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Urinary Excretion of 3,N4-Etheno-2′-deoxycytidine in Humans as a Biomarker of Oxidative Stress: Association with Cigarette Smoking Hauh-Jyun Candy Chen,* Chan-Fu Wu, Chia-Liang Hong, and Chia-Ming Chang Department of Chemistry and Biochemistry, National Chung Cheng University, 160 San-Hsing, Ming-Hsiung, Chia-Yi 62142, Taiwan Received October 2, 2003
Smokers are known to have elevated levels of lipid peroxidation, a form of oxidative stress. Etheno DNA adduct formation can originate from endogenous lipid peroxidation or from exogenous exposure of carcinogens. Using a modified stable isotope dilution GC/negative ion chemical ionization/MS assay originally developed for urinary 3,N4-ethenocytosine (Cyt), the nucleoside 3,N4-etheno-2′-deoxycytidine (dCyd) was detected for the first time in human urine. The presence of dCyd in human urine was confirmed by LC/electrospray ionization/tandem MS. Concentrations of dCyd in the 24 h urine samples from healthy individuals not occupationally exposed to industrial chemicals were in the range between 0 and 0.80 nM. A statistically significant correlation was established between cigarette smoking and urinary excretion of dCyd after being adjusted for creatinine (p ) 0.004). Furthermore, the urinary total antioxidant capacity was found to correlate inversely with the dCyd levels (r ) -0.50, p ) 0.02). The results indicate that urinary dCyd may provide a valuable noninvasive biomarker for oxidative DNA damage.
Introduction The damage of DNA forming DNA adducts initiates multistep carcinogenesis processes. Exocyclic etheno DNA adducts, Ade,1 Cyt, and 1,N2-Gua and Gua, were originally considered to derive from exposure to environmental carcinogens, such as the occupational carcinogen vinyl chloride, the food contaminant ethyl carbamate (urethane), and their metabolites (1, 2). Lately, these etheno adducts are recognized to form endogenously from aldehydic lipid peroxidation end products (3, 4), from oxidative degradation of the sugar backbone of DNA (5), or from ascorbate-dependent decomposition of lipid hydroperoxides (6). Furthermore, etheno adducts are associated with DNA damage due to chronic infections and inflammation since they are produced in nitric oxideinduced lipid peroxidation (7). HNE is one of the major R,β-unsaturated aldehydic products of lipid peroxidation (8, 9). Upon further oxidation, HNE can be epoxidized to 2,3-epoxy-4-hydroxynonanal via autoxidation (10) or by biological oxidants such as hydrogen peroxide and fatty acid hydroperoxides (11). 2,3-Epoxy-4-hydroxynonanal is a potent tumorinitiating agent (12), and it cannot be detoxified by
human epoxide hydrolase (13). Thus, this epoxy aldehyde might be an important contributor to the endogenous formation of etheno DNA adducts (14, 15). Elevated levels of etheno DNA adducts were found in cancer-prone tissues, suggesting the correlation between this promutagenic lesion with the risk of cancers. For example, elevated levels of Ade and Cyt were found in the liver of patients with Wilson’s disease and primary hemochromatosis, whose lipid peroxidation levels are also higher than normal individuals (16). In addition, a high dietary intake of ω-6 polyunsaturated fatty acids resulted in high levels of Ade and Cyt in white blood cell DNA of females but not of males (17). In patients with familial adenomatous polyposis, higher levels of Ade and Cyt were observed in colonic polyps as compared to unaffected colon tissue (18). These etheno adducts are very mutagenic in mammalian cells blocking DNA polymerases and leading to misincorporations (19-21). As lipid peroxidation is implicated in tumorigenesis, it has been postulated that DNA damage caused by these aldehydic products plays a very important role in carcinogenesis (3, 22). Thus, etheno adducts might be valid biomarkers for cancer risk assessment (4, 23).
