comet assay singh et al in 1988comet assay singh et al in 1988

The emitted light from individual cells passed an emission filter (long pass 525nm). For all quartiles and comet parameters, the post-hoc pairwise comparisons indicate that there is no difference between the treatments control 1, control 2, -ala and visible light 30 min. Unable to load your collection due to an error, Unable to load your delegates due to an error. In alkaline conditions the DNA double helix is denatured and the nucleoid becomes single stranded. The model image is an 8-bit image with available grey values 0255. We would therefore recommend EB as the first choice of stain for high throughput genetic toxicological studies. Basic Clin Pharmacol Toxicol. The Comet Assay has mainly remained an assay of academic and scientific interest until quite recently. Tice,R.R., Agurell,E., Anderson,D., Burlinson,B., Hartmann,A., Kobayashi,H., Miyamae,Y., Rojas,E., Ryu,J. II. Mutat Res. In some software packages, the user can interactively draw rectangles that define the head and the tail ROI (a; box segmentation). 2012 Apr 20;7:61. doi: 10.1186/1748-717X-7-61. An image is composed of small dots called pixels. Singh NP, McCoy MT, Tice RR, Schneider EL. P. Duez and others, Statistics of the Comet assay: a key to discriminate between genotoxic effects, Mutagenesis, Volume 18, Issue 2, March 2003, Pages 159166, https://doi.org/10.1093/mutage/18.2.159. From these data, we can conclude that the 75th percentile offers at least equal, and in some cases higher, sensitivity as the median; however, this statistical parameter tends to saturate for higher stress levels (Table I). The computation procedure includes the application of linear regression. 8). The https:// ensures that you are connecting to the ,Double Stranded Sperm DNA Breaks, Measured by Comet Assay, Are Associated with Unexplained Recurrent Miscarriage in Couples without a Female Factor, Last edited on 15 September 2022, at 20:21, Learn how and when to remove this template message, "A simple technique for quantitation of low levels of DNA damage in individual cells", "Single Cell Gel/Comet Assay: Guidelines for in vitro and in vivo Genetic Toxicology Testing", 10.1002/(SICI)1098-2280(2000)35:3<206::AID-EM8>3.0.CO;2-J, "Evaluation of DNA damage using single-cell gel electrophoresis (Comet Assay)", "The comet assay for DNA damage and repair: principles, applications, and limitations", "The alkaline comet assay: towards validation in biomonitoring of DNA damaging exposures", "DNA damage and repair following treatment with ionizing radiation", https://www2.le.ac.uk/departments/csmm//SCG%20Electrophoresis.pdf, "Clinical significance of sperm DNA damage in assisted reproduction outcome", "The effects of ascorbate and @-tocopherol supplementation in vitro on DNA integrity and hydrogen peroxide-induced DNA damage in human spermatozoa", "Emerging applications of the single cell gel electrophoresis (Comet) assay. This can be performed by manual scoring or automatically by imaging software.[4][5]. Food Chem Toxicol 2007. Introduction The Comet Assay or single cell gel electrophoresis(SCGE) assay is a rapid, sensitive and relatively simplemethod for detecting DNA damage at the level ofindividual cells (Singh et al.1988; Comet Assay in-terest group website: http://cometassay.com/). 2001a, b). government site. An official website of the United States government. Undamaged DNA strands are too large and do not leave the cavity, whereas the smaller the fragments, the farther they are free to move in a given period of time. determination of the cell cycle status of cells by measuring their DNA content) that may be important in the characterisation of the genotoxicity of some compounds. Radiat Res 2003;159:4956. Relative sensitivity of fish and mammalian cells to sodium arsenate and arsenite as determined by alkaline single cell gel electrophoresis and cytokinesis-block micronucleus assay. (1988), with modifications. Chou PH, Chen CH, Chen KY, Ko FC, Tsai TY, Yeh YP. In order to determine if the use of the median or 75th percentile affects the sensitivity of the comet procedure, we compared the slopes of the relationships comet metric vesus dose of light, described in Table I; a statistical parameter will effectively be considered more sensitive if small