BIOMONITORING OF GENOTOXIC AND CYTOTOXIC CHANGES …

BIOMONITORING OF GENOTOXIC AND CYTOTOXIC

CHANGES AMONG PATIENTS EXPOSED TO DIGITAL

RADIOGRAPHS - A COMPARATIVE STUDY

DISSERTATION

Submitted to The Tamil Nadu Dr. M.G.R Medical University in

partial fulfillment of the requirement for the degree of

MASTER OF DENTAL SURGERY

BRANCH IX

ORAL MEDICINE AND RADIOLOGY

2012 - 2015

CERTIFICATE

This is to certify that this dissertation titled “Biomonitoring of

Genotoxic and Cytotoxic Changes Among Patients Exposed to Digital

Radiographs – A Comparative Study” is a bonafide research work done

by Dr. Meera Mathai under our guidance during her Post Graduate study

during the period of 2012-2015 under THE TAMIL NADU Dr. M.G.R.

MEDICAL UNIVERSITY, CHENNAI, in partial fulfillment for the degree

of MASTER OF DENTAL SURGERY IN ORAL MEDICINE AND

RADIOLOGY, BRANCH IX. It has not been submitted (partial or full) for

the award of any other degree or diploma.

Dr. TATU JOY. E M.D.S

Guide

Professor and HOD

Department of Oral Medicine and Radiology

Sree Mookambika Institute of Dental Sciences,

Kulasekharam.

Dr. HEMA. G M.D.S

Co- Guide

Reader

Department of Oral Medicine and Radiology


Kulasekharam.

SREE MOOKAMBIKA INSTITUTE OF DENTAL

SCIENCES, KULASEKHARAM

ENDORSEMENT BY THE PRINCIPAL / HEAD OF THE INSTITUTION

This is to certify that this dissertation titled “Biomonitoring of

Genotoxic and Cytotoxic Changes among Patients Exposed to Digital

Radiographs – a Comparative Study” is a bonafide research work done by

Dr. Meera Mathai under the guidance of Dr. Tatu Joy E MDS, Professor

and Head, Department of Oral Medicine and Radiology, Sree Mookambika

Institute of Dental Sciences, Kulasekharam.

Dr. Elizabeth Koshi MDS

PRINCIPAL

Sree Mookambika Institute of Dental Sciences

V.P.M Hospital Complex

Padanilam, Kulasekharam.

KanyaKumari District.

Tamil Nadu - 629 161

ACKNOWLEDGEMENT

I extend my profound gratitude to my guide Dr. Tatu Joy E MDS, Professor

& Head of the Department for his invaluable guidance, co-operation, constant

encouragement and immense patience with me in every step of this endeavor. I thank

him for the trust he had in me that made me to complete this work.

I am thankful to my co- guide Dr. Hema G MDS Reader, Department of Oral

Medicine & Radiology for the support she showed me in completing this work.

Am grateful to Dr. Shashi Kiran M MDS Reader, Department of Oral

Medicine & Radiology for his constant enthusiasm, strive for perfection, and patience

he showed me for writing this study. With comforting words and smile, he persuaded

us to develop our presentation skill.

I take this opportunity to express my sincere gratitude to my teachers Dr.

Eugenia Sherubin, Dr. Raghupathy for the constant encouragement throughout the

course of this study

I would like to acknowledge the help & support given by Dr. Velayuthan

Nair, chairman and Dr. Rema V Nair director Sree Mookambika Institute of Dental

Sciences without which my study would not have been possible

It is my utmost privilege to acknowledge Dr. Issac Joseph Head of

Department Oral Pathology & Microbiology for his kindness by permitting me to use

the facilities in his department.

I am indebted to Dr. Deepa A G, Senior lecturer Department of Oral

pathology & microbiology for helping me with the study.

Am grateful to Dr. Pratheesh Sathyan Reader Department of Oral pathology

& Microbiology for his valuable suggestions in this work.

I thank Kalavathy, in the department of Oral Pathology & Microbiology for

helping me in the study.

I thank my batch mates, Dr. Redwin Dhas Manchil, Dr. Vineetha

Vijayakumar for their support and encouragement.

I would like to thank my juniors Dr. Indu Krishna, Dr. Melbia Shiny for

helping me to get through difficult times and for all emotional support and caring.

I thank Dr. George Jacob Dr. Gomakumar and Dr. Arun Mohan, my

fellow PGs in the department of Oral Pathology & Microbiology for being helpful,

and providing me with the support and encouragement for fulfilling thesis work.

I would like to thank Dr. Sainul Abideen Reader INDIAN INSTITUTE OF

SCIENCE AND RESEARCH for his immense kindness that made me possible to

write the thesis by providing me the articles.

I would like to thank Dr. Anand Reader Department of Community Dentistry,

Koorg Institute of Dental Science for helping me with my statistical work.

Words are less to express my deep gratitude to Emily Andrews, first best

friend then my mother, my loving father P. J. Mathai and caring brother Dr. Kiran

Mathai for their support and encouragement they have given me throughout my life.

I would like to thank my friend Dr. Arya Ravisankar for her immense faith

and support and positive energy she filled in me. I would extend my sincere gratitude

to Dr. Swathy Anand Senior lecturer Rajas Dental college for his support and

suggestions for writing this study.

I thank almighty for his blessings he had given on me in my life.

CONTENTS

NO TITLE PAGE NO

1 List of abbreviations i - ii

2 List of tables iii - iv

3 List of graphs v

4 List of colour plates vi

5 List of annexures vii

6 Abstract viii - ix

7 Introduction 1-3

8 Aim and objectives 4

9 Review of literature 5 -33

10 Materials and methods 34 - 42

11 Results and observations 43 - 53

12 Discussion 54 - 60

13 Summary and conclusion 61

14 Bibliography x - xxii

15 Annexures

i

LIST OF ABBREVEATIONS

PAP: Papanicolaou stain

MN: Micronucleus

NCRP: National Council on Radiation Protection and Measurements

FISH: Fluorescent insitu hybridization

MGG: May-Gruwald Giemsa

KR: Karyorrhexis

PN: Pyknosis

KL: Karyolysis

CC: Condensed chromatin

SCC: Squamous cell carcinoma

TB: Toluidine blue

FNAC: Fine Needle Aspiration Cytology

OSMF: Oral Sub Mucous Fibrosis

PMDs: Potentially Malignant Diseases

CBCT: Cone Beam Computed Tomography

CT: Computed Tomography

IOPA: Intra Oral Periapical radiograph

OPG: Orthopantomograph

ii

C2H5N+: Acrylonitrite group

D.P.X: Din butyl phthate in Xylene

SPSS: statistical package for social sciences

iii

LIST OF TABLES

Table No Title of the table

Table -1 Distribution of study subjects according to age and gender

Table - 2 Total No of cells having Micronucleus before and after

radiation/100 cells

Table – 3 Total No of Micronucleus before and after radiation/100

cells

Table – 4 Mean value & standard deviation of MN assay

Table – 5 Total No of cells having Karyorrhexis before and after

radiation/100 cells

Table – 6 Total No of cells having Karyolysis before and after

radiation/100 cells

Table – 7 Total No of cells having Pyknosis before and after

radiation/100 cells

Table – 8 Mean value & standard deviation of cytological changes

iv

Table – 9 Gender variation in micronucleus count

Table – 10 Gender wise comparison of micronucleus

Table – 11 Gender wise comparison of Karyorrhectic cell count

Table – 12 Gender wise comparison of Karyorrhexis

Table – 13 Gender wise comparison of karyolitic cell count

Table – 14 Gender wise comparison of karyolysis

Table – 15 Gender wise comparison of Pyknotic cell count

Table – 16 Gender wise comparison of pyknosis

v

LIST OF GRAPHS

Graph No

TITLE OF THE GRAPH

Graph - 1

Distribution of study subjects according to gender and age

Graph - 2

Mean changes in Genotoxic and Cytotoxic parameters

before and after radiation exposure among study subjects

Graph - 3

Gender wise comparison for micronucleus before and after

radiation exposure

Graph - 4

Gender wise comparison for Karyorrhexis before and after

radiation exposure

Graph - 5

Gender wise comparison for Karyolysis before and after

radiation exposure

Graph - 6

Gender wise comparison for Pyknosis before and after

radiation exposure

vi

LIST OF COLOUR PLATES

Color Plate No

Title of Color Plate

CP - 1

Armamentarium for sample collection

CP - 2

Planmeca Prolin XC machine

CP - 3

RAPID PAPTM

staining kit

CP - 4

Stained slides in slide box

CP- 5

Light microscope with AP viewer

vii

LIST OF ANNEXURES

Annexure No Title of Annexure

Annexure -1 Certificate from Institutional Human Ethics

Committee

Annexure - 2 Patient information sheet

English

Malayalam

Tamil

Annexure - 3 Patient consent form

English

Malayalam

Tamil

Annexure - 4 Case record form

Annexure - 5 Study observation sheet

ABSTRACT

Abstract

viii

Background of the study

The discovery of X- rays is considered as the discovery of the 20th

century. It

has its boon as it is an important tool for diagnosis and treatment in medical and

dental practice. However its bane remains as ionizing radiation is a well known

mutagen and carcinogen for the human population. It is largely known that there is no

safety in radiation doses and that the biological effects of exposure received would be

accumulated through time. In order to detect the effects of low dose ionizing radiation

in diagnostic radiology various sensitive analysis are needed.

Aim

The aim of this study is the evaluation of genotoxicity and cytotoxicity in

patients after exposure to digital radiographs.

Materials & methods

Clinically healthy patients who required orthodontic treatment were selected.

As a part of their treatment plan, they were exposed to x rays for making

orthopantomogram and lateral cephalogram. Smears were taken from the buccal

mucosa before exposure and 10±2 days after exposure. The smears were fixed and

stained with DNA specific PAP stain using RAPID PAPTM

staining kit. 100 cells were

analyzed on each slide under 40 × magnification using light microscope with the help

of AP viewer.

Abstract

ix

1. For the evaluation of Genotoxicity, presence of micronucleus was estimated.

2. For the determination of cytotoxicity, parameters taken were Karyorrhexis,

karyolysis and pyknosis.

Results

There was a significant increase in the number of micronuclei after radiation

exposure which indicates that the x rays can produce genotoxic damage to the cells.

There was also an increase in the number of other nuclear alterations after exposure

which indicated the increased cytotoxicity produced by radiation. There is no gender

predilection found in this study.

Conclusion

Radiography is one of the most valuable diagnostic tool used in

comprehensive dental care for diagnosis, treatment planning and for follow up. This

study concluded that even low level ionizing radiation can induce both genotoxic and

cytotoxic damage to the cells. Hence radiographs should be advised only when it is of

utmost necessity.

KEYWORDS: Ionizing radiation, Micronucleus, Karyorrhexis, Karyolysis, Pyknosis

INTRODUCTION

Introduction

1

Carcinogenesis is a multistep process governed by genetic or epigenetic

mechanisms and signaling pathways and in at least any one of those steps there will

be a change in cellular behavior and morphology due to mutations related to the

control of cell division, cell death and metastatic potential. Among the multiple

mutations found in human cancers such as gene amplification, chromosome

alterations and translocations, point mutation is very important.1 When the normal

function of DNA repair is altered as a result of mutation by various agents, the risk of

malignant transformation increases. Among these agents, ionizing radiation forms the

bulk of contribution to human exposure because of its wide use for diagnostic and

therapeutic purposes.

X rays are potent mutagenic agent capable of inducing both gene mutation and

chromosomal aberrations. They act directly on DNA molecule or indirectly through

the formations of reactive compounds that interact with DNA. In spite of their

mutagenic potential, X- rays are an important tool in diagnosing disease and are used

in both medical and dental practice. Taking the strong evidence for relationship

between DNA damage and carcinogenesis into considerations, it would be useful to

know the harmful effects induced by X- rays.

Accumulating evidence suggests that the radiographs which are widely used

for diagnosis in medical and dental practice can induce cytotoxic effects and cause

DNA damage. The nucleus (including its genetic material) is more radiosensitive than

the cytoplasmic structures of the cell. Therefore the elucidation of the genotoxic

effects induced by radiography is relevant to identify and minimize potential risk to

the patient.

Introduction

2

Research on these issues has to be primarily addressed on reduction in the

radiation dose to the patient. Although it is generally accepted that there is no safe

level of radiation exposure, the possible risk associated with the exposure to X-rays

must be outweighed against the clinical benefits. With this in mind, various methods

have been developed to detect radiation effects of low dose radiographic exposure.2

Biomonitoring studies have been used in health sciences for many years to

assist in diagnosing and staging diseases as well as to evaluate the risk assessment.

They give information concerning environmental exposure and susceptibility. These

studies are divided into three groups; first to define the exposure to mutagenic and/or

carcinogenic agents, second to show the biological effects on target tissues and third

to give information about the individual susceptibility.

Cytogenetic biomonitoring has been used in health sciences for many years in

diagnosing and staging diseases, as well as in risk assessment, and provides

information concerning levels of risk and susceptibility status. A variety of assays has

been proposed as potential tools in cytogenetic biomonitoring studies, including those

that assess metaphase chromosomal aberrations, sister chromatid exchanges and

deoxyribonucleic acid (DNA) damage and repair. However, these methods are

typically laborious and time-consuming or require highly trained technicians to

accurately read and interpret slides.

Biomarkers are biologic parameters that provide information about a

physiologic or pathologic state of an individual or population. National institute of

health defined the term biomarker as a characteristic that is objectively measured and

evaluated as an indicator of normal biological process, pathological processes or

pharmacological responses to a therapeutic intervention or other health care

Introduction

3

interventions.3 Molecular epidemiology research focuses on three types of

biomarkers; biomarker of exposure (chromosomal aberrations, micronucleus, sister

chromatid exchange), biomarker of susceptibility (genetic polymorphism) and

biomarkers of disease( tumor biomarker).4

Micronuclei and other nuclear abnormalities are biomarker that offers

additional endpoints for possible evaluation of chromosomal instability and different

cell death events (Karyorrhexis, pyknosis). Micronucleus can be evaluated in many

tissues involving any dividing cells like bladder, esophagus, bronchial nasal and

buccal mucosa.

