Chewing habits are very common in India and abroad from a

longer period. Tobacco is being commercially manufactured and

distributed in different forms. Presently a number of chewing products

are available in the market having contents of betel quid i.e. areca nut,

chatechu and lime. Chewing mixes without tobacco are termed as 'Pan

Masala' and with tobacco as 'Gutkha'. Areca nut is a main component

of gutkha which is able to causes oral sub-mucous fibrosis (OSMF)

(Tilakaratne et al, 2005). OSMF is incurable disease and finally leads to

oral cancer (Murti et al, 1985). After long time of smoking, adverse

effects are seen but in case of gutkha users, OSMF develops within a

very short span of time (Babu et al, 1996). The intake of gutkha and

OSMF is very common in young person (Gupta et al,1998). Pan masala

contains arecanut as one of its ingredients and is also unfit for health

due to its mutagenic, genotoxicand carcinogenic properties. Areca nut

increases the chances of formation of Pre-cancerous lesion and oral

sub-mucous fibrosis.

The main carcinogens in pan masala and gutkha are derived

from their ingredients; areca nut, catechu, lime and tobacco.

i. Lime: Reactive oxygen species generation (ROS) in oral

cavity is favored by alkaline condition build up by Ca(OH)₂ in slaked lime. Lime is responsible for causing irritation and

hyperplasia of the oral mucosa (Dunham et al, 1974).

ii. Arecanut: It contains a number of phenolic compounds,

which are responsible for development of proliferative

lesions (Bhide et al, 1984).

iii. Tobacco: The leaching of various nitrosamines has been

reported from tobacco when kept in mouth (Nair et al,

1985).

iv. Catechu: Tannin and polyphenols are the main constituents

of catechu. Foods those are rich in tannins, have high

incidence of oesophageal cancer (Morton, 1972).

1

Mutagenic property of catechu has been shown Stitch et al

and clastogenicity by Giri et al.

Genomic damage is probably the most important fundamental

cause of developmental and degenerative disease. It is also well

established that genomic damage is produced by environmental

exposure to genotoxins, medical procedures (e.g. radiation and

chemicals), micronutrient deficiency (e.g. folate), lifestyle factors (e.g.

alcohol, smoking, drugs, and stress), and genetic factors such as

inherited defects in DNA metabolism and/or repair (Holland et al,

2008). Cytogenetic markers like chromosomal aberration (CAs), sister

chromatid exchanges (SCEs), and micronuclei (MN) are sensitive

indicators of genetic damage.

Micronuclei are small chromatin bodies that appear in the

cytoplasm by the condensation of acrocentric chromosomal fragments

or by whole chromosomes, lagging behind the cell division. Thus it is

the only biomarker that allows the simultaneous evaluation of both

clastogenic and aneugenic effects in a wide range of cells, which are

easily detected in interphase cells (Kayal et al, 1993). In humans, MN

can be easily assessed in erythrocyte, lymphocytes and exfoliated

epithelial cells (e.g. oral, urothelial, nasal) to obtain a measure of

genome damage induced in vivo.

Buccal cells are the first barrier for the inhalation or ingestion

route and are capable of metabolizing proximate carcinogens to react

products. Approximately 90% of human cancers originate from

epithelial cells (Rosin, 1992).

The oral epithelium is composed of 4 strata of structural,

progenitor, and maturing cell populations including the lamina propria

(connective tissue), the basal layer (stratum basale), prickle cell layer

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

finger like structures called “rete pegs” projects up from the lamina

propria into the epidermal layer producing an undulating basal cell

2

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 the exfoliate into the buccal cavity. Some of these cells may

degenerate into cells with condensed chromatin, fragmented nuclei

(karyorrhectic cells), pyknotic nuclei, or completely loss their nuclear

material (karyolitic or ghost cell) (Tolbert et al, 1992). In rare cases

some cells may be blocked in a binucleated stage or may exhibit

nuclear buds (also known as broken eggs in buccal cells) a biomarker

of genome damage (e.g. MN, nuclear buds) and cell death (e.g.

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

buccal cell systems.

Lifestyle and Cancer:-

Lifestyle influences a person's risk for cancer by generating

growth-promoting signals that affect cells primed to become

cancerous, or that already are cancerous. What primes those cells to

become cancerous in the first place are changes in their genes.

3

Figure 1:-Risk factors (BBC News 07-Dec-2012)

Overall, environmental factors, defined broadly to include

tobacco use, diet, infectious diseases, chemicals, and radiation, are

believed to cause between 75 and 80 percent of all cancer cases in the

United States. Tobacco use, including cigarettes, cigars, chewing

tobacco, and snuff, can cause cancers of the lung, mouth, throat,

larynx, bladder, kidney, esophagus, and pancreas. Smoking alone

causes one-third of all cancer deaths in the India. Heavy consumption

of alcohol has also been shown to increase the risk of developing

cancer of the mouth, pharynx, larynx, esophagus, liver, and breast.

Tobacco use is a major cause of lung, lip, mouth, larynx, and throat

cancer, and is a contributing cause of many other cancers in India.

Molecular Mechanism of Cancer:-

The disease caused by an uncontrolled division of abnormal cells

in a part of the body and are able to invade other tissues (metastasis).

During metastasis, malignant cells travel among tissues via the

circulatory and/or lymphatic system. Unregulated cell growth and

metastasis are caused by mutations in the genes (DNA) of proteins

4

involved in the regulation of the cell cycle. Agents that cause DNA

damage leading to the transformation of a cell are called carcinogens.

Cancers result from a series (progression) of gene mutations that

typically involve two categories of function: promotion of cell division

and inactivation of cell cycle suppression. Proto-oncogenes are normal

genes that promote cell growth and mitosis, whereas tumor suppressor

genes discourage cell growth. Proto-oncogenes can be mutated by

carcinogenic agents to become oncogenes. This type of mutation

usually has a dominant effect only one of the cell’s two gene copies

and undergo change and altered the gene called oncogene, the normal

allele being a proto-oncogene (Fialkow, 1976). The second is to make

an inhibitory gene inactive. This type of mutation usually has a

recessive effect both the cell's gene copies must be inactivated or

deleted to free the cell of the inhibition and the lost gene is called

tumor suppressor gene.

Oral Cancer:-

Oral cancer is a subtype of head and neck cancer is any

cancerous tissue growth located in the oral cavity. It may arise as a

primary lesion originating in any of the oral tissues, by metastasis from

a distant site of origin, or by extension from a neighboring anatomic

structure, such as the nasal cavity. There are several types of oral

cancers, but around 90% are squamous cell carcinomas originating in

the tissues that line the mouth and lips. Oral or mouth cancer most

commonly involves the tongue. It may also occur on the floor of the

mouth, cheek lining, gingiva (gums), lips, or palate.

Causes of Oral Cancer:-

Oral cancer most commonly occurs in middle-aged and older

individuals. From an epidemiological and clinicopathological

perspective, “oral cancer” can be divided into three categories:

5

carcinomas of the oral cavity proper, carcinomas of the lip vermilion,

and carcinomas arising in the oropharynx. Intraoral and oropharyngeal

tumors are more common among men than women, with a male:

female ratio of over 2:1 (Neville et al; 2002).

Around 75 percent of oral cancers are linked to modifiable

behaviors such as tobacco use and excessive alcohol consumption.

Other factors include poor oral hygiene, irritation caused by ill-fitting

dentures and other rough surfaces on the teeth, poor nutrition, and

some chronic infections caused by bacteria or viruses (Krivitsky, A.,

and Aalam, A.A.).

