Molecular Mechanism Cancer
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Transcript of Molecular Mechanism Cancer
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).
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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
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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.
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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
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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:
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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.
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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.
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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
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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
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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 :-
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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).
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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.
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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
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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
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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
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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
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(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
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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.
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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
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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
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