Article Cite This: J. Agric. Food Chem. 2018, 66, 1258−1263
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Urinary Methyleugenol-deoxyadenosine Adduct as a Potential Biomarker of Methyleugenol Exposure in Rats Yukun Feng,† Saide Wang,† Hui Wang,† Ying Peng,*,† and Jiang Zheng*,‡,† †
Wuya College of Innovation, Shenyang Pharmaceutical University, Shenyang, Liaoning 110016, People’s Republic of China State Key Laboratory of Functions and Applications of Medicinal Plants, Key Laboratory of Pharmaceutics of Guizhou Province and Guizhou Medical University, Guiyang, Guizhou 550004, People’s Republic of China
‡
ABSTRACT: Methyleugenol (ME), a natural ingredient of several herbs and spices used in the human diet, is hepatocarcinogenic in rodents. Following metabolic activation to the reactive carbocation intermediate, ME can bind covalently to DNA, which is directly associated with its carcinogenicity. In this work, a non-invasive approach to determine ME exposure was established by monitoring the urinary N6-(methylisoeugenol-3′-yl)-2′-deoxyadenosine (ME-dA) adduct. The developed method entails liquid−liquid extraction enrichment of urinary ME-dA, incorporation of deuterated ME-dA as an internal standard, and analysis by liquid chromatography coupled tandem mass spectrometry. Male rats (10−12 weeks, 180−200 g) were treated (p.o.) with ME, and ME-dA was excreted in urine in a dose- and time-dependent manner. The non-invasive approach enabled us to successfully determine exposure to ME-containing herbs and spices. These results suggest that ME-dA can potentially serve as an effective biomarker of ME exposure in rats. It is expected that the developed approach of detecting urinary ME-dA will facilitate the investigation of ME carcinogenesis. KEYWORDS: methyleugenol-deoxyadenosine adduct, biomarker, urine, LC−MS/MS, spices
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INTRODUCTION Methyleugenol (ME, 4-allyl-1,2-dimethoxybenzene), an allylalkoxybenzene compound, is a natural ingredient present in a variety of herbs and spices.1 It also has been found in blackberry essence, bananas, black pepper, bilberries, and walnuts.2 Essential oils and extracts of spices rich in ME have been approved for commercial use as fragrance and flavoring agents. As a result of its widespread use in flavoring food and beverages, the general population is exposed to ME at a certain level ranging from 14 to 217 μg kg−1 of body weight (bw) day−1 mainly through ingestion of dietary products, herbs, spices, and their essential oils on a daily basis.3 However, on the basis of sufficient evidence of carcinogenicity from studies in experimental animals, ME was classified by the National Toxicology Program (NTP) as a “reasonably anticipated to be human carcinogen”.4 ME-induced toxicities are believed to be associated with its metabolic activation.5−7 Many studies demonstrated that, after hydroxylation and sulfation, ME bioactivated to the corresponding carbocation intermediate could result in the formation of ME−DNA adducts in liver and extrahepatic tissues of rodents (primarily cecum, kidney, and stomach).8−10 It has been confirmed that the abundance of hepatic ME−DNA adducts was directly associated with its carcinogenicity.11,12 In general, ME−DNA adducts involved the binding of the 3′-C atom of ME to the exocyclic amino groups of purine bases. N2(Methylisoeugenol-3′-yl)-2′-deoxyguanosine (ME-dG) and N6(methylisoeugenol-3′-yl)-2′-deoxyadenosine (ME-dA) as ME− DNA adducts formed in vivo have been identified by mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy.13 ME−DNA adducts are also abundant in human tissue samples after exposure to ME. Tremmel and co-workers reported the detection of ME−DNA adducts in surgical human © 2018 American Chemical Society
