Respirometric Screening and Characterization of Mitochondrial Toxicities Induced by ToxCast Chemicals Steven O. Simmons The views expressed in this presentation are those of the author[s] and do not necessarily reflect the views or policies of the U.S. Environmental Protection Agency. Office of Research and Development September 3, 2018 National Center for Computational Toxicology
Mitochondria as Targets of Toxicity Mitochondria are critical in eukaryotic cells because they generate >90% of the cellular supply of ATP • • Also key to regulating cell cycle/growth, differentiation and apoptosis • Many chemicals are known to impair mitochondrial function through various mechanisms: • Electron transport chain (ETC; Complexes I-IV) inhibition • Uncoupling and Ionophores Phosphorylation (Complex V) inhibition • • Transport inhibition (ATP) • Kreb cycle inhibitors • Disease states associated with genetic mitochondrial disorders provide insights about possible adverse outcomes In many of these cases, mitochondria have normal morphology- the impact is functional, not structural • • Current ToxCast/Tox21 high-throughput test methods typically use immortalized/tumor cells (Warburg Effect) cultured in high-glucose medium (Crabtree Effect), and thus are impervious to mitochondrial insult • ToxCast/Tox21 mitochondrial assays have focused on two endpoints: mitochondrial mass (swelling) and mitochondrial membrane potential (MMP) These assay use dye probes to measure structural mitochondrial defects due primarily to membrane changes • and are not sensitive to chemicals that impair mitochondrial function through other mechanisms (i.e. ETCi) • The Seahorse XF Analyzer platform measures mitochondrial function, so it is sensitive to most mechanisms of disruption
Seahorse XF Platform Two fluorophores measure: 1) dissolved O 2 and 2) pH Direct, non-invasive analyte measurement of oxidative phosphorylation and glycolysis in real time
Basal Respiration Cytoplasm H + H + H + H + H + H + H + H + H + H + H + H + Mitochondrial H + H + H + Intermembrane e - Space I ATP e - - O 2 e - Co Q Cyt C synthase IV - O 2 III ADP H + ATP II Pi Mitochondrial H + H + 2H 2 O Matrix NADH O 2 NAD+ Glycolysis Krebs Cycle
ATP Synthase Inhibition Cytoplasm H + H + H + H + H + H + H + H + H + H + H + H + H + H + H + H + H + H + H + H + H + H + H + H + H + H + H + H + H + H + H + H + H + Mitochondrial H + H + H + H + H + H + Intermembrane H + H + H + H + H + e - Space H + I e - - O 2 e - Co Q V Cyt C IV - O 2 III H + ATP II Mitochondrial H + H + 2H 2 O Matrix NADH O 2 NAD+ Glycolysis Krebs Cycle
Uncoupling Respiration from ATP Synthesis Cytoplasm H + H + H + H + H + H + H + H + H + H + H + H + Mitochondrial H + H + H + Intermembrane e - Space I U U U U U e - - O 2 e - Co Q U V Cyt C IV - O 2 III H + H + ATP II Mitochondrial H + H + 2H 2 O Matrix H + NADH H + O 2 H + H + NAD+ H + H + H + H + H + Glycolysis Krebs Cycle
ETC Inhibition Cytoplasm Mitochondrial Intermembrane Space I U U U U U Co Q U V Cyt C IV - O 2 III ATP II Mitochondrial H + H + 2H 2 O Matrix NADH NAD+ Glycolysis Krebs Cycle
Seahorse Screening Project- Tiered Overview 78 total plates 51 total plates Data Analysis Data Analysis 1 conc, N=3 7 concs, N=3 ETC inhibitor yes 100µM 0.1-100µM Active Uncoupler Confirmation 1,051 samples 246 samples ATP synthase i Redox cycling no Post-hoc Cytotoxic Cell Viability Inactive 805 samples Electron Flow Assay • Human HepG2 hepatocellular carcinoma cells (50% glycolytic) • Screening assay comprised of 4 temporal windows separated by 3 sequential injections (reagent additions): - Port A: DMSO (vehicle), Fenpyroximate (ETCi), 2,4-Dinitrophenol (Uncoupler), Blinded Test Samples - (Basal Respiration) - Port B: 250nM FCCP - (Maximal Respiration) - Port C: 1uM Rotenone + 1uM Antimycin A - (Inhibited Respiration) • Total assay time is > 75 minutes. Cell viability was measured on cells at conclusion of Seahorse run (mutli-conc plates only) • Each assay plate accepted/rejected on 5 QC criteria: - %CV (DMSO) - rZ’ OCR ↓ (DMSO/FENP) - AC50 FENP - rZ’ OCR ↑ (DMSO/DNP) - AC50 DNP
ToxCast Screening Protocol • Replaced Oligomycin injection Redox-cyclers with test compounds/controls • Used variation in vehicle (DMSO) response to establish activity Uncouplers thresholds (cut-offs) Redox-cyclers • Tracked activity throughout time course of assay to identify potential mitochondrial toxicants (single concentration) and then ETC to confirm activity and define inhibitors Redox-cyclers mechanism (concentration- ETC inhibitors Cytotoxic response) ATP synthase Cytotoxic • Anticipated that most actives Cytotoxic would decrease OCR
Cytotoxicity Filtering Number of concs filtered by spid
Binning Actives by Mechanism Conc x yes Filter Out sample is cytotoxic? Uncoupler (16) no yes Fit each window x yes ETC inhibitor direction to TCPL models (125) no ETC inhibitors confirm Uncouplers Electron Flow Assay Inactive yes no (57) no ATP synthase yes Inhibitor (40) no ATP synthase inhibitors Redox-cyclers yes Redox Cycler (4) *probe interference (2)
Example: Uncoupler AC 50 = 2.16uM • Used as a systemic fungicide in crop protection products • Inhibits all developmental stages of fungi by influencing respiration in the mitochondrial cytochrome bc 1 complex (III)
Example: Electron Transport Chain Inhibitor AC 50 = 2.35uM Used as a fungicide against certain • fungal diseases in wheat and barley • Blocks the electron transport between cytochrome b and cytochrome c1 (complex III)
Example: ATP Synthase Inhibitor AC 50 = 3.62uM • Metabolite (hydrolysis) of plasticizer di- 2-ethylhexyl phthalate (DEHP) • Suspected androgen disruptor No known mitochondrial action •
Confirmation: Electron Flow Assay Azide Rotenone Antimycin A Cytoplasm H + H + H + H + H + H + H + H + H + H + H + H + Mitochondrial H + H + H + Intermembrane e - U Space U U I A TP O 2- e - U U e - Co Q U Cyt C synthase IV O 2- III ADP H + ATP II Pi H + Mitochondrial H + H + H + 2H 2 O H + Matrix NADH H + H + H + O 2 H + FAD NAD+ TMPD Pyruvate FADH 2 Malate Ascorbate Succinate → Fumarate • Permeabilized HepG2 cells • Fully uncoupled with FCCP
Enhanced ETC Inhibitor Detection: Rotenone
Acknowledgements NCCT Hayley Ryskoski (U of Texas) Katie-Paul Friedman Danielle Suarez (NHEERL/EPHD) NHEERL Dan Hallinger (NHEERL/TAD) simmons.steve@epa.gov 20
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