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Carcinogenesis Advance Access originally published online on July 6, 2005
Carcinogenesis 2006 27(1):146-151; doi:10.1093/carcin/bgi177
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Carcinogenesis vol.27 no.1 © Oxford University Press 2005; all rights reserved.

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

Mu-Rong Chao, Chien-Jen Wang 1, Louis W. Chang 1 and Chiung-Wen Hu 2, *

Department of Occupational Safety and Health, Chung Shan Medical University, No. 110, Sec.1, Chien-Kuo N Road, Taichung 402, Taiwan and 1 Division of Environmental Health and Occupational Medicine, National Health Research Institutes, 100 Shih-Chuan 1st Road, Kaohsiung, Taiwan and 2 Department of Public Health, Chung Shan Medical University, no. 110, Sec. 1, Chien-Kuo N Road, Taichung 402, Taiwan

* To whom correspondence should be addressed. Tel: +886 4 24730022, ext. 11835; Fax: +886 4 23248179; Email: windyhu{at}csmu.edu.tw


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 References
 
Previous studies demonstrated the presence of unknown direct-acting ethylating agents arising from cigarette smoke. We hypothesized that such agents would also lead to ethylation of guanine in DNA followed by depurination/repair and excretion of N7-ethylguanine (N7-EtG) in urine. In this study, a highly specific and sensitive liquid chromatographic/tandem mass spectrometric (LC/MS/MS) method was firstly developed for measuring urinary N7-EtG. With the use of an isotope internal standard (15N5-N7-EtG) and on-line enrichment techniques, the detection limit of this method was estimated as 0.59 pg/ml (0.33 pmol) on-column. This method was then applied to measure urinary samples obtained from 35 non-smokers and 32 smokers with dietary control. The results showed that the mean urinary levels of N7-EtG were 85.5 ± 105 and 28.1 ± 19.4 pg/mg creatinine for smokers and non-smokers, respectively. Smokers had about three times higher level of N7-EtG than non-smokers (P < 0.005). It was further noted that the urinary level of N7-EtG was significantly associated with cotinine for smokers (r = 0.49, P < 0.005). Taken together, this is the first study that demonstrated the presence of N7-EtG in urine, and that cigarette smoke was highly responsible for the increased urinary excretion of N7-EtG. This non-invasive measurement of urinary N7-EtG would be useful for the surveillance of ethylating agent exposure and its associated cancer risk in the future.

Abbreviations: ACN, acetonitrile; BMI, body mass index; FA, formic acid; HPLC, high performance liquid chromatography; LC/MS/MS, liquid chromatography with tandem mass spectrometry; LOD, limit of detection; N3-EtA, N3-ethyladenine; N7-EtG, N7-ethylguanine; O4-EtT, O4-ethylthyminine; TSNA, tobacco-specific nitrosamine


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Supplementary material
 References
 
Cigarette smoke is causally associated with the occurrence of several human cancers at different sites including lung, oral, bladder, oropharyngeal, hypopharyngeal, laryngeal and pancreatic cancers (1). A number of cigarette smoke constituents are known to be carcinogenic such as tobacco-specific nitrosamine (TSNA). TSNAs are metabolically activated to alkylating agents that are capable of reacting with DNA to form adducts (2). If these DNA adducts are not repaired properly, they could lead to miscoding, resulting in a permanent mutation. For example, the tobacco-specific 4-methylnitrosamino-1-3-pyridyl-1-butanone and N-nitrosodiethylamine are metabolically activated to methylating and ethylating agents, respectively, and further lead to the formation of methylated and ethylated DNA adducts. These alkylated adducts have been suggested to be partially responsible for the incidence of lung cancer in smokers (3). However, ethylating agents are thought to be more potent carcinogens compared with analogous methylating agents since the resulting ethylated DNA adducts are removed less efficiently than methylated adducts (4,5).

