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Articles Halothane-Induced Liver Injury in Outbred Guinea Pigs: Role of Trifluoroacetylated Protein Adducts in Animal Susceptibility Mohammed Bourdi,*,† Hamid R. Amouzadeh,†,‡ Thomas H. Rushmore,§ Jackie L. Martin,†,| and Lance R. Pohl† Molecular and Cellular Toxicology Section, Laboratory of Molecular Immunology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892-1760, Department of Drug Metabolism, Merck Research Laboratories, West Point, Pennsylvania 19486-0004, and Department of Anesthesiology and Critical Care Medicine, The Johns Hopkins Medical Institutions, Baltimore, Maryland 21287 Received November 30, 2000

Halothane causes a mild form of liver injury in guinea pigs that appears to model the hepatotoxicity seen in approximately 20% of patients treated with this drug. In previous studies, it was concluded that the increased susceptibility of some outbred guinea pigs to halothaneinduced liver injury is not caused by their inherent ability to metabolize halothane to form toxic levels of trifluoroacetylated protein adducts in the liver. In this study, we reevaluated the role of trifluoroacetylated protein adducts in halothane-induced liver injury in guinea pigs. Male outbred Hartley guinea pigs were treated with halothane intraperitoneally. On the basis of serum alanine aminotransferase levels and liver histology, treated animals were designated as being susceptible, mildly susceptible, or resistant to halothane. Immunoblot studies with the use of anti-trifluoroacetylated antibodies showed that susceptible guinea pigs for the most part had higher levels of trifluoroacetylated protein adducts in the liver 48 h after treatment with halothane than did less susceptible animals. In support of this finding, the level of trifluoroacetylated protein adducts detected immunochemically in the sera of treated guinea pigs correlated with sera levels of alanine aminotransferase activity. In addition, the levels of cytochrome P450 2A-related protein but not those of other cytochrome P450 isoforms, measured by immunoblot analysis with isoform-specific antibodies, correlated with the amount of trifluoroacetylated protein adducts detected in the livers of guinea pigs 8 h after halothane administration. The results of this study indicate that the susceptibility of outbred guinea pigs to halothane-induced liver injury is related to an enhanced ability to metabolize halothane in the liver to form relatively high levels of trifluoroacetylated protein adducts. They also suggest that cytochrome P450 2A-related protein might have a major role in catalyzing the formation of trifluoroacetylated protein adducts in the liver of susceptible guinea pigs. Similar mechanisms may be important in humans.

Introduction The inhalation anesthetic halothane (CF3CHClBr) is known to cause both a mild and severe form of hepatotoxicity in patients (1). The milder form of hepatotoxicity occurs in approximately 20% of patients receiving halothane anesthesia and usually resolves with minimal toxicity. In contrast, the severe form of hepatotoxicity * To whom correspondence should be addressed: Molecular and Cellular Toxicology Section, Laboratory of Molecular Immunology, National Heart, Lung, and Blood Institute, Building 10, Room 8N110, Bethesda, MD, 20892-1760. Telephone: (301) 402-7223. Fax: (301) 480-4852. E-mail: [email protected]. † National Institutes of Health. ‡ Current address of the author: Center for Drug Evaluation and Research, Food and Drug Administration, Rockville, MD. § Merck Research Laboratories. | The Johns Hopkins Medical Institutions. 1 Abbreviations: TFA, trifluoroacetyl; ALT, alanine aminotransferase; GRP94, glucose-regulated protein 94; GRP78, glucose-regulated protein 78; ERp72, endoplasmic reticulum protein 72; CRT, calreticulin; P59, carboxylesterase; PDI, protein disulfide isomerase; P58, isoform of PDI.

(halothane hepatitis) occurs infrequently and can lead to fulminant hepatic failure and death. The mechanism of halothane hepatitis is of clinical interest because halothane is still widely used in adults throughout the world and is the agent of choice for children in the United States (2-4). Moreover, it is thought that the newer inhalation anesthetics, enflurane (CHF2OCF2CHFCl), isoflurane (CHF2OCHClCF3), and desflurane (CHF2OCHFCF3), may cause hepatitis by a similar mechanism (5). Clinical and experimental findings have indicated that halothane hepatitis may have an immunopathological basis (2, 6, 7). For example, patients diagnosed with halothane hepatitis often have symptoms of a druginduced allergic reaction that can include fever, rash, arthralgias, peripheral blood eosinophilia, and/or hepatic eosinophilia (6). The majority of halothane hepatitis patients also have antibodies in their sera that can react with one or more liver microsomal antigens, either in their native state (autoantigens) or after they have been

