Electrochemical Detection of anti-Benzo [a] pyrene Diol Epoxide DNA

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Electrochemical Detection of anti-Benzo[a]pyrene Diol Epoxide DNA Damage on TP53 Codon 273 Oligomers Jennifer E. Satterwhite, Amanda M. Pugh, Allison S. Danell, and Eli G. Hvastkovs* Department of Chemistry, 300 Science and Technology Building, East Carolina University, Greenville, North Carolina 27858, United States

bS Supporting Information ABSTRACT: DNA damage from (þ/
)-anti-benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE) at a hotspot TP53 gene sequence was electrochemically detected. BPDE was exposed to gold electrode immobilized double-stranded DNA oligomers followed by voltammetric measurements in the presence of redox-active C12H25V2þC6H12V2þC12H25 (V2þ = 4,40 -bipyridyl or viologen, C12-viologen). Square wave voltammograms from BPDE-exposed DNA-modified electrodes showed the emergence of a C12-viologen
DNA complex at
0.37 V versus Ag/AgCl. The peak current intensity of this redox wave was dependent on both BPDE concentration and exposure time. Controls with alternate xenobiotics and DNA sequences showed this redox wave to be primarily due to BPDE damage at the wild-type DNA sequence. The detection limit was determined to be approximately 170 nM BPDE. Mass spectrometry and UV thermal melting experiments provided insight into the BPDE reaction and mirrored the sensor results. This report demonstrates that an electrochemical hybridization sensor can be used to detect sequence-related xenobiotic DNA damage.

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iving organisms are continually exposed to chemicals that originate outside the body, or xenobiotics. Metabolism processes that attempt to facilitate elimination of these molecules can introduce reactivity in a process called bioactivation.1
3 Reactive species can react with and damage biomaterial, such as DNA and proteins.2,3 Damaged DNA is termed genotoxicity, and some genotoxic lesions are not effectively or correctly repaired.4 Persistent DNA adducts cause errors upon replication, which eventually result in permanent mutations.5
7 Bioactivated xenobiotics typically do not damage DNA at random; rather specific sites within the genome are attacked.8 These sites are termed “hotspots.” A well-studied example of this involves benzo[a]pyrene (BP), a xenobiotic introduced into the body primarily via cigarette smoking.9,10 BP is metabolized and adducts DNA at site-specific locations within the TP53 gene.10 TP53 codes for the p53 protein, which is involved in cellular apoptosis.11
14 Analysis of TP53 DNA that was exposed to the ultimate BP metabolite (þ)-anti-benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE) showed that BPDE specifically adducted guanines at codons 157, 248, and 273.10 Damage at these genetic sites affects transcription by altering the amino acids that form the p53 DNA binding region.15 DNA mutations at these particular codons are prevalent in many cancers.8,9,15 Hotspot elucidation and detection is not a trivial task. Typical methods to perform this analysis involve exposing DNA or cells to a xenobiotic of interest, followed by DNA separation, enzymatic digestion, specialized polymerase chain reaction (PCR) amplification protocols, gel separation, and sequencing.6,7,10,16
20 Higher throughput methods have been developed to detect DNA damage,21 r 2011 American Chemical Society

but these typically are selective for general genotoxicity types, i.e., oxidative damage,22 xenobiotic adduct,23,24 or assay the production of reactive metabolites.25
28 Detailed disease etiological information can be assessed by determining definitive genetic damage sites;10 therefore, newer strategies must be explored to couple DNA sequence genotoxicity specificity to a highthroughput platform. Electrochemical hybridization sensors provide a convenient DNA analysis platform.29,30 Short DNA sequences can be detected based on specific recognition of two complementary strands, and detection can take place via numerous electrochemical transduction approaches. Very sensitive approaches utilize elaborate signal amplification schemes involving enzyme or nanoparticle modifications.31 A facile way to electrochemically monitor DNA hybridization is by detecting redox-active molecules that specifically interact with DNA.31,32 Typically, this entails detecting an intercalating or a groove-binding molecule that provides a characteristic signal in the presence of doublestranded DNA (dsDNA). This method is not the most sensitive in regards to complementary target detection.31 Rather, the benefits of this approach lie in the ability to glean valuable DNA structural information. The Barton group has effectively demonstrated how base pairs that form the DNA π-stack affect electrode communication to a distally located redox-active intercalator.33,34 Mismatches35 and protein binding interactions36 can alter π-stacking and affect Received: November 23, 2010 Accepted: March 23, 2011 Published: March 23, 2011 3327

