Chem. Res. Toxicol. 1993,6, 786-793
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An Immunochemical Approach of Identifying and Characterizing Protein Targets of Toxic Reactive Metabolites Lance R. Pohl' Laboratory of Chemical Pharmacology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892 Received August 31, 1993
Introduction Reactive metabolites are known to cause toxicities produced by many drugs, environmental chemicals, and endogenous molecules (1-10). But the mechanisms by which they lead to toxicity has rarely been understood. One important reason for this is that relatively little is known of the identity of tissue protein targets of reactive metabolites. In the past the major way the structures of toxic reactive metabolites were determined and covalent adducts detected was by the use of radiolabeled parent compounds. This radiochemical approach, however, has not been used as successfully for the identification of the specific protein targets of reactive metabolites for several reasons. First, it requires the synthesis of a radiolabeled parent compound, which in many cases may be very difficult to accomplish. Alternatively, the compound may be purchased from a chemical or drug company, but this can be quite expensive. If instead the radiochemical is obtained as a gift, it often may not be of sufficient quantity that will be required for the isolation of protein adducts in uiuo. The second problem is that the radiochemical compound may not be of high enough specific activity for the detection of protein adducts in tissues or in fractions eluting from columns during purification that have been analyzed by the high-resolution separation method of sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE).l The third disadvantage of the radiochemical approach for the detection of macromolecular targets of reactive metabolites is that it involvesthe hazards and restrictions of working with radiochemical isotopes. More recently, an immunochemical approach has been developed for the sensitive detection, identification, and characterization of protein targets of toxic reactive metabolites. This method, which does not require the use of radioactive-labeled compounds, has, as its basis, specific antibodies that will recognizethe reactive metabolite, when it is covalently bound to tissue protein. In order to develop such antibodies, it is necessary to know the structure and chemistry of the reactive metabolite. This information often already exists in the literature or can be predicted, based upon the large number of mechanistic studies that have been done on the identification and chemistry of reactive metabolites (11). Once the identity of a reactive
* Address correspondence to Laboratory of Chemical Pharmacology, NHLBI, NIH, Building 10, Room 8N 115, Bethesda, MD 20892. Tel: 301-496-4841; Fax: 301-402-0171. 'Abbreviations: SDS/PAGE, sodium dodecyl sulfate/polyacrylamide gel electrophoresis; ELISA, enzyme-linked immunosorbent assay; KLH, keyhole limpet hemocyanin; RSA, rabbit serum albumin; TFA, trifluoroacetylated; TFEC, S-(1,1,2,2-tetrafluoroathyl)-~-cysteine; CTFC, S-(2chloro-1,1,2-tnfluor~thyl)-~-cysteine; NAPQI,N-acetyl-p-benzoquinone imine;APAS, acetaldehyde-protein adducts, NSAIDs, nonsteroidal antiinflammatory drugs; UDPGT, UDP-glucuronosyltransferase; UDP-GA, UDP-glucuronic acid; MDA, malondialdehyde; HNE, 4-hydroxy-2nonenal; LDL, low-density lipoprotein.
