Formation of Quinonoid-Derived Protein Adducts in the Liver and

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Formation of Quinonoid-Derived Protein Adducts in the Liver and Brain of Sprague-Dawley Rats Treated with 2,2′,5,5′-Tetrachlorobiphenyl Po-Hsiung Lin,† Ramiah Sangaiah, Asoka Ranasinghe, Patricia B. Upton, David K. La, Avram Gold, and James A. Swenberg* Department of Environmental Sciences and Engineering, University of North Carolina, Chapel Hill, North Carolina 27599-7400 Received February 14, 2000

A possible role for metabolic activation of 2,2′,5,5′-tetrachlorobiphenyl (TCB) to quinonoid metabolites was investigated in vitro in rat liver microsomes and in vivo in male SpragueDawley rats. Incubation of TCB with phenobarbital-induced rat liver microsomes resulted in metabolism of TCB to 3-hydroxy-TCB (3-OH-TCB) and 3,4-dihydroxy-TCB (3,4-diOH-TCB), which were further oxidized to form a reactive intermediate that bound to liver proteins. The predominant species observed in the Raney nickel assay for cysteinyl adducts was identified as 3,4-diOH-TCB, consistent with an adduct having the structure 5-cysteinyl-3,6-dichloro-4(2′,5′-dichlorophenyl)-1,2-benzoquinone. This adduct may arise via the Michael addition of the sulfhydryl group of cysteine to 3,6-dichloro-4-(2′,5′-dichlorophenyl)-1,2-benzoquinone (Cl4PhBQ). Metabolism of 3-OH-TCB by phenobarbital-induced microsomes in the presence of either NADPH or cumene hydroperoxide as a cofactor resulted in the formation of adducts. Dosedependent formation of cysteinyl adducts was observed in liver cytosolic protein from rats treated with a single dose of TCB (0-200 mg/kg) by gavage. By regression analysis, the TCB adducts decayed with a half-life of 2.03 ( 0.131 days (mean ( SE), which is ∼2.5-fold shorter than the endogenous half-life for liver cytosolic protein in rat liver, suggesting adduct instability. Saturable formation of TCB adducts was observed in liver cytosolic protein of rats receiving multiple doses of TCB over 5 days. The levels of Cl4PhBQ-derived adducts were 2.1-fold greater than the estimated steady-state levels predicted by the single-dose treatment [97.7 ( 13.2 vs 45.7 ( 3.73 (pmol/g)/(mg/kg of body weight)], suggesting induction of metabolism. A single cysteinyl adduct, inferred to be 5-cysteinyl-3,6-dichloro-4-(2′,5′-dichlorophenyl)-1,2-benzoquinone, was detected in brain cytosolic protein of rats treated with multiple doses of TCB with levels of 15.2 (pmol/g)/(mg/kg of body weight). Implied involvement of a reactive quinone in the liver and brain of TCB-treated rats supports the idea that quinonoid metabolites may be important contributors to PCB-derived oxidative damage to genomic DNA.

Introduction Polychlorinated biphenyls (PCBs)1 are known environmental pollutants. To date, evidence indicates that PCBs are rodent hepatocarcinogens (1, 2). Evidence suggests that induction of the aryl hydrocarbon (Ah)-receptor signal transduction pathway by coplanar PCB congeners followed by metabolism, oxidative events, and tumor promotion may be associated with the development of PCB-induced liver tumors (1, 3-5). Recently, the metabolic studies of lower chlorinated PCBs (mono- and * To whom correspondence should be addressed. Telephone: (919) 966-6142. Fax: (919) 966-6123. † Current address: Department of Environmental Engineering, National Chung-Hsing University, Taichung, Taiwan. 1 Abbreviations: 2′,5,5′-Cl PhBQ-Y , 2-cysteinyl- or 2,6-dicysteinyl3 n 2′,5,5′-Cl3PhBQ adduct (n ) 1 or 2); 2,2′,5-Cl3PhBQ-Yn, 5-cysteinylor 5,6-dicysteinyl-2,2′,5′-Cl3PhBQ adduct (n ) 1 or 2); 2,5-Cl2PhBQYn, 2′,5′-dichloro-2,5-dicysteinyl-3,4-dihydroxybiphenyl or 2′,5′-dichloro3,4-dihydroxy-2,5,6-tricysteinylbiphenyl adduct (n ) 2 or 3); 3,4,6-[2H3]3′,4′-diOH-TCB, 3,4,6-[2H3]-3′,4′-dihydroxy-2,2′,5,5′-tetrachlorobiphenyl; 3,4-diOH-TCB, 2,2′,5,5′-tetrachloro-3,4-dihydroxybiphenyl; 3-OH-TCB, 2,2′,5,5′-tetrachloro-3-hydroxybiphenyl; Ah, aryl hydrocarbon; Cl4PhBQ, 3,6-dichloro-4-(2′,5′-dichlorophenyl)-1,2-benzoquinone; Cl4PhBQY, 5-cysteinyl-3,6-dichloro-4-(2′,5′-dichlorophenyl)-1,2-benzoquinone; PCBs, polychlorinated biphenyls; TCB, 2,2′,5,5′-tetrachlorobiphenyl.

