Quantitation and mapping of aflatoxin B1-induced DNA damage in genomic DNAusing aflatoxin B1-8,9-epoxide and microsomal activation systems

Aflatoxin BI (AFB1) is a mutagenic and carcinogenic mycotoxin which may pla y a role in the etiology of human liver cancer. In vitro studies have shown that AFB1 adducts form primarily at the N7 position of guanine. Using quan titative PCR (QPCR) and ligation-mediated PCR (LMPCR), we have mapped total AFB1 adducts in genomic DNA treated with AFB1-8,9-epoxide and in hepatocyt es exposed to AFB1 activated by rat liver microsomes or human liver and ent erocyte microsomal preparations. The p53 gene-specific adduct frequencies i n DNA, modified in cells with 40-400 mu M AFB1, were 0.07-0.74 adducts per kilobase (kb). In vitro modification with 0.1-4 ng AFB1-8,9-epoxide per mic rogram DNA produced 0.03-0.58 lesions per kb. The adduct patterns obtained with the epoxide and the different microsomal systems were virtually identi cal indicating that adducts form with a similar sequence-specificity in vit ro and in vivo. The lesions were detected exclusively at guanines with a pr eference towards GpG and methylated CpG sequences. The methods utilizing QP CR and LMPCR thus provide means to assess gene-specific and sequence-specif ic AFB1 damage, The results also prove that microsomally-mediated damage is a suitable method for avoiding manipulations with very unstable DNA-reacti ve metabolites and that this damage can be detected by QPCR and LMPCR. (C) 1999 Elsevier Science B.V. All rights reserved.