| Abstract|| |
Context: This study was carried out on the assumption that oral mucosal cells might show DNA damage in oral squamous cell carcinoma (OSCC).
Aims: To evaluate the extent of DNA damage in oral smears of patients with OSCC and determine correlation if any of the extent of DNA damage to TNM staging of oral cancer.
Settings and design: A randomized controlled study at a regional cancer centre was designed for this project. Smears were taken from lesion proper of 30 patients with OSCC and from the buccal mucosa of 30 normal healthy volunteers.
Materials and methods: Collected cells were centrifuged and single-cell gel electrophoresis (SCGE) assay was performed. DNA damage was visualized under a fluorescent microscope.
Statistical analysis used : Mean DNA damage levels of both the groups were measured and statistically analyzed with students' test. The extent of DNA damage was correlated with the TNM stages by employing the one way ANOVA 'F' technique.
Results: High statistical significance (P < 0.0001) was found in DNA damage levels between control and study groups. A stepwise increase in DNA damage levels with high statistical significance (P < 0.005) was also found between all the TNM stages.
Conclusions: Statistically significant increased DNA damage levels in OSCC patients and their correlation to clinical staging suggest that comet assay may be used effectively to assess the prognosis of OSCC.
Keywords: Comet assay, DNA damage, oral cancer, oral squamous cell carcinoma, single-cell gel electrophoresis assay, single stranded DNA damage
|How to cite this article:|
Bhagwath S S, Chandra L. Assessing extent of single stranded DNA damage in oral mucosal cells of patients with oral squamous cell carcinoma and its correlation with TNM staging. Indian J Dent Res 2014;25:555-8
According to the assessment of WHO, oral cancer is the third most common malignancy in males and sixth most common malignancy in females. Among all the malignancies affecting oral cavity, oral squamous cell carcinoma (OSCC) is the most common.  It is postulated that one out of every three patients diagnosed with oral cancer would die within 5 years of detection.  Therefore, the prevention of oral cancer is a desirable goal, as is the case with its early diagnosis and treatment.
|How to cite this URL:|
Bhagwath S S, Chandra L. Assessing extent of single stranded DNA damage in oral mucosal cells of patients with oral squamous cell carcinoma and its correlation with TNM staging. Indian J Dent Res [serial online] 2014 [cited 2019 Jul 16];25:555-8. Available from: http://www.ijdr.in/text.asp?2014/25/5/555/147075
Although histopathological findings of suspected cancer remain the only method of confirming malignancy, there are many other advanced methods such as cellular proliferation studies, cytophotometry, analysis of cell products, blood antigens, cell surface carbohydrate fractions, trace elements in circulating blood, oncogenic expression DNA cytology, and assessing DNA damage. The various methods to detect DNA damage include-alkaline sucrose sedimentation, DNA unwinding and hydroxyapatite chromatography, filter elution, Nucleoid sedimentation, microelectrophoresis, nick translation, DNA precipitation assay, pulse field gel electrophoresis, and fluctuation spectroscopy.
A sensitive technique for measuring and analyzing the DNA damage in eukaryotic cells is the single-cell gel electrophoresis assay (SCGE) or Comet assay. ,, This technique has been widely used to study DNA damage and repair induced by radiation and during carcinogenesis, determining the genotoxicity, biomonitoring, and analysis of irradiated foods. The SCGE assay has also been employed to assess DNA damage in peripheral blood leukocytes (PBL) of patients with OSCC,  epithelial dysplasia,  and in tobacco users.  These studies as well as findings of increased DNA damage in PBL in patients with cervical carcinoma lend support to a systemic host response. 
The principle of SCGE assay is that the presence of breaks in the DNA releases the super coiling, so that the loops, instead of being constrained within the bounds of nuclear matrix are free to extend outside. The lysis of DNA at a neutral pH results in double stranded breaks, whereas lysis at an alkaline pH results in denaturation of cellular RNA and detection of single stranded breaks, facilitating easier detection. , On electrophoresis, the single stranded breaks are drawn towards the anode as a 'tail' and therefore this assay is also referred to as Comet assay. Measurement of this tail is an index of DNA damage. Although the DNA damage in PBL with OSCC, epithelial dysplasia, in tobacco users and also in exfoliated buccal cells in tobacco users has been assayed,  the possible DNA damage in exfoliated cells of patients with OSCC has not been studied. This study was planned to fill in this void.
| Materials and methods|| |
For the purpose of this study, patients who were diagnosed as having OSCC formed the study group. Those patients without any systemic disease and tobacco habituation and with a clinically normal oral mucosa formed the control group. Patient's oral smears were collected from the lesional tissue of 30 OSCC patients after histopathological confirmation and evaluation according to Broader's grading system. Oral smears were also collected from the buccal mucosa of 30 persons of the control group. A written and signed informed consent was taken from each subject. This study was reviewed and approved by the institutional ethical board.
