Role of Phenoxyl Radicals in DNA Adduction by ... - ACS Publications

Chem. Res. Toxicol. 2005, 18, 771-779

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Role of Phenoxyl Radicals in DNA Adduction by Chlorophenol Xenobiotics Following Peroxidase Activation Jian Dai, Amy L. Sloat, Marcus W. Wright, and Richard A. Manderville* Department of Chemistry, Wake Forest University, Winston-Salem, North Carolina 27109-7486 Received January 7, 2005

Chlorophenol (CP) toxins are classified as probable human carcinogens and are known to undergo bioactivation to generate benzoquinone (BQ) electrophiles that react covalently with biopolymers. Recently, we characterized the ability of pentachlorophenol (PCP) to react covalently with deoxyguanosine (dG) following treatment with horseradish peroxidase (HRP)/ H2O2 or myeloperoxidase to yield a C8-dG oxygen (O)-adduct that suggested the intermediacy of the pentachlorophenoxyl radical in covalent bond formation [Dai, J., Wright, M. W., and Manderville, R. A. (2003) Chem. Res. Toxicol. 16, 817-821]. Investigations currently focus on a wider range of CP substrates (PCP, 2,4,6-trichlorophenol (2,4,6-TCP), 2,4,5-TCP, and 2,4-dichlorophenol (2,4-DCP)) to establish their reactivity toward dG and duplex DNA (calf thymus (CT)) following activation by HRP/H2O2, as a representative peroxidase system. Our data show that chlorophenoxyl radicals may either react directly with dG and CT-DNA to form C8-dG O-adducts in an irreversible process or couple to yield 1,4-BQ electrophiles that react with dG to afford adducts of the benzetheno variety. These results are the first to establish the in vitro relevance of C8-dG O-adducts of phenolic toxins. The 1H NMR chemical shifts and reactivity of the benzetheno adducts favor 4′′-hydroxy-1,N2-benzetheno-dG adduct assignment, which is in contrast to other literature which has assigned the 1,4-BQ-dG adduct as 3′′-hydroxy1,N2-benzetheno-dG. Overall, the results from this current study have provided new insights into peroxidase-mediated activation of CP substrates and have strengthened the hypothesis that direct reactions of phenoxyl radicals with DNA contribute to peroxidase-driven toxic effects of phenolic xenobiotics.

Introduction Phenols are ubiquitous substances that possess numerous biological activities. Vitamin E and related phenols containing electron-releasing alkyl and methoxy groups possess antioxidant properties that are beneficial to human health (1). However, phenols with electronwithdrawing substituents may display deleterious prooxidant properties that are thought to contribute to aging and disease (2). This pro-oxidant activity is initiated through the 1-electron (e)1 oxidation of phenols into reactive phenoxyl radical intermediates that oxidize essential thiols, such as glutathione (GSH) (3), and generate reactive oxygen species that damage lipids (4), proteins (5), and DNA (6). Such phenoxyl radicals may also react directly with DNA to form covalent DNA adducts, an event that may initiate tumorigenesis (7). Recent findings from our work on DNA adduction by chlorophenol (CP) xenobiotics highlight their ability to react covalently with the C8 site of deoxyguanosine (dG) * To whom correspondence should be addressed. Present address: Department of Chemistry, University of Guelph, Guelph, Ontario, Canada N1G 2W1. Phone: (519) 824-4120, ext 53963. Fax: (519) 766-1499. E-mail: [email protected]. 1 Abbreviations: e, electron; BQ, benzoquinone; HRP, horseradish peroxidase; CP, chlorophenol; PCP, pentachlorophenol; OTA, (N-{[(3R)5-chloro-8-hydroxy-3-methyl-1-oxo-7-isochromanyl]carbonyl}-3-phenylL-alanine); PCP-dG, oxygen (O)-bonded pentachlorophenol-deoxyguanosine adduct; HMQC, heteronuclear multiple-quantum coherence; HMBC, heteronuclear multiple-bond connectivity.

following activation by peroxidase enzymes or redoxactive transition metals (8-11). Given that phenols are known to undergo peroxidase-mediated oxidation into phenoxyl radicals (12), and C8-dG adduction is in keeping with the susceptibility of the C8 site to radical attachment, as exemplified by 8-oxo-dG formation by hydroxyl radical (6), our studies suggest the intermediacy of the phenoxyl radical in covalent bond forming reactions with DNA. As illustrated in Scheme 1, the ambident reactivity of phenoxyl radicals at C8-dG would yield O-adducts, as well as ortho and para C-adducts. Our data show that O-attachment is favored for pentachlorophenol (PCP; Chart 1) (11), while the natural fungal carcinogen ochratoxin A (OTA; Chart 1) favors para C-attachment (8-10), possibly due to the ortho-flanking carbonyl groups that hinder O-attack. We have now extended our studies on peroxidasemediated dG adduction by PCP (11) and have included dG adduction by 2,4,6-trichlorophenol (2,4,6-TCP), 2,4,5-TCP, and 2,4-dichlorophenol (2,4-DCP) (Chart 1), which, together with PCP, are listed by the U.S. Environmental Protection Agency (EPA) as priority pollutants (13). We have also determined whether the pentachlorophenoxyl radical reacts with an actual duplex DNA substrate to form an O-bonded C8-dG adduct. The results of these studies are presented in this paper, which provides new insights into DNA adduction by CP xenobiotics following peroxidase activation. Here we demon-

10.1021/tx0500023 CCC: $30.25 © 2005 American Chemical Society Published on Web 03/25/2005

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Chem. Res. Toxicol., Vol. 18, No. 4, 2005 Scheme 1. Ambident Reactivity of Phenoxyl Radical toward dG

Chart 1

strate for the first time that chlorophenoxyl radicals can react directly with duplex DNA to form C8-dG O-adducts. In a competitive process, chlorophenoxyl radical coupling yields 1,4-BQ electrophiles, the expected CP liver metabolites (14), that react with dG to form benzetheno adducts. The implications of these findings are discussed.

