Detection of Adducts of Bromobenzene 3,4-Oxide with Rat Liver

14 Jan 1999 - Department of Medicinal Chemistry, University of Kansas, Lawrence, Kansas .... dine (Kirkegaard and Perry), BioMax MS autoradiography fi...
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Chem. Res. Toxicol. 1999, 12, 159-163

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Detection of Adducts of Bromobenzene 3,4-Oxide with Rat Liver Microsomal 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 24, 1998

The hepatotoxicity of bromobenzene (BB) has been attributed to covalent modification of cellular proteins by reactive metabolites generated during its oxidative biotransformation. Much of the net covalent binding which occurs originates via quinone metabolites, but bromobenzene 3,4-oxide (BBO), which is the reactive metabolite thought to be most significant toxicologically, also arylates protein side chains, although to a lesser extent. To facilitate the detection, isolation, and identification of rat liver proteins specifically adducted by BBO, we raised polyclonal antibodies capable of recognizing S-(p-bromophenyl)cysteine moieties (anti-BP) by immunizing rabbits with p-bromophenylmercapturic acid conjugated to keyhole limpet hemocyanin. The antiserum had a high titer, showed a high specificity for hapten in competitive ELISA with hapten analogues, and performed well in Western blot experiments using synthetically haptenized control proteins. When used for Western analysis of protein fractions from in vitro incubations of rat liver microsomes with [14C]BB, affinity-purified anti-BP recognized a limited number of bands, each of which also contained 14C. One of these bands corresponds to hydrolase B, a nonspecific esterase known to contain one free sulfhydryl group and previously shown to be a target protein for [14C]BB metabolites.

Introduction The hepatotoxicity of bromobenzene (BB)1 has been associated with the covalent modification of cellular proteins by reactive intermediates generated during its biotransformation (1-3). Brodie et al. initially speculated that bromobenzene 3,4-oxide (BBO) was the reactive metabolite responsible for protein alkylation (1), but subsequent work suggested that quinone metabolites might also be involved (4-7). More recently, detailed analysis of liver proteins from rats treated with bromobenzene provided definitive evidence for the alkylation of protein-SH groups by several quinone and bromoquinone metabolites of bromobenzene (8, 9), and for the alkylation of cysteine (8-10), histidine (11), and lysine (11) side chains by BBO. However, only three protein targets for reactive BB metabolites have been identified to date; they include a subunit of a cytosolic glutathione transferase (12) and two closely related isoforms of a microsomal esterase (13). Administration of [14C]-p-bromophenol to rats also results in extensive covalent labeling of liver proteins (14). Presumably, much of this is due to 1,4-benzoquinone (BQ), a known major metabolite of p-bromophenol (15). Despite this covalent binding, the striking observation * To whom correspondence should be addressed. Telephone: (785) 864-3750. Fax: (785) 864-5326. E-mail: [email protected]. 1 Abbreviations: BB, bromobenzene; BBO, bromobenzene 3,4-oxide; BSA, bovine serum albumin; DMSO, dimethyl sulfoxide; EDC, 1-[3′(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride; ELISA, enzyme-linked immunosorbent assay; KLH, keyhole limpet hemocyanin; KPBS, potassium phosphate-buffered saline (composition varies; see the text); KPBS-T, potassium phosphate-buffered saline with Tween-20 (composition varies; see the text); PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate.

