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ORIGINAL ARTICLE
Year : 2019  |  Volume : 22  |  Issue : 8  |  Page : 1041-1048

Evaluation of cell and DNA damage induced by panoramic radiography


1 Department of Oral and Maxillofacial Radiology, Faculty of Dentistry, Institute of Oncology, Istanbul University, Istanbul, Turkey
2 Departments of Basic Medical Sciences and Biophysics, Faculty of Medicine, Institute of Oncology, Istanbul University, Istanbul, Turkey
3 Department of Oncologic Cytology, Institute of Oncology, Istanbul University, Istanbul, Turkey
4 Department of Oral and Maxillofacial Radiology, Faculty of Dentistry, Okan University, Istanbul, Turkey

Date of Acceptance05-Mar-2019
Date of Web Publication14-Aug-2019

Correspondence Address:
Dr. H Cakir Karabas
Istanbul University, Faculty of Dentistry, Department of Oral and Maxillofacial Radiology 34093, Capa/Fatih, Istanbul
Turkey
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/njcp.njcp_675_18

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   Abstract 


Background: X-rays are potential mutagenic agents that can cause both the gene mutations and chromosomal aberrations. Aims: In this study, the micronucleus (MN) test and the comet assay methods are implemented in order to observe the damage that can occur in the cell nucleus and in the structure of DNA of the patients who underwent a panoramic examination. Methods and Materials: In our study, buccal mucosa swabs were obtained just before the radiography and 2 weeks after the radiography from 30 volunteer patients who had to take radiographs due to dental diagnosis. Changes in the nuclei of 1,000 cells of each swab sample had been counted under a light microscope and recorded. Besides, 100 cells of each other swab samples were analyzed by the comet assay. Comet assay parameters namely tail length and percentage of DNA in tail, which indicate the level of DNA damage were analyzed and compared in both groups. Statistical analysis was performed by using the Statistical Package for the Social Sciences (Version 21). Results: In our study, the results of percentage of DNA in tail and tail length before and after X-ray exposure were statistically significant (P < 0.001). Likewise, increase in the MN frequency observed in buccal mucosa cells after X-ray exposure was found significant (P < 0.001). Conclusions: As a result, panoramic radiographs taken during dental diagnosis and treatment cause cytotoxicity and DNA damage in oral mucosal cells. Panoramic radiographs should be applied only when necessary, using an accurate radiographic technique and radioprotection criteria.

Keywords: Cell injury, comet assay, DNA damage, micronucleus test, panoramic radiography


How to cite this article:
Karabas H C, Ozcan I, Sener L T, Guler S D, Albeniz I, Erdem T L. Evaluation of cell and DNA damage induced by panoramic radiography. Niger J Clin Pract 2019;22:1041-8

How to cite this URL:
Karabas H C, Ozcan I, Sener L T, Guler S D, Albeniz I, Erdem T L. Evaluation of cell and DNA damage induced by panoramic radiography. Niger J Clin Pract [serial online] 2019 [cited 2019 Sep 21];22:1041-8. Available from: http://www.njcponline.com/text.asp?2019/22/8/1041/264423




   Introduction Top


Physical, chemical, and biological environmental agents may interact with DNA and induce mutations. X-rays are one of those potent mutagenic agents which are able to induce both gene mutations and chromosomal aberrations. They affect either directly the DNA molecule or indirectly by forming reactive compounds that interact with DNA molecule.[1] The examples of DNA damage include DNA single- and double-strand breaks, deletion or insertion of base pairs and DNA–protein crosslinks.[2] Most DNA damages are fixed by DNA repair mechanisms. But if the damages are heavy or repair mechanisms are inadequate, DNA damages may lead upto cell death. Unrepaired DNA damage is believed to have an effective role in the etiology of various diseases, most cancers, infertility, and aging.[3] However, X-rays are significant tool for the diagnosis of diseases and are frequently used in dental applications, although they have mutagenic potential and are a type of ionizing radiation.

