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THUETT-DISSERTATION.Pdf COMPARATIVE NEUROTOXICITY OF METHYLMERCURY AND MERCURIC CHLORIDE IN VIVO AND IN VITRO A Dissertation by KERRY ALMEDA THUETT Submitted to the Office of Graduate Studies of Texas A&M University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY August 2009 Major Subject: Toxicology COMPARATIVE NEUROTOXICITY OF METHYLMERCURY AND MERCURIC CHLORIDE IN VIVO AND IN VITRO A Dissertation by KERRY ALMEDA THUETT Submitted to the Office of Graduate Studies of Texas A&M University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Approved by: Co-Chairs of Committee, Louise C. Abbott Evelyn Tiffany-Castiglioni Committee Members, Wei-Jung A. Chen Timothy D. Phillips Chair of Toxicology Faculty, Robert C. Burghardt August 2009 Major Subject: Toxicology iii ABSTRACT Comparative Neurotoxicity of Methylmercury and Mercuric Chloride In Vivo and In Vitro. (August 2009) Kerry Almeda Thuett, B.S.; M.S., Texas Tech University Co-Chairs of Advisory Committee: Dr. Louise C. Abbott Dr. Evelyn Tiffany-Castiglioni It is impossible to remove methylmercury (MeHg) from biological systems because MeHg is found throughout our environment in many fresh and salt water fish. The consumption of fish is important to human nutrition and health. The mechanism of MeHg neurotoxicity must be understood to minimize adverse exposure consequences. The dissertation objective was to: 1) compare mechanisms of MeHg neurotoxicity between animals exposed as adults and those exposed during gestation, and 2) develop an in vitro test model of in vivo MeHg exposure. Total mercury (Hg) levels in tissue / cells were determined by combustion / trapping / atomic absorption. Cell death was determined by Fluoro-Jade histochemical staining and activated caspase 3 immunohistochemistry for in vivo studies, and Trypan blue exclusion, lactate dehydrogenase activity, and cytotoxicity assays for in vitro studies. Mitochondrial membrane potential (MMP), intracellular calcium ion 2+ concentration ([Ca ]i), and production of reactive oxygen species (ROS) were determined using fluorescence microscopy or microplate reader assays. Young adult C57Bl/6 mice were exposed to a total dose of 0, 1.0, or 5.0 mg/kg body weight MeHg divided over postnatal days (P)35 to 39. Pregnant female mice were exposed to a total does of 0, 0.1, or 1.0 mg/kg body weight MeHg divided over gestational days (G)8 to 18. SY5Y cells were exposed to 0, 0.01, 0.1, or 1.0 µM MeHg or HgCl2 for 24, 48, or 72 hours. iv Total Hg in brains of young adult mice, mouse pups, and SY5Y cells accumulated in a dose-dependent manner. Cell death increased in SY5Y cells exposed to the highest concentrations of MeHg and HgCl2 used in this study. Cell death increased in the molecular and granule cerebellar cell layers of young adult mice exposed to the highest doses of MeHg used in this study. P0 mouse pups showed no increase in cell death within the cerebellum following MeHg exposure. Cerebella of mice at P10 exhibited decreased dying cells only in the external germinal layer. Low concentrations of MeHg affected MMP in both in vivo and in vitro studies, but did not result in decreased MMP typically associated with higher MeHg 2+ concentrations. [Ca ]i was increased throughout the in vivo experiments in an age- , sex- and brain region-dependent manner. Generation of ROS was decreased in both in vivo and in vitro studies with both the MeHg and HgCl2 (in vitro) treatments. In summary, low and moderate MeHg exposure, both in vivo and in vitro, altered mitochondrial function, Ca2+ homeostasis, and ROS differently than what is reported in the literature for higher MeHg exposure concentrations. SY5Y cells were sensitive to low-levels of MeHg and HgCl2 and responded similarly to cells in the whole animal studies, thus making SY5Y cells realistic candidates for mechanistic MeHg studies. Cell culture and whole animal neuronal functional studies at chronic low-level MeHg exposure are limited. These data suggest that low-levels of MeHg may affect neuronal function. Therefore, further chronic low-level MeHg neuronal functional studies are warranted. v DEDICATION There are people in my life that I love more than life itself. They mean more to me than any degree I could earn, more than any praise or accolade I will ever receive. To these pillars upon whom my dissertation is founded, I dedicate these words: “God is our refuge and strength, a very present help in trouble.” (Psalms 46:1) “Come to Me, all who are weary and heavy-laden, and I will give you rest.” (Matthew 11:28) “Every good thing bestowed and every perfect gift is from above, coming down from the Father of lights, with whom there is no variation, or shifting shadow.” (James 1:17) “In all your ways acknowledge Him, and He will make your paths