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Respirometric Screening and Characterization of Mitochondrial Toxicities Induced by ToxCast Chemicals
Citation:
Simmons, Steve. Respirometric Screening and Characterization of Mitochondrial Toxicities Induced by ToxCast Chemicals. Presented at European Society of Toxicology (Eurotox), Brussels, N/A, BELGIUM, September 02 - 05, 2018. https://doi.org/10.23645/epacomptox.7078166
Impact/Purpose:
Many chemicals in the environment disrupt mitochondrial function and can subsequently cause liver, kidney and heart toxicity. A modified version of the Seahorse XF Analyzer mitochondrial stress test was developed to screen 1,051 chemical samples comprising the ToxCast Phase I and II chemical libraries using a tiered approach in human hepatocellular carcinoma (HepG2) cells. The data confirms that previous dye-based mitochondrial toxicity assays underestimated the number and potency of mitochondrial toxicants among the ToxCast Phase I and II chemicals.
Description:
Many chemicals in the environment disrupt mitochondrial function and can subsequently cause liver, kidney and heart toxicity. Current high-throughput screening approaches to identify mitochondrial toxicants use fluorescent dye-based endpoints that adeptly detect structural changes to the inner mitochondrial membrane and/or mitochondrial mass, but fail to adequately detect chemicals that impair mitochondrial function through other mechanisms such as inhibition of the electron transport chain (ETC). A modified version of the Seahorse XF Analyzer mitochondrial stress test was developed to screen 1,051 chemical samples comprising the ToxCast Phase I and II chemical libraries using a tiered approach in human hepatocellular carcinoma (HepG2) cells. The initial single-concentration (100 µM final) screen identified 246 putative mitochondrial toxicants (23.4%). Subsequent concentration-response screening followed by a post hoc cytotoxicity assay confirmed the mitochondrial-disrupting activity first tier actives and provided potency data. Tracking the dose-dependent effects of the test chemicals as the assay temporally progressed from basal respiration through uncoupling and culminating with complete ETC inhibition permitted the classification mitochondrial toxicants into thee function bins: ETC inhibitors (e.g. rotenone), uncouplers, and ATP synthase inhibitors (e.g. oligomycin). The mutli-concentration profiling also helped to identify two distinct classes of confounding false positive test chemicals: cytotoxic chemicals that mimicked ETC inhibitors, and redox-cycling chemicals that increased non-mitochondrial oxygen consumption mimicking uncouplers. ETC inhibitors were further confirmed and characterized using a modified substrate oxidization assay in permeabilized HepG2 cells to identify which ETC complexes were targeted. The data confirms that previous dye-based mitochondrial toxicity assays underestimated the number and potency of mitochondrial toxicants among the ToxCast Phase I and II chemicals. This abstract does not necessarily reflect US EPA policy.
URLs/Downloads:
DOI: Respirometric Screening and Characterization of Mitochondrial Toxicities Induced by ToxCast Chemicals
EUROTOX_10_MITO_SIMMONS_V1.PDF (PDF, NA pp, 2813.735 KB, about PDF)