Bax-mediated mitochondrial outer membrane permeabilization (MOMP), distinct from the mitochondrial permeability transition, is a key mechanism in diclofenac-induced hepatocyte injury: Multiple protective roles of cyclosporin A

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Abstract

Diclofenac, a widely used nonsteroidal anti-inflammatory drug, has been associated with rare but severe cases of clinical hepatotoxicity. Diclofenac causes concentration-dependent cell death in human hepatocytes (after 24–48 h) by mitochondrial permeabilization via poorly defined mechanisms. To explore whether the cyclophilin D (CyD)-dependent mitochondrial permeability transition (mPT) and/or the mitochondrial outer membrane permeabilization (MOMP) was primarily involved in mediating cell death, we exposed immortalized human hepatocytes (HC-04) to apoptogenic concentrations of diclofenac (> 500 μM) in the presence or absence of inhibitors of upstream mediators. The CyD inhibitor, cyclosporin A (CsA, 2 μM) fully inhibited diclofenac-induced cell injury, suggesting that mPT was involved. However, CyD gene silencing using siRNA left the cells susceptible to diclofenac toxicity, and CsA still protected the CyD-negative cells from lethal injury. Diclofenac induced early (9 h) activation of Bax and Bak and caused mitochondrial translocation of Bax, indicating that MOMP was involved in cell death. Inhibition of Bax protein expression by using siRNA significantly protected HC-04 from diclofenac-induced cell injury. Diclofenac also induced early Bid activation (tBid formation, 6 h), which is an upstream mechanism that initiates Bax activation and mitochondrial translocation. Bid activation was sensitive to the Ca2+ chelator, BAPTA. In conclusion, we found that Bax/Bak-mediated MOMP is a key mechanism of diclofenac-induced lethal cell injury in human hepatocytes, and that CsA can prevent MOMP through inhibition of Bax activation. These data support our concept that the Ca2+–Bid–Bax–MOMP axis is a critical pathway in diclofenac (metabolite)-induced hepatocyte injury.

Introduction

Diclofenac is a frequently used nonsteroidal anti-inflammatory drug that is generally perceived to be a safe and well tolerated medication. However, due to its large consumer population worldwide, diclofenac has been reported to cause significant adverse effects, including mild hepatic injury and rare, but severe idiosyncratic hepatotoxicity (Boelsterli, 2003, Daly et al., 2007).

At present, the underlying mechanisms of diclofenac-induced low-incidence/high-severity hepatotoxicity are incompletely understood because the susceptibility factors that can modulate the penetrance and expressivity of diclofenac-associated idiosyncratic reactions are currently not known (Boelsterli, 2003). Before a rationale-based search for determinants of susceptibility can be initiated, one must, however, understand the inherent toxic potential of diclofenac and identify the intrinsic molecular mechanisms underlying diclofenac-induced hepatocellular injury. Unfortunately, suitable animal models are currently not available, due in part to the extreme sensitivity of rodents to diclofenac-induced gastrointestinal toxicity, which is a limiting factor in mechanistic toxicological studies. In contrast, human hepatocytes lend themselves ideally for mechanistic studies, and two key mechanisms causally involved in diclofenac toxicity have been identified in vitro; first, metabolic bioactivation at the proximal end of the cascade of events (Tang, 2003, Lim et al., 2006) and, second, the induction of apoptosis towards the distal end (Gomez-Lechon et al., 2003a, Tsutsumi et al., 2004). However, the signaling pathways linking the drug (metabolites) with the downstream executors of cell death have not been well understood, although this would be important in view of potential therapeutic intervention.

Traditionally, mitochondrial dysfunction has been implicated as a possible mode of action in diclofenac hepatotoxicity. This concept was primarily based on experimental evidence obtained from isolated mitochondria and cultured liver cells (Masubuchi et al., 2000, Masubuchi et al., 2002, Gomez-Lechon et al., 2003b). For example, diclofenac has been shown to dissipate the mitochondrial inner transmembrane potential (ΔΨm) due to its protonophoretic activity (Masubuchi et al., 2000). Uncoupling of oxidative phosphorylation (OXPHOS) from ATP synthesis eventually leads to a decrease in ATP synthesis. However, we have recently shown that this mechanism is not directly related to the cytotoxic effects of diclofenac in human hepatocytes, as it is the parent drug that causes uncoupling, while it is an oxidative metabolite that causes apoptosis (Lim et al., 2006).

