Quantitative Measurement of DNA Adducts Using Neutral Hydrolysis

Department of Pharmacology, University of Connecticut Health Center, Farmington, Connecticut 06032. Neutral hydrolysis and LCrMS/MS analysis of 6-nm-t...
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Anal. Chem. 2005, 77, 2056-2062

Quantitative Measurement of DNA Adducts Using Neutral Hydrolysis and LC-MS. Validation of Genotoxicity Sensors Maricar Tarun† and James F. Rusling*,†,‡

Department of Chemistry, 55 North Eagleville Road, University of Connecticut, Storrs, Connecticut 06269-3060, and Department of Pharmacology, University of Connecticut Health Center, Farmington, Connecticut 06032

Neutral hydrolysis and LC-MS/MS analysis of 6-nm-thick DNA-polyion films used in voltammetric genotoxicity screening sensors showed that concentrations of N7guanine DNA adducts with methyl methanesulfonate and styrene oxide increased with incubation time with the same trends as found for sensor response. Results show that the genotoxicity sensors can be used to estimate relative DNA damage rates for chemical toxicity screening. Neutral thermal hydrolysis provided a relatively clean sample matrix allowing quantitative estimates of nucleobase adducts after several minutes of incubation with damage agents. In addition, an approximate standardization procedure for neutral thermal hydrolysis was developed and validated that avoids need for a pure standard and should be useful in cases where nucleobase adduct standards are unavailable or where their identities are unknown. The large number of new drug and agricultural chemical candidates synthesized each year must be screened for potential human toxicity before commercialization, placing a huge burden on time-consuming, expensive biological testing. Clearly, rapid, inexpensive, in vitro methods to screen new chemicals for toxicity at early stages of discovery would be very useful. Genotoxicity is a major toxic mechanism that involves the reaction of an electrophilic site in the molecule with a nucleobase in DNA, often producing a covalently bound DNA adduct.1-4 Nucleobase adducts may initiate complex processes leading to mutagenesis and carcinogenesis.5 Thus, detection of DNA adducts is an important tool for predicting human cancer risk.6 Conventional methods include 32P-postlabeling with detection limits approaching 1 * Corresponding author. E-mail: [email protected]. † University of Connecticut. ‡ University of Connecticut Health Center. (1) Singer, B.; Grunberger, D. Molecular Biology of Mutagens and Carcinogens; Plenum: New York, 1983. (2) Farmer, P. B. Toxicol. Lett. 2004, 149, 3-9. (3) McConnell, E. E.; Swenberg, J. A. CRC Crit. Rev. Toxicol. 1994, 24, S49S55. (4) Nestmann, E. R.; Bryant, D. W.; Carr, C. J.; Fennell, T. T.; Gorelick, N. J.; Gallagher, J. E.; Swenberg, J. A.; Williams, G. M. Regul. Toxicol. Pharmacol. 1996, 24, 9-18. (5) Vodicka, P.; Koskinen, M.; Arand, M.; Oesch, F.; Hemminki, K. Mutat. Res. 2002, 551, 239-254. (6) Hemminki, K.; Koskinen, M.; Rajaniemi, H.; Zhao, C. Regul. Toxicol. Pharmacol. 2002, 32, 264-275.

