Anal. Chem. 2005, 77, 1361-1367
Evaluating Enzymes That Generate Genotoxic Benzo[a]pyrene Metabolites Using Sensor Arrays Bingquan Wang,† Ingela Jansson,‡ John B. Schenkman,‡ and James F. Rusling*,†,‡
Department of Chemistry, University of Connecticut, U-60, 55 North Eagleville Road, Storrs, Connecticut 06269-3060, and Department of Pharmacology, University of Connecticut Health Center, Farmington, Connecticut 06032
Arrays with individually addressable, demountable electrodes coated with ultrathin DNA/enzyme films were evaluated to estimate relative rates of genotoxic bioactivation of benzo[a]pyrene (BP) for several different enzymes simultaneously. Specifically, cytochrome (cyt) P450cam, cyt P40 1A2, and myoglobin in the array were activated with H2O2 to metabolize BP to genotoxic metabolites. DNA damage by the metabolites was detected by increases in square wave voltammetric oxidation peaks using Ru(bpy)32+ as catalyst. Cyt P450cam and cyt P450 1A2 showed 3-fold higher activity for genotoxic bioactivation of BP than myoglobin. The ability of the arrays to generate and detect metabolite-based DNA damage simultaneously for several enzymes is a rapid and promising approach to identify and characterize enzymes involved in genotoxicity of drugs and pollutants. Bioactivation of lipophilic pollutants and drugs to toxic metabolites in the liver by cytochrome P450 (cyt P450) enzymes represents a major mechanism of genotoxicity.1-5 c-DNA-directed expression has enabled human liver cyt P450 enzymes to be investigated individually. Over 20 different human cyt P450s with different activities have been expressed.6 Polymorphism and chemical enzyme induction can lead to interindividual differences in levels and distributions of different cyt P450s in humans. These interindividual differences could predispose certain individuals to a higher risk of pollutant-caused disease, drug toxicity, or both.7,8 * To whom correspondence should be addressed. E-mail: James.Rusling@ uconn.edu. † University of Connecticut. ‡ University of Connecticut Health Center. (1) Travis, C. C.; Hester, S. T. Environ. Sci. Technol. 1991, 25, 815-817. (2) Jacoby, W. B., Ed. Enzymatic Basis of Detoxification; Academic: New York, 1980; Vols. I and II. (3) Singer, B.; Grunberger, D. Molecular Biology of Mutagens and Carcinogens, Plenum: New York, 1983. (4) Schenkman, J. B., Greim, H., Eds. Cytochrome P450; Springer-Verlag: Berlin, 1993. (5) Ortiz de Montellano, P. R., Ed. Cytochrome P450, Plenum: New York, 1995. (6) (a) Gonzalez, F. J.; Gelboin, H. V. Environ. Health Perspect. 1992, 89, 8185. (b) Guengerich, F. P. Life Sci. 1992, 50, 1471-1487. Guengerich, F. P. Am. Sci. 1993, 81, 440-447. (7) (a) Guengerich, F. P. Asia Pac. J. Pharmacol. 1990, 5, 327-345. (b) Guengerich, F. P. Toxicol. Lett. 1994, 70, 133-138. (c) Guengerich, F. P.; Parikh, A.; Turesky, R. J.; Josephry, P. D. Mutat. Res. 1999, 428, 115-124. (d) Guengerich, F. P. Chem. Res. Toxicol. 2001, 14, 611-650. (8) (a) Gonzalez, F. J. Trends Pharm. Sci, 1992, 13, 346-352. (b) Gonzalez, F. J. In New Horizons in Biological Dosimetry; Gladhill, B. L., Mauro, I., Eds.; Wiley-Liss: New York, 1991; pp 11-20. 10.1021/ac0485536 CCC: $30.25 Published on Web 02/03/2005
© 2005 American Chemical Society
Thus, there is a clear need to identify which human cyt P450s actively generate DNA-reactive metabolites for environmental toxicology and drug development. Rapid detection of DNA damage by enzyme-generated metabolites can be used as the basis for screening the genotoxicity of new organic chemicals and drugs. One approach is reaction of DNA with metabolites, followed by analysis of the hydrolyzed DNA sample by liquid chromatography or capillary electrophoresis coupled to mass spectrometry.9,10 These methods provide a wealth of detailed molecular information on DNA damage, but routine high-throughput toxicity screening may be limited by analysis time and cost. Conversely, electrochemical DNA detection offers a variety of simple strategies to detect DNA damage and hybridization.11-18 One of the more sensitive electroanalytical approaches for DNA detection was first reported by Thorp et al., who showed that ruthenium tris(2,2′-bipyridyl) [Ru(bpy)32+] oxidizes guanine bases in DNA and oligonucleotides in the electrochemical catalytic pathway shown in Scheme 1 where G ) guanine:19
Scheme 1 Ru(bpy)32+ T Ru(bpy)33+ + e-
(at electrode)
(1)