SCGE assay was carried out for both these groups. The DNA damage was quantified using a fluorescent microscope following the method outlined by Singh et al.  and the results were statistically analyzed and evaluated. The technique employed by the authors to assess DNA damage is that developed by Singh et al.  that is a modification of original procedure of Ostling and Johanson.  In order to improve the sensitivity for detection of DNA damage in isolated cells, Ostling and Johanson developed a microgel electrophoresis technique. The cells were embedded in agar on a microscope slide and were lysed by detergents and salts at high concentrations. Later, electrophoresis was carried out under neutral conditions at 5 V/cm. This caused some of the DNA stained with acridine orange to stretch out as a tail in the direction of the anode. However, the neutral conditions employed for lysis and electrophoresis by Ostling and Johanson permit the detection of double stranded DNA breaks only and do not allow the detection of single stranded DNA breaks. 
In SCGE assay, the electrophoresis is performed for 15 min under high pH with the power supply adjusted to 20 V and 400 mA. Thus, alkali labile sites as well as frank breaks are revealed. Comets form as broken ends of a negatively charged DNA molecule that becomes free and migrate in the electric field toward the anode. The intensity of fluorescence in the tail relative to the head provides information of the number of single-stranded DNA breaks. Thus, the concept of stretching and migration of the separated strands are generally considered to explain the patterns of DNA migration observed in the comet assay.  For visualization of DNA damage, the slides after electrophoresis were stained with ethidium bromide and observed at 250× magnification using a fluorescent microscope equipped with an excitation of filter of 515-560 nm and a barrier of 590 nm. Using an oculometer grid fitted to the eye piece, the total length and the diameter was measured. The tail length was calculated by subtracting the diameter from the length. Twenty five randomly selected cells per slide were analyzed and mean calculated.
The DNA damage was compared between control and study groups employing the students' test. DNA damage was also compared between the various TNM stages of OSCC by employing the one way ANOVA 'F' technique for significant difference between the various TNM stages of OSCC.
| Results|| |
DNA damage levels between patients of the control group were compared with those of the study group and assessed using the SCGE assay. Smears were taken from lesion proper in the 30 patients of the study group and from the buccal mucosa of 30 normal and healthy volunteers. The results were statistically analyzed and tabulated in [Table 1].
|Table 1: Comparison of DNA damage in normal subjects (control) and patients with oral squamous cell carcinoma (study) |
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The DNA damage levels ranged from 0.55-1.19 μm in normal patients [Figure 1] and from 1.88-10.32 μm in patients with oral squamous cell carcinoma [Figure 2]. The mean was 0.8616 and 3.874 μm with a standard deviation of 0.8142 and 2.5205 μm [Table 1].
|Figure 1: Photomicrograph of DNA of a lysed cell of patient with clinically normal oral mucosa, viewed under fluorescent microscope at ×250 magnification after staining with ethidium bromide|
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Statistical significance was observed for DNA damage levels between normal subjects and patients with oral squamous cell carcinoma employing the students' 't' test [Table 1]. The results were found to be statistically highly significant (P < 0.0001).
|Figure 2: Photomicrograph of DNA of a lysed cell of patient with moderately differentiated squamous cell carcinoma, viewed under fluorescent microscope at ×250 magnification after staining with ethidium bromide|
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Patients having OSCC were divided into four stages, namely stage I, II, III, and IV, following the TNM staging. Out of 30 patients, OSCC, five were in stage I, eleven in stage II, eight in stage III, and six in stage IV [Table 2].
|Table 2: Mean and standard deviation of DNA damage levels in different clinical stages of oral squamous cell carcinoma |
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The DNA damage levels ranged from 1.88-2.8 μm in stage I, 2.4-6.6 μm in stage II, 2.12-6.92 μm in stage III, and 2.08-10.32 μm in stage IV, with a mean of 2.312, 3.171, 3.490, and 6.89 μm respectively. The standard deviations were 0.366, 1.439, 1.971, and 3.710 μm, respectively [Table 2]. The DNA damage was altered in all four clinical stages of OSCC. In order to analyze multiple variance between different clinical stages of OSCC, one way ANOVA 'F' technique was applied. A high significance at P < 0.005 was observed between the various stages [Table 3].
|Table 3: Comparison of DNA damage levels between different clinical stages of OSCC |
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| Discussion|| |
A central feature of cancer development is DNA damage. Different carcinogens attack different sites on the DNA resulting in accumulation of cellular and molecular changes, thereby progressively altering the DNA. The resultant DNA damage can include single and double stranded breaks, DNA-protein cross links, loss of bases from DNA backbone, and modified base residues. 
