Download Micronucleus Assay for Evaluation of Genotoxicity in Potentially

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10.5005/jp-journals-10011-1103
Micronucleus Assay for Evaluation of Genotoxicity in Potentially Malignant and Malignant Disorders
ORIGINAL ARTICLE
Micronucleus Assay for Evaluation of
Genotoxicity in Potentially Malignant and
Malignant Disorders
1
1
Professor and Head, Department of Oral Medicine and Radiology, Rama Dental College, Hospital and Research Center
Kanpur, Uttar Pradesh, India
2
3
4
Parvathi Devi, 2Thimmarasa VB, 3Vishal Mehrotra, 4Pallak Arora
Professor, Department of Oral Medicine and Radiology, Rama Dental College, Hospital and Research Center
Kanpur, Uttar Pradesh, India
Senior Lecturer, Department of Oral Medicine and Radiology, Rama Dental College, Hospital and Research Center
Kanpur, Uttar Pradesh, India
Postgraduate Student, Department of Oral Medicine and Radiology, Rama Dental College, Hospital and Research Center
Kanpur, Uttar Pradesh, India
Correspondence: Parvathi Devi, Professor and Head, Department of Oral Medicine and Radiology, Number-1260, 46th Cross
1st Stage, Kumara Swami Layout, Bengaluru-560078, Karnataka, India, e-mail: [email protected]
ABSTRACT
Oral cancer is a common malignancy, ranking first among all cancers in Western and Asian countries. It is preceded by some benign
lesions or conditions, which are termed precancerous. Only one-third of people at the precancerous stage of disease succumb to cancer,
it would be of practical importance to identify individuals at risk among them. Biomarkers, instruments of individual tumor prevention, help
to detect high-risk patients. The induction of micronucleus is considered to be an effective biomarker of diseases. In the recent past, a great
deal of enthusiasm was raised by application of the micronucleus test to assess DNA damage in human population. The present study is
aimed at the evaluation of frequency of micronuclei in smears of oral exfoliated cells. A total of 33 patients with potentially malignant
(leukoplakia, OSMF, lichen planus) and malignant oral epithelial diseases from the department of oral medicine and radiology were considered
as study group and compared with 33 age and sex matched healthy controls. Micronucleus frequencies were found higher in diseased
patients than in control subjects. Hence, concluded that the micronucleus assay can be used as a prognostic indicator in potentially
malignant and malignant disorders.
Keywords: Micronuclei, Exfoliative cytology, Oral cancer, Precancerous lesions.
INTRODUCTION
Carcinogenesis is a multistep process characterized by genetic,
epigenetic and phenotypic changes. Such changes involve
genetic damage, mutation in critical genes related to the control
of cell division, cell death, metastatic potential and activation
of signaling or metabolic pathways that give the cells favorable
growth and survival characteristics.1 Many chemical, physical
and biological environmental agents are able to interact with
DNA to induce mutations. When the normal function of DNA
repair genes and/or cell proliferation and differentiation control
genes is lost as a consequence of mutations, the risk of cancer
development increases.2 To evaluate genetic instability, there
are biomarkers that predict if a premalignant lesion or condition
is likely to develop into an aggressive metastasizing tumor.3
Micronuclei and cytoplasmic fragments of DNA have been
reported as markers for high cancer risk as they arise in response
to carcinogens. They arise from acentric fragments or whole
chromosomes, which are not included into the main nuclei of
the daughter cells. The formation of micronuclei can be induced
by substances that cause chromosome breakage (clastogens) as
well as by agents that affect the spindle apparatus (aneugens).1
They can be detected in exfoliated cells and used as an indicator
of recent DNA injury within oral mucosa. The frequency of
micronucleated exfoliated cells elevates in human tissues, which
appear to be the main targets of carcinogens, and from which
carcinomas arise.4 The assay is reliable and technically easy to
perform, noninvasive and sensitive with limited cost.5 With this
view in mind, the present study was carried out to assess the
levels of micronuclei in oral exfoliative cytology of healthy
control subjects and diseased patient.
AIMS AND OBJECTIVES
1. To evaluate the frequency of micronuclei in smears of oral
exfoliated cells from healthy control subjects and potentially
malignant and malignant disorders.
2. Comparison of micronucleus frequencies between the
control group and diseased patients.
