Aim: To assess the potential for mitochondrial toxicity in an in vitro experimental system.

Background information: Disruption of mitochondrial homeostasis is a known cellular mechanism associated to adverse drug reactions which can impact on drug attrition. Early assessment of potential for mitochondrial toxicity of individual compounds or lead pharmaceutical series early in the drug discovery process increases the chance of successfully selecting those with better safety characteristics.

Several mammalian cell lines developed for in vitro culture have metabolically adapted to grow under anaerobic and hypoxic conditions by use of culture media with high glucose concentrations. As consequence, most of their energy is generated from glycolysis instead of mitochondrial oxidative phosphorylation (the “Crabtree” effect), thereby reducing cell susceptibility to mitochondrial toxicants. Replacing glucose by galactose in the culture media avoids the Crabtree effect and increases the cells’ dependence to generate ATP by mitochondrial oxidative phosphorylation. By comparing the cytotoxic effect of the exposure to a test item in culture media containing glucose with that of a culture media containing galactose the potential deleterious effect on mitochondrial function can be assessed.

In this assay, cell cytotoxicity is assessed by means of the MTT assay, based on the ability of living functional cells to metabolically cleave the yellow coloured and water-soluble tetrazolium ring (MTT), into purple coloured non-water-soluble formazan crystals, this being an indicator of the integrity and functional level of the mitochondria.