Dissertation “COMPARISON OF SERUM MALONDIALDEHYDE LEVELS IN PATIENTS WITH ORAL SUBMUCOUS
“COMPARISON OF SERUM MALONDIALDEHYDE LEVELS IN PATIENTS WITH ORAL SUBMUCOUS FIBROSIS AND CONTROL GROUP – A HOSPITAL BASED STUDY” By DR.ALAKA HEBBAR REG. NO. IG0208001 Dissertation Submitted to K.L.E University, Belgaum, Karnataka In partial fulfillment Of the requirements for the degree of MASTERS OF DENTAL SURGERY In ORAL MEDICINE AND RADIOLOGY (BRANCH – IX) Under the guidance of DR. ARVIND SHETTI M.D.S Professor DEPARTMENT OF ORAL MEDICINE AND RADIOLOGY KLE VISHWANATH KATTI INSTITUTE OF DENTAL SCIENCES BELGAUM, KARNATAKA MAY 2011 I K.L.E UNIVERSITY, BELGAUM KARNATAKA DECLARATION BY THE CANDIDATE I hereby declare that this dissertation/thesis entitled “COMPARISON OF SERUM MALONDIALDEHYDE LEVELS IN PATIENTS WITH ORAL SUBMUCOUS FIBROSIS AND CONTROL GROUP – A HOSPITAL BASED STUDY” is a bonafide and genuine research work carried out by me under the guidance of DR.ARVIND SHETTI M.D.S Professor and Head, Department of Oral Medicine and Radiology, K.L.E. V.K Institute of Dental Sciences. DATE: PLACE: Belgaum DR.ALAKA HEBBAR II K.L.E UNIVERSITY, BELGAUM KARNATAKA CERTIFICATE BY GUIDE This is to certify that the dissertation entitled “COMPARISON OF SERUM MALONDIALDEHYDE LEVELS IN PATIENTS WITH ORAL SUBMUCOUS FIBROSIS AND CONTROL GROUP – A HOSPITAL BASED STUDY” is a bonafide research work done by DR.ALAKA HEBBAR, in partial fulfillment of the requirement for the degree of MASTERS OF DENTAL SURGERY in Oral Medicine and Radiology. DATE: PLACE: Belgaum DR.ARVIND SHETTI M.D.S Professor, Department of Oral Medicine & Radiology, KLES VK Institute of Dental Sciences, Belgaum III K.L.E UNIVERSITY, BELGAUM KARNATAKA CERTIFICATE BY CO-GUIDE This is to certify that the dissertation entitled “COMPARISON OF SERUM MALONDIALDEHYDE LEVELS IN PATIENTS WITH ORAL SUBMUCOUS FIBROSIS AND CONTROL GROUP – A HOSPITAL BASED STUDY” is a bonafide research work done by DR.ALAKA HEBBAR, in partial fulfillment of the requirement for the degree of MASTERS OF DENTAL SURGERY in Oral Medicine and Radiology. DATE: PLACE: Belgaum DR.P.B.DESAI MD Professor and Head Department of Biochemistry, JN Medical College, Belgaum. IV K.L.E UNIVERSITY, BELGAUM KARNATAKA ENDORSEMENT BY THE HOD, PRINCIPAL/HEAD OF THE INSTITUTION This is to certify that the dissertation entitled “COMPARISON OF SERUM MALONDIALDEHYDE LEVELS IN PATIENTS WITH ORAL SUBMUCOUS FIBROSIS AND CONTROL GROUP – A HOSPITAL BASED STUDY” is a bonafide research work done by DR.ALAKA HEBBAR, post graduate student under the guidance of DR.ARVIND SHETTI M.D.S Professor, Dept. of Oral Medicine and Radiology, K.L.E V.K Institute of Dental Sciences, Belgaum. HEAD OF DEPARTMENT PRINCIPAL DR.VAISHALI KELUSKAR M.D.S Professor and Head Department of Oral Medicine and Radiology, K.L.E. V.K Institute of Dental Sciences, KLE University Belgaum -590010. DR.ALKA D.KALE M.D.S Principal K.L.E. V.K Institute of Dental Sciences, K.L.E University Belgaum-590010. Date: Place: Belgaum Date: Place: Belgaum V K.L.E UNIVERSITY, BELGAUM KARNATAKA COPYRIGHT DECLARATION BY THE CANDIDATE I hereby declare that K.L.E University, Belgaum, Karnataka shall have the rights to preserve, use and disseminate this dissertation/thesis titled “COMPARISON OF SERUM MALONDIALDEHYDE LEVELS IN PATIENTS WITH ORAL SUBMUCOUS FIBROSIS AND CONTROL GROUP – A HOSPITAL BASED STUDY” in print or electronic format for academic/research purpose. DATE: PLACE: Belgaum DR.ALAKA HEBBAR © KLE University, Belgaum, Karnataka [Established under section 3 of the UGC Act, 1956 vide GOI. Notification No.F.9-19/2000-U.3(A)] VI ACKNOWLEDGEMENT “A teacher is a mother of knowledge” “Gurur Brahma, Gurur Vishnu, Gurur Devo Maheshwaraha, Gurur Sakshaath Parabrahma tasmai Shree Gurave Namaha” “Teaching is a profession that teaches all the other professions” I dedicate these words to express my profound respect, deep sense of gratitude and indebtness to my beloved teacher Dr. Vaishali Keluskar M.D.S Professor and Head, Department of Oral Medicine, Diagnosis and Radiology, K.L.E. V.K Institute of Dental Sciences, Belgaum, for her immense support, patient hearing and constant motivation which boosted up my confidence during tough times. “A Good teacher Explains… A Superior teacher Demonstrates… A Great teacher Inspires” A teacher is like a compass that activates the magnets of curiosity, knowledge and wisdom amongst the people. Words are indeed inadequate to express my heartfelt gratitude and indebtedness to my guide Dr. Arvind Shetti M.D.S Professor and, Department of Oral Medicine, Diagnosis and Radiology, K.L.E.’s Vishwanath Katti Institute of Dental Sciences, Belgaum. He with his ever inspiring presence, critical observation and deep understanding helped me to shape my ideas in a coherent fashion. Without his enthusiastic guidance, constant encouragement, constructive criticism and valuable suggestions, it would not have been possible for me to complete this study. VII “Good teaching is more a giving of right questions than a giving of right answers” I owe an immense debt of gratitude to Dr. Anjana Bagewadi, M.D.S Professor, Department of Oral Medicine, Diagnosis and Radiology, K.L.E.’s Vishwanath Katti Institute of Dental Sciences, Belgaum, for her comforting presence and ever helpful hand during the study. “Teachers open the door, but you must enter by yourself” Words do not suffer to express my sincere and humble gratitude to my respected teachers Dr. Renuka Ammanagi M.D.S and Dr. Zameera Naik M.D.S Reader, Department of Oral Medicine and Radiology, K.L.E.’s Vishwanath Katti Institute of Dental Sciences, Belgaum, for their valuable suggestions during the study. I am highly obliged to our beloved Principal Dr. Alka. D. Kale M.D.S Professor, Department of Oral Pathology and Microbiology, K.L.E.’s Vishwanath Katti Institute of Dental Sciences, Belgaum for allowing me to utilize the clinical material, facilities in this institution and also providing me with all the necessary support whenever needed. I take the opportunity to express my profound gratefulness to Dr. P . B. Desai M.D. Professor and Head, Department of Biochemistry, Jawaharlal Nehru Medical College, Belgaum, for his constant encouragement and help in conducting the biochemical analysis during the period of my study. I extend my heartfelt thanks to Dr.Vasanti Jirge, M.D.S Dr. Shivyogi M.D.S and Dr. Ambika M.D.S Senior lecturer and for their valuable suggestions during my study. I express my sincere thanks to Professor Dr.H.N. Nagaraj and Professor Mr. Mallapur for providing help in statistical analysis for this study. VIII “Side by side or miles apart, dear friends are always close to the heart” I wish to acknowledge and thank my colleagues, Dr. Archana, Dr. Kumud, Dr. Vishlesh, Dr. Poonam, Dr. Ashish, Dr. Ankur, Dr. Sapna, Dr.Anshul, Dr. Rakhi, Dr.Nidhi, Dr. Ishita, Dr. Hina, Dr. Sugandha and Dr. John for their cooperation and help throughout the course of my study. “The virtue of parents is in itself a great legacy” I am blessed to have loving families, who have been a constant support and encouragement to me through thick and thin. I am infinitely obliged to express my feeling of pride for my most cherished treasure – My Father Dr. H.R.Hebbar, mother Dr. Pushpagandhini and sister Malavika Hebbar, for their love, affection, support and advice. I am thankful to my aunties Sumangala Bha, Anuradha Navada and my uncles H.N. Krishnaraj and Ramachandra Bhat who always stood by me and supported me in this endeavour. I am thankful to my friends Dr. Shweta Advani and Dr. Raveena Thapar and Dr. Tejaswini Gauripur and all those people who have offered me genuine words of advice and encouragement during the period of my study. This list will be incomplete if I do not acknowledge the debt that I owe to all the subjects who consented to be part of the study. Lastly and above all, I thank God Almighty for making all these wonderful people happen to me and giving me strength and courage to complete this project. Dr. ALAKA HEBBAR. IX Dedicated to my beloved Parents and Grandmother X LIST OF ABBREVIATIONS ANOVA One way Analysis of Variance OSMF Oral Submucous Fibrosis ROS Reactive Oxygen Species MDA Malondialdehyde H2O2 Hydrogen Peroxide RAR Retinoic Acid Receptor PAS Periodic Acid Schiff ANA Antinuclear Antibody SMA Anti Smooth muscle Antibody CTLA Cytotoxic T-lymphocytes Associated Antigen Ig Immunoglobulin SGOT Serum Glutamic Oxaloacetic Transaminase SGPT Serum Glutamic Pyruvic Transaminase RBC Red Blood Cells HLA Human Leukocyte Antigen WHO World Health Organization O2 Oxygen PR Pyrogallol red H2 Hydrogen HOCl Hypochlorous acid SOD Superoxide Dismutase NADPH Nicotinamide Adenosine Dinucleotide Phosphate MPO Myeloperoxidase Fe Iron XI DNA Deoxyribonuclic Acid UV Ultraviolet • OH Hydroxyl Radical NO Nitric Oxide PUFA Poly Unsaturated Fatty Acid ATP Adenosine Triphosphate LPO Lipid Peroxidation GECA Anti Gastric Perietal Cell Antibodies M Macroglobulin HOO• Hydroperoxyl Radical ROO• Lipid Peroxyl Radical R• Free Radical MPO MyeloPeroxidase O2•- Superoxide Radical RSH Thiol Compounds CCl4 Carbon Tetrachloride • CCl3 Trichloromethyl radical CH2 Methylene ROOH Hydroperoxide R-OOH Lipid hydroperoxide ALE Advanced lipoxidation end products AGE Advanced glycation end products TBA Thiobarbituric Acid TBARS Thiobarbituric acid reactive substances GR Glutathione Reductase XII GPX Glutathione Peroxidase G6PDH Glucose-6 Phosphate Dehydrogenase GST Glutathione-S-transferase M1 G Melondialdehyde guanine COX2 Cyclooxygenase 2 M1-dG Melondialdehyde deoxyguanosine NO3 Nitrate NO2 Nitrite HCl Hydrochloric acid XIII ABSTRACT Background and Objectives: Oral submucous fibrosis is a chronic, insidious and debilitating condition with a high malignant potential and a high prevalence rate in India and South East Asian countries. Studies have shown that the process of carcinogenesis occurs in OSMF by the generation of reactive oxygen species (ROS), which act by initiating lipid peroxidation (LPO). Malondialdehyde (MDA) is one of the important and easily detectable biomarker of LPO and a indicator of oxidative stress. The objective of this study was to assess MDA levels in the serum of OSMF patients and to compare it with the Control group. Method: 36 patients with OSMF and 24 age matched controls were included in the study and the serum MDA levels were estimated using Thiobarbituric acid (TBA) reagent. Results: There was a statistically significant difference in serum MDA levels among the patients with OSMF and the control group. Serum MDA levels varied in different stages of OSMF also. The levels were increased in stage III OSMF followed by stage II and stage I. Interpretation & Conclusion: It is evident from the present study that serum MDA levels were significantly increased in patients with OSMF indicating the increased amount of oxidative stress. Keywords: OSMF, Malondialdehyde, Oxidative stress, lipid peroxidation XIV TABLE OF CONTENTS SI. NO. PARTICULARS PAGE 01 INTRODUCTION 1 02 AIM AND OBJECTIVES 3 03 REVIEW OF LITERATURE 4 04 METHODOLOGY 49 05 RESULTS 58 06 DISCUSSION 66 07 CONCLUSION 74 08 SUMMARY 75 09 BIBLIOGRAPHY 76 10 ANNEXURES ANNEXURE - I: PROFORMA 90 ANNEXURE - II: CONSENT FORM 92 ANNEXURE - III: MASTER CHART- CASES 93 ANNEXURE - IV: MASTER CHART – CONTROLS 95 XV LIST OF TABLES SI. NO. TABLES PAGE NO. 01 Laboratory procedure for MDA estimation by TBA method 53 02 Mean value for age among various groups 58 03 ANOVA Test for ages among different study groups 59 04 Mean value for MDA among various groups 59 05 ANOVA Test for MDA among various groups 60 06 Mean MDA value comparison 61 07 Multiple comparisons of serum MDA values in various groups 61 using Tukey Kramer Test XVI LIST OF GRAPHS SI. NO. GRAPHS PAGE NO. 01 Distribution of number of subjects in various study groups 63 02 Distribution of subjects in various study groups according to age 63 03 Mean age of patients among different study groups 64 04 Mean serum malondialdehyde levels among various study groups 64 05 Comparison of mean MDA levels among various study groups 65 XVII LIST OF FIGURES SI. FIGURES NO. PAGE NO. 01 Molecular pathogenesis of OSMF (1) 14 02 Molecular pathogenesis of OSMF (2) 15 03 Molecular pathogenesis of OSMF (3) 16 04 Molecular pathogenesis of OSMF (4) 16 05 The cellular sources of free radicals 24 06 Generation of oxygen free radicals in respiratory burst 27 07 Lipid peroxidation process 34 08 Structure of malondialdehyde 37 09 Complex multifactorial nature of oxidative damage to cells 39 10 Equipments used in the clinical examination of the patients 54 11 Reagents used for the estimation of serum malondialdehyde 54 12 Boiling water bath 55 13 Centrifuge machine 55 14 Spectrophotometer 56 15 Blanching on labial mucosa 56 16 Blanching on buccal mucosa 57 17 Shrunken uvula 57 18 Reduced mouth opening 57 19 Reduced tongue protrusion 57 XVIII “COMPARISON OF SERUM MALONDIALDEHYDE LEVELS IN PATIENTS WITH ORAL SUBMUCOUS FIBROSIS AND CONTROL GROUP – A HOSPITAL BASED STUDY” By REG. NO. IG0208001 Dissertation Submitted to K.L.E University, Belgaum, Karnataka In partial fulfillment Of the requirements for the degree of MASTERS OF DENTAL SURGERY In ORAL MEDICINE AND RADIOLOGY (BRANCH – IX) DEPARTMENT OF ORAL MEDICINE AND RADIOLOGY KLE VISHWANATH KATTI INSTITUTE OF DENTAL SCIENCES BELGAUM, KARNATAKA MAY 2011 I K.L.E UNIVERSITY, BELGAUM KARNATAKA ENDORSEMENT BY THE HOD, PRINCIPAL/HEAD OF THE INSTITUTION This is to certify that the dissertation entitled “COMPARISON OF SERUM MALONDIALDEHYDE LEVELS IN PATIENTS WITH ORAL SUBMUCOUS FIBROSIS AND CONTROL GROUP – A HOSPITAL BASED STUDY” is a bonafide research work done by Candidate Reg. No. IG0208001 of Oral Medicine and Radiology, K.L.E V.K Institute of Dental Sciences, Belgaum. HEAD OF DEPARTMENT DR.VAISHALI KELUSKAR M.D.S Professor and Head Department of Oral Medicine and Radiology, K.L.E. V.K Institute of Dental Sciences, KLE University Belgaum -590010. PRINCIPAL DR.ALKA. D.KALE M.D.S Principal K.L.E. V.K Institute of Dental Sciences, K.L.E University Belgaum-590010. Date: Place: Belgaum Date: Place: Belgaum II LIST OF ABBREVIATIONS ANOVA One Way Analysis of Variance OSMF Oral Submucous Fibrosis ROS Reactive Oxygen Species MDA Malondialdehyde H2O2 Hydrogen Peroxide RAR Retinoic Acid Receptor PAS Periodic Acid Schiff ANA Antinuclear Antibody SMA Anti Smooth muscle Antibody CTLA Cytotoxic T-lymphocytes Associated Antigen Ig Immunoglobulin SGOT Serum Glutamic Oxaloacetic Transaminase SGPT Serum Glutamic Pyruvic Transaminase RBC Red Blood Cells HLA Human Leukocyte Antigen WHO World Health Organization O2 Oxygen PR