dichlorobiphenyls) suggested the formation of redoxactive quinonoids as sources of oxidative stress (4, 6). The role of quinonoid metabolites in carcinogenesis by PCBs has not, however, been established. 2,2′,5,5′-Tetrachlorobiphenyl (TCB), a prototypical orthosubstituted PCB congener, is one of the major components of commercially available PCB mixtures. TCB causes various effects in rodents, including neurotoxicity and liver tumor promotion (7, 8). The toxic pathways arising from TCB exposure are believed to be independent of the Ah receptor. TCB is readily metabolized in rodents, with a half-life of 2-3 days in mice (9). Metabolism of TCB in mammals is primarily mediated by the phenobarbital-inducible forms of liver microsomal enzymes, predominantly the cytochrome P450 2B subfamily, to 2,2′,5,5′-tetrachloro-3-hydroxybiphenyl (3-OHTCB), which is subsequently oxidized to 2,2′,5,5′-tetrachloro-3,4-dihydroxybiphenyl (3,4-diOH-TCB) (7, 10, 11). Chlorinated hydroquinones and catechols have been detected as the reactive intermediates of PCBs (12). Previous investigations into the metabolism of the lower chlorinated PCB, 4-chlorobiphenyl, pointed to the production of hydroquinones and catechols, which are sub-

10.1021/tx000030f CCC: $19.00 © 2000 American Chemical Society Published on Web 07/27/2000

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sequently oxidized to semiquinones and quinones (6). PCB-derived quinones and semiquinones are known to bind covalently to cellular macromolecules (4, 13). Covalent binding of TCB to proteins and RNA following activation by monkey liver microsomes has been reported (14); the adducts, however, have not been characterized. Whether the TCB-derived reactive semiquinone or quinone species could account for the covalent binding to macromolecules has yet to be investigated. We used TCB as a model compound to examine the hypothesis that quinone metabolites are reactive species following PCB metabolism. A procedure to characterize and quantitate the cysteinyl adducts of TCB quinone in proteins by the Raney nickel technique (15-19) has been developed. This protein adduct assay has been used to investigate the formation of chlorinated quinonoid adducts following metabolism of TCB in liver microsomes in vitro and in the liver and brain of rats receiving both single and multiple doses of TCB. The stability and pseudo-steady-state levels of TCB quinone-protein adducts in livers of rats treated with a single dose of TCB have been compared to results obtained in the multipledose study.

Materials and Methods Chemicals. Procedures for the synthesis of TCB and metabolites 3-OH-TCB, 3,4,6-[2H3]-3′,4′-dihydroxy-2,2′5,5′-tetrachlorobiphenyl (3,4,6-[2H3]-3′,4′-diOH-TCB), 3,4-dihydroxy2,2′,5,5′-tetrachlorobiphenyl, 3,4-dihydroxy-2,2′,5-trichlorobiphenyl, 3,4-dihydroxy-2′,2,5′-trichlorobiphenyl, 2,5-dichloro-3′,4′dihydroxybiphenyl, and 4-phenyl-1,2-benzoquinone are described elsewhere (R. Sangaiah, P. H. Lin, A. Gold, J. A. Swenberg, manuscript in preparation). Briefly, TCB (99%, GC/ MS) was prepared from 2,2′,5,5′-tetrachlorobenzidine by the method of Hutzinger et al. (37). 3-OH-TCB (95%, GC/MS) was synthesized by coupling 2,5-dichlorophenol with 2,5-dichloroaniline by the method of Gardner et al. (38) and was further purified by thin-layer chromatography and high-pressure liquid chromatography. Dimethoxy derivatives of biphenyls were synthesized by the Cadogan diaryl coupling of a chlorinated aniline with a chlorinated anisole. Dihydroxy derivatives of biphenyls (>90%, GC/MS) were synthesized by demethylation of the corresponding dimethoxy derivatives as described by Bauer et al. (20) and Safe et al. (21). Liver microsomes derived from male Sprague-Dawley rats treated with corn oil or phenobarbital were purchased from In Vitro Technologies, Inc. (Baltimore, MD). All other chemicals were purchased from Sigma, Aldrich, or Fisher and used as received unless stated otherwise. Protein Adduct Analysis. All cysteinyl adducts were assayed by the Raney nickel procedure described by Lin et al. (18), with the modifications shown in Figure 1. Isolated protein (0.510 mg) was dissolved in Bis-Tris buffer (0.15 M, pH 7.0) containing ascorbic acid (0.12 M), protein-bound internal standards (including 200 µg of 3,4,6-[2H3]-3′,4′-diOH-TCB, and 20 µg of 4-phenylcatechol), and protease (Pronase E; 20% w/w) in a final volume of 2 mL. The medium was incubated at 37 °C for 12 h with constant shaking. The protein digest was cooled in an ice bath, and 0.35 mL of ascorbic acid (0.9 M) was added. After the pH was adjusted to 3 with H2SO4, the medium was extracted six times with 2 volumes of MTBE to remove unbound contaminants. Organic solvent was removed under a stream of nitrogen. After the addition of 2,5-dichloro-2′,3′-dihydroxybiphenyl (200 pg) and Raney nickel (80 mg/mL), the medium (pH 3-4) was vigorously shaken at room temperature in the presence of ascorbic acid for 10 min, cleaving sulfur-bound adducts to yield chlorinated catechols. The adducts were isolated by extraction with 2 × 2 volumes of MTBE. The organic extracts were combined, reduced in volume under a stream of nitrogen,