changes in light dosage cause significantly larger changes in the response function (Causon, 1997). Raisuddin S, Jha AN. Moreover, cells treated with high doses of gamma radiations and processed immediately gave a similar response. In the reproducibility study, nine cell treatments (two controls and seven different photodynamic stresses) were performed on independent samples and analysed in triplicate electrophoresis runs, two slides replicating each treatment in each electrophoresis. In all our studies (up to stress levels leading to a median 70% tail DNA), both tail DNA and Olive tail moment essentially reach the same conclusions and no obvious differences have been seen between the two parameters with regard to the investigated statistics. These tests are clearly overly sensitive, which is most probably due to the high number of comets compared (n100). HMBA is a phenolic acid, known for its antioxidant, antifungal, and hepatoprotective property. A simple technique for the quantitation of low levels of DNA damage in individual cells. Kruman II, Kumaravel TS, Lohani A, Pedersen WA, Cutler RG, Kruman Y, et al. Please enable it to take advantage of the complete set of features! Based on their observation, they concluded that 50m gives the best resolution of the method used. and transmitted securely. Careers. Further retrospective analysis from in vitro and in vivo experiments using chemicals also suggested that OTM and % Tail DNA gave good correlations with the dose of genotoxic agents used and were the most reliable comet measurements. head and tail). They concluded that only a few comet measurements provided by the image analysis software correlated well with gamma radiation dose. 1). Reliable Comet Assay measurements for detecting DNA damage induced by ionising radiation and chemicals. (, Anderson,D., Yu,T.W., Phillips,B.J. It is based on nuclear decompaction followed by electrophoresis and visualization of individual spermatozoa. Bookshelf [15] For example, Swain and Rao, using the comet assay[16] reported marked increases in several types of DNA damages in rat brain neurons and astrocytes during aging, including single-strand breaks, double-strand breaks and modified bases (8-OHdG and uracil). Briefly, cells are suspended in low melting point agarose on a microscope slide. In an in vitro study the cells would be exposed to a test agent typically UV light, ionising radiation, or a genotoxic chemical to induce DNA damage in the encapsulated cells. The kinetics of repair of oxidative DNA damage (strand breaks and oxidised pyrimidine) in human cells. Tail length increases with damage initially and then reaches a maximum Advantage of Comet Assay 1. The single cell gel electrophoresis assay (comet assay): a European review. Mutat Res 1995;339:3759. The comet parameters considered in this study were tail DNA and Olive tail moment, the product of tail DNA and the distance between the head and tail centres of gravity. Our attempts to estimate the within-electrophoresis reproducibility (MannWhitney test for each pair of duplicate slides) met with the same problem. Comet Assay - an overview | ScienceDirect Topics Zar,J.H. Meaningful statistics (median and 75th percentile) were consequently extracted from repeated experiments and found suitable for comparing stress conditions in an ANOVA and in a trend analysis; the 75th percentile is theoretically more sensitive but tends to more rapidly saturate at extensive stress levels. The size and shape of the comet and the distribution of DNA within the comet correlate with the extent of DNA damage (Fairbairn et al. PMC et al., 1997; De Boeck et al., 2000) tests; the KolmogorovSmirnov test has also been applied to pooled data from several experiments (Bergqvist et al., 1998), resulting in very high numbers of points which may distort the statistical treatment. The image of a real comet on the microscope slide is shown in (a). A glass cover slip is held at an angle and the mono-suspension applied to the point of contact between the coverslip and the slide. [1988; Radiat Res 112:86-94], the comet assay has been widely used in genetic toxicology, environmental biomonitoring, molecular and human epidemiology, and clinical investigations. Environ Mol Mutagen 2000;35:20621. 