Abstract
The nonsteroidal anti-inflammatory drug diclofenac causes a rare but potentially fatal hepatotoxicity that may be associated with the formation of reactive metabolites. In this study, three glutathione (GSH) adducts, namely 5-hydroxy-4-(glutathion-S-yl)diclofenac (M1), 4′-hydroxy-3′-(glutathion-S-yl)diclofenac (M2), and 5-hydroxy-6-(glutathion-S-yl)diclofenac (M3), were identified by liquid chromatography-tandem mass spectrometry analysis of bile from Sprague-Dawley rats injected i.p. with a single dose of diclofenac (200 mg/kg). These adducts presumably were formed via hepatic cytochrome P-450 (CYP)-catalyzed oxidation of diclofenac to reactive benzoquinone imines that were trapped by GSH conjugation. In support of this hypothesis, M1, M2, and M3 were generated from diclofenac in incubations with rat liver microsomes in the presence of NADPH and GSH. Increases in adduct formation were observed when incubations were performed with liver microsomes from phenobarbital- or dexamethasone-treated rats. Adduct formation was inhibited by polyclonal antibodies against CYP2B, CYP2C, and CYP3A (40–50% inhibition at 5 mg of IgG/nmol of CYP) but not by an antibody against CYP1A. Maximal inhibition was obtained when the three inhibitory antibodies were used in a cocktail fashion (70–80% inhibition at 2.5 mg of each IgG/nmol of CYP). These data suggest that diclofenac undergoes biotransformation to reactive metabolites in rats and that CYP isoforms of the 2B, 2C, and 3A subfamilies are involved in this bioactivation process. With respect to CYP2C isoforms, rat hepatic CYP2C7 and CYP2C11 were implicated as mediators of the bioactivation based on immunoinhibition studies using antibodies specific to CYP2C7 and CYP2C11. Screening for GSH adducts also was carried out in human hepatocyte cultures containing diclofenac, and M1, M2, and M3 again were detected. It is possible, therefore, that reactive benzoquinone imines may be formed in vivo in humans and contribute to diclofenac-mediated hepatic injury.
Diclofenac is a nonsteroidal anti-inflammatory drug that in rare cases causes severe liver injury (Breen et al., 1986; Helfgott et al., 1990; Purcell et al., 1991). The hepatotoxicity has been described as idiosyncratic in nature and possibly associated with metabolism of the drug (Banks et al., 1995;Boelsterli et al., 1995). Conjugation with glucuronic acid represents a major biotransformation pathway for diclofenac and the resulting acyl glucuronide has been implicated as a mediator of the hepatotoxicity (Boelsterli et al., 1995). With immunochemical detection, diclofenac was found to form protein adducts in the liver of treated mice and rats (Pumford et al., 1993; Hargus et al., 1994) as well as in hepatocyte cultures (Kretz-Rommel and Boelsterli, 1993; Gil et al., 1995). The formation of diclofenac-protein adducts was dependent upon uridine diphosphate glucuronosyltransferase activity and the covalently modified proteins were shown to retain the glucuronic acid moiety (Hargus et al., 1994; Kretz-Rommel and Boelsterli, 1994). In rat liver, the protein targets for diclofenac binding included 110-, 140-, and 200-kDa plasma membrane proteins and a 60-kDa microsomal protein. The 110-kDa protein was identified as dipeptidyl peptidase IV (Hargus et al., 1995). Data also were obtained that suggest that reactive metabolite(s) of diclofenac could be generated through a cytochrome P-450 (CYP)1-mediated pathway (Kretz-Rommel and Boelsterli, 1993; Shen et al., 1997a, b). For example, a 51-kDa protein was found to be modified covalently in male rats dosed with diclofenac and this modification, which also was observed in incubations with rat liver microsomes, was a NADPH-dependent process (Hargus et al., 1994; Shen et al., 1997a). The 51-kDa protein subsequently was identified as CYP2C11 (Shen et al., 1997a). In rat hepatocyte cultures, the diclofenac-induced leakage of lactate dehydrogenase was reduced markedly in the presence of CYP2C-selective inhibitors, suggesting that a CYP2C enzyme(s) was (were) involved in the drug-associated cytotoxicity (Kretz-Rommel and Boelsterli, 1993).
