The Covalent Binding of [14C]Acetaminophen to Mouse Hepatic

The supernatant was then treated with (NH4)2SO4 (25% satn) to separate out the ... Finally, the antibodies were dissolved in K2HPO4 (10 mM; pH 7.5) co...
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Chem. Res. Toxicol. 1996, 9, 1176-1182

The Covalent Binding of [14C]Acetaminophen to Mouse Hepatic Microsomal Proteins: The Specific Binding to Calreticulin and the Two Forms of the Thiol:Protein Disulfide Oxidoreductases LingXiang Zhou,† Bruce A. McKenzie,‡ Eric D. Eccleston, Jr.,‡ Sri Prakash Srivastava,§,| NengQain Chen,§ Richard R. Erickson,⊥ and Jordan Loyal Holtzman*,†,§,# Research and Medical Services, Veterans Affairs Medical Center, Minneapolis, Minnesota 55417, and Departments of Medicine and Pharmacology and The Institute of Human Genetics, University of Minnesota, Minneapolis, Minnesota 55455 Received April 22, 1996X

Numerous in vitro studies have indicated that acetaminophen is activated by mouse hepatic microsomal cytochrome P450 to form N-acetylbenzoquinone imine. This in turn covalently binds through a Michael addition to protein sulfhydryl and amino groups. Although acetaminophen adducts of several cytosolic proteins have been purified after its administration in vivo, no adducts of specific microsomal proteins have been reported. We find that, after the in vitro incubation of mouse hepatic microsomes with [ring-14C]acetaminophen in the presence of an NADPH generating system, 95% of the bound radioactivity was associated with adducts to three intraluminal microsomal proteins: calreticulin and the two forms of thiol:protein disulfide oxidoreductase, Q2 and Q5. The acetaminophen bound to 0.35, 1.32, and 0.25 mol/ mol of the three proteins, respectively. Sequencing of the 14C-labeled tryptic peptides indicated that the acetaminophen bound to lysine 103 of Q2, lysines 202, 209 or 210 and 354 of Q5 and lysines 233 or 239 of calreticulin. No adducts of cysteine residues were observed. Our data might suggest that acetaminophen hepatotoxicity results from the formation of the reactive metabolite within the endoplasmic reticulum. This then binds to these essential proteins and blocks the posttranslational modification of secretory and membrane proteins. This inhibition could then lead to cellular injury and death.

Introduction Studies from Gillette’s and our laboratory have indicated that, during the in vitro incubation of acetaminophen in the presence of NADPH, the acetaminophen covalently binds to mouse hepatic microsomal proteins (1, 2). This binding is presumably mediated through the formation of the reactive metabolite, N-acetylbenzoquinone imine (NABQI)1 (3, 4). Furthermore, our studies have suggested that this binding to microsomal proteins may be the best in vitro index of in vivo toxicity (2, 4). Two groups, Hinson and Pumford’s and Khairallah and Cohen’s, have examined the covalent binding of acetaminophen to hepatic proteins (5-7). By utilizing antibodies to the bound acetaminophen, both groups have identified * Address all correspondence to: Jordan L. Holtzman, M.D., Ph.D., Chief, Section on Therapeutics (111T), Veterans Affairs Medical Center, One Veterans Dr., Minneapolis, MN 55417. Telephone: (612) 725-2000 ext. 3616; Fax: (612) 725-2093; E-Mail: holtz003@ maroon.tc.umn.edu. † Department of Medicine, University of Minnesota. ‡ The Institute of Human Genetics, University of Minnesota. § Department of Pharmacology, University of Minnesota. | Present address: Department of Biochemistry, University of Michigan, Ann Arbor, MI 48109-0606. ⊥ Research Service, Veterans Affairs Medical Center. # Medical Service, Veterans Affairs Medical Center. X Abstract published in Advance ACS Abstracts, September 15, 1996. 1 Abbreviations: FPLC ) fast protein liquid chromatography; GSH ) reduced glutathione; NABQI ) N-acetylbenzoquinone imine; PBS ) phosphate buffered NaCl; PEG ) poly(ethylene glycol) 6000; PVDF ) poly(vinyl difluoride) membranes; Q2 ) an isoform of thiol:protein disulfide oxidoreductase; Q5 ) an isoform of thiol:protein disulfide oxidoreductase; SDS-PAGE ) sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

