810
Chem. Res. Toxicol. 1998, 11, 810-815
Quantitation of 1,N6-Ethenoadenine in Rat Urine by Immunoaffinity Extraction Combined with Liquid Chromatography/Electrospray Ionization Mass Spectrometry Ten-Yang Yen,† Sharon Holt, Ramiah Sangaiah, Avram Gold, and James A. Swenberg* Department of Environmental Sciences and Engineering and Curriculum in Toxicology, University of North Carolina, Chapel Hill, North Carolina 27599-7400 Received January 27, 1998
A fast, highly specific analytical method was developed to quantify 1,N6-ethenoadenine (A) in urine of rats. A is a highly mutagenic DNA adduct generated by vinyl chloride (VC) exposures as well as endogenously from lipid peroxidation. A was concentrated through extraction from rat urine by immunoaffinity chromatography and quantitated by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS). The average A recovery by immunoaffinity extraction was 66%. The LC/ESI-MS selected-ion monitoring (SIM) of the response ratio of A to its isotopically labeled internal standard [15N5]A was linear (r2 ) 0.999) and reproducible from 0.15 to 30 pmol/injection. The detection limit obtained in the routine analysis of urine of unexposed rats was 270 fmol/sample with a signal-to-noise ratio (S/N) 3:1. The concentration of endogenous A was determined to be 21.6 ( 14.8 pmol/mL (3 rats). Following portal injection of chloroethylene oxide (CEO; the putative active metabolite of VC), the rate of A excretion in urine was greatest from 0 to 24 h, with ∼90% of the CEOinduced A excreted. By 132 h, the excretion of A was similar to pretreatment amounts. The accuracy of the quantitation was 107 ( 6% (n ) 4), established by analyzing urine of an unexposed rat spiked with authentic A. These data indicate that the LC/ESI-MS with immunoaffinity extraction method is precise and accurate for A quantification. The measurement of A in urine provides a potential biomarker for exposure to chemicals and processes that form this adduct.
Introduction The chemical modification of DNA by chemical carcinogens or their metabolites can be the initial step of chemical carcinogenesis (1). Therefore quantitation of DNA adducts provides a rational assessment of human risk associated with exposure to exogenous and/or endogenous carcinogens (1-5). The DNA adducts induced by vinyl chloride (VC)1 are of particular interest because VC is a widely used compound in the production of plastics and is a known human and rodent carcinogen (6). In mammalian systems, VC is oxidized by cytochrome P450 2E1 to chloroethylene oxide (CEO), which rearranges to chloroacetaldehyde (CAA) (6, 7). Both CEO and CAA can react with the DNA bases to form covalent adducts. In vitro reaction of CEO with calf thymus DNA resulted in the formation of N7-(2-oxoethyl)guanine (OEG) as the major DNA adduct along with minor amounts of etheno adducts such as 1,N6-ethenoadenine (A) and N2,3-ethenoguanine (7). While OEG does not show mispairing in DNA templates, the etheno adducts * Corresponding author. Tel: 919-966-6139. Fax: 919-966-6123. † Current address: Department of Chemistry and Biochemistry, San Francisco State University, San Francisco, CA 94132. 1 Abbreviations: VC, vinyl chloride; CEO, chloroethylene oxide; CAA, chloroacetaldehyde; OEG, N7-(2-oxoethyl)guanine; A, 1,N6-ethenoadenine; LC/ESI-MS, liquid chromatography/electrospray ionization-mass spectrometry; CID, collision-induced dissociation; SIM, selected-ion monitoring; RIC, reconstructed ion chromatogram.
