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The ability of the carcinogenic environmental toxin pentachlorophenol (PCP, 1) to react ... adduct(s) was observed from HRP/H2O2 and PCP in the presen...
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Chem. Res. Toxicol. 2003, 16, 817-821

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Communications An Oxygen-Bonded C8-Deoxyguanosine Nucleoside Adduct of Pentachlorophenol by Peroxidase Activation: Evidence for Ambident C8 Reactivity by Phenoxyl Radicals Jian Dai, Marcus W. Wright, and Richard A. Manderville* Department of Chemistry, Wake Forest University, Winston-Salem, North Carolina 27109-7486 Received April 23, 2003

The ability of the carcinogenic environmental toxin pentachlorophenol (PCP, 1) to react with DNA bases has been assessed using MS and NMR. Treatment of PCP (100 µM) with horseradish peroxidase (HRP/H2O2) or myeloperoxidase (MPx/H2O2, from human leukocytes) in the presence of excess deoxyguanosine (dG, 2 mM) led to the isolation and identification of the oxygenbonded C8-dG nucleoside adduct 4. The reaction was absolutely specific for dG; no detectable adduct(s) was observed from HRP/H2O2 and PCP in the presence of deoxyadenosine, deoxycytidine, or thymidine. Formation of 4 was also specific for peroxidase activation that is known to oxidize PCP into the phenoxyl radical. Treatment of PCP/dG with rat liver microsomes (RLM) failed to generate 4; instead, an adduct derived from the benzoquinone electrophile tetrachloro-1,4-benzoquinone (chloranil) was observed in the extracted ion chromatogram from the RLM/NADPH-treated PCP/dG sample. The adduct 4 is the first structurally characterized O-bonded phenolic DNA nucleoside adduct and highlights the ambident electrophilicity of phenoxyl radicals (O- vs C-) in reaction at C8 of dG, as we have previously demonstrated that the para-chlorophenolic toxin, ochratoxin A (2), reacts at C8 of dG to give the C-bonded adduct 3 via the intermediacy of the OTA phenoxyl radical. Given that PCP is known to induce DNA adduct formation in vivo and human exposure has been linked to incidences of leukemia, the adduct 4 could play a key role in PCP-mediated carcinogenesis.

Introduction PCP1 (1, Figure 1) is listed by the U.S. Environmental Protection Agency as a priority pollutant (1). It is classified as a group 2B environmental carcinogen by the International Agency for Research on Cancer (2). Thus, there is much interest in understanding the mechanism(s) of PCP-mediated carcinogenesis (3-5) and in the development of green chemical processes for its degradation (6, 7). The prooxidant properties of PCP and other phenolic toxins are thought to contribute to their carcinogenic mechanisms by facilitating oxidative stress, oxidative DNA damage, and direct DNA adducts (3-5, 8). Chronic exposure of rats to PCP generates DNA adducts in the liver, as evidenced by the 32P-postlabeling assay (3). One of the adducts was found to comigrate with an adduct * To whom correspondence should be addressed. Tel: (336)758-5513. Fax: (336)758-4656. E-mail: [email protected]. 1Abbreviations: PCP, pentachlorophenol; OTA, ochratoxin A (N{[(3R)-5-chloro-8-hydroxy-3-methyl-1-oxo-7-isochromanyl]carbonyl}-3phenyl-L-alanine); HRP, horseradish peroxidase; MPx, myeloperoxidase; SCE, saturated calomel electrode; RLM, rat liver microsomes; DMSO, dimethyl sulfoxide; TBI, three channel (1H, 13C, X) broad band inverse; BBO, broad band observe; COSY, correlation spectroscopy; DQF, double quantum filtered; HMQC, heteronuclear multiple quantum coherence; HMBC, heteronuclear multiple bond connectivity; NOESY, nuclear Overhauser enhancement spectroscopy.

