Apyrimidinic Lesions in DNA with High

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Determination of Apurinic/Apyrimidinic Lesions in DNA with High-Performance Liquid Chromatography and Tandem Mass Spectrometry Kenneth P. Roberts,*,† Justin A. Sobrino,† Julie Payton,† Lavinnia B. Mason,† and Robert J. Turesky‡,§ Department of Chemistry and Biochemistry, The UniVersity of Tulsa, Tulsa, Oklahoma 74104, and National Center for Toxicological Research, U.S. Food and Drug Administration, Jefferson, Arkansas 72079 ReceiVed September 14, 2005

A new method has been developed to accurately measure apurinic and apyrimidinic (AP) DNA damage sites, which are lesions in DNA formed by loss of a nucleobase from oxidative stress or carcinogen adducts. If AP sites are left unrepaired (or if improperly repaired), these sites can lead to DNA mutations that may ultimately result in the formation of cancer. Hence, detection of AP sites may provide a useful indicator of exposure and susceptibility to chemical carcinogens and oxidative stress. AP detection is currently accomplished by immunodetection methods using an aldehyde reactive probe [Nakamura, J., Walker, V. E., Upton, P. B., Chiang, S.-Y., Kow, Y. W., and Swenberg, J. A. (1998) Cancer Res. 58, 222-225; Atamna, H., Cheung, I., and Ames, B. N. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 686-691]; however, these approaches lack the specificity required for unequivocal identification of the AP site. Therefore, we have developed an accurate method based on mass spectrometry detection of AP sites from AP DNA that have been prelabeled with O-4-nitrobenzylhydroxylamine (NBHA). Once labeled and once the excess labeling agent has been removed, enzymatic digestion of DNA to monomeric subunits can be accomplished, followed by isolation and detection with high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS). Optimization and validation of the experimental procedures and detection limits have been established using a model DNA oligomer (11-mer) containing uracil. Enzymatic removal of uracil with uracil glycosylase generates welldefined AP sites in both single- and double-stranded DNA. The addition of NBHA labels the AP site in the oligomer, creating a labeled 11-mer. HPLC-ESI-MS/MS in the negative ionization mode was used to monitor and confirm binding of NBHA to the AP oligomer. The NBHA-tagged oligomer underwent endo- and exonuclease digestion to the 5′-deoxyribose monophosphate (5′-dRp) level, thereby releasing free 5′-dRp-NBHA. The 5′-dRp-NBHA product was partially purified by solid phase extraction and quantified by LC-MS/MS using several transitions of the deprotonated molecule ([M - H]- at m/z 363) and isotopically labeled 5′-dRp-NBHA as an internal standard. Further experiments with 5′,3′-bisphosphatedeoxyribose and heat/acid-treated calf thymus DNA showed similar labeling, digestion, and detection results. Initial results show a quantification limit with 100 µg of DNA to be 100 fmol (three abasic sites per 107 bases). Introduction Apurinic/apyrimidinic (AP) sites in DNA are one of the most prevalent types of DNA damage and are often the consequence of spontaneous hydrolysis of the N-glycoside bond from exposure to chemical carcinogens, irradiation, oxidative stress, or through enzymatic processes in base excision repair pathways (1, 2). The result is loss of a nucleobase, leaving a ribosyl moiety that is in equilibrium between a ring-closed and a ring-opened aldehydic structure. The ring-opened structure accounts for approximately 5% of the equilibrium concentration (3). If incorrectly (or inefficiently) repaired, AP sites may lead to genetic mutations and to the development of cancer (4). What is more, reports have shown that overexposure to certain environmental toxicants may induce formation of high levels * To whom correspondence should be addressed. Fax: 918-631-3404. E-mail: [email protected]. † The University of Tulsa. ‡ U.S. Food and Drug Administration. § Current address: Division of Environmental Disease and Prevention, NYS Department of Health, Albany, NY 12201-0509.

of AP sites beyond the reported basal level of one AP site in 105 bases (5, 6), which could exceed the capacity of error-free repair (7-9). Hence, detection of AP sites may provide a useful dosimeter of exposure and susceptibility to toxicants and various forms of oxidative stress. Several approaches for AP site detection have been utilized including radiometric labeling of the AP site with 14C-methoxyamine, 14C-phenylhydrazine, and 14C-semicarbazide (10-12); HPLC with 32P-postlabeling (13); diaminopurine tethered to acridine for NMR detection of single-strand breaks attributed to AP sites (14); fluorimetric labeling of AP sites with rhodamine and dansyl probes (15); a colorimetric ELISA assay based on formation of a Schiff base with O-4-nitrobenzyl hydroxylamine (NBHA) (16); atomic force microscopy of probed AP sites (17); and, most commonly, colorimetric detection with an aldehyde reactive probe (ARP) (3, 6, 18-23). In the ARP approach, the aldehyde group of the AP site is covalently labeled with specially designed aminooxy functionality at one end of the ARP probe to form a Schiff base at the AP site of intact DNA. The opposing end of ARP contains a

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Figure 1. General overview of detecting AP sites by HPLC-ESI-MS/MS. Upon loss of a DNA base by a chemical carcinogen or other mechanisms of oxidative stress, the aldehydic position of the ring-opened ribosyl moiety is labeled with NBHA. After digestion to the monophosphate by a three-enzyme system, identification and qunantification are accomplished by HPLC-UV and HPLC-ESI-MS/MS.

biotin moiety for avidin recognition in subsequent slot-blot detection schemes. AP site determinations with ARP, utilizing a commercially available kit (Dojindo Molec. Tech., Gaithersburg, MD), can be accomplished within the detection limits of this commercial ELISA type assay (one AP site per 105 base pairs), and one AP site per 107 base pairs has been reported using an ARP-based slot-blot method (6). Although commercialization and sufficient detection levels have been demonstrated with ARP, these approaches lack specificity and, hence, the potential mislabeling of DNA oxidation products other than AP sites (6, 12, 23, 24). In addition, noncovalent intercalation of the ARP probe within DNA may occur, resulting in further overestimation of AP sites. With this, researchers have recently begun to utilize mass spectrometry as a more accurate analysis of oxidative products of DNA damage, either by monitoring apurinic sites by 14C-labeling with accelerator mass spectrometry detection (12) or by using tandem mass spectrometry to measure oxidation products of deoxyribose that have been labeled with ARP (23). Detection limits were one AP site in 106 nucleotides and two adducts in 108 guanines, respectively. In our laboratories, we have developed a method for measuring AP sites based on chromatographic separation and electrospray mass spectrometry detection of AP sites labeled with NBHA. Once AP sites are labeled in DNA and the excess labeling agent has been removed, enzymatic digestion of DNA to monomeric subunits can be accomplished, liberating the NBHA-labeled AP site. Following digestion, high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) is used for the resolution and unambiguous mass identification of AP site labeled with NBHA. A general overview of this methodology is shown in Figure 1. In this study, proof-of-concept and detection limits were established using a model DNA oligomer (11-mer) containing uracil. Enzymatic removal of uracil with uracil glycosylase generates well-defined AP sites in both single- and double-stranded DNA (25-27). The addition of the NBHA probe molecule tags the AP site in the oligomer, creating a labeled 11-mer. HPLCESI-MS/MS in the negative ionization mode was used to monitor and confirm binding of NBHA to the AP oligomer.

