Mississippi State University Scholars Junction Theses and Dissertations Theses and Dissertations 8-10-2018 Determination of Age-Related Differences in Activation and Detoxication of Organophosphates in Rat and Human Tissues Edward Caldwell Meek Follow this and additional works at: https://scholarsjunction.msstate.edu/td Recommended Citation Meek, Edward Caldwell, "Determination of Age-Related Differences in Activation and Detoxication of Organophosphates in Rat and Human Tissues" (2018). Theses and Dissertations. 1339. https://scholarsjunction.msstate.edu/td/1339 This Dissertation - Open Access is brought to you for free and open access by the Theses and Dissertations at Scholars Junction. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of Scholars Junction. For more information, please contact [email protected]. Template A v3.0 (beta): Created by J. Nail 06/2015 Determination of age-related differences in activation and detoxication of organophosphates in rat and human tissues By TITLE PAGE Edward Caldwell Meek A Dissertation Submitted to the Faculty of Mississippi State University in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Environmental Toxicology in the College of Veterinary Medicine Mississippi State, Mississippi August 2018 Copyright by COPYRIGHT PAGE Edward Caldwell Meek 2018 Determination of age-related differences in activation and detoxication of organophosphates in rat and human tissues By APPROVAL PAGE Edward Caldwell Meek Approved: ____________________________________ Janice E. Chambers (Major Professor) ____________________________________ John Allen Crow (Committee Member) ____________________________________ Stephen B. Pruett (Committee Member) ____________________________________ George Eli Howell, III (Committee Member) ____________________________________ Russell Carr (Committee Member / Graduate Coordinator) ____________________________________ Mark L. Lawrence Associate Dean College of Veterinary Medicine Name: Edward Caldwell Meek ABSTRACT Date of Degree: August 10, 2018 Institution: Mississippi State University Major Field: Environmental Toxicology Major Professor: Dr. Janice E. Chambers Title of Study: Determination of age-related differences in activation and detoxication of organophosphates in rat and human tissues Pages in Study 168 Candidate for Degree of Doctor of Philosophy The mechanism of toxic action for organophosphates (OPs), originally developed as insecticides, is the persistent inhibition of acetylcholinesterase (AChE) resulting in accumulation of acetylcholine and subsequent hyperstimulation of the nervous system. Many OPs require bioactivation via cytochromes P450 to oxon metabolites which are anticholinesterases. Organophosphates display a wide range of acute toxicities. Differences in the OPs’ chemistries results in differences in the compounds' metabolism and toxicity. Acute toxicities of OPs appear to be principally dependent on compound specific efficiencies of detoxication, and less dependent upon efficiencies of bioactivation and sensitivity of AChE. Esterases, such as carboxylesterase (CaE) and butyrylcholinesterase (BChE), play a prominent role in OP detoxication. Organophosphates can stoichiometrically inhibit these enzymes, removing OPs from circulation thus providing protection for the target enzyme, AChE. This in vitro study investigated: 1) age-related sensitivity of AChE, BChE and CaE to structurally different OPs in rat tissues; 2) interspecies and intraspecies differences in bioactivation and detoxication of the OP insecticide malathion in rat and human hepatic microsomes; and 3) interspecies and intraspecies differences in sensitivity of AChE from erythrocyte ghost preparations to malaoxon. Sensitivities of esterases to 12 OPs was assessed by IC50s. The OPs displayed a wide range of AChE IC50s (low nM-µM) with no differences among ages; however, the CaE IC50s generally increased with age (up to 100-fold) reflecting greater protection in adults. Kinetic analysis of the bioactivation of malathion to the anticholinesterase metabolite, malaoxon, was measured in hepatic microsomes from rats (adult) and humans (various ages) of both sexes. No statistical interspecies (rat and human) or intraspecies (among humans) differences were found. The CaE degradation of malathion and malaoxon was determined in the microsomal samples using indirect measurements. No interspecies or intraspecies differences were found; however, CaE activity in rat microsomes was significantly higher than in humans. Inhibition of AChE by malaoxon was analyzed kinetically in erythrocyte ghost preparations from rats (adults) and humans (three age groups) of both sexes. No statistical interspecies or intraspecies differences were