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Cancer Research 67, 9024, October 1, 2007. doi: 10.1158/0008-5472.CAN-07-2245
© 2007 American Association for Cancer Research
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Priority Reports

Glucuronidation of Nicotine and Cotinine by UGT2B10: Loss of Function by the UGT2B10 Codon 67 (Asp>Tyr) Polymorphism

Gang Chen1,2, Andrea S. Blevins-Primeau1,3, Ryan W. Dellinger1,3, Joshua E. Muscat1,2 and Philip Lazarus1,2,3

1 Cancer Prevention and Control Program, Penn State Cancer Institute and Departments of 2 Public Health Sciences and 3 Pharmacology, Penn State University College of Medicine, Hershey, Pennsylvania

Requests for reprints: Philip Lazarus, Division of Population Sciences and Cancer Prevention, Department of Pharmacology, Penn State Cancer Institute, Penn State College of Medicine, MC-H069, 500 University Drive, Hershey, PA 17033. Phone: 717-531-5734; Fax: 717-531-0480; E-mail: plazarus{at}psu.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Nicotine, the major addicting agent in tobacco and tobacco smoke, undergoes a complex metabolic pathway, with ~22% of nicotine urinary metabolites in the form of phase II N-glucuronidated compounds. Recent studies have shown that UGT2B10 is a major enzyme involved in the N-glucuronidation of several tobacco-specific nitrosamines. In the present study, microsomes of UGT2B10-overexpressing HEK293 cells exhibited high N-glucuronidation activity against both nicotine and cotinine with apparent KM's that were 37- and 3-fold lower than that observed for microsomes of UGT1A4-overexpressing cells against nicotine and cotinine, respectively. The KM of microsomes from wild-type (WT) UGT2B10-overexpressing cells for nicotine and cotinine was similar to that observed for human liver microsomes (HLM) against both substrates. The level of glucuronidated nicotine or cotinine in 112 HLM samples was correlated with UGT2B10 genotype; the levels of nicotine- and cotinine-glucuronide were 21% to 30% lower in specimens from subjects with the UGT2B10 (*1/*2) genotype compared with specimens from subjects with the WT UGT2B10 (*1/*1) genotype; a 5- and 16-fold lower level of nicotine- and cotinine-glucuronide formation, respectively, was observed in HLM from subjects with the UGT2B10 (*2/*2) genotype. In contrast to the relatively high activity observed for cells overexpressing WT UGT2B10 in vitro, little or no glucuronidation was observed for microsomes from cells overexpressing the UGT2B10*2 variant against either nicotine or cotinine. These data suggest that UGT2B10 is the major hepatic enzyme involved in nicotine/cotinine glucuronidation and that the UGT2B10*2 variant significantly reduces nicotine- and cotinine-N-glucuronidation formation and plays an important role in nicotine metabolism and elimination. [Cancer Res 2007;67(19):9024–9]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Tobacco smoking causes 500,000 deaths annually in the United States, and nicotine is the single most important pharmacologic agent responsible for tobacco addiction. The most abundant nicotine metabolite is cotinine, which is further metabolized to 3'-hydroxycotinine and other compounds. Nicotine, cotinine, and 3'-hydroxycotinine undergo further phase II detoxification reactions by conjugation with glucuronic acid via catalysis by the UDP-glucuronosyltransferase (UGT) family of enzymes. Up to 31% of nicotine urinary metabolites are in the form of phase II glucuronidated compounds, with nicotine-glucuronide, cotinine-glucuronide, and trans-3'-hydroxycotinine glucuronide comprising the majority of these conjugates (1). Both cotinine and nicotine are glucuronidated on the nitrogen of the pyridine ring, and N-glucuronidation of both compounds is observed in human liver microsomes (HLM) and in the urine of smokers (2–5). Whereas N-glucuronidation of 3'-hydroxycotinine was observed in HLM, only its O-glucuronide was detected in the urine of smokers (6).

There is a high correlation between the in vivo urinary ratio of nicotine-glucuronide/(unconjugated + nicotine-glucuronide) to the ratio of cotinine-glucuronide/(unconjugated + cotinine-glucuronide) in smokers (7). The in vivo urinary nicotine-glucuronide ratio is only moderately correlated with 3'-hydroxycotinine-glucuronide, suggesting that different UGT enzymes are responsible for the glucuronidation of nicotine and cotinine versus 3'-hydroxycotinine.

