\S -L\- o OxyGEN TOXICITY AND RAUATION lfrl.JuRy TO THE PULMONARY SySTEM A THESIS SUBMIT:I-ED FOR THE DEcnEE OF DOCTOR OF PHILOSOPHY BY GEoFFREy MCLENNAN, MBBS, FRACP,SBSTJ MnncH 1997 FRONT PAGES A-C TITLE A INDEX B ABBREVIATTONS c FoREwono 1-2 ACKNOWLEDGEMENTS 3-4 ACADEMIC STETT SUPPORTED 5 DEDICATION 6 DECLARAT1ON 7 ABSTRACT a- ro CHAPTER 1 - INTRODUCTION 11-53 CHAPTER 2 _ PULMONRRY OXYCEN TOXCITY - EFFEcT or ETvTRoNMENTAL AND INHALED GAS TEVPENNTUNE 54-A5, INTRoDUCTION 55 METHODS 55-58 RESULTS 5.9-82 DIScUSSION a3-a5 CHAPTER 3 - EFFECT OF INTRAPERITONEALLYADMINISTERED SUPEROXIDE DISMUTASE ON PULMOT.IRRY DRIT¡RGE RESULTING FROM HYPEROXIA a6- 103 INTRoDUCTION a7-aa METHODS a9-92 RESULTS 92- rOO DISCUSSION rol - 103 CHAPTER 4 - ALVEOLáR MACROPHAGE FUNCTION _ DIFFERENCES BETWEEN NON€MOKERS AND SMOKERS, AND OBSERVATIONS ON PHAGOCYTOSIS OF RAT COMPARED TO HUMAN CELLS 1c4- 134 INTRoDUCTION 105- 110 METHODS 111 -112 RESULTS 113- 131 DIScUSSION 132- 134 CHAPTER 5 - THE ROLE OF OXYGEN DERIVED FREE RADTCALS IN RADIATION INDUCED DAMAGEAND DEATH OF NON.DIVIDING EUKARYOTIC CELLS r35- 155 TNTRODUCTTON 136 MATERIALS AND METHODS 137 - 139 RESULTS 14c-- 152 DIScUSSIoN 153- 155 c 6 - CoNcLUStoN 15,6 - 167 c _ BIBLIOGRAPHY 168- 184 Page : B ABBREV NAME ABBREV NAME PAM PULMoNARYALVEOLAR LTso LENGTH OF TIME TO SOYO DEATH MACRoPHAGE BAL B RO NCH OALVEOLAR L-AVAGE TEi,l TRerusvrssroN ELEcTRoN MIcRoScoPY soD SupERoxIoE DISMUTASE cOz CARBoN DIoXIDE ATP AD ENosrNE TRTPHoSPHATE ADCC ANTIBODY DEPENDENT CELLULAR CYTOTOXICITY Oz' SUPEROXIDE ANION NíEM MoDIFIED EAGLES MEDIUM Hzoz HYDROGEN PEROXIDE NNaOH NoRMAL SODIUM HYDROXIDE HO- HYDRoXYL RADICAL EDTA DISODIUM EtHvUeTepIAMINETETRA ACETATE ODFR OXYGEN DERIVED FNEE RNOICNI DETAPAC DIETHYLENE- TRIEIN EPENTAAC ETIC ACID PMN PoLYMoRPHoNUCLEAR PBS PHoSPHATE.BUFFERED SALINE NEUTROPHIL DNA D ESoXYRIBO NUCLEIC ACID Fe++ FERRIc Mn MANGANESE fs+++ FERROUS CulZn Coppgn./ZrNc ARDS ADULT RESPIRATORY DISTRESS SYNDRoME Hz0 WATER Fi0z I NSPÍ RED OXYCEI\ PERCENTAGE TLC ToTRI- LUNG CAPACITY IPF IDIoPATHIC PULMoNARY FrnRosrs -NO IGM IMMUNoGLOBULIN M NITRIC OXIDE oNoo- PEROXYNITRITE RADICAL Pa0z PARTIAL PRESSURE ARTERIAL OXYGEN Hg HAEMoGLOBIN P(A-a)02 ARTERIAL-oXYGEN GRADIENTS pac02 PARTIAL PRESSURE ARTERIAL CARBoN DIOXIDE Page: C FOREWORD These studies have taken a number of years to complete, but the findings remain relevant. This work commenced almost 20 years ago, when I left Australia to work at the University of lowa in full-time bench research. At that stage I had completed my FRACP, and had completed clinical training in pulmonary diseases. After two years at the University of lowa, I returned to South Australia to a full time staff physician post in Thoracic Medicine at the Royal Adelaide Hospital. Research was discouraged within the Department of Thoracic Medicine, with clinical work being given absolute priority. Nevertheless, I wrote two seed grants that were funded by external bodies, and continued to pursue some of the unfinished work from lowa. Mr. Grantley Gill, Reader in Surgery, generously provided laboratory space for me at the University of Adelaide. I established a small laboratory and continued observations in human PAM function. I was successful with NH&MRC funding, and laboratory space in the Hanson Center, within the Royal Adelaide Hospital, was then made available to me. With limited time available for research, I put effort into supporting my own Ph.D. students and Advanced Trainees in Thoracic Medicine, including providing external funding support for salaries and consumables where necessary, as well as daily supervision. At about this time, I also realized that a larger funding base for lung research in Australía was required. To that end I established the Australian Lung Foundation, with support of colleagues within the Thoracic Society of Australia and New Zealand, the British Lung Foundation, and Bedford lndustries in South Australia, as well as business friends and associates. This effort, which I had to initiate from the very beginning, in an Australian economy during a downturn in economic activity, was much greater than I had realized at the beginning, taking six very full years of constant hard work. The result is a solid well- organized research funding body for the benefit of all in Australia. The Australian Lung Foundation is now a very active supporter of lung research in Australia. However, this effort did greatly restrict the time available for my own thesis preparation. However, I did finally take accumulated vacation time, and over this 3 month period, finished most of the statistical calculations. At that point I was ready to complete my thesis, but unfortunately I developed a malignant soft tissue sarcoma. This required wide surgical excision, and subsequent extensive radiation therapy. This further reduced my efforts towards my own Ph.D. thesis presentation, although I continued very actively supporting my own Ph.D. students, and Advanced Trainees in Pulmonary Medicine. lndeed, it took several years to