SEARCH

SEARCH BY CITATION

Keywords:

  • cytochrome P450;
  • genetic polymorphism;
  • genotype;
  • interindividual difference;
  • nicotine metabolism;
  • phenotype;
  • poor metabolizer

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results and Discussion
  6. References

Aims  Previously, we determined the phenotyping of in vivo nicotine metabolism and the genotyping of the CYP2A6 gene (CYP2A6*1 A, CYP2A6*1B, CYP2A6*2, CYP2A6*3, CYP2A6*4 and CYP2A6*5 ) in 92 Japanese and 209 Koreans. In the study, we found one Korean and four Japanese subjects genotyped as CYP2A6*1B/CYP2A6*4 who revealed impaired nicotine metabolism, although other many heterozygotes of CYP2A6*4 demonstrated normal nicotine metabolism (CYP2A6*4 is a whole deletion type). After our previous report, several CYP2A6 alleles, CYP2A6*6 (R128Q), CYP2A6*7 (I471T), and CYP2A6*8 (R485L), have been reported. The purpose of the present study was to clarify whether the impaired nicotine metabolism can be ascribed to these CYP2A6 alleles. Furthermore, we also determined whether the subjects possessing CYP2A6*1×2 (duplication) reveal higher nicotine metabolism.

Methods  Genotyping of CYP2A6 alleles, CYP2A6*6, CYP2A6*7, CYP2A6*8, and CYP2A6*1×2 was determined by PCR.

Results  The five poor metabolizers were re-genotyped as CYP2A6*7/CYP2A6*4, suggesting that a single nucleotide polymorphism (SNP) causing I471T decreases nicotine metabolism in vivo. Furthermore, we found that two subjects out of five with a lower potency of nicotine metabolism possessed SNPs of CYP2A6*7 and CYP2A6*8 simultaneously. The novel allele was termed CYP2A6*10. In the 92 Japanese and 209 Koreans, the CYP2A6*6 allele was not found. The allele frequencies of CYP2A6*7, CYP2A6*8, and CYP2A6*10 were 6.5%, 2.2%, and 1.1%, respectively, in Japanese, and 3.6%, 1.4%, and 0.5%, respectively, in Koreans. The CYP2A6*1×2 allele was found in only one Korean subject (0.5%) whose nicotine metabolic potency was not very high.

Conclusions  It was clarified that the impaired in vivo nicotine metabolism was caused by CYP2A6*7 and CYP2A6*10 alleles.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results and Discussion
  6. References

Cytochrome P450 (CYP) is a superfamily of haemoproteins, many of which can metabolize xenobiotics such as procarcinogens, drugs, and environmental pollutants. CYP2A6 is a major hepatic member of the family in humans, which metabolizes pharmaceutical agents such as coumarin (+) - cis - 3,5 - dimethyl - 2 - (3 - pyridyl) thiazolidin-4-one hydrochloride (SM-12502), methoxyflurane, halothane, losigamone, letrozole, valproic acid, disulfiram, fadrozole, and activates some procarcinogens such as 4-methylnitrosoamino-1-(3-pyridyl)-1-butanone and N-nitrosodiethylamine [1]. Especially, CYP2A6 is a major metabolic enzyme of nicotine. Nicotine is metabolized to cotinine by CYP2A6 [2], and cotinine is further metabolized to trans-3′-hydroxycotinine [3]. Nicotine metabolism is known to show large interindividual differences [4, 5]. Previously, we established a phenotyping method of in vivo nicotine metabolism [6]. Furthermore, we determined the relationship between the interindividual differences in nicotine metabolism and genetic polymorphism of the CYP2A6 gene (CYP2A6*1 A, CYP2A6*1B, CYP2A6*2, CYP2A6*3, CYP2A6*4, and CYP2A6*5) in 92 Japanese and 209 Koreans [7, 8]. The CYP2A6*1 A is a wild type of the CYP2A6 gene. The CYP2A6*1B allele has a gene conversion with CYP2A7 in the 3′-untranslated region [9]. The CYP2A6*2 allele has a single amino acid substitution (L160H) and encodes an unstable and catalytically inactive enzyme [10]. The CYP2A6*3 allele has gene conversions with CYP2A7 in exons 3, 6, and 8 [11], proposed to be inactive. The CYP2A6*4 allele deletes the whole CYP2A6 gene [12, 13]. The CYP2A6*5 allele has a point mutation in exon 9 leading to a single amino acid substitution (G479V) as well as gene conversion in the 3′-untranslated region [9]. In our previous studies [7, 8], seven Japanese and five Korean subjects were phenotyped as poor metabolizers of cotinine formation from nicotine with probit analyses. Among them, three Japanese and four Korean subjects who were absolutely deficient in cotinine formation were genotyped as CYP2A6*4/CYP2A6*4. The other poor metabolizers whose nicotine metabolism was impaired were genotyped as CYP2A6*1B/CYP2A6*4, indicating the possibility of the presence of some other mutation(s) in the CYP2A6*1B allele in those subjects. Therefore, the alleles had been tentatively termed CYP2A6*1B(unknown)/CYP2A6*4[8].

