Genetics and Molecular Biology, 32, 1, 155-158 (2009) Copyright © 2009, Sociedade Brasileira de Genética. Printed in Brazil www.sbg.org.br Short Communication Application of the comet assay in erythrocytes of Oreochromis niloticus (Pisces): A methodological comparison Cintya A. Christofoletti1, José Augusto O. David2 and Carmem S. Fontanetti1 1Departamento de Biologia, Instituto de Biociências, Universidade Estadual Paulista “Júlio de Mesquita Filho”, Rio Claro, SP, Brazil. 2Departamento de Genética, Instituto de Biociências, Universidade Federal do Pará, Belém, PA, Brazil. Abstract The present study applied the comet assay to erythrocytes of Oreochromis niloticus with the aim of improving proto- cols to detect DNA damage in these cells, by using two distinct pHs (pH = 12.1 and pH > 13) and evaluating whether there is a correspondence between silver and ethidium bromide staining. Comets were visually examined and, the frequency of cells with and without damage was obtained, as well as the distribution of classes and scores. By using the Kruskal-Wallis test, our results revealed that pH 12.1 is more effective, although both pHs can be used. Our find- ings also suggest that silver staining can substitute ethidium bromide, an expensive and highly toxic stain that re- quires specific equipment for examination. Key words: comet assay, ethidium bromide, silver staining, tilapia. Received: April 2, 2008; Accepted: September 5, 2008. The development of new methodologies and the ap- sensitivity of the blood cells of these animals to genotoxic plication of more sensitive assays to detect genotoxicity in effects (Padrangi et al., 1995; Belpaeme et al., 1998; Gon- different samples have been the subject of several scientific tijo et al., 2003). Thus, the aim of the present study was to studies on environmental monitoring (Al-Sabti and Metcal- apply the comet assay to erythrocytes of specimens of the fe, 1995; Lemos et al., 2005). The Comet Assay, or single fish Oreochromis niloticus, in order to improve protocols cell gel electrophoresis, is a sensitive genotoxicological for analyzing DNA damage in these cells, by using two dis- method for assessing DNA damage in single cells, allowing tinct pHs, and evaluate whether there is a correspondence for the quantification of DNA breaks and alkaline labile between silver and ethidium bromide staining. sites. Compared with other genotoxicity tests, the advan- For the bioassay, two 20 L aquaria were filled with tages of the comet assay are the detection of slight DNA water from an artesian well, this then being aerated for 48 h damage, the low number of cells required, low cost, preci- and maintained at a room temperature ranging from 20 to sion, ease of application, reproducibility, and short period 23 °C, prior to the experiment. Five specimens of of time to conduct the experiment (Belpaeme et al., 1998; Oreochromis niloticus (Perciformes, Cichilidae), com- Tice et al., 2000; Bücker et al., 2006). monly known as Nile tilapia, were placed in the aquaria (ta- This technique is the result of studies undertaken by ble 1). Fishes were obtained from a fish tank of the Institute Östling and Johanson, who developed the methodology of of Biosciences of the São Paulo State University, Rio Claro DNA electrophoresis in micro-gel, and those by Singh et al. campus. The fishes were not fed during the experiment and (1988), who improved the technique. Currently, several in- were exposed to a 14:10 h light/dark cycle, under constant ternational research groups have published recommenda- aeration. These were then divided into two groups, treated tions describing protocols and criteria for the comet assay, and non-treated. Non-treated fishes were injected intra- aiming at establishing high standards to obtain valid, repro- peritonially with saline solution (30 mL of physiological ducible, and accurate data (Klaude et al., 1996; Brendler- solution/50 g of fish) 72 h prior to the end of the experi- Schwaab et al., 2005; Di-Paolo, 2006). ment. The treated group received an intraperitonial injec- The comet assay has been successfully applied in tion of cyclosphosphamide (20 mg/mL) (30 mL of cyclo- erythrocytes of several fish species, thereby showing the sphosphamide /50 g of fish), also 72 h prior to the end of the experiment. Send correspondence to Carmem S. Fontanetti. Departamento de Biologia, Instituto de Biociências, Universidade Estadual Paulista The comet assay was undertaken according to Singh “Júlio de Mesquita Filho”, Av. 24-A, 1515, 13506-900 Rio Claro, SP, et al. (1988) and Villela et al. (2006), with certain modifica- Brazil. E-mail: [email protected]. tions, as follows. Slides were previously covered with 1.5% 156 Christofoletti