|Year : 2011 | Volume
| Issue : 4 | Page : 221-224
Assessment of electron and gamma-induced dna damage in human peripheral blood by alkaline comet assay
Praveen Joseph1, Narayana Yerol1, Rajesha Nairy1, Ganesh Sanjeev1, NN Bhat2
1 Department of Studies in Physics, Mangalore University, Mangalagangotri, RPAD, India
2 Bhabha Atomic Research Centre (BARC), Mumbai, India
|Date of Web Publication||17-Jan-2013|
Department of Studies in Physics, Mangalore University, Mangalagangotri, RPAD
Source of Support: None, Conflict of Interest: None
In the present study, the effect of electron and gamma irradiation on the induction of DNA damage in human peripheral blood cells was investigated using comet assay. Blood samples were irradiated with an 8 MeV pulsed electron beam at a dose rate of 100 Gy min -1 . Gamma irradiation was carried out at a dose rate of 2 Gy min -1 using 60 Co gamma source. The total dose delivered to the samples was varied from 0 to 4 Gy. Samples were maintained at 0° C before irradiation, and the comet assay was carried out immediately after irradiation. Electrophoresis was performed at a field strength of 0.74 V cm -1 for 25 min at 4°C. A dose-dependent increase in DNA damage was observed. From the observed DNA damage, the relative biological effectiveness (RBE) for electron radiation with reference to gamma radiation on induction of DNA damage has been calculated.
Keywords: Comet assay, DNA, electron beam, microtron accelerator, relative biological effectiveness
|How to cite this article:|
Joseph P, Yerol N, Nairy R, Sanjeev G, Bhat N N. Assessment of electron and gamma-induced dna damage in human peripheral blood by alkaline comet assay. Radiat Prot Environ 2011;34:221-4
|How to cite this URL:|
Joseph P, Yerol N, Nairy R, Sanjeev G, Bhat N N. Assessment of electron and gamma-induced dna damage in human peripheral blood by alkaline comet assay. Radiat Prot Environ [serial online] 2011 [cited 2020 Jun 6];34:221-4. Available from: http://www.rpe.org.in/text.asp?2011/34/4/221/106072
| 1. Introduction|| |
The ionizing radiation, which deposits energy by its interaction with biological material, is a potent inducer of DNA damage due to both free radical formation and direct DNA interactions.  Ionizing radiation is a potent inducer of DNA damage because it causes single and double-strand breaks, alkali-labile sites, base damage, and cross-links. The single cell gel electrophoresis (comet assay) is a sensitive method to evaluate DNA damage at single cell level. It provides a very sensitive method for detecting strand breaks and measuring DNA damages in single cells.
Radiation plays an important role in life science and medicine. Equal physical doses of different types of radiation do not produce equal biological effects because of differences in their energy deposition patterns. This difference must be considered in clinical applications that use alternative modalities to photons. This is taken into account by the concept of relative biological effectiveness (RBE). The consideration of RBE ensures that radiation oncologists can benefit from the large clinical experience gained with photon beams.
Therefore, in the present study, the alkaline comet assay has been applied to assess the radiation-induced DNA damage in human peripheral blood exposed to electron radiation from microtron accelerator and gamma radiation from 60 Co source. The RBE for electron radiation with reference to gamma radiation in the induction of DNA damage was calculated from the observed DNA damage.
| 2. Materials and Methods|| |
2.1. Sample preparation and irradiation
Peripheral blood samples were collected from a 28-year-old male donor having no pre-history of radiation by venipuncture in heparinized vials. 1 ml of blood was cultured with 9 ml of culture medium (RPMI-1640, Sigma) supplemented with 10% fetal calf serum (Himedia). Radiation treatment has been carried out using electron beam from microtron accelerator at Mangalore University. It is a pulsed mode circular accelerator offering an electron beam with a maximum pulse current of 50 mA and pulse duration of 2.5 μs, the details of which are given elsewhere.  Dosimetry and irradiation of the blood samples were carried out at 30 cm from the beam exit point of the accelerator. Samples were irradiated at the center of a 4 cm × 4 cm area with a uniform dose rate.  For irradiating the samples, eppendorf tubes of 1.5 ml volume were used. Since the range of the electrons in water phantoms was measured as 3 cm at the sample position,  the electron dose distribution within the sample was nearly uniform. Gamma irradiation was carried out at a dose rate of 2 Gy min -1 using 60 Co gamma source. The total dose delivered to the samples was varied from 0 to 4 Gy. Samples were mainted at 0 °C before irradiation, and the comet assay was carried out immediately after irradiation. The experiments were repeated 3 times under identical conditions.
