RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,
KARNATAKA, BANGALORE.
ANNEXURE-II
PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION
1. / NAME OF THE CANDIDATE AND ADDRESS (IN BLOCK LETTERS) / Dr.SATYAJIT .S. TOPAJICHE,POST GRADUATE STUDENT,
SRI HASANAMBA DENTAL COLLEGE AND HOSPITAL,
VIDYANAGAR,
HASSAN-573201, KARNATAKA.
2. / NAME OF THE INSTITUTION / SRI HASANAMBA DENTAL COLLEGE AND HOSPITAL,
VIDYANAGAR,
HASSAN-573201,
KARNATAKA.
3. / COURSE OF STUDY AND SUBJECT / MASTER OF DENTAL SURGERY IN ORAL PATHOLOGY AND MICROBIOLOGY.
4. / DATE OF ADMISSION TO COURSE / 25- 05 - 2012
5. / TITLE OF THE TOPIC / “GENOTOXIC EFFECT OF METHYL METHACRYLATE AND NICKEL COBALT CHROMIUM IN DENTAL LAB TECHNICIANS-A MICRONUCLEI AND CYTOMORPHOMETRIC STUDY OF BUCCAL MUCOSAL CELLS.”
6.
7.
8. /
BRIEF RESUME OF THE INTENDED WORK:
6.1 Need for the study:A person spends, on average, one-third of his life at his workplace and therefore the environment in which he works can be a major factor in determining health.1 Methyl methacrylate (MMA) has been implicated as primary irritant and sensitizer, which can cause allergic eczematous reaction on the oral mucosa. To date, there is growing concern that MMA may produce genetic damage by inducing mutation.2
Nickel and hexavalent chromium (soluble/insoluble) compounds have been classified as human carcinogens by the International Agency for Research in Cancer (IARC,1990).3
Micronucleus test in the buccal epithelial cells has been used to study the genotoxic effects in humans exposed to carcinogenic substances and is being increasingly accepted as a reliable biomarker of genotoxicity in occupationally exposed groups.3
Cytomorphometric analysisviaoralbrush biopsy is a valuable adjunct to biopsy & Cytoplasmic Diameter, Nuclear Diameter andmicronuclei evaluationserves as important diagnostic markers.5
Buccal Epithelial Cells (BEC) are the first barrier for the inhalation or ingestion route and are capable of metabolizing proximate carcinogens to reactive products.7
This study aims to determine the extent of genotoxic damage in relation to MMA and nickel-cobalt-chromium in dental laboratory technicians via micronuclei induction test and cytomorphometric analysis .
6.2Review of Literature:
In a study conducted in 2011 on 84 tannery workers exposed to chromium using micronucleus test and it was found that the workers exposed to chromium revealed a significant micronuclei frequency when compared to the ones who were unexposed. 5
In another study conducted in 2012 on 100 electroplaters exposed to nickel and chromium based on duration of exposure using micronuclei test and they found that there was a significant micronuclei frequency when compared to the ones who were unexposed and also there was a significant increase in the micronuclei frequency in those who were exposed for a longer duration.3
A study was done to assess the genetic damage by methyl methacrylate employing in vitro mammalian test system and was concluded that methyl methacrylate is a cytotoxic and genotoxic agent causing toxic reactions and adverse effects on oral mucosa.2
A study in 2010 concluded that detection of micronuclei and their assay is an upcoming research domain in the field of cancer prevention and therapeutics. These miniature nuclear offshoots if properly identified can turn out to be important biomarkers with huge potential in screening and predicting patients with oral precancers and also can act as risk assessors in patient’s ongoing treatment for invasive cancers.6
A cytomorphometric study in 40 premalignant and malignant disorders concluded that a decrease in the mean cytoplasmic diameter of exfoliated buccal mucosal cells could serve as an early indicator of dysplastic change especially in lesions which appear histologically benign.4
6.3 OBJECTIVES OF THE STUDY:
1. To examine the exfoliated buccal mucosal cells for the presence of micronuclei in the dental lab technicians exposed to methylmethacrylate and nickel-cobalt-chromium.
2. To examine the cytomorphometric changes in buccal exfoliated cells of dental lab technicians exposed to methylmethacrylate and nickel-cobalt-chromium.
: The following parameters will be determined in each cell in cytomorphometric analysis:
· Cytoplasmic area(CA)
· Nuclear area(NA)
· Nuclear/Cytoplasmic Ratio
All the above parameters to be examined in 2 groups
Group 1: 25 Dental Laboratory Technicians
Group 2: 25 Healthy volunteers.
MATERIALS AND METHODS :
7.1 Source of data:
Samples will be collected from dental laboratory technicians from various institutions and laboratories in Karnataka.
Samples for the healthy volunteers will be collected from patients visiting the department of Oral Pathology at Sri Hasanamba Dental College and Hospital.
Materials:
· Cytobrush.
· Glass slide with coverslips.
· Fixative. ( 95% methanol)
· Stains ( micronuclei test- Fuelgen reaction with schiffs reagent.
cytomorphometric analysis- Papanicolau’s stain)
· Light microscopy.
· Image analysis software for cytomorphometry Image ProgRes C3 (Version 7).
