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Vol. 288, Issue 1, 65-72, January 1999
Unidad de Hepatología Experimental, Centro de Investigación, Hospital Universitario "La Fe", Valencia, Spain (R.J., M.J.G.-L., R.B., J.V.C.); Departamento de Bioquímica, Facultad de Medicina, Universidad de Valencia, Valencia, Spain (R.B., R.J., J.V.C.); and Departamento de Parasitología y Biología Celular, Facultad de Ciencias Biológicas, Universidad de Valencia, Valencia, Spain (X.P.)
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Abstract |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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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 primary 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.
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Introduction |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Diclofenac
is a frequently prescribed nonsteroidal anti-inflammatory 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., 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, which could be related to drug toxicity, has been reported
(Miyamoto et al., 1997).
Diclofenac undergoes hepatic metabolism both in rat and human
hepatocytes, and the main biotransformation reactions (aromatic hydroxylations and conjugations at various sites of the molecule) are
common to several animal species (Riess et al., 1978; Stierling et al.,
1979). In human liver microsomes, the major oxidative metabolic pathway
is the formation of 4'-hydroxydiclofenac (4'-OHdic) by
CYP2C9 (Smith and Jones, 1992; Leemann et al., 1993). Formation of 5-hydroxydiclofenac (5-OHdic), 3'-hydroxydiclofenac, 4',5-dihydroxydiclofenac, and 3'-hydroxy-4'-methoxydiclofenac has also
been reported in humans, but to a much lesser extent (Riess et al.,
1978; Faigle et al., 1988). In the rat, 4'-OHdic together
with 5-OHdic are the major urine metabolites (Stierling et
al., 1979).
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. 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, in particular with the formation of 5-OHdic and N,5-dihydroxydiclofenac (N,5-(OH)2dic). Both metabolites can easily interchange by oxidation and reduction and both cause a continuous consumption of NADPH. Thus, in addition to the effects of diclofenac and its major metabolites on mitochondrial function, the existence of a futile red-ox cycle could also contribute to the mechanism of toxicity.
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Materials and Methods |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Materials.
Diclofenac sodium salt, flurbiprofen, proadifen,
-naphthoflavone, phenytoin (5,5-diphenyl-hydantoin), maleic acid
diethyl ester (DEM),
L-buthionine-[S,R]-sulfoximine
(BSO), buthylated hydroxytoluene (BHT), deferoxamine mesylate,
nifedipine, (±)-verapamil, rhodamine 123, and
o-phthaldialdehyde were obtained from Sigma Chemical Co.
(St. Louis, MO). Malondialdehyde (MDA) bis(dimethylacetal) was obtained
from Merck (Darmstadt, Germany). Fluo-3-AM and pluronic F-127
were obtained from Molecular Probes, Inc. (Eugene, OR). 
-Glucuronidase/arylsulfatase, collagenase, the Glucose GOD-Perid test, and the ATP bioluminescence CLS test were obtained from Boehringer Mannheim (Mannheim, Germany). Calf serum was obtained from
GIBCO (Paisley, UK), and culture media were obtained from Flow Labs.
(Irvine, CA). Diclofenac metabolites (4'-OHdic, 5-OHdic, and
4',5-OHdic) were synthesized as described (Bort et al.,
1996). All other reagents used in this study were of analytical grade.
Cell Cultures. MDCK, FaO, and HepG2 cells were cultured in Dulbecco's minimal essential medium supplemented with 10% of fetal calf serum and containing 50 µg of streptomycin/ml and 50 mU of penicillin/ml. Cells were routinely seeded in 3.5-cm plates at a density of 14 × 103 cells in 0.1 ml medium per well and used 24 h later (75% monolayer confluence).
