Influence of UGT2B10 Genotype on Urinary Excretion of 4

Feb 20, 2018 - Masonic Cancer Center, University of Minnesota, Minneapolis, Minnesota 55455, United States. ‡ Epidemiology Program, University of Ha...
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Cite This: Chem. Res. Toxicol. 2018, 31, 168−175

Influence of UGT2B10 Genotype on Urinary Excretion of 4‑(Methylnitrosamino)-1-(3-pyridyl)-1-butanol-N-glucuronide by African American Smokers Sharon E. Murphy,*,† Linda B. von Weymarn,† Marc Parenteau,† Irina Stepanov,§ Maarit Tiirikainen,‡ Loic LeMarchand,‡ and Sungshim L. Park¶ †

Department of Biochemistry, Molecular Biology and Biophysics and Masonic Cancer Center, University of Minnesota, Minneapolis, Minnesota 55455, United States § Masonic Cancer Center, University of Minnesota, Minneapolis, Minnesota 55455, United States ‡ Epidemiology Program, University of Hawaii Cancer Center, Honolulu, Hawaii 96813, United States ¶ Department of Preventive Medicine, Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, Los Angeles, California 90089, United States S Supporting Information *

ABSTRACT: At similar smoking levels, African American’s lung cancer risk is as much as twice that of whites. We hypothesized that racial/ethnic differences in UDP-glucuronosyltransferase (UGT)-catalyzed glucuronidation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), a detoxication pathway for the tobacco-specific lung carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) may contribute to this variable risk. UGT2B10 catalyzes NNAL-N-glucuronidation, and a UGT2B10 splice variant is common among African Americans. Smokers from two independent studies were genotyped for this variant (rs116294140) and an Asp67Tyr variant (rs61750900), and urinary NNAL and NNAL-glucuronide concentrations were quantified. In the first, no significant differences in NNAL-N-glucuronidation between African Americans (n = 257) and whites (n = 354) or between homozygous carriers of UGT2B10 variants (genetic score 2) and noncarriers (score 0) were detected. However, total NNAL glucuronidation by score 2 compared to score 0 smokers was lower (68.9 vs 71.2%, p < 0.0001). For NNAL-N-glucuronide to be more precisely quantified in a second study, a sensitive high-resolution LC-MS/ MS-based method, which separated NNAL, NNAL-O-glucuronide, and NNAL-N-glucuronide prior to analysis, was developed. In this study, the excretion of total NNAL (free plus glucuronides) by African American (n = 52) and white (n = 54) smokers was not different; however, total NNAL glucuronidation by African Americans (64.0%) was slightly less than by whites (68.3%, p = 0.05). The mean NNAL-N-glucuronidation by African Americans was much lower than for whites (14 vs 24.9%, p < 0.00001), but the NNAL-O-glucuronidation was greater (50.0 vs 43.3%, p = 0.013). UGT2B10 genotype influenced NNAL-N-glucuronidation; the geometric mean percentage N-glucuronidation was 22.5% for smokers with genetic score 0 (n = 57) and 11.2% for score 2 (n = 11). In summary, the high prevalence of a UGT2B10 splice variant among African Americans results in lower NNAL-Nglucuronidation but only a small decrease in total NNAL glucuronidation. Therefore, despite the significant contribution of UGT2B10 to NNAL-N-glucuronidation, the UGT2B10 genotype does not play a large role in NNAL detoxication. Any decrease in N-glucuronidation was accompanied by a parallel increase in O-glucuronidation.

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INTRODUCTION

levels of cigarettes smoked per day (10−15), the lung cancer risk of African Americans is twice that of whites.4 This discrepancy in risk appears in part to be explained by a greater intensity of smoking per cigarette by African Americans compared to white smokers.5,6 However, racial/ethnic differences in tobacco carcinogen metabolism, activation or detoxication, may also play a role.