Ru(bpy)33+ + DNA(G) f Ru(bpy)32+ + DNA(G•) + H+ (2) Conversion of Ru(bpy)33+ to Ru(bpy)32+ by DNA in eq 2 provides a greatly enhanced catalytic oxidation current in voltammetry by maintaining levels of Ru(bpy)32+ at the electrode. Guanine is the most easily oxidized DNA base. In double-stranded (ds) DNA, (9) Cadet, J.; Weinfeld, M. Anal. Chem. 1993, 65, 675A-682A. (10) Deforce, D. L. D.; Ryniers, F. P. K.; Van den Eeckout, E. G.; Lemiere, F.; Esmans, E. L. Anal. Chem. 1996, 68, 3575-3584. (11) Palecek, E. Electroanalysis 1996, 8, 7-14. (12) Mikkelsen, S. R. Electroanalysis 1996, 8, 15-19. (13) Thorp, H. H. Trends Biotechnol. 1998, 16, 117-121. (14) Wang, J. Chem. Eur. J. 1999, 5, 1681-1685. (15) Palacek E.; Fojta, M. Anal. Chem. 2001, 73, 74A-83A. (16) Palecek, E.; Fojta, M.; Tomschik, M.; Wang, J. Biosens. Bioelectron. 1998, 13, 621-628. (17) (a) Johnston, D. H.; Glasgow, K. C.; Thorp, H. H. J. Am. Chem. Soc. 1995, 117, 8933-8938. (b) Napier, M. E.; Thorp, H. H. Langmuir 1997, 13, 63426344. (c) Yang, I. V.; Thorp, H. H. Inorg. Chem. 2000, 39, 4969-4976. (18) Wang, J.; Rivas, G.; Ozsoz, M.; Grant, D. H.; Cai, X.; Parrado, C. Anal. Chem. 1997, 69, 1457-1460. (19) (a) Weatherly, S. C.; Yang, I. V.; Thorp, H. H. J. Am. Chem. Soc. 2001, 123, 1236-1237. (b) Weatherly, S. C.; Yang, I. V.; Armistead, P. A.; Thorp, H. H. J. Phys. Chem. B 2003, 107, 372-378.
Analytical Chemistry, Vol. 77, No. 5, March 1, 2005 1361
bases are protected by base-pairing and oxidation is relatively slow, while guanine in single-stranded (ss) DNA is more accessible and is ∼200-fold more reactive toward oxidation.17 We employed this strategy with soluble and polymeric catalysts to detect DNA damage from styrene oxide,20-22 which forms covalent adducts with guanine and adenine moieties that disrupt the double helix, increase the DNA oxidation rate, and enhance the catalytic signal. For optimum efficiency, sensors for genotoxicity screening should produce the metabolites as well as detect the resulting DNA damage. We recently used cyt P450 enzymes and other iron heme proteins in ultrathin films with DNA on single electrodes to generate styrene oxide from styrene, detected the reaction of the styrene oxide with DNA, and validated styrene oxide adduct formation on the DNA with LC-MS.22-24 As a prototype array strategy to investigate genotoxic bioactivation from several cyt P450 isoforms simultaneously, we tested arrays of eight individually addressable electrodes coated with enzyme-DNA films and control films. Benzo[a]pyrene (BP) was chosen as a model for the polycyclic aromatic hydrocarbons (PAHs) as its mutagenic and tumorigenic activity has been studied extensively in vitro and in vivo.25-28 PAHs are widely found in cigarette smoke, soot, grilled meat, and automobile exhaust.29 They are bioactivated in the human liver by cyt P450 monooxygenases to highly reactive diol epoxides,30-32 which bind chemically to cellular DNA and can cause gene mutation, deletion, and cancer.28,33 Reactions between these metabolites and DNA lead to structural changes that can serve as biomarkers for genetic disease.34,35 Benzo[a]pyrene undergoes metabolic biotransformation to a variety of oxy metabolites, including four enantiomers of benzo[a]pyrene diol epoxide (BPDE) with different tumorigenic activities. Chirality is important, and the (+)-anti-BPDE enantiomer is more mutagenic in mammalian cells and highly tumorigenic in mice.36,37 In this paper, ds-DNA films were first assembled on electrode arrays using layer-by-layer electrostatic adsorption, and the result(20) Zhou, L.; Rusling, J. F. Anal. Chem. 2001, 73, 4780-4786. (21) Mugweru, A.; Rusling, J. F. Anal. Chem. 2002, 74, 4044-4049. (22) Wang, B.; Rusling J. F. Anal. Chem. 2003, 75, 4229-4235. (23) Zhou, L.; Yang, J.; Estavillo, C.; Stuart, J. D.; Schenkman, J. B.; Rusling, J. F. J. Am. Chem. Soc. 2003, 125, 1431-1436. (24) Mugweru, A.; Jing Yang, J.; Rusling, J. F. Electroanalysis 2004, 16, 11321138. (25) Cheng S. C.; Hilton, B. D.; Roman, J. M.; Dipple, A. Chem. Res. Toxicol. 1989, 2, 334-340. (26) Matter, B.; Wang, G.; Jones, R.; Tretyakova, N. Chem. Res. Toxicol. 2004, 17, 731-741. (27) Balu, N.; Padgett, W. T.; Lambert, G. R.; Swank, A. E.; Richard, A. M.; Nesnow, S. Chem. Res. Toxicol. 2004, 17, 827-838. (28) Osborne, M. R., Crosby, N. T., Eds. Benzopyrenes; Cambridge University Press: Cambridge, 1987. (29) Strauss, B. S. Cancer Res. 1992, 52, 249-253. (30) Conney, A. H. Cancer Res. 1982, 42, 4875-4917. (31) Singer, B.; Grunberger, D. Molecular Biology of Mutagens and Carcinogens; Plenum Press: New York, 1983. (32) Harvey, R. G. Polycyclic Aromatic Hydrocarbons: Chemistry and Carcinogenicity; Cambridge University Press: Cambridge, 1991. (33) Hecht, S. S. J. Natl. Cancer Inst. 1999, 91, 1194-1210. (34) Hemminki, K. Carcinogenesis 1993, 14, 2007-2012. (35) Cantoreggi, S.; Lutz, W. K.; Hemminki, K. Carcinogenesis 1993, 14, 355360. (36) Buening, M. K.; Wislocki, P. G.; Levin, W.; Yagi, H.; Thakker, D. R.; Akagi, H.; Koreeda, M.; Jerina, D. M.; Conney, A. H. Proc. Natl. Acad. Sci. U.S.A. 1978, 75, 5358-5361. (37) Slaga, T. J.; Bracken, W. J.; Gleason, G.; Levin, W.; Yagi, H.; Jerina, D. M.; Conney, A. H. Cancer Res. 1979, 39, 67-71.
