FUNCTIONAL ANALYSIS OF THREE CYP1A2 VARIANTS FOUND IN A JAPANESE POPULATION Yoshiro Saito, Nobumitsu Hanioka, Keiko Maekawa, Takashi Isobe, Yumi Tsuneto, Ryosuke Nakamura, Akiko Soyama, Shogo Ozawa, Toshiko Tanaka-Kagawa, Hideto Jinno, Shizuo Narimatsu, and Jun-ichi Sawada Project Team for Pharmacogenetics (Y.S., K.M., R.N., A.S., S.O., H.J., J.S.), Division of Biochemistry and Immunochemistry (Y.S., K.M., R.N., J.S.), Division of Pharmacology (S.O.), Division of Environmental Chemistry (T.T.-K., H.J.), National Institute of Health Sciences, Tokyo, Japan; and Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University, Okayama, Japan (N.H., T.I., Y.T., S.N.). Received May 30, 2005; accepted September 8, 2005 ABSTRACT: Human cytochrome P450 1A2 (CYP1A2) catalyzes the metabolism of many important drugs and environmental chemicals. We previ- ously reported three naturally occurring genetic polymorphisms (125C>G, Pro42Arg, CYP1A2*15; 1130G>A, Arg377Gln, *16; and 1367G>A, Arg456His, *8) found in a Japanese population. In this study, these variant enzymes were expressed in Chinese hamster V79 cells, and their mRNA and protein expression levels as well as catalytic activities were determined. All three variant enzymes showed reduced protein expression levels (66% for Pro42Arg and approximately 30% for Arg377Gln and Arg456His) compared with that of the wild type (WT) without any change in mRNA expression levels. Kinetic analysis for 7-ethoxyresorufin O-deethylation re- vealed that V max and V max /K m of all three variants were less than 3 and 1% of the WT, respectively, although the K m value was signif- icantly increased only in the Arg377Gln variant (approximately a 9-fold increase). Markedly reduced activities of the three variants were also observed for phenacetin O-deethylation. In the reduced CO difference spectral analysis using recombinant proteins pro- duced in the Sf21/baculovirus system, the peak at 450 nm seen in the WT protein was hardly observed in the three variants, suggest- ing marked reductions in their hemoprotein formation. These re- sults suggest that Pro42, Arg377, and Arg456 are critical amino acids for the production of catalytically active CYP1A2 holoen- zyme. Human cytochrome P450 (P450) enzymes catalyze the metabolism of a wide variety of clinically, physiologically, and toxicologically important compounds (Lewis, 2001). The human CYP1A subfamily consists of CYP1A1 and CYP1A2. The former is expressed mainly in extrahepatic tissues, and the latter is almost exclusively expressed in the liver. CYP1A2 is responsible for the oxidative metabolism of drugs such as theophylline, mexiletine, and phenacetin (Distlerath et al., 1985; Sarkar and Jackson, 1994; Nakajima et al., 1998). This enzyme has also been shown to be involved in the metabolic activa- tion of carcinogenic arylamines to produce reactive intermediates (Eaton et al., 1995). Up to 60-fold interindividual variation in the CYP1A2 activity has been reported (Shimada et al., 1994; Saruwatari et al., 2002). Also, approximately 15- and 40-fold interindividual variations in CYP1A2 mRNA and protein expression levels have been observed in the human liver (Ikeya et al., 1989; Guengerich et al., 1999). These interindividual differences are likely to influence the drug metabolism and to be associated with drug efficacy and safety and cancer suscep- tibility caused by procarcinogens. Environmental factors have been thought to influence the interindividual differences. Cigarette smoking and intake of oral contraceptive steroids are well established modifiers of CYP1A2 activity (Rasmussen et al., 2002). However, it has been suggested that approximately 35 to 75% of the interindividual vari- ability in CYP1A2 activity is due to genetic factors (Kendler and Prescott, 1999; Rasmussen et al., 2002). Thus, several researchers have focused their efforts on the identification of CYP1A2 genetic variants. To date, 23 CYP1A2 haplotypes, including nine subtypes, have been publicized on the Human Cytochrome P450 Allele Nomencla- ture Committee home page (http://www.imm.ki.se/CYPalleles/ cyp1a2.htm). Since CYP1A2 is inducible by environmental factors, many investigators have tried to identify single nucleotide polymor- phisms (SNPs) in the transcriptional regulatory regions: the distal enhancer region, the promoter region, noncoding exon 1, and intron 1. CYP1A2*1C (-3860GA) was reported to be associated with de- creased enzyme inducibility in Japanese smokers (Nakajima et al., 1999). CYP1A2*1F (-163CA), located in intron 1, has been sug- gested to be linked with a higher enzyme inducibility in white smok- ers (Sachse et al., 1999). Recently, Aklillu et al. reported that the CYP1A2*1K haplotype (-739TG, -729CT, and -163CA; all Y.S., N.H., and K.M. contributed equally to this work. This study was supported in part by the Program for the Promotion of Funda- mental Studies in Health Sciences, Health and Labor Sciences Research Grants from the Ministry of Health, Labor and Welfare, and a Research Grant from the Japan Health Sciences Foundation. Article, publication date, and citation information can be found at http://dmd.aspetjournals.org. doi:10.1124/dmd.105.005819. ABBREVIATIONS: P450, cytochrome P450; SNP, single nucleotide polymorphism; WT, wild type; RT, reverse-transcription; PCR, polymerase chain reaction; HPLC, high-performance liquid chromatography; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. 0090-9556/05/3312-1905–1910$20.00 DRUG METABOLISM AND DISPOSITION Vol. 33, No. 12 Copyright © 2005 by The American Society for Pharmacology and Experimental Therapeutics 5819/3064686 DMD 33:1905–1910, 2005 Printed in U.S.A. 1905 at ASPET Journals on January 14, 2016 dmd.aspetjournals.org Downloaded from