Identification of the major hepatic DNA adduct formed by the food mutagen 2-amino-9H-pyrido[2,3-b]indole (A alpha C).

2-Amino-9H-pyrido[2,3-b]indole (A alpha C) is among the most prevalent heterocyclic amines detected in grilled or panfried meat; it was shown to be carcinogenic in mice, to induce preneoplastic foci in rat liver, and to form covalent DNA adducts in vitro and in vivo. The corresponding nitro compound 2-nitro-9H-pyrido[2,3-b]indole (N alpha C) was prepared and shown to be a direct acting mutagen in the Salmonella assay, while the amino compound required external metabolic activation with rat liver homogenate (S9). When A alpha C was incubated with S9 in the presence of calf thymus DNA, one major DNA adduct spot was detected upon 32P-postlabeling analysis. This adduct comigrated on ion-exchange TLC and reversed-phase HPLC with the major adduct detected in primary hepatocytes treated with A alpha C. In DNA isolated from livers of male F344 rats treated with 800 and 160 ppm, the formation of the same major adduct was observed with relative adduct levels of 20.6 +/- 9.6 and 1.4 +/- 1.1 adducts/10(8), respectively, as determined with the butanol extraction variant of the 32P-postlabeling assay. No DNA adducts were detected in liver DNA from rats treated with 32 ppm A alpha C or control animals. The major adduct spot was eluted and hydrolyzed and the modified base characterized by chromatographic and UV spectral comparison with a synthetic standard synthesized from acetylated guanine N3-oxide and A alpha C. Electrospray mass spectrometry and 1H- and 13C-NMR spectroscopy provided further evidence for the major adduct as N2-(guanin-8-yl)-2-amino-9H-pyrido[2,3-b]indole. A alpha C is formed especially in high-temperature preparation of food and may contribute considerably to the human carcinogenic risk that might be imposed by heterocyclic amines.