Ser-249 p53 mutations in plasma DNA of patients with hepatocellular carcinoma from The Gambia.

BACKGROUND: A selective mutation, an arginine-to-serine substitution in codon 249, of the p53 gene has been identified as a "hotspot" mutation in hepatocellular carcinoma (HCC). This mutation occurs in populations that are exposed to aflatoxins and have a high prevalence of hepatitis B virus carriers. We evaluated whether this mutation could be detected in cell-free DNA isolated from the plasma of subjects from The Gambia to detect this mutation that is strongly associated with HCC. METHODS: Fifty-three patients with HCC, 13 patients with cirrhosis, and 53 control subjects were prospectively recruited from The Gambia. Sixty patients, of non-African origin, with various liver pathologies were also selected from France. DNA was extracted and purified from 200-microL aliquots of plasma. The Ser-249 p53 mutation was detected by restriction endonuclease digestion of polymerase chain reaction products from exon 7 and was confirmed by direct sequencing of the amplified DNA. RESULTS: The Ser-249 p53 mutation was detected in plasma DNA from 19 (36%) of the 53 patients with HCC, two (15%) of the 13 patients with cirrhosis, and three (6%) of the 53 control subjects. This mutation was not detected in any plasma DNA from the European patients. The adjusted odds ratio for having the mutation was 16.4 (95% confidence interval = 3.0-90.5) for patients with HCC compared with the control subjects. CONCLUSION: The Ser-249 p53 mutation in plasma DNA is strongly associated with HCC in Gambian patients. This mutation was also detected at a much lower prevalence in plasma DNA from Gambian patients with cirrhosis and in Gambian control subjects, findings that may lead to the earlier detection of HCC. Use of the Ser-249 p53 mutation should facilitate further molecular epidemiologic studies on the development of HCC.

Expression of cytochrome P450 2A5 in preneoplastic and neoplastic mouse liver lesions.

Cytochrome P450 (CYP) 2A5 is involved in the metabolism of carcinogens like aflatoxin B1 and N-nitrosodiethylamine (NDEA), and CYP2A5 levels are increased in some pathological states of the liver (e.g., infectious hepatitis and porphyria). We analyzed the expression of CYP2A5 during experimental liver carcinogenesis in three different mouse strains (C3H/He, C57BL/6J, and B6C3F1) with immunohistochemical techniques and in situ hybridization. In normal liver, CYP2A5 protein and mRNA were detected in centrilobular hepatocytes only. Phenobarbital treatment increased the number of CYP2A5-positive centrilobular hepatocytes and the CYP2A5-positive areas were extended into the middle zone in all strains, but periportal hepatocytes remained negative. Fifty percent of the spontaneous foci in untreated mice, over 90% of the foci in mice treated with NDEA or phenobarbital and all of the hepatocellular adenomas and carcinomas displayed positive immunostaining and a strong CYP2A5 mRNA signal by in situ hybridization. In the liver tumors metastasized to the lung, expression of CYP2A5 had largely disappeared. CYP2A5 expression in neoplastic and putative preneoplastic lesions, although sometimes heterogeneous, was apparently independent of the typical zonal expression pattern in normal tissue. As expected, the C57BL/6J mice developed fewer foci and tumors than the C3H/He and B6C3F1 mice, but the phenotype of CYP2A5 overexpression was similar in all the strains. Our data suggest that the increased expression of CYP2A5 may play an important role in the development of liver cancer in mice and may be used as a novel marker for spontaneous and NDEA-induced mouse liver foci.

Liver injury and expression of cytochromes P450: evidence that regulation of CYP2A5 is different from that of other major xenobiotic metabolizing CYP enzymes.

The purpose of this study was to find out how liver injury caused by two well-known hepatotoxins, chloroform and thioacetamide, alters the expression of hepatic xenobiotic metabolizing cytochrome P450 (CYP) enzymes of DBA/2N mice. Dose-dependent toxic effects of the two hepatotoxins were verified by histological examination. Along with the toxicity, intense staining of immunoreactive material was detected in the centrilobular zone, with anti-CYP2A5 antibody in hepatic tissue. This apparent increase in the expression of Cyp2a-5 was verified by Northern blot and Western blot analyses and by determining the enzymatic activity, coumarin 7-hydroxylase, in hepatic tissue. The results suggest that liver injury due to these hepatotoxins increases the expression of Cyp2a-5 and that the expression is pretranslationally regulated. The increased expression of Cyp2a-5 is in contrast with that of other xenobiotic metabolizing CYPs because a dose-[dose-dependent] dependent decrease of the total hepatic P450 content and either a decrease or no change in the levels of CYP1A, 2B, 2C, 2E1, and 3A4 were observed. The results suggest that essential differences exist in the regulation of CYP2A5 and other major xenobiotic metabolizing CYP enzymes and that in a damaged liver CYP2A5 may be a major catalyst of xenobiotic metabolism.