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

Previously we showed that the oxidant chemical, tert-butylhydroperoxide (t- BuOOH), induces a mitochondrial permeability transition (MPT) in intact hep atocytes, causing lethal cell injury. Here, we investigated the role of mit ochondrial free Ca2+ in t-BuOOH cytotoxicity to 1-day-cultured rat hepatocy tes using confocal microscopy of autofluorescence and parameter-indicating fluorophores, t-BuOOH (100 mu mol/L) caused an early increase of mitochondr ial free Ca2+, as assessed by confocal microscopy of Rhod-2 fluorescence. I ncreased mitochondrial Ca2+ was followed by onset of the MPT, as evidenced by permeation of cytosolic calcein into mitochondria and loss of the mitoch ondrial membrane potential-indicating dye, tetramethylrhodamine methylester , Preincubation with an intracellular Ca2+ chelator (BAPTA-AM and its deriv atives) 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 occurre d when loading of the Ca2+ chelator was predominantly mitochondrial, The an tioxidant, desferal, also diminished increased mitochondrial Ca2+ after t-B uOOH and prevented cell death. We conclude that oxidative stress induced by t-BuOOH enhances mitochondrial Ca2+ uptake, leading to increased matrix Ca 2+, increased ROS formation, onset of the MPT, and cell death.