Detection and simultaneous quantification of three smoking-related ethylthymidine adducts in human salivary DNA by liquid chromatography tandem mass spectrometry

Toxicology Letters 224 (2014) 101–107

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Detection and simultaneous quantification of three smoking-related ethylthymidine adducts in human salivary DNA by liquid chromatography tandem mass spectrometry Hauh-Jyun Candy Chen ∗ , Chin-Ron Lee Department of Chemistry and Biochemistry, National Chung Cheng University, 168 University Road, Ming-Hsiung, Chia-Yi 62142, Taiwan

h i g h l i g h t s • • • •

First simultaneous analysis of three ethylthymidine adducts in human salivary DNA by mass spectrometry. Ethylthymidine adduct levels are higher in smokers than nonsmokers. Ethylthymidine adduct levels are associated with cigarette smoking. Ethylthymidine adducts are not detectable in nonsmokers.

a r t i c l e

i n f o

Article history: Received 12 August 2013 Received in revised form 4 October 2013 Accepted 7 October 2013 Available online 18 October 2013 Keywords: DNA adduct Ethylthymidine Nanoflow LC Mass spectrometry Saliva

a b s t r a c t Smoking cigarette increases levels of certain ethylated DNA adducts in certain tissues and urine. Cigarette smoking is a major risk factor of various cancers and DNA ethylation is involved in smoking-related carcinogenesis. Among the ethylated DNA adducts, O2 -ethylthymidine (O2 -edT) and the promutagenic O4 -ethylthymidine (O4 -edT) are poorly repaired and they can accumulate in vivo. Using an accurate, highly sensitive, and quantitative assay based on stable isotope dilution nanoflow liquid chromatography–nanospray ionization tandem mass spectrometry (nanoLC–NSI/MS/MS), O2 -edT, N3 edT (N3 -ethylthymidine), and O4 -edT adducts in human salivary DNA were simultaneous detected and quantified. Saliva is easily accessible and available and it can be a potential target in searching for noninvasive biomarkers. Under the highly selected reaction monitoring (H-SRM) mode, salivary samples from 20 smokers and 13 nonsmokers were analyzed. Starting with 50 ␮g of DNA isolated from about 3.5 mL of saliva, levels of O2 -edT, N3 -edT, and O4 -edT in 20 smokers’ salivary DNA samples were 5.3 ± 6.2, 4.5 ± 5.7, 4.2 ± 8.0 in 108 normal nucleotides, respectively, while those in 13 nonsmokers were non-detectable. In addition, statistically significant correlations (p < 0.0001) were observed between levels of O2 -edT and N3 -edT ( = 0.7388), between levels of O2 -edT and O4 -edT ( = 0.8839), and between levels of N3 -edT, and O4 -edT ( = 0.7835). To the best of our knowledge, this is the first report of detection and quantification of these three ethylthymidine adducts in human salivary DNA, which might be potential biomarkers for exposure to ethylating agents and possibly for cancer risk assessment. © 2013 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Cigarette smoking is a leading cause of lung cancer as more than 80% of lung cancer patients are tobacco smokers or ex-smokers. Cigarette smoke contains thousands of chemicals and more than sixty of them are carcinogens (Hecht, 2012). Some of these chemicals are direct acting alkylating agents and some require metabolic activation to the ultimate carcinogens, such as polycyclic aromatic hydrocarbons and tobacco-specific nitrosamines. Formation of

∗ Corresponding author. Tel.: +886 5 242 8176; fax: +886 5 272 1040. E-mail address: [email protected] (H.-J.C. Chen). 0378-4274/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.toxlet.2013.10.002

carcinogen–DNA adducts, if not repaired efficiently, can cause genetic instability and mutation during replication and eventually lead to cancer formation. Thus, DNA adducts have been used as biomarkers of exposure to carcinogens and risk of cancer (Jarabek et al., 2009; Vineis and Perera, 2000). Increased levels of ethylated DNA adducts in tissues and urine of smokers suggest that DNA ethylation is associated with cigarette smoking (Chao et al., 2006; Chen et al., 2012; Godschalk et al., 2002a,b; Kopplin et al., 1995; Prevost and Shuker, 1996; Prevost et al., 1993). Ethylation of DNA bases gives N3 -ethyladenine, O6 and N7 -ethylguanine as well as O2 -, O4 -, and N3 -ethylthymine in humans (Anna et al., 2011; Bronstein et al., 1992; Chao et al., 2006; Chen et al., 2007, 2012; Kopplin et al., 1995; Prevost and