The NBHA-tagged oligomer underwent endo- and exonuclease digestion to release free (E/Z)-5-(4-nitrobenzyloxyimino)-2,3dihydroxypentyl dihydrogen phosphate, referred to hereafter as 5′-deoxyribose monophosphate-NBHA (5′-dRp-NBHA). The 5′dRp-NBHA product was partially purified by solid phase extraction (SPE) and quantified by HPLC-MS/MS using several transitions of the deprotonated molecule ([M - H]- at m/z 363) with isotopically enriched 5′-dRp-NBHA as an internal standard. Further experiments were conducted with other model AP precursors such as 5′,3′-bisphosphate-deoxyribose and heat/acidtreated calf thymus (CT) DNA in order to establish optimal experimental conditions, controls, and limits of detection.

Materials and Methods Materials. CT DNA (sodium salt of type I), 2′-deoxyadenosine 5′-monophosphate sodium salt (5′-dAMP), 2′-deoxyguanosine 5′-monophosphate sodium salt (5′-dGMP), 2′-deoxyguanosine 3′-monophosphate sodium salt (3′-dGMP), 2′-deoxyguanosine (dG), guanidine (G), ammonium acetate, sodium dihydrogen phosphate, magnesium chloride, HEPES, 1,1,1,3,3,3-hexafluoro2-propanol (HFIP), deoxyribonuclease I (DNase I) from bovine pancreas, EDTA, BisTris, Tris-HCl, methoxyamine, N2-isobutyril-dG, 1,3dicyclohexyl-carbodiimide (DCC), barium cyanoethyl phosphate, NBHA, dithiothreitol, triethylamine (TEA), 200 proof ethanol, HPLC grade methanol and acetonitrile, Dowex resin 50WX8 (50-100 mesh, H form), and anhydrous pyridine were obtained from Sigma-Aldrich-Fluka-Supelco (St. Louis, MO). Uracil DNA glycosylase was purchased from New England Biolabs (Ipswich, MA). Nuclease P1 was obtained from Roche (Nutley, NJ), and snake venom phosphodiesterase I (PDase I) was from Worthington (Lakewood, NJ). Sodium chloride, zinc chloride, glacial acetic acid, hydrochloric acid, potassium hydroxide, sodium hydroxide, and ammonium hydroxide were obtained from Fisher Scientific (Fairlawn, NJ). SPE tC18 cartridges were purchased from Waters (Milford, MA). Synthetic single-stranded and double-stranded oligomers were obtained with PAGE purity from Integrated DNA Technologies (Coralville, IA). U-13C5-dGMP internal standard was obtained from (Isotec, St. Louis, MO) and had a ∼99% isotopic purity, as determined by mass spectrometry. HPLC-UV Instrumentation. Experiments were conducted either on a Varian ProStar Quaternary HPLC system with UV/vis diode array detection (Palo Alto, CA) or on an Agilent 1100 binary system

302 Chem. Res. Toxicol., Vol. 19, No. 2, 2006 with UV/vis diode array detection (Palo Alto, CA). Unless otherwise stated below, the separation columns of choice were 4.6 mm × 250 mm Supelco C18-DB and Supelco Discovery C18, with inline C18 Supelco guard columns. Injection of sample onto the HPLC was either manual or by the use of an autosampler. All solvents were of HPLC grade and/or prepared with doubly deionized ultrahigh purity (18 MΩ) water. Mobile phase buffers were filtered through 0.45 µm nylon filters and degassed by sonication prior to use. Unless otherwise stated, the optimal HPLC-UV/vis conditions for separations were to utilize a solvent gradient beginning with 100% 50 mM NaH2PO4 (pH 4.5) for 3 min followed by a linear gradient to 100% of 66% acetonitrile at 13 min, and holding to 16 min before returning to 100% NaH2PO4 for reequilibration. The detection wavelength was 274 nm. A constant flow rate of 1.0 mL per min was maintained. In cases where subsequent MS/MS was to be performed, the phosphate buffer was replaced with 50 mM ammonium acetate (pH 6.8). HPLC-ESI-MS/MS Instrumentation. Chromatography was accomplished with a capillary HPLC system from LC Packings/ Dionex (Amsterdam, Netherlands) and comprised of an UltiMate quaternary pump, Switchos II column switching system, and a Famos autosampler. A Waters Symmetry Column (1 mm × 5 cm, 4 µm particle size with 80 Å pore size) was used for chromatographic separations using mobile phases of 0.1% formic acid (A) and 0.1% formic acid in acetonitrile (B). Elution was carried out by holding A constant for 4 min followed by a linear gradient to 100% B in 15 min at a flow rate of 50 µL/min. Typical injection volumes were 1-5 µL. Detection and quantification of DNA digestion products were accomplished by ESI-MS/MS with a Waters Micromass Quattro Ultima triple quadrupole mass spectrometer (Manchester, U.K.). Unless otherwise stated, qualitative and quantitative detection were done in negative ionization mode using full scan and product ion mass spectral transitions. The monitored transitions for 5′-dRpNBHA were m/z 363 f 97 and m/z 363 f 79, and for the 13C internal standard, the transitions were m/z 368 f 97 and m/z 368 f 79. The dwell time for the selected reaction monitoring transitions of 5′-dRp-NBHA was 0.1 s. The cone voltage was 40 V, the capillary voltage was 3.0 kV, hexapoles 1 and 2 were 0 V, and the collision energy was set to 21 eV. Source and desolvation temperatures were 110 and 300 °C, respectively, with cone gas and desolvation flow rates of 70 and 500 L/h, respectively. Argon was used as the collision gas at a pressure of 2.5 mTorr. Synthesis of 5′-dRp-NBHA. The procedure used for synthesis of 5′-dRp-NBHA was carried out with either 5′-dAMP or 5′-dGMP due to the lack of commercially available 5′-dRp at the time of this study. In brief, to 100 mg of dAMP in 10 mL of H2O was added 173 µL of concentrated acetic acid and hydrolyzed at 65 °C for 70 min. Upon cooling, NBHA label could be added directly to the hydrolysis mixture (see below), or if need be, 5′-dRp was purified. The latter was accomplished with 3 cc Waters brand tC18 Sep-Pak SPE cartridges. Cartridges were conditioned with 2.0 mL of methanol followed by 2.0 mL of water. Three milliliters of hydrolysis product was then loaded on the cartridges, and 1 mL fractions were collected. No additional elution solvent was used. Purified 5′-dRp eluted in fractions 1 and 2 as determined by mass spectrometry. Fractions 1 and 2 were collected, and solvent was removed by evaporation under reduced pressure. However, in most cases, NBHA was added directly to the hydrolysis mixture without SPE purification. This was done by adding 1.8 mM NBHA (final concentration) to the hydrolysis mixture and reacting for 2 h at room temperature, protected from light with aluminum foil. After centrifugation to remove insoluble nucleobase (e.g., Ade), SPE was performed with Waters tC18 3 cc cartridges. Prior to sample loading, SPE cartridges were equilibrated with 2.0 mL of methanol, followed by 2.0 mL of 2.0 mM NaH2PO4 (pH 4.5). One milliliter of the hydrolysis supernatant was added to a single SPE cartridge, and 0.5 mL fractions were collected. A stepwise solvent gradient was added in 1.0 mL increments that included 2.0 mM NaH2PO4, 75% 2.0 mM NaH2PO4: 25% methanol, 50% NaH2PO4:50% methanol, 25% NaH2PO4:75%