liver samples from 121 subjects, and Monien et al. detected these adducts in 10 out of 10 human lung samples.14,15 Hence, ME−DNA adducts can been considered as effective biomarkers for DNA damage induced by ME. Currently, studies on ME−DNA adducts focus intensively on detection of DNA isolated from liver, lung, and kidney tissues. However, the tissue sampling process involves sacrificing the laboratory rodents, and these tissues are not typically available for human biomonitoring studies. Evidence has shown that the DNA adducts may be repaired by the nucleotide excision repair (NER) mechanism.16−19 Modified deoxynucleosides as degradation products of DNA adducts were eliminated via urination, and the deoxynucleoside adduct level in urine is an indicator of the balance between adduct formation and the repair system in response to the chemical damage at a certain point in time. Certain deoxynucleoside adducts in urine have served as biomarkers for carcinogen exposure and evaluation of cancer risk.20 We reasoned that ME−DNA adducts would be repaired and the corresponding deoxynucleoside adducts would be excreted in urine, and urinary ME-dA may be employed as a biomarker of ME exposure. The major objective of the present study was to define the correlation of ME or ME-containing herbs and spices exposure with the urinary excretion of ME-dA. It is anticipated that MEdA can serve as an effective biomarker of ME exposure and that the developed approach will facilitate the understanding of carcinogenicity of ME. Received: Revised: Accepted: Published: 1258
November 6, 2017 January 3, 2018 January 12, 2018 January 12, 2018 DOI: 10.1021/acs.jafc.7b05186 J. Agric. Food Chem. 2018, 66, 1258−1263
Article
Journal of Agricultural and Food Chemistry
Figure 1. Representative MRM chromatograms of ME-dA (m/z 428.2 → 177.1) and IS (m/z 431.2 → 180.1) obtained from analysis of (A) blank urine sample, (B) urine sample collected from a rat 12 h after an oral administration of ME, (C) blank urine sample spiked with ME-dA, and (D) urine sample spiked with IS. The MS/MS spectrum and proposed fragment assignments of (E) ME-dA and (F) IS.
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prepared by methylation of phenolic hydroxyl of eugenol using CD3I as a methylating agent, according to our reported method.21 High-Performance Liquid Chromatography−Tandem Mass Spectrometry (HPLC−MS/MS) Instrumentation and Analytical Conditions. The analysis was performed on an AB SCIEX Instruments 5500 triple quadrupole mass spectrometer (Applied Biosystems, Foster City, CA, U.S.A.) interfaced online with a 1260 infinity system (Agilent Technologies, Santa Clara, CA, U.S.A.). HPLC separation was carried out by gradient elution from a BDS HYPERSIL C18 ODS column (5.0 μm, 150 × 4.6 mm, Thermo, San Jose, CA, U.S.A.) with a flow rate of 0.8 mL/min of mobile phases, including 0.1% (v/v) formic acid in acetonitrile (A) and 0.1% (v/v) formic acid in water (B). Gradient elution was employed as follows: 20% A at 0−1 min, 20−40% A at 1−10 min, 40−90% A at 10−12 min, 90−20% A at 12−14 min, and 10% A at 14−17 min. ME-dA was analyzed in positive ion mode by multiple-reaction monitoring (MRM) scanning. The optimized mass spectrometric instrument parameters obtained after tuning were as follows: curtain gas (CUR), gas 1 (GS1), and gas 2 (GS2) were 35, 50, and 50 psi; ion source temperature (TEM) was at 650 °C; and ion spray voltage (IS) and entrance potential (EP) were 5500 and 10 V, respectively. The characteristics of ion pairs [corresponding to declustering potential (DP), collision energy (CE), collision cell exit potential (CXP)] were m/z 428.2 → 177.1 (80, 35, and 10) for ME-dA and m/z 431.2 → 180.1 (80, 35, and 10) for IS. Animals and Treatment. Male Sprague Dawley rats (180−200 g) were purchased from the Animal Center of Shenyang Pharmaceutical University (Shenyang, China). Rats had free access to food and water
MATERIALS AND METHODS
