Detection of Benzoquinone Adducts to Rat Liver Protein Sulfhydryl

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Chem. Res. Toxicol. 1997, 10, 1407-1411

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Detection of Benzoquinone Adducts to Rat Liver Protein Sulfhydryl Groups Using Specific Antibodies Elizabeth M. Rombach and Robert P. Hanzlik* Department of Medicinal Chemistry, University of Kansas, Lawrence, Kansas 66045-2506 Received July 25, 1997X

Benzoquinone is an electrophilic metabolite of bromobenzene and other simple aromatic compounds of toxicological interest including benzene, phenol, hydroquinone, and acetaminophen. In reacting with proteins benzoquinone shows great selectivity for Michael addition to sulfhydryl groups and formation of S-(2,5-dihydroxyphenyl) protein adducts. To facilitate the specific detection and eventual isolation and identification of such adducted proteins, we prepared an antiserum capable of recognizing hydroquinone moieties by immunizing rabbits with keyhole limpet hemocyanin modified with 3-[(2,5-dihydroxyphenyl)thio]propanoyl groups as haptens. The antiserum had a high titer and showed high specificity for hapten in competitive ELISA with hapten analogues. In Western blot experiments the antiserum detected not only synthetically haptenized control proteins but also several proteins from rat liver microsomes that had been incubated in vitro with [14C]bromobenzene. This binding was completely blocked by free hapten, showing that it was hapten-specific. Each of the microsomal protein bands detected in the Western blots also contained radioactivity, but not all radioactive protein bands reacted with antibody. This antiserum should prove useful in exploring the role of protein arylation by benzoquinone in cytotoxic responses to its metabolic precursors.

Introduction The hepatotoxicity of bromobenzene, like that of many other simple organic compounds, has been strongly correlated with its metabolic activation and the subsequent covalent binding of reactive metabolites to cellular proteins. It is widely believed that this covalent binding is an important causative factor in the subsequent cytotoxicity observed, but only recently has the chemistry of the adduct-forming process started to become clear. Bromobenzene gives rise to a number of reactive metabolites including two arene oxides and several quinones and bromoquinones, all of which have been observed to alkylate protein sulfhydryl groups (1). Additional evidence indicates bromobenzene 3,4-oxide (structure 3 in Scheme 1) also alkylates histidine and lysine side chains in rat liver proteins in vivo (2). In vivo, the major bromobenzene-derived adduct to protein-SH groups arises via its electrophilic metabolite 1,4-benzoquinone (4) (1), which is formed by oxidative debromination of p-bromophenol (3, 4). Although it is believed that bromobenzene 3,4-oxide is a more significant hepatotoxin than benzoquinone (5), the latter is also of toxicological interest as a metabolite of benzene, phenol, and hydroquinone (6) and of acetaminophen (7). Much less is known about the protein targets of reactive bromobenzene metabolites. Early work by Aniya and Anders (8) identified glutathione transferase as a cytosolic target for [14C]BB1 metabolites. More recently we identified two isozymes of a nonspecific carboxylesterase (hydrolases A and B) in liver microsomes as in vitro * Address correspondence to this author. Tel: 785-864-3750. Fax: 785-864-5326. E-mail: [email protected]. X Abstract published in Advance ACS Abstracts, November 15, 1997. 1 Abbreviations: BB, bromobenzene; BQ, benzoquinone; DEAE, (diethylamino)ethyl Sepharose; DTT, dithiothreitol; ECL, enhanced chemilumenescence; HQ, hydroquinone; HRP, horseradish peroxidase; KLH, keyhole limpet hemocyanin; KPBS(-T), potassium phosphatebuffered saline (containing Tween-20); MT, metallothionein; PAGE, polyacrylamide gel electrophoresis; PVDF, poly(vinylidene difluoride); SDS, sodium dodecyl sulfate.

