Maddalena 2014 Abstract MiP2014
|Experimental considerations for evaluating bioenergetic effects of a mitochondria-targeted compound, TPP-IOA.
Triphenylphosphonium-conjugated imidazole-substituted oleic acid (TPP-IOA) is an inhibitor of cytochrome c peroxidase activity that appears to initiate apoptotic cell death via cytochrome c release. As a molecule that interacts directly with cytochrome c, TPP-IOA may also affect mitochondrial respiration. We are evaluating the dose-effect relationships of TPP-IOA’s interactions with mitochondria.
Although original data came from cells maintained in glucose media in culture, the interpretation of these data is limited by the fact that under these conditions the cells derive little ATP from oxidative phosphorylation and have low mitochondrial densities. We are revisiting the effects of TPP-IOA on mitochondrial respiration and apoptotic cell death in glucose-free media, in which energy is derived from galactose and glutamine and cells are highly reliant on oxidative phosphorylation. Cells growing under these conditions are highly sensitive to drugs that may perturb oxidative phosphorylation. We are also evaluating the effects of 3-hydroxypropyl-TPP (TPP), the TPP moiety of TPP-IOA, on mitochondrial function in this system and in isolated mitochondria. At concentrations that effectively inhibit mitochondrial-mediated cell death in cells, TPP-IOA exerted significant effects on oxidative phosphorylation, e.g. reducing the respiratory control ratio. Conjugation to TPP is frequently used to promote accumulation of molecules in mitochondria. However, since TPP and linker molecules can themselves have effects on mitochondrial function it is necessary to evaluate these independently, an experimental control that is often not performed. We find similar effects of TPP and TPP-IOA on mitochondrial OXPHOS, suggesting that it is the TPP moiety that interferes with normal mitochondrial function in this assay.
Taken together, these preliminary data indicate the importance of (i) choosing appropriate cell culture conditions that promote reliance on oxidative phosphorylation to evaluate the effects of molecules that are targeted to mitochondria, and (ii) including experimental controls of TPP, since this moiety and linker group can have significant effects on mitochondrial function.
Labels: MiParea: Respiration, Pharmacology;toxicology
Preparation: Isolated mitochondria
Coupling state: OXPHOS
Event: C3, Poster MiP2014
1-Dep Biol Sc; 2-Dep Chem; 3Centre Biotechnology; Brock Univ, St. Catharines, Canada. – [email protected]