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Characterization of a Deoxyguanosine Adduct of Tetrachlorobenzoquinone: Dichlorobenzoquinone-1,N2-etheno-2′-deoxyguanosine Thu N. T. Nguyen,† Anthony D. Bertagnolli,† Peter W. Villalta,‡ Philippe Bu¨hlmann,§ and Shana J. Sturla*,†,‡ Department of Medicinal Chemistry, College of Pharmacy, The Cancer Center, and Department of Chemistry, University of Minnesota, Minneapolis, Minnesota 55455 Received July 25, 2005
Pentachlorophenol (PCP), a widespread environmental pollutant that is possibly carcinogenic to humans, is metabolically oxidized to tetrachloroquinone. DNA adducts attributable to tetrachloroquinone have been observed previously in vitro and detected in vivo. In addition, an unidentified adduct in these studies coeluted with the product of the reaction of deoxyguanosine (dG) and tetrachlorobenzoquinone (Cl4BQ). We have synthesized, isolated, purified, and characterized the predominant adduct formed from the reaction of dG and Cl4BQ. The preparation of a 13C-labeled version of this adduct facilitated its structural characterization. On the basis of 1H NMR, 13C NMR, MS, IR, UV, and cyclic voltammetry, we propose that the adduct is a dichlorobenzoquinone nucleoside in which two chlorine atoms in Cl4BQ have been displaced by reaction at the 1- and N2-positions of dG. The 1H and 13C NMR chemical shifts are consistent with the dichlorobenzoquinone assignment. In contrast, under standard analytical conditions, LC-MS data are consistent with a reduced hydroquinone structure, similar to what may be expected based on results from other chloroquinones. Data from the present study indicate that this reduction could be occurring in the electrospray ionization source and that the initial product of the reaction of dG and Cl4BQ is a dichlorobenzoquinone. The results of this study contribute to the hypothesis that direct reactions between chlorophenols and DNA may play a role in the toxic effects of chlorophenols and indicate a potential difference in reactivity and biological influence between PCP and other less substituted chlorophenols or phenols.
Introduction Chlorophenols have been used extensively as biocides and wood preservatives, and they contaminate groundwater, drinking water, and food (1-3). They are cytotoxic (4) and possible human carcinogens (5). Metabolic activation of chlorophenols can result in DNA modification through the formation of covalent DNA adducts. If a DNA adduct is formed in a critical gene, carcinogenic mutations may result (6). Pentachlorophenol (PCP)1 was one of the most commonly used biocides before applications were limited in 1987, and in 2002, an estimated 11 million pounds were produced (7). Biotransformation routes of PCP in mammals have been characterized, and Scheme 1 illustrates three modes of DNA damage that can result from PCP * To whom correspondence should be addressed. E-mail: sturl002@ umn.edu. † Department of Medicinal Chemistry. ‡ The Cancer Center. § Department of Chemistry. 1 Abbreviations: Cl BQ, 2,3,5,6-tetrachlorobenzo-1,4-quinone (p4 chloranil); Cl2BQ-dG, 3-(2′-deoxyribosyl)-3H-benzo[d]imidazo[1,2-f]purin-6,9,11-trione (dichlorobenzoquinone-1,N2-etheno-2′-deoxyguanosine); Cl4HQ, 2,3,5,6-tetrachlorobenzene-1,4-diol; Cl2HQ-dG, 3-(2′-deoxyribosyl)-3H-dihydrobenzo[d]imidazo[1,2-f]purin-11(5H)-one; CV, cyclic voltammetry; dG, 2′-deoxyguanosine; ESI, electrospray ionization; EtO, acetyl acetate; IARC, International Agency for Research on Cancer; MS/MS, tandem mass spectrometry; PCP, pentachlorophenol; ROS, reactive oxygen species.
