INTRODUCTION

The diagnostic radiation associated with orthodontic care has public health significance due to the high and increasing prevalence of orthodontic treatment, especially in young people.1 This significance exists because ionizing radiation is able to cause single- and double-strand breaks and DNA-protein crosslinks.2 When normal functioning of DNA repair genes and/or cell proliferation and differentiation control genes is lost as a consequence of mutations, the risk of cancer development increases.3

The genetic damage caused by genotoxic agents, such as ionizing radiation, can be measured using biomonitoring tests, and the micronucleus (MN) test is a very reliable assay for evaluating mutagenicity. This test is based on the formation of micronuclei from particles of chromatin material that, as a result of chromosome breakage or spindle dysfunction, do not migrate to the poles during anaphase and are not incorporated into the telophase nuclei of the dividing cell and result in the formation of one or more small satellite nuclei in the cytoplasm of the daughter cells.4

The MN test utilizes a well-established protocol that is performed in human peripheral blood lymphocyte cultures. MN evaluations are made in buccal epithelial exfoliated cells (BEC) as well, and this test is considered to be the least invasive method available with which to measure DNA damage in humans.5 As a result of its ability to assess the activity of many chemical or physical carcinogenic and mutagenic agents in situ, the MN test in the BEC is the choice of many recent human biomonitoring studies, including those involving the following: alcohol,6 tobacco,7 oral cancer and other oral pathologies,8 and patients undergoing radiotherapy2 or chemotherapy.9

With regard to ionizing radiation in dentistry, the MN test in the BEC was utilized to evaluate the panoramic dental radiograph,3,10–16 the lateral digital radiograph,17 mandibular cone beam computed tomography (CBCT),18 and orthodontic radiographs.19,20 In general, these studies revealed only cytotoxic effects,10–12,14–19 and only one study3 showed mutagenic results after exposure to the dental X-ray. None of the studies pointed out the effects of CBCT utilized for orthodontic planning in the BEC of children. In this investigation, the frequencies of the micronucleated cells (mutagenicity) in the buccal mucosa of individuals exposed to diagnostic methods used in orthodontic planning were compared using CBCT or a radiographic protocol. To monitor cytotoxic effects, karyorrhexis, pyknosis, and karyolysis were also evaluated in this setting. Finally, a comparison between the alterations caused by these exams was realized.

MATERIALS AND METHODS

Data Analysis

All slides were analyzed by an experienced and blinded cytopathologist. The micronucleated cells (measure of DNA damage) were scored according to the criteria described by Sarto et al.21 For cytotoxicity, the following nuclear alterations were considered, as described by Tolbert et al.22: pyknosis, karyolysis, and karyorrhexis (Figure 1a–d). A total of 1000 cells were assessed per person in this study for the micronucleus frequency and other parameters of cytotoxicity. The results were calculated by assessing % of altered cells only. Results are expressed in percentages. Similar analyses were established in previous published studies.11,14,16,19,20

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RESULTS

According to the Kappa test, the concordance was adequate (Kappa value  =  0.752). Table 1 shows the frequency of MNC (mutagenicity) and other nuclear alterations (cytotoxicity) in children undergoing radiographs or the CBCT necessary for orthodontic treatment. Before X-ray exposure, the mean frequency of MNC was 0.025% for the CBCT group and 0.008% for the Radiographic Set group. No statistically significant differences (P > .05) were noted after ionizing radiation exposure, showing no mutagenic effect. However, a significant increase in other nuclear alterations was observed after these exams, specifically, karyorrhexis, pyknosis, and karyolysis, evidencing cytotoxicity (P ≤ .001 for the CBCT group and P ≤ .007 for the Radiographic Set group). This increase was greater in the CBCT group (P ≤ .044). These data are summarized in Table 1. None of the evaluated children were exposed to other known genotoxic agents.

Table 1.  Frequency (%) of Micronucleated Cells (MNC) and Other Nuclear Alterations (Karyorrhexis, Pyknosis, and Karyolysis) in Children Undergoing Radiographs or Cone Beam Computed Tomography (CBCT) and Differences Between “After” and “Prior to” Exams Periods (Values Are Means ± Standard Deviation [SD]; ^*, **, and *** Were Significant)

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DISCUSSION

Buccal epithelial cells represent a preferred target site for early genotoxic events induced by carcinogenic agents entering the body via inhalation and ingestion.5 Add to that the knowledge that oral malignant neoplasms are the sixth most common neoplasm in the world,23 and 90% of all oral human cancers originate from epithelial cells.24 These facts highlight the advantage of the MN assay, an in vivo exam that elucidates the effects of toxic agents directly on a target tissue, the buccal epithelium. The limited cost, ease of counting, person-time required, and precision obtained from scoring large numbers of cells15 improve the popularity of this noninvasive method.

Damages that lead to the formation of micronuclei take place in the basal layer of the epithelial tissue, where cells undergo mitosis. The rapid turnover of epithelial tissues brings the cells to the surface, where they exfoliate.25 In general, cells take 7–16 days to emerge to the surface and exfoliate.26 For this reason, exfoliated oral mucosa cells were collected immediately before ionizing radiation exposure and after 10 days, in accordance with similar studies.3,11,13 This period allowed time for the basal layer that was exposed to radiation to mature and be collected when exfoliated.

