DNA Films and Capillary LC

Anal. Chem. 2006, 78, 624-627

Genotoxicity Screening Using Biocatalyst/DNA Films and Capillary LC-MS/MS Maricar Tarun,† Besnik Bajrami,† and James F. Rusling*,†,‡

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

Detection of DNA adducts can serve as a basis for genotoxicty screening of new chemicals and drugs. We report here a simple, sensitive procedure for this purpose using films containing DNA and a biocatalyst to mimic the metabolic action of human liver cytochrome P450s. DNA adducts formed from an in-situ-generated toxic metabolite (styrene oxide) were detected at subpicomole levels after neutral thermal hydrolysis of the DNA films and analysis with capillary liquid chromatography with online column preconcentration and MS/MS detection. An on-line column switching system allowed for increased sample loading volume and analyte preconcentration. This approach provides an estimate of the relative rate of DNA damage. Advanced chemical synthesis methods including combinatorial techniques have led to an explosion into the millions in the number of compounds produced each year for possible pharmaceutical, agricultural, personal care, and dietary applications. This has created a need for rapid toxicity screening of new chemicals at an early stage in their commercial development to complement conventional microbiology and animal toxicity testing. Genotoxic compounds or their metabolites react with DNA to form nucleobase adducts, which may initiate processes leading to mutagenesis and carcinogenesis.1-4 Formation of DNA adducts has been shown to be an accurate and reliable predictor of the carcinogenicity of xenobiotics.5-7 Films containing DNA and human liver cytochrome (cyt) P450 enzymes or other heme proteins as models can serve as a basis for genotoxicity screening, and we have employed them as the active elements in nonspecific voltammetric screening sensors.8 These enzymes convert lipophilic pollutants and drugs to potentially genotoxic metabolites * Corresponding author. E-mail: [email protected]. † University of Connecticut. ‡ University of Connecticut Health Center. (1) Farmer, P. B. Toxicol. Lett. 2004, 149, 3-9. (2) Sharma, R. A.; Farmer, P. B. Clin. Cancer Res. 2004, 10, 4901-4912. (3) Baird, W. M.; Mahadeva, B. Mutat. Res. 2004, 547, 1-4. (4) Vodicka, P.; Koskinen, M.; Arand, M.; Oesch, F.; Hemminki, K. Mutat. Res. 2002, 551, 239-254. (5) (a) La, D. K.; Swenberg, J. A. Mutat. Res. 1996, 365, 129-146. (6) Farmer, P. B. Toxicol. Lett. 2004, 149, 3-9. (7) Hemminki, K.; Koskinen, M.; Rajaniemi, H.; Zhao, C. Regul. Toxicol. Pharmacol. 2000, 32, 264-275. (8) (a) Zhou, L.; Yang, J.; Estavillo, C.; Stuart, J. D.; Schenkman, J. B.; Rusling, J. F. J. Am. Chem Soc. 2003, 125, 1431-1436. (b) Wang, B.; Jansson, I.; Schenkman, J. B.; Rusling, J. F. Anal. Chem. 2005, 77, 1361-1367. (c) Wang, B.; Rusling, J. F. Anal. Chem. 2003, 75, 4229-4235.

