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 Table of Contents  
ORIGINAL ARTICLE
Year : 2018  |  Volume : 7  |  Issue : 2  |  Page : 50-55

Evaluation of DNA Damage in Peripheral Blood Leukocytes in Oral Potentially Malignant and Malignant Disorders by Comet Assay


1 Department of Oral and Maxillofacial Pathology, Maulana Azad Institute of Dental Sciences, New Delhi, India
2 Department of Microbiology, Maulana Azad Medical College, New Delhi, India

Date of Web Publication8-Mar-2018

Correspondence Address:
Dr. Garima Rawat
A-4/F-1, A-block, Dilshad Garden, New Delhi – 110 095
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ccij.ccij_66_17

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  Abstract 


Purpose: Oral squamous cell carcinoma (OSCC) and potentially malignant disorders (PMD) are associated with DNA damage which can be caused by exposure to carcinogens, genotoxins, or increased oxidative stress. Early detection and assessment of the amount of DNA damage using a biomarker such as a comet assay can prove to be extremely beneficial for the patients. The present study evaluated the efficacy of comet assay in assessing DNA damage in peripheral blood leukocytes (PBLs) in oral potentially malignant and malignant disorders. Materials and Methods: The study included fifty-five patients each of leukoplakia, oral submucous fibrosis (OSMF), and OSCC along with fifty-five healthy individuals as control. The patients with deleterious oral habits were categorized into smokeless, smoked, and mixed habit groups. DNA damage was evaluated by measuring the mean tail length (μm). Results: An increased mean tail length (μm) and higher DNA damage were found in OSCC (22.4335 ± 1.52341), and there was a progressive stepwise increase in mean tail length from control (6.8307 ± 0.84261) to PMD (leukoplakia [13.0022 ± 0.74316]; OSMF [10.6085 ± 0.88140]) to OSCC. Although there was a significant increase in the DNA damage in different habit groups (smokeless [14.9380 ± 5.18516]; smoked [15.4947 ± 4.59589], and mixed [16.3650 ± 5.62407]) compared to controls, there was no significant difference between the habit groups. Conclusion: Thus, comet assay technique can be used as a sensitive and reliable indicator for DNA damage evaluation.

Keywords: Comet assay, DNA damage, oral squamous cell carcinoma, peripheral blood leukocytes


How to cite this article:
Rawat G, Urs AB, Chakravarti A, Kumar P. Evaluation of DNA Damage in Peripheral Blood Leukocytes in Oral Potentially Malignant and Malignant Disorders by Comet Assay. Clin Cancer Investig J 2018;7:50-5

How to cite this URL:
Rawat G, Urs AB, Chakravarti A, Kumar P. Evaluation of DNA Damage in Peripheral Blood Leukocytes in Oral Potentially Malignant and Malignant Disorders by Comet Assay. Clin Cancer Investig J [serial online] 2018 [cited 2018 Nov 21];7:50-5. Available from: http://www.ccij-online.org/text.asp?2018/7/2/50/226847




  Introduction Top


In 2005, the World Health Organization (WHO) recommended that the term “potentially malignant disorders (PMDs)” may be used as not all the lesions and conditions described under this term may transform to cancer.[1],[2] The most common PMDs affecting the oral cavity include leukoplakia, erythroplakia, and oral submucous fibrosis (OSMF).[1],[2] Almost 30%–80% of the oral malignancies arise from PMDs such as leukoplakia and OSMF. Malignant transformation rates for leukoplakia and OSMF range from 0.13% to 17.5% and 2.3% to 7.6%, respectively.[3] Oral squamous cell carcinoma (OSCC) is the most common malignant disorder of the oral cavity, accounting for over 90% of all malignant neoplasms in this region.[4],[5],[6]

OSCC has a multifactorial etiology. The various factors implicated in the etiopathogenesis of OSCC include smoking, chewing betel quid/tobacco, and alcohol intake separately or synergistically, viruses, genetic factors, environmental factors, and gene-environment interactions.[7] The above-mentioned deleterious habits may induce oxidative stress or generation of reactive oxygen species (ROS) which leads to cellular damage, as well as DNA damage.[8],[9] The DNA damage may occur in the form of DNA breaks, double-strand breaks (DSB) or single-strand breaks (SSBs), alkali-labile sites (ALS), and micronucleus formation which elevate chromosomal aberrations.[10]

Various methods for evaluation of DNA damage known as genotoxicity assays used in the past include single cell gel electrophoresis (SCGE) or comet assay and micronucleus assay (MN assay). Among these, comet assay (SCGE) is widely accepted as an in vitro and in vivo genotoxicity test.[9],[11]

