Indian Journal of Dental Research

: 2012  |  Volume : 23  |  Issue : 5  |  Page : 574--578

Analysis of micronuclei in buccal epithelial cells in patients subjected to panoramic radiography

Manjushri Waingade, Raghavendra S Medikeri 
 Sinhgad Dental College and Hospital, Vadgaon (Bk), Pune, Maharashtra, India

Correspondence Address:
Manjushri Waingade
Sinhgad Dental College and Hospital, Vadgaon (Bk), Pune, Maharashtra


Context: Ionizing radiation is a well-known carcinogen in humans. Chromosomal aberrations and formation of micronuclei in cell cytoplasm are early biological evidence of carcinogenesis. Aims: This study was undertaken to assess the genotoxic effect of panoramic radiography in the buccal epithelial cells. Materials and Methods: The study included 60 healthy individuals (median age 23.5 years; age range 12-65 years) who underwent panoramic radiographic examination. Exfoliated buccal epithelial cells were obtained immediately before and 10 days after radiation exposure. The cells were stained with Giemsa and evaluated for micronuclei by scoring 1000 cells per sample. Statistical analysis used: The paired «SQ»t «SQ» test was used to find out the significance of difference in the number of micronuclei before and after x-ray exposure. The Karl Pearson correlation coefficient was used to find out the correlation between age and micronucleated cell frequencies and number of micronucleus per 1000 cells. The ANOVA test was used to find out if there were significant differences in micronucleated cell frequencies between different age-groups. Student«SQ»s unpaired «SQ»t«SQ» test was used to find out the significance of difference in micronucleated cell frequencies and number of micronucleus per 1000 cells between genders. Results: The paired «SQ»t«SQ» test showed that micronucleated cell frequencies (P = 0.02) and number of micronucleus per 1000 cells (P = 0.047) were significantly higher after radiographic exposure. The mean number of micronucleated cells before and after radiation exposure were 0.48 ± 0.14 and 0.51 ± 0.15, respectively. There was statistically significant increase in the frequency of micronuclei in buccal epithelial cells after exposure to panoramic radiography. The correlation of micronucleus frequency with age and gender was statistically nonsignificant. Conclusions: The results indicate that panoramic radiography may induce genotoxic effects in buccal epithelial cells. Considering this risk, panoramic radiography should be used cautiously.

How to cite this article:
Waingade M, Medikeri RS. Analysis of micronuclei in buccal epithelial cells in patients subjected to panoramic radiography.Indian J Dent Res 2012;23:574-578

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Waingade M, Medikeri RS. Analysis of micronuclei in buccal epithelial cells in patients subjected to panoramic radiography. Indian J Dent Res [serial online] 2012 [cited 2021 Mar 1 ];23:574-578
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Full Text

Damage to the genome is probably the most important and fundamental cause of developmental anomalies and degenerative diseases. [1] It has been established that genomic damage is produced by genotoxic substances, medical procedures (radiation, chemicals), micronutrient deficiency (folic acid), lifestyle factors (alcohol, smoking, stress, drugs), and genetic factors such as defects in metabolism and/or in repair of DNA. [1],[2],[3] Human biomonitoring has become a central tool in environmental and occupational medicine and in research in the identification, control, and prevention of population exposure to potentially harmful compounds. [4]

Owing to its ability to deposit energy within the cells, ionizing radiation has some unique characteristics as a mutagenic and carcinogenic agent, and there is no doubt about the risk that exposure to high doses of ionizing radiation poses for human health. [5],[6] Such radiation genotoxic effects have been detected in peripheral blood lymphocytes in patients exposed to medical diagnostic procedures, occupationally exposed workers, and survivors of atomic bombings. [7],[8],[9],[10],[11]

Many approaches and techniques have been developed for the monitoring of human populations exposed to various mutagens. [4] The analysis of micronuclei (MN) has become a standard approach for the assessment of chromosomal damage in human populations. This test was proposed in 1983 and continues to gain in popularity as a biomarker of genetic damage. It can be used as an early indicator of the development of long-term health problems. [1],[12] Scoring of MN is usually performed in peripheral blood lymphocytes but MN can also be relatively easily scored in other cell types relevant for human biomonitoring, such as fibroblasts, exfoliated epithelial cells (from buccal or nasal mucosa or bladder cells in urine), and in erythrocytes. [4]

Panoramic radiography is widely used to complement clinical examination and is considered less harmful than performing several periapical radiographs. [7],[13] During panoramic radiography, buccal epithelial cells (BEC) are a primary target for radiation and so can be used for monitoring human exposure. [14],[15] The advantages with BEC are that they can be easily and rapidly sampled, do not have to be cultivated, and do not require stimulation or metaphase preparations, as is necessary with peripheral lymphocytes. The application of the MN test in BEC has been considered as a sensitive tool for biomonitoring genetic damage in populations exposed to several genotoxic agents. [13],[14],[15],[16],[17],[18] Therefore, we used the MN test to assess whether panoramic radiography induces carcinogenic changes in the BEC.

