Synthesis and Characterization of Polycyclic ... - ACS Publications

Chem. Res. Toxicol. 1999, 12, 237-246

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Synthesis and Characterization of Polycyclic Aromatic Hydrocarbon o-Quinone Depurinating N7-Guanine Adducts† Kirsten D. McCoull,‡ Diane Rindgen,§ Ian A. Blair,§ and Trevor M. Penning*,‡ Department of Pharmacology and Center for Cancer Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6084 Received August 4, 1998

Polycyclic aromatic hydrocarbons (PAHs) are environmental pollutants which may cause cancer and require metabolic activation to exert their carcinogenic effects. One pathway of activation involves the dihydrodiol dehydrogenase-catalyzed oxidation of non-K region transdihydrodiols to yield catechols, which autoxidize to form reactive o-quinones. As a step toward identifying the spectrum of PAH o-quinone-DNA adducts that may form in biological systems, depurinating PAH o-quinone-guanine adducts were synthesized. Naphthalene-1,2-dione, phenanthrene-1,2-dione, and benzo[a]pyrene-7,8-dione were reacted with 5 equiv of 2′deoxyguanosine (dGuo) under acidic conditions (1:1 acetic acid/water). The products were purified by reversed-phase HPLC, characterized by a combination of UV spectroscopy, electrospray ionization/tandem mass spectrometry, and high-field proton nuclear magnetic resonance spectroscopy, and identified as 7-(naphthalene-1,2-dion-4-yl)guanine (MH+, m/z 308), 7-(phenanthrene-1,2-dion-4-yl)guanine (MH+, m/z 358), and 7-(benzo[a]pyrene-7,8-dion-10-yl)guanine (MH+, m/z 432), respectively. Reaction at N7 of dGuo leads to cleavage of the glycosidic bond, producing depurinating adducts. Reaction of phenanthrene-1,2-dione with calf thymus DNA led to the formation of the corresponding depurinating adduct. The loss of modified bases in DNA generates apurinic sites which, if unrepaired, can lead to mutations and thus cellular transformation. These synthesized PAH o-quinone-N7-guanine adducts can be used as standards to identify such adducts in vitro and in vivo.

Introduction (PAHs)1

Polycyclic aromatic hydrocarbons are environmental pollutants found, for example, in car exhaust fumes and tobacco smoke, and may be causative agents in human cancer (1). To exert their carcinogenic effects, PAHs require metabolic activation to electrophilic species which can modify DNA (1), providing routes to “change of function” mutations in proto-oncogenes and tumor suppressor genes. Many human tumors contain oncogenic ras activated by point mutations in the 12th codon arising from G to T transversions (2, 3). Characteristic of many lung cancers, for which PAHs are likely causative agents, are mutations in the p53 tumor suppressor gene (4) at codons 157, 248, and 273 (5). The majority of these mutations are G to T transversions (4). Any explanation † A preliminary account of this work was presented at the 89th annual meeting of the American Association for Cancer Research, New Orleans, LA, March 28 to April 1, 1998. * To whom correspondence should be addressed: Department of Pharmacology, University of Pennsylvania School of Medicine, 3620 Hamilton Walk, Philadelphia, PA 19104-6084. Fax: (215) 573-2236. E-mail: [email protected]. ‡ Department of Pharmacology. § Center for Cancer Pharmacology. 1 Abbreviations: BP, benzo[a]pyrene; anti-BPDE, (()-trans-7,8dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene; BP-diol, (()-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene; BPQ, benzo[a]pyrene-7,8-dione; CID, collision-induced dissociation; DD, dihydrodiol dehydrogenase (trans-1,2-dihydrobenzene-1,2-diol dehydrogenase, EC 1.3.1.20); D2O, deuterium oxide; dGuo, 2′-deoxyguanosine; ESI/MS/ MS, electrospray ionization/tandem mass spectrometry; NPQ, naphthalene-1,2-dione; ODS, octadecylsilane; PAH, polycyclic aromatic hydrocarbon; PQ, phenanthrene-1,2-dione.

of the molecular origins of cancer must provide a mechanism to account for these mutations. Metabolic activation of benzo[a]pyrene (BP), a representative PAH, occurs via several pathways (Scheme 1). One pathway involves the formation of diol epoxides (6). Epoxidation of BP, catalyzed by cytochrome P450 (P450), and subsequent hydrolysis catalyzed by epoxide hydrolase, yield a trans-dihydrodiol, (()-trans-7,8-dihydroxy7,8-dihydrobenzo[a]pyrene (BP-diol). A second P450catalyzed epoxidation yields (()-trans-7,8-dihydroxy-anti9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE). anti-BPDE forms predominantly stable adducts with nucleic acids, e.g., by reaction with the N2 amino group of the guanine moiety (7). Stable anti-BPDE-DNA adducts activate proto-oncogenic ras via a G to T transversion (8), and anti-BPDE selectively targets hot spots mutated in the p53 tumor suppressor gene in lung cancer patients (5). Stable adducts can give rise to these mutations, e.g., by translesional synthesis (9), and provide a route to the mutations observed in cancer. PAHs are also activated to radical cations by oneelectron oxidation, catalyzed by cytochrome P450 or peroxidases (10). Radical cations form mainly depurinating adducts with DNA, e.g., N7-guanine, C8-guanine, N7adenine, and N3-adenine adducts (11, 12), in which the modified base is lost from the DNA following hydrolysis of the glycosidic bond. When mouse skin papillomas were initiated by PAHs, the level of depurinating adducts derived from radical cations correlated with the A to T or G to T transversions observed in the c-H-ras oncogene

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238 Chem. Res. Toxicol., Vol. 12, No. 3, 1999

McCoull et al.

