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ORIGINAL ARTICLE
Year : 2012  |  Volume : 4  |  Issue : 2  |  Page : 124-128

Biomonitoring of genotoxic and cytotoxic effects of gingival epithelial cells exposed to digital panoramic radiography


1 Department of Oral Medicine and Radiology, Oxford Dental College and Hospital, Bangalore, Karnataka, India
2 Department of Oral Medicine and Radiology, Vishnu Dental College and Hospital, Bhimavaram, Andhra Pradesh, India
3 Department of Oral and Maxillofacial Surgery, AME'S Dental College Hospital and Research Centre, Raichur, Karnataka, India

Date of Web Publication17-Jan-2013

Correspondence Address:
Yadavalli Guruprasad
Department of Oral and Maxillofacial Surgery, AME'S Dental College Hospital and Research Centre, Raichur - 584 103, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0975-8844.106207

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  Abstract 

Objective: The aim of this study was to evaluate genotoxic and cytotoxic effects of low level ionizing radiation used in digital panoramic radiography on gingival epithelial cells. Materials and Methods: We included 50 healthy individuals advised for digital panoramic radiography for diagnostic purpose were included in this study. Demographic data and personal history of all subjects were recorded in a proforma before the examination. Gingival epithelial cells were obtained by gentle scraping with a modified cytobrush immediately before X-ray exposure and 10 ± 2 days later. Cytological preparations were stained according to the Feulgen/fast green method and analyzed under a light microscope. Micronuclei and degenerative nuclear alterations (pyknosis, karyolysis, karyorrhexis and condensed chromatin) were scored. Results: The frequency of formation of micronuclei was not significant with regard to age, gender and after exposure to digital panoramic radiography ( P = 0.276). However this study showed significant increase in the frequencies of nuclear alterations like karyorrhexis, pyknosis, condensed chromatin, karyolysis and indicative of cell death ( P < 0.001). Conclusion: Panoramic radiographic examination does not induce genotoxic effect like micronuclei, but it does induce cytotoxic effects leading to cell death.

Keywords: Chromosomal aberration, gingival epithelial cells, micronucleus test, panoramic radiography, radiation hazard


How to cite this article:
Pai A, Sharma RC, Naik RM, Guruprasad Y. Biomonitoring of genotoxic and cytotoxic effects of gingival epithelial cells exposed to digital panoramic radiography. J Orofac Sci 2012;4:124-8

How to cite this URL:
Pai A, Sharma RC, Naik RM, Guruprasad Y. Biomonitoring of genotoxic and cytotoxic effects of gingival epithelial cells exposed to digital panoramic radiography. J Orofac Sci [serial online] 2012 [cited 2019 Sep 15];4:124-8. Available from: http://www.jofs.in/text.asp?2012/4/2/124/106207


  Introduction Top


Ionizing radiations (X-rays) are potent mutagenic agent capable of inducing both mutation and chromosomal aberrations. [1] For cancer prevention in workers chronically exposed to low level ionizing radiation in nuclear power plants, occupational medicine applies essentially biomonitoring of exposure and effects. [2] To date, a variety of assays have been proposed in biomonitoring studies, including those that assess metaphase chromosomal aberrations, sister chromatid exchanges, DNA damage and host cell reactivation. However, these methods are typically difficult and time-consuming or require highly qualified technicians to accurately read and interpret slides. For these reasons simple test like application of the micronucleus test to uncultured exfoliated cells has been encouraged. [3]

Making an allowance for the strong evidence of the relationship between DNA damage and carcinogenesis, the objective of the present study was to evaluate genotoxic and cytotoxic effects of low level ionizing radiation used in digital panoramic radiography on gingival epithelial cells using micronucleus test.


  Materials and Methods Top


A comparative study of gingival epithelial cells obtained from normal subjects by exfoliative cytology to identify the potential role of digital panoramic radiographic exposure towards genotoxicity and cytotoxicity was performed.

