Determination of in Vitro-and in Vivo-Formed DNA Adducts of 2-Amino

ylimidazo[4,5-f]quinoline (IQ) from in vitro and in vivo sources. Constant neutral ... enabled sensitive and specific detection of IQ adducts in vitro...
0 downloads 0 Views 263KB Size
Download PDF
Chem. Res. Toxicol. 1999, 12, 1019-1027

1019

Determination of in Vitro- and in Vivo-Formed DNA Adducts of 2-Amino-3-methylimidazo[4,5-f]quinoline by Capillary Liquid Chromatography/Microelectrospray Mass Spectrometry Eric T. Gangl,† Robert J. Turesky,‡ and Paul Vouros*,† Barnett Institute and Department of Chemistry, Northeastern University, Boston, Massachusetts 02115, and Nestle Research Centre, Nestec Ltd., Vers-chez-les-Blanc, 1000 Lausanne 26, Switzerland Received April 8, 1999

Capillary liquid chromatography/microelectrospray mass spectrometry has been applied to the detection of deoxyribonucleoside adducts of the food-derived mutagen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) from in vitro and in vivo sources. Constant neutral loss (CNL) and selective reaction monitoring (SRM) techniques with a triple-quadrupole mass spectrometer enabled sensitive and specific detection of IQ adducts in vitro and in animals. Detection of 1 adduct in 104 unmodified bases is achieved using CNL scanning detection, while the lower detection limits using SRM approach 1 adduct in 107 unmodified bases using 300 µg of DNA. The DNA adducts N-(deoxyguanosin-8-yl)-2-amino-3-methylimidazo[4,5-f]quinoline (dG-C8IQ) and 5-(deoxyguanosin-N2-yl)-2-amino-3-methylimidazo[4,5-f]quinoline (dG-N2-IQ) were detected in kidney tissues of chronically treated cynomolgus monkeys at levels and in proportions consistent with previously published 32P-postlabeling data [Turesky, R. J., et al. (1996) Chem. Res. Toxicol. 9, 403-408]. Thus, capillary tandem LC/MS is a highly sensitive technique, which can be used to screen for DNA adducts in vivo.

Introduction Diet is believed to be an important factor in the etiology of human cancer, and the presence of potential carcinogens in foods is a major concern for human health (1, 2). Heterocyclic aromatic amines (HAAs)1 are one class of carcinogenic chemicals found in cooked beef, fish, and poultry, which are receiving widening attention (3). These chemicals induce tumors at multiple sites in rodents (4), and one HAA, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), is a potent hepatocarcinogen in non-human primates (5). Risk assessment of potential genotoxic human carcinogens requires not only measurement of the extent of exposure but also the detection of genetic damage, such as through DNA adduct formation (2, 6, 7). IQ and other HAAs covalently bind to DNA following metabolism by cytochrome P450 1A2-mediated N-oxidation (8, 9). Fur* To whom correspondence should be addressed: Department of Chemistry, 102 Hurtig Hall, Northeastern University, 360 Huntington Ave., Boston, MA 02115. Phone: (617) 373-2794. Fax: (617) 373-2693. † Northeastern University. ‡ Nestec Ltd. 1 Abbreviations: HAAs, heterocyclic aromatic amines; IQ, 2-amino3-methylimidazo[4,5-f]quinoline; ESI, electrospray ionization; dG, 2′deoxyguanosine; dA, 2′-deoxyadenosine; dC, 2′-deoxycytidine; dT, 2′deoxythymidine; SRM, selected reaction monitoring; CNL, constant neutral loss; NHOHIQ, 2-(hydroxyamino)-3-methylimidazo[4,5-f]quinoline; dG-C8-IQ, N-(deoxyguanosin-8-yl)-2-amino-3-methylimidazo[4,5f]quinoline; dG-N2-IQ, 5-(deoxyguanosin-N2-yl)-2-amino-3-methylimidazo[4,5-f]quinoline; APCI, atmospheric pressure chemical ionization; DAD, diode array detector; MeIQx, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline; PhIP, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine; 4-ABP, 4-aminobiphenyl; MNSPD, micrococcal nuclease spleen phosphodiesterase; CI, chemical ionization; AMS, accelerator mass spectrometry.

ther metabolic activation of the N-hydroxy metabolites may occur via N,O-acetyltransferase or sulfotransferase to generate the reactive N-acetoxy and N-sulfate analogues, respectively, which readily bind to DNA (5, 8, 1012). The two major DNA adducts of IQ synthesized from the reactive N-acetoxy intermediate are formed at the C8 and N2 positions of the guanine base to give N-(deoxyguanosin-8-yl)-2-amino-3-methylimidazo[4,5-f]quinoline (dG-C8-IQ) and 5-(deoxyguanosin-N2-yl)-2-amino-3methylimidazo[4,5-f]quinoline (dG-N2-IQ) (13) (Figure 1). Both adducts have also been detected in rodents and nonhuman primates (14). Currently, 32P-postlabeling methods are the mainstay of DNA adduct detection (15-17). Their high sensitivity has kept this technique at the forefront of the DNA adduct detection field. However, in the absence of authentic reference compounds, this methodology provides no structural information about the adducts. With the advancement of soft ionization techniques such as electrospray (ESI) and atmospheric pressure chemical ionization (APCI), mass spectrometry is receiving more attention in the arena of DNA adduct detection and characterization. Several groups, including ours, have investigated the advantages of using on-line electrospray mass spectrometry to detect DNA adducts in vitro (1820). In the end, however, in vivo adduct detection by LC/ MS methodologies has been a major challenge due to the very low level of DNA adduct formation. With new developments in the area of electrospray ionization, namely, those of nanospray and microspray, detection limits have reached into the high-attomole to low-femtomole range (21-24). With this, the prospects

10.1021/tx990060m CCC: $18.00 © 1999 American Chemical Society Published on Web 09/23/1999

1020

Chem. Res. Toxicol., Vol. 12, No. 10, 1999

Gangl et al.