Figure 1. Chemical structures of PCP (1), OTA (2), and their C8-dG adducts.

induced by the benzoquinone electrophile tetrachloro-1,4benzoquinone (chloranil) that is generated by cytochrome P450 (CYP450) metabolism of PCP (3-5, 9). Recent in vitro studies (10) show that adduct levels (36 ( 9/105 nucleotides) by 100 µM PCP following activation by HRP/ H2O2 are 30-fold higher than levels induced by microsomes and 10-fold higher than levels induced by 5 mM chloranil (4, 10). However, the structures of such DNA

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adducts have not been characterized and their role in PCP-mediated carcinogenesis is not known. Recently, we presented evidence that the carcinogenic para-chlorophenolic mycotoxin OTA (2, Figure 1) forms the carbon-bonded (C-bonded) C8-deoxyguanosine (dG) nucleoside adduct 3 following oxidative activation by redox active transition metals (Fe and Cu) and HRP/H2O2 (11). The identification of the OTA-dG adduct 3 was surprising, as it suggested the intermediacy of the OTA phenoxyl radical in reaction with dG, not the anticipated benzoquinone electrophile (12). This hypothesis stemmed from the well-known susceptibility of the C8-position of dG to radical attack, as amply proven through formation of the hydroxyl radical-derived lesion, 8-oxodeoxyguanosine (13), coupled with the fact that in aqueous buffer OTA undergoes a 1e oxidative process at ∼0.8 V vs SCE and ∼1.04 V vs NHE (14). Our findings with OTA prompted investigation of PCP reactivity toward the DNA nucleosides to determine whether it would also yield a C-bonded C8-dG adduct. Presently, we show definitive evidence that PCP reacts with dG to form the O-bonded C8-dG adduct 4 (Figure 1) in a facile and efficient manner by oxidative activation with HRP/H2O2 and MPx (MPx/H2O2, from human leukocytes). The adduct 4 is the first structurally characterized O-bonded phenolic DNA nucleoside adduct and highlights the ambident electrophilicity of phenoxyl radicals (O- vs C-) in reaction at C8 of dG.

Experimental Procedures Caution: The work described involves the synthesis and handling of hazardous agents and was therefore conducted in accordance with NIH guidelines for the Laboratory use of Chemical Carcinogens (15). Materials. PCP (1), dG, 2′-deoxyadenosine (dA), 2′-deoxycytidine (dC), thymidine (dT), HRP (type VI), MPx, NADP, glucose-6-phosphate dehydrogenase, and glucose-6-phosphate were purchased from Sigma-Aldrich and used as received. Aroclor 1254-induced Sprague-Dawley RLM was obtained from IN VITRO Technologies and used without further purification. Acetonitrile (ACN, HPLC grade), DMSO (freshly distilled), hydrogen peroxide (30%), and formic acid (90%, purified) were purchased 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 PCP (50 mM) and dG (500 mM) were prepared in freshly distilled DMSO. NADPH regenerating system (NRS) was made fresh according to the literature procedure (16). LC/MS Analyses. LC/MS analyses were performed on an Agilent 1100 series LC/MSD SL 00045 Trap system with an atmosphere pressure chemical ionization (APCI) interface. Samples (20 µL) were injected into the LC/MSD system through an Agilent 1100 series autosampler. Separations were carried out on a 5 µm Agilent ZORBAX SB-C18 column (4.6 mm × 150 mm) at 25 °C, controlled by an Agilent 1100 series thermostat, with 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 was used for most analyses and consisted of 65/35 solvent A/B for 3 min followed by a linear gradient to 20/80 solvent A/B in 17 min and then an isocratic elution at 20/80 A/B for 3 min. HPLC method II was employed in the initial studies of HRP/H2O2 and RLM/NADPH systems and consisted of 95/5 solvent A/B for 5 min followed by a linear gradient to 20/80 solvent A/B in 25 min and then an isocratic elution at 20/80 A/B for 3 min. The flow was directed to the APCI source after passing through an Agilent 1100 series Diode Array detector (detection at 280 nm). The LC/MSD trap was