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that can react with DNA.9 Detection and measurement of these DNA adducts is, thus, useful for predicting human cancer risk.10 We previously detected and measured DNA adducts in polycation-DNA films that were incubated with suspected human carcinogens by conventional LC-MS/MS.11,12 These films did not contain enzymes or bioactivating catalysts. In the present Correspondence, we report for the first time the sensitive measurement of major DNA adducts of a genotoxic agent generated in situ in biocatalyst/DNA films. We employed capillary liquid chromatography (LC)-tandem mass spectrometry (CapLCMS/MS) using on-line analyte preconcentration. The method mimics a major toxicity pathway in the human liver and provides molecular signatures of DNA damage. For proof of concept, we used styrene, which is metabolized to styrene-7,8-oxide (SO)13,14 mainly in the human liver by cytochrome P450 enzymes. Styrene oxide forms adducts with DNA and is a potential human carcinogen.13,15 The iron heme protein myoglobin (Mb) activated by hydrogen peroxide can serve as an “enzyme mimic” in thin films to produce liver metabolites.8,16 We have used films of Mb, polymers, and DNA to catalyze reactions that produced DNA-damaging metabolites. Herein, we build on that concept to quantify the amount of DNA adducts formed using capillary liquid chromatographytandem mass spectrometry. Mb in Mb/DNA film was activated by H2O2 to convert styrene to styrene oxide.17 Styrene oxide then reacted with DNA in the film forming covalently bound adducts, ∼90% of which is N7-guanine styrene oxide (N7-GSO).15b N7-GSO (9) (a) Schenkman, J. B., Greim , H., Eds. Cytrochrome P450; Springer-Verlag: Berlin, 1993. (b) Ortiz de Montellano, P. R., Ed. Cytochrome P450; Plenum: New York, 1995. (c) Singer, B.; Grundberger, D. Molecular Biology of Mutagens and Carcinogens; Plenum: New York, 1983. (10) Hemminki, K.; Koskinen, M.; Rajaniemi, H.; Zhao, C. Regul. Toxicol. Pharmacol. 2002, 32, 264-275. (11) Tarun, M.; Rusling, J. F. Anal. Chem. 2005, 77, 2056-2062. (12) Yang, J.; Wang, B.; Rusling, J. F. Mol. Biosyst. 2005, 1, 251-259. (13) Teixeira, J. P.; Gaspar, J.; Silva, S.; Torres, J.; Silva, S. N.; Azevedo, M. C.; Neves, P.; Laffon, B.; Me´ndez, J.; Gonc¸ alves, C.; Mayan, O.; Farmer, P. B.; Rueff, J. Toxicology 2004, 195, 231-242. (14) Laffon, B.; Perez-Cadahia, B.; Pasaro, E.; Mendez, J. Toxicology 2003, 186, 131-141. (15) (a) Vodicaka, P.; Hemminki, K. Carcinogenesis 1988, 9, 1657-1660. (b) Koskinen, M.; Vodicka, P.; Hemminki, K. Chem.-Biol. Interact. 2000, 124, 13-27. (c) Koskinen, M.; Vodickova, L.; Vodicka, P.; Warner, S. C.; Hemminki, K. Chem.-Biol. Interact. 2001, 138, 111-124. (16) (a) Lvov, Y. M.; Lu, Z.; Schenkman, J. B.; Zu, X.; Rusling, J. F. J. Am. Chem. Soc. 1998, 120, 4073-4080. (b) Zu, X.; Lu, Z.; Zhang, Z.; Schenkman, J. B.; Rusling, J. F. Langmuir 1999, 15, 7372-7377. (17) Ortiz de Montellano, P. R.; Catalano, C. E. J. Biol. Chem. 1985, 260, 92659271. 10.1021/ac0517996 CCC: $33.50