Recently, several new CYP2A6 alleles have been reported [14–16]. The CYP2A6*6 allele has a point mutation in exon 3 leading to a single amino acid substitution (R128Q) [14]. It has been reported that the coumarin 7-hydroxylase activity of the CYP2A6.6 enzyme expressed in insect cells with baculovirus was significantly reduced. The CYP2A6*7 and CYP2A6*8 alleles have point mutations in exon 9, each leading to a single amino acid substitution, I471T and R485L, respectively [15]. These mutations link to CYP2A6*1B. The CYP2A6.7 enzyme expressed in E. coli are almost lacking in nicotine C-oxidase activity and show reduced coumarin 7-hydroxylase activity. However, the effects of the CYP2A6*8 allele on CYP2A6 activity are still unclear. The CYP2A6*1×2 allele has a CYP2A6 gene duplication [16]. This allele is considered to be the reciprocal product of the CYP2A6*4 allele after the unequal crossover event between the 3′-flanking region of the CYP2A6 and CYP2A7 genes. It has been reported the smokers who possess the CYP2A6*1×2 allele appeared to show higher cotinine concentrations in plasma than do homozygotes of CYP2A6*1[16]. In addition, a single nucleotide polymorphism (SNP) in exon 5 (T670C) leading to a single amino acid substitution of S224P has been reported [17]. The effects of the SNP on the CYP2A6 activity are unknown. In the present study, to clarify further the relationships between nicotine metabolism and genetic polymorphism in the CYP2A6 gene, we determined several CYP2A6 alleles recently reported in the subjects whose in vivo nicotine metabolism had been previously phenotyped [7, 8].

Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results and Discussion
  6. References

Chemicals and reagents

Takara LA Taq DNA polymerase and Takara Ex Taq DNA polymerase were purchased from Takara (Kyoto, Japan), and Taq DNA polymerase was obtained from Greiner Japan (Tokyo, Japan). Restriction enzymes were purchased from Toyobo (Osaka, Japan), Takara, or New England Biolabs (Beverly, MA). All other chemicals and solvents were of the highest grade commercially available.

Phenotyping of in vivo nicotine metabolism

In our previous studies [7, 8], 92 Japanese and 209 Korean subjects were phenotyped for in vivo nicotine metabolism. Briefly, the subjects chewed one piece of nicotine gum (Nicorette®, containing 2 mg nicotine, Pharmacia & Upjohn Co, Tokyo, Japan) for 30 min, chewing for 10 s per 30 s. Blood samples were collected from a cubital vein just before and 2 h after the start of chewing. The concentrations of nicotine and cotinine in the plasma samples were determined by h.p.l.c. as described previously [18]. The cotinine/nicotine ratio of the plasma concentration was calculated as an index of nicotine metabolism. Probit transformations of the data were conducted by plotting the cotinine/nicotine ratios of the plasma concentration against their corresponding percent areas under the normal probability curve on probability paper [19]. The cotinine/nicotine ratios of the plasma concentration were ranked from the lowest to the highest value, and the percent area under the normal probability curve was calculated for each data point. The percent area under the normal probability curve was 100 × i/(n + 1), where i is the rank of the data point and n is the sample size.

