Use of DNA Repair Enzymes in Electrochemical Detection of Damage

Electrochemical measurements at mercury or solid amal- gam electrodes offer a highly sensitive detection of DNA strand breaks. On the other hand, ...
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Anal. Chem. 2005, 77, 2920-2927

Use of DNA Repair Enzymes in Electrochemical Detection of Damage to DNA Bases in Vitro and in Cells Katerˇina Cahova´-Kucharˇı´kova´, Miroslav Fojta,* Toma´sˇ Mozga, and Emil Palecˇek

Institute of Biophysics, Academy of Sciences of the Czech Republic, Kralovopolska 135, 612 65 Brno, Czech Republic

Electrochemical measurements at mercury or solid amalgam electrodes offer a highly sensitive detection of DNA strand breaks. On the other hand, electrochemical detection of damage to DNA bases at any electrode is usually much less sensitive. In this paper, we propose a new voltammetric method for the detection of the DNA base damage based on enzymatic conversion of the damaged DNA bases to single-strand breaks (ssb), single-stranded (ss) DNA regions, or both. Supercoiled DNA exposed to UV light was specifically cleaved by T4 endonuclease V, an enzyme recognizing pyrimidine dimers, the major products of photochemical DNA damage. Apurinic sites (formed in dimethyl sulfate-modified DNA) were determined after treating the DNA with E. coli exonuclease III, an enzyme introducing ssb at the abasic sites and degrading one of the DNA strands. The ssb or ssDNA regions, or both, were detected by adsorptive transfer stripping alternating current voltammetry at the mercury electrode. This technique offers much better sensitivity and selectivity of DNA base damage detection than any other electrochemical method. It is not limited to DNA damage in vitro, but it can detect also DNA base damage induced in living bacterial cells. Interactions of DNA with chemical or physical agents occurring in the environment may result in changes of the genetic information (mutations) and subsequently in serious health disorders.1,2 Screening of DNA damage in persons exposed to potentially genotoxic agents, detection of DNA damaging species in the environment, water, food, etc., and toxicity testing of newly synthesized chemicals are therefore important for human health protection. Methods currently used in DNA damage analysis usually involve hydrolysis of the genetic material followed by chromatographic separation and determination of damaged entities.3-9 These methods, although highly sensitive, are rather laborious and time-consuming. Other approaches are therefore sought that are based on detection of changes in entire DNA molecules resulting from their interactions with genotoxic agents. Among other methods (electrophoretic techniques including plasmid relaxation10-12 or comet assays,3,5,13-18 immunoassays,8,9,19-21 * Corresponding author. Tel.: +4205 41517197. Fax: +4205 41211293. E-mail: [email protected]. (1) Friedberg, E. C. Nature 2003, 421, 436-440. (2) Scharer, O. D. Angew. Chem., Int. Ed. 2003, 42, 2946-2974.

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PCR,22 etc.), electrochemical analysis has proven useful in detecting DNA damage.23-28 DNA is an electroactive and surface-active substance yielding analytically valuable electrochemical signals.23-25 Adenine, cytosine, and guanine undergo redox processes at the mercury electrodes while guanine and adenine are oxidizable at carbon and some other solid electrodes. In addition, adsorption/desorption processes of DNA at the mercury electrodes provide distinct (3) Collins, A.; Gedik, C.; Vaughan, N.; Wood, S.; White, A.; Dubois, J.; Rees, J. F.; Loft, S.; Moller, P.; Cadet, J.; Douki, T.; Ravanat, J. L.; Sauvaigo, S.; Faure, H.; Morel, I.; Morin, M.; Epe, B.; Phoa, N.; Hartwig, A.; Schwerdtle, T.; Dolara, P.; Giovannelli, L.; Lodovici, M.; Olinski, R.; Bialkowski, K.; Foksinski, M.; Gackowski, D.; Durackova, Z.; Hlincikova, L.; Korytar, P.; Sivonova, M.; Dusinska, M.; Mislanova, C.; Vina, J.; Moller, L.; Hofer, T.; Nygren, J.; Gremaud, E.; Herbert, K.; Lunec, J.; Wild, C.; Hardie, L.; Olliver, J.; Smith, E. Free Radical Biol. Med. 2003, 34, 1089-1099. (4) Collins, A. R.; Cadet, J.; Moller, L.; Poulsen, H. E.; Vina, J. Arch. Biochem. Biophys. 2004, 423, 57-65. (5) Guetens, G.; De Boeck, G.; Highley, M.; van Oosterom, A. T.; de Bruijn, E. A. Crit. Rev. Clin. Lab. Sci. 2002, 39, 331-457. (6) England, T. G.; Jenner, A.; Aruoma, O. I.; Halliwell, B. Free Radical Res. 1998, 29, 321-330. (7) Bykov, V. J.; Kumar, R.; Forsti, A.; Hemminki, K. Carcinogenesis 1995, 16, 113-118. (8) Stiborova, M.; Rupertova, M.; Hodek, P.; Frei, E.; Schmeiser, H. H. Collect. Czech. Chem. Commun 2004, 69, 476-498. (9) Sun, X.; Nair, J.; Bartsch, H. Chem. Res. Toxicol. 2004, 17, 268-272. (10) Boublikova, P.; Vojtiskova, M.; Palecek, E. Anal. Lett. 1987, 20, 275-291. (11) Fojta, M.; Palecek, E. Anal. Chim. Acta 1997, 342, 1-12. (12) Fojta, M.; Kubicarova, T.; Palecek, E. Electroanalysis 1999, 11, 1005-1012. (13) Collins, A. R. Mol. Biotechnol. 2004, 26, 249-261. (14) Faust, F.; Kassie, F.; Knasmuller, S.; Boedecker, R. H.; Mann, M.; MerschSundermann, V. Mutat. Res. Rev. Mutat. 2004, 566, 209-229. (15) Collins, A. R. Am. J. Clin. Nutr. 2005, 81, 261S-267S. (16) Olive, P. L.; Durand, R. E.; Raleigh, J. A.; Luo, C.; Aquino-Parsons, C. Br. J. Cancer 2000, 83, 1525-1531. (17) Pouget, J. P.; Douki, T.; Richard, M. J.; Cadet, J. Chem. Res. Toxicol. 2000, 13, 541-549. (18) Cadet, J.; D’Ham, C.; Douki, T.; Pouget, J. P.; Ravanat, J. L.; Sauvaigo, S. Free Radical Res. 1998, 29, 541-550. (19) Wang, H. L.; Lu, M. L.; Mei, N.; Lee, J.; Weinfeld, M.; Le, X. C. Anal. Chim. Acta 2003, 500, 13-20. (20) Peccia, J.; Hernandez, M. Appl. Environ. Microb. 2002, 68, 2542-2549. (21) Cooke, M. S.; Podmore, I. D.; Mistry, N.; Evans, M. D.; Herbert, K. E.; Griffiths, H. R.; Lunec, J. J. Immunol. Methods 2003, 280, 125-133. (22) Kumar, A.; Tyagi, M. B.; Jha, P. N. Biochem. Biophys. Res. Commun. 2004, 318, 1025-1030. (23) Palecek, E.; Fojta, M.; Jelen, F.; Vetterl, V. In The Encyclopedia of Electrochemistry; Bard, A. J., Stratsmann, M., Eds.; Wiley-VCH: Weinheim, 2002; Vol. 9 (Bioelectrochemistry), pp 365-429. (24) Fojta, M. Electroanalysis 2002, 14, 1449-1463. (25) Fojta, M. Collect. Czech. Chem. Commun. 2004, 69, 715-747. (26) Erdem, A.; Ozsoz, M. Electroanalysis 2002, 14, 965-974. (27) Palecek, E.; Fojta, M. Anal. Chem. 2001, 73, 74A-83A. (28) Mascini, M. Pure Appl. Chem. 2001, 73, 23-30. 10.1021/ac048423x CCC: $30.25

