title: CI-1010 induced opening of the mitochondrial permeability transition pore precedes oxidative stress and apoptosis in SY5Y neuroblastoma cells.
abstract: The hetero-bifunctional nitroimidazole radiosensitizer CI-1010, R-alpha-[[(2-bromoethyl)-amino]methyl]-2-nitro-1H-imidazole-1-ethanol monohydrobromide, causes selective irreversible apoptotic loss of retinal photoreceptor cells in vivo. The human neuroblastoma cell line, SH-SY5Y, was used as a neuronotypic model of CI-1010-mediated retinal degeneration. Exposure to CI-1010 for 24 h induced apoptosis in neuroblastoma cells, as determined by histopathological and ultrastructural analysis and by TUNEL technique. CI-1010 causes a dose-dependent decrease in cell viability in SY5Y cells, as measured by the reduction of MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. Superoxide dismutase reduced loss of cell viability following CI-1010 treatment suggesting an oxidative stress-mediated mechanism of toxicity. The effects of CI-1010 on mitochondrial membrane potential and intracellular levels of reactive oxygen species were assessed in live SY5Y cells by confocal microscopy using the fluorescent dyes, tetramethylrhodamine methyl ester and 5,6-carboxy-2',7'-dihydrodichlorofluorescein diacetate. CI-1010 caused a rapid depolarization of mitochondria in SY5Y cells followed by an increase in ROS. Both CI-1010-induced mitochondrial depolarization and subsequent increases in ROS were prevented by pretreatment with either the permeability transition pore inhibitor, cyclosporin A (CsA), and by the antioxidant, alpha-tocopherol. However, CsA and alpha-tocopherol were unable to prevent apoptosis in CI-1010-treated cells, suggesting the influence of additional mechanism(s) of CI-1010-induced toxicity. This study evaluates intracellular oxidative stress associated with pore opening prior to apoptosis and provides evidence in support of a mitochondrial mechanism of CI-1010-induced neuronal cell death.
authors.affiliation: Toxicology Program, Department of Environmental Health Sciences, The University of Michigan, 1420 Washington Heights, SPH II Ann Arbor, MI 48109-2029, USA.
authors.forename: Terry J
authors.initials: TJ
authors.lastname: Miller
authors.forename: Amanda D
authors.initials: AD
authors.lastname: Phelka
authors.forename: Ronald B
authors.initials: RB
authors.lastname: Tjalkens
authors.forename: Lloyd A
authors.initials: LA
authors.lastname: Dethloff
authors.forename: Martin A
authors.initials: MA
authors.lastname: Philbert
mesh_terms: D000975:Antioxidants / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D001932:Brain Neoplasms / Q000378:metabolism* / Q000648:ultrastructure; D016923:Cell Death / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D053938:DNA Fragmentation / Q000187:drug effects; D005434:Flow Cytometry; D006801:Humans; D020287:In Situ Nick-End Labeling; D008564:Membrane Potentials / Q000187:drug effects; D008854:Microscopy, Electron; D008928:Mitochondria / Q000187:drug effects* / Q000648:ultrastructure; D009447:Neuroblastoma / Q000378:metabolism* / Q000648:ultrastructure; D009593:Nitroimidazoles / Q000494:pharmacology*; D018384:Oxidative Stress / Q000502:physiology*; D010539:Permeability / Q000187:drug effects; D010786:Photoreceptor Cells / Q000187:drug effects / Q000502:physiology; D011838:Radiation-Sensitizing Agents / Q000494:pharmacology*; D017382:Reactive Oxygen Species / Q000378:metabolism; D013778:Tetrazolium Salts; D013844:Thiazoles; D014407:Tumor Cells, Cultured; D014810:Vitamin E / Q000494:pharmacology
chemical_list: D000975:Antioxidants; D009593:Nitroimidazoles; D011838:Radiation-Sensitizing Agents; D017382:Reactive Oxygen Species; D013778:Tetrazolium Salts; D013844:Thiazoles; C067834:RB 6145; D014810:Vitamin E; C022616:thiazolyl blue