metabolite has been determined or predicted, the next step involves the preparation of an immunogenic haptencarrier protein conjugate, consisting of multiple copies of the reactive metabolite or a derivative of it coupled to a carrier protein. Although very specific monoclonal antibodies may be produced against the bound haptens, polyclonalantibodies should be prepared initially, not only because it takes less time and energy, but also because polyclonal antibodies will likely be directed against multiple epitopes of the bound reactive metabolite and therefore may react with other structurally similar covalently bound reactive metabolites of the compound being studied. Usually within 8-12 weeks, polyclonal antisera can be developed against the covalently bound reactive metabolite. The most common way of monitoring the formation of the antibodies directed against a covalently bound reactive metabolite is by an enzyme-linked immunosorbent assay (ELISA). The test antigen that is reacted with the antisera should not be the hapten-carrier conjugate that was used for the immunization. Instead, it should be an antigen consisting of the reactive metabolite or its derivative covalently coupled to a carrier protein that is structurally different from the carrier protein that was used for the immunization procedure. For example, if the carrier protein used for immunization was keyhole limpet hemocyanin (KLH), then the carrier protein used for the ELISA procedure could be rabbit serum albumin (RSA). The reason for doing this is that when animals are immunized with hapten-carrier conjugates, antibodies may be raised against the carrier protein as well as against the covalently bound hapten. Antibodies directed against the carrier protein would increase the level of background signal and therefore make it difficult in some cases to detect antihapten antibodies. It is necessary to go one step further to show that the antibodies are directed against the covalently bound hapten. This is done by a competitive ELISA method. In this procedure, it is determined whether amino acid derivatives of the reactive metabolite can inhibit the interaction of the antisera with the test hapten-carrier conjugate. This approach may also provide information about the amino acid residue in the carrier protein, where the reactive metabolite is covalently bound. Once an antisera has been raised against a covalently bound metabolite, it can be used in conjunction with several immunochemical techniques for detecting, identifying, and characterizing protein targets of the metabolite. For example, the antibodies can be used in ELISA procedures for detecting protein adducts in tissues, subcellular fractions, or in column fractions. The apparent monomeric molecular masses of the adducts in these samples can be determined by probing immunoblots of them with the antisera. The cellular and subcellular
This article not subject to U S . Copyright. Published 1993 by the American Chemical Society
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localization of the adducts in tissues can be determined by immunohistochemical application of the antisera. Very importantly, the antisera can also be used for the purification of adducts by immunoaffinity chromatography.
Protein Adducts Associated with Halothane Hepatitis Several studies have suggested that the idiosyncratic hepatitis caused by the inhalation anesthetic halothane may be due to immune reactions directed against liver proteins that have been altered by halothane (for reviews see refs 12-15). The most informative findings that have led to this conclusiod have come from immunoblotting studies. In these investigations, serum antibodies from halothane hepatitis patients were found to recognize polypeptide fractions of 100,76,59,57, and 54 kDa in liver microsomes of humans (16)and animals ( 1 7-19) that had been treated with halothane. Patients’ sera differed markedly in patterns of polypeptide recognition, with the 100- and 76-kDa antigens being recognized most commonly. Subcellular fraction studies revealed that the antigens recognized by the patients’ sera were concentrated in the microsomal fraction of the liver (17). The reactions with these protein fractions were not seen when the sera of control patients or patients suffering from other forms of hepatitis were used in place of the halothane hepatitis patients’ sera. Moreover, very little if any reaction occurred when the liver microsomes used for the immunoblotting studies were from sources other than halothanetreated humans or animals. These findings indicated that halothane treatment had somehow altered the liver microsomal proteins to produce neoantigens that were recognized by the patients’ serum antibodies. Since halothane is not chemically reactive under physiological conditions, it appeared that a reactive metabolite of halothane was most likely responsible for the recognition of the liver microsomal proteins by the halothane patients’ serum antibodies. In this regard, studies indicated that this metabolite was trifluoroacetyl chloride [CF&(O)ClI formed from the oxidative metabolism of halothane by cytochromes P-450 (Figure 1A) (20).For example, it was found with the use of an ELISA procedure that two of six patients diagnosed as having halothane-associated massive liver cell necrosis contained serum antibodies that reacted with trifluoroacetylated (TFA) RSA (21). This finding was consistent with the idea that the hapten-carrier conjugates recognized by the halothane hepatitis patients’ serum antibodies might be formed by the reaction of endoplasmic microsomalproteins with CF3COC1. In order to test this idea, an antibody was raised against the TFA hapten by immunizing rabbits with TFA-RSA, which was formed by the reaction of RSA with S-ethyl trifluorothiolacetate (Figure 1B) (22). This reagent mainly reacts with lysine residues of proteins, which were assumed to be the stable site of adduct formation of CF&(O)Cl with tissue proteins. Immunoblotting studies with microsomes from rats (18)and humans (14)that had been treated with halothane revealed that the protein fractions recognized by the patients’ serum antibodies also reacted with the anti-TFA antibodies. When the TFA hapten was removed from the liver microsomes by treatment with 1M piperidine, the patients’ antibody recognition of the microsomal proteins was abolished (18).This showed that the TFA moiety was required for antibody recognition by the patients’ serum antibodies. The patients’ antibodies,
A F Br I 1 F-C-C-CI 1 1 F H
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Figure 1. (A) Pathway for the metabolic activationof halothane by cytochromes P-450 to form trifluoroacetyl chloride and the covalent bonding of this metaboliteto lysine residues of proteins (P).(B)Pathway for the synthesis of TFA-RSA immunogen that was used for the production of antibodies that recognized trifluoroacetylchloride bound to cellular proteins. (C) Pathway for the metabolic activation of TFEC by cysteine conjugate @-lyase to form difluorothionoacetylfluoride and the covalent bonding of this metaboliteto lysine residues of proteins. (D) Pathway for the metabolic activation of CTFC by cysteine conjugate 8-lyase to form chlorofluorothionoacetyl fluoride and the covalent bonding of this metabolite to lysine residues of proteins.
