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 Table of Contents  
ORIGINAL ARTICLE
Year : 2021  |  Volume : 33  |  Issue : 1  |  Page : 16-21

Genotoxic and cytotoxic biomonitoring in patients exposed to panoramic dental radiography: Comparison between five different age groups


1 Department of Oral Medicine and Radiology, SRM Dental College, Chennai, Tamil Nadu, India
2 Department of Oral Pathology and Microbiology, SRM Dental College, Chennai, Tamil Nadu, India

Date of Submission28-Jun-2020
Date of Decision04-Feb-2021
Date of Acceptance05-Feb-2021
Date of Web Publication26-Mar-2021

Correspondence Address:
Dr. S Anbumeena
Department of Oral Medicine and Radiology, SRM Dental College, Ramapuram, Chennai - 600 089, Tamil Nadu
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jiaomr.jiaomr_124_20

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   Abstract 


Introduction: Radiography is an inseparable tool in dentistry. Radiation can induce DNA damage as well as cellular damage. Aims and Objectives: To estimate the genotoxicity and cytotoxicity after taking Orthopantomograph (OPG). To compare the result between five different age groups. Materials and Methods: Buccal mucosal smears were collected from the participants and stained using the PAP method and PAS method. It was evaluated for genotoxicity and cytotoxicity. Settings and Design: The study group consisted of 60 participants. They are divided into five different groups with 12 in every single group. Statistical Analysis Used: Wilcoxon Signed Ranks Test was used within groups. Kruskal–Wallis test was used for continuous data. Results: The mean of cytotoxicity after taking OPG was significantly increased (P < 0.05). Though there was an increase in micronuclei expression, no statistically significant difference was evidenced (P > 0.05). Conclusion: The result of our study reveals that OPG did not cause any chromosomal damage, but it is capable of inducing cytotoxicity in buccal mucosal cells in all age groups.

Keywords: Cytotoxicity, genotoxicity, micronucleus, OPG


How to cite this article:
Anbumeena S, Kannan A, Krithika C L, Vasanthi V. Genotoxic and cytotoxic biomonitoring in patients exposed to panoramic dental radiography: Comparison between five different age groups. J Indian Acad Oral Med Radiol 2021;33:16-21

How to cite this URL:
Anbumeena S, Kannan A, Krithika C L, Vasanthi V. Genotoxic and cytotoxic biomonitoring in patients exposed to panoramic dental radiography: Comparison between five different age groups. J Indian Acad Oral Med Radiol [serial online] 2021 [cited 2021 Apr 13];33:16-21. Available from: https://www.jiaomr.in/text.asp?2021/33/1/16/312191




   Introduction Top


Genotoxicity is described as the damaging impact on a cell's hereditary material (DNA, RNA) by altering its integrity.[1] X-rays can cause aberration in the chromosome and mutation in the gene.[2] Given the strong association between cancer formation and DNA damage, it will be beneficial to know the level of genetic and cellular damage resulting from X-rays.[3]

X-rays can cause cytotoxicity as well. It is essential to study cytotoxicity while studying genotoxicity because the incidence of the nuclear abnormalities indicating cytotoxicity was similar to or greater than that of genotoxicity in the oral cavity.[4]

A biomarker is a reliable tool for assessing the risk factor associated with various diseases.[5]

Many studies have monitored these effects using peripheral lymphocytes, they are inappropriate for monitoring effects owing to dental X-rays.[6] The buccal mucosa is under direct exposure to dental X-rays. Thus, buccal cell micronucleus assay is an excellent and most apt biomarker for assessing these effects because of dental X-rays.[7]

The detrimental effects of radiation vary with age.[8] About the current study, most reference studies on buccal cell micronucleus assay failed to demonstrate their influence on age. Hence, this study aimed to assess and compare the cytotoxic and genotoxic effects of radiation in buccal mucosal exfoliated cells owing to OPG in different age groups.


   Materials and Methods Top


Study design

The present study was performed in the department of Oral Medicine and Radiology, of a dental school in Tamil Nadu, India after getting approval from the Institutional Ethical Committee (SRMDC/IRB/2017/MDS/No.902) and written informed consent from the patients. The procedures followed were by the ethical standards of the Helsinki Declaration of 1975, as revised in 2000. The study group included 60 patients who required OPG for their dental treatment and they were not exposed solely for this study purpose. Participants were recruited for the study after satisfying the exclusion and inclusion criteria. They were divided into five groups based upon the age. We obtained a sample size of 60 with 12 in each group. The sample size was calculated by keeping the power as 99% and alpha error value as 1%.

