Metabolic Activation of PCBs to Quinones: Reactivity toward Nitrogen

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Chem. Res. Toxicol. 1996, 9, 623-629

623

Metabolic Activation of PCBs to Quinones: Reactivity toward Nitrogen and Sulfur Nucleophiles and Influence of Superoxide Dismutase Anthony R. Amaro,† Greg G. Oakley,† Udo Bauer,‡ H. Peter Spielmann,§ and Larry W. Robertson*,† Graduate Center for Toxicology, University of Kentucky Medical Center, 306 Health Sciences Research Building, Lexington, Kentucky 40536-0305, and Department of Biochemistry, University of Kentucky Medical Center, Lexington, Kentucky 40536 Received June 29, 1995X

Polychlorinated biphenyls (PCBs) may undergo cytochrome P-450-catalyzed hydroxylations to form chlorinated dihydroxybiphenyl metabolites. When the hydroxyl groups are ortho or para to each other, oxidation to a quinone may be catalyzed by peroxidases present within the cell. In order to study the reactivity of PCB-derived quinones, selected chlorophenyl 1,2- and 1,4-benzoquinones were synthesized and characterized, including their reduction potentials against a saturated calomel electrode. Two quinones, 4-(4′-chlorophenyl)-1,2-, and 4-(3′,4′dichlorophenyl)-1,2-benzoquinone, were obtained via the oxidation of the corresponding dihydroxybiphenyls with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone. Six 1,4-benzoquinones were synthesized via the Meerwein arylation: 2-(2′-chlorophenyl)-1,4-, 2-(3′-chlorophenyl)1,4-, 2-(4′-chlorophenyl)-1,4-, 2-(2′,5′-dichlorophenyl)-1,4-, 2-(3′,4′-dichlorophenyl)-1,4-, and 2-(3′,5′-dichlorophenyl)-1,4-benzoquinone. As a model study, the rate of reactivity of 2-(4′chlorophenyl)-1,4-benzoquinone toward the nitrogen nucleophiles glycine, L-arginine, L-histidine- and L-lysine was determined under pseudo-first-order conditions at pH 7.4. The rate constants ranged from 0.45 to 0.75 min-1 M-1. Higher rates were obtained under conditions of higher pH. Two reaction products were identified as the 5- and 6-ring addition products in the ratio of 1:4. In contrast, the reaction of 2-(4′-chlorophenyl)-1,4-benzoquinone with the sulfur nucleophiles glutathione or N-acetyl-L-cysteine was instantaneous. The major product of the reaction of glutathione with 2-(4′-chlorophenyl)-1,4-benzoquinone was also the 6-ring addition product. The hydroquinone thioether could be enzymatically reoxidized to the quinone thioether. Also, the influence of atmospheric oxygen and superoxide dismutase on the rates of the following horseradish peroxidase/H2O2-catalyzed oxidations was investigated: 3,4dichloro-2′,5′-dihydroxybiphenyl to 2-(3′,4′-dichlorophenyl)-1,4-benzoquinone and 3,4-dichloro3′,4′-dihydroxybiphenyl to 4-(3′,4′-dichlorophenyl)-1,2-benzoquinone. While the presence or absence of atmospheric oxygen did not alter the rates of the oxidation reactions, the presence of superoxide dismutase signficantly increased the rates of both oxidation reactions, having the greater effect on the oxidation of the 1,4-hydroquinone. These data show that PCB-derived quinones react with both nitrogen and sulfur nucleophiles of the cell and may explain, in part, the toxic effects of individual PCBs and PCB formulations, such as glutathione depletion, oxidative stress, and cell death.

Introduction Polychlorinated biphenyls (PCBs)1 were large scale industrial chemicals which were used in diverse applications, such as in dielectric fluids, in transformers and capacitors, in hydraulic fluids, and as sealants (1). The physical properties which made PCBs ideal for industry have also resulted in worldwide environmental contamination (2). While PCBs with multiple halogens per * Address correspondence to this author at the Graduate Center for Toxicology, 306 Health Sciences Research Bldg., University of Kentucky, Lexington, KY 40536-0305. Telephone: (606) 257-3952; Fax: (606) 323-1059; E-mail: [email protected]. † The Graduate Center for Toxicology. ‡ Current address: Department of Chemistry, University of Oulu, Fin-90570 Oulu, Finland. § Department of Biochemistry. X Abstract published in Advance ACS Abstracts, March 15, 1996. 1 Abbreviations: PCB, polychlorinated biphenyl; SOD, superoxide dismutase; HRP, horseradish peroxidase; GSH, glutathione; NAC, N-acetyl-L-cysteine; FAB/MS, fast atom bombardment mass spectrometry; EI-MS, electron impact mass spectrometry; SCE, saturated calomel electrode; TEAP, tetraethylammonium perchlorate; NMR, nuclear magnetic resonance.

