Article pubs.acs.org/crt
Potential Association of Urinary N7‑(2-Carbamoyl-2-hydroxyethyl) Guanine with Dietary Acrylamide Intake of Smokers and Nonsmokers Chih-Chun Jean Huang,†,‡ Chia-Fang Wu,‡,§ Wei-Chung Shih,∥ Yu-Syuan Luo,∥ Ming-Feng Chen,‡ Chien-Ming Li,‡ Saou-Hsing Liou,‡ Wen-Sheng Chung,⊥ Su-Yin Chiang,*,# and Kuen-Yuh Wu*,∥ †
Department of Food Science, National Pingtung University of Science and Technology, Neipu, Pingtung 91201, Taiwan Division of Environmental Health and Occupational Medicine, National Health Research Institutes, Zhunan Town, Miaoli County 35053, Taiwan § Graduate Institute of Occupational Safety and Health and Department of Occupational Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan ∥ Department of Public Health & Institute of Occupational Medicine and Industrial Hygiene, College of Public Health, National Taiwan University, Taipei 10055, Taiwan ⊥ Department of Applied Chemistry, National Chiao Tung University, Hsinchu 30010, Taiwan # School of Chinese Medicine, China Medical University, Taichung 404, Taiwan ‡
S Supporting Information *
ABSTRACT: Acrylamide (AA), a rodent carcinogen, is widely used in industry and present in cigarette smoke as well as in foods processed at high temperatures. The metabolic activation of AA to glycidamide (GA) could be critical for AA carcinogenicity since GA causes DNA adduct formation in vivo. N7-(2-carbamoyl-2-hydroxyethyl) guanine (N7-GAG), the most abundant DNA adduct of AA, is subjected to spontaneous and enzymatic depurination and excreted through urine. Urinary N7-GAG analysis can confirm AA genotoxicity and identify active species of AA metabolites in humans, thereby serving as a risk-associated biomarker for molecular epidemiology studies. This study aimed to develop an isotopedilution solid-phase extraction liquid chromatography tandem mass spectrometry method to comparatively analyze urinary N7-GAG levels in nonsmokers and smokers. Urinary N-acetyl-S-(propionamide)-cysteine (AAMA), a metabolite of AA, was also analyzed as a biomarker for current AA exposure. Urinary N7-GAG was quantified by monitoring m/z 239 → 152 for N7-GAG and m/z 242 → 152 for 13C3-labeled N7-GAG under positive electron spray ionization and multiple reaction mode. The median urinary N7-GAG level was 0.93 μg/g creatinine in nonsmokers (n = 33) and 1.41 μg/g creatinine in smokers (n = 30). Multiple linear regression analysis of data revealed that N7-GAG levels were only significantly associated with AAMA levels. These results demonstrate that urinary N7GAG of nonsmokers and smokers is significantly associated with a very low level of dietary AA intake, assessed by analyzing urinary AAMA.
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was the most affected organ in B6C3F1 mice treated with AA.12 Epidemiology studies have been inconclusive to AA carcinogenicity because of its insignificant contribution to increases in cancer incidence in heavily exposed workers and populations with very low dietary exposure.14−19 Therefore, AA has been classified as a probable human carcinogen (Group 2A) by the International Agency for Research on Cancer.1 Upon AA absorption, AA can be metabolically activated to glycidamide (GA) by cytochrome P450 2E1.20,21 Both AA and GA can be detoxified by glutathione-S-transferase to form
INTRODUCTION Acrylamide (AA) is a highly water-soluble industrial chemical with an α,β-unsaturated carbonyl group. In addition to the widespread applications in industry and biotechnology laboratories,1 AAs were reported to be present in foods processed at high temperatures2−5 and in mainstream cigarette smoke, with contents ranging from 1.1 to 2.34 μg/cigarette.6 The general public may be at risk of AA exposure. AA induces neurotoxicity and reproductive toxicity, such as infertility, embryo implantation losses, and reduced postnatal survival.7−11 AA causes cancers, such as mammary fibroadenomas, thyroid follicular cell adenomas, and testicular mesotheliomas in rats.11−13 A recent bioassay reported that the Harderian gland © 2014 American Chemical Society