* To whom correspondence should be addressed. Fax: (886)5-2721040. E-mail:
[email protected]. 1 Abbreviations: CID, collision-induced dissociation; Ade, 1,N6ethenoadenine; Cyt, 3,N4-ethenocytosine; dAdo, 1,N6-etheno-2′-deoxyadenosine; dCyd, 3,N4-etheno-2′-deoxycytidine; 1,N2-Gua, 1,N2ethenoguanine; Gua, N2,3-ethenoguanine; ESI, electrospray ionization; HNE, trans-4-hydroxy-2-nonenal; MS/MS, tandem mass spectrometry; NICI, negative ion chemical ionization; 8-oxo-dG, 8-oxo-7,8-dihydro2′-deoxyguanosine; 8-oxo-Gua, 8-oxo-7,8-dihydroguanine; PFB, pentafluorobenzyl; SIM, selective ion monitoring; SPE, solid phase extraction; MRM, multiple reaction monitoring; TAC, total antioxidant capacity.
The measurement of urinary oxidative DNA adducts, such as 8-oxo-dGuo, can provide a noninvasive biomarker for monitoring the level of in vivo oxidative stress (24). The presence of etheno base adducts, including Ade (25), Cyt (26), 1,N2-, and N2,3-Gua (27), has been reported in healthy human urine. Even though levels of Ade and Cyt in pancreatic and pulmonary DNA were found to be similar between smokers and nonsmokers (28, 29), urinary excretion of Ade and Cyt bases was found to correlate with cigarette smoking (25, 30).
10.1021/tx0342013 CCC: $27.50 © 2004 American Chemical Society Published on Web 05/20/2004
Cigarette Smoking and Urinary dCyd
To the best of our knowledge, the only literature reporting detection of the etheno deoxyribonucleoside adduct in biological fluids is for urinary dAdo in Japanese women using immunoprecipitation/HPLCfluorescence detection, and its level is associated with salt-induced inflammation and lipid peroxidation (31). In this study, we detect and quantify dCyd nucleoside in urine samples from 23 volunteers not occupationally exposed to industrial chemicals. The urinary dCyd concentrations were determined by the assay modified from the isotope dilution GC/NICI/MS method originally developed for Cyt (26), and the association between urinary dCyd excretion and cigarette smoking is investigated. Urinary excretion of dCyd is found to correlate inversely with urinary TAC, and urinary dCyd is thus considered a potential noninvasive biomarker for oxidative DNA damage.
Materials and Methods Materials. PFB bromide (PFB-Br), diisopropylethylamine, anhydrous methanol, and anhydrous phosphorus pentoxide were obtained from Aldrich Chemical Co. (Milwaukee, WI). 3,N4Ethenocytocine was purchased from Sigma Chemical Co. (St. Louis, MO). dCyd and [13C4,15N3]Cyt were synthesized as reported previously from [13C9,15N3]cytidine (Cambridge Isotope Laboratories, Andover, MA) (26). Bond Elut C18-OH and Si SPE columns (500 mg, 3 mL) were from Varian (Harbor City, CA). Urine Pretreatment. Urine collected over a 24 h period was stored as 1.0 mL aliquots in 1.5 mL Eppendorff tubes in the -84 °C freezer. To examine the possibility that the analyte is contained in the precipitate, two procedures are employed after defrosting the samples in an ice-water bath. In procedure A, the sample was centrifuged at 23000g for 25 min at 0 °C. Alternatively, the sample was diluted 1:1 (v/v) with lithium acetate (100 mM, pH 6.5), incubated at 37 °C for 10 min, and followed by centrifugation at 5000g for 10 min at room temperature (procedure B) (32, 33). The precipitate was discarded, and the leftover samples were not reused. The creatinine contents were analyzed by a picric acid method (34). Adduct Enrichment by C18-OH SPE Column. Each new batch of SPE columns was tested for consistency in the elution pattern with 1.0 µg each of standard Cyt and dCyd before use for urine samples. After elution with the conditions described below, the fractions were collected every 3 mL. These fractions were evaporated and analyzed by reversed phase HPLC with photodiode array detection as described previously (26). Assay Procedures. 1. Separation by C18-OH SPE Column. The supernatant (0.1 mL) of the pretreated urine sample was added to 0.1 mL of potassium phosphate buffer (50 mM, pH 7.0) and [13C4,15N3]Cyt (1.0 ng), and the mixture was loaded on a C18-OH SPE column preconditioned with 15 mL of methanol, followed by 15 mL of water. After the sample was loaded and eluted, the column was washed with 12 mL of water, followed by 3 mL of 10% methanol in water. The fraction containing Cyt was eluted with 3 mL of 15% methanol in water (30), and dCyd was collected with 3 mL of 30% methanol solution in separated 4 mL silanized glass vials. The two fractions were evaporated under vacuum. The fraction containing Cyt was dried over phosphorus pentoxide for derivatization, and the dCyd fraction was hydrolyzed to Cyt as described below. 