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) 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). Mutagenic property of catechu has been shown Stitch et al and clastogenicity by Giri et al.
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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 p roducing 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
2

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 growthpromoting 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.

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 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 cells 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 inhibition and the lost gene is called tumor suppressor gene. of the

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: 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
5

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.

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.
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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. 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
7

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 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 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 N9

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 :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 4ounce 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 drinkers risk to reduce to that of someone who has neve r 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).

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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).

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
11

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.

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 exoral 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.

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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 disease (Kramer et al, 1978). In the evaluation of clinical features of leukoplakias, the following 3 types were taken into consideration: i) ii) iii) The homogeneous type, The ulcerated type, and 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 grayishyellow 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

13

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 isnt 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 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 welldeveloped 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).
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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 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

15

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 (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.

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Subjects:A total number of 20 individuals (both male & female)were 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. ii. iii. iv. v. vi. vii. Name Place Age Sex Habit (gutkha/pan/tobacco/Khaini/smoking/alcohol etc.) Duration of use Consumption per day

Sample Collection:For sample collection following materials are required, i. ii. iii. iv. Instruments used to obtain sample (wooden spatula or coverslip) Clean grease free microscopic slide of good quality A marker pen for marking of slides 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. iii. v. 70% alcohol or methanol Hematoxylin Absolute alcohol ii. iv. vi. 50% alcohol Alcoholic eosin 0.3% acid alcohol

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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 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.
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Procedure for Staining:i. ii. After fixation the slide was washed in distill water. Then the slide was stained with Harris haematoxylin for 10min for nuclear stain. iii. It was rinsed in distill water to prevent smear being washed off the slides. iv. v. vi. It is differentiated with 0.3% acid alcohol. It was again rinsed with running tap water. 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.

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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 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

20

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 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

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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 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

22

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 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
23

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. 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.

24

SL NO.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

AGE
47 49 25 59 43 53 45 40 58 60 66 56 42 47 50 38 37 60 42 28

SEX
M M M M M M M M M F M F M M F M M M M M

HABIT
ALCOHOL, CIGARETTE, PAN GUTKHA, CIGARETTE,ALCOHOL GUTKHA, CIGARETTE PAN, GUDAKHU GUTKHA, CIGARETTE,ALCOHOL, PAN PAN, GUTKHA PAN, PAN MASALA (PUDIYA) PAN, GUTKHA, CIGARETTE, ALCOHOL,ALL TYPES GUTKHA GUDAKHU GUDAKHU GUDAKHU PAN, HARIDAKHANDI GUTKHA, PAN,HARIDAKHANDI, KHAINI (NASA) GUDAKHU ALL PAN, PAN MASALA,HARIDAKHANDI GUDAKHU CIGARETTER, ALCOHOL,PAN MASALA GUTKHA, CIGARETTE

APPROX. DURATION OF USE IN YEARS

20 10 10 25 25 10 30 18 15 50 40 40 24 30 30 15 35 40 20 10

Table no. 1:- Data representing age, Habits of tobacco & duration of use in years
25

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

26

(a)

(b)

(c)

(d)

(e)
27

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

28

(f)

(g)

(h)

(i)

(j)
29

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

30

(k)

(l)

(m)

(n)

(o)
31

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

32

(p)

(q)

(r)

(s)

(t)
33

Dura tion of use

MN per 1000 cells

Pyknoti c cells per 1000 cells

Nuclear change per 1000 cells

Kryolytic cells per 1000 cells

Tadpole shaped nucleus cells per 1000 cells

Binucle ated cells per 1000 cells 4

Grand total

0-10

35

52

14

25

132

10-20

20

61

13

21

27

11

153

20-30

23

71

24

22

37

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 Sum square between nuclear damaged cells Degree of freedom for SSC Sum square between duration of use Degree of freedom for SSR Sum square of total

= = = = = =

28,706 12,797.87 5 1447.57 4 15,761

34

Source of variation

SS (Sum of squares)

DF (Degree of freedom)

MSS (Mean sum of squares)

Calculated value of F (V.R)

Tabulate d value of F (0.05)

Between nuclear damaged cells Between duration of use

12,797.87

V1 = 5

2559.7

33.75

(5,20) = 2.7

1447.57

V2 = 4

361.9

4.77

(4,20)= 2.9

Error

1516.47

V3 = 20

75.82

Total

15,761

29

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

Standard deviation of MNs Mean Coefficient of variation Standard deviation of Pyknotic cells Mean Coefficient of variation

(S1) (X1) (CV1) (S2) (X2) (CV2)

= = = = = =

7.85 28.2 0.2785 20.144 74.4 0.2707

35

Standard deviation of Nuclear change Mean Coefficient of variation Standard deviation of Karyolytic cells Mean Coefficient of variation Standard deviation of tadpole nucleus Mean Coefficient of variation

(S3) (X3) (CV3) (S4) (X4) (CV4) (S5) (X5) (CV5)

= = = = = = = = = = = =

14.412 21.8 0.6611 3.768 20.2 0.1865 5.814 31.6 0.1840 4.219 9.4 0.488

Standard deviation of Binucleated cells ( S6) Mean Coefficient of variation (X6) (CV6)

36

Figure 5:- Number of nuclear damaged cells counted per 1000 cells, depicts that with increase in duration of use number of nuclear damaged cell increases.

37

Figure 6:- Coefficient of variation of different types of nuclear damage indicating lesser the coefficient variation more the occurring frequency.

38

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. The significance though is unknown but may be indicative of Cytokinesis.

39

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 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
40

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). 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).

41

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.

42

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51