|Year : 2021 | Volume
| Issue : 4 | Page : 372-378
Evaluation of genomic damage from buccal epithelial cells in patients subjected to cone beam computed tomography
Gutta Mounika1, Koduri Sridevi1, Buduru Krishnaveni1, Nalli Prasanth Kumar1, Harika Naidu1, Bonthagarla Krishna Sahi2
1 Department of Oral Medicine and Radiology, Lenora Institute of Dental Sciences, Rajahmundry, Andhra Pradesh, India
2 Department of Oral Medicine and Radiology, GSL Dental College and Hospital, Rajahmundry, Andhra Pradesh, India
|Date of Submission||25-Mar-2021|
|Date of Decision||16-Aug-2021|
|Date of Acceptance||28-Sep-2021|
|Date of Web Publication||27-Dec-2021|
Dr. Gutta Mounika
Post Graduate Student, Department of Oral Medicine and Radiology, Lenora Institute of Dental Sciences, Rajahmundry, Andhra Pradesh
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: Radiography forms an important and integral part in diagnosis which is used in the oral and maxillofacial region to give an appropriate diagnosis and treatment planning. But, diagnostic radiations also induce some amount of cell damage at cytogenetic levels, depending on the dosage of the radiation administered. The ionizing radiation which has been emitted during these procedures has deleterious effects on the DNA and induces cell death. Aim: To evaluate the genomic damage from buccal epithelial cells in patients subjected to cone-beam computed tomography (CBCT). Materials and Methods: Patients were divided into two groups with 30 subjects in each group. Group-1 included the subjects advised for single CBCT exposure and Group-2 included the subjects advised for double CBCT exposure. Exfoliated buccal epithelial cells were collected immediately before the exposure, 15 days, and 30 days after the CBCT exposure. The cytological smears were examined to detect the micronucleus and pyknotic nucleus. Results: The mean of the micronucleus and pyknotic nucleus obtained from group-2 (double exposure) subjects was significantly higher in males than group-1 (single exposure) subjects with a P value of 0.0001 during pre-exposure, 15 days, and 30 days after exposure. Conclusion: Genomic damage does take place due to cone-beam computed tomography. So, CBCT should be cautiously used when necessary and it cannot be considered as a risk-free procedure.
Keywords: Buccal epithelial cells, CBCT, micronucleus, papanicolaou stain, pyknotic nucleus
|How to cite this article:|
Mounika G, Sridevi K, Krishnaveni B, Kumar NP, Naidu H, Sahi BK. Evaluation of genomic damage from buccal epithelial cells in patients subjected to cone beam computed tomography. J Indian Acad Oral Med Radiol 2021;33:372-8
|How to cite this URL:|
Mounika G, Sridevi K, Krishnaveni B, Kumar NP, Naidu H, Sahi BK. Evaluation of genomic damage from buccal epithelial cells in patients subjected to cone beam computed tomography. J Indian Acad Oral Med Radiol [serial online] 2021 [cited 2022 May 21];33:372-8. Available from: https://www.jiaomr.in/text.asp?2021/33/4/372/333881
| Introduction|| |
The fortuitous chance discovery of X-rays by Sir Wilhelm Conrad Roentgen on November 8, 1895, revolutionized the practice of medicine and made radiology an important diagnostic method in modern medicine As a transition from two dimensional (2D) to three dimensional (3D) procedures, Computed Tomography (CT), Cone-beam computed tomography (CBCT) and Magnetic resonance imaging (MRI) ameliorated the field of Oral Maxillofacial Radiology to determine a definite diagnosis and interventional procedures pertaining to the maxillofacial region.,
As most of the diagnostic radiological modalities or aids use ionising radiation, they may result in cell damage leading to genotoxicity or cytotoxicity. The radiation effects of low dose diagnostic radiographic exposures and the resultant genomic damage can be assessed and evaluated by numerous methods, wherein the sensitive analysis and specific approach being the micronucleus (MN) test.,
Buccal Micronucleus Cytome (BMCyt) assay was first proposed in 1983 to determine the presence of micronucleus. The micronuclei are the small extranuclear cytoplasmic DNA bodies that are induced in cells by numerous genotoxic agents that damage chromosomes.,
As the literature shows, there are few studies regarding the genetic damage from the buccal epithelial cells, the present study was undertaken to evaluate the genomic damage from buccal epithelial cells with an attempt to know the effect of radiation exposure in patients subjected to cone-beam computed tomography scans.
| Materials and Methods|| |
The present study was conducted in the Department of Oral Medicine and Radiology, Rajahmundry, East Godavari district, Andhra Pradesh, India after getting approval from the Institutional Ethical Committee (103/IEC/LIDS/2018) and written informed consent from the patients. The procedures followed the ethical standards of the Helsinki Declaration of 1975, as revised in 2000. A total of 60 patients who were advised for CBCT exposure were enrolled for the study. These patients were divided into two groups comprising of Group-1 with single exposure and Group-2 with double exposure with 30 subjects in each group. The sample size was calculated based on the pilot study conducted on 30 patients of which 60% of them showed the presence of micronucleus and pyknotic nucleus. The sample size was calculated by using the formula
S1: Standard Deviation in the first group
S2: Standard Deviation in the second group
SP: Sample size
So, a total of 60 sample size was included in the study.
