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.

“This book should prove a useful reference for students, researchers and biomedical science professionals working with this species as an evolving model for toxicity assessment.”  (British Toxicology Society New, 1 November 2012)

• Preface xi Contributors xiiiAcknowledgments xvii1. The Reproductive Biology and Spawning of Zebrafish in Laboratory Settings 11.1 Introduction 11.2 Overview of Zebrafish Reproductive Biology and Behavior 21.3 Spawning Techniques and Technology 51.4 Determining Factors for Reproduction in Laboratory Stocks of Zebrafish 81.5 Conclusions 11References 112. Developmental Toxicity Assessment in Zebrafish 152.1 Introduction 152.2 Methods 162.3 Results 202.4 Discussion 23References 253. Use of Emerging Models for Developmental Toxicity Testing 273.1 Importance of Assessing Developmental Toxicity 273.2 Current Methods for Assessing Developmental Toxicity 273.3 Use of Emerging Models for Developmental Toxicity Testing 283.4 New Guidelines for Chemical Testing Using Zebrafish 293.5 Conclusions 42References 434. Assessment of Drug-Induced Cardiotoxicity in Zebrafish 454.1 Introduction 454.2 Zebrafish Heart 454.3 Summary of Cardiotoxicity Study Design and Results 474.4 Materials and Methods 474.5 Results 504.6 Conclusions 54References 545. Cardiotoxicity Studies in Zebrafish 555.1 Introduction 555.2 Repolarization Toxicity 565.3 Initial Screening: Bradycardia 565.4 High-Resolution Assays of Repolarization 595.5 Future Directions 61References 626. In Vivo Recording of the Adult Zebrafish Electrocardiogram 656.1 Introduction 656.2 Optimization of Zebrafish Electrocardiogram Recording 666.3 Basic Intervals 686.4 Drug Effects 696.5 Conclusions 71References 727. Hematopoietic and Vascular System Toxicity 737.1 Introduction 737.2 Hematopoiesis and Vascular Development in the Zebrafish 737.3 Morphological and Functional Assays to Assess Toxicity 777.4 Summary 85Acknowledgment 85References 858. Hepatotoxicity Testing in Larval Zebrafish 898.1 Introduction: The Larval Zebrafish Model 898.2 Liver Development 898.3 Hepatic Gene Knockdown and Mutation 918.4 Hepatotoxicity Testing in Drug Discovery 928.5 Phenotypic-Based Larval Zebrafish Hepatotoxicity Screens 938.6 Secondary and Mechanistic Liver Assays 968.7 Conclusions 99References 999. Whole Zebrafish Cytochrome P450 Assay for Assessing Drug Metabolism and Safety 1039.1 Introduction 1039.2 Background and Significance 1049.3 Materials and Methods 1059.4 Results 1079.5 Conclusions 113Acknowledgment 113References 11310. Methods for Assessing Neurotoxicity in Zebrafish 11710.1 Introduction 11710.2 Limitations of Current Neurotoxicity Testing 11810.3 Assessing Neurotoxicity in Zebrafish 11810.4 Summary 130Acknowledgments 131References 13111. Zebrafish: A Predictive Model for Assessing Cancer Drug-Induced Organ Toxicity 13511.1 Introduction 13511.2 Materials and Methods 13611.3 Results 13911.4 Conclusions 149Reference 14912. Locomotion and Behavioral Toxicity in Larval Zebrafish: Background, Methods, and Data 15112.1 Introduction 15112.2 Background 15212.3 Locomotion 15312.4 Zebrafish Models 15412.5 Analyzing Larval Locomotion 15512.6 Chemical Effects on Larval Locomotion 15812.7 Conclusions 161Acknowledgments 162References 16213. Zebrafish: A Predictive Model for Assessing Seizure Liability 16513.1 Introduction 16513.2 Materials and Methods 16713.3 Results 17113.4 Conclusions 174References 17514. Zebrafish: A New In Vivo Model for Identifying P-Glycoprotein Efflux Modulators 17714.1 Introduction 17714.2 Materials and Methods 17914.3 Results 18214.4 