Rat hepatocytes were obtained from 200- to 300-g Sprague-Dawley male rats by perfusion of the liver with collagenase as described in detail elsewhere (Gómez-Lechón et al., 1984). Human hepatocytes were obtained from small liver biopsies from patients undergoing cholecystectomy, after informed consent. The patients had not received
any medication for at least a week before surgery. Cell suspensions
were obtained as described in detail elsewhere
(Gómez-Lechón et al., 1990). Hepatocytes were seeded on
fibronectin-coated culture plates (3.5 g/cm2) at
a density of 80 × 103
cells/cm2. Unattached cells were removed by
changing the medium 1 h after seeding.
Evaluation of Toxicity of Diclofenac to Hepatocytes.
Increasing concentrations of the drug in phosphate-buffered saline
(PBS) were added to cultures after medium renewal. After a 24-h
incubation, microtiter plates were washed twice with 50 µl of
PBS/well, and cytotoxicity was assessed either by measuring the loss of
intracellular lactate dehydrogenase (Ponsoda et al., 1991) or by the
3-[4,5-dimethylthiazol-2-yl]-3,5-diphenylformazan (MTT) test
(Carmichael et al., 1987).
). The gluconeogenic rate was determined from the
rate of glucose production by hepatocytes.
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., 1985).
Glutathione (GSH) was measured by a fluorimetric reaction with
o-phthaldialdehyde adapted to microscale measurements (Jover et al., 1992). Briefly, cells were detached and homogenized by ultrasound. The resulting homogenates were deproteinized and
centrifuged in the same 96-well plate. Aliquots of the supernatant were
transferred to microtiter plates and allowed to react with
o-phthaldialdehyde. Fluorescence was measured in a multiwell
plate fluorimeter (excitation filter: 355 ± 35 nm; emission
filter: 460 ± 25 nm). Known amounts of GSH (0-5000 pmol/well)
were used as reference standards.
Lipid peroxidation was determined by measuring the generation of MDA in
the culture media by the fluorimetric reaction with thiobarbituric acid
(Castell et al., 1997). After incubation for 60 min in a boiling bath,
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. MDA bis(dimethylacetal) diluted in culture medium was used as standard. Results were referred to intracellular protein content.
To monitor intracellular calcium
([Ca++]i), cells
previously incubated with diclofenac were loaded with 5 µM of
Fluo-3-AM 0.075% (w/v) pluronic F-127 in culture medium for 30 min at
37°C. The cells were washed three times with HEPES-buffered
Krebs-Henseleit solution (10 mM HEPES, pH = 7.4, 25°C)
containing 1% bovine serum albumin, and immediately thereafter
fluorescence was measured with a multiwell plate fluorimeter (Cytofluor
2350, Millipore; excitation, 485 ± 22 nm; emission, 530 ± 30 nm) (Jover et al., 1993). Fluorescence readings were calibrated to
calculate [Ca++]i. First
the hydrolysed, cytosolic Fluo-3 was released out of cells into medium
containing 2 mM CaCl2 by treating cells with 30 µM digitonin for 4 min, and fluorescence
(Fmax) was recorded. Next, 10 mM EGTA was
added to quench Ca++, and fluorescence
(Fmin) was again recorded.
[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., 1989), according to the following equation:
[Ca++]i = Kd × ((F-Fmin)/(Fmax
F)).
ATP synthesis was determined in cultured hepatocytes incubated in
HEPES-buffered saline medium with glucose as the only energetic substrate and 500 µM diclofenac. At regular time intervals, cells were washed with PBS and homogenized in 1 ml of 3%
HClO4 at 0°C. Samples were centrifuged (5 min × 9000g) and the supernatant kept at 4°C until
analysis. The ATP concentration of the diluted samples (1:4900) was
measured using the luciferin/luciferase assay according to the
manufacturer's instructions. Bioluminescence was quantified in a Lumat
LB luminescence photometer (Berthold GmbH, Wildbad, Germany).
Mitochondrial membrane potential was assayed in the same conditions as
ATP, but rhodamine 123 (final concentration 1 µM) was also added.