are highly promutagenic and thus considered of greater biological significance (8). It has also been found that the etheno adducts can be formed endogenously through lipid peroxidation (9) and by the reaction of mucochloric acid, a genotoxic compound found in chlorinated drinking water, with nucleosides (10). These results suggest that etheno adducts may be potential biomarkers for monitoring human exposure to both exogenous and endogenous carcinogens. Several analytical methods, including 32P-postlabeling and mass spectrometric assays, have been developed to measure etheno adducts in vivo (9, 11-13). A major challenge for these assays is the enrichment of adducts in the presence of a 106-8-fold excess of normal DNA bases. Extraction by immunoaffinity chromatography combined with 32P-postlabeling has been demonstrated as a simple and ultrasensitive assay for DNA adducts (one adduct per 109 bases) (9, 12, 14). However, a major drawback of this assay for quantifying DNA adducts is inaccuracy arising from variable sample recovery in the immunoaffinity extraction step (12). Isotope dilution mass spectrometry employs internal standards labeled with stable isotopes to compensate for variation in the analyte recovery during sample preparation and is therefore more accurate for DNA adduct quantitation (11, 13-20). We had previously reported the use of immunoaffinity columns and HPLC with fluorescence detection
S0893-228x(98)00019-8 CCC: $15.00 © 1998 American Chemical Society Published on Web 07/20/1998
LC/MS Quantitation of 1,N6-Ethenoadenine in Urine
of A in urine (21). In this study, we demonstrate that A excreted in rat urine can be rapidly and accurately quantified by combining immunoaffinity chromatography for sample extraction with liquid chromatography/electrospray ionization-mass spectrometry (LC/ESI-MS) for analyte quantitation with recovery controlled by isotope dilution mass spectrometry.
Experimental Procedures Caution: Chloroethylene oxide (CEO) is a suspect carcinogen and should be handled with protective clothing in a wellventilated hood. Materials. HPLC grade water and methanol (MeOH) were obtained from Fisher Scientific (Fair Lawn, NJ) and Mallinckrodt Chemical Co. (Paris, KY), respectively. A chemical standard was purchased from Sigma (St. Louis, MO). Other chemicals utilized were analytical grade and were obtained from Fisher Scientific. [15N5]-1,N6-Ethenoadenine ([15N5]A) was synthesized from [15N5]adenosine (Cambridge Isotopes, Andover, MA) in two steps by deglycosylation using 0.1 N HCl followed by reaction with chloroacetaldehyde in the presence of acetate buffer (22). CEO was synthesized in-house by photochlorination of ethylene oxide (23). EA Sample Assay. Male Fischer 344 rats were purchased from Charles River Breeding Laboratories (Raleigh, NC) and were provided with a standard NIH diet and water ad libitum. Before the dosing experiment, urine was collected to serve as the control. Rats were dosed intraportally with 8.5 mg of CEO/ kg of body weight to selectively alkylate the liver. Urine was collected at 4, 6, 12, 24, 72, or 132 h after treatment and was stored at -70 °C prior to A analysis. Immunoaffinity columns used for A extraction were prepared according to a method developed in our laboratory (12). Briefly, A-specific monoclonal antibody (EM-A-1; provided by Prof. Manfred F. Rajewsky, Essen, Germany) columns were bound to gel of protein A-Sepharose CL-4B (Pharmacia Biotechnology, Piscataway, NJ). Each column was packed with 0.5 mL of resin bed volume, equilibrated in PBS buffer (0.137 M NaCl, 2.68 mM KCl, 6.41 mM Na2HPO4, and 0.88 mM KH2PO4) with 0.02% sodium azide to prevent bacterial growth, and