that p-bromophenol is not hepatotoxic (14) has cast doubt on the role of quinone metabolites and their covalent binding in the mechanism of toxicity of bromobenzene. This suggests that if identification of protein targets for reactive bromobenzene metabolites is to be pursued as a means of elucidating mechanisms of bromobenzeneinduced toxicity, it would be important to distinguish those proteins specifically alkylated by BBO from those alkylated by quinone metabolites. To help make this distinction, we raised antibodies capable of detecting proteins bearing hydroquinone-like S-(2,5-dihydroxyphenyl)cysteine neoantigens. This antibody (anti-HQ) was shown in Western blotting to detect specifically and selectively a subset of those proteins that become radiolabeled during incubation of rat liver microsomes with [14C]bromobenzene and an NADPHgenerating system (16). We subsequently analyzed liver microsomal proteins from rats treated in vivo with [14C]bromobenzene and observed overall patterns of protein radiolabeling and reaction with anti-HQ antibody similar to those seen in the in vitro experiment (unpublished results). In this paper, we report the generation and characterization of a polyclonal antibody (anti-BP) which selectively and specifically recognizes S-(p-bromophenyl)cysteine moieties (viz. structures 1-4 in Figure 1). Using this antibody, we probed by Western blotting the same mixture of 14C-labeled rat liver microsomal proteins used in our earlier work with the anti-HQ antibody. The results show that significantly fewer proteins contain S-(p-bromophenyl)cysteine moieties than contain S-(2,5dihydroxyphenyl)cysteine moieties. In agreement with earlier alkaline permethylation results (8, 9), the overall level of p-bromophenylthio epitopes is much lower than

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Figure 1. Structures of target neoantigen 1, immunogen 2, model adduct 3, and competing ligands 4-13.

the level of 2,5-dihydroxyphenylthio epitopes. It is anticipated that this anti-BP antibody will aid efforts to detect and identify target proteins likely to be causally involved in bromobenzene hepatotoxicity.

Experimental Section Sources of Reagents. Electrophoresis and blotting reagents and Affigel 102 were purchased from Bio-Rad. Super Signal Ultra reagent, the BCA protein assay kit, and horseradish peroxidase-conjugated goat anti-rabbit IgG (H & L) were purchased from Pierce. Keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), metallothionein, Freund’s adjuvants, normal goat serum, and all chemicals used in the preparation of buffers were purchased from Sigma. Other reagents and supplies were purchased as indicated: Immunolon 4 96-well microtiter plates (Fisher), 3,3′,5,5′-tetramethylbenzidine (Kirkegaard and Perry), BioMax MS autoradiography film (Kodak), 1-[3′-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (EDC) (Aldrich), and Ultra Gold XR scintillation fluid (Packard). L-(p-Bromophenyl)mercapturic acid was synthesized as described previously (17). Synthesis of Immunogen 2 and Preparation of Antisera. Activation of l-(p-bromophenyl)mercapturic acid (4) for coupling to KLH was achieved by adding EDC (127 µmol) slowly to a stirred solution of 4 (63 µmol) in 8 mL of 50% (v/v) DMSO in sodium phosphate buffer (10 mM, pH 5.0). After being stirred for 10 min, this mixture was added dropwise to a stirred solution of 20 mg of keyhole limpet hemocyanin in 10 mL of sodium phosphate buffer (0.2 M, pH 8.0). The reaction mixture was incubated for 48 h while it was mixed gently, dialyzed against distilled H2O (7 × 1000 mL), and lyophilized to yield the final KLH-hapten adduct 2. Alkaline permethylation analysis (8, 9) of the product indicated a label density of 7 mol of hapten per mole of protein. Rabbits were immunized with 100 µg of 2 injected subcutaneously as an emulsion with complete Freund’s adjuvant, after which they were boosted twice at 2 week

