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Chem. Res. Toxicol. 1997, 10, 420-423
Cytochrome P4502C11 Is a Target of Diclofenac Covalent Binding in Rats Sijiu Shen,*,† Sally J. Hargus,†,‡ Brian M. Martin,§ and Lance R. Pohl† Molecular and Cellular Toxicology Section, Laboratory of Molecular Immunology, National Heart, Lung and Blood Institute, and Clinical Neurosciences Branch, National Institute of Mental Health, NIH, Bethesda, Maryland 20892 Received September 17, 1996X
Diclofenac antiserum was previously developed and used to detect protein adducts of metabolites of dichlofenac in livers of mice and rats. In this study, the antibody has been used to facilitate the purification of a major 51 kDa microsomal adduct of diclofenac from the liver microsomes of male rats that were treated with diclofenac. The adduct was identified as male-specific cytochrome P4502C11 based on its N-terminal amino acid sequence, reaction with a cytochrome P4502C11 antibody, and by its absence from liver microsomes of diclofenactreated female rats. When diclofenac was incubated with liver microsomes of control rats in the presence of NADPH, only the 51 kDa adduct was produced. The formation of the adduct was inhibited by a cytochrome P4502C11 monoclonal antibody, but not by reduced glutathione or N-R-acetyl-L-lysine. No adduct was detected when diclofenac was incubated with liver microsomes from female rats. Moreover, adduct formation in vivo appeared to lead to a 72% decrease in the activity of cytochrome P4502C11. The results indicate that cytochrome P4502C11 metabolizes diclofenac into a highly reactive product that covalently binds to this enzyme before it can diffuse away and react with other proteins.
Introduction Diclofenac is a nonsteroidal anti-inflammatory drug (NSAID)1 that is widely used for the treatment of rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, and acute muscle pain conditions (1). In rare cases, it can cause severe hepatic injury (2-10). Although the mechanism of diclofenac hepatotoxicity is still not clear, it is thought that covalently modified proteins may be important in causing the toxicity either directly or by eliciting an immune response (11). To study this possibility, we developed a polyclonal antibody that could be used to detect protein adducts of diclofenac by immunoblotting. Adducts of 50, 70, 110, and 140 kDa were found in liver homogenates of mice treated with diclofenac (12), while those of 50, 110, 140, and 200 kDa were subsequently found in the liver homogenates of diclofenac-treated rats (13). Subcellular fractionation studies revealed that the 50 kDa adduct was concentrated in the microsomes, while the 110, 140, and 200 kDa adducts were in the plasma membrane fraction of rat liver (13). Moreover, the 50 kDa adduct was formed by a P450-dependent pathway, while the 110, 140, and 200 kDa adducts were formed by UGT-dependent pathways (13). Other researchers have subsequently developed diclofenac antisera and have detected major protein * To whom correspondence should be addressed at the Molecular and Cellular Toxicology Section, NHLBI, NIH, Building 10, Room 8N110, Bethesda, MD 20892-1760. Telephone: 301-496-4841. Fax: 301-480-4852. † National Heart, Lung and Blood Institute. ‡ Present address: Department of Toxicology & Pathology, 3M Pharmaceuticals, 3M Center, Building 270-3S-05, St. Paul, MN 551441000. § National Institute of Mental Health. X Abstract published in Advance ACS Abstracts, March 15, 1997. 1 Abbreviations: NSAID, nonsteroidal anti-inflammatory drug; P450, cytochrome P450; UGT, uridine diphosphate glucuronosyltransferase; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; GSH, reduced glutathione; NAL, N-R-acetyl-L-lysine; PVDF, poly(vinylidene difluoride).
