UNIVERSITY OF CALIFORNIA SAN DIEGO Establishment and Validation of the Freshwater Planarian, Dugesia japonica, as an Alternative Animal Model for Developmental Neurotoxicology using Organophosphorus Pesticides A dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in Biology by Danielle Hagstrom Committee in charge: Professor Eva-Maria Schoetz Collins, Chair Professor James Posakony, Co-Chair Professor Palmer Taylor Professor Robert Tukey Professor Jing Wang Professor Deborah Yelon 2018 Copyright Danielle Hagstrom, 2018 All rights reserved. The Dissertation of Danielle Hagstrom is approved, and it is acceptable in quality and form for publication on microfilm and electronically: Co-Chair Chair University of California San Diego 2018 iii TABLE OF CONTENTS Signature Page………………………………………………………………………………... iii Table of Contents……………………………………………………………………………... iv List of Figures……………………………………………………………………………........ v List of Tables………………………………………………………………………………..... vii Acknowledgements………………………………………………………………………....... viii Vita…………………………………………………………………………………………… xii Abstract of the Dissertation…………………………………………………………………… xiii Chapter 1: Planarian brain regeneration as a model system for developmental neurotoxicology………….……………………………………………………………………. 1 Chapter 2: Freshwater planarians as an alternative animal model for neurotoxicology………. 38 Chapter 3: Multi-behavioral endpoint testing of an 87-chemical compound library in freshwater planarians………………………………………………………………………………………. 83 Chapter 4: Comparative analysis of zebrafish and planarian model systems for developmental neurotoxicity screens using an 87-compound library………………………………………….. 136 Chapter 5: Planarian cholinesterase: in vitro characterization of an evolutionarily ancient enzyme to study organophosphorus pesticide toxicity and reactivation………………………………... 169 Chapter 6: Planarian cholinesterase: molecular and functional characterization of an evolutionarily ancient enzyme to study organophosphorus pesticide toxicity………………………………… 198 Chapter 7: Comparative analysis of the mechanisms of organophosphorus pesticide developmental neurotoxicity in freshwater planarians........…………………………………………………… 240 Chapter 8: Conclusion and future outlook……………………………………………………... 274 Appendix: Studying planarian regeneration aboard the International Space Station within the Student Space Flight Experimental Program………………………………………………….. 281 iv LIST OF FIGURES Figure 1.1. Comparison of the two scoring systems by Grebe and Schaeffer (GS-system) (Grebe & Schaeffer 1991) and Wu et al (Wu, Chen, et al. 2012).……………………………………… 8 Figure 1.2. Examples of planarian morphological readouts and body shapes…………………... 12 Figure 1.3. Overview of behavioral assays employed in the literature to quantify neuronal function after toxicant exposure…………………………………………………………………………. 14 Figure 1.4. Morphological and anatomical readouts of developmental neurotoxicity in planarians. …………………………………………………………………………………………………. 18 Figure 2.1. Overview of assay………………………………………………………………….. 54 Figure 2.2. Viability of full and regenerating worms…………………………………………… 56 Figure 2.3. Unstimulated behavior of toxicant-exposed full and regenerating worms………… 60 Figure 2.4. Regeneration is generally unaffected by toxicant exposure………………………... 64 Figure 2.5. Effects on brain morphology………………………………………………………. 67 Figure 2.6. Temperature sensing assay………………………………………………………… 71 Figure 2.7. Effect and potency of all toxicants on ten quantitative endpoints………………….. 73 Figure 3.1. Overview of planarian screening platform………………………………………… 103 Figure 3.2. Lethality and eye regeneration endpoints………………………………………...... 105 Figure 3.3. Unstimulated behavior: gliding and resting……………………………………….. 108 Figure 3.4. Comparison of time-points and worm types for unstimulated behavior hits………. 110 Figure 3.5. Stimulated behaviors………………………………………………………………. 111 Figure 3.6. Comparison of shared hits in stimulated vs unstimulated behaviors………………. 115 Figure 3.7. Analysis of LOEL by endpoint…………………………………………………….. 117 Figure 3.8. Summary of screening results for regenerating tail………………………………... 118 Figure 3.9. Summary of screening results in full planarians…………………………………… 119 Figure 4.1. Comparison of screening schemes in the zebrafish and planarian systems………... 148 v Figure 4.2. Summary of (A) zebrafish and (B) planarian hits in each endpoint class………… 150 Figure 4.3. Comparison of active hits in the zebrafish and regenerating planarian screens……. 152 Figure 4.4. Physicochemical properties of the NTP 87-compound library…………………….. 154 Figure 4.5. Inter-relationship between 28 chemicals, zebrafish and planarian assay endpoints and study types in ToxRefDB……………………………………………………………………… 157 Figure 5.1. DjChE shows kinetic characteristics intermediate to mammalian AChE and BChE. 179 Figure 5.2. Inhibition by classic reversible quaternary and uncharged amine inhibitors………. 