Chem. Res. Toxicol. 1996, 9, 1159-1166
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Human Cytochrome P450 2E1 Is a Major Autoantigen Associated with Halothane Hepatitis Mohammed Bourdi,*,† Weiqiao Chen,‡ Raimund M. Peter,‡ Jackie L. Martin,§ Jeroen T. M. Buters,| Sidney D. Nelson,‡ 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, Department of Medicinal Chemistry, University of Washington, Seattle, Washington 98195, The Department of Anesthesiology and Critical Care Medicine, The Johns Hopkins Medical Institutions, Baltimore, Maryland 21287, and Laboratory of Molecular Carcinogenesis, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892 Received May 14, 1996X
Autoantibodies against specific human cytochrome P450s have been found in the sera of patients suffering from a variety of diseases, including those caused by drugs. In the cases of tienilic acid- and dihydralazine-induced hepatitis, patients have serum autoantibodies directed against cytochromes P450 2C9 and P450 1A2, respectively. In the present study, we have found that 25 of 56 (45%) patients diagnosed with halothane hepatitis have autoantibodies that react with human cytochrome P450 2E1 that was purified from a baculovirus expression system. The autoantibodies inhibited the activity of cytochrome P450 2E1 and appeared to be directed against mainly conformational epitopes. In addition, because cytochrome P450 2E1 became trifluoroacetylated when it oxidatively metabolized halothane, it is possible that the covalently altered form of cytochrome P450 2E1 may be able to bypass the immunologic tolerance that normally exists against cytochrome P450 2E1. A similar mechanism may explain the formation of autoantibodies that have been found against other cellular targets of the reactive trifluoroacetyl chloride metabolite of halothane.
Introduction The elucidation of the mechanism of a rare (approximately 1 in 35,000 patients) but severe form of hepatitis caused by the inhalation anesthetic halothane (CF3CHClBr) 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 U.S. (see refs 1 and 2 for reviews). Moreover, it is thought that the newer inhalation anesthetics, enflurane (CHF2OCF2CHFCl), isoflurane (CHF2OCHClCF3), and desflurane (CHF2OCHFCF3), may cause hepatitis by a similar mechanism (see ref 3 for a review). Clinical and immunological findings have indicated that halothane hepatitis may have an immunopathological basis (see refs 1 and 2 for reviews). For example, a unique characteristic of most patients diagnosed with halothane hepatitis is the presence of serum antibodies that react with liver microsomal proteins that have been acylated by the trifluoroacetyl chloride (CF3COCl) metabolite of halothane. These trifluoroacetyl (TFA)1 -labeled proteins have been purified from the livers of halothane-treated rats and identified as TFA-neoantigens of 100 kDa (94 kDa glucose-regulated protein, GRP94) (4), 82 kDa (78 kDa glucose-regulated protein, GRP78) (5), 80 kDa (a 72 kDa endoplasmic reticulum protein of unknown function, ERp72) (6), 63 kDa (calreticulin) (7), 59 kDa (carboxyl* To whom correspondence should be addressed at the Molecular and Cellular Toxicology Section, Laboratory of Molecular Immunology, National Heart, Lung, and Blood Institute, Building 10, Room 8N110, Bethesda, MD 20892-1760. Tel: 301-496-4841; Fax 301-480-4852. † National Heart, Lung, and Blood Institute, National Institutes of Health. ‡ University of Washington. § The Johns Hopkins Medical Institutions. | National Cancer Institute, National Institutes of Health. X Abstract published in Advance ACS Abstracts, September 15, 1996.