S0893-228x(96)00167-1
adducts of diclofenac of 50, 60, 80, and 126 kDa in homogenates of cultured rat hepatocytes (14, 15), a major adduct of 60 kDa in homogenates of cultured human hepatocytes (15), and major adducts of 60 and 80 kDa in liver homogenates of rats treated with diclofenac (14). It is not yet known whether the various antisera recognize the same adducts. The 110 kDa adduct was recently identified as dipeptidyl peptidase IV (16), a serine exopeptidase that preferentially cleaves dipeptides from the N-terminus of proteins and peptides containing proline as the penultimate amino acid residue (17). Although the physiological role of dipeptidyl peptidase IV in plasma membranes of rat liver remains to be determined, the activity of this enzyme was inhibited by diclofenac treatment. These findings have suggested that diclofenac may cause hepatotoxicity either by inactivating dipeptidyl peptidase IV and/or by inducing immune reactions against covalently altered dipeptidyl peptidase IV. In this study, we have identified the major 50 kDa (now more accurately defined as the 51 kDa) protein target of diclofenac in rat liver microsomes as cytochrome P4502C11. This enzyme metabolizes diclofenac into a reactive metabolite(s) that covalently bind(s) to P4502C11 and appears to inactivate it.
Experimental Procedures Materials. Chemicals were obtained from the following commercial sources: sodium salt of diclofenac, benzphetamine, and NADPH from Sigma (St. Louis, MO); BCA Protein Assay Reagent kit from Pierce Chemical (Rockford, IL); goat antirabbit IgG (peroxidase conjugated) from Boehringer Mannheim (Indianapolis, IN); DEAE-52 from Whatman (Clifton, NJ); hydroxylapaptite from Bio-Rad (Richmond, CA); polyethylene glycol 8000 from USB (Cleveland, OH); enhanced chemiluminescence Western blotting reagents from Amersham (Arlington Heights, IL); reduced glutathione (GSH) from Acros (New
This article not subject to U.S. Copyright.
Published 1997 by the American Chemical Society
Covalent Binding of Diclofenac to P4502C11 Jersey); N-R-acetyl-L-lysine (NAL) from ICN Biochemicals (Cleveland, OH); Emulgen 911 was a gift from Kao Corp. (Tokyo, Japan). Rabbit anti-P4502C11 serum that was used for immunoblotting was obtained from Affinity Bioreagents, Inc. (Neshanic Station, NJ). Inhibitory P4502C11 monoclonal antibody (ascites, 1-68-11) and control antibody (ascites) used in immunoinhibition studies were gifts from Dr. Harry V. Gelboin of the National Cancer Institute. Diclofenac antiserum was raised in rabbits as previously described (12). Isolation of Microsomes. Male Sprague-Dawley rats (175200 g; Taconic Farms, Germantown, NY) were treated with 200 mg/kg diclofenac intraperitoneally. After 4 h, rats were killed by decapitation. Livers were immediately removed and homogenized in ice-cold 0.1 M Tris-acetate (pH 7.5), containing 1 mM EDTA and 0.25 M sucrose. The microsomes were isolated by differential centrifugation, suspended in 10 mM Tris-acetate (pH 7.5), containing 1 mM EDTA and 20% (v/v) glycerol (buffer A), and stored at -80 °C. Purification of 51 kDa Diclofenac Adduct from Liver Microsomes. The procedure employed was that used for the purification of P450 from rat liver microsomes by Waxman (18). Briefly, microsomes were solubilized with 3 mg of sodium cholate/mg of microsomal protein, and proteins were fractionated with polyethylene glycol 8000. The 10-16% fraction was applied to a DEAE-52 