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Analytical Chemistry electrochemical signals. Wong and Gooding electrochemically assayed cisplatin-induced DNA morphology changes at oligomers containing intrastrand guanine dinucleotide (GG) sequences.37 Cisplatin binding at these sequences kinked the DNA helix, which affected the π-stack leading away from the electrode and the electrochemical response to the intercalator at the tethered DNA 30 -end. DNA oligomers have been detected indirectly on gold electrodes employing redox-active diviologen molecules that bind to DNA in a structure-specific manner.38
40 C12H25V2þC6H12V2þ C12H25 (V2þ = 4,40 -bipyridyl or viologen, termed C12-viologen from here on) was used to distinguish dsDNA from ssDNA38 and also distinguish cDNA
PNA from mismatched DNA
PNA hybrids.39 In the presence of perfectly matched dsDNA or PNA
DNA hybrids, C12-viologen produced dual-wave voltammetry originating from two C12-viologen
DNA bound forms.39 Evidence suggested that the two electrochemical waves were from a minor groove bound C12-viologen population and a second electrostatic bound form.38 Electrochemical signals changed dramatically in the presence of mismatched hybrids or ssDNA indicating that C12-viologen voltammetry was dependent on the DNA helical structure.39 The main analytical focus presented here is to detect carcinogenic BPDE-related DNA damage at a specific gene sequence. Several different types of DNA adducts form from BPDE exposure based on its stereochemistry. Some of these result in an intercalated pyrenyl moiety within the DNA π-stack, but the most mutagenic adduct results from exposure of (þ)-anti-BPDE forming a minor groove located adduct with minimal base pair or base stacking alterations.41 On the basis of its DNA structuredependent voltammetry, it was hypothesized that C12-viologen could be used to detect structure altering xenobiotic DNA adducts. C12-viologen is not known to be a classic DNA intercalator,38,39 and it may be effective facilitating detection of DNA adducts that do not dramatically alter the base-pairing interior. We indeed demonstrate that C12-viologen square wave voltammetry can be used to detect BPDE induced damage at a known TP53 gene sequence hotspot. In addition, we demonstrate BPDE binding selectivity at this DNA sequence. UV
vis thermal melting and mass spectrometry experiments were performed to support the electrochemical sensor findings. Overall, we present a biophysical
analytical method to detect and study an important biological genotoxic process involving a wellknown carcinogen that damages DNA at specific genomic sites.

’ EXPERIMENTAL SECTION Materials. C12-viologen was synthesized according to a previously published procedure.38 1H NMR (600 MHz, Varian) was employed to assess purity, which was determined to be >99%. Benzo[a]pyrene-r-7,t-8-dihydrodiol-t-9,10-epoxide ((), (anti) (anti-BPDE, BPDE) was obtained from the NCI Chemical Carcinogen Reference Standards repository (item L0137), Midwest Research Institute (Kansas City, MO). All DNA oligomers (see below) were purchased from IDT DNA Technologies (Coralville, IA). BP, styrene oxide (SO), mercaptohexanol (MCH), Tris
HCl, Tris base, KH2PO4, K2HPO4, and THF were obtained from SigmaAldrich. All other chemicals were reagent grade and used as received. Solution Preparation. DNA oligomer sequences used in this study were the following: wt.273, 50 -TTT GAG GTG CGT GTT TGT GCC-30 ; wt.273 complement, 50 -GGC ACA AAC ACG CAC CTC AAA-30 ; con.273, 50 -TTT GAG GTG CCT GTT

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TGT GCC-30 ; con.273 complement, 50 -GGC ACA AAC AGG CAC CTC AAA-30 . The wt.273 and con.273 oligomers were both purchased as unmodified and with 50 -thiol modifications. Thiolated oligomers are shipped as disulfides for stability and were reduced following IDT supplied instructions prior to use. The 1 mM stock solutions for all sequences were prepared in 10 mM Tris, 50 mM NaCl, 1 mM EDTA, pH 8.0 (TE buffer). Thiolated DNA aliquots were diluted to 1.5 μM in 1 M H2PO4
/HPO42
buffer, 1 mM EDTA, pH 7.4 (immobilization buffer). Unmodified DNA aliquots were diluted to 2.0 μM in 10 mM Tris, 1 M NaCl, 1 mM EDTA, pH 7.4 (hybridization buffer). All aqueous solutions were made with 18 mΩ 3 cm deionized (DI) water. All DNA was stored at
20 °C prior to use. BPDE is a known carcinogen, and styrene oxide is a suspected carcinogen. Both were handled using proper personal protective equipment. BPDE was distributed into amber vials under a N2 atmosphere and kept sealed at
20 °C before use. Fresh stock solutions were prepared in the amber vials with THF for each day performing damage studies. Fresh BP stock solutions in THF were also prepared each day. Styrene oxide solutions were prepared in electrochemical buffer (see below) and used as needed. Electrode Preparation. Gold 2 mm diameter electrodes used in this study were purchased from CH Instruments (Austin, TX). Electrodes were polished and electrochemically cleaned following a previously reported procedure.38 Typically, this results in a surface roughness factor of 1.3
1.6 and an effective electrode area of 0.04
0.05 cm
2. Freshly cleaned electrodes were immediately rinsed with absolute ethanol, dried under a stream of N2 ,and exposed to a thiolated DNA aliquot for 15 s. The electrode was rinsed with immobilization buffer and DI H2O and exposed to 5 μM MCH in deionized water for 1 h. Following MCH exposure, the electrode was rinsed with DI H2O and hybridization buffer and exposed to an aliquot of the unmodified cDNA oligomer for 1.5 h at 37 °C. The electrode was then rinsed with hybridization buffer followed by immersion in 10 mM Tris, 10 mM NaCl, pH 7.4 (electrochemical buffer) at 37 °C for 30 min to remove nonspecifically bound DNA. Electrochemical Experiments. All electrochemical measurements were performed on a CH Instruments 660A workstation (Austin, TX). DNA-modified electrodes were connected to the potentiostat and placed in electrochemical buffer along with saturated Ag/AgCl reference and Pt counter electrodes. The electrochemical buffer was aggressively purged with N2 before all electrochemical runs. DNA surface coverage (Γ) was verified employing a previously established procedure using ruthenium hexamine (Ru(NH3)62þ/3þ) that binds to the electrode-immobilized DNA.32 The electrode preparation procedure routinely provided dsDNA Γ ∼1.5
2.0  1012 dsDNA oligomers cm
2. After Γ determination, the electrode was rinsed in 2 M NaCl and electrochemical buffer and placed back into the electrochemical cell in 10 mL of fresh electrochemical buffer. Ten micromolar C12-viologen was added to the electrochemical buffer. At this concentration, dual-wave voltammetry in cyclic voltammetry (CV) and square wave voltammetry (SWV) experiments were clearly evident. The following parameters were employed for the following techniques: cyclic voltammetry (scan from 0.1 to
0.7 V, 100 mV/s), square wave voltammetry (
0.2 to
0.75 V scan, 4 mV step, 25 mV amplitude, 2 Hz frequency), chronocoulometry (0 to
0.7 V pulse, 0.25 s pulse width, 0.01 sample interval). DNA Damage with BPDE. After obtaining the baseline SWV signal, electrodes were removed from the cell and exposed to 3328

