Metabolic Activation of Diclofenac by Human ... - ACS Publications

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Chem. Res. Toxicol. 1999, 12, 214-222

Metabolic Activation of Diclofenac by Human Cytochrome P450 3A4: Role of 5-Hydroxydiclofenac Sijiu Shen,†,‡ Michael R. Marchick,† Margaret R. Davis,§ George A. Doss,| and Lance R. Pohl*,† Molecular and Cellular Toxicology Section, Laboratory of Molecular Immunology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892, Department of Drug Metabolism, Merck Research Laboratories, West Point, Pennsylvania 19486, and Department of Drug Metabolism, Merck Research Laboratories, Rahway, New Jersey 07065 Received October 26, 1998

Cytochrome P450 2C11 in rats was recently found to metabolize diclofenac into a highly reactive product that covalently bound to this enzyme before it could diffuse away and react with other proteins. To determine whether cytochromes P450 in human liver could catalyze a similar reaction, we have studied the covalent binding of diclofenac in vitro to liver microsomes of 16 individuals. Only three of 16 samples were found by immunoblot analysis to activate diclofenac appreciably to form protein adducts in a NADPH-dependent pathway. Cytochrome P450 2C9, which catalyzes the major route of oxidative metabolism of diclofenac to produce 4′-hydroxydiclofenac, did not appear to be responsible for the formation of the protein adducts, because sulfaphenazole, an inhibitor of this enzyme, did not affect protein adduct formation. In contrast, troleandomycin, an inhibitor of P450 3A4, inhibited both protein adduct formation and 5-hydroxylation of diclofenac. These findings were confirmed with the use of baculovirusexpressed human P450 2C9 and P450 3A4. One possible reactive intermediate that would be expected to bind covalently to liver proteins was the p-benzoquinone imine derivative of 5-hydroxydiclofenac. This product was formed by an apparent metal-catalyzed oxidation of 5-hydroxydiclofenac that was inhibited by EDTA, glutathione, and NADPH. The p-benzoquinone imine decomposition product bound covalently to human liver microsomes in vitro in a reaction that was inhibited by GSH. In contrast, GSH did not prevent the covalent binding of diclofenac to human liver microsomes. These results suggest that for appreciable P450-mediated bioactivation of diclofenac to occur in vivo, an individual may have to have both high activities of P450 3A4 and perhaps low activities of other enzymes that catalyze competing pathways of metabolism of diclofenac. Moreover, the p-benzoquinone imine derivative of 5-hydroxydiclofenac probably has a role in covalent binding in the liver only under the conditions where levels of NADPH, GSH, and other reducing agents would be expected to be low.

Introduction Diclofenac (Figure 1) is a nonsteroidal anti-inflammatory drug (NSAID)1 that is widely used for the treatment of osteoarthritis and rheumatoid arthritis, ankylosing spondylitis, and acute muscle pain conditions (1). In rare cases, it can cause severe hepatic injury (2, 3). Although the etiology of this toxicity is not known, clinical evidence suggests that it may be due to an immune (4-7) or a nonimmune mechanism (3, 8-11). In both cases, covalent modification of liver proteins may play an important role in the etiology of diclofenac hepatotoxicity (12). Several protein covalent adducts of diclofenac have been detected in liver tissues of mice and rats and in primary cultures * To whom correspondence should be addressed: Molecular and Cellular Toxicology Section, NHLBI, NIH, Building 10, Room 8N110, Bethesda, MD 20892-1760. Telephone: (301) 496-4841. Fax: (301) 4804852. E-mail: [email protected]. † National Institutes of Health. ‡ Present address: Department of Pathology, The Pennsylvania State University School of Medicine, P.O. Box 850, Hershey, PA 17033. § Merck Research Laboratories, West Point, PA. | Merck Research Laboratories, Rahway, NJ. 1 Abbreviations: NSAID, nonsteroidal anti-inflammatory drug; P450 reductase, cytochrome P450 reductase; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Figure 1. Structure of diclofenac.

of rat and human hepatocytes, using immunochemical approaches (13-18) and radiolabeled diclofenac (15, 19, 20). A 110 kDa target protein of diclofenac, which appeared to be formed from an acyl glucuronide metabolite(s) of diclofenac, was identified as the plasma membrane protein dipeptidyl peptidase IV, a serine exopeptidase (21). In addition, the activity of the enzyme was

10.1021/tx9802365 CCC: $18.00 © 1999 American Chemical Society Published on Web 01/14/1999