© 2005 American Chemical Society Published on Web 03/26/2005

tensammetric peaks. Some of these signals (namely, cathodic and capacitive peaks measured at the mercury electrodes) respond sensitively to changes in the DNA structure including those related to DNA damage.23-25 We showed that formation of DNA single- or double-strand breaks can be detected using supercoiled (sc) DNA and alternating current (ac) voltammetry at the hanging mercury drop (HMDE),11,29 mercury film (MFE),30 or silver solid amalgam (AgSAE)31,32 electrodes. Ac voltammetric responses of DNAs containing free ends (i.e., the strand breaks) at these electrodes qualitatively differ from those of the scDNA whose molecules are covalently closed: while linear (lin) or open circular (oc) DNAs produce a tensammetric (ac voltammetric) peak 3, the scDNA does not.11 Thus, any scissions of the scDNA strands (caused by, for example, γ-radiation, chemical agents, or enzymes) can be sensitively monitored through the peak 3 intensity.11,12,23-25 HMDE, MFE, or AgSAE modified with scDNA can be utilized as easy-to-prepare electrochemical sensors for the DNA cleaving agents. A variety of genotoxic agents induce damage to DNA base residues.1,2 Some of the base modifications may result in formation of new electroactive species providing specific peaks different from intrinsic signals of intact DNA. For example, 8-oxoguanine (one of the most abundant products of oxidative DNA damage in vivo) has been detected via its oxidation signal at carbon electrodes.33,34 In other cases, electroactive sites of the DNA bases may be lost due to modification of the base moiety, subsequently resulting in diminishing of the respective intrinsic DNA signals (such as guanine anodic peaks at both carbon and mercury electrodes).24,25,35-39 This approach, however, exhibits inherently low sensitivity because a relatively large portion of guanine residues (exceeding standard deviation of the assay) has to be damaged to gain a significant decrease of an initially large signal. Moreover, some biologically important nucleobase modifications (such as formation of thymine dimers upon UV irradiation1,2) may not directly affect DNA electrochemical responses. Such lesions may be detected electrochemically provided that they are connected with distortions of the DNA double helix, resulting in exposure of the adjacent electroactive bases.24,25,40 This principle, combined with electrocatalytic guanine oxidation,41,42 has been utilized in (29) Fojta, M.; Kubicarova, T.; Palecek, E. Biosens. Bioelectron. 2000, 15, 107115. (30) Kubicarova, T.; Fojta, M.; Vidic, J.; Havran, L.; Palecek, E. Electroanalysis 2000, 12, 1422-1425. (31) Fadrna, R.; Cahova-Kucharikova, K.; Havran, L.; Yosypchuk, B.; Fojta, M. Electroanalysis 2005, 17, 452-459. (32) Kucharikova, K.; Novotny, L.; Yosypchuk, B.; Fojta, M. Electroanalysis 2004, 16, 410-414. (33) Langmaier, J.; Samec, Z.; Samcova, E. Electroanalysis 2003, 15, 1555-1560. (34) Brett, A. M. O.; Piedade, J. A. P.; Serrano, S. H. P. Electroanalysis 2000, 12, 969-973. (35) Mascini, M.; Palchetti, I.; Marrazza, G. Fresenius J. Anal. Chem. 2001, 369, 15-22. (36) Jelen, F.; Tomschik, M.; Palecek, E. J. Electroanal. Chem. 1997, 423, 141148. (37) Marin, D.; Perez, P.; Teijeiro, C.; Palecek, E. Biophys. Chem. 1998, 75, 8795. (38) Wang, J.; Chicharro, M.; Rivas, G.; Cai, X. H.; Dontha, N.; Farias, P. A. M.; Shiraishi, H. Anal. Chem. 1996, 68, 2251-2254. (39) Wang, J.; Rivas, G.; Ozsos, M.; Grant, D. H.; Cai, X. H.; Parrado, C. Anal. Chem. 1997, 69, 1457-1460. (40) Vorlickova, M.; Palecek, E. Int. J. Radiat. Biol. 1974, 26, 363-372. (41) Thorp, H. H. In Long-Range Charge Transfer in DNA; Topics in Current Chemistry 237; Springer-Verlag: Heidelberg, 2004; pp 159-181. (42) Popovich, N.; Thorp, H. Interface 2002, 11, 30-34.

