Access the full text.
Sign up today, get DeepDyve free for 14 days.
Loading next page...
/lp/springer-journals/interaction-between-cytochrome-p450-2a6-and-catechol-o-V10PEYqMHU
- Publisher
- Springer Journals
- Copyright
- Copyright © 2017 by The Author(s)
- Subject
- Biomedicine; Neurosciences; Neurology; Behavioral Therapy; Psychiatry
- eISSN
- 1744-9081
- DOI
- 10.1186/s12993-017-0127-2
- pmid
- 28472995
- Publisher site
- See Article on Publisher Site
Abstract
Background: Although some effects of gene–gene interactions on nicotine–dopamine metabolism for smok ‑ ing behavior have been reported, polymorphisms of cytochrome P450 (CYP) 2A6 and catechol‑ O‑ methyltransferase (COMT ) have not been studied together to determine their effects on smokers. The aim of this study was to investigate the effects of the interaction between the CYP 2A6 and COMT genes on smoking behavior in young Taiwanese men. Results: A self‑ report questionnaire regarding smoking status was administered to 500 young men. Polymorphisms of the CYP 2A6 and COMT genes as well as urinary nicotine and urinary cotinine levels were determined. The odds ratio for starting smoking was significantly lower in subjects carrying a CYP2A6 low activity/variant COMT rs4680 genotype than in those possessing a CYP2A6 wild‑ type/variant COMT rs4680 genotype (0.44, 95% confidence inter ‑ val = 0.19–0.98, P = 0.043). Comparisons of Fagerstrom Test for Nicotine Dependence (FTND), Physiological Cigarette Dependence Scale (PCDS), and Cigarette Withdrawal symptoms (CWS‑ 21) among the smokers with different CYP2A6/ COMT polymorphisms were not significantly different. The adjusted urinary nicotine concentrations were not sig‑ nificantly different between the two groups carrying different genotypes. The adjusted urinary cotinine level was significantly different between the COMT rs4680 wild‑ type group and COMT rs4680 variant group [92.46 ng/μL vs. 118.24 ng/μL (median value), P = 0.041] and between the COMT rs4680 wild‑ type/COMT rs165599 variant group and COMT rs4680 variant/COMT rs165599 variant group (97.10 ng/μL vs. 122.18 ng/μL, P = 0.022). Conclusions: These findings suggest that a single nucleotide polymorphism (rs4680) of the COMT gene and the interaction between the CYP 2A6 and COMT genes affect smoking status in young Taiwanese men. Keywords: Catechol‑ O‑ methyltransferase, Cotinine, Cytochrome P450 2A6, Nicotine, Smoking status Background factors have a greater influence on smoking cessation Tobacco smoking is a multi-factorial behavior with both than do environmental factors [2]. Genetic factors are genetic and environmental determinants [1]. Genetic responsible for 30–50% of the variance in the risk of withdrawal symptoms, 40–75% of the variation in smok- ing initiation, 50% of the variance in cessation success, *Correspondence: ching.shan.h@gmail.com; plchen@ctust.edu.tw and 70–80% of the variation in smoking maintenance [1]. Wei‑ Chih Ou and Yi‑ Chin Huang contributed equally to this work Ching‑ Shan Huang and Pei‑ Lain Chen contributed equally to this work Genetic risk information enhances the motivation for Department of Medical Laboratory Science and Biotechnology, Central smoking cessation [3]. Therefore, assessment of genetic Taiwan University of Science and Technology, No. 666 Buzih Road, Beitun background could be a promising tool to understand District, Taichung City 40601, Taiwan Administration Center for Research and Education, Changhua Christian smoking risk and to guide the selection of the most effec - Hospital, Changhua, Taiwan tive cessation treatment for an individual smoker. Full list of author information is available at the end of the article © The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Ou et al. Behav Brain Funct (2017) 13:8 Page 2 of 10 Nicotine is the major psychoactive ingredient in affected smoking intensity in young men [14]. Those tobacco, and it modulates dopamine activity in the mid- findings encouraged us to perform more genetic studies brain, which contributes to the development and main- of Taiwanese smokers. In relation to nicotine–dopamine tenance of rewarding behaviors such as smoking [4]. metabolism, smoking status has been reported to corre- Smokers modulate their smoking to maintain brain nico- late better with some gene–gene interactions than with tine levels within a certain concentration range, and fac- a single gene only. These interactions include COMT and tors that alter nicotine clearance affect smoking behavior MAOA [15, 16], MAOA and CYP2A6 [17], CYP2A6 and [4]. Individuals who eliminate nicotine rapidly are less the nicotine acetylcholine receptor gene [18, 19], and likely to achieve low craving scores even after smoking CYP 2A6 and DRD2 TaqIA [20]. However, the effects of freely [4]. Consequently, genetic polymorphisms in both CYP2A6 and COMT together have not been explored, nicotine metabolism and dopamine catabolism genes and we hypothesized that a single polymorphism as well influence smoking status, interact with each other to as an interaction of the two genes could be involved in result in risk modulation, and affect smoking cessation smoking status. therapies. In women not using oral contraceptives, nicotine and The cytochrome P450 (CYP ) 2A6 gene, located on cotinine clearance is 13 and 24% higher, respectively, than chromosome 19q12-q13.2, consists of nine exons. It in men [6]. Sex differences exist for