Bioactivation of Diclofenac by Human P450 3A4

inhibited by diclofenac treatment, suggesting that the dipeptidyl peptidase IV adduct may have either a direct role in the etiology of diclofenac hepatitis or possibly an indirect role, by eliciting an immune response against hepatocytes containing this adduct or other adducts on the surface of their plasma membranes. In this regard, it has been shown that diclofenac-treated mouse hepatocytes carried antigenic determinants that were recognized by T cells derived from mice that had been immunized with conjugates of diclofenac and keyhole limpet hemocyanin (22). More recently, a 51 kDa microsomal adduct of diclofenac was identified in rat liver as cytochrome P450 2C11 (23). P450 2C11 catalyzed adduct formation and was inactivated during the process. Similar results were previously found with tienilic acid, which suggested another possible mechanism for diclofenac-induced hepatotoxicity. Tienilic acid, a uricosuric diuretic, causes an idiosyncratic hepatitis that appears to have an immunopathological basis (24-26). This drug, like diclofenac, is activated by P450 2C11 and forms covalent adducts with the enzyme (27). P450 2C9, which is 76% identical and 85% homologous with that of P450 2C11 (28), catalyzes a similar reaction (29). It has been proposed that tienilic acid adducts of P450 2C9 are immunogenic in those patients that develop tienilic acid-induced hepatitis, and are responsible for the induction of the serum antibodies that react with P450 2C9 and cross-react with P450 2C11 and its tienilic acid adduct (27). On the basis of the findings with tienilic acid and the fact that P450 2C9 was known to have a major role in the oxidative metabolism of diclofenac (30, 31), it seemed possible that P450 2C9 might metabolically activate diclofenac into a reactive metabolite(s), which may have a role in diclofenac hepatitis in humans. To test this idea, we investigated the mechanism of covalent binding of diclofenac to human liver microsomes. The results indicate, however, that diclofenac is metabolically activated predominantly by P450 3A4 instead of by P450 2C9 in human liver microsomes, to form protein adducts.

Experimental Procedures Materials. Sodium salt of diclofenac, NADPH, and all the P450 chemical inhibitors were from Sigma (St. Louis, MO). GSH was from Acros (Pittsburgh, PA). The BCA Protein Assay Reagent kit was from Pierce Chemical Co. (Rockford, IL). NADH, goat anti-rabbit IgG (peroxidase-conjugated) was from Boehringer Mannheim (Indianapolis, IN). Enhanced chemiluminescence Western blotting reagents were from Amersham (Arlington Heights, IL). Goat anti-rat P450 1A1 serum, control insect cell microsomes, insect cell microsomes containing overexpressed human P450 3A4, cytochrome P450 reductase (P450 reductase), and cytochrome b5, and insect cell microsomes containing overexpressed human P450 2C9 and P450 reductase were purchased from Gentest Co. (Woburn, MA). Goat antirabbit P450 2E1 serum was from D. R. Koop of Oregon Health Sciences University. Rabbit anti-human P450 2C9 was from J. M. Lasker of the Mount Sinai School of Medicine. Mouse antiP450 3A4 Mab(275-1-2) was from H. V. Gelboin of the National Cancer Institute. Supelcosil high-performance liquid chromatography (HPLC) columns were from Supelco Inc. (Bellefonte, PA). Diclofenac antisera were raised in rabbits as previously described (13). Instrumentation and Analytical Methods. Chromatographic separations of diclofenac from its microsomal metabolites were performed with the use of a Waters Instrument (Millipore Corp., Milford, MA), consisting of a 600E gradient

