University of New Hampshire University of New Hampshire Scholars' Repository Doctoral Dissertations Student Scholarship Fall 2006 Cellular uptake and actions of bilberry anthocyanins in retinal pigment epithelial cells Paul Everett Milbury Jr. University of New Hampshire, Durham Follow this and additional works at: https://scholars.unh.edu/dissertation Recommended Citation Milbury, Paul Everett Jr., "Cellular uptake and actions of bilberry anthocyanins in retinal pigment epithelial cells" (2006). Doctoral Dissertations. 345. https://scholars.unh.edu/dissertation/345 This Dissertation is brought to you for free and open access by the Student Scholarship at University of New Hampshire Scholars' Repository. It has been accepted for inclusion in Doctoral Dissertations by an authorized administrator of University of New Hampshire Scholars' Repository. For more information, please contact [email protected]. CELLULAR UPTAKE AND ACTIONS OF BILBERRY ANTHOCYANINS IN RETINAL PIGMENT EPITHELIAL CELLS BY PAUL EVERETT MILBURY JR., M.S., CFII Baccalaureate Degree in Chemistry, University of New Hampshire, 1972 Master’s Degree in Animal Sciences, University of New Hampshire, 1978 DISSERTATION Submitted to the University of New Hampshire in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in the Animal and Nutritional Sciences September, 2006 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. UMI Number: 3231358 INFORMATION TO USERS The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleed-through, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. ® UMI UMI Microform 3231358 Copyright 2006 by ProQuest Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, Ml 48106-1346 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. This dissertation has been examined and approved. ( i..- 1) i.i i'. ( ____________________________________ dissertation Director, "loanne Curran-Celentano, Ph.D., Professor of Nutritional Sciences Dennis J. BobilyapPh.D., Associate Professor of Nutritional Sciences 1 Samuel C. Smith, Ph.D., Professor Emeritus Andrew P. Laudano, Ph.D., Associate Professor of Biochemistry and Molecular Biology Arthur F. Stucchi, Ph.D., Affiliate Associate Professor of Animal and Nutritional Sciences ! J Y~ v Date Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ACKNOWLEDGEMENTS I would like to thank the members of my dissertation committee Joanne Curran- Celentano, Dennis Bobilya, Samuel Smith, Andrew Laudano, and Arthur Stucchi for their guidance, patience, and willingness to serve on this unique endeavor to earn a doctoral degree at the University of New Hampshire. I have greatly benefitted from my interactions with each of them throughout this project. This endeavor was indeed unique in that I undertook this task of earning a doctorate late in my research career. I also wanted to maintain my current research position. This presented many opportunities but also presented significant challenges that required flexibility. I would especially like to express my sincere appreciation to Joanne Curran-Celentano for her willingness and courage to seek the means to make this dissertation and degree effort possible. Without her willingness to consider new approaches to fulfilling degree requirements this project would not have happened. I would also like to thank Jeffrey Blumberg, my supervisor at the Human Nutrition Research Center on Aging at Tufts University. This endeavor would also not have been possible without his blessing. His willingness to permit me to write much of this research into a USDA CRIS proposal and to allow me to pursue assistance and funding by mentoring in the Tufts Summer Scholar program resulted in funds to support the research presented in this dissertation. My wife, Carol, played an important role in getting me through this process. She helped edit this dissertation. More importantly, however, she tolerated my varied moods and the long hours and late evenings I spent on my academic class work, this research, and my day job. Her love and support sustained me throughout this project. iii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TABLE OF CONTENTS ACKNOWLEDGEMENTS..................................................................................................iii LIST OF TABLES...................................................................................................................v LIST OF FIGURES............................................................................................................... vi LIST OF ABBREVIATIONS............................................................................................ viii ABSTRACT............................................................................................................................. xi CHAPTER PAGE INTRODUCTION....................................................................................................................1 I. VACCINUM MYRTILLUS: THE BILBERRY....................................................... 5 The Botany of the Vaccinium Genus ......................................................................5 Bilberry as Food and Traditional Medicine ........................................................... 6 Bilberry: the Vision Legend .................................................................................. 9 II. AGE-RELATED MACULAR DEGENERATION...............................................11 Pathophysiology of AMD ......................................................................................11 The Role of RPE in AMD ......................................................................................12 RPE Death by Apoptosis .......................................................................................14 Anthocyanins and Apoptosis ................................................................................ 23 Oxidative Stress, Redox Biology, Aging and AMD ........................................... 26 Epidemiology and Intervention Studies in AMD ................................................34 III. BILBERRY ANTHOCYANINS...............................................................................47 Anthocyanins as Antioxidants .............................................................................. 47 Enzyme Activity and Cell Signaling Effects ....................................................... 53 IV. METHODS AND MATERIALS...............................................................................59 Materials and Chemicals........................................................................................59 Chromatography, Polyphenolic, and Glutathione Assays .................................. 59 RPE Cell Culture Model Characterization ........................................................... 62 Redox Status and Apoptosis Assay ......................................................................64 Protein and mRNA Analysis ................................................................................. 68 V. RESULTS..................................................................................................................... 73 Bilberry Extract.......................................................................................................73 RPE in Vitro Model and Anthocyanin Cellular Uptake ..................................... 76 RPE Oxidative Stress and Apoptosis ....................................................................90 Bilberry Extract Induction of HO-1 and GSTP1 .................................................97 VI. DISCUSSION AND CONCLUSIONS................................................................... 103 Effects on RPE Apoptosis and Phase II Up-regulation .................................... 105 Future Direction of Research .............................................................................. 133 LIST OF REFERENCES.................................................................................................. 139 iv Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF TABLES Table 1. Heme oxygenase and glutathione S-transferase Lux Primer 71 definitions and sequences Table 2. M/Z chart for LC/MS/MS selected ion analysis of anthocyanin 88 metabolites Table 3. RPE days in culture versus H 2 O2 LD50 determined by 90 MTT, JC-1, and ATP assays Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF FIGURES Figure 1. Basic flavonoid and anthocyanin structure 47 Figure 2. HPLC/ECD analysis of bilberry anthocyanin-rich extract 73 Figure 3. UV 520 nm spectograxn of anthocyanins from bilberry extract 74 Figure 4. Fractionation of bilberry extract 75 Figure 5. RPE cells grown on Transwell membranes for 10 days 76