Formation of depurinating N3adenine and N7guanine adducts after reaction of 1,2-naphthoquinone or enzyme-activated 1,2-dihydroxynaphthalene with DNA

Chemico-Biological Interactions 165 (2007) 175–188

Formation of depurinating N3adenine and N7guanine adducts after reaction of 1,2-naphthoquinone or enzyme-activated 1,2-dihydroxynaphthalene with DNA Implications for the mechanism of tumor initiation by naphthalene Muhammad Saeed, Sheila Higginbotham, Eleanor Rogan, Ercole Cavalieri ∗ Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, 986805 Nebraska Medical Center, Omaha, NE 68198-6805, United States Received 17 August 2006; received in revised form 4 December 2006; accepted 5 December 2006 Available online 16 December 2006

Abstract Naphthalene is considered by the US Environmental Protection Agency to be a carcinogenic compound based on inhalation studies in rats. The primary metabolite of naphthalene is naphthalene 1,2-arene oxide. This unstable intermediate can lead to formation of 1-naphthol and naphthalene-1,2-dihydrodiol. Secondary metabolites include 1,2-dihydroxynaphthalene (1,2-DHN), which can be further oxidized to 1,2-naphthoquinone (1,2-NQ). Based on the metabolism of naphthalene and its similarity to the metabolic activation of carcinogenic natural estrogens, synthetic estrogens and benzene, we hypothesize that naphthalene is activated to initiate cancer by reaction of 1,2-NQ with DNA to form the depurinating adducts 1,2-DHN-4-N3Ade and 1,2-DHN-4-N7Gua. These adducts were synthesized by reaction of 1,2-NQ with Ade or dG in acetic acid/water/DMF (1:1:1). 1,2-NQ was reacted with DNA, and the depurinating 1,2-DHN-4-N3Ade and 1,2-DHN-4-N7Gua adducts were analyzed by ultraperformance liquid chromatography/tandem mass spectrometry and HPLC with electrochemical detection. After the reaction of 1,2-NQ with DNA, the N3Ade and N7Gua adducts were found. Similarly, when 1,2-DHN was activated by tyrosinase in the presence of DNA, higher amounts of the N3Ade and N7Gua adducts were detected. These same adducts were also formed when 1,2-DHN was activated by prostaglandin H synthase or 3-methylcholanthrene-induced rat liver microsomes in the presence of DNA. These depurinating adducts are analogous to those obtained from the ortho-quinones of natural estrogens, synthetic estrogens and benzene. These results suggest that reaction of ortho-quinones with DNA by 1,4-Michael addition is a general mechanism of weak carcinogenesis that occurs with naphthalene and a number of other aromatic compounds. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Metabolic activation; Tumor initiation; Depurinating DNA adducts

Abbreviations: Ade, adenine; COSY, chemical shift correlation spectroscopy; dA, deoxyadenosine; dG, deoxyguanosine; 1,2-DHN, 1,2dihydroxynaphthalene; FAB-MS/MS, fast-atom bombardment-tandem mass spectrometry; Gua, guanine; HMBC, heteronuclear multiple bond correlation; HSQC, heteronuclear single quantum coherence; MC, 3-methylcholanthrene; MRM, multiple reaction monitoring; 1,2-NQ, 1,2naphthoquinone; PDA, photodiode array; TFA, trifluoroacetic acid; UPLC–MS/MS, ultraperformance liquid chromatography–tandem mass spectrometry ∗ Corresponding author. Tel.: +1 402 559 7237; fax: +1 402 559 8068. E-mail address: [email protected] (E. Cavalieri). 0009-2797/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.cbi.2006.12.007

176

M. Saeed et al. / Chemico-Biological Interactions 165 (2007) 175–188

1. Introduction Naphthalene, a condensed two-benzene ring aromatic hydrocarbon, is a component of coal tar, coal tar products (e.g., creosote) and moth repellents, and is extensively used as an intermediate in the production of plasticizers, resins, insecticides and surface-active agents [1]. It is a ubiquitous pollutant found mainly in ambient air and to a minor extent in effluent water. Although the major contributors of naphthalene in air are coal and oil gasification sites, a significant amount is also released as a pyrolytic product of both mainstream and side stream tobacco smoke. Human exposure to naphthalene was corroborated when it was found in nearly 40% of human fat samples tested [2] and in 75% of human breast milk samples [3]. These facts argue that the U.S. population is exposed to this compound, and it has been included as one of 189 hazardous air pollutants under the Clean Air Act Amendments of 1990 (Title III) of the Environmental Protection Agency [4]. Until recently, naphthalene in the environment was thought to present no significant cancer risk to humans, in spite of the reported cancer cases in employees working in a naphthalene purification plant in the former East Germany [5,6]. Mice inhaling naphthalene chronically (10 or 30 ppm) exhibited diverse effects, including inflammation of the nose, metaplasia of the olfactory epithelium, and hyperplasia of the respiratory epithelium [7–9]. No neoplastic effects in male mice were noted. Female mice, however, showed a slight increase in alveolar/bronchiolar adenomas and carcinomas at the highest exposure level. More recently, the U.S. National Toxicology Program released the results of a 2-year bioassay study with rats exposed to doses of 10, 30 or 60 ppm naphthalene [10,11], show-

ing a concentration-dependent increase in adenomas of the respiratory epithelium of the nose and of neuroblastomas of the olfactory epithelium. These results are considered to be significant in rodent studies and have raised concerns about naphthalene as a potential human carcinogen [1]. As for many chemical carcinogens, the toxicity of naphthalene is dependent on its metabolic activation. Studies conducted in vitro and in vivo demonstrated that the first step in the metabolic conversion of naphthalene is the cytochrome P450-dependent formation of the 1,2epoxide (Fig. 1) [7,12–18]. This compound is unstable at physiological pH [14,15] and can either react with glutathione to form glutathione conjugates or convert to the metabolites 1-naphthol by chemical isomerization or naphthalene 1,2-dihydrodiol by epoxide hydrolase [16,17]. The conversion to 2-naphthol can occur after ␤-elimination of the sulphated/glucuronidated conjugate of naphthalene 1,2-dihydrodiol (not shown in Fig. 1). Naphthalene 1,2-dihydrodiol [19–21] or 1naphthol [22] can be further metabolically oxidized to 1,2-dihydroxynaphthalene (1,2-DHN) or its oxidized product, 1,2-naphthoquinone (1,2-NQ). 1,2-NQ has been reported to be the metabolite that binds covalently to proteins [22,23]. Being electrophilic in nature, 1,2-NQ can also bind covalently to the cellular nucleophiles of DNA and RNA. Analogously to catechol estrogen quinones derived from natural estrogens [24–29], synthetic estrogens [30,31], and benzene [32], we hypothesize that 1,2-NQ can react with DNA preferably at the N3 position of adenine and the N7 position of guanine, leading to the formation of the corresponding depurinating adducts (Fig. 2). Apurinic sites generated by depurination are

Fig. 1. Metabolic activation of naphthalene to the electrophilic 1,2-naphthoquinone.