Received: July 7, 2014 Published: November 26, 2014 43
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Ammonium formate (AF) was bought from Fluka Biochemika (Steinheim, Germany). Formic acid (FA) was obtained from Riedelde Haën (Seelze, Germany). Water was purified using a Millipore Milli-RO/Milli-Q system (Bedford, MA). Syntheses of N7-GAG and 13C3-Labeled N7-GAG. To synthesize the N7-GAG, 102.7 mg of 2′-deoxyguanosine was first dissolved in 7.5 mL of water and 0.5 mL of FA, and then 1 g of GA was added into the solution.24,25 The mixture was stirred at room temperature overnight and heated in a water bath at 45 °C for 3 h. The mixture was purified by using an HPLC-UV system, which consisted of an autosampler (L-7250, Hitachi, Ltd., Tokyo, Japan), a pump (L7100, Hitachi) delivering an isocratic mobile phase of 86% ACN/ 0.01% FA at a flow rate of 3 mL/min through an analytical column (Diol-60, 150 × 10 mm, 5 μm, Thermo Electron Corporation, San Jose, CA), and a UV detector (L-7420, Hitachi) set at 254 nm. The fraction containing N7-GAG was collected from retention time at 16 to 17 min by using a fraction collector (L-5200, Hitachi) and dried in a rotor evaporator. The residue was dissolved in DMSO-d6 and characterized with 1H and 13C NMR (Varian-Unity INOVA-500 MHz spectrometer). 13C3-labeled N7-GAG was synthesized and purified with the same HPLC-UV system to serve as an internal standard. Study Subjects. This study was permitted by the Human Ethics Committee of the National Health Research Institutes, Taiwan. All study subjects were provided with written consent for their participation in this study. Spot urine samples were collected from each study subject, and questionnaires were used to collect their background information including age, gender, dietary intake, smoking status, lifestyle, occupational AA exposure history, betel-nut chewing habit, tea consumption, exercise habit, medical history, and medication records.22 A total of 63 subjects were recruited with 33 self-reported nonsmokers and 30 being smokers with no history of occupational AA exposure. They were 20 to 38 years old (median: 22), with a body mass index (BMI) ranging from 17.3 to 27.7 (median: 22.1). All urine samples were stored at −80 °C until used for analysis of N7-GAG, AAMA, cotinine, or creatinine. Solid-Phase Extractions for Urine Sample Cleanup. Prior to the N7-GAG analysis with LC-MS/MS, solid-phase extraction (SPE) using different combinations of SPE cartridges was studied for sample cleanup. The two-step cleanup procedures were developed with a combination of the NH2 and MCX SPE cartridges. Two hundred and 50 microliters of urine sample was spiked with 25 μL of 13C3-labeled N7-GAG (100 ng/mL) and mixed very well, and then 2.25 mL of ACN was added to precipitate proteins. The solution was centrifuged at 3000g for 10 min, and the supernatant was transferred into an NH2 SPE cartridge (500 mg, 3 c.c., Waters Corporation, Milford, MA) preconditioned with 3 mL of ACN. Then, the sample was washed with 2 mL of 80% ACN/0.1% FA and was eluted with 4 mL of 70% ACN/ 0.1% FA. The eluate added with 80 μL of FA and was then loaded into a Waters Oasis MCX cartridge (60 mg, 3 c.c.) pretreated with 3 mL of ACN followed by 3 mL of 70% ACN/2% FA. After the cartridge was washed with 2 mL of 70% ACN/2% FA and 2 mL of MeOH, the sample was eluted with 1.5 mL of 0.5% NH4OH in MeOH. The eluate was dried under a vacuum, and the residue was raised in 125 μL of 2 mM AF/3.6 mM FA. Finally, the sample was ready for analysis of N7GAG with online LC-MS/MS. Online LC-MS/MS for N7-GAG Determination. After being cleaned up with two-step SPE, 50 μL of the sample was injected into an online SPE