Immunochemical Visualization and Identification of Rat Liver Proteins

Matthew Reed , Michael Monske , Fredine Lauer , Stephen Meserole , Jerry Born , Scott Burchiel ... Matthew Reed , Hideji Fujiwara , David C Thompson...
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Chem. Res. Toxicol. 1997, 10, 1109

1109

Immunochemical Visualization and Identification of Rat Liver Proteins Adducted by 2,6-Di-tert-butyl-4-methylphenol (BHT) Matthew Reed and David C. Thompson* Department of Medical Pharmacology & Toxicology, Texas A&M University Health Science Center, College Station, Texas 77843-1114 Received July 16, 1997X

Several alkylphenols (e.g., 2,6-di-tert-butyl-4-methylphenol, BHT) form reactive quinone methide intermediates (e.g., 2,6-di-tert-butyl-4-methylene-2,5-cyclohexadienone, BHT-QM) upon oxidation by cellular enzymes. In order to pursue the role of protein alkylation in alkylphenol toxicity, we used an immunochemical approach to identify protein targets alkylated by BHT. Synthetic BHT-N-acetylcysteine (BHT-NAC) was coupled to keyhole limpet hemocyanin and used as an antigen from which polyclonal antibodies were raised in New Zealand white rabbits. Rabbit serum contained an antibody which was highly specific for BHT-NAC, as determined by competitive ELISA. The BHT antibody was used as a probe to look for the presence of BHT-protein adducts in in vitro incubations with rat liver microsomes or tissue slices and also in vivo in liver tissue from male Sprague-Dawley rats exposed to BHT. Western blotting of protein gels revealed BHT-dependent protein alkylation over a wide molecular weight range. Prominent recurrent bands were observed at approximately 34.5, 52, 64.5, 74, and 97 kDa. Detection of adducts was inhibited in microsomal incubations by cytochrome P450 inhibitors, deuterated BHT, and the omission of NADPH. Similar protein alkylation patterns were observed in rat liver microsomes exposed to synthetically prepared BHT-QM as in the enzymemediated incubations. In rats gavaged with up to 1000 mg/kg BHT, the amount of protein alkylation observed was maximal at 24 h postdosing and was dose-dependent. Two alkylated proteins were isolated and identified by N-terminal sequencing: a mitochondrial β-oxidation enzyme, enoyl-CoA hydratase, and a plasma membrane/cytoskeletal linker protein from the ezrin/moesin/radixin family.

Introduction Many alkylphenols form quinone methides upon oxidation by cytochrome P450 (1-3). The formation of these reactive intermediates has been implicated in processes that ultimately lead to cytotoxicity, including the depletion of intracellular glutathione and the alkylation of cellular proteins (4, 5). Butylated hydroxytoluene1 (2,6di-tert-butyl-4-methylphenol, BHT) is a well-known and well-studied alkylphenol (6). It is primarily used by the food and cosmetic industries as an antioxidant and preservative in consumer goods. Even though BHT is relatively harmless at low doses, high doses have been shown to cause pulmonary toxicity in mice (7-9) and hepatotoxicity and hemorrhagic death in rats (10-12). In addition, BHT and BHTOOH (2,6-di-tert-butyl-4hydroperoxy-4-methyl-2,5-cyclohexadienone), a hydroperoxy metabolite of BHT, act as tumor promoters in mouse lung and mouse skin, respectively (13-15). Each of these effects of BHT has been linked to the formation and reactivity of the quinone methide metabolite. * Address all correspondence to this author. Tel: 409-845-8740. Fax: 409-845-0699. E-mail: [email protected]. X Abstract published in Advance ACS Abstracts, September 15, 1997. 1 Abbreviations: BHA, butylated hydroxyanisole, 2[3]-tert-butylanisole; BHT, butylated hydroxytoluene, 2,6-di-tert-butyl-4-methylphenol; BHTOOH, hydroperoxy BHT, 2,6-di-tert-butyl-4-hydroperoxy-4-methyl2,5-cyclohexadienone; BHT-QM, 2,6-di-tert-butyl-4-methylene-2,5-cyclohexadienone; BSA, bovine serum albumin; ECL, enhanced chemiluminescence; ELISA, enzyme-linked immunosorbent assay; Hepes, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; HRP, horseradish peroxidase; KLH, keyhole limpet hemocyanin; NAC, N-acetylcysteine; PAGE, polyacrylamide gel electrophoresis; PVDF, polyvinylidene difluoride; SDS, sodium dodecyl sulfate; SKF 525-A, 2-(diethylamino)ethyl 2,2′-diphenylvalerate.

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Although much work has been done elucidating the mechanisms of BHT toxicity in various target tissues, and specifically on the role of a quinone methide metabolite in BHT-induced pulmonary and liver toxicity, the relationship between alkylation of specific proteins and cell viability has not been addressed. Covalent binding of BHT metabolites to cellular macromolecules has been reported in a number of studies. Covalent binding of BHT to protein is metabolism-dependent and occurs in target tissues in both mice and rats (16-18). A pathway for the cytochrome P450-dependent oxidation of BHT to a reactive quinone methide intermediate and subsequent reaction with a nucleophilic protein residue is shown in Figure 1. Nakagawa et al. (19-22) observed that BHT was bound primarily to cysteine residues in liver proteins isolated from BHT-treated rats. However, these investigators did not attempt to identify specific proteins which were alkylated by BHT. In light of the recent advances in the use of immunochemical methods as a means of studying protein alkylation by reactive intermediates, such as halothane and acetaminophen (23), we have used a similar ap-