The comet assay or SCGE is a sensitive and rapid technique for quantifying and analyzing DNA damage in individual cells.[12],[13],[14]

The assay was first developed by Ostling and Johansson in 1984 and later modified by Singh et al. in 1988. It depends on the partial unwinding of the supercoiled DNA in agarose-embedded microscopic slides, which allows the DNA to be drawn out toward the anode under electrophoresis, forming “comet-like” images as seen under fluorescence microscopy. The relative amount of DNA in the comet tail indicates DNA break frequency.[13],[15] A cell with DNA damage appears as a “comet”, and undamaged cell appears as a “halo.” The head of the comet is composed of intact DNA while the tail is composed of damaged DNA (SSBs or DSBs). The comet tail length is directly proportional to the amount of DNA damage.[16] This assay can be used on any eukaryotic cell type that can be obtained as a single cell or nuclear suspension.[13] Many genotoxicity tests evaluate the tissue thought to be the primary site of metabolism lymphocytes are the most commonly used cells in comet assay to assess DNA damage.

The aim of the present study was to evaluate the efficacy of comet assay in assessing DNA damage in peripheral blood leukocytes (PBLs) in oral potentially malignant and malignant disorders.


  Materials and Methods Top


Ethics

This study was conducted in the Department of Oral Pathology and Microbiology, Maulana Azad Institute of Dental Sciences, New Delhi, India. The study was approved by the Institutional Ethical Committee Board.

Study design

This prospective study was designed to evaluate the efficacy of comet assay in PBLs in assessing DNA damage in oral potentially malignant and malignant disorders.

Patient selection

Clinically and histopathologically confirmed patients with leukoplakia, OSMF, and OSCC in the age range from 18 to 80 years were included in the study. The patients were selected after obtaining written informed consent from each. A total of 220 patients with leukoplakia (Group B; n = 55), OSMF (Group C; n = 55), and oral squamous cell carcinoma (Group D; n = 55) along with healthy age- and sex-matched individuals (control, Group A; n = 55) were included in the study.

The patients with habits were categorized into three habit groups:

Smokeless (consuming tobacco, gutkha, pan, or supari), smoked (consuming cigarette or bidi), and mixed (both smoked and smokeless forms).

Patients not willing to participate, suffering from any infectious or contagious disease, with any other white patch such as candidiasis, oral lichen planus and lichenoid reaction and previous history of surgery, radiotherapy or chemotherapy, or any vitamin, or dietary supplement use were not included in the study.

Sample collection

Three milliliters of peripheral blood were withdrawn from each patient and controls under aseptic conditions in sterile tubes containing EDTA anticoagulant. Blood samples were stored at −80°C till used.

Comet assay

Slides were dipped in 1% normal melting point agarose (NMPA) and allowed to dry at 37°C. Once NMPA solidified, 80 μl of peripheral blood was diluted with 80 μl of (×1) phosphate buffered saline and added to a microcentrifuge tube containing an equal volume (160 μl) of low-melting-point agarose (LMPA, 37°C). This mixture of gel (LMPA) and blood was placed over the previously NMPA-coated slides. A coverslip was placed carefully over the slide so that a uniform layer over the NMPA coat is formed avoiding trapping of air bubbles. The slides were kept over an ice pack to solidify the gel for 10–15 min. Then, the coverslip was carefully removed, and 100 μl of LMPA was added over the gel mixture layer. A fresh coverslip was placed and again kept over an ice pack to solidify the gel for 10–15 min. The coverslip was finally removed, and the slide was dipped into lysis buffer solution and refrigerated for 24 h. After lysis at 4°C, DNA was allowed to unwind under alkaline conditions. The slides were allowed to stay in the cold (4°C) alkaline buffer (pH >13) for 20 min to unwind DNA strands and expose the ALS (alkali unwinding). Electrophoresis was performed for 20 min under alkaline conditions in the refrigerator (4°C) at 280 mA and 24 V (~0.74 V/cm). The slides were gently picked up from the alkaline electrophoresis buffer and placed on a staining tray. Then, the slides were carefully flooded three times with a neutralizing buffer (pH 7.5) for 5 min each. Following neutralization, the slides were stained using staining solution of ethidium bromide. The DNA comets were visualized using a fluorescent microscope. The images were captured, and measurements of tail lengths were done using Image J software (NIH, MD, USA). A total of 100 randomly selected cells per slide were analyzed. The DNA damage was evaluated by calculating the length of the comet tail for each cell, and the mean tail length was assessed.