10.1021/tx000244x CCC: $20.00 © 2001 American Chemical Society Published on Web 03/22/2001

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trifluoroacetylated (TFA)1 (neoantigens) by the trifluoroacetyl chloride (CF3COCl) reactive metabolite of halothane (1). It is thought that the TFA-neoantigens are the immunogens responsible for the induction of not only these antibodies but also T cells that have been associated with halothane hepatitis (8, 9). Several of the TFA-neoantigens associated with halothane hepatitis have been identified. They include glucoseregulated protein 94 (GRP94) (1), glucose-regulated protein 78 (GRP78) (10), endoplasmic reticulum 72 (ERp72) (11), calreticulin (CRT) (12), a carboxylesterase (P59) (13), protein disulfide isomerase (PDI) (14), an apparent isoform of protein disulfide isomerase (P58) (15), UDP-glucose:glycoprotein glucosyltransferase (16), and P450 2E1 (17, 18), which has a major role in the oxidative metabolism of halothane in forming CF3COCl (17, 19). Other halothane hepatitis patients have been reported to have autoantibodies in their sera that react with components of the 2-oxoacid dehydrogenase complexes (20) and additional undefined autoantigens (21). Although attempts have been made to develop an animal model of halothane hepatitis that has an immunopathological basis caused by specific antibodies and/ or T cells directed against liver TFA-protein targets of the CF3COCl reactive metabolite of halothane, no one has accomplished this task to date (22-24). However, studies in guinea pigs have provided important clues regarding the mechanism of halothane hepatitis. This species is the only animal studied to date in which halothane can cause hepatotoxicity without the requirements of extensive pretreatments and manipulations such as exposing animals to hypoxic conditions (25). It is thought that the guinea pig is a model of the milder, self-limiting form of liver injury that occurs in approximately 20% of patients treated with halothane (26, 27). This form of liver injury, however, may also be a prerequisite for the subsequent development of severe liver damage caused by immunopathological mechanisms. The reason for this is that injury to hepatocytes would allow adequate levels of the TFA-neoantigens, which are concentrated in the endoplasmic reticulum and are not secreted (28), to be picked up by professional antigen-presenting cells. These cells may subsequently have a role in activating antigenspecific naive B and T cells to produce pathogenic antibodies or cytotoxic T cells, respectively, in susceptible individuals (29). In the study presented here, we have reinvestigated the guinea pig model of halothane-induced liver injury in an effort to better understand the mechanism of this toxicity and the role that it may have in the development of halothane hepatitis. In contrast to earlier studies (30, 31), we have found that susceptible guinea pigs have higher levels of TFA-protein adducts in their livers than resistant animals and that elevated levels of P450 2Arelated protein and not those of P450 2E1 may account for these differences in TFA-protein adduct formation. In addition, we have found that several intact TFAprotein adducts are released into blood from damaged hepatocytes where they may be taken in by professional antigen-presenting cells either in the liver or at other sites to initiate primary immune reactions against these antigens.

ketamine HCl (Fort Dodge Laboratories, Fort Dodge, IA), and anti-rabbit IgG (peroxidase-conjugated), anti-rabbit IgG (phosphatase-conjugated), and an alkaline phosphatase substrate mixture consisting of p-nitrophenyl phosphate in diethanolamine buffer (Bio-Rad, Richmond, CA). NADPH (Sigma, St. Louis, MO), Vectastain anti-rabbit IgG and anti-mice IgG (peroxidase-conjugated) ABC kits (Vector Laboratories, Burlingame, CA), coumarin, 7-hydroxycoumarin, and chlorzoxazone (Sigma), and enhanced chemiluminescence reagents (Amersham, Arlington Heights, IL) were used. MEM, fetal bovine serum, a nonessential amino acid solution, a sodium pyruvate solution, Geneticin (G-418), penicillin G sodium, streptomycin sulfate, L-glutamine, (Gibco-BRL, Grand Island, NY), protease inhibitor cocktail tablets (Roche Molecular Biochemicals, Indianapolis, IN), and TCH (defined serum replacement) and xylazine HCI (ICN Biomedicals, Aurora, OH) were used. The ImmunoPure protein A IgG orientation kit (Pierce, Rockford, IL), the alanine aminotransferase (ALT) kit (Sigma), a Centricon-10 concentrator (Amicon, Beverly, MA), and a Quick-Clone cDNA library (Clonetech, Palo Alto, CA) were used. Mouse antihuman P450 2A6 and goat anti-rat P450 1A1, 2B1, 3A1, and 2C11 antibodies (GENTEST Corp., Woburn, MA) and rabbit anti-mouse GRP94 antibody (StressGen, Victoria, BC) were used.