Experimental Procedures Caution: The work described involves the handling of hazardous agents and was therefore conducted in accordance with NIH Guidelines for the Laboratory Use of Chemical Carcinogens (1981, NIH Publication No. 81-2385). Materials. PCP, 2,4,6-TCP, 2,4,5-TCP, 2,4-DCP, 2,6-DCBQ, 2,5-DCBQ, 2-CBQ, dG, calf thymus DNA (CT-DNA), and horse radish peroxidase (HRP; type VI) were purchased from SigmaAldrich and used as received. Acetonitrile (ACN; HPLC grade), dimethyl sulfoxide (DMSO), H2O2 (30%), and formic acid (90%, purified) were obtained from Fisher Scientific. Deionized water from a Milli-Q system (Millipore) was used for HPLC analysis and preparation of all aqueous solutions. Stock solutions of chlorophenols (5 and 50 mM), chlorobenzoquinones, and dG (500 mM) were prepared in freshly distilled DMSO. LC/MS Analyses. LC/MS analyses were performed on an Agilent 1100 series LC/MSD_SL Trap system with an atmospheric pressure chemical ionization (APCI) interface. Samples (20 µL) were injected into the LC/MSD system through an Agilent 1100 series thermostated autosampler. Separations were carried out on a 5 µm Agilent ZORBAX SB-C18 column (4.6 × 150 mm) at 25 °C at a flow rate of 0.75 mL/min using HPLC method I (or II). Two mobile-phase solvents were used in these methods. Solvent A was 0.1% formic acid in deionized water. Solvent B was 0.1% formic acid in ACN. HPLC method I: 80/ 20 A/B for 5 min and then ramp to 25/75 in 20 min by a linear gradient followed by an isocratic elution at 25/75 for 3 min. HPLC method II: 98/2 A/B for 10 min followed by a linear gradient to 20/80 A/B in 25 min. The flow was directed to the APCI source after passing through an Agilent 1100 series diode array detector. The LC/MSD trap was operated at APCI negative ionization mode (APCI-) with a corona current of 20000 nA and a heating temperature of 400 °C. The gas (N2)

Dai et al. for the nebulizer was set at 60 psi. The flow rate and temperature for dry gas (N2) were 5 L/min and 350 °C, respectively. Data were acquired over the m/z range of 80-800 under normal scan resolution (13000 (m/z)/s). Data acquisition was started at 3 min after injection to minimize the sample solvent effect to the baseline. Data analyses were performed using LC/MSD Trap Software 4.1 DataAnalysis Version 2.1 (build 49). NMR Parameters. All spectra were collected on a Bruker Avance 500 DRX equipped with a 5 mm TBI probe for inverse detected experiments and a 5 mm BBO probe for 13C observation. 1H and 13C spectra were recorded at 500.1 and 125.5 MHz, respectively. Data collection was done at 298 K in DMSO-d6 in a 5 mm Shigemi NMR tube. The 1H and 13C 1-D spectra were collected with 128 and 20 K scans, respectively. The 2-D gradient-selected spectra were collected with 1024 points in the F2 dimension and 512 points in the F1 dimension: gp-COSY (16 scans), gp-HMQC (128 scans), gp-HMBC (128 scans). pKa Determinations. The proton affinities for the 1,N2-benzetheno-dG adducts over a pH range of 4-12.5 were determined spectrophotometrically at 25 °C in aqueous media containing 0. 1 M NaCl and 1 vol % DMSO. Absorption measurements were obtained by a Hewlett-Packard (HP-8453) spectrophotometer equipped with a thermostated compartment. All samples were stirred by a magnetic stir bar within the cell. The pH measurements were obtained at 25 °C on an Orion model 210A pH meter using a Beckman semi micro glass electrode (S101A). Calibrations were performed using commercial buffers (BDH, pH 4.00, 7.00, and 10.00, all (0.01). An initial UV-vis spectrum was followed by subsequent addition of 0.10-0.010 M NaOH; pKa values were determined from the plot of log(ionization ratio) vs pH (15). Reactions of Chlorophenols with dG. Reaction mixtures (1 mL total volume) of chlorophenols (10 or 100 µM) and various molar equivalents of dG (0.5-400 equiv) in 100 mM phosphate buffer (pH 7.4) were incubated with HRP (2.5 units/mL) in the presence of H2O2 (100 µM) at 37 °C for 1 h. Aliquots (20 µL) were analyzed by LC/MS using HPLC method I. Semipreparative Separation. Semipreparative collection was performed on a Hitachi 7400 series HPLC with an L7455 diode array detector. Samples (1 mL) were injected, and separations were carried out on a 5 µm Phenomenex C-8 column (250 × 10 mm) at ambient temperature with a 5.00 mL/min flow rate. Methods III and IV, used for adduct isolation, have two solvents. Solvent C was 5 mM ammonium formate in deionized water. Solvent D was ACN. Method III: 90/10 C/D for 5 min, then ramp to 70/30 in 15 min and then to 20/80 in 5 min, followed by isocratic separation at 20/80 for 3 min, and return to 90/10 in 2 min. Method IV: 85/15 C/D for 5 min, then ramp to 75/25 in 15 min and then to 20/80 in 5 min, followed by isocratic separation for 3 min, and return to 85/15 in 2 min. Method V also used two solvent systems. Solvent A was 0.1% formic acid in deionized water. Solvent B was 0.1% formic acid in ACN. Method V: 97/3 A/B for 5 min, then ramp to 80/20 in 15 min and then to 30/70 in 5 min, followed by 3 min of isocratic separation, and return to 97/3 in 2 min. The adduct peak was collected and concentrated under diminished pressure. The light tan adducts were obtained after freeze-drying three subsequent times on a Freezon 4.5 LABCONO system. Preparation and Isolation of 2′′-Chloro-4′′-hydroxy1,N2-benzetheno-dG, 3. Reaction mixtures (1 and 5 mL) of 2,6-DCBQ (100 µM and 2 mM) and 20 molar equiv of dG in 50/50 DMSO/phosphate buffer (200 mM, pH 7.4) were allowed to react for 30 and 120 min, respectively, at 37 °C. LC/MS analysis using method I verified adduction prior to semipreparative isolation. Isolation was performed using method III, and the adduct peak was collected at 22 min. 1H NMR (DMSOd6, ppm): δ 12.99 (br s, 1H, NH), 9.80 (br s, 1H, ArOH), 8.14 (s, 1H, H8), 8.00 (d, J ) 1.97 Hz, 1H, H5′′), 6.90 (d, J ) 1.97 Hz, 1H, H3′′), 6.30 (t, J ) 6.9 Hz, 1H, H1′), 5.34 (d, J ) 3.94 Hz, 1H, 3′-OH), 5.05 (br s, 1H, 5′-OH), 4.42 (s, 1H, H3′), 3.63, 3.56 (m, 2H, H5′,5′′), 2.65, 2.30 (m, 2H, H2′,2′′). 13C NMR (DMSO-d6, ppm): δ 154.87, 151.53, 151.25, 150.33 (C4), 136.58