regain excellent health after this serious illness. Even at this time, the active discouragement of my research continued in the Department of Thoracic Medicine at the Royal Adelaide Hospital. Throughout all of this period, I d¡d continue with a strong time and effort commitment to community service through volunteer work with the St John Ambulance Brigade, as Corp Surgeon in South Australia. I feel very fortunate to have been able to complete my thesis, by virtue of recently being in a very supportive environment in the Pulmonary Division at the University of lowa, ln this environment I am currently very well funded with external grants and am actively pursuing several of the areas arising from this thesis. Page:2 ACKNOWLEDGEMENTS The work towards this thesis began in the same year the motion picture 'Star Wars' was first released, and is being submitted in the year that Star Wars is being re-released. Over this span of time there have been a number of people who have contributed to the work in this thesis, some of whom have gone from being colleagues to very close friends. Throughout this time my family have been entirely supportive of all of my initiatives and in particular of the research and educational effort, some of which is included in this thesis. This particularly applies to my wife, Christine, who not only has assisted with the preparation and presentation of papers, grants, abstracts and the thesis, but has also been happy to participate in, and sometimes lead, the voyage of discovery embodied in this thesis, a voyage not just in science but in attitudes, ideas, cultures and technology. Throughout the thesis there have been two primary supervisors, Professor Ann Autor, initially at the University of lowa, and currently at the University of British Columbia, and Professor Barry Vernon-Roberts at the University of Adelaide. Professor Autor was instrumental in providing research space and support in lowa in the early stages of this thesis, as well as providing guidance and an opportunity to study pulmonary toxicity and alveolar macrophage function. Professor Vernon-Roberts has been fundamentally important in the later stages of the research and thesis development. Without support from both of these mentors this work would not have been possible. ln the early stages in lowa, Professor George Bedell (Director, Pulmonary Medicine) provided essential time for bench research, and my family and I remain always appreciative of his kindness and generosity (and that of Miriel) towards us. Dr Jeffrey Stevens Ph.D. (post-doc in the Autor lab) introduced me, on a daily basis, to bench research with a great degree of patience, practical advice and helpful criticism. The radiation experiments were supported with practical help and enthusiasm, by now Professor Larry Oberley, at the University of lowa. The transmission electron microscopy Page : 3 was practically helped by now Professor Kenneth Moore, and Dr Eugene Shih patiently supported the scanning electron microscopy, also at the University of lowa. The initial BAL studies were supported by Dr Autor, and were performed by me in the University of lowa. On my return to Adelaide, after a period of time, Mr. Grantley Gill, Reader, Department of Surgery, University of Adelaide, kindly provided laboratory space. This allowed for further BAL and alveolar macrophage work. During this time laboratory Mr. Neville DeYoung ably and enthusiastically provided supervision for research staff, and the work on phagocytosis and other alveolar macrophage functions continued using the technical expertise of Ms. Karen Martin. During this time, as well, I began much more work with protein chemistry in the lung, with very strong support by Robert Walsh, Clinical Chemistry, at the lnstitute of Medical and Veterinary Science. I am indebted to Bob for not only his enthusiastic support, but his enormous technical skill and knowledge, manifest by early protein chemistry, and also with subsequent work. Mark Stevens, Cytologist, also at the lnstitute of Medical and Veterinary Science, has been extremely helpful in corroborating BAL cell counts and differentials in the normal volunteers, and in providing reports on the cell counts for the patient studies. I would also ackknowledge the patient help from the University of Adelaide, in allowing me to complete this thesis. I have had the oppoftunity also to have strong and sincere personal support over the course of this project from Dr. Michael Drew at the Royal Adelaide Hospital and in the last few years from Mr. Bill Menzel AOM (and his wife Gerda). Finally, Professor Gary Hunninghake (and Margie) at the University of lowa, continue to be significant in providing great personal and professional support. Page:.4 Academic Staff Supported Ph.D. students Mr. A. Wozniak, Completed and Awarded 1994 Ms. T.J. Dillon, Completed and Awarded 1995. Mr. R.L. Walsh, ln Progress. Ms. R. Uppaluri, Completed and awarded 1997 Advanced Trainees in Thoracic Medicine Dr. M. Chia, Research Fellow with Dr.