MATERIALS AND METHODS
In the present study, 33 patients with potentially malignant
(leukoplakia, lichen planus, OSMF) and malignant oral
epithelial diseases (oral squamous cell carcinoma), from the
Journal of Indian Academy of Oral Medicine and Radiology, April-June 2011;23(2):97-100
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Parvathi Devi et al
department of oral medicine and radiology were considered as
study group and compared with 33 age and sex matched healthy
controls with no history of tobacco consumption. All the subjects
were administered a standardized questionnaire to obtain any
history of relevant risk factors and addiction. Written consents
were taken for the procedures to be carried out on them
subsequently. Potentially malignant and malignant disorders
were histopathologically proved. The subjects for the study were
grouped into three categories: (1) Healthy subjects with no oral
lesions (n = 33), (2) patient with potentially malignant disorders
(n = 23), (3) patients with oral squamous cell carcinoma
(n = 10).
Subjects were asked to rinse their mouth with water to swab
or gently scrape the mucosa to remove debris. Oral mucosal
cells were scraped from buccal mucosa of control group and
from lesional tissues using a premoistened metal spatula
(Fig. 1). The samples were placed in tubes containing 25 ml of
buffer solution (0.1MEDTA, 01M Tris and 02M NaCl) pH 7.
The cells were washed thrice in the buffer solution by
centrifugation at 800 rpm for 5 minutes and slides for
microscopic analyses were prepared. Cell suspension was
dropped on to clean slides and cell density was checked using
a light microscope. The slides were then allowed to dry and
then fixed in 80% cold methanol. The cells were then stained
with 10% Giemsa solution and were mounted with cover
glass using DPX mountant. The frequency of micronuclei in
epithelial cells was evaluated by scoring 1000 cells on each
slide (Fig. 2). The scoring was done according to the criteria
established by Tolbert et al.6
RESULTS
The data collected was statistically analyzed using SPSS 16
version software. ANOVA and Newman- Keuls tests were used
for comparison. Table 1 shows the distribution of study subjects
according to the age group, ranging from 18 to 70 years in
precancerous group, 28 to 70 years in cancerous group and
18 to 70 years in control group. Among the gender distribution,
out of 23 patients with potentially malignant disorder
(precancer), 15 were males and eight were females and out of
10 malignant (cancerous) patients, six were males and four were
females. Among 33 control subjects, 19 were males and
14 were females (Fig. 3).
The mean percentage of micronuclei in precancerous group
was 0.12% and it was 0.45% in the malignant group, and in the
control group it was 0.06%. A significant (p < 0.05) stepwise
increase was found in the percentage of micronucleated cells
and micronuclei from control to precancer patients, and from
precancer to cancer patients. Pairwise comparision of groups
was done by Newman-Keuls test. While comparing cancer,
precancer and cancer versus control group, p-value was
significant at the level of 0.0001, and while comparing precancer
versus control group, p-value was highly significant at the level
of 0.0231 (Table 2 and Fig. 4).
DISCUSSION
The suggested criteria for identifying MN are:
a. Rounded smooth perimeter suggestive of a membrane
b. Less than one-third diameter of the associated nucleus, but
large enough to discern shape and color
c. Staining intensity similar to that of nucleus
d. Texture similar to that of the nucleus
e. Same focal plane as nucleus and
f. Absence of overlap with, or bridge to, the nucleus.
The MN test has been receiving increasing attention as a rapid,
simple and sensitive short-term assay for studying the effects
of environmental genotoxicants.7 Micronucleated cell indexes
are thought to reflect genomic instability.8 Biomonitoring of
the changes in patients with diagnosed diseases or pathological
changes that may lead to the development of cancer and other
illnesses is becoming increasingly popular, and may be the most
rapidly growing area of application of the MN assay to epithelial
cells.9 Micronuclei are suitable internal dosimeters for revealing
tissue specific genotoxic damage in individuals exposed to
carcinogenic mixtures.10 Epithelial cells are highly proliferative
and are the origin of more than 90% of all human cancers.
Therefore, the application of micronucleus test in epithelial cells
Fig. 1: Scraping from right buccal mucosa in squamous cell carcinoma
Fig. 2: Cell with micronuclei
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Micronucleus Assay for Evaluation of Genotoxicity in Potentially Malignant and Malignant Disorders
is considered to be a sensitive tool for biomonitoring the genetic
damage in human population.