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adduct/1010 unmodified DNA bases,7 and liquid chromatography (LC) coupled to electrospray ionization mass spectrometry (LCMS)8 for detection in the picogram range (1 adduct in 107-109 bases7).9 32P-Postlabeling gives no structural information and employs radioactive phosphorus, complex enzyme reactions, and variable labeling efficiency.10 On the other hand, LC-MS offers excellent sensitivity and selectivity, especially in the single ion recording (SIR), multiple reaction monitoring (MRM), and single reaction monitoring modes. Collision-induced dissociation (CID) can be used for structural identification. LC-MS enables quantitative determination and structural characterization of DNA adducts. Niessen,9 Andrews et al.,8 and Koc and Swenberg11 have recently reviewed the analysis of DNA adducts by MS. Applications include quantitation of nucleobase adducts with ethylene oxide,29 2-amino3,4-dimethylimidazo[4,5-f]quinoxaline,12 acetaldehyde,13 butadi(7) Turesky, R. J.; Vouros, P. J. Chromatogr., B 2004, 802, 155-166. (8) Andrews, C. L.; Vouros, P.; Harsch, A. J. Chromatogr., A 1999, 856, 515526. (9) Niessen, W. M. A. J. Chromatogr., A 1999, 856, 179-197. (10) Inagaki, S.; Esaka, Y.; Deyashiki, Y.; Sako, M.; Goto, M. J. Chromatogr., B 2003, 987, 341-347. (11) Koc, H.; Swenberg, J. A. J. Chromatogr., B 2002, 778, 323-343. (12) Paehler, H.; Richoz, J.; Soglia, J.; Vouros, P.; Turesky, R. J. Chem. Res. Toxicol. 2002, 15, 551-561. (13) Inagaki, S.; Esaka, Y.; Deyashiki, Y.; Sako, M.; Goto, M. J. Chromatogr., B 2003, 987, 341-347. (14) Koc, H.; Tretyakova, N. T.; Walker, V. E.; Henderson, R. F.; Swenberg, J. A. Chem. Res. Toxicol. 1999, 12, 566-574. (15) Doerge, D. R.; Churchwell, M. I.; Fang, J.-L.; Beland, F. A. Chem. Res. Toxicol. 2000, 13, 1259-1264. (16) da Costa, G. G.; Marques, M. M.; Beland, F. A.; Freeman, J. P.; Churchwell, M. I.; Doerge, D. R. Chem. Res. Toxicol. 2003, 16, 357-366. (17) van den Driessche, B.; Lemiere, F.; van Dongen, W.; Esmans, E. L. J. Chromatogr., B 2003, 785, 21-37. (18) Churchwell, M. I.; Beland, F. A.; Doerge, D. R. Chem. Res. Toxicol. 2002, 15, 1295-1301. (19) (a) Zhou, L.; Rusling, J. F. Anal. Chem. 2001, 73, 4780-4786. (b) Yang, J.; Zhang, Z.; Rusling, J. F. Electroanalysis 2002, 14, 1494-1500. (20) Mugweru, A.; Rusling, J. F. Anal. Chem. 2002, 74, 4044-4049. (21) Zhou, L.; Yang, J,; Estavillo, C.; Stuart, J. D.; Schenkman, J. B.; Rusling, J. F. J. Am. Chem. Soc. 2003, 125, 1431-1436. (22) (a) Wang, B.; Rusling, J. F. Anal. Chem. 2003, 75, 4229-4235. (b) Rusling, J. F. Biosens. Bioelectron. 2004, 20, 1022-1028. (23) (a) Johnston, D. H.; Glasgow, K. C.; Thorp, H. H. J. Am. Chem. Soc. 1995, 117, 8933-8938. (b) Ontko, A. C.; Armistead, P. M.; Kircus, S. R.; Thorp, H. H. Inorg. Chem. 1999, 38, 1842-1846. (c) Yang, I. V.; Thorp, H. H. Inorg. Chem. 2000, 39, 4969-4976. (24) Humphreys, W. G.; Kim, D.-H.; Guengerich, F. P. Chem. Res. Toxicol. 1991, 4, 445-453. (25) Mathison, B. H.; Murphy, S. E.; Shank, R. C. Toxicol. Appl. Pharmacol. 1994, 127, 91-98. 10.1021/ac048283r CCC: $30.25