The formula used for calculating the sample size is





Where,

: Standard Deviation in the first group

: Standard Deviation in the second group

: Mean difference between the samples

α: Significance level

1-β : Power

Inclusion criteria

Normal, healthy participants having good oral hygiene were included in the study and incorporated in Group 1: Age between 6 and 11 years (children), Group 2: Age between 12 and 18 years (Adolescence), Group 3: Age between 19 and 39 years (Young Adulthood), Group 4: Age between 40 and 60 years (Middle adulthood), and Group 5: Age above 60 years (Late adulthood).

Exclusion Criteria

Included participants who were smokers, with any systemic or genetic disease, carcinoma patients, acute dental infection, maxillofacial trauma, pregnancy, and any clinically visible oral lesions.

Collection of the cells

Participants were requested to rinse the oral cavity with normal water to eliminate any debris or food particles that will hinder during the analysis. With a moist wooden spatula, exfoliated cells were collected by gently scraping the buccal mucosa in a rolling motion and it was immediately smeared onto the center of the slide. Then the slide was coded. Smears were dried in air and then fixed with 80% ethanol. For every participant, two slides were prepared (one slide for PAP stain and another is stained with PAS Stain). Now, the participants were subjected to OPG. The panoramic dental radiographs were performed with X Mind Trium Aceton equipment system 65–79 kV/8 mA/12 s. After which the participants were recalled for the second collection of cells within 7–10 days. The participants were requested not to get exposed to any kind of medical radiation in this period.

Scoring principles

Cells were examined under 10x, 40x, 100x magnification using Olympus BX51 Research Microscope to evaluate cytotoxicity as well as genotoxicity. Tolbert's criteria[4] were applied for the scoring of the micronucleus. The count of micronucleus was taken into consideration for evaluating genotoxicity while the incidences of karyolysis, pyknosis, as well as karyorrhexis were evaluated for estimating the cytotoxicity. Both the PAP Stain and PAS Stain were observed using the same procedure for assessment of genotoxicity as well as cytotoxicity. The slides were evaluated by an experienced oral pathologist. The observer was blinded with the time of collection of samples and the demographic details. The values were plotted on the table and statistical analysis was performed to extract the results.

Statistical analysis

All statistical analysis was performed using SPSS, version 17. Wilcoxon Signed Ranks test was used within groups. Kruskal–Wallis test was used for continuous data. A two-sided P value < 0.05 was considered statistically significant.


   Results Top


In the PAP group, while comparing pre-exposure with post-exposure smears, there was an increase in the mean value of micronuclei from 9.73 to 9.85, pyknosis increased from 35.17 to 45.67, karyolysis increased from 11.17 to 22.00, and karyorrhexis increased from 19.00 to 32.33, respectively. Though there was an increase in the incidence of micronuclei it was not statistically significant with a P- value of 0.925, indicating the absence of significant genotoxicity. The P- value for pyknosis was 0.001, karyorrhexis was 0.000, and karyolysis was 0.000. These values were statistically significant indications of cytotoxicity [Table 1] [Figure 1].
Table 1: Comparison between pre exposure and post exposure values of PAP Smear group using Wilcoxon Signed Ranks test

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Figure 1: Graphical representation of comparison between pre exposure and post exposure values in PAP Smear

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To analyze the influence of age, each parameter like micronucleus, karyolysis, pyknosis, and karyorrhexis were compared between five different groups. The P- value for micronucleus was 0.427, pyknosis was 0.394, karyolysis was 0.133, and karyorrhexis was 0.489. These values were statistically not significant which showed that age does not influence genotoxic and cytotoxic changes following OPG [Table 2] [Figure 2].
Table 2: Kruskal Wallis test for comparison of the variable between groups in PAP Smear

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Figure 2: Graphical representation of the comparison of karyolysis, karyorrhexis, pyknosis and micronucleus among different age groups in PAP smear

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Similarly, In the PAS group, while comparing pre-exposure with post-exposure smears, there was an increase in the mean value of micronuclei from 5.42 to 6.60, pyknosis increased from 22.17 to 31.67, karyorrhexis increased from 16.50 to 25.50, and karyolysis increased from 6.50 to 19.00. Though there was an increase in the incidence of micronuclei it was not statistically significant with a P- value of 0.192, indicating the absence of significant genotoxicity. The P- value for pyknosis was 0.004, karyorrhexis was 0.000, and karyolysis was 0.000. These values were statistically significant suggestive of cytotoxicity [Table 3] [Figure 3].
Table 3: Comparison between pre exposure and post exposure values of PAS Smear group using Wilcoxon Signed Ranks test

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Figure 3: Graphical representation of comparison between pre exposure and post exposure values in PAS smear

