Assessment of ToxCast Phase II for Mitochondrial Liabilities Using a High-Throughput Respirometric Assay

Area of Science:

• Toxicology
• Mitochondrial Biology
• Cell Biology

Background:

• High-throughput screening for mitochondrial toxicants often uses immortalized cell lines and focuses on membrane potential, which may be insufficient.
• Previous methods may not capture diverse mitochondrial dysfunction mechanisms.

Purpose of the Study:

• To screen a large chemical library for mitochondrial toxicants using primary renal proximal tubule cells (RPTC).
• To compare the sensitivity of RPTC and immortalized human renal (HK2) cells to mitochondrial toxicants.
• To identify mechanisms and criteria for assessing chemical mitochondrial liabilities.

Main Methods:

• Screened 676 compounds from the ToxCast Phase II library against primary RPTC using Seahorse Extracellular Flux Analyzer.
• Assessed cytotoxicity, electron transport chain (ETC) inhibition, and uncoupling effects.
• Compared RPTC findings with results from immortalized HK2 cells exposed to a subset of compounds.

Main Results:

• 73% of tested compounds showed mitochondrial toxicity in RPTC, predominantly ETC inhibition.
• Primary RPTC identified 491 mitochondrial toxicants, including 3 cytotoxicants, 110 ETC inhibitors, and 5 uncouplers.
• Immortalized HK2 cells were significantly less sensitive, identifying only 6% of compounds as mitochondrial toxicants, with limited overlap in identified inhibitors and uncouplers.

Conclusions:

• Primary RPTC are more sensitive to a broader range of mitochondrial toxicants than immortalized HK2 cells.
• Immortalized cell lines may underestimate mitochondrial toxicity, necessitating caution in their use for screening.
• Findings provide valuable data for predicting and assessing mitochondrial liabilities of chemicals.