A Mitochondria-Targeted Nitroxide/Hemigramicidin S Conjugate Protects Mouse Embryonic Cells Against Gamma Irradiation

Abstract

Purpose-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 γ-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 and Materials-Cells were incubated with 5-125 before (10 minutes) or after (1 hour) γ-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 μM) effectively suppressed γ-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 γ-irradiated cells. In addition, 5-125 enhanced and prolonged γ-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.

Designing inhibitors of cytochrome c/cardiolipin peroxidase complexes: mitochondria-targeted imidazole-substituted fatty acids

“…We further attempted to validate the significance of the chemical/biochemical and computational predictions using a model of irradiation induced apoptosis in MECs [53] treated with TPP- n- ISAs. Two apoptosis hallmarks, i.e.…”

Oxidative lipidomics of hyperoxic acute lung injury: mass spectrometric characterization of cardiolipin and phosphatidylserine peroxidation

“…Recently, we (29,74) have developed mitochondria-targeted electron scavengers, conjugates of GS with nitroxide radicals (GS-nitroxides), capable of preventing reactive oxygen species production and CL peroxidation in mitochondria. We reasoned that GS-nitroxides may be effective in suppressing hyperoxia-induced phospholipid peroxidation in MLEC.…”

Interplay between bax, reactive oxygen species production, and cardiolipin oxidation during apoptosis

“…Recently, we reported that mitochondria targeted electron scavengers -hemigramicidin S conjugated nitroxides -prevented both superoxide accumulation and CL peroxidation and apoptosis initiated by ActD or irradiation without affecting Bax translocation [27,28]. Combined with the results of this work showing that Bax/Bak translocation precedes superoxide production and CL peroxidation and that genetic ablation of Bax/Bak inhibited ActD and rotenone induced CL oxidation and apoptosis, we speculate that CL peroxidation not only correlates but is likely causatively and time-dependently related to mitochondrial relocation of Bax/Bak.…”