© 2005 American Chemical Society Published on Web 02/15/2005

ene,14 urethane,15 tamoxifen,16 melphalan,17 and heterocyclic aromatic amines.7 DNA adducts formed by oxidative stress were identified using MRM.18 We recently described prototype sensors designed for simple, inexpensive, rapid screening of chemical toxicity based on electrochemical measurement of DNA damage by a chemical or its metabolites.19-22 In the most sophisticated version, ultrathin films of double-stranded (ds)-DNA and enzymes assembled on pyrolytic graphite (PG) electrodes produce metabolites from the test chemical via enzyme catalysis, and then catalytic square wave voltammetry (SWV) is used to detect DNA damage resulting from the metabolites.21,22 Voltammetric oxidation of guanines23 in DNA is catalyzed by soluble Ru(bpy)32+ or poly(vinylpyridine)-Ru(bpy)22+ metallopolymers in the film. Alternatively, DNA-polyion films can be incubated with compounds that react directly with DNA. The initial goal of the work described herein was quantitative confirmation that DNA damage rates were in fact being measured by our toxicity sensors. Our previous work used total DNA hydrolysis with capillary electrophoresis19 and LC-MS21 to confirm that nucleobase adducts were indeed formed with damage agents such as styrene oxide and methylating agents under sensor incubation conditions. However, the sensitivity of the separation methods was less than that of the toxicity sensors, and we were unable to compare relative DNA damage rates with sensor responses on the same time scale. Compared to complete hydrolysis of DNA before analysis, neutral thermal hydrolysis greatly improves LC-MS sensitivity for the nucleobase adducts so that meaningful measurements can be made after 5 min of DNA incubation, comparable to the time scale on which our sensor signals develop. Neutral thermal hydrolysis selectively releases guanine adducts alkylated at the N7 position and adenine adducts alkylated at N3 by simply heating DNA at neutral pH and has been used to detect a variety of DNA adducts.14,24-34 In complete acid hydrolysis of DNA, the phosphodiester bond undergoes hydrolysis, accompanied by the cleavage of the N-glycosidic bond between the base and the deoxyribose. The hydrolysate contains phosphoric acid, deoxyribose, and all damaged and undamaged nucleobases. On the other hand, neutral thermal hydrolysis involves only the breaking of glycosidic bonds between chemically altered purine bases and (26) Rios-Blancos, M. N.; Plna, K.; Faller, T.; Kessler, W.; Hakanson, K.; Kreuzer, P. E.; Ranasinghe, A.; Filser, J. G.; Segerback, D.; Swenberg, J. A. Mutat. Res. 1997, 380, 179-197. (27) Pauwels, W.; Veulemans, H. Mutat. Res. 1998, 418, 21-33. (28) (a) Tretyakova, N.; Lin, Y.; Sangaiah, R.; Upton, P. B.; Swenberg, J. A. Carcinogenesis 1997, 18, 137-147. (b) Tretyakova, N. T.; Sangaiah, R.; Yen, T.-Y.; Swenberg, J. A. Chem. Res. Toxicol. 1997, 10, 779-785. (29) Leclercq, L.; Laurent, C.; DePauw, E. Anal. Chem. 1997, 69, 1952-1955. (30) Ham, A.-J. L.; Ranasinghe, A,; Morinello, J.; Nakamura, J.; Upton, P. B.; Johnson, F.; Swenberg, J. A. Chem. Res. Toxicol. 1999, 12, 1240-1246. (31) Kelly, J. D.; Shah, D.; Chen, F.; Wurderman, R.; Gold, B. Chem. Res. Toxicol. 1998, 11, 1481-1486. (32) (a) Wang, M.; Upadhyaya, P.; Dinh, T. T.; Bonilla, L. E.; Hecht, S. S. Chem. Res. Toxicol. 1998, 11, 1567-1573. (b) Wang, M.; McIntee, E. J.; Shi, Y,; Cheng, G.; Upadhyaya, P.; Villalta, P. W.; Hecht, S. S. Chem. Res. Toxicol. 2001, 14, 1435-1445. (33) (a) Upadhyaya, P.; Sturla, S. J.; Tretyakova, N.; Ziegel, R.; Villalta, P. W.; Wang, M.; Hecht, S. S. Chem. Res. Toxicol. 2003, 16, 180-190. (b) Wang, M.; Cheng, G.; Shi, Y.; McIntee, E. J.; Villalta, P. W.; Upadhyaya, P.; Hecht, S. S. Chem. Res. Toxicol. 2003, 16, 616-626. (34) Booth, E. D.; Kilgour, J. D.; Robinson, S. A.; Watson, W. P. Chem.-Biol. Interact. 2004, 147, 195-211.