2007) and chemo-sensitivity (Smith et al. Since the protocol was published by Singh et al. The .gov means its official. As a large number of factors could influence the shape, size, identification and determination of induced damage, which includes the scoring criteria, staining techniques, selection of parameters (whilst using the software packages) and appearance of hedgehog or clouds, this article aims (a) to provide an overview of evolution of measurements of DNA damage using the Comet Assay and (b) to summarise and critically analyse the advantages and disadvantages of different approaches currently being adopted whilst using this assay. Smith AJO, Almeida GM, Thomas AL, Jones GD. Only the DNA of the cell remains, and unravels to fill the cavity in the agarose that the whole cell formerly filled. We demonstrate in the present paper that the reduction of data to representative non-parametric statistics (essentially the medians) is an effective tool to compare samples, either by analysis of repeated experiments or by trend analysis. The determination of damage is carried out either through visual scoring of cells (after classification into different categories on the basis of tail length and shape) or by using different commercially available or public domain software (which automatically recognise the extent of damage). Gichner T, Patkova Z, Szakova J, Demnerova K. Cadmium induces DNA damage in tobacco roots, but no DNA damage, somatic mutations or homologous recombination in tobacco leaves. Fluorescence spectroscopy and microscopy (Duez et al., 2001) confirmed that upon -ala incubation (300 M, 3 h), porphyrins are effectively induced in P388D1 cells; under these conditions, a 30 min photodynamic treatment allows the survival of >80% of the cells for UVA and 100% for visible light (Duez et al., 2001). Disclaimer. There are still several issues to be resolved before the comet assay is accepted as a standard assay for detecting DNA damage and repair in a single cell. Intracellular DNA damage by lysine-acetylene conjugates. 2009 Jan-Feb;681(1):3-12. doi: 10.1016/j.mrrev.2008.03.005. The normality of distributions was visually appreciated from normal probability plots and objectively evaluated by the ShapiroWilk W-test (Shapiro and Wilk, 1965) using the Analyse-it software (Analyse-It Software Ltd, Leeds, UK, http://www.analyse-it.com). In a typical Comet Assay, electrophoresis methods and differences in cell preparations create a significant source of variation for the measurements. Unable to load your collection due to an error, Unable to load your delegates due to an error. Single cell gel/Comet Assay: guidelines for in vitro and in vivo genetic toxicology testing. Clipboard, Search History, and several other advanced features are temporarily unavailable. This was a neutral version of the Comet Assay, and interestingly, they used quite sophisticated techniques of image analysis for quantification of the comets, using acridine orange (AO) as the DNA binding dye (Fig. 3), but they did not calculate the total score for each gel. 8600 Rockville Pike Basic Statistical Theory. et al., 1998; Rojas et al., 1999). and transmitted securely. (, Olive,P.L., Johnston,P.J., Banath,J.P. Therefore, the amount of DNA that leaves the cavity is a measure of the amount of DNA damage in the cell. and Durand,R.E. Lee RF, Steinert S. Use of the single cell gel electrophoresis/Comet Assay for detecting DNA damage in aquatic (marine and freshwater) animals. Help! (1995) is becoming popular especially in biomonitoring and DNA repair studies. 2011 Feb;8(2):540-53. doi: 10.3390/ijerph8020540. Breast Cancer Res Treat 2005;91:8993. Dhawan & Anderson (2009): The Comet Assay in Toxicology. These statistics notably present a low sensitivity to the few extreme values that can be encountered in comet populations (the so-called apoptotic cells). The Alkaline Comet Assay: Towards Validation in Biomonitoring of DNA This assay was later modified by two groups, Singh et al. The comet assay is a versatile technique for detecting damage and with adjustments to the protocol can be used to quantify the presence of a wide variety of DNA altering lesions (damage). Exp Cell Res 1988;175:18491. Different software packages use different approaches to detect the head and the tail (Fig. This enables the DNA supercoils to get relaxed and unwind