International Journal of Research in Medical Sciences
Agrawal V et al. Int J Res Med Sci. 2016 Aug;4(8):3130-3133
www.msjonline.org pISSN 2320-6071 | eISSN 2320-6012
DOI: http://dx.doi.org/10.18203/2320-6012.ijrms20162236
Research Article
Evaluation of micronucleus frequency in oral exfoliated
buccal mucosa cells of smokers and tobacco chewers:
a comparative study
Vandana Agrawal*, Ishan Dubey, K. B. Mishra
Department of Pathology, L.N. Medical College & Research Centre, Bhopal, India
Received: 25 June 2016
Accepted: 12 July 2016
*Correspondence:
Dr. Vandana Agrawal,
E-mail: vandanadoc78@gmail.com
Copyright: © the author(s), publisher and licensee Medip Academy. This is an open-access article distributed under
the terms of the Creative Commons Attribution Non-Commercial License, which permits unrestricted non-commercial
use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
Background: Oral cancer is one of the tenth most common cancers in the world and constitutes the third most
important group of malignancies in India. Majority of these cancers are diagnosed at an advanced stage resulting in
poor prognosis and survival rates among patients. Hence early diagnosis of oral cancers seems to be the need of the
hour. Analysis of exfoliated buccal cell micronuclei (MN) is a sensitive method of monitoring genetic damage. The
present study aims to compare the frequency of micronucleus in tobacco chewers and smokers and thus evaluate the
genotoxic potential of these habits.
Methods: The study was conducted on 60 subjects, divided into 3 groups each of 20 subjects. Group 1: with history
of chewing tobacco, group 2: with history of chewing tobacco and smoking, group 3: healthy subjects without any
habits as controls. Oral exfoliated cells were obtained from buccal mucosa of the subjects, 2 slides were prepared
from each subject stained with Giemsa and H&E stain respectively. Atleast 1000 cells were examined for each subject
and micronuclei frequency was scored according to criteria of Tolbert et al.
Results: The mean number of micronuclei was 18.5±9.5 in tobacco chewers, 19.1±9.2 in chewers with smoking habit
and 8.2±5.6 in controls. Bonferroni multiple comparisons amongst these three groups showed the mean difference of
micronuclei to be highly significant when chewers and chewers with smoking habit were compared to controls.
Similarly based on the duration of addiction, a highly significant difference was noted in no. of micronucleated cells
in subjects addicted to tobacco for more than 15 years.
Conclusions: Tobacco can cause and increase the rate of nuclear anomalies in both smoking and smokeless forms.
Thus oral mucosal micronuclei frequency can be used as a marker of epithelial carcinogenic progression.
Keywords: Exfoliated cells, Micronuclei, Genotoxic, Tobacco users
INTRODUCTION Indians chew tobacco rather than smoke it, especially in
rural areas due to which 75,000 to 80,000 new oral cancer
Oral cancer is one of the tenth most common cancer in cases had been identified in 2012 and these proportions
the world and constitutes the third most important group are expected to rise further by 2025.2,3 Majority of these
of malignancies in India.1 Nearly 1,30,000 Indians die cancers are diagnosed at an advanced stage resulting in
due to tobacco related oral cancer. The International poor prognosis and survival rate among patients. Hence
Agency for Research on oral cancer (IARC) regards the early diagnosis of oral cancer seems to be the need of
chewing of tobacco to be a known human carcinogen, the hour.
which has a role in multistage progression of oral cancer.2
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Agrawal V et al. Int J Res Med Sci. 2016 Aug;4(8):3130-3133
Analysis of exfoliated buccal cell micronuclei (MN) is a scraped cells were placed onto pre cleaned slides. 2 slides
sensitive method of monitoring genetic damage in human were prepared for each subject .One Slide was air dried
population. First proposed by Stitch et al this test still and stained with geimsa and other wet fixed in 95%
continues to gain popularity as a biomarker of genetic alcohol and stained with H & E stain. Atleast 1,000 cells
damage due to its low cost, minimal invasiveness and were examined for each subject and MN frequency
ease of storage and slide preparation.4,5 scored according to the criteria of Tolbert et al.