Roberts et al. methanol, and 100% methanol. Purified 5′-dRp-NBHA eluted in fractions eight and nine, which were combined and evaporated to dryness. HPLC-UV diode array detection was utilized to monitor hydrolysis, NBHA labeling, and purity of the SPE fractions using HPLC conditions described above. The characterization of the 5′-dRp-NBHA product was characterized by full scan product ESI-MS spectra, both positive and negative ionization mode, and by NMR, which showed the presence of E/Z isomers. For completeness, control studies were conducted with dG, 3′-dGMP, and commercial 5′-deoxyribose. Hydrolysis, NBHA labeling, HPLC, and mass spectrometry monitoring were achieved in the same manner as that described above for the hydrolysis product of 5′-GMP (data not shown). Synthesis of 5′,3′-Bis-phosphodeoxyguanosine (pdGp). Barium cyanoethyl phosphate (1.4 g) was added to 150 mL of water and stirred. Barium cyanoethyl phosphate is only partially soluble in water. Dissolution was optimized by alternating between sonication and magnetic stirring for approximately 30 min. The solution remained cloudy. A 1 cm × 20 cm glass column was filled with approximately 10 mL of Dowex resin and washed with 5 resin volumes of 1 M HCl and then rinsed with 5× resin volumes of water (the resin appeared brown). A 1 M concentration of pyridine was passed through the column (5 resin volumes) until the resin was yellowish. Next, the column was washed with 8× bed volume using high purity water to remove excess pyridine. Immediately thereafter, the barium cyanoethyl phosphate solution was passed through the column at a rate of 1-2 mL/min, and the effluent was collected into a 300 mL round-bottom flask. After evaporation of the effluent to an oily residue [bis(pyridinium)cyanoethyl phosphate] by rotoevaporation, further drying was accomplished by overnight desiccation under vacuum until a final residue weight of approximately 0.8 g was obtained. The amount of N2-isobutyril-dG added was calculated to be 1/5 equivalents of the bis(pyridinium)cyanoethyl phosphate residue. N2-Isobutyryl-dG was dissolved in 2.0 mL of anhydrous pyridine (pyridine saturated with molecular sieves). The amount of DCC used was calculated as 10 equiv of N2-isobutyril-dG, e.g., 5 mol of bis(pyridinium)cyanoethyl phosphate to 1 mol of N2-isobutyryl-dG to 10 mol of DCC. The reaction took place by adding the N2-isobutyril-dG solution to a 30 mL round-bottom flask that contained the bis(pyridinium)cyanoethyl phosphate, followed by gentle stirring for 3 min. Next, DCC solid was added, along with enough anhydrous pyridine to give a final volume of 10 mL. The solution remained turbid, as DCC is not highly soluble in this milieu. The reaction was allowed to run overnight at room temperature with stirring. The reaction was quenched by addition of 15 mL of water and stirring for 5 min. After vacuum filtration and washing the precipitate with 5 mL of water, the aqueous filtrate was evaporated to a final volume of 0.5 mL. Deprotection of N2 and PO4 was accomplished with the addition of 10 mL of 2 M NH4OH for 4-8 h in a hot water bath at 55-60 °C with stirring. Deprotection kinetics were monitored by HPLC-UV diode array detection (see instrumentation details above). The mobile phase components were 100 mM ammonium acetate (pH 6.8) (A) and 100% methanol (B). Solvent B was increased from 0 to 50% B in 25 min, and to 100% B at 28 min, and then held at 100% B until 30 min. HPLC absorbance detection was accomplished at 256 nm. Under these conditions, deprotected pdGp eluted nearly at the dead volume. Upon completion of the deprotection step, ammonium hydroxide was removed by evaporation. The oily residue remaining was dissolved in a minimum volume of water and purified by SPE. The SPE procedure was accomplished by loading 0.3 mL of the dissolved product onto SPE cartridges (3 cc t-C18 SepPaks, Waters) preconditioned with 5 mL of methanol, followed by 5 mL of water. One milliliter fractions were collected using water as the SPE mobile phase. pdGp eluted in fraction 1 at approximately 95% purity as determined by HPLC-UV/vis and ESI-MS/MS. The final pdGp product was evaporated to dryness and stored at -80 °C. Synthesis of 5′,3′-pdRp-NBHA. Prior to NBHA labeling of pdGp, acid hydrolysis was performed to remove guanine. This was achieved by dissolving the oily pdGp product in a minimum volume