deoxyribose moieties. This provides a hydrolysate with a large fraction of nucleobase adducts that can be determined using LC with electrospray ionization (ES) mass spectrometry. In this paper, we combine the simplicity and specificity of neutral thermal hydrolysis with the selectivity of LC-ES-MS to determine N7-guanine adducts in ultrathin sensor films of polycations and DNA assembled or carbon cloth after they were incubated with styrene oxide or methyl methanesulfonate (MMS). We incubated the DNA films with damaging agents under the same conditions as in sensor experiments and then determined the DNA adducts formed in the film using LC-MS after neutral hydrolysis. Results confirmed the formation of N7-guanine adducts at similar relative rates as signal increases detected by the toxicity sensors. We also present a semiquantitative standardization method that may be used for the quantitation of DNA adducts for which standards are not available or are unknown. EXPERIMENTAL SECTION Chemicals. Double-stranded salmon testes (st) DNA (∼2K base pairs, 41.2% G/C) and 7-N-methylguanine were from Sigma. Poly(diallyldimethylammonium chloride), styrene oxide (SO), MMS, formic acid, and tris(2,2′-bipyridyl)dichlororuthenium(II) hexahydrate were from Aldrich. All other chemicals used were analytical reagent grade. Neutral Thermal Hydrolysis. Hydrolysis times were optimized to provide large amounts of adducts compared to unreacted guanine and adenine. Aqueous 2 mg mL-1 DNA solutions were incubated with SO (1% dispersion by volume) or 2 mM MMS for 30 min and then heated at 100 °C for different times. The damaged DNA was preconcentrated using Centricon filters with cutoff mass of 30 000 (Amicon, Beverly, MA), and the resulting solution was injected onto the LC. The efficiency of the hydrolysis was assessed by comparing the amounts of adducts in the neutral hydrolysate and the retentate as measured by LC. After neutral thermal hydrolysis and subsequent filtration, the retentate was subjected to complete acid hydrolysis using formic acid and heating to 140 °C for 30 min.19 This procedure released the remaining adducts and unreacted DNA bases in the retentate, which was then analyzed by LC. These studies resulted in the optimized hydrolysis times below. DNA-polyion films were immersed in water after incubation and submitted to neutral thermal hydrolysis by boiling for 15 min for SO incubations and 7 min for the MMS incubations. The hydrolysate was filtered, preconcentrated, and then analyzed by LC-MS. Film Assembly. Alternate layers of PDDA and DNA were assembled on carbon cloth by electrostatic adsorption.35,36 A 10 cm × 10 cm carbon cloth (graphitized spun yarn carbon fabric, Zoltec) was washed with water, immersed in acetone for 15 min, and ultrasonicated for 15 min. The carbon cloth was then (35) Lvov, Y. M.; Lu, Z.; Schenkman, J. B.; Zu, X.; Rusling, J. F. J. Am. Chem. Soc. 1998, 120, 4073-4080. (36) (a) Lvov, Y. In Protein Architecture: Interfacing Molecular Assemblies and Immobilization Biotechnology; Lvov, Y., Mohwald, H., Eds.; Marcel Dekker: New York, 2000; pp 125-167. (b) Rusling, J. F. In Protein Architecture: Interfacing Molecular Assemblies and Immobilization Biotechnology; Lvov, Y., Mohwald, H., Eds.; Marcel Dekker: New York, 2000; pp 337-354. (c) Lvov, Y. In Handbook Of Surfaces And Interfaces Of Materials, Vol. 3. Nanostructured Materials, Micelles and Colloids; Nalwa, R. W., Ed.; Academic Press: San Diego, 2001; pp 170-189.

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alternately dipped into solutions of 2 mg mL-1 PDDA (+ 50 mm NaCl) and 2 mg mL-1 ds-DNA in pH 7.1 TRIS buffer and washed with water between adsorption steps to make (PDDA/DNA)2 films of ∼6-nm nominal thickness as estimated previously.19 Incubation of Films with DNA Damaging Agents. Safety note: Styrene oxide and methyl methanesulfonate are suspected carcinogens. All procedures were done while wearing gloves and under closed hoods. Films were incubated in stirred vessels at 37 °C containing saturated SO (1% dispersion by volume, 85 mM) or 2 mM MMS. SO incubations were done at pH 5.5, the pH at which the reaction rate between SO and DNA was found to be maximum.37,38 MMS incubations were at pH 6.5.22 Films were washed by dipping in ethyl acetate for the SO incubation, and in phosphate buffer for MMS incubations, before being subjected to neutral thermal hydrolysis. Similar methods were used to construct films on ordinary PG.19 Liquid Chromatography-Mass Spectrometry. High-pressure liquid chromatography (Perkin-Elmer) with diode array detection was done using an Ultra C18 (Restek) reversed-phase column (250 × 4.6 mm, particle size 5 µm). A binary gradient consisting of 10 mM ammonium acetate, pH 5, and methanol was used for SO nucleobase adducts, and 10 mM ammonium acetate pH 5 and 50:50 methanol/water for MMS adducts, both at the rate of 0.7 mL min-1. A microsplitter valve (Upchurch Scientific) delivered 5% of the flow to the mass spectrometer. Injection volume was 20 µL. Electrospray ionization mass spectrometry (ESI-MS) employed a Quattro II (Micromass) with an electrospray source. Ion source temperature was 120 °C, with nitrogen as nebulizing gas. Spectra were obtained at a low cone voltage (15 kV) in the positive ion mode (ES+). Identification of adduct peaks was done using the tandem mass spectrometry methods termed MRM and daughter ion analysis (DAU) at cone voltage of 15 V, collision energy of 15 eV, and collision gas (Ar) pressure 5 × 10-3 mbar. Quantitation of adducts was done by integrating SIR peaks. Standardization for Quantitation of N7-SO-Gua. The following standardization procedure allowed us to estimate amounts of the N7-guanine adducts in the absence of a pure standard. We employed the MMS adduct for validation since a N-7methylguanine (N7-metGua) standard was commercially available for comparison. Thus, we prepared a stock solution of N7metGua by incubating MMS and guanine for 8 h at 37 °C. At the end of the incubation, we assumed that

