Detection and Quantification of 1,N6-Ethenoadenine in Human Urine

Camila Carriãao Machado Garcia , José Pedro Friedmann Angeli , Eduardo Alves de Almeida , Marisa Helena Gennari de Medeiros , Paolo Di Mascio. 2011 ...
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Chem. Res. Toxicol. 2003, 16, 1099-1106

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Detection and Quantification of 1,N6-Ethenoadenine in Human Urine by Stable Isotope Dilution Capillary Gas Chromatography/Negative Ion Chemical Ionization/Mass Spectrometry Hauh-Jyun Candy Chen* and Wei-Loong Chiu Department of Chemistry and Biochemistry, National Chung Cheng University, 160 San-Hsing, Ming-Hsiung, Chia-Yi 62142, Taiwan Received March 24, 2003

1,N6-Ethenoadenine (Ade) is a promutagenic lesion detected in tissue DNA; it has been shown that Ade can be repaired by human DNA glycosylases, and it is expected to be excreted in urine. In this paper, we present for the first time detection and accurate quantification of Ade in human urine samples by a highly sensitive and specific stable isotope dilution gas chromatography/negative ion chemical ionization/mass spectrometric assay (GC/NICI/MS). Analysis by GC/NICI/MS includes adduct enrichment by a solid phase extraction column, followed by electrophore labeling and postderivatization cleanup. Using selective ion monitoring mode, the assay allows quantification of 0.5 pg of Ade in as little as 0.1 mL of the urine sample, which is equivalent to corresponding concentration quantification limit of 31 pM. Using this assay, concentrations of Ade in the 24 h urine samples of 23 healthy individuals were determined, which ranged from 0 to 124 pg/mL. After we adjusted for creatinine, a statistically significant correlation was found between Ade excretion and cigarette smoking in males (p ) 0.03). Thus, this stable isotope dilution GC/NICI/MS assay offers a sensitive and accurate quantification of urinary Ade as a potential biomarker for oxidative damage of DNA and repair.

Introduction Humans are exposed to many exogenous carcinogens and endogenous reactive species derived from normal cellular metabolism, both of which are capable of damaging cells and tissues. Formation of exocyclic 1 DNA adducts, including Ade, Cyt, and N2,3-ethenoguanine, in vivo was originally shown to result from exposure of exogenous carcinogens, such as the industrial chemical vinyl chloride (1, 2) and the food contaminant ethyl carbamate (urethane) (3, 4). In addition to exogenous chemicals (5), recent evidences showed endogenous sources of -DNA adducts due to lipid peroxidation and oxidative stress might play an important role in carcinogenesis (6-8). Although Ade induces low mutation frequency in bacteria (9, 10), it is highly mutagenic in mammalian cells (10). Thus, accurate quantification of this adduct is essential for its use as a biomarker in cancer etiology and risk assessment. Analysis of Ade in DNA has been performed by immunoaffinity/32P-postlabeling (11, 12), isotope dilution GC/NICI/MS (13, 14), and LC/ESI/MS/ MS (15). -DNA adducts are known to be repaired by separate base excision repair enzymes (reviewed in 16), and their repair efficiencies by human DNA glycosylases vary (17). For example, a mammalian m3A-DNA glycos* To whom correspondence should be addressed. Tel: (886)5-2428176. Fax: (886)5-272-1040. E-mail: [email protected]. 1 Abbreviations: , etheno; Ade, 1,N6-ethenoadenine; Cyt, 3,N4ethenocytosine; ESI, electrospray ionization; MS, mass spectrometry; MS/MS, tandem mass spectrometry; NICI, negative ion chemical ionization; PFB, pentafluorobenzyl; SIM, selective ion monitoring; SPE, solid-phase extraction; %RSD, percentage relative standard deviation.