Chem. Res. Toxicol., Vol. 12, No. 2, 1999 215 system controller and a 490E variable-wavelength detector. The data were collected with a PE Nelson 900 series interface and Turbochrom 4.0 software (Cupertino, CA). Analytical separations were performed on a Supelcosil LC-18 column (25 cm × 4.6 mm, 5 µm). After injection of 200 µL of a sample, compounds were eluted with a linear gradient from 50% solvent A [0.1% (v/v) acetic acid in water] and 50% solvent B [0.1% (v/v) acetic acid in methanol] to 85% solvent B over the course of 30 min, followed by 100% solvent B for 15 min at a flow rate of 1 mL/ min. The fractions eluting from the analytical HPLC column were identified as diclofenac (retention time of 34 min), 4′hydroxydiclofenac (retention time of 25-26 min), 5-hydroxydiclofenac (retention time of 26-27 min), and 5-hydroxydiclofenac-p-benzoquinone imine decomposition product (retention time of 19-20 min). These assignments were made by UV, MS, and NMR analyses of samples preparatively collected from a Supelcosil LC-18 column (25 cm × 10 mm, 5 µm). Samples (1 mL) were injected onto the column, and products were eluted from the column with the same gradient that was used in analytical separation, but at a flow rate of 2.5 mL/min. Fractions were collected, dried under vacuum in a SpeedVac concentrator from Savant (Farmingdale, NY), and stored at -80 °C until analyses were done. Liquid chromatography/mass spectrometry (LC/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS) were carried out with a Hewlett-Packard HP-1090 gradient HPLC system that was interfaced with a Finnigan TSQ 7000 tandem mass spectrometer. Samples were injected onto a Beckman Ultrasphere narrow-bore C18 column (15 cm × 2.0 mm, 5 µm) using a mobile phase consisting of solvent C [0.1% (v/v) formic acid in water] and solvent D [0.1% (v/v) formic acid in acetonitrile], at a flow rate of 0.2 mL/min. The gradient used was as follows: isocratic condition of 20% solvent D for 10 min, followed by a linear increase to 90% solvent D over the course of 35 min. The column was equilibrated for 15 min with 20% solvent D prior to each injection. Mass spectral analyses were carried out using electrospray ionization (ESI) in the positive ion mode. The ESI ionizing voltage was maintained at 5.5 kV for all analyses. MS/MS was based on collision-induced dissociation (CID) of ions entering the rf-only octapole region where argon was used as the collision gas at a pressure of 1.8 mTorr. The collision offset was cycled continuously from -15 to -25 to -35 eV for all MS/MS analyses. NMR spectra were recorded in CD3OD on a Varian Unity 500 MHz spectrometer. Chemical shifts are given in ppm and were referenced to a residual solvent signal at 3.3 ppm. Samples sizes ranged from 20 to 40 µg. UV absorption spectra were recorded with the use of a HP 8453 spectrophotometer from Hewlett-Packard (Wilmington, DE) with methanol as a solvent. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting were conducted as previously reported unless otherwise stated (23). The immunoblots were developed with the enhanced chemiluminescence detection method, according to the manufacturer’s instructions, and where indicated, they were scanned with a Personal Laser Densitometer and the data analyzed with ImageQuanNT software, both from Molecular Dynamics (Sunnyvale, CA). Protein concentrations were determined by the BCA assay with bovine serum albumin as the standard according to the manufacturer’s instructions. P450 content was measured by the method of Omura and Sato (32). Experimental data are presented as the average of duplicate determinations that did not vary by more than 10%. Linear regression analyses and statistical analyses were carried out with Prism (version 2.01) and StatMate (version 1.0), respectively (GraphPad Software, San Diego, CA). Preparation of Rat and Human Liver Microsomes. Rat liver microsomes were prepared from male Sprague-Dawley rats (175-200 g; Taconic Farms, Germantown, NY) as previously described and stored at -80 °C (23). Human liver samples were acquired from J. W. Harris, A. Rahman, and M. E. Fitzsimmons

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Figure 2. Formation of diclofenac-labeled protein adducts in human liver microsomes. Human liver microsomes (HM) from 16 individuals or male rats (rat LM) were incubated with diclofenac in the absence [rat LM(1)] or presence of NADPH. Samples (200 µg) were analyzed for diclofenac protein adduct formation by immunoblotting with diclofenac antiserum. of the Food and Drug Administration. The tissues were originally collected by them under the auspices of the Washington Regional Transplant Consortium (Washington, DC). As previously described, the samples were immediately transported to the laboratory on ice, sliced into cubes in a bath of cold Eurocollins transplantation buffer, and stored at -80 °C, until microsomes were prepared (33). Metabolism and Protein Covalent Binding of Diclofenac in Vitro. Unless otherwise stated, liver microsomal proteins from rats or humans (5 mg/mL) were incubated with 100 mM potassium phosphate (pH 7.4), 1 mM EDTA, 2 mM NADPH, and 1 mM diclofenac in a total volume of 0.5 mL at 37 °C for 4 h. When inhibitors of P450 were used, the reaction mixtures were preincubated with the inhibitors at 37 °C for 3 min, before the addition of diclofenac to start the reactions. All P450 inhibitors were dissolved in ethanol except for diethyl dithiocarbamate, which was dissolved in water. The final concentration of ethanol in the reaction mixtures was 2%. For microsomal studies with baclovirus-expressed P450s, 0.4 nmol/mL P450 and 5 mM NADPH were used. Reactions to be analyzed for protein adducts of diclofenac were stopped by addition of an equal volume of SDS-PAGE sample buffer to the reaction mixtures, followed by boiling for 3 min, SDS-PAGE, and immunoblotting with diclofenac antiserum. When metabolites of diclofenac were being investigated, the reactions were stopped by the addition of 1/4 volume of acetonitrile. The samples were then centrifuged at 100000g for 10 min with the use of a Beckman Optima TLX ultracentrifuge, followed by filtration of the supernatants through a 0.45 µm HM filter (Millipore, Yonezawa, Japan), and storage of the filtrates at -80 °C, until they were analyzed by HPLC. Characterization of 4′-Hydroxy- and 5-Hydroxydiclofenac Isolated by Preparative HPLC from Incubations of Diclofenac with Rat Liver Microsomes. 4′-Hydroxydiclofenac: λmax 272 nm; 1H NMR δ 7.16 (dd, J ) 7.5, 1.5 Hz, H-6), 6.95 (dt, J ) 7.9, 1.4 Hz, H-4), 6.86 (s, H3′ + H5′), 6.74 (dt, J ) 7.6, 1.3 Hz, H-5), 6.22 (dd, J ) 8.2, 1.3 Hz, H-3), 3.62 (s, CH2). MS/MS product ion scanning results of 35Cl2 [M + H]+ (m/z 312), 35Cl37Cl [M + H]+ (m/z 314), and 37Cl2 [M + H]+ (m/z 316) were as follows, respectively: m/z 294, 296, and 298 (M + H - H2O), 266, 268, and 270 (M + H - H2CO2), 233 (268 35Cl•), 232 (268 - H35Cl and 270 - H37Cl), 231 (266 - 35Cl•), and 230 (266 - H35Cl and 268 - H37Cl). 5-Hydroxydiclofenac: λmax 278 nm; 1H NMR δ 7.30 (d, J ) 8.0 Hz, H-3′ + H-5′), 6.90 (t, J ) 8.0 Hz, H-4′), 6.69 (d, J ) 2.8 Hz, H-6), 6.45 (dd, J ) 8.5, 2.8 Hz, H-4), 6.30 (d, J ) 8.5 Hz,