straight.” (Proverbs 3:6) Drawn from The Holy Bible, New American Standard Version. Ronald and Nancy, you are the greatest parents ever given to any child. Trevor, Jodi, Dalton and Caelan, I love you all; we need to play more Guitar Hero! Casey Donnelly and Tim Knight, you are the best; thanks for the many fun celebrations. Deanna Mueller Keene, Catherine Mueller Glaze, and Jennafer Todd Chaddrick, you are my very best friends, and I thank you endlessly for keeping in touch when I had retreated from the world. Sarah Wills, I am a better person because of you, and I am grateful. Ian Murray, thank you for being a supportive scientist and friend; thanks also for bringing Kevin to Salsa class. Kevin Joy, where did you come from?! You entered my life at a time that was most needed; I know, without a doubt, that you were sent from God to ensure that my Ph.D was completed. Nick Garcia, Nickercizing with the Nick-elodeon was a daily respite from the tedium of research; as you abused my body, you freed my mind; because of your excellent skills and training, I am stronger and healthier today than I have ever been in my life. vi ACKNOWLEDGEMENTS I would like to thank my committee co-chairs, Dr. Louise Abbott and Dr. Evelyn Castiglioni, and my committee members, Dr. Wei-Jung Chen and Dr. Tim Phillips for their guidance and support throughout the course of this research. These individuals were consistently supportive, patient, and understanding as I made my way through the process of attaining a Ph.D. Their direction and expertise in whole animal, cell culture, statistical, and environmental toxicological research played a pivotal role in my success. Thanks also go to Dr. Robert Burghardt and Dr. Rola Baroumi in the College of Veterinary Medicine and Biomedical Sciences Image Analysis lab for their mentoring, time, and attention. These two individuals and the equipment they provided were instrumental in my successful and proper completion of over half of my dissertation research. Dr. Yongchang Qian, was a continual support and source of knowledge during my cell culture research. He provided mentoring as well as the space and supplies necessary for the in vitro portion of my project. He and Ying Zheng, together helped make my cell culture research a success. Throughout my time at Texas A&M, several undergraduate students participated in portions of my research. Heather Thompson, Rene Ramon, Susan Hernandez, Raul Grajeda, Francisco Gomez, Ian Windsor, Tania Kanadan, and Devang Ghandi have earned my deepest gratitude for their eagerness to work, willingness to learn, and dedication to the end goal. I would like to thank Sang-Soep Nahm, Tamy Frank-Cannon, Sairam Bellum, Bhupinder Bawa, Sarah Wills, and Pei-San Huang for their expertise, kindness, and ultimately, their friendship. These lab colleagues taught me techniques and skills and provided helping hands when needed. vii TABLE OF CONTENTS Page ABSTRACT .............................................................................................................. iii DEDICATION .......................................................................................................... v ACKNOWLEDGEMENTS ...................................................................................... vi TABLE OF CONTENTS .......................................................................................... vii LIST OF FIGURES ................................................................................................... ix LIST OF TABLES .................................................................................................... xiv CHAPTER I INTRODUCTION ................................................................................ 1 Hippocampal Formation ................................................................. 1 Cerebellum ..................................................................................... 13 Central Nervous System Development .......................................... 27 Mitochondria .................................................................................. 37 Reactive Oxygen Species ............................................................... 43 Calcium .......................................................................................... 47 Neurotoxicology ............................................................................. 51 Developmental Neurotoxicology ................................................... 56 Artifacts of Cell Culture ................................................................. 58 Mercury .......................................................................................... 59 Objective of This Dissertation ........................................................ 74 II SUBCELLULAR
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