however, did not appear to be directed solely against the TFA hapten, but instead against epitopes that consisted of the TFA hapten and undefined specific structural features of the individual carrier proteins. This conclusion was based upon the finding that the patients’ sera often differed in their patterns of protein recognition and by the discovery that the reaction of the patients’ antibodies with the TFA-microsomal proteins was only partially inhibited by the hapten derivative N‘-TFA-L-lysine, whereas it nearly abolished the binding of the anti-TFA antibodies to the TFA-liver microsomal proteins (18,23). These results also indicated that the CF3C(O)Clmetabolite of halothane preferentially formed stable covalent bonds with lysine residues of microsomal proteins, a finding that was recently confirmed by 19Fnuclear magnetic resonance studies (24). Purification and characterization of several of the TFAproteins from rat liver microsomes has been accomplished with the use of the anti-TFA antibodies. The 59-matarget protein was purified by affinity chromatography on an anti-TFA column and has been identified as a carboxylesterase (23). The anti-TFA antibodies were used to monitor the purification of several other TFA-proteins by traditional chromatographic procedures. The 100-kDa protein was identified as ERp99, which is identical to a 94-kDa glucose-regulated protein, and endoplasmin (25); the 76-kDa protein fraction was resolved into two protein targets of CF&(O)Cl, an80-kDaprotein, which is identical to ERp 72 (26),and an 82-kDa protein, which corresponds to a 78-kDa glucose-regulated protein, also known as BiP (27);the 57-kDa target protein is identical to protein disulfide isomerase ( 28);and the 54-kDa protein target appears to correspond to an unidentified form of cytochromes P-450 (20). Two additional protein targets of CF&(O)Cl were discovered during the purification of the TFA-proteins: a 63-kDa protein target, which corresponds to calreticulin (29),and a 58-kDa protein (30,31),which may be a member of the PDI family (321,a cellular protease (33),or a carnitine medium/long-chain acyltransferase (34). The purified rat liver TFA-proteins appear to correspond, at least in part, to the TFA-carrier proteins detected
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by the serum antibodies of the halothane hepatitis patients on immunoblots of liver microsomes from halothanetreated animals and humans. For example, it was found in an ELISA study that the sera of 10 halothane hepatitis patients reacted with several of t h e purified r a t TFA-proteins: TFA-100 kDa (9 of lo), TFA-80 kDa (5 of lo), TFA-63 kDa (1of lo), TFA-59 kDa (2 of lo), TFA-58 kDa (5 of lo), and 57 kDa (2 of 10) (35).
