lt+''1 "c{-( Trrn EnrNCr Or SHORT.TERM PNNTNNATMENT WITH PnnOxISOME PNOI,TTERATORS oN THE Acurn ToxrcrrY oF V¡.nrous ToxrcANTS, INcruorNG Pnn¡,cETAMoL Feticity April Nich olls-Grzem s ki B. Sc . (Hons- M elb) Thesis submitted for the degree of Doctor of Philisophy in Department of Clinical and Experimental Pharmacology, Faculty of Medicine, University of Adelaide June, 1998 Hou to get there Go to tlrc end of tlrc pathuntil gou get to the gate Go throughthe gate and head straight out towards the horizon Keep going towards the horizon Sit doutn ønd haue a rest euery now and again Ehfi keep on goíng Just keep onwithit Keep on going as far as Aou can Tltøt's how gou get there Michael Leunig TABLE OF'CONTENTS Table of Contents................ Abstract CHAPTER 1 INtRo¡ucrroN 1.1 Discovery of peroxisome proliferators 1 I.2 The Peroxisome................ 1 1.2.1 Discovery of the Peroxisome 1 1.2.2 Morphology of the Peroxisome.... 2 1.2.3 Biochemical and cellular properties of the peroxisome..................... J 1.3. What initiates a proliferation of hepatic peroxisomes?......... 5 1.4. Are all species responsive to PxPs? 5 1.4.1 Non-primate Species 5 1.4.2 Primate species 6 1.4.3 Humans 6 1.5 Mechanism of Peroxisome Proliferation.. 7 1.5.1 The Peroxisome Proliferator Activated Receptor... 7 1.5.1.1 Subtypes of the PP4R.............. 8 1.5.1.2 Do PxP directly interact with the PPAR? 8 1.5.1.3 What are the endogenous ligands for the PPAR? 9 I.5.1.4 Does variation in the PPAR underlie species differences? .... 9 I.5.2 A role for substrate overload in peroxisome proliferation? 9 1.6 The effects of exposure to PxPs 10 1.6. 1 Long term exposure: Hepatòcarcinogenesis 11 1.6.1.1 Induction of cell proliferation . 11 1.6.1.2 Oxidative Stress Theory...... 12 1.6.1.3 A combination of cell proliferation and oxidative stress ? .. 13 1.6.2 Short term exposure: Peroxisome proliferation .. T4 1.6.2.1 Morphological effects I4 1.6.2.2 Biochemical changes as a result of peroxisome proliferation. 15 1.7 The effects of short term PxP pretreatment on biotransformation T6 1.7.1 Alterations in Phase I pathways T6 1.7 .2 Alterations in Phase II conjugation pathways ..................... T7 1.7 .2.1 Glutathione S-transferases . 18 I.7 .2.2 UDP-glucuronyl transferase 18 1.7 .2.3 Sulphotransferases I9 1.7 .2.4 Epoxide Hydralase..... I9 1.7.3 Alteration in antioxidant eîzyme defences t9 1 1.7 .3.1 Glutathione peroxidase ........ ..20 1.7.4.2 Superoxide dismutase ..20 1.7.4 Alterations in cellular antioxidants .... ..21 1.7 .4.1 Glutathione... ..21 L7 .4.2 a-tocopherol and ascorbic acid.. .21 1.7.4.3 Metallothioneins and other antioxidants .22 1.7.5 Conclusion: Potential effects of PxP on xenobiotic metabolism... .22 1.8 Evidence that PxP alter susceptibility to hepatotoxicity? .24 I . 9 Paracetamol - model hepatotoxicant ............ .26 1.9. 1 Possible mechanisms of AAP hepatotoxicity .......... l.9.1. 1 Protein binding..... L9.L2 Disruption of intracellular calcium homoeostasis......... 1.9. 1.3 Mitochondrial dysfunction......... 