Rajiv Gandhi University of Health Science, Bangalore,
Karnataka.
MDS
Oral Medicine & Radiology
Synopsis for Registration of Dissertation.
M R Ambedkar Dental College and Hospital
#1/36, Cline Road, Cooke Town
Bangalore, Karnataka- 560005
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES
KARNATAKA, BANGALORE.
ANNEXURE II
SYNOPSIS FOR REGISTRATION OF DISSERTATION
1 / NAME OF THE CANDIDATE AND ADDRESS / Dr. Naveen Kumar. N.Dept. Of Oral Medicine & Radiology
M. R. Ambedkar Dental College & Hospital
#1/36, Cline Road ,Cooke Town
Bangalore-560005.
2 / NAME OF INSTITUTION / M. R. Ambedkar Dental College & Hospital
#1/36, Cline Road ,Cooke Town
Bangalore-560005.
3 / COURSE OF STUDY AND SUBJECT / M.D.S
ORAL MEDICINE & RADIOLOGY
4 / DATE OF ADMISSION / 25/05/11
5 / TITLE OF THE TOPIC:
EVALUATION OF MICRONUCLEUS ASSAY IN ORAL EXFOLIATED BUCCAL CELLS IN POTENTIALLY MALIGNANT DISORDERS AND ORAL CANCER.
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8. / BRIEF RESUME OF WORK:
NEED FOR THE STUDY:
Oral cancer is one of the ten most common among all cancers and oral squamous cell carcinoma is the most common of all oral malignancies which is generally preceded by some benign lesion or condition which is termed as precancerous. Biological behavior of these precancerous lesions is unpredictable as some precancer may progress to invasive malignancy.
The oral epithelial cells represent a preferred target site for earlier genotoxic events induced by carcinogenic agents entering the body via inhalation and ingestion. Buccal mucosa cells are the first barrier which are capable of metabolizing carcinogens to reactive products.
Many chemical, physical and biological environmental agents are able to interact with DNA to induce genetic damage. The chromosomal damage leading to micronuclear formation occurs during the division of cells from the basal layer of the oral epithelium that is observed later in exfoliated cells. Micronuclei originate from chromosome fragments or whole chromosomes that lag behind at anaphase during nuclear division and are excluded from the reforming nuclei. They are observed in cytoplasm as micronuclei and these can be assessed easily in the exfoliated buccal cells.
The presence of micronuclei reflects a genotoxic exposure leading to DNA mutation resulting in cancer development. This detection of frequency of micronuclei from exfoliated buccal cells will be arguably the least invasive, simple and technically easy to perform. This method of assessing the genetic damage in potentially malignant disorders (lesions and conditions) and oral cancer may enable to provide counselling, prevention and thereby reducing the costly and painful
Treatment later on and number of deaths. With the above knowledge the purpose of the study is to
Evaluate the risk assessment of micronuclei frequency in exfoliated buccal cells for biomonitoring potentially malignant disorders and oral cancer.
REVIEW OF LITERATURE:
Halder A et al (2004): Conducted a study on Comparative Study of Exfoliated Oral Mucosal Cell Micronuclei Frequency in Normal, Precancerous and Malignant Epithelium. In this study 50 patients with precancerous or malignant oral epithelial lesions were compared with 50 age and sex matched healthy controls without any oral lesions. The micronuclei assay in exfoliated cells is an innovative genotoxicity technique, which holds promise for the study of epithelial carcinogens. Micronuclei are suitable internal dosimeters for revealing tissue specific genotoxic damage in individuals exposed to carcinogenic mixtures. They concluded that the gradual increase in micronuclei counts from normal mucosal to precancerous lesions to carcinoma suggested a link of this biomarker with neoplastic progression. Analysis of exfoliated buccal cell micronuclei (MN) is a sensitive method of monitoring genetic damage in the human population.
Cerqueira EMM et al (2008): Conducted a study on Genotoxic effects of X-rays on keratinized mucosa cells during panoramic dental radiography. 40 healthy individuals who underwent this procedure for diagnostic purposes on request from their dentists agreed to participate in this study. Epithelial gingival cells were obtained from the keratinized mucosa of the upper dental arcade by gentle scraping with a cervical brush immediately before exposure and 10 days later. Nuclear projections (broken eggs) and degenerative nuclear alterations (pyknosis, karyolysis, karyorrhexis and condensed chromatin) were scored. The frequency of micronuclei was significantly higher after exposure (P < 0.05), as were the frequencies of nuclear alterations indicative of apoptosis (P < 0.001). These results indicated that X-ray radiation emitted during panoramic dental radiography induced a genotoxic effect on epithelial gingival cells that increased the frequency of chromosomal damage and nuclear alterations indicative of apoptosis.
Palve DH, Tupkari JV (2008): Conducted a study on clinico-pathological correlation of micronuclei in oral squamous cell carcinoma by exfoliated cytology. This study comprised of 30 cases of histologically diagnosed squamous cell carcinoma and 20 healthy control subjects without any habits of consumption of tobacco, other tobacco related substances, or other such substances. Micronulei 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 frequencies were found higher in squamous cell carcinoma patients than in control subjects. Micronuclei frequencies were also found to be raised with increasing histological grades of squamous cell carcinoma. The micronucleus frequencies were found to increase from grade I to grade II, the difference being statistically highly significant (p<0.001). The micronuclei frequency in grade III was observed to be higher than that in grade II.
