Oxidative status and genomic damage in an obesity model Oxidativer Status und Genom-Schäden in einem Adipositas-Modell Doctoral thesis for a doctoral degree at the Graduate School of Life Sciences, Julius-Maximilians-Universität Würzburg, Section Biomedicine submitted by Ezgi Eylül Bankoglu from Samsun, Turkey Würzburg, 2016 Submitted on: Members of the Promotionskomitee: Chairperson: Prof. Dr. Jörg Schultz Primary Supervisor: Prof. Dr. Helga Stopper Supervisor (Second): Prof. Dr. Tcholpon Djuzenova Supervisor (Third): Prof. Dr. Ute Hentschel Humeida Supervisor (Fourth): Prof. Dr. Jörg Vienken Date of Public Defence: Date of Receipt of Certificates: Table of Contents LIST OF ABBREVIATIONS ............................................................................................... V 1 INTRODUCTION ........................................................................................................ 1 Obesity and its prevalence .................................................................................... 1 Oxidative stress and genomic damage in obesity ................................................. 3 Endogenous reactive oxygen species (ROS) sources ............................................. 4 1.3.1 Mitochondria ........................................................................................................ 5 1.3.2 NADPH oxidase (NOX) enzyme family .................................................................. 7 Obesity and cancer incidence .............................................................................. 10 Insulin signaling and type 2 diabetes mellitus (T2DM) ....................................... 12 DNA damage and cancer ..................................................................................... 15 Impact of weight change in cancer ..................................................................... 17 Bariatric surgery as therapeutic option for weight control and obesity related diseases ............................................................................................................... 18 1.8.1 Gastric banding .................................................................................................. 19 1.8.2 Vertical sleeve gastrectomy ............................................................................... 19 1.8.3 Biliopancreatic diversion with a duodenal switch .............................................. 19 1.8.4 Roux-en-Y gastric bypass surgery ...................................................................... 20 2 OBJECTIVES ............................................................................................................ 21 3 MATERIALS AND METHODS .................................................................................... 22 Materials .............................................................................................................. 22 3.1.1 Cell line and cell culture reagents ...................................................................... 22 3.1.2 Animals ............................................................................................................... 22 3.1.3 Chemicals and reagents ..................................................................................... 22 3.1.4 Antibodies .......................................................................................................... 23 Methods............................................................................................................... 23 I 3.2.1 Experimental design I ......................................................................................... 23 3.2.1.1 Rodent obesity model ............................................................................... 23 3.2.1.2 Surgery and perioperative care ................................................................. 25 3.2.1.2.1 RYGB surgery ..................................................................................... 25 3.2.1.2.2 Sham surgery ..................................................................................... 26 3.2.1.3 Metabolic measurement ........................................................................... 26 3.2.1.4 Detection of ROS on cryosections by DHE staining ................................... 27 3.2.1.5 Western Blot analysis ................................................................................ 27 3.2.1.6 Genotoxicity tests ...................................................................................... 28 3.2.1.6.1 Isolation of primary cells for alkaline comet assay............................ 28 3.2.1.6.2 Alkaline comet assay.......................................................................... 28 3.2.1.6.3 Detection of phosphorylated H2AX sites .......................................... 29 3.2.1.7 Urine analysis ............................................................................................ 31 3.2.1.7.1 TBARS assay ....................................................................................... 31 3.2.1.7.2 Quantification of urinary DNA and RNA oxidation biomarkers in urine by LC/MS/MS .................................................................................... 32 3.2.2 Experimental design II ........................................................................................ 33 3.2.2.1 Whole-body Pten haplodeficient mouse model ....................................... 34 3.2.2.2 In vitro hepatocyte model to investigate the insulin-mediated genotoxicity ............................................................................................... 35 3.2.2.2.1 Cell culture and treatment conditions............................................... 35 3.2.2.2.2 Vitality test ......................................................................................... 35 3.2.2.2.3 Intracellular ROS analysis by DHE staining ........................................ 36 3.2.2.2.4 Alkaline comet assay.......................................................................... 36 3.2.2.2.5 Cytokinesis-block micronucleus (CBMN) assay ................................. 36 3.2.3 Statistical analysis .............................................................................................. 38 4 RESULTS ................................................................................................................. 39 II Results of the Zuckerfa/fa rat obesity model ........................................................ 39 4.1.1 Body weight, daily food intake and metabolic parameters ............................... 39 4.1.2 Oxidative stress and genomic damage in kidneys of obese Zuckerfa/fa rats ...... 44 4.1.2.1 Oxidative stress ......................................................................................... 44 4.1.2.2 Heat shock protein expression .................................................................. 45 4.1.2.3 DNA damage induction ............................................................................. 47 4.1.2.4 Correlation between DNA-damage and basal plasma insulin level .......... 49 4.1.3 Oxidative stress and genomic damage in livers of obese Zuckerfa/fa rats .......... 51 4.1.3.1 Oxidative stress ......................................................................................... 51 4.1.3.2 Heat shock proteins expression ................................................................ 52 4.1.3.3 DNA damage induction ............................................................................. 54 4.1.3.4 Correlation between DNA-damage and basal plasma insulin level .......... 56 4.1.4 Oxidative stress and genomic damage in colons of obese Zuckerfa/fa rats ........ 58 4.1.4.1 Oxidative stress ......................................................................................... 58 4.1.4.2 Heat shock protein expression .................................................................. 59 4.1.4.3 DNA damage induction ............................................................................. 61 4.1.4.4 Correlation between DNA-damage and basal plasma insulin level .......... 63 4.1.5 Urine analysis of obese Zuckerfa/fa rats .............................................................. 64 4.1.5.1 Lipid peroxidation products ...................................................................... 64 4.1.5.2 RNA oxidation biomarker .......................................................................... 67 4.1.5.3 DNA oxidation biomarkers ........................................................................ 69 Results of insulin-mediated oxidative stress and genomic damage ................... 73 4.2.1 Modulation of insulin mediated oxidative stress and genomic damage in vitro ................................................................................................................. 73 4.2.1.1 Insulin induced oxidative stress in immortalized human hepatocytes ..... 73 4.2.1.2 Insulin induced genomic damage in immortalized human hepatocytes .. 75 III 4.2.2 Influence of deficiency of Pten, a protein in the insulin signaling, on oxidative stress and genomic damage in vivo ................................................................... 79 4.2.3 Oxidative stress and genomic damage in the liver of a whole-body Pten haplodeficient mouse model .............................................................................. 79 4.2.3.1 Oxidative stress ......................................................................................... 79 4.2.3.2 Heat shock protein expression .................................................................