2. Hydrolysis of EdCyd. The fraction collected for dCyd was added to [13C4,15N3]Cyt (1.0 ng) and hydrolyzed in 88% formic acid at 120 °C for 60 min to release Cyt. The hydrolysate was evaporated and dried over phosphorus pentoxide under vacuum. 3. Derivatization and GC/NICI/MS Analysis of PFBECyt. The dried hydrolysate of the dCyd fraction was derivatized and cleaned up using procedures previously reported (26). The GC/NICI/MS analysis was performed using a HewlettPackard 6890 GC with 5973 MSD mass selective detector with
Chem. Res. Toxicol., Vol. 17, No. 7, 2004 897 the NICI source (Agilent Technologies, Palo Alto, CA). SIM at m/z 134 and 141 was used to monitor and quantify PFB-Cyt and [13C4,15N3]PFB-Cyt for the respective [M - 181]- fragment ions as reported (26) with modification that the oven temperature of GC was held at 50 °C for the first 2 min, then raised to 300 °C at a gradient of 5 °C/min, and maintained at 300 °C for 5 min. Assay Calibration. One nanogram of [13C4,15N3]Cyt was added as an internal standard to each sample containing various amounts of dCyd ranging from 0, 3, 5, 10, 20, 50, 100, and 250 pg. The samples were processed through the same procedures for urine samples, i.e., acid hydrolysis, pentafluorobenzylation, Si SPE purification, and GC/NICI/MS analysis. The quantification of dCyd was based on intrapolation of the ratio of the peak area of PFB-Cyt vs that of [13C4,15N3]PFB-Cyt to the calibration curve obtained from at least duplicated experiments. Direct Pentafluorobenzylation of EdCyd. A silanized 4 mL vial containing 25 pg of dCyd and 1.0 ng of [13C4,15N3]Cyt was dried over phosphorus pentoxide, derivatized with PFBBr, purified by a Si SPE column, and analyzed by GC/NICI/MS under SIM mode as described above. LC/ESI/MS/MS Analysis of EdCyd. The urine sample collected from the C18-OH SPE column was evaporated, reconstituted in 0.01% acetic acid (20 µL), and injected into a liquid chromatography system consisting of a Hitachi L-7000 pump system, a Rheodyne injector, and a reversed phase C18 column [Prodigy ODS (3), 2.0 mm × 150 mm, 3 µm, Phenomenex, Torrance, CA] eluting with 25% methanol in 0.01% acetic acid with a flow rate of 0.2 mL/min. The elution was connected without splitting to a triple quadrupole mass spectrometer (Quattro Ultima, Micromass, Manchester, U.K.) equipped with an ESI interface. A voltage of 2.5 kV was applied to the electrospray needle. N2 was used as the desolvation gas (500 L/h) to help nebulization and as the nebulization gas (100 L/h) to help desolvation and to stabilize the spray. The source temperature was at 120 °C, and the stainless steel capillary was heated to 350 °C to obtain optimal desolvation. Argon was used as the collision gas in MS/MS experiments. The cone voltage was 10 V. In the MRM experiment, the precursor [M + H]+ ion for dCyd was generated in the ESI source under the positive ion mode in quadrupole 1 (Q1). The precursor ion [M + H]+ for the deoxynucleoside dCyd (m/z 252) was dissociated in a collision cell (quadrupole 2) yielding product ion [B + H]+ for the nucleobase Cyt (m/z 136), which was analyzed in quadrupole 3 (Q3). For the MRM transition of dCyd (m/z 252 f m/z 136), analysis was performed in Q1 and in Q3 with a collision energy of 20 V. The daughter ion spectrum was obtained by scanning from m/z 100 to m/z 300. Masurement of Urinary TAC. The procedure was modified from the published methods (35, 36) using hydrogen peroxide, methemoglobin, and 2,2′-azinobis(3-ethylbenzothiazoline6-sulfonate) (30). Quantification was based on the standard curve constructed from various amounts of uric acid ranging from 0 to 300 µM. Urinary levels of TAC were average values from four replicated experiments. Statistical Analysis. All results are reported as the means ( standard deviation (SD). Statistical analysis of two groups was performed by the Mann-Whitney U-test and p e 0.05 was considered significant. The Spearman rank correlation was used to calculate the correlation coefficient. GraphPad InStat version 3.00 for Windows 95 (GraphPad Software, San Diego, CA; www.graphpad.com) was used for these analyses.