H-3), 3.59 (s, CH2). MS/MS product ion scanning results of 35Cl2 [M + H]+ (m/z 312), 35Cl37Cl [M + H]+ (m/z 314), and 37Cl2 [M + H]+ (m/z 316) were similar to those of 4-hydroxydiclofenac. These results are consistent with previous spectral assignments made for these metabolites of diclofenac (34). Chemical Decomposition of 5-Hydroxydiclofenac. 5-Hydroxydiclofenac (20-50 µg), purified by preparative HPLC from the incubation mixture of diclofenac with rat liver microsomes, was dissolved in phosphate-buffered saline (10 mM, pH 7.4) and incubated at 37 °C for 16 h in the absence or presence of 1 mM NADPH, GSH, or EDTA. The samples were stored at -80 °C, until they were analyzed by HPLC. The major decomposition product was isolated from the reaction mixture by preparative HPLC and characterized by MS, NMR, and UV spectrometry. MS/MS product ion scanning results of 35Cl2 [M + H]+ (m/z 310), 35Cl37Cl [M + H]+ (m/z 312), and 37Cl2 [M + H]+ (m/z 314) were as follows, respectively: m/z 292, 294, and 296 (M + H H2O), 264, 266, and 268 (M + H - H2CO2), 231 (266 - 35Cl• and 268 - 37Cl•), 230 (266 - H35Cl and 268 - H37Cl), 229 (264 - 35Cl•), 228 (264 - H35Cl), and 166 (M + H - dichlorophenyl moiety). 1H NMR δ 7.43 (d, J ) 8.0 Hz, H-3′ + H-5′), 7.12 (t, J ) 8.0 Hz, H-4′), 6.66 (d, J ) 9.8 Hz, H-3), 6.65 (d, J ) 2.2 Hz, H-6), 6.44 (dd, J ) 9.8, 2.2 Hz, H-4), 3.65 (s, CH2). When 10 µL of 2-mercaptoethanol was added to the NMR tube containing the decomposition product of 5-hydroxydiclofenac, a new product was rapidly formed, which had the following 1H NMR spectrum: δ 7.33 (d, J ) 8.1 Hz, H-3′ + H5′), 6.94 (t, J ) 8.1 Hz, H-4′), 6.76 (d, s, H-6), 6.47 (d, s, H-3). The aliphatic signals were obscured by the excess of 2-mercaptoethanol in the sample. λmax ) 267 nm (shoulder at 276 nm).

Results Formation of Protein Adducts of Diclofenac in Vitro in Human Liver Microsomes. When liver microsomes from 16 individuals were incubated with 1 mM diclofenac and NADPH, protein adducts of diclofenac were detected at appreciable levels by immunoblot analysis with diclofenac antisera in only three of the reaction mixtures (Figure 2, HM9, HM13, and HM16). The pattern of protein labeling was similar in the three samples, with each showing several covalently modified proteins. No protein adducts were detected in the reaction mixtures when the incubations were conducted without NADPH or with 0. 1 mM instead of 1 mM diclofenac


Figure 3. Effect of several inhibitors of cytochromes P450 on the formation of diclofenac-labeled protein adducts in human liver microsomes. HM9 was incubated with diclofenac in the absence (lane 1) or presence of NADPH (lane 2) and in the presence of the following inhibitors of P450s: SKF525A (1.0 mM), nonspecific inhibitor (lane 3); sulfaphenazole (50 µM), inhibitor of P450 2C9/10 (lane 4); troleandomycin (50 µM), inhibitor of P450 3A4 (lane 5); quinidine (100 µM), inhibitor of P450 2D6 (lane 6); 4-methylpyrazole (200 µM), inhibitor of P450 2E1 (lane 7); diethyl dithiocarbamate (20 µM), inhibitor of P450s 2A6 and 2E1 (lane 8); and R-naphthoflavone (100 µM), inhibitor of P450 1A1/2 (lane 9). Samples (200 µg) were analyzed for diclofenac protein adduct formation by immunoblotting with diclofenac antiserum.

