Mitochondria targeting of non-peroxidizable triphenylphosphonium conjugated oleic acid protects mouse embryonic cells against apoptosis: role of cardiolipin remodeling.

Peroxidation of cardiolipin in mitochondria is essential for the execution of apoptosis. We suggested that integration of oleic acid into cardiolipin generates non-oxidizable cardiolipin species hence protects cells against apoptosis. We synthesized mitochondria-targeted triphenylphosphonium oleic acid ester. Using lipidomics analysis we found that pretreatment of mouse embryonic cells with triphenylphosphonium oleic acid ester resulted in decreased contents of polyunsaturated cardiolipins and elevation of its species containing oleic acid residues. This caused suppression of apoptosis induced by actinomycin D. Triacsin C, an inhibitor of acyl-CoA synthase, blocked integration of oleic acid into cardiolipin and restored cell sensitivity to apoptosis.

A mitochondria-targeted triphenylphosphonium-conjugated nitroxide functions as a radioprotector/mitigator.

Removal of excessive mitochondrial reactive oxygen species by electron scavengers and antioxidants is a promising therapeutic strategy to reduce the detrimental effects of radiation exposure. Here we exploited triphenylphosphonium (TPP) cation as a means to target nitroxide radicals to mitochondria. We synthesized a library of TPP-conjugated nitroxides and tested their radioprotective effects in gamma-irradiated mouse embryo cells and human epithelial BEAS-2B cells. Cells were incubated with conjugates either before or after irradiation. We found that [2-(1-oxyl-2,2,6,6-tetramethyl-piperidin-4-ylimino)-ethyl]-triphenyl-phosphonium (TPEY-Tempo) significantly blocked radiation-induced apoptosis as revealed by externalization of phosphatidylserine on the cell surface and inhibition of cytochrome c release from mitochondria. Using electron paramagnetic resonance, we showed that TPEY-Tempo was integrated into cells and mitochondria, where it underwent one-electron reduction to hydroxylamine. TPEY-Tempo acted as an electron scavenger that prevented superoxide generation and cardiolipin oxidation in mitochondria. Finally, TPEY-Tempo increased the clonogenic survival rate of irradiated cells. The cellular integration efficiencies of nonradioprotective TPP conjugates, including Mito-Tempo (Alexis, San Diego, CA), were markedly lower, although these homologues were integrated into isolated succinate-energized mitochondria to a similar extent as TPEY-Tempo. We conclude that mitochondrial targeting of TPP-conjugated nitroxides represents a promising approach for the development of novel radioprotectors.

Mitochondrial targeting of electron scavenging antioxidants: Regulation of selective oxidation vs random chain reactions.

Effective regulation of highly compartmentalized production of reactive oxygen species and peroxidation reactions in mitochondria requires targeting of small molecule antioxidants and antioxidant enzymes into the organelles. This review describes recently developed approaches to mitochondrial targeting of small biologically active molecules based on: (i) preferential accumulation in mitochondria because of their hydrophobicity and positive charge (hydrophobic cations), (ii) binding with high affinity to an intra-mitochondrial constituent, and (iii) metabolic conversions by specific mitochondrial enzymes to reveal an active entity. In addition, targeted delivery of antioxidant enzymes via expression of leader sequences directing the proteins into mitochondria is considered. Examples of successful antioxidant and anti-apoptotic protection based on the ability of targeted cargoes to inhibit cytochrome c-catalyzed peroxidation of a mitochondria-specific phospholipid cardiolipin, in vitro and in vivo are presented. Particular emphasis is placed on the employment of triphenylphosphonium- and hemi-gramicidin S-moieties as two effective vehicles for mitochondrial delivery of antioxidants.

Mass-spectrometric analysis of hydroperoxy- and hydroxy-derivatives of cardiolipin and phosphatidylserine in cells and tissues induced by pro-apoptotic and pro-inflammatory stimuli.

Oxidation of two anionic phospholipids--cardiolipin (CL) in mitochondria and phosphatidylserine (PS) in extramitochondrial compartments--is important signaling event, particularly during the execution of programmed cell death and clearance of apoptotic cells. Quantitative analysis of CL and PS oxidation products is central to understanding their molecular mechanisms of action. We combined the identification of diverse phospholipid molecular species by ESI-MS with quantitative assessments of lipid hydroperoxides using a fluorescence HPLC-based protocol. We characterized CL and PS oxidation products formed in a model system (cyt c/H(2)O(2)), in apoptotic cells (neurons, pulmonary artery endothelial cells) and mouse lung under inflammatory/oxidative stress conditions (hyperoxia, inhalation of single walled carbon nanotubes). Our results demonstrate the usefulness of this approach for quantitative assessments, identification of individual molecular species and structural characterization of anionic phospholipids that are involved in oxidative modification in cells and tissues.