Study group

Gingival epithelial cells obtained from normal subjects prior to digital panoramic radiographic exposure was defined as controls (n = 50) and gingival epithelial cells obtained 10 days after exposure to digital panoramic radiography from same normal subjects was considered as cases (n = 50). Healthy individuals between the age group of 20-30 years advised for digital orthopantomograph were included in this study. The individuals were divided into two groups with age < 25 years and age group > 25 years. Patients below the age of 20 years and above 40 years, patients with the habit of chronic smoking, alcoholism, use of alcohol containing mouth washes and individuals with oral mucosal lesions (leukoplakia, erythroplakia, oral submucous fibrosis, lichen planus, tobacco pouch keratosis etc) were excluded from the study.

Study setting

The study was approved by institutional review board in 2009. All the data of individuals were recorded in a proforma pertaining to demographic data, habit of smoking, alcoholism, and oral hygiene habits (use of alcohol containing mouthwash) before to digital panoramic radiographic exposure. Study protocol was explained and informed consent was obtained from all individuals. Cells were obtained from the gingiva (keratinised mucosa) of 50 healthy subjects satisfying the inclusion criteria. These subjects were then subjected to undergo digital panoramic dental radiographs by PLANMECA Proline XC digital orthopantomographic system with the parameters 64-70 Kvp 12 mA for 18 seconds with the effective dose of 5-14 μsev. The subjects were then asked to revisit dental OPD after 10 days for obtaining post exposure cytological smear.

Cell collection and slide preparation

Exfoliated gingiva cells were collected immediately before x-ray exposure and 10 days after exposure. After rinsing the mouth with water, exfoliated cells from the upper dental arch was collected using a modified cytobrush. Cells were then transferred to a test tube containing saline solution and centrifuged (1000 rpm) for 5 minutes. The supernatant saline was discarded then the cell deposit was collected using micropipette and dropped on to pre-cleaned slides following which smear was made, air dried and fixed using 95% ethyl alcohol. Samples were briefly placed in 5 M Hcl at room temperature for 15 min, rinsed in distilled water for 10-15 min, stained using Schiff's reagent for 90 min and counterstained with fast Green 1% for 1 min. The demonstration of DNA is by the Feulgen technique, which will demonstrate the sugar deoxyribose and by the methyl green pyronin technique in the phosphate combines with the basic dye methyl green at an acid ph.

Determination of cytological analysis

A minimum of 1000 cells was studied by blind analysis. Abnormalities were identified under the light microscope (×400). Micronuclei in cells, pyknosis, karyorrhexis, karyolysis and condensed chromatin were confirmed by observing them in oil immersion at ×1000 magnification. Subjects were then asked to revisit dental OPD after 10 days for obtaining post exposure cytological smear [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5] and [Figure 6]. Statistical analysis was carried out using descriptive statistics, independent sample T test and paired T test.
Figure 1: Photomicrograph showing differentiated epithelial cells (×400)

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Figure 2: Photomicrograph showing ×400 group of intermediate cells with desmosomal attachments, arrow head demonstrating micronucleus (MN) (×400)

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Figure 3: Photomicrograph showing three micro nuclei (MN) in a cell (×1000)

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Figure 4: Photomicrograph of condensed chromatin (×400)

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Figure 5: Photomicrograph demonstrates cell with karyolysis and pyknosis (×1000)

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Figure 6: Photomicrograph of cell showing fragmentation of nucleus suggestive of Karyorrhexis (×1000)

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  Results Top


In the present study the mean age of the individuals was 23 yrs, with the gender distribution of 14 males and 36 females.