M. Saeed et al. / Chemico-Biological Interactions 165 (2007) 175–188

177

Fig. 2. Proposed mechanism of cancer initiation by estradiol and naphthalene. The presumed major pathway of naphthalene to naphthoquinone is shown.

mutagenic and may lead to initiation of cancer after accumulation of mutations in critical genes controlling cell division [28,29,33]. In this article we provide the first evidence concerning the ability of 1,2-NQ and enzymatically activated 1,2-DHN to react with DNA and form depurinating and stable adducts. The depurinating adducts formed were identified and quantified by using the standard synthesized adducts 1,2-DHN-4-N3Ade and 1,2-DHN-4-N7Gua. Furthermore, the kinetics of depurination were also investigated. Depurination of the N3Ade adduct is rapid, whereas that of the N7Gua adduct is slower. These findings may have important implications for understanding the etiology of naphthalene carcinogenesis. 2. Materials and methods Caution. Catechols and quinones are hazardous chemicals and should be handled according to NIH guidelines [34]. 2.1. Chemicals, reagents, and enzymes 1,2-NQ, citric acid, deuteriated DMSO, DMF and adenine (Ade) were purchased from Aldrich Chemical Co. (Milwaukee, WI). Deoxyguanosine (dG) and DNA were purchased from USB (Cleveland, OH). Prostaglandin H synthase was purchased from Cayman Chemicals (Ann Arbor, MI). Tyrosinase, 3methylcholanthrene (MC)-induced rat liver microsomes,

methemoglobin, arachidonic acid, ammonium acetate and deoxyadenosine (dA) were purchased from Sigma (St. Louis, MO). 1,2-DHN was synthesized by reducing 1,2-NQ with NaBH4 in ethanol [35]. All chemicals were used as such without further purification. 2.2. Instrumentation (1) UV. The UV spectra were obtained during HPLC by using a photodiode array (PDA) detector, Waters 996 (Milford, MA) for all synthesized compounds. HPLC separations were monitored at 254 nm. (2) NMR. NMR spectra were recorded on a Varian Unity-Inova 500 instrument operating at a resonance frequency of 499.8 MHz for 1 H and 125.6 MHz for 13 C spectra at 25 ◦ C. Samples were dissolved in 600 ␮L of DMSO-d6 and referenced to the solvent signals at 2.5 ppm for 1 H and 39.7 ppm for 13 C. All 2D experiments were performed by using the standard Varian software (VNMR v6.1c). For 2D experiments, relaxation delays of 1.2–2 s were used; 128–512 t1 increments and 2048 complex data points in t2 were recorded for a spectral width of 8000 Hz in two dimensions. 1 H–1 H correlations were recorded by using correlation spectroscopy (COSY) techniques. In pulsed field gradient (PFG) 1 H–13 C heteronuclear single quantum coherence (HSQC) and heteronuclear multiple bond correlation (HMBC) sequences, delays were optimized for

178

M. Saeed et al. / Chemico-Biological Interactions 165 (2007) 175–188

coupling constants around 140 and 8 Hz, respectively. (3) Mass spectrometry (MS). Fast atom bombardment tandem mass spectrometry (FAB-MS/MS) was conducted at the Nebraska Center for Mass Spectrometry (University of Nebraska-Lincoln) using a MicroMass (Manchester, England) AutoSpec high resolution magnetic sector mass spectrometer. The instrument was equipped with an orthogonal acceleration time-of-flight serving as the second mass spectrometer. Xenon was admitted to the collision cell at a level to attenuate the precursor ion signal by 75%. Data acquisition and processing were accomplished by using OPUS software that was provided by the manufacturer (Microcasm). Samples were dissolved in 5–10 ␮L of CH3 OH; 1 ␮L aliquots were placed on the sample probe tip along with 1 ␮L of a 1:1 mixture of glycerol/thioglycerol. (4) Ultra performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS). Samples were analyzed by a Waters Acquity UPLC equipped with a MicroMass QuattroMicro triple stage quadrupole system (Waters, Milford, MA). The 10 ␮L injections were carried out on a Waters Acquity UPLCTM BEHC18 column (1.7 ␮m, 1 mm × 100 mm). The instrument was operated in positive electrospray ionization mode. All aspects of system operation, data acquisition and processing were controlled using QuanLynx v4.0 software (Waters). The column was eluted starting with 5% CH3 CN in H2 O (0.1% formic acid) for 1 min at a flow rate of 150 ␮L/min; then raised to 55% CH3 CN in 10 min. Ionization was achieved by using the following settings: capillary voltage 3 kV; cone voltage 15–40 V; source block temperature 100 ◦ C; desolvation temperature 200 ◦ C with a nitrogen flow of 400 L/h. Nitrogen was used as both the desolvation and auxillary gas. MS/MS conditions were optimized prior to analysis using pure standards of 1,2-DHN-4-N3Ade and 1,2-DHN-4-N7Gua. Argon was used as the collision gas. Three-point calibration curves were run for each standard. Triplicate samples were analyzed for each data point. (5) HPLC. Purification of adducts was conducted by using preparative HPLC on a Waters DeltaPrep system equipped with a 2478 dual wavelength UV detector. About 10 mL of reaction mixture was ˚ loaded on a Luna-2 C-18 column (10 ␮m, 120 A, 21.2 mm × 250 mm, Phenomenex, Torrance, CA) and eluted with a mobile phase consisting of 10% CH3 CN in H2 O [0.4% trifluoroacetic acid (TFA)] which was increased linearly up to 40% CH3 CN

in 20 min, and to 100% in the next 5 min at a flow rate of 25 mL/min. Analyses of reaction mixtures were performed on a Waters 2690 (Alliance) Separations Module equipped with a Waters 996 PDA interfaced to a Digital Venturis Fx 5100 computer. A 50 ␮L aliquot of a reaction mixture was ˚ eluted on a Luna-2 C-18 column (5 ␮m, 120 A, 250 mm × 4.6 mm, Phenomenex), using a linear gradient of 10% CH3 CN/90% H2 O (0.4% TFA), which was changed to 40% CH3 CN in 20 min and then to 100% CH3 CN in 5 min at a flow rate of 1 mL/min. The eluants were monitored at 254 nm. Analyses of depurinating adducts were conducted on an HPLC system equipped with dual ESA Model 580 solvent delivery modules, an ESA Model 540 auto-sampler and a 12-channel CoulArray electrochemical detector (ESA, Chelmsford, MA). The two mobile phases used were (A) CH3 CN:CH3 OH:buffer:H2 O (15:5:10:70) and (B) CH3 CN:CH3 OH:buffer:H2 O (50:20:10:20). The buffer was composed of a mixture of 105 g citric acid, 78 g ammonium acetate in triple-distilled H2 O, and the pH was adjusted to 3.6 with acetic acid. The 50 ␮L injections were carried out on a Phenomenex Luna-2 C-18 ˚ 4.6 mm × 250 mm) were initially column (5 ␮m, 120 A, eluted isocratically at 90% A/10% B for 15 min, followed by a linear gradient to 90% B in the next 10 min at the rate of 1 mL/min. The serial array of 12 coulometric electrodes was set at potentials of −50, −10, 10, 50, 100, 150, 200, 250, 300, 350, 400 and 450 mV. The system was controlled and the data were acquired and processed using the CoulArray software package. Peaks were identified by both retention time and peak height ratios between the dominant peak and the peaks in the two adjacent channels. The depurinating adducts were quantified by comparison of peak response ratios with known amounts of standards. 2.3. Synthesis of standard adducts 2.3.1. Reaction of 1,2-NQ with dG, Ade or dA To a stirred solution of Ade (405 mg, 3.0 mmol), dG (450 mg, 1.5 mmol) or dA (807 mg, 3.0 mmol) in acetic acid/H2 O (1:1, 15 mL) was added a solution of 1,2-NQ (50 mg, 0.3 mmol) in DMF (7.5 mL) at room temperature. The reaction mixture was stirred at the same temperature for 12 h and filtered to remove any insoluble material. Analysis and purification of adducts were performed on HPLC, as described above.

