UNIVERSITY OF CINCINNATI Date:___________________ I, _________________________________________________________, hereby submit this work as part of the requirements for the degree of: in: It is entitled: This work and its defense approved by: Chair: _______________________________ _______________________________ _______________________________ _______________________________ _______________________________ Toxicogenetic Studies in Drosophila: Using Fruit Flies to Study Arsenic Toxicity A dissertation submitted to The Graduate School of the University of Cincinnati In partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY (Ph.D.) In the Department of Molecular Genetics, Biochemistry, and Microbiology of the College of Medicine 2008 By Jorge Gerardo Muñiz Ortiz B.S., University of Dayton, 2002 Committee Chair: Iain L. Cartwright, Ph.D. Abstract Arsenic contamination of drinking water supplies around the world is considered the worst environmental disaster of recent times. Chronic consumption of arsenic can lead to an array of serious pathological outcomes, in some of which methylation of the metal may be a crucial component in determining toxicity. Differential responsiveness within human populations suggests inter-individual genetic variation also plays an important role. We have used Drosophila melanogaster as a model to study arsenic response pathways because of unrivalled access to varied genetic approaches and significant overlap with many aspects of mammalian physiology and disease phenotypes. Genetic analysis of various strains exhibiting relative susceptibility or resistance to arsenite toxicity resulted in the identification of a chromosomal region able to confer a differential response phenotype. We created fly lines harboring small, overlapping deficiencies in this region and found that relative arsenite sensitivity arose when the glutathione synthetase (GS) gene dose was reduced by half. Knock-down of GS expression by RNA interference both in S2 cells and in vivo led to highly enhanced arsenite sensitivity. These analyses provide genetic proof that an optimally functioning glutathione (GSH) biosynthetic pathway is required for a robust defense against arsenite. Moreover, they unexpectedly highlight a step previously considered to be without regulatory significance; the implications of this are discussed in the context of GSH supply and demand under arsenite-induced stress. Recent work has shown that Drosophila does not possess an arsenic methylation pathway comparable to the human. Since methylated arsenicals (MAs) may be key players in the carcinogenic activity of arsenic, we have “humanized” Drosophila through the introduction of the human arsenic(III) methyltransferase (hAS3MT) gene expressed under easily manipulated regulatory control. Transgenic flies can be induced to express an antigenically cross-reactive form with arsenic iii methyltransferase activity of the hAS3MT enzyme and its expression does not affect the development or viability when exposed or unexposed to arsenic. Preliminary results using an in vivo genotoxicity assay have shown that production of MAs induces tumorigenesis in Drosophila. This model is ready for use in exploring mechanisms of arsenic genotoxicity and/or carcinogenicity in many informative genetic backgrounds, as well as the effects of different polymorphic variants of AS3MT found in human populations. iv v Dedication To my late abuela Epi, the matriarch and rock behind the Ortiz family, for teaching me what the term family really means and for teaching me how to make paper boats and paper airplanes. And To my wife, Deborah María Sánchez Aceituno, who taught me to stand up for what I believe in and to surround myself with those who improve my quality of life. And To my dad, Papi, Viejo, Jorge Muñiz Morales, who taught me to never ever give up and how to always be a gentleman. And To my mom, Mami, Carmen Belén Ortiz Rivera, who taught me that nobody is better than me, but I’m not better than anybody and to shine by my own light. And To my brother, Javy, Jorge Javier Muñiz Ortiz, because even though we have been far apart for so long, it is as if we are not. And Para mi Isla del Encanto, Puerto Rico, que esto sea una fuente de inspiración para los que sueñan… (For my Island of Enchantment, Puerto Rico, may this be a fountain of inspiration for those who dream…) vi Acknowledgements First and foremost, I would like to thank my advisor, Dr. Iain L. Cartwright because even when it seemed that no progress was being made, we were able to move forward because of the psychological support he provided. I owe you everything I’ve learned throughout my graduate career, for challenging me and for always wanting what is best for me as a scientist. Thank you for your support in every endeavor I wanted to undertake, especially when pledging and when planning to go to conferences. Who thought that Sir Iain from England, now an American citizen, the foremost figure in the English language, would be so patient with me, a native Spanish speaker who uses English to survive. To my committee members, Drs. Gary E. Dean, thank you for challenging me, Dan J. Hassett, thank you for expecting plenty of me, Anil G. Menon, thank you for your positive feedback, it made me feel good, and Alvaro Puga, thank you for your expertise on arsenic. I want to thank the Shull lab for teaching me Northern analysis, the Miller lab for teaching me Western analysis, the DeKoter lab for teaching me real time PCR, Weiss, Stringer, Thompson, Kovall, Herr, Shertzer, and Caruso labs for use of their equipment. Thanks also go to the Air Force Research Lab for use of their equipment and their friendship. I want to express my sense of gratitude to Dr. Richard M. Weinshilboum for providing the antibody against the human hAS3MT, Dr. David J. Thomas for technical advice on use of antibodies and Dr. James Lessard for providing the antibodies against actin (there’s plenty for the whole department for an immeasurable amount of years). I want to thank Dr. Bryan Mackenzie for his help on statistical analyses. I would like to thank my wife, Debbie, for lighting a match under me and helping me deal with the pressures of lab work and morally helping me push through the hard times when it vii seemed that graduation was more than 10 years away. I can finally be grateful for receiving a Ph.D., but without your love I have nothing. May the adventures we live from now on mark our future and leave a mark for generations to come. My family, Papi, Mami and Javy, for their great support even though sometimes it is hard when they are miles and miles away. For always being there since the day I left home to pursue a career en el Norte. However, with the advancement of cellular phone communication devices, just a second away, hopefully they feel as if they have personally witnessed every failure and every glory. Los quiero mucho, les debo la vida que vivo y para ustedes le dejo la muestra de mi esfuerzo y mis riesgos que nunca fueron en vano. To my student colleagues, postdocs, faculty and office employees, past and present, which helped me throughout my graduate career, from simple experiments to complicated matters of life. I would also like to acknowledge Dan Kane for his summer work on the FLP- FRT strains and Mayank Patel for the time he kept our flies well fed. To the MolGen Softball Team, the Banana Slugs Softball Team and Los Caimanes de Cincinnati Professional Softball Team, for providing me with an athletic avenue in order to vent my frustrations. To my best friends outside of science, who gave me an escape from the daily routine in the lab: Luis, Tommy, Millo y Pancho. viii Table of Contents Abstract............................................................................................................................. iii Dedication ......................................................................................................................... vi Acknowledgements ......................................................................................................... vii Table of Contents ............................................................................................................. ix List of Abbreviations and Symbols .............................................................................. xiii List of Figures and Tables............................................................................................. xvi Chapter I. An Introduction to Arsenic and Drosophila ............................................1 History of Arsenic..................................................................................................1 Arsenic Worldwide and Health Effects................................................................1 Toxicology and Mode of Action............................................................................3 1. Oxidative Stress.....................................................................................3 1.1.1. Production of ROS..............................................................3 1.1.2. ROS-induced DNA Damage...............................................6 1.1.3. Interaction of Arsenic-induced ROS with Signaling Pathways ..............................................................................7 1.2. Production of Reactive Arsenical Species............................8 2. Inhibition of DNA Damage Repair......................................................9 3. Epigenetic Alterations ........................................................................10