Molecular Studies in Mercury Toxicity Using X-ray Absorption Spectroscopy and High Energy Resolution Fluorescence Detection X-ray Absorption Spectroscopy A Dissertation Submitted to the College of Graduate and Postdoctoral Studies In Partial Fulfillment of the Requirements For the Degree of Doctor of Philosophy In the Department of Geological Sciences University of Saskatchewan Saskatoon, SK, Canada By Susan Nehzati Copyright Susan Nehzati, April, 2019. All rights reserved. PERMISSION TO USE In presenting this dissertation in partial fulfilment 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 dissertation in any manner, in whole or in part, for scholarly purposes may be granted by the professor or professors who supervised my dissertation work or, in their absence, by the Head of the Department or the Dean of the College in which my dissertation work was done. It is understood that any copying or publication or use of this dissertation 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 dissertation. DISCLAIMER Reference in this dissertation to any specific commercial products, process, or service by trade name, trademark, manufacturer, or otherwise, does not constitute or imply its endorsement, recommendation, or favoring by the University of Saskatchewan. The views and opinions of the author expressed herein do not state or reflect those of the University of Saskatchewan, and shall not be used for advertising or product endorsement purposes. Requests for permission to copy or to make other uses of material in this dissertation in whole or part should be addressed to: Head of the Department of Geological Sciences University of Saskatchewan 114 Science Place Saskatoon, Saskatchewan, Canada S7N 5E2 i OR Dean College of Graduate and Postdoctoral Studies University of Saskatchewan 116 Thorvaldson Building, 110 Science Place Saskatoon, Saskatchewan, Canada S7N 5C9 ii ABSTRACT Molecular understanding of mercury compounds is of considerable interest as the element is pervasive in the environment and certain chemical forms are highly toxic to humans at very low doses. Chalcogenides have high affinity for mercury and one in particular, selenium, may play a major role in the mechanisms involved with mercury toxicity in vivo. In this dissertation, conventional X-ray absorption spectroscopy (XAS) and high-energy resolution fluorescence detection X-ray absorption spectroscopy (HERFD XAS) have been employed to identify the chemical coordination of mercury with various ligand types relevant to mercury toxicity. XAS and density functional theory (DFT) calculations were used to identify the molecular structures responsible for the drastic changes in toxicity in zebrafish larvae exposed to a common hypopigment-inducer 1-phenyl-2-thiourea. Next, XAS and X-ray fluorescence imaging (XFI) were used to investigate the fate of mercury localization in zebrafish larvae following treatment with the dithiol 1,3-benzene-diamido-2-carboxylethanethiol. During the course of exploring the molecular structure of various mercury-ligand complexes, it was evident that current methods to probe the electronic structure of mercury compounds are met with challenges, and capabilities to observe richer spectroscopy would significantly benefit molecular studies of mercury toxicity. Hence, the quest to develop methods for enhanced chemical characterization using HERFD XAS was pursued to reveal the potential of increased chemical specificity and improved concentration sensitivity of this technique in natural relevant levels of a series of mercury and selenium compounds. The results present substantial improvements in chemical characterization of mercury and selenium compounds for applications in the study of mercury toxicity. HERFD XAS was thereupon employed as a complementary tool to conventional XAS in the investigations of species identification of mercury and selenium compounds in cases of biological organisms. XAS, HERFD XAS, and quantum-mechanical/molecular-mechanical (QM/MM) studies were employed to examine the interaction of Hg(II) with 1-methylthymine and a canonical DNA helix to understand this mode of biological coordination of Hg(II). Finally, mammalian tissue is examined to reveal unreported chemical species in a historical specimen showing signs of organomercury poisoning from Minamata Bay, Japan. iii ACKNOWLEDGMENTS I would like to thank my advisors, Dr. Graham George and Dr. Ingrid Pickering for their guidance and support throughout this journey. For their contributions to my growth as an academic researcher, I will be forever grateful. To my committee members, Dr. Jonathan Dimmock, Dr. Lee Wilson, and Dr. Derek Peak for their thoughtful inquiry and inspiration. To Dr. Erik Nelson for sponsoring me as an Exchange Scholar at the Stanford Synchrotron Radiation Lightsource (SSRL) – without which I would not have been able to carry out much of my research. Thank you to the overwhelmingly supportive staff of SSRL – Dr. Cynthia Patty, Dr. Matthew Latimer, Dr. Dimosthenis Sokaras, Dr. Thomas Kroll, and Dr. Sam Webb. To Dr. Mariam Alaverdashvili, Dr. Julien Cotelesage, Dr. Natasha Dolgova, Marlon Forsberg, Dr. Mark Hackett, Ashley James, Dr. Tracy MacDonald, and Dr. Jake Pushie for their technical coaching, comedy, wisdom and encouragement along the way. Acknowledgements for work conducted at national research facilities are detailed in individual chapters (Stanford Synchrotron Radiation Lightsource, Chapters 5, 6, 7, 8, 9, 10; Advanced Photon Source, Chapter 5). I would also like to acknowledge the funding support which has allowed me to carry out this research – the Dr. Rui Feng Scholarship, Natural Sciences and Engineering Research Council of Canada (NSERC), Canadian Institutes of Health Research (CIHR), and CIHR – Training in Health Research Using Synchrotron Techniques (CIHR–THRUST). This dissertation is dedicated to my family – my father who has encouraged me to explore; my mother who has given me my strength; my brother for his love and support. iv TABLE OF CONTENTS PERMISSION TO USE ................................................................................................................... i ABSTRACT ................................................................................................................................... iii ACKNOWLEDGMENTS ............................................................................................................. iv TABLE OF CONTENTS ................................................................................................................ v LIST OF TABLES .......................................................................................................................... x LIST OF FIGURES ....................................................................................................................... xi LIST OF ABBREVIATIONS ...................................................................................................... xiv CHAPTER 1. Introduction.............................................................................................................. 1 1.1 Mercury Toxicity ........................................................................................................... 2 1.1.1 Elemental Mercury ............................................................................................ 3 1.1.2 Inorganic Mercury ............................................................................................. 3 1.1.3 Organic Mercury................................................................................................ 4 1.2 Minimizing of Mercury Exposure in Biological Organisms ......................................... 5 1.3 Selenium ........................................................................................................................ 8 1.3.1 Selenium Sources .............................................................................................. 8 1.3.2 Selenium Biochemistry...................................................................................... 9 1.3.3 Selenium and Mercury..................................................................................... 10 1.4 Analytical Methods in Molecular Toxicology ............................................................. 11 1.4.1 Nuclear Magnetic Resonance (NMR) Spectroscopy ....................................... 12 1.4.2 Vibrational Spectroscopy ................................................................................ 13 1.4.3 X-ray Absorption Spectroscopy (XAS) ........................................................... 14 1.4.4 General Remarks ............................................................................................. 15 1.5 Research Objectives ..................................................................................................... 16 CHAPTER 2. Introduction to Synchrotron Radiation Spectroscopic Methods ............................ 18 2.1 X-ray Absorption Spectroscopy (XAS) ....................................................................... 18 2.1.1 X-ray Absorption and X-ray Absorption Spectroscopy Theory ...................... 20 2.1.2 Strengths and Limitations