M. Saeed et al. / Chemico-Biological Interactions 165 (2007) 175–188

2.3.1.1. Reaction with dG: 1,2-DHN-4-N7Gua. Yield (10%); UV (215.5, 242.6, 336.3). 1 H NMR (ppm) δ 11.72 (br s, 1 H, NH-1-Gua, exchangeable with D2 O), 9.79 (s, 1 H, Ar–OH, exchangeable with D2 O), 9.49 (s, 1 H, Ar–OH, exchangeable with D2 O), 8.64 (s, 1 H, H-8 Gua), 8.13 (d, J = 8.3 Hz, 1 H, H-8), 7.46 (dd, J = 8.3, 8.3 Hz, 1 H, H-7), 7.32 (s, 1 H, H-3), 7.29 (t, J = 8.3, 8.3 Hz, 1 H, H-6), 7.27–7.33 (br s, 2 H, NH2 Gua, exchangeable with D2 O), 7.21 (d, J = 8.3 Hz, 1 H, H-5). FAB-MS: m/z 310.0895 [(M + H)+ C15 H12 N5 O3 , calc. 310.0862]. 2.3.1.2. Reaction with Ade: 1,2-DHN-4-N3Ade. Yield (8%); UV (213.2, 233.1, 260.3, 301.3, 338.7). 1 H NMR (ppm) δ 9.83 (s, 1 H, Ar–OH, exchangeable with D2 O), 9.49 (s, 1 H, Ar–OH, exchangeable with D2 O), 8.42 (s, 1 H, H-8-Ade), 8.15 (d, J = 8.5Hz, 1 H, H-8), 8.13 (s, 1 H, H-2-Ade), 7.85 (br s, 2 H, NH2 -Ade, exchangeable with D2 O), 7.46 (dd, J = 8.5, 7.8 Hz, 1 H, H-7), 7.29 (s, 1 H, H-3), 7.26 (dd, J = 8.0, 7.8 Hz, 1 H, H-6), 7.03 (d, J = 7.8 Hz, 1 H, H-5). FAB-MS: m/z 294.0946 [(M + H)+ C15 H12 N5 O2 , calc. 294.0913]. 2.3.1.3. 1,2-DHN-4-N6 Ade. Yield (9%); UV (213.2, 233.1, 341.1). 1 H NMR (ppm) δ 10.00 and 9.73 (2 × s, 2 H, 2 × Ar–OH, exchangeable with D2 O), 8.59 (s, 1 H, H-2-Ade), 8.56 (s, 1 H, H-8-Ade), 8.21 (d, J = 8.5 Hz, 1 H, H-8), 7.56 (s, 1 H, H-3), 7.51 (dd, J = 8.5, 7.8 Hz, 1 H, H-7), 7.34 (dd, J = 8.0, 7.8 Hz, 1 H, H-6), 7.23 (d, J = 7.8 Hz, 1 H, H-5). FAB-MS: m/z 294.0957 [(M + H)+ C15 H12 N5 O2 , calc. 294.0913]. 2.3.1.4. 1,2-DHN-4-N1Ade. Yield (20%); UV (229.6, 275.7, 338.7). 1 H NMR (ppm) δ 10.00 (br s, 1 H, Ar–OH, exchangeable with D2 O), 9.69 (br s, 1 H, Ar–OH, exchangeable with D2 O), 9.54 and 9.29 (s, 2 H, NH2 Ade, exchangeable with D2 O), 8.89 (s, 1 H, H-2-Ade), 8.49 (s, 1 H, H-8-Ade), 8.19 (d, J = 8.5Hz, 1 H, H-8), 7.52 (s, 1 H, H-3), 7.48 (t, J = 8.5 Hz, 1 H, H-7), 7.28 (t, J = 8.5 Hz, 1 H, H-6), 7.22 (d, J = 7.8 Hz, 1 H, H-5). FAB-MS: m/z 294.0949 [(M + H)+ C15 H12 N5 O2 , calc. 294.0913]. 2.3.1.5. Reaction with dA: 1,2-DHN-4-N6 dA. Yield (10%). 1 H NMR (ppm) δ 10.02 (br s, 1 H, Ar–OH, exchangeable with D2 O), 9.74 (br s, 1 H, Ar–OH, exchangeable with D2 O), 8.81 (s, 1 H, H-8-dA), 8.69 (s, 1 H, H-2-dA), 8.50 (br s, 1 H, 6-NH-dA, exchangeable with D2 O), 8.20 (d, J = 8.30 Hz, 1 H, H-8), 7.58 (s, 1 H, H-3), 7.51 (dd, J = 7.32, 7.80 Hz, 1 H, H-6/7), 7.34 (dd, J = 7.32, 7.80 Hz, 1 H, H-6/7), 7.23 (d, J = 8.30 Hz, 1 H, H-5), 6.48 (t, J = 6.35 Hz, 1 H, H-1 -dA), 5.43 (br

179

s, 1 H, OH-dA, exchangeable with D2 O), 5.09 (br s, 1 H, OH-dA, exchangeable with D2 O), 4.46 (m, 1 H, H-3 -dA), 3.94 (m, 1 H, H-4 -dA), 3.62 and 3.44 (m, 2 H, H2 -5 -dA), 2.73 and 2.51 (m, 2 H, H2 -2 -dA). FAB-MS: m/z 410.1410 [(M + H)+ C20 H20 N5 O5 , calc. 410.1386]. 2.3.2. Kinetics of the appearance and disappearance of 1,2-DHN-4-N7dG adduct To a stirred solution of dG (90 mg, 0.3 mmol) in acetic acid/H2 O (1:1, 3 mL) was added a solution of 1,2-NQ (10 mg, 0.06 mmol) in DMF (1.5 mL). The pH of this solution was ∼2. The reaction mixture was stirred either at room temperature (22 ◦ C) or at 37 ◦ C. For reactions at pH 4.6, dG (90 mg) was stirred in 0.1 M phosphate buffer (pH 4.6, 3 mL) with a solution of 1,2-NQ (10 mg) in DMF (1.5 mL) at either 22 ◦ C or 37 ◦ C. Aliquots of the reaction mixture were removed after 0, 0.5, 1, 2, 5, 7, 10, and 15 h and analyzed quickly on HPLC and mass spectrometry. Identification of 1,2-DHN-4-N7dG adduct was confirmed by isolating the labile peak by preparative HPLC, recording its molecular weight by mass spectrometry and observing its conversion into the N7Gua adduct by analytical HPLC. Quantification was carried out by comparing the area under the peak of the N7dG adduct with that of the standard depurinating 1,2-DHN-4-N7Gua adduct. The rationale for this comparison derives from the identical molar extinction coefficients of both adducts. 2.3.3. Covalent binding of 1,2-NQ to DNA A solution of 1,2-NQ (5 mg, 32 ␮mol) in 500 ␮L DMSO was mixed with DNA (3 mM in 0.067 M sodium potassium phosphate buffer, pH 7.0) in 10 mL total reaction mixture and incubated at 37 ◦ C for 10 h. After incubation, DNA was precipitated with two volumes of ethanol, and the supernatant, containing depurinating adducts, was evaporated to dryness. The residue was dissolved in 10 mL of a mixture of DMF/CH3 OH, filtered and injected to preparative HPLC as described before. Blind fractions were collected one minute before and after the pre-established retention times of the standard depurinating adducts. The retention times of the adducts were established after injecting a mixture of standard 1,2-DHN-4-N3Ade and 1,2-DHN-4-N7Gua on the HPLC column and recording the retention times for both adducts. The collected fractions were evaporated and the residue reconstituted in 200 ␮L of 50% CH3 OH in 0.1 M NH4 OAc (pH 4.4). This solution (100 ␮L) was analyzed by HPLC connected to the electrochemical detector and 10 ␮L was analyzed on UPLC–MS/MS. The levels of depurinating adducts were determined by