Figure 1. Proposed pathway of cytochrome P450-dependent activation of BHT in rat liver to a quinone methide and subsequent covalent binding to tissue proteins. © 1997 American Chemical Society

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proach to isolate an antiserum that is specific for BHTalkylated protein from both in vitro and in vivo experiments. It is the purpose of this study to present data which indicate that selective protein alkylation occurs in rat liver tissue exposed to BHT under a variety of conditions. The isolated antiserum was highly specific for BHT as indicated by competitive enzyme-linked immunosorbent assay (ELISA) and the lack of ability to detect proteins alkylated by structurally related alkylphenols. This antiserum was used to isolate and identify two proteins alkylated by BHT. The BHT antiserum described in this report will be useful in future studies to identify additional target proteins that are of toxicological relevance in the various deleterious effects mediated by BHT and, more generally, in quinone methidemediated toxicity.

Materials and Methods Materials. BHT, 4-methylphenol, 2-methoxy-4-allylphenol, BHA (2[3]-tert-butylanisole), and most other chemicals and cofactors for incubations were obtained from Sigma Chemical Co. (St. Louis, MO), except as noted below. Piperonyl butoxide, 2,4,6-trimethylphenol, 2-tert-butylphenol, 4-allyl-2,6-dimethoxyphenol, and 2,4-dimethylphenol were purchased from Aldrich (Milwaukee, WI), while 2-(diethylamino)ethyl 2,2′-diphenylvalerate (SKF 525-A) was obtained from Research Biochemicals International (Natick, MA), and 2,6-di-tert-butyl-4-ethylphenol and 6-tert-butyl-2,4-dimethylphenol were purchased from Pfaltz & Bauer (Waterbury, CT). Deuterated BHT (2,6-di-tert-butyl4-[R,R,R-d3]methylphenol) was available from a previous study (24) where it was synthesized as described by Mizutani et al. (25). Reanalysis by electron impact MS confirmed the stability of the deuterated compound: m/z (relative intensity) 223 (M+, 32), 208 (100), estimated content of hydrogen in methyl group 2.9%. Hydroperoxy BHT (BHTOOH, 2,6-di-tert-butyl-4-hydroperoxy-4-methyl-2,5-cyclohexadienone) was synthesized according to the method of Kharasch and Joshi (26). Materials for polyacrylamide gel electrophoresis (PAGE), transfer for Western blotting, and molecular weight standards were obtained from Novex (San Diego, CA). Visualization of Western blots was carried out using an enhanced chemiluminescence (ECL) detection kit in combination with horseradish peroxidase (HRP)coupled goat, anti-rabbit IgG from Amersham (Arlington Heights, IL). Conjugate Preparation and Immunization. In order to prepare a suitable immunogen for the preparation of polyclonal antisera, a BHT-N-acetylcysteine (BHT-NAC) conjugate was synthesized according to the method described by Bolton et al. (27) with the exception that N-acetylcysteine was used instead of glutathione. The structure of the BHT-NAC conjugate was confirmed by proton NMR (500 MHz) in deuterated dimethyl sulfoxide: δ 1.36 (s, 18H, tert-butyl), 1.83 (s, 3H, acetyl), 2.68 (dd, 1H, cys β), 2.90 (dd, 1H, cys β′), 3.61 (dd, 2H, benzylic CH2), 4.20 (m, 1H, cys R), 7.01 (s, 2H, ArH). BHT-NAC was subsequently coupled to keyhole limpet hemocyanin (KLH) using an Imject Immunogen Conjugation kit obtained from Pierce (Rockford, IL). The structure of the BHT-NAC-KLH antigen is shown in Figure 2. The antigen was lyophilized and dissolved in sterile Complete Freund’s Adjuvant, and 500 µg was injected into a pathogen-free New Zealand white rabbit. Subsequent injections of 250 µg in Incomplete Freund’s Adjuvant were given 2 and 4 weeks later. Serum was collected beginning 6 weeks after the initial injection. All antibody work, including immunization of rabbits, handling, and serum collection, was carried out at the Department of Veterinary Sciences, M. D. Anderson Cancer Center (Bastrop, TX). Serum samples were stored in small aliquots at -80 °C. Characterization of Anti-BHT Antibody Specificity by Competitive ELISA. A competitive ELISA was used to elucidate the specificity of the polyclonal antisera for BHT. Bovine serum albumin (BSA) was reacted directly with synthetic

