Critical role of free cytosolic calcium, but not uncoupling, in mitochondrial permeability transition and cell death induced by diclofenac oxidative metabolites in immortalized human hepatocytes

Toxicology and Applied Pharmacology 217 (2006) 322 – 331 www.elsevier.com/locate/ytaap

Critical role of free cytosolic calcium, but not uncoupling, in mitochondrial permeability transition and cell death induced by diclofenac oxidative metabolites in immortalized human hepatocytes Miao Shan Lim a , Priscilla L.K. Lim a , Rashi Gupta a , Urs A. Boelsterli a,b,⁎ a

Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117597, Singapore b Department of Pharmacy, Faculty of Science, National University of Singapore, Singapore 117597, Singapore Received 15 August 2006; revised 26 September 2006; accepted 28 September 2006 Available online 5 October 2006

Abstract Diclofenac is a widely used nonsteroidal anti-inflammatory drug that has been associated with rare but serious hepatotoxicity. Experimental evidence indicates that diclofenac targets mitochondria and induces the permeability transition (mPT) which leads to apoptotic cell death in hepatocytes. While the downstream effector mechanisms have been well characterized, the more proximal pathways leading to the mPT are not known. The purpose of this study was to explore the role of free cytosolic calcium (Ca2+c) in diclofenac-induced cell injury in immortalized human hepatocytes. We show that exposure to diclofenac caused time- and concentration-dependent cell injury, which was prevented by the specific mPT inhibitor cyclosporin A (CsA, 5 μM). At 8 h, diclofenac caused increases in [Ca2+]c (Fluo-4 fluorescence), which was unaffected by CsA. Combined exposure to diclofenac/BAPTA (Ca2+ chelator) inhibited cell injury, indicating that Ca2+ plays a critical role in precipitating mPT. Diclofenac decreased the mitochondrial membrane potential, ΔΨm (JC-1 fluorescence), even in the presence of CsA or BAPTA, indicating that mitochondrial depolarization was not a consequence of the mPT or elevated [Ca2+]c. The CYP2C9 inhibitor sulphaphenazole (10 μM) protected from diclofenac-induced cell injury and prevented increases in [Ca2+]c, while it had no effect on the dissipation of the ΔΨm. Finally, diclofenac exposure greatly increased the mitochondria-selective superoxide levels secondary to the increases in [Ca2+]c. In conclusion, these data demonstrate that diclofenac has direct depolarizing effects on mitochondria which does not lead to cell injury, while CYP2C9-mediated bioactivation causes increases in [Ca2+]c, triggering the mPT and precipitating cell death. © 2006 Elsevier Inc. All rights reserved. Keywords: Calcium; Diclofenac; Hepatotoxicity; Immortalized human hepatocytes; Mitochondria; Permeability transition; Reactive oxygen species; Uncoupling

Introduction Diclofenac is a widely used nonsteroidal anti-inflammatory drug. It is generally safe but, in view of the large consumer population worldwide, the drug has been associated with significant adverse effects including gastrointestinal bleeding Abbreviations: [Ca2+]c, free cytosolic calcium; CsA, cyclosporin A; ΔΨm, mitochondrial inner transmembrane potential; JC-1, 5,5′,6,6′-tetrachloro1,1′,3,3′-tetraethylbenzimidazol-carbocyanine iodide; mPT, membrane permeability transition; ox-phos, oxidative phosphorylation. ⁎ Corresponding author. National University of Singapore, Dept Pharmacology, Molecular Toxicology Lab, MD2, 18 Medical Drive, Singapore 117597, Singapore. Fax: +65 6873 7690. E-mail address: [email protected] (U.A. Boelsterli). 0041-008X/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.taap.2006.09.012

and ulceration, but also small increases in plasma alanine aminotransferase (ALT) activity, suggesting mild hepatic injury. In rare cases, diclofenac causes more severe idiosyncratic hepatotoxicity (Banks et al., 1995; Boelsterli et al., 1995; Boelsterli, 2003). The mechanisms underlying diclofenac liver liability have not been established as the susceptibility factors determining the human health risk are currently unknown. However, a number of studies have addressed the hazard of diclofenac in cellular models. For example, reactive metabolites have been invoked to account for some of these effects (Kretz-Rommel and Boelsterli, 1993; Tang, 2003); in fact, diclofenac can be glucuronidated to a protein-reactive acyl glucuronide that can reach high concentrations in the biliary tract due to active uphill secretion from the hepatocytes into the

