Chem. Res. Toxicol. 1993,6, 147-150
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Communications Immunochemical Detection of Liver Protein Adducts of the Nonsteroidal Antiinflammatory Drug Diclofenac Neil R. Pumford,tJ Timothy G. Myers,+Julio C. Davila,? Robert J. Highet,s and Lance R. Pohl*ft Laboratory of Chemical Pharmacology and Laboratory of Biophysical Chemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892 Received November 11, 1992
Serious idiosyncratic hepatic injury has been associated with the use of many nonsteroidal antiinflammatory drugs, including the widely prescribed agent diclofenac. In order to investigate the possibility that covalent protein adducts of reactive metabolites of diclofenac might be responsible for the hepatotoxicity produced by this drug, we have developed a polyclonal antibody that can recognize such adducts in tissues. Immunoblotting revealed that protein adducts of reactive metabolites of diclofenac of 50,70,110, and 140 kDa were formed in the livers of mice treated with diclofenac. In the future, it will be determined whether these adducts can cause hepatotoxicity by either a hypersensitivity or metabolic mechanism. Similar approaches may be used to study the protein adducts and mechanisms of hepatotoxicity of other nonsteroidal antiinflammatory drugs.
Introduction Serious idiosyncratichepatic injury has been associated with the use of many nonsteroidalantiinflammatorydrugs (NSAIDs),land some agents have even been withdrawn from the market because of this problem (1). It is not known how this class of drugs causes hepatotoxicity. One of the most widely prescribed NSAIDs is diclofenac (Figure 1A). Although this drug is relatively safe, several cases of severe and even fatal hepatotoxicity have been reported to occur while patients were taking it (2-9). Some of the studies suggest a hypersensitivitybasis for the toxicity (3, 7,9),while others favor a metabolic mechanism (2,443). Both mechanisms of hepatotoxicity, however, could be attributed to the covalent modification of tissue proteins by reactivemetabolitesof diclofenac(10-13). For example, the hypersensitivity mechanism of toxicity might be due to an immune response against the covalentadducts, while the metabolic mechanism of toxicity might be due to the alteration of a vital cellular function, as a consequence of protein adduct formation. In order to investigate these possibilities,we have developed a polyclonal antibody that recognizes covalently bound metabolites of diclofenac in immunoblots of liver tissue. In the future, it will be
* Correspondence and reprint requests should be addressed to this author at the Laboratory of Chemical Pharmacology, NHLBI, NIH, Building 10, Room 8N 115, Bethesda, MD 20892. Telephone: 301-4964841; Fax: 301-402-0171. + Laboratory of Chemical Pharmacology. f Present address: Division of Toxicology,Department of Pharmacology and Toxicology, Mail Slot 638,Universityof Arkansas for Medical Sciences, Little Rock, AR 72206. 8 Laboratory of Biophysical Chemistry. 'Abbreviations: NSAIDs,nonsteroidalantiinflammatorydrugs;KLH, keyhole limpethemocyanin;EDC, l-ethyl-3-[3-(dimethylamino)propyllcarbodiimidehydrochloride;PBS, phosphate-bufferedsaline;RSA, rabbit serum albumin; ELISA, enzyme-linked immunosorbent assay; NP40, Nonidet P40; FCS, fetal calf serum; CI/MS, chemical ionization mass spectrometry; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
B
---
Protein-xH lX=O.S,NH)
coon
I
COOH
Figure 1. (A) Structures of diclofenac and its lysine derivative. (B) Possible pathways of covalent binding to tissue proteins of acyl glucuronide metabolites of NSAIDs.
determined whether these adducts can cause hepatotoxicity by either a hypersensitivityor metabolic mechanism.
