The role of TRPM2 channels in oxidative stress-induced liver damage Ehsan Kheradpezhouh Discipline of Physiology, School of Medical Sciences University of Adelaide Submitted for the degree of Doctor of Philosophy December 2014 Contents List of Abbreviations ....................................................................................................... i Abstract ........................................................................................................................... v Declaration of Originality ............................................................................................vii Acknowledgements ......................................................................................................... x Chapter 1: Research Background ................................................................................. 1 1.1 Introduction ............................................................................................................ 1 1.2 Oxidative Stress ...................................................................................................... 4 1.2.1 Systems Eliminating Oxidative Compounds ................................................... 5 1.2.2 Cellular and Molecular Targets of Oxidative Stress ....................................... 6 1.2.3 Mechanisms of Oxidative Stress Mediated Cellular Damage ......................... 8 2+ 1.3 Oxidative Damage and Ca Signalling................................................................ 11 1.4 TRP Channels in Oxidative Stress ....................................................................... 12 1.5 TRPM2 Channels ................................................................................................. 15 1.5.1 History ........................................................................................................... 15 1.5.2 TRPM Subfamily .......................................................................................... 16 1.5.3 TRPM2 Channel Isoforms and Variants ....................................................... 16 1.5.3.1 TRMP2-S ................................................................................................ 18 1.5.3.2 SSF-TRPM2 ............................................................................................ 18 1.5.3.3 TRPM2-ΔN ............................................................................................. 19 1.5.3.4 TRPM2-ΔC ............................................................................................. 19 1.5.3.5 TRPM2-ΔNΔC ........................................................................................ 19 1.5.4 TRPM2 Structure .......................................................................................... 19 1.5.4.1 TRPM2 Channel Topology ..................................................................... 19 1.5.4.2 Nudix Box and NUDT9-H ...................................................................... 22 1.5.5 Cellular localizations of TRPM2 channel ..................................................... 23 1.5.6 TRPM2 Channel Activation .......................................................................... 24 1.5.6.1 TRPM2 Gating........................................................................................ 24 1.5.6.2 TRPM2 Channel Activators ................................................................... 24 1.5.6.3 Direct TRPM2 Activators ....................................................................... 25 1.5.6.4 Indirect TRPM2 Channel Activators ...................................................... 28 1.5.7 TRPM2 Channel Blockers ............................................................................. 30 1.5.7.1 ACA......................................................................................................... 31 1.5.7.2 Fenamates............................................................................................... 31 1.5.7.3 Clotrimazole and other Azoles ............................................................... 32 1.5.7.4 2-APB ..................................................................................................... 33 1.5.8 The Role of TRPM2 Channels in oxidative stress-related pathologies ........ 33 1.6 Oxidative Stress and Liver Diseases .................................................................... 37 1.7 Acetaminophen ..................................................................................................... 40 1.7.1 History of discovery ...................................................................................... 40 1.7.2 Acetaminophen Pharmacokinetics ................................................................ 41 1.7.3 Acetaminophen Pharmacodynamics ............................................................. 42 1.7.4 Acetaminophen Overdose and Liver Damage ............................................... 44 1.7.5 Oxidative Damage in Acetaminophen Toxicity ............................................ 46 1.7.6 Current Clinical Treatment of Acetaminophen Toxicity, Advantages and Disadvantages ............................................................................................... 50 1.7.7 Antioxidants in Treatment of Acetaminophen Toxicity ................................ 51 1.7.8 Role of Ca2+ in Acetaminophen-induced Hepatocellular Damage ................ 52 1.7.9 Evidence for Possible Involvement of TRPM2 Channels in Acetaminophen Toxicity ............................................................................... 53 1.8 Conclusions and Project Aims .............................................................................. 54 Chapter 2: The Role of the TRPM2 Channel in Acetaminophen-mediated 2+ [Ca ]c rise in rat hepatocytes ...................................................................................... 56 2.1 Introduction .......................................................................................................... 56 2.2 Methods and Materials ......................................................................................... 58 2.2.1 Chemicals ...................................................................................................... 58 2.2.2 Animals ......................................................................................................... 58 2.2.3 Solutions ........................................................................................................ 58 2.2.4 Hepatocyte Isolation and Culture .................................................................. 59 2.2.5 Calcium Imaging ........................................................................................... 59 2.2.6 Immunofluorescence Imaging ....................................................................... 60 2.2.7 Western Blotting ............................................................................................ 62 2.2.8 Reverse Transcription Polymerase Chain Reaction (RT-PCR) and Quantitative RT-PCR .................................................................................... 63 2.2.9 Patch-clamp Recording ................................................................................. 66 2.2.10 TRPM2 Knocked Down (KD) Hepatocytes ................................................ 66 2.2.11 HEK 293T Cells Culture and Transfection ................................................. 66 2.2.12 Statistical Analysis ...................................................................................... 67 2.3 Results .................................................................................................................. 67 2.3.1 Expression of functional TRPM2 channel in rat hepatocytes ....................... 67 2.3.2 Activation of Ca2+ entry and non-selective cation current in rat hepatocytes in response to treatment by H2O2 and acetaminophen .............. 73 2.3.3 siRNA-mediated knock down of TRPM2 protein attenuates H2O2- and acetaminophen-induced Ca2+ entry and the cation current ........................... 76 2+ 2.3.4 H2O2 and acetaminophen-induced Ca entry requires ADPR ...................... 79 2.4 Discussion ............................................................................................................. 80 Chapter 3: The role of TRPM2 Channel in acetaminophen-induced Hepatocellular Damage ................................................................................................ 88 3.1 Introduction .......................................................................................................... 88 3.2 Methods and Materials ......................................................................................... 89 3.2.1 Chemicals ...................................................................................................... 89 3.2.2 Animals ......................................................................................................... 90 3.2.3 Solutions ........................................................................................................ 90 3.2.4 Estimation of the number of dead cells using Trypan blue. .......................... 90 3.2.5 Induction of In Vivo Acetaminophen Toxicity in Mice................................. 91 3.2.6 Blood Liver Enzymes Assay ......................................................................... 91 3.2.7 Histopathology .............................................................................................. 92 3.2.8 Hepatocyte Isolation and Culture .................................................................