Lately, several pieces of evidence have suggested that there could be uncharacterized ethylating agents present in cigarette smoke, since increased levels of N3-ethyladenine (N3-EtA) have been reported in the urine of smokers as compared with non-smokers (6–8). Increased urinary excretion of N3-EtA most probably resulted from reaction of an ethylating agent with adenine in nucleic acids followed by depurination or repair. Moreover, O4-ethylthyminine (O4-EtT) levels have been found to be higher in lung DNA of smokers than of non-smokers (9). Carmella et al. (10) also demonstrated elevated levels of N-terminal N-ethylvaline in hemoglobin of smokers, supporting the proposal that cigarette smoke contains an, as yet, unidentified ethylating agent. Recently, Singh et al. (11) have clearly confirmed the presence of direct-acting ethylating agents in cigarette smoke by treating calf thymus DNA directly with cigarette smoke. The formation of N7-ethylguanine (N7-EtG) was observed in vitro and was highly correlated with the number of cigarettes treated. However, the identity of the direct-acting ethylating agent remains unknown.

A variety of analytical techniques have been developed to detect alkylated DNA adducts, such as high performance liquid chromatography (HPLC)/UV, HPLC/fluoresence, HPLC/electrochemical detection, immunoslot blot or gas chromatography with mass spectrometry (GC–MS) (8,12,13). Although these methods have been successful, they have drawbacks such as labor intensiveness, necessity for chemical derivatization, low sensitivity or a limited specificity due to the possible interferences arising from complex biological matrix (i.e. crude urine). Recently, liquid chromatography with tandem mass spectrometry (LC/MS/MS) has become a powerful technology to overcome the sensitivity and specificity issues in analysis of DNA adducts. Accurate quantitation of extremely low concentrations of adducted bases has frequently relied on the use of non-radioactive isotope-labeled standards to compensate for loss of analyte during sample preparation, which has been the most critical step to eliminate the matrix effect for analysis of modified bases by mass spectrometry (MS) (14,15). Moreover, on-line sample extraction using column-switching device is an extremely useful technique to get the biological samples prepared automatically for LC/MS methods (16). Its advantages include less ion suppression, relatively short run time as well as higher sensitivity and selectivity, especially for urine samples containing a considerable amount of cross-reacting substances.

As the N7 position of guanine is the predominant reaction site (5), the urinary N7-EtG could have the potential to be a representative biomarker for assessing the exposure to ethylating agents. However, to our knowledge, no analytical method has ever been presented to date that enables monitoring ultra-trace level of N7-EtG in urine. In this study, an isotope dilution LC/MS/MS with on-line enrichment method was firstly developed for quantitation of urinary N7-EtG. By using this method, we investigated if exposure to cigarette smoke resulted in increased levels of urinary N7-EtG.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Supplementary material
 References
 
Chemicals
Solvents and salts were of analytical grade. Reagents were purchased from the indicated sources: 15N5-deoxyguanosine (15N5-dG) and D3-cotinine (Cambridge, Andover, MA); N7-EtG (Merck, Darmstadt, Germany); diethylsulfate, N,N-dimethylacetamide and cotinine (Sigma, St Louis, MO).

Urinary samples
Urine samples were obtained from 67 apparently healthy males (32 regular smokers and 35 non-smokers) with dietary control for 4 days. We used a questionnaire to obtain subject age, body mass index (BMI) and the smoking status (self-reported daily cigarette consumption). Urine samples were kept at 4°C during sampling and stored at –20°C prior to analysis. Determination of creatinine was performed by a routine procedure in a local hospital.