stored at 4 °C before use. Urine was mixed with an equal volume of PBS buffer (0.1-0.3 mL), and [15N5]A (15 pmol) was added for quantitation. The mixtures were loaded onto immunoaffinity columns followed by washing with 5 mL of PBS buffer and 5 mL of water and then eluted with 5 mL of 50% MeOH/water. The eluent was dried in a SpeedVac (Savant Instruments, Farmingdale, NY) and resuspended in 80 µL of water before LC/ESI-MS analysis. Immunoaffinity columns were regenerated between the experiments by consecutive washing with 10 mL of water, 10 mL of 0.1 M formic acid, 10 mL of water, and 10 mL of PBS/sodium azide. Between assays, columns were filled with 1 mL of PBS/sodium azide and stored at 4 °C. LC/ESI-MS Analyses. Aqueous solution (5-20 µL) from resuspended eluent was injected via a Valco injector (Valco Instruments, Houston, TX) and analyzed by LC/ESI-MS. LC/ ESI-MS analyses were conducted using a Pharmacia liquid chromatographic system with dual pumps #2248 (Pharmacia LKB Biotechnology, Uppsala, Sweden) coupled to a Finnigan 4000 quadrupole mass spectrometer (Finnigan-MAT, San Jose, CA) retrofitted with a pneumatic electrospray source (Analytica of Branford, Branford, CT). The LKB pumps were operated at a flow rate of 0.4 mL/min. After the high-pressure mixer, the solvent (water and/or methanol) was passed through a microflow processor (LC Packings, San Francisco, CA). One outlet of the microflow processor was connected to the waste receptacle and the other to a capillary C18 column (150- × 0.8-mm i.d., Hypersil, 3-µm particle size; LC Packings) with a flow rate of 20 µL/min. A length of fused-silica capillary (30-cm × 50-µm i.d., 375-µm o.d.) directed the eluent of the capillary column to the electrospray needle. Chromatographic separations were accomplished by increasing the mobile phase from 97% water/3% MeOH to
Chem. Res. Toxicol., Vol. 11, No. 7, 1998 811 50% water/50% MeOH in 11 min, then to 2% water/98% MeOH in 6 min, and holding at 98% MeOH for 3 min. A voltage of 2.85 kV was applied to the electrospray needle, and 70 psi nebulizer gas (N2) was employed to stabilize the spray. The voltage difference between the exit of the glass capillary and the first skimmer in the differential pumping region was optimized at 130 V for the signal of the [M + H]+ ion of A and at 180 V for collision-induced dissociation (CID) to generate the fragment ions from the [M + H]+ ion. Data were acquired and processed by a Technivent Vector 2 data system (Prolab Resources, Madison, WI). Full-scan mass spectra were obtained by scanning from m/z 15 to 350 in 2 s. For selected-ion monitoring (SIM) analysis, two ions (m/z 160 and 165) were monitored with a dwell time of 0.7 s. Quantification of A was accomplished by measuring the peak areas of the [M + H]+ ion of A (m/z 160) and the [M + H]+ ion of [15N5]A (m/z 165) in standard solutions containing 150, 300, 750, 1500, 3000, 6000, and 30 000 fmol of A and 1500 fmol of [15N5]A per injection. The calibration curve was constructed by plotting the ratio of the areas of the chromatographic peaks of A to [15N5]A against the molar ratios of A to [15N5]A. The equation of calibration curve was determined by linear regression on the data (least-squares method). After every fifth sample, a standard calibration solution was analyzed to ensure the quality of the analyses. The relative difference among these standards throughout the day was less than 15%. For every eight samples, one method blank containing 15 pmol of [15N5]A was analyzed to check for carryover of analyte from previous experiments. A was not detectable in method blanks. The calibration curve was also performed with immunoaffinity columns.