 Materials and Methods

This study enrolled 60 healthy individuals who had been subjected to panoramic radiograph. Individuals requiring panoramic radiography for further dental treatment were considered eligible for inclusion in the study. Subjects who did not present with any clinical abnormalities in their oral mucosa were included in the study. All subjects filled up a questionnaire before the x-ray examination. With this questionnaire we collected data regarding age, genetic disorders, previous exposure to diagnostic x-rays, vaccinations, medications, tobacco chewing, smoking, alcohol consumption, radiotherapy/chemotherapy for cancer, any occupational or environmental exposure to tobacco, presence of any syndromes, history of cardiovascular or neurodegenerative diseases, and presently malnourished. Subjects taking any medications or having history of exposure to any kind of radiation for the 12 months prior to the study were excluded from the study. The subjects were informed of the objectives of the study and all gave written informed consent to participate in this study. The institutional ethical committee approved the research procedures used in this study.

The selected subjects were divided into four groups according to their ages: 11-20 years, 21-30 years, 31-40 years, and ≥41 years. The panoramic dental radiographs were taken with a Gendex Orthoralix system 9200, using settings of 60-80 kV, 10 mA, 12 s, with an output dose rate of 0.325 m Gy/s.

Collection of cells and slide preparation

The BEC were collected twice for each subject: immediately before radiation exposure and 10 days after. For collecting the sample of BEC, a small-headed toothbrush was gently but firmly rotated with a circular motion against the inside of both cheeks. The head of the toothbrush was then immersed in a conical tube containing 25 ml of buccal cell buffer solution (0.1M EDTA, 0.01M Tris-HCl, 0.02M NaCl, pH 7.0). The cells were then washed twice by centrifugation for 10 min to remove bacteria and cell debris that can complicate scoring.

An adequate cell suspension was dropped onto clean slides and cell density was checked using a light microscope. The slides were allowed to dry, fixed in 80% cold methanol (0°C) for 30 min, and air-dried overnight at room temperature. The cells were then stained with 10% Giemsa solution and mounted with cover slip using DPX (A mixture of Distyrene, a plasticizer, and xylene) mountant. Slides with significant number of bacteria on them were excluded from scoring.

Cytological analysis

All slides were observed under a light microscope, using 100× magnification. The frequency of MN in BEC was evaluated by scoring 1000 cells on each slide. Scoring was done by two senior pathologists, according to the criteria established by Tolbert et al.[19]

Criteria to be satisfied by the cell to be scored:

Intact cytoplasm and relatively flat cell position on the slideLittle or no overlap with adjacent cellsLittle or no debrisNucleus normal and intact; nuclear perimeter smooth and distinct

Criteria for identifying MN:

Rounded smooth perimeter, suggestive of a membraneLess than a third the diameter of the associated nucleus, but large enough to discern shape and colorStaining intensity similar to that of nucleusTexture similar to that of the nucleusSame focal plane as nucleusAbsence of overlap with, or bridge to, the nucleus [19]

Statistical analysis

Data was collected and analyzed using Statistical Package for Social Sciences (SPSS ® version 11.0, Chicago, USA). The paired 't' test was used to find out significance of differences between micronucleated cell frequencies (MCF) and MN per 1000 cells before and after x-ray exposure. The Karl Pearson correlation coefficient 'r' was used to find out the correlation between age and MCF and MN per 1000 cells. One-way analysis of variance (ANOVA) was used to find out if there were significant differences in MCF between different age-groups. The Student's unpaired 't ' test was used to assess the significance of difference between genders with respect to MCF and MN per 1000 cells.