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bile canaliculi (Kretz-Rommel and Boelsterli, 1994; Boelsterli, 2002). In addition, diclofenac has also been shown to be oxidatively metabolized by CYP2C9/19 to mono- or dihydroxylated derivatives, and these ring-hydroxylated metabolites have been suggested to be involved in producing oxidant stress and glutathione adducts due to their ability to form benzoquinone imines with the amino group in the para position (Shen et al., 1999; Tang et al., 1999a; Poon et al., 2001). However, the role of these metabolites has primarily been studied in rat hepatocytes, and their significance in diclofenac-associated liver injury in humans has remained unknown. Apart from these possible mechanisms that involve reactive intermediates, diclofenac has also been demonstrated to target mitochondria and induce mitochondrial dysfunction (Masubuchi et al., 1998; Gomez-Lechon et al., 2003b; Inoue et al., 2004). Specifically, in isolated rat or mouse liver mitochondria, diclofenac readily uncouples oxidative phosphorylation (ox-phos), leading to a collapse of the mitochondrial inner transmembrane potential (ΔΨm). Furthermore, diclofenac can induce the mitochondrial permeability transition (mPT) in isolated mitochondria (Masubuchi et al., 2002; Gomez-Lechon et al., 2003a), which may lead to the release of proapoptotic factors into the cytosol and nucleus and activate the cell death pathways. Indeed, in-depth studies have characterized the diclofenac-mediated activation of caspase cascades in hepatocytes and have shown that caspase-8 and -9 are apical caspases that activate executioner caspase-3 and lead to apoptotic cell death (Gomez-Lechon et al., 2003b). While these distal pathways are well known, relatively little is known about the more proximal mechanisms of diclofenac-induced cell injury. For example, the mPT can be triggered by a number of stress factors. Among these factors are not only reactive oxygen species (ROS) and increases in Ca2+, but also thiol-reactive metabolites that can oxidize critical sulfhydryl groups in the protein components that are part of the mPT pore (Petronilli et al., 1994; He and Lemasters, 2002). In addition, the role of diclofenac metabolic activation and their relationship to cell injury has not been adequately addressed. Since most of the studies on the induction of apoptosis were performed in isolated mitochondria or cultured rat hepatocytes, it is important to consider the species-specific bioactivation pathways and to explore the upstream mechanisms in human hepatocytes. The purpose of this study was therefore to examine the role of [Ca2+]c and oxidant stress in diclofenac-induced mitochondrial toxicity in an immortalized human hepatocellular cell line (HC-04) (Sattabongkot et al., 2006). We chose this metabolism-competent cell line to obtain a consistent experimental background, as primary human hepatocytes exhibit great variation in CYP expression. The second aim of the study was to explore whether oxidative metabolites of diclofenac were involved. While we found that diclofenac readily causes uncoupling, it is the oxidative metabolites that cause increased [Ca2+]c, which is a critical factor for the initiation of the mPT and cell death in these immortalized human hepatocytes.

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Methods Chemicals. All chemicals were purchased from Sigma (St. Louis, MO) unless otherwise stated. Culture of human immortalized HC-04 cells. HC-04 cells (Siam Life Science Ltd, Bangkok, Thailand) were grown in Hepatocyte Basal Medium (HBM) and supplemented with Hepatocyte Culture Medium Bulletkit (Cambrex, Baltimore, MD), supplemented with 10% fetal bovine serum (Hyclone, Logan, UT). Primaria flasks (Falcon) were coated with 0.01 mg/ml each of fibronectin and BSA as well as 0.03 mg/ml collagen in HBM for 2 h at 37 °C. The cells were maintained at 37 °C in a humidified incubator with 95% atmospheric air and 5% CO2. The medium was replaced two times a week, and cells were passaged every 4–5 days at a 1:3 ratio. The cells were plated and grown to subconfluency for 24 h before exposure to drugs. For fluorescence assays that involved the use of a microplate reader, the cells were seeded in black 96-well plates with μ-clear bottom (Greiner Bio-One, Longwood, FL) at a density of 30 × 104/ml. Exposure to drugs in vitro. All drugs were either directly added to the culture medium or dissolved in DMSO and then added to the culture medium (final DMSO concentration 0.1% v/v). This low concentration of DMSO has no apparent effects on CYP2C9 activity in human hepatocytes (Easterbrook et al., 2001). Exposure to drugs was done in serum-free culture medium to avoid differential binding to plasma proteins. Measurement of cell injury. Lactate dehydrogenase (LDH) activity was determined spectrofluorometrically with a test kit (Promega, Madison, WI). In the presence of LDH, resazurin is converted to resorufin which was quantified in a microplate reader (excitation 560 nm, emission 590 nm). Enzyme activity in the medium was sampled at the indicated time points and expressed as percentage of total intracellular and extracellular LDH activity at each of the respective time points. Alternatively, to detect cell injury, we used Sytox Green (Cambrex), a high affinity nucleic acid stain that easily penetrates cells with a compromised plasma membrane but not that of live cells. Drug-exposed cells were stained with 1 μM Sytox Green for 15 min at 37 °C. Next, the cells were washed once with PBS and maintained in HBSS supplemented with 20 mM HEPES. Lethally injured cells were detected by laser scanning confocal microscopy. To detect apoptotic features of hepatocellular nuclei (chromatin condensation), drug-exposed cells were fixed in methanol for 5 min, air-dried, and rinsed twice in PBS before being stained with Acridine Orange (1:1000 in PBS) for 15 min in the dark (Schoemaker et al., 2004). Fluorescent nuclei were evaluated by fluorescence microscopy. Determination of mitochondrial superoxide production. To detect increased ROS production in mitochondria, we used the novel cell-permeable fluorogenic probe, MitoSOX Red (Invitrogen), which consists of dihydroethidine coupled to triphenylphosphonium cation via a hexyl linker. Being a lipophilic cation, MitoSOX Red is selectively targeted to mitochondria where it specifically reacts with superoxide anion. Diclofenac-preexposed cells were loaded with MitoSOX Red (5 μM) for 10 min at 37 °C, washed with PBS, and the fluorescence was determined at 510 nm (excitation) and 580 nm (emission). Rotenone (50 μM) was used as a positive control. Measurement of mitochondrial membrane potential. JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazol carbocyanine iodide, Molecular Probes, Eugene, OR) is a cationic dye that selectively accumulates within the mitochondrial matrix. In the presence of a highly negative transmembrane potential, JC-1 forms red fluorescent J-aggregates (emission 590 nm), while under depolarized conditions it exists as green monomers (emission 530). These characteristics make JC-1 a more sensitive probe for mitochondrial membrane potential measurements than other dyes because fluorescence is not dependent on mitochondrial size, shape, or density. Cell cultures were incubated with 2 μM JC-1 dissolved in HBBS supplemented with 20 mM HEPES for 45 min at 37 °C, and the ratio of red to green fluorescence was determined. CCCP (carbonyl cyanide