Several well-established methods have been used for evaluating the mutagenic potential of environmental agents. Today, the micronucleus (MN) test is considered a very reliable assay for detecting and evaluating DNA damage.[1] During cell division, some chromosome fragments or whole chromosomes are not included in the main daughter nuclei and they are called micronuclei. Therefore, the MN test is able to measure both chromosome breakage and chromosome loss and is a sensitive indicator of DNA damage.[4] MN test can be applied in the evaluation of the genotoxic effects of chemical agents on human peripheral blood lymphocytes, bone marrow, and buccal mucosa cells.[5] It is possible to increase the sensitivity of the MN test by taking degenerative nuclear alterations indicative of apoptosis (karyorrhexis, pyknosis, and condensed chromatin) and necrosis (karyorrhexis, karyolysis, pyknosis, and condensed chromatin) into consideration.[1]

Besides, comet assay, also named as the single-cell gel electrophoresis assay, is a visual technique that is widely used in the measurement of DNA damage.[6] DNA molecules with different molecular weights and electric charges under alkaline conditions act at different speed under the electric field. During electrophoresis, undamaged DNAs move without losing their integrity, whereas denatured DNA fragments migrate out of the cell nucleus of the damaged DNAs. DNA damage is visualized as an increased migration of genetic material named “comet tail” from the nucleus called “comet head.”[7] Comet assay is basically the quantification of the obtained images under a light microscope.

Panoramic radiography is a commonly used technique by dentists in the diagnosis of dental diseases. A single tomographic image of the facial structures including both the maxillary and mandibular dental arches and their supporting structures is produced.[8]

The aim of the study is to evaluate the genotoxic effects induced by X-ray radiation during panoramic dental radiography in buccal mucosa cells by using the micronuclei test and the comet assay.


   Subjects and Methods Top


Subjects

In this study, buccal mucosa swabs were obtained from 30 volunteer patients who applied to the Department of Oral and Maxillofacial Radiology Clinic, which is also functioning as an Oral Diagnosis Clinic. Those volunteers were selected among patients with complaints of impacted third molar or temporomandibular joint, but without any infection, abscess, etc., in their mouth. Informed written consent was obtained from the volunteer patients prior to their enrolment in the study, according to the ethical guidelines of the 2008 declaration of Helsinki. With the help of the questionnaire, volunteers were selected among patients who do not consume tobacco and alcohol, do not regularly use oral antiseptic solutions, do not have systemic diseases, and have not exposed to radiography (especially head and neck imaging) within the last year. Patients who comply with these criteria were involved in the study and requested to take panoramic radiography by our clinic. Swabs were taken from the same patients just before the imaging and 2 weeks later. Because of the patients who did not come back for the second part of the study (2 weeks after the radiography) and due to the sample sensitiveness required by laboratory stages, we worked with nearly 100 patients in 9 months. The panoramic dental radiographs were taken with a Kodak 8000 Digital Panoramic System, using settings of 66-74 kV, 5-8 mA, and 13.1-13.9 s.

The study was approved by the Clinical Research Ethics Committee of Medical School (protocol number: 2013/685).

Cell collection and slide preparation

Prior to cell sampling, patients washed out their mouths three times with tepid water to remove dead exfoliated cells. Buccal mucosa swabs were obtained with the help of a cytobrush just before the radiography and 2 weeks after the radiography. Cytological smears were prepared on clean slides and cells were fixed in 96% ethyl alcohol. The standard method of Papanicolaou Stain protocol was applied to the smears. Changes in the nuclei (pyknosis, karyolysis, karyorrhexis, and MN) of 1,000 cells of each swab sample had been counted under a light microscope and recorded.