Materials and Methods

Figure 1. Chemical structures of IQ and DNA adducts dGC8-IQ and dG-N2-IQ. The C8 adduct is drawn in the syn conformation, and the N2 adduct is drawn in the anti conformation.

of detecting in vivo DNA adducts by mass spectrometry have become more realistic. As a consequence of these advancements in nanoflow ionization techniques, a renewed interest has been spurred in the area of capillary chromatography. During the past decade, several groups have developed and evaluated nanoflow chromatographic methods such as open-tubular LC, tightly packed capillary column LC, and partially packed capillary LC (2527). Although these techniques never flourished, they do have niche applications. With compatible flow rates, the coupling of capillary LC to nano- or microelectrospray is a good marriage of sensitivity and specificity that has begun to receive attention (28-31). Several groups have recently illustrated the usefulness of these capillary LC/ MS/MS on-line techniques with proteins and other biologically related analytes (29, 32, 33). On the basis of 32P-postlabeling assays conducted by TLC and HPLC, the dG-C8-IQ and dG-N2-IQ adducts appear to be the most prevalent lesions in rodents and non-human primates treated with IQ (14). The purpose of this work is twofold: to demonstrate the feasibility of detecting DNA adducts from both in vitro reactions and living systems and to explore the degree of corroboration between 32P-postlabeling (the currently accepted method for DNA adduct analysis) and microLC/MS techniques, both in vitro and in living systems. Herein we report the development and implementation of an analytical system that enables the characterization of dG-C8-IQ and dGN2-IQ from in vitro and in vivo sources by capillary separation techniques coupled to mass spectrometry.

Chemicals. Caution: IQ is carcinogenic to rodents and should be handled carefully. All water used was purified with a Milli-Q water filtration system (Millipore, Bedford, MA). DNase I (type IV, from bovine pancreas), alkaline phosphatase (type II-S, Escherichia coli), phosphodiesterase (type VII, Crotalus atrox venom), calf thymus DNA type I, formamide, deoxyguanosine, and acetic acid were obtained from Sigma (St. Louis, MO). [2-14C]IQ (10 mCi/mmol) and azido-IQ were obtained from Toronto Research Chemical Inc. (North York, ON). All solvents were HPLC grade unless otherwise specified. Methanol was obtained from EM Science (Gibbstown, NJ), and acetonitrile was obtained from J. T. Baker (Phillipsburg, PA). Isolute C18 end-capped (EC) cartridges were obtained from Jones Chromatography (Lakewood, CO). The capillary LC stationary phase was obtained from Phenomenex (Torrance, CA). Adduct Synthesis and HPLC Purification. The dG-C8IQ and dG-N2-IQ adduct standards were synthesized by reacting the photoactivated azido-IQ with dG as previously described (34). [2-14C]IQ-modified calf thymus DNA was prepared by reacting NHOH-IQ with calf thymus DNA in the presence of acetic anhydride according to the methods described in ref 34. The level of adduct modification is estimated to be 9.3 adducts in 104 3′dNp molecules. dG-N2-IQ and dG-C8-IQ comprised 4.3 and 71% of the total radioactivity, respectively (34). The adducts of calf thymus DNA modified with IQ were quantified by both 32P-postlabeling and liquid scintillation counting. The estimates of both methods were in excellent agreement and within 5% of each other (13, 34). Final purification of the dG-C8-IQ and dG-N2-IQ adduct standards was accomplished on a reversed-phase C18 column (4.6 cm × 15 cm × 5 µm Supelco LC 18 DB column, Supelco, Bellefonte, PA). The conditions were as follows. Solvent A was 50 mM ammonium acetate in water, and solvent B was 100% MeOH. With a flow rate of 1 mL/min, the gradient started at 15% B and was brought to 100% B over the course of 40 min. The eluents were monitored by diode array detection from 200 to 600 nm with a chromatographic trace monitored at 267 and 254 nm. The collected fractions had diode array spectra similar to those previously reported (34). Additional experiments by MS/ MS were carried out to ensure their composition (see below). The fractions of the dG-C8-IQ and dG-N2-IQ standards were lyophilized and stored at 5 °C. Since dG-N2-IQ and dG-C8-IQ were the only compounds in which there was interest, no attempts were made to characterize the remaining reaction products. Animals and Treatments. Cynomolgus monkeys (Macaca fuscicularis), born in a closed colony and mother-reared, were used for the study. Animals were housed in an AAALACaccredited facility at Hazleton Laboratories under contract to the National Cancer Institute and in compliance with The Guide for the Care and Use of Laboratory Animals. The protocol was reviewed and approved by the NIH Animals Care and Use Committee. Details on the housing and diet were previously reported (5, 35, 36). Animals received either a single oral dose of IQ (20 mg/kg) or five doses per week at a dose of 10 or 20 mg/kg of body weight for 3.6 years. In all cases, the final dose of IQ was administered 24 h prior to autopsy. Animals were sacrificed with an overdose of sodium pentobarbital. 32P-Postlabeling. The procedures for the 32P-postlabeling experiments, isolation of DNA, and enzymatic digestion of IQDNA adducts with MNSPD were carried out as previously described (13). In this study, only the kidney tissues were examined (14, 17). Digestion and Sample Workup Procedure for LC/MS. All treated and untreated DNA from in vitro and in vivo systems underwent the same digestion and workup procedure. For LC/ MS analyses, the DNA was cleaved to the deoxyribonucleoside level. For in vitro and in vivo experiments, 0.3-0.4 mg of DNA was dissolved in 600 µL of 5 mM Tris (pH 7.5)/10 mM MgCl2 and thoroughly sonicated. Then, 0.02 mg of DNase I (dissolved