Inclusion Criteria: Patients who were advised for CBCT exposure.
Exclusion criteria: Adverse habits like smoking and alcohol consumption, pan or gutka chewing, occupations that are associated with chemicals, malignancies, previous history of radiotherapy or chemotherapy, alcohol-containing mouthwashes, and patients subjected to CBCT or any other ionizing radiation one month prior to the study.
All the subjects who participated in the study were explained about the study in their known language and the consent was taken. Patients were made comfortably seated on a dental chair and the detailed history along with the demographic data was recorded. A thorough clinical examination was carried out under artificial illumination and the initial smear was made.
Scanning protocol: Patients were subjected to CBCT using I MAX Touch 3D CBCT machine having the parameters of tube voltage of 76 kVp, a tube current of 9 mA with the exposure time of 8 seconds, a field of view (FOV) measuring 9.3 × 8.3 cm and a focal spot of 0.5 mm with a single 360-degree rotation after following radiologic safety measures.
Sample collection and slide preparation: Patients were asked to gently rinse the mouth with water and exfoliated buccal epithelial cells were collected with cytobrush [Figure 1] prior, 15 days, and 30 days after the single and double CBCT exposures from Group-1 and Group-2 patients respectively. The obtained sample by cytobrush was smeared on a glass slide [Figure 2]. Then, the prepared slides with smears were marked and fixed with 95% alcohol and then subjected to papanicolaou staining. PAP stain was preferred in the present study, as it consists of a nuclear stain that stains all nuclear DNA, both intranuclear and extranuclear. It also consists of 2 counterstains that make the cytoplasm transparent and well-demarcated when compared with the other stains.
Cytological analysis: The stained and fixed slides were examined in randomly selected microscopic fields at a magnification of 400x for the presence of micronucleus and pyknotic nucleus from the exfoliated cells [Figure 3], [Figure 4], [Figure 5]. At least 500 exfoliated cells in each smear were counted in a zigzag manner starting from one end of the slide and approaching the other end.
|Figure 4: a and b: Buccal epithelial cells depicting the micronuclei under 400x magnification|
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|Figure 5: Buccal epithelial cell depicting the pyknotic nucleus under 400x magnification|
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Scoring criteria: Cells were examined under 100x magnification using Labomed Binocular Research Microscope to evaluate the genomic damage. Tolbert's criteria for evaluating the micronucleus in which the MN will be less than 1/5th to 1/3rd diameter of the main nucleus and is on the same plane of focus with the main nucleus, having a smooth oval or round shape, same color, texture, and refraction as the main nucleus, and it should be clearly separated from the main nucleus. The count of micronucleus was taken into consideration for evaluating genotoxicity while the incidence of pyknosis, was evaluated for estimating the cytotoxicity. The slides were evaluated by an experienced oral pathologist. The values were plotted on the table and statistical analysis was performed to extract the results.
The statistical analysis was performed using SPSS (Statistical package for social sciences) version 20.0. One-way ANOVA was applied for the determination of comparison of groups. Independent t-test and Levene's test for equality of variances were applied for gender analysis. A P value of < 0.05 was considered to be statistically significant.
| Results|| |
The current study was performed on 60 healthy patients with ages ranging from 24–50 years, among which 27 (45%) were males with mean age of 36.17 ± 7.648 and 33 (55%) were females with mean age of 36.30 ± 8.867 [Table 1].
[Table 2] depicts the micronucleus frequency in groups 1 and 2 seen at the time of pre-exposure, on the 15th day, and 30th day after exposure. There was increased micronucleus count observed in both groups 15 days after exposure when compared with pre-exposure. However, it was observed that on the 30th day after exposure, the micronucleus count was reduced when compared with the 15th day after exposure [Table 2]. The test showed that there was a statistically significant difference with P value of 0.000 in both group-1 and group-2 and there was no statistically significant difference in micronucleus count from 15 days after exposure to 30 days after exposure. However, the genomic damage was found to be significantly higher in group-2 when compared with group-1 individuals reflecting the fact that the double exposure causes significant genomic damage than the single exposure.
