Mitochondrial integrity and antioxidative enzyme efficiency in fischer rats effects of ageing and epigallocatechin 3 gallate intervention 1

MITOCHONDRIAL INTEGRITY AND ANTIOXIDATIVE ENZYME EFFICIENCY IN FISCHER RATS: EFFECTS OF AGEING AND EPIGALLOCATECHIN-3GALLATE INTERVENTION QINGYING MENG (BACHELOR OF SCIENCE) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF OTOLARYNGOLOGY YONG LOO LIN SCHOOL OF MEDICINE，NATIONAL UNIVERSITY OF SINGAPORE 2007 ACKNOWLEDGEMENTS This work was conducted through a joint program between the National University of Singapore (NUS) and Institute of Bioengineering and Nanotechnology (IBN), the Agency for Science, Technology and Research (A*STAR) from January 2004 to December 2007 In the words as follows, I would like to take the opportunity to thank everybody who helped and supported me in this journey To begin with, I express my sincere gratitude to my supervisor, Dr Runsheng Ruan, Assistant Professor, for giving me an opportunity to carry out the PhD program in his well-equipped laboratory at IBN, introducing me to the study of aging mechanisms, and providing me guidance during the graduation periods I sincerely thank Dr Chidambaram Natesa Velalar, Senior Research Scientist, IBN, for his guidance and patient training of me towards the becoming of a researcher I also thank him for his kind review of the manuscripts for journal papers and this PhD thesis I would like to thank Prof Jackie Y Ying, Executive Director, and Ms Noreena AbuBakar, Director, IBN, and Dr Thomas Loh, Assistant Professor, Head of the Department of Otolaryngology, Yong Loo Lin School of Medicine, NUS, for their concurrence to perform my graduation studies through their institution and department I sincerely thanks IBN, A*STAR for financially supporting the i project; Biological Resource Center, A*STAR for housing and taking care of the experimental animals Special thanks go to Doctor to be Jie Chen, for his collaboration in the same project of aging mechanism studies I truly appreciate his keenness of insight in the research I would like to thank my friends Kok Heng Foong, Brian Tan, Myranda Lee, Yunshi Wang, Dr Seong Loong Lo for being kind and affectionate friends I would also like to thank my other colleagues in IBN for the good working environment and my friends in both NUS and NTU for their fellowship I wish to extend my thanks to my teachers in the School of Life Sciences, Nanjing University for introducing me to the world of biological sciences Specially, I would like to thank Professor Qiang Xu and my seniors Jun Wang and Ying Zhao for their truthful help during my honor’s project and preparing me for the next step of my PhD training I also thank my precious friend far away, Bin Zhou, for her consistent encouragement and forever trustfulness At the end, I express my heartfelt thanks to my dear family I thank my parents for providing me the best environment in a family and sharing all my joys and sorrows in life I have no words to appreciate the love and support I got from them ii TABLE OF CONTENTS ACKNOWLEDGEMENTS …………………………………………………… i TABLE OF CONTENTS ……………………………………………….….…iii ABSTRACT ………………………………………………………………… ix PAPER PREPARED FROM THIS THESIS ……………………… ……xii CONFERENCE CONTRIBUTIONS FROM THIS THESIS ………… xiii LIST OF TABLES ……………………………………………………… …xiv LIST OF FIGURES ………………………………………………… ………xv ABBREVIATIONS …………………………………………………… …xviii Introduction ……………………………………………………………………1 1.1 Definition of aging ………………………………… ……………… ….1 1.2 Mechanisms of aging ………………………… …………………………2 1.2.1 The free radical theory of aging ……………………………….……2 1.2.2 The mitochondrial theory of aging ….………………………………3 1.2.3 The cross-linking theory of aging …………… ……………………3 1.2.4 The immunological theory of aging …………………………… …6 1.2.5 The telomere theory of aging ………………………………….