cravings, affect, and is involved in producing a 494-amino-acid protein preference for immediate smoking after cue exposure that oxidizes coumarin, nicotine, and tobacco-specific [21]. Because Taiwanese men smoke significantly more nitrosamines [5]. CYP2A6 is the primary human enzyme cigarettes than Taiwanese women [12, 22], only men were involved in nicotine metabolism [4]. CYP2A6 catalyzes invited to participate in our study. In this study, the poly- the C-oxidation of nicotine to the inactive metabolite morphisms CYP2A6*1A (wild-type), CYP 2A6*1B (poly- cotinine and the subsequent conversion of cotinine into morphism of faster nicotine clearance) [23], CYP2A6*4C trans-3′-hydroxycotinine [4]. CYP2A6 is the most studied (the most studied polymorphism of decreased nico- enzyme involved in both adult and adolescent smokers tine metabolism) [24], COMT Val/Met (rs4680) [9], and [6]. The results of studies among Taiwanese individuals COMT rs165599 [10] were determined. Our objectives indicate that the variant status of CYP2A6 is different were to investigate the effects of the interaction between from that among other ethnic groups [7]. Therefore, we the CYP 2A6 and COMT genes and their association with hypothesized that, for Taiwanese individuals, polymor- smoking risk in young Taiwanese men. phisms in the CYP2A6 gene that affect smoking status could be different from those in other ethnic groups. Methods The catechol-O-methyltransferase (COMT) gene is Participants and procedures located on chromosome 22q11.21, has eight exons, and This cross-sectional study was advertised to all students produces a 271-amino-acid protein that metabolizes cat- at both Chang Jung Christian University and Central echolamines [8]. Low enzyme activity of the Met allele Taiwan University of Science and Technology. The vol - at codon 108/158 (in the rs4680 polymorphism) of the unteers contacted the authors of the study, and conveni- COMT gene, which encodes a key enzyme involved in the ence samples were then screened for eligibility at the metabolic inactivation of dopamine, has been associated health centers of the two universities. All study subjects with nicotine dependence [9]. Another polymorphism provided written, informed consent at the beginning of of COMT, rs165599, has been related to the response to study, and the study was approved by the review board bupropion therapy for smoking cessation [10]. However, of Chang Jung Christian University (CJCU-99004) and those associations have been inconsistent among ethnic Central Taiwan University of Science and Technology groups [10, 11]. Thus, it seems necessary to perform a (CTUST-99016). The study was conducted in accordance genetic study of smoking status for each ethnicity. with Good Clinical Practice procedures and the Declara- Smoking addiction is currently a significant social tion of Helsinki. problem in Taiwan [12]. Nonetheless, very few genetic We administered a self-report questionnaire to all of investigations of nicotine–dopamine metabolism and the study subjects [25–27]. The questionnaire included smoking status among Taiwanese individuals have been demographic data, smoking background and status. performed. Recently, for Taiwanese smokers in a group of In addition, the questionnaire included the fagerstrom methadone maintenance patients, polymorphisms of the test for nicotine dependence (six-item FTND) [25], μ-opioid receptor gene were associated with the plasma the 15-item short form of the Physiological Cigarette concentration of cotinine [13]. Very recently, our team Dependence Scale (PCDS) derived from the 30-item reported that the interaction of the dopamine D2 recep- PCDS [27], and the Withdrawal Symptoms Scale (Cig- tor (DRD2) TaqIB and monoamine oxidase A (MAOA) arette Withdrawal Scale, CWS-21) [26, 27]. These Ou et al. Behav Brain Funct (2017) 13:8 Page 3 of 10 questionnaires used biomarkers (nicotine and cotinine assays. The DNAs of CYP2A6*1A/*1A, CYP2A6*1A/*1B, concentrations) as validation, with no over- or under- CYP2A6*1B/*1B, CYP2A6*1A/*4C, and CYP2A6*1B/*4C, exaggeration. Never smokers were defined as persons which have been found in Taiwanese and Chinese pop- who had never smoked in their lifetime. Current smok- ulations, were identified by DNA sequencing [29, 30]. ing was defined as ever smoking cigarettes on 1 or more Additional file 1: Table S1 lists the result. days of the past 30 days. Ever smokers were defined as For COMT rs4680, the forward primer 5′-CTGTGGCT persons who smoked at one time, had quit, and were not ACTCAGCTGTG-3′ and reverse primer 5′-CCTT currently smoking. The current smokers were divided TTTCCAGGTCTGACAA-3′ were used to amplify a into two groups according to their intensity of cigarette 169-bp fragment [10]. Using N1a III as the restriction smoking: light smokers and heavy smokers, depend- enzyme, three (114, 32, and 23-bp), four (96, 32, 23, and ing on number of cigarettes per day lower than or equal 18-bp), and five (114, 96, 32, 23, and 18-bp) fragments to (or higher than) the average value of all the smokers, were obtained for the G/G, A/A, and G/A genotypes, respectively. The exclusion criteria were (1) a history of respectively [10]. For COMT rs165599, the forward diagnosed mental health disease or cancer, (2) alcoholism primer 5′-CATTCAAAGCTCCCCTTGAC-3′ and or drug abuse, (3) severe communication problems, or (4) reverse primer 5′-GGGAGTAGG-GAAGGAGATGC-3′ terminal illness. When the survey was completed, blood were utilized to amplify a 301-bp fragment [32]. Using and urine samples were obtained at a university center Msp I as the restriction enzyme, one (301-bp), two (166 by trained research assistants (licensed nurses or medical and 135-bp), and three (301, 166, and 135-bp) fragments technologists). were obtained from the A/A, G/G, and A/G genotypes, respectively [32]. Determination of CYP2A6 and COMT polymorphisms In this study, to determine the genotypes of the vari- Determination of urinary nicotine and cotinine ants of interest, total genomic DNA was isolated from To evaluate the effects of genetic polymorphisms on blood cells using a blood DNA isolation kit (Favorgen, the metabolism of nicotine and cotinine, urinary nico- Ping-Tung, Taiwan). Five milliliters of whole blood with tine and cotinine levels of smokers were measured. Gas EDTA as an anticoagulant were required. Approximately chromatography–mass spectrometry (GC/MS) was per- 40 alleles of the CYP2A6 gene have been identified [28]. formed as previously described [33], with the follow- However, only the wild-type (CYP2A6*1A) and two ing difference: GC–MS analyses were performed on a highly prevalent (>10%) variants of CYP2A6*1B (approxi- ThermoElectron DSQII quadrupole mass spectrometer mately 45%) [29] and CYP2A6*4C (approximately 15%) connected directly to a ThermoElectron focus gas chro - [30], which have been observed in Chinese individuals, matograph and an autosampler AS 3000 (Thermo Elec - were determined in this project. tron Corporation, Dreieich, Germany). All urine samples The polymorphisms of CYP2A6 were identified were stored at −20 °C before analysis. All of the analyses using polymerase chain reaction-restriction fragment were performed in duplicate and repeated if values dif- length polymorphism (PCR–RFLP) [31]. We designed fered by >10%. Tobacco cigarettes currently smoked in the forward primer 5′-CACCGAAGTGTTCCCTATG Taiwan contain 0.57–0.64 mg of nicotine per cigarette on CTG-3′ and reverse primer 5′-TGTAAAATGGGCATG average [12]. The nicotine and cotinine concentrations in AACGCCC-3′ according to the GenBank accession sys- the urine of each subject were divided by the number of tem. PCR was performed with a thermal cycler (Bio-Rad, daily cigarettes smoked and defined as the adjusted nico - Carlsbad, CA, USA). The PCR conditions were as fol - tine and adjusted cotinine concentration. lows: first cycle, denaturation at 94 °C for 3 min; cycles 2–31, denaturation at 94 °C for 1 min, annealing at 55 °C The effects of gene polymorphism interactions on smoking for 1 min, and elongation at 72 °C for 2 min, with a final behaviors extension for 7 min at 72 °C. We detected a 1332-base The CYP2A6 genotypes consisted of wild-type (*1A/*1A), pair (bp) fragment on a 1% agarose gel after electropho- high -activity (*1A/*1B and *1B/*1B), and low-activity resis at 100 V for 60 min. Using BstU-I as the restriction (*1A/*4C, *1B/*4C, and *4C/*4C) genotypes. The COMT enzyme, one (1332-bp) and two (291 and 1041-bp) frag- rs4680 genotypes consisted of wild-type (G/G) and vari- ments were obtained for CYP2A6*1A and CYP2A6*non- ant (G/A and A/A). The COMT rs165599 genotypes 1A, respectively. Using the restriction enzyme Bsu36-I, consisted of wild-type (A/A) and variant (A/G and G/G) three fragments (104, 437, and 792-bp) were observed genotypes. To assess the interaction between the effects for CYP2A6*1A or CYP2A6*1B. Four fragments (64, of CYP2A6 and COMT gene polymorphisms on smok- 104, 437, and 728-bp) were found for CYP2A6*4C. ing behaviors, multiple models were used to analyze the Positive controls were run for each of the genotyping CYP2A6, COMT rs4680, and COMT rs165599 data: (1) Ou et al. Behav Brain Funct (2017) 13:8 Page 4 of 10 single gene (model 1: CYP2A6) or single SNP (model 2: fit a normal distribution, the Mann–Whitney U test with COMT rs4680, COMT rs165599, respectively); (2) 2 SNPs Bonferroni adjustment was utilized to compare the data (model 3: COMT rs4680 and COMT rs165599); and (3) between the two groups (Table 3). All data were analyzed multiple genes (models 4–7). We evaluated the effect of using SPSS (version 18.0 software for Windows, SPSS the interactions between different gene combinations on Inc., Chicago, IL). smoking status (Table 2), smoking intensity (Additional file 2: Table S2), nicotine dependence (FTND), physiolog- Results ical cigarette dependence (PCDS), nicotine toxicity and We recruited a total of 500 men aged 20–25 years. withdrawal symptoms (CWS-21) (Additional file 3: Table Analysis of the questionnaires revealed that there were S3), and urine nicotine/cotinine concentration (Table 3) 219 never smokers, 261 current smokers, and 20 ever in young men. smokers. Due to the small sample size (moderate power analysis in general, α = 0.05 and 80% power requires 200 Statistical analysis samples), the 20 ever smokers were excluded from fur- To evaluate the effect of the genetic variants on smok - ther study. Mean age did not differ between the 261 cur - ing status and smoking intensity, this study assigned an rent smokers and the 219 never smokers (22.6 ± 1.69 vs. odds ratio (OR) as 1 to subjects carrying the wild-type. 22.4 ± 1.42, t = 1.371, P = 0.176, data not shown). The Mantel–Haenszel Chi square test was utilized to cal - The distributions of CYP2A6 and COMT polymor- culate the ORs and their 