Buccal epithelial cells provide an alternative source of tissue for human

monitoring to occupational and environmental toxic exposures. This tissue is under

direct radiation exposure during panoramic radiography and lateral cephalometric

radiography and in this way it is a primary target for radiation induced damage.

Furthermore this tissue has an advantage of rapid and easy sampling by brushing the

buccal mucosa. Accurately the presence of micronucleus is observed in exfoliated

epithelial tissues which are derived from the basal layer where the cell division takes

place and they migrate towards the surface within 5-14 days. In this manner, the

epithelial tissues can reflect damage occurred at this time.3

As there is a strong association between DNA damage and carcinogenesis, the

present study is aimed to evaluate the genotoxic and Cytotoxic effects of low level

ionizing radiation used in dental radiography on buccal epithelial cells by a simple

technique.

AIM & OBJECTIVES

Aim & Objectives

4

AIM

1. To compare the genotoxic and Cytotoxic changes after exposure to low level

diagnostic ionizing radiation

OBJECTIVES

1) To assess the number of micronuclei before and after digital radiographs

2) To assess Karyorrhexisbefore and after digital radiographs.

3) To assess karyolysisbefore and after digital radiographs.

4) To assess pyknosis before and after digital radiographs.

5) To assess the variation of these factors based on gender.

REVIEW OF LITERATURE

Review of Literature

5

The study of DNA damage in exfoliated cells collected from the oral cavity holds

great promise as a minimally invasive method for monitoring populations exposed to

genotoxic agents. The presence of micronuclei (MN) and other nuclear anomalies within

these cells has been known to be associated with genetic defects in genome maintenance,

accelerated ageing, exposure to genotoxic agents, oral cancer risk and neurodegenerative

diseases and was also used in chemo preventive studies.5

The first case of human injury was reported in the literature just a few months

following Roentgen's original paper in 1895 announcing the discovery of x-rays. As early

as 1902, the first case of x-ray induced cancer was reported in the literature.6

Radiation damage starts at the cellular level. The mechanism by which the

damage occurs can happen via

(a) direct action

(b) indirect action

A. Direct Action

Radiation may impact the DNA directly, causing ionization of the atoms

in the DNA molecule. This ionization results in the breakage of the macromolecule‟s

chemical bonds causing them to become abnormal structures which may lead to adverse

chemical reactions. Subsequent chromosomal damage includes abnormal replication, cell

death or temporary damage. If the radiation directly affects the somatic cells, the effects

on the DNA could result in radiation induced malignancy. If damage is to the


6

reproductive cells, the result could be radiation induced congenital abnormality. This can

be visualized as a “direct hit” by the radiation on the DNA, and thus is a fairly

uncommon occurrence due to the small size of the target; the diameter of the DNA helix

is only about 2 nm.

B. Indirect Action

In the second scenario, the radiation interacts with non-critical target atoms or

molecules, usually water. This results in the production of free radicals, which are atoms

or molecules that have an unpaired electron and thus are highly reactive. These free

radicals can then attack critical targets such as the DNA. Because they are able to diffuse

only some distance in the cell, the initial ionization event does not have to occur so close

to the DNA in order to cause damage. Thus, damage from indirect action is much more

common than damage from direct action, especially for radiation that has a low specific

ionization.

When the DNA is attacked, either via direct or indirect action, damage is caused

to the strands of molecules that make up the double-helix structure. Most of this damage

consists of breaks in only one of the two strands and is easily repaired by the cell, using

the opposing strand as a template. If, however, a double-strand break occurs, the cell has

much more difficulty repairing the damage and may make mistakes. This can result in

mutations, or changes to the DNA code, which can result in consequences such as cancer

or cell death. Double-strand breaks occur at a rate of about one double-stand break to 25

single-strand breaks. Thus, most radiation damage to DNA is reparable.


7

The general population is exposed to various types of radiation, mainly natural

and manmade radiation. Natural radiation is caused by terrestrial sources and cosmic

sources. Sources of manmade radiation includes medical and diagnostic treatment,

consumer and industrial products.7

AVERAGE ANNUAL EFFECTIVE DOSE OF IONIZING RADIATION.7

Source Dose [µSv]

Natural

Cosmic 0.4

Terrestrial

External 0.5

Radon 1.2

Other 0.3

Manmade

Medical X-ray diagnosis 2

Nuclear medicine 0.5

Other consumer products 0.08

Occupational 0.01

Fallout 0.01

Nuclear fuel cycle <0.01

Dental radiology ≤0.01


8

DOSE LIMITS

After recognizing harmful effects of radiation and risks involved in the use

National Council on Radiation Protection and Measurements [NCRP] and International

Council on Radiological Protection has established guidelines for the limitations of

radiation received by both occupationally exposed to individuals and public.

EEFECTIVE DOSE FROM DIAGNOSTIC X-RAY EXAMINATIONS AND

EQUIVALENT BACKGROUND EXPOSURE.7

EXAMINATION EFFECTIVE DOSE

[µSv]

Equivalent background

exposure [days]

Extra oral

Panoramic 9-26 1-3

Cephalometric 3-6 0.5-1

CBCT [Galileo‟s] 70 9

CT 2000 243

Plain Skull 70 9

CHROMOSOMAL ABBERATIONS

To study the carcinogenic potential of ionizing radiation, Chromosomal alteration

is another important tool. Lea et al in 1946 had mentioned the chromosomal aberrations

due to ionizing radiations with the data obtained in plant cells and Drosophila. The types

and frequencies of induced chromosomal aberrations depend on the mutagen used and the

stage of the cell cycle treated.


9

Ionizing radiations produce chromosomal aberrations in G 1 stage of cell cycle.

These chromosomal aberrations have been used to monitor human exposure to genotoxic

agents and in patients treated with cytotoxic drugs in peripheral lymphocytes. Structural

chromosome aberrations arise from direct DNA breakage, replication on a damaged DNA

template or inhibition of DNA synthesis and may involve both chromatids of the

chromosome or only one chromatid of the chromosome.

MICRONUCLEUS

The action of carcinogens can induce chromosomal instabilities such as deletions,

translocations, gain or loss of entire chromosomes, contributing to the development of

malignant cellular processes.8 Micronucleus (MN) is a small additional nucleus and is

readily identifiable by light microscopy. During the last few decades, it has generally

been used as a biomarker of chromosomal damage, genome instability and cancer risk.

The MN test provides a reliable measure of chromosome breakage and chromosome

loss.9 The level of baseline chromosome damage in untreated cancer patients and also in

various preneoplastic conditions is much higher than in cancer-free controls. Therefore,

MN scoring can be used as a bio-marker to identify different pre-neoplastic conditions

much earlier than the manifestations of clinical features and might specifically be

exploited in the screening of high-risk population for a specific cancer.10

The

micronucleus (MN) test has been extensively used in a variety of investigations to

understand the basic mechanisms underlying Genotoxicity.11


10

HISTORICAL PERSPECTIVE

In the late 1800s and early 1900s Howell and Jolly described Feulgen – positive

nuclear bodies in human reticulocytes, known as Howell – Jolly bodies and representing

the chromosomes separated from the mitotic spindle.12

The micronucleus (MN) assay

was used for the first time in vitro in radiation experiments with roots of Vicia faba by

Evans et al. in 1959 and for in vivo bone marrow cell studies by Schmid et al in the early

seventies.13, 14

In the early 1970s the term micronucleus test was suggested for the first time by

Boller and Schmidt and Heddle who showed that this assay provided a simple method to

detect the genotoxic potential of mutagens after in vivo exposure of animals using bone

marrow erythrocytes.4 In 1982, the suitability of MN test for human biomonitoring

studies was first described by Stich & co- workers who used exfoliated cells of buccal

mucosa. They postulated that this test system can be used to study genotoxic effects in all

human tissues from which exfoliated cells can be obtained.15

A few years later it was

shown by Countryman and Heddle that peripheral blood lymphocytes could be used for

the micronucleus approach and they recommended using micronuclei as a biomarker in

testing schemes.

The Human Micronucleus [HUMAN] Project is an international collaborative

project aimed at studying the micronucleus frequency in human populations and

assessing the effects of protocol and scoring criteria on the values obtained. It was

established in 1997 and currently involves more than 35 laboratories worldwide. It has

three main goals; (a) compilation and comparison of base line micronucleus frequencies


11

in human population to establish “normal base-line frequencies of DNA damage and

determine the main demographic, environmental and methodological variables that

impact on this index; (b) comparison of variable methods used to measure MN

frequencies in human blood and epithelial cells to identify the important methodological

variables and establish standard protocols to enable more reliable comparison of data

among laboratories and among populations; and (c) to establish prospective

epidemiological studies aimed at determining whether the MN frequency predicts risk of

cancer and other degenerative diseases associated with DNA damage and

ageing.16,17,18,19,20,21

The oral epithelium is composed of four strata of structural, progenitor, and

maturing cell populations, that is, the basal cell layer (stratum basale), prickle cell layer

(stratum spinosum), and the keratinized layer at the surface. A series of finger-like

structures called "rete pegs" project up from the lamina propria into the epidermal layer

producing an undulating basal cell layer effect. The oral epithelium maintains itself by

continuous cell renewal whereby new cells produced in the basal layer by mitosis migrate

to the surface replacing those that are shed. The basal layer contains the stem cells that

may express genetic damage (chromosome breakage or loss) as MN during nuclear

division. The daughter cells, which may or may not contain MN, eventually differentiate

into the prickle cell layer and the keratinized superficial layer, and then exfoliate into the

buccal cavity. Some of these cells may degenerate into cells with condensed chromatin,

fragmented nuclei (Karyorrhectic cells), Pyknotic nuclei, or completely lose their nuclear

material (karyolitic or "ghost" cells). In rare cases, some cells may be blocked in a


12

binucleated stage or may exhibit nuclear buds (also known as "broken eggs" in buccal

cells), a biomarker of gene amplification.

These biomarkers of genome damage (Eg: MN, nuclear buds) and cell death (Eg:

apoptosis, karyolysis) can be observed in both the lymphocyte and buccal cell systems,

and thus provide a more comprehensive assessment of genome damage and MN in the

context of cytotoxicity and cytostatic effects. Micronuclei derived from acentric

chromosome fragment can be distinguished from those derived from whole chromosome

by FISH using a pancentromeric probe, which will detect the centromere in the

micronucleus. Thus, clastogenic and aneugenic events can be detected by the MN

technique.22

In various studies which evaluates MN in patients undergoing radiotherapy in

head and neck region, the most striking increase in the cytogenetic damage [150-300

MN/1000cells] was observed in early phase of treatment where they are exposed to a

cumulative dose of 3400-4000 cGy.23

Other authors reported 68 MN/1000 cells after

2000 cGy and 16 MN/ 1000 cells after treatment with 1000 cGy for 3 weeks.24, 25

Theories of Origin of MN

There are two predominant mechanisms leading to the formation of MN in a

mitotic cell: chromosomal breakage and dysfunction of the mitotic apparatus. Clastogens

induce chromosome breaks and yield acentric fragments. These chromosomal fragments

are directly included into micronuclei. In other mechanism, aneugenic agents prevent the

formation of the spindle apparatus during mitosis. As a result of it the whole

chromosomes lag behind at anaphase. The chromosome is surrounded by the nuclear


13

envelope, forming micronuclei. Therefore the daughter cells have micronuclei containing

whole chromosomes.

Besides these two important mechanisms, MN may be formed due to broken

anaphase bridges. This may be because of dicentric chromatids, intermingled ring

chromosomes or union of sister chromatids. MN may not always represent the loss of

chromosomal material as it may be also be formed due to amplified DNA budding.

Causes of MN Formation

There are several of causes of MN formation. This may be noted spontaneously in

the normal healthy individual due to exposure to environmental pollutants, radiation, bio-

hazard materials, and drugs, other poisonous chemicals and free-radical injuries. The

other causes of MN formation are long standing chronic inflammation, heavy metal

poisoning, chemotherapy, radiation injuries, and various preneoplastic and neoplastic

conditions. Besides that a large number of genetic diseases, infections, and nutritional

deficiency are also responsible for MN formation. Direct DNA damage or breakage,

chromosomal aberrations, mitotic apparatus dysfunctions, and interference with DNA

synthesis are the possible explanations of MN formation. The mechanism by which the

chromosomes or chromatids getting eliminated from the daughter cells is not understood

fully.26

It may be probably those displaced at metaphase and gets predispose to “lag” at

this phase of cell division after other chromatids have moved to the other spindle poles.27

It has also been shown that some micronuclei can perform DNA synthesis and mitotic

condensation synchronously with the main nucleus further producing chromosome loss

and gain at successive mitotic cycles.28


14

CATEGORY ETIOLOGY EXPLANATIONS

Spontaneous Healthy persons Exposure to environmental

pollutants, food/drink

habits etc.

Chronic

inflammation

Tobacco, Para-amino hippuric

acid, Ethylene oxide,

Formaldehyde, Crohn‟s disease,

oral Lichen

Planus etc.

Free radical injuries, Metabolic

dysfunction, Mitotic

spindle dysfunction

Genotoxicity Anti-neoplastic drugs, Pesticide Direct DNA damage, cell cycle

inhibition, mitotic

spindle toxins

Chemotherapy Cancer patients As above

Radiation injuries Increased MN frequency after

radiotherapy,

radiation-induced cancers

Direct DNA damage/ breakage,

accumulated in

consecutive populations

Pre-neoplastic

conditions

Oral submucosal fibrosis,

cervical intra-epithelial

lesions etc.

Chromosomal aberrations,

chromosome loss/breakage,

mitotic apparatus dysfunctions,

aneuploidy,

genetic instability

Neoplastic

conditions

Cervical carcinoma, oral

carcinomas, breast

Primary or secondary DNA

damage, Aneuploidy,


15

carcinoma urothelial cancer,

Colon cancer

mitotic dysfunction genetic

instability

Genetic diseases Down syndrome, Xeroderma

pigmentosa

Various chromosomal aberrations,

DNA damage/breakage, genetic

instability

Infective Genital Chlamydiasis,

schistosoma haematobium

infection, Human papilloma

virus infection

Mechanisms exactly not known;

possibly interfere

With cellular division and

produce free radicals leading to

DNA damage. HPV is a DNA

virus and interferes with genes

involved in cell cycle.