Rombach and Hanzlik intervals with the same immunogen emulsified in incomplete Freund’s adjuvant. Antiserum (anti-BP) was collected 14 days after the last boost and assayed for binding to model adduct 3 in ELISA. Synthesis of Coating Antigen 3. As described above, EDC (317 µmol) was added slowly to a stirred solution of L-(pbromophenyl)mercapturic acid (157 µmol) dissolved in 16 mL of 50% (v/v) DMSO in sodium phosphate buffer (10 mM, pH 5.0). After addition was complete, the mixture was stirred for 10 min and added dropwise to a stirred solution of bovine serum albumin (50 mg) in 20 mL of sodium phosphate buffer (0.2 M, pH 8.0). This mixture was then incubated for 48 h while it was mixed gently, dialyzed against distilled H2O (7 × 2000 mL), and lyophilized to yield 66 mg of BSA-hapten adduct 3. Alkaline permethylation analysis (8, 9) of the product indicated a label density of 37 mol of hapten per mole of protein. Enzyme-Linked Immunosorbent Assay. The serum of rabbits immunized with 2 was analyzed for anti-bromophenyl (anti-BP) antibody titer by ELISA employing 3 as the solid phase antigen. Wells of 96-well Immunolon 4 microtiter plates were coated by addition of 100 µL of 3 at 5, 1, 0.5, 0.1, and 0.05 µg/mL in KPBS buffer [25 mM potassium phosphate and 150 mM NaCl (pH 7.5)]. The wells were then blocked with 200 µL aliquots of 5% (w/v) 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, and 0.5% poly(vinyl alcohol) (pH 7.4)], 100 µL aliquots of anti-BP antiserum serially diluted from 1/1000 to 1/150000 in KPBS-T were added and the plates were incubated for 1 h at 37 °C. Wells were again washed and preincubated for 20 min with KPBS-T containing 1% (v/v) normal goat serum and 1% (w/v) nonfat dry milk, followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit 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. The plates were read at 495 nm. Competitive ELISAs were performed as above except that wells were coated with 0.05 µg of 3 and the anti-BP antiserum was diluted to 1/20000 and preincubated overnight at 4 °C in the presence of competing ligands 4-13 at concentrations of 0.001-1000 µM. In parallel control experiments, anti-BP antiserum preincubated similarly but without competing ligands showed no diminution in effective antibody titer or binding. Purification of Hapten Specific Anti-pBrPMA Abs. AntiBP antibodies were purified for use in Western blot analysis by adsorption on a (p-bromophenyl)mercapturic acid affinity column prepared as follows. EDC (57 mg, 300 µmol) was added slowly to a stirred solution of l-(p-bromophenyl)mercapturic acid (4; 47.8 mg, 150 µmol) in a mixture of DMSO (1 mL) and potassium phosphate buffer (1 mL, 20 mM, pH 5.0), and the solution was incubated for 30 min at ambient temperature, after which the insoluble fraction was removed by centrifugation. The clarified supernatant was added dropwise to 20 mL of a 50% suspension of Affigel 102 in potassium phosphate buffer (0.2 M, pH 7.5) containing DMSO (50% v/v). The slurry was incubated at ambient temperature for 3 h while it was mixed gently on a tube rotator, after which it was poured into a column and washed with 3 column volumes of 100% DMSO (to remove any unbound hapten) and 6 volumes of buffer [25 mM potassium phosphate and 150 mM NaCl (pH 7.5)], after which the column was washed sequentially with 20 mL volumes of Tris buffer (10 mM, pH 7.5), Tris buffer containing NaCl (0.5 M), glycine buffer (100 mM, pH 2.5), Tris buffer (10 mM, pH 8.8), and aqueous triethylamine (100 mM, pH 11.5). Finally, the column was equilibrated in Tris buffer (10 mM, pH 7.5) prior to being used. For antibody purification, a 2 mL aliquot of anti-BP antiserum was diluted to 20 mL with Tris buffer (10 mM, pH 7.5) and loaded onto the L-(p-bromophenyl)mercapturic acid column (column volume of 10 mL). The column was washed sequentially with 20 mL volumes of Tris buffer (10 mM, pH 7.5), Tris buffer containing NaCl (0.5 M), glycine buffer (100 mM, pH 2.5), Tris buffer (10 mM, pH 8.8), and finally aqueous triethylamine (100