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biphenyl are more resistant to chemical and biologic attack, PCBs with fewer halogens may have measurable rates of metabolism (3). Cytochrome P-450 metabolism of aromatic hydrocarbons, including halogenated benzenes and halogenated biphenyls, often involves a hydroxylation reaction, the introduction of an OH functionality into the molecule. When two hydroxyl groups are introduced ortho or para to each other, further oxidation by peroxidases within the cell will lead to quinone metabolites. Considerable data support the occurrence of catechols and hydroquinone metabolites of PCBs. These have been detected in in vivo (4,5) and in vitro (6,7) studies. We have recently shown that three peroxidases (myelo- and horseradish peroxidase and prostaglandin (H) synthase) (8) will catalyze the further oxidation of such PCBderived catechols and hydroquinones to quinoid species, which bind to deoxynucleotides (9) and DNA (8). The occurrence of PCB quinone metabolites and their potential reactivity toward cellular nucleophiles is therefore of current interest. © 1996 American Chemical Society

624 Chem. Res. Toxicol., Vol. 9, No. 3, 1996

Figure 1. Structures of the synthetic chloroquinones.

Quinones make up a large class of compounds with diverse biological activity. They can be found in many animal and plant cells and are widely used as anticancer, antibacterial, or antimalarial drugs as well as fungicides. The cytotoxicity of quinones can be attributed to their binding with cellular nucleophiles such as protein and nonprotein sulfhydryls and/or their ability to redox cycle with the creation of oxidative stress (10). We are currently investigating the metabolic activation of lower chlorinated biphenyls to dihydroxy compounds and their subsequent oxidation to the quinone/semiquinones. In an effort to better understand the reactivity of PCB quinone metabolites, we have synthesized a series of PCB-1,4-benzoquinones and two 1,2-benzoquinones for our studies (Figure 1). These compounds have allowed us to determine their reactivity with nitrogen and sulfur nucleophiles at physiological pH. We have also studied the role of oxygen and superoxide dismutase (SOD) on the horseradish peroxidase (HRP)/H2O2-catalyzed oxidation reactions of lower chlorinated dihydroxybiphenyls to PCB quinones.

Materials and Methods Chemicals and Reagents. Horseradish peroxidase (type 6) (EC 1.11.1.7, Type VI), superoxide dismutase (EC 1.15.1.1), glutathione, and N-acetyl-L-cysteine were purchased from Sigma Chemical Co. (St. Louis, MO). Ethylenediamine was purchased from Aldrich Chemical Co. All other reagents were of the highest purity available and were from Fisher Chemical (Cincinnati, OH). Caution: Synthetic PCBs and metabolites should be considered potentially toxic and hazardous and therefore should be handled in an appropriate manner. Dihydroxybiphenyl and Quinone Syntheses. We have previously reported the synthesis, isolation, and characterization of the chlorinated dihydroxybiphenyls (11). The chlorophenylsubstituted 1,2-benzoquinones were synthesized by oxidation of the respective catechols (12) using 2,3-dichloro-5,6 dicyano1,4-benzoquinone (Aldrich Chemical Co.), while the 1,4-benzoquinones were synthesized using the Meerwein arylation as described by Brassard et al. (13) from 1,4-benzoquinone and