Scheme 1. Proposed Pathways of Metabolic Activation of Polycyclic Aromatic Hydrocarbons

(12). The loss of depurinating adducts leaves behind apurinic sites which, if unrepaired, leads to the preferential insertion of deoxyadenosine opposite that site (13). This results in an A to T or G to T transversion upon DNA replication. Thus, depurinating adducts provide an alternative means to the mutations observed in cancer. A third pathway of PAH activation involves the oxidation of trans-dihydrodiols to o-quinones catalyzed by dihydrodiol dehydrogenase (DD) (14, 15). This enzyme suppresses the formation of diol epoxides by diverting PAH trans-dihydrodiols to catechols (Scheme 1). Autoxidation of the catechol produces reactive and redox active o-quinones, via an o-semiquinone anion radical intermediate. For example, rat and human DDs convert the proximate carcinogen BP-diol to benzo[a]pyrene-7,8-dione (BPQ) (15, 16), a reaction that also occurs in whole cells (isolated rat hepatocytes) (17). Formation of PAH o-quinones by this pathway could result in a spectrum of DNA modifications (Scheme 2).

First, PAH o-quinones could form stable and depurinating adducts by Michael addition. We have shown that [1,3-3H2]BPQ forms stable dGuo adducts with calf thymus and plasmid DNA (18), but no information exists about the formation of depurinating adducts. Second, PAH o-quinones can enter futile redox cycles to generate reactive oxygen species (ROS) (hydroxyl radical, hydrogen peroxide, and superoxide anion radical), which can lead either to the formation of oxidatively damaged bases, e.g., 8-hydroxy-dGuo, or to hydroxyl radical-mediated strand scission which yields base propenals. It is known that BPQ will promote hydroxyl radical-mediated strand scission of double-stranded oligonucleotides (19), and phage (19) and hepatocyte DNA (20), most likely via a Criegee rearrangement (21). The pathway of PAH activation via o-quinones is also reminiscent of the activation of estrogens via catechol estrogens. In the latter pathway, depurinating adducts derived from estrogen o-quinones have been synthesized

PAH o-Quinone-N7-Guanine Adducts

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Scheme 2. Spectrum of DNA Modifications That Could Result from Polycyclic Aromatic Hydrocarbon o-Quinones

(22) and detected both in vitro by reaction of the oquinones with calf thymus DNA and in vivo in rat mammary glands (23). These depurinating adducts provide a route to G to T transversions (by misreplication of unrepaired apurinic sites) in proto-oncogenes and tumor suppresser genes (23). In this study, we use PAH o-quinones that are produced by the DD pathway to synthesize depurinating adducts with dGuo. These adducts can be used as standards to detect their prevalence in biological systems. In addition, we document the formation of depurinating adducts when a non-K region PAH o-quinone was reacted with calf thymus DNA. The synthesis and characterization of PAH o-quinone-guanine standards and their detection in vitro represent an important first step toward determining the contribution of o-quinones to the DNA adduct profile observed with PAHs.

Experimental Procedures Caution: The work described involves the synthesis and handling of hazardous agents and was therefore conducted in accordance with the NIH Guidelines for the Laboratory Use of Chemical Carcinogens. Materials. The o-quinones, naphthalene-1,2-dione (NPQ), phenanthrene-1,2-dione (PQ), and BPQ, were synthesized according to published procedures (24-26). The purity of the o-quinones was checked by 1H NMR spectroscopy and reversedphase HPLC and found to be >99% in the case of NPQ and >95% for PQ and BPQ. 2′-Deoxyguanosine monohydrate and calf thymus DNA (type I, sodium salt “highly polymerized”) were

purchased from Sigma Chemical Co. (St. Louis, MO) and used without further purification. HPLC. Reversed-phase HPLC was performed using a Beckman 125 pump and a Beckman 166 variable-wavelength UV/ vis detector set to 254 nm for the NPQ and BPQ adducts and 290 nm for the PQ adducts. Analytical separations were carried out using a PhaseSeparations Spherisorb ODS-2 column (250 mm × 4.6 mm i.d., 5 µm) at a flow rate of 1 mL/min using the following mobile phases: for reactions with NPQ, 10% methanol/water for 10 min, followed by a linear gradient to 100% methanol over the course of 30 min; for reactions with PQ, 30% methanol/water for 10 min, followed by a linear gradient to 100% methanol over the course of 35 min; and for reactions with BPQ, 20% methanol/ water for 10 min, and then a linear gradient to 60% methanol/ water over the course of 60 min, followed by a linear gradient to 100% methanol over the course of 20 min. Semipreparative HPLC purification of the NPQ-guanine adduct was achieved using a Whatman Partisil ODS column (500 mm × 9.4 mm i.d., 10 µm) at a flow rate of 3 mL/min and a mobile phase of 20% methanol/water for 10 min, followed by a linear gradient to 100% methanol over the course of 60 min. Semipreparative HPLC purification of the PQ-guanine adduct was achieved using a YMC-Pack ODS-AQ column (250 mm × 10 mm i.d., 5 µm) at a flow rate of 4.7 mL/min. For the first step in the purification, the mobile phase consisted of 30% methanol/water for 10 min, followed by a linear gradient to 90% methanol/water over the course of 40 min. The pooled fractions were further purified using a mobile phase of 10% acetonitrile/ water for 10 min, and then a linear gradient to 50% acetonitrile/ water over the course of 40 min, followed by a linear gradient to 80% acetonitrile/water over the course of 15 min.