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3-chlorophenylhydrazone, 100 μM), a standard uncoupler of ox-phos, was used as a positive control. Determination of intracellular free calcium. Fluo-4-acetoxymethyl ester (AM) (Molecular Probes) is a cell-permeant fluorophore that can be used to measure the temporal changes in cytosolic free calcium concentrations ([Ca2+]c). Fluo-4-AM passively diffuses across the plasma membrane and, once inside the cell, is hydrolyzed, causing the free Fluo-4 to be trapped in the cytosol. In contrast to Fluo-3, Fluo-4 offers a brighter emission signal upon binding to Ca2+. Briefly, an aliquot of the AM stock solution in DMSO was mixed with an equal amount of Pluronic (Molecular Probes) and added to the assay buffer consisting of HBSS, 20 mM HEPES, pH 7.4, and 2.5 mM probenecid (Molecular Probes), to give a final Fluo-4 concentration of 10 μM. Pluronic helps to disperse the nonpolar AM ester in aqueous media, while probenecid (an inhibitor of organic anion transporters) reduces leakage of Fluo-4. At the desired time points following exposure of cells to diclofenac, the supernatant from each well was removed and the cells were incubated with Fluo4-AM for 1 h at 37 °C. This was followed by a 15-min period at room temperature to ensure complete hydrolysis of the AM ester bond. The change in fluorescence (excitation 485 nm, emission 520 nm) was monitored in a fluorescence microplate reader. Ionomycin calcium salt (20 μM, Molecular Probes) was used as a positive control. Statistical evaluation. All measurements were done in triplicate in three independent experiments. The data were expressed as means ± SD and analyzed by ANOVA/Tukey–Kramer multiple comparison test (InStat, GraphPad). P values of <0.05 were considered significant.

Results Diclofenac induces mPT-mediated cell injury in cultured human HC-04 cells Exposure of HC-04 cells to diclofenac resulted in time and concentration-dependent cell injury, as assessed by LDH release into the extracellular medium (Fig. 1). Diclofenac at concentrations ≥ 500 μM resulted in significant cytotoxicity as compared to solvent controls, which became apparent at a late time point (48 h). This delayed toxicity suggests that time-dependent intracellular signaling pathways were activated, or that genera-

Fig. 1. Time- and concentration-dependent induction of lethal cell injury following exposure to diclofenac. HC-04 cells were treated with the indicated concentrations of diclofenac (in 0.1% DMSO), and LDH activity in the extracellular medium was measured at the indicated time points. Total LDH (100%) is the extracellular plus intracellular LDH activity at each time point. Data are mean ± SD of 3 independent experiments. *P ≤ 0.05 vs. solvent control.