Amaro et al. diazotized chloroanilines (Aldrich Chemical Co.). The 1H and 13C NMR spectra reported in this section were recorded on either a Varian VXR-400S or a Varian Gemini-200 spectrometer by using CDCl3 and acetone-d6 (Aldrich Chemical Co.) as solvents and tetramethylsilane (Aldrich Chemical Co.) as an internal standard. GC/MS spectra were recorded on a Finnigan INCOS 50 using a fused silica capillary column (DB-5MS 15 m × 0.25 mm and OV-1, 25 m, J&W Scientific, Folsom, CA). The PCB-1,2-benzoquinones were unstable, so their derivatives with ethylenediamine were synthesized (14). Ethylenediamine (1 µL) was added to the quinone 1 (1 mg) dissolved in absolute ethanol (500 µL) and was stirred at room temperature for 1 min. After the addition of ethylenediamine, the yellowcolored solution turned to a dark orange-brown color. This procedure was repeated for quinone 2. The samples were submitted for GC/EI-MS analysis. 4-(4′-Chlorophenyl)-1,2-benzoquinone from 4-chloro-3′,4′-dihydroxybiphenyl (1). 1H NMR (400 MHz, acetone-d6): δ ) 6.48-6.62 (m, 3H), 7.24-7.62 (m, 4H); MS (EI) m/z (relative intensity) ) 243 (100) [M+ + 1], 208 (5) [M+ - Cl] 152 (25), [M+ - Cl, C2H4N2]. Mass spectral data reported for ethylenediamine derivative of quinone. 4-(3′,4′-dichlorophenyl)-1,2-benzoquinone from 3,4-dichloro3′,4′-dihydroxybiphenyl (2). 1H NMR (400 MHz, acetone-d6): δ ) 6.52 (dd, J ) 10.4, 0.9 Hz , 1H, 6′-H), 6.59 (dd, J ) 2.4, 0.9 Hz , 1H, 3′-H), 7.40 (dd, J ) 10.4, 2.4 Hz , 1H, 5′-H), 7.48 (dd, J ) 8.5, 2.4 Hz , 1H, 6′-ArH), 7.60 (d, J ) 8.5 Hz , 1H, 5′-ArH), 7.74 (d, J ) 2.4 Hz , 1H, 2′-ArH); 13C NMR (100 MHz, acetoned6): δ ) 126.32, 128.89, 131.04, 131.67, 133.96, 135.73, 136.38, 140.09, 140.10, 148.26, 179.83, 179.94; MS (EI) m/z (relative intensity) ) 277 (100) [M+ + 1], 186 (8), [M+ - Cl, C2H4N2], 151(5) [M+ - 2 Cl, C2H4N2]. Mass spectral data reported for ethylenediamine derivative of quinone. 2-(2′-Chlorophenyl)-1,4-benzoquinone from 1,4-benzoquinone and 2-chloroaniline (3) mp 80-81 °C (acetone). 1H NMR (200 MHz, CDCl3): δ ) 6.78-6.81 (m, 1H), 6.86-6.90 (m, 2H), 7.197.51 (m, 4H); 13C NMR (50 MHz, CDCl3): δ ) 126.82, 129.86, 130.64, 130.75, 132.41, 133.12, 135.03, 136.41, 136.82, 146.16, 185.05, 187.24; MS (EI) m/z (relative intensity) ) 218 (50) [M+], 190 (5) [M+ - CO], 183 (100), [M+ - Cl], 155 (20) [M+ - CO, Cl], 127 (8) [M+ - 2 CO, Cl]. 2-(3′-Chlorophenyl)-1,4-benzoquinone from 1,4-benzoquinone and 3-chloroaniline (4) mp 144-145 °C (acetone). 1H NMR (200 MHz, CDCl3):δ ) 6.81-6.92 (m, 3H), 7.34-7.49 (m, 4H); 13C NMR (50 MHz, CDCl3): δ ) 127.35, 129.24, 129.78, 130.11, 133.15, 134.27, 134.53, 136.33, 136.99, 144.60, 186.04, 187.20; MS (EI) m/z (relative intensity) ) 218 (61) [M+], 190 (12), [M+ - CO], 183 (100) [M+ - Cl], 155 (25) [M+ - CO, Cl], 127 (15) [M+ - 2 CO, Cl]. 2-(4′-Chlorophenyl)-1,4-benzoquinone from 1,4-benzoquinone and 4-chloroaniline (5) mp 130-132 °C (acetone). 1H NMR (200 MHz, CDCl3): δ ) 6.80-6.92 (m, 3H), 7.43 (s, 4H); 13C NMR (50 MHz, CDCl3):δ ) 128.90, 130.56, 131.07, 132.69, 136.37, 136.63, 137.05, 144.82, 186.28, 187.25; MS (EI) m/z (relative intensity) ) 218 (82) [M+], 190 (12), [M+ - CO], 183 (100) [M+ - Cl], 155 (19) [M+ - CO, Cl], 127 (15) [M+ - 2 CO, Cl]. 2-(2′,5′-Dichlorophenyl)-1,4-benzoquinone from 1,4-benzoquinone and 2,5-dichloroaniline (6) mp 114-116 °C (acetone). 1H NMR (200 MHz, CDCl ): δ ) 6.78-6.81 (m, 1H), 6.83-6.95 3 (m, 2H), 7.21-7.24 (m, 1H), 7.33-7.43 (m, 2H); 13C NMR (50 MHz, CDCl3): δ ) 130.55, 130.70, 130.99, 131.55, 132.88, 133.83, 135.33, 136.55, 136.77, 144.97, 184.56, 186.81; MS m/z (relative intensity) ) 252 (49) [M+], 217 (100), [M+ - Cl], 189 (25) [M+ - CO, Cl], 161 (8) [M+ - 2 CO, Cl], 126 (12) [M+ - 2 CO, 2 Cl]. 2-(3′,4′-Dichlorophenyl)-1,4-benzoquinone from 1,4-benzoquinone and 3,4-dichloroaniline (7) mp 163-164 °C (acetone). 1H NMR (200 MHz, CDCl ): δ ) 6.82-6.94 (m, 3H), 7.31 (dd, J 3 ) 8.3, 2.0 Hz, 1H, 6′-ArH), 7.53 (d, J ) 8.3 Hz, 1H, 5′-ArH), 7.61 (d, J ) 2.0 Hz 1H, 2′-ArH); 13C NMR (50 MHz, CDCl3): δ ) 128.41, 130.62, 131.11, 132.47, 133.07, 133.15, 134.78, 136.48, 137.03, 143.69, 185.84, 186.96; MS m/z (relative intensity) ) 252 (52) [M+], 217 (100), [M+ - Cl], 189 (15) [M+ - CO, Cl], 161 (7) [M+ - 2 CO,Cl], 126 (12) [M+ + 2 CO, 2 Cl].

