Accurate assessment of mitochondrial toxicity is essential for the safe development of pharmaceuticals, prevention of unwanted toxic side effects that lead to drug withdrawals, and understanding the aetiology of mitochondrial-related diseases, such as Parkinson’s. The ever increasing awareness of mitochondrial involvement in disease processes, toxicity and cell death and has led to significant interest in the development of high throughput assays to identify mitochondrial function and morphology.

Mitochondrial function

Mitochondria are often described as the ‘powerhouse of cells’ as they generate 95% of cellular energy in the form of adenosine triphosphate (ATP). ATP is produced by oxidative phosphorylation during aerobic respiration, a process which is dependent upon the electrochemical gradient across the inner mitochondrial membrane. Mitochondria also have key functions in cellular calcium homeostasis, lipid metabolism, steroid hormone synthesis, redox signaling and cell death (Figure 1).

Mitochondrial toxicity can have a wide range of consequences including cell injury, cell death and even organ failure and death. Although mechanisms and consequences vary depending upon the cause, oxidative phosphorylation is often targeted leading to loss of ATP production. Figure 1 highlights some characteristics of mitochondrial toxicity and dysfunction. Permeabilization of the outer mitochondrial membrane is classed as the point of no return for the cell as this leads to the release of mitochondrial proteins, such as cytochrome c, into the cytoplasm, activation of apoptosis and ultimately cell death.

Figure 1. Mitochondrial function and dysfunction

Mitochondrial disruption assays

Historically, mitochondrial toxicity assays relied on measurements of activity using an electrode to measure oxygen consumption of cells. However, these types of assays do not lend themselves well to high throughput drug screening which has led to the development of new imaging based methods to identify mitochondrial disruption in vitro. These in vitro experiments typically require that cell cultures are treated with a compound of interest prior to fluorescence labelling, imaging and analysis.

Mitotoxicity is often subtle and difficult to detect in vitro as the immortalized cell lines used are adapted to growth in anaerobic conditions, deriving energy from glycolysis, rather than oxidative phosphorylation. Replacing glucose in culture media with galactose can increase cellular reliance on oxidative phosphorylation, rendering cells more susceptible to cell death via mitochondrial disruption. The results can then be compared to those in traditional glucose medium (the ‘glu/gal’ assay) to identify toxic compounds.

There are now a wide range of fluorescent probes available that can detect more subtle markers of mitochondrial toxicity or distress, including loss of membrane potential, changes in pH, changes in calcium levels and Reactive Oxygen Species (ROS)/superoxide generation. Additional information can be gained by the assessment of mitochondrial morphology including size, length, shape and fragmentation; machine learning algorithms are often employed to simplify this type of classification process.

Example Fluorescent Probes

Membrane potential s (Δψm) dye: Rhodamine 123, tetramethylrhodamine ethyl ester (TMRE) and the fixable CMXRos are examples of cationic membrane potential dependent probes whose fluorescent intensity represents the condition of the mitochondria (Lu, 2018; Poot, 1996). Some dyes are suited to measuring loss of Δψm, such as THE JC-1/9/10 fluorescent dyes (Sivandzade, 2019). Inside the mitochondria, these probes exist either as monomers and fluoresce green (low membrane potential) or aggregates and fluoresce red (high membrane potential). Therefore the loss of red/orange fluorescence coincides with loss of membrane potential.

Calcium indicators: Increases in mitochondrial calcium levels are thought to cause the mitochondrial permeability transition pore (mPTP) to open, which dissipates the proton gradient and compromises the membrane potential as well as allowing mitochondrial proteins to enter the cytoplasm. Calcium levels can be detected by fluorescent mitochondrial calcium indicators, such as Rhod-2 AM (Perez, 2018). Cellular calcium indicators such as calcein-AM can also be used to assess mPTP opening by combining with cytoplasmic cobalt quenching, such as that used in the calcein/cobalt mPTP assay (Petronilli, 1999).

ROS detectors: Mitochondrial superoxide builds up due to the incomplete oxidation of oxygen during respiration causing oxidative stress. This can be detected by mitochondrial superoxide detectors such as mitosoxtm (Dikalov, 2014; Davidson, 2018).

Cytochrome c release: Increases in mitochondrial permeability leads to cytochrome c release into the cytoplasm, which can then activate apoptosis. Cytochrome c can be detected by immunofluorescence staining or stable cell lines genetically engineered to express fluorescent cytochrome c (Matapurkar, 2006; Willhite, 2003). Alternatively, new nanosensors, such as the nitrogen doped carbon dots, are being developed that permit direct detection of cytochrome c release (Zhang H. Z., 2018).

Lactate accumulation. When mitochondrial function is impaired, cells shift away from oxidative phosphorylation towards glycolytic activity. This leads to an increase in lactate accumulation in the cytoplasm, increased oxygen consumption rate (OCR) and extracellular acidification of the surrounding culture media. This traditionally involved repeated measurements of extracellular pH and oxygen consumption. Alternatively, to visualize this directly, methods use cell lines expressing a genetically encoded FRET sensor for lactate, called Laconic, to identify increases in lactate accumulation (Contreras-Baeza, 2019).

Applications of assays

Assessment of mitochondrial disruption is essential in a variety of different fields including drug development, neurodegenerative research, aging and cancer.

Neurons are particularly susceptible to mitochondrial disease because of their reliance on ATP production and mitochondrial buffering of calcium. Mitochondrial disruption is implicated in many neurological and neurodegenerative diseases, such as Parkinson’s, Huntington’s, Alzheimer’s, Epilepsy and Schizophrenia. For example, in Parkinson’s disease models, MPP+ toxicity is thought to occur via its accumulation in the mitochondria and subsequent ATP depletion, ROS induction and subsequent cell death. Protecting the function of mitochondria can therefore offer an effective therapeutic approach.

Mitochondrial dysfunction is a major cause of unwanted drug induced toxicity. In fact, mitochondrial toxicity is one of the leading causes of post-market drug withdrawals, such as for Troglitazone and Cerivastatin. Therefore, early profiling of drug candidates for mitochondrial toxicity is an essential component in drug discovery to prevent approval of toxic compounds, unnecessary side effects for patients and in the worst cases, unnecessary deaths.

In drug screening, there are often huge numbers of compounds that must be assessed in a fast, accurate and high throughput manner. High content screening can be employed to examine large numbers of compounds with multiple toxicity markers, including mitochondrial dysfunction and cell viability, using multi-well plates (i.e 96 or 384 wells). In order to get an accurate picture of mitochondrial function it is necessary to also acquire information on the number of mitochondria, the number of cells and cell cycle stages. Automated analysis is desirable to permit fast analysis of these parameters to give accurate results with minimal user input and quickly identify drug candidates based on their safety profiles.


Mitochondria are essential for normal functioning of cells; their disruption can lead to changes in energy production, calcium homeostasis, induction of ROS, and ultimately cell death. This has been implicated in many disease processes, such as Parkinson’s, and the post-market withdrawal of a host of drugs. Accurately assessing mitochondrial toxicity is now recognized as essential for preventing safety issues during drug development and approval. The use of specialized mitochondrial toxicity assays in combination with other fluorescent labelling methods and cytotoxicity markers is instrumental in the safe and effective generation of new drug therapies.



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