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Chart 1. Top View of the Sensor Array Setupa
a Each of eight PG Electrodes (CH 1-8) are coated with appropriate films and linked to one of the eight channels of the multipotentiostat, with common counter and reference electrodes. Individual electrodes are removable for polishing and incubations. They are reinserted into the array for electrochemical analyses.
ing sensor array was shown to detect DNA damage after incubation with BPDE. Then, cyt P450cam, cyt P450 1A2, and myoglobin were incorporated into DNA films on the array electrodes to generate BPDE from benzo[a]pyrene. The resulting DNA damage from metabolites generated in situ on the different array elements was detected simultaneously using catalytic square wave voltammetry. EXPERIMENTAL SECTION Chemicals and Materials. Salmon testes (ST) ds-DNA was from Sigma (2000 average base pairs, 41.2% G/C). Horse heart myoglobin (Mb) from Sigma (MW 17 400) was dissolved in pH 5.5 buffer and filtered through an Amicon YM30 membrane (30 000 MW cutoff). 38 Pseudomonas putida Cyt P450cam, MW 46 500, was expressed in Escherichia coli DH5R containing P450cam cDNA and purified and characterized as reported previously.39 cDNA-expressed human Cyt P450 1A2, MW 52 000, was similarly expressed, isolated, and purified from E. coli using a published method.40 Poly(diallydimethylammonium chloride) (PDDA) and BP were from Aldrich. Anti-benzo[a]pyrene-7,8-diol9,10-expoxide (BPDE) was a racemic mixture of (+)-anti-BPDE and (-)-anti-BPDE obtained from the NCI chemical carcinogen reference standard repository (Midwest Research Institute, Kansas City, MI). Water was treated with a Hydro Nanopure system to specific resistivity of >18 MΩ cm. All other chemicals were reagent grade. Voltammetry. CH Instruments model 1030 multipotentiostat was used for square wave (SWV) and cyclic voltammetry (CV). The electrochemical cell employed a saturated calomel reference electrode (SCE), a Pt wire counter electrode, and array of eight film-coated working electrode disks (diameter 3 mm) of ordinary basal plane pyrolytic graphite (PG, Advanced Ceramics). (Chart 1). SWV conditions were 4-mV step height, 25-mV pulse height, (38) Nassar, A.-E. F.; Willis, W. S.; Rusling, J. F. Anal. Chem. 1995, 67, 23862392. (39) Zhang, Z.; Nassar, A.-E. F.; Lu, Z.; Schenkman, J. B.; Rusling, J. F. J. Chem. Soc., Faraday Trans. 1997, 93, 1769-1774. (40) Fisher, C. W.; Caudle, D. L.; Martin-Wixtrom, C. FASEB J. 1992, 6, 759764.