Metabolic Megaloblastic anemia, vitamin

deficiencies

Interference with DNA synthesis

Details of the Methodology of MN Scoring on Smear

Either MGG or Papanicolaou‟s stained smear can be used for MN scoring. All the

cells with intact cell membrane should be included. Degenerated cells, cells with

obscured or altered morphology and large cell clusters or clumped groups should better

be avoided. Bi or multinucleated cells may show MN and should be counted and given a

score of one. Cells with multiple MN should be carefully looked for a possibility of

keratohyaline granules and if MN is confirmed morphologically, it should be given a

score of one. Overall score is usually expressed as number of micronucleated cells per

1,000 or 100 cells. With strict criteria, MN can be identified with confidence in

Papanicolaou‟s stained smear.26


16

Morphology of Micronucleus

1 Location

Intra cytoplasmic near the main nucleus; generally within inner half of

cytoplasm. If the main nucleus is oval or spindly, MN is generally close

to one end of it. MN is always separated from the main nucleus.

2 Size 1/16th to 1/3rd the diameter of the main nucleus

3 Staining Generally same intensity or of more intensity with the main nucleus.

Occasionally paler.

4 Texture Same as the main nucleus. May be more clumped. Perimeter is smooth

suggesting a membrane. Non-refractile.

5 Shape Mostly oval or round; may be pyramidal, hemispheric, elliptical,

cylindrical or very rarely irregular.

6 Number Generally occurs singly in a cell. Very occasionally double. Triple or

more not seen or rare.

7 Others Plane of focus coincides or nearly same as the main nucleus.

8 Cells of

occurrence

Seen in the benign-appearing cells as well as frankly malignant or

dysplastic cell

9 Mimickers Stain deposits, bacteria, nuclear dusts, clumped cytoplasmic fragments,

partial Karyorrhexis or necrotic nucleus, carried over nuclear fragment

from other cells.


17

MN SCORING CRITERIA

In 1976, Heddle introduced the scoring of micronucleus in lymphocytes after

exposure to genotoxic agents.29

Criteria for identifying micronuclei as given by Heddle

& Countryman (1976) are:

1. Diameter less than 1/3rd the main nucleus.

2. Non-refractility (to exclude small stain particles).

3. Color same as or lighter than the nucleus (to exclude large stain particles).

4. Location within 3 or 4 nuclear diameters of a nucleus; and not touching the

nucleus (to make frequency measurements meaningful).

5. No more than 2 micronuclei associated with one nucleus.

Belien et al [1995] also tried to make a standardization protocol for the counting of

micronuclei in exfoliated buccal cells.30

Paige E. Tolbert et al [1992] developed Criteria for the inclusion in the total cell

count which are the following: (1) cytoplasm intact and lying relatively flat; (2) little or

no overlap with adjacent cells; (3) little or no debris; and (4) nucleus normal and intact,

nuclear perimeter smooth and distinct.31

In order for a cell to be considered micronucleated, the cell must satisfy the above

criteria regarding inclusion in the total cell count and the putative micronucleus is

required to meet the following criteria: Fenech et al (2003)


18

1. The diameter of the MN should be less than 1/3rd

of the main nucleus

2. MN should be separate or marginally overlap from the main nucleus as long as

there is a clear identification of the nuclear boundary.

3. Micronucleus have similar staining as the main nucleus

Nuclear Abnormalities in Buccal Cells

Besides the presence of MN, Tolbert et al in 1991 described other nuclear

abnormalities that can occur not only during physiological cellular differentiation but also

during cell death with DNA damage.25

In the cells these changes are produced in the

nuclei, it can be easily differentiated from the normal cells. These nuclear abnormalities

[NA] are Karyorrhexis (KR), Pyknotic nuclei (PN), karyolysis (KL), condensed

chromatin (CC) and nuclear buds. Among the various abnormalities the present study

focused on Karyorrhexis, karyolysis and pyknosis. The mechanisms by which each of

these abnormalities produced or its biological meaning is still unknown. Raj et al in 2001

described MN and NA presence has an effect on dose response after radiotherapy in

patients with oral Squamous cell carcinoma and suggested the use of these biomarkers to

assess the radio sensitivity.32

These NA are considered to be a biomarker for DNA

damage (MN), cell death evidence (CC, KR, PN, KL), different stages of necrosis

indicators (PN, KR, KL) and identifying response to cell damage (PN).3


19

Karyorrhectic cell

The word is derived from Greek word ‘karyon’ meaning seed or nucleus and

‘rhexis’ meaning bursting. This is a destructive fragmentation of the nucleus of a dying

cell, where its chromatin is distributed irregularly throughout the cytoplasm. The cells

are characterized by more extensive appearance of nuclear chromatin aggregation. It is

usually preceded by pyknosis and can occur as a result of either apoptosis, senescence or

necrosis.

Pyknotic cell

The word pyknosis is derived from the Greek words ‘pyknono’ meaning to

condense or to thicken up. It is the irreversible condensation of chromatin in the nucleus

of a cell undergoing necrosis or apoptosis. The cells are characterized by a small

shrunken nucleus with a high density of nuclear material that is uniformly but intensely

stained. The nuclear diameter is usually 1/3rd

– 2/3rd

of that of a normal nucleus

Karyolitic cell

The word Karyolysis is derived from the Greek Word „karyon’ meaning seed or

nucleus and ‘lyein’ meaning to separate. There is a complete dissolution of the chromatin

of dying cells due to enzymatic degradation by endonucleases. It is usually preceded by

Karyorrhexis as a result of necrosis. The nucleus is completely depleted of DNA and

appears as a ghost like image that has no staining.


20

The International Collaborative Project on Micronucleus Frequency in Human

Populations was organized to collect data on micronucleus [MN] frequencies in different

human populations and different cell types in 1999 & 2003. The information will be used

to:

1. Determine the extent of variation of „normal‟ values for different laboratories and

the influence of other factors potentially affecting baseline MN frequency. Eg:

age, gender and life-style.

2. Provide information on the effect of experimental protocol variations on MN

frequency measurements.

3. Design and test optimal protocols for the different cell types.

4. Determine the extent to which MN frequency is a valid biomarker of ageing and

risk for diseases such as cancer.

B.J. Majer et al [2001] reviewed the use of the micronucleus assay with

exfoliated epithelial cells as a biomarker for monitoring individuals at elevated risk of

genetic damage and in chemoprevention trials and concluded that the MEC assay is a

useful biomarker for the detection of human cancer risk in organs.15

Baseline frequencies for micronucleated cells in the BM are usually within the

0.5-2.5 Micronuclei/ 1,000 cells range. Cells with multiple micronuclei are rare in healthy

subjects but become more common in individuals exposed to radiation or other genotoxic

agents.


21

PAP STAIN

It is a multi-chromatic staining cytological technique developed by George

Papanicolaou in 1942 and subsequently modified by him in 1952 and 1960.

Classically it consists of 5 dyes and 3 solutions. A haematoxylin used for staining

nucleus. Orange – G counter stain to stain keratin. Second counter stain consists of 2

dyes, Eosin Y to stain superficial epithelial squamous cells. Light Green SF which stains

cytoplasm of other cells including non-keratinized squamous cells. Advantages of this

staining technique are chromatin patterns are well visible in it. It also has increased

cytoplasmic transparency. Even though the conventional staining method takes 40

minutes rapid PAP staining kits are available which finishes total staining in 3 minutes.

MICRONUCLEUS IN TOBACCO USERS

A study was conducted in 1987 in 50 clinically normal individuals and divided

into those having smoking habit and non- smokers. The frequencies of micronuclei

resulting from chromosome were about double in smokers as compared with non-

smokers and the frequencies of micronucleus from spindle disturbances were

insignificant in them.24

In 1997 a study was carried out in 54 clinically healthy individuals to analyze the

genotoxic effects of smokeless tobacco, smoking and non-smokers/non-users. It was

found that the number of micronucleus was significant in smokeless tobacco users and

smokers with similar results.33


22

A study was conducted in 2001 among patients using khat (Catha Edulis- a

psycho stimulant plant leaf) consumers, tobacco users and alcohol habits and found an

increase in the number of micronucleus. They also added that the number was higher in

khat users and use of tobacco and alcohol had an added effect in the course of genetic

damage.34

A study carried out in 2011, among patients with pan chewing habits and gutkha

habit to find out the genotoxic effects on these agents using an MN assay found a

significant increase in the micronucleus and chromosomal aberration in those patients

and suggested the increased chances of cancer development in these patients.35

A cohort study was done in 2012, to assess the correlation of frequency of

micronucleus in smokers with the duration of habit. They found that the frequency of

micronucleus was higher in smokers than non- smokers and a direct correlation of

number of micronucleus with the duration of habit noted as their number increase with

duration of habit.36

A study done in 2013 to find out the alterations in buccal cells due to alcohol,

tobacco and combination of two. They found a significant increase in the number of

micronucleus in the patients with tobacco and alcohol habit than in control group. There

is no significant difference when compared with alcohol alone group with control group

and the results showed that these habits can produce cellular alterations which may

cumulatively lead to carcinomatous changes.37

A study conducted in 10 tobacco users in

the same year, to evaluate the presence of micronuclei and other nuclear abnormalities

and concluded that age, duration of exposure and other factors like smoking and intake of


23

alcohol affects the frequencies of nuclear abnormalities. They found no increase in the

number of micronucleus in this patients.38

A study was carried out in 2013 to find out the Genotoxicity associated with betel

quid chewers. They grouped 50 individuals as those with betel quid habit alone, betel

quid with tobacco and healthy individuals. They concluded that the number of

micronucleus was higher in individuals with both betel quid and tobacco habit followed

by tobacco habit alone. In contrast to other studies, in their study, there was a higher

frequency of micronucleus among males compared with females.39

MICRONUCLEUS IN CANCER PATIENTS

A cohort study was conducted in 2003 in occupationally exposed workers using

MN in peripheral blood lymphocytes as a cancer biomarker and found that smoking habit

has no influence on the frequency of MN frequency.40

A comparative study was carried out in 2004 in normal, pre malignant and

malignant epithelium using exfoliated buccal cells and found an increase in their number

from normal to premalignant then to malignant epithelium.41

In 2005 a study was carried among oral Squamous cell carcinoma patients. They

found that the number of micronucleus was high at the time of diagnosis and there was an

increase in their number immediately after exposure to radiation therapy and suggested

the use of micronucleus assay as a marker to identify the tumor sensitivity.42


24

A study was conducted in 2007 among cancer patients, patients in pre-cancer

stage and normal patients using MN assay. A step wise increase in the number of MN

from normal to premalignant and from premalignant to malignant patients was noted.

They concluded that the MN assay can be used as a prognostic indicator in this

lesions.43

In the same year; a study was conducted among patients with erosive and

atrophic lichen planus for the assessment of micronucleus in exfoliated cells and found a

significant increase in the MN frequency in these lesions than in normal individuals.44

In 2010, a study was conducted among 20 patients who had white lesions in the

oral cavity. All the lesions were stained with Toluidine Blue and samples were taken for

cytological evaluation. It is concluded that the frequency of MN were higher in those

patients, irrespective of TB staining.45

In 2010, a prospective study was conducted among breast cancer patients with

benign cases as control group. The patients were diagnosed by FNAC. They used

Acridine Orange, a fluorescent dye for the identification of micronucleus and found an

increase in their frequency.46

A study conducted in 2011 among patients who have potentially malignant

disorders and malignant lesions the presence of micronucleus and found that the number

of micronucleus was higher in malignant group than in patients who are diagnosed with

potentially malignant disorders. However, the number was higher than the clinically

healthy individuals who are the control group.47


25

In 2014 a study was conducted among normal patients, leukoplakia, and

Squamous cell carcinoma using PAP staining and found that MN were higher in SCC

than in leukoplakia and healthy control. Hence, MN assay can be used as an important

biomarker for cytogenetic damage in oral leukoplakia and SCC.48

A study done among patients who are having tobacco related potentially

malignant disorders using MN assay. The sample was collected from the exfoliated

buccal epithelial cells of 8 leukoplakia, 7 OSMF patients and 15 healthy controls and

found a significant increase in their number in patients diagnosed with PMDs compared

to control group.49

MICRONUCLEUS IN OTHER CLINICAL SETTINGS

In 2001 a study was carried among 49 male workers who are exposed to

pesticides to assess the genotoxic effects of these chemicals with the help of MN assay in

peripheral blood lymphocytes and buccal epithelial cells. The results showed no increase

in the number of micronucleus in the samples collected from both sites. No correlation

was drawn with smoking and other habits.50

In the year 2010 a study was conducted among 23 chronic denture stomatitis

patients and analyzed the cytological changes comparing with the control group. The

results showed an increase in the cytological changes among these patients.51


26

A study was done in 2011 among patients wearing dental prosthesis having white

lesions in the buccal mucosa. These lesions were stained with Toluidine Blue and were

categorized into positive and negative. The frequency of micronucleus increased in white

lesions but the increase in number was irrespective of vital staining.45

A study was carried among children in the same year, to evaluate the genotoxic

effects of various dental materials through MN assay. Buccal epithelial cells were

collected from children of various ages ranging from 4yrs to 12 years, who were

undergoing same type of dental treatment. Cells were collected before and at the end of

treatment. There was a significant increase in the number of bi-nucleated cells after the

treatment with cement based dental materials and monomer. They concluded that the

combination of cement-based dental material with monomers increases the cytotoxic

action of dental materials.52

In the same year, a study was done among full time tannery workers for the

assessment of Genotoxicity using MN assay. The samples were collected from buccal

epithelial cells. There was a significant increase in the MN frequency in them. There is a

direct relationship between the duration of exposure, smoking and alcoholism with that of