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Analytical Chemistry 50 μL electrochemical buffer containing a desired BPDE concentration. BPDE was diluted from a THF stock solution for each timed damage run (see above). Damage reactions were performed at 37 °C for a desired period of time, after which the electrode was rinsed with electrochemical buffer and placed back into the C12-viologen electrochemical solution cell. Similar protocols were employed for BP and SO damage experiments. For nonelectrode DNA damage control experiments, 500 μM dsDNA was reacted with 1.25 mM BPDE or 10% SO in 40 μL total volume for 60 min at 4 °C in the dark. The reaction took place either in electrochemical buffer or 50 mM ammonium acetate buffer, pH 6.8. DNA was removed from the reaction medium using Millipore 3K 0.5 mL Amicon Ultra Centrifugal filter units centrifuging at 14 000g for 30 min. This results in a small residual volume in the filter tip containing the DNA. DNA was collected according to the manufacturer protocol, and the concentration was verified using a Thermo Nanodrop 2000c spectrophotometer. UV
Vis Studies. Five micromolar DNA solutions in the respective buffer (50 μL total) were added into thermostatted quartz cells (Starna Cells, Valencia, CA) and placed in a Varian Cary 300 Bio UV
vis spectrophotometer with a temperature controller. The samples were heated from 35 to 75 °C at a rate of 1.00 °C/min with a data collection interval of 0.5 °C. Electrospray Ionization Mass Spectrometry (ESI-MS) Analysis. DNA samples were diluted to 20 μM in 50:50 methanol
water. ESI-MS was conducted on an Esquire 3000plus quadrupole ion trap mass spectrometer (Bruker Daltonics, Billerica, MA), which has unit mass resolution. Ions were created in negative mode ESI using a 3 kV potential difference between the emitter and source aperture. Nitrogen was used to aid in nebulization and desolvation, and heating parameters were varied from 50 to 300 °C depending on the analysis. Data Analysis. OriginPro 8 graphing software was used for all data analysis.

’ RESULTS Genotoxicity Detection at the TP53 Codon 273 Gene. DNA damage from BPDE exposure was detected at an immobilized DNA 21-mer sequence spanning TP53 codons 270
276 (named wt.273 from here on). This particular DNA sequence was chosen as it contains a single previously identified BPDE reaction hotspot at the codon 273 guanine.10 Initial experiments were conducted in order to determine if DNA damage could be detected using C12-viologen with a DNA hybridization sensor platform. Background C12-viologen SWV and CV at immobilized wt.273 were obtained to provide the initial electrochemical signal, representing 0 s damage. CVs showing that the dual-wave C12-viologen response occurs only at a dsDNA (wt.273)-modified Au electrode under these conditions are shown in Supporting Information Figure S1. This response is not seen at a bare or MCH-modified surface. The wt.273-modified electrodes were then exposed to BPDE solutions to test the time-dependent electrochemical response. After selected reaction time periods, the electrode was rinsed and placed in buffer containing C12-viologen for electrochemical monitoring. C12-viologen voltammetry in the presence of DNA provides information related to the DNA structure38,39 and how this structure is affected by BPDE exposure. Figure 1 shows raw (inset) and background-subtracted SWV responses at immobilized wt.273 exposed to 100 μM BPDE for time periods up to 30 min. The background-subtracted voltammetric

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Figure 1. Background-subtracted SWV showing C12-viologen reduction on a wt.273-modified Au electrode upon 100 μM BPDE exposure (10 mM Tris, 10 mM NaCl, pH 7.4). BPDE exposure from 15 s to 30 min causes current increase at
0.37 V denoted by the arrow. Raw SWV plots are shown in the inset. The background was corrected by subtracting the response at 0 s BPDE (black inset).