ylase excises Ade and Gua, but it is not effective for Cyt (18, 19). After they are repaired by DNA Nglycosylases, the excised DNA adducts are excreted in urine as free bases. Although the 32P-postlabeling technique is highly sensitive, it cannot be applied for analysis of free adducted bases or nucleosides since its methodology is based on labeling the 3′-momophosphate with radioactive ATP. The urinary -adduct was first detected in high levels upon exposure of rats to chloroethylene oxide, the metabolite of vinyl chloride, which led to a 50-fold increase of urinary Ade concentration using HPLC with fluorescence detection (20). On the other hand, an endogenous level of Ade in the urine of untreated rats was determined by immunoaffinity extraction combined with LC/ ESI/MS under SIM mode (21). Levels of 8-oxoguanine and its nucleoside in tissue DNA and in urine are higher in rodents than in humans due to the higher specific metabolic rate of rodents (22, 23). Because urinary levels of Ade are expected to be lower in human than in rodents, a high sensitivity assay is thus demanded for analysis of human samples. We have demonstrated the presence of Cyt in human urine using a highly sensitive and specific stable isotope dilution GC/NICI/MS assay (24). Recently, N2,3-ethenoguanine and 1,N2-ethenoguanine were both detected in human urine using LC with MS/MS (25). Because Ade is a preferred substrate for human 3-methyladenine DNA glycosylase (26), Ade might be present in human urine as a consequence of base excision repair mechanism. A plausible endogenous source of -DNA adducts is lipid peroxidation (8), and smokers are shown to have elevated

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lipid peroxidation (27) and oxidative DNA damage (28) as compared to nonsmokers. In this study, the 24 h urine samples from healthy individuals not occupationally exposed to industrial chemicals are analyzed to examine the effect of cigarette smoking on urinary excretion of Ade using a stable isotope dilution GC/NICI/MS assay modified from the two methods developed for Ade in DNA (13) and Cyt in urine (24).

Materials and Methods Chemicals. Standard Ade and Cyt were from Sigma Chemical Co. (St. Louis, MO). 2,3,4,5,6-PFB bromide, diisopropylethylamine, anhydrous methanol, and anhydrous phosphorus pentoxide were obtained from Aldrich Chemical Co. (Milwaukee, WI). Bond Elut C18-OH SPE columns (500 mg, 3 mL) were from Varian (Harbor City, CA). Strata Si SPE columns (500 mg, 3 mL) were from Phenomenex (Torrance, CA). The stable isotope [13C1,15N2]adenine (8-13C, 98%; 6-amino, 78%; 1-15N, 22%; 9-15N, 98%) was purchased from Cambridge Isotope Laboratories (Andover, MA). [15N5]Ade was synthesized as described (13). Synthesis of Stable Isotope-Labeled EAde Internal Standard. The isotope standard [13C1,15N2]Ade was synthesized from reaction of 2-chloroacetaldehyde (76 µL, 470 µmol) with [13C1,15N2]adenine (0.5 mg, 3.62 µmol) in a total volume of 1.3 mL at pH 4.5 at 60 °C for 4 h. The reaction mixture was neutralized and purified with a C18-OH SPE (500 mg, 3 mL) preconditioned with 15 mL of methanol, followed by 15 mL of water. After the sample was loaded, the SPE column was washed with 12 mL of water and the fraction containing [13C1,15N2]Ade was eluted by 3 mL of 35% methanol. The eluant was dried and quantified based on the molar UV absorbance of Ade on the reversed phase HPLC equipped with Hitachi L-7000 pump system with a D-7000 interface, a Rheodyne injector, a L-7450A photodiode array detector (Hitachi Ltd., Tokyo, Japan), and a Rheodyne injector connected to a Prodigy ODS (3) 250 mm × 4.6 mm 5 µ column (Phenomenex). The chromatographic system was performed with a linear water and acetonitrile gradient: 0-5 min, 0% acetonitrile; 5-20 min, 0-50% acetonitrile at a flow rate of 1.0 mL/min. The stable isotope [13C1,15N2]Ade was obtained in 30% yield as quantified by HPLC using the molar UV absorbance of commercially available Ade standard. The identity and isotopic purity of [13C1,15N2]Ade were further confirmed by GC/NICI/MS after pentafluorobenzylation. Adduct Enrichment by C18-OH SPE Column. Before application to urine samples, each batch of SPE columns was tested for consistency in their elution pattern using a standard solution containing 1.0 µg of Ade. A C18-OH SPE column was conditioned with 15 mL of MeOH, followed by 24 mL of water. After the volume of the sample was eluted, the column was eluted with 12 mL of water, 3 mL of 10% methanol in water, and followed by 3 mL of 15, 20, 25, 30, or 35% methanol in water. The fractions were collected every 3 mL in an 8 mL silanized glass vial. The fractions were evaporated and analyzed by reversed phase HPLC with photodiode array detection described above. The fraction containing Ade was found to elute with 3 mL of 35% methanol in water. Sample Preparation. Urine samples stored at the -80 °C freezer as 1.0 mL aliquots in 1.5 mL Eppendorff tubes were defrosted in an ice-water bath and centrifuged at 15 000g for 10 min at 4 °C. The precipitate was discarded, and the left over samples were not reused. The creatinine contents were analyzed by a picric acid method (29). A 0.1 mL aliquot of the supernatant was added to 0.1 mL of potassium phosphate buffer (50 mM, pH 7.0) to adjust the pH, followed by addition of the internal standard [13C1,15N2]Ade (2.0 ng). The adduct was enriched by applying the mixture to a C18-OH SPE column preconditioned with 15 mL of MeOH, followed by 24 mL of water. After the sample was applied, the column was washed with 12 mL of water, followed by 3 mL of 10% methanol in water. The fraction