Reed and Thompson

Figure 2. Structure of synthetic BHT-NAC-KLH antigen. BHT-QM [prepared as described by Bolton et al. (27)] to prepare a suitable antigen for the ELISA experiments. Each well of a 96-well ELISA plate was coated with 50 µL of BHT-BSA (dissolved in 0.1 M carbonate buffer, pH 9.6) at a final concentration of 500 ng of protein/well. The plate was allowed to dry overnight at 37 °C. Plates were subsequently washed three times with distilled water; then each well was individually blocked with 1% BSA borate buffer (1% BSA, 0.12 M NaCl, 0.05% Tween 20, 1 mM EDTA, 0.1 M borate, pH 8.5) for 30 min. After blocking, plates were washed three times with distilled water, wrapped in a paper towel, inverted, and vigorously tapped on a cushion of paper towels to remove water from each well. BHT antisera were diluted to a final concentration of 1:250 in borate blocking buffer and added 1 to 1 (1:500 final antisera dilution) to solutions of serially diluted competitive inhibitors. Inhibitors tested included BHT, 2-tert-butyl-4,6-dimethylphenol, 4-methylphenol, 2-tert-butylphenol, NAC, and BHT-NAC. Following a 1 h incubation at 25 °C, 50 µL of each antibody/inhibitor solution was added to duplicate wells of a “blocked” ELISA plate. Plates were allowed to incubate at room temperature for 2 h; the wells were emptied, washed with distilled water, and dried as above. After 10 additional min of blocking, followed by washing and drying, 50 µL of HRP-coupled anti-rabbit IgG solution (diluted 1:1000 in borate blocking buffer) was added to each well and allowed to incubate at room temperature for 2 h. Again, plates were washed and dried. Finally, 50 µL of 3,3′,5,5′-tetramethylbenzidine solution (10 mg/mL 3,3′,5,5′tetramethylbenzidine in distilled water, diluted 1:99 in 0.1 M acetate buffer, pH 5.6) was added to each well and allowed to incubate for 30 min. Subsequently, 50 µL of 2 M H2SO4 was added to each well and the absorbance read at 450 nm on an ELISA plate reader. Microsomal Incubations. Microsomes from male Sprague-Dawley rats (200-225 g) were prepared as previously described (28). Incubations contained an NADPH-regenerating system (1 mM NADP+, 5 mM isocitric acid, 0.2 unit/mL isocitric dehydrogenase), 5 mM MgCl2, and 2 mg/mL microsomal protein in 0.1 M Tris buffer (pH 7.5). Incubations were carried out at 37 °C for 6 h. All test compounds were used at a final concentration of 1 mM unless stated otherwise. Alternatively, some microsomal incubations contained a combination of 100 µM BHA, 100 µM BHT, and 1 mM BHTOOH in place of BHT and NADPH. The combination of a peroxide (BHTOOH) and phenol (BHA) has been shown to activate BHT to quinone methide via a peroxidase-dependent mechanism (29). All reactions were terminated and the proteins precipitated with icecold acetone, in order to extract unreacted BHT and metabolites. The protein mixture was centrifuged at 10000g for 10 min, and acetone was removed by aspiration. Thereafter, protein was resuspended in ice-cold acetone, sonicated, and processed, as described, two additional times. Afterward, protein concentration was determined by the Lowry method (30). Liver Slice Incubations. Precision-cut rat liver slices were prepared as previously described (5). Briefly, each slice was loaded onto a stainless steel screen, inserted into a Teflon cylinder, and placed into an incubation vial that contained 2.5 mL of incubation medium (Krebs/Hepes buffer, pH 7.4). Individual vials were separated into two groups of 12 slices (control and BHT-treated, respectively). BHT was added at a final

Selective Protein Alkylation by BHT in Rat Liver concentration of 1 mM, using dimethyl sulfoxide (1%, v/v) as the vehicle. Both groups were incubated at 37 °C for 3 and 6 h. Slices were removed, grouped collectively into vials containing ice-cold acetone, sonicated, and processed as above. In Vivo Studies. Male Sprague-Dawley rats (200-225 g) were gavaged with corn oil (vehicle control, 1 mL volume/rat) or 500-1000 mg/kg BHT dissolved in corn oil. Animals were sacrificed at various time points up to 36 h; the livers were removed and homogenized in 0.25 mL of sucrose, 0.1 M Tris buffer (pH 7.5). The whole liver homogenate was filtered through three layers of cheesecloth and fractionated into nuclear (700g pellet), mitochondrial (10000g pellet), microsomal (100000g pellet), and cytosolic (100000g supernatant) fractions by differential centrifugation. Nuclear fractions were further purified as described by Jones et al. (31). Each fraction was resuspended in buffer and processed as described above. Gel Electrophoresis and Western Blotting. Liver proteins were separated by molecular weight and analyzed by using one-dimensional sodium dodecyl sulfate (SDS)-PAGE as described by Laemmli (32). Samples of liver protein were combined with 2× conjugation buffer, boiled for 10 min, loaded into individual wells (25 µg/well) of a 4-20% Tris/glycine gel, and run at a constant 140 V for 1.5 h. Gels were removed and proteins transferred to polyvinylidene difluoride (PVDF) membranes at 30 V constant overnight (15-16 h). Membranes were removed and placed in blocking buffer (0.05 M Tris, pH 8.0, 0.2 M NaCl, 1% casein, 0.01% Thimersol). After blocking for 1-3 h, individual membranes were incubated for 1 h in BHT specific antisera diluted 1:50 with blocking buffer. The membrane was removed and washed for 3 × 1 min periods in wash buffer (0.05 M Tris, pH 8.0, 0.2 M NaCl, 0.05% Tween 20) followed by 3 × 1 min washes with distilled water. Membranes were subsequently incubated for 1 h in horseradish peroxidase-coupled, goat anti-rabbit IgG (diluted 1:1000 in blocking buffer). After three brief 10 s washes in wash buffer and three brief distilled water washes, the membranes were visualized by using an ECL detection kit and by exposing membranes to NEF 486 autoradiography film (New England Nuclear, Boston, MA). The developed film images were scanned into a computer and analyzed densitometrically using scanning software (1-D scan/ 0-D scan) from Scanalytics (Billerica, MA). Purification and Identification of BHT-Alkylated Proteins. The 34 kDa protein was partially purified from BHTtreated (1000 mg/kg) whole liver homogenate by ammonium sulfate precipitation followed by a combination of ion exchange and hydroxyapatite column chromatography. Rat liver was homogenized in buffer containing 0.5% Triton X-100, 300 mM NaCl, 1 mM EGTA, 0.25 mM phenylmethanesulfonyl fluoride, 10 µM E64 [trans-(epoxysuccinyl)-L-leucinamido(4-guanidino)butane], 1 mM benzamide, and 20 mM Tris-Cl, pH 7.4. The mixture was stirred over ice for 60 min and centrifuged at 20000g for 20 min. The supernatant was brought to 40% ammonium sulfate, and the precipitate was removed by centrifugation at 12000g for 20 min. The precipitate was resuspended and solubilized in buffer (10 mM imidazole, 20 mM dithiothreitol, pH 6.7) and dialyzed against the same buffer overnight at 10 °C. The solution was clarified at 30000g and then applied to an Econo-Pac CHT-II hydroxyapatite column (Bio-Rad Laboratories, Hercules, CA) that was preequilibrated with 100 mM potassium phosphate (pH 7.0). Fractions were eluted with a 40 mL linear gradient from 100 to 800 mM potassium phosphate, and 1 mL fractions were collected. Fractions containing the protein of interest were identified by Western blotting using the BHT antisera. Fractions rich in the 34 kDa protein were pooled and dialyzed overnight at 10 °C against 10 mM bis-Tris propane buffer (pH 6.7) containing 20 mM NaCl and 1 mM dithiothreitol. The solution was further applied to an Econo-Pac High-Q column (Bio-Rad Laboratories), developed with a 40 mL linear gradient of 20 mM to 1 M NaCl, and collected in 1 mL fractions. Fraction 3, which contained the desired protein, was concentrated to about 300 µL. An aliquot (12.5 µL) was then applied to a 10% polyacrylamide gel, electrophoresed, transferred to PVDF membrane, and