Apurinic/Apyrimidinic Lesions in DNA of water to which 0.5 M HCl was added at a 20:1 ratio, i.e., 20 parts HCl to 1 part pdGp. Hydrolysis was carried out at 37 °C for 12 h or until hydrolysis was complete, as monitored by HPLCUV. HPLC conditions were as follows: 50 mM NH4Ac (pH 6.8) (A) was held for 3 min with a linear gradient to 100% of 66% acetonitrile (B) in 10 min, returning to solvent A in 20 min. HPLCUV absorbance was monitored at 274 nm. Upon complete hydrolysis, a 10× mole excess of NBHA was added to the solution. The labeling was allowed to proceed for approximately 3 h at room temperature with stirring. Purification of the pdRp-NBHA product was realized by semiprep-HPLC (Supleco, C18 10 mm × 250 mm) using a 2.0 mL injection volume, at a flow rate of 2.5 mL/min under isocratic conditions. Characterization of 5′,3′-pdGp starting material and the 5′,3′-pdRp-NBHA product was accomplished by negative mode ESI-MS/MS. AP Sites in a Single-Stranded Oligomer (11-mer) and NBHA Labeling. AP sites can be “cleanly” prepared in synthetic oligomers that contain uracil. The following is a modification of a previously described procedure for generation of AP sites in oligomers (26). Our adapted procedure involved enzymatic removal of uracil with uracil DNA glycosylase (UDG) from a single-stranded uracil-oligo (11-mer), with the sequence, 5′-GCCGT-U-AGGTA-3′. The parent mass of the oligo is 3358.2 g/mol. In short, the procedure used 1.79 mg of single-stranded oligo in 400 µL of UDG buffer (40 mM HEPES, 70 mM NaCl, and 2 mM EDTA, pH 7.5). To the oligo solution was added 157 µL of UDG at 2000 units/mL (1.7 nmol oligomer:1 unit enzyme). After 1 h of incubation with slight agitation at 37 °C, the reaction was quenched by placing the incubation tube on ice. Next, 533 µL of NBHA in UDG buffer was added, i.e., adding 10× stoichiometric excess NBHA. Monitoring of a uracil removal by UDG and subsequent labeling with NBHA at room temperature was accomplished by HPLC-UV, injecting 20 µL, and detecting at 256 nm. To monitor the reaction kinetics of uracil removal, HPLC-UV was employed. Twenty microliters of the incubation mixture was injected, with detection at 256 nm. To provide suitable HPLC conditions that would not hamper ESI efficiency, we made adaptations to a previously described procedure for oligomer separations (26). The same LC-18-DB column as described above was used for the separations. A two-solvent gradient, 400 mM hexafluoro2-propanol (HFIP), pH adjusted to 7.0 with triethyamine (TEA) (A), and 800 mM HFIP, pH adjusted to 7.0 with TEA/100% methanol (1:1, v/v%) (B), was used for complete separation of the uracil oligomer, the AP oligomer product, and the NBHA-labeled AP oligomer. A linear gradient from 100% A to 100% B over 22 min, and held at 100% B until 25 min, followed by return to 100% A at 27 min, was employed. For subsequent DNA digestion to the nucleotide level, the labeled oligo was HPLC purified and then lyophilized to dryness. MS showed the labeled oligo to be stable to this process (data not shown). Final concentrations of the purified NBHA-AP oligo were determined by UV spectrometry, using the extinction coefficient of the parent oligo 108300 L/mol cm. The concentration was measured to be 0.48 mM, dissolved in 500 µL of 5 mM BisTris, pH 7.1. Complete characterization of uracil oligomer, the AP oligo, and the NBHA-AP oligo was accomplished by negative mode ESI-MS. Generation of AP Sites in CT DNA and NBHA Labeling. AP sites were created under acidic conditions with adaptations of previously described procedures (28). In our study, 5.0 mg of CT DNA was dissolved in 5.0 mL of 5.0 mM methoxyamine, which was in 10 mM Tris-HCl (pH 7.2 with KOH). Methoxyamine was allowed to cap preexisting AP sites by incubation for 2 h at 37 °C. After cooling on ice, 100 µL of 5 M NaCl was added prior to precipitation in 15 mL of cold ethanol, followed by washing the DNA pellet 3× with 15 mL of 70% ethanol. Further removal of methoxyamine was achieved by redissolving the washed pellet in 5.0 mL of 10 mM Tris-HCl (pH 7.2 with KOH), addition of 100 µL of 5 M NaCl, and reprecipitation in 15 mL of cold ethanol. Again, the DNA was washed 3× with 15 mL of 70% ethanol and dried under nitrogen.

Chem. Res. Toxicol., Vol. 19, No. 2, 2006 303 For comparison to methoxyamine, sodium borohydride was also used to reduce the number of preexisting AP sites in CT DNA according to previously described methods (3). Stock solutions of 0.1 mg/mL CT DNA were dissolved in deionized water and dialyzed overnight at 4 °C against 10 mM sodium phosphate, pH 11.5. The DNA solution was then treated with 0.1 M sodium borohydride at room temperature for 2 h and neutralized with 0.05 M HCl. Excess sodium borohydride was removed from the DNA by overnight dialysis against 10 mM sodium phosphate buffer (pH 7.5) at 4 °C. The DNA was then chilled on ice. A 100 µL amount of 5 M NaCl was added, and the AP DNA precipitated with 15 mL of cold ethanol. The AP DNA was then washed 3× with cold 70% ethanol and dried under nitrogen. The CT DNA was then resuspended in 5.0 mL of sodium citrate buffer (10 mM sodium citrate, 10 mM NaH2PO4, and 10 mM NaCl, pH 5.0) and incubated at 70 °C for specific times to achieve desired DNA damage levels (28). The reaction was stopped by rapid chilling on ice. A 100 µL amount of 5 M NaCl was added, and the AP DNA precipitated with 15 mL of cold ethanol. The AP DNA was then washed 3× with cold 70% ethanol and dried under nitrogen. Labeling of AP DNA with NBHA was carried out by dissolving the AP DNA pellet in 2.5 mL of 10 mM Tris-HCl (pH 7.2 with KOH). Upon dissolution, 2.5 mL of 2 mM NBHA in 10 mM TrisHCl (pH 7.2 with KOH) was added. The solution was then incubated at 37 °C for 1 h. Following incubation and chilling on ice, 100 µL of 5 M NaCl was added, and the labeled AP DNA was precipitated with 15 mL of cold ethanol. After washing the DNA 3× with cold 70% ethanol, the pellet was dried with N2 and redissolved in 5.0 mL of 5.0 mM BisTris, pH 7.1. The final DNA concentration was determined by UV spectrophotometry. Enzyme Digestion of NBHA-Labeled AP DNA. Digestion of the labeled DNA to the nucleotide level was done with a three enzyme system and using 13C-dRp-NBHA as an internal standard for quantification. The procedure was an adaptation of a previously described procedure for digestion of DNA adducted with chemical carcinogens (29). As an example assay, to 5.0 mL of a 1 mg/mL (5.0 mM BisTris, pH 7.1) solution from the labeling procedure above were added 500 µL of 100 mM MgCl2 and 30 µL of 1.0 µM 13C-dRp-NBHA, [M - H]- 368. To this solution, 50 µL of DNase I [10 mg/mL saline (in 0.9% NaCl)] was added, followed by incubation at 37 °C for 1.5 h. Next, 75 µL of Nuclease P1 (1 mg/mL in 1 mM ZnCl2) was added, followed by incubation for 3 h at 37 °C. Finally, 4 units of PDase I (type II) (1 unit in 1.0 mL water) was added, followed by incubation for 13 h. Upon completion of incubation, the digestion mixture was chilled on ice and then centrifuged at 4000 rpm for 10 min to remove excess protein prior to evaporation to dryness. Isolation of the digested 5′-dRPNBHA product was accomplished by SPE conditions described above for the standard. HPLC-UV or HPLC-MS/MS conditions described above for characterization of the standard were then utilized to determine the presence and quantity of 5′-dRp-NBHA from the digest (see the Results section for further details). Limit of Detection. Absolute detection limits for HPLCMS/MS were established by isotope dilution quantification of the NBHA-labeled (and HPLC-purified) AP oligo described (see above). In a 15 mL conical tube, 29.4 µL of 0.17 mg/mL oligo solution in 5 mM BisTris (pH 7.1) was added and diluted to 5.0 mL with 1 mg/mL CT DNA (5 mM BisTris (pH 7.1) stock solution. This provided the equivalent of one AP site in 103 bases. A series of serial dilutions were performed with CT DNA stock solution to provide AP sites to unmodified DNA base ratios of 1:104, 1:105, 1:106, and 1:107. A control experiment was performed on the CT DNA stock solution where no NBHA-AP oligo was added. To 5.0 mL of each of these solutions was added 30 µL of 1.0 µM 13C-dRp-NBHA, [M - H]- 368. This gave the equivalent of two AP sites in 106 deoxyribonucleotides. Next, enzyme digestion was performed simultaneously on each dilution (and control) under digestion procedures described above. After digestion, each dilution was processed by SPE and lyophilized to dryness. Prior to HPLCMS/MS analysis, each sample was reconstituted in 900 µL water/ 100 µL acetonitrile.