LC/MS Analysis of DNA Adducts

Chem. Res. Toxicol., Vol. 12, No. 10, 1999 1021

Figure 2. Schematic of the capillary LC/microelectrospray MS/MS system consisting of an Hewlett-Packard 1100 HPLC apparatus, an LC Packings Acurate splitter, a Valco C4 internal loop injector, a 10 cm × 75 µm × 5 µm C18 column, a custom-built microelectropray interface, and a Finnigan TSQ 700 triple-quadrupole mass spectrometer. in 5 mM Tris/10 mM MgCl2) was added, and DNA was digested for 5 h at 37 °C. Afterward, 1.0 unit of alkaline phosphatase (used as provided) and 0.0072 unit of phosphodiesterase (dissolved in 5 mM Tris/10 mM MgCl2) were added, and the digestion was continued for an additional 18 h at 37 °C. Then, 3 volumes of cold ethanol was added, and the solution was centrifuged for 5 min to remove salt and protein. The supernatant was retrieved and concentrated by vacuum centrifugation (Savant model SC110A) to 100 µL. Water (2 mL) was added, and the DNA adducts were purified by solid-phase extraction [100 mg of C18 (EC), isolute SPE], where the cartridge was conditioned with methanol (5 mL), followed by water (5 mL). After application of the digest mixture, the cartridge was washed with 10% methanol to remove nonmodified nucleosides and salt, and the adducts were subsequently eluted with methanol. The efficiency of adduct recoveries from the solid-phase extraction procedure has not been established. Column Packing for Capillary LC. Capillary LC was performed on a 10 cm × 0.075 mm i.d. (0.150 mm o.d.) fused silica column filled with 5 µm nucleosil C18 100 Å packing material. The columns were packed in-house using fused silica capillaries (Polymicro Technologies, Phoenix, AZ) based on a previously described protocol (37). The only exception was the use of a polymer end frit rather than a sintered silica frit. Briefly, the frit was made by first mixing 15 µL of potassium silicate (Kasil-1, PQ Corp., Valley Forge, PA) and 85 µL of formamide. The resulting solution was vortexed for 1 min and then centrifuged for an additional 5 min. Fused silica of the desired size (∼25 cm long) was then dipped into the mixture, and via capillary action, the solution filled approximately 1-3 cm of the capillary. After the capillary had been baked for 1 h at 100 °C, the frit was cut so that it was only 2-3 mm long. The frit was conditioned with 50 µL each of 1 M NH4NO3, 1 M HCl, H2O, and then CH3CN. The columns were then packed. The final column length was ca. 18 cm with a packing length of 10 cm (8 cm unfilled fused silica capillary). The resulting columns were used without a precolumn or entrance frit. Capillary LC. A schematic of our capillary LC/µMS system is depicted in Figure 2. Solvent was delivered using an unmodified Hewlett-Packard (Palo Alto, CA) HP 1100 binary pump with nanoliter flow rates achieved by coupling the pump effluent to a precolumn splitter (Acurate, IC-400 Var, LC Packings, San Francisco, CA). The nanoliter flow calibrator provided with the Acurate splitter was used to deliver a precolumn split flow rate of an estimated 0.250 µL/min. The HPLC effluent flow rate was kept constant at 0.380 mL/min, resulting in a split ratio of ca. 1400/1. An injector equipped with a 0.200 µL internal loop (model C4, Valco Instruments Co. Inc., Houston, TX) was used

for all injections where 1.5 µL of sample was loaded into the injector to ensure complete filling of the loop. In effect, this ensures that 0.2 µL of volume is loaded on-column. The HPLC registered a starting back-pressure of 165 bar (85% A/15% B) and a peak back-pressure of 255 bar (50% A/50% B). It was determined that the delay time between the programmed gradient change and the point at which it reached the column was 4.6 min with the injector left in the inject position. All mobile-phase solvents were degassed with helium for at least 30 min prior to use. The column was kept at ambient temperature (25 °C) during analysis. A binary gradient system composed of acetic acid in water and acetic acid in methanol was used for all capillary separations. For the in vitro and serially diluted in vitro samples, solvent A was 0.2% CH3COOH in H2O and solvent B was 0.2% CH3COOH in CH3OH. A gradient separation was used where the level of B went from 15 to 50% in 7 min. In the case of the in vivo samples, solvent A was 0.2% CH3COOH in H2O and solvent B was 0.1% CH3COOH in CH3OH. A step gradient was employed where the level of B was taken from 15 to 50% in 0.01 min. Microelectrospray Mass Spectrometry. Mass spectrometric measurements were conducted with a TSQ 700 triplequadrupole mass spectrometer (Finnigan MAT, San Jose, CA). The mass spectrometer was calibrated using the recommended MRFA-myoglobin solution. To improve selectivity and sensitivity, constant neutral loss (CNL) and selected reaction monitoring (SRM) scans were employed. To analyze a class of compounds (e.g., those containing a 116 Da ribose), the CNL scan mode is useful. In this mode, both the first and third quadrupoles scan over a given mass range while the second quadrupole is used as a collision chamber. However, the third quadrupole scans are offset by exactly -116 Da from those of the first quadrupole. Therefore, only compounds that lose 116 Da in the collision cell are allowed to pass through the final mass analyzer and onto the detector. In cases where there is a specific target analyte, SRM scanning techniques are employed. This mode of detection involves the selective passage of a parent ion through the first quadrupole. The parent ion is then fragmented in the second quadrupole, and a targeted fragment mass is allowed to pass through the final quadrupole (38). For MS/MS experiments, argon was used as the collision gas. The collision cell was kept at a pressure of 2.2 mTorr for product ion spectra and a pressure of 0.9 mTorr for CNL and SRM scans. Collision energies ranged from -29 V for CNL and SRM scans to -35 V for product ion spectra. The scan rates were kept at one scan per second for SRM and product ion scans. CNL scan rates varied from one to two per second depending on the scan range (maximum scan rate of 400 amu/s). For CNL experiments, the mass spectrometer

1022

Chem. Res. Toxicol., Vol. 12, No. 10, 1999

Gangl et al.

Figure 3. (A) Product ion scans of dG-C8-IQ and dG-N2-IQ. (B) Product ion scan of aglycon dG-C8-IQ and dG-N2-IQ (m/z 348). Adducts were dissolved in 80% CH3CN, 20% H2O, and 0.1% acetic acid and infused at a rate of 0.300 µL/min with a liquid sheath flow rate of 0.300 µL/min composed of 80% CH3CN, 20% H2O, and 0.1% CH3COOH. was scanned from a parent mass of m/z 220 to 520. The electron multiplier voltage was set at 1000 V for routine experiments and increased to 1400 V for in vivo samples and serially diluted in vitro samples. The ESI source inlet supplied with the instrument was replaced with an integrated capillary LC-ESI system designed and constructed in-house (Figure 2). The system was designed for compactness and ease of use. It consisted of an injector fitted with a 0.200 µL internal loop, an XYZ positioner (FP-2 Newport, Irvine, CA), and an Upchurch cross (P 730 Upchurch Scientific, Oak Harbor, WA) all mounted on a rail mount system [9742(M) New Focus, Inc., Sunnyvale, CA] fitted to the TSQ 700 instrument. The high-voltage connection was made through one side of the PEEK cross via a platinum wire, while the other side was used to deliver the sheath flow. The column ran axially through the PEEK cross to the stainless steel tip (30 gauge, Small Parts Inc., Sunny Lakes, FL) where the electrospray process took place directly from the end of the column. Spraying directly from the end frit of the capillary has an advantage in that any peak broadening due to postcolumn volumes and connections is removed. The tip was positioned 3 mm away from the heated capillary orifice (175 °C) and operated in positive ion mode at 2.7 kV. Although the ESI process was selfsustaining under these conditions, a flow of nitrogen at a rate of 0.30 L/min was used to guide the evaporating droplets into the MS system. A sheath flow rate of 0.250 µL/min (70% CH3CN, 30% H2O, and 0.1% CH3COOH) was delivered using a syringe pump (model 980532, Harvard Apparatus, Holliston, MA) and a 50 µL syringe. To maximize the sensitivity for the detection of the IQ adducts during in vivo and in vitro analysis, tuning was performed on the dG-C8-IQ adduct (m/z 464). The compound was infused at a concentration of 1 × 10-5 M in 75% CH3OH, 25% H2O, and 0.1% CH3COOH using the microelectrospray interface. In addition, experiments were carried out to determine the ideal collision energy needed to obtain the maximum ion intensity from the loss of the deoxyribose moiety in the SRM scan (see the discussion below). On the day of in vivo sampling, system and procedural blanks were taken. These blanks were analyzed to eliminate false positives derived from carryover in the analytical system, digestion enzymes, solvents, pipetting equipment, and other workup apparatus. In addition, the reference standards were

