Immunochemical Detection of Quinol−Thioether-Derived Protein

Chem. Res. Toxicol. 1998, 11, 1283-1290

1283

Immunochemical Detection of Quinol-Thioether-Derived Protein Adducts Heather E. Kleiner,†,‡ Maria I. Rivera,†,§ Neil R. Pumford,| Terrence J. Monks,† and Serrine S. Lau*,† Division of Pharmacology and Toxicology, College of Pharmacy, The University of Texas at Austin, Austin, Texas 78712, and Division of Toxicology (Mail Slot 638), University of Arkansas Medical Sciences, 4301 Markham Street, Little Rock, Arkansas 72205 Received June 10, 1998

Glutathione (GSH) conjugates of hydroquinone (HQ) and 2-bromohydroquinone (2-BrHQ) produce severe renal proximal tubular necrosis in rats. Since the reactivity of quinones lies, in part, in their ability to alkylate proteins, our goal was to develop an immunochemical method with which to investigate the role of protein adduct formation in quinone-thioether-mediated toxicity. An immunogen was synthesized by coupling 2-bromo-6-(N-acetylcystein-S-yl)hydroquinone (2-BrHQ-NAC) to keyhole-limpet hemocyanin (KLH). Anti-2-BrHQ-NAC-KLH antibodies were raised in rabbits and purified by affinity chromatography. Antibody binding to the 2-BrHQ-NAC epitope was confirmed by competitive enzyme-linked immunosorbent assay (ELISA) with a bovine serum albumin conjugate of 2-BrHQ-NAC. Affinity-purified anti-2BrHQ-NAC-KLH antibodies recognized adducted proteins in the kidneys of rats treated with HQ, 2-BrHQ, 2-bromo-bis(glutathion-S-yl)hydroquinone, 2-(glutathion-S-yl)hydroquinone, 2,5bis(glutathion-S-yl)hydroquinone, and 2,3,5-tris(glutathion-S-yl)hydroquinone. Immunoreactive proteins were found in all renal subcellular fractions of 2-BrHQ-treated rats, and the distribution of adducts was similiar to that obtained by quantifying 2-Br[14C]HQ covalent adducts. Western blot analysis revealed that three proteins, at 42, 46, and 79 kDa, were adducted by all the compounds examined. The identification of these adducted proteins will be required to assess their significance in quinol-thioether-mediated nephrotoxicity.

Introduction (GSH)1

Glutathione conjugates of hydroquinone (HQ) and 2-bromohydroquinone (2-BrHQ) are selectively toxic to renal proximal tubule cells in rats (1-3). Quinone reactivity resides in their ability to redox cycle and * To whom all correspondence should be addressed: Division of Pharmacology and Toxicology, College of Pharmacy, The University of Texas at Austin, Austin, TX 78712. Telephone: (512) 471-5190. Fax: (512) 471-5002. E-mail: [email protected]. † The University of Texas at Austin. ‡ Present address: The University of Texas M. D. Anderson Cancer Center, Science Park Research Division, P.O. Box 389, Smithville, TX 78957. Telephone: (512) 237-9441. Fax: (512) 237-2444. E-mail: [email protected]. § Present address: Laboratory of Drug Discovery Research and Development, National Cancer Institute, FCRDC, Building 560, 3260, Frederick, MD 21702-1201. | University of Arkansas Medical Sciences. 1 Abbreviations: ABTS, 2,2′-azino-di(3-ethylbenzthiazoline sulfonate); 2-BrHQ, 2-bromohydroquinone; 2Br6GHQ, 2-Bromo-6-(glutathion-S-yl)hydroquinone; 2-BrHQ-NAC, 2-bromo-6-(N-acetyl-L-cysteinS-yl)hydroquinone; BrBGHQ, 2-bromo-3,5(6)-bis(glutathion-S-yl)hydroquinone; BSA, bovine serum albumin; BUN, blood urea nitrogen; CTFC, S-(2-chloro-1,1,2-trifluoroethyl)-L-cysteine; ECL, enhanced chemiluminescence; EDC, 1-ethyl-3-(3-diamethylaminopropyl)carbodiimide hydrochloride; ELISA, enzyme-linked immunosorbent assay; γ-GT, γ-glutamyltranspeptidase; GSH, reduced glutathione; GHQ, 2-(glutathion-S-yl)hydroquinone; 2,5-BGHQ, 2,5-bis(glutathion-S-yl)hydroquinone; TGHQ, 2,3,5-tris(glutathion-S-yl)hydroquinone; HCFC-123, hydrochlorofluorocarbon-123; HEPES, N-(2-hydroxyethyl)piperazineN′-2-ethanesulfonic acid; ic, intracardiac; ip, intraperitoneal; iv, intraveinous; KLH, keyhole-limpet hemocyanin; LDH, lactate dehydrogenase; MW, molecular mass; NAC, N-acetylcysteine; PBS, phosphatebuffered saline; PMSF, phenylmethanesulfonyl fluoride; sc, subcutaneous; SDH, succinate dehydrogenase; SDS, sodium dodecyl sulfate; TBS, tris-buffered saline; TFEC, S-(1,1,2,2-tetrafluoroethyl)L-cysteine.