The CYP-catalyzed biotransformation of diclofenac gives rise to two monohydroxylated metabolites, namely, 4′-hydroxydiclofenac and 5-hydroxydiclofenac (Leemann et al., 1993; Shen et al., 1997b). It was proposed that the 5-hydroxy derivative underwent further oxidation to a putative benzoquinone imine (Shen et al., 1997b). By virtue of its electrophilic nature, this intermediate could react with proteins or cellular glutathione (GSH). From an analytical point of view, characterization of the GSH adduct(s) of such reactive metabolites would provide insight into the nature of the short-lived electrophilic species (Baillie and Davis, 1993). In this paper, we describe the identification of GSH-conjugated metabolites in the bile from rats treated with diclofenac. The role of rat hepatic CYP enzymes in the formation of these metabolites was investigated using rat liver microsomes and antibodies against CYP enzymes and screening for GSH adducts also was carried out in human hepatocyte cultures treated with diclofenac.
Materials and Methods
Chemicals.
Diclofenac, dimethyl sulfoxide, GSH, and NADPH were purchased from Sigma Chemical Co. (St. Louis, MO). Trifluoroacetic acid (TFA) was obtained from Fisher Scientific (Fair Lawn, NJ) and bis(pinacolato)diboron was purchased from Strem Chemical (Newburyport, MA). All other chemicals were obtained from Aldrich Chemical Co. (Milwaukee, WI). BondElut C18 solid phase extraction cartridge columns were obtained from Varian Chromatography Systems (Walnut Creek, CA).
Instrumentation and Analytical Methods.
Liquid chromatography-tandem mass spectrometry (LC/MS/MS) was carried out on a SCIEX API III+ tandem mass spectrometer (Perkin-Elmer, Toronto, Canada) interfaced to a HPLC system consisting of two LC-10A pumps and a static-bed mixer (Shimadzu Scientific Instruments, Kyoto, Japan). LC/MS/MS experiments were performed with an ion spray interface with positive ion detection at a voltage of 5 kV. The orifice potential was 65 V. Collision-induced dissociation (CID) used argon as the collision gas at a thickness of 1.3 × 1014 atoms/cm2 and the collision energy was 30 eV. Chromatography was performed on a Zorbax Rx-C8 column (4.6 × 250 mm, 5 μm; DuPont, Wilmington, DE) and samples were delivered at a flow rate of 1 ml/min with a 1:25 split. The mobile phase consisted of aqueous acetonitrile containing 10% methanol and 0.05% TFA and was programmed by a linear increase from 10 to 70% acetonitrile during a 30-min period.
HPLC purification of synthetic products was performed either on a Dynamax SD-200 liquid chromatograph (Rainin Instruments, Woburn, MA) with a Zorbax RX-C18 column (21.2 × 250 mm, 5 μm) at a flow rate of 20 ml/min or a LC-10AD liquid chromatograph (Shimadzu Scientific Instruments) with a Zorbax Rx-C8 column (4.6 × 250 mm, 5 μm) at a flow rate of 1 ml/min. UV detection was at 210 nm.
NMR spectra of synthetic products were obtained on either a Varian Inova400 or a Varian Inova500 spectrometer operating at 400 and 500 MHz, respectively, and chemical shifts were expressed relative to tetramethylsilane.
Synthesis of Diclofenac Metabolites.4′-Hydroxy-3′-(glutathion-S-yl)diclofenac(4′-OH-3′-GS-diclofenac, M2).
4′-Hydroxydiclofenac was generated from diclofenac in incubations with a baculovirus-insect cell line expressing CYP2C9. The product was purified by the Dynamax HPLC system (Rainin Instruments; mobile phase, isocratic 35% acetonitrile containing 0.1% TFA; retention time, 65 min). LC/MS, m/z 312 (MH+).1H NMR (CD3OD), δ 3.6 (s, CH2COOH); 6.22 (d, J = 8.5 Hz, 3-CH); 6.74 (t, J = 8.5 Hz, 5-CH); 6.86 (s, 3′ and 5′-CH); 6.94 (t, J = 8.5 Hz, 4-CH); 7.16 (d, J = 8.5 Hz, 6-CH).