Scheme 1. PCP Metabolites and Formation of DNA Adducts
exposure (8). Hepatic cytochrome P450s oxidize PCP to tetrachlorohydroquinone (Cl4HQ), tetrachlorocatechols, and tetrachloroquinones. Peroxidases oxidize chlorophenols to the corresponding phenoxyl radicals. Enzymatic transformation to reactive chlorinated organic metabolites is also coupled with the generation of reactive oxygen species (ROS) that can result in oxidative damage to DNA. Pathways characteristic of DNA damage caused by ROS include base oxidation, deoxyribose damage, strand breaks, and abasic sites. Oxidative lesions are a subject of intense investigation, but less is known regarding the structure and biological influence of direct adducts of chlorophenols. Chronic exposure of rats to PCP results in increased levels of 8-oxo-dG (9), as well as increases in DNA adducts detected by 32P-postlabeling (9). Similarly, chlorocatechols and Cl4HQ have been found to induce DNA
10.1021/tx050204z CCC: $30.25 © 2005 American Chemical Society Published on Web 10/21/2005
Chloroquinone Adducts Scheme 2. Benzetheno-dG Adduct Results from Benzoquinone, a Metabolite of Benzene
damage, as gauged by increases of aldehydic DNA lesions (10, 11). These effects are potentiated by the degree of chlorination and are promoted by Cu(II)/NADP(H) (10). In addition to oxidative damage, 32P-postlabeling supports the formation of four major adducts (11) in calf thymus DNA and human HeLa S3 tumor cells treated with Cl4HQ (11, 12). These adducts were stable to neutral thermal hydrolysis, and the aldehyde reactive probe-slotblot assay demonstrated an associated increase in abasic sites and levels of 8-oxo-dG. Data strongly supported that these abasic sites are derived from ROS damage, rather than decomposition of directly formed adducts (11). Furthermore, conditions that promote redox cycling [Cu(II)/NADP(H)] enhance levels of abasic sites (11). The phenoxyl radical formed by oxidation of PCP (Scheme 1) reacts directly with 2′-deoxyguanosine (dG) to generate adducts containing an aryl ether linkage (13). The formation of DNA adducts in vivo has been observed after exposure to the quinone metabolite 1,4tetrachlorobenzoquinone (Cl4BQ) (9). Quinones are reactive electrophiles with the capacity to alkylate biological nucleophiles and lead to cancer (14). For example, the accumulation of p-benzoquinone in bone marrow is thought to be associated with benzene-induced leukemias (15). p-Benzoquinone reacts with dG to form the benzetheno adduct 1 (Scheme 2). The analogous PCP metabolite, Cl4BQ, forms a mixture of adducts with sulfhydryl groups in proteins, as reported for hemoglobin and albumin samples (16). As determined by 32P-postlabeling, the reaction of calf thymus DNA with Cl4BQ produced four major adducts that were stable to neutral thermal hydrolysis but whose structures were not characterized (11). Furthermore, a DNA adduct detected in the liver of rats after chronic PCP exposure coeluted with an adduct formed from reaction with Cl4BQ, but its structure was not determined (9). This quinone-derived adduct was also observed in liver DNA from mice exposed to PCP (15 mg/kg/day) for 7 days but at higher levels (8 adducts/ 107 nucleotides) (9). To identify a potential DNA adduct resulting from in vivo metabolic activation of PCP to Cl4BQ, we report here characterization of the primary product of the in vitro reaction of dG with Cl4BQ and its chemical stability and discuss how this adduct differs from previously identified dG-quinone reaction products.
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 (17). Apparatus and Assay Conditions. HPLC analysis of reaction mixtures was carried out using an Agilent 1100 HPLC with a diode array detector. The HPLC column used was a Phenomenex Luna 5µ C18(2) 100A 250 mm × 4.60 mm (Phenomenex, Torrance, CA). Chromatography solvents used were 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). The gradient system used a flow rate of 0.5 mL/min and