development of a sensor for genotoxicity testing.43-45 Generally, the electrochemical determination of damaged bases in DNA is much less sensitive than determination of strand breaks by means of the above-mentioned peak 3 (affording detection of 1 strand break among ∼2 × 105 intact sugar-phosphate bonds11) due to a large background signal produced by the undamaged DNA.24,25,30,46 Conversion of damaged bases into the strand breaks (using specific DNA repair enzymes) may thus result in a great increase of sensitivity of electrochemical detection of the damaged bases. Cells possess enzymatic machineries taking care of their genetic stability.2 Damaged DNA is repaired to prevent accumulation of mutations and to preserve the DNA ability to replicate and express the genetic information. Damaged entities are usually recognized and cut out by specific enzymes (N-glycosylases, endonucleases), followed by degradation and resynthesis of a part of the DNA strand next to the lesion (base or nucleotide excision repair mechanisms).2 In the initial steps, abasic sites (deoxyribose moieties lacking the base residues), single-strand breaks (ssb), or both are formed at the sites of base DNA damage. Some of the DNA repair enzymes are commercially available and can be utilized for analytical purposes (including Escherichia coli exonuclease III recognizing abasic sites, T4 endonuclease V specific for UV radiation-induced pyrimidine dimers, and others).3,16-18 Utilization of the DNA repair enzymes in connection with, for example, the comet assay resulted in a considerable improvement of sensitivity and specificity of the DNA damage detection.3-5,15-18 In this paper, we utilized DNA repair enzymes endonuclease V and exonuclease III to convert DNA base damage induced by an alkylating agent dimethyl sulfate or by ultraviolet radiation, respectively, into ssb. The latter were detected by ac voltammetry at the HMDE. We show that this approach allows for significant improvement of electrochemical detection of the base damage. In addition, a simple method for the detection of damage to DNA bases in cells exposed to a genotoxic agent is described. EXPERIMENTAL SECTION Material. ScDNA of plasmid pBSK(-) was isolated from chloramphenicol-amplified E. coli TOP10 cells using Qiagen plasmid preparation kit followed by two isopycnic centrifugations in CsCl/ethidium bromide gradient, extraction with cold butan1-ol, extensive dialysis against 10 mM Tris, 1 mM EDTA buffer pH 7.9 (TE buffer), and ethanol precipitation. T4 endonuclease V (endoV) and E. coli exonuclease III (exoIII) were purchased from Epicentre and from Takara, respectively. Other chemicals were of analytical grade. Irradiation of DNA or Bacterial Cells with UV Light. This procedure was performed with a 254-nm UV lamp (Desaga) from a distance of 4 cm. DNA was irradiated in clear polypropylene Eppendorf tubes containing 50 µL of 50 µg mL-1 scDNA solution in 100 mM Tris-HCl, pH 7.6. The bacteria were irradiated in 1-mL aliquots of the cell culture (in the LB medium) spread on a Petri dish (diameter 5 cm). For more details, see Results and Discussion. DNA Modification with Dimethyl Sulfate. The scDNA (50 µg mL-1) was shaken with the dimethyl sulfate (DMS, 8-40 mM) (43) Rusling, J. F. Biosens. Bioelectron. 2004, 20, 1022-1028. (44) Yang, J.; Zhang, Z.; Rusling, J. F. Electroanalysis 2002, 14, 1494-1500. (45) Wang, B. Q.; Rusling, J. F. Anal. Chem. 2003, 75, 4229-4235. (46) Cai, X.; Rivas, G.; Farias, P. A. M.; Shiraishi, H.; Wang, J.; Fojta, M.; Palecek, E. Bioelectrochem. Bioenerg. 1996, 40, 41-47.