Chem. Res. Toxicol., Vol. 10, No. 10, 1997 1111 stained with Coomassie R-250. The 34 kDa band was excised and the N-terminus microsequenced at the Macromolecular Structure Facility, Department of Biochemistry, Michigan State University. The 76 kDa protein was isolated from BHT-treated (1000 mg/ kg) rat liver cytosol. Cytosol was stirred in a glass beaker over ice until the temperature reached 0-5 °C. Acetone (-20 °C) was added slowly, in 5 mL increments, up to a final concentration of 50% (v/v). The solution was spun for 10 min at 10000g, the supernatant was discarded, and the pellet was redissolved in 0.01 M Tris-HCl, pH 7.5; 1 mL of the protein solution was subjected to ion exchange chromatography using a 5 mL EconoPac High-Q ion exchange cartridge (Bio-Rad Laboratories). The 76 kDa protein was eluted in 1.5 mL fractions using 0.01 M Tris-buffered HCl run over a linear NaCl gradient from 0 to 1.0 M over 50 min. Fractions rich in the 76 kDa protein were pooled and concentrated using 30 kDa ultracentrifuge tubes (Micron Separations, Inc., Westboro, MA). The concentrated sample was run on a single-lane, 6% polyacrylamide minigel and then transferred to a PVDF membrane for 6 h at 125 mV constant voltage in transfer buffer containing 0.012 M Tris base and 0.1 M glycine, pH 8.3. The membrane was then stained with Coomassie R-250, and the protein band corresponding to the BHT-modified 76 kDa protein was excised and the Nterminus sequenced as described above. In order to confirm the identity of ezrin, mouse anti-ezrin monoclonal antibody (Chemicon International, Temecula, CA) was used in Western blotting experiments performed under the same conditions described above except that HRP-coupled, anti-mouse antibody (donkey) was used as the secondary antibody.

Results Specificity of BHT Antisera. In order to prepare polyclonal antisera which would recognize proteins alkylated by BHT, a BHT-NAC conjugate was prepared based on literature evidence that protein adducts involve the attachment of nucleophilic (mainly thiol) residues to the benzylic carbon of BHT (21, 33). The BHT-NAC conjugate was coupled to KLH, and the resulting complex was used for immunization of rabbits. The structure of the BHT-NAC-KLH conjugate is shown in Figure 2. Rabbit antisera contained an antibody which was specific for BHT as determined by a competitive ELISA. Synthetic BHT-QM was added directly to bovine serum albumin (BSA), and the resulting conjugate (BHT-BSA) was used as a solid-phase antigen for the ELISA. Rabbit antisera recognized BHT-BSA but not BSA alone. As shown in Figure 3, BHT-NAC was the most effective inhibitor of the binding of BHT antibody with BHT-BSA, with an IC50 of 1.3 × 10-6 M. BHT also effectively inhibited binding, although the IC50 (2.7 × 10-4 M) was approximately 2 log units higher than that of BHT-NAC. In order to gain some insight into the epitope recognized by the BHT antibody, we tested several structural analogs of BHT in the competitive ELISA. Analogs with longer 4-alkyl chains (2,6-di-tert-butyl-4-ethylphenol and 2,6-di-tertbutyl-4-isopropylphenol) had similar inhibitory potencies as BHT (not shown). However, removal of one of the tertbutyl groups (2-tert-butylphenol) resulted in loss of inhibitory potency as did replacement of one of the tertbutyl groups with a methyl group (2-tert-butyl-4,6dimethylphenol). Removal of both tert-butyl groups (4methylphenol) resulted in an even lower inhibitory potency. NAC alone had no effect on binding at concentrations up to 6 × 10-3 M (not shown). In some cases IC50s could not be determined because of solubility limitations at high concentrations. In addition, nonspecific binding may occur at high concentrations. Other phenolic compounds tested in this ELISA which had little

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Figure 3. Competitive inhibition of binding of anti-BHT serum antibodies to the solid-phase antigen BHT-BSA. The inhibitors used in the competitive ELISA were BHT-NAC (O), BHT (b), 2-tert-butyl-4,6-dimethylphenol (9), 2-tert-butylphenol (0), and 4-methylphenol (4).