Jindal C, Palaskar S (2010): Conducted study on evaluation of micronuclei using papanicolaou and M G giemsa stain in individuals with different tobacco habits. Using 45 subjects (15 smoker, 15 smokeless tobacco and 15 non users) were examined. Two cytological smears were taken from the normal buccal mucosa and which were fixed and stained with pap stain and remaining 45 smears were fixed and stained with MGG. They found micronucleus frequency decreased in smokeless tobacco chewers than in smokers and concluded that pap is a better stain for counting micronuclei due to the fact that micronuclei were easily seen in the clear cytoplasm in regard to MGG smears where bacteria and cell debris mask the effect of micronucleus.
Devi P, Thimmarasa VB, Mehrotra V, Arora P (2011): Conducted a study on micronucleus assay for evaluation of genotoxicity in potentially malignant and malignant disorders. With a sample of 33 patients with potentially malignant and malignant oral epithelium diseases and compared with 33 age and sex matched healthy controls. They found a significant (P<0.005) stepwise increase in the percentage of micronuclei cells and micronuclei from control to precancer patients, and from precancer to cancer patients. He concluded that micronucleus frequency in oral exfoliated cells was significantly increased in malignant and potentially malignant group as compared to the control group. The induction of micronucleus is considered to be an effective biomarkers of diseases.
AIMS AND OBJECTIVES:
The present study is designed with the following aims:
1. To evaluate the number of micronuclei in oral exfoliated buccal cells from controls, potentially malignant disorders and oral cancer.
2. Comparison of micronucleus frequency between controls, potentially malignant disorders and oral cancer.
3. To ascertain the reliability of micronucleus as a prognostic biomarker for early detection of oral cancer in high risk group.
MATERIALS AND METHODS:
SOURCE OF DATA:
In this study a sample size of 45 cases will be selected. The subjects for the study will be grouped into 3 groups :15 cases of healthy individuals with no oral lesions as controls, 15 cases of potentially malignant disorders (leukoplakia, lichen planus, oral submucous fibrosis) and 15 cases of oral cancer(oral squamous cell carcinoma). In case of group 2 and group 3, clinical diagnosis will be confirmed by histopathological study. Age ranging from 18-60 and above from the department of oral medicine and radiology, M R Ambedkar dental college and hospital Bangalore.
METHOD OF COLLECTION OF DATA:
The subjects will be clinically examined with standardized questionnaire to obtain the relevant habits and informed consent will be obtained. The subjects will be asked to rinse the mouth with water to remove debris, oral mucosal cells are scraped from buccal mucosa of control group and from lesional tissues using separate pre-moistened wooden spatula. These scraped cells will be smeared over a precleaned microscope slide. These smears will be fixed with papanicolaou stain. Presence and frequency of micronucleus will be observed using light microscope using magnification of 40x and 100x.
Criteria used for identification of micronuclei by Tolbert et al:
a. Rounded smooth perimeter suggestive of a membrane.
b. Less than one-third diameter of the associated nucleus, but large enough to discern shape and colour.
c. Staining intensity similar to that of nucleus.
d. Texture similar to that of nucleus.
e. Same focal plane as nucleus.
f. Absence of overlap with, or bridge to the nucleus.
The data collected will be subjected to statistical analysis using ANOVA.
DOES THE STUDY REQUIRE ANY INVESTIGATIONS TO BE CONDUCTED ON PATIENTS OR OTHER HUMANS OR ANIMALS?
Yes
HAS ETHICAL CLEARANCE BEEN OBTAINED FROM YOUR INSTITUTION IN CASE OF 7.3?
Yes
LIST OF REFERENCES :
1) Halder A, Chakraborty T, Mandal K, Gure P K, Das S, Raychowdhury R et al. Comparative Study of
Exfoliated oral mucosal Cell Micronuclei Frequency in Normal, Precancerous and Malignant Epithelium. International journal of human genetics 2004; 4(4):257-260.
2) Cerqueira EMM, Meirele J R C, Lopes MA, Junqueira JC, Filho Gomes IS, Trindade S et al.
Genotoxic effects of X-rays on keratinized mucosa cells during panoramic dental radiography. Dentomaxillofacial Radiology 2008; 37:398–403.
3) Palve DH, Tupkari JV. Clinico-pathological correlation of micronuclei in oral squamous cell carcinoma by exfoliative cytology. Journal of oral and maxillofacial pathology 2008; 12(1):2-7.
4) Jindal C, Palaskar S. Evaluation of micronuclei using papanicolaou and may grunwald giemsa stain in
Individuals with different tobacco habits-A comparative study. Journal of clinical and Diagnostic research 2010; 4; 3607-3613.
5) Devi P, Thimmarasa VB, Mehrotra V, Arora P. Micronucleus assay for evaluation of genotoxicity in
Potentially malignant and malignant disorders. Journal of Indian academy of oral medicine and radiology 2011; 23:97-100.