Figure 1. Scheme of the Raney nickel assay for measuring TCB-derived quinone adducts. dried over anhydrous sodium sulfate, and derivatized with heptafluorobutyrylimidazole chloride (HFB). The HFB derivatives were dissolved in hexane, washed with deionized water, and analyzed by GC/HRMS. In this procedure, 4-phenyl-1,2benzoquinone-modified liver cytosolic protein was used as a dual protein-bound internal standard, and 2,5-dichloro-2′,3′-dihydroxybiphenyl served as an unbound internal standard. GC/MS Analysis. Analytes were quantitated on an HP 5890 gas chromatograph interfaced to a VG70-250 SEQ hybrid mass spectrometer in the NCI mode. One microliter of the solution to be analyzed was injected onto an EC-5 fused silica column (30 m × 0.32 mm × 1 µm, Alltech). The helium head pressure was maintained at 10 psi. The injection port and the initial column temperatures were 250 and 75 °C, respectively. After 2 min, the column temperature was ramped to 300 °C at 10 °C/ min and held for 3 min. The mass resolving power was set at 10 K, and the ion source temperature was 250 °C. Methane (3 × 10-5 mbar) served as the reagent gas. The emission current was 0.5 mA. Fragment ions of HFB-derivatized analytes (loss of one HFB group from molecular ions) were monitored by GC/ HRMS in the NCI mode. The following ions were selectively monitored: [2H3]-3′,4′-diOH-TCB (m/z 523.8929), 3,4-diOH-TCB (m/z 518.8774), 3,4-dihydroxy-2′,2,5′-trichlorobiphenyl and 3,4dihydroxy-2′,5,5′-trichlorobiphenyl (m/z 482.9193), and 2,5dichloro-2′,3′-dihydroxybiphenyl and 2,5-dichloro-3′,4′-dihydroxybiphenyl (m/z 448.9582) and 4-phenylcatechol (m/z 381.0362). The limit of detection for both quinone and semiquinone adducts was estimated to be 10 pmol/g of protein when a 10 mg portion of protein was assayed. All data are expressed as means ( the standard error. For the characterization of products following Raney nickel cleavage of adducted proteins, samples of liver cytosolic proteins obtained from male Sprague-Dawley rats treated with a single

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dose of TCB (10 mg each of six groups of three rats treated with TCB at 50, 100, and 200 mg/kg of body weight by gavage) were assayed by the Raney nickel procedure as described above to cleave the adducts in the reduced form as catechol derivatives in the presence of ascorbic acid. The cleaved adducts were derivatized by HFB. The HFB derivatives of these 18 samples were combined and analyzed by GC/MS in the full scan mode. Mass spectrometer resolution was set to 1K, and mass spectra were acquired by scanning from m/z 100 f 900 in the EI mode and from m/z 100 f 750 in the NCI mode. Standard Curves for Quantitation of Protein Adducts. (1) Preparation of 4-Phenylcatechol Protein-Bound Internal Standards. An internal standard was prepared by reacting 4-phenyl-1,2-benzoquinone (1 mM in acetone) with the rat liver cytosolic fraction in PBS (50 mM, pH 7.4). After incubation for 15 min at 37 °C, the reaction was terminated by placing the mixture in an ice bath. Liver cytosolic proteins were isolated from incubates as described below. 4-Phenylcatechol protein-bound internal standards were assayed by the Raney nickel procedure as described above with the addition of synthetic catechol standards after protein digestion. Following treatment with Raney nickel (to liberate 4-phenylcatechol), the adducts and synthetic catechol standards were isolated by extraction with MTBE and derivatized with HFB. The HFB derivatives were analyzed by GC/HRMS. The peak area ratios of added catechol to 4-phenylcatechol.were plotted against the quantity of added catechols. Correlation coefficients of the standard curves (r2) were between 0.966 and 0.994. (2) Preparation of 3,4,6-[2H3]-3′,4′-diOH-TCB ProteinBound Internal Standard. 3,4,6-[2H3]-3′,4′-diOH-TCB proteinbound internal standard was prepared by incubating liver cytosolic proteins from untreated male Sprague-Dawley rats with the 3,4,6-[2H3]-3′,4′-diOH-TCB (5 mg in 100 µL of acetone), Tris-HCl (50 mM, pH 9), horseradish peroxidase (5 units/mL), and hydrogen peroxide (1 mM) at 37 °C for 1 h. The reaction was terminated by placing the mixture in an ice bath. Liver cytosolic proteins were isolated from incubates as described below. 3,4,6-[2H3]-3′,4′-diOH-TCB protein-bound internal standard was assayed by the Raney nickel procedure as described above with the addition of 4-phenylcatechol protein-bound internal standards after protein digestion. Following treatment with Raney nickel (to liberate 4-phenylcatechol), the adducts and synthetic catechol standards were isolated by extraction with MTBE and derivatized with HFB. The HFB derivatives were analyzed by GC/HRMS. The peak area ratio of protein-bound 3,4,6-[2H3]-3′,4′-diOH-TCB to protein-bound 4-phenylcatechol standard was determined and the 3,4,6-[2H3]-3′,4′-diOH-TCB protein-bound standard quantitated from the standard curve developed for 3,4-diOH-TCB by interpolation. (3) Quantitation of Protein-Bound Metabolites. Standard curves (0.991 < r2 < 0.999) were prepared from putative metabolites over a 0-10 ng range by extracting the unbound synthetic standards from solutions similar in composition to experimental samples and using 3,4,6-[2H3]-3′,4′-diOH-TCBbound liver cytosolic protein as the protein-bound internal standard. Peak area ratios of the HFB-derivatized analytes and [2H3]-3′,4′-diOH-TCB added as internal standard were used in deriving standard curves. Standard curves for each adduct were prepared by plotting the quantity of added metabolite against peak area ratio of metabolite and 3,4,6-[2H3]-3′,4′-diOH-TCB protein-bound internal standard. Formation of TCB-Derived Protein Adducts in Liver Cytosolic Proteins in Vitro. The formation of protein adducts of 3,6-dichloro-4-(2′,5′-dichlorophenyl)-1,2-benzoquinone (Cl4PhBQ) in vitro was investigated by incubating 3,4-diOH-TCB with liver cytosol derived from untreated male Sprague-Dawley rats in the presence of horseradish peroxidase (HRP) and H2O2 as described above. In a separate experiment, TCB and 3-OHTCB were metabolically activated by a microsomal activation system to form adducts with cysteinyl residues in liver cytosolic