180

M. Saeed et al. / Chemico-Biological Interactions 165 (2007) 175–188

comparing peak heights with known adduct standards. Recovery of the depurinating adducts in the reaction mixtures was assessed by spiking a known amount of standard 1,2-DHN-4-N3Ade and 1,2-DHN-4-N7Gua in a reaction mixture containing only DNA. The DNA was precipitated immediately and the supernatant was processed for depurinating adducts as described above. Under these conditions, the recoveries of the adducts were from 80 to 95%. Precipitated DNA from each experiment was processed for 32 P-post-labeling analysis of stable adducts [36] after purification of the DNA. The purified DNA was hydrolyzed with micrococcal nuclease, spleen phosphodiestrase and nuclease P1. The labeled nucleotides were separated by using polyacrylamide gel electrophoresis [37]. Four bands were observed; they were cut out of the gel and the 32 P counted. 2.3.4. Kinetics of DNA depurination 1,2-NQ (2.5 mg, 16 ␮mol) in 500 ␮L DMSO was mixed with DNA (3 mM in 0.067 M sodium potassium phosphate buffer, pH 7.0) in 10 mL of total reaction mixture and incubated at 37 ◦ C for 0.5, 1, 2, 3, 4, 5, 6, 7, 9, 10, 15, 20, or 24 h. DNA at each time point was precipitated by ethanol and the supernatant processed for measurement of the level of depurination by UPLC–MS/MS and HPLC with electrochemical detection. 2.3.5. Covalent binding of 1,2-DHN to DNA in the presence of air or an enzyme 1,2-DHN (2.5 mg in 500 ␮L DMSO) was reacted with 3 mM calf thymus DNA (pH 7.0) in 10 mL reaction mixtures in the presence of air. Alternatively, 1,2-DHN was bound to DNA in 10 mL reaction mixtures catalyzed by tyrosinase, prostaglandin H synthase, or MC-induced rat liver microsomes during 10 h of incubation at 37 ◦ C. In the tyrosinase experiments, the mixture containing 3 mM calf thymus DNA in 0.067 M sodium–potassium phosphate (pH 7.0), 2.5 mg of 1,2-DHN (in 500 ␮L of DMSO) and 1 mg of enzyme (2577 units) was incubated at 37 ◦ C for 10 h. For prostaglandin H synthase-catalyzed reactions, the mixture containing 3 mM DNA, 2.5 mg of 1,2-DHN (in 500 ␮L of DMSO), 1 mL of methemoglobin (1.47 mg/mL in 75 mM KH2 PO4 , pH 7.5), 152 ␮L of arachidonic acid (commercial 100 mg/mL solution in ethanol) and 800 ␮L of prostaglandin H synthase (400 units) was incubated at 37 ◦ C for 10 h. For the MC-induced microsomes, the reaction mixture containing 3 mM DNA in 150 mM Tris-HCl (pH 7.5), 150 mM KCl, 5 mM MgCl2 , 2.5 mg of 1,2-DHN (in 500 ␮L of DMSO), 10 mg of microsomal protein, and NADPH (0.6 mM) was incubated at 37 ◦ C for 10 h. After the

incubation period, DNA was precipitated with 2 volumes of ethanol, and processed for 32 P-post-labeling analysis of stable adducts [36,37]. The supernatant was used for the analysis of depurinating adducts, as described above. Control reactions were carried out under identical conditions either with no enzyme or no cofactor. 3. Results and discussion 3.1. Synthesis and structure elucidation of DNA adducts Previous studies from our laboratory have demonstrated that ortho-quinones, derived from natural estrogens [24–29], synthetic estrogens [30,31] and benzene [32], react with dG predominantly at the N-7 position and with Ade at the N-3 position, leading to the formation of depurinating adducts. Analogously, 1,2-NQ was reacted with 5 equiv. of dG at room temperature for 12 h (Fig. 3). Analysis by reverse phase HPLC during this time indicated the formation of three compounds detected in the adduct area of the chromatogram. However, analysis after 12 h indicated the disappearance of the middle peak corresponding to the labile 1,2-DHN4-N7dG adduct (see below for details), leaving a major adduct, 1,2-DHN-4-N7Gua (10%), and a minor adduct, 1,2-NQ-4-N7Gua (0.7%). The structures of the synthesized adducts were elucidated based on detailed NMR spectroscopy and mass spectrometry (MS) as described below. Purification of adducts was attempted by using a solid phase extraction cartridge, as described [38]. However, HPLC analysis of the sample after elution from the cartridge showed only one compound, with complete elimination of the major adduct, 1,2-DHN-4-N7Gua, and exclusive formation of the oxidized adduct, 1,2NQ-4-N7Gua. This indicated that the use of solid phase extraction facilitates the autoxidation of the catechol adduct to the corresponding quinone adduct. Therefore, we decided to skip the extraction step and employed preparative reverse phase chromatography directly after filtration of the reaction mixture. The HPLC conditions for both analytical and preparative scales are described in Section 2. Along with recovered starting material, two products could be isolated, eluting at retention times of 7 and 9 min and identified as 1,2-NQ-4-N7Gua (0.7%) and 1,2-DHN-4-N7Gua (10%), respectively. The recovered starting material (52%) was recycled to increase the overall yield of the reaction. Preliminary NMR and MS analyses of 1,2-NQ4-N7Gua indicated that it is the same compound

M. Saeed et al. / Chemico-Biological Interactions 165 (2007) 175–188

181

Fig. 3. Synthesis of standard adducts by reaction of 1,2-naphthoquinone with dG.