Pohl A
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Other Uses of the Anti-TFA Antibodies The anti-TFA antibodies have been used to solve several additional problems. In immunohistochemistry studies, the antibodies were employed to show that the perivenous region of the liver lobule was the major site of trifluoroacetylation of liver proteins and that surface macromolecules were also labeled by the trifluoroacetyl chloride metabolite of halothane (22). Western blotting and immunohistochemical studies have revealed that small levels of TFA-proteins are formed in extrahepatic tissues including the testes (20),kidney (36,37),heart (38),lung (37), and respiratory and olfactory epithelium of nasal tissue (37). Moreover, in the liver, low levels have been discovered in Kupffer cells, which may have a role in the development of the immune responses of the halothane hepatitis patients against the TFA-proteins (39). The anti-TFAantibodies have been used to detect target proteins of reactive metabolites of other halogenated hydrocarbons that form TFA adducts or adducts that are structurally similar to the TFA adducts. For example, HCFC-123 (CF&HC12), which has been developed as a replacement for ozone depleting chlorofluorocarbons, produced nearly identical patterns and levels of TFAliver proteins as that of halothane (24). Much lower levels of hepatic TFA-proteins were found after rats were exposed to HCFC-124 (CF3CHFCl) or HCFC-125 (CF3CHF2) treatments (40). These results indicated that because HCFC-124 and HCFC-125 produced lower TFA-protein concentrations, they may be safer chlorofluorocarbon alternatives than HCFC-123. Similarly,the protein targets of the reactive acyl halide metabolite [CHF20CFzC(O)FI of the inhalation anesthetic enflurane (CHF20CF2CHFCl) have been found to be similar to those of halothane (41, 42). In this case, the anti-TFA antibodies and serum antibodies of the halothane hepatitis patients cross-reacted with CHFzOCF2CO adducts. The anti-TFA antibodies have been shown to crossreact with CHFzCS adducts formed from the reaction of tissue proteins with the difluorothionoacetylating metabolite [CHFzC(S)FI of the nephrotoxicagent S-(1,1,2,2tetrafluoroethy1)-L-cysteine(TFEC) (Figure 1C). When rats were treated with TFEC, immunohistochemical analysis revealed that CHFzCS adducts in the kidney were localized to the damaged areas of proximal tubules (43). Immunoblotting of subcellular fractions showed that major targets of CHF2C(S)F were proteins in the mitochondria (43-45), the organelle considered to be primary target of toxicity of TFEC, and other cysteine conjugate metabolites. These proteins have been reported to have apparent monomeric masses of 99 kDa (45), 87 kDa (43), 84 kDa (45),80 kDa (44),79 kDa (43),66 kDa ( 4 5 ) , 6 1kDa (43), 52 kDa (45), 48 kDa (451,and 40 kDa (44). Very recently, N-terminal amino acid sequence analyses of the purified 84-, 66-, and 42-kDa proteins indicate that these proteins are members of the heat shock protein 70 and 60 families, and aspartate aminotransferase, respectively ( 4 5 ) . It
C=O
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0 C
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Figure 2. (A) Pathway for the metabolic activation of acetaminophen by cytochromes P-450to form NAPQI and the covalent bonding of this metabolite to cysteine residues of proteins (P). (Band C)Alternative pathwaysfor the synthesis of immunogens that were used for the production of antibodies that recognized NAPQI covalently bound to cellular proteins.
is thought that the adducts of these proteins may have a role in the development of the kidney toxicity produced by TFEC (45). They may also have a role in the kidney toxicity produced by other cysteine conjugate metabolites, (CTFC). such as S-(Zchloro-1,1,2-trifluoroethyl)-~-cysteine The protein targets of CHClFC(S)F, formed from CTFC (Figure lD), also have been found to cross-react with the anti-TFA antibodies and appear to be similar in apparent monomeric molecular mass, at least in part, to those found with TFEC (46). Like CF&(O)Cl, CHF&(S)F ( 4 7 , 4 8 ) and CHFClC(S)F (48) were found to form stable covalent bonds preferentially with lysine residues of proteins.
Protein Adducts Associated with Acetaminophen Hepatotoxicity The reactive metabolite that causes the hepatotoxicity produced by an overdose of acetaminophen is N-acetylp-benzoquinone imine (NAPQI). This metabolite is formed from the oxidation of acetaminophen by cytochromes P-450 (49, 50) and appears to bind covalently predominantly to cysteine residues of proteins (Figure 2A) (51,521. On the basis of these findings, two approaches have been used to raise antibodies that can detect this reactive metabolite when it is bound covalently to tissue proteins [Figure 2B (53)and Figure 2C (54)l. Antibodies have been successfully raised by both of these methods and have been used to detect adducts of NAPQI in tissues immunohistochemically (55,561,by ELISA methods ( 5 3 , and by immunoblotting (56,58,59). The immunoblotting studies have revealed that several proteins are targets of NAPQI. In mouse liver, major targets are a microsomal protein of 44 kDa and a cytosolic protein of 55-58 kDa
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H
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CH3-C-N-Lys-P I I H H
B
R
CH3-C-H
H
P + NaBH4 or NaBH3CN )
CH3-C-N-Lys-P I I H H
Figure 3. (A) Pathway for the metabolic activation of ethanol by cytochromes P-450 or alcohol dehydrogenase to form acetaldehyde and the covalent bonding of this metabolite to lysine residues of proteins (P) as a Schiff base derivative or a more stable reduced N-ethyl derivative of lysine. (B)Pathways for the synthesesof immunogenic acetaldehydeadducts of proteins (P)that were used for the production of antibodies that recognized acetaldehyde bound to cellular proteins.