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Scheme 1. Metabolic Activation and Covalent Binding of Bromobenzene

targets for [14C]BB metabolites (9). The structure of the adducts formed to these proteins is not known, but the majority of the radioactivity is associated with the minor isozyme, hydrolase B. It is also interesting to note that both GST and hydrolase B are sulfhydryl proteins and that while GST is a target protein for the reactive metabolite of acetaminophen (10), the hydrolase is a target for halothane metabolites (11). To facilitate the identification of proteins specifically bearing adducts of benzoquinone to sulfhydryl groups, we have developed a polyclonal antibody capable of recognizing S-(2,5-dihydroxyphenyl) moieties such as 6. Herein we report the characterization of this antibody and its use to detect selectively rat liver microsomal proteins bearing benzoquinone-derived adducts generated by metabolic activation of bromobenzene in vitro. © 1997 American Chemical Society

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Experimental Section Sources of Reagents. Electrophoresis and blotting reagents were purchased from BioRad, N-succinimidyl 3-(2-pyridyldithio)propionate, Super Signal Ultra, Bradford reagent, and horseradish peroxidase-conjugated goat anti-rabbit IgG (H & L) were purchased from Pierce, and keyhole limpet hemocyanin, metallothionein, Freund’s adjuvants, normal goat serum, Sephadex G-25, and all chemicals used in the preparation of buffers were purchased from Sigma. Other reagents were obtained as follows: 3,3′,5,5′-tetramethylbenzidine (Kirkegaard and Perry), Immunolon 4 96-well microtiter plates (Fisher), BioMax MS autoradiography film (Kodak), Ultra Gold XR scintillation fluid (Packard), and DEAE Sephacel (Pharmacia). Synthesis of Immunogen and Preparation of Antisera. N-Succinimidyl 3-(2-pyridyldithio)propionate (SPDP; 10 mg, 32 µmol) in 200 µL of DMSO was added slowly to 10 mg of KLH in 0.65 mL of buffer (20 mM potassium phosphate, 150 mM NaCl, 1 mM EDTA, pH 7.2) followed by stirring for 1 h at room temperature, after which the mixture was passed through a Sephadex G-25 column equilibrated with 25 mM potassium phosphate (pH 6.0) containing 150 mM NaCl. The disulfide bonds were reduced by incubation for 1 h at room temperature in the presence of 50 mM DTT, after which the small molecules were removed by passage through G-25. The protein concentration and its free sulfhydryl content were quantitated using Bradford reagent and Ellman’s reagent, respectively. Finally, a stock solution of benzoquinone in acetonitrile (37 mM; 0.75 mol of BQ/mol of protein-SH) was added to generate the final KLH-hapten adduct 8, which was used immediately. Rabbits were immunized with 100 µg of 8 [containing an estimated 12 nmol of (dihydroxyphenyl)thio adducts] injected subcutaneously as an emulsion with complete Freund’s adjuvant, after which they were boosted twice at 2-week intervals with the immunogen emulsified in incomplete Freund’s adjuvant. Antiserum was collected 14 days after the last boost and assayed for binding to model adduct 9 in ELISA. Synthesis of S-(2,5-Dihydroxyphenyl)metallothionein (9) and Its Oxidized Form (10). Antigen 9 was generated by addition of 0.146 µmol of benzoquinone (4 µL of a 0.0366 M solution in acetonitrile) to a rapidly stirred solution of metallothionein (150 µg, 0.022 µmol) in 0.5 mL of 0.1 M acetic acid. Following the same procedure but using 40 mol of BQ/mol of MT afforded the oxidized adduct 10 directly (12). The reactions were complete within 1 min, and the products were used immediately. Enzyme-Linked Immunosorbent Assay. The serum of rabbits immunized with 8 was analyzed for anti-hydroquinone antibody activity by ELISA employing 9, 10, and unmodified metallothionein as solid-phase antigens. Wells of 96-well Immunolon 4 microtiter plates were coated by addition of 2 µg of the desired antigen in 100 µL of KPBS buffer (25 mM potassium phosphate, 150 mM NaCl, pH 6.0). The wells were blocked with 200-µL aliquots of 5% nonfat dry milk in KPBS containing 0.02% Tween-20 (KPBS-T). After the wells were blocked and washed with wash buffer [25 mM Hepes, 150 mM NaCl, 0.02% Tween20, 0.5% poly(vinyl alcohol), pH 7.4], 100-µL aliquots of antiHQ antiserum, serially diluted from 1/500 to 1/32000 in KPBST, were added and the plates were incubated for 1 h at 37 °C. Wells were washed and preincubated for 20 min with KPBS-T containing 1% (w/v) nonfat dry milk and 1% (v/v) normal goat serum, followed by incubation with HRP-conjugated goat antirabbit IgG diluted 1/4000 in KPBS-T. After a final wash the bound secondary antibody was detected with 3,3′,5,5′-tetramethylbenzidine in the presence of H2O2 by measurement of absorbance at 495 nm. The competitive ELISA was performed as above except that wells were coated with 1 µg of 9 and the anti-HQ antiserum, diluted 1/10000 in KPBS-T buffer containing 1% (w/v) nonfat dry milk, was preincubated overnight at 4 °C in the presence of varying concentrations of competing ligands 11-16 before use. Parallel control experiments employing anti-HQ antiserum preincubated similarly but without competing ligands added showed no diminution in antibody titer or binding.