Chen and Chiu containing Ade was eluted with 3 mL of 35% methanol in water and collected in an 8 mL silanized glass vial. The eluant was evaporated under vacuum by a centrifuge concentrator, dried over phosphorus pentoxide, and derivatized by PFB-Br (5 µL) in the presence of diisopropylethylamine (7 µL) in 0.1 mL of anhydrous methanol at 40 °C for 1 h (13). After the reaction mixture was evaporated to dryness in a vacuum, it was dissolved in dichloromethane (0.2 mL) and purified by a Si SPE column preconditioned with 15 mL of dichloromethane. The column was washed with 3 mL of 1% methanol in dichloromethane (v/v), followed by 1 mL of 5% methanol in dichloromethane (v/v). The fraction containing PFB-Ade was eluted with 2 mL of 5% methanol in dichloromethane (v/v) and collected in a 4 mL silanized glass vial. The eluant was evaporated by a centrifuge concentrator, transferred to a silanized insert, and evaporated to dryness. The residue was dissolved in 10 µL of acetone, and 1 µL of aliquot was analyzed by GC/NICI/MS with SIM at m/z 158 and 161 for PFB-Ade and [13C1,15N2]PFB-Ade, respectively. GC/NICI/MS Analysis of PFB-EAde. GC/NICI/MS experiments were performed as reported for PFB-Cyt (24). The analysis was conducted using a Hewlett-Packard 6890 GC with 5973 MSD mass selective detector with the NICI source (Agilent Technologies, Palo Alto, CA). In the SIM mode, the filament was operated at 120 eV with the ion source at 150 °C. The analyses were carried out with a cool-on-column inlet, a precolumn (J&W, 1.0 m, 0.53 mm, deactivated silica), and a HP5MS capillary column (Hewlett-Packard, 0.25 mm × 30 m, 0.25 µm film thickness) inserted into the ion source. Methane (99.999% pure) was the reagent gas with a flow rate of 2.0 mL/ min, and the pressure at the ion gauge was 2.2 × 10-4 Torr. Helium was used as the carrier gas (99.999% pure) at a flow rate of 1.2 mL/min. The oven temperature was held at 50 °C for 2 min and then raised to 300 °C at 10 °C/min (gradient A) or at 7 °C/min (gradient B). Gradient B was only used in control urine spiked with 0.5 pg of Ade. The rest of the analysis throughout this study was performed with gradient A. PFBAde and [13C1,15N2]PFB-Ade were detected and quantified at m/z 158 and 161, respectively, under the SIM mode for the [M - 181]- fragment ions. The quantification of Ade was based on intrapolation of the ratio of the peak area of PFB-Ade vs [13C1,15N2]PFB-Ade on the respective calibration curves. Calibration Curves. The stock solutions of Ade and [13C1,15N2]Ade (1.0 mg/mL) in water were stored at -80 °C. Sample solutions for calibration were freshly prepared by diluting the stock solutions in water for each analysis. Samples with various amounts of Ade ranging from 0, 0.5, 1.0, 5, 10, and 50 pg each were added [13C1,15N2]Ade (2.0 ng) as internal standards. The samples were processed through the same procedures, i.e., C18-OH SPE enrichment, pentafluorobenzylation, Si SPE purification, and GC/MS analysis. The equations of the calibration curves were obtained by linear regression. EAde-Spiked Urine Samples. 1. Spiked Control Urine. To 0.1 mL of a nonsmoker’s urine, in which Ade was not detectable, was added 0.5 pg of Ade and 2.0 ng of [13C1,15N2]Ade and processed through the assay procedures described above. 2. Reverse Addition. To 0.1 mL of a smoker’s urine was added 1.0 ng of Ade and processed through the assay procedures described above, including C18-OH SPE enrichment, pentafluorobenzylation, Si SPE purification, and GC/MS analysis under SIM mode as described above. Pentafluorobenzylation of 1,N6-Etheno-2′-deoxyadenosine. A silanized 4 mL vial containing 29 pg of 1,N6-etheno2′-deoxyadenosine and 1.0 ng of [15N5]Ade was dried over phosphorus pentoxide, derivatized with PFB-Br, purified by a Si SPE column, and analyzed by GC/NICI/MS under SIM mode as described above. Statistical Analysis. All results are reported as the means ( standard deviation (SD). Statistical analysis of the two groups was performed by the Mann-Whitney U-test. Spearman rank correlation was used to calculate correlation coefficients. Multiple regression analysis was performed to examine the contri-