The micronucleus is defined as a microscopically visible The suspected nucleus is required to meet the following
round or oval cytoplasmic chromatin mass next to the criteria in order to be considered as Micronucleus: (a)
nucleus.6 They originate from aberrant mitoses and rounded, smooth perimeter suggestive of membrane; (b)
consist of acentric chromosomes that have failed to less than third the diameter of the main nucleus, but large
incorporate into daughter nuclei during mitosis, events enough to discriminate shape and color; (c) staining
thought to be associated with increased risk for cancer.7 intensity similar to that of nucleus; (d) same focal plane
The frequency of occurrence of micronuclei is a measure as nucleus.
of chromosome breakage in early cell divisions and the
number of micronuclei is known to increase with Statistical analysis
carcinogenic stimuli, long before the development of
clinical symptoms.8 Statistical analysis was done using SPSS version 20. The
findings are presented as mean and standard deviation.
Thus the present study aims to compare the frequency of To analyse the mean difference of micronuclei among the
micronucleus in tobacco chewers and cigarette smokers three groups, one way ANOVA was performed and p
and thus evaluate the genotoxic potential of these habits. value of less than 0.05 was considered statistically
significant.
METHODS
RESULTS
This cross sectional study was carried out in Department
of Pathology, L. N. Medical College, Bhopal, India. The All the subjects included in the present study were males.
study population comprised of a total of 60 subjects, No significant difference was observed regarding the age
divided into three groups - Group 1: comprising of 20 across the 3 study groups (Table 1).
subjects with a history of chewing tobacco, Group 2:
comprising of 20 subjects with a history of chewing Table 1: Characterization of study subjects.
tobacco and cigarette smoking and Group 3: comprising
of 20 age and sex matched healthy subjects without any Groups n=60 Age in years (Mean±2SD)
habits as controls. A written informed consent was taken Chewers 20 44.3±12.1
from all the subjects. The study was approved by the Chewers with
institutional ethical committee. 20 42.8±13.7
smoking habit
Controls 20 41.0±14.4
Inclusion criteria Chewers: Person chewing mixture of betel leaf, areca nut and
tobacco. Controls : Non chewers non-smokers.
The inclusion criteria for smokers and tobacco chewers
were the use of cigarettes and/or tobacco for atleast last Figure 1-3 represents the frequency distribution of
six months. micronucleated cells in each of the 20 subjects across the
3 study groups.
Exclusion criteria
Subjects with a history of recent viral infection.
Subjects with use of antibiotics within 2 months
before sample collection.
Subjects with recent history of exposure to potential
genotoxic agents, including X rays, chemotherapy
and potential occupational exposures.
Subjects who are chronic alcoholics.
Subjects with any oral pathological lesions
Sample collection, staining and cytological analysis
Prior to sampling each subject was asked to rinse his Figure 1: Frequency distribution of micronucleated
mouth thoroughly with tap water. Oral exfoliated cells cells in buccal mucosa smears of each of the 20
were scraped from the buccal mucosa of control and subjects (tobacco chewers).
study group with a moistened wooden spatula. The
International Journal of Research in Medical Sciences | August 2016 | Vol 4 | Issue 8 Page 3131
Agrawal V et al. Int J Res Med Sci. 2016 Aug;4(8):3130-3133
comparisons amongst the two tobacco exposed groups
(chewers and chewers with smoking habit) showed no
significant difference in the mean micronucleated cell
count (Table 3).
Figure 2: Frequency distribution of micronucleated
cells in buccal mucosa smears of each of the 20
subjects (tobacco chewers with smoking).
The mean no. of micronucleated cells in the tobacco
exposed group was found to be significantly higher as Figure 3: Frequency distribution of micronucleated
compared to the control group (p<0.05) (Table 2). cells in buccal mucosa smears of each of the
Similarly the difference in the mean micronucleated cell 20 subjects (controls).
count between the cases and controls was also highly
significant (P<0.001). In an attempt to examine the impact of the duration of
exposure of tobacco on micronucleus frequency a
Bonferroni multiple comparisons showed the difference comparison was made between subjects having a period
in the mean micronucleated cell count to be highly of addiction >15 years and <15 years. A highly
significant when the tobacco exposed group was significant difference was seen between the two groups
compared to the controls. However intragroup (Table 4).
Table 2: Comparison of mean no. of micronucleated cells across 3 study groups by one way ANOVA.
Characteristic Chewers Chewers with smoking habit Controls P- value
No. of micronucleated
18.5±9.5 19.1±9.2 8.2±5.6 0.000 S
cells/1000cells
All values in the three groups are reported as Mean+2SD.