Communications operated at APCI negative ionization mode (APCI-) with a corona current of 20 000 nA and a heating temperature of 400 °C. The gas (N2) 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 80800 under normal scan resolution (13 000 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 NMR spectra were collected on a Bruker 500 DRX equipped with a 5 mm TBI probe for inverse detected experiments and a 5 mm BBO probe for 13C observation. All data processing used Bruker XWinNMR 2.6. Data collection was at 298 K in DMSO-d6 (99.9%) using a 5 mm Shigemi NMR tube. The 1H dimensions were set to a sweep width of 12.6 ppm, and 13C dimensions were set at 250 ppm. The one-dimensional (1D) 1H and 13C required 16K and 17K scans, respectively. All two-dimensional (2D) spectra were collected with 2K points in F2 (direct dimension) and 512 points in F1 (indirect dimension). The number of scans in the 2D experiments varied as follows: gs-COSY (8 scans), gs-HMQC (32 scans), gs-HMBC (128 scans), 600 ms NOESY (64 scans), and gs-DQFCOSY (64 scans). All 2D spectra were processed to 1K in the F2 and F1 dimensions. The 600 ms NOESY and DQFCOSY allowed unambiguous assignment of the exchangeable protons. All coupling constants were measured from the 1D 1H spectrum, and spin simulation was used to verify couplings. Reaction of PCP with dG. 1. HRP Activation. Reaction mixtures (1 mL total volume) of PCP (1, 100 µM) and various molar equivalents of dG (0.5-40 equiv) in 100 mM phosphate buffer (pH 7.4) were incubated with HRP type VI (25 units/mL) in the presence of H2O2 (1 mM) at 37 °C for 60 min (up to 24 h). Aliquots (20 µL) were analyzed by LC/MS. Incubations of reaction mixtures (1 mL total volume) of PCP (1, 100 µM) and dG (2mM) in 100 mM phosphate buffer (pH 7.4) with various amounts of HRP (0.5 to 25 units/mL) in the presence (or absence) of H2O2 (100 µM to 1 mM) were carried out and analyzed in a similar manner. Incubations of reaction mixtures (1 mL total volume) of PCP (1, 100 µM) and HRP (type VI, 25 units/mL)/H2O2 (1 mM) in 100 mM phosphate buffer (pH 7.4) were performed in the presence of other DNA nucleosides (2 mM, dA, dC, or dT), and aliquots (20 µL) were analyzed by LC/ MS. 2. MPx Activation. Reaction mixtures (1 mL total volume) of PCP (1, 100 µM) and dG (2 mM) in 100 mM phosphate buffer (pH 7.4) were incubated with MPx (0.5-10 units/mL) in the presence of H2O2 (100 µM) at 37 °C for at least 30 min (up to 24 h). Aliquots (20 µL) at various incubation times were analyzed by LC/MS. 3. RLM Activation. Reaction mixtures (1 mL total volume) of PCP (1, 100 µM) and dG (2 or 4 mM) in 100 mM phosphate buffer (pH 7.4) were incubated at 37 °C for 60-90 min with RLM (1 mg/mL) in the presence of an NRS (100 µM). Aliquots (20 µL) were analyzed by LC/MS. Incubation of reaction mixtures of PCP (100 µM)/dG (2 mM) without RLM and PCP with RLM were carried out and analyzed in a similar manner. Preparation and Isolation of PCP-dG Adduct (4). A reaction mixture (100 mL total volume) of 100 µM PCP (1) and 40 molar equiv of dG in 50 mM phosphate buffer (pH 7.4, containing 1% DMSO) was incubated at 37 °C in the presence of HRP type VI (12.5 units/mL)/H2O2 (500 µM) for 18 h. The reaction mixture was concentrated by removal of water on a rotovaporator at 37 °C. A mixture of 25% DMSO/75% H2O was added, and then, the reaction mixture was centrifuged. The PCP-dG adduct 4 was isolated from the supernatant using a Hitachi 7000 HPLC system. Semipreparatory HPLC was performed on this system with a 5 µm Phenomenex Kromasil C8 column (10.00 mm × 250 mm) at a flow rate of 5.00 mL/min using the following mobile phase: 70/30 solvent A/B for 5 min and then to 20/80 solvent A/B in 15 min by a linear gradient

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Figure 3. (a) 1H NMR spectrum (500 MHz) of the PCP-dG adduct 4 in DMSO-d6. (b) 13C NMR spectrum of 4 in DMSO-d6.