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Shuker, 1996; Prevost et al., 1993). Among them, O6 -ethylguanine and O4 -ethylthymidine (O4 -edT) are miscoding lesions formed in rats treated with diethylnitrosamine, but only O4 -edT accumulates in the rat liver (Scherer et al., 1980; Swenberg et al., 1984). O6 -Ethylguanine is repaired efficiently, while O2 -edT and O4 -edT accumulate as persistent DNA lesions in rat and human cells (Bronstein et al., 1992; Den Engelse et al., 1987; Scherer et al., 1980; Swenberg et al., 1984; Thomale et al., 1990). Among the ethylated DNA adducts, N3 -ethyladenine and N7 -ethylguanine are detected in human liver and leukocyte DNA as well as in smokers’ urine partly due to spontaneous depurination (Balbo et al., 2011; Chao et al., 2006; Chen and Liu, 2013; Chen et al., 2007; Kopplin et al., 1995; Prevost and Shuker, 1996). Recently, N7 -ethylguanine was detected in calf thymus DNA treated with extracts of areca nut, suggesting that ethylating agents are also present in areca nut extracts (Hu and Chao, 2012). Animal studies showed that the miscoding O4 -edT is closely associated with cancer (Swenberg et al., 1984; Thomale et al., 1990), but limited information is available in humans (Verna et al., 1996). In DNA from normal lung tissue adjacent to tumors of lung cancer patients, levels of O4 -edT are higher in smokers than in nonsmokers (Anna et al., 2011; Godschalk et al., 2002b). DNA from saliva (oral fluid) has been used in forensic sciences (Walsh et al., 1992) and genomic DNA typing analysis (Lum and Le Marchand, 1998). Salivary DNA originates predominantly from the gingival crevice-originated leukocytes and epithelial buccal cells (Osswald et al., 2003), both are short-lived cells (Ashkenazi and Dennison, 1989). The oral cavity is directly exposed to carcinogens in cigarette smoke and in areca nuts; damage in salivary DNA is correlated with oral cancer (Prasad et al., 1995; Proia et al., 2006; Wong, 2006). Because of the trace amounts of DNA adduct and the inaccessibility of human tissues, there is a need to establish noninvasive DNA adducts as biomarkers in surrogate tissues or biological fluids to assess DNA damage in the body. Saliva is readily available and is a surrogate fluid of blood that mirrors the whole body condition (Chiappin et al., 2007; Wong, 2006), supported by the concentrations of certain metabolites appropriately reflecting their levels in plasma (Alvarez-Sanchez et al., 2012; Takeda et al., 2009). Only until recently that measurement of salivary DNA adducts was performed on heterocyclic aromatic amine-induced adducts (Bessette et al., 2010) and on exocyclic propano and etheno adducts (Chen and Lin, 2011). The rapid development of analytical technology has enabled characterization and quantification of trace levels of DNA adducts in biological tissues and fluids (Himmelstein et al., 2009). In this study, a highly accurate, specific and sensitive method based on stable isotope dilution nanoflow liquid chromatography–nanospray ionization tandem mass spectrometry (nanoLC–NSI/MS/MS) is used for detection and quantification of O2 -, N3 -, and O4 -ethylthymine (edT) simultaneously in human salivary DNA. Levels of these three edT adducts in smokers and nonsmokers are compared.

Table 1 Characteristics of the study population. Smokers (n = 20)

Nonsmokers (n = 13)

Mean ± SD Age (year) Weight (kg) Height (cm) BMI Cigarettes smoked per daya Years smokeda Smoking indexa , b a b

27.2 75.1 174.2 24.8 13.5 6.8 86.5

± ± ± ± ± ± ±

10.4 10.1 4.3 3.3 7.1 (8–40) 5.0 (1–20) 66.0 (15–260)

22.0 ± 79.2 ± 174.2 ± 26.0 ± – – –

2.9 11.9 5.6 2.6

The ranges are expressed in parentheses. Smoking index is the number of cigarettes smoked per day × years of smoking.

