Zebrafish – Methods for Assessing Drug Safety and Toxicity

Patricia McGrath , MBA, is President and CEO of Phylonix Pharmaceuticals, Inc. She has some twenty-five years of experience in high-tech and biotechnology management. Phylonix Pharmaceuticals develops and markets novel in vivo zebrafish-based assays for therapeutic and industrial screening for cancers, central nervous system and cardiovascular diseases, apoptosis, and toxicity.

Preface xi Contributors xiii Acknowledgments xvii 1. The Reproductive Biology and Spawning of Zebrafish in Laboratory Settings 1 1.1 Introduction 1 1.2 Overview of Zebrafish Reproductive Biology and Behavior 2 1.3 Spawning Techniques and Technology 5 1.4 Determining Factors for Reproduction in Laboratory Stocks of Zebrafish 8 1.5 Conclusions 11 References 11 2. Developmental Toxicity Assessment in Zebrafish 15 2.1 Introduction 15 2.2 Methods 16 2.3 Results 20 2.4 Discussion 23 References 25 3. Use of Emerging Models for Developmental Toxicity Testing 27 3.1 Importance of Assessing Developmental Toxicity 27 3.2 Current Methods for Assessing Developmental Toxicity 27 3.3 Use of Emerging Models for Developmental Toxicity Testing 28 3.4 New Guidelines for Chemical Testing Using Zebrafish 29 3.5 Conclusions 42 References 43 4. Assessment of Drug-Induced Cardiotoxicity in Zebrafish 45 4.1 Introduction 45 4.2 Zebrafish Heart 45 4.3 Summary of Cardiotoxicity Study Design and Results 47 4.4 Materials and Methods 47 4.5 Results 50 4.6 Conclusions 54 References 54 5. Cardiotoxicity Studies in Zebrafish 55 5.1 Introduction 55 5.2 Repolarization Toxicity 56 5.3 Initial Screening: Bradycardia 56 5.4 High-Resolution Assays of Repolarization 59 5.5 Future Directions 61 References 62 6. In Vivo Recording of the Adult Zebrafish Electrocardiogram 65 6.1 Introduction 65 6.2 Optimization of Zebrafish Electrocardiogram Recording 66 6.3 Basic Intervals 68 6.4 Drug Effects 69 6.5 Conclusions 71 References 72 7. Hematopoietic and Vascular System Toxicity 73 7.1 Introduction 73 7.2 Hematopoiesis and Vascular Development in the Zebrafish 73 7.3 Morphological and Functional Assays to Assess Toxicity 77 7.4 Summary 85 Acknowledgment 85 References 85 8. Hepatotoxicity Testing in Larval Zebrafish 89 8.1 Introduction: The Larval Zebrafish Model 89 8.2 Liver Development 89 8.3 Hepatic Gene Knockdown and Mutation 91 8.4 Hepatotoxicity Testing in Drug Discovery 92 8.5 Phenotypic-Based Larval Zebrafish Hepatotoxicity Screens 93 8.6 Secondary and Mechanistic Liver Assays 96 8.7 Conclusions 99 References 99 9. Whole Zebrafish Cytochrome P450 Assay for Assessing Drug Metabolism and Safety 103 9.1 Introduction 103 9.2 Background and Significance 104 9.3 Materials and Methods 105 9.4 Results 107 9.5 Conclusions 113 Acknowledgment 113 References 113 10. Methods for Assessing Neurotoxicity in Zebrafish 117 10.1 Introduction 117 10.2 Limitations of Current Neurotoxicity Testing 118 10.3 Assessing Neurotoxicity in Zebrafish 118 10.4 Summary 130 Acknowledgments 131 References 131 11. Zebrafish: A Predictive Model for Assessing Cancer Drug-Induced Organ Toxicity 135 11.1 Introduction 135 11.2 Materials and Methods 136 11.3 Results 139 11.4 Conclusions 149 Reference 149 12. Locomotion and Behavioral Toxicity in Larval Zebrafish: Background, Methods, and Data 151 12.1 Introduction 151 12.2 Background 152 12.3 Locomotion 153 12.4 Zebrafish Models 154 12.5 Analyzing Larval Locomotion 155 12.6 Chemical Effects on Larval Locomotion 158 12.7 Conclusions 161 Acknowledgments 162 References 162 13. Zebrafish: A Predictive Model for Assessing Seizure Liability 165 13.1 Introduction 165 13.2 Materials and Methods 167 13.3 Results 171 13.4 Conclusions 174 References 175 14. Zebrafish: A New In Vivo Model for Identifying P-Glycoprotein Efflux Modulators 177 14.1 Introduction 177 14.2 Materials and Methods 179 14.3 Results 182 14.4 Conclusions 188 Acknowledgment 188 References 189 15. Assessment of Effects on Visual Function in Larval Zebrafish 191 15.1 Introduction 191 15.2 Development of Visual System in Zebrafish 192 15.3 Methods for Assessing Visual Function in Larval Zebrafish 194 15.4 Conclusions 202 References 202 16. Development of a Hypoxia-Induced Zebrafish Choroidal Neovascularization Model 205 16.1 Introduction 205 16.2 Materials and Methods 207 16.3 Results 210 16.4 Discussion 215 Acknowledgments 217 References 217 17. Zebrafish Xenotransplant Cancer Model for Drug Screening 219 17.1 Introduction 219 17.2 Background and Significance 219 17.3 Materials and Methods 221 17.4 Results 226 17.5 Conclusions 230 References 231 18. Zebrafish Assays for Identifying Potential Muscular Dystrophy Drug Candidates 233 18.1 Introduction 233 18.2 Materials and Methods 238 18.3 Results 242 18.4 Discussion 251 Acknowledgment 252 References 252 19. Cytoprotective Activities of Water-Soluble Fullerenes in Zebrafish Models 257 19.1 Introduction 257 19.2 Materials and Methods 261 19.3 Results 265 19.4 Discussion 277 19.5 Conclusions 278 Acknowledgments 278 References 279 20. Fishing to Design Inherently Safer Nanoparticles 283 20.1 Introduction 283 20.2 Application of Embryonic Zebrafish 284 20.3 Tier 1: Rapid Toxicity Screening 286 20.4 Tier 2: Cellular Toxicity and Distribution 288 20.5 Tier 3: Molecular Expression 289 20.6 Embryonic Zebrafish Data to Design "Safer" Nanoparticles 291 20.7 Conclusions 292 References 292 21. Radiation-Induced Toxicity and Radiation Response Modifiers in Zebrafish 295 21.1 Introduction 295 21.2 Materials and Methods 296 21.3 Validation of Zebrafish Embryos as a Model System for Radiation Protectors/Sensitizers 298 21.4 Gross Morphological Alterations Associated with Radiation Exposure 299 21.5 Radiation-Associated Apoptosis Incidence 299 21.6 Radiation-Associated Gastrointestinal Toxicity 300 21.7 Radiation-Associated Nephrotoxicity 301 21.8 Ototoxicity in Irradiated Zebrafish 301 21.9 Radiation Protectors in Zebrafish 302 21.10 Summary 303 References 304 22. Caudal Fin Regeneration in Zebrafish 307 22.1 Introduction 307 22.2 Signaling and Epimorphic Regeneration 308 22.3 Caudal Fin Architecture 308 22.4 Stages of Epimorphic Regeneration 309 22.5 Methodology 311 22.6 Strategies Used to Manipulate Gene Function During Fin Regeneration 312 22.7 The Larval Fin Regeneration Model 316 22.8 Summary 317 Acknowledgments 317 References 317 Index 321