responses indicate that the C12-viologen
DNA environment changes with BPDE exposure in a time-dependent fashion. The most prevalent potential change is the newly emergent peak at
0.37 V versus Ag/AgCl (Figure 1 arrow). This peak is shifted positive with respect to the signals seen in the presence of undamaged DNA. The emergence of this peak suggests that the BPDE reaction affects the DNA oligomer structure, which in turn alters how C12-viologen interacts with the oligomer. The rise of a positive-shifted peak indicates that a less stable oxidized C12-viologen
DNA complex is formed. The significance of this and possible reasons for the positive potential shift are discussed in more detail below. The
0.37 V peak emerges versus lower background and therefore was used as a quantitative metric to assay BPDE exposure at wt.273. Chronocoulometric analysis demonstrated that the electrochemical response is not due to large-scale BPDEinduced DNA denaturation. Surface-bound C12-viologen concentration (Γ) remained constant throughout BPDE exposure conditions (Supporting Information, Figure S2). Additionally, Ru(NH3)63þ showed very little SWV changes due to BPDE exposure when used as an indirect DNA detection redox probe (Supporting Information, Figure S3). Ru(NH3)63þ has been shown to associate with DNA exclusively in an electrostatic manner.32 These data confirm that the SWV responses shown in Figure 1 are due to perturbations in the DNA helix that alter C12viologen voltammetry. The magnitude of peak current (Ip) increase at
0.37 V was found to be dependent on both DNA sequence and xenobiotic. Figure 2 shows background SWV responses for wt.273 exposed to BPDE, BP, or SO for 1 min. Additionally, BPDE was exposed to a control DNA sequence containing a cytosine
guanine substitution at the codon 273 location (this sequence is named con.273 from here on). The plot shows that the C12-viologen reduction current that emerges after xenobiotic exposure is dramatically muted in situations where BPDE is not exposed to wt.273. Overall, 3329

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Analytical Chemistry the control plots demonstrate that BPDE
DNA damage at this DNA oligomer can be detected using indirect redox reporter C12-viologen to gauge DNA structure. Kinetic Responses. Electrochemical peak current response at
0.37 V was found to be dependent on BPDE concentration. This is shown in Figure 3a. At 1 min BPDE exposure, the
0.37 V peak height increases with BPDE concentration from 10 to 200 μM. The peak potential undergoes additional positive shift at higher BPDE concentrations. This is likely due to C12viologen aggregation at these highly damaged oligomers forcing oxidized viologen (V2þ) centers to come in close intramolecular contact. The positive shift in formal potential denotes stability for the reduced cation radical viologen (V•þ) under these conditions. This process is discussed in detail below. Figure 3b shows the time-dependent response for
0.37 V Ip as a function of BPDE concentration. The BPDE
DNA reaction is faster at elevated BPDE reaction concentrations as demonstrated by the increasing initial slopes shown in Figure 3b. The
0.37 V peak current versus time comparing BPDE and BP exposure to wt.273 and BPDE to con.273 is shown in Supporting

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Information Figure S4. Taken together, Figure 3 and Supporting Information Figure S4 data demonstrate that the DNA damage reaction is kinetically related to the BPDE concentration based on the following: rate ¼ k0 ½BPDE where k0 is the pseudo-first-order rate constant (k0 = k[DNA]). Table 1 summarizes apparent k0 data for wt.273 exposed to BPDE, BP, and SO as well as con.273 exposed to BPDE. These apparent rate constants were acquired by using the initial slopes of the plots in Figure 3b and Supporting Information Figure S4 that are proportional to the initial rates of these reactions. It is clear that the signal increase over time is dependent on xenobiotic and the DNA sequence used. The BPDE reaction rate is significantly faster at wt.273 versus con.273. This suggests that the wt.273 sequence, and specifically the presence of the central guanine base, affects the BPDE reaction rate. This result was expected based on previous reports studying the solution reaction of polynucleotides with BPDE. BPDE has been shown to initially intercalate within DNA base pairs before either covalently adducting DNA or undergoing hydrolysis to tetraols.42 The intercalative binding affinity has actually been shown to be stronger in (dA
dT) 3 (dT
dA) polynucleotides, but the rate of covalent attachment was shown to be much faster when guanine was present in the DNA sequence.43 On the basis of the muted response using BP, the
0.37 V peak is not due to an intercalative process that merely distorts the DNA helix. The use of con.273 also provides support for this argument. BP and BPDE are both expected to intercalate into con.273 with comparable affinity based on favorable hydrophobic stacking interactions.43 The analytical con.273 signal is muted, lending support that covalent BPDE
DNA adducts influence the C12-viologen Table 1. Apparent Rate Constants for Xenobiotics Exposed to DNA-Modified Au Surfacesa

Figure 2. Background-subtracted SWV comparison showing C12viologen reduction on a wt.273-modified Au electrode exposed to 100 μM BPDE (black), 200 μM BP (blue), or 10% SO (red) or a con.237modified electrode exposed to 200 μM BPDE (green) (all 1 min exposure, 10 mM Tris, 10 mM NaCl, pH 7.4).

