Mutagenesis 26, 461471 (2011): https://doi.org/10.1093/mutage/ger005, Slyskova, J. et al. A bootstrap replication number of 10,000 were employed with a 95% confidence interval. Association between age and repair of oxidatively damaged DNA in human peripheral blood mononuclear cells. Health Perspect. If the epigenetic processes are not correctly regulated, it may lead to changes in DNA methylation and histone modification patterns that disrupt important cellular processes, including gene expression, DNA repair and tumor suppression (Walsh and Xu, 2006; Li et al., 2007; Brooks et al., 2010; Tost, 2010). Fikrova, P. et al. It is also observed that among the colorectal cancer cell lines, cisplatin affects HCT-15 more than the others. AOPWiki https://aopwiki.org/aops/46. doi: 10.1016/j.mrgentox.2011.05.010, Azqueta, A., Gutzkow, K. B., Priestley, C. C., Priestley, C. C., Meier, S., Meier, S.,et al. Crebelli, R. et al. Keywords: Hematopoietic stem cells, DNA damage, Double-strand breaks, Neutral comet assay Go to: Background Even though theoretically the percentage tail DNA following MspI treatment represents all of the 5-CCGG-3 sites in the DNA, it is important to note that when using MspI and HpaII only the methylated cytosines outside of CpG islands are quantified as these enzymes tend to mainly recognize sequences outside of CpG islands. All authors read and approved the final manuscript. GERSTEL Twisters were . A: Untreated cell line after alkaline lysis. Setayesh, T. et al. An optimized comet-based in vitro DNA repair assay to assess base and nucleotide excision repair activity. 56, 97110 (2015). Slides were observed under a UV microscope. Appl. Moller, P. et al. Med. Single Cell Gel lectrophoresis - also known as a comet assay - is used to detect DNA damage at the level of individual eukaryotic cells. Comet Assay The Comet Assay, also called single cell gel electrophoresis (SCGE), is a sensitive and rapid technique for quantifying and analyzing DNA damage in individual cells. Biophys. In short, HepG2 cells were exposed to the demethylating agent 5-Aza-dcR for 24 h as exposure to this demethylating agent causes a decrease in the percentage global DNA methylations. Single cell gel electrophoresis combined with lesion-specific enzymes to measure oxidative damage to DNA. Res. A: Control HCT-15 cell line without cisplatin. Athas, W. F., Hedayati, M. A., Matanoski, G. M., Farmer, E. R. & Grossman, L. Development and field-test validation of an assay for DNA repair in circulating human lymphocytes. Vodicka, P. et al. The comet assay has a long history of being used to assess the effects of various endogenous and exogenous substances on DNA damage and repair. In May 2023, Frontiers adopted a new reporting platform to be Counter 5 compliant, in line with industry standards. 5:215. doi: 10.3389/fgene.2014.00215. Aging and defense against generation of 8-oxo-7,8-dihydro-2-deoxyguanosine in DNA. 22, 303324 (1976). doi: 10.1016/j.mrrev.2008.11.001, van Dyk, E., Steenkamp, A., Koekemoer, G., and Pretorius, P. J. Health Perspect. The enzyme reaction was performed at 37C for 1 h followed by heat inactivation (65C for 15 min). Med. The use of the comet assay over other methods such as CEA for the measurement of global DNA methylation offers the advantage that it is less expensive. Cell Res. PLoS ONE 8:e79044. The use of methylation sensitive restriction endonucleases can modify the traditional alkaline comet assay to be methylation sensitive. Azqueta, A. et al. The principle of the comet assay is that unfragmented DNA maintains a well-organized structure in the nucleus, but becomes disrupted when the cell is damaged. Overview of the procedure. The half-maximal inhibitory concentration IC 50 of the compounds was determined and used to perform the comet assay, which was carried out to reveal the photo-induced DNA damaging ability of the compounds of individual cells. 47, 608615 (2009). (2012). Microelectrophoretic study of radiation-induced DNA damages in individual mammalian cells. (2014) . Phosphate buffered saline (PBS, pH 7.4, 0.01 M) and neutral red dye were obtained from Sigma-Aldrich. 