Figure 4. Western blot of rat liver microsomal proteins alkylated by BHT. Incubations were carried out for 6 h at 37 °C and contained 2 mg/mL microsomal protein, an NADPHregenerating system, and 1 mM BHT in 0.1 M Tris buffer (pH 7.5). Microsomal proteins were separated by SDS-PAGE and probed with BHT specific antisera. Lanes: (A) control (no NADP+), (B) complete reaction, (C) deuterated BHT, (D) BHT + 1 mM SKF 525-A, and (E) BHT + 1 mM piperonyl butoxide.

or no ability to recognize the BHT antibody included 2,6dimethoxyphenol, 2,4-dimethylphenol, and 2-tert-butyl4-methylphenol (results not shown). These results demonstrate that the 2,6-di-tert-butylphenol moiety is essential for recognition by the BHT antibody. Immunochemical Detection of BHT-Protein Adducts in Rat Liver Microsomal Incubations. The ability of the polyclonal BHT antisera to recognize rat liver protein alkylated during in vitro incubations with BHT was determined using the Western blotting technique. Proteins were separated by SDS-PAGE, subsequently transferred to PVDF membranes, and incubated with BHT antisera. Alkylated proteins were visualized using chemiluminescence techniques. Figure 4 is a Western blot of proteins from rat liver microsomal incubations. The complete reaction (lane B) contained microsomal protein, BHT, and an NADPH-regenerating

Reed and Thompson

Figure 5. Western blot of alkylphenol-treated rat liver microsomal protein. Microsomal incubations were carried out as described in Figure 3 with 1 mM BHT or other alkylphenols and probed with BHT specific antisera. Lanes: (A) control (no BHT), (B) BHT, (C) 2,6-di-tert-butyl-4-ethylphenol, (D) 6-tertbutyl-2,4 dimethylphenol, (E) 2-tert-butyl-4-methylphenol, (F) 2,6-dimethoxy-4-allylphenol, (G) 2-methoxy-4-allylphenol, (H) 2,4,6-trimethylphenol, (I) 2,4-dimethylphenol, and (J) 4-methylphenol.

system. Lane A represents a control reaction in which NADP+ was omitted. Little or no background binding was observed with the BHT antisera. In the complete reaction (lane B) several proteins were recognized by the antisera. These had approximate molecular weights of 34.5, 52, 64, 74, and 97 kDa. Other bands appear to be present also, although they are less prominent. In some cases, such as with the band at 52 kDa, there appear to be multiple bands present at a given molecular weight. In Figure 4, for example, there appears to be an additional band at 50 kDa, just below the 52 kDa protein. Substitution of deuterated BHT, in which deuterium atoms replace each of the three protons on the 4-methyl group, for BHT in the reaction showed a modest decrease in the amount of protein alkylation (lane C). Densitometric analysis indicated a 32% reduction in the total amount of binding detected in lane C compared to lane B. This is consistent with previous reports which show that deuterated BHT is less toxic and forms quinone methide at a slower rate than BHT (25). In addition, the presence of the cytochrome P450 inhibitors piperonyl butoxide or SKF 525-A almost completely abolished protein binding (lanes D and E). Piperonyl butoxide inhibited total protein binding by 92%, while SKF 525-A inhibited protein binding by 96%. Several p-alkylphenols are known to be activated by cytochrome P450 to reactive intermediates which alkylate cellular proteins. The ability of the BHT antibody to detect protein alkylation by a number of these compounds is illustrated in Figure 5. Lane A is a control microsomal reaction (without BHT), while lane B is the same reaction containing BHT. Lanes C-J represent identical reactions containing various other alkylphenols. Of the alkylphenols tested, only 2,6-di-tert-butyl-4-ethylphenol (lane C) formed protein adducts which were detected by the BHT antibody. However, the total amount of binding was greatly reduced (86%) compared with BHT. This reduction is consistent with observations that this alkylphenol causes less toxicity in mice than BHT (34, 35), that it is metabolized to a quinone methide in rat liver

Selective Protein Alkylation by BHT in Rat Liver

Figure 6. Western blot of rat liver microsomal protein modified by BHT-QM. Three separate activation systems were used to generate quinone methide. Lanes: (A) control (no BHT), (B) cytochrome P450-dependent system (BHT + NADPH), (C) peroxidase-dependent system (BHTOOH, BHT, and BHA), and (D) direct addition of synthetic quinone methide.

