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Andrew Collins - One of the best experts on this subject based on the ideXlab platform.
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THE Comet Assay: a biomonitoring tool for nutraceutical research
2020Co-Authors: Vwc Wong, Yim Tong Savio Szeto, Andrew Collins, Iris F. F. BenzieAbstract:The Comet Assay is a relatively simple, but sen- sitive and well validated tool for measuring strand breaks in DNA in single cells. Cells are embedded in a thin layer of agarose on a microscope slide and lysed with detergent and high salt solution. This procedure also removes pro- teins and histones, leaving a nucleoid from each embedded cell lying within a cavity in the gel. The presence of breaks in DNA causes a local relaxation in the supercoiled loops of DNA in the nucleoid. When a small electrical charge is passed through the gel, the relaxed areas of the DNA loops are pulled towards the anode, forming a Comet 'tail', the DNA in the nucleoid being the Comet 'head'. Comets are visualized by fluorescent microscopy, and the amount of DNA in the tail, relative to the head, is proportional to the amount of strand breaks. Cells can be incubated in vitro with an agent of interest prior to the Comet Assay, and the resulting DNA damage can then be measured. The effect of a 'challenge' to DNA after treated cells have been exposed to a putative protective agent can also be investigated, and cells (usually lymphocytes) can be collected pre- and post- supplementation with an agent of interest to assess possible genoprotective or genotoxic effects. There are various ver- sions of the Comet Assay, and these make it a highly versa- tile biomonitoring tool for nutraceutical research. In this review, the principles and applications of the Comet Assay are described and its application in nutraceutical research highlighted.
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the next three decades of the Comet Assay a report of the 11th international Comet Assay workshop
Mutagenesis, 2017Co-Authors: Gudrun Koppen, Andrew Collins, Gunnar Brunborg, Amaya Azqueta, Bertrand Pourrut, Sabine A S LangieAbstract:: The International Comet Assay Workshops are a series of scientific conferences dealing with practical and theoretical aspects of the Comet Assay (single-cell gel electrophoresis)-a simple method for detecting DNA strand breaks. The first paper describing such an Assay was published over 30 years ago in 1984 by Swedish researchers O. Ostling and K. J. Johanson. Appropriately, the theme for the 2015 meeting was looking to the future: 'The Next 3 Decades of the Comet Assay'. The programme included 25 oral and 43 poster presentations depicting the latest advances in technical developments as well as applications of the Comet Assay in genotoxicity testing (in vitro and in vivo) and biomonitoring of both humans and the environment. Open discussion sessions based on questions from the participants allowed exchange of practical details on current Comet Assay protocols. This report summarises technical issues of high importance which were discussed during the sessions. We provide information on ways to improve the Assay performance, by testing for cytotoxicity, by using reference samples to reduce or allow for inter-experimental variation, and by standardising quantification of the damage, including replicates and scoring enough Comets to ensure statistical validity. After 30 years of experimentation with the Comet Assay, we are in a position to control the important experimental parameters and make the Comet Assay a truly reliable method with a wealth of possible applications.
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can the Comet Assay be used reliably to detect nanoparticle induced genotoxicity
Environmental and Molecular Mutagenesis, 2015Co-Authors: Hanna L Karlsson, Andrew Collins, Sebastiano Di Bucchianico, Maria DusinskaAbstract:The Comet Assay is a sensitive method to detect DNA strand breaks as well as oxidatively damaged DNA at the level of single cells. Today the Assay is commonly used in nano-genotoxicology. In this review we critically discuss possible interactions between nanoparticles (NPs) and the Comet Assay. Concerns for such interactions have arisen from the occasional observation of NPs in the “Comet head”, which implies that NPs may be present while the Assay is being performed. This could give rise to false positive or false negative results, depending on the type of Comet Assay endpoint and NP. For most NPs, an interaction that substantially impacts the Comet Assay results is unlikely. For photocatalytically active NPs such as TiO2, on the other hand, exposure to light containing UV can lead to increased DNA damage. Samples should therefore not be exposed to such light. By comparing studies in which both the Comet Assay and the micronucleus Assay have been used, a good consistency between the Assays was found in general (69%); consistency was even higher when excluding studies on TiO2 NPs (81%). The strong consistency between the Comet and micronucleus Assays for a range of different NPs—even though the two tests measure different endpoints—implies that both can be trusted in assessing the genotoxicity of NPs, and that both could be useful in a standard battery of test methods. Environ. Mol. Mutagen. 56:82–96, 2015. © 2014 Wiley Periodicals, Inc.