Results EdCyd Analysis Using GC/NICI/MS. The GC/NICI/ MS assay for Cyt (26) was modified to analyze both Cyt and dCyd in the same urine sample, and the procedures are depicted in Scheme 1. Incorporation of the stable isotope [13C4,15N3]Cyt as internal standards for monitoring both Cyt and the hydrolyzed dCyd provides high
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Scheme 1. Procedures of GC/NICI/MS Analysis of ECyt and EdCyd in Urine
Figure 1. Calibration curve for the GC/NICI/MS analysis of dCyd. Samples containing various amounts (0-250 pg) of dCyd were added to a fixed amount of [13C4,15N3]Cyt (1.0 ng) and subjected to the assay procedures described in the Materials and Methods (r2 ) 0.9984). The data are combined from at least separate experiments in duplicates. The ratio of each analyte to the internal standard was calculated based on the peak areas.
specificity and accurate quantification of these two analytes in the complex mixture of urine samples. Separation and enrichment of Cyt and dCyd in urine samples could be achieved using a reversed phase C18-OH SPE column. The isotope standard [13C4,15N3]Cyt was added to 0.1 mL of urine. After the sample was loaded, it was washed with water to remove the very polar components in urine, followed by 10% aqueous methanol with which Cyt did not elute. The fraction containing Cyt was collected by eluting with 15% aqueous methanol, whereas the dCydcontaining fraction was collected by the subsequent elution of 30% aqueous methanol. The dCyd-containing fraction was added isotope standard [13C4,15N3]Cyt, followed by formic acid hydrolysis to release Cyt from dCyd. After complete removal of formic acid by evaporation, both Cyt-containing fractions were dried and derivatized with the electrophore, PFB-Br, before GC/ NICI/MS analysis. The retention time of PFB-Cyt in GC is the same as that of [13C4,15N3]PFB-Cyt due to their identical chemical properties. Nonetheless, PFBCyt and [13C4,15N3]PFB-Cyt can be distinguished by MS. The ideal assay for dCyd should incorporate the stable isotopomer of dCyd in its analysis. Unfortunately, the precursor for dCyd synthesis, the isotopmer of dCyd, which is at least 3 mass units higher than dCyd, is not available to us. Although this is not the most straightforward method to analyze dCyd, it is a good alternative to hydrolyze dCyd to Cyt and monitor the Cyt isotopomer. Sample Pretreatment. To examine the possibility that the analyte is contained in the precipitate, as reported for 8-oxo-dGuo (32, 33), urinary dCyd levels were analyzed with two different sample pretreatment procedures before C18-OH SPE enrichment. Urine samples were centrifuged with high speed at a low temperature (procedure A) or diluted with buffer and
incubated to release the analyte from the precipitate, followed by centrifugation with low speed at room temperature (procedure B). As compared to procedure A, no increase was observed for adduct levels obtained using procedure B. In the three samples analyzed, dCyd concentrations from duplicated experiments were determined as 0.460, 0.470, and 0.802 using procedure A and 0.454, 0.445, and 0.799 using procedure B, respectively. It is probably due to the low levels of dCyd as compared to 8-oxo-dGuo. Furthermore, this assay used a reversed phase SPE column, derivatization, and postderivatization cleanup by a normal phase SPE column. These procedures can remove most interference materials