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 Table of Contents  
ORIGINAL ARTICLE
Year : 2022  |  Volume : 34  |  Issue : 2  |  Page : 188-192

Comparison between micronuclei and AgNORs in assessing the short-term genotoxic effects of panoramic radiography on oral mucosa: A cross-sectional study


1 Department of Oral Medicine & Radiology, TMDCRC, Teerthanker Mahaveer University, Moradabad, Uttar Pradesh, India
2 Department of Oral Pathology & Microbiology, TMDCRC, Teerthanker Mahaveer University, Moradabad, Uttar Pradesh, India
3 Department of Oral Pathology, Govt. Dental College and Hospital, Ahmedabad, 380016, Gujarat, India
4 Department of Oral Pathology & Microbiology, Seema Dental College and Hospital, Rishikesh, Uttarakhand, India

Date of Submission11-Sep-2021
Date of Decision20-May-2022
Date of Acceptance20-May-2022
Date of Web Publication22-Jun-2022

Correspondence Address:
Shilpa Dutta Malik
Department of Oral Pathology & Microbiology, TMDCRC, Teerthanker Mahaveer University, Moradabad, Uttar Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jiaomr.jiaomr_264_21

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   Abstract 


Context: Accumulated evidence suggests that X-radiations can induce genotoxic effects and are not safe at any particular radiation dose level. Various assays have been proposed as potential tools in cytogenetic biomonitoring studies, but they have limitations. Aims of the Study: To assess the genotoxic effects of panoramic radiography on oral mucosa by estimating micronuclei count (using acridine orange) and AgNORs (using silver stain) from cytological smear cells and to compare these two biomarkers. Material and Methods: 100 subjects who underwent panoramic radiography for dental treatment with pre-defined inclusion and exclusion criteria formed the sample for this cross-sectional study. Two smears were prepared using a cytobrush from the buccal mucosa wet fixed using 95% ethyl alcohol. After 10 days and 40 days, the subjects were recalled for a similar repetition of smear preparations from the same site. Before all the procedures, ethical approval was obtained from the institute's ethical committee where the study was conducted, and written informed consent was taken from all the patients before they participated in the study. Statistical Analysis: The results obtained were further assessed using one-way analysis of variance (ANOVA) and Post Hoc Tukey's HSD for inter and intra observational comparisons. Results: Radiation exposure from panoramic radiography showed a statistically significant increase in micronuclei. However, the AgNOR count remained unchanged after radiation exposure. Conclusion: MN count is better for detecting the short-term genotoxic effect of panoramic radiation exposure on buccal mucosal cells than AgNORs.

Keywords: AgNOR, genotoxicity, micronuclei, panoramic radiography


How to cite this article:
Malik SD, Malik U, Pillai J, Sharma S, Singh M, Lehri S. Comparison between micronuclei and AgNORs in assessing the short-term genotoxic effects of panoramic radiography on oral mucosa: A cross-sectional study. J Indian Acad Oral Med Radiol 2022;34:188-92

How to cite this URL:
Malik SD, Malik U, Pillai J, Sharma S, Singh M, Lehri S. Comparison between micronuclei and AgNORs in assessing the short-term genotoxic effects of panoramic radiography on oral mucosa: A cross-sectional study. J Indian Acad Oral Med Radiol [serial online] 2022 [cited 2022 Dec 7];34:188-92. Available from: http://www.jiaomr.in/text.asp?2022/34/2/188/347920




   Introduction Top


Imaging with X-rays plays a pivotal role in diagnosis and treatment planning and is of great help to health care professionals in the same regard.[1] The radiation dose level is determined based on a balance between clinical benefits and harmful effects. Panoramic radiography is widely used as a supplement to oro-dental examination as it is considered less harmful (26 μSv) compared to full mouth intraoral periapical radiographs (33 μSv) due to the lower exposure values (60-90 Kvp, 2- 15 mA.[2] Nuclear alterations, pyknosis, condensed chromatin, and karyorrhexis should be observed as genotoxic effects in cells exposed to radiation.

A micronucleus (MN) is formed when a damaged chromosome and its resultant fragments fail to be included in either of the two daughter nuclei. The genetic material left behind forms a “micronucleus” in new generation cells. Since the MN frequency test is a good marker for DNA damage of the cells, the MN induction test has largely been used as a sensitive genotoxicity assay under in vivo and in vitro conditions.[3]

Many studies have demonstrated that ionizing radiation may induce DNA damage and genomic alteration.[4],[5] DNA damage includes single and double-strand breaks and DNA protein crosslinks which induce cellular death. Such genomic instabilities are considered a major cause of developmental and degenerative disorders. Genomic damages can be assessed and evaluated by numerous methods.[6] One of the most popular and sensitive methods is the micronucleus test.

