Proc. Natl. Acad. Sci. USA Vol. 86, pp. 322-326, January 1989 Medical Sciences Human anti-endoplasmic reticulum antibodies in sera of patients with halothane-induced hepatitis are directed against a trifluoroacetylated carboxylesterase (drug hypersensitivity/neoantigens/metabolism) H. SATOH*, B. M. MARTINt, A. H. SCHULICK*, D. D. CHRIST*, J. G. KENNA*, AND L. R. POHL*t *Laboratory of Chemical Pharmacology, National Heart, Lung and Blood Institute, and tClinical Neuroscience Branch, National Institute of Mental Health, National Institutes of Health, Bethesda, MD 20892 Communicated by Allan H. Conney, September 28, 1988 (receivedfor review July 5, 1988)
ABSTRACT Previous studies have demonstrated that pa- covalently modified by the reactive TFA halide metabolite of tients with halothane-induced hepatitis have serum antibodies halothane (11). that are directed against novel liver microsomal neoantigens To investigate the role of the halothane-induced neoanti- and have suggested that these neoantigens may play an immu- gens in the pathogenesis of halothane hepatitis, a general nopathological role in development of the patients' liver dam- approach for their purification and characterization has been age. These investigations have further revealed that the anti- developed and utilized to identify one of them. bodies are directed against distinct polypeptide fractions (100 kDa, 76 kDa, 59 kDa, 57 kDa, 54 kDa) that have been covalently modified by the reactive trifluoroacetyl halide me- MATERIALS AND METHODS tabolite of halothane. In this paper, the trifluoroacetylated Purification of 59-kDa-TFA Protein from Halothane- (TFA) 59-kDa neoantigen (59-kDa-TFA) recognized by the Treated Rats by Immunoaffmnity and Anion-Exchange HPLC. patients' antibodies was isolated from liver microsomes of Specific anti-TFA IgG (12) was purified from antisera derived halothane-treated rats by chromatography on an immunoaf- from rabbits immunized with TFA rabbit serum albumin (13) finity column of anti-TFA IgG. Antibodies were raised against as described. Anti-TFA IgG (250 mg) was coupled to Affi-Gel the 59-kDa-TFA protein and were used to purify the native 10 (25 ml) according to the manufacturer's instructions protein from liver microsomes ofuntreated rats. Based upon its (Bio-Rad) and packed into a chromatography column (1.6 cm apparent monomeric molecular mass, NH2-terminal amino x 13 cm). acid sequence, catalytic activity, and other physical properties, Male Sprague-Dawley rats were treated with halothane the protein has been identified as a previously characterized and after 12 hr liver microsomes were prepared as described microsomal carboxylesterase (EC 3.1.1.1). A similar strategy elsewhere (12). The microsomes (-3 g) from 20 rats were and characterize neoantigens associated solubilized by stirring gently for 1 hr at 40C in 200 ml of 10 mM may be used to purify potassium phosphate (pH 7.4) containing 0.2 mM EDTA, with other drug toxicities that are believed to have an immu- 0.5% (wt/vol) sodium cholate, 0.2% (vol/vol) Emulgen 911, nopathological basis. 20% (vol/vol) glycerol, and a mixture ofproteinase inhibitors (aprotinin, 87 ,g/ml; leupeptin, 0.7 ,ug/ml; pepstatin A, 0.7 It has been estimated that between 3% and 25% of all drug ,ug/ml; and trypsin inhibitor, 50 ,ug/ml) (buffer A). After toxicities, which can include anaphylaxis, serum sickness, centrifugation at 105,000 x g for 90 min, the supernatant was asthma, urticaria, dermatitis, fever, hemolytic anemia, applied (10 ml/hr) at 4°C to the anti-TFA IgG affinity column, thrombocytopenia, granulocytopenia, hepatitis, nephritis, which had been equilibrated with buffer A. The column was vasculitis, pneumonitis, and lupus-erythematosus-like syn- washed (1 ml/min) with 3 column volumes of 100 mM drome, are due to hypersensitivity (allergic) reactions (1). potassium borate (pH 8.4) containing 1 M KCI, 0.2 mM Although most of these drug-induced hypersensitivities have EDTA, and the proteinase inhibitors (buffer B). The TFA been presumed to be mediated by immunogens formed by the proteins were eluted (1 ml/min) from the