Role of Phenoxyl Radicals in DNA Adduction (C8), 129.17, 115.76, 115.58, 115.55, 111.99 (C3′′), 101.14 (C5′′), 87.70 (C4′), 82.97 (C1′), 70.64 (C3′), 61.58 (C5′), 39.40 (C2′). UVvis (λmax, nm): 272; 333. pKa: 7.13, 9.49. LC/MS (ES-, ion, m/z): [M - H]- ) 390. Preparation and Isolation of 3′′-Chloro-4′′-hydroxy1,N2-benzetheno-dG, 11. Adduct 11 was prepared and isolated using the procedures described for adduct 3 with the exception that isolation was performed using method IV and the adduct peak was collected at 13 min. 1H NMR (DMSO-d6, ppm): δ 12.53 (br s, 1H, NH), 10.10 (br s, 1H, ArOH), 8.25 (s, 1H, H5′′), 8.11 (s, 1H, H8), 7.36 (s, 1H, H2′′), 6.27 (t, J ) 6.92 Hz, 1H, H1′), 5.31 (s, 1H, 3′-OH), 5.11 (br s, 1H, 5′-OH), 4.40 (s, 1H, H3′), 3.86 (s, 1H, H4′), 3.60, 3.55 (m, 2H, H5′,5′′), 2.65, 2.26 (m, 2H, H2′,2′′). 13C NMR (DMSO-d6, ppm): δ 149.7 (C4), 147.3, 136.7 (C8), 126.4, 117.1, 116.3, 111.5 (C2′′), 103.4 (C5′′), 87.8 (C4′), 83.3 (C1′), 70.8 (C3′), 61.6 (C5′), 39.5 (C2′). The remaining 13C peaks could not be determined. UV-vis (λmax, nm): 272; 334. pKa: 7.05, 9.51. LC/MS (ES-, ion, m/z): [M - H]- ) 390. Methylation of 3 To Yield 2′′-Chloro-4′′-methoxy-1,N2benzetheno-3′,5′-OMe-dG, 12. To a 15 mL DMSO solution of 3 (38.1 mg) were added excess NaH and 50 µL of CH3I. Following a 3 h period at ambient temperature, the mixture was concentrated under vacuum. LC/MS analysis using method I showed the presence of a major adduct with [M - H]- ) 432, which suggested attachment of three methyl groups to adduct 3 (i.e., 390 + 3(15) - 3H ) 432). Semipreparative isolation using method V followed by NMR (NOESY, COSY) identified the isolated adduct as 2′′-chloro-4′′-methoxy-1,N2-benzetheno-3′, 5′-OMe-dG, 12. 1H NMR (DMSO-d6, ppm): δ 8.32 (s, 1H, NH), 8.06 (d, J ) 2.3 Hz, 1H, H5′′), 7.85 (s, 1H, H8), 6.93 (d, J ) 2.3 Hz, 1H, H3′′), 6.22 (t, J ) 8.79 Hz, H1′), 4.09 (m, 2H), 3.81 (s, 3H, 4′′-OCH3), 3.58, 3.52 (m, 2H, H5′,5′′), 3.34 (s, 3H, 3′-OCH3), 3.32 (s, 3H, 5′-OCH3), 2.76, 2.43 (m, 2H, H2′,2′′). pKa: 6.58. LC/MS (ES-, ion, m/z): [M - H]- ) 432. Reaction of PCP with CT-DNA. A reaction mixture (1 mL total volume) of PCP (100 µM) and CT-DNA (1 mg/mL) in 100 mM phosphate buffer (pH 7.4) was incubated at 37 °C for 24 h in the presence of HRP (25 units/mL)/H2O2 (1 mM). The reacted CT-DNA was precipitated (1 mL of 2% LiClO4/acetone), pelleted (centrifuge), and then washed with 3× (1 mL) 70% ethanol. The pellet was dissolved in 20 mM Tris-HCl (pH 7.0)/5 mM MnCl2, and then digested enzymatically by incubation at 37 °C with DNase I (200 units, 4 h), nuclease P1 (55 units, 16 h), and alkaline phosphatase (125 units, 6 h). The hydrolysate was concentrated and analyzed by LC/MS using HPLC method II.

