Evaluation of Genotoxic Effects of Panoramic Dental Radiography on Cells of Oral Mucosa by Micronucleus Assay and Evaluation of Time Period Required by Cells of Oral Mucosa to Recover from the Genotoxic Effects

INTRODUCTION

On November 8, 1895, Wilhelm Conrad Roentgen discovered a mysterious type of rays which he later called as “X-rays.” This remarkable scientific achievement has had an effect on medicine and science that has been matched by only few other advances.¹

But, what was not realized was that these X-rays were potentially life-threatening in their carcinogenic potential. Thus, the principle of ALARA, an abbreviation for as low as reasonably achievable, was laid down.

X-rays being ionizing radiation are well known as mutagens and carcinogens in the human population. Radiation genotoxic effects following low-dose medical exposure have been detected in both patients and exposed workers.² Genetic alterations, such as chromosomal aberrations and formation of micronuclei in cell cytoplasm, are the early biological effects of carcinogenesis.²

Since 1937, micronuclei have been regarded as indicators for genotoxic exposure of target tissues.³

These micronuclei, which are cytoplasmic fragments of DNA, have been reported as markers for high cancer risk as they arise in response to carcinogens. They can be detected in exfoliated cells and used as an indicator of recent DNA injury within the oral mucosa.⁴ Hence, the quantitative detection of micronuclei is widely used for the analysis of cytogenetic damage.⁵

To detect micronuclei, human peripheral blood lymphocytes have been most frequently used cells for cytogenetic monitoring. However, this appears to be an inappropriate cell system for monitoring the genotoxic effects because radiation-induced damage is not seen significantly in them since they are not the primary target for radiation in the oral cavity.²

The buccal epithelium cells provide an excellent alternative source of tissue for human monitoring to occupational and environmental toxic exposures. This is because tissue is under direct radiation exposure and is easy attainable.

The key advantage of the oral epithelial micronucleus assay is the relative ease of obtaining the tissue, the limited costs, and the lesser time required. Besides, the precision of the results obtained from scoring larger numbers of cells is very accurate.³

In dentistry, digital panoramic radiography is an integral part of dental radiology. However, despite the fact of the genotoxic and mutagenic effects of radiation during panoramic radiography being well known, it is encouraging to know that the cells, during the post-exposure period, do tend to recover from the radiation-induced effects to normalcy.

This brings in to mind the question, as to how much is the time period required post-exposure of one panoramic radiograph and for the cells to recover before another panoramic radiograph can be advised.

With these points in mind, this study has been undertaken to evaluate the genotoxic effects of X-rays on epithelial cells during panoramic dental radiography and to evaluate the time required by the cells of the oral mucosa to recover from the genotoxic effects.

MATERIALS AND METHODS

In this clinically oriented histopathologic study, 100 patients of either sex reporting to the Department of Oral Medicine and Radiology of Maharishi Markandeshwar College of Dental Sciences and Research, Mullana, were selected. The patients were regular outpatients who visited the department, and orthopantomograph (OPG) was required to be done as a part of routine diagnosis. All the selected patients were informed and explained about the study, and a signed informed consent was obtained. A clinical examination was then carried out.

The selected 100 patients were divided into two age groups. In group I, there were 50 subjects of age ranging from 15 years to 25 years. This included 29 female subjects and 21 male subjects. In group II, there were 50 subjects of age ranging from 40 years to 50 years. This included 27 female subjects and 23 male subjects.

CLINICAL EXAMINATION

The patients who were included in the study were those who had not undergone any diagnostic radiograph in the past 6 months. They should not have undergone any radiotherapy. Patients should not be suffering from any kind of malignancy of any part of the body, and the oral cavity should be free of any pre-malignant condition or lesion. Furthermore, there should be no history of tobacco consumption in any form whether smoking or chewing along with no history of alcohol consumption. It was also ascertained that all the included patients were not working in radiation facilities of any kind so as to reduce false-positive findings.