Lin et al. protein. The microsomal incubation system contained 50 mM phosphate-buffered saline solution (pH 7.4), untreated rat liver cytosolic fraction (15 mg of protein/mL), 16 mM KCl, 4 mM MgCl2, and 2 mg of microsomal protein/mL. After a 5 min preincubation at 37 °C, 1 mM NADPH, 50 µM TCB, and 3-OHTCB in dimethyl sulfoxide were added to the samples which were incubated for 30 min. HRP (5 units/mL) and H2O2 (1 mM) were added to some of the samples and allowed to react for an additional 30 min. The reaction was terminated by chilling in an ice bath and addition of ascorbic acid (10 mM). Liver cytosolic proteins were isolated as described below. Administration of TCB to Animals. Male Sprague-Dawley rats (14 weeks old) were purchased from Charles River Breeding Laboratory (Raleigh, NC). For the single-dose study, six groups of three rats were treated with TCB at 50, 100, of 200 mg/kg of body weight by gavage. Animals were sacrificed 24 or 72 h after administration. Control animals were given corn oil (5 mL/kg of body weight) and sacrificed 24 h after administration. For the multiple-dose study, four groups of four rats were given a daily dose of TCB at 50, 100, and 200 mg/kg of body weight for 5 days by gavage. Control animals were given corn oil only. The animals were sacrificed 24 h after the final administration. Immediately following collection of blood by cardiac puncture, livers (perfused with phosphate-buffered saline) and brains were excised and frozen at -80 °C until further processing. Isolation of Cytosolic Protein from Liver and Brain. Liver and brain tissue (forebrain and midbrain) were homogenized in ice-cold PBS (pH 7.4) and centrifuged at 1000g and 4 °C for 15 min to pellet the nuclear fraction. The 1000g supernatant was centrifuged at 105000g and 4 °C for 70 min to pellet the mitochondrial and microsomal fractions. The 105000g supernatant was dialyzed (molecular mass cutoff of 6000-8000 Da) against 3500 mL of deionized water containing 1 mM ascorbic acid at 4 °C with four changes of water. Liver and brain cytosolic proteins were recovered via lyophilization. Statistical Analysis. All data are expressed as estimated means ( SEs. The production of liver protein adducts per unit dose of TCB was estimated by the least-squares regression analysis. The coefficients of determination (r2) for the leastsquares regression analysis of the rates of formation of adducts (r2) were between 0.697 and 0.949.

Results Characterization of Cysteinyl Adducts of TCB. Formation and structure of cysteinyl adducts of TCB were investigated in liver cytosolic protein from SpragueDawley rats treated with a single oral dose of TCB (one each of six groups of three rats treated with TCB at 50, 100, and 200 mg/kg of body weight by gavage). Cysteinyl adducts were released by catalytic cleavage with Raney nickel, the resulting catechols derivatized by HFB and analyzed by GC/EI/MS and GC/NCI/MS. In an effort to establish structures of the catechols released by Raney nickel, analytical standards of several putative chlorinated catechol analytes were synthesized (22) for comparison of mass spectra and retention times. Figures 2-4 demonstrate that the mass spectra of adducts cleaved from proteins match those of the analytical standards 3,4diOH-TCB, 3,4-dihydroxy-2,2′,5′-trichlorobiphenyl, and 2,5-dichloro-3′,4′-dihydroxybiphenyl, respectively. Note that the mass spectrum for 3,4-dihydroxy-2′,5,5′-trichlorobiphenyl was not shown. These catechols are consistent with the formation of cysteinyl adducts of TCB quinone via reaction with protein and/or nonprotein thiols. The presence of 3,4-diOH-TCB as a cleavage product suggests 5-cysteinyl-3,6-dichloro-4-(2′,5′-dichlorophenyl)1,2-benzoquinone (Cl4PhBQ-Y, where Y represents protein and/or nonprotein thiol) as the structure of the adduct, consistent with generation of 3,6-dichloro-4-(2′,5′-


Figure 2. Mass spectra of the HFB derivative of 3,4-diOHTCB derived from (A) an authentic analytical standard analyzed by GC/MS/EI in the full scan mode (m/z 100-900), (B) an authentic analytical standard analyzed by GC/MS/NCI in the full scan mode (m/z 100-750), and (C) a sample of liver cytosolic proteins obtained from male Sprague-Dawley rats treated with a single dose of TCB (one each of six groups of three rats treated with TCB at 50, 100, and 200 mg/kg of body weight by gavage) analyzed under NCI mode. Liver proteins were reacted with Raney nickel to cleave the adducts in the reduced form as catechol derivatives in the presence of ascorbic acid. The cleaved adducts were derivatized by heptafluorobutyrylimidazole chloride (HFB) and analyzed by GC/MS/NCI in the full scan mode (m/z 100-750).

