Contribution of increased mitochondrial free Ca2+ to the mitochondrial permeability transition induced by tert-butylhydroperoxide in rat hepatocytes

Hepatology. 1999 May;29(5):1523-31. doi: 10.1002/hep.510290521.


Previously, we showed that the oxidant chemical, tert-butylhydroperoxide (t-BuOOH), induces a mitochondrial permeability transition (MPT) in intact hepatocytes, causing lethal cell injury. Here, we investigated the role of mitochondrial free Ca2+ in t-BuOOH cytotoxicity to 1-day-cultured rat hepatocytes using confocal microscopy of autofluorescence and parameter-indicating fluorophores. t-BuOOH (100 micromol/L) caused an early increase of mitochondrial free Ca2+, as assessed by confocal microscopy of Rhod-2 fluorescence. Increased mitochondrial Ca2+ was followed by onset of the MPT, as evidenced by permeation of cytosolic calcein into mitochondria and loss of the mitochondrial membrane potential-indicating dye, tetramethylrhodamine methylester. Preincubation with an intracellular Ca2+ chelator (BAPTA-AM and its derivatives) partially blocked the late phase of mitochondrial NAD(P)H oxidation after t-BuOOH, but failed to prevent the early oxidation of mitochondrial NAD(P)H. Ca2+ chelation also prevented the increase of mitochondrial Ca2+, generation of mitochondrial reactive oxygen species (ROS), onset of the MPT, and subsequent cell death. Confocal images showed that protection occurred when loading of the Ca2+ chelator was predominantly mitochondrial. The antioxidant, desferal, also diminished increased mitochondrial Ca2+ after t-BuOOH and prevented cell death. We conclude that oxidative stress induced by t-BuOOH enhances mitochondrial Ca2+ uptake, leading to increased matrix Ca2+, increased ROS formation, onset of the MPT, and cell death.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Aminoquinolines / pharmacology
  • Animals
  • Calcium / metabolism*
  • Cell Death / drug effects
  • Chelating Agents / pharmacology
  • Deferoxamine / pharmacology
  • Egtazic Acid / analogs & derivatives
  • Egtazic Acid / pharmacology
  • Liver / cytology
  • Liver / drug effects*
  • Liver / metabolism*
  • Male
  • Mitochondria, Liver / metabolism*
  • NADP / metabolism
  • Oxidation-Reduction / drug effects
  • Permeability
  • Rats
  • Rats, Sprague-Dawley
  • Reactive Oxygen Species / metabolism
  • Time Factors
  • tert-Butylhydroperoxide / pharmacology*


  • Aminoquinolines
  • Chelating Agents
  • Reactive Oxygen Species
  • 1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester
  • Egtazic Acid
  • NADP
  • Quin2-acetoxymethyl ester
  • tert-Butylhydroperoxide
  • Deferoxamine
  • Calcium