Download dissertation – synopsis

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