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and purified some of the proteins which form adducts. The first group have immunized animals with NABQIprotein adducts, while the latter group have utilized p-aminobenzoic acid adducts as the antigen for preparing the antibodies. Both groups have then used their respective antibodies to detect adducts after the in vivo administration of high doses of acetaminophen. They have reported that acetaminophen binds to a large number of proteins in all cellular fractions (8, 9), but both groups have specifically identified adducts of 44 and 56 kDa cytosolic proteins (5-7). Khairallah and Cohen’s group has suggested that these proteins are derived from the endoplasmic reticulum (4, 5). The 56 kDa protein has been identified as a Se binding protein (5, 7). One problem with these studies is that it is difficult both to quantitate which proteins are most affected by the toxic metabolite and to determine which amino acids of the target proteins have formed the adducts. Furthermore, the specific functions of these proteins have not yet been elucidated. Hence, it not clear what effect the formation of these adducts has on cellular metabolism. As noted above, our studies and those from Gillette’s laboratory have suggested that the in vivo hepatotoxicity of acetaminophen may best be correlated with the in vitro covalent binding of the drug to hepatic microsomal proteins (2, 4). In particular, we have found that the chronic administration of ethanol to mice nearly doubled both the in vivo hepatotoxicity of acetaminophen and the in vitro covalent binding of the drug to hepatic microsomal proteins, but had a much smaller effect on the over all oxidative metabolism of the drug (4; Zhou et al., © 1996 American Chemical Society

Acetaminophen Binding to Microsomal Proteins

manuscript submitted).2 These data support a model for acetaminophen toxicity in which cytochrome P450 catalyzes the formation of NABQI. This metabolite then covalently binds to critical microsomal proteins, and it is this binding which then initiates the hepatocellular damage. In order to further study this possible mechanism for the toxicity of acetaminophen, we have, in the current in vitro study, incubated hepatic microsomes from male mice with an NADPH generating system and [ring-14C]acetaminophen. We then solubilized the washed microsomes, purified the adducted proteins, and identified both the adducted proteins and the specific amino acids to which the NABQI had bound. We have found that in the microsomes 95% of the covalently bound acetaminophen is associated with adducts to three intralumenal microsomal proteins: calreticulin and the two forms of thiol:protein disulfide oxidoreductase, Q2 and Q5 (10). The latter two proteins are also called protein disulfide isomerases (11). Furthermore, we have examined the specific tryptic peptides which have the bound radioactivity and found that these peptides contain no cysteines, indicating that acetaminophen binds primarily to these three proteins through the -amino groups of lysines or to histidines rather than the thiol groups of cysteines.

Chem. Res. Toxicol., Vol. 9, No. 7, 1996 1177 Scheme 1. Purification of Acetaminophen Adducts to Mouse Microsomal Proteins from the 16% PEG Supernatant Fraction