Genotyping of CYP2A6 alleles

Genomic DNA was extracted from peripheral lymphocytes using a Puregene DNA isolation kit (Gentra Systems, Minneapolis, MN). Primers used in the present study are shown in Table 1. The genotyping of CYP2A6*1B, CYP2A6*2, CYP2A6*3, CYP2A6*4, and CYP2A6*5 was previously performed in our laboratory [7, 8]. In present study, genotyping of CYP2A6*1×2, *6, *7, *8 was performed for all subjects. The genotyping of T670C (S224P) was performed for only 92 Japanese. The CYP2A6*7 allele was genotyped according to the method reported by Ariyoshi et al.[15] with two-step polymerase chain reaction (PCR). For the first PCR, primers of 2A6ex8F and 2A6R1 were used, and for the second PCR, primers 2 A-wt or 2 A-mt and 2A6R2 were used. The CYP2A6*1×2 allele was genotyped by the method previously reported by Rao et al.[16]. The primers 2A6ex8F or 2A7ex8F and 2A7R1 were used.

Table 1.  Sequence of the primers used in the present study.
PrimersSequence
  1. a From Oscarson et al.[20]. bFrom Ariyoshi et al.[21]. cFrom Fernandez-Salguero et al.[11]. dFrom Ariyoshi et al.[15]. eFrom Oscarson et al.[13]. fFrom Oscarson et al.[9].

2A6ex1a5′-GCTGAACACAGAGCAGATGTACA-3′
2A6-UTRAS1b5′-TGTAAAATGGGCATGAACGCCC-3′
2A6 E3Fc5′-GCGTGGTATTCAGCAACGGG-3′
2A6 E3Rc5′-TCGTCCTGGGTGTTTTCCTTC-3′
2 A-wtd5′-CTCCCAGTCACCTAAGGACAT-3′
2 A-mtd5′-CTCCCAGTCACCTAAGGACAC-3′
2A6R1e5′-GCACTTATGTTTTGTGAGACATCAGAG  ACAA-3′
2A6R2e5′-AAAATGGGCATGAACGCCC-3′
2A6ex8Fe5′-CCAGCACTTCCTGAATGAG-3′
2A6*8-wt5′-CTTTGCCACGATCCCACG-3′
2A6*8-mt5′-CTTTGCCACGATCCCACT-3′
2A6 int45′-CCAATCCAGCCTCGTTTAA-3′
2A6 int55′-AGGGTTAATTTGAATGGGC-3′
2A7ex8Fe5′-CATTTCCTGGATGAC-3′
2A7R1f5′-GCACTTATGTTTTGTGAGACATCAGATA  GAG-3′

In a previous report by Kitagawa et al.[14], for the detection of CYP2A6*6, PCR products of exon 3 were digested with Msp I with the PCR-PFLP method. The restriction enzyme Msp I digests PCR products derived from CYP2A6*1 but not those derived from CYP2A6*6 and CYP2A7. Therefore, if exon 3 in the CYP2A7 gene were nonspecifically amplified in the PCR, the digestion pattern with Msp I would lead to a misclassification of CYP2A6*6. Therefore, we improved the PCR-restriction fragment length polymorphism (RFLP) method as described below, using a restriction enzyme of Aor51H I which is specific for CYP2A6*6. In the first PCR reaction, regions from exon 1 to the 3′-untranslated region of the CYP2A6 gene were specifically amplified with the primers of 2A6ex1 and 2A6-UTRAS1. The first LA-PCR product was used as a template for the second PCR with the primers of 2A6 E3F and 2A6 E3R. These PCR conditions were described previously [6]. A PCR product of 202 bp was digested with Aor51H I. The digested fragments were analysed in a 3.5% NuSieve 3 : 1 gel and stained with ethidium bromide. The wild type of the CYP2A6 gene yielded a 202-bp fragment and the CYP2A6*6 allele yielded 159 and 43 bp fragments (Figure 1a).

