Analysis of Ethylated Thymidine Adducts in Human Leukocyte DNA by

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Analysis of Ethylated Thymidine Adducts in Human Leukocyte DNA by Stable Isotope Dilution Nanoflow Liquid Chromatography− Nanospray Ionization Tandem Mass Spectrometry Hauh-Jyun Candy Chen,* Yi-Ching Wang, and Wen-Peng Lin Department of Chemistry and Biochemistry, National Chung Cheng University, 168 University Road, Ming-Hsiung, Chia-Yi 62142, Taiwan S Supporting Information *

ABSTRACT: Studies showed that levels of ethylated DNA adducts in certain tissues and urine are higher in smokers than in nonsmokers. Because cigarette smoking is a major risk factor of various cancers, DNA ethylation might play an important role in cigarette smoke-induced cancer formation. Among the ethylated DNA adducts, O2-ethylthymidine (O2-edT) and O4-ethylthymidine (O4-edT) are poorly repaired and are accumulated in the body. In addition, O4-edT possesses promutagenic properties. In this study, we have developed a highly sensitive, accurate, and quantitative assay for simultaneous detection and quantification of O2-edT, N3-edT (N3-ethylthymidine), and O4-edT adducts by isotope dilution nanoflow liquid chromatography−nanospray ionization tandem mass spectrometry (nanoLC−NSI/MS/MS). Under the highly selected reaction monitoring (H-SRM) mode, the detection limit of O2-edT, N3-edT, and O4-edT injected on-column was 5.0, 10, and 10 fg, respectively. The quantification limit for the entire assay was 50, 100, and 100 fg of O2-edT, N3-edT, and O4-edT, respectively, corresponding to 1.1, 2.3, and 2.3 adducts in 109 normal nucleotides, respectively, starting with 50 μg of DNA (from 1.5−2.0 mL of blood). Levels of O2-edT, N3-edT, and O4-edT in 20 smokers’ leukocyte DNA were 44.8 ± 52.0, 41.1 ± 43.8, 48.3 ± 53.9 in 108 normal nucleotides, while those in 20 nonsmokers were 0.19 ± 0.87, 4.1 ± 13.3, and 1.0 ± 2.9, respectively. Levels of O2-edT, N3-edT, and O4-edT in human leukocyte DNA are all significantly higher in smokers than in nonsmokers, with p values of 0.0004, 0.0009, and 0.0004, respectively. Furthermore, levels of O2-edT show a statistically significant association (γ = 0.4789, p = 0.0327) with the smoking index in smokers. In the 40 leukocyte DNA samples, the extremely significant statistical correlations (p < 0.0001) are observed between levels of O2-edT and O4-edT (γ = 0.9896), between levels of O2-edT and N3-edT (γ = 0.9840), and between levels of N3-edT and O4-edT (γ = 0.9901). To our knowledge, this is the first mass spectrometrybased assay for ethylated thymidine adducts. Using this assay, the three ethylated thymidine adducts were detected and quantified for the first time. Therefore, this highly sensitive, specific, and accurate assay should be clinically feasible for simultaneous quantification of the three ethylated thymidine adducts as potential biomarkers for exposure to ethylating agents and for cancer risk assessment.

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bases, including N3 of adenine, O6 and N7 of guanine, and O2, O4, and N3 of thymine (Scheme 1).8−13,16 Among the ethylated DNA base adducts, O6-ethylguanine and O4-ethylthymidine (O4-edT) are miscoding lesions and they are related to cancer formation in animal studies.17,18 On the other hand, O6-ethylguanine can be efficiently repaired but not O2-ethylthymidine (O2-edT) or O4-edT.18−22 Both O2-edT and O4-edT accumulate as persistent DNA lesions.18−20 3-Ethyladenine and N7-ethylguanine undergo spontaneous depurination and they have been detected in smokers’ urine.9−11 Levels of urinary 3-ethyladenine are higher in smokers than in nonsmokers.9,10 N7-Ethylguanine in urine and in the liver are shown to be higher in smokers than in nonsmokers.11,12 A positive correlation was obtained between levels of O4-edT and PAH−DNA adducts in lung DNA of lung cancer patients, implying that both adducts are formed from cigarette smoke as

umans are constantly exposed to various chemicals, which modify DNA structure, form a plethora of DNA adducts, and cause mutation and possible cancer formation if these DNA adducts are not efficiently repaired. DNA adduct levels in a tissue reflect the balance between adduct formation and the repair capacity of the tissue upon chemical damage. Because DNA adduction is involved in the initiation stage of carcinogenesis, DNA adducts have been used as biomarkers of exposure to carcinogens and cancer risk assessment.1 Alkylating agents are commonly found in the environment. For instance, cigarette smoke contains polycyclic aromatic hydrocarbons (PAHs) and tobacco-specific nitrosamines, which are potent alkylating agents after metabolized by cytochrome P450 enzymes.2,3 Direct-acting nonbulky alkylating agents are also present in cigarette smoke. Methylated and ethylated DNA adducts have been identified in human tissues and urine.4−13 Unlike methylated DNA adducts, levels of ethylated DNA adducts have been shown to be higher in smokers than in nonsmokers in some studies.5,6,9−13 Ethylation of DNA can take place on the phosphate backbones14,15 as well as on the © 2012 American Chemical Society