Chem. Res. Toxicol., Vol. 18, No. 11, 2005 1771 consisted of 95% A, 5% for 5 min, followed by a 20 min linear gradient to 5% A, 95% B that was held for 15 min. Preparative scale purification was carried out on a Biotage Sp1 highperformance flash chromatography system (Biotage, Charlottesville, VA) equipped with a Biotage C18 reverse phase Flash column (25 + M). Chromatography solvents were water (A) and acetonitrile (B). This system was preequilibrated with 100% A for 3 min at 25 mL/min. After sample introduction, the column was eluted with 100% A for 3.3 min followed by a linear gradient to 80% A, 20% B over 23.1 min. LC-electrospray ionization (ESI)MS was carried out with a Finnigan LCQ Deca instrument (Thermo Finnigan LC/MS Division, San Jose, CA) interfaced with a Waters Alliance 2690 HPLC multisolvent delivery system and equipped with an SPD-10A UV detector (Shimadzu Scientific Instruments, Columbia, MD) using the HPLC solvent elution system and column as described above. The ESI source was set in negative ion mode as follows: voltage, 6 kV; current, 4 µA; and capillary temperature, 275 °C. MS data were collected for a mass range of 100-1000 amu. Tandem mass spectrometry (MS/MS) data were acquired with the following parameters: isolation width, 1.5 amu; normalized collision energy, 40%; activation Q, 0.25; and activation time, 30 ms. Xcalibur version 1.4 SRI was used to simulate calculated MS spectra. 1H NMR spectra were recorded in d6-DMSO using a Mercury 300 or Inova (Varian, Inc., Palo Alto, CA) spectrometer operating at 300 or 500 MHz, respectively, at 25 °C. 13C NMR of 5 was recorded using the Inova spectrometer (125 MHz). The IR spectrum was recorded in KBr using a Nicolet Prote´ge´ 460 ESP (Thermo Electron Corporation, Madison, WI). Cyclic voltammograms (CV) were measured with a CYSY-1 electroanalysis station (Cypress Systems, Lawrence, KS), a platinum wire auxiliary electrode, a Ag/AgCl reference electrode, and a gold disk working electrode (Bioanalytical Systems, West Lafayette, IN) that was polished with wet 0.3 µm alumina slurry (Alpha Micropolish #2, Buehler, Lake Bluff, IL) on a felt pad, rinsed with water, and cleaned in a sonicator directly before use. Solutions were purged with argon for at least 15 min prior to CV measurements. Chemicals and Enzymes. All reagents were used as supplied without further purification. dG (manufactured under Fluka label) was obtained from Sigma Aldrich Chemical Co. (Milwaukee, WI). 13C6-Cl4BQ was obtained from Cambridge Isotope Laboratories, Inc. (Andover, MA). 7,8-Dichloro-3-(2′-deoxyribofuranos-1-yl)-3H-imidazo[4′,5′:4,5]pyrimido[1,2-a]benzimidazole-6,9,11(5H)-trione (5). To a 20 mL scintillation vial with a magnetic stir bar were added dG (12.6 mg, 0.045 mmol), Cl4BQ (16.1 mg, 0.062 mmol), and DMSO (1 mL) resulting in the formation of a yellow solution. Immediately thereafter, 5.25 mL of 100 mM Na3PO4 buffer, pH 7.4, was added, producing a cloudy gray solution. The reaction mixture was heated (65 °C) for 2 h producing a clear black solution, which was then concentrated to a volume of approximately 3 mL using a rotary evaporator. This crude mixture was purified in two equal portions using the Biotage system described above. The reaction was performed several times giving an average yield of 10.5 mg (53% yield) of the title compound that was isolated as a green/black solid that produced a deep green solution in water. λmax (340 nm); mp > 200 °C dec. HRMS calcd for C16H10O6N5Cl2-, 438.00066; found, 438.0014. ESI MS m/z 438.3. MS/MS m/z (relative intensity): 438.3 (100), 322.3 (49), 286.2 (18). 1H NMR (300 MHz, d6-DMSO): δ 8.04 (s, 1H, H8); 6.25 (t, J ) 7.5 Hz, 1H, 1′CH); 5.29 (d, J ) 3.6 Hz, 1H, OH); 5.16 (t, J ) 6.0 Hz, 1H, OH); 4.39 (m, 1H, 3′CH); 3.85 (m, 1H, 4′CH); 3.60 (m, 1H, 5′CH2a); 3.53 (m, 1H, 5′CH2b); 2.95 (br, 1H, NH); 2.70 (m, 1H, 2′CH2a); 2.22 (ddd, J ) 12.9, 6.0, 3.0 Hz, 1H, 2′CH2b). 13C NMR (125 MHz, d6-DMSO): δ 176.5, 158.0; 156.4 (C6); 153.2 (C2); 151.4; 149.8 (C4); 141.0, 138.4 (C8); 130.4, 119.1 (C5); 117.4; 88.5 (C4′); 84.3 (C1′); 71.7 (C3′); 62.7 (C5′); (the C2′ peak was obscured by DMSO at 40.5 ppm). IR (KBr, cm-1): υ 3356, 2924, 1684, 1635. (254 nm) ) 4 × 104 M-1 cm-1. 13C -7,8-Dichloro-3-(2′-deoxyribofuranos-1-yl)-3H-imi6 dazo[4′,5′:4,5]pyrimido[1,2-a]benzimidazole-6,9,11(5H)-trione (13C6-5). The title compound was prepared using the same
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Figure 2. Benzetheno adducts 2 and 3 and hypothetical angular p-quinone adduct 4.
