Influence of CYP2A6*4 Genotypes on Maternal Serum Cotinine Among Chinese Nonsmoking Pregnant Women
Chuanbo XieXiaozhong WenPeng DingTao LiuYanhui HeZhongzheng NiuJianmiao LinShixin YuanXiaoling GuoDeqin JiaWeiqing Chen
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Serum cotinine is a common biomarker for smoking and secondhand smoke (SHS) exposure, but it can be affected by the activity of nicotine-metabolizing enzymes. This study investigated the influence of CYP2A6*4 genotypes on serum cotinine among nonsmoking pregnant women.We analyzed the data from 545 Chinese nonsmoking pregnant women in a case-control study on SHS exposure and birth outcomes in southern China. Participants self-reported their status and duration of SHS exposure during pregnancy right after delivery in hospital. Research staff used polymerase chain reaction to genotype CYP2A6*4 and enzyme-linked immunosorbent assay to measure cotinine levels in maternal serum samples collected before delivery. We stratified women by their self-reported SHS exposure status and CYP2A6*4 genotypes and then compared their median levels of serum cotinine.Among women who self-reported non-SHS exposure (n = 317), the median serum cotinine levels were 2.83ng/ml for those with CYP2A6*1/*1 genotype, 1.39ng/ml for CYP2A6*1/*4, and 0.77ng/ml for CYP2A6*4/*4, respectively. Among women who self-reported SHS exposure (n = 228), the median cotinine levels were 3.32ng/ml for those with CYP2A6*1/*1 genotype, 2.38ng/ml for CYP2A6*1/*4, and 1.56ng/ml for CYP2A6*4/*4, respectively. Strikingly, self-reported SHS-exposed women with CYP2A6*1/*4 or CYP2A6*4/*4 genotype had significantly lower (rather than higher) median cotinine levels than self-reported non-SHS-exposed women with CYP2A6*1/*1 genotype (p = .012).CYP2A6*4 genotype is associated with lower serum cotinine among Chinese nonsmoking pregnant women. Measuring CYP2A6*4 genotype may help to improve the validity of SHS exposure measurement by serum cotinine in pregnant women and possibly also in other nonpregnant populations.Keywords:
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Of the various biochemical markers used to validate the smoking status of a person, nicotine and continine are considered as good markers for both active and passive smoking. In the present study an attempt was made to estimate urinary levels of nicotine and cotinine in healthy individuals from north India using different types of tobacco to identify and validate the smoking status.Twenty four hour urine sample of 130 healthy volunteers (smokers=70, passive smokers=20, tobacco chewers=20, non smokers=20) were analyzed by high-pressure liquid chromatography (HPLC) assay. Smokers were divided into different groups, viz., cigarette, bidi and hooka smokers.The mean values of nicotine (ng/ml) and cotinine (ng/ml) in urine were highest in cigarette smokers (nicotine=703.50+/-304.34; cotinine=2736.20+/-983.29), followed by hooka smokers (nicotine 548.0+/-103.47 and cotinine 2379.0+/-424.25), and bidi smokers (nicotine=268.53+/-97.62, cotinine=562.60+/-249.38). There was no correlation of nicotine or cotinine values with smoking index. In passive smokers (nicotine=109.75+/-22.33, cotinine=280.75+/-86.30) and in nonsmokers, the values were much lower (nicotine=55.00+/-13.71, cotinine=7.30+/-2.47) compared to smokers. In tobacco chewers, the values for nicotine and cotinine were 447.75+/-145.09 and 2178.30+/-334.29 respectively.All forms of tobacco users had significantly higher values compared to passive smokers and nonusers. Thus, cotinine and nicotine levels in urine may be considered as good indicators to assess the exposure to tobacco in our population.
