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Real-Time Assessment of Mitochondrial Toxicity in HepG2 Cells Using the Seahorse Extracellular Flux Analyzer
Jether Amos Espinosa1, Grace Pohan1, Michelle R Arkin1
1Small Molecule Discovery Center and Department of Pharmaceutical Chemistry, University of California, San Francisco, California.
Abstract:
The liver is the primary organ responsible for drug detoxification. Drug-induced liver injury (DILI) is a leading cause of attrition during drug development and is one of the main reasons that drugs are withdrawn from the market. Hence, the prevention of DILI plays a central role in the overall drug-discovery process. Most of the liver's energy supply comes in the form of adenosine triphosphate (ATP), which is largely generated by mitochondria. This article describes the evaluation of drug-induced mitochondrial dysfunction using the Seahorse Extracellular Flux Analyzer (Agilent). The described protocols detail the accurate measurement of ATP production rate in HepG2 cells after exposure to a panel of potentially toxic compounds. This assay measures changes in extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) as indicators of glycolysis and mitochondrial respiration-the two major energy-generating pathways in a cell. This assay provides a useful model to predict mitochondrial dysfunction-mediated DILI. © 2021 Wiley Periodicals LLC. Basic Protocol: Measurement of cellular ECAR, OCR, and ATP production in live HepG2 cells Support Protocol 1: Culturing and maintaining of HepG2 cells Support Protocol 2: Determining optimal cell density per well.
Insights
This study introduces a method to assess drug-induced liver injury by measuring cellular energy production in HepG2 cells. The Seahorse Analyzer quantifies mitochondrial dysfunction, aiding in the prediction of drug toxicity during development.
Area of Science:
- Hepatology
- Toxicology
- Biochemistry
Background:
- Drug-induced liver injury (DILI) is a significant challenge in drug development and market withdrawal.
- Mitochondria are crucial for liver energy metabolism, primarily through adenosine triphosphate (ATP) generation.
- Mitochondrial dysfunction is a key mechanism underlying DILI.
Purpose of the Study:
- To evaluate drug-induced mitochondrial dysfunction as a predictor of DILI.
- To establish a protocol for measuring ATP production rate in HepG2 cells exposed to toxic compounds.
- To utilize the Seahorse Extracellular Flux Analyzer for assessing cellular energy metabolism.
Main Methods:
- Utilized HepG2 cells, a human liver cell line.
- Employed the Seahorse Extracellular Flux Analyzer to measure extracellular acidification rate (ECAR) and oxygen consumption rate (OCR).
- Quantified ATP production rate following exposure to a panel of potentially toxic compounds.
Main Results:
- The assay accurately measured changes in ECAR and OCR, reflecting glycolysis and mitochondrial respiration.
- Demonstrated the capability to assess ATP production rate in response to drug exposure.
- Established a model for predicting mitochondrial dysfunction-mediated DILI.
Conclusions:
- The Seahorse assay provides a reliable method for evaluating drug-induced mitochondrial dysfunction.
- This approach can serve as a valuable tool in predicting DILI early in the drug discovery process.
- Optimizing HepG2 cell culture and density is crucial for accurate assay results.
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