k0 (s
1)

xenobiotic

DNA sequence

BPDE

wt.273

0.015

BPDE

con.273

7.5  10
4

BP

wt.273

3.1  10
4

SO

wt.273

ndb

Initial rate determined by initial electrochemical response at
0.37 V vs Ag/AgCl. b Initial rate unable to be determined. a

Figure 3. (a) Background-subtracted SWV comparison showing C12-viologen reduction on a wt.273-modified Au electrode exposed to varying BPDE concentrations for 1 min (10 mM Tris, 10 mM NaCl, pH 7.4). (b) Ip vs BPDE exposure time for electrodes exposed to different BPDE concentrations. 3330

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Figure 4. Typical thermal denaturation curves monitoring 260 nm UV absorbance for (a) wt.273 and (b) con.273 DNA sequences before (red dash) and after (solid black) exposure to BPDE. Conditions: 25
75 °C, 1 °C min
1, 50 mM ammonium acetate, pH 6.8.


0.37 V electrochemical response. These adducts form on wt.273 at faster rates compared to con.273. MS data discussed below confirms that covalent DNA adducts are formed on the DNA oligomers from BPDE exposure. The rate of the BPDE
DNA reaction is therefore heavily influenced by the presence of the codon 273 guanine in wt.273 oligomers. Analytical Response. An approximate limit of detection (LOD) was determined by monitoring the sensor response at lower BPDE concentrations. The background-subtracted
0.37 V SWV Ip at 30 s BPDE exposure exhibited a linear relationship from 5 to 100 μM [BPDE] (Supporting Information, Figure S5). The slope of this region from the least-squares fit indicates that the LOD is approximately 170 nM BPDE at 3:1 signal-to-noise. It is important to note that this sensor is not technically designed to detect trace amounts of solution PAH but, rather, to elucidate the reaction at specific prone DNA sequences. In this light, the LOD determined is on par with other sensors designed to provide general DNA damage information (i.e., no sequence specificity).23 Other electrochemistry-based immunoassay approaches exhibiting low-nanomolar detection limits have been explored to detect DNA adducts from BPDE exposure.44,45 In general, immunoassay approaches provide enhanced LOD sensitivity compared to indirect redox detection due to secondary binding interactions and catalytic detection schemes.31 In addition, the reaction environment employed in this study affects our LOD. The BPDE
DNA reaction under aqueous conditions is complex resulting in some non-DNAdamaging BP products (discussed below),23 which assuredly impacts our ability to detect BPDE adducts derived from very dilute solutions. Thermal Melting Studies. Thermal melting UV spectrometry experiments were utilized to study the BPDE reaction with different DNA sequences in more detail. In particular, we were interested in DNA oligomer stability before and after BPDE exposure. The wt.273 and con.273 sequences were exposed to BPDE solutions for 60 min followed by filtration procedures to separate DNA from the reaction medium. UV absorbance at 260 nm was monitored while increasing the temperature for each DNA solution. The dsDNA duplex denatures at elevated temperature resulting in respective ssDNA that have stronger 260 nm absorbance. The first derivative maximum(a) for a melting profile plot provides an approximate melting temperature (Tm), and the Tm change (ΔTm) along with the Tm curve shape provide insight into the stability of the dsDNA due to a binding or reaction process.46 Here, ΔTm provides a measure of induced dsDNA duplex instability from BPDE exposure. Figure 4 shows thermal

Figure 5. Mass spectra showing the charge states detected for BPDEexposed wt.273. The black diamond (() denotes ss1 singly adducted with BPDE. The open diamond ()) denotes singly adducted ss2 with BPDE. The lower spectrum expands m/z 630
685 from the top spectrum, showing the 10
charge state and sodium adduction. See the Experimental Section for ss1 and ss2 sequences.

melting responses for either wt.273 or con.273 that was both nonexposed and exposed to BPDE. Figure 4a demonstrates that the Tm for wt.273 decreased 2.5 ( 0.1 °C upon BPDE exposure denoting formation of a less stable dsDNA duplex. The stability decrease results from bulky PAH adduction that alters DNA stability. The use of ammonium acetate buffer produced larger ΔTm presumably due to the lack of buffer reacting with BPDE. The wt.273 exposed to BPDE under Tris buffer conditions exhibited ΔTm =
1.1 ( 0.1 °C (Supporting Information, Figure S6). BPDE exposed to con.273 does not produce significant ΔTm in either buffer, but a destabilizing effect was slightly more prevalent when using ammonium acetate. The BPDE-exposed samples exhibit broader phase transitions in both Figure 4 plots, which is indicative of destabilization.46 The melting data provides further evidence that the codon 273 guanine present in wt.273 is a primary BPDE binding location. Additionally, SO exposure to wt.273 under similar conditions produced negligible ΔTm. (Supporting Information, Figure S7). These data demonstrate that SO adducts are not highly DNA destabilizing. Overall, both the BPDE and SO thermal melting data are consistent with electrochemical responses. Another important aspect of these data is that BPDE exposure does not result in denaturation of dsDNA. The bulky BPDE
nucleobase adducts that form on the oligomers are not sufficient to completely disrupt the ability of the oligomers to form helices. This result is important as it lends evidence that electrochemical signals shown in Figures 1
3 are due to an alternate C12viologen
DNA complex rather than dsDNA dehybridization. Mass Spectrometry Studies. Mass spectrometry experiments were performed to study DNA oligomers exposed to BPDE or SO. It was of interest to determine the number of adducts formed on DNA oligomers via xenobiotic exposure in order to gain insight into the damage reaction. Figure 5 is a mass spectrum showing the charge states found in a typical BPDE reaction with 3331