McCoull et al.

Scheme 3. Reaction of Naphthalene-1,2-dione with 2′-Deoxyguanosine

Mass Spectrometry. The mass spectrometric data were acquired on either a Finnigan TSQ7000 triple-quadrupole mass spectrometer or a Finnigan LCQ ion trap instrument equipped with a Finnigan electrospray source. On-line chromatography was performed using a Waters Alliance 2690 HPLC system employing the aforementioned HPLC conditions. A postcolumn split was used which generated a 400 µL/min flow to the instrument source with 600 µL/min diverted to a Hitachi variable-wavelength UV/vis detector. In addition, a postcolumn solution of 50% methanol/water with 1% acetic acid was introduced into the source at 100 µL/min to assist analyte ionization. All collision-induced dissociation (CID) spectra were acquired in the positive ion mode with the optimal collision conditions individually determined for the different analytes. UV Spectroscopy. UV spectra were obtained on-line using a Perkin-Elmer Binary LC Pump 250 and a Perkin-Elmer LC480 Auto Scan diode array detector with the aforementioned HPLC conditions. NMR Spectroscopy. 1H NMR spectra were obtained on a Bruker AM-500 spectrometer operating at 500 MHz, using Me2SO-d6 as the solvent. All chemical shifts (δH) are in ppm downfield from tetramethylsilane. The spectra were referenced to residual protonated solvent residues as internal standards; for Me2SO, δH ) 2.52 ppm. Coupling constants J are described to the nearest 0.1 Hz. The deuterium oxide (D2O) exchange experiment was performed by adding 2 drops of D2O to the sample, shaking, then reacquiring the spectrum after 4 h. Synthesis of 7-(Naphthalene-1,2-dion-4-yl)guanine. An orange solution of NPQ (10 mg, 63 µmol) in acetonitrile (2 mL) was added to a stirred solution of dGuo (90 mg, 316 µmol) in a 1:1 mixture of acetic acid and water (4 mL). After the mixture was stirred at room temperature for 18 h, aliquots were removed for analysis by HPLC, MS, and UV spectroscopy, and the solvent was evaporated under reduced pressure to yield a brown solid. The crude reaction product was partially purified by solid-phase extraction. In a representative procedure, 10 mg of crude product was dissolved in 1% acetic acid/water (1 mg/mL) and loaded onto a Waters C18 Sep-Pak Classic cartridge which had been conditioned by washing with methanol (10 mL) followed by 1% acetic acid/water (10 mL). After being loaded, the cartridge was washed successively with 1% acetic acid/water (30 mL) and water (3 mL). The product was then eluted with 75% methanol/water (10 mL). The eluents from several similar Sep-Pak purifications were combined; the methanol was evaporated under reduced pressure, and the remaining aqueous solution was lyophilized to give a brown solid (9 mg). This product was dissolved in 20% methanol/water and further purified by semipreparative HPLC. After removal of the solvent, a brown solid was obtained, which was characterized by NMR and UV spectroscopy. Reaction of Naphthalene-1,2-dione with Guanine. An orange solution of NPQ (5.0 mg, 32 µmol) in acetonitrile (1 mL) was added to a stirred suspension of guanine (24 mg, 158 µmol) in a 1:1 mixture of acetic acid and water (2 mL). The resulting mixture was stirred at room temperature for 18 h and the solvent evaporated under reduced pressure to give a light brown solid. The crude product was analyzed by reversed-phase HPLC. Synthesis of 7-(Phenanthrene-1,2-dion-4-yl)guanine. A solution of dGuo (34 mg, 120 µmol) in a 1:1 mixture of acetic acid and water (2 mL) was added to a stirred red suspension of PQ (5 mg, 24 µmol) in acetonitrile (1 mL). The reaction mixture

