Excision Repair of Aflatoxin B^DNA Adducts in Human Fibroblasts1

The processing of covalent aflatoxin B, (AFBi)-DNA adducts was investigated in confluent normal fibroblasts (NF) and xeroderma pigmentosum skin fibroblasts of Complementation Group A (XPA) following treatment with rat liver microsomeactivated AFB, for 30 min. Following rapid DMA isolation at slightly acidic pH by a new filter technique, more than 90% of the adducts corresponded to 2,3-dihydro-2-(A/7-guanyl)-3-hydroxy-AFBi (AFBi-N7-Gua) according to the analysis of acid hydrolysates by high-pressure liquid chromatography. The changes in adduct concentration and composition were com pared between DNA isolated immediately following AFB, treat ment and incubated at neutrality in vitro and DNA in situ in the cell isolated after different lengths of incubation. The following conclusions were reached from the observed differences in the kinetics of adduct removal from free DNA and DNA in situ in NF and XPA: (a) AFB,-N7-Gua is removed spontaneously and enzymatically in NF but probably only spontaneously in XPA. This result suggests that these lesions are removed via nucleofide excision repair in NF; (b) the putative 2,3-dihydro-2-(/V5formyl)-2',5',6'-triamino-4'-oxo-/V5-pyrimidyl)-3-hydroxyaflatoxin B! is formed in a secondary reaction from AFBi-N7-Gua in vitro and in situ in the cell. It accumulates more rapidly and to a greater extent in XPA than in NF and persists in both cell types over prolonged periods. The reaction of AFB,-/V7-Gua to 2,3-dihydro-2-(W5-formyl)-2',5',6'-triamino-4'-oxo-W5-pyrimidyl)-3-hydroxyaflatoxin B, represents the transformation of a repairable lesion to a nonrepairable, persistent lesion.