and 15-Hz frequency. Catalytic SWV was done in 50 µM Ru(bpy)32+ in 10 mM pH 5.5 acetate buffer containing 0.5 M NaCl. Solutions were purged with purified nitrogen, and a nitrogen atmosphere was maintained subsequently. Film Construction. PG electrodes were abraded on 400-grit SiC paper and then ultrasonicated in water and ethanol for 30 s. Alternate layer-by-layer electrostatic adsorption was used to assemble DNA or enzyme films on each freshly prepared PG surface in the array.20,41 Optimal conditions were derived from previous work.22,42 Layers of PDDA, ds-DNA, and enzyme were adsorbed alternately from 25-µL droplets placed on each of the PG array elements for 15 min, rinsing with water between adsorption steps. Adsorbate solutions were as follows: (a) 2 mg mL-1 PDDA in 50 mM NaCl; (b) 2 mg mL-1 DNA in 5 mM pH 7.1 Tris buffer containing 0.50 M NaCl; (c) 3 mg mL-1 Mb in 10 mM pH 5.5 acetate buffer; (d) 1 mg mL-1 cyt P450cam in 10 mM pH 5.5 acetate buffer; and (e) 1 mg mL-1 cyt P450 1A2 in 10 mM pH 5.5 buffer. DNA films with no enzyme are denoted in order of layer deposition as (PDDA/DNA)2, while enzyme-DNA films are denoted as PDDA/DNA/(enzyme/DNA)2. Incubation of DNA Films with BPDE. The array of (PDDA/ DNA)2-coated electrodes was first immersed in an electrochemical cell containing 50 µM Ru(bpy)32+ to measure initial SWV peak currents (Ip,i). Then these electrodes were detached from the Teflon array holder and incubated in thermostated reactors at 37 °C containing 35 µM BPDE (or 35 µM BP for controls) in pH 7.1 Tris buffer. Individual electrodes were incubated for different times. After washing with water, the electrodes were reassembled into the array and transferred into an electrochemical cell containing Ru(bpy)32+ to record the final SWV peak currents (Ip,f). Incubation of Enzyme-DNA Films with BP. Four (enzyme/ DNA)2 electrodes were first scanned by SWV in electrochemical cell containing 50 µM Ru(bpy)32+ to obtain average initial peak currents (Ip,i). Fresh (enzyme/DNA)2 electrodes were then incubated in buffer containing 50 µM BP and 1 mM H2O2 at 25 °C in 10 mL of pH 7.1 Tris buffer containing 50 mM NaCl.22,42 After washing with water, these electrodes were reassembled into the array and immersed in buffer containing 50 µM Ru(bpy)32+ to record final SWV peak currents (Ip,f). Safety Note: BP and BPDE are suspected human carcinogens. Gloves were worn, and all weighing and manipulations were done under a closed hood. All reactions were carried out in closed vessels. Incubation of DNA Solutions with BPDE. The 2 mg mL-1 ST-DNA in pH 7.1 tris-HCl buffer (5 mM) was incubated with 200 µM BPDE for 48 h at 37 °C, and then unreacted BPDE was extracted with ethyl acetate (3×) and diethyl ether (2×). The DNA solution was vacuum-dried, and the residue was dissolved in formic acid following a previously reported DNA hydrolysis (41) (a) Lvov, Y., Mohwald, H., Eds. Protein Architecture: Interfacing Molecular Assemblies and Immobilization Biotechnology; Marcel Dekker: New York, 2000; pp 125-167. (b) Rusling, J. F., Lvov, Y., Mohwald, H., Eds. Protein Architecture: Interfacing Molecular Assemblies and Immobilization Biotechnology; 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. (42) (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. (c) Joseph, S.; Rusling, J. F.; Lvov, Y. M.; Freidberg, T.; Fuhr, U. Biochem. Pharmacol. 2003, 65, 1817-1826.
Figure 1. Square wave voltammetry of eight (PDDA/DNA)2 films in the array in pH 5.5 acetate buffer + 0.5 M NaCl containing 50 µM Ru(bpy)32+. SWV amplitude, 25 mV; frequency, 15 Hz; step, 4 mV. Baselines are offset for clarity.
procedure.22 The solutions were heated at 150 °C for 40 min and then dried. The residue was dissolved in pH 4.5 ammonium acetate and filtered through 0.2-µm filters (GHP, Waters) before LC-MS. Liquid Chromatography-Mass Spectrometry. A PerkinElmer LC with diode array (255 and 280 nm) and mass spectrometer (Micromass, Quattro II) detection was used. The HPLC column was Restek Ultra C-18 reversed phase (i.d. 2.1 mm, length 10 cm, particle size 5 µm). Solvents were (A) 10 mM pH 4.5 ammonium acetate and (B) 99.95% methanol with 0.05% acetate acid. Solvent program was as follows: initial hold 10% B for 5 min (T ) 5); 10-min linear gradient to 50% B (T ) 15); 10-min linear gradient to 75% B (T ) 25); 15-min hold at 75% B (T ) 40); 5-min linear gradient to 10% B (T ) 45); ending with 5-min hold at 10% B (T ) 50). Single ion reaction spectra were at cone voltage 15 V with an electrospray voltage of 3.5 kV in the positive ion mode (ESI). RESULTS Array Reproducibility. Figure 1 shows SWVs of the eight (PDDA/DNA)2 films in pH 5.5 acetate buffer containing 50 µM Ru(bpy)32+. Oxidation peaks were found at 1.05 V versus SCE resulting from the catalytic oxidation of guanines on DNA in the films by Ru(bpy)32+(eqs 1 and 2). Average peak current was 13.2 ( 1.4 µA, indicating reasonable electrode-to-electrode reproducibility, with variance due mainly to reproducibility of electrode areas and film formation. Detecting DNA Damage from BPDE. We first assessed the ability to detect DNA damage from BPDE by catalytic SWV as well as the reusability of (PDDA/DNA)2 films. After incubation in buffer for 10 min, a film was scanned twice in 50 µM Ru(bpy)32+ (Figure 2). The second SWV of this electrode gave a smaller peak current than the first scan. The same film was then incubated in 35 µM BPDE for 10 min, and SWV was recorded again. This last peak current was significantly larger than that of the second scan and was even larger than the peak in the first scan. All of the array electrodes exhibited similar phenomena, demonstrating that the films can be employed for at least three uses in this type of application. Analytical Chemistry, Vol. 77, No. 5, March 1, 2005
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Table 1. Comparison of Enzyme Loading and Relative Turnover Rates myoglobin cyt 450cam cyt 450 1A2 Γ,a nmol cm-2 relative turnover rate (nmol of enzyme)-1 min-1
0.26 0.92
0.060 3.0
0.054 3.5
a Surface concentration of enzyme estimated by integration of slow scan rate CVs.
