In vitro recapitulating of TP53 mutagenesis in hepatocellular carcinoma associated with dietary aflatoxin B1 exposure.

Abstract

Dietary exposure to aflatoxin B(1) (AFB(1)), in addition to other known factors, increases risk for human hepatocellular carcinoma (HCC). HCCs from AFB(1)-exposed individuals frequently have distinct TP53 mutations, such as G to T transversions in the second guanine of codon 249 (AGG to AGT), and a characteristic mutational spectrum predominated by G:C to T:A mutations. To recapitulate the distinctive features of TP53 mutations in AFB(1)-associated HCC, we investigated AFB(1)-induced DNA adduction in relation to mutagenesis in transgenic mouse fibroblasts exposed to AFB(1) in vitro. Immunodotblot determination of DNA adducts in the overall genome of AFB(1)-exposed cells revealed the dose-dependant formation of persistent imidazole ring-opened AFB(1)-DNA adducts. DNA footprinting analysis of the cII transgene in AFB(1)-exposed cells verified the dose-dependent and sequence-specific formation of DNA adducts. The preferential formation of AFB(1)-induced DNA adducts along the cII transgene was almost exclusively localized to guanine-containing sequences encompassing CpG dinucleotides. Mutation analysis of the cII transgene in AFB(1)-exposed cells revealed a dose-dependent induction of cII mutant frequency (P < .001) and a unique induced mutational spectrum characterized by predominant induction of G:C to T:A transversions that occurred within CpG sequence contexts. Notably, codons 42 and 45 of the cII transgene, which have identical sequence contexts to that of codon 249 of human TP53, constituted 2 frequently mutated sites in AFB(1)-exposed cells that contained the G to T transversion signature mutation at their third base positions. In this model system, AFB(1)-induced DNA adduction and mutagenesis recapitulate the unique mutational features of TP53 in AFB(1)-associated human HCC.