Materials and Methods Materials. The animals used in these studies were 25 g CF-1 male mice purchased from Harlan Laboratories. [ring-14C]Acetaminophen (29,735-6) was obtained from Sigma Chemical Co. (St. Louis, MO). Just prior to use, it was purified by thin layer chromatography on silica gel GF plates (Analtech, Newark, DE) with methanol/water (70:30). The acetaminophen band was scraped and eluted with methanol. The methanol was removed in a stream of nitrogen. NADP+, gucose 6-phosphate, glucose6-phosphate dehydrogenase, sodium dodecyl sulfate (SDS), acrylamide, and a series of peptide molecular weight standards, including (Try-Gly-Gly) (T-9005) (MW ) 295.3 Da), leucine enkephalin (L-9133) (MW ) 555.6 Da), pepstatin A (P-4265) (MW ) 685.9 Da), adrenocorticotropic hormone (fragment 1124) (A-2532) (MW ) 1652.1 Da), bradyykinin (B-3259) (MW ) 1660.2 Da), and [Ala-OH21]conantokin-T (C-2176) (MW ) 2683.8 Da), were purchased from Sigma Chemicals. All other chemicals were ACS reagent grade. Enzyme Preparation and Incubation. The mice were killed by decapitation with a guillotine. The livers were removed and quickly cooled on ice. They were homogenized in KCl-Tris (150 mM:50 mM; pH 7.4) (3 mL/g of liver). The homogenates were centrifuged at 9000g for 15 min, and the supernatants were centrifuged at 140000g for 45 min. The pellets were resuspended in KCl-Tris and recentrifuged. The microsomes (3 mg of protein/mL) were incubated with [ring14C]acetaminophen (1 µM; 107 total dpm added), NADP+ (0.4 mM), glucose 6-phosphate (5.5 mM), and glucose-6-phosphate dehydrogenase (0.6 unit/mL) in KCl-Tris-MgCl2 (75 mL) (150 mM:50 mM:5 mM; pH 7.4) for 20 min at 37 °C with constant shaking in air. In preliminary studies we have found that these conditions are within the linear range for both time and protein concentration (Zhou et al., manuscript submitted).2 The incubations were terminated by cooling on an ice bath. The samples were then centrifuged at 140000g for 45 min and washed with KCl-Tris. Protein Purification, The microsomes were solubilized in 0.3% Na-cholate and the labeled proteins purified by a modification of our previously described procedure (Schemes 1 and 2) (12). In this purification the solubilized microsomes were 2 Zhou, L.-X., Erickson, R. R., Peterson, F. J., and Holtzman, J. L. (1996) Kinetic Studies Demonstrating the Formation of Multiple Pools of N-Acetyl-p-Benzoquinone Imine During Cysteine Conjugation and Protein Binding of Acetaminophen by Hepatic Microsomes from Male Mice. Biochim. Biophys. Acta (submitted).

Scheme 2. Purification of Acetaminophen Adducts to Mouse Microsomal Proteins from the 16% PEG Pellet Fraction

initially precipitated with poly(ethylene glycol) 6000 (PEG) (16% w/v final concentration). About half of the radioactivity was found in the supernatant and half in the pellet. The supernatant was then treated with (NH4)2SO4 (25% satn) to separate out the PEG. The aqueous phase was chromatographed on an FPLC MonoQ column (1 cm × 10 cm) (Pharmacia, Piscataway, NJ) with a NaCl gradient (0-1 M). The radioactive peaks were rechromatographed on a MonoQ column (0.5 cm × 5 cm) with a shallower gradient (0-0.3 M). The radioactive proteins were finally purified by chromatofocusing on a MonoP column (0.5 cm × 5 cm) (Pharmacia) (pH gradient 4.0-7.0).

