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Biochemistry of nicotine metabolism and its relevance to lung cancer

Open AccessPublished:April 28, 2021DOI:https://doi.org/10.1016/j.jbc.2021.100722
      Nicotine is the key addictive constituent of tobacco. It is not a carcinogen, but it drives smoking and the continued exposure to the many carcinogens present in tobacco. The investigation into nicotine biotransformation has been ongoing for more than 60 years. The dominant pathway of nicotine metabolism in humans is the formation of cotinine, which occurs in two steps. The first step is cytochrome P450 (P450, CYP) 2A6–catalyzed 5′-oxidation to an iminium ion, and the second step is oxidation of the iminium ion to cotinine. The half-life of nicotine is longer in individuals with low P450 2A6 activity, and smokers with low activity often decrease either the intensity of their smoking or the number of cigarettes they use compared with those with “normal” activity. The effect of P450 2A6 activity on smoking may influence one's tobacco-related disease risk. This review provides an overview of nicotine metabolism and a summary of the use of nicotine metabolite biomarkers to define smoking dose. Some more recent findings, for example, the identification of uridine 5′-diphosphoglucuronosyltransferase 2B10 as the catalyst of nicotine N-glucuronidation, are discussed. We also describe epidemiology studies that establish the contribution of nicotine metabolism and CYP2A6 genotype to lung cancer risk, particularly with respect to specific racial/ethnic groups, such as those with Japanese, African, or European ancestry. We conclude that a model of nicotine metabolism and smoking dose could be combined with other lung cancer risk variables to more accurately identify former smokers at the highest risk of lung cancer and to intervene accordingly.

      Keywords

      Abbreviations:

      FMO (flavin monooxygenase), NNK (4-(methylnitrosamino-1-(3-pyridyl)-butanone)), P450,CYP (cytochrome P450), UGT (uridine 5′-diphospho-glucuronosyltransferaseglucuronosyltransferase)
      In the United States, an estimated 235,760 new lung cancer cases will be diagnosed, and 131,880 individuals will die from the disease in 2021 (
      • Siegel R.L.
      • Miller K.D.
      • Fuchs H.E.
      • Jemal A.
      Cancer statistics, 2021.
      ). Worldwide, lung cancer is the leading cause of cancer death, accounting for 11.6% of the 9.6 million estimated deaths in 2018 (
      • Bray F.
      • Ferlay J.
      • Soerjomataram I.
      • Siegel R.L.
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      ). Cigarette smoking is the cause of as much as 90% of this cancer (
      International Agency for Research on Cancer
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      United States Department of Health and Human Services
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      • Ma J.
      • Soerjomataram I.
      • Flanders W.D.
      • Brawley O.W.
      • Gapstur S.M.
      • Jemal A.
      Proportion and number of cancer cases and deaths attributable to potentially modifiable risk factors in the United States.
      ). However, only 11 to 24% of smokers will develop lung cancer, and for the same reported lifetime quantity of cigarettes, lung cancer risk differs by racial/ethnic group (
      • Murphy S.E.
      • Park S.L.
      • Balbo S.
      • Haiman C.A.
      • Hatsukami D.K.
      • Patel Y.
      • Peterson L.A.
      • Stepanov I.
      • Stram D.O.
      • Tretyakova N.
      • Hecht S.S.
      • Le Marchand L.
      Tobacco biomarkers and genetic/epigenetic analysis to investigate ethnic/racial differences in lung cancer risk among smokers.
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      • Stram D.O.
      • Park S.L.
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      • Patel Y.
      • Hecht S.S.
      • Le Marchand L.
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      ). Nicotine, the primary psychoactive compound present in tobacco, is responsible for maintaining smoking behaviors (
      • Benowitz N.L.
      Pharmacological aspects of cigarette smoking and nicotine addiction.
      ), and individual variation in nicotine metabolism is an important contributor to racial/ethnic and individual differences in lung cancer risk (
      • Murphy S.E.
      • Park S.L.
      • Balbo S.
      • Haiman C.A.
      • Hatsukami D.K.
      • Patel Y.
      • Peterson L.A.
      • Stepanov I.
      • Stram D.O.
      • Tretyakova N.
      • Hecht S.S.
      • Le Marchand L.
      Tobacco biomarkers and genetic/epigenetic analysis to investigate ethnic/racial differences in lung cancer risk among smokers.
      ).
      Nicotine is not a carcinogen but is arguably the compound present in tobacco with the greatest influence on a smoker's cancer risk. Nicotine sustains tobacco addiction and continued smoking (
      • Hecht S.S.
      Tobacco smoke carcinogens and lung cancer.
      ,
      United States Department of Health and Human Services
      Mechanisms of cancer induction by tobacco smoke.
      ,
      • Benowitz N.L.
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      ,
      • Hatsukami D.K.
      • Luo X.
      • Jensen J.A.
      • al'Absi M.
      • Allen S.S.
      • Carmella S.G.
      • Chen M.
      • Cinciripini P.M.
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      • Lane T.
      • Le C.T.
      • Leischow S.
      • Luo K.
      • et al.
      Effect of immediate vs gradual reduction in nicotine content of cigarettes on biomarkers of smoke exposure: A randomized clinical trial.
      ). Upon inhalation, nicotine enters the circulation by way of the lungs. It then travels to the brain where it readily diffuses into the tissue and stereoselectively binds to nicotinic cholinergic receptors. This results in the release of dopamine, which mediates the pleasurable experience of smoking. The time between inhaling a puff of tobacco smoke and the release of dopamine is a few seconds. Each puff contains more than 70 identified carcinogens, many of which contribute to the risk of a smoker developing lung cancer (
      United States Department of Health and Human Services
      Mechanisms of cancer induction by tobacco smoke.
      ,
      • Hecht S.S.
      Research opportunities related to establishing standards for tobacco products under the Family Smoking Prevention and Tobacco Control Act.
      ). Biological effects of the noncarcinogenic toxicants present in tobacco smoke are also involved. These cocarcinogenic and tumor-promoting compounds contribute to the well-established mechanism of tobacco carcinogenesis (
      • Hecht S.S.
      Tobacco smoke carcinogens and lung cancer.
      ,
      United States Department of Health and Human Services
      Mechanisms of cancer induction by tobacco smoke.
      ). However, the entire process is dependent on nicotine. When the nicotine content of a cigarette is reduced below an addictive level, very few individuals continue to smoke these cigarettes (
      • Hatsukami D.K.
      • Luo X.
      • Jensen J.A.
      • al'Absi M.
      • Allen S.S.
      • Carmella S.G.
      • Chen M.
      • Cinciripini P.M.
      • Denlinger-Apte R.
      • Drobes D.J.
      • Koopmeiners J.S.
      • Lane T.
      • Le C.T.
      • Leischow S.
      • Luo K.
      • et al.
      Effect of immediate vs gradual reduction in nicotine content of cigarettes on biomarkers of smoke exposure: A randomized clinical trial.
      ,
      • Hatsukami D.K.
      Reducing nicotine in cigarettes to minimally addictive levels: A new frontier for tobacco control.
      ).
      The study of nicotine metabolism began primarily in the purview of chemists, who identified, characterized, and quantified nicotine metabolites in the blood and urine of multiple species. This effort took off in the late 1950s and early 1960s. In 1959, cotinine was identified as the principal nicotine metabolite in the urine of smokers (
      • Bowman E.R.
      • Turnbull L.B.
      • McKennis H.,J.
      Metabolism of nicotine in the human and excretion of pyridine compounds by smokers.
      ). While a hydroxycotinine metabolite was detected in the early study, it was more than 25 years later that trans 3′-hydroxycotinine was characterized and found to be the major urinary nicotine metabolite (
      • Neurath G.B.
      • Dunger M.
      • Orth D.
      • Pein F.G.
      Trans-3'-hydroxycotinine as a main metabolite in urine of smokers.
      ). About 5 years after that, cotinine glucuronide was identified and determined to be equally or more abundant than cotinine in a smoker's urine (
      • Byrd G.D.
      • Chang K.M.
      • Greene J.M.
      • deBethizy J.D.
      Evidence for urinary excretion of glucuronide conjugates of nicotine, cotinine, and trans-3'-hydroxycotinine in smokers.
      ,
      • Benowitz N.L.
      • Jacob III, P.
      • Fong I.
      • Gupta S.
      Nicotine metabolic profile in man: Comparison of cigarette smoking and transdermal nicotine.
      ). The quantification of cotinine plus these two metabolites led to the realization that cotinine formation by nicotine 5′-oxidation was the critical pathway for the elimination of nicotine in smokers (Fig. 1). Prior to this, the N-oxidation of nicotine was believed to be as important or possibly more important than 5′-oxidation in the detoxification of nicotine.
      Figure thumbnail gr1
      Figure 1Nicotine metabolism pathways in smokers. The compounds with boxed names have been quantified as urinary metabolites in smokers. The percentages are estimated levels in smokers who are not deficient in P450 2A6 or UGT2B10 activity (
      • Hukkanen J.
      • Jacob III, P.
      • Benowitz N.L.
      Metabolism and disposition kinetics of nicotine.
      ,
      • Upadhyaya P.
      • Hecht S.S.
      Quantitative analysis of 3'-hydroxynorcotinine in human urine.
      ,
      • Hecht S.S.
      • Carmella S.G.
      • Murphy S.E.
      Effects of watercress consumption on urinary metabolites of nicotine in smokers.
      ,
      • Borrego-Soto G.
      • Perez-Paramo Y.X.
      • Chen G.
      • Santuario-Facio S.K.
      • Santos-Guzman J.
      • Posadas-Valay R.
      • Alvarado-Monroy F.M.
      • Balderas-Renteria I.
      • Medina-Gonzalez R.
      • Ortiz-Lopez R.
      • Lazarus P.
      • Rojas-Martinez A.
      Genetic variants in CYP2A6 and UGT1A9 genes associated with urinary nicotine metabolites in young Mexican smokers.
      ). ∗4-hydroxy-4-(3-pyridyl)butanoic acid (hydroxy acid), 4-oxo-4-(3-pyridyl)butanoic acid (keto acid), 4-(methylamino)-1-(3-pyridyl)-1-butanone (aminoketone); ∗∗Norcotinine is a product of P450 2A6–catalyzed cotinine metabolism, but norcotinine was not present in the urine of individuals who were administered cotinine.
      The next phase of the study of nicotine metabolism, the characterization of important enzyme catalysts, brought biochemists and pharmacologists into the field. These investigations began with microsomal studies in the 1970s and 80s (
      • Murphy P.J.
      Enzymatic oxidation of nicotine to nicotine delta1'(5') iminium ion.
      ,
      • Castagnoli N.
      • Shigenaga M.K.
      • Carlson T.
      • Trager W.F.
      • Trevor A.
      The in vitro metabolic fate of (S)-nicotine.
      ,
      • Kyerematen G.A.
      • Vesell E.S.
      Metabolism of nicotine.
      ,
      • Gorrod J.W.
      • Schepers G.
      Biotransformation of nicotine in mammalian systems.
      ). Also, in the 1980s, the addictive nature of nicotine became well recognized (
      • Benowitz N.L.
      Pharmacological aspects of cigarette smoking and nicotine addiction.
      ). This knowledge, combined with the realization that most smokers metabolize more than 80% of the nicotine they consume to cotinine, led to the hypothesis that the activity of the enzyme responsible for nicotine 5′-oxidation would influence smoking behaviors (
      • Benowitz N.L.
      • Jacob III, P.
      • Sachs D.P.
      Deficient C-oxidation of nicotine.
      ). Early studies confirmed that the catalyst was a cytochrome P450 (P450, CYP) enzyme (
      • Murphy P.J.
      Enzymatic oxidation of nicotine to nicotine delta1'(5') iminium ion.
      ,
      • Gorrod J.W.
      • Schepers G.
      Biotransformation of nicotine in mammalian systems.
      ) but not until 1996 was P450 2A6 identified as the primary human enzyme responsible for nicotine 5′-oxidation (
      • Cashman J.R.
      • Park S.B.
      • Yang Z.C.
      • Wrighton S.A.
      • Jacob III, P.
      • Benowitz N.L.
      Metabolism of nicotine by human liver microsomes: Stereoselective formation of trans-nicotine N'-oxide.
      ,
      • Nakajima M.
      • Yamamoto T.
      • Nunoya K.
      • Yokoi T.
      • Nagashima K.
      • Inoue K.
      • Funae Y.
      • Shimada N.
      • Kamataki T.
      • Kuroiwa Y.
      Role of human cytochrome P4502A6 in c-oxidation of nicotine.
      ). This discovery resulted in numerous studies that investigated the relationship of genetic variants of CYP2A6 to smoking behavior (
      • Murphy S.E.
      Nicotine metabolism and smoking: Ethnic differences in the role of P450 2A6.
      ). A number of these have confirmed an association between CYP2A6 genotype and cigarettes per day.
      The relative risk of lung cancer is significantly influenced by smoking dose, and CYP2A6 variants that affect smoking would be expected to influence the risk. Many, but not all epidemiological studies have reported an association between CYP2A6 variants and the risk of a smoker to develop lung cancer (
      • Liu T.
      • Xie C.B.
      • Ma W.J.
      • Chen W.Q.
      Association between CYP2A6 genetic polymorphisms and lung cancer: A meta-analysis of case-control studies.
      ). The quantitation of nicotine metabolites as biomarkers of tobacco exposure in lung cancer cases has confirmed that the relationship of CYP2A6 genotype to lung cancer risk is mediated by the effect of variation in nicotine metabolism on smoking dose, as illustrated in Figure 2. The first studies to demonstrate this were carried out in populations with a high prevalence of CYP2A6 nonfunctional variants, but now there is good evidence that CYP2A6 genotype contributes to a smoker's lung cancer risk in a number of different populations.
      Figure thumbnail gr2
      Figure 2Proposed relationship of CYP2A6 diplotype to smoking intensity and cancer.
      There are more than 40 million smokers in the United States (
      • Cornelius M.E.
      • Wang T.W.
      • Jamal A.
      • Loretan C.G.
      • Neff L.J.
      Tobacco product use among adults — United States, 2019.
      ) and more than a billion worldwide (https://www.who.int/tobacco/global_report/2017/en/). Accurate measures of tobacco smoke exposure could improve lung cancer risk prediction and target smoking cessation interventions to those most at risk. In the United States today, more than 60% of lung cancers occur in former smokers (
      • Siegel R.L.
      • Miller K.D.
      • Fuchs H.E.
      • Jemal A.
      Cancer statistics, 2021.
      ). These individuals remain at an elevated risk of lung cancer more than 20 years after quitting, therefore identifying those at the greatest risk is critical to appropriately target lung cancer screening efforts (
      • Tindle H.A.
      • Stevenson Duncan M.
      • Greevy R.A.
      • Vasan R.S.
      • Kundu S.
      • Massion P.P.
      • Freiberg M.S.
      Lifetime smoking history and risk of lung cancer: Results from the Framingham Heart study.
      ). Current recommendations for screening are based on age and smoking history, measured in pack years (the number of years smoked times reported cigarettes per day). Cigarettes per day is an imprecise measure of smoking dose exposure (
      • Joseph A.M.
      • Hecht S.S.
      • Murphy S.E.
      • Carmella S.G.
      • Le C.T.
      • Zhang Y.
      • Han S.
      • Hatsukami D.K.
      Relationships between cigarette consumption and biomarkers of tobacco toxin exposure.
      ,
      • Blank M.D.
      • Breland A.B.
      • Enlow P.T.
      • Duncan C.
      • Metzger A.
      • Cobb C.O.
      Measurement of smoking behavior: Comparison of self-reports, returned cigarette butts, and toxicant levels.
      ). In studies presented here on the relationship of nicotine metabolism to lung cancer, we illustrate how biomarkers of nicotine metabolism and tobacco exposure provide significantly better measures of smoking. These biomarkers have been key to elucidating our understanding of some of the observed racial/ethnic differences in lung cancer risk. The challenge now is to apply our knowledge of nicotine metabolism and smoking dose biomarkers to the lung cancer risk of former smokers. One approach is to develop a genetic model of P450 2A6-mediated nicotine metabolism to better predict smoking exposure in Former smokers. The predicted smoking dose derived from this model could be used to improve current lung cancer risk prediction models to identify former smokers at the highest risk of developing lung cancer.
      The goal of this review is to highlight the metabolic pathways and nicotine-related biomarkers that are critical to understanding the contribution of nicotine metabolism to the etiology of lung cancer in smokers. The most recent comprehensive review of nicotine metabolism was published 15 years ago by Hukkanen et al. (
      • Hukkanen J.
      • Jacob III, P.
      • Benowitz N.L.
      Metabolism and disposition kinetics of nicotine.
      ). Key findings since then, including the identification of the uridine 5′-diphospho-glucuronosyltransferase (UGT), 2B10 as the catalyst of nicotine and cotinine N-glucuronidation, in vitro studies of P450 2A6–catalyzed nicotine, nicotine Δ1′,5′-iminium ion, and cotinine metabolism, and variation in the relative abundance of nicotine metabolic pathways in different racial/ethnic groups, will also be discussed. A more complete characterization of nicotine metabolic pathways by racial/ethnic group has contributed to our understanding of the variable incidence of lung cancer in smokers from these groups.