Besides, epithelial cells of buccal mucosa were also taken from each patient for comet assay study. The brushes were afterward stirred in 30 ml solution, which was obtained by adding DMEM F12 (Dulbecco's Modified Eagle's medium) over 10% fetal bovine serum, 1% penicillin--streptomycin, and transported within 30 min to the laboratory for further processing. A well-defined protocol of another study was applied by the relevant laboratory.[9] Hundred cells of each swab samples were analyzed by comet assay method and have been evaluated by a computer program named “comet score.” Tail length and percentage of DNA in tail, comet parameters which indicate the level of DNA damage, were analyzed and compared in both groups.

Statistical analysis

Basic statistical parameters were used including mean, standard deviation, median, minimum, maximum, percentile, and score. Wilcoxon test was applied for the comparison of variables before and after the procedure. When comparing the variables with regard the two groups, independent samples t-test was used for the variables compatible with the normal distribution, and Mann-Whitney U test for the variables those are not compatible with the normal distribution. Statistically significant level was considered as P < 0.05.


   Results Top


The age of the included patients was statistically analyzed and the maximum was 46, the minimum was 20, and mean was calculated as 23. Nine women (30%) and 21 men (70%) were included in the study and the result revealed that there was no statistically significant difference between sex and DNA damage.

The difference between the medians of the variables with regard to percentage of DNA in tail before and after the panoramic radiography was found statistically significant (P < 0.001) [Table 1].
Table 1: The statistical evaluation of % DNA in tail before and after exposure

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The difference between the medians of the variables with regard to tail length before and after the panoramic radiography was found statistically significant (P < 0.001) [Table 2].
Table 2: The statistical evaluation of tail length before and after exposure

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Below, two images of buccal swab cells are given: The cells obtained before X-ray exposure reveal normal, undamaged DNA structure [Figure 1]a, whereas the comet heads and comet tails are easily recognizable after X-ray exposure [Figure 1]b.
Figure 1: (a) All cells with undamaged DNA structure before X-ray exposure (on the left). (b) The cells with damaged DNA structure (comet head and comet tail) after X-ray exposure (on the right)

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The difference between the medians of the variables with regard to MN before and after the panoramic radiography was found statistically significant (P < 0.001). Under the light microscope, MN image can be seen in the cytoplasm of the cell. [Figure 2]. Regarding other changes in the cell nucleus related to cytotoxicity, statistically significant increases (P < 0.001) were observed concerning karyorrhexis [Figure 3] and karyolysis [Figure 4]; however, the increase in pyknosis [Figure 5] was not statistically significant (P = 0.765) [Table 3].
Figure 2: Micronucleus in the cytoplasm of the cell

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Figure 3: Karyorrhexis

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Figure 4: Karyolysis

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Figure 5: Karyolysis on the left side and pyknosis on the right side

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Table 3: The statistical evaluation of changes in the cell nucleus before and after exposure

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   Discussion Top


X-rays with known mutagenic and carcinogenic effects can induce cytotoxicity and can cause DNA damage.[10],[11],[12] Because of the genetic material it contains, the nucleus is more sensitive to radiation than the cytoplasmic structure of the cell.[13],[14] When ionizing radiation interacts with biological molecules, it forms reactive ions, causing single- or double-strand breaks, base damage or loss, and DNA forming interstrand crosslinks or DNA-protein crosslinks.[11],[15]

Relevant genotoxic effects can cause mutations by making changes in the number and structure of chromosomes.[13] As a result of the mutation, if DNA repair genes and/or cell proliferation and differentiation control genes lose their functionality, the risk of cancer development increases.[1],[13],[16],[17],[18]

It should be noted that there is not any safe level for radiation exposure, in fact, its accumulation over time can cause biological effects, frequent and low exposure may increase the frequency of chromosomal aberrations, and low-dose medical exposure may cause genotoxic effects on both patients and workers.[19],[20],[21],[22],[23],[24],[25] A sensitive analysis and a specific approach are needed to detect the effects of low-dose diagnostic radiographic exposure.[10]

The MN test, as a highly reliable technique for the evaluation of chromosomal damage, is in the standard genotoxicity test system and can be both applied in vivo and in vitro on all cell types undergoing mitotic division.[1],[26],[27] In our study, it is applied in vivo on the buccal cell.