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esterase) (8), 58 kDa (an apparent isoform of protein disulfide isomerase (PDI)) (9), and 57 kDa (PDI) (10). Results from other studies indicate that similar proteins are present in the livers of humans, and they appear to become TFA-labeled after patients have been administered halothane (11). Therefore, it is possible that human liver orthologues of the rat TFA-proteins represent the immunogens driving the immune responses of the halothane hepatitis patients. The antibody responses of the halothane hepatitis patients are more complicated than initially realized. It has been found that many of the halothane hepatitis patients also have serum autoantibodies that react with one or more of the native forms of PDI (10), PDI isoform (12), a carboxylesterase (13), and ERp72 (6). In addition, some halothane hepatitis patients have serum antibodies that react with the E2 subunits of the pyruvate dehydrogenase complex, the 2-oxoglutarate dehydrogenase complex, and the branched chain 2-oxoacid dehydrogenase complex, and protein X, which is a constituent of the pyruvate dehydrogenase complex (14). Others may have autoantibodies directed against additional autoantigens (15). In this regard, it was anticipated that halothane hepatitis patients might have autoantibodies directed against P450 2E1, the major form of P450 in the liver that metabolizes halothane to CF3COCl (16). This idea was based upon the observation that many autoantigens 1 Abbreviations: TFA, trifluoroacetylated; ERp72, a 72 kDa endoplasmic reticulum protein; PDI, protein disulfide isomerase; P450, cytochrome P450; DLPC, L-R-dilauroyl-sn-glycero-3-phosphocholine; G-6-P, glucose 6-phosphate; G6P-DH, glucose-6-phosphate dehydrogenase; 6-OH-OZ, 6-hydroxychlorzoxazone; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; SDS/PAGE, sodium dodecyl sulfate/polyacrylamide gel electrophoresis; CAH, chronic active hepatitis; Th, T helper; MHC, major histocompatibility complex.
© 1996 American Chemical Society
1160 Chem. Res. Toxicol., Vol. 9, No. 7, 1996
associated with halothane hepatitis are targets of CF3COCl (10, 12, 6) and the assumption that P450 2E1 should also be labeled by this metabolite. Indeed, we now report that 45% of the halothane hepatitis patients have serum autoantibodies that react with purified human cytochrome P450 2E1 (P450 2E1). The autoantibodies appeared to be directed against mainly conformational epitopes of P450 2E1 and may have been formed from the interaction of the immune system with TFA-labeled P450 2E1.
Experimental Procedures Materials. Chemicals were purchased from the following sources: goat anti-human IgG (phosphatase conjugated) and sheep anti-goat IgG (phosphatase conjugated) were from Boehringer Mannheim (Indianapolis, IN); alkaline phosphatase substrate mixture consisting of p-nitrophenyl phosphate in diethanolamine buffer was purchased from Biorad Laboratories (Richmond CA); chlorzoxazone was obtained from Sigma (St. Louis, MO); 6-hydroxychlorzoxazone (6-OH-OZ) and 5-fluoro2(3H)-benzoxazolinone were synthesized as previously reported (17). Anti-TFA serum was prepared by immunizing rabbits with TFA-rabbit serum albumin as previously described (18). Goat anti-rabbit P450 2E1 was obtained from Dr. Dennis R. Koop (Department of Pharmacology, Oregon Health Science University, Portland, OR). Human P450 3A4 was expressed in SF9 insect cells and purified as previously reported (19). Escherichia coli strain C-1A carrying plasmid pOR263, which encodes for rat cytochrome P450 reductase, was obtained from Dr. Charles B. Kasper (McArdle Lab, University of WisconsinsMadison, Madison, WI). The enzyme was purified from the bacterial cultures, according to a published procedure (20). A full length cDNA clone of human cytochrome b5 was obtained from Dr. R. Kato (Department of Pharmacology, Keio University, Tokyo). The 5′ and 3′ ends of the clone were modified by polymerase chain reaction to incorporate a NcoI and HindIII site, respectively. The modified cDNA was cloned into the prokaryotic expression vector pSE420 (Invitrogen, San Diego) and expressed in Escherichia coli strain DH5R (Gibco-LifeTechnologies, Gaithersburg, MD) in TB broth containing 200 µg of ampicillin/mL and 0.5 mM