Rombach and Hanzlik Animals and Microsome Preparation. Male SpragueDawley rats (180 g; Charles River Laboratories, Wilmington, MA) were housed in a temperature- and humidity-controlled room with a 12-h light/dark cycle and ad libitum access to food and water. After acclimating for at least 3 days, the animals were given daily ip injections of sodium phenobarbital (80 mg/ kg) in 0.9% saline (1.0 mL/kg). After the third dose, food was withheld overnight, and the next morning the rats were killed by decapitation under CO2 narcosis. Their livers were removed, chilled, and homogenized in ice-cold buffer (50 mM potassium phosphate, 0.15 M KCl, 1 mM EDTA, 0.5 mM DTT, pH 7.4; 4 mL/g of tissue). The homogenate was successively centrifuged at 3000g (10 min), 12000g (20 min), and 100000g (60 min), and the resulting microsomal pellet was homogenized in 0.1 M sodium pyrophosphate buffer, pH 8.2 (2 mL/g of tissue) followed by centrifugation at 100000g. The final microsomal pellet was resuspended in buffer (100 mM potassium phosphate, 20% glycerol, 0.5 mM EDTA, pH 7.4; 0.2 mL/g of tissue) and stored at -70 °C. The protein content of the microsomal preparation was determined by Bradford assay, and the cytochrome P450 and b5 contents were determined by difference spectra according to Omura and Sato (13). Conduct of Incubations. Incubations were carried out in 120-mL glass-stoppered Erlenmeyer flasks for 90 min at 37 °C in a shaking water bath. They contained 120 mg of microsomal protein, 80 µmol of [14C]bromobenzene (5.17 Ci/mol, delivered in 400 µL of acetonitrile), and a freshly prepared NADPHgenerating system (consisting of 40 µmol of NADP, 400 µmol of glucose-6-phosphate, and 40 units of glucose-6-phosphate dehydrogenase predissolved in 1.0 mL of incubation buffer), all in a final volume of 40 mL of 100 mM potassium phosphate buffer, pH 7.4, containing 1 mM EDTA. Incubations were terminated by chilling the incubation flask in an ice bath. The microsomes were separated from the incubation mixture by centrifugation at 100000g and washed to remove unbound label by two cycles of homogenization in buffer (50 mM potassium phosphate, pH 7.4) followed by centrifugation at 100000g. Protein Solubilization and Fractionation. The washed microsomal pellet was resuspended in 20 mL of buffer [50 mM potassium phosphate, 20% (v/v) glycerol, 0.1 mM PMSF, 0.1 mM DTT, pH 7.4], and the suspension was stirred on ice while sufficient sodium cholate stock solution (20%, w/v) was added dropwise to achieve a final cholate concentration of 2%. The mixture was stirred for 1 h on ice after which insoluble material was removed by centrifugation at 100000g for 1 h. The buffer of the supernatant obtained after centrifugation was replaced with loading buffer [20 mM potassium phosphate, pH 7.4, containing 20% (v/v) glycerol, 1.0 mM EDTA, 1.0 mM DTT, and 0.2% (v/v) Triton X-100] by ultrafiltration (PM10, Amicon). The resulting clear supernatant was loaded at a flow rate of 10 mL/h onto a 60-mL DEAE Sephacel column which had been equilibrated with loading buffer. The column was washed with loading buffer (240 mL) until the radiolabel in the eluate was e 2 times background signal. Retained proteins were then eluted from the column in stepwise fashion using equilibrating buffer made 0.05, 0.1, 0.15, 0.2, 0.3, 0.5, and 1.0 M in NaCl. Column fractions (4 mL) were pooled based on their content of radiolabel and protein, and the pooled fractions were concentrated by ultrafiltration using a PM10 membrane and stored at -20 °C until used. To locate radiolabeled proteins, aliquots from concentrated pooled fractions were separated by SDS-PAGE and the proteins transferred electrophoretically to PVDF membranes. The membranes were then stained for protein with 0.4% Coomassie brilliant blue R-250 in methanol/acetic acid/water (80:5:15, v/v), destained in the same solvent, and subjected to phosphorimaging analysis for detection of 14C using a Molecular Dynamics storage phosphor screen, scanning unit, and software. Western Blot Analyses. Adduct-bearing rat liver proteins were separated using a modified SDS-PAGE procedure in which the pH of the 12% separating gel was 7.0 (rather than the more customary 8.8) to prevent autooxidation of the HQ adducts such as 7 and 9. Two identical gels were always run in parallel. After SDS-PAGE the proteins were electrophoretically transferred to nitrocellulose membranes using standard