Synthesis of 15N5-N7-ethylguanine stable isotope internal standard
15N5-N7-ethylguanine (15N5-N7-EtG) was synthesized as described by Gombar et al. (17) with several modifications. Briefly, 10 mg of dimethylsulfate and 5 mg of 15N5-dG were dissolved in 100 µl of N,N-dimethylacetamide. The reaction mixture was stirred and incubated for 6 h at room temperature. Celite of 5 mg was added and the mixture was centrifuged. The pH of supernatant was adjusted to 8.0 with NH4OH and diluted to 300 µl with acetone. The precipitate, 15N5-N7-ethyldeoxyguanosine was collected by centrifugation, washed once with ice-cold ethanol and once with ether, and allowed to dry overnight. The 15N5-N7-ethyldeoxyguanosine was hydrolysed in 1 N HCl at 80°C for 30 min. Ammonium hydroxide was added dropwise until the 15N5-N7-EtG precipitated. The product was collected by centrifugation, washed twice with ice-cold water, and dried under vacuum in a SpeedVac. The 15N5-N7-EtG was then purified by the semi-preparative HPLC system, confirmed by MS analysis, and quantified using unlabeled N7-EtG.

Urinary sample pretreatment using off-line solid-phase extraction (off-line SPE)
Urine samples were thawed at room temperature and centrifuged at 10 000 g for 5 min. To a quantity of 0.5 ml of urine were added 2.5 ml of deionized water (DIW) and 100 µl of 15N5-N7-EtG solution (0.43 ng/ml) for use as an internal standard. The urine mixture was loaded onto a Sep-Pak C18 cartridge (500 mg/3 ml, Waters Milford, MA, USA) preconditioned with 5 ml methanol and 5 ml DIW. The cartridge was then washed with 5 ml of 5% methanol and eluted with 3 ml of 40% methanol. The eluate was dried under vacuum and redissolved in 300 µl of 96% acetonitrile (ACN) containing 0.1% formic acid (FA) ready for LC/MS/MS analysis.

Analysis of urinary N7-EtG using an isotope dilution LC/MS/MS method with on-line enrichment
After pretreatment with off-line SPE, the solution containing N7-EtG was then analysed by LC/MS/MS coupled with an on-line enrichment system using an automatic column-switching device. The column-switching device was described in detail in a previous publication (18). In brief, it consisted of a switching valve (two-position microelectric actuator; Valco, Houston, TX) and a Nucleosil NH2 cartridge [35 x 4.6 mm inner diameter (i.d.), 10 µm]. The switching valve function was controlled using PE-SCIEX control software Analyst. Table I summarizes the detailed column-switching operation sequence showing the LC gradients used during the on-line enrichment and the analytical procedures. When the switching valve was at position A, 100 µl of pretreatment urine sample was loaded onto the cartridge using an autosampler (PE series 200, PerkinElmer, Norfolk, CT), and a quaternary pump (PE series 200, PerkinElmer) delivered by 0.1% of FA in 96% ACN at a flow rate of 1 ml/min as the loading and washing buffer (Solvent A). The cartridge was flushed with the loading buffer for 2 min followed by valve switching to injection position (position B) to inject the sample into the LC system. At the fourth minute, the valve was switched back to position A and the cartridge was washed using a mobile phase (Eluent A) with linear gradient from 100% of 96% ACN with 0.1% FA (Solvent A) to 100% of 50% ACN with 0.1% FA (Solvent B) from third minute to tenth minute, and back to 100% of Solvent A in 1 min for equilibration of the cartridge and preparation for the next analysis. The total run time was 15 min.


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  		Table I. Timetable for on-line enrichment and analytical procedure

 
The HPLC system consisted of two series 200 micro-pumps and a series 200 autosampler (PerkinElmer, Boston, MA), with a Polyamine-II endcapped HPLC column (150 x 4.6 mm i.d., 5 µm, YMC) and a guard column (10 x 2 mm i.d., YMC). As shown in Table I, gradient mode was used to achieve the separation of analytes using Eluent B. After automatic sample cleanup for 2 min (second to fourth minute), the sample was automatically eluted from the trap column into the analytical column. The mobile phase was 90% ACN containing 0.1% FA (Solvent C) and delivered at a flow rate of 1 ml/min for 3 min, then varied to 80% ACN with 0.1% FA (Solvent D) within 10 min and rapidly back to Solvent C with a linear gradient in 1 min.