Experimental Procedures Reagents. Reagents were purchased from the following sources: halothane (Halocarbon Labs Inc., Hackensack, NJ),

Methods for raising antisera against the TFA-hapten (32), rat P59 (13), CRT (12), PDI (14), P58 (33), ERp72 (11), GRP78 (10), P450 reductase (34), and human P450 2E1 (17) have been described previously. Preparation of affinity-purified anti-TFAIgG has been described elsewhere (35). Halothane was distilled and prepared as a 21.5% solution in sesame oil. Animals. Male outbred Hartley guinea pigs (Charles River Labs,Wilmington, MA) of similar age and weight (650-750 g) were maintained in our animal facility according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals (36) and were used approximately 10 days after arrival. Animals were treated with either sesame oil or halothane (10 mmol/kg) intraperitoneally, and after 8 or 48 h, they were anesthetized with a mixture of xylazine (3 mg/kg) and ketamine (25 mg/kg) intramuscularly. Blood was collected by cardiac puncture and allowed to clot for approximately 2 h at room temperature and then overnight at 4 °C. Sera were prepared; a portion of each sample was used for ALT measurements, while the remainder was frozen in liquid nitrogen and stored at -80 °C until analyses were carried out. Livers were removed, and a portion was fixed in buffered formalin and embedded in paraffin by American Histolabs (Gaithersburg, MD). Sections (5 µm thick) either were mounted onto glass slides and stained with hematoxylin and eosin or were mounted on poly(L-lysine)treated glass slides and used for immunohistochemical analyses following a previously described procedure (37). The remaining portion of the livers was homogenized in ice-cold 100 mM Trisacetate (pH 7.5), containing 250 mM sucrose, 2 mM EDTA, and protease inhibitors (1 tablet in 50 mL of Tris buffer). Homogenates were snap frozen in liquid nitrogen and stored at -80 °C until they were subjected to SDS-PAGE and immunoblot analyses. Determination of the Extent of Liver Damage. Guinea pigs with serum ALT levels less than 2 times or greater than 5 times those of control animals, 48 h after halothane treatment, were classified as being either resistant or susceptible, respectively, to halothane-induced liver injury, as described previously (31, 38). Animals with ALT that fell between these two limits were considered mildly susceptible. Liver injury was confirmed histologically as reported previously (25, 39). Enzyme-Linked Immunosorbent Assay of TFA-Protein Adducts in Sera. The level of TFA-protein adducts in guinea pig sera 48 h after halothane treatment was measured by a previously described ELISA method (11, 17). Briefly, the wells of a microtiter plate were coated overnight at 4 °C with guinea pig serum (diluted 1:2 in PBS). The TFA-protein adducts in the sera were detected with the use of anti-TFA sera diluted 1:1000.