Nucleolar Organizer Regions (NORs) are loops of ribosomal DNA that occur in the cells' nucleoli on the short arms of the acrocentric chromosomes 13, 14, 15,21, and 22. The interphasic NORs can be visualized at the light microscopic level by using a silver reaction that stains the NORs' acidic proteins on routinely prepared histopathological and cytological samples. After silver staining, the AgNORs can be identified as black dots throughout the nucleolar area. In quantitative terms, the number of AgNORs per nucleus suggests it to be a marker of the proliferative activity of the cell. Thus, qualitatively (based on the shape, size, and distribution pattern), AgNOR acts as a marker of pre-malignant or malignant change and can also be considered a useful adjunct to identifying changes.[7] Oral epithelial cells provide an ideal source of oral tissue for assessing genotoxic changes using various staining techniques.[6] The present study was designed to assess the genotoxic effects of panoramic radiography on oral mucosa by estimating the MN and AgNORs on buccal mucosal cells.

Aim and Objectives of the Study

To assess the genotoxic effects of panoramic radiography on oral mucosa by estimating micronuclei (MN) & AgNORs on buccal mucosal cells to assess micronuclei and AgNOR count in Acridine orange and a silver-stained cytological smear of buccal mucosa respectively before radiographic exposure. And to assess MN and AgNOR count in Acridine orange and a silver-stained cytological smear of buccal mucosa, respectively, after 10 days and 40 days of radiographic exposure.

Study design

100 subjects were taken up for the study, calculated using SPSS 21.0 software. Ethical clearance for the same was taken from the ethical committee of Seema Dental College & Hospital (the institute has not provided an Ethical Clearance Number). Procedures were performed as per Helsinki declaration. These subjects were divided into two groups, with one aged between 15-25 years (group I) and the other the 40-50 years of age (group II) 9 to observe the age-related genotoxic effects in the younger and groups.

Inclusion criteria are not clearly stated.

Inclusion criteria

One hundred otherwise healthy subjects who reported to the outpatient department of Seema Dental College and Hospital, Rishikesh, for diagnostic panoramic radiographs for further dental treatment were included in this study after obtaining written informed consent.

Exclusion criteria

Those patients who had diagnostic radiographs of the maxillofacial region in the previous six months, malignancy relating to any part of the body, including pre-malignant lesions or conditions in the oral cavity, and history of tobacco or alcohol consumption in any form were excluded as subjects in this study.


   Materials and Methods Top


Before sample collection, patients were asked to rinse their mouths with 1% acetic acid. Then two smears were prepared using a cytobrush from the buccal mucosa and were wet fixed using 95% ethyl alcohol. One of the smears was stained with Acridine orange for the assessment of micronuclei, and the other smear was stained with silver staining using the Ploton[8] method for AgNORs. Digital panoramic radiographs were taken with the digital panoramic machine “Kodak 8000C”. After 10 days and 40 days (for assessing long-term effects), the patients were called for subsequent smear preparation.

Was no radiation exposure on these days. The evaluation and the comparison of the same were made. Acridine orange stained smears were evaluated at (×100) magnification under a fluorescent microscope. In the present study, the criteria used to identify micronuclei were suggested by Sarto et al.[8] Micronuclei were scored only when chromatin structure and color intensity were similar to or weaker than the main nucleus; borders were assessed. The evaluation excluded dead or degenerating cells (karyolysis, karyorrhexis, pyknosis, and nuclear fragmentation). Micronuclei appeared in the form of discrete green dots present in the orange cytoplasm of exfoliated cells [Figure 1]. A total of 100 cells per slide were counted in all the cases. The one-step silver-staining technique was used as described by Ploton et al. (1986).[9] The staining solution (0.3 ml) was poured immediately on each slide, and the slide was left for 14 to 20 minutes at room temperature to achieve the desired intensity of the reaction. Nucleolar organizer region counting was done according to the Crocker et al. method.[10] Using a binocular microscope, all the smears were examined under ×1000 magnification in oil immersion. AgNORs were distinctly visible as dark brown to black dots or blebs of varying size in the brown stained nucleus on a pale yellow background of the cells [Figure 2]. A total of 100 cells per slide were counted, and total numbers of AgNORs were recorded. The number of cells with more than 5 and more than 3 AgNORs/100 cells was also recorded.
Figure 1: The silver stained cytological smear show Agnors in nucleus

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Figure 2: The acridine orange smear shows micronuclei in cytoplasm

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Statistical analysis

The results were tabulated and assessed statistically using SPSS v 21.0 software (IMB Corp., Armonk, NY). The results obtained were further assessed using one-way analysis of variance (ANOVA) and Post Hoc Tukey's HSD for inter and intra observational comparisons.


   Results Top


In the present study, the total number of MN (100 cells per slide) was counted in both the age groups at various time intervals (0 days, 10 days, and 40 days).