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To analyze the influence of age, each parameter like micronucleus, karyolysis, pyknosis, and karyorrhexis were compared between five different groups. The P- value for micronucleus was 0.432, pyknosis was 0.505, karyolysis was 0.500, and karyorrhexis was 0.200. These values were statistically not significant which showed that age does not influence genotoxic and cytotoxic changes following OPG [Table 4] [Figure 4].
Table 4: Kruskal Wallis test for comparison of the variable between groups in PAS Smear

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Figure 4: Graphical representation of the comparison of karyolysis, karyorrhexis, pyknosis and micronucleus among different age groups in PAS smear

Click here to view



   Discussion Top


The oral cavity serves as an initial contact medium and imparts an initial defense against pathogens. 60% of oral epithelium is composed of non-keratinized stratified squamous cells which permit easy uptake of stain, in turn facilitating the exact evaluation of morphological changes of the nuclei. Hence in this study, OPG radiation related changes were observed in oral cavity epithelial cells.[5]

The traditional biomonitoring methods need skilled technicians, time consuming, and laborious.[9] The buccal cell micronucleus assay is simple, accurate, non invasive, painless, quick, affordable, and repeated sampling can be done.[10]

Holland et al. stated that after ionizing radiation exposure, expression of micronucleus in buccal mucosal cells takes a minimum of about 5–7 days to a maximum of 21 days.[11] Riberio et al. reported that the turnover time for the oral cavity epithelial cells is 7–16 days and the highest number of micronucleus was anticipated from 1 week to 3 weeks after a genetic insult. Taking these factors into consideration a time interval of about 7–10 days was given for the second collection.[12]

Pan Yang et al. stated that the radiation sensitivity of the various cells in the oral cavity like exfoliated cells of buccal mucosal, tongue, and gingiva had no statistically significant difference.[13] Sunitha et al. stated that after taking full mouth radiographs, the incidence of micronucleus and other nuclear changes were elevated in exfoliated cells of buccal mucosa when compared to gingival epithelial cells. The non-keratinized epithelium has a rapid turnover which makes them more prone to damage.[14] Considering these buccal mucosal cells were used for observing genetic and cytologic damage.

Mohan et al. concluded in their study that the genetic damage caused by Intra Oral Periapical Radiograph (IOPA) is higher compared to OPG. The cellular damage caused by OPG was higher compared to IOPA.[2] Naveena et al. study showed that bitewing radiograph results in three times rise in micronuclei level. Similarly, digital OPG results in two times rise in micronuclei level. Even though both the OPG and bitewing result in elevation of micronuclei they stressed the fact that an increase in an exposure time of radiation and a decrease in scattered radiation result in the formation of more micronuclei.[15] Sandhu et al. compared the genotoxicity caused during obtaining conventional and digital OPG radiographic examination and found out that higher genotoxicity was resulted because of conventional OPG.[16]

Gang Li et al. showed that there was no evidence of changes except for karyolytic cells when the radiation was less than 1 mGy.[17] Raj et al. concluded that micronucleus can be effectively used as a biomarker for evaluating radiosensitivity of the cell.[18]

The current study revealed an increase in the level of micronuclei after OPG exposure, but the value was not statistically significant which was similar to the result of Eman A El-Ashiry,[19] Popova,[20] Poonam Agarwal,[21] and Madhavan.[22] On contrary, few studies like Mohan,[2] Arora,[23] Sandhu,[16] Naveena Preethi,[15] Antonia[8] showed a statistically significant rise in the micronuclei mean value after exposure to OPG. Studies done by Angelieri,[24] Lorenzoni,[25] Ribeiro,[3] Haghgoo,[26] Ribeiro[12] showed no change in the level of micronuclei after radiation exposure.[14]

Antonio et al. said that the occurrence of micronuclei depends upon the dose, radiation type, and sensitivity of that tissue to radiation.[8] The upsurge in the incidence of micronuclei will not inevitably result in the appearance of a new premalignant lesion or cancer yet it indicates the genotoxic nature of that particular agent.[13]

With regards to cytotoxicity there exists a statistically significant change consistent with the study of Poonam Agarwal,[21] Haghgoo,[26] Mohan,[2] Antonia,[8] Angelieri,[24] Eman A El-Ashiry,[19] Lorenzoni,[25] Madhavan R.[22]

In this study, a comparative assessment of genotoxicity and cytotoxicity in OPG between the five different age groups was carried out. The results of our study show no difference between five age groups which was in the same way as the result of the study done by Gang li and[17] Haghgoo.[26] Arora et al. concluded in their study that only in gingival epithelial cells there exists a correlation of age with genotoxicity and cytotoxicity.[23]

Limitations and future prospects

In the present study, effect of OPG on buccal mucosal cells were only evaluated but the effect of other radiographs on epithelial cells from different sites were not evaluated which is beyond the perspective of this study.