amount of N7-metGua adduct formed ) initial amount of Gua - final amount of Gua N7-metGua adducts were identified using LC-MS/MS. Various concentrations of this N7-metGua “standard” from the reaction solution were prepared and injected onto LC-MS. Peaks were integrated using SIR mode at m/z 166 [N7-metGua + H+]. Solutions with the same concentrations were prepared from the authentic N7-metGua, and SIR peak areas were integrated for comparison. We effected a similar procedure to quantitate the SO adduct N7-SO-Gua. A stock solution of SO-Gua was obtained by (37) Mbindyo, J.; Zhou, L.; Zhang, Z.; Stuart, J. D.; Rusling, J. F. Anal. Chem. 2000, 72, 2059-2065. (38) Rusling, J. F.; Zhou, L.; Munge, B.; Yang, J.; Estavillo, C.; Schenkman, J. B. Faraday Discuss. 2000, 116, 77-87.

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incubating guanine in SO for 8 h at 37 °C. Assuming that the only reaction that occurred was the formation of adducts with guanine, then

amount of SO-Gua adduct formed ) initial amount of Gua - final amount of Gua SO-Gua peaks were verified using tandem MS. SO-Gua solutions of different concentrations were analyzed by LC-MS under the SIR mode, with m/z 272 (protonated SO-Gua). Comparative samples were also analyzed after total acid hydrolysis of the DNA.19 Voltammetry. The procedure for voltammetric sensors was described previously.19 Styrene oxide incubations of the sensors were done in 10 mM acetate buffer + 50 mM NaCl at pH 5.5. MMS incubations were done with 10 mM phosphate buffer + 50 mM NaCl, pH 6.5. SWV was done with (PDDA/DNA)2 films on both carbon cloth (1 cm × 1 cm) and PG (Advanced Ceramics, A ) 0.16 cm2). RESULTS Optimization of Neutral Thermal Hydrolysis. Heating DNA samples to 100 °C for long periods releases not only adducts of guanine at the N7 position and adenine adducts at the N3 position but unreacted guanine and adenine as well. Neutral thermal hydrolysis at 100 °C for 15 min gave large fractions of nucleobase SO adducts with the smallest amount of unreacted guanine and adenine. For MMS, an optimized hydrolysis was achieved by heating at 100 °C for 7 min. We determined the efficiency of hydrolysis, defined as

% efficiency ) 100 × amount of N7-Gua adduct in filtrate of neutral hydrolysate total amount of N7-gua adduct After neutral thermal hydrolysis, filtration of the neutral hydrolysate through MW cutoff filter of 30 000 resulted in filtrate that contains predominantlyr N7-guanine and N3-adenine adducts. The retentate contains the depurinated DNA backbone. Total acid hydrolysis of the retentate was used to release all remaining adducts and unreacted bases. The total amount of N7-guanine adduct was obtained as the sum of the N7 adducts in the filtrate and the hydrolyzed retentate. For SO, the efficiency was 94 ( 2%. That is, most of the N7-SO-Gua adduct was released by the neutral hydrolysis. The efficiency for MMS was 45 ( 4%. Hydrolysis of the MMS-incubated samples for 15 min gave higher efficiency, but also significant amounts of unreacted nucleobases bases, which was undesirable. Identification of Adducts in Films by LC-MS. DNApolyion films on carbon cloth incubated in MMS and SO solutions were submitted to neutral thermal hydrolysis. We analyzed the hydrolysate for the N7-guanine adducts, which are the major DNA adducts for MMS and SO.39-41 CID of authentic N7-metGua gave a major fragment of m/z 149 corresponding to the loss of NH3, the same as for an MMS-incubated DNA neutral hydrolysate (Figure 1). The MRM transition m/z 166 (N7-metGua) f 149 (Gua) for the MMS-incubated neutral hydrolysate gave a peak at (39) Vodicaka, P.; Hemminki, K. Carcinogenesis 1988, 9, 1657-1660.

Figure 2. Partial LC-MS/MS chromatogram of 100 mM N7metGua and neutral hydrolysate of DNA film incubated in 2 mM MMS at 37 °C for 30 min. MRM for the transition m/z 166 (N7-metGua) f 149 (Gua).