dichlorophenyl)-1,2-benzoquinone (Cl4PhBQ) which reacts with sulfhydryl groups by Michael addition. 3,4Dihydroxy-2,2′,5′-trichlorobiphenyl in the protein digests suggests Raney nickel cleavage of 5-cysteinyl- or 5,6dicysteinyl-2,2′,5′-Cl3PhBQ adducts (2,2′,5-Cl3PhBQ-Yn, n ) 1 or 2). 3,4-Dihydroxy-2′,5,5′-trichlorobiphenyl could be derived from 2-cysteinyl or 2,6-dicysteinyl-2′,5,5′-Cl3PhBQ adducts (2′,5,5′-Cl3PhBQ-Yn, n ) 1 or 2) and 2,5dichloro-3′,4′-dihydroxybiphenyl from 2′,5′-dichloro-2,5dicysteinyl-3,4-dihydroxybiphenyl or 2′,5′-dichloro-3,4dihydroxy-2,5,6-tricysteinylbiphenyl adducts (2,5-Cl2PhBQYn, n ) 2 or 3). Thus, multi-sulfhydryl-substituted adducts of Cl4PhBQ can exist in liver proteins. A proposed pathway for the formation of Cl4PhBQ-derived cysteinyl adducts is given in Figure 5. Formation of TCB-Derived Adducts with Liver Proteins in Vitro. Activation of TCB and 3-OH-TCB was investigated in the presence of NADPH and/or cumene hydroperoxide or in the presence of HRP and H2O2 using Sprague-Dawley rat liver microsomes (unin-


Figure 3. Mass spectra of the 3,4-dihydroxy-2,2′,5′-trichlorobiphenyl-HFB derivative derived from (A) an authentic analytical standard analyzed by GC/MS/EI in the full scan mode (m/z 100-900), (B) an authentic analytical standard analyzed by GC/ MS/NCI in the full scan mode (m/z 100-750), and (C) a sample of liver cytosolic proteins obtained from male Sprague-Dawley rats treated with a single dose of TCB (one each of six groups of three rats treated with TCB at 50, 100, and 200 mg/kg of body weight by gavage). Liver proteins were reacted with Raney nickel to cleave the adducts in the reduced form as catechol derivatives in the presence of ascorbic acid. The cleaved adducts were derivatized by heptafluorobutyrylimidazole chloride (HFB) and analyzed by GC/MS/NCI in the full scan mode (m/z 100750).

duced and phenobarbital-induced) and untreated liver cytosol. Table 2 summarizes adduct levels. Incubation of TCB (50 µM) with uninduced rat liver microsomes and NADPH cofactor resulted in no detectable adducts in liver cytosolic protein. In the presence of NADPH cofactor, adducts were generated in relative amounts: Cl4PhBQ-Y > 2,2′,5-Cl3PhBQ-Yn > 2,5,5′-Cl3PhBQ-Yn (Figure 6A). The presence of HRP and cumene hydroperoxide, or HRP and H2O2, did not strongly affect adduct levels or profiles. No adducts were detected when cumene hydroperoxide alone was added to phenobarbitalinduced microsomes. The formation of adducts by phenobarbital-induced microsomes in the presence of NADPH suggests that the cytochrome P450 2B subfamily is responsible for the activation of TCB. With 3-OH-TCB (50 µM) as the substrate, phenobarbital-induced liver microsomes with NADPH cofactor generated four cysteinyl adducts (Figure 6B). Levels of these adducts were in the following order: Cl4PhBQ-Y > 2,2′,5-Cl3PhBQ-Yn > 2,5-Cl2PhBQ-Yn > 2,5,5′-Cl3PhBQ-Yn. Addition of HRP and H2O2 to the microsomal

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Figure 5. Proposed pathway for the formation of cysteinyl adducts of Cl4PhBQ in liver proteins. Note that Y-SH represents proteins and/or nonprotein thiols. Table 2. Formation of Cysteinyl Adducts of Quinonoid Metabolites of TCB in Liver Cytosolic Proteins Following Microsome- and Peroxidase-Mediated Activation of TCB and 3-OH-TCBa

Figure 4. Mass spectra of the 2′,5′-dichloro-3,4-dihydroxybiphenyl-HFB derivative derived from (A) an authentic analytical standard analyzed by GC/MS/EI in the full scan mode (m/z 100900), (B) an authentic analytical standard analyzed by GC/MS/ NCI in the full scan mode (m/z 100-750), and (C) a sample of liver cytosolic proteins obtained from male Sprague-Dawley rats treated with a single dose of TCB (one each of six groups of three rats treated with TCB at 50, 100, and 200 mg/kg of body weight by gavage). Liver proteins were reacted with Raney nickel to cleave the adducts in the reduced form as catechol derivatives in the presence of ascorbic acid. The cleaved adducts were derivatized by heptafluorobutyrylimidazole chloride (HFB) and analyzed by GC/MS/NCI in the full scan mode (m/z 100-750). Table 1. Cysteinyl Adducts in Liver Cytosolic Proteins Derived from 3,4,6-[2H3]-3′,4′-Dihydroxy-2,2′5,5′-tetrachlorobiphenyl Protein-Bound Internal Standarda adduct

adduct level (SE) (nmol/g)