Figure 2. (a) TIC (baseline subtracted, APCI- mode) from incubation of PCP (100 µM) and dG (2 mM) 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). (b) APCI- spectrum of the PCP-dG adduct. followed by an isocratic elution for 3 min at 20/80 solvent A/B (A: 0.1% formic acid in H2O; B: 0.1% formic acid in ACN). The reaction and HPLC separations were repeated three times. The PCP-dG adduct 4 was obtained as an off-white solid after removal of HPLC solvents on a freeze-dry system (Freezon 4.5, LABCONCO). 1H NMR (DMSO-d6, δ, ppm): 10.86 (b, NH), 6.59 (b, NH2), 6.26 (app t, H1′, J1′2′ ) 7.71, J1′2′′ ) 6.60 Hz), 5.31 (b, 3′OH), 4.81 (b, 5′OH), 4.34 (ddd, H3′, J3′2′ ) 6.17, J3′2′′ ) 3.08, J3′4′ ) 3.09 Hz), 3.81 (td, H4′, J4′5′5′′ ) 6.16 Hz), 3.49 (m, H5′,5′′), 2.94 (ddd, H2′, J2′2′′ ) 13.15 Hz), 2.21 (ddd, H2′′). 13C NMR (DMSO-d6, δ, ppm): 155.7, 153.8,150.4, 147.0 (C8), 145.4, 131.5, 131.0, 127.6, 110.4, 87.6 (C4′), 82.0 (C1′), 71.0 (C3′), 62.2 (C5′), 36.5 (C2′). UV-vis: 212, 246, 283 nm. MS (APCI-): [M - H]) 528; [M - Cl - H]- ) 493; [M - 2Cl - 2H]- ) 457; [M PCP + OH - H]- ) 281.

Results and Discussion

Figure 4. TIC (baseline subtracted, APCI-) from incubations of PCP (100 µM) and dG (2 mM) in 100 mM phosphate buffer (pH 7.4) at 37 °C in the presence of (a) HRP (type VI, 2.5 units/ mL)/H2O2 (100 µM) for 60 min; (b) MPx (2.5 units/mL)/H2O2 (100 µM) for 60 min; and (c) RLM (1 mg/mL)/NADPH (100 µM) for 90 min.

The reaction of PCP (100 µM) with DNA nucleosides (2 mM) was initially investigated using HRP/H2O2 as oxidation catalyst and atmospheric pressure chemical ionization (negative mode, APCI-) MS. Figure 2a shows the total ion chromatogram (TIC) following 1 h of incubation of PCP/dG in 100 mM phosphate buffer (pH 7.4) at 37 °C. Interestingly, a single major product eluting at ∼8.5 min was detected. Its APCI- spectrum (Figure 2b) contained the five chlorine isotope pattern of PCP and had a molecular ion at [M - H]- ) 528, suggesting attachment of dG with loss of two protons (i.e., PCP(264) + dG(267) - 2H ) 529). Its MS/MS spectrum exhibited ions at m/z 493 ([M - Cl - H]-), 457 ([M - 2Cl - H]-), and 281 ([M - PCP + OH - H]-). Further experiments showed the reaction to be absolutely specific for dG; no detectable adduct(s) was observed from HRP/H2O2 and PCP in the presence of dA, dC, or dT (not shown). Semipreparative scale reactions yielded the PCP-dG adduct as a white solid; its 1H NMR spectrum in DMSO-

d6 is shown in Figure 3a. The key findings from the 1H NMR spectrum include the loss of the C8 proton from dG, the loss of the PCP OH proton, and the downfield shift of H2′ from ∼2.5 ppm in dG to 2.94 ppm in the PCP-dG adduct (1H assignments were aided by COSY and NOESY experiments). The 13C NMR spectrum shown in Figure 3b was consistent with PCP attachment with fourteen resonances (i.e., G (5 peaks), PCP (4 peaks), and deoxyribose (dR, 5 peaks) ) 14) at δ 155.7, 153.8, 150.4, 147.0 (C8), 145.4, 131.5, 131.0, 127.6, 110.4, 87.6 (C4′), 82.0 (C1′), 71.0 (C3′), 62.2 (C5′), and 36.5 (C2′) ppm. The sugar carbons were assigned by HMQC, while the HMBC spectrum showed a correlation between the sugar H1′ and C8 at 147.0 ppm, which is ∼11 ppm downfield of C8 in dG, as expected for replacement of the C8 H-atom by an O-atom. These NMR results coupled with the MS data (Figure 2) confirmed C8 attachment by the O-site of PCP to yield the adduct 4 (Figure 1).