MN frequency.53

A study was conducted in 2012, among 10 chronic Periodontitis and 10

aggressive Periodontitis patients to find out DNA damage. They evaluated the presence

of micronucleus in peripheral lymphocytes but there was no correlation between two

groups.54


27

MICRONUCLEUS IN IONIZING RADIATION

In the year 2000 a study carried among hospital workers who are occupationally

exposed to ionizing radiation using micronucleus centromere assay in peripheral

lymphocytes and found a higher number in them. In the studied population, smoking did

not yield any significant correlation.55

In the same year, a study was conducted among

workers in radiation department, to estimate the extent of exposure to workers using

peripheral lymphocytes. Even though the number of micronucleus was higher than the

control group, the annual dose of exposure was below the whole body exposure per

year.56

A similar study was conducted in 2002 aimed at preventing the risk of cancer

from exposure to ionizing radiation among hospital workers occupationally exposed to

these kinds of radiations. But even though the number of MN was higher than the control

group, it was not statistically significant. They also found that there is an increase in the

MN frequency in workers who are exposed to the smoking.57

In order to assess occupationally induced chromosomal damage, a study was

conducted among 29 industrial radiographers in 2002 and found an increase in the

number of micronucleated cells in them than control group. The increase in the number

of micronucleus correlates with age and it did not have any correlation with the duration

of exposure.58

A study was carried out in 2004, to find out the genotoxic effects of ionizing

radiation in patients exposed to dental panoramic radiographs. The results suggest that

the ionizing radiation induces a cytotoxic effect by increasing apoptosis and other nuclear


28

alterations and the number of micronucleus was insignificant. In addition, they concluded

that the micronucleus improves the sensitivity in the detection of genotoxic effects.59

A study conducted in 2005, to assess the frequency of micronuclei (MN), which is

an indicator of DNA damage, in a population exposed to low levels of ionizing radiation

and found a significantly higher MN frequency than control group. In contradiction to

other studies they found that the smoking status did not affect micronuclei frequency

either in exposed workers or controls, while age was associated with increased MN

frequency in the exposed only.60

A study was conducted in 32 non- smokers who had been subjected to panoramic

radiographs to assess the possible genotoxic effect of radiation exposure for dental

diagnostic purpose and no significant increase in the frequency of cells with micronuclei

and total number of micronuclei after panoramic tomography was detected. But a

significant correlation between the age of investigated subjects and the initial frequency

of micronuclei in buccal cells was observed.61

In the same year, nuclear changes in tongue epithelial cells following panoramic

radiography was studied and the radiation emitted during panoramic radiographs

increased the number of nuclear anomalies (except micronuclei) in exfoliated cells of the

lateral border of the tongue. This effect was more pronounced when the patients were

exposed to a repeat radiograph, without however implying the increased risk of

irreversible tissue damage.62


29

In the same year, a study was carried out among children following exposure to

radiation during dental radiography to evaluate DNA damage and cellular death.

Exfoliated cells are taken from buccal mucosa for cytological analysis. The results

indicates that the panoramic radiography might not induce chromosomal damage, but

may be cytotoxic.63

In 2008, s study was carried out to find out variation in the DNA damage

[micronucleus] and cellular changes like pyknosis, Karyorrhexis, karyolysis in adult and

children who are undergoing diagnostic radiographs. The results showed no significant

variation in the number of micronucleus and other nuclear alterations among adult and

children. The frequency of nuclear alterations except micronucleus was increased in both

groups after exposure.64

A study was done to assess the genotoxic effects of individuals who are subjected

to CBCT in 2010. They also assessed the other nuclear alterations like Karyorrhexis,

karyolysis and pyknosis. The results showed no significant increase in the MN frequency.

But the number of other nuclear alterations got increased to a significant level. They

concluded that cone beam CT may not be a factor that induces chromosomal damage, but

it is able to promote cytotoxicity.65

In the same year, a study was carried out in 18 healthy individuals who had taken

lateral and frontal radiographs for orthodontic purpose and evaluated DNA damage

(micronucleus) and cellular death (pyknosis, karyolysis and Karyorrhexis) in exfoliated

buccal mucosa cells and after the analysis of results, they concluded that the radiography


30

may not be a factor in inducing chromosomal damage, but it does promote cytotoxicity as

there is an increase in the nuclear alterations except micronucleus.66

In the same year, DNA damage and cellular death in oral mucosa was evaluated

by comparing the samples taken from buccal mucosa and lateral border of tongue. The

samples were taken from both smokers and non- smokers. They found that X-ray

exposure were able to increase nuclear alterations closely related to cytotoxicity. The

most pronounced effects were seen in lateral border of tongue of smokers. They

concluded that the panoramic radiography is able to induce cellular death in both buccal

mucosa and tongue. The lateral border of tongue is more sensitive to cytotoxic insult

induced by ionizing radiation combined with continuous cigarette smoke exposure.67

The genotoxic effect of panoramic radiographs in children was evaluated in the

same year from the cytological analysis of buccal epithelial cells of 20 healthy children

who underwent panoramic dental radiography for diagnostic purpose. The cells were

attained with Feulgen reaction. There was no significant Genotoxicity seen. But there was

a significant increase in the Karyorrhexis and pyknosis, while karyolysis did not show

significant difference.68

In the year 2011, DNA damage and cellular death in exfoliated buccal mucosal

from individuals following digital lateral radiography was evaluated. The results pointed

out no statistically significant difference of MN in oral mucosal cells. But it was able to

increase other nuclear alterations like Karyorrhexis, karyolysis, pyknosis which are

closely related to cytotoxicity.69


31

In the year 2012, a study was done to evaluate genotoxic and cytotoxic effects of

low level ionizing radiation used in digital panoramic radiography on gingival epithelial

cells. The frequency of formation of micronuclei was not significant with regard to age,

gender and after exposure to digital panoramic radiography. However this study showed

significant increase in the frequencies of nuclear alterations like Karyorrhexis, pyknosis,

condensed chromatin, karyolysis and indicative of cell death.70

In the same year a similar study was carried out but on buccal epithelial cells.

They assessed the MN frequency and other nuclear projections (buds and broken eggs)

and degenerative nuclear alterations (condensed chromatin, Karyorrhexis, karyolysis).

The results showed significant increase in MN frequency and other nuclear alterations.

They also showed that these alterations are higher in females than that of males.71

A study done in the same year in 60 clinically healthy individuals who underwent

panoramic radiography to evaluate the genotoxic effects using MN assay. The results

showed an increased frequency of micronucleus after exposure to ionizing radiation.

They concluded that considering the risk, panoramic radiographs should be advised

carefully.72

In the same year a study was conducted among clinically healthy children

undergoing orthodontic treatment. They were advised to take a complete set of

orthodontic radiographs, which includes lateral cephalographs, posteroantero-

cephalographs, panoramic radiographs, IOPA full set and bitewing. The cells were

analyzed for the genotoxic and cytotoxic damage. No significant increase in the MN


32

frequency was seen. But there was rise in other nuclear alterations like Karyorrhexis,

karyolysis, pyknosis.73

A study was done in the same year to compare the effects of different types of

radiographic techniques using buccal cytochrome assay. The study comprised of 90

patients who are grouped into 3. First being patients taking IOPA radiographs, second

was those taking digital OPG and third were patients who are taking conventional OPG.

But the results came similar to the other studies which showed an increase in the

cytotoxicity without increase in MN frequency.74

In the same year a study was done to compare the effects of X- rays on gingival

and buccal epithelial cells using MN assay. 80 clinically healthy individuals were

selected for the study. Cells were collected from the maxillary anterior gingiva and

buccal mucosa. The slides were stained using PAP stain. Significant increase in the

number of micronucleus was noted in the anterior gingiva while buccal mucosa showed

no increase in the MN count. They concluded that the radiographs should be taken with

adequate protection measures and only when potential benefits outweigh the risk factor.2

In 2013, a comparative study was done using CBCT and conventional radiograph

for the assessment of genotoxic and Cytotoxic effects. The buccal mucosal cells were

collected from the patient before and 10 days after exposure. After the cytological

analysis, there is no increase in the number of MN which was indicator of the

Genotoxicity seen in either group, while there is a significant increase in the cytological

change. CBCT group presented a greater increase in the cell death than conventional


33

radiographic group. They concluded that this variation may be due to increase exposure

of CBCT used for taking radiographs for orthodontic planning.75

A study conducted in 2014 to evaluate the possible genotoxic effects of

panoramic radiation by assessing the frequency of micronuclei formation in the

exfoliated buccal epithelium. They found that the X-radiation emitted during panoramic

radiography does induce some genotoxic changes in the form of increased frequency of

micronuclei in target buccal epithelial cells.76

A similar study to evaluate the possible genotoxic effects of routinely used

panoramic exposure in exfoliated buccal cells and keratinized gingiva which is measured

by micronucleus formation and concluded that the genotoxic effects of radiation exposure

from panoramic radiography showed a statistically significant increase in the MN

frequency in the buccal epithelial cells. But the correlation of age and the MN frequency

was noted in keratinized gingival cells. No such correlation was noted in buccal epithelial

cells.77

MATERIALS & METHODS

Materials & Methods

34

The study was planned & conducted in the department of Oral Medicine &

Radiology, Sree Mookambika Institute of Dental Sciences to find out the genotoxic and

cytotoxic changes among patients exposed to digital radiographs.

SAMPLE SIZE & ITS CHARACTERISTICS

The patients for the study were selected among the outpatients who visited the

department of Oral Medicine and Radiology. The study group comprised of clinically

healthy subjects who needed orthodontic treatment. Sample consists of 127 patients.

Sample size was calculated using software GPower version 3.1.7.

a. Inclusion criteria:

Patients undergoing digital radiographic examination for

orthodontic purposes

b. Exclusion criteria:

Patients undergoing radiographic examination for reasons other

than orthodontic treatment

Patients of age > 40 yrs

Patients who have a history of radiation exposure within the last

one year

Materials & Methods

35

Patients with any pre - malignant lesions/ conditions on the buccal

mucosa

Patients with malignant lesions

Patients with ulceration on the buccal mucosa

Patients with habits of smoking, alcoholism or tobacco chewing

Patients wearing prosthesis

Patients with any other clinical abnormalities on the buccal mucosa

After screening, information on the nature and potential benefits of the study was

explained to the patient. They were made to understand that the participation in

this study was purely voluntary and they had the option of exiting the study at any

point of time. And among the consenting volunteers who fit the inclusion and

exclusion criteria were selected. A formal informed written consent was taken

from all of them. A study protocol was approved by the Research Committee and

the Institutional Human Ethics Committee of Sree Mookambika Institute of

Medical Sciences. Kulasekharam. A detailed case history of the patient was taken

and a thorough clinical examination was done and recorded. Serial number was

also assigned to each patient.

Materials & Methods

36

Armamentarium

a) For sample selection and case history

A pair of sterile disposable gloves and disposable mouth masks

Stainless steel kidney tray, mouth mirror, straight probe and

tweezer

Glass tumbler with water

b) For taking digital radiographs

Planmeca digital orthopantomograph machine Prolin XC

c) For sample collection

Microscopic slide

Sterile wooden spatula

Bio spray in the RAPID – PAPTM

kit for fixing the sample

d) For staining the sample slide

RAPID – PAPTM

staining kit

Coplin jars

Filter/tissue paper

Cover slip

e) For cytological examination

Light microscope

AP View software

Materials & Methods

37

Brief procedure:

Sample collection

Patients were asked to rinse their mouth with water before making digital

radiographs. This helps to remove any food or artifacts that may interfere with the

analysis. Buccal epithelial cells were obtained using brush biopsy technique using a

wooden spatula. Oral mucosa is the first line of contact with hazardous agents and the

first barrier against potential carcinogens and is therefore susceptible to damage by these

agents before reflecting its effects systemically. It also provides an easy access to

samples collection. This technique is minimally invasive and painless and for these

reasons it is well tolerated among patients. After drying, the slides were fixed using

Biofix spray which comes along with the RAPID – PAP kit.

Digital orthopantomographs and lateral cepholograms were taken for the above

subjects with exposure of 68kV; 11mA; 18sec for orthopantomograph and 68kv 5mA;

17sec for lateral cepholograms.

The turnover rate for the appearance of MN in exfoliated buccal cells in otherwise

normal cell after exposure to an acute genotoxic event like ionizing radiation is estimated

to be between 8-12 days. The patient was recalled after 10±2 days and the sample was

collected again from the buccal mucosa using the same procedure because peak

expression of MN may vary depending on the effects of particular DNA damage on basal

cell turnover rate.

Materials & Methods

38

Nearly 60% of oral mucosal surface is stratified non-keratinized epithelia, which

allows cells in the most superficial layer to maintain their nuclei well defined and almost

intact which favours colorant absorption, ease of observation, proper identification of

nucleus and morphological characteristic of cell with the help of microscope.

Staining of slides

The colorant used should be basic and must have a high affinity to DNA in order

to obtain a contrast so that artifacts can be easily differentiated. In this study DNA

specific Papanicolaou stain was used. It is an acidic – basic stain which can produce a

contrast between the cytoplasm and nucleus and there by helps in reducing the bias

produced when non specific DNA stains are used.

The sample slides were stained using RAPID PAPTM

Papanicolaou’s stain kit.

It is a user friendly PAP staining kit by BIOLAB DIAGNOSTICS. Unlike the

conventional PAP staining, this kit allows the staining of slides in 3 minutes. It requires

minimum skills and laboratory aids for good results.

The RAPID – PAPTM

staining kit consists of

Biofix spray : for cell fixation

RAPID - PAPTM

NUCLEAR STAIN Haematoxylin solution

RAPID – PAPTM

CYTOPLASM STAIN OG-6 Solution [2A]

Materials & Methods

39

RAPID – PAPTM

CYTOPLASM STAIN Light Green SF-

Eosin[2B]

D.P.X GLASS MOUNTING MEDIUM

RAPID – PAPTM

DEHYDRANT Propanol

XYLENE

RAPID – PAPTM

WASH BUFFER Scotte’s tap water buffer

Haematoxylin nuclear stain is a natural stain which has an affinity for chromatin,

attaching to sulfate group in DNA molecule. It acts as acidic stain for basic

neutroproteins. Its pH is 2.5-3

Scotte’s tap water buffer: the pH of this solution varies from 8-8.5. This subjects

haematoxylin to alkaline conditions and changes its color from red to blue.