……6 1.2.6 The wear and tear theory of aging …………………………… ……7 1.2.7 Longevity genes ………………………………………………….…7 1.3 The free radical theory of aging …………………………………… ……9 1.3.1 Free radicals …………………………………………………… …9 1.3.2 Resource of ROS ………………………………………… ………11 1.3.3 Oxidative damages …………………………………… …………11 iii 1.3.3.1 Lipid peroxidation ………………………………….……12 1.3.3.2 Change of protein structure ……………………… ……13 1.3.3.3 DNA damage ……………………………………….……13 1.3.4 Other functions of ROS ……………………………………………15 1.3.5 Antioxidant defense systems ………………………………………15 1.3.5.1 Antioxidative enzymes ……………………………… …16 1.3.5.2 Non-enzymatic molecules ………………………….……16 1.3.6 The repair system ……………………………………………….…16 1.3.6.1 Lipid repair ………………………………………………17 1.3.6.2 Protein repair ………………………………….…………17 1.3.6.3 DNA repair ………………………………………………18 1.3.7 Synthesis: Interaction of ROS generation, defense and repair systems ……………………………………….………………….21 1.4 The mitochondrial theory of aging ………………………………………22 1.4.1 The pivotal role of mitochondria in aerobic organisms ……… …22 1.4.2 Mitochondria electron transport chain …………………………….23 1.4.3 Mitochondria genome …………………… ………………………25 1.4.4 MtDNA damage …………………………………… ……………26 1.4.5 MtDNA repair …………………………………………………… 28 1.4.6 Other forms of mitochondria damage ………………… …………29 1.4.6.1 Lipid oxidative damage …………………………….……29 1.4.6.2 Protein oxidative damage ……………………… ………29 1.4.6.3 Enlarged volume …………………………………………30 1.4.6.4 Mitochondrial turnover ………………………… ………31 iv 1.5 Anti-aging strategies ……………………………………………… ……31 1.5.1 Calorie restriction …………………………………………….……32 1.5.2 Gene manipulation …………………………………… …………33 1.5.3 Dietary nutrition supplements ………………………………… …34 1.5.3.1 Antioxidant ………………………………………………34 1.5.3.2 Flavonoids and EGCG ………………………………… 35 1.5.3.3 Other forms of nutrition supplements ………… ………37 1.5.3.4 Side effects ………… ………………… …………… 38 Research objectives ……………………………………………………… …39 2.1 Hypothesis ……………………………………………………….………39 2.2 Objectives …………………………………………………… …………42 2.2.1 Study of mitochondrial integrity and antioxidant defense system efficiency in the young and old rats ………………………… …43 2.2.2 In vitro evaluation of the anti-aging effect of EGCG ………… …43 2.2.3 In vivo evaluation of the anti-aging effect of EGCG ……… ……44 2.3 Significance and applications ……………………………………………44 Materials and methods …………………………………………………….…45 3.1 Part one: Age-related changes in mitochondrial function and antioxidative enzyme activity in Fischer 344 rats ………………………………………… …45 3.1.1 Animals ………………………………………….……….…… …45 3.1.2 Mitochondria isolation from liver and brain ………………………46 3.1.3 Mitochondrial membrane potential measurement ….…….…… …47 3.1.4 Mitochondria respiratory activity test …………………………… 47 3.1.5 Antioxidative enzyme activity assays …………………………… 48 v 3.1.6 Western bolt …………………………………………………… 48 3.1.7 Statistics ………………………………………………………… 49 3.2 Part two: Effects of epigallocatechin-3-gallate on mitochondrial integrity and antioxidative enzyme activity in aging process of human fibroblast … .49 3.2.1 Cell culture and EGCG preparation … 49 3.2.2 Cell viability … .50 3.2.3 Radical scavenging activity 51 3.2.4 Determination of H2O2 .51 3.2.5 Intracellular ROS 51 3.2.6 Mitochondrial potential 52 3.2.7 Quantitative real-time PCR 53 3.2.8 Antioxidative enzyme activity … 55 3.2.9 Senescence status … .55 3.2.10 Statistics … 56 3.3 Part three: Regulation of age-related oxidative damage, mitochondrial integrity and antioxidative enzyme activity in Fischer 344 rats by the supplementation of the antioxidant epigallocatechin-3-gallate .56 3.3.1 Animal 56 3.3.2 Histopathology observation 57 3.3.3 Preparation of leukocytes and plasma … 58 3.3.4 Evaluation of