Experimental Procedures Materials. Chemicals were obtained from the following sources: keyhole limpet hemocyanin (KLH) and 1-ethyl-3-[3(dimethy1amino)propyllcarbodiimidehydrochloride(EDC)from Pierce Chemical Co. (Rockford, IL), and the sodium salt of diclofenac and Nu-acetyl-L-lysinefrom Sigma (St. Louis, MO). Immunogen Preparation. Diclofenacwas covalently coupled to KLH by a two-step conjugation method (14). EDC (288 mg, 1.5 mmol) in 7.5 mL of 20 mM sodium phosphate (pH 5.0) was added to 2.5 mL of a methanolic solution of the sodium salt of diclofenac (95.6mg, 0.30mmol). After 2 min,the reactionmixture was addedto KLH (40mg, in 20 mL of 160 mM sodium phosphate, pH 8) and incubated overnight at room temperature. The conjugate was dialyzed against 3 L of phosphate-bufferedsaline
This article not subject to U.S.Copyright. Published 1993 by the American Chemical Society
148 Chem. Res. Toxicol., Vol. 6, No. 2, 1993
(PBS),with three changes. Diclofenac was coupled to rat serum albumin (RSA) in a similar manner, except that 0.06 mmol of diclofenac was used for this reaction. Preparation and Detection of Anti-Diclofenac Serum Antibodies. Two female New Zealand White rabbits (2.5 kg, Dutchland, Denver, PA) were immunized with 500 mg of the diclofenac-KLH conjugate in 2 volumes of Freund's complete adjuvant, subcutaneously at 20 sites along the back and intramuscularly in the right and left hind quarters. After 4 weeks, rabbits were boosted in a similar manner with 500 pg of diclofenacKLH in Freund's incomplete adjuvant, and sera were collected after 2 weeks. Anti-diclofenac antibodies were determined by an enzyme-linked immunosorbent assay (ELISA), with the use of diclofenac-RSA as the test antigen. All incubations were done at room temperature unless otherwise stated. Antigen (20 ng) diluted in 50 pL of 60 mM sodium carbonate (pH 9.6) was applied to the wells of microtiter plates (Immulon 2, Dynatech, Chantilly, VA). After overnight incubation a t 4 "C, the wells were washed four times with 0.05% Nonidet P40 (NP40) in PBS (washing buffer). Excess binding sites were blocked with 5% fetal calf serum (FCS) in PBS (FCS-PBS) for 1 h, followed by addition of 100 pL of anti-diclofenac serum diluted in 2% FCS-PBS. After incubation for 2 h, the wells were washed four times. Goat antirabbit alkaline phosphatase conjugate (100 pL) (Gibco BRL, Gathersburg, MD), diluted 1:3000 in 2 % FCS-PBS, was added to the wells and incubated for 1 h. After washing, 100 pL of substrate solution @-nitrophenyl phosphate, Bio-Rad, Richmond, CA, prepared according to manufacturer's insert) was added and the reaction was allowed to develop for 1h. Product absorbance was measured a t 410 nm. A competitive ELISA was performed essentially as described above, except that the anti-diclofenac serum was diluted to 1 : l O 000 in a solution of PBS, containing 5 % methanol and various concentrations of inhibitors. Synthesis of Derivatives of Diclofenac. (A) Diclofenac methyl ester was synthesized by dissolving the sodium salt of diclofenac (2 g, 6.3 mmol) in 50 mL of methanol and adding to this solution concentrated sulfuric acid (1mL). The resulting suspension was stirred overnight a t room temperature. One volume of HzO was added, and the precipitate was filtered and washed with HzO. The isolated white solid (quantitative yield) was characterized by GC-CIIMS [miz 311 (MH+,base peak)], and by 1H and 13C NMR: NMR spectra were obtained with the use of a Varian XL300 spectrometer on samples 50-100 mM in deuteriochloroform. Assignments were based on homo- and heteronuclear correlation spectra and established substituent effects (15). The position numbers conform to a previously established convention of designating the aromatic carbon atoms and substituents of diclofenac and its derivatives (16). 'H NMR: 6 = 7.35 ppm (d, 8 Hz, H-3',5'), 7.22 (dd, 8, 2, H-6), 7.12 (td, 8, 2, H-4), 6.97 (t, 8, H-49, 6.92 (td, 8, 2, H-5), 6.54 (dd, 8, 2, H-3), 3.82 (5, CX-H~), 3.76 (5, CH3). 