In many Asian cultures chewing betel, paan and Areca is known

to be a strong risk factor for developing oral cancer. In India where

such practices are common, oral cancer represents up to 40% of all

cancers, compared to just 4% in the UK. Some oral cancers begin as

leukoplakia a white patch (lesion), red patches (erythroplakia) or non-

healing sores that have existed for more than 14 days. In Indian

subcontinent Oral Submucousa fibrosis is very common. This condition

is characterized by limited opening of mouth and burning sensation on

eating of spicy food. This is a progressive lesion in which the opening of

the mouth becomes progressively limited, and later on even normal

eating becomes difficult.

Genes & Oral Cancer:-

Oncogene can encode growth factor receptor act on internal

signaling molecule and regulate DNA transcription factor. Once

mutated these gene product may not monitor mitosis as they should

resulting in neoplastic transformation. These oncogenes that have

significance in oral cancer are H-ras, C-myc and C-erb B-oncogene.

There is also a family of transforming growth factor that can modulate

cell growth. EGF, TGF-alpha and TGF-beta, all of which have been

implicated in oral cancer.

6

Normal cell growth

Figure 2:- Function of Oncogene and protooncogene

The role of altered cell proliferation and its effect on genetic

change during premalignant progression are related to local expression

of growth factor. Neoplastic cells have been shown to become

unresponsive to growth regulation by TGF-beta. Inactivation of tumor

suppressor genes may lead to a similar effect on neoplastic

progression. These data suggest that multiple mutation of certain

oncogene and tumor suppressor genes are necessary step in the oral

cancer process.

DNA changes are responsible for causing cells of the oral cavity

and oropharynx to become cancerous. One of the changes often found

in DNA of oral cancer cells is a mutation of the p53 gene. The protein

produced by this gene normally works to prevent cells from growing

too much and helps to destroy cells with DNA damage too extensive for

the cells to repair. Damage to p53 DNA can lead to increased growth of

abnormal cells and formation of cancers. Another DNA change found in

some oral cancers is that DNA from a papillomavirus (HPV) becomes

mixed together with the patient's own DNA. Some parts of the HPV

DNA instruct the cells to produce proteins that inactivate the p53

protein (Oral Cancer Study 2012).In addition to the human HPV, other

viruses such as HSV and adeno-virus are responsible for oral cancer.

7

HPV and HSV are most likely synergetic virus involved in human oral

cancer.

Neoplasia is a pathology disturbance of growth characterized by

an excessive and unceasing proliferation of cells. Some neoplasm is

benign because they grow slowly and remain so localized that the

patient usually experiences little difficulty from them. Others are

malignant or cancer tend to proliferate rapidly where the underline

tissue and metastasizing throughout the body that unless successfully

treated, they eventually cause death to the host (Harris et al, 1969).

Factors responsible for Oral Cancer:-

In India about 80% cancers are intra-oral cancer, rest are others.

Most of the people of India are in the habit of chewing tobacco in the

form of Khaini, betel, gutkha, etc. and in habit of alcohol consumption

is one of the reasons for oral cancer. Many factors are responsible for

oral cancer in human beings who are addicted to these narcotic things.

Some narcotic things are described below.

A. Tobacco Smoking:-

Tobacco smoke contains dozens of known carcinogens. The risk

of oral cancer and premalignant lesions increases with the amount of

tobacco consumed and the duration of tobacco use. This increased risk

holds for all types and uses of tobacco, whether it is smoked as a

cigarette, cigar, pipe or bidi, or used smokeless as a chew, plug or

snuff.

i)Bidi Smoking:-

The Indian form of cigarette is known as bidi, a smoke for the

common man in the country. It is made by rolling with the fingers 0.25

to 0.5 g. of tobacco flakes in a rectangular piece of dried leaf of

temburni (Diospyrosmelan-oxylon). Leaves of other genera and species

such as Bauhinia racemosa, Bauhinia vahlii, Buteafrondosa, and

Castanopsisindica are also used for wrapping the tobacco. Only two are

8

grown in India-namely, Nicotianatabacum and Nicotianarustica. Both

types are used for making bidi, as well as for chewing. Bidi smoking

can cause cancers of respiratory and digestive sites, including mouth,

oropharynx, larynx, lung, esophagus, and stomach (Sanghvi et al,

1955).

ii) Cigar & Pipe Smoking:-

A cigar is a product made of tobacco leavesor parts of leaves

rolled together and covered with a binder and an outer wrapper made

of natural or reconstituted tobacco. Some small cigars are similar in

size to a cigarette and may include a filter.

Chutta is a more coarsely prepared cheroot and is often smoked

with the burning end inside the mouth.

Chilum is a conical clay pipe, usually about 10 cm long. The

narrow lower end is put to the mouth, sometimes wrapped in a small

piece of wet cloth which acts as a filter (Khanolkar, 1959).

Hooka (an Indian pipe), the tobacco smoke is filtered through

water that is kept in a special receptacle and may contain aromatic

substances.

Hookli is a clay pipe with a rather short stem, varying from 7 cm

to 10 cm long and used in Gujarat (Mehta et al, 1969).

B. Smokeless Tobacco:-

a. Chewing:-

i. Betel Quid:-

The BQ is a mixture of areca nut (Areca catechu), catechu

(Acacia catechu) and slaked lime (calcium oxide and calcium

hydroxide) wrapped in a betel leaf (Piper betel).Condiments,

sweetening agents and spices may be added according to individual

preferences. In India, most habitual chewers of BQ add tobacco. The

9

bolus formed by chewing the preparation is either spat out, swallowed,

or kept in the mouth for hours, sometimes even during sleep. BQ

chewing has been related mainly to oral, pharyngeal and esophageal

cancer (IARC, 1985, 2004).

Commercial betel quid substitutes are pan masala and gutkha.

Pan Masala is basically a preparation of areca nut, catechu, cardamom,

lime and a number of natural and artificial perfuming and flavoring

materials.

ii. Gutkha :-

Gutkha is a variant of pan masala, in which in addition to these

ingredients flavored chewing tobacco is added (Thomas et al, 2009). It

is a powdery, granular white substance placed between lips and gum

under the tongue. Within moments, the Gutkha begins to dissolve and

turn deep red in color. It impacts upon its user “abuzz” somewhat more

than that of tobacco. It is used by millions of adults & its use can begin

at a very young age (Gupta et al, 1990).

b. Snuffing:-

Snuff may be moist or dry. Moist snuff is usually taken orally .This

product is sold in small round cans, in which the snuff is loosely

packed, or in small, tea-bag-like sachets. Dry snuff, which is less

commonly used, is usually inhaled through the nose.

The chemical carcinogens in smokeless tobacco include

polynuclear aromatic hydrocarbons (usually benzo[a]pyrene), polonium

210, and N-nitrosamines. Other chemicals include radium-226 and

lead-210.There is an association between the tobacco-specific N-

nitrosamines in smokeless tobacco and cancers of the upper digestive

tract (esophagus and stomach) and mouth (Hoffmann et al, 1994;

Winn, 1993).

c. Alcohols :-

10

Alcohol consumption is also a strong risk factor for oral

cancer and premalignant lesions. The risk increases with

increased consumption and duration of use of alcohol. Typically,

one 8-ounce glass of beer, one 4-ounce glass of wine and 1

ounce of spirits have equal amounts of alcohol (Franceschi et al,

2000). In many studies, heavy drinking is defined as consumption

of more than 14 to 21 drinks per week. Again, the risk of oral

cancer decreases when alcohol is no longer consumed, but it

takes many years for a drinker’s risk to reduce to that of

someone who has never been a drinker (Laronde et al, 2008).