      Overview of nicotine metabolism

      Two nicotine metabolism pathways are common to all mammals, 5′-oxidation, and N-oxidation. Studies have been carried out in many mammalian systems, from humans to mice (
      • Gorrod J.W.
      • Schepers G.
      Biotransformation of nicotine in mammalian systems.
      ,
      • Hukkanen J.
      • Jacob III, P.
      • Benowitz N.L.
      Metabolism and disposition kinetics of nicotine.
      ,
      • McKennis H.
      • Turnbull L.B.
      • Bowman E.R.
      γ-(3-Pyridyl)-γ-methylaminobutyric acid as a urinary metabolite of nicotine.
      ,
      • Hucker H.B.
      • Gillette J.R.
      • Brodie B.B.
      Cotinine: An oxidation product of nicotine formed by rabbit liver.
      ,
      • Turner D.M.
      The metabolism of [14C] nicotine in the cat.
      ), and more than 20 nicotine metabolites have been identified (Fig. 1). Several comprehensive reviews are available that discuss much of this work (
      • Kyerematen G.A.
      • Vesell E.S.
      Metabolism of nicotine.
      ,
      • Gorrod J.W.
      • Schepers G.
      Biotransformation of nicotine in mammalian systems.
      ,
      • Hukkanen J.
      • Jacob III, P.
      • Benowitz N.L.
      Metabolism and disposition kinetics of nicotine.
      ,
      • Nakayama H.
      Nicotine metabolism in mammals.
      ,
      • Neurath G.B.
      Aspects of the oxidative metabolism of nicotine.
      ).
      In humans, nicotine is metabolized by three primary pathways: P450-catalyzed 5′-oxidation, UGT-catalyzed N-glucuronidation, and flavin monooxygenase (FMO)–catalyzed N′-oxidation (Fig. 1). The Δ1′,5′-iminium ion product of nicotine 5′-oxidation is further metabolized to cotinine. The formation of cotinine is quantitatively the most important nicotine metabolism pathway (
      • Hukkanen J.
      • Jacob III, P.
      • Benowitz N.L.
      Metabolism and disposition kinetics of nicotine.
      ). Three minor pathways: methylation of the pyridine nitrogen to the nicotine isomethonium ion, 2′-oxidation, and oxidative N-demethylation also contribute to nicotine metabolism.
      Cotinine, like nicotine is metabolized by three major pathways: 3′-oxidation to trans 3′-hydroxycotinine, cotinine N-glucuronidation, and cotinine N-oxidation (Fig. 1). In contrast to nicotine, the N-oxidation of cotinine occurs on the pyrrolidine nitrogen not the pyridine nitrogen, and the catalyst of this reaction is a P450 enzyme not an FMO (
      • Hukkanen J.
      • Jacob III, P.
      • Benowitz N.L.
      Metabolism and disposition kinetics of nicotine.
      ). 3′-Hydroxycotinine is further metabolized to its O-glucuronide conjugate. Minor metabolites of cotinine include 5′-hydroxycotinine and norcotinine.

      Nicotine metabolites excreted by smokers

      In addition to cotinine, 12 urinary nicotine metabolites have been identified (
      • Kyerematen G.A.
      • Vesell E.S.
      Metabolism of nicotine.
      ,
      • Hukkanen J.
      • Jacob III, P.
      • Benowitz N.L.
      Metabolism and disposition kinetics of nicotine.
      ,
      • Neurath G.B.
      Aspects of the oxidative metabolism of nicotine.
      ,
      • Upadhyaya P.
      • Hecht S.S.
      Quantitative analysis of 3'-hydroxynorcotinine in human urine.
      ). The pathways that give rise to these metabolites are presented in Figure 1, and the names of the compounds are boxed. A 14th possible urinary metabolite, 5′-hydroxycotinine, is shown; the name is in a dashed box since the concentration of this compound in urine has only been reported in a review article (
      • Neurath G.B.
      Aspects of the oxidative metabolism of nicotine.
      ). The estimated percent of each metabolite in the urine of smokers who are not deficient in P450 2A6 or UGT2B10 are presented. These estimates are updated based on more recent data and slightly modified from those presented in the review by Hukkanen et al. (
      • Hukkanen J.
      • Jacob III, P.
      • Benowitz N.L.
      Metabolism and disposition kinetics of nicotine.
      ,
      • Upadhyaya P.
      • Hecht S.S.
      Quantitative analysis of 3'-hydroxynorcotinine in human urine.
      ,
      • Hecht S.S.
      • Carmella S.G.
      • Murphy S.E.
      Effects of watercress consumption on urinary metabolites of nicotine in smokers.
      ,
      • Borrego-Soto G.
      • Perez-Paramo Y.X.
      • Chen G.
      • Santuario-Facio S.K.
      • Santos-Guzman J.
      • Posadas-Valay R.
      • Alvarado-Monroy F.M.
      • Balderas-Renteria I.
      • Medina-Gonzalez R.
      • Ortiz-Lopez R.
      • Lazarus P.
      • Rojas-Martinez A.
      Genetic variants in CYP2A6 and UGT1A9 genes associated with urinary nicotine metabolites in young Mexican smokers.
      ). The importance of P450 2A6 and UGT2B10, the predominant catalysts of nicotine and cotinine oxidation and N-glucuronidation, to overall nicotine metabolism is discussed later in separate sections.
      In smokers, eight metabolites (nicotine N-oxide, nicotine glucuronide, cotinine, cotinine glucuronide, cotinine N-oxide, 3′-hydroxycotinine, 3′-hydroxycotinine glucuronide, and 4-hydroxy-4-(3-pyridyl)butanoic acid (hydroxy acid)) plus unmetabolized nicotine account for >90% of the nicotine dose (
      • Benowitz N.L.
      • Jacob III, P.
      • Fong I.
      • Gupta S.
      Nicotine metabolic profile in man: Comparison of cigarette smoking and transdermal nicotine.
      ,
      • Hukkanen J.
      • Jacob III, P.
      • Benowitz N.L.
      Metabolism and disposition kinetics of nicotine.
      ,
      • Borrego-Soto G.
      • Perez-Paramo Y.X.
      • Chen G.
      • Santuario-Facio S.K.
      • Santos-Guzman J.
      • Posadas-Valay R.
      • Alvarado-Monroy F.M.
      • Balderas-Renteria I.
      • Medina-Gonzalez R.
      • Ortiz-Lopez R.
      • Lazarus P.
      • Rojas-Martinez A.
      Genetic variants in CYP2A6 and UGT1A9 genes associated with urinary nicotine metabolites in young Mexican smokers.
      ,
      • Hecht S.S.
      • Hatsukami D.K.
      • Bonilla L.E.
      • Hochalter J.B.
      Quantitation of 4-oxo-4-(3-pyridyl)butanoic acid and enantiomers of 4-hydroxy-4-(3-pyridyl)butanoic acid in human urine: A substantial pathway of nicotine metabolism.
      ). The other five nicotine metabolites that have been quantified each account for <1 or 2% of the nicotine metabolites excreted by a smoker (
      • Hukkanen J.
      • Jacob III, P.
      • Benowitz N.L.
      Metabolism and disposition kinetics of nicotine.
      ,
      • Upadhyaya P.
      • Hecht S.S.
      Quantitative analysis of 3'-hydroxynorcotinine in human urine.
      ,
      • Wei B.
      • Feng J.
      • Rehmani I.J.
      • Miller S.
      • McGuffey J.E.
      • Blount B.C.
      • Wang L.
      A high-throughput robotic sample preparation system and HPLC-MS/MS for measuring urinary anatabine, anabasine, nicotine and major nicotine metabolites.
      ). These minor metabolites are discussed briefly here. Nornicotine and norcotinine are both found in the urine of smokers, but norcotinine was not detected in the urine of individuals administered cotinine (
      • Bowman E.R.
      • Mc Jr., K.H.
      Studies on the metabolism of (-)-cotinine in the human.
      ). However, Hukkanen et al. (
      • Hukkanen J.
      • Jacob III, P.
      • Benowitz N.L.
      Metabolism and disposition kinetics of nicotine.
      ) reported in unpublished data that smokers administered D4-cotinine excreted D4-norcotinine. Also, norcotinine is a major product of P450 2A6–catalyzed cotinine metabolism in vitro (
      • Brown K.M.
      • von Weymarn L.B.
      • Murphy S.E.
      Identification of N-(hydroxymethyl)-norcotinine as a major product of cytochrome P450 2A6, but not cytochrome P450 2A13-catalyzed cotinine metabolism.
      ). Norcotinine is found in the urine of dogs administered nornicotine, and nornicotine is a very minor metabolite of P450 2A6–catalyzed nicotine metabolism (
      • Wada E.
      • Bowman E.R.
      • Turnbull L.B.
      • Mc Jr., K.H.
      Norcotinine (desmethylcotinine) as a urinary metabolite of nornicotine.
      ,
      • Murphy S.E.
      • Raulinaitis V.
      • Brown K.M.
      Nicotine 5'-oxidation and methyl oxidation by P450 2A enzymes.
      ). It is unclear from these data if the norcotinine excreted by smokers is a product of cotinine or nornicotine metabolism; both pathways are presented in Figure 1. Nornicotine is present in tobacco smoke, and a portion of the nornicotine in smokers is from that exposure (
      • Benowitz N.L.
      • Jacob III, P.
      • Fong I.
      • Gupta S.
      Nicotine metabolic profile in man: Comparison of cigarette smoking and transdermal nicotine.
      ). The urinary concentration of the nicotine isomethonium ion was quantified in smoker's urine by Neurath et al. (
      • Neurath G.B.
      • Dunger M.
      • Orth D.
      • Pein F.G.
      Trans-3'-hydroxycotinine as a main metabolite in urine of smokers.
      ) but has rarely been measured by others. The most recently identified nicotine metabolite, 3′-hydroxynorcotinine (
      • Upadhyaya P.
      • Hecht S.S.
      Quantitative analysis of 3'-hydroxynorcotinine in human urine.
      ), could be a product of norcotinine oxidation (Fig. 1), or it might form by the demethylation of 3′-hydroxycotinine. It is unknown if one or both pathways occur in smokers.
      4-Oxo-4-(3-pyridyl)butanoic acid (keto acid), the precursor of hydroxy acid, is a minor urinary nicotine metabolite in smokers, but the sum of these two acids accounts for as much as 15% of the nicotine dose excreted (
      • Hecht S.S.
      • Carmella S.G.
      • Murphy S.E.
      Effects of watercress consumption on urinary metabolites of nicotine in smokers.
      ,
      • Borrego-Soto G.
      • Perez-Paramo Y.X.
      • Chen G.
      • Santuario-Facio S.K.
      • Santos-Guzman J.
      • Posadas-Valay R.
      • Alvarado-Monroy F.M.
      • Balderas-Renteria I.
      • Medina-Gonzalez R.
      • Ortiz-Lopez R.
      • Lazarus P.
      • Rojas-Martinez A.
      Genetic variants in CYP2A6 and UGT1A9 genes associated with urinary nicotine metabolites in young Mexican smokers.
      ,
      • Hecht S.S.
      • Hatsukami D.K.
      • Bonilla L.E.
      • Hochalter J.B.
      Quantitation of 4-oxo-4-(3-pyridyl)butanoic acid and enantiomers of 4-hydroxy-4-(3-pyridyl)butanoic acid in human urine: A substantial pathway of nicotine metabolism.
      ,
      • Rangiah K.
      • Hwang W.T.
      • Mesaros C.
      • Vachani A.
      • Blair I.A.
      Nicotine exposure and metabolizer phenotypes from analysis of urinary nicotine and its 15 metabolites by LC-MS.
      ). In dogs and rats, keto acid and hydroxy acid are metabolites of cotinine and are proposed to form from 5′-hydroxycotinine or norcotinine (
      • Hukkanen J.
      • Jacob III, P.
      • Benowitz N.L.
      Metabolism and disposition kinetics of nicotine.
      ,
      • McKennis H.
      • Schwartz S.L.
      • Turnbull L.B.
      • Tamaki E.
      • Bowman E.R.
      The metabolic formation of gamma-(3-pyridyl)-gamma-hydroxybutyric acid and its possible intermediarty role in the mammalian metabolism of nicotine.
      ), but neither norcotinine nor hydroxy acid has been detected in humans administered cotinine (
      • Bowman E.R.
      • Mc Jr., K.H.
      Studies on the metabolism of (-)-cotinine in the human.
      ,
      • McKennis H.
      • Schwartz S.L.
      • Turnbull L.B.
      • Tamaki E.
      • Bowman E.R.
      The metabolic formation of gamma-(3-pyridyl)-gamma-hydroxybutyric acid and its possible intermediarty role in the mammalian metabolism of nicotine.
      ,
      • Hecht S.S.
      • Hochalter J.B.
      • Villalta P.W.
      • Murphy S.E.
      2'-Hydroxylation of nicotine by cytochrome P450 2A6 and human liver microsomes: Formation of a lung carcinogen precursor.
      ). Human liver microsomal metabolism of nicotine by 2′-oxidation generates keto acid (
      • Hecht S.S.
      • Hochalter J.B.
      • Villalta P.W.
      • Murphy S.E.
      2'-Hydroxylation of nicotine by cytochrome P450 2A6 and human liver microsomes: Formation of a lung carcinogen precursor.
      ); therefore, this pathway of keto acid and hydroxy acid formation is illustrated in Figure 1.
      In smokers not deficient in P450 2A6 activity, 75% to 80% of the nicotine dose is metabolized to cotinine and its metabolites (
      • Benowitz N.L.
      • Jacob III, P.
      • Fong I.
      • Gupta S.
      Nicotine metabolic profile in man: Comparison of cigarette smoking and transdermal nicotine.
      ,
      • Hukkanen J.
      • Jacob III, P.
      • Benowitz N.L.
      Metabolism and disposition kinetics of nicotine.
      ,
      • Murphy S.E.
      • Park S.S.
      • Thompson E.F.
      • Wilkens L.R.
      • Patel Y.
      • Stram D.O.
      • Le Marchand L.
      Nicotine N-glucuronidation relative to N-oxidation and C-oxidation and UGT2B10 genotype in five ethnic/racial groups.
      ). However, each smoker's urinary nicotine metabolite profile depends on the relative abundance and activity of the enzymes involved. Significant differences in the relative frequency of genetic variants of P450 2A6 (gene CYP2A6) across racial/ethnic groups (Fig. 3A and Table 1) result in variation in the urinary nicotine metabolite profile (Fig. 3B) of smokers from these groups (
      • Murphy S.E.
      • Park S.S.
      • Thompson E.F.
      • Wilkens L.R.
      • Patel Y.
      • Stram D.O.
      • Le Marchand L.
      Nicotine N-glucuronidation relative to N-oxidation and C-oxidation and UGT2B10 genotype in five ethnic/racial groups.
      ). Smokers of Japanese ancestry have a high frequency of low or no activity of CYP2A6 alleles, and this is reflected in the reduced proportion of nicotine metabolized by 5′-oxidation. In individuals who are homozygous for CYP2A6∗4, a deletion allele (Fig. 3B), nicotine 5′-oxidation is a minor pathway, and the percentage of nicotine excreted unchanged increases, as does nicotine metabolism by N-glucuronidation and/or N-oxidation (
      • Murphy S.E.
      • Park S.S.
      • Thompson E.F.
      • Wilkens L.R.
      • Patel Y.
      • Stram D.O.
      • Le Marchand L.
      Nicotine N-glucuronidation relative to N-oxidation and C-oxidation and UGT2B10 genotype in five ethnic/racial groups.
      ,
      • Yamanaka H.
      • Nakajima M.
      • Nishimura K.
      • Yoshida R.
      • Fukami T.
      • Katoh M.
      • Yokoi T.
      Metabolic profile of nicotine in subjects whose CYP2A6 gene is deleted.
      ,
      • Park S.L.
      • Tiirikainen M.I.
      • Patel Y.M.
      • Wilkens L.R.
      • Stram D.O.
      • Le Marchand L.
      • Murphy S.E.
      Genetic determinants of CYP2A6 activity across racial/ethnic groups with different risks of lung cancer and effect on their smoking intensity.
      ).
      Figure thumbnail gr3
      Figure 3The distribution of CYP2A6 haplotypes and urinary nicotine metabolites in African American, White, and Japanese American smokers in the multiethnic cohort. A, CYP2A6 haplotypes described by eight single nucleotide polymorphisms and two copy number variants (∗12 and ∗4) and listed in the order of predicted metabolic activity (normal [green] to nonfunctional or deleted [red]). The allele nomenclature is as described at https://www.pharmvar.org/htdocs/archive/cyp2a6.htm. Figure copyright © 2016, Oxford University Press, reused with permission (
      • Park S.L.
      • Tiirikainen M.I.
      • Patel Y.M.
      • Wilkens L.R.
      • Stram D.O.
      • Le Marchand L.
      • Murphy S.E.
      Genetic determinants of CYP2A6 activity across racial/ethnic groups with different risks of lung cancer and effect on their smoking intensity.
      ). B, the proportion of nicotine metabolized by C-oxidation, N-glucuronidation, and N-oxidation in three racial/ethnic groups. The values for each slice of the pie are the mean percentage of the compounds excreted relative to total nicotine equivalents for African Americans (n = 364), Whites (n = 437), Japanese Americans (n = 674), and smokers homozygous for the CYP2A6 ∗4 allele (n = 34) who are all Japanese American (
      • Murphy S.E.
      • Park S.S.
      • Thompson E.F.
      • Wilkens L.R.
      • Patel Y.
      • Stram D.O.
      • Le Marchand L.
      Nicotine N-glucuronidation relative to N-oxidation and C-oxidation and UGT2B10 genotype in five ethnic/racial groups.
      ,
      • Park S.L.
      • Tiirikainen M.I.
      • Patel Y.M.
      • Wilkens L.R.
      • Stram D.O.
      • Le Marchand L.
      • Murphy S.E.
      Genetic determinants of CYP2A6 activity across racial/ethnic groups with different risks of lung cancer and effect on their smoking intensity.
      ). The figure is modified from the study by Murphy et al., 2014 (
      • Murphy S.E.
      • Park S.S.
      • Thompson E.F.
      • Wilkens L.R.
      • Patel Y.
      • Stram D.O.
      • Le Marchand L.
      Nicotine N-glucuronidation relative to N-oxidation and C-oxidation and UGT2B10 genotype in five ethnic/racial groups.
      ).
      Table 1CYP2A6 variants with an allele frequency greater than 1% in one of five populations
      AlleleDefining variantVariant typeFunctional consequence (in vivo unless noted)Allele frequency in various populations (%)
      Data presented are from Zhou et al., 2017 (122) who derived the frequencies from exome sequencing data of 56,945 provided by the Exome Aggregate Consortium, except data for ∗1A and ∗1H, which are from the studies of Bloom et al., 2012 (165) and Park et al., 2016 (52). ∗1B and ∗1X2 are from the study of Tanner et al., 2017 (121) and ∗12 references (52, 121, 165). Values are bolded when that group has a more than twofold greater frequency than any other group. The groups are Europeans (EUR), Africans (AFR), East Asians (EAS), South Asians (SAS), and admixed Americans (AMR). Frequencies for CYP2A6∗1 do not consider variants of ∗1A, ∗1B, and ∗1H.
      EURAFREASSASAMR
      ∗1None64.665.130.865.671.9
      ∗1Ars113711551 G>A, synonymousReduced mRNA expression and reduced activity202013NA14
      ∗1B58 bp gene conversion 3′ UTRIncreased mRNA stability301530NANA
      ∗1Hrs61663607−745 A>Glower mRNA expression in vitro11821NA4
      ∗2rs1801272Missense (L160H)No activity2.30.501.11.2
      ∗4Whole gene deletionNo activity11.51774
      ∗7rs5031016Missense I471TMuch reduced activity<0.1<0.112.90.30.3
      ∗9rs28399468Disrupts TATA boxReduced activity11.18.32314.413.8
      ∗12Hybrid allele, exon 1 and 2 from CYP2A7 (
      • Park S.L.
      • Tiirikainen M.I.
      • Patel Y.M.
      • Wilkens L.R.
      • Stram D.O.
      • Le Marchand L.
      • Murphy S.E.
      Genetic determinants of CYP2A6 activity across racial/ethnic groups with different risks of lung cancer and effect on their smoking intensity.
      ,
      • Tanner J.A.
      • Tyndale R.F.
      Variation in CYP2A6 activity and personalized medicine.
      ,
      • Bloom A.J.
      • Harari O.
      • Martinez M.
      • Madden P.A.
      • Martin N.G.
      • Montgomery G.W.
      • Rice J.P.
      • Murphy S.E.
      • Bierut L.J.
      • Goate A.
      Use of a predictive model derived from in vivo endophenotype measurements to demonstrate associations with a complex locus, CYP2A6.
      )
      No activity1–3<11.4NA3
      ∗14rs28399435, rs1137115Missense (S29N), 51 G>ANo change activity1.40.8<0.13.51.2
      ∗17rs28399454Missense (V365M)Reduced activity011.2000.6
      ∗18rs1809810Missense (Y392F)Reduced activity in vitro1.50.61.21.41.1
      ∗19rs5031016, rs1809010Missense (I471T, Y392F)Reduced activity<0.1<0.11.20.30.3
      ∗20Frameshift exon 4Truncated protein, little activity01.50NANA
      ∗21rs6413474Missense (K476R)Reduced in vitro2.80.2<0.11.90.3
      ∗23rs56256500Missense (R203C)Reduced activity00<0.1<0.1
      ∗25rs28399440Missense (F118L)Reduced activity01.4000
      ∗28rs28399440, rs8192730Missense (N418D, E419D)Reduced activity02.00.1<0.10.2
      ∗35rs143731390 (rs61736436)
      Earlier publications refer to this rs number (123, 165); the frequencies reported by Zhou et al., 2017 (122) are much different than those reported by Tanner et al., 2017 (121).
      Missense (N438Y)Reduced activity14.95.912.83.74.5
      ∗1X2Whole gene duplicationIncreased metabolism~1%0<0.5NANA
      NA, not available.
      a Data presented are from Zhou et al., 2017 (
      • Zhou Y.
      • Ingelman-Sundberg M.
      • Lauschke V.M.
      Worldwide distribution of cytochrome P450 alleles: A meta-analysis of population-scale sequencing projects.
      ) who derived the frequencies from exome sequencing data of 56,945 provided by the Exome Aggregate Consortium, except data for ∗1A and ∗1H, which are from the studies of Bloom et al., 2012 (
      • Bloom A.J.
      • Harari O.
      • Martinez M.
      • Madden P.A.
      • Martin N.G.
      • Montgomery G.W.
      • Rice J.P.
      • Murphy S.E.
      • Bierut L.J.
      • Goate A.
      Use of a predictive model derived from in vivo endophenotype measurements to demonstrate associations with a complex locus, CYP2A6.
      ) and Park et al., 2016 (
      • Park S.L.
      • Tiirikainen M.I.
      • Patel Y.M.
      • Wilkens L.R.
      • Stram D.O.
      • Le Marchand L.
      • Murphy S.E.
      Genetic determinants of CYP2A6 activity across racial/ethnic groups with different risks of lung cancer and effect on their smoking intensity.
      ). ∗1B and ∗1X2 are from the study of Tanner et al., 2017 (
      • Tanner J.A.
      • Tyndale R.F.
      Variation in CYP2A6 activity and personalized medicine.
      ) and ∗12 references (
      • Park S.L.
      • Tiirikainen M.I.
      • Patel Y.M.
      • Wilkens L.R.
      • Stram D.O.
      • Le Marchand L.
      • Murphy S.E.
      Genetic determinants of CYP2A6 activity across racial/ethnic groups with different risks of lung cancer and effect on their smoking intensity.
      ,
      • Tanner J.A.
      • Tyndale R.F.
      Variation in CYP2A6 activity and personalized medicine.
      ,
      • Bloom A.J.
      • Harari O.
      • Martinez M.
      • Madden P.A.
      • Martin N.G.
      • Montgomery G.W.
      • Rice J.P.
      • Murphy S.E.
      • Bierut L.J.
      • Goate A.
      Use of a predictive model derived from in vivo endophenotype measurements to demonstrate associations with a complex locus, CYP2A6.
      ). Values are bolded when that group has a more than twofold greater frequency than any other group. The groups are Europeans (EUR), Africans (AFR), East Asians (EAS), South Asians (SAS), and admixed Americans (AMR). Frequencies for CYP2A6∗1 do not consider variants of ∗1A, ∗1B, and ∗1H.
      b Earlier publications refer to this rs number (
      • Wassenaar C.A.
      • Zhou Q.
      • Tyndale R.F.
      CYP2A6 genotyping methods and strategies using real-time and end point PCR platforms.
      ,
      • Bloom A.J.
      • Harari O.
      • Martinez M.
      • Madden P.A.
      • Martin N.G.
      • Montgomery G.W.
      • Rice J.P.
      • Murphy S.E.
      • Bierut L.J.
      • Goate A.
      Use of a predictive model derived from in vivo endophenotype measurements to demonstrate associations with a complex locus, CYP2A6.
      ); the frequencies reported by Zhou et al., 2017 (
      • Zhou Y.
      • Ingelman-Sundberg M.
      • Lauschke V.M.
      Worldwide distribution of cytochrome P450 alleles: A meta-analysis of population-scale sequencing projects.
      ) are much different than those reported by Tanner et al., 2017 (
      • Tanner J.A.
      • Tyndale R.F.
      Variation in CYP2A6 activity and personalized medicine.
      ).