Worldwide, in 2012 there were over 1.6 million deaths from lung cancer.1 In the United States, lung cancer is the leading cause of cancer-related death; its incidence is second only to breast cancer in woman and prostate cancer in men.2 Smoking cigarettes is the main cause of lung cancer, accounting for 90% of the deaths from this disease in the U.S. However, only 11−24% of smokers develop lung cancer, and for the same quantity of cigarettes smoked, the risk of lung cancer varies by racial/ethnic group.3 For similar low © 2018 American Chemical Society

Received: September 22, 2017 Published: February 20, 2018 168

DOI: 10.1021/acs.chemrestox.7b00264 Chem. Res. Toxicol. 2018, 31, 168−175

Article

Chemical Research in Toxicology Among the more than 70 carcinogens in tobacco smoke, tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3pyridyl)butanone (NNK) is considered an important causative agent for lung cancer. As the result of numerous studies in laboratory animals and epidemiological investigations, NNK was classified as a human lung carcinogen by the International Agency for Research on Cancer.7 In smokers, NNK is metabolized by three pathways, N-oxidation, α-hydroxylation, and reduction to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL, Scheme 1).8 NNAL is carcinogenic, and like NNK, it is

We hypothesize that, analogously, African American smokers may have decreased NNAL-N-glucuronidation and potentially decreased detoxication of the tobacco-specific lung carcinogen NNK. In the study presented here, we report on the influence of the UGT2B10 genotype on NNAL glucuronidation in African American compared to white smokers.

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EXPERIMENTAL PROCEDURES

Study Population and Urine Samples. Urine samples analyzed were from two studies both approved by the University of Minnesota Institutional Review Board. The subjects of Study 1 are the Multiethnic Cohort (MEC)22 participants who were current smokers at the time of (overnight or first morning) urine collection (n = 2393) for whom cotinine and total nicotine equivalents (TNE, the sum of total cotinine, total nicotine, total 3-hydroxycotinine, and nicotine N-oxide) were previously quantified and with TNE values >1.27 nmol/mL.18 “Total” refers to the analyte and its glucuronide conjugate. UGT2B10 genotype was available on 2239 of these subjects,18 and among these samples, 1901 had available measures for all three biomarkers: total NNAL, NNAL, and NNAL-N-gluc.5 In addition, for this study’s analyses, if total NNAL was 10 pmol/mL (extreme outliers, n = 10) samples were excluded, leaving 1754 samples. Study 2 consisted of 106 subjects (54 white and 52 African Americans) for whom 24 h urine was collected. These were baseline samples collected for a study of NNK metabolism in which current smokers with a smoking history of at least 10 cigarettes daily for 1 year or more were recruited. Subjects were asked to collect a 24 h urine sample and bring it to a research clinic at the University of Minnesota. Urine samples were stored at −20 °C before analysis. UGT2B10 Genotyping. For the MEC study, UGT2B10 genotypes were extracted from Illumina 1 M imputed data.23 Genotyping for two UGT2B10 variants in Study 2 participants was performed at University of Hawaii Cancer Center Genomics Shared Resources. The UGT2B10 splice variant (rs116294140/rs2942857) and the Asp67Tyr variant (rs61750900) were genotyped using either lymphocyte or oral cell DNA and custom designed (Dr. Tiirikainen) TaqMan allele discrimination assays (Applied Biosystems, Carlsbad, CA). The genotype frequencies were consistent with Hardy−Weinberg equilibrium in each ethnic group (P > 0.05). Concordance rate across the ∼5% blinded duplicate samples genotyped with the study samples was 100%. The genotyping call rate was 99%. Analysis of NNAL, NNAL-N-gluc, and NNAL-O-gluc. The urinary concentrations of NNAL and its glucuronide conjugates for the subjects of Study 1 were quantified previously.24 For the NNAL-glucuronide analysis, three independent aliquots were processed for each urine sample. One aliquot was untreated; one was base treated to release NNAL from NNAL-N-gluc, and one was treated with ß-glucuronidase to release NNAL from both NNAL-N-gluc and NNAL-O-gluc. The NNAL concentration in each aliquot was quantified by LC-MS-MS, and the concentration of NNAL-N-gluc was calculated by subtracting the concentration of free NNAL in the untreated aliquot from the NNAL in the base-treated aliquot, and the concentration of NNAL-O-gluc was calculated by subtracting the NNAL in the base-treated sample from that in the ß-glucuronidase-treated aliquot. As noted above, only samples with total NNAL concentrations greater than 0.45 pmol/mL were included in the analysis. This was done to minimize the error in the calculation of the NNAL glucuronides, which would be greatest when two small values are subtracted. For Study 2, a new method was developed that used a single aliquot of urine and separated NNAL, NNAL-N-gluc, and NNAL-O-gluc on a solid phase extraction (SPE) column (Figure 1). In the development of this method, a number of SPE columns were tested with various different elution protocols for (1) the retention of NNAL-N-gluc on the column and (2) the complete separation of NNAL-N-gluc, NNAL, and NNAL-O-gluc. The NNAL-N-gluc was not retained on any of the 9 SPE columns tested (Phenomenex Strata X and Strata XAW and Strata XA, Waters Oasis MAX and MCX, Resprep C18, Hypersep NH2 and CN, Sola WCX, Bondelute PBA). Conditions were defined to efficiently and reproducibly separate NNAL-N-gluc, NNAL, and NNAL-O-gluc in