Antibodies to Bromobenzene Epoxide Adducts mM, pH 11.5). Fractions containing protein (A280) were assayed for protein content using BCA reagent and for binding to 3 using ELISA. The highest-affinity anti-BP antibodies eluted from the column in the 100 mM triethylamine fractions; they were pooled, dialyzed against 25 mM KPBS buffer, aliquoted into small vials, and stored frozen at -20 °C for later use in Western blot analyses. Western Blot Analysis Using Rabbit Anti-pBrPMA Abs. Proteins were separated by SDS-PAGE on a 4 to 20% gradient mini-gel and electrophoretically transferred to PVDF membranes using standard conditions. The membranes were blocked for 1 h with KPBS-T containing 5% (w/v) nonfat dry milk and incubated for 2 h with anti-BP antibody diluted to 2.7 µg of protein/mL in KPBS-T containing 1% (w/v) nonfat dry milk. The membranes were washed (3 × 15 min and 2 × 5 min) with KPBS-T and incubated for 20 min in KPBS-T containing 1% (v/v) normal goat serum and 1% (w/v) nonfat dry milk and then for 2 h with horseradish peroxidase-conjugated goat anti-rabbit IgG (diluted 1/100000) in KPBS-T containing 1% (v/v) normal goat serum and 0.1% (w/v) nonfat dry milk. After being washed with KPBS-T, the bound antibody was detected by exposing the gel to Super Signal Ultra reagent and then to BioMax MS autoradiography film. Competitive Western blot analyses were performed in a similar manner except that the anti-BP antibody was preincubated overnight at 4 °C with 1000 µM 4. Radiolabeled proteins on Western blot membranes were detected by phosphorimaging of the membrane using a Molecular Dynamics storage phosphor screen, scanning unit, and software. Treatment of Animals and Preparation of Microsomes. Male Sprague-Dawley rats (180 g, Charles River Laboratories, Wilmington, MA) were housed in a temperature- and humiditycontrolled room with a 12 h light/dark cycle and ad libitum access to food and water. After being acclimated for at least 3 days, 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 and microsomes prepared as described previously (13, 16). Incubations and Protein Purification. Incubations and protein purification were carried out as previously described (13, 16). In brief, a mixture containing microsomal protein (120 mg), [14C]BB (80 µmol, 5.17 Ci/mol, delivered in 400 µL of acetonitrile), and a freshly prepared NADPH-generating system in a final volume of 40 mL of buffer [100 mM potassium phosphate and 1 mM EDTA (pH 7.4)] was incubated at 37 °C for 90 min. Incubations were terminated by chilling the incubation flask in an ice bath. The microsomes were collected by centrifugation at 100000g and washed by two cycles of homogenization in 50 mM potassium phosphate (pH 7.4), followed by centrifugation at 100000g. The proteins were detergent-solubilized and fractionated by DEAE or High Q chromatography as described previously (13, 16).

Results and Discussion The hepatotoxicity observed in mammals treated with bromobenzene correlates with and has been attributed to the covalent modification of hepatocellular proteins by chemically reactive bromobenzene metabolites, especially BBO. To distinguish target proteins containing BBO adducts from those containing other types of adducts (e.g., quinone-derived), we generated antibodies to the p-bromophenylthio epitope (viz. 1-4) by immunization of New Zealand white rabbits with (p-bromophenyl)mercapturic acid-KLH conjugate 2. To characterize the resulting anti-BP antiserum, we evaluated its reaction at dilutions from 1/1000 to 1/150000 using 0.05-5.0 µg of coating antigen 3 in ELISA. These data (not shown) indicated optimum results were obtained using 0.05 µg

Chem. Res. Toxicol., Vol. 12, No. 2, 1999 161 Table 1. Effects of Competing Ligands on Anti-BP Antibody Binding to (p-Bromophenyl)mercapturic Acid Conjugated to Bovine Serum Albumin in ELISA ligand

inhibition of antibody binding

4 5 6 7 8 9 10 11 12 13

IC50 ) 1.7 µM IC50 ) 12 µM 19% inhibition at 1000 µM 42% inhibition at 1000 µM 7% inhibition at 1000 µM no inhibition at 1000 µM no inhibition at 1000 µM no inhibition at 1000 µM 43% inhibition at 1000 µM IC50 ) 280 µM