Statistical analysis

The mean values, standard deviation, and ranges (maximum and minimum) were calculated for each variable. The resulting data were analyzed using SPSS software, version 20 (Armonk, NY: IBM Corp). Data were expressed as a mean ± standard deviation. Differences between different variables were analyzed using parametric Student's t-test and analysis of variance (ANOVA). The correlation was calculated using the Pearson's correlation. A value of P ≤ 0.05 was considered to be statistically significant.


  Results Top


The peripheral blood samples were collected from a total of 220 individuals which included 55 patients each of leukoplakia, OSMF, OSCC, and age- and sex-matched healthy controls. Distribution of patients and controls according to age, gender, and habit was studied [Table 1].
Table 1: Demographic data of patients included in the study

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Comet assay was performed on PBLs of all the participants. The DNA damage (mean tail length) in the PBLs in leukoplakia, OSMF, OSCC, and control groups was assessed, and results of each are illustrated in [Figure 1], [Figure 2], [Figure 3], [Figure 4]. The mean tail length (μm) of PBLs in leukoplakia, OSMF, and OSCC was compared with control [Table 2].
Figure 1: Nonfragmented and undamaged DNA in peripheral blood leukocytes of control

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Figure 2: DNA damage in the form of comet in peripheral blood leukocytes of leukoplakia

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Figure 3: DNA damage in peripheral blood leukocytes of oral submucous fibrosis

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Figure 4: DNA damage in the form of comet in peripheral blood leukocytes of oral squamous cell carcinoma

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Table 2 Comparison of mean tail length of PBLs between different study groups

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Comparison of mean tail length of PBLs in study groups and controls was done using ANOVA test [Table 3].
Table 3: P values obtained by comparison of mean tail length of PBLs between different study groups

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Comparison of mean tail length of PBLs between different habit groups and control was done using Student's t-test [Table 4].
Table 4: Comparison of mean tail length of PBLs between different habit groups and control

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Comparison of mean tail length of PBLs between different habit groups was carried out using ANOVA [Table 5].
Table 5: P values obtained by comparison of mean tail length of PBLs between different habit groups

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  Discussion Top


Prolonged exposure to carcinogenic agents induces oxidative stress or ROS generation that is genotoxic and cytotoxic to human cells can cause damage leading to genetic alterations. Accumulation of these genetic alterations may initiate the development of premalignant disorders and subsequently OSCC.[8] When the amount of ROS generated in the cells is high, it leads to cellular damage, as well as DNA damage.[8],[9] The DNA damage may occur in the form of DNA breaks, DSB or SSBs, ALS, and micronucleus formation which elevate chromosomal aberrations.[10] The ROS also affect the DNA repair mechanisms essential for maintenance of DNA integrity and prevention of cancer.[8] Thus, the progression of OSCC from PMDs is a multistep process.[3],[5] The common PMDs such as leukoplakia and OSMF have malignant transformation rates in the range of 0.13%–17.5% and 2.3%–7.6%, respectively.[3]

The comet assay or SCGE has become increasing popular in assessing the DNA damage due to its rapidity, sensitivity, inexpensiveness, and requirement of little biological material.[17],[18],[19],[20] Comet assay depends on the partial unwinding of the supercoiled DNA in agarose-embedded microscopic slides, which allows the DNA to be drawn out toward the anode under electrophoresis, forming “comet-like” images as seen under fluorescence microscopy.[13],[15] A cell with DNA damage appears as a “comet” and undamaged cell appears as a “halo”. The head of the comet is composed of intact DNA while the tail is composed of damaged DNA (SSBs or DSBs). The comet tail length is directly proportional to the amount of DNA damage.[21] The most commonly used cells in human biomonitoring studies are peripheral blood lymphocytes.

The age of patients in the study ranged from 18 to 80 years with the mean age for controls, leukoplakia, OSMF, and OSCC being 36.33 years, 43.54 years, 38.95 years, and 49.05 years, respectively. About 79% of the patients included in the study were males. Other studies had a comparable demographic profile with a predominance of males.[3],[7]

Among the patients in the study groups, there was a predominance of smokeless tobacco usage (48.2%) among the study groups, with mixed tobacco (both smokeless and smoked), and smoked form accounting for 18.2% and 8.6% of the patients, respectively.