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Affinity Purification of TFA-Protein Adducts from Sera. An ImmunoPure protein-A IgG orientation kit was used for the purification of TFA-protein adducts from the sera of two guinea pigs following the manufacturer’s instructions. In short, affinity-purified anti-TFA-IgG (4 mg) was cross-linked to 2 mL of immobilized protein-A to form an anti-TFA affinity column. Guinea pig serum (2 mL), isolated 48 h after halothane treatment, was diluted with equal parts of 10 mM Tris-HCI (pH 7.5) and incubated with unmodified protein-A (2 mL) for 1 h at room temperature to extract the IgG fraction from serum. The extracted serum sample was loaded onto an anti-TFA affinity column, which was washed several times with 10 mM Tris-HCI (pH 7.5) until the baseline absorbance at 280 nm was reached. TFA-protein adducts were eluted from the column with 5 mL of ImmunoPure elution buffer [0.1 M glycine-HCl (pH 2.8)], neutralized with 1 M Tris-HCl (pH 9.5) (50 µL per 1 mL of fraction collected), and concentrated to 1 mL with the use of a Centricon-10 concentrator. The concentrated samples were snap frozen in liquid N2 and stored at -80 °C until they were subjected to SDS-PAGE and immunoblot analyses. Cloning of P450s 2E1 and 2A6. The coding sequence for P450 2E1 was amplified from a Quick-Clone cDNA library using a forward primer (5′-GATCGAGAATTCCACCATGTCTGCCCTCGGAGTGAC-3′) and a reverse primer (5′-AGCTAGGAATTCAGATCTCATGAGCGGGGAATGACACA-3′) (a consensus Kozak sequence was included in the forward primer, and is underlined). The PCR fragment was cloned into pCRII (Invitrogen Corp., Carlsbad, CA), and the plasmid was transformed into Escherichia coli. Several clones were recovered, and the entire cDNA fragment was sequenced from both directions. The sequence was confirmed to be identical to that reported by Song et al. (40) (GanBank accession no. J02625). A single clone was chosen and the cDNA recovered by digestion with EcoRI. The fragment was inserted into the expression vector pcDNA3 (Invitrogen Corp.) after digestion with the same restriction enzyme. After transformation into E. coli, several clones were recovered and sequenced from both ends to ensure that the cDNA was inserted in the proper orientation. A pUC9 plasmid containing the coding sequence for P450 2A6 (GanBank accession no. XI3897) was obtained from F. Gonzalez and H. Gelboin (National Cancer Institute, Bethesda, MD). The entire coding region was excised from pUC9 by XbaI and EcoRI digestion. The fragment was inserted into the expression vector pcDNA3 after digestion with the same restriction enzymes. After transformation into E. coli, several clones were recovered and sequenced from both ends to ensure that the cDNA was inserted in the proper orientation. Stable Expression of Human P450s in HepG2 Cells. Human HepG2 cells were maintained in MEM as previously described (41). Stable cell lines were established for each of the human P450s after transfection and selection by G-418 (42). Briefly, cells were plated in 25 cm2 flasks (approximately 1 × 104 cells), and after 24 h, cells in 24 plates were transfected with one of the expression constructs described above (10 µg of DNA per flask). Twenty-four hours later, the medium was changed and supplemented with G-418 (50 µg/µL) to a final concentration of 800 µg/mL. The medium was changed every fourth day. The cells were allowed to grow in the selection medium for 4 weeks, at which time individual clonal expansions (clones) were visible. Twenty clones were recovered from the flasks and grown to confluence in 48-well plates. At confluence, each clone was tested for P450 activity by exposing the clone to a specific substrate for 16 h (50 µM coumarin for P450 2A6 and 50 µM chlorzoxazone for P450 2E1). After exposure for 16 h to the specific substrate, the medium was recovered and monitored for both the parent and products using a generic high-pressure liquid chromatography method (43). Clones that exhibited activity toward the specific substrate were expanded to 25 cm2 flasks and stored in liquid N2 for further use. The level of expression of each of the human P450s was estimated by immunoblot analyses to be approximately 30 pmol of P450 per 106 cells.

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Figure 1. ALT levels in the sera of guinea pigs 48 h after treatment with halothane. Guinea pigs 1-7 were treated with 10 mmol of halothane/kg of body weight in sesame oil. Number 8 represents the mean ALT value for three guinea pigs that were treated with the vehicle sesame oil. Using the classification presented in Experimental Procedures, animals 2 and 4-6 were designated as susceptible to halothane-induced liver injury while animals 1 and 3 were designated as resistant. Animal 7 was classified as mildly susceptible to the hepatotoxic effect of halothane. Detection of TFA-Protein Adducts of Halothane in HepG2 Cells Expressing Human P450 2E1 or P450 2A6. Cells were plated in 160 cm2 culture flasks (approximately 4 × 106 cells) containing 20 mL of MEM, supplemented with 10% (v/v) fetal bovine serum, nonessential amino acids (0.1 mM), sodium pyruvate (1 mM), Geneticin (G148) (800 mg/mL), penicillin G sodium (100 mg/mL), streptomycin sulfate (0.25 mg/ mL), and L-glutamine (2 mM) (MEM complete medium). Cells were grown in a humidified atmosphere of 5% CO2 at 37 °C. Two days before the addition of halothane to the flasks, the medium was replaced with MEM complete medium that lacked G148 and antibiotics. Immediately before the addition of halothane to the flasks, the concentration of fetal bovine serum in the medium was reduced to 3% (v/v), and the medium was supplemented with 2% (v/v) TCH replacement serum (44). Cells (50% confluent) were then treated with halothane at the indicated times and concentrations and were incubated at 37 °C with the flask caps tightened to minimize evaporation of halothane. After incubation, cells were washed with PBS (2 times), collected, and centrifuged for 5 min at 1000 rpm. Pellets were sonicated for 20 s, homogenized in ice-cold 100 mM Tris-acetate (pH 7.5), containing 250 mM sucrose, 2 mM EDTA, and protease inhibitors (1 tablet in 50 mL of homogenization buffer), and stored at -80 °C until they were used. Other Methods. SDS-PAGE, immunoblotting, and laser densitometry of immunoblots were used as previously described (28). The coumarin 7-hydroxylation assay was carried out as previously described (45). Regression and statistical analyses were carried out with the use of Prism version 3.00 for Windows (GraphPad Software, San Diego, CA).