      Nicotine metabolism to cotinine and trans 3′-hydroxycotinine

      When nicotine is inhaled, vaped, absorbed from a patch, or swallowed from smokeless tobacco or nicotine gum, it is metabolized relatively quickly to cotinine (
      • Hukkanen J.
      • Jacob III, P.
      • Benowitz N.L.
      Metabolism and disposition kinetics of nicotine.
      ,
      • Benowitz N.L.
      • Hukkanen J.
      • Jacob III., P.
      Nicotine chemistry, metabolism, kinetics and biomarkers.
      ). Metabolism occurs predominantly in the liver (
      • Hukkanen J.
      • Jacob III, P.
      • Benowitz N.L.
      Metabolism and disposition kinetics of nicotine.
      ). In human liver microsomes, P450 2A6 is the key catalyst in the transformation of nicotine to cotinine (
      • Cashman J.R.
      • Park S.B.
      • Yang Z.C.
      • Wrighton S.A.
      • Jacob III, P.
      • Benowitz N.L.
      Metabolism of nicotine by human liver microsomes: Stereoselective formation of trans-nicotine N'-oxide.
      ,
      • Nakajima M.
      • Yamamoto T.
      • Nunoya K.
      • Yokoi T.
      • Nagashima K.
      • Inoue K.
      • Funae Y.
      • Shimada N.
      • Kamataki T.
      • Kuroiwa Y.
      Role of human cytochrome P4502A6 in c-oxidation of nicotine.
      ,
      • Messina E.S.
      • Tyndale R.F.
      • Sellers E.M.
      A major role for CYP2A6 in nicotine C-oxidation by human liver microsomes.
      ). P450 2A6 catalyzes the 5′-oxidation of nicotine to the Δ1′,5′-iminium ion, which is then oxidized to cotinine (Fig. 1). The oxidation of the iminium ion may be catalyzed by a cytosolic aldehyde oxidase or by P450 2A6 (
      • Murphy S.E.
      • Raulinaitis V.
      • Brown K.M.
      Nicotine 5'-oxidation and methyl oxidation by P450 2A enzymes.
      ,
      • Brandänge S.
      • Lindblom L.
      The enzyme “aldehyde oxidase” is an iminium oxidase. Reaction with nicotine delta 1'(5') iminium ion.
      ,
      • Gorrod J.W.
      • Hibberd A.R.
      The metabolism of nicotine-delta 1'(5')-iminium ion, in vivo and in vitro.
      ,
      • von Weymarn L.B.
      • Retzlaff C.
      • Murphy S.E.
      CYP2A6 and CYP2A13-catalyzed metabolism of the nicotine delta 1'(5') iminium ion.
      ). In the study of microsomal metabolism of nicotine, cytosol is often added as a source of aldehyde oxidase. However, human liver microsomes and P450 2A6 convert nicotine to cotinine in the absence of cytosol (
      • Murphy S.E.
      • Raulinaitis V.
      • Brown K.M.
      Nicotine 5'-oxidation and methyl oxidation by P450 2A enzymes.
      ,
      • Dicke K.
      • Skrlin S.
      • Murphy S.E.
      Nicotine and 4-(methylnitrosamino)-1-(3-pyridyl)-butanone (NNK) metabolism by P450 2B6.
      ), and P450 2A6 catalyzes the oxidation of the Δ1′,5′-iminium ion to cotinine (
      • von Weymarn L.B.
      • Retzlaff C.
      • Murphy S.E.
      CYP2A6 and CYP2A13-catalyzed metabolism of the nicotine delta 1'(5') iminium ion.
      ). Therefore, aldehyde oxidase is not required to metabolize nicotine to cotinine in vitro and may not be necessary in vivo. P450 2B6 is the only other human enzyme that catalyzes the 5′-oxidation of nicotine, but it is a much less efficient enzyme than P450 2A6 (
      • Dicke K.
      • Skrlin S.
      • Murphy S.E.
      Nicotine and 4-(methylnitrosamino)-1-(3-pyridyl)-butanone (NNK) metabolism by P450 2B6.
      ,
      • Yamazaki H.
      • Inoue K.
      • Hashimoto M.
      • Shimada T.
      Roles of CYP2A6 and CYP2B6 in nicotine C-oxidation by human liver microsomes.
      ,
      • Bloom A.J.
      • Wang P.F.
      • Kharasch E.D.
      Nicotine oxidation by genetic variants of CYP2B6 and in human brain microsomes.
      ). Interestingly, P450 2B6 does not convert nicotine to cotinine in the absence of cytosol (
      • Dicke K.
      • Skrlin S.
      • Murphy S.E.
      Nicotine and 4-(methylnitrosamino)-1-(3-pyridyl)-butanone (NNK) metabolism by P450 2B6.
      ). That is, P450 2B6, unlike P450 2A6, does not catalyze the oxidation of the Δ5′(1′)-iminium ion. In vitro, both enzymes catalyze a small amount of nicotine demethylation (
      • Dicke K.
      • Skrlin S.
      • Murphy S.E.
      Nicotine and 4-(methylnitrosamino)-1-(3-pyridyl)-butanone (NNK) metabolism by P450 2B6.
      ,
      • Yamanaka H.
      • Nakajima M.
      • Fukami T.
      • Sakai H.
      • Nakamura A.
      • Katoh M.
      • Takamiya M.
      • Aoki Y.
      • Yokoi T.
      CYP2A6 and CYP2B6 are involved in nornicotine formation from nicotine in humans: Interindividual differences in these contributions.
      ). When P450 2A6 is present, P450 2B6 appears to contribute little to hepatic nicotine metabolism, either 5′-oxidation or N-demethylation (
      • Dicke K.
      • Skrlin S.
      • Murphy S.E.
      Nicotine and 4-(methylnitrosamino)-1-(3-pyridyl)-butanone (NNK) metabolism by P450 2B6.
      ,
      • Yamanaka H.
      • Nakajima M.
      • Fukami T.
      • Sakai H.
      • Nakamura A.
      • Katoh M.
      • Takamiya M.
      • Aoki Y.
      • Yokoi T.
      CYP2A6 and CYP2B6 are involved in nornicotine formation from nicotine in humans: Interindividual differences in these contributions.
      ).
      The metabolism of cotinine to 3′-hydroxycotinine is also catalyzed by P450 2A6, although less efficiently than the 5′-oxidation of nicotine (
      • Brown K.M.
      • von Weymarn L.B.
      • Murphy S.E.
      Identification of N-(hydroxymethyl)-norcotinine as a major product of cytochrome P450 2A6, but not cytochrome P450 2A13-catalyzed cotinine metabolism.
      ,
      • Nakajima M.
      • Yamamoto T.
      • Nunoya K.
      • Yokoi T.
      • Nagashima K.
      • Inoue K.
      • Funae Y.
      • Shimada N.
      • Kamataki T.
      • Kuroiwa Y.
      Characterization of CYP 2A6 involved in 3'-hydroxylation of cotinine in human liver microsomes.
      ). No other human P450 has been identified as a catalyst of this reaction, and little or no 3′-hydroxycotinine is excreted as a nicotine metabolite by individuals who do not express P450 2A6 (Fig. 3B) (
      • Nakajima M.
      • Yamagishi S.
      • Yamamoto H.
      • Yamamoto T.
      • Kuroiwa Y.
      • Yokoi T.
      Deficient cotinine formation from nicotine is attributed to the whole deletion of the CYP2A6 gene in humans.
      ). Therefore, the ratio of 3′-hydroxycotinine to cotinine (in urine, plasma, and saliva) has been characterized as a measure of P450 2A6 activity in smokers and is referred to as the nicotine metabolism ratio (
      • Hukkanen J.
      • Jacob III, P.
      • Benowitz N.L.
      Metabolism and disposition kinetics of nicotine.
      ,
      • Benowitz N.L.
      • Swan G.E.
      • Jacob III, P.
      • Lessov-Schlaggar C.N.
      • Tyndale R.F.
      CYP2A6 genotype and the metabolism and disposition kinetics of nicotine.
      ,
      • St Helen G.
      • Novalen M.
      • Heitjan D.F.
      • Dempsey D.
      • Jacob III, P.
      • Aziziyeh A.
      • Wing V.C.
      • George T.P.
      • Tyndale R.F.
      • Benowitz N.L.
      Reproducibility of the nicotine metabolite ratio in cigarette smokers.
      ).