(results not shown). The latter finding indicated that the Km for the covalent binding of diclofenac to HM9, HM13, and HM16 was relatively high. In contrast, only one major protein adduct was detected at 51 kDa in the reaction mixture of 1 mM diclofenac with liver microsomes from male rats (Figure 2, lane 2). This protein was previously identified as P450 2C11 and was found to catalyze its own covalent alteration (23). Sulfaphenazole, quinidine, and 4-methylpyrazole, inhibitors of P450s 2C9, 2D6, and 2E1, respectively (35), did not block protein adduct formation in HM9 (Figure 3, lanes 4, 6, and 7, respectively). In contrast, troleandomycin, an inhibitor of P450 3A4 (35), blocked the formation of protein adducts (Figure 3, lane 5) to nearly undetectable levels, as did the nonspecific inhibitor of P450, SKF-525A (Figure 3, lane 3) (36). Diethyl dithiocarbamate and R-naphthoflavone, inhibitors of P450s 2E1 and 2A6 and 1A1/A2, respectively (36), inhibited protein adduct formation to a much smaller degree than did troleandomycin (Figure 3, lanes 8 and 9, respectively). The levels of protein adduct formation in samples HM9, HM13, and HM16 also correlated with the relative levels of immunoreactive P450 3A4 in the samples, but not with those of P450s 2C9, 1A, and 2E1 (Figure 4). Moreover, insect cell microsomes containing overexpressed human P450 3A4 metabolized diclofenac to form protein adducts (Figure 5, lane 4), while those containing human P450 2C9 did not appear to catalyze this reaction (Figure 5, lane 6). Formation of 5-Hydroxydiclofenac in Vitro in Human Liver Microsomes. It seemed possible that 5-hydroxydiclofenac, a minor oxidative metabolite of

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Figure 4. Relative levels of immunoreactive P450s in human liver microsomes. Samples (20 µg) of human liver microsomes (HM) from 16 individuals were analyzed for relative levels of immunoreactive P450s 3A4, 2C9, 1A, and 2E1 by immunoblotting with protein specific antibodies, followed by densitometric analyses.

Figure 5. Formation of diclofenac-labeled protein adducts in insect cell microsomes containing overexpressed human P450 3A4. Control insect cell microsomes (lanes 1 and 2) and insect microsomes containing overexpressed P450 3A4, P450 reductase, and cytochrome b5 (lanes 3 and 4) or P450 2C9 and reductase (lanes 5 and 6) were incubated with diclofenac in the absence or presence of NADPH. Samples (80 µg) were analyzed for diclofenac protein adduct formation by immunoblotting with diclofenac antiserum.

diclofenac (37, 38), may have a role in the covalent binding of diclofenac to human liver microsomes. This idea was based upon the earlier finding that 5-hydroxydiclofenac was unstable at neutral pH and could be


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Figure 7. Correlation of the 5-hydroxylation of diclofenac with relative levels of immunoreactive P450 3A4 in human liver microsomes. Liver microsomes from 16 individuals were incubated with diclofenac in the presence of NADPH. The 5-hydroxylation of diclofenac was measured by HPLC, and the relative levels of immunoreactive P450s 1A, 2C9, 2E1, and 3A4 were determined by immunoblotting with protein specific antibodies, followed by densitometric analyses. The correlation coefficients and statistical significance are indicated (n ) 16).

Figure 6. Formation of diclofenac metabolites by human liver microsomes or insect cell microsomes containing overexpressed human P450s. HM4 (A and B), HM9 (C), and insect microsomes containing overexpressed P450 3A4, P450 reductase, and cytochrome b5 (D) or P450 2C9 and P450 reductase (E) were incubated in the absence (A) or presence (B-E) of NADPH. Reaction mixtures were analyzed by HPLC. DF corresponds to diclofenac.

stabilized with ascorbic acid (38, 39). It was anticipated that because of its chemical structure, it might undergo air oxidation to form a p-benzoquinone imine reactive intermediate. To test this hypothesis, the oxidative metabolism of diclofenac by the human microsomes was investigated and the chemical properties of 5-hydroxydiclofenac were studied. When diclofenac was incubated with HM4 and NADPH, one major metabolite was detected by HPLC (Figure 6B). The product was identified as 4′-hydroxydiclofenac, the major human oxidative metabolite of diclofenac (37, 38), which is known to be formed from P450 2C9 in human liver microsomes (30, 31). Its structural assignment was based upon the facts that it had the same retention time as a sample of 4′-hydroxydiclofenac that was purified and characterized from rat liver microsomes, its formation was inhibited by sulfaphenazole (results not shown), and it was formed as the major metabolite of diclofenac in insect cell microsomes containing overexpressed human P450 2C9 (Figure 6E).