Results and Discussion In a study of the factors affecting LC/ESI-MS response of N2,3-ethenoguanine (13), we found that a linear gradient program used with a water/methanol mobile phase in the absence of modifier (buffer or acid) provided sufficient separation and optimal signal for the [M + H]+ ion in LC/ESI-MS analyses. For etheno adducts, better analyte response was obtained with the skimmer offset voltage of the ESI source set at 130 V, rather than the normal setting at 80 V. Using the capillary column described above, the detection limit of etheno adducts was about 150 fmol with S/N ) 15:1. Figure 1a-c shows the results of LC/ESI-MS analyses of A standards. The base peak of the full-scan mass spectrum of A is the [M + H]+ ion at m/z 160 with a retention time of 13.1 ( 0.5 min (Figure 1a,b). When ESI-MS was performed under conditions promoting CID in ESI source, three useful fragment ions at m/z 106, 119, and 133 were generated from the [M + H]+ ion of A (Figure 1c). The proposed fragmentation pathway (Figure 1c) was supported by the fragmentation pattern (m/z 109, 123, and 137) of the [M + H]+ ion of [15N5]A under the same CID conditions (spectrum not shown). One of the most difficult challenges for analyzing DNA adducts in biological media is the isolation of analyte from the large excess of normal bases and other potential interferences. Immunoaffinity chromatography on reusable columns containing monoclonal antibodies provides a rapid, highly specific method for extraction of analyte from complex biological matrixes. Our laboratory and others have shown that immunoaffinity chromatography coupled with 32P-postlabeling affords an ultrasensitive bioassay for detecting A at less than 4 adducts/109 parent nucleotides (9, 12). However, quantitation by immunoaffinity/32P-postlabeling must include careful calibration for analyte recovery since variation in sample
812 Chem. Res. Toxicol., Vol. 11, No. 7, 1998
Figure 1. LC/ESI-MS analysis of A (21 pmol): (a) reconstructed ion chromatogram at m/z 160; (b) backgroundsubtracted mass spectrum of A; (c) collision-induced dissociation mass spectrum of A.
recovery between columns, or between experiments in different environments, will result in significant inaccuracy. We measured A recovery from immunoaffinity columns using isotope dilution LC/ESI-MS. Sample recovery was found to be strongly affected both by the recovery from the immunoaffinity column and by analyte adsorption on sample preparation tubes. To measure recovery for the extraction step, authentic A (1.5-60 pmol) or [15N5]A (15 pmol) was loaded onto different sets of immunoaffinity columns and eluted according to the procedures described in Experimental Procedures. A 10µL aliquot of an 80-µL sample solution was mixed with 1.37 pmol of A or 1.5 pmol of [15N5]A and injected for LC/ESI-MS analysis with SIM. The recovery was determined by LC/ESI-MS measurement of the ratio of areas
Yen et al.
of the chromatographic peak of the extracted analyte to the spiked internal standard. A recovery for two different sets of freshly prepared immunoaffinity columns was 72 ( 8% (6 columns) and 62 ( 11% (10 columns). A recovery from a set of 8-month-old immunoaffinity columns used for 10 previous assays was 32 ( 8% (10 columns). For one set of immunoaffinity columns from which recovery initially averaged 55%, analyte could not be detected in a second run. These data demonstrate that A recovery from immunoaffinity columns degrades over time, and in some cases the immunoaffinity columns may completely lose activity for extraction of analyte. Thus, analyte quantification employing immunoaffinity chromatography for sample extraction must be controlled for sample recovery, and isotope dilution mass spectrometry is an ideal technique for this purpose. Gas chromatography/mass spectrometry (GC/MS) in combination with immunoaffinity extraction has been shown to be able to accurately quantitate O6-butylguanine and 3-alkyladenines in biological matrixes (19, 20). To measure analyte loss during sample preparation by adsorption on the walls of culture tubes, we prepared three sets of 10- × 130-mm tubes: borosilicate glass tubes with and without silanization and polypropylene