Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2021 Mar;1(3):e75.
doi: 10.1002/cpz1.75.

Real-Time Assessment of Mitochondrial Toxicity in HepG2 Cells Using the Seahorse Extracellular Flux Analyzer

Jether Amos Espinosa  1 , Grace Pohan  1 , Michelle R Arkin  1 , Sarine Markossian  1   2
Affiliations

Real-Time Assessment of Mitochondrial Toxicity in HepG2 Cells Using the Seahorse Extracellular Flux Analyzer

Jether Amos Espinosa et al. Curr Protoc. 2021 Mar.

Erratum in

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injury (DILI) is a leading cause of attrition during drug development and is one of the main reasons that drugs are withdrawn from the market.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.

Keywords: ATP production; ECAR; HepG2; OCR; PER; Seahorse; drug-induced liver injury (DILI); glycolysis; mitochondrial respiration; oxidative phosphorylation.

PubMed Disclaimer

References

Literature Cited

References
    1. Chan, R., & Benet, L. Z. (2017). Evaluation of DILI predictive hypotheses in early drug development. Chemical Research in Toxicology, 30(4), 1017-1029. doi: 10.1021/acs.chemrestox.7b00025.
    1. Depaoli, M. R., Karsten, F., Madreiter-Sokolowski, C. T., Klec, C., Gottschalk, B., Bischof, H., … Malli, R. (2018). Real-time imaging of mitochondrial ATP dynamics reveals the metabolic setting of single cells. Cell Reports, 25(2), 501-512.e3. doi: 10.1016/j.celrep.2018.09.027.
    1. Duewelhenke, N., Krut, O., & Eysel, P. (2007). Influence on mitochondria and cytotoxicity of different antibiotics administered in high concentrations on primary human osteoblasts and cell lines. Antimicrobial Agents and Chemotherapy, 51(1), 54-63. doi: 10.1128/AAC.00729-05.
    1. Fu, D., & Lippincott-Schwartz, J. (2018). Monitoring the effects of pharmacological reagents on mitochondrial morphology. Current Protocols in Cell Biology, 79(1), e45. doi: 10.1002/cpcb.45.
    1. Gaignard, P., Liere, P., Thérond, P., Schumacher, M., Slama, A., & Guennoun, R. (2017). Role of sex hormones on brain mitochondrial function, with special reference to aging and neurodegenerative diseases. Frontiers in Aging Neuroscience, 9, 406. doi: 10.3389/fnagi.2017.00406.
Internet Resource
    1. https://www.agilent.com/cs/library/usermanuals/public/103591-400_Seahors...

LinkOut - more resources