Micronuclei Count as an Indicator for Cytotoxic Damage in Tobacco Users.
Background: Tobacco usage in the smoked and smokeless forms has reached epidemic proportions in the Asian sub continent often leading to oral cancer which is the sixth most common cause of cancer related deaths globally. Micronuclei (MN) count is a promising indicator for the cytotoxic effects of tobacco usage. Our study was aimed at establishing a correlation between the MN counts among various tobacco habits.
Methods: Exfoliated cells were collected from a total of 400 individuals consisting of 100 individuals each of smokers, gutkha chewers, khaini chewers and controls. PAP staining was done and the number of cells with micronuclei was counted under 40 X, by two independent examiners. The mean MN count was compared using the ANOVA test for statistical analysis.
Results: Significant increase in micronuclei count was observed in individuals with tobacco habit. Mean value of epithelial cells with micronuclei +/- SD was 1.58 +/- 0.24 for controls, 7.51 +/- 0.59 for smokers, 13.95 +/- 0.97 for khaini chewers and 15.45 +/- 1.17 for gutkha users.
Conclusion: The MN count can be used as an early indicator for susceptibility to Oral squamous cell carcinoma (OSCC), as a non invasive early detection tool for mass screening, for patient education as well as to check for efficacy of treatment.
Micronuclei; Exfoliated cells; Smokers; Khaini users; Gutkha users.
Use of tobacco in smoked and smokeless forms is widely prevalent in all parts of the world and reaches epidemic proportions in the Asian sub continent. Tobacco may be smoked or used alone/mixed with additives and chewed/kept it the buccal/labial sulcus.
Oral cancer is the 11th most common cancer worldwide  and among the top ten most common in India . Globally, oral cancer is the sixth most common cause of cancer associated deaths .Greater than 90% of oral malignancies arise from the epithelium and a vast majority is associated with tobacco habit.
The oral cavity has the distinction of being the only mucosal site
that can be examined visually with the naked eye to see for any
morphological changes due to tobacco habits. The emphasis on
early diagnosis and treatment depends on the morphological alter
Micronuclei (MN) have been defined as a microscopically visible,
round to oval cytoplasmic chromatin masses next to the nucleus
. MN formation is the result of segregation defects due to chromosomal instability causing chromatin to be excluded from the reforming nucleus .
Our study was aimed at assessing the extent of MN formation in
individuals with tobacco habits and to establish a correlation between the various habits and to determine the existing cytotoxic
damage present even in the absence of significant clinical manifestations that were noticeable to the individual, thereby giving a false sense of well being.
Materials & methods
All individuals gave a written consent for participation. All individuals were interviewed for awareness of presence of any lesion associated with the habit, type of habit, duration and intensity of habit, dietary habits, systemic and local disease history and family history.
400 individuals consisting of 100 each of controls, smokers, khaini chewers and gutkha users were included in our study
a. Smokers who were smoking 5 or more, filtered cigarettes per day for a minimum of 1 year.
b. Tobacco users who were using khaini, 1 packet or more per day for more than 6 months.
c. Gutkha users who were using flavored gutkha 1 packet or more per day for more than 6 months.
d. Control individuals were healthy individuals who had never consumed tobacco in any form.
a. Any history of chronic systemic disease eg. Diabetes, hypertension, heart disease
b. Any history of medication for chronic illness or recent antibiotic
c. Any history of oral lesions eg. Recurrent aphthus, herpes or poor oral hygiene
d. Present history of stress
e. Any history of radiation or chemotherapy.
f. Malnutrition and vitamin deficiency
g. Tea drinkers having more than two cups of tea per day.
To avoid any confounding factors individuals using alcohol based
products or consuming alcohol more than 60 ml twice a month
were not included in the study. Any individual having multiple
habits were also not included.
Before sampling, all individuals were asked to rinse thoroughly
with tap water. Exfoliated cells were collected from the buccal
mucosa of controls, site of placement in khaini or gutkha users
and palatal mucosa of smokers. The cells were collected by gently
rubbing the mucosa with a pre moistened wooden spatula. The
cells were spread onto pre cleaned glass slides, allowed to air dry and fixed with Biofix spray (Biolab Diagnostics (I) Pvt Ltd) and then stained with Rapid - PAP stain (Biolab Diagnostics (I) Pvt Ltd). 1000 cells per slide were counted under high power (x40)
using the battle field method (Fig 1). Only cells which were not fragmented and not overlapping were counted. 2 blind examiners carried out the count.