Figure 1. (A) HPLC analysis with UV detection (254 nm) of the reaction of Cl4BQ with dG. The retention time of Cl4BQ in this system is 29 min. (B) Corresponding UV spectrum at pH 4 overlaid with a spectrum obtained at pH 7. Because the adduct is not stable to base, a UV could not be obtained at a higher pH for comparison. procedure described for the preparation of 5, from dG (11.5 mg, 0.04 mmol) and 13C6-Cl4BQ (11.8 mg, 0.05 mmol). After isolation and purification as described for 5, the labeled adduct was obtained as a green solid. 1H NMR matched 5. ESI MS m/z 446.3. MS/MS m/z 446.3 (100%), 330.3 (23%). 13C NMR (125 MHz, d6-DMSO, 13C labeled positions): δ 176.5 (dddd, J ) 52, 50, 25, 8 Hz, C3′′), 158.0 (dddd, J ) 80, 52, 20, 5, 2 Hz, C2′′); 151.4 (dddd, J ) 70, 59, 8, 5 Hz, C6′′); 141.0 (dddd, J ) 82, 70, 13, 8, 2 Hz, C5′′); 130.4 (dddd, J ) 82, 50, 20, 8 Hz, C4′′), 117.4 (dddd, J ) 80, 59, 25, 13 Hz, C1′′). Thermal Stability of 5. To a solution of 5 (125 µL, 3.3 mM in 20% DMSO/phosphate buffer) was added 125 µL of an acidic (0.2 M HCl), basic (0.2 M NaOH), or neutral (H2O) solution. The resulting solutions were placed at 4, 37, or 85 °C. At 20 min intervals over a 180 min period, samples were neutralized to pH 7.0 with an equal volume of acidic, basic, or aqueous solution and prepared for HPLC analysis. To a 1.5 mL HPLC vial, 1 mL of distilled water was added followed by 100 µL of neutralized sample and 50 µL of an injection standard (0.1 mg/mL 3,5dinitrobenzoic acid in water). HPLC injection volumes were 100 µL. Rate constants were determined by linear regression of a plot of the ln[5] vs time.
Results An aqueous solution prepared from the crude mixture resulting from the reaction of dG with Cl4BQ in phosphate buffer (18), pH 7.4, at 37 °C was analyzed by HPLC with UV detection. The disappearance of dG and Cl4BQ was associated with the appearance of a new product (Figure 1A). The UV spectrum corresponding to the major peak (Figure 1B) at pH 4 displayed a long wavelength λmax (340 nm) consistent with 1,N2-etheno and benzetheno type dG adducts (19-21). To generate sufficient quantities of the compound for full chemical characterization, reaction conditions (time and temperature) were optimized, resulting in the procedure described in the Experimental Procedures (65 °C for 2 h). Initial LC-ESI-MS analysis in negative ion mode under standard conditions (chromatography solvents buffered with 0.1% formic acid; ESI source voltage, 6 kV; see Supporting Information for data) of the reaction product described above gave rise to an adduct that had a
nominal mass of [M - H]- ) 440, consistent with the hydroquinone structure 3 (Figure 2). We found, however, that by arraying the source voltage between 6 and 2 kV the predominant ion measured switched from [M - H]) 440 to [M - H]- ) 438. The MS data in Figure 3 were acquired by infusion of the same sample with the source voltage set at either 6 or 2 kV. This phenomenon was observed for a solution that contained formic acid. If the sample was prepared without formic acid, an increase in the 440 peak was still observed but to a lesser extent. We concluded that an original adduct with [M - H]- ) 438 was being reduced to a secondary product of [M H]- ) 440, i.e., 3. This reaction may occur in the spectrometer source (22) at high voltages in the presence of formic acid or on the analytical HPLC column. Figure 3B shows the expansion of [M - H]- ) 438 with multiple peaks from the chlorines isotopes. A mixture of [M - H]) 440 and [M - H]- ) 438 is present in Figure 3E. Although [M - H]- ) 440 is the major product, adjusting the voltage does not solely produce the hydroquinone. In both cases, the mass envelopes are characteristic of the isotopic distribution expected for a dichloride. MS/MS analysis of the 438 peak produced ions with mass-tocharge ratios of 322, corresponding to loss of deoxyribose, and 286, corresponding to loss of HCl and deoxyribose (Figure 4). A transformation consistent with the initial formation of quinone adduct, 5 (Scheme 3) with [M - H]- ) 438, is illustrated in Scheme 3. Angular adduct 4 (Figure 2) would also be consistent with the present MS data. If the