title: Detection of Redox Imbalance in Normal Lymphocytes with Induced Mitochondrial Dysfunction - EPR Study.
abstract: The present study describes a new approach for direct imaging of redox status in live cells using paramagnetic spin-probes, which allows evaluation of the level of oxidative stress due to overproduction of superoxide. The method is based on redox cycling of cell/mitochondria-penetrating nitroxide radicals (e.g. mito-TEMPO) and their electron-paramagnetic resonance (EPR) contrast, which makes them useful molecular sensors for analysis of redox status and oxidative stress in cells and tissues. Oxidative stress was induced in normal human lymphocytes by treatment with 2-methoxyestradiol and rotenone (ME/Rot) at different concentrations. This combination provokes mitochondrial dysfunction, which is accompanied by overproduction of superoxide. The EPR measurements were performed in dynamics on X-Band spectrometer after addition of mito-TEMPO to cell suspensions. The intensity of the EPR signal in untreated cells decreased significantly, which indicates a conversion of paramagnetic mito-TEMPO to its non-contrast diamagnetic form (hydroxylamine - mito-TEMPOH) due to reduction. In ME/Rot-treated cells, the signal decreased more slowly and to a lower level with increasing the concentration of ME/Rot. These data indicate an induction of oxidative stress in the cells in a concentration-dependent manner. A very good positive correlation between the intensity of EPR signal of mito-TEMPO and the intracellular level of superoxide was found, analyzed by conventional dihydroethidium test (R=0.9143, p<0.001). In conclusion, our study demonstrated that cell-penetrating paramagnetic spin-probes, such as mito-TEMPO, are valuable tools for EPR imaging of the superoxide level in live cells, as well as for EPR imaging of mitochondrial dysfunction and metabolic activity, accompanied by superoxide imbalance.
authors: Medical Faculty, Trakia University, Stara Zagora, Bulgaria.
authors: Ekaterina
authors: E
authors: Georgieva
authors: Medical Faculty, Trakia University, Stara Zagora, Bulgaria.|Institute of Biophysics & Biomedical Engineering, Bulgarian Academy of Sciences, Sofia, Bulgaria.
authors: Zhivko
authors: Z
authors: Zhelev
authors: Department of Molecular Imaging and Theranostics, National Institute of Radiological Sciences, National Institute for Quantum and Radiological Science and Technology, Chiba, Japan.
authors: Ichio
authors: I
authors: Aoki
authors: Department of Molecular Imaging and Theranostics, National Institute of Radiological Sciences, National Institute for Quantum and Radiological Science and Technology, Chiba, Japan bakalova.rumiana@qst.go.jp.|Medical Faculty, Sofia University St. Kliment Ohridski, Sofia, Bulgaria.
authors: Rumiana
authors: R
authors: Bakalova
authors: Department of Molecular Imaging and Theranostics, National Institute of Radiological Sciences, National Institute for Quantum and Radiological Science and Technology, Chiba, Japan.
authors: Tatsuya
authors: T
authors: Higashi
mesh_terms: D000077584:2-Methoxyestradiol; D000328:Adult; D004578:Electron Spin Resonance Spectroscopy; D004958:Estradiol / Q000031:analogs & derivatives; D006801:Humans; D008214:Lymphocytes / Q000378:metabolism*; D008928:Mitochondria / Q000378:metabolism*; D009943:Organophosphorus Compounds / Q000494:pharmacology*; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D010880:Piperidines / Q000494:pharmacology*; D012402:Rotenone; D013481:Superoxides / Q000378:metabolism
chemical_list: C555916:MitoTEMPO; D009943:Organophosphorus Compounds; D010880:Piperidines; D012402:Rotenone; D013481:Superoxides; D004958:Estradiol; D000077584:2-Methoxyestradiol
keywords: Nitroxides; electron paramagnetic resonance imaging; mitochondrial dysfunction; superoxide