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Mitochondrial Toxicity Glu/Gal Assay (MitoTox Glo)

Mitochondrial dysfunction due to drug-induced effects, has been highlighted as a potential contributor to a number of organ toxicities including those associated with cardiac, muscular and liver systems (1).

Mitochondrial toxicity has been indicated as a contributing cause of DILI and the pre- and post-market withdrawal of several high profile pharmaceuticals as well as predominant liability in a number of pharmaceuticals with Black Box Warnings and precautionary drug labelling (2,3). Several drugs have been implicated in mitochondrial liabilities such as Amiodarone, Troglitazone, Tolcapone and Cerivastatin, which have also been indicated in other contributing DILI mechanisms (3,4).

In eukaryotic mammalian cells, mitochondria are the principal contributors for over 90 % of cellular energy in the form of ATP and are essential for both cellular metabolism and synthesis processes. Many immortalised cell models used in drug discovery in vitro studies, have metabolic adaptations for growth under non in vivo-like conditions, allowing for growth in glucose-rich media, harnessing cellular energy from glycolysis and not oxidative phosphorylation.

This process is termed the Crabtree effect (5) and has been indicated as reducing the effects of mitochondrial toxicants. By replacing glucose growth media with galactose, cellular metabolism is switched from glycolysis to oxidative phosphorylation, enhancing cellular susceptibility to mitochondrial toxicants.

The Mitochondrial Tox Glo assay provides multiplexed output for ATP depletion and cytotoxicity (membrane integrity) providing greater sensitivity over other mitochondrial toxicity assays as ATP depletion, and hence mitochondrial dysfunction can be observed in the absence of cell death (3).

Sygnature Discovery’s DMPK group assesses mitochondrial toxicity in HepG2 cell model (alternative cell lines could be available upon request).



Culture media Glucose (DMEM consisting of 25 mM Glucose)

Galactose (DMEM consisting of 10 mM Galactose)

Cell culture HepG2 cells cultured in Glucose or Galactose media respectively
Test compound titrations Half log dilutions from 100 µM – 0.0003 µM, final DMSO concentration 0.3 %. (Alternative regimes available)
Standard assay formation 96-well microplate format – 3 x test compounds, 1 x mitochondrial toxicity control and 1 x non tox control in duplicate
Required article concentration 200μL of a 100 mM DMSO stock solution or 2mg of solid
Method of analysis Cells are analysed for ATP depletion and increase in cytotoxicity (membrane integrity)
Results/data delivery EC50 for ATP and Cytotoxicity,   ratios for EC50 ATP glu/gal and EC50 CYTO gal /

EC50 ATP gal. Mitochondrial tox risk rating



EC50 ATP glu/gal >2 indicates potential mitochondrial toxicity.

EC150 CYTOgal/gal >2 indicates mitochondrial toxicity precedes cell death.


Figure 1. The effect of compound exposure on ATP depletion and cytotoxicity (membrane integrity) in HepG2 cells.  Cells were incubated with test compounds for 90 minutes in glucose or galactose containing medium and the effect on ATP depletion (closed circles, glucose; open circles, galactose) and membrane integrity (closed triangles, glucose; open triangles, galactose) were assessed.  Values represent mean +/- S.E.M. N=2 experiments in conducted in duplicate.




Table 1. EC50 values ratios and mitochondrial risk prediction for test compounds.

About Us

The DMPK & Physical Sciences department at Sygnature Discovery is dedicated to understanding and optimising the absorption, distribution, metabolism and excretion of drug candidates by working in close partnership with clients and other departments within Sygnature to provide successful optimisation strategies.

We have extensive know-how and expertise to provide well validated, state-of-the-art assays and a comprehensive applied consultancy service for interpretation of the in vitro ADME and in vivo PK data.

Our corporate vision is to accelerate the discovery of new medicines, from the laboratory into development to treat patients.

Our DMPK mission is to deliver tailored DMPK expertise through innovation, quality and commitment.



  1. Dykens JA, Marroquin LD, Will Y. Strategies to reduce late-stage drug attrition due to mitochondrial toxicity. Expert Rev Mol Diagn. 2007 Mar 1;7(2):161–75.
  2. Labbe G, Pessayre D, Fromenty B. Drug-induced liver injury through mitochondrial dysfunction: mechanisms and detection during preclinical safety studies. Fundam Clin Pharmacol. 2008 Aug;22(4):335–53.
  3. Kamalian L, Chadwick AE, Bayliss M, French NS, Monshouwer M, Snoeys J, et al. The utility of HepG2 cells to identify direct mitochondrial dysfunction in the absence of cell death. Toxicol Vitr. 2015;29(4):732–40.
  4. Dykens JA, Will Y. The significance of mitochondrial toxicity testing in drug development. Drug Discov Today. 2007;12(17-18):777–85.
  5. Marroquin LD, Hynes J, Dykens JA, Jamieson JD, Will Y. Circumventing the Crabtree Effect: Replacing Media Glucose with Galactose Increases Susceptibility of HepG2 Cells to Mitochondrial Toxicants. Toxicol Sci . 2007 Jun 1;97 (2 ):539–47.



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