generate an oxidative stress (4) and/or to alkylate protein and nonprotein sulfhydryls such as GSH (5). With respect to protein alkylation, the degree of 2-BrHQinduced nephrotoxicity correlates positively with the amount of covalently adducted proteins present in renal tissue (6). However, the mechanistic role of protein alkylation in quinone-thioether-mediated toxicity remains to be determined. Identification of covalently adducted proteins is necessary as a first step in determining their role in the development of toxicity, and immunochemical techniques provide a sensitive and selective approach to this problem. The availability of antibodies that recognize chemically adducted proteins has facilitated the identification of proteins covalently adducted by reactive metabolites of halothane, acetaminophen, and diclofenac (7-11). For example, the 44 kDa acetaminophen-binding protein in mouse liver and kidney is glutamine synthetase (12, 13), and the 50 kDa mitochondrial acetaminophen-binding protein in mouse liver is glutamate dehydrogenase (14). Immunodetection also showed that diclofenac covalently binds to a 110 kDa protein in rat liver identified as dipeptidyl peptidase IV (15). Also, using anti-dichloroacetyl antibodies, Halmes and co-workers (16) determined that trichloroethylene binds covalently to a 50 kDa hepatic microsomal protein that comigrates with cytochrome P450 2E1. Recently, an immunochemical method was developed that detects in vitro bromobenzene-derived adducts to rat liver protein sulfhydryl groups (17). However, antibodies that identify in vivo covalent protein

10.1021/tx980134e CCC: $15.00 © 1998 American Chemical Society Published on Web 10/29/1998

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adducts of the quinol-thioethers have not yet been reported. The goal of the present studies was therefore to develop antibodies that would specifically recognize proteins adducted by quinone-thioethers. We report on the isolation, characterization, and purification of antibodies raised against a 2-BrHQ-NAC-KLH antigen, and the ability of these antibodies to recognize immunoreactive proteins in the kidney of quinol- and quinol-thioethertreated rats.

Experimental Procedures Caution: HQ, bromohydroquinone, quinol-thioethers, and acrylamide are hazardous and should be handled carefully. Chemicals. 2-Bromo[14C]hydroquinone (20.1 mCi/mmol) was purchased from Dupont (Wilmington, DE). 2-BrHQ and sucrose were products of ICN Biomedicals, Inc. (Cleveland, OH). Silver(II) oxide was purchased from Aldrich Chemicl Co., Inc. (Milwaukee, WI). Antipain, aprotinin, bovine serum albumin (BSA), N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid (HEPES), merthiolate, N-acetylcysteine (NAC), leupeptin, phenylmethanesulfonyl fluoride (PMSF), Tween 20, and Tris were purchased from Sigma Chemical Co. (St. Louis, MO). Freund’s adjuvant was obtained from Life Technologies, Inc. (Grand Island, NY). Keyhole-limpet hemocyanin (KLH) and 1-ethyl3-(3-diamethylaminopropyl)carbodiimide hydrochloride (EDC) were purchased from Pierce (Rockford, IL). Horseradish peroxidase-conjugated affinity-purified goat anti-rabbit IgG (H and L chain) and ELISAmate kits, which included BSA blocking reagent, imidazole-buffered Tween 20, 2,2′-azino-di(3-ethylbenzthiazoline sulfonate) (ABTS), sodium dodecyl sulfate (SDS), and hydrogen peroxide, were products of Kirkegaard and Perry Inc. (Gaithersburg, MD). EAH Sepharose 4B was obtained from Pharmacia Biotech Inc. (Piscataway, NJ). Casein and SDS were purchased from BDH Laboratory Supplies (Poole, England). Enhanced chemiluminescent reagents (ECL) and Hyperfilm ECL were purchased from Amersham Life Science (Arlington Heights, IL). Protogel (acrylamide/bisacrylamide) was a product of National Diagnostics (Atlanta, GA). Low- and high-range molecular mass markers were supplied by Bio-Rad Laboratories (Hercules, CA). 2-Bromo-6-(N-acetylcystein-S-yl)HQ (2-BrHQNAC), 2-(glutathion-S-yl)hydroquinone (GHQ), 2,5-bis(glutathionS-yl)hydroquinone (2,5-BGHQ), 2,3,5-tris(glutathion-S-yl)hydroquinone (TGHQ), 2-bromo-6-(glutathion-S-yl)hydroquinone (2Br6GHQ), and 2-bromo-3,5(6)-bis(glutathion-S-yl)hydroquinone (BrBGHQ) were synthesized as previously described (1, 2, 18, 19). Animals. Male New Zealand white rabbits (5-6 lb) were obtained from Myrtle Rabbitry (Thompson Station, TN) and were housed at the University of Texas M. D. Anderson Cancer Center-Science Park, Veterinary Division (Bastrop, TX), by contract for immunization (see Immunization of Rabbits below). Male Fischer 344 rats (116-135 g) were purchased from Harlan Sprague-Dawley (Indianapolis, IN). Animals were allowed food and water ad libitum prior to and during experiments. Synthesis of Antigens. 2-Bromo-6-(N-acetylcystein-S-yl)[14C]hydroquinone (2-Br-[14C]-HQ-NAC) was synthesized according to Hill et al. (20) with minor modifications. KLH or BSA derivatives of 2-Br-[14C]-HQ-NAC were synthesized as follows. EDC (100.6 mM) was mixed with 8.19 mM 2-BrHQNAC in 20 mM HEPES buffer (pH 4.9) immediately followed by the addition of 2 mg of KLH or BSA for 90 min, in a total volume of 0.7 mL. The mixture was dialyzed overnight against three changes (800 mL each) of 20 mM HEPES buffer (pH 4.9) at 4 °C. A large-scale nonradioactive synthesis was prepared for immunizations and assays. The protein concentration was determined by the method of Lowry et al. (21), using BSA as a standard. Immunization of Rabbits. Two rabbits were pre-bled and injected subcutaneously (sc) in 10 sites on the back with 100 µg