2.3. 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 13 male nonsmokers. The information on age, weight, height, and smoking status of the study-subjects is listed in Table 1. 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 approved by the Institutional Review Board of the National Chung Cheng University (IRB No. 100112902). 2.4. Isolation of salivary DNA The subjects were asked not to eat any food 1 h before saliva collection. They brushed their teeth with toothpaste and rinsed their mouths thoroughly and saliva was collected under nonstimulated conditions. The saliva was isolated by a Blood DNA Extraction Midiprep System (Viogen, Sunnyvale, CA) following the previously reported procedures (Chen and Lin, 2011). The amount of DNA was quantified by a NanoDrop 1000 photometer (J&H Technology Co., Ltd., Wilmington, DE). The purity of DNA was confirmed by the absorbance ratio of A260 /A280 being between 1.8 and 1.9. The entire extraction procedures yielded an average of 13.6 ± 2.5 ␮g (mean ± SD) of DNA per milliliter saliva with a range of 10.0–20.3 ␮g/mL. 2.5. Assay procedures Typically, salivary DNA (50 ␮g) was added 100 pg each of [13 C10 ,15 N2 ]O2 edT, [13 C10 ,15 N2 ]N3 -edT, and [13 C10 ,15 N2 ]O4 -edT as internal standards and enzyme hydrolyzed to the nucleosides following the previously reported procedures (Dolan and Pegg, 1985). The adducts were enriched by a reversed phase C18-OH solidphase extraction (SPE) column, evaporated to dryness, dissolved in 10 ␮L of 0.1% acetic acid, and 2 ␮L of the aliquot was analyzed by nanoLC–NSI/MS/MS under the H-SRM mode using the equipment and conditions previously described (Chen et al., 2012). The transition was monitored from the parent ion [M+H]+ focused in quadrupole 1 (Q1) and dissociated in a collision cell (Q2) with a collision energy of 10 V, to yield the product ion analyzed in quadrupole 3 (Q3). The dwell time was 0.1 s and the mass width of Q1 and Q3 was 0.2 and Q3 0.7 m/z, respectively. The H-SRM method 1 monitored O2 -edT, 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. For [13 C10 ,15 N2 ]O2 -edT, [13 C10 ,15 N2 ]N3 -edT, and [13 C10 ,15 N2 ]O4 -edT, Q1 and Q3 were at m/z 283.1 and m/z 162.1, respectively. The H-SRM 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−C2 H4 ]+ ) in Q3 at m/z 127.1, respectively. For [13 C10 ,15 N2 ]O2 -edT, [13 C10 ,15 N2 ]N3 -edT, and [13 C10 ,15 N2 ]O4 -edT, Q1 and Q3 were at m/z 283.1 and m/z 134.1, respectively.

2. Materials and methods

2.6. Assay calibration

2.1. Chemicals and reagents

The solutions containing 100 pg each of [13 C10 ,15 N2 ]O2 -edT, [13 C10 ,15 N2 ]N3 -edT, and [13 C10 ,15 N2 ]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 and the fraction containing these adducts was evaporated and reconstituted in 10 ␮L of 0.1% acetic acid (pH 3.2), and 2 ␮L of the aliquot was subjected to the nanoLC–NSI/MS/MS analysis.

Enzymes used for DNA hydrolysis were obtained from Sigma Chemical Co. (St. Louis, MO) except that alkaline phosphatase was from Calbiochem Chemical Co. (La Jolla, CA). [13 C10 ,15 N2 ]2 -Deoxythymidine was from Cambridge Isotope Laboratories (Andover, MA). All reagents are of reagent grade or above.

2.7. Statistical analysis 2.2. Synthesis of isotope-labeled internal standards The isotope-labeled standards [13 C10 ,15 N2 ]O2 -edT, [13 C10 ,15 N2 ]N3 -edT, and [13 C10 ,15 N2 ]O4 -edT were obtained from reaction of [13 C10 ,15 N2 ]2 -deoxythymidine with N-ethyl-N-nitrosourea and purified and quantified as reported (Chen et al., 2012).

