cytochrome-c-t and idebenone

Digitonin solubilizes mitochondrial membrane, breaks the integrity of the respiratory chain and releases two mobile redox-active components: coenzyme Q (CoQ) and cytochrome c (cyt c). In the present study we report the inhibition of glycerol-3-phosphate- and succinate-dependent oxygen consumption rates by digitonin treatment. Our results show that the inhibition of oxygen consumption rates is recovered by the addition of exogenous synthetic analog of CoQ idebenone (hydroxydecyl-ubiquinone; IDB) and cyt c. Glycerol-3-phosphate oxidation rate is recovered to 148 % of control values, whereas succinate-dependent oxidation rate only to 68 %. We find a similar effect on the activities of glycerol-3-phosphate and succinate cytochrome c oxidoreductase. Our results also indicate that succinate-dependent oxidation is less sensitive to digitonin treatment and less activated by IDB in comparison with glycerol-3-phosphate-dependent oxidation. These findings might indicate the different mechanism of the electron transfer from two flavoprotein-dependent dehydrogenases (glycerol-3-phosphate dehydrogenase and succinate dehydrogenase) localized on the outer and inner face of the inner mitochondrial membrane, respectively.

Topics: Animals; Cytochromes c; Digitonin; Disease Models, Animal; Dose-Response Relationship, Drug; Glycerolphosphate Dehydrogenase; Glycerophosphates; Hyperthyroidism; Kinetics; Male; Mitochondria, Liver; Mitochondrial Membranes; Oxidation-Reduction; Oxygen Consumption; Rats; Rats, Wistar; Recovery of Function; Succinate Cytochrome c Oxidoreductase; Succinic Acid; Ubiquinone

Idebenone is a coenzyme Q10 analog and an antioxidant that has been used clinically to treat Friedreich Ataxia. Being an antioxidant, idebenone could have potential therapeutic potential to treat other neurodegenerative diseases such as Parkinson's disease in which oxidative stress plays a role in their pathogenesis. But whether idebenone can be used to treat Parkinson's disease has not been evaluated. In this study, we found that exposure of the dopaminergic neuroblastoma SHSY-5Y cells to 1-10 μM idebenone for 72 h had no effect on the cell viability revealed by trypan blue exclusion assay and MTT assay. However, cells exposed to 25 μM or higher concentrations of idebenone showed extensive trypan blue-positive staining and significant reduction in cell viability revealed by MTT assay indicating that most of the cells were no longer viable. Idebenone-induced cell death was characterized by genomic DNA fragmentation and accumulation of cytochrome c in the cytosol indicating that the death was apoptotic in nature. In addition, idebenone induced an increase in the total RNA of the pro-apoptosis protein BAX, it also increased the caspase-3 activity in the cell lysates when compared with the untreated control cells or cells exposed to 10 μM or lower concentrations of idebenone. The detrimental effect of idebenone was attenuated by glutathione, an antioxidant, suggesting that oxidative stress contributed to the idebenone-induced cell death. In conclusion, our results suggest that antioxidant idebenone induced apoptosis when used in high concentrations.

Topics: Antioxidants; Apoptosis; Caspase 3; Cell Line, Tumor; Cell Survival; Cytochromes c; DNA Fragmentation; Dopamine; Dose-Response Relationship, Drug; Glutathione; Humans; Neuroblastoma; Tetrazolium Salts; Thiazoles; Time Factors; Trypan Blue; Ubiquinone