column with 200 ml covalent interaction of a reactive drug metabolite with tissue of 20 mM NM-TFA-L-lysine (TFA-Lys) in 10 mM potassium carrier macromolecules (2-6), it is only in the case ofhepatitis phosphate (pH 7.4) containing 0.1 mM EDTA, 20% (vol/vol) caused by the inhalation of anesthetic halothane that this glycerol, and 0.5% (wt/vol) sodium cholate. The eluent was mechanism has been supported substantially by experimental concentrated, dialyzed against 20 mM Tris acetate (pH 7.5) evidence. containing 0.2% (vol/vol) Lubrol PX (buffer C), and injected Previous studies have demonstrated that the majority of onto a Bio-Gel TSK DEAE-5-PW (7.5 mm x 7.5 cm) HPLC halothane hepatitis patients have unique serum antibodies column (Bio-Rad). Elution, monitored at 280 nm, was at a that react with novel neoantigens in livers of animals (1, 7, 8) flow rate of 1 ml/min with a 90-min solvent program con- and humans (9) treated with halothane and have suggested sisting of an initial 60-min linear gradient of buffer C to 35% that these neoantigens may play an immunopathological role buffer D (buffer C containing 0.8 M sodium acetate), followed in development of the patients' liver damage. Characteriza- by a 15-min linear gradient to 100% buffer D, and an tion of these neoantigens by immunoblotting with hapten- additional 15 min at 100%Wo buffer D. The 59-kDa-TFA protein specific anti-trifluoroacetyl (TFA) antibodies and sera from was isolated in the eluent from the HPLC column in a yield several halothane hepatitis patients has revealed that they of 1.3 mg. correspond to distinct liver microsomal protein fractions (100 Purification of 59-kDa Native Protein from Rats by Immu- kDa, 76 kDa, 59 kDa, 57 kDa, 54 kDa) (10, 11) that have been noaffiity and Anion-Exchange HPLC. The purification pro- cedure was similar to that described for the purification ofthe The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviation: TFA, trifluoroacetyl. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 322 Downloaded by guest on September 24, 2021 Medical Sciences: Satoh et al. Proc. Natl. Acad. Sci. USA 86 (1989) 323
59-kDa-TFA protein. In short, antisera were raised against with the hapten derivatives TFA-Lys and N6-acetyl-L-lysine the purified 59-kDa-TFA protein by injecting a female New (Ac-Lys) have been described in detail elsewhere (10-12, 16). Zealand White rabbit with 100 pug of the protein in an equal Comparative TFA Labeling of Liver Microsomal Proteins volume of Freund's complete adjuvant i.m. and s.c. at After the Administration ofHalothane, Ethyl Trifluoroacetate, several sites. After 4 weeks, a booster of 100 ,g ofthe protein Trifluoroethanol, or Sodium Trifluoroacetate. Halothane (10 was administered by i.v. injection and antisera were collected mmol/kg), ethyl trifluoroacetate (5 mmol/kg), or trifluoro- weekly for 5 weeks. Anti-59-kDa-TFA IgG (462 mg) was ethanol (5 mmollkg), dissolved in sesame oil, was adminis- coupled to Affi-Gel 10 and packed into a column. Liver tered i.p. to rats and sodium trifluoroacetate (1.6 mmol/kg, microsomes from 20 untreated male rats were solubilized and dissolved in water) was given by gavage. After 12 hr, rats loaded onto the affinity column. The column was washed were killed and TFA proteins in liver microsomes were with 1 column volume of buffer A and 3 column volumes of detected by immunoblotting as described (12). buffer B containing 0.5% (wt/vol) sodium cholate, and the Other Methods. Carboxylesterase (EC 3.1.1.1) enzyme 59-kDa protein was eluted with 3 column volumes of 2 M activity was determined spectrophotometrically with p- KSCN (pH 7.5). After dialysis and concentration, the 59-kDa nitrophenyl acetate as substrate according to the method of protein was further purified by HPLC on the Bio-Gel TSK McLean et al. (17). Deglycosylation with endoglycosidase H DEAE-5-PW column. A 20-min solvent elution program was was performed as described by Harano et al. (18). Protein used consisting of an initial 9 min with buffer C, followed by was determined according to the method of Lowry et al. (19) a 10-min linear gradient