The patients who were included in the study, based on the above-mentioned criteria, were verbally explained about the study, and their written consent was obtained.

The subjects were made to sit comfortably on the dental chair, equipped with artificial illumination. Their data such as name, age, sex, address, etc. were recorded in the proforma by a single observer. The complete case history and clinical findings of the subject were recorded. The clinical examination was carried out with sterile mouth mirror and probe under artificial illumination by the same observer.

PROCEDURE

PREPARATION OF SLIDE

Before taking the sample, the patient was asked to rinse his/her mouth. The scrapings were taken from the relevant sites with the help of a wooden spatula. The scrapings obtained were then transferred to clean slides.

STAINING PROCEDURE

• The slides were air-dried and wet fixed in 100% alcohol for 20 minutes.
• The slides were hydrated in 95% alcohol followed by 80% alcohol. This was then followed by hydration in 70% alcohol and finally by hydration in 50% alcohol. Each hydration period was of for 2 minutes.
• The slides were then rinsed in water for 5 minutes.
• After this, the slides were stained in Harris’s hematoxlin for 5 minutes.
• Then, the slides were rinsed in water for 5 minutes.
• Next, the slides were differentiated in 0.5% aqueous hydrochloric acid for approximately 5 seconds.
• The slides were rinsed in water for 5 minutes.
• The slides were rinsed in 95% alcohol for 2 minutes.
• The slides were stained in Orange Green 6 for 1 minute.
• The slides were rinsed in 95% alcohol for 2 minutes.
• The slides were stained in Eosin Azure 36 for 30 seconds.
• The slides were rinsed in 100% alcohol for 1 minute.
• The slides were immersed in xylene for 10 minutes.
• The slides were then mounted using Distyrene Plasticizer and Xylene (DPX) solution.

The patients were recalled on the 10th day for the second smear and on the 21st day for the third smear. The same staining procedure was then repeated on the 10th day and 21st day.

RESULTS

Patients were divided into two groups according to age. Group I included patients ranging from ages 15 years to 25 years, whereas group II included patients with ages ranging from 40 years to 50 years. The mean age of group I was 21.06, and the mean age of group II was 45.14.

In group I, there were 51.8% females and 47.7% males. In group II, there were 56% females and 44% males.

Table 1 depicts the total micronucleus frequency in groups I and II seen at the time of pre-exposure, on the 10th day post-exposure, and on the 21st day post-exposure. There was increased micronucleus count observed in both keratinized and non-keratinized mucosa on the 10th day post-exposure when compared with micronucleus cell count at the time of pre-exposure. However, it was observed that on the 21st day post-exposure, the micronucleus count reduced when compared with the 10th day post-exposure (Table 1).

The test results showed that there was a significant difference with p value of 0.002 between the micronuclei count of keratinized mucosa seen at the time of pre-exposure and on the 10th day post-exposure in group I. But no statistically significant difference was seen in the micronucleus frequency between micronucleus cell count at the time of pre-exposure and on the 21st day post-exposure in keratinized mucosa, denoting that the micronucleus frequency reduced on the 21st day post-exposure.

In group II, a statistically significant difference with p value of 0.001 was seen in the micronuclei count of keratinized mucosa at the time of pre-exposure and on the 10th day post-exposure. Besides, a statistically significant difference with p value of 0.004 was seen in the micronuclei count of keratinized mucosa at the time of pre-exposure and on the 21st day post-exposure. However, this difference was not highly statistically significant.

In non-keratinized mucosa, in group I, no statistically significant difference was seen in the micronuclei count at the time of pre-exposure and on the 10th day post-exposure. However, it was seen that the micronuclei frequency in group I returned to baseline value on the 21st day post-exposure.

In group II, no statistically significant differences were noted at the time of pre-exposure and on the 10th day post-exposure as well as on the 21st day post-exposure. However, the micronuclei frequency did reduce on the 21st day post-exposure when compared with the micronucleus cell count on the 10th day post-exposure.