The sample eluted from the HPLC system was introduced into a TurboIonspray source installed on an API 3000 triple-quadrupole mass spectrometer (Applied Biosystems, Foster City, CA), operated in positive mode with a needle voltage of 5.5 kV, nitrogen as the nebulizing gas, and the turbogas temperature set at 500°C. Data acquisition and quantitative processing were accomplished using AnalystTM software, Ver. 1.1 (Applied Biosystems). The fragmentation pattern of N7-EtG in this study (data not shown) was consistent with that reported by Singh et al. (11). The transition of the [M + H]+ precursor ethylated guanine ion to guanine (m/z 152 or 157) resulted in the product ion with the highest intensity. Therefore, the samples were analysed in positive-ionization MS/MS multiple reaction monitoring (MRM) mode for the [M + H]+ ion to guanine base [B + H]+ transitions of N7-EtG (m/z 180 to 152) and 15N5-N7-EtG (m/z 185 to 157). The dwell time of analyte was set to 150 ms and internal standard was set to 100 ms. Nebulizer and curtain gas flow rates were set to 15. Collision-assisted-dissociation (CAD) gas and turbogas were set at 6 and 8, respectively. Collision energy was set at 25 eV with nitrogen as collision gas. Resolution was set at 0.7 Th (FWHM) for both Q1 and Q3.

Analysis of urinary cotinine
Urinary cotinine was measured by an isotope dilution LC/MS/MS method. The urinary sample was purified using a liquid–liquid extraction method described by Ceppa et al. (19) with several modifications. Briefly, a total of 1 ml urine, 2 ml of DIW and 200 µl of internal standard (D3-cotinine, 40 ng/ml) was added to 13 ml screw capped glass tubes. After the addition of 0.7 ml of 5 N KOH and 3 ml of dichloromethane, tubes were sealed hermetically, shaken for 15 min and centrifuged for 10 min at 10 000 g. The aqueous phase was then discarded and the clear organic phase was pipetted out. After evaporation to dryness under a stream of nitrogen, residues were then redissolved in 400 µl of 5% methanol with 20 mM ammonium acetate.

An aliquot of 20 µl was injected into the same LC/MS/MS as described above. The analytical column was Purospher STAR RP-18 (55 x 4 mm i.d., 3 µm, Merck). Gradient mode was used to achieve the separation of analytes using mixtures of mobile phase A (5% methanol with 20 mM ammonium acetate) and mobile phase B (95% methanol with 0.1% FA) at a flow rate of 600 µl/min. The following gradient was run: 0–2.5 min: 0% B; 2.5–4.0 min: 40% B; 4.0–4.1 min: 100% B; 4.1–5.0 min: 100% B; 5.0–5.1 min: 0% B; 5.1–8.0 min: 0% B.

As for the MS condition, the mass spectrometer was operated in the MRM mode. The transition of m/z 177 to 80 was determined for cotinine and the corresponding transition of m/z 180 to 80 for D3-cotinine. The dwell time was set to 150 ms. Nebulizer and curtain gas flow rates were set to 10, CAD gas and turbogas were set at 10 and 8, respectively, and a source heater probe temperature at 300°C.

Statistical analysis
The data were analysed using a SAS statistical package (SAS, version 8.2). Concentrations of urinary N7-EtG and cotinine measured by LC/MS/MS were logarithm transformed to normalize their distributions before statistical analysis. A Student's t-statistic was used to compare urinary concentrations of N7-EtG and cotinine between non-smokers and smokers. Pearson's correlation coefficients were used to study the relationship of urinary N7-EtG concentrations to cotinine concentrations or self-reported daily cigarette consumption. In multiple linear regression models, the relationships of urinary N7-EtG concentrations to cotinine concentrations or daily cigarettes consumption were investigated after adjusting for other variables (i.e. age and BMI).