Pyrogallol red H2 Hydrogen HOCl Hypochlorous acid SOD Superoxide Dismutase NADPH Nicotinamide Adenosine Dinucleotide Phosphate MPO Myeloperoxidase Fe Iron III DNA Deoxyribonuclic Acid UV Ultraviolet • OH Hydroxyl Radical NO Nitric Oxide PUFA Poly Unsaturated Fatty Acid ATP Adenosine Triphosphate LPO Lipid Peroxidation GECA Anti Gastric Perietal Cell Antibodies M Macroglobulin HOO• Hydroperoxyl Radical ROO• Lipid Peroxyl Radical R• Free Radical MPO MyeloPeroxidase O2•- Superoxide Radical RSH Thiol Compounds CCl4 Carbon Tetrachloride • CCl3 Trichloromethyl radical CH2 Methylene ROOH Hydroperoxide R-OOH Lipid hydroperoxide ALE Advanced lipoxidation end products AGE Advanced glycation end products TBA Thiobarbituric Acid TBARS Thiobarbituric acid reactive substances GR Glutathione Reductase IV GPX Glutathione Peroxidase G6PDH Glucose-6 Phosphate Dehydrogenase GST Glutathione-S-transferase M1 G Melondialdehyde guanine COX2 Cyclooxygenase 2 M1-dG Melondialdehyde deoxyguanosine NO3 Nitrate NO2 Nitrite HCl Hydrochloric acid V ABSTRACT Background and Objectives: Oral submucous fibrosis is a chronic, insidious and debilitating condition with a high malignant potential and a high prevalence rate in India and South East Asian countries. Studies have shown that the process of carcinogenesis occurs in OSMF by the generation of reactive oxygen species (ROS), which act by initiating lipid peroxidation (LPO). Malondialdehyde (MDA) is one of the important and easily detectable biomarker of LPO and a indicator of oxidative stress. The objective of this study was to assess MDA levels in the serum of OSMF patients and to compare it with the Control group. Method: 36 patients with OSMF and 24 age matched controls were included in the study and the serum MDA levels were estimated using Thiobarbituric acid (TBA) reagent. Results: There was a significant difference in serum MDA levels among the patients with OSMF and the control group. Serum MDA levels varied in different stages of OSMF. Serum MDA levels were found to be highest in stage III OSMF followed by stage II and stage I. Interpretation & Conclusion: It is evident from the present study that serum MDA levels were significantly increased in patients with OSMF indicating the increased amount of oxidative stress. Keywords: OSMF, Malondialdehyde, Oxidative stress, lipid peroxidation VI TABLE OF CONTENTS SI. NO. PARTICULARS PAGE 01 INTRODUCTION 1 02 AIM AND OBJECTIVES 3 03 REVIEW OF LITERATURE 4 04 METHODOLOGY 49 05 RESULTS 58 06 DISCUSSION 66 07 CONCLUSION 74 08 SUMMARY 75 09 BIBLIOGRAPHY 76 10 ANNEXURES ANNEXURE - I: PROFORMA 90 ANNEXURE - II: CONSENT FORM 92 ANNEXURE - III: MASTER CHART- CASES 93 ANNEXURE - IV: MASTER CHART – CONTROLS 95 VII LIST OF TABLES SI. NO. TABLES PAGE NO. 01 Laboratory procedure for MDA estimation by TBA method 53 02 Mean value for age among various groups 58 03 ANOVA Test for ages among different study groups 59 04 Mean value for MDA among various groups 59 05 ANOVA Test for MDA among various groups 60 06 Mean MDA value comparison 61 07 Multiple comparisons of serum MDA values in various groups 62 using Tukey Kramer Test VIII LIST OF GRAPHS SI. NO. GRAPHS PAGE NO. 01 Distribution of number of subjects in various study groups 63 02 Distribution of subjects in various study groups according to age 64 03 Mean age of patients among different study groups 64 04 Mean serum malondialdehyde levels among various study groups 65 05 Comparison of mean MDA levels among various study groups 65 IX LIST OF FIGURES SI. NO. FIGURES PAGE NO. 01 Molecular pathogenesis of OSMF (1) 14 02 Molecular pathogenesis of OSMF (2) 15 03 Molecular pathogenesis of OSMF (3) 16 04 Molecular pathogenesis of OSMF (4) 16 05 The cellular sources of free radicals 24 06 Generation of oxygen free radicals in respiratory burst 27 07 Lipid peroxidation process 34 08 Structure of malondialdehyde 37 09 Complex multifactorial nature of oxidative damage to cells 39 10 Equipments used in the clinical examination of the patients 54 11 Reagents used for the estimation of serum malondialdehyde 54 12 Boiling water bath 55 13 Centrifuge machine 55 14 Spectrophotometer 56 15 Blanching on labial mucosa 56 16 Blanching on buccal mucosa 57 17 Shrunken uvula 57 X 18 Reduced mouth opening 57 19 Reduced tongue protrusion 57 XI Introduction INTRODUCTION Oral submucous fibrosis is a chronic insidious and progressive disease involving oral mucosa. Recent studies reveal that there is an increase prevalence of Oral submucous fibrosis in different states of India.1 This condition has been shown to be precancerous2 and carries a high risk for malignant conversion even after the cessation of areca nut use, which is known to play a major role in the development of the disease.3 There is no report suggesting spontaneous regression nor there is any no effective or widely accepted treatment, making this condition worse.4 Epidemiological studies have shown that the process of carcinogenesis occurs by the generation of Reactive Oxygen Species (ROS).5 Free radicals can be defined as molecules or molecular fragments with an unpaired electron which imparts certain characteristics to the free radicals such as reactivity.7,8 Reactive free radicals are able to produce chemical modifications and damage proteins, lipids, carbohydrates and neucleotides in the tissues. Reactive oxygen species (ROS) may damage the cells by initiation of lipid peroxidation (LPO)6 that causes profound alteration in the structural integrity and functions of the cell membranes. Free radical induced LPO has been implicated in the pathogenesis of several diseases. The concentration of LPO product malondialdehyde (MDA) is most widely used in this regard. The etiology and malignant transformation potential of Oral submucous fibrosis is poorly understood. Literature available suggests it to be multifactorial in origin, which includes arecanut chewing, heredity, nutritional deficiencies and immunological factors. 1 Introduction OSMF being the potentially malignant condition and associated with carcinogens like tobacco was thought to have some relation with ROS. Hence this study was designed to estimate serum LPO product malondialdehyde in various stages of OSMF patients and to compare the same with control group. 2 Aim and Objective AIM AND OBJECTIVES AIM: 1. To estimate and compare the levels of serum lipid peroxidation product malondialdehyde in patients with oral submucous fibrosis and the control group. OBJECTIVES: 1. To estimate serum lipid peroxidation product malondialdehyde levels in patients with oral submucous fibrosis. 2. To estimate serum malondialdehyde levels in age & sex matched controls with chewing habit without the disease. 3. To estimate serum malondialdehyde levels in age and sex matched healthy controls without chewing habit. 4. To compare serum malondialdehyde levels in patients with oral submucous fibrosis & controls. 3 Review of literature ORAL SUBMUCOUS FIBROSIS Oral submucous fibrosis is a chronic debilitating disease of the oral cavity. It is a disease predominantly of Indian population, although there is worldwide distribution. It is a well recognized potentially malignant condition which predominantly affects the oral cavity, but may extend to pharynx, oesophagus and even to larynx. It is characterized by juxta-epithelial inflammatory reaction followed by fibro-elastic changes in the lamina propria with epithelial atrophy leading to stiffness of oral cavity and causing trismus and inability to eat. OSMF also results in difficulty in speech, swallowing, pain in the throat and relative loss of auditory acquity.9 HISTORICAL REVIEW Oral submucous fibrosis has been well established in medical literature since the time of Sushruta – a renowned Indian physician who lived in the era of 2500-3000 B C. He described a condition resembling OSMF and named it as ‘VIDHARI’ having features of progressive narrowing of mouth, depigmentation of oral mucosa and pain on taking food.10 Schwartz (1952) was the first person to describe OSMF as a fibrosing condition among five Indian women from Kenya and east Africa and named it as “atophica idiopathica tropica mucosae oris”.11 Joshi S G (1953) first described the condition in India and suggested the name “submucous fibrosis” of palate and pillars’12. Lal D (1953) suggested the notation “diffuse OSMF” to denote the complete replacement of the subepithelial layer of dense, acellular, nonelastic collagenous material with collections of lymphocytes and plasma cells.13 4 Review of literature Sui P (1954) termed it as “idiopathica scleroderma of mouth”.14 Paymaster J C (1956) was the first to describe it as a premalignant condition and its association in development of slow growing squamous cell carcinoma in one third of the cases of OSMF in Mumbai.15 Rao B N (1962) termed it as “idiopathic palatal fibrosis” in his study of 46 cases of palatal fibrosis in Hyderabad.16 Pindborg J J et al (1965) in their study of OSMF among 100 South Indians with oral cancer reported the finding of OSMF in 40 out of 100 patients.17 Mohd. Akbar (1976) in his study reported the occurrence of OSMF associated with leukoplakia of buccal mucosa in 4 cases and of tongue in 1 case.18 George Laskaris et al (1981) reported a case of OSMF in a 67 year old Greek female who later developed squamous cell carcinoma of the tongue which later progressed to a fetal termination.19 Pindborg J J et al (1984) found that the rate of malignant transformation to be 4.5% out of 89 patients with the disease in Ernakulum district, Kerala17. Murti P R et al (1985) in their seventeen year old follow up study of OSMF noted that the rate of malignant transformation was 4.5 at the end of 15 years and 7.6 at the end of 17 years, they have stressed that OSMF had a high degree of malignant potential.20 DEFINITIONS According to WHO (1978), OSMF is defined as “A slow growing progressive disease in which fibrous bands form in the blanched oral mucosa resulting in severe restriction of movement of mouth”. 5 Review of literature The most accepted definition is the one stated by Pindborg and Sirsat (1966) “OSMF is an insidious chronic disease affecting any part of oral cavity and sometimes pharynx although occasionally preceded by and/ or associated with juxta epithelial inflammatory reaction followed by fibroelastic changes in the lamina propria with epithelial atrophy leading to stiffness of oral mucosa and causing trismus and inability to eat”.9 EPIDEMIOLOGY Worldwide estimates in 1996 indicated that this disease affected 2.5 million people. In 2002 statistics from India alone were about 5 million people that is 0.5% of the Indian population. OSMF is very common in South East Asian countries such as Bangladesh, Bhutan, Pakistan, India, Sri Lanka. The prevalence rate of OSMF in these countries ranges from 0 to 1.2%. Only in India incidence is 0.2 to 0.5%. Prevalence is higher in south compared to north India. The incidence in Ernakulum was reported as 8 per 1 lakh men and 19 per 1 lakh women.5 ETIOLOGY AND RISK FACTORS Although various etiological agents are proposed, the exact etiology of oral submucous fibrosis has not yet been identified. Various etiological agents and predisposing factors have been studied and current evidence suggests that arecoline in areca nut plays a major role in initiating the disease process. However role of different etiological agents and factors so far studied are: Chillies Genetic Predisposition 6 Review of literature Tobacco and areca nut Immunological factors Nutritional factors Autoimmune mechanism More recently collagen related genes are implicated in the susceptibility and pathogenesis of OSMF. i) Chillies OSMF is more prevalent in South India and Sri Lanka where chillies are eaten dried, powdered or raw at meal.25,23 The chillies which contain capsaicin promote changes of OSMF. A study showed chilli extract to be mutagenic and it has been found to enhance the tumorogenicity of tobacco in experimental animals.23,24 ii) Arecanut Chewing of areca nut wrapped in betel leaf and smeared with the paste of crude lime and spices was incriminated in one study as the causative agent. The pathological role was attributed to high tannic acid, slaked lime, and the continuous and prolonged action of nicotine contained in the betel nut, these have neurotropic effect on the oral mucosa.14,21 Experimental evidence for the etiological role of betel nut in OSMF came from in vitro studies in which it was shown that ethanolic extracts of the nut stimulated collagen synthesis in human fibroblasts.15 In a study on histological effect of arecoline on the palate and buccal mucosa of 28 vistar rats of varying periods of time was observed. It was noted that OSMF of palate 7 Review of literature was produced by the habit of chewing betel nut. Based on the findings it was suggested that arecoline played an important role in the causation of OSMF of palate in human being also.26, 22 The pharmacological effect of different betel nut preparations was studied in relation to OSMF. The variations in nut alkaloids and tannin content probably due to plant variability and different cooking procedures were noted. Thus variations in the pharmacologically active constituents of the betel nut may contribute to the regional differences in the incidence of the disease.27 iii) Nutritional deficiency OSMF has been proposed as an Asian version of sideropenic dysphagia wherein the chronic iron deficiency leads to mucosal susceptibility to irritants such as chilli and areca nut. Deficiency of vitamins and iron has been implicated as being of etiological importance in the oral submucous fibrosis.28 iv) Genetic predisposition In a study on HLA frequencies in 50 OSMF and 50 controls were studied, three antigens (A10, B7, and DR3) were significantly raised in the patients suggesting genetic linkage of the disease.13 Researchers analyzed the expression of retinoic acid receptor (RAR beta) and p53 by immunohistochemistry in 50 cases of OSMF and 30 histologically normal oral tissues. The study concluded that altered expression of either of these proteins in majority of OSMF cases or associated with disease pathogenesis. Follow up is required to determine if these cases harboring concomitant alterations in (RAR beta) and p53 are at a high risk of transition to malignancy.28 8 Review of literature v) Immunological factors OSMF have similarity in clinical presentation to collagen disorders like scleroderma that has an autoimmune pathogenesis. The term ‘idiopathic scleroderma of the mouth’ has been coined for it because of the same.13 Histologically eosinophilic material