Tobacco and alcohol consumption work together synergistically,

increasing the risk of oral cancer to more than 30 time that of those

who do not smoke or drink. Heavy drinkers and smokers are also more

likely to be diagnosed with late-stage disease. Ceasing to use tobacco

and alcohol greatly reduces the risk of developing oral cancer and

premalignant lesions (Laronde et al, 2008)

d. Human Papilloma Virus:-

Having human papilloma virus (HPV) is a strong risk

factor for oral cancers, especially when the lingual and

palatine tonsils, the soft palate and the base of the tongue

are involved. Of the more than 120 types of HPV, only a

few are high-risk factors for oral cancer, primarily HPV-16

and HPV-18.The combination of smoking and HPV infection

and of alcohol and HPV infection may have an additive

effect (Smith et al, 2004).

e. Other Issues:-

Studies of the role of marijuana in oral cancer are

scarce. Marijuana smoke contains many of the same

carcinogens found in tobacco smoke and has 4 times the

tar burden (Larondeet al, 2008).

11

Development of Cancer:-

Cancer is a multistep process. In Oral cancer, the cancer

develops in stepwise manner. i.e.

Inflammation

Leukoplakia

Erythroplakia

Dysplasia

Hyperplasia

Squamous cell carcinoma

Carcinoma in situ

Invasive Carcinoma

Figure 3:- Stages showing development of Oral cell Carcinoma

I. Inflammation:-

Inflammation is further categorized into non-specific inflammation &

specific inflammation. Inflammation of the mucous lining of any of the

structures in the mouth, which may involve the cheeks, gums, tongue,

lips, and roof or floor of the mouth. It is stimulated by chemical factors

released by injured cells and serves to establish a physical barrier

against the spread of infection, and to promote healing of any

damaged tissue following the clearance of pathogens.

a) Non-specific Inflammation:-

There is no definite cause for non-specific inflammation

or any specific agent, sore in mouth or even taking hot tea

may cause inflammation.

12

b) Specific Inflammation:-

It is caused by some specific agents such as parasite,

fungus, etc.

c) Fungal Infection:-

In some ways fungus may share with other factors produce

oral diseases due to unhygienic habits

&nutrition.Acitonomycosis& candidiasis are commonly involving

the oral mucosa. Cervico facial actionomycosis is the commonest

form of the disease developing of angle of the mandible.

d) Candidiasis:-

It is caused by Candida albicans& other candida species

which are normal body flora found on the skin, mouth, vagina

and intestines. Candida infection has various manifestations

depending on the site for ex-oral candidiasis (thrush) present as

raised white plaques on the oral mucosa, tongue or gums.

e) Parasitic Infection:-

Entamoebagingivalis smear in over 60% of patients with

poor dentition & oral hygiene. Morphologically the parasites are

similar to Entamoebahistolytica.

f) Herpetic Stomatitis:-

It as an acute disease occurring in infants & young

children. It is caused by herpes simplex virus cause-stress,

emotional upsets, and upper respiratory infection.

II. Leukoplakia:-

The term leukoplakia was first used by Schwimmer in 1877 to

describe a white lesion of the tongue, which probably represented a

syphilitic glossitis (Schwimmer et al, 1877). As defined by the World

Health Organization, leukoplakia is “a white patch or plaque that

cannot be characterized clinically or pathologically as any other

13

disease (Kramer et al, 1978). In the evaluation of clinical features of

leukoplakias, the following 3 types were taken into consideration:

i) The homogeneous type,

ii) The ulcerated type, and

iii) The speckled type.

The homogeneous type is characterized by raised plaque

formation consisting of plaques or groups of plaques varying in size

and with irregular edges. These lesions are predominantly white, but

may have areas of a grayish-yellow color.

The ulcerated type of leukoplakia gives the impression that

ulceration has been caused by trauma either of chewing or of burning

in cases of reverse smoking. The affected area is usually uniformly red,

but yellowish areas of fibrin may be present.

The speckled leukoplakia has the characteristics of white patches

on an erythematous base (Mehta et al, 1969). The factor that

potentiate that risk include tobacco, micro-organism including viruses,

nutrition & actinic radiation.

Two specific tobacco-related lesions of the oral mucosa, nicotine

stomatitis and tobacco pouch keratosis, have often been included

under the broad umbrella of leukoplakia (Neville et al, 2002).

a) Nicotine Stomatitis:-

Nicotine stomatitis is a thickened, hyperkeratotic

alteration of the palatal mucosa that is most frequently

related to pipe smoking, but milder examples can also

develop secondary to cigar smoking or, rarely, from cigarette

smoking (Neville et al, 2002; Kramer et al,1978).

The term nicotine stomatitis is actually a misnomer

because it isn’t the nicotine that causes the changes; the

changes are caused by the intense heat generated from the

smoking. Nicotine stomatitis is seen more often in pipe

14

smokers because of the great amount of heat that is

generated from the pipe stem (Neville et al, 2002).

b) Tobacco Pouch Keratosis:-

Another specific tobacco-related oral mucosal alteration

occurs in association with smokeless tobacco use, either from

snuff or chewing tobacco. Such lesions typically occur in the

buccal or labial vestibule where the tobacco is held, but they

can also extend onto the adjacent gingiva and buccal mucosa.

Early lesions may show slight wrinkling that disappears when

the tissues are stretched. Advanced lesions exhibit greatly

thickened zones of grayish white mucosa with well-developed

folds and fissures. Tobacco pouch keratoses can occur at any

age, even in children and adolescents. Smokeless tobacco

keratoses are seen with some degree of frequency in older

women, who may have started their snuff-dipping habit in

early childhood (Smith et al, 1970).

III. Erythroplakia:-

Oral erythroplakia occurs most frequently in older men and

appears as a red macule or plaque with a soft, velvety texture. The

floor of mouth, lateral tongue, retro-molar pad, and soft palate are the

most common sites of involvement (Nevilleet al, 2002). All

erythroplakia cases showed some degree of epithelial dysplasia; 51

percent showed invasive squamous cell carcinoma, 40 percent were

carcinoma in situ or severe epithelial dysplasia, and the remaining 9

percent demonstrated mild-to-moderate dysplasia. Therefore, true

clinical erythroplakia is a much more worrisome lesion than leukoplakia

(Mashberg et al, 1995).

IV. Dysplasia:-

Oral dysplasia are the classic cytologic abnormalities associated

with most epithelial atypias : based layer hyperchromatism a typical

mitosis, altered nuclear cytoplasmic ratio, loss of cellular polarity

15

nuclear pleomorphism, hyperchromatic nucleoli & basal layer

hyperplasia (Blozis, 1972).

V. Hyperplasia:-

Inflammatory papillary hyperplasia most often occurs on the oral

cavity in association with an ill-fitting maxillary denture or in

association with poor oral hygiene.

Papillary hyperplasia present as a proliferation of multiple

exophyte papillary projections, supported by a connective tissue core

that is nearly always chronically inflamed.

VI. Squamous Cell Carcinoma:-

It is of three types;

a) Well differentiated squamous cell carcinoma:-

The cells are shed in single or in sheets with marked

variation in size & shape(polygonal, spindle, tadpole or pearl

formation). The nuclei, always irregular can be pyknotic or

vesicular. The chromatin when discernible is irregularly clumped

with pointed projection and occasional prominent nucleoli. The

nuclear membrane is usually thick, irregular in outline with

multiple indentations. Binucelation or multi-nucleation

occasionally seen. The cytoplasm of the cell thick and occasional

keratohyalinic granular precipitate forming perinuclear ring.

Enucleated heavily keratinized (ghost like) cells are common in

very well differentiated carcinomal (Achieve of oncology, 2000).

b) Poor differentiated squamous cell carcinoma:-

In the neoplasm the cells shed singly, in clusters (80%) or

in sheets (20%). They have scanty to adequate non-keratinized

cytoplasm & are evenly stained deep blue purple often with

indistinct borders. Their nuclei are hyperchromatic & centrally

placed the chromatin shows coarse but irregular pattern with

large reddish nucleoli in more than 60% of cells. Variable mounts

of inflammatory cells, degenerate cellular debris and protein

deposits are usually found in the background of the smear

16

(Koller, 1963).

c) Verrucous Carcinoma:-

Verrucous carcinoma is a low grade variant of oral

squamous cell carcinoma and comprises approximately 3% of all

primary invasive carcinoas of the oral mucosa (Bouquot, 1998).