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Figure 2. Heat/acid hydrolysis of 5′-dAMP or 5′-dGMP to 5′-dRp and subsequent labeling with NBHA. The reaction is shown in part A. (B) HPLC-UV monitoring of the hydrolysis kinetics up to 65 min as shown in chromatograms a (0 min), b (15 min), c (35 min), d (50 min), and e (65 min). (C) HPLC-UV monitoring of kinetics of labeling 5′-dRp with NBHA. Chromatogram a is 5′-dRp only, which has no observable chromophore, and chromatogram b is that of NBHA labeling agent only. The kinetics of labeling 5′-dRp with NBHA are shown in chromatograms c-f (t ) 0, 50, 100, and 130, respectively). Inset: online absorbance comparison of 5′-dRp-NBHA (274 nm) with NBHA (272 nm). HPLC conditions: 4.6 mm × 250 mm LC-C18-DB (Supelco); solvent A, 400 mM HFIP (pH 7.0) with TEA; solvent B, 800 mM HFIP/100% MeOH (1:1, v/v%); gradient: 0-100% B in 22 min, holding B at 100% to 25 min and then back to 100% A at 27 min for reequilibration. HPLC conditions: 4.6 mm × 250 mm LC-C18-DB (Supelco); solvent A, 50 mM ammonium acetate (pH 6.8); solvent B, 66% acetonitrile; gradient: 100 to 75% A in 5 min, to 100% B in 10 min, holding for 15 min, to 100% A at 20 min for reequilibration. λabs ) 274 nm.

Results and Discussion Synthesis and Characterization of 5′-dRp-NBHA Standard. The general scheme of acid-hydrolyzed 5′-dGMP or 5′dAMP, and subsequent labeling with NBHA, is shown in part A of Figure 2. As determined by HPLC-UV, complete hydrolytic cleavage of adenine was observed at 65 min under acidic conditions (∼pH 3) at 70°C (see part B). Although 5′-dRp was not detectable by UV, observance of free adenine (7.2 min) in the chromatogram and the decrease in absorbance of 5′-dAMP indicated successful hydrolysis. Purification of 5′-dRp proved successful by SPE, as evidenced by no detectable peaks on the HPLC chromatogram for SPE fractions 1 and 2 and as confirmed by negative mode MS/MS, [M - H]- at m/z 213 (data not shown). HPLC-UV was used to monitor the NBHA-labeling kinetics of purified 5′-dRp as shown in part C of Figure 2. Purified 5′dRp is shown in chromatogram a (with no chromophore, it has no UV absorbance). The chromatogram shows no presence of hydrolytically cleaved adenine or unreacted dAMP. The NBHA standard (chromatogram b) elutes at 13 min, and labeling of 5′-dRP is indicated by the presence of a new peak at 11 min with the simultaneous reduction of the NBHA peak. As shown in the figure inset, the on-line absorbance of 5′-dRp-NBHA (dotted line) differs from that of NBHA (solid line) by a slight red shift from 272 to 274 nm. Under these conditions, formation of the 5′-dRp-NBHA product was complete in approximately 2 h as shown in chromatograms c-f in the figure. Synthesis of the 5′-dRp-NBHA product was further confirmed by HPLC fractionation and isolation of the 11 min peak of the chromatogram for NMR and mass spectrometry analyses. All protons in the NMR spectrum were readily assigned, and the presence of the E/Z isomers (ratio of 45 to 55%, respectively) of 5′-dRpNBHA was clearly revealed (data not shown). The 5′-dRpNBHA standard was determined to be stable for at least 48 h at 37 °C in 10 mM Tris Tris-HCl (pH 7.2 with KOH) as monitored by HPLC-UV (data not shown).

Mass spectrometry characterization of the 5′-dRp-NBHA standard was accomplished by electrospray ionization in both positive and negative ionization modes. Although parent masses were clearly revealed in both modes of ionization, with [M H]- and [M + H]+ detected at m/z 363 and 365, respectively, it was evident that there is much less background noise in the negative ionization mode under these solvent conditions (data not shown). The negative ionization mode was chosen for future identification and quantification of AP sites. Product ion spectra in negative mode and the fragmentation scheme for 5′-dRpNBHA standard and 13C-labeled internal standard are shown in Figure 3A,B, respectively. Distinctive fragment ions are observed at m/z 210, 151, 97, and 79 as illustrated in the figure. With the 13C-labeled isotope remaining on the ribosyl portion of the 5′-dRp-NBHA standard, an increase in 5 Da was clearly observed at m/z for [M - H]- (368) and the fragment ion generated at m/z of 215 from loss of the nitrobenzyl alcohol from cleavage of the N-O bond. Similar phosphonate fragments (m/z 79 and 97) were generated for both species. Synthesis of 5′,3′-pdGp and 5′,3′-pdRp-NBHA. Initial testing and validation of AP site detection were accomplished with the model compound 5′,3′-pdGp. Success of the reaction (see Materials and Method section for reaction details) was concluded by monitoring the final deprotection kinetics with HPLC-UV diode array detection. Deprotection of N2 and PO4 with 2 M NH4OH was readily achievable as evidenced by the formation of 5′,3′-pdGp in the HPLC separation at 3 min (data not shown). Deprotection was completed in approximately 9 h. SPE purification of the 3 min peak yielded approximately 95% purity of 5′,3′-pdGp as confirmed by negative mode ESI-MS/ MS with [M - H]- at 426 (data not shown). For completeness, enzymatic synthesis of 5′,3′-pdGp was performed. For total conversion of 5′,3′-pdGp to 5′,3′-pdRp, heating under acidic conditions was allowed to take place overnight. Completion of hydrolysis to 5′,3′-pdRp was determined by the loss of 5′,3′-pdGp in the HPLC chromatogram (data not shown).



Figure 3. Fragmentation scheme and product ion (MS/MS) spectra in negative mode for 5′-dRp-NBHA (A) and the internal standard 13C-5′-dRpNBHA (B). See the Materials and Methods for MS details.