tion of (a) reactive intermediate(s) was required before the toxic response becomes manifest. Fluorescence microscopical analysis using Sytox Green confirmed the presence of increased numbers of injured cells (Fig. 2). Furthermore, Acridine Orange staining revealed the formation of nuclei with condensed chromatin exhibiting bright fluorescence, typical for apoptotic hepatocytes (Fig. 2). To ascertain whether cell death induced by these high concentrations of diclofenac was mediated by mitochondria, the extent of LDH release was determined in the presence or absence of low (non-toxic) concentrations of the specific mPT inhibitor CsA (5 μM). Fig. 3 demonstrates that in the presence of 1 mM diclofenac CsA fully prevented cell injury and that the extracellular LDH activity was similar to that in the solvent control, providing strong evidence for a role of mitochondrial permeabilization in diclofenac-induced cell death. Diclofenac-induced increase in [Ca2+]c is an upstream mediator of the mPT To analyze possible treatment-related temporal changes in the free cytosolic calcium concentrations, we measured [Ca2+]c with the cell-permeant and highly specific calcium probe Fluo4. As shown in Fig. 4, diclofenac caused significant increases in the fluorescence signal. The calcium signal was transient; at 1 mM diclofenac the peak occurred early (8 h) and was back to baseline levels at 24 h; at 500 μM diclofenac the increase in [Ca2+]c peaked at 32 h. The quantitative increases in cytosolic calcium concentrations were comparable to those induced by the positive control, ionomycin (Fig. 4). Importantly, the transient increase in calcium clearly preceded the manifestation of overt cell injury. The reason for the transient nature of the rise in cytosolic calcium and its recovery is not known but could be explained by a possible sequestration of Ca2+ by ER or plasma membrane calcium export pumps, or by mitochondrial uptake of Ca2+ via the calcium uniporter (MCU) in mitochondria whose inner transmembrane potential was still intact. If increased [Ca2+]c were causally involved in triggering the mPT, then sequestration of Ca2+ should protect from diclofenac cell injury. To this end, we concomitantly exposed cells to diclofenac and BAPTA (a cell-permeable Ca2+ chelator with high specificity and selectivity for calcium). Indeed, BAPTA (25 μM) prevented the diclofenac (1 mM)induced increase in LDH release (Fig. 5). Alternatively, the presence of CsA (which protected from cell injury) did not affect the diclofenac-induced increases in [Ca2+]c (diclofenac 1 mM, 156.1 ± 14.9% of solvent control; diclofenac + CsA, 173.2 ± 9.9% of solvent control; no significant difference). This indicates that the increases in [Ca2+]c is an upstream event and causally related to the induction of the mPT, and not simply a downstream consequence of mitochondrial injury. BAPTA, although widely used as a calcium chelator, has also been described to be an iron chelator (Britigan et al., 1998). Therefore, to make sure that the cytoprotective effects of BAPTA against diclofenac were indeed due to removal of calcium and not to sequestration of iron (which would inhibit possible Fenton-type reactions), we exposed cells to diclofenac

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Fig. 2. Nuclear staining of lethally injured cells following exposure to diclofenac. HC-04 cells were treated with 1 mM diclofenac (A, C, D, E) or solvent alone (B, F, G, H) for 48 h and stained with Acridine Orange or Sytox Green. Arrows show chromatin condensation and apoptotic nuclei. Original objective magnification ×100 (A, B) or ×40 (C–H).

in the presence or absence of the ferric ion chelator deferoxamine (DFO) or the ferrous ion chelator, 2,2′-bipyridyl (BIP). DFO and BIP are known as potent iron chelators that can inhibit Fentontype reactions because the iron chelate prevents single-electron exchange with hydrogen peroxide (Galey, 1997; Stäubli and Boelsterli, 1998). As shown in Fig. 6, neither DFO nor BIP attenuated the extent of diclofenac-induced cytotoxicity, suggesting that iron is not primarily involved and that the protective effect afforded by BAPTA was likely mediated by calcium sequestration. In line with this conclusion is the fact that the affinity of BAPTA for Fe2+ (which makes up the redox-active labile iron pool in hepatocytes) is approximately one order of magnitude lower than that for Ca2+ (Britigan et al., 1998). Diclofenac increases mitochondrial superoxide anion levels

Fig. 3. Effects of mPT inhibition on diclofenac-induced lethal cell injury. HC-04 cells were exposed to diclofenac in the presence or absence of CsA, and LDH release was determined after 60 h. Data are mean ± SD of 3 independent experiments. *P ≤ 0.05 vs. solvent control; #P ≤ 0.05 vs. diclofenac alone.