liver and kidney functions 58 3.3.5 Determination of H2O2 in the plasma .60 3.3.6 Mitochondrial potential 60 vi 3.3.7 Lipid peroxidation in the plasma 60 3.3.8 8-OHdG in the plasma 61 3.3.9 Mitochondrial DNA deletion 61 3.3.10 Antioxidative enzyme activities .62 3.3.11 Antioxidative enzyme gene expressions 62 3.3.12 Microarray and data analysis 63 3.3.13 Statistics 64 Main results ……………………………………………………………… …64 4.1 Part one: Age-related changes in mitochondrial function and antioxidative enzyme activity in Fischer 344 rats (Paper I) …… …64 4.1.1 Introduction ……………………………………… ………64 4.1.2 Results …………………………………………………… 67 4.1.3 Discussions ……………… ………………………………74 4.1.4 Summaries …………………………………………………78 4.2 Part two: Effects of EGCG on mitochondrial integrity and antioxidative enzyme activity in aging process of human fibroblast (Paper II) ……………………………………………………………79 4.2.1 Introduction ……………………… ………………………79 4.2.2 Results …………………………………… ………………81 4.2.3 Discussions ……………………………………… ………93 4.2.4 Summaries …………………………………………………96 4.3 Part three: Regulation of age-related oxidative damage, mitochondrial integrity and antioxidative enzyme activity in Fischer 344 rats by the supplementation of the antioxidant EGCG (Paper III) ………………… ………97 vii 4.3.1 Introduction ………………………… ……………………97 4.3.2 Results …………………………………………… ………99 4.3.3 Discussions …………………………….…………………119 4.3.4 Summaries ……………………………… ………………123 Conclusions and future perspectives ………………………………………124 5.1 Introduction ……………………………………………….……………124 5.2 Summary of the important findings ………………………….…………127 5.3 Future perspectives ………………………….…………………….……130 References ……………………………………………………… …………133 viii ABSTRACT Generally, the “senescence” or the “replicative senescence” explains the cellular aging, while the “aging” describes the getting older of the whole organism Aging is usually associated with the changes typically in the system's deterioration over time, but aging itself is not a result of diseases According to the free radical theory of aging, aging can be resulted from oxidative damages by reactive oxygen species (ROS), which include free radicals and their derivatives of the non-free radical mimics Oxidative damages could be partially overcome through caloric restriction (CR), transgenic manipulation, pharmacological and dietary antioxidant interventions Although CR is the only proven anti-aging regimen, the antioxidant system is also supposed to control the aging process by scavenging ROS and improving mitochondrial integrity Particularly, in the aging process, when the enzymatic antioxidant system gradually loses its activity to protect against oxidative damages, the supplementation of non-enzymatic antioxidants could surrogate to deal with the oxidative stress and probably reinforce the enzymatic antioxidant system, thus ameliorating aging and aging-related disorders In order to prove the hypothesis, we firstly investigated the most likely results of aging such as the dysfunction of mitochondria by determining the mitochondrial respiration control ratio and membrane potential in the liver and brain of young and old male Fischer 344 rats Such mitochondrial malfunctions in the old rats was associated with the decrease in activities of the antioxidative enzymes including catalase (CAT), glutathione peroxidase (GPx), Cu/Zn-superoxide ix dismutase (Cu/Zn-SOD, SOD1) and Mn-SOD (SOD2), as well as their protein expressions Thus, the unfavorable release and accumulation of ROS in the cells and tissues as well as the concurrent malfunctions of mitochondria and antioxidant enzymes most likely resulted in the aging process of the old rats Secondly, we systematically studied the effects of antioxidant, epigallocatechin-3gallate (EGCG), intervention on aging and