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in 100 µL of 100 mM Tris, pH 7.6 at 20 °C. Prior to the enzymatic treatment, the DNA was precipitated with ethanol. Cleavage of UV-Irradiated DNA with Endov. DNA (20 µg mL-1) was incubated with 1 unit of the endoV per milliliter in 100 mM KCl, 50 mM Tris, 1 mM EDTA, pH 7.07. More details are given in Results and Discussion. Cleavage of DMS-Treated DNA with ExoIII. The ethanolprecipitated DNA samples were dissolved in 50 µL of deionized water and diluted twice into 50 mM Tris-HCl, pH 8.0, 5 mM MgCl2, and 10 mM 2-mercaptoethanol containing 14 units of the exoIII/ mL. After the enzymatic digestion, NaCl was added to each sample (final concentration 0.2 M). E. coli Cultivation and Plasmid DNA Minipreparation. The E. coli cells (TOP10 strain transformed with pBSK(-)) were cultivated for 12 h at 37 °C in 100 mL of LB medium (1% Trypton, 1% NaCl, 0.5% yeast extract) with 1 µΜ ampicilin. After UV irradiation (vide supra), the bacteria were lysed and plasmid DNA was isolated using Qiagen-tip 20 columns according to the user’s manual, precipitated with ethanol, and dissolved in 50 µL of 10 mM Tris, 1 mM EDTA, pH 7.8. Prior to the electrochemical analysis, NaCl was added to each sample (final concentration 0.2 M). Electrochemical Measurements. All measurements were performed at room temperature in the adsorptive transfer stripping (AdTS; “medium exchange”) mode. DNA was adsorbed at the electrode surface from 3-µL aliquots for 60 s, followed by successive washing of the electrode by deionized water and by background electrolyte solution. The measurements were performed with an Autolab analyzer (EcoChemie, The Netherlands). Ac voltammetric measurements were carried out at HMDE (surface area 0.4 mm2) in 50 mM Na2HPO4, 0.3 M NaCl. The following settings were chosen: frequency 230 Hz, peak-to-peak amplitude 10 mV, in-phase component, scan rate 20 mV s-1, initial potential -0.6 V, and final potential -1.6 V (deaeration of the background electrolyte was not necessary when tensammetric DNA responses at mercury electrodes were measured). Squarewave voltammetric (SWV) measurements were performed at a homemade pyrolytic graphite electrode (PGE; basal plane orientation, geometric area 21 mm2; the electrode surface was renewed by peeling the top graphite layer off as in ref 47) in 0.2 M sodium acetate, pH 5.0, using the following parameters: initial potential 0 V, pulse amplitude 25 mV, frequency 200 Hz, and potential step 5 mV. In all measurements, Ag/AgCl/3 M KCl was used as a reference and platinum wire as an auxiliary electrode. Gel Electrophoresis. The 5-µL aliquots of the plasmid DNA minipreparations were loaded on 1% agarose gel containing 40 mM Tris-acetate, 1 mM EDTA, and 0.3 µg mL-1 ethidium bromide. Voltage density was 3-4 V cm-1, and time of electrophoresis was ∼40 min. RESULTS AND DISCUSSION Principles. Tensammetric and reduction electrochemical signals yielded by DNA at mercury electrodes in neutral or weakly alkaline media are uniquely dependent on accessibility of base residues.23-25 Electrochemical responses of native (doublestranded, ds) and denatured (single-stranded, ss) DNAs at the mercury electrodes greatly differ, allowing for determination of (47) Fojta, M.; Havran, L.; Kizek, R.; Billova, S. Talanta 2002, 56, 867-874.

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Figure 1. Scheme of enzymatic conversion of DNA base damage to ssb- or ssDNA regions. (A). Due to exposure of DNA to UV light, pyrimidine dimers are formed as one of the major products.1,2 These lesions are recognized by T4 endoV,48 which cleaves N-glycosidic bond of the 5′-pyrimidine in the adduct (i), followed by cleavage of the 3′-phosphodiester bond of the same nucleotide, thus forming the ssb (ii). (B). Methylation of guanine at N7 facilitates spontaneous release of the base from DNA (a).2,49,51 The resulting apurinic sites are substrates for E. coli exoIII,49,50 which introduces ssb next to the apurinic site (b). The same enzyme then degrades one strand in dsDNA, thus generating a ssDNA region (c). Both ssb and ss regions can be sensitively detected using ac voltammetry at mercury or amalgam electrodes.23-25,32

small amounts of ssDNA in dsDNA samples (including enzymatically generated ss regions within dsDNA molecules). Moreover, responses of the dsDNA are strongly changed upon introduction of ssb.11 We have shown that these entities represent centers of potential-induced unwinding of the DNA double helix at the mercury surface.23,25 During a slow potential scan through the socalled region U (-1.2 V), DNA molecules possessing free ends (oc- or linDNA) are partially unwound (denatured) and the resulting ss regions can be detected using specific signals (e.g., ac voltammetric peak 3).11,12,23-25 Intact dsDNA lacking strand termini (such as plasmid covalently closed circular scDNA), whose denaturation is restricted, does not yield the peak 3. The latter signal therefore indicates formation of single- or double-strand breaks that can be determined with a high sensitivity (one break among more than 2 × 105 intact phosphodiester bonds11). Here we show that damage to DNA bases can be easily converted into strand breaks by means of endonucleases involved in DNA repair and the strand breaks determined electrochemically (without removing the enzyme from the mixture). Figure 1 shows a scheme of enzymatic conversions of base damage to ssb or ssDNA regions, or both, by T4 endoV or by E. coli exoIII used in this work. EndoV (Figure 1A) recognizes cyclobutane pyrimidine dimers, one of the major products of UV-light induced DNA