and makes possible the detection of alkali labile sites and single-strand breaks in DNA during electrophoresis. Bookshelf government site. In this approach, for example, if 100 comets are scored and each comet assigned a value of 0 to 4 according to its class, the total score for the sample gel will be between 0 and 400 arbitrary units. Visual scoring is rapid as well as simple and should appeal to those exploring the usefulness of the technique without the need to invest in expensive analytical equipment or software packages (Collins 2004). Baseline and oxidatively damaged DNA in end-stage renal disease patients on varied hemodialysis regimens: a comet assay assessment. and Wallin,H. The comet assay is also referred to as the single cell gel electrophoresis (SCGE) assay. The single cell gel electrophoresis (comet) assay is a technically simple and fast method that d. The Alkaline Comet Assay: Towards Validation in Biomonitoring of DNA Damaging Exposures - Mller - 2006 - Basic & Clinical Pharmacology & Toxicology - Wiley Online Library Skip to Article Content Skip to Article Information An image of a comet is composed of separate pixels (a), where each pixel has a grey value, as shown in (b) for a small image area outlined in red in (a). 1988). A recent review details the many compounds investigated by the Comet assay, which notably include metals, pesticides, opiates, nitrosamines and anticancer drugs (Rojas et al., 1999). McKenna DJ, Rajab NF, McKeown SR, McKerr G, McKelvey-Martin VJ. Accurate identification of clouds comes with practice and experience; hence, it is important that clouds are identified correctly whilst scoring slides using image analysis systems. O-hydroxylamine-coupled alkaline gel electrophoresis assay for the detection and measurement of DNA single-strand breaks. (1993) Complete Business Statistics, 2nd Edn. Accessibility The agarose forms a matrix of carbohydrate fibres that encapsulate the cells, anchoring them in place. Collins AR, Duthie SJ, Dobson VL. in 1990. Mutat Res 2004;559:4957. et al., 1994) or measured from morphological parameters obtained by image analysis and integration of intensity profiles (Olive et al., 1990; Kent et al., 1995; Bocker et al., 1997; Olive, 1999). The Comet Assay: A comprehensive review. (B) Visible light; 75th percentile of the tail DNA distributions. An SAS/AF-based program, "Comet Assay," was developed for this computation and is freely available on the Internet. Vol. It was first developed by stling & Johansson in 1984 and later modified by Singh et al. Although the Comet assay is able to detect genotoxic effects of chemical and physical agents, only chemical substances and environmental complex mixtures will be considered in this review. The position of the fluorescent hybridisation spots in the comet head or tail indicates whether the sequence of interest lies within or in the vicinity of a damaged region of DNA. Second, the Comet assay (Singh et al., 1988) has the unique feature that it can distinguish between single and double stranded DNA breaks (ssSDF and dsSDF respectively) when it is performed under alkaline or neutral conditions. The stronger the signal from the migrated DNA the more damage there is present. It is useable in almost any cell type and applicable to both eukaryotic and prokaryotic organisms. Mutat Res 2006;612:84104. Biochem Biophys Res Commun 1984;123:2918. in 1988. and Sarasin,A. Comets is an anthology of science fiction short stories edited by Isaac Asimov, Martin H. Greenberg and Charles G. Waugh as the fourth volume in their Isaac Asimov's Wonderful Worlds of Science Fiction series. An expert panel at an International Workshop on Genotoxicity Test Procedures (IWGTP) held in Washington, DC, in 1999, identified minimal experimental and methodological standards necessary to ensure that the results of the Comet Assay studies would be acceptable as being informative by knowledgeable scientists and regulatory authorities in this field (Tice et al. (1999) Practical Nonparametric Statistics, 3rd Edn. Although these software packages may differ slightly the way they calculate DNA damage, the underlying principles of image analysis are the same (Vilhar 2004; http://www.botanika.biologija.org/exp/comet/Comet-principles). B Optimal concentration of DNA binding dye is used and