LC-MS/MS system programmed as summarized in Supporting Information, Table S1 for N7-GAG analysis. The automated sample cleanup system was assembled as the previously described.34 It consisted of an autosampler (PE Series 200, PerkinElmer, Norfolk, CT), a switching valve (Two-position Microelectric Actuator, Valco, Houston, TX), a C18 cleanup cartridge (Inertsil ODS-3, 4.6 × 33 mm, 5 μm, GL Sciences Inc., Tokyo, Japan), and two sets of LC pumps, a quaternary pump and a micropump (PE Series 200, PerkinElmer). After injection, the sample was delivered to the cleanup cartridge with the mobile phase A (2 mM AF/3.6 mM FA), which served as a loading and washing solution, at a flow rate of 1 mL/min for 3 min. Then, a gradient was adopted for analysis of N7-
glutathione conjugates, which are further metabolized to their corresponding mercapturic acids and excreted through urine. Urinary mercapturic acids of AA (AAMA) and GA have been analyzed to demonstrate similar metabolic pathways between humans and rodents and validated as biomarkers for AA exposure.22,23 If not detoxified, GA can react with DNA bases to form DNA adducts. Nine DNA adducts have been identified from the in vitro reaction of GA with 2′-deoxydadenosine, 2′deoxyguanosine, cytidine, and thymidine.24−27 Liquid chromatography tandem mass spectrometry (LC-MS/MS) methods were developed to analyze GA-induced DNA adducts in rodents treated with AA or GA. Only N7-(2-carbamoyl-2hydroxyethyl)-guanine (N7-GAG) and N3-(2-carbamoyl-2hydroxyethyl)-adenine (N3-GAA) were detectable in neonatal and adult mice treated with AA or GA. N7-GAG was the most abundant adduct, with levels approximately 100-fold greater than those of N3-GAA. A superlinear dose−response for N7GAG in the tissues of adult mice treated with AA exhibited metabolic saturation.26 Although the GA-induced DNA adducts were relatively evenly distributed in the brains, testes, and livers of rats treated with AA, N3-GAA was more rapidly removed from the tissues than N7-GAG.26 The contents of N7-GAG and N3-GAA in the tissues of wild-type mice treated with AA were 52- to 66-fold greater than those in the tissues of CYP2E1-null type mice treated with AA, suggesting that CYP2E1 is the primary enzyme that metabolically activates AA to GA.28 The accumulation of N7-GAG in the testes of young rats was much higher than that in adult rats, suggesting that the testis is more vulnerable to AA genotoxicity in young rats than in adult rats.29 Female Sprague−Dawley (SD) rats treated with 0.1 to 100 μg/kg of AA generate N7-GAG levels equivalent to the low end of the background levels in humans.30 The chronic exposure to AA at doses as low as 1 μg/kg/day in mice caused increases in DNA damage in male germ cells, suggesting that chronic exposure to AA at doses equivalent to those experienced by humans might cause DNA damage in the reproductive organs of mice.31 N7-GAG has not been analyzed in DNA from human tissues. However, it may be spontaneously or enzymatically depurinated to form apurine sites in the DNA backbone and can be excreted in the urine. Urine samples are easy to assess with noninvasive methods, and the analysis of urinary DNA adducts has been validated to confirm the active species of carcinogens and to serve as exposure- and probable cancer risk-associated biomarkers in molecular epidemiology studies.32,33 Therefore, we aimed to develop an isotope-dilution liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method to analyze N7-GA-GAG in urine samples collected from smokers and nonsmokers. Urinary AAMA and cotinine were previously measured with LC-MS/MS to assess the current exposure to AA and smoking status in our study subjects.22,23 Statistical analyses of the levels of urinary N7-GAG with those of urinary AAMA and cotinine further elucidate the association between urinary N7-GAG and dietary AA intake of our study subjects.