IPTG at 37 °C for 16 h. A spheroblast fraction containing the membrane bound form of human cytochrome b5 was obtained according to Holsman et al. (21) and further purified by previously described methods (22, 23). Microsomes were prepared as previously described from human liver sample H17C (17) . Expression and Purification of Human P450 2E1. A full length cDNA clone of human P450 2E1 was obtained from human liver HL118 (Human Liver Bank, Program Project Grant, Departments of Medicinal Chemistry and Pharmaceutics, University of Washington).2 The coding region of the cDNA was identical to the published cDNA sequence of human CYP2E1 (GenBank/EMBL Accession Number J02625) apart from three silent mutations as follows: G to C at position 18, T to C at position 105, and C to T at position 516 (relative to the start codon). The cDNA was cloned into the EcoRI restriction site of transfer vector pBacPAK8 (Clontech, Palo Alto) and used to prepare a recombinant baculovirus in TN5 (Tricoplusia ni) insect cells. To prepare the expressed P450 2E1, TN5 cells were infected with the recombinant baculovirus, and hemin (5 µg/ mL) was added 24 h post-infection. The infected cells were harvested 72 h post-infection. Identity of P450 2E1 protein was verified by immunoblotting with a polyclonal anti-rat P450 2E1 antibody (24). For purification of the enzyme, cell pellets were solubilized with 100 mM sodium phosphate (pH 7.4), containing 20% (v/v) glycerol, 1 mM DTT, 0.2 mM PMSF, 1 mM EDTA, and 1% (w/v) sodium cholate, and were precipitated with 6% (v/v) poly(ethylene glycol) (PEG). The supernatant of the PEG 2 W. Chen, R. M. Peter, S. McArdle, K. E. Thummel, R. O. Sigle, and S. D. Nelson, submitted manuscript.
Bourdi et al. precipitation was loaded onto an octyl Sepharose CL-4B hydrophobic column. P450 2E1 was eluted with 10 mM potassium phosphate buffer (pH 7.4), containing 20% (v/v) glycerol, 0.1 mM EDTA, and 1% (w/v) Emulgen 911, and loaded directly onto a DEAE-Sepharose column that was equilibrated with 10 mM potassium phosphate buffer (pH 7.4), containing 20% (v/v) glycerol, 0.1 mM EDTA, 0.6% (w/v) Emulgen 911 (w/v), and 0.2% (w/v) sodium cholate. P450 2E1 did not bind to the DEAESepharose column and was eluted directly onto a hydroxylapatite column. P450 2E1 was eluted with 300 mM potassium phosphate (pH 7.4), containing 20% (v/v) glycerol, 0.1 mM EDTA, and 1.5% (w/v) sodium cholate, and was dialyzed against 100 mM potassium phosphate (pH 7.4), containing 0.1 mM EDTA and 20% (v/v) glycerol prior to storage at -80 °C. The specific content of purified human P450 2E1 was 12 nmol/mg of protein. Reaction of Patients’ Serum Antibodies with Human P450 2E1. The reactions of serum antibodies with purified human P450 2E1 (1 µg/well) were measured by a previously described enzyme-linked immunosorbent assay method (ELISA) (6). Sera were obtained from patients with a clinical diagnosis of halothane hepatitis and from various control groups of individuals as described in detail elsewhere (25, 26). Briefly, the sera of 55 patients with unexplained hepatitis following halothane anesthesia were negative for serological markers of hepatitis A and B infection, cytomegalovirus, and Epstein-Barr virus, and none had received any potentially hepatotoxic drugs or had a history of alcohol excess. Sera were also collected from the following groups of patients that did not have halothane hepatitis. These patients included those with multiple halothane exposures without developing evidence of liver dysfunction (n ) 10), normal subjects (n ) 26), anesthesiologists and operating and recovery room nurses (n ) 36), patients with primary biliary cirrhosis (n ) 17), patients with acute fulminant liver disease of unknown etiology (n ) 21), and patients with viral hepatitis, who had been exposed to halothane (n ) 9). Sera from patients with hepatotoxicity, following exposure to the fluorinated inhaled anesthetics enflurane (n ) 8), isoflurane (n ) 8), or desflurane (n ) 3), were also tested. Immunoinhibition of P450 2E1 Activity by Halothane Hepatitis Patients’ Sera. Incubations were conducted with human liver microsomes (25 pmol of P450) or diluted homogenates of TN5 cells containing expressed human P450 2E1 (15 pmol) that was reconstituted by mixing with 