that behave like the analyte in reversed phase HPLC with electrochemical or mass detection (32, 33). Detection Limits of EdCyd by GC/NICI/MS Analysis. The calibration curve for dCyd was linear, ranging from 3.0 (12 fmol) to 250 pg (Figure 1), revealing a concentration quantification limit of 0.12 nM. The correlation coefficient (R2) was 0.9998 with a control ratio of peak areas being 0.0025 (Figure 2a). The slope of the linearly regressed curve showed that dCyd was converted to Cyt quantitatively. Unlike Ade, Cyt is stable under harsh acidic conditions. The limit of quantification for dCyd by GC/NICI/MS was comparable to that for analysis of dCyd in tissue DNA (9.2 fmol) using LC/ESI/ MS/MS (37, 38), and it was much lower than that for analyzing dCyd by HPLC with fluorescence detection (5.9 pmol) (26). The recovery of the entire assay was 35%. The interday relative standard error of the assay was 3.6% (n ) 3), and the intraday relative standard error was 5.2% (n ) 4). GC/NICI/MS Analysis of EdCyd. The GC/NICI/MS chromatograms of the dCyd fraction in the urine sample of a nonsmoker are shown in Figure 2b. The [13C4,15N3]PFB-Cyt peak of 32.05 min at m/z 141 unambiguously located the PFB-Cyt peak of 32.03 min at m/z 134 in the complex chromatogram. The concentration of dCyd in this urine sample was calculated to be 36 pg/mL after intrapolation into the calibration curve. Unlike the malondialdehyde-2′-deoxyguanosine adduct with which electrophoric derivatization was accompanied by depurination (39), no reaction took place when direct electrophore derivatization of dCyd was carried out as an attempt to avoid the hydrolysis step (data not shown).
Cigarette Smoking and Urinary dCyd
Figure 2. GC/NICI/MS chromatogram of the dCyd fraction in (a) the control and (b) the urine of a nonsmoker. The dCyd fraction collected from a C18-OH SPE column was hydrolyzed by 88% formic acid before derivatization with PFB-Br as described in the Materials and Methods. The concentration of dCyd in this sample was calculated as 36 pg/mL (0.144 nM).
LC/ESI/MS/MS Analysis of EdCyd. To confirm the detection of dCyd by the GC/NICI/MS method, the urinary dCyd fraction enriched by a C18-OH SPE
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Figure 3. LC/ESI/MS/MS analysis of dCyd. (a) MRM transition (m/z 252 f m/z 136) chromatogram of a smoker’s urine sample. (b) Daughter ion spectrum of the peak at 5.08 min of the urine sample of panel a. (c) Daughter ion spectrum of the peak at 5.07 min of a nonsmoker’s urine sample.
column was analyzed by the highly specific LC/ESI/MS/ MS using a triple quadrupole instrument. In the MRM experiment, the precursor ion [M + H]+ for dCyd was generated in the ESI source under the positive ion mode and focused in quadrupole 1. The precursor ion [dCyd + H]+ at m/z 252 was dissociated in a collision cell (quadrupole 2) yielding product ion [M - deoxyribose + H]+ (i.e., [Cyt + H]+) at m/z 136, which was analyzed in
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Table 1. Characteristics of the Study Population mean ( SD (range) sex (male/female) age (years) cigarettes/day smoking years smoking indexb
smokers (n ) 10)
nonsmokers (n ) 13)
10/0 43 ( 16 (24-68) a 15 ( 6 (5-25) 22 ( 16 (3-50) 331 ( 253 (15-750)
10/3 31 ( 9 (20-45)
a Results are expressed as means ( SD. The ranges are expressed in parentheses. b Smoking index ) number of cigarettes smoked per day × years of smoking.