      P450 2A6–catalyzed metabolism of nicotine, Δ1′(5′)iminium ion, and cotinine in vitro

      P450 2A6–catalyzed nicotine metabolism has typically been quantified by measuring the formation of cotinine in the presence of aldehyde oxidase (
      • Nakajima M.
      • Yamamoto T.
      • Nunoya K.
      • Yokoi T.
      • Nagashima K.
      • Inoue K.
      • Funae Y.
      • Shimada N.
      • Kamataki T.
      • Kuroiwa Y.
      Role of human cytochrome P4502A6 in c-oxidation of nicotine.
      ,
      • Hecht S.S.
      • Hochalter J.B.
      • Villalta P.W.
      • Murphy S.E.
      2'-Hydroxylation of nicotine by cytochrome P450 2A6 and human liver microsomes: Formation of a lung carcinogen precursor.
      ,
      • Messina E.S.
      • Tyndale R.F.
      • Sellers E.M.
      A major role for CYP2A6 in nicotine C-oxidation by human liver microsomes.
      ,
      • Yamazaki H.
      • Inoue K.
      • Hashimoto M.
      • Shimada T.
      Roles of CYP2A6 and CYP2B6 in nicotine C-oxidation by human liver microsomes.
      ). However, the nicotine Δ1′,5′-iminium ion has been quantified directly as the major product of P450 2A6–catalyzed nicotine metabolism in both heterologous expression systems and with human liver microsomes (
      • Murphy S.E.
      • Raulinaitis V.
      • Brown K.M.
      Nicotine 5'-oxidation and methyl oxidation by P450 2A enzymes.
      ,
      • von Weymarn L.B.
      • Brown K.M.
      • Murphy S.E.
      Inactivation of CYP2A6 and CYP2A13 during nicotine metabolism.
      ). Additional metabolites were also identified. The products of methyl oxidation and 2′-oxidation, 4-(methylamino)-1-(3-pyridyl)-1-butanone aminoketone, and nornicotine were minor metabolites of P450 2A6–catalyzed metabolism (Fig. 1) (
      • Murphy S.E.
      • Raulinaitis V.
      • Brown K.M.
      Nicotine 5'-oxidation and methyl oxidation by P450 2A enzymes.
      ). Aminoketone was also a minor metabolite of human liver microsomal nicotine metabolism (
      • Murphy S.E.
      • Raulinaitis V.
      • Brown K.M.
      Nicotine 5'-oxidation and methyl oxidation by P450 2A enzymes.
      ,
      • Hecht S.S.
      • Hochalter J.B.
      • Villalta P.W.
      • Murphy S.E.
      2'-Hydroxylation of nicotine by cytochrome P450 2A6 and human liver microsomes: Formation of a lung carcinogen precursor.
      ). The amount of nicotine 2′-oxidation relative to 5′-oxidation was from <2% to 11% in these reactions. The higher percentage of nicotine metabolism by 2′-oxidation was estimated from the ratio of amino ketone to cotinine formation in the presence of cytosol (
      • Hecht S.S.
      • Hochalter J.B.
      • Villalta P.W.
      • Murphy S.E.
      2'-Hydroxylation of nicotine by cytochrome P450 2A6 and human liver microsomes: Formation of a lung carcinogen precursor.
      ). However, based on later experiments (
      • Murphy S.E.
      • Raulinaitis V.
      • Brown K.M.
      Nicotine 5'-oxidation and methyl oxidation by P450 2A enzymes.
      ), it is likely that in the original experiment, not all the iminium ions were converted to cotinine. Therefore, total 5′-oxidation accounted for a higher percentage of nicotine metabolism than what was reported (
      • Hecht S.S.
      • Hochalter J.B.
      • Villalta P.W.
      • Murphy S.E.
      2'-Hydroxylation of nicotine by cytochrome P450 2A6 and human liver microsomes: Formation of a lung carcinogen precursor.
      ), and the relative amount of 2′-oxidation was closer to 1 to 2%. In the earlier publication, a relatively large peak at the correct retention time of nicotine Δ1′,5′-iminium ion was observed in the HPLC chromatograms from which aminoketone and cotinine were quantified. The overwhelming predominance of 5′-oxidation relative to 2′-oxidation is supported by the X-ray structure of nicotine complexed with P450 2A6 (
      • DeVore N.M.
      • Scott E.E.
      Nicotine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone binding and access channel in human cytochrome P450 2A6 and 2A13 enzymes.
      ). In this structure, nicotine is oriented with the 5′-pyrrolidine carbon within 4 Å of the heme; the 2′-carbon is much further away.
      The oxidation of nicotine Δ1′,5′-iminium ion to cotinine by a P450 enzyme was first observed in experiments on nicotine metabolism with rabbit liver microsomes (
      • Shigenaga M.K.
      • Trevor A.J.
      • Castagnoli N.
      Metabolism-dependent covalent binding of (S)-[5-3H]nicotine to liver and lung microsomal macromolecules.
      ). In more recent experiments, cotinine was quantified as the major product of P450 2A6–catalyzed metabolism of the iminium ion (
      • von Weymarn L.B.
      • Retzlaff C.
      • Murphy S.E.
      CYP2A6 and CYP2A13-catalyzed metabolism of the nicotine delta 1'(5') iminium ion.
      ). A minor product of this reaction was tentatively identified as 2′-hydroxy-3′,4′-dehydronicotine or a related isomer, which would dehydrate to β-nicotyrine. Interestingly, cotinine and cotinine metabolites were also detected as products of P450 2A6–catalyzed nicotine Δ1′,5′-iminium ion, even at short incubation times. The cotinine metabolites detected were 3′-hydroxycotinine, norcotinine, and N-hydroxynorcotinine (Fig. 1). Additional experiments supported the hypothesis that the majority of the 3′-hydroxycotinine formed was the product of the sequential metabolism of the nicotine Δ1′,5′-iminium ion (
      • von Weymarn L.B.
      • Retzlaff C.
      • Murphy S.E.
      CYP2A6 and CYP2A13-catalyzed metabolism of the nicotine delta 1'(5') iminium ion.
      ).
      The in vitro metabolism of nicotine and the iminium ion support the argument that the urinary products of nicotine 2′-oxidation, hydroxy acid, and keto acid, and the product of methyl oxidation, nornicotine, may depend on P450 2A6 activity in smokers (
      • Murphy S.E.
      • Raulinaitis V.
      • Brown K.M.
      Nicotine 5'-oxidation and methyl oxidation by P450 2A enzymes.
      ,
      • Hecht S.S.
      • Hochalter J.B.
      • Villalta P.W.
      • Murphy S.E.
      2'-Hydroxylation of nicotine by cytochrome P450 2A6 and human liver microsomes: Formation of a lung carcinogen precursor.
      ,
      • von Weymarn L.B.
      • Retzlaff C.
      • Murphy S.E.
      CYP2A6 and CYP2A13-catalyzed metabolism of the nicotine delta 1'(5') iminium ion.
      ). The relationship of P450 2A6 activity to the formation of these metabolites has not been investigated. Characterizing whether smokers who have no CYP2A6 activity excrete hydroxy acid, which is a relatively abundant nicotine metabolite, would allow a more complete phenotyping of smokers for P450 2A6 activity.
      The in vitro metabolism of cotinine by CYP2A6 results in the formation of three products; the major product is N-(hydroxymethyl)norcotinine not 3′-hydroxycotinine (
      • Brown K.M.
      • von Weymarn L.B.
      • Murphy S.E.
      Identification of N-(hydroxymethyl)-norcotinine as a major product of cytochrome P450 2A6, but not cytochrome P450 2A13-catalyzed cotinine metabolism.
      ). This is surprising given that in smokers norcotinine is a minor nicotine metabolite. One explanation for the discrepancy in the relative abundance of these two pathways in vivo and in vitro may be that a majority of the 3′-hydroxycotinine formed in vivo is from sequential metabolism of the nicotine Δ1′,5′-iminium ion (
      • von Weymarn L.B.
      • Retzlaff C.
      • Murphy S.E.
      CYP2A6 and CYP2A13-catalyzed metabolism of the nicotine delta 1'(5') iminium ion.
      ). That is, much of the cotinine product of the iminium ion does not leave the active site prior to further metabolism to 3′-hydroxycotinine.

      Nicotine N-oxide and FMO3

      FMO3 is the catalyst of nicotine N-oxidation in smokers (
      • Hukkanen J.
      • Jacob III, P.
      • Benowitz N.L.
      Metabolism and disposition kinetics of nicotine.
      ,
      • Park S.B.
      • Jacob III, P.
      • Benowitz N.L.
      • Cashman J.R.
      Stereoselective metabolism of (S)-(-)-nicotine in humans: Formation of trans-(S)-(-)-nicotine N-1'-oxide.
      ). The primary product of this reaction in vitro is trans nicotine N-oxide, which is the only isomer detected in smoker's urine (
      • Park S.B.
      • Jacob III, P.
      • Benowitz N.L.
      • Cashman J.R.
      Stereoselective metabolism of (S)-(-)-nicotine in humans: Formation of trans-(S)-(-)-nicotine N-1'-oxide.
      ,
      • Hinrichs A.L.
      • Murphy S.E.
      • Wang J.C.
      • Saccone S.
      • Saccone N.
      • Steinbach J.H.
      • Goate A.
      • Stevens V.L.
      • Bierut L.J.
      Common polymorphisms in FMO1 are associated with nicotine dependence.
      ). Genetic variants in FMO3 influence, albeit to a small extent, variance in the percentage of nicotine metabolized to cotinine (
      • Bloom A.J.
      • Murphy S.E.
      • Martinez M.
      • von Weymarn L.B.
      • Bierut L.J.
      • Goate A.
      Effects upon in-vivo nicotine metabolism reveal functional variation in FMO3 associated with cigarette consumption.
      ). In vitro FMO1 is a similar or more efficient catalyst of nicotine N-oxidation than FMO3, but both cis and trans nicotine N-oxide are products of FMO1 metabolism (
      • Hinrichs A.L.
      • Murphy S.E.
      • Wang J.C.
      • Saccone S.
      • Saccone N.
      • Steinbach J.H.
      • Goate A.
      • Stevens V.L.
      • Bierut L.J.
      Common polymorphisms in FMO1 are associated with nicotine dependence.
      ,
      • Perez-Paramo Y.X.
      • Chen G.
      • Ashmore J.H.
      • Watson C.J.W.
      • Nasrin S.
      • Adams-Haduch J.
      • Wang R.
      • Gao Y.T.
      • Koh W.P.
      • Yuan J.M.
      • Lazarus P.
      Nicotine-N'-oxidation by flavin monooxygenase enzymes.
      ). FMO3 is primarily expressed in the liver, whereas FMO1 is expressed in extrahepatic tissues, including the brain (
      • Zhang J.
      • Cashman J.R.
      Quantitative analysis of FMO gene mRNA levels in human tissues.
      ). FMO1 does not contribute significantly to total nicotine N-oxidation in smokers, but it may contribute to nicotine metabolism in the brain. Human brain tissue has FMO activity, and microsomes prepared from human brain catalyze both cis and trans oxidation of nicotine but with different kinetic parameters (
      • Bhagwat S.V.
      • Bhamre S.
      • Boyd M.R.
      • Ravindranath V.
      Cerebral metabolism of imipramine and a purified flavin-containing monooxygenase from human brain.
      ,
      • Teitelbaum A.M.
      • Murphy S.E.
      • Akk G.
      • Baker T.B.
      • Germann A.
      • von Weymarn L.B.
      • Bierut L.J.
      • Goate A.
      • Kharasch E.D.
      • Bloom A.J.
      Nicotine dependence is associated with functional variation in FMO3, an enzyme that metabolizes nicotine in the brain.
      ). These data support a role for both FMO1 and FMO3 in nicotine metabolism in the brain. Variation in the activity of these enzymes might influence nicotine addiction, by contributing to brain nicotine concentration and interaction with nicotinic cholinergic receptors. Interestingly, a genome-wide association study did report a significant association between single nucleotide polymorphisms in FMO1 and nicotine dependence (
      • Hinrichs A.L.
      • Murphy S.E.
      • Wang J.C.
      • Saccone S.
      • Saccone N.
      • Steinbach J.H.
      • Goate A.
      • Stevens V.L.
      • Bierut L.J.
      Common polymorphisms in FMO1 are associated with nicotine dependence.
      ). In addition, polymorphisms in FMO3 were associated with cigarettes per day and nicotine dependence (
      • Bloom A.J.
      • Murphy S.E.
      • Martinez M.
      • von Weymarn L.B.
      • Bierut L.J.
      • Goate A.
      Effects upon in-vivo nicotine metabolism reveal functional variation in FMO3 associated with cigarette consumption.
      ,
      • Teitelbaum A.M.
      • Murphy S.E.
      • Akk G.
      • Baker T.B.
      • Germann A.
      • von Weymarn L.B.
      • Bierut L.J.
      • Goate A.
      • Kharasch E.D.
      • Bloom A.J.
      Nicotine dependence is associated with functional variation in FMO3, an enzyme that metabolizes nicotine in the brain.
      ).