to 12% buffer D. The 59-kDa protein with bovine serum albumin as a standard. was isolated in the eluent from the HPLC column in a yield of 2.7 mg. NH2-Terminal Amino Acid Sequence Analysis. Samples of RESULTS the 59-kDa protein were transblotted to polyvinylidene di- Purification of the 59-kDa-TFA and Native 59-kDa Proteins. fluoride membranes and sequenced as described by Mat- As previously shown (11), 12 hr after the administration of sudaira (14) with modifications as reported by Martin et al. halothane to rats, protein fractions of 100 kDa, 76 kDa, 59 (15). Automated Edman degradation was conducted employ- kDa, 57 kDa, and 54 kDa are among the major TFA labeled ing an Applied Biosystems model 470 A gas-phase sequencer constituents in liver microsomes, with the 59-kDa fraction with an on-line model 120 A phenylthiohydantoin (PTH) being the most prominent TFA component (Fig. 1A, lane 1). amino acid analyzer. Normal program 03R PTH was em- The TFA proteins were separated from other microsomal ployed as provided by Applied Biosystems. proteins (Fig. 1A, lane 2) by binding them selectively to an NaDodSO4/PAGE and Immunoblotting with Anti-TFA IgG affinity column of anti-TFA IgG and then, after thorough and Human Sera. Procedures for polypeptide electrophoretic washing of the column, eluting them selectively with the separation, staining, transfer, and immunoperoxidase detec- hapten derivative TFA-Lys (Fig. 1A, lane 3). Further sepa- tion with anti-TFA IgG and human sera from halothane ration by HPLC anion-exchange chromatography (Fig. 1B) hepatitis patients as well as conditions for antibody blocking resulted in the purification of the 59-kDa-TFA protein (Fig. A B
kDa 0 -100- - 00 -76- _ UJ 1 259- z T -54- m 0 U) m
1 2 3 4
0 10 20 30 40 50 60 70 80 90 RETENTION TIME (Min) FIG. 1. Purification of 59-kDa-TFA protein from liver microsomes by immunoaffinity and anion-exchange HPLC 12 hr after administration of halothane to rats. (A) Lane 1, immunoperoxidase staining with anti-TFA IgG of a NaDodSO4/polyacrylamide gel blot of the liver microsomes before purification. Lanes 2-4, NaDodSO4/polyacrylamide gels stained with Coomassie blue. Lane 2, liver microsomes before purification; lane 3, mixture ofTFA proteins that were isolated from liver microsomes by chromatography on an anti-TFA IgG affinity column; lane 4, 59-kDa-TFA (59-TFA) that was purified from other TFA proteins by HPLC anion-exchange column as indicated in B. (B) Further purification of59-kDa-TFA by anion-exchange HPLC. The 59-kDa-TFA (59-TFA) protein eluted from the column between 23 and 28 min. Preliminary results indicate that the 100-kDa-TFA and 76-kDa-TFA proteins eluted from the column in the broad fraction between 70 and 90 min. Downloaded by guest on September 24, 2021 324 Medical Sciences: Satoh et al. Proc. Natl. Acad. Sci. USA 86 (1989) 1A, lane 4). The number ofTFA moieties bound to the 59-kDa Table 1. Comparison of NH2-terminal sequences of the 59-kDa protein remains to be determined. protein with that of rat and rabbit carboxylesterases Polyclonal antibodies raised against the 59-kDa-TFA pro- tein selectively recognized not only the 59-kDa-TFA protein Cycle 59-kDa Esterase in liver microsomes of halothane-treated rats and a number protein Rat* Rabbitt halothane-treated patient but also the native 59-kDa protein 1 Tyr Tyr His in liver microsomes of untreated rats, as determined by 2 Pro Pro Pro immunoblotting (results not shown). Based upon these find- 3 Ser Ser Ser ings, the native 59-kDa protein was isolated from control rat 4 Ser Ser Ala liver microsomes by affinity chromatography on an anti-59- 5 Pro Pro Pro kDa-TFA column (Fig. 2A, lane 1). It was further purified by 6 Pro Pro Pro HPLC anion-exchange chromatography (Fig. 2B) to apparent 7 Val Val Val homogeneity (Fig. 2A, lane 2). 