It was found that the mean tail length (μm) was significantly increased in OSCC (22.4335 ± 1.52341), leukoplakia (13.0022 ± 0.74316), and OSMF (10.6085 ± 0.88140) compared to controls (6.8307 ± 0.84261 μm). The increased comet tail lengths in all study groups compared to controls depicts the presence of DNA damage in the PBLs of these patients. DNA damage can occur in the form of SSBs, ALS, and cross-linking.[22] The generation of ROS and exposure to genotoxins causes DNA breaks and reduced DNA repair capacity. These genotoxins attack different sites on the DNA leading to the accumulation of DNA damage.[23] During oxidative stress, the damage is characterized by the presence of oxidized purines or pyrimidines. All these significantly contribute to increase in DNA damage which increases the risk of cancer.[18],[24]

The amount of DNA damage was greatest in OSCC patients as this group had the maximum mean tail length followed by leukoplakia and OSMF. There was a significant stepwise increase in DNA damage in the PBLs from control to precancer patients and from precancer to oral cancer patients. The DNA repair systems protect the integrity of the genome so that any deficiency in DNA repair leads to increased DNA damage and development of cancer.[25]

Similar to our results, Mukherjee et al. found that the mean (± standard deviation [SD]) tail length of OSCC (24.95 ± 5.09 μm) and leukoplakia (12.96 ± 2.68 μm) was significantly greater than in controls (8.54 ± 2.55 μm, P < 0.05). In leukoplakia, the mean (± SD) tail length was significantly greater as compared to OSMF (11.03 ± 5.92 μm).[7] Our study also showed similar results. Thus, the DNA damage in blood cells as measured by comet assay is significantly greater in oral cancer and leukoplakia as compared to OSMF.

Various other authors in the past have obtained comparable results. These studies have shown that comet tail length was highest in OSCC patients, lesser in leukoplakia, OSMF patients, and lowest in controls.[3],[26] Vellappally et al. in their study demonstrated that the mean tail length for leukoplakia patients with moderate-to-severe dysplasia (1.25 ± 0.14 μm) was significantly more than controls (0.31 ± 0.10 μm). Thus, the DNA damage in blood cells evaluated by SCGE is greater in leukoplakia than in controls, and deleterious oral habits are associated with greater DNA damage.[27]

Cancer patients have maximum DNA damage as depicted by the greatest mean tail length of the comet in lymphocytes. This has been observed in cancers other than those of the oral cavity.[25],[28],[29],[30],[31] Udumudi et al. in cervical cancer patients and Lou et al. in lung cancer patients found significantly higher mean tail moment compared to controls.[32],[33] Likewise, the comet assay results in patients with squamous cell carcinoma of the head and neck were similar.[34]

On comparing the mean tail length of PBLs in different habit groups with the control group of no habit, highly significant results were obtained in this study.

Guttikonda et al. found similar results wherein the mean tail length was highest in tobacco habituated patients with OSCC (25.375 μm) followed by tobacco habituation but with clinically normal mucosa (2.5833 μm), and it was least in healthy individuals (2.18 μm).[35] Many other investigators have investigated the effect of habit on mean tail length and have obtained comparable results.[11],[36]

Tobacco smoking and smokeless tobacco are important etiologic factors leading to oral cancer. These products are composed of carcinogens such as polyaromatic hydrocarbons nitrosamines and aromatic amines. These carcinogenic agents after deactivation in the liver are converted into electrophilic intermediates which in turn react with DNA to form covalently bound adducts. The formation of DNA adducts and the resulting mutations are responsible for oncogene activation and inactivation of tumor suppressor genes, leading to cancer. Few authors have reported the presence of these DNA adducts in smokers.[23],[37],[38]

On the contrary, Hoffmann and Speit showed no significant difference in DNA damage between smokers and nonsmokers. It was concluded that cigarette smoking had no effect on the amount of DNA damage in peripheral blood cells.[39] Further, Betti et al. proved that there was no correlation between comet tail length in smokers and the number of cigarette smoked per day.[16] No correlation between the length of the comet and the number of cigarettes or the frequency of smoking was detected by Frenzilli et al.[40] and Mohankumar et al.[38] These findings can be explained by assuming that the single DNA strand breaks can be induced in leukocytes also by free radicals generated due to the inflammation normally present in smokers. This reaction is independent of the amount of cigarette smoked and is related to individual susceptibility. The DNA SSBs induced by agents such as hydrogen peroxide are quickly repaired.[16],[38]


  Conclusion Top


The present study attempted to evaluate the efficacy of comet assay in assessing the DNA damage in PBLs in oral potentially malignant and malignant disorders. An increased tail length and higher DNA damage were associated with OSCC, and a progressive stepwise increase in tail length was observed from control to PMD to OSCC. Our study has shown promising results, and hence, comet assay of PBLs can be used effectively in early detection of oral PMDs and malignant disorders.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

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

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]


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