also re-examined to establish the analyte retention time under the specified conditions. Care was taken to inject a dilute sample such that the system was not contaminated before analysis of the in vivo sample. The procedural blank was prepared by analyzing unmodified calf thymus DNA that had been carried through the entire workup procedure. The system blank was composed of an injection of 10% CH3OH.

Results Analysis of Standards and Optimization of Detection Conditions. The electrospray mass spectra of the synthetic dG-C8-IQ and dG-N2-IQ standards were examined first and are presented in Figure 3. As expected, the product ion scans of the [M + H]+ ions (m/z 464) of the two isomers are quite similar. Typical of adducts of this type, a major fragmentation pathway involves the loss of the deoxyribose group (116 Da) from [M + H]+ which yields an ion of m/z 348 (38). Since the AH2+ ([MH - deoxyribose + H]+) ions are so abundant following CID, constant neutral loss (CNL) and selected reaction monitoring (SRM) scanning techniques are particularly sensitive for compounds containing a ribose group. The facile loss of the deoxyribose group enables sensitive and specific screening of DNA bases in mixtures with and without covalent modifications. However, to obtain more informative collisional data for the standards, nozzle-skimmer fragmentation conditions were used to induce loss of the ribose group in-source. The product ion spectra of the aglycon ions of the two IQ adducts are shown in Figure 3B. Unique to the individual isomers are the relatively higher intensities of the peaks at m/z 331 in the spectrum of the dG-N2-IQ adduct and of the m/z 303 ion in that of the dG-C8-IQ adduct. The more favorable loss of NH3 (17 Da) from the aglycon ion of m/z 348 in the spectrum of dG-N2-IQ as opposed to that of the C8 adduct has been reported for a variety of related compounds. This fragmentation may be used on a diagnostic basis to differentiate isomeric compounds. In CI, the elimination of NH3 from nucleic acids has been

LC/MS Analysis of DNA Adducts

Chem. Res. Toxicol., Vol. 12, No. 10, 1999 1023

Figure 4. Capillary LC/microelectrospray MS/MS using SRM scanning of the m/z 464 to 348 transition. Injection of 0.2 µL of reference standards dG-N2-IQ and dG-C8-IQ. LC conditions were as follows: (A) 0.2% CH3COOH in H2O and (B) 0.2% CH3COOH in CH3OH. The gradient was from 15 to 50% B over the course of 7.00 min (NL represents the value of the normalized ion intensity).

shown to originate in part from loss of the endocyclic nitrogen as shown in Figure 3 (39). Suggested fragmentations to yield these ions are indicated in Figure 3. Analysis of IQ Adduct Standards by Capillary LC/ µESI-MS. Commencement of the gradient at 15% MeOH allows for the IQ adduct peaks to be effectively compressed at the head of the column while also allowing for less hydrophobic components to elute before the peaks of interest. Focusing is required because an injection of 0.200 µL onto a 75 µm capillary column represents a disproportionately large injection volume. This injection size is analogous to injecting 1 mL onto a 4.6 mm LC column running at a rate of 1 mL/min. This focusing strategy improves detection limits by narrowing peak width and negating dispersion effects introduced by the 8 cm of unpacked capillary between the point of injection and the head of the column. Using a steep methanol gradient, the two adducts were separated in less than 15 min (Figure 4). Consistent with earlier observations for the purification of the standards by HPLC, the dG-N2-IQ adduct elutes before dG-C8-IQ. It should be pointed out that some peak tailing due to the basic nature of the analytes is observed under the capillary LC conditions. Active silanol groups on the untreated inner wall of the fused silica capillary and in the stationary phase provide a source of undesirable interaction that causes some peak asymmetry problems. The relatively high concentration of acetic acid in the mobile phase assists in reducing the extent of these interactions. Analysis of an in Vitro Reaction Mixture. An in vitro reaction mixture was prepared by reacting Nacetoxy-IQ with 1 mg/mL calf thymus DNA as previously described (34). Following removal of the bulk of unmodified DNA by solid-phase extraction, a sample enriched with DNA adducts was isolated and reconstituted in a final volume of 6 µL, comprised of 1 µL of MeOH and 5 µL of H2O. Subsequently, 1.5 µL of this solution was injected into the capillary HPLC/µESI-MS/MS system and analyzed using the CNL (loss of 116 Da) scanning mode. This mode of detection precludes the possibility of detecting any depurination products, if present. The reconstructed ion chromatograms (Figure 5) exhibit several parent peaks that may be associated with the loss

Figure 5. Signals obtained from the analysis of digested in vitro reaction material by capillary LC/microelectrospray MS/ MS. Traces A-F represent extracted ion chromatograms of m/z 228, 242, 252, 266, 268, and 464, respectively. LC conditions were as follows: (A) 0.2% CH3COOH in H2O and (B) 0.2% CH3COOH in CH3OH. The gradient was from 15 to 50% B over the course of 7.00 min.

