Diclofenac Toxicity to Hepatocytes

0022-3565/98/2881-0065$03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Copyright © 1998 by The American Society for Pharmacology and Experimental Therapeutics JPET 288:65–72, 1998 Vol. 288, No. 1 Printed in U.S.A. Diclofenac Toxicity to Hepatocytes: A Role for Drug Metabolism in Cell Toxicity1 ´ ´ ´ ´ ROQUE BORT, XAVIER PONSODA, RAMIRO JOVER, M. JOSE GOMEZ-LECHON and JOSE V. CASTELL Unidad de Hepatologıa Experimental, Centro de Investigacion, Hospital Universitario “La Fe”, Valencia, Spain (R.J., M.J.G.-L., R.B., J.V.C.); ´ ´ Departamento de Bioquımica, Facultad de Medicina, Universidad de Valencia, Valencia, Spain (R.B., R.J., J.V.C.); and Departamento de ´ Parasitologıa y Biologıa Celular, Facultad de Ciencias Biologicas, Universidad de Valencia, Valencia, Spain (X.P.) ´ ´ ´ Accepted for publication July 21, 1998 This paper is available online at http://www.jpet.org ABSTRACT Diclofenac, a 2-arylacetic acid, nonsteroidal anti-inflammatory drug, has been reported to cause adverse hepatic effects in certain individuals. To discriminate among possible mechanisms of hepatotoxicity, we examined the effects of diclofenac on human and rat hepatocytes and hepatic cell lines (HepG2, FaO), investigated the major biochemical events in the course of diclofenac cytotoxicity (calcium homeostasis, lipid peroxidation, and mitochondrial dysfunction), and investigated whether cytotoxicity could be related to drug metabolism by cytochrome P-450. Acute diclofenac-induced toxicity in hepatocytes was preluded by a decrease in ATP levels, whereas no significant oxidative stress (decrease in glutathione and lipid peroxidation) or increase in intracellular calcium concentration could be observed at early incubation stages. Diclofenac was more cytotoxic to drug metabolizing cells (rat and human pri- mary cultured hepatocytes) than to nonmetabolizing cell lines (HepG2, FaO). Despite the fact that diclofenac itself was effective in impairing ATP synthesis by mitochondria, we found evidence that toxicity was also related to drug metabolism and was reduced by the addition of cytochrome P-450 inhibitors (proadifen and ketoconazole) to culture medium. The in vitro cytotoxicity correlated well with the formation by hepatocytes of 5-hydroxydiclofenac and, in particular, N,5-dihydroxydiclofenac, a minor metabolite first characterized in this article. Hepatic microsomes showed the ability to both oxidize 5-hydroxydiclofenac to N,5-dihydroxydiclofenac and back reduce the latter to 5-hydroxydiclofenac with the consumption of NADPH. The experimental results suggest that the toxic effect of diclofenac on hepatocytes may be caused by drug-induced mitochondrial impairment, together with a futile consumption of NADPH. Downloaded from jpet.aspetjournals.org by guest on April 26, 2011 Diclofenac is a frequently prescribed nonsteroidal antiinflammatory drug. Although diclofenac-associated hepatitis was thought to be rare, there are clinical reports of severe hepatic reactions associated with their use (Helfgott et al., 1990; Sallie, 1990; Ouellette et al., 1991; Purcell et al., 1991). In addition to this, there are indications that borderline increases in serum transaminases occur in approximately 15% of patients taking the drug regularly, which suggests that diclofenac-associated hepatotoxicity might be more common than previously recognized (Ciccolunghi et al., 1978; Iveson et al., 1990). Although in some case reports, the adverse hepatic effects of diclofenac showed features compatible with a drug hypersensitivity reaction (Breen et al., 1986; Schapira et al., 1986; Salama et al., 1991; Romano et al., Received for publication April 8, 1998. 1 This study was supported, in part, by the BIOMED II Research Program of the European Union and the Fondo de Investigaciones Sanitarias, Spanish Ministry of Health. R.B. was recipient of a predoctoral fellowship from the Consellerıa de Educacion y Ciencia, Generalitat Valenciana. ´ ´ 1994), the clinical findings in other patients appear to be more consistent with a direct toxic effect of the drug or a drug metabolite (Helfgott et al., 1990; Iveson et al., 1990; Sallie, 1990; Scully et al., 1993). The mechanism by which diclofenac causes liver alterations in certain individuals is not yet fully understood, and both the formation of a toxic metabolite and covalent binding of the drug to hepatic proteins have been invoked to explain its toxicity. Diclofenac was found to generate protein adducts in the livers of treated mice as well as in rat hepatocytes via protein acylation by the drug glucuronide (Pumford et al., 1993). In vitro experiments with cultured rat hepatocytes have shown, however, that the covalent binding of diclofenac is neither the only nor the major cause of acute cytotoxicity (Kretz-Rommel and Boelsterli, 1993). Moreover, previous work has suggested that diclofenac is cytotoxic to rat hepatocytes after cytochrome P-450 (CYP)-mediated metabolism (Schmitz et al., 1992; Jurima-Romet et al., 1994). Recently, the formation of reactive metabolite(s) by drug oxidation, ABBREVIATIONS: BHT, buthylated hydroxytoluene; BSO, L-buthionine-[S,R]-sulfoximine; CYP, cytochrome P-450; MDA, malondialdehyde; DEM, maleic acid diethyl ester; MTT, 3-[4,5-dimethylthiazol-2-yl]-3,5-diphenylformazan; PBS, phosphate-buffered saline; 4 -OHdic, 4 -hydroxydiclofenac; 5-OHdic, 5-hydroxydiclofenac; N,5-(OH)2dic, N,5-dihydroxydiclofenac; [Ca ]i, intracellular calcium; GSH, glutathione; HPLC-MS, high-performance liquid chromatography-mass spectrometry. 65 1993). Downloaded from jpet. 505 which could be related to drug toxicity. After a 24-h incubation. 1997). MO). Results were referred to intracellular protein content. 1989). [Ca ]i was calculated for any previous fluorescence signal (F) using a Kd of 400 nM for the Fluo-3-Ca complex (Minta et al. (Irvine.. Unattached cells were removed by changing the medium 1 h after seeding.. Faigle et al. rhodamine 123.R]-sulfoximine (BSO). Wildbad. 