(56,58,59). Recently the 55-58-kDa protein target has been purified and identified as a selenium binding protein (60,61).
Protein Adducts Associated with Ethanol Hepatotoxicity Alcoholic liver disease is a major cause of morbidity and mortality. In spite of extensive investigations, the mechanism by which alcohol produces hepatitis remains unknown (62,63).The possibility that this disease may have an immune basis has received considerable attention during the last several years (62-64).Several studies have shown that 38-74% of alcoholic hepatitis patients have antibodies in their sera that react with the surface of hepatocytes of rabbits that had been treated with alcohol (65-68).No ethanol-related antibodies were found in the sera of normal individuals or in patients with other types of acute or chronic liver disease. These and other findings have led to an increased interest in identifying the liver adducts of alcohol that are responsible for the patients immune responses. It appears that acetaldehyde is responsible for the formation of neoantigens recognized by the patients' antibodies. For example, treatment of rabbits with 4-methylpyrazole (an inhibitor of alcohol dehydrogenase), or with disulfiram (an inhibitor of aldehyde dehydrogenase), decreased and increased, respectively, the amount of reaction of the patients' antibodies with the rabbit hepatocytes (68). Acetaldehyde has been shown to react in vitro with a variety of proteins including albumin, plasma proteins, erythrocyte membrane proteins, hepatic microsomal proteins, hemoglobin, and tubulin (69,70). I t forms both unstable and stable adducts (Figure 3A) (71).The unstable adduct appears to be a Schiff base formed on the a-amino group of N-terminal amino acids or the e-amino group of lysine residues, because they can be converted to stable N-ethyl derivatives by treatment with sodium borohydride or sodium cyanoborohydride (69).Not as much is known about the structures of the stable adducts, except in the case of hemoglobin, where they appear to be imidazolidinone derivatives of the N-terminal valine residues of both a and B chains (70).Moreover, it is thought that the Schiff base derivative is reduced in the body to a N-ethyl derivative of lysine, possibly by ascorbate or NADH (71). In this regard, acetaldehyde-protein adducts (APAS), formed from the reaction of acetaldehyde with KLH or other proteins in the presence of sodium borohydride or
sodium cyanoborohydride (Figure 3B), have been used as immunogens to raise antibodies that recognize APAS in tissues. A 37-kDa neoantigen in the liver of rats fed alcohol chronically has been detected by immunoblotting liver subcellular fractions with anti-APAS antibodies (72,73). The adduct was found in cytosol,but not in the microsomal or mitochondrial fractions of the liver. An apparently identical 37-kDa adduct is formed when primary cultures of rat hepatocytes are exposed to ethanol for several days (74).It appears that acetaldehyde is responsible for the formation of the 37-kDa neoantigen, because the amount of the protein increases when the acetaldehyde dehydrogenase inhibitor cyanamide is added either to the diet of rats (73)or the culture media of hepatocytes exposed to ethanol (74). Recent amino sequence analysis of the purified 37-kDa protein suggested that the protein is a member of the aldehyde/aldose reductase family (75).The same anti-APAS antibodies have been used to detect APAS on the surface of hepatocytes (76). In contrast to these findings, other investigators have detected different adducts in the livers of animals treated with ethanol, with the antibodies that they have raised against APAS. In one study, several major APAS of 14, 17, 26, 35, 46, 55, 59 and 81 kDa were detected in the cytosol of livers of rats fed ethanol (77).Other researchers have discovered a 52-kDa neoantigen in the liver microsomesof alcohol-fed rats (78).The protein was isolated by immunoaffinity chromatography on a column of Sepharose-conjugated anti-APAS IgG and was found to correspond to cytochrome P-4502E1, the microsomal enzyme that is induced by ethanol and oxidizes it to acetaldehyde. Although it is not clear why the antibodies raised against APAS by different laboratories appear to have different specificities, it may be due, at least in part, to differences in the nature of the hapten-carrier conjugates that were used as immunogens to raise the APAS antisera.