was stirred at room temperature, and aliquots were removed for analysis by HPLC, MS, and UV spectroscopy after 18 and 36 h. After 36 h, the solvent was evaporated in vacuo to yield a brown solid, which was partially purified by semipreparative HPLC using a methanol/water gradient. The products of four such reactions were combined, and further purified by semipreparative HPLC using an acetonitrile/water gradient. After removal of the solvent, a brown solid was obtained, which was characterized by NMR and UV spectroscopy. Synthesis of 7-(Benzo[a]pyrene-7,8-dion-10-yl)guanine. A solution of dGuo (13 mg, 44 µmol) in a 1:1 mixture of acetic acid and water (0.5 mL) was added to a stirred purple suspension of BPQ (2.5 mg, 9 µmol) in Me2SO (1.5 mL). The reaction mixture was sealed to prevent solvent evaporation, and then stirred at 37 °C for 5 days, during which time aliquots were removed periodically for analysis by HPLC, MS, and UV spectroscopy. Reaction of Phenanthrene-1,2-dione with Calf Thymus DNA. Method A. A solution of PQ (6.5 mg, 31.3 µmol) in Me2SO (0.5 mL) was added to a solution of calf thymus DNA (10 mg) in water (4.5 mL). The resulting mixture was stirred in a sealed flask in the dark for 18 h at 37 °C. After being cooled to room temperature, the reaction mixture was diluted to a total volume of 8 mL with 10% Me2SO/water, and then filtered through four Centricon-10 filters (Millipore Corp.) to remove the DNA. The filtrates were combined and concentrated on a vacuum centrifuge (Savant) to a total volume of approximately 1.5 mL and analyzed by MS. Method B was identical to method A except that after 18 h at 37 °C the reaction mixture was subjected to thermal hydrolysis at 100 °C for 30 min and then cooled on ice prior to DNA removal.

Results Formation of Depurinating NPQ Adducts with dGuo. Initial attempts to form PAH o-quinone depurinating adducts focused on NPQ, since this o-quinone can be prepared in larger quantities than PQ or BPQ. Previously, estrogen 3,4-o-quinones formed in situ by chemical oxidation of catechol estrogens were shown to react with dGuo under acidic reaction conditions to produce depurinating adducts (22). Consequently, NPQ was reacted with 5 equiv of dGuo in a 1:1:1 mixture of acetonitrile, acetic acid, and water at room temperature for 18 h (Scheme 3). Analysis by reversed-phase HPLC indicated that virtually all of the NPQ had been consumed. When THF was used in place of acetonitrile, a larger amount of NPQ (tR ) 33.7 min) remained (Figure 1A). Only one product was detected (tR ) 26.5 min) in addition to a small amount of guanine (tR ) 17.3 min), presumably produced by hydrolysis of dGuo under the acidic reaction conditions. Since dGuo had been used in excess, a large amount remained unreacted (tR ) 14.4 min). Analysis by HPLC/electrospray ionization/mass spectrometry (HPLC/ESI/MS) in conventional full scan mode showed a protonated molecular ion (MH+) at m/z 308, at



Figure 2. Collision-induced dissociation spectrum of the m/z 308 (MH+) ion of the naphthalene-1,2-dione-guanine adduct (tR ) 26.5 min in Figure 1A), obtained on a Finnigan TSQ7000 instrument.

Figure 3. 1H NMR spectrum of 7-(naphthalene-1,2-dion-4-yl)guanine (500 MHz, Me2SO-d6).

Figure 1. (A) HPLC separation of the products obtained from the reaction of naphthalene-1,2-dione with 2′-deoxyguanosine in 1:1:1 THF/CH3CO2H/H2O. (B) UV spectra of guanine, naphthalene-1,2-dione, and the adduct formed. (C) HPLC separation of the products obtained from the reaction of naphthalene-1,2dione with guanine in 1:1:1 CH3CN/CH3CO2H/H2O.

a retention time characteristic of the major reaction product (data not shown). This molecular ion corresponded to an NPQ-guanine adduct, and the product ions obtained upon CID of the MH+ ion were consistent with this structure (Figure 2). For example, a fragment ion at m/z 152 (protonated guanine) was observed, confirming the presence of guanine. In addition, product ions derived from the o-quinone were detected at m/z 129 (MH+ - guanine, CO) and 101 (MH+ - guanine, 2CO). The formation of these product ions can be rationalized by cleavage of the bond linking the o-quinone and guanine moieties, accompanied by the elimination of one or two molecules of carbon monoxide from the quinone portion, a known pathway of o-quinone fragmentation (27). Other fragment ions observed were consistent with losses from either the guanine or the quinone portion of the adduct, i.e., m/z 280 (MH+ - CO), 263 (MH+ - CO, NH3), 237 (MH+ - CO, HNCO; or MH+ - NH3, 2HCN), and 183 (C10H5O2NC+). There was no mass spectrometric

Table 1. 1H NMR Spectroscopic Data for 7-(Naphthalene-1,2-dion-4-yl)guaninea δ (ppm) 6.38 (br s, 2H) 6.69 (s, 1H) 7.18 (d, 1H) 7.66-7.77 (m, 2H) 8.09 (d, 1H) 8.25 (s, 1H) 10.99 (br s, 1H) a

J (Hz)

7.8 7.3

assignment Gua 2-NH2 (D2O exchangeable) 3-CH 5-CH 6-CH, 7-CH 8-CH Gua 8-CH Gua 1-NH (D2O exchangeable)

At 500 MHz in Me2SO-d6.

