Radiol Oncol 2009; 43(2): 97-107. doi:10.2478/v10019-009-0015-y research article Usage of the standard and modified comet assay in assessment of DNA damage in human lymphocytes after exposure to ionizing radiation Morana Mikloš, Goran Gajski, Vera Garaj - Vrhovac Institute for Medical Research and Occupational Health, Mutagenesis Unit, Zagreb, Croatia Background. Human organisms are extremely sensitive to ionizing radiation, which has the strong genoto- xic effect on the DNA molecule. The aim of the present study was to detect the type of DNA damage and cell death caused by ionizing radiation as well as the sensitivity of the standard and modified comet assay. Methods. The effect of gamma radiation (0.1 Gy and 4 Gy) on human lymphocytes was observed using the standard alkaline and Fpg-modified comet assay with ability to detect oxidized purines as well as with the DNA diffusion test. Results. Parameters of the standard comet assay showed significantly higher values in samples exposed to 4 Gy than in samples exposed to radiation dose of 0.1 Gy and control sample. The Fpg-modified comet assay showed significantly higher values already at dose of 0.1 Gy as the result of oxidative DNA damage. The DNA diffusion test showed that gamma rays lead to apoptosis more often than to necrosis. Conclusions. This observation suggests that the standard alkaline and Fpg-modified comet assays as well as the DNA diffusion test are reliable techniques for estimation of DNA damage and form of the cell death caused by gamma radiation in vitro. In addition Fpg-modified comet assay prove to be more sensitive for detection of gamma irradiation induced DNA damage than the standard assay. Key words: human peripheral blood lymphocytes; ROS; gamma radiation; alkaline comet assay; Fpg- modified comet assay; diffusion test Introduction and medicine. People live and work near nuclear facilities and places of testing nu- Ionizing radiation is nowadays omnipres- clear weapon and also use nuclear (gamma) ent in human lives because of the increas- radiation in medical purposes. In each ing development of technology, industry case, human organism bears the strong genotoxic effect making the structure of the Received 21 January 2009 DNA molecule unstable and causing the Accepted 19 March 2009 genesis of many changes in it. 1 The damage Correspondence to: dr. Vera Garaj-Vrhovac, Institute of the genetic material caused by ionizing for Medical Research and Occupational Health, radiation is one of the best precondition in- Mutagenesis Unit, Ksaverska cesta 2, 10000 Zagreb, Croatia. Tel. +385 1 4673 188; Fax. +385 1 4673 303; dicator for development of malign diseases E-mail: [email protected] such as breast, gall-bladder or thyroid can- 98 Mikloš M et al. / Comet assay in assessment of DNA damage cer. 2,3 Gamma radiation affects the DNA granular DNA with a dense central zone structure directly, causing strand breaks, and a lighter and hazy outer zone, giving or indirectly causing cleavage of the water the overall appearance of a halo. 26 molecules and damaging the DNA mol- Peripheral blood lymphocytes were ex- ecule by reactive oxygen species (ROS). 4 posed to gamma radiation doses of 0.1 Strand breaks and oxidative damage in the Gy and 4 Gy in vitro. In that manner, the DNA causes further cell death in the form aim of this study was to detect the type of of apoptosis or necrosis. 5 These changes DNA damage caused by ionizing radiation. can be examined at human lymphocytes Considering that, two forms of the comet using different cytogenetic techniques. assay, standard alkaline and Fpg-modified The most frequently used cytogenetic tech- were used. In addition, sensitivity of both niques are the comet assay, micronucleus techniques toward different doses of gam- test, chromatid exchange test, chromosom- ma radiation was measured. The DNA dif- al aberrations test and fluorescence in situ fusion test was also used to detect the form hybridization (FISH) test. 6-10 of the cell death caused by gamma radiation. The comet assay (SCGE; single cell gel electrophoresis) is a rapid, simple, visual and sensitive technique for measuring and Materials and methods analyzing DNA damage at the single cell level. 6,11-17 This technique can be performed Blood sampling at the level of individual cells and requires a small number of cells per sample. Single The study was performed on peripheral cells can be used in in vivo and in vitro as blood samples obtained from a healthy well as in biomonitoring of population female non-smoking donor (age 24 years). exposed to radiation or chemical muta- The donor was not exposed to ionizing ra- gens. 6,18,19 The comet assay detects single diation, vaccinated or used medicals for a and double stranded breaks at the level of year before blood sampling. Whole venous DNA molecule, sites of incomplete repair, blood was collected under sterile conditions alkali labile sites, DNA-DNA and DNA- in heparinised vacutainer tubes (Becton protein cross-links. Besides, the comet as- Dickinson, NJ, USA) containing lithium say can be used for detection of the level of heparin as anticoagulant. After collection, the DNA fragmentation in apoptosis. 