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Reference cells and ploidy in the Comet Assay
Frontiers in Genetics, 2015Co-Authors: Gunnar Brunborg, Andrew Collins, Kristine B Gutzkow, Anne Graupner, Ann-karin OlsenAbstract:In the Comet Assay, single cells are analyzed with respect to their level of DNA damage. Discrimination of the individual cell or cell type based on DNA content, with concomitant scoring of the DNA damage, is useful since this may allow analysis of mixtures of cells. Different cells can then be characterized based on their ploidy, cell cycle stage, or genome size. We here describe two applications of such a cell type-specific Comet Assay: (i) Testicular cell suspensions, analyzed on the basis of their ploidy during spermatogenesis; and (ii) reference cells in the form of fish erythrocytes which can be included as internal standards to correct for inter-Assay variations. With standard fluorochromes used in the Comet Assay, the total staining signal from each cell – whether damaged or undamaged – was found to be associated with the cell’s DNA content. Analysis of the fluorescence intensity of single cells is straightforward since these data are available in scoring systems based on image analysis. The analysis of testicular cell suspensions provides information on cell type specific composition, susceptibility to genotoxicants, and DNA repair. Internal reference cells, either untreated or carrying defined numbers of lesions induced by ionizing radiation, are useful for investigation of experimental factors that can cause variation in Comet Assay results, and for routine inclusion in experiments to facilitate standardization of methods and comparison of Comet Assay data obtained in different experiments or in different laboratories. They can also be used - in combination with a reference curve - to quantify the DNA lesions induced by a certain treatment. Fish cells of a range of genome sizes, both greater and smaller than human, are suitable for this purpose and they are inexpensive.
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The Comet Assay: a heavenly method!
Mutagenesis, 2014Co-Authors: Andrew CollinsAbstract: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. Innovative methods are described, technical issues are explored, and guidelines are given for venturing into relatively new or unexploited areas of research. The popularity of the Comet Assay in a historical context is illustrated by a bibliometric survey.
Maria Dusinska - One of the best experts on this subject based on the ideXlab platform.
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can the Comet Assay be used reliably to detect nanoparticle induced genotoxicity
Environmental and Molecular Mutagenesis, 2015Co-Authors: Hanna L Karlsson, Andrew Collins, Sebastiano Di Bucchianico, Maria DusinskaAbstract:The Comet Assay is a sensitive method to detect DNA strand breaks as well as oxidatively damaged DNA at the level of single cells. Today the Assay is commonly used in nano-genotoxicology. In this review we critically discuss possible interactions between nanoparticles (NPs) and the Comet Assay. Concerns for such interactions have arisen from the occasional observation of NPs in the “Comet head”, which implies that NPs may be present while the Assay is being performed. This could give rise to false positive or false negative results, depending on the type of Comet Assay endpoint and NP. For most NPs, an interaction that substantially impacts the Comet Assay results is unlikely. For photocatalytically active NPs such as TiO2, on the other hand, exposure to light containing UV can lead to increased DNA damage. Samples should therefore not be exposed to such light. By comparing studies in which both the Comet Assay and the micronucleus Assay have been used, a good consistency between the Assays was found in general (69%); consistency was even higher when excluding studies on TiO2 NPs (81%). The strong consistency between the Comet and micronucleus Assays for a range of different NPs—even though the two tests measure different endpoints—implies that both can be trusted in assessing the genotoxicity of NPs, and that both could be useful in a standard battery of test methods. Environ. Mol. Mutagen. 56:82–96, 2015. © 2014 Wiley Periodicals, Inc.
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On the search for an intelligible Comet Assay descriptor.
Frontiers in Genetics, 2014Co-Authors: Peter Moller, Maria Dusinska, Gudrun Koppen, Steffen Loft, Clara Ersson, Andrew CollinsAbstract:The Comet Assay has developed over the past 30 years and today, a variety of different DNA lesions and DNA repair can be measured by different versions of the Assay (Collins, 2004). In the final step of the method, an image resembling a Comet with a head (the nuclear core) and a tail (consisting of mainly single stranded DNA that has migrated out from the cell nuclei) is analyzed. The magnitude of the Comet's DNA-tail provides information about the level of DNA lesions in the cell. The results from Comet Assay analyses are reported using different descriptors, the most frequently used being percentage of DNA in the tail (%T), tail length and tail moment (the product of %T and tail length). These descriptors can be reported in different ways, i.e., as means, medians or as distribution patterns. To compile the information on the migration of thousands of Comets into a single value that is meaningful to convey to other researchers, is difficult. The solution has been practical and controlled by those researchers with the longest experience with the Comet Assay. In this opinion paper, we revisit the search for a commonly accepted descriptor for DNA damage measured by the Comet Assay. We define the “best” Comet Assay descriptor as a measurement that best describes the migration of DNA in each Comet in the agarose, fits the distribution of Comets in the gel, and conveys the technical measurement of Comets as a descriptor that other researchers can understand. It should be emphasized that we do not embark on a mission to promote only one Comet Assay descriptor.