Table 3: Bonferroni multiple comparisons across 3 groups.
Mean difference Standard error Significance
Chewers Chewers with smoking habit 0.550 2.9 0.997
Chewers Controls 10.30 2.5 0.001*
Chewers with smoking
Controls 10.85 2.4 0.000*
habit
*The mean difference is significant at the 0.05 level.
Table 4: Comparison of mean no. of micronucleated cells with duration of addiction.
Period of addiction Period of addiction
Characteristic P- value
( <15 years) (>15 years)
No. of micronucleated
15.21±8.3 25.5±7.0 0.000 S
cells/1000cells
All values are reported as Mean±2SD.
DISCUSSION agents was first proposed by Stich et al. The micronuclei
(MN) assay is potentially an excellent biomarker to detect
The use of the micronucleus test on exfoliated cells from chromosome loss or malfunction of mitotic spindle which
oral epithelium with the aim of undertaking bio- is caused by aneugenic mechanisms. The efficacy of this
monitoring on human populations exposed to genotoxic test for this purpose has been highlighted in many
studies.7,9,10
International Journal of Research in Medical Sciences | August 2016 | Vol 4 | Issue 8 Page 3132
Agrawal V et al. Int J Res Med Sci. 2016 Aug;4(8):3130-3133
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This study showed that tobacco in both smoking and S, et al. The HUMN and HUMNxL international
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buccal mucosa cells as evidenced by a higher MN assays in lymphocytes and buccal cells--past,
frequency in tobacco chewers and tobacco chewers with present and future. Mutagenesis. 2011;26:239-45.
smoking habit. These results are in full agreement with 6. Schimd W. The micronucleus test. Mut Res.
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other studies.12-14
7. Tolbert PE, Shy CM, Allen JW. Micronucleus and
other nuclear anomalies in buccal smears: Methods
The synergistic effect from the habits of chewing and development . Mut Res. 1991;271:69-77.
smoking tobacco on micronucleus induction in oral 8. Stich HF, Rosin MP, Vallejera MO. Reduction with
epithelial cells was investigated by Kassie et al the vitamin A and beta-carotene administration of
findings of which corroborate with our study. 15 proportion of micronucleated buccal mucosal cells
in Asian betal nut and tobacco chewers. Lancet.
Furthermore the study also showed the micronucleus 1984;1:1204-6.
frequency to be significantly higher in persons with a 9. Cavallo D, Ursini CL, Perniconi B, Francesco AD,
longer duration of exposure to tobacco as observed by Giglio M, Rubino FM, Marinaccio A, Iavicoli S.
Caplash et al in his study. Evaluation of genotoxic effects induced by exposure
to antineoplastic drugs in lymphocytes and
The results obtained from this study in which occurrences exfoliated cells of oncology nurses and pharmacy
of micronuclei in relation to the habits of smoking and employees. Mutation Research. 2005;587:45-51.
chewing tobacco were investigated, the accumulated 10. Salama SA, Serrana M, Au WW. Biomonitoring
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MW, et al. Tobacco smoking-response genes in
CONCLUSION
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37.
Tobacco can cause and increase the rate of nuclear 12. Gabriel HE, Crott JW, Ghandour H, Dallal GE,
anomalies in both smoking and smokeless forms. Thus Choi SW, Keyes MK, Jang H. Chronic cigarette
oral mucosal micronuclei frequency can be used as a smoking is associated with diminished folate status,
marker of epithelial carcinogenic progression. Simplicity, altered folate form distribution, and increased
accuracy, multipotentiality and large tissue applicability genetic damage in the buccal mucosa of healthy
of the MN technology made it attractive in the past and adults. Am J Clin Nutr. 2006;41:835-41.
will ensure a key role in the evaluation of mutagenicity 13. Veerachari U, Venkatesh SH, Yadavand A,
and primary prevention in the future. Narayanappa R. Biomonitoring genetic instability in
normal healthy population using a simple
Funding: No funding sources cytogenetic marker-micronucleus test. International
Conflict of interest: None declared Multidiscipl Res J. 2011;1(2):01-09.
Ethical approval: The study was approved by the 14. Naderi NJ, Farhadi S, Sarshar S. Micronucleus
Institutional Ethics Committee assay of buccal mucosa cells in smokers with the
history of smoking less and more than 10 years.
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