Antibody Detection of Benzoquinone-Protein Adducts

Figure 1. Anti-hydroquinone antibody binding to control antigens in ELISA. conditions. For Western analysis one of the membranes was blocked for 1 h with 5% (w/v) nonfat dry milk in KPBS-T. The membranes were then incubated for 1 h with 1/5000 diluted anti-HQ antiserum [in KPBS-T, pH 7.0, 1% (w/v) nonfat dry milk] which had been preincubated overnight at 4 °C in either the absence or presence of a large excess (1000 µM) of hapten 11. Each membrane was then washed five times with KPBST, preincubated 20 min with KPBS-T containing 1% (v/v) normal goat serum and 1% (w/v) nonfat dry milk, and incubated for 1 h with HRP-goat anti-rabbit IgG diluted 1/100000 in KPBS-T containing 1% (v/v) normal goat serum and 0.1% (w/v) nonfat dried milk. After a final wash, bound antibody was detected using Peirce’s Super Signal Ultra ECL reagent. After Western analysis, radiolabeled proteins were detected by phosphorimaging of the same membrane using a Molecular Dynamics storage phosphor screen, scanning unit, and software.

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Figure 2. Inhibition of anti-hydroquinone antibody binding by small competing ligands. (See Chart 1 for structures.)

Chart 1. Structures of Target Antigen 7, Immunogen 8, Model Adducts 9 and 10, and Competing Ligands 11-16

Results and Discussion The single most abundant type of reactive metabolite adduct to protein-SH groups in the livers of BBintoxicated rats contains the S-(2,5-dihydroxyphenyl) moiety shown in structure 6 of Scheme 1 (1). Such adducts have also been detected in hemoglobin and albumin of rats exposed to benzene (14) and in albumin of rats exposed to acetaminophen (7). Their detection typically requires sensitive GC/MS analysis of adducts released by treating macroscopic amounts of protein (>10 mg) with alkali and methyl iodides (1) or with Raney nickel (14). To facilitate the rapid, sensitive, and specific detection of proteins bearing S-(2,5-dihydroxyphenyl) adducts, an antiserum to chemically synthesized immunogen 8 was raised in New Zealand white rabbits. Antibody specificity and titer were analyzed by titration of the antiserum against 2 µg of solid-phase metallothionein bearing the target hapten adduct as in adduct 9, the corresponding oxidized form as in adduct 10, or the unmodified metallothionein in ELISA (Chart 1). As shown in Figure 1, the antiserum was specific for S-(2,5dihydroxyphenyl)metallothionein (9) and did not bind to either its oxidized form (10) or unmodified metallothionein. The dramatically reduced affinity of the antibody toward the quinoid form 10, as compared to the hydroquinoid form 9, may stem from the very different hydrogen-bonding capabilities of phenolic hydroxyl groups versus carbonyl groups. A similar contrast is seen by comparing results with 13 versus 14-16 (see below). The specificity of the antibody-hapten reaction was next analyzed in a competitive ELISA by preincubation of the antiserum with varying concentrations of haptens 11-16 before analysis against microtiter wells coated