Kleiner et al. of 2-BrHQ-NAC-KLH diluted 1:2 in Complete Freund’s Adjuvant. The rabbits were boosted 2 weeks later using Incomplete Freund’s Adjuvant. The rabbits were boosted at 2 week intervals for a total of four boosts followed by monthly booster injections and collections of serum to determine antibody titer. Affinity Purification of Antibodies. A 2-BrHQ-NACamino Sepharose 4B affinity column was constructed as follows; 192 mg of 2-BrHQ-NAC was dissolved in 10 mL of HEPES (20 mM, pH 4.5). The carbodiimide coupling agent EDC was added in powder form to the ligand at a final concentration of 0.1 M. This solution was added to 5 mL of drained EAH Sepharose 4B and mixed for 4 h at room temperature. The pH was maintained between 4.5 and 6 during the reaction with the addition of 1 N HCl. The gel was washed alternatively three times each with 0.1 M acetate buffer (pH 4) and 0.1 M Tris-HCl buffer (pH 8), each containing 0.5 M sodium chloride. Washes were saved to quanititate the amount of 2-BrHQ-NAC remaining. The gel was washed with ultrapure water, packed into a 10 mL glass chromatography column, and stored in PBS containing 0.02% merthiolate. A 1/10 scale synthesis using 2-Br-[14C]-HQ-NAC was first performed to confirm the epitope density. Rabbit serum was first partially purified by ammonium sulfate precipitation to isolate IgG (22) and then immunoaffinity purified at 4 °C on the 2-BrHQ-NAC-amino Sepharose column as follows. The column (9 mL) was equilibrated with 45 mL of 10 mM Tris buffer (pH 7.5). Ammonium sulfate-precipitated serum diluted 1:1 in 10 mM Tris buffer (pH 7.5, 4.5 mL) was applied to the column. The column was eluted with at least 90 mL of 10 mM Tris (pH 7.5) and then with at least 450 mL of 10 mM Tris buffer (pH 7.5) containing 500 mM NaCl. Fractions (3 mL) were collected and monitored at 280 nm to detect elution of protein. Specific antibodies were eluted with 0.1 M glycine buffer (pH 3) into tubes containing 1 M Tris buffer (pH 8) to neutralize the pH. BSA (0.5 mg/mL) was added as a stabilizer; antibodies were dialyzed against three 1 L changes of PBS, lyophilized to dryness, and reconstituted in 1/10 of the volume in ultrapure water. Antibody titer and purity were determined in the noncompetitive ELISAs and Western blot assays prior to use in any experiments. ELISAs. Rabbit serum was tested for antibody activity directed against the hapten by a noncompetitive ELISA. Reagent volumes were all 50 µL per well, and assays were conducted at room temperature, with shaking. Polystyrene ELISA plates (96-well) (Corning, Corning, NY) were coated with 4 µg/mL 2-BrHQ-NAC-BSA. Plates were blocked with 300 µL of 0.1% BSA for 30 min at room temperature, or at 4 °C overnight. Serial dilutions of antibodies [dissolved in phosphatebuffered saline [PBS, 10 mM sodium phosphate buffer (pH 7.4) containing 140 mM sodium chloride]] were incubated on the plate for 1 h, and then washed five times with 300 µL of imidazole-buffered saline containing 0.02% Tween 20. A 1:1000 dilution (100 ng/mL in PBS) of anti-rabbit secondary antibody was incubated on the plate for 1 h and washed five times, and 0.3 g/L ABTS peroxidase substrate and 0.01% hydrogen peroxide were added for color development. Reactions were terminated after 30 min with 1% SDS, and the absorbance at 405 nm was determined with a microtiter plate reader (Bio Tek Instruments, Inc., Winooski, VT). Backgrounds (PBS instead of primary antibody) were subtracted from experimental readings. Radiolabel Covalent Binding Study. Male Fischer 344 rats were treated with 2-Br-[14C]-HQ (0.9 mmol/kg, 2200 dpm/ nmol) (dissolved in 2:1 v/v PBS/methanol) ip. Control rats were treated with vehicle only. At appropriate time points, rats were euthanized by cervical dislocation, and the kidneys were perfused with ice-cold isolation buffer [20 mM Tris buffer (pH 7.4) containing 250 mM sucrose, 20 mM HEPES, 0.1 mM PMSF, and 1 mM EDTA]. Kidneys were quickly removed and papillae dissected out, and the remaining tissue was homogenized 1:10 (w/v) in isolation buffer. Subcellular fractions were isolated by differential centrifugation (described below). The extent of covalent binding to each tissue was determined by SDS equilibrium dialysis (23). The amount of radioactivity in each