and marked increase in PAS positive material with metachromasia in the ground substance, indicative of presence of fibroid like material as seen in number of cases of connective tissue disorders involving dense collagen proliferation is seen. The ultrastructural changes were also similar to those seen in rheumatoid arthritis and scleroderma.26 In view of the association of the human leukocyte antigen (HLA) DR3 with scleroderma a study was conducted and increase in DR3 antigen among 44 patients with OSMF was noted. An increase in serum IgG immunoglobulins and auto antibodies were also noted among these subjects.13 Incidence of auto antibodies in 109 Tiwanese patiens with OSMF was studied and a high frequencies of antinuclear antibodies (ANA) (23.9%), anti-smooth muscle antibodies (SMA) (23.9%) and anti gastric parietal cell antibodies (GECA) (14.7%) was observed compared to that of healthy controls(9.2, 7.3 and 5.5%) respectively suggesting that OSMF may be an autoimmune disease.30 In a recent study the role of cytotoxic T- lymphocytes – associated antigen 4 (CTLA4) in the maintenance of immune tolerance was highlighted. A higher frequency of G allele at position +49 in exon 1 of CTLA-4 was noticed in 64 OSMF patients as compared to controls. Such defect in cellular immunity is also seen in scleroderma as a result of autoimmune phenomenon. The authors thus suggested CTLA-4 polymorphism might play a role in susceptibility to OSMF.31 9 Review of literature In a review regarding pathogenesis of OSMF multifactorial etiology was proposed stating the role of genetic alteration, carcinogen like areca nut, tobacco, nutritional factors and immunological factors.32 The serum protein, ascorbic acid, iron and tissue collagen were studied in OSMF patients. The patients were categorized into mild, moderate and severe based on the severity of the clinical manifestation. Biopsy samples were obtained for tissue collagen estimation. Authors reported significant rise of protein and tissue collagen, early albumin: globulin ratio was decreased with significantly raised levels of globulin while ascorbic acid and iron content was decreased.33 In a study quantifying the number of mast cells in different grades of oral submucous fibrosis it was found that in grade I at an average 4.5 mast cells per unit microfield were present, in grade II 4.9 cells per unit microfield and 0.5 cells per unit microfield in grade III cases as compared to the average number of mast cells per unit microfield in normal buccal mucosa which is 1.02.34 It was later hypothesized that sensitized mast cells coming in contact with arecoline act as an allergen resulting in degranulation causing release of histamine and heparin which produces fibrosis. It was further suggested that OSMF may be hypersensitivity type I reaction to arecoline and the reaction represents a genetically controlled predisposition to the production of specific IgE antibodies.34 Hypersensitivity caused by local irritants and the resultant persistent juxtaepithelial inflammatory response may act as initiating factors. Thus defective inflammatory reparative response culminates in fibrotic healing.35 Increase in the serum mucoprotien and mucopolysaccharide levels in patients with OSMF has been observed. It was suggested that these represent the reactants in the active stage of the disease where breakdown of tissues and collagen degeneration is 10 Review of literature occurring. Rise in anti-streptolysin titre suggested a possible role of immunological response in the form of a localized collagen disorder to streptococcal toxicity in the etiology of oral submucous fibrosis.36 One of the studies conducted in 34 patients of OSMF and 20 age and sex matched controls showed elevated levels of total globulin in OSMF patients and this was highly significant when compared to control group. The author also reported a significant increase in total immunoglobulin levels in OSMF patients as compared to control group. The IgG fraction showed a significant increase in OSMF than controls but IgA levels were comparable to control group. IgM did not show any alterations in OSMF. Thus in view of hyperglobulinemia and hyperimmunoglobulinemia the author concluded that OSMF may be an autoimmune disorder.37 A study was conducted to know the possible rate of immunological factors in the OSMF by the evaluation of the 113 OSMF cases and 25 controls, male: female ratio was 1.5:1. Serum IgA, IgG and IgM levels were elevated significantly as compared to the controls. Circulating autoantibodies and tissue deposited antibodies were also found in 33% and 40% of patients and serum globulin levels in 47% of patients, thus indicating a immunological basis.38 vi) Immunoglobulins According to WHO, immunoglobulins are the proteins of animal origin endowed with antibody activity and for certain other proteins related to them by chemical structure. The definition includes, besides antibody globulins, the subnormal proteins found in myeloma, macroglobulinemia, cryoglobulinemia and naturally occurring subunits of immunoglobulins. 11 Review of literature They are synthesized by plasma cells and to some extent by lymphocytes and constitute 20-25% of the total serum proteins. Immunoglobulins are glycoproteins, each molecule consisting of 2 pairs of polypeptide chains of different sizes and is the basis of classifying 5 types, i.e. IgG, IgA, IgM, IgD and IgE in order of decreasing concentration in the serum vii) Arecoline and immunity Short term effects of general parameters on the adrenal and the lymphoid organs were screened to explore in detail its immunomodulatory influence in murine model system. Arecoline was administered subcutaneously to male mice at subtoxic dose levels (5, 10, 20 mg/Kg body weight) for 1, 2 and 3 weeks on a daily basis, while total protein, albumin, glucose, phosphatase and hemoglobin concentrations were not altered, increase in SGOT and SGPT levels were observed at the high dose. The white and red blood cell counts decreased in a dose dependent manner. These observations demonstrated the effect of arecoline on the lymphoid organs which may be due its direct action or through the elevation of corticosterone39. B cell mediated immune response in mice after short term exposure to arecoline examined to explore its modulatory influence on B cell mediated immune response in murine model system. The in vivo and in vitro effects of arecoline were evaluated at a subtoxic concentration. Arecoline exposure for a week involved the dose dependent effect on primary antibody forming cells to the sheep RBC’s with the maximum reduction at the dosage of 20 mg/Kg bw, a moderate reduction at 10mg/Kg body weight and no effect at 5 mg/Kg body weight was observed. HA and HL titres to sheep RBC’s were suppressed markedly at arecoline dosage of 20mg/Kg body weight 12 Review of literature and moderately at a dose of 10mg/Kg body weight, given daily for 1, 2 and 3 weeks. The inhibitory effect of arecoline was not dependent on the duration of treatment.40 viii) Allergic role The presence of eosinophilic cells as constant finding in most of the histologic pictures of OSMF made to remark that the condition is probably an allergic reaction. It was hypothesized that sensitized mast cells coming in contact with arecoline act as an allergen resulting in degranulation causing release of histamine and heparin results in fibrosis. It was further suggested that OSMF may be hypersensitivity type I reaction to arecoline and the reaction represents a genetically controlled predisposition to the production of specific IgE antibodies. Thus only those individuals who are active to sensitive ingredients of betel nut are affected. This also could explain why majority of betel nut chewers in India don’t suffer from OSMF. Hypersensitivity caused by local irritants and the resultant persistent juxtaepithelial inflammatory reparative response culminating in fibrotic healing. ix) Defective iron metabolism The first reported case of anemia in patients of OSMF was described by Moosa and Madan. It is postulated that the changes brought about by anemia in tissues would make the individuals more susceptible to OSMF. It is a well recognized fact that the histological changes observed in oral mucosa of patients with OSMF lack of iron in the tissue would also result in the improper function of vascular channels and concomitant decrease in the vascularity. The percolation of ester of arecoline would be made easier which in turn results in fibrosis. It is also noted that occurrence of iron deficiency anemia could be due to the clinical nature of OSMF. The initial burning sensation, vesiculations and ulcerations render the consumption of solid food difficult. 13 Review of literature This lack of consumption of normal diet would possibly initiate anemia in females. After frank establishment of the lesion anemia may be further perpetuated by inadequate intake of food due to fibrosis and trismus. Thus the relationship of iron deficiency anemia with OSMF seems to be a vicious circle. PATHOGENESIS The most important histopathological characteristic of OSMF is the deposition of collagen in the submucosa. The arecanut component of betel quid especially alkaloid called arecoline plays a role in pathogenesis of OSMF by causing an abnormal increase in collagen production. The flavanoid component is believed to have some direct effect on the collagen metabolism. Alkaloid exposure to buccal mucosal fibroblast results in accumulation of collagen. A decreased degradation of collagen due to increased cross-linking of fibers and reduced collagenase activity are found in OSMF mucosa compared to normal oral mucosa. The etiopathogenesis of OSMF is simplified by the following flow charts.41 Oral Mucosa Increased susceptibility due to deficiency of iron and Vit B12 Betel quid habit Duration and frequency of the habit Constant irritation Chronic inflammation Activated T-cell and macrophages at site Increase in cytokines- IL6, TNF, IF-α Increase in growth factor- TGF-β Figure No.1: Molecular Pathogenesis of Oral Submucous Fibrosis 14 Review of literature TGF-β Procollagen activation PNP BMP1/PCP Increased Procollagen ProLOX Increased collagen (soluble form) LOX Increased copper in arecanut Increase LOX Increased collagen (insoluble form) Increase in collagen production Figure No.2: Molecular Pathogenesis of Oral Submucous Fibrosis Pro-LOX: Pro Lysyl oxidase LOX: Lysyl oxidase PNP: Pro-collagen N-Protein PCP: Pro-collagen C-Protein BMP: Bone Morphogenic Protein 1 15 Review of literature TGF-β Activation of TMP gene Actvation of PAI gene Increase TMPs Increase PAI plasminogen Inhibits activated collagenases procollagenase Decrease in collagenase activity plasmin collagenase Flavinoids in arecanut Decrease in collagen degradation Figure No.3: Molecular Pathogenesis of Oral Submucous Fibrosis Decreased collagen destruction Increased collagen production Increased collagen insoluble form – Cross linking of insoluble collagen Fibrosis Oral Submucous Fibrosis Figure No.4: Molecular Pathogenesis of Oral Submucous Fibrosis 16 Review of literature CLINICAL FEATURES The majority of patients of OSMF are between the age group of 20 to 40 years. The most frequent location is the buccal mucosa and retromolar area. It also involves the palate, faucial pillars, uvula, tongue and labial mucosa. In some cases even the floor of the mouth and gingival are involved. SYMPTOMS The symptoms of OSMF can be broadly divided under two headings Prodromal symptoms Later symptoms Prodromal symptoms: The onset of this condition is insidious and is often of 2-5 years duration. The most common initial symptom is burning sensation of the mouth often experienced when the patient is eating hot and spicy food. The other frequent early symptom is blisters, ulcerations or recurrent stomatitis. Excessive salivation, defective gustatory sensation and dryness of the mouth may also occur in early stages of the disease. Loss of taste sensation may be due to reduced contact surface of the tongue mucosa during chewing or due to persistent fibrosis of the taste fibres.42,32 Later symptoms: After varying periods of time, in some cases a few years after the appearance of initial symptoms, patient complains of stiffness of certain areas of the oral mucosa leading to difficulty in mouth opening, inability to blow, difficulty in swallowing. Pain in the ear is the later symptom of the disease. This is attributed to pharyngeal fibrosis and occlusion of “eustachian tube”. Later nasal twang of the voice may develop.16 17 Review of literature SIGNS The most common and earliest sign is blanching of the mucosa caused by impairment of local vascularity. The blanched mucosa becomes opaque and white. The whitening often takes place in spots so that the mucosa acquires a marble like appearance. As the disease progresses the mucosa becomes stiff and the vertical fibrous bands appear. Labial mucosa is blanched, rubbery and is characterized by the presence of the fibrous bands around the rima oris. In severe cases opening of the mouth is altered to an elliptical shape and lips are difficult to evert. Soft palate and uvula- they become fibrotic and clear delineation of the soft and hard palate is seen. Mobility of the soft palate is restricted. Uvula when involved is shrunken and in extreme cases they become bud shaped. Tongue- the initial change is depapillation, usually in the lateral margins of the tongue. The surface of the tongue becomes smooth, its mobility also decreases. Floor of the mouth becomes elastic and rubbery. Gingival when affected becomes fibrotic, blanched and inelastic. Associated features: Hyperpigmentation or loss of pigmentation is very common in association with OSMF.43 It is usually found in the areas of redness in the soft palate, the anterior faucial pillars, buccal mucosa or the mucosal surface of the lip particularly lower labial mucosa. The vesicles are painful and they soon rupture leaving behind superficial ulcerations.17 Ulcerations often develop in advanced cases. The epithelium becomes atrophic which render it fragile and vulnerable to ulcerations. Petechiae or small raised reddish blue spots which sometimes occur in OSMF they are most common on the tongue, labial mucosa and buccal mucosa due to focal vasodialation.35 18 Review of literature CLINICAL STAGING OF OSMF Various staging/grading for OSMF are given by many authors which are as below: I .Pindborg (1989)44, divided OSMF based on the physical findings into three stages as follows: a) Stage I: Stomatitis includes erythematous mucosa, vesicles, mucosal