Formation of 2-amino-α-carbolines in pan-fried poultry and 32P-postlabeling analysis of DNA adducts

Amino-alpha-carbolines are food-derived genotoxic heterocyclic aromatic amines (HAAs) that have been shown to be carcinogenic in rodents, to induce preneoplastic foci in rat liver and to be active in a number of genotoxicity tests, inducing gene mutations, DNA strand breaks and cell transformation. 2-Amino-alpha-carboline (A alpha C) has been estimated to account for about 20% of the carcinogenic HAAs that are ingested with the diet. Using the tandem cartridge solid-phase extraction procedure we were able to show that in pan-fried poultry the proportion of A alpha C and its 3-methyl derivative (MeA alpha C) account for 15% of the HAA fraction. Covalent DNA adducts are considered to be the primary lesions in chemical carcinogenesis. Both A alpha C and MeA alpha C have been shown to induce covalent DNA adducts in vitro and in vivo. Formation of DNA adducts was investigated by P-32-postlabelling analysis in vitro in primary rat hepatocytes treated with 10-500 mu M A alpha C or MeA alpha C. While for MeAnC-derived adducts the highest response was obtained with the nuclease P1 enhancement procedure, AaC adducts were most efficiently detected upon P-32-postlabelling preceded by a cartridge enrichment step. Adduct levels detected were dose dependent but decreased for the higher doses due to cytotoxic effects. We have characterised the major DNA adducts of A alpha C and MeA alpha C as deoxyguanosine derivatives with the amino-alpha-carboline bound via the 2-amino group to the C8 position of guanine.