A minor metabolite of diclofenac, with a slightly longer retention time than that of 4′-hydroxydiclofenac, was detected in the incubation mixture with HM4. The metabolite was formed at considerably higher levels when diclofenac was incubated with HM9 (Figure 6C). The only other microsomal samples producing levels of this metabolite comparable to that of HM9 were HM13 and HM16 (results not shown). On the basis of its comigration in several HPLC solvent systems with a standard purified and characterized from rat liver microsomes, the metabolite was identified as 5-hydroxydiclofenac. Since the form(s) of P450 responsible for the formation of 5-hydroxydiclofenac had not been determined yet, additional studies were carried out. The 5-hydroxylation of diclofenac was inhibited by troleandomycin, but not by inhibitors of other forms of P450 used in this study (results not shown), and the level of hydroxylation correlated with the relative levels of P450 3A4 in the human microsomal samples (Figure 7). Moreover, when diclofenac was incubated with insect cell microsomes containing overexpressed P450 3A4, 5-hydroxydiclofenac was formed as the major metabolite (Figure 6D). Identification of the Chemical Decomposition Product of 5-Hydroxydiclofenac as a p-Benzoquinone Imine Derivative. When 5-hydroxydiclofenac, purified from incubations of diclofenac with liver microsomes of male rats, was incubated at 37 °C for 16 h in an atmosphere of air, it decomposed into one major product, as measured by HPLC analysis (Figure 8B). The formation of the product was inhibited by GSH, EDTA, or NADPH (Figure 8C-E, respectively) or an atmosphere of argon (results not shown). MS, NMR, and UV spectrometric analyses of the purified decomposition product were consistent with a structure corresponding to that of the p-benzoquinone imine derivative of 5-hydroxydiclofenac. For example, the (M + H)+ ion multiplets in the mass spectra at m/z 310, 312, and 314, representing pseudomolecular ions con-



Figure 9. Proposed origins of product ions generated upon CID of the three chlorine-containing molecular ions of the pbenzoquinone imine decomposition product of 5-hydroxydiclofenac. Panels A-C contain the proposed product ions produced upon CID of the 35Cl2-, 35Cl37Cl-, and 37Cl2-containing (M + H)+ ions, respectively.

Figure 8. Decomposition of 5-hydroxydiclofenac. 5-Hydroxydiclofenac (A) was incubated at 37 °C for 16 h in phosphatebuffered saline in the absence (B) or presence of GSH (C), EDTA (D), or NADPH (E). The reaction mixtures were analyzed by HPLC. DF corresponds to diclofenac.

taining 35Cl2, 35Cl37Cl, and 37Cl2 atoms, respectively, corresponded to the molecular mass of 5-hydroxydiclofenac minus two hydrogen atoms. Product ions generated upon CID of the (M + H)+ ion multiplets provided strong evidence for the p-benzoquinone imine structural assignment of the decomposition product, particularly the non-chlorine containing ion at m/z 166 that was interpreted as a quinone iminium ion (Figure 9). These results were confirmed by 1H NMR analysis of the purified decomposition product, which showed resonances consistent with protons attached to the 3, 4, and 6 carbons of a p-benzoquinone imine moiety (Figure 10b). Moreover, when 2-mercaptoethanol was added to the NMR tube with the decomposition product, several changes in the spectrum occurred, which were consistent with the expected addition reaction of a mercaptan with a p-benzoquinone imine (40). The doublet of doublets corresponding to the proton attached to the 4 carbon disappeared, while the doublets representing the protons attached to the 3 and 6 carbons collapsed into singlets (Figure 10c). Further proof of the p-benzoquinone imine nature of the decomposition product was derived from its UV absorption spectrum, which had an absorption maximum at 267 nm, similar to the 263 nm absorption maximum of the p-benzoquinone imine oxidation product of acetaminophen (41).

Figure 10. 1H NMR spectra of (a) 5-hydroxydiclofenac, (b) the p-benzoquinone imine decomposition product of 5-hydroxydiclofenac, and (c) the product of the reaction of 2-mercaptoethanol with the p-benzoquinone imine decomposition product.