Conclusions 188Acknowledgment 188References 18915. Assessment of Effects on Visual Function in Larval Zebrafish 19115.1 Introduction 19115.2 Development of Visual System in Zebrafish 19215.3 Methods for Assessing Visual Function in Larval Zebrafish 19415.4 Conclusions 202References 20216. Development of a Hypoxia-Induced Zebrafish Choroidal Neovascularization Model 20516.1 Introduction 20516.2 Materials and Methods 20716.3 Results 21016.4 Discussion 215Acknowledgments 217References 21717. Zebrafish Xenotransplant Cancer Model for Drug Screening 21917.1 Introduction 21917.2 Background and Significance 21917.3 Materials and Methods 22117.4 Results 22617.5 Conclusions 230References 23118. Zebrafish Assays for Identifying Potential Muscular Dystrophy Drug Candidates 23318.1 Introduction 23318.2 Materials and Methods 23818.3 Results 24218.4 Discussion 251Acknowledgment 252References 25219. Cytoprotective Activities of Water-Soluble Fullerenes in Zebrafish Models 25719.1 Introduction 25719.2 Materials and Methods 26119.3 Results 26519.4 Discussion 27719.5 Conclusions 278Acknowledgments 278References 27920. Fishing to Design Inherently Safer Nanoparticles 28320.1 Introduction 28320.2 Application of Embryonic Zebrafish 28420.3 Tier 1: Rapid Toxicity Screening 28620.4 Tier 2: Cellular Toxicity and Distribution 28820.5 Tier 3: Molecular Expression 28920.6 Embryonic Zebrafish Data to Design “Safer” Nanoparticles 29120.7 Conclusions 292References 29221. Radiation-Induced Toxicity and Radiation Response Modifiers in Zebrafish 29521.1 Introduction 29521.2 Materials and Methods 29621.3 Validation of Zebrafish Embryos as a Model System for Radiation Protectors/Sensitizers 29821.4 Gross Morphological Alterations Associated with Radiation Exposure 29921.5 Radiation-Associated Apoptosis Incidence 29921.6 Radiation-Associated Gastrointestinal Toxicity 30021.7 Radiation-Associated Nephrotoxicity 30121.8 Ototoxicity in Irradiated Zebrafish 30121.9 Radiation Protectors in Zebrafish 30221.10 Summary 303References 30422. Caudal Fin Regeneration in Zebrafish 30722.1 Introduction 30722.2 Signaling and Epimorphic Regeneration 30822.3 Caudal Fin Architecture 30822.4 Stages of Epimorphic Regeneration 30922.5 Methodology 31122.6 Strategies Used to Manipulate Gene Function During Fin Regeneration 31222.7 The Larval Fin Regeneration Model 31622.8 Summary 317Acknowledgments 317References 317Index 321

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.

“This book should prove a useful reference for students, researchers and biomedical science professionals working with this species as an evolving model for toxicity assessment.”  (British Toxicology Society New, 1 November 2012)

• Preface xi Contributors xiiiAcknowledgments xvii1. The Reproductive Biology and Spawning of Zebrafish in Laboratory Settings 11.1 Introduction 11.2 Overview of Zebrafish Reproductive Biology and Behavior 21.3 Spawning Techniques and Technology 51.4 Determining Factors for Reproduction in Laboratory Stocks of Zebrafish 81.5 Conclusions 11References 112. Developmental Toxicity Assessment in Zebrafish 152.1 Introduction 152.2 Methods 162.3 Results 202.4 Discussion 23References 253. Use of Emerging Models for Developmental Toxicity Testing 273.1 Importance of Assessing Developmental Toxicity 273.2 Current Methods for Assessing Developmental Toxicity 273.3 Use of Emerging Models for Developmental Toxicity Testing 283.4 New Guidelines for Chemical Testing Using Zebrafish 293.5 Conclusions 42References 434. Assessment of Drug-Induced Cardiotoxicity in Zebrafish 454.1 Introduction 454.2 Zebrafish Heart 454.3 