THE ROLE OF LOCAL AND PERIPHERAL ANTIOXIDANTS IN THE PATHOGENESIS OF CHRONIC PERIODONTITIS IN SMOKERS by NICOLA ANN LING-MOUNTFORD A thesis submitted to The University of Birmingham for the degree of MASTER OF PHILOSOPHY School of Dentistry The University of Birmingham October 2009 University of Birmingham Research Archive e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder. ABSTRACT Chronic periodontitis is one of the most common chronic inflammatory diseases of man and accounts for 60% of tooth loss. It is initiated by the subgingival biofilm and in susceptible individuals an abnormal inflammatory-immune response fails to resolve the inflammation and leads to destruction of the supporting tissues and the teeth. Risk factors for periodontitis may be systemic or local and of the systemic risk factors the most significant is smoking. Periodontitis patients appear to express a hyper-inflammatory phenotype involving excess or prolonged production of enzymes and reactive oxygen species (ROS) from cells of the innate immune response (primarily neutrophils). Neutrophil hyper-reactivity and hyperactivity, with respect to ROS production, has been demonstrated by several authors. Consistent with the exaggerated ROS production is the depletion of antioxidant defences against ROS within the periodontal pockets. Cigarette smoke is also reported to increase the oxidative burden and deplete antioxidant defences, but no data are available on gingival crevicular (GCF) antioxidant levels in smokers compared to non-smokers. This thesis explores the total antioxidant capacity (TAOC) of GCF and plasma in smokers and non-smokers with periodontitis and analyses the impact of smoking on the outcomes of periodontal therapy and upon local and peripheral antioxidant status in both groups. The working hypothesis is that an important mechanism underpinning the increased prevalence of periodontitis in smokers involves reduction of antioxidant defences due to smoking and thus increased oxidative stress and tissue damage. The cross-sectional data presented here suggest that smokers with periodontitis have a further compromise in GCF TAOC compared to age-, gender- and disease-matched non-smokers with periodontitis. The longitudinal data presented within this thesis suggest that the compromised GCF TAOC concentration seen in periodontitis irrespective of smoking status is likely to result from the inflammatory lesion, rather than predisposing to it. Moreover, the impact of the periodontal inflammation upon TAOC compromise appears more dominant than the effects of smoking. DEDICATION This thesis is dedicated to my husband, Rich, for all his unfailing support and love during this period of study and to my newly arrived daughter, Isobel, for giving me the sense of urgency to complete this work. Nic Ling-Mountford October 2009 ACKNOWLEDGEMENTS I would like initially to thank my supervisor, Professor Iain Chapple, for his preliminary research and continued enthusiasm for chemiluminescence and for his continual support and encouragement, without which this thesis would not have been possible. I would also like to thank Dr John Matthews and his team for their invaluable assistance with the laboratory element of this thesis, in particular Dr Kevin Carter. Their meticulous attention to detail and scientific principles in the instruction of the assay, storage and running of samples, ensured this research was carried out successfully. I would like to extend my thanks to all the staff of the Unit of Periodontology and the Birmingham Dental Hospital, particularly to those who were willing to be volunteers and provide blood and gingival crevicular fluid