Covalent Binding by Quinone-Thioethers subcellular fraction was determined by liquid scintillation spectroscopy. Isolation of Subcellular Fractions. Kidneys were perfused with ice-cold isolation buffer [20 mM Tris buffer (pH 7.4) containing 250 mM sucrose, 20 mM HEPES, 0.1 mM PMSF, and 1 mM EDTA] via the aorta. Kidneys were quickly removed and placed in ice-cold isolation buffer, and renal papillae were dissected and discarded. All procedures were conducted at 4 °C. The remaining renal tissue was homogenized 1:3 (w/v) in isolation buffer, and nuclei, mitochondria, plasma membrane, endoplasmic reticulum, and cytosol-enriched fractions were isolated by differential centrifugation. Mitochondria, endoplasmic reticulum, cytosol, and plasma membrane-enriched fractions were isolated following the procedure of Kinsella et al. (24) with minor modifications. Isolation of kidney nuclei was conducted by a combination of procedures (25-27). Protein concentrations were determined by the method of Lowry (21) using BSA as a standard. Aliquots of tissue were stored at -80 °C for later immunochemical analysis. To determine the enrichment of subcellular fractions, the following assays were performed: succinate dehydrogenase (mitochondria), γ-glutamyl transpeptidase (γ-GT) (plasma membrane), NADPH-cytochrome c reductase (microsomes), and lactate dehydrogenase (LDH) (cytosol). Succinate dehydrogenase activity was measured only in freshly isolated mitochondria. All other fractions could be kept frozen at -20 °C for several weeks. Succinate dehydrogenase was assayed by the procedure of Sottocasa et al. (28) in which enzyme activity is measured by following the reduction of cytochrome c at 550 nm in the presence of succinate. γ-GT activity was determined as described in Sigma Technical Bulletin 545 using l-γ-glutamyl-p-nitroanilide as the substrate. One unit of γ-GT is defined as 1 nmol of p-nitroaniline formed per minute at 25 °C. NADPH-cytochrome c reductase was analyzed spectrophotometrically by following the reduction of cytochrome c at 550 nm (25 °C). The assay mixture contained, in a total volume of 3 mL, 1 µmol of NaCN, 0.1 µmol of oxidized cytochrome c, and 0.2 M potassium phosphate. The reaction was initiated by the addition of 0.4 µmol of NADPH. LDH was measured by following the oxidation of NADH (0.23 mM) at 340 nm in the presence of pyruvate (0.6 mM). Treatment of Rats and Preparation of Renal Cytosolic Fractions. Male Fischer 344 rats were treated with the following compounds: 2-BrHQ (0.9 mmol/kg, ip; dissolved in 2:1 v/v PBS/methanol), BrBGHQ (60 µmol/kg, tail vein, iv; dissolved in PBS), HQ (4.5 mmol/kg, gavage; dissolved in corn oil), GHQ (200 µmol/kg, tail vein, iv; dissolved in PBS), 2,5-BGHQ (100 µmol/kg, tail vein, iv; dissolved in PBS), or TGHQ (20 µmol/kg, tail vein, iv; dissolved in PBS). Control rats were treated with vehicle only. Animals were housed in stainless steel metabolism cages, and urine was collected in light-protected tubes on ice. At 2 h after dosing, rats were anesthetized with equithesin (35 mg/kg sodium pentobarbital and 140 mg/kg chloral hydrate, ip), and urine from the bladder was pooled and combined with the urine already collected. The kidneys were perfused with ice-cold isolation buffer [20 mM Tris buffer (pH 7.4) containing 250 mM sucrose, 20 mM HEPES, 0.1 mM PMSF, 1 mM EDTA, and aprotinin, antipain, and leupeptin each at 5 mg/L each]. Kidneys were quickly removed and papillae dissected out, and the remaining tissue was homogenized 1:5 (w/v) in isolation buffer. The homogenates were centrifuged at 10000g for 20 min. The 10000g supernatants were further centrifuged at 100000g for 1 h to obtain microsomes and 100000g supernatant (cytosol). Western Blot Analysis. Proteins were resolved by the procedure of Laemmli (29). Samples were first diluted in Trisbuffered saline (TBS) [20 mM Tris buffer (pH 7.4) containing 500 mM NaCl], and 24% (w/v) SDS and 30% (v/v) glycerol were added, followed by the addition of 2× sample buffer [0.125 M Tris, 10% (w/v) SDS, 40% (v/v) glycerol, 0.04% (w/v) bromophenol blue, and 80 mM dithiothreitol], resulting in final concentrations of 0.042 M Tris buffer (pH 6.8), 5% (w/v) SDS, 20% (v/v) glycerol, 0.013% (w/v) bromophenol blue, and 26.6 mM dithiothreitol. Samples were heated to 100 °C for 2 min, and 15-250 µg of