Summary of Cardiotoxicity Study Design and Results 474.4 Materials and Methods 474.5 Results 504.6 Conclusions 54References 545. Cardiotoxicity Studies in Zebrafish 555.1 Introduction 555.2 Repolarization Toxicity 565.3 Initial Screening: Bradycardia 565.4 High-Resolution Assays of Repolarization 595.5 Future Directions 61References 626. In Vivo Recording of the Adult Zebrafish Electrocardiogram 656.1 Introduction 656.2 Optimization of Zebrafish Electrocardiogram Recording 666.3 Basic Intervals 686.4 Drug Effects 696.5 Conclusions 71References 727. Hematopoietic and Vascular System Toxicity 737.1 Introduction 737.2 Hematopoiesis and Vascular Development in the Zebrafish 737.3 Morphological and Functional Assays to Assess Toxicity 777.4 Summary 85Acknowledgment 85References 858. Hepatotoxicity Testing in Larval Zebrafish 898.1 Introduction: The Larval Zebrafish Model 898.2 Liver Development 898.3 Hepatic Gene Knockdown and Mutation 918.4 Hepatotoxicity Testing in Drug Discovery 928.5 Phenotypic-Based Larval Zebrafish Hepatotoxicity Screens 938.6 Secondary and Mechanistic Liver Assays 968.7 Conclusions 99References 999. Whole Zebrafish Cytochrome P450 Assay for Assessing Drug Metabolism and Safety 1039.1 Introduction 1039.2 Background and Significance 1049.3 Materials and Methods 1059.4 Results 1079.5 Conclusions 113Acknowledgment 113References 11310. Methods for Assessing Neurotoxicity in Zebrafish 11710.1 Introduction 11710.2 Limitations of Current Neurotoxicity Testing 11810.3 Assessing Neurotoxicity in Zebrafish 11810.4 Summary 130Acknowledgments 131References 13111. Zebrafish: A Predictive Model for Assessing Cancer Drug-Induced Organ Toxicity 13511.1 Introduction 13511.2 Materials and Methods 13611.3 Results 13911.4 Conclusions 149Reference 14912. Locomotion and Behavioral Toxicity in Larval Zebrafish: Background, Methods, and Data 15112.1 Introduction 15112.2 Background 15212.3 Locomotion 15312.4 Zebrafish Models 15412.5 Analyzing Larval Locomotion 15512.6 Chemical Effects on Larval Locomotion 15812.7 Conclusions 161Acknowledgments 162References 16213. Zebrafish: A Predictive Model for Assessing Seizure Liability 16513.1 Introduction 16513.2 Materials and Methods 16713.3 Results 17113.4 Conclusions 174References 17514. Zebrafish: A New In Vivo Model for Identifying P-Glycoprotein Efflux Modulators 17714.1 Introduction 17714.2 Materials and Methods 17914.3 Results 18214.4 Conclusions 188Acknowledgment 188References 18915. Assessment of Effects on Visual Function in Larval Zebrafish 19115.1 Introduction 19115.2 Development of Visual System in Zebrafish 19215.3 Methods for Assessing Visual Function in Larval Zebrafish 19415.4 Conclusions 202References 20216. Development of a Hypoxia-Induced Zebrafish Choroidal Neovascularization Model 20516.1 Introduction 20516.2 Materials and Methods 20716.3 Results 21016.4 Discussion 215Acknowledgments 217References 21717. Zebrafish Xenotransplant Cancer Model for Drug Screening 21917.1 Introduction 21917.2 Background and Significance 21917.3 Materials and Methods 22117.4 Results 22617.5 Conclusions 230References 23118. Zebrafish Assays for Identifying Potential Muscular Dystrophy Drug Candidates 23318.1 Introduction 23318.2 Materials and Methods 23818.3 Results 24218.4 Discussion 251Acknowledgment 252References 25219. Cytoprotective Activities of Water-Soluble Fullerenes in Zebrafish Models 25719.1 Introduction 25719.2 Materials and Methods 26119.3 Results 26519.4 Discussion 27719.5 Conclusions 278Acknowledgments 278References 27920. Fishing to Design Inherently Safer Nanoparticles 28320.1 Introduction 28320.2 Application