The mitochondrial toxicity assays provide a means to measure mitochondrial dysfunction due to the toxic effect of a test compound. We provide mitochondrial toxicity assays for all small molecule formulations such as pharmaceuticals, industrial chemicals and consumer products.
Our mitochondrial toxicity assays use a genetically modified version of the immortalized human liver cell line HepaRG™, created with our proprietary CompoZr® zinc finger nuclease (ZFN) technology. Apart from fresh human hepatocytes, HepaRG™ cells are the most metabolically active liver cell line described to date and have the potential for use as a viable surrogate in many functional liver assays, including toxicity testing, with none of the drawbacks of limited availability and donor-to-donor variation.
Evaluating mitochondrial toxicity is an important component in the overall assessment of drug safety. This is due in part to the recognition that mitochondrial dysfunction is a contributor to compound attrition and post-market drug withdrawals (e.g., nefazodone and troglitazone). In the past, these types of effects were often missed in part because they are subtle and only manifest themselves over time; thus, they do not lead to overt histopathology within the time frame of most pre-clinical studies. A second reason is that most early discovery toxicity screens use highly proliferative immortalized cell lines, which are adapted for rapid growth under hypoxic and acidic conditions. These cell lines derive almost all of their energy from glycolysis rather than mitochondrial oxidative phosphorylation, and thus the common use of high glucose media with these cell lines masks the effects of potential mitochondrial toxicants. This is referred to as the Crabtree effect and can be addressed by comparing the cytotoxic effect of drug candidates in glucose versus galactose supplemented media. Another common mechanism of mitochondrial toxicity is the loss of mitochondrial membrane potential.
We offers two format for assessing mitochondrial toxicity:
The mitochondrial membrane potential assay uses JC-10 to screen for mitochondrial depolarization in cell lines. JC-10 (a more water-soluble analog of JC-1) is a cationic dye that accumulates in energized mitochondria. At high membrane potentials, the dye aggregates yielding a red/orange-colored emission, while at low membrane potentials the dye exists as a monomer and emits a green fluorescence. Mitochondrial membrane potential can therefore be monitored by following the loss of red/orange fluorescence. FCCP (carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone), an uncoupler of oxidative phosphorylation, is used as a positive control.