GraphPad InStat version 3.00 for Windows 95, GraphPad Software (San Diego, CA, http://www.graphpad.com) was used for statistical analysis. The nonparametric Spearman correlation was used to analyze salivary levels of O2 -edT, N3 -edT, and O4 -edT between each adduct and the number of cigarette smoked per day or the smoking index (number of cigarette per day × years smoked).

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103

O N HO

N

OEt

O H

H

OH

H

µ

H

O2-ethylthymidine (O2-edT) O NEt HO

N

O

O H

H

H

OH

H

H

N3-ethylthymidine (N3-edT)

OEt NH HO

N

O

O H

H

H

OH

H

H

O4-ethylthymidine (O4-edT) Fig. 1. Structures and assay procedures for the three ethylthymidine adducts in salivary DNA.

3. Results 3.1. Analysis of edT in human salivary DNA The study subjects were asked not to eat any food at least 1 h before saliva collection. Saliva was collected after the subjects brushed their teeth and rinsed their mouths thoroughly. These precautions prevent contamination of salivary DNA from food and bacteria and the salivary DNA collected is mainly from leukocytes and epithelial buccal cells (Klinkhamer and Mitchell, 1979; Osswald et al., 2003). Fig. 1 outlines the assay procedures for the simultaneous detection and quantification of the three ethylthymidine adducts. After saliva samples were collected under unstimulated conditions, DNA was isolated by a DNA isolation kit originally intended for isolation of DNA from blood with modifications (Chen and Lin, 2011). The entire extraction procedures yielded an average of 13.6 ± 2.5 ␮g (mean ± SD) of DNA per milliliter saliva and ranged from 10.0 to 20.3 ␮g/mL in 33 samples. The purity of DNA was confirmed by the absorbance ratio of A260 /A280 being between 1.8 and 1.9. Fifty micrograms of DNA was added stable isotope-labeled standards of the analytes, i.e. [13 C10 ,15 N2 ]O2 -edT, [13 C10 ,15 N2 ]N3 -edT, and [13 C10 ,15 N2 ]O4 -edT, as internal standards and subjected to enzyme hydrolysis. The adducted nucleosides were enriched by a reversed phase solid-phase extraction (SPE) column and the fraction containing O2 -edT, N3 -edT, and O4 -edT and their isotopomers was analyzed by nanoLC–NSI/MS/MS under the highly selective reaction monitoring (H-SRM) mode.

Fig. 2 shows typical nanoLC–NSI/MS/MS chromatograms of edT regioisomers at H-SRM transitions of the parent ion [M+H]+ at m/z 271.1 to the daughter ion without the deoxyribose moiety [M+H−116]+ ([M+H−dR]+ ) at m/z 155.1 (top panel). For the isotopelabeled standards [13 C10 ,15 N2 ]edT, the parent and daughter ions were at m/z 283.1 and m/z 162.1, respectively (second panel). O2 edT, N3 -edT, and O4 -edT eluted at retention time of 16.0, 20.4, and 22.9 min, respectively. A second set of H-SRM transitions were monitored at m/z 271.1 to the ion losing the deoxyribose and ethylene moiety [M+H−144]+ ([M+H−dR−C2 H4 ]+ ) at m/z 127.1 for edT (third panel) and those for [13 C10 ,15 N2 ]edT were at m/z 283.1 to m/z 134.1 (lowest panel) to provide additional evidence (as qualifier) for the presence of edT in the sample. The signal intensity of edT using the first set of H-SRM transitions is about three times of that using the second set of transitions. Therefore, the first set of H-SRM transitions is used for quantification of edT throughout this study. The calibration curves (Fig. 3) were constructed by plotting the peak area ratios of each edT isomer versus its stable isotopelabeled standard [13 C10 ,15 N2 ]edT (100 pg) to the various amounts of edT standard added (0.05–50 pg). The linear regression lines passed through the origin because no signals were observed without addition of any edT standards. Good linearity was achieved with the equations for O2 -edT, N3 -edT, and O4 -edT being y = 0.0099x, y = 0.0095x, and y = 0.0097x with the correlation coefficient (R2 ) of 0.9988, 0.9998, and 0.9991, respectively. The lower limit of quantification (LLOQ), defined as the lowest amount that shows linearity in the calibration curve, was 50 fg for O2 -edT and N3 -edT, and 100 fg