      Glucuronide conjugation of nicotine, cotinine, and 3-hydroxycotinine

      Prior to the structural characterization of the quaternary N1-glucuronide of nicotine (
      • Byrd G.D.
      • Caldwell W.S.
      • Bhatti B.S.
      • Ravard A.
      • Crooks P.A.
      Determination of nicotine N-1-glucuronide, a quaternary N-glucuronide conjugate, in human biological samples.
      ), several investigators quantified the presence of a nicotine glucuronide in smokers (
      • Byrd G.D.
      • Chang K.M.
      • Greene J.M.
      • deBethizy J.D.
      Evidence for urinary excretion of glucuronide conjugates of nicotine, cotinine, and trans-3'-hydroxycotinine in smokers.
      ,
      • Benowitz N.L.
      • Jacob III, P.
      • Fong I.
      • Gupta S.
      Nicotine metabolic profile in man: Comparison of cigarette smoking and transdermal nicotine.
      ,
      • Hukkanen J.
      • Jacob III, P.
      • Benowitz N.L.
      Metabolism and disposition kinetics of nicotine.
      ,
      • Davis R.A.
      • Curvall M.
      Determination of nicotine and its metabolites in biological fluids:in vivo studies.
      ) from the urinary nicotine concentrations before and after treatment with β-glucuronidase. Cotinine N-glucuronide has similarly been characterized and quantified in smokers (
      • Byrd G.D.
      • Chang K.M.
      • Greene J.M.
      • deBethizy J.D.
      Evidence for urinary excretion of glucuronide conjugates of nicotine, cotinine, and trans-3'-hydroxycotinine in smokers.
      ,
      • Benowitz N.L.
      • Jacob III, P.
      • Fong I.
      • Gupta S.
      Nicotine metabolic profile in man: Comparison of cigarette smoking and transdermal nicotine.
      ,
      • Hukkanen J.
      • Jacob III, P.
      • Benowitz N.L.
      Metabolism and disposition kinetics of nicotine.
      ,
      • Davis R.A.
      • Curvall M.
      Determination of nicotine and its metabolites in biological fluids:in vivo studies.
      ,
      • Caldwell W.S.
      • Greene J.M.
      • Byrd G.D.
      • Chang K.M.
      • Uhrig M.S.
      • deBethizy J.D.
      • Crooks P.A.
      • Bhatti B.S.
      • Riggs R.M.
      Characterization of the glucuronide conjugate of cotinine: A previously unidentified major metabolite of nicotine in smokers' urine.
      ). In 2007, two research groups independently identified UGT2B10 as a catalyst of nicotine and cotinine N-glucuronidation (
      • Chen G.
      • Blevins-Primeau A.S.
      • Dellinger R.W.
      • Muscat J.E.
      • Lazarus P.
      Glucuronidation of nicotine and cotinine by UGT2B10: Loss of function by the UGT2B10 codon 67 (Asp>Tyr) polymorphism.
      ,
      • Kaivosaari S.
      • Toivonen P.
      • Hesse L.M.
      • Koskinen M.
      • MH C.
      • Finel M.
      Nicotine glucuronidation and the human UDP-glucuronosyltransferase UGT2B10.
      ). Prior to this discovery, UGT1A4, which also catalyzes these reactions, was believed to be the catalyst in smokers (
      • Hukkanen J.
      • Jacob III, P.
      • Benowitz N.L.
      Metabolism and disposition kinetics of nicotine.
      ,
      • Kuehl G.E.
      • Murphy S.E.
      N-Glucuronidation of nicotine and cotinine by human liver microsomes and heterologously-expressed UDP-glucuronosyltransferases.
      ,
      • Nakajima M.
      • Tanaka E.
      • Kwon J.T.
      • Yokoi T.
      Characterization of nicotine and cotinine N-glucuronidations in human liver microsomes.
      ). However, UGT2B10 is a more efficient catalyst than is UGT1A4, and it is the enzyme responsible for nicotine and cotinine glucuronidation in human liver microsomes and in smokers (
      • Murphy S.E.
      • Park S.S.
      • Thompson E.F.
      • Wilkens L.R.
      • Patel Y.
      • Stram D.O.
      • Le Marchand L.
      Nicotine N-glucuronidation relative to N-oxidation and C-oxidation and UGT2B10 genotype in five ethnic/racial groups.
      ,
      • Chen G.
      • Blevins-Primeau A.S.
      • Dellinger R.W.
      • Muscat J.E.
      • Lazarus P.
      Glucuronidation of nicotine and cotinine by UGT2B10: Loss of function by the UGT2B10 codon 67 (Asp>Tyr) polymorphism.
      ,
      • Kaivosaari S.
      • Toivonen P.
      • Hesse L.M.
      • Koskinen M.
      • MH C.
      • Finel M.
      Nicotine glucuronidation and the human UDP-glucuronosyltransferase UGT2B10.
      ,
      • Berg J.Z.
      • Mason J.
      • Boettcher A.J.
      • Hatsukami D.K.
      • Murphy S.E.
      Nicotine metabolism in African Americans and European Americans: Variation in glucuronidation by ethnicity and UGT2B10 haplotype.
      ).
      Two UGT2B10 variants that code for nonfunctional enzyme have been characterized (
      • Murphy S.E.
      • Park S.S.
      • Thompson E.F.
      • Wilkens L.R.
      • Patel Y.
      • Stram D.O.
      • Le Marchand L.
      Nicotine N-glucuronidation relative to N-oxidation and C-oxidation and UGT2B10 genotype in five ethnic/racial groups.
      ,
      • Chen G.
      • Blevins-Primeau A.S.
      • Dellinger R.W.
      • Muscat J.E.
      • Lazarus P.
      Glucuronidation of nicotine and cotinine by UGT2B10: Loss of function by the UGT2B10 codon 67 (Asp>Tyr) polymorphism.
      ). Both significantly impact the extent of nicotine and cotinine glucuronidation in smokers (
      • Murphy S.E.
      • Park S.S.
      • Thompson E.F.
      • Wilkens L.R.
      • Patel Y.
      • Stram D.O.
      • Le Marchand L.
      Nicotine N-glucuronidation relative to N-oxidation and C-oxidation and UGT2B10 genotype in five ethnic/racial groups.
      ,
      • Berg J.Z.
      • Mason J.
      • Boettcher A.J.
      • Hatsukami D.K.
      • Murphy S.E.
      Nicotine metabolism in African Americans and European Americans: Variation in glucuronidation by ethnicity and UGT2B10 haplotype.
      ,
      • Berg J.Z.
      • von Weymarn L.B.
      • Thompson E.T.
      • Wickham K.M.
      • Weisensel N.A.
      • Hatsukami D.K.
      • Murphy S.E.
      UGT2B10 genotype influences nicotine glucuronidation, oxidation and consumption.
      ,
      • Chen G.
      • Giambrone Jr., N.E.
      • Dluzen D.F.
      • Muscat J.E.
      • Berg A.
      • Gallagher C.J.
      • Lazarus P.
      Glucuronidation genotypes and nicotine metabolic phenotypes: Importance of functional UGT2B10 and UGT2B17 polymorphisms.
      ). White smokers homozygous for an Asp67Tyr UGT2B10 variant (rs6175900) excreted little if any cotinine or nicotine glucuronide (
      • Chen G.
      • Giambrone Jr., N.E.
      • Dluzen D.F.
      • Muscat J.E.
      • Berg A.
      • Gallagher C.J.
      • Lazarus P.
      Glucuronidation genotypes and nicotine metabolic phenotypes: Importance of functional UGT2B10 and UGT2B17 polymorphisms.
      ), whereas heterozygous white smokers excreted about half as much of each glucuronide (
      • Berg J.Z.
      • von Weymarn L.B.
      • Thompson E.T.
      • Wickham K.M.
      • Weisensel N.A.
      • Hatsukami D.K.
      • Murphy S.E.
      UGT2B10 genotype influences nicotine glucuronidation, oxidation and consumption.
      ,
      • Chen G.
      • Giambrone Jr., N.E.
      • Dluzen D.F.
      • Muscat J.E.
      • Berg A.
      • Gallagher C.J.
      • Lazarus P.
      Glucuronidation genotypes and nicotine metabolic phenotypes: Importance of functional UGT2B10 and UGT2B17 polymorphisms.
      ). However, this variant, with a frequency of ∼10% in white versus ∼5% in African Americans, does not account for the significantly lower nicotine and cotinine glucuronidation in African American compared with white smokers reported more than 20 years ago (
      • Berg J.Z.
      • Mason J.
      • Boettcher A.J.
      • Hatsukami D.K.
      • Murphy S.E.
      Nicotine metabolism in African Americans and European Americans: Variation in glucuronidation by ethnicity and UGT2B10 haplotype.
      ,
      • Benowitz N.L.
      • Perez-Stable E.J.
      • Fong I.
      • Modin G.
      • Herrera B.
      • Jacob III, P.
      Ethnic differences in N-glucuronidation of nicotine and cotinine.
      ). A UGT2B10 splice variant (rs2942857) has now been identified that explains the decreased nicotine and cotinine glucuronidation in African American smokers (
      • Murphy S.E.
      • Park S.S.
      • Thompson E.F.
      • Wilkens L.R.
      • Patel Y.
      • Stram D.O.
      • Le Marchand L.
      Nicotine N-glucuronidation relative to N-oxidation and C-oxidation and UGT2B10 genotype in five ethnic/racial groups.
      ,
      • Wang H.
      • Park S.L.
      • Stram D.O.
      • Haiman C.A.
      • Wilkens L.R.
      • Hecht S.S.
      • Kolonel L.N.
      • Murphy S.E.
      • Le Marchand L.
      Associations between genetic ancestries and nicotine metabolism biomarkers in the Multiethnic Cohort study.
      ,
      • Patel Y.M.
      • Stram D.O.
      • Wilkens L.R.
      • Park S.S.
      • Henderson B.E.
      • Le Marchand L.
      • Haiman C.A.
      • Murphy S.E.
      The contribution of common genetic variation to nicotine and cotinine glucuronidation in multiple ethnic/racial populations.
      ). The frequency of the splice variant is 37% in individuals of African ancestry compared with <1% of whites (
      • Murphy S.E.
      • Park S.S.
      • Thompson E.F.
      • Wilkens L.R.
      • Patel Y.
      • Stram D.O.
      • Le Marchand L.
      Nicotine N-glucuronidation relative to N-oxidation and C-oxidation and UGT2B10 genotype in five ethnic/racial groups.
      ,
      • Wang H.
      • Park S.L.
      • Stram D.O.
      • Haiman C.A.
      • Wilkens L.R.
      • Hecht S.S.
      • Kolonel L.N.
      • Murphy S.E.
      • Le Marchand L.
      Associations between genetic ancestries and nicotine metabolism biomarkers in the Multiethnic Cohort study.
      ,
      • Patel Y.M.
      • Stram D.O.
      • Wilkens L.R.
      • Park S.S.
      • Henderson B.E.
      • Le Marchand L.
      • Haiman C.A.
      • Murphy S.E.
      The contribution of common genetic variation to nicotine and cotinine glucuronidation in multiple ethnic/racial populations.
      ,
      • Fowler S.
      • Kletzl H.
      • Finel M.
      • Manevski N.
      • Schmid P.
      • Tuerck D.
      • Norcross R.D.
      • Hoener M.C.
      • Spleiss O.
      • Iglesias V.A.
      A UGT2B10 splicing polymorphism common in African populations may greatly increase drug exposure.
      ). About 30% of African Americans carry neither the splice nor the Asp67Tyr variant compared with >80% of whites (Fig. 4A; genetic score 0) (
      • Murphy S.E.
      • Park S.S.
      • Thompson E.F.
      • Wilkens L.R.
      • Patel Y.
      • Stram D.O.
      • Le Marchand L.
      Nicotine N-glucuronidation relative to N-oxidation and C-oxidation and UGT2B10 genotype in five ethnic/racial groups.
      ,
      • Chen G.
      • Dellinger R.W.
      • Gallagher C.J.
      • Sun D.
      • Lazarus P.
      Identification of a prevalent functional missense polymorphism in the UGT2B10 gene and its association with UGT2B10 inactivation against tobacco-specific nitrosamines.
      ). Almost 20% of African Americans have no functional UGT2B10 enzyme (Fig. 4A; genetic score 2). Among smokers from five different racial/ethnic groups those who are heterozygous for either the splice or the Asp67Tyr variant excreted 50% less nicotine and cotinine glucuronide than individuals who carried neither variant (Fig. 4B; genetic score 1 versus 0) (
      • Murphy S.E.
      • Park S.S.
      • Thompson E.F.
      • Wilkens L.R.
      • Patel Y.
      • Stram D.O.
      • Le Marchand L.
      Nicotine N-glucuronidation relative to N-oxidation and C-oxidation and UGT2B10 genotype in five ethnic/racial groups.
      ). Smokers homozygous for either variant (genetic score 2), more than 80% of whom are African American, excreted little or no nicotine or cotinine N-glucuronide. Interestingly, the relative glucuronidation of cotinine in African Americans carrying one or neither variant alleles is significantly lower (p < 0.05) than in whites with these genotypes (Fig. 4), possibly because of the presence of other UGT2B10 variants in African Americans that have yet to be characterized (
      • Murphy S.E.
      • Park S.S.
      • Thompson E.F.
      • Wilkens L.R.
      • Patel Y.
      • Stram D.O.
      • Le Marchand L.
      Nicotine N-glucuronidation relative to N-oxidation and C-oxidation and UGT2B10 genotype in five ethnic/racial groups.
      ).
      Figure thumbnail gr4
      Figure 4The distribution of UGT2B10 variants and nicotine and cotinine glucuronidation phenotypes for the five racial/ethnic groups in the multiethnic cohort. A, the section of the pies represents the proportion of smokers with no (blue), one (orange), or two (gray) UGT2B10 alleles that code for no functional enzyme as described by genetic scores 0, 1, and 2. Genetic score 0, rs61750900 GG and rs2942857 AA; score 1, rs61750900 GT or rs2942857 CA; score 2, rs61750900 TT or rs2942857 CC, or both rs61750900 GT and rs2942857 CA. B, the urinary ratio of cotinine glucuronide to cotinine and nicotine glucuronide to nicotine excreted by smokers by UGT2B10 genetic score. The values, geometric means, and 95% confidence interval are adjusted for age, sex, creatinine, body mass index, and race. When n <10 for score 2, whites (n = 1), Japanese American (n = 2), Hawaiian (n = 2), and Latinos (n = 7), arithmetic means are presented and no confidence interval. The data used to create Figures A and B are from Table S3 of Murphy et al., 2014 (
      • Murphy S.E.
      • Park S.S.
      • Thompson E.F.
      • Wilkens L.R.
      • Patel Y.
      • Stram D.O.
      • Le Marchand L.
      Nicotine N-glucuronidation relative to N-oxidation and C-oxidation and UGT2B10 genotype in five ethnic/racial groups.
      ).
      The glucuronide conjugate of 3′-hydroxycotinine excreted by smokers is the O-glucuronide. No 3′-hydroxycotinine N-glucuronide is found in the urine of smokers even though human liver microsomes catalyze both O- and N-glucuronidation of 3-HCOT (
      • Hukkanen J.
      • Jacob III, P.
      • Benowitz N.L.
      Metabolism and disposition kinetics of nicotine.
      ,
      • Kuehl G.E.
      • Murphy S.E.
      N-Glucuronidation of trans-3'-hydroxycotinine by human liver microsomes.
      ). UGT1A9, UGT2B7, and UGT2B17 are all catalysts of 3′-hydroxycotinine O-glucuronidation (
      • Chen G.
      • Giambrone N.E.
      • Lazarus P.
      Glucuronidation of trans-3'-hydroxycotinine by UGT2B17 and UGT2B10.
      ,
      • Yamanaka H.
      • Nakajima M.
      • Katoh M.
      • Kanoh A.
      • Tamura O.
      • Ishibashi H.
      • Yokoi T.
      Trans-3'-hydroxycotinine O- and N-glucuronidations in human liver microsomes.
      ). In smokers, UGT2B17 contributes significantly to the glucuronidation of 3′-hydroxycotinine. Individuals who are homozygous for a UGT2B17 deletion allele excrete much less 3HCOT glucuronide than do individuals who carry two functional alleles (
      • Murphy S.E.
      • Park S.S.
      • Thompson E.F.
      • Wilkens L.R.
      • Patel Y.
      • Stram D.O.
      • Le Marchand L.
      Nicotine N-glucuronidation relative to N-oxidation and C-oxidation and UGT2B10 genotype in five ethnic/racial groups.
      ,
      • Chen G.
      • Giambrone N.E.
      • Lazarus P.
      Glucuronidation of trans-3'-hydroxycotinine by UGT2B17 and UGT2B10.
      ). The frequency of the UGT2B17 deletion is from 75 to 85% in Asian populations (
      • Xue Y.
      • Sun D.
      • Daly A.
      • Yang F.
      • Zhou X.
      • Zhao M.
      • Huang N.
      • Zerjal T.
      • Lee C.
      • Carter N.P.
      • Hurles M.E.
      • Tyler-Smith C.
      Adaptive evolution of UGT2B17 copy-number variation.
      ). Either UGT1A9 or UGT2B7 must contribute to the remaining glucuronidation of 3′-hydroxycotinine that occurs in these smokers.

