Mitochondrial Toxicity

CXR has the ability to run a large number of in vitro mitochondrial toxicity assays.

The optimal choice of test systems and endpoints we would recommend depends very much on the stage of development, number of compounds, and whether there is any particular concern around specific modes of toxicity or tissue/organ toxicity.

The following assays are currently available (not HTS) in isolated mitochondria and/or cell cultures (primary or immortalised):

ATP concentration
Fluorescence/spectrophotometric

Respiration and respiratory control (ratio of state3/state 4)
Oxygen consumption (oxygen electrode and spectrophotometer)

Mitochondrial membrane potential
Fluorescence/spectrophotometric

Mitochondrial swelling (isolated mitochondria only)
Spectrophotometric

Reactive Oxygen Species and GSH concentrations
Fluorescence/spectrophotometric

Fatty acid beta-oxidation (usually in isolated mitochondria)
14C-fatty acid metabolism

Apoptosis
TUNEL or caspase 3 release, e.g. in cultured hepatocytes

Assaying dysfunction in isolated mitochondria

The simplest and most revealing test for energetic dysfunction in isolated mitochondria is to measure mitochondrial respiratory control. Absolute values of respiration rates in different states and conditions, and qualitative measurements of protomotive force (pmf), can clarify mechanisms, but the best way to gain mechanistic insight is to simultaneously titrate both respiration rate and pmf and plot the kinetics of the different modules of oxidative phosphorylation.

Candidate assays of the amounts or activities of specific complexes and enzymes, mitochondrial morphology and responses to specific stressors can be useful to test specific hypotheses, but should generally be held in reserve and not used as the primary assay for mitochondrial dysfunction.

Assaying mitochondrial dysfunction in intact cells

Again, the measurement of cell respiratory control is the single most useful general test of mitochondrial function in cells. The experiment is easy to perform using the appropriate apparatus, and yields information that allows a quick, simple and full assessment of the bioenergetic status of the cells: how fast they are turning over ATP, how well-coupled their mitochondria are, and how much spare respiratory capacity they have available to deal with energetic demands.

Measurements of mitochondrial membrane potential are subject to several pitfalls that need to be understood, but can also be very useful. The best way to gain mechanistic insight is to monitor both respiration and potential, to assay the mitochondrial proton circuit in situ. Other indirect assays of mitochondrial function in cells, such as measurements of dye reduction or total cell ATP, can be problematic.

Key references:

Brand and Nicholls (2011). Assessing mitochondrial dysfunction in cells. Biochem. J. (2011) 435, 297–312

Marroquin et al (2007). Circumventing the Crabtree Effect: Replacing Media Glucose with Galactose Increases Susceptibility of HepG2 Cells to Mitochondrial Toxicants. TOXICOLOGICAL SCIENCES 97(2), 539–547

For further information about our mitochondrial toxicity services, or to discuss a specific issue, please contact us.