Measurements of DNA damage on silver stained comets using free Internet software

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1 Mutation Research 627 (2007) Short communication Measurements of DNA damage on silver stained comets using free Internet software Omar Garcia a,, Ivonne Romero b, Jorge E. González a, Tania Mandina a a Centro de Proteccion e Higiene de las Radiaciones Havana, Calle 20 No 4113 e/41 y 47 Playa, AP 6195, La Habana, Cuba b Facultad de Biología, Universidad de la Habana, La Habana, Cuba Received 22 June 2006; received in revised form 17 November 2006; accepted 24 November 2006 Available online 3 January 2007 Abstract Silver stain offers the possibility to stain comets permanently, but up to now it was impossible to measure the majority of the comet parameters, because the distinction between head and tail was not recognised by software. Here, we report a silver staining protocol that allows the measurement of comet parameters using the free Internet software CASP. We validated the silver stain protocol by comparing the behaviour of the parameter % DNA in tail in silver and fluorescent stained comets. The range of % DNA in tail for different visual categories of damage in silver stained comets was similar to that reported with fluorescence staining. The range was for category 0 (no damage), <1%; category 1 (low damage), 1 25%; category 2 (medium damage), >25 45%; category 3 (high damage), >45 70%; category 4 (very high damage), >70%. The mean of % DNA in tail in silver stained comets was also similar to that reported with fluorescence staining. The mean was for category 0, 0.4 ± 0.34%; category 1, 12 ± 7%; category 2, 37 ± 4%; category 3, 57 ± 5% and category 4, 83 ± 6%. Others comet parameters such as tail length, tail moment and Olive tail moment can be also measured. The silver staining protocol reported here opens new opportunities for those working in the assay without fluorescent microscope as the measurement of comet parameters using free Internet software and conventional microscope becomes possible Elsevier B.V. All rights reserved. Keywords: Comet assay; Silver staining; DNA in tail; Free Internet software 1. Introduction The comet assay (single cell gel electrophoresis) is widely used to evaluate for DNA damage and repair in eukaryotic cells. The popularity of this test is due to its sensitivity, relatively low cost and simplicity. The most common way to visualise the comets is by using a fluorescent dye such as ethidium bromide, DAPI, acridine orange, propidium iodide, etc. These dyes are easy to use, as they do not require special treatment Corresponding author. Tel.: ; fax: addresses: omar@cphr.edu.cu (O. Garcia), ivonne@cphr.edu.cu (I. Romero), jorgee@cphr.edu.cu (J.E. González), tania@cphr.edu.cu (T. Mandina). of gel or time-consuming procedures, but the use of fluorescent microscope represent for less developed laboratories a serious limitation for the use of the assay. Silver stain offers the possibility to stain comets and visualise it using conventional microscope. Silver stain has been used to stain both proteins and DNA in polyacrylamide gels and during the 80 s it became possible to use this stain to quantitate the amount of protein and DNA in agarose gels [1 3]. Nevertheless, to date the application of silver stain to the comet assay has been very limited. The most probable reason for this is that in the early silver staining protocols the quality of the comet images was not as good as when fluorescent dyes were used. In fact, in silver stained comets, it was impossible or difficult to distinguish between comet /$ see front matter 2006 Elsevier B.V. All rights reserved. doi: /j.mrgentox

2 O. Garcia et al. / Mutation Research 627 (2007) head and tail, and consequently the only comet parameter possible for measurement was total DNA migration [4] or visual classification of comets [5 7]. In addition for some silver staining protocols, the background was excessive. We recently published a silver staining method that permits a better distinction between the comet head and tail. With this protocol, we visually classified comets into five categories according to the relative proportion of DNA in tail and head with the expression of DNA damage in arbitrary units (AU) [8,9]. We tried to measure comets stained according to this protocol using various free Internet image analysis programmes (CometScore, ScionImage and CASP), but because of the poor contrast between the head and tail, the software was unable to identify unequivocally between these regions, particularly in the most damaged comets. Nevertheless, after ours initial experiences, the software CASP was chosen as the most convenient for our