et al. Table 1 - Mean and standard deviation of the weight and size of fishes used in the comet assay for both treatments. Treatments Weight (g) Size (cm) Non-treated 37.1 ± 10.60 13.24 ± 1.66 Treated 41.34 ± 8.01 13.98 ± 0.82 normal melting point agarose. Cardiac punctures were per- formed and 0.3 mL of blood was drawn from the speci- mens. The assay was done in triplicate and later evaluated regarding the effect of different pHs and staining. After car- m Figure 1 - Comet assay applied in erythrocytes of O. niloticus using pH diac punctures, 5 L of the blood sample were diluted in 12.1 stained with ethidium bromide (A. Class 0, B. Class 1, C. Class 2) and 1000 mL of PBS. 10 mL of cell suspension with 120 mLof silver (D. Class 0, E. Class 1, F. Class 2). 0.5% low melting point agarose at 37 °C, were layered on previously prepared slides, cover-slipped and placed in the refrigerator for 5 min, for solidification of the gel. The then washed again three times with distilled water and after cover-slips were then removed and the slides immersed in a allowed to dry at room temperature. They were later exam- freshly-prepared lysis solution (1 mL of Triton-X 100, ined under a light microscope at 400x magnification. 20 mL of DMSO and 79 mL of lysis stock solution: 2.5 M One hundred comets per slide were examined using NaCl, 100 mM EDTA and 10 mM Tris, pH 10.0-10.5) for a visual classification based on the migration of DNA frag- minimum of at least one hour and a maximum of two, at ments of the nucleus. Comets were classified into four 4 °C. After lysis, slides were transferred to a horizontal classes: class 0 (no damage), class 1 (little damage), class 2 electrophoresis tank. For the first two runs, alkaline solu- (medium damage), and class 3 (extensive damage). Data tion was added (300 mM NaOH and 1 mM EDTA, were presented as the frequency of damaged cells, class dis- pH = 12.1) for 20 min. Upon completion of the relaxing tribution, and damage scores, calculated as the sum of cells time, slides were subjected to electrophoresis for 15 min at in each class and the total number of cells in each class mul- 21 V and 270 mA (0.8 V.cm-1). The same procedures were tiplied by the number of classes (0-3). Scores ranged from followed for the last run, although an alkaline buffer zero (all cells with no damage - 0x100) to 300 (all cells with (NaOH 300 mM and 1 mM EDTA, pH > 13) was used in maximum damage 3x100), according to Rigonato et al. this case. All procedures were carried out under indirect (2005). Statistical analysis was performed using the Krus- light. After electrophoresis, slides were carefully removed kal-Wallis test, with the level of significance set at p < 0.05. from the tank. Neutralization was performed through three The results obtained here for the comet assay in baths of 5 min with a neutral buffer (0.4 M Tris-HCl, pH Oreochromis niloticus erythrocytes showed similar images 7.5) to remove salts and detergents. Slides were then al- (Figure 1) and score (Table 2) for both staining procedures lowed to dry at room temperature. when pH 12.1 was used in the assay. For ethidium bromide staining (Figure 1A-C), slides In the untreated group, a cell statistical difference was were fixed with 100% ethanol for 10 min and allowed to observed between pH 12.1 and pH > 13 (Table 2). When dry at room temperature. These were then stained with an pH > 13 was used in this group, the number of class 0 com- aqueous solution of ethidium bromide (0.02 mg/mL) before ets decreased while class 1 comets revealed a significant examination under a fluorescence microscope (Leica increase (Table 2). This can probably be due to, the expres- DMLB), filter B-34 (excitation: i = 420 nM; barrier: sion of alkali-labile sites that occur only with pH > 13. I = 520 nM), magnification 1000x. Similar levels of damage were registered in treated For silver staining (Figure 1D-F), slides were im- group cells at pH 12.1 and pH > 13 (Table 2). The effect of mersed in a fixative solution (15% trichloroacetic acid, 5% treatment could be measured with both staining and pH, al- zinc sulfate, 5% glycerol), for 10 min in a vertical tank, though the difference between treated and untreated groups rinsed three times with distilled water and allowed to dry was greater at pH 12.1, probably because of the expression overnight at room temperature. They were then rehydrated of different DNA damage in pH > 13. for 5 min with distilled water and stained with 36 mL of so- According to Padrangi et al. (1995) and Lemos et al. lution A (5% sodium carbonate) and 54 mL of solution B (2005), different tissues of aquatic organisms can be used (0.1% ammonium nitrate, 0.1% silver nitrate, 0.1% silico- for the comet assay.