© 2006 American Chemical Society Published on Web 12/13/2005

on the DNA was released by neutral thermal hydrolysis of the incubated film11 and determined by CapLC-MS/MS. This method provided molecularly specific quantitative information on rates of nucleobase adduct formation. EXPERIMENTAL SECTION Chemicals. Double-stranded salmon testes (st) DNA (∼2K base pairs, 41.2% G/C), horse heart myoglobin (MW 17 400), and 7-N-methylguanine were from Sigma. Styrene, styrene oxide, poly(diallyldimethylammonium chloride), and polystyrene sulfonate (PSS) were from Aldrich. All other chemicals used were analytical reagent grade. Mb-DNA Film Construction. Layers of Mb and DNA were assembled on carbon cloth by alternate electrostatic adsorption.18 A 10 × 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 dipped into 2 mg mL-1 polydiallyldimethylammonium chloride (PDDA) for 15 min, then alternately dipped into solutions of 2 mg mL-1 ds-DNA in pH 7.1 TRIS buffer and 3 mg mL-1 Mb in pH 5.5 acetate buffer, with washing in water between adsorption steps to make PDDA/DNA(Mb/DNA)2 films of nominal thickness ∼30 nm as estimated previously.8d Although these films are formed one layer at a time, the final structures feature considerable layer intermixing.18 Incubation with Styrene and H2O2. Safety note: Styrene and styrene oxide are suspected carcinogens. All procedures were performed while wearing gloves and under closed hoods. Mb-DNA films were incubated in stirred vessels at 37 °C containing 1% styrene (by vol) and 1 mM H2O2 in pH 5.5 acetate buffer, the pH at which the reaction rate between styrene oxide and DNA is optimum19 and Mb turnover rates are relatively large.16 Films were dipped into hexane to remove organic reactant and products before being subjected to neutral thermal hydrolysis. Neutral Thermal Hydrolysis. After incubation with styrene, films were heated in boiling water for 15 min, then the solution was immersed in ice. The cooled hydrolysate was filtered through Centricon filters with cutoff mass of 3000 Da (Amicon, Beverly, MA), and the resulting solution was injected onto the CapLC. CapLC-MS/MS. The trapping column (Atlantis dC18, 23.5 mm, 0.18 mm i.d., 5 µm particle size) and analytical column (Atlantis dC18, 150 mm, 300 µm i.d., 5 µm particle size) were from Waters (Milford, MA). The capillary LC system (CapLC, Waters, Milford, MA) was equipped with column switching, which allowed the selective capture of nucleobase adducts in the trapping column, while the unmodified nucleobases were directed to waste. A 10-µL portion of the sample was loaded into the trapping column at a flow rate of 4.25 µL min-1 and flushed with 90:10 ammonium acetate (10 mM pH 5)-methanol at a flow rate of 40 µL min-1. Five loading and washing cycles were made before the columnswitching valve was switched so that the trapping column was in-line with the analytical column. The trapped adducts were backflushed onto the analytical column for separation and detection. Elution at the analytical column was achieved at a flow rate of (18) 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. (19) Rusling, J. F.; Zhou, L.; Munge, B.; Yang, J.; Estavillo, C.; Schenkman, J. B. Faraday Discuss. 2000, 116, 77-87.

Figure 1. General design of genotoxicity screening method. (A) Conceptual view of enzyme/DNA film constructed layer-by-layer on a polydiallyldimethylammonium chloride (PDDA) underlayer on carbon cloth. The Mb/DNA film is incubated with styrene and H2O2 to produce styrene oxide, which reacts with DNA. (B) Outline of the procedure for the analysis of DNA adducts on the Mb/DNA film after incubation.

4.25 µL min-1 with the following gradient: (A, 10 mM acetate buffer, pH 5; B, methanol) 10 min, 10% A; 10 min, 10-20% A; 10 min, 20% A; 5 min, 20-10%A; 5 min, 10% A. Electrospray ionization mass spectrometry (ESI-MS) employed a Micromass Quattro II (Beverly, MA) with electrospray source operated in the positive ion mode (ES+). Samples were analyzed using single reaction monitoring (SRM) with a cone voltage of 15 V, collision energy of 15 eV, and collision gas (Ar) pressure of 5 × 10-3 mbar. Validation of in Situ Styrene Oxide Formation by GC. To prove that styrene oxide was formed in these incubations, alternate layers of PSS and Mb were assembled on top of a PDDA layer on carbon cloth. The Mb/PSS film was incubated in styrene and H2O2 for 10 min, after which the film and incubation solution were extracted with hexane. The organic layer was then reduced in volume under N2, and styrene oxide was determined using gas chromatography (HP 6980) on an RTX-5 column (30 m, 0.32 mm i.d.) as described previously.20 RESULTS The design of the genotoxicity screening procedure is shown in Figure 1. The incubation of the Mb/DNA film in styrene and H2O2 mimics the action of cyt P450 enzymes. Mb converts styrene (20) Estavillo, C.; Lu, Z.; Jansson, I.; Schenkman, J. B.; Rusling, J. F. Biophys. Chem. 2003, 104, 291-296.

