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 National Center for Computational Toxicology September 3, 2018 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 O2 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 Intermembrane H Space e- - - e- ATP O2I Co Q e O - Cyt C synthase H+ 2 IV ADP III ATP Pi Mitochondrial II H+ + Matrix 2HH2O NADH
O2 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+ Space e- H+ - - e- O2I Co Q e - Cyt C V + O2 IV H III ATP Mitochondrial II H+ + Matrix 2HH2O NADH
O2 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 Intermembrane H Space e- U - - U U U OI e- e U U 2 Co Q - Cyt C V + O2 IV + H III H ATP Mitochondrial II + H 2HH+O Matrix 2 + NADH H H+ O2 + + NAD+ H + H H+ H H+ H+ H+
Glycolysis Krebs Cycle
ETC Inhibition
Cytoplasm
Mitochondrial Intermembrane Space U U I U U U U Co Q - Cyt C V O2 IV III ATP Mitochondrial II H+ + Matrix 2HH2O NADH
NAD+
Glycolysis Krebs Cycle
Seahorse Screening Project- Tiered Overview
51 total plates Data Analysis 78 total plates Data Analysis
1 conc, N=3 yes 7 concs, N=3 ETC inhibitor 100µM Active 0.1-100µM Uncoupler Confirmation 1,051 samples 246 samples ATP synthase i no Post-hoc Redox cycling Cell Viability Cytotoxic 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) - AC50FENP - rZ’OCR↑ (DMSO/DNP) - AC50DNP 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 ETC (single concentration) and then inhibitors Redox-cyclers to confirm activity and define ETC inhibitors Cytotoxic mechanism (concentration- ATP synthase response) Cytotoxic • Anticipated that most actives Cytotoxic would decrease OCR Cytotoxicity Filtering
Number of concs filtered by spid Binning Actives by Mechanism
yes Conc x Filter Out sample is cytotoxic? Uncoupler (16) no yes Fit each window x direction to TCPL models yes ETC inhibitor no (125) ETC inhibitors Uncouplers confirm Electron Flow Assay Inactive yes no (57)
no
yes ATP synthase no Inhibitor (40)
ATP synthase inhibitors
Redox-cyclers yes Redox Cycler (4) *probe interference (2) Example: Uncoupler
AC50 = 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 = 2.35uM • Used as a fungicide against certain 50 fungal diseases in wheat and barley • Blocks the electron transport between cytochrome b and cytochrome c1 (complex III) Example: ATP Synthase Inhibitor
AC50 = 3.62uM
• Metabolite (hydrolysis) of plasticizer di- 2-ethylhexyl phthalate (DEHP) • Suspected androgen disruptor • No known mitochondrial action Confirmation: Electron Flow Assay
Rotenone Antimycin A Azide Cytoplasm
H+ + + + H H H H+ H+ + H+ + H+ H H + H + H+ Mitochondrial + H + H Intermembrane H Space e- U - - - U U AT P U O2I Co Q e e U U O - Cyt C synthase H+ 2 IV ADP + III ATP Pi Mitochondrial H II H+ H+ + H+ Matrix 2HH2O NADH H+ + + H O2 H H+ NAD+ FAD TMPD Pyruvate FADH2 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)