13CN M R 6 = 172.5 (C=O), 142.7 (C-2), 137.8 (C-1'), 130.8 (C-6), 129.5 (C-2',6'), 128.8 (C3',5'), 128.0 (C-4), 124.1 (C-1), 124.0 (C-4'), 121.9 (C-5), 118.1 (C-3), 52.3 (CHs), 38.5 (C-CX). (B) Nt-Diclofenac-Ne-acetyl-L-lysine conjugate was prepared by suspending the sodium salt of diclofenac (1.26 g, 4 mmol) in HzO (50 mL) and adding to it EDC (0.84 g, 4.4 mmol). After the mixture was stirred for approximately 30 s,N*-acetyl-~-lysine (3.74 g, 20 mmol) was added, and the stirring was continued overnight a t room temperature. The reaction mixture was acidified with acetic acid to approximately pH 3-4 and extracted with ethyl acetate (25 mL) five times. The ethyl acetate extracts were combined, washed once with water, and dried over anhydrous sodium sulfate. After ethyl acetate was removed by rotary evaporation, the residue was dissolved in methanol (10 mL) and the Nc-diclofenac-N'-acetyl-L-lysineconjugate was purified from reactants and other products by HPLC on a Whatman M9 10/50 ODs-3 column (9.4 mm X 50 cm, Whatman, Hillsboro, OR), with the use of a 40-min linear solvent gradient from 85% methanol/ H 2 0(1:l) (solvent I)/15% methanol (solvent 11)to 100% solvent I1 with 1% acetic acid throughout, at a flow rate of 4 mL/min. The N-diclofenac-No-acetyl-L-lysine conjugate eluted from the
Communications
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101 10-11
,
, 10.9
1;-7
'
10.5
'
I
10.3
Inhibitor Concentration (M)
Figure 2. Competitive inhibition of binding of anti-diclofenac serum antibodies to the solid-phase antigen diclofenac-RSA. The inhibitors used in the competitive ELISA were WdiclofenacW-acetyl-L-lysine (e),diclofenac (O), diclofenac methyl ester (A),Nm-acetyl-L-lysine(e), dichlorophenol (+), dichloroaniline (A),and phenylacetamide (0). column a t approximately 20 min and was recovered as a white solid (10% yield) after removal of solvent by rotary evaporation. FAB/MS: m/z 466 (MH+), 468 ([MH + 219,488 (MNa+),490 ([MNa 219,504 (MK+),506 ([MK + 219; 'H N M R 6 = 7.30 (d, 8 Hz, H-3',5'), 7.13 (d, 8, H-6), 7.06 (t, 8, H-4), 6.94 (t,8, H-47, 6.86 (t, 8, H-5), 6.78 (d, CY-NH), 6.44, (d, 8, H-3), 6.40 (br, NH), 4.43 (br, Lys H-2), 3.66 (s, a-Hz), 3.15 (br, Lys H-6), 1.94 (s, AcCH3), 1.73 (br, Lys H-3), 1.43 (br, Lys H-5), 1.32 (br, Lys H-4). 13C NMR: 6 = 174.7, 172.4, 171.5 (CEO'S), 142.9 (C-2), 137.5 (C-I.'), 130.8 (C-6), 130.2 (C-2',6'), 128.9 (C-3',5'), 128.1 (C-4), 124.5 (C-4'),124.4 (C-l), 121.6 (C-5), 117.4 (C-3),52.5 (LYSC-2), 40.7 (C-a),39.2 (LYSC-6),31.2 (LYSC-3), 28.8 (LYSC-5), 22.8 (Ac CH3), 22.2 (LYSC-4). Immunochemical Detection of Diclofenac Adducts in Liver Tissue of Mice. Female B6C3F1 mice (2 months old, NCI, Frederick, MD) were administered a solution of the sodium salt of diclofenac in warm distilled water (40 "C, 0.01 mL/g animal weight) at doses of 100, 200, or 300 mg/kg, by intraperitoneal injection. Control animals received only distilled water. After 8 h, livers were removed and homogenized in 3 volumes of icecold 100 mM Tris-acetate (pH 7.4) containing 250 mM sucrose and 1 mM EDTA. The samples were stored at -80°C until analyzed. Proteins were separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE), transferred to nitrocellulose, and immunochemically detected with anti-diclofenac serum antibodies, following reported procedures (I7). Other Methods. Proteins were determined according to the method of Lowry et al. (18) with bovine serum albumin as standard.
+
Results In order to prepare polyclonal antibodies that would recognize protein adducts of reactive metabolites of diclofenac, rabbits were immunized with a diclofenac conjugate that was synthesized by coupling the carboxyl group of diclofenac to the lysine residues of KLH. In an ELISA, the antibodies reacted with a solid-phase antigen of diclofenac coupled to RSA, but not to RSA alone. In a competitive ELISA, diclofenac and its methyl ester derivative were at least 3 orders of magnitude more effective as inhibitors of the reaction of the antibodies with diclofenac-RSA than was phenylacetamide, dichloroaniline, or dichlorophenol (Figure 2). Moreover, Nediclofenac-Na-acetyl-L-lysine(Figure 1A) was a more potent and complete inhibitor than was either diclofenac or its methyl ester. These findings indicated that the antibodies could be used to detect covalently bound metabolites of diclofenac in tissues. Indeed, immunoblotting revealed that the anti-diclofenac antibodies could
Chem. Res. Toxicol., Vol. 6, No. 2, 1993 149
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B
C
D
A B C
E
D
E
140e
1 IO+
14-
11-
70-b
70-
50+
Figure 3. Detection of protein adducts of reactive metabolites of diclofenac in mouse liver homogenates by immunoblotting. Eight hours after treatment of mice, proteins in liver homogenates were separated by SDS-PAGE (200 pg/lane) and stained with Coomassie Blue dye (lane E) or transferred to nitrocellulose and probed with anti-diclofenac serum antibodies (1/5Oodilution) (lanes A-D). Lane A, mice treated with water vehicle only; lanes B-E, mice treated with diclofenac, 100,200,300,and 300 mg/kg, respectively.