Chemicals and Reagents. ME (≥99.0%), eugenol (≥99.0%), and 2′-deoxyadenosine (dA, ≥99.0%) were purchased from Tokyo Chemical Industry (Shanghai) Development Co., Ltd. (Shanghai, China). CD3I (≥99.0%) was obtained from Shanghai Bodi Chemical Technology Co., Ltd. (Shanghai, China). Formic acid was acquired from Fisher Scientific (Springfield, NJ, U.S.A.). N-Bromosuccinimide (NBS) was purchased from Aladdin Reagent Co., Ltd. (Shanghai, China). Asari Radix, Acori Tatarinowii Rhizome, and Myristicae Semen were obtained from Tong-Ren-Tang Pharmacy (Shenyang, China). Shi San Xiang was purchased from Zhumadian Wang Shouyi Shi San Xiang Multiflavoured Spice Group Co., Ltd. (Henan, China). All organic solvents were obtained from Fisher Scientific (Springfield, NJ, U.S.A.). All reagents and solvents were either analytical or highperformance liquid chromatography (HPLC) grade. Chemical Synthesis. ME-dA was synthesized by bromination of ME, followed by reaction with dA. Briefly, a solution of NBS (0.15 g, 0.85 mmol) in CH2Cl2 (4.0 mL) was slowly added to a solution of ME (0.14 g, 0.77 mmol) in 4.0 mL of CH2Cl2. After stirring for 2 h at 40 °C, the solution was concentrated to dryness, reconstituted in 1.0 mL of methanol, mixed with a dA solution (0.25 g, 1.0 mmol, dissolved in 10 mL of water), and stirred for 2 h at 60 °C. The resulting mixture was concentrated to dryness, reconstituted with acetonitrile/water (50:50, v/v), centrifuged, and submitted to HPLC for purification. The purified product was submitted to mass spectrometry and NMR for characterization. Synthesis of deuterium-labeled d3-ME-dA as the internal standard (IS) was started by preparation of d3-ME. d3-ME was 1259
DOI: 10.1021/acs.jafc.7b05186 J. Agric. Food Chem. 2018, 66, 1258−1263
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Journal of Agricultural and Food Chemistry and were housed in a temperature-controlled (22 ± 4 °C) facility with a 12 h dark/light cycle for at least 5 days after receipt and before treatment. All animal studies were performed according to procedures approved by the Ethics Review Committee for Animal Experimentation of Shenyang Pharmaceutical University. Propanediol as a 20% aqueous solution was used as the vehicle for ME. Rats were treated with ME at doses of 1, 5, or 25 mg/kg by gavage (n = 4). In another study, rats (n = 4) were administered with the dose of 5 mg/kg of ME at 12 h per interval in 3 consecutive days. In a separate study, rats were randomly divided into five groups, and each group contained four animals. Each group of rats was treated with a single dose of vehicle, Asari Radix extracts (0.3 g/kg), Acori Tatarinowii Rhizoma extracts (1.0 g/kg), Myristicae Semen extracts (1.0 g/kg), or Shi San Xiang extracts (2.0 g/kg). In all studies, urine samples from the day prior to dosing and 12 h intervals post-dosing were collected over ice. During sample collection, the urine was filtered through a gauze filter to remove pieces of feces and crumbs of food pellets. The walls of the metabolic cages were rinsed with distilled water, and the washes were added to the main portion of collected urine. The collected urine samples were processed immediately for LC−MS/MS quantification of urinary ME-dA. Standard Solution Preparation. An appropriate amount of MEdA was weighed and dissolved in methanol to produce the stock solution to obtain a concentration of 1.25 mg/mL. The stock solution was further diluted with methanol to obtain working solutions at six concentration levels. The IS working solution (450 ng/mL) was prepared by dissolving d3-ME-dA with methanol. Assay standard samples for construction of a six-point calibration curve were prepared by spiking blank rat urine with the corresponding working solutions (100 μL) and IS (100 μL), and the final concentrations of the calibration standards were 0.25, 0.5, 1.25, 2.5, 6.25, and 12.5 ng/mL. All of the solutions were stored in a refrigerator at 4 °C. Urine Sample Preparation. The collected urine samples (1.0 mL) were spiked with 100 μL of IS solution, followed by mixing with 2.0 mL of methanol, vortexing for 3 min, and centrifuging at 16000g for 5 min at 4 °C. The supernatants were collected and evaporated to dryness under a nitrogen gas stream. The resulting residue was reconstituted with 1.0 mL of H2O and then extracted with 3.0 mL of ethyl acetate. The organic phase was collected and evaporated to dryness. Each dried residue was reconstituted with 100 μL of the incipient mobile phase, and 10 μL of the supernatant was injected to LC−MS/MS for analysis.