Received: December 21, 2011 Accepted: January 23, 2012 Published: January 24, 2012 2521

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etry (nanoLC−NSI/MS/MS) under the highly selected reaction monitoring (H-SRM) mode. This assay is used for detection and quantification of these three ethylated dT adducts in leukocyte DNA from healthy individuals, and their levels are compared with smokers and nonsmokers.

Scheme 1. Structures of Ethylated Base Adducts

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EXPERIMENTAL SECTION Caution: N-Ethyl-N-nitrosourea is a mutagen and carcinogen, and iodoethane is a highly toxic volatile chemical. They should be handled using gloves and safety glasses in a well-ventilated hood. Materials. Calf thymus DNA and all the enzymes used for DNA hydrolysis were obtained from Sigma Chemical Co. (St. Louis, MO) except that alkaline phosphatase was a photodiode array detector (L-7450A), from Calbiochem Chemical Co. (La Jolla, CA). [13C10,15N2]2′-Deoxythymidine was purchased from Cambridge Isotope Laboratories (Andover, MA). All reagents are of reagent grade or above. HPLC−UV Conditions. The HPLC−UV system consisted of a Hitachi L-7000 pump system with a photodiode array detector (L-7450A), a D-7000 interface (Hitachi, Tokyo, Japan), a Rheodyne injector, and a reversed phase C18 column [Gemini C18(2), 4.6 mm × 250 mm, 5 μm, 100 Å (Phenomenex, Torrance, CA)]. It was eluted at a flow rate of 1.0 mL/min with a linear gradient from 0 to 100% aqueous methanol from 0 to 30 min. The methanol concentration was maintained at 100% for 10 min before conditioning with 100% water for 20 min. NMR Spectrometer. 1H and 13C NMR spectra were recorded on a Varian-Unity INOVA-500 MHz NMR spectrometer. Chemical shifts are presented in parts per million from the internal standard tetramethylsilane. Synthesis of O2-edT, N3-edT, and O4-edT Standards. The procedures reported by Robins and co-workers were followed.26 N-Ethyl-N-nitrosourea (150 mg, 1.28 mmol) was dissolved in 1.0 mL of 40% aqueous KOH and 1.5 mL of diethyl ether and stirred vigorously at 0 °C for 20 min to generate diazoethane. To the upper layer of the solution was added 2′-deoxythymidine (1.0 mg, 4.13 nmol) in 0.7 mL of methanol and stirred at room temperature for 1 h. The mixture was subjected to HPLC−UV and collected from 15 to 16.5 min for O2-edT, from 17 to 18.5 min for N3-edT, and from 19 to 20.5 min for O4-edT. The collected fractions were evaporated to dryness. O2-edT: 1H NMR (500 MHz, D2O) δ 7.78 (s, 1H, H-6), 6.28 (t, 1H, H-1′), 4.41 (q, 1H, H-3′, 2H, H-8), 3.99 (q, 1H, H-4′), 3.80 (dd, 1H, H-5′), 3.71 (dd, 1H, H-5″), 2.39 (m, 2H, H-2′), 1.88 (s, 3H, H-7), 1.34 (t, 3H, H-9). 13C NMR (500 MHz, D2O) δ 175.3 (C-4), 156.07 (C-2), 136.58 (C-6), 116.42 (C-5), 87.08 (C-4′), 86.82 (C-1′), 70.20 (C-3′), 66.27 (C-5′), 61.04 (C-8), 39.47 (C-2′), 13.47 (C-7), 12.83 (C-9). Yield: 22%. N3-edT: 1H NMR (500 MHz, D2O) δ 7.59 (s, 1H, H-6), 6.27 (t, 1H, H-1′), 4.41 (td, 1H, H-3′), 3.97 (q, 1H, H-4′), 3.89 (q, 2H, H-8), 3.79 (dd, 1H, H-5′), 3.71 (dd, 1H, H-5″), 2.32 (m, 2H, H-2′), 1.86 (s, 3H, H-7), 1.11 (t, 3H, H-9). Yield: 53%. O4-edT: 1H NMR (500 MHz, D2O) δ 7.84 (s, 1H, H-6), 6.22 (t, 1H, H-1′), 4.40 (td, 1H, H-3′), 4.34 (q, 2H, H-8), 4.02 (q, 1H, H-4′), 3.82 (dd, 1H, H-5′), 3.73 (dd, 1H, H-5″), 2.42 (m, 1H, H-2′), 2.26 (m, 1H, H-2′), 1.93 (s, 3H, H-7), 1.33 (t, 3H, H-9). 13C NMR (500 MHz, D2O) δ 171.58 (C-4), 157.77 (C-2), 140.38 (C-6), 107.74 (C-5), 86.97 (C-4′), 86.56 (C-1′), 70.41 (C-3′), 64.40 (C-5′), 61.18 (C-8), 39.66 (C-2′), 13.49 (C-7), 11.47 (C-9). The 1H and 13C NMR spectra data of O4-edT are in agreement with those reported in the literature for