detect protein adducts of diclofenac in the livers of mice 8 h after treatment with diclofenac. Adducts of approximately 50,70,110,and 140 kDa were formed in a dosedependentmanner with increasingamountsof diclofenac, but were not detected in liver homogenates of control mice (Figure3). The specificityof the immunoblotting analysis for detecting diclofenacadducts was confirmed by showing that the immunochemical reactions could be inhibited by diclofenacor Nz-diclofenac-Nu-acetyl-L-lysine, but not by Nu-acetyl-L-lysine(Figure 4). No immunochemical staining of liver proteins was observed when diclofenac was added to liver homogenates of control mice (results not shown),a fiiding consistent with the idea that the detected adducts were produced by the covalent reaction of liver proteins with a reactive metabolite of diclofenac. To rule out the possibility that the covalent reaction of a reactive metabolite of diclofenac with tissue proteins occurred during the heating of the liver homogenates in SDS-PAGE sample buffer prior to immunoblotting, samples were also prepared at room temperature and analyzed by immunoblotting. Adducts were still observed under these conditions of analysis (results not shown).
Discussion In this study, we have found that a reactive metabolite or metabolites of diclofenac covalently binds to liver proteins of mice. The formation of the adducts appeared to be highly selective, because most proteins in the liver did not react immunochemically with the anti-diclofenac antibodies (Figure 2). At the present time, it remains to be determined what reactive metabolite of diclofenac is responsible for the adduct formation. Diclofenac acyl glucuronide, however, is a likely metabolite of diclofenac that could have been responsible, at least in part, for the formation of the liver adducts (16, 19). It has been suggested that acyl glucuronide metabolites of NSAIDs might mediate some of the toxicities produced by this class of compounds (20-22). The formation of acyl glucuronidesof NSAIDs is catalyzed by microsomal UDP-
Figure 4. Inhibition of immunoblot detection of protein adducta of reactive metabolites of diclofenac in mouse liver homogenates Eight hours by diclofenac and N-diclofenac-Ncl-acetyl-L-lysine. after treatment of mice with diclofenac (300mg/kg), proteins in liver homogenates were separated by SDS-PAGE (75pg/lane), transferred to nitrocellulose, and probed with anti-diclofenac serum antibodies (1/500dilution) in the absence (lane A) or presence of diclofenac (1 mM, lane B), N-diclofenac-Ncl-acetylL-lysine (0.1 mM, lane C),or Na-acetyl-L-lysine (1 mM, lane D). Lane E was an immunoblot incubated without anti-diclofenac serum antibodies to serve as a control.
glucuronosyltransferase (UDPGT, EC 2.4.1.17) and involves the conjugation of a carboxyl functional group with glucuronic acid (Figure 1B)(23). Acyl glucuronide metabolites are chemically reactive and can covalently bind to tissue proteins by transacylation (20-22). Alternatively, they may undergo acyl migration within the glucuronic acid molecule, prior to covalent binding to proteins by glycosylation (22). In both cases, the NSAIDs would be covalently linked to proteins as acyl derivatives. Since the anti-diclofenac antibodies were raised against an acyl conjugate of diclofenac, they would be expected to bind to either of these classesof adducts. In this regard, another possible metabolite of diclofenac that might lead to the transacylation of proteins is diclofenac acyl-CoA (24). It is possible that other metabolites of diclofenac might also covalently bind to liver proteins and be recognized by the anti-diclofenac antibodies. In the future, it should be possible to use the antidiclofenac antibodies to determine whether similar covalent adducts of reactive metabolites of diclofenac are formed in the livers of diclofenac hepatitis patients. The antibodies could also be used to purify the liver adducts by immunoaffinity chromatography or to follow their purification by other forms of chromatography (25). Once this has been done, it can be determined whether diclofenac hepatitis patients have developed hypersensitivity reactions against the purified protein adducts (26). Altematively, after the protein targets of dichlofenac have been identified, it could be determinedwhether their biological activities are altered by adduct formation. Similar approaches may be used to study the molecular basis of other toxicities produced by diclofenac, such as hemolyticanemia (27),thrombocytopenia (28), and agranulocytosis (29).
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