1988). Hepatocytes were seeded on fibronectin-coated ´ culture plates (3.5-dimethylthiazol-2-yl]-3. 1992). The ATP concentration of the diluted samples (1:4900) was measured using the luciferin/luciferase assay according to the manufacturer’s instructions. Diclofenac undergoes hepatic metabolism both in rat and human hepatocytes. The gluconeogenic rate was determined from the rate of glucose production by hepatocytes.5-diphenylformazan (MTT) test (Carmichael et al. 1993). the fluorochrome formed was extracted with n-butanol and the fluorescence of the organic phase was measured at 530 25 nm excitation and 590 35 nm emission. Millipore. Calf serum was obtained from GIBCO (Paisley. After incubations at different times. FaO. The patients had not received any medication for at least a week before surgery. deferoxamine mesylate. but to a much lesser extent (Riess et al. has been reported (Miyamoto et al.5-diphenyl-hydantoin). -naphthoflavone. Increasing concentrations of the drug in phosphate-buffered saline (PBS) were added to cultures after medium renewal. cells were detached and homogenized by ultrasound. the major oxidative metabolic pathway is the formation of 4 -hydroxydiclofenac (4 -OHdic) by CYP2C9 (Smith and Jones. and HepG2 cells were cultured in Dulbecco’s minimal essential medium supplemented with 10% of fetal calf serum and containing 50 g of streptomycin/ml and 50 mU of penicillin/ml. Germany). 1987). 1984). (St. the Glucose GOD-Perid test. 5-OHdic. Fluo-3-AM and pluronic F-127 were obtained from Molecular Probes.. buthylated hydroxytoluene (BHT). 485 22 nm. Despite the fact that diclofenac itself was effective in impairing ATP synthesis by mitochondria. according to the following equation: [Ca ]i Kd ((F-Fmin)/ (Fmax F)). Human hepatocytes ´ ´ were obtained from small liver biopsies from patients undergoing cholecystectomy.. and fluorescence (Fmin) was again recorded. The synthesis and secretion of albumin by hepatocytes was monitored by a competitive enzyme-linked immunosorbent assay in aliquots of culture medium taken at regular time intervals (Castell et al. CA). Briefly.. pH 7. in addition to the effects of diclofenac and its major metabolites on mitochondrial function.. Louis. maleic acid diethyl ester (DEM). Fluorescence readings were calibrated to calculate [Ca ]i. 1990).. . 3 -hydroxydiclofenac. -Glucuronidase/arylsulfatase. cells previously incubated with diclofenac were loaded with 5 M of Fluo-3-AM 0. and the main biotransformation reactions (aromatic hydroxylations and conjugations at various sites of the molecule) are common to several animal species (Riess et al. Germany). Thus. Inc. Cell suspensions were obtained as described in detail elsewhere (Gomez´ Lechon et al.. ATP synthesis was determined in cultured hepatocytes incubated in HEPES-buffered saline medium with glucose as the only energetic substrate and 500 M diclofenac. and 3 -hydroxy-4 -methoxydiclofenac has also been reported in humans. Glutathione (GSH) was measured by a fluorimetric reaction with o-phthaldialdehyde adapted to microscale measurements (Jover et al.5-(OH)2dic). Leemann et al. Cell Cultures. 4 . Germany). as described in detail previously (Castell et al. Diclofenac metabolites (4 -OHdic. The cells were washed three times with HEPES-buffered Krebs-Henseleit solution (10 mM HEPES. and culture media were obtained from Flow Labs.org by guest on April 26. At regular time intervals. Rat hepatocytes were obtained from 200. 25°C) containing 1% bovine serum albumin. Lipid peroxidation was determined by measuring the generation of MDA in the culture media by the fluorimetric reaction with thiobarbituric acid (Castell et al.075% (w/v) pluronic F-127 in culture medium for 30 min at 37°C. the existence of a futile red-ox cycle could also contribute to the mechanism of toxicity. The effects of diclofenac on gluconeogenesis were investigated in glycogen-depleted hepatocytes incubated with the drug and with lactate as the only gluconeogenic precursor. Stierling et al. microtiter plates were washed twice with 50 l of PBS/ well. After incubation for 60 min in a boiling bath. Samples were centrifuged (5 min 9000g) and the supernatant kept at 4°C until analysis. Bioluminescence was quantified in a Lumat LB luminescence photometer (Berthold GmbH. flurbiprofen. 288 Evaluation of Toxicity of Diclofenac to Hepatocytes. L-buthionine-[S. Known amounts of GSH (0 –5000 pmol/well) were used as reference standards. 530 30 nm) (Jover et al. collagenase.aspetjournals... and 4 . 1985). cytosolic Fluo-3 was released out of cells into medium containing 2 mM CaCl2 by treating cells with 30 M digitonin for 4 min. proadifen. 1979). Formation of 5-hydroxydiclofenac (5-OHdic).. and immediately thereafter fluorescence was measured with a multiwell plate fluorimeter (Cytofluor 2350. Mitochondrial membrane potential was assayed in the same conditions as ATP.5-dihydroxydiclofenac (N. emission. 4 -OHdic together with 5-OHdic are the major urine metabolites (Stierling et al. and o-phthaldialdehyde were obtained from Sigma Chemical Co. in particular with the formation of 5-OHdic and N.. 1996).. 1978. but rhodamine 123 (final concentration 1 M) was also added. emission filter: 460 25 nm).. MDCK.66 Bort et al. Diclofenac sodium salt. 1992. To monitor intracellular calcium ([Ca ]i).1 ml medium per well and used 24 h later (75% monolayer confluence). 10 mM EGTA was added to quench Ca . OR)... excitation. phenytoin (5. In the rat. and cytotoxicity was assessed either by measuring the loss of intracellular lactate dehydrogenase (Ponsoda et al. Aliquots of the supernatant were transferred to microtiter plates and allowed to react with o-phthaldialdehyde. (Eugene. UK). Cells were routinely seeded in 3. First the hydrolysed. Both metabolites can easily interchange by oxidation and reduction and both cause a continuous consumption of NADPH. 2011 Materials and Methods Materials.. Malondialdehyde (MDA) bis(dimethylacetal) was obtained from Merck (Darmstadt. 