Alcohol acts a dehydrant which helps to minimize cellular distortion, reduce cell

lose from glass slide.

OG-6: it is an acidic dye that demonstrates attraction to the basic proteins like

parakeratin. It is a monochromatic stain which colors keratin brilliant green. The effect of

OG - 6 is evident if the smear has keratinized cells.

Light Green SF- Eosin:

Eosin gives pink color to cytoplasm of mature squamous cells and nucleoli.

Materials & Methods

40

Light green stains cytoplasm of metabolically active cells like parabasal cells,

intermediate squamous cells in blue. It has a C2H5N+ reactive group which possesses an

affinity for RNA of ribosomes.

Xylol: It acts as a clearing agent. It is a colorless, chemically non- reactive and has almost

the same refractive index as that of glass. Thus it helps to give transparency to image.

D.P.X Mountant: it acts as a permanent bond between the slide and cover slip, protects

the cell material from air drying and shrinkage. It also acts as a seal against oxidation and

fading of stain.

Reagent Preparation: cytoplasm stain is prepared by mixing equal volume of cytoplasm

stain 2A and 2B. It is then stored in an air tight bottle to prevent contamination from

water and other pollutants.

Steps in slide staining using RAPID PAPTM

kit

1. Dip the fixed smear for 3 minutes in tap water and blot the excess water

from the slide.

2. Dip 60 seconds in PAPID PAPTM

Nuclear Stain.

3. Add 3 drops of Scott Tap Water buffer and wash after 10 seconds. Blot

out the excess water from the slides.

4. Dip with the two changes in RAPID PAPTM

dehydrant for 30 seconds

each.

Materials & Methods

41

5. Dip in 45 seconds in working cytoplasm stain.

6. Wash in tap water and blot out excess water from the slide

7. Repeat dehydration in a second bath of RAPID PAPTM

dehydrant for 30

seconds and dry.

8. Dip in xylene, dry and mount with cover glass using a drop of D.P.X.

Cytological examination

The stained slides are examined using light microscope first in 10X magnification

and then 40X magnification. AP viewer software is used for the visualization of cells and

for taking photomicrographs. The cells are selected for scoring as per Tolbert’s criteria

(1992) (1) cytoplasm intact and lying relatively flat; (2) little or no overlap with adjacent

cells; (3) little or no debris; and (4) nucleus normal and intact, nuclear perimeter smooth

and distinct.

The micronucleus is identified with the help of the suggested criteria by Fenech et

al 2003

1. The diameter of the MN should be less than 1/3 rd

of the main nucleus

2. MN should be separate or marginally overlap from the main nucleus as

long as there is a clear identification of nuclear boundary.

3. MN have similar staining as the main nucleus

Materials & Methods

42

Apart from micronucleus, other cellular alterations are considered for the

cytological analysis. Among the various cellular alterations, the present study analyzed

the presence of Karyorrhexis, karyolysis, pyknosis.

Karyorrhectic cell: cells are characterized by more extensive appearance of nuclear

chromatin aggregation leading to fragmentation and eventual disintegration of nucleus

Pyknotic cell: cells are characterized by a small shrunken nucleus with a high density of

nuclear material that is uniformly but intensely stained. The nuclear diameter is usually

1/3rd

– 2/3rd

of normal nucleus

Karyolitic cell: nucleus id completely depleted of DNA and apparent as a ghost like

image that has no staining.

In the present study, with strict criteria, 100 cells in each slide were analyzed for

the presence of micronucleus and other nuclear alterations. The results were sent for

statistical analysis.

COLOUR PLATES

Colour plates

CP - 2: PLANMECA PROLIN XC MACHINE

CP – 1: ARMAMENTARIUM FOR SAMPLE COLLECTION

Colour plates

CP – 3: RAPID PAPTM

STAINING KIT

CP – 4: STAINED SLIDES IN SLIDE BOX

Colour plates

CP – 5: LIGHT MICROSCOPE & AP VIEWER

RESULTS & OBSERVATIONS

Results & Observations

43

The purpose of the study was to evaluate the genotoxic and Cytotoxic changes

after exposure to low level ionizing radiation. The study comprises of evaluating the

Genotoxicity by micronucleus assay and cytotoxicity by the presence of Karyorrhexis,

karyolysis, pyknosis before exposure and 10 days after exposure.

Statistical analysis: The data was expressed in means and standard deviation.

SPSS version 17.0 was used for statistical analysis. Paired t test was applied to find

statistical significant within the groups. P≤ 0.05 considered statistically significant.

Two samples were taken from 128 patients, one before exposure and one 10±2

days after exposure. In the total sample, 22 patients were males and 105 patients were

females. Total number of cells among all the samples having micronucleus was found

to be 245 before exposure and 1240 after exposure. Total number of micronucleus in

245 cells was 272 and in 1240 was 1575. A comparison of micronucleus was made

before and after exposure using paired t test. The mean value before exposure was

1.92±1.93 and after 9.76±6.70. The total number of cells showing Karyorrhexis was

392 before exposure and 462 after exposure. The mean value was 3.08±3.26 before

and 3.63±3.23 after exposure. The total number of cells showing karyolysis was 661

before and 881 after exposure. The mean value was 5.27±4.53 before and 6.38±5.44

after exposure. The total number of cells showing pyknosis was 815 before and 1011

after exposure. It has a mean value of 6.41± 4.36 before and 7.96±6.31 after exposure.

Gender wise comparison was made for each parameter. Among males the total

number of micronucleated cells was 33 before and 212 after exposure. The number of

Karyorrhectic cells was 51 before and 93 after exposures. The number cells showing

karyolysis was 116 before and 115 after exposure to X-rays. The number of Pyknotic

cells was 163 and 156 before and after exposure. Among females, the total number of


44

micronucleus was 212 before exposure and 1028 after exposure with a mean of 2.01

and 9.76 respectively. The number of cells showing Karyorrhexis was 341 and 369

before and after exposure with a mean of 3.24 and 3.51 respectively. Number of

karyolitic cells in females before and after exposure was noted as 545 and 696 and the

mean values were 5.19 and 6.62. The number of Pyknotic cells was counted and it

was 652 and 855 before and after exposure with a mean of 6.2 before exposure and

8.14 after exposure.

Overall it was seen that both cytotoxic and genotoxic effects were increased

following radiation exposure.

TABLE 1: Distribution of study subjects according to age and gender

Gender Total number Mean Age t test p value and

significance

Males 22 21.95±2.33

1.147

0.253

NS Females 105 21.46±1.68

Total 127 21.55±1.81

P<0.05, NS – Non Significant

Micronucleus analysis

Samples collected from 127 patients, before and 10±2 days after exposure

were analyzed for the presence of micronucleus. 100 cells were examined in each

slide. The number of micronucleated cells were counted. Micronuclear cell frequency

was also noted along with it.


45

Light microscope study

IMAGE 1: SHOWING NORMAL CELL AT 40 × MAGNIFICATION

IMAGE2: SHOWING MICRONUCLEATED CELL AT 40 × MAGNIFICATION


46

IMAGE 3: SHOWING MICRONULEATED CELL WITH TWO

MICRONULCEUS AT 40 × MAGNIFICATION

IMAGE 4: SHOWING MICRONLCEATED CELL WITH MULTIPLE

MICRONUCLEUS AT 40 × MAGNIFICATION


47

Image 1 shows the photomicrograph of a normal cell in 40 × magnification. It

is used as a standard for the size of normal nucleus. Image 2, image 3, image 4 are the

photomicrograph taken using AP viewer in the same magnification, which showed the

presence of micronucleus [single or multiple].

TABLE: 2 Total No of cells having Micronucleus before and after radiation/100 cells

Total No of cells having MN before

radiation

Total No of cells having MN after

radiation

245 1240

TABLE: 3 Total No of Micronucleus before and after radiation/100 cells

Total No of MN before radiation Total No of MN after radiation

272 1575

TABLE: 4 Mean value & standard deviation of MN assay

Comparison Mean Std. Deviation Paired t value P value

MICRO

NUCLEUS

Before 1.9291 1.93621

12.132 0.000

HS After 9.7638 6.70046

Table 2 shows the number of micronucleated cells while table 3 shows the

number of micronucleus seen in examination of all the samples taken before and after

radiation exposure. Table 4 shows the mean values and standard deviation of the

micronucleus examined. There is a steady increase in the number of micronucleated


48

cell after radiation with highly significant P value of 0.000. Micronuclear cell

frequency also showed an increase after radiation exposure.

Other nuclear alterations

Samples taken from the patients before and 10±2 days after radiation exposure

are analyzed for other nuclear anomalies. In the present study examination of

Karyorrhexis, karyolysis and pyknosis was done among 100 cells in each slide.

IMAGE 5: SHOWING KARYORRHEXIS OF THE NUCLEUS IN 40 ×

MAGNIFICATION

TABLE: 5 Total No of cells having Karyorrhexis before and after radiation/100 cells

Total No of cells having Karyorrhexis

before radiation

Total No of cells having Karyorrhexis

after radiation

392 462


49

IMAGE 6: SHOWING KARYOLYSIS IN CELL AT 40× MAGNIFICATION

TABLE: 6 Total No of cells having Karyolysis before and after radiation/100 cells

Total No of cells having Karyolysis

before radiation

Total No of cells having Karyolysis

after radiation

661 881

TABLE: 7 Total No of cells having Pyknosis before and after radiation/100 cells

Total No of cells having Pyknosis

before radiation

Total No of cells having Pyknosis after

radiation

815 1011


50

IMAGE7: SHOWING PYKNOTIC NUCLEUS AT 40 × MAGNIFICATION

Image 5, image 6, image7 are the photomicrographs taken with the help of AP viewer

in 40 × magnification for showing nuclear alterations like Karyorrhexis, karyolysis

and pyknosis.

TABLE 8: Mean value & standard deviation of cytological changes

Comparison Mean Std.

Deviation

Paired t

value

P value

KARYORRHEXIS Before 3.0866 3.26361

1.372 0.173

NS After 3.6378 3.23343

KARYOLYSIS Before 5.2047 4.53442

1.906 0.059

NS After 6.3858 5.44824

PYKNOSIS Before 6.4173 4.36334

2.304 0.023

S After 7.9606 6.31501


51

Table 8 shows the mean and standard deviation of other nuclear alterations.

There is a statistically significant increase in the pyknosis after radiation with a P

value of 0.023. The other nuclear alterations like Karyorrhexis, karyolysis also shows

an increase after radiation even though it is not statistically significant.

Gender wise comparison was made for micronucleus, Karyorrhexis, karyolysis

and pyknosis.

TABLE: 9 Gender variations in micronucleus count

GENDER MN cell count before exposure MN cell count after exposure

MALES 33 212

FEMALES 212 1028

TABLE: 10 Gender wise comparison of micronucleus

GENDER MN before exposure MN after exposure

MALES 1.5 9.636

FEMALES 2.019 9.76


52

TABLE: 11 Gender wise comparison of Karyorrhectic cell count

GENDER Karyorrhectic cell count

before exposure

Karyorrhectic cell count after

exposure

MALES 51 93

FEMALES 341 369

TABLE: 12 Gender wise comparison of Karyorrhexis

GENDER Karyorrhexis before exposure Karyorrhexis after exposure

MALES 2.318 4.227

FEMALES 3.247 3.514

TABLE: 13 Gender wise comparison of karyolitic cell count

GENDER Karyolitic cell count before

exposure

Karyolitic cell count after

exposure

MALES 116 115

FEMALES 545 696


53

TABLE: 14 Gender wise comparison of karyolysis

GENDER Karyolysis before exposure Karyolysis after exposure

MALES 5.272 5.227

FEMALES 5.19 6.628

TABLE: 15 Gender wise comparison of Pyknotic cell count

GENDER Number of Pyknotic cell

before exposure

Number of Pyknotic cell after

exposure

MALES 163 156

FEMALES 652 855

TABLE: 16 Gender wise comparison of pyknosis

GENDER Pyknosis before exposure Pyknosis after exposure

MALES 7.409 7.09

FEMALES 6.209 8.142

GRAPHS

Graphs

Graph1: Distribution of study subjects according to gender and age

Graph 2: Mean changes in Genotoxic and Cytotoxic parameters before

and after radiation exposure among study subjects

0

20

40

60

80

100

120

Males Females

22

105

21.95 21.46

Co

un

t

Gender

Total number

Mean Age

0

1

2

3

4

5

6

7

8

9

10

Before After Before After Before After Before After

MICRO NUCLEUS KARYOHEXIS KARYOLYSIS PYKNOSIS

1.9291

9.7638

3.0866 3.6378

5.2047

6.3858 6.4173

7.9606

Co

un

t

Graphs

Graph 3: Gender wise comparison for micronucleus before and after

radiation exposure

Graph 4: Gender wise comparison for Karyorrhexis before and after

radiation exposure

0

1

2

3

4

5

6

7

8

9

10

Before radiation exposure After radiation exposure

1.5

9.636

2.019

9.79

Me

an C

ou

nt

Males

Females

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

Before radiationexposure

After radiation exposure

2.318

4.227

3.247 3.514

Me

an C

ou

nt

Males

Females

Graphs

Graph-5: Gender wise comparison for Karyolysis before and after

radiation exposure

Graph – 6: Gender wise comparison for Pyknosis before and after

radiation exposure

0

1

2

3

4

5

6

7

Before radiationexposure

After radiation exposure

5.272 5.227 5.19

6.628 M

ean

Co

un

t

Males

Females

0

1

2

3

4

5

6

7

8

9

Before radiation exposure After radiation exposure

7.409 7.09

6.209

8.142

Me

an C

ou

nt

Males

Females

DISCUSSION

Discussion

54

Cancer is a complex disease caused by altered gene expression resulting in

abnormal uncontrolled growth of tissue. The mutations in various genes causes

acceleration in cell division and inhibition of apoptosis, which is programmed cell death.