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Supplementary material
 References
 
Analysis of N7-EtG in human urine using LC/MS/MS
A typical LC/MS/MS chromatogram for N7-EtG and 15N5-labeled N7-EtG in the urine of a non-smoker was shown in Figure 1. The positive electrospray ionization mass spectrum of N7-EtG contained a precursor ion at m/z 180.0 and a product ion at m/z 152.0; a precursor ion at m/z 185.0 and product ion at m/z 157.0 characterized the 15N5-N7-EtG. The precision of the present method was evaluated by performing repeated determinations of N7-EtG in three different urine samples (see Table II). The coefficients of variation were 0.7–4.5% and 2.0–11.3% for within-run and between-run precision tests, respectively.



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  		Fig. 1. Determination of urinary N7-EtG from a non-smoker by LC/MS/MS coupled with on-line analyte enrichment.

 

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  		Table II. Precision of isotope dilution LC/MS/MS with on-line enrichment for analysis of urinary N7-EtG

 
A linear calibration curve ranging from 5 to 200 pg/ml was obtained using aqueous standard solutions; each calibration solution contained 100 µl of 0.43 ng/ml 15N5-N7-EtG, and yielded an equation for the line y = 2.0748x – 0.0029 (r2 = 0.9995). The limit of detection (LOD), defined on the basis of a signal-to-noise (S/N) ratio of 3, was calculated to be 0.59 pg/ml on-column (0.33 fmol in a 100 µl injection volume) directly using diluted standard solutions.

Urinary excretion of N7-EtG and cotinine in smokers and non-smokers
Subject characteristics, urinary N7-EtG and cotinine concentrations were summarized in Table III. It was found that smokers and non-smokers were similar in age and BMI. Smokers have a mean age of 25.4 with a mean BMI value of 22.3 while non-smokers have a mean age of 23.1 with a mean BMI value of 21.9. As for the urinary N7-EtG, the urinary levels of N7-EtG in the present study were all above the detection limit of 0.59 pg/ml. After adjustment by creatinine, smokers had a mean urinary N7-EtG concentration of 85.5 ± 105 pg/mg creatinine and non-smokers had a mean concentration of 28.1 ± 19.4 pg/mg creatinine. Smokers had significantly higher levels of urinary N7-EtG than non-smokers (P < 0.005). Moreover, urinary levels of cotinine observed in smokers (1187 ± 1152 ng/mg creatinine) were significantly higher than those of non-smokers (8.5 ± 5.5 ng/mg creatinine; P < 0.005).


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  		Table III. Overall characteristics of the study subjects

 
Correlation between urinary N7-EtG and cotinine for smokers
As shown in Figure 2, the correlation between urinary N7-EtG and cotinine was investigated for smokers. Urinary N7-EtG concentrations were found to be highly associated with urinary cotinine concentrations for smokers (Pearson's correlation coefficient r = 0.49, P < 0.005). The correlation between urinary N7-EtG and self-reported daily cigarette consumption was also studied. No significant correlation was observed between the urinary N7-EtG concentrations and the self-reported daily cigarette consumption (Pearson's correlation coefficient r = 0.23, P = 0.21). In multiple linear regressions (see Table IV), the correlation between urinary N7-EtG and cotinine was not confounded by other variables, including age and BMI (P < 0.0001). There was no correlation between urinary N7-EtG and self-reported daily cigarette consumption after adjustment for age and BMI (P = 0.30).



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  		Fig. 2. Correlation between the urinary levels of N7-EtG and cotinine for smokers. See on-line Supplementary material for a color version of this figure.

 

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  		Table IV. Multiple regression for urinary N7-EtG

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Supplementary material
 References
 
A highly specific and sensitive LC/MS/MS method for the detection of N7-EtG in human urines has been firstly established. With the use of isotope internal standard and on-line solid-phase extraction system, this method was proven to exhibit an extremely low LOD of 0.59 pg/ml on-column (0.33 fmol) and made the trace levels of N7-EtG detectable in complex urine matrix.