Ade in Human Urine by GC/MS

Chem. Res. Toxicol., Vol. 16, No. 9, 2003 1101

Scheme 1. Analysis of EAde in Human Urine by Stable Isotope Dilution GC/NICI/MS

bution of two or more factors. GraphPad InStat version 3.00 for Windows 95, GraphPad Software (San Diego, CA; www.graphpad.com) was used for these analyses.

Results The procedures for analyzing Ade in the urine sample are described in Scheme 1. Typically, the urine sample was added to stable isotope [13C1,15N2]Ade as the internal standard, and adduct enrichment by a C18-OH SPE column, pentafluorobenzylation, postderivatization cleanup by a Si SPE column, and GC/NICI/MS analysis were performed. Adduct Enrichment by SPE. Urine is composed of a complex mixture of cellular metabolites. To maximize the efficiency in purification and enrichment of Ade in urine, conditions were optimized using a reversed phase nonendcapped C18-OH SPE column. To confirm the consistency in the elution pattern, each batch of SPE columns was tested with standard Ade. The optimized condition for eluting Ade was found to be 35% aqueous methanol after removing the very polar components by washing with water followed by 10% aqueous methanol, and the nonpolar compounds retained in the column were discarded. The recovery of Ade after the C18-OH SPE column was 86% as analyzed by reversed phase HPLC with photodiode array detection (data not shown). HPLC analysis showed no matrix effect when standard Ade was added to urine samples. Therefore, 0.1 mL of the 24 h urine sample was neutralized and added to stable isotope standards [13C1,15N2]Ade and enriched by a disposable C18-OH SPE column. The collected fraction was dried and derivatized before GC/NICI/MS analysis. Adduct Quantification by the GC/MS Assay. Incorporation of stable isotope of the analyte as internal standard for mass spectrometric analysis offers the advantage of identification and accurate quantification of the analyte in the complex samples. Because the isotope standard is chemically identical to the analyte,

Figure 1. Pentafluorobenzylation of [13C1,15N2]Ade. Two nanograms of [13C1,15N2]Ade was pentafluorobenzylated, purified by a Si SPE column, and analyzed by GC/NICI/MS analysis under SIM mode as described in the Experimental Section. The peak of 23.33 min at m/z 161 is [13C1,15N2]PFB-Ade.