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The Comet Assay as a tool for human biomonitoring studies: The ComNet Project
Mutation Research - Reviews in Mutation Research, 2014Co-Authors: Andrew Collins, Marek Kruszewski, Iris Benzie, Vanessa Valdiglesias, Alok Dhawan, Maria Dusinska, Emilio Rojas, Gudrun Koppen, Peter Moller, Erdem CoskunAbstract:The Comet Assay is widely used in human biomonitoring to measure DNA damage as a marker of exposure to genotoxic agents or to investigate genoprotective effects. Studies often involve small numbers of subjects, and design may be sub-optimal in other respects. In addition, Comet Assay protocols in use in different laboratories vary significantly. In spite of these difficulties, it is appropriate to carry out a pooled analysis of all available Comet Assay biomonitoring data, in order to establish baseline parameters of DNA damage, and to investigate associations between Comet Assay measurements and factors such as sex, age, smoking status, nutrition, lifestyle, etc. With this as its major objective, the ComNet project has recruited almost 100 research groups willing to share datasets. Here we provide a background to this project, discussing the history of the Comet Assay and practical issues that can critically affect its performance. We survey its diverse applications in biomonitoring studies, including environmental and occupational exposure to genotoxic agents, genoprotection by dietary and other factors, DNA damage associated with various diseases, and intrinsic factors that affect DNA damage levels in humans. We examine in depth the quality of data from a random selection of studies, from an epidemiological and statistical point of view. © 2013 Elsevier B.V.
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the Comet Assay as a tool for human biomonitoring studies the comnet project
Mutation Research-reviews in Mutation Research, 2014Co-Authors: Andrew Collins, Vanessa Valdiglesias, Alok Dhawan, Maria Dusinska, Iris F. F. Benzie, Emilio Rojas, Gudrun Koppen, Peter Moller, Marcin Kruszewski, Erdem CoskunAbstract:The Comet Assay is widely used in human biomonitoring to measure DNA damage as a marker of exposure to genotoxic agents or to investigate genoprotective effects. Studies often involve small numbers of subjects, and design may be sub-optimal in other respects. In addition, Comet Assay protocols in use in different laboratories vary significantly. In spite of these difficulties, it is appropriate to carry out a pooled analysis of all available Comet Assay biomonitoring data, in order to establish baseline parameters of DNA damage, and to investigate associations between Comet Assay measurements and factors such as sex, age, smoking status, nutrition, lifestyle, etc. With this as its major objective, the ComNet project has recruited almost 100 research groups willing to share datasets. Here we provide a background to this project, discussing the history of the Comet Assay and practical issues that can critically affect its performance. We survey its diverse applications in biomonitoring studies, including environmental and occupational exposure to genotoxic agents, genoprotection by dietary and other factors, DNA damage associated with various diseases, and intrinsic factors that affect DNA damage levels in humans. We examine in depth the quality of data from a random selection of studies, from an epidemiological and statistical point of view.
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The Comet Assay in human biomonitoring: Gene-environment interactions
Mutagenesis, 2008Co-Authors: Maria Dusinska, Andrew R. CollinsAbstract:The Comet Assay is the method of choice for measuring DNA damage, of various sorts, in human cells such as lymphocytes obtained in the course of population-based studies of environmental and occupational exposure to different genotoxic agents, including radiation, chemicals and oxidative stress. It is noted for its versatility and the breadth of its possible applications. In terms of simplicity, cost, small amount of material required, sensitivity and reliability, the Comet Assay in its various modifications has few serious competitors. When standardized and validated, the Comet Assay can provide invaluable information in the areas of hazard identification and risk assessment of environmental and occupational exposure, diseases linked with oxidative stress (e.g. diabetes and cardiovascular disease), nutrition, monitoring the effectiveness of medical treatment and investigating individual variation in response to DNA damage that may reflect genetic or environmental influences. The information obtained could lead to individual advice on lifestyle changes to promote health and especially on relative risks of genotoxic exposure to environmental pollution.
Peter Moller - One of the best experts on this subject based on the ideXlab platform.