Results and Discussion Reactions with 2′-Deoxyguanosine. As demonstrated previously (11), PCP (100 µM) in the presence of 2 mM dG and HRP (2.5 units/mL)/H2O2 (100 µM) yields an O-bonded C8-dG adduct (PCP-dG) as the sole detectable adduct following 1 h of incubation in 100 mM phosphate buffer (pH 7.4) at 37 °C. The O-adduct possessed UV absorbances at 212, 246, and 283 nm with a molecular ion at [M - H]- ) 528 having five chlorine atoms, with the most abundant isotope at m/z 530. In contrast, activation of PCP with rat liver microsomes failed to yield PCP-dG and instead generated trace amounts of an adduct with [M - H]- ) 440 that was found to coelute with an unidentified adduct induced by reaction of dG with authentic tetrachloro-1,4-benzoquinone (chloranil). In terms of HRP/H2O2 activation, the results with PCP suggested that the other CPs (2,4, 6-TCP, 2,4,5-TCP, and 2,4-DCP, Chart 1) would similarly react with dG to yield C8 O-adducts. To test this hypothesis, CP/dG reactions were carried out using conditions analogous to the PCP/dG reaction and were analyzed by LC/MS. Here it was expected that 2,4,6-TCP

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Figure 1. Extracted ion chromatograms (EICs, APCI-) from incubations of 100 µM chlorophenols and 2 mM dG in 100 mM phosphate buffer (pH 7.4) at 37 °C for 1 h in the presence of HRP (2.5 units/mL)/H2O2 (100 µM): (a) PCP, EIC 530, 440; (b) 2,4,6-TCP, EIC 460, 390; (c) 2,4,5-TCP, EIC 460, 390; (d) 2,4-DCP, EIC 426, 390. O-adduct ) oxygen-bonded C8-dG adduct, and Q-adduct ) quinone adduct of dG.

Figure 2. (a) In-line UV spectrum of the Q-adduct derived from 2,4,6-TCP (100 µM) and dG (2 mM) following 1 h of incubation in 100 mM phosphate buffer (pH 7.4) at 37 °C in the presence of HRP (2.5 units/mL)/H2O2 (100 µM). (b) APCI- spectrum of the Q-adduct in (a) with [M - H]- ) 390/392 showing CID of the parent ion to yield the ion at m/z 274/276 for loss of deoxyribose.

and 2,4,5-TCP would yield an O-adduct with a molecular ion at [M - H]- ) 460 while the O-adduct from 2,4-DCP would have a molecular ion at [M - H]- ) 426. Figure 1 highlights the LC/MS results from analyses of the various CP/dG reactions. Interestingly, under conditions favorable for O-adduct formation by PCP (Figure 1a) (11), the only other CP to yield detectable levels of O-adduct was 2,4,5-TCP (Figure 1c). While 2,4, 6-TCP (Figure 1b) did not yield O-adduct, it generated adduct levels comparable to those of PCP; levels for 2,6-DCP (Figure 1d) and 2,4,5-TCP were 3-30-fold lower, respectively. The major adduct detected for 2,4,6-TCP, 2,4,5-TCP, and 2,4,-DCP, labeled Q-adduct in Figure 1, had UV absorbances at ∼275 and 334 nm with a molecular ion at [M - H]- ) 390/392 with a single chlorine isotope, as exemplified in Figure 2 for the Q-adduct derived from 2,4,6-TCP/dG/HRP. The UV spectrum in Figure 2a is analogous to the spectrum reported for a 1,N2-benzetheno-dG adduct generated from 1,4-benzoquinone (BQ)/dG (16). Thus, it was possible to confirm Q-adduct identity through reaction of dG with authentic CBQ. For example, the Q-adduct from 2,4, 6-TCP (Figure 1b) comigrated with the major adduct

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Dai et al. Table 1. Spectral Data for CP-dG Adduct Formation CP PCP 2,4,6-TCP 2,4,5-TCP 2,6-DCP

Figure 3. Extracted ion chromatograms (EIC, APCI-) from incubations of 10 µM CPs and 4 mM dG in 100 mM phosphate buffer (pH 7.4) at 37 °C for 1 h in the presence of HRP (2.5 units/mL)/H2O2 (100 µM)L (a) 2,4,6-TCP, EIC 460, 390; (b) 2,4,5-TCP, EIC 460, 390; (c) 2,4-DCP, EIC 426, 390.