Figure 5. Correlation between urinary dCyd and Cyt concentrations.
Figure 4. Distribution of urinary levels of dCyd in smokers and nonsmokers.
quadrupole 3. For the MRM transition of dCyd, analysis was performed at m/z 252 in quadrupole 1 to m/z 136 in quadrupole 3. The limit of detection under MRM for dCyd was 20 pg (80 fmol). The MRM chromatogram of dCyd in a nonsmoker’s urine showed a peak at 5.08 min (Figure 3a). The daughter ion spectrum was obtained by scanning the peak under the CID. In the daughter ion spectra of the dCyd peaks, the molecular [dCyd + H]+ ion at m/z 252 and the fragment [Cyt + H]+ ion at m/z 136 were both present in the five urine samples analyzed, including smokers and nonsmokers (Figure 3b,c). This analysis could not provide quantitative information of dCyd because no internal standard was incorporated. Nonetheless, direct analysis of urinary dCyd without derivatization by the highly specific MS/MS unambiguously confirmed the presence of dCyd in human urine samples. Adduct Levels and Statistical Analysis. The study population consists of 10 male smokers, 10 male nonsmokers, and three female nonsmokers, and the characteristics of the subjects are listed in Table 1. No heavy smokers were included, according to the numbers of cigarettes per day and the smoking index, defined as the number of cigarettes smoked per day times the years of smoking. Urinary concentrations of dCyd determined by the GC/NCI/MS assay are in a range between 0 and 0.80 nM in the 23 samples. Among them, dCyd concentrations in five out of the 13 nonsmokers are below the quantification limits of the assay. Urinary concentrations
Figure 6. Correlation between urinary dCyd levels with TAC in 23 human urine samples.
of dCyd are higher in smokers than nonsmokers (0.45 ( 0.17 vs 0.16 ( 0.17 nM). Statistical analysis shows that urinary excretion of dCyd is associated with cigarette smoking whether it is adjusted for creatinine concentration, body weight, or both (Table 2). The distribution of dCyd among smokers and nonsmokers is shown in Figure 4. A statistically significant association between concentrations of dCyd and Cyt (Spearman correlation coefficient r ) 0.4257, p ) 0.0428) is found in subjects of the same population, of which the values have been reported recently (30) (Figure 5). The urinary TAC level provides a convenient measure for antioxidant defenses among individuals (36). Interestingly, an inverse association is observed between levels of dCyd and TAC of the individuals, with a Spearman correlation coefficient of -0.4959 (p ) 0.0161) (Figure 6).
Discussion The presence of deoxyribonucleoside adducts in urine might emanate from the action of nucleotide excision repair (NER) enzymes on damaged cellular DNA, from its formation in the deoxyribonucleotide pools, or from hydrolysis of DNA of dead cells (40). It has been shown
Table 2. Levels of EdCyd in Human Urine Samples and Statistical Data mean ( SD (range) dCyd (nM) dCyd/creatinine (nmol/g) dCyd/body weight (pM/kg) dCyd/body weight/creatinine (pmol/kg/g) Cyt (nM)b
smokers (n ) 10)
nonsmokers (n ) 13)
p valuea
0.45 ( 0.17 (0.29-0.80) 0.46 ( 0.21 (0.27-0.96) 5.7 ( 2.1 (3.5-10.5) 5.9 ( 2.6 (3.5-12.6) 1.1 ( 1.2 (0.10-4.24)
0.16 ( 0.17 (0-0.47) 0.21 ( 0.27 (0-0.98) 2.5 ( 2.7 (0-8.2) 3.3 ( 4.2 (0-15) 0.18 ( 0.18 (0.02-0.71)
0.0029c 0.0043c 0.0084c 0.0256c 0.0019c
a The p values were obtained by comparing adduct levels among smokers vs nonsmokers using the nonparametric Mann-Whitney U-test. b Recalculated from a previous report (29). c p < 0.05.