This tumor occurs often in older men, although many examples

have also been documented in older women in areas of the

country where the habit of snuff dipping has been popular among

women (Brown et al, 1965 and McCoy, 1981).

VII. Carcinoma In Situ (CIS):-

The tendency of the oral in situ cells to shed as single cells,

rather than a sheet& of their cytoplasm to be slightly scantier and

more granular. The amount of inflammatory (polymorphonuclear) cells

& cellular debris is less abundant than in invasive of mature

inflammatory lymphocytes may be seen (Singleton et al, 1968).

The present investigation was undertaken to find and the

use of tobacco in different forms like Gutkha, bidi, cigarette, pan

masala, gudakhu, Khaini etc. The uses of betel pan with tobacco as

evidenced have little carcinogenic effect due to the presence of

allylbenzene. But the other forms of tobacco cause oral submucousa

fibrosis and periodontal carcinoma.

Subjects:-

A total number of 20 individuals (both male & female)were

17

included in the study of the effect of Gutkha, Pan, and Cigarette etc. on

the oral cell. Of these, 17 (mean age 47 years) were from urban area.

The rest 3 (mean age 47 years) were from rural area. After the scrape

was taken a few questions were interviewed such as,

i. Name

ii. Place

iii. Age

iv. Sex

v. Habit (gutkha/pan/tobacco/Khaini/smoking/alcohol etc.)

vi. Duration of use

vii. Consumption per day

Sample Collection:-

For sample collection following materials are required,

i. Instruments used to obtain sample (wooden spatula or

coverslip)

ii. Clean grease free microscopic slide of good quality

iii. A marker pen for marking of slides

iv. Name, age, sex, habits and other data were recorded in

separate sheets

Chemicals Required:-

For the study of effect of tobacco on buccal cells, following

chemicals are required,

i. 70% alcohol or methanol ii. 50% alcohol

iii. Hematoxylin iv. Alcoholic eosin

v. Absolute alcohol vi. 0.3% acid alcohol

Procedure For Obtaining Smear:-

Oral mucosa cells were collected from each subject using a

small square coverslip (22mm) or wooden spatula gently from the oral

18

mucosa of inner side of cheeks. Before sampling, each individual rinsed

his or her mouth thoroughly with tap water.

Preparation of Smear:-

After scrapping was obtained the exfoliated buccal mucosa

cells were placed on to the pre-cleaned slide and spread the scraped

specimen uniformly over the surface of previously labeled slide & kept

for drying. The main aim of the smear was to obtain a monolayer of

cells spread uniformly over the entire surface of the slide.

Precautions:-

While taking smear following care should be taken,

i. Mouth of the individual should be cleaned properly with tap

water before the sample was taken. So that extra foreign

bodies cannot mix with the buccal cells.

ii. The slides were labeled with identifying numbers & name

informs of code, data before the smear was taken.

iii. Smear should be spread uniformly. Care was taken to avoid

too thick or too thin smear.

Methods:-

Fixation of smear:-

As soon as the specimen was spread, the smear was

allowed to dry for few minutes. For fixation of the smear, the slide was

immersed in 70% alcohol or in methanol. Smears were kept in that

condition for a minimum of 15 minutes for fixation.

Staining Procedure:-

Slides of each individual were stained by

Hematoxylin & Eosin stain to observe cytomorphology.

Procedure for Staining:-

i. After fixation the slide was washed in distill water.

ii. Then the slide was stained with Harris haematoxylin for

10min for nuclear stain.

19

iii. It was rinsed in distill water to prevent smear being washed

off the slides.

iv. It is differentiated with 0.3% acid alcohol.

v. It was again rinsed with running tap water.

vi. To prepare permanent slides, the slides were undergone

for alcoholic gradation of 50% & 70% for 5 minutes each.

vii. Then the slides were stained with ethyl eosin for 2 minutes

for cytoplasmic stain.

viii. Eosin stained slides were washed in 70% alcohol to remove

excess eosin.

ix. Then the slides were dehydrated in absolute alcohol and

kept for drying and observed under microscope with

magnification of 100x with oil immersion & photography of

different nuclear change cells were taken in USB device in

computer.

Out of 20 subjects whose case history have been recorded;

12 subjects were regular use of pan or betel quid with mixtures

of paan masala, Gutkha which is a preparation of crushed betel nut

tobacco & sweet flavors. They were also consuming Khaini which

20

contain tobacco, lime oil, menthol, and contain added flavors &

chewing tobacco. Besides these they were also using Gudakhu which is

in the form of paste like solution containing tobacco and some flavors.

8 subjects were consuming regularly smoking tobacco like

cigarettes, bidi containing pure tobacco. They also have alcohol

addiction. In addition to smoking tobacco they were also the users of

smokeless tobacco like Gutkha and Paan.

Initially the cells were scored according to the foci per subject for

all the various cells types outlined in the buccal cytome assay. The

consisted of cells containing MNs, nuclear changes like nuclear buds,

change in the shapes of nucleus and binucleates& the cell death

parameter condensed chromatin (dysplasia), karyorrhectic, pyknotic

and karyolitic cells.

Subjects with different habits show different types of cells like

normal cells with other abnormal cells. The abnormal cells include the

MNs, binucleate, pyknotic cells, nuclear changes, condensed chromatin

in which chromatin material coarsely granular & clumped on one side

of nuclear membrane, karyorrhectic and karyolytic cells.

Criteria For Identifying & Scoring Cell Types In Buccal

Epithelial Cells:-

Cancer cells characterized by an irregular configuration nuclear

enlargement with alteration of nuclear cytoplasmic ratio, irregular

distribution of chromatin in combination with variation in chromatin

particle size & multiple, irregular, micronuclei. Since any tissue is

susceptible to neoplastic transformation and since tumor often closely

resemble such tissue of origin, Cancer cells tend to be larger than

normal cells to have irregular outlines with bizarre forms & to show

pleomorphism giant cells often with multiple nuclei and unusual shapes

are common in highly malignant cancer. Degenerative changes such as

vacuolization are common in both cytoplasm and nucleus. The

nucleolus is usually more prominent than in normal cells and more

than one is often observed. A greater proportion of the cells appear to

21

in normal or abnormal mitosis (Sachs et al, 1971, 1972).

The criteria were outlined by Tolbert et al for buccal cytome

assay.The criteria are intended to classify BCs into categories that

distinguish between ‘normal’ cells and cells that are considered

‘abnormal’, based on nuclear morphology. These abnormal nuclear

morphologies are thought to be indicative of DNA damage or cell death

(Thomaset al, 2007).

Figure 4:- Buccal cytome model showing cellular relationship

Source:British Dental Journal, 1992

A. Basal Cells:-

The nuclear to cytoplasm ratio is larger than that in differentiated

BCs that are derived from basal cells. Basal cells have a uniformly

stained nucleus and they are smaller in size when compared to

differentiated BCs. Basal cells can contain MNs and were scored in the

22

assay.

B. Normal Differentiated Cells:-

These cells have a uniformly stained nucleus that is usually oval or

round in shape. They are distinguished from basal cells by their larger

size and by a smaller nuclear to cytoplasmic ratio. No other DNA-

containing structures apart from the nucleus are observed in these

cells. These cells are considered to be terminally differentiated relative

to basal cells because no mitotic cells are observed in this population.

C. Cells with Micronuclei:-

These cells are characterized by the presence of both a main

nucleus and one or more smaller nuclei called MNs.The MNs are usually

round or oval in shape and their diameter may range between 1/3 and

1/16 the diameter of the main nucleus.MNi have the same staining

intensity and texture as the main nucleus. Most cells with MNi will

contain only one MN but it is possible to find cells with two or more

MNi.Cells with multiple MNi are rare in healthy subjects but become

more common in individuals exposed to radiation or other genotoxic

agents.The MNs must be located within the cytoplasm of the cells. The

presence of MNs is indicative of chromosome loss or fragmentation

occurring during previous nuclear division (Fenech et al, 1986). Cells,

which are pyknotic (i.e., shrunken nuclei), and have condensed

chromatin or karyorrhecticnuclei, are not scored for MNi.