12, 27832796. Comet assaycomparison of treatment with (a) etoposide, (b) bleomycin, and (c) EMS. Cell Biol. Immunol. Open Access Danielsen, P. H. et al. DNA migration in an electric field, supposed proportional to strand breakage, is a proposed estimation of genotoxicity. Gaivo, I., Rodrguez, R. & Sierra, L. M. Use of the comet assay to study DNA repair in Drosophila melanogaster. In the experiments described here, a medium-throughput comet assay and methylation sensitive comet assay are combined to produce a methylation sensitive medium-throughput comet assay to measure changes in the global DNA methylation pattern in individual cells under various growth conditions. These modifications range from altering the pH of the electrophoresis buffer (Calini et al., 2002), to exposing cells to various chemicals to assess the DNA repair capacity, to treatment of nucleoids with restriction enzymes (Andersson and Hellman, 2005) and even protein extracts to assess the effect of a given substance on DNA repair (Collins et al., 2001; van Dyk et al., 2010). Clin. Physical Form. In breast cancer, an anthracycline antibiotic is also used; it inhibits the enzyme topoisomerase II, thus inhibiting transcription. Tissue differences in BER-related incision activity and non-specific nuclease activity as measured by the comet assay. The influence of scoring method on variability in results obtained with the comet assay. It is used in humans and other mammals. Guarnieri, S. et al. Redox and epigenetic regulation of the APE1 gene in the hippocampus of piglets: the effect of early life exposures. Mutagen. Other techniques are based on molecular imaging, such as 18F-fluorodeoxyglucose-positron emission tomography (18-FDG-PET) or magnetic resonance imaging (MRI) [22], but these methods have common adverse effects. The adaption of the comet assay to measure global methylation relies on the isoschizomeric properties of the two restriction enzymes: MspI and HpaII. Dusinska, M. et al. Comet assay lysis solution and flare slides were purchased from R&D systems (Minneapolis, USA). Following the repair phase, a 50 l aliquot of the cell sample was mixed with 100 l of 0.5% low melting point agarose (LMPA) maintained at 40C. 208, 7681 (2012). Res. Proc. Cancer 1975, 36: 881892. Mutagenesis 32, e24 https://academic.oup.com/mutage/article/32/6/e1/4844756#121612377 (2018). 301, 283315. Adv Intern Med 1950, 4: 175. J. Nutr. PubMed Part of 601, 5060 (2008). DNA methylation in pre-diagnostic serum samples of breast cancer cases: results of a nested case-control study. The comet assay is an affordable and flexible method which can be easily adapted for the measurement of global DNA methylation. In the low-throughput method the enzyme/buffer mixture is spread over the entire gel, a glass cover slide is placed over the frosted glass slide and it is then incubated in a damp plastic container. Res. First, we need high amounts of proteins, which mean a lot of genetic material. Moreover, the compounds' effects on antioxidant defense systems of enzymes in cells: superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx . The Comet assay (or Single Cell Gel Electrophoresis assay) is a sensitive technique to detect DNA damage at the level of an individual cell. Oxidatively damaged DNA and its repair after experimental exposure to wood smoke in healthy humans. & Johnson, R. T. Correlations of DNA damage and repair with nuclear and chromosomal damage in HeLa cells caused by methylnitrosamides. (2002). Mutagenesis 16, 297301 (2001). If material is not included in the articles Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. The comet assay is a simple and cost effective technique, commonly used to analyze and quantify DNA damage in individual cells. 400, 190194. The global 5-CCGG-3 methylation can be calculated by the HpaII/MspI ratio. The results for the medium-throughput methylation sensitive comet assay and the CEA following 5-Aza-dcR treatment are similar due to the fact that the enzyme digestion conditions are nearer to that recommended by the manufacturer and closer to the conditions used in the CEA. DNA methylation patterns and epigenetic memory. Exposure to ultrafine particles from ambient air and oxidative stress-induced DNA damage. DNA damage and repair activity after broccoli intake in young healthy smokers. Occupational exposure to mineral fibres. After incubation, the frosted glass plate was removed from the gasket and placed in electrophoresis buffer at 4C. USA 94, 80168020 (1997). the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Collins, A. R. et al. Single Cell Gel lectrophoresis - also known as a comet assay - is used to detect DNA damage at the level of individual eukaryotic cells. Lab. Mutat. (Sigma-Aldrich, catalog number: A9414) Seakem Agarose (Ozyme, catalog number: LON50004) PBS (Ca2+ and Mg2+-free phosphate-buffered saline) 5 N NaOH; 0.5 M EDTA disodium salt solution (pH 8) Trisma . Mutat. An 50 l aliquot of the cell sample was mixed with 100 l (1520 cells/l) of 0.5% low-melting-point agarose (LMPA; Fermentas) followed by the application of 100 l of this solution to a frosted glass slide that had been pre-coated with a thin layer of 1% high-melting-point agarose (HMPA; SigmaAldrich). Conversely, MspI will digest methylated DNA, but only 5-CmCGG-3 and not 5-mCCGG-3 (Tost and Gut, 2010). 