95% confidence intervals (CIs) phisms are shown in Table 1. Using the frequencies of (Table 2; Additional file 2: Table S2). To compare the CYP2A6*1A/*1A, CYP2A6*1A/*1B, and CYP2A6*1B/*1B significance of FTND, PCDS, and CWS-21 and to com - as examples, the distributions of CYP2A6 genotypes pare the urinary nicotine and cotinine clearance among agreed with Hardy–Weinberg equilibrium: P = 0.713 subjects carrying different CYP2A6 and COMT polymor- and 0.332 for current smokers and never smokers, phisms, Student’s t test or analysis of variance (ANOVA), respectively. The distribution of COMT rs165599 geno- as appropriate, was applied to compare quantitative data. types also agreed with Hardy–Weinberg equilibrium: A P value of <0.05 was defined as statistically significant P = 0.966 and 0.668 for current smokers and never for each analysis (Additional file 3: Table S3). When the smokers, respectively. The distribution of COMT rs4680 result of ANOVA was statistically significant, multiple genotypes did not agree with Hardy–Weinberg equilib- comparisons were followed by application of Scheffé’s rium: P = 0.003 and 0.012 for current smokers and never post hoc test. However, if the data in any group did not smokers, respectively. Table 1 Distribution of CYP2A6 and COMT polymorphisms Genotypes Current smokers Hardy–Weinberg Never smokers Hardy–Weinberg [n = 261 (n%)] equilibrium [n = 219 (n%)] equilibrium a a CYP2A6 P = 0.713 P = 0.332 1A/1A 63 (24.1) 46 (21.0) 1A/1B 100 (38.3) 88 (40.2) 1B/1B 44 (16.9) 31 (14.2) 1A/4C 24 (9.2) 25 (11.4) 4C/4C 4 (1.5) 3 (1.37) 1B/4C 26 (10.0) 26 (11.9) b b COMT rs4680 P = 0.003 P = 0.012 G/G 147 (56.3) 113 (51.6) G/A 73 (28.0) 69 (31.5) A/A 41 (15.7) 37 (16.9) c c COMTrs165599 P = 0.966 P = 0.668 A/A 61 (23.4) 45 (20.6) A/G 130 (49.8) 112 (51.1) G/G 70 (26.8) 62 (28.3) For CYP2A6 1A/1A, 1A/1B, 1B/1B For COMT rs4680 For COMT rs165599 Ou et al. Behav Brain Funct (2017) 13:8 Page 5 of 10 Table 2 Odds ratios for the effects of CYP2A6 and COMT polymorphisms on smoking status Genotypes interaction Current smokers N = 261 Never smokers N = 219 OR (95% CI) P Model 1 CYP2A6 Wild type 63 46 1.0 High activity 144 119 0.88 (0.56–1.39) 0.591 Low activity 54 54 0.73 (0.43–1.25) 0.249 Model 2 COMT COMT rs4680 Wild type 147 113 1.0 Variant 114 106 0.83 (0.58–1.19) 0.301 COMT rs165599 Wild type 61 45 1.0 Variant 200 174 0.85 (0.55–1.31) 0.458 Model 3 COMT rs4680/COMT rs165599 Wild type/wild type 27 13 1.0 Wild type/variant 120 100 0.58 (0.28–1.18) 0.128 Variant/wild type 34 32 0.51 (0.23–1.16) 0.107 Variant/variant 80 74 0.52 (0.25–1.08) 0.078 Model 4 COMT rs4680 wild type CYP2A6 wild type 35 25 1.0 CYP2A6 high activity 76 65 0.84 (0.45–1.54) 0.563 CYP2A6 low activity 36 23 1.12 (0.54–2.33) 0.765 Model 5 COMT rs4680 variant CYP2A6 wild type 28 21 1.0 CYP2A6 high activity 68 54 0.94 (0.48–1.84) 0.867 CYP2A6 low activity 18 31 0.44 (0.19–0.98) 0.043 Model 6 COMT rs165599 wild type CYP2A6 wild type 15 8 1.0 CYP2A6 high activity 33 30 0.59 (0.22–1.58) 0.289 CYP2A6 low activity 13 7 0.99 (0.28–3.48) 0.988 Model 7 COMT rs165599 variant CYP2A6 wild type 48 38 1.0 CYP2A6 high activity 111 89 0.99 (0.59–1.64) 0.961 CYP2A6 low activity 41 47 0.69 (0.38–1.26) 0.224 Mantel–Haenszel Chi square test The genotypes of (1) CYP2A6: wild-type (*1A/*1A), high activity (*1A/*1B and *1B/*1B), low activity (*1A/*4C, *1B/*4C, and *4C/*4C); (2) COMT rs4680: wild-type (G/G), variant (G/A and A/A); and (3) COMT rs165599: wild-type (A/A), variant (A/G and G/G) As shown in Table 2, after using never smokers as the The average number of cigarettes per day among the reference group, the OR was significantly lower in the 261 current smokers was 10. There were 127 heavy subjects carrying the genotype of CYP2A6 low activity/ smokers and 131 light smokers (data for three smok- variant COMT rs4680 than in those possessing the geno- ers were missing). With light smokers as the reference type of CYP2A6 wild-type/variant COMT rs4680 (0.44, group, 15 ORs for heavy smoking were not statistically 95% CI 0.19–0.98, P = 0.043). The other 14 ORs were not significant (P = 0.061–0.112, data in Additional file 2: statistically significant (P = 0.078–0.988). Table S2). Ou et al. Behav Brain Funct (2017) 13:8 Page 6 of 10 Table 3 Comparisons of adjusted urinary nicotine concentration and adjusted urinary cotinine concentration among subjects (N = 122) carrying different CYP2A6 and COMT polymorphisms N Adjusted urinary nicotine, Adjusted urinary cotinine, ng/μL median (min–max) ng/μL median (min–max) Model 1 CYP2A6 Wild type 17 86.23 (6.14–296.36) 120.90 (20.39–485.32) High activity 76 70.45 (6.28–1117.87) 111.08 (8.34–822.11) Low activity 29 61.59 (15.28–456.87) 75.87 (10.87–266.44) P NS NS Model 2 COMT COMT rs4680 Wild type 66 63.86 (6.28–646.38) 92.46 (8.34–506.97) Variant 56 67.87 (6.14–1117.87) 118.24 (13.59–822.11) P NS 0.041 COMT rs165599 Wild type 27 68.10 (12.42–212.74) 89.42 (23.06–218.62) Variant 95 64.11 (6.14–1117.87) 107.24 (8.34–822.11) P NS NS Model 3 COMT rs4680/COMT rs165599 Wild type/wild type 13 89.29 (12.42–212.74) 79.37 (23.06–218.62) Wild type/variant 53 63.61 (6.28–646.38) 97.10 (8.34–506.97)* Variant/wild type 14 67.87 (15.28–177.61) 95.86 (32.18–202.0) Variant/variant 42 69.46 (6.14–1117.87) 122.18 (13.59–822.11)* P NS Model 4 COMT rs4680 wild type CYP2A6 wild type 9 95.04 (25.04–296.36) 105.70 (53.48–485.32) CYP2A6 high activity 39 72.99 (6.28–646.38) 97.45 (8.34–506.97) CYP2A6 low activity 18 42.54 (19.38–371.29) 60.20 (10.87–266.44) P NS NS Model 5 COMT rs4680 variant CYP2A6 wild type 8 74.91 (6.14–217.41) 141.53 (20.39–228.68) CYP2A6 high activity 37 68.10 (17.68–1117.87) 115.78 (13.59–822.11) CYP2A6 low activity 11 66.13 (15.28–456.87) 161.65 (39.19–224.53) P NS NS Model 6 COMT rs165599 wild type CYP2A6 wild type 3 95.04 (62.36–188.57) 102.30 (79.37–120.90) CYP2A6 high activity 17 67.64 (12.42–212.74) 89.42 (32.18–218.62) CYP2A6 low activity 7 78.05 (15.28–177.61) 63.55 (23.06–202.00) P NS NS For the 261 current smokers, FTND, PCDS, and was not significantly different (P = 0.224–0.911, 0.054– CWS-21 were analyzed for 181, 227, and 210 subjects, 0.700, and 0.075–0.836, respectively) (data in Additional respectively, because of missing data. Each of the eight file 3: Table S3). comparisons for FTND, PCDS, and CWS-21 among the Of the 261 current smokers, 122 subjects provided groups with different CYP2A6/COMT polymorphisms urine samples for the determination of nicotine and Ou et al. Behav Brain Funct (2017) 13:8 Page 7 of 10 Table 3 continued N Adjusted urinary nicotine, Adjusted urinary cotinine, ng/μL median (min–max) ng/μL median (min–max) Model 7 COMT rs165599 variant CYP2A6 wild type 14 74.92 (6.14–296.36) 141.53 (20.39–485.32) CYP2A6 high activity 59 72.99 (6.28–1117.87) 111.09 (8.34–822.11) CYP2A6 low activity 22 59.62 (19.38–456.87) 81.66 (10.87–266.44) P NS NS NS not statistically significant Mann–Whitney U test or Kruskal–Wallis test with Bonferroni adjustment * Significantly different from the (COMT rs4680 wild-type/COMT rs165599 variant) group versus the (COMT rs4680 variant/COMT rs165599 variant) group by Scheffe’s Post hoc test, P value = 0.022 cotinine levels by GC/MS. The values for adjusted nico - COMT rs4680 genotype. The CYP2A6 and COMT genes tine concentration and adjusted cotinine concentration were reported to be associated with smoking status. were not normally distributed in either genotype group. CYP2A6*4C is a whole-deletion type, decreased nico- The SD was too high: near the mean value or even greater tine metabolism polymorphism [34]. In Japanese adults than the mean value. Therefore, the Mann–Whitney U and young Japanese students, the frequency of the test or Kruskal–Wallis test with Bonferroni adjustment CYP2A6*4C gene was significantly higher among non- was used for comparison of the values of adjusted nico- smokers than smokers [34, 35]. Among Chinese males, tine concentration and adjusted cotinine concentration participants with the CYP2A6*4C genotype had a lower between groups carrying different genotypes. As shown risk of smoking initiation and smoking persistence than in Table 3, the adjusted urinary nicotine concentration those with the CYP2A6*1/CYP2A6*1 genotype [17]. For did not differ significantly in any of the comparisons. people living in southern China, reduced metabolic func- The median adjusted urinary cotinine concentration dif - tion of CYP2A6 in smokers appears to be associated with fered between the COMT rs4680 wild-type group and the fewer cigarettes smoked, later initiation of smoking regu- COMT rs4680 variant group (92.46 ng/μL vs. 118.24 ng/ larly, shorter smoking duration, and lower likelihood of μL, P = 0.041) and between the COMT rs4680 wild- smoking cessation [36]. For Caucasian individuals, the type/COMT rs165599 variant group and the COMT CYP2A6 slow inactivator genotype increased the risk of rs4680 variant/COMT rs165599 variant group (97.10 ng/ nicotine dependence when smoking was initiated dur- μL vs. 122.18 ng/μL, P = 0.022). The other comparisons ing adolescence. However, it reduced the risk of smoking were not statistically significant. initiation, lowered cigarette consumption, and decreased the duration of smoking among adult dependent smokers Discussion [37]. The continued effect of slow metabolism on reduc - All subjects in this study were young adult university stu- ing cigarette consumption, throughout the smoking his- dents. Alcohol and drug abusers were excluded. There - tory of people with CYP2A6 slow inactivators, may affect fore, the effects of age, education status and alcohol and tolerance and withdrawal mechanisms among these drug interactions on smoking were absent. individuals [37]. Smokers with CYP2A6 slow inactiva- Our results show that the distribution of COMT rs4680 tors smoke fewer cigarettes and tend to be less depend- does not agree with Hardy–Weinberg equilibrium, ent on nicotine than smokers with normal activity alleles consistent with reports that Asiatic individuals have [38]. With respect to smoking initiation, adolescents with Hardy–Weinberg disequilibrium of the COMT rs4680 slower activity alleles may progress to nicotine depend- polymorphism [8]. The main finding of this study is that ence more slowly than normal metabolizers [38]. Very among adult males with variant COMT rs4680, the sub- recently, researchers reported that CYP2A6 slow metab- jects carrying the low-activity genotype of CYP2A6 have olism was associated with increased adolescent smok- a 0.44-fold lower risk of starting smoking than those ing cessation in Caucasian individuals [39]. The ORs for possessing the wild-type genotype of CYP2A6. In other current smoking are reportedly higher in COMT rs4680 words, the OR is 2.27-fold higher in subjects carrying G/G (the high activity allele) carriers than in those pos- the CYP2A6 wild-type/variant