samples. The support in recruitment and screening of periodontal patients from the dental staff, especially Mr Paul Weston, Mr Mike Milward and Dr Gareth Brock, was invaluable. My special thanks also to the nursing staff the Periodontal Department, particularly Miss Keeley Askey, for their clinical assistance with recording data. I gratefully acknowledge the financial support received from The Oral & Dental Research Trust in association with Colgate-Palmolive in the form of the “Colgate- Palmolive PCD Research Award 2004”, which aided the laboratory fees. Finally I would like to thank Mr Mike Milward for his friendship, encouragement and for being a good listener, which was much appreciated. CONTENTS: Page Introduction 1.1. Inflammatory periodontal disease 2 1.2. Classification of inflammatory periodontal diseases 3 1.3. The aetiology and pathogenesis of gingivitis and periodontitis 4 1.4. Dental plaque and the microbiology of periodontitis 5 1.4.1. Putative pathogens 6 1.4.2. Microbial virulence factors 8 1.5. Host response and its contribution to the aetiology of 10 periodontitis 1.5.1. Mechanisms of tissue damage 12 1.6. The threat of oxygen 13 1.6.1 Basic free radical reactions 14 1.6.2. Atomic and molecular oxygen 15 1.6.3. Origins and formation of reactive oxygen species 17 1.6.4. Reactive oxygen species 18 1.6.4.1. Singlet oxygen 19 1.6.4.2. Superoxide 19 1.6.4.3. Hydrogen peroxide 20 1.6.4.4. Hydroxyl radical 22 1.6.5. Sources of Fenton-reactive metal iron in vivo 23 1.6.6. Nitrogen-derived free radicals 23 1.6.6.1. Nitric oxide 24 1.6.6.2. Peroxynitrite 24 1.6.7. Sulphur derived free radicals 25 1.7. The role of ROS in tissue damage 25 1.7.1. Lipid peroxidation 25 1.7.2. Protein oxidation 27 1.7.3. Nucleic damage 28 1.7.4. Carbohydrate damage 28 1.8. Role of ROS in periodontal disease 29 1.8.1. Direct actions of ROS in periodontal destruction 29 1.8.2. Indirect action of ROS in periodontal destruction 30 1.8.3. ROS produced by neutrophils 31 1.8.4. Other sources of ROS in the periodontal tissues 32 1.9. Host defence against free radicals – Antioxidants 33 1.9.1. Superoxide dismutase 37 1.9.2. Catalase 37 1.9.3. Glutathione 38 1.9.4. Ascorbic acid (vitamin C) 39 1.9.5. α-Tocopherol (vitamin E) 40 1.9.6. Carotenoids (vitamin A) 41 1.9.7. Co-enzymes Q10 42 1.9.8. Uric acid 42 1.9.9. Polyphenols 43 1.10. Concept of “total antioxidant capacity” 43 1.11. Smoking 44 1.11.1. Smoking as a risk factor for periodontitis 44 1.11.2. The effects of smoking on ROS production from neutrophils 47 1.11.3 The effects of smoking on antioxidant status 47 1.12. Measuring ROS and antioxidant status in biological samples 48 1.12.1. Total antioxidant capacity methodology 49 1.12.1.2. Assays for measuring water-soluble TAOC 50 1.12.1.3. Assays for measuring lipid-soluble TAOC 52 1.13. Gingival crevicular fluid 53 1.14. Immediate background and aim of the study 54 1.14.1. Objectives 55 Materials & Methods Materials 57 2.1. Standard solutions and equipment for antioxidant assay 57 2.1.1. Phosphate buffered saline containing bovine serum albumin 57 (PBS-BSA) – Running buffer 2.1.2. Synthesis and preparation of signal reagent 57 2.1.2.1. Signal reagent powder constituents 57 2.1.2.2. Signal reagent buffer preparation 58 2.1.2.3. Preparation of working signal reagent 58 2.1.3. Horseradish peroxidase stick solution 50lU/ml 59 2.1.4. Standard ECL assay mix 59 2.1.5. Trolox standards (80µM) for assay calibration 60 2.1.6. BioOrbit 1250 Luminometer 60 2.2. Classification and categorisation of patients 61 2.2.1. Indices of health & disease 61 2.2.1.1. Periodontal probing depths 61 2.2.1.2. Recession 61 2.2.1.3. Clinical attachment levels 