Protein Adducts Associated with Diclofenac Hepatotoxicity Serious idiosyncratic hepatic injury has been associated with the use of many nonsteroidal anti-inflammatory drugs (NSAIDs), and some agents have even been withdrawn from the market because of this problem (79).One of the most widely prescribed NSAIDs is diclofenac. Although relatively safe, several cases of severe and even fatal hepatotoxicity have been reported to occur while patients were taking this drug (80-87). Some of the studies suggest a hypersensitivity basis for the toxicity (81,85,87), while others favor a metabolic mechanism (80,82-84).In order to investigate the possibility that diclofenac-protein conjugates might have a role in the hepatotoxicity produced by diclofenac, an antisera was developed for detecting adducts of diclofenac in liver tissue. The development of the antisera was based upon the suggestion that acyl glucuronide metabolites of NSAIDs might mediate some of the toxicities produced by this class of compounds (88-90). The formation of acyl glucuronides of NSAIDs is catalyzed by microsomal UDPglucuronosyltransferase (UDPGT; EC 2.4.1.17) and involves the conjugation of a carboxyl functional group with glucuronic acid derived from UDP-glucuronic acid (UDPGA) (Figure 4) (91).Acyl glucuronide metabolites are chemically reactive and can covalently bind to tissue
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790 Chem. Res. Toxicol., Vol. 6, No. 6, 1993
r
A c y l migration
H N - Prore in
pr0tein.N
Figure 4. Possible pathways for the metabolic activation of diclofenac by UDPGT to form a 1-0-acylglucuronide or possibly rearranged acyl glucuronides and the covalent bonding of these metabolites to proteins.
1 Carbodiimide Activation
2 Coupling to KLH Cl
Figure 5. Pathway for the synthesis of diclofenacKLH immunogen that was used for the production of antibodies that recognized diclofenac covalently bound to cellular proteins. proteins by transacylation (88-90). Alternatively, they may undergo acyl migration within the glucuronic acid molecule, prior to covalent binding to proteins by glycosylation (90).In both cases, the NSAIDs would be covalently linked to proteins as acyl derivatives. Therefore, diclofenac was coupled to KLH through its carboxyl group, and anti-haptenantibodies were raised against this product (Figure 5 ) (92). Since diclofenac was able to inhibit, at least in part, the reaction of the antisera with an antigen formed from the conjugation of diclofenac to RSA, it appeared that the antisera contained antibodies that would be expected to react with both types of glucuronide adducts as well as other possible covalent adducts of diclofenac that retained structural features of the diclofenac molecule. With the use of these antibodies, four major protein adducts of 50, 70, 110, and 140 kDa were detected in immunoblots of liver homogenates of mice treated with diclofenac (92). The 110-kDa adduct was found to be concentrated in the plasma membrane fraction of liver cells (93). This finding was corroborated by the immunohistochemicalevidence of diclofenacadduct accumulation in canalicular plasma membranes in the perivenous region of the liver lobule (93). The role that the adducts may have in the hepatotoxicity produced by diclofenac is currently being studied.