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Figure 6. Mass spectra showing charge states from BPDE-exposed (a) ss1 and (b) ss2 (individual ssDNA making up wt.273). The black diamonds (() and ((() denote single- and double-BPDE-adducted ss1, respectively. The open diamond ()) denotes single-BPDE-adducted ss2. The inset in panel a shows Naþ adduction leading to spectra complexity.

wt.273. The spectrum has added complexity due to incomplete desalting (Figure 5 expanded view). In order to promote efficient desolvation leading to larger signals and a clearer charge state distribution, solvent and MS conditions were employed that induce duplex dehybridization into two ssDNA (termed ss1 and ss2 = wt.273 and complementary wt.273 outlined in the Experimental Section). The spectrum shows four distinct species were present in the recovered solution: two unreacted ssDNA species and two distinct BPDE-adducted ssDNA species. The first two represent individual ss1 and ss2 that form the original hybridized wt.273 oligomer. The second two are ss1 and ss2 each with a single BPDE adduct. Peaks at m/z 665 and 678 are definitively from BPDE exposure, as wt.273 exposed to similar reaction conditions excluding BPDE resulted in MS with no BPDE adduction peaks. This result is shown in Supporting Information Figure S8. No intact dsDNA evidence is seen; however, the thermal melting data discussed previously showed that the solution contained dsDNA before exposure to MS conditions. Additionally, only singly adducted ssDNA species are seen in the spectrum. This result can be interpreted in one of two ways. One possibility is the formation of a single adduct on either ssDNA per dsDNA oligomer. Upon dehybridization, this would result in a mixture of both unreacted ss1 and ss2 in combination with a singly adducted ssDNA. Another possibility is that one BPDE adduct could form on each ssDNA forming the dsDNA oligomer, or two BPDE per dsDNA. This interpretation would suggest that the MS shown in Figure 5 reflects a majority of unreacted dsDNA. Mass spectra shown in Figure 6 show the results when BPDE was exposed to individual ss1 or ss2 that form the wt.273 duplex. On the basis of the corresponding unreacted ssDNA peaks in these spectra, some unreacted dsDNA would be expected upon BPDE exposure. Additionally, it has been shown that the primary reaction pathway for BPDE under aqueous

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conditions is hydrolysis to form tetraols. This reaction is facilitated by DNA.42 In addition, it has also been shown that BPDE will form a single adduct at short DNA oligomers with related sequences as that used here.41 Therefore, the Figure 5 m/z data are consistent with BPDE reacting with equal probability at either ss1 or ss2 resulting in a singly adducted ssDNA. Unreacted ssDNA originates from the unreacted ssDNA on each hybrid or two ssDNA from a completely unreacted hybrid. Also, despite some evidence of doubly adducted ss1 (Figure 6a), no doubly adducted ss1 or ss2 was detected in the dsDNA spectrum (Figure 5). Two BPDE adducts per dsDNA would likely render the 21-mer very unstable, while the thermal melting data suggests hybridization is retained at room temperature. Overall, a discrete number of adducts are formed per oligomer under the employed reaction conditions. BPDE has been shown to primarily adduct guanine. There are 11 total guanine sites in the wt.273 oligomer. The data presented here demonstrate that BPDE does not equally target each potential adduct site. Under these reaction conditions, BPDE exhibits definite reaction specificity at wt.273 oligomers and reacts at a specific site within this sequence. BPDE likely targets the codon 273 guanine in these reactions. Evidence supporting this site specificity comes from studies employing con.273 oligomers. Supporting Information Figure S9 shows a mass spectrum where con.273 was exposed to BPDE under identical conditions. The charge state distribution exhibits primarily unreacted individual ssDNA. Lower BPDE adduct levels are seen in the spectrum, showing that the reaction happens more efficiently at wt.273. These data are consistent with the thermal melting experiments that showed only slight con.273 destabilization upon BPDE exposure. The only difference between con.273 and wt.273 sequences is cytosine substitution at the codon 273 guanine base in con.273. This is strong evidence that the codon 273 guanine is the primary damage site for BPDE adduction in the oligomers. Similar experiments were performed exposing wt.273 to SO. Mass spectra for these experiments are shown in Supporting Information Figure S10. The spectra show that one SO adduct is seen on each ssDNA and two on each wt.273 oligomer. These data demonstrate that there is an SO
DNA reaction and that it also generates a discriminate number of adducts. This lends evidence that a SO
DNA reaction takes place on the electrode surface, and lack of electrochemical signal arising from these reacted surfaces shows that the sensor is sensitive to specific adduct types. This is an interesting finding in itself and is discussed further below.

