Methods for Assessing Cytochrome C Oxidase Inhibitors and Potential Antidotes
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Title Page Methods for Assessing Cytochrome c Oxidase Inhibitors and Potential Antidotes by Kristin L. Frawley AS, Community College of Allegheny County, 2008 BS, Point Park University, 2010 MPH, University of Pittsburgh, 2013 Submitted to the Graduate Faculty of the Department of Environmental & Occupational Health Graduate School of Public Health in partial fulfillment of the requirements for the degree of Doctor of Public Health University of Pittsburgh 2019 Committee Membership Page UNIVERSITY OF PITTSBURGH GRADUATE SCHOOL OF PUBLIC HEALTH This dissertation was presented by Kristin L. Frawley It was defended on August 6, 2019 and approved by Dissertation Advisor: Jim Peterson, PhD, Professor, Department of Environmental and Occupational Health, Graduate School of Public Health, University of Pittsburgh Dissertation Co-Advisor: Aaron Barchowsky, PhD, Professor, Department of Environmental and Occupational Health, Graduate School of Public Health, University of Pittsburgh Linda Pearce, PhD, Professor, Department of Environmental and Occupational Health, Graduate School of Public Health, University of Pittsburgh Joel Haight, PhD, Professor, Industrial Engineering, Swanson School of Engineering, University of Pittsburgh ii Copyright © by Kristin L. Frawley 2019 iii Jim Peterson, PhD Aaron Barchowsky, PhD Methods for Assessing Cytochrome c Oxidase Inhibitors and Potential Antidotes Kristin L. Frawley, DrPH University of Pittsburgh, 2019 Abstract The Countermeasures Against Chemical Terrorism (CounterACT) Program, sponsored by the U.S. Department of Homeland Security, seeks to promote and support research aimed at finding new (therapeutics) pharmaceuticals that are antidotal toward toxicants considered likely to pose significant terrorist threat. More specifically, this means countermeasures to toxicants that can be easily prepared from readily available precursors in quantities suitable for inflicting mass casualties on civilian and/or military targets. The challenge to Public Health issued by the CounterACT program is to identify and develop antidotes to any such toxicants deemed to be of particular concern, including the mitochondrial poisons sulfide, cyanide and azide. Ideally, in addition to efficacy, the antidotes should be stable enough for stockpiling and safe enough for self- administration. Herein, various biological systems mimicking some relevant aspects of acute poisonings in humans have been tested for their usefulness as experimental “models” suitable for examining toxic mechanisms and assessing the efficacies of putative antidotes. Larvae of the greater wax moth (Galleria mellonella) are shown to be a convenient and inexpensive invertebrate model for investigating the action of some mitochondrial poisons and their antidotes. The acute toxicities of sulfide, cyanide and azide have been studied together with the ameliorating effects of sodium nitrite and a cobalt-based scavenging agent. The results obtained with the larvae are iv compared to findings employing a cultured mammalian cell line (bovine pulmonary artery endothelial cells) and rodents (Swiss-Webster mice). The Galleria mellonella larvae are argued to be an extremely useful intact organism for (i) pre-screening putative antidotes for efficacy and (ii) circumventing any confounding effects that can arise in some studies of intracellular processes due to the presence of blood in intact vertebrates. v Table of Contents Preface .......................................................................................................................................... xv 1.0 Introduction ............................................................................................................................. 1 1.1 Mitochondria ................................................................................................................... 1 1.2 Cytochrome c Oxidase ................................................................................................... 4 1.3 The Inhibitors and the Putative Antidotes ................................................................... 5 1.3.1 Sulfide, Cyanide and Azide ................................................................................ 5 1.3.2 Putative Antidotes ............................................................................................... 9 1.4 Typical Models Used for Toxicological Experiments ................................................ 11 1.4.1 Cell Models ........................................................................................................ 12 1.4.2 Drosophila melanogaster ................................................................................... 13 1.4.3 Zebrafish ............................................................................................................ 14 1.4.4 Mouse Model ..................................................................................................... 14 1.4.5 Galleria mellonella ............................................................................................. 16 1.5 Overall Objective of the Dissertation ......................................................................... 21 2.0 Sulfide Toxicity and Its Modulation by Nitric Oxide in Bovine Pulmonary Artery Endothelial Cells ......................................................................................................................... 24 2.1 Abstract ......................................................................................................................... 25 2.2 Introduction .................................................................................................................. 26 2.3 Experimental Procedures ............................................................................................ 28 2.3.1 Chemicals ........................................................................................................... 28 2.3.2 Animals and Sulfide Exposure ......................................................................... 28 vi 2.3.3 Cells and Cell Culture ....................................................................................... 29 2.3.4 Hydrogen Sulfide Toxicity in BPAEC ............................................................. 29 2.3.5 Cellular Assays .................................................................................................. 30 2.3.6 Western Blotting ............................................................................................... 31 2.3.7 Respirometric Experiments ............................................................................. 32 2.3.8 Instrumentation ................................................................................................. 33 2.3.9 Data Analysis ..................................................................................................... 33 2.4 Results and Discussion ................................................................................................. 34 2.4.1 Intact Mice ......................................................................................................... 34 2.4.2 Cultured Cells .................................................................................................... 37 2.4.3 Respirometric Measurements .......................................................................... 45 2.5 Conclusion ..................................................................................................................... 48 2.5.1 Post-Acute Toxicity ........................................................................................... 48 2.5.2 An Approach to Antidotes? .............................................................................. 49 2.6 Supplemental Materials and Figures .......................................................................... 51 3.0 Results of Toxicant/Antidote Testing in a Mouse Model .................................................. 53 3.1 Introduction .................................................................................................................. 53 3.1.1 Sulfide Toxicity Testing .................................................................................... 53 3.1.2 Cyanide Toxicity Testing .................................................................................. 55 3.1.3 Azide Toxicity Testing ...................................................................................... 57 3.2 Summary ....................................................................................................................... 59 4.0 Assessing Modulators of Cytochrome c Oxidase Activity in Galleria mellonella Larvae .......................................................................................................................................... 60 vii 4.1 Abstract ......................................................................................................................... 61 4.2 Introduction .................................................................................................................. 62 4.3 Materials and Methods ................................................................................................ 65 4.3.1 Reagents ............................................................................................................. 65 4.3.2 G. mellonella Larvae Exposures ...................................................................... 66 4.3.3 Righting-Recovery Testing ..............................................................................