Abstract
Objectives
The aim of the present study was to evaluate DNA damage (micronucleus) and cellular death (pyknosis, karyolysis and karyorrhexis) in exfoliated buccal mucosa cells from individuals following radiography.
Methods
Lateral and frontal cephalometric X-ray and panoramic dental X-rays were taken of a total of 18 healthy patients (6 male and 12 female) referred for orthodontic therapy. Exfoliated oral mucosa cells were collected immediately before X-ray exposure and after 10 days.
Results
The results revealed no statistically significant difference (P > 0.05) in the frequency micronucleated oral mucosa cells after X-ray exposure. However, X-ray was able to increase other nuclear alterations closely related to cytotoxicity, such as karyorrhexis, pyknosis and karyolysis.
Conclusions
Data indicated that exposure to certain radiography may not be a factor in inducing chromosomal damage, but it does promote cytotoxicity.
Keywords: buccal mucosa cells, radiographic documentation, micronucleus test
Introduction
Radiography is one of the most valuable diagnostic tools used in comprehensive dental care. Orthodontic radiographs carried out in radiological clinics are essential for diagnosis, planning and control of orthodontic treatment.1 For example, a lateral cephalometric X-ray must be obtained for all patients, before the start of treatment, when the information obtained from this film is expected to benefit or enhance the formulation of the patient's diagnosis and treatment plan.2 In the same way, a panoramic X-ray must be obtained for all patients, before the start of treatment, unless there are other suitable radiographs, such as full-mouth periapical radiography, that will enable the orthodontic practitioner to formulate an appropriate diagnosis and treatment plan.2 Frontal cephalometric analysis is also a valuable complementary examination in establishing the correct diagnosis and orthodontic planning.3 At present, lateral and frontal cephalograms are considered mandatory in orthodontic therapy.
Although it is generally accepted that there is no safe level of radiation exposure, the possible risk associated with exposure to X-rays must be compared against the benefits of clinical interpretation.4 It is well known that ionizing radiation damages DNA, including single and double strand breaks, and DNA protein cross-links.5 The application of validated biomarkers helps to delineate the continuum of events between exposure and resulting disease; identify smaller exposures to specific agents; enhance group risk monitoring and assessment; and reveal toxicological mechanisms by which an exposure and a disease are related.6 To date, a variety of assays have been proposed as potential biomarkers, including those that assess metaphase chromosomal aberrations, sister chromatid exchanges and host cell reactivation. However, these methods are typically laborious and time consuming or require highly trained technicians to accurately read and interpret slides. For these reasons, there was a great deal of enthusiasm regarding the application of the micronucleus test to uncultured exfoliated cells.7 A micronucleus arises from acentric fragments or whole chromosomes, which are not included in the main nuclei of the daughter cells. The formation of micronuclei can be induced by substances that cause chromosome breakage (clastogens) as well as agents that affect the spindle apparatus (aneugens).8 As a result, the present study was undertaken to investigate the frequency of micronucleated cells in oral mucosa from individuals following orthodontic radiography. To monitor cytotoxic effects, pyknosis, karyolysis and karyorrhexis were also evaluated. Such data will contribute to a better understanding of the outcomes on the cellular system following radiography.
Materials and methods
Participants
The participants of this study comprised 18 healthy volunteers (6 male and 12 female) with a mean age of 14.2 ± 1.4 years referred for orthodontic therapy at the Department of Orthodontics, Methodist University, Brazil. All the participants underwent lateral and frontal cephalometric X-rays and panoramic dental radiography prior to orthodontic therapy. A patient history was taken including gender, age, habits and exposure to genotoxic agents. All panoramic dental radiographs were requested by an orthodontist and were performed with Siemens Orthophos equipment (Erlangen, Germany), system 250–71 kV/15 mA/14 s/110 mGy/cm2. Informed consent was obtained from the participants or from their parents or guardians. All individuals were non-smokers.
The research was approved by the Institutional Ethics Committee of the Methodist University UMESP, Brazil.
Micronucleus test in oral mucosa cells
Damage that leads to the formation of micronuclei takes place in the basal layer of the epithelial tissue where cells undergo mitosis. Rapid turnover of epithelial tissues brings the cells to the surface where they exfoliate. As a result, the maximal rate of micronuclei formation in exfoliated cells is seen 1–3 weeks after exposure to the genotoxic agent.9,10 For this reason exfoliated oral mucosa cells were collected immediately before X-ray exposure and 10 days after. After rinsing the mouth with tap water, cells were obtained by scraping the right or left cheek mucosa with a moist wooden spatula. Cells were then transferred to a tube containing saline solution, centrifuged (800 rpm) for 5 min, fixed in 3:1 methanol/acetic acid and dropped onto pre-cleaned slides. Air-dried slides were then stained using the Feulgen/Fast-Green method and examined under a light microscope at ×400 magnification to determine the frequency of micronucleated cells as described elsewhere.8 2000 cells were scored from each patient on both sampling occasions (before and after X-ray exposure).
Data analysis
Micronuclei were scored according to the criteria described by Sarto et al11 as a parameter of DNA damage (mutagenicity). For cytotoxicity, the nuclear alterations pyknosis, karyolysis and karyorrhexis, were considered. Results were expressed as a percentage. This is the same analysis established in a previous study conducted by our research group.12
Statistical methods
The Wilcoxon test for dependent samples was used to compare the frequency of micronuclei and other cellular alterations among the before and after X-ray exposure samples using SigmaStat software, version 1.0 (Jadel Scientific, Rafael, CA). The level of statistical significance was 5%.