Statistical analysis was done using the ANOVA test.
(Rapid PAP stain X40) Increased number of Micronuclei observed in exfoliated cells obtained from the buccal mucosa of individuals with the habit of Tobacco smoking and chewing.
There was observed a statistically significant increase in MN
count when controls were compared to individuals with a history
of tobacco use. The number of MN increased with an increase in
duration and intensity of habit.
Case selection has to be done keeping in mind that confounding
factors have the potential to induce expression of MN, such as;
exposure to physical and chemical mutagens, radiation, consumption
of alcohol, tea and coffee, food habits, stress, medication, viral infections suffered in the last 3 months, vaccination & hereditary diseases, use of anticonvulsants & antibiotics, oral infections, pregnant or lactating women.
Collection of cell samples can be done using metal spatula, moistenedwooden spatula, cotton swab, tooth brushes or cytobrushes. Though cytobrushes have shown ideal results, the cost factor makes the wooden spatula suitable for large number of samples. Casterelli et al have observed that vigorous scraping lead to higher MN count suggesting a decreasing gradient of MN presence from the basal to the superficial layers .
Various staining procedures have been used such as acridine orange, propidium iodide, Giemsa and PAP. However PAP staining
remains the preferred stain due to its DNA specificity.
Criteria for identification of MN were first given by Heddle &
Countryman in 1976 as:
1. Diameter less than 1/3rd the main nucleus
2. Non – refractivity (to exclude small stain particles)
3. Colour same as or lighter than the nucleus (to exclude large
4. Location within 3 or 4 nuclear diameters of a nucleus (to make
frequency measurements meaningful)
5. No more than 2 MN associated with one nucleus.
Numerous studies have been done on MN count and most substantiate
that there is an increase in MN formation due to tobacco
habits however the quantification varies significantly. The increase
in MN count varies from 2 to 8 times when control group count
is compared to tobacco users. Similarly, the count of the control
group itself varies from 0.39  to 2.70 . Such findings further
underline the significance of standardization of protocols for MN
count to be used for effective comparison between study populations.
We found a significant increase in MN frequency in tobacco users
(Fig 1) when compared to controls. Contrary to the findings of
Sarto et al and Piyathilake et al, we found that the MN formation
was more in users of smokeless tobacco as compared to individuals
with smoked tobacco usage [10, 11]. An increase in MN count was also associated with smokeless tobacco as studied by Desai et al, Roberts DM and Stich et al, however not in accordance with Ozkul et al, who found no statistically significant variation between users of smoked and smokeless forms of tobacco [12, 13, 14, 15]. We also found a positive correlation between MN frequency and gutkha chewing, which was increased when compared to controls.
Sufficient evidence exists that tobacco in smoked or smokeless
form causes cytogenic and genotoxic damage to epithelial cells.
The usage of tobacco products in any form is detrimental to oral
health. The tobacco users have to be made aware of these dangers
and there can be no better way than to practically demonstrate the
effects of tobacco.
Being non invasive, economical & rapid, easy to carry out & highly
reproducible, the MN count can be repeatedly obtained from the
same patient for longitudinal studies and for checking the efficacy
of treatment. Moreover it can be used as a reliable tool for mass
screening & early detection, patient education & motivation.
Compliance with Ethical Standards:
Disclosure of potential Conflicts of Interest:
The author of this article has not received any research grant,
remuneration, or speaker honorarium from any company or
committee whatsoever, and neither owns any stock in any company.
The author declares that she does not have any conflict of
Research involving human participants and /or animals:
All procedures performed on the patients (human participants)
involved were in accordance with the ethical standards of the
institution and/or national research committee, as well as with the
1964 Helsinki declaration and its later amendments and comparable
This article does not contain any new studies
with human participants or animals performed by the author.
Informed consent was obtained from all the
individual participants in this study.
This study was not funded by any organization / society
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