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Updated: Apr 22, 2026
Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue
Published on: March 23, 2015
Profiling of the Tox21 chemical collection for mitochondrial function to identify compounds that acutely decrease
Matias S Attene-Ramos1, Ruili Huang, Sam Michael
1National Center for Advancing Translational Sciences, National Institutes of Health (NIH), Department of Health and Human Services (DHHS), Bethesda, Maryland, USA.
Background:
Mitochondrial dysfunction has been implicated in the pathogenesis of a variety of disorders including cancer, diabetes, and neurodegenerative and cardiovascular diseases. Understanding whether different environmental chemicals and druglike molecules impact mitochondrial function represents an initial step in predicting exposure-related toxicity and defining a possible role for such compounds in the onset of various diseases.
Objectives:
We sought to identify individual chemicals and general structural features associated with changes in mitochondrial membrane potential (MMP).
Methods:
We used a multiplexed [two end points in one screen; MMP and adenosine triphosphate (ATP) content] quantitative high throughput screening (qHTS) approach combined with informatics tools to screen the Tox21 library of 10,000 compounds (~ 8,300 unique chemicals) at 15 concentrations each in triplicate to identify chemicals and structural features that are associated with changes in MMP in HepG2 cells.
Results:
Approximately 11% of the compounds (913 unique compounds) decreased MMP after 1 hr of treatment without affecting cell viability (ATP content). In addition, 309 compounds decreased MMP over a concentration range that also produced measurable cytotoxicity [half maximal inhibitory concentration (IC50) in MMP assay/IC50 in viability assay ≤ 3; p < 0.05]. More than 11% of the structural clusters that constitute the Tox21 library (76 of 651 clusters) were significantly enriched for compounds that decreased the MMP.
Conclusions:
Our multiplexed qHTS approach allowed us to generate a robust and reliable data set to evaluate the ability of thousands of drugs and environmental compounds to decrease MMP. The use of structure-based clustering analysis allowed us to identify molecular features that are likely responsible for the observed activity.
Insights
Researchers screened thousands of chemicals to find those impacting mitochondrial membrane potential (MMP). This study identified specific compounds and structural features that decrease MMP, aiding in predicting chemical toxicity and disease links.
Area of Science:
- Toxicology
- Mitochondrial Biology
- Chemical Screening
Background:
- Mitochondrial dysfunction is linked to diseases like cancer, diabetes, and neurodegeneration.
- Environmental chemicals and drugs can impact mitochondrial function, influencing toxicity and disease onset.
Purpose of the Study:
- Identify specific chemicals and structural features that alter mitochondrial membrane potential (MMP).
- Predict chemical toxicity and potential roles in disease pathogenesis.
Main Methods:
- Utilized a multiplexed quantitative high-throughput screening (qHTS) assay measuring MMP and adenosine triphosphate (ATP) content.
- Screened the Tox21 library of ~8,300 unique chemicals at 15 concentrations in triplicate using HepG2 cells.
- Employed informatics and structure-based clustering analysis.
Main Results:
- Approximately 11% of compounds (913 unique) decreased MMP without affecting cell viability (ATP).
- 309 compounds decreased MMP at concentrations also causing cytotoxicity.
- Over 11% of structural clusters were enriched for MMP-decreasing compounds.
Conclusions:
- The multiplexed qHTS approach generated a reliable dataset for evaluating MMP-decreasing compounds.
- Structure-based clustering identified molecular features responsible for observed MMP changes.