ulcers, melanotic mucosal pigmentation and mucosal petechiae b) Stage II: Fibrosis occurs in healing vesicles and healing ulcers, which is the hallmark of this stage. Early lesions demonstrates the blanching of the oral mucosa Oral lesions include vertical and circular fibrous bands in association with the blanched mucosa. Specific findings include reduction of mouth opening, stiff and small tongue, blanched and leathery floor of the mouth, fibrotic and depigmented gingiva, rubbery soft palate with decreased mobility, blanched and atrophic tonsils, shrunken bud like uvula and sunken cheeks not commensurate with age and nutritional status. c) Stage III: Sequelae of OSMF are as follows Leukoplakia is found in more than 25% of individuals with OSMF Speech and hearing deficits may occur because of involvement of tongue and the eustachian tube. II. LAI D R45 (1995) gave the clinical staging of OSMF based on the interincisal distance Group A : Mouth opening greater than 35mm Group B : Mouth opening between 30-35mm Group C : Mouth opening between 20-30mm 19 Review of literature Group D : Mouth opening less than 20 mm III. S M. Haider47 et al (2000) gave clinical staging based on the presence of fibrous bands on various anatomic sites and gave functional staging based on the mouth opening Clinical staging: Stage 1: Fibrous bands only Stage 2: Faucial and buccal bands Stage 3: Faucial, buccal and labial bands Functional staging: Stage 1: Mouth opening >20mm Stage 2: Mouth opening 11-19mm Stage 3: Mouth opening <10mm IV. Bailoor DN and Nagesh KS48 (2001) suggested the following stages - - Stage I: Early OSMF Mild blanching Normal mouth opening (35-45mm) Normal tongue protrusion (5-6cm) Burning sensation on taking only hot or spicy food Stage II: Moderate OSMF Moderate to severe blanching Mouth opening and tongue protrusion reduced by 33% 20 Review of literature - Burning sensation even in the absence of stimuli Palpable bands present Unilateral or bilateral lymphadenopathy Stage III: Severe OSMF Severe burning sensation Mouth opening and tongue protrusion reduced by 66% Ulcerative lesions may appear on the cheek Thick palpable fibrous bands Lymphadenopathy bilaterally evident V. Ranganathan K46 et al (2001) used a baseline study on the mouth opening parameters of normal patients and divided the OSMF patients as: Grade I: Only symptoms, with no demonstrable restriction of mouth opening Grade I: Limited mouth opening and above Grade III: Mouth opening less than 20mm Grade IV: Advanced OSMF with limited mouth opening. Precancerous and cancerous changes seen throughout the mucosa. VI. Rajendran R35 (2003) reported the clinical features of OSMF as follows Early OSMF: Burning sensation in the mouth, blisters especially on the palate, ulcerations or recurrent generalized inflammation of oral mucosa, excessive salivation, defective gustatory sensation and dryness of mouth. 21 Review of literature Advanced OSMF: Blanched and slightly opaque mucosa, fibrous bands in the buccal mucosa running in vertical direction. Palate and the faucial pillars are the areas first involved. There is gradual impairment of tongue movement and difficulty in mouth opening. VII. Sani N K42 and Bhama L K (2006) gave clinical staging based on the involvement of different anatomical areas Early: Fibrous bands present, no trismus, no involvement of tongue Moderate: Trismus present but no gross tongue involvement, fibrosed soft palate Advanced : Marked trismus, marked fibrosis stiff soft palate and restricted tongue movements OSMF advanced with limited mouth opening. Precancerous and cancerous changes seen throughout the mucosa In this study 2.34%, 40%, 10% and 26.67% of the biopsies were in stage I, stage II, stage III and stage IV respectively.51 MALIGNANT POTENTIAL Oral cancer originates in submucous fibrosis from diverse intra-oral location, without any noticeable predilection for any particular site.48 Atrophic epithelium first becomes hyperkeratotic and later, intracellular edema and basal cell hyperplasia develops eventually, following epithelial atypia with moderate epithelial hyperplasia and then carcinoma can develop at any time.49 To substantiate the precancerous nature of the condition, following points are noted, high occurrence of sub mucous fibrosis in oral 22 Review of literature cancer patient, higher incidence of oral cancer in patient with OSMF, histological diagnosis of carcinoma, without the clinical suspicion of it, higher prevalence of leukoplakia among OSMF patients, higher frequency of epithelial dysplasia. The WHO Collaborating Centre for Oral Precancerous Lesions has concluded that although OSMF predisposes to cancer, it is not absolutely conclusive. It is highly probable that such relationship does exist. Following facts support this hypothesis:51 1) The frequency of oral leukoplakia in OSMF patient is 6-8 times higher than control group. 2) Carcinoma patients exhibiting OSMF have a frequency of fibrosis in general population. 3) Immunological alterations observed in OSMF are almost similar to that observed in oral cancer. ROLE OF OXIDATIVE STRESS IN ORAL SUBMUCOUS FIBROSIS FREE RADICALS A free radical is a species capable of independent existence that contains one or more unpaired electrons. An unpaired electron is being one that is alone in the orbital.52 The unpaired electron gives certain characteristic properties to the free radical such as paramagnetism. The chemical reactivity of free radicals is usually high. They may be positively charged, negatively charged or electrically neutral.53 A free radical is conventionally represented by a superscript dot (R•). A compound becomes free radical by gaining an additional electron as in the case of reduction of molecular oxygen to superoxide anion radical (O2•). Other free radicals 23 Review of literature are hydroxyl radical (•OH), hydroperoxyl radical (HOO•), lipidperoxyl radical (ROO•). The sequential univalent reduction steps of oxygen may be represented as.54 O2 (+)e- O2•- (+)e-,2H H2O2 (+)e-, H+ • OH (+)e-, H+ H2O -H2O Generation of free radicals: Free radicals may be formed by; Cleavage of covalent bond of a normal molecule Loss of single electron from a normal molecule Addition of single molecule to the normal molecule Sources of free radicals: Oxidants related to human diseases are derived from three sources Those generated via normal intracellular biological processes but in a exaggerated, inappropriate fashion or in a milieu where the normal defences that serve to protect the tissue are inadequate. Those released by inflammatory cells into their local environment. Figure No.5: The Cellular Sources of Free Radicals 24 Review of literature Those secondary to xenobiotics, either because the xenobiotics include oxidants (eg: inhaled oxidant gases) or because these xenobiotics induce oxidant generation within cells, eg: drugs that injure tissues through oxidant mechanisms.55 1 Endogenous sources: a) Endogenous sources of free radicals include those that are generated and act intracellularly and those that are formed within the cell and are released into the surrounding area.56 Oxidation-reduction reactions generate free radicals constantly within the body. These can be mediated by the action of the enzymes or non-enzymatically, often through the redox chemistry of transition metal ions.57 Oxidants and electron transport systems are prime, continuous sources of intracellular reactive oxygen free radicals. Electron transfer from transition metals such as iron to oxygen can initiate free radical reactions. 56 An important source of superoxide anion radical is the “univalent leak” of superoxide anion radical from the mitochondrial electron transport system.58 b) Intracellular free radicals are generated from the auto oxidants and consequent inactivation of the small molecules such as reduced flavins and thiols, and from the activity of certain oxidases, cycloxygenases, lipoxygenases, dehydrogenases and peroxidases.56 A variety of enzyme systems catalyze the univalent reduction of the molecular oxygen to superoxide anion radical. Such univalent reduction of molecular oxygen also occurs in vivo in non-enzymatic electron transfer oxidation-reduction reactions. 25 Review of literature (E.g: hydroquinone + O2 superoxide + O2•- + H+) and during auto oxidation reactions including those that involve catecholamines, flavins and reduced ferridoxins.59 c) NADPH oxidase in the inflammatory cells (neutrophils, eosinophils, monocytes and macrophages) produces superoxide anion by the process of respiratory burst during phagocytosis. The superoxide is converted to hydrogen peroxide and to hypochlorous acid (HOCl) with the help of superoxide dismutase (SOD) and myeloperoxidase (MPO). This superoxide and hypochlorous ions are the final effectors of bactericidal action. The gene for myeloperoxidase is chromosome 17. The enzyme myeloperoxidase has a molecular weight 156 Kilo Dalton and contains two iron atoms per molecule. This is deliberate production of the free radicals by the body. About 10% of oxygen uptake by macrophage is used for free radical generation. Along with the activation of macrophages, the consumption of the oxygen by the cell is increased drastically; this is called the respiratory burst.60 (Figure 6) d) Hydroxyl radicals are formed in the “Fenton reactions” whenever hydrogen peroxide comes into contact with ferrous or cupric ions. An iron catalyzed Haber-weiss type of reaction may also form this radical, the net effect of which is an interaction between hydrogen peroxide and superoxide anion radical in the presence of traces of transition metal ions to form hydroxyl radical. Finally the OH• .radical is also the product of ionizing reaction.59, 61 Fe2+ + H2O2 O2•- + H2O2 Fe3+ + •OH + OH- (Fenton Reaction) Fe salt catalyst O2 + •OH + OH- (Haber Weiss reaction) 26 Review of literature The chelated Fe3+ can be reduced to Fe2+ by thiols, ascorbate and most of other reductants. Fe2+ can then auto oxidize producing O2•. Figure No. 6: Generation of oxygen free radicals in respiratory burst 2 Exogenous sources: a) Cigarette smoke contains high concentration of various free radicals other toxic compounds such as carbon tetrachloride, drugs and inhalation of the pollutants, anaesthetics, organic solvents, hypertoxic environments and pesticides will increase the production of free radicals.56 b) Ionizing radiation damages the tissue by producing haemolytic fission of background water. H2O (gamma, UV radiation) H• + OH• c) Light of appropriate wavelengths can cause excitation of oxygen in presence of photosensitizers to produce singlet oxygen. 27 Review of literature Sites of free radical generation: Main sites of free radical generation are mitochondria, lysosomes, peroxisomes, nuclei, endoplasmic reticulum, plasma membrane and the cytosol.56 i) Endoplasmic reticulum, nuclear membrane and electron transport systems: Free radicals produced by the endoplasmic reticulum and nuclear membrane can undergo both intraorganelle and cytosolic reactions. In case of nuclear membrane generated radicals DNA would be particularly susceptible to free radical damage. ii) Plasma membrane: Plasma membrane is the site of action of extracellularly generated free radicals. They must cross the plasma membrane before reacting with other cell components and may initiate toxic reactions at the membrane. The unsaturated fatty acids present in the membrane and transmembrane proteins containing oxidizable amino acids are susceptible to free radical damage. Increased membrane permeability caused by lipid peroxidation or oxidation of structurally important proteins can cause breakdown of transmembrane ion gradients, resulting in loss of secondary functions and inhibition of integrated cellular metabolic processes. The interior of the biological membranes is hydrophobic and O2•- produced in the environment could be extremely damaging. Much of the O2•- generated within the cells comes from membrane bound systems and it is certainly possible that some of it is formed in the membrane interior.62 iii) Peroxisomes: Peroxisomes are the potential sources of cellular hydrogen peroxide because of the high concentration of oxidases. 28 Review of literature REACTIVE OXYGEN SPECIES (ROS) Oxygen derived free radicals and other non radical species are collectively known as reactive oxygen species (ROS). Several reactive oxygen species are known, prominent among them include: i) Superoxide radical (O2•- ): This ROS is formed when oxygen takes up one electron and as leaks in the mitochondrial electron transport but its formation is easily increased with exogenous components (redox cycling compounds) are present. Its first production site is the internal mitochondrial membrane (NADH ubiquinone reductase and ubiquinone cytochrome-c-reductase).63 This species is reduced and forms hydrogen peroxide (H2O2). The production of superoxide radicals at the membrane level (NADPH oxidase) is initiated in specialized cells with phagocytic functions (macrophages) and contributes to their bactericidal action (oxidative burst).64 The flavin cytosolic enzyme xanthine oxidase found in quite all tissues and in milk fat globules generates superoxide radicals from hypoxanthine and oxygen and is supposed to be at the origin of vascular pathologies. ii) Hydrogen peroxide (H2O2): Hydrogen peroxide is mainly produced by enzymatic reactions. These enzymes are located in the microsomes, peroxisomes and mitochondria. Even in normal conditions the hydrogen peroxide production is relatively important and leads to a constant cellular concentration between 10-9 and 10-7 M, in plant and animal cells superoxide dismutase is able to produce H2O2 by dismutation of O2.