evidence of the formation of the corresponding catechol adduct. The UV spectrum of the adduct (λmax ) 251 and 286 nm) showed a composite of the characteristic absorbances assigned to guanine (λmax ) 248 and 274 nm) and NPQ (λmax ) 252 nm) (Figure 1B). The 1H NMR spectrum (Figure 3) of the purified adduct supported its identification as an N7-guanine adduct of NPQ. Proton assignment (Table 1) was achieved by comparing the chemical shifts, signal multiplicities, and coupling constants in the adduct spectrum with those of NPQ and dGuo in the same solvent, and by a D2O exchange experiment. The spectrum clearly demonstrated the absence of the sugar group of dGuo. The absence of the quinone 4-CH signal (a doublet centered


at 7.68 ppm in NPQ) and the presence of the quinone 3-CH signal at 6.69 ppm as a singlet (rather than as a doublet centered at 6.42 ppm in NPQ) confirmed that the guanine group was attached at the 4-position of the o-quinone. The NMR spectrum also provided information with regard to the site of attachment at guanine. The guanine N1, N2, and C8 proton resonances were clearly visible at 10.99, 6.38, and 8.25 ppm, respectively, indicating that addition could not have occurred at these positions. Reaction at N7 (or N9) leads to cleavage of the glycosidic bond. For dGuo to act as the nucleophile, it is more likely that an N7 rather than an N9 adduct would be formed. N9 adducts could arise if guanine rather than dGuo acts as the nucleophile. This is unlikely under our reaction conditions. First, after 18 h a significant amount of dGuo remained; i.e., very little was hydrolyzed to guanine. Second, when NPQ was reacted with guanine under the same reaction conditions, very little adduct formation was observed (Figure 1C). Thus, reaction of NPQ with dGuo under acidic conditions led to the formation of a depurinating adduct, identified as 7-(naphthalene-1,2-dion-4-yl)guanine. When NPQ was reacted with 2′-deoxyadenosine under the same reaction conditions, no detectable product was observed by HPLC. This may be due to steric hindrance at N7 of adenine, since calculations of the interactions in the transition states for reaction of methanediazonium ions with different sites in guanine and adenine have demonstrated that for reaction at the adenine N7 site, the N6 amino group offers steric hindrance to the attacking methanediazonium ion (28). Formation of Depurinating PQ Adducts with dGuo. Attempts were also made to synthesize depurinating adducts with PQ because this non-K region o-quinone, like BPQ, contains a bay region which could influence reactivity. PQ was reacted with dGuo under conditions similar to those described for the reaction of NPQ with dGuo (1:1:1 acetonitrile/acetic acid/water). However, due to the lower solubility of PQ, the reaction mixture was more dilute (1.3 times more solvent was used), and a small amount of PQ remained undissolved. Analysis of the reaction by reversed-phase HPLC demonstrated that one major product was formed with a retention time of 32.5 min (Supporting Information). In addition to the expected unreacted dGuo (tR ) 5.1 min), some unreacted PQ (tR ) 42.1 min) also remained after 36 h. By substituting THF for acetonitrile, we could obtain a higher concentration of PQ in the reaction mixture. However, a significant amount of unreacted PQ was present 24 h into the reaction, demonstrating the lower reactivity of this o-quinone compared to NPQ. The UV spectrum of the product (λmax ) 273 and 295 nm) resembled the spectra of guanine and PQ (λmax ) 290 nm) (Supporting Information). Full scan analysis by HPLC/ESI/MS revealed a prominent ion at m/z 358, at a retention time corresponding to the major reaction product (data not shown). This MH+ ion was consistent with a PQ-guanine adduct; i.e., a depurinating adduct had been formed. The product ion profile obtained upon CID of the m/z 358 ion (Figure 4) was similar to that observed for the MH+ ion of 7-(naphthalene-1,2-dion-4-yl)guanine (shown in Figure 2). An ion corresponding to protonated guanine (m/z 152) was observed, as were ions derived from PQ, i.e., m/z 207 (MH+ - guanine) and 179 (MH+ - guanine, CO). Com-

McCoull et al.

Figure 4. Collision-induced dissociation spectrum of the m/z 358 (MH+) ion of the phenanthrene-1,2-dione-guanine adduct, obtained on a Finnigan LCQ ion trap.

mon losses from guanine as well as the quinone were again observed: m/z 330 (MH+ - CO), 316 (MH+ - NH2CN), 287 (MH+ - CO, HNCO), and 273 (MH+ - NH2CN, HNCO). As the NPQ- and PQ-guanine adducts were formed under identical conditions, it is likely that they have analogous structures; i.e., the adduct formed by reaction of PQ with dGuo is most likely an N7-guanine adduct of the o-quinone, 7-(phenanthrene-1,2-dion-4-yl)guanine. This structural assignment was supported by the 1H NMR spectrum of the purified adduct, although several resonances were broad, even at an elevated temperature (110 °C). The spectrum clearly demonstrated the absence of the sugar group of dGuo, and revealed the presence of a vinyl proton (quinone 3-CH) as a singlet at 6.09 ppm. The N1 and N2 resonances of guanine were observed at 10.51 and 5.92 ppm, respectively, being identified as NH by their absence when the spectrum was acquired under conditions of water suppression. Although the breadth of several peaks precluded accurate assignment of the other resonances, the number of proton resonances observed confirmed the presence of the guanine C8 proton. Thus, analogous to the NPQ adduct of guanine, the PQ-guanine adduct that was synthesized was found to be an N7 adduct formed by the addition of guanine to the C4 position of PQ, and is 7-(phenanthrene-1,2-dion4-yl)guanine. Formation of Depurinating BPQ Adducts with dGuo. BPQ is the most relevant PAH o-quinone since it is the product of the DD-catalyzed oxidation of the proximate carcinogen BP-diol (15, 16). Several attempts were made to react BPQ with dGuo using acetonitrile/ acetic acid/water and THF/acetic acid/water mixtures at room temperature or 40 °C, but these failed to yield any detectable product as determined by HPLC. This was presumed to be due to the very low solubility of BPQ in these reaction mixtures. To improve the solubility of BPQ and still maintain acidic conditions, the final solvent mixture consisted of 6:1:1 Me2SO/acetic acid/water (2.4 times more solvent than was used in the reaction of NPQ with dGuo). Under these reaction conditions, one major product (tR ) 75.9 min) and one minor product (tR ) 77.1 min) were detected by HPLC after 24 h, although considerable amounts of unreacted BPQ and dGuo remained, even after reaction for 5 days (Supporting Information).