its retention time in the GC chromatogram is virtually the same as the analyte and they only differ in the mass detection under the SIM mode of the MS. Another advantage of using isotope standard is that the subnanogram quantity of the added isotope standard serves as carrier for trace amounts of the analyte, allowing its accurate quantification. Electrophore labeling of DNA adducts provides very high sensitivity in NICI/MS (reviewed in 30). In the stable isotope dilution GC/NICI/ MS assay, the limit of detection (LOD) for PFB-Ade and PFB-Cyt injected on the column was 10 (30 amol) (13) and 1.0 fg (3.2 amol) (24), respectively. Using the conditions and the benchtop GC/MS instrument for PFB-Cyt, the on-column detection limit of standard PFB-Ade is improved 5-fold (2.0 fg, 5.9 amol). A postderivatization cleanup using a Si SPE column is required before GC/MS analysis to maintain the lifetime of the GC column. Derivation of Ade with PFBBr in the presence of diisopropylethylamine results in formation of diisopropylethylammonium bromide salt, which is retained in the Si SPE column. The contribution of Ade in the synthesized isotope [13C1,15N2]Ade is merely detectable, as evidenced by pentafluorobenzylation of [13C1,15N2]Ade itself. No PFBAde was detected at 23.33 min under m/z 158 (Figure 1). Positive control experiments with isotopes only were carried out to measure the background level of the entire assay system. The area ratio of the control samples containing solely [13C1,15N2]Ade was 0.0001, and the calibration curve was linear from 0.5 (limit of quantification, LOQ) to 50 pg with a correlation coefficient (γ2) of 0.9977 (Figure 2). Levels of Ade in urine were quantified according to the calibration curve. The blank run with solvent was performed before each sample run to ensure

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Figure 2. Calibration curve for the GC/NICI/MS analysis of Ade. Samples containing various amounts (0-50 pg) of Ade were added to a fixed amount of [13C1,15N2]Ade (2.0 ng) and subjected to the assay procedures described in the Experimental Section (r2 ) 0.9977). 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. Table 1. Precision of the GC/MS Assay for Urinary EAde adduct levels samples

day 1

RSD (%) (n ) 5) day 2

no. 1 no. 2 no. 3

73.7 ( 3.0 (4.0%) 50.8 ( 3.8 (7.2%) 33.2 ( 2.7 (8.2%)

76.9 ( 5.3 (5.3%) 53.5 ( 3.5 (3.5%) 34.3 ( 2.5 (7.3%)

no carryover from the previous sample. Because 0.1 mL of urine was used throughout the assay, the standard curve was suitable for quantifying Ade concentrations ranging from 5.0 to 500 pg/mL and the LOQ is equivalent to a concentration quantification limit of 5.0 pg/mL or 31 pM Ade. To confirm the value of LOQ, 0.5 pg of Ade was added to 0.1 mL of a control urine sample in which Ade was not detectable (Figure 3A). The concentration of Ade in this spiked urine was determined as 6.1 ( 0.6 pg/mL from five replicates with the %RSD of 10.5%. Using a relatively slow temperature gradient (gradient B), the peak of 30.82 min at m/z 158 was unambiguously quantified (Figure 3B). It is likely that the control urine sample contains approximately 1 pg/mL of Ade, which is below the quantification limit of this assay. Injecting 1/10 of the entire sample to GC/MS unavoidably increases the LOQ 10-fold. The big difference between LOD and LOQ is mainly due to the background level in the control samples containing only the isotope. Observing a background level is not unusual in ultrasensitive analysis. A possible source of contamination might be the centrifuge concentrator since it is also used to evaporate samples for a standard curve, which contains high levels of Ade standard. Three samples at the lower quartile, middle quartile, and upper quartile with five replicates were performed in five replicates on three separate days to obtain intraday and interday validation. The results are shown in Table 1. Both intraday and interday %RSD have the trend to increase with decreasing adduct concentrations. The intraday %RSD ranges from 1.2 to 8.2% and those for interday are between 3.3 and 9.4%. Artifactual Formation of EAde? To examine the possible artifactual formation of Ade during sample purification and derivatization, two experiments were performed. First, a copious amount of Ade (1.0 ng),

day 3

interday variation RSD (%)