The study included 60 subjects: 41 females (68.33%) and 19 males (31.67%) [Table 1]. The mean age of the study population was 27.63 ± 10.93 years (males 28.26 ± 12.11; females 27.34 ± 10.50 years). There was statistically significant increase in MCF (P = 0.02) and MN per 1000 cells (P < 0.047) after panoramic radiographic exposure [Table 2] and [Table 3].{Table 1}{Table 2}{Table 3}

We examined the MCF and MN per 1000 cells in the different age-groups before and after x-ray exposure; we found an increase in MCF after radiation in all age-groups, but this was not statistically significant. Further comparison of MCF and MN per 1000 cells in different age-groups by one-way ANOVA test showed statistically nonsignificant difference both before (F = 0.51; P=.68) and after radiation exposure (F = 0.52; P = 0.67). There was no statistically significant difference in the MN per 1000 cells between the different age-groups (F = 0.69; P = 0.56). Similar results were obtained after x-ray exposure (F = 0.91; P = 0.44).

Karl Pearson's correlation coefficient showed statistically nonsignificant correlation of age with MCF and MN per 1000 cells [Table 4]. We used the unpaired 't' test to compare the MCF and MN per 1000 cells before x-ray with that after x-ray, in both males and females. The mean of MCF in males and females before exposure was 0.52 ± 0.14 and 0.46 ± 0.14, respectively, and after exposure it was 0.55 ± 0.18 and 0.50 ± 0.13, respectively. The mean number of MN per 1000 cells in males and females before exposure was 0.60 ± 0.25 and 0.54 ± 0.25, respectively, and after exposure it was 0.66 ± 0.27 and 0.58 ± 0.20, respectively. These differences were not statistically significant in either males or females.{Table 4}

A total of 120000 cells were scored in 60 subjects, 60000 cells before radiation exposure and 60000 cells after radiation exposure. A total of 287 cells contained MN before x-ray exposure, which increased to 307 after exposure. The percentage of MCF before and after exposure was 0.48% and 0.51%, respectively. There were 335 MN per 1000 cells before exposure, whereas after exposure there were 363 MN per 1000 cells. The percentages of MN per 1000 cells [(number of MN / 1000 BEC) × 100] before and after exposure were 0.56% and 0.61%, respectively.


Ionizing radiation is well known to induce a wide spectrum of DNA lesions, either directly by energy absorption or indirectly through the production of reactive free radicals. [6] When cells are exposed to low doses of ionizing radiation, double-strand break formation is one of the most important kinds of damage observed. If the repair is either wrong or not possible, the affected cell is meant to die or remain damaged. [5]

Epithelial kinetics plays an important role in the interpretation of the results. According to Angelieri et al. and Ribeiro et al., the ideal interval between the collection of specimens for cytopathology is 7-21 days. [3],[16],[17] A 10-day interval was adopted in the present study because chromosomal damage leading to MN formation occurs in dividing cells from the basal layer of the oral epithelium but is only observed later in exfoliated cells after the differentiation. The turnover of this epithelium is rapid (7-16 days) and thus the maximal rate of formation of MN is expected at 1-3 weeks after the exposure to a genotoxic agent. [3],[13],[15],[20]

In a given population, the detection of an elevated MN frequency indicates an increased risk of carcinogenesis. [17] We found a statistically significant increase in MCF and MN per 1000 cells after exposure to panoramic radiography, i.e. from 0.48% to 0.51% (P = 0.0205) and from 0.56% to 0.61% (P = 0.0500), respectively.

Some cells showed one MN (0.41%), two MN (0.05%), or three MN (0.01%) before exposure to radiation, which after exposure increased to 0.43%, 0.07%, and 0.08%, respectively. Additionally, after radiation exposure, a few cells showed five MN (0.002%), which was not observed before radiation exposure. The most important observation in our study during the interpretation of slides was the presence of more than two MN within a cell. Therefore, MCF and the number of MN per 1000 cells were calculated separately. This finding has not been reported in previous studies in this field.

Several staining methods have been used for evaluation of MN. Although DNA-specific stains are preferred for staining MN and other nuclear anomalies, the most commonly used staining procedure for identifying DNA of the nucleus and MN is the Feulgen stain, followed by a counterstain with fast green to delineate the cell cytoplasm. [12],[21],[22],[23],[24] Feulgen stain is favored by many investigators because of its DNA specificity and because the cytoplasm is clear and transparent with this stain, which enables the easy identification of MN. However, the staining procedure is a relatively lengthy procedure; it is also technique sensitive and may lead to underscoring of MN. [3] Therefore, in our study, we used Giemsa stain for assessing the MN frequency.