adduct

tRa (min)

[M - H]-

λmax (nm)

yieldb (%)

oxygen quinone oxygen quinone oxygen quinone oxygen quinone

16.9 NDc 13.5 6.3 14.5 6.3 12.5 6.3

528d ND 460 390 460 390 426 390

283, 246 ND 279,250 334, 275, 235 275 334, 272 279, 250 334, 275

34 ND ND 47 ND 1 ND 13

a HPLC conditions: 80/20 (0.1% formic acid in H O/0.1% formic 2 acid in ACN) for 5 min and then ramp to 25/75 in 20 min by a linear gradient followed by an isocratic elution at 25/75 for 3 min using a 5 µm Agilent ZORBAX SB-C18 column (4.6 × 150 mm) at 25 °C at a flow rate of 0.75 mL/min. b Yields are for the 100 µM CP reaction and are estimated from the reported PCP-dG yield (34%), using the relative integrated area ratio. c ND ) not determined. d Calculated on the basis of 35Cl.

Scheme 2. Proposed Pathways for Peroxidase-Mediated Oxidation of Chlorophenols

derived from 2,6-DCBQ/dG; the same Q-adduct was derived from reaction of 2-CBQ with dG (i.e., Figure 1d). For 2,4,5-TCP (Figure 1c), the Q-adduct comigrates with an adduct from 2,5-DCBQ/dG; structural assignments of the various Q-adducts is presented and discussed in the following section. Inspection of the literature on the oxidation of halogenated phenols by peroxidase enzymes (17-21) suggests that a 1,4-BQ electrophile may arise from chlorophenoxyl radical formation by the steps outlined in Scheme 2. In the case of PCP, Kazunga and co-workers presented evidence that the final quinone product, chloranil, is an artifact of extraction and analytical methods and that the principal product is the ether 2,3,4,5,6-pentachloro-4pentachlorophenoxy-2,5-cyclohexadienone formed by pentachlorophenoxyl radical coupling (21). This suggested a common mechanism for HRP/H2O2-mediated oxidation of a CP involving bimolecular radical coupling for 1,4-BQ formation. It also suggested the possibility that the results presented in Figure 1 reflected differences in the rates of bimolecular chlorophenoxyl radical coupling and that lowering the concentration of the parent CP to retard radical coupling would favor C8-dG O-adduct formation for 2,4,6-TCP, 2,4,5-TCP, and 2,4-DCP. Figure 3 shows extracted ion chromatograms (EICs) from incubations of 10 µM CPs and 4 mM dG in 100 mM phosphate buffer (pH 7.4) at 37 °C for 1 h in the presence

Figure 4. 1H NMR (500 MHz) spectra in DMSO-d6 of (a) 2′′-chloro-4′′-hydroxy-1,N2-benzetheno-dG, 3, and (b) 3′′chloro-4′′-hydroxy-1,N2-benzetheno-dG, 11.

of HRP (2.5 units/mL)/H2O2 (100 mM). Now all three CPs generated the anticipated C8-dG O-adduct as the major detectable lesion, showing UV and elution time (tR) data consistent with those previously observed for PCP-dG that had been fully characterized by NMR (11). For 2,4, 5-TCP (Figure 3b) the O-adduct was the only detectable adduct, while 2,4,6-TCP (Figure 3a) and 2,4-DCP (Figure 3c) still formed detectable levels of Q-adduct. The results from LC/MS analyses of the CP/dG reactions are summarized in Table 1. Q-Adduct Identity. Figure 4a shows the 500 MHz 1H NMR spectrum in DMSO-d6 of the Q-adduct isolated from

Role of Phenoxyl Radicals in DNA Adduction


Figure 5. UV-vis/pH titration of 2′′-chloro-4′′-hydroxy-1,N2benzetheno-dG, 3, at 25 °C in H2O containing 0.1 M NaCl and 1 vol % DMSO: (a) pH range 4-9; (b) pH range 9-12.5.

reaction of 2,6-DCBQ/dG. HMQC and HMBC spectra provided unambiguous assignment of the sugar proton/ carbon resonances. The HMBC spectrum also provided unambiguous assignment of the H8 proton at 8.11 ppm by way of a correlation between H1′ (6.30 ppm) and C8 at 136.58 ppm. The two remaining aromatic protons at 8.00 and 6.90 ppm were observed as doublets, showed a meta coupling constant, J ) 1.97 Hz, and are labeled H5′′ and H3′′ in the spectrum. Figure 4b shows the 1H NMR spectrum of the major Q-adduct isolated from reaction of 2,5-DCBQ with dG. This adduct showed spectral features similar to those in Figure 4a with the exception that the aromatic peaks labeled H5′′ (8.25 ppm) and H2′′ (7.36 ppm) were not coupled. The pH-dependent UV spectra of the Q-adducts were determined at 25 °C in aqueous media. The UV titrations showed two distinct regions, each containing characteristic sets of isosbestic points, as shown in Figure 5 for the Q-adduct derived from 2,6-DCBQ/dG. The first deprotonation (Figure 5a) caused the 334 nm absorbance of the neutral ligand (L) to shift to 353 nm with reduced intensity, while the second deprotonation (Figure 5b) caused a further red shift to yield an absorbance at 368 nm for the deprotonated ligand (L2-). From Figure 5a, a pKa value of 7.13 was determined, while from Figure 5b the second pKa was 9.49; the corresponding values for the Q-adduct derived from 2,5-DCBQ/dG were 7.07 and 9.51. For reaction of dG with 1,4-BQ the mechanism in Scheme 3 for 3′′-hydroxy-1,N2-benzetheno-dG (1) formation has been proposed (16, 22-24). However, definitive evidence for adduct structure has not been presented, and the alternative mechanism outlined in Scheme 4 is possible and involves initial Schiff base formation from the N2 atom of dG and the carbonyl carbon and subsequent nucleophilic attack of the endocyclic N1 on the vinyl carbon to yield 4′′-hydroxy-1,N2-benzetheno-dG (2). On the basis of analogy to etheno ()-dG formation