78.6 ( 1.2 (1.2%) 54.7 ( 2.9 (2.9%) 39.5 ( 2.9 (7.4%)

3.3 3.7 9.4

instead of [13C1,15N2]Ade, was added to a urine sample as an internal standard (reverse addition) and processed through the same assay procedures. Adenine is present in urine at micromolar concentrations (31). If the assay procedures lead to artifactual formation of PFB-Ade, it will be detected. At m/z 161, no peak corresponding to [13C1,15N2]PFB-Ade at 22.85 min was detected (Figure 4). We also tried to clarify if Ade detected in urine is derived from cleavage of the glycosidic bond in1,N6etheno-2′-deoxyadenosine during derivatization as reported for malondialdehyde-2′-deoxyguanosine adduct (32). No peak corresponding to PFB-Ade was detected at m/z 158 (Figure 5). The result showed that 1,N6etheno-2′-deoxyadenosine did not give rise to PFB-Ade after the assay procedures, which might be due to the stronger glycosidic linkages in 2′-deoxyadenosine as compared to 2′-deoxyguanosine nucleosides. Adduct Levels. Figure 6 showed the representative GC/NICI/MS chromatograms of the urine analysis of a smoker and a nonsmoker. Under the SIM mode, the PFB-Ade peaks monitoring at m/z 158 show identical retention times as those for the [13C1,15N2]PFB-Ade peak at m/z 161. The peak of 23.12 min at m/z 158 in Figure 6A represents 1.8 pg (5.3 fmol) of PFB-Ade, and the peak at 23.84 min in Figure 6B represents 0.64 pg (1.9 fmol) of PFB-Ade. The results correspond to an Ade concentration of 84 pg/mL in the smoker and 30 pg/mL in the nonsmoker. Levels of Ade in the 24 h urine samples from 10 smokers and 13 nonsmokers were determined by isotope dilution GC/NICI/MS in this pilot study. Our previous study showed that Cyt was detected at high levels in two smokers using 1.0 mL of urine. Because injection of 1/10 of the processed sample led to overloading of the GC column, only 1/100 of the sample was injected (24). In this study, 0.1 mL of urine was used and 1/10 of the processed sample was injected to GC/MS for analysis. All

Ade in Human Urine by GC/MS

Chem. Res. Toxicol., Vol. 16, No. 9, 2003 1103

Figure 4. GC/NICI/MS chromatograms of a smoker’s urine spiked with 1.0 ng of Ade in the absence of [13C1,15N2]Ade. A smoker’s urine (0.1 mL) was added to Ade (1.0 ng), enriched with a C18-OH SPE column, derivatized with PFB-Br, purified by a Si SPE column, and analyzed by GC/NICI/MS under SIM mode as described in the Experimental Section. The peak of 22.85 min at m/z 158 is PFB-Ade.

Figure 3. GC/NICI/MS chromatograms of (A) the control urine and (B) the control urine spiked with 0.5 pg of Ade in the presence of [13C1,15N2]Ade. Samples were added [13C1,15N2]Ade (2.0 ng), enriched with a C18-OH SPE column, derivatized with PFB-Br, purified by a Si SPE column, and analyzed by GC/ NICI/MS under SIM mode as described in the Experimental Section. The peak of 22.51 min at m/z 158 in panel A is [13C1,15N2]PFB-Ade, eluting with temperature gradient A. The peak of 30.83 min at m/z 161 in panel B is [13C1,15N2]PFB-Ade, eluting with temperature gradient B.

of the samples are accurately quantified with the intraday and interday precision of 1.8 and 1.6%, respectively. Characteristics of the study population are listed in Table 2. No heavy smokers were included in this study according to the smoking index, which is defined as the number of cigarettes smoked per day times years of smoking.

Figure 5. GC/NICI/MS chromatograms for pentafluorobenzylated 1,N6-etheno-2′-deoxyadenosine in the presence of [15N5]Ade. 1,N6-Etheno-2′-deoxyadenosine (29 pg) and [15N5]Ade (1.0 ng) were evaporated and derivatized with PFB-Br, purified by a Si SPE column, and analyzed by GC/NICI/MS under SIM mode as described in the Experimental Section. The peak at 22.90 min at m/z 161 is [15N5]PFB-Ade.

