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

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, ΔΨ_m (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 ΔΨ_m. 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.

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 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 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 (ΔΨ_m). 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.