1991) or by the 3-[4. we found evidence that toxicity was also related to drug metabolism. Fluorescence was measured in a multiwell plate fluorimeter (excitation filter: 355 35 nm. 1978. In human liver microsomes..5 g/cm2) at a density of 80 103 cells/cm2. The fluorescence of samples was measured (excitation. medium was removed and monolayers were processed to measure fluorescence in the mitochondrial compartment as described elsewhere (Nieminen et al. cells were washed with PBS and homogenized in 1 ml of 3% HClO4 at 0°C. 1997).. The resulting homogenates were deproteinized and centrifuged in the same 96-well plate. nifedipine. MDA bis(dimethylacetal) diluted in culture medium was used as standard.5-cm plates at a density of 14 103 cells in 0. 1990). and fluorescence (Fmax) was recorded. 1985). Next. ( )verapamil.4. 1979). after informed consent. All other reagents used in this study were of analytical grade. Vol.5-OHdic) were synthesized as described (Bort et al. We have investigated the acute effects of diclofenac on the viability and functionality of cultured hepatocytes to discriminate between possible mechanisms of toxicity and found that a decrease in ATP levels preluded cell death.5dihydroxydiclofenac. and the ATP bioluminescence CLS test were obtained from Boehringer Mannheim (Mannheim.to 300-g Sprague-Dawley male rats by perfusion of the liver with collagenase as described in detail elsewhere (Gomez-Lechon et al. 25% acetonitrile) was delivered at 1 ml/min.. containing 1 mM EDTA and 0. acetate buffer 0. rat hepatocytes. 30 mU of arylsulfatase/ml. 1 mM EDTA. 1987) and finally resuspended in 0. and extracted with ethyl acetate as described above. 10 mM Tris. Phase Separations Ltd. Ar-CH2-CO-. Downloaded from jpet. The reaction was stopped by the addition of 1 ml of cold acetonitrile. diclofenac was added to hepatocyte cultures and incubated for 20 h.4 min. To estimate IC10 and IC50 values (concentrations that produce a 10% and 50% inhibitory effect. and homogenized in 4 ml/g ice-cold homogenization buffer (0. Similar results were obtained (data not shown) when the leakage of intracellular lactate dehydrogenase was determined as a parameter for cell viability. Metabolism of Diclofenac. MA).org by guest on April 26. 3 mM magnesium chloride. final concentration) and after 5 min.2 mM). Rat microsomes were obtained by homogenization of liver samples in 0. The UV-(4 -OHdiclactam: max 239 and 5-OHdic-lactam: max 250 nm). The column effluent was also monitored at 282 nm using a photodiode detector (M996. Assays were performed in a buffer containing 75 mM Tris-hydrochloride buffer. Biotransformation of the drug was studied both in vitro and in vivo. and 500 g of microsomal protein.1 M. 6H. . m. the typical sigmoid dose-effect curves were linearized using the LOGIT transformation. The reaction was stopped by adding acetonitrile to the samples (1:1. 5 mM Tris. CA) of the main metabolites were obtained and compared with chemically synthesized standards.5-mM solution in PBS) was added to samples as an internal standard. Waters Scientific.. rat hepatocytes cultured in the presence of 10 M proadifen. The microsomal fraction was stored in the same buffer containing 30% v/v glycerol. and MDCK) were cultured in microwells and incubated with increasing concentrations of diclofenac. Values are the mean of quintuplicated wells S. For in vivo studies. 1980). The mobile phase was changed to 63% 20 mM ammonium acetate buffer (pH 6. The statistical significance of the experimental data was analyzed by the Student’s t test.25 M potassium phosphate buffer. resuspended in methanol. m. pH 4. Statistical Analysis.1-mm C-18 reversed-phase column (Simmetry. pH 7. 50 4. v/v). 1 mM EDTA. to SpragueDawley rats (40 mg/kg) and urine was collected over cold (0°C) acetate buffer (0.4) per gram of liver. as well as in the nonhepatic cell line MDCK. pH 7. 1H NMR(4 -OHdic-lactam: 3. s 2H. the solution was brought to dryness. of a representative culture. Waters).25 M sucrose.. cytotoxicity was also evaluated in human and rat hepatomas. Metabolites were routinely analyzed by high-performance liquid chromatography (HPLC) in deconjugated and nondeconjugated samples (Leemann et al. pH 7.1 M. 2011 Results Cytotoxicity of Diclofenac on Hepatic and Nonhepatic Cells. 7. The supernatant was diluted with 100 mM phosphate buffer (pH 7. Mechanisms of Diclofenac Hepatotoxicity 67 aromatic) and mass spectra (m/z 293. Diclofenac eluted at 29. Isolation of Mitochondria from Rat Liver. The mobile phase (75% triethanolamine 0. Figure 1 shows that hepatoma Fig. 9000 rpm).6-mm C-18 reverse-phase column (Spherisorb ODS 5 m. pH 4. Human. For in vitro studies. mass spectra. 1). 4 mM EDTA. diclofenac was administered i. followed by differential ultracentrifugation (Boobis et al.7.5 mM malate.4) to reach 25% (v/v) acetonitrile.8.1998 nm. Upon completion of the reaction (5 h at room temperature). washed.. 7. Flurbiprofen (2 l of a 4.aspetjournals. Identification of Diclofenac Metabolites. The lactams of 4 -OHdic and 5-OHdic were synthesized by reacting the corresponding hydroxyderivative of diclofenac with a 10% molar excess of dicyclohexyl carbodiimide in dry chloroform.D. Metabolites were extracted from acidified urine with ethyl acetate either before or after deconjugation. pH 7. ATP was measured as described above. Samples were analyzed by HPLC as described above. Aliquots of culture medium were enzymatically deconjugated (50 mU of -glucuronidase/ml. and analyzed by HPLC-mass spectrometry (MS) (Waters Alliance equipped with a therma beam detector.4. Controls were incubated either without NADPH or without microsomes. Milford. respectively. Each determination was done in four plates (eight wells for cytotoxicity experiment) from each culture. ATP Measurement in Isolated Mitochondria.p.3 ml/min. Varian Gemini 300 MHz spectrometer.7 ml of the assay solution (0. 