This is one of the few studies reported in the literature that correlates the influence of age on genotoxicity and cytotoxicity induced by OPG. Moreover, to our knowledge, this is the first study to be carried out in a wider age range and a larger sample which will act as evidence in future results.


   Conclusion Top


The results of this study will aid in a better understanding of radiation induced changes on the buccal mucosal cells after OPG among different age groups. Buccal cell micronucleus assay can be used effectively as a biomarker to study genotoxic and cytotoxic damage in the oral cavity. Though there is the absence of genotoxicity, there is evidence of cytotoxicity. Dental X-rays must be taken only when they were extremely needed obeying the ALARA principle. Age does not influence genetic and cytotoxic damage caused by OPG. To generalize our results, further studies must be performed with a larger sample at the multicentric level.

Declaration of patient consent

The authors certify that they have obtained all appropriate patient consent forms. In the form, the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.

Acknowledgements

I am grateful to Dr. P.H. Raghuram, Dr. M.S. Anandhi and Dr.R. Kanmani for their ever willingness to help and for being a source of inspiration for successful completion of this study.

It is with great privilege and honour that I convey my sincere and heartful gratitude to Dr.R. Ramya, Dr.A. Ramesh Kumar and Dr. Swarnalatha under whose guidance, mentoring made it possible to complete my study.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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Shah SU. Importance of genotoxicity and S2A guidelines for genotoxicity testing for pharmaceuticals. IOSR J Pharm Biol Sci 2012;1:43-54.  Back to cited text no. 1
    
2.
Mohan N, Ravikumar PT, Madhumitha C. Genotoxic and cytotoxic effects following dental and panoramic radiography. Indian J Oral Sci 2016;7:92.  Back to cited text no. 2
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Ribeiro DA, De Oliveira G, De Castro GM, Angelieri F. Cytogenetic biomonitoring in patients exposed to dental X-rays: Comparison between adults and children. Dentomaxillofac Radiol 2008;37:404-7.  Back to cited text no. 3
    
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Tolbert PE, Shy CM, Allen JW. Micronuclei and other nuclear anomalies in buccal smears: Methods development. Mutat Res 1992;271:69-77.  Back to cited text no. 4
    
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Torres-Bugarín O, Zavala-Cerna MG, Nava A, Flores-García A, Ramos-Ibarra ML. Potential uses, limitations, and basic procedures of micronuclei and nuclear abnormalities in buccal cells. Dis Markers 2014;2014:956835.  Back to cited text no. 5
    
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Fenech M, Bolognesi C, Kirsch-Volders M, Bonassi S, Zeiger E, Knasmuller S, et al. Harmonisation of the micronucleus assay in human buccal cells--a Human Micronucleus project initiative commencing in 2007. Mutagenesis 2007;22:3-4.  Back to cited text no. 7
    
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Antonio EL, Nascimento AJ, Lima AA, Leonart MS, Fernandes Â. Genotoxicity and cytotoxicity of x-rays in children exposed to panoramic radiography. Rev Paul Pediatr 2017;35:296-301.  Back to cited text no. 8
    
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Holland N, Bolognesi C, Kirsch-Volders M, Bonassi S, Zeiger E, Knasmueller S, et al. The micronucleus assay in human buccal cells as a tool for biomonitoring DNA damage: The HUMN project perspective on current status and knowledge gaps. Mutat Res 2008;659:93-108.  Back to cited text no. 11
    
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Ribeiro DA. Cytogenetic biomonitoring in oral mucosa cells following dental X-ray. Dentomaxillofac Radiol 2012;41:181-4.  Back to cited text no. 12
    
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Yang P, Hao S, Gong X, Li G. Cytogenetic biomonitoring in individuals exposed to cone beam CT: Comparison among exfoliated buccal mucosa cells, cells of tongue and epithelial gingival cells. Dentomaxillofac Radiol 2017;46:20160413.  Back to cited text no. 13
    
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Kesidi S, Maloth KN, Reddy KV, Geetha P. Genotoxic and cytotoxic biomonitoring in patients exposed to full mouth radiographs–A radiological and cytological study. J Oral Maxillofac Radiol 2017;5:1-6.  Back to cited text no. 14
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[PUBMED]  [Full text]  
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Arora P, Devi P, Wazir SS. Evaluation of genotoxicity in patients subjected to panoramic radiography by micronucleus assay on epithelial cells of the oral mucosa. J Dent (Tehran) 2014;11:47-55.  Back to cited text no. 23
    
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    Figures

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

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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