|Table 2: Micronucleus count on pre exposure, 15 days and 30 days after exposure among group - 1 & group - 2|
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[Table 3] depicts the pyknotic nucleus frequency in groups 1 and 2 seen at the time of pre-exposure, 15th day, and 30th day after exposure. There was increased pyknotic nucleus count observed in both groups, 15 days after exposure when compared with pyknotic nucleus count at the time of pre-exposure. It was observed that on the 30th day after exposure, the pyknotic nucleus count was reduced when compared with the 15th day after exposure. The test showed that there was a statistically significant difference with P value of 0.001 in group-1 and 0.000 in group-2. The statistically significant increase in the pyknotic nucleus 15 days after radiation exposure reflected the fact that the radiation caused by the single exposure and double exposure to CBCT scan induces genomic damage. However, the genomic damage was found to be significantly higher in group-2 when compared with group-1 individuals. [Table 3]
|Table 3: Pyknotic nucleus count on pre exposure, 15 days and 30 days after exposure among group - 1 & group - 2|
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Another component of this study was gender-related increase in the micronucleus and pyknotic nucleus in males and females among group-1 and group-2 individuals. A statistically significant difference was found from pre-exposure, 15 days and 30 days after exposure in males with a P value of 0.019 and 0.036 in group-1 individuals and 0.016 and 0.007 in group-2 individuals. The statistically significant difference in the micronucleus count 15 days after radiation exposure between both the genders reflected the genomic damage caused by single and double exposure was significantly higher in males. [Table 4]
|Table 4: Gender distribution of mean micronucleus count on pre exposure, 15 days and 30 days after exposure in group - 1 and group-2|
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The present study showed a statistically significant difference in group-2 with an increase in the pyknotic nucleus in males during pre-exposure, 15 days, and 30 days after exposure. The statistically significant difference in the pyknotic nucleus 15 days after radiation exposure between both the genders reflected the genomic damage caused by single and double exposure was significantly higher in males with the P values of 0.049 and 0.013 in group-1 and group-2 individuals. [Table 5]
|Table 5: Gender distribution of mean pyknotic nucleus count on pre exposure, 15 days and 30 days after exposure in group - 1 and group-2|
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In comparison of micronucleus and pyknotic nucleus, micronucleus was found to be higher in males in group-2 individuals in patients subjected to CBCT for evaluating the genomic damage. Also, the pyknotic nucleus has more significant values among males in group-2 individuals. The results depicted highly significant values implying that the micronucleus and pyknotic nucleus increases with the amount of radiation exposure.
| Discussion|| |
Radiography is one of the most valuable diagnostic tools used in comprehensive dental care. Radiographic methods in the field of dentistry pertaining to oral and maxillofacial radiology are CT and CBCT in addition to conventional two-dimensional imaging modalities. CBCT is widely used in maxillofacial imaging because of the less radiation dose, detailed 3D images over the region of interest with good quality by allowing volumetric analysis and lower cost when compared with CT. However, excessive exposures to these imaging modalities also may cause untoward effects which may lead to genotoxicity. As a result, the different adverse effects which can be encountered in the oral cavity initially are xerostomia, oral mucositis, radiation-related dental caries, changes in the dental hard tissues, such as initial enamel demineralization and finally osteoradionecrosis.,
According to the International Commission on Radiological Protection and the National Council on Radiation Protection and Measurement, it has been demonstrated that <100 mGY and 1 mSV2 per annual dose are sufficient enough to cause biological damage in oral mucosal cells.
Various studies conducted by Naveena Preethi, F Angelieri, Mahima Sandhu, Pallak Arora, and S Anbumeena evaluated the genotoxicity in the exfoliated buccal epithelial cells in individuals exposed to two-dimensional imaging like panoramic radiography whereas Santosh Palla et al., Juliana BM da Fonte et al., Soha Basha, V Carlin et al., Diego Coelho Lorenzoni, Pan Yang and Farhadi S evaluated the genotoxicity in the exfoliated buccal epithelial cells in individuals exposed to three-dimensional imaging like CT and CBCT.
Ionizing radiation is known to induce a wide spectrum of DNA changes either directly or indirectly in the form of energy absorption, ionization, and production of reactive free radicals. Genotoxicity could be the result of damage to the DNA as reported in cases of radiation-induced cell death that might lead to mutations, the key step in carcinogenesis.
Micronucleus arises from acentric fragments which are not included in the main nuclei of the daughter cells and the formation of micronuclei can be induced by substances that cause chromosome breakage as well as by the agents that affect the spindle apparatus. Micronucleus may be used as a marker to evaluate the radiation effects of low-dose diagnostic radiographic exposures.
Micronucleus can be detected in exfoliated cells by a sensitive method known as Buccal Micronucleus Cytome (BMCyt) assay and used as an indicator of recent DNA injury within the oral mucosa and hence, the quantitative detection of micronucleus is widely used for the analysis of cytogenetic damage. The advantage of BMCA has the relative ease of obtaining the tissue, limited cost, and less time.