image

Figure 1. Genotyping of CYP2A6*6, CYP2A6*8 and SNP of T670C (S224P). (a) Schematic PCR-RFLP patterns for detection of the CYP2A6*6 allele. (b) Schematic AS-PCR patterns for detection of the CYP2A6*8 allele. wt, PCR product obtained by wild-specific primer; mt, PCR product obtained by mutant-specific primer. (c) Schematic PCR-RFLP patterns for the detection of SNP of T670C (S224P).

Download figure to PowerPoint

An allele specific (AS)-PCR method for the detection of the CYP2A6*8 allele was developed in the present study. The first PCR was performed with primers of 2A6ex8F and 2A6R1 according to the method of Oscarson et al.[13]. The first PCR products were used as a template for the second PCR with the primers of 2A6*8-wt or 2A6*8-mut and 2A6R2. The reaction mixture contained the first PCR product (1.0 µl), 1 X PCR buffer (Greiner), 1.5 mm MgCl2, 0.25 mm dNTPs, 0.4 µm each primer, and 1 U of Taq DNA polymerase (Greiner) in a final volume of 25 µl. After an initial denaturation at 94 °C for 3.0 min, the amplification was performed by denaturation at 94 °C for 15 s, annealing at 58 °C for 30 s, and extension at 72 °C for 1 min for 18 cycles. The expected size of the PCR product was 394 bp. An aliquot (5 µl) of the PCR product was analysed by electrophoresis using 0.8% agarose gel (Figure 1b).

Genotyping method for the detection of T670C (S224P) reported by Daigo et al.[17] was developed in our laboratory with PCR-RFLP. CYP2A6 specific PCR was accomplished with the primers of 2A6 int4 and 2A6 int5. The reaction mixture contained approximately 50 ng genomic DNA, 1 X PCR buffer (Takara), 0.25 mm dNTP, 0.4 µm each primer, 1 U of Ex Taq polymerase (Takara) in a final volume of 25 µl. After an initial denaturation at 94 °C for 3 min, the amplification was performed by denaturation at 94 °C for 1 min, annealing at 56 °C for 1 min, and extension at 72 °C for 1 min for 30 cycles. A PCR product of 532 bp was digested with Mbo II restriction enzyme. The digestion patterns were determined by electrophoresis using 2% agarose gel. The wild type of the CYP2A6 gene (T/T) yielded 263, 228, and 41 bp fragments and the mutant type (C/C) yielded 491 and 41 bp fragments (Figure 1c).

DNA sequencing

To confirm the presence of CYP2A6*10, DNA sequencing analysis was performed. The PCR product with the primers of 2A6ex8F and 2A6R1 from the subject who was genotyped as a heterozygote of CYP2A6*10/CYP2A6*4 was subcloned into pT7Blue T-vector (Novagen, Madison, WI). The plasmid DNA was purified by QIAGEN Plasmid Midi kit (QIAGEN, Valencia, CA) and submitted to DNA sequencing using a Thermo Sequenase Cy5.5 Dye Terminator Cycle Sequencing kit (Amersham Pharmacia Biotech, Buckinghamshire, UK) with T7F primer (Amersham Pharmacia Biotech). DNA sequences were analysed on a Long-Read Tower DNA sequencer (Amersham Pharmacia Biotech).

Data analysis

Ethnic differences in metabolic ratios in the different genotype groups were tested by Mann–Whitney U-test. P < 0.05 was considered statistically significant.