Figure 2. SWVs of the same (PDDA/DNA)2 film after incubation in buffer and 35 µM BPDE for 10 min.
Figure 3. Influence of incubation time with 35 µM BPDE on average SWV current ratio of (PDDA/DNA)2 films (n ) 4) in pH 5.5 buffer containing 50 µM Ru(bpy)32+. Also shown are controls representing the incubation of (PDDA/DNA)2 films with 35 µM BP. Error bars represent standard deviations for n ) 4.
The smaller peak for the second scan is caused by consumption of guanine in the SWV analysis because of irreversible guanine oxidation.20 However, the peak current greatly increased after incubation in BPDE for 10 min. In later studies with BPDE as the direct DNA damage reagent, one new film was first incubated in the control buffer without BPDE to get initial current (Ip,i) and then incubated with BPDE to obtain the final current (Ip,f). Presenting the data as the ratio Ip,f/Ip,i compensates partly for electrode-to-electrode variability. Figure 3 shows the Ip,f/Ip,i ratios of (PDDA/DNA)2 films after incubations in BPDE solutions and controls. Incubating with BP did not influence the peak currents significantly (see Figure S1, Supporting Information, for raw data), and BP controls gave nearly the same peak currents as in buffer alone. When films were incubated in 35 µM BPDE, the peak currents were larger than that of the controls and increased with time over 30 min. Relatively small error bars indicate that the time course of the DNA reaction can be readily monitored using the Ip,f/Ip,i ratio. Compared with a smaller slope for incubation of similar films for 8-10 mM styrene oxide,20 the larger slope for 35 µM BPDE found here suggests a faster reaction rate for DNA with BPDE than with styrene oxide. 1364 Analytical Chemistry, Vol. 77, No. 5, March 1, 2005
Bioactivating Benzo[a]pyrene with Enzyme/DNA Films. Cytochrome P450s are known to metabolize BP to BPDE.43,44 Our previous results with olefinic molecules showed that myoglobin and cyt P450s in thin films activated by small amounts of H2O2 can catalyze epoxidations,22,23,42,45,46 as well as produce known drug metabolites.42c We incorporated cyt P450cam, cyt P4501A2, and myoglobin (Mb mimics the oxidative catalysis of cyt P450s) in the DNA films to metabolize BP. In these experiments, if the same film was used to obtain the initial control current and then incubated in 50 µM BP and 1 mM H2O2, the final current was smaller than the initial current. This suggests that the relatively small amount of BPDE generated from BP in the protein-DNA films may produce DNA damage in lesser amounts than the DNA consumed during oxidative SWV analysis. Thus, in the following studies, electrodes were analyzed by SWV only once, before or after bioactivation. The enzymes in the PDDA/DNA/(enzyme/DNA)2 films were characterized by their known electrochemical properties.22-24,42,45 For example, pairs of reduction-oxidation peaks corresponding to the reversible MbFeIII/MbFeII conversion in PDDA/DNA/ (Mb/DNA)2 films were observed with a midpoint potential of -0.3 V under anaerobic conditions (Figure S2, Supporting Information) as reported previously.42 A surface concentration of 0.26 nmol cm-2 was estimated for Mb by integration (Table 1) of the CV reduction peak at low scan rates where nonideal thin-film voltammetry47 was found. Similar data were collected for films containing cyt P450s. Electroactivity of these films is illustrated by SWV peaks at about -0.27 V versus SCE corresponding to their heme FeIII/FeII redox couples (Figure S3, Supporting Information).45,46 Our previous results showed that less than a few millimolar hydrogen peroxide does not significantly damage ds-DNA over the incubation times used in this work.22,23 Thus, 1 mM H2O2 was used to activate the enzymes. Figure 4 shows the SWVs of eight PDDA/DNA/(Mb/DNA)2 films in the array in pH 5.5 buffer containing 50 µM Ru(bpy)32+ after incubations with 50 µM BP and 1 mM H2O2 for different times. Two electrodes served as replicates for each incubation time. Each replicate pair of electrodes gave similar catalytic peak heights at 1.05 V. The peak (43) Shimada, T.; Martin, M. V.; Pruess-Schwartz, D.; Marnett, L. J.; Guengerich, F. P. Cancer Res 1989, 49, 6304-6312. (44) Vahakangas, K.; Raunio, H.; Pasanen, M.; Sivonen, P.; Park, S. S.; Gelboin, H. V.; Pelkonen, O. J. Biochem. Toxicol. 1989, 4, 79-86. (45) Munge, B.; Estavillo, C.; Schenkman, J. B.; Rusling, J. F. ChemBiochem 2003, 4, 82-89. (46) Estavillo, C.; Lu Z. Q.; Jansson, I.; Schenkman, J. B.; Rusling, J. F. Biophys. Chem. 2003, 104, 291-296. (47) (a) Rusling, J. F.; Zhang, Z. In Handbook of surfaces and interfaces of materials; Nalwa, H. S, Ed.; Academic Press: New York, 2001, (b) Rusling J. F.; Zhang Z. In Biomolecular Films; Rusling, J. F. Ed.; Marcel Dekker: New York, 2003; pp 1-64.