The quantitative measurement of DNA damage in tumor cells is therefore an important prognostic indicator for patients with malignancy. Many methods are available to detect and quantify DNA single and double stranded breaks, both in vivo and in vitro. However, since many agents induce 5-2000 fold more single stranded breaks of DNA, techniques to measure single stranded breaks of DNA are definitely more sensitive to detect DNA damage. The SCGE assay, also known as comet assay is a recent, use simple and sensitive technique to evaluate single stranded breaks.
Malini et al.  using comet assay showed that persons with cervical carcinomas demonstrated increased DNA damage levels in PBLs as compared to normal individuals. Malini et al.  also demonstrated a significant stepwise increase in the mean DNA damage in the PBLs from normal subjects in the control group to patients with precancerous lesions of cervix showing different grades of dysplasia.
Venkateswara et al.  using SCGE assay demonstrated a significant increase in DNA damage level in PBLs of patients with OSCC.
A study to evaluate DNA damage in PBLs of patients with oral leukoplakia showed a stepwise increase in DNA damage from the control group to patients with different grades of dysplasia.  In leukoplakic lesions without epithelial dysplasia, statistical significance was found when compared to controls. This result obtained by comet assay showed it to be a sensitive technique to identify DNA damage in PBLs even before morphological changes became apparent.
Rekha et al.  employing the comet assay to detect DNA damage in PBLs of tobacco users found statistically significant DNA damage levels in tobacco users when compared with control group patients. The study also revealed statistically significant DNA damage levels in tobacco users with normal oral mucosa and with oral cancer and also between patients with OSCC and the control group. Rojas et al.  employing the comet assay have evaluated the DNA damage in exfoliated buccal mucosal cells of smokers and nonsmokers. A statistically significant mean DNA damage was found in the smoker group, when compared with the control group. Rojas et al.  also showed statistically significant DNA damage levels in exfoliated tear duct epithelial cells in patients exposed to environmental pollutants. This study was carried out on the assumption that oral mucosal cells could show the DNA damage in oral cancer, as they were shown by Rojas et al. to manifest DNA damage in tobacco users, which is a prime cause of oral cancer.
For this study, 30 patients with OSCC were selected after histopathological confirmation using Broader's histopathological grading system. DNA damage was quantitatively assessed by performing SCGE assay of exfoliated cells of patients of control as well as the study group. The exfoliated cells were first centrifuged along with animal tissue culture medium and then spread on half-frosted glass slides and SCGE assay was performed. To visualize the single-stranded breaks of DNA, the slides after electrophoresis were stained with ethidium bromide and viewed under fluorescent microscope at 250 × magnification. The mean DNA damage levels in these patients were compared with DNA damage levels in 30 normal, healthy volunteers without tobacco habituation and systemic illness. The mean DNA damage in patients with OSCC was found to be 3.874 μm and this when compared to mean DNA damage in control group patients (0.8616 μm) was found to be highly statistically significant (P < 0.0001).
An attempt was made to evaluate DNA damage in relation to clinical TNM staging. Patients with OSCC were staged clinically using the TNM staging system and into four stages, namely stage I, II, III, and IV.
The DNA damage levels were altered in all the stages of OSCC and showed a stepwise increase from stage I to stage IV indicating that DNA strands breaks increased in direct relation to TNM stage of OSCC. On comparison between the stages, the DNA damage levels in oral mucosal cells were found to be statistically highly significant at P < 0.005 between the various stages.
These preliminary findings suggest that the DNA damage levels in oral mucosal cells could be an important prognostic indicator and can be utilized to supplement the clinical staging and histopathological grading to assess the prognosis of OSCC.
| Conclusions|| |
This study was carried out on the assumption that oral mucosal cells might show DNA damage in OSCC. The assumption was based on DNA damage observed in exfoliated buccal mucosal cells of smokers and DNA damage in mucosal cells of cervical cancer.
The findings of statistically significant increased DNA damage levels in oral mucosal cells in OSCC patients and their correlation to clinical staging suggest that comet assay may be used effectively to assess the prognosis of OSCC. However, a more detailed study involving a larger sample is needed on order to corroborate these findings.
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S S Bhagwath
Department of Basic Sciences, College of Dentistry, University of Ha'il, Kingdom of Saudi Arabia
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3]