aging-related factors such as ROS accumulation, mitochondrial integrity and antioxidative enzyme activities in human diploid fibroblast (HDF), a well-known in vitro model for aging studies EGCG was firstly evaluated for its cytotoxicity, the pro- and anti- oxidative properties Then it was further determined that HDF treated with EGCG considerably increases CAT, SOD1, SOD2 and GPx gene expressions as well as their enzyme activities, thus protecting HDF against exogenous H2O2-induced oxidative damage, accompanied with decreased intracellular ROS accumulation, well-maintained mitochondrial potential and mitochondrial DNA integrity both in the short-term and long-term interventions, as well as a more juvenile cell status observed in the long-term treatment Finally, the results from the in vitro model were further extended to the in vivo model to evaluate the effects of EGCG on the middle-aged male Fischer 344 rats In addition to the improvement of kidney functions by elevating creatinine clearance, high dose EGCG also significantly decreased the levels of H2O2, MDA and 8-OHdG in the plasma, maintained the mitochondrial membrane potential of the peripheral lymphocytes, and lessened the deletion of ND4 region from x mtDNA in the liver Such protection effects of high dose EGCG against oxidative stress were comparable with the effects of CR intervention The global gene expression profiling also provided the most possible mechanisms underlying the amelioration of aging process by EGCG in the rats In conclusion, based on the observations from these studies, EGCG can be considered to be used as an antiaging reagent in the future xi PAPER PREPARED FROM THIS THESIS This thesis is based on the following scientific papers, which are referred to in the text by their roman numerals I Meng Q, Wong YT, Chen J and Ruan R (2007); Age-related changes in mitochondrial function and antioxidative enzyme activity in Fischer 344 rats; Mechanisms of Ageing and Development, 128(3):286-92 II Meng Q, Velalar CN and Ruan R, (2008); Effects of epigallocatechin-3-gallate on mitochondrial integrity and antioxidative enzyme efficiency in aging process of human fibroblast; Free Radical Biology and Medicine, 44(6):1032-41 III Meng Q, Velalar CN and Ruan R, (2008); Mitochondrial integrity, antioxidative enzyme efficiency and age related oxidative damage in Fischer 344 rats with 6-month supplementation of antioxidant epigallocatechin-3-gallate; Rejuvenate Research, 11(3):649-660 xii CONFERENCE CONTRIBUTIONS FROM THIS THESIS I Meng Q and Ruan R; Abstract accepted for The Global Enterprise for MicroMechanics and Molecular Medicine (GEM4) Conference on Cancer in conjunction with International Conference on Materials for Advanced Technologies (ICMAT) 2007, July 1st-6th, 2007, Suntec International Convention and Exhibition Centre, Singapore II Meng Q and Ruan R; Effects of epigallocatechin-3-gallate in mitochondrial integrity and antioxidative enzyme efficiency in the aging process, poster presentation at American Aging Association - 36th Annual Meeting, 1st-4th June 2007, Marriott Plaza San Antonio, San Antonio, Texas, USA III Meng Q and Ruan R; Mitochondrial integrity and antioxidative enzyme efficiency in aging process and green tea catechin intervention; oral presentation in the 2nd Institute of Bioengineering and Nanotechnology Graduate Student Symposium, on 18th May 2007, Aspiration Theatrette, Matrix, Biopolis, Singapore IV Meng Q and Ruan R.; Age related mitochondria function and antioxidant enzyme activities in Fisher-344 rats; poster presentation at Combined Scientific Meeting (CSM 2005), 4th-6th November, 2005, Raffles City Convention Centre, Singapore xiii LIST OF TABLES Table The identified longevity genes in the S cerevisiae, C elegans, D melanogaster and M musculus ………………………………………………… Table The major ROS formation in biological systems …………………… 10 Table Major known repair proteins or pathways for principle oxidative DNA base lesions …………………………………………………………………… 20 Table The sequences of primers used in the quantitative real-time PCR analysis ……………………………………………………………….