Assessment of Radiation Induced DNA Damage in Human Peripheral Blood Lymphocytes Using COMET Assay

The study was conducted to assess DNA damage in peripheral blood lymphocytes of cancer patients put on radiotherapy, medical workers occupationally exposed to ionizing radiations and control group of normal healthy individuals. The blood samples were collected from 20 cancer patients undergoing radiotherapy in Government Rajindra Hospital, Patiala, 16 medical workers from Radiology and Radiotherapy department of the Government Rajindra hospital, Mata Kaushalya hospital, T.B. hospital, Patiala and 10 normal healthy individuals from Punjabi University, Patiala, India. The DNA damage was evaluated by using alkaline COMET assay, the damage was assessed from two COMET parameters i.e. mean COMET tail length and frequency of cells showing migration. It was found that all the cancer patients undergoing radiotherapy as well as the medical workers, who were occupationally exposed to ionizing radiations for a variable period of time showed DNA damage, whereas none of the control subjects showed any damage. The comparison of DNA damage among the cancer patients and medical workers revealed highly significant differences. On the basis of results obtained, it could be said that the exposure to acute high doses of radiations cause greater DNA damage in comparison to chronic low doses of radiations. Key-wordsSingle cell gel electrophoresis, Micronuclei, Thermoluminescent dosimeter, Aberration INTRODUCTION The maintenance of DNA from one generation to the next is one of the primary goals of our biological systems. Any gross change in its structure, base sequence is referred as DNA damage, which can pose serious biological changes in the body. Detection of DNA damage in individual cells first reported in 1978 . The damage was quantified by measuring the ratio of green fluorescence (indicating double strand DNA) to red fluorescence (indicating single strand DNA). The evaluation of the DNA damage plays an important role in genotoxic studies. The techniques utilized to detect DNA damage in humans have involved cytogenetic evaluation of chromosomal aberrations, sister chromatid exchanges in mitogen stimulated peripheral Access this article online Quick Response Code Website: www.ijlssr. com DOI: 10.21276/ijlssr.2017. 3.4. 17 blood lymphocytes, Micronuclei assay, immuno-assays and single cell gel electrophoresis (COMET assay). The technique known as SCGE was developed in 1984 with the introduction of micro electrophoresis, which was later modified in 1988 . Single cell gel electrophoresis is one of the most commonly used assays for the population biomonitoring to identify the DNA instability. It is a simple, rapid, visual and sensitive technique to detect DNA single strand breaks, double strand breaks and alkali labile sites/damage. This technique was based on the principle that DNA is negatively charged and when the electric field is applied, it moves towards the anode. Singh modified the technique of electrophoresis originally given by Ostling . They replaced the neutral buffer an with alkaline buffer (pH>13) during electrophoresis in order to detect the presence of single strand breaks and alkali labile damage in individual cells. The technique has been used to evaluate both DNA damage and repair in the cancer cell lines, cancer patients undergoing radiotherapy/chemotherapy, occupationally exposed workers etc. Though radiotherapy is a powerful weapon against cancer and has great significance, but the radiations also produce marked toxicity at the cancer sites, ultimately resulting in the DNA damARTICLE RESEARCH Int. J. Life Sci. Scienti. Res., 3(4) JULY 2017 Copyright © 2015-2017| IJLSSR by Society for Scientific Research is under a CC BY-NC 4.0 International License Page 1209 age leading to second cancer. Several studies used the COMET assay to evaluate DNA damage in cancer cell lines [5-7] in cancer patients undergoing radiotherapy [6,8] and in workers occupationally exposed to radiations. The majority of such studies have indicated radiation induced DNA damage in various cancer cell lines and in peripheral blood lymphocytes of cancer patients put on radiotherapy . An inverse correlation was found between cell survival and COMET tail length . Even when the peripheral blood lymphocytes of healthy individuals were exposed to varying doses of X-rays or γ-radiations, comets were formed, indicating DNA damage which was correlated with the extent of the dose. Higher doses induced greater DNA damage . Work done on DNA repair revealed quick repair of single strand breaks (SSBs) in comparison to double strand breaks (DSBs) . A significant increase in the damage was reported in the workers, who were occupationally exposed to varying doses of radiations . MATERIALS AND METHODS SubjectsDuration of the study was one year. The study was conducted on 20 cancer patients undergoing radiotherapy (group A), 16 workers occupationally exposed to radiations (group B) and 10 healthy subjects (group C). Group A subjects were selected from Government Rajindra Hospital, Patiala. Only those patients who had undergone at least 5 cycles of radiotherapy were selected for the study. Group B workers were selected from T.B. Hospital and Government Rajindra Hospital, Patiala, workers working on X-ray machine or handling machines for at least 5 years. All the workers wore individual TLD (Thermoluminescent dosimeter) badges during their work. The TLD badges were replaced after every 3 months and were sent to BARK, Bombay for estimating radiation exposures. The accumulative dosimeter readings of the exposed workers during the year prior to the study were recorded from the hospital records. Blood SamplesAbout 0.5 ml of whole blood was collected from fingertip using a sterilized lancet from selected subjects. Blood samples were collected in eppendorf tubes containing one drop of EDTA solution. The tubes were numbered and stored in refrigerator till use. COMET AssayThe alkaline COMET assay was performed on lymphocytes under alkaline conditions according to Singh in 1988 with slight modifications . An aliquot of 40 μl of whole blood was used to assess the DNA damage. Slides were prepared in duplicates per sample. Slides were covered with normal melting point agarose (NMPA) and dries for further use. For the second layer 25 μl of whole blood was added to 75 μl of 0.5% low melting point agarose (LMPA) and pipetted out on the precoated slides. The slides were covered with coverslips for the proper spreading of the cells. Further the slides were allowed to gel in the 4oC for 30 minutes. The coverslips were removed and third layer of 110 μl of LMPA was pipetted out on the slides and allowed to gel at 4oC for 30 minutes. After third layering, the uncovered slides were immersed in freshly prepared chilled lysing solution and refrigerated for 2 hours. Slides were then placed in the alkaline electrophoretic buffer (E-buffer, pH=13) for 20 minutes to allow unwinding of the DNA. Electrophoresis was conducted for 25 minutes at 25V adjusted to 300 mA by raising or lowering the buffer level in the tank. Slides were then drained, placed on a tray and washed thrice with neutralizing buffer (N-buffer, pH= 7.5). Finally, all the slides were stained with silver nitrate in the presence of dim light so as to minimize artefactual DNA damage, further the slides were dried and viewed under a microscope. Analysis was performed by using 10x and 40x objectives Zeiss microscope. A total of 100 individual cells was screened per sample (50 cells per slide). An undamaged cell resembles an intact nucleus without a tail and a damaged cell has the appearance of a comet. The length of the DNA migrated in the comet tail, which is an estimate of DNA damage was measured by using an ocular micrometer. Quantification of DNA damage for each cell was calculated as given in the formula: Comet tail length (μm)= Maximum tail length–Head