Carcinogenesis, Vol 18, 1291-1297, Copyright © 1997 by Oxford University Press
K Mace, F Aguilar, JS Wang, P Vautravers, M Gomez-Lechon, FJ Gonzalez, J Groopman, CC Harris and AM Pfeifer
Epidemiological evidence has been supporting a relationship between dietary
aflatoxin B1 (AFB1) exposure, development of human primary hepatocellular
carcinoma (HCC) and mutations in the p53 tumor suppressor gene. However,
the correlation between the observed p53 mutations, the AFB1 DNA adducts
and their activation pathways has not been elucidated. Development of
relevant cellular in vitro models, taking into account species and tissue
specificity, could significantly contribute to the knowledge of
cytotoxicity and genotoxicity mechanisms of chemical procarcinogens, such
as AFB1, in humans. For this purpose a non-tumorigenic SV40-immortalized
human liver epithelial cell line (THLE cells) which retained most of the
phase II enzymes, but had markedly reduced phase I activities was used for
stable expression of the human CYP1A2, CYP2A6, CYP2B6 and CYP3A4 cDNA. The
four genetically engineered cell 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 hepatocytes. The T5-1A2, T5-2A6, T5-2B6 and T5-3A4 cell
lines exhibited a very high sensitivity to the cytotoxic effects of AFB1
and were approximately 125-, 2-, 2- and 15-fold, respectively, more
sensitive than the control T5-neo cells, transfected with an expressing
vector which does not contain CYP450 cDNA. In the CYP450-expressing cells,
nanomolar doses of AFB1-induced DNA adduct formation including
AFB1-N7-guanine, -pyrimidyl and -diol adducts. In addition, the T5-1A2
cells showed AFM1-DNA adducts. At similar levels of total DNA adducts, both
the T5-1A2 and T5-3A4 cells showed, at codon 249 of the p53 gene, AGG to
AGT transversions at a relative frequency of 15x10(-6). In contrast, 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 position of the codon
250. No significant AFB1-induced p53 mutations could be detected in the
T5-2A6 cells. Therefore, the differential expression of specific CYP450
genes in human hepatocytes can modulate the cytotoxicity, DNA adduct levels
and frequency of p53 mutations produced by AFB1.
ARTICLES
Aflatoxin B1-induced DNA adduct formation and p53 mutations in CYP450- expressing human liver cell lines
Nestle Research Centre, Department of Life Sciences, Lausanne, Switzerland.
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