17, 20-22 blood was divided into a large number of In addition; particular enzymes such as for- samples. All experiments were conducted mamidopyrimidine glycosilase can be used on peripheral blood lymphocytes cultivated for detection of oxidative damage at the lev- at 37°C in an atmosphere of 5% CO 2 in air. el of the DNA molecule caused by ROS. 23-25 The DNA diffusion assay, a simple, sen- Exposure conditions sitive, and reliable cytogenetic method is often used for quantification of apoptosis, The whole blood samples were irradi- is based on the principle that nuclear DNA ated with gamma radiation on the ice. of apoptotic cells have abundant alkali- As a source of radiation Gammacell 220 labile sites and under alkaline conditions (Institute “Ruñer Bošković”, Zagreb, small pieces of DNA thus generates diffuse Croatia) was used. Vacutainers (volume in agarose, giving the appearance of a halo 5 cm 3) containing blood samples were ex- if stained with a sensitive fluorescent dye. posed to radiation doses defined as doses Apoptotic cells show a circular gradient of in the water, but irradiation was performed Radiol Oncol 2009; 43(2): 97-107. Mikloš M et al. / Comet assay in assessment of DNA damage 99 in the air. Samples were irradiated with 10% dimethyl sulfoxide (Kemika, Zagreb, radiation doses of 0.1 Gy and 4 Gy that Croatia). The slides were then placed on is equal to radiation periods of 32.3 s and a horizontal gel electrophoresis tank. The 23 min and 9 s at temperature of 21°C. unit was filled with electrophoresis buffer 27 Significance of the absorbed dose was 3%. (0.3 M NaOH, 1 mM Na 2EDTA; pH 13) and the To get homogenate samples, they were slides were placed in this alkaline buffer for stirred after irradiation, cooled to 4°C, 20 min. Denaturation and electrophoresis transported to the laboratory on ice and were performed at 4°C under dim light. processed as quickly as possible. Electrophoresis was carried out for 20 min at 25 V (300 mA). After electrophoresis the slides were rinsed with neutralization Determination of cell viability buffer (0.4 M Tris–HCl, pH 7.5). Each slide The indices of cell viability and necro- was stained with ethidium bromide (20 µg/ sis were obtained from differential stain- ml) and covered with a coverslip. The slides ing with acridine orange and ethidium were then stored in sealed boxes at 4°C until bromide, using fluorescence microscopy. 28 analysis. Lymphocytes were isolated using a modifi- cation of the Ficoll-Histopaque centrifuga- Fpg-modified comet assay tion method. 29 The slides were prepared using 200 µl of human peripheral blood For evaluation of possible oxidative DNA- lymphocytes and 2 µl of stain (acridine damaging effect of gamma radiation and orange and ethidium bromide, both diluted to test the sensitivity of the technique, in PBS). A total of 100 cells were analyzed modified version of the comet assay was to determine the percentage of viable cells performed using an Fpg FLARE™ assay using an Olympus AX-70 microscope with kit (Trevigen Inc, Gaithersburg, USA) with 60× magnification and 515–560 nm fluo- some modification. 30 Within the kit the rescence filters. The cells were classified manufacturer provided all the reagents according the following description: live used. Fully-frosted microscopic slides were cells with a functional membrane, with prepared. Each slide was covered with uniform green staining of the nucleus and 1% normal melting point (NMP) agarose necrotic cells with uniform red staining of (Sigma). After solidification, the gel was the nucleus. scraped off the slide. The slides were then coated with 0.6% NMP agarose. A low melt- ing point (LMP) agarose was melted and The alkaline comet assay stabilized in a water bath at 37°C. For each To evaluate DNA damage after irradiation sample and control, 5 µl of cell homogenate and to test sensitivity of the technique to- was mixed with 100 µl of LMP agarose and wards gamma radiation the comet assay was placed on the slides. After 10 min of solidi- carried out under alkaline conditions, basi- fication on ice, the slides were covered with cally as described by Singh et al. 14 Fully frost- 0.5% LMP agarose. The slides were then ed slides were covered with agarose (Sigma) immersed in a pre-chilled lysis solution containing the whole blood sample. The slides and kept in a refrigerator at 2°C for 60 min. were then immersed for 1 h in lysis solution Followed the immersion in the buffer, three (2.5 M NaCl, 100 mM Na 2EDTA, 10 mM times for 15 min.