Characterization of the major DNA adduct formed by the food mutagen 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeAalphaC) in primary rat hepatocytes.

Abstract

Cooking of proteinous food results in the formation of heterocyclic amines. Among these, 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeAalphaC) has been identified as a mutagenic pyrolysis product of soya protein and has been detected in grilled or pan fried meat. It was subsequently proven to be carcinogenic in mice and, recently, in rats and to form covalent DNA adducts in vitro and in vivo. The corresponding nitro compound, 2-nitro-3-methyl-9H-pyrido[2,3-b]indole (MeNalphaC), was prepared and shown to be a direct acting mutagen in the Ames Salmonella reversion assay. When MeNalphaC was chemically reduced in the presence of DNA a major DNA adduct was detected using the 32P-postlabelling assay. This major adduct was characterized by UV spectroscopy and mass spectrometry as N-(deoxyguanosin-8-yl)-2-amino-3-methyl-9H-pyrido[2,3-b]indole. This structure was corroborated by identification of the modified base as a guanine moiety modified at the C8 position as judged by chromatographic and spectral comparison with a standard synthesized from acetylated guanine-N3-oxide and MeAalphaC was characterized by UV/Vis and 1H NMR spectroscopy and mass spectrometry. Treatment of primary rat hepatocytes with MeAalphaC (100 microM, 24 h) resulted in adduct levels of 9.8 fmol/microg DNA as determined by 32P-postlabelling analysis. Using HPLC analysis, two major 32P-labelled adducts were observed accounting for 80 and 13% of total binding respectively. The major adduct was chromatographically indistinguishable from the synthetic deoxyguanosine-C8 adduct using both ion-exchange thin layer or reversed-phase HPLC. Although MeAalphaC is formed only in low p.p.b. levels in cooked food, the contribution to the human carcinogenic risk that might be imposed by heterocyclic amines is not to be neglected.