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Figure 1. Cytotoxicity and ATP production are measured in cancerous and normal hepatocytes using a known inducer of cellular necrosis. (All figures: BioTek Instruments)   

Mitochondria are responsible for more than 90% of a cell’s energy production via ATP (adenosine triphosphate) generation, in addition to playing a significant role in respiration and many signaling events within most eukaryotic cells. These intracellular powerhouses range in size and quantity within each cell depending on the organism and overall cell function.

Mitochondria consist of a semi-permeable outer membrane, a thin inter-membrane space where oxidative phosphorylation occurs, an impermeable inner membrane that is intricately folded to create layered compartments—or christae—and the matrix that contains ATP-producing enzymes and the organelle’s own independent genome. Each section has a highly specialized function, and any impairment within the organelle can lead to disease or disorders within the overall organism.

Mitochondrial dysfunction may be inherited, arise spontaneously, or develop as a result of drug toxicity. Inherited mitochondrial disorders can play a role in prevalent diseases such as cardiac disease and diabetes, and can also result in rare diseases such as Pearson syndrome or Leigh’s disease. Mitochondrial toxicity as a result of pharmaceutical use may damage key organs, such as the liver and heart. For example, nefazodone—a depression treatment—was withdrawn from the U.S. market after it was shown to significantly inhibit mitochondrial respiration in liver cells, leading to liver failure. Troglitazone, an anti-diabetic and anti-inflammatory, was withdrawn from all markets after research concluded that it caused acute mitochondrial membrane depolarization, also leading to liver failure. Drug recalls are costly to a manufacturer’s bottom line and reputation, and more importantly, can be harmful or even fatal to users. As drug discovery continues to evolve, much lead compound research now includes careful review of its interaction and potential toxicity with mitochondria.

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Figure 2. Primary hepatocytes grown in either high glucose or no glucose in the presence of the mitotoxicant antimycin show significant decrease in ATP production. The HepG2 cancer cell line grown in no glucose media displays a similar ATP decrease, while those grown in high glucose media have very little ATP decrease, thus representative of the Crabtree effect.   

Cell-based mitochondrial assays in microplate format may include mitochondrial membrane potential, total energy metabolism, oxygen consumption, and metabolic activity; and offer a truer environment for mitochondrial function in the presence of drug compounds compared to isolated mitochondria-based tests. Combining more than one assay in a multiplex format increases the amount of data per well while decreasing data variability arising from running the assays separately. The aggregated data also provides a more encompassing analysis of the drug’s effect on mitochondria than a single test.

One example, when testing compound effects on mitochondria, would be to measure cell membrane integrity as a function of cytotoxicity and mitochondrial function via ATP production concurrently, thus distinguishing between compounds that exhibit mitochondrial toxicity versus overt cytotoxicity. General cytotoxicity is characterized by a decrease in ATP production and a loss of membrane integrity whereas mitochondrial toxicity results in decreased ATP production with little to no change in membrane integrity. The assay’s efficiency is further enhanced via automation.

Robotic instrumentation ensures repeatable operation within the microplate wells when performing tasks such as cell dispensing, serial titration and transfer of compounds, and reagent dispensing. Additionally, by automating tasks within the assay process, researchers are free to attend to other tasks, reducing overall active time spent on the assay. Multi-mode microplate readers are compact instruments that can detect both fluorescent and luminescent signals. In addition, an automated process—including liquid handling and detection—can increase throughput capacity compared to manual methods.

Cytotoxicity in primary suspension hepatocytes, as well as immortalized human liver carcinoma cell line (HepG2), was first assessed by using a fluorogenic peptide substrate to measure the presence of a protease associated with necrosis; a measure of dead cell protease activity. As the substrate cannot cross live cell membranes, viable cells produce no signal. As shown in Figure 1, digitonin, a detergent known to permeabilize plasma membranes and cause cellular necrosis, was tested on HepG2 cells and primary hepatocytes. The resulting cytotoxicity assay signal increase in both cell lines, combined with decreased ATP concentrations, confirms the ability to measure digitonin’s necrosis-inducing characteristics.

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Figure 3. Z’-factor validation data using the ATP detection assay.   

After cytotoxicity is measured, an ATP detection reagent is added to the prepared cells, the cells are lysed, and an emitted luminescent signal is measured that is proportional to ATP levels as a measure of mitochondrial function. Per Figure 2, ATP production, as a function of the detected luminescent signal, substantially decreases in the hepatocyte and non-glucose/non-serum HepG2 cells (top graphs, bottom left graph). Cytotoxicity data proves that toxicity from antimycin is due to the compound impeding mitochondrial function, and not a result of primary necrosis.

An important note when measuring ATP production is the phenomenon known as the Crabtree effect. While primary cells rely on mitochondrial oxidative phosphorylation to generate ATP, cancer cells and other highly proliferative cells rely instead on glycolysis to produce ATP when grown using typical high glucose media. This mechanism can cause false negative results in the assay even when the tested drug normally induces mitochondrial toxicity in vivo as demonstrated via HepG2 cells grown in high glucose/serum containing media (Figure 2, bottom right). Only when glucose is substituted with galactose in culture will the cancer cells revert back to ATP via oxidative phosphorylation and offer a truer indication of toxic potential in vivo.

Z’-factor assays were performed to validate the automated HepG2 and primary hepatocyte assays using antimycin as the control mitotoxicant.  Per Figure 3, ATP detection assay Z’-factor values are indicative of a useful assay. (Zhang JH, et al. J Biomol Screen. 1999; 4(2):67.) The difference in the signal-to-background ratio seen between the two cell models is due to the inability of antimycin to completely kill the hepatocytes, which leads to a higher signal from the negative control.

Along with testing whole cells, and prior to testing organs and complete systems, it is vitally important to understand how the compound interacts with mitochondria and other intracellular organisms.

Multiplexed cell-based mitochondrial assays increase sample throughput and decrease variability, costs, and overall time for project completion. Automating the process with robotic instrumentation allows for rapid compound profiling, repeatability, further throughput increase, and decreased per-assay and overall project time.

The authors would like to thank Tim Moeller, Celsis IVT(Baltimore, Md.) and Tracy Worzella, Promega Corp. (Madison, Wis.) for their contribution to this research.

About the authors
Brad Larson is a principal scientist at BioTek Instruments, where he optimizes drug discovery assay chemistries on BioTek’s microplate instrumentation including kinase, GPCR, histone deacetylase, drug metabolism, and toxicity assays. Peter Banks is scientific director at BioTek Instruments; providing key scientific leadership and direction in emerging trends, opportunities and scientific discovery across the company’s global business platforms.

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