tested to enrich analytes. Ethyl acetate showed better extraction efficiency than the others; therefore, ethyl acetate was selected as the extraction solvent. Various volumes of ethyl acetate in the range of 1.0−5.0 mL were investigated to optimize the extraction efficiency. The extraction efficiency increased with the increase of the volume applied until 3.0 mL was employed. Calibration Curve, Lower Limit of Quantification, and Recovery. The typical chromatograms from LC−MS/MS analysis of ME-dA and d3-ME-dA are displayed in Figure 1. No such peak responsible for ME-dA was observed in the urine samples collected from vehicle-treated rats. A calibration curve was established by computing the regression line of the peak area ratios (Y) of ME-dA to d 3 -ME-dA versus the concentrations (X) of ME-dA using a 1/x2 weighted least squares linear regression model. The typical regression equation with a correlation coefficient (r) was Y = 0.2234X − 0.002 with good linearity (r = 0.9996) (Figure 2). The lower
Figure 2. Calibration curve of ME-dA with IS.
limit of quantification (LLOQ) determining the sensitivity of the method was assessed as the lowest concentration on the calibration curve and as the concentration level with a signal-tonoise (S/N) ratio of 10. The LLOQ of ME-dA was 0.25 ng/ mL. The extraction recovery was evaluated by comparing peak areas obtained from extracted spiked samples to those of the post-extracted spiked samples at a corresponding concentration. The extraction efficiency of ME-dA was determined by analyzing six replicate samples at a final concentration of 2.5 ng/mL. The average of extraction recovery was 80.0%, and the relative standard deviation (RSD) value was 6.6%. The data obtained from the validation procedure suggest that this method exhibited good linearity and acceptable recovery. Urinary ME−DNA Adduct Study in Rats. ME-dA was detected in the urine samples of ME-treated animals, and the approach developed in the present study enabled us to quantify urinary ME-dA. The amount of the adduct excreted in urine was found to be proportional to the doses of ME applied in rats (Figure 3). Interestingly, a plateau of urinary ME-dA content was observed in animals after the fourth consecutive administration at the dose of 5 mg/kg. The level of urinary ME-dA was undetectable (under the limit of quantification) 60 h after the last administration (Figure 4). It has been reported that metabolic activation of ME leads to the formation of mutagenic DNA adducts.22 On the basis of NER mechanisms, ME−DNA adduction can be repaired and the corresponding deoxynucleoside adducts were eliminated via urination (Scheme 1). Urinary deoxynucleoside or nucleobase adducts derived from some important carcinogens, such as aflatoxin B1, benzo[a]pyrene, benzene, and aristolochic acid, have been proposed to be valuable non-invasive biomarkers of