Holland et al. stated that after ionizing radiation exposure, expression of micronucleus in buccal mucosal cells takes a minimum of about 5–7 days to a maximum of 21 days. Ribeiro et al. reported that the turnover time for the oral cavity epithelial cells is 7–16 days and the highest number of micronucleus was anticipated from 1 week to 3 weeks after a genetic insult. Taking these factors into consideration a time interval of about 10–15 days was given for the second collection.
With the above background and reported studies from the literature, the present prospective study was conducted to evaluate the effect of radiation on genomic damage from the buccal epithelial cells in patients subjected to cone-beam computed tomography scans.
In the present study, BEC could be considered as a preferred sample to accurately reflect the cytotoxic changes and genomic instabilities in epithelial tissues. This could be due to the high turnover rate of the BEC that brings the cells to the surface and the vicinity of the BEC to be directly in the field and the primary beam of radiation exposure. Also, the BEC has a continuous cell renewal, where new cells are produced in the basal layer by mitosis and migrate to the surface by replacing the cells for shedding.
The mean for micronucleus and pyknotic nucleus showed a statistically significant difference during pre-exposure to 15 days after exposure in both the groups with higher significance in group-2. There was no statistically significant difference from 15 days after exposure to 30 days after exposure representing the genomic damage from the buccal epithelial cells in patients subjected to CBCT. Though it causes genomic damage within 15 days after exposure, the significant decrease in the micronucleus count in the present study suggests the decrease in the effect of radiation on genomic damage from the buccal epithelial cells.
Our study was in accordance with the reports by Mahima Sandhu and Ivleen Kaur et al. which showed a significant increase in the micronucleus, evaluated 15 days after exposure. The present study reported the mean of the pyknotic nucleus in group-1 and group-2 individuals with a statistically significant difference thereby revealing the increased frequency of the exfoliated buccal epithelial cells after 15 days of exposure to CBCT.
Pan Yang et al. stated that the radiation sensitivity of the various cells in the oral cavity like exfoliated cells of buccal mucosa, tongue, and gingiva had no statistically significant difference. Eman A El-Ashiry stated that after taking full mouth radiographs, the incidence of micronucleus and other nuclear changes were elevated in exfoliated cells of buccal mucosa when compared to gingival epithelial cells.
The results of the present study indicated that the increase in the frequency of micronucleus and nuclear alterations such as pyknosis were significantly related to gender. The mean micronucleus and pyknotic nucleus count were found to be significantly higher in males at all three intervals of collection of samples. However, by comparing the groups, group-2 individuals showed significantly higher micronucleus and pyknotic nucleus when compared with group-1. The studies conducted by Pallak Arora and Soha Basha did not show statistically significant differences regarding the frequency of micronucleus and pyknotic nuclei among genders.
The significant difference in males could be due to some lifestyle changes like smoking, alcohol consumption, exposure to drugs and chemicals, some disease conditions and infections, body mass index, and variations in the amount of radiation exposure.
Finally, the genomic damage through evaluation of micronucleus and pyknotic nucleus from buccal epithelial cells was found to be significantly higher from pre-exposure to 15 days and 30 days after exposures that can increase the chromosomal alteration. In spite of all the radiation protection measures taking into consideration by adhering to the ALARA principle, it can be stated that CBCT should be cautiously used following the rationale for diagnosis and treatment plan.
- Low sensitivity and limited accuracy associated with a pap smear,
- Pap staining is a screening test that must be followed up with more specialized diagnostic tests.
- In the present study, the effect of CBCT on buccal mucosal cells was only evaluated but the effect of other radiographs on epithelial cells from different sites was not evaluated which is beyond the perspective of this study.
Future study prospects
Genomic damage analyses are crucial and the vision for the future. It has the potential to analyze the premalignant lesions and conditions at an early stage which occur due to the radiation effects before entering into a malignant stage. This is one of the few studies reported in the literature that correlates the genomic damage before exposure, 15 days, and 30 days after the exposure induced by CBCT. However further studies with larger sample sizes are needed to strengthen the results of the present study.
| Conclusion|| |
The results of the present study will aid in better understanding of radiation induced changes on the buccal mucosal cells after single and double exposure to CBCT. Buccal cell micronucleus assay can be used effectively as a biomarker to study genomic damage in the oral cavity. Dental X-rays must be taken only when they were extremely needed obeying the ALARA principle. However further more periodic studies among different sample population groups with larger sample size are required to validate and confirm whether evaluation of micronucleus, pyknotic nucleus from the buccal epithelial cells could be considered as a biomarker for the evaluation of genomic damage in cone beam computed tomography exposed individuals.
Declaration of patient consent
The authors certify that they have obtained all appropriate patient consent forms. In the form, the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]