CANCER GENOMICS & PROTEOMICS 1: 53-58 (2004) Altered Levels of Cytochrome P450 Genes in Hepatitis B or C Virus-infected Liver Identified by Oligonucleotide Microarray NORIO IIZUKA1,2, MASAAKI OKA1, YOSHIHIKO HAMAMOTO3, NAOHIDE MORI1, TAKAO TAMESA1, AKIRA TANGOKU1, TAKANOBU MIYAMOTO3, SHUNJI UCHIMURA3, HIRONOBU NAKAYAMA4, KENJI HAMADA4 and HISAFUMI YAMADA-OKABE4 1Department of Surgery II and 2Department of Bioregulatory Function, Yamaguchi University School of Medicine, 1-1-1 Minami-kogushi, Ube, Yamaguchi 755-8505; 3Department of Computer Science and Systems Engineering, Faculty of Engineering, Yamaguchi University, 2-16-1 Tokiwadai, Ube, Yamaguchi 755-8611; 4Pharmaceutical Research Department 4, Kamakaura Research Laboratories, Chugai Pharmaceutical Co. Ltd., 200 Kajiwara, Kamakura, Kanagawa 247-8530, Japan Abstract. Background: Molecular pathogenesis of hepatocellular (1,2). However, the genetic basis underlying these diseases carcinoma (HCC) remains to be clarified. Many studies with DNA remains unclear. To overcome this dilemma, many chip technology have revealed altered levels of several cytochrome researchers have applied DNA chip technology to elucidate P450 (CYP) family genes in human HCC. However, little is known genome-wide changes in these diseases (3-13). Interestingly, about their alterations in hepatitis B virus (HBV)- and hepatitis C as summarized in Table I, commonly found in those studies virus (HCV)-infected livers. Materials and Methods: We here used are altered levels of several cytochrome P450 family genes high-density oligonucleotide arrays to evaluate alterations of CYP (CYPs), such as CYP2E, CYP2A6 and CYP2A7, in HCC genes in five of each HBV- or HCV-infected livers in comparison versus non-cancerous liver. with six normal livers. We extracted the data of 32 CYP genes from The cytochrome P450 system is a group of enzymes that are those of 12600 genes. Results: Among these 32 CYPs, expression responsible for metabolizing many endogenous and exogenous levels of four genes were insignificant. The expressions of CYP1B1 substances including xenobiotics or carcinogens into more and CYP3A7 were up-regulated, whereas the expressions of 12 hydrophilic substances (14). This means that altered levels of other CYPs, including CYP2A6, CYP2A7 and CYP2C19, were CYPs might be related to hepatocarcinogenesis. Some types of down-regulated in HBV- and/or HCV-infected livers compared CYPs are likely to be involved in rat hepatocarcinogenesis (15). with normal livers. Conclusion: These data will allow us to better It has also been shown that the genetic polymorphisms of understand the roles of CYPs in the pathogenesis of HBV- and CYP2E, CYP2D6 and CYP2C19 are associated with the HCV-related HCCs. development of HCC (16, 17). However, it remains unclear how these CYPs are involved in HBV- or HCV-related Hepatocellular carcinoma (HCC) is a common cause of hepatocarcinogenesis. To elucidate their roles in these cancer death throughout the world (1,2). Chronic infection diseases, it is essential to clarify expression patterns of CYP with hepatitis B or C virus (HBV or HCV) is the most genes in HBV- or HCV-infected livers that represent clearly established risk factor for HCC. The majority of HCC hypercarcinogenic states for HCC. For this purpose, we tested can be attributed to infection with either of the two viruses oligonucleotide microarrays with RNA isolated from human livers that were uninfected or infected with HBV or HCV, and we identified CYP genes that were differentially expressed Abbreviations: CYP, cytochrome P450; HCV, hepatitis C virus; between uninfected and infected liver tissues. In combination HCC, hepatocellular carcinoma. with gene expression profiles of CYPs in HCC (3-13), the possible roles in pre-cancerous livers are discussed. Correspondence to: Prof. Masaaki Oka, Department of Surgery II, Yamaguchi University School of Medicine, 1-1-1 Minami-Kogushi, Materials and Methods Ube, Yamaguchi 755-8505, Japan. Tel/Fax:+81 836 22 2262, e- mail: [email protected] Patients. Non-tumourous liver samples were obtained from six patients who underwent hepatic resection for benign or metastatic Key Words: DNA chip, cytochrome P450, HBV, HCV, hepatocellular liver tumours. All six patients showed liver function values within carcinoma. normal limits. Their livers were histologically normal and were 1109-6535/2004 $2.00+.40 53 CANCER GENOMICS & PROTEOMICS 1: 53-58 (2004) Table I. CYPs expressed differentially in human hepatocellular carcinoma. Cytochrome p450 (CYP) Condition Type of HCC Ref. CYP2C, CYP2E down-regulation in tumour vs. normal liver HBV-associated HCC Lau et al., 2000 (Ref.3) CYP2C down-regulation in tumour vs. normal liver HCC* Shirota et al., 2001 (Ref.4) CYP2B, CYP2C, CYP17 down-regulation