Figure 6. Benzetheno-dG adduct structure and models for adduct assignments.

Scheme 3. Postulated Mechanism for 3′′-Hydroxy-1,N2-benzetheno-dG (1) Formation

Scheme 4. Mechanism for 4′′-Hydroxy-1,N2-benzetheno-dG (2) Formation

(25-27), isomeric angular N2,3-adducts are also possible. Thus, 2,6-DCBQ/dG could conceivably form 2′′-chloro-4′′hydroxy-1,N2-benzetheno-dG (3), 5′′-chloro-3′′- hydroxy1,N2-benzetheno-dG (4), 2′′-chloro-4′′-hydroxy-N2,3- ben-

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zetheno-dG (5), and 5′′-chloro-3′′-hydroxy-N2,3-benzethenodG (6) (Figure 6). While our spectral evidence (Figures 4 and 5) also does not permit rigorous proof of structure, the strong pH dependence of the electronic spectra depicted in Figure 5 favors the linear 1,N2-isomers 3 and 4 over the angular N2,3-adducts 5 and 6 (25, 26). For linear 1,N2-benzetheno adduct formation, previously reported 1H NMR chemical shifts for the aromatic protons of 1 in DMSO-d6 (16) are shown in Scheme 3. Note that H2′′ with a meta coupling constant of ∼2 Hz that is flanked by the OH and NH groups is downfield at δ 8.02 ppm. For adduct 2 depicted in Scheme 4, H5′′ would be the downfield proton with a meta coupling constant of ∼2 Hz. The pathway in Scheme 4 is analogous to the Nenitzescu indole synthesis wherein a 1,4-BQ condenses with an alkyl 3-aminocrotonate (28, 29). Here it has been shown that formylation at C9 of 7 to yield 8 (see Figure 6 for structures) causes the C8 H-atom to shift downfield from 7.2 to 8.05 ppm. This change in chemical shift has been ascribed as an example of diamagnetic anisotropic deshielding in the benzenoid ring by a carbonyl substituent in an adjacent ring of a pyrroloindole (29). For the 6-hydroxyindole derivative 9 the C4 H-atom is downfield at 7.92 ppm, while the C7 H-atom that is flanked by the OH and NH groups resonates at 6.85 ppm (30). Given that 8 and 9 are structurally similar to the phenolic moiety of 2 (Scheme 4) and 1 (Scheme 3), respectively, and thus can be viewed as chemical shift models for these adducts, it is clear that the NMR chemical shifts favor 2 adduct assignment over 1 adduct assignment because the downfield shift of H5′′ in 2 can be explained by the deshielding effect of the C6 carbonyl of the dG moiety (29). It is also worthy to note that the pathway in Scheme 4 is consistent with the mechanism of 1,N2--dG formation from 2-haloacetaldehydes, where it has been unequivocally proven by NMR labeling studies that the exocyclic amine N2 reacts with the aldehyde (27). Furthermore, indoles that have the phenolic OH and NH atoms in a 1,4 arrangement and include 5-hydroxytryptamine (serotonin) are known to undergo 2H+/2e oxidations to form quinone imine intermediates that react with H2O to generate catechols (31). That the catechol 3′′,4′′-dihydroxy-1,N2-benzetheno-dG 3′-monophosphate (10) (Figure 6) has been isolated and characterized (32) from reaction of 1,4-BQ with 3′-MPdG is completely consistent with initial formation of 2 followed by its 2H+/ 2e oxidation to form a quinone imine intermediate that reacts with H2O to yield 10. On the basis of the above arguments, the adduct isolated from the 2,6-DCBQ/dG reaction was assigned as 3 (Figure 6), and the downfield doublet at 8.00 ppm in Figure 4a was assigned to H5′′ on the basis of its shift being influenced by the C6 carbonyl moiety (29). The corresponding adduct from the 2,5-DCBQ/dG reaction was assigned as 3′′-chloro-4′′-hydroxy-1,N2-benzethenodG (11; Figure 6). The UV-vis pH titrations (Figure 5 for 3) suggest that 3 and 11 have NH and phenolic pKa values of 7.13/9.49 and 7.07/9.51, respectively. For structurally similar compounds the NH pKa of 1,N2--dG is 9.2 (26) and the pKa of m-CP is ∼8.97 (33). These values are nearly identical, so unambiguous assignment of the pKa values could not initially be made. However, treatment of 3 with NaH/CH3I afforded 12 (Figure 6) in which the phenolic moiety is methylated. This adduct showed a single pKa at 6.58, establishing the first pKa at ∼7 as

