Aflatoxin B1 formamidopyrimidine adducts are preferentially repaired by the nucleotide excision repair pathway in vivo

Aflatoxin B1 (AFB1), the most potent member of the aflatoxin family of hepatocarcinogens, upon metabolic activation reacts with DNA and forms a population of covalent adducts. The most prevalent adduct, 8,9-dihydro-8-(N7-guanyl-)-9-hydroxyaflatoxin (AFB1-N7-dG), as well as the AFB1 formamidopyrimidine adduct (AFB1-FAPY), resulting from imidazole ring opening of the major adduct, are thought to be responsible for mutations caused by AFB1. The AFB1-N7-dG lesions are rapidly removed in Escherichia coli and mammals, whereas the AFB1-FAPY lesions persist in mammalian cells, which along with the higher stability of this lesion suggests that AFB1-FAPY might significantly contribute to the observed toxicity and carcinogenicity of AFB1 in higher organisms. Other workers have shown in vitro evidence that AFB1-FAPY lesions are substrates for both nucleotide excision repair (NER) and base excision repair (BER). The present study, done in vivo, utilized a modified host cell reactivation assay and showed that AFB1-FAPY lesions are preferentially repaired in E.coli by NER. Comparisons of repair in wild-type, NER-deficient (uvrA), BER-deficient (mutM) and NER/BER double mutant E.coli strains transformed with plasmids enriched for AFB1-N7-dG or AFB1-FAPY lesions indicate that both lesions are efficiently repaired by NER-proficient cells (both wild-type and BER-deficient strains). We have found that the level of activity of the reporter gene is significantly affected by the presence of either lesion in NER-deficient strains due to the lack of repair. This effect is similar in NER-deficient and NER/BER-deficient strains indicating that BER (specifically in the strains we investigated) does not contribute significantly to the repair of these lesions in vivo. Consistent with this finding, in vitro analysis of AFB1-FAPY adduct excision by purified MutM and its functional analog human 8-oxoguanine DNA glycosylase using site-specifically modified oligonucleotides indicates that this lesion is a poor substrate for both proteins compared with canonical substrates for these enzymes, such as 7,8-dihydro-8-oxoguanine and methylformamidopyrimidine.