Reactivity of PCB Quinones 2-(3′,5′-Dichlorophenyl)-1,4-benzoquinone from benzoquinone and 3,5-dichloroaniline (8) mp 102-104 °C (acetone). 1H NMR (200 MHz, CDCl3): δ ) 6.82-6.94 (m, 3H), 7.35-7.48 (m, 3H); 13C NMR (50 MHz, CDCl ): δ ) 127.62, 130.00, 133.63, 135.31, 3 135.36, 136.47, 136.99, 143.53, 185.53, 186.82; MS m/z (relative intensity) ) 252 (72) [M+], 217 (40), [M+ - Cl], 189 (8) [M+ CO, Cl], 161 (5) [M+ - 2 CO,Cl], 126 (11) [M+ + 2 CO, 2 Cl]. Determination of Oxidation/Reduction Potentials. All quinone biphenyls (5 mM in DMSO) were diluted to 1 mM solutions with dry DMSO to a final volume of 5 mL. Tetraethylammonium perchlorate (TEAP, 0.1 mM) was added to the solution as a supporting electrolyte. The working electrode was glassy carbon and platinum wire was used as the counter electrode. All peak potentials were measured against a saturated calomel electrode (SCE). The solutions were purged with N2 for 10 min prior to recording the cyclic voltammogram at a sweep rate of 100 mV/s on a BAS 100A Electrochemical Analyzer. Reactivity with Nucleophiles. A. Kinetic analysis of amino acid addition to 2-(4′-chlorophenyl)-1,4-benzoquinone. The rates for selected amino acids were determined following the decrease in absorbance of the 2-(4′-chlorophenyl)-1,4-benzoquinone chromophore at 380 nm ( ) 1430 M-1 cm-1). Reactions, run under pseudo-first-order conditions, were initiated by adding 5 µL of a 25 mM solution of the quinone in DMSO to 995 µL of a buffer solution (12 mM amino acid in 50 mM phosphate, pH 7.4) in a quartz cuvette. The absorbance of each reaction was monitored on a Shimadzu MPS-2000 UV-vis spectrophotometer at 25 °C for 15 min. All plots of ln At versus time were linear, indicating that each reaction followed pseudofirst-order kinetics. Pseudo-first-order rate constants were estimated for each reaction from the slope of the regression lines fit to each plot. Rate constants were normalized to second-order with units of min-1 M-1 after dividing by the amino acid concentration (12 mM). B. Analysis of nucleophilic addition of thiols to 2-(4′-chlorophenyl)-1,4-benzoquinone and subsequent reoxidation of the thiol-hydroquinone adducts. Reactions were initiated under second-order conditions with the addition of 10 µL of 25 mM glutathione or N-acetyl-L-cysteine to 10 µL of 25 mM 2-(4′chlorophenyl)-1,4-benzoquinone in 970 µL of 50 mM phosphate buffer solution. The reaction was monitored over the range of 650 nm to 350 nm. Horseradish peroxidase (5 µL of 0.25 units/ µL) and 0.1 M H2O2 (5 µL) were added to the cuvette and the reaction was monitored over the same wavelength range and recorded. Reaction of 2-(4′-Chlorophenyl)-1,4-benzoquinone (5) with Glutathione (GSH). A procedure was employed similar to that described by Eckert et al. (15). To a solution of 2-(4′chlorophenyl)-1,4-benzoquinone (4.5 mmol), dissolved in 2.5 mL of tetrahydrofuran, 120 mmol of glutathione was added in 10 mL of 0.1 M sodium acetate, pH 9.0. The reaction, which was allowed to run for 5 min at room temperature, was accompanied by a slight color change from orange to light yellow. Purification of the crude product was achieved with a Sephadex G-10 column (1 × 25 cm) using water as the solvent. The glutathione adducts were eluted from the column in two faint but distinct yellow bands. The purity of the fractions from the column was determined by HPLC chromatography (Shimdzu HPLC system fitted with a 3 mm × 25 cm Upchurch C-18 column) and UVVis spectroscopy. The major glutathione conjugate was separated using a linear gradient of 40-100% acetonitrile containing 0.1% trifluoroacetic acid over 30 min at a flow rate of 0.3 mL/ min (Rt ) 11.5 min). 1H NMR spectra were obtained with a Varian Inova 500 MHz spectrometer and are reported in part per million referenced to the residual HDO peak in the spectra. 1H NMR of the major glutathione adduct of 4′-chloro-1,1′-biphenyl-2,5-quinone (500 MHz, D2O, 25 °C) δ ) 2.11 (m, 2H, CH2), 2.46 (t, J ) 7.5 Hz, 2H, CH2), 3.23 (dd, J ) 8.3, 14.3 Hz, 1H, CH2), 3.38 (dd, J ) 5, 14.3 Hz, 1H, CH2), 3.76 (t, J ) 6.5 Hz, 1H, CH), 3.82 (s, 2H, CH2), 4.48 (dd, J ) 5, 8.3 Hz, 1H, CH), 6.81 (d, J ) 2.9 Hz, 1H, CH), 7.01 (d, J ) 2.9 Hz, 1H, CH), 7.48 (m, 4H, CH). 13C NMR