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Figure 1. Proposed structure of the immunogen, 2-bromo-6(N-acetyl-L-cystein-S-yl)hydroquinone-keyhole-limpet hemocyanin. protein/lane was loaded onto a 14 cm × 14 cm × 1.5 mm (l x h x w) resolving gel in a vertical slab apparatus (model SE 600, Hoefer Scientific Instruments, San Francisco, CA). Proteins were loaded at 100 V (constant voltage) through the 3% (w/v) acrylamide stacking gel and resolved at 250 V through the 10% (w/v) acrylamide resolving gel. Proteins were transferred to nitrocellulose (0.45 µm, Bio-Rad Laboratories) electrophoretically (30) in 0.015 M Tris and 0.12 M glycine buffer (pH 8.3) containing 20% (v/v) reagent grade methanol at 400 mA (constant current) for 1.5 h using a Bio-Rad Trans-Blot cell. Duplicate gels were stained with 0.05% (w/v) Coomassie Blue. After transfer, gels were stained with Coomassie Blue to ensure efficiency of transfer. Only blots with efficient transfer were immunostained. The immunoblot procedure was derived from the method described by Kenna and co-workers (31). Western blots were marked by lane, and the lanes containing molecular mass markers were cut off and stained with Amido Black. Immunoblots were first blocked for 2 s in 0.0002% (w/v) poly(vinyl alcohol) and then blocked for 1 h in blocking buffer [0.01 M Tris and 0.15 M sodium chloride buffer (pH 7.6) containing 0.02% (w/v) merthiolate and 2.5% casein]. Blots were incubated overnight at 4 °C with affinity-purified rabbit anti-BrHQ-NAC antibodies diluted 1:20 in washing buffer [0.01 M Tris and 0.15 M sodium chloride buffer (pH 7.6) containing 0.02% (w/v) merthiolate and 0.5% casein]. Blots were then washed for 5 min (room temperature) in detergent buffer (washing buffer containing 0.5% Triton X-100 and 0.1% SDS), rinsed with water three times, and washed with washing buffer twice for 5 min each. Blots were incubated with goat anti-rabbit IgG (peroxidase-labeled) diluted 1:2000 in washing buffer for 1.5 h at room temperature. Blots were washed for 5 min in detergent buffer, rinsed three times in water, washed for 5 min in washing buffer, rinsed three times with water, washed for 5 min in Tris-saline [0.05 M Tris and 0.20 M NaCl (pH 7.4)], rinsed three times in water, and then incubated for 1 min in ECL solution. Blots were exposed to Hyperfilm ECL for 1-5 min, and the film was developed. Western blots were scanned using a UMAX UC630 MaxColor scanner (UMAX Data System, Inc., Industrial Park Hsinchu, Taiwan). Scans were analyzed using NIH Image software. The molecular masses of unknown proteins were extrapolated from standard curves generated on a logarithmic scale using the molecular mass markers (range of 31-97 kDa).

Results Proposed Structure of Antigens. An immunogen that contains the 2-BrHQ hapten was synthesized by using a carbodiimide reagent to couple 2-BrHQ-NAC to a carrier protein, KLH (Figure 1). The coupling reagent activates carboxyl groups to allow peptide bond formation with primary amino groups. The carboxyl group of the cysteine residue is free to bind with the amino group of lysine residues of KLH, which is rich in lysine residues. The epitope density from two separate syntheses was 53 and 160 nmol of 2-Br-[14C]-HQ-NAC bound per nanomole of KLH using an average MW of 1 × 106 kDa for KLH. A BSA conjugate of 2-BrHQ-NAC was synthesized for use as a testing antigen to screen the antibodies for specificity

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Figure 2. 2-Bromo-6-(N-acetylcystein-S-yl)hydroquinone-amino Sepharose affinity purification of rabbit anti-2-BrHQ-NACKLH antibodies. The data represent the absorbance at 280 nm of fractions (3 mL each) purified on a 2-BrHQ-NAC-amino Sepharose affinity column. The column was washed with 10 mM Tris buffer (pH 7.5) (a), followed by 10 mM Tris buffer (pH 7.5) containing 500 mM NaCl (b), and the anti-2-BrHQ-NAC antibodies were eluted with 0.1 M glycine buffer (pH 3) (c).

against the 2-BrHQ-NAC portion of the antigen. The molar ratio of 2-Br-[14C]-HQ-NAC to BSA was 11:1 (165 nmol of 2-Br-[14C]-HQ-NAC bound per milligram of BSA). Affinity Purification of Antibodies. Antibodies were purified using a 2-BrHQ-NAC-amino Sepharose affinity column (Figure 2). The epitope density of the gel was determined, in a 1/10 scale synthesis, to be 1200 nmol of 2-Br-[14C]-HQ-NAC per milliliter of drained gel. The specific antibodies retained a titer of at least 1:10000 as determined by ELISA, whereas fractions resulting from the washing steps had a titer lower than 1:2000 (data not shown), despite their higher protein concentrations, as indicated by a higher absorbance at 280 nm (Figure 2). Determination of Antibody Specificity. Antibody binding to antigen was dependent on both the concentration of antigen and the concentration of serum (data not shown). After the fifth inoculation, rabbit titer was at least 1:30000 (Figure 3) against 2-BrHQ-NAC-BSA (4 µg/mL). Preimmune serum (serum from a rabbit prior to inoculation with antigen) showed negligible activity (0.3% of that of anti-2-BrHQ-NAC serum) (Figure 3) against 2-BrHQ-NAC-BSA (4 µg/mL). Plates coated with BSA alone (4 µg/mL) (Figure 3) resulted in minimal color development (0.1% of that of plates coated with 2-BrHQ-NAC-BSA). These results indicate that the antibodies recognize the 2-BrHQ-NAC epitope. After determination of optimum conditions in noncompetititve ELISAs (Figure 3), competitive ELISAs were conducted to further confirm antibody specificity. Antigen at decreasing concentrations was preincubated with the antibodies prior to addition to the ELISA plate, which was coated with 2-BrHQ-NAC-BSA (4 µg/mL). The concentration of 2-BrHQ-NAC-BSA (in solution) necessary to inhibit 50% antibody binding (IC50) to the ELISA plate was 15 ng/well (Figure 4). BSA, N-acetyl-L-cysteine, or N-acetyl-L-lysine failed to inhibit antibody binding at concentrations as high as 5000, 16 000, or 18 000 ng/well, respectively. These data eliminate the possibility that the antibodies recognize the carrier protein (BSA) or the peptide linker portions of the antigen (N-acetyl-L-cysteine or N-acetyl-L-lysine). Isolation of Subcellular Fractions. The degree of enrichment of each subcellular fraction was determined