In the present study, the oral mucosal micronucleus
frequency in the control population was 0.06%. In subjects with
potentially malignant disorders, the micronucleus frequency was
0.12% and in the cancer patients, the micronucleus frequency
was 0.45%. In our study, a stepwise increase in percentage of
micronuclei was observed from control to precancer patients
and from precancer to cancer patients. These observations
indicate cytogenetic damage of the oral epithelium. Halder
et al10 conducted a similar study and found the mean percentage
of micronuclei in precancerous group was 0.63% and in
cancerous lesions was 1.36%. However, Casartelli et al,11 Palve
and Tupkari et al12 concluded that there was a gradual increase
in micronucleus counts from normal mucosal, precancerous
lesions and squamous cell carcinoma whereas Ghosh and Parida
(1995) obtained 50 smears from various tribes consuming active
tobacco and alcohol in Orissa and reported micronucleus
frequency to be 7.37%. There is marked difference between
the micronucleus frequency in our group and that found by
Ghosh and Parida. However, the population from which their
subjects were drawn was held to be at higher risk of oral cancer.
Saran et al3 conducted a similar study to explore risk assessment
of oral cancer in patients with precancerous states and found a
significant increase in the percentage of micronuclei from
control (0.16%) to precancer patients (0.20%) and from
precancer to cancer (0.25%). It is evident that our findings are
most comparable with Saran et al.3
Screening of individuals who are at high-risk of malignant
transformation is more pivotal in preventing and reducing the
number of deaths than the costly and painful treatment later on.
From the present study, it is evident that the individual cancer
risk was predicted on the basis of increased percentage of
micronuclei in the oral epithelial cells and it helps in identifying
those patients with precancer who were at high-risk of
developing oral cancer. As most oral cancers are presumed to
Table 1: Distribution of patients with oral cancer, precancer and controls based on age groups
Groups
Cancer
Precancer
Control
Total
18-27
%
0
3
19
22
0.00
13.04
57.58
33.33
28-37
1
7
5
13
%
38-47
%
48-70
%
Total
10.00
30.43
15.15
19.70
5
9
4
18
50.00
39.13
12.12
27.27
4
4
5
13
40.00
17.39
15.15
19.70
10
23
33
66
Table 2: Comparison of oral cancer, precancer and controls with respect to percentage of micronuclei (% MN cells) by
ANOVA test and Newman-Keuls test
Groups
n
Means
Std. dev.
Cancer
Precancer
Control
10
23
33
0.4500
0.1174
0.0576
0.0972
0.0778
0.0663
F-value (ANOVA)
p-value
105.1014
0.0000*
Pairwise comparison of groups by Newman-Keuls test
Cancer vs precancer
Cancer vs control
Precancer vs control
p = 0.0001*
p = 0.0001*
p = 0.0231*
*Significant
Fig. 3: Distribution of patients with oral cancer, precancer and
controls based on gender
Fig. 4: Comparison of oral cancer, precancer and controls with
respect to percentage of micronuclei (% MN 0.45 cells)
Journal of Indian Academy of Oral Medicine and Radiology, April-June 2011;23(2):97-100
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Parvathi Devi et al
originate from precancerous lesions or conditions, it is highly
desirable to identify high-risk individuals and counsel them.3
4.
CONCLUSION
The mean micronucleus frequency in oral exfoliated cells was
significantly increased in malignant and potentially malignant
group as compared to the control group. Thus, from the present
study it is evident that the percentage of micronuclei is uniformly
elevated in diseased patients, suggesting a strong cytogenetic
damage of the oral epithelium. Therefore, MN assay in
exfoliated cells holds promise as a site-specific biomarker of
exposure to genetic toxins, and for cancer it can be used as a
screening prognostic and educational tool in community center
of oral precancer and cancer.
However, despite the considerable potential of MN assay
for biomonitoring, there is diversity of possible methodological
variables, and hence their impact on assay performance. So,
further research including larger sample, exploring and
addressing sources of variability like strict adoption of optimal
scoring criteria, etc. in the assay, are necessary to confirm the
findings.
5.
6.
7.
8.
9.
10.
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