AFLATOXIN B-1-INDUCED DNA ADDUCT FORMATION AND P53 MUTATIONS IN CYP450-EXPRESSING HUMAN LIVER-CELL LINES

Epidemiological evidence has been supporting a relationship between di etary aflatoxin B-1 (AFB(1)) exposure, development of human primary he patocellular carcinoma (HCC) and mutations in the p53 tumor suppressor gene. However, the correlation between the observed p53 mutations, th e AFB(1) DNA adducts and their activation pathways has not been elucid ated. Development of relevant cellular in vitro models, taking into ac count species and tissue specificity, could significantly contribute t o the knowledge of cytotoxicity and genotoxicity mechanisms of chemica l procarcinogens, such as AFB(1), in humans. For this purpose a non-tu morigenic SV40-immortalized human liver epithelial cell line (THLE cel ls) which retained most of the phase II enzymes, but had markedly redu ced phase I activities was used for stable expression of the human CYP 1A2, CYP2A6, CYP2B6 and CYP3A4 cDNA. The four genetically engineered c ell lines (T5-1A2, T5-2A6, T5-2B6 and T5-3A4) produced high levels of the specific CYP450 proteins and showed comparable or higher catalytic activities related to the CYP450 expression when compared to human he patocytes. The T5-1A2, T5-2A6, T5-2B6 and T5-3A4 cell lines exhibited a very high sensitivity to the cytotoxic effects of AFB(1) and were ap proximately 125-, 2-, 2- and 15-fold, respectively, more sensitive tha n the control T5-neo cells, transfected with an expressing vector whic h does not contain CYP450 cDNA. In the CYP450-expressing cells, nanomo lar doses of AFB(1)-induced DNA, adduct formation including AFB(1)-N-7 -guanine, -pyrimidyl and -diol adducts. In addition, the T5-1A2 cells showed AFM(1)-DNA adducts. At similar levels of total DNA adducts, bot h the T5-1A2 and T5-3A4 cells showed, at codon 249 of the p53 gene, AG G to AGT transversions at a relative frequency of 15x10(-6). In contra st, only the T5-3A4 cells showed CCC to ACC transversion at codon 250 at a high frequency, whereas the second most frequent mutations found in the T5-1A2 cells were C to T transitions at the first and second po sition of the codon 250. No significant AFB(1)-induced p53 mutations c ould be detected in the T5-2A6 cells. Therefore, the differential expr ession of specific CYP450 genes in human hepatocytes can modulate the cytotoxicity, DNA adduct levels and frequency of p53 mutations produce d by AFB(1).