Chem. Res. Toxicol., Vol. 9, No. 3, 1996 625 of the major glutathione adduct of 4′-chloro-1,1′-biphenyl-2,5quinone (125.7 MHz, D2O, 25 °C) δ ) 30.51, 35.79, 46.17, 57.78, 58.42, 122.75, 124.58, 125.38, 133.14, 134.34, 135.26, 137.71, 140.56, 150.84, 153.73, 176.77, 177.10, 178.17, 179.17. Fast atom bombardment (FAB/MS) spectra were determined on a Kratos Concept 1H double focussing mass spectrometer. Reaction of 2-(4′-Chlorophenyl)-1,4-benzoquinone (5) with Glycine. 2-(2′-Chlorophenyl)-1,4-benzoquinone (4.5 mmols), dissolved in 5 mL of tetrahydrofuran, was added dropwise to 2.5 mL of 0.1 M phosphate (pH 9.0) containing 4.5 mmol of glycine. A dark purple color developed immediately. After 1 h the pH of the reaction mixture was adjusted to 7, and the mixture was extracted with diethyl ether. The ether phase was discarded. The aqueous layer, after the pH was adjusted to 2, was extracted again with ether, which efficiently extracted the purple product. The product was washed by extraction into water (pH 7) and into ether (pH 2) two additional times. The product was dried and further purified on a sephadex G-10 column (2 × 55 cm) in methanol. The dark purple product, which eluted in 5 mL fractions (11-25), showed two peaks in reverse-phase HPLC using the conditions described (Rt ) 18.0 min and Rt ) 20.0 min). These were also characterized by 500 MHz NMR. 1H NMR of the major glycine adduct of 4′-chloro-1,1′-biphenyl2,5-quinone (500 MHz, D2O, 10 °C) δ ) 3.88 (s, overlapped with minor isomer, 2H, CH2), 5.41 (d, J ) 3 Hz, 1H, CH), 6.69 (d, J ) 3 Hz, 1H, CH), 7.47 (m, 4H, CH). 13C NMR of the major glycine adduct of 4′-chloro-1,1′-biphenyl-2,5-quinone (125.7 MHz, CD3OD/D2O 3:1, 0 °C) δ 48.29, 98.82, 130.66, 130.85, 132.88, 133.52, 133.84, 137.84, 138.41, 144.21, 150.69, 176.35, 184.43, 188.80. 1H NMR of the minor glycine adduct of 4′-chloro-1,1′-biphenyl2,5-quinone (500 MHz, D2O, 10 °C) δ ) 3.88 (s, overlapped with major isomer, 2H, CH2), 5.44 (s, 1H, CH), 6.70 (s, 1H, CH), 7.44 (m, 4H, CH). Horseradish Peroxidase-Catalyzed Oxidation of 3,4Dichloro-3′4′- and 3,4-Dichloro-2′,5′-dihydroxybiphenyls). Reactions were performed in 100 mM sodium citrate (pH 7.4) at 37 °C under the following conditions: 0.05 mM 3,4-dichloro3′4′-dihydroxybiphenyl or 3,4-dichloro-2′,5′-dihydroxybiphenyl; 2.3 nM HRP; 0.5 mM H2O2; ( 300 units of SOD. The formation of 2-(3′,4′-dichlorophenyl)-1,4-benzoquinone or 4-(3′,4′-dichlorophenyl)-1,2-benzoquinone was monitored by measuring absorbance changes at 365 ( ) 2716 M-1 cm-1) or 323 nm ( ) 5604 M-1 cm-1), respectively. Anaerobic conditions (oxygen exclusion) were achieved by bubbling N2 through the solution for 10 min before the reaction while a constant flow of N2 was blown over the cuvette during the reaction.