damage. The enzyme primarily attacks the N-glycosidic bond of the 5′-pyrimidine, followed by cleavage of the 3′-phosphodiester bond of the same nucleotide.48 ExoIII (Figure 1B) is a multifunctional enzyme that can introduce ssb at sites of missing bases (acting as AP-endonuclease).49,50 The abasic sites are intermediates of DNA repair pathways involving N-glycosylases (enzymes removing damaged bases from the N-glycosidic bond) or may be formed due to spontaneous DNA depurination. Release of the purine bases is often facilitated by chemical modification of the purine moieties, for example, by methylation of N7 position in guanine residue by DMS (Figure 1B).2,49,51 After formation of the sb, the exoIII can degrade one DNA strand from its 3′-OH end as a processive exonuclease, leaving the ss region within the doublestranded molecule.49,50 Both ssb- and ssDNA regions resulting from the enzymatic digestion of DNA containing the base lesions can be detected by ac voltammetry at HMDE. None of the enzymes can attack intact DNA, thus offering a specific response for the given lesion. Detection of DNA Base Damage in Vitro. (1) Pyrimidine Dimers. ScDNA of plasmid pBSK(-) was exposed to UV light (dose 5.2 J cm-2) and subsequently treated with the endoV. After the enzymatic digestion, the samples were analyzed by AdTS ac voltammetry without removing the enzyme (Figure 2A). The irradiated and enzymatically cleaved DNA yielded voltammograms characteristic for DNA containing free ends, i.e., showing a broad peak 1 at -1.11 V, a shoulder corresponding to peak 2 at -1.26 V, and particularly a well-developed peak 3 at -1.36 V (Figure 2A, curve d). Control scDNA (neither UV-irradiated nor treated with the endoV, curve a) also yielded peak 1 and peak 2, but only a small inflection appeared at a potential corresponding to peak 3 (most likely due to a small amount of ocDNA present in the scDNA preparation). After UV irradiation (without the subsequent endoV cleavage), the voltammogram of scDNA did not change significantly (Figure 2A, curve c). Treatment of the nonirradiated scDNA with the endoV resulted in a slight increase of the peak 3 intensity, suggesting that a small portion of the scDNA molecules was cleaved by the enzyme (Figure 2A, curve b). Intensity of peak 3 yielded by the irradiated and endoV-cleaved DNA was remarkably dose-dependent (Figure 2B). Up to ∼5 J cm-2, the peak 3 height increased almost linearly (slope 20 nA cm2 J-1). For higher UV doses, the peak 3 intensity tended to level off. Silver solid amalgam electrode modified with mercury meniscus (m-AgSAE)32,52,53 yielded a similar dependence on the UV dose as HMDE (Figure 2B). When the endoV treatment was omitted, irradiation caused no significant changes in the ac voltammogram shape (not shown; in difference to ionizing radiation,10,11 low doses of UV light do not induce strand breaks). Previously we have shown10,11 that sensitivity of detection of the ssb formed in very low amounts of the plasmid DNA molecules can be remarkably increased by selective DNA denaturation prior (48) Schrock, R. D., 3rd; Lloyd, R. S. J. Biol. Chem. 1993, 268, 880-886. (49) Blackburn, M. G.; Gait, M. J. Nucleic Acids in Chemistry and Biology; IRL Press: New York, 1990. (50) Rogers, S. G.; Weiss, B. In Methods in Enzymology; Grosmann, L., Moldave, K., Eds.; Academic Press: New York, 1980; Vol. 65, pp 201-211. (51) Boturyn, D.; Constant, J. F.; Defrancq, E.; Lhomme, J.; Barbin, A.; Wild, C. P. Chem. Res. Toxicol. 1999, 12, 476-482. (52) Yosypchuk, B.; Heyrovsky, M.; Palecek, E.; Novotny, L. Electroanalysis 2002, 14, 1488-1493. (53) Yosypchuk, B.; Novotny, L. Electroanalysis 2002, 14, 1733-1738.

Figure 2. (A) Effects of UV irradiation, endoV treatment, or both on DNA ac voltammetric responses: control (undamaged) scDNA (a); undamaged scDNA incubated with endoV (b); scDNA exposed to UV light (c); DNA exposed to UV light followed by the enzymatic treatment (d). The DNA solution (50 µg mL-1 in 100 mM Tris-HCl, pH 7.6) was irradiated with a dose 5.2 J cm-2 and cleaved with endoV (1 unit mL-1) for 30 min. Then, NaCl was added (to a final concentration of 0.2 M), and the AdTS ac voltammetric measurement was performed (for more details, see the Experimental Section). (B) Dependence of the DNA peak 3 height on the dose of UV irradiation. After exposure to the UV light, the DNA was treated with the endoV as in (A). The AdTS ac voltammetric responses were measured on HMDE (b) or on a m-AgSAE (4). Inset: details of baseline-subtracted peak 3 (measured at the HMDE) yielded by sc DNA UV-irradiated with a dose 0.04 J cm-2 (a); the same DNA, exposed to 95 °C for 120 s (b); the UV-irradiated DNA after the endoV treatment (c); endoVcleaved and thermally denatured DNA (d).

to adsorption at the electrode. When a mixture of sc and oc (or linear) DNA is exposed to 95 °C for a short time (120 s), only the DNAs with free ends are irreversibly denatured while the scDNA retains its double-helical structure. By means of ac voltammetry, the denatured (ss) DNA can be determined in large (100-fold) excesses of the scDNA with a high sensitivity. Here we used this procedure for detection of DNA damage induced by a UV dose of 0.04 J cm-2. Such a low dose caused only negligible peak 3 after the endoV treatment when the denaturation step was omitted (inset in Figure 2B, curve c). On the other hand, after the thermal denaturation, a large peak 3 appeared (curve d). Although small peak 3 increase was observed also after heating of enzymatically uncleaved DNA (curve b), no matter whether exposed to the UV light or not (probably due to the presence of a small amount of the nicked ocDNA in the scDNA sample), the thermal treatment resulted in a strong selective enhancement of the signal yielded by the damaged, endoV-treated DNA. Analytical Chemistry, Vol. 77, No. 9, May 1, 2005

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Figure 3. Dependence of voltammetric DNA signals on the time of endoV treatment: peak 3 (at HMDE), UV-irradiated DNA (5.2 J cm-2) (b); peak 3, nonirradiated DNA (2); peak Gox (due to electrochemical oxidation of DNA guanine residues measured by AdTS square-wave voltammetry on pyrolytic graphite electrode), UV-irradiated DNA (9). Potential of the peak Gox was +1.11 V, and its heights plotted in the graph were multiplied by a factor of 0.1. For other details, see Figure 2.