the main exposure source.5 Furthermore, a study demonstrated significantly elevated levels of N-terminal N-ethylvaline in hemoglobin of smokers compared to nonsmokers.23 These results lead to the postulation that ethylated DNA adducts originate from ethylating agents in cigarette smoke. Evaluation of the roles of ethylated DNA adducts in carcinogenesis requires highly sensitive, specific, qualitative, and quantitative methods for their analyses in vivo. Urinary 3ethyladenine was analyzed by gas chromatography with electron impact ionization mass spectrometry (GC-EI/MS).8−10 Analyses of N7-ethylguanine in human urine and liver samples were performed by liquid chromatography with electrospray ionization tandem mass spectrometry (LC−ESI/MS/MS).11,12 These mass spectrometry-based studies used the stable isotope of the analyte as the internal standard, and these methods are referred to as stable isotope dilution mass spectrometry (SIDMS) methodology,24,25 which provides the highest possible specificity for quantitative measurements. Accurate quantification of trace amounts of analyte in a complex matrix requires an appropriate internal standard. An isotopomer that is structurally identical to the analyte is the ideal internal standard because it has identical chemical and physical properties as those of the analyte except for the mass. The stable isotope-labeled internal standard also serves as a carrier for the minute amounts of analyte going through the sample processing steps. The isotope-labeled standard can be used to identify the analyte peak in the complicated chromatogram for the analyte based on the nearly identical retention times in the chromatograms of the analyte and the isotope-labeled standard. Hitherto, there is no MS-based method available for analyzing ethylated thymine adducts. The mutagenic O4-edT has been detected and quantified in liver, but not leukocyte, from healthy individuals,4 in lung tissue from cancer patients,5 in cells from the lower respiratory tract,6 and from smokers’ lung tissue.13 Measurement of O4-edT in these studies was performed by the 32 P-postlabeling techniques. Unlike the SID-MS-based methodology, the internal standards used in these 32P-postlabeling assays5,6,13 are not similar to the analyte in terms of the structure or the retention properties on high-pressure liquid chromatography (HPLC) and thin layer chromatography (TLC). In this study, we developed a highly sensitive and quantitative assay for simultaneous detection and quantification of ethylthymidine (dT) adducts, including O2-edT, N3edT, and O4-edT, by stable isotope dilution nanoflow liquid chromatography−nanospray ionization tandem mass spectrom2522

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Figure 1. HPLC−UV chromatogram of reaction mixture of dT with N-ethyl-N-nitrosourea.

containing DNA (50 μg), the internal standards, and DNase 1 (10.2 units) in 10 mM sodium citrate (pH 6.5) and 10 mM MgCl2 was incubated at 37 °C for 1 h. To the reaction mixture was then added phosphodiesterase I (0.0016 unit) and alkaline phosphatase (0.03 unit) in 0.1 M Tris buffer (pH 8.0), and the mixture was incubated at 37 °C for 18 h. Adduct Enrichment. The hydrolysate was filtered through a 0.22 μm Nylon syringe filter and then purified using a solidphase extraction (SPE) column [Bond Elut Bonded phase C18OH, nonend-capped, 500 mg, 3 mL, Varian (Harbor City, CA)] after conditioning with 15 mL of methanol, followed by 15 mL of water. After sample loading, the SPE column was washed with 12 mL of H2O and 3 mL of 15% aqueous MeOH, and the three ethylated adducts were eluted with 3 mL of 40% aqueous MeOH. The fraction containing these adducts was dried, redissolved in 10 μL of 0.1% acetic acid (pH 3.2), and passed through a 0.22 μm Nylon syringe filter. A volume of 2 μL of the processed sample was subjected to the nanoLC− NSI/MS/MS analysis described below. NanoLC−NSI/MS/MS Analysis. A 2 μL injection loop was connected to a six-port switching valve injector into an LC system consisting of an UltiMate 3000 Nano LC system (Dionex, Amsterdam, The Netherlands) and a reversed phase column packed in-house (75 μm × 11 cm, Magic C18AQ, 5 μm, 200 Å, Michrom BioResource, Album, CA). The pump output (30 μL/min) was split before the injection port to a flow rate of 300 nL/min. Mobile phase A was 0.1% acetic acid (pH 3.2), and mobile phase B was 0.1% acetic acid in acetonitrile. The elution started with a linear gradient of 10% mobile phase B to 100% mobile phase B from 0 to 30 min and maintained at 100% B in the next 10 min. The effluent was subjected to analysis by a triple quadrupole mass spectrometer, TSQ Quantum Ultra EMR mass spectrometer (Thermo Electron Corp., San Jose, CA), equipped with a nanospray ionization (NSI) interface under the positive-ion mode for the NSI-MS/MS. The spray was monitored by a built-in CCD camera. The spray voltage was 1.5 kV, and the source temperature was at 270 °C. Argon was used as the collision gas in MS/MS experiments, the pressure of

