The comet assay is a sensitive and rapid method for dna strand break detection in individual cells, and the year 2009 represents the 25th anniversary of the first description of this methodology. Additional information is needed before the assay can be critically evaluated for its utility in genetic toxicology. The vast number of chemicals existing or being added into the environment, have globally aroused great concern regarding their adverse effects in human population. Development and validation of sensitive and better test systems which can assess th. It is useable in almost any cell type and applicable to both eukaryotic and prokaryotic organisms. A versatile tool for assessing dna damage alok dhawan, mahima bajpayee and devendra parmar new chemicals are being added each year to the existing burden of toxic substances in the environment. As per the ich recommendations, the comet assay combined with micronucleus assay can be performed as part of the initial test battery after performing the ames assay. Save up to 80% by choosing the etextbook option for isbn. This assay is used in diverse fields ranging from clinical applications, human monitoring and environmental toxicology through to genetic toxicity testing.
Unlike the other assays described above, the comet assay measures transient genetic damage that is not a fixed change to the dna. Because collembolans have a high content of chitin, a new methodology was. The comet assay in toxicology by andreas hartmann overdrive. The comet assay is a singlecell gel electrophoresis method that can measure a variety of types of dna damage, and repair of damage, in individual cells. The comet assay is a sensitive and rapid method for dna strand break detection in individual cells, and the year 2009 represents the 25th anniversary of. H2ax foci analysis, however, bear the ability to directly assess the damage on a single cell level. Initially, the comet assay protocol was adapted to the rtg. The purpose of the comet assay is to identify substances that cause dna. Application of in vitro comet assay for genotoxicity testing. In vivo mammalian alkaline comet assay introduction 1. In vivo comet data the in vivo comet assay examines the ability of substances to cause dna damage in cells from a variety of different tissues in an organism, such as stomach, liver, lung, or brain. Compared to the transgenic rodent mutation assay, the in vivo comet assay does not need special expensive transgenic animals and has the potential to be integrated into subchronic toxicology studies and be combined with the standard micronucleus test 6, 7. The comet assay performed under alkaline conditions ph is considered the optimal version for identifying agents with genotoxic activity.
The comet assay is a wellpublished method for measuring cellular dna damage. The comet assay is a common follow up to a positive in vitro assay such as the chromosome aberration, mouse lymphoma andor in vitro micronucleus assay. Frontiers the comet assay and its applications in the field. Metafer 1 is the perfect system for automated, highthroughput comet assay analyses. Since singh and colleagues, in 1988, launched to the scientific community the alkaline single cell gel electrophoresis scge protocol, or comet assay, its uses and applications has been increasing.
Use of micronucleus and comet assay to evaluate evaluate the. Rodent alkaline single cell gel electrophoresis comet assay. The purpose of the comet assay is to identify substances that cause dna damage. This updated and revised edition of the comet assay in toxicology provides the latest information on this important tool. Mar 16, 2017 the present study shows the comet assay technique being successfully applied for the first time to one of the most widely used soil organisms in standardized ecotoxicological tests, folsomia candida, providing a step forward in assessing the genotoxicity induced by xenobiotics. An exposure period of 2 h was found to be optimal, because dna damage decreased when exposure was prolonged. The thematic 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. This multiauthor book will serve as both a reference and a guide to investigations in the biomedical, biochemical and pharmaceutical sciences. Therefore, trevigen recommends the use of gloves, lab coats, and eye protection while using any of these chemical reagents. The comet assay in toxicology 2nd edition pdf is written by diana anderson, diana anderson, alok dhawan, alok dhawan, andrew collins, michael plewa, tomas gichner, narendra singh, declan mckenna, ashutosh kumar, s piperakis, maria dusinska, emilio rojas, siegfried knasmueller, giada frenzilli, sheena lewis, andreas hartmann, mark browne, d lovell and you can. The comet assay is a versatile, reliable, costefficient, and fast technique for detecting dna damage and repair in any tissue. The contributions to this special issue of mutagenesis have been selected to cover the main research areas served by the comet assay, namely genotoxicology, environmental toxicology, human biomonitoring and fundamental investigations into mechanisms of dna damage and repair.