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Scheme 1. Proposed Pathways for the Ambident Reactivity of para-Chlorophenoxyl Radicals at the C8-Position of dG

Additional experiments were carried out to determine whether the O-bonded adduct 4 could be formed by alternative enzyme systems. Shown in Figure 4a is the PCP/dG/HRP reaction in which the yield of 4 was ca. 34%, based on a five point calibration curve using our authentic sample of 4. Treatment of PCP (100 µM)/dG (2 mM) with 2.5 units/mL MPx/H2O2 (100 µM) also yields 4 (Figure 4b) but ca. 10-fold lower yield than the HRP/ H2O2 system (Figure 4a). However, treatment of PCP/ dG with RLM shown in Figure 4c failed to generate 4; instead, an adduct with [M - H]- ) 440 was detected in the extracted ion chromatogram (EIC) (data not shown). While the structure of this adduct was not characterized, it was found to coelute with an adduct induced by reaction of dG with authentic chloranil. These observations are consistent with studies showing that peroxidase treatment yields the PCP phenoxyl radical (17) that reacts with dG to give 4, while liver enzymes convert PCP into chloranil (3-5, 9) that reacts with dG by an alternative mode of binding. That PCP is a substrate for MPx is highly significant given that exposure to PCP has been linked to incidences of leukemia (2). It has been proposed that benzene, via its phenolic metabolite, is a leukemogen because phenol is oxidized by intracellular MPx to its phenolic radical, which could be a key player in benzene carcinogenesis (18). In Scheme 1, pathways for ambident reactivity of parachlorophenoxyl radicals at C8 of dG are depicted. The O-site of the phenoxyl radical (generated by peroxidase/ H2O2) is shown reacting at C8 to yield the N-radical intermediate with the sp3-bound C8-atom; H-atom abstraction (by a phenoxyl radical) would furnish the O-adduct, i.e., 4. In contrast, C-attack would generate the corresponding quinoidal intermediate; loss of HCl would furnish the O-radical that would require an H-atom to give the C-adduct, i.e., 3. It is worthy to note that the oxidation potential of dG at 1.29 V vs NHE (19) is higher than that of PCP (E ) 0.99 V vs NHE (20)) and OTA (14). Thus, the phenoxyl radicals of these toxins cannot oxidize isolated dG to dG+• prior to attachment at C8. However, in duplex DNAs where π-stacking of purines stabilizes G+• (13), phenoxyl radicals of PCP and OTA could indeed cause spontaneous oxidation of G in

G-rich regions of DNA. Phenoxyl radical coupling with G+• would yield cationic intermediates with an sp3-bound C8-atom; loss of H+ would generate the C8-adducts. From the pathways outlined in Scheme 1, it is anticipated that O-attachment would be kinetically favored; O-adduct formation by PCP to give 4 was noted in the TIC (Figure 2a) following 1 h of incubation with HRP/ H2O2, while C-adduct formation by OTA to yield 3 (Figure 1) was observed in the EIC following 24 h of incubation at 37 °C (11). For OTA (11), O-attachment must be disfavored due to steric (flanking carbonyl moieties) or electronic factors (delocalization of the OTA phenoxyl radical). Binding of the phenolic O-site to a transition metal would also be expected to preclude O-attachment. In this regard, Ni(salens) that form phenoxyl radicals upon oxidation are reported to act like OTA and favor C-adduct formation (21). Current efforts are focused on determining the factors that govern ambident reactivity of phenoxyl radicals toward the C8-position of dG and the biological significance of adducts 3 and 4. In summary, the O-bonded C8-dG nucleoside adduct 4 of PCP (1) has been definitively identified by MS and NMR and is formed by peroxidase (HRP, MPx) activation. The adduct 4 is the first structurally characterized O-bonded DNA nucleoside adduct of a chlorophenolic toxin and demonstrates the ambident reactivity of phenoxyl radicals toward the C8-position of dG. Currently, the impact of C8-dG (O- and C-bonded) adducts of phenolic species on DNA structure, DNA repair, aging, and disease is an open area of investigation.

Acknowledgment. We are grateful to the National Institutes of Health (R01- CA080787) for financial support of this research.

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