Figure 3 shows dependence of the peak 3 height on the time of enzymatic hydrolysis for the UV-irradiated (5.2 J cm-2) and nonirradiated scDNA. With the irradiated DNA, dependence of the peak height on the cleavage time followed a hyperbolic shape, displaying a steep increase for times up to 10 min and then tending to level off. Thus, most UV-induced pyrimidine dimers were converted to the ssb in the first 10-15 min of the enzymatic treatment under the given conditions. In contrast, digestion of the nonirradiated scDNA was completed in 5 min of the enzymatic treatment (the peak 3 height did not change for longer incubation times, Figure 3). Such behavior suggests that this small peak 3 increase was due to a certain amount of pyrimidine dimers present in the scDNA (accidentally induced by sunlight or UV rays from fluorescent lamps in the laboratory) rather than to nonspecific DNA cleavage (by, for example, an endonuclease contaminating the endoV preparation or by the endoV itself accidentally attacking undamaged DNA). Wang et al. reported39 on detection of UV-induced DNA damage via measurements of guanine oxidation signal (peak Gox) at carbon screen-printed electrodes. In their work, dose-dependent decrease of the peak Gox intensity in the UV-irradiated DNA was observed due to conversion of the guanine bases into electroinactive 2,6-diamino-4-hydroxy-5-formamidopyrimidine and its release from the DNA.39 We measured the DNA peak Gox at a PGE. Under the given conditions, the UV radiation had no measurable effect on the height of peak Gox (at +1.11 V) for doses up to 7.8 J cm-2; for a dose of 13 J cm-2, the peak Gox decrease did not exceed 15% (signal intensity of undamaged DNA taken as 100%, not shown). Cleavage of the UV-irradiated DNA with endoV did not result in significant changes of the peak Gox intensity (Figure 3). This observation was in agreement with previous data showing that measurements at carbon electrodes cannot provide information about formation of small number of breaks in long DNA molecules and do not allow for differentiation between sc, oc, and lin DNAs.30,46 (2) Apurinic Sites. The scDNA was incubated with 16 mM dimethyl sulfate for 30 min, followed by ethanol precipitation and 2924 Analytical Chemistry, Vol. 77, No. 9, May 1, 2005

Figure 4. (A) Effects of scDNA modification with dimethyl sulfate, treatment with exoIII, or both on intensity of peak 3 measured at the HMDE: control (undamaged) scDNA (a); undamaged scDNA treated with exoIII (b); scDNA modified with DMS (c); DMS-modified scDNA treated with the exoIII (d). The DNA (50 µg mL-1) was incubated with 16 mM DMS at 20 °C for 30 min. After the ethanol precipitation, the DNA was subsequently treated with exoIII (14 units mL-1) for 30 min and analyzed by AdTS ac voltammetry as in Figure 2. Baselinesubtracted sections of the ac voltammograms are shown. Inset: dependence of the peak 3 height on the DMS concentration (other conditions the same). (B) Schematic representation of intensities of AdTS SWV peak Gox yielded at the PGE by intact control scDNA, the same DNA treated with 16 mM DMS (30 min), and the DMStreated DNA, then cleaved with exoIII. Height of the control scDNA was taken as 100%. For other details, see (A) and Figure 2.

dissolving of the DNA in deionized water. Then, the DNA was treated with the exoIII and analyzed by AdTS ACV (Figure 4A). DMS-untreated scDNA did not yield the peak 3, no matter whether it was treated with the exoIII or not (curves a and b in Figure 4). DNA modified with DMS but not cleaved with the enzyme produced a small peak 3 (Figure 4A, curve c), probably due to some extent of random spontaneous breakage of the scDNA chains during its incubation with the methylating agent. After exoIII cleavage of the DMS-modified DNA, a large peak 3 appeared (Figure 4A, curve d), in agreement with DNA nicking at the apurinic sites and subsequent formation of ssDNA stretches. Intensity of the peak 3 yielded by the DMS-modified and exoIIIdigested DNA increased with the DMS concentration (Figure 4A, inset). Between 0 and 16 mM DMS, the peak 3 height increased almost linearly (slope ∼19 nA mM-1). At higher DMS concentrations, the dependence followed a less steep trend (slope ∼3.3 nA mM-1). Without the enzymatic digestion, only a small peak 3 was detected regardless of the DMS concentration (for 40 mM DMS reaching 4% of the peak intensity yielded by the exoIII-cleaved DNA, not shown). In general, peak 3 intensities observed after the digestion of DMS-treated DNA with exoIII were remarkably

Figure 5. Dependence of the DNA peak 3 height on the time on exoIII treatment: scDNA modified with 40 mM DMS (b); control scDNA (2). For other details, see Figure 4.

higher than those obtained as responses to UV-induced DNA damage after the endoV cleavage (Figures 2 and 3). This observation accords with different products of DNA cleavage by the two enzymes (ssb in dsDNA molecules or ssDNA stretches for endoV and exoIII, respectively; see Figure 1) and suggests a possible utilization of the exoIII as a tool of amplification of the strand breaks signal (see below). It has been previously shown that chemical modification of guanine residues in DNA can result in diminishing of the guanine signals at mercury or carbon electrodes.23,24 For example, treatment of denatured calf thymus DNA with 1% (∼80 mM) DMS caused ∼60% decrease of the peak G measured at HMDE after 37 min of incubation.36 We measured peak Gox using AdTS SWV at the PGE after 30-min modification of the scDNA with 16 mM DMS. Under these conditions, no significant changes of the peak Gox intensity were observed (Figure 4B). After cleavage of the DMS-treated DNA with the exoIII, intensity the peak Gox increased by ∼25% (Figure 4B) in agreement with formation of the ssDNA regions (Figure 1B). Compared to this only partial signal increase, the ac voltammetric measurements at the HMDE (or m-AgSAE; not shown) offered much more pronounced differences between the responses of the DMS-modified DNA cleaved with the exoIII and signals obtained for undamaged or enzyme-untreated DNA (Figure 4A). For DNA modified with 40 mM DMS, the peak 3 height was measured as a function of the exoIII cleavage time (Figure 5). The dependence of peak 3 height on the enzymatic hydrolysis time followed a saturation curve reaching maximum signal intensity after 15 min of DNA cleavage. No significant changes in the peak 3 height were observed with the DMS-untreated DNA even after 30 min of the exoIII treatment. It can be concluded that, similarly as in the case of detection of the UV-induced lesions (see Pyrimidine Dimers), combination of exoIII cleavage and ac voltammetric measurements at the mercury electrode provides specific response for apurinic sites in DNA. (3) Specificity of the Technique. The DNA peak 3 (Figure 2A) is a signal responding to interruptions of the DNA sugarphosphate backbone, regardless of how the strand breaks were formed. The signal itself therefore does not allow differentiation between various genotoxic agents whose action results in the DNA