in tumour vs. normal liver HBV-associated HCC Xu et al., 2001 (Ref.5) CYP2C9, CYP2E, CYP4F2 down-regulation in tumour vs. normal liver HCC** Xu et al., 2001 (Ref.6) CYP2A6, CYP2C8, CYP2C9 down-regulation in tumour vs. normal liver HCC* Okabe et al., 2001 (Ref.7) CYP2E down-regulation in HBV-associated HCC CYP2A6, CYP2B, CYP8B1 down-regulation in tumour vs. normal liver HCC** Tackels-Horne et al., 2001(Ref.8) CYP2A7, CYP2E down-regulation in tumour vs. normal liver HBV-associated HCC Iizuka et al., 2002 (Ref.9) CYP2A7, CYP2E down-regulation in tumour vs. normal liver HCV-associated HCC CYP2C8, CYP27A1 up-regulation in tumour vs. normal liver HCC** Li et al., 2002 (Ref.10) CYP3A3, CYP27A1 down-regulation in tumour with venous invasion HCC** Chen et al., 2002 (Ref.11) CYP2A7 down-regulation in metastatic HCC HBV-associated HCC Cheung et al., 2002 (Ref.12) CYP2A6, CYP2A7, down-regulation during tumour progression HCC* Chuma et al., 2003 (Ref.13) CYP2A13, CYP2C8 *Samples consisting of HBV- and HCV-associated HCCs, ** Sample labels are unclear seronegative for both HBs antigen and HCV antibody. Five HBV- of arbitrary expression units was less than 40 by Affymetrix) infected and five HCV-infected liver samples were obtained from the and were thus considered insignificant. Levels of the remaining non-tumourous areas of ten patients with hepatocellular carcinoma. 28 genes were higher than those of the four genes and were Among the ten liver samples, four were histopathologically diagnosed as chronic hepatitis and six as liver cirrhosis. All sixteen patients considered biologically significant. Of these 28, levels of enrolled in this study underwent uniform premedication before CYP1B1 and CYP3A7 were up-regulated and levels of 12 CYP surgery and general anesthesia with 1.7% sevoflurane and 60% genes, including CYP2A subfamily (2A4, 2A6 and 2A7) and nitrous oxide. None of them underwent specific therapies. Informed CYP2C19 were down-regulated in HBV- and/or HCV-infected consent in writing was obtained from all patients before surgery. The livers compared with HBV-/HCV-double-negative livers. study protocol was approved by the Institutional Review Board for Levels of CYP3A7 were significantly higher in HCV-infected Human Use of the Yamaguchi University School of Medicine, Japan. liver than in HBV-infected and HBV-/HCV-double-negative Oligonucleotide microarray and gene selection. Extraction of RNA, livers. Levels of CYP27A1 were significantly lower in HBV- syntheses of cDNA and cRNA, and oligonucleotide microarray infected liver than in HCV-infected and HBV-/HCV-double- screening were performed as described previously (9, 18, 19). In the negative livers. Expression levels of the remaining 14 CYP present study, to obtain expression data of many CYPs, large-size genes were unchanged by either HBV or HCV infection. DNA chips (huU95A DNA chips; Affymetrix, Santa Clara, CA, USA) containing 12600 genes were used for microarray Discussion experiments. We extracted the data of 32 CYPs from those of 12600 genes. For statistical analysis of gene expression, ANOVA with Fisher’s projected least significant difference (PLSD) test was Many studies have revealed the relationship between CYPs and performed with the use of StatView 5.0 software (Abacus Concepts, human hepatocarcinogenesis (17,20,21). Chau et al. showed Berkeley, CA, USA). A p<0.05 was judged as significant. that poor metabolizer phenotype caused by the mutation of the CYP2C19 gene in cirrhotic patients with HCV infection is Results associated with a high risk for developing HCC (17). CYP27A, which is known as a D3-25 hydroxylase, was found to be We examined the differential expression of 32 CYP genes significantly up-regulated in HCC (20). CYP1A1 polymorphism (Figure 1 and Table II) on the basis of the oligonucleotide is an important modulator of the hepatocarcinogenic effect of microarray data. Levels of expression of four genes were very tobacco-derived polycyclic aromatic hydrocarbons (21). Given low in normal, HBV-positive and HCV-positive livers (average the fact that CYPs are large family genes consisting of many 54 Iizuka et al: Expression Profile of CYP in Human Liver Figure 1. Gene expression patterns of the 32 CYP genes. Color display of expression of 32 CYP genes in normal livers (Liver 64-69), HBV-infected livers (HBV 53-57) and HCV-infected livers (HCV 49, 58-60 and 62). Red color represents relatively high expression and green relatively low expression. The accession number of each gene was obtained from PubMed (http://www3.nibi.nlm. nih.gov/PubMed/) or the TIGR database (http://www.tigr. org/tdb/hgi/searching/ reports.html). 55 CANCER GENOMICS & PROTEOMICS 1: 53-58 (2004) Table II. Summary of the 32 CYP genes expressed in human livers. GB number