Results and Discussion Synthesis. A modified version of the Meerwein arylation was used to synthesize a series of mono- and dichlorophenyl-substituted-1,4-benzoquinones (13). The benzoquinones were recrystallized from acetone in 6070% yields and characterized using 1H NMR, 13C NMR, and GC/MS. Three of these compounds, 4-(2′,5′-dichlorophenyl)-1,4-, 4-(3′,4′-dichlorophenyl)-1,4-, and 4-(3′,5′dichlorophenyl)-1,4-benzoquinone have not been previously reported. The 1,2-benzoquinones, 4-(4′-chlorophenyl)-1,2- and 4-(3′,4′-dichlorophenyl)-1,2-benzoquinone, were synthesized from the corresponding dihydroxybiphenyls using 2,3-dichloro-5,6-dicyano-1,4-benzoquinone as the oxidizing agent. The yields ranged from 20-30%, and attempts to recrystallize these compounds resulted in a loss of product due to polymerization and/ or decomposition (i.e., insoluble black precipitate). The 1,2-benzoquinones were analyzed with GC/MS, but the molecular ion peak was two mass units higher than expected. Apparently, these compounds were reduced on the GC stationary phase, and the eluted compound was the reduced (catechol) species. We derivatized the

626 Chem. Res. Toxicol., Vol. 9, No. 3, 1996

Amaro et al. Table 2. Peak Potentials of Chlorinated Quinone Biphenyls vs a Saturated Calomel Electrode benzoquinones

Ep,c1 (mV)a

Ep,a1 (mV)a

Ep,c2 (mV)b

.(mV)

Ep,a2

4-(4′-chlorophenyl)-1,24-(3′,4′-dichlorophenyl)-1,22-(2′-chlorophenyl)-1,42-(3′-chlorophenyl)-1,42-(4′-chlorophenyl)-1,42-(2′,5′-dichlorophenyl)-1,42-(3′,4′-dichlorophenyl)-1,42-(3′,5′-dichlorophenyl)-1,4-

-262 -228 -405 -384 -392 -366 -362 -380

-185 -160 -315 -317 -327 -293 -293 -311

-1018 -983 -1229 -1136 -1081 -1159 -1090 -1135

-890 -865 -1103 -1061 -1012 -1068 -993 -1059

b

aE 2- b p,c1 and p,a1 - semiquinone + e ) (hydroquinone) . Ep,c2 and p,a2 - quinone + e- ) semiquinone.

Figure 2. UV-visible spectrum of 2-(4′-chlorophenyl)-1,4benzoquinone (A), the reaction product(s) of 2-(4′-chlorophenyl)1,4-benzoquinone with glutathione at pH 7.4 (B), and the reoxidation of the 2-(4′-chlorophenyl)-1,4-benzoquinone thioether with horseradish peroxidase and H2O2 at pH 7.4 (C). Table 1. Rates of Reactivity of 2-(4′-Chlorophenyl)-1,4-benzoquinone with Selected Nucleophiles at pH 7.4 nucleophile L-arginine

glycine L-histidine L-lysine glutathione N-acetyl-L-cysteine

K (min-1 M-1) 0.45 0.62 0.75 0.64 instantaneousa instantaneousa

a Instantaneous reaction. The rate could not be measured under pseudo-first-order or second-order conditions. Results represent means of three experiments.

quinones with ethylenediamine to obtain a stable product (15). The GC showed only one product for each reaction, and a corresponding [M+ + 1] peak was obtained for each compound using electron impact mass spectrometry. The [M+ + 1] peak may be a result of the protonation (i.e., self-chemical ionization) of the basic sites on the derivatives. Kinetics and Absorption Spectral Changes during Amino Acid Addition to PCB Quinones. The quinone 2-(4′-chlorophenyl)-1,4-benzoquinone was selected as a model compound to study the reactivity of PCB quinones with nitrogen nucleophiles. This quinone has a distinct absorption maximum at 380 nm (Figure 2, spectrum A) which facilitated the monitoring of the reactions with glycine, L-arginine, L-histidine, and L-lysine. All reactions were conducted in the presence of a 99-fold excess of the amino acid, and all reactions followed pseudo-first-order kinetics. The rate constants for each reaction are rather small (Table 1) signifying the lack of nucleophilicity of the amino acids at pH 7.4; the protonated form of the amino group(s) will predominate under these conditions. A hypochromic shift of the quinone absorbance at 380 nm was the only significant change observed in the UV-vis spectrum at pH 7.4. When the ratio of amino acid/quinone was reduced to 10:1 and the pH of the reaction was increased to 9.0, the absorbance changes were more pronounced as shown in Figure 3. The reaction of 2-(4′-chlorophenyl)-1,4-benzoquinone and glycine was monitored every 5 min for 45 min. The original quinone absorbance (380 nm) decreased while a concomitant hypsochromic shift from 380