Results TFA-Protein Adducts in the Liver. It has been previously reported that approximately 50% or more of outbred guinea pigs are susceptible to developing liver injury 48 h after treatment with halothane (25, 30, 39, 46). In agreement with these findings, we found that four of seven male Hartley guinea pigs were susceptible (animals 2 and 4-6) while two of seven guinea pigs (animals 1 and 3) were resistant to the hepatotoxic effects of halothane on the basis of elevations in their serum ALT levels (Figure 1) and liver histology. One guinea pig (animal 7) was considered mildly susceptible on the basis of its ALT level (Figure 1) and liver histology. Immunoblot analyses of proteins from liver homogenates of these animals with anti-TFA sera revealed that all of the susceptible guinea pigs except for animal 4 had much higher levels of TFA-protein adducts in the liver than


Figure 2. Immunoblot detection of TFA-protein adducts in liver homogenates of guinea pigs 48 h after treatment with halothane. The homogenates in lanes 1-7 were isolated, respectively, from the numbered guinea pigs described in the legend of Figure 1. Lane R represents a typical immunoblot of a male Sprague-Dawley rat liver homogenate 48 h after treatment with 10 mmol of halothane/kg of body weight in sesame oil, intraperitoneally. Each lane was loaded with 50 µg of protein, and TFA-protein adducts were detected with anti-TFA sera.

those of the two halothane resistant animals and that of the mildly susceptible one (Figure 2). In addition, the level of TFA-protein adducts in the liver of male rats (Figure 2, lane R), which are resistant to the hepatotoxic effects of halothane when exposed to this drug under a normal atmosphere of air and without prior inductions of P450s (47), was approximately 1 order of magnitude lower than those of guinea pigs as previously reported (48). Immunohistochemical findings, with liver sections from the animals described in the legend of Figure 1, confirmed the immunoblot results by showing that susceptible guinea pig 6 had levels of TFA-protein adducts in the perivenous zones of the liver considerably higher than those of mildly susceptible animal 7 (panels A and C of Figure 3, respectively) and the resistant animals (results not shown). Moreover, the immunochemical staining in the section of susceptible guinea pig 6 was uniform throughout the perivenous zones (Figure 3A), while most of the staining in the liver section of susceptible guinea pig 4 was concentrated in the periphery of the perivenous zones (Figure 3B). These differences might be attributed to the greater loss of TFA-protein adducts from damaged cells in the regions closest to the periveins in the liver of guinea pig 4 than those of guinea pig 6 and could possibly account for the relatively low levels of immunoreactive TFA-protein adducts seen in the immunoblot of guinea pig 4 (Figure 2). No immunochemical staining was observed in the section of the vehicle-treated animal (Figure 3D).