Previous studies conducted to identify the UGT isoforms that glucuronidate nicotine and cotinine suggested that UGT1A4 was the primary enzyme responsible for the glucuronidation of these compounds (8). Whereas other studies of overexpressed UGT1A4 showed no nicotine and cotinine glucuronidation activity (9, 10), imipramine, a UGT1A4 substrate, was shown to inhibit the glucuronidation of nicotine and cotinine in HLM (10).

UGT2B10 was shown recently to exhibit high N-glucuronidation activity against several tobacco-specific nitrosamines (TSNA), including 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol, N'-nitrosonornicotine, N'-nitrosoanabasine, and N'-nitrosoanatabine.4 As nicotine and cotinine are structurally related to TSNAs, we hypothesized that UGT2B10 also plays an important role in nicotine and cotinine glucuronidation. The goal of the present study was to examine the activity of UGT2B10 against these agents and study the potential effects of the recently identified functional UGT2B10 codon 67 polymorphism on nicotine/cotinine glucuronidation activities in UGT2B10-overexpressing cell lines and HLM as a measure of glucuronidation phenotype.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
[14C]UDPGA (200 mCi/mmol) was purchased from American Radiolabeled Chemicals. The high-performance liquid chromatography (HPLC) scintillation solution, Ecoscint Flow, was purchased from National Diagnostics. Nicotine, cotinine, alamethicin, ß-glucuronidase, anti-calnexin antibody, and bovine serum albumin were from Sigma-Aldrich. DMEM, Dulbecco's PBS (minus calcium-chloride and magnesium-chloride), fetal bovine serum, penicillin-streptomycin, and geneticin (G418) were all purchased from Invitrogen. The bicinchoninic acid (BCA) protein assay kit was purchased from Pierce. All other chemicals were purchased from Fisher Scientific.

Normal human liver tissue specimens and matching genomic DNA samples from 78 samples were described previously (11). The number of specimens used in the current study was expanded to 112 and obtained in the same manner as the original 78 specimens. All 112 subjects were Caucasians; gender was known for 95 of the 112 subjects, and 41% of these individuals were female. The liver microsomes were prepared using differential centrifugation as described previously (12) and stored (10–20 mg protein/mL) at –80°C. Microsomal protein concentrations were measured using the BCA assay. The cells used for overexpressing UGTs 1A4 and 2B10 have also been described previously (13).4 Cell microsomes were prepared essentially as described above for liver microsomes by resuspending pelleted cells in TBS [25 mmol/L Tris-HCl (pH 7.4), 138 mmol/L NaCl, 2.7 mmol/L KCl] and subjecting them to three rounds of freeze-thaw before gentle homogenization and differential centrifugation.

Glucuronidation activities of HLM or microsomes from UGT-overexpressing cell lines were determined as described previously4 after an initial incubation of HLM (2–5 µg protein) or UGT-overexpressing cell microsomes (40 µg protein) with alamethicin (50 µg/mg protein) for 15 min in an ice bath. Incubations (4 µL) were then done at 37°C for 2 h, a time that was found in previous studies to be within the linear range of product formation for the UGTs tested in this study. Reactions were terminated by the addition of equal volume of cold acetonitrile. After centrifugation at 13,000 x g for 10 min, the supernatants were diluted to 50 µL with water.

Samples (50 µL) were analyzed for glucuronide formation by HPLC using a Beckman Coulter System Gold 126 Solvent Module HPLC system equipped with an automatic injector (model 508), a UV detector operated at 254 nm (model 166), and a radioactive flow detector with 1,000 µL flow cell (INUS Systems). HPLC was done using a Synergi Fusion-RP-80 4 µm column (4.6 x 250 mm; Phenomenx) and an Aquasil 5 µm C18 analytic column (4.6 x 250 mm; Thermo) in series. The gradient elution conditions were as follows: starting with 100% buffer A [100 mmol/L NH4Ac (pH 5.0)] for 5 min for nicotine glucuronide formation or for 2 min for cotinine glucuronide formation, a subsequent linear gradient to 78% buffer B (90% acetonitrile, 10% water) over 10 min was done and then maintained at 78% buffer B for 10 min. The elution flow rate was 1 mL/min and the scintillation solution flow rate was 1.5 mL/min. The amount of N-glucuronide formed was calculated based on the ratio of the radioactivity of the N-glucuronide versus total radioactivity. Nicotine- and cotinine-N-glucuronides were confirmed by sensitivity to ß-glucuronidase as described previously (14). As controls, glucuronidation assays were regularly done using HLM and untransfected HEK293 microsomes as positive and negative controls, respectively, for glucuronidation activity. Cell line experiments were always done in triplicate in independent assays; HLM glucuronidation assays were repeated for 20% of all samples to monitor experimental variation.