found. These results suggest the age-related differences in acute toxicities of OPs in mammals is primarily a result of their detoxication capacity. DEDICATION I dedicate this work to my family. The love and support you have given me through the years have helped me become the person I am today and accomplish my educational goals. The sacrifices you have made for me are great and for that I am eternally grateful. To my parents, John and Louise Meek, I couldn’t have asked for better a better mother and father and I miss you every day. You instilled in me faith, ethics, and hard work and I hope I made you proud. To my brother, the Meek boys, will always be best friends and I know you will always be there if I need anything. To my wife, Denise and step-son Chase, you are a blessing every day. Your love and support drive me to be a better man and I strive every day to be a better husband and father. I love you very much. To my grandparents and all my aunts and uncles that shaped my childhood, thank you. I wouldn’t take anything for growing up in a rural area with all my family and the lessons they taught me. Finally, I would also like to dedicate this work to Jan Chambers for seeing a little potential in me and believing in me and Howard Chambers a great scientist and a better friend. ii ACKNOWLEDGEMENTS First, I would like to thank God for the strength and guidance throughout my life and for bringing all the people into my life that have helped me along the way. I want to thank my parents for instilling in me faith and the work ethic that drives me every day. I appreciate the sacrifices they made so that I could pursue my education. I would like to thank my brother for always being there when I needed anything; you and I will always be best friends. I would like to thank my wife, Denise, for believing in me and encouraging me to pursue my doctoral degree. I would like to thank my step-son, Chase, for always being there with a smile and telling me “I know you will do good.” I would like to thank all the Center for Environmental Health Sciences faculty, staff and students for all their help and support through the years. I consider it an honor and a privilege to work with such a great group of people. I would especially like to thank Dr. Janice Chambers for believing in me and allowing me to be a part of your laboratory. Thank you for mentoring me and allowing me to further my education while working full time. I have learned more than just toxicology from you. Thank you for allowing me to participate in writing grants, professional meetings, and training of staff and students. All of these efforts have given me greater perspective on how to run a successful research program and for that I am extremely grateful. I appreciate all the encouragement, support and friendship through the years. I would like to thank all of my committee members for their advice and guidance as I developed my research project, iii conducted my experiments, and authored my dissertation. I have a great amount of respect for each of you and, I am honored to have you on my committee. Dr. Russell Carr, thank you for all your help over the years especially teaching me techniques for in vivo studies. I have enjoyed working with you and appreciate your friendship and everything you have done for me. Dr. Allen Crow, I have enjoyed working with you on many research projects over the last few years. Your knowledge of physiology and clinical experience have brought a unique perspective to many of our projects and I have learned a lot from you. Thank you for your help and advice with my kinetic calculations as well as your friendship. Dr. Stephen Pruett, thank you for agreeing to be a late addition to my committee and being very accommodating, especially with your busy schedule. Thanks for your leadership as our department head and the encouragement you given me pursuing my doctoral degree. Dr. Trey Howell, thanks for also agreeing to serve on my committee late in the process. I have enjoyed working with you on several projects over the last few years and appreciate your advice and friendship. Finally, I want to say a special thanks to my former committee member Dr. Howard Chambers for his many hours of mentorship and troubleshooting experiments. I miss our conversations on science and life and your sense of humor. Again, thanks to everyone for serving on my committee for their friendship and mentorship. Thanks to the administrative support staff that have helped me with many things through the years including Jeanne Whitehead, Tia Perkins, and Toni Roberson. Thank you to the National Institute of Health for the financial support of the first specific aim of this research (R01 ES011287). Thank you to Exponent/FMC for