To determine whether a particular isoform(s) of P450 might be responsible for the relatively high levels of TFAprotein adducts seen in the livers of susceptible guinea pigs (Figure 2), immunoblotting was repeated with both TFA and isoform-specific P450 antisera and liver homogenates isolated from another group of guinea pigs that were killed 8 h after treatment with halothane. This time point was chosen instead of 48 h to limit the loss of proteins from damaged hepatocytes. As can be seen in Figure 4A, guinea pigs 15 and 16 and to a lesser extent guinea pig 17 had levels of immunoreactive TFA-protein adducts in liver homogenates relatively higher than those of guinea pigs 11-14. Of all the P450s that were studied, only the relative levels of immunoreactive P450 2Arelated protein, which cross-reacted with mouse-antihuman P450 2A6 sera, were comparable with those of the TFA-protein adducts found in the liver homogenates (Figure 4B). Indeed, statistical analyses revealed that the band intensities of immunoreactive P450 2A-related protein in liver homogenates and liver microsomes correlated with the levels of immunoreactive TFA-protein adducts (Figure 5) and coumarin 7-hydroxylase activity (r2 ) 0.78, P < 0.05), respectively. Moreover, when halothane was incubated with HepG2 cells containing stably expressed human P450 2A6, immunoblot analyses of cell lysates showed that TFA-protein adducts were formed in a concentration-dependent manner (Figure 6, lanes 2-4). Most of the major TFA-protein adducts in the immunoblot were tentatively identified by their comigration with proteins that reacted immunochemically with antibodies raised against protein targets of the reactive metabolite of halothane that had been isolated and identified from rat liver. In addition, it appeared that P450 2A6 also became TFA-labeled when it metabolized halothane (Figure 6, lanes 2-4 and 5). The pattern of TFA-labeled proteins was very similar to that seen in the immunoblot of the cell lysate of HepG2 cells containing stably expressed human P450 2E1 that had been incubated with halothane (Figure 6, lane 7). The only major difference was that in this case P450 2E1 appeared to become TFA-labeled when it metabolized halothane (Figure 6, lanes 6 and 7). TFA-Protein Adducts in the Sera. TFA-protein adducts were also immunochemically detected in the sera of guinea pigs, as described in the legend of Figure 1, by an ELISA procedure 48 h after halothane administration (Figure 7). The highest levels were found in the sera of animals 2 and 4-6, which had been previously found to be susceptible to halothane-induced liver injury (Figure 1). Moreover, a statistically significant correlation existed between the level of TFA-protein adducts in the sera and the amount of liver injury (r2 ) 0.96, P ) 0.0002). A similar result was found when the study was repeated with a larger group of animals. In this case, 11 of 18 guinea pigs (61%) were classified as being susceptible to halothane-induced liver injury, while one was mildly susceptible (6%) and six were resistant (33%) to halothane (Figure 8A). Again, a statistically significant correlation existed between the level of TFA-protein adducts in the sera and the amount of liver injury (Figure 8B). The nature of the TFA-protein adducts in the sera could not be determined directly by immunoblot analysis, due to interference caused by large amounts of constitutive serum proteins in the samples. To overcome this problem, TFA-protein adducts were isolated from the sera

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Figure 3. Immunohistochemical detection of TFA-protein adducts in liver sections of guinea pigs 48 h after treatment with halothane. The liver sections were obtained from animals described in the legend of Figure 1. Panels A and B show liver sections from susceptible guinea pigs 6 and 4, while panel C shows a liver section from mildly susceptible guinea pig 7. Panel D shows a liver section from a vehicle-treated guinea pig. TFA-protein adducts were detected with anti-TFA sera.

of susceptible and mildly susceptible guinea pigs 5 and 7 (described in the legend of Figure 1), respectively, with the use of anti-TFA affinity columns, prior to immunoblot analysis with anti-TFA sera. This procedure permitted the detection of several TFA-protein adducts in the sera of susceptible guinea pig 5 (Figure 9A, lane 3). Most of the TFA-protein adducts appeared to correspond to those adducts found in the liver homogenate of this animal (Figure 9A, lane 1) and were tentatively identified by their reaction with protein-specific antibodies (Figure 9B, lanes 1-3). In contrast, none of these TFA-protein adducts were detected in the serum of mildly susceptible guinea pig 7 (panels A and B of Figure 9, lane 4).

Discussion It is well established that the oxidative metabolism of halothane in forming trifluoroacetyl chloride and TFAlabeled protein adducts has a role in the etiology of liver injury caused by halothane in guinea pigs (26, 49, 50). In contrast, studies have indicated that this pathway of metabolism of halothane does not contribute to the variable susceptibility of outbred guinea pigs to halothane-induced liver injury in guinea pigs (30, 31). For example, susceptible and resistant guinea pigs did not differ in the levels of trifluoroacetic acid, a hydrolysis product of trifluoroacetyl chloride, measured in plasma 1 h after the administration of halothane (30). Guinea pigs also did not differ in the levels and activities of hepatic P450 2E1 (31), which is a major isoform of P450 in the liver of rats and humans that oxidatively metabolizes halothane (19, 51, 52). Although our results also showed that the levels of P450 2E1 in the livers of guinea