the center of gravity of DNA distribution is properly defined. The brighter and longer the tail, the higher the level of damage. Toxicol Lett 2004;146:1518. Federal government websites often end in .gov or .mil. Upon porphyrin induction, a significant relationship appears between DNA damage and light dose for both quartiles and comet parameters (P < 0.05). Analysis of Experimental Models by Least Squares. https://doi.org/10.1007/s10565-007-9043-9, DOI: https://doi.org/10.1007/s10565-007-9043-9. Speit G, Schtz P, Bonzheim I, Trenz K, Hoffmann H. Sensitivity of the FPG protein towards alkylation damage in the comet assay. Biotechniques 1999;27:92630. The first group performed electrophoresis under highly alkaline conditions (pH > 13). . [1988], its use has increased in different topic areas: clinical applications, human monitoring, radiation biology, genetic toxicology, and genetic ecotoxicology. Detection of genetic instability at HER-2/neu and p53 loci in breast cancer cells using Comet-FISH. On the other hand, the proposed statistics may not be appropriate to the data distribution patterns; parametric tests indeed assume Gaussian distributions, whereas non-parametric tests require essentially similar distribution curve shapes (Zar, 1996; Conover, 1999; Statsoft Inc., 1999). Singh NP, McCoy MT, Tice RR, Schneider EL. The comet assay is an extremely sensitive DNA damage assay. Under these circumstances, it is advisable to use % Tail DNA for regulatory purposes and for inter-laboratory comparisons. Tice RR, Furedi-Machacek M, Satterfield D, Udumudi A, Vasquez M, Dunnick JK. Non-parametric comparisons of samples were by the KruskalWallis test (Conover, 1999). Epub 2023 Jan 27. Comet Assay analysis of DNA strand breaks after exposure to the DNA De Boeck,M., Touil,N., De Visscher,G., Aka Vande,P. Careers. Tin Stars. [18] [19] [20] [21] However, various phenolic compounds are known for their anticancer property. Santos S, Ferreira T, Almeida J, Pires MJ, Colao A, Lemos S, Gil da Costa RM, Medeiros R, Bastos MMSM, Neuparth MJ, Abreu H, Pereira R, Pacheco M, Gaivo I, Rosa E, Oliveira PA. Mar Drugs. The slides are put in lysing buffer to allow the DNA to unwind and then in electrophoresis buffer. eCollection 2022. The latter function, however, presents a singularity for low values of the parameter (n < 2) (Bock et al., 1998), precluding a direct comparison of stressed and unstressed samples. Comet assay in genetic ecotoxicology: a review The Comet assay, also called the single cell gel electrophoresis (SCGE) assay or microgel electrophoresis (MGE) assay, primarily measures DNA strand breakage in single cells. Moreover, visual scoring has the potential to be used for inter-laboratory comparisons. It is based on the capacity of negatively charged loops/fragments of DNA to be pulled through an agarose gel in response to an electric field, appearing like a 'comet.' The COMET assay: single-cell analysis of DNA damage PubMedGoogle Scholar. P1-0212). Singh NP, Stephens RE, Schneider EL. liver unscheduled DNA synthesis assay) in regulatory genotoxicological studies with in vivo Comet assay. The assay has also been successfully implemented in plant cells under laboratory conditions (Gichner et al. Analysis of DNA alkylation damage and repair in mammalian cells by the comet assay. Electrophoresis at high pH results in structures resembling comets, observed by fluorescence microscopy; the intensity of the comet tail relative to the head reflects the number of DNA breaks. Epub 2008 Mar 30. [2][3], The comet assay (single-cell gel electrophoresis) is a simple method for measuring deoxyribonucleic acid (DNA) strand breaks in eukaryotic cells. We considered the response of each of four DNA damage parameters: tail moment, tail length, tail DNA, and tail inertia for constructing the dose-time-response surface. The Comet Assay was first introduced by Ostling and Johanson in 1984. Schematic diagram explaining how concentration of DNA binding fluorescent dye may affect Comet Assay measurements using image analysis. This site needs JavaScript to work properly. 2000). 98 Citations Metrics Abstract The comet assay is a versatile, reliable, cost-efficient, and fast technique for detecting DNA damage and repair in any tissue. Genotoxicological studies in aquatic organisms: An overview.