The amount of mutations accumulated within cancer cells cannot be explained solely by

the normal point mutation rate. There are several factors which can induce these kinds of

mutations and ionizing radiation is one among them. Because cancer may take several

years to develop and it is not always practicable to do epidemiological studies over such

long periods, use of biomarkers for the analysis of DNA damage can be used as a

predictive indicator of cancer risk.78

In medical and dental practice, x rays are used for diagnosis and treatment. It is a

well known fact that the ionizing radiations can induce DNA damage including double or

single strands breakage or DNA protein cross link damage. The specific character of

ionizing radiation which differentiates it from other physical carcinogens is its ability to

penetrate cells and deposit energy within the cells in random fashion, unaffected by usual

cellular barriers when presented to chemical agents. In this way, all cells in the body are

susceptible to damage caused by ionizing radiation. The amount of damage is related to

the physical parameters that determine the radiation dose received by particular tissue or

cell. By 1980, radiation induced cancer (in vivo) and cellular transformation (in vitro)

were well established.6 Considering the association of ionizing radiation and

carcinogenesis, various analyses have been introduced to detect the effects of low level

ionizing radiation used in diagnostic radiology.

Discussion

55

The purpose of the present study was to evaluate the genotoxic and Cytotoxic

changes in patients after exposure to low level ionizing radiation.

Various biomarkers like assessment of metaphase chromosomal aberrations, sister

chromatid exchange can be used to determine the effects of ionizing radiation. But these

methods are very technique sensitive, expensive, time consuming and required expertise

to interpret the slides. Hence application of micronucleus test and other nuclear

anomalies in uncultured exfoliated cells has received great interest. These biomarkers

offer additional end points for possible evaluation of chromosomal instability, gene

amplification and different cell death events.

MN is formed during the transition metaphase-anaphase of the mitosis and they

are chromosomes left out as a result of mitotic apparatus damage [aneuploidogenic

effect] or chromosome fragments without centromere [clastogenic damage]. In both

cases, these genetic materials were unable to incorporate into daughter cells and can be

visible as a round or oval cytoplasmic chromatin masses next to the nucleus. They are

identified using the criteria put forward by Fenech et al in 2003.

Cytokinesis – Block MN assay in lymphocytes is the most popular technique of

MN assay. But lymphocytes must be stimulated to undergo mitosis and the changes are

visible at a later phase. But in case of diagnostic radiology in dental practice, the target

tissue of interest is buccal epithelial tissue. Epithelial cells need not be stimulated. These

cells show genotoxic effects within 1-3 weeks earlier than the lymphocytes. Epithelial

cells have high proliferative capacity and more than 90% of human cancers are epithelial

in origin. Therefore the application of MN test in epithelial cells is considered to a

Discussion

56

sensitive tool for assessing genotoxicity. Cells of buccal mucosa have limited DNA repair

capacity when compared to lymphocytes and therefore more accurately reflect genomic

instability. It is simple non- invasive and has good patient compliance.11

The samples for the study were taken before exposure and 10±2 days after

exposure. The harmful effects of ionizing radiation take place in the basal layer of

epithelium where cells undergo mitosis. Rapid turnover of the epithelial tissue brings

these cells to the surface when they exfoliate. Hence maximal rate of micronucleus

formation in exfoliated cells is seen 1-3 weeks after exposure to genotoxic agents.64

For

this reason a period of 10±2 days after exposure was adopted for the study.

Several staining methods have been used for the evaluation of micronucleus.

Although DNA specific stains are preferred for staining MN, the most commonly used

staining procedure is Feulgen staining reaction. However the staining procedure is

lengthy and also technique sensitive and may lead to under scoring of MN. In the present

study, PAP stain is used. It is done using RAPID- PAP TM

kit. It is rapid, easy to read and

the fixative used has a bactericidal effect and the nuclear stain stains both intranuclear

and extra nuclear DNA and cytoplasmic stain helps to maintain the transparency of the

cytoplasm and makes boundaries well demarcated.79

The added advantage is that it can

differentiate between cytoplasm of non- keratinized and keratinized cells where it appears

as blue/ green in former and pink/orange in latter. The nucleus appears blue/black.80

The present study consists of individuals with age ranging from 19-27years with a

mean of 21±1.8 and showed no statistical significance with respect to age. [P=0.253] This

was in accordance with a previous study conducted in Brazilian population with a mean

Discussion

57

age of 21 years.81

The influence of tobacco and alcohol is considered as a relevant factor.

In the study done by Stich & Rosin a synergistic effect in the formation of MN frequency

was noted in these patients.23

Thus in the present study all the patients recruited did not

have any habits. Based on the recent studies done by Shally Khanna et al on oral

squamous cell carcinoma patients and Uma et al on potentially malignant disorders,

where they found an increase in the number of micronucleus in these conditions, they are

excluded from the study.48, 49

A study done by Pollyanna Francielli et al among patients

wearing prosthesis who presented with white lesions on the buccal mucosa also showed

an increase in MN frequency.45

Hence these patients are also excluded from the study to

reduce bias. It has been postulated that as age advances, there is a progressive increase in

the spontaneous chromosome instability, decline in the DNA repair capacity and

increased chromosome loss. This leads to increase in the MN frequency as the age

advances.61

So patients of age more than 40 years were also not includes in the study.

A comparison of the MN values before and 10 days after radiation exposure was

calculated among 100 cells in each slide. There is a significant increase in the MN count

after exposure to ionizing radiation. Both the number and mean of micronucleated cells

was 245 before and after 1240 and 1.93±1.94 before and 9.76±6.70 after exposure with a

highly significant P value (P value: 0.000) This correlates with the studies done by

Sabrina Angeli.60

The Micronuclear cell frequency is increased [before: 272; after:

1575]. This result is comparable to the studies done by Manjushree Waingadae.72

Similar studies conducted by Popova et al and Fernanda Angelieri to find out the

genotoxic effects of low level ionizing radiation on buccal mucosa by evaluating the MN

frequencies before exposure as 0.04±0.06% & 2.34 ±1.49% respectively and after

Discussion

58

exposure as 0.05±0.06% and 2.81±1.64% respectively.61, 63

There is no gender

predilection noted in the present study. Even though, this result does not correlates with

the studies done by Ramakrishna Mahadevan, where a female predominance is noted,

present study result correlates with the study done by Anuradha et al.70, 71

The higher

frequency of micronucleus in buccal epithelial cells observed after X ray exposure

corroborates the data in the review done by D A Ribeiro et al.82

in which X ray radiation

are reported to be effective in inducing genetic damage. A high radiation dose of the

patients [panoramic radiograph and lateral cephalographs] could be a reason for the

positive result.

Apart from MN presence, other nuclear anomalies like Karyorrhexis, karyolysis,

pyknosis were also determined. Tolbert et al described these nuclear alterations as

phenomenon that can occur not only during physiological cellular differentiation but also

during cell death with DNA damage.25

Mechanisms leading to the production of these

abnormalities are still unknown. Among the various nuclear abnormalities, Karyorrhexis,

karyolysis and pyknosis are considered to be biomarker for cell death evidence, different

stages of necrosis indicators and identifying the response to cell damage respectively.

Karyorrhectic cell shows the destructive fragmentation of the nucleus where the

chromatin is distributed irregularly through the cytoplasm. Karyolysis is the complete

dissolution of chromatin in dying cells. The cell appears without nucleus like a ghost

image without staining. Pyknosis is the irreversible condensation of chromatin in the

nucleus of degenerating cell. The diameter of the nucleus is 1/3rd

– 2/3rd

of the normal

nucleus.

Discussion

59

The number of cells undergoing Karyorrhexis, karyolysis, and pyknosis is

estimated in 100 cells from each slide. For Karyorrhexis, the mean value was 3.08±3.26

before exposure and 3.63±3.23 after exposure and for karyolysis the mean values are

5.20±4.53 before exposure and 6.39±5.45 after exposure. There is an increase in these

nuclear abnormalities after exposure to ionizing radiation even though it is not very

statistically significant (P value:1.73 [Karyorrhexis] & 0.056 [karyolysis]) But it was in

contrast with the study done by Ana Elisa where a significant increase in the number of

Karyorrhectic cell were noted.62

The increase in karyolysis was not of much statistical

significance, which suggests that the cellular response to X rays does not induce a

cytotoxic effect that lead to necrosis. This results correlates with the study of Cerqueira

et al in 2004.59

But the mean value of pyknosis was 6.42±4.36 before exposure and

7.96±6.32 after 10 days. It has a P value of 0.02 which is statistically significant. These

result correlates with the study done by Anuradha et al where a significant increase in

the pyknosis is noted (P<0.001).70

The results suggest that since pyknosis is a nuclear

alteration indicative of apoptosis, there are multiple routes that leads cell to apoptosis.

But the study done by Cerqueira et al did not find significant increase in pyknosis in

exfoliated cells after exposure.59

But in the present study there is an overall increase in

the nuclear alterations after radiation exposure. Analogous results were described by

other authors. Considered in-toto such results supports the notion that low level ionizing

radiations are cytotoxic agents.

Discussion

60

This study has some areas which leave room for debate. Staining with Feulgen stain

which is recommended by most authors was not attempted as we felt that the PAP stain

has certain advantages which have been discussed. Other genotoxic factors and nuclear

alterations like condensed chromatin and nuclear buds etc were not assessed in the study.

Further larger studies are recommended to consolidate the findings and rectify the few

shortcoming of the present study.

SUMMARY & CONCLUSION

Summary & Conclusion

61

The objective of the study was to evaluate the genotoxic and cytotoxic changes

after low level ionizing radiation. Buccal epithelial cells were stained with RAPID

PAPTM

kit, before and 10 days after exposure. Genotoxic effect was evaluated by micro

nucleus assay which is found to be high after exposure. The various other nuclear

alterations like Karyorrhexis, karyolysis and pyknosis are evaluated and there is an

increase in these parameters after exposure. The result depends on the type, amount and

radiation dose absorbed and the individual’s capacity to withstand the action of genotoxic

agents. Future studies should evaluate populations according to age, different cell

collection procedures, DNA staining procedures and type of radiographs. The possibility

of other genotoxic factors and other nuclear changes should also be investigated.

Since the use of radiographs has become more frequent in dental practice as a

diagnostic measure, treatment planning or for follow up, their indication should always

follow the concept of maximum benefit with minimum risk. Our study indicates the

presence of genotoxic and cytotoxic changes after exposure. Radiographs should be made

carefully avoiding retakes and thereby decreasing the radiation dose thus reducing risk of

cytogenic damage. Thus based on the present study we cannot consider exposure from

low level ionizing radiation as risk –free and should be used only when necessary. Based

on the results of the study, low level ionizing radiation do carry their own level of risk in

inducing cellular and genetic changes and should be used with caution and only as and

when necessary.

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http://www.ncbi.nlm.nih.gov/pubmed?term=Dwarakanath%20BS%5BAuthor%5D&cauthor=true&cauthor_uid=11423343

http://www.ncbi.nlm.nih.gov/pubmed?term=Parmar%20VS%5BAuthor%5D&cauthor=true&cauthor_uid=11423343

http://www.ncbi.nlm.nih.gov/pubmed?term=Malik%20S%5BAuthor%5D&cauthor=true&cauthor_uid=11423343

http://www.ncbi.nlm.nih.gov/pubmed?term=Adhikari%20JS%5BAuthor%5D&cauthor=true&cauthor_uid=11423343

http://www.ncbi.nlm.nih.gov/pubmed?term=Adhikari%20JS%5BAuthor%5D&cauthor=true&cauthor_uid=11423343

http://www.ncbi.nlm.nih.gov/pubmed?term=Tyagi%20YK%5BAuthor%5D&cauthor=true&cauthor_uid=11423343

http://www.ncbi.nlm.nih.gov/pubmed?term=Goel%20S%5BAuthor%5D&cauthor=true&cauthor_uid=11423343

http://www.ncbi.nlm.nih.gov/pubmed?term=Gupta%20K%5BAuthor%5D&cauthor=true&cauthor_uid=11423343

http://www.ncbi.nlm.nih.gov/pubmed?term=Bose%20M%5BAuthor%5D&cauthor=true&cauthor_uid=11423343

http://www.ncbi.nlm.nih.gov/pubmed?term=Olsen%20CE%5BAuthor%5D&cauthor=true&cauthor_uid=11423343

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Hadjidekova, D Ziya and VGHadjidekova. Micronucleus Test in Buccal

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ANNEXURES

CONSENT FORM PART 1 OF 2

INFORMATION FOR PARTICIPANTS OF THE STUDY

1. Name of the Principal Investigator:

Meera Mathai

Post Graduate student

Department of Oral Medicine and Radiology,


Kulasekharam, KanyaKumari District-629161

2.Name of the Guide: Dr. P.G.Agnihothri M.D.S Professor & H.O.D

Department of oral medicine and Radiology.

Sree Mookambika Institute of Dental Sciences.


Dear Volunteers,

We welcome you and thank you for your keen interest in participation in this

research project. Before you participate in this study, it is important for you to understand

why this research is being carried out. This form will provide you all the relevant details of

this research. It will explain the nature, the purpose, the benefits, the risks, the discomforts,

the precautions and the information about how this project will be carried out. It is important

that you read and understand the contents of the form carefully. This form may contain

certain scientific terms and hence, if you have any doubts or if you want more information,

you are free to ask the study personnel or the contact person mentioned below before you

give your consent and also at any time during the entire course of the project.

2. Name of the Co-Guide: Dr. Hema.G. MDS.

Reader

Department of oral medicine and Radiology.

Sree Mookambika Institute of Dental Sciences.


4.Institute:


V.P.M Hospital complex, Padanilam, Kulasekharam,

Kanyakumari – 629161

Tamilnadu

5. Title of the study:

“Biomonitoring of genotoxic and cytotoxic effects among patients exposed to digital

radiographs- a comparitative study .”

6 .Background information:

You are undergoing orthodontic treatment in which radiographs are

compulsory and in our study we are making you as a subject taking two salivary

samples without causing any discomfort to you people. The analysis of sample gives

an idea about your exposure to x-rays and gives a precaution about your exposure in

future.