’ DISCUSSION Current change at
0.37 V versus Ag/AgCl (Figure 1) indicates that BPDE adduction alters wt.273 morphology and alters the C12viologen
DNA complex. We previously reported that C12-viologen voltammetry in the presence of dsDNA was dependent to some extent on the helix morphology.38,39 Cyclic voltammograms showed that two electrochemical waves were seen when C12viologen was reduced in the presence of dsDNA. A sample CV that is shown in Supporting Information Figure S1 shows this dual-wave voltammetry in the presence of dsDNA. The negative-shifted peak (∼
0.6 V vs Ag/AgCl) was due to face-to-face oriented π-dimers from singly reduced cation radical viologen (V•þ) species bound within the DNA minor groove, whereas the more positive peak (∼
0.45 V vs Ag/AgCl) was thought to be due to a nonspecifically electrostatic-bound complex formed along the anionic DNA duplex backbone. Data presented here suggest that it is the nonspecific 3332

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Analytical Chemistry electrostatically bound complex (at approximately
0.45 V) that is most influenced by the BPDE reaction giving rise to the
0.37 V peak. This indicates that this C12-viologen species undergoes an altered bound environment related to the BPDE
DNA adduction process. BPDE primarily reacts at the exocyclic amine N2-guanine site involved in base pairing to a complementary cytosine.41 The eventual DNA
BPDE structure is dependent on the BPDE enantiomer exposed to the guanine. (þ)-anti-BPDE and (
)anti-BPDE enantiomers covalently adduct the exocyclic guanine amine causing minimal base-pairing disruptions resulting in an adduct where the BPDE pyrenyl ring system lies in the minor groove.41 Syn-BPDE enantiomers form intercalative adducts with DNA oligomers at guanine sites.41 The placement of the bulky hydrophobic adduct in the minor groove exposed to the aqueous exterior is a free energy trade off for alleviated steric constraints and base pair retention associated with the anti-BPDE adducts.41 C12-viologen has large hydrophobic alkyl regions, which likely experience favorable interactions with exposed pyrenyl moieties on DNA. These interactions may result in accumulation of C12viologen at adduct sites external to the DNA helix. C12-viologen accumulation at these sites is accompanied by the concurrent loss of what was previously assigned as externally electrostatic DNA bound viologen. Therefore, the hydrophobic BPDE adduct displaces some C12-viologen that was primarily bound electrostatically to DNA. The positive formal reduction potential shift equates to stabilization of V•þ in C12-viologen bound to DNA. In other words, the oxidized viologen form (V2þ) is destabilized due to replacement of ion pairing with hydrophobic interactions. Although further study is warranted, a possible explanation for the
0.37 V peak is the formation of face to face V•þ π-dimer complexes formed when C12-viologen is reduced. Previous reports employing viologen self-assembled monolayers (SAM) showed that, upon reduction, these complexes form if V2þ moieties are electrode bound at sufficiently elevated concentrations.47 Additional hydrophobicity imparted by BPDE damage may cause C12viologen aggregation to reach this critical concentration resulting in dimer formation upon reduction. In our previous work, we assigned negative-shifted voltammetric peaks to V•þ π-dimer formation. The negative formal potential shift was thought to be due to additional stability oxidized V2þ species encounter bound within the minor groove.38 The
0.37 V peak seen at BPDE-exposed wt.273 is positive-shifted compared to the formal reduction potential (
0.45 V vs Ag/AgCl). A positive potential shift would be expected if C12viologen π-dimers form at external oligomer locations where BPDE adducts protrude from the minor groove. These locations would not offer V2þ stabilization, such as minor groove or electrostatic interactions. The positive formal potential shift (ΔEf = 80 mV) and resulting π-dimer formation free energy (ΔGform =
2FΔEf, F = 9.65  104 C mol
1, ΔGform =
15 kJ mol
1) mimics the responses of n-alkyl viologen SAMs.47 The calculated ΔGform is also consistent with the free energy of solution-phase cation radical viologen π-dimer formation providing more evidence that this process accounts for the positive-shifted redox wave at
0.37 V.47,48 Another explanation that may be considered to account for this redox peak is the formation of pyrene
bipyridinium π-electron donor
acceptor complexes. It is unlikely, however, that these complexes would result in positive-shifted redox potentials. Viologen is a strong π-acceptor, and oxidized V2þ would be stabilized in a π-donor BPDE complex. Viologen π-acceptors in dendrimers exhibited negative formal redox potential shifts upon forming complexes with π-donors.49 The observed positive redox