Cl4PhBQ-Y 2,2′,5-Cl3PhBQ-Yn 2,5,5′-Cl3PhBQ-Yn 2,5-Cl2PhBQ-Yn total adducts

66.1 (3.02) 3.94 (0.143) 0.833 (0.063) NDb 70.9 (3.03

a Synthesized by incubating liver cytosolic proteins derived from untreated male Sprague-Dawley rats with 3,4,6-[2H3]-2,2′,5,5′tetrachloro-3′,4′-dihydroxybiphenyl (5 mg in 100 µL of acetone), Tris-HCl (50 mM, pH 9), horseradish peroxidase (5 units/mL), and hydrogen peroxide (1 mM) at 37 °C for 1 h. Proteins were isolated as described in Materials and Methods. b ND, not detected.

mixture resulted in a 20% reduction of the adduct levels. With cumene hydroperoxide cofactor alone, all Cl4PhBQderived adducts were observed in liver cytosolic proteins in the following relative quantities: 2,2′,5-Cl3PhBQ-Yn > Cl4PhBQ-Y > 2,5-Cl2PhBQ-Yn > 2,5,5′-Cl3PhBQ-Yn.

conditions DMSO PB-induced microsomes TCB (50 µM) noninduced microsomes PB-induced microsomes 3-OH-TCB (50 µM) PB-induced microsomes

adduct levelb (nmol/g of protein)

NADPH

NDc

NADPH

ND

NADPH NADPH, HRP, and H2O2

2.97 (0.502) 2.53 (0.460)

NADPH NADPH, HRP, and H2O2 cumene hydroperoxide

47.7 (7.46) 30.6 (5.50) 25.0 (3.01)

a The microsomal incubation system contained 50 mM phosphatebuffered saline (pH 7.4), untreated rat liver cytosolic fraction (15 mg of proteins/mL), 16 mM KCl, 4 mM MgCl2, and 2 mg of microsomal proteins/mL. After a 5 min preincubation at 37 °C, 1 mM NADPH or cumene hydroperoxide, 50 µM TCB, and 3-OHTCB (prepared in dimethyl sulfoxide) were added to the samples, and allowed to incubate for 30 min. HRP (5 units/mL) and H2O2 (1 mM) were added to some of the samples and allowed to continue for an additional 30 min. The reactions were terminated by chilling in an ice bath and with the addition of ascorbic acid (10 mM). Liver cytosolic proteins were recovered as described in Materials and Methods. b Mean ( standard error (n ) 3). c ND, not detected.

Cumene hydroperoxide thus appears to serve as a cofactor for the liver microsomal cytochrome P450 2B isozymes in activating 3-OH-TCB to Cl4PhBQ. Formation of TCB-Derived Adducts in Liver Protein in Vivo. The formation of adducts in liver cytosolic protein was investigated in male SpragueDawley rats following administration of TCB at a single dose (0-200 mg/kg of body weight) or in multiple doses (0-200 mg/kg of body weight, one dose per day, 5 days).


Figure 6. Formation of cysteinyl adducts of TCB-derived quinone in liver cytosolic proteins following incubation with (A) TCB (50 µM) or (B) 3-OH-TCB (50 µM) in the presence of PBinduced Sprague-Dawley rat liver microsomes (1 mg/mL) and NADPH (1 mM) at 37 °C for 30 min followed by incubation with horseradish peroxidase (10 units) with H2O2 (1 mM) for an additional 30 min.


Figure 8. Formation of Cl4PhBQ-derived cysteinyl adducts (A) 2,2′,5-Cl3PhBQ-Yn and (B) 2,5,5′-Cl3PhBQ-Yn in liver cytosolic proteins following a single oral administration of TCB to Sprague-Dawley rats. Note that mean values and standard errors are shown for three animals per group. Table 3. Regression Analysis of Cysteinyl Adducts (Mean ( SE) [(Picomoles per Gram)/(Milligram per Kilogram of Body Weight)] in Liver Cytosolic Proteins from Male Sprague-Dawley Rats Treated with a Single Oral Dose of TCB (0-200 mg/kg of Body Weight) and Sacrificed 24 and 72 h after Treatment adduct

24 h

72 h


13.3 (0.763) 1.45 (0.130) 0.243 (0.024) 0.576 (0.066)b 15.6 (0.777)

6.62 (0.319) 0.766 (0.050)a 0.096 (0.013) 0.399 (0.034)b 7.88 (0.325)

a Estimated by least-squares regression analysis at PCP doses between 0 and 100 mg/kg of body weight. b Estimated as the mean of the adduct levels vs the corresponding administered dose of TCB.

Figure 7. Formation of Cl4PhBQ-derived cysteinyl adducts (A) Cl4PhBQ-Y and (B) 2,5-Cl2PhBQ-Yn in liver cytosolic proteins following a single oral administration of TCB to Sprague-Dawley rats. Note that mean values and standard errors are shown for three animals per group.