D. Cells with nuclear buds:-

Cells with nuclear buds contain nuclei with an apparent sharp

constriction at one end of the nucleus suggestive of a budding process,

i.e., elimination of nuclear material by budding. In the original Tolbert

et al. publication (Tolbert et al, 1992). These were referred to as

‘broken egg’ cells. The nuclear bud (NBUD) and the nucleus are usually

in very close proximity and appear to be attached to each other. The

nuclear bud has the same morphology and staining properties as the

nucleus; however, its diameter may range from a half to a quarter of

that of the main nucleus. The mechanism leading to nuclear bud

23

formation is not known but it may be related to the elimination of

amplified DNA or DNA repair (Thomas et al, 2009).

E. Binucleated cells:-

Binucleated cells are cells containing two main nuclei instead of

one. The nuclei are usually very close and may touch each other and

usually have the same morphology as that observed in normal cells.

The significance of these cells is unknown, but they are probably

indicative of failed cytokinesis following the last nuclear division.

F. Condensed Chromatin cells:-

Condensed Chromatin cells show a roughly striated nuclear pattern in

which the aggregated chromatin is intensely stained.In these cells it is

apparent that chromatin is aggregating in some regions of the nucleus

while being lost in other areas. When chromatin aggregation is

extensive the nucleus may appear to be fragmenting (Wyllie, 1981).

These cells may be undergoing early stages of apoptosis, although this

has not been shown conclusively. These cells as well as karyorrhectic

cells invariably result in fragmented nuclei, leading to eventual

disintegration, and sometimes appear to contain bodies similar to MNi,

but these are not scored as MNi in the assay as their origin cannot be

accurately determined (Thomas et al, 2009).

G. Karyorrhectic cells:-

Karyorrhectic cells have nuclei that are characterized by more

extensive nuclear chromatin aggregation relative to condensed

chromatin cells. They have a densely speckled nuclear pattern

indicative of nuclear fragmentation leading to the eventual

disintegration of the nucleus.These cells may be undergoing a late

stage of apoptosis, but this has not been conclusively proven. These

cells should not be scored for MNi in the assay.

H. Pyknotic cells:-

Pyknotic cells are characterized by a small shrunken nucleus, with a

high density of nuclear material that is uniformly but intensely stained.

24

The biological significance of the pyknotic cells and the mechanism

leading to their formation are unknown, but it is thought that these

cells may be undergoing a unique form of cell death; however, the

precise mechanism remains unknown. They may represent an

alternative mechanism of nuclear disintegration that is distinct from

the process leading to the condensed chromatin and karyorrhectic cell

death stages (Holland et al, 2008 and Chen et al, 2006).

I. Karyolytic cells:-

Karyolytic cells are cells in which the nucleus is completely depleted

of DNA and is apparent as a ghost-like image that has no Haemotoxylin

staining (Wyllie, 1981 and Tolbert, 1991) Therefore, these cells appear

to have no nucleus and represent a very late stage in the cell death

process.

Statistical Analysis:-

Two-way of variance (ANOVA) was used to determine the

significance of the cellular parameters measured between duration of

the habit used and the nuclear damage like MNs, Pyknotic, Nuclear

changes, Karyolytic cells, Tadpole shaped nucleus and binucleated

cells. The significance was accepted at F < 0.05.

25

Table no. 1:- Data representing age, Habits of tobacco & duration of use in years

26

SL NO. AGE SEX HABIT

APPROX. DURATION OF USE IN

YEARS

1 47 MALCOHOL, CIGARETTE,

PAN20

2 49 MGUTKHA,

CIGARETTE,ALCOHOL10

3 25 M GUTKHA, CIGARETTE 10

4 59 M PAN, GUDAKHU 25

5 43 MGUTKHA,

CIGARETTE,ALCOHOL, PAN

25

6 53 M PAN, GUTKHA 10

7 45 MPAN, PAN MASALA

(PUDIYA)30

8 40 MPAN, GUTKHA,

CIGARETTE, ALCOHOL,ALL TYPES

18

9 58 M GUTKHA 15

10 60 F GUDAKHU 50

11 66 M GUDAKHU 40

12 56 F GUDAKHU 40

13 42 M PAN, HARIDAKHANDI 24

14 47 MGUTKHA,

PAN,HARIDAKHANDI, KHAINI (NASA)

30

15 50 F GUDAKHU 30

16 38 M ALL 15

17 37 MPAN, PAN

MASALA,HARIDAKHANDI35

18 60 M GUDAKHU 40

19 42 MCIGARETTER,

ALCOHOL,PAN MASALA20

20 28 M GUTKHA, CIGARETTE10

LEGEND

Fig. a : Cell Having Single Micronuclei of 20 Years Of

Duration of Use

Fig. b : Cell Having Two Micronuclei of 10 Years Of

Duration of Use

Fig. c : Cell with Micronuclei of 10 Years Of

Duration of Use

Fig. d : Karyorrhectic Cell with two micronuclei of 40 Years

of Duration of Use

Fig. e : Pyknotic cell of 20 Years Of

Duration of Use

27

(b)

(c) (d)

(e)

28

LEGEND

Fig. f : Clear Pyknotic Cell of 10 Years Of

Duration of Use

Fig. g : Cell with Concave Type Nucleus of 18 Years Of

Duration of Use

Fig. h : Cell with Kidney Shaped Nucleus of 18 Years Of

Duration of Use

Fig. i : Cell with Elongated Nucleus of 18 Years Of

Duration of Use

Fig. j : Cell with Tadpole Shaped Nucleus of 20 Years Of

Duration of Use

29

(f)

(g)

(h) (i)

(j)

30

LEGEND

Fig. k : Cell with Nuclear Bud of 20 Years Of

Duration of Use

Fig. l : Cell with Fragmented Nucleus of 20 Years Of

Duration of Use

Fig. m : Tadpole Shaped Nucleus of 10 Years Of

Duration of Use

Fig. n : Dumbled Shaped Nucleus of 10 Years Of

Duration of Use

Fig. o : Lobbed Nucleus of 35 Years Of

Duration of Use

31

(k) (l)

(m) (n)

(o)

32

LEGEND

Fig. p : Cell with Tadpole Shaped nucleus of 35 Years Of

Duration of Use

Fig. q : Moderate Dysplasia Showing Fragmented Nucleus of

35 Years Of Duration of Use

Fig. r : Karyolytic Cell of 30 Years Of

Duration of Use

Fig. s : Indistinct Binucleated Cell of 20 Years Of

Duration of Use

Fig. t : Distinct Binucleated Cell of 20 Years Of

Duration of Use

33

(p) (q)

(r) (s)

(t)

34

Duration of use

MN per 1000

cells

Pyknotic cells

per 1000 cells

Nuclear

change per 1000 cells

Kryolytic cells

per 1000 cells

Tadpole shaped nucleus cells per

1000 cells

Binucleated cells per

1000 cells

Grand

total

0-10 35 52 2 14 25 4 132

10-20 20 61 13 21 27 11 153

20-30 23 71 24 22 37 6 183

30-40 25 85 37 20 31 12 210

40-50 38 103 33 24 38 14 250

141 372 109 101 158 47 928

Table no. 2:- Different types of Nuclear damages studied per 1000 cells with respect to the duration of use in different individuals

Correction Factor = 28,706

Sum square between nuclear damaged cells = 12,797.87

Degree of freedom for SSC = 5

Sum square between duration of use = 1447.57

Degree of freedom for SSR = 4

Sum square of total = 15,761

Source of

variation

SS(Sum of squares

)