experimental conditions. We then focused our efforts on improving the staining protocol. The efficiency of the silver stain pattern varies with the duration of the development period and with the concentration of various reagents [1,2]. Thus, we modified our previous staining protocol, and particularly these two variables, until we obtained a contrast between head and tail that could be recognised by the software for all categories of visual comet classification. We emphasise the measurement of % DNA in tail as this parameter is generally recommended for expressing the results of the comet assay [10]. Additionally, the range of % DNA in tail and the mean of % DNA in tail have been reported for the five categories of visual classification of comets stained with fluorescent dyes [11 13]. We used these values as references to validate the results obtained with the modified silver staining protocol. This evaluation demonstrates that the % DNA in tail can be measured in silver stained comets. Others comet parameters such as tail length, tail moment and Olive tail moment can be also measured. This new staining protocol opens new opportunities for those interested in performing the assay without a fluorescent microscope as the measurement of comet parameters using free Internet software and conventional microscope becomes possible. 2. Materials and methods 2.1. Induction of DNA damage and comet assay DNA damage was induced in human lymphocytes by gamma irradiation. A blood sample was collected by a finger prick of a female non-smoker aged 22 years, and irradiated with doses of 0, 0.25, 0.5, 1.0 and 2.0 Gy, using a 60 Co source with 0.5 Gy/min dose rate at room temperature. After irradiation the blood was kept in ice. The aim of the irradiation was to obtain enough quantities of all comets categories. A total of two slides per dose were obtained, and three experiences were performed in three different days. Lymphocytes were isolated after irradiation to simulate in the most realistic way the in vivo irradiation exposure. The slides were prepared and the comet assay was performed as described previously [8,9]. Briefly: to prepare each slide, approximately 30 l of blood plus 1 ml phosphate buffered saline (PBS) were mixed in a 1.5 ml Eppendorf tube, and left on ice for 30 min. The lymphocytes were isolated using Histopaque After isolation lymphocytes were embedded in 140 l of 1% low melting point agarose in PBS at 37 C. Cells were then transferred as two roughly equal drops to microscope slides (frosted at one end), precoated with 1% normal agarose and dried. Each drop was covered with an 18 mm 18 mm coverslip and left in the refrigerator for 5 min, after which the coverslip was removed. Basic steps of the assay were performed at 4 C as follow (a) lysis: 1 h, in lysis buffer (2.5 M NaCl, 0.1 M EDTA, 10 mm Tris, 1% (v/v) Triton X-100, ph 10), (b) alkaline unwinding: 40 min in electrophoresis solution (0.3 M NaOH, 1 mm EDTA, ph 14), (c) electrophoresis: 30 min, 300 ma, 30 V, 1 V/cm and (d) neutralization: 5 min; three times in neutralising buffer (0.4 M Tris ph 7.5) Modification of the silver staining protocol The modifications in the protocol published previously [8,9] were: (a) the concentration of the solution B (see below for definition) was reduced by 50%, (b) the staining time was reduced to 20 min, (c) the staining was conducted without shaking and (d) the solutions were stored at 4 C and the staining, using the cold solutions took place at room temperature (25 C). Briefly the full staining protocol is as follows. After electrophoresis and neutralisation, slides were (a) washed twice with deionised water, (b) dried overnight at 37 C, (c) fixed for 10 min in fixation solution (15% trichloroacetic acid, 5% zinc sulphate heptahydrate, 5% glycerol), (d) washed twice with deionised water, (e) dried h at 37 C, (f) re-hydrated for 5 min in deionised water, (g) placed back-to-back in a horizontal staining jar, (h) stained for 20 min at room temperature (25 C) in the dark, without shaking, using 100 ml of freshly prepared stain solution comprising 34 ml of vigorously mixed stock solution B (0.05% ammonium nitrate, 0.05% silver nitrate, 0.125% tungstosilicic acid, 0.075% formaldehyde, v/v) and 66 ml of stock solution A (5% sodium carbonate) prepared the same day as the staining, (i) washed 2 3 times with deionised water, (j) immersed 5 min in a stop solution (acetic acid 1%) and (k) air-dried. The solutions, except the water for washing and the stop solution were used at 4 C. The solution B was prepared from convenient stocks solutions of ammonium nitrate, silver nitrate and tungstosilicic acid. These stocks solutions can be maintain at 4 C for several weeks.