Fifty-eight milligrams of silver(I) oxide was added to 3 mg of 4′-hydroxydiclofenac in 1 ml of benzene. The reaction mixture was stirred at room temperature for 22 h and the resulting solution was treated with 36 mg of GSH in 1 ml of phosphate buffer (pH 7.4). The two-phase reaction mixture was stirred vigorously at 37°C for 8 h. The aqueous phase was separated and the crude product purified by the Shimadzu HPLC system (mobile phase, aqueous acetonitrile containing 10% methanol and 0.05% TFA, linear increase from 10% to 70% acetonitrile during a 30 min period; retention time, 14 min). LC/MS: m/z 617 (MH+). [1H] NMR (D2O), δ 2.0 to 2.2 (m, Glu, CH2); 2.4 to 2.5 (m, Glu, CH2); 3.2 to 3.3 and 3.4 to 3.6 (m, Cys, CH2); 3.7 to 3.9 (m, Gly, CH2, Glu, CH, CH2COOH); 4.3 (m, Cys, CH); 6.36 (d, J = 8.4 Hz, 3-CH); 6.94 (t, J = 8.4 Hz, 5-CH); 7.16 (t, J = 8.4 Hz, 4-CH); 7.19 (s, 5′-CH); 7.28 (d, J = 8.4 Hz, 6-CH).
5-Hydroxy-4-(glutathion-S-yl)diclofenac (5-OH-4-GS-diclofenac; M1) and 5-hydroxy-6-(glutathion-S-yl)diclofenac (5-OH-6-GS-diclofenac; M3).
A 877-mg quantity of iodine monochloride in 4 ml of acetic acid was added to 1.6 g of diclofenac in 35 ml of acetic acid. The mixture was stirred for 1.5 h and then mixed with 6 ml of 20% aqueous sodium persulfate. The solid product, 5-iododiclofenac, was collected and washed with 8 ml of 50% ethanol.
Seventy milligrams of bis(pinacolato)diboron, 5.6 mg of palladium(II) chloride, and 74 mg of potassium acetate under nitrogen were added to 5.5 mg of 5-iododiclofenac in dimethyl sulfoxide. The mixture was stirred at 60–70°C for 15 h, acidified to pH 3 to 4, and extracted with ethyl acetate. The product, 5-(tetramethyldioxaboron)diclofenac, was purified by the Dynamax HPLC system (mobile phase, isocratic 47% acetonitrile containing 0.1% TFA; retention time, 45 min).
Seven milligrams of sodium bicarbonate and 0.2 ml of acetone were added to 4 mg of 5-(tetramethyldioxaboron)diclofenac in 0.5 ml of 1.2% aqueous sodium hydroxide. The mixture was cooled to 0°C and a total of 0.5 ml of 6 mg of Oxone in 0.4 mM EDTA solution was added dropwise. The mixture was stirred for an additional 5 min at 0°C, mixed with 21 ml of 20% aqueous sodium persulfate, acidified to pH 2 to 3, and extracted with ethyl acetate. The product, 5-hydroxydiclofenac, was purified by the Dynamax HPLC system (mobile phase, isocratic 37% acetonitrile containing 0.1% TFA; retention time, 14.3 min). LC/MS,m/z 312 (MH+);1H NMR (CD3OD): δ 3.6 (s, CH2COOH); 6.31 (d, J = 8.5 Hz, 3-CH); 6.46 (dd, J = 1.3 and 8.5 Hz, 4-CH); 6.70 (d, J = 1.3 Hz, 6-CH); 6.92 (t, J = 8.0 Hz, 4′-CH); 7.31 (d, J = 8.0 Hz, 3′ and 5′-CH).