displayed an MH+ ion at m/z 358 and exhibited a retention time consistent with that of the standard (Table 2). In addition, the major product ions of the MH+ ion were identical to those of the standard, and their relative intensities were similar (Table 2). Under the conditions of the analysis, no depurinating adduct was observed in the sample which had not been subjected to thermal hydrolysis. This suggests that the rate of spontaneous depurination is low and the levels of adducts formed by this route are beneath the detection limit of the method. A similar set of parallel reactions were performed using a solvent mixture of 13.5% acetonitrile/water, and again, PQ-N7-guanine was only observed in the filtrate of the reaction mixture which had been subjected to thermal hydrolysis (data not shown). Figure 5. Collision-induced dissociation spectrum of the m/z 432 (MH+) ion of the benzo[a]pyrene-7,8-dione-guanine adduct, obtained on a Finnigan LCQ ion trap.

Mass spectrometric analysis revealed an MH+ ion at m/z 432 for the major adduct peak (tR ) 75.9 min) (data not shown), consistent with a depurinating adduct. CID of the m/z 432 ion (Figure 5) generated the quinonederived fragment ions at m/z 281 (MH+ - guanine) and 253 (MH+ - guanine, CO). Additional product ions, consistent with the proposed structure, were observed at m/z 415 (MH+ - NH3), 404 (MH+ - CO), 389 (MH+ HNCO), 362 (MH+ - CO, NH2CN), and 308 (MH+ - CO, NH2CN, 2HCN). Since the fragmentation pattern is similar to that observed with the NPQ-N7-guanine and PQ-N7-guanine adducts, and 1H NMR spectroscopy supports these assignments, it is likely that an N7guanine adduct was formed with BPQ, i.e., 7-(benzo[a]pyrene-7,8-dion-10-yl)guanine. The UV spectrum of this adduct (λmax ) 240 and 350 nm) again showed similarities with respect to the spectra of guanine and BPQ (λmax ) 347 nm) (Supporting Information). Attempts to obtain sufficient quantities of this adduct for 1H NMR by scaling up the reaction were unsuccessful due to the low yield of the reaction and the instability of the adduct during HPLC purification. Complete characterization of the minor adduct (tR ) 77.1 min) was not possible, due to the low level of compound generated in the reaction mixture. Although the mass spectrometric data were inconclusive (data not shown), the UV spectrum (λmax ) 248, 281, 334, and 349 nm) (Supporting Information) suggested that it was a catechol adduct, since it resembled the UV spectrum of 7,8-dihydroxybenzo[a]pyrene obtained previously (29). Formation of Depurinating Adducts with Calf Thymus DNA. To determine whether PAH o-quinones could form depurinating adducts with DNA, PQ was reacted with calf thymus DNA in 10% Me2SO/water at 37 °C for 18 h, and the reaction products were analyzed by HPLC/ESI/MS/MS. Two reactions were performed in parallel and worked up differently. In the first instance, the reaction mixture was filtered to remove the DNA. In the second instance, the reaction mixture was subjected to thermal hydrolysis, a gentle procedure used to release any N7-substituted guanine that remained bound to deoxyribose (30), and the DNA was subsequently removed by filtration. In both instances, the filtrates were analyzed using HPLC/ESI/MS/MS and compared with the standard. Analysis of the DNA reaction which had undergone thermal hydrolysis demonstrated the presence of 7-(phenanthrene-1,2-dion-4-yl)guanine. The product