O4-edT27 and O4-edT 3′-monophosphate, respectively.6 Yield: 24%. ESI+ MS spectra for O2-edT, N3-edT, and O4-edT: m/z 271 ([M + H]+) are shown in Figure 1S in the Supporting Information. The collision-induced dissociation mass spectra of O2-edT, N3-edT, and O4-edT at 10 eV showed m/z 155 ([M + H − dR]+), while m/z 127 ([M + H − dR − C2H4]+) were shown at 25 eV. Synthesis of [13C10,15N2]O2-edT, [13C10,15N2]N3-edT, and 13 [ C10,15N2]O4-edT Standards. The procedures were the same as those for O2-edT, N3-edT, and O4-edT described above. ESI+ MS spectra for [13C10,15N2]O2-edT, [13C10,15N2] N3-edT, and [13C10,15N2]O4-edT showed m/z 283 ([M + H]+). Collision-induced dissociation mass spectra of [13C10,15N2]O2edT, [13C10,15N2]N3-edT, and [13C10,15N2]O4-edT showed m/z 162 ([M + H − dR]+) at 10 eV and m/z 134 ([M + H − dR − C2H4]+) at 25 eV. Extraction of DNA from Blood. Freshly drawn human blood was stored in the presence of 10% (v/v) citrate-dextrose solution (ACD) as an anticoagulant and stored at 4 °C. An aliquot of 1.1 mL of the blood/anticoagulant solution was subjected to the Blood Genimic Midi kit (Viogen, Sunnyvale, CA) following the protocol described previously.28 The amount of DNA was quantified by a NanoDrop 1000 photometer (J&H Technology Co., Ltd., Wilmington, DE). The purity of DNA was checked by the absorbance ratio A260/A280 being between 1.8 and 2.0. Iodoethane-Treated Calf Thymus DNA. To a solution containing calf thymus DNA (1.0 mg) in 583 μL of water was added iodoethane (20 μL, 0.25 mmol) in 50 μL of N,Ndimethylformamide (DMF). The mixture was stirred vigorously at 58 °C for 12 h, and the final pH was 7.0. The solution was extracted with dichloromethane (10 mL), followed by hexane (10 mL) to remove iodoethane. Enzyme Hydrolysis of DNA. To the DNA solution was added 100 pg each of [13C10,15N2]O2-edT, [13C10,15N2]N3-edT, and [13C10,15N2]O4-edT and subjected to enzyme hydrolysis. The hydrolysis procedures modified from the published procedures previously used for O4-methyl dT.29 A solution 2523

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Table 1. Precision in Quantification of O2-edT, N3-edT, and O4-edT by nanoLC−NSI/MS/MS Analysis adducts levels (adducts/108 nucleotides) mean ± SD (RSD, %)a,b O2-edT N3-edT O4-edT

day 1

day 2

day 3

interday variation

5.7 ± 0.5 (9.16%) 77.2 ± 2.4 (3.1%) 8.0 ± 0.7 (8.6%)

6.0 ± 0.2 (2.8%) 81.8 ± 2.7 (3.3%) 7.9 ± 0.6 (6.5%)

5.2 ± 0.2 (4.0%) 70.5 ± 0.5 (0.6%) 7.7 ± 0.4 (4.9%)

5.6 ± 0.4 (7.2%) 76.5 ± 0.07 (7.4%) 7.9 ± 0.02 (1.9%)

Each experiment started with 50 μg of iodoethane-treated calf thymus DNA, and an equivalent of 10 μg of DNA hydrolysate was subjected to the nanoLC−NSI/MS/MS analysis. bAdduct levels are presented as mean ± standard deviation (SD) from triplicate experiments. The percentage standard deviation (RSD) is expressed in parentheses. a

approved by the Institutional Review Board of the National Chung Cheng University (IRB No. 100112902). Statistical Analysis. GraphPad InStat version 3.00 for Windows 95, GraphPad Software (San Diego, CA, www. graphpad.com) was used for statistical analysis. The nonparametric Mann−Whitney test was used to analyze levels of O2edT, N3-edT, and O4-edT in human leukocyte DNA samples between the 20 smokers and 20 nonsmokers as well as between each adduct and the smoking index (number of cigarette per day × years smoked) in smokers. The correlation between two adducts in these 40 samples was performed by linear regression.