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MATERIALS AND METHODS
Chemicals. GA and 13C3-labeled GA were purchased from Toronto Research Chemicals Inc. (North York, ON, Canada). Cotinine was obtained from Sigma (St. Louis, MO), and its deuterium analogue was obtained from Isotec (Miamisburg, OH). Methanol (MeOH), acetonitrile (ACN), and other solvents were of HPLC grade and were purchased from Mallinckrodt Baker Inc. (Phillipsburg, NJ). 44
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Figure 1. MS/MS product ion scan spectra of (A) N7-GAG and (B) its internal standard marked with an * for the position labeled with 13C. GAG from 3 to 3.1 min with the 95% mobile phase A and 5% mobile phase B (47.5% ACN/47.5% MeOH/10 mM AF), which was held for 1.5 min. At 4.5 min, the switch valve was switched to injection position, and the sample washed out from the cartridge was directed to the analytical column (Atlantis dC18, 2.1 × 150 mm, 3 μm, Waters Corporation). At 6 min, the valve was switched back to the beginning position. N7-GAG was eluted and delivered to the mass spectrometer at a retention time of 8.6 min. The analytical column was then cleaned with 100% mobile phase B, and the mobile phase was set back to the initial conditions ready for the next run. After cleaning, the analytical column was conditioned with initial mobile phase A before the injection of the next sample. The analytical column was connected to a triple quadrupole mass spectrometer with a TurboIonSpray source (API 3000, Applied Biosystems/MDS SCIEX, Foster City, CA), which was operated under positive mode with an ion spray voltage set at 5500 V. Nitrogen was used as the turbo gas with the temperature set at 400 °C, and as the nebulizer gas, curtain gas, and collision gas at settings of 8, 8, and 12, respectively. The multiple reaction monitoring (MRM) mode was operated to monitor the ion mass transitions of m/z 239 → 152 for N7-GAG with the dwell time set at 150 ms and of m/z 242 → 152 for N7-13C3GAG with the dwell time set at 100 ms. The calibration curves of N7-GAG were established for the concentrations ranging from 1, 2, 5, 8, 10, and 16 μg/L of N7-GAG prepared in the mobile phase and urine collected from a nonsmoker without any AA occupational exposure history and spiked with 2.5 ng of N7-13C3GAG (20 μg/L).
All data acquisition and quantitative processing were controlled by Analyst software, version 1.1 (Applied Biosystems). Statistic Methods. The Mann−Whitney U test was used to testify the differences in age, BMI, creatinine, N7-GAG, AAMA, and cotinine between the smokers and nonsmokers. The Fisher exact test and the Chi-square test were used to testify the significant differences in the categorical data between the two study groups. Spearman correlation coefficient was calculated to testify the association between urinary N7-GAG and independent variables. Simple linear regression was also used to study the linear association between urinary N7-GAG and independent variables. Multiple linear regression was used to adjust the confounding factors. All p-values were two-sided. Statistical analyses were conducted using the Statistical Package for the Social Sciences (SPSS), version 11.0 (SPSS Inc., Chicago, IL, USA).
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RESULTS Characterization of Purified N7-GAG. The purified N7GAG and its isotope analogue were characterized using electrospray ionization MS/MS operated in positive ion mode. Their molecular ions [M + H]+ at m/z 239 and 242, respectively, were the predominant ions in the Q1 mass spectra. In the product ion spectra, the most abundant product ion observed was m/z 152 for both N7-GAG at m/z 239 and N7-13C3GAG at m/z 242 (Figure 1). The positive MRM mode 45
dx.doi.org/10.1021/tx500265p | Chem. Res. Toxicol. 2015, 28, 43−50
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Figure 2. Representative LC-MS/MS chromatograms generated from analyzing a urine sample collected from a smoker by monitoring the ion mass transitions at m/z 239 → 152 for N7-GAG and m/z 242 → 152 for N7-13C3GAG.