of 3/well and a 1/20000 dilution of antiserum from rabbit 26 (one of four immunized with 2). To examine the specificity of the anti-BP antibodies, competitive ELISAs were performed using antiserum diluted 1/20000 and incubated overnight in the presence of competing ligands 4-13 at concentrations of 0.0011000 µM prior to analysis for binding to 3; the results are listed in Table 1. As anticipated, the target hapten, (p-bromophenyl)mercapturic acid (4), is a very efficient inhibitor of antibody binding, having an IC50 value of 1.7 µM (170 pmol/well). However, the antibody is sensitive to even minor structural changes in the p-bromophenylthio epitope. Merely relocating the bromine atom to the meta position, as in 5, causes a 7-fold drop in affinity, while moving it to the ortho position (6) or removing it altogether (7) causes much larger decreases. Removal of the sulfur atom from 4 to create 8, or replacing it with an amine function as in 12, all but abolishes antibody recognition, indicating that the sulfur moiety per se is a significant part of the overall epitope. Aromatic structures found in the naturally occurring aromatic amino acids tyrosine (9) and phenylalanine (10), or in benzoquinone-cysteine adducts such as 11, are not detectably recognized by the anti-BP antibody. Finally, p-bromophenyl adducts of BBO with lysine (12) or histidine (13) interact relatively weakly with the antibody. This feature of the antibody is not necessarily undesirable, however, as the purpose of the antibody is to detect proteins bearing BBO adducts, not just BBO-cysteine adducts similar to 4. As expected, the anti-BP antiserum performed well in Western blot experiments using protein conjugate 3 as a positive control (data not shown). When used for Western analysis of proteins from rat liver microsomes incubated in vitro with [14C]bromobenzene, however, it failed to give any positive responses, even though p-bromophenyl-S-protein adducts were shown to be present in the mixture by means of alkaline permethylation analysis. Since the latter analyses are usually carried out on a scale of g10 mg of protein while SDS-PAGE/ Western analyses often involve only a few micrograms of protein per spot, and since p-bromophenyl-S-protein adducts typically comprise only 1% of all adducts, we surmised that the failure of our anti-BP antiserum to detect adducts on microsomal proteins directly was most likely due to a very low absolute abundance of the epitopes of interest in the sample on the blot. To overcome this suspected limitation, the anti-BP antibodies were affinity-purified from the antiserum for use in Western blotting (see above) and solubilized microsomal proteins were subjected to fractionation by either DEAE or High Q chromatography [for details of

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Figure 2. Phosphorimaging and Western blotting analysis of microsomal proteins fractionated by DEAE column chromatography (fraction 1-4). Proteins were separated on SDS-PAGE in a 12% running gel, transblotted onto nitrocellulose, and probed by Western analysis (panel B) using affinity-purified anti-BP antibody. The phosphorimage of the same blot is shown in panel A. A blot of a duplicate gel that was probed using antibody preincubated with hapten 4 is shown in panel C. The blots are shown at actual size.