COMT rs4680 genotype sessing the COMT rs4680 variant among healthy Cauca- than in those possessing the CYP2A6 low activity/variant sian men of Croatian origin [40], Americans of European Ou et al. Behav Brain Funct (2017) 13:8 Page 8 of 10 ancestry [41], and Japanese men [15]. On the other hand, Conclusions the COMT rs4680 variation (G/A or A/A, the low-activ- A single nucleotide polymorphism (rs4680) in the COMT ity alleles) was associated with nicotine dependence in gene and the interaction between the CYP2A6 and men and women of African-American and European- COMT genes affect smoking status in young Taiwanese American descent [9], smoking severity among Chinese men. Effective smoking prevention and cessation inter - male smokers [42], heaviness of smoking in Caucasian vention programs are required to reduce smoking among pregnant women [43], and susceptibility to cigarette university students [46]. We found that the interaction smoking among Thai males [11]. Our results demonstrate of the low-activity CYP2A6 genotype and low-activity for the first time that the combination of the low-activity COMT genotype is associated with the risk of starting CYP2A6 genotype and low-activity COMT genotype is smoking. In addition, interaction of the DRD2 TaqIB and associated with the risk of starting smoking. MAOA genes also affects smoking intensity in Taiwanese We also found that the adjusted urinary cotinine con- young men [14]. This knowledge is useful for develop - centration was higher in subjects with low-activity ing an approach to reducing smoking among Taiwanese COMT genes than in those with high-activity COMT university students. A clearer understanding of the rela- genes. Additionally, it was higher in subjects with the tive roles of genetic and non-genetic factors in the initia- COMT rs4680 variant/COMT rs165599 variant than in tion of smoking could have implications for the design of those with the COMT rs4680 wild-type/COMT rs165599 smoking prevention programs [47]. variant. The urinary cotinine concentration is a reliable Additional files easy-to-use marker for plasma levels of cotinine and the sum of nicotine metabolites in smokers [44]. Therefore, Additional file 1: Table S1. Results of PCR–RFLP fragments for CYP2A6. our findings indicate that cotinine and the sum of nico - Additional file 2: Table S2. Odds ratios for the effect of CYP2A6 and tine metabolites are metabolized more slowly in subjects COMT. carrying the low-activity genotype than in those possess- Additional file 3: Table S3. Comparisons of FTND, PCDS and CWS‑21 ing the high-activity genotype. However, such a difference among subjects carrying different CYP2A6 and COMT polymorphisms. did not affect FTND, PCDS, or CWS-21 scores, and it was not related to the risk of heavier smoking. The smok - ing intensity of the university students was lower (aver- Abbreviations CYP2A6: cytochrome P450 2A6; COMT: catechol‑O‑methyltransferase; FTND: age cigarettes/day = 10 and average FTND score = 3.7) Fagerstrom Test for Nicotine Dependence; PCDS: Physiological Cigarette than that of Taiwanese adult smokers, e.g., average ciga- Dependence Scale; CWS: Cigarette Withdrawal Symptoms Scale. rettes/day = 24 and average FTND score = 7.1 for Chi- Authors’ contributions nese adults [42]. This finding may be the reason, at least Conceived and designed the experiments: WCO, PLC, CSH. Performed the in part, that the low activity genotypes of CYP2A6 and experiments: WCO, PLC, MHL, YJC, CNL, and MCC. Analyzed the data: CLH, COMT were associated with high nicotine dependence YCC,YCH. Wrote the paper: CSH, PLC, YCH. All authors read and approved the final manuscript. scores and heavier smoking, as we reported in this study. For Taiwanese university students, the primary reason Author details for the first contact with smoking was curiosity. Anxiety, Department of Medical Laboratory Science and Biotechnology, Central Tai‑ wan University of Science and Technology, No. 666 Buzih Road, Beitun District, avoiding stress, and the difficulties of smoking cessation Taichung City 40601, Taiwan. Department of Nursing, Chang Jung Christian explained continuing smoking behavior among university University, Tainan, Taiwan. Administration Center for Research and Education, students [45]. The environmental factors may be more Changhua Christian Hospital, Changhua, Taiwan. Company Limited of Ditech Enterprise, Taipei, Taiwan. Division of Infectious Diseases, Jen‑Ai Hospital, predominant than certain genetic factors (e.g., polymor- Taichung, Taiwan. phisms of CYP2A6 and COMT genes) among Taiwanese smokers in the university setting. Acknowledgements We express our deep appreciation to the National Science Council of Taiwan There are several limitations to our study. The percent - (Grant NSC–100–2320–B–371–001), Changhua Christian Hospital, Taiwan age of female smokers in the Taiwanese population is low, (Grant CCH–ICO10008). only 4.4%, and they are difficult to recruit as participants. Competing interests In our report, study participation was restricted to young The authors declared that they have no competing interests. male Taiwanese smokers. Although the total number of study subjects was relatively large, the numbers of indi- Availability of data and materials The authors confirm that all data and materials underlying the findings are viduals with some of the genotypes were too small to fully available