The hepatic cytochrome P4502A6 (CYP2A6) enzyme mediates the oxidative metabolism of several procarcinogens that have liver as their primary target. Mouse models indicate that liver tumors invariably overexpress CYP2A forms, and that inflammation and cirrhosis may regulate the CYP2A expression pattern. In this study, the distribution of the CYP2A6 protein was investigated in a series of 24 human hepatocellular carcinoma (HCC) samples by immunohistochemical analysis. A polyclonal antibody was raised in chicken against CYP2A5, the mouse orthologue of CYP2A6. The antibody was characterized and found to be specific for CYP2A members. In DBA/2 mouse liver, a strong increase of CYP2A5 protein amount, localized in the perivenous region, occurred in response to treatment with pyrazole. In human HCC samples, overexpression of CYP2A6 protein was associated with the presence of chronic inflammation and cirrhosis. CYP2A6 protein was observed in 9 of 16 (56%) of samples with non-neoplastic hepatocytes and in 10 of 24 (42%) HCC samples. The staining for CYP2A6 protein was very heterogeneous in tumor cells, suggesting that increased expression of CYP2A6 occurred in a distinct subpopulation of neoplastic cells. In Kaplan-Meyer survival analysis, there was a tendency toward a more favorable prognosis in patients with CYP2A6positive tumors in comparison with patients with CYP2A6negative tumors. These data suggest that, in human HCC, in contrast to mouse liver tumors, CYP2A6 overexpression is not an invariable phenotype. (HEPATOLOGY 1998;27:427-432.) Cytochrome P450 (CYP) enzymes mediate the oxidative metabolism of numerous exogenous and endogenous compounds. 1 It is well established that several CYP forms participate in the chemical carcinogenesis process by metabolically activating procarcinogens to reactive, DNA-binding intermediates. 2 The human CYP2A6 is one such form. 3 Like most other CYPs, CYP2A6 is most abundant in liver, and its levels in extrahepatic tissues are very low. 4,5 The CYP2A6 enzyme catalyses the metabolic activation of several procarcinogens and promutagens, including the liver-specific carcinogen aflatoxin B 1 , 6-8 several nitrosamines, 9-11 and 1,3butadiene. 12 CYP2A6 is also a major catalyst in the oxidative metabolism of nicotine and cotinine, 13 as well as several pharmaceuticals. 14 The regulation of expression of members in the CYP2A subfamily differs markedly from other CYP forms. Studies on CYP2A5, the mouse orthologue of CYP2A6, have revealed that it is inducible by a variety of structurally dissimilar compounds, including phenobarbital, some heavy metals, heterocyclic nitrogen-containing agents, and various hepatotoxins including cocaine. 3,15,16 Animal studies have also shown that the expression of hepatic CYP2As is up-regulated by various types of biological insults, such as infestation with the liver fluke Opisthorchiasis viverrini 17 and integration of hepatitis B virus (HBV) to hepatocyte DNA. 18 In these animal models, CYP2A induction is associated with chronic inflammation in the liver tissue. In addition, liver tumors in mice contain highly elevated levels of CYP2A5. 19,20 Recent studies indicate that human liver CYP2A6 is also induced by O. viverrini infestation, 21 and an elevated level of CYP2A6 protein was found in hepatocytes expressing the HBV surface antigen. 22 CYP2A mRNA has been shown to be expressed at higher levels in hepatocytes adjacent to areas of fibrosis in cirrhotic livers, 10 but no information exists on CYP2A6 regulation in human liver tumors. This study was performed to find out whether human hepatocellular carcinomas (HCC) exhibit elevated levels of CYP2A6 protein and to assess whether hepatic inflammatory processes affect liver CYP2A6 content. To achieve this goal, a polyclonal antibody against mouse CYP2A5 was developed and used in immunohistochemical analysis of a series of human liver samples obtained from HCC patients.
Methods
Materials. Male (range, 7 and 12 wks) DBA/2Kuo mice were obtained from the National Laboratory Animal Center, Kuopio, Finland. The mice were housed in plastic cages, with aspen chips as bedding, in a 14-hour light/10-hour dark cycle. Pyrazole, a strong and specific inducer of CYP2A5, 23 was intraperitoneally administered to the mice at a dose of 200 mg/kg once a day for 3 days. Liver sections from these mice were used as positive controls in immunohistochemistry.
Sections from 24 HCC cases, resected between 1983 and 1994, were collected from the files of the Department of Pathology, Oulu University Hospital. All of the sections had been fixed in 10% neutral formalin and embedded in paraffin wax. The diagnosis of all cases was based on conventional light microscopy according to the criteria of the World Health Organization. 24 Ten of them were grade Abbreviations: CYP, cytochrome P450; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; COH, coumarin 7-hydroxylase.