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The residence times of nicotine and its metabolites in rat brain after acute peripheral nicotine administration were determined. We hypothesize that nicotine metabolites will reach pharmacologically significant concentrations in brain. Cotinine, nornicotine, and norcotinine were structurally identified by dual label radiochemical and gas chromatography-mass spectrometric analysis as biotransformation products of nicotine present in rat brain after s. c. injection of S(-)-nicotine. Two unidentified minor metabolites were also detected in brain. The half-lives in brain of nicotine metabolites were determined after a single s.c. injection of [2'-(14)C]-(+/-)nicotine (0.8 mg/kg) and analysis of radiolabeled metabolites by high pressure-liquid radiochromatography. The brain half-lives of nicotine, cotinine, and nornicotine were 52, 333, and 166 min, respectively. Peak brain concentrations of nicotine metabolites were 300, 70, and 7 nM for cotinine, nornicotine, and norcotinine, respectively. Even with potential accumulation of cotinine in brain after chronic nicotine administration, it is likely that the brain concentration of cotinine will be insufficient to produce neuropharmacological effects resulting from activation of nicotinic receptors to induce dopamine release. Conversely, the concentration of nornicotine in brain after acute nicotine approaches the range found to be neuropharmacologically active. It is likely that nornicotine will accumulate in brain on chronic nicotine administration based on the brain half-life of this metabolite. Importantly, nornicotine is also a major alkaloidal component of tobacco. Thus, as a consequence of tobacco use, alkaloidal and metabolically formed nornicotine may reach concentrations in brain sufficient to produce pharmacological effects.
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Abstract Nicotine is the major alkaloid present in tobacco and the most frequently determined compound as a biomarker of tobacco exposure in both smokers and non-smokers exposed to environmental tobacco smoke. Current knowledge on the human metabolism and disposition kinetics of nicotine is reviewed, together with methods for the determination of nicotine and various metabolites in different human biological fluids and matrices. Only short-term biomarkers of nicotine exposure exist and long-term biomarkers of exposure such as the incorporation of nicotine and cotinine into human hair, toenails and deciduous teeth require further investigation. Determination of ‘nicotine boost’, the difference in blood nicotine concentrations that occur after smoking a single cigarette, provides an experimental indication of individual smoking behaviour, but is unsuitable for population studies. The determination of nicotine plus multiple phase I and phase II metabolites in 24-hour urine, often expressed as ‘nicotine equivalents’, provides the most accurate way to determine exposure to nicotine in smokers; however, few laboratories are equipped to perform the complex analysis required for this purpose. Nicotine equivalents can be used to estimate the uptake of nicotine from a cigarette in both individuals and in population studies. Despite recent advancements in analytical methodology and the possibility of determining multiple nicotine metabolites in various biological fluids, determination of cotinine, the major metabolite of nicotine, is likely to remain the most commonly used approach to assess exposure to tobacco smoke in both smokers and non-smokers. Representative data for cotinine in blood, saliva and urine of smokers and non-smokers are presented.
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Serum cotinine is a common biomarker for smoking and secondhand smoke (SHS) exposure, but it can be affected by the activity of nicotine-metabolizing enzymes. This study investigated the influence of CYP2A6*4 genotypes on serum cotinine among nonsmoking pregnant women.We analyzed the data from 545 Chinese nonsmoking pregnant women in a case-control study on SHS exposure and birth outcomes in southern China. Participants self-reported their status and duration of SHS exposure during pregnancy right after delivery in hospital. Research staff used polymerase chain reaction to genotype CYP2A6*4 and enzyme-linked immunosorbent assay to measure cotinine levels in maternal serum samples collected before delivery. We stratified women by their self-reported SHS exposure status and CYP2A6*4 genotypes and then compared their median levels of serum cotinine.Among women who self-reported non-SHS exposure (n = 317), the median serum cotinine levels were 2.83ng/ml for those with CYP2A6*1/*1 genotype, 1.39ng/ml for CYP2A6*1/*4, and 0.77ng/ml for CYP2A6*4/*4, respectively. Among women who self-reported SHS exposure (n = 228), the median cotinine levels were 3.32ng/ml for those with CYP2A6*1/*1 genotype, 2.38ng/ml for CYP2A6*1/*4, and 1.56ng/ml for CYP2A6*4/*4, respectively. Strikingly, self-reported SHS-exposed women with CYP2A6*1/*4 or CYP2A6*4/*4 genotype had significantly lower (rather than higher) median cotinine levels than self-reported non-SHS-exposed women with CYP2A6*1/*1 genotype (p = .012).CYP2A6*4 genotype is associated with lower serum cotinine among Chinese nonsmoking pregnant women. Measuring CYP2A6*4 genotype may help to improve the validity of SHS exposure measurement by serum cotinine in pregnant women and possibly also in other nonpregnant populations.