-, thus contributing to the lowering of oxidative reactions. The neutral combination dismutase and catalase 29 Review of literature contributes to remove H2O2 and thus has a true cellular antioxidant activity. H2O2 is also able to diffuse easily through membranes.65 iii) Hydroxyl radical (•OH) : In the presence of Fe2+, H2O2 produces the very active species •OH by Fenton reaction. Fe2+ + H2O2 Fe3+ + • OH + OH- This iron-catalyzed decomposition of oxygen peroxide is considered the most prevalent reaction in the biological systems and the source of various deleterious lipid peroxidation products. iv) Nitric oxide (NO•): Nitric oxide is produced in vascular endothelium. This species is not too reactive (poorly oxidizing function), it reacts readily with O2•- and gives the extremely reactive peroxynitrite (ONOO-). This ROS is naturally found in activated macrophages66 and endothelial cells67 and considered as an active agent in several pathologies based on inflammation, organ reperfusion and also may play an important role in atherosclerosis. v) Singlet oxygen (1O2 ): This chemical form of oxygen is not a true radical but is reported to be an important ROS in reactions related to ultraviolet exposition (UVA 320-400 nm). Its toxicity is reinforced when appropriate photoexcitable compounds (sensitizers) are present with molecular oxygen.68 Several natural sensitizers are known to catalyze oxidative reactions such as tetrapyroles (bilirubin), flavins, chlorophyll, hemoprotiens and reduced pyridine molecules (NADH), some of these sensitizers are also found in 30 Review of literature foods and cosmetics. Some are used for therapeutic purposes (anti cancer treatment) and are sensitive to visible light. The presence of metal contributes to increase the production of single oxygen as well as anion superoxide, and thus accelerates the oxidation of unsaturated lipid generating hydroperoxides. It has been suggested that the singlet oxygen may be formed during the degradation of lipid peroxides and thus may cause the production of other peroxide molecules. This singlet oxygen formation may account for the chemiluminescence observed during lipid peroxidation.69 vi) Thiyl radical: Thiol compounds (RSH) are frequently oxidized in the presence of iron or copper ions. RSH + Cu2+ RS• + Cu+ + H + These thiyl radicals have strong reactivity in combining with oxygen.70, 71 RS• + O2 RSO•2 Furthermore they are able to oxidize NADH into NAD, ascorbic acid and to generate various free radicals (•OH and O2.•-). These thiyl radicals may also be formed by homolytic fission of disulfide bonds in protiens. vii) Carbon centered radicals: The formation of this reactive free radical is observed in calls treated with carbon tetrachloride (CCl4). The action of the cytochrome P450 systems generates the trichloromethyl radical (•CCl3) which is able to react with oxygen to give several peroxyl radicals (i.e. •O2CCl3).72 31 Review of literature DAMAGE PRODUCED BY FREE RADICALS Free radicals are extremely reactive. Their mean effective nidus of action is only 30Ao. and their half life is only a few milliseconds. When a free radical reacts with a normal compound, other free radicals are generated. This chain reaction leads to thousands of events. Peroxidation of polyunsaturated fatty acids (PUFA) severely damages the cell membrane leading to loss of membrane functions like absoption, secretion etc. almost all biological macromolecules are damaged by the free radicals, e.g. Peroxidation of PUFA in plasma membrane Oxidative inactivation of sulfhydryl containing enzymes Polysaccharide depolymerisation and DNA breaks DNA damage may directly cause inhibition of protein and enzyme synthesis indirectly it also causes cell death or mutation and carcinogenesis. Lipid peroxidation and consequent degradation product such as MDA seen in biological fluids. Their effect in the serum is often employed to assess the oxidant stress.73 Proteins: Protein molecules undergo substantial modifications through reactive reactions with free radicals. Proteins containing tryptophan, tyrosine, phenylalanine, histamine, methionine and cysteine can undergo free radical mediated amino acid modifications. Free radicals promote sulfhydryl mediated cross linkage of such labile amino acids as well as cause fragmentation of the polypeptide chains. Oxidative modifications enhance degradation of critical enzymes by cytosolic neutral proteases.74 Enzymes 32 Review of literature undergo cross-linking with resulting increase in molecular weight, each enzyme cross-links with their neighbors in a random destructive reaction. The normal precision arrangement of protein and enzymes in subcellular membrane and organelles is badly disrupted and their biological properties are lost or impaired75. Carbohydrates: Advances in free radical chemistry indicate that no biological substructure is impervious to free radical attack. Therefore it is most important that glucose and other related monosaccharides undergo when conditions are appropriate.74 Hyaluronic acid undergoes polymer fragmentation following exposure to free radical systems, which leads to destabilization of connective tissue and loss of synovial fluid viscosity. Nucleic acids: DNA is readily attacked by oxidizing radicals if they are found in its vicinity has been clearly demonstrated by radiation biologists. It must therefore be considered as a vulnerable and important target. Cell mutation and death from ionizing radiation is primarily due to free radical reactions with DNA. Cell death and mutations arising from free radicals generated during normal metabolism have also been ascribed to reactions with DNA.76 Lipids: All of the major classes of biomolecule may be attacked by free radicals but lipids are probably the most susceptible.57 Cell membranes are the rich sources of polyunsaturated fatty acids. Biomembrane and organelles are the major sites of lipid peroxidation damage. Major constituents of the biological membranes are lipids and 33 Review of literature proteins. Lipid peroxidation can damage membrane protein as well as lipids.62 The membrane fluidity is due to the presence of polyunsaturated fatty acid side chain in many membrane lipids, which lower the melting point of the interior membrane. Lipid peroxidation decreases membrane fluidity. The conditions which favor lipid peroxidation are: i) A high degree of unsaturation in the lipid substrate ii) A rich supply of oxygen and iii) The presence of traditional metal catalysts77 LIPID PEROXIDATION Lipid peroxidation is defined as “oxidative deterioration of polyunsaturated lipids. Lipid peroxidation is particularly damaging because it proceeds as a self perpetuating chain reaction”.78 Figure No. 7: The Lipid Peroxidation Process 34 Review of literature Figure illustrates lipid peroxidation. The reaction is initiated by an existing free radical (X•), by light or by metal ions. Malondialdehyde is only formed by fatty acids with three or more double bonds, and is used as a measure of lipid peroxidation together with ethane from the terminal two Carbon of α-3 fatty acids and pentane from terminal five carbon of α - 6 fatty acids.79 Peroxidation of polyunsaturated fatty acids usually involves three operationally defined processes.62 1. Initiation phase: During this phase the primary event is the abstraction of the hydrogen atom from bisallylic site of PUFA. Initiation peroxidation sequence in membrane or PUFA74 is due to the attack of any species that has sufficient reactivity to abstract a hydrogen atom from methylene (CH2) group. This leaves behind an unpaired electron on the carbon – CH-. The carbon radical tends to be stabilized by the molecular rearrangement to produce a conjugated diene, which then easily reacts with an oxygen molecule to give peroxy radical, R-CO•. the presence of the redox active metals such as iron or copper can facilitate the initiation process.62 ROOH + Metal (n)+ X• + RH ROO• + Metal(n-1)+ + H+ R• + XH 2. Propagation phase: During this phase lipid peroxidation relies on the interaction of molecular oxygen with carbon-centered free radicals to form lipid hydroperoxides.74 The peroxy radical abstract a hydrogen atom from another lipid molecule and once the process begins it tends to continue. The peroxy radical combines with the hydrogen atom that it 35 Review of literature abstracts to give lipid hydroperoxides R-OOH. A probable alternative fate of the peroxy radicals is to form cyclic peroxides. With the help of metal catalysts the decomposition of the hydroperoxides results in the formation of alkoxyl or peroxyl radicals. These radicals are capable of further reactions and thus the propagation of lipid peroxidation continues.62, 74 R• + O2 ROO• ROO• + RH ROOH + R• 3. Termination phase: The propagation reactions of lipid peroxidation will not proceed very far before they meet a protein molecule., which can then be attacked and damaged, in addition aldehyde can attack amino groups on the protein molecule to form both intramolecular cross links and also cross links between different protein molecules, e.g: malondialdehyde. Any kind of lipid free radical can react with a lipid peroxyl radical to give non-initiating and non-propagating species. ROO• + ROO• ROO• + R R• + R • RO-OR + O2 ROOR RR TOXIC EFFECTS OF LIPID PEROXIDATION: The uncontrolled peroxidation of bio-membranes can lead to profound effects on membrane structure and function and may be sufficient to cause cell death.53 The toxic products generated during lipid peroxidation may be involved in damage to specific protein and transport system critical to cell function.82 Malondialdehyde produced by lipid peroxidation can cause cross linking and polymerization of 36 Review of literature membrane components. This can alter the intrinsic membrane properties such as ion transport, enzyme activity. Because malondialdehyde is diffusible, it will also react with nitrogenous bases of DNA81 lipid hydroperoxides can directly inhibit enzymes.82 MALONDIALDEHYDE Malondialdehyde is the organic compound with the formula CH2 (CHO)2. The structure of this species is more complex than this formula suggests. This reactive species occurs naturally and is a marker for oxidative stress. IUPAC name[hide] Propanedial Figure No 8: Structure of malondialdehyde Structure and synthesis : Malondialdehyde mainly exists in the enol form: CH2(CHO)2 → HOCH=CH-CHO In organic solvents, the cis isomer is favored, whereas in water the trans-isomer predominates. 37 Review of literature Malondialdehyde is a highly reactive compound that is not typically observed in pure form. In the laboratory it can be generated in situ by hydrolysis of 1,1,3,3tetramethoxypropane, which is commercially available.83 It is easily deprotonated to give the sodium salt of the enolate (m.p. 245 °C). Biochemistry: Reactive oxygen species degrade polyunsaturated lipids, forming malondialdehyde.84 This compound is a reactive aldehyde and is one of the many reactive electrophile species that cause toxic stress in cells and form covalent protein adducts which are referred to as advanced lipoxidation end products (ALE), in analogy to advanced glycation end-products (AGE).85 The production of this aldehyde is used as a biomarker to measure the level of oxidative stress in an organism.86, 87 Malondialdehyde reacts with deoxyadenosine and deoxyguanosine in DNA, forming DNA adducts, primarily M1G, which is mutagenic.88 The guanidine group of arginine residues condense with MDA to give 2-aminopyrimidines. Human ALDH1A1 aldehyde dehydrogenase is capable of oxidising malondialdehyde. MDA is reactive and potentially mutagenic. OXIDATIVE STRESS The occurrence of ROS known as pro-oxidants is an attribute of normal aerobic life. The steady state formation of pro-oxidants is balanced by similar rate of their consumption by antioxidants that are enzymatic and/or non-enzymatic. “Oxidative stress” results from imbalance in this pro-oxidant-antioxidant equilibrium in favour of the pro-oxidants.in the context of an increased oxidant burden and/or decreased 38 Review of literature antioxidant defense, the oxidants are free to interact with and modify normal biological components. When these components are critical and extent of modification sufficient injury becomes manifest at the tissue level. Figure depicts some of the mechanisms by which oxidative stress causes tissue injury.92 DNA DNA DAMAGE DAMAGE OXIDATIVE STRESS Increased lipid peroxidation GSH DEPLETION DIRECT DAMAGE TO PROTEINS CYTOSKELETAL DAMAGE RISE IN INTRACELLULAR FREE Ca2+ INHIBITION OF ATP SYNTHESIS MEMBRANE BLEBBING NAD(H) DEPLETION POLY(ADP) RIBOSE SYNTHETASE ACTIVATION RISE IN INTRACELLULAR FREE IRON MEMBRANE PEROXIDATION AND DESTRUCTION Increased damage to DNA, proteins and lipids Metal ion release into surrounding tissue, injury to adjacent cells Figure No.9: Complex Multifactorial Nature of Oxidative Damage to Cells 39 Review of literature MALONDIALDEHYDE IN HEALTH AND DISEASES M. Nishi et al (1986) examined changes in the lipid peroxide levels in the submandibular gland, blood, and liver after low-dose x-ray irradiation of rats. The lipid peroxide level in the blood plasma was determined according to Yagi (1976). The lipid peroxide concentration was calculated in terms of mol malondialdehyde/mL of blood. The lipid peroxide levels in the blood plasma of the irradiated group were generally higher than those of controls. Significant differences were seen at two hours, seven days, and 14 days after irradiation.93 Balwant Rai (2007) conducted a study on salivary lipid peroxidation product malondialdehyde in periodontal diseases. Lipid peroxidation product MDA was analyzed in 25 patients of periodontal disease and 30 healthy subjects served as controls. Significantly elevated levels of salivary MDA was observed in periodontitis as compared to controls (p<0.05). These findings indicate a role of free radicals in its pathogenesis.94 Joseph A. Knight et al (1987) measured lipoperoxides, as malondialdehyde (MDA), by liquid chromatography in plasma from 230 male and 148 female adult blood donors, to establish reliable reference values and to compare possible sex-, age, and specimen related differences. Their studies showed that men have higher MDA concentrations in plasma [0.60 (0.21) µmol/L] than do women [0.54 (0.20) µmol/L] (P <0.05), older men have higher values [0.67 (0.20) µmol/L] than younger men [0.58 (0.17) µmol/L] (p <0.05), and older women have higher values [0.57 (0.20) µmol/L] than young women [0.47 (0.17) µmol/L] (P<0.001). These age-related results support 40 Review of literature earlier studies in experimental animals that lipid peroxidation increases with increasing age.95 J Khalili et al (2008) carried out a study on salivary MDA level in clinically healthy and periodontal diseased individuals. MDA levels in saliva were measured in 104 subjects aged between 18-65 years. Three groups with different degrees of severity of generalized chronic periodontitis were established, 30 patients in early stage , 30 with moderate disease and 14 with severe disease. And 30 with healthy periodontium served as controls, a significant increase in the salivary MDA level existed in the samples obtained from the three groups of the patients compared to control subjects.96 MA Carbonneau et al (1991) performed assay of free and total malondialdehyde (MDA) in human serum and plasma from healthy subjects and from patients with high risk of lipoperoxidation. Free MDA averaged 0.042 