      Nicotine metabolites as biomarkers of tobacco exposure

      Cigarettes are designed to efficiently deliver nicotine to the user. Therefore, nicotine itself or its metabolites are arguably the most reliable biomarkers for smoking exposure (
      • Benowitz N.L.
      • Hukkanen J.
      • Jacob III., P.
      Nicotine chemistry, metabolism, kinetics and biomarkers.
      ). Nicotine has a relatively short half-life (1–2 h). Whereas, cotinine has a half-life of 8 to 30 h (
      • Hukkanen J.
      • Jacob III, P.
      • Benowitz N.L.
      Metabolism and disposition kinetics of nicotine.
      ), and the presence of cotinine in either blood or urine is widely used as a biomarker of tobacco exposure (
      • Benowitz N.L.
      • Bernert J.T.
      • Foulds J.
      • Hecht S.S.
      • Jacob P.
      • Jarvis M.J.
      • Joseph A.
      • Oncken C.
      • Piper M.E.
      Biochemical verification of tobacco use and abstinence: 2019 update.
      ). Cotinine is the most abundant nicotine metabolite present in blood but typically accounts for <15% of the nicotine dose excreted in urine. The most commonly used urinary biomarker of tobacco exposure is total nicotine equivalents, which is the sum of the molar concentrations of nicotine and six metabolites.
      Both blood and urine cotinine concentrations are affected by individual differences in nicotine and cotinine metabolism. One smoker who metabolizes either compound more or less efficiently than another smoker will likely have different levels of cotinine for the same nicotine dose. This is particularly important in the study of racial/ethnic differences in tobacco-related disease risk, since the frequencies of UGT2B10 and CYP2A6 alleles vary by racial/ethnic group (Figs. 3A and 4A). In addition, it has been recognized for many years that African Americans, who are active smokers or who are exposed to secondhand tobacco smoke, have significantly higher cotinine concentrations than whites with similar exposure (
      • Wagenknecht L.E.
      • Cutter G.R.
      • Haley N.J.
      • Sidney S.
      • Manolio T.A.
      • Hughes G.H.
      • Jacobs D.R.
      Racial differences in serum cotinine levels among smokers in the Coronary Artery Risk Development in (Young) Adults study.
      ,
      • Wagenknecht L.E.
      • Manolio T.A.
      • Sidney S.
      • Burke G.L.
      • Haley N.J.
      Environmental tobacco smoke exposure as determined by cotinine in black and white young adults: The CARDIA study.
      ,
      • Perez-Stable E.J.
      • Herrera B.
      • Jacob III, P.
      • Benowitz N.L.
      Nicotine metabolism and intake in black and white smokers.
      ,
      • Benowitz N.L.
      • Bernert J.T.
      • Caraballo R.S.
      • Holiday D.B.
      • Wang J.
      Optimal serum cotinine levels for distinguishing cigarette smokers and nonsmokers within different racial/ethnic groups in the United States between 1999 and 2004.
      ).
      With the exception of smokers who carry two CYP2A6 deleted alleles, UGT2B10 variants and cotinine glucuronidation levels influence blood cotinine concentrations significantly more than does P450 2A6 activity (
      • Murphy S.E.
      • Sipe C.J.
      • Choi K.
      • Raddatz L.M.
      • Koopmeiners J.S.
      • Donny E.C.
      • Hatsukami D.K.
      Low cotinine glucuronidation results in higher serum and saliva cotinine in African American compared to White smokers.
      ,
      • Sipe C.J.
      • Koopmeiners J.S.
      • Donny E.C.
      • Hatsukami D.K.
      • Murphy S.E.
      UGT2B10 genotype influences serum cotinine levels and is a primary determinant of higher cotinine in African American smokers.
      ). At similar levels of smoking, individuals who are homozygous for the CYP2A6∗4 deletion allele have significantly lower plasma cotinine levels than do individuals who carry CYP2A6 alleles coding for fully functional enzymes (
      • Yamanaka H.
      • Nakajima M.
      • Nishimura K.
      • Yoshida R.
      • Fukami T.
      • Katoh M.
      • Yokoi T.
      Metabolic profile of nicotine in subjects whose CYP2A6 gene is deleted.
      ,
      • Nakajima M.
      • Yamagishi S.
      • Yamamoto H.
      • Yamamoto T.
      • Kuroiwa Y.
      • Yokoi T.
      Deficient cotinine formation from nicotine is attributed to the whole deletion of the CYP2A6 gene in humans.
      ). However, in smokers with reduced P450 2A6 activity, the relationship of enzyme activity to plasma cotinine levels is complicated since both the formation of cotinine and its further metabolism is mediated by P450 2A6. Cotinine clearance, not formation, appears to dominate this process since plasma cotinine concentrations tend to be higher in individuals with detectable but reduced P450 2A6 activity (
      • Zhu A.Z.
      • Renner C.C.
      • Hatsukami D.K.
      • Swan G.E.
      • Lerman C.
      • Benowitz N.L.
      • Tyndale R.F.
      The ability of plasma cotinine to predict nicotine and carcinogen exposure is altered by differences in CYP2A6: The influence of genetics, race, and sex.
      ). A significant amount of cotinine is metabolized by UGT2B10-catalyzed glucuronidation, and total cotinine clearance is 50% lower in individuals who are homozygous for the UGT2B10 splice variant compared with those who do not carry this allele (
      • Taghavi T.
      • St Helen G.
      • Benowitz N.L.
      • Tyndale R.F.
      Effect of UGT2B10, UGT2B17, FMO3, and OCT2 genetic variation on nicotine and cotinine pharmacokinetics and smoking in African Americans.
      ). The decreased clearance of cotinine in these smokers results in 45% higher serum cotinine levels (
      • Sipe C.J.
      • Koopmeiners J.S.
      • Donny E.C.
      • Hatsukami D.K.
      • Murphy S.E.
      UGT2B10 genotype influences serum cotinine levels and is a primary determinant of higher cotinine in African American smokers.
      ) (Fig. 5; genetic score 0 versus 2). Smokers who are heterozygous for either the UGT2B10 splice variant or the Asp67Tryr variant have on average 19% higher plasma cotinine concentrations. These differences are observed after adjustment for smoking dose (cigarettes per day and total nicotine equivalents) and P450 2A6 activity (
      • Sipe C.J.
      • Koopmeiners J.S.
      • Donny E.C.
      • Hatsukami D.K.
      • Murphy S.E.
      UGT2B10 genotype influences serum cotinine levels and is a primary determinant of higher cotinine in African American smokers.
      ).
      Figure thumbnail gr5
      Figure 5Determinants of serum cotinine. Values, adjusted for age, gender, and each of the other covariates, except UGT2B10 genetic score (0 versus 1), are the increase in the geometric mean cotinine concentration for the change in the covariant in a population of African American (n = 289) and non-Hispanic White smokers (n = 627). Genetic score 0, rs61750900 GG and rs2942857 AA; score 1, rs61750900 GT or rs2942857 CA; score 2, rs61750900 TT or rs2942857 CC, or both rs61750900 GT and rs2942857 CA. The data are from Table 3 and Table S2 in the study by Sipe et al. (
      • Sipe C.J.
      • Koopmeiners J.S.
      • Donny E.C.
      • Hatsukami D.K.
      • Murphy S.E.
      UGT2B10 genotype influences serum cotinine levels and is a primary determinant of higher cotinine in African American smokers.
      ).
      UGT2B10 genotype has a greater effect on plasma cotinine than does a significant increase in smoking does. An increase of five cigarettes per day or a total nicotine equivalent increase of 20 nmol/ml results in only a 9% change in mean plasma cotinine (Fig. 5). The UGT2B10 genotype effect is also greater than that observed for changes in P450 2A6 activity. A 0.1 change in the 3′-hydroxycotinine/cotinine ratio results in a 6% difference in plasma cotinine (Fig. 5). A 0.1 to 0.2 lower plasma ratio has been observed in smokers heterozygous for CYP2A6 variant alleles ∗9, ∗2, and ∗4 (
      • Benowitz N.L.
      • Swan G.E.
      • Jacob III, P.
      • Lessov-Schlaggar C.N.
      • Tyndale R.F.
      CYP2A6 genotype and the metabolism and disposition kinetics of nicotine.
      ,
      • Swan G.E.
      • Lessov-Schlaggar C.N.
      • Bergen A.W.
      • He Y.
      • Tyndale R.F.
      • Benowitz N.L.
      Genetic and environmental influences on the ratio of 3'hydroxycotinine to cotinine in plasma and urine.
      ,
      • El-Boraie A.
      • Taghavi T.
      • Chenoweth M.J.
      • Fukunaga K.
      • Mushiroda T.
      • Kubo M.
      • Lerman C.
      • Nollen N.L.
      • Benowitz N.L.
      • Tyndale R.F.
      Evaluation of a weighted genetic risk score for the prediction of biomarkers of CYP2A6 activity.
      ).
      Plasma cotinine concentrations are on average 50% higher in African Americans than in whites at the same levels of smoking, and much of this difference can be explained by the prevalence of UGT2B10 splice variant in African Americans (
      • Sipe C.J.
      • Koopmeiners J.S.
      • Donny E.C.
      • Hatsukami D.K.
      • Murphy S.E.
      UGT2B10 genotype influences serum cotinine levels and is a primary determinant of higher cotinine in African American smokers.
      ). Recognizing the contribution of UGT2B10 genotype to serum (or saliva) cotinine levels is critical to understanding the relationship of an individual's cotinine level to smoking exposures and ultimately the relationship to lung cancer risk. This is true both in smokers and secondhand smoke–exposed individuals, since those with no UGT2B10 activity would appear to have a greater exposure based on cotinine levels.
      UGT2B10 genotype also has a significant impact on urinary cotinine levels. UGT2B10-deficient smokers have significantly higher levels of urinary cotinine, whereas total cotinine levels, the sum of cotinine and cotinine glucuronide, are lower compared with smokers with “normal” UGT2B10 activity (
      • Murphy S.E.
      • Park S.S.
      • Thompson E.F.
      • Wilkens L.R.
      • Patel Y.
      • Stram D.O.
      • Le Marchand L.
      Nicotine N-glucuronidation relative to N-oxidation and C-oxidation and UGT2B10 genotype in five ethnic/racial groups.
      ,
      • Murphy S.E.
      • Sipe C.J.
      • Choi K.
      • Raddatz L.M.
      • Koopmeiners J.S.
      • Donny E.C.
      • Hatsukami D.K.
      Low cotinine glucuronidation results in higher serum and saliva cotinine in African American compared to White smokers.
      ,
      • Sipe C.J.
      • Koopmeiners J.S.
      • Donny E.C.
      • Hatsukami D.K.
      • Murphy S.E.
      UGT2B10 genotype influences serum cotinine levels and is a primary determinant of higher cotinine in African American smokers.
      ). Despite some limitations of cotinine as a biomarker of tobacco exposure, several studies have established plasma cotinine and urinary total cotinine levels as predictors of lung cancer risk after adjustment for cigarettes per day (
      • Boffetta P.
      • Clark S.
      • Shen M.
      • Gislefoss R.
      • Peto R.
      • Andersen A.
      Serum cotinine level as predictor of lung cancer risk.
      ,
      • Thomas C.E.
      • Wang R.
      • Adams-Haduch J.
      • Murphy S.E.
      • Ueland P.M.
      • Midttun O.
      • Brennan P.
      • Johansson M.
      • Gao Y.T.
      • Yuan J.M.
      Urinary cotinine is as good a biomarker as serum cotinine for cigarette smoking exposure and lung cancer risk prediction.
      ,
      • Timofeeva M.N.
      • McKay J.D.
      • Smith G.D.
      • Johansson M.
      • Byrnes G.B.
      • Chabrier A.
      • Relton C.
      • Ueland P.M.
      • Vollset S.E.
      • Midttun O.
      • Nygard O.
      • Slimani N.
      • Romieu I.
      • Clavel-Chapelon F.
      • Boutron-Ruault M.C.
      • et al.
      Genetic polymorphisms in 15q25 and 19q13 loci, cotinine levels, and risk of lung cancer in EPIC.
      ,
      • Yuan J.M.
      • Koh W.P.
      • Murphy S.E.
      • Fan Y.
      • Wang R.
      • Carmella S.G.
      • Han S.
      • Wickham K.
      • Gao Y.T.
      • Yu M.C.
      • Hecht S.S.
      Urinary levels of tobacco-specific nitrosamine metabolites in relation to lung cancer development in two prospective cohorts of cigarette smokers.
      ,
      • Yuan J.M.
      • Gao Y.T.
      • Murphy S.E.
      • Carmella S.G.
      • Wang R.
      • Zhong Y.
      • Moy K.A.
      • Davis A.B.
      • Tao L.
      • Chen M.
      • Han S.
      • Nelson H.H.
      • Yu M.C.
      • Hecht S.S.
      Urinary levels of cigarette smoke constituent metabolites are prospectively associated with lung cancer development in smokers.
      ,
      • Yuan J.M.
      • Gao Y.T.
      • Wang R.
      • Chen M.
      • Carmella S.G.
      • Hecht S.S.
      Urinary levels of volatile organic carcinogen and toxicant biomarkers in relation to lung cancer development in smokers.
      ,
      • Larose T.L.
      • Guida F.
      • Fanidi A.
      • Langhammer A.
      • Kveem K.
      • Stevens V.L.
      • Jacobs E.J.
      • Smith-Warner S.A.
      • Giovannucci E.
      • Albanes D.
      • Weinstein S.J.
      • Freedman N.D.
      • Prentice R.
      • Pettinger M.
      • Thomson C.A.
      • et al.
      Circulating cotinine concentrations and lung cancer risk in the Lung Cancer Cohort Consortium (LC3).
      ). However, these studies were carried out in populations exclusively or predominately of similar racial/ethnic ancestry, whites, or Chinese.
      Total nicotine equivalents levels are a better biomarker of recent nicotine intake and tobacco exposure than is cotinine since the concentration is not influenced by nicotine metabolism (
      • Benowitz N.L.
      • Bernert J.T.
      • Foulds J.
      • Hecht S.S.
      • Jacob P.
      • Jarvis M.J.
      • Joseph A.
      • Oncken C.
      • Piper M.E.
      Biochemical verification of tobacco use and abstinence: 2019 update.
      ,
      • Scherer G.
      • Engl J.
      • Urban M.
      • Gilch G.
      • Janket D.
      • Riedel K.
      Relationship between machine-derived smoke yields and biomarkers in cigarette smokers in Germany.
      ,
      • Benowitz N.L.
      • St Helen G.
      • Nardone N.
      • Cox L.S.
      • Jacob P.
      Urine metabolites for estimating daily intake of nicotine from cigarette smoking.
      ). The benefit of total nicotine equivalents is that it is composed of the products of all the major nicotine metabolism pathways, nicotine N-oxide, nicotine N-glucuronide, cotinine, cotinine N-glucuronide, 3′-hydroxycotinine, and 3′-hydroxycotinine glucuronide (Fig. 1). The sum of these six compounds plus nicotine account for 85 to 90% of a smoker's nicotine dose (
      • Benowitz N.L.
      • Jacob III, P.
      • Fong I.
      • Gupta S.
      Nicotine metabolic profile in man: Comparison of cigarette smoking and transdermal nicotine.
      ,
      • Hukkanen J.
      • Jacob III, P.
      • Benowitz N.L.
      Metabolism and disposition kinetics of nicotine.
      ). Nicotine N-oxide is not always included in the measurement of total nicotine equivalents, and other more minor metabolites such as nornicotine, norcotinine, and cotinine N-oxide may be added (
      • Benowitz N.L.
      • St Helen G.
      • Nardone N.
      • Cox L.S.
      • Jacob P.
      Urine metabolites for estimating daily intake of nicotine from cigarette smoking.
      ,
      • Wang L.
      • Bernert J.T.
      • Benowitz N.L.
      • Feng J.
      • Jacob 3rd, P.
      • McGahee E.
      • Caudill S.P.
      • Scherer G.
      • Scherer M.
      • Pluym N.
      • Doig M.V.
      • Newland K.
      • Murphy S.E.
      • Caron N.J.
      • Sander L.C.
      • et al.
      Collaborative method performance study of the measurement of nicotine, its metabolites, and total nicotine equivalents in human urine.
      ). Hydroxy acid, which makes up a significant proportion of the excreted nicotine dose (Fig. 1), is not currently included in total nicotine equivalents. The influence of variation in nicotine metabolism on the relative amount of hydroxy acid excreted by a smoker is unknown. As discussed previously, the most likely source of hydroxy acid in urine is as the product of nicotine 2′-oxidation. In vitro, this reaction is catalyzed by P450 2A6, albeit to a much lesser extent than 5′-oxidation. Other P450 enzymes, possibly P450 3A4, which catalyzes the 2′ hydroxylation of the structurally similar compound N′-nitrosonornicotine (
      • Patten C.J.
      • Smith T.J.
      • Tynes R.
      • Friesen M.
      • Lee J.
      • Yang C.S.
      • Murphy S.E.
      Evidence for cytochrome P450 2A6 and 3A4 as major catalysts for N'-nitrosonornicotine a-hydroxylation in human liver microsomes.
      ), may catalyze nicotine 2′-oxidation. It would be interesting to determine if the amount of hydroxy acid excretion is influenced by CYP2A6 genotype.
      Total nicotine equivalents and cotinine are not reliable biomarkers of tobacco exposure in individuals who use other nicotine-containing products, such as electronic (e) cigarettes and nicotine replacement therapies that are often used as a means to reduce tobacco use. Two biomarkers that can distinguish cigarette smoking from other sources of nicotine are NNAL, a metabolite of the tobacco-specific lung carcinogen 4-(methylnitrosamino-1-(3-pyridyl)-butanone) (NNK), and cyanoethyl mercapturic acid, a metabolite of acrylonitrile (
      • Hecht S.S.
      Progress and challenges in selected areas of tobacco carcinogenesis.
      ,
      • Luo X.
      • Carmella S.G.
      • Chen M.
      • Jensen J.A.
      • Wilkens L.R.
      • Le Marchand L.
      • Hatsukami D.K.
      • Murphy S.E.
      • Hecht S.S.
      Urinary cyanoethyl mercapturic acid, a biomarker of the smoke toxicant acrylonitrile, clearly distinguishes smokers from nonsmokers.
      ). Acrylonitrile is a volatile toxicant found in substantial quantities in cigarette smoke. There is little human exposure to acrylonitrile, other than from tobacco, and cyanoethyl mercapturic acid has recently been validated as an excellent biomarker to distinguish e-cigarette use from smoking (
      • Luo X.
      • Carmella S.G.
      • Chen M.
      • Jensen J.A.
      • Wilkens L.R.
      • Le Marchand L.
      • Hatsukami D.K.
      • Murphy S.E.
      • Hecht S.S.
      Urinary cyanoethyl mercapturic acid, a biomarker of the smoke toxicant acrylonitrile, clearly distinguishes smokers from nonsmokers.
      ).

