INTRODUCTION:

Radiations are an important part of modern medicine, which is absolutely necessary. X rays, also known as radiographs are principal diagnostic methods in all fields of medicine as well as in dentistry. Along with a thorough clinical oral examination they provide a clear picture of the diseased site. X rays are valuable diagnostic, prognostic and treatment planning tool in dental practice. Apart from this, X-rays can help in identifying diseases and developmental problems before those become serious issues¹. X rays basically are high form of ionizing electromagnetic radiation that can cause potential damage to exposed cells. The cells when exposed to X rays are found to undergo mutational as well as chromosomal changes¹. These changes can cause dysplasia of the cells resulting in cancer^2,3. Hence, though X-rays are widely used in dental practice, considerable concern prevails with regard to the potential harmful effects associated with radiation exposure as there is no safe margin of dosage.

Genomic damage is one of the most important fundamental causes of developmental and degenerative disease⁴. It occurs due to exposure to genotoxins, medical procedure, lifestyle factors and genetic causes⁵. So in the present scenario, it is important to study the genotoxicity of eukaryotic cells for which micronucleus (MN) assay provides the base. Micronuclei are anomaly structures often found in eukaryotic cells and are indicative of genotoxicity. When chromosomal fragments or the whole chromosome lags behind in the anaphase stage of cell division due to any physical or chemical damage, a microneucleus is formed⁶. The increase in number of micronuclei has been detected in various cancers of oral cavity, oesophagus and bladder which had made these microstructures an important diagnostic biomarker for dysplasia and metaplasia^7,8,9. Moreover identification if micronuclei are relatively easy and cost effective. Therefore, micronuclei scoring can be used as a biomarker to identify different pre-neoplastic conditions much earlier than the manifestations of clinical features¹⁰.

Numerous studies have evaluated the effects of dental x rays on the buccal epithelial cells using micronuclei assay in pediatric subjects^2,11,12. Minimal data is available on adult population^13,14,15. To our knowledge, literature search revealed minimal data on the effect oral x rays on the buccal epithelial cells on basis of micronuclei counts in Middle Eastern region. In lieu with the above the present study was undertaken to explore the genotoxicity of cells obtained from the buccal mucosa in patients who were exposed to dental X-rays using micronuclei analysis.

MATERIAL AND METHODS:

Study Design:

The study was initiated after approval from the Research Ethics Committee, Ajman University, UAE (F-H-18-05-03). Patients reporting to the outpatient department at the College of Dentistry, Ajman University requiring radiological investigation were selected for the study. The study objective and procedure were explained thoroughly to the patients and sample was collected after obtaining a written informed consent with signature from patients for this study. Healthy patients between the ages of 6 to 75 years and with no previous exposure to radiation of the head and neck region were included in the study. Patients exposed to radiographs in the past six months prior to sample collection and any visible changes in buccal mucosa would lead to exclusion from the study.

All subjects underwent a routine oral clinical examination and subjects with any visible or symptomatic change in the buccal mucosa were excluded. Based on the inclusion and exclusion criteria a total of 116 subjects, were recruited, Based on the smoking status, the included subjects were nonsmokers.

Sample collection:

Prior to X-ray exposures, the subjects were asked to rinse their mouth thoroughly with normal drinking water to remove any debris or smear layer. Exfoliated buccal mucosal cells for the MN analysis was obtained by scraping the buccal mucosa (both right and left side) with a disposable tongue blades. The sample was collected at 4 time points: Before exposure, immediately after exposure, 8 days after exposure and 14 days after exposure. The collected sample was transferred to glass slides and were allowed to dry. Then the cells were stained with Papanicolaou (PAP) staining procedure and placed in a tray to dry for 15 minutes. The efficacy of the staining were checked at 100X and 400X magnification.

Degenerative nuclear alterations study:

The nuclear alterations were studied under transmitted light microscopy. The nuclei and micronuclei were magenta in color, whereas the cytoplasm appeared pale blue/green. The total smears obtained were stained under PAP stain and the study of presence of degenerative nuclear alterations (Pyknotic, Karyorhexis and Karyolysis) were studied under microscope (Leica corporation) at 400x magnification. The frequency of cells was counted in 1000 cells per patient's sample.

Statistics:

The Shapiro-Wilk was used to determine the sample distribution. Data was not normally distributed. Mean of the total number of cells at each stage was determined. The Friedman’s two way Analysis of variance was used to compare the occurrences of cell changes before exposure, after exposure, 8 day after exposure and 14 days after radiation exposure. The level of significance was set at 5%. The inter-rater reliability (ICC Coefficient) of the histological examination was established by having another observer examine all the prepared slides. The data was analyzed using IBM Statistics software, v. 26.0 (IBM Corporation, Armonk, NY; formerly SPSS Inc., Chicago, IL) was used.