DF(Degre

e of freedo

MSS(Mean sum of squares

Calculated value

of F (V.R)

Tabulated

value of F

35

m) ) (0.05)

Between

nuclear damaged cells

12,797.87

V1 = 5 2559.7 33.75(5,20) =

2.7

Between

duration of use

1447.57V2 = 4

361.9 4.77(4,20)=

2.9

Error 1516.47V3 = 20

75.82

Total 15,76129

Table no. 3:- ANOVA table showing significant change in nuclear damage with increase in duration of use

Standard deviation of MNs (S1) = 7.85

Mean (X1) = 28.2

Coefficient of variation (CV1) = 0.2785

Standard deviation of Pyknotic cells (S2) = 20.144

Mean (X2) = 74.4

Coefficient of variation (CV2) = 0.2707

36

Standard deviation of Nuclear change (S3) =

14.412

Mean (X3) = 21.8

Coefficient of variation (CV3) = 0.6611

Standard deviation of Karyolytic cells (S4) = 3.768

Mean (X4) = 20.2

Coefficient of variation (CV4) = 0.1865

Standard deviation of tadpole nucleus (S5) = 5.814

Mean (X5) = 31.6

Coefficient of variation (CV5) = 0.1840

Standard deviation of Binucleated cells ( S6) = 4.219

Mean (X6) = 9.4

Coefficient of variation (CV6) = 0.488

37

38

Figure

5:-

N

um

ber

of

nucl

ear

dam

aged c

ells

cou

nte

d p

er

10

00

cells

, depic

ts t

hat

wit

h

incr

ease

in d

ura

tion o

f use

num

ber

of

nu

clear

dam

aged c

ell

incr

ease

s.

39

Figure

6:-

Coeffi

cien

t of

vari

ati

on o

f diff

ere

nt

types

of

nucl

ear

dam

age indic

ati

ng less

er

the c

oeffi

cient

vari

ati

on m

ore

the o

ccurr

ing f

req

uency

.

The various forms of smokeless tobacco are chewed, sucked, or

applied to teeth or gum. Gutkha, a dry preparation commercialized

since 1975, containing areca nut, slaked lime, catechu, condiment. The

habit of chewing tobacco is increasing because of free availability,

cheaper rate. Studies have confirmed that the use of tobacco is

harmful for oral cavity causing OSF & periodontal condition

(Gajalakshmi et al).

Frequency of chewing rather than total duration of the habit was

directly related to oral submucousa fibrosis (Hazare et al, 1998; Shah

et al, 1998)

The present study indicates the distinction between normal cells

and cells that are considered abnormal based on nuclear morphology.

These abnormal nuclear morphologies are thought to be indicative of

DNA damage or cell death. The cells as described that the normal

differentiated cells have uniformly stained nucleus usually oval or

round shaped. The abnormal cells with the presence of both the main

nucleusand one or more small nuclei called MNs (Fig. a, Fig. b, Fig. c,

Fig. d). The presence of MNs is indicative of chromosome loss or

fragmentation occurring during previous nuclear division (French, M. &

Morley, A.A., 1986).Cells with condensed chromatin or karyorrhectic

cells was not scored for MNs.

Cell with nuclear buds (Fig. k) that these also have nuclei with an

apparent sharp constriction at one end i.e. suggesting the elimination

of nuclear material by budding. The diameter of the bud may range

from a half to quarter of that of the main nucleus. The mechanism

leading to this morphology may be due to elimination of amplified DNA

or DNA-repair complete (Fenech,M. and Coot, J.W., 2002).Fig. s & Fig. t

shows that are with two nuclei i.e. bi-nucleated instead of one. The

nuclei are very close to each other or may be touching to each other.

The nuclei usual have the same morphology as that of normal cells.

40

The significance though is unknown but may be indicative of

Cytokinesis.

Fig. d, Fig. l, Fig q shows karyrrhectic cells characterized by the

more extensive appearance of nuclear chromatin aggregation leading

to fragmentation or dis-integration of the nucleus.

Occurrence of pyknotic cells(Fig. e,Fig. f) are characterized by a

small sunken nucleus with a high density of nuclei material. The

nucleus diameter is usually one to two-third of the nucleus of normal

differentiated cells. The significance of these cells are unknown but

may lead to a form cell-death.

The karyolitic cells appear in which the nucleus is completely

depilated of DNA and appear as a ghost like image(Fig. r). It is probable

that these cells represent a very late stage of cell-death process.

From some studies it was found that financial status of a person

also affects the nuclear damage with respect to the duration of use.

Individuals from poor family have higher risk towards OSMF and

leukoplakias where as individuals from rich background were able to

resist themselves and gets less affected than the poor individual from

pre-cancerous lessons though they intake almost same amount of

tobacco.

The comparative result of different nuclear abnormalities with

the duration of use of tobacco indicates that the frequency of

occurrence of micronuclei increased as duration of use increased. From

the result it was found that the table value of F for V1 = 5 and V3 = 20

at 5% level of significance is 2.7. The calculated value is greater than

the table value i.e. 33.75. From this it is concluded that the different

types of nuclear damages differ significantly. The critical value of F for

V2 = 4 and V3 = 20 at 5% level of significance is 2.9. The calculated

value is greater than this. The calculated value is 4.77. Hence it shows

there is significant difference between the duration of use. It means

with increase of duration of use, the nuclear damage increases. As it is

41

shown in figure 6 the number of pyknotic cells is higher in respect to

other type of cells. Next to pyknotic cells, MNs & tadpole shaped

nucleus damage in more number. The number increases significantly

with duration of use. Also it shows that with increase of duration of use

tobacco the occurrence of damaged cells increases. Figure 7 shows

the coefficient of variation of different types of damaged cells. When

coefficient of variation increases, the occurrence frequency of nuclear

damaged cells decreases. Hence from figure 7 it is concluded that

karyolitic cells, MNs, tadpole shaped nucleus & pyknotic cells occur

more frequently than that of binucleated cells & nuclear shape

changed cells.

Anil et al, 2011 revealed that an increase in micro-nuclei in OSS

passion and emphasized that Gutkha chewing habit in younger age

increased the chances of malignant transformation. Babul et al, 1996

pointed that factors may be responsible on the addition of tobacco

content and the absence of Beetle leaf and its contents and much

higher dry-weight of pan masala or gutkha. Increased frequencies of

micro-nucleated cells in exfoliated buccal mucosa and high ingredients

of CA and CSE in peripheral lymphocytes have been observed in uses

of pan masala with and without tobacco in comparison with control

individuals (Dave et al, 1991;Trivedi,1992;Yadav&Chadha,

2002;Beena& Patel, 2009).

A significant increase in frequencies of chromosomal aberration

and micro-nuclei was observed in bone marrow cells after a day

dependent treatment with pan masala or gutkha (Majidar et al,2009).

In vitro use of aqueous extract of pan masala in Chinese hamster ovary

cells show significant increase of chromosomal aberration and micro-

nuclei (Patel et al, 1994; Adhyaryu et al,1989). Similarly increase in

frequency of micro nucleated buccal mucosa cells in individuals

consuming tobacco were observed by Gandhi &Kaur,(2000), Siddique

et al, (2008) and Fareed et al, (2011).

42

Age play an important role in these cytogenetic markers.

Similarly role of age on the frequencies of the spontaneous micro

nuclear formation was earlier reported (Tile &Stelaw,1985; French

&Morely,1985; Ghosh et al,1990; Yadav and Chadha,2002).

The present investigation indicated as alarming condition of use

of gutkha, panmasala and other type of tobacco product. It can rapidly

devastate the oral mucosa showing carcinogenic effect. So this is the

high time to put ban on the manufacture and selling of such products

and the acceleration of the programmes for elimination of use of these

products.