8 Val Val Val Characterization of the 59-kDa Protein as a Carboxylester- 9 Asn Asn Asp ase. The NH2-terminal amino acid sequence of the 59-kDa 10 Thr Thr Thr protein was identical to a previously characterized rat micro- 11 Val Val somal serine-type carboxylesterase of apparent monomeric 12 Lys Lys molecular mass of59 kDa (18) and highly similar to a reported 13 Gly Gly 60-kDa rabbit liver microsomal serine-type carboxylesterase 14 Lys Lys (20) (Table 1). Further analysis of our 59-kDa protein re- 15 Val Val vealed other similarities to those reported for the rat car- *Data taken from Harano et al. (18). boxylesterase. It is an apparent trimer and is a high-mannose tData taken from Korza and Ozols (20). type of glycoprotein that can be deglycosylated by endogly- cosidase H to a peptide ofapparent monomeric size of57 kDa polypeptide fractions of 100 kDa, 76 kDa, and 54 kDa and, (18, 21) (results not shown). Moreover, the 59-kDa-TFA when control microsomes were tested by immunoblotting, protein hydrolyzed p-nitrophenyl acetate at a rate of 63.6 staining ofthe 100-kDa, 76-kDa, 59-kDa, or 54-kDa polypep- ,umol/min per mg, which was comparable to the rates tide fractions was negligible (Fig. 3A, serum from patient 1, reported for the rat 59-kDa carboxylesterase (44.4 ,umoUmin lane SES MS). per mg) (21). This activity was abolished by the serine Antibodies in the serum from patient 1 reacted strongly esterase and peptidase inhibitor phenylmethylsulfonyl fluo- with purified 59-kDa-TFA (Fig. 3A, lane 59-TFA) but negli- ride, as observed with the rat 59-kDa carboxylesterase (18). gibly with native 59 kDa (Fig. 3A, lane 59). Sera from three Immunoblotting the 59-kDa-TFA and Native 59-kDa Pro- additional halothane hepatitis patients, previously found to teins with Sera from Halothane Hepatitis Patients. The sera of react with a 59-kDa-TFA fraction in liver microsomes from five halothane hepatitis patients were previously reported to halothane-treated rats (11), also reacted with the 59-kDa- contain antibodies that reacted with a 59-kDa-TFA polypep- TFA protein, but negligibly if at all with the native 59-kDa tide fraction in liver microsomes from halothane-treated rats protein (Fig. 3A, sera from patients 2-4). The immunoblot in (11). This is illustrated by the immunoblot in Fig. 3A (serum Fig. 3A, stained with TFA hapten-specific rabbit IgG instead from patient 1, lane HAL MS). As found earlier (11), this ofhuman sera, confirms that 59-kDa-TFA contained the TFA patient's serum antibodies also recognized additional TFA moiety. A B 59k Da
0Co 00
z 59kDa- iitm m 0 (I) co
1 2
0 5 I 12 0 5 10 15 20 RETENTION TIME (Min) FIG. 2. Purification of 59-kDa protein from liver microsomes of untreated rats by immunoaffinity and anion-exchange HPLC. (A) Lanes 1 and 2, NaDodSO4/polyacrylamide gels stained with Coomassie blue. Lane 1, partially purified 59-kDa protein that was isolated from liver microsomes by chromatography on an anti-59-kDa-TFA IgG affinity column; lane 2, 59-kDa protein that was purified further by anion-exchange HPLC as indicated in B. (B) Further purification of the 59-kDa protein by anion-exchange HPLC. The protein eluted from the column between 8 and 11 min. Downloaded by guest on September 24, 2021 Medical Sciences: Satoh et al. Proc. Natl. Acad. Sci. USA 86 (1989) 325
4' "I,<"" halothane hepatitis patients are not directed against the TFA A 01-3)
59kDa - DISCUSSION One of the major reasons why relatively little progress has been made in the understanding of how drugs produce allergic reactions is that no drug-induced immunogen has Control TFA-Lys AC-Lys previously been identified (1). In the present study, it has been shown how immunogens involved in drug hypersensi- A,< tivities may be purified and identified when the structure of C a covalently bound drug metabolite is known and serum antibodies directed against drug-induced neoantigens are available. A neoantigen recognized by antibodies in the sera of several halothane hepatitis patients was isolated from liver microsomes of halothane-treated rats by chromatography on 59kDa- " an immunoaffinity column of anti-TFA IgG and identified as a 59-kDa-TFA carboxylesterase. The immunogen responsi- ble for the induction ofthe anti-59-kDa-TFA antibodies in the 2 2 2 patients' sera is presumably a human 59-kDa-TFA carboxyl- Control TFA-Lys AC-Lys esterase. This conclusion is based upon the fact that