and 0.043 µmol/L, respectively, in serum and plasma from healthy subjects. Free MDA increased significantly in the plasma from cancer patients (0.270 ± 0.047 µmol/L) and from hemodialyzed patients (0.214 ± 0.035 µmol/L).97 Marie-Jeanne Richard et al (1992) determined the MDA-TBA complex by HPLC method. The normal range (2.51 ± 0.25 µmol/L) determined in 32 normal subjects showed that there existed no sex-related difference for TBARS: 2.57 (0.28) in men versus 2.44 (0.20) µmoI/L in women.98 41 Review of literature Flemming N et al (1997) carried out a study to generate reliable reference intervals for plasma malondialdehyde. Analysis of variance was used to reveal relations between normalized plasma MDA concentration in the three 20-year age groups and gender. The analysis revealed a significant effect from gender (p = 0.033), but apparently not from age (P = 0.109). No major interaction occurred between gender and age (p = 0.103). Men had slightly but significantly higher plasma MDA concentrations than women. In the reference sample group, 92 were smokers and 122 were nonsmokers. Smokers had a significantly higher plasma MDA concentration (mean 0.66 µmol/L) than nonsmokers (mean 0.60 µmol/L) (p = 0.05). Correlation analysis revealed an association between plasma MDA and the number of hours of daily exposure to cigarette smoke (r = 0.162, p = 0.03), but we found no clear correlation between plasma MDA and the number of cigarettes smoked (r = - 0.065, p = 0.55). Plasma MDA was significantly correlated with weekly alcohol consumption (r = 0.153, p = 0.03).9 F Farinati et al (1998) in a study involving 88 consecutive outpatients, undergoing endoscopy for upper gastrointestinal symptoms, divided the patients into histological subgroups: 27 with chronic non-atrophic gastritis, 41 with atrophic gastritis, six with gastric cancer, and 14 unaffected controls. No significant difference was observed in thiobarbituric acid reactive substance (TBARS) concentrations in the above mentioned four groups of patients, nor was there any difference in patients with no, mild, moderate, or severe intestinal metaplasia (no or mild: 25.7 (4.4), moderate, 30.1 (5.5), severe: 25.4 (4.3) mol/g). However, patients with high grade intestinal metaplasia (moderate and severe cases considered together) showed a trend towards 42 Review of literature higher TBARS concentrations (28.1 (12.8) versus 24.3 (11.1) mol/g). Similarly, no difference in TBARS concentrations was detected in patients with no versus moderate or severe disease activity (20 .8 (8.6) versus 26.0 (12.9) and 26.8 (12.3) mol/g, respectively) or in patients with no, moderate, or severe H pylori infection (no: 23.5 (7.8), moderate: 29.4 (14.4), severe: 27.8 (8.8) mol/g; NS). However, H pylori positive patients had significantly higher TBARS concentrations overall (29.1 (13.4) versus 21.5 (8.7) mol/g, p=0.04).100 G. Gitanjali et al (1999) estimated the level of vitamin E and serum MDA in patients with cervical carcinoma receiving radical radiotherapy. Control group comprised of 50 healthy, nonsmoking women. Study group was divided into two subgroups of 25 each. Group I was given vitamin E supplementation (100 mg orally daily) in addition to radical radiotherapy. Group II was given placebo. There was no statistically significant difference in the mean serum MDA levels of healthy controls (3.40 ± 0.75 mole/ml) and the 50 patients taken together (3.54 ± 1.89 mol/ml). In group I after completion of radical radiotherapy and vitamin E supplementation, the mean serum vitamin E levels increased from 9.17 ± 2.69 to 10.88 ± 2.63 µg/ml (p < 0.02) and the mean serum MDA levels decreased from 3.19 ± 1.02 to 0.56 mol/ml (p < 0.01). In group II the mean serum vitamin E levels increased from 9.18 ± 3.14 to 11.3 ± 2.81 µg/ml (p < 0.01) while serum MDA levels decreased from a mean value of 3.88 ± 1.66 mol/ml to a mean of 3.23 ± 1.53 mol/ml (p < 0.04). The difference in serum MDA level in group I and II was statistically significant with p < 0.05.101 43 Review of literature Sabitha KE et al (1999) studied the effect of radiation on oral cancer patients using activities of superoxide dismutase (SOD), catalase, glutathione peroxidase (GPX), glutathione reductase (GR), glutathione-S-transferase (GST), glucose-6phosphate dehydrogenase (G6PDH) and levels of malondialdehyde (MDA). The levels of MDA showed a significant increase in untreated and radiation treated oral cancer patients (p < 0.01 and p < 0.001 respectively) when compared with normal subjects. Radiation treated patients had higher MDA levels than untreated patients. The activities of red blood cell hemolysate antioxidant enzymes such as SOD, catalase, GPX, GR, GST and G6PDH showed a significant decrease, representing the lack of antioxidant defense. Radiation induces lipid peroxidation by inactivating the antioxidant enzymes, thereby rendering the system inefficient in management of the free radical attack. Thus, the degree of radiation affects the extent of the depression of the antioxidant enzyme activities and increases lipid peroxidation.102 Talia Weinstein et al (2000) studied extracellular and intracellular antioxidant defence mechanisms 3 months after recovery from haemolysis in haemodialysis patients. In 29 patients and 20 controls serum MDA was measured as a marker of oxidative stress. In the haemodialysis group, MDA levels were higher than the controls (2.37±0.07 versus 0.97±0.10 mol/ml; p<0.0001. There was a correlation between MDA levels and the number of years patients were treated by dialysis (r=0.43; P<0.02).103 Unal Sahin et al (2001) randomly selected 47 male patients with lung cancer (37 smokers, 10 nonsmokers) in the study group and 35 healthy subjects (20 smokers, 44 Review of literature 15 nonsmokers) were taken as controls. MDA levels levels were studied by flourometric determination of thiobarbituric acid reactive substances in serum based on the reaction between Malondialdehyde and thiobarbituric acid described by Wasowicz et al. The MDA levels of the study and control groups were 20.5 ± 7.9 and 12.6 ± 7.1 mol/ml with statistically significant difference (p < 0.001). MDA levels of smoker and nonsmoker lung cancer patients were 22.5 ± 8.0 and 16.1 ± 6.7 mol/ml, (p < 0.001).104 Ricky A. Sharma et al (2001) tested the hypothesis that cycloxygenase-2 (COX-2) activity in human colon cells results in formation of MDA and generation of malondialdehyde-guanine (M1G) adducts. Levels of M1G correlated significantly (r = 0.98, p < 0.001) with those of intracellular MDA determined colorimetrically in the four malignant cell types, but neither parameter correlated with expression of COX-2 or prostaglandin biosynthesis. Malondialdehyde treatment of human nonmalignant colon epithelial cells resulted in a doubling of M 1G levels. These results show for the first time in human colon cells that COX-2 activity is associated with formation of the endogenous mutagen, MDA. Moreover, they demonstrate the correlation between MDA concentration and M1G adduct levels in malignant cells.105 Simon M. Everett et al (2001) measured levels of malondialdehydedeoxyguanosine (M1-dG), a DNA adduct derived from lipid peroxidation, using the immunoslot-blot technique alongside mucosal MDA and plasma, mucosal, and gastric juice ascorbic acid and total vitamin C. They studied patients with normal and H. pylori-infected mucosa and followed H. pylori infected patients for up to 12 months 45 Review of literature after eradication of the organism. The MDA concentration in H. pylori-positive patients were higher [123.7 mol/g (93.5–157.2)] than H. pylori negative patients [82.5 mol/g (59.5–104.7)] (p <0.001). After successful eradication of H. pylori from 42 patients. MDA concentrations fell (from 134.9 to 106.2 mol/g; p = 0.007), and gastric juice ascorbic acid and total vitamin C both increased significantly 6 months after successful eradication of H. pylori (from 6.7 to 13.0 µg/ml for ascorbic acid; from 9.4 to 17.0 µg/ml for total vitamin C; p < 0.001 for both). No changes were seen in plasma or antral ascorbic acid or total vitamin C. Despite these potentially favorable changes, there was no change in M1-dG concentration 6 months after successful eradication of H. pylori. Thirteen evaluable patients who had received eradication therapy returned at 12 months, all of whom were H. pylori negative. There were no significant changes between 6 and 12 months for gastric juice, antral and plasma ascorbic acid, and total vitamin C or for MDA. Twenty-one patients received placebo eradication therapy. Data were available for 12 of these patients at 6 months and for 8 patients at 12 months, all of whom remained H. pylori positive. MDA levels did not change significantly between pretreatment and 6 months (from 95.9 to 103.6 mol/g; p = 0.5) but surprisingly, increased significantly by 12 months (from 95.9 mol/g at pretreatment to 178.7 mol/g at 12 months; p < 0.001).106 Patait M (2002) estimated serum MDA level in patients with oral cancer undergoing radiotherapy with and without antioxidant therapy. He found that the mean serum MDA level in the pre-radiotherapy study group was 0.598 ± 0.1609 µmol/L which was significantly higher than the control group 0.3084 ± 0.1016 µmol/L. The mean serum MDA level in the study group after radiotherapy without 46 Review of literature taking antioxidant was 0.792 ± 0.1157 µmol/L which was significantly higher as compared to pre-radiotherapy mean serum MDA level of 0.598 ± 0.1609 µmol/L. The mean serum MDA level in the study group after radiotherapy taking antioxidant therapy was 0.763 ± 0.0953 µmol/L which was significantly higher as compared to pre-radiotherapy mean serum MDA level of 0.596 ± 0.0952 µmol/L. No correlation in lipid peroxides level was observed between degree of differentiation of malignant lesion with or without antioxidant therapy. Thus he stated that the mean serum MDA level increases in oral squamous cell carcinoma patients as compared to the healthy individuals and MDA level further increases after radiotherapy.107 Ebubekir Bakan et al (2002) investigated the levels of malondialdehyde and total nitrite (NO2–) plus nitrate (NO3–) marker for nitric oxide (NO•) generation in gastric carcinoma and to correlate their levels with the cancer stage. The pretreatment plasma samples were obtained from 38 patients with gastric cancer (seven patients at stage II, 19 at stage III and 12 at stage IV). MDA was measured by the thiobarbituric acid method. The levels of plasma MDA, NO• and NO3– were significantly higher in patients with gastric cancer compared with the healthy control group. Higher levels of MDA, NO• and NO3– were observed as the stage of the disease increased. MDA level in control group (n = 24) was 5.4 ± 1, in stage II (n = 7) was 6.7 ± 1.0 (p< 0.01), in stage III (n = 19) was 7.7 ± 1.5 (p< 0.001), in stage IV (n = 12) was 7.8 ± 1.3 (p< 0.001) and total (n = 38) was 7.6 ± 1.4 (p < 0.001). Thus they found that increased NO• production and MDA levels were present in plasma of patients with gastric cancer. These increases can be associated with the oxidant–antioxidant status in these patients.108 47 Review of literature Soma Gupta et al (2004) estimated the levels of MDA and antioxidants in plasma and erythrocytes of 34 cases of oral submucous fibrosis. They found that plasma MDA level was increased in all grades of oral submucous fibrosis cases (3.3 + 0.4 mole/ml, p < 0.001) as compared to controls (2.4 + 0.5 mole/ml), plasma β carotene and vitamin E levels were decreased significantly in oral submucous fibrosis (81.7 + 14.3 µg/100 ml, p < 0.001; 9.3 + 0.9 mg/L, p < 0.01 respectively) with respect to healthy controls (110 + 20.8 µg/100 ml and 10.1 + 1.2 mg/L). The mean MDA level was more in OSMF grade II (3.5 + 0.8 mol/ml, p < 0.001) than in grade III (3.3 + 0.1 mol/ml, p < 0.001) and grade I (3.1 +0.8 mol/ml, p <0.001). The decrease in β-carotene and vitamin E was found to be more significant in OSMF grade II and III than in grade I. After 6 weeks of oral administration of beta-carotene and vitamin E, patients showed increase in plasma levels of these two antioxidants along with decrease in MDA level associated with clinical improvement.109 48 Methodology MATERIALS AND METHODS SOURCE OF DATA: Patients reporting to the Department of Oral Medicine and Radiology, KLE’S.V.K. Institute of Dental Sciences, Belgaum, with clinically diagnosed Oral submucous fibrosis were selected in the study after obtaining an informed consent. METHOD OF COLLECTION OF DATA: This study included two main groups: 1) Clinically diagnosed cases of OSMF (36) 2) Age and sex matched healthy control group (12) 3) Age and sex matched chewer control group (12) Diagnosis of OSMF was done on the basis of history and characteristic clinical features based on case history proforma designed for the same. OSMF was divided clinically into three stages as per the criteria described by Bailoor DN (1993). Age and sex matched controls with tobacco and arecanut chewing habit and without the habit were selected. After explaining about the study to the subjects, an informed consent was obtained as per the consent form attached. Subjects were grouped according to age. All cases and controls underwent detailed history, thorough oral examination and serum malondialdehyde estimation. SELECTION CRITERIA: Inclusion criteria: 1) 36 clinically diagnosed cases of Oral submucous fibrosis 12 patients with Grade I Oral submucous fibrosis (GROUP 1) 12 patients with Grade II Oral submucous fibrosis (GROUP 2) 49 Methodology 12 patients with Grade III Oral submucous fibrosis (GROUP 3) 2) 24 age & sex matched healthy individuals, 12 among them were chewers and 12 non chewers, without any oral & systemic diseases (CONTROL). 12 chewer controls (GROUP 4) 12 non-chewer controls (GROUP 5) Exclusion criteria: Patients with any systemic diseases/conditions which may alter serum malondialdehyde levels will be excluded from the study like Patients with chronic systemic diseases like Tuberculosis, AIDS, Diabetes etc. Patients with chronic renal failure and on hemodialysis Patients with carcinoma Patients on drug therapies such as antioxidants, anticonvulsants, broad spectrum antibiotics and systemic steroids. ARMAMENTARIUM: For clinical examination of the patient: - Dental chair with light source - Mouth mirror - Probe - Tweezer - Gauze - Gloves - Mouth mask - Scale - Divider 50 Methodology For the collection of blood sample: - Disposable syringe - Gauze - Antiseptic - Test tube - Tourniquet For separating serum and storage: - Pipette - Pipette tips - Centrifuging machine - Storage vial with a lid - Refrigerator For estimation of malondialdehyde: - Gloves - Test tubes - Test tube stand - Pipette - Boiling water bath - Centrifuging machine - Spectrophotometer METHOD FOR COLLECTION OF BLOOD SAMPLE: A detailed medical and dental history was recorded as per the case history proforma, clinical examination was carried out and clinical diagnosis of Oral submucous fibrosis 51 Methodology was made. Patients were categorized into 3 stages according to Nagesh and Bailoor classification. Later the purpose and procedure was explained to the patient and patient’s consent was obtained. 