Selenium mediated arsenic toxicity modifies cytotoxicity, reactive oxygen species and phosphorylated proteins 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 in the Department of Chemistry of the College of Arts and Sciences by Karnakar Reddy Chitta Bachelor of Pharmacy, Nagarjuna University 2004 M.S., Chemistry, Western Illinois University 2007 M.S., Chemistry, University of Cincinnati 2011 Committee Chair: Joseph. A. Caruso, Ph.D. i Abstract of dissertation The effect of selenium on modulating arsenic cytotoxicity is well known in mammals, but not well understood. Cell cytotoxicity and reactive oxygen (ROS) changes were performed in combinations of As(III) and selenomethionine (SeMet) toxic mixes on, HEK 293, human embryonic kidney cells. Cell growth is readily restored from 20% to 60% when switching from 30 M As(III) as toxin to a mix of 30 M As(III) and 100 M SeMet. As(III) alone triggers ROS formation, primarily hydrogen peroxide, in a concentration dependent manner as observed through changes in the fluorescence from 2’,7’-dichlorofluorescin diacetate. Importantly, SeMet induces lower ROS levels at the same concentrations used to modulate As(III) cytotoxicity (IC50). Elevated ROS is important to As(III) cytotoxicity and minimizing it is essential to the SeMet modulating function. Changes in cell signaling, through analysis of signaling changes via differential protein phosphorylation to uncover molecular level changes occurring in HEK 293 human kidney cells as SeMet modulates the As(III) cytotoxicity. To discover changes in the phosphoproteome, cells were incubated under three conditions: 30 M As(III), 100 M SeMet, and 30 M As(III)+ 100 M SeMet. After total protein isolation the three samples were separated into fractions using size exclusion chromatography by detecting 31P+. Each sample was analyzed for the phosphorylated peptides by enzymatic digestion, selective enrichment of phosphorylated peptides via TiO2, followed by nanoLC-ESIMS. Phosphorylated proteins unique to the As(III)/SeMet mixture were then identified. The molecular level changes to the cells show uniquely that the As(III)/SeMet mixture details proteins involved in ROS detoxification, cell cycle arrest, and protein/DNA damage. This study shows that SeMet not only lowers the total ii amount of ROS in a cell but also confers upon HEK 293 cells the ability to detoxify. Thus, SeMet is not only a potent antioxidant in this system, but induces molecular changes that confer survival. iii iv Acknowledgements I am dedicating this dissertation to my parents Koti Reddy Chitta and Venkata Lakshmi Chitta. I would like to thank them a lot for allowing me to pursue my goals and supporting me throughout these years. Their unconditional love always makes me feel indebted for them. I would like to acknowledge my brother Venugopal Reddy Chitta for sharing my happiness and sadness. This journey of mine is not possible without the families - love, support and encouragement. I want to express my deep heartfelt gratitude to Professor Joseph A Caruso; I along with many people call him by “DOC”. It was for me more than pleasure to work with him and would be honored to graduate as his student. The joy and enthusiasm I had working with him is great. He is unconditionally excellent as advisor, great person by nature and his motivation is contagious. When I was going through extremely hardships in the middle of my Ph.D, he gave me not only hope but chance to work with him. The freedom he provides in the project along with his valuable guidance is always unforgetful. I can probably write another thesis, how I am indebted to doc for opportunities he has provided me, so I would stop now. I would specially thank Judy Caruso for kindness, hospitality and treating me as a part of family. I would specially thank Dr.Edward J.Merino for his invaluable suggestions, collaborator and being a co-advisor in my work. I am grateful to Professor Thomas H.Ridgway for serving in my committee more than 5 years and guiding me all of the time. v I would like to thank my dearest friend Phani Chand Kodali for all his help and support for this long time. I would like to extend my special greeting to Dr.Julio Alberto Landero for teaching me some mass spectrometry in my initial days and being a helpful guide. I would like to acknowledge the past and present members of the Caruso group for their support and friendship. Many thanks to, Dr. Karolin Kroening, Dr. Qilin Chan, Dr.Irena, Dr.Uma Tiwari, Dr.Necate Kaval, Dr.Eme Amba, Dr. Yaofang Zhang, Dr. Renee Easter, Dr. Brittany Catron, Dr. Cheolho Yoon, Dr. Daniel Persson, Dr. Pablo Pacheco, Traci Hanley, Ryan Saadawi, Anna Daigle, Christopher Medley, Nicole Hanks, Keaton Nahan, Kaitlyn Taylor, Morwina Solivia, Tiffany Bell, Josh Rohman. vi Table of Contents Abstract of dissertation Acknowledgements Chapter 1: Introduction………………………………………………………………………………….1 1.1 Arsenic, Arsenic toxicity, Selenium, Arsenic and Selenium antagonism....2 1.1.1 Arsenic………………………………………………………………………………………………2 1.1.2 Arsenic toxicity………………………………………………………………………………….3 1.1.3 Selenium……………………………………………………………………………………………4 1.1.4 Arsenic and selenium antagonism……………………………………………………..5 1.2 Phosphorylation……………………………………………………………………………………….5 1.3 Metallomics………………………………………………………………………………………………8 1.3.1 Introduction to Metallomics………………………..