Figure 6. Specificity of the electrochemical base damage detection using the DNA repair enzymes. The bars represent intensities of the DNA peak 3 obtained after subsequent treatment of scDNA with agents and enzymes, as indicated below the graph. UV irradiation, dose 0.22 J cm-2; DMS modification, 16 mM, 30 min; time of enzymatic treatment, 30 min with each enzyme. For other details, see Figures 2 and 4.

strand breaking. On the other hand, the DNA repair enzymes exhibit specificities toward certain types of the base lesions. Combination of the enzymatic approaches with the electrochemical detection can thus be utilized for identification of individual types of base damage. This is demonstrated in Figure 6. DNA damaged by the UV radiation yielded a well-developed peak 3 after incubation with the endoV but not with exoIII (Figure 6). By analogy, the DMS-modified DNA produced a large peak 3 specifically after digestion with exoIII but not with endoV (Figure 6). UV-irradiated DNA treated subsequently with exoIII and endoV yielded peak 3 intensity similar to the same DNA cleaved solely with endoV. Notably, when UV-irradiated DNA was digested with endoV followed by exoIII, a significant increase of the peak 3 height was observed, compared to the same DNA cleaved with endoV only (Figure 6). This effect was due to exoIII exonucleolytic action on ocDNA molecules into which ssb were introduced by the endoV (Figure 1). Conversion of the ssb into the ss stretches results in enhanced accessibility of more DNA base residues for contact with the electrode surface and thus in more intense peak 3. Such enhancement of the strand break signal may, in principle, be utilized in combination with other endonucleases (including deoxyribonuclease I; not shown). On the other hand, cleavage of randomly formed ocDNA by the exoIII may lead to a higher background signal (see Figures 4-6). Detection of DNA Base Damage in Cells. To detect DNA damage in vivo, the DNA repair enzymes have been used in connection with electrophoretic techniques such as the comet assay.5,17 Other techniques developed for analysis of DNA base damage in living organisms (besides the techniques based on the complete DNA hydrolysis3,5,18,54) involve, for example, application of fluorescent probes,51 immunoassays,20,55 or biotin-avidin tech(54) Helbock, H. J.; Beckman, K. B.; Shigenaga, M. K.; Walter, P. B.; Woodall, A. A.; Yeo, H. C.; Ames, B. N. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 288293. (55) Chen, B. X.; Kubo, K.; Ide, H.; Erlanger, B. F.; Wallace, S. S.; Kow, Y. W. Mutat. Res. 1992, 273, 253-261.

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Figure 7. Detection of damage to DNA isolated from UV lightexposed bacterial cells. (A) Scheme of the experiment. Aliquots of E. coli bacterial culture bearing the plasmid pBSK(-) were exposed to the UV radiation, followed by lysis of the cells and isolation of the plasmid scDNA using the Qiagen-tip 20 columns (i, ii). After elution and ethanol precipitation, the DNA was treated with endoV (iii) and analyzed by AdTS ac voltammetry at the HMDE (iv). (B) Effect of the irradiation time on the peak 3 height. AdTS ac voltammetric responses of the DNA were recorded without any enzymatic treatment (white columns) or after the endoV cleavage (gray columns). Inset i: AdTS ac voltammograms of plasmid DNA isolated from nonirradiated cells (curve 1) or isolated from cell exposed to the UV light (2.6 J cm-2) and subsequently treated with the endoV (curve 2). Inset ii: ethidiumstained agarose gel after electrophoresis of DNA isolated from nonirradiated cells (lanes 1 and 2) or from cells irradiated with a dose 5.2 J cm-2 (lanes 3 and 4). The samples were loaded on the gel without endoV treatment (lanes 1 and 3) or after the endoV cleavage (lanes 2 and 4). For other details, see Figure 2.

nology-based assays.56 On the other hand, in contrast to increasing number of papers concerning electrochemical detection of interactions of purified DNAs with a variety of genotoxic substances in vitro,23,24,26 to our knowledge there are no reports on direct electrochemical analysis of DNA exposed to various agents in the cells. Here we used a simple DNA minipreparation procedure based on commercially available kits to isolate plasmid DNA from bacterial cells exposed to UV light for subsequent electrochemical analysis (Figure 7A). E. coli cells transformed with the pBSK(-) plasmid were cultivated for 12 h (for more details, see Experimental Section). The 1-mL aliquots of cell culture were exposed to UV radiation. Then, DNA was isolated using the Qiagen-tip 20 columns followed by ethanol precipitation. The isolated scDNA was treated with endoV and analyzed by AdTS ac voltammetry at the HMDE as in section Pyrimidine Dimers. Inset i in Figure 7B shows typical ac voltammograms of scDNA (isolated from untreated cells, curve 1) and of DNA containing (56) Ide, H.; Akamatsu, K.; Kimura, Y.; Michiue, K.; Makino, K.; Asaeda, A.; Takamori, Y.; Kubo, K. Biochemistry 1993, 32, 8276-8283.