Kleiner et al.

Figure 3. Noncompetitive ELISA for determining the titer of rabbit anti-2-BrHQ-NAC antibodies against 2-BrHQ-NAC-BSA or BSA. The data represent the averages of two wells each in the noncompetitive ELISA. Color development in the absence of rabbit antibody (background) was subtracted from each value.

Figure 4. Competitive ELISA using 2-BrHQ-NAC-BSA (4 µg/ mL) as a coating antigen. The data represent the average of two or three wells each in competitive ELISAs. Color development in the absence of rabbit antibody (background) was subtracted from each value.

by measuring the activity of enzymes specific for each fraction (Table 1). Enrichment factors with respect to the homogenate were 7, 3, 5, and 2 for γ-GT, NADPHcytochrome c reductase, succinate dehydrogenase, and LDH, respectively. Enzyme activity in the fractions to which the enzyme is not specifically associated was similar or lower than homogenate values. Immunochemical and Radiochemical Determination of 2-BrHQ-Derived Protein Adducts. Thirty minutes after administration of 2-Br-[14C]-HQ to rats, the highest amount of covalently bound material was located in the cytosolic fraction, followed by the microsomal and plasma membrane fractions (Figure 5). After 1 h, the amount of radiolabel associated with proteins in the cytosol decreased, whereas that in the nuclear fraction increased, and reached a maximum at 4 h. The levels of covalently adducted proteins were lowest in mitochondria. Proteins immunoreactive with antibodies directed against 2-BrHQ-NAC-KLH were also identified in renal

Covalent Binding by Quinone-Thioethers

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Table 1. Enrichment of Renal Subcellular Fractionsa subcellular fraction homogenate I II mitochondrial I II membranal I II cytosolic I II

γ-GT

NADPH-cyt c reductase

SDH

LDH

1.62 ( 0.43 0.81 ( 0.12

7.84 ( 1.68 8.05 ( 0.25

67.0 ( 15 21.4 ( 0.65

1.55 ( 0.24 0.85 ( 0.03

1.77 ( 0.17 1.58 ( 0.29

10.3 ( 1.12 11.8 ( 1.24

342.0 ( 17 138.0 ( 0.89

0.09 ( 0.01 0.01 ( 0.00

11.4 ( 3.8b 9.99 ( 1.06

20.7 ( 0.99b 27.3 ( 3.57

38.2c 34.2 ( 4.7

0.95 ( 0.24 1.32 ( 0.15

0.03 ( 0.00 0.02 ( 0.01

1.66 ( 0.19 0.53 ( 0.25

0.41 ( 0.03 0.50 ( 0.03

2.88 ( 0.07 2.43 ( 0.10

a γ-GT activity is nanomoles of p-nitroaniline formed per minute per microgram of protein. NADPH-cyt c reductase activity is nanomoles of cyt c reduced per minute per milligram of protien. Succinate dehydrogenase activity (SDH) is nanomoles of cyt c reduced per minute per milligram of protein. Values are representative of two separate isolations (I and II). Data are mean ( SE (n ) 3). The activity of each marker enzyme in the subcellular fraction where the enzyme is specifically located was significantly higher than the activity in the other fractions (p < 0.05). b n ) 2. c n ) 1.

Figure 5. Subcellular distribution of 2-Br-[14C]-HQ equivalents in renal subcellular fractions after treatment of male Fischer 344 rats with 2-bromo[14C]hydroquinone (0.9 mmol/kg, 2200 dpm/nmol): rat kidney nuclei (black bars), mitochondria (crosshatched bars), plasma membranes (white bars), microsomes (differentially shaded bars), or cytosol (uniformly shaded bars). The data represent means ( SE (n ) 4).