characterized by McCoull et al., as a sole product of the reaction [38]. In addition to the quinone adduct, we were able to isolate and characterize the catechol adduct. The partial oxidation of the catechol adduct to the quinone adduct, in the reaction mixture, was due to the 1,2-NQ, which was reduced to 1,2-DHN, as observed on HPLC. This was previously observed for the N6 Ade adduct of hexestrol-3 ,4 -quinone [39]. In that case, only the quinone adduct was observed. Moreover, treatment with NaBH4 completely reduced 1,2-NQ-4-N7Gua to 1,2-DHN-4-N7Gua. The 1,2-NQ-4-N7Gua is formed by autoxidation of 1,2-DHN-4-N7Gua during purification. In fact, 1,2-DHN is not very stable and tends to autoxidize in the presence of air. FAB-MS of 1,2-DHN-4-N7Gua showed a protonated molecular ion [M + H]+ at m/z 310, which is consistent with the addition of 1,2-DHN to the Gua moiety. The 1 H NMR spectrum of the adduct showed two exchangeable phenolic protons resonating at 9.79 and 9.49 ppm, along with the characteristic chemical shifts of the naphthalene structure. The spectrum also demonstrated the absence of the deoxyribose moiety of dG and the presence of H-8, NH-1 and exocyclic NH2 , resonating at 8.64, 11.72 and 7.32 ppm, respectively, thus confirming the identifica-

tion of the adduct as N7Gua. The attachment of the Gua moiety at C-4 of the naphthalene structure was established as follows: (1) two doublets in the first ring of the naphthalene moiety were replaced with one singlet resonating at 7.32 ppm, indicating the bond is at either C-3 or C-4. (2) The doublet at 7.21 ppm was significantly shifted upfield, relative to that in the parent 1,2-DHN (i.e., 7.70 ppm) due to its location in the shielding zone of the Gua C8-N9 double bond. (3) Finally, in the long range HMBC experiment, H-8 of Gua showed a correlation with C-4 at 122.4 and not at 118.0 ppm, assigned to C-3, thus ruling out the C-3 position as the site of attachment. The reaction of 1,2-NQ with Ade (Fig. 4) afforded three major adducts, as well as some minor humps on HPLC (not shown). Based on their on-line UV spectra, we assumed these minor compounds (∼10% of the total adduct yield) were the oxidized adducts of the major catechol adducts. This assumption was based on our previous observation of such oxidized adducts formed in the reaction between dG and 1,2-NQ. A brief treatment with NaBH4 removed these oxidized adducts, rendering the original major adducts as the only products of the reaction. Purification by preparative HPLC and structure

182

M. Saeed et al. / Chemico-Biological Interactions 165 (2007) 175–188

Fig. 4. Synthesis of standard adducts by reaction of 1,2-naphthoquinone with Ade or dA.

elucidation by NMR and MS yielded the major adduct (20%) as 1,2-DHN-4-N1Ade and two other adducts as 1,2-DHN-4-N3Ade (8%) and 1,2-DHN-4-N6 Ade (9%). FAB-MS analysis of all three compounds produced the same [M + H]+ at m/z 294.0946, corresponding to the proposed elemental composition. The 1 H NMR spectrum of the major compound, 1,2DHN-4-N1Ade, showed four proton signals resonating at 10.00, 9.69, 9.54 and 9.29 ppm, which disappeared after D2 O exchange. Thus, two of them were assigned to the phenolic OH’s and the remaining two were assigned as NH protons of Ade. This compound was initially thought to be 1,2-DHN-4-N6 Ade, in which the exocyclic NH2 group was attached to the naphthalene moiety at C-4. However, further detailed 2D NMR experiments, including HSQC and HMBC, ruled out this compound being 1,2-DHN-4-N6 Ade. In the HMBC experiment, a correlation between H-2 of Ade at 8.89 ppm with C-4 of naphthalene at 123.2 ppm suggested the compound to be either an N3Ade or N1Ade adduct. Previous results from our laboratory have shown the splitting of the NH2 signal into two separate singlets when the bond is at the N-1 position of Ade [40,41]. On this basis, the structure was assigned as 1,2-DHN-4-N1Ade. The presence of two characteristic doublets and two triplets at 8.19, 7.22, 7.48 and 7.28 ppm, respectively, indicated that the protons of ring B of the naphthalene moiety remained unattached after nucleophilic reaction of Ade with 1,2-NQ. However, the appearance of one singlet at 7.52 ppm indicated the attachment of Ade at ring A of the naphthalene moiety. Assignment of this proton as H-3 was proposed based on the 1 H/13 C correlation between this proton and two carbons at C-10 (124.5 ppm) and C-1 (140.8 ppm) in an HMBC experiment with the adduct.

The 1 H NMR spectrum of 1,2-DHN-4-N6 Ade showed two sharp singlets (10.00, 9.73 ppm) and two broad singlets at 9.78, and 8.37 ppm, which disappeared after D2 O exchange. The two sharp singlets at 10.00 and 9.73 ppm were assigned to the phenolic OH’s and the remaining two at 9.78 and 8.37 ppm were assigned as the N-9 and exocyclic N6 H protons of Ade. All other chemical shifts were assigned with the help of detailed 1D and 2D NMR data. The 1 H NMR spectrum of 1,2-DHN-4-N3Ade showed a two-proton broad singlet resonating at 7.85 ppm, assigned as the exocyclic NH2 group of Ade, based on its disappearance after D2 O exchange. All other chemical shifts were assigned with the help of detailed 1D and 2D NMR data. In the reaction between 1,2-NQ and dA (Fig. 4), the reaction mixture was treated with NaBH4 to remove the minor oxidized adducts and then subjected to preparative HPLC. The major adduct formed was elucidated by NMR and MS and found to be 1,2-DHN-4-N6 dA. FAB-MS analysis of the adduct showed an [M + H]+ at m/z 410, which is consistent with the addition of 1,2DHN to the dA moiety. Moreover, the 1 H NMR showed signals for the deoxyribose unit, indicating the formation of an adduct containing a stable glycosyl bond. Assignments of all chemical shifts were carried out as described above for reaction between 1,2-NQ and Ade. 3.2. Kinetics of the loss of the deoxyribose moiety After 2 h, the reaction of 1,2-NQ with dG in a mixture of DMF/water/acetic acid (1:1:1) afforded three adducts, namely, 1,2-DHN-4-N7dG, 1,2-DHN-4-N7Gua and 1,2NQ-4-N7Gua as shown in Fig. 5A. When the reaction mixture was analyzed by HPLC after 12 h, the mid-

M. Saeed et al. / Chemico-Biological Interactions 165 (2007) 175–188

183

Fig. 5. Reaction of 1,2-NQ with dG. (A) HPLC showing the presence of 1,2-NQ-4-N7Gua, 1,2-DHN-4-N7dG and 1,2-DHN-4-N7Gua after 2 h, (B) presence of 1,2-DHN-4-N7Gua after 12 h, and (C) MS/MS of 1,2-DHN-4-N7dG.