Results and Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results and Discussion
  6. References

The cotinine/nicotine ratios of the different CYP2A6 genotype groups are shown in Table 2. As we previously reported [8], in total subjects, Korean subjects (8.73 ± 11.88) revealed a significantly (P < 0.005) higher metabolic ratio than Japanese subjects (3.78 ± 3.09). Furthermore, in the genotypes of CYP2A6*1 A/CYP2A6*1 A (7.42 ± 6.56 vs 4.13 ± 3.00, P < 0.005), CYP2A6*1 A/CYP2A6*1B (10.4 ± 16.89 vs 6.05 ± 3.99, P < 0.05), CYP2A6*1B/CYP2A6*1B (12.53 ± 9.70 vs 4.94 ± 3.49, P < 0.005), CYP2A6*1 A/CYP2A6*4 (4.79 ± 3.17 vs 2.55 ± 1.22, P < 0005), and CYP2A6*1 A/CYP2A6*7 (6.27 ± 4.76 vs 1.47 ± 0.92, P < 0.05), the mean metabolic ratios in the Koreans were significantly higher than those in the Japanese. The allele frequencies of CYP2A6 in Japanese and Koreans are also shown in Table 2. Among the present 92 Japanese and 209 Koreans, no CYP2A6*6 allele was found. Three DNA samples genotyped as heterozygotes of CYP2A6*6 by their method were kindly provided by Drs Kitagawa and Kawamoto (University of Occupational and Environmental Health, Kitakyusyu, Japan). However, these DNA samples were not genotyped as CYP2A6*6 with our improved genotyping method using Aor51H I that is specific for the CYP2A6*6 allele. In their analysis [14], only just a part of exon 3 from the PCR product was analysed. In contrast, in our genotyping method, after the almost complete CYP2A6 gene (from exon 1 to 3′-untranslated region) was amplified, exon 3 was amplified with the first PCR product as a template. Therefore, the possibility cannot be excluded that the SNP in CYP2A6*6 allele might be from a certain pseudogene, because the CYP2A6*6 allele was not amplified with our improved PCR method. Previously, Kitagawa et al.[14] reported that the allele frequency of CYP2A6*6 was 0.4% in 894 Japanese studied. Our contradictory results might be due to the small sample size compared with their study.

Table 2.  Cotinine/nicotine ratios of the plasma concentration and allele frequencies of CYP2A6 gene in 92 Japanese and 209 Koreans.
GenotypeCotinine/nicotine ratios of the plasma concentration
JapaneseKoreans
CYP2A6*1 A/CYP2A6*1 A4.13 ± 3.00 (n = 22) 7.42 ± 6.56 (n = 41)**
CYP2A6*1 A/CYP2A6*1B6.05 ± 3.99 (n = 14)10.34 ± 16.89 (n = 79)*
CYP2A6*1 A or CYP2A6*1B(n = 0)12.54 (n = 1)
/CYP2A6*1 × 2
CYP2A6*1B/CYP2A6*1B4.94 ± 3.49 (n = 12)12.53 ± 9.70 (n = 27)*
CYP2A6*1 A/CYP2A6*42.55 ± 1.22 (n = 15) 4.79 ± 3.17 (n = 19)*
CYP2A6*1B/CYP2A6*44.49 ± 2.66 (n = 10) 8.10 ± 4.85 (n = 16)*
CYP2A6*4/CYP2A6*40.00 ± 0.00 (n = 3) 0.00 ± 0.00 (n = 4)*
CYP2A6*1 A/CYP2A6*5(n = 0) 5.68 (n = 1)
CYP2A6*1B/CYP2A6*5(n = 0)14.82 (n = 1)
CYP2A6*1 A/CYP2A6*71.47 ± 0.92 (n = 3) 6.27 ± 4.76 (n = 7)*
CYP2A6*1B/CYP2A6*73.74 (n = 1)13.35 (n = 1)
CYP2A6*7/CYP2A6*71.10 (n = 1) 0.92 (n = 2)
CYP2A6*7/CYP2A6*40.79 ± 0.62 (n = 5) 2.08 (n = 2)
CYP2A6*1 A/CYP2A6*83.37 (n = 2) 4.52 ± 2.95 (n = 3)
CYP2A6*1B/CYP2A6*84.35 (n = 2)10.33 ± 8.81 (n = 3)
CYP2A6*7/CYP2A6*100.73 (n = 1) 1.75 (n = 1)
CYP2A6*10/CYP2A6*40.09 (n = 1) 0.07 (n = 1)
Total3.78 ± 3.09 (n = 92) 8.73 ± 11.88 (n = 209)**
AlleleNumber of alleles (%)
Japanese (n = 184)Koreans (n = 418)
  1. Data are expressed as mean ± s.d. *P < 0.05, **P < 0.005, from Japanese subjects with each genotype by Mann–Whitney U-test.