Fifty-eight milligrams of silver(I) oxide was added to 3 mg of 5-hydroxydiclofenac in 1 ml of benzene. The reaction mixture was stirred at room temperature for 22 h and the resulting solution was treated with 36 mg of GSH in 1 ml of phosphate buffer (pH 7.4). The two-phase reaction mixture was stirred vigorously at 37°C for 8 h. Two products (M1 and M3) were purified by the Shimadzu HPLC system (mobile phase, aqueous acetonitrile containing 10% methanol and 0.05% TFA, linear increase from 10% to 70% acetonitrile during a 30-min period; retention time, 13.8 min for M1 and 15.0 min for M3). M1, LC/MS, m/z 617 (MH+);1H NMR (CD3OD), δ 2.0 to 2.1 (m, Glu, CH2), 2.3 to 2.5 (m, Glu, CH2), 3.1 to 3.2, and 3.4 to 3.5 (m, Cys, CH2), 3.7 to 3.9 (m, Gly, CH2, Glu, CH, CH2COOH), 4.5 to 4.6 (m, Cys, CH), 6.54 (s, 3-CH), 6.82 (s, 6-CH), 7.0 (t, J = 8.5 Hz, 4′-CH), 7.37 (d, J = 8.5 Hz, 3′ and 5′-CH). M3, LC/MS: m/z 617 (MH+); 1H NMR (CD3OD): δ 2.1 to 2.2 (m, Glu, CH2), 2.4 to 2.6 (m, Glu, CH2), 3.2 to 3.3, 3.4 to 3.6 (m, Cys, CH2), 3.6 to 3.8 (m, Gly, CH2, Glu, CH, CH2COOH), 4.3 to 4.4 (m, Cys, CH), 6.44 (d, J = 8.5 Hz, 3-CH), 6.70 (d, J = 8.5 Hz, 6-CH), 6.98 (t, J = 8.0 Hz, 4-CH), 7.34 (d, J = 8.0 Hz, 3′ and 5′-CH).
Animal Experiments.
Experiments were performed according to procedures approved by the Merck Institutional Animal Care and Use Committee. Male and female Sprague-Dawley rats purchased from Harlan Laboratories (Indianapolis, IN) and weighing 270 to 360 g were allowed free access to commercial rat chow and water. They were anesthetized with sodium pentobarbital (nembutal) and their bile ducts were cannulated with PE-10 tubing. Bile was collected before treatment. An aqueous solution (pH 7) of diclofenac was then administered at either 10 or 200 mg/kg by i.p. injection and bile was collected for an additional 8 h.
A group of seven female rats was dosed with aqueous phenobarbital (PB) at 100 mg/kg per day for 3 days. Another group of 20 male rats was dosed with dexamethasone (Dex) in 2% Tween 80 at 200 mg/kg/day for 3 days. These rats were used for the isolation of liver microsomes.
Biological Preparations.
Rat liver microsomes were isolated by differential centrifugation (Raucy and Lasker, 1991) and pooled from 1) 40 naive male rats (control); 2) 30 naive female rats; 3) 7 PB-treated rats; and 4) 20 Dex-treated rats, respectively.
Polyclonal antibodies directed against rat CYP enzymes were prepared in rabbits by immunization with the electrophoretically homogeneous proteins (Bandiera and Dworschak, 1992; Levine et al., 1998; Wong and Bandiera, 1998). Monospecific antibodies were prepared by passing the polyspecific antibodies through a series of columns containing partially purified CYP enzymes excluding those CYPs the antibodies were raised against (Bandiera and Dworschak, 1992; Levine et al., 1998).
Incubations with Rat Liver Microsomes.
Diclofenac and GSH in phosphate buffer (pH 7.4) were added to rat liver microsomes (0.5–2.0 nmol of CYP/ml) suspended in 0.1 M phosphate buffer (pH 7.4) containing EDTA (1 mM). Substrate concentrations were 1, 5, 10, 25, 50, 200, and 1000 μM and the GSH concentration was 5 mM. The mixture was incubated at 37°C for 5 min before adding NADPH in phosphate buffer (1 mg/ml final concentration) to initiate the reaction. After an additional 30-min incubation, the reaction was quenched with 10% aqueous TFA.