the apparent monomeric molecular masses of the halothane- FIG. 3. Recognition ofpurified 59-kDa-TFA by antibodies in sera induced neoantigens detected in liver microsomes of rats of patients with halothane hepatitis after NaDodSO4/PAGE and (11), rabbits (10), and humans (9), by immunoblotting with immunoblotting. (A) Liver microsomes (20 ,ug per lane) from halo- sera thane-treated (HAL MS) or control (sesame oil, SES MS)-treated from halothane hepatitis patients, are very comparable rats or purified 59-kDa-TFA (59-TFA) or native 59-kDa protein (59) in size and the fact that a 59-kDa protein has been detected (2 ,ug per lane) were probed for immunoreactivity with sera from four in liver microsomes of a patient by immunoblotting with patients with halothane hepatitis or with hapten-specific anti-TFA rabbit antibodies directed against the rat 59-kDa-TFA pro- IgG. Sera from three of these patients were previously found by tein. A similar approach could be used to purify and char- NaDodSO4/PAGE and immunoblotting to contain antibodies recog- acterize the neoantigens present in livers ofhalothane-treated nizing the following liver microsomal TFA neoantigenic fractions in humans, when sufficient amounts oftissue become available. addition to the 59-kDa-TFA protein: 100, 76, and 54 kDa (patient 1); The purified halothane-induced neoantigens will have sev- 100 kDa (patient 2); 100 and 76 kDa (patient 3) (11). (B) Recognition eral important applications. It will be possible to test directly of the 59-kDa-TFA protein by serum antibodies from patient 1 the between (Control lane) was partially inhibited by preincubation of the serum relationship sensitization to the neoantigens and for 90 min with 1 mM hapten derivative TFA-Lys (TFA-Lys lane) but the pathogenesis of halothane hepatitis by immunizing ani- not by 1 mM Ac-Lys (AC-Lys lane). (C) In contrast, 1 mM TFA-Lys mals with the neoantigens prior to challenging them with did not affect the recognition of the 59-kDa-TFA protein by serum halothane (1, 8, 11). Ifthis approach is successful, then it will antibodies from patient 2. Results similar to these were found with be possible to explore whether the hepatocellular damage the sera from patients 3 and 4 (data not shown). caused by halothane is mediated by specific antibodies or sensitized T cells (1). The reactions of the antibodies in the sera of patients 2-4 A related problem that can be addressed with the purified with 59-kDa-TFA (Fig. 3C, Control) were not inhibited by neoantigens concerns the reasons for them becoming immu- preincubation ofthe sera with the hapten derivative TFA-Lys nogens in the first place. One contributing factor may be the (Fig. 3C, TFA-Lys) or Ac-Lys (Fig. 3C, AC-Lys). The same number ofcovalently bound TFA moieties. Epitope mapping concentration of TFA-Lys, but not Ac-Lys, partially inhib- ofthe neoantigens with antibodies and T cells from halothane ited recognition of 59-kDa-TFA by antibodies in serum from hepatitis patients may help define other structural features patient 1 (Fig. 3B). In contrast, we have demonstrated that make the TFA proteins immunogenic. previously that recognition of TFA microsomal proteins by Another factor that could possibly determine whether a hapten-specific anti-TFA antibodies is inhibited nearly com- TFA protein becomes an immunogen is its rate of degrada- pletely by TFA-Lys but not by Ac-Lys (11, 12). These results tion. Those altered proteins that are degraded very rapidly confirm the idea that the antibodies in the sera of most may not become exposed to the immune system in sufficient Downloaded by guest on September 24, 2021 326 Medical Sciences: Satoh et al. Proc. Nati. Acad. Sci. USA 86 (1989) concentrations to be effective immunogens. It is believed that Weck, A. L. & Bundgaard, H. (Springer, Berlin), pp. 3-36. this may be a contributing factor as to why cytochrome P450, 6. Park, B. K., Coleman, J. W. & Kitteringham, N. R. (1987) which can become labeled with the TFA moiety (26) and may Biochem. Pharmacol. 36, 581-590. 