2 molar equiv of rat cytochrome P450 reductase and human cytochrome b5 and 0.4% (w/v) sodium cholate in a minimum volume for 10 min at room temperature; the final concentration of sodium cholate in the reaction mixture was 0.02% (w/v). The samples were incubated in the presence of patient’s serum (2 µL) and potassium phosphate (100 mM, pH 7.4) in a total volume of 430 µL, for 30 min at room temperature. Chlorzoxazone (1 mM) was added, followed by incubation for 3 min at 37 °C. The reactions were started by the addition of a NADPH generating system, which consisted of 13.7 mM G-6-P, 0.66 mM NADP+, and 2.8 IU of G6P-DH. After 10 min of incubation, the reactions were stopped and the samples were processed and analyzed for 6-OH-OZ by HPLC as previously reported (17). In addition, immunoinhibition titration studies were conducted with diluted homogenates of TN5 cells, containing expressed P450 2E1 (15 pmol). The amount of patient’s serum used for these studies was 0.3, 0.5, 1.0, 2.0, or 4.0 µL. TFA Labeling of Human P450 2E1. Reconstituted purified human P450 2E1 (200 pmol) was incubated in a sealed container (7 mL of air head space) with halothane (2 µL), a NADPH generating system, and 100 mM potassium phosphate (pH 7.4) in a final volume of 1 mL for 60 min at 37 °C. Reactions were also conducted with diluted homogenates of TN5 cells containing expressed and reconstituted P450 2E1 (200 pmol) and halothane (2 µL) in a total reaction volume of 2 mL. Both reaction mixtures were analyzed by immunoblotting with anti-TFA serum. Other Methods. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE) and immunoblotting were done as
P450 2E1 Autoantibodies in Halothane Hepatitis
Figure 1. Purified human P450 2E1 and 3A4. SDS/PAGE gel stained for protein with Coomassie Blue. Molecular mass standards (lane 1), purified human P450 2E1 (lane 2; 2 µg of protein), and purified human P450 3A4 (lane 3; 2 µg of protein).
Figure 2. ELISA screening of patients’ sera for autoantibodies that react with purified native human P450 2E1. Sera (diluted 1/100) from the following sources were tested for their reaction against 1 µg of purified human P450 2E1: 55 patients with halothane hepatitis (group A), 19 patients with hepatitis caused by other fluorinated inhalation anesthetics (group B), 9 halothane exposed patients with viral hepatitis (group C), 17 patients with primary biliary cirrhosis (group D), 21 acute fulminant hepatitis patients (group E), 10 halothane exposed patients that did not develop hepatitis (group F), 36 anesthesiology personnel (group G), and 26 normal subjects (group H). A reaction was considered positive when the absorbance at 405 nm was greater than 2 standard deviations above the mean value (represented by the line) obtained from the sera of the 26 normal subjects. previously reported (27). Protein concentrations were determined with the BCA protein assay kit of Pierce Chemical (Rockford, IL) with bovine serum albumin as a standard.
Results Reaction of Halothane Hepatitis Patients’ Serum Antibodies with Purified Human P450 2E1. Human P450 2E1 was purified from a baculovirus expression system (Figure 1, lane 2) and was used in an ELISA procedure for determining the presence of P450 2E1 autoantibodies in the sera of halothane hepatitis patients and in the sera of other groups of individuals. Twentyfive of 55 (45%) halothane hepatitis patients tested positive for antibodies by this assay (Figure 2, group A). In contrast, only 3 of 83 (4%) of the sera from patients diagnosed with other forms of liver disease (Figure 2, groups C, D, E, and F) and those patients exposed to halothane, who did not develop hepatitis (Figure 2, group H), reacted with P450 2E1. Interestingly, 11 of 36 (31%) of a group of anesthesiology personnel, who were subclinically exposed to low levels of anesthetic agents from
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Figure 3. ELISA screening of halothane hepatitis patients’ sera for autoantibodies that react with purified native P450 2 E1 or P450 3A4. Sera (1/100 dilution) from 20 halothane hepatitis patients were tested for their reaction against 1 µg of purified native human P450 2E1 or purified native P450 3A4. Reactions of the patients’sera were significantly greater with P450 2E1 (P