microsomes at a slower rate than BHT,2 and that this quinone methide is less reactive than BHT-QM (36). Each of the other alkylphenols had modified or lost one or both of the two tert-butyl groups in the 2 and 6 positions. Although each of these compounds is metabolized to reactive intermediates which are capable of alkylating liver proteins, the BHT antibody did not detect any binding. This further supports the ELISA results (Figure 3) regarding the specificity of the antibody. To confirm the role that metabolic activation plays in the alkylation of microsomal protein by BHT and to further indicate that an electrophilic quinone methide is responsible for protein alkylation, comparisons were made between the binding profiles of enzymatically generated quinone methide and the direct addition of synthetic quinone methide to microsomal protein (Figure 6). Lane A shows a control reaction, while lane B shows the cytochrome P450-dependent alkylation profile of microsomal proteins by BHT. Lane C is a peroxidasedependent activation system for BHT which also generates the quinone methide intermediate. Lane D is the result of the direct addition of synthetic quinone methide. With each system, whether an enzymatic or nonenzymatic system was used to generate the quinone methide, similar binding profiles were observed. This confirms that the quinone methide is directly responsible for the protein alkylation by BHT. Detection of BHT-Alkylated Proteins in Vivo in Rat Liver and in Vitro in Rat Liver Slices. Liver proteins were isolated from rats treated with BHT to ascertain whether the antibody could detect protein alkylation in vivo. A dose of BHT sufficient to cause liver necrosis (1000 mg/kg) was administered by gavage to rats, and liver proteins were isolated 24 h later. Subcellular fractions of liver proteins were prepared and the results are shown in Figure 7. Lane A is total liver homogenate from a control rat, while B is the same from a BHT-treated rat. Lanes C and D are the microsomal and cytosolic fractions, respectively. Lane E is mitochondrial protein, while lane F represents nuclear protein. Protein binding was observed in all subcellular fractions; however, the greatest amount of binding was detected in the microsomal fraction. Prominent bands were 2

M. Reed and D. Thompson, unpublished observation.

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Figure 7. Detection of BHT-alkylated protein in vivo and in liver slices. Lanes B-F contain liver protein from a rat treated with 1000 mg/kg BHT and sacrificed 24 h later. Lanes: (A) control rat liver (corn oil), (B) whole liver homogenate, (C) microsomes, (D) cytosol, (E) mitochondria, and (F) nucleus. Lanes G-I contain total cellular proteins from rat liver slices incubated with 1 mM BHT for up to 6 h. Lanes: (G) control (no BHT), (H) BHT (3 h), and (I) BHT (6 h).

observed at the same molecular weights as seen in the in vitro microsomal incubations. Additional high molecular weight bands (>100 kDa) which react with the BHT antibody were also apparent in the microsomal fraction. In the cytosolic fraction, three prominent immunoreactive bands were observed at 34.5, 64.5, and 74 kDa. These bands may represent related proteins which are present in more than one intracellular compartment, or different proteins entirely. Lower amounts of protein binding were observed in both the mitochondrial and nuclear subcellular fractions. In both the mitochondrial and nuclear fractions immunoreactive bands were observed at 34.5 and 52 kDa. The 34.5 kDa protein gave a particularly strong signal in each of the subcellular fractions. The ability of the BHT antibody to detect protein alkylation in intact cells was assessed using precisioncut rat liver slices. Slices were incubated with 1 mM BHT for up to 6 h. Lanes G-I in Figure 7 represent total cellular proteins isolated from liver slices at 0, 3, and 6 h. No immunoreactive bands were observed in control slices or in slices incubated for 3 h. However, at the 6 h time point bands at 34.5, 52, and 74 kDa became visible. Dose and Time Dependence of Protein Alkylation in Vivo. The dose dependence of liver protein alkylation in rats gavaged with BHT is illustrated in Figure 8. Rats were gavaged with 0, 500, 750, or 1000 mg/kg BHT; 24 h later the liver proteins were isolated and analyzed as described above. No immunoreactive bands were seen in the control rats or rats dosed with 500 mg/kg BHT. At the 750 mg/kg BHT dose several bands were visible in both the cytosolic and microsomal fractions, in particular a microsomal band at 74 kDa. The 1000 mg/kg BHT dose gave a more prominent response in the intensity of staining. In Figure 9 the time dependence of protein alkylation is shown in rats gavaged with 1000 mg/kg BHT. Immunoreactive bands begin to appear at 18 h postdosing and are most prominent at 24 h. The intensity of staining decreased at the 36 h time point. Partial Purification and Identification of the 34 and 76 kDa Proteins. The 34 kDa protein was partially purified from whole liver homogenate by ammonium sulfate precipitation followed by a combination of ion

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Reed and Thompson

Figure 8. Dose-dependent detection of BHT-mediated protein alkylation in rat liver. Rats were gavaged with 0-1000 mg/kg BHT, and cytosolic and microsomal liver proteins were isolated after 24 h. Dose is indicated along the top of the figure: 0, vehicle control; 500, 500 mg/kg BHT; 750, 750 mg/kg BHT; and 1000, 1000 mg/kg BHT. For each dose the left lane represents cytosolic and the right lane microsomal proteins.

Figure 9. Time-dependent detection of BHT-mediated protein alkylation in rat liver. Rats were gavaged with 1000 mg/kg BHT, and cytosolic and microsomal liver proteins were isolated at the times indicated. Time (h) is indicated along the top of the figure. At each time point the left lane represents cytosolic and the right lane microsomal proteins.

exchange and hydroxyapatite column chromatography. The partially purified protein gave a solitary band at 34 kDa using Coomassie R-250 and also reacted strongly with BHT antisera (Figure 10). The N-terminus of the protein was microsequenced, and the following 14 amino acid residues were obtained: GANFQYIITEKKGK. A search of protein data banks revealed a 100% match with rat mitochondrial enoyl-CoA hydratase. The 76 kDa protein was purified from rat liver cytosol by acetone precipitation and ion exchange chromatography. Following SDS gel separation and transfer, BHTtreated rat liver cytosol yielded a partially purified protein sample that contained a highly immunoreactive band at approximately 74-76 kDa (Figure 11). This protein was separated by greater than 2% of the gel length from its nearest contaminant, lane A. Excision and N-terminal sequencing yielded the following 20 amino acid sequence: KPINVRVTTMDAQLEFAIQP. A search of protein data banks revealed that the protein was nearly identical to one of three proteins from the band 4.1 superfamily and the TERM (talin, ezrin, radixin,

