EFFECTS OF INORGANIC MERCURY ON DEVELOPING ZEBRAFISH (DANIO RERIO) LARVAE A Thesis Submitted to the College of Graduate Studies and Research In Partial Fulfillment of the Requirements For the Degree of Doctor of Philosophy In the Toxicology Graduate Program University of Saskatchewan Saskatoon By TRACY CLARYLINDA MACDONALD Copyright Tracy Clarylinda MacDonald, September, 2015. All rights reserved. PERMISSION TO USE STATEMENT In presenting this thesis in partial fulfillment of the requirements for a postgraduate degree from the University of Saskatchewan, I agree that the Libraries of this University may make it freely available for inspection. I further agree that permission for copying of this thesis in any manner, in whole or in part, for scholarly purposes may be granted by the professor or professors who supervised my thesis work or, in their absence, by the Head of the Department or the Dean of the College in which my thesis work was done. It is understood that any copying or publication or use of this thesis or parts thereof for financial gain shall not be allowed without my written permission. It is also understood that due recognition shall be given to me and to the University of Saskatchewan in any scholarly use which may be made of any material in my thesis. Requests for permission to copy or to make other use of material in this thesis in whole or part should be addressed to: Chair of the Toxicology Graduate Program Toxicology Centre University of Saskatchewan 44 Campus Drive Saskatoon, Saskatchewan, Canada S7N 5B3 i ABSTRACT Mercury (Hg) compounds are some of the most toxic compounds of any heavy metal on earth. Due to long-range transport from point sources Hg can be found world-wide in air, soil, water, and living organisms. Mercury compounds can cause a number of adverse effects, with the unborn fetus, infants, and children being most susceptible. Zebrafish (Danio rerio) are an excellent vertebrate model system for toxicological studies, including developmental effects. The overall objective of this research was to investigate the effects of inorganic forms of Hg in zebrafish larvae. Unique accumulation patterns were observed using synchrotron X-ray fluorescence imaging after zebrafish were exposed to one of four Hg compounds (i.e. mercuric chloride, mercury bis-L-cysteineate, methylmercury chloride, methylmercury L-cysteineate). Specifically, we noted chemical form dependant and tissue-specific Hg accumulation including the sensory cells of the olfactory epithelia and the neuromasts. Phenylthiourea (PTU) is commonly used to block zebrafish melanogenesis to generate transparent larvae to aid with enhanced visualization of immunohistochemical and vital stains. It was determined that PTU dramatically alters Hg toxicity through chemical interaction with Hg so that further studies were conducted in the absence of PTU. To investigate the effects of Hg on primary neurons, the immunohistochemistry protocol using anti-acetylated tubulin was performed and the results demonstrated that mercuric chloride damages primary neurons particularly in the olfactory pits. To study potential detoxification of Hg in zebrafish we examined the efficacy of two sequestration agents, dimercaptosuccinic acid and alpha lipoic acid, as well as endogenous selenium. The levels of Hg were not significantly lower following treatment with either sequestration agent under the conditions used in this research. Previous work examining the antagonistic relationship between Hg and selenium has been conducted by dosing animals with both Hg and selenium (Se). We discovered a mixed chalcogenide of the general form HgSxSe(1-x) forming in vivo following exposure to mercuric chloride without the addition of selenium. Indeed the selenium may have been remobilized from natural stores in the pigment spots. The research presented herein demonstrates that the target tissues for Hg depend strongly on chemical form. In particular inorganic Hg can accumulate in a number of important tissues ii including sensory systems. The formation of insoluble and non-toxic HgSxSe(1-x) in zebrafish larvae suggests that endogenous selenium may play critical roles in modulating toxicity. iii ACKNOWLEDGEMENTS My thesis work would not have been possible without guidance, patience, and support from a number of people. I am very grateful for my supervisors, Dr. Graham George and Dr. Pat Krone, who gave me the opportunity to complete my PhD in their research groups. They challenged and encouraged me to become a better scientist. I would also like to thank my committee members, Dr. Barry Blakley, Dr. Dave Janz, Dr. Phyllis Paterson, and Dr. Ingrid Pickering, who helped me improve my research by always leaving their doors open when I needed advice and suggestions. I am also appreciative of the beamline support provided by Dr. Robert Gordon and Dr. Sam Webb. I would like to acknowledge the support I received from the Natural Sciences and Engineering Research Council of Canada (NSERC), the Canadian Institutes of Health Research (CIHR), and CIHR-THRUST. I owe a debt of gratitude to all of my friends and colleagues for their ongoing encouragement and for making my graduate studies an enjoyable experience. I would like to thank Dr. Gosia Korbas for taking me under her wing and teaching me about many aspects of her research. I would also like to thank my group members and friends, especially Dr. Brian Bewer, Tanner Bodnarik, Dr. Sally Caine, Dr. Julien Cotelesage, Dr. Mark Hackett, Ashley James, Eden Marchand, Susan Nehzati, Kurt Nienaber, Dr. Jake Pushie, Kelly Summers, Nicole Sylvain, Dr. Cynthia Swan, Mylyne Tham, and Justin Tse, for providing their scientific expertise, advice, and thoughtful comments. Finally, for their unconditional love and support throughout my education, I would like to send a special thank-you to my grandma, my parents, and my sister. iv DEDICATION I dedicate my dissertation to my grandpa, Harold Campbell (1920-2014), who taught me the importance of pausing and “havin’ a look” to appreciate the world around me. My achievements were made possible by his constant love and encouragement. His accomplishments and the person he was will continue to inspire me. v TABLE OF CONTENTS PERMISSION TO USE STATEMENT .......................................................................................... i ABSTRACT .................................................................................................................................... ii ACKNOWLEDGEMENTS ........................................................................................................... iv DEDICATION ................................................................................................................................ v TABLE OF CONTENTS ............................................................................................................... vi LIST OF TABLES .......................................................................................................................... x LIST OF FIGURES ....................................................................................................................... xi LIST OF ABBREVIATIONS ...................................................................................................... xiv CHAPTER 1. INTRODUCTION ................................................................................................... 1 1.1. Mercury ......................................................................................................................... 1 1.1.1. Types of Mercury Compounds ......................................................................... 1 1.1.2. Sources .............................................................................................................. 2 1.1.3. Absorption, Distribution, and Excretion of Mercury ........................................ 6 1.1.4. Effects of Mercury on Organisms ..................................................................... 7 1.1.5. Mercury and Selenium ...................................................................................... 9 1.2. Zebrafish ..................................................................................................................... 11 1.2.1. A Model System for Studying Toxins ............................................................ 11 1.2.2. An Introduction to the Olfactory System ........................................................ 12 1.2.3. An Introduction to the Lateral Line System ................................................... 14 1.2.4. Potential Effects on Developing Sensory Systems ......................................... 15 1.3. Synchrotron Techniques ............................................................................................. 16 1.3.1. X-ray Fluorescence Imaging ........................................................................... 16 1.3.1.1. Overview of X-ray Fluorescence Imaging ....................................... 16 1.3.1.2. Advantages of X-ray Fluorescence Imaging .................................... 17 1.3.1.3. Disadvantages of X-ray Fluorescence Imaging ............................... 18 1.3.1.4. Potential Complementary Techniques ............................................. 18 1.3.2. X-ray Absorption Spectroscopy ...................................................................... 20 1.4. Research Objectives and Null Hypotheses ................................................................. 21 1.4.1. Objectives ......................................................................................................