Levels for two male nonsmokers are below the LOQ. The highest concentration in nonsmokers belongs to a female. Because there are no female smokers in the study population, statistical comparisons are performed between male smokers and male nonsmokers. As shown in Table 3, a statistically significant correlation is observed

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Among nonsmokers, the average level of Ade normalized for creatinine excretion in the three women is found to be much higher than the 10 men (577 vs 135 pmol/g creatinine). It might be partially due to the lower creatinine levels in women than in men. Thus, urinary excretion of Ade in 10 male smokers is 401 ( 115 pmol/g creatinine (mean ( SE), while that for the 10 male nonsmokers is 135 ( 44 pmol/g creatinine (mean ( SE). Using the nonparametric Mann-Whitney U-statistics, male smokers excrete significantly more Ade than male nonsmokers (p ) 0.03). It is possible that more significant association will be found in large-scale epidemiological studies since smokers recruited in this pilot study were light to moderate smokers and no heavy smokers were included. We also examine the association between normalized Ade excretion with age and body weight. However, no significant correlation was observed when these two factors were analyzed independently on male smokers and male nonsmokers using nonparametric Spearman correlation. Multiple regression analysis of both age and body weight on male smokers and male nonsmokers also did not show statistically significant correlations.

Discussion

Figure 6. GC/NICI/MS analysis of PFB-Ade in (A) a smoker’s urine and (B) a nonsmoker’s urine. Urine (0.1 mL) was added [13C1,15N2]Ade (2.0 ng), enriched with a C18-OH SPE column, derivatized with PFB-Br, purified by a Si SPE column, and analyzed by GC/NICI/MS under SIM mode as described in the Experimental Section. The peak of 23.12 min at m/z 158 in panel A represents 1.8 pg (5.3 fmol) of PFB-Ade, and the peak of 23.84 min at m/z 158 in panel B represents 0.64 pg (1.9 fmol) of PFB-Ade. The results correspond to an Ade concentration of 84 (A) and 30 pg/mL (B). Table 2. Characteristics of the Study Population

sex (male/female) age (years) cigarettes/day smoking years smoking indexb

smokers (n ) 10) mean ( SE (range)

nonsmokers (n ) 13) mean ( SD (range)

10/0 43 ( 5 (24-68)a 15 ( 2 (5-25) 22 ( 5 (3-50) 331 ( 84 (15-750)

10/3 31 ( 3 (20-45)

a Results are expressed as mean ( standard error (SE). The ranges are expressed in parentheses. b Smoking index ) number of cigarettes smoked per day × years of smoking.

between the concentration of Ade and cigarette smoking. The two-tailed p value is 0.0052 comparing Ade concentrations between male smokers and male nonsmokers.

Cigarette smoking is the cause of several human cancers, including lung, esophageal, bladder, and pancreas (33). In addition, smokers are shown to have increased levels of lipid peroxidation as compared to nonsmokers (27). -Adducts are derived from endogenous lipid peroxidation, and their levels were found to be elevated in cancer prone tissues (8), suggesting that these promutagenic lesions could drive cells to malignancy. No differences were found in levels of Ade and Cyt in pancreatic DNA between smokers and nonsmokers (34). In the measurement of DNA adducts for humans exposed to carcinogens, DNA extracted from target tissues, obtained from necropsy or during surgery, or circulating blood cells as surrogate tissues have been used. These methods limit the number of samples available for analysis. Unlike measurement of tissue DNA adducts, analysis of adducts in urine is noninvasive and urinary adduct levels reflect DNA repair, diet, and cell death (35). However, the profile of adduct formation in the deoxynucleotide pool with the methylating agent is different from that in DNA (36). In addition, adducts derived from RNA modification are not recognized the same way as adducts on DNA. For example, methylated adducts in liver RNA of hamsters exposed to the methylating agent are not repaired (37). Although it remains to be proven, it is very unlikely that 1,N6-ethenoadenosine-containing RNA is repaired by DNA glycosylases responsible for excision of 1,N6-etheno-2′-deoxyadenosinecontaining DNA. Spontaneous depurination of Ade from 1,N6-ethenoadenosine is much more difficult than from 1,N6-etheno-2′-deoxyadenosine since the glycosidic linkage in riboses is stronger than that in 2′-deoxyriboses. Our results showed that 1,N6-etheno-2′-deoxyadenosine does not contribute to the formation of PFB-eAde in the assay. Therefore, the possibility that RNA or the nucleotide pools as a source of Ade in urine is excluded provisionally. The influence of dietary factors on tissue -adduct formation has been investigated. High intake of ω-6 polyunsaturated fatty acid markedly increased levels of