In immunoinhibition experiments, rat liver microsomes (0.5 nmol of CYP/ml) were preincubated with each antibody (0.5, 1.0, 2.5, and 5.0 mg of IgG/nmol of CYP) for 15 min at room temperature. Control incubations contained IgG from untreated rabbits. Substrate concentration was 5 μM. Diclofenac, GSH, and NADPH were added thereafter and the incubations were performed in a manner similar to that described above.
Incubations with Human Hepatocyte Cultures.
The human liver samples were obtained from the Pennsylvania Regional Tissue Bank (Exton, PA). An agreement was made between the tissue bank and Merck & Co. for research use of the samples. The death of one donor, a 25-year-old male (ID no. 1130971), had been caused by an accident; the second donor, a 65-year-old male (ID no. 0514981), died from anoxia. Hepatocytes were isolated based on a two-step perfusion procedure (Pang et al., 1997). Upon isolation, the hepatocytes were seeded on Matrigel-coated 6-well plates at 2 × 106 cells/ml and cultured in Willams’ medium containing 0.1 μM Dex for 48 h before metabolic studies.
Diclofenac dissolved in dimethyl sulfoxide was added to the hepatocyte cultures to give a final drug concentration of 300 μM. Dimethyl sulfoxide concentration was 0.1% (v/v). After incubation for 24 h, the culture medium was acidified with 10% aqueous TFA.
Detection of GSH-Conjugated Metabolites.
Rat bile (200 μl) was acidified with 10% aqueous TFA and precipitates were removed via centrifugation at 13,600g for 5 min. Aliquots of bile (20–50 μl) were injected onto the Zorbax Rx-C8 column and analyzed by LC/MS/MS. Metabolites were identified based on their fragmentation upon CID and on their HPLC retention times compared with those of reference compounds obtained by synthesis.
Samples from microsomal incubations or from hepatocyte cultures were applied to a C18 extraction cartridge column that was prewashed with methanol and water. The column was washed consecutively with water and methanol. The methanol eluate was evaporated to dryness under a stream of nitrogen and the residue was reconstituted in 300 μl of 60% aqueous acetonitrile containing 0.05% TFA. An 80-μl aliquot of the solid phase extracts was injected onto the Zorbax Rx-C8 column and analyzed by LC/MS/MS. Identification of the metabolites was based on multiple reaction monitoring detection of four transitions, namely, m/z 617 → 542, 617 → 488, 617 → 342, and 617 → 324.
Results
Characterization of the GSH Adducts Formed In Vivo in Rats.
LC/MS/MS screening for GSH adducts was performed by constant neutral loss scan monitoring of ions that lose 129 Da upon CID (Baillie and Davis, 1993). Three components that exhibited this response were detected in a bile sample from the rat treated with diclofenac (Fig. 1) and were assigned as diclofenac metabolites based on their characteristic chlorine isotope cluster (m/z 617/619). The associated parent ions were all at m/z 617, consistent with these metabolites being GSH adducts of hydroxydiclofenac. The metabolites were arbitrarily designated as M1, M2, and M3 according to their relative HPLC retention times.
LC/MS/MS detection of the GSH adducts of diclofenac by constant neutral loss scanning (loss of 129 Da).
An aliquot of bile (50 μl) from the rat treated with diclofenac (200 mg/kg) was acidified and injected onto a Zorbax C8 column.
Subsequent CID of the MH+ ions at m/z617 produced product ions at m/z 542 and 488 resulting from neutral losses of glycine (75 Da) and pyroglutamate (129 Da), respectively (Fig. 2). These neutral losses are characteristic for xenobiotic GSH adducts (Baillie and Davis, 1993). One major common fragment ion in the spectra of adducts was found atm/z 342, which could be derived from cleavage adjacent to the thioether moiety and charge retention on the aromatic moiety (Fig.2). An additional loss of water from the m/z 342 ion would give rise to the observed product ion at m/z 324.
a, LC/MS/MS product ion spectra of isomeric GSH adducts detected in bile from rats treated with diclofenac at 200 mg/kg: A, 5-OH-4-GS-diclofenac (M1) and B, 5-OH-6-GS-diclofenac (M3).