After incubations at different times, medium was removed and
monolayers were processed to measure fluorescence in the mitochondrial compartment as described elsewhere (Nieminen et al., 1990). The fluorescence of samples was measured (excitation, 505 nm; emission, 523 nm) in a Hitachi F-2000 fluorescence spectrophotometer (Hatachi Scientific Instruments, Mountain View, CA) .
Isolation of Mitochondria from Rat Liver.
Sprague-Dawley
rats were euthanized and their livers immediately removed, washed, and
homogenized in 4 ml/g ice-cold homogenization buffer (0.25 M sucrose, 5 mM Tris, 1 mM EDTA, pH 7.4). Mitochondria were isolated by
centrifugation in sucrose as described elsewhere (Cain and Skilleter,
1987) and finally resuspended in 0.7 ml of the assay solution (0.25 M
sucrose, 10 mM Tris, 1 mM EDTA, 5 mM KH2PO4, 2 mM MgCl2, pH 7.4) per gram of liver.
ATP Measurement in Isolated Mitochondria. A mitochondrial suspension (final concentration 2 mg/ml protein) containing 2.5 mM ADP and variable concentrations of diclofenac or its metabolites was preincubated at 30°C for 5 min. Then substrates were added (2.5 mM glutamate, 2.5 mM malate, final concentration) and after 5 min, 100 µl was taken and dropped on 900 µl of boiling buffer (100 mM Tris, 4 mM EDTA, pH 7.75). ATP was measured as described above.
Metabolism of Diclofenac.
Biotransformation of the drug was
studied both in vitro and in vivo. For in vitro studies, diclofenac was
added to hepatocyte cultures and incubated for 20 h. Aliquots of
culture medium were enzymatically deconjugated (50 mU of
-glucuronidase/ml, 30 mU of arylsulfatase/ml; acetate buffer 0.1 M,
pH 4.5) for 4 h at 37°C. The reaction was stopped by adding
acetonitrile to the samples (1:1, v/v). The resulting precipitate was
removed by centrifugation (10 min, 9000 rpm). The supernatant was
diluted with 100 mM phosphate buffer (pH 7.4) to reach 25% (v/v)
acetonitrile. Flurbiprofen (2 µl of a 4.5-mM solution in PBS) was
added to samples as an internal standard.
). Samples (20 µl) were injected into a
200 × 4.6-mm C-18 reverse-phase column (Spherisorb ODS 5 µm,
Phase Separations Ltd., Queensferry, UK) furnished with a
precolumn (ODS 5 µm; 50 × 4.6 mm). The mobile phase (75%
triethanolamine 0.02% in 100 mM phosphate buffer pH 7.4, 25%
acetonitrile) was delivered at 1 ml/min. The column effluent was
monitored at 282 nm. Diclofenac eluted at 12.2 min and flurbiprofen at
7.4 min.
Identification of Diclofenac Metabolites. Culture supernatants were collected, deconjugated, and extracted with ethyl acetate as described above. The organic extracts were dried under vacuum, resuspended in methanol, and analyzed by HPLC-mass spectrometry (MS) (Waters Alliance equipped with a therma beam detector; Waters Scientific, Milford, MA). Samples (25 µl) were injected into a 150 × 2.1-mm C-18 reversed-phase column (Simmetry; Waters). A new HPLC method was developed to adjust it to this specific detector. The mobile phase was changed to 63% 20 mM ammonium acetate buffer (pH 6.5)/37% methanol and it was delivered at 0.3 ml/min. The column effluent was also monitored at 282 nm using a photodiode detector (M996; Waters). Diclofenac eluted at 29.8 min. The UV, mass spectra, and 1H NMR (d6-acetone, Varian Gemini 300 MHz spectrometer, Palo Alto, CA) of the main metabolites were obtained and compared with chemically synthesized standards.