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Urinary cotinine, one of the main metabolites of nicotine, has been widely used as a biomarker for assessment of direct or passive exposure to cigarette smoke. However, there is wide variation of the cotinine level among smokers who smoke the same number of cigarettes. To use urinary cotinine as a proper exposure-biomarker for cigarette smoke, interindividual variations of cotinine formation must be considered. Therefore, we studied the effects of genetic polymorphisms in drug metabolic enzymes on urinary cotinine levels among 190 male Japanese smokers (ages 19-66 years; mean, 40.6 years). Genetic polymorphisms in cytochrome P-450s (CYP1A1, CYP2A6, CYP2E1), and aldehyde dehydrogenase 2 (ALDH2) were determined by analyzing DNA isolated from peripheral blood. Cotinine in morning spot urine was analyzed by high-performance liquid chromatography. Lifestyle, i.e., smoking, alcohol consumption, and intake of coffee or tea, was examined using a questionnaire. The number of cigarettes smoked and CYP2A6 polymorphism were significantly associated with the urinary cotinine level. Especially, the urinary cotinine levels was drastically lower in CYP2A6-deleted homozygous (CYP2A6*4/*4) subjects than in CYP2A6*1 allele-positive subjects. The polymorphism in the CYP2E1 5'-flanking region was related to the urinary cotinine level in intermediate smokers (who smoke 11-20 cigarettes/day; P < 0.01). Polymorphisms in CYP1A1 or ALDH2, and consumption of alcohol, coffee, or tea were not associated with the urinary cotinine level.
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Abstract CYP2A6 inactivates nicotine to cotinine and cotinine to 3-hydroxycotinine. We investigated which of plasma nicotine and metabolites were most related to CYP2A6 genotype and smoking levels. We assessed demographic and smoking histories in 152 Caucasian ad libitum smokers, measured breath carbon monoxide (CO) levels, and determined plasma nicotine, cotinine, and 3-hydroxycotinine by high-performance liquid chromatography and CYP2A6 genotypes by PCR. Cigarettes per day was most closely related to CO (r = 0.60, P < 0.001) followed by plasma cotinine (r = 0.53, P < 0.001), whereas plasma cotinine was most strongly correlated with CO levels (r = 0.74, P < 0.001), confirming that cotinine is a good indicator of smoking levels; this was not limited by CYP2A6 variants. 3-Hydroxycotinine/cotinine is reported to be a good marker of CYP2A6 activity, and we found that the 3-hydroxycotinine/(cotinine + nicotine) ratio was most correlated with CYP2A6 genotype (r = 0.38, P < 0.001). Inclusion of the CYP2A6*12A allele strengthened the correlation (r = 0.46, P < 0.001), suggesting that the identification of novel alleles will continue to improve this relationship. Nicotine metabolism is slower in smokers, and we have shown that CYP2A6 is reduced by nicotine treatment in monkeys. Here, we found that plasma nicotine levels were inversely correlated with CYP2A6 activity (3-hydroxycotinine/cotinine, r = −0.41, P < 0.001) among those without CYP2A6 variants, suggesting a reduction in metabolism with higher nicotine levels. Together, these findings (a) confirm the use of plasma cotinine and CO as indicators of Caucasians' smoking levels, and that this is not limited by CYP2A6 genetic variation; (b) indicate that 3-hydroxycotinine/cotinine and 3-hydroxycotinine/(cotinine + nicotine) are moderately good indicators of the CYP2A6 genotype; and (c) support that nicotine exposure may reduce its own metabolism. (Cancer Epidemiol Biomarkers Prev 2006;15(10):1812–9)
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Urinary cotinine, one of the main metabolites of nicotine, has been widely used as a biomarker for assessment of direct or passive exposure to cigarette smoke. However, there is wide variation of the cotinine level among smokers who smoke the same number of cigarettes. To use urinary cotinine as a proper exposure-biomarker for cigarette smoke, interindividual variations of cotinine formation must be considered. Therefore, we studied the effects of genetic polymorphisms in drug metabolic enzymes on urinary cotinine levels among 190 male Japanese smokers (ages 19–66 years; mean, 40.6 years). Genetic polymorphisms in cytochrome P-450s ( CYP1A1 , CYP2A6 , CYP2E1 ), and aldehyde dehydrogenase 2 ( ALDH2 ) were determined by analyzing DNA isolated from peripheral blood. Cotinine in morning spot urine was analyzed by high-performance liquid chromatography. Lifestyle, i.e. , smoking, alcohol consumption, and intake of coffee or tea, was examined using a questionnaire. The number of cigarettes smoked and CYP2A6 polymorphism were significantly associated with the urinary cotinine level. Especially, the urinary cotinine levels was drastically lower in CYP2A6- deleted homozygous ( CYP2A6\*4/\*4 ) subjects than in CYP2A6*1 allele-positive subjects. The polymorphism in the CYP2E1 5′-flanking region was related to the urinary cotinine level in intermediate smokers (who smoke 11–20 cigarettes/day; P < 0.01). Polymorphisms in CYP1A1 or ALDH2 , and consumption of alcohol, coffee, or tea were not associated with the urinary cotinine level.