to 340 nm occurred within the first 10 min of the reaction. A new absorbance also appeared at about 500 nm which was attributed to the formation of a new product. The glycine-quinone adduct was isolated as a 4:1 mixture of isomers. There were only two protons present on the quinone ring of both of the isomers. This forces the conclusion that the glycine added 1,4 to the ring and did not form the expected 1,2 imino-quinone addition product. The major isomer was identified as the 1,2,3,5substituted product by the presence of a 3 Hz coupling between the protons on the quinone ring. This is consistent with an aromatic meta coupling between the two protons. The minor product shows no resolvable coupling between the two protons on the quinone ring. This is consistent with a 1,2,4,5-substitution pattern where the two remaining protons are para to each other. On the basis of (1) extraction procedure which would favor a carboxylic acid, (2) NMR data showing ring addition, with a 1,2,3,5- and a 1,2,4,5-substitution patterns for the major and minor products, respectively, (3) quinone carbon chemical shifts, and (4) UV/vis spectra consistent with ortho-iminoquinones, we have assigned an iminoquinone structure to the products. Similar reactions involving both ortho- and para-quinones have also produced iminoquinone products (16-20). Absorption Spectral Changes during the Glutathione-Reductive Addition to PCB Quinones. The nucleophilic addition of the sulfur nucleophile glutathione (GSH) to 2-(4′-chlorophenyl)-1,4-benzoquinone proceeded rapidly and was over within seconds. The spectral changes corresponding to this reaction under aerobic conditions involved a decrease in the absorbance due to 2-(4′-chlorophenyl)-1,4-benzoquinone at 380 nm (Figure 2; spectrum A) and the formation of a new peak at 310 nm (Figure 2, spectrum B). The latter absorption peak reached its maximal intensity within the first minute and was attributed to glutathionyl-2-(4′-chlorophenyl)-1,4hydroquinones. The products from this reaction were analyzed using FAB/MS. The data confirm the presence of monoglultathionyl-hydroquinone adducts with a molecular ion peak at [M - 1 ) 524] (Figure 4). Crude reaction products also showed a molecular ion peak corresponding to diglutathionyl-hydroquinone adduct(s) (data not shown). The products from spectrum B were subsequently treated with HRP/H2O2, and within 15 s the clear solution had acquired an orange color. The absorbance at 310 nm rapidly decreased, and the formation of a new peak was observed at 365 nm (Figure 2, spectrum C); this latter absorption peak is attributed to the oxidation of the glutathionyl-hydroquinones to corresponding glutathionyl-quinone adducts. The reaction with N-acetyl-L-cysteine gave similar spectral changes.

Reactivity of PCB Quinones

Chem. Res. Toxicol., Vol. 9, No. 3, 1996 627

Figure 3. UV-visible spectra depicting the reaction of 2-(4′-chlorophenyl)-1,4-benzoquinone with glycine at pH 9.0. The reaction was monitored every 5 min for 45 min. The concentration of the quinone was 125 µM, while the concentration of glycine was 1250 µM, giving a 1:10 ratio. Arrows (at 340, 380, and 500 nm) indicate the wavelengths at which the greatest change in absorbance was seen. The structures represent tautomers of the two major products of the reaction.

Figure 4. Fast atom bombardment mass spectrum of the 6-glutathionyl-2-(4′-chlorophenyl)-1,4-hydroquinone.

Under reaction conditions optimized for the mono adducts (cf. Materials and Methods), the major product of the reaction of 2-(4′-chlorophenyl)-1,4-benzoquinone with glutathione shows only two protons present on the quinone ring of the major isolated product leading to the conclusion that a 1,4-Michael addition has taken place on the quinone ring. The product exhibits a 2.9 Hz coupling between the two protons on the quinone ring. This is consistent with a 1,2,3,5-substitution pattern where the two protons are meta- to each other. PCB-hydroquinone/glutathionyl adduct(s) formed after 1,4-Michael addition behave chemically similar to other GSH-hydroquinone adducts; these compounds are also susceptible to oxidation and the formation of quinonethioethers (21). The reaction of PCB-derived quinoid metabolites with GSH and the further oxidation of the glutathionyl-hydroquinone adducts may result in a depletion of GSH levels in exposed cells. Alternatively, (PCBquinone)-glutathione conjugates may participate in redox

cycling with formation of reactive oxygen species. Both effects would result in an increased oxidative stress (due to depletion of radical scavengers and/or production of free radicals in exposed cells) with possible damage to cellular macromolecules (i.e., lipids, protein, or DNA) and even cell death. Several studies have reported a reduction in hepatic GSH levels following PCBs administration (22,23). Horseradish Peroxidase-Mediated Quinone Formation in the Absence and Presence of SOD. Chlorinated biphenyls may be hydroxylated to dihydroxy compounds and subsequently oxidized to quinones by various peroxidases present in cells (cf. Introduction). Peroxidases are one-electron transfer enzymes which oxidize catechols and hydroquinones with the abstraction of 1 e- to yield semiquinones (24). Further oxidation to quinones occurs by one of two routes. The semiquinone can disproportionate to give the quinone and hydroquinone (eq 1) or semiquinones can react directly with

