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DISSERTATION – SYNOPSIS Dr. SMIT SINGLA Post graduate student Department of Oral Medicine and Radiology A .J. INSTITUTE OF DENTAL SCIENCES, MANGALORE 2009 – 2010 Rajiv Gandhi University of Health Sciences, Karnataka. Bangalore ANNEXURE II PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION Dr. SMIT SINGLA 1. Name of the Candidates and POST GRADUATE STUDENT Address DEPARTMENT OF ORAL MEDICINE AND RADIOLOGY A.J. INSTITUTE OF DENTAL SCIENCES, MANGALORE 2. Name of the institution 3. Course of study and subject 4. Date of admission to course A.J. INSTITUTE OF DENTAL SCIENCES, MANGALORE Master of Dental Surgery ORAL MEDICINE AND RADIOLOGY 20 May 2009 Title of the topic: 5. MICRONUCLEUS ASSAY – AN EARLY DIAGNOSTIC TOOL TO ASSESS GENOTOXIC CHANGES IN TOBACCO AND RELATED HABITS BRIEF RESUME OF THE INTENDED WORK 6. 6.1 Need for the study Oral cancer is by far the most common oral mucosal malignant disease. Though the diagnosis of oral cancer seldom presents difficulty, it is the early detection, cancer staging and histological grading that are more important for prognosis. As documented the phenotypic changes of oral cancer is always preceded by genetic damages accumulated due to various carcinogens. It is the need of the hour, to detect these genetic fingerprinting at the earliest, even before dysplastic changes is attributed to the cell. Among the various non-invasive early detection tools, the use of Micronuclei (MN) as a marker for the above effect has been well documented and has a sensitivity and specificity of 94% and 100% respectively1. Micronuclei are induced in cells by a variety of substances, like ultraviolet radiation, infrared rays, X-radiations, and chemicals. Among them tobacco- specific nitrosamines have been reported to be potent mutagenic agents which are thought to be responsible for the induction of chromosomal aberrations resulting in production of micronuclei. As documented the effects of smokeless tobacco and tobacco smoking is varied. Hence an appropriate tool for early detection of tobacco related genotoxic effects on a cell even before phenotypic changes of cancer are evident1. Micronuclei are extranuclear cytoplasmic bodies, caused due to double strand chromosomal aberrations and detected through appropriate staining procedure. These damaged chromosomes, in the form of acentric chromatids or chromosome fragments, lag behind in anaphase when centric elements move towards the spindle poles. The lagging elements are included in the daughter cells too, but a considerable portion is transformed into one or several secondary nuclei, which are as a rule, much smaller than the principal nucleus and are therefore called micronuclei2, 3. The detection of such micronuclei can be done in various types of tissues from the body (RBC, reticulocytes, lymphocytes and exfoliated epithelial cells). The exfoliated cell Micronucleus assay has an advantage over the widely used Micronucleus test in lymphocytes because, an epithelial cell unlike a lymphocyte, do not need to be stimulated to undergo mitosis since they are a constantly dividing pool of cells4. The use of buccal exfoliated cells in the detection of genotoxic effects of tobacco and related products can be used as a mass screening procedure for the early detection of dysplastic changes and oral cancer. 