anion exchange column (2.5 × 35 cm) that was equilibrated with 10 mM potassium phosphate (pH 7.4), containing 0.1 mM EDTA, 20% (v/v) glycerol, 0.5% (w/v) sodium cholate, and 0.2% (v/v) Emulgen 911 (buffer B). The column was washed first with a half column volume of buffer B and then with 3-4 volumes of buffer B plus 20 mM potassium chloride. The fractions eluting from the column were monitored by their absorption at 417 nm, SDS-PAGE, and immunoblotting. Fractions containing the 51 kDa diclofenac adduct were pooled, dialyzed in 10 mM potassium phosphate (pH 7.4), containing 0.1 mM EDTA, 20% (v/v) glycerol, and 0.2% (v/v) Emulgen 911 (buffer C), and applied to a hydroxylapatite column (1.4 × 6 cm) that was equilibrated with buffer C. After the column was washed with 1 column volume of buffer C, proteins were eluted with a linear gradient of 10-100 mM potassium phosphate in buffer C. Fractions containing the 51 kDa adduct were pooled, concentrated with an Amicon ultrafiltration cell using a PM 30 membrane (Danvers, MA), dialyzed in 50 mM potassium phosphate (pH 7.4), and stored at -80 °C. N-Terminal Amino Acid Sequence Determination of 51 kDa Adduct. Approximately 20 µg of partially purified 51 kDa adduct was fractionated by SDS-PAGE and transferred to Immobilon PVDF. The N-terminal amino acid sequence of the fraction corresponding to the 51 kDa adduct was determined by automated Edman degradation following a previously described procedure (19). Covalent Binding of Diclofenac to Rat Microsomal Proteins in Vitro. Liver microsomal proteins (2.5 mg in 120 µL of buffer A) from saline-treated rats were incubated with 100 mM potassium phosphate (pH 7.4), 1 mM EDTA, and 1 mM diclofenac, in the presence or absence of 5 mM NADPH, GSH, or NAL in a total volume of 0.5 mL at 37 °C for 4 h. In immunoinhibition studies, microsomes (2.5 mg in 120 µL of buffer A) were allowed to incubate with 20 or 40 µL of monoclonal antibodies (ascites, 30 mg/mL) at room temperature for 10 min before the addition of the other reaction components. All the reactions were stopped by freezing the reaction mixtures in liquid nitrogen and storing them at -80 °C, until they were analyzed by SDS-PAGE and immunoblotting. Immunoblotting. SDS-PAGE and immunoblotting were conducted as previously reported (13). The immunoblots were incubated with 1/10 000, 1/2000, and 1/10 000 dilutions of diclofenac antiserum, P4502C11 antiserum, and anti-rabbit IgG (peroxidase conjugated), respectively, and were developed with enhanced chemiluminescence detection according to the manufacturer’s instructions. Other Methods. Protein concentrations were determined by the BCA assay with bovine serum albumin as standard, according to the manufacturer’s instructions. Benzphetamine
Chem. Res. Toxicol., Vol. 10, No. 4, 1997 421
Figure 1. Immunochemical detection of diclofenac adducts in liver microsomes of rats treated with diclofenac. Four hours after male and female rats were administered saline or declofenac (200 mg/kg intraperitoneally), liver microsomes were isolated and analyzed by immunoblotting (100 µg) with diclofenac antiserum. Lane 1, microsomes from saline-treated male rats; lanes 2 and 3, microsomes from male and female rats, respectively, treated with diclofenac. demethylation assay was performed according to the procedure described by Ryan et al. (20). P450 content of rat liver microsomes was measured by the method of Omura and Sato (21).