Figure 2. Assay procedures for O2-edT, N3-edT, and O4-edT in DNA samples by stable isotope dilution nanoLC−NSI/MS/MS.

the collision cell was 1.5 mTorr, and the collision energy was 10 V. The adduct-enriched sample was analyzed by nanoLC− NSI/MS/MS with the transition from the parent ion [M + H]+ focused in quadrupole 1 (Q1) and dissociated in a collision cell (Q2), yielding the product ion, which was analyzed in quadrupole 3 (Q3) under the highly selective reaction monitoring (H-SRM) mode with the mass width of Q1 = 0.2 m/z and Q3 = 0.7 m/z, and a dwell time of 0.1 s. Two H-SRM method monitoring methods were employed: method 1 monitored O2edT,N3-edT, and O4-edT in Q1 at m/z 271.1 and the daughter ion [M + H − 116]+ ([M + H − dR]+) in Q3 at m/z 155.1, respectively, with a collision energy of 10 V. For [13C10,15N2] O2-edT, [13C10,15N2]N3-edT, and [13C10,15N2]O4-edT, Q1 and Q3 were at m/z 283.1 and m/z 162.1, respectively, with a collision energy of 10 V. Method 2 monitored O2-edT,N3-edT, and O4-edT in Q1 at m/z 271.1 and the daughter ion [M + H − 144]+ ([M + H − dR − C2H4]+) in Q3 at m/z 127.1, respectively, with a collision energy of 25 V. For [13C10,15N2] O2-edT, [13C10,15N2]N3-edT, and [13C10,15N2]O4-edT, Q1 and Q3 were at m/z 283.1 and m/z 134.1, respectively, with a collision energy of 25 V. The collision-induced dissociation mass spectra of O4-edT and [13C10,15N2]O4-edT are shown in Figure 3S in the Supporting Information. Calibration Curves. The solutions containing 100 pg each of [13C10,15N2]O2-edT, [13C10,15N2]N3-edT, and [13C10,15N2] O4-edT with various amounts (0, 0.05, 0.1, 0.2, 0.5, 1.0, 10, and 50 pg) of the O2-edT, N3-edT, and O4-edT were prepared. Each sample went through a C18-OH SPE column as described above. The fraction containing these adducts was evaporated and reconstituted in 10 μL of 0.1% acetic acid (pH 3.2), and 2 μL of the aliquot was subjected to the nanoLC−NSI/MS/MS analysis described above. Study-Subjects. The subjects of this study were healthy individuals recruited from employees and students of the National Chung Cheng University, including 20 male smokers and 20 nonsmokers (14 male and 6 female). The mean age was 21.9 ± 5.2 (S.D.) for smokers and 25.3 ± 6.5 for nonsmokers. The information on age, gender, and smoking status of the studysubjects is listed in Table 1S in the Supporting Information. The subjects received a written warranty stating that the information was for research purposes only and that their personal information would be kept confidential. This study is

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RESULTS AND DISCUSSION Synthesis and Characterization of Standards. Standard O2-edT, N3-edT, and O4-edT were synthesized from 2′deoxythymidine with diazoethane generated in situ from Nethyl-N-nitrosourea under alkaline conditions (Figure 1S, Supporting Information).26 As analyzed by HPLC with photodiode array detection, these three ethylated regioisomers have distinctive UV spectra30 and retention time (Figure 1). These standards were also characterized by their ESI mass spectra, the collision-induced dissociation spectra, and their NMR spectra. Representative mass spectra were shown in Figures 2S and 3S in the Supporting Information. Method Development. The entire assay procedure includes (1) addition of internal standards [13C10,15N2]O2edT, [13C10,15N2]N3-edT, and [13C10,15N2]O4-edT to DNA, (2) enzyme hydrolysis, (3) adduct enrichment by a reversed phase solid-phase extraction (SPE) column, and (4) nanoLC−NSI/ MS/MS analysis under the H-SRM mode (Figure 2). Method Performance and Validation. In this study, the highly sensitive nanoflow liquid chromatography is coupled to a triple quadrupole mass spectrometer monitored under the highly selective reaction monitoring (H-SRM) mode, which both quadrupole 1 and 3 are in the static mode selecting specific m/z values for the precursor and daughter ions, respectively. Currently, it is a trend to use nanobore LC column with nanospray ionization MS/MS for quantification of trace amounts of adducts in DNA samples so that the assay sensitivity is increased and subsequently the amount of DNA required is reduced.25,28,31−33 The combination of the nanoflow LC system with nanospray ionization MS/MS is the most sensitive setup. To increase the specificity of the assay, the HSRM mode with a narrow window (0.2 Da) for the precursor ion was used. It allows fewer ions passing through quadrupole 1 and thus lowers the background signals and is especially useful 2524