these two chromatographic separations and the pooling of various fractions, see refs 16 and 13, respectively]. Fractions enriched in 14C-adducted proteins were pooled, concentrated, and examined via SDS-PAGE. In all cases, duplicate gels were run simultaneously and the proteins transblotted to nitrocellulose or PVDF membranes. One membrane was probed with affinity-purified anti-BP antibodies, and as a control, the other was probed with the same antibodies after preincubation with ligand 4. Although many of the various pools of column fractions contained 14C adducts, several of which reacted with antibodies against HQ-type adducts related to ligand 11 (13), only two of 18 pools of DEAE column fractions (pool 1-4, eluted with 0.05 M NaCl, and pool 5-1, eluted with 0.3 M NaCl) and one of four pools of High Q column fractions (pool 1, the nonretained column flow-through) contained proteins specifically recognized in Western blots by affinity-purified anti-BP antibodies. Figure 2 shows results from Western blot analysis of DEAE column fraction 1-4 made using the purified antiBP antibodies and the phosphorimage analysis of the same membrane. As seen in panel B, the antibody reacted with two protein bands in this fraction, and the reaction was inhibited when the antibody was preincubated with ligand 4 (panel C). Significantly, both of the bands detected by the antibody are radiolabeled (panel A), confirming that they do contain bromobenzene-derived residues. In contrast, the most densely 14C-labeled band and several faintly 14C-labeled bands in this fraction are not detected by anti-BP antibodies. Figure 3 shows results from Western and phosphorimaging analysis of DEAE fraction 5-1. There is a strong antibody reaction in a broad band around 67 kDa (panel B). This reaction is completely blocked by preincubation of the antibody with ligand 4 (panel C), but the phosphorimage (panel A) shows only a slight amount of radioactivity above background in this region. Two other protein bands around 42 and 32 kDa are also detected by the antibody (panel B). These bands are clearly radiolabeled, and the extent of their reaction with antibody is significantly diminished by preincubating the antibody with ligand 4. As a final example, the reaction of affinity-purified anti-BP with proteins in the pool 1 fraction from the High

Rombach and Hanzlik

Figure 3. Phosphorimaging and Western blotting analysis of microsomal proteins fractionated by DEAE column chromatography (fraction 5-1). Proteins were separated on SDS-PAGE in a 12% running gel, transblotted onto nitrocellulose, and probed by Western analysis (panel B) using affinity-purified anti-BP antibody. The phosphorimage of the same blot is shown in panel A. A blot of a duplicate gel that was probed using antibody preincubated with hapten 4 is shown in panel C. The blots are shown at actual size.

Figure 4. Phosphorimaging and Western blotting analysis of microsomal proteins fractionated by High Q column chromatography (pool 1). Proteins were separated on triplicate SDSPAGE gels using a 7.5% running gel to enhance separation of proteins in the 45-66 kDa range (13). Proteins were transferred to nitrocellulose for Western analysis using affinity-purified anti-BP antibody (panel B) and antibody preincubated with hapten 4 (panel C). Proteins from the third gel were transferred to a PVDF membrane and stained with Coomassie dye (panel D). The phosphorimage of the nitrocellulose blot in panel B is shown in panel A. The single arrowhead points to the band for hydrolase A; double arrowheads point to the band for hydrolase B. The blots are shown at actual size.

Q column is shown in Figure 4. This fraction is especially interesting because it contains two esterase isozymes shown previously to be target proteins for reactive metabolites of [14C]bromobenzene, namely, hydrolase A (57 kDa) and hydrolase B (59 kDa) (13). Figure 4 shows a Coomassie-stained blot of an SDS-PAGE analysis of this fraction; protein bands previously shown (13) to contain hydrolase A and hydrolase B are indicated by the single and double arrowheads, respectively. In the Western analysis of this fraction (panel B), two protein bands near 58 and 48 kDa are seen to react strongly with affinity-purified anti-BP, and these reactions are almost completely blocked by ligand 4 (panel C). The fact that the upper band in panel B contains hydrolase B is supported by the phosphorimage (panel A) which shows