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the ssb (isolated from cells exposed to a dose 2.6 J cm-2 of UV light and subsequently cleaved by the endoV, curve 2), indicating that the simple DNA purification protocol was sufficient for preparation of DNA samples suitable for the ac voltammetric measurements. Intensity of peak 3 measured after the endoV treatment remarkably increased with the UV light dose (Figure 7B). Interestingly, remarkable intensities of the peak 3, corresponding to 50 and 43% of the endoV-cleaved samples, were measured also in the enzyme-untreated DNA samples isolated from cells irradiated with 2.6 and 5.2 J cm-2. This indicated that significant portion of the plasmid DNA molecules contained strand interruptions, ss regions, or both, most likely due to action of intracellular endonucleases involved in repair of the UV-induced DNA damage1,2 The electrochemical data correlated well with the results of agarose gel electrophoresis, showing increasing amounts of nicked circular DNA upon cell exposure to the UV light and the enzymatic DNA treatment (Figure 7B, inset ii). Using the same protocol (but involving exoIII DNA treatment instead of endoV), we were able to detect apurinic sites formed in the plasmid DNA in DMS-treated bacteria as well (data not shown). Comparison with Other Techniques. Sensitivity (limit of detection) of the proposed technique toward the DNA base lesions is determined by the sensitivity of electrochemical detection of the strand breaks. Using ss plasmid DNA for calibration, we have previously shown11 that formation of a ssb can be detected in ∼1% of scDNA (when 50-100 µg of scDNA/mL is used and provided that the nicked DNA is selectively denatured prior to analysis, see section Pyrimidine Dimers) with a relative standard deviation below 10%. For a 3-kb plasmid DNA, this corresponds to recognition of 4-5 ssb/106 intact phosphodiester bonds (nucleotides). The same limit of detection can be reached for the base lesions after their quantitative enzymatic conversion to the ssb. Considering the DNA amount used for one analysis (3 µL of solution containing 20 µg of 3-kb DNA/mL, i.e., 60 ng), our technique allows detection of ∼1 fmol of the pyrimidine dimers (or other base lesions). Similar detection limits were reported for other techniques used to monitor UV-induced DNA damage such as mass spectrometry,57 alkaline gel electrophoresis,58 ELISA,21 or 32P-postlabeling7 (see Table 1). Although selectivity of the electrochemical technique (i.e., its ability to recognize damaged nucleotides among normal ones) is still below the limit allowing detection of background DNA damage in vivo (for oxidative DNA damage reported at levels of ∼10-6 lesions/nucleotide3,4), it is well competitive with other techniques in monitoring of induced DNA damage. It uses no DNA labeling, and compared to the electrophoretic methods, it is faster and offers easier quantifiable data. When compared to the plasmid relaxation assay in agarose gels, the electrochemical technique is not only by 1 order of magnitude more sensitive to small levels of DNA damage11 but also responds to formation of additional strand breaks even after complete conversion of sc- into ocDNA.11,12 In contrast to electrochemical techniques based on changes of guanine redox responses,24,25,30,36,38,39,46 the ac voltammetric measurements at mercury or solid amalgam electrodes are inherently more sensitive (by orders of magnitude) to formation of small number of the (57) Podmore, I. D.; Cooke, M. S.; Herbert, K. E.; Lunec, J. Photochem. Photobiol. 1996, 64, 310-315. (58) Freeman, S. E.; Blackett, A. D.; Monteleone, D. C.; Setlow, R. B.; Sutherland, B. M.; Sutherland, J. C. Anal. Biochem. 1986, 158, 119-129.

Table 1. Comparison of Sensitivities of Various Techniques Used for Detection of Pyrimidine Dimers in UV Light-Exposed DNA sensitivity technique

ref

molar amount of pyrimidine dimer

dimers detectable per undamaged nucleotides

mass spectrometry ELISA alkaline gel electrophoresis 32P postlabelinga ac voltammetry

57 21 58

20-50 fmol 0.9 fmol tenths of femtomoles

6-15 per 106 2 per 107 ∼1 per 106

7 this work, 11

femtomolesa ∼1 fmol

not reported† 4-5 per 106

a In recent applications of the 32P-postlabeling techniques, limits of detection of DNA adducts at the level of hundreds of attomoles or of one adduct per 109-1010 nucleotides have been reached.8,9

lesions (due to zero signal of the undamaged scDNA) and do not require involvement of electroactive base residues in the DNA damage. CONCLUSIONS Besides DNA hybridization sensors,23,27,59-61 considerable progress has recently been attained in development of electrochemical sensors for DNA damage, which may in future serve as environmental guardians, tools for toxicity testing, for monitoring of exposure of humans to genotoxic substances, etc.24-26,38,43-45,62 In this paper, we present a novel technique of DNA base damage detection based on combination of enzymatic cleavage of the damaged DNA with voltammetric analysis. While the enzymatic treatment provides specific recognition of the given lesion (Figure 1), the ac voltammetric measurement at the mercury electrode offers highly sensitive detection of the resulting DNA strand breaks.11,24,25 Such a high sensitivity has never been attained with (59) Drummond, T. G.; Hill, M. G.; Barton, J. K. Nat. Biotechnol. 2003, 21, 11921199. (60) Gooding, J. J. Electroanalysis 2002, 14, 1149-1156. (61) Wang, J. Anal. Chim. Acta 2002, 469, 63-71. (62) Labuda, J.; Buckova, M.; Heilerova, L.; Silhar, S.; Stepanek, I. Anal. Bioanal. Chem. 2003, 376, 168-173.

any non-mercury electrode23,24,26 (see Figures 3 and 4). The inconvenience of HMDE for DNA sensors can be overcome by using silver solid amalgam electrodes31,32 (Figure 2). In addition, in this paper we used for the first time the electrochemical technique to analyze DNA isolated from cells exposed to a damaging agent. Our results (Figure 6) suggest that using a simple protocol combining plasmid DNA minipreparation with the voltammetric detection, it is possible to monitor not only DNA damage itself, but also incomplete DNA repair sites1,2,13 (more results will be published elsewhere). ACKNOWLEDGMENT This work was supported by a grant 203/04/1325 from the Grant Agency of the Czech Republic, by a purpose endowment from the Ministry of Industry and Trade of the Czech Republic (project 1H-PK/42), and a grant Z 5004920. The authors thank Dr. Ludek Havran for critical reading of the manuscript.

Received for review October 26, 2004. Accepted March 2, 2005. AC048423X

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