Aflatoxin B1 induces the transversion of G-->T in codon 249 of the p53 tumor suppressor gene in human hepatocytes.

Abstract

Hepatoceliular carcinoma (HCC) represents a major cause of mortality in certain areas ofthe world. Contamination offood with the mycotoxin aflatoxin B1 (AFB1) has been implicated as an etiological factor in certain regions of eastern Asia and subSaharan Africa (1, 2). Indeed, recent studies suggest that hepatitis B virus and AFB1 may exert a synergistic effect (3). Approximately 50%6 of HCC in high AFB1 regions (4, 5), but only 20%o in low AFB1 regions, harbors mutations in the p53 tumor suppressor gene, and the spectrum of mutations is quite different (6, 7). More than half of the tumors from high AFB1 regions contain G --T transversions in the third position of codon 249 (AGG), resulting in the replacement of arginine by serine (4-8). In contrast, mutations are distributed throughout the highly conserved domains IV and V of p53 in HCC from low AFB1 regions and no prevalence of G --T transversions is observed (9, 10). These results indicate that the substitution of arginine 249 by serine in the p53 protein is not required for hepatocarcinogenesis in man. Though G --T transversions are in agreement with the mutational specificity of AFB1 (11, 12) and other carcinogens forming bulky DNA adducts (13,14), there is no convincing explanation for the prevalence of mutations in codon 249, which almost never harbors mutations in other forms of human cancer. Many factors determine the mutability of a particular gene sequence, in addition to the chemical properties of the ultimate mutagen, and it is difficult to predict on the basis of model experiments. Among them, sequence context, local DNA conformation, chromatin structure, transcriptional activity, and repairability are recognized to play a role. Therefore, we have directly evaluated the mutability to The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.AFB1 of codons 247-250 in human hepatocarcinoma cells HepG2 by genotypic analysis using Msp I and Hae III restriction fragment length polymorphism/polymerase chain reaction (RFLP/PCR) (15-17). Our results indicate that AFB1 induces the transversion of G --T in the third position of codon 249, with the highest frequency producing the same mutation that is found almost exclusively in HCC from high AFB1 regions in east Asia and Africa. Our results support the notion that AFB1 represents a causative carcinogen in hepatocarcinogenesis in these regions of the world. MATERIALS AND METHODSHepG2 human hepatocarcinoma cells were grown in minimum essential medium supplemented with 10% fetal calf serum to =60% confluency before treatment for 30 min with AFB1 (0.5 ug/ml) in the presence of rat liver microsomes as described (18)(19)(20). The medium was replaced and cell growth continued for 0 hr or for 96 hr, respectively. Despite AFB1 treatment, the cell number doubled within 96 hr. Control cultures were sham-treated only with rat liver microsomes.

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.…”

“…Aguilar et al (1994) have shown a higher relative abundance of the 249 ser mutant liver cells, in nontumorous liver, by using a highly sensitive genotypic mutation assay. They suggested that this early mutational event may be due to the high mutability of the third base at codon 249 to AFB 1 , as suggested by previous in vitro studies in human liver cells (Aguilar et al, 1993;Cerutti et al, 1994). Another possible explanation might be that the 249 ser mutant p53 protein may provide a special growth and/or survival advantage to these liver cells (Puisieux et al, 1995).…”

The mouse equivalent of the human p53ser249 mutation p53ser246 enhances aflatoxin hepatocarcinogenesis in hepatitis B surface antigen transgenic and p53 heterozygous null mice

“…Rat liver microsome-activated AFB1 preferentially induces the transversion of G=ϾT in the third position of codon 249 in human HCC cells (HepG2). 36 However, AFB1 also induces G=T and C=A transversions into adjacent codons, albeit at lower frequencies.…”

p53 As a mutagen test in breast cancer

“…Dimethylbenzanthracene (DMBA) causes primarily base pair substitutions, predominantly A:T 3 T:A (44%) and G:C 3 T:A (24%) [Manjanatha et al, 2000]. In human populations, signature mutations associated with specific exogenous mutagens have been observed in samples of somatic mutations in the p53 tumor-suppressor gene [Harris and Hollstein, 1993;Hussain and Harris, 1999], for example, G 3 T transversions in lung cancers of cigarette smokers [Chiba et al, 1990], C 3 T and CC 3 TT tandem dipyrimidine transitions in skin cancers associated with excessive sun exposure [Brash et al, 1991], and G 3 T transversions at arginine 249 in liver cancers associated with aflatoxin exposure [Bressac et al, 1991;Hsu et al, 1991;Scorsone et al, 1992;Aguilar et al, 1993;Li et al, 1993]. Such signature mutations can be detected in the mutation pattern with high sensitivity.…”