Antibodies to Bromobenzene Epoxide Adducts

the two hydrolase isozymes, indicated by single and double arrowheads, respectively, resolved as separate radioactive bands. Hydrolase B has one free sulfhydryl group, while hydrolase A, which is ca. 3 times as abundant as hydrolase B in rat liver microsomes, has none (18). Despite its lower abundance, the sulfhydryl-containing hydrolase B becomes radiolabeled by bromobenzene metabolites to a much greater extent than hydrolase A (13). At present, we cannot exclude the possibility that a portion of the net covalent aduction of hydrolase B can be attributed to quinone metabolites of bromobenzene, but the fact that the specificity of our anti-BP antibody includes the sulfur atom of the p-bromophenylthio moiety allows us to conclude that the unique sulfhydryl group of hydrolase B is a quantitatively significant target for BB 3,4-oxide. Further experiments will be needed to evaluate the potential toxicological significance of this covalent binding reaction. Hydrolase A was recently shown to be a target for reactive metabolites of the thiolcarbamate herbicide molinate (19). In this case, the enzyme was inactivated, presumably by carbamoylation of its active site serine nucleophile, by S-oxidized metabolites of molinate. This inactivation was suggested to underlie metabolic disturbances in steroid and lipid metabolism induced by molinate and, possibly, its cytotoxic effects. On the other hand, there is no detectable loss of hydrolase B activity in microsomes labeled with BB metabolites by incubation with BB in vitro (13). This is consistent with its mechanism of action as a serine hydrolase of the “B-esterase” type and with the fact that B-esterase enzymes are not significantly inhibited by mercury compounds (20-22). On the surface, then, it would appear that alkylation of the single sulfhydryl group on hydrolase B by BBO is probably without toxic consequences for the cell; it may even constitute a form of detoxication by sparing other more important target proteins from alkylation by BBO. In conclusion, we have produced a high-titer polyclonal antibody that specifically recognizes p-bromophenylthio moieties derived via the alkylation of rat liver protein SH groups by bromobenzene 3,4-oxide. When used in Western blot experiments after affinity purification, this antibody recognizes a small subset of the many hepatic proteins labeled with metabolites of [14C]bromobenzene, and the reaction is specifically blocked by preincubating the antibody with free hapten. One of the few adducted proteins it recognizes proved to be hydrolase B, a rat liver microsomal protein previously identified as a target for [14C]bromobenzene metabolites. Hydrolase B hydrolyzes a number of physiological as well as xenobiotic esters, but since it does not appear to be inactivated by alkylation with bromobenzene metabolites, this alkylation seems unlikely to play an important role in the mechanism of bromobenzene-induced cytotoxicity. Nevertheless, it remains likely that some of the proteins modified by chemically reactive metabolites of bromobenzene do play a role in cytotoxicity. Antibodies that specifically recognize protein adducts derived from bromobenzene 3,4oxide may become a significant tool for finding and identifying those particular proteins.

Acknowledgment. We thank Dr. Larry Hall for performing the phosphorimage analyses mentioned herein and in our previous papers and Dr. Yakov Koen for helpful discussions and technical advice. Financial sup-

Chem. Res. Toxicol., Vol. 12, No. 2, 1999 163

port for this work was provided in part by NIH Grant GM-21784.