dle peak corresponding to 1,2-DHN-4-N7dG was absent (Fig. 5B), and only two peaks could be seen. To identify the initially formed labile adduct, an aliquot of the reaction mixture was directly infused into the mass spectrometer after 2 h and parent molecular ions were scanned. A molecular ion peak at m/z 426 indicated the presence of an adduct in which the deoxyribose unit is still bonded to the Gua moiety. We tentatively named this adduct 1,2-DHN-4-N7dG, based on the following observations: (a) the reaction mixture after 2 h was subjected to preparative HPLC, the labile peak was collected and analyzed quickly on MS by direct infusion; it produced an ion at m/z 426, corresponding to [1,2DHN-4-N7dG]+ , which upon further fragmentation by MS/MS gave a daughter ion at m/z 310 (Fig. 5C), corresponding to 1,2-DHN-4-N7Gua, i.e., the adduct without the deoxyribose moiety; (b) when the isolated peak was re-injected in analytical HPLC, the labile adduct was completely converted to 1,2-DHN-4-N7Gua; (c) we were unable to record the NMR spectrum of the 1,2-DHN-4-N7dG adduct because it decomposed during evaporation/lyophilization of the solvent. Analogous N7dG adducts have been described in similar studies of adducts formed by natural and synthetic estrogens [42]. Next, we undertook the kinetics of formation and decomposition of the labile 1,2-DHN-4-N7dG adduct. The reaction was conducted at different pHs (1.9 and 4.6) and different temperatures (22 and 37 ◦ C). Aliquots of the reaction mixtures were analyzed by HPLC at different time points, as shown in Fig. 6. The greatest amount

of 1,2-DHN-4-N7dG adduct was formed at the lower temperature (22 ◦ C) and lower pH (1.9) (Fig. 6A). This phenomenon may be explained by assuming a relative stabilization of the glycosyl bond at lower temperature so that more of the N7dG adduct accumulated over time. Alternatively, more of the N7dG adduct may have formed at lower pH in the acid-catalyzed Michael addition. When the temperature was raised to 37 ◦ C keeping the pH fixed at 1.9, a high rate of loss of deoxyribose was observed. Even though the level of N7dG adduct at 37 ◦ C was not as high as that at 22 ◦ C (Fig. 6A), a relatively high level of the 1,2-DHN-4-N7Gua adduct was observed (Fig. 6B). This indicates a faster 1,4-Michael addition (first step), as well as faster loss of deoxyribose from the labile 1,2-DHN-4-N7dG adduct. Increasing the pH to 4.6 afforded a much lower yield of 1,2-DHN4-N7Gua at both the lower (22 ◦ C) and higher (37 ◦ C) temperature (Fig. 6B). Again, this can be explained by assuming less efficient 1,4-Michael addition at the higher pH. 3.3. Covalent binding of 1,2-NQ or enzyme-activated 1,2-DHN to DNA Formation of depurinating adducts was observed by reacting 1,2-NQ or enzyme-activated 1,2-DHN with calf thymus DNA at 37 ◦ C for 10 h. The supernatant containing the depurinating DNA adducts was analyzed by HPLC with electrochemical detection and by UPLC–MS/MS. Quantitation of the adducts was car-

184

M. Saeed et al. / Chemico-Biological Interactions 165 (2007) 175–188

Fig. 6. (A) Formation and disappearance of 1,2-DHN-4-N7dG and (B) formation of 1,2-DHN-4-N7Gua from 1,2-DHN-4-N7dG at various temperatures and pHs.

ried out by monitoring one of the major fragments of 1,2-DHN-4-N7Gua (m/z 310 > 185.9) for the N7Gua adduct and of 1,2-DHN-4-N3Ade (m/z 294 > 231) for the N3Ade adduct, as shown in Figs. 7 and 8, respectively. Reaction of 1,2-NQ with DNA produced the depurinating adducts 1,2-DHN-4-N7Gua and 1,2DHN-4-N3Ade at similar levels (3.1 ± 0.7 and 2.5 ± 0.9 ␮mol/mol DNA-P, respectively, Table 1). The potential of 1,2-DHN for autoxidation was assessed by reacting 1,2-DHN with DNA in the presence of air. We observed formation of depurinating adducts at low levels, i.e., 0.5 ± 0.1 ␮mol N3Ade/mol DNA-P and 0.4 ± 0.1 ␮mol N7Gua/mol DNA-P. Oxidation of 1,2-DHN in the presence of tyrosinase (with oxygen of the air as cofactor) was found to be highly efficient, producing 25.6 ± 2.1 ␮mol N3Ade/mol DNA-P and 17.5 ± 1.0 ␮mol N7Gua/mol DNA-P. The level of depurinating adducts was intermediate after activation

of 1,2-DHN with prostaglandin H synthase (and arachidonic acid as cofactor) and lower with MC-induced rat liver microsomes (and NADPH). The lower level of formation of depurinating adducts with the latter enzymes may be due to the in situ reaction of 1,2-NQ with proteins. The stable adducts were quantified by the 32 P-postlabeling method [36,37]. In each case, four adduct bands were detected upon separation by polyacrylamide gel electrophoresis. The total amounts of stable DNA adducts were similar to those of the depurinating adducts, except when 1,2-DHN was activated with tyrosinase, which formed much higher levels of depurinating adducts (Table 1). Although exact quantification was not achieved due to differences in adduct recovery, the pattern of adduct formation suggests that depurinating adducts are an important fraction of total adducts.

M. Saeed et al. / Chemico-Biological Interactions 165 (2007) 175–188

185

Fig. 7. Multiple reaction monitoring (MRM) profile of the standard 1,2-DHN-4-N7Gua and 1,2-DHN-4-N7Gua formed in tyrosinase-catalyzed reaction of 1,2-DHN with calf thymus DNA. The upper spectrum is the MS/MS of the biologically formed adduct.

Fig. 8. Multiple reaction monitoring (MRM) profile of the standard 1,2-DHN-4-N3Ade and 1,2-DHN-4-N3Ade formed in tyrosinase-catalyzed reaction of 1,2-DHN with calf thymus DNA. The upper spectrum is the MS/MS of the biologically formed adduct.

186

M. Saeed et al. / Chemico-Biological Interactions 165 (2007) 175–188

Table 1 Formation of adducts after reaction of 1,2-NQ with DNAa Compound

Depurinating adducts (␮mol/mol DNA-P) 1,2-DHN-4-N3Ade

1,2-NQ 1,2-DHN Air Tyrosinase Prostaglandin H synthase MC-induced rat liver microsomes a b

2.5 ± 0.9 0.5 25.6 3.7 0.8

± ± ± ±

0.1 2.1 0.3 0.3

Stable adducts (␮mol/mol DNA-P)

1,2-DHN-4-N7Gua 3.1 ± 0.7

1.6 ± 0.9

± ± ± ±

NDb 2.5 ± 0.3 4.0 ± 0.3 3.1 ± 0.8

0.4 17.5 6.4 1.0

0.1 1.0 0.1 0.4

These results are the average of three reactions. Not determined.

Fig. 9. Rate of depurination of 1,2-DHN-4-N3Ade and 1,2-DHN-4N7Gua from DNA after reaction of 1,2-NQ with DNA.

estrogens [24–27,42], the catechol quinones of the synthetic estrogens diethylstilbestrol and its hydrogenated derivative hexestrol [30,31,42] and the catechol quinone of the leukemogen benzene [32,43]. The apurinic sites formed by the depurinating DNA adducts may be converted by error-prone base excision repair [28,29,44] into mutations that can initiate cancer. This unifying mechanism renders plausible the hypothesis that naphthalene initiates cancer via the same mechanism of activation, in which 1,2-NQ is its ultimate carcinogenic metabolite.

