Aflatoxin B1-induced DNA adduct formation and p53 mutations in CYP450- expressing human liver cell lines

Epidemiological evidence has been supporting a relationship between dietaryaflatoxin B1 (AFB1) exposure, development of human primary hepatocellularcarcinoma (HCC) and mutations in the p53 tumor suppressor gene. However,the correlation between the observed p53 mutations, the AFB1 DNA adductsand their activation pathways has not been elucidated. Development ofrelevant cellular in vitro models, taking into account species and tissuespecificity, could significantly contribute to the knowledge ofcytotoxicity and genotoxicity mechanisms of chemical procarcinogens, suchas AFB1, in humans. For this purpose a non-tumorigenic SV40-immortalizedhuman liver epithelial cell line (THLE cells) which retained most of thephase II enzymes, but had markedly reduced phase I activities was used forstable expression of the human CYP1A2, CYP2A6, CYP2B6 and CYP3A4 cDNA. Thefour genetically engineered cell lines (T5-1A2, T5-2A6, T5-2B6 and T5-3A4)produced high levels of the specific CYP450 proteins and showed comparableor higher catalytic activities related to the CYP450 expression whencompared to human hepatocytes. The T5-1A2, T5-2A6, T5-2B6 and T5-3A4 celllines exhibited a very high sensitivity to the cytotoxic effects of AFB1and were approximately 125-, 2-, 2- and 15-fold, respectively, moresensitive than the control T5-neo cells, transfected with an expressingvector which does not contain CYP450 cDNA. In the CYP450-expressing cells,nanomolar doses of AFB1-induced DNA adduct formation includingAFB1-N7-guanine, -pyrimidyl and -diol adducts. In addition, the T5-1A2cells showed AFM1-DNA adducts. At similar levels of total DNA adducts, boththe T5-1A2 and T5-3A4 cells showed, at codon 249 of the p53 gene, AGG toAGT transversions at a relative frequency of 15x10(-6). In contrast, onlythe T5-3A4 cells showed CCC to ACC transversion at codon 250 at a highfrequency, whereas the second most frequent mutations found in the T5-1A2cells were C to T transitions at the first and second position of the codon250. No significant AFB1-induced p53 mutations could be detected in theT5-2A6 cells. Therefore, the differential expression of specific CYP450genes in human hepatocytes can modulate the cytotoxicity, DNA adduct levelsand frequency of p53 mutations produced by AFB1.