7.Aims and Objectives:

1. To compare genotoxic and cytotoxic before and after radiation

2. To study the changes in the micronuclei before and after radiation

3. To compare whether there is any cytological changes before and after

radiation

8.Scientific justification of the study:

Exposure to x-rays are hazardous to body and public will be under impression

that even minor expoure radiation is hazardous. Here we are trying to establish that

though the patient is exposed to radiation, the exposure is going to be minimum with

minimum damages to human being. Here an attempt is made to explain individuals,

with a detailed experiments to appreciate that the toxicity will be minimum.

9.Procedure for the study:

• Once you are enrolled into the study a roll no will be implemented to represent the

name.

• Patients are advised to wash their mouth with water to remove debris from the site.

• Buccal epithelial cells are obtained using brush biopsy technique using a soft bristle

tooth brush, immediately before exposure and 10 days after exposure.

• The solution will be sent for cytological evaluation.

10.Expected risks for the participants:

• Abrasion to buccal mucosa.

11.Expected benefits of research for the participants:

• You will not be required to pay for the lab tests.

• You can enquire about the outcome of the procedures and your details.

12.Maintenance of confidentiality:

• You have the right to confidentiality regarding the privacy of your medical

information

(Personal details, results of physical examinations, investigations, and your

medical history).

• By signing this document, you will be allowing the research team investigators, other

study Personnel, sponsors, institutional ethics committee and any person or agency

required by law to view your data, if required.

• The results of clinical tests and therapy performed as part of this research may be

included in your medical record.

• The information from this study, if published in scientific journals or presented at

scientific meetings, will not reveal your identity.

13.Why have I been chosen to be in this study?

Chosen because of groping under the inclusion and exclusion criteria

Need of good sampling size

No invasive procedure that harm your health and helps in diagnosis and

helpful for the society

14.How many people will be in the study?

126 15.Agreement of compensation to the participants (In case of a study

related injury): Patient will be taken care in case of complication and medical treatment will

be provided in the institution.

16.Anticipated prorated payment, if any, to the participant(s) of the

study: No

17.Can I withdraw from the study at any time during the study

period?

• The participation in this research is purely voluntary and you have the right to

withdraw from this study at any time during the course of the study without giving

any reasons.

• However, it is advisable that you talk to the research team prior to stopping

information.

18.If there is any new findings/information, would I be informed? Yes

19.Expected duration of the participant’s participation in the study:

1 year

20.Any other pertinent information: Not applicable

21. Whom do I contact for further information?

Place: Kulasekharam

Date:

Signature of Principal Investigator

Signature of the participant

For any study related queries, you are free to contact :

Meera Mathai

Post Graduate student.

Department of Oral Medicine and Radiology,


Kulasekharam, KanyaKumari District-629161.

Mobile No: 08281538491

[email protected]

CONSENT FORM

PART 2OF 2

PARTICIPANT CONSENT FORM

The details of the study has been explained to me in writing

and the details have been fully explained to me. I am aware that the results of the e

study may not be directly beneficial to me but will help in the advancement or medical

sciences. I confirm that the understood the study had the opportunity to ask questions. I

understood that my participation in the study is voluntary and that I am free to withdraw

it any time, without giving any reason, without the medical cure that will normally be

provided by the hospital being affected I agree not to restrict the use of any data or

results that arise from this study provided such a use is only for scientific purpose I

have been given an information sheet giving details of the study. I fully consent to

participate in the study titled “ Biomonitoring of genotoxic and cytotoxic changes

among patients exposed to digital radiographs”- a comparative study

Serial / ref no

Name of the participant: Address of the Participant:

Contact number of the Participant:

Signature/Thumb impression of the participant/ Legal guardian

Witnesses:

1.

2.

Date:

Place: Kulasekharam

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8. KthjWw \S¯phm\pÅ \ymboIcWw: tdUntbj³ a\pjycnÂ

A]ISImcnbmWv F¶v ]e ]T\§fneqsSbpw sXfnbn¡s¸«n«p-v.

\yq¢nbknepÅ ]e Xc nepÅ amä§Ä P\nXnI XIcmdpIfntebv¡v

shfn w hoip¶hbmWv. Cu ]T\w FIvkvtd FSp¡p¶Xpaqeap-mIp¶

amä§Ä P\nXnIXe nepw IWnImXe nepw ]cntim[n¡p¶p.

3

9. ]T\coXn :

1) ]T\¯nsâ `mKambn hyàn¡v Hcp \¼À \ÂIp¶XmWv.

2) \n§fpsS apgph³ hnhc§fpaS§nb tIkv lnÌdnbnÂ

tcJs¸Sp¯p¶XmWv.

3) Bhiym\pkcWambn NnX§Ä FSp¡mhp¶XmWv.

4) shÅw D]tbmKn¨v hmbv hr¯nbm¡m\mhiys¸Sp¶p

5) arZphmb Hcp _jv D]tbmKn¨v IhnfnÂ \n#o¶pw \n§fpsS

Dan\ocnsâ Hcp km¼nÄ FSp¡p¶p.

6) CtX coXnbnÂ ]¯p Znhk¯n\ptijw c-mw kmw]nÄ

FSp¡p¶p.

7) tijw AXv ]cntim[\IÄ¡mbn Ab¡p¶p.

10) ]Xo£n¡p¶ A]ISkm²yXIÄ

sNdnb coXnbnÂ thZ\ (achn¸n¡m\pÅ acp¶pIÄ

D]tbmKn¨v Cu thZ\bpw CÃm¯m¡mhp¶XmWv.

11) ]Xo£n¡mhp¶ ]tbmP\§Ä

1) em_v sSÌpIÄ¡v \n§Ä XpI \ÂtI-XnÃ

2) \n§Ä¡v Cu KthjW nsâ hnhc§Ä Xnc¡mhp¶XmWv.

12) hyànhnhc§fpsS kzImcyX:

1) tcmKhnhc§fpw aäv hyànhnhc§fpw kzImcyambn

kq£n¡s¸Sp¶Xmbncn¡pw

2) Cu t^md¯nÂ H¸nSp¶Xv hgn \nbaw A\pimkn¡p¶

coXnbnÂ ]T\¯nÂ DÄs¸Sp¶ hyànIÄ¡v \n§fpsS

hnhc§Ä ]cntim[n¡mhp¶XmWv.

4

3) Cu ]T\¯nsâ hnhc§Ä imkvXm\p]m[nIfmb

]kn²oIcW§fntem, IqSn BtemN\Ifntem

shfns¸Sp¯t¼mÄ \n§fpsS kzImcyX

kq£n¡s¸Sp¶XmWv

13) F´psIm-v \n§Ä XncsªSp¡s¸«p?

1) ]T\¯n\v \Ã iXam\w BfpIÄ BhiyamWv.

2) ]e Iq«n¡pd¨nepIÄs¡mSphnÂ \n§Ä DÄs¸Sp¶

hn`mKs¯ XncsªSp¯p

3) \n§fpsS klIcWw aqew kaql¯n\v klmbhpw

\·bpw D -mIp¶p.

14) FX BfpIÄ Cu ]T\¯nÂ DÄs¸Sp¶p?126

15) \jvS]cnlmc DS¼Sn? (]T\hpambn _Ôs¸«v Fs´¦nepw]cn¡p - mbmÂ)

]T\hnt[bambn GsX¦nepw Xc nÂ tcmKw k¦oÀ®ambmÂtcmKnsb Cu Øm]\ nÂ hnZKv² NnInÕ¡p hnt[b\m¡p¶XmWv.

16) GsX¦nepw hn[¯nÂ thX\w e`n¡ptam?CÃ

17) Ft¸mÄ thWsa¦nepw F\n¡v Cu ]T\ nÂ \n¶v ]n³amdmtam?

ImcWw hyàam¡msX Ft¸mÄ thWsa¦nepw \n§Ä¡v Cu]T\¯nÂ \n¶pw ]n·mdmhp¶XmWv. F¦nepw AXn\v ap³]mbnKthjIcpambn kwkmcn¡p¶Xv \ÃXmWv.

6

18) ]T\hpambn _Ôs¸« Fs´¦nepw ]pXnb hnhc§Ä Ds-¦nÂFs¶ Adnbn¡p¶XmtWm?

AsX

19) ]Xo£n¡p¶ ]T\Imemh[n ?Hcp hÀjw

20) asäs´¦nepw hnhcw ?CÃ

21) hnhc§Ä¡v _Ôs¸tS-Xv Bsc?

tUm.aocm a¯mbn_ncpZm\ć _ncpZ hnZymÀ°n/\n

_ncpZm\ć _ncpZ hnZymÀ°n/\n

Un¸mÀ«vsaâv Hm^v HmdÂ saUnkn³ Bâv tdUntbmfPn



t^m¬ tUm.aocm a¯mbn þ 08281538491

C.sabnÂ,sF.Un.: [email protected]

Øew: ][m\ KthjIsâ H¸v

XobXn k¶² tkhI³/ tkhI

7

k½X]Xw þ 2

Cu ]T\¯nepÅ Fsâ ]¦mfn¯w kz´w XmÂ]cy]Imcw

amXamsW¶pw, Ft¸mÄ thWsa¦nepw tNmZy§Ä _Ôs¸«htcmSv

tNmZn¡msa¶pw, Cu ]T\¯nÂ \n¶pw ImcWw tcJs¸Sp¯msX

Ft¸mÄ thWsa¦nepw F\n¡v ]n³hm§msa¶pw Rm³ a\Ênem¡p¶p.

Cu ]T\mhkm\w shfs¸Sp¶ AdnhpIfpw, tcJIfpw, imkvX]camb

Dt±i§Ä¡p D]tbmKn¡m³ Rm³ k½Xn¡p¶p. ]Tt\mt±iw

hnhcn¡p¶ hniZmwi§Ä \ÂInbn«p-v.

“ ------------------þtcmKnIfnÂ UnPnäÂ FIvkvtdbv¡p tijapÅ P\nXnI Xe¯nepw,

IWnIXe¯nepw DÅ amä§fpsS \nÀ®bw” F¶ Cu ]T\hpambn

klIcn¡m³ Fsâ ]cn]qÀ® k½Xw Adnbn¡p¶p.

tUm.aocm a¯mbn

t]cv (k¶² tkhI³/tkhI) k¶² tkhIsâtbm/

_Ôs¸Sm\pÅ \¼À tkhIbpsStbm hnemkw

km£nIÄ H¸v/hncÂ ASbmfw

(kz´w/\nba]cmb

1. kwc£I³)

2.

XobXn:

Øew:

Jl◊Rp T•Ym

Tœß I - 2

BnÆp Te˙LtTY⁄dLÙ] RLYp T•Ym

1. BnYÙ[o

U⁄. ¡WÙ UjRÙn ÿ’L˚X ThPRÙ¨

KWp ˘U•£u Utflm ˙W•˙VÙX¥

c ÍLÙm©LÙ Tp U⁄j’YU˚] LpÌ¨, œX˙NLWm.

Lu≤VÙœU¨.

2. YØLÙh•

U⁄. ©.¥. Ad°≤ ˙LÙjß¨

˙TWÙ£¨Vo, R˚X˚UVÙ[o



Lu≤VÙœU¨.

3. ’˚Q YØLÙh•

U⁄. ˙aUÙ. ¥.

√Po (’˚Q ˙TWÙ£¨Vo)



Lu≤VÙœU¨.

4. Bn‹ ®˚XVm

c ÍLÙm©LÙ Tp U⁄j’YU˚] LpÌ¨, œX˙NLWm,

Lu≤VÙœU¨ UÙYhPm. R™rSÙ”.

5. R˚Xl◊ : •¥hPp F”Lßo ÍXm HtT”m Àu Utflm ß—dL∞u UÙt\eL˚[ Jl©h” TÙojRp.

Ußl©tœ¨V Ru]ÙoYX˙W,

RÙeLs CkR BnÆp Te˙LtL YkR˚Udœ Su±, CkR BnÆp Te˙LtTRtœ

ÿu CkR BnÆu ÿd°Vj’Ym Utflm Ru˚U A±k’ ˘LÙs[ ˙Yi”m. CkR

T•Ym BnÆu ÿ›ÆTWeL˚[Ÿm EeLfidœ ˘R¨Ædœm. BnÆp Te˙LtTRtœ

ÿu CkR T•Yj˚R ÿ›Y’m T•j’ ◊¨k’ ˘LÙiP ©u Te˙LtL ˙Yi”m. CkR

T•Yjßp £X U⁄j’Y Utflm A±ÆVp ˘NÙtLs Es[]. BRXÙp G˙R‡m

Nk˙RLeLs HtThPÙp CkR T•Yjßu Cflß´p œ±l©h”s[ ST¨Pm BnÆp

Te˙LtTRtœ ÿu ˘R∞‹ ˘T\ ˙Yi”m

6. BnÆu ©uTXm :

•¥hPp F”Lß¨]Ùp Y⁄m Lßo≈f— LiQm Utflm Dü ˘NpL∞u ®⁄‹⁄L˚[

TÙßj’ ˘Np˚X UWQU˚PV ˘Nn°\’. G]˙Y CR˚] U]ßp ˘LÙi” •¥hPp F”Lß˚W

GfN¨d˚LŸPu TVuT”jR ˙Yi”m.

7. œ±d˙LÙs :

• F”Lßo ˘NnYRtœ ÿu Utflm ©u Àu Utflm ß—L∞u HtT”m UÙt\eL˚[ Jl©h”

TÙojRp.

• F”Lßo £°h˚Ndœ ÿu Utflm ©u ®Ÿd- V≥p HtT”m UÙt\eLs.

• F”Lßo ÍXm ˘NpL∞p H˙R‡m UÙt\eLs HtT”Y˚R LiP±Rp.

8. A±ÆVp NÙufl (©uTXm) :

Lßo≈f— U≤R C]jßtœ ATÙVUÙ] Jufl. •.Gu.H. A[Æp Y⁄°u\ L⁄‹dœ¨V

UÙt\m ˘Np ˙NRÙWjßu BWmT NÙu\Ùœm. CkR Bn‹ œ˚\kR F”Lß¨]Ùp LiQjßp

Y⁄°u\ ˘Np Utflm ß—dL∞u UÙt\j˚R LiP±V ˘NnVlT”°\’.

