|Year : 2017 | Volume
| Issue : 1 | Page : 82-86
Modified comet assays for the detection of cyclobutane pyrimidine dimers and oxidative base damages
Ganesan Muthusamy, Agilan Balupillai, Kanimozhi Govindasamy, Karthikeyan Ramasamy, Veeramani Kandan Ponniresan, Illiyas Magbool Malla, Rajendra Prasad Nagarajan
Department of Biochemistry and Biotechnology, Annamalai University, Chidambaram, Tamil Nadu, India
|Date of Web Publication||1-Feb-2017|
Rajendra Prasad Nagarajan
Department of Biochemistry and Biotechnology, Annamalai University, Chidambaram, Tamil Nadu
Source of Support: None, Conflict of Interest: None
The comet assay (also known as single-cell gel electrophoresis) is a technique for the detection of DNA damage at the level of the individual cell. It is a versatile, relatively simple to perform and sensitive method. Although most investigations make use of its ability to measure DNA single-strand breaks, modifications to the method allow detection of cyclobutane pyrimidine dimers (CPDs), crosslinks, base damage, and apoptotic nuclei. Many investigators also interested in examining the DNA damage as a function of time after exposure to a known genotoxic agent. Here, we present a procedure of comet assay for the detection of DNA strand breaks, base damages, and CPDs that can be used to measure DNA damage during toxicity, oxidative stress, and ultraviolet radiation exposure and it can be applied in human toxicological biomonitoring scenarios.
Keywords: Cyclobutane pyrimidine dimers, DNA base damages, DNA damage, modified comet assay, single-strand breaks
|How to cite this article:|
Muthusamy G, Balupillai A, Govindasamy K, Ramasamy K, Ponniresan VK, Malla IM, Nagarajan RP. Modified comet assays for the detection of cyclobutane pyrimidine dimers and oxidative base damages. J Radiat Cancer Res 2017;8:82-6
|How to cite this URL:|
Muthusamy G, Balupillai A, Govindasamy K, Ramasamy K, Ponniresan VK, Malla IM, Nagarajan RP. Modified comet assays for the detection of cyclobutane pyrimidine dimers and oxidative base damages. J Radiat Cancer Res [serial online] 2017 [cited 2019 Sep 17];8:82-6. Available from: http://www.journalrcr.org/text.asp?2017/8/1/82/199312
| Introduction|| |
Many methods have been developed to understand the effect of genotoxicity in the cellular system. The comet assay, also referred to as single-cell gel electrophoresis, has been extensively used in assessing genotoxicity in cells exposed to various toxicants, including radiation.,, Comet assay was first introduced by Ostling and Johanson, later it has been modified by Singh et al., The term “comet assay” was first given by Olive et al. The basic of this assay was to combine DNA gel electrophoresis with fluorescence microscopy to understand migration of DNA strands from damaged cells. This technique is based on the electrophoresis of single nucleoids (DNA attached to the nuclear matrix after cell lysis and stripping of histones), giving a comet-like image with the intensity of the tail depending on the frequency of breaks which relax supercoiling and allow migration of the DNA loops containing the breaks. Computer software tools are used to detect the level of DNA damage by measuring the extent of tail formation as percentage of DNA present in the comet tail.
| Modified Comet Assays|| |
Comet assay under neutral conditions allows the detection of DNA double-strand breaks which is most relevant to ionizing radiation exposure. In the neutral variant, the molecule of DNA is preserved as a double-stranded structure which leads to uncovering of double-stranded DNA breaks. In the alkaline variant of the method, the denaturing step allows to reveal simultaneously double- and single-stranded DNA breaks, as well as alkali labile sites, which leads to the general assumption that the alkaline assay is much more sensitive in comparison with its neutral variant in DNA damage detection.
Since Singh et al. introduced the alkaline comet assay, its uses and applications have been increasing. The research areas of its current employment in the evaluation of genetic toxicity are vast, either in vitro or in vivo, both in the laboratory and in the environment, terrestrial or aquatic. Moreover, scientists modified the assay protocols for the analysis of unexplored DNA damages during neutral and alkaline comet assays. The most recent developments concern the adoption of the enzyme-linked assay (digestion with lesion-specific repair endonucleases) and prediction of the ability to repair of oxidative DNA damage, which is becoming a widespread approach in human biomonitoring. These modified assays detect genotoxic/carcinogenic potential of environmental chemicals and ultraviolet radiations (UVR) and could be useful in the study of base excision repair (BER) and nucleotide excision repair (NER) mechanisms. Modifications to the alkaline comet assay using lesion-specific endonucleases can detect DNA bases with oxidative damage and bulky DNA damages. After alkaline lysis, comet slides were washed and then the cells were incubated with appropriate enzyme under 22 mm × 22 mm cover slips in an optimized condition. [Table 1] illustrates the list of endonucleases and their target DNA damages.