62 2.2.1.4. Bleeding scores 62 2.2.1.5. Plaque scores 62 2.2.1.6. Radiographs 63 2.2.2. Volunteer groups 63 2.2.2.1. Smokers with chronic periodontitis 63 2.2.2.2 Inclusion criteria 64 2.2.2.3. Exclusion criteria 64 2.2.2.4. Non-smokers with chronic periodontitis – Group A 64 2.2.2.4.1. Non-smokers with chronic periodontitis – Group B 65 2.2.2.5. Periodontally healthy subjects – Group A 65 2.2.2.5.1. Periodontally healthy subjects – Group B 65 Methods 67 2.3. Treatment regime 67 2.3.1. Smoking cessation 67 2.4. Clinical sample collection protocols 68 2.4.1. Plasma 68 2.4.2. GCF sampling 68 2.4.2.1. Index sites 69 2.4.2.2. Deep sites 70 2.4.2.3. Shallow sites 70 2.4.2.4. GCF volume determination 70 2.4.2.5. Periotron 8000 operating procedure 71 2.4.2.6. GCF sample elution and storage 72 2.5. Total antioxidant capacity (TAOC) assay methodology 73 2.5.1. Introduction to chemiluminescence as a diagnostic tool 73 2.5.2. Enhanced chemiluminescence assays 74 2.5.2.1. Mechanism of enhanced chemiluminescence 74 2.5.2.2. Application of the ECL system as an antioxidant assay: 75 inhibition by radical scavengers 2.5.2.3. BioOrbit 1250 Luminometer protocol 76 2.5.2.4. Preparation of working solution for the assay 76 2.5.2.5. Influence of the test solution (GCF & Plasma) upon ECL 77 2.5.2.6. Definition of T10% 77 2.5.2.7. Total antioxidant assay calibration and sample volumes 78 2.5.2.8 Calculation of total antioxidant capacity 78 2.6. Statistical analysis of results 79 Results & Discussion 3. Cross-sectional investigations: 81 Local and peripheral total antioxidant capacity (TAOC) 3.1. Context 81 3.2. Clinical and demographic data 82 3.3. GCF samples 84 3.3.1. Smokers with periodontitis versus group A (index sites - 84 patient as unit of analysis) 3.3.2. Smokers with periodontitis versus group B (deep sites - 86 diseased site as unit of analysis) 3.4. Total antioxidant capacity (TAOC) 87 3.4.1. GCF TAOC per 30 second sample of smokers with 87 periodontitis versus group A (patient as unit of analysis) 3.4.1.2. GCF TAOC concentration of smokers with periodontitis 87 versus group A (patient as unit of analysis) 3.4.2. GCF TAOC per 30 second sample of smokers with 88 periodontitis versus group B (diseased site as unit of analysis) 3.4.2.1. GCF TAOC concentration of smokers with periodontitis 88 versus group B (diseased site as unit of analysis) 3.4.3. Plasma TAOC 89 3.5. Summary of findings 92 3.6. Discussion 92 4. Longitudinal investigations: 97 Local and peripheral total antioxidant capacity 4.1. Context 97 4.2. Clinical and demographic data 97 4.3. Total antioxidant capacity (TAOC) 104 4.3.1. GCF TAOC per 30 second sample of smokers with 104 periodontitis versus group A (patient as unit of analysis) 4.3.1.2 GCF TAOC concentration of smokers with periodontitis 105 versus group A (patient as unit of analysis) 4.3.2. GCF TAOC per 30 second sample of smokers with 106 periodontitis versus group B (diseased site as unit of analysis) 4.3.2.1 GCF TAOC concentration of smokers with periodontitis 106 verses group B (diseased site as unit of analysis) 4.3.3. Plasma TAOC 107 4.4. Summary of findings 109 4.5. Discussion 109 5. Site specific longitudinal investigations: 115 Local total antioxidant capacity 5.1. Context 115 5.2. Clinical and demographic data 115 5.3. Total antioxidant capacity (TAOC) 118 5.3.1. GCF TAOC per 30 second 118 5.3.2 GCF TAOC concentration 119 5.4. Discussion 121 General Discussion and conclusions 6. General Discussion and conclusions 124 Appendices after page 129 Periotron calibration – volumes “look-up” table Publications arising directly from this thesis Publication arising indirectly from this thesis References