5 ml of blood was drawn by vena puncture from the OSMF patients and controls under aseptic precautionary measures. The subject was made to sit in a comfortable position, the patient’s arm was stretched and tourniquet was applied above the right cubital fossa. Around 5 ml of blood was withdrawn from the cubital vein under aseptic condition in a disposable syringe. The blood was then put into a 10 ml glass test tube. Then the test tube along with blood was subjected to centrifugation for 4-5 mins at 2500 rpm. Later the test tube was withdrawn from the centrifugation machine and with the help of a pipette serum was separated in a vial and stored in the refrigerator until it was transferred to the biochemical laboratory. The above procedure was repeated for all the patients. ESTIMATION OF MALONDIALDEHYDE (MDA) IN SERUM110 Principle: The reaction depends on the formation of pink colored complex between malonaldehyde and thiobarbituric acid (TBA) having an absorption of maximum at 532nm. Preparation of thiobarbituric acid reagent: - 75ml thiobarbituric acid, 15gm trichloroacetic acid, 2.08ml – 0.2N HCl All the above mentioned components were mixed and volume made upto 100ml with distilled water. 52 Methodology Procedure: TABLE 1: Blank (ml) Test (ml) Serum - 0.75 Distilled water 0.75 - Thiobarbituric acid reagent 3 3 - Keep the test tubes in boiling water bath for 15 minutes. - Cool, centrifuge for 10 minutes at 10,000 r.p.m. - Read absorbance of supernatants of blank and test immediately at 535nm. 0.75ml of serum was mixed with 3 ml of thiobarbituric acid in a test tube and the test tube was kept in a boiling water bath for 15 mins, then it was removed from the boiling water bath and was centrifuged for 10 mins at 10,000 rpm. The supernatant fluid was separated and is read under spectrophotometer at 535nm immediately. Calculation: Malondialdehyde (nano moles/ ml) = Absorbance of test X total volume Nanomolar extension of coefficient X sample volume X 100 = Absorbance of test X 3.75 1.56 X 105 X 0.75 X 100 = Absorbance of test X 3205 100 53 Methodology ARMAMENTARIUM USED Figure No. 10: EQUIPMENTS USED IN CLINICAL EXAMINATION OF THE PATIENTS Figure No. 11: REAGENTS USED FOR ESTIMATION OF SERUM MALONDIALDEHYDE 54 Methodology Figure No. 12: BOILING WATER BATH Figure No. 13: CENTRIFUGE MACHINE 55 Methodology Figure No. 14: SPECTROPHOTOMETER Figure No. 15: BLANCHING ON LABIAL MUCOSA 56 Methodology Figure No. 16: BLANCHING ON BUCCAL MUCOSA Figure No. 18: REDUCED MOUTH OPENING Figure No. 17: SHRUNKEN UVULA Figure No. 18: REDUCED TONGUE PROTRUSION 57 Results & Observation RESULTS AND OBSERVATION A total of 60 patients were included in the study. The study group comprised of all male patients. The age of the patients varied between 18 years to 58 years. The patients were divided into five groups with 12 patients in each group. There were 36 OSMF patients who were divided into three groups Grade I, Grade II and Grade III. There were 24 controls who were divided under two groups i.e. tobacco chewers without OSMF or any other premalignant lesion/condition and healthy controls without chewing habit. Table 2: Mean value for age among various groups Level Minimum Median Maximum Mean SD Chewer controls 21 31.5 48 32.0833 9.3172 Healthycontrols 22 34 45 33.6667 8.49996 OSMF stage I 25 32 40 32.6667 4.6775 OSMF stage II 18 26.5 58 30.5000 10.7153 OSMF stage III 22 32.5 46 32.3333 7.8547 The above table shows that mean value of age among the five groups are homogenous Conclusion: the age distributions are very similar (p-value for comparing the mean ages = 0.9235). Since they are all males, the groups are gender and age matched. 58 Results & Observation Table 3: ANOVA test for ages among various groups One-way analysis of variance was used to compare the means of the ages of the five groups. Source DF Sum of Mean Square F Ratio Prob > F Inf. Squares Group 4 63.3333 15.8333 0.2215 0.9253 NS Error 55 3931.9167 71.4894 - - - C. Total 59 3995.2500 - - - - From the above table of one way ANOVA the ages in the five groups are homogenous Table 4: Mean value of MDA among various groups Means and SD: Level Number Mean SD Std Err Mean Chewer controls 12 5.7250 0.90174 0.26031 Healthy controls 12 4.4017 1.65257 0.47706 OSMF Stage I 12 10.8458 1.21897 0.35189 OSMF Stage II 12 14.1708 1.11169 0.32092 OSMF Stage III 12 14.9817 1.40018 0.40420 59 Results & Observation The above table shows that highest mean serum MDA level observed in group three followed by group 2, group 1, chewer controls and healthy controls. For healthy controls the mean value is 4.4017. Table 5: ANOVA test for MDA among various groups One way analysis of variance was used to test the equality of the mean MDA values for the five groups and there was a significant difference (p<0.0001). Then Tukey-Kamer test was used at a level of significance of 0.05 to simultaneously test the equality of any two pairs of groups. The conclusion is that there is no significant difference between Stage III and II (group A), Stage I is significantly different from the rest (group B), and there is no significant difference between the healthy and chewer controls (group C). See the table below with highlighted p-values for the pair wise comparisons. There is also a table below that gives means, standard deviations, and standard errors for each of the groups. There is also a 95% confidence interval for the mean in that table. Source DF Sum of Squares Mean Square F Ratio Prob > F Inf. Group 4 1110.4972 277.624 168.7404 <.0001* S Error 55 90.4901 1.645 - - - C. Total 59 1200.9873 - - - - 60 Results & Observation The p-value in the above table indicates non homogenicity in the means of five groups. To make pair-wise comparisons Tukey-Kramer Test was conducted and following table is obtained. Table 6: Mean comparisons Comparisons for all pairs using Tukey - Kamer HSD Level Mean OSMF Stage III A 14.981667 OSMF Stage II A 14.170750 OSMF Stage I B 10.845833 Chewer controls C 5.725000 Healthy controls C 4.401667 Levels not connected by same letter are significantly different. 61 Results & Observation TABLE 7: Multiple comparison of serum MDA among various groups using Tukey-Kramer test Level - Level Difference Std Err Dif p-Value Inf. OSMF Stage III Healthy controls 10.58000 0.5236530 <.0001* S OSMF Stage II Healthy controls 9.76908 0.5236530 <.0001* S OSMF Stage III Chewer controls 9.25667 0.5236530 <.0001* S OSMF Stage II Chewer controls 8.44575 0.5236530 <.0001* S OSMF Stage I Healthy controls 6.44417 0.5236530 <.0001* S OSMF Stage I Chewer controls 5.12083 0.5236530 <.0001* S OSMF Stage III OSMF Stage I 4.13583 0.5236530 <.0001* S OSMF Stage II OSMF Stage I 3.32492 0.5236530 <.0001* S Chewer controls Healthy controls 1.32333 0.5236530 0.0993 NS OSMF Stage III OSMF Stage II 0.81092 0.5236530 0.5361 NS 62 Results & Observation The above table shows that in all the pair wise comparison there is statistically significant difference in all except last two rows that is between chewer control and healthy controls, between Stage II and Stage III. Graph 1: Number of patients among various groups 14 No. of patients 12 12 12 12 12 12 10 8 6 Series 1 4 2 0 Grade I Grade II Grade III Chewer Cnt Healthy Cnt Study Groups Graph 2: Distribution of patients in study groups in different age 30 24 25 No. of patients 20 15 18 15 Series 1 10 5 2 1 0 16-25 26-35 36-45 46-55 56-65 Age in years 63 Results & Observation Graph 3: Mean age among patients of different study groups 34 33.667 33.5 33 32.667 32.333 Mean age 32.5 32 32.003 31.5 31 30.5 30.5 Series 1 30 29.5 29 28.5 Grade I Grade II Grade III Healthy controls Chewer cintrols Study Groups Graph 4: Mean serum MDA levels among various study groups 16 14.1708 14.9817 14 Mean MDA levels 12 10.4858 10 8 6 5.725 Column1 4.4017 4 2 0 Healthy Cnt Chewer Cnt Grade I Grade II Grade III Study groups 64 Results & Observation Graph 5: One way analysis of MDA by group The above graph shows the equality of the mean MDA values in five groups which was tested using ANOVA test. It also shows the degree of equality among any two pairs of the groups which is denoted by circles. IMPRESSION: At the end of the study it was observed that there was significant rise in serum MDA levels in patients with Oral submucous fibrosis with a maximum increase in Stage III compared to that of control group. 65 Discussion DISCUSSION Oral submucous fibrosis is a chronic irreversible disease, etiology of which is poorly understood. It is mainly found in Indian population and other South East Asian countries like Sri Lanka, Pakistan, Bangladesh and Nepal. OSMF affects about 0.2 1.2% of Indian population attending dental clinics. OSMF is also seen in Indians living in other countries like Kenya, Malaysia, Uganda, South Africa, Fiji and UK. Cases have also been reported from ethnic groups in Taiwan, Thailand and Vietnam. The disease is predominant in males in their second and third decades of life. The disease leads to fibroelastic transformation of the lamina propria and epithelial atrophy of the oral mucosa. Later the mucous membrane becomes stiff leading to trismus. Although the etiology is not known it has been postulated that the disease is caused by an irritation of the oral submucosa by irritants such as tannins in betel nut and capsaicin in chillies. Immunological, genetic and environmental factors have also been thought to be contributing factors for the disease. Clinical research in the area of lipid peroxidation has been hampered by the lack of a valid biomarker. One of the most frequently used biomarkers providing an indication of the overall lipid peroxidation level is the concentration of malondialdehyde, one of the several byproducts of lipid peroxidation processes.16 Malondialdehyde is a naturally occurring endogenous product of lipid peroxidation and prostaglandin biosynthesis, but is mutagenic and tumorigenic. The MDA value in blood is a measure for the ability of the body to handle the oxidative stress it is exposed to. Disturbance of balance between oxidative processes and antioxidative defenses causes the oxidative stress that can damage proteins, lipids, polysaccharides and nucleic acids. The most frequently used test is the measurement of MDA by the 66 Discussion TBA reaction. Here, TBA reacts with MDA to form TBA: MDA adduct, which absorbs maximally at 535 nm. In this study this coloured complex was measured by photospectrometer. This method is of particular interest because of its procedural simplicity and nanomolar sensitivity. This study included 36 subjects with oral submucous fibrosis and 24 subjects without OSMF as controls. In the OSMF group the largest number of patients belonged to the age group of 20-45. This finding correlates with the report of Chiang CP et al (2002)111, Anuradha CD et al (1993)112 and Gupta S et al (2004).113 The mean age in our study was 32.3 years which was similar to Ranganathan K et al (2004)114 who reported a mean age of 32.4 years. Most of the patients in our study were in the second and third decade of life with 15 patients (25%) in the age group 16-25 years, 24 patients (40%) in the age group of 26-35 years, 18 patients (30%) belonged to the age group of 36-45 years and 3 patients (5%) were above 46 years. This is in agreement with Chaturvedi VN et al (1991)115 and Haider SM et al (2000).47 The higher prevalence of OSMF patients in younger age group is explained by popularity of refined areca nut products, which are readily available and introduction of betel nut chewing habit at an early age. OSMF is seen to be more prevalent among males as per the observation of Kiran K et al (2007)116, Hazare VK et al (2007)117, Chaturvedi VN and Marathe NG (1988)118, Shah N et al (1994)40 and Ranganathan K et al (1004).114 The sex incidence ratio varies from study to study due to varying habits in various populations. As the habit of chewing betel nut is less prevalent among females in and around Belgaum city, only male patients with OSMF were included in our study to maintain homogeneity with respect to gender. 