You are here:
Mitochondrial-Disrupting ToxCast Chemicals Inhibiting the Electron Transport Chain
Citation:
Hallinger, D., H. Ryskoski, AND Steve Simmons. Mitochondrial-Disrupting ToxCast Chemicals Inhibiting the Electron Transport Chain. Presented at Society of Toxicology 58th Annual Meeting & ToxExpo, Baltimore, Maryland, March 10 - 14, 2019. https://doi.org/10.23645/epacomptox.7847768
Impact/Purpose:
Method development to identify toxicants that elicit adverse effects by mitochondrial disruption.
Description:
Mitochondria generate the majority of cellular ATP through aerobic respiration and are central to cell proliferation, metabolic regulation, and apoptosis. Impaired mitochondrial function is linked to disease states such as Parkinson’s disease and metabolic syndrome; therefore, identifying mitochondrial-disrupting chemicals is important. We screened ToxCast Phase I/II chemicals using a modified Agilent Seahorse mitochondrial stress assay protocol with hepatocellular carcinoma HepG2 cells to identify compounds that altered basal, maximal or inhibited oxygen consumption rates (OCR). Of 1,051 blinded test samples, 805 were inactive in a single-concentration format while the remaining 246 samples were re-evaluated in a multiple-concentration format. These data were used to classify active chemicals as electron transport chain inhibitors (ETCi), uncouplers, or ATP synthase inhibitors. The largest classification group was ETCi with more than 120 chemicals. A subset of putative ETCi were mechanistically characterized using an electron flow assay to elucidate the inhibited ETC complex. Cells were permeabilized using a reagent that leave mitochondrial membranes intact and permitted the direct exposure to twenty suspected ETCi, each at three concentrations (31µM, 64µM, and 100µM). Mitochondria were fully uncoupled with carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP) to maximize OCR and initially provided glutamate and malate as complex I substrates. OCR was measured then challenged with three sequential injections: 1) suspected ETCi, 2) rotenone (complex I inhibitor)/succinate (complex II substrate), and 3) antimycin A (complex III inhibitor)/ascorbate and TMPD (complex IV substrates). The ETCi mechanism of each test chemical was elucidated using OCR recovery data. We determined that six chemicals specifically blocked complex I, three were specific to complex II/III, and five inhibited multiple ETC complexes. This effort is the first of its kind to identify mechanisms of putative ETCi identified using a screening-based approach and will be expanded to include all identified ETCi with the ToxCast Phase I/II chemicals. This abstract does not necessarily reflect US EPA policy.
URLs/Downloads:
DOI: Mitochondrial-Disrupting ToxCast Chemicals Inhibiting the Electron Transport Chain
HALLINGER SOT POSTER_FINAL_M.PDF (PDF, NA pp, 1970.129 KB, about PDF)