Apart from Ca2+, another key stimulator of the mPT is oxidant stress. One of the factors that can produce oxidant stress-mediated apoptosis is increased mitochondrial production of superoxide anion radicals (Takeyama et al., 1993; Conde de la Rosa et al., 2006). Therefore, we measured the levels of O2U−

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Fig. 4. Time- and concentration-dependent effects of diclofenac on cytosolic free Ca2+ concentrations ([Ca2+]c). HC-04 cells were exposed to diclofenac for the indicated time periods, washed, and loaded with Fluo-4-AM as described in methods. Ionomycin exposure (positive control) was for 3 h only due to cytotoxicity at later time points. Fluorescence was expressed as percentage of solvent control (100%) at each time point. Data are mean ± SD of 3 independent experiments. *P ≤ 0.05 vs. solvent control.

in the presence of diclofenac as a function of time, using the mitochondria-targeting fluorogenic probe MitoSOX Red. Diclofenac induced a clear concentration-dependent increase in superoxide net production (Fig. 7). While the changes in fluorescence after 500 μM diclofenac were not significant (except one time point), the mitochondrial signal after 1 mM diclofenac was approximately 6-fold higher than that in solvent controls. This became evident after 16 h exposure and reached a maximum after 32 h. Thus, the ROS signal clearly appeared later in time than the increased [Ca2+]c signal. To determine whether the increased superoxide levels were a consequence of the dysregulated Ca2+ homeostasis, we measured superoxide levels in cells exposed to combined diclofenac and BAPTA (Fig. 8). The Ca2+ chelator protected in part from the increase in ROS, but Ca2+ could not fully account for the oxidant stress. However, combined exposure to diclofenac and CsA did not attenuate the ROS levels as compared to diclofenac alone,

Fig. 5. Effects of Ca2+ chelation on diclofenac-induced lethal cell injury. HC-04 cells were exposed to diclofenac in the presence or absence of BAPTA for 60 h. LDH release was determined in the supernatant. Data are mean ± SD of 3 independent experiments. *P ≤ 0.05 vs. solvent control; #P ≤ 0.05 vs. diclofenac alone.

Fig. 6. Effects of the ferric ion chelator deferoxamine (DFO) or the ferrous ion chelator 2,2′-bipyridyl (BIP) on diclofenac-induced cell injury. HC-04 cells were exposed to 1 mM diclofenac in the presence or absence of 100 μM DFO or 100 μM BIP for 48 h. DFO or BIP were added from stock solutions in DMSO (final DMSO concentration 0.1%). LDH release was determined in the supernatant. Data are mean + SD of 3 independent experiments. *P ≤ 0.05 vs. solvent control.

indicating that the oxidant stress was not primarily a consequence of the mPT (Fig. 8). Inhibition of diclofenac bioactivation by sulphaphenazole also protected in part from the increase in mitochondrial oxidant stress, implicating a causal role of the diclofenac oxidative metabolites. Collectively, these data indicate that apparent increase in mitochondria-specific U O2 − occurred secondary to [Ca2+]c increases, suggesting that mitochondrial oxidant stress was a consequence rather than the cause for the changes in calcium homeostasis. Diclofenac decreases the mitochondrial membrane potential independently of [Ca2+]c and cell injury Diclofenac decreased the mitochondrial inner transmembrane potential (ΔΨm) in a concentration-dependent manner, attaining 66% inhibition from the baseline value at 1 mM (Fig. 9). The extent of depolarization was similar to that induced by CCCP, which was used as a positive control. This putative uncoupling effect (Petrescu and Tarba, 1997; Uyemura et al.,

Fig. 7. Time- and concentration-dependent effects of diclofenac on mitochondrial superoxide levels. Fluorescence was expressed as percentage of solvent control (100%) at each time point. Data are mean ± SD of 3 independent experiments. *P ≤ 0.05 vs. solvent control.

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Fig. 8. Effects of CsA, BAPTA, or sulphaphenazole on diclofenac-induced mitochondrial superoxide levels. Rotenone was used as a positive control. Cells were exposed to diclofenac for 24 h. Fluorescence was expressed as percentage of solvent control (100%) at each time point. Data are mean ± SD of 3 independent experiments. *P ≤ 0.05 vs. solvent control; #P ≤ 0.05 vs. diclofenac alone.

1997; Moreno-Sanchez et al., 1999; Masubuchi et al., 2002) occurred at an early time point (16 h) and clearly preceded the manifestation of cell injury. Because a collapse of the ΔΨm could also occur as a consequence of mitochondrial membrane permeabilization (including mPT), we determined the extent of depolarization caused by diclofenac in the presence or absence of CsA. Fig. 10A demonstrates that CsA did not affect the diclofenac alone-induced decrease in JC-1 fluorescence, indicating that ΔΨm is not a consequence of mPT. Instead, it could even be an effect entirely unrelated to the mPT. Next, to explore whether the collapse in ΔΨm was the result of diclofenac-induced increases in [Ca2+]c, cells were exposed to diclofenac in the presence of BAPTA. Fig. 10B shows that depolarization by diclofenac remained unaffected by BAPTA, indicating that membrane depolarization is either an upstream event or not causally related to calcium at all.