tubes, each tube containing 15 pmol of [15N5]A and 5 mL of 50% MeOH/water. After thorough mixing, the sample solution was dried by SpeedVac, and the residue was resuspended in 80 µL of water. A 10-µL aliquot of each solution was spiked with 1.37 pmol of A and then analyzed by LC/ESI-MS. [15N5]A recovery was 44 ( 8% for nonsilanized glass tubes, 48 ( 4% for silanized glass tubes, and 58 ( 12% for polypropylene tubes (n ) 3). Adsorption of trace amounts of analyte on the walls of capillary tubing has been reported by Andren and coworkers (24). Preadsorption of other compounds on the walls of sample tubes was reported to minimize analyte adsorption. We have similarly observed that addition of 200 pmol of arginine to the sample solution increased [15N5]A recovery from 44% to 57% for samples in nonsilanized glass tubes (n ) 2) and from 50% to 55% in a silanized glass tube. The higher analyte recovery for samples previously run through immunoaffinity columns may result from a similar preadsorption effect by adventitious compounds eluted from the columns. Figure 2 shows the calibration curve of the A assay as a plot of the concentration ratio of A to [15N5]A versus the ratio of the areas of the chromatographic peaks of m/z 160 (A) to 165 ([15N5]A). The calibration mixtures consisted of [15N5]A (0.15 pmol/µL) and A ranging from 0.015 to 3 pmol/µL (0.15-30 pmol/injection). The calibration curve, constructed from seven different concentration points, was reproducible and linear with r2 ) 0.999. The standard deviation at each concentration of calibration standard was less than 15%, indicating that the assay is precise. We also found that the calibration curve for standards processed through the immunoaffinity column was similar to that of direct analyses by LC/ ESI-MS (r2 ) 0.997, standard deviation of measurements less than 15%). The procedure for the analysis of A in rat urine by immunoaffinity-LC/ESI-MS is the same as that for standards. Rat urine, 200-300 µL for unexposed rats or 100 µL for CEO-treated rats, was prepared and loaded onto the immunoaffinity column. A was eluted and quantified by LC/ESI-MS as described in Experimental Procedures. We were able to process 8-16 samples
LC/MS Quantitation of 1,N6-Ethenoadenine in Urine
Figure 2. Calibration graph for A obtained from the direct analysis of standard solutions (O) and the analysis of standard solutions processed through immunoaffinity columns (2).
Chem. Res. Toxicol., Vol. 11, No. 7, 1998 813
Figure 4. SIM chromatograms of urine of a CEO-treated rat (100 µL), 6 h after CEO exposure, spiked with 15 pmol of [15N5]A analyzed m/z ) 160 for A and m/z )165 for [15N5]A.
Figure 5. Graph of the accumulation of A plotted as a function of time after CEO exposure.
Figure 3. Selected-ion monitoring (SIM) chromatograms of urine of an unexposed rat (300 µL) spiked with 15 pmol of [15N5]A analyzed m/z ) 160 for A and m/z )165 for [15N5]A. The endogenous A in this urine sample is 20.6 pmol/mL.
simultaneously. This procedure should be readily adaptable to automation for increasing sample throughput. Precision and accuracy of the immunoaffinity-LC/ESIMS method was investigated by analysis of urine of unexposed rats. In replicate assays of the urine of 3 rats, A was detected at levels of 4.02 ( 0.09 (n ) 3), 20.6 ( 1.1 (n ) 3), and 40.2 ( 2.8 pmol/mL (n ) 4), respectively. When the adduct concentrations were adjusted to 24-h urine volumes, the daily excretion of A was 72, 206, and 482 pmol. The reconstructed ion chromatograms (RIC) of SIM at m/z 160 (A) and 165 ([15N5]A) for analysis of urine of an unexposed rat are shown in Figure 3. Standard deviations of less than 10% for individual animals indicate that the immunoaffinity-LC/ESI-MS method is precise. Accuracy was determined by spiking a known amount of A (7.5 pmol) into urine samples of an unexposed rat. The increase in A concentration was determined to be 8.04 ( 0.45 pmol (n ) 4) giving an accuracy of 107 ( 6%. The detection limit obtained in the routine analysis of urine of unexposed rats was 270 fmol of A with a signal-to-noise ratio (S/N) 3:1. The quantity of A excreted in urine of CEO-treated rats rapidly increased for a few hours after exposure.