In Group I, at 0 days, the mean number of micronuclei was found to be 6.96 (±3.03) (with a range of 2- 17).

At 10 days, the mean number of micronuclei was 8.96 (±3.09) (with a range of 3-17). At 40 days, the mean number of micronuclei was 11.1 (±14.09) (with a range of 3-24).

In Group II (40-50 years), at 0 days, the mean number of micronuclei was found to be 11 (±4.14) (with the range of 3-20); at 10 days, mean the number of micronuclei was observed to be 1.72 (±4.55) (with the range of 4-28), and at 40 days, the mean number of micronuclei was 4.86 (±5.72) (with a range of 7-39). Hence the mean number of micronuclei increased from 0 days to 40 days in both the age groups [Table 1] and [Graph 1].
Table 1: Distribution of Micronuclei in GROUP I (15-25 years) and GROUP II (40-50 Years) Age Group at Various Time Intervals

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The overall outcome of the study was tabulated and analyzed using the Unpaired t-test. When two age groups were compared using an unpaired t-test for the changes in micronuclei and AgNOR, it was found that micronucleus was a better marker to assess the genotoxicity. The P value (0.0057) was significant for changes regarding micronuclei, recorded in both the groups between 0 to 10 days [Table 2]. Similarly, the values were tabulated and analyzed within the groups at various time intervals (table).
Table 2: comparison between two age groups for change in micronuclei and AgNOR-‘unpaired t test’

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   Discussion Top


X-radiations are a potent mutagenic agent capable of inducing gene mutations and chromosomal aberrations. In the oral cavity, exfoliated cells from the oral epithelium appear to be target cells for this X-ray exposure. High proliferative activity, greater surface area, and non-keratinization of buccal mucosal cells make them vulnerable to change in response to any insult or trauma in the oral cavity. Buccal epithelial tissue is under direct radiation exposure and thus is a primary target for radiation-induced damage. Reports have shown that buccal cells have limited DNA repair capacity relative to peripheral blood lymphocytes and, therefore, may more accurately reflect genomic instability in epithelial tissues. Micronucleus, which can be used to assess chromosomal damage, is a small intranuclear DNA structure separated from the main nucleus of the basal cell layer. According to the literature, micronucleus formations can be observed at 10 days post-exposure. The daughter cells containing these micronuclei migrate upwards through the epithelium and exfoliate in the oral cavity, which can be seen between 1 and 3 weeks after exposure to the genotoxic effect. Thus the number of micronuclei in these exfoliated cells can be used as a biomarker to monitor genetic damage in humans. Although long-term follow-ups, it becomes of utmost importance to assess any increase in genotoxic changes related to radiation as the stochastic effect of radiation is always late in its manifestation. Still, due to time constraints, a follow up period of 40 days was kept in order to assess changes that may have long-term implications, and further studies which can assess these changes at longer intervals become necessary in order to assess the stochastic effects related to radiation.[11],[12],[13],[14],[15] It has been suggested that the number of AgNOR in interphase nuclei may reflect the state of activation and/or the degree of malignant transformation of certain tissues. Certain reports indicate that the AgNOR number reflects the degree of cellular proliferation, activation, and differentiation, thus contributing to diagnosis. The number of AgNOR in a nucleus depends on the number of transcriptionally active NORs and the degree to which these active sites associate, giving rise to larger AgNOR.[16] Hence, the AgNOR evaluation was also done in the present study to assess the genotoxic changes caused by panoramic radiography on exfoliated buccal cells.

This study showed an increased number of micronuclei in the older age group as compared to the younger age group at different time intervals. Furthermore, this increase was found to be statistically highly significant with increasing age This was in accordance with Sheikh et al. 2012,[11] who further explained that this effect could be due to genetic instability, which may enhance the hypothesized progressive chromosome instability related to the aging process. Our results were in contrast with the study conducted by Pai et al. 2012[12] and Cerqueira EM 2008[1] who independently found that there was no statistically significant difference between micronuclei frequency and age. This insignificance was attributed to the mean age of 23 years and 26 years respectively in the above studies. Chromosomal damage leading to micronucleus formation occurs during the division of cells from the basal layer of the oral epithelium, but it is only observed later in exfoliated cells, between 1 week and 3 weeks after exposure to a genotoxic agent.[6],[7],[8] Day 10 was chosen on the basis of the fast turnover in epithelial cell kinetics (from 7–16 days). The rationale behind choosing the 40th day for sample collection was to study the long-term toxic effect of radiation.[12] While no significant difference was found in micronuclei frequency from time intervals of (0-10 and 10-40 days). In the present study, the total number of AgNOR (100 cells per slide) were counted in all the cases of Group I (15-25 years) and Group II (40-50 years of age) at various time intervals. A significant change in the number of micronuclei from 0-10 days was observed. An insignificant difference in micronuclei was observed on the 10th and 40th day in both the age groups 15-25 years and 40-50 years). An increase in the mean number of AgNOR from 0 days to 40 days in both the age groups was observed but there was no statistically significant difference in AgNOR count before and after exposure.