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In studying the effects of exposure to secondhand smoke (SHS), concentrations of cotinine (the proximate metabolite of nicotine), measured in plasma, urine or saliva, are widely used as indicators of nicotine exposure [1]. In interpreting the concentrations of cotinine in non-smokers relative to concentrations in smokers, the assumption is made that the pharmacokinetics of nicotine and cotinine are independent of the level of nicotine exposure. Published pharmacokinetic data on nicotine and cotinine are based on administration of nicotine and cotinine to smokers and non-smokers in doses consistent with cigarette smoking, which are much higher than those relevant to SHS [2]. It is possible that the kinetics and metabolism of nicotine are different at low doses compared with higher doses. It is reported, for example, that the half-life of cotinine is longer after cessation of SHS exposure than after cessation of active smoking [3]. Nicotine is metabolized to cotinine primarily by the liver enzyme CYP2A6 [4]. The same enzyme also metabolizes cotinine to trans-3′-hydroxycotinine. The clearances of nicotine and cotinine and the activity of CYP2A6 are quite variable among individuals [5, 6]. Genetic variation explains some of the variability, with a number of CYP2A6 gene variants known to be associated with reduced enzymatic activity resulting in slow metabolism of nicotine and cotinine [7]. The level of enzymatic activity could influence whether the nicotine clearance is dose-dependent in a particular person. For example, dose-dependence is more likely when enzyme activity low. For this reason, we also assessed CYP2A6 genotype to examine its influence on dose-dependent pharmacokinetics. We conducted a study of the disposition kinetics of nicotine and cotinine at doses that are consistent with intake of nicotine during SHS exposure. Sixteen healthy non-smokers (eight males, eight females) participated in a three dose randomized, crossover study. Subjects came to the clinical research centre at San Francisco General Hospital on three occasions during which they received intravenous infusions of deuterium labelled nicotine (nicotine-3′,3′-dideuteronicotine) and cotinine (cotinine-2,4,5,6-d4), in doses of 0.05 µg kg−1, 0.1 µg kg−1 and 0.2 µg kg−1, infused over 60 min. Infusions were initiated at 07.00 h after an overnight fast, and blood samples were obtained over the subsequent 24 h. A blood sample was also obtained for genotyping for variants of the CYP2A6 gene. Plasma nicotine concentrations were measured by GC-MS [8], modified for MS/MS determination using a triple quadrupole instrument to improve sensitivity. Cotinine was determined by LC-MS/MS [9]. Lower limits of quantitation were 0.1 ng ml−1 for both analytes. These subjects were genotyped for prevalent CYP2A6 alleles of known impact on rates of metabolism, *2,*4H,*7- *10,*12,*17,*20,*23–26 and *35[10–12]. Clearance, elimination half-life and steady-state volume of distribution were estimated using WinNonlin. The extent of conversion of nicotine to cotinine was estimated using the plasma concentrations of cotinine d2 (generated from nicotine d2), dose of nicotine d2 and the clearance of cotinine D4 using the following equation: fc = (AUCCOT–d2)/(dosenic–d2) × (CLCOT–d4). Statistical comparison of pharmacokinetic analyses across different dose groups was conducted by two-way repeated measures analysis of variance, comparing doses of nicotine and genotype group (wild type vs. variant). The 16 subjects averaged 34 years of age (range 20–56 years). Eight subjects were Caucasian, three Asian, three mixed race, one African American and one Hispanic. The disposition kinetics of nicotine and cotinine at the three doses are shown in Table 1. There were no significant differences by dose for any of the pharmacokinetic parameters. Eleven of the subjects had *1/*1 CYP2A6 genotype. The remaining subjects had *1/*9 (two subjects), *1/*7,*1/*12 and *1/*4H (one subject each). Subjects with variant