6H.15 M KCl. The resulting precipitate was removed by centrifugation (10 min. cell viability was determined by the MTT test. 523 nm) in a Hitachi F-2000 fluorescence spectrophotometer (Hatachi Scientific Instruments. Palo Alto.8 min. and cell lines (FaO.25 M sucrose. emission.9.5)/37% methanol and it was delivered at 0.E.5) for 4 h at 37°C. 5-OHdic-lactam: 3. Waters). and the reaction was started by the addition of NADPH (final concentration 1. and the IC values were interpolated mathematically. s 2H. 1993). Each experiment was done in at least three different cultures.2 min and flurbiprofen at 7. Culture supernatants were collected. Mitochondria were isolated by centrifugation in sucrose as described elsewhere (Cain and Skilleter. molecular peak for both derivatives) were consistent with the assigned chemical structure of the two cyclic diclofenac derivatives. HepG2. 5 mM KH2PO4. Samples (20 l) were injected into a 200 4. 1. The column effluent was monitored at 282 nm. To elucidate whether these effects were hepatocyte-specific. Queensferry. After 24 h. Sprague-Dawley rats were euthanized and their livers immediately removed. filtered to remove the dicyclohexyl urea formed.75). pH 7. dissolved in ethyl acetate. and 1H NMR (d6-acetone. as assessed by the MTT test (Fig. deconjugated. The UV.4 – 6. Mountain View. and the results shown are the mean value S.M.4 – 6. A new HPLC method was developed to adjust it to this specific detector. 4 -OHdic and 5-OHdic were prepared as described in detail previously (Bort et al. Diclofenac eluted at 12.5 mM glutamate. aromatic. 2. and crystallized by the addition of petroleum ether. respectively). The estimated IC50 values for rat and human hepatocytes (concentration causing 50% cell death) were closely related: 392 34 M (n 16) and 331 7 M (n 6). Then substrates were added (2. Samples (25 l) were injected into a 150 2.25.02% in 100 mM phosphate buffer pH 7.6 mm). 1996). A mitochondrial suspension (final concentration 2 mg/ml protein) containing 2. Microsome Incubations.4. Cytotoxicity of diclofenac in different cellular systems. Samples were preincubated for 5 min at 37°C.5) during the next 20 h.4). 2 mM MgCl2.8. CA) .5 mM ADP and variable concentrations of diclofenac or its metabolites was preincubated at 30°C for 5 min. UK) furnished with a precolumn (ODS 5 m. Ar-CH2-CO-. 100 l was taken and dropped on 900 l of boiling buffer (100 mM Tris. Diclofenac showed a characteristic concentration-dependent cytotoxicity when added to cultured human or rat hepatocytes. The organic extracts were dried under vacuum. versus control at 392 34 M). 3C). 2.D. 3D). 1993) was moderately effective in decreasing diclofenac cytotoxicity (IC50 415 92 and 507 30 M. N. In parallel. lipid peroxidation. 1 mM t-butyl hydroperoxide (t-BOOH) was added to cells.. GSH was measured in cells incubated with diclofenac or the GSH inhibitor BSO. also reduced diclofenac toxicity significantly (IC50: 514 38. Diclofenac was added to hepatocytes and aliquots of culture medium were taken at regular time intervals. Downloaded from jpet. with concentration-toxicity curves shifting to higher concentrations of the drug (i. ATP dropped to 10% of initial levels after 120 min of exposure to the drug. Vol. We also investigated the effects of diclofenac on intracellular GSH levels.S. of a representative culture. 2011 Fig. we found evidence showing a good correlation between cytotoxicity (IC50) and the extent of diclofenac metabolization (r 0. Neither BSO nor DEM (500 M) increased the toxicity of diclofenac (IC50 368 12 and 367 47 M. the metabolism of diclofenac was investigated by HPLC analysis in cell culture media incubated with 250 M diclofenac. As expected. 150 – 200% over controls.). the presence of BSO caused a rapid GSH depletion in cells. hepatomas.). As a positive control. addition of 1 mM t-butyl hydroperoxide resulted in an immediate. Correlation between cytotoxicity and drug metabolization.e. For evaluation of mitochondrial membrane potential. In agreement with these findings. fast depletion of ATP in hepatocytes kept in glucose as the only energy source. D. cell viability of the same experiment (LDH) is also depicted. which inhibits the 2C and 3A subfamilies. higher IC50 values) but more sensitive than the nonhepatocyte cell line MDCK (908 56 M. n 4 and FaO 682 64 M. confidence interval. For a better interpretation of results. dramatic increase in thiobarbituric-reactive substances (Fig. however. [Ca ]i was fluorimetrically measured at regular time intervals (C) in cells incubated with the diclofenac or t-butyl hydroperoxide. An IC50 concentration of diclofenac did not cause significant changes in intracellular GSH during the 5 h following incubation (Fig. Plates were washed and cells detached with 3% HClO4. Rat hepatocytes were incubated with different concentrations (100 –750 M) of diclofenac and the cytotoxicity was expressed as IC50. changes in [Ca ]i. caused an important. Fig. we observed a gradual increase in [Ca ]i (ca. n 3. p . Values represented as mean of five wells S. cells were incubated with rhodamine 123.org by guest on April 26. After reaction with thiobarbituric acid. In control experiments. The possibility that calcium could be involved in the early stages of toxicity by diclofenac was also investigated. data not shown). HPLC analysis of culture medium after a 24-h incubation with a subtoxic concentration of the drug (100 M) showed that rat and human hepatocytes metabolized diclofenac at similar rates (310 and 340 pmol min 1 mg 1. [Ca ]i.95.005. 3. Diclofenac. mitochondrial membrane potential. Jover et al. The differences in diclofenac toxicity observed in primary cultured hepatocytes. In agreement with this . Evaluation of diclofenac toxicity to hepatocytes. a broad CYP inhibitor.68 Bort et al. fluorescence was determined and expressed as nanomoles of MDA. and ATP content of cultured rat hepatocytes. diclofenac (400 M) was added to cultured rat hepatocytes. Fig. GSH content. Analysis of toxicologically relevant biochemical parameters in hepatocytes exposed to diclofenac (GSH levels. regression. respectively). 