Discussion One goal of this work was to synthesize PAH o-quinone depurinating adducts as chemical standards to detect the prevalence of these adducts in biological systems. Depurinating adducts of dGuo with NPQ, PQ, and BPQ were prepared for the first time, by reaction under acidic conditions similar to those used to prepare depurinating adducts by reaction of estrogen o-quinones with deoxyribonucleosides (22). The acid polarizes the o-quinones, increasing the positive character at C4 for NPQ and PQ, and at C10 for BPQ, thus facilitating attack by nucleophiles at these positions. Since the pKa (N7) of dGuo is 2.31 (31), the use of 50% aqueous acetic acid (pH ∼1.3) and an organic solvent ensures that a portion of the dGuo N7 atoms remain unprotonated as required for nucleophilic addition. 1 H NMR spectroscopy confirmed that the compound formed by reaction of NPQ with dGuo was an NPQguanine adduct and supported addition of the N7 group of guanine at the C4 position of NPQ. In particular, the presence of the guanine C8 proton confirmed that addition had not occurred at this site. The electrospray/ tandem mass spectrometric data were consistent with an NPQ-guanine structure, and indeed, following literature precedent (32-34), showed characteristics of reaction at the N7 group of guanine. High-energy tandem mass spectrometry has been used to distinguish between N7and C8-guanine adducts of BP (32, 33) and dibenzo[a,l]pyrene (34). For example, the N7 adducts fragmented mainly at the carcinogen-base bond, yielding product ions consisting only of carcinogen fragments, while the C8 adducts yielded a greater abundance of product ions derived by cleavage through the imidazole ring of guanine. Fragmentation of the MH+ ion of 7-(naphthalene1,2-dion-4-yl)guanine (Figure 2) also yielded more product ions derived by cleavage of the o-quinone-guanine bond (m/z 129 and 101) than those formed by cleavage through the imidazole ring (m/z 183). Thus, although an NPQ-C8-guanine standard was not available for comparison, the fragmentation pattern observed for 7-(naphthalene-1,2-dion-4-yl)guanine is characteristic of N7 addition to o-quinones. Mass spectrometry confirmed that the products of reaction of PQ and BPQ with dGuo were also PQguanine and BPQ-guanine adducts, respectively. In the CID spectra of the MH+ ions of the PQ-guanine and BPQ-guanine adducts (Figures 4 and 5, respectively), fragmentation patterns similar to those observed for the NPQ-N7-guanine adduct were detected. The most no-


McCoull et al.

Table 2. LC/MS Retention Times and Mass Spectral Fragmentation Patterns for 7-(Naphthalene-1,2-dion-4-yl)guanine, 7-(Phenanthrene-1,2-dion-4-yl)guanine, and 7-(Benzo[a]pyrene-7,8-dion-10-yl)guanine Formed under Various Reaction Conditions reaction product NPQ-N7-guanine (standard) PQ-N7-guanine (standard) PQ-N7-guanine (from DNA) BPQ-N7-guanine (standard)

tR (min) MH+ (m/z) 21.8 21.3 22.0 69.5

308 358 358 432

major product ions (m/z) and their relative intensities (CID MH+) 280 (92%), 263 (100%), 237 (56%), 183 (8%), 152 (6%), 129 (73%), 101 (5%) 330 (100%), 287 (2.3%), 273 (1.6%), 207 (1.6%), 179 (3.8%) 330 (100%), 287 (2.1%), 273 (1.2%), 207 (1.8%), 179 (3.6%) 415 (1.6%), 404 (5.0%), 389 (0.4%), 362 (0.7%), 308 (2.0%), 281 (0.25%), 253 (2.8%)

table similarities included the observation of fragment ions derived by cleavage through the o-quinone-guanine bonds and the lack of fragmentation arising from cleavage through the imidazole ring. This suggests that the three adducts have analogous structures, i.e., that the PQ-guanine and BPQ-guanine adducts are also N7 addition products. 1H NMR spectroscopy of the PQguanine adduct supported its structure as a PQ-N7guanine adduct. No 1H NMR spectrum was available for the BPQ-guanine adduct. It should be noted however that BPQ exhibits structural features similar to those of PQ, i.e., the presence of a bay region, and the BPQguanine adduct MH + ion displayed a fragmentation profile analogous to those of the MH+ ions of the PQN7-guanine and NPQ-N7-guanine adducts. The structures of the latter two adducts were confirmed by 1H NMR experiments, suggesting that the BPQ-guanine adduct is also an N7 addition product. The formation of PAH o-quinone-guanine adducts can be rationalized from the proposed mechanism (Scheme 4). Adduct formation is believed to occur by Michael addition of dGuo to the o-quinone to give, upon tautomerization, a catechol adduct as the initial product. However, the catechol adduct is then oxidized by air or unreacted o-quinone (35) to yield a quinone adduct as the final product. The electron donating properties of the nitrogen group in the catechol adduct lower the o-quinone adductcatechol adduct redox potential relative to the o-quinonecatechol redox potential. Thus, the o-quinone can oxidize the intermediate catechol adduct to the quinone adduct, itself being reduced to the corresponding catechol in the process (35). Attack by the N7 group of dGuo will lead to a depurinating adduct, since addition at this position destabilizes the glycosidic bond. Although a concerted depurination mechanism is shown for simplicity, it most likely occurs in a stepwise manner, since PQ-N7-guanine adducts could only be detected following thermal hydrolysis of calf thymus DNA. As the number of rings in the o-quinone reactant increased in the order NPQ < PQ < BPQ, the amount of adduct formed decreased (Figure 1A and Supporting Information). This could be due to a number of factors. First, reaction conditions could influence product yield. The solubility of the o-quinones in the reaction mixtures decreased as the number of rings increased, and BPQ was not completely dissolved in the reaction mixtures which yielded some adduct. In addition, due to the poor solubility of BPQ, (i) a different organic solvent was used (Me2SO as opposed to acetonitrile or THF) and (ii) the reaction of dGuo with BPQ was conducted under more dilute conditions than with the other two quinones. Both solvent and concentration would be expected to influence the rate of reaction. Second, differences exist in the inherent reactivity of the three o-quinones. The site of addition in PQ and BPQ is the sterically hindered bay region, and this may slow