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Figure 3. NanoLC−NSI/MS/MS chromatograms of O2-edT, N3-edT, and O4-edT in human leukocyte DNA (sample no. 20) analyzed under the HSRM mode.

0.9989, 0.9985, and 0.9978 for O2-edT, N3-edT, and O4-edT, respectively (Figure 4S, Supporting Information). The calibration curves pass through the origin because no adducts were detectable in the sample containing only the isotopes. The accuracy of the assay was validated by adding known amounts of O2-edT (3.0, 6.0, and 15 pg), N3-edT (35, 70, and 175 pg), and O4-edT (3.0, 6.0, and 15 pg) to iodoethanetreated calf thymus DNA (10 μg) in the presence of untreated calf thymus DNA (40 μg) and analyzing for their levels. Linear regression gave a y-intercept of 2.15, 28.8, and 3.42 pg for O2edT, N3-edT, and O4-edT, respectively, with high correlation coefficients (Figure 5S, Supporting Information). These amounts are very close to 2.22, 29.9, and 3.27 pg of O2-edT, N3-edT, and O4-edT, respectively, in 50 μg of iodoethane-treated calf thymus DNA without the addition of standards, which correspond to 5.2, 70.5, and 7.7 adducts in 108 total normal nucleotides. The precision of the assay was evaluated by analyzing O2-edT, N3-edT, and O4-edT in iodoethane-treated calf thymus DNA (5 times diluted 50 μg in total) in triplicates, and the experiments were performed on three different days following the optimized assay procedures. The results showed that the relative standard deviations for both intraday and interday analyses were within 10% (Table 1), revealing the high reproducibility of the assay. Analysis of Human Leukocyte DNA. Quantification of DNA adducts in leukocyte DNA isolated from human blood represents a low invasive biomarker to reflect the extent of DNA damage in the whole body. Because the DNA adduct levels in blood tend to be lower than in other tissues due to its relatively short lifetime, a highly sensitive and specific assay is demanded to examine the possibility of using leukocyte as the surrogate tissue for biomonitoring. Figure 3 shows representative nanoLC−NSI/MS/MS chromatograms of the H-SRM transitions for O2-edT, N3-edT, and O4-edT and their isotope-labeled internal standards. The three adducts were clearly detected in both H-SRM transitions

in the analysis of a complex mixture. The accuracy, precision, and reproducibility of the analysis were monitored by the isotopomers of the analytes as internal standards added at the beginning of the assay. Two H-SRM transitions were employed, i.e., monitoring the parent ion [M + H]+ at m/z 271.1 to the daughter ion [M + H − 116]+ ([M + H − dR]+) in Q3 at m/z 155.1 (method 1) and to [M + H − 144]+ ([M + H − dR − C2H4]+) in Q3 at m/z 127.1 (method 2). Because the signal intensity of these adducts obtained using method 1 is three times of that using method 2, method 1 is used throughout the study for quantification of these adducts. The detection limits of O2-edT, N3-edT, and O4-edT injected on-column was 5.0 fg (18.5 amol) for O2-edT and that for N3edT and O4-edT was 10 fg (37 amol). The calibration curves were obtained by mixing a fixed amount of [13C10,15N2]O2-edT, [13C10,15N2]N3-edT, and [13C10,15N2]O4-edT (100 pg each) with various amounts of standard O2-edT, N3-edT, and O4-edT adducts, following the SPE enrichment step, and analyzed by the nanoLC−NSI/MS/MS. The quantification limit for the entire assay, defined as the lowest amount of analyte showing linearity, was 50, 100, and 100 fg of O2-edT, N3-edT, and O4edT, respectively, corresponding to 1.2, 2.3, and 2.3 adducts in 109 normal nucleotides, respectively, starting with 50 μg of DNA. That means 1.5−2 mL of blood is required for the assay as a range of 25−35 μg/mL DNA is isolated from freshly drawn blood. Because the maximal volume injected is 2 μL in this nanoLC system, 10 μL of solvent was used to reconstitute the enriched sample. The quantification limit is at least 5 times greater than that of the on-column detection limit due to the fact that only one-fifth of the reconstituted solution of the enriched sample was injected into the nanoLC−NSI/MS/MS. The calibration curves demonstrated good linearity with the correlation coefficient (R2) of 1.0000 for all three adducts in the range from the quantification limit to 50 pg. In the range of the quantification limit to 1.0 pg, the correlation coefficient was 2525

