Published OnlineFirst September 28, 2010; DOI: 10.1158/0008-5472.CAN-09-4582 Published OnlineFirst on September 28, 2010 as 10.1158/0008-5472.CAN-09-4582 Prevention and Epidemiology Cancer Research Glucuronidation Genotypes and Nicotine Metabolic Phenotypes: Importance of Functional UGT2B10 and UGT2B17 Polymorphisms Gang Chen1,2, Nino E. Giambrone, Jr.1,3, Douglas F. Dluzen1,3, Joshua E. Muscat1,2, Arthur Berg1,2, Carla J. Gallagher1,2, and Philip Lazarus1,2,3 Abstract Glucuronidation is an important pathway in the metabolism of nicotine, with previous studies suggesting that ∼22% of urinary nicotine metabolites are in the form of glucuronidated compounds. Recent in vitro stud- ies have suggested that the UDP-glucuronosyltransferases (UGT) 2B10 and 2B17 play major roles in nicotine glucuronidation with polymorphisms in both enzymes shown to significantly alter the levels of nicotine- glucuronide, cotinine-glucuronide, and trans-3′-hydroxycotinine (3HC)–glucuronide in human liver micro- somes in vitro. In the present study, the relationship between the levels of urinary nicotine metabolites and functional polymorphisms in UGTs 2B10 and 2B17 was analyzed in urine specimens from 104 Caucasian smokers. Based on their percentage of total urinary nicotine metabolites, the levels of nicotine-glucuronide and cotinine-glucuronide were 42% (P < 0.0005) and 48% (P < 0.0001), respectively, lower in the urine from smokers exhibiting the UGT2B10 (*1/*2) genotype and 95% (P < 0.05) and 98% (P < 0.05), respectively, lower in the urine from smokers with the UGT2B10 (*2/*2) genotype compared with the urinary levels in smokers having the wild-type UGT2B10 (*1/*1) genotype. The level of 3HC-glucuronide was 42% (P < 0.001) lower in the urine from smokers exhibiting the homozygous UGT2B17 (*2/*2) deletion genotype compared with the levels in urine from wild-type UGT2B17 subjects. These data suggest that UGTs 2B10 and 2B17 play im- portant roles in the glucuronidation of nicotine, cotinine, and 3HC and suggest that the UGT2B10 codon 67 SNP and the UGT2B17 gene deletion significantly reduce overall glucuronidation rates of nicotine and its ma- jor metabolites in smokers. Cancer Res; 70(19); 7543–52. ©2010 AACR. Introduction form of phase II glucuronidated compounds, with nicotine- N-glucuronide (nicotine-Gluc), cotinine-N-glucuronide Tobacco smoking causes 500,000 deaths annually in the (cotinine-Gluc), and cotinine-3′-O-glucuronide (3HC-Gluc) United States, and nicotine is the pharmacologic agent re- comprising the majority of these conjugates (3). Both cotinine sponsible for tobacco addiction (1). The analysis of urinary and nicotine are glucuronidated on the nitrogen of the pyri- nicotine metabolite profiles indicates that ∼70% to 80% of dine ring, and N-glucuronidation of both compounds is ob- nicotine is metabolized to cotinine and then cotinine is fur- served in human liver microsomes (HLM) and in the urine ther metabolized to trans-3′-hydroxycotinine (3HC) and other of smokers (4–7). Approximately 90% of the systemic dose compounds (ref. 2; see Fig. 1). Hepatic metabolism of nicotine of nicotine is excreted through the urine, and although both to cotinine and then to 3HC is catalyzed primarily by CYP2A6 O-glucuronidation and N-glucuronidation of 3HC were ob- (3). Nicotine, cotinine, and 3HC undergo further phase II de- served in HLM, only its O-glucuronide, 3HC-Gluc, was detected toxification reactions by conjugation with glucuronic acid via in the urine of smokers (8). catalysis by the UDP-glucuronosyltransferase (UGT) family of A high correlation exists between the in vivo urinary ratio enzymes. Up to 31% of nicotine urinary metabolites are in the of nicotine-Gluc/(nicotine + nicotine-Gluc) and the ratio of cotinine-Gluc/(cotinine + cotinine-Gluc) in smokers. By con- trast, the in vivo urinary nicotine-Gluc/(nicotine + nicotine- Authors' Affiliations: 1Molecular Epidemiology and Cancer Control Program, Penn State Cancer Institute and Departments of 2Public Gluc) ratio is only moderately correlated with the ratio of Health Sciences and 3Pharmacology, Penn State University College of 3HC-Gluc/(3HC + 3HC-Gluc; ref. 2). This suggests that the Medicine, Hershey, Pennsylvania glucuronidation of nicotine/cotinine versus 3HC may be via Corresponding Author: Philip Lazarus, Department of Pharmacology, different enzyme pathways. Previous studies have also shown Division of Population Sciences and Cancer Prevention, Penn State Can- cer Institute, Penn State University College of Medicine, 500 University a high correlation between the glucuronidation of nicotine Drive, Hershey, PA 17033. Phone: 717-531-5734; Fax: 717-531-0480; and cotinine in HLM in vitro and that the hepatic UGT2B10 E-mail: [email protected]. was the major enzyme responsible for their glucuronidation; doi: 10.1158/0008-5472.CAN-09-4582 no correlation exists between either nicotine-Gluc or cotinine- ©2010 American Association for Cancer Research. Gluc formation and 3HC-Gluc formation in HLM in vitro www.aacrjournals.org 7543 Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2010 American Association for Cancer Research. Published OnlineFirst September 28, 2010; DOI: 10.1158/0008-5472.CAN-09-4582 Chen et al. Figure 1. Major nicotine metabolites in the urine of smokers. (9, 10). Whereas earlier studies identified UGTs 2B7 and significant decrease in the O-glucuronidation of NNAL (12). 