1178 Chem. Res. Toxicol., Vol. 9, No. 7, 1996 The 16% PEG pellet (10 mg of protein/mL) was suspended in Tris-acetate buffer (20 mM; pH 7.4) containing EDTA (1 mM), DTT (0.1 mM,) and glycerol (20%) (Scheme 2). The suspension was dialyzed overnight; Emulgen 911 (18 mg/mg of protein) was added; and the preparation was stirred for 2 h at 4 °C. The samples were chromatographed on a MonoQ column in Tris-HCl (100 mM; pH 7.4) with a NaCl gradient (0-1 M). The radioactive peak was rechromatographed on a MonoQ column (1 cm × 10 cm) in Tris-HCl (100 mM; pH 7.4) with a shallower NaCl gradient (0-0.5 M). Finally, the radioactive peak was chromatographed on an octyl-Sepharose column (1 cm × 10 cm) in Tris-HCl (100 mM; pH 7.4) with a cholate gradient (0-0.5%). Electrophoresis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed by the method of Laemmli (12) with a 4% stacking gel and 12% separating gel. The gels were stained with silver stain. Autoradiograms were obtained after the proteins had been separated by SDS-PAGE and transferred to a PVDF membrane (Immobilon-P, Millipore, Bedford, MA) in a glycine-Tris buffer (192 mM:25 mM; pH 8.6). X-ray film (XAR-5; Eastman Kodak, Rochester, NY) was exposed to the membranes for 10-20 h. The membranes were then stained with Coomassie Blue to identify the protein bands. Antibody Studies. Polyclonal antibodies to the purified proteins were developed in laying hens as described by Damiani et al. (13). Purified Q2, Q5, and calreticulin were prepared as previously described (10, 14). The protein (2 µg) in a mixture of NaCl-Tris (0.15 M:20 mM; pH 7.4) (0.1 mL) and complete Freund’s adjuvant (0.1 mL) was injected into the hens at several subcutaneous and intramuscular sites. At 2 and 3 weeks, the immunizations were repeated except with incomplete Freund’s adjuvant. Beginning 1 month after the first injection, the eggs were collected daily. The yolks were separated and diluted in NaCl-Na2HPO4 (0.1 M:10 mM; pH 7.5) (PBS). Most egg yolk proteins were precipitated with PEG (final concentration of 3.5%) in PBS. The suspension was centrifuged for 20 min at 12000g, and the antibodies were precipitated with PEG (final concentration of 11%), stirred for 30 min at 4 °C, and centrifuged. The precipitate was redissolved in PBS and reprecipitated three times. Finally, the antibodies were dissolved in K2HPO4 (10 mM; pH 7.5) containing NaN3 (0.01%) and stored at -80 °C. For immunoblots we have found that the PEG fraction gave satisfactory results so that it was unnecessary to further purify the antibodies by column chromatography. Immunoblotting was performed according to the transblotting procedures described by Towbin et al. (15). An alkaline phosphatase reaction was utilized to develop the color. The indicator dye was a combination of nitroblue tetrazolium and 5-bromo4-chloro-3-indolyl phosphate (Bio-Rad, Richmond, CA). Peptide Sequencing after Trypsin Digestion of the Radioactive Proteins. The radioactive proteins were digested with trypsin (1 µg/50 µg of protein) for 24 h at 37 °C in NH4HCO3 (100 mM). The peptides were purified by HPLC chromatography on a C18 column (2.1 mm × 25 cm) (Vydac, Hesperia, CA) by the method of Stone et al. (16). In this procedure the digest was placed on the column with an aqueous elution buffer of trifluoroacetic acid (0.06%) and eluted at 0.5 mL/min in trifluoroacetic acid (0.052%) with a 0-70% acetonitrile gradient (50 mL). The peaks were detected at 215 nm. We next chromatographed the radioactive peaks from the C18 column on a TSK G2500 PWxL gel filtration column (6 mm; 7.8 mm × 30 cm) (TosoHaas, Montgomeryville, PA) with a mobile phase of acetonitrile (45%) containing trifluoroacetic acid (0.1%) in water (55%) at a flow rate of 0.3 mL/min. The peaks were detected at 215 nm. The molecular weights versus the retention times were calibrated with a set of peptides standards (MW ) 165-2685 Da). The radioactive peaks were then sequenced. Other Assays. Protein concentrations were determined by the instant dye method with bovine serum albumin as the standard (Bio-Rad). The radioactivity in the protein and peptide samples was determined by liquid scintillation counting. The samples were first dissolved in Solvable (0.5 mL) (NEN/DuPont, Boston, MA). After incubation overnight at room temperature,

Zhou et al.

Figure 1. Chromatographic profile from a MonoQ column of the 16% PEG supernatant (A) and pellet (B) fractions of microsomes incubated with [14C]acetaminophen. Solid lines are the absorbance at 280 nm, the dashed lines are the cpm, and the dotted lines are the [NaCl]. H2O2 (30%; 10 µL) was added to decolorize the sample and a scintillation fluid was added (10 mL) (Ultimata Gold, Packard Instrument, Des Plains, IL). The radioactivity was determined in a Tri-Carb 1900CA β-liquid scintillation counter (Packard Instrument). The protein and peptide sequences were determined at the Microchemical Facilities of the University of Minnesota on a Model 477A protein sequencer (Applied Biosystems, Foster City, CA). All samples contained 30-50 pmol of the protein or peptide. We feel that these sequences were unequivocal since at each step of the Edman degradation only one amino acid gave a significant peak.