3.4. Kinetics of DNA depurination Acknowledgements The rate of depurination of DNA adducts was determined by reacting 1,2-NQ with DNA at 37 ◦ C and pH 7.0 and analyzing the depurinating adducts at different time points, i.e., 0.5, 1, 2, 3, 4, 5, 7, 9 and 10 h (Fig. 9). At each time point, the DNA was precipitated with 2 vol. of ethanol and the supernatant was analyzed by UPLC–MS/MS. Depurination of the N3Ade adduct was almost complete after 1 h; after that it remained almost constant. The depurination of the N7Gua adduct, however, was found to be slower and was complete in 3 h. The slower depurination of the N7Gua adduct is common for natural and synthetic estrogens [26,42], and benzene [43]. 4. Conclusions 1,2-NQ and enzymatically activated 1,2-DHN, both secondary metabolites of naphthalene [19,22,23], react with DNA by 1,4-Michael addition to form specifically the depurinating N7Gua and N3Ade adducts, as well as unidentified stable adducts. Furthermore, the N3Ade adduct depurinates instantaneously, whereas the N7Gua adduct depurinates with a half-life of ca. 1.5 h. These chemical and biochemical properties are similar to those of the catechol-3,4-quinones of the natural

This research was supported by U.S. Public Health Service Grant P01 CA49210. Core support at the Eppley Institute was provided by Grant P30 CA36727 from the National Cancer Institute. References [1] IARC, IARC Monographs on the Evaluation of Carcinogenic Risks to Human: Some Traditional Herbal Medicines, Some Mycotoxins, Naphthalene and Styrene, vol. 82, Lyon, France, 2002, pp. 367–435. [2] J.S. Stanley, Broad Scan Analysis of the FY82 National Human Adipose Tissue Survey Specimens, Executive Summary (EPA560/5-86-035), Environmental Protection Agency, vol. 1, Office of Toxic Substances, Washington, DC, 1986. [3] E.D. Pellizzari, T.D. Hartwell, B.S.H. Harris III, R.D. Waddell, D.A. Whitaker, M.D. Erickson, Purgeable organic compounds in mother’s milk, Bull. Environ. Contam. Toxicol. 28 (1982) 322–328. [4] Environmental Protection Agency. Clean Air Act Amendments of 1990 Conference Report to Accompany S. 1630 (Report No. 101-952), Washington, DC, Office of Pollution Prevention and Toxics, 1990. [5] O. Wolf, Cancers in Chemical Workers in a Former Naphthalene Purification Plant, vol. 31, Dt. Gesundh, Wesen, 1976, pp. 996–999. [6] O. Wolf, Carcinoma of the larynx in naphthalene purifiers, Z. Get. Hyg. 24 (1978) 737–739.

M. Saeed et al. / Chemico-Biological Interactions 165 (2007) 175–188 [7] A. Buckpitt, B. Boland, M. Isbell, D. Morin, M. Shultz, R. Baldwin, K. Chan, A. Karlsson, C. Lin, A. Taff, J. West, M. Fanucchi, L. Van Winkle, C. Plopper, Naphthalene-induced respiratory tract toxicity: metabolic mechanisms of toxicity, Drug Metab. Rev. 34 (2002) 791–820. [8] National Toxicology Program (NTP), Toxicology and Carcinogenesis Studies of Naphthalene (CAS No. 91-20-3) in B6C3F1 Mice (inhalation studies), Technical Report Series No. 410, NIH Publication No. 92-3141; U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health, Research Triangle Park, NC, 1992. [9] K.M. Abdo, S.L. Eustis, M. McDonald, M.P. Joiken, B. Adkins Jr., J.K. Haseman, Naphthalene: a respiratory tract toxicant and carcinogen for mice, Inhal. Toxicol. 4 (1992) 393–409. [10] K.M. Abdo, S. Grumbein, B.J. Chou, R. Herbert, Toxicity and carcinogenicity study in F344 rats following 2 years of wholebody exposure to naphthalene vapors, Inhal. Toxicol. 13 (2001) 931–950. [11] National Toxicology Program (NTP), Toxicology and Carcinogenesis Studies of Naphthalene (CAS No. 91-20-3) in F344/N Rats (Inhalation Studies), CNTP Technical Report Series No. 500; NIH Publication No. 01-4434, Research Triangle Park, NC, 2000. [12] A.R. Buckpitt, N. Castagnoli Jr., S.D. Nelson, A.D. Jones, L.S. Bahnson, Stereoselectivity of naphthalene epoxidation by mouse, rat and hamster pulmonary, hepatic and renal microsomal enzymes, Drug Metab. Disp. 15 (1987) 491–498. [13] D.M. Jerina, J.W. Daly, B. Witkop, Z. Zaltzman-Nirenberg, S. Udenfriend, 1,2-naphthalene oxide as an intermediate in the microsomal hydroxylation of naphthalene, Biochemistry 9 (1970) 147–156. [14] P.J. Van Bladeren, K.P. Vyas, J.M. Sayer, D.E. Ryan, P.E. Thomas, W. Levin, D.M. Jerina, Stereoselectivity of cytochrome P-450c in the formation of naphthalene and anthracene 1,2-oxides, J. Biol. Chem. 259 (1984) 8966–8973. [15] S. Kanekal, C. Plopper, D. Morin, A. Buckpitt, Metabolism and cytotoxicity of naphthalene oxide in the isolated perfused mouse lung, J. Pharmacol. Exp. Ther. 256 (1991) 391–401. [16] A.R. Buckpitt, L.S. Bahnson, Naphthalene metabolism by human lung microsomal enzymes, Toxicology 41 (1986) 333–341. [17] M.D. Tingle, M. Pirmohamed, E. Templeton, A.S. Wilson, S. Madden, N.R. Kitteringham, B.K. Park, An investigation of the formation of cytotoxic, genotoxic, protein-reactive and stable metabolites from naphthalene by human liver microsomes, Biochem. Pharmacol. 46 (1993) 1529–1538. [18] E. Boyland, P. Sims, Metabolism of polycyclic compounds, Biochem. J. 68 (1958) 440–447. [19] T.E. Smithgall, R.G. Harvey, T.M. Penning, Spectroscopic identification of ortho-quinones as the products of polycyclic aromatic trans-dihydrodiol oxidation catalyzed by dihydrodiol dehydrogenase, J. Biol. Chem. 263 (1988) 1814–1820. [20] R.M. Turkall, G.A. Skowronski, A.M. Kadry, M.S. AbdelRahman, A comparative study of the kinetics and bioavailability of pure and soil-absorbed naphthalene in dermally exposed male rats, Arch. Environ. Toxicol. 26 (1994) 504–509. ˚ Gustafsson, B. Gustafsson, Catabolism [21] J. Bakke, C. Struble, J.-A. of premercapturic acid pathway metabolites of naphthalene to naphthols and methylthio-containing metabolites in rats, Biochemistry 82 (1985) 668–671. [22] M. d’Arcy Doherty, R. Makowski, G.G. Gibson, G.M. Cohen, Cytochrome P-450 dependent metabolic activation of 1-naphthol to naphthoquinones and covalent binding species, Biochem. Pharmacol. 34 (1985) 2261–2267.