2). respectively. whereas -naphthoflavone-specific CYP1A inhibitor did not apparently modify cytotoxicity (IC50: 392 81. significantly reduced the toxicity of the drug (IC50: 489 32. ATP content was measured by means of luminescence production by luciferase. which in these experiments began to be noticeable 120 min after addition of the drug (Fig. Moreover.). and the results expressed as percentage of metabolized drug. Major Biochemical Events in Course of Diclofenac Toxicity. This rapid ATP depletion preceded cell death. respectively). and ATP) revealed that upon incubation of cells with toxic concentrations of the drug. p . dashed line.. n 2).aspetjournals. B. Ketoconazole (25 M). no significant accumulation of MDA was found in the medium after several hours. Addition of 25 M verapamil or 100 M nifedipine to culture medium (concentrations effectively blocking calcium influx in cells. 3A). Solid line.005).001. After much longer incubation times ( 6 h). 3B). The addition of an IC50 concentration of diclofenac did not result in an increase in intracellular Ca during the first 120 min of incubation (Fig. 288 cells were less sensitive to diclofenac than primary cultures of hepatocytes (HepG2 763 61 M. Remaining fluorescence in medium was measured fluorimetrically as described in Materials and Methods. and nonhepatic cells could reflect the different sensitivity of cell lines versus hepatocytes to the drug or to differences in the ability of these cells to metabolize diclofenac. we did not observe a decrease in toxicity (increased IC50) when cells were incu- Fig. in parallel. Incubation of rat hepatocytes with a noncytotoxic concentration (10 M) of proadifen. whereas cell lines were unable to metabolize it to a measurable extent (data not shown). In fact. bated in the presence of the antioxidant BHT (50 M) or 10 mM deferoxamine (data not shown. p . 1). Effects of diclofenac on lipid peroxidation. and 1H-NMR spectra of chemically synthesized metabolites. the M peak of this metabolite suggested the existence of two hydroxy substituents. both in culture media and in vivo. Analysis by HPLC of culture media of hepatocytes incubated with diclofenac allowed the identification of several metabolites. N. two specific hepatic functions that require substantial amounts of ATP. A peak with a retention time of 3. The molecular ion peak (M ) was at m/z 327.5-(OH)2dic unequivocally demonstrated the cellular formation of this metabolite (Table 3).5-(OH)2dic. 5A). the IC50 correlated with 5-OHdic (r 0. 5-OHdic was added to urine of untreated rats and processed as described in Materials and Methods. which could be attributable either to the drug or to any of its metabolites. In control experiments. The IC50 were 110 12 and 108 9 M.4 – 6. 2H. Effects of Diclofenac and Its Metabolites on Hepatocyte Mitochondrial Function.1998 Mechanisms of Diclofenac Hepatotoxicity 69 finding.5-(OH)2dic structure to this metabolite (Fig. Fig. 5B) and with N. A minor peak (retention time 2. 4). A poor correlation was observed in the case of the major metabolite 4 -OHdic (r 0. To exclude an artifactual formation of N. administration of diclofenac. we investigated the role of diclofenac and each metabolite on mitochondria by monitoring the synthesis of ATP. Conversely. 2011 Fig. This metabolite was found in culture media mainly in its conjugated form ( 95%).5 min were identified as lactams of 4 -OHdic and 5-OHdic.5-(OH)2dic is found only as a conjugate. respectively. In control experiments. As shown in Fig. as compared with that recorded for 5-OHdic.2 M). by comparison of the UV-.5-(OH)2dic formation (r 0. points to the proximal N as the site of oxidation.68 s.5-(OH)2dic was observed. and its concentration is greater inside cells than in culture medium (3. the presence of conjugated N. no spontaneous formation of N. and the presence of isotopic peaks at m/z 329 and 331 confirmed the presence of two Cl.52. In view of the fact that ATP depletion was paralleled by a decrease in mitochondrial membrane potential. This inhibition was noticeable at subcytotoxic concentrations of diclofenac.9 min) was present with variable intensity in the different culture supernatants assayed. while the peak with a retention time of 5. which suggests that conjugation may be crucial for this metabolite to escape from the cell. 6H) and ethylenic H ( 3. its occurrence was investigated in rat urine 20 h after i. 5. we assigned the N.org by guest on April 26. which excludes an aromatic hydroxylation. the cytotoxicity of diclofenac (IC50) was determined under an array of different incubation conditions (100 –750 M drug in the presence or absence of selective CYP inhibitors) and was correlated with the amount of metabolites formed by cells incubated with 250 M diclofenac.92.p. In the experiment shown in Fig. When compared with the mass spectra of diclofenac.1 min was identified as 5-OHdic. both in free and conjugated forms.84. Ar-CH2COOH) as 5-OHdic. 6. the mitochondrial ATP synthesis was effectively impaired by 30 M diclofenac (concentration found in the portal blood of rats given the drug Downloaded from jpet. the displacement to a high field of the signal of H(3). Other minor peaks at 10. and the most relevant spectral features are summarized in Table 2 and Fig. 4.5-(OH)2dic in the course of in vitro experiments. 4. The compound was analyzed by HPLC-MS and 1H NMR. 5B).4 m. Approximately 28% of the in vivo administered diclofenac was found in urine as hydroxylated metabolites.aspetjournals. mass-. According to the results shown in Fig. the decrease in ATP was an early event in diclofenac toxicity.8 min was assigned to 4 -OHdic. increasing diclofenac in culture medium resulted in a concentration-dependent inhibition of gluconeogenesis and albumin synthesis. Metabolism of Diclofenac. 5-OHdic).4 and 13. Structure and mass spectra of N. . The 1H NMR spectra of the purified metabolite (Table 1) showed the same number of aromatic H (7. In addition to 4 -OHdic and 5-OHdic. Fig. Based on these data. Fig. 4D. Interestingly. The compound showed the features of an acid and a UV maximum displacement to 268 nm (versus 282 nm.2 versus 0. the metabolism of diclofenac was investigated by HPLC analysis in 24-h cell culture media incubated with 250 M diclofenac. control C. Iveson et al. 10. when the NADPH content of the incubation mixture was monitored. 