Figure 4. HPLC-UV chromatogram of synthesized 5′,3′-pdRp-NBHA (A). Products and starting materials are labeled on the chromatogram. HPLC conditions are identical to those used in the separation shown in Figure 2. Inset: Online HPLC diode array spectra for 5′,3′-pdGp (dashed line, λmax ) 251 nm), Gua (solid line, λmax ) 246 nm), 5′,3′-pdRp-NBHA (dotted line, λmax ) 274 nm), and unreacted NBHA (dashed dotted line, λmax ) 272 nm). (B) Negative mode ESI-MS/MS results for 5′,3′-pdRp-NBHA with [M - H]- of 442.8 m/z. MS/MS conditions are identical to those described in Figure 5.

Subsequent labeling of 5′,3′-pdRp with NBHA was carried out in situ. As indicated in the chromatogram (A) of Figure 4, 5′,3′pdRp-NBHA elutes at approximately 10 min with the hydrolyzed Gua product and excess NBHA eluting at 8.7 and 13.5 min, respectively. As shown in the inset of part A, normalized online UV data allowed for spectral distinction between reactants and products with 5′,3′-pdGp (dashed line) having a maximum at 251 nm with Gua (solid line) reaching a plateau at 246 nm. Moreover, 5′,3′-pdRp-NBHA (dotted line) is red-shifted by 2 nm with respect to unreacted NBHA (dashed dotted line) from 272 to 274 nm. This allowed for cursory identification of the in situ hydrolysis and labeling products based on both retention

time and online UV data. However, for completeness, each product was further identified by negative mode MS/MS, an example of which is shown in part B of Figure 6 where the product ion mass spectrum of 5′,3′-pdRp-NBHA reveals an [M - H]- of 443 and an indicative fragmentation signature. Because of the small size of the 5′,3′-pdRp-NBHA moiety, initial development of enzyme digestion methodologies was only conducted with the single exonuclease enzyme, PDase I. Using procedures adapted from previous studies on carcinogen-DNA adducts (29), 3′-phosphate digested away from 5′,3′-pdRpNBHA after 24 h. Production of 5′-dRp-NBHA was confirmed by HPLC-UV and MS/MS (data not shown).


Roberts et al.

Figure 5. NBHA labeling of an AP oligomer. (A) Chromatogram a shows the AP oligomer (15.7 min peak) at t ) 0, prior to the addition of NBHA. As shown in chromatogram b, at t ) 30 min, the occurrence of the NBHA-labeled oligo is observed at 17 min in the chromatogram. At 1 h, almost all of the original oligomer has been labeled with NBHA, as shown in chromatogram c. Under these conditions, complete loss of the AP oligo was observed in 2 h (chromatogram d). HPLC conditions: 4.6 mm × 250 mm LC-C18-DB (Supelco); solvent A, 400 mM HFIP (pH 7.0) with TEA; solvent B, 800 mM HFIP/100% MeOH (1:1, v/v%); gradient: 0-100% B in 22 min, holding B at 100% to 25 min and then back to 100% A at 27 min for reequilibration. (B) Negative mode ESI-MS of a uracil-containing oligomer with a base peak at [M - 5H]5- of 670.6 m/z with other ionization states noted. (C) Negative mode ESI-MS of AP oligomer created by removing uracil with UDG and revealing a base peak at [M - 5H]5- of 651.8 m/z. Other ionization states are also noted. (D) Negative mode ESI-MS of NBHA-labeled AP oligomer with a base peak at [M - 5H]5- of 681.8 m/z. MS instrument parameters: capillary, -2.66 kV; cone, -40 V; hex 1, 17.7 V; aperture, 0.0 V; hex 2, 0.0 V; source temperature, 120 °C; desolvation temperature, 250 °C; cone gas flow, 90 L/h; desolvation gas flow, 290 L/h; LM 1 resolution, 15.0; HM 1 resolution, 15.0; ion energy 1, 0.7; entrance, 50; collision, 2; exit, 50; LM 2 resolution, 15.0; HM 2 resolution, 15.0; ion energy, 21.9; and multiplier, -669 V.

Figure 6. Results for the enzymatic digestion of the NBHA-labeled AP oligomer with DNase I, nuclease P1, and PDase I. (A) The top chromatogram shows the HPLC-UV results for the digested NBHA-labeled AP oligomer, where, along with the four unmodified mononucleotides, 5′-dRp-NBHA is clearly present at ∼11 min in the chromatogram and has the same retention time as that of the 5′-dRp-NBHA standard (bottom chromatogram). HPLC-UV conditions are the same as those used in Figure 2. Inset: Online UV spectrum of the 11 min peak in the top chromatogram (solid line) clearly matching the standard (dotted line). (B) Negative mode ESI-MS/MS of the 11 min peak in the top chromatogram, which has the identical MS/MS signature of the 5′-dRp-NBHA standard with an [M - H]- at 363.2 and characteristic fragments at 210, 97, and 79 m/z. MS conditions are identical to those used for Figure 3 data.

Identification of AP Sites in a Single-Stranded Oligomer. With success using a model dimer AP DNA system, further

investigation into a slightly larger model system was undertaken. Enzymatic removal of uracil with uracil DNA glycosylase



Figure 7. Results for AP sites generated and labeled in CT DNA. (A) Chromatogram a is the product of enzymatic digestion of the NBHA-labeled AP DNA, and chromatogram b is the standard of 5′-dRp-NBHA. Chromatogram a shows a match in retention time to that of the 5′-dRp-NBHA standard at approximately 13 min. HPLC conditions: 4.6 mm × 250 mm LC-C18-DB (Supelco); solvent A, 50 mM NaH2PO4 (pH 4.5); solvent B, 100 mM NaH2PO4/MeOH (1:1, v/v%); gradient 100% A to 3 min to 100% B in 10 min, hold 100% B to 15 min, returning to 100% A at 20 min for reequilibration. λabs ) 274 nm. Inset: Online HPLC-UV data for the 13 min peak of the DNA digest (solid line) as compared to the 13 min peak of the 5′-dRp-NBHA standard (dotted line). (B) Negative mode ESI-MS/MS of the 13 min peak of the digest. The indicative [M - H]- and fragments of 5′-dRp-NBHA are clearly revealed. MS conditions are the same as those used for Figure 3 data.