Scheme 1. NNK Metabolism and NNAL Glucuronidation Pathways

metabolically activated by P450-catalyzed α-hydroxylation.8 Detoxication of NNAL, which occurs by UDP-glucuronosyltransferase (UGT)-catalyzed glucuronidation, is also an important pathway for the detoxication of NNK. Both NNAL-Nglucuronide (NNAL-N-gluc) and NNAL-O-glucuronide (NNAL-O-gluc) are excreted in smokers’ urine (Scheme 1). UGT2B7, UGT2B17, and UGT1A9 are catalysts of NNAL-Oglucuronidation,9−11 whereas UGT2B10 and UGT1A4 catalyze NNAL-N-glucuronidation.12,13 However, it has been reported that UGTB10 is a more efficient catalyst of NNAL-N-glucuronidation and that this enzyme may be the more important catalyst in smokers because smokers who carry the Asp67Tyr UGT2B10 variant excreted lower levels of NNAL-N-gluc.11,13 Nicotine and its primary metabolite cotinine are also metabolized by N-glucuronidation of the pyridine ring. In vitro, both UGT2B10 and UGT1A4 are catalysts of these reactions.14−16 However, in smokers, UGT2B10 is the only enzyme that contributes significantly to cotinine and nicotine N-glucuronidation.17,18 Individuals who have no functional UGT2B10 enzyme excrete little if any cotinine or nicotine glucuronide.18 Two single nucleotide polymorphisms in UGT2B10, a missense mutation, Asp67Tyr, and a splice variant, result in the expression of no functional enzyme.14,18,19 Smokers who are heterozygous for these variant alleles excrete ∼50% as much nicotine and cotinine glucuronide as do smokers who carry neither allele.17,18 The UGT2B10 splice variant (rs116294140) has an allele frequency of between 35 and 39% in African Americans,18−20 and its prevalence in African Americans results in significantly lower levels of nicotine and cotinine glucuronidation, and this contributes to the higher plasma cotinine concentrations typically observed in African American smokers compared to whites.21 169

DOI: 10.1021/acs.chemrestox.7b00264 Chem. Res. Toxicol. 2018, 31, 168−175

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Chemical Research in Toxicology