1A9 to be important in the glucuronidation of 3HC, more Together, these data suggest that both polymorphisms could recent studies have shown that UGT2B17 exhibits the high- significantly alter how nicotine is metabolized in vivo. est activity against 3HC in vitro.4 The goal of the present study was to assess the levels of A functional polymorphism (rs61750900) at codon 67 nicotine and a panel of nicotine metabolites [cotinine, 3HC, (Asp > Tyr) of the UGT2B10 gene with an allelic prevalence nicotine-Gluc, cotinine-Gluc, 3HC-Gluc, nicotine N′-oxide, of ∼10% in Caucasians has been reported (9). The UGT2B10Tyr cotinine N-oxide, and 4-hydroxy-4-(3-pyridyl)-butanoic acid variant was shown to be associated with significantly re- (4HPBA)] in the urine of smokers and to determine whether duced N-glucuronidation of nicotine and cotinine in HLM the relative levels of urinary nicotine-Gluc, cotinine-Gluc, and and UGT2B10-overexpressing cell lines in vitro (9, 11). The 3HC-Gluc are associated with the functional polymorphisms UGT2B10Tyr variant was also shown to be associated with in the UGT2B10 and UGT2B17 genes in vivo. significantly reduced N-glucuronidation of 4-(methylnitrosa- mino)-1-(3-pyridyl)-1-butanol (NNAL), the major metabolite of the nicotine-derived tobacco-specific nitrosamine 4- Materials and Methods (methylnitrosamino)-1-(3-pyridyl)-1-butanone, in HLM and in UGT2B10-overexpressing cell lines in vitro (11). Similarly, Chemicals a polymorphic whole-gene deletion of the UGT2B17 gene, Nicotine and creatinine were purchased from Sigma- with an allelic prevalence of ∼30% in Caucasians, has been Aldrich. Cotinine, 3HC, nicotine-Gluc, cotinine-Gluc, 3HC-Gluc, shown to be associated with a significant decrease in the nicotine N′-oxide, cotinine N-oxide, and deuterium-labeled in- 4 O-glucuronidation of 3HC in HLM in vitro. In similar studies, ternal standards including nicotine-methyl-D3, cotinine- this polymorphism was also shownto be associated with a methyl-D3, 3HC-methyl-D3, nicotine(methyl-D3)-glucuronide, ′ cotinine(methyl-D3)-glucuronide, cotinine(methyl-D3)-3 -O- ′ glucuronide, nicotine N -oxide-methyl-D3, cotinine N-oxide- 4 Chen and Lazarus, unpublished results. methyl-D3, and creatinine-D3 were purchased from Toronto 7544 Cancer Res; 70(19) October 1, 2010 Cancer Research Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2010 American Association for Cancer Research. Published OnlineFirst September 28, 2010; DOI: 10.1158/0008-5472.CAN-09-4582 UGT Polymorphisms and Nicotine Metabolism Research Chemicals, Inc.. All other chemicals were purchased consisting of an Acquity UPLC pump, an autosampler, an from Fisher Scientific. ACQUITY UPLC BEH HILIC (2.1 mm × 100 mm, 1.7 μm par- ticle size; Waters Corporation) column at 30°C, and an Ac- Subjects and samples quity TQ tandem mass spectrometer (Waters Corporation). Spot urine specimens and matching genomic DNA from UPLC was performed at a flow rate of 0.4 mL/min using the buccal cells were from 107 subjects that were a subset of following conditions: 1.5 minutes in 20% solvent A, a linear the group of healthy subjects that were recruited as controls gradient for 1 minute to 100% solvent A, and 3 minutes in as part of a case-control study conducted at the H. Lee Moffitt 100% solvent A, where solvent A is 5 mmol/L NH4AC (pH Cancer Center (Tampa, FL) from 2000 to 2003, as previously 6.7) and 50% acetonitrile (v/v) and solvent B is 5 mmol/L described (13). All of the 107 subjects in the present study NH4AC (pH 6.7) and 90% acetonitrile (v/v). The injection vol- were Caucasian and were self-reported current smokers. ume of each prepared urine sample was 5 μL, which repre- The age of the subjects ranged from 30 to 74 years with an sented 0.25 μL of urine collected from each subject. To verify average of 56.8 years, and 43% (n = 46) were female. All urine the validity of values obtained from the analysis of one sam- and DNA samples were stored at −80°C. ple per subject, six sample preparations and analyses were carried out on urine specimens from five individual subjects. Sample preparation The interassay coefficient of variation was <10% for all five A 10-μL aliquot of each urine sample was added to 5 μLof subjects. a mixture of deuterium-labeled internal standards, which The Waters Acquity TQ tandem mass spectrometer was included nicotine-methyl-D3, nicotine-oxide-methyl-D3, cotinine- equipped with an electrospray ionization probe operated in methyl-D3, cotinine-N-oxide-methyl-D3, 3HC-methyl-D3, nico- the positive ion mode, with capillary voltage at 0.64 kV.