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Figure 3. Partial LC-MS/MS chromatogram of PDDA/DNA/ (Mb/DNA)2 films incubated in styrene and 1 mM H2O2 using single reaction monitoring (SRM) for the transition m/z 272 (SO-Gua) to m/z 152 (Gua). Five 10-µL injections onto the trapping column for preconcentration and then elution in the analytical column at flow rate 4.25 µL min-1.

Figure 2. Daughter ion (DAU) chromatogram (A) and mass spectrum (B) of 1.2 µM standard GSO solution. Injection volume ) 0.82 µL, directly injected onto the analytical column (no trapping column) at a flow rate of 4.25 µL min-1.

to styrene oxide,17 a DNA alkylating agent. The amount of N7guanine-styrene oxide (N7-GSO) formed from the alkylation of DNA by the in-situ-generated styrene oxide with DNA on the film was measured using CapLC-MS/MS after neutral hydrolysis. The use of column switching in CapLC allowed for on-line sample cleanup and analyte preconcentration21 (see the Supporting Information, Figure S1). MS data were acquired in the single reaction monitoring (SRM) mode, providing enhanced sensitivity and selectivity. The SRM transition was selected from the results of collision-induced dissociation (CID) of a standard GSO solution prepared by incubating guanine with styrene oxide as previously described.11 The resulting solution is a mixture of GSO isomers, and the concentration is obtained by assuming that the only reaction that occurred was the formation of adducts with guanine. The amount of GSO adduct formed is taken as the initial amount of Gua minus the final amount of Gua. The SRM mass chromatogram and spectrum of 1.2 µM GSO are shown in Figure 2. The major fragment ion resulting from the CID of GSO was m/z 152 (protonated guanine), corresponding to the loss of styrene oxide. (21) Van den Driessche, B.; Lemie`re, F.; Van Dongen, W.; Van der Linden, A.; Esmans, E. L. J. Mass Spectom, 2004, 39, 29-37.

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Thus, the SRM transition m/z 272 (GSO) f m/z 152 (Gua) was used for the quantitation of GSO adducts on the Mb/DNA films. Films were incubated with styrene and H2O2 for different lengths of time, then subjected to neutral thermal hydrolysis to release N7-GSO adducts from the damaged DNA. Without the trapping column, no SRM peak was detected. This is because of the very small amount of N7-GSO present in the sample and limited sample volume that could be injected onto the analytical column. The use of a trapping column permitted larger injection volumes and effective preconcentration of the analyte, allowing the detection of subpicomole amounts of N7-GSO, which was captured in the trapping column while the unmodified guanine was sent to waste. The flow was then changed so that the trapping column was in-line with the analytical column, and the trapped N7-GSO was backflushed onto the analytical column. The detection limit of the method, measured as 3 times the average noise, was ∼30 fmol GSO. Figure 3 shows that SRM peak areas for N7-GSO increased with increasing incubation time, indicating increasing DNA damage as the films were incubated with styrene for longer periods of time. DNA/Mb films incubated in toluene, which is not oxidized by Mb under these conditions,8a did not give any SRM peak for the GSO f Gua transition. Control incubations with styrene but without H2O2, and with H2O2 but without styrene did not give SRM peaks, suggesting that the GSO f SO transition was, indeed, due to the in-situ-generated styrene oxide reacting with DNA in the film. To estimate the amount of N7-GSO formed on the film, a calibration plot of SRM peak area vs GSO concentration was used, using stock GSO solutions prepared as described previously.11 Figure 4 shows the amount of GSO detected at different incubation times. Films incubated for as short as 1 min gave detectable amounts of GSO, 0.37 pmol GSO. The amount of GSO detected increased with increasing incubation time. The slope of the amount of GSO plotted against reaction time constitutes an effective DNA damage rate of 0.57 nM guanine min-1.

Figure 4. Amount of GSO formed in PDDA/DNA/(Mb/DNA)2 films after incubation with 1% styrene oxide and 1 mM H2O2.