3 188 O. Garcia et al. / Mutation Research 627 (2007) Measurement of DNA damage using the software CASP The software CASP was downloaded from casp.of.pl [14]. Thresholds of CASP parameters were adjusted to obtain the optimal values for our staining protocol. The selected parameters were: Head center threshold (HCT) = 0.95, Comet thresholds (CT) = 0.05, Head threshold (HT) = 0.05, Tail threshold (TT) = 0.1 and profile 1. One hundred of each class of visually classified comets from category 0 (undamaged, no discernible tail) to category 4 (almost all DNA in tail), according to the criteria described previously [8], were selected for image analysis using CASP. First of all the comet parameter recorded was the % DNA in tail for the reasons explained in the introduction; even though the program offers other comet parameters (tail length, tail moment, etc.) as well (see Section 3.3). The images were captured using an Olympus microscope BH2-RFCA with a Sony camera SSC-C370 and digitised using the ATI video player software. The slides were analysed using 100 magnification. Previously we compared 100 comets of different categories using 100 and 200 magnification and obtained similar values of % DNA in tail, for all categories of comets (Pearson r = with p = ). The use of 100 magnification allows a faster analysis of gels Comparison of results obtained with silver and fluorescence stained comets The range and mean of % DNA in tail obtained for each category of comet was compared with previously published ranges for the same categories of comet obtained in human lymphocytes [11,13], while the range were compared to that obtained for Jurkat cells or isolated nuclei from rat cardiac or skeletal muscle [12]. 3. Results and discussion Examples of images obtained with the old and the new silver staining protocols are provided in Fig. 1. As can be seen, the new staining conditions allow for a clearer distinction of comet head and tail. This contrast is recognised by the software CASP for all comet categories. In all slides the contrast was similar. The critical point then was to evaluate whether the deposition of silver ions was proportional to DNA quantity. Silver stain is suitable for the detection of both native and denatured DNA in a quantitative manner using an internal standard in the gel [2]. The % DNA in tail is the fraction of DNA in tail relative to the whole image. To estimate the % DNA in tail, the software uses as reference the background and the intensity in comet head and tail; consequently, if the deposition of silver ions is proportional to DNA Fig. 1. Examples of images obtained with the old (left) and the new (right) silver staining protocol. The numbers 3 and 4 indicate the comet category. The software CASP generates the frame to limit the comet area, the line to limit the tail length, the circle to identify the comet head and the cross to identify the head center. The small rectangle at the bottom of each picture is generated by CASP as background reference. In the left pictures the software was unable to identify the comet head. In the right picture the software correctly identify comet head. Magnification 200. quantity, the % DNA in tail using silver staining or fluorescent dye should be similar. As can be seen below, we obtained similar results with silver stain to those obtained previously with three different fluorescent dyes, three different image analysis softwares, and considering two different parameters, the range of % DNA in tail and the mean of % DNA in tail in the five categories of comets. Additional the proportionality of silver deposition on DNA was verified by plotting the % DNA in tail versus the total DNA, on a cell-by-cell basis, for all comet categories (see Fig. 2). Slight increase in total DNA contents with an increasing fraction of DNA in tail was found. Opposite trend was reported in DAPI stained comets [10]. These differences can be explained considering the Fig. 2. Relationship between total DNA content and percentage of DNA in tail. DNA content was calculated as the sum of pixel intensities. A total of 100 comets were included in the graphic.

4 O. Garcia et al. / Mutation Research 627 (2007) Table 1 Percentage of DNA in tail for different categories of comets obtained in the present work and published previously Comet category Percentage of DNA in tail according to: Percentage of DNA in tail [present work] Noroozi et al. a [11] Yasuhara et al. b [12] Range Mean ± S.D [0 1) 0.4 ± [1 25) 12 ± [25 45) 37 ± [45 70) 57 ± 5 4 > >70 83 ± 6 a Electrophoresis conditions: 25 V for 30 min, ph 13. Amperage no reported. b Electrophoresis conditions: 1 V/cm for 20 min in neutral buffer. Amperage no reported. DNA structure in tail and the staining peculiarities with DAPI and silver. DNA in tail should be single stranded and seems to be a halo of relaxes loops. DAPI binds to the major groove, and fluorescence should be dependent on double stranded structure, so the higher the proportion of DNA in tail, the less the total fluorescence detected in the whole comet should be [10]. Silver stain allow the effective detection of both single and double stranded DNA [2] and the relaxed loops should allow better deposition of silver in the single stranded DNA presented in the tail, so the higher the proportion of DNA in tail, the more the total DNA detected in the whole comet should be The range of percentage of DNA in tail for the five categories of visual classification of comets The range of % DNA in tail for the different visual classes of comets and the comparison with previously published results, are provided in Table 1. The values obtained for silver stained comets are essentially similar to those reported