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

Abstract

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.

TP53 mutations and hepatocellular carcinoma: insights into the etiology and pathogenesis of liver cancer

“…In vitro studies exposing human liver cell lines to AFB 1 presented the same 249 ser mutational pattern of TP53 (Aguilar et al, 1993;Mace et al, 1997), as it has been reported in the epidemiological studies ( Figure 1c). There are at least two possible explanations for these findings.…”

Desacetyl-Diltiazem Displays Severalfold Higher Affinity to CYP2D6 Compared with CYP3A4

“…THLE cells, obtained by transfection of the simian virus 40 large tumor antigen gene into primary hepatocytes (Pfeifer et al, 1995), did not express several important P450 isoenzymes, but clones expressing CYP3A4 (T5-3A4) or CYP2D6 (T5-2D6) were produced by subsequent transfection (Mace et al, 1997). Cell-culturing details are presented elsewhere (Molden et al, 2000).…”

Aflatoxin variations in maize flour and grains collected from various regions of Kenya

“…These compounds are immunosuppressive, carcinogenic, teratogenic and mutagenic, and acute exposure to high levels of aflatoxin leads to aflatoxicosis, which can result in rapid death from liver failure. Symptoms of acute aflatoxicosis include, but are not limited to, swollen stomach, fatigue, swollen legs, and eyes turning yellowish [11,12]. In 2004, during the worst known outbreak of aflatoxicosis in Kenya, 317 cases were reported of which 125 died [13].…”