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Table 2. Levels of O2-edT, N3-edT, and O4-edT in Human Leukocyte Samples adduct levels (adducts in 108 nucleotides)b,c mean ± SD (%RSD) sample a

1

2a 3a 4a 5a 6a 7a 8a 9a 10a 11a a

12

13a 14a

O2-edT

N3-edT

1.6 ± 0.02 (1.4%) NDd

21.8 ± 0.3 (1.4%) 4.6 ± 0.1 (2.5%) 3.6 ± 0.3 (8.3%) 54.4 ± 5.3 (9.8%) 6.0 ± 0.07 (1.2%) 1.6 ± 0.07 (4.2%) 4.8 ± 0.1 (2.8%) 5.2 ± 0.5 (9.5%) 141 ± 4 (2.6%) 6.11 ± 0.50 (8.2%) 98.8 ± 0.4 (0.4%) 40.0 ± 3.3 (8.3%) 22.3 ± 0.8 (5.1%) NDd

2.7 ± 0.2 (6.4%) 73.4 ± 2.7 (3.6%) 5.2 ± 0.1 (2.7%) 2.6 ± 0.1 (5.1%) 3.8 ± 0.02 (0.6%) 6.4 ± 0.2 (2.5%) 179 ± 2 (1.3%) NDd 121 ± 3 (2.6%) 47.8 ± 1.9 (4.0%) 26.2 ± 0.8 (3.2%) 5.6 ± 0.4 (7.0%) 43.1 ± 1.1 (2.6%) 98.0 ± 3.7 (3.7%) 115 ± 8.4 (7.3%) 81.5 ± 1.1 (1.3%) 69.3 ± 0.3 (0.5%) 14.9 ± 1.4 (9.5%) NDd

O4-edT 2.5 ± 0.2 (6.0%) NDd 3.0 ± 0.2 (4.6%) 68.0 ± 1.8 (2.7%) 6.3 ± 0.3 (4.1%) 1.6 ± 0.04 (2.3%) 3.5 ± 0.2 (5.7%) 2.9 ± 0.1 (3.9%) 171 ± 6 (3.3%) NDd

23

3.9 ± 0.2 (4.4%) NDd

24

NDd

25

NDd

NDd

26−40 mean ± SD smokers (n = 20) mean ± SD nonsmokers (n = 20)

NDd 44.8 ± 52.0

NDd 41.1 ± 43.8

2.8 ± 0.2 (8.6%) 12.5 ± 0.07 (0.6%) 2.9 ± 0.2 (6.9%) 2.1 ± 0.1 (6.2%) NDd 48.3 ± 53.9

0.19 ± 0.87

4.1 ± 13.3

1.0 ± 2.9

a

16

17a 18a 19a 20a 21 22

provided additional evidence for their identity in a qualitative sense. Starting from 50 μg of leukocyte DNA, only an equivalent of 10 μg of DNA hydrolysate was loaded on the column and analyzed by nanoLC−NSI/MS/MS. The level of O2-edT, N3-edT, and O4-edT in this sample was 14.8, 13.0, and 30.3 in 108 normal nucleotides. In the 40 human leukocyte samples analyzed, only 5 out of 20 nonsmokers had detectable amounts of one or more edT adducts. Table 2 summarizes the levels of O2-edT, N3-edT, and O4-edT in these 40 human leukocyte samples from healthy volunteers. The nonparametric Mann−Whitney test was used to compare adduct levels between smokers and nonsmokers because the adduct levels are not normally distributed. Although the number of subjects is limited, levels of O2-edT, N3-edT, and O4-edT in human leukocyte DNA are all significantly higher in smokers than in nonsmokers, with p values of 0.0004, 0.0009, and 0.0004, respectively. The mean levels of O2edT, N3-edT, and O4-edT in 20 smokers’ leukocyte DNA were 44.8 ± 52.0, 41.1 ± 43.8, 48.3 ± 53.9 in 108 normal nucleotides, while those in 20 nonsmokers were 0.19 ± 0.87, 4.1 ± 13.3, and 1.0 ± 2.9, respectively. All the RSD for each adduct in all the samples were within 10%, suggesting the excellent reproducibility and precision of the assay. Among the three ethylated dT adducts, most reports focused on O4-edT and less was known for O2-edT, while N3-edT was the least studied adduct. The mutagenic O4-edT was detected in the liver of healthy individuals but was below the detection limit of approximately 0.2−0.8 × 10−8 thymine in the leukocyte DNA by a 32P-postlabeling-based assay.5 Levels of O2-edT and N3-edT in human samples have never been determined previously. This assay provides valuable information on levels of these three adducts and permits comparison of them in the same DNA sample. Levels of these three adducts in these 40 samples show extremely significant statistical correlations (p < 0.0001) with each other. Using linear regression, the correlation coefficient (γ) between levels of O2-edT and O4-edT, between levels of O2-edT and N3-edT, and between levels of N3-edT and O 4 -edT was 0.9896, 0.9840, and 0.9901, respectively. Furthermore, levels of O2-edT in smokers show a statistically significant association (γ = 0.4789, p = 0.0327) with the smoking index, defined as the number of cigarettes smoked per day times the number of years smoked (Figure 4). It is convincing that ethylated DNA adducts originate from ethylating agents in cigarette smoke.9−13 Cigarette smoke contains a relatively high concentration of ethylamine, 34 which might be nitrosylated to produce ethyl diazonium ion, an