without restriction. All relevant data are within the paper. reach statistically significant power. Therefore, further studies with a larger sample size and that include female Consent for publication Informed consent to publish was obtained from the patients in this research. smokers are needed. Ou et al. Behav Brain Funct (2017) 13:8 Page 9 of 10 Ethics approval and consent to participate 16. Shiels MS, Huang HY, Hoffman SC, Shugart YY, Bolton JH, Platz EA, This study was approved by the review board of Chang Jung Christian Uni‑ Helzlsouer KJ, Alberg AJ. A community‑based study of cigarette smoking versity (CJCU‑99004) and the ethics board of the Central Taiwan University of behavior in relation to variation in three genes involved in dopamine Science and Technology (CTUST‑99016). It was conducted in accordance with metabolism: Catechol‑O‑methyltransferase (COMT ), dopamine beta‑ Good Clinical Practice procedures and the Declaration of Helsinki. hydroxylase (DBH) and monoamine oxidase‑A (MAO ‑A). Prev Med. 2008;47:116–22. Funding 17. Tang X, Guo S, Sun H, Song X, Jiang Z, Sheng L, Zhou D, Hu Y, Chen D. This study was financially supported by Central Taiwan University of Science Gene‑ gene interactions of CYP2A6 and MAOA polymorphisms on smok‑ and Technology (Grant CTU103–P–20, CTU103‑P ‑21). ing behavior in Chinese male population. Pharmacogenetics Genom. 2009;19:345–52. 18. Rodriguez S, Cook DG, Gaunt TR, Nightingale CM, Whincup PH, Day Publisher’s Note IN. Combined analysis of CHRNA5, CHRNA3 and CYP2A6 in relation to Springer Nature remains neutral with regard to jurisdictional claims in pub‑ adolescent smoking behaviour. J Psychopharmacol. 2011;25:915–23. lished maps and institutional affiliations. 19. Wassenaar CA, Dong Q, Wei Q, Amos CI, Spitz MR, Tyndale RF. Relationship between CYP2A6 and CHRNA5‑ CHRNA3‑ CHRNB4 variation and smoking Received: 30 September 2016 Accepted: 26 April 2017 behaviors and lung cancer risk. J Natl Cancer Inst. 2011;103:1342–6. 20. Ohmoto M, Takahashi T, Kubota Y, Kobayashi S, Mitsumoto Y. Genetic influence of dopamine receptor, dopamine transporter, and nicotine metabolism on smoking cessation and nicotine dependence in a Japa‑ nese population. BMC Genet. 2014;15:151. References 21. Doran N. Sex differences in smoking cue reactivity: craving, nega‑ 1. Quaak M, van Schayck CP, Knaapen AM, van Schooten FJ. Genetic varia‑ tive affect, and preference for immediate smoking. Am J Addict. tion as a predictor of smoking cessation success. A promising preven‑ 2014;23:211–7. tive and intervention tool for chronic respiratory diseases? Eur Respir J. 22. Huang CL, Cheng CP, Huang HW. The development of a Chinese‑ 2009;33:468–80. language instrument to measure social smoking motives among male 2. Kim YS, Ko H, Yoon C, Lee DH, Sung J. Social determinants of smok‑ Taiwanese smokers. J Transcult Nurs. 2013;24:371–7. ing behavior: the healthy twin study, Korea. J Prev Med Public Health. 23. Mwenifumbo JC, Lessov‑Schlaggar CN, Zhou Q, Krasnow RE, Swan GE, 2012;45:29–36. Benowitz NL, Tyndale RF. Identification of novel CYP2A6*1B variants: the 3. Wright AJ, French DP, Weinman J, Marteau TM. Can genetic risk informa‑ CYP2A6*1B allele is associated with faster in vivo nicotine metabolism. tion enhance motivation for smoking cessation? An analogue study. Clin Pharmacol Ther. 2008;83:115–21. Health Psychol. 2006;25:740–52. 24. Schoedel KA, Hoffmann EB, Rao Y, Sellers EM, Tyndale RF. Ethnic variation 4. Tsuang MT, Francis T, Minor K, Thomas A, Stone WS. Genetics of smoking in CYP2A6 and association of genetically slow nicotine metabolism and and depression. Hum Genet. 2012;131:905–15. smoking in adult Caucasians. Pharmacogenetics. 2004;14:615–26. 5. Di YM, Chow VD, Yang LP, Zhou SF. Structure, function, regulation and 25. Huang CL, Lin HH, Wang HH. The psychometric properties of the Chinese polymorphism of human cytochrome P450 2A6. Curr Drug Metab. version of the fagerstrom test for nicotine dependence. Addict Behav. 2009;10:754–80. 2006;31:2324–7. 6. Benowitz NL, Hukkanen J, Jacob P. 3rd. Nicotine chemistry, metabolism, 26. Lu CC, Lin HH, Chen CJ, Huang CL. Psychometric testing of the Chinese kinetics and biomarkers. Handb Exp Pharmacol. 2009;192:29–60. version of the dimensions of Tobacco Dependence Scale. J Clin Nurs. 7. Mwenifumbo JC, Myers MG, Wall TL, Lin SK, Sellers EM, Tyndale RF. Ethnic 2009;18:2470–7. variation in CYP2A6*7, CYP2A6*8 and CYP2A6*10 as assessed with a 27. Huang CL, Lin HH, Wang HH. Cigarette dependence questionnaire: novel haplotyping method. Pharmacogenetics Genom. 2005;15:189–92. development and psychometric testing with male smokers. J Adv Nurs. 8. Jimenez‑ Jimenez FJ, Alonso‑Navarro H, Garcia‑Martin E, Agundez JA. 2010;66:2341–9. COMT gene and risk for Parkinson’s disease: a systematic review and 28. Raunio H, Rahnasto‑Rilla M. CYP2A6: genetics, structure, regulation, and meta‑analysis. Pharmacogenetics Genom. 2014;24:331–9. function. Drug Metabol Drug Interact. 2012;27:73–88. 9. Beuten J, Payne TJ, Ma JZ, Li MD. Significant association of catechol‑ 29. Nurfadhlina M, Foong K, Teh LK, Tan SC, Mohd Zaki S, Ismail R. O‑methyltransferase (COMT ) haplotypes with nicotine dependence in CYP2A6 polymorphisms in Malays, Chinese and Indians. Xenobiotica. male and female smokers of two ethnic populations. Neuropsychophar‑ 2006;36:684–92. macology. 2006;31:675–84. 