Formation of Protein Adducts of 5-Hydroxydiclofenac and Its p-Benzoquinone Imine Derivative in Vitro in Human Liver Microsomes. When 5-hydroxydiclofenac was incubated with HM9, several protein adducts were detected by immunoblot analysis with


Figure 11. Covalent binding of 5-hydroxydiclofenac and its p-benzoquinone imine decomposition product to human liver microsomes. HM9 was incubated with diclofenac (1 mM, lanes 1-3), 5-hydroxydiclofenac (100 nM, lanes 4-6), 4′-hydroxydiclofenac (100 nM, lanes 7 and 8), or the p-benzoquinone imine decomposition product of 5-hydroxydiclofenac (50 nM, lanes 9-11) in the absence or presence of NADPH and/or GSH at 37 °C for 4 h. Samples (62 µg from the 5-hydroxydiclofenac decomposition reaction mixtures and 100 µg from all others) were analyzed for protein adduct formation by immunoblotting with diclofenac antiserum.

diclofenac antisera (Figure 11, lane 4). The inclusion of NADPH or GSH in the reaction mixture blocked the formation of the protein adducts (Figure 11, lanes 5 and 6, respectively). A similar pattern of protein adducts was formed, but at higher levels, when the p-benzoquinone imine derivative of 5-hydroxydiclofenac was incubated with HM9 in the absence of NADPH or GSH (Figure 11, lane 9). In contrast, diclofenac formed protein adducts with HM9 in a NADPH-dependent reaction (Figure 11, lane 2) that was not inhibited by GSH (Figure 11, lane 4). No protein adducts were detected in the incubations of 4′-hydroxydiclofenac with HM9 (Figure 11, lanes 7 and 8).

Discussion Although diclofenac can cause a dose-dependent toxicity to cultures of rat hepatocytes, which appears to be mediated, at least in part, by an oxidative metabolite(s) (19, 42-45), it remains a mystery how the severe form of diclofenac-induced idiosyncratic hepatotoxicity develops in humans. One of the major reasons for this is that the toxicity has not yet been produced in animals. Nevertheless, like many idiosyncratic reactions, it is thought that diclofenac-induced hepatotoxicity may be due to a toxic reactive metabolite(s) that either disrupts important cellular processes, possibly by covalently altering essential proteins, or leads to the formation of protein adducts that induce immune attack against liver tissue (12). In either case, there must be some reason(s) why the hepatotoxicity is host-dependent. Although numerous explanations are possible for the idiosyncratic nature of both nonimmune and immune mechanisms of diclofenacinduced hepatotoxicity, one possible contributing factor might be the aberrant capacity to produce high levels of a reactive metabolite(s) in the liver (46). In this study, we have shown for the first time that P450 in human liver microsomes can activate diclofenac into a reactive metabolite(s), which binds covalently to

Shen et al.

microsomal proteins (Figure 2). This observation may help explain, at least in part, how protein adducts of diclofenac were formed in primary cultures of human hepatocytes (16). The finding that only three of 16 human microsomal samples (HM9, HM13, and HM16) activated diclofenac appreciably in a NADPH-dependent pathway to form protein adducts indicated that these samples contained an elevated level of a P450 isoform(s) that catalyzed this reaction. We originally thought that the covalent binding of diclofenac to human liver microsomal proteins might be catalyzed by P450 2C9, because the rat orthologue of this enzyme, P450 2C11, catalyzed the covalent binding of diclofenac to rat liver microsomes (23). Several findings of this study, however, do not support this idea. The first indication that P450 2C9 might not have a major role in the metabolic activation of diclofenac in human liver microsomes was the labeling pattern of protein adducts observed in the immunoblot of liver microsomes from the incubations with diclofenac. Multiple protein adducts of diclofenac were detected in the microsomal reactions with human liver microsomes, while only one adduct was detected in those of rat liver microsomes (Figure 2). This finding suggested that the reactive metabolite(s) of diclofenac formed by P450s in human liver microsomes was longer-lived than that formed in rat liver microsomes and was able to diffuse away from the site of its formation. Other evidence included the findings that protein adduct formation was not blocked by the P450 2C9 inhibitor sulfaphenazole (Figure 3, lane 4), the level of formation did not correlate with the relative levels of immunoreactive P450 2C9 in the human microsomal samples HM9, HM13, and HM16 (Figure 4), and the protein adduct was not detected in incubations of insect cell microsomes containing overexpressed human P450 2C9 (Figure 5, lane 6). Moreover, 4′-hydroxydiclofenac, which is formed from a P450 2C9catalyzed reaction (Figure 6E) (30, 31) and is the major oxidative metabolite of diclofenac (Figure 6B,C) (37, 38), did not appear to bind covalently to liver proteins, when it was incubated with human liver microsomes (Figure 11, lanes 7 and 8). The results of this study are consistent instead with P450 3A4 having a major role in the formation of protein adducts of diclofenac in human liver microsomes. Indeed, protein adduct formation of diclofenac was inhibited substantially by the P450 3A4 inhibitor troleandomycin (Figure 3, lane 5); the level of formation correlated with the relatively high levels of immunoreactive P450 3A4 in samples HM9, HM13, and HM16 (Figure 4), and the protein adduct was catalyzed by overexpressed human P450 3A4 in insect cell microsomes (Figure 5, lane 4). The findings that diethyl dithiocarbamate and R-naphthoflavone, inhibitors of P450s 2E1 and 2A6 and 1A1/ A2, respectively (36), blocked protein adduct formation to a small extent (Figure 3, lanes 8 and 9, respectively) suggested that other forms of P450 may have a partial role in the covalent binding of diclofenac to human liver microsomes. P450 2E1 was probably not involved in the covalent binding, because 4-methylpyrazole, an inhibitor of this enzyme, did not decrease the level of adduct formation (Figure 3, lane 7). There is the possibility, however, that at the concentrations used in our study diethyl dithiocarbamate and R-naphthoflavone may have partially inhibited P450 3A4 (47).