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Fig. 2. Chromatograms of ethylthymidine adducts in human salivary DNA of a smoker analyzed by nanoLC–NSI/MS/MS under the H-SRM mode.

for O4 -edT, corresponding to 1.2, 1.2, and 2.3 O2 -edT, N3 -edT, and O4 -edT in 109 normal nucleotides, respectively, using 50 ␮g of DNA. After intrapolation of the peak area ratio into the calibration curve, the adduct levels from the sample of a smoker, shown in Fig. 2,

are determined as 14.1, 8.7, and 13.4 in 108 normal nucleotides for O2 -edT, N3 -edT, and O4 -edT, respectively. The reproducibility in quantification of edT in triplicated samples was revealed by the average relative standard deviation (RSD)

Fig. 3. Calibration curve of O2 -edT, N3 -edT, and O4 -edT by stable isotope dilution nanoLC–NSI/MS/MS under H-SRM transitions method 1.

H.-J.C. Chen, C.-R. Lee / Toxicology Letters 224 (2014) 101–107 Table 2 Levels of O2 -edT, N3 -edT, and O4 -edT in human salivary DNA.

105

Table 3 Statistical correlation between edT levels in human salivary DNA samples.a , b

Adduct levels (adduct in 108 normal nucleotides)a , b Mean ± SD (%RSD)

O2 -edT 2

Samplec

O2 -edT

N3 -edT

O4 -edT

1* 2* 3* 4* 5* 6* 7* 8* 9* 10* 11* 12* 13* 14* 15–20* 21–33 Mean ± SD Smoker (n = 20) Nonsmoker (n = 13)

NDd 1.2 ± 0.1 (7.1%) 14.1 ± 0.9 (5.2%) 21.2 ± 2.0 (9.2%) 3.5 ± 0.1 (3.2%) 8.1 ± 0.5 (2.6%) 6.6 ± 0.6 (5.7%) 2.9 ± 0.1 (1.2%) 3.4 ± 0.3 (9.2%) 0.2 ± 0.0 (7.6%) 15.5 ± 0.9 (6.0%) 13.9 ± 0.4 (3.0%) 6.2 ± 0.6 (9.5%) 9.4 ± 0.5 (5.7%) ND ND 5.3 ± 6.2

6.2 ± 0.4 (6.3%) 17.3 ± 1.0 (5.9%) 8.7 ± 0.8 (9.8%) 19.8 ± 1.1 (5.7%) ND 7.1 ± 0.5 (5.1%) 4.4 ± 0.3 (5.1%) 1.8 ± 0.1 (2.5%) 3.3 ± 0.3 (2.2%) 6.6 ± 0.3 (3.1%) 9.5 ± 0.1 (5.2%) 4.5 ± 0.2 (4.3%) ND ND ND ND 4.5 ± 5.7

1.7 ± 0.2 (7.5%) 1.9 ± 0.1 (5.2%) 13.4 ± 1.1 (7.6%) 29.7 ± 2.9 (9.8%) 5.2 ± 0.1 (2.5%) 8.3 ± 0.5 (5.2%) 3.4 ± 0.3 (9.4%) 2.2 ± 0.2 (6.2%) 1.5 ± 0.1 (7.5%) ND 8.9 ± 0.7 (7.7%) 4.0 ± 0.3 (8.6%) ND 3.6 ± 0.1 (1.7%) ND ND 4.2 ± 8.0

ND

ND

ND

a Each experiment started with 50 ␮g of human salivary DNA, and an equivalent of 10 ␮g of DNA hydrolysate was subjected to the nanoLC–NSI/MS/MS analysis. b Adduct levels are presented as mean ± standard deviation (SD) from triplicate experiments. The percentage standard deviation (RSD) is expressed in parentheses. c Smokers are marked by *. d ND, not detectable.