Figure 10. Purification of the 32 kDa BHT-alkylated protein: (top) Coomassie R-250 stain of protein fractions from ion exchange and hydroxyapatite columns and (bottom) Western blot of the same fractions using the BHT antiserum.

moesin) family of proteins. The 74-76 kDa protein was found to have the most sequence homology with radixin and ezrin followed by moesin, a family of plasma membrane/ cytoskeletal linker proteins involved with connecting F-actin to the plasma membrane. Sequence homology was identical except for one or two amino acids at the N-terminus. These differences are most likely due to species differences. The protein’s identity was confirmed using an anti-ezrin antibody (although raised against ezrin, the antibody is unable to differentiate among ezrin, moesin, and radixin).

Discussion In this study, we isolated a polyclonal antiserum which recognized proteins alkylated by BHT. The antiserum was highly specific for BHT and did not recognize protein adducts from a number of other structurally similar alkylphenols. The ELISA inhibition studies indicated that the antiserum required the presence of the 2,6-ditert-butylphenol moiety for recognition. The 4-alkyl group (methyl in the case of BHT) was less important as a recognition epitope as the antiserum also recognized protein adducts from 4-ethyl-2,6-di-tert-butylphenol. Background levels of binding were very low, probably a result

Selective Protein Alkylation by BHT in Rat Liver

Figure 11. Purification of the 76 kDa rat cytosolic BHTalkylated protein: (lane A) Coomassie R-250 stain of a PVDF transfer of the proteins eluted from a Q sepharose column and (lanes B and C) identical transfers probed by Western blotting using BHT (lane B) or ezrin (lane C) antibodies.

of the fact that the tert-butyl moiety is uncommon in vivo. The antiserum recognized alkylated protein in rat liver following either in vitro or in vivo exposure to BHT. Studies with inhibitors and deuterated BHT, as well as a comparison of the protein binding profiles of enzymeactivated BHT with the direct addition of synthetic quinone methide, indicated that protein alkylation was a result of the cytochrome P450-dependent formation of a reactive quinone methide metabolite. Numerous previous studies support the importance of a quinone methide metabolite in mediating the diverse toxic effects of BHT in various tissues (9, 10, 15, 25, 27). The 32% reduction in protein binding observed in the experiments comparing deuterated BHT with BHT translates to an isotope effect of 1.47. This is similar to the inhibitory effect of deuteration on the metabolism of BHT to BHT-QM (isotope effect of 1.69) reported by Mizutani et al. (25). The similarity in magnitude between the effects of deuteration on the formation of quinone methide and protein binding further links these two events and suggests that a high percentage of quinone methide formed becomes covalently bound to protein. The antiserum was used to detect, by immunoblotting, a wide range of protein adducts derived from BHT. In microsomal incubations we observed five prominent immunoreactive bands corresponding to proteins with approximate molecular weights of 34.5, 52, 64, 74, and 97 kDa. Bands representing the 74 and 97 kDa proteins reacted the most intensely with the antiserum. Even in the presence of cytochrome P450 inhibitors, a small amount of alkylation was detected on these two proteins. A similar pattern of protein alkylation was also observed in liver proteins isolated from tissue slices incubated with 1 mM BHT and from rats treated with 1000 mg/kg BHT. In subcellular localization studies, binding was detected in all fractions of liver isolated from rats treated in vivo

Chem. Res. Toxicol., Vol. 10, No. 10, 1997 1115

with BHT. The amount of binding was greatest in the microsomal fraction followed by cytosol, mitochondrial, and nuclear fractions. Our data correlate well with the original observations of BHT-induced rat liver damage reported by Nakagawa et al. (10). In this study BHT produced liver necrosis in rats only at high doses (1000 mg/kg). A dose of 500 mg/ kg caused a decrease in hepatic glutathione levels but was not sufficient to elicit hepatic necrosis or changes in serum transaminase levels. The nadir of hepatic glutathione concentrations occurred 6 h after administration of BHT. With a dose of 1000 mg/kg BHT, significant elevations in serum transaminase levels were observed at 12 and 24 h time points. Similarly, Powell and Connolly (11) reported a lack of liver necrosis in rats 36 h following an oral dose of 500 mg/kg BHT, unless glutathione levels were depleted with buthionine sulfoximine or cytochrome P450 enzymes were induced with phenobarbital. In the present experiments, we were able to detect significant protein alkylation only at doses of 750 mg/kg and above. In addition, protein alkylation was most apparent between 18 and 24 h postdosing. Nucleophilic addition of reactive intermediates, such as BHT-quinone methide, to specific protein targets may be a consequence of several factors related to either the electrophile or the target protein. These include factors related to differences in chemical reactivity, steric accessibility, or active site proximity. Skipper (37) recently suggested a two-step model of covalent adduct formation in which the first step of the process involves the “docking” of an electrophile in a “receptor” or “binding” region of a protein. The second step involves the actual addition reaction resulting in the formation of an adduct. Since the rates of association/dissociation for the first step are much faster than the known rates of addition reactions in the second step, it is likely that addition reactions will occur primarily on amino acid residues which are present in or near the “binding” region. Therefore, proteins which have “binding” regions for a given electrophile will be preferentially alkylated. Proteins alkylated by BHT may have tertiary structures or sites which are particularly well adapted for binding the tert-butyl moiety or other part of the molecule. The specificity of BHT for individual proteins is strongly supported by the observations that the same proteins were alkylated under the several activation scenarios used in the present study. For example, whether BHT was activated in microsomal incubations in the presence or absence of cytosolic proteins, or whether BHT-QM was added directly to the incubation mixture, the same alkylation pattern resulted. In addition, a similar pattern of alkylation was observed in liver slices and in livers of rats exposed to BHT. The 34.5 and 76 kDa proteins alkylated by BHT were partially purified and identified by their N-terminal sequences. The 34.5 kDa protein was identified as mitochondrial enoyl-CoA hydratase. The enzyme contains 261 amino acid residues and has a calculated molecular mass of 32 kDa. This enzyme functions as part of the β-oxidation pathway and catalyzes the addition of water to trans-∆-enoyl-CoA to yield 3-hydroxyacyl-CoA. The same enzyme activity is also present in peroxisomes; however, the hydration reaction is catalyzed by a bifunctional enzyme of 78 kDa, which also has 3-hydroxyacylCoA dehydrogenase activity. The cytosolic 76 kDa protein band was identified as either ezrin, moesin, or radixin. These proteins are part