Results After solubilization of the microsomes with cholate and precipitation of the proteins with PEG, there were approximately equal amounts of radioactivity in the supernatant and the PEG pellet fractions (3830 total cpm and 3728 total cpm, respectively) (Scheme 1). All of the 14 C in the 16% PEG supernatant was found in two proteins with Mr’s ) 55 and 58 kDa (Figure 1A). These proteins were purified to homogeneity as determined by silver staining on SDS-PAGE (Figure 2). The N-terminal amino acid sequences of these two proteins indicated that they were the thiol:protein disulfide oxidoreductase, isoform Q5, and calreticulin, respectively. In support of these observations, on immunoblotting these proteins reacted appropriately with antibodies to calreticulin and Q5, respectively (Figure 3). The [ring-14C]acetaminophen

Acetaminophen Binding to Microsomal Proteins

Figure 2. SDS-PAGE of the purified Q2, Q5, and calreticulin with bound [ring-14C]acetaminophen. The gel was silver stained. Lanes 1 and 6, standards; lane 2, microsomes; lane 3, Q2; lane 4, Q5; lane 5, calreticulin.

Figure 3. Immunoblots of the SDS-PAGE of mouse microsomes and the purified Q2, Q5, and calreticulin with bound [ring-14C]acetaminophen. Lanes 1, 6, and 11 calreticulin; lanes 2, 7, and 12, Q5; lanes 3, 8, and 13, Q2; lanes 4, 9, and 14, microsomes; lanes 5, 10, and 15, standards. Antibodies to Q2, Q5, and calreticulin were applied to the three blots. Lanes 1-5 were reacted with anti-Q2; lanes 6-10 were reacted with antiQ5; and lanes 11-15 were reacted with anti-calreticulin.

bound was 0.25 and 0.34 mol/mol of protein for the 55 and 58 kDa proteins, respectively. These results have been replicated over a dozen times with several different batches of microsomes. One unexpected observation in this portion of the study was that the band in the purified preparation which reacted with the antibody to calreticulin (Figure 3, lane 11) had the expected Mr ) 58 kDa that we and others have observed for this protein (17). Furthermore, this Mr is close to the molecular weight predicted from the protein sequence derived from the cDNA (18). On the other hand, the major immunoreactive band in the intact microsomes had a Mr ) 90 kDa (Figure 3, lane 14). The microsomal component in this band is most likely a membrane bound protein, calnexin. The lumenal portion of this protein has a high degree of homology to calreticulin (19). Hence, it would be expected that polyclonal antibodies to the latter would react with the former. The high intensity of the calnexin band would suggest that this protein is present in much higher concentrations in the intact endoplasmic reticulum than is the calreticulin. The purification of the 14C-binding proteins in the pellet indicated that there was essentially only a single protein with bound radioactivity (Figure 1b) (Scheme 2). This protein was purified to homogeneity as determined by SDS-PAGE with silver staining (Figure 2). The

Chem. Res. Toxicol., Vol. 9, No. 7, 1996 1179

Figure 4. Autoradiogram of the SDS-PAGE of the purified Q2, Q5, and calreticulin with bound [ring-14C]acetaminophen. Lanes 1 and 6, 14C-labeled protein standards; lane 2, microsomes; lane 3, Q2; lane 4, Q5; lane 5, calreticulin.

N-terminal amino acid sequence was identical to that of form Q2 of the thiol:protein disulfide oxidoreductase we have previously purified (10). In line with this identification, the purified protein reacted to antibodies to Q2 (Figure 3). This protein had 1.32 mol of bound [14C]acetaminophen/mol of protein. The three proteins showed single bands by autoradiography after SDS-PAGE (Figure 4). No radioactivity was observed in the crude microsomes (lane 2). This is not surprising since, after resolution of the labeled proteins, the specific activity of the purified fractions was 2 orders of magnitude greater than that observed in the intact microsomes. Since the same amount of protein was applied to each lane of the gel, the total amount of radioactivity in the crude fraction was well below the sensitivity of the detection system. We digested the [14C]-labeled proteins with trypsin and isolated the labeled peptides by HPLC on a C18 column. When we further purified the radiolabeled peptide fractions by gel filtration chromatography, we found that the fraction obtained from Q2 was sufficiently homogeneous to give a meaningful sequence (Figure 5A), but those from Q5 (Figure 5B) and calreticulin (data not shown) had multiple peaks with similar concentrations. We determined the N-terminal amino acid sequence of each of the radiolabeled peptides obtained after the gel filtration chromatography (Table 1). Only a single amino acid could be detected at each step of the Edman degradation for all of these purified radiolabeled peptides. We were able to sequence 6-12 of the residues from each peptide. All of these residues were identical to the amino acids for these peptides reported in the published sequences for each of the three proteins. The only exception was for lysine 354 of Q5 that gave a thiohydantoin derivative which could not be identified. Presumably this was the acetaminophen adduct of the lysine which has been identified to be at this position. On the other hand, the amino acids before and after this residue were identical to the residues reported in the published sequence for this protein. We identified the C-terminal residue based on the known trypsin cleavage specificity in which only the peptide bonds between K-X and R-X are cleaved. The only exception to this specificity is that the bonds between K-P and R-P are not cleaved (20). We have also presumed that trypsin will not cleave a K-X bond if the -amino group of the lysine is blocked. With these caveats in mind, our data indicate that none of the radiolabeled peptides contained a cysteine residue. Hence, the ad-