1990. we investigated possible linkages between drug metabolism and cell injury to better understand the mechanisms of hepatotoxicity. 1990. The ATP concentration was measured using the luciferin/luciferase assay according to the manufacturer’s instructions.41.5 ) 1H (4 ) 1H (6) 1H (4) 1H (3) 5-OHdic N.43. whereas the incubation of N. 2011 Fig. control B.7) 6. 7A). Among the several biochemical parameters examined.7) metha (J 2.5 mM glutamate and 2. proadifen B. Under the same experimental conditions. Purcell et al.5 mM malate) and after 5 min 100. 3D). 1990.. 7. Downloaded from jpet. Most interestingly. doublet metha (J 2. control E.5-(OH)2dic to 5-OHdic. a biphasic behavior was observed: a rapid NADPH decrease that coincided with the reduction of most N.7) 6. doublet ortho (J 8. Rat hepatocytes were incubated with differents concentrations (100 –750 M) of diclofenac in the presence or absence of CYP inhibitors and the cytotoxicity was expressed as IC50.7) a N..7) 6. phenytoin B. 1991. We examined whether 5-OHdic and N.68. dashed line. Discussion Diclofenac is an anti-inflammatory drug for which a certain number of severe adverse hepatic reactions have been reported (Ciccolunghi et al. incubation of 4 OHdic did not result in either metabolism or in NADPH consumption (Fig. 1990. regression. 1993). the equilibrium between the two metabolites was reached. control A. 7C).5-(OH)2dic by Liver Microsomes. This reduction did not take place in the absence of either NADPH or microsomes. In the present study. Interconversion between 5-OHdic and N. Sallie.65. 12. 1990. and the formation of 4 -OHdic (A) and 5-OHdic and N..40.l samples of the incubation mixture were dropped in 900 l of boiling buffer (100 mM Tris.5-(OH)2dica compared to 5-OHdic Aromatic Hydrogens Compound 2H (3 . Scully et al. Ouellette et al.5-(OH)2dic (B) correlated with the cytotoxicity determined for each cell culture.10.org by guest on April 26. rather than a drug-allergy mechanism (Helfgott et al. double doublet ortho (J 8. doublet ortho (J 7. could also indirectly alter the energetic status of cells. proadifen D. followed by a sustained NADPH consumption once . doublet ortho (J 7. These results. In parallel. 6.8) 7. 5.5-(OH)2dic (Fig. pH 7. phenytoin D. whose formation correlated with the toxicity of diclofenac.. 1990. as well as by its hydroxylated metabolites.7) 6. Effects of diclofenac and its metabolites on hepatocyte mitochondrial function. 1. 9.01. ATP was the earliest affected by the cytotoxic concentrations in short-term experiments (Fig. 8. Sallie. 1991. Incubation of 5-OHdic with rat liver microsomes in aerobic conditions resulted in the formation of N. 2-ketoconazole B. 288 TABLE 1 Relevant proton chemical shifts of aromatic hydrogens of N.5-(OH)2dic 7.75). Fig. Vol. Solid line...40. 1996). Bioluminescence was quantified in a Lumat LB luminescence photometer.59.5-(OH)2dic.5 mM ADP and 30 M diclofenac or diclofenac metabolites was preincubated for 5 min at 30°C.76.5-(OH)2dic in presence of microsomes and NADPH yielded 5-OHdic (Fig.. control D.8) 7. ketoconazole E. 7B). doublet metha (J 2.5-(OH)2dic was purified from rat urine after extraction with ethyl acetate and purification by thin-layer chromatography. 11. 1993). doublet ortho (J 8.8) 6. triplet ortho (J 7..8) 7. 4 mM EDTA. Then substrates were added (2. Helfgott et al. 4. Tabata et al. Parallel to this. ketoconazole D. 6. a decrease in mitochondrial membrane potential was observed.70 Bort et al. confidence interval.7) metha (J 2. together with the previously reported protective effect of orally. 3. 5. Correlation between cytotoxicity and diclofenac metabolites formation. A mitochondrial suspension (final concentration 2 mg/ml protein) containing 2.7) 6. 1978. Iveson et al.. Detailed analysis of some individual case reports revealed clinical features that could be compatible with a direct toxic effect of diclofenac (or any of its metabolites).. triplet ortho (J 7. Scully et al.aspetjournals. double doublet ortho (J 8. The fact that it appeared only as a conjugate reinforced this hypothesis and could also indicate that this compound escapes from the hepatocyte only after conjugation. 2011 Rat hepatocytes Culture media Rat urine 3.aspetjournals. The authors also reported that hepatic microsomes could oxidize 5-OHdic to the same iminoquinone. 1).. 229. and the lack of a clear effect on the cytotoxicity of radical scavengers (BHT) and inhibitors of the formation of active oxygen species (deferoxamine). and the slight decrease in cell toxicity (not significant) that we observed agrees with this interpretation. 201. N. Also the control and 4 -OHdic incubations are depicted..5 (100%)a 1 0. 267. The experiments also revealed a possible link between drug metabolism and toxicity to hepatocytes. we have found that the concentration of this metabolite inside cells is approximately 16 times grater than in culture medium. excluded an artifactual. On the other hand. Tabata et al. fructose on diclofenac toxicity (Ponsoda et al. Second. TABLE 2 In vitro and in vivo metabolism of diclofenac Sample N. The presence of N.5(OH)2dic would render the hypothesized iminoquinone. noncellular formation of the metabolite (Table 2).8 (100%) 59 14 (56%) 80 24 (85%) 38 17 (34%) 20 9 (66%) 16 3 Data represent relative abundance of metabolites in each analyzed sample. Third. 1992). 3C). 