| T4 Endonuclease V-Modified Alkaline Comet Assay|| |
Bulky DNA damages can also be assessed using T4 endonuclease V (T4endoV)-modified alkaline comet assay. The T4endoV-modified comet method was performed with specific refinements. The predominant form of DNA damages induced by ultraviolet B (UVB; 280 - 320 nm) radiation is CPDs. The concentration and incubation period for T4endoV will vary with cell types. Karbaschi et al. employed 0.1 U/µL and 60 min incubation for HaCaT cells and 0.02 U/µL plus 60 min for HDFa cells in a humidified atmosphere to explore UV-induced CPDs formation., Decome et al. evaluated photolyase repair activity in human keratinocytes after a single dose of UVB irradiation using the T4endoV comet assay. The authors found that UVB irradiation increased the single-strand breaks (SSB) level in keratinocytes and additional T4endoV treatment enhanced this SSB level by 1.5–2.0-fold confirming that CPDs were the major base modifications generated by UVB irradiation. Langie et al. demonstrated that T4endoV assay can be applied in molecular epidemiological studies to assess interindividual differences in NER mechanism.
UVR may also generate the formation of (apurinic/apyrimidinic sites) AP sites, which may be detected by the AP lyase activity of T4endoV, which could lead to an overestimation of CPDs. However, it is possible to discriminate between AP sites and CPDs by plotting data derived from the alkaline comet assay alone (which representing all frank strand breaks and those induced by the action of high pH on AP and other alkali-labile sites) versus in conjunction with the T4endoV (which includes all of the former strand breaks, plus those induced by the enzyme). This is a well-established approach to determine levels of strand breaks and alkali-labile sites [Figure 1].
|Figure 1: Simplified mechanism of (a) T4 endonuclease V-modified alkaline comet assay and (b) human 8-oxoguanine DNA glycosylase-1-modified comet assay|
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| DNA Glycosylases-Modified Comet Assays|| |
Collins developed an elegant comet assay-based method to measure phenotypic differences in BER that was found to be applicable in molecular epidemiological studies. This alternative approach involves measurement of the capacity of human lymphocyte extracts to perform the initial step of BER, that is, damage incision, on DNA substrates carrying 8-hydroxydeoxyguanosine lesions. Further, base damages in the DNA can be assessed using human 8-oxoguanine DNA glycosylase 1 (hOGG-1)-modified comet assay with specific refinements. HOGG-1 recognizes 8-oxo-7,8-dihydroguanine (8-oxoGua) and 8-oxoGua, but also two other hydroxyl radical-induced products, 2,6-diamino-4-hydroxy-formamidopyrimidine (FapyGua) and to a much lesser extent, FapyAde. The hOGG-1-modified comet assay has recently widely adopted by the human biomonitoring researchers [Figure 1]. Karbaschi et al. employed 3.2 U/mL of hOGG-1 and 45 min incubation in a humidified atmosphere for the detection of 8-oxoGua in UV-irradiated cells. Pothmann et al. analyzed local genotoxic effects of multiwalled carbon nanotubes in the lung, kidney, and liver cells by both standard and hOGG-1-modified comet assay. Galic et al. employed hOGG-1-modified comet assay for the evaluation of glass-ionomer cements biocompatibility in human cells.
When associated with DNA repair glycosylases such as the Escherichia coli formamidopyrimidine-DNA glycosylase (Fpg) and the E. coli endonuclease III (endo III) which recognize modified purines and pyrimidines, respectively, the method allows the detection of base damage., It was possible using a calibration of the modified comet assay to measure very low levels of DNA damage such as 8-oxoGua. A modified comet assay using endo III and Fpg enzymes showed that pyrimidines and purines were oxidatively damaged during silver nanoparticles exposure. DNA damage and repair status in patients with Fabry disease has been investigated using endo III and Fpg enzymes. [Table 2] illustrates different version of modified comet assays intended to detect various types of DNA damages.
|Table 2: Different version of modified comet assays intended to detect various types of DNA damages|
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| Assay Procedure|| |
Materials and reagents
- Low melting point agarose (LMPA) (Sigma)
- Microgel electrophoresis slides (Trevigen)
- Horizontal gel electrophoresis apparatus (Biorad)
- Fluorescence microscope (Nikon)
- 1X phosphate-buffered saline (PBS)
- Lysis solution: Ingredients per 1000 mL (2.5 M NaCl, 100 mM EDTA, 10 mM Tri base, 1% Triton X-100, and 10% DMSO). Cool the prepared lysis solution to 4°C for at least 20 min before use. The addition of DMSO is optional and is required only for samples containing heme, such as blood cells or tissue samples
- Comet low melting (LM) agarose: The comet 0.5%–0.7% LMPA is ready to use once molten. Loosen the cap to allow for expansion then heat the bottle in a 90°C–100°C water bath for 5 min, or until the agarose is molten. Caution: Microwaving is not recommended. Place the bottle in a 37°C water bath for at least 10 min to cool
- Alkaline unwinding and electrophoresis solution pH 13 (200 mM NaOH, 1 mM EDTA), Prepare a stock solution of 500 mM EDTA, pH 8. For 1 L of electrophoresis solution
- Neutralization buffer: Add 0.4 M Tris to 800 mL dH2O, adjust pH to 7.5 with concentrated (>10 M) HCl. Adjust the volume to 1000 mL with dH2O, store at room temperature
- Staining Solution: Ethidium bromide or propidium iodide (20 μg/mL)
- Endonucleases: T4endoV at 0.1 U/µL for 60 min, hOGG1 at 3.2 U/mL for 45 min, endo III at 10 U/mL for 45 min, at 37°C in a humidified atmosphere.