7. Neuberger, J. & Kenna, J. G. (1987) Clin. Sci. 72, 263-270. correspond to the 54-kDa-TFA neoantigen (11), is, at most, 8. Satoh, H., Davies, H. W., Takemura, T., Gillette, J. R., only a minor immunogen. Maeda, K. & Pohl, L. R. (1987) in Progress in Drug Metabo- Probably one of the most important factors in determining lism, eds. Bridges, J. W., Chasseaud, L. F. & Gibson, G. G. the immunogenic potential of a TFA-altered protein, assum- (Taylor & Francis, Philadelphia), Vol. 10, pp. 187-206. ing that it can accumulate to adequate concentrations and be 9. Kenna, J. G., Neuberger, J. M. & Williams, R. (1988) Hepa- recognized by the immune system, will be its efficiency of tology, in press. contacting the immune system. This will be important not 10. Kenna, J. G., Neuberger, J. & Williams, R. (1987) J. Pharma- only for the initial induction of an immune response but also col. Exp. Ther. 242, 733-740. for the subsequent development ofimmune-mediated cellular 11. Kenna, J. G., Satoh, H., Christ, D. D. & Pohl, L. R. (1988) J. damage (1). Although TFA adducts have been detected Pharmacol. Exp. Ther. 245, 1103-1109. 12. Christ, D. D., Satoh, H., Kenna, J. G. & Pohl, L. R. (1988) immunochemically on the outer surface of isolated hepato- Drug Metab. Dispos. 16, 135-140. cytes (13), where they could conceivably come in contact 13. Satoh, H., Fukuda, Y., Anderson, D. K., Ferrans, V. J., with the immune system, their composition and the mecha- Gillette, J. R. & Pohl, L. R. (1985) J. Pharmacol. Exp. Ther. nisms by which they reached this site remain to be deter- 233, 857-862. mined (8). 14. Matsudaira, P. (1987) J. Biol. Chem. 262, 10035-10038. A practical application of the purified halothane-induced 15. Martin, B. M., Tsuji, S., La Marca, M. E., Maysak, K., neoantigens will be for the development of ELISA methods Eliason, W. & Ginns, E. I. (1988) DNA 7, 99-105. for the detection of individuals sensitized to halothane that 16. Christ, D. D., Kenna, J. G., Kammerer, W., Satoh, H. & Pohl, are L. R. (1988) Anesthesiology, in press. more specific and sensitive than procedures currently 17. McLean, L. R., Demel, R. A., Socorro, L., Shinomiya, M. & available (7, 8, 10, 11, 27). This could prevent patients Jackson, R. L. (1986) Methods Enzymol. 129, 738-763. sensitized to halothane from being reexposed to this agent or 18. Harano, T., Miyata, T., Lee, S., Aoyagi, H. & Omura, T. (1988) to other drugs that might elicit cross-sensitization, such as the J. Biochem. 103, 149-155. structurally related inhalation anesthetic enflurane (12, 16). 19. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951) J. Biol. Chem. 193, 265-275. We thank Dr. James R. Gillette for critically reviewing the 20. Korza, G. & Ozols, J. (1988) J. Biol. Chem. 263, 3486-3495. manuscript. D.D.C. was supported by a National Research Service 21. Mentlein, R., Heiland, S. & Heymann, E. (1980) Arch. Bio- Award (ES05368) from the National Institute of Environmental chem. Biophys. 200, 547-559. Health Sciences, and J.G.K. was supported in part by a grant from 22. Sipes, I. G., Gandolfi, A. J., Pohl, L. R., Krishna, G. & the Wellcome Trust, U.K. Brown, B. R., Jr. (1980) J. Pharmacol. Exp. Ther. 214, 716- 720. 23. Faed, E. M. (1984) Drug Metab. Rev. 15, 1213-1249. 1. Pohl, L. R., Satoh, H., Christ, D. D. & Kenna, J. G. (1988) 24. Caldwell, J. & Marsh, M. V. (1983) Biochem. Pharmacol. 32, Annu. Rev. Pharmacol. Toxicol. 28, 367-387. 1667-1672. 2. Parker, C. W. (1977) in Drug Design and Adverse Reactions, 25. Fraser, J. M. & Kaminsky, L. S. (1987) Toxicol. Appl. Phar- eds. Bundgaard, H., Juul, P. & Kofod, H. (Academic, New macol. 89, 202-210. York), pp. 153-164. 26. Satoh, H., Gillette, J. R., Davies, H. W., Schulick, R. D. & 3. Parker, C. W. (1982) Pharmacol. Rev. 34, 85-104. Pohl, L. R. (1985) Mol. Pharmacol. 28, 468-474. 4. De Weck, A. L. (1983) in Allergic Reactions to Drugs, eds. De 27. Hubbard, A. K., Roth, T. P., Gandolfi, A. J., Brown, B. R., Weck, A. L. & Bundgaard, H. (Springer, Berlin), pp. 75-133. Jr., Webster, N. R. & Nunn, J. F. (1988) Anesthesiology 68, 5. Schneider, C. H. (1983) inAllergic Reactions to Drugs, eds. De 791-7%. Downloaded by guest on September 24, 2021