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RESULTS AND DISCUSSION Characterization of Synthetic ME-dA. Synthetic ME-dA was characterized by MS and 1H NMR. The high-resolution mass spectrum showed [M + H]+ ions at m/z 428.1933, which corresponded to the formula C 12 H26N 5 O5 (calculated: 428.1928). The 1H NMR data and MS identities of synthetic ME-dA were consistent with published results.13 NMR characterization of ME-dA: 1H NMR (DMSO-d6, 600 MHz), δ 2.25 (m, 1H, H2′′′a), 2.59 (dt, 1H, H2′′′b), 3.50 (m, 1H, H5′′′a), 3.62 (m, 1H, H5′′′b), 3.70 (s, 3H, OCH3), 3.72 (s, 3H, OCH3), 3.85 (dd, 1H, H4′′′, J = 4.0 and 1.2 Hz), 4.23 (S, 2H, H3′), 4.39 (m, 1H, H3′′′), 5.30 (m, 1H, OH-5′′′), 5.41 (dd, 1H, OH-3′′′, J = 5.2 and 2.7 Hz), 6.25 (m, 1H, H2′), 6.33 (dd, 1H, H1′′′, J = 6.3 and 7.5 Hz), 6.42 (d, 1H, H1′, J = 15.9 Hz), 6.84 (m, 1H, H6, or H5), 6.87 (m, 1H, H5, or H6), 6.98 (d, 1H, H3, J = 1.3 Hz), 8.06 (bs, 1H, H6′′), 8.20 (bs, 1H, H2′′), 8.33 (s, 1H, H8′′). Optimization of Sample Preparation. The performance of the sample preparation method was based on the recovery of ME-dA in the urine matrix and the loss of signal intensity caused by ion suppression. To reduce the matrix effect, the urine samples were initially precipitated by methanol, and urinary ME-dA was then enriched by liquid−liquid extraction (LLE). Dichloromethane, ethyl acetate, and n-hexane were 1260
DOI: 10.1021/acs.jafc.7b05186 J. Agric. Food Chem. 2018, 66, 1258−1263
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Journal of Agricultural and Food Chemistry
galangal, fennel, pepper, and nutmeg. Asari Radix and Acori Tatarinowii Rhizome are representative Chinese herbs rich in ME and have a long history of use in the clinic. We examined the urinary excretion of ME-dA in rats after exposure to those model ME-containing herbal medicines or spices. The contents of ME in the extracts of Asari Radix, Acori Tatarinowii Rhizome, nutmeg, and Shi San Xiang tested were 1.01, 0.84, 1.23, and 0.96 mg/g, respectively. Rats were orally administered with the corresponding extracts individually at 0.3, 1.0, 1.0, and 2.0 g/kg, respectively, and urinary ME-dA was analyzed and quantified. As expected, ME-dA was detected in the urine samples of all rats after exposure to the individual extracts. Additionally, the content of urinary ME-dA was found to be almost proportional to the content of ME detected in the extracts administered in the animals (Figure 5). These findings indicate that ME-dA would be a potential biomarker for ME exposure.
Figure 3. Dose-dependent and time-course changes in the levels of urinary ME-dA. Rats were dosed orally with ME at 1, 5, and 25 mg/kg. Urine samples of 12 h intervals were collected through the experiment. The data represent the mean ± standard deviation (SD) for four rats.
Figure 4. Urinary excretion of ME-dA in rats treated with ME. Rats were orally dosed with ME at 5 mg/kg at 12 h per interval in 3 consecutive days. Urine samples of 12 h intervals were collected through the experiment. The data represent the mean ± SD for four rats.
Figure 5. Levels of urinary ME-dA in Asari Radix (0.3 g/kg), Acori Tatarinowii Rhizome (1.0 g/kg), Myristicae Semen (1.0 g/kg), and Shi San Xiang (2.0 g/kg) dosed rats. Urine samples at 12 h post-dosing were collected. The data represent the mean ± SD for four rats.