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the Comet Assay ready for 30 more years
Mutagenesis, 2018Co-Authors: Peter MollerAbstract:: During the last 30 years, the Comet Assay has become widely used for the measurement of DNA damage and repair in cells and tissues. A landmark achievement was reached in 2016 when the Organization for Economic Co-operation and Development adopted a Comet Assay guideline for in vivo testing of DNA strand breaks in animals. However, the Comet Assay has much more to offer than being an Assay for testing DNA strand breaks in animal organs. The use of repair enzymes increases the range of DNA lesions that can be detected with the Assay. It can also be modified to measure DNA repair activity. Still, despite the long-term use of the Assay, there is a need for studies that assess the impact of variation in specific steps of the procedure. This is particularly important for the on-going efforts to decrease the variation between experiments and laboratories. The articles in this Special Issue of Mutagenesis cover important technical issues of the Comet Assay procedure, nanogenotoxicity and ionising radiation sensitivity on plant cells. The included biomonitoring studies have assessed seasonal variation and certain predictors for the basal level of DNA damage in white blood cells. Lastly, the Comet Assay has been used in studies on genotoxicity of environmental and occupational exposures in human biomonitoring studies and animal models. Overall, the articles in this Special Issue demonstrate the versatility of the Comet Assay and they hold promise that the Assay is ready for the next 30 years.
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Statistical analysis of Comet Assay results
Frontiers in Genetics, 2014Co-Authors: Peter Moller, Steffen LoftAbstract:The Comet Assay can distinguish small differences in DNA damage between different samples of cells, implying that statistical tests are important to assess whether this occurs by chance. Excellent scholarly papers with concise descriptions of statistical analysis and recommendations for tests have been published (Lovell et al., 1999; Lovell and Omori, 2008). We often come across publications that unfortunately have not taken advantage of statistical models in design and analysis of Comet Assay results. The present commentary is based on the notion that statistical analysis of Comet Assay data should not be complicated, but consideration of statistical analysis before carrying out the experiments typically makes it much easier to analyse the results.
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On the search for an intelligible Comet Assay descriptor.
Frontiers in Genetics, 2014Co-Authors: Peter Moller, Maria Dusinska, Gudrun Koppen, Steffen Loft, Clara Ersson, Andrew CollinsAbstract:The Comet Assay has developed over the past 30 years and today, a variety of different DNA lesions and DNA repair can be measured by different versions of the Assay (Collins, 2004). In the final step of the method, an image resembling a Comet with a head (the nuclear core) and a tail (consisting of mainly single stranded DNA that has migrated out from the cell nuclei) is analyzed. The magnitude of the Comet's DNA-tail provides information about the level of DNA lesions in the cell. The results from Comet Assay analyses are reported using different descriptors, the most frequently used being percentage of DNA in the tail (%T), tail length and tail moment (the product of %T and tail length). These descriptors can be reported in different ways, i.e., as means, medians or as distribution patterns. To compile the information on the migration of thousands of Comets into a single value that is meaningful to convey to other researchers, is difficult. The solution has been practical and controlled by those researchers with the longest experience with the Comet Assay. In this opinion paper, we revisit the search for a commonly accepted descriptor for DNA damage measured by the Comet Assay. We define the “best” Comet Assay descriptor as a measurement that best describes the migration of DNA in each Comet in the agarose, fits the distribution of Comets in the gel, and conveys the technical measurement of Comets as a descriptor that other researchers can understand. It should be emphasized that we do not embark on a mission to promote only one Comet Assay descriptor.
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The Comet Assay as a tool for human biomonitoring studies: The ComNet Project
Mutation Research - Reviews in Mutation Research, 2014Co-Authors: Andrew Collins, Marek Kruszewski, Iris Benzie, Vanessa Valdiglesias, Alok Dhawan, Maria Dusinska, Emilio Rojas, Gudrun Koppen, Peter Moller, Erdem CoskunAbstract:The Comet Assay is widely used in human biomonitoring to measure DNA damage as a marker of exposure to genotoxic agents or to investigate genoprotective effects. Studies often involve small numbers of subjects, and design may be sub-optimal in other respects. In addition, Comet Assay protocols in use in different laboratories vary significantly. In spite of these difficulties, it is appropriate to carry out a pooled analysis of all available Comet Assay biomonitoring data, in order to establish baseline parameters of DNA damage, and to investigate associations between Comet Assay measurements and factors such as sex, age, smoking status, nutrition, lifestyle, etc. With this as its major objective, the ComNet project has recruited almost 100 research groups willing to share datasets. Here we provide a background to this project, discussing the history of the Comet Assay and practical issues that can critically affect its performance. We survey its diverse applications in biomonitoring studies, including environmental and occupational exposure to genotoxic agents, genoprotection by dietary and other factors, DNA damage associated with various diseases, and intrinsic factors that affect DNA damage levels in humans. We examine in depth the quality of data from a random selection of studies, from an epidemiological and statistical point of view. © 2013 Elsevier B.V.