with 1 µg of the target antigen 9. As shown in Figure 2, the anti-HQ antibodies were inhibited most efficiently by the target hapten, (dihydroxyphenyl)mercapturic acid (11), which had an IC50 of 0.25 µM. Interestingly, the anti-HQ antibody also recognized the brominated analogue 12, which had an IC50 of 2 µM. Even hydroquinone itself (13) showed an IC50 value of 9 µM, but removal of the hydroxyl moieties from the target hapten, as in 14, eliminated antibody recognition altogether (IC50 . 1000 µM). Finally, neither N-acetyltyrosine (15) nor (p-bromophenyl)mercapturic acid (16), a model for epoxidederived adduct 5, was recognized by the antibodies. Western blot analysis of bromobenzene-adducted microsomal proteins using the anti-HQ antiserum was complicated by the sensitivity of the dihydroxyphenyl moiety toward oxidation to the corresponding quinone under the mild basic conditions (pH 8.8) routinely used for SDS-PAGE. Oxidation of the dihydroxyphenyl group interferes with recognition by the anti-HQ antibodies, as indicated by the results from the ELISA shown in Figure

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Rombach and Hanzlik

Figure 3. Analysis of fractionated microsomal proteins by phosphorimaging and Western blotting. DEAE column fractions 2-1, 2-2, and 3-1, along with model adduct 9 (lane MT) as a positive control, were separated on SDS-PAGE and probed by Western analysis (panel B) and phosphorimaging of the same blot (panel A). A duplicate gel was run and probed using antiserum preincubated with hapten 11 (panel C).

Figure 4. Analysis of fractionated microsomal proteins by phosphorimaging and Western blotting. DEAE column fractions 0-1, 1-1, 1-3, and 1-4 were separated on SDS-PAGE and probed by Western analysis (panel B) and phosphorimaging of the same blot (panel A). A duplicate gel was run and probed using antiserum preincubated with hapten 11 (panel C).

Figure 5. Analysis of fractionated microsomal proteins by phosphorimaging and Western blotting. DEAE column fractions 4-1, 4-2, 4-3, 5-3, 6-1, and 7 were separated on SDS-PAGE and probed by Western analysis (panel B) and phosphorimaging of the same blot (panel A). A duplicate gel was run and probed using antiserum preincubated with hapten 11 (panel C).

1. To avoid oxidation of the hapten moiety, proteins were separated by SDS-PAGE using gels prepared at pH 7.0 rather than at pH 8.8. This seemingly small change in pH had a major effect on the course of the electrophoresis, with a marked reduction in separation of proteins of MW g 45 kDa. While the modified SDS-PAGE made it possible to detect the positive control antigen 9 in Western blot analysis quite well, the antibodies failed to detect such adducts in samples taken directly from in vitro microsomal incubations with bromobenzene. This failure may have been due to a low concentration of the specific adduct versus the other adducts within the protein bands. To overcome this problem, microsomes incubated in vitro with [14C]BB were detergent-solubilized and the proteins fractionated by DEAE chromatography (see Table 1). Pooled DEAE column fractions were then examined by both Western blot analysis to detect the specific adducts and 14C-phosphorimaging to detect all adducts. In all cases duplicate gels were run and the proteins transblotted to nitrocellulose membranes. One membrane was then probed with antiserum, and as a control, the other

was probed using antibody that had been preincubated with hapten 11. It was therefore possible to compare directly, for the same blot, the detection of specific adducts by Western blotting versus general detection by phosphorimaging of BB-derived radioactivity. The clearest example of specific Ab recongnition of BBderived S-(dihydroxyphenyl) adducts to microsomal proteins can be seen in Figure 3. As shown in panel A, the phosphorimage analysis indicates that lanes 2-1 and 2-2 each contain two bands of protein which bear significant radiolabel while lane 3-1 contains only one such band. Based on their similarities in molecular weight and the fact that the three lanes shown represent sequential fractions from the DEAE column, it is likely that the band at ca. 60000 MW in lanes 2-1 and 2-2 contains primarily the same protein(s). In contrast, the protein bands at ca. 50 kDa in all three lanes must contain some differently radiolabeled proteins, because only the two bands in lanes 2-2 and 3-1 react with antibody (panel B). Preincubation of the antibody with hapten 11 abolishes reaction toward these bands and toward the positive control protein S-(2,5-dihydroxyphenyl)metal-