4'-OHdic and 5-OHdic were prepared as described in detail previously (Bort et al., 1996). 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. Upon completion
of the reaction (5 h at room temperature), the solution was brought to
dryness, dissolved in ethyl acetate, filtered to remove the
dicyclohexyl urea formed, and crystallized by the addition of petroleum
ether. The UV-(4'-OHdic-lactam: 
max
239 and 5-OHdic-lactam: max 250 nm), 1H NMR- (4'-OHdic-lactam: 
3.7, s 2H, Ar-CH2-CO-, 7.4-6.8, m,
6H, aromatic; 5-OHdic-lactam: 3.9, s 2H,
Ar-CH2-CO-, 7.4-6.8, m, 6H, aromatic)
and mass spectra (m/z 293, molecular peak for both
derivatives) were consistent with the assigned chemical structure of
the two cyclic diclofenac derivatives.
Microsome Incubations.
Rat microsomes were obtained by
homogenization of liver samples in 0.25 M potassium phosphate buffer,
pH 7.25, containing 1 mM EDTA and 0.15 M KCl, followed by differential
ultracentrifugation (Boobis et al., 1980). The microsomal fraction was
stored in the same buffer containing 30% v/v glycerol. Assays were
performed in a buffer containing 75 mM Tris-hydrochloride buffer, pH
7.4, 3 mM magnesium chloride, and 500 µg of microsomal protein.
Samples were preincubated for 5 min at 37°C, and the reaction was
started by the addition of NADPH (final concentration 1.2 mM). The
reaction was stopped by the addition of 1 ml of cold acetonitrile.
Controls were incubated either without NADPH or without microsomes.
Samples were analyzed by HPLC as described above.
Statistical Analysis. Each experiment was done in at least three different cultures. Each determination was done in four plates (eight wells for cytotoxicity experiment) from each culture, and the results shown are the mean value ± S.E.M. To estimate IC10 and IC50 values (concentrations that produce a 10% and 50% inhibitory effect, respectively), the typical sigmoid dose-effect curves were linearized using the LOGIT transformation, and the IC values were interpolated mathematically. The statistical significance of the experimental data was analyzed by the Student's t test.
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Results |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Cytotoxicity of Diclofenac on Hepatic and Nonhepatic Cells. Diclofenac showed a characteristic concentration-dependent cytotoxicity when added to cultured human or rat hepatocytes, as assessed by the MTT test (Fig. 1). Similar results were obtained (data not shown) when the leakage of intracellular lactate dehydrogenase was determined as a parameter for cell viability. The estimated IC50 values for rat and human hepatocytes (concentration causing 50% cell death) were closely related: 392 ± 34 µM (n = 16) and 331 ± 7 µM (n = 6), respectively. To elucidate whether these effects were hepatocyte-specific, cytotoxicity was also evaluated in human and rat hepatomas, as well as in the nonhepatic cell line MDCK. Figure 1 shows that hepatoma cells were less sensitive to diclofenac than primary cultures of hepatocytes (HepG2 763 ± 61 µM, n = 4 and FaO 682 ± 64 µM, n = 3,), with concentration-toxicity curves shifting to higher concentrations of the drug (i.e., higher IC50 values) but more sensitive than the nonhepatocyte cell line MDCK (908 ± 56 µM, n = 2).
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1·mg1,
respectively), whereas cell lines were unable to metabolize it to a
measurable extent (data not shown). Incubation of rat hepatocytes with
a noncytotoxic concentration (10 µM) of proadifen, a broad CYP
inhibitor, significantly reduced the toxicity of the drug
(IC50: 489 ± 32, p < .005;
Fig. 1). Ketoconazole (25 µM), which inhibits the 2C and 3A
subfamilies, also reduced diclofenac toxicity significantly
(IC50: 514 ± 38, p < .005), whereas -naphthoflavone-specific CYP1A inhibitor did not
apparently modify cytotoxicity (IC50: 392 ± 81, N.S.). Moreover, we found evidence showing a good correlation between cytotoxicity (IC50) and the extent of
diclofenac metabolization (r = 0.95, p < .001; Fig. 2).