The differences in radiation dose, the frequency of exposure, sampling from different anatomic sites, evaluated cell types, different preparation procedures, number of cells included, criteria for MN scoring, characteristics of populations, and including different ethnic groups may affect the MN test results.[13],[28],[29],[30]

The studies in the literature on patients who received radiotherapy,[31],[32] occupationally exposed (in low doses but frequently) workers [33],[34], and patients who received single dental radiography [10],[11] reveal that MN formation and cell death increase in parallel to increase in radiation dose. For example, in one study,[35] 68 micronuclei were detected in 1,000 cells after 2,000 cGy exposure, whereas in another study,[36] 16 micronuclei were counted in 1,000 cells after 1,000 cGy exposure.

It is suggested to count at least 1,000 cells in the MN study, but to increase the number up to 2,000-3000 cells if significant increase cannot be observed within the first 1,000 cells.[28],[36],[37] Furthermore, the sensitivity of the MN test can be increased by including degenerative nuclear alterations indicative of apoptosis (karyorrhexis, pyknosis, and condensed chromatin) and necrosis (karyorrhexis, karyolysis, pyknosis, and condensed chromatin) into the study.[1],[36],[38],[39] Thus, in our study, 1,000 cells were counted and it was enough, and analysis of karyorrhexis, pyknosis, and karyolysis were also included to increase MN sensitivity.

Since the MN test is a visual technique, the evaluation may vary from person to person.[28] Thus, all the samples in our study were evaluated by a single person.

In the literature, there are some studies which reveal a significant relationship between the MN test and sex,[26],[27] whereas some others cannot prove that relationship.[1],[10] In our study, an important relationship was not found between age and the MN test results.

Smoking may increase DNA damage. The number of cigarettes per day and types of smoking like chewing may affect the MN increase.[1],[28] But, it should also be kept in mind that there are some studies without evidence for a relationship between smoking and the MN increase.[7] The same results (positive and no relation) are also valid for alcohol consumption and MN increase.[40],[41],[42] For these reasons, tobacco and alcohol consumers are excluded from our study.

There are some studies that reveal the relation between the use of regular oral antiseptic solution and the frequency of MN incidence, whereas there are some studies that cannot prove that relation.[1] For this reason, patients regularly using oral antiseptic solutions were not included in our study.

Regarding the studies on MN frequency changes before and after X-ray exposure, despite the studies lacking to show a relation between MN frequency increase and exposure,[10],[15],[19] most studies manifest that X-ray exposure, even in low doses, may induce DNA damage.[1],[27],[31],[43] This study also reveals that there is a statistically significant difference (P < 0.001) in MN frequency increase before and after the exposure. Besides, statistically significant increases were observed in this study concerning karyorrhexis and karyolysis (P < 0.001); however, the increase in pyknosis was not statistically significant (P = 0.765).

Pyknosis is a cellular alteration which is an indicator of both necrosis and apoptosis. Therefore, the result in this study suggests that there are multiple pathways leading the cell to apoptosis, and preferably, pyknosis is developed in the apoptotic process.[1]

Epithelial cells show a high level of proliferation and account for more than 90% of all human cancers. MN test application on epithelial cells is used as a sensitive tool for following-up genetic damage in the human population since epithelial cells can easily be collected from various anatomic sites and are the first point of contact for interaction with chemical agents.[4],[10] Besides, it is also known that cell types with the ability to repair DNA damage efficiently cause lower levels of permanent damage to the cell.[44],[45] When compared with peripheral blood lymphocytes, buccal cells with their relatively limited DNA repair capacities have been reported to more accurately reflect genomic instability in epithelial tissues.[30],[44] These cells are exposed to direct radiation in the panoramic radiographic examination and are the main target for radiation-induced damage. In addition, the related cells can easily be obtained from the mouth with a brush.[10],[13] For these reasons, buccal cells were used in our study.