In previous studies, several attempts have been made to monitor the formation of N7-EtG in biological samples. Singh et al. (12) and van Delft et al. (20) developed HPLC-electrochemical detection methods for N7-EtG and resulted in LODs of 2000 and 114 fmol, respectively. Immunochemical methods have also been used for N7-EtG analysis with LODs ranged from 190 to 3800 fmol per 100 µg of DNA (21). Kato et al. (22) established a two-step HPLC with 32P-postlabeling method for detection of N7-EtG in human tissues and reported a LOD of 0.76 fmol using 100 µg of DNA. Despite being time consuming and requiring large amounts of radioactivity, the 32P-postlabeling approach, has the advantage of high sensitivity.

Interestingly, there has not been much effort toward the development of quantitative assays using MS for this important ethylated DNA adduct (16). The use of LC/MS/MS has been recently proposed for the analysis of N7-EtG with a LOD of 2.0 fmol in calf thymus DNA (11). It offers numerous advantages over the 32P-postlabeling assays for the detection of N7-EtG and encounters problems of dealing with radioactive materials and low labeling efficiencies due to depurination. Apparently, our newly developed on-line sample enrichment coupled to isotope dilution LC/MS/MS method has an even better sensitivity (0.33 fmol). Moreover, a lower amount of DNA needed could be expected if this method was further applied in the determination of N7-EtG in DNA. It has been suggested that there were 0.9–3.6 N7-EtdG per 107 dGp in non-tumorous lung tissues (23), which was equivalent to 1.4–5.5 fmol in 20 µg DNA. This indicated that the LC/MS/MS method described here is totally capable of quantitating the level of N7-EtG in human tissues and could be a useful tool for surveillance of ethylating agent exposure.

Among the DNA ethylation products, N7-EtG is thought to be a better biomarker for assessing human exposure to ethylating agents. Since N7 position of guanine is the most nucleophilic site in DNA, N7-EtG is the most abundant base damage in ethylated DNA and could be quantitated more easily. Although this adduct is not considered to be promutagenic, it may result in the formation of apyrimidic sites by spontaneous depurination thus altering the fidelity of DNA replication. In addition, N7-EtG is relatively slowly repaired compared with O6-ethylguanine (O6-EtG) and N3-EtA (i.e. t1/2 in vivo: N7-EtG, 168 h; O6-EtG, 72 h; 3-EtA, 7.5 h) and could increase genomic instability and contribute to lung cancer risk in smokers (24,25).

This is the first study assessing ethylating agent exposure from cigarette smoke by measuring urinary N7-EtG. In the present study, smokers had an elevated urinary N7-EtG of 85.5 ± 105 pg/mg creatinine while non-smokers had a mean level of 28.1 ± 19.4 pg/mg creatinine. Smokers had about three times higher level of N7-EtG than non-smokers, suggesting that the increased urinary N7-EtG was formed from cigarette smoke as the main exposure source. Elevated urinary excretion of ethylated DNA adducts in smokers has been investigated in previous studies. Kopplin et al. (7) collected 24 h urine samples and reported a urinary N3-EtA excretion of ~20 and ~120 ng/day for non-smokers and smokers, respectively, roughly equal to 13–29 and 72–161 pg/mg creatinine after adjustment by a normal range of daily creatinine excretion (800–1800 mg/day) (26). Prevost and Shuker (8) also collected 24-h urine samples of smokers and found a urinary level of N3-EtA ~40 ng/day on non-smoking days (roughly equal to 22–50 pg/mg creatinine) and 100 ng/day on smoking days (roughly equal to 56–125 pg/mg creatinine). It was interesting to note that the excretion levels of N7-EtG in the present study were found to be somewhat close to those of N3-EtA reported previously, although it has been indicated that N7-EtG is the most abundant base damage in ethylated DNA. Possible explanations for this discrepancy include differences in experimental setting (smoking status, dietary control and analytical method) and/or differences in rates of ethylated adducts excreted into the urine (N7-EtG versus N3-EtA). Moreover, our data also indicated that N7-EtG was detectable in all urines of non-smokers. The source of these background levels could be from environment, diet or environmental tobacco smoke (9).

