mitotempo and Necrosis

We previously reported that delayed treatment with Mito-tempo (MT), a mitochondria-targeted superoxide dismutase mimetic, protects against the early phase of acetaminophen (APAP) hepatotoxicity by inhibiting peroxynitrite formation. However, whether this protection is sustained to the late phase of toxicity is unknown. To investigate the late protection, C57Bl/6J mice were treated with 300 mg/kg APAP followed by 20 mg/kg MT 1.5 h or 3 h later. We found that both MT treatments protected against the late phase of APAP hepatotoxicity at 12 and 24 h. Surprisingly, MT-treated mice demonstrated a significant increase in apoptotic hepatocytes, while the necrotic phenotype was observed almost exclusively in mice treated with APAP alone. In addition, there was a significant increase in caspase-3 activity and cleavage in the livers of MT-treated mice. Immunostaining for active caspase-3 revealed that the positively stained hepatocytes were exclusively in centrilobular areas. Treatment with the pan-caspase inhibitor ZVD-fmk (10 mg/kg) 2 h post-APAP neutralized this caspase activation and provided additional protection against APAP hepatotoxicity. Treatment with N-acetylcysteine, the current standard of care for APAP poisoning, protected but did not induce this apoptotic phenotype. Mechanistically, MT treatment inhibited APAP-induced RIP3 kinase expression, and RIP3-deficient mice showed caspase activation and apoptotic morphology in hepatocytes analogous to MT treatment. These data suggest that while necrosis is the primary cause of cell death after APAP hepatotoxicity, treatment with the antioxidant MT may switch the mode of cell death to secondary apoptosis in some cells. Modulation of mitochondrial oxidative stress and RIP3 kinase expression play critical roles in this switch.

Topics: Acetaminophen; Acetylcysteine; Animals; Antioxidants; Apoptosis; Caspase 3; Chemical and Drug Induced Liver Injury; Hepatocytes; Male; Mice, Inbred C57BL; Necrosis; Organophosphorus Compounds; Piperidines; Receptor-Interacting Protein Serine-Threonine Kinases

Generation of excessive reactive oxygen species (ROS) leads to mitochondrial dysfunction, apoptosis, and necrosis in renal ischemia-reperfusion (IR) injury. Previously we showed that lentiviral vector-mediated overexpression of superoxide dismutase-1 (SOD1) in proximal tubular epithelial cells (LLC-PK(1)) reduced cytotoxicity in an in vitro model of IR injury. Here, we examined the effects of SOD1 overexpression on mitochondrial signaling after ATP depletion-recovery (ATP-DR). To examine the role of mitochondrial ROS, a subset of cells was treated with the mitochondrial antioxidant MitoTEMPO. ATP-DR-mediated increase in mitochondrial calcium, loss of mitochondrial membrane potential, and increase in mitochondrial permeability transition pore (MPTP) were attenuated by SOD1 and MitoTEMPO (P<0.01). SOD1 prevented ATP-DR-induced mitochondrial Bax translocation, although the release of proapoptotic proteins from mitochondria was not prevented by SOD1 alone and required the presence of both SOD1 and MitoTEMPO. SOD1 suppressed the increase in c-jun phosphorylation, suggesting that JNK signaling regulates Bax translocation to mitochondria via ROS. ATP-DR-mediated changes in MPTP and mitochondrial signaling increased necrosis and apoptosis, both of which were partially attenuated by SOD1 and MitoTEMPO. These studies show that SOD1 and MitoTEMPO preserve mitochondrial integrity and attenuate ATP-DR-mediated necrosis and apoptosis.

Topics: Adenosine Triphosphate; Animals; Antioxidants; Apoptosis; Cyclic N-Oxides; Mitochondria; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Mitochondrial Proteins; Mitogen-Activated Protein Kinase 8; Necrosis; Organophosphorus Compounds; Phosphorylation; Piperidines; Proto-Oncogene Proteins c-jun; Superoxide Dismutase; Superoxide Dismutase-1; Swine