RESULTS AND DISCUSSION:

The total number of samples used in this study are 116 including both females and males. 35 of the patients underwent radiographic exposure for cone beam computed tomography (CBCT) images while the remaining 81 were exposed for orthopantomogram (OPG) images. The mean age of the sample was 31.8 + 13.28 years. 69.8 % of patients were selected for OPG while the rest were done with CBCT.

The mean number of nuclear alterations were significantly higher immediately after exposure and 14 days post exposure to radiation in both OPG as well as CBCT. The number of of multinucleated cells from before exposure, immediately after exposure to 14 days post exposure in the patients who underwent panoramic radiographic exposure was 0.74 + 1.45, 7.5 + 4.69, 15.79 + 5.67 while that of CBCT exposure was 0.89 + 1.88, 6.95 + 4.66 and 15.37 + 5.87 respectively (Table 1).

There was no statistically significant differences among cell turnover between both the radiographic exposures (p=0.458). Pyknotic, karyorrhectic and karyolytic cells were identified (Figure 1).

Carcinogenesis can be indicated by MN assay at early stage. Ionizing radiation is very important part in both medical and dental practices for diagnosis of diseases instead of its mutagenic and carcinogenic properties^16,17. In this study, buccal mucosa cells were used as they are relatively easy to collect. Also, in most of human cancers, the genotoxicity occurs either by inhalation or ingestion of genotoxicants. The study of genetic defects in humans has shown to be displayed better through buccal mucosal cell study^18,19. The former well-established criteria for identifying MN lacks cell inclusion criteria. The inclusion of cells for study of MN frequency was later developed. Scoring of cells protocol developed later which is most commonly used and implemented in this study too^6,20,21,22. For the evaluation of micronucleus, several staining methods are available. Mostly DNA-specific stains such as Feulgen stain is preferred for staining MN for its DNA specificity nature. But this is a lengthy, sensitive process and may lead to under scoring of MN2^23,24. Hence, in the present study, Giemsa stain was used for assessing the micronuclei.

In a study, it was found that the frequency of karyorrhectic, pyknotic and karyolytic cells were significantly increased after 2-dimensional planar radiographic examination^25,26,27,28. However, in low dose group of the studied samples in another study, only the frequency of karyolytic cells were significantly increased after 2-plananr radiographic examinations²⁹. The significant differences between the frequency of pyknotic and karyolytic cells before and after the dental X-ray examinations were also studied in large dose groups indicating that dental X-ray examination promote cytotoxicity of oral mucosa cells. In the present study it was found that in large data groups, there was too no statistical significant increase in micronuclei numbers. However, the micronucleus assay provides the base to prevent and treat cancer at an early stage. This can be an important biomarker for screening and identifying of patients with potentially malignant lesions. It is important to study genotoxicity and to prevent them in perspective ways^30,31,32.

CONCLUSION:

From the results of the present study it was concluded that as there is no difference in micronuclei number before and after panoramic radiographs, hence dental X-rays should be prescribed only when absolutely necessary. X rays may be a cause of cytotoxicity but don’t have any mutagenic effects in the oral mucosal cells. Furthermore, greater emphasis should be given to detect the genotoxic human activity, for the prevention of serious diseases and detection of high-risk patients.

ACKNOWLEDGEMENT:

This work was done from the internal research grant of Ajman University (REF: IRG-2018-A-DEN-01). We are grateful to DGSR, Ajman University for supporting the study. We are thankful to the Dean, College of Dentistry, Ajman University for providing us facility for the research work. We are also thankful to Dr Rawan Oueis and Dr Sangeetha (University of Sharjah), oral pathologist for their support in this research.

CONFLICT OF INTEREST:

Authors declare no conflict of interest.

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Received on 25.08.2020 Modified on 29.01.2021

Research J. Pharm. and Tech 2021; 14(11):5845-5848.

DOI: 10.52711/0974-360X.2021.01017

Before Exposure			Immediately after exposure		14 days after exposure
Cell types	OPG	CBCT	OPG	CBCT	OPG	CBCT
Pyknotic cells	0.55 ± 0.86	0.6 ± 1.05	5.42 ± 2.84	5.21 ± 2.88	11.87 ± 3.37	12.04 ± 3.36
Karyolytic cells	0.11 ± 0.32	0.16 ± 0.45	1.1 ± 0.94	0.9 ± 0.94	2.19 ± 1.14	1.88 ± 1.33
Karyorrhectic cells	0.08 ± 0.27	0.13 ± 0.38	0.98 ± 0.91	0.84 ± 0.84	1.73 ± 1.16	1.45 ± 1.18