Mutations in the p53 tumor suppressor gene in tree shrew hepatocellular carcinoma associated with hepatitis B virus infection and intake of aflatoxin B1.

Infection with hepadnaviruses and exposure to aflatoxin B1 (AFB1) are considered to be major risk factors in the development of hepatocellular carcinoma (HCC) in humans. A high rate of p53 mutations at codon 249 has been reported in these tumors. The tree shrew (Tupaia belangeri chinensis) is a useful animal model for the development of HCC after human hepatitis B virus (HBV) infection or AFB1 treatment. Therefore, it was of particular interest to determine whether the p53 gene in tree shrew HCCs associated with HBV infection and/or with exposure to AFB1 is affected in the same manner as in human HCCs. We determined the tree shrew p53 wild-type nucleotide sequences by RT-PCR and automatic DNA-sequencing. Tree shrew wild-type p53 sequence showed 91.7 and 93.4% homologies with human p53 nucleotide and amino acids sequences, respectively, while it showed 77.2 and 73.7% homologies in mice. One HCC and normal liver tissue from AFB1 treated and one HCC from AFB1- and HBV-treated tree shrew showed no change in p53 sequences, while three HCCs from AFB1- and HBV-treated tree shrews showed point mutations in p53 sequences. One HCC showed point mutations at codon 275, which is on the DNA-binding domain of p53 gene, which might be a cause of gain-of-function during the development of HCC. As a result, our finding indicates that tree shrews exposed to AFB1 and/or HBV had neither codon 249 mutations nor significant levels of other mutations in the p53 gene, as is the case with humans.

Synergistic effect of hepatitis B virus and aflatoxin B1 in hepatocarcinogenesis in tree shrews.

An animal experiment with tree shrews was performed to detect the synergistic effects of hepatitis B virus (HBV) infection and dietary aflatoxin B1 (AFB1) in hepatocarcinogenesis. Adult healthy tree shrews (Tupaia belangeri chinensis) were divided into four groups: Group A (HBV + AFB1)--animals were infected with human HBV serum at first, then fed AFB1 diluted with milk, 150 ug/kg.bw/day, 6 days/week for 105 weeks. Group B (HBV)--animals were infected with human HBV as Group A, but no AFB1 treatment. Group C (AFB1)--animals were treated with AFB1 as Group A but no HBV infection. Group D--animals were treated neither with human HBV nor AFB1. During the experiment, blood samples and liver biopsies were taken regularly from all animals in each group. All the animals were sacrificed on the 160th week when the experiment ended. The samples of sera and liver tissues were checked for HBV markers and histological changes. Hepatocellular carcinomas (HCCs) were found only in Group A and Group C, with incidences of 67% and 30% respectively. The average time for HCC occurrence in Group A and Group C was 120.3 +/- 16.6 and 153.3 +/- 5.8 weeks respectively (P < 0.01). Even though no HCC occurred in Group B, 1 animal which died before the end of the experiment showed two large hepatocellular nodules. These results showed that there is synergistic effect between HBV and AFB1 in tree shrews' hepatocarcinogenesis, even though the hepatocarcinogenic effect played by HBV alone is rather weak.