CYP2A6*1 A78 (42.4)192 (45.9)
CYP2A6*1B51 (27.7)155 (37.1)
CYP2A6*2 0 (0) 0 (0)
CYP2A6*3 0 (0) 0 (0)
CYP2A6*437 (20.1) 46 (11.0)
CYP2A6*5 0 (0) 2 (0.5)
CYP2A6*6 0 (0) 0 (0)
CYP2A6*712 (6.5) 15 (3.6)
CYP2A6*8 4 (2.2) 6 (1.4)
CYP2A6*10 2 (1.1) 2 (0.5)

As reported by Ariyoshi et al.[15], we confirmed that the T1412C mutation in the CYP2A6*7 allele was not found on the CYP2A6*1 A allele. In the present study, the allele frequencies of CYP2A6*7 in the Japanese and the Koreans were 6.5% and 3.6%, respectively. Although the allele frequency of CYP2A6*7 was previously reported to be 15.7%[15], the inconsistency in the allele frequencies would be due to the small sample size analysed by Ariyoshi et al.[15] (only 21 subjects). Ariyoshi et al.[15] also reported the presence of CYP2A6*8 allele. However, the frequency of the CYP2A6*8 allele is unknown. Therefore, in the present study, the genotyping method for CYP2A6*8 was developed to determine the allele frequency. The allele frequencies of CYP2A6*8 in the Japanese and the Koreans were 2.2% and 1.4%, respectively. We also confirmed that the G1454T mutation in the CYP2A6*8 allele was not found on the CYP2A6*1 A allele. In the process of performing the CYP2A6*7 and CYP2A6*8 genotyping, two subjects were genotyped as CYP2A6*7/CYP2A6*4 and CYP2A6*8/CYP2A6*4 as well. Since the CYP2A6*4 allele is a whole deletion type, the results suggested the presence of an allele possessing two SNPs of T1412C (CYP2A6*7) and G1454T (CYP2A6*8) simultaneously on an allele. The novel allele was termed as CYP2A6*10. Therefore, the two subjects were re-classified as CYP2A6*10/CYP2A6*4. Furthermore, since two other subjects were genotyped as homozygotes of CYP2A6*7 as well as heterozygotes of CYP2A6*8, they were re-classified as CYP2A6*7/CYP2A6*10. We confirmed the DNA sequence of CYP2A6*10 from DNA samples from a subject genotyped as CYP2A6*10/CYP2A6*4 (Figure 2). The allele frequencies of CYP2A6*10 in the Japanese and the Koreans were 1.1% and 0.5%, respectively.

image

Figure 2. The electrophoregram of CYP2A6 gene sequence of exon 9 in a subject genotyped as CYP2A6*10/CYP2A6*4. Arrows indicate the nucleotides substituted in the CYP2A6*10 allele.

Download figure to PowerPoint

Rao et al.[16] reported that the frequency of the CYP2A6*1×2 was 1.7% (5/296) in Caucasians. In the present study, one Korean subject possessed the CYP2A6*1×2 allele. Therefore, the frequency was 0.5% (1/209) in Koreans. We first found the CYP2A6*1×2 allele in an Oriental. However, there was no CYP2A6*1×2 allele in Japanese subjects. Finally, one Japanese subject was heterozygote of T670C (S224P) allele.