Animals were sacrificed after 24 and 72 h in the singledose study and after 24 h following the final treatment in the multiple-dose study. (1) Single-Dose Study. The dependence of adduct levels in liver protein on TCB dose is shown in Figures 7 and 8. Linear relationships were observed over the dose range of 0-200 mg/kg of body weight for most of the Cl4PhBQ-derived adducts of liver cytosolic proteins. Nonlinear relationships were observed in the single-dose

study for 2,5-Cl2PhBQ-Yn and 2,2′,5-Cl3PhBQ-Yn at 72 h after administration, with a less than proportional production of adducts observed. Similar to the in vitro activation of 3-OH-TCB, adduct levels were ordered as follows: Cl4PhBQ-Y > 2,2′,5-Cl3PhBQ-Yn > 2,5-Cl2PhBQYn > 2,5,5′-Cl3PhBQ-Yn. Results of least-squares regression of adduct levels versus TCB dose over linear ranges are given in Table 3. (2) Prediction of the Steady-State Levels of TCBDerived Quinone Adducts in the Liver. Following a single dose of TCB, levels of all Cl4PhBQ-derived adducts were 2-fold greater at 24 h than at 72 h. If pseudo-firstorder elimination of total Cl4PhBQ-derived adducts ([RY]) after a single administration of TCB is assumed, the concentration of adducts with time is given by the relationship

d[RY] ) -kae[RY] dt where kae is the pseudo-first-order elimination rate constant of all Cl4PhBQ-derived adducts. A value for kae of 0.341 ( 0.022 day-1 with a corresponding half-life of

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Table 4. Estimates of the Rates of Production of Cysteinyl Adducts (Means ( SE) [(Picomoles per Gram)/ (Milligrams per Kilogram of Body Weight)] in Liver and Brain Cytosolic Proteins from Male Sprague-Dawley Rats Treated with Multiple Oral Doses of TCB (0-200 mg/kg of Body Weight; One Dose per Day for 5 days) and Sacrificed 24 h after Treatmenta adduct

liver

brain


82.7 (13.1) 8.91 (1.16) 2.53 (0.429) 3.53 (0.473) 97.7 (13.2)

15.2 (1.44) NDb ND ND 15.2 (1.44)

a Estimated as the mean of the adduct levels vs the corresponding administered dosage of TCB. b ND, not detected.

2.03 ( 0.131 days was estimated. The steady-state level A [(nanomoles per gram of protein)/(milligrams per kilogram of body weight)], of all Cl4PhBQ-derived adducts in Sprague-Dawley rats after chronic administration of TCB, can be predicted from the following expression (23):

A)

[RY]a kae

where [RY]a represents the rate of production of adducts per day [(nanomoles per gram of protein)/(milligrams per kilogram of body weight)/day]. From Table 3, the value of [RY]a is estimated to be 15.6 ( 0.777 (nmol/g of protein)/(mg/kg of body weight)/day. On the basis of a single oral administration of TCB, the value of A for all Cl4PhBQ-derived adducts following a chronic treatment with TCB can be estimated to be 45.7 ( 3.73 (nmol/g of protein)/(mg/kg of body weight). (3) Multiple-Dose Study. Adduct levels in liver cytosolic protein in the multiple-dose regimen are given as a function of TCB dose in panels A and B of Figure 9. The relationship between adduct levels and dose was nonlinear at doses above 100 mg/kg of body weight, with a less than proportional production of adduct. Adduct levels were ordered as follows: Cl4PhBQ-Y > 2,2′,5-Cl3PhBQ-Yn > 2,5-Cl2PhBQ-Yn = 2,5,5′-Cl3PhBQ-Yn. The average level of formation of total Cl4PhBQ-derived adducts per unit dose of TCB was estimated to be 97.7 ( 13.2 (pmol/g)/(mg/kg of body weight) (Table 4). Formation of TCB-Derived Adducts in Brain Cytosolic Protein in Vivo. The formation of TCB quinone in rat brain was investigated in cytosolic protein isolated from the forebrain and midbrain of SpragueDawley rats receiving multiple doses (one dose per day, 5 days) of TCB or corn oil. Assay by the Raney nickel procedure confirmed the presence of 6-cysteinyl-3,4diOH-TCB in brain cytosolic protein of the treated rats (Figure 9A). No adduct was detected in the corn oil control. This result suggests the formation of Cl4PhBQ in rat brain, with subsequent binding to cysteinyl residues of brain cytosolic protein.

Discussion The objective of this study was to investigate the possible formation of protein adducts of TCB via quinone intermediates. Covalent adducts with proteins were generated in vitro in rat liver microsomes and in vivo in male Sprague-Dawley rats following administration of single or multiple doses of TCB. Phenobarbital-induced rat liver microsomes metabolized TCB to a reactive

Figure 9. Formation of Cl4PhBQ-derived cysteinyl adducts (A) Cl4PhBQ-Y in liver and brain cytosolic proteins following multiple administrations of TCB (one dose per day for 5 days) to Sprague-Dawley rats and (B) 2,2′,5-Cl3PhBQ-Yn, 2,5,5′-Cl3PhBQ-Yn, and 2,5-Cl2PhBQ-Yn in liver cytosolic proteins following multiple administrations of TCB (one dose per day for 5 days) to Sprague-Dawley rats. Note that mean values and standard errors are shown for four animals per group.

