Contribution of Increased Mitochondrial Free Ca2+ to the Mitochondrial Permeability Transition Induced By Tert –Butylhydroperoxide in Rat Hepatocytes

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Abstract
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 μmol/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.

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