Human biomonitoring studies in buccal cells involve several confounding factors, such as age, lifestyle, oral hygiene (eg, mouth rinse utilization), dental health, and smoking and alcohol use.5 These factors were controlled in our study. The sample comprised only children between 8 and 15 years of age with suitable oral hygiene and dental health. Children are minimally affected by confounders such as cigarette smoking, drinking habits, occupational exposure, and lifestyle (mainly dietary factors), which are factors of great concern in adults.27 Moreover, each patient was considered to serve as his own control. Therefore, any effect of other genotoxic agents must have been present in the first cell count. Therefore, potential differences between the first and second counts can be attributed to radiation.12 Some studies have pointed toward a relationship between age and MN occurrence,13,27 whereas others have not.3,14 As a result of the homogeneity in casuistic, it was not possible to correlate the frequency of MNCs with age in this setting.

Mutagenicity can be effectively assessed by the MN assay.2,5 The MNC frequencies were not significantly different before and after X-ray exposure in our sample. These results contrast with those of other authors,2,3,28,29 who reported higher rates of chromosomal aberrations subsequent to X-ray exposure. However, despite the larger radiation dose in our investigation, many similar studies10–16 involving dental radiographs, PAN or LAT only, showed similar results (ie, no mutagenic characteristic was evidenced by the MN test). Similarly, recent studies with adult patients undergoing orthodontic radiographs19 and CBCT showed no mutagenic effects by the MN test.18 The large increase in MNC after the radiographs were taken was not statistically significant because the distribution of these cells was not homogeneous in the sample. This is evident by the high standard deviation in this group before and after the examination.

Differences in radiation dose, frequency of exposition, type of cells evaluated, and site of collected cells may influence the results of the MN test. Some authors investigated patients undergoing radiotherapy five to six times per week for 5–7 weeks,2,28 others observed effects of frequent occupational exposition to low doses of X-ray,29 and still others pointed out results of only one dental radiographic exposure,3,10–17,19 and the literature shows that MN, MNC,30 and cellular death12 increase with radiation dose. With regard to the different cells employed in the MN assay, radiotherapy is a potent clastogenic agent in circulating lymphocytes and BEC of head-and-neck cancer patients.2 However, lymphocytes are more sensitive than BEC when detecting chromosomal aberrations caused by anticancer drugs.9,26 Additionally, in the cytogenetic studies of dental radiograph effects, different sites were selected for collection of buccal epithelial cells: buccal cheek mucosa,10,11,13,15–17 lateral border of the tongue,12,16 and keratinized mucosa of the upper dental arch.3 One study16 showed that the lateral border of the tongue is a more sensitive site with regard to cytotoxic insult induced by ionizing radiation combined with continuous cigarette smoke exposure when compared with the cheek buccal mucosa. The unique research that revealed the genetic damage capacity of PAN was contained in the unique study3 that utilized keratinized mucosa of the upper dental arch. These facts emphasize the need for more comparisons between different buccal sites, and they help us to explain the divergence found in the studies of radiation biological effects. Based on our findings, we assume that there are no mutagenic effects related to radiographs or CBCT utilized for orthodontic planning in children.

Researchers10,22 have called attention to nuclear changes other than MN that characterize cellular death and may increase the sensitivity of tests to detect genotoxicity. Thus, cytotoxic effects were investigated through the frequencies of karyorrhexis, karyolysis, and pyknosis. Contrary to genetic damage, cellular death was induced by radiographs and CBCT, as indicated by the statistically significant differences (P ≤ .001 for the CBCT group and P ≤ .007 for the Radiographic Set group) between values before and after X-ray exposure, in agreement with the findings of other studies.10,11,14,19 These results reinforce the idea that X-rays are cytotoxic, and based on the knowledge that cytotoxicity interferes with micronucleus induction because some MNC are inevitably lost after a cytotoxic insult,11 the lack of mutagenic effects of X-ray can be accepted.

Nevertheless, repeated exposure to cytotoxic agents can result in chronic cell injury, compensatory cell proliferation, hyperplasia, and, ultimately, tumor development. These cytotoxic/nongenotoxic agents act by interfering with molecules intimately involved in cell growth and cell death. Increased cell proliferation appears to be a unifying feature of epigenetic carcinogens. Proliferation may increase the risk of mutation within target cells and may also be important in selective clonal expansion of initiated cells.31

Comparisons show that the CBCT group presented a larger increase in cell death than the Radiographic Set group. This might have happened once the CBCT group received a greater radiation dose due to the protocols used in orthodontic treatment planning for this research, which were in accordance with the recent radiation doses described in the literature. The total radiation doses in the Radiographic Set group varies between 195 and 205 µSv, and in the CBCT group (FOV22 + FOV13) they varied between 287 and 304 µSv.32–34 The fact that cytotoxicity is closely related to the amount of radiation received was shown in patients exposed to a repetition of a PAN (14–24 µSv).12

CONCLUSIONS

• CBCT or radiographs that are requested for orthodontic planning can induce cytotoxic effects in oral mucosa cells.

• The CBCT group experienced more cell death, and that might have happened once this group received a greater radiation dose due to the protocols used in orthodontic planning for this study.

• Considering that cellular death and nongenotoxic mechanisms of carcinogenesis are closely related, CBCT or radiographs should be used when essential to treatment planning following the ALARA (As Low As Reasonably Achievable) principle, because cellular death was induced.