Proteins Posttranslationally Modified by Products of Oxidative Stress Oxidative stress, produced by normal cellular metabolism, xenobiotics, or pathological states, such as reperfussion injury, may lead to the posttranslational modification of proteins by at least two mechanisms. In the first process, metal-catalyzed mixed-function oxidation types
of reactions lead to the formation of reactive oxygen radicals, presumably hydroxyl radical ( 3 , 4 ) . This species is believed to cause the fragmentation of proteins, the cross-linking of proteins through -S-S- and -Tyr-Tyrbonding, and the introduction of carbonyl groups into amino acid residues of proteins (3, 4, 94). The second pathway of the posttranslational modification of cellular proteins by oxidative stress products involvesthe reaction of proteins with aldehyde lipid peroxidation products, such as malondialdehyde (MDA) or 4-hydroxy-2-nonenal(HNE) (5-7, 95-97). It is thought that one or both of these pathways of protein modification may have a role in protein turnover and aging and in many diseases, such as atherosclerosis, neurological disorders, cataractogenesis, ischemia reperfusion tissue damage, and others (3-10,98). I t is also conceivable that these same covalently modified proteins may be recognized as foreign proteins in certain individuals and be responsible, at least in part, for the onset of immune-mediated reactions (99)and a variety of pathological states. Although several spectrophotometric and radiochemical assays have been developed to detect carbonyl groups in oxidized proteins (94) and aldehyde adducts of proteins (97),they have not been useful for the identification and characterization of individually modified protein in a mixture of cellular proteins. In this regard, two recent studies have shown that this problem can be overcome with the use of specific antibodies. In one of the studies, the investigators developed an immunochemical method of detecting oxidized proteins, containing carbonyl groups. Their approach involved first reacting the modified proteins with (2,4-dinitrophenyl)hydrazineto give the corresponding hydrazone derivatives (Figure 6A) (100). This reaction is the basis for the spectrophotometric determination of carbonyl content in oxidatively modified proteins (94). The method, however, was made more specific and sensitive than the spectrophotometric method by next fractionating the derivatized proteins by SDS/ PAGE, transferring the separated proteins electrophoretically to nitrocellulose, and detecting those proteins containing the (2,4-dinitrophenyl)hydrazonemoiety with 2,4-dinitrophenyl antiserum that was commercially obtained. The specificity of the procedure was confirmed
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0 2 L
O2N.
lmmunoblotwith Anti-Z,4-ONPH Abs
Detection of Proteins Containing Carbonyl Groups
Figure 6. (A) Immunochemical method for the detection of oxidized proteins containing carbonyl groups. 'Anti-2,4-DNP Abs" corresponds to 2,4-dinitrophenyl antisera. (B)Reaction of HNE with proteins, where X represents the NH group of the side chains of lysine and histidine and the S atom of a cysteine residue, respectively.
by showing that the immunochemical reactions could be blocked by 2,4-dinitrophenol. Although this method was used in this study to detect only carbonyl-containing products of the oxidation of bovine serum albumin by radiolysis and metal-catalyzed reactions, it appears to have important applications for the study of proteins that are oxidatively modified in vivo. In the other study, investigators have developed an antisera that can detect cellular proteins that have been covalently modified by HNE (101). The antibody was prepared by immunizing rabbits with the product of the reaction of KLH with HNE. Previous studies have shown that a major pathway of reaction of HNE with proteins is by a Michael addition mechanism with histidine (95), lysine (96),or cysteine residues (Figure 6B)(93, although tyrosine and arginine residues may also react with HNE (7). Since the antibody reaction with HNE-modified proteins could be completely inhibited by HNE-Nacetylhistidine, HWN-acetyllysine, or HNE-glutathione, it appeared that the antigenic determinant recognized by the antibody was the HNE moiety. This was an important finding because it suggested that the antisera could recognize HNE bound to many different amino acid residues. When primary rat liver hepatocytes where exposed to HNE or oxidative stress with tert-butyl hydroperoxide or metal-catalyzed oxidation systems, the HNE-specific antibody reacted selectively with a number of HNE-protein adducts in immunoblot analyses of crude liver homogenates. Other investigators have raised antibodies against HNE-modified low-density lipoprotein (LDL) and have used these antibodies to detect HNEmodified apolipoprotein B in oxidized LDL (7) and in atherosclerotic lesions (9,IO). Similarly, MDA-modified LDL has been found in atherosclerotic lesions (9,101 and in the serum of patients with cardiovascular diseases (8), with the use of antibodies that have been raised against MDA-modified LDL.
Conclusion Due to the advent of the immunochemical approach for identifying the targets of toxic reactive metabolites, in the next five years, many more of the protein targets of
toxic reactive metabolites will be identified. The next major problem to solve will be the role that the target proteins have in the toxicities produced by reactive metabolites. If the target proteins are found to have important cellular functions, perhaps reactive metabolites will be shown to alter these activities. Alternatively, if the toxicity being studied has an immune basis, the protein targets may be the immunogens that lead to the immunopathology. In contrast, the protein targets may be scavengers of reactive metabolites and protect cells from toxic reactive metabolites. In order to solve these problems in the future, chemical toxicologists will have to become proficient in cellular biology, immunology, and molecular biology.
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