43

1. Ackerman, L.V., (1948): Verrucous carcinoma of the oral cavity.

Surgery; 23:670-678.

2. Alsayyad, J., Hamadeh, R., (2007): Cancer Incidence among the

Bahraini Population: Five-Year (1998-2002) Experience. Ann

Saudi Med; 27(4):251-8.

3. Babu, S., Sesikeran, B., and Bhat, R.V., (1996): Oral fibrosis

among teenagers chewing tobacco, areca nut and pan masala.

Lancet, 348: 692.

4. Bhide, S.V., Gothoskar, S.V., and Shivapurkar, H.H., (1984):

Arecolinetumorigenecity in Swiss strain mice on normal and vitamin

B deficient diet. J.Cancer.Res.Clin.Oncol., 107: 169-171.

5. Blozis, G.G., (1972): The value of exfoliated cytology in the

diagnosis of oral cancer. Int. Dent. J,22:481-488.

6. Bouquot, J.E., (1998): Oral verrucous carcinoma. Incidence in two

US populations. Oral Surg Oral Med Oral Pathol Oral RadiolEndod;

86:318-324.

7. British Dental Journal, (1992): 173, 53. Page.55

8. Chen, C., et al, (2006): Cytogenetic damage in buccal epithelia

and peripheral lymphocytes of young healthy individuals exposed to

ozone. Mutagenesis21,131–137.

9. Conrad D.R., Molecular Mechasacnisms of Cancer, (1990).

10. Dunham, L.J., Sheets, R.H., and Morton,J.F., (1974): Proliferative

lesions in check pouch and esophagus of hamsters treated with

plants from Curacao Netherlands Antilles, J. Natl. Cancer Inst., 53:

1259-1269.

44

11. Etemadi, A., Sadjadi, A., Semnani, S.H., et al, (2008): Cancer

Registry in Iran: A Brief Overview. Arch Iran Med, 11(5): 577-580.

12. Farahmand,M., Almasi-Hashiani,A., and HosseinFallahzade,M.,

(2013): Epidemiology of cancer of liver and intrahepatic bile

ducts based on fars province cancer registry’s data, Zahedan

journal of research in medical sciences, Feb; 15(2): 86-89.

13. Fareed, M., Afzal, M., Siddique Y.H., (2011): Micronucleus

investigation in buccal mucosal cells among pan masala/gutkha

chewers and its relevance for oral cancer, Biology and Medicine,

Vol3(2):8-15.

14. Fenech, M.,&Crott, J.W., (2002): Micronuclei, nucleoplasmic

bridges and nuclear buds induced in folic acid deficient human

lymphocytes-evidence for breakage-fusion-bridge cycles in the

cytokinesis-block micronucleus assay.Mutat. Res.504, 131–136.

15. Fenech, M., and Morley, A. A., (1986): Cytokinesis-block

micronucleus method in human lymphocytes: effect ofin vivo ageing

and low dose X-irradiation. Mutat. Res., 161, 193–198.

16. Fialkow, P.J., (1976): Clonal origin of human tuomors, Biochem,

Biophys, 458:283-321.

17. Franceschi, S., Levi, F., Dal Maso, L., Talamini, R., Conti, E., Negri,

E., and other, (2000): Cessation of alcohol drinking and risk of

cancer of the oral cavity and pha-rynx’, Int J Cancer; 85(6):787–9.

18. Giri, A.K., Bannerjee, T.S., Talukder, G., and Sharma, A., (1988):

Induction of sister chromatid exchange and dominant lethal

mutation by ‘Katha’ (catechu) in male mice. Cancer Lett, 38:357-

364.

19. Grady, D., Greene, J., Daniels, T.E., et al, (1990): Oral mucosal

lesions found in smokeless tobacco users. J Am Dent Assoc;121:117-

123.

45

20. Greer,R.O.Jr, Poulson,T.C., (1983): Oral tissue alter-ations

associated with the use of smokeless tobacco by teen-agers. Part I.

Clinical findings. Oral Surg Oral Med Oral Pathol;56:275-284.

21. Gupta, P.C., Hammer, J.E., andMurti, P.R., (1990): Control of

tobacco – Related cancers and other diseases, Tata institute of

fundamental research: Oxford University.

22. Gupta, P.C., Sinor, P.N., and Bhonsle, R.B., (1998): Oral

submucousa fibrosis in India: A new epidemic? Natl.Med. J.India,

11:113-6.

23. Harris, H.,Muller, O.J., et al., (1969): Suppression of malignancy

by cell fusion, Nature, 238:363-368.

24. Hashibe, M., Brennan, P., Benhamou, S., Castellsague, X., Chen,

C., Curado, M., and others, (2007): Alcohol drinking in never users of

tobacco, cigarette smoking in never drinkers, and the risk of head

and neck cancer: pooled analysis in the International Head and Neck

Cancer Epidemiology Consortium.JNatl Cancer Inst; 99(10):777–89

25. Hashibe, M., Ford, D.E., Zhang, Z.F.,(2002): Marijuana smoking

and head and neck cancer.JClinPharmacol; 42(11 Suppl):103S–7S.

26. Hazare, V.K., Goele, R.R., Gupta, P.C., (1998): Oral Submucousa

Fibrosis arecanut and pan masala use: a cause control study; Natl.

Med. J. India; 11:299.

27. Hoffmann, D., Brunnemann, K.D., et al., (1994): Tobacco-specific

N-nitrosamines and Areca-derived N-nitrosamines:chemistry,

biochemistry, carcinogenicity, and relevance to humans, J Toxicol

Environ Health; 41:1-52.

28. Holland., N., et al., (2008): The micronucleus assay in human

buccal cells as a tool for biomonitoring DNA damage: the HUMN

project perspective on current status and knowledge gaps, Mutat.

Res.659, 93–108.

29. Kaugars, G.E., Riley, W.T., Brandt, R.B., et al, (1992): The

prevalence of oral lesions in smokeless tobacco users and an

evaluation of risk factors. Cancer;70:2579-2585.

46

30. Khanolkar, V. R., (1959): Oral cancer in India, ActaUn.Znt. Cancr.

15:67-77.

31. Koller, P.C., (1963): The nucleus of the cancer cell exp cell Res.

[Suppl]9:3.

32. Kramer, I.R., Lucas, R.B., Pindborg, J.J., et al, (1978): WHO

Collaborating Centre for Oral Precancerous Lesions. Definition of

leukoplakia and related lesions: An aid to studies on oral pre-cancer.

Oral Surg Oral Med Oral Pathol;46:518-539.

33. Krivitsky, A., Aalam, A., Oral cancer Examination, (2010)..

34. Laronde, D.M., Hislop, T.G., et al, (2008): Oral Cancer: Just the

Facts, Clinical Practice-jcda, April, Vol. 74, No. 3, PP. 269-272

35. Lingen, M., Sturgis, E.M., Kies, M.S., (2001): Squamous cell

carcinoma of the head and neck in nonsmokers: clinical and biologic

characteristics and implications formanagement.CurrOpinOncol;

13(3):176–82.

36. Main, J.H., Lecavalier, D.R., (1988): Smokeless tobacco and oral

disease, J Can Dent Assoc; 54:586- 91.

37. Mashberg, A., Samit, A., (1995): Early diagnosis of asymptomatic

oral and oropharyngeal squamous cancers. CA Cancer J Clin;45:328-

351.

38. Mayne, S., Morse, D., Winn, D., (2006): Cancers of the oral cavity

and pharynx. In: Schottenfeld, D., Fraumeni, J., Jr, editors. Cancer

epidemiology and prevention. 3rd ed. New York: Oxford University

Press; p. 674–96.

39. McCoy, J.M., Waldron, C.A., (1981): Verrucous carcinoma of the

oral cavity.A review of forty-nine cases. Oral Surg Oral Med Oral

Pathol; 52:623-629.