Figure 1. Identification of N7-metGua adduct in DNA films incubated with MMS. (A) LC-MS/MS chromatogram of neutral hydrolysate from DNA film incubated in 2 mM MMS at 37 °C for 30 min (DAU, parent ion m/z 166, collision energy 15 eV). Collection of MS data started 20 min after LC injection of sample. (B) Daughter ion spectrum. Major fragment is m/z 149, corresponding to [M NH3]+.

retention time within day-to-day experimental error of the authentic N7-metGua solution (Figure 2). CID daughter ion chromatograms and spectra of SO-incubated neutral hydrolysates verified the formation of N7-SO-Gua in films, is shown in Figure 3. The parent ion with m/z 272 corresponds to the protonated SO-Gua species, and the mass chromatogram showed two peaks (Figure 3A). The mass spectra (Figure 3B,C) have guanine as the main fragment, m/z 152 (corresponds to protonated guanine). MS chromatograms obtained in the SIR and MRM modes also gave two peaks. Standardization of LC-MS. A stock solution of N7-metGua was made by incubating guanine in MMS at 37 °C for 8 h. The concentration of N7-metGua was estimated as the difference between initial and final guanine concentrations, assuming that the only products are isomers of N7-metGua. Different concentrations of N7-metGua were prepared from a 1.29 mM stock solution. Similar concentrations of authentic N7-metGua were prepared and subjected to LC-MS in SIR mode, with m/z 162 (protonated N7-metGua). Peak areas of N7-metGua prepared by incubating guanine with MMS were plotted against areas for the authentic standard N7-metGua (Figure 4). The y-intercept (40) (a) Kumar, R.; Vodicka, P.; Peltoren, K.; Hemminki, K. Carcinogenesis 1997, 18, 407-414. (b) Koskinen, M.; Vodicka, P.; Hemminki, K. Chem.-Biol. Interact. 2000, 124, 13-27. (c) Koskinen, M.; Calibiro, D.; Hemminki, K. Chem.-Biol. Interact. 2000, 126, 201-213. (d) Koskinen, M.; Vodickova, L.; Vodicka, P.; Warner, S. C.; Hemminki, K. Chem.-Biol. Interact. 2001, 138, 111-124. (41) Glaab, W. E.; Tindall, K. R.; Skopek, T. R. Mutat. Res. 1999, 427, 67-78.

was 3202 ( 3180, and the slope was 0.95 ( 0.12. t-Tests at 95% confidence limits showed that the slope and intercept do not differ significantly from ideal full correlation values of 1 and 0, respectively. We conclude that there were no systematic differences between calibration plots prepared from synthesized and authentic N7-metGua. Applying the same procedure for the quantitation of N7-SOGua in DNA films, guanine solution of known concentration was reacted with SO at 37 °C for 8 h, and the concentration of guanine remaining was determined. Assuming that the only products are the different isomers of SO-Gua, the amount of SO-Gua (not just the N7 isomer) is the difference between the initial and final amounts of guanine. Our stock solution then contains unreacted guanine and the isomers of SO-Gua. Tandem mass spectrometry (MRM, transition m/z 272 (SO-Gua) f 152 (Gua)) was used to verify the SO-Gua peaks in the stock solution (Figure 5). There were five peaks that correspond to SO-Gua, consistent with previous reports identifying four isomeric forms of N7-SO-Gua as well as adducts of SO at N2 and O6 positions.39,40 The stock solution used to make calibration plots was 0.856 mM SO-Gua. N7-metGua and N7-SO-Gua Adducts in DNA Films. DNA films incubated with MMS and SO showed increasing SIR peak areas (m/z 162 for N7-metGua, m/z 272 for N7-SO-Gua) with increasing incubation time (Figure 6). To quantitate the amounts of these adducts, standard solutions of N7-SO-Gua and N7-metGua were used to make calibration plots. For both MMS and SO, the amount of N7-guanine adduct increased with increasing incubation time (Figure 7). Controls films incubated in buffer without MMS or SO did not show any peaks. Voltammetry of Damaged DNA Films. We previously showed that voltammetry employing Ru(bpy)32+ as an oxidation catalyst can be used to detect DNA damage in (PDDA/DNA)2 films on PG electrodes.19,22 Since our LC-MS analyses focused on (PDDA/DNA)2 films on carbon cloth to obtain a large surface area for the films, we wanted to confirm that (PDDA/DNA)2 films on PG and carbon cloth gave similar detection trends upon incubation with damage agent. Figures S1 and S2 (Supporting Information) show that SWV of DNA films on carbon cloth and Analytical Chemistry, Vol. 77, No. 7, April 1, 2005

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Figure 4. SIR peak areas (m/z 162) of N7-metGua solutions prepared by incubating guanine and MMS (y-axis) plotted against authentic areas for standard N7-metGua solutions (x-axis). Error bars are smaller than circle diameters. Numbers above the points are picomoles of N7-metGua in 20-µL injections. A t-test at 95% confidence limits showed no systematic differences between the calibration plots.