previously for the same categories of comets stained with two different dyes, ethidium bromide [11] and SYBR Gold [12] and analysed by two different software, the BRS2 Image Analyser (Imaging Research Inc.) [11] and the Scion Image downloaded from pages/download now.htm [15]. These data refute the idea that % DNA in tail cannot be comparable between laboratories due to differences between comet programs and calibration. In this particular situation, the electrophoresis conditions (voltage and time) in the three compared reports were similar, a fact that may be important when comparing results The mean of percentage of DNA in tail for the five categories of visual classification of comets The mean of % DNA in tail for the different visual classes of comets are provided in Table 1. The previous published values, derived from a graphic reported in literature [13] were for category 0 around 6%, for category 1 around 16%, for category 2 around 32%, for category 3 around 51%, and for category 4 around 84%. The mean of % DNA in tail for category 0 reported in literature [13] is slightly higher than in the present work, and is also out of published ranged (see Table 1). This may be explained by differences between scorers in the visual criteria used for identification of comets, an effect that is probably most important at low levels of DNA damage [8]. For the remaining categories, the mean of % DNA in tail is similar to those reported here (see Table 1). In conclusion, the values obtained by us are very close to those reported previously for the same categories of comets from human lymphocytes stained with DAPI and analysed by the software Kinetic Imaging Comet 2.2. The electrophoresis conditions in the two compared reports were identical. Table 2 Tail length, tail moment and Olive tail moment for different categories of comets Comet category Tail length ( m) Tail moment Olive tail moment Range Mean ± S.D. Range Mean ± S.D. Range Mean ± S.D ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 7.4

5 190 O. Garcia et al. / Mutation Research 627 (2007) Others comet parameters for the five categories of visual classification of comets Tail length, tail moment and Olive tail moment, are also popular parameters in the comet assay. These parameters are provided in the Table 2 for different categories of comets. Previous reports of these parameters for the five categories of comets were not found in the literature. As can bee see, some overlap is presenting in different ranges reflecting the fact that tail length and percentage of DNA in tail are not necessary associated. 4. Conclusion The modifications of the silver staining protocol allow the use of the software CASP combined with conventional microscope to measure the most common parameters used in comet assay to express DNA damage. It is particular important, the possibility to measure % of DNA in tail probably the most useful parameter in the comet assay. This procedure offers an opportunity for those working with the assay without fluorescent microscopy and should contribute to a more extensive use of the assay. Acknowledgements The editorial assistance of Andrew Collins is greatly appreciated. This work was supported by the project PI/CPHR/04-01 from the Centre from Radiation Protection and Hygiene. References [1] S. Peats, Quantitation of protein and DNA in silver-stained agarose gels, Anal. Biochem. 140 (1984) [2] M. Gottlieb, M. Chavko, Silver staining of native and denatured eucaryotic DNA in agarose gels, Anal. Biochem. 165 (1987) [3] C.R. Merril, Silver staining of proteins and DNA, Nature 343 (1990) [4] P. Reinhardt-Poulin, J.R. McLean, Y. Deslauriers, W. Gorman, S. Cabat, M. Rouabhia, The use of silver-stained comets to visualize DNA damage and repair in normal and xeroderma pigmentosum fibroblasts after exposure to simulated solar radiation, Photochem. Photobiol. 71 (2000) [5] H. Cerda, H. Delincée, H. Haine, H. Rupp, The DNA comet assay a rapid screening technique to control irradiated food, Mutat. Res. 375 (1997) [6] S.B. Nadin, L.M. Vargas-Roig, D. Cioca, A silver staining method for single-cell gel assay, J. Histochem. Cytochem. 49 (2001) [7] N. Kizilian, R.C. Wilkins, P. Reinhardt, C. Ferrarotto, J.R.N. McLean, J.P. McNamee, Silver-stained comet assay for detection of apoptosis, Biotechniques 27 (1999) [8] O. García, T. Mandina, A.I. Lamadrid, A. Díaz, A. Remigio, Y. González, J. Piloto, J.E. González, A. Alvarez, Sensitivity and variability on visual scoring comets. Results of the slide scoring excercise with the use of silver stained comets, Mutat. Res. 556 (2004) [9] O. García, T. Mandina, DNA damage evaluated by the comet assay in lymphocytes of children with 137 Cs internal contamination caused by the Chernobyl accident, Mutat. Res. 565 (2005) [10] A.R. Collins, The comet assay for DNA damage and repair: principles, applications and limitations, Mol. Biotech. 26 (2004) [11] M. Noroozi, W.J. Angerson, M.E.J. Lean, Effects of flavonoids and Vitamin C on oxidative DNA damage to human lymphocytes, Am. J. Clin. Nutr. 67 (1998) [12] Sh. Yasuhara, Y. Zhu, T. Matsui, N. Tipirneni, Y. Yasuhara, M. Kaneki, A. Rosenzweig, J.A.J. Martyn, Comparison of comet assay, electron microscopy and flow cytometry for detection of apoptosis, J. Histochem. Cytochem. 51 (2003) [13] A.R. Collins, A.-G. Ma, S.J. Duthie, The kinetics of repair of oxidative DNA damage (strand breaks and oxidised pyrimidines) in human cells, Mutat. Res. (DNA Repair) 336 (1995) [14] K. Konca, A. Lankoff, A. Banasik, H. Lisowska, T. Kuszewski, S. Gozdz, Z. Koza, A. Wojcik, A cross-platform public domain PC image-analysis program for the comet assay, Mutat. Res. 534 (2003) [15] Sh. Yasuhara, Personal communication.