exposure.23−26 As such, a possible finding of urinary deoxynucleoside adducts of ME would be an important contribution to the biomarker of exposure to ME. The most important feature of the urinary DNA adduct biomarkers, besides the possibility of non-invasive sampling, is their capability to reflect the extent of compound-specific damage to DNA. To investigate the carcinogenesis of ME, we sought to develop a non-invasive approach for the sensitive assessment of ME exposure by quantifying urinary-excreted deoxynucleoside adducts of ME. Human exposure to ME primarily occurs via ingestion of herbs and spices.2 It is anticipated that ME-dA could be employed as a reliable biomarker after ME-containing herbs and spices exposure. Nutmeg is a common ME-containing spice used in the human diet. Shi San Xiang, awarded “China TimeHonored Brand”, is a very popular multi-flavored natural seasoning. It consists of many spices, such as clove, cinnamon,
It is worth noting that no urinary ME-dG was detected until the oral dose of ME reached 25 mg/kg. Surprisingly, the level of ME-dG was lower than that of ME-dA in the urine, which is inconsistent with the report that the level of ME-dG was 50− 70-fold higher than that of ME-dA in liver and other tissues of animals given ME.27 A possible explanation could be the poor efficiency for repair of ME-derived DNA adduction at dG. Glutathione (GSH) conjugation is a prominent detoxification pathway of metabolism of electrophilic species.28 Our early study demonstrated that a high dose (100 mg/kg) of ME was required to see urinary GSH and related conjugates derived from electrophilic metabolites of ME in rats.29 The present study showed high abundance of ME-dA in urine of rats given ME at 5 mg/kg. This could result from high volume of ME-
Scheme 1. Proposed Pathway for the Formation of Urinary ME-dA
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DOI: 10.1021/acs.jafc.7b05186 J. Agric. Food Chem. 2018, 66, 1258−1263
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methyleugenol to 1′-hydroxymethyleugenol in Fischer 344 rat and human liver microsomes. Carcinogenesis 1997, 18, 1775−1783. (6) National Toxicology Program (NTP). NTP Toxicology and Carcinogenesis Studies of Methyleugenol (CAS NO. 93-15-2) in F344/n Rats and B6C3F1 Mice (Gavage Studies); NTP: Research Triangle Park, NC, 2000; https://ntp.niehs.nih.gov/ntp/htdocs/lt_rpts/tr491.pdf (accessed Oct 29, 2017). (7) Groh, I. A.; Rudakovski, O.; Gründken, M.; Schroeter, A.; Marko, D.; Esselen, M. Methyleugenol and oxidative metabolites induce DNA damage and interact with human topoisomerases. Arch. Toxicol. 2016, 90, 2809−2823. (8) Ellis, J. K.; Carmichael, P. L.; Gooderham, N. J. DNA adduct levels in the liver of the F344 rat treated with the natural flavour methyl eugenol. Toxicology 2006, 226, 73−74. (9) Herrmann, K.; Engst, W.; Meinl, W.; Florian, S.; Cartus, A. T.; Schrenk, D.; Appel, K. E.; Nolden, T.; Himmelbauer, H.; Glatt, H. Formation of hepatic DNA adducts by methyleugenol in mouse models drastic decrease by Sult1a1 knockout and strong increase by transgenic human SULT. Carcinogenesis 2014, 35, 935−941. (10) Herrmann, K.; Engst, W.; Florian, S.; Lampen, A.; Meinl, W.; Glatt, H. The influence of the SULT1A status−wild-type, knockout or humanized−on the DNA adduct formation by methyleugenol in extrahepatic tissues of mice. Toxicol. Res. 2016, 5, 808−815. (11) Waddell, W. J.; Crooks, N. H.; Carmichael, P. L. Correlation of Tumors with DNA Adducts from Methyl Eugenol and Tamoxifen in Rats. Toxicol. Sci. 2004, 79, 38−40. (12) Paini, A.; Scholz, G.; Marin-Kuan, M.; Schilter, B.; O’Brien, J.; van Bladeren, P. J.; Rietjens, I. M. Quantitative comparison between in vivo DNA adduct formation from exposure to selected DNA-reactive carcinogens, natural background levels of DNA adduct formation and tumour incidence in rodent bioassays. Mutagenesis 2011, 26, 605−618. (13) Herrmann, K.; Engst, W.; Appel, K. E.; Monien, B. H.; Glatt, H. Identification of human and murine sulfotransferases able to activate hydroxylated metabolites of methyleugenol. Mutagenesis 2012, 27, 453−462. (14) Tremmel, R.; Herrmann, K.; Engst, W.; Meinl, W.; Klein, K.; Glatt, H.; Zanger, U. M. Methyleugenol DNA adducts in human liver are associated with SULT1A1 copy number variations and expression levels. Arch. Toxicol. 