of 8.0%, 4.9%, and 6.5% for O2 -edT, N3 -edT, and O4 -edT, respectively. The accuracy and precision of this assay has been validated (Chen et al., 2012). The nanoLC–NSI/MS/MS method used in this study was proven to be highly accurate and precise in measuring these three edT adducts in human leukocyte DNA (Chen et al., 2012). Incorporation of stable isotope-labeled standards of the analytes as internal standards accurately monitors the recovery in each step of the procedures and reflects matrix-induced ion suppression in the mass spectrometry analysis. Coupling nanoflow LC with nanospray ionization source on a triple quadrupole mass spectrometer monitoring fragmentation of the parent ion at quadrupole 1 to the daughter ion at quadrupole 3 offers the highest sensitivity possible. The highly selective reaction monitoring (H-SRM) mode with a narrow m/z window of 0.2 Da at quadrupole 1 provides extra specificity of the SRM transitions. 3.2. Levels of edT in human salivary DNA Saliva samples from 20 male smokers and 13 male nonsmokers were analyzed and their levels listed in Table 2. Levels of O2 -edT, N3 -edT, and O4 -edT in 20 smokers’ salivary DNA were 5.3 ± 6.2, 4.5 ± 5.7, and 4.2 ± 8.0 in 108 normal nucleotides, respectively. Levels of these three adducts in 6 out of the 20 smokers and all 13

O -edT N3 -edT O4 -edT

N3 -edT c

0.7388

Spearman  b Spearman  b a b c d

Number of cigarettes smoked per day 0.3791 (0.0296)c Smoking Indexd 0.4904 (0.0038)c

0.8839c 0.7835c

a

n = 33. Correlation coefficient () was obtained by the nonparametric Spearman correlation. c The two-tailed p value < 0.0001. b

nonsmokers were below the LLOQ and considered non-detectable (ND). Statistically significant correlations were observed between levels of O2 -edT and O4 -edT (correlation coefficient  = 0.8839), between levels of O2 -edT and N3 -edT ( = 0.7388), and between levels of N3 -edT, and O4 -edT ( = 0.7835), as shown in Table 3. These correlations are also observed in human leukocyte DNA (Chen et al., 2012). Moreover, salivary levels are associated with the number of cigarette smoked per day in a statistically significant manner with correlation coefficient  of 0.3791, 0.4250, and 0.3489 for O2 -edT, N3 -edT, and O4 -edT, respectively. The smoking index, defined as the number of cigarettes smoked per day times the number of years smoked, in these subjects also shows statistically significant correlation with adduct levels; correlation coefficient is 0.4904, 0.3465, and 0.3789 for O2 -edT, N3 -edT, and O4 -edT, respectively (Table 4). In this study, there are no heavy smokers in the smoking subjects recruited based on the average number of cigarettes smoked per day (±SD) of 13.5 ± 7.1 (range: 8–40) and the average smoking index (±SD) of 87 ± 66 (range: 20–260). Stronger correlations are expected if heavy smokers are included. 4. Discussion Although cytochrome P450 enzymes are contained in salivary glands and buccal mucosa cells (Kragelund et al., 2008; Spivack et al., 2004; Vondracek et al., 2001), metabolic activation of nitrosamines, such as N-nitrosodiethylamine and N-nitrosoethylmethylamine, by these cytochrome P450 enzymes is unlikely to contribute significantly to DNA ethylation due to their low concentrations in cigarette smoke (Hoffmann et al., 2001; Scherer et al., 1980). On the other hand, convincing evidences suggest that ethylated DNA adducts are derived from direct-acting ethylating agents in cigarette smoke (Anna et al., 2011; Chao et al., 2006; Chen and Liu, 2013; Chen et al., 2007, 2012; Kopplin et al., 1995; Prevost and Shuker, 1996), such as ethylamine, which could be nitrosylated to produce ethyl diazonium ion, an effective ethylating agent (Schmeltz et al., 1977). Salivary edT levels measured in this study are about 10 times lower than those reported in leukocyte DNA of smokers (Chen et al., 2012). This observation can be reasoned by the fact that the turnover time of oral epithelia and gingival crevice-originated leukocytes, cells which salivary DNA is isolated from, is much

Table 4 Statistical correlation between edT levels and cigarette smoking.a , b O2 -edT

O4 -edT

N3 -edT

O4 -edT

0.425 (0.0137)c

0.3489 (0.0466)c

0.3465 (0.0482)c

0.3789 (0.0297)c

n = 33. Correlation coefficient () was obtained by the nonparametric Spearman correlation. The two-tailed p value is shown in the parenthesis. Smoking index is the number of cigarette smoked per day × years smoked.