30. Oscarson M, McLellan RA, Gullsten H, Yue QY, Lang MA, Bernal ML, Sinues 10. Berrettini WH, Wileyto EP, Epstein L, Restine S, Hawk L, Shields P, Niaura R, B, Hirvonen A, Raunio H, Pelkonen O, Ingelman‑Sundberg M. Characteri‑ Lerman C. Catechol‑O‑methyltransferase (COMT ) gene variants predict sation and PCR‑based detection of a CYP2A6 gene deletion found at a response to bupropion therapy for tobacco dependence. Biol Psychiatry. high frequency in a Chinese population. FEBS Lett. 1999;448:105–10. 2007;61:111–8. 31. Chiang CT. The polymorphisms of CYP2A6 among Taiwanese lung cancer 11. Suriyaprom K, Tungtrongchitr R, Harnroongroj T. Impact of COMT Val patients. Master’s thesis. Chunghua University ( Tainan, Taiwan). 2006. 108/158 Met and DRD2 Taq1B gene polymorphisms on vulnerability to http://handle.ncl.edu.tw/11296/ndltd/29131916603407156420. Accessed cigarette smoking of Thai males. J Mol Neurosci. 2013;49:544–9. 24 Apr 2016. 12. Health Promotion Administration. Taiwan tobacco control: annual report 32. Chan RC, Chen RY, Chen EY, Hui TC, Cheung EF, Cheung HK, Sham P, Li 2012. 2013. http://health99.hpa.gov.tw/media/public/zip/21747.zip. T, Collier D. The differential clinical and neurocognitive profiles of COMT Accessed 24 Apr 2016. SNP rs165599 genotypes in schizophrenia. J Int Neuropsychol Soc. 13. Chen Y T, Tsou HH, Kuo HW, Fang CP, Wang SC, Ho IK, Chang YS, Chen 2005;11:202–4. CH, Hsiao CF, Wu HY, et al. OPRM1 genetic polymorphisms are associ‑ 33. Dempsey D, Tutka P, Jacob P 3rd, Allen F, Schoedel K, Tyndale RF, Benowitz ated with the plasma nicotine metabolite cotinine concentration in NL. Nicotine metabolite ratio as an index of cytochrome P450 2A6 meta‑ methadone maintenance patients: a cross sectional study. J Hum Genet. bolic activity. Clin Pharmacol Ther. 2004;76:64–72. 2013;58:84–90. 34. Iwahashi K, Waga C, Takimoto T. Whole deletion of CYP2A6 gene 14. Huang CL, Ou WC, Chen PL, Liu CN, Chen MC, Lu CC, Chen YC, Lin MH, (CYP2A6AST;4C) and smoking behavior. Neuropsychobiology. Huang CS. Eec ff ts of interaction between dopamine D2 receptor and 2004;49:101–4. monoamine oxidase a genes on smoking status in young men. Biol Res 35. Waga C, Iwahashi K. CYP2A6 gene polymorphism and personality traits Nurs. 2015;17:422–8. for NEO‑FFI on the smoking behavior of youths. Drug Chem Toxicol. 15. Tochigi M, Suzuki K, Kato C, Otowa T, Hibino H, Umekage T, Kato N, Sasaki 2007;30:343–9. T. Association study of monoamine oxidase and catechol‑O‑methyl‑ 36. Liu T, David SP, Tyndale RF, Wang H, Zhou Q, Ding P, He YH, Yu XQ, Chen W, transferase genes with smoking behavior. Pharmacogenetics Genom. Crump C, et al. Associations of CYP2A6 genotype with smoking behaviors 2007;17:867–72. in southern China. Addiction. 2011;106:985–94. Ou et al. Behav Brain Funct (2017) 13:8 Page 10 of 10 37. O’Loughlin J, Paradis G, Kim W, DiFranza J, Meshefedjian G, McMillan‑ 43. Munafo MR, Freathy RM, Ring SM, St Pourcain B, Smith GD. Association Davey E, Wong S, Hanley J, Tyndale RF. Genetically decreased CYP2A6 and of COMT Val(108/158)Met genotype and cigarette smoking in pregnant the risk of tobacco dependence: a prospective study of novice smokers. women. Nicotine Tob Res. 2011;13:55–63. Tob Control. 2004;13:422–8. 44. Nagano T, Shimizu M, Kiyotani K, Kamataki T, Takano R, Murayama N, 38. Gold AB, Lerman C. Pharmacogenetics of smoking cessation: role of Shono F, Yamazaki H. Biomonitoring of urinary cotinine concentrations nicotine target and metabolism genes. Hum genet. 2012;131(6):857–76. associated with plasma levels of nicotine metabolites after daily cigarette 39. Chenoweth MJ, O’Loughlin J, Sylvestre MP, Tyndale RF. CYP2A6 slow smoking in a male Japanese population. Int J Environ Res Public Health. nicotine metabolism is associated with increased quitting by adolescent 2010;7:2953–64. smokers. Pharmacogenetics Genom. 2013;23:232–5. 45. Wang KY, Yang CC, Chu NF, Wu DM. Predictors of cigarette smoking 40. Nedic G, Nikolac M, Borovecki F, Hajnsek S, Muck‑Seler D, Pivac N. behavior among military university students in Taiwan. J Nurs Res. Association study of a functional catechol‑O‑methyltransferase poly‑ 2009;17:161–9. morphism and smoking in healthy Caucasian subjects. Neurosci Lett. 46. Eticha T, Kidane F. The prevalence of and factors associated with current 2010;473:216–9. smoking among College of Health Sciences students, Mekelle University 41. Redden DT, Shields PG, Epstein L, Wileyto EP, Zakharkin SO, Allison DB, in northern Ethiopia. PLoS ONE. 2014;9:e111033. Lerman C. Catechol‑O‑methyl‑transferase functional polymorphism and 47. Wilkinson AV, Swartz MD, Yu X, Spitz MR, Shete S. Cigarette experimenta‑ nicotine dependence: an evaluation of non replicated results. Cancer tion and the population attributable fraction for associated genetic and Epidemiol Biomark Prev. 2005;14:1384–9. non‑ genetic risk factors. PLoS ONE. 2013;8:e53868. 42. Guo S, da Chen F, Zhou DF, Sun HQ, Wu GY, Haile CN, Kosten TA, Kosten TR, Zhang XY. Association of functional catechol O‑methyl transferase (COMT ) Val108Met polymorphism with smoking severity and age of smoking initiation in Chinese male smokers. Psychopharmacology. 2007;190:449–56. Submit your next manuscript to BioMed Central and we will help you at every step: • We accept pre-submission inquiries • Our selector tool helps you to find the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit
Journal
Behavioral and Brain Functions – Springer Journals
Published: May 4, 2017