Figure 6. SWVs of PDDA/DNA/(Cyt P450cam/DNA)2 films in 50 µM Ru(bpy)32+ after incubations with 50 µM BP and 1 mM H2O2 for 10 min. Also shown are controls after incubation in 1 mM H2O2 or buffer for 10 min.
Figure 4. SWVs of PDDA/DNA/(Mb/DNA)2 array elements in 50 µM Ru(bpy)32+ after incubation with 50 µM benzo[a]pyrene and 1 mM H2O2 for different times. A dashed line and a solid line of the same color belong to replicates incubated for the same time.
Figure 7. Influence of incubation time with 50 µM benzo[a]pyrene and 1 mM H2O2 on the peak current ratios from SWV of PDDA/DNA/ (enzyme/DNA)2 films. Control is PDDA/DNA/(Mb/DNA)2 film in 50 µM benzo[a]pyrene alone (4 replicates for Mb films, 3 each for cyt P450 films). Error bars represent standard deviations for n ) 4. Figure 5. Influence of incubation time with 50 µM benzo[a]pyrene and 1 mM H2O2 on SWV catalytic peak current in 50 µM Ru(bpy)32+ for PDDA/DNA/(Mb/DNA)2 films (n ) 4). Controls represent the incubation of PDDA/DNA/(Mb/DNA)2 films with 50 µM benzo[a]pyrene but no H2O2. Error bars represent standard deviations for n ) 4.
currents after incubation in 50 µM BP and 1 mM H2O2 were larger than for the controls incubated without H2O2 for equivalent times. Moreover, peak heights increased markedly with increment in the incubation time of the films and reproducibility was very good. Figure 5 shows the peak responses of (Mb/DNA)2 films after incubation in 50 µM BP and 1 mM H2O2 for different times. Average SWV peak currents for (Mb/DNA)2 films increased with incubation time. Control incubations in either hydrogen peroxide or BP alone gave no significant increases in peak current, which were similar to those of new films after incubation in buffer. DNA films containing cyt P450cam and cyt P450 1A2 gave qualitatively similar results to Mb films. Figure 6 shows SWVs of PDDA/DNA/(cyt P450cam/DNA)2 films after incubations for 10 min. Compared with controls, the peaks at 1.05 V increased markedly after incubation with BP and H2O2. The control in 1 mM H2O2 alone gave no significant increase in peak current compared to control in buffer. Figure 7 compares the change in peak ratios for DNA/enzyme films after incubation with BP and H2O2. For cyt P450cam and
cyt P450 1A2, a relatively rapid increase in SWV peak current was found for the first 5 min, followed by a slower increase from 5 to 30 min. The initial slope of peak ratio versus time was largest for Mb. Cyt P450 1A2 gave a slightly larger initial slope than cyt P450cam. The rate of increase for cyt P450 1A2 was ∼22% less with Mb. As discussed later, part of the slope difference represents a difference in enzyme reactivity and part a difference in relative amounts of enzyme in the films. Surface concentrations (Γ) of enzyme in the films (Table 1) allowed estimates of the specific reactivity of each enzyme to produce DNA-reactive metabolites. Comparisons of Γ-values with quartz crystal microbalance measurements of total enzyme in similar films23 suggest that the voltammetrically measured values are good estimates for total enzyme concentration in these ultrathin films. Thus, relative in vitro metabolic turnover rates were obtained using the initial slopes of the SWV peak ratios versus incubation time plots (Figure 7) divided by nanomoles of enzyme in each film (Table 1). Results suggest that cyt P450cam and cyt P450 1A2 have significantly higher activities than Mb for metabolizing BP to toxic metabolites. LC-MS Analysis of DNA-BPDE Adducts. The formation of BPDE-guanine adducts under our reaction conditions was confirmed by LC-MS (Figure 8). Incubations for 48 h were necessary to obtain sufficient adducts for analysis. After incubation Analytical Chemistry, Vol. 77, No. 5, March 1, 2005
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Figure 8. LC-ESI-MS traces of the reaction mixtures with BPDE at 37 °C. (A) 0.3 mg mL-1 guanine incubated with 200 µM BPDE for 48 h. Output displayed as total ion current at m/e 152 (guanine + H+) and m/e 454 (guanine-BPDE + H+). (B) 2 mg mL-1 of ST dsDNA incubated with and without 200 µM BPDE for 48 h and then acid-hydrolyzed. Output displayed as ion current at m/e 454 only.