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shorter than that of leukocytes in blood (Ashkenazi and Dennison, 1989; Klinkhamer and Mitchell, 1979; Osswald et al., 2003). As a consequence, these adducts do not accumulate in salivary DNA. Thus, saliva may be a feasible target for monitoring recent status of smoking-associated DNA damage. Salivary DNA is conveniently available and it has been used as a target of biomarkers associated with breast and oral cancer (Pfaffe et al., 2011; Proia et al., 2006). This assay requires only 50 ␮g of DNA isolated from about 3.5 mL of saliva. To the best of our knowledge, this is the first report of detection and quantification of three ethylthymidine adducts in human salivary DNA. The results implies that analysis of ethylthymine adducts in salivary DNA should provide valuable noninvasive biomarkers for exposure to ethylating agents in tobacco and for evaluation of their role in carcinogenesis. 5. Conclusions Using this highly accurate, sensitive and specific stable isotope dilution nanoLC–NSI/MS/MS assay, three ethylthymine adducts, including O2 -edT, N3 -edT, and O4 -edT, in human salivary DNA were simultaneously analyzed. Adduct levels in smokers’ samples were quantified, while those in 13 nonsmokers were non-detectable, using 50 ␮g of DNA isolated from about 3.5 mL of saliva. Levels of adducts correlate significantly with each other. To the best our knowledge, this is the first report of detection and quantification of these three ethylthymidine adducts in human salivary DNA. These ethylthymines should render valuable noninvasive biomarkers for tobacco-induced DNA damage in intervention studies. Conflict of interest statement The authors declare that there are no conflicts of interest. Acknowledgements This work was supported by National Science Council of Taiwan (Grants NSC 97-2113-M-194-007-MY3 and NSC 100-2113-M-194002-MY3) and National Chung Cheng University (to H.-J.C.C.). References Alvarez-Sanchez, B., Priego-Capote, F., Luque de Castro, M.D., 2012. Study of sample preparation for metabolomic profiling of human saliva by liquid chromatography-time of flight/mass spectrometry. Journal of Chromatography A 1248, 178–181. Anna, L., Kovacs, K., Gyorffy, E., Schoket, B., Nair, J., 2011. Smoking-related O4ethylthymidine formation in human lung tissue and comparisons with bulky DNA adducts. Mutagenesis 26, 523–527. Ashkenazi, M., Dennison, D.K., 1989. A new method for isolation of salivary neutrophils and determination of their functional activity. Journal of Dental Research 68, 1256–1261. Balbo, S., Villalta, P.W., Hecht, S.S., 2011. Quantitation of 7-ethylguanine in leukocyte DNA from smokers and nonsmokers by liquid chromatography– nanoelectrospray-high resolution tandem mass spectrometry. Chemical Research in Toxicology 24, 1729–1734. Bessette, E.E., Spivack, S.D., Goodenough, A.K., Wang, T., Pinto, S., Kadlubar, F.F., Turesky, R.J., 2010. Identification of carcinogen DNA adducts in human saliva by linear quadrupole ion trap/multistage tandem mass spectrometry. Chemical Research in Toxicology 23, 1234–1244. Bronstein, S.M., Skopek, T.R., Swenberg, J.A., 1992. Efficient repair of O6ethylguanine, but not O4-ethylthymine or O2-ethylthymine, is dependent upon O6-alkylguanine-DNA alkyltransferase and nucleotide excision repair activities in human cells. Cancer Research 52, 2008–2011. Chao, M.R., Wang, C.J., Chang, L.W., Hu, C.W., 2006. Quantitative determination of urinary N7-ethylguanine in smokers and non-smokers using an isotope dilution liquid chromatography/tandem mass spectrometry with on-line analyte enrichment. Carcinogenesis 27, 146–151. Chen, H.J., Lin, W.P., 2011. Quantitative analysis of multiple exocyclic DNA adducts in human salivary DNA by stable isotope dilution nanoflow liquid chromatography–nanospray ionization tandem mass spectrometry. Analytical Chemistry 83, 8543–8551.

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