The comet assay in toxicology 2nd edition pdf is written by diana anderson, diana anderson, alok dhawan, alok dhawan, andrew collins, michael plewa, tomas gichner, narendra singh, declan mckenna, ashutosh kumar, s piperakis, maria dusinska, emilio rojas, siegfried knasmueller, giada frenzilli, sheena lewis, andreas hartmann, mark browne, d lovell and you can download for free. The comet assay in toxicology issn by andreas hartmann contributor sheena e. Journal of toxicology and environmental health, part a. Despite similarities with other eukaryotic systems, namely animal models, the comet assay protocols for plants take into account relevant differences. Comet assay is rapid results are available in days, simple to perform, requires small amounts of the test substance 2550 mg, and can be performed in almost any eukaryotic cell different animal target organs.
Amaya azqueta, isabele campos costaamaral and andrew r. The physical, chemical, and toxicological properties of the products contained within the cometassay kit may not have been fully investigated. Our comet assays provide a fast, convenient way to screen for general dna damage, regardless of the source. The comet assay is a common follow up to a positive in vitro assay such as the chromosome aberration, mouse lymphoma andor invitro micronucleus assay. Download the comet assay in toxicology 2nd edition pdf free.
Development and applications of the comet fish assay for the study of dna damage and repair. The comet assay in toxicology is the first book of its kind to be devoted exclusively to the comet assay and its applications as an important tool in modern toxicology. The comet assay in toxicology 1st edition 9780854041992. Pdf the comet assay and its applications in the field of. Since then, the neutral comet assay is mostly used.
Alkaline comet assay for assessing dna damage in individual. Download the comet assay in toxicology 2nd edition pdf. Under alkaline conditions ph, the comet assay can detect single and double stranded breaks, resulting, for example, from direct interactions with dna, alkali labile sites or as a consequence of transient dna strand breaks resulting from dna excision repair. The software provides the same analysis options as metafer 1, but it is also available as a standalone system. Due to its simplicity and sensitivity, and the small number of cells required to obtain robust results, the use of plant comet assay has drastically increased in the last decade. Invitro methods are being developed with the aim of future validation. Frontiers the comet assay and its applications in the.
This protocol describes several procedures for preparing high quality frozen tissue samples at the time of necropsy for use in the comet assay to assess dna damage. Environmental and molecular mutagenesis, 35 3, 206221. Highthroughput measurement of dna breaks and oxidised bases with the comet assay. Use of micronucleus and comet assay to evaluate evaluate the genotoxicity of oregano essential oil origanum vulgare l. In vivo mammalian alkaline comet assay introduction. Comet assay single cell gel electrophoresis sigmaaldrich. Vasquez, roland frotschl, in genetic toxicology testing, 2016. Continue to access rsc content when you are not at your institution. In vitro toxicology in vitro comet assay background information protocol in addition to the capability of the comet assay to identify dna damage at the single cell level, other significant advantages include its sensitivity for detecting low levels of dna damage, the requirement for only small numbers of cells per sample, its ease of application. As part of the toxicology portfolio, metasystems has automated both assays with metafer.
Jun 27, 2006 the comet assay with multiple mouse organs. Detection of dna damage at the level of an individual eukaryotic cell warrants high significance in the fields of toxicology, pharmaceuticals, genotoxicity testing, environmental human biomonitoring, diagnosis of genetic disorders. Pdf the comet assay in male reproductive toxicology. The comet assay is a versatile and sensitive method for the evaluation of dna damages and dna repair capacity at singlecell level. The first reports on the use of comet assay in plants date from the 1990s e. The compromised integrity of the genetic material has been known to cause cancer.
The assay is based on the electrophoretic separation of relaxed dna loops and fragments that migrate from nucleoids of lysed cells. The most widely used method for assessment of dna damage is the alkaline comet assay. The expert panel reached a consensus that the optimal version of the comet assay for identifying agents with genotoxic activity was the alkaline ph. Experiences with the in vivo and in vitro comet assay in. Application of in vitro comet assay for genotoxicity. In addition we used assays that reflect other pathways of cytotoxicity such as the ldh assay membrane damage, caspase 37 apoptosis, adenosine triphosphate levels atp, metabolic competency of. The alkaline ph comet assay guidelines developed by the expert panel represent a work in progress.