In this study, slides with significant number of bacteria on them were excluded from scoring, and if at all bacteria were present they were differentiated by morphology and staining characteristics. Also, buccal cell buffer solution helps to inactivate endogenous DNAases present in the oral cavity and to remove bacteria and cell debris that could complicate scoring. [25]

The individual genetic variability determines the genotoxic response to mutagens. In addition to other constitutional factors, the most consistent demographic variables influencing the MN frequency are age and gender. [3],[26] A progressive increase in spontaneous chromosome instability/chromosomal loss due to the ageing process is associated with the accumulation of DNA damage due to an age-related decline in DNA repair capacity. [27] Some studies have found positive association of age with MN count, [12],[14],[28] while others have not. [29] However, the relationship between age and MN count has been reported more consistently in lymphocytes than in exfoliated BEC. [27] This work assessed the frequency of MN and did not find any statistically significant difference between different age-groups in MN frequency. This probably may be due to the small size of the study population (especially of subjects above 41 years), which could have decreased the statistical power.

From the scientific literature it is known that age has a strong effect on MN frequency in adult life, especially in females, followed by decline above 60 years. [15],[30] We did not find any statistically significant correlation between gender and MN count, and Cerquira et al.[29] and Popova et al.[14] have reported results that are concordant with ours.

Analysis of MN was done according to the criteria given by Tolbert et al.[19] and Sarto et al.[12] In the literature, increased frequency of other nuclear anomalies, including karyorrhexis, condensed chromatin, and pyknosis, have been reported in populations exposed to carcinogens, indicating adverse cellular reaction and/or the presence of a surveillance mechanism to eliminate cells with genetic damage. [3],[15],[17],[25],[29],[31] As suggested by Tolbert et al.[19] and Angelieri et al., [16] if these cytotoxic effects are included for assessment of cytogenetic damage it is possible to increase the sensitivity of biomonitoring studies.

During the interpretation of slides, some cytotoxic effects like 'broken eggs,' karyorrhexis, and karyolysis were reported. However, we did not consider these in the assessment of the genotoxic effect as some authors have suggested that they are associated with normal epithelial differentiation. [13],[29]

The population characteristics and methodological aspects like differences in sites, collection of cells, fixing techniques, various staining procedures, number of cells counted, and scoring criteria for MN, etc., may affect the results. [3] Genetic variation in DNA repair genes may influence individual DNA repair capacity and risk of developing cancer. [6] Similar to previous studies done in different populations, this study done in an Indian population showed that even the low levels of radiation exposure associated with panoramic radiography may cause genetic changes in the BEC.

In this study, radiation exposure from panoramic radiography was shown to induce a statistically significant increase in the frequency of MN, suggesting genotoxic effects in the BEC. However, no significant correlation was observed between MN frequency and age or gender. [13] The radiation exposure required to cause clinically observable changes in the epithelium depends on various factors, including radiation dose, duration of exposure, time of cell division and susceptibility of cells. Further longitudinal research is necessary, addressing the sources of variability, such as differences in methodology, adoption of strict criteria for identification and scoring of micronuclei, and /or population characteristics.

A statistically significant outcome indicates that there is likely to be some relationship between the variables. The use of significance tests to evaluate differences between preexposure measurements and postexposure measurements is of limited value. Firstly, the test provides no information on the variability of response to panoramic radiation exposure within the sample. Secondly, a statistically significant increase in MN does not necessarily indicate a clinical significance. Statistically significant effects refer to real differences as opposed to ones that are illusionary, questionable, or unreliable. Statistical significance implies that the observed difference is not simply a chance finding. [32] On the other hand, clinical significance refers to the benefits derived from any observed difference, its impact on patients or clinician, or its ability to make a difference in people's lives. The variations between studies with respect to MN are difficult to interpret clinically due to complex interactions between environment and genotype within the matrix of growth dynamics, development, and adaptation. All these processes may have significant impact on the level of genome damage measurable by the MN assay. Information about health status and infectious diseases are also important for interpretation of MN assay results. The dynamics of cell division in children may be different from that in adults. Cell proliferation may vary by age and in different cell types. This information is important to the timing of the cell collection to assess exposure of genotoxicity events.

Thus, at the present time, it is difficult to define the real biological significance of the observed variations in MN frequencies in BEC after panoramic radiation exposure. Standardization of the protocols and improvement of study design, large prospective studies combining exposure data with comprehensive assessment of lifestyle factors and health status, and inclusion of other nuclear anomalies may prove to be vital benefit in assessing cytotoxic damage. Novel molecular technologies that are being introduced present a very promising line of future mechanistic research of MN.


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