40. Mehta, F.S., Pindborg, J.J., Gupta, P.C., Daftary, D.K.,

(1969):Epidemiologic and histologic study of oral cancer and

47

leukoplakia among 50,915 villagers in india, oral cancer and

leukoplakia in india, Vol. 24, No. 4, PP. 832-849.

41. Michelle Roberts Health reporter, BBC News 07-Dec (2012).

42. Mohan, R., Cancer In India, (2010).

43. Morton, J.F., (1972): Further associated of plant tannis and

human cancer. Crude Drug. Res., 12:1829-1841.

44. Mucci, L., Adami, H., (2002): Oral and pharyngeal cancer. In:

Adami H, Hunter D, Trichopoulos D, editors. Textbook of cancer

epidemiology. New York: Oxford University Press; p. 115–32.

45. Murti, P.R., Bhonsle, R.B., andPindborg, J.J., (1985): Malignant

transformation rate in oral submucousa fibrosis over a 17-year

period. Commun. Dent. Oral.Epidemiol., 13: 340-1.

46. Nair, J., Ohshima,H., Friesen,M., Croisy,A., Bhide, S.V., and

Bartsch, H., (1985): Tobacco-specific and betel nut-specific N-

nitrosocompunds: occurrence in saliva and urine of betel quid

chewers and formation in vitro by nitrosation of betel quid

Carcinogenesis, 6:295-303.

47. Nair, U., Bartsch, H., et al., (2004): Alert for an epidemic of oral

cancer due to use of the betel quid substitutes gutkha and pan

masala: a review of agents and causative mechanisms,

Mutagenesis, vol. 19, no. 4, pp. 251-262.

48. National Cancer Control Program, (1996): India: DGHS, MHF and

W, Govt. of India, New Delhi.

49. Nersesyan, A.K., (2005): Nuclear buds in exfoliated human cells.

Mutat. Res.588, 64–68.

50. Neville, B.W., Damm, D.D., Allen, C.M., et al., (2002): Oral &

maxillofacial pathology. 2nd ed. Phila., PA: Saunders;337-369.

48

51. Neville, B.W., Day, T.A., (2002): Oral Cancer and Precancerous

Lesions, CA Cancer J Clin; 52:195-215.

52. Quon, H., Hershock, D., Feldman, M., Sewell, D., Weber, R.,

(2004): Cancer of the head and neck. In: Abeloff M, Armitage J,

Niederhuber J, Kastan M, McKenna W,editors. Clinical oncology. 3rd

ed. Orlando: Churchill Livingstone, an imprint of Elsevier; p. 1499–

500.

53. Sachs, L., Robinowitz, S., et al., (1972) : The formation of variants

with reversion of properties of transformed cells VII, Chromosomal

number and reversion in subtetraploid variant, Int. J. Cancer, 10:9-

13.

54. Sanghvi, L. D., Rao, K. C. M., et al, (1955): Smoking And Chewing

Of Tobacco in Relation To Cancer Of The Upper Alimentary Tract,

British Medical Journal, pp. 1111-1114.

55. Schwimmer, E., (1877): Die idiopathischenSchleim-hautplaques

der Mundhöhle (Leukoplakia buc-calis). Arch DermatSyph;9:570-

611.

56. Sellappa, S., Balkrishnan, M., Vellingiri, B., (2009): Mixture of

betel leaf, areca nut and tobacco chewing is a risk factor for

cytogenetic damage in construction workers from south india, Braz J

Oral Sci; 8(3):145-148.

57. Shah, N., Sharma, P.P., (1998): Role of chewing and smoking

habits in the etiology of oral submucousa fibrosis: a cause control

study; J. oral. Patho. Med.; 27:459-9.

58. Shimizu, N., Itoh, N., Utiyama, H.,& Wahl, G.M., (1998): Selective

entrapment of extrachromosomally amplified DNA by nuclear

budding and micronucleation during S phase.J. Cell Biol.140, 1307–

1320.

59. Shimizu, N., Kamezaki, F.,&Shigematsu, S., (2005): Tracking of

microinjected DNA in live cells reveals the intracellular behavior and

49

elimination of extrachromosomal genetic material.Nucl. Acids

Res.33, 6296–6307.

60. Singleton, H., et al, (1968): Human cervical intraepithelial

neoplasia Fine structure of dysplasia & carcinoma in situ cancer

Res., 28:695.

61. Smita, J., Mohammad, A., Yasir, H.S., (2011): Genotoxic Effects of

Pan Masala and Gutkha: A review, World Journal of Zoology

6(3):301-306.

62. Smith, E.M., Ritchie, J.M., Summersgill, K.F., Hoffman, H.T., Wang,

D.H., Haugen, T.H., and other, (2004): Human papillomavirus in oral

exfoliated cells and risk of head and neck cancer.JNatl Cancer Inst;

96(6):449–55.

63. Smith, J.F., Mincer, H.A., Hopkins, K.P., et al, (1970): .Snuff-

dipper’s lesion. A cytological and patholog-ical study in a large

population.ArchOtolaryngol;92:450-456.

64. Spouge, J.D.,(1973) : Oral Pathology St. LowisMoshy

65. SRS based abridged life tables 1990–94 and 1991–95. SRS

Analytical Studies, 1998, 1.

66. Stitch, H.F., and Stich, W., (1982): Chromosome damaging

activity of saliva of betel nut and tobacco chewers. CacerLett.,

15:193-202.

67. The Health Consequences of Using Smokeless Tobacco: A Report

of the Advisory Committee to the Surgeon General, (1983):

Washington, DC. US Dept. of Health and Human Services.

68. Thomas, P., Hecker, J., Faunt, J., Fenech, M.,(2007): Buccal

micronucleus cytome biomarkers may be associated with

Alzheimer’s disease, Mutagenesis, vol. 22, no. 6, pp. 371–379.

69. Thomas, P., Holland, N.,Bolognesi, C., Kirsch-Volders, M., Bonassi,

S.,Zeiger. E., Knasmueller, S., Fenech, M.,(2009): Buccal

micronucleus cytome assay, Nature Protocols, VOL.4. NO.6, PP. 825-

837.

50

70. Tilakarantne, W.M., and. Klinikowski, M.F., (2005): Review on

etiology and pathogensis, Oral Oncol.42:561-568.

71. Tolbert, P. E., Shy, C. M., and Allen, J. W., (1991): Micronuclei and

other nuclear anomalies in buccal smears: a field test in snuff

users.Am. J.Epidemiol., 134, 840–850.

72. Tolbert, P.E., Shy, C.M. & Allen, J.W., (1992): Micronuclei and

other nuclear anomalies in buccal smears: methods

development.Mutat. Res.271, 69–77.

73. Walsh, P.M., Epstein, J.B., (2000): The Oral Effects of Smokeless

Tobacco, Journal de l’Associationdentairecanadienne, Vol. 66, No 1,

PP. 22-25.

74. Weignberg, R.A., September (1996): How Cancer Arises, Scientific

American, Vol 75, No. 3, pp. 31-40.

75. Werning, J.W., May 16, (2007): Oral cancer: diagnosis,

management, and rehabilitation. p. 1.isbn 978-1-58890-309-9.

76. What’s New In Oral Cancer Research, Oral Cancer Study (2012).

77. Winn, D.M., (1993): Surveillance of and knowledge about cancer

associated with smokeless tobacco use’, Smokeless Tobacco or

Health: An International Perspective. Bethesda, Md: National Cancer

Institute, National Institutes of Health, 11-18. DHHS Publication NIH

93-3461.NCI Smoking and Tobacco Control Monograph No. 2

78. Wyllie, A.H., (1981): Cell Death: A New Classification Separating

Apoptosis from Necrosis9–34 (Chapman and Hall Ltd, London).

51

27,29,31,33

37,38

1-26,28,30,32,34-36,39-50

52