preferential incorporation of dCMP, the correct base, opposite the lesion. When the 5′ flanking base to the lesion was C, A or T, the mutational frequency was under 2.1%. When G flanked to the lesion, the mutational frequency was slightly increased to 4.2%. Misincorporation of dAMP, dTMP, and/or dGMP varied depending on the 5′ flanking base. When dG-N2-AAN was positioned at codon 61 of noncoding strand of human c-Ha-ras1 gene (5′TCCTCCTXGCCTCTC, where X is dG-N2-AAN), the mutational frequency was 6.7%; G → T transversions (4.7%), followed by G → A transition (2.0%), were observed. These results demonstrated that dG-N2-AAN is a weak mutagenic lesion in mammalian cells. The influence of 5′ flanking sequence context was observed on the mutational frequency and specificity of this adduct.