7. In a recent article.1998 Mechanisms of Diclofenac Hepatotoxicity 71 Fig. parallel to a decrease in cell viability. A decrease in ATP impairs plasma membrane and endoplasmic reticulum ATPdependent calcium pumps and leads to a sustained rise in intracellular free calcium (Nicotera et al.5-(OH)2dic (Fig.. First of all.5-(OH)2dic was incubated in presence of microsomes and NADPH and the formation of 5-OHdic followed by HPLC (B). and inhibition of drug metabolism resulted in a decrease in toxicity (Fig. 1995).. Miyamoto et al. C. analysis of culture media of hepatocytes revealed more precisely that cytotoxicity to hepatocytes was related to the formation of 5-OHdic and N. which is in agreement with previous reports (Schmitz et al. Pure 5-OHdic was incubated in the presence of microsomes and the formation of N. 2). as well as in urine of rats given diclofenac.5-(OH)2dic 4 -OHdic 5-OHdic n Downloaded from jpet. close to the portal concentration in the rat (30 M.5-(OH)2dic and in culture media of cells [m/z: 309 (M ). The chemical structure of this new metabolite was identified by the combined use of HPLC-MS. or compounds depleting intracellular GSH (BSO. In fact. lead us to consider a mitochondrial bioenergetic dysfunction to be one of the possible events causing diclofenac toxicity. was detected in preparations of N. caused a 50% inhibition of two characteristic ATP-consuming hepatocyte functions. [Ca ]i levels did not increase during the early stages of cell exposure to diclofenac (Fig. (1997) using MS-MS. did not support the hypothesis that this mechanism is a key event in diclofenac toxicity. and 166]. Several facts support this assessment.e.. the ability of cells to metabolize diclofenac (human rat [tmt] FaO HepG2 MDCK) was inversely related to the IC50 values. . and chemical data. Only after a longer incubation of cells with diclofenac did [Ca ]i increase moderately.org by guest on April 26.5-(OH)2dic in culture medium. It is thus conceivable that the decrease in cellular ATP observed in hepatocytes incubated with diclofenac could be at the root of the late [Ca ]i elevation. GSH and MDA levels) were observed. UV spectra. (1997) reported the formation of an iminoquinone by HOCl oxidation of diclofenac in activated neutrophils. Indeed. a Data in parentheses represent the approximate percentage of conjugated metabolite.5-(OH)2dic was monitored by HPLC analysis (A). the consumption of NADPH was monitored by HPLC analysis of the same samples as in B. We believe this event may be simply concomitant with cell death. the fact that no significant changes in oxidative stress parameters (i.5-(OH)2dic and 5-OHdic by liver microsomes. Interconversion of N. 5B). 195. 1996). namely gluconeogenesis and albumin synthesis.2 2. 1H NMR (Table 1). DEM). The fact that subcytotoxic concentrations of diclofenac (100 M). there was a direct correlation between cytotoxicity and the extent of diclofenac metabolization by hepatocytes (Fig. This compound shows strong chemical similarities with the one reported in this study: dehydration of N. Both are “service” functions of hepatocytes and have in common the fact that they are not essential for hepatocyte survival but both require substantial amounts of ATP. B. which was trapped with GSH. a compound showing similar fragments by HPLC-MS (electron impact fragmentation) than those obtained by Miyamoto et al. would favor this hypothesis. 1995). Nicotera P. a metabolite that is not further metabolized by microsomes. 288 term tolerability study of up to two years with diclofenac sodium (Voltaren). Sallman A. Di Fonso M.Castell@uv. Sulc M. Br J Clin Pharmacol 9:11–19. Ponsoda X. McGee JO’D and Chapman RWG (1990) Diclofenac associated hepatitis. and hypochlorous acid. Gut 32:1381–1385.. Cancer Res 47:936 –942. Transon C and Dayer P (1993) Cytochrome-P450TB (CYP2C): A major monooxygenase catalyzing diclofenac 4 . Oguri K and Yoshimura H (1988) Enzymic reduction of Nhydroxyamphetamine: the role of electron transfer system containing cytochrome b5. Toxicol In Vitro 8:55– 66. Toxicol In Vitro 9:439 – 444. Faigle JW. Miguel Angel Miranda for his skillful advice in mass spectrometry and 1H NMR data interpretation. Schapira D. Bottcher I. Chem Res Toxicol 10: 414 – 419. we observed that the hydroxylamine generated by oxidation of 5-OHdic with microsomes (Fig. Sandberg-Cook J. Cain K and Skilleter DN (1987) Preparation and Use of Mitochondria in Toxicological Research in Biochemical Toxicology: A Practical Approach (Snell K and Mullock B eds) pp 217–254. Bellomo G and Orrenius S (1992): Calcium-mediated mechanisms in chemically induced cell death. 7C suggest: addition of N. Saunders JH and Davies DS (1980) Monooxygenase activity of human liver in microsomal fractions of needle biopsy specimens. Xenobiotica 9:601– 610. Chaudri HA. J Biol Chem 265:2399 –2408. In Vitro 20:826 – 832. J Hepatol 11:281. Kao JPY and Tsien RY (1989) Fluorescent indicators for cytosolic calcium based on rhodamine and fluorescein chromophores. Kriemler HP. Schlumpf E. Ryley NG.5-(OH)2dic to 5-OHdic. 1992). Postgrad Med J 62:63– 65. Gores GJ. Gazdar AF. Lagarde S and West AB (1991) Reversible hepatitis associated with diclofenac. Unidad de Hepatologıa Ex´ ´ perimental. Lepper H and Estler CJ (1995) Failure of calcium antagonistic agents to prevent hepatotoxicity induced by diclofenac. Highet RJ and Pohl LR (1993) Immunochemical detection of liver protein adducts of the non-steroidal anti-inflammatory drug diclofenac. It is thus suggestive to speculate that one factor contributing to the idiosyncratic nature of diclofenac hepatotoxicity could be the different expression of the CYPs metabolizing the drug into reactive/toxic metabolites. J Pharmacol Exp Ther 252:442– 447. Clarke D and Barr RJ (1993) Diclofenac induced hepatitis: three cases with features of autoimmune chronic active hepatitis. in addition to the inhibitory effects that diclofenac and its metabolites show on mitochondrial ATP synthesis. Pumford NR. Gomez-Lechon MJ and Castell JV (1996) Metabolism ´ ´ of aceclofenac in humans. DeGraff WG. Castell. submitted for publication). Crawford K and Huang HS (1994) Comparative cytotoxicity of non-steroidal anti-inflammatory drugs in primary cultures of rat hepatocytes.5-(OH)2dic nor 5-OHdic showed much higher toxicity to mitochondria than diclofenac. Ouellette GS. Oxford. Salama A. Biochem Pharmacol 37:368 –370. SVS. Vol. Montoya A. Helfgott SM. Herman B and Lemasters JJ (1990) Toxic injury from mercuric chloride