adduct had structure 4, the N-1H resonance would be expected in the range of 12-14 ppm and the 1H resonance from the anomeric position would be expected to shift significantly because of its proximity to the quinone moiety. Furthermore, UV absorption shifts have been compared for angular and etheno type adducts, and the angular adducts display a low-energy UV band that is shifted toward a shorter wavelength by about 30-50 nm (21). Finally, if the adduct had structure 4, it would likely undergo depurination upon neutral thermal hydrolysis, which was not observed. While these data are consistent with structure 5, one cannot unequivocally rule out 4 as a possibility. Proton NMR analysis of 5 resulted in a spectrum similar to dG, with slight variation in chemical shifts, as indicated in Table 1. The N-H signals in dG (10.64 and 6.47 ppm) were absent in 5. For other positions, changes in chemical shift values are observed relative to dG, but the spectrum lacks characteristic new signals, making the characterization of an adduct generated from Cl4BQ challenging relative to its un-, mono-, di-, and trichlorinated analogues. 13 C chemical shifts provided more conclusive structural information. We synthesized 5 using commercially obtained 13C6-Cl4BQ. The resulting 13C NMR (Figure 5)
Chloroquinone Adducts
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Figure 3. ESI-MS analysis of 5 at varying source voltage. (A) Full MS scan, 2 kV; (B) expansion of A; and (C) calculated MS for C16H10Cl2N5O6- (M - H for Cl2BQ-dG). (D) Full MS scan, 6 kV; (E) expansion of D; and (F) calculated MS for C16H12Cl2N5O6- (M H for Cl2HQ-dG). Isotopic distribution is consistent with dichloride formulation.
Figure 5. 13C NMR (125 MHz, d6-DMSO) of 13C-labeled 5 illustrating the six 13C-labeled carbons with chemical shifts consistent with the quinone structure and 13C-13C coupling. Table 1. Comparison of Chemical Shifts (ppm) of 1H NMR Peaks (d6-DMSO) in 5 with Analogous Resonances in dG (26)
Figure 4. MS/MS analysis of 5 obtained from infusion of a sample that does not contain formic acid and with the source voltage at 2 kV. Product ions are consistent with loss of deoxyribose and loss of HCl.
Scheme 3. Reaction of Cl4BQ with dG Produces the Dichlorobenzoquinone Adduct 5
provided structural information from chemical shift values and 13C-13C coupling constants (23). Six 13C resonances were observed at chemical shifts characteristic of an unsymmetric quinone. For reference, the values of the C1 13C shift (d6-DMSO) of benzoquinone and Cl4BQ are 187 and 170 ppm, respectively, and the analogous C2 values are 136 and 140 ppm (24). The
proton
dG
5
8 1′ 2′a 2′b
7.92 (s) 6.11 (dd, J ) 7.9, 6.0 Hz) 2.50 (ddd) 2.19 (ddd, J ) 13.1, 5.7, 3.0 Hz) 4.33 (m) 3.80 (td) 3.52 (m) 5.27 (d, J ) 5.5 Hz) 5.10 (t, J ) 3.9 Hz)
8.04 (s, 1H) 6.25 (t, J ) 7.5 Hz) 2.70 (m) 2.22 (ddd, J ) 12.9, 6.0, 3.0 Hz) 4.39 (m) 3.85 (m) 3.60-3.53 (m) 5.29 (d, J ) 3.6 Hz) 5.16 (t, J ) 6.0 Hz)
3′ 4′ 5′ OH
values of the 13C chemical shifts in Cl4HQ are 144 and 121 ppm (d6-DMSO). The 13C resonance value of 176.5 ppm in 5 is characteristic of a conjugated carbonyl. The second carbonyl resonance displays a significantly higher field chemical shifts151.4 ppmswhich can be rationalized on the basis of the contribution of the resonance structure illustrated in Scheme 4. Furthermore, the imine character of the C2′′-position illustrated in this resonance structure supports the low fields158.0 ppms shift of the C2′′-position relative to the other olefin-like positions at 141.0, 130.4, and 117.4 ppm. Surprisingly, the C2′′ resonance is lower field than the C6′′-position, which was assigned based on analysis of 13C-13C coupling constants as illustrated in Table 2. The distribution of
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Scheme 4. Dichlorobenzoquinone Adduct 5, Corresponding 13C Chemical Shift Values, Proposed Contributing Resonance Structure 6, and Potential Tautomeric Structures 7 and 8
Scheme 5. Proposed Mechanism for the Formation of 5a
Table 2. 13C-13C Coupling Constants (Hz) Determined from Analysis of the 13C Peaks for 13C6-5a C2′′
C3′′
C4′′
C5′′
C6′′
80
25 52
4 20 50
13 2 8 82
59 5