subcellular fractions (Figure 6). Consistent with the relative cellular distribution of radioactivity at each time point, immunoreactivity was highest in the cytosolic fraction and lowest in the nuclear fractions at 1 h. By 2 h, immunoreactivity was highest in the nuclear fraction and lowest in the mitochondrial and plasma membrane fractions. By 4 h, immunoreactivity remained highest in the nuclear fraction but lowest in the cytosolic fraction. There was negligible antibody binding to tissue isolated from untreated rats (data not shown). Western Blot Analysis of Immunoreactive Protein Adducts. Western blot analysis of kidney cytosol from male Fischer 344 rats treated with nephrotoxic doses of either 2-BrHQ, HQ, or their corresponding GSH conjugates revealed numerous immunostaining proteins, with three predominant immunostaining proteins at approximately 42, 46, and 79 kDa (Figure 7). Preincubation of antibodies with 0.1 mg/mL 2-BrHQ-NAC-BSA prevented antibody binding to adducted proteins (data not shown), and renal tissue from rats treated with the vehicle alone showed no detectable immunostaining (Figure 7), suggesting that the antibodies recognized adducts specific to the treatment group. Although the pattern of immunostaining was similar in all treatment groups, the intensity of staining was dependent upon the particular quinol-thioether administered to the rats.

Figure 6. Immunochemical detection of 2-Br-[14C]-HQ protein adducts in renal subcellular fractions after treatment of male Fischer 344 rats with 2-bromo[14C]hydroquinone (0.9 mmol/kg, 2200 dpm/nmol). ELISA plates were coated with 0.8 µg of protein/well of rat kidney nuclei (black bars), mitochondria (cross-hatched bars), plasma membranes (white bars), microsomes (differentially shaded bars), or cytosol (uniformly shaded bars). Rabbit anti-2-BrHQ-NAC IgG was diluted 1:100. The data represent means ( SE (three rats per group) of duplicate wells in noncompetitive ELISAs. Backgound (absorbance in the absence of primary antibody and absorbance from untreated tissue) was subtracted from each reading.

Thus, to achieve a similar intensity of immunostaining in all treatment groups, different amounts of protein were loaded onto the gel; 15 µg of 2-BrHQ-treated rat kidney cytosol gave an intensity of immunostaining similar to that of 250 µg of BrBGHQ or TGHQ. The intensity of staining for the different treatment groups when the same amount of protein (100 µg) was loaded on the gel was in the following order: 2-BrHQ > 2,5-BGHQ > HQ > GHQ > BrBGHQ ) TGHQ (data not shown).

Discussion We have successfully raised antibodies directed against 2-BrHQ-NAC-KLH that recognize quinol- and quinolthioether-derived protein adducts. The antibodies recognize the 2-BrHQ-NAC epitope (Figure 4) and crossreact with adducted proteins derived from metabolites of HQ, 2-BrHQ, BrBGHQ, GHQ, 2,5-BGHQ, and TGHQ (Figure 7). Antibodies directed against a halothane metabolite (7) also recognize in vivo covalent protein adducts of both halothane (2-bromo-2-chloro-1,1,1-trifluoroethane) and hydrochlorofluorocarbon-123 (HCFC-

1288 Chem. Res. Toxicol., Vol. 11, No. 11, 1998

Figure 7. Immunochemical detection of covalent protein adducts of HQ, 2-BrHQ, or their corresponding GSH conjugates in male Fischer 344 rat kidney cytosol. The doses were as follows: 2-BrHQ (0.9 mmol/kg, ip), BrBGHQ (60 µmol/kg, tail vein, iv), HQ (4.5 mmol/kg, gavage), GHQ (200 µmol/kg, tail vein, iv), 2,5-BGHQ (100 µmol/kg, tail vein, iv), or TGHQ (20 µmol/ kg, tail vein, iv). Renal cytosolic fractions were isolated 2 h after dosing; proteins were separated on a 10% SDS-PAGE gel, transferred to nitrocellulose, and probed with anti-2-BrHQ-NAC rabbit antibodies (1:20 dilution) with enhanced chemiluminescent detection. Duplicate gels were stained with Coomassie Blue: (left) untreated rat kidney cytosol stained with Coomassie Blue (100 µg of protein/lane) and (right) Western blot immunostained with anti-2-BrHQ-NAC antibodies. The amount of protein loaded on the Western blot for each treatment group was as follows: 15, 250, 50, 50, 25, 250, and 250 µg/lane for 2-BrHQ, BrBGHQ, HQ, GHQ, 2,5-BGHQ, TGHQ, and vehicle control, respectively. At least two rats per treatment were assayed by Western blot analysis, but represenatative samples are shown for illustrative purposes.

123) (2,2-dichloro-1,1,1-trifluoroethane) (32) in rat liver. These two chemicals differ by only one halogen group (Cl for HCFC-123 and Br for halothane) and are bioactivated by cytochrome P450 to form identical trifluoro-N-acetylated lysine adducts on proteins (32). Rat kidney cytosolic proteins at approximately 36, 42, 58, 76, and 94 kDa are similarly adducted by either halothane or HCFC-123 treatment in vivo (32). These anti-halothane metabolite antibodies also cross-react with covalent protein adducts derived from the nephrotoxic cysteine conjugates S-(1,1,2,2-tetrafluoroethyl)-L-cysteine (TFEC) and S-(2chloro-1,1,2-trifluoroethyl)-L-cysteine (CTFC) (33). The pattern of immunoreactive proteins in renal subcellular fractions was similar to that found by radiochemical analysis (Figures 5 and 6). Western blot analysis of kidney cytosol from Fischer 344 rats pretreated with nephrotoxic doses of 2-BrHQ, HQ, or their corresponding GSH conjugates (Figure 7) revealed at least three common predominant immunostaining proteins at approximately 42, 46, and 79 kDa, although the intensity of immunostaining against tissue from the different treatment groups varied. No immunoreactive proteins were found in cytosol obtained from untreated