- Keep lysis solution and cool at 4°C for at least 20 min before use
- Prepare LM agarose in a beaker of boiling water for 5 min. Place bottle in a 37°C water bath for at least 20 min to cool. The temperature of the agarose is critical or the cells may undergo heat shock
- Clean the slides with ethanol and make sure slides are dust free. Note: If dust present in the slides, it will interfere in final results
- Take 100 μL LM agarose, add 5–10 μL of treated cells (minimum 10,000 cells should be taken), mix well, and apply over the slide. If necessary, use side of pipette tip to spread agarose/cells over sample area to ensure complete coverage of the sample area. If sample is not spreading evenly, warm the slide at 37°C before application. Note: Do not allow the gel into solidify and prepare freshly each time running of experiments
- Place slides flat at 4°C in ice or in refrigerator for 45 min. A 0.5 mm clear ring appears at edge of slide area. Slides should be labeled before storage
- Immerse slides in 4°C lysis solution for 60 min. The longer the exposure to alkali, the greater the expression of alkali-labile damage
- Enzyme treatment (either hOGG1/T4endoV/endo III): Prepare 1X enzyme reaction buffer. Wash slides in a staining jar with enzyme buffer three times 5 min each wash. Dab off excess liquid with a tissue. Place 50 µL of the enzyme solution (hOGG1 or T4endoV or endonuclease III) alone onto gel surface and cover with a 22 mm × 22 mm cover slip
- Put slides in a moist box and incubate at 37°C for 30 min. While the amount of enzyme required should be determined for each cell type
- Drain excess enzyme buffer from slides and immerse in freshly prepared alkaline unwinding solution, pH >13 for 20 min. Wear gloves when preparing or handling this solution. Gently, take the slides from alkaline unwinding buffer. Incubate 40 min at 4°C before beginning electrophoresis
- Add ~850 ml cold alkaline electrophoresis solution, place slides in electrophoresis slide tray (slide label adjacent to black cathode) and cover with slide tray overlay. Set power supply to 25 volts (0.74 V/cm) and apply voltage for 20 min. Adjust the current to 300 mA by raising or lowering the buffer level. A lower voltage and a longer electrophoresis time may allow for increased sensitivity. Different gel boxes will require different voltage settings to correct for the distance between the anode and the cathode
- After turning off the power, gently lift the slides from the buffer and keep in a neutralization buffer for few minutes, then wash gently with twice in PBS for 5 min. Train the buffer and repeat the procedure two to three times. Note: Wash the slides properly without any background and getting good clarity results
- Dry samples at 37°C for 15 min. Drying brings all the cells in a single plane to facilitate observation. Samples may be stored at room temperature, with desiccant before scoring at this stage
- Stain the slides with 50 μL of 1X propidium iodide or ethidium bromide, leave for 5 min and then dipped in chilled dH2O to remove excess stain. Place a coverslip over the gel and score immediately.
For CPDs and 8-OGH measurement, the assays have to be carried out in the presence and absence if of respective enzymes as these enzymes also explore AP sites. The difference between alkaline comet assay and enzyme-modified assay will exactly give the amount of CPDs and oxidized bases present in the given cell types. For visualization DNA damage, observations are made with fluorescent-stained DNA using 40X objective in fluorescent microscope. In general, 50–100 randomly selected cells are analyzed per sample. ScoreComets, OpenComet, Komet 7, Comet Assay IV, and CASP labs (free software available at http://www.casplab.com/) are few software used to assess the quantitative extent of DNA damage in the cells by measuring length of DNA migration and percentage of migrated DNA. [Table 3] illustrates different comet measurements and their definitions. [Figure 2] illustrates measurement of comet attributes (head DNA, comet tail length, tail moment [TM], and olive TM [OTM]) using CASP software. These software measure head DNA, tail DNA, tail length, etc., the program calculates the TM and the OTM.
|Figure 2: (a) Normal cells with intact nuclei. (b) Ultraviolet-B exposed cells with comet tail. (c) Measurement of cyclobutane pyrimidine dimers attributes (head DNA, comet tail length, tail moment, and olive tail moment) using CASP software|
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| Troubleshooting|| |
- Ensure that agarose is fully dissolved and the concentration has not been altered (e.g., by evaporation)
- If slides are not handled gently during lysis and electrophoresis and gel falls off the slide
- Evaluate cell viability. If an enzyme disaggregation method is used, try reducing exposure time or enzyme concentration
- If the untreated cells show comet tails, DNA in the cells is damaged. Repeat the assay in dark light.
- During short electrophoresis, fragments are not separated, this assay provides no information about fragment size
- Interpretation of comet results is complicated by the fact that there is no simple relationship between the amount of DNA damage caused by a specific chemical and the biological impact of that damage.
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Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3]
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