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Major Biochemical Events in Course of Diclofenac Toxicity. Analysis of toxicologically relevant biochemical parameters in hepatocytes exposed to diclofenac (GSH levels, lipid peroxidation, changes in [Ca++]i, and ATP) revealed that upon incubation of cells with toxic concentrations of the drug, no significant accumulation of MDA was found in the medium after several hours. In control experiments, addition of 1 mM t-butyl hydroperoxide resulted in an immediate, dramatic increase in thiobarbituric-reactive substances (Fig. 3A). In agreement with these findings, we did not observe a decrease in toxicity (increased IC50) when cells were incubated in the presence of the antioxidant BHT (50 µM) or 10 mM deferoxamine (data not shown.).
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) was moderately effective
in decreasing diclofenac cytotoxicity (IC50 = 415 ± 92 and 507 ± 30 µM, respectively).
Diclofenac, however, caused an important, fast depletion of ATP in
hepatocytes kept in glucose as the only energy source. ATP dropped to
10% of initial levels after 120 min of exposure to the drug. This
rapid ATP depletion preceded cell death, which in these experiments
began to be noticeable 120 min after addition of the drug (Fig. 3D). In
agreement with this finding, increasing diclofenac in culture medium
resulted in a concentration-dependent inhibition of gluconeogenesis and
albumin synthesis, two specific hepatic functions that require
substantial amounts of ATP. This inhibition was noticeable at
subcytotoxic concentrations of diclofenac. The
IC50 were 110 ± 12 and 108 ± 9 µM, respectively.
Metabolism of Diclofenac. Analysis by HPLC of culture media of hepatocytes incubated with diclofenac allowed the identification of several metabolites. A peak with a retention time of 3.8 min was assigned to 4'-OHdic, while the peak with a retention time of 5.1 min was identified as 5-OHdic, by comparison of the UV-, mass-, and 1H-NMR spectra of chemically synthesized metabolites. Other minor peaks at 10.4 and 13.5 min were identified as lactams of 4'-OHdic and 5-OHdic.
A minor peak (retention time 2.9 min) was present with variable intensity in the different culture supernatants assayed. This metabolite was found in culture media mainly in its conjugated form (>95%). The compound was analyzed by HPLC-MS and 1H NMR, and the most relevant spectral features are summarized in Table 2 and Fig. 4. The molecular ion peak (M+) was at m/z 327, and the presence of isotopic peaks at m/z 329 and 331 confirmed the presence of two Cl. When compared with the mass spectra of diclofenac, the M+ peak of this metabolite suggested the existence of two hydroxy substituents. The 1H NMR spectra of the purified metabolite (Table 1) showed the same number of aromatic H (7.4-6.4 m, 6H) and ethylenic H ( 3.68 s, 2H,
Ar-CH2-COOH) as 5-OHdic, which
excludes an aromatic hydroxylation. Interestingly, the displacement to
a high field of the signal of H(3), as compared with that
recorded for 5-OHdic, points to the proximal N as the site
of oxidation. The compound showed the features of an acid and a UV
maximum displacement to 268 nm (versus 282 nm, 5-OHdic).
Based on these data, we assigned the
N,5-(OH)2dic structure to this
metabolite (Fig. 4).
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Effects of Diclofenac and Its Metabolites on Hepatocyte
Mitochondrial Function.
According to the results shown in Fig. 4D,
the decrease in ATP was an early event in diclofenac toxicity, which
could be attributable either to the drug or to any of its metabolites.
In view of the fact that ATP depletion was paralleled by a decrease in
mitochondrial membrane potential, we investigated the role of
diclofenac and each metabolite on mitochondria by monitoring the
synthesis of ATP. As shown in Fig. 6, the
mitochondrial ATP synthesis was effectively impaired by 30 µM
diclofenac (concentration found in the portal blood of rats given the
drug orally; Tabata et al., 1996), as well as by its hydroxylated
metabolites.