The above results denote that ionizing radiation produces genotoxic effects on both keratinized and keratinized mucosa, but the effects are more marked in keratinized mucosa. The results also showed that the effects of ionizing radiation reduced 3 weeks after exposure.

Table 2 depicts the total frequency of micronuclei in the total sample seen at the time of pre-exposure, on the 10th day post-exposure, and on the 21st day post-exposure in both keratinized and non-keratinized mucosa. The test results showed a statistically significant difference between micronucleus cell count at the time of pre-exposure and on the 10th day post-exposure with p value of 0.020 in cells of non-keratinized mucosa (Table 2). A statistically significant difference was seen as well in cells of keratinized mucosa on comparing them with micronucleus cell count at the time of pre-exposure and on the 10th day post-exposure. These values implied that genotoxic effects do take place in oral epithelial cells exposed to panoramic radiation. However, this effect is more pronounced in cells of keratinized mucosa when compared with non-keratinized mucosa.

On comparison of the frequency of micronuclei at the time of pre-exposure and on the 21st day post-exposure in the non-keratinized mucosa, no statistically significant difference was noted. This implied that since the frequency of micronuclei reduced on the 21st day post-exposure and returned to the baseline value, the genotoxic effects taking place in the non-keratinized mucosa were reversed 21 days post-exposure, whereas on comparing the frequency of micronucleated cells of keratinized mucosa at the time of pre-exposure and on the 21st day post-exposure, it was seen that the frequency of micronuclei did reduce 21 days post-exposure. However, the decrease was not statistically significant. This shows that the genotoxic effects taking place in cells of keratinized mucosa were not completely reversed 21 days post-exposure to panoramic radiation.

Table 3 depicts the variation in the micronucleus count in group I and group II. To evaluate the association of age with the micronucleus count, both the groups were compared using the Wilcoxon signed-rank test and Mann–Whitney test. The test results depicted highly significant values implying that the micronucleus frequency increases with age (Table 3).

DISCUSSION

Genomic damage is probably the most important fundamental cause of developmental and degenerative diseases. Micronucleated cell indexes may reflect genomic instability, although the mechanisms are not exactly known.⁶ Overall, the detection of an elevated frequency of micronuclei in a given population indicates increased risk of cancer.⁷ Hence, in this study, the genotoxic effects of X-ray exposure during panoramic dental radiography were evaluated first at the time of pre-exposure, on the 10th day post-exposure, and on the 21st day post-exposure by evaluating number of micronuclei. Day 10 was chosen on the basis of the fast turnover in epithelial cell kinetics which ranges from 7 days to 16 days.^4,8 Day 21 was chosen so as to evaluate whether the ionizing effects of panoramic radiography in oral mucosal cells were reversible or not. Although the effective radiation dosage from an OPG is quite less (ranges from 4 to 30 μSv), still the literature reveals that there is no such term as “SAFE DOSAGE.” Henceforth, this study was conducted to evaluate the effect of radiation dosage of OPG on micronuclear cells of oral cavity.

Various studies indicate that smoking and alcohol consumption produce genotoxic effects causing induction of micronuclei formation.^8–10 Moreover, since micronuclei are markers of neoplastic progression, their frequency increases in pre-cancerous and cancerous lesions.¹¹ Therefore, the sample included in this study consisted only of subjects with healthy normal mucosa with no history of tobacco and alcohol consumption or presence of any cancerous or pre-cancerous lesions.

The method of staining used was PAP staining method as it is easy to perform and is a standardized method. Ayyad et al.¹² found that PAP stain is the preferred method in field studies for scoring and detecting micronuclei in cells of buccal mucosa, as micronuclei were seen easily in transparent cytoplasm.

For scoring of micronuclei, the criteria developed by Tolbert et al.¹³ were followed.

In this study, the mean frequency of micronuclei in the non-keratinized cells seen 21 days post-exposure was 1.90. On comparing this value with frequency of micronuclei at the time of pre-exposure, no statistically significant difference was seen. The number of micronuclei seen 10 days post-exposure was 207 and 21 days post-exposure was 190. There was reduction of micronuclei frequency post-exposure with the number of micronuclei returning to the baseline value at the time of pre-exposure which was 186.