180 Figure 5.3. Oxime elicited reactivation of diazinon oxon inhibited DjChE activity using 4 mM (A) 2-PAM or (B) RS194B)………………………………………………………………………. 183 Figure 5.4. DjChE is efficiently reactivated by NaF……………………………………........... 184 Figure 6.1. Candidate DjChEs show characteristics of both AChE and BChE……………….... 213 Figure 6.2. Homology modeling of planarian cholinesterase protein structure……………....... 214 Figure 6.3. Planarian cholinesterases are expressed in the nervous system…………………..... 215 Figure 6.4. Planarian cholinesterases co-localize with each other and Djchat in the medial arc of the brain……………………………………………………………………………………….. 216 Figure 6.5. Inhibition of DjChE decreases sensitivity to heat stress…………………………… 218 Figure 6.6. Diazinon and physostigmine, but not DjChE knockdown, increase worm adhesion (“stickiness”)………………………………………………………………………………….. 222 Figure 7.1. Body shape classifications in the morphology assay………………………………. 253 Figure 7.2. Regenerating planarian toxicological profiles…………………………………….. 256 Figure 7.3. Full planarian toxicological profiles………………………………………………. 264 vi LIST OF TABLES Table 1.1. Mechanistic pathways tested in planarian toxicology………………………………. 23 Table 2.1. Chemicals and concentration ranges tested………………………………………..... 44 Table 2.2. LC50 values after 2, 4, 8, or 15 days of exposure for full and regenerating worms…. 57 Table 2.3. Comparison of LC50 values for planarians with zebrafish and nematodes………….. 74 Table 2.4. Comparison of LOEL values of tested chemicals in planarians with previous studies in zebrafish and nematodes………………………………………………………………………. 75 Table 3.1. Summary of statistical testing……………………………………………………… 101 Table 3.2. Summary of percentage of actives observed in different toxicant classes in all endpoints for either full worms (F) or regenerating tails (R)……………………………………………… 120 Table 3.3. Developmentally selective chemicals ……………………………............................ 121 Table 3.4. Comparison of results with previous planarian studies……………………………... 125 Table 3.5. Summary of the strengths and weaknesses of the planarian toxicology system……. 127 Table 4.1. Classes of endpoints used in the two systems………………………………………. 144 Table 5.1. IC50 (M) of reversible inhibitors for 0.1 and 1 mM ATCh and BTCh……………… 181 Table 5.2. Rates of inhibition by OP oxons……………………………………………………. 182 Table 5.3. Rates of inhibition by carbamylating agents………………………………………... 182 Table 7.1. Chemicals tested in this screen……………………………………………………... 248 Table 7.2. Most sensitive endpoints affected by each chemical in regenerating planarians…… 257 Table 7.3. Developmental selectivity scores, quantified as the log(LOELfull/LOELregen), for endpoints shared in both worm-types………………………………………………………….. 265 vii ACKNOWLEDGEMENTS I would like to acknowledge Professor Eva-Maria S. Collins for her amazing support as the chair of my committee and as a fantastic mentor. I would also like to thank my committee members for all of their support and guidance through my graduate work. I would like to acknowledge Siqi Zhang for being my partner through most of my research. Our well-oiled teamwork was instrumental for our success. Her creation of the planarian medium-throughput screening platform was essential to my work. Chapter 1, in full, is a reprint of the material as it appears in Hagstrom, Danielle; Cochet- Escartin, Olivier; and Collins, Eva-Maria S. “Planarian brain regeneration as a model system for developmental neurotoxicology”, Regeneration, vol. 3, 2016. The final version is available online at: https://onlinelibrary.wiley.com/doi/full/10.1002/reg2.52. The authors retain copyright of this manuscript, which is an open access article permitting the use herein. Danielle Hagstrom, Olivier Cochet-Escartin, and Eva-Maria S. Collins co-wrote and edited the manuscript. Danielle Hagstrom was the primary investigator and author of this paper. Chapter 2, in full, is a reformatted reprint of the material as it appears in Hagstrom, Danielle; Cochet-Escartin, Olivier; Zhang, Siqi; Khuu, Cindy; and Collins, Eva-Maria S. “Freshwater planarians as an alternative animal model for neurotoxicology”, Toxicological Sciences, vol. 147, 2015. The version of record is available online at: https://academic.oup.com/toxsci/article/147/1/270/1642148. Use of this manuscript in the dissertation herein is covered by the rights permitted to the authors by Oxford Journals. Danielle Hagstrom, Olivier Cochet-Escartin, and Eva-Maria S. Collins designed the experiments and co- wrote the manuscript. Danielle Hagstrom, Olivier Cochet-Escartin, and Siqi Zhang designed and performed the experiments, analyzed,