   Conclusion Top


MN count is a better method for detecting short-term genotoxic effect of panoramic radiation exposure on buccal mucosal cells than AgNORs).

Limitations and future prospects

The present study was conducted using the micronucleus test. The micronucleus assay detects chromosomal damage only, it becomes essential to employ other methodologies for evaluating genotoxicity induced by dental X-rays, especially those able to detect DNA single- and double-strand breaks, DNA adducts, point mutations, and others. This type of approach is particularly important because cytotoxicity may cause an underestimation of the micronucleus frequency due to cell death. Moreover, further studies evaluating cell-cycle control are also important to clarify the effect of dental X-rays on oral mucosa cells at the cellular and molecular levels. For this purpose, in vitro studies should be conducted to better understand the role of cell signaling pathways in oral mucosa cells.

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

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

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Cerqueira EM, Meireles JR, Lopes MA, Junqueira VC, Gomes-Filho IS, Trindade S, et al. Genotoxic effects of X-rays on keratinized mucosa cells during panoramic dental radiography. Dentomaxillofac Radiol 2008;37:398-403.  Back to cited text no. 1
    
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Moll MA, Seuthe M, von See C, Zapf A, Hornecker E, Mausberg RF, et al. Comparison of clinical and dental panoramicfindings: A practice-based crossover study. BMC Oral Health 2013;13:48.  Back to cited text no. 2
    
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Chang WP, Kirsch-Volders M, Holland N, Bonassi S, Zeiger E; HUman MicronNucleus project. HUMN project: Detailed description of the scoring criteria for the cytokinesis- block micronucleus assay using isolated human lymphocyte culture. Mutat Res 2003;534:65-75.  Back to cited text no. 3
    
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Chiu KY, Loke SL, Wong KK. Improved silver technique for showing nucleolar organiser regions in paraffin wax sections. J Clin Pathol 1989;42:992-4.  Back to cited text no. 4
    
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Ogden GR, Cowpe JG, Green MW. Quantitative exfoliative cytology of normal buccal mucosa: Effect of smoking. J Oral Pathol Med 1990;19:53-5.  Back to cited text no. 5
    
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Sarto F, Finotto S, Giacomelli L, Mazzotti D, Tomanin R, Levis AG. The micronucleus assay in exfoliated cells of the human buccal mucosa. Mutagenesis 1987;2:11-7.  Back to cited text no. 8
    
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Ploton D, Menager M, Jeannesson P, Himber G, Pigeon F, Adnet JJ. Improvement in the staining and in the visualization of the argyrophilic proteins of the nucleolar organizer region at the optical level. Histochem J 1986;18:5-14.  Back to cited text no. 9
    
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Crocker J, Boldy DA, Egan MJ. How should we count AgNORS? Proposals for a standardized approach. J Pathol 1989;158:185-8.  Back to cited text no. 10
    
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Sheikh S, Pallagatti S, Grewal H, Kalucha A, Kaur H. Genotoxicity of digital panoramic radiography on oral epithelial tissues. Quintessence Int 2012;43:719-25.  Back to cited text no. 11
    
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Pai A, Sharma RC, Naik RM, Guruprasad Y. Biomonitoring of genotoxic and cytotoxic effects of gingival epithelial cells exposed to digital panoramic radiography. J Orofac Sci 2012;4:124-8.  Back to cited text no. 12
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Madhavan R, Kumaraswamy M, Kailasam S, Kumar SM. Genetic damage in exfoliated cells from oral mucosa of individuals exposed to X-rays after panoramic radiograph: A cross-sectional study. J Indian Acad Oral Med Radiol 2012;24:102-5.  Back to cited text no. 13
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Jindal S, Chauhan I, Grewal HK. Alteration in buccal mucosal cells due to the effect of tobacco and alcohol by assessing the silver-stained nucleolar organiser regions and micronuclei. J Cytol Indian Acad Cytol 2013;30:174-8.  Back to cited text no. 14
    
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Sandhu M, Mohan V, Kumar JS. Evaluation of genotoxic effect of X-rays on oral mucosa during panoramic radiography. J Indian Acad Oral Med Radiol 2015;27:25-8.  Back to cited text no. 15
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Agarwal P, Vinuth DP, Haranal S, Thippanna CK, Naresh N, Moger G. Genotoxic and cytotoxic effects of X-ray on buccal epithelial cells following panoramic radiography: A pediatric study. J Cytol 2015;32:102-6.  Back to cited text no. 16
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