(UDG) from a single-stranded uracil-oligo (11-mer), with the sequence 5′-GCCGT-U-AGGTA-3′ provided a defined source of an AP site within a sequence. Removal of uracil was complete in less than 1 h using instructions provided by the manufacturer. As shown in Figure 5, MS was used to monitor the untreated oligomer [M - 5H]5- at m/z 670.6 and following removal of uracil, [M - 5H]5- at m/z 651.8, which matched exactly with the calculated mass of the two oligomer species (3357.4 and 3264.1 g/mol, respectively). With an AP oligomer (5′-GCCGT-dR-AGGTA-3′) in place, NBHA labeling and enzymatic liberation of 5′-dRp-NBHA were conducted and then monitored by HPLC-UV. As shown in part A of Figure 5, after 2 h under these conditions, labeling was complete as evidenced by the loss of the AP oligomer chromatographic peak at 15.7 min and the formation of the NBHA-labeled AP oligomer peak at 17 min in the chromatogram. Separation of the labeled and unlabeled oligomer was afforded by utilizing HFIP and TEA as mobile phase additives. HFIP is purported to reduce the number of charged states on the oligomer (30), while TEA aids in the chromatographic separation of phosphate bearing molecules through an ionpairing mechanism (31, 32). MS analysis of the HPLC-purified NBHA-AP oligomer product measured an [M - 5H]5- of 681.8, in agreement with the calculated value. MS results for the uracilcontaining oligomer, AP oligomer, and the labeled AP oligomer are shown in Figure 5B-D, respectively. Chromatographic results of the enzymatic digestion of the NBHA-labeled AP oligomer with DNase I (1.5 h), nuclease P1 (3 h), and PDase I (10 h) are shown in Figure 6A. As shown, along with the four unmodified mononucleotides, 5′-dRp-NBHA is clearly present and has an identical retention time (11 min) to that of the 5′-dRp-NBHA standard. In addition, as shown in the inset, the online UV spectrum (solid line) clearly matches that of the standard (dotted line), and MS/MS (part B) confirms the identity of the 11 min peak as 5′-dRp-NBHA with an

[M - H]- at 365 and characteristic fragment ions at m/z 210, 97, and 79. Identification of AP Sites in CT DNA. Using previously described conditions, one AP site in 104 bases was generated in CT DNA. Once AP lesions were generated, sites were labeled with NBHA (see Materials and Methods section), and the NBHA-labeled AP DNA was ethanol extracted and washed to remove any unreacted NBHA. Digestion of the labeled AP DNA was accomplished using conditions similar to those of the AP oligomer (see above). HPLC-UV results of the enzyme digestion are shown in Figure 7. As seen in part A, in comparison to the standard (chromatogram b), there is clear evidence of 5′-dRpNBHA in chromatogram a with the corresponding peak at approximately 13 min. Furthermore, as shown in the inset, the online UV spectra from each standard (dotted line) are identical to that from the chromatogram of the DNA digest (solid line). However, complete identification is shown in part B of the figure where MS/MS results show that the 13 min peak of the digest has the indicative [M - H]- and fragments of 5′-dRp-NBHA. Interestingly, the ratio of the E/Z isomers in CT DNA does not match that of the 5′-dRp-NBHA standard, suggesting an energetic predominance for a particular isomer in intact DNA. With the relatively large concentration of undamaged mononucleotides present and other additives associated with enzyme digestion, a control sample where no NBHA was added was chromatographed to rule out the possibility of a similarly eluting peak at 13 min. With HPLC-UV, there were no detectable background species in this elution window (data not shown). To attain sufficient detectability under online HPLC-ESI-MS/ MS detection conditions, tC18 SPE was successfully utilized to reduce the large levels of background mononucleoties and other undesired digestion components that are incompatible with low-level HPLC-ESI-MS/MS analyses (data not shown). Optimization of the above methodology revealed that 1.0 mM final concentration of NBHA was sufficient for 5.0 mg of CT


Roberts et al.

m/z 368 f 97 (chromatogram d). Coelution of 5′-dRp-NBHA and 13C-5′-dRp-NBHA was easily observed at 6.6 min. Interesting, as shown in chromatogram b, an unknown substance was revealed at 6.9 min when monitoring the 363 f 97 of 5′-dRpNBHA. Regardless, the significant shift in retention time excluded this substance from being 5′-dRp-NBHA or internal standard; therefore, no attempts were made to identify the substance. Under these conditions, a quantification limit was established with 100 µg of DNA to be 100 fmol (three AP sites per 107 bases) as per the calibration data established from serial dilutions of the labeled AP oligo.

Conclusion Figure 8. HPLC-ESI-MS/MS results for limits of detection of 5′-dRpNBHA by monitoring transitions from m/z 363 f 79 (chromatogram a), m/z 363 f 97 (chromatogram b), and for the 13C-5′-dRp-NBHA internal standard, m/z 368 f 79 (chromatogram c) and m/z 368 f 97 (chromatogram d). HPLC-MS/MS conditions: Phenomenex Synergi Hydro-RP 1 mm × 150 mm column using (A) 2.5 mM ammonium acetate and (B) acetonitrile containing 2.5 mM ammonium acetate (pH 6.8), 100% A for 1 min, and then to 100% B at 10 min at a flow rate of 50 µL/min; injection volume of 5 µL; MicroMass Ultima Quattro Triple Quadrupole ESI MSCapillary voltage, 3.00 kV; cone voltage, 40 V; hex 1 and hex 2, 0 V; desolvation temperature, 300 °C; source temperature, 110 °C; cone gas, 70 L/h; desolvation gas, 500 L/h; collision, 21 V; and CID gas, argon at 2.5 mTorr.

DNA and that labeling of AP sites was complete within 30 min when incubated at 37 °C. In addition, it was evidenced that optimal recovery of 5′-dRp-NBHA was achieved under these conditions (e.g., 5.0 mg of DNA) when the PDase I concentration was increased by a factor of 10, from 0.4 units previously reported (29), to 4.0 units. This is likely due to the refractory nature of the 5′-dRp-NBHA subunit within the DNA structure. Moreover, increasing the PDase I digestion period from 10 to 13 h further maximized the release of 5′-dRp-NBHA, although no increase was observed beyond 13 h. In addition, 5′-dRpNBHA demonstrated no degradation under these conditions when digestions were carried out to 24 h (data not shown). 5′-dRp-NBHA Limit of Detection. In studies using commercial CT DNA starting material (5.0 mg), the background level of AP sites detected in negative control samples (DNA pretreated with methoxyamine or NaBH4 to mask preexisting AP sites) was two AP sites in 106 bases by HPLC-ESI-MS/MS using 13C-5′-dRp-NBHA as the internal standard (data not shown). This background level of AP sites could be attributed to a variety of sources including ethanol precipitation/washing steps used for removing the pretreatment agents, dissolved oxygen in the reaction milieu, or from water hydrolysis during the 30 min of incubation with NBHA at 37 °C. Attempts to generate controls of commercial DNA with absolute zero levels of AP sites were unsuccessful. However, absolute detection levels were determined by means of isotope dilution using a series of NBHA-AP oligomer dilutions with CT DNA at AP damage levels of one AP site per 104-107 bases, giving a linear response (R2 > 0.99) as quantified with 13C-5′-dRp-NBHA as the internal standard (see Materials and Methods). Quantification by isotope dilution has the distinct advantage of utilizing an internal standard with a nearly identical MS response to that of the analyte, which provides a high degree of certainty in quantitative determinations. As an example, Figure 8 shows chromatograms that were generated for one AP site in 106 bases by monitoring transitions for 5′-dRp-NBHA from m/z 363 f 79 (chromatogram a) and m/z 363 f 97 (chromatogram b) and for the 13C-5′-dRp-NBHA internal standard using m/z 368 f 79 (chromatogram c) and