2.2. Comet assay
After irradiation, to quantify the DNA damage, the comet assay (single cell gel electrophoresis) was carried out under alkaline conditions, by the methods outlined by Singh et al.  100 μl of cell suspension in complete media was mixed with 100 μl of low melting point agarose (0.75% in PBS) at 37 °C and layered on frosted slides pre-coated with 200 μl of normal melting point agarose (1.5%). Cover slips were immediately placed, and the slides were put to 4° C. After solidification, a layer of 200 μl of low melting point agarose (0.75%) was added to the slides. The cover slips were removed, and slides were placed in a chilled lysing solution containing 2.5 M NaCl, 100 mM Na 2 -EDTA with freshly added 1% Triton X-100 and 10% DMSO, for 12 h at 4° C. The slides were then removed from the lysing solution and washed with alkaline buffer and placed on a horizontal electrophoresis tank filled with freshly prepared alkaline electrophoresis buffer (300 mM NaOH, 1 mM Na 2 EDTA, pH ≥13.0). The slides were equilibrated in the same buffer for 30 min, and electrophoresis was carried out at 0.74 Vcm -1 , 300 mA for 25 min. After electrophoresis, the slides were washed gently with 0.4 M Tris base buffer (pH 7.4) to remove the alkali. The slides were stained with ethidium bromide and visualized at 40× magnification using Olympus BX 51 fluorescent microscope. Jenoptik C5 cooled CCD camera were attached to the microscope, and camera control software used was ProgRes Capture Pro 2.8.
Images of 50 cells per slide were acquired using the digital imaging system and stored. For comparison of DNA damage, the quantitative measurement of the olive tail moment (OTM) was carried out using Comet Assay Software Project (CASP). OTM is the product of percentage of damaged DNA (TDNA) and tail length (TL), which is an internationally most accepted parameter for comparing the DNA damage. 
2.3. Statistical analysis
Statistical analysis of the data generated from the comet assay was carried out by importing the result file to the software Microcal Origin Version 8. Comet parameter OTM was used for comparison of data between control and radiation exposed groups. Data are presented as the mean ± standard deviation (S.D.). The differences in DNA damage between control and irradiated groups and between two irradiated groups were analyzed using Student's t-test. Differences where P < 0.05 were considered to be statistically significant.  To calculate the RBE, the iso-effective doses obtained from the fitted curves were compared between the electron beam and gamma rays.
| 3. Results and Discussion|| |
3.1. DNA damage
In the preset study, the induction of DNA strand breaks in human peripheral blood after electron, and gamma irradiation has been investigated using comet assay in alkaline condition. The optimized and standardized comet assay protocol was used in order to analyze the amount of induced DNA damage in single cells. In the present study, different comet parameters were recorded using Comet Assay Software Project (CASP), and to describe the DNA damage, the OTM is used. [Figure 1] shows images of two cells analyzed with the comet assay. Cell scoring showed no DNA migration in unirradiated cell, which in fact appeared as nucleoids. Cells with few strand breaks have a spherical shape, while those with a high frequency of strand breaks present a tail of DNA streaming out from the nucleoid, forming a comet-like appearance. In irradiated cells, DNA fragments migrated out of the head to form the comet tail, thus increasing both comet length and percentage of DNA in the tail.