The spectra were obtained by CID of the MH+ ions atm/z 617 and the proposed origins of key fragment ions are as indicated. b, LC/MS/MS product ion spectrum of a GSH adduct detected in bile from rats treated with diclofenac at 200 mg/kg: 4′-OH-3′-GS-diclofenac (M2). The spectrum was obtained by CID of the MH+ ion at m/z 617 and the proposed origins of key fragment ions are as indicated.
Further structural assignment for the metabolites was obtained via comparison with synthetic reference compounds. Thus, identical MS/MS fragmentation patterns and HPLC retention times were observed for M1 and 5-OH-4-GS-diclofenac. The same held true for M2 and 4′-OH-3′-GS-diclofenac and for M3 and 5-OH-6-GS-diclofenac.
Metabolite M2 was detected only in bile from rats treated with a high dose of diclofenac (200 mg/kg), whereas M1 and M3 were found in rats treated with either the low or high dose (10 or 200 mg/kg). Similar results were observed for both male and female rats.
Metabolite Formation in Incubations with Rat Liver Microsomes.
The detection by LC/MS/MS of diclofenac metabolites formed in microsomal incubations was based on multiple reaction monitoring of four characteristic mass transitions. These transitions coincided with the HPLC retention times for M1, M2, and M3 upon analysis of samples derived from incubations of diclofenac with rat liver microsomes fortified with NADPH and GSH (Fig. 3). The metabolite profiles were similar throughout a wide range of substrate concentrations from 1 to 1000 μM (Fig. 3). Adduct M2 was a minor metabolite in incubations at all substrate concentrations. The formation of M1 and M3 increased with increasing substrate concentration, reaching a plateau at approximately 5 μM diclofenac (data not shown). Similar to results observed in vivo, the GSH adducts were detected in incubations containing liver microsomes isolated from either male or female rats.
LC/MS/MS detection of GSH adducts in incubations containing diclofenac, rat liver microsomes, NADPH, and GSH.
Four mass transitions were used as criteria for metabolite identification: m/z 617 → 524, 617 → 488, 617 → 342, and 617 → 324. Substrate concentrations were A, 1 μM and B, 1000 μM.
As compared with liver microsomes from untreated rats, an approximately 2-fold increase in the yield of metabolites was observed when diclofenac (50 μM) was incubated with liver microsomes isolated from PB- or Dex-treated rats. The comparison was made based on microsomal protein concentrations.
Metabolite M2 also was detected in incubations of 4′-hydroxydiclofenac with rat liver microsomes in the presence of NADPH and GSH; M1 and M3 were formed in incubations containing 5-hydroxydiclofenac (data not shown).
Immunoinhibition of Rat Hepatic CYP-Mediated Bioactivation.
The formation of metabolites in incubations containing 5 μM diclofenac and 0.25 nmol of rat liver microsomal CYP (untreated) was linear over a period of 30 min, with M1 and M3 being the predominant products. Inhibition experiments were performed using 30 min as the end point and the percentage of inhibition was calculated based on decreases in the formation of M1 and M3 relative to controls that lacked the antibody.
Metabolite formation in incubations with rat liver microsomes was inhibited by polyclonal antibodies against CYP2B, CYP2C, and CYP3A (Fig. 4), whereas no effect was observed with the antibody against CYP1A (Table 1). For liver microsomes from male rats, maximal inhibition occurred at the highest IgG concentration used (Fig. 4, top). For liver microsomes from female rats, maximal inhibition was achieved at approximately 2.5 mg of IgG/ nmol of CYP (Fig. 4, bottom). When antibodies against CYP2B, CYP2C, and CYP3A were used in a cocktail fashion, the inhibition was 70 to 80%, which was greater than that achieved by any single antibody (Table 1).
Inhibition of diclofenac metabolism by polyclonal antibodies against CYP enzymes in incubations with liver microsomes isolated from untreated male rats (top) and female rats (bottom).
Percentage of control was calculated based on decreases in the formation of M1 and M3.