References (1) Brodie, B. B., Reid, W. D., Cho, A. K., Sipes, G., Krishna, G., and Gillette, J. R. (1971) Possible mechanism of liver necrosis caused by aromatic organic compounds. Proc. Natl. Acad. Sci. U.S.A. 68, 160-164. (2) Reid, W. D., and Krishna, G. (1973) Centrolobular Hepatic Necrosis Related to Covalent Binding of Metabolites of Halogenated Aromatic Hydrocarbons. Exp. Mol. Pathol. 18, 80-99. (3) Jollow, D. J., Mitchell, J. R., Zampaglione, N., and Gillette, J. R. (1974) Bromobenzene-Induced Liver Necrosis. Protective Role of Glutathione and Evidence for 3,4-Bromobenzene Oxide as the Hepatotoxic Metabolite. Pharmacology 11, 151-169. (4) Hesse, S., Wolff, T., and Mezger, M. (1980) Involvement of phenolic metabolites in the irreversible protein-binding of 14Cbromobenzene catalysed by rat liver microsomes. Arch. Toxicol. Suppl. 4, 358-362. (5) Narasimhan, N., Weller, P. E., Buben, J. A., Wiley, R. A., and Hanzlik, R. P. (1988) Microsomal metabolism and covalent binding of [3H/14C]-bromobenzene. Evidence for quinones as reactive metabolites. Xenobiotica 18, 491-499. (6) Buben, J. A., Narasimhan, N., and Hanzlik, R. P. (1988) Effects of Chemical and Enzymic Probes on Microsomal Covalent Binding of Bromobenzene and Derivatives. Evidence for Quinones as Reactive Metabolites. Xenobiotica 18, 501-510. (7) Weller, P. E., Narasimhan, N., Buben, J. A., and Hanzlik, R. P. (1988) In Vitro Metabolism and Covalent Binding Among OrthoSubstituted Bromobenzenes of Varying Hepatotoxicity. Drug Metab. Dispos. 16, 232-237. (8) Slaughter, D. E., and Hanzlik, R. P. (1991) Identification of epoxide- and quinone-derived bromobenzene adducts to protein sulfur nucleophiles. Chem. Res. Toxicol. 4, 349-359. (9) Slaughter, D. E., Zheng, J., Harriman, S., and Hanzlik, R. P. (1993) Identification of Covalent Adducts to Protein Sulfur Nucleophiles by Alkaline Permethylation. Anal. Biochem. 208, 288-295. (10) Weller, P. E., and Hanzlik, R. P. (1991) Isolation of S-(Bromophenyl)cysteine Isomers from Liver Proteins of BromobenzeneTreated Rats. Chem. Res. Toxicol. 4, 17-20. (11) Bambal, R. B., and Hanzlik, R. P. (1995) Bromobenzene-3,4-Oxide Alkylates Histidine and Lysine Side Chains of Rat Liver Proteins in Vivo. Chem. Res. Toxicol. 8, 729-735. (12) Aniya, Y., McLenithan, J. C., and Anders, M. W. (1988) Isozyme Selective Arylation of Cytosolic Glutathione S-Transferase by [14C]Bromobenzene Metabolites. Biochem. Pharmacol. 37, 251-257. (13) Rombach, E. M., and Hanzlik, R. P. (1998) Identification of a Rat Liver Microsomal Esterase as a Target Protein for Bromobenzene Metabolites. Chem. Res. Toxicol. 11, 178-184. (14) Monks, T. J., Hinson, J. A., and Gillette, J. R. (1983) Bromobenzene and p-bromophenol toxicity and covalent binding in vivo. Life Sci. 30, 841-848. (15) Zheng, J., and Hanzlik, R. P. (1992) Dihydroxylated Mercapturic Acid Metabolites of Bromobenzene. Chem. Res. Toxicol. 5, 561567. (16) Rombach, E. M., and Hanzlik, R. P. (1997) Detection of Benzoquinone Adducts to Rat Liver Protein-SH Groups Using Specific Antibodies. Chem. Res. Toxicol. 10, 1407-1411. (17) Zheng, J., and Hanzlik, R. P. (1992) Bromo(monohydroxy)phenyl mercapturic acids. A new class of mercapturic acids from bromobenzene treated rats. Drug Metab. Dispos. 20, 688-694. (18) Yan, B., Yang, D., Brady, M., and Parkinson, A. (1994) Rat Kidney Carboxylesterase. Cloning, Sequencing, Cellular Localization, and Relationship to Rat Liver Hydrolase. J. Biol. Chem. 269, 2968829696. (19) Jewell, W. T., and Miller, M. G. (1998) Identification of a Carboxylesterase as the Major Protein Bound by Molinate. Toxicol. Appl. Pharmacol. 149, 226-234. (20) LaDu, B. N., and Snady, H. (1971) Esterases of Human Tissues. Handb. Exp. Pharmacol. 28-2, 477-499. (21) Dueker, S. R., Lame´, M. W., and Segal, H. J. (1992) Hydrolysis of Pyrrolizidine Alkaloids by Guinea Pig Hepatic Carboxylesterases. Toxicol. Appl. Pharmacol. 117, 116-122. (22) Dueker, S. R., Lame´, M. W., and Segal, H. J. (1992) Hydrolysis of Pyrrolizidine Alkaloids by Guinea Pig Hepatic Carboxylesterases. Toxicol. Appl. Pharmacol. 117, 1407-1411.

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