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Interconversion between 5-OHdic and N,5-(OH)2dic by Liver Microsomes. We examined whether 5-OHdic and N,5-(OH)2dic, whose formation correlated with the toxicity of diclofenac, could also indirectly alter the energetic status of cells. Incubation of 5-OHdic with rat liver microsomes in aerobic conditions resulted in the formation of N,5-(OH)2dic (Fig. 7A), whereas the incubation of N,5-(OH)2dic in presence of microsomes and NADPH yielded 5-OHdic (Fig. 7B). This reduction did not take place in the absence of either NADPH or microsomes. Most interestingly, when the NADPH content of the incubation mixture was monitored, a biphasic behavior was observed: a rapid NADPH decrease that coincided with the reduction of most N,5-(OH)2dic to 5-OHdic, followed by a sustained NADPH consumption once the equilibrium between the two metabolites was reached. Under the same experimental conditions, incubation of 4'-OHdic did not result in either metabolism or in NADPH consumption (Fig. 7C).
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Discussion |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Diclofenac is an anti-inflammatory drug for which a certain number
of severe adverse hepatic reactions have been reported (Ciccolunghi et
al., 1978; Helfgott et al., 1990; Iveson et al., 1990; Sallie, 1990;
Ouellette et al., 1991; Purcell et al., 1991; Scully et al., 1993).
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), rather than a drug-allergy
mechanism (Helfgott et al., 1990; Iveson et al., 1990; Sallie, 1990;
Scully et al., 1993). In the present study, we investigated possible
linkages between drug metabolism and cell injury to better understand
the mechanisms of hepatotoxicity.
Among the several biochemical parameters examined, ATP was the
earliest affected by the cytotoxic concentrations in short-term experiments (Fig. 3D). Parallel to this, a decrease in mitochondrial membrane potential was observed. These results, together with the
previously reported protective effect of fructose on diclofenac toxicity (Ponsoda et al., 1995), lead us to consider a mitochondrial bioenergetic dysfunction to be one of the possible events causing diclofenac toxicity. The fact that subcytotoxic concentrations of
diclofenac (100 µM), close to the portal concentration in the rat (30 µM; Tabata et al., 1996), caused a 50% inhibition of two characteristic ATP-consuming hepatocyte functions, namely
gluconeogenesis and albumin synthesis, would favor this hypothesis.
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.
On the other hand, the fact that no significant changes in oxidative stress parameters (i.e., GSH and MDA levels) were observed, and the lack of a clear effect on the cytotoxicity of radical scavengers (BHT) and inhibitors of the formation of active oxygen species (deferoxamine), or compounds depleting intracellular GSH (BSO, DEM), did not support the hypothesis that this mechanism is a key event in diclofenac toxicity.
[Ca++]i levels did not
increase during the early stages of cell exposure to diclofenac (Fig.
3C), which is in agreement with previous reports (Schmitz et al.,
1995). Only after a longer incubation of cells with diclofenac did
[Ca++]i increase
moderately, parallel to a decrease in cell viability. We believe this
event may be simply concomitant with cell death, and the slight
decrease in cell toxicity (not significant) that we observed agrees
with this interpretation. A decrease in ATP impairs plasma membrane and
endoplasmic reticulum ATP-dependent calcium pumps and leads to a
sustained rise in intracellular free calcium (Nicotera et al., 1992).
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.
The experiments also revealed a possible link between drug
metabolism and toxicity to hepatocytes. Several facts support this assessment. First of all, the ability of cells to metabolize diclofenac (human 
rat [tmt] FaO > HepG2 MDCK) was
inversely related to the IC50 values, and
inhibition of drug metabolism resulted in a decrease in toxicity (Fig.
1). Second, there was a direct correlation between cytotoxicity and the
extent of diclofenac metabolization by hepatocytes (Fig. 2). Third,
analysis of culture media of hepatocytes revealed more precisely that
cytotoxicity to hepatocytes was related to the formation of
5-OHdic and
N,5-(OH)2dic (Fig. 5B).