6.2 REVIEW OF LITERATURE : A. Paige E. Tolbert, Carl M. Shy and James W. Allen (1991)4 established a revised protocol for the exfoliated cell micronucleus assay was field-tested in a population exposed to a genotoxic agent, snuff, at level associated with a significant increase in cancer risk. The standard assay involves examination of epithelial smears to determine the prevalence of micronucleated cells, an indication of chromosome breakage or mitotic interference. B. Paige E. Tolbert, Carl M. Shy and James W. Allen(1992)3 evaluated the exfoliated cell micronucleus assay involving microscopic analysis of epithelial smears to determine the prevalence of micronucleation, an indicator of structural or numerical chromosome aberrations. Refinement in micronucleus scoring criteria and the inclusion of other nuclear anomalies in the scoring system were proposed. C. Devendra Devendre H Palve, Jagdish V Tupkari.(2008)2 investigated that micronuclei were good prognostic indicators in oral Squamous cell carcinoma. Micronucleus frequencies in oral exfoliated cells stained with papanicolaou stain were counted and correlated with the histopathological grades and clinical stages of Squamous cell carcinoma patients. They were also compared with healthy control subjects. Micronuclei (MN) frequencies were also found higher in Squamous cell carcinoma patients than in control subjects. MN frequencies were also found to be raised with increasing histological grades of Squamous cell carcinoma. D. Sudha Sellappa, Mythili Balakrishnan, Sangeetha Raman and Subashini (2009)1 evaluation of micronuclei in buccal mucosa of healthy individuals from southern India, who were regularly chewing a mixture of betel leaf, areca nut and tobacco. The mean percentage of MN was 1.90±1.03 in chewers, 2.00±1.12 in chewers with smoking habits and 0.81±0.66 in controls. There was no significant difference between the mean percentages of the two experimental groups. It can be concluded that a mixture of betel leaf, areca nut, and tobacco is unsafe for oral health. E. Beena P. Patel, Pina J. Trivedi, Manisha M. Brahmbhatt, Shilin N. Shukla, Pankaj M. Shah and Sonal R. Bakshi (2009)5 analysed the tobacco related genotoxic effects in chewers monitoring micronuclei (MN) and chromosomal aberrations. The biomarker was compared with non chewer to predict the risk of genotoxicity. . 6.3 OBJECTIVES OF THE STUDY: 1. To determine the specificity and sensitivity and consequent early detection of dysplastic changes through MN assay in tobacco and related habits. 2. To compare MN frequency among subjects, chewing tobacco only, chewing and smoking tobacco only, and chewing, smoking with alcohol, and to co-relate with control subjects. 7 MATERIAL AND METHODS : Evaluation of micronucleated cell from buccal exfoliated cell using Acridine Orange fluorescent Staining. 7.1 SOURCE OF SAMPLE : Buccal smear will be made after obtaining a suitable consent form healthy patients who are visiting the department of Oral Medicine and Radiology, A.J. Institute of Dental Sciences, having habits of smoking, chewing tobacco and alcohol and those without any habit. CLINICAL INCLUSION CRITERIA: 1. Patient should be healthy without any clinical lesion in oral cavity. 2. Patient must have history of chewing, smoking tobacco and alcoholism. INCLUSION CRITERIA FOR TOTAL CELL COUNT: 1. No debris. 2. No overlap with adjacent cells. 3. Cytoplasm intact and lying relatively flat. 4. Nucleus normal and intact with nuclear perimeter smooth and distinct. EXCLUSION CRITERIA: 1. Under or followed vitamins and antioxidants supplementation. 2. Under or followed radiation therapy. 7.2 METHODS OF COLLECTION OF DATA METHODOLOGY: Patient selection The sample will be selected from the healthy patients visiting to the Department of Oral Medicine and Radiology. A sought of information on inclusion and exclusion criteria obtained. Subjects will be studied in the following groups with 20 samples in each group. Group 1: chewing tobacco only. Group 2: chewing and smoking. Group 3: chewing, smoking and alcohol. Group 4: control. Collection of exfoliated cells Subjects will be asked to rinse their mouth gently with water. Mucosal cells will be scraped from buccal mucosa using a slightly moistened wooden spatula. The cells will be immediately smeared on pre-cleaned microscopic slides.3, 5 Just prior to drying; the smears were fixed with commercially available spray fixative (available as BIOFIX). Then slides will be coded to ensure observer blindness4 and will be fixed in 100% alcohol. Staining procedure ACRIDINE ORANGE SOLUTION This technique requires a pH of 6.0 for the differential staining of RNA and DNA. Formalin- fixed material does not stain satisfactorily, neither does tissue fixed in Bouin’s solution; alcohol is the fixative of choice6. 