Figure 4 shows a RIC of SIM at m/z 160 (A) and 165 ([15N5]A) for urine of a CEO-treated rat 12 h postexposure. The amount of A (154 pmol) detected in this urine sample of a CEO-treated rat is about 10-fold higher than that of spiked internal standard [15N5]A (15 pmol) and is sufficient for detection by a full-scan mass spectrum. At low skimmer offset voltage (130 V), the base peak of the [M + H]+ ion of A was detected at 13 min (result was not shown). Increasing the skimmer offset voltage from 130 to 180 V to promote CID in the ESI source resulted in a full-scan mass spectrum of the chromatographic peak corresponding to m/z 160 with fragment ions of m/z 106, 119, and 133, identical to the spectrum generated by authentic A (Figure 1c). We also observed a peak at m/z 165 representing the [M + H]+ ion of [15N5]A. These data indicate that A was excreted in the urine of unexposed rats but increased markedly in the urine of CEO-treated rats. The time course for the accumulation of A in urine of CEO-treated rats is shown in Figure 5. Accumulation was calculated by the amount of A measured in each sample multiplied by the total amount of rat urine collected in each time interval. At zero time, A accumulation was based on the A measured in the urine of unexposed rats. Between 4 and 24 h postexposure, the amount of A in urine became rapidly elevated. The amount of A excreted in urine did not significantly increase from 24 to 132 h postexposure. An interesting observation was that the concentration of A in urine of
814 Chem. Res. Toxicol., Vol. 11, No. 7, 1998
a rat 132 h after CEO exposure (17.9 pmol of A/mL) was nearly identical to the average concentration of endogenous A in rat urine (21.6 ( 14.8 pmol of A/mL, for 3 rats). Accumulation curves obtained from other rats also showed similar rapid elevation of A in urine during the 24 h after CEO exposure. Our data suggest that A formed by CEO exposure is rapidly excreted in urine. The excretion rate for A is similar to that of 3-methyladenine produced by methylating agents (25). While A is formed in CEO-treated calf thymus DNA in ∼30-fold greater amounts than G (26), tissue measurements of these two promutagenic etheno adducts have shown the reverse, with G greater than A (27). This difference is likely to be due to differences in DNA repair. Dosanjh et al. demonstrated that both adducts were excised by cell-free extracts of the cloned human 3-methyladenine DNA glycosylase but that A was excised with nearly 10 times greater efficiency (28). The present demonstration of rapid elimination of A in urine is consistent with efficient base excision repair. In earlier studies using immunoaffinity-HPLC with fluorescence detection for A in urine, hepatic DNA adducts were shown to decrease rapidly during the same period that urinary adducts increased (21). However, it is not possible to exclude other sources such as RNA and nucleotide, nucleoside, and base pool adduction. Nevertheless, urinary excretion of A provides an excellent new means to monitor processes that form this adduct in vivo. Additional studies will be needed to fully understand the contributions of different sources of A. The quantitation of DNA adducts in complex biological matrixes is a challenging task. In this study, monoclonal antibody immunoaffinity chromatography was used to extract A from rat urine. Two potential sources of error were identified, loss of analyte to adsorption on the walls of sample tube and degradation of the activity of the immunoaffinity columns. Although the recovery of A from new immunoaffinity columns was shown to be efficient; the activity of immunoaffinity columns was, in some cases, rapidly degraded. These results suggest that it is important to constantly monitor column performance and to add a cleanup step to urine sample preparation to prevent column degradation. The results of this work suggest that the excretion of A in urine can serve as a biomarker of exposure to agents and processes that result in the formation of A. Measurement of DNA adducts in urine is a noninvasive method that may serve to monitor acute carcinogen exposure or whole body burden (29). Because certain adducts can be generated from either exogenous or endogenous sources and excreted into urine, use of ultrasensitive methods of detection and quantitation requires that basal levels first be identified in a control population.
Acknowledgment. This research was supported by NIEHS Grant P42 ES05948. We thank Ms. S.-Y. Chiang, Ms. D. Burgin, and Mr. B. Aron for processing samples. Prof. M. F. Rajewsky and Dr. P. Lorenz are thanked for providing the monoclonal antibody for the immunoaffinity column.
References (1) Bartsch, H., Hemminki, K., and O’Neill, I. K., Eds. (1988) Methods for detecting DNA damaging agents in humans: Application in cancer epidemiology and prevention, IARC Scientific Publications No. 89, Lyon, France.