The frequency of micronuclei in the age group < 25years was 0.0004% and 0.0005% before and after exposure respectively. Whereas the mean micronuclei in age group > 25 years was 0.00061% and 0.00064% before and after exposure to radiographs respectively suggesting no statistical difference between the two groups are shown in [Table 1].
Table 1: Association of age and mean frequency of MN and other nuclear alteration in gingival epithelial cells of patients before and after panoramic radiography

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There was no association of gender in inducing micronuclei and other chromosomal aberrations before and after exposure to radiography as shown in [Table 2].
Table 2: Association of gender and mean frequency of MN and other nuclear alteration in gingival epithelial cells of patients before and after panoramic radiography

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The frequency of micronuclei and other nuclear alterations are shown in [Graph 1] [Additional file 1]. The distribution of micronuclei before exposure was 0.0046%, no statistical difference was found after exposure (P > 0.001). In contrast there was increase in other nuclear alterations.

The distribution of mean micronuclei (MN) and other nuclear alteration in the pre exposure group was 0.0046% and 0.2622% respectively. However there was mild increase in MN and other nuclear alterations, the mean of which was 0.0062%, 0.364% respectively. The P value for micronuclei distribution with respect to pre and post exposure to radiation was 0.031, whereas the P value for other nuclear alterations and differentiated cells in pre and post exposure was < 0.001. This suggested that even though there was overall increase in mean MN% it was not statistically significant, on the other hand there was overall increase in other nuclear alterations with P value < 0.001 suggestive of cytotoxic effects.


  Discussion Top


X-rays have been widely used for diagnosis in medical and dental practices and ionizing radiation is a well known mutagen and carcinogen in the human population. [4] Chromosomal aberrations are a frequent and significant response on exposure to mutagenic agents. They are of significance from the stand point of inherited human disease and have been implicated, at least indirectly, in carcinogenesis. Micronucleus has been reported as a rapid assay for chromosomal damage which measures accurate X-ray induced chromosomal damage. [5]

The micronucleus is formed due to chromosomal damage in the basal cells of the epithelium. When these cells divide, chromosomal fragments (or entire chromosomes which lack attachment to the spindle apparatus) lag behind and are excluded from the main nuclei in the daughter cells. These fragments form their own membranes and appear as Feulgen-specific bodies, termed micronucleus in the cell cytoplasm. Such micronuclei are helpful in assessing the genotoxicity. [4],[5]

Buccal epithelial tissue is under direct radiation exposure for any oral and maxillofacial radiography and in that way it is the primary target for radiation-induced damage. The genotoxic and cytotoxic effects during panoramic radiography were evaluated immediately before and 10 ± 2 days after using micronucleus test. Chromosomal damage leading to micronucleus formation occurs during the division of cells from the basal layer of the oral epithelium, but it is only observed later in exfoliated cells, between 1 week and 3 weeks after exposure to a genotoxic agent. [6],[7],[8] Day 10 was chosen on the basis of the fast turnover in epithelial cell kinetics (from 7-16 days).

Present study consisted individuals with age ranging from 20-30 years with a mean age of 23 years and showed no statistical significance with respect to age (P=0.911). This was in accordance with a previous study conducted in Brazilian population with the mean age group of 21 years. [9],[10]

The genocytotoxicity of X-ray exposure are known to be varying with respect to gender of an individual. [10] In present study there were a total number of 36 females and 14 males amongst 50 individuals selected randomly showed no statistical significance in genotoxic and cytotoxic effect of X-rays which is in accordance with previous literature (P>0.05).

In the present study the decrease in the number of micronuclei count was due to decrease in the effective dose (5-14μsev) of digital panoramic radiography which is in accordance with the study conducted EMM Cerqueira et al[10] and Angelieri et al[11] when compared to effective dose of conventional panoramic radiography (21.4μSv). [7],[11]

The amount of radiation exposure in dental diagnostic radiography is insignificant when compared to the above said values. The occurrence of pyknosis and karyolysis was increased after exposure (P<0.001). In the Pyknosis is a nuclear alteration indicative of apoptosis and this result suggests that there are multiple routes that lead the cell to apoptosis. However a study conducted by EMM Cerqueira et al[10],[11] did not detect any increase in pyknosis in exfoliated cells from the oral mucosa. The occurrence of condensed chromatin and karyolysis was also increased after exposure to panoramic radiographic exposure (P<0.001) which is in accordance to other studies which suggested overall increase in other nuclear alterations in the cells. [10],[12] It is possible that cytotoxic effects by apoptosis may be related to cell death under normal conditions in gingival epithelial cells.