125 ± 4 (3.4%) 52.8 ± 4.4 (8.2%) 31.1 ± 2.2 (7.0%) 2.8 ± 0.3 (9.5%) 55.2 ± 1.7 (3.0%) 110 ± 2.4 (2.2%) 141 ± 4.1 (2.9%) 84.8 ± 2.9 (3.4%) 73.6 ± 1.1 (1.5%) 30.3 ± 2.7 (9.0%) NDd

45.2 ± 1.3 (2.9%) 95.6 ± 0.4 (0.4%) 115 ± 2.5 (2.2%) 79.2 ± 4.5 (6.1%) 63.7 ± 2.1 (3.2%) 13.0 ± 0.4 (3.2%) 18.5 ± 1.0 (5.6%) 4.7 ± 0.2 (4.8%) 57.9 ± 1.9 (3.3%) NDd

15a

Figure 4. Correlation between smoking index and O2-edT levels in smokers.

Smokers. bEach experiment started with 50 μg of human leukocyte DNA and an equivalent of 10 μg of DNA hydrolysate was subjected to the nanoLC−NSI/MS/MS analysis. cAdduct levels are presented as mean ± standard deviation (SD) from triplicate experiments. The percentage standard deviation (RSD) is expressed in parentheses. d Not detectable. a

method 1 (top panel) and method 2 (the third panel). Although method 1 was used for adduct quantification due to its higher sensitivity, the presence of these adducts using method 2 2526

dx.doi.org/10.1021/ac203405y | Anal. Chem. 2012, 84, 2521−2527

Analytical Chemistry

Article

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effective ethylating agent. N-Nitrosodiethylamine and Nnitrosoethylmethylamine are also possible ethylating agents present in cigarette smoke, albeit in relatively low concentrations.18,35 Therefore, this assay should be valuable in investigating the importance of ethylated dT adducts in the mechanism of cigarette smoke-induced carcinogenesis.

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CONCLUSIONS To our knowledge, this is the first mass spectrometry-based assay for ethylated dT adducts. This method is used for detection and simultaneous quantification of three ethylated dT regioisomers O2-edT, N3-edT, and O4-edT in 50 μg of human leukocyte DNA. Levels of O2-edT, N3-edT, and O4-edT in 40 human leukocyte DNA samples are associated with cigarette smoking in a statistically significant manner. Statistically significant correlation was observed between levels of O2-edT, N3-edT, and O4-edT. The requirement for the small amount of sample (50 μg of DNA from 1.5−2 mL of blood) makes this assay clinically feasible in measuring ethylated dT adduct levels in human leukocyte DNA as low invasive biomarkers for DNA damage resulting from exposure to ethylating agents and in understanding their roles in cancer risk evaluation and prevention.

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ASSOCIATED CONTENT

S Supporting Information *

(1) Schemes for synthesis of O2-edT, N3-edT, and O4-edT, (2) ESI(+) mass spectra, (3) collision-induced dissociation mass spectra of O2-edT, N3-edT, and O4-edT, (4) the calibration curves for O2-edT, N3-edT, and O4-edT, and (5) age, gender, and smoking status of study-subjects. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: (886) 5-242-8176. Fax: (886) 5-272-1040. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the National Science Council of Taiwan (Grant NSC 97-2113-M-194-007-MY3) and National Chung Cheng University (to H.-J.C.C.).

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NOTE ADDED AFTER ASAP PUBLICATION This paper was published on the Web on February 13, 2012. The description for the reversed phase column packed in-house was revised, and another text correction was made. The corrected version was reposted on February 27, 2012.

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dx.doi.org/10.1021/ac203405y | Anal. Chem. 2012, 84, 2521−2527