1.9.1.4 Oxidative damage 1.10 Conclusion CHAPTER 2 ESTRSTTSHMENT OF A MURINE MoDEL oF PERoXISoME PRoLIpSRATToN 2.1 INTRODUCTION JJ 2.2 ]ù/4.ATERIALS AND METHODS............... 34 2.2.1 Chemicals.. 34 2.2.2 Animals ............ 34 2.2.3 Preparation of mouse feed containing Silvex 35 2.2.4 Animal Treatment ................ 35 2.2.5 Tissue Sampling 35 2.2.6 Enryme Assays:... 36 2.2.6.1 Palmitoyl CoA Oxidase ...... 36 2.2.6.2 Catalase 36 2.2.7 Histolo gical identifi cation of peroxisomes 37 2.2.8 Statistical Analysis............. 37 2.3 RESULTS ............. 37 2.4 DISCUSSION 40 CHAPTER 3 ETT.BCT OF PEROXISOME PROI-F.SRATION oN THE AcUTE ToxcITy oF THE MoDEL HgparoroxICANT, PRnacBravtol. 3.1 INTRODUCTION 42 3.2 MATERIALS AND METHODS..... 44 3.2.1 Chemicals.......... 44 3.2.2 Animal treatment. .44 3.2.3 Collection of blood for plasma enzyme analysis 44 3.2.4 Microscopic analysis of liver samples 45 3.2.5 Plasma Enzyme Assays............. ..45 3.2.5.1 Alanine Aminotransferase.. 45 3.2.5.2 Sorbitol Dehydrogenase (SDH) ..45 3.2.5.3 Lactate Dehydrogenase (LDH) . 45 3 .2.6 Statistical Analysis........... ..46 ii 3.3 RESULTS .46 3.3.1 Effect of pretreatment with three different PxP on the acute toxicity of a single dose of 44P............... 3.3.2 Effect of pretreatment with the peroxisome proliferator clofibrate on the dose response curve of AAP toxicity. 48 3.4 DISCUSSION 51 3 .4. 1 Selection of a plasma marker enzyme for quantitation of hepatotoxicity ... 51 3.4.2 Effect of pretreatment with clofibrate on the hepatotoxicity of AAP 51 CHAPTER 4 INVBSTICRTIONS INTO THE DOSB-RSSPONSE AND TEMPoRAL RpI-RTIoNSHIPS BET'wEEN CLo¡.ISRATE INDUcED PenoxIsoN4E PRoLIFERATIoN AND PRoTECTIoN FRoM PeReceTRMoL-INDUcED HEPAToToXICITY. 4.1 INTRODUCTION 53 4.2 MATEzuALS AND METHODS 54 4.2.1 Materials........... 54 4.2.2 Cloftbrate dosing protocols........ 54 4.2.3 Animal sampling 55 4.2.3.1 Subgroup A: Quantitation of peroxisome proliferation 55 4.2.3.2 Subgroup B: Investigation of AAP toxicity 55 4.2.4 Bíochemical Assays 55 4.2.4.1 Lauric Acid o-hydroxylation. .... 56 4.2.4.2 Glutathione level ........ 56 4.2.5 Statistics .......... 57 4.3 RESULTS 57 4.3.1 Time and dose dependency of peroxisome proliferation by clofibrate.............. 57 4.3 .1 .l Dose response relationship.................. 57 4.3.I.2 Time course of peroxisome proliferation........ 59 4.3.2 Dose and time dependency of clofibrate induced hepatoprotection against AAP 63 4.4 DISCUSSION 65 CHAPTER 5 TTTS F.aTB oF PARACETAMoL IN MICE PRBTRgRTED wITH CI.oTISRATB. 5.1 INTRODUCTION 70 5.2 MATERIALS AND METHODS....................... 72 5.2.1 Materials... 72 5.2.2 Animal treatment regimen 72 5 .2.3 Microsomal study : Metabolism of AAP ...... 73 5.2.3 .l Microsome preparation and incubation............... IJ 5.2.3.2 HPLC Analysis of the GSH:AAP conjugate 73 5.2.4 Analysis of urinary AAP metabolite formation.. 73 5.2.4.I Sample collection........ 73 5.2.4.2 HPLC analysis of urinary AAP metabolites.............. 