Yen et al. (2) Fedtke, N., and Swenberg, J. A. (1991) Quantitative analysis of DNA adducts: the potential for mass spectrometric techniques. In Molecular Dosimetry and Human Cancer: Analytical, Epidemiological and Social Consideration (Groopman, J. D., and Skipper, P. L., Eds.) pp 171-188, CRC Press, Boca Raton, FL. (3) Wishnok, J. S. (1992) Environmental carcinogens: in vivo monitoring using GC/MS. Anal. Chem. 64, 1126A-1135A. (4) Poirier, M. C., and Beland, F. A. (1992) DNA adduct measurements and tumor incidence during chronic carcinogen exposure in animal models: Implications for DNA adduct-based human cancer risk assessment. Chem. Res. Toxicol. 5, 749-755. (5) Marnett, L. J., and Burcham, P. C. (1993) Endogenous DNA adducts: potential and paradox. Chem. Res. Toxicol. 6, 771-785. (6) Laib, R. J. (1986) The role of cyclic base adducts in vinyl-chlorideinduced carcinogenesis: studies on nucleic acid alkylation in vivo. In The role of cyclic nucleic acid adducts in carcinogenesis and mutagenesis (Singer, B., and Bartsch, H., Eds.) pp 101-108, IARC Scientific Publications No. 70, Oxford University Press, London. (7) Guengerich, F. P. (1992) Roles of the vinyl chloride oxidation products 2-chlorooxirane and 2-chloroacetaldehyde in the in vitro formation of etheno adducts of nucleic acid bases. Chem. Res. Toxicol. 5, 2-5. (8) Barbin, A., Laib, R., and Bartsch, H. (1985) Lack of miscoding properties of 7-(2-oxoethyl)guanine, the major vinyl chloride DNA adduct. Cancer Res. 45, 2440-2444. (9) Ghissassi, F. E., Barbin, A., Nair, J., and Bartsch, H. (1995) Formation of 1,N6-ethenoadenine and 3,N4-ethenocytosine by lipid peroxidation products and nucleic acid bases. Chem. Res. Toxicol. 8, 278-283. (10) Kronberg, L., Sjo¨holm, R., and Karlsson, S. (1992) Formation of 3,N4-ethenocytitidine, 1,N6-ethenoadenosine and 1,N2-ethenoguanosine in reactions of mucochloric acid with nucleosides. Chem. Res. Toxicol. 5, 852-855. (11) Fedtke, N., Boucheron, J. A., Walker, V. E., and Swenberg, J. A. (1990) Vinyl chloride-induced DNA adducts. I: Quantitative determination of N2,3-ethenoguanine based on electrophore labeling. Carcinogenesis 11, 1279-1285. (12) Misra, R. R., Chiang, S.-Y., and Swenberg, J. A. (1994) A comparison of two ultrasensitive methods for measuring 1,N6etheno-2′-deoxyadenosine and 3,N4-etheno-2′-deoxycytidine in cellular DNA. Carcinogenesis 15, 1647-1652. (13) Yen, T.-Y., Christova-Gueorguieva, N. I., Scheller, N., Holt, S., Swenberg, J. A., and Charles, M. J. (1996) Quantitative analysis of the DNA adduct N2,3-ethenoguanine using liquid chromatography/electrospray ionization mass spectrometry. J. Mass Spectrom. 31, 1271-1276. (14) Chiarelli, M. P., and Lay, J. O., Jr. (1992) Mass spectrometry for the analysis of carcinogen-DNA adducts. Mass Spectrom. Rev. 11, 447-493. (15) McCloskey, J. A., and Crain, P. F. (1992) Progress in mass spectrometry of nucleic acid constituents: analysis of xenobiotic modifications and measurements at high mass. Int. J. Mass Spectrom. Ion Processes 118/119, 593-615. (16) Chaudhary, A. K., Nokubo, M., Marnett, L. J., and Blair, I. A. (1994) Analysis of the Malondialdehyde-2′-deoxyguanosine adduct in rat liver DNA by gas chromatography/electron capture negative chemical ionization mass spectrometry. Biol. Mass Spectrom. 