These results show that the genotoxic effects caused by panoramic imaging in exposed cells of buccal mucosa were reversed 21 days later post-exposure. This can be attributed to the fact that the turnover rate of cells of the buccal mucosa is a mean of 14 days.¹⁴

In case of keratinized cells obtained from the gingiva from the total sample, it was seen that the mean of micronuclei before exposure to panoramic imaging was 1.61 and 10 days post-exposure was 2.14. This increase was statistically significant with a p value of 0.001. This increase in micronuclei frequency showed that genotoxic effects of panoramic radiation occurred in the gingiva as well.

This increase was also seen in studies carried out by Cerqueira et al.¹⁵ and Sheikh et al.¹⁶

In this study, the mean of the micronuclei frequency in the keratinized mucosa seen 21 days post-exposure was 1.93. Although there was a decrease in the micronucleus frequency on the 21st day post-exposure when compared with the 10th day post-exposure, the micronucleus count did not return the baseline value at the time of pre-exposure. On comparison with the baseline frequency of micronuclei, this increase still remained statistically significant with the p value of 0.004. This showed that the genotoxic effects on gingival epithelial cells were still evident 21 days post-exposure. This can be explained by the regional differences in the patterns of epithelial maturation which appear to be associated with different turnover rates when compared with the non-keratinized buccal epithelium which turns over faster than the keratinized gingival epithelium.¹⁴

In this study, it was seen that radiation exposure due to panoramic radiography was capable of inducing genotoxic effects in the cells of both the keratinized and the non-keratinized mucosa. Although it does not cause irreversible tissue damage, it was also seen that the effect is more profound on the cells of the keratinized mucosa. This is because the rate of cell proliferation and turnover is highest for cells in the thin non-keratinized regions than for the thicker keratinized regions, such as the palate and the gingival.¹⁴ This difference has important implications for healing and for the rate of recovery of the tissue from radiation-induced damage.

Another finding of this study was an age-related increase in the micronuclei count in the older age group when compared with the younger group. This study also confirms the increase of basal level of micronuclei with age. It is important to bear in mind that the genotoxic response to mutagenic agent exposure depends on individual genetic variability as well.¹⁷ There was no significant decrease in micronuclei in this age group 21 days post-exposure in older age group. This denotes that there is a decrease in the reversal capability of genotoxic effects in older age indicating impairment of DNA repair mechanism with age. Hence, this study confirms the role of age as a confounding factor on the baseline as well as post-exposure frequency of micronuclei.

However, it should be taken into consideration that each patient has a unique level of contact with genotoxic agents capable of producing changes in cells in the oral mucosa. The results reported by different studies vary because genotoxic effects depend on the type, amount, and radiation dose absorbed; on the type of cell affected; and on the individual’s capacity to withstand the action of genotoxic agents.^18,19

Biomonitoring studies of populations exposed to X-rays are quite difficult and rather specific because each population is exposed to different doses of radiation. This could explain why some studies find an increase in genetic damage in populations exposed to X-rays.²⁰

As seen according to this study, panoramic imaging does induce genotoxic effects on oral epithelial cells of keratinized and non-keratinized mucosa leading to formation of micronuclei. Therefore, panoramic dental radiography should only be requested when necessary because it cannot be considered as a risk-free procedure. Even though the risks associated are small, they should not be overlooked.

CONCLUSION

This study concludes that panoramic dental radiography might induce chromosomal damage, thus emphasizing weighing the benefits against the risks. This study also stresses to follow adequate radiation protection measures and avoiding retakes while taking these radiographs.

Furthermore, it can be concluded that a time period of around 21 days could be given in between two consecutive panoramic radiographs so as to allow the cells of oral mucosa to repair the incurred DNA damage caused due to the radiation. This study also confirms the usefulness of the micronucleus assay in biomonitoring studies.

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