Results Identification of the 51 kDa Adduct as Covalently Modified P4502C11. As previously reported (13), a major adduct of diclofenac of approximately 51 kDa was detected by immunoblotting with diclofenac antiserum in liver microsomes of male rats that had been treated with diclofenac (Figure 1, lane 2). An additional, less intense band was also detected with an apparent molecular mass of approximately 53 kDa. In contrast, only the 53 kDa adduct was found in liver microsomes of female rats that had been treated with diclofenac (Figure 1, lane 3). These results suggested that the major microsomal diclofenac protein adduct of 51 kDa might be a male-specific form of P450. To test this idea, the 51 kDa adduct was purified from liver microsomes of male rats by a procedure previously used for that of malespecific cytochrome P4502C11 (18). After DEAE-52 anion exchange and hydroxylapatite chromatography, a partially purified 51 kDa protein adduct (Figure 2, lanes 3 and 4) was transferred to a PVDF membrane and sequenced by Edman degradation. The N-terminal amino acid sequence, MDPVLVLVLT, was 100% identical to that of cytochrome P4502C11 (22). No other rat protein was found with this N-terminal sequence. This assignment was confirmed by showing that specific antiP4502C11 serum reacted with the partially purified 51 kDa adduct (Figure 2, lane 5). Formation of the 51 kDa Adduct in Vitro in Rat Liver Microsomes. The 51 kDa adduct was formed in vitro when rat liver microsomes from untreated male rats were incubated with diclofenac in the presence of NADPH (Figure 3). The formation of the adduct was inhibited by a specific inhibitory P4502C11 monoclonal antibody,
422 Chem. Res. Toxicol., Vol. 10, No. 4, 1997
Figure 2. Purification and immunochemical characterization of microsomal 51 kDa diclofenac adduct. Four hours after male rats were administered diclofenac (200 mg/kg intraperitoneally), liver microsomes were isolated, and the 51 kDa adduct was purified by DEAE-52 anion exchange and hydroxylapatite chromatography. Lanes 1-3, SDS-PAGE gel stained with Coomassie blue dye: lane 1, molecular mass standards; lane 2, 100 µg of microsomal protein before purification; lane 3, 10 µg of purified 51 kDa adduct. Lanes 4 and 5, immunoblots of 10 µg of purified 51 kDa adduct probed with diclofenac and P4502C11 antisera, respectively.
Shen et al.
Figure 4. Effect of N-R-acetyl-L-lysine or glutathione on the formation of the 51 kDa diclofenac adduct in rat liver microsomes. Rat liver microsomes from saline-treated male or female rats were incubated with diclofenac (1 mM) in the absence or presence of NADPH, and 5 mM GSH or NAL. Samples (200 µg) were analyzed by immunoblotting with diclofenac antiserum. Table 1. Inhibition of Benzphetamine Demethylation in Liver Microsomes of Male Rats by Diclofenac Treatment in Vivoa activity % cytochrome P450 nmol of HCHO gender treatment (nmol/mg of protein) nmol-1 min-1 inhibition male male female female
Figure 3. Inhibition of microsomal 51 kDa adduct formation in vitro by P4502C11 monoclonal antibody. Rat liver microsomes from saline-treated male rats were incubated with diclofenac (1 mM) in the absence or presence of NADPH, and in the absence or presence of P4502C11 inhibitory monoclonal antibody or control monoclonal antibody. Samples (100 µg of protein) were analyzed by immunoblotting with diclofenac antiserum.
but not by a control antibody (Figure 3) or by 5 mM GSH or NAL (Figure 4). Moreover, the 53 kDa adduct (Figure 1) was not formed in vitro in a NADPH-dependent reaction, indicating that an enzyme other than or in addition to liver cytochrome P450 had a role in the formation of this adduct. No adduct was detected in the incubations with liver microsomes from female rats (Figure 4). Inhibition of P4502C11 Activity by Diclofenac. Diclofenac treatment did not have a significant effect on the specific content of total P450 in liver microsomes of either male or female rats (Table 1). In contrast, diclofenac treatment decreased the benzphetamine demethylation activity by 23.3% in liver microsomes of male rats, but not in female rats. Using a specific inhibitory monoclonal antibody against P4502C11, it was found that 33.5% of benzphetamine demethylation in the microsomes of the male rats was contributed by P4502C11 (Table 2). This finding indicated that approximately 72% of benzphetamine demethylation activity catalyzed by P4502C11 was inhibited by diclofenac treatment.
saline diclofenac saline diclofenac
0.44 ( 0.05 0.47 ( 0.09 0.41 ( 0.04 0.45 ( 0.04
8.68 ( 0.15 6.66 ( 0.16b 5.12 ( 0.12 5.25 ( 0.13
0 23.3 0 0
a Male and female rats (3 in each group) were administered saline or dichlofenac (200 mg/kg) intraperitoneally. After 4 h, liver microsomes were isolated, and total P450 content and benzphetamine demethylation activity were determined. The results represent the means ( standard deviation of three determinations. b Significantly different from the male rats administered saline (p < 0.01).