Fig. 10. Effects of mPT inhibition or Ca2+ chelation on ΔΨm. HC-04 cells were exposed to diclofenac for 16 h in the presence or absence of CsA (A) or BAPTA (B). Cells were then loaded with JC-1 and fluorescence measured and expressed as percentage of solvent control (100%). Data are mean ± SD of 3 independent experiments. *P ≤ 0.05 vs. solvent control.

Mechanistic role of oxidative diclofenac metabolites

Fig. 9. Effects of diclofenac on the mitochondrial inner transmembrane potential (ΔΨm). HC-04 cells were exposed to diclofenac for the indicated time periods, washed, and loaded with JC-1 as described in Materials and methods. Fluorescence was expressed as percentage of solvent control (100%) at each time point. CCCP was used as a positive control. Data are mean ± SD of 3 independent experiments. *P ≤ 0.05 vs. solvent control.

To investigate whether oxidative metabolites of diclofenac, rather than the parent compound, were involved in the mitochondrial toxicity, and to explore their mechanistic role, we used the specific CYP2C9 inhibitor, sulphaphenazole. Inclusion of sulphaphenazole (10 μM) in the incubation medium largely prevented diclofenac-induced cytotoxicity (Fig. 11). These findings indicate that oxidative metabolites catalyzed by CYP2C9 (including 4′-hydroxy and 5-hydroxy diclofenac, the major oxidative metabolites in humans), rather than the parent compound, greatly contribute to cell injury. If CYP2C9-catalyzed metabolites were responsible for the elevated [Ca2+]c (which in turn leads to mitochondrial injury), then chemical inhibition of metabolism should attenuate increases in [Ca 2+ ]c. Indeed, sulphaphenazole prevented diclofenac-induced increases in [Ca2+]c (Fig. 12A). Again, this strongly suggests that the oxidative metabolites, and not diclofenac itself, trigger the mPT. Consistent with this

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Fig. 11. Effects of CYP2C inhibition on diclofenac-induced lethal cell injury. HC-04 cells were exposed to diclofenac for 60 h in the presence or absence of sulphaphenazole. LDH activity was determined in the supernatant. Data are mean ± SD of 3 independent experiments. *P ≤ 0.05 vs. solvent control; # P ≤ 0.05 vs. diclofenac alone.

These conclusions were derived from a number of observations. Firstly, sequestration of [Ca2+]c by the cellpermeable and highly selective Ca2+ chelator BAPTA greatly attenuated diclofenac-induced cell injury (but did not prevent the collapse of ΔΨm). These data are in line with earlier studies (Masubuchi et al., 2002) who showed that the calcium channel blocker verapamil attenuated diclofenacinduced toxicity to isolated rat hepatocytes. Secondly, the increase in [Ca2+]c preceded the onset of cell death, and inhibition of the mPT by CsA left the increases in calcium unaffected. This indicates that the increase in [Ca2+]c is a cause rather than a consequence of mPT. Thirdly, blocking the CYP2C9-mediated oxidative bioactivation of diclofenac fully prevented the increase in [Ca2+]c and protected from cell death, suggesting that one or several metabolites may be responsible for initiating mPT. Furthermore and importantly, this indicates that the rise in intracellular calcium (associated with oxidative metabolites) occurs independently of the

hypothesis is the findings that sulphaphenazole did not prevent the collapse of ΔΨm caused by diclofenac (Fig. 12B). Collectively, the data suggest that it is the parent compound that causes uncoupling and dissipation of the ΔΨm, but that this alone is not sufficient to trigger mPT and cell injury. Instead, CYP2C9-mediated metabolites independently cause increases in [Ca2+]c and initiate the mPT and cell death. Discussion The aim of this study was to explore the upstream mechanisms of diclofenac-induced cell injury in cultured immortalized human hepatocytes. In particular, we investigated the roles of cytosolic free calcium ([Ca2+]c) and mitochondrial depolarization in triggering the more downstream events leading to the mPT and lethal cell injury. An important question was whether these potential changes were dependent on CYPmediated oxidative biotransformation of diclofenac. We found that diclofenac at high (supratherapeutic) concentrations depolarized mitochondria but that this temporal impairment of energy homeostasis alone did not lead to significant cell injury. Independently of its uncoupling effects, diclofenac was bioactivated by CYP2C9 to oxidative intermediates which, in contrast to the parent compound, did not cause uncoupling but which were associated with increases in [Ca2+]c. These early increases in calcium were causally associated with the induction of the mPT. These results confirm and extend earlier reports showing that diclofenac in rat or human hepatocytes was able to induce mPT and apoptosis (Masubuchi et al., 2002; Cantoni et al., 2003; Gomez-Lechon et al., 2003a, 2003b). However, while these earlier reports focused on the characterization of the molecular events distal to the induction of mPT, the more upstream mechanisms (role of reactive metabolites) have remained largely unexplored. Here, we show that elevated [Ca2+]c plays a pivotal role in triggering the mitochondrial pathways of cell death in immortalized human hepatocytes.