Inhibition by anti-CYP antibodies of the metabolic activation of diclofenac in incubations with rat liver microsomes1-a
The metabolism of diclofenac was inhibited by a monospecific antibody against CYP2C7 (Fig. 5). In this case, maximal inhibition occurred at the highest IgG concentration used (Fig. 5 and Table 1). Diclofenac metabolism in incubations with liver microsomes from male rats also was inhibited by a monospecific antibody against CYP2C11 (Fig. 5 and Table1). No effect was observed with the antibody against CYP2C11 when liver microsomes from female rats were used (Fig. 5). This finding is consistent with the specificity of the antibody, because CYP2C11 is known to be expressed only in male rats.
Inhibition of diclofenac metabolism by monospecific antibodies against CYP2C7 and CYP2C11 in incubations with liver microsomes isolated from untreated male rats (top) and female rats (bottom).
Percentage of control was calculated based on decreases in the formation of M1 and M3.
Formation of GSH Adducts in Human Hepatocyte Cultures.
Similarly, the detection of diclofenac metabolites in hepatocyte cultures was based on MS/MS multiple reaction monitoring coupled with HPLC separation. Hepatocytes isolated from the two human liver samples exhibited a viability of greater than 80% as determined by the trypan blue exclusion test. Metabolites M1, M2, and M3 were detected readily in incubations of diclofenac with human hepatocyte cultures (Fig. 6).
LC/MS/MS detection of GSH adducts in human hepatocyte cultures treated with diclofenac.
Four mass transitions were used as criteria for metabolite identification: m/z 617 → 524, 617 → 488, 617 → 342, and 617 → 324. The donor was a 25-year-old male.
Discussion
Protein adducts have been identified in rats treated with diclofenac, but information on the structures of the reactive intermediates formed via CYP-mediated oxidation of diclofenac has been elusive. In this study, LC/MS/MS data were obtained indicating the presence of GSH-conjugated metabolites in bile from either male or female rats dosed with diclofenac. These conjugated metabolites subsequently were identified as 5-OH-4-GS-diclofenac (M1), 4′-OH-3′-GS-diclofenac (M2), and 5-OH-6-GS-diclofenac (M3).
Two types of intermediate electrophiles that potentially could react with GSH during the metabolism of diclofenac are benzoquinone imines (Brune and Lindner, 1992) and arene oxides (Blum et al., 1996; SchemeFS1). Analysis of the metabolites formed in vivo in rats suggested that diclofenac most likely was oxidized on the dichloroaniline ring to the 1′,4′-benzoquinone imine that was trapped by GSH because only M2 was present in the bile. A similar process could occur on the phenylacetic acid moiety to give M1 and M3 via the common intermediate diclofenac-2,5-quinone imine (Scheme FS1). This hypothesis was further supported by the fact that M2 was synthesized chemically from 4′-hydroxydiclofenac and M1 and M3 were prepared from 5-hydroxydiclofenac. In vitro incubation of 4′-hydroxydiclofenac with rat liver microsomes in the presence of NADPH and GSH resulted in the formation of M2, whereas incubation of 5-hydroxydiclofenac produced M1 and M3. The 4′- and 5-hydroxylated derivatives are viewed as the biological precursors of benzoquinone imines. Differences in the formation of M1 and M3 (Table 1) are most likely the result of preferential attack by GSH on one of the reactive centers of diclofenac-2,5-quinone imine.
Proposed metabolic pathways leading to the formation of GSH-conjugated metabolites of diclofenac through oxidative biotransformation.
It is proposed that the metabolic activation of diclofenac through an oxidative pathway is catalyzed by CYP enzymes (Scheme FS1). Consistent with this hypothesis was the observation that the formation of GSH adducts in incubations with rat hepatic microsomes from either sex was NADPH-dependent and was inhibited by antibodies against CYP enzymes. Inhibitory antibodies included polyclonal antibodies against CYP2B, CYP2C, and CYP3A but not an antibody against CYP1A, suggesting that the bioactivation process was catalyzed by CYP isoforms in rat hepatic 2B, 2C, and 3A subfamilies. Among these enzymes, CYP2B1/2 are inducible upon treatment of rats with PB and CYP3A1/2 can be induced by Dex as well as by PB (Correia, 1995). These data are in accord with the observations that adduct formation was higher in incubations with liver microsomes from PB- or Dex-treated rats.