of a deoxyribose group (116 Da). Due to incomplete removal of the bulk normal DNA, the protonated forms of dT, dC, dA, and dG are detected; these ions are found at m/z 228, 242, 252, and 268, respectively. In addition, an a priori unexpected [M + H]+ peak at m/z 266 was observed at an elution time of 6.47 min. Attempts to obtain CID data for this ion yielded only one major fragment ion at m/z 150 corresponding to an adenosine base modified by 14 Da. Other minor peaks in the CID spectrum matched the product ions obtained from deoxyadenosine (data not shown). It is reasonable to assume that this compound corresponds to a methylated dA, and can likely be attributed to N6-Me-dA. This modification of dA has been reported to occur naturally in some sources of DNA (40). This adduct is also observed in the control digest of calf thymus DNA, and it was therefore deemed to be of no relevance to the in vitro reaction. Analysis of the DNA control sample by CNL shows the presence of the four unmodified bases as well as the proposed N6-Me-dA (i.e., traces A-E in Figure 5; data not shown). The detection of protonated IQ adducts of m/z 464 eluting with retention times corresponding to those of the reference dG-C8-IQ and dG-N2-IQ is shown in the bottom trace of Figure 5. The region corresponding to the retention time of the dG-N2-IQ adduct was magnified to render the peak more visible. This result establishes the presence of IQ deoxyguanosine adducts from in vitro reactions. In addition, the ratio of the dG-N2-IQ to dGC8-IQ recovered from IQ-modified calf thymus DNA is in good agreement with results previously published (34). On the basis of liquid scintillation counting and 32Ppostlabeling, the level of [14C]IQ bound to DNA was estimated to be 9.3 base modifications per 104 unmodified

1024

Chem. Res. Toxicol., Vol. 12, No. 10, 1999

Gangl et al.

Figure 7. 32P-Postlabeling analysis of 30 µg of the IQ-DNA adduct from kidney tissue of non-human primate 24 h after a single oral dose of IQ (A) (20 mg/kg of body weight) and from kidney tissue of non-human primate undergoing carcinogen bioassay for 3.6 years (B).

Figure 6. Three repeat injections (B-D) of in vitro reaction material diluted with calf thymus DNA to a level of 1 modified base in 106 unmodified bases. Trace A is a blank injection of 10% CH3COOH in H2O. Samples were analyzed by capillary LC/microelectrospray MS/MS using SRM scanning of the m/z 464 to 348 transition. Signals were normalized to an ion intensity of 1.9 × 102. LC conditions were as follows: (A) 0.2% CH3COOH in H2O and (B) 0.2% CH3COOH in CH3OH. The gradient was from 15 to 50% B over the course of 7.00 min.

bases (13, 34). The dG-C8-IQ adduct accounts for approximately 71% of the total 14C-bound radioactivity and 32P-postlabeled material, and dG-N2-IQ accounts for approximately 5% of it. On the basis of this level of IQ adduct modification, our methodology using CNL scanning in conjunction with capillary LC is able to detect approximately 1 base modification in 104 unmodified bases per 300 µg of DNA (i.e., ca. 30 ng or 65 pmol of adduct). Consistent with prior 32P-postlabeling accounts, adduction at the other three nucleobases was not detected in these analyses. The in vitro reaction mixture of 1 adduct in 103 normal bases was serially diluted with unmodified calf thymus DNA to levels of modification containing approximately one adduct in 104, 105, and 106 normal bases, respectively, to establish the lower detection limits by LC/MS/MS. Each solution (300 µg of DNA) was subjected to digestion, cleanup by solid-phase extraction, and analysis by LC/ MS/MS using SRM of the transition from m/z 464 to 348. Results of triplicate injection of samples which contained 300 µg of DNA starting material and a total adduct content of 1 adduct in 106 unmodified bases are shown in Figure 6. Using the liquid scintillation counting and HPLC data as a basis for adduct levels (34), these chromatograms reflect an on-column injection of ca. 17 fmol of total IQ adduct (this assumes 100% digestion efficiency and adduct recovery). Thus, if it is assumed that 5% of the total adduct content is the dG-N2-IQ compound (34), these chromatograms reflect a detection

limit of approximately 1 fmol on-column. This figure also demonstrates the repeatability of the retention times and peak areas. The peak area variability is 21% RSD for the C8 adduct, and the retention variability is 2% RSD for both adducts. In addition, analysis of the samples containing 1 adduct in 103, 104, and 105 normal bases yielded roughly linear responses, indicating the feasibility of quantitative analysis using this general approach (data not shown). The lower level (1 in 106) of adduct modification approaches the levels estimated in vivo in chronically treated non-human primates based upon 32P-postlabeling data (34). In Vivo Sample Characterization by 32P-Postlabeling and LC/MS/MS. Two kidney tissue samples from non-human primates were examined for their IQ adduct content. The first sample was from a monkey sacrificed 24 h after a single administration of IQ (20 mg/kg of body weight). The second sample was from a monkey given an IQ diet over a period of 3.6 years at a dose of 20 mg/ kg of body weight 5 days per week for a cumulative dose of 37.5 g of IQ at the time of sacrifice (17). 32P-Postlabeling data for each sample reveal the presence of two major spots, which comigrate with the synthetic isomeric 3,5-bisphosphate DNA adducts of dG-C8-IQ and dG-N2IQ (Figure 7). On the basis of postlabeling, dG-N2-IQ and dG-C8-IQ were detected at levels of 84 ( 23 and 336 ( 107 adducts per 109 normal nucleotides, respectively, in the animal treated with a single dose. In the chronically treated animal, the dG-N2-IQ adduct predominated and levels were estimated to be 2.4 ( 0.5 adducts per 106 normal nucleotides, while the amount of dG-C8-IQ was estimated to be 0.8 ( 0.2 adduct per 106 normal nucleotides (15, 17). When the postlabeling efficiency and DNA adduct recovery are taken into account (44-59%), the actual adduct level is approximately 2-fold higher than these values (17). The predominance of the dG-N2-IQ isomer in the kidney tissue of the chronically exposed animal represents a reversal of the dG-N2-IQ versus dGC8-IQ ratio typically encountered in the chemical synthesis of these adducts with reactive IQ intermediates in in vitro reactions, or following a single in vivo dose

LC/MS Analysis of DNA Adducts

Figure 8. Capillary LC/microelectrospray MS/MS using SRM scanning of the m/z 464 to 348 transition. The top trace depicts injection of dG-C8-IQ and dG-N2-IQ standard adducts. The bottom trace depicts 10% methanol. LC conditions were as follows: (A) 0.2% CH3COOH in H2O and (B) 0.1% CH3COOH in CH3OH. The step gradient was from 15 to 50% B over the course of 0.01 min.