9. YØÿ˚\ :

CkR BnÆp øeLs Te˙Lt˙\©u ReLfidLÙ] R≤ CXdL Gi YZeLlT”m.

RiΩWÙp YÙn˘LÙlT∞j’ —jRm ˘Nn’ ˘LÙs[‹m.

™⁄’YÙ] ©Wx ÍXm YÙn Li]jßp C⁄k’ ˘NpL˚[ F”Lßo £°h˚Ndœ ÿu

Utflm Tj’ SÙhLfidœ ©u‡m G”dL ˙Yi”m.

˘NpL∞u UÙt\eLs Bn‹ ®˚XVjßp LiP±VlT”m.

10. GßoU˚\ Æ˚[‹Ls :

• LiQjßp EWÙn‹

11. HtT”m Su˚ULs :

• BnÆtLÙL TQm G’‹m RW ˙R˚Y´p˚X

• BnÆu ÿ•‹L˚[ øeLs A±k’ ˘LÙs[XÙm.

12. RLY- u TÙ’LÙl◊ :

EeLfi˚PV RLYpLs A˚]j’m TÙ’LÙdLlT”m GkR ®˚X´¤m

˘Y∞´PlTPÙ’.

øeLs CkR BnÆp Te˙Lt\©u GeL[’ BnYÙ[o EeLfi˚PV RLYpL˚[

˘R¨k’ ˘LÙs[ ÿ•Ÿm. ˙U¤m ˙R˚Y HtThPÙp GjßdLp L™h•´Pÿm

LÙi©dLlT”m.

BnÆu ÿ•‹Ls EeLfi˚PV U⁄j’Y RLYp T•Yjßp (˙LÙl©p) Tß‹

˘NnVlT”m.

CkR Bn‹ ÿ•‹ ˘Y∞´PlT”m ˘TÙ›’ EeL[’ RLYpLs ˘Y∞´PlTPÙ’.

13. CkR BnÆp øeLs ˙NoYRtLÙ] LÙWQm?

øeLs CkR BnÆu ˙Nol◊ Utflm Æ”l◊ Yœl◊L∞u Es A˚UVlT”YRÙp

BnÆu ˙R˚YVÙ] UÙß¨dLÙL‹m

NÍLjßu Ut\m U⁄j’Y ÿu˙]t\jßtLÙL

14. BnÆu UÙß¨ Gi¶d˚L ? 126

15. SxP¬” :

BnÆp HtT”m UÙt\eLfidLÙ] £°h˚N Bn‹ ®˚XVjßp YZeLlT”m.

16. SxP¬” TQm : TQm G’‹m YZeLTPÙ’.

17. BnÆp C⁄k’ GkR ˙SWjß¤m GkR LÙWQÿm Cu± ÆXLXÙm

18. BnÆu ÿu˙]t\jßp HtT”m UÙt\eLs ˘R¨ÆdLlTP ˙Yi”m

19. BnÆu LÙX AYLÙNm? J⁄ Bi”

20. ˙Yfl GkR RLYpLfim ˙R˚Y Cp˚X

21. ˘RÙPo◊ ˘LÙs[ ˙Yi•V STo :

CPm :

BnYÙ[o ˚L˘VÙlTm

˙Rß :

Teœ˘TflTYo ˚L˘VÙlTm

U⁄. ¡WÙ UjRÙn

ÿ’L˚X ThPRÙ¨,

JWp ˘U•£u Utflm ˙W•˙VÙX¥,


˘RÙ˚X˙T£ Gi : 08281538491

™u AgNp : [email protected]

Jl◊Rp T•Ym

TÙLm : 2

Teœ˘TflTYü Jl◊Rp T•Ym

CkR BnÆu ÿ› ÆTWeLfim G›j’ ÍXUÙL‹m, ÆŸÙL‹m, Gu≤Pm

·\lThP’. G]dœ CkR BnÆu Æ˚[‹Ls GkR TV‡m RWÆp˚X Gu\Ù¤m, U⁄j’Y

A±ÆV- u ˙UmTÙh•tLÙL TVuT”jRlT”m Gufl A±˙Yu. SÙu CkR BnÆp GkR

®oTkRÿm CpXÙUp Teœ˘Tfl°˙\u. ˙U¤m CkR BnÆu GpXÙ ÆßLs Utflm

Æßÿ˚\L˚[Ÿm A±˙Yu. GkR Nk˙RÙLjßtœm ˙LsÆ ˙LhL˙YÙ ApX’ BnÆp

C⁄k’ ÆXL˙YÙ GpXÙ E¨˚ULfim G]dœ Ei” Gufl A±˙Yu. CkR BnÆu

Æ˚[‹Ls GkR A±ÆVp TVuTÙh•tœm TVuT”jR G]dœ R˚P´p˚X. G]dœ CRtœ

ÿuTÙL BnÆu RLYp T•Ym ˘LÙ”dLlThP’. G]dœ CkR BnÆp Teœ˘T\ ÿ›

NmURm.

BnÆu ˘TVo :

•¥hPp F”Lßo ÍXm HtT”m Àu Utflm ß—dL∞u UÙt\eL˚[

Jl©h” TÙojRp.

œ±l◊ Gi :

˘TVo :

ÿLY¨ :

˘RÙ˚X˙T£ Gi : ˚L˘VÙlTm

NÙh£ : 1

2

CPm :

˙Rß :

CASE RECORD FORM

Date of examination: OP File no.:

Personal details:

1. Name : 2. Age: 3. Sex: 4. Educational status: 5. Occupation : 6. Income : ( per year)

7. Address:

Ph no:

8. Number of family members: 9. Habits :

Do you? Yes NoSmoke Chew tobacco Alcohol

10. Any systemic diseases?

Disease Yes No On treatment? Diabetic Hypertensive CAD Stroke Hypercholesterolemia TB Hypothyroid BA Epilepsy

11. Presenting complaint:

12. History of presenting complaints:

13. Family history

14. Past history: Do you have? Yes No radiation exposure within one year

prosthetic rehabilitation Pre-malignancy/ malignancy Hospital admissions Any other abnormalities in buccal mucosa

15. General Examination:

Pulse rate

Respiratory rate

Temperature

BP

JVP

Signs of Yes No Pallor Jaundice Cyanosis Clubbing Pedal oedema Regional lymphadenopathy

Local examination: Any abnormalities in Yes No Gingiva Buccal mucosa Labial mucosa Palate Tongue Floor of the mouth

Check list: Information sheetConsent form Personal details Biopsy number (before radiation)Biopsy number (after radiation)

NO

AGE

SEX

MN BEFORE

MN AFTER

KR BEFORE

KR AFTER

KL BEFORE

KL AFTER

PN BEFORE

PN AFTER

1 21yrs F 4 15 1 4 0 2 5 4 2 20yrs F 2 15 5 9 10 9 10 2 3 23yrs F 3 12 0 6 17 8 3 5 4 21yrs M 1 10 3 5 5 6 11 8 5 21yrs M 0 15 0 8 5 3 8 8 6 20yrs F 5 12 4 4 5 5 7 9 7 19yrs M 1 4 1 0 20 12 4 4 8 20yrs F 4 9 3 12 3 6 9 8 9 24yrs M 2 15 0 10 0 9 9 2

10 23yrs F 0 1 1 1 0 20 3 6 11 21yrs F 2 5 0 3 5 7 1 12 12 24yrs F 2 4 0 2 14 20 0 2 13 20yrs F 3 5 1 1 6 27 2 0 14 21yrs F 2 4 3 2 8 8 4 11 15 23yrs F 0 3 0 0 8 14 5 4 16 20yrs F 0 14 0 4 5 2 7 7 17 24yrs M 1 2 2 3 9 8 7 7 18 24yrs F 0 9 0 2 8 8 3 8 19 20yrs F 4 0 6 2 3 5 5 18 20 21yrs F 2 6 0 3 0 18 5 12 21 21yrs F 4 4 5 3 9 1 3 9 22 20yrs M 0 7 0 3 2 6 5 3 23 23yrs F 0 3 2 1 9 7 6 5 24 21yrs F 3 8 1 2 13 13 1 5 25 24yrs F 3 13 2 4 3 5 10 4 26 23yrs F 5 6 3 1 0 1 13 3 27 23yrs F 11 0 11 9 0 34 5 2 28 24yrs F 9 9 9 0 3 2 5 10 29 23yrs F 7 14 3 2 3 3 2 7 30 20yrs F 1 9 0 0 4 6 16 0 31 21yrs M 1 8 0 17 2 1 7 1 32 27yrs M 3 5 6 4 4 16 5 5 33 27yrs M 3 11 6 0 4 8 5 5 34 21yrs F 2 12 3 4 5 11 3 0 35 22yrs F 3 6 13 1 0 4 6 5 36 22yrs M 3 6 0 7 6 4 7 5 37 22yrs M 4 7 0 4 2 3 4 6 38 23yrs F 1 3 4 1 2 6 4 5 39 20yrs M 2 3 5 2 1 1 1 14 40 20yrs F 0 7 0 1 3 10 2 4 41 24yrs F 3 5 10 4 3 7 3 7 42 23yrs F 2 1 0 0 6 1 14 3 43 22yrs F 0 9 0 10 3 7 2 7 44 21yrs F 2 16 4 3 2 4 2 4 45 22yrs F 4 1 1 6 3 10 2 18 46 23yrs F 1 12 6 3 3 3 10 4 47 22yrs F 2 4 1 0 5 1 6 7 48 23yrs F 2 16 1 11 3 13 2 2 49 23yrs F 3 8 7 6 5 13 4 750 19yrs F 8 2 5 0 1 7 13 11 51 22yrs F 0 11 1 6 0 14 6 8

VALUES OF STUDY

52 23yrs F 2 3 4 3 0 5 0 6 53 23yrs F 1 14 0 6 0 5 8 11 54 23yrs F 1 11 4 3 6 6 7 16 55 24yrs F 2 4 1 0 0 13 13 4 56 23yrs F 1 21 2 0 11 1 7 6 57 20yrs F 0 8 3 0 8 7 5 2 58 19yrs F 1 1 1 3 0 7 18 15 59 20yrs F 1 8 4 0 4 8 10 4 60 20yrs F 1 12 2 4 6 3 11 10 61 20yrs F 2 3 2 3 8 3 5 9 62 20yrs F 1 8 4 5 1 5 14 2 63 23yrs F 0 13 5 3 1 3 4 6 64 20yrs F 0 6 3 6 7 9 8 7 65 25yrs M 3 19 4 6 10 1 10 9 66 20yrs F 3 16 6 7 9 3 14 11 67 20yrs F 1 6 5 2 4 8 18 7 68 20yrs F 6 9 4 3 13 0 10 18 69 20yrs F 2 5 7 0 3 3 21 10 70 20yrs F 0 4 2 1 2 0 4 1 71 19yrs M 0 4 1 2 3 2 23 12 72 23yrs M 2 11 8 0 20 5 2 7 73 23yrs F 1 5 9 7 4 0 2 4 74 22yrs F 7 12 24 4 6 9 3 4 75 20yrs F 2 11 1 0 25 3 0 1 76 20yrs F 2 12 4 3 5 3 8 3 77 20yrs F 3 2 3 4 19 12 2 3 78 23yrs F 4 3 5 0 2 7 4 4 79 22yrs F 2 21 2 0 9 1 6 6 80 21yrs F 0 10 4 2 17 7 1 10 81 21yrs F 2 5 0 1 6 4 8 11 82 20yrs F 2 6 4 4 7 13 4 2 83 23yrs F 1 7 2 2 3 5 3 17 84 23yrs F 1 12 0 6 6 15 4 9 85 23yrs F 3 11 1 8 3 5 4 5 86 23yrs F 2 13 3 0 7 10 2 8 87 23yrs F 2 25 12 14 0 6 4 4 88 23yrs F 2 18 3 5 3 2 4 24 89 22yrs F 1 2 2 1 6 4 2 13 90 19yrs F 1 7 2 5 4 4 2 14 91 19yrs F 1 27 1 4 4 4 6 15 92 21yrs F 2 3 0 5 4 4 6 14 93 22yrs F 3 13 4 7 6 3 8 7 94 19yrs F 2 10 3 3 3 10 8 3 95 22yrs M 0 6 0 5 3 7 5 27 96 19yrs F 0 6 4 5 5 3 5 17 97 19yrs F 2 7 5 7 7 3 3 20 98 20yrs F 2 11 1 9 3 5 7 23 99 19yrs F 0 7 0 3 4 10 4 19

100 20yrs F 1 24 5 9 3 0 3 10 101 20yrs F 3 5 7 3 5 5 4 25 102 22yrs F 0 33 3 0 5 9 6 13 103 22yrs M 0 12 2 7 2 3 8 9 104 22yrs F 2 25 1 2 3 4 4 0

105 21yrs F 1 6 6 2 13 5 14 3 106 23yrs M 1 13 0 5 1 5 15 3 107 23yrs F 0 19 3 2 12 2 3 3 108 28yrs F 0 10 5 2 2 3 6 2 109 23yrs F 2 12 0 2 4 2 7 3 110 22yrs F 0 22 3 3 2 2 7 3 111 22yrs F 0 3 1 3 5 8 13 4 112 23yrs F 0 32 2 2 5 2 6 5 113 19yrs F 2 11 5 2 7 2 12 6 114 19yrs M 0 10 4 2 10 1 9 4 115 20yrs M 5 4 4 1 0 1 8 4 116 20yrs F 1 5 1 0 4 6 5 4 117 22yrs M 1 31 3 0 3 3 7 3 118 20yrs M 0 9 2 2 4 10 3 10 119 20yrs F 3 13 0 2 1 0 7 3 120 24yrs F 0 3 4 2 7 22 4 2 121 21yrs F 1 6 0 4 2 6 10 36 122 20yrs F 3 12 3 0 5 1 0 7 123 19yrs F 0 21 2 7 3 9 9 12 124 23yrs F 0 18 5 9 6 2 10 11 125 23yrs F 6 11 6 3 8 4 12 18 126 20yrs F 1 15 3 9 8 12 5 9 127 19yrs F 2 11 2 10 4 7 9 26

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