The in vivo alkaline single cell gel electrophoresis assay, also called alkaline comet assay is a method measuring dna strand breaks in eukaryotic cells. The molluscan digestive gland, the analogous of the vertebrate liver and therefore of high relevance in toxicological studies, was shown to yield levels of single strand breakage likely too high from autolytic processes for a valid application of the comet assay without proper cell sorting and viability check refer to raimundo et al. The present study shows the comet assay technique being successfully applied for the first time to one of the most widely used soil organisms in standardized ecotoxicological tests, folsomia candida, providing a step forward in assessing the genotoxicity induced by xenobiotics. The comet assay in toxicology issues in toxicology. The alkaline comet assay is capable of detecting dna double. Because collembolans have a high content of chitin, a new. In 2014, an oecd test guideline was approved for the rodent comet assay, thus increasing the acceptability of the assay for use in regulatory testing of genotoxic potential. It further focuses on in vivo and in vitro comet assay studies with sperm and a comparison of this assay with other assays measuring germ cell genotoxicity. The comet assay serves as an important tool in early drug development as a mechanistic and genotoxic predictor tice et al. Evaluation of genotoxicity by comet assay singlecell gel.
With an overdrive account, you can save your favorite libraries for ataglance information about availability. Comet assay with the fish cell line rainbow trout gonad. A positive and a vehicle control group are also used. Evaluating in vitro dna damage using comet assay protocol. Each treated group is composed of a minimum of 5 animals of one sex or of each sex as appropriate. Blois meadow business centre, steeple bumpstead haverhill, suffolk cb9 7bn united kingdom tel. Comet assay or single cell gel electrophoresis assay detects single or double strand breaks measured at the individual cell level. The assay is a simple, sensitive rapid and visual technique for the quantitative and qualitative assessment of dna damage in single cells. The comet assay in toxicology 1st edition by andreas hartmann and publisher royal society of chemistry. Use of micronucleus and comet assay to evaluate evaluate. Pdf the comet assay, also known as the singlecell gel electrophoresis assay, is a method of detecting dna damage in virtually any. The thematic areas of its current employment in the. The comet assay can be used to evaluate the genotoxicity of many test articles including, but not limited to, industrial chemicals, pharmaceuticals, nanoparticles, consumer products, medical devices, environmental contaminants, complex mixtures, and impurities.
The comet assay was first developed by ostling and johanson in 1984 by demonstrating the migration of dna fragments from nuclei under a neutral condition 1. The comet assay, also known as single cell gel electrophoresis scre, helps scientists to determine whether there has been deoxyribonucleic acid dna damage to a single cell from apoptosis cell. As a consequence, many sophisticated techniques including ames assay, in vitro and in vivo toxicology tests, and comet assay have been developed to assess the chemicals potential to cause dna damage that may lead to cancer. Choose from the standard 3well comet assay kit or the higherthroughput 96well comet assay kit.
As such, this is one of the techniques used in the area of cancer research for the evaluation of genotoxicity and effectiveness of chemoprevention. The single cell gel electrophoresis assay scge, also known as comet assay is an uncomplicated and sensitive technique for the detection of dna damage at the level of the individual eukaryotic cell. Chapter 1 the comet assay in toxicology rsc publishing. Draft oecd guideline for the testing of chemicals in.
Use of frozen tissue in the comet assay for the evaluation. Plant comet assay has been applied to an increasing variety of adverse conditions. Use of frozen tissue in the comet assay for the evaluation of. The in vivo alkaline single cell gel electrophoresis assay, also called alkaline comet assay is a method measuring dna strand breaks in eukaryotic cells each treated group is composed of a minimum of 5 animals of one sex or of each sex as appropriate. Pdf use of the comet assay in environmental toxicology. Comet assay and foci dna damage quantification metasystems. It is now in widespread use in genetic toxicology and oncology. We used the mtt assay from the battery of cytotoxicity assays described in iso 10993, part 59, to assess the cytotoxicity of lonp. 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. The expert panel reached a consensus that the optimal version of the comet assay for identifying agents with. The comet assay, also called the singlecell gel electrophoresis scge assay, is a simple, rapid, and sensitive technique for detecting deoxyribonucleic acid dna damage at the level of individual eukaryotic cells.124 175 163 553 305 587 1273 1403 168 564 243 1424 400 582 1168 1400 148 283 1538 1618 1405 328 1139 682 1202 751 1476 1461 138 1305 1318 544 845 956 509 289 234 519 49 467 1275