(Figure 7), where the dG-C8-IQ adduct predominates (14, 17, 34). In line with our objective of establishing a semiquantitative comparison between LC/MS and 32Ppostlabeling, the kidney tissues were examined with regard to the relative ratios of these two adducts. To improve the overall analyte detectability [signal/ noise (S/N)], a number of changes were made to the in vitro/standard analysis conditions. First, to improve ionization efficiency, a mobile phase with a lower HOAc content was used, thereby reducing ion suppression effects. Moreover, the new mobile phase provided conditions for sharpening the LC peak of the N2-labeled isomer, thereby enhancing analyte flux into the ion source. Accordingly, a mixture of adduct standards was analyzed to establish the retention times of the adducts. Figure 8 (top trace) shows the separation of the adducts from injection of a reference sample containing approximately 10 fmol of each isomer. A system blank is shown in the lower trace. With the final chromatographic conditions in place, the in vivo samples were prepared. DNA from kidney tissue of monkeys exposed to both a single dose and chronic administration of IQ was digested, and the adduct fraction was reconstituted into a 6 µL volume as described in Materials and Methods. Representative chromatograms from a 1.5 µL injection of the single administration and chronic exposure samples are shown in traces B and C, respectively (Figure 9). For comparison purposes, the two sample chromatograms (traces B and C) and that of the procedural blank (Figure 9, trace A) are displayed on the same scale. Injections of the single dose (Figure 9, trace B) failed to show a definitive signal for either the dG-N2-IQ or the dG-C8IQ adduct. According to 32P-postlabeling data, the respective dG-N2-IQ and dG-C8-IQ adduct contents are on the order of 84 and 336 adducts per 109 nucleotides, respectively (ca 0.4 fmol of adduct on-column). Therefore, the adduct levels appear to be below the detection limits of LC/MS analysis based upon the results obtained from the serial dilution of the in vitro samples (see Figure 6). However, this sample serves as a form of control for the analysis of tissues from the chronic exposure sample. The LC/MS/MS chromatogram generated from the chronic exposure sample provides unequivocal evidence for both the dG-C8-IQ and dG-N2-IQ adducts in the kidney tissue (Figure 9, trace C). Here the dG-C8-IQ and dG-N2-IQ

Chem. Res. Toxicol., Vol. 12, No. 10, 1999 1025

Figure 9. Capillary LC/microelectrospray MS/MS using SRM scanning of the m/z 464 to 348 transition. (A) Procedural blank digest of 0.387 mg of calf thymus DNA. (B) Digest of 0.403 mg of the IQ-DNA adduct from kidney tissue of non-human primate 24 h after a single oral dose of IQ (20 mg/kg of body weight). (C) Digest of 0.368 mg of DNA from kidney tissue of non-human primate undergoing carcinogen bioassay. LC conditions were as follows: (A) 0.2% CH3COOH in H2O and (B) 0.1% CH3COOH in CH3OH. The step gradient was from 15 to 50% B over the course of 0.01 min.

adducts are observed with S/N ratios of 7/1 and 79/1, respectively. Duplicate analysis of this chronic exposure in vivo sample demonstrated an area repeatability of 5% RSD for the dG-N2-IQ adduct (data not shown). Comparisons of analyte response between the standards, the in vitro samples, and the in vivo samples by LC/MS and radiolabeling methods are in good agreement. Collectively, the numbers suggest that we are just above the detection limits for the analysis of the single-dose in vivo sample (ca. 300 µg of DNA) containing 1 modification in 108. Similarly, this data suggests a comfortable detection (S/N approaching 100/1) of the adducts in the same amount of DNA containing 1 modification in 106. In fact, the experimental result is a S/N ratio of 79/1 for the dGN2-IQ adduct. In addition, the peak areas indicate that the ratio of dG-N2-IQ to dG-C8-IQ adducts in the DNA of the kidney tissue from chronically treated animals is approximately 4/1. This ratio is similar to the relative amounts estimated by 32P-postlabeling (17).

Discussion In recent years, LC/MS/MS has assumed a prominent role in bioanalytical chemistry in terms of structure characterization, trace level detection, and quantitation. However, in the field of DNA adducts, the analytical methodology has been dominated by the use of radiochemical methods, specifically 32P-postlabeling. This has been largely due to the low level of incidence of these biomarkers in living systems. The ability to detect less than 1 adduct in 108-1010 normal bases has thus far made 32P-postlabeling the method of choice in most applications. On the other hand, 32P-postlabeling suffers from several drawbacks, including the need for reference standards, the inability to characterize unknown adducts, and its susceptibility to false positives or false negatives. To a certain extent, even the more sensitive and emerging technique of accelerator mass spectrometry (AMS) (41) suffers from many of the same disadvantages. These drawbacks can be overcome using a technique that can combine structural specificity along with high sensitivity. ESI-MS and ESI-MS/MS can meet these requirements, particularly when coupled with microseparation techniques such as LC or capillary electrophoresis (CE). Some key attributes of LC/MS/MS include exceptional selectiv-

1026

Chem. Res. Toxicol., Vol. 12, No. 10, 1999

ity and specificity, corroborative evidence from characteristic retention times, the potential for quantitative analysis using isotopically labeled internal standards (42, 43), and removal of the need to work with radioisotopes and adduct analysis without resorting to often cumbersome chemical derivatization reactions. In view of these aforementioned features, a number of laboratories, including our own, have been interested in bridging the gap between “classical” LC/MS/MS and radiolabeling methods. Lagging in this regard has been information pertaining to the suitability of LC/MS to the analysis of DNA adducts in biological samples. Such capability should provide for a relatively unequivocal identification of a given DNA adduct(s). This work on the analysis of IQ adducts of deoxyguanosine in kidney tissues from non-human primates demonstrates the feasibility of attaining this goal. The results show the ability to screen for unknown adducts at levels of less than 1 modification in 104 normal bases using CNL and to detect 1 targeted adduct in 107 normal bases using SRM. This level of analysis is accomplished using ca. 300 µg of DNA. Recently, LC/MS analyses were successfully conducted on the dG-C8 adduct of 4-aminobiphenyl in rodents (42), further demonstrating the power of this technique for the detection and characterization of DNA adducts. The work described here discusses the stages of development for DNA adduct detection. This includes the synthesis of standards, use of the latter compounds to optimize chromatographic and detection conditions, evaluation of detection limits in vitro, and, finally, application of the methodology to an in vivo system. Parallel radiolabeling studies were used to either support or assess the validity of the LC/MS/MS analyses. The levels of detection demonstrated in these LC/MS/MS experiments were achieved, mainly, by optimizing the front end of the analytical system, i.e., the coupling of microLC with microspray, as well as the handling of the DNA from tissue samples for analyte recovery. Further improvements in sensitivity and detection limits are anticipated by increasing sampling efficiency in the injection (current sampling efficiencies are a mere 3%), by exploring techniques that will improve chromatographic performance and ionization efficiency, and by application of state-of-the-art MS instrumentation. On that basis, it may be possible to reliably detect IQ adducts from singledose experiments (1 adduct in 108-109 normal bases) and to determine dose-response relationships from low-dose, single-dose, or chronic exposures. Mass spectrometric ionization and detection methods are in a very dynamic state of growth, and it is reasonable to expect further improvements in LC/MS instrumentation technology. While it is debatable whether LC/MS techniques will achieve the sensitivity of current radiochemical detection methods (it is certainly unlikely to reach the sensitivity of AMS), in the least, sensitive LC/ MS assays should enable a parallel use of the different techniques and a strong role of mass spectrometry for confirmation of adduct structures and recognition of new adducts. The work described in this paper demonstrates an effective parallel use of 32P-postlabeling and mass spectrometry for the in vivo detection and quantitation of DNA adducts. The general principles and approaches outlined here provide a basis for future related applications.