|Figure 1: Comet assay image of DNA under normal and irradiated conditions|
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Results of the alkaline comet assay have been summarized in [Figure 2], and it shows the effect of different dose of electron and gamma radiation on human peripheral blood cells. In the [Figure 2], the OTM values are normalized with control value to avoid initial variations in different experiments. A significant increase in DNA strand breaks as indicated by an increase in OTM was observed for all the doses employed. The dose response of DNA damage of human blood cells after electron irradiation has been fitted by an error-weighted minimum Chi 2 method to a linear-quadratic model represented by OTM electron = c + αD + βD 2 where D represents the dose in Gy, c is the background DNA damage, and α and β are linear and quadratic coefficients, respectively, and the coefficients α and β of the dose response curve were thus estimated. The variation in OTM with dose after electron irradiation is fitted to, OTM electron =1 + (4.14 ± 1) D + (1.16 ± 0.29) D 2 (R 2 = 0.99, chi 2 = 1.76). The value of the coefficients α and β were found to be 4.14 Gy -1 and 1.16 Gy -2 , respectively, for electron radiation; the corresponding values for gamma radiation were 4.57 ± 0.65 Gy -1 and 0.43 ± 0.19 Gy -2 (R 2 = 0.99, Chi 2 = 0.74). From these data, it is clear that, the gamma dose response on DNA damage induction is almost linear with a smaller quadratic component (α/β =10.6), but for electron irradiation, the response is linear quadratic with a larger quadratic component. This can be attributed to the high dose rate irradiation (100 Gy min -1 ) using electron, whereas the dose rate for gamma irradiation was 2 Gy min -1 . The larger quadratic component in the case of electron irradiation could be possibly due to the lesser time available for repair during irradiation as the given dose is delivered at a higher dose rate compared to gamma irradiation.
|Figure 2: Variation of olive tail moment with dose for human peripheral blood cells after electron and gamma irradiation|
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3.2. Relative biological effectiveness
From the [Figure 2], it is clear that there is a difference in the response of human peripheral blood cells towards 8 MeV electrons and 60 Co gamma rays when DNA damage induction was studied as an endpoint, and this difference can be estimated in terms of RBE. The RBE was determined from the doses of gamma rays and electrons resulting in the same DNA damage, and it was calculated from the [Figure 2] for different DNA damage levels and shown in [Table 1]. From the table, it can be seen that at all dose points, the RBE was found to be higher than unity, indicating electrons are more effective in inducing DNA damage per unit absorbed dose compared to gamma rays. The average RBE has been calculated, and it is found to be 1.20 ± 0.04. In addition, the value of RBE determined as the ratio of α coefficients of the dose-response curves of electrons and gamma rays was found to be 1.1. Santhosh et al.  has reported an RBE value of 1.12 ± 0.27 for 8 MeV electrons in comparison to the 60 Co gamma rays in MN induction. Therefore, the observed RBE of electron beam on DNA damage induction is comparable with those observed in MN induction.
|Table 1: The relative biological effectiveness of electron with respect to gamma in inducing DNA damage in human peripheral blood cells measured at different olive tail moment values|
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| 4. Conclusions|| |
The DNA damage in human blood cells increases with absorbed dose of electron and gamma radiation. Electrons are more effective in inducing DNA damage per unit absorbed dose compared to gamma rays. The RBE for 8 MeV electron radiation estimated from the comet assay is comparable to that obtained by MN assay.
| References|| |
|1.||Da Cruz AD, Glickman BW. Nature of mutation in the human hprt gene following in vivo exposure to ionizing radiation of cesium-137. Environ Mol Mutagen 1997;30:385-95. |
|2.||Siddappa K, Ganesh S, Balakrishna KM, Ramamurthi SS, Soni HC, Shrivastava P, et al. Variable energy microtron for R and D work. Radiat Phys Chem 1998;51:441-2. |
|3.||Gupta BL, Narayan GR, Nilekani SR, Bhat RM, Kaul A, Bemalkhedkar MM, et al. Preliminary dosimetry studies for microtron using chemical dosimetry. Radiat Prot Environ 1999;22:169-74. |
|4.||Singh NP, Mc Coy MT, Tice RR, Schneider EL. A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res 1988;175:184-91. |
|5.||Chaubey RC, Bhilwade HN, Rajagopalan R, Bannu S, Kulgod SV, Chadda VK. SCGE-Pro: A Fluorescence based Digital Imaging System developed for measuring DNA damage using Single Cell Gel Electrophoresis (Comet assay). BARC Newsletter. In: Kumar V, editor. Mumbai, India: Bhabha Atomic Research Center; 2000. p. 1-13. |
|6.||Chaubey RC, Bhilwade HN, Rajagopalan R, Bannur SV. Gamma ray induced DNA damage in human and mouse leucocytes measured by SCGE-Pro: A software developed for automated image analysis and data processing for Comet assay. Mutat Res 2001;490:187-97. |
|7.||Acharya S, Sanjeev G, Bhat NN, Siddappa K, Narayana Y. Relative biological effectiveness for the induction of micronuclei in human blood lymphocytes after in-vitro irradiation with 8 MeV electrons from Microtron. Radiat Protect Environ 2006;29:45-8. |
[Figure 1], [Figure 2]
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