CHARACTERIZATION OF NOVEL CYP 2 A 6 POLYMORPHIC ALLELES ( CYP 2 A 6 * 18 AND CYP 2 A 6 * 19 ) THAT AFFECT ENZYMATIC ACTIVITY

Genetic polymorphisms of CYP2A6 gene are known as a causal factor of the interindividual differences in nicotine metabolism. We found three novel CYP2A6 alleles. The CYP2A6*18A allele has a single nucleotide polymorphism (SNP) of A5668T (A1175T, Y392F) in exon 8. The CYP2A6*18B allele has synonymous SNPs of G51A (G51A), T5684C (T1191C), and T5702C (T1209C) in addition to A5668T (A1175T, Y392F). The CYP2A6*19 allele has the SNPs of A5668T (A1175T, Y392F), T6354C (intron 8), and T6558C (T1412C, I471T) as well as the conversion with the CYP2A7 sequence in the 3 -untranslated region, in which the latter two changes correspond to CYP2A6*7. Ethnic differences in the frequencies of these alleles were observed between whites, African-Americans, Japanese, and Koreans. Wild or variant CYP2A6 (CYP2A6*18, CYP2A6*19, and CYP2A6*7) were expressed in Escherichia coli. For coumarin 7-hydroxylation and 5-fluorouracil formation from tegafur, the Km values were increased, and Vmax values were decreased in CYP2A6.18 compared with those in CYP2A6.1, resulting in decreased clearance to 50 and 35% of that of the wild type, respectively. The Km and Vmax values for nicotine C-oxidation were both increased, resulting in no change of clearance. In CYP2A6.19, the effects on the coumarin 7-hydroxylation and 5-fluorouracil formation (increased Km and decreased Vmax) were prominent, resulting in decreased clearance to 8% of those of the wild type. For nicotine C-oxidation, the Km and Vmax values were both decreased, resulting in decreased clearance to 30% of that of the wild type. The changes of the kinetics in CYP2A6.19 were similar to those in CYP2A6.7. In vivo nicotine metabolism was evaluated in whites (n 56) and Koreans (n 40). Although the CYP2A6*18 and CYP2A6*19 alleles were found only heterozygously, a subject with CYP2A6*7/CYP2A6*19 showed a lower cotinine/nicotine ratio of the plasma concentration compared with homozygotes of the CYP2A6*1A, supporting the in vitro results that the CYP2A6*19 allele leads to decreased enzymatic activity. Cytochrome P450 (P450) is responsible for the metabolism of endogenous and exogenous compounds. CYP2A6 is one of the major isoforms in humans and is well known to catalyze nicotine C-oxidation (Nakajima et al., 1996). CYP2A6 also metabolizes coumarin, tegafur, valproic acid, halothane, and aflatoxin B1. In addition, it can activate tobacco-specific nitrosamines such as 4-methylnitrosoamino1-(3-pyridyl)-1-butanone and N-nitrosodiethylamine (Oscarson, 2001). There are genetic polymorphisms in the CYP2A6 gene. Several CYP2A6 alleles, such as CYP2A6*2 (Yamano et al., 1990), CYP2A6*4 (Oscarson et al., 1999b; Nunoya et al., 1999a,b), CYP2A6*5 (Oscarson et al., 1999a), CYP2A6*6 (Kitagawa et al., 2001), CYP2A6*7 (Ariyoshi et al., 2001), CYP2A6*9 (Pitarque et al., 2001), CYP2A6*10 (Yoshida et al., 2002), CYP2A6*11 (Daigo et al., 2002), CYP2A6*12 (Oscarson et al., 2002), and CYP2A6*17 (Fukami et al., 2004) have been reported to show decreased enzymatic activity. We have developed a simple phenotyping method of nicotine metabolism using nicotine gum (Nakajima et al., 2000a, 2001) and have demonstrated that the genetic polymorphisms of the CYP2A6 gene could account for most of the large interindividual differences in nicotine metabolism. In addition, some research groups have reported an association between genetic polymorphisms of CYP2A6 and smoking behavior or lung cancer risk (Miyamoto et al., 1999; Minematsu et al., 2003; Fujieda et al., 2004). These findings would reflect the relatively high frequencies (10–20%) of CYP2A6*4, CYP2A6*7, and CYP2A6*9 in Asian populations (Nakajima et al., 2002; Yoshida et al., 2003). However, the results from such studies in white Europeans have been inconclusive and controversial (Loriot et al., 2001; Schulz et al., 2001) because of the low frequencies (1–7%) of defective alleles (Schoedel et al., 2004). In the present study, we found two novel CYP2A6 alleles (CYP2A6*18A and CYP2A6*18B) containing a single nucleotide polymorphism (SNP) that leads to an amino acid change of Y392F (Fig. 1) from Caucasians. From Korean subjects, the CYP2A6*18A allele and another novel allele, CYP2A6*19, possessing SNPs that cause amino acid changes of Y392F as well as I471T, were found (Fig. 1). The amino acid change of I471T in CYP2A6.7 is already This study was supported in part by a grant from Japan Health Sciences Foundation with Research on Health Science Focusing on Drug Innovation, by an SRF grant for biomedical research in Japan, and by Philip Morris Incorporated. Article, publication date, and citation information can be found at http://dmd.aspetjournals. org. doi:10.1124/dmd.105. 004994. ABBREVIATIONS: P450, cytochrome P450 enzyme; SNP, single nucleotide polymorphism; 5-FU, 5-fluorouracil; PCR-RFLP, polymerase chain reaction-restriction fragment length polymorphism; PCR, polymerase chain reaction; bp, base pair(s); AS-PCR, allele specific-polymerase chain reaction; NPR, NADPH-P450 reductase; HPLC, high-performance liquid chromatography. 0090-9556/05/3308-1202–1210$20.00 DRUG METABOLISM AND DISPOSITION Vol. 33, No. 8 Copyright © 2005 by The American Society for Pharmacology and Experimental Therapeutics 4994/3043249 DMD 33:1202–1210, 2005 Printed in U.S.A. 1202 at A PE T Jornals on Jne 9, 2017 dm d.aspurnals. org D ow nladed from