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potential shift described here must account for a V•þ formation driving force. This driving force is the overlap of two singly occupied π* orbitals from individual V•þ groups oriented in a face-to-face manner.47 Controls demonstrated that the
0.37 V SWV peak was due to a covalent BPDE adduct on wt.273. When wt.273 was replaced with con.273 on the electrode, the electrochemical signal exhibited an approximate 4-fold decrease (Figure 2). This is consistent with the codon 273 guanine as the primary BPDE adduct site in the oligomers employed in this study. Additionally, alternate xenobiotics did not produce similar electrochemical results. Several reports have shown that nonmetabolized BP conclusively intercalates into DNA.50
52 BP does not contain electrophilic functional groups that react at nucleophilic nucleobase sites. Electrochemical signals seen when BPDE was exposed to wt.273 were therefore not due to an intercalation event. Styrene oxide is known to covalently adduct DNA bases at several sites, but primarily at the N7 guanine location producing protrusions into the major groove.53 Xenobiotics producing such adducts can be toxic due to their prevalence in abasic site formation;54 however, major groove oriented SO adducts only slightly disrupt the helix and are thought to be nonmutagenic.55 Therefore, SO toxicity is muted compared to highly toxic carcinogens that disrupt base pairing and minor groove regions.54 The lack of electrochemical signal increase for SO-exposed DNA indicates that less toxic major groove hydrophobic entities do not promote C12-viologen binding. The smaller SO adduct is essentially buried within the major groove of the helix55 and may not promote sufficient hydrophobicity to cause C12-viologen aggregation. This is consistent with the proposed minor groove location of C12-viologen and the location of the eventual C12-viologen complex that results in the positive-shifted redox potential upon BPDE damage. Few reports have elucidated SO
DNA sequence specificity. Stone and co-workers looked at the mutagenicity of N6 SO adducts in N-ras protooncogene sequences.55,56 In general, their findings suggest some sequence effects for SO binding based on favorable interactions within the major groove environment. Fundador et al. showed that SO targeted a polyguanine site in a DNA sequence that codes for the cyt P450 1B1 gene.57 Data presented here show that only two SO adducts are seen on each dsDNA, or one per ssDNA, with no other population evidence. This demonstrates that SO does not indiscriminately target random guanine or adenine sites. Spectroscopic data were consistent with the electrochemical sensor responses. BPDE exposed to wt.273-modified surfaces produced large SWV Ip increases, whereas these signals were significantly muted in the presence of con.273-modified electrodes. MS experiments showed that a discrete number of adducts form on the wt.273 oligomer. Less adduct formation is observed for con.273 exposed to BDPE relative to wt.273. The only difference between the two oligomers is the substitution of guanine for cytosine at the codon 273 site (eighth nucleobase 50 on ss1). This suggests that the primary BPDE reaction at wt.273 is the codon 273 site. Overall, the sensor and spectroscopic data are consistent with sequence-specific DNA damage detection, with solid evidence toward site-specific DNA damage detection at the codon 273 guanine. An extensive research effort has shown that BPDE reacts at DNA based on sequence effects.8,9,18 BPDE has been found to react preferentially at 50 -CpG-30 sequences. TP53 codon 273 exhibits this dinucleotide sequence. However, naked DNA free from epigenetic modifications and protein associations is not the typical state 3333

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Analytical Chemistry found in vivo, and this is especially true for the TP53 gene.8,58,59 Along these lines, it has been shown that cytosine methylation at the 5-carbon plays a vital role in site-specific DNA adduction by BPDE.58 Although this study was not focused on the inclusion of epigenetic methylation modifications, we are currently exploring how cytosine methylation affects BPDE adduct formation leading to detectable and quantifiable electrochemical responses. As constructed, however, the sensor can elucidate BPDE DNA damage at this particular sequence, which is important in that many xenobiotic reaction hotspots are primarily DNA sequence dependent and secondarily influenced by epigenetic concerns.60

’ CONCLUSIONS A hybridization sensor was used to detect DNA damage from carcinogenic BPDE exposure at a DNA oligomer encompassing TP53 codon 273. BPDE-induced DNA structural changes were detected monitoring SWV responses from redox-active C12-viologen. BPDE exposure caused a new peak at
0.37 V versus Ag/AgCl. Current increases at this potential were most likely caused by reduced viologen dimerization due to C12-viologen accumulation at damaged DNA sites. The sensor was able to detect adducts from relatively low BPDE concentrations and was selective for covalent BPDE adducts at a sequence consisting of codon 273 from the p53 gene. Very little
0.37 V peak current was seen employing controls studying nonmetabolized BP or SO, both of which orient differently within the DNA duplex compared with BPDE. Additionally, DNA sequence was paramount to produce the
0.37 V peak. Switching the centralized codon 273 guanine with cytosine effectively muted the electrochemical signal. Spectroscopic UV thermal melting and MS experiments were conducted that provided evidence supporting the electrochemical sensor results detecting DNA damage at this DNA oligomer. MS showed that a discrete number of BPDE or SO adducts were formed on each dsDNA, demonstrating sequence preference for the xenobioitcs employed in the study. In this manner, a hybridization sensor was used to detect DNA damage from a known carcinogen at a known hotspot sequence. ’ ASSOCIATED CONTENT

bS

Supporting Information. Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected].

’ ACKNOWLEDGMENT This work was financially supported by the North Carolina Biotechnology Center, East Carolina Thomas Harriot College of Arts and Sciences, and East Carolina Office of Research and Graduate Studies. This project was performed, in part, using compound(s) provided by the National Cancer Institute’s Chemical Carcinogen Reference Standards repository operated under contract by Midwest Research Institute, no. N02-CB-66600. We thank Drs. Anne Spuches (Nanodrop) and Andrew Morehead (N2 atmosphere drybox) at ECU for graciously allowing us to use their equipment.

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