2017, 91, 3329−3339. (15) Monien, B. H.; Schumacher, F.; Herrmann, K.; Glatt, H.; Turesky, R. J.; Chesné, C. Simultaneous Detection of Multiple DNA Adducts in Human Lung Samples by Isotope-Dilution UPLC-MSMS. Anal. Chem. 2015, 87, 641−648. (16) Lukin, M.; Zaliznyak, T.; Johnson, F.; de los Santos, C. Structure and stability of DNA containing an aristolactam II-dA lesion: Implications for the NER recognition of bulky adducts. Nucleic Acids Res. 2012, 40, 2759−2770. (17) Sidorenko, V. S.; Yeo, J. E.; Bonala, R. R.; Johnson, F.; Schärer, O. D.; Grollman, A. P. Lack of recognition by global-genome nucleotide excision repair accounts for the high mutagenicity and persistence of aristolactam-DNA adducts. Nucleic Acids Res. 2012, 40, 2494−2505. (18) Yan, S.; Wu, M.; Buterin, T.; Naegeli, H.; Geacintov, N. E.; Broyde, S. Role of Base Sequence Context in Conformational Equilibria and Nucleotide Excision Repair of Benzo[a]pyrene Diol Epoxide-Adenine Adducts. Biochemistry 2003, 42, 2339−2354. (19) Geacintov, N. E.; Broyde, S.; Buterin, T.; Naegeli, H.; Wu, M.; Yan, S.; Patel, D. J. Thermodynamic and structural factors in the removal of bulky DNA adducts by the nucleotide excision repair machinery. Biopolymers 2002, 65, 202−210. (20) Vineis, P.; Perera, F. DNA adducts as markers of exposure to carcinogens and risk of cancer. Int. J. Cancer 2000, 88, 325−328. (21) Feng, Y.; Wang, H.; Wang, Q.; Huang, W.; Peng, Y.; Zheng, J. Chemical interaction of protein cysteine residues with reactive metabolites of methyleugenol. Chem. Res. Toxicol. 2017, 30, 564−573. (22) Cartus, A. T.; Herrmann, K.; Weishaupt, L. W.; Merz, K. H.; Engst, W.; Glatt, H.; Schrenk, D. Metabolism of methyleugenol in liver microsomes and primary hepatocytes: Pattern of metabolites,
derived DNA adduction relative to that arising from GSH conjugation. This observation could be interpreted by concepts of hard and soft electrophiles/nucleophiles.30−32 Briefly, hard electrophiles (alkyl carbonium ion) react predominantly with hard nucleophiles (amino group of purine bases) rather than soft nucleophiles (sulfhydryl group of cysteine or GSH). To summarize, urinary ME-dA is better than the corresponding GSH conjugates as a biomarker for ME exposure. In conclusion, ME-dA could be excised from ME−DNA adducts by the NER mechanism and the urinary excretion of ME-dA occurred in a time- and dose-dependent manner. Results from this study indicate that it is possible to monitor ME exposure non-invasively by quantifying urinary-excreted ME-dA. The work suggests that urinary ME-dA may be a valuable biomarker for monitoring ME exposure. The developed analytical approach was sensitive and selective and will facilitate the investigation of the mechanisms of ME carcinogenesis.
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AUTHOR INFORMATION
Corresponding Authors
*Telephone: +86-24-23986361. Fax: +86-24-23986510. E-mail:
[email protected]. *Telephone: +86-24-23984215. Fax: +86-24-23986510. E-mail:
[email protected]. ORCID
Jiang Zheng: 0000-0002-0340-0275 Funding
This work was supported in part by the National Natural Science Foundation of China (81430086, 81373471, and 81773813). Notes
The authors declare no competing financial interest.
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ABBREVIATIONS USED ME, methyleugenol; dA, 2′-deoxyadenosine; dG, 2′-deoxyguanosine; GSH, glutathione; NER, nucleotide-excision repair; NBS, N-bromosuccinimide; NMR, nuclear magnetic resonance; LLE, liquid−liquid extraction; LLOQ, lower limit of quantification; CE, collision energy; CXP, cell exit potential; DP, declustering potential; EP, entrance potential; MRM, multiple-reaction monitoring
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REFERENCES
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DOI: 10.1021/acs.jafc.7b05186 J. Agric. Food Chem. 2018, 66, 1258−1263
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DOI: 10.1021/acs.jafc.7b05186 J. Agric. Food Chem. 2018, 66, 1258−1263