pigs did not differ appreciably among animals (Figure 4B), they do indicate that susceptibility and resistance to halothane-induced liver injury in guinea pigs could be attributed to differences in the oxidative metabolism of halothane. Indeed, susceptible guinea pigs had levels of TFA-protein adducts in their livers and sera higher than those of resistant or mildly susceptible animals, 48 h after halothane administration (Figures 2, 7, and 8). One possible reason for this discrepancy is that trifluoroacetic acid was assessed in the plasma of susceptible and resistant guinea pigs 1 h after halothane administration, when the metabolism of halothane could be inhibited by high concentrations of halothane (53). This idea is supported in part by another study where it was found that most guinea pigs with severe liver damage at 48 h had levels of trifluoroacetic acid in the urine 24 h after the administration of halothane higher than those of animals that were less susceptible to halothane-induced liver injury (25). Our findings suggest that variations in P450 2Arelated protein instead of P450 2E1 may account for the differences in the amounts of TFA-protein adducts found in the livers of susceptible and resistant guinea pigs. Immunoblot analyses of guinea pig liver homogenates, 8 h after halothane administration, revealed that only the levels of P450 2A-related protein, detected with antihuman P450 2A6 antibodies, and not those of other isoforms of P450s that were immunochemically measured, correlated strongly with the levels of TFA-protein adducts (Figure 5). A correlation was also found between the levels of P450 2A-related protein and the rate of



Figure 6. Immunoblot detection of TFA-protein adducts in cell lysates of HepG2 cells containing stably expressed human P450 2A6 or P450 2E1 after incubation with halothane. Each lane was loaded with 30 µg of protein and was immunoblotted with anti-TFA, anti-P450 2A6, or anti-P450 2E1 sera. The tentative identification of several of the TFA-protein adducts was determined by their comigration in immunoblots with P450 2A6, P450 2E1, GRP94, GRP78, ERp72, CRT, and PDI, which were detected with protein-specific antibodies.

Figure 4. Immunoblot detection of TFA-protein adducts and specific isoforms of P450 in liver homogenates from another group of seven guinea pigs 8 h after treatment with halothane. Each lane was loaded with 50 µg of protein. Panel A was immunoblotted with anti-TFA sera, while panel B was immunoblotted with antisera directed against various isoforms of P450 and P450 reductase.

Figure 5. Correlation of the levels of P450 2A-related protein and TFA-protein adducts in liver homogenates of guinea pigs 8 h after treatment with halothane. The plot was made from the results of laser densitometric scans of immunoblots of the TFA-protein adducts and P450 2A-related protein in liver homogenates of the seven guinea pigs described in the legend of Figure 4.

coumarin 7-hydroxylation in the liver, which is a catalytic measure of P450 2A6 activity (45). These results are supported by previous reports where coumarin 7-hy-

Figure 7. ELISA determination of the levels of TFA-protein adducts in the sera of guinea pigs 48 h after treatment with halothane. The sera were isolated from the guinea pigs described in the legend of Figure 1.

droxylation activity in the liver of guinea pigs was found to vary considerable among animals (54), and P450 2A6 was shown to metabolize halothane oxidatively in vitro to form trifluoroacetic acid (51). In addition, we showed that P450 2A6, like P450 2E1, could metabolize halothane to form TFA-protein adducts when expressed stably in HepG2 cells (Figure 6). Therefore, these findings indicate that the P450 2A-related protein in the liver of guinea pigs is a homologue of human liver P450 2A6 and appears to account for the increased levels of TFA-protein adducts found in the livers of susceptible guinea pigs. Several P450 2A isoforms have been characterized in humans, mice, and rats (55, 56), but less is known about P450 2A isoforms in guinea pigs. It has been shown by immunoblot analyses that rat polyclonal P450 2A1 (45) and human polyclonal P450 2A6 (54) antisera recognized three and two proteins in the liver of guinea pigs, respectively, suggesting that guinea pigs may have more than one isoform of P450 2A expressed in the liver. More studies are needed to characterize the isoforms of P450 2A that oxidatively metabolize halothane in the liver of guinea pigs to form TFA-protein adducts. Other studies have suggested that susceptible guinea pigs develop liver injury when they are exposed to halothane because they are more sensitive to hepatic is-

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Figure 8. Correlation of the levels of TFA-protein adducts and ALT values in the sera of another group of guinea pigs 48 h after treatment with halothane. (A) ALT levels in the sera of 18 guinea pigs (21-38) treated with 10 mmol of halothane/kg of body weight in sesame oil. Using the classification presented in Experimental Procedures, 11 animals were classified as being susceptible, one was mildly susceptible, and six were resistant to halothane-induced liver injury. (B) Correlation of the levels of TFA-protein adducts and ALT values in the sera of the 18 guinea pigs. TFA-protein adducts in the sera were assessed by the ELISA.