………….54 Table The weights of animals, the major organs, and the food consumptions ………………………………………………………… ……….101 Table The histopathology observations in the liver and kidney …….………102 Table Liver and kidney functions …………………………………… ……105 Table Global views of transcriptional changes induced by EGCG supplementation and caloric restriction ……………………………………… 116 Table Genes expression with more than 1.5-fold change vs control in response to low dose and high dose EGCG interventions …………………………… …117 xiv LIST OF FIGURES Figure Cross-linking between glucose and a lysine amino acid in a protein molecule ………………………………………………………………………… Figure Chemical structures of some stable oxidative DNA base lesions …….14 Figure Interaction of ROS generation, defense, and repair system ………… 21 Figure Mammalian electron transport chain complexes (I–V) ……………….24 Figure The schematic representation of RCR measurement …………………24 Figure Human mitochondrial genome ……………………………………… 25 Figure Mitochondrial and nuclear DNA repair pathways ……………… ….28 Figure The structure of (-)-epigallocatechin-3-gallate (EGCG) …………… 36 Figure The schematic representation of the hypothesis of this study …… …40 Figure 10 Fluorescence image demonstrating mitochondrial membrane potential in the liver of young (A) and old (B) rats ……………………………………… 68 Figure 11 Mitochondrial membrane potential in the liver and brain of young and old rats … .70 Figure 12 Mitochondrial respiratory control ratio in the liver and brain of young and old rats …………………………………………………………………… 70 Figure 13 (A) Total SOD, SOD1 and SOD2 activities, (B) GPx and CAT activity, in the liver, kidney, brain and heart of young and old rats ………………………72 xv Figure 14 Western blot analysis of antioxidative enzyme protein expressions Western blot image representing the antioxidative enzyme protein expressions in the (A) liver and (B) kidney of young and old rats; (C) and (D) quantification of the protein expressions in the liver, kidney, brain and heart of young and old rats ………………………………………………………………………… … 73 Figure 15 Cytotoxicity of EGCG on HDF (A) Cell viability of young HDF; (B) LC50 for both young and old HDF …………………………………………… 82 Figure 16 Anti- and pro-oxidant effects of EGCG in MEM in the presence and absence of HDF (A) the ability of various concentrations of EGCG to scavenge the stable radical DPPH in MEM at different time points; (B) EGCG-induced H2O2 in the absence of HDF; (C) EGCG-induced H2O2 in the presence of HDF ……………………………………………………………………… ……84 Figure 17 Protective effects of EGCG against H2O2 induced oxidative stress on young HDF (A) Cell viability; (B) the representative pictures showing the cell viability; (C) cell proliferation for continuous days after exposure to 100 μM and (D) 200 μM H2O2; (E) intracellular ROS; (F) mitochondrial potential ………….86 Figure 18 Effects of EGCG in (A) antioxidative enzyme gene expressions and (B) enzyme activities in the short term treatment ……………………………………89 Figure 19 Effects of EGCG on the middle-aged HDF in the long term treatment, including (A) intracellular ROS; (B) mitochondrial potential; (C) mitochondrial DNA integrity; (D) antioxidative enzyme gene expressions and (E) enzyme activities ………………………………………………………… …………… 91 Figure 20 Effects of EGCG on cellular senescence of middle-aged HDF in the long term treatment ……………………………………………….