Table 2. Inhibition of Benzphetamine Demethylation in Liver Microsomes of Male Rats by P4502C11 Monoclonal Antibodya activity antibodies anti-P4502C11 none 5 µL 10 µL 20 µL control antibody none 5 µL 10 µL 20 µL
nmol of HCHO nmol-1 min-1
% inhibition
8.67 ( 0.16 5.77 ( 0.12 5.92 ( 0.08 5.85 ( 0.09
0 33.5b 31.7b 32.5b
8.90 ( 0.15 9.25 ( 0.05 8.93 ( 0.04 9.48 ( 0.04
0 0 0 0
a Benzphetamine demethylation activity was measured in liver microsomes from saline-treated rats in the presence or absence of P4502C11 monoclonal antibody or control monoclonal antibody. The results represents the means ( standard deviation of three determinations. b Significantly different from the reactions that did not contain P4502C11 monoclonal antibody (p < 0.01).
Discussion In this study we have identified the major 51 kDa microsomal adduct of diclofenac in the livers of male rats as gender-specific P4502C11 (18, 22). This assignment
Covalent Binding of Diclofenac to P4502C11
was based on the amino acid sequence analysis of its N-terminus, by its reaction with P4502C11 antibodies (Figure 2), and by its absence from liver microsomes of diclofenac-treated female rats (Figures 1 and 4). The finding that an inhibitory P4502C11 monoclonal antibody blocked the formation of the 51 kDa adduct in vitro (Figure 3) indicates that P4502C11 catalyzed the formation of the reactive metabolite(s) that subsequently became covalently bound to this enzyme in vivo. Although the identity of the reactive intermediate(s) responsible for adduct formation remains to be determined, it appears to be highly reactive. For example, when diclofenac was incubated with rat liver microsomes of male rats, the reactive metabolite(s) became covalently bound exclusively to a 51 kDa protein, presumably P4502C11, and could not be chemically trapped (Figure 4). The results of previous chemical stability studies have suggested that the reactive metabolite(s) of diclofenac might be bound to either an aspartate or a glutamate residue of the 51 kDa microsomal protein (13). If the metabolite(s) was (were) bound to E104, E203, E300, D360, or D468 of P4502C11, amino acid residues that are well conserved and are near or in the potential substrate recognition sites (22, 23), it may explain how diclofenac inhibited the activity of P4502C11 (Tables 1 and 2). Cytochrome P4502C9 is the major form of P450 in human liver microsomes that oxidatively metabolize diclofenac (24, 25). Since this enzyme shows 76% identity and 85% homology with that of P4502C11 (26), it is conceivable that a reactive metabolite(s) of diclofenac might also become covalently bound to P4502C9 in humans. If this occurs, the covalently modified P4502C9 might have a role in the development of the idiosyncratic hepatotoxicity produced by diclofenac. This suggestion is based upon previous studies done with tienilic acid. The hepatotoxicity caused by this drug is thought to have an immunopathological mechanism, in part, because many patients with this toxicity have serum autoantibodies that react with P4502C9 (27) and cross-react with rat P4502C11 (28). The autoantibodies are believed to be induced by adducts of P4502C9 that are formed when P4502C9 metabolizes tienilic into reactive metabolite(s) (27). Whether or not similar P4502C9 autoantibodies are found in the sera of diclofenac hepatitis patients remains to be determined.
Acknowledgment. We thank John W. George and Michael R. Marchick for their assistance in this work.
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