Figure 3. Identification of N7-SO-Gua adduct in DNA films incubated in SO. (A) LC-MS/MS chromatogram of neutral hydrolysate from DNA film incubated in 1% SO at 37 °C for 30 min (DAU, parent ion m/z 272,collision energy 15 eV). Collection of MS data started 25 min after LC injection of sample. (B) Daughter ion spectrum of peak 1. (C) Daughter ion spectrum of peak 2. Major fragment for both peaks is m/z 152, corresponding to protonated guanine.

PG incubated with MMS or SO at 37 °C both give increasing peaks at -1.05 V versus SCE with increasing incubation times, consistent with earlier results. Backgrounds were much larger on the carbon cloth and peak increases were relatively smaller, showing that PG gives superior performance in these toxicity sensors. Figure 8 compares the amount of adduct found by LC-MS in the films with the voltammetric sensor response as final peak current after incubation divided by initial peak current of the sensor before incubation. Identical incubation conditions and times were used for the sensors and for films prepared for LC-MS. Similar increasing trends in adduct amounts from LC-MS and sensor response for both electrode materials were found. Furthermore, in the early time ranges, approximate initial slopes (up to 10 min) of the sensors versus incubation time were 0.139/min for MMS and 0.128/min for SO. Dividing by the respective concentrations of damage agents during incubation gave 0.070 2060 Analytical Chemistry, Vol. 77, No. 7, April 1, 2005

Figure 5. Partial LC-MS/MS chromatogram of 8.56 µM SO-Gua. MRM for the transition m/z 272 (SO-Gua) f 152 (Gua).

min-1 mM-1 for MMS and 0.0015 min-1 mM-1 consistent with a larger rate of damage for MMS than for SO. DISCUSSION The results in Figure 8 clearly demonstrate that voltammetric toxicity sensors based on DNA damage detection give quantitative correlations with measured concentrations of individual nucleobase adducts in DNA films incubated with damage agents. Furthermore, slopes of sensor response versus incubation time corrected for reactant concentration were 0.070 min-1 mM-1 for MMS and 0.0015 min-1 mM-1 consistent with a larger rate of DNA damage for MMS than for SO. This again correlates with the LCMS results showing (Figure 7) much larger amounts and formation rates for nucleobase adducts for MMS incubation compared to SO, also taking into account that the efficiency of neutral hydrolysis for N7-SO-Gua adducts is much better than for N7MetGua.

Figure 8. Comparisons of electrochemical toxicity sensor and LCMS data. Peak current ratio Ip,f/Ip,i for DNA damage sensors consisting of (PDDA/DNA)2 films on carbon cloth (0) and PG (b), and pmoles of nucleobase adducts found by LC-MS (O) after incubation with (A) 2 mM MMS, and (B) 1% SO (by volume about 10 mM in saturated solution). Figure 6. Partial LC-MS chromatograms of neutral hydrolysates from DNA films: (A) incubated in 2 mM MMS at 37 °C. SIR of m/z 166, corresponding to protonated N7-metGua; (B) incubated in 1% SO at 37 °C. SIR of m/z 272, corresponding to protonated SO-Gua.

Figure 7. Amounts of N7-metGua and N7-SO-Gua in DNA films incubated in 2 mM MMS and 1% SO (∼10 mM in saturated solution), respectively.