…………… 92 Figure 21 Concentration of H2O2 in the plasma of middle-aged male Fischer 344 rats ……………………………………… ……………………………………106 Figure 22 Mitochondrial membrane potential of lymphocytes of middle-aged male Fischer 344 rats ………………………………………………………… 106 xvi Figure 23 Lipid peroxidation (A) and 8-OHdG (B) in the plasma of middle-aged male Fischer 344 rats ………………………………………………… ………108 Figure 24 Mitochondrial DNA deletion in the liver of middle-aged male Fischer 344 rats …………………………………………………………….……… .109 Figure 25 Antioxidative enzyme activities in the liver (A), kidney (B), heart (C) and brain (D) of middle-aged male Fischer 344 rats …………………… ….111 Figure 26 Antioxidative enzyme gene expressions in the liver (A), kidney (B), heart (C) and brain (D) of middle-aged male Fischer 344 rats …………… ….113 Figure 27 Venn diagram analysis of gene profiling in the middle-aged male Fischer 344 rats ………………………………….………………………… …115 xvii ABBREVIATIONS 4-HNE 4-hydroxynonenal 5-OHdC hydroxy-2'-deoxycytosine, 8-oxo-dA 5-OHdU hydroxy-2'-deoxyuracil, 8-oxo-dA 8-iso-PGF2α 8-iso-prostaglandin F2α 8-OHdA hydroxy-2'-deoxyadenine, 8-oxo-dA 8-OHdG 8-hydroxy-2'-deoxyguanosine, 8-oxo-dG ALT alanine aminotransferase ANT adenine nucleotide transporter AP alkaline phosphatase AP site apurinic and apyrimidinic site AP-1 activator protein-1 ARE antioxidant responsive element AST asparate aminotransferase C elegans Caenorhabditis elegans CAT catalase Complex I NADH dehydrogenase Complex II succinate dehydrogenase Complex III cytochrome bc1 complex Complex IV cytochrome c oxidase Complex V Fo-F1 ATP synthase COX-2 cycloxygenase-2 xviii CR calorie restriction D melanogaster Drosophila melanogaster DPPH 2,2-diphenyl-2-picrylhydrazyl hydrate EGCG epigallocatechin-3-gallate ETC electron transport chain FADH2/FAD flavine adenine dinucleotide Fapy-dA and -dG formamidopyrimidines FDG fluorescein di-β-D-galactopyranoside GH growth hormone GPx glutathione peroxidase GSH/GSSH glutathione GST glutathione S-transferase H2DCF-DA 2’,7’-dichlorofluorescin diacetate HDF human diploid fibroblast HRP horseradish peroxidase IGF-1 insulin/insulin-like growth factor iNOS inducible-nitric oxide synthetase MDA malondialdehyde MEM minimum essential medium MFI median fluorescence intensity M musculus Mus musculus mtDNA mitochondrial DNA NADH/NAD+ nicotinamide adenine dinucleotide xix nDNA nuclear DNA NF-κB nuclear factor-kappa B NOAEL no-observed adverse effect level Nrf2 NF-E2-related factor-2 OGG1 8-oxoguanine DNA glycosylase PDL population doubling level PLA2 phospholipase A2 PTP permeability transition pore RCR respiratory control ratio RFU relative fluorescence unit Rho123 rhodamine123 ROS reactive oxygen species S cerevisiae Saccharomyces cerevisiae SA-βG senescence-associated β-galactosidase SOD1 superoxide dismutase 1, cytosolic-SOD, Cu/Zn SOD SOD2 superoxide dismutase , mitochondrial-SOD, Mn SOD TBARS thiobarbituric acid reactive substances TCA cycle tricarboxylic acid cycle, Krebs cycle, citric acid cycle TF transcription factors TNF-α Tumor Necrosis Factor alpha UCP uncoupling protein X-Gal 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside XO xanthine oxidase xx ... 61 3. 3 .10 Antioxidative enzyme activities .62 3. 3 .11 Antioxidative enzyme gene expressions 62 3. 3 .12 Microarray and data analysis 63 3 .3. 13 Statistics 64 Main results... activity in Fischer 34 4 rats; Mechanisms of Ageing and Development, 12 8 (3) :286-92 II Meng Q, Velalar CN and Ruan R, (2008); Effects of epigallocatechin- 3- gallate on mitochondrial integrity and antioxidative. .. peroxidation ………………………………….…? ?12 1 .3. 3.2 Change of protein structure ……………………… …… 13 1 .3. 3 .3 DNA damage ……………………………………….…… 13 1 .3. 4 Other functions of ROS …………………………………………? ?15 1 .3. 5 Antioxidant defense