of guanine with BPDE, analysis by LC-MS gave a large peak at 7.7 min at m/e 152 (Figure 8A), corresponding to [guanine + H]+. Another peak at m/e 454 was observed at ∼23 min, which corresponds to [BPDE-guanine + H]+, confirming the formation of BPDE-guanine adduct. In addition, ST ds-DNA was incubated with BPDE for 48 h, and the subsequently acid hydrolyzed sample showed a large peak at 23 min at m/e 454 (Figure 8B), corresponding to [BPDE-guanine + H]+. This peak was not found in DNA controls incubated without BPDE. To further confirm the presence of BPDE-guanine adduct, tandem mass spectrometry with collision-induced dissociation was used to generate fragment ions from the parent ion m/e 454. The expected daughter peaks48,49 [G + H]+ at m/e 152 and [BPDE + H]+ at m/e 303 were observed, confirming the formation of BPDEguanine adduct under our experimental conditions. DISCUSSION Results in Figures 2-7 show clearly that BP metabolitegenerated damage caused to ds-DNA in enzyme/DNA films can be detected by catalytic SWV. No significant current increases were observed in control experiments without BPDE, with 1 mM H2O2 alone, or with BP alone (Figures 3, 5, and 7). Formation of BPDE-guanine adducts under our reaction conditions was (48) Wang, J. J.; Marshall, W. D.; Law, B.; Lewis, D. M. Int. J. Mass Spectrom. 2003, 230, 45-55. (49) Barry, J. P.; Norwood, C.; Wouros, P. Anal. Chem. 1996, 68, 1432-1438.
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demonstrated by LC-MS on the DNA samples after incubation with BPDE (Figure 8). After reaction of the DNA in the films with BPDE, the SWV peak current increases presumably because of disruption of the double helix by the reactions of DNA with BPDE.50 Similar reasoning explained reported increases in catalytic SWV peaks after incubation of DNA films with DNA-reactive epoxides and methylating agents.20-24 BPDE binds covalently to N2 groups of guanine or N6 groups of adenine residues in DNA, and at least four different BPDE-DNA adducts have been identified.25,26,51 Furthermore, based on linear dichroism and other spectroscopic data, it was suggested that the hydrophobic flat aromatic ring of BPDE interacts noncovalently with ds-DNA,52 including by intercalation between adjacent base pairs.53 In ds-DNA, guanines are relatively protected from reactions with oxidants. However, strong interactions between BPDE and DNA, in particular the formation of bulky BPDE-nucleobase adducts, are likely to expose additional guanine residues in DNA to the oxidant Ru(bpy)33+, causing faster oxidation rates resulting in larger SWV peak current than with intact ds-DNA. Thus, damaged DNA exhibits larger catalytic SWV peaks than intact ds-DNA. With increasing incubation time, more BPDE-DNA adducts are presumably formed, giving larger catalytic peak currents. In our previous toxicity sensor studies, similar DNA films were incubated with 8-10 mM styrene oxide to obtain somewhat smaller catalytic peak ratios per unit incubation time,20-22 In the present work, BPDE at the very low concentration of 35 µM provided larger peak currents than styrene oxide for comparable incubation times (Figure 3) with all other conditions the same. This result suggests that BPDE reacts significantly faster than styrene oxide with DNA and is a more potent genotoxic agent, in agreement with previous results utilizing PCR amplification.54 We reported previously that the activation of layered polyion films of various cyt P450s and other iron heme proteins with H2O2 produces styrene oxide from styrene.23.24,42,45,46 Furthermore, peroxides have been widely used to initiate the catalytic reactions of cyt P450s.55 Cumene hydroperoxide has been used to mediate BP metabolite formation in microsomal cyt P450s.56 Cyt 450s serve as monooxygenases with the ability to catalyze the epoxidation reaction. The iron heme in the enzyme is oxidized by peroxide to a presumed oxyferryl radical intermediate, which transfers an oxygen atom to the olefinic double bond.42,45,46 BP-induced cyt P450s in mouse and human liver cells convert BP to a number of metabolites, including the DNA-reactive BPDE43,44,57 in a complex biochemical process. BP is initially oxidized into BP-4,5-diol, BP-7,8-diol, and BP-9,10- diol and other (50) Adams, S. P.; Laws, G. M.; Storer, R. D.; Deluca, J. G.; Nichols; W. W. Mutat. Res. 1996, 368, 235-248. (51) Geacintov, N. E.; Cosman, M.; Hingerty, B. E.; Amin, S. Broyde, S.; Patel, D. J. Chem. Res. Toxicol. 1997, 10, 111-146. (52) Geacintov, N. E.; Yoshida, H.; Ibanez, V.; Harvey, R. G. Biochem. Biophys. Res. Commun. 1981, 100, 1569-1577. (53) MacLeod, M. C.; Selkirk, J. K. Carcinogenesis 1982, 3, 287-292. (54) Laws, G. M.; Skopek, T. R.; Reddy, M. V.; Storer, R. D.; Glaab, W. E.; Mutat. Res. 2001, 484, 3-18. (55) Werringloer, J.; Kawano, S.; Estabrook, R. W. In Microsomes, drug oxidation and chemical carcinogenesis; Coon, M. J., Ed.; Academic Press: New York, 1980; pp 403-406. (56) Winston, G. W.; James, M. O.; Jewell, C. S. E. Polycyclic Aromat. Compd. 1993, 3, 1079-1086. (57) McManus, M. E.; Burgess, W. M.; Veronese, M. E. Cancer Res. 1990, 50, 3367-3376.