Antibody Detection of Benzoquinone-Protein Adducts Table 1. Summary of DEAE Fractionation of Adducted Microsomal Proteins column conditions

column results

[NaCl] (M)

fraction numbers

pool number

0a 0.05 0.05 0.05 0.05 0.1 0.1 0.15 0.15 0.2 0.2 0.2 0.3 0.3 0.3 0.5 0.5 1.0

20-65a 71-76 77-78 79-82 83-90 91-98 100-117 118-122 123-134 136-142 144-147 148-151 152-157 158-159 160-173 174-176 177-183 184-202

0-1a 1-1 1-2 1-3 1-4 2-1 2-2 3-1 3-2 4-1 4-2 4-3 5-1 5-2 5-3 6-1 6-2 7-1

a

14C

eluted (nCi) 2027 15.8 5.4 20.7 31.4 50.6 47.9 19.4 61.3 16.8 12.8 8.1 8.1 18.9 60.5 10.4 2.2 10.5

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In conclusion, results presented above demonstrate that we have produced a high-titer polyclonal antibody that specifically recognizes hydroquinone moieties covalently adducted to protein sulfhydryl groups. Such adducts arise via Michael addition of protein-SH groups to benzoquinone, a reactive electrophilic metabolite of benzene, phenol, hydroquinone, acetaminophen, and bromobenzene. In Western blots these antibodies recognize a small subset of those microsomal proteins which become radiolabeled upon incubation in vitro with [14C]bromobenzene, and the binding is specifically inhibited by excess free hapten. Efforts to identify these proteins are currently underway in our laboratory.

Acknowledgment. We thank Nathan Parker for skilled technical assistance with the ELISA experiments. Financial support for this work was provided by NIH Grant GM-21784.

Column-loading flow-through.

lothionein (Figure 3, panel C). Finally, since the antibody fails to recognize the 60-kDa protein bands in lanes 2-1 and 2-2, the radioactivity associated with these bands must be attributed to protein adducts other that those formed by reaction of the benzoquinone metabolite of bromobenzene with protein sulfhydryl groups. Figure 4 shows a similar analysis of antibody detection of S-(dihydroxyphenyl) protein adducts versus detection of total adducts by 14C-phosphorimaging applied to earlier fractions from the DEAE column. All four fractions show a major 14C-containing band at ca. 50 kDa (panel A). All four of these bands are detected by the anti-HQ Ab (panel B), but not if the Ab is preincubated with hapten (panel C), indicating that antibody recognition of these proteins is hapten-specific. In addition it appears that across these fractions the intensity of antibody binding does not correlate with 14C content, suggesting that at this level of protein fractionation, the 50-kDa band may also contain adducts not recognized by the antibody. A further example of differential detection of adducts by antibody versus phosphorimaging methods may be seen in the band at ca. 55 kDa in lane 1-3 of Figure 4. Although this band, indicated with an arrowhead in panel A, has a low but definite 14C content, it shows a strong antibody reaction (panel B) which is blocked by preincubating the antibody with hapten (panel C). The band at ca. 58 kDa in lane 1-1 of panel C is the only example we have seen of antibody binding to protein in the presence of hapten and probably represents a nonspecific cross-reacting antibody present in the antiserum used. A third example comparing antibody versus phosphorimaging detection of microsomal proteins adducted by bromobenzene metabolites is shown in Figure 5. Most obvious are two dark bands near 46 kDa in lanes 4-2 and 4-3 of panel B, which correspond to weakly radiolabeled bands indicated by arrowheads in these lanes of panel A. Several additional antibody-detected bands also appear in lanes 4-1, 5-3, and 7, all of which contain low but definite levels of 14C. None of these bands are detected by antibody preincubated with hapten (panel C). Finally, the four most-densely 14C-labeled bands in panel A (lanes 4-1, 4-2, 4-3, and 5-3) are not detected by the antibody and must therefore contain adduct species other than the S-(2,5-dihydroxyphenyl) protein type.

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