Fig. 12. Effects of CYP2C inhibition on diclofenac-induced increases in [Ca2+]c and decreases in ΔΨm. HC-04 cells were exposed to diclofenac for 8 h (A) or 16 h (B) in the presence or absence of sulphaphenazole. The cells were then loaded with Fluo-4 (A) or JC-1 (B), and fluorescence was determined and expressed as percentage of solvent control (100%). Data are mean ± SD of three independent experiments. *P ≤ 0.05 vs. solvent control; #P ≤ 0.05 vs. diclofenac alone.

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collapse of the membrane potential which is caused by the parent compound only, due to its protonophoretic activity (Masubuchi et al., 1998). Normally, low levels of calcium have a global positive effect on mitochondria because many mitochondrial functions are regulated by Ca2+ (including stimulation of ATP synthesis). However, higher levels of intramitochondrial Ca2+ potentiates binding of cyclophilin D to the matrix side of the mPT pore, in particular the adenine nucleotide translocase (ANT) (Crompton et al., 1998). Opening of the mPT can thus be induced by intramitochondrial Ca 2+, alone or aided by other factors including prooxidants. Thus, the diclofenac metabolite-induced increase in [Ca2+]c could be sufficient to sensitize mitochondria to permeabilization. An unanswered question is how the diclofenac metabolite(s) induce increases in [Ca2+]c. It has been suggested that, due to depolarization of mitochondria, Ca2+ leaks out (Luo et al., 1997). Theoretically, the diclofenac (parent compound)induced depolarization may contribute to the increased [Ca2+]c levels as the uncoupled mitochondria feature a reduced ability to take up and retain Ca2+ due to the dissipation of the inside-negative inner transmembrane potential, which is the driving force for Ca2+ uptake into the matrix. However, our data provide evidence that this mechanism is of minor importance (Fig. 12A). Another possibility is that because the 4′-hydroxylated and 5-hydroxylated diclofenac metabolites can be further metabolized to the respective intermediate (Shen et al., 1999; Tang et al., 1999a, 1999b; Poon et al., 2001), ROS may be generated through redox cycling via the semiquinone radical. Similarly, a minor metabolite (N,5-dihydroxy diclofenac) may induce oxidant stress through consumption of NADPH (Bort et al., 1998). These imbalances in the redox status may then inactivate critical sulfhydryl groups including those of Ca2+ pumps. It has become clear that ROS and Ca2+, together with ATP and other factors, form a complex network with each having the ability to control the others (Brookes et al., 2004). Here, however, we clearly show that the increases in [Ca2+]c occur prior to the apparent massive increases in ROS. The consequences of increased Ca2+c can of course include activation of pathways distinct from the mPT. For example, intracellular calpains, which are activated by Ca2+, can contribute to cell injury and even propagate cell injury after removal of the toxicant (Limaye et al., 2003). However, since CsA in this study blocked the mPT and fully prevented cell injury while leaving the increased [Ca2+]c unaffected, mitochondria-independent pathways may not be critically involved in diclofenac-induced toxicity in human hepatocytes. We also provide evidence for a significant increase in the net production of superoxide anion caused by high concentrations of diclofenac. This is in line with previous findings indicating that ROS are generated in hepatocytes (Cantoni et al., 2003; Gomez-Lechon et al., 2003b). However, in these earlier studies, the increases in ROS were relatively small, and the source of ROS was not determined. In particular, data with dichlorofluorescein have to be analyzed with caution as