products. Then BP-7,8- diol is further oxidized to 9,10-oxides and anti- and/or syn-BPDE. Among the four BPDEs produced, the (7R,8S,9S,10R) enantiomer anti-BPDE is generally formed to the greatest extent and shows the highest carcinogenic activity.58 The key experimental results for enzyme/DNA arrays are that SWV peak currents incubated in benzo[a]pyrene and hydrogen peroxide increased with reaction time (Figures 4-7) consistent with DNA damage. Taken in the context of the above discussion of the literature and with results in Figure 3, our data suggest that Mb, cyt P450cam, and cyt P450 1A2 in the films metabolize BP in the presence of H2O2, and that the in situ generated metabolites, presumably rich in BPDE, cause DNA damage. Cyt P450 enzyme distributions show interindividual variability in humans, and different cyt P450s may be involved in the metabolism of specific chemical procarcinogens.59 Thus, it is important to know which enzymes bioactivate specific molecules for genotoxicity, as it may be a basis for individually variable toxic effects. Figure 7 compares the enzyme specificity toward in vitro BP metabolism causing DNA damage. The initial rate of increase in the SWV peak ratio for cyt P450cam and cyt P450 1A2 was ∼22% less than that with Mb films. However, because of their larger molecular size compared to Mb, the surface concentration (nmol cm-2) of cyt P450cam in the enzyme/DNA films is only ∼1/5 of that of Mb as reported proviously.23 The surface concentration of cyt P450 1A2 was slightly smaller than that of cyt P450cam (Table 1). Relative in vitro turnover rates suggest that cyt P450cam and cyt P450 1A2 have considerably higher activities for generating genotoxic BP metabolites than Mb. On the other hand, given the relative standard deviation of the SWV data of (10%, the activity of cyt P450 1A2 can be considered similar to that of cyt P450cam, or at best only slightly larger. Shimada et al.43,60 reported that cyt P450 1A1 and 1B1 had similar catalytic specificities toward BP, whereas cyt P450 1A2 and 3A4 showed much lower rates for BP bioactivation. Cyt P450 2E1 was almost inactive in the activation of polycyclic aromatic hydrocarbons. Results in Table 1 are in general agreement with these previous findings. That is, cyt P450 1A2 and cyt P450cam have somewhat similar activities for generation of genotoxic metabolites, while bioactivation of BP by the enzyme mimic Mb is much slower In general, the present work demonstrates the advantages of individually addressable electrode arrays and DNA/enzyme films (58) Sticha, K. R. K.; Staretz, M. E.; Wang, M.; Liang, H.; Kenney, P. M. J.; Hecht S. S. Carcinogenesis 2000, 21, 1711-1719. (59) Guengerich, F. P. Carcinogenesis 2000, 21, 345-351. (60) Shimada, T.; Oda, Y.; Gillam, Elizabeth M. J.; Guengerich, F. P.; Inoue, K. Drug Metab. Dispos. 2001, 29, 1176-1182.
for bioactivated toxicity studies: (i) experimental throughput is increased; (ii) incorporated replicate elements and controls built into arrays provide statistically relevant results more rapidly; (iii) arrays can be used to simultaneously measure differences in genotoxic metabolic activity of a series of enzymes. The method has sufficient sensitivity and is rapid as shown by obtaining easily measurable DNA damage rates within ∼5 min at micromolar BP concentration for the relatively low activity cyt P450s used here and, for Mb, a cyt P450 mimic with less than 1/3 the activity of the cyt P450s used. In the present case, we employed eight-electrode arrays for proof of concept because of commercial availability of an eightelectrode potentiostat. In future practical applications, larger arrays could easily be used. Further, the repetitive features of layer-bylayer film construction suggest that film formation can be automated.41 In summary, electrode arrays featuring enzyme/DNA films were used to estimate genotoxic bioactivation of benzo[a]pyrene for several different enzymes simultaneously. These toxicity sensor arrays hold promise for screening new chemicals for potential genotoxicity and identifying specific enzymes that generate genotoxic metabolites. Efforts are underway in our laboratories to validate the methodology with a library of procarcinogens. Future toxicity array sensors may involve all the important human liver cyt P450s on larger microarrays. ACKNOWLEDGMENT This work was supported by U.S. PHS Grant ES03154 from the National Institute of Environmental Health Sciences (NIEHS), NIH, USA. BPDE was generously provided by the National Cancer Institute’s chemical carcinogen reference standards repository operated under contract by Midwest Research Institute, N02-CB07008. The authors thank David Osier for construction of electrode arrays, and Carmelita Estavillo, Jing Yang, and Maricar Tarun for experimental assistance. SUPPORTING INFORMATION AVAILABLE Three additional figures presenting the peak current change after BPDE incubation and voltammetry of enzymes in the films. This material is available free of charge via the Internet at http:// pubs.acs.org. Received for review September 28, 2004. Accepted December 16, 2004. AC0485536
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