Results
The frequency of micronucleated cells in patients undergoing cephalometric and panoramic radiography can be seen in Table 1. Before X-ray exposure, the mean frequency of micronucleated cells was 0.02%. There was no significant statistical difference (P > 0.05) after X-ray exposure. However, an increase of other nuclear alterations after X-ray exposure was observed as shown by the frequency of karyorrhexis, pyknosis and karyolysis. The data are summarized in Table 1.
Table 1. Frequency of micronucleated cells (MNC) and other nuclear alterations (karyorrhexis, pyknosis and karyolysis) in orthodontic patients undergoing X-ray exposure.
| Groups | MNC (%) |
Other nuclear alterations* (%) |
||
| No. of individuals | Mean ± SD | No. of individuals | Mean ± SD | |
| Prior to X-ray exposure | 18 | 0.2 ± 0.1 | 18 | 8.5 ± 3.2 |
| After X-ray exposure | 18 | 0.2 ± 0.1 | 18 | 14.4 ± 4.8† |
*Karyorrhexis, pyknosis and karyolysis
†P < 0.05 when compared to individuals prior to X-ray exposure
To compare the data with accuracy, all patients included in this study were non-smokers. In addition, 12 of the participants used oral antiseptic solutions regularly. Daily alcohol consumption was not recorded in this study because of possible recall bias phenomenon.
Discussion
The aim of this study was to use the micronucleus test to assess chromosome damage and/or cellular death in individuals who had undergone radiography by means of cephalometric and panoramic radiographs. To the best of our knowledge this approach has not been addressed in the literature before.
Micronucleus assay in exfoliated buccal mucosa cells has been used systematically in genetic biomonitoring of populations exposed to several genotoxic chemicals, such as tobacco products, pesticides and alcohol.13–15 The key advantage of the micronucleus assay is the relative ease of scoring, the limited costs, time efficiency and the precision obtained from scoring larger numbers of cells.
Micronucleated cell indexes are thought to reflect genomic instability.16 Detection of an elevated frequency of micronuclei in a given population indicates an increased risk of cancer.17 It was surprising that micronucleus frequency was not significantly different before and after X-ray exposure in this trial; however, such findings are fully in line with other authors.18–20 Conversely, some articles have reported higher rates of cytogenetic damage induced by X-ray.21 Biomonitoring studies of populations exposed to X-rays can be difficult and rather specific owing to the different doses of radiation each population is exposed to. This could explain why some studies have found increased genetic damage in populations exposed to X-rays. Based on our results, we postulate the lack of clastogenic and/or aneugenic effects are related to dental panoramic radiography and lateral or frontal cephalometric X-ray exposure in healthy individuals.
To monitor cytotoxic effects the frequency of karyorrhexis, karyolysis and pyknosis was evaluated in this study. Despite the lack of cytogenetic damage our results demonstrated that panoramic and cephalometric radiographs induced cellular death as demonstrated by the statistically significant differences (P < 0.05) between values before X-ray exposure compared with after. Analogous results have been reported by others.18,20,21 Taken as a whole these results support the notion that X-ray is a cytotoxicant agent. It is important to stress that cytotoxicity does interfere with micronucleus induction, since some micronucleated cells are inevitably lost after cytotoxic insult, confirming, therefore, the lack of mutagenic effect induced by X-ray. Nevertheless, it has been postulated that repeated exposure to cytotoxicants can result in chronic cell injury, compensatory cell proliferation, hyperplasia and ultimately tumour development.22 In fact, a correlation between cell proliferation and induction of cancer is assumed.23 It is likely that proliferation increases the risk of mutations within target cells, and is important in selective clonal expansion of (exogenously or endogenously) initiated cells from pre-neoplastic foci and eventually tumours.22
In human cytogenetic studies some confounding factors need to be considered. Viruses, alterations in the immune system, failures in DNA repair system and inter-individual variations have already been associated with an increased frequency of chromosome aberration.24 Furthermore, an age-related increase of micronuclei has been postulated in participants of a similar age.24 The influence of tobacco smoke has also been considered as a relevant factor.11 Thus, all adults recruited to participate in this study were non-smokers. The mutagenic potential of alcohol is controversial and quite complicated to interpret using the micronucleus assay in exfoliated cells. For example, in two reports almost all participants consumed alcohol and tobacco and, therefore, the influence of the individual factors could not be elucidated.25 In the another study, no genotoxic effect of alcohol was found.26 In a study by Stich and Rosin27 the effects of alcohol consumption, cigarette smoking and a combination of the two were examined. A synergistic effect of alcohol and nicotine was observed, but the two drugs alone did not cause an elevation of micronuclei frequencies.
In conclusion, the results of the present study suggest that radiography is able to induce cytotoxicity but not mutagenic effects in oral mucosa cells; therefore, radiographs should be used only when necessary. Further studies are necessary to confirm these findings.
Acknowledgments
This work was supported by grants from FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo, Grant number: 07/01228-4). Daniel A Ribeiro is a recipient of the CNPq fellowship.
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