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Zhou et al.

Figure 5. Chromatograms of the gel filtration chromatography of (A) Q2 tryptic peptide peak #10 and (B) Q5 tryptic peptide peak #8. Table 1. The [14C]-Labeled Peptides Obtained after the in Vitro Incubation of Mouse Hepatic Microsomes with [ring-14C]Acetaminophen

a The molecular weights were estimated by gel filtration chromatography. b The molecular weights were calculated from the sequences of the adducted peptides obtained after gel filtration chromatography. c We have not given the published sequences for these peptides since they were identitical to our observed sequences. d The first numeral (Arabic) is the number of the peptide obtained from the C18 column, while the second numeral (Roman) is the number of the peptide obtained from the gel filtration column. e Residue 354 did not give an identifiable thiohydantoin derivative even though residues 353 and 355 gave the expected amino acids. Presumably, the reported lysine at 354 was adducted with the acetaminophen metabolite which gave a previously unidentified peak.

ducts probably formed with the other major nucleophile found in proteins, the -amino groups of one or more of the lysines before the first trypsin cleavage site, i.e., the first arginine or unadducted lysine after the N-amino acid terminal residue of the peptide (Table 1). As noted above, this concept is supported by our observation that the lysine at 354 in Q5 gave an unidentifiable thiohydantoin derivative. Presumably this was the acetaminophen adduct of the lysine reported to be at this position. For some of the peptides there were two lysines which met these criteria (Table 1). It is also possible that the adducts may have formed with histidyl residues 356 of Q2 and 242 of calreticulin. Such histidine adducts were reported by Bambal and Hanzlik (21) during the metabolism of bromobenzene. Finally, even if we were to assume that the peptides had been cleaved at the next set of K-X or R-X bonds in the sequence, the published sequences indicate that there still would have been no cysteines to react with the metabolite of acetaminophen. Hence, it would appear that for all of these microsomal proteins the NABQI bound to lysine or histidyl rather than cysteine residues.

Discussion Our current data indicate that the cytochrome P450 dependent metabolism of acetaminophen with isolated

microsomes leads to the formation of adducts with only three, intralumenal, proteins: Q2, Q5, and calreticulin. If this adduct formation is the initial event in the hepatotoxicity of acetaminophen, then these data would suggest that this toxin interferes with one of two critical pathways. First, the endoplasmic reticulum is the major, intracellular, Ca2+ storage site and is important in the modulation of the cytosolic [Ca2+] (22). Furthermore, Q2, Q5, and calreticulin are major proteins involved in Ca2+ sequestration by the endoplasmic reticulum (10, 14, 26). In particular, calreticulin is the major Ca2+-binding protein of this organelle (14) while Q2 and Q5 modulate the oxidation-reduction status of the sulfhydryls of the microsomal Ca2+ binding proteins (23). Hence, impairment of the activities of these proteins might lead to the disruption of cellular Ca2+ homeostasis and thereby cause cellular injury (23, 24). In support of this model some workers have observed increased cytosolic [Ca2+] in hepatocytes incubated with toxic concentrations of acetaminophen (25-27). On the other hand, other workers have found that the increase is a result, and not the cause, of cellular injury (28, 29). An alternative metabolic pathway which could be disrupted is the synthesis of plasma membrane proteins. These proteins are synthesized within the lumen of the