With respect to CYP2C isoforms, male-specific CYP2C11 was shown previously to be modified covalently by unknown reactive metabolites of diclofenac (Shen et al., 1997a). In this study, CYP2C11 was found to be involved in catalyzing the oxidation of diclofenac to reactive benzoquinone imines because the formation of GSH adducts was inhibited by the antibody against CYP2C11. It is tempting to speculate that the benzoquinone imine intermediates may serve to arylate CYP2C11. Similarly, CYP2C7 also was implicated in diclofenac bioactivation based on immunoinhibition studies with the antibody against CYP2C7. Whether CYP2C7 is modified by the reactive metabolite(s) remains to be determined.
It is of interest to note that the GSH-conjugated metabolites were detected in both male and female rats as well as in incubations with liver microsomes from rats of either sex. In other words, gender difference was not apparent in terms of the CYP-catalyzed bioactivation of diclofenac in rats as measured by GSH adduct formation. In an earlier study, the CYP-mediated formation of a 51-kDa protein adduct was found to occur only in male rats treated with diclofenac and in vitro protein adduct formation was not attenuated by adding GSH to the incubations (Shen et al., 1997a). Further investigation is warranted to determine whether the formation of GSH conjugated metabolites and the formation of protein adducts share the same bioactivation pathway(s).
Primary cell cultures are widely regarded as good models for the corresponding in vivo systems (Li and Kedderis, 1997). In this investigation, the GSH adducts detected in rats also were identified in human hepatocyte cultures containing diclofenac, suggesting that the CYP-mediated bioactivation of diclofenac could occur in vivo in humans. The resulting electrophilic benzoquinone imines probably are capable of arylating proteins, potentially leading to toxic consequences either by altering protein functions or by provoking immune responses. Alternatively, the reactive intermediates could be scavenged by their reaction with GSH. However, GSH conjugation not only represents a detoxification process but may also lead to depletion of cellular GSH pools. The resulting GSH deficiency, caused by the exposure to reactive compounds, is known to result in cell injury and death due largely to the impaired antioxidant function of the GSH redox system (Reed, 1990). In view of the idiosyncratic nature of diclofenac-mediated hepatotoxicity, potential damage caused by reactive metabolites through GSH depletion should be considered, particularly in patients who, because of genetic and/or environmental factors, may be already compromised with respect to GSH.
In summary, three GSH adducts were identified in bile from rats treated with diclofenac. The metabolites most likely were formed through rat hepatic CYP-catalyzed oxidation of diclofenac to putative benzoquinone imine intermediates followed by conjugation with GSH. These findings may be relevant to diclofenac hepatotoxicity because the same adducts were detected in human hepatocyte cultures incubated with the drug. Current studies are in progress to investigate the involvement of human hepatic CYP isoforms in diclofenac bioactivation.
Acknowledgments
We thank Dr. Anthony Y. H. Lu (Rutgers University) and Regina W. Wang (Merck Research Laboratories) for valuable discussions.
Footnotes
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Send reprint requests to: Dr. Wei Tang, Department of Drug Metabolism, Merck & Co., P.O. Box 2000, RY80L-109, Rahway, NJ 07065. E-mail: wei_tang{at}merck.com
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A preliminary account of this study was presented at Experimental Biology, San Francisco, CA, April 1998.
- Abbreviations used are::
- CYP
- cytochrome P-450
- CID
- collision-induced dissociation
- Dex
- dexamethasone
- GSH
- glutathione
- LC/MS/MS
- liquid chromatography-tandem mass spectrometry
- M1
- 5-OH-4-GS-diclofenac or 5-hydroxy-4-(glutathion-S-yl)diclofenac
- M2
- 4′-OH-3′-GS-diclofenac or 4′-hydroxy-3′-(glutathion-S-yl)diclofenac
- M3
- 5-OH-6-GS-diclofenac or 5-hydroxy-6-(glutathion-S-yl)diclofenac
- PB
- phenobarbital
- TFA
- trifluoroacetic acid
- Received September 4, 1998.
- Accepted December 2, 1998.
- The American Society for Pharmacology and Experimental Therapeutics
