The Role of Coenzyme Q10 in Statin Treated Zebrafish (Danio rerio)

ii Résumé . iii List of Figures . vii List of Tables . ix List of Abbreviations . x Acknowledgements . xii Chapter 1 – General Introduction . 1 1.1. Rationale for the Study . 1 1.2. Statins . 2 1.2.1. Overview . 2 1.2.2. General characteristics and mode of action of statin drugs . 3 1.2.3. Atorvastatin . 4 1.2.4. Statin absorption, distribution, metabolism, and excretion . 6 1.2.5. Statin environmental fate and uptake by fish . 8 1.2.6. Past studies on statins and fish . 11 1.3. The Biochemical Significance of CoQ10 and its Relation to Statins . 13 1.3.1. Overview . 13 1.3.2. Functional description of CoQ10 . 13 1.3. 3. Therapeutic uses of CoQ10 . 15 1.4. Literature Review on Statin Effects and Key genes Involved in Muscle Atrophy . 21 1.5. Zebrafish as a Model Organism . 23 1.6. Hypotheses and Objectives of this Study . 24 Chapter 2 – Materials and Methods . 26 2.1. Chemicals . 26 2.2. Fish . 26 2.3. Experiments Using Zebrafish Embryos/Larvae . 27 2.3.1. Embryo Collection . 27 2.3.2. Drug Exposure . 27 2.3. 3. Heart rate . 28 2.3. 4. Response to a tactile stimulus . 29 v 2.3. 5. Spontaneous displacement . 29 2.3. 6. TUNEL and ROS Assay . 30 2.3. 6.1. Reactive oxygen species (ROS) detection . 30 2.3. 6.2. TUNEL assay . 31 2.4. Experiments Using Adult Zebrafish . 32 2.4.1. Swimming behavior . 33 2.4.2. Swim tunnel test . 34 2.4.4. Staining and Tissue Histology . 37 2.4.4.1. Hematoxylin and eosin staining . 37 2.4.4.3. TUNEL Assay . 38 2.5. Enzyme Activities . 38 2.5. 1. Cytochrome oxidase (COX) (E.C. 1.9.3.1) . 39 2.5. 2. Citrate synthase (CS) (E.C. 2.3.3.1) . 39 2.5. 3. Lactate dehydrogenase (LDH) (E.C. 1.1.1.27) . 40 2.6. Molecular Procedures . 40 2.6.1. RNA extraction . 40 2.6.2. cDNA synthesis . 41 2.6. 3. Quantitative real-time PCR (qPCR) . 42 2.7. Statistical Analysis . 43 Chapter 3 – Results . 44 3.1. Zebrafish embryos/larvae . 44 3.1.1. Effects of ATV on mortality, pericardial sac edema, and heart rate . 44 3.1.2. Effects of ATV on locomotor behavior . 45 3.1.3. Reactive oxygen species (ROS) generation and apoptosis . 46 3.1.5. Effects of ATV on molecular markers of muscle atrophy . 47 3.2. Effects of ATV on Zebrafish Adults . 48 3.2.1. Effects of ATV on mortality . 48 3.2.2. Effects of ATV on adult behavior . 48 3.2.3. Effects of ATV on muscle histology and apoptosis . 49 3.2.4. Effects of ATV on enzyme activities associated with oxidative capacity and mitochondrial function . 49 3.2.5. Effects of ATV on molecular markers of muscle atrophy . 50 Chapter 4 – Discussion and Conclusion . 73 4.1. Zebrafish embryos/larvae . 73 4.1.1. Effects of ATV on mortality, pericardial sac edema, and heart rate . 73 vi 4.1.2. Effect of ATV on larval behavior . 74 4.1.3. Reactive oxygen species (ROS) generation and apoptosis . 77 4.1.4. Effects of ATV on enzymes activity associated with oxidative capacity and mitochondrial function . 78 4.1.5. Effects of ATV on molecular markers of muscle atrophy . 79 4.2. Effects of ATV on Zebrafish Adults . 81 4.2.1. Effects of ATV on adult behaviors . 81 4.2.2. Muscle staining . 82 4.2.3. Effect of ATV on enzymes activity associated with oxidative capacity and mitochondrial function . 83 4.2.4. Effects of ATV on molecular markers of muscle atrophy . 85 4.3. General Conclusions . 86 4.4. Prospective Future Research . 87 4.5. Significance . 89 References . 91 Appendix A – Matlab Commends . 107 Appendix B – Python Commends . 111 Appendix C – Protein Content and Cell size . 120