CYP2A6 alleles had on average lower clearances of nicotine and cotinine and fractional conversion of nicotine to cotinine, but differences were not significant except for fractional conversion at the lowest dose of nicotine (Table 1). Among those with variant CYP2A6 alleles, there was no evidence of dose-dependence differences in nicotine or cotinine pharmacokinetics. Our study provides novel data on the pharmacokinetics of intravenous nicotine and cotinine dosed at levels that are consistent with exposure to SHS in non-smokers. We found no evidence of dose-related differences in nicotine and cotinine kinetics and found that the average pharmacokinetics were similar to those observed in smokers receiving higher doses of nicotine and cotinine [2, 6]. The presence of variant CYP2A6 alleles was associated with slower metabolism of nicotine and cotinine as expected, but there was no evidence of dose-dependent kinetics in these subjects either. In conclusion, we found that the disposition kinetics of nicotine and cotinine were similar at low levels of exposure compared with high levels of exposure, without evidence of dose-dependent kinetics. Cotinine concentrations measured in people with SHS exposure can be interpreted similarly to those of active smokers in estimating daily intake of nicotine. Dr Benowitz is a consultant to several pharmaceutical companies that market medications to aid smoking cessation and has served as a paid expert witness in litigation against tobacco companies. Dr Tyndale holds shares in Nicogen Research. No Nicogen funds were used in this work and others affiliated with Nicogen did not review the manuscript. Dr Tyndale has also been a paid consultant for Novartis and McNeil. We thank Brenda Herrera for assistance in the clinical studies, Minjiang Duan and Lita Ramos for performing analytical chemistry, Ewa Hoffmann and Qian Zhou for genotyping, Faith Allen for data management and Marc Olmsted for editorial assistance. The work was supported by the Flight Attendants Medical Research Institute and US Public Health Service grants DA12393 and U01 DA 020830 and R25 CA113710 from the National Institute on Drug Abuse, National Institutes of Health and MOP86471 and TMH-109787 from the CIHR, a CRC and CAMH.
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Cotinine, a nicotine metabolite, is a biomarker of tobacco, nicotine, and carcinogen exposure. However, a given cotinine level may not represent the same tobacco exposure; for example, African-Americans have higher cotinine levels than Caucasians after controlling for exposure.Cotinine levels are determined by the amount of cotinine formation and the rate of cotinine removal, which are both mediated by the enzyme CYP2A6. Because CYP2A6 activity differs by sex (estrogen induces CYP2A6) and genotype, their effect on cotinine formation and removal was measured in nonsmoking Caucasians (Study 1, n = 181) infused with labeled nicotine and cotinine. The findings were then extended to ad libitum smokers (Study 2, n = 163).Study 1: Reduced CYP2A6 activity altered cotinine formation less than cotinine removal resulting in ratios of formation to removal of 1.31 and 1.12 in CYP2A6 reduced and normal metabolizers (P = 0.01), or 1.39 and 1.12 in males and females (P = 0.001), suggesting an overestimation of tobacco exposure in slower metabolizers. Study 2: Cotinine again overestimated tobacco and carcinogen exposure by 25% or more in CYP2A6 reduced metabolizers (≈2-fold between some genotypes) and in males.In people with slower relative to faster CYP2A6 activity, cotinine accumulates resulting in substantial differences in cotinine levels for a given tobacco exposure.Cotinine levels may be misleading when comparing those with differing CYP2A6 genotypes within a race, between races with differing frequencies of CYP2A6 gene variants (i.e., African-Americans have higher frequencies of reduced function variants contributing to their higher cotinine levels), or between the sexes.
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