7.2 Method of collection of data:
The data will be obtained from those subjects satisfying the criteria, as listed below
INCLUSION CRITERIA:
1. Dental laboratory technicians exposed to methylmethacrylate and nickel-cobalt-chromium for a minimum period of five years.
EXCLUSION CRITERIA:
1. Subjects with a history of tobacco usage in any forms.
2. Subjects with a history of alcohol intake.
3. Subjects with any premalignant/ malignant disorders.
4. Subjects with any systemic disorders.
5. Subjects under long term antibiotic or steroid therapy.
PROCEDURE:
An approval from the institutional ethical committee shall be obtained before beginning the study and all the participants will be given full information about the purpose of the study and informed consent will be taken before undertaking the study.
The sample will be collected from 15 dental lab technicians and 15 healthy volunteers . Smears will be obtained from the buccal mucosa from all the subjects.
Before taking the smear the individuals will be instructed to rinse the mouth with distilled water to remove the debris, after which the scrapings will be obtained. The surface of the buccal mucosa is rolled or scraped uniformly with cytobrush, smear will be prepared with the collected epithelial cells on an appropriately labeled glass slide and fixed with commercially available spray fixative (95% methanol) . Two cytologic smears will be obtained from each subject.
The fixed slides will be stained by:
1. Fuelgen reaction using schiffs reagent for micronuclei assessment.
2. Papanicolau’s stain for cytomorphometric analysis.
All the slides will be assessed for -
1) Micronuclei assessment – the slides prepared will be examined under binocular light microscope to note the frequency of micronuclei formation. Only cells which are non-fragmented, non-accumulated and non-overlaid will be examined.
2) Cytomorphometric analysis- the slides prepared will be examined under a binocular light microscope. 50 cells on each slide will be randomly selected for examination. The images of the cytologic fields will be captured at a higher magnification(400) to measure nuclear and cytoplasmic areas. The image analysis system Image ProgRes C3 (Version 7) will be used to enhance precision.
The following parameters will be determined in each cell:
· Cytoplasmic area(CA)
· Nuclear area(NA)
· Nuclear/Cytoplasmic Ratio.
Statistical Analysis
The statistical analysis will be done using Unpaired‘t’ test.
7.3. Does the study require any investigations or interventions to be conducted on patients or other humans or animals? If so please describe briefly.
YES.
Patients will be asked to rinse their mouth in distilled water before cell collection. Buccal mucosal cells will be collected by scraping the buccal mucosa with the help of cytobrush.
7.4 Has ethical clearance been obtained from your institution in case of 7.3?
YES.
LIST OF REFERENCES:
1. Danadevi K, Rozati R, Banu BS, Grover P. Genotoxic evaluation of welders occupationally exposed to chromium and nickel using the Comet and micronucleus assays. Mutagenesis 2004;19:35±41.2. Yang HW, Chou LS, Chou MY, Chang. Assessment of genetic damage by methyl methacrylate employing in vitro mammalian test system. Biomaterials 2003;24:2909-2914Y.
3. Qayyum S, Ara A, Usmani JA. Effect of nickel and chromium exposure on buccal cells of electroplaters. Toxicology and Industrial Health 2012;28:74-82.
4. Hegde V. Cytomorphometric analysis of squames from oral premalignant and malignant lesions. J Clin Exp Dent 2011;3:e441-4.
5. Sellappa S, Prathyumnan S, Joseph S, Keyan KS. Micronucleus Test in Exfoliated Buccal Cells from Chromium Exposed Tannery Workers. International Journal of Bioscience, Biochemistry and Bioinformatics 2011;1:58-62.
6. Jois HS, Kale AD, Kumar MKP. Micronucleus as Potential Biomarker of Oral Carcinogenesis. IJDA 2010;2:197-202.
7. Mahimkar MB, Samant TA, Kannan S, Patil T. Influence of genetic polymorphisms on frequency of micronucleated buccal epithelial cells in leukoplakia patients. Oral Oncol 2010;46:761–766.
9. / SIGNATURE OF THE CANDIDATE / (Dr. SATYAJIT. S. TOPAJICHE )
10. / REMARKS OF THE GUIDE
11.
12. / NAME & DESIGNATION OF
(IN BLOCK LETTERS)
11.1 GUIDE
11.2 SIGNATURE
11.3 CO-GUIDE (IF ANY)
11.4 SIGNATURE
11.5 HEAD OF THE DEPARTMENT
11.6 SIGNATURE
REMARKS OF THE CHAIRMAN
& THE PRINCIPAL
12.1 SIGNATURE
/Dr.SARVANI MURTHY M.D.S,
PROFESSOR AND HEAD,DEPARTMENT OF ORAL PATHOLOGY AND MICROBIOLOGY,
SRI HASANAMBA DENTAL COLLEGE AND HOSPITAL,
HASSAN – 573201.
Dr.SARVANI MURTHY M.D.S,
PROFESSOR AND HEAD,DEPARTMENT OF ORAL PATHOLOGY AND MICROBIOLOGY,
SRI HASANAMBA DENTAL COLLEGE AND HOSPITAL,
HASSAN – 573201.
Dr.S. RAVINDRA M.D.S.
PRINCIPAL,
SRI HASANAMBA DENTAL COLLEGE AND HOSPITAL,HASSAN – 573201.