The chemical structure of this new metabolite was identified by the
combined use of HPLC-MS, 1H NMR (Table 1), UV
spectra, and chemical data. In a recent article, Miyamoto et al. (1997)
reported the formation of an iminoquinone by HOCl oxidation of
diclofenac in activated neutrophils. The authors also reported that
hepatic microsomes could oxidize 5-OHdic to the same
iminoquinone, which was trapped with GSH. This compound shows strong
chemical similarities with the one reported in this study: dehydration
of N,5-(OH)2dic would render
the hypothesized iminoquinone. In fact, a compound showing similar
fragments by HPLC-MS (electron impact fragmentation) than those
obtained by Miyamoto et al. (1997) using MS-MS, was detected in
preparations of N,5-(OH)2dic
and in culture media of cells [m/z: 309 (M+), 267, 229, 201, 195, and 166].
The presence of N,5-(OH)2dic in culture medium, as well as in urine of rats given diclofenac, excluded an artifactual, noncellular formation of the metabolite (Table 2). 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. Indeed, we have found that the concentration of this metabolite inside cells is approximately 16 times grater than in culture medium.
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Despite the fact that ATP is the most relevant biochemical event
observed, it is noteworthy that neither
N,5-(OH)2dic nor
5-OHdic showed much higher toxicity to mitochondria than
diclofenac, as the data shown in Fig. 5B anticipated. Looking for other
possible links between metabolite formation and cytotoxicity, we
observed that the hydroxylamine generated by oxidation of
5-OHdic with microsomes (Fig. 7A) could be backreduced
to generate 5-OHdic in the presence of active microsomes
and NADPH (Fig. 7B). Cellular enzymes able to carry out both types of
reaction are known to exist (Yamada et al., 1988; Clement and Kunze,
1992). If both reactions take place simultaneously, this would allow
N,5-(OH)2dic and
5-OHdic to enter a futile cycle resulting in NADPH oxidation
by O2. This is what the data presented in Fig. 7C
suggest: addition of
N,5-(OH)2dic to microsomes
resulted first in a rapid decrease in NADPH by the reduction of
N,5-(OH)2dic to
5-OHdic, followed by a sustained decrease in the nucleotide concentration when both metabolites had reached equilibrium (Fig. 7B).
This is not the case for 4'-OHdic, a metabolite that is not
further metabolized by microsomes, and its formation by cells does not
apparently correlate with cytotoxicity. The feasibility of this futile
cycle is further supported by the fact that
N,5-(OH)2dic tends to
accumulate inside the cell. Consequently, in addition to the inhibitory
effects that diclofenac and its metabolites show on mitochondrial ATP
synthesis, futile depletion of NADPH by
5-OHdic/N,5-(OH)2dic
oxido-reduction could be at the root of the mechanism of hepatocyte
toxicity induced by diclofenac.
Variable formation of 5-OHdic and
N,5-(OH)2dic is observed in
cultures of hepatocytes from different human donors when incubated with
diclofenac, and recent evidence from our laboratory suggests that this
could be due to a different expression of CYP2C19, which is involved in
the 5-hydroxylation of diclofenac in the liver (R. Bort, submitted for
publication). There is great variability in the expression of this
enzyme in humans (Relling et al., 1990). 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.
| |
Acknowledgments |
|---|
We thank Carmen Lorenzo and Epifanía Belenchón for their expert technical help. The assistance of Dr. Luis Martínez in conducting spectroscopic analysis is acknowledged. We are indebted Dr. Miguel Angel Miranda for his skillful advice in mass spectrometry and 1H NMR data interpretation.
| |
Footnotes |
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Accepted for publication July 21, 1998.
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 Consellería de Educación y Ciencia, Generalitat Valenciana.
Send reprint requests to: Dr. José V. Castell, Unidad de Hepatología Experimental. Centro de Investigación, Hospital Universitario "La Fe", SVS. Avda. Campanar 21, E-46009 Valencia, Spain. E-mail: Jose.Castell{at}uv.es
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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.
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References |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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