Gangl et al.

Acknowledgment. This work was supported by a grant from the National Institutes of Health (1RO1CA 69390). The provision of non-human primate tissue samples by Dr. E. G. Snyderwine and Dr. R. H. Adamson (National Institutes of Health, Bethesda, MD) is greatly appreciated. We also acknowledge Dr. Andreas Harsch for the synthesis of the reference standards. This is contribution 762 from the Barnett Institute.

References (1) Ames, B. N., Gold, L. S., and Willett, W. C. (1995) The causes and prevention of cancer. Proc. Natl. Acad. Sci. U.S.A. 92, 52585265. (2) Wogan, G. N. (1992) Molecular epidemiology in cancer risk assessment and prevention: recent progress and avenues for future research. Environ. Health Perspect. 98, 167-178. (3) Stavric, B. (1994) Biological significance of trace levels of mutagenic heterocyclic aromatic amines in human diet: a critical review. Food Chem. Toxicol. 32, 977-994. (4) Wakabayashi, K., Nagao, M., Esumi, H., and Sugimura, T. (1992) Food-derived mutagens and carcinogens. Cancer Res. 52, 2092s2098s. (5) Adamson, R. H., Thorgeirsson, U. P., Snyderwine, E. G., Thorgeirsson, S. S., Reeves, J., Dalgard, D. W., Takayama, S., and Sugimura, T. (1990) Carcinogenicity of 2-amino-3-methylimidazo[4,5-f]quinoline in nonhuman primates: induction of tumors in three macaques. Jpn. J. Cancer Res. 81, 10-14. (6) 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. (7) Badawi, A. F., Stern, S. J., Lang, N. P., and Kadlubar, F. F. (1996) Cytochrome P-450 and acetyltransferase expression as biomarkers of carcinogen-DNA adduct levels and human cancer susceptibility. Prog. Clin. Biol. Res. 395, 109-140. (8) Kato, R. (1986) Metabolic activation of mutagenic heterocyclic aromatic amines from protein pyrolysates. Crit. Rev. Toxicol. 16, 307-348. (9) Vineis, P., and McMichael, A. (1996) Interplay between heterocyclic amines in cooked meat and metabolic phenotype in the etiology of colon cancer. Cancer Causes Control 7, 479-486. (10) King, R. S., Kadlubar, F. F., and Turesky, R. J. (1999) In Vivo Metabolism of Heterocyclic Aromatic Amines. In Heterocyclic Amines: Food Borne Carcinogens (Nagao, M., and Sugimura, T., Eds.) John Wiley & Sons Ltd., Chichester, Sussex, England (in press). (11) Turesky, R. J., Lang, N. P., Butler, M. A., Teitel, C. H., and Kadlubar, F. F. (1991) Metabolic activation of carcinogenic heterocyclic aromatic amines by human liver and colon. Carcinogenesis 12, 1839-1845. (12) Turesky, R. J., Constable, A., Richoz, J., Varga, N., Markovic, J., Martin, M. V., and Guengerich, F. P. (1998) Activation of heterocyclic aromatic amines by rat and human liver microsomes and by purified rat and human cytochrome P450 1A2. Chem. Res. Toxicol. 11, 925-936. (13) Turesky, R. J., and Markovic, J. (1994) DNA adduct formation of the food carcinogen 2-amino-3-methylimidazo[4,5-f]quinoline at the C-8 and N2 atoms of guanine. Chem. Res. Toxicol. 7, 752761. (14) Turesky, R. J., Box, R. M., Markovic, J., Gremaud, E., and Snyderwine, E. G. (1997) Formation and persistence of DNA adducts of 2-amino-3-methylimidazo[4,5-f]quinoline in the rat and nonhuman primates. Mutat. Res. 376, 235-241. (15) Randerath, K., Randerath, E., Agrawal, H. P., Gupta, R. C., Schurdak, M. E., and Reddy, M. V. (1985) Postlabeling methods for carcinogen-DNA adduct analysis. Environ. Health Perspect. 62, 57-65. (16) Yamashita, K., Umemoto, A., Grivas, S., Kato, S., Sato, S., and Sugimura, T. (1988) Heterocyclic amine-DNA adducts analyzed by 32P-postlabeling method. Nucleic Acids Symp. Ser. 19, 111114. (17) Turesky, R. J., Gremaud, E., Markovic, J., and Snyderwine, E. G. (1996) DNA adduct formation of the food-derived mutagen 2-amino-3-methylimidazo[4,5-f]quinoline in nonhuman primates undergoing carcinogen bioassay. Chem. Res. Toxicol. 9, 403-408. (18) Rindgen, D., Turesky, R. J., and Vouros, P. (1995) Determination of in vitro formed DNA adducts of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine using capillary liquid chromatography/electrospray ionization/tandem mass spectrometry. Chem. Res. Toxicol. 8, 1005-1013.