The proportion of the dose of diclofenac metabolized by P450 3A4 in vivo could be theoretically increased not only by high levels of P450 3A4, which can be induced by other drugs (35) and hormones (48), but also by low levels of other enzymes that also metabolize diclofenac. For example, the 4′-hydroxylation of diclofenac can be inhibited by other drugs (49). Alternatively, it is known that an allelic form of P450 2C9 exists that cannot 4′hydroxylate diclofenac at an appreciable rate (31). Similarly, if the UGT-catalyzed acyl glucuronidation of diclofenac (37) was impaired as well, then even more of the diclofenac would be available for metabolic activation by P450 3A4. Perhaps, only when several of these conditions occur simultaneously in an individual can the metabolic activation of diclofenac by P450 3A4 lead to liver damage. It initially appeared that 5-hydroxydiclofenac may have a role in the covalent binding of diclofenac to human liver microsomes, because this metabolite was formed at the highest levels in HM9, HM13, and HM16 (Figure 6) and appeared to be formed by a P450 3A4-catalyzed reaction (Figures 6D and 7) that was inhibited by troleandomycin. Moreover, 5-hydroxydiclofenac oxidized in aqueous solution to form a reactive p-benzoquinone imine derivative (Figures 8-10) that reacted with HM9 to produce nearly the same pattern of protein adducts as those formed in the incubations with diclofenac (Figure 11). Other findings, however, indicated that 5-hydroxydiclofenac and its decomposition product did not have a role in the covalent binding of diclofenac to human liver microsomes, under the conditions of our incubations. For example, the covalent binding of diclofenac to HM9 was dependent upon the presence of NADPH and was not inhibited by GSH (Figure 11, lanes 1-3). In contrast, NADPH and GSH inhibited the covalent binding of 5-hydroxydiclofenac and its p-benzoquinone imine decomposition product to proteins in HM9 (Figure 11, lanes 4-6 and 9-11, respectively). NADPH and GSH, as a result of their reducing properties, could have possibly blocked the covalent binding of 5-hydroxydiclofenac to proteins in HM9 by preventing its chemical oxidation to the reactive p-benzoquinone imine decomposition product (Figure 8). Alternatively, if the p-benzoquinone imine was formed in the reaction mixture, by either chemical oxidation or P450-mediated oxidation of 5-hydroxydiclofenac analogous to the dehydrogenation of acetaminophen for forming a p-benzoquinone imine derivative, it might have formed a conjugate with GSH, as indicated by its reaction with 2-mercaptoethanol (Figure 10c) or might have been reduced back to 5-hydroxydiclofenac by NADPH, before it could react with microsomal proteins (40). In this regard, it has been reported that the p-benzoquinone imine derivative of 5-hydroxydiclofenac could be trapped with glutathione, when diclofenac was incubated with rat liver microsomes in the presence of NADPH and glutathione (50, 51), or reduced to 5-hydroxyldiclofenac by ascorbic acid (51). These findings suggest that the p-benzoquinone imine derivative of 5-hydroxydiclofenac may have a role in the covalent binding of diclofenac to proteins in the liver or possibly other sites only under conditions of oxidative stress, where the levels of NADPH, GSH, and other reducing agents may be low. For example, it was recently found that diclofenac could be oxidized by activated neutrophils to form the p-benzoquinone imine decomposition product of 5-hydroxydiclofenac (51). The reaction


appeared to be catalyzed by myeloperoxidase, and 5-hydroxydiclofenac was an apparent intermediate in the reaction. It was suggested that similar reactions may occur in the bone marrow and result in the formation of protein adducts of the p-benzoquinone imine reactive intermediate at this site, which may have a role in the development of diclofenac-induced blood dyscrasias (51). If this were the case, then it follows that P450 3A4 might have a role in the hematological toxicities associated with diclofenac. Nevertheless, the results of another recent study indicate that P450 2C9 and UGT(s) may have role in a case of diclofenac-induced immune hemolytic anemia (52). It was discovered that antibodies in the serum of the patient reacted with red blood cells that had been treated specifically with the 4′-hydroxydiclofenac acyl glucuronide metabolite of diclofenac. No other metabolites of diclofenac that were tested had this activity.

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