in rat hepatocytes. Venuti A and Barone C (1994) Positivity of patch tests in cutaneous reaction to diclofenac. which is involved in the 5-hydroxylation of diclofenac in the liver (R. Gomez-Lechon MJ. Carmichael J. In Vitro Cell & Dev Biol 26:67–74. Scully LJ. Spain. Biochemical characterization and comparison with in vivo. Life Sci 52:29 –34. Miyamoto G. Jose V. IRL Press. Davila JC. ´ Avda. and recent evidence from our laboratory suggests that this could be due to a different expression of CYP2C19. Kelly PMA. Theobald W and Wagner J (1978) Pharmacokinetics and metabolism of the anti-inflammatory agent voltaren. Jover R. Larrauri A. Trowell JM. Godbillon J. Leemann T.aspetjournals. Stierlin H. Ponsoda X. Gimenez P. Breen EG. Ciccolunghi SN. This is what the data presented in Fig. Gerardin A. Slitzky BE. We are indebted Dr. Lopez P. Despite the fact that ATP is the most relevant biochemical event observed. Faigle JW. I. ´ ´ Castell JV. Sallmann A. Iveson TJ. There is great variability in the expression of this enzyme in humans (Relling et al. Annu Rev Pharmacol Toxicol 32:449 – 470. followed by a sustained decrease in the nucleotide concentration when both metabolites had reached equilibrium (Fig. Moppert J. Chiarelli C.5-(OH)2dic and 5-OHdic to enter a futile cycle resulting in NADPH oxidation by O2. Aoyama T. Henry D and Melville G (1991) Diclofenac hepatitis. Gates JA. Gut 27:1390 –1393. 5B anticipated. Lopez P and Gomez-Lechon MJ (1985) Effects of ´ ´ ´ benorylate and impacina on the metabolism of cultured hepatocytes. Yamada H. Minta A. futile depletion of NADPH by 5-OHdic/N. Faigle JW. Kretz-Rommel A and Boelsterli UA (1993) Diclofenac covalent protein binding is dependent on acyl glucuronide formation and is inversely related to P450mediated acute cell injury in cultured rat hepatocytes. Schmitz G. it is noteworthy that neither N. Isolation and identification of principal metabolites. Xenobiotica 18:1191–1197. myeloperoxidase. Dig Dis Sci 38:744 –751. J Hepatol 10:85– 89. Gottsche B and Mueller-Eckhardt C (1991) Autoantibodies and drug. McNicholl J. Brodie MJ. Luis Martınez in conduct´ ing spectroscopic analysis is acknowledged. Variable formation of 5-OHdic and N. Schubiger BI and Reddrop R (1978) Report on a long Send reprint requests to: Dr. Baba T. Sippel H. Richter WJ. Castell JV. Cellular enzymes able to carry out both types of reaction are known to exist (Yamada et al. Xenobiotica 15:743–749. Hospital Universitario “La Fe”. Ponsoda X. 7A) could be backreduced to generate 5-OHdic in the presence of active microsomes and NADPH (Fig. Drug Metab Dispos 24:834 – 841. Garcovich A. Myers TG.es . Jover R. ´ Schmid K. 7B). Fletcher DR.72 Bort et al. McCabe J and Stevens FM (1986) Fatal hepatitis associated with diclofenac. E-mail: Jose. Stauffert I. 7B). Larrauri A. Alt KO and ¨ Winkler T (1979) Biotransformation of diclofenac sodium (Voltaren) in animals and man. Bort R. 1990). Centro de Investigacion. Yamaoka K. Gomez-Lechon MJ. Looking for other possible links between metabolite formation and cytotoxicity. Schneider W. Acknowledgments Downloaded from jpet. Riess W. Kung W. J Clin Gastroenterol 13:205–210. If both reactions take place simultaneously. Clement B and Kunze T (1992) The reduction of 6-N-hydroxylaminopurine to adenine by xanthine oxidase. Bort. Minna JD and Mitchell JB (1987) Evaluation of a tetrazolium-based semiautomated colorimetric assay: Assessment of chemosensitivity testing. London. Toxicol Appl Pharmacol 120:155–161. Gomez-Lechon MJ. Jover R. Drug Metab Dispos 24:216–220. Smith DA and Jones BC (1992) Speculations on the substrate structure-activity relationship (SSAR) of cytochrome P450 enzymes. Zahid N and Uetrecht JP (1997) Oxidation of diclofenac to reactive intermediates by neutrophils. J Am Med Assoc 264:2660 –2662.5-(OH)2dic to microsomes resulted first in a rapid decrease in NADPH by the reduction of N. Fabra R and Castell JV (1990) Culture of human hepatocytes from small surgical liver biopsies. Academic Press. Donato T. The assistance of Dr. Gonzalez FJ and Meyer UA (1990) Tolbutamide and mephenytoin hydroxylation by human cytochrome P450s in the CYP2C subfamily.org by guest on April 26. Bort R. Montoya A. Dawson TL.5-(OH)2dic oxido-reduction could be at the root of the mechanism of hepatocyte toxicity induced by diclofenac. Allergy 49:57–59. 2011 We thank Carmen Lorenzo and Epifanıa Belenchon for their ex´ ´ pert technical help.5-(OH)2dic tends to accumulate inside the cell. Romano A. Bassan L. J Hepatol 14:408 – 409. Campanar 21. Stierling H. Biochem Pharmacol 46:1967–1974. Kahn GC. References Boobis AR. E-46009 Valencia. Nieminen AL. Purcell P. Degen P. Lepper H and Estler CJ (1992) Toxicity of diclofenac to isolated hepatocytes. Gomez-Lechon MJ and Castell JV (1993) Cocaine hepatotoxicity: ´ ´ two different toxicity mechanisms for phenobarbital-induced and non-induced rat hepatocytes. Scand J Rheumatol Suppl 22:116 –121.or metabolite-dependent antibodies in patients with diclofenac immune haemolysis. Carrasco E. Kriemler HP. Pietrantonio F.. This is not the case for 4 -OHdic. Relling MV. as the data shown in Fig. Gomez-Lechon MJ and Castell JV (1995) Molecular ´ ´ mechanism of diclofenac hepatotoxicity: association of cell injury with oxidative metabolism and decrease in ATP levels. Biochem Pharmacol 44:2089–2098. and its formation by cells does not apparently correlate with cytotoxicity. in In vitro Methods in Pharmaceutical Research (Castell JV and Gomez-Lechon MJ eds) pp 375– 410. Clement and Kunze. Ponsoda X and Bort R (1997) In vitro investigation of the ´ ´ molecular mechanisms of hepatotoxicity. this would allow N. Consequently. Nahir AM and Scharf Y (1986) Diclofenac-induced hepatotoxicity.hydroxylation in human liver. Schmitz G. Biochem Pharmacol 44:1501–1509. Ponsoda X. Trul´ ´ ´ ´ lenque R. The feasibility of this futile cycle is further supported by the fact that N. 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Zakim D and Nestler J (1990) Diclofenac-associated hepatotoxicity.