Scholars' Mine Doctoral Dissertations Student Theses and Dissertations Fall 2015 Effects of n-acetylcysteine amide in preventing/treating cataracts Sri Krishna Yasaswi Maddirala Follow this and additional works at: https://scholarsmine.mst.edu/doctoral_dissertations Part of the Biochemistry Commons, and the Chemistry Commons Department: Chemistry Recommended Citation Maddirala, Sri Krishna Yasaswi, "Effects of n-acetylcysteine amide in preventing/treating cataracts" (2015). Doctoral Dissertations. 2452. https://scholarsmine.mst.edu/doctoral_dissertations/2452 This thesis is brought to you by Scholars' Mine, a service of the Missouri S&T Library and Learning Resources. This work is protected by U. S. Copyright Law. Unauthorized use including reproduction for redistribution requires the permission of the copyright holder. For more information, please contact [email protected]. EFFECTS OF N-ACETYLCYSTEINE AMIDE IN PREVENTING/TREATING CATARACTS by SRI KRISHNA YASASWI MADDIRALA A DISSERTATION Presented to the Faculty of the Graduate School of the MISSOURI UNIVERSITY OF SCIENCE AND TECHNOLOGY In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY in CHEMISTRY 2015 Approved by Nuran Ercal, Advisor Shubhender Kapila Prakash Reddy Klaus Woelk Melanie Mormile iii ABSTRACT Cataract, the opacification of an eye lens, is a common pathological abnormality of the lens accounting for approximately 50% of all blindness. The only effective treatment currently available for a cataract is the surgical removal of the affected lens and replacement with an artificial lens for the restoration of vision. Although, cataract surgery is considered to be a very successful procedure in terms of visual outcome, the cost of surgery, need for trained personnel and surgeons, and postsurgical complications, limit the worldwide availability and accessibility of this procedure. Hence, alternative preventive and treatment procedures are worthy of investigation. The lens depends on a balanced redox state for maintaining its transparency, and a high content of glutathione (GSH) in the lens is believed to play a key role in doing so. Several studies have reported that oxidative stress plays an important role in the etiology of cataract development and, therefore, the present study has sought to evaluate the efficacy of a thiol antioxidant, (R) - N-acetylcysteine amide (NACA), in preventing/reversing cataracts. To investigate NACA’s ability to provide therapeutic benefits for cataracts, three different experimental models were utilized. The first was an ex-vivo cataract model, where culturing the rat lenses in dexamethasone resulted in posterior cataracts. The second was an in vivo mouse model, where injection of acetaminophen caused cataracts. The third model was a rat in vivo model where injection of sodium selenite generated nuclear cataracts. Treatment with NACA in each model helped to decrease the severity of cataracts. In summary, the results from this study suggest that NACA can potentially be developed into a promising therapeutic option for prevention and reversal of cataract formation. iv ACKNOWLEDGMENTS I would like to take this opportunity to thank those who have made my dissertation possible. I would not have come to U.S. without the acceptance of my Master. I would not have been able to complete my dissertation without the guidance of my committee members, help from friends, and support from my family. I would like to express my deepest gratitude to my advisor, Dr. Nuran Ercal, for her excellent guidance, caring, patience, and providing me with an excellent atmosphere for doing research. I would like to thank Dr. Eylem Y. Pinarci and Dr. Humeyra Karacal, two outstanding ophthalmologists who provided me with insight and knowledge about the anatomy of the eye, cataract formation and grading as well as training in the tedious procedure of properly dissecting areas of interest from the eye. I would also like to thank Dr. Shubhender Kapila for allowing me to access his wealth of knowledge and helping me develop a strong background in biochemistry, and analytical methodology. I would like to thank Dr. Shakila Tobwala without whom this dissertation wouldn’t have been possible. She has been a big help in improving my lab techniques and writing skills. I also would like to thank Rakesh Kacham, Ahdab Khayyat, Hsiu-jen Wang, Weili Fan, Joshua Carey, Xinsheng Zhang and Adam Martin along with the undergraduate researchers who assisted in taking care of the animals used for this study. Special thanks goes to Dr. David E. Falkenhain and Dr. V.E. Falkenhain for providing us with the Slit-lamp Microscope. I would also like to thank my parents, younger sister, relatives and my friends. They were always a source of encouragement, wishing me the best with my goals. I also would like to thank Barbara Harris for editing this dissertation. v TABLE OF CONTENTS Page ABSTRACT…………………………………………………………………………….. iii ACKNOWLEDGMENTS ................................................................................................. iv LIST OF ILLUSTRATIONS ............................................................................................. ix LIST OF TABLES ............................................................................................................. xi LIST OF ABBREVIATIONS ........................................................................................... xii SECTION 1. INTRODUCTION ....................................................................................................... 1 2. REVIEW OF LITERATURE ...................................................................................... 5 2.1. CATARACT ........................................................................................................ 5 2.2. OXIDATIVE STRESS ....................................................................................... 10 2.2.1. Reactive Oxygen Species (ROS). ............................................................ 10 2.2.2. Lipid Peroxidation (LPO). ....................................................................... 11 2.2.3. Protein Oxidation. .................................................................................... 13 2.3. ANTIOXIDANT DEFENSE .............................................................................. 13 2.3.1. Antioxidant Enzymes. .............................................................................. 15 2.3.2. Small Antioxidants................................................................................... 17 2.4. CALPAIN ACTIVATION ................................................................................. 20 2.5. N-ACETYLCYSTEINE AMIDE (NACA). ....................................................... 24 3. METHODS ............................................................................................................... 27 3.1. HPLC ANALYSIS ............................................................................................. 27 3.1.1. Determination of GSH. ............................................................................ 27 vi 3.1.2. Measurement of Total GSH and GSSG. .................................................. 27 3.1.3. Determination of MDA. ........................................................................... 29 3.2. SPECTROPHOTOMETRIC STUDIES ............................................................. 30 3.2.1. GR Activity. ............................................................................................. 30 3.2.2. Thioltransferase (Ttase) Activity. ............................................................ 30 3.2.3. Determination of Calcium Concentration. ............................................... 31 3.2.4. Total Protein Determination. ................................................................... 31 3.3. GEL ELECTROPHORETIC STUDIES ............................................................ 32 3.3.1. Casein Zymography. ................................................................................ 32 3.3.2. Preparation of Water-Soluble Lens Proteins............................................ 33 3.3.3. SDS-PAGE of Lens Soluble Proteins. ..................................................... 33 3.3.4. Western Blot of m-Calpain. ..................................................................... 33 3.4. CATARACT EVALUATION ........................................................................... 34 3.5. WATER CONTENT .......................................................................................... 35 3.6. STATISTICAL ANALYSIS .............................................................................. 35 4. N-ACETYLCYSTEINE AMIDE PROTECTS AGAINST DEXAMETHASONE- INDUCED CATARACT RELATED CHANGES IN CULTURED RAT LENSES ........................................................................................................... 36 4.1. EXPERIMENTAL DESIGN .............................................................................. 37 4.2. RESULTS ........................................................................................................... 38 4.2.1. Prevention of Dex-Induced Cataracts by NACA. .................................... 38 4.2.2. Effects of NACA on Intracellular GSH Levels in Dex-Treated Cultured Rat Lens….. ................................................................................................ 38 4.2.3. Effect of NACA on Oxidized Glutathione (GSSG) Levels and GSH/GSSG Ratio in Dex-Treated Cultured Rat Lens. .............................. 39 vii 4.2.4.