Kleiner et al.

animals, further verifying the specificity of the antibodies toward the quinol/quinone epitope. Differences in the pattern of immunostaining seen with the different treatment groups (Figure 7) are probably due to a variety of factors. The number of immunostained proteins found after administration of either 2-BrHQ or hydroquinone is greater than the number of proteins immunostained after administration of the conjugates. This makes sense, because 2-BrHQ and hydroquinone are metabolized to a variety of GSH conjugates, all of which have the potential to form covalent adducts with protein. In contrast, the pattern of immunostained proteins observed after administration of TGHQ is relatively simple, presumably because these proteins represent specific targets for metabolites of this conjugate. Consistent with this view, the pattern of immunostained proteins seen after administration of 2,5DGHQ is a little more complex than that seen with TGHQ, but less complex than that for either GHQ or HQ. In addition, it is likely that the ability of anti-2-BrHQNAC-KLH antibodies to recognize protein adducts derived from the different nephrotoxicants varies. Thus, treatment of rats with 2-BrHQ or HQ results in a spectrum of protein adducts derived from all possible thioether-derived metabolites, whereas treatment with BrBGHQ results in adducts derived only from bis-thiol metabolites. The number of adducted proteins should decrease as the degree of thiol substitution increases. Hence, the complexity and intensity of staining are dependent upon the spectrum of adducted proteins recognized by the polyclonal antibody and the dose of the compound administered. Antibody affinity should be higher for the conjugates exhibiting structures closer to the original epitope, 2-BrHQ-NAC, and lower for the more highly substituted thiol conjugates. Since common immunostaining proteins were visualized in rat kidney cytosol by Western blot analysis (Figure 7) (i.e., 42, 46, and 79 kDa) after treatment with the various structural analogues, there may be common protein targets adducted by the different quinones and/ or quinone-thioethers. The hepatotoxicants bromobenzene and acetaminophen bind covalently to a common 58 kDa liver cytosolic protein in mice (34), suggesting that this protein may be a common target for reactive electrophiles. A common protein target may serve a detoxication function, neutralizing reactive electrophiles and preventing them from damaging critical cellular targets. The 58 kDa acetaminophen-binding protein, which is rich in nucleophilic cysteine residues (10, 35), may serve to trap electrophiles (34). The nonhepatotoxic m-hydroxy derivative of acetaminophen, [14C]-3′-hydroxyacetanilide, also binds covalently to hepatic proteins to an extent similar to that of acetaminophen, and may also bind to the 58 kDa acetaminophen-binding protein (36). Thus, either the 58 kDa acetaminophen-binding protein is not critical for cell viability or factors other than covalent binding, such as protein-thiol oxidation, may be involved in acetaminophen hepatotoxicity (36). However, other investigators showed that immunodetection of 3′-hydroxyacetanilide binding proteins in mouse liver demonstrates very little binding to a 56 kDa cytosolic protein, suggesting loss of labile adducts during electrophoresis (37). Interestingly, methyleugenol (3,4-dimethoxyallylbenzene), which is a natural food flavoring that can produce hepatotoxicity and carcinogenicity in rodents (38, 39), covalently binds to an ∼44 kDa liver microsomal

Covalent Binding by Quinone-Thioethers

protein in rats, and like the ∼44 kDa acetaminophen liver microsomal binding protein, this protein appears to be a peripheral membrane protein (i.e., it can be extracted from the endoplasmic reticulum using 0.1 M sodium carbonate) (40). In a rat liver microsomal incubation with [14C]bromobenzene, 46 and 50 kDa proteins have been recognized by anti-HQ antibodies which are selective for S-2,5-dimethoxyphenyl moieties (17). It is interesting that 42 and 46 kDa proteins were detected in the cytosolic fraction in this study (Figure 7). Whether HQ, 2-BrHQ, or their corresponding GSH conjugates also bind cellular targets common to acetaminophen or bromobenzene is currently not known. In conclusion, we have developed an immunochemical method for detecting in vivo quinol-thioether-derived protein adducts. Affinity-purified polyclonal antibodies were specific for the 2-BrHQ-NAC epitope, did not recognize nonadducted proteins, and gave results comparable to those from radiochemical analysis. Antibodies cross-reacted with the multisubstituted quinol-thioethers, and will serve as effective probes for studying the role of alkylation in quinone-thioether-mediated nephrotoxicity. Identification of the proteins specifically adducted by these conjugates is required before their significance in nephrotoxicity can be assessed, and such studies are ongoing.

Acknowledgment. This work was supported in part by an award from the National Institute of General Medical Sciences [GM39338 (S.S.L.)] and the National Institute of Environmental Health Sciences to T.J.M. (ES07359). H.E.K. was a recipient of the CIBA-Geigy Graduate Student Fellowship sponsored by the Society of Toxicology, and a Toxicology Training Fellowship (ES 07247). We also thank Dr. Dennis Johnston for his help with the analysis of Western blots using NIH image analysis supported by Center Grant ES 07784.

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