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

Toxicol Appl Pharmacol. 2006 Dec 15;217(3):322-31. doi: 10.1016/j.taap.2006.09.012. Epub 2006 Oct 5.


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 (Ca(2+)(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 microM). At 8 h, diclofenac caused increases in [Ca(2+)](c) (Fluo-4 fluorescence), which was unaffected by CsA. Combined exposure to diclofenac/BAPTA (Ca(2+) chelator) inhibited cell injury, indicating that Ca(2+) plays a critical role in precipitating mPT. Diclofenac decreased the mitochondrial membrane potential, DeltaPsi(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 [Ca(2+)](c). The CYP2C9 inhibitor sulphaphenazole (10 microM) protected from diclofenac-induced cell injury and prevented increases in [Ca(2+)](c), while it had no effect on the dissipation of the DeltaPsi(m). Finally, diclofenac exposure greatly increased the mitochondria-selective superoxide levels secondary to the increases in [Ca(2+)](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 [Ca(2+)](c), triggering the mPT and precipitating cell death.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Anti-Inflammatory Agents, Non-Steroidal / metabolism
  • Anti-Inflammatory Agents, Non-Steroidal / toxicity*
  • Apoptosis / drug effects
  • Aryl Hydrocarbon Hydroxylases / antagonists & inhibitors
  • Aryl Hydrocarbon Hydroxylases / biosynthesis
  • Calcium / metabolism*
  • Cell Line, Transformed
  • Cyclosporine / pharmacology
  • Cytochrome P-450 CYP2C9
  • Cytosol / drug effects*
  • Cytosol / metabolism
  • Diclofenac / metabolism
  • Diclofenac / toxicity*
  • Dose-Response Relationship, Drug
  • Drug Antagonism
  • Egtazic Acid / analogs & derivatives
  • Egtazic Acid / pharmacology
  • Hepatocytes / drug effects*
  • Hepatocytes / metabolism
  • Hepatocytes / pathology
  • Humans
  • Membrane Potentials / drug effects
  • Mitochondria, Liver / drug effects*
  • Mitochondrial Membranes / drug effects*
  • Oxidation-Reduction
  • Permeability
  • Sulfaphenazole / pharmacology
  • Superoxides / metabolism
  • Uncoupling Agents


  • Anti-Inflammatory Agents, Non-Steroidal
  • Uncoupling Agents
  • Sulfaphenazole
  • Superoxides
  • Diclofenac
  • Egtazic Acid
  • Cyclosporine
  • CYP2C9 protein, human
  • Cytochrome P-450 CYP2C9
  • Aryl Hydrocarbon Hydroxylases
  • 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid
  • Calcium