of Embryonic Zebrafish 28420.3 Tier 1: Rapid Toxicity Screening 28620.4 Tier 2: Cellular Toxicity and Distribution 28820.5 Tier 3: Molecular Expression 28920.6 Embryonic Zebrafish Data to Design “Safer” Nanoparticles 29120.7 Conclusions 292References 29221. Radiation-Induced Toxicity and Radiation Response Modifiers in Zebrafish 29521.1 Introduction 29521.2 Materials and Methods 29621.3 Validation of Zebrafish Embryos as a Model System for Radiation Protectors/Sensitizers 29821.4 Gross Morphological Alterations Associated with Radiation Exposure 29921.5 Radiation-Associated Apoptosis Incidence 29921.6 Radiation-Associated Gastrointestinal Toxicity 30021.7 Radiation-Associated Nephrotoxicity 30121.8 Ototoxicity in Irradiated Zebrafish 30121.9 Radiation Protectors in Zebrafish 30221.10 Summary 303References 30422. Caudal Fin Regeneration in Zebrafish 30722.1 Introduction 30722.2 Signaling and Epimorphic Regeneration 30822.3 Caudal Fin Architecture 30822.4 Stages of Epimorphic Regeneration 30922.5 Methodology 31122.6 Strategies Used to Manipulate Gene Function During Fin Regeneration 31222.7 The Larval Fin Regeneration Model 31622.8 Summary 317Acknowledgments 317References 317Index 321

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.

“This book should prove a useful reference for students, researchers and biomedical science professionals working with this species as an evolving model for toxicity assessment.”  (British Toxicology Society New, 1 November 2012)

• Preface xi Contributors xiiiAcknowledgments xvii1. The Reproductive Biology and Spawning of Zebrafish in Laboratory Settings 11.1 Introduction 11.2 Overview of Zebrafish Reproductive Biology and Behavior 21.3 Spawning Techniques and Technology 51.4 Determining Factors for Reproduction in Laboratory Stocks of Zebrafish 81.5 Conclusions 11References 112. Developmental Toxicity Assessment in Zebrafish 152.1 Introduction 152.2 Methods 162.3 Results 202.4 Discussion 23References 253. Use of Emerging Models for Developmental Toxicity Testing 273.1 Importance of Assessing Developmental Toxicity 273.2 Current Methods for Assessing Developmental Toxicity 273.3 Use of Emerging Models for Developmental Toxicity Testing 283.4 New Guidelines for Chemical Testing Using Zebrafish 293.5 Conclusions 42References 434. Assessment of Drug-Induced Cardiotoxicity in Zebrafish 454.1 Introduction 454.2 Zebrafish Heart 454.3 Summary of Cardiotoxicity Study Design and Results 474.4 Materials and Methods 474.5 Results 504.6 Conclusions 54References 545. Cardiotoxicity Studies in Zebrafish 555.1 Introduction 555.2 Repolarization Toxicity 565.3 Initial Screening: Bradycardia 565.4 High-Resolution Assays of Repolarization 595.5 Future Directions 61References 626. In Vivo Recording of the Adult Zebrafish Electrocardiogram 656.1 Introduction 656.2 Optimization of Zebrafish Electrocardiogram Recording 666.3 Basic Intervals 686.4 Drug Effects 696.5 Conclusions 71References 727. Hematopoietic and Vascular System Toxicity 737.1 Introduction 737.2 Hematopoiesis and Vascular Development in the Zebrafish 737.3 Morphological and Functional Assays to Assess Toxicity 777.4 Summary 85Acknowledgment 85References 858. Hepatotoxicity Testing in Larval Zebrafish 898.1 Introduction: The Larval Zebrafish Model 898.2 Liver Development 898.3 Hepatic Gene Knockdown and Mutation 918.4 Hepatotoxicity Testing in Drug Discovery 928.5 Phenotypic-Based Larval Zebrafish Hepatotoxicity Screens 938.6 Secondary and Mechanistic Liver Assays 968.7 Conclusions 99References 999. Whole Zebrafish Cytochrome P450 Assay for Assessing Drug Metabolism and