Dai et al. Scheme 5. Deprotonation of 2′′-Chloro-4′′-hydroxy-1,N2-benzetheno-dG (3)

deprotonation of the NH atom, while the second pKa at ∼9.5 is deprotonation of the phenolic oxygen atom. As summarized in Scheme 5 for deprotonation of 3, resonance stabilization of the monoanion (L-) from loss of the NH proton generates an oxyanion at C6, which has a structure fully conjugated with the phenolic ring. This conjugative stabilization provides a rationale for the lower NH pKa of 3/11 versus 1,N2--dG (26). In Table 2 is given a compilation of the NMR data and protonation constants for adducts 2, 3, and 10-12. Reaction of PCP with CT-DNA. While ample evidence is available for the in vitro relevance of 1,N2-benzetheno adduct formation (22, 24, 32), to our knowledge MS evidence for C8-dG O-adduct formation by a phenolic xenobiotic has not been previously reported. To determine whether a C8-dG O-adduct forms with a duplex DNA substrate, CT-DNA (1 mg/mL) was treated with PCP (100 µM) in phosphate buffer (100 mM, pH 7.4) at 37 °C for 24 h in the presence of HRP (type VI, 25 units/mL)/H2O2 (1 mM). The reacted DNA was precipitated, enzymatically digested, and analyzed by LC/ MS. Figure 7 shows the LC/MS results of the digested CT-DNA, where panel a shows unmodified DNA bases, panel b the EIC of m/z 530 (the major isotope of PCPdG), and panel c the EIC of authentic PCP-dG. The HPLC retention time, MS/MS fragmentation, and parent ion of the PCP-dG adduct in panel b were identical to those of the authentic adduct standard in panel c; the other peak in panel b lacks a chlorine isotope pattern and was not characterized. Previous 32P-postlabeling experiments show that adduct levels by 100 µM PCP in the presence of CT-DNA (1 mg/mL) following HRP/H2O2 activation (3600 ( 940 adducts/107 nucleotides (34)) are 30-fold higher than levels induced by microsomes and 10-fold higher than levels induced by 5 mM chloranil itself (35). The data presented in Figure 7 imply that the pentachlorophenoxyl radical reacts with CT-DNA to form the O-bonded PCP-dG adduct, which contributes to adduct levels induced by PCP following peroxidase activation. Reaction Pathways and Biological Relevance. The reaction of a CP with dG following activation by HRP/H2O2 is summarized in Scheme 6. The reaction is initiated by the production of the chlorophenoxyl radical, which may either react directly with dG to furnish the C8-dG O-adduct in an irreversible process (path A) or self-couple to yield the 1,4-BQ electrophile, which reacts with dG to give 4′′-hydroxy-1,N2-benzetheno-dG (path B). In the current study we have examined HRP/H2O2mediated dG reactions of PCP, 2,4,6-TCP, 2,4,5-TCP, and 2,4-DCP. At 100 µM CP, PCP reacts directly with dG via



Table 2. Aromatic 1H Resonances and Acidity Constants for Benzetheno Adducts R2′′

R3′′

10 3

7.230, d J ) 8.6a 6.92, s Cl

11 12

6.93, s Cl

6.866, dd J ) 2.3, 8.6 OH 6.90, d J ) 1.97 Cl 6.93, d J ) 2.3

2

R4′′ OH OH OH OH OCH3

pKa1

pKa2

R5′′

H8

solvent

8.022, d J ) 2.3 8.11, s 8.00, d J ) 1.97 8.25, s 8.06, d J ) 2.3

8.124, s

DMSO

ND

ND

16

8.16, s 8.14, s

CD3OD DMSO

ND 7.13b ( 0.04

ND 9.49c ( 0.12

32 this work

8.11, s 7.85, s

DMSO DMSO

7.05 ( 0.04 6.58d

9.51 ( 0.11

this work this work

a Chemical shifts are in parts per million; coupling constants are in hertz. determinations. c Phenolic pKa. d Value from one determination.

b

ref

NH pKa, standard deviation from three independent pKa

Table 3. Parameters for Phenols in Aqueous Solutiona phenol

pKa

Ered°(pH 12.0)b

σ+

PhOH PCP 2,4,6-TCP 2,4,5-TCP 2,4-DCP 4-MeOPhOH

9.98 4.74 5.99 6.72 7.90 10.20

0.86 0.99 0.90 0.90 0.88 0.58

0.00 1.1 0.30 0.61 0.21 -0.78

a Data taken from ref 33. b Estimated reduction potential of the phenoxyl radical in volts vs NHE.

Figure 7. LC/MS analysis of enzymatically digested CT-DNA (1 mg/mL) that had been treated with PCP (100 µM), HRP (25 units/mL), and H2O2 (1 mM) for 24 h at 37 °C in 100 mM phosphate buffer, pH 7.4: (a) chromatogram of UV detector response (254 nm); (b) EIC (APCI-) of m/z 530, most abundant isotope of PCP-dG; (c) EIC of m/z 530 for authentic PCP-dG.