intermediate that formed adducts with sulfhydryl groups from which chlorinated catechols were released on reductive cleavage by Raney nickel. Similar in vitro adducts, giving identical cleavage products, were obtained from oxidation of 3,4-diOH-TCB in the presence of HRP and H2O2. These results are consistent with microsomal metabolism of TCB to 3,4-diOH-TCB followed by oxidation to Cl4PhBQ by HRP and H2O2. Formation of adducts would occur by Michael addition of protein and/or nonprotein sulfhydryls via multiple substitution reactions. Superoxide anion, derived from the oxidase activity of cytochrome P450 2B, may also catalyze the conversion of 3,4-diOH-TCB to Cl4PhBQ since the absence of HRP and H2O2 in the incubates did not affect the production of TCB adducts (Table 2). The observation that the oxidase activity of liver microsomes, including NADPH oxidation, and production of superoxide anion and H2O2, was increased by pretreatment of animals with phenobarbital supports the role of superoxide anion as a catalyst in the oxidation of 3,4-diOH-TCB (24-26). A scheme representing the activation pathway and adduct formation is given in Figure 10. Consistent with observations on metabolic activation of TCB, the activation of 3-OH-TCB occurred in the presence of NADPH or cumene hydroperoxide in the absence of HRP (Table 2). This finding is in accord with reports that in the microsomal metabolism of chlorinated phenols (27-29), cytochromes P450 serve dual roles in the conversion of chlorinated phenols to quinones. It is likely that NADPH-dependent cytochrome P450 monooxygenase activity mediates the oxidation of 3-OH-TCB to 3,4-diOH-TCB which is further oxidized to Cl4PhBQ in the presence of reactive oxygen species. On the basis of the ratio of adducts generated from TCB and 3-OHTCB as substrates, conversion of 6-8% of TCB to 3-OHTCB can be estimated from Table 2. This result is


Figure 10. Metabolic pathways of TCB leading to the formation of 3-OH-TCB, 3,4-diOH-TCB, Cl4PhBQ, and the respective semiquinones (Cl4PhSQ) mediated by microsomal cytochrome P450 2B and peroxidase in the presence of NADPH and/or cumene hydroperoxide.

comparable with that observed by Preston and his associates (30), who reported that close to 15% of TCB was converted by phenobarbital-induced rat liver microsomes to 3-OH-TCB. The nature of TCB-derived cysteinyl adducts in liver and brain cytosolic protein is consistent with Cl4PhBQ as a reactive intermediate in rats following administration of TCB. In general, the dose dependence of adduct formation was linear in liver with a single dose (Figures 7 and 8) and nonlinear with a less-than-proportional production of adducts in both liver and brain protein for multiple doses at levels of >100 mg/kg/dose (Figure 9). In contrast to liver, only a single cleavage product, corresponding to the adduct Cl4PhBQ-Y, was detected in rat brain. Low levels of nonprotein thiols present in the brain may contribute to the predominant formation of this monosubstituted adduct of Cl4PhBQ. Additionally, this finding implies that 2,2′,5-Cl3PhBQ-Yn and 2,5,5′Cl3PhBQ-Yn are products of multiple reactions of Cl4PhBQ with sulfhydryls of protein and nonprotein thiols. The stability of TCB adducts in Sprague-Dawley rats was estimated by measuring adduct levels 24 and 72 h following administration of a single dose of TCB. Adduct levels declined over the course of the experiment at rates which were comparable among the different adducts with the exception of 2,5-Cl2PhBQ-Yn (Figures 7 and 8). The estimated half-life of TCB adducts (2.03 ( 0.131 days) is about 2.5-fold shorter than the turnover rate of liver cytosolic protein (t1/2 ) 5.1 days; 31). This observation suggests that the adducts are unstable in vivo, perhaps


due to continued reaction with nonprotein thiols in a manner similar to that observed for pentachlorophenolderived quinone adducts in liver protein (17). Steadystate adduct levels measured in rat liver following administration of multiple doses of TCB were 2.1-fold higher than predicted on the basis of the single-dose study. This suggests self-induced metabolism of TCB, likely involving cytochrome P450 2B. Overall, results from in vivo metabolism of TCB in rats were consistent with the observation that non-arene oxide aromatic ring hydroxylation is the major metabolic pathway of TCB (30). It has been reported that the 3,4-oxide of TCB is a reactive metabolite of TCB (32, 33). Reactions of the 3,4oxide of TCB with cysteine and glutathione give 3- and 4-(cystein-S-yl)-TCB as the major products (33). We believe that, although less favorable, formation of the 3,4oxide of TCB and the subsequent formation with sulfhydryls on liver proteins cannot be ruled out. Induction of single-strand breaks in DNA of human lymphoblastoid cells by TCB has been reported (34). Chlorinated quinones are known to undergo redox cycling with generation of reactive oxygen species with consequent induction of oxidative DNA damage (35). Recent reports of quinone-mediated toxicities indicate that pentachlorophenol-derived hydroquinone and quinone cause oxidative damage in genomic DNA in the presence of metal ions and/or reducing equivalents (19, 36). A number of promutagenic DNA lesions were identified, including abasic sites and 8-hydroxy-2′-deoxyguanosine. The implied intermediacy of quinones in the liver and brain of TCB-treated rats in this study supports the suggestion that quinone metabolites are also significant contributors to PCB-derived oxidative DNA damage and possibly to PCB-induced liver carcinogenesis in rodents.

Acknowledgment. We gratefully acknowledge the assistance of Dr. Kevin McDorman and Dr. Jun Nakamura with the animal experiments. This work was supported by the National Institute of Environmental Health Sciences through Grants P42ES05948, T32ES07126, and F32ES05868.

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Formation of Quinonoid-Derived Protein Adducts in the Liver and

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