regression and show excellent linearity; y = 0.040x + 0.002 (r2 = 0.999) for the mobile phase and y = 0.036x + 0.004 (r2 = 0.998) for urine. These two regression lines were compared, and their slopes were not significantly different (p = 0.629), suggesting that the effects of the urine matrix was insignificant. The detection limit of N7-GAG was 0.1 ng/mL (20 fmol) in the mobile phase on-column (signal-to-noise ratio = 3.3) and 0.25 ng/mL in urine (signal-to-noise ratio = 3.9). Analysis of Urinary N7-GAG in Smokers and Nonsmokers. The demographic characteristics of the study subjects, as categorized by either self-reported smokers (2−40 cigarettes/day) or nonsmokers, are depicted in Table 1. Their corresponding creatinine levels were 1.74 ± 0.51 [mean ± SD] and 1.59 ± 0.61, respectively. Their urinary N7-GAG, AAMA or cotinine levels were presented by adjustment with the creatinine level. These two groups also showed similar ages, BMI levels, exercise habits, and medication background. However, a greater number of smokers chewed betel nuts and drank tea, which are common in Taiwan, compared with the nonsmokers (p < 0.001 and p = 0.035, respectively). The urinary N7-GAG levels in the study subjects were then quantitated with our newly developed method by using the calibration curve constructed with a urine matrix. Figure 2 shows representative chromatograms of N7-GAG generated from an analysis of a urine sample collected from a smoker and demonstrates that our method possesses desirable sensitivity to accurately quantify urinary N7-GAG. For the self-reported smokers, the mean urinary N7-GAG level was 2.01 (0.92−5.11) (5th−95th percentile) ng/mL urine or 1.41 (0.61−6.22) μg/g creatinine, which was not significantly different from 1.50 (0.63−5.97) ng/mL urine or 0.93 (0.36−2.97) μg/g creatinine of the nonsmokers (p = 0.148 and p = 0.099, respectively) (Table 1). The smoking status of the study subjects was confirmed by the urinary cotinine contents, which showed that the smokers had significantly greater urinary cotinine levels than the nonsmokers (p < 0.001). Furthermore, greater urinary
was then applied for quantitation by monitoring the ion mass transitions of m/z 239 → 152 for N7-GAG and m/z 242 → 152 for the internal standard N7-13C3GAG. The NMR spectra of the purified N7-GA-GAG were consistent with those previous reported.26−30 Optimization of Solid-Phase Extraction for Urine Samples. Prior to the LC-MS/MS analysis, the urine samples were cleaned up using two-step SPE procedures. Figure 2 shows the representative LC chromatograms generated from the analysis of N7-GAG in a urine sample collected from a nonsmoker. The very clean chromatograms demonstrated that the two-step SPE and one-step online SPE are needed for the accurate quantitation of urinary N7-GAG. Method Validation. To evaluate the method performance, N7-GAG standard at a concentration of 5 ng/mL was spiked in the mobile phase and in a urine sample collected from a nonsmoker, and the standard solutions were used for the twostep SPE cleanup and online LC-MS/MS analysis. The precision, expressed as the relative standard deviation, was evaluated by analyzing these samples repeatedly within 1 day (4 replicates for the mobile phase and 3 replicates for the urine sample), and the entire experiment was also repeated on 3 different days. The intraday and interday variations for N7GAG in the mobile phase were calculated to be 8.5 and 6.5%, respectively, and those in urine were 3.7 and 2.3%, respectively. A carryover effect from sample to sample was not observed. The mean accuracy, defined as the percentage ratio of the spiked N7-GAG concentration calculated from the calibration curve over the expected spiked concentration in the mobile phase and urine matrices, was found to be 104.7 and 100.7%, respectively. The calibration curves were established by analyzing the standard solutions at 1, 2, 5, 8, 10, and 16 ng/L of N7-GAG prepared in the mobile phase or urine sample collected from a nonsmoker and spiked with 2.5 ng of N7-13C3GAG (20 ng/ mL). The obtained calibration curves were analyzed with linear 46
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Table 1. Urinary N7-GAG, AAMA, and Cotinine Concentrations Adjusted and Not Adjusted with Creatinine Level of the Study Subjects and Their Demographic Data Categorized as Self-Reported Smokers or Non-Smokers nonsmokers (n = 33) age (yr) BMI (kg/m2) creatinine (g/L urine)
smokers (n = 30)
Mean ± (SD) 22.9 ± 2.8 24.6 ± 5.7 22.0 ± 2.1 22.0 ± 3.2 1.7 ± 0.5 1.59 ± 0.6
p-value 0.856a 0.709a 0.299a
Median (5th−95th Percentile) N7-GAG μg/L urine μg/g creatinine AAMA μg/L urine μg/g creatinine cotinine μg/L urine μg/g creatinine betel-nut chewing no yes tea consumption no yes medication no yes regular exercise no yes a
1.50 (0.63−5.97) 0.93 (0.36−2.97)
2.01 (0.92−5.11) 1.41 (0.61−6.22)
0.148a 0.099a
112 (81.9−168) 68.5 (34.0−144)
152 (92.2−222) 98.8 (61.9−215)