1116 Chem. Res. Toxicol., Vol. 10, No. 10, 1997

of the band 4.1 superfamily and are involved with the linking of cytoskeleton, namely, F-actin, to the plasma membrane (38). All three proteins have similar Nterminal sequences. Of the three proteins, moesin is an unlikely candidate for being the 76 kDa BHT binding protein since it has not been identified as a significant component of rat hepatocytes. Moesin is localized on the apical membrane of endothelial cells. On the other hand, ezrin is present in substantial concentrations in epithelial cells of the bile duct, while radixin is associated with hepatocyte microvilli (38). The association of ezrin with the bile duct epithelia, which are lined with numerous microvilli, does not lend itself to being a mechanistic possibility for the induction of liver necrosis by BHT. However, early studies looking at the metabolism of BHT found that metabolites were actively transported within the enterohepatic cycle and BHT-QM has been isolated from the bile of rats (39). If BHT-QM is present in significant concentrations in the bile, then it is reasonable to assume that cellular proteins from associated microvilli might be alkylated, including ezrin. However, due to the increased concentration of radixin and the active presence of metabolizing machinery within the hepatocyte, it seems probable that the protein identified as the 76 kDa BHT binding protein is most likely radixin. The alkylation of either of these proteins (enoyl-CoA hydratase and radixin) by BHT-QM may at least theoretically contribute to the hepatotoxicity caused by the parent compound. The alkylation of a mitochondrial protein is consistent with reports of the inhibition of mitochondrial function by BHT. BHT has been shown to rapidly deplete ATP levels in isolated hepatocytes and to inhibit respiratory control in isolated rat liver mitochondria (40). In addition, the quinone methide of another alkylphenol (2,6-dimethoxy-4-allylphenol) has recently been shown to directly inhibit mitochondrial respiration in isolated rat liver mitochondria (41). Alkylation of enoyl-CoA hydratase by BHT may therefore contribute to the initial or continued depletion of ATP levels through inhibition of the β-oxidation pathway. If radixin is a primary target of BHT alkylation, then the relationship between radixin, the plasma membrane, and the cytoskeleton is an interesting one. The formation of membrane blebs is an early event in hepatocyte toxicity for many compounds, including BHT, although the exact cause of membrane blebs is unknown (40, 42). Most recent studies suggest that plasma membrane cytoskeletal attachments are the most likely cause of plasma membrane perturbations (43-45). The disassembly of these proteins from the lipid bilayer could lead to the formation of blebs. For example, microfilament disruptions by cytochalasins and phalloidins produce hepatocellular blebs (43, 46) as do large doses of germander (47). The inhibition of F-actin formation through ATP depletion is also known to cause bleb formation (48). Changes in the phosphorylation status of proteins, free calcium, and thiol oxidation can also lead to bleb formation (42). Radixin, ezrin, and moesin bind actin at the carboxyl terminus and the plasma membrane at the amino terminus (49). The interaction at the plasma membrane is possibly with a membrane protein known as CD44 (50). These proteins contain a highly conserved phosphorylation site. These factors leave a wide area of possibilities for the disruption of plasma membraneradixin-cytoskeleton (F-actin) interaction. In addition, alkylating quinones have been shown to interfere with critical thiol groups of the cytoskeleton leading to bleb

Reed and Thompson

formation (51). Similar events may possibly initiate bleb formation and lead to the hepatocellular necrosis seen with high doses of BHT. In summary, we have presented evidence that we can detect the presence of BHT-protein adducts in rat liver tissue by use of a polyclonal antibody. Several specific protein adducts were observed at 34.5, 52, 64.5, 74, and 97 kDa in various subcellular fractions, and two of the alkylated proteins were identified. Studies are currently in progress to determine the consequences of alkylation to the function of these proteins, as well as attempting to isolate and identify the other protein targets. The identification of these proteins and their cellular functions may lead to a more fundamental understanding of the mechanism of alkylphenol-mediated toxicity as well as greater insight into general aspects of the role of quinone methide-mediated protein alkylation in toxicology.

Acknowledgment. Support for this work was provided by NIH Grant ES06016.

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