2 semiquinone a quinone + hydroquinone

(1)

oxygen (autoxidation), generating both the quinone and superoxide anion radicals (eq 2).

semiquinone + O2 a quinone + O2- + H+

(2)

The oxidation of the 3,4-dichloro-3′,4′-dihydroxybiphenyl and 3,4-dichloro-2′,5′-dihydroxybiphenyl by HRP/ H2O2 was monitored on the UV-vis spectrometer by following the formation of the quinone over a 50 s period (Figures 5 and 6, respectively). The initial experiment was conducted under anaerobic conditions (exclusion of oxygen) to establish a baseline rate of quinone formation.

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Figure 5. Formation of 2-(3′,4′-dichlorophenyl)-1,4-benzoquinone from 3,4-dichloro-2′,5′-dihydroxybiphenyl in the presence and absence of superoxide dismutase and oxygen. The reaction was monitored for 50 s. Results represent means + SD of three experiments.

Figure 6. Formation of 4-(3′,4′-dichlorophenyl)-1,2-benzoquinone from 3,4-dichloro-3′,4′-dihydroxybiphenyl in the presence and absence of superoxide dismutase. The reaction was monitored for 50 s. Results represent means + SD of three experiments.

The experiment was repeated in the presence of atmospheric oxygen, and there was no significant rate change. This indicates that the forward reaction in eq 2 is negligible unless SOD is added. It has been previously reported that the rate of 1,4benzoquinone formation increased when SOD was added (25) while there was no increase in 1,2-benzoquinone formation from catechol in the presence of SOD (26). We found a significant increase in the rate of chlorophenylsubstituted 1,4-benzoquinone formation after SOD was added under aerobic conditions as well as an increase in 1,2-benzoquinone formation using alkyl- and chlorophenyl-substituted catechols as substrates. Apparently alkyl or phenyl substituents on catechols enhance the rate of 1,2-benzoquinone formation. This trend has also been described for alkyl-substituted 1,4-benzoquinones (27). The reduction of oxygen is thermodynamically favorable for both the 1,2-benzoquinones and 1,4-benzoquinones considering the reduction potential of O2/O2•- ) -179 mV at neutral pH (28) and the Ey2 of the Q/Q•- couple for the 4-(3′,4′-dichlorophenyl)-1,2-benzoquinone and the 2-(3′,4′-dichlorophenyl)1,4-benzoquinone are -924 and -1042 mV, respectively. However, the rate of 1,4benzoquinone formation was significantly increased, whereas only a minor increase in 1,2-benzoquinone formation was observed in the presence of SOD. If the equilibrium in eq 1 favors disproportionation versus comproportionation, then the overall rate should not be significantly altered once SOD is added. This could explain why the 1,2-benzoquinone formation was not drastically altered during the first 30 s of the reaction (Figure 6). Thus, 1,2-benzoquinones might undergo disproportionation more readily, whereas comproportionation of the hydroquinone and quinone is favored for the 1,4-benzoquinones. In separate experiments we have observed that 1,4and 1,2-quinones (HRP/H2O2 generated) participate in a redox cycle with the production of O2-. The 1,4-benzoquinones exhibited the highest rate of reduction of acetylated cytochrome c, whereas the 1,2, benzoquinones showed only a slight or no redox activity.2 This would agree with the fact that 1,2-benzoquinones do not readily react with O2 while 1,4-benzoquinones can react directly with O2, generating both the quinone and superoxide anion radicals.

In conclusion, these studies demonstrate that PCB quinones react slowly with nitrogen nucleophiles, such as glycine, L-arginine, L-histidine, and L-lysine, at physiological pH. The sulfur nucleophiles, N-acetyl-L-cysteine and glutathione, react instantaneously to form the corresponding hydroquinone adducts. These adducts may be reoxidized and add a second moiety. Through these mechanisms, PCB metabolites may bind to cellular macromolecules and cause GSH depletion, oxidative stress, and other quinone-mediated toxicities.

2

Unpublished results from our laboratory.

Acknowledgment. A.R.A. and G.G.O. have contributed equally to this project, and both should be considered as first authors. This work is supported by NIH Grant CA 57423. A.R.A. and G.G.O. are supported by NIEHS Training Grant ES 07266. The authors thank Dr. A. Daniel Jones from the Facility of Advanced Instrumentation at the University of California, Davis for his helpful comments and suggestions, Dr. Jan Pyrek of the University’s Life Sciences Mass Spectrometry facility for the mass spectral analyses, Dr. John P. Selegue, Department of Chemistry, for helpful discussions, and Wendell Neeley, Amanda Varner, Timothy Twaroski, and Chris Girard for technical assistance.

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