Ade in Human Urine by GC/MS

Chem. Res. Toxicol., Vol. 16, No. 9, 2003 1105 Table 3. Levels of EAde in Human Urine Samples

Ade (pg/mL) rangec Ade/creatinine (pmol/g)b rangec

smokers all male (n ) 10)b

nonsmokers male (n ) 10)b

56 ( 12 20-124c 401 ( 115 110-1292c

18 ( 5 0-42c 135 ( 44 0-370c

p valuea 0.005d 0.03d

nonsmokers female (n ) 3)b 59 ( 30 30-101 577 ( 344 331-1060c

a The two-tailed p values were obtained by comparing adduct levels between male smokers and male nonsmokers using the nonparametric Mann-Whitney U-test. b Results are expressed as mean ( SE. The level for each sample was obtained from at least four repeated measurements. c The numbers in the parentheses represent ranges of Ade levels. d p < 0.05.

Ade and Cyt in white blood cells (WBC) in women at high risk for breast cancer (38) but not in healthy female volunteers (39). Vegetable or vitamin E consumption contributes to lower Ade levels in WBC DNA of healthy females (39). Ethanol increased Ade and Cyt levels in rat hepatic DNA approximately 2-fold (40). Urinary Ade excretion in human could reveal a level of oxidative DNA damage of the body due to endogenous and dietary factors. Large interindividual variations have been demonstrated for repair of Ade-containing oligonucleotide, presumably by DNA glycosylases (41). A sex specific effect on Ade and Cyt in WBC DNA showed that levels of these two -adducts were elevated in females, but not males, after intake of ω-6 polyunsaturated fatty acid (38). The effect was possibly due to estrogen metabolism. Hanaoka et al. reported a positive association between levels of 1,N6-etheno-2′-deoxyadenosine with salt excretion and ω-6 polyunsaturated fatty acid intake in postmenopausal women (42). 1,N6-Etheno-2′-deoxyadenosine is thus considered a potential biomarker of DNA damage derived from salt-induced inflammation and lipid peroxidation. Excretion of 1,N6-etheno-2′-deoxyadenosine might derive from the deoxynucleotide pool and/or the possible nucleotide excision repair pathways, while the known base excision repair glycosylases contribute to urinary Ade base excretion (17, 18, 26). The average level of 1,N6-etheno-2′-deoxyadenosine in Hanaoka’s study (12-226 pmol/48 h or ca. 4-75 pM) (42) was much lower than that of Ade (0-124 pg/mL or 0-780 pM) found in this study. Furthermore, levels of Ade excretion determined in this study are in the same range as Cyt in urine of two smokers (0.8 nM) (24) and 1,N2-ethenoguanine and N2,3-ethenoguanine isomers in human urine (0.3-8 nM) (25). Assuming that the contributions from the deoxynucleotide pool and diet are equal, it is very likely that base excision repair is the predominant pathway for repair of Ade-containing damage in human. In conclusion, this study is the first paper detecting the promutagenic Ade in human urine. The levels of Ade in male smokers’ urine are significantly higher than male nonsmokers within the limited number of subjects. The stable isotope dilution GC/NICI/MS assay provides a useful tool to quantify a noninvasive biomarker for oxidative DNA damage in molecular epidemiology to assess carcinogenic risks from various lifestyles and in cancer prevention studies.

Acknowledgment. This work was supported by grants from the National Science Council of Taiwan (to H.-J.C.C.). We thank all of the participants for donating the urine samples and Mr. Chia-Liang Hong for helpful technical assistance.

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