Putative UGT2B10-catalyzed nicotine- and cotinine-glucuronide peaks were collected by HPLC essentially as described above, with collected fractions dried and resuspended in methanol. An Applied Biosystems 4000 Q Trap LC/MS/MS mass spectrometer was used to characterize individual glucuronides as described previously (8).

RFLP analysis was done using the HinfI restriction enzyme as described previously to detect the UGT2B10 codon 67 polymorphism.4 Genotyping was done for subjects from whom liver specimens were obtained and for a subset (n = 784) of healthy subjects serving as controls recruited as part of a case-control study conducted at the H. Lee Moffitt Cancer Center (Tampa, FL) from 2000 to 2003 (15).

Kinetic constants were determined using Prism version 4.01 software (GraphPad Software). The rate of nicotine- and cotinine-glucuronide formation in HLM was compared by gender and UGT2B10 codon 67 genotype [homozygotes, heterozygotes, and wild-type (WT)] by trend test and Student's t test using SPSS statistical software (version 15.0, SPSS, Inc.).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
To determine whether UGT2B10 exhibited activity against nicotine and cotinine similar to that for UGT2B10 against TSNAs,4 glucuronidation assays using microsomes prepared from a WT UGT2B10-overexpressing cell line were done. UGT2B10 exhibited significant N-glucuronidation activity against both nicotine and cotinine (Fig. 1 ). A putative glucuronide peak was observed by HPLC for assays using UGT2B10-overexpressing microsomes at retention times of 16.6 and 13.5 min for nicotine (Fig. 1A) and cotinine (Fig. 1B), respectively, times that were identical to that observed for UGT1A4 and HLMs. Both peaks were sensitive to treatment with ß-glucuronidase (data not shown), indicating the presence of a glucuronide conjugate. Whereas previous studies have indicated that glucuronidation activity for UGT1A4 against both nicotine and cotinine was only observed in incubations done at pH 8.9 (8), glucuronidation activity was observed for microsomes from both UGT2B10- and UGT1A4-overexpressing cells when assays were done at either pH 7.4 or pH 8.9 in the present study (data not shown). The putative UGT2B10-catalyzed nicotine- and cotinine-glucuronide peaks were analyzed by tandem mass spectrometry and were shown to fragment to a mass of 163 and 177, respectively (data not shown), which is consistent with that observed in previous studies (8).


Figure 1
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  		Figure 1. HPLC analysis of nicotine- and cotinine-glucuronide formation by microsomes of UGT2B10- and UGT1A4-overexpressing cells and HLM. Incubations were done using 40 µg of total microsomal protein for UGTs 1A4- and 2B10-overexpressing cells or 5 µg of total microsomal protein for HLM at 37°C for 2 h with 4 mmol/L [14C]UDPGA and 5 mmol/L substrate. HPLC was done as described in Materials and Methods. A, nicotine; B, cotinine.

 
Kinetic analysis was done for UGT2B10- and UGT1A4-overexpressing cell lines. Microsomes from UGT2B10-overexpressing cells were significantly more active as determined by in vitro KM assessment than microsomes from cells overexpressing UGT1A4 against both nicotine and cotinine (Table 1 ). The apparent KM values for nicotine and cotinine were 37- and 3-fold lower, respectively, for UGT2B10 than for UGT1A4. The higher affinity exhibited by UGT2B10 was observed whether assays were done at pH 7.5 or pH 8.9, with >200-fold lower KM observed for both UGTs 2B10 and 1A4 against nicotine in glucuronidation assays done at pH 8.9 (data not shown). The apparent KM's for UG2B10 against both nicotine and cotinine approached that observed for HLMs (Table 1).


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  		Table 1. Kinetic analysis of UGT1A4-, UGT2B10-, and HLM-induced glucuronidation of nicotine and cotinine

 
Previous studies showed that the glucuronidation of nicotine was inhibited by imipramine in HLM (10). To determine whether imipramine is a substrate and potential competitive inhibitor of UGT2B10, glucuronidation assays similar to those described above for nicotine and cotinine were done with imipramine as substrate. Imipramine was shown to be a substrate for UGT2B10 in the present study, with an apparent KM of 49 µmol/L. This compared with a KM of 763 µmol/L for UGT1A4 (data not shown).