Safety 1039.1 Introduction 1039.2 Background and Significance 1049.3 Materials and Methods 1059.4 Results 1079.5 Conclusions 113Acknowledgment 113References 11310. Methods for Assessing Neurotoxicity in Zebrafish 11710.1 Introduction 11710.2 Limitations of Current Neurotoxicity Testing 11810.3 Assessing Neurotoxicity in Zebrafish 11810.4 Summary 130Acknowledgments 131References 13111. Zebrafish: A Predictive Model for Assessing Cancer Drug-Induced Organ Toxicity 13511.1 Introduction 13511.2 Materials and Methods 13611.3 Results 13911.4 Conclusions 149Reference 14912. Locomotion and Behavioral Toxicity in Larval Zebrafish: Background, Methods, and Data 15112.1 Introduction 15112.2 Background 15212.3 Locomotion 15312.4 Zebrafish Models 15412.5 Analyzing Larval Locomotion 15512.6 Chemical Effects on Larval Locomotion 15812.7 Conclusions 161Acknowledgments 162References 16213. Zebrafish: A Predictive Model for Assessing Seizure Liability 16513.1 Introduction 16513.2 Materials and Methods 16713.3 Results 17113.4 Conclusions 174References 17514. Zebrafish: A New In Vivo Model for Identifying P-Glycoprotein Efflux Modulators 17714.1 Introduction 17714.2 Materials and Methods 17914.3 Results 18214.4 Conclusions 188Acknowledgment 188References 18915. Assessment of Effects on Visual Function in Larval Zebrafish 19115.1 Introduction 19115.2 Development of Visual System in Zebrafish 19215.3 Methods for Assessing Visual Function in Larval Zebrafish 19415.4 Conclusions 202References 20216. Development of a Hypoxia-Induced Zebrafish Choroidal Neovascularization Model 20516.1 Introduction 20516.2 Materials and Methods 20716.3 Results 21016.4 Discussion 215Acknowledgments 217References 21717. Zebrafish Xenotransplant Cancer Model for Drug Screening 21917.1 Introduction 21917.2 Background and Significance 21917.3 Materials and Methods 22117.4 Results 22617.5 Conclusions 230References 23118. Zebrafish Assays for Identifying Potential Muscular Dystrophy Drug Candidates 23318.1 Introduction 23318.2 Materials and Methods 23818.3 Results 24218.4 Discussion 251Acknowledgment 252References 25219. Cytoprotective Activities of Water-Soluble Fullerenes in Zebrafish Models 25719.1 Introduction 25719.2 Materials and Methods 26119.3 Results 26519.4 Discussion 27719.5 Conclusions 278Acknowledgments 278References 27920. Fishing to Design Inherently Safer Nanoparticles 28320.1 Introduction 28320.2 Application of Embryonic Zebrafish 28420.3 Tier 1: Rapid Toxicity Screening 28620.4 Tier 2: Cellular Toxicity and Distribution 28820.5 Tier 3: Molecular Expression 28920.6 Embryonic Zebrafish Data to Design “Safer” Nanoparticles 29120.7 Conclusions 292References 29221. Radiation-Induced Toxicity and Radiation Response Modifiers in Zebrafish 29521.1 Introduction 29521.2 Materials and Methods 29621.3 Validation of Zebrafish Embryos as a Model System for Radiation Protectors/Sensitizers 29821.4 Gross Morphological Alterations Associated with Radiation Exposure 29921.5 Radiation-Associated Apoptosis Incidence 29921.6 Radiation-Associated Gastrointestinal Toxicity 30021.7 Radiation-Associated Nephrotoxicity 30121.8 Ototoxicity in Irradiated Zebrafish 30121.9 Radiation Protectors in Zebrafish 30221.10 Summary 303References 30422. Caudal Fin Regeneration in Zebrafish 30722.1 Introduction 30722.2 Signaling and Epimorphic Regeneration 30822.3 Caudal Fin Architecture 30822.4 Stages of Epimorphic Regeneration 30922.5 Methodology 31122.6 Strategies Used to Manipulate Gene Function During Fin Regeneration 31222.7 The Larval Fin Regeneration Model 31622.8 Summary 317Acknowledgments 317References 317Index 321