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Table 3. Levels of Selected DNA Adducts in Healthy Human Urine Samplesa levelb (smokers, pM)
adduct
levelb (nonsmokers, pM)
450 ( 170 (10) (range 290-800) 1100 ( 1200 (10) (range 100-4240)
dCyd Cyt
160 ( 170 (13) (range 0-470) 180 ( 180 (13) (range 20-710) ca. 4-75d (59 females) 12-226 pmol/48 he 113 ( 31 (10 males) (range 0-260) 370 ( 190 (3 females) (range 190-630)
dAdo Ade
352 ( 75 (10) (range 126-780)
Gua 1,N2-Gua 8-oxo-dGuo
Cyt > Ade (Table 3). Adducts derived from oxidative DNA damage
c
are in general higher in smokers than in nonsmokers except 8-oxo-dGuo and Ade in females. Interestingly, urinary levels of Ade and Cyt are higher than those of dAdo and dCyd, implicating that BER is the major repair pathway while NER is a minor or back-up repair pathway for both Ade and Cyt, like 8-oxo-Gua (54). Measurement of these lesions in urine should provide noninvasive biomarkers for oxidative DNA damage and cancer chemoprevention studies. Smoking is a severe oxidative stress, and high levels of oxidants are contained in cigarette smoke (55), which contributes to one-third of human cancer (56) and is the known cause of lung cancer (57). Levels of 8-oxo-Gua in leukocyte DNA and in urine were elevated in smoking lung cancer patients and healthy smokers as compared to nonsmokers. It is likely due to a deficient BER mechanism in cancer patients (53). Deficiency in NER is known to be responsible for various cancers, including smoking-related cancers (58). Our recent paper reported that the average urinary Cyt level in smokers was significantly higher than that in nonsmokers (30). In the present study, a statistically significant association is observed between urinary excretion of dCyd nucleoside and cigarette smoking. Furthermore, the inverse association between levels of dCyd and urinary TAC implies that dCyd can serve as a promising biomarker for oxidative stress. Urinary levels of dCyd also correlate with Cyt levels in the study population. In addition to cigarette smoking, factors affecting urinary excretion of DNA adducts have been investigated. Exposure to air polluted with car exhaust increased urinary 8-oxo-Gua levels (59), while gender and body mass index were shown to affect 8-oxo-dGuo concentrations (60). Both urinary 8-oxo-Gua and 8-oxo-dGuo levels in human are not dependent on diet (51). On the other hand, urinary dAdo excretion is influenced by saltinduced inflammation, ω-6 polyunsaturated fatty acid intake, and lipid peroxidation (31). Speina et al. recently compared levels of Ade and Cyt in lung and leukocyte DNA, and no differences in Ade and Cyt levels between tumor and nonaffected lung tissues were found in cancer patients (61). Moreover, adduct levels in leukocyte DNA were higher and the activities of BER in leukocytes and lung tissues were lower in lung cancer patients than healthy volunteers. However, urinary levels of Ade and Cyt or their nucleosides in these subjects were not analyzed, nor were the NER repair activities.
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The level of adducted bases in tissue DNA indicates a concentration measurement in specific tissue or cells at the moment of sampling, whereas the excretion of the repaired adducts in urine represents the average rate of damage in the total body (52). Our isotope dilution GC/ NICI/MS methods enable measurement of not only steady state levels of the promutagenic Cyt in tissue DNA (26) but also urinary concentrations of Cyt and dCyd in the same urine of an individual. The combination of these data with activities of BER and NER should provide valuable information for the elucidation between etheno adducts and cancer risk. Avoidance of smoking, increased consumption of fruits and vegetables, and control of infections should have a major effect on reducing incidences of cancer (62). Thus, this assay allows further investigations on the qualitative and quantitative importance of oxidative DNA damage and carcinogenesis in humans and it should be valuable in elucidating possible preventive measures (63).
Acknowledgment. This work was supported by grants from the National Science Council of Taiwan and from National Chung Cheng University (to H.-J.C.C.). We thank Tai-Chun Lin for initiating this study.
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