0.1% aqueous acridine orange. Before use dilute one part stains with 10 part of pH 6.0 0.06M phosphate buffer to give a 0.01% solution. BUFFER pH 6.0 phosphate buffer7. DIFFERENTIATOR 0.1 M calcium chloride ( 11.099g calcium chloride in 100 ml distilled water)6. Technique 1. Take alcohol-fixed smear to distilled water. 2. Rinse in 1% acetic acid for a few seconds and in two changes of distilled water over 1 minute. 3. Stain in the diluted acridine orange solution at pH 6.0 for 3 minutes. 4. Rinse in pH 6.0 buffer for 1 minutes. 5. Differentiate in the 0.1 M calcium chloride solution for ½-1 minute. 6. Wash in phosphate buffer and mount in the same. DNA will be stained in yellow- green. RNA, some mucins will be stained in red6. INTERPRETATION OF RESULTS: 100 cells from each sample will be focused under fluorescent microscope and number of Micronucleated cells (MNC) will be counted by a single observer. 7.3 Does the study requires any investigations or interventions to be conducted on patients or other humans or animals ? If So, please describe briefly. Not applicable 7.4 Has ethical clearance been obtained from your institution in case of 7.3 ? Obtained INVESTIGATION DESIGN 80 Samples DOUBLE BLIND STUDY 20 chewing tobacco 20 chewing & smoking 20 Chewing, smoking & alcohol Buccal exfoliated cells Acridine orange staining EXAMINATION UNDER FLUORESCENT MICROSCOPE Results 20 Control group 8. LIST OF REFERENCES : 1. Sudha Sellappa, Mythili Balakrishnan, Sangeetha Raman and Subashini. Induction of micronuclei in buccal mucosa on chewing a mixture of betel leaf, areca nut and tobacco. J. Oral Science, 2009; 51(2):289-292. 2. Devendre H Palve, Jagdish V Tupkari. Clinico-pathological correlation of micronuclei in oral Squamous cell carcinoma by exfoliative cytology. J. Oral Maxillofac Pathol, 2008; 12:2-7. 3. Paige E. Tolbert, Carl M. Shy and James W. Allen. Micronuclei and other nuclear anomalies in buccal smears: methods development. Mutation Research, 1992; 271:69-77. 4. E. Tolbert, Carl M. Shy and James W. Allen. Micronuclei and other nuclear Paige anomalies in buccal smears: A field test in snuff users. American J Epidemiology 1991; 134(8): 840-850. 5. Beena P. Patel, Pina J. Trivedi, Manisha M. Brahmbhatt, Shilin N. Shukla, Pankaj M. Shah and Sonal R. Bakshi. Micronuclei and Chromosomal aberrations in healthy tobacco chewers and control: A study from Gujarat, India. Arch Oncol, 2009; 17(12): 7-10. 6. Handbook of Histopathological and Histochemical Techniques (1974). Culling, C.F.A. 3rd Edn. London; Buttterworths. (Page no. - 30) 7. Practical Heamatology (2001).S M Lewis, B J Bain and I Bates 9th Edn. Churchill, Livingstone.(Page no.- 606) 9. Signature of Candidate 10. Remarks of the guide : Dr. VATHSALA Name & Designation of 11. PROFESSOR & HOD DEPARTMENT OF ORAL MEDICINE AND 11.1 Guide : RADIOLOGY A. J. INSTITUTE OF DENTAL SCIENCES. 11.2 Signature : Dr. RAGHAVENDRA KINI 11.3 Co-Guide PROFESSOR DEPARTMENT OF ORAL MEDICINE AND RADIOLOGY A.J. INSTITUTE OF DENTAL SCIENCES. 11.4 Signature : 11.5 Head of Department Dr. VATHSALA PROFESSOR & HOD DEPARTMENT OF ORAL MEDICINE AND RADIOLOGY A. J. INSTITUTE OF DENTAL SCIENCES. 11.6 Signature : 12. 12.1 Remarks of the Chairman & Principal : Dr. B SURESHCHANDRA PRINCIPAL 12.2 Signature