In situ conversion of styrene to styrene oxide was confirmed by incubating Mb/PSS films with styrene and H2O2. After incubation, the solution and film were extracted with hexane to obtain the unreacted styrene and the styrene oxide that had been formed. Gas chromatography of the organic layer showed that after 10 min of incubation, 8.2 nmol of styrene oxide was found (see Supporting Information, Figure S2). DISCUSSION Results described above suggest that enzyme/DNA films incubated with organic chemicals and analyzed by CapLC-MS/ MS after neutral hydrolysis can provide a relatively simple and fast in vitro genotoxicity screening procedure. The reaction mimics the bioactivation of lipophilic compounds by cytochrome P450 enzymes in the human liver.9 Styrene produced styrene oxide, which reacts with DNA in the film to give N7-GSO (Figure 3). The SRM peak area increased with increasing incubation time of the Mb/DNA films (Figure 4), demonstrating the ability to estimate relative DNA damage rates at very low levels of damage, that is, subpicomoles. We view the biocatalyst/DNA films as an important feature of our approach, since the metabolites are formed in a thin film with large effective concentrations of DNA. This provides a high probability of reaction of the metabolites with DNA as they diffuse out of the films past the DNA. The importance of this feature can be illustrated by comparing films with and without biocatalyst that were used previously for our electrochemical toxicity sensors that measure DNA damage. For sensors employing DNA/PPDA films, (22) Zhou, L.; Rusling, J. F. Anal. Chem. 2001, 73, 4780-4786. (23) Munge, B.; Estavillo, C.; Schenkman, J. B.; Rusling, J. F. ChemBiochem. 2003, 4, 82-89. (24) Hemminki, K. Chem.-Biol. Interact. 1991, 77, 39-50.

∼10 mM styrene oxide solutions were required to provide a given sensor response after 5 min incubation.22 For Mb/DNA or cyt P450/DNA film sensors activated by 0.2 mM H2O2, similar responses were found after 5 min of incubation with styrene,8a even though only nanomolar quantities of styrene oxide were detected in external solutions.23 Thus, the film method is expected to be superior to toxicity screening approaches using dissolved DNA and enzyme, in which dilution of the DNA and metabolite make the damage reactions relatively slow. Further, in the present method, simple heating and filtering selectively isolates major damaged nucleobases and leaves the biocatalyst and DNA backbone behind, simplifying sample workup prior to LC-MS. Styrene oxide forms nine adducts with guanine.24 This explains the presence of more than one peak in the DAU chromatogram of a standard GSO solution (Figure 2A). The fragmentation pattern (Figure 2B) observed is typical of N7-alkyated guanines, with protonated guanine as the major fragment. Sample preconcentration using on-line column switching was essential to measure the small amount of GSO formed in short reaction times. This method allowed five 10-µL injections onto the trapping column before backflushing all the trapped N7-GSO from the five injections onto the analytical column. At 1 min of incubation of the Mb/DNA film in styrene and H2O2, 0.37 pmol of N7-GSO was detected, suggesting the possibility of very sensitive genotoxicity screening. The detection limit was ∼30 fmol. The approximate rate of DNA damage by styrene was found to be 0.57 nM guanine min-1. We previously showed that rates of DNA damage in thin films of DNA are well-correlated with measures of animal genotoxicity for several carcinogens.11,12 In summary, we report herein a sensitive in-vitro genotoxicity screening procedure using biocatalyst/DNA films and CapLCMS/MS capable of measuring subpicomole levels of adduct and estimating relative rates of DNA damage. This procedure is amenable to the use of human metabolic enzymes, such as cyt P450s, which will be more relevant to direct genotoxicity screening. Films of these enzymes and DNA have been made in a similar way.8 We are currently pursuing such applications for more complex metabolic systems. ACKNOWLEDGMENT This work was supported by U.S. PHS Grant No. ES03154 from the National Institute of Environmental Health Sciences (NIEHS), National Institutes of Health, Washington, DC. SUPPORTING INFORMATION AVAILABLE Two additional figures showing the schematic of the on-line column-switching system and the gas chromatogram for the determination of styrene oxide. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review October 7, 2005. Accepted November 23, 2005. AC0517996

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