The reported LD50 for the rat was 225 mg/kg for MMS and 2000 mg/kg for SO. Further, TDLo, the lowest dose to produce carcinogenic or neoplastigenic effects, for MMS in mouse is 11 g/kg of body weight, while for SO it is 74 g/kg of body weight.42

These biological toxicity data correlate with the much greater rate of N7-metGua formation compared to N7-SO-Gua estimated by LC-MS (Figure 7) as well as DNA damage rates monitored by the sensors (Figure 8). Taken together, these results clearly demonstrate that voltammetric toxicity sensors featuring DNA films do indeed detect relative DNA damage rates. Previous work showed that SO forms several adducts with DNA, but the major adduct was N7-SO-Gua at 93% of total alkylation products.40b Thus, N7-SO-Gua is a good marker for DNA damage. Neutral thermal hydrolysis avoided interferences in the MS analysis from large amounts of intact nucleobases and increased sensitivity compared to enzyme or acid hydrolysis of DNA.19,21 These total hydrolysis procedures are very timeconsuming and result in samples containing the desired nucleobase adducts along with a large fraction of unreacted bases. Neutral thermal hydrolysis, on the other hand, selectively releases N7-guanine and N3-adenine adducts under mild conditions in 7-15 min. The N7-SO-Gua adduct is easily separated from the depurinated DNA using molecular weight 30 000 cutoff filters. The resulting filtrate can be injected directly into the LC-MS. We were able to detect and quantitate nucleobase adducts in films incubated for as short as 5 min. (42) Toxicity data from http://hazard.com/msds/that quotes original literature sources.

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We also developed a procedure that can be used to estimate DNA adducts in the absence of authentic standards. This should be especially valuable in cases where the identities of the nucleobase adducts are unknown, e.g., from unknown metabolites, or where many nucleobase adducts are formed. Stock solutions of adducts are prepared by incubating guanine (or other DNA bases) with the DNA damaging agent, and the concentration of this stock solution is estimated from the difference between initial and final guanine concentrations. Figure 4 shows that stock solutions of N7-metGua prepared in this way gave calibration plots with no systematic differences from an authentic N7metGua standard. An SO-Gua solution prepared by reaction of guanine and SO confirmed five prominent peaks by LC-MS/MS (Figure 5) for SO-Gua. This shows that there are at least five SO-Gua isomers from the reaction of guanine and SO, consistent with previous findings in which reaction of SO with deoxyguanosine monophosphate produced four isomeric forms at the N7 position, as well as adducts formed at N2 and O6 positions.40a Incubation of the films with SO followed by neutral hydrolysis gave two major N7-SOGua derivatives (Figure 6B). The identity of the adducts formed on the DNA films was verified using tandem mass spectrometry prior to quantitation. The major adduct in the reaction between DNA and MMS is N7metGua.41 The fragmentation pattern of the standard N7-metGua was consistent with previously reported pattern, the major adduct being m/z 149, which corresponds to loss of NH3.43,44 With the mass spectrometer monitoring the m/z 166 (N7-metGua) f 149 (Gua) transition, the neutral hydrolysate gave a peak that eluted at the same time as the standard N7-metGua solution, confirming (43) Porcelli, B.; Muraca, L. F.; Frosi, B.; Marinello, E.; Vernillo, R.; de Martino, A.; Catinella, S.; Traldi, P. Rapid Commun. Mass Spectrom. 1997, 11, 398404. (44) Lafaye, A.; Junot, C.; Ramounet-Le Gall, B.; Fritsch, P.; Tabet, J.-C.; Ezan, E. Rapid Commun. Mass Spectrom. 2003, 17, 2541-2549.

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the presence of N7-metGua in the neutral hydrolysate. The N7SO-Gua adduct gave a major fragment, m/z 152 (protonated guanine), that is typical of N7-alkylated guanines.28a,b Tandem mass spectrometry results showed two peaks for N7SO-Gua (Figures 3A and 6B). It is likely that these peaks correspond to the R and β isomers of N7-SO-Gua. Kumar et al.40a and Koskinen et al.40b positively identified these isomeric N7SO-Gua adducts by using 32P-postlabeling and ESI-MS, respectively. In summary, neutral hydrolysis and LC-MS/MS analysis of 6-nm-thick DNA films used in voltammetric genotoxicity sensors showed that concentrations of N7-guanine adducts with MMS and SO in the DNA increased with incubation time with the same trends as found for sensor response. Results suggest that the genotoxicity sensors as well as LC-MS/MS can be used to estimate relative DNA damage rates in the range of low picomoles of nucleobase adduct min-1. In addition, an approximate standardization procedure applicable to neutral thermal hydrolysis was reported and validated and should be useful in cases in where nucleobase adduct standards are unavailable or where their identities are unknown. ACKNOWLEDGMENT This work was supported by U.S. PHS Grant ES03154 from the National Institute of Environmental Health Sciences (NIEHS), NIH. SUPPORTING INFORMATION AVAILABLE Two additional figures presenting the peak current changes of sensors after incubations. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review November 19, 2004. Accepted January 12, 2005. AC048283R