10.1148/radiol.12111177, Olive PL, Banth JP: The comet assay: a method to measure DNA damage in individual cells. The comets are classified according to extent of DNA in tail and a value 0-4 is given (0: almost no DNA in tail, 4: almost the entire DNA in tail). Furthermore, the use of Western Blot technique is intended for a limited number of proteins. Mol. 12, 10071010 (2013). 99, 10181024 (2008). Dose-limiting side effects, such as myelosuppression and cardiotoxicity, have been reported after treatment with doxorubicin [18]. The original low-throughput and modified medium-throughput comet assays were then compared. Available for Sale. Mutagen. Associated Data Data Availability Statement Go to: Abstract The comet assay is a versatile, simple, and sensitive gel electrophoresis-based method that can be used to measure and accurately quantify DNA damage, particularly single and double DNA strand breaks, in single cells. The versatility of the comet assay allows introduction of various modifications to the basic technique. Ramos, A. doi: 10.4161/epi.1.1.2587, Li, Y. Q., Zhou, P. Z., Zheng, X. D., Walsh, C. P., and Xu, G. L. (2007). Collins, A. R. (2009). Cell Dev. Between HCT-15 and LoVo, which both represent colon adenocarcinoma, completely different data were observed. Overexposure can lead to leukopenia, anemia and thrombocytopenia, while chromosomal abnormalities, hepatotoxicity, neurotoxicity and cardiac irregularities have been reported; both its hematologic and gastrointestinal side effects are usually dose-dependent. Cancer Res. Inter-individual differences in repair of DNA base oxidation, measured in vitro with the comet assay. In Vitro 27, 768773. Mutat. Mutagenesis 28, 673681 (2013). After incubation and removal of the coverslips, the slides were put into the electrophoresis tank and covered with electrophoresis buffer (5 mol/L NaOH and 0.4 mol/L EDTA). Exp. Both techniques are non-invasive, however 18-FDG-PET involves exposure to ionizing radiation and the MRI environment may cause harm in patients by using strong magnetic fields and radiowaves. No. J. Wildl. Boer, K., Isenmann, S. & Deufel, T. Strong interference of hemoglobin concentration on CSF total protein measurement using the trichloroacetic acid precipitation method. P.V. Acta 1219, 724727 (1994). Food Res. Borghini, A., Roursgaard, M., Andreassi, M. G., Kermanizadeh, A. Tormey DC: Combined chemotherapy and surgery in breast cancer: a review. A.A. thanks the Ministry of Economy, Industry and Competitiveness (Ramn y Cajal programme, RYC2013-14370) of the Spanish Government for personal support. Molecular cloning and functional expression of a human cDNA encoding the antimutator enzyme 8-hydroxyguanine-DNA glycosylase. Enzymatic MPG DNA repair assays for two different oxidative DNA lesions reveal associations with increased lung cancer risk. Received: 28 April 2014; Accepted: 20 June 2014; Published online: 07 July 2014. Commun. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate. 262, 624628. Relationship between the capacity to repair 8-oxoguanine, biomarkers of genotoxicity and individual susceptibility in styrene-exposed workers. DNA repair as a human biomonitoring tool: comet assay approaches. Performance of the comet assay in a high-throughput version. Cell Res. Surg Oncol 2007, 16: 299310. Nutrients 5, 12001217 (2013). doi: 10.1101/gad.947102, Brooks, J. D., Cairns, P., Shore, R. E., Klein, C. B., Wirgin, I., Afanasyeva, Y.,et al. Their viability was analyzed with propidium iodide staining after 24hrs of incubation (NucleoCounter NC-100, Chemometec). This method can be used also to study genetic instability, or genotoxicity [24, 25]. Cells were cultured in 12-well plates (3513, Corning) in different concentrations (0.01-50M) for each drug. 642, 3742 (2008). Formation of methylation patterns in the mammalian genome.