Scheme 4. Proposed Mechanism of Polycyclic Aromatic Hydrocarbon o-Quinone-N7-Guanine Depurinating Adduct Formation

the rate of reaction. Electronic effects related to the increase in the number of aromatic rings may also play a role. For example, the cations obtained upon protonation of the o-quinones under the acidic reaction conditions


experience an increase in stability in the order NPQ < PQ < BPQ due to an increased level of delocalization of the positive charge around the larger aromatic ring systems. This leads to lower reactivity as the number of rings increases. Indeed, previous studies have shown that the rate of addition of nucleophiles to BPQ is slower than for addition to NPQ (36). BPQ is also expected to have a lower redox potential than NPQ or PQ, since there will be greater delocalization of the lone electron in the o-semiquinone radical as the size of the ring system increases. In fact, electron paramagnetic resonance measurements indicate that the o-semiquinone anion radical of BPQ is more stable, i.e., longer-lived, than the osemiquinone anion radical of NPQ.2 Thus, although the initial 7,8-dihydroxy-BP-guanine adduct could be oxidized to the BPQ-guanine adduct by either oxygen or unreacted BPQ (35), this event is less favored than in reactions with NPQ and PQ. If unreacted BPQ acts as the oxidant of the guanine adduct, then 7,8-dihydroxybenzo[a]pyrene will be formed, and its reoxidation back to BPQ by oxygen will also be slower. Any BPQ-derived o-semiquinone radical formed during the reoxidation process might have a lifetime long enough to react with dGuo. This could lead to isomeric products, e.g., C8 adducts. Interestingly, studies have shown that the o-semiquinone anion radical of estrone-3,4-o-quinone reacts with dGuo to form stable N2 and depurinating C8 adducts, but not N7 adducts (37). 4-Hydroxyequilenin, a metabolite of the estrogen replacement formulation Premarin, reacts with dGuo, 2′-deoxyadenosine, and 2′deoxycytidine to form stable cyclic adducts, and these reactions are also believed to proceed via an o-semiquinone radical (38, 39). The formation of PAH o-quinone depurinating adducts in vitro was confirmed when PQ was reacted with calf thymus DNA. The detection of PQ-N7-guanine adducts by HPLC/ESI/MS/MS demonstrated that non-K region PAH o-quinones can covalently modify DNA to yield depurinating adducts, resulting in the formation of apurinic sites. The chemical reaction of BPQ with dGuo was the slowest of the three o-quinones, raising issues of biological relevance. However, the BPQ-N7-guanine adduct could be detected by HPLC within 24 h, suggesting that when BPQ encounters DNA, apurinic sites could form after short exposure times. When [1,3-3H2]BPQ was reacted with calf thymus DNA or plasmid DNA, stable dGuo adducts were formed within 18 h (18), and depurinating adducts may form over the same time course. In these experiments, the supernatant was not analyzed for depurinating adducts. Further, when [1,3-3H2]BPQ was incubated with isolated rat hepatocytes, 30% of the radioactivity was sequestered into the cell pellet within 30 min, and 30 ( 17 adducts per 106 DNA base pairs were observed in the DNA precipitate. However, no stable adducts were observed after degradation of the DNA to its constituent deoxyribonucleosides, suggesting that the adducts were unstable (20). The sequestering of 30% of the radioactivity into the cell pellet suggests that BPQ is found in association with cellular macromolecules, increasing the likelihood of a reaction between the quinone and DNA. Thus, the slow reaction rates observed in preparing synthetic standards may have no bearing on the rate of reaction between a quinone and DNA. 7,2

S. T. Ohnishi and T. M. Penning, unpublished results.


12-Dimethylbenz[a]anthracene is a very potent carcinogen, and its corresponding quinone, 7,12-dimethylbenz[a]anthracene-3,4-dione, is much more reactive as a Michael acceptor than BPQ (36). In fact, 7,12-dimethylbenz[a]anthracene-3,4-dione is as reactive as NPQ with select nucleophiles (36). It is likely that the amount of depurinating adduct formed will thus vary with the reactivity of the PAH o-quinone, and this could impact biological significance. Depurinating adducts of three PAH o-quinones with dGuo have now been synthesized, and detection of depurinating adducts upon reaction of PQ with calf thymus DNA has been demonstrated. Identification of these adducts in biological systems is part of an ongoing study designed to evaluate the contribution of PAH o-quinones in the metabolic activation of PAHs. To further address the biological significance of the DD pathway, whole-cell studies are needed. Cell lines that are stably transfected with rat and human DDs have been established (40), and the detection of quinone-N7guanine adducts upon exposure of the cells to transdihydrodiols will be pursued. Importantly, the reaction of PAH o-quinones with DNA is reminiscent of the proposed metabolic activation of catechol estrogens in which o-quinones form depurinating DNA adducts (23), thus providing a route to mutations via misreplication at apurinic sites. Depurinating adducts formed by reaction of PAH o-quinones with DNA could provide an analogous route to the mutations observed in cancers that arise from exposure to PAHs.

Acknowledgment. This work was supported by Grants CA39504 (to T.M.P.) and CA65878 (to I.A.B.) from the National Cancer Institute and an NRSA Fellowship (to D.R.). We thank Dr. G. Furst for assistance with NMR experiments. Supporting Information Available: HPLC separation and UV characterization of the PQ-N7-guanine and BPQ-N7guanine adducts formed upon reaction of the o-quinones with dGuo. This material is available free of charge via the Internet at http://pubs.acs.org.

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