We have successfully developed a method for unambiguous identification and quantification of AP sites by HPLC-UV and HPLC-ESI-MS/MS. AP sites were readily detectable in model systems of mononucleotides, a bis-phospho-dG species, a uracilcontaining oligomer, and CT DNA. Optimal conditions were established for labeling the AP site, digesting the site to the monophosphate level, and then detection with HPLC-UV and HPLC-ESI-MS/MS. By combining retention time information provided by HPLC, the optical signature of online UV detection, and the unequivocal information of MS and MS/MS fragmentation, this method has the distinct advantage of authenticating AP site formation by measuring the direct presence of 5′-dRpNBHA rather than indirect evidence from ELISA type methodologies used thus far. Moreover, this method utilizes a relatively inexpensive labeling agent, NBHA, which also stabilizes the AP site and aids in detection. What is more, due to its relatively small size, it is believed that this labeling agent will have relatively little hindrance to the AP site as compared to other large-sized fluorescent AP site labels previously studied (15). The latter is the subject of future investigations. Further studies will also involve controlled in vitro and in vivo studies of rat liver/kidney from animals exposed to chemical carcinogens with their suspected mode-of-action being linked to the formation of apurinic sites, e.g., dibenzo[a,l]pyrene (9). Of particular concern will be the issue of artifactual formation of AP sites from the testing methodology itself. Although the current background level of artifactual AP sites that we have observed is very low at two AP sites per 106 nucleotides with commercial DNA treated with methoxyamine, the use of alternative DNA handling procedures and the labeling of AP sites prior to isolation of the nuclear pellet will likely reduce these values even further. Moreover, further studies will include consideration into the potential bias of AP site determinations from potential loss of AP sites due to hydrolysis/βelimination and to the creation of AP sites from spontaneous removal of unstable DNA bases. It is anticipated that this methodology will be useful in determining precarcinogenic AP levels that may only be slightly elevated above basal, when small amounts of DNA are available. Moreover, the versatility of this approach will likely aid in the basic studies of AP site formation and repair as related to the fundamental elucidation of base excision repair. Acknowledgment. We thank Dr. Fred Kadlubar for his guidance in synthesizing 5′,3′-pdGp. We also thank Dr. Richard Beger for his NMR analysis of the 5′-dRp-NBHA standard, and we thank Mr. Ricky Holland for his assistance with the mass spectrometry. In addition, we thank the Office of Research at the University of Tulsa for their financial support of this research. The views presented in this article do not necessarily represent those views of the U.S. Food and Drug Administration.



References

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(1) Loeb, L. A., and Preston, B. D. (1986) Mutagenesis by apurinic/ apyrimidinic sites. Annu. ReV. Genet. 20, 201-230. (2) Lindahl, T. (1990) Repair of intrinsic DNA lesions. Mutat. Res. 238, 305-311. (3) Kow, Y. W., and Dare, A. (2000) Detection of abasic sites and oxidative DNA base damage using an ELISA- like assay. Methods (San Diego, CA, U.S.) 22, 164-169. (4) Kunkel, T. A., Schaaper, R. M., James, E., and Loeb, L. A. (1983) Infidelity of DNA replication as a basis of mutagenesis. Basic Life Sci. 23, 63-82. (5) Helbock, H. J., Beckman, K. B., Shigenaga, M. K., Walter, P. B., Woodall, A. A., Yeo, H. C., and Ames, B. N. (1998) DNA oxidation matters: The HPLC-electrochemical detection assay of 8-oxo-deoxyguanosine and 8-oxo-guanine. Proc. Natl. Acad. Sci. U.S.A. 95, 288293. (6) Nakamura, J., and Swenberg, J. A. (1999) Endogenous apurinic/ apyrimidinic sites in genomic DNA of mammalian tissues. Cancer Res. 59, 2522-2526. (7) Devanesan, P. D., RamaKrishna, N. V., Padmavathi, N. S., Higginbotham, S., Rogan, E. G., Cavalieri, E. L., Marsch, G. A., Jankowiak, R., and Small, G. J. (1993) Identification and quantitation of 7,12dimethylbenz[a]anthracene-DNA adducts formed in mouse skin. Chem. Res. Toxicol. 6, 364-371. (8) Chakravarti, D., Mailander, P. C., Li, K.-M., Higginbotham, S., Zhang, H. L., Gross, M. L., Meza, J. L., Cavalieri, E. L., and Rogan, E. G. (2001) Evidence that a burst of DNA depurination in SENCAR mouse skin induces error-prone repair and forms mutations in the H-ras gene. Oncogene 20, 7945-7953. (9) Chakravarti, D., Mailander, P. C., Cavalieri, E. L., and Rogan, E. G. (2000) Evidence that error-prone DNA repair converts dibenzo[a,l]pyrene-induced depurinating lesions into mutations: formation, clonal proliferation and regression of initiated cells carrying H-ras oncogene mutations in early preneoplasia. Mutat. Res. 456, 17-32. (10) Coombs, M. M., and Livingston, D. C. (1969) Reaction of apurinic acid with aldehyde reagents. Biochim. Biophys. Acta 174, 161-173. (11) Talpaert-Borle, M., and Liuzzi, M. (1983) Reaction of apurinic/ apyrimidinic sites with [14C]methoxyamine. A method for the quantitative assay of AP sites in DNA. Biochim. Biophys. Acta 740, 410-416. (12) Zhou, X., Liberman, R. G., Skipper, P. L., Margolin, Y., Tannenbaum, S. R., and Dedon, P. C. (2005) Quantification of DNA strand breaks and abasic sites by oxime derivatization and accelerator mass spectrometry: Application to γ-radiation and peroxynitrite. Anal. Biochem. 343, 84-92. (13) Weinfeld, M., Liuzzi, M., and Paterson, M. C. (1990) Response of phage T4 polynucleotide kinase toward dinucleotides containing apurinic sites: Design of a 32P-postlabeling assay for apurinic sites in DNA. Biochemistry 29, 1737-1743. (14) Fkyerat, A., Demeunynck, M., Constant, J. F., Michon, P., and Lhomme, J. (1993) A new class of artificial nucleases that recognize and cleave apurinic sites in DNA with great selectivity and efficiency. J. Am. Chem. Soc. 115, 9952-9959. (15) Boturyn, D., Constant, J. F., Defrancq, E., Lhomme, J., Barbin, A., and Wild, C. P. (1999) A simple and sensitive method for in vitro quantitation of abasic sites in DNA. Chem. Res. Toxicol. 12, 476482. (16) Chen, B. X., Kubo, K., Ide, H., Erlanger, B. F., Wallace, S. S., and Kow, Y. W. (1992) Properties of a monoclonal antibody for the detection of abasic sites, a common DNA lesion. Mutat. Res. 273, 253-261. (17) Sun, H. B., Qian, L., and Yokota, H. (2001) Detection of abasic sites on individual DNA molecules using atomic force microscopy. Anal. Chem. 73, 2229-2232.

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Apyrimidinic Lesions in DNA with High

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