chemia (31, 57), cholestasis (38), and disturbances in calcium homeostasis (38, 39) than resistant guinea pigs. Although our findings indicate that TFA-protein adducts are a contributing factor to the susceptibility of guinea pigs to halothane-induced liver injury, it remains to be determined whether they cause liver injury by initiating these or other pathological processes. Several of the TFAprotein adducts, however, were tentatively identified in liver homogenates of guinea pigs as stress proteins, based upon their immunoreactivity with both anti-TFA and protein-specific antibodies (Figure 9). They include P58, PDI, CRT, ERp72, GRP78, and GRP94, which have been previously identified in the livers of rats treated with halothane (1). These proteins, which are concentrated in the lumen of the endoplasmic reticulum, have roles in protein folding and in calcium homeostasis (58-60). Consequently, any decrease in the levels or activities of these ER stress proteins may predispose the cells to injury (60-62). In this regard, susceptible and mildly susceptible guinea pigs were found to have the same levels of ER stress proteins in their livers (Figure 9B, lanes 1 and 2). This result is consistent with studies in rat hepatocytes where it was found that TFA labeling of the endoplasmic reticulum stress proteins did not alter their rates of degradation (28). Nevertheless, these results do not exclude the possibility that TFA labeling of the endoplasmic reticulum stress proteins can inhibit their activities and contribute to liver injury. One or more of these proteins are also targets of reactive metabolites

Figure 9. Immunoblot detection of TFA-protein adducts in the sera of guinea pigs 48 h after treatment with halothane. TFAprotein adducts were isolated by affinity chromatography from the sera of susceptible and mildly susceptible guinea pigs 5 and 7, respectively, described in the legend of Figure 1. Then isolated TFA-protein adducts were immunoblotted with either anti-TFA sera (A) or antisera against GRP94, GRP78, ERp72, CRT, P59, or PDI (B). Lanes 1 and 3 were loaded with proteins from liver homogenate and serum, respectively, from guinea pig 5, while lanes 2 and 4 were loaded with proteins from liver homogenate and serum, respectively, from guinea pig 7. Lanes 1 and 2 were loaded with 25 µg of protein, and lanes 3 and 4 were loaded with 15 µL of affinity-purified TFA-protein adducts from guinea pig sera.

of acetaminophen (63), hydrochlorofluorocarbons (64), sulfamethoxazole (65), and thioacetamide (66), which also cause hepatotoxicity. The finding of intact TFA-protein neoantigens in the sera of susceptible guinea pigs, which appeared to be released from damaged hepatocytes (Figure 9), is a potentially important finding because it may explain how these endoplasmic reticulum proteins can interact with cells of the immune system to induce immune responses. In this regard, anti-TFA antibodies have been found in the sera of guinea pigs treated with halothane (24). It



has been suggested that the antigen-presenting cells mediating the formation of these antibodies are Kupffer cells. This idea is based upon the finding of TFA-protein neoantigens in Kupffer cells isolated from halothanetreated guinea pigs (67) and the observation that these cells could induce the proliferation of autologous splenic lymphocytes (27). Presumably, Kupffer cells had taken up the TFA-protein neoantigens that were released from injured hepatocytes because they did not form detectable levels of these neoantigens when incubated with halothane (48). Even though the TFA-protein neoantigens appear to be immunogenic and induce the formation of anti-TFA antibodies in susceptible guinea pigs, they do not lead to immunopathology and halothane hepatitis (24, 27). Indeed, when guinea pigs were treated multiple times with halothane, the severity of liver injury and the level of anti-TFA antibodies remained constant (24). It appeared that the animals could only mount a primary immune response. Perhaps, immunosuppressive factors were preventing the increase in anti-TFA antibody titers and/or possibly the formation of cytotoxic T cells that could mediate immunopathological reactions against the TFA-protein neoantigens in the liver. In this regard, we recently found a high correlation between ALT levels and anti-inflammatory cytokines IL-10 and IL-4 in the sera of guinea pigs treated with halothane (68). In summary, the results of this study indicate that the level of TFA-protein adducts formed in the liver of outbred guinea pigs has a role in determining the susceptibility of an animal to halothane-induced liver injury and suggest that a P450 2A-related protein may have a role in catalyzing the formation of these adducts. They also suggest that the release of TFA-protein adducts into the blood stream from damaged hepatocytes may be a pathway by which these adducts can interact with cells of the immune system to initiate immunopathological reactions. Similar mechanisms may be important in the development of halothane hepatitis in humans.

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Halothane-Induced Liver Injury in Outbred Guinea Pigs: Role of

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