Cotinine is one of the major metabolites of nicotine. Because cotinine has a longer elimination half-life (~20 h compared with 2 h for nicotine), the measurement of cotinine in body fluids has been widely used as a reliable biomarker to estimate active smoking behavior. It was suggested that cotinine levels in non-smokers were <30 ng/mg creatinine while passive/light smokers (<5 cigarettes/day) and regular smokers were 30–100 and 100–7000 ng/mg creatinine, respectively (27,28). In our study, the smokers and non-smokers had mean urinary levels of cotinine of 1187 ng/mg creatinine (range from 63 to 5138) and 8.5 ng/mg creatinine (range from 1.5 to 23.1), respectively, which was consistent with a previously reported range. A highly positive correlation between the urinary N7-EtG and cotinine (r = 0.49, P < 0.005) for smokers was observed in this study and was not confounded by other variables including age and BMI. Our results support the proposal that cigarette smoke resulted in increased DNA ethylation, followed by depurination/repair and excretion of N7-EtG in urine. It has been shown that cigarette smoke contains ethylating agents such as N-nitrosodiethylamine and N-nitrosoethylmethylamine occurring in concentrations of 3 and 13 ng/cigarette, respectively (29). However, at such low levels, these promutagens could not account for the formation of ethylated DNA adducts and protein adducts observed in smokers (3). A good correlation between the urinary N7-EtG and cotinine may also provide the evidence for the presence of direct-acting ethylating agents in cigarette smoke, implying that smoking individuals are exposing themselves to ethylating agents regardless of their capacity for metabolism and will be ultimately affected by the smoking behavior (highly associated with nicotine intake) (11). Unfortunately, the direct-acting ethylating agents still required to be further characterized.

There was no significant correlation between the self-reported daily cigarette consumption and the urinary N7-EtG levels (r = 0.23, P = 0.21). One possible explanation for the lack of correlation was that self-report on smoking status was insufficient due to recall bias, unwilling disclosure of smoking habits, invalid reported number of cigarettes consumed, and various cigarette brands containing ethylating agents at different concentrations (30,31).

In conclusion, we developed an isotope dilution LC/MS/MS method for determining urinary N7-EtG. With the use of on-line enrichment techniques, the urinary N7-EtG could be further purified/enriched to make this DNA adduct detectable with high selectivity and specificity. By applying this method, we further confirmed the presence of N7-EtG in urine and that cigarette smoke was highly responsible for the increased urinary excretion of N7-EtG. The results of this study supported the proposal that cigarette smoke contains unidentified ethylating agents, which were involved in DNA damage. It is believed that such non-invasive measurement of urinary N7-EtG could serve as a useful biomarker for assessing ethylating agent exposure and its associated cancer risk.


   Supplementary material
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
References
 
Supplementary material can be found at: http://www.carcin.oxfordjournals.org/


   Acknowledgments
 
This study was supported by a grant from National Science Council, Republic of China (Grant NSC 93-2320-B-040-026). The authors thank the Division of Environmental Health and Occupational Medicine core facility of National Health Research Institute for providing LC/MS/MS and technical assistance. Authors also thank Dr K.Y.Wu for his valuable suggestions and Mr Y.J.Lu for his help in sample preparation.

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 References
 

  1. IARC (1986) IARC Monographs on the evaluation of carcinogenic risk of chemicals to humans. Volume 38: Tobacco Smoking. International Agency for Research on Cancers, Lyon, France.
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Received April 30, 2005; revised June 14, 2005; accepted June 28, 2005.


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C.-W. Hu, C.-J. Wang, L. W. Chang, and M.-R. Chao
Clinical-Scale High-Throughput Analysis of Urinary 8-Oxo-7,8-Dihydro-2'-Deoxyguanosine by Isotope-Dilution Liquid Chromatography-Tandem Mass Spectrometry with On-Line Solid-Phase Extraction
Clin. Chem., July 1, 2006; 52(7): 1381 - 1388.
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