(30 mg, Phenomenex, Torrance, CA) that had been conditioned with 1 mL of methanol (MeOH), 0.5 mL of water, and 0.5 mL of 25 mM ammonium acetate pH 8.5. NNAL and its glucuronides were eluted with 2 mL of water (2 mL), 1 mL of MeOH, and 0.5 mL of 5% formic acid in MeOH. The flow through and water fraction containing NNAL-N-gluc were pooled (fraction 1). NNAL was eluted with MeOH (fraction 2), and NNAL-O-gluc was eluted with formic acid/MeOH (fraction 3). The volume of fraction 1 was reduced to ∼1.5 mL under a gentle stream of N2. Fractions 2 and 3 were each evaporated to dryness under a stream of N2 and resuspended in 1 mL of phosphate buffered saline. Fractions 1 and 3 were incubated with ß-glucuronidase (E. coli Sigma, 1600 Units) at 37 °C overnight. Then, the free NNAL present in each fraction was quantified by the method previously described by Carmella et al.24 with the following modifications: (1) Elution from the MCX 96-well plates was with 95:5 methanol/NH4OH, and eluted samples were dried under a stream of N2 and resuspended in 40 μL of 10 mM ammonium acetate, and (2) LC-MS/MS analysis was on a high-resolution/high-massaccuracy instrument with nanoflow LC. Each sample (1 μL) was injected onto a nanocolumn (75 μm ID, 10 cm length, 15 μm orifice) created by hand packing a commercially available fused-silica emitter (New Objective, Woburn MA) with Luna C18-bonded separation media (Phenomenex, Torrance, CA). NNAL was eluted with a mobile phase of 80% 5 mM ammonium acetate:20% acetonitrile and a flow rate of 1 μL/min for the first 5 min followed by a flow rate of 300 nL/min and analyzed by nano electrospray mass spectrometry on an LTQ Orbitrap Velos instrument (Thermo Scientific, Waltham, MA). The electrospray source voltage was set at 2.2 kV. The capillary temperature was 350 °C, and the S-Lens RF level was set at 66%. HRMS/MS monitoring was carried out at m/z 210.12 → m/z 149.0835 (NNAL), m/z 213.14 → m/z 149.0835 (d3-NNAL), m/z 215.16 → m/z 151.0962 (d5-NNAL), and m/z 216.14 → m/z 155.1037

Figure 1. Outline of the high-resolution nano-LC-MS/MS method for the analysis of NNAL, NNAL-O-glucuronide, and NNAL-N-glucuronide in smoker’s urine. aSPE = solid phase extraction. three fractions on the Strata XA column. The complete separation of each metabolite from the other was confirmed by adding a known amount of one metabolite at increasing concentrations (up to 2.6 pmol/mL) to nonsmokers urine and confirming that after β-glucuronidase treatment NNAL was detected only in the correct fraction (Figure 2). The method developed was as follows: Urine (200 μL) was mixed with 500 μL of 25 mM ammonium acetate pH 8.5 containing three internal standards (0.5 pmol 13C6-NNAL, 0.7 pmol d3-NNAL-N-gluc, and 0.4 pmol d5-NNAL-O-gluc) and loaded onto a Strata-XA column

Figure 2. High-resolution nano-LC-ESI-MS/MS chromatograms obtained from the analysis of NNAL in fractions 1−3 (Figure 1) of a smoker’s urine sample with three internal standards added (13C6-NNAL, d3-NNAL-N-gluc, and d5-NNAL-O-gluc). Monitoring was for (A) 210.12 → 149.08349 (NNAL), (B) 213.14 → 149.08349 (d3-NNAL), (C) 215.14 → 151.09623 (d5-NNAL), and (D) 216.14 → 155.10365 (13C6-NNAL). 170

DOI: 10.1021/acs.chemrestox.7b00264 Chem. Res. Toxicol. 2018, 31, 168−175

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Chemical Research in Toxicology

Table 1. Mean NNAL-Glucuronides as a Proportion of Total NNAL Stratified by Race/Ethnicity for Study 1, the Multiethnic Cohorta

NNAL glucuronides1 p-value (compared to whites) NNAL-O-glucuronides p-value (compared to whites) NNAL-N-glucuronides p-value (compared to whites) ratio of O-/N-glucuronideb p-value (compared to whites)

African Americans N = 297

Native Hawaiians, N = 246

whites, n = 354

Latinos, N = 362

Japanese Americans, N = 504

0.74 (0.73−0.75) 0.006 0.54 (0.52−0.55) 0.008 0.20 (0.19−0.21) 0.38 2.99 (2.68−3.34) 0.25

0.69 (0.68−0.70) 0.0001 0.46 (0.45−0.48)