…………………………………….8 1.3.2 Metallomics approach……………………………………………………………………….8 1.3.3 Molecular Mass Spectrometry for metallomics and metal-binding proteins……………………………………………………………………………………………………10 1.4 Inductively Coupled Plasma Mass Spectrometry (ICPMS)………………………11 1.4.1 Introduction to ICPMS……………………………………………………………………..11 1.4.2 Advantages and limitations of ICPMS…………………………………..………….14 1.5 High Performance Liquid Chromatography (HPLC)……………..………………….15 1.5.1 HPLC-ICPMS…………………………………………………………………………………….17 1.6 References………………………………………………………………………………………………18 vii Chapter 2: Selenium mediated arsenic toxicity modifies cytotoxicity, reactive oxygen species and phosphorylated proteins……………………………………………..22 2.1 Abstract…………………………........……………………………………………………………….23 2.2 Introduction………………………………..…………………………………………………………25 2.3 Experimental………………………………………………………………………………………….29 2.3.1 Reagents…………………………………………………………………………………………29 2.3.2 Cell culture, cell cytotoxicity and cell lysate…………………………………….30 2.3.3 ROS detection…………………………………………………………………………………31 2.3.4 Size Exclusion Chromatography (SEC)……………………………………………..31 2.3.5 Inductively Coupled Plasma Mass Spectrometry (ICPMS)……..………..32 2.3.6 ESI-MS/MS, NanoLC-Chip and Tryptic digestion……………………….………32 2.4 Results…………………………………………………………………………………………….………34 2.4.1 Effect of SeMet on arsenic cytotoxicity……………………………………..…….34 2.4.2 Effect of SeMet on the As(III) generated ROS…………………………………..35 2.4.3 Using SEC-ICPMS as part of the identification scheme……………………..37 2.4.4 Identification of -casein using phosphochip®………..……………………...41 2.4.5 Identification of phosphorylated proteins using the phosphochip®…41 2.5 Conclusion………………………………..…………………………………………………………...50 2.6 Acknowledgements…………………………………………………………………………….….51 2.7 References..................................................................................................52 Chapter 3: Identification of selenium-containing proteins in HEK 293 kidney cells using multiple chromatographies, LC-ICPMS and nano-LC-ESIMS……………….61 3.1 Abstract………………………………………………………………………………………………….62 viii 3.2 Introduction…………………………………………………………………………………………..63 3.3 Experimental………………………………………………………………………………………….65 3.3.1 Reagents…………………………………………………………………………………………65 3.3.2 Cell culture and Cell lysate………………………………………………………………66 3.3.3 Size Exclusion Chromatography (SEC)……………………………………………..66 3.3.4 Inductively Coupled Plasma Spectrometry (ICPMS)…………………………67 3.3.5 Capillary Reversed Phase Liquid Chromatography (capRPLC)……..…..67 3.3.6 Tryptic digestion, ESI-MS and Nano-LC-Chip……………………………………68 3.4 Results and Discussion…………………………………………………………………………..69 3.4.1 capRPLC………………………………………………………………………………………….72 3.4.2 Identification of Se-containing proteins………………………………..………..74 3.5 Conclusion……………………………………………………………………………………………..78 3.6 Acknowledgements………………………………..……………………………………………..79 3.7 References……………………………………………………………………………………………..80 Chapter 4: Separation of Peptides by HPLC using a Surface Confined Ionic Liquid Stationary Phase………………………………………………………………………………………….84 4.1 Abstract………………………………………………………………………………………………….86 4.2 Introduction……………………………………………………………………………………………89 4.3 Experimental……………………………………………………………………………..…………..89 4.3.1 Materials…………………………………………………………………………………………89 4.3.2 Methods………………………………………………………………………………………….90 4.3.2.1 Stationary Phase Synthesis……………………..……………………………………90 ix 4.3.2.2 HPLC analysis…………………………………………………………….……………….90 4.3.2.3 Capillary Electrophoresis Analysis……………………………………………….91 4.4 Results and Discussion………………………………………………………………………….91 4.4.1 Post-void volume eluting peptides……………………………………..………….92 4.4.1.1 Effect of organic modifier on enkephalin retention………..…………..92 4.4.1.2 Effect of TFA……………………………………………………………………………….95 4.4.2 Pre-void volume eluting peptides…………………………………………………..97 4.5 Conclusions…………………………………………………………………………………………100 4.6 Acknowledgements…………………………………………………………………………….101 4.7 References……………………………………………..…………………………………………..102 Chapter 5: Preliminary work with Human Keratinocytes, Conclusions and Future work………………………………………………………………………………………………………….104 5.1 Preliminary work with Human Keratinocytes