Scheme 6. Summary of HRP/H2O2-Mediated Reaction of a CP with dG

path A (Scheme 6), while 2,4,6-TCP and 2,4-DCP react with dG via path B. In contrast, 2,4,5-TCP showed production of both C8-dG O-adduct and benzetheno adduct, suggesting competition between the two dG reaction pathways outlined in Scheme 6. At 10 µM CP, all CPs tested reacted directly with dG to yield the O-adduct as the major adduct (Figure 3), although 2,4, 6-TCP and 2,4-DCP still generated detectable levels of benzetheno adduct from production of 2,6-DCBQ and 2CBQ, respectively. At the higher CP concentration (100 µM), the observed differences in product distribution may be ascribed to differences in rate constant for the phenoxyl radical bimolecular self-reaction (path B). If the

bimolecular self-reaction is favorable, then path B competes effectively with path A at 100 µM CP substrate, while if the self-reaction is not favorable, then path A dominates. Increased Cl substitution would be expected to inhibit the bimolecular self-reaction due to steric and/or electronic repulsion. In this regard, the bimolecular self-reaction rate constant for phenoxyl radical in H2O is 2.6 × 109 M-1 s-1 (33), and it has a half-life of ∼60 µs in CCl4 (36). In contrast, the pentachlorophenoxyl radical has a half-life of 30-45 min in aqueous buffer, as determined from direct detection by EPR spectroscopy (18). That the CP substrates behave similarly at 10 µM and react with dG to form the O-adduct (path A, Scheme 6) highlights the effects of reaction conditions on the nature of the reaction products. Since the rate of phenoxyl radical coupling (path B) is proportional to the square of the phenoxyl radical concentration, relative rates of competing reactions of the phenoxyl radical (path A) will decrease to a much lesser extent as the rate of radical formation decreases. In this example the concentration of the parent CP was lowered; the same effect may have been achieved by lowering the enzyme concentration. Thus, increased Cl substitution on the parent phenol and pathways that diminish phenoxyl radical concentration should inhibit phenoxyl radical coupling and enable the radical time to diffuse away from its site of formation and react with biopolymers. It has also been observed that remote substituent effects of phenoxyl radicals exhibit a linear correlation with Brown σ+ constants, as opposed to the Hammett σ constant, because of the through-resonance between the electron-deficient phenoxyl radical and the ring substituent (37). Table 3 gives pKa values, standard reduction potentials (Eredο) for the phenoxyl radical/phenolate ion couples at pH 12, and σ+ values that are available in the literature for the CP substrates used in this study along with phenol (PhOH) and 4-MeOPhOH for comparison (33). A high value of σ+ suggests that the radical is localized on the oxygen atom, while the negative value for 4-MeOPhOH (-0.78) suggests extensive radical stabilization by the 4-MeO substituent. Not surprisingly, the fully substituted analogue PCP has the highest σ+ value (1.1) and is predicted to be the most reactive O-centered

778


radical followed by 2,4,5-TCP (σ+ ) 0.61), 2,4,6-TCP (σ+ ) 0.3), and 2,4-DCP (σ+ ) 0.21). Our data show that the larger value of σ+ for the CP gives a greater proportion of C8-dG O-adduct, i.e., path A (Scheme 6), while a lower value makes path B competitive at 100 µM CP. It will be interesting to extend this study and include additional phenols, even those with electron-donating groups that possess negative σ+ values, and determine whether a correlation of σ+ with C8-dG adduct (O versus C) formation can be established for a greater range of phenols. That PCP was found to react with CT-DNA to generate the C8-dG O-adduct PCP-dG establishes the in vitro relevance of C8-dG adducts of phenolic toxins. We have also recently demonstrated that OTA (Chart 1) reacts covalently with dG to form the corresponding C8-dG C-adduct (8-10). Other literature suggests that this reactivity is general. For example, Ni(salens) that form phenoxyl radicals upon oxidation are reported to act like OTA and favor para C-adduct formation; binding of the phenolic O-site to a transition metal would preclude O-attachment (38). 4-Hydroxytamoxifen (4-OHTAM), a metabolite of the clinically used drug tamoxifen, is known to form a single DNA adduct following uterine peroxidase activation (39), and benzene is proposed to cause leukemia through metabolism to phenol with subsequent phenoxyl radical formation and covalent reaction with DNA (40). Finally, the tyrosyl radical that initiates protein-protein cross-links (41) is also thought to play a key role in protein-DNA adduction (42), which may contribute to aging and cancer. Our data strengthen the hypothesis that direct reactions of phenoxyl radicals with DNA contribute to peroxidase-driven toxic effects of phenolic xenobiotics (40), and our current efforts are focused on the synthesis of C8-dG adducts (O and C) for incorporation into duplex DNAs to determine their impact on DNA structure in an effort to gain an understanding of their biological consequences. In summary, we have demonstrated that peroxidaseactivated CP substrates afford phenoxyl radicals that may either react directly with dG to yield C8-dG Oadducts or self-couple to give 1,4-BQ electrophiles that react with dG to yield 4′′-hydroxy-1,N2-benzetheno-dG adducts. Our work provides the first in vitro evidence for C8-dG O-adducts of phenolic toxins by demonstrating that PCP reacts directly with CT-DNA to give the O-adduct PCP-dG following activation by HRP/H2O2. In the present paper we also present new evidence that supports 4′′-hydroxy-1,N2-benzetheno-dG adduct formation by 1,4-BQ electrophiles; all previous literature has assigned the dG adduct as 3′′-hydroxy-1,N2-benzethenodG, which is not consistent with 1H NMR chemical shifts and reaction chemistry for this family of cyclic adducts.

Acknowledgment. Financial support from the Lance Armstrong Foundation and the EPA is acknowledged. R.A.M. also acknowledges the helpful comments provided by the reviewers of this manuscript.

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