Recent haplotyping studies have indicated the presence of a single missense polymorphism at codon 67 of the UGT2B10 gene that is highly correlated with UGT2B10 glucuronidation activity against TSNAs.4 In an analysis of 784 healthy Caucasian subjects, the prevalence of the UGT2B10*2 variant allele was 9.1%. The prevalence of the UGT2B10 codon 67 polymorphism in this population was consistent with that expected by Hardy-Weinberg equilibrium (P = 0.266).

Similar to that observed for TSNAs, the glucuronidation of nicotine by the UGT2B1067Tyr variant was barely detectable in vitro; no detectable glucuronidation activity was observed for the UGT2B1067Tyr variant against cotinine. This contrasts with the relatively high level of activity observed for WT UGT2B10 67ASP against both compounds (Table 1). Due to its overall low activity, a KM could not be ascertained for the UGT2B1067Tyr variant against either substrate.

To determine whether an association was observed for the UGT2B10 codon 67 polymorphism and the glucuronidation of nicotine and cotinine in HLM, the formation of nicotine- and cotinine-glucuronide was examined in a series (n = 112) of microsomal specimens prepared from normal human liver tissue from individual subjects. The rate of nicotine-glucuronide formation was strongly correlated (r = 0.76) with cotinine-glucuronide formation in this series of HLM (Fig. 2 ). No significant difference in levels of nicotine- or cotinine-glucuronide formation was observed in HLM after stratification of the entire population by gender. As shown in Fig. 3 , there was a significant (P < 0.01) trend toward decreased glucuronidation activity against both nicotine and cotinine in HLM from subjects with an increasing number of the variant UGT2B10 allele (termed the UGT2B10*2 allele). Eighty-two percent (n = 92) of the subjects for whom HLMs were analyzed were homozygous WT UGT2B10 (*1/*1). There was a significant (P < 0.05) 21% decrease in nicotine glucuronidation activity in HLM from subjects with the heterozygous UGT2B10 (*1/*2) genotype (n = 18) and a significant (P < 0.001) 5-fold decrease in activity in HLM from subjects homozygous for the UGT2B10*2 allele (n = 2), compared with subjects with the homozygous WT UGT2B10 (*1/*1) genotype. Similarly, a significant (P < 0.001) 30% decrease in cotinine glucuronidation activity was observed in HLM from subjects with the heterozygous UGT2B10 (*1/*2) genotype and a significant (P < 0.001) 16-fold decrease in activity in HLM from subjects with the homozygous UGT2B10 (*2/*2) genotype, compared with subjects with the homozygous WT UGT2B10 (*1/*1) genotype. Of the 95 subjects for whom gender was known, 44%, 33%, and 0% of the subjects exhibiting the UGT2B10 (*1/*1), UGT2B10 (*1/*2), and UGT2B10 (*2/*2) genotypes, respectively, were female. There was no association between levels of nicotine- or cotinine-glucuronide formation and UGT2B10 genotype after stratification by gender.


Figure 2
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  		Figure 2. Correlation between rate of nicotine- and cotinine-glucuronide formation in HLM. Glucuronidation assays were done as described in the legend of Fig. 1 for 112 HLM specimens as described in Materials and Methods.

 

Figure 3
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  		Figure 3. Nicotine-N-glucuronide (A) and cotinine-N-glucuronide (B) formation in HLM from subjects stratified by copy number of UGT2B10*2. The number of subjects within each genotype group of 0, 1, and 2 copies of the UGT2B10*2 allele are 92, 18, and 2, respectively. Columns, mean of glucuronide formation; bars, SE. *, P < 0.05; **, P < 0.01, a significant decrease in rate of nicotine- or cotinine-glucuronide formation was observed when compared with the UGT2B10 (*1/*1) genotype group.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This is the first report examining the role of UGT2B10 in nicotine metabolism. Whereas both UGTs 1A4 and 2B10 exhibited N-glucuronidation activity in the present study, UGT2B10 exhibited significantly greater activity against both nicotine and cotinine than UGT1A4. Like UGT1A4, UGT2B10 is a hepatic enzyme (16). The fact that the KM of liver microsomes for nicotine and cotinine approached the KM's observed for UGT2B10 against these two compounds in vitro suggests that UGT2B10 is the dominant enzyme involved in the glucuronidation of these compounds in liver. This is supported by the fact that the UGT2B10 codon 67 polymorphism effectively reduces overall liver glucuronidation of both nicotine and cotinine to 5- and 16-fold, respectively, in subjects homozygous for the UGT2B1067Tyr variant. In addition, the high correlation between nicotine- and cotinine-glucuronide formation observed in HLM in this study indicates that the two substrates are glucuronidated by the same enzymes, primarily UGT2B10. The fact that imipramine was a good substrate for UGT2B10 is consistent with the fact that imipramine was able to block nicotine and cotinine glucuronidation in HLM in previous studies (10). Although O-glucuronidation of 3'-hydroxycotinine is important in the overall nicotine metabolism pathway, its N-glucuronide has not been detected in smokers' urine (3) and has therefore not been screened in the present study.