74 5.2.5 Radiolabelled AAP experiment t4 I4C-AAP 5.2.5.I Preparation and administration of 74 5.2.5.3 Tissue sampling, preparation and radioactivity counting 74 5.2.6 Effect of AAP on mitochondrial oxidative phosphorylation. ... l6 iii 5.2.6.l Mitochondrial preparation... 76 5.2.6.2 Mitochondrial analysis 76 5.2.7 Effect of AAP intoxication on microsomal enzyme activity 77 5.2.8 Statistical Analysis... t8 5.3 RESULTS 78 5.3.1 Microsomal formation of GSH:AAP conjugate invitro. 18 5.3.2 Analysis of urinary metabolites of AAP following clofibrate treatment. 80 5.3.3 Investigation of the systemic fate and cellular binding of AAP using oC-AAÞ radiola'belled' 82 5.3.3.1 Urinary excretion of total AAP following clofibrate pretreatment in the mouse.82 5 .3.3.2 Effect of clofibrate treatment on the distribution of AAP to extrahepatic tissue. 83 5.3.3.3 Subcellular localisation of binding in the liver .................83 5.3.4 Investigation of mitochondrial effects of 44P....... .................85 5.3.5 Glutamine synthetase .... ......87 5.4 DISCUSSION ......88 5.4.1 Investigation of the bioactivation of AAP by hepatic microsomes .. ......88 5.4.2 Urinary metabolites of AAP .............. .....,89 5 .4.2.1 Glucuronide and sulphate metabolites ........... ......89 5 .4.2.2 Glutathione-derived metabolites ......90 5.4.2.3 Unchanged parent compound. ......91 5.4.3 The fate of radiolabelled '4C-AAP ......92 5.4.4 Effects of AAP intoxication on cellular targets .... ......93 5.4.4.1 Mitochondria.. ......93 5 .4.4.2 Microsomal glutamine synthetase. ..... ......94 5.4.5 Conclusion... ......94 CHAPTER 6 INVoLVEMENT oF GLUTRTUIoNE IN THE PROTECTIVE EFFECT OF CLOFIBRATE TOWARD THE Acure HgpATOTOXICITY OF PARACETAMOL. 6.1 INTRODUCTION ........;......... ...................96 6.2 MATEzuALS AND METHODS............... ..................99 6.2.1 Chemicals.. 99 6.2.2 Animals and treatment regimen ............100 6.2.3 Measurement of hepatic GSH following clof,rbrate treatment...... ............100 6.2.4 Depletion of hepatic GSH by AAP ............100 6.2.5 Assays for various GSH related enzymes ..100 6.2.5 .1 Glutathione S-transferase determination....... ..100 6.2.5 .2 Glutathione peroxidase determination .......... ..101 6.2.5.3 Glutathione reductase activity determination ..101 6.2.6 Depletion of hepatic GSH by various xenobiotics ..r02 6.2.6.1 Depletion of hepatic GSH by DEM ..t02 6.2.6.2 Depletion of hepatic GSH by 8SO......... r02 6.2.7 Determination of AAP toxicity following prior GSH depletion.. t02 6.2.7.l Depletion of GSH by DEM with AAP challenge r02 6.2.7.2 Depletion of GSH by BSO with AAP challenge .... t02 6.2.8 Indices of toxicity t02 1V 6.2.8.1 Hepatotoxicity: SDH. .t02 6.2.8.2 Renal toxicity: BUN ........ .103 6.2.9 Statistical Analysis ...103 6.3 RESULTS.... 103 6.3.2 Depletion of hepatic GSH by AAP t04 6.3.2.1 Time based analysis 104 6.3.2.2 Dose based analysis 105 6.3.3 Effect of clofibrate treatment on GSH related enzymes 106 6.3.4 Effect of GSH depletion by DEM on the toxicity of AAP 108 6.3.4.1 Depletion of hepatic GSH by DEM 108 6.3.4.2 Effect of DEM on GSH depletion and AAP hepatotoxicity in mice following treatment with clofibrate .