      Nicotine metabolism, smoking behavior, and CYP2A6 variants

      A smoker's rate of nicotine clearance contributes significantly to nicotine intake and in turn their smoking behavior; that is the number of cigarettes they smoke and how they smoke them (
      • Benowitz N.L.
      • Hukkanen J.
      • Jacob III., P.
      Nicotine chemistry, metabolism, kinetics and biomarkers.
      ). The relationship of nicotine clearance to nicotine intake was first quantitatively documented in a pharmacokinetic study of Chinese American smokers (
      • Benowitz N.L.
      • Perez-Stable E.J.
      • Herrera B.
      • Jacob III, P.
      Slower metabolism and reduced intake of nicotine from cigarette smoking in Chinese-Americans.
      ), and a few years later, the direct relationship of nicotine clearance to CYP2A6 genotype was confirmed (
      • Benowitz N.L.
      • Swan G.E.
      • Jacob III, P.
      • Lessov-Schlaggar C.N.
      • Tyndale R.F.
      CYP2A6 genotype and the metabolism and disposition kinetics of nicotine.
      ).
      There are numerous CYP2A6 variants, many of which significantly impact nicotine metabolism (
      • Tanner J.A.
      • Chenoweth M.J.
      • Tyndale R.F.
      Pharmacogenetics of nicotine and associated smoking behaviors.
      ,
      • Tanner J.A.
      • Tyndale R.F.
      Variation in CYP2A6 activity and personalized medicine.
      ,
      • Zhou Y.
      • Ingelman-Sundberg M.
      • Lauschke V.M.
      Worldwide distribution of cytochrome P450 alleles: A meta-analysis of population-scale sequencing projects.
      ). The Human Cytochrome 450 Nomenclature database lists more than 75 different CYP2A6 alleles (https://www.pharmvar.org/htdocs/archive/cyp2a6.htm). Variants with a minor allele frequency of >1% in any of the five major populations are listed in Table 1. These include single nucleotide changes, small deletions and insertions, gene deletions and duplications, and gene hybrids (
      • Wassenaar C.A.
      • Zhou Q.
      • Tyndale R.F.
      CYP2A6 genotyping methods and strategies using real-time and end point PCR platforms.
      ). Importantly, the prevalence of the individual alleles varies by racial/ethnic group. The deletion allele, CYP2A6∗4, is prevalent in East Asian populations, with an allele frequency of up to 20%. Other variants are found exclusively in one population, for example, CYP2A∗17 (allele frequency 11%) is found only in individuals of African ancestry and CYP2A6∗7 (allele frequency 13%) only in East Asians (
      • Park S.L.
      • Tiirikainen M.I.
      • Patel Y.M.
      • Wilkens L.R.
      • Stram D.O.
      • Le Marchand L.
      • Murphy S.E.
      Genetic determinants of CYP2A6 activity across racial/ethnic groups with different risks of lung cancer and effect on their smoking intensity.
      ,
      • Zhou Y.
      • Ingelman-Sundberg M.
      • Lauschke V.M.
      Worldwide distribution of cytochrome P450 alleles: A meta-analysis of population-scale sequencing projects.
      ).
      The effects of the 11 amino acid substitutions present in CYP2A6 variants (Table 1) on enzyme activity are not all well characterized. The L160H change in CYP2A6∗2 results in no functional enzyme since this variant does not incorporate heme (
      • Yamano S.
      • Tatsuno J.
      • Gonzalez F.J.
      The CYP2A3 gene product catalyzes coumarin 7-hydroxylation in human liver microsomes.
      ). Several of the other amino acid substitutions affect the kinetic parameters of nicotine 5′-oxidation. Among these, the most significant impact is for I471T (CYP2A6∗7) and V365M (CYP2A6∗17), which have decreased intrinsic clearance of 83% and 69%, respectively (
      • Yamamiya I.
      • Yoshisue K.
      • Ishii Y.
      • Yamada H.
      • Chiba M.
      Effect of CYP2A6 genetic polymorphism on the metabolic conversion of tegafur to 5-fluorouracil and its enantioselectivity.
      ,
      • Han S.
      • Choi S.
      • Chun Y.J.
      • Yun C.H.
      • Lee C.H.
      • Shin H.J.
      • Na H.S.
      • Chung M.W.
      • Kim D.
      Functional characterization of allelic variants of polymorphic human cytochrome P450 2A6 (CYP2A6∗5, ∗7, ∗8, ∗18, ∗19, and ∗35).
      ,
      • Fukami T.
      • Nakajima M.
      • Yoshida R.
      • Tsuchiya Y.
      • Fujiki Y.
      • Katoh M.
      • McLeod H.L.
      • Yokoi T.
      A novel polymorphism of human CYP2A6 gene CYP2A6∗17 has an amino acid substitution (V365M) that decreases enzymatic activity in vitro and in vivo.
      ,
      • Al Koudsi N.
      • Ahluwalia J.S.
      • Lin S.K.
      • Sellers E.M.
      • Tyndale R.F.
      A novel CYP2A6 allele (CYP2A6∗35) resulting in an amino-acid substitution (Asn438Tyr) is associated with lower CYP2A6 activity in vivo.
      ). Notably, the closely related P450 2A13 has a methionine residue at position 365, and this change contributes to the larger active site of P450 2A13 compared with P450 2A6 and influences the binding of nicotine (
      • DeVore N.M.
      • Scott E.E.
      Nicotine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone binding and access channel in human cytochrome P450 2A6 and 2A13 enzymes.
      ). The R203C substitution in CYP2A6∗23 was reported to have similar activity to CYP2A6∗17; however, no kinetic parameters have been reported (
      • Ho M.K.
      • Mwenifumbo J.C.
      • Zhao B.
      • Gillam E.M.
      • Tyndale R.F.
      A novel CYP2A6 allele, CYP2A6∗23, impairs enzyme function in vitro and in vivo and decreases smoking in a population of Black-African descent.
      ). The arginine at position 203 is present in a substrate recognition region of P450 2A6 (
      • Tiong K.H.
      • Yiap B.C.
      • Tan E.L.
      • Ismail R.
      • Ong C.E.
      Functional characterization of cytochrome P450 2A6 allelic variants CYP2A6∗15, CYP2A6∗16, CYP2A6∗21, and CYP2A6∗22.
      ). The amino acid changes in CYP2A6∗35 (N438Y) and CYP2A6∗18 (Y392F) have not been shown to significantly affect kcat/Km values for nicotine 5′-oxidation, although modest changes in both Km and kcat have been reported for each (
      • Han S.
      • Choi S.
      • Chun Y.J.
      • Yun C.H.
      • Lee C.H.
      • Shin H.J.
      • Na H.S.
      • Chung M.W.
      • Kim D.
      Functional characterization of allelic variants of polymorphic human cytochrome P450 2A6 (CYP2A6∗5, ∗7, ∗8, ∗18, ∗19, and ∗35).
      ,
      • Al Koudsi N.
      • Ahluwalia J.S.
      • Lin S.K.
      • Sellers E.M.
      • Tyndale R.F.
      A novel CYP2A6 allele (CYP2A6∗35) resulting in an amino-acid substitution (Asn438Tyr) is associated with lower CYP2A6 activity in vivo.
      ,
      • Fukami T.
      • Nakajima M.
      • Higashi E.
      • Yamanaka H.
      • Sakai H.
      • McLeod H.L.
      • Yokoi T.
      Characterization of novel CYP2A6 polymorphic alleles (CYP2A6∗18 and CYP2A6∗19) that affect enzymatic activity.
      ). No change in the kinetic parameters for nicotine 5′-oxidation were reported for CYP2A6∗21 (K476R) (
      • Tiong K.H.
      • Yiap B.C.
      • Tan E.L.
      • Ismail R.
      • Ong C.E.
      Functional characterization of cytochrome P450 2A6 allelic variants CYP2A6∗15, CYP2A6∗16, CYP2A6∗21, and CYP2A6∗22.
      ). The rate of nicotine 5′-oxidation catalyzed by either CYP2A6∗28 and CYP2A6∗25 at 30 or 300 μM nicotine was no different than that of P450 2A6 (
      • Mwenifumbo J.C.
      • Al Koudsi N.
      • Ho M.K.
      • Zhou Q.
      • Hoffmann E.B.
      • Sellers E.M.
      • Tyndale R.F.
      Novel and established CYP2A6 alleles impair in vivo nicotine metabolism in a population of Black African descent.
      ). The kinetic parameters for these two variants have not been reported. The phenylalanine at position 118 is located in the highly conserved substrate recognition site-1 region of the CYP2A family, and mutations at this position, including F118L, affect the substrate specificity of flavonoids (
      • Tiong K.H.
      • Yiap B.C.
      • Tan E.L.
      • Ismail R.
      • Ong C.E.
      Functional characterization of cytochrome P450 2A6 allelic variants CYP2A6∗15, CYP2A6∗16, CYP2A6∗21, and CYP2A6∗22.
      ,
      • Uno T.
      • Obe Y.
      • Ogura C.
      • Goto T.
      • Yamamoto K.
      • Nakamura M.
      • Kanamaru K.
      • Yamagata H.
      • Imaishi H.
      Metabolism of 7-ethoxycoumarin, safrole, flavanone and hydroxyflavanone by cytochrome P450 2A6 variants.
      ,
      • Uno T.
      • Ogura C.
      • Izumi C.
      • Nakamura M.
      • Yanase T.
      • Yamazaki H.
      • Ashida H.
      • Kanamaru K.
      • Yamagata H.
      • Imaishi H.
      Point mutation of cytochrome P450 2A6 (a polymorphic variant CYP2A6.25) confers new substrate specificity towards flavonoids.
      ). Therefore, in most cases, in vitro nicotine metabolism by variant P450 2A6 enzymes parallels what has been observed in vivo (Table 1). However, it is important to recognize that an individual may carry multiple single nucleotide polymorphisms in CYP2A6, and each may differentially affect the catalytic activity as well as the expression of the enzyme.
      Several studies have observed a relationship between cigarettes per day and CYP2A6 genotype (
      • Ariyoshi N.
      • Miyamoto M.
      • Umetsu Y.
      • Kunitoh H.
      • Dosaka-Akita H.
      • Sawamura Y.
      • Yokota J.
      • Nemoto N.
      • Sato K.
      • Kamataki T.
      Genetic polymorphism of CYP2A6 gene and tobacco-induced lung cancer risk in male smokers.
      ,
      • Fujieda M.
      • Yamazaki H.
      • Saito T.
      • Kiyotani K.
      • Gyamfi M.A.
      • Sakurai M.
      • Dosaka-Akita H.
      • Sawamura Y.
      • Yokota J.
      • Kunitoh H.
      • Kamataki T.
      Evaluation of CYP2A6 genetic polymorphisms as determinants of smoking behavior and tobacco-related lung cancer risk in male Japanese smokers.
      ,
      • Pan L.
      • Yang X.
      • Li S.
      • Jia C.
      Association of CYP2A6 gene polymorphisms with cigarette consumption: A meta-analysis.
      ,
      • Wassenaar C.A.
      • Dong Q.
      • Wei Q.
      • Amos C.I.
      • Spitz M.R.
      • Tyndale R.F.
      Relationship between CYP2A6 and CHRNA5-CHRNA3-CHRNB4 variation and smoking behaviors and lung cancer risk.
      ). Many of these are in East Asian populations, which have a greater than 60% prevalence of functionally deficient CYP2A6 alleles (Table 1) (
      • Zhou Y.
      • Ingelman-Sundberg M.
      • Lauschke V.M.
      Worldwide distribution of cytochrome P450 alleles: A meta-analysis of population-scale sequencing projects.
      ). CYP2A6 genotype has also been shown to influence smoking intensity (mean and total puff volume) (
      • Strasser A.A.
      • Malaiyandi V.
      • Hoffmann E.
      • Tyndale R.F.
      • Lerman C.
      An association of CYP2A6 genotype and smoking topography.
      ). Total nicotine equivalent levels are related to CYP2A6 genotype (
      • Park S.L.
      • Tiirikainen M.I.
      • Patel Y.M.
      • Wilkens L.R.
      • Stram D.O.
      • Le Marchand L.
      • Murphy S.E.
      Genetic determinants of CYP2A6 activity across racial/ethnic groups with different risks of lung cancer and effect on their smoking intensity.
      ,
      • Yuan J.M.
      • Nelson H.H.
      • Butler L.M.
      • Carmella S.G.
      • Wang R.
      • Kuriger-Laber J.K.
      • Adams-Haduch J.
      • Hecht S.S.
      • Gao Y.T.
      • Murphy S.E.
      Genetic determinants of cytochrome P450 2A6 activity and biomarkers of tobacco smoke exposure in relation to risk of lung cancer development in the Shanghai Cohort study.
      ). Both total nicotine equivalents and cigarettes per day are influenced by a smoker's P450 2A6 activity and therefore by their ability to metabolize nicotine (
      • Park S.L.
      • Tiirikainen M.I.
      • Patel Y.M.
      • Wilkens L.R.
      • Stram D.O.
      • Le Marchand L.
      • Murphy S.E.
      Genetic determinants of CYP2A6 activity across racial/ethnic groups with different risks of lung cancer and effect on their smoking intensity.
      ,
      • Chenoweth M.J.
      • Tyndale R.F.
      Pharmacogenetic optimization of smoking cessation treatment.
      ). Several investigators are working to develop genetic risk models to predict the nicotine metabolism (
      • Buchwald J.
      • Chenoweth M.J.
      • Palviainen T.
      • Zhu G.
      • Benner C.
      • Gordon S.
      • Korhonen T.
      • Ripatti S.
      • Madden P.A.F.
      • Lehtimaki T.
      • Raitakari O.T.
      • Salomaa V.
      • Rose R.J.
      • George T.P.
      • Lerman C.
      • et al.
      Genome-wide association meta-analysis of nicotine metabolism and cigarette consumption measures in smokers of European descent.
      ,
      • Loukola A.
      • Buchwald J.
      • Gupta R.
      • Palviainen T.
      • Hallfors J.
      • Tikkanen E.
      • Korhonen T.
      • Ollikainen M.
      • Sarin A.P.
      • Ripatti S.
      • Lehtimaki T.
      • Raitakari O.
      • Salomaa V.
      • Rose R.J.
      • Tyndale R.F.
      • et al.
      A genome-wide association study of a biomarker of nicotine metabolism.
      ). Ideally, these models could provide a better reflection of lifetime daily smoking than self-reported cigarettes per day or a single total nicotine equivalent measurement.