LC/MS Analysis of DNA Adducts (19) Vanhoutte, K., Van Dongen, W., Hoes, I., Lemiere, F., Esmans, E. L., Van Onckelen, H., Van den Eeckhout, E., van Soest, R. E., and Hudson, A. J. (1997) Development of a nanoscale liquid chromatography/electrospray mass spectrometry methodology for the detection and identification of DNA adducts. Anal. Chem. 69, 3161-3168. (20) Leclercq, L., Laurent, C., and De Pauw, E. (1997) Highperformance liquid chromatography/electrospray mass spectrometry for the analysis of modified bases in DNA: 7-(2-hydroxyethyl)guanine, the major ethylene oxide-DNA adduct. Anal. Chem. 69, 1952-1955. (21) Valaskovic, G. A., Kelleher, N. L., Little, D. P., Aaserud, D. J., and McLafferty, F. W. (1995) Attomole-sensitivity electrospray source for large-molecule mass spectrometry. Anal. Chem. 67, 3802-3805. (22) McCormack, A. L., Schieltz, D. M., Goode, B., Yang, S., Barnes, G., Drubin, D., and Yates, J. R., III (1997) Direct analysis and identification of proteins in mixtures by LC/MS/MS and database searching at the low-femtomole level. Anal. Chem. 69, 767-776. (23) Wilm, M., Shevchenko, A., Houthaeve, T., Breit, S., Schweigerer, L., Fotsis, T., and Mann, M. (1996) Femtomole sequencing of proteins from polyacrylamide gels by nano-electrospray mass spectrometry. Nature 379, 466-469. (24) Morris, H. R., Paxton, T., Panico, M., McDowell, R., and Dell, A. (1997) A novel geometry mass spectrometer, the Q-TOF, for lowfemtomole/attomole-range biopolymer sequencing. J. Protein Chem. 16, 469-479. (25) de Wit, J. S., Parker, C. E., Tomer, K. B., and Jorgenson, J. W. (1987) Direct coupling of open-tubular liquid chromatography with mass spectrometry. Anal. Chem. 59, 2400-2404. (26) Novotny, M. (1988) Recent advances in microcolumn liquid chromatography. Anal. Chem. 60, 500A-510A. (27) Karlsson, K. E., and Novotny, M. (1988) Separation efficiency of slurry-packed liquid chromatography microcolumns with very small inner diameters. Anal. Chem. 60, 1662-1665. (28) Wu, J. T., Huang, P., Li, M. X., Qian, M. G., and Lubman, D. M. (1997) Open-tubular capillary electrochromatography with an online ion trap storage/reflectron time-of-flight mass detector for ultrafast peptide mixture analysis. Anal. Chem. 69, 320-326. (29) Davis, M. T., Stahl, D. C., Hefta, S. A., and Lee, T. D. (1995) A microscale electrospray interface for on-line, capillary liquid chromatography/tandem mass spectrometry of complex peptide mixtures. Anal. Chem. 67, 4549-4556. (30) Parker, C. E., Perkins, J. R., Tomer, K. B., Shida, Y., and O’Hara, K. (1993) Nanoscale Packed Capillary Liquid ChromatographyElectrospray Ionization Mass Spectrometry: Analysis of Penicillins and Cephems. J. Chromatogr. 616, 45-57. (31) Robins, R. H., and Guido, J. E. (1997) Design, Construction and Application of a Simple Packed Capillary High Performance Liquid Chromatography/Electrospray Mass Spectrometry System. Rapid Commun. Mass Spectrom. 11, 1661-1666.

Chem. Res. Toxicol., Vol. 12, No. 10, 1999 1027 (32) Banks, J. F. (1997) Protein analysis by packed capillary liquid chromatography with electrospray ionization and time-of-flight mass spectrometry detection. J. Chromatogr. A 786, 67-73. (33) Coutant, J. E., Chen, T.-M., and Ackerman, B. L. (1990) Interfacing Microbore and Capillary Liquid Chromatography to Continuous-flow Fast Atom Bombardment Mass Spectrometry for the Analysis of Glycopeptides. J. Chromatogr. 529, 265-275. (34) Turesky, R. J., Rossi, S. C., Welti, D. H., Lay, J. O., Jr., and Kadlubar, F. F. (1992) Characterization of DNA adducts formed in vitro by reaction of N-hydroxy-2-amino-3-methylimidazo[4,5f]quinoline and N-hydroxy-2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline at the C-8 and N2 atoms of guanine. Chem. Res. Toxicol. 5, 479-490. (35) Adamson, R. H., Takayama, S., Sugimura, T., and Thorgeirsson, U. P. (1994) Induction of hepatocellular carcinoma in nonhuman primates by the food mutagen 2-amino-3-methylimidazo[4,5-f]quinoline. Environ. Health Perspect. 102, 190-193. (36) Thorgeirsson, U. P., Dalgard, D. W., Reeves, J., and Adamson, R. H. (1994) Tumor incidence in a chemical carcinogenesis study of nonhuman primates. Regul. Toxicol. Pharmacol. 19, 130-151. (37) Tomer, K. B., Moseley, M. A., Deterding, L. J., and Parker, C. E. (1995) Capillary Liquid Chromatography/Mass Spectrometry. Mass Spectrom. Rev. 13, 431-457. (38) Wolf, S. M., and Vouros, P. (1994) Application of capillary liquid chromatography coupled with tandem mass spectrometric methods to the rapid screening of adducts formed by the reaction of N-acetoxy-N-acetyl-2-aminofluorene with calf thymus DNA. Chem. Res. Toxicol. 7, 82-88. (39) Crow, F. W., Tomer, K. B., Gross, M. L., McCloskey, J. A., and Bergstrom, D. E. (1984) Fast atom bombardment combined with tandem mass spectrometry for the determination of nucleosides. Anal. Biochem. 139, 243-262. (40) Matthews, C. K., and Van Holde, K. E. (1996) Biochemistry, 2nd ed., The Benjamin/Cummings Publishing Co. Inc., Menlo Park, CA. (41) Turteltaub, K. W., Mauthe, R. J., Dingley, K. H., Vogel, J. S., Frantz, C. E., Garner, R. C., and Shen, N. (1997) MeIQx-DNA adduct formation in rodent and human tissues at low doses. Mutat. Res. 376, 243-252. (42) Beland, F. A., Doerge, D. R., Churchwell, M. I., Poirier, M. C., Schoket, B., and Marques, M. M. (1999) Synthesis, characterization, and quantitation of a 4-aminobiphenyl-DNA adduct standard. Chem. Res. Toxicol. 12, 68-77. (43) Tretyakova, N., Chiang, S. Y., Walker, V. E., and Swenberg, J. A. (1998) Quantitative analysis of 1,3-butadiene-induced DNA adducts in vivo and in vitro using liquid chromatography electrospray ionization tandem mass spectrometry. J. Mass Spectrom. 33, 363-376.

TX990060M