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this fluorophore could also be oxidized by non-specific mechanisms including diclofenac radicals (Galati et al., 2002) or even cytochrome c during its apoptotic release (Burkitt and Wardman, 2001). Here, using a mitochondria-targeting probe, we show for the first time that the source of ROS induced by diclofenac is mitochondria. The lack of initial production of superoxide anion in this study is commensurate with the generally accepted concept that uncouplers like diclofenac decrease rather than increase mitochondrial ROS generation because the production of ROS is exponentially dependent on ΔΨm (Brookes et al., 2004). In contrast, the massive production of superoxide in the second phase, following disruption of [Ca2+]c homeostasis, could be the result of bioactivation of diclofenac by a mechanism distinct from CYP29 and even further promote mitochondrial damage via mPT in a vicious cycle. A key conclusion from our studies is that uncoupling and depolarization induced by nonmetabolized diclofenac is not responsible for the observed mPT and cell injury. Instead, we show that oxidative metabolites are causally involved in mPT and lethal cell injury. This can also explain why the cytotoxic effects become apparent after a delay (> 48 h). So far, the role of electrophilic diclofenac metabolites in the precipitation of liver injury has not been clear. However, it has been well known that there are two distinct pathways of diclofenac bioactivation (Tang, 2003). The first one is acyl glucuronidation, leading to the protein-reactive 1-O-acyl glucuronide (Kretz-Rommel and Boelsterli, 1994). Furthermore, this glucuronide can react with glutathione leading to a reactive diclofenac-S-acyl-glutathione. This thioester has been shown to be even more reactive than the acyl glucuronide (Grillo et al., 2003a, 2003b). The second pathway involves ring hydroxylation to the 4′-hydroxy derivative (catalyzed by CYP2C9) or the 5-hydroxy metabolite (subject to CYP3A4 cooperativity). These two p-hydroxy metabolites can form putative iminoquinone intermediates, the hypothetical formation of which was based on identification of the respective glutathione adducts (Shen et al., 1999; Tang et al., 1999a; Poon et al., 2001). While the role of these metabolites in the toxicity of diclofenac via generation of oxidant stress is still unclear, we provide evidence that the oxidative metabolites are involved in the generation of increased [Ca2+]c, and that this is a critical step in triggering the mPT and ensuing cell death. That diclofenac, similarly to many other NSAIDs, is an uncoupler of ox-phos has been demonstrated earlier (Uyemura et al., 1997; Moreno-Sanchez et al., 1999; Masubuchi et al., 2000). This most likely occurs through the protonophoretic activity of the weakly acidic and lipophilic diclofenac, translocating protons across the inner mitochondrial membrane (Boelsterli, 2003). In one of these studies (Masubuchi et al., 2000), it also became clear that it is the parent compound and not the oxidative metabolites that were responsible for the uncoupling effects. However, it has been postulated that uncoupling may lead to the opening of the mPT pore. Here, we clearly demonstrate that uncoupling per se does not lethally injure the hepatocytes but that depolarization and mPT are two

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Fig. 13. Proposed sequence of events and mechanisms involved in the mitochondrial toxicity of diclofenac. The parent compound penetrates into mitochondria and dissipates the ΔΨm, which causes decreases in ATP production but which does not lead to cell injury. On the other hand, that portion of the parent compound that escapes glucuronidation is gradually metabolized by CYP2C9 to hydroxylated metabolites, which may be further bioactivated to reactive iminoquinone intermediates, resulting in enhanced superoxide production and increased [Ca2+]c levels. This rise in [Ca2+]c in turn initiates the mPT and ultimately lethal cell injury. The highly increased levels of mitochondrial superoxide anion is, in part, the consequence of the increases in [Ca2+]c, and not only a result of mitochondrial permeabilization.

distinct pathways. It is therefore possible that with increasing metabolization of the parent compound over time the uncoupling effect would become weaker and result in a partial restoration of the ΔΨm, which in turn would facilitate mitochondrial uptake of Ca2+. The net increases in Ca2+ could then trigger the mPT (Byrne et al., 1999). Based on all the experimental evidence, we propose the following sequence of events occurring in immortalized human hepatocytes exposed to diclofenac (Fig. 13): The portion of diclofenac that escapes glucuronidation induces depolarization in mitochondria (by uncoupling), which causes a drop in ATP but which per se does not kill the cells. Independently, diclofenac is converted to oxidative metabolites which cause a transient increase in [Ca2+]c. These elevated calcium levels trigger the mPT, resulting in the release of proapoptotic factors and the precipitation of lethal cell injury. This may be promoted by the great increase in mitochondrial ROS. It must be stressed that the diclofenac concentrations used in this in vitro study are very high and cannot be compared with the in vivo situation (where unknown but high portal concentrations, enterohepatic cycling, high plasma protein binding, and rapid clearance of diclofenac all confound the picture). This is in agreement with the clinical picture, where the vast majority of patients do not develop any signs of hepatotoxicity at therapeutic dosage of diclofenac. Thus, the overall purpose of this study was not to mimic a clinically relevant situation but rather to identify a hazard and to push the concentration to a limit where diclofenac induces toxicity in these human hepatocytes in order to delineate the molecular mechanisms. In conclusion, we identify a clear mitochondrial hazard by diclofenac oxidative metabolites and demonstrate that mitochondrial depolarization by diclofenac can be “uncoupled” from the induction of mPT and the downstream events

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