  Conclusion Top


Results of the present study indicate that at low dose of digital panoramic radiographic exposure used for diagnostic purpose had no genotoxic effect however could induce cytotoxic effects suggesting apoptosis.


  Acknowledgment Top


The authors wish to thank Dr. N. Chaitanya Babu, Professor and Dr. K. Vinod Kumar, Assistant Professor, Department of Oral Pathology and Microbiology for valuable contribution in cytological analysis.

 
  References Top

1.Touil N, Vande Aka P, Buchet JP, Thierens H, Volders MK. Assessment of genotoxic effects related to chronic low level exposure to ionising radiation using biomarkers for DNA damage and repair. Mutagenesis 2002;17:223-33.  Back to cited text no. 1
    
2.Belien JA, Copper MP, Braakhuis BJ, Snow GB, Baak JP. Standardization of counting micronuclei: Definition of a protocol to measure genotoxic damage in human exfoliated cells. Carcinogenesis 1995;16:2395-400.  Back to cited text no. 2
    
3.Jois HS, Kale AD, Mohan Kumar KP. Micronucleus as potential biomarker of oral carcinogenesis. Indian J Dent Advancements 2010;2:1-5.  Back to cited text no. 3
    
4.Popova L, Kishkilova D, Hadjidekova VB, Hristova RP, Atanasov P, Hadjidekova VV, et al. Micronucleus test in buccal epithelium cells from patients subjected to panoramic radiography. Dentomaxillofac Radiol 2007;36:168-71.  Back to cited text no. 4
    
5.Stich HF, Stich W, Parida BB. Elevated frequency of micronucleated cells in the buccal mucosa of individuals at high risk for oral cancer: Betel quid chewers. Cancer Letters 1982;17:125-32.  Back to cited text no. 5
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6.Ribeiro DA, Oliveira GD, 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. 6
    
7.Bolognesi C, Lando C, Forni A, Landini E, Scarpato R, Migliore L, et al. Chromosomal damage and ageing: Effect on micronuclei frequency in peripheral blood lymphocytes. Age Ageing 1999;28:393-7.  Back to cited text no. 7
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8.Pastor S, Gutie×rrez S, Creus A, Xamena N, Piperakis S, Marcos R. Cytogenetic analysis of Greek farmers using the micronucleus assay in peripheral lymphocytes and buccal cells. Mutagenesis 2001;16:539-45.  Back to cited text no. 8
    
9.Cerqueira EM, Meireles JR, Lopes MA, Junqueira VC, Filho IS, Trindade S, et al. Genotoxic effects of X-rays on keratinized mucosa cells during panoramic dental radiography. Dentomaxillofac Radiol 2008;37:398-403.  Back to cited text no. 9
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10.Angelieri F, Carlin V, Saez1 DM, Pozzi R, Ribeiro DA. Mutagenicity and cytotoxicity assessment in patients undergoing orthodontic radiographs. Dentomaxillofac Radiol 2010;39:437-40.  Back to cited text no. 10
    
11.Miyaji CK, Colus IM. Cytogenetic biomonitoring of Brazilian dentists occupationally exposed to low doses of X-radiation. Pesqui odontol Bras 2002;16:196-201.  Back to cited text no. 11
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12.Bolognesi C, Lando C, Forni A, Landini E, Scarpato R, Migliore L, et al. Chromosomal damage and ageing: Effect on micronuclei frequency in peripheral blood lymphocytes. Age and ageing 1999;28:393-7.  Back to cited text no. 12
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
 
 
    Tables

  [Table 1], [Table 2]



 

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Abstract
Introduction
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