23, 457-464. (17) Farmer, P. B., and Sweetman, G. M. A. (1995) Mass spectrometric detection of carcinogen adducts. J. Mass Spectrom. 30, 13691379. (18) Teixeira, A. J. R., Ferreira, M. R., van Dijk, W. J., van de Werken, G., and de Jong, J. M. (1995) Analysis of 8-hydroxy-2′-deoxyguanosine in rat urine and liver DNA by stable isotope dilution gas chromatography/mass spectrometry. Anal. Biochem. 226, 307319. (19) Bonfanti, M., Magagnotti, C., Galli, A., Bagnati, R., Moret, M., Gariboldi, P., Fanelli, R., and Airoldi, L. (1990) Determination of O6-butylguanine in DNA by immunoaffinity extraction/gas chromatography-mass spectrometry. Cancer Res. 50, 6870-6875. (20) Prevost, V., Shuker, D. E. G., Friesen, M. D., Eberle, G., Rajewsky, M. F., and Bartsch, H. (1993) Immunoaffinity purification and gas chromatography-mass spectrometric quantification of 3-alkyladenines in urine: metabolism studies and basal excretion levels in man. Carcinogenesis 14, 199-204. (21) Holt, S., Yen, T.-Y., Sangiah, R., and Swenberg, J. A. (1995) Detection of 1,N6-ethenoadenine excreted in rat urine after chloroethylene oxide exposure. Toxicologist 15, 77. (22) Sattsangi, P. D., Leonard, N. J., and Frihart, C. R. (1977) 1,N2ethenoguanosine and N3,3-ethenoguanosine. Synthesis and comparison of electronic spectral properties of these linear and angular triheterocycles related to the Y bases. J. Org. Chem. 42, 3292-3296.
LC/MS Quantitation of 1,N6-Ethenoadenine in Urine (23) Guengerich, F. P., Crawford, W. M., Jr., and Watanabe, P. G. (1979) Activation of vinyl chloride to covalently bound metabolites: roles of 2-chloroethylene oxide and 2-chloroacetaldehyde. Biochemistry 18, 5177-5182. (24) Andren, P. E., Emmett, M. R., and Caprioli, R. M. (1994) Microelectrospray: zeptomole/attomole per microliter sensitivity for peptides. J. Am. Soc. Mass Spectrom. 5, 867-869. (25) Hanski, C., and Lawley, C. (1985) Urinary excretion of 3-methyladenine and 1-methylnicotinamide by rats, following administration of [methyl-14C]methyl methane sulphonate and comparison with administration of [14C]methionine or formate. Chem.-Biol. Interact. 55, 225-234. (26) Mu¨ller, M., Belas, F. J., Blair, I. A., and Guengerich, F. P. (1997) Analysis of 1,N2-ethenoguanine and 5,6,7,9-tetrahydro-7-hydroxy9-oxoimidazol[1,2-a]purine in DNA treated with 2-chloroxirane
Chem. Res. Toxicol., Vol. 11, No. 7, 1998 815 by high performance liquid chromatography/electrospray mass spectrometry and comparison of amounts to other DNA adducts. Chem. Res. Toxicol. 10, 242-247. (27) Swenberg, J. A., Fedtke, N., Ciroussel, F., Barbin, A., and Bartsch, H. (1992) Etheno adducts formed in DNA of vinyl chloride-exposed rats are highly persistent in liver. Carcinogenesis 13, 727-729. (28) Dosanjh, M., Chenna, E., Kim, E., Fraenkel-Conrat, H., Samson, L., and Singer, B. (1994) All four known cyclic adducts formed in DNA by the vinyl chloride metabolite chloroacetaldehyde are released by a human DNA glycosylase. Proc. Natl. Acad. Sci. U.S.A. 91, 1024-1028. (29) Shuker, D. E. G., and Farmer, P. B. (1992) Relevance of urinary DNA adducts as markers of carcinogen exposure. Chem. Res. Toxicol. 5, 450-460.
TX9800194
