A mitochondria-targeted nitroxide/hemigramicidin S conjugate protects mouse embryonic cells against gamma irradiation.
OBJECTIVE
To evaluate the in vitro radioprotective effect of the mitochondria-targeted hemigramicidin S-conjugated 4-amino-2,2,6,6-tetramethyl-piperidine-N-oxyl (hemi-GS-TEMPO) 5-125 in gamma-irradiated mouse embryonic cells and adenovirus-12 SV40 hybrid virus transformed human bronchial epithelial cells BEAS-2B and explore the mechanisms involved in its radioprotective effect.


METHODS
Cells were incubated with 5-125 before (10 minutes) or after (1 hour) gamma-irradiation. Superoxide generation was determined by using dihydroethidium assay, and lipid oxidation was quantitated by using a fluorescence high-performance liquid chromatography-based Amplex Red assay. Apoptosis was characterized by evaluating the accumulation of cytochrome c in the cytosol and externalization of phosphatidylserine on the cell surface. Cell survival was measured by means of a clonogenic assay.


RESULTS
Treatment (before and after irradiation) of cells with 5-125 at low concentrations (5, 10, and 20 mum) effectively suppressed gamma-irradiation-induced superoxide generation, cardiolipin oxidation, and delayed irradiation-induced apoptosis, evaluated by using cytochrome c release and phosphatidylserine externalization. Importantly, treatment with 5-125 increased the clonogenic survival rate of gamma-irradiated cells. In addition, 5-125 enhanced and prolonged gamma-irradiation-induced G(2)/M phase arrest.


CONCLUSIONS
Radioprotection/mitigation by hemi-GS-TEMPO likely is caused by its ability to act as an electron scavenger and prevent superoxide generation, attenuate cardiolipin oxidation in mitochondria, and hence prevent the release of proapoptotic factors from mitochondria. Other mechanisms, including cell-cycle arrest at the G(2)/M phase, may contribute to the protection.
Jiang
Jianfei
J
Center for Medical Countermeasures Against Radiation, University of Pittsburgh, Pittsburgh, PA 15219, USA.
Belikova
Natalia A
NA
Hoye
Adam T
AT
Zhao
Qing
Q
Epperly
Michael W
MW
Greenberger
Joel S
JS
Wipf
Peter
P
Kagan
Valerian E
VE
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D002308:Cardiolipins / Q000378:metabolism; D002455:Cell Division / Q000187:drug effects / Q000502:physiology; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D003497:Cyclic N-Oxides / Q000378:metabolism / Q000494:pharmacology*; D045304:Cytochromes c / Q000378:metabolism; D053595:Embryonic Stem Cells / Q000187:drug effects* / Q000378:metabolism / Q000528:radiation effects; D004996:Ethidium / Q000378:metabolism; D016195:G2 Phase / Q000187:drug effects / Q000502:physiology; D005720:Gamma Rays; D006801:Humans; D051379:Mice; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism*; D010718:Phosphatidylserines / Q000378:metabolism; D011092:Polyethylene Glycols / Q000494:pharmacology; D011832:Radiation Injuries / Q000517:prevention & control*; D017382:Reactive Oxygen Species / Q000378:metabolism; D013482:Superoxide Dismutase / Q000494:pharmacology; D013481:Superoxides / Q000378:metabolism; D013997:Time Factors
D002308:Cardiolipins; D003497:Cyclic N-Oxides; C527676:Hemi-GS-TEMPO 5-125; D010718:Phosphatidylserines; D017382:Reactive Oxygen Species; D013481:Superoxides; D011092:Polyethylene Glycols; D045304:Cytochromes c; D013482:Superoxide Dismutase; C063471:polyethylene glycol-superoxide dismutase; D004996:Ethidium