In our previous studies [7, 8], one Korean and four Japanese subjects who had been genotyped as CYP2A6*1B/CYP2A6*4 were phenotyped as poor metabolizers of nicotine. In the present study, these subjects were re-genotyped as CYP2A6*7/CYP2A6*4 or CYP2A6*10/CYP2A6*4 (Figure 3). It has been reported that CYP2A6.7 expressed in E. coli lacked in vitro nicotine C-oxidase activity [15]. Therefore, the present study could directly prove that the CYP2A6*7 allele decreases the CYP2A6 activity in vivo. The nicotine metabolism potencies in two subjects genotyped as CYP2A6*10/CYP2A6*4 (cotinine/nicotine ratio, 0.07 and 0.09) were lower than those in the three subjects genotyped as CYP2A6*7/CYP2A6*4 (cotinine/nicotine ratio, 0.24, 0.32, and 0.46), indicating the SNP of CYP2A6*8 (G1454T) might also decrease the CYP2A6 activity (Table 2). However, there was no in vitro study to determine the effect of SNP of CYP2A6*8 allele on the CYP2A6 activity. Furthermore, in the present study, there was no subject homozygous for CYP2A6*8/CYP2A6*8 and heterozygous for CYP2A6*8/CYP2A6*4. Thus, the effects of SNP of CYP2A6*8 on CYP2A6 activity in vivo are still unclear. In our previous studies [7, 8], the antimode was determined as 0.6 of cotinine/nicotine ratio by the probit analysis. However, the present study revealed that the extensive metabolizers near to the antimode who were genotyped as CYP2A6*7/CYP2A6*7 (cotinine/nicotine ratio, 0.82, 1.02, and 1.10), CYP2A6*7/CYP2A6*10 (cotinine/nicotine ratio, 0.73 and 1.75), or CYP2A6*7/CYP2A6*4 (cotinine/nicotine ratio, 0.76, 1.46, 1.48, and 3.40) showed a lower potency of nicotine metabolism (Figure 3, Table 2). The discordance of genotype and phenotype in these subjects possibly suggested that the phenotyping of in vivo nicotine metabolism might be affected by some factor(s) other than genetic factors, such as diet or environmental factors.

image

Figure 3. Probit analysis for the cotinine/nicotine ratio of the plasma concentration 2 h after chewing one piece of nicotine gum in 92 Japanese and 209 Koreans. The abscissa denotes the cotinine/nicotine ratio of the plasma concentration in different individuals. The ordinate represents the percent area under the normal probability curve for each data point. The arrow represents the antimode of the probit plot.

Download figure to PowerPoint

In the present study, one Korean subject possessing the CYP2A6*1×2 allele did not show the highest potency of nicotine metabolism. It has been reported that smokers who possess the CYP2A6*1×2 allele have higher plasma cotinine levels than homozygous wild type smokers [16]. However, their phenotyping was performed under a condition in which the nicotine intake was not controlled. Therefore, it is still unclear whether the existence of the CYP2A6*1×2 allele could increase nicotine metabolism.

In conclusion, we clarified that impaired nicotine metabolism is due to the genetic polymorphism of CYP2A6 gene. The CYP2A6 enzymatic activity is lost in the subjects homozygous for either CYP2A6*4, CYP2A6*7, or CYP2A6*10, or heterozygous for these alleles in combination.