Chromosomal damage leading to MN 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.[1],[10],[11],[44] Therefore, in this study, the genotoxic effects of X-ray exposure during panoramic dental radiography were evaluated immediately before and after 2 weeks after the exposure.

Interaction of ionizing radiation with cell DNA leads to various primary lesions.[43],[46] In this context, comet analysis is a widely used tool for detecting DNA damage.

Alkaline comet assay allows detection of single- and double-strand breaks, incomplete excision repair sites, and crosslinking, whereas comet made at neutral pH basically detects double-strand breaks and cross-linking.[47]

Studies comparing comet analysis with MN test indicate that MN test is less sensitive in assessing the DNA damage potential and that sensitivity difference is due to the differences in the outputs of the related tests. Exposure to X-rays, especially at low doses, may cause DNA single-strand breaks, but not MN formation. For this reason, at low doses, comet assay is also required in addition to MN test.[43] In this context, our study included the comet analysis in addition to the MN test, as our study generally examined the effects of low-dose panoramic radiography on cell DNA and nuclei.

The overall stages in the comet assay are well defined and many laboratories have developed their own methods for lysis electrophoresis or DNA staining. Different evaluation parameters have been used in different studies.[48],[49] Although Singh [50] used tail length, Olive et al.[51] used the tail moment. Colins et al.[52] defined a visual counting method and graded comets from 0-4. However, in studies involving multiple electrophoreses in order to reduce the variability in the results, it is recommended to use the “percentage of DNA in the tail.”[47] The percentage of DNA in the tail is directly proportional to the DNA strand break.[53] Disadvantages of the comet assay include the need to assess many cells in order to achieve reliable results, technical variability, and interpretation differences.[6] In our study, 100 cells were counted from each sample and the entire study was conducted by one evaluater to avoid possible differences in interpretation and technical differences. The percentage of DNA in the tail and tail length were evaluated together as an evaluation parameter. No sex-related difference was observed with regard to the percentage of DNA in the tail and tail length. However, the percentage of DNA in the tail (P < 0.001) and tail length (P < 0.001) of the cells before and after panoramic radiography were statistically significant.

Although comet assay is able to detect short-term repairable DNA damage, MN test allows detecting the damage at the chromosomal level associated with health risks leading to more severe genomic instability.[54],[55],[56] The newly formed MN either will be deported from the cell, thus leaving the cell deficient in terms of genetic material, or will lead the cell to apoptosis. Therefore, contrary to the primary damage detected with comet assay, a MN presence in the cell indicates an unrepairable genomic instability. If primary DNA damages are not repaired, genomic damage may turn into mutations and aberrations in the chromosomal structure. Such aberrations will also result in MN formation at later stages of cell division.[56]


   Conclusions Top


To sum up, panoramic radiographs taken during dental diagnosis and treatment causes cytotoxicity and DNA damage in oral mucosal cells. Although simple DNA damages can be fixed by repair mechanisms, in case of a problem in repair mechanisms, repair becomes impossible.

Monitoring the development process from DNA damage to cancer formation is crucial. Our study reveals the DNA damage, but does not provide any information with regard to the development process leading to cancer formation. Therefore, an additional study that will be done 6 months and 1 year after the panoramic radiography will further complement our study in better understanding the development path.

It is generally accepted that ionizing radiation has an inducing role in cancer formation. Although this study supports this view, in the presence of various environmental factors, any cancer type is not solely dependent on ionizing radiation. Nonetheless, panoramic radiographs should be applied only when necessary using an accurate radiographic technique and radioprotection criteria. And, instead of panoramic radiography, periapical radiography should be preferred where periapical radiography is sufficient (in single-tooth treatments or dental treatments involving a section of the jaw).

Financial support and sponsorship

This study was financially supported by the Istanbul University Scientific Research Projects, Turkey.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]



 

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