Acetaminophen Binding to Microsomal Proteins

endoplasmic reticulum. After the formation of the basic peptide chain, these proteins must undergo a number of posttranslational modifications (30). These include the N-glycosylation of an asparagine, the alignment of the peptide chain into the proper configuration, and the formation of disulfide bonds. Recent studies with mammalian forms of calreticulin and Q5 have indicated that they play a major role in the catalysis of these steps (31, 32). Furthermore, studies in yeast have indicated that the thiol:protein disulfide oxidoreductases are critical for survival (33-35) and also appear to play major roles in all three of these processes, (11, 36-38). These observations would suggest that any toxin which inactivates Q2, Q5, and/or calreticulin could block the final synthesis of membrane proteins and thereby cause cellular injury. On the other hand, some agents, such as 3-hydroxyacetanilide (39) and methoxychlor (40), are not hepatotoxic but also show protein binding during microsomal metabolism. This would suggest that one test of our proposed model would be to determine whether these agents form adducts with the same proteins as hepatotoxins. In line with this approach, we have examined the covalent binding of methoxychlor to rat hepatic microsomes utilizing our current protocol (41). We found that it only binds to iodothyronine 5′-monodeiodinase, type I, a protein which is 98% homologous with Q5 (42). Since methoxychlor is not an hepatotoxin and does not bind to Q2, Q5, or calreticulin, these studies lend support to our suggestion that binding to these three proteins is critical in initiating toxicity. It is noteworthy that Pohl’s group (43) has found that the major oxidative metabolite of halothane, trifluoroacetyl chloride, also binds to Q2, Q5, and calreticulin (43-45). They began their studies by purifying hepatic proteins which reacted with antibodies obtained from patients with a history of halothane hepatitis. These antibodies reacted with a number of proteins, but primarily with the trifluoroacetyl adducts of Q2, Q5, and calreticulin (45). Yet it is generally felt that, unlike our model for acetaminophen toxicity, halothane hepatotoxicity is due to an immune response to these protein adducts rather than being due to a direct toxic effect (43, 46, 47). Hence it would appear that, even though the two agents show the same pattern of protein adduct formation, the subsequent toxic events are different. One problem with our observations on acetaminophen is that neither Cohen and Khairallah’s group nor Hinson and Pumford’s group have ever observed adduct formation by acetaminophen to these three proteins. Furthermore, when we forwarded samples of our labeled proteins to both groups, neither were able to observe any immunological reaction with their antibodies, even though the samples contained amounts of adducts which were well above the minimum detectable levels for their assays.3,4 These data would suggest that their antibodies do not react with -lysyl adducts of acetaminophen. Alternatively, it is possible that the radioactivity which we have observed in these proteins is not associated with the an acetaminophen adduct, but rather is due to a minor contaminant in the preparation or results from an exchange or recycling reaction, as is commonly seen with 3H-labeled compounds. Neither possibility seems likely since we have used [ring-14C]acetaminophen and taken 3 Personal communication: Edward Khairallah, Department of Molecular and Cellular Biology, University of Connecticut, Storrs, CT. 4 Personal communication: Neil Pumford, Division of Toxicology, University of Arkansas Medical Sciences, Little Rock, AK.

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great care to purify the radioactive compound just before use. This latter step should eliminate any contaminants which may have formed due to the radioactive degradation of the substrate. Furthermore, we have found that the activity required NADPH, that product formation was linear with time, and that unlabeled acetaminophen blocked adduct formation (2). These results would indicate that the binding is associated with a metabolic process and is specific for acetaminophen. It is of interest that, contrary to the usual assumption and the observations of Hoffmann et al. (48) with albumin, the adducts which we have observed were to -amino groups of lysines and not to the thiols of cysteines. This observation would suggest that the reactive metabolite, NABQI, forms adducts with the polar groups on the surface of the nearest protein to the site of its formation and that the thiol groups of the lumenal proteins are primarily present as disulfides. As a result, they are not available to react with the NABQI. In summary, our data would suggest that the hepatotoxicity of acetaminophen might be initiated by the covalent binding of a reactive metabolite, NABQI, to Q2, Q5, and calreticulin. The denaturation of these proteins may then impair the posttranslational modification of plasma membrane proteins. This could lead to a failure to replace plasma membrane proteins as they turn over and cause a loss of membrane integrity. Finally, this loss of membrane integrity might then lead to cell death.

Acknowledgment. This study was supported in part by the General Medical Research Service of the Department of Veterans Affairs and USPHS Grant RO1 ES 03731.

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