A previous report showed that UGT1A4-overexpressing baculosomes are active against both nicotine and cotinine, with activity observed only at pH 8.9; no activity was observed at physiologic pH (8). N-glucuronidation activity was observed against both compounds when assays were done at pH 7.4 as well as pH 8.9 in the present study. This contrast is likely due to the different source of overexpressed UGT1A4 in the two studies (baculosomes versus overexpressing mammalian cells). Baculosomal UGTs may lack the necessary post-translational modifications necessary for optimal activity for all UGTs (17), and this may be the case for UGT1A4 against these substrates. The fact that UGT1A4 was considered inactive in a second study (10) is likely due to methodologic issues including assay sensitivity.

The prevalence of the UGT2B10*2 variant allele is relatively high in Caucasians (9.1%). Therefore, this polymorphism may have an important overall effect on nicotine metabolism and potentially nicotine addiction. In the urine of smokers, up to 5% of absorbed nicotine is metabolized to form nicotine-glucuronide and up to 10% remains as unmetabolized nicotine (1, 18). Therefore, an increase in overall unconjugated nicotine could occur due to the resulting near-inactivation of the UGT2B10 enzyme in people homozygous for the UGT2B1067Tyr variant. In addition, cotinine-glucuronide comprises up to 17% of the total urinary nicotine metabolites in smokers. Therefore, subjects with the UGT2B1067Tyr variant would also have significantly higher levels of cotinine. This would also likely result in higher absolute levels of nicotine in subjects with one or more low-activity UGT2B10 alleles assuming the rate of metabolism of nicotine to cotinine and the rate of metabolism of cotinine to trans-3'-OH-cotinine remains constant.

The effects of this polymorphism could be most pronounced, however, in subjects with a deleterious CYP2A6 polymorphism because this is the major hepatic enzyme involved in metabolism of nicotine to its nonaddictive metabolite, cotinine. It has been shown that ~20% of Caucasians and African Americans as well as 70% of Asians exhibit a poor CYP2A6 metabolizing enzyme phenotype (activity decreased by at least 25%; ref. 19). For example, in smokers homozygous for the CYP2A6*4 deletion allele and who therefore have no active CYP2A6 enzyme, the levels of urinary nicotine-glucuronide and unconjugated nicotine were shown to be as high as 45% and 55%, respectively, of total absorbed nicotine (20). In subjects with a poor CYP2A6 metabolizing enzyme phenotype, the levels of nicotine would likely increase to even higher internal levels if that individual also had one or more UGT2B10*2 variant alleles. Therefore, the UGT2B10 codon 67 polymorphism could be an important modifier of nicotine metabolism and addiction. Further studies examining urinary nicotine metabolites in smokers of different combined CYP2A6 and UGT2B10 genotypes will be required to better assess these possibilities.


   Acknowledgments
 
Grant support: NIH Public Health Service grants P01-CA68384 and R01-DE13158 (P. Lazarus) and a formula grant under the Pennsylvania Department of Health's Health Research Formula Funding Program (State of PA, Act 2001-77—part of the PA Tobacco Settlement Legislation; P. Lazarus).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank the Tissue Procurement Facility at the H. Lee Moffitt Cancer Center for tissue procurement for these studies and Functional Genomics Core Facility and the Molecular Biology Core Facility at the Penn State College of Medicine for DNA genotyping and sequencing services.


   Footnotes
 
4 G. Chen, et al. Loss of function of the codon 67 (Tyr) variant in tobacco-specific nitrosamine glucuronidation. Submitted for publication. Back

Received 6/19/07. Revised 8/ 2/07. Accepted 8/14/07.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online