67 Discussion All the patients in our study had atleast one of the arecanut based chewing habits. Similar observations were made by Shear et al (1967)119, Van Wyk CW et al (1990)120. Arecanut chewing is identified as the most important etiological factor. Some researchers noticed OSMF in patients without any oral habits, such as 0.12% reported by Gupta S (2004)109 and 1.81% reported by Shah N (1998)121 but this was not consistent with our study. This suggests that betel nut chewing could contribute to the development of oral cancer, but requires a longer period of time to produce the changes in the oral epithelium. Whereas subjects who chewed tobacco with betel leaf and betel nut developed carcinoma within a shorter time span, indicating that tobacco and betel leaf/betel nut together can induce malignant changes earlier than betel nut alone. Betel leaf and tobacco quid not only irritates the oral mucosa mechanically, but the chemical irritation (nicotine and arecoline predominantly) is probably much greater because of the higher concentration of the chemicals kept for prolonged periods at one site. According to M.C. Downer122, only heavy smoking (> 20 cigarettes per day) produced a significant odds ratio. One patient who had habit of alcohol (wine/whisky) intake along with tobacco, betel nut and cigarette developed precancerous changes in oral mucosa followed by oral cancer within a shorter period of time (8 years) and also showed high serum MDA level (43.60 nmol/ml). Literature reveals that the process of carcinogenesis occurs by generation of reactive oxygen species, which act by initiating lipid peroxidation. The present study has revealed an intriguing aspect of tumor biochemistry. This study showed significant differences between serum MDA levels in oral submucous fibrosis [14.1708 (± 0.32092) nmol/ml], chewer controls [5.7250 (± 0.26031) nmol/ml] and 68 Discussion healthy controls [4.4017 (±0.47706) nmol/ml] (p < 0.01). Serum MDA increased in oral submucous fibrosis and chewer controls as compared to normal healthy individuals. In cancers other than oral cancer, MDA levels showed marked increase.104, 107 According to Unal Sahin et al33 MDA levels are increased significantly in lung cancer (20.5 ± 7.9 nmol/ml) as compared to healthy controls (12.6 ± 7.1 nmol/ml) (p < 0.001). Ebubekir Bakan et al108 stated that the levels of plasma MDA were significantly higher in patients with gastric cancer (7.6 ± 1.4 nmol/ml) as compared to the healthy controls (5.4 ± 1 nmol/ml). In a study on cervical carcinoma by G. Gitanjali et al102, there was no statistically significant difference in the mean serum MDA levels of healthy controls (3.40 ± 0.75 nmol/ml) and patients with cervical carcinoma (3.54 ± 1.89 nmol/ml). Other than precancer and cancer, literature reported increased MDA levels in chronic obstructive pulmonary disease, H. pylori-infected gastric mucosa35, during cardiopulmonary bypass123 and during hemodialysis.105, 97 Though radiotherapy is one of the clinical means by which oral cancer can be treated, many biochemical complications, such as damage to cellular DNA and membrane structures can occur. Both Sabitha K. E. et al102 and Patiat M108 showed significant increase in MDA levels in untreated and radiation treated oral cancer patients when compared with normal subjects. Radiation treated patients had higher MDA levels (0.792 ± 0.1157 nmol/ml) than untreated patients (0.598 ± 0.169 nmol/ml) (p < 0.001). According to Soma et al38 MDA levels in oral sub mucous fibrosis (3.3 + 0.4 nmol/ml) were more as compared to healthy controls (2.4 + 0.5) (p < 0.001). In our 69 Discussion study serum MDA levels was found to be increased in oral submucous fibrosis [14.1708 (± 0.32092) nmol/ml] as compared to normal individuals [4.4017 (±0.47706) nmol/ml] and the increase was statistically significant (p < 0.01). This shows a possibility of the role of reactive oxygen species in etiopathogenesis of oral submucous fibrosis. An attempt was made to study the level of oxidative stress in different stages of OSMF. In our study serum MDA levels in stage I OSMF was [10.8458 (± 0.35189) nmol/ml], MDA levels in stage II OSMF was found to be [14.1708 (± 0.32092) nmol/ml] and in stage III OSMF it was [14.9817 (± 0.40420) nmol/ml]. This shows that the oxidative stress increases as the disease progresses reflecting the amount of tissue damage, our study results also showed statistically significant difference in MDA level of grade I and stage III OSMF (p <0.0001)as well as stage I and stage II OSMF (p <0.0001) but there was no statistical significance between stage II and stage III disease (p =0.5361), which is in correlation with the study conducted by Soma Gupta et al (2004) where plasma MDA levels were found to be increased in all stages of OSMF (mean = 3.3 + 0.4 nmol/ml) compared to healthy controls (mean= 2.4 +0.5 nmol/ml) and the increase was statistically significant (p <0.001). It is established that the lipid peroxidation increases with severity of the disease reflecting the extent of the tissue injury. Their results also showed that mean MDA levels were more in OSMF stage II than in stage I (3.1 + 0.8 nmol/ml). However it did not vary much between grade II(3.5 + 0.8 nmol/ml) and grade III (3.3 + 0.1 nmol/ml). Further attempt was made to study the association of lipid peroxidation and the habit of either betel nut or betel leaf chewing, or tobacco chewing, in normal subjects. In the present study 12 controls had betel nut or tobacco chewing habit 70 Discussion without OSMF and 12 were healthy controls without chewing habit. The MDA levels were increased in chewer controls [5.7250 (± 0.26031) nmol/lm] as compared to the healthy nonchewer controls [4.4017 (± 0.47706) nmol/ml] though the increase was not statistically significant (p = 0.0993). Cigarette smoke, in both the gaseous phase and condensed particles, contains alkenes, nitrosamines, aromatic and heterocyclic hydrocarbons and amines. In addition, it is an excellent source of ROS, such as hydroxyl radicals, superoxides and peroxides that are capable of initiating or promoting oxidative damage. Unal Sahin et al104 has reported increased MDA levels in smokers (22.5 ± 8.0 nmol/ml) than non smokers (16.1 ± 6.7 nmol/ml) (p < 0.001) with lung cancer. Abraham Z. Reznick et al124 elucidated the outcome of interaction of between cigarette smoke and oral salivary peroxidase in smokers and nonsmokers. After smoking a single cigarette, a sharp drop of oral peroxidase activity was observed in both groups: 42.5% in smokers and 58.5% in nonsmokers (p < 0.05). After 30 minutes, the level of activity returned to 90 - 100% of the pre smoking level, presumably due to the secretion of new saliva into the oral cavity. The oral peroxidase activity loss was accompanied by increased carbonylation of the salivary proteins, an indicator of the oxidative damage to proteins. According to Flemming Nielsen et al99 daily smokers had a slightly higher average concentration of plasma MDA than nonsmokers, and plasma MDA correlated with daily exposure to cigarette smoke. Jason D. Morrow125 proved that Plasma levels of lipid peroxidation product (F2-isoprostanes) were significantly higher in the smokers as compared to the nonsmokers. One study by Wendy Y. Craig et al126 was in contrast with the above studies, which stated that there was no strong relation between lipid peroxide level (determined by TBARS level) and cigarette smoke exposure, suggesting that certain interactions related to oxidation status are not measurable in the 71 Discussion serum compartment. They further stated that serum copper was the major determinant of serum lipid peroxidation status, indicating that it contributes to lipid peroxidation in vivo. Flemming Nielsen et al99 demonstrated a positive correlation between plasma MDA and alcohol consumption. No major interaction occurred between gender and age. Men had slightly but significantly higher plasma MDA concentrations than women. Reaction of the ROS with cellular DNA results in oxidative damage, including a number of oxidized bases, single-strand breaks and/or alkali-labile lesion. These types of DNA damage are considered to be crucial in cancer development, thus providing an additional possible mechanism for an apparent association between tobacco chewing, smoking and mouth, lung, pharynx, esophagus, bladder and cervical cancers. Laura J. Niedernhofer et al127 proved that MDA-induced DNA damage is mutagenic in human cells. Yujing Zhang et al128 showed that MDA–DNA adducts may serve a biomarker of DNA damage by lipid peroxidation induced endogenously or exogenously in oral mucosal cells. Chiara Leuratti et al129 showed that malondialdehyde - deoxyguanosine (M1-dG), a DNA adduct derived from lipid peroxidation plays a role in human colorectal carcinogenesis, in combination with other genetic and environmental factors. Within-subjects and day-to-day variations of serum MDA levels indicated that further studies have to be carried out with larger sample size. However, on a group basis, the present data support that serum MDA levels may be a potential biomarker for oxidative stress. Still much work is required to study the role of reactive oxygen 72 Discussion species and lipid peroxidation in oral precancer and cancer. Studies should be undertaken in more number of subjects to compare age and sex related differences. 73 Conclusion CONCLUSION The present study showed significant increase in the serum malondialdehyde levels, which is a reliable marker and product of lipid peroxidation and this was increased in all grades of Oral submucous fibrosis and the maximum being in Grade III. The levels of serum malondialdehyde in healthy controls and chewer controls were significantly low. There was no statistically significant difference between the serum MDA levels between healthy controls and chewer controls. This increase in the serum MDA levels is indicative of increased damage to the tissues caused by lipid peroxidation due to free radical; it reflects the increased oxidative stress in the tissues. To conclude the present study suggests a possibility of ROS playing a part in aetiopathogenesis of the disease. Exogenous supplementation of non-enzymatic antioxidants may decrease the damage to oral mucosa by quenching and preventing the free radical action which are responsible for the disease process and malignant conversion. 74 Summary SUMMARY The present study was conducted to estimate serum lipid peroxidation product malondialdehyde levels in 36 patients with OSMF and 24 age and sex matched chewer and non-chewer controls without OSMF. Alterations in the serum malondialdehyde levels were assessed using conventional thiobarbituric acid method, using a photospectrometer and reading are calculated. The statistical analysis was done using Tukey Kamer test and ANOVA test. The result of our study depicted the following outcome. There was a significant rise in the serum malondialdehyde levels in patients with OSMF and controls who were arecanut chewers. The maximum rise of serum malondialdehyde levels were seen in Grade III OSMF, followed by Grade II, Grade I, chewer controls and finally the healthy controls. There was no significant difference in serum malondialdehyde levels among chewer controls and healthy controls and also between Grade II and Grade III OSMF. From the present study it is evident that lipid peroxidation product malondialdehyde level rises in patients with Oral submucous fibrosis. Malondialdehyde is an indicator of oxidative stress which in turn indicates increased damage caused by ROS. 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Carcinogenesis 2002; 23(1): 207-211. 129. Leuratti C, Watson MA, Deag EJ, Welch A, Singh R, Gottschalg E, Marnett LJ, Atkin W, Day NE, Shuker DG and Bingham SA. Detection of malondialdehyde DNA adducts in human colorectal mucosa - Relationship with diet and the presence of adenomas. Cancer Epidemiol Biomarkers Prev 2002; 11: 267-273. 88 Bibliography 130. Soma Gupta, M.V.R. Reddy and B.C Harinath. Role of oxidative stress & antioxidants in aetiopathogenesis & management of oral submucous fibrosis. Indian Journal of Clinical Biochemistry 2000; 19(1): 133-141. 89 Annexure ANNEXURE – I DEPARTMENT OF ORAL MEDICINE AND RADIOLOGY, K.L.E.’S VISHWANATH KATTI INSTITUTE OF DENTAL SCIENCES NEHRU NAGAR, BELGAUM. “COMPARISON OF SERUM MALONDIALDEHYDE LEVELS IN PATIENTS WITH ORAL SUBMUCOUSFIBROSIS AND CONTROL GROUP- A HOSPITAL BASED STUDY”. CASE HISTORY PROFORMA Patient’s Name: Case no: Age: Date: Sex: Occupation: Address: Chief complaint: History of present illness: Past History: - Medical History: - Dental History: Personal history: Habits: Guthka Tobacco Betel quid Cigarette smoking Beedi smoking Duration Frequency 90 Annexure Extra oral examination: TMJ: Lymphnodes: Intra oral examination: Hard tissue examination: Soft tissue examination: Buccal mucosa Palate Tongue Floor of mouth Lips uvula Mouth opening Tongue protrusion Provisional Diagnosis: Investigations: Treatment plan: 91 Annexure ANNEXURE – II CONSENT FORM DEPARTMENT OF ORAL MEDICINE AND RADIOLOGY, INSTITUTE OF DENTAL SCIENCES, BELGAUM “COMPARISON OF SERUM MALONDIALDEHYDE LEVELS IN PATIENTS WITH ORAL SUBMUCOUSFIBROSIS AND CONTROL GROUP- A HOSPITAL BASED STUDY”. I, _________________________________ aged__________ years has been informed about my involvement in the study. 1. I agree to give my personal details like Name Age, Sex, Address, Past Dental and Medical History and any other details required for the study to the best of my knowledge. 2. I will cooperate with the dentist for examination and also for various investigations. 3. I permit the operator to utilize the information given by me and the results obtained from this study for presentation and publication. 4. I permit the dentist to take my photographs, collect blood to utilize it for the study and presentation purpose. 5. I am participating in this study with my own wish and will and the dentist has explained the nature and the effect of the procedure in my vernacular language. Name of the doctor: Name of the patient: Name of the witness: Signature of Patient: Signature of witness: Date: Date: 92 Annexure ANNEXURE – III MASTER CHART – CASES CASES: OSMF STAGE I Sl. No. NAME AGE SEX Hb% MDA levels 1 Vital Gundu Yonus Vinod Adivappa Mumtaz Amrut Gangadhar Basalinga Ravsaheb Jyothiba harishchandra 37yrs 40yrs 29yrs 25yrs 30yrs 32yrs 26yrs 35yrs 32yrs 32yrs 37yrs 37yrs M M M M M M M M M M M M 11.47gm% 11.08gm% 10.69% 9.66gm% 12.30gm% 9.68gm% 7.31gm% 12.3gm% 9.40gm% 12.12gm% 9.68gm% 12.30gm% 10.96 10.86 11.43 8.67 10.93 12.64 9.93 9.96 10.16 12.13 12.65 9.83 2 3 4 5 6 7 8 9 10 11 12 CASES: OSMF STAGE II Sl. No. NAME AGE SEX Hb% MDA levels 1 Somanath M Ramappa Pramod Imthiyaz ajay H Immansaab Abhishek Thosif Akram Elig Liaquat Shantinath 23yrs 58yrs 25yrs 29yrs 24yrs 26yrs 18yrs 27yrs 26yrs 31yrs 38yrs 41yrs M M M M M M M M M M M M 11.3gm% 10.23gm% 9.32gm% 10.16gm% 10.28gm% 10.61gm% 9.69gm% 11.11gm% 9.68gm% 9.71gm% 9.70gm% 9.61gm% 14.129 2 3 4 5 6 7 8 9 10 11 12 13.98 12.81 14.62 12.13 14.64 13.62 15.67 13.62 16.15 13.96 14.97 93 Annexure CASES: OSMF STAGE III Sl. No. NAME AGE SEX Hb% MDA levels 1 Shrinivas Mahesh Anarnath Shafaj Gangappa Ravi Rphan Krishna Gajanan Issrani Arif Sharrieff 29yrs 36yrs 22yrs 32yrs 35yrs 44yrs 46yrs 28yrs 33yrs 37yrs 24yrs 22yrs M M M M M M M M M M M M 10.93gm% 11.42gm% 9.35gm% 9.17gm% 10.26gm% 10.24gm% 10.10gm% 9.12gm% 7.69gm% 9.36gm% 9.24gm% 8.36gm% 15.28 13.39 14.33 18.21 16.23 14.31 15.21 13.16 14.45 13.93 15.16 16.12 2 3 4 5 6 7 8 9 10 11 12 94 Annexure ANNEXURE –I V MASTER CHART – CONTROLS HEALTHY CONTROLS Sl. No. NAME AGE SEX Hb% MDA levels 1 Yusuf Praveen Jyotjiba mahaveer Krishna Basappa Nagappa Deepak Gopal ganpathi Ramesh Jagendra 22YRS 24yrs 32yrs 36yrs 41yrs 45yrs 25yrs 32yrs 36yrs 24yrs 45yrs 42yrs M M M M M M M M M M M M 12.37gm% 12.31gm% 13.06gm% 13.12gm% 12.40gm% 10.12gm% 12.06gm% 11.62gm% 12.13gm% 13.48gm% 12.43gm% 12.33gm% 5.89 3.85 4.49 3.95 3.39 3.86 2.56 4.32 2.68 4.48 4.52 8.83 2 3 4 5 6 7 8 9 10 11 12 CHEWER CONTROLS Sl. No. NAME AGE SEX Hb% MDA levels 1 mallappa Dattu Naziyak shubhalaxman Vihaba padmaraj Santosh Yash raja K S uttam A mallappa Manjith 22yrs 31yrs 26yrs 41yrs 48yrs 21yrs 26yrs 35yrs 37yrs 21yrs 45yrs 32yrs M M M M M M M M M M M M 10.36gm% 10.38gm% 11.14gm% 12.36gm% 13.64gm% 12.28gm% 12.23gm% 15.16gm% 12.24gm% 12.11gm% 11.22gm% 13.06gm% 5.12 6.11 6.11 6.25 6.12 5.51 4.5 4.43 6.91 6.93 6.22 4.49 2 3 4 5 6 7 8 9 10 11 12 95
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