187

[23] M. Flueraru, A. Chichirau, L.L. Chepelev, W.G. Willmore, T. Durst, M. Charron, L.R. Barclay, J.S. Wright, Cytotoxicity and cytoprotective activity in naphthalenediols depends on their tendency to form naphthoquinones, Free Radic. Biol. Med. 39 (2005) 1368–1377. [24] E.L. Cavalieri, D.E. Stack, P.D. Devanesan, R. Todorovic, I. Dwivedy, S. Higginbotham, S.L. Johansson, K.D. Patil, M.L. Gross, J.K. Gooden, R. Ramanathan, R.L. Cerny, E.G. Rogan, Molecular origin of cancer: catechol estrogen-3,4-quinones as endogenous tumor initiators, Proc. Natl. Acad. Sci. USA 94 (1997) 10937–10942. [25] K.-M. Li, R. Todorovic, P. Devanesan, S. Higginbotham, H. Kofeler, R. Ramanathan, M.L. Gross, E.G. Rogan, E.L. Cavalieri, Metabolism and DNA binding studies of 4-hydroxyestradiol and estradiol-3,4-quinone in vitro and in female ACI rat mammary gland in vivo, Carcinogenesis 25 (2004) 289–297. [26] M. Zahid, E. Kohli, M. Saeed, E. Rogan, E. Cavalieri, The greater reactivity of estradiol-3,4-quinone vs estradiol-2,3-quinone with DNA in the formation of depurinating adducts: implications for tumor-initiating activity, Chem. Res. Toxicol. 19 (2006) 164–172. [27] E. Cavalieri, E. Rogan, D. Chakaravarti, The role of endogenous catechol quinones in the initiation of cancer and neurodegenerative diseases, in: H. Sies, L. Packer (Eds.), Methods in Enzymology, Quinones and Quinone Enzymes, Part B, vol. 382, Elsevier, Duesseldorf, Germany, 2004, pp. 293–319. [28] D. Chakravarti, P. Mailander, K.-M. Li, S. Higginbotham, H. Zhang, M.L. Gross, E. Cavalieri, E. Rogan, Evidence that a burst of DNA depurination in SENCAR mouse skin induces error-prone repair and forms mutations in the H-ras gene, Oncogene 20 (2001) 7945–7953. [29] P.C. Mailander, J.L. Meza, S. Higginbotham, D. Chakravarti, Induction of A.T to G.C mutations by erroneous repair of depurinated DNA following estrogen treatment of the mammary gland of ACI rats, J. Steroid Biochem. Mol. Biol. 101 (2006) 204–215. [30] M. Saeed, S.J. Gunselman, S. Higginbotham, E. Rogan, E. Cavalieri, Formation of the depurinating N3Adenine and N7Guanine adducts by reaction of DNA with hexestrol-3 ,4 -quinone or enzyme activated 3 -hydroxyhexestrol. Implications for a unifying mechanism of tumor initiation by natural and synthetic estrogens, Steroids 70 (2005) 37–45. [31] M. Saeed, E. Rogan, E. Cavalieri, Mechanism of tumor initiation by the human carcinogen diethylstilbestrol: the defining link to natural estrogens, Proc. Am. Assoc. Cancer Res. 46 (2005) 2129. [32] E.L. Cavalieri, K.-M. Li, N. Balu, M. Saeed, P. Devanesan, S. Higginbotham, J. Zhao, M.L. Gross, E.G. Rogan, Catechol orthoquinones: the electrophilic compounds that form depurinating DNA adducts and could initiate cancer and other diseases, Carcinogenesis 23 (2002) 1071–1077. [33] Z. Zhao, W. Kosinska, M. Khmelnitsky, E.L. Cavalieri, E.G. Rogan, D. Chakravarti, P. Sacks, J.B. Guttenplan, Mutagenic activity of 4-hydroxyestradiol, but not 2-hydroxyestradiol, in BB2 rat embryonic cells, and the mutational spectrum of 4hydoxyestradiol, Chem. Res. Toxicol. 19 (2006) 475–479. [34] NIH Guidelines for the Laboratory Use of Chemical Carcinogens, NIH Publication No. 81-2385, US Government printing Office, Washington, DC, 1981. [35] K.L. Platt, F. Oesch, Efficient synthesis of non-K-region trans-dihydro diols of polycyclic aromatic hydrocarbons from o-quinones and catechols, J. Org. Chem. 48 (1983) 256–268. [36] L. Chen, P.D. Devanesan, S. Higginbotham, F. Ariese, R. Jankowiak, G.J. Small, E.G. Rogan, E.L. Cavalieri, Expanded analysis of benzo[a]pyrene-DNA adducts formed in vitro and in

188

[37]

[38]

[39]

[40]

M. Saeed et al. / Chemico-Biological Interactions 165 (2007) 175–188 mouse skin: their significance in tumor initiation, Chem. Res. Toxicol. 9 (1996) 897–903. I. Terashima, N. Suzuki, S. Shibutani, 32 P-postlabeling/ polyacrylamide gel electrophoresis analysis: application to the detection of DNA adducts, Chem. Res. Toxicol. 15 (2002) 305–311. K.D. McCoull, D. Rindgen, I.A. Blair, T.M. Penning, Synthesis and characterization of polycyclic aromatic hydrocarbon o-quinone depurinating N7-guanine adducts, Chem. Res. Toxicol. 12 (1999) 237–246. S.T. Jan, P.D. Devanesan, D.E. Stack, R. Ramanathan, J. Byun, M.L. Gross, E.G. Rogan, E.L. Cavalieri, Metabolic activation and formation of DNA adducts of hexestrol, a synthetic nonsteroidal carcinogenic estrogen, Chem. Res. Toxicol. 11 (1998) 412–419. P.P.J. Mulder, L. Chen, B.C. Sekhar, M. George, M.L. Gross, E.G. Rogan, E.L. Cavalieri, Synthesis and structure determination of the adducts formed by electrochemical oxidation of 1,2,3,4-tetrahydro-7,12-dimethylbenz[a]anthracene in the presence of deoxyribo-nucleosides or adenine, Chem. Res. Toxicol. 9 (1996) 1264–1277.

[41] K.-M. Li, M. Byun, M.L. Gross, D. Zamzow, R. Jankowiak, E.G. Rogan, E.L. Cavalieri, Synthesis and structure determination of the adducts formed by electrochemical oxidation of dibenzo[a,l]pyrene in the presence of adenine, Chem. Res. Toxicol. 12 (1999) 749–757. [42] M. Saeed, M. Zahid, S.J. Gunselman, E. Rogan, E. Cavalieri, Slow loss of deoxyribose from the N7deoxyguanosine adducts of estradiol-3,4-quinone and hexestrol-3 ,4 -quinone. Implications for mutagenic activity, Steroids 70 (2005) 29–35. [43] A. Raghavanpillai, K. Olson, E. Rogan, E. Cavalieri, Slow loss of deoxyribose from N7deoxyguanosine adducts of catechol quinone: possible relevance for mutagenic activity, Proc. Am. Assoc. Cancer Res. 45 (2004) 1553. [44] D. Chakravarti, P.C. Mailander, E.L. Cavalieri, E.G. Rogan, Evidence that error-prone DNA repair converts dibenzo[a,l]pyreneinduced depurinating lesions into mutations: formation, clonal proliferation and regression of initiated cells carrying H-ras oncogene mutations in early preneoplasia, Mutat. Res. 456 (2000) 17–32.