This study was supported by an SRF Grant for Biomedical Research in Japan, a grant from Japan Health Sciences Foundation with Research on Health Science focusing on Drug Innovation, and by Philip Morris Incorporated. We thank Mr Brent Bell for reviewing this article.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results and Discussion
  6. References
  • 1
    Oscarson M. Genetic polymorphisms in the cytochrome P450 2A6 (CYP2A6 ) gene: implications for interindividual differences in nicotine metabolism. Drug Metab Dispos 2001; 29: 9195.
  • 2
    Nakajima M, Yamamoto T, Nunoya K-I, et al. Role of human cytochrome P4502A6 in C-oxidation of nicotine. Drug Metab Dispos 1996; 24: 12121217.
  • 3
    Nakajima M, Yamamoto T, Nunoya K-I, et al. Characterization of CYP2A6 involved in 3′-hydroxylation of cotinine in human liver microsomes. J Pharmacol Exp Ther 1996; 277: 10101015.
  • 4
    Cholerton S, Arpanahi A, McCracken N, et al. Poor metabolisers of nicotine and CYP2D6 polymorphism. Lancet 1994; 343: 6263.
  • 5
    Benowitz NL, Jacob PIII, Sachs DPL. Deficient C-oxidation of nicotine. Clin Pharmacol Ther 1995; 57: 590594.
  • 6
    Nakajima M, Yamagishi S-I, Yamamoto H, et al. Deficient cotinine formation from nicotine is attributed to the whole deletion of the CYP2A6 gene in humans. Clin Pharmacol Ther 2000; 67: 5769.
  • 7
    Nakajima M, Kwon J-T, Tanaka N, et al. Relationship between interindividual differences in nicotine metabolism and CYP2A6 genetic polymorphism in humans. Clin Pharmacol Ther 2001; 69: 7278.
  • 8
    Kwon J-T, Nakajima M, Chai S, et al. Nicotine metabolism and CYP2A6 allele frequencies in Koreans. Pharmacogenetics 2001; 11: 317323.
  • 9
    Oscarson M, McLellan RA, Gullstén H, et al. Identification and characterisation of novel polymorphisms in the CYP2A locus: implications for nicotine metabolism. FEBS Lett 1999; 460: 321327.
  • 10
    Yamano S, Tatsuno J, Gonzalez FJ. The CYP2A3 gene product catalyzes coumarin 7-hydroxylation in human liver microsomes. Biochemistry 1990; 29: 13221329.
  • 11
    Fernandez-Salguero P, Hoffman SMG, Cholerton S, et al. A genetic polymorphism in coumarin 7-hydroxylation: sequence of the human CYP2A genes and identification of variant CYP2A6 alleles. Am J Hum Genet 1995; 57: 651 660.
  • 12
    Nunoya K-I, Yokoi T, Takahashi Y, et al. Homologous unequal cross-over within the human CYP2A gene cluster as a mechanism for the deletion of the entire CYP2A6 gene associated with the poor metabolizer phenotype. J Biochem 1999; 126: 402407.
  • 13
    Oscarson M, McLellan RA, Gullstén H, et al. Characterisation and PCR-based detection of a CYP2A6 gene deletion found at a high frequency in a Chinese population. FEBS Lett 1999; 448: 105110.
  • 14
    Kitagawa K, Kunugita N, Kitagawa M, Kawamoto T. CYP2A6*6, a novel polymorphism in cytochrome P450 2A6, has a single amino acid substitution (R128Q) that inactivates enzymatic activity. J Biol Chem 2001; 276: 1783017835.
  • 15
    Ariyoshi N, Sawamura Y, Kamataki T. A novel single nucleotide polymorphism altering stability and activity of CYP2A6. Biochem Biophys Res Commun 2001; 281: 810814.
  • 16
    Rao Y, Hoffmann E, Zia H, et al. Duplications and defects in the CYP2A6 gene: identification, genotyping, and in vivo effects on smoking. Mol Pharmacol 2000; 58: 747755.
  • 17
    Daigo S, Takahashi Y, Ariyoshi N, et al. Genotyping of CYP2A6 gene in patient whose pharmacokinetics of tegafur was abnormal. Xenobio Metab Dispos 2000; 15: S209.
  • 18
    Nakajima M, Yamamoto T, Kuroiwa Y, Yokoi T. Improved highly sensitive method for determination of nicotine and cotinine in human plasma by high-performance liquid chromatography. J Chromatogr B 2000; 742: 211215.
  • 19
    Finney DJ. Probit Analysis. London: Cambridge University Press, 1964.
  • 20
    Oscarson M, Gullstén H, Rautio A, et al. Genotyping of human cytochrome P450 2A6 (CYP2A6), a nicotine C-oxidase. FEBS Lett 1998; 438: 201205.
  • 21
    Ariyoshi N, Takahashi Y, Miyamoto M, et al. Structural characterization of a new variant of the CYP2A6 gene (CYP2A6*1B) apparently diagnosed as heterozygotes of CYP2A6*1A and CYP2A6*4C Pharmacogenetics 2000; 10: 687693.