      Relationship of CYP2A6 and nicotine metabolism to lung cancer

      As discussed, variations in P450 2A6–catalyzed nicotine metabolism influence smoking intensity. P450 2A6 is also one of several catalysts of the bioactivation of the tobacco-specific lung carcinogen, NNK (
      • Dicke K.
      • Skrlin S.
      • Murphy S.E.
      Nicotine and 4-(methylnitrosamino)-1-(3-pyridyl)-butanone (NNK) metabolism by P450 2B6.
      ,
      • Jalas J.R.
      • Hecht S.S.
      • Murphy S.E.
      Cytochrome P450 enzymes as catalysts of metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a tobacco-specific carcinogen.
      ,
      • Wong H.,L.
      • Zhang X.
      • Zhang Q.Y.
      • Gu J.
      • Ding X.
      • Hecht S.S.
      • Murphy S.E.
      Metabolic activation of the tobacco carcinogen 4-(methylnitrosamino)-(3-pyridyl)-1-butanone by cytochrome p450 2A13 in human fetal nasal microsomes.
      ). Many studies, including the first to report an association between CYP2A6 genotype and lung cancer (
      • Miyamoto M.
      • Umetsu Y.
      • Dosaka-Akita H.
      • Sawamura Y.
      • Yokota J.
      • Kunitoh H.
      • Nemoto N.
      • Sato K.
      • Ariyoshi N.
      • Kamataki T.
      CYP2A6 gene deletion reduces susceptibility to lung cancer.
      ), hypothesized that decreased activation of NNK in smokers with decreased P450 2A6 activity protects smokers from lung cancer risk. While this may be one mechanism by which CYP2A6 genotype is associated with lung cancer risk, the direct relationship of NNK bioactivation to lung cancer risk has not been demonstrated. In contrast, much evidence has established a strong connection from CYP2A6 genotype to nicotine metabolism to smoking dose and lung cancer risk (
      • Murphy S.E.
      Nicotine metabolism and smoking: Ethnic differences in the role of P450 2A6.
      ,
      • Yuan J.M.
      • Nelson H.H.
      • Butler L.M.
      • Carmella S.G.
      • Wang R.
      • Kuriger-Laber J.K.
      • Adams-Haduch J.
      • Hecht S.S.
      • Gao Y.T.
      • Murphy S.E.
      Genetic determinants of cytochrome P450 2A6 activity and biomarkers of tobacco smoke exposure in relation to risk of lung cancer development in the Shanghai Cohort study.
      ,
      • Park S.L.
      • Murphy S.E.
      • Wilkens L.R.
      • Stram D.O.
      • Hecht S.S.
      • Le Marchand L.
      Association of CYP2A6 activity with lung cancer incidence in smokers: The Multiethnic Cohort study.
      ). The significantly lower dose of carcinogens received by smokers who carry variant CYP2A6 alleles (
      • Park S.L.
      • Tiirikainen M.I.
      • Patel Y.M.
      • Wilkens L.R.
      • Stram D.O.
      • Le Marchand L.
      • Murphy S.E.
      Genetic determinants of CYP2A6 activity across racial/ethnic groups with different risks of lung cancer and effect on their smoking intensity.
      ), not decreased bioactivation of NNK, is likely the far greater contributor to their lower lung cancer risk.
      A number of epidemiology studies have confirmed the protective effect of the CYP2A6∗4 deletion allele on a smoker's lung cancer risk (
      • Liu T.
      • Xie C.B.
      • Ma W.J.
      • Chen W.Q.
      Association between CYP2A6 genetic polymorphisms and lung cancer: A meta-analysis of case-control studies.
      ,
      • Ariyoshi N.
      • Miyamoto M.
      • Umetsu Y.
      • Kunitoh H.
      • Dosaka-Akita H.
      • Sawamura Y.
      • Yokota J.
      • Nemoto N.
      • Sato K.
      • Kamataki T.
      Genetic polymorphism of CYP2A6 gene and tobacco-induced lung cancer risk in male smokers.
      ,
      • Fujieda M.
      • Yamazaki H.
      • Saito T.
      • Kiyotani K.
      • Gyamfi M.A.
      • Sakurai M.
      • Dosaka-Akita H.
      • Sawamura Y.
      • Yokota J.
      • Kunitoh H.
      • Kamataki T.
      Evaluation of CYP2A6 genetic polymorphisms as determinants of smoking behavior and tobacco-related lung cancer risk in male Japanese smokers.
      ,
      • Miyamoto M.
      • Umetsu Y.
      • Dosaka-Akita H.
      • Sawamura Y.
      • Yokota J.
      • Kunitoh H.
      • Nemoto N.
      • Sato K.
      • Ariyoshi N.
      • Kamataki T.
      CYP2A6 gene deletion reduces susceptibility to lung cancer.
      ,
      • Hosono H.
      • Kumondai M.
      • Arai T.
      • Sugimura H.
      • Sasaki T.
      • Hirasawa N.
      • Hiratsuka M.
      CYP2A6 genetic polymorphism is associated with decreased susceptibility to squamous cell lung cancer in Japanese smokers.
      ,
      • Liu Y.L.
      • Xu Y.
      • Li F.
      • Chen H.
      • Guo S.L.
      CYP2A6 deletion polymorphism is associated with decreased susceptibility of lung cancer in Asian smokers: A meta-analysis.
      ,
      • Johani F.H.
      • Majid M.S.A.
      • Azme M.H.
      • Nawi A.M.
      Cytochrome P450 2A6 whole-gene deletion (CYP2A6∗4) polymorphism reduces risk of lung cancer: A meta-analysis.
      ). A meta-analysis of nine case control studies reported a more than 60% reduction in lung cancer incidence among subjects with no functional P450 2A6 (genotype CYP2A6∗4/∗4 or CYP2A∗2/∗2) compared with those who carry neither of these alleles (
      • Ariyoshi N.
      • Miyamoto M.
      • Umetsu Y.
      • Kunitoh H.
      • Dosaka-Akita H.
      • Sawamura Y.
      • Yokota J.
      • Nemoto N.
      • Sato K.
      • Kamataki T.
      Genetic polymorphism of CYP2A6 gene and tobacco-induced lung cancer risk in male smokers.
      ,
      • Fujieda M.
      • Yamazaki H.
      • Saito T.
      • Kiyotani K.
      • Gyamfi M.A.
      • Sakurai M.
      • Dosaka-Akita H.
      • Sawamura Y.
      • Yokota J.
      • Kunitoh H.
      • Kamataki T.
      Evaluation of CYP2A6 genetic polymorphisms as determinants of smoking behavior and tobacco-related lung cancer risk in male Japanese smokers.
      ,
      • Miyamoto M.
      • Umetsu Y.
      • Dosaka-Akita H.
      • Sawamura Y.
      • Yokota J.
      • Kunitoh H.
      • Nemoto N.
      • Sato K.
      • Ariyoshi N.
      • Kamataki T.
      CYP2A6 gene deletion reduces susceptibility to lung cancer.
      ,
      • Hosono H.
      • Kumondai M.
      • Arai T.
      • Sugimura H.
      • Sasaki T.
      • Hirasawa N.
      • Hiratsuka M.
      CYP2A6 genetic polymorphism is associated with decreased susceptibility to squamous cell lung cancer in Japanese smokers.
      ,
      • Tan W.
      • Chen G.F.
      • Xing D.Y.
      • Song C.Y.
      • Kadlubar F.F.
      • Lin D.X.
      Frequency of CYP2A6 gene deletion and its relation to risk of lung and esophageal cancer in the Chinese population.
      ,
      • Kamataki T.
      • Nunoya K.
      • Sakai Y.
      • Kushida H.
      • Fujita K.
      Genetic polymorphism of CYP2A6 in relation to cancer.
      ,
      • Rotunno M.
      • Yu K.
      • Lubin J.H.
      • Consonni D.
      • Pesatori A.C.
      • Goldstein A.M.
      • Goldin L.R.
      • Wacholder S.
      • Welch R.
      • Burdette L.
      • Chanock S.J.
      • Bertazzi P.A.
      • Tucker M.A.
      • Caporaso N.E.
      • Chatterjee N.
      • et al.
      Phase I metabolic genes and risk of lung cancer: Multiple polymorphisms and mRNA expression.
      ,
      • Tamaki Y.
      • Arai T.
      • Sugimura H.
      • Sasaki T.
      • Honda M.
      • Muroi Y.
      • Matsubara Y.
      • Kanno S.
      • Ishikawa M.
      • Hirasawa N.
      • Hiratsuka M.
      Association between cancer risk and drug-metabolizing enzyme gene (CYP2A6, CYP2A13, CYP4B1, SULT1A1, GSTM1, and GSTT1) polymorphisms in cases of lung cancer in Japan.
      ,
      • Islam M.S.
      • Ahmed M.U.
      • Sayeed M.S.
      • Maruf A.A.
      • Mostofa A.G.
      • Hussain S.M.
      • Kabir Y.
      • Daly A.K.
      • Hasnat A.
      Lung cancer risk in relation to nicotinic acetylcholine receptor, CYP2A6 and CYP1A1 genotypes in the Bangladeshi population.
      ). The lung cancer cases in most of these studies included a mixture of smokers and never smokers (typically defined as smoking <100 per lifetime) and, as would be expected, the significance of the protective effect was attenuated if it was not stratified by smoking status (
      • Liu Y.L.
      • Xu Y.
      • Li F.
      • Chen H.
      • Guo S.L.
      CYP2A6 deletion polymorphism is associated with decreased susceptibility of lung cancer in Asian smokers: A meta-analysis.
      ,
      • Johani F.H.
      • Majid M.S.A.
      • Azme M.H.
      • Nawi A.M.
      Cytochrome P450 2A6 whole-gene deletion (CYP2A6∗4) polymorphism reduces risk of lung cancer: A meta-analysis.
      ). A few studies have found no relationship between CYP2A6 genotype and lung cancer risk (
      • Tan W.
      • Chen G.F.
      • Xing D.Y.
      • Song C.Y.
      • Kadlubar F.F.
      • Lin D.X.
      Frequency of CYP2A6 gene deletion and its relation to risk of lung and esophageal cancer in the Chinese population.
      ,
      • Loriot M.A.
      • Rebuissou S.
      • Oscarson M.
      • Cenee S.
      • Miyamoto M.
      • Ariyoshi N.
      • Kamataki T.
      • Hemon D.
      • Beaune P.
      • Stucker I.
      Genetic polymorphisms of cytochrome P450 2A6 in a case-control study on lung cancer in a French population.
      ,
      • Wang H.
      • Tan W.
      • Hao B.
      • Miao X.
      • Zhou G.
      • He F.
      • Lin D.
      Substantial reduction in risk of lung adenocarcinoma associated with genetic polymorphism in CYP2A13, the most active cytochrome P450 for the metabolic activation of tobacco-specific carcinogen NNK.
      ,
      • Ezzeldin N.
      • El-Lebedy D.
      • Darwish A.
      • El Bastawisy A.
      • Abd Elaziz S.H.
      • Hassan M.M.
      • Saad-Hussein A.
      Association of genetic polymorphisms CYP2A6∗2 rs1801272 and CYP2A6∗9 rs28399433 with tobacco-induced lung cancer: Case-control study in an Egyptian population.
      ). However, the majority, if not all, of these negative studies are due to the inclusion of never smokers or to an insufficient number of cases. The latter is a significant challenge, particularly in populations with relatively low frequencies of CYP2A6 variant alleles. CYP2A6 alleles that code for little or no active protein are relatively common in individuals of East Asian ancestry but are rare in whites (Table 1 and Fig. 3A).
      A large early study in Japanese reported that subjects who carried one or more CYP2A6 variant alleles (∗4, ∗7, ∗9, or ∗10) smoked significantly fewer cigarettes per day than those who carried none of these alleles (
      • Fujieda M.
      • Yamazaki H.
      • Saito T.
      • Kiyotani K.
      • Gyamfi M.A.
      • Sakurai M.
      • Dosaka-Akita H.
      • Sawamura Y.
      • Yokota J.
      • Kunitoh H.
      • Kamataki T.
      Evaluation of CYP2A6 genetic polymorphisms as determinants of smoking behavior and tobacco-related lung cancer risk in male Japanese smokers.
      ). However, even after adjustment for cigarette consumption, the risk for lung cancer was significantly lower for smokers who carried the variant alleles. The authors suggested, as others have, that the additional effect of CYP2A6 genotype on lung cancer risk was due to a reduction in P450 2A6–mediated bioactivation of NNK. A more likely explanation is that cigarettes per day is a poor measure of smoking dose, and that a smoker's CYP2A6 genotype is “correcting” for differences in nicotine and carcinogen consumption per cigarette. That is, smokers who carry variant CYP2A6 alleles may smoke each cigarette less intensely than other smokers, inhaling less often or less deeply. More recent studies that use nicotine and carcinogen biomarkers to measure smoking dose support this hypothesis (
      • Yuan J.M.
      • Nelson H.H.
      • Butler L.M.
      • Carmella S.G.
      • Wang R.
      • Kuriger-Laber J.K.
      • Adams-Haduch J.
      • Hecht S.S.
      • Gao Y.T.
      • Murphy S.E.
      Genetic determinants of cytochrome P450 2A6 activity and biomarkers of tobacco smoke exposure in relation to risk of lung cancer development in the Shanghai Cohort study.
      ,
      • Park S.L.
      • Murphy S.E.
      • Wilkens L.R.
      • Stram D.O.
      • Hecht S.S.
      • Le Marchand L.
      Association of CYP2A6 activity with lung cancer incidence in smokers: The Multiethnic Cohort study.
      ,
      • Yuan J.M.
      • Nelson H.H.
      • Carmella S.G.
      • Wang R.
      • Kuriger-Laber J.
      • Jin A.
      • Adams-Haduch J.
      • Hecht S.S.
      • Koh W.P.
      • Murphy S.E.
      CYP2A6 genetic polymorphisms and biomarkers of tobacco smoke constituents in relation to risk of lung cancer in the Singapore Chinese Health study.
      ,
      • Patel Y.M.
      • Park S.L.
      • Han Y.
      • Wilkens L.R.
      • Bickeboller H.
      • Rosenberger A.
      • Caporaso N.
      • Landi M.T.
      • Bruske I.
      • Risch A.
      • Wei Y.
      • Christiani D.C.
      • Brennan P.
      • Houlston R.S.
      • McKay J.
      • et al.
      Novel association of genetic markers affecting CYP2A6 activity and lung cancer risk.
      ).
      In a study of Shanghai Chinese, the direct relationship of CYP2A6 genotype to nicotine metabolism, smoking dose, and lung cancer risk was demonstrated (
      • Yuan J.M.
      • Nelson H.H.
      • Butler L.M.
      • Carmella S.G.
      • Wang R.
      • Kuriger-Laber J.K.
      • Adams-Haduch J.
      • Hecht S.S.
      • Gao Y.T.
      • Murphy S.E.
      Genetic determinants of cytochrome P450 2A6 activity and biomarkers of tobacco smoke exposure in relation to risk of lung cancer development in the Shanghai Cohort study.
      ). In this cohort, CYP2A6 genotype predicted P450 2A6 activity, measured as the urinary ratio of 3′-hydroxycotinine to cotinine, and the risk of lung cancer was 30% lower for predicted poor metabolizers compared with other groups (Fig. 6). The protective effect of CYP2A6 genotype on lung cancer risk was no longer significant when adjusted for total nicotine equivalents, since nicotine consumption was mediating this association. In a study of similar design in Singapore Chinese, CYP2A6 genotype was also associated with both total nicotine equivalent levels and a reduced risk of developing lung cancer (
      • Yuan J.M.
      • Nelson H.H.
      • Carmella S.G.
      • Wang R.
      • Kuriger-Laber J.
      • Jin A.
      • Adams-Haduch J.
      • Hecht S.S.
      • Koh W.P.
      • Murphy S.E.
      CYP2A6 genetic polymorphisms and biomarkers of tobacco smoke constituents in relation to risk of lung cancer in the Singapore Chinese Health study.
      ).
      Figure thumbnail gr6
      Figure 6Nicotine metabolism phenotype by CYP2A6 diplotype in the Shanghai cohort. The values are geometric means of the urinary total trans-3′-hydroxycotinine (3HC) to total cotinine ratio for smokers with different CYP2A6 genotype that are grouped by metabolizer phenotype (
      • Yuan J.M.
      • Nelson H.H.
      • Butler L.M.
      • Carmella S.G.
      • Wang R.
      • Kuriger-Laber J.K.
      • Adams-Haduch J.
      • Hecht S.S.
      • Gao Y.T.
      • Murphy S.E.
      Genetic determinants of cytochrome P450 2A6 activity and biomarkers of tobacco smoke exposure in relation to risk of lung cancer development in the Shanghai Cohort study.
      ). Figure copyright © 2015, John Wiley and Sons, reused with permission.
      Only a handful of studies in non-Asian populations have found a significant relationship between CYP2A6 genotype and lung cancer risk (
      • Wassenaar C.A.
      • Dong Q.
      • Wei Q.
      • Amos C.I.
      • Spitz M.R.
      • Tyndale R.F.
      Relationship between CYP2A6 and CHRNA5-CHRNA3-CHRNB4 variation and smoking behaviors and lung cancer risk.
      ,
      • Rotunno M.
      • Yu K.
      • Lubin J.H.
      • Consonni D.
      • Pesatori A.C.
      • Goldstein A.M.
      • Goldin L.R.
      • Wacholder S.
      • Welch R.
      • Burdette L.
      • Chanock S.J.
      • Bertazzi P.A.
      • Tucker M.A.
      • Caporaso N.E.
      • Chatterjee N.
      • et al.
      Phase I metabolic genes and risk of lung cancer: Multiple polymorphisms and mRNA expression.
      ,
      • Wassenaar C.A.
      • Ye Y.
      • Cai Q.
      • Aldrich M.C.
      • Knight J.
      • Spitz M.R.
      • Wu X.
      • Blot W.J.
      • Tyndale R.F.
      CYP2A6 reduced activity gene variants confer reduction in lung cancer risk in African American smokers--findings from two independent populations.
      ). In a large population-based study of just over 3000 smokers, CYP2A6∗2 genotype, which codes for nonfunctional enzyme, was inversely associated with lung cancer risk (
      • Rotunno M.
      • Yu K.
      • Lubin J.H.
      • Consonni D.
      • Pesatori A.C.
      • Goldstein A.M.
      • Goldin L.R.
      • Wacholder S.
      • Welch R.
      • Burdette L.
      • Chanock S.J.
      • Bertazzi P.A.
      • Tucker M.A.
      • Caporaso N.E.
      • Chatterjee N.
      • et al.
      Phase I metabolic genes and risk of lung cancer: Multiple polymorphisms and mRNA expression.
      ). In a case control study in light smokers of European ancestry, fewer lung cancer cases carried one or more CYP2A6 variant (∗2, ∗4, ∗9, or ∗12), than did controls (
      • Wassenaar C.A.
      • Dong Q.
      • Wei Q.
      • Amos C.I.
      • Spitz M.R.
      • Tyndale R.F.
      Relationship between CYP2A6 and CHRNA5-CHRNA3-CHRNB4 variation and smoking behaviors and lung cancer risk.
      ). CYP2A6 genotype was also associated with lung cancer in two African American cohorts (
      • Wassenaar C.A.
      • Ye Y.
      • Cai Q.
      • Aldrich M.C.
      • Knight J.
      • Spitz M.R.
      • Wu X.
      • Blot W.J.
      • Tyndale R.F.
      CYP2A6 reduced activity gene variants confer reduction in lung cancer risk in African American smokers--findings from two independent populations.
      ). Male smokers in these cohorts with one or more copies of CYP2A6∗2, ∗4, ∗9, ∗17, ∗25, ∗26, ∗27, ∗31, or ∗35 (Table 1) were less likely to have lung cancer compared with those who carried none of these alleles.
      The observed variation in lung cancer risk by racial/ethnic group (
      • Murphy S.E.
      • Park S.L.
      • Balbo S.
      • Haiman C.A.
      • Hatsukami D.K.
      • Patel Y.
      • Peterson L.A.
      • Stepanov I.
      • Stram D.O.
      • Tretyakova N.
      • Hecht S.S.
      • Le Marchand L.
      Tobacco biomarkers and genetic/epigenetic analysis to investigate ethnic/racial differences in lung cancer risk among smokers.
      ,
      • Stram D.O.
      • Park S.L.
      • Haiman C.A.
      • Murphy S.E.
      • Patel Y.
      • Hecht S.S.
      • Le Marchand L.
      Racial/ethnic differences in lung cancer incidence in the Multiethnic Cohort study: An update.
      ,
      • Haiman C.A.
      • Stram D.O.
      • Wilkens L.R.
      • Pike M.C.
      • Kolonel L.N.
      • Henderson B.E.
      • Le Marchand L.
      Ethnic and racial differences in the smoking-related risk of lung cancer.
      ) is based on reported cigarettes per day. However, cigarettes per day is a crude and often poor measure of tobacco exposure (
      • Joseph A.M.
      • Hecht S.S.
      • Murphy S.E.
      • Carmella S.G.
      • Le C.T.
      • Zhang Y.
      • Han S.