More recently, diclofenac has been demonstrated to induce the mitochondrial permeability transition (mPT) (Masubuchi et al., 2002), a phenomenon referring to an abrupt transition in permeability of the mitochondrial inner membrane via opening of a proteinaceous megapore consisting of the adenine nucleotide translocator (ANT), the voltage-dependent anion channel (VDAC), cyclophilin D (CyD), and other accessory proteins (Bernardi and Petronilli, 1996, Lemasters, 1998). Sustained opening of the mPT pore can trigger a cascade of events, starting with osmotic swelling of the mitochondria, due to redistribution of solutes between cytosol and the matrix, followed by rupture of the outer mitochondrial membrane and release of proapoptotic factors such as cytochrome c into the cytosol, thus activating cell death pathways that lead to apoptosis or necrosis (Green and Reed, 1998, Green and Kroemer, 2004). We and others have recently confirmed the involvement of mitochondrial permeabilization, presumably mPT, in diclofenac-induced cell death (Masubuchi et al., 2002, Lim et al., 2006). This conclusion was primarily inferred from the protective effects of cyclosporin A (CsA), based on its “specific” binding to and inhibition of cyclophilin D (CyD) (Crompton et al., 1998), which resulted in rescuing human hepatocytes from diclofenac-induced cell death (Lim et al., 2006). However, the specificity of the effects of CsA has been questioned; for example, it has been reported that CsA can act more proximally by preventing cell death through inhibition of Bax activity (Marzo et al., 1998, Pastorino et al., 1998) suggesting a possible key role of Bax in diclofenac-induced cell injury. Indeed, upon translocation to the mitochondria, Bax can interact with components of the pore such as ANT and VDAC, thus regulating mPT (Marzo et al., 1998, Shimizu et al., 1999). The extent, however, to which mPT contributes to diclofenac-induced cell injury is still unknown.

Another function of Bax is a key regulatory role in inducing the mitochondrial outer membrane permeabilization (MOMP), a process distinct from the mPT (Green and Reed, 1998). Recent evidence has revealed that upon translocation of activated Bax to mitochondria, monomers of Bax and Bak (another proapoptotic Bcl-2 family protein localized on the outer mitochondrial membrane) can oligomerize to form large channels and induce formation of pores (Green and Kroemer, 2004, Er et al., 2006). These channels can allow apoptogenic factors and proteins sequestered in the mitochondrial intermembrane space to be released into the cytosol, resulting in apoptosis via activation of caspases and apoptosomes (Green and Reed, 1998). To date, little is known about the involvement of MOMP in diclofenac-induced cell injury. Hence, the aim of this study was to define the roles of Bax and its upstream regulator, Bid, in diclofenac-induced mitochondrial toxicity in hepatocytes because such key regulators could become potential therapeutic targets. In particular, we hypothesized that the Bax/Bak pathway could initiate MOMP. We found that the Bid–Bax–MOMP axis is indeed a major mechanistic pathway in diclofenac-induced lethal cell injury and that Bax may be a key target to prevent these toxic effects.

Section snippets

Culture of immortalized human HC-04 cells and exposure to drugs in vitro

HC-04 cells (Siam Life Science Ltd, Bangkok, Thailand), metabolically competent immortalized human hepatocytes (Lim et al., 2007b), were cultured in Hepatocyte Basal Medium (HBM) supplemented with Hepatocyte Culture Medium (HCM) Bulletkit (Cambrex, USA) and 10% fetal bovine serum. Primaria flasks (Falcon) were pre-coated with 0.03 mg/ml of collagen solution, 0.01 mg/ml of fibronectin and 0.01 mg/ml of bovine serum albumin in HBM for 2 h at 37 °C before seeding of cells. The cells were

Diclofenac induces CsA-sensitive delayed cell injury in human hepatocytes

First, we confirmed earlier results demonstrating that exposure of the metabolically competent immortalized human hepatocytes (HC-04) (Lim et al., 2006) to diclofenac caused time- and concentration-dependent cell injury. Based on LDH release as an indicator of cell injury, diclofenac at > 500 μM resulted in marginal cytotoxicity at 24 h and severe cell injury at 48 h as compared to solvent controls (Fig. 1A). This apparent delayed toxicity suggests that the manifestation of the toxic response

Discussion

The objectives of this study were (1) to elucidate the molecular signaling pathways that link diclofenac-induced Ca2+ increases and oxidant stress (upstream events) with the execution of mitochondria-mediated lethal cell injury in human hepatocytes, and (2) to find a way to specifically block these major pathways, and thus prevent toxicity. We had hypothesized that the proapoptotic Bcl-2 family protein, Bax, which is involved in the mPT, could also activate MOMP, which is an alternative mode of

Acknowledgments

This work was supported by grants from the Biomedical Research Council, Singapore (R-184-000-096-305, to U.A.B.), National Medical Research Council, Singapore (R-184-000-080-214, to U.A.B.), and NUS Office of Life Sciences Toxicology Program (R-184-000-079-712, to U.A.B.), and the Boehringer Ingelheim Endowed Chair in Mechanistic Toxicology at the University of Connecticut.

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