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the Comet Assay as a tool for human biomonitoring studies the comnet project
Mutation Research-reviews in Mutation Research, 2014Co-Authors: Andrew Collins, Vanessa Valdiglesias, Alok Dhawan, Maria Dusinska, Iris F. F. Benzie, Emilio Rojas, Gudrun Koppen, Peter Moller, Marcin Kruszewski, Erdem CoskunAbstract:The Comet Assay is widely used in human biomonitoring to measure DNA damage as a marker of exposure to genotoxic agents or to investigate genoprotective effects. Studies often involve small numbers of subjects, and design may be sub-optimal in other respects. In addition, Comet Assay protocols in use in different laboratories vary significantly. In spite of these difficulties, it is appropriate to carry out a pooled analysis of all available Comet Assay biomonitoring data, in order to establish baseline parameters of DNA damage, and to investigate associations between Comet Assay measurements and factors such as sex, age, smoking status, nutrition, lifestyle, etc. With this as its major objective, the ComNet project has recruited almost 100 research groups willing to share datasets. Here we provide a background to this project, discussing the history of the Comet Assay and practical issues that can critically affect its performance. We survey its diverse applications in biomonitoring studies, including environmental and occupational exposure to genotoxic agents, genoprotection by dietary and other factors, DNA damage associated with various diseases, and intrinsic factors that affect DNA damage levels in humans. We examine in depth the quality of data from a random selection of studies, from an epidemiological and statistical point of view.
Gudrun Koppen - One of the best experts on this subject based on the ideXlab platform.
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the next three decades of the Comet Assay a report of the 11th international Comet Assay workshop
Mutagenesis, 2017Co-Authors: Gudrun Koppen, Andrew Collins, Gunnar Brunborg, Amaya Azqueta, Bertrand Pourrut, Sabine A S LangieAbstract:: The International Comet Assay Workshops are a series of scientific conferences dealing with practical and theoretical aspects of the Comet Assay (single-cell gel electrophoresis)-a simple method for detecting DNA strand breaks. The first paper describing such an Assay was published over 30 years ago in 1984 by Swedish researchers O. Ostling and K. J. Johanson. Appropriately, the theme for the 2015 meeting was looking to the future: 'The Next 3 Decades of the Comet Assay'. The programme included 25 oral and 43 poster presentations depicting the latest advances in technical developments as well as applications of the Comet Assay in genotoxicity testing (in vitro and in vivo) and biomonitoring of both humans and the environment. Open discussion sessions based on questions from the participants allowed exchange of practical details on current Comet Assay protocols. This report summarises technical issues of high importance which were discussed during the sessions. We provide information on ways to improve the Assay performance, by testing for cytotoxicity, by using reference samples to reduce or allow for inter-experimental variation, and by standardising quantification of the damage, including replicates and scoring enough Comets to ensure statistical validity. After 30 years of experimentation with the Comet Assay, we are in a position to control the important experimental parameters and make the Comet Assay a truly reliable method with a wealth of possible applications.
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On the search for an intelligible Comet Assay descriptor.
Frontiers in Genetics, 2014Co-Authors: Peter Moller, Maria Dusinska, Gudrun Koppen, Steffen Loft, Clara Ersson, Andrew CollinsAbstract:The Comet Assay has developed over the past 30 years and today, a variety of different DNA lesions and DNA repair can be measured by different versions of the Assay (Collins, 2004). In the final step of the method, an image resembling a Comet with a head (the nuclear core) and a tail (consisting of mainly single stranded DNA that has migrated out from the cell nuclei) is analyzed. The magnitude of the Comet's DNA-tail provides information about the level of DNA lesions in the cell. The results from Comet Assay analyses are reported using different descriptors, the most frequently used being percentage of DNA in the tail (%T), tail length and tail moment (the product of %T and tail length). These descriptors can be reported in different ways, i.e., as means, medians or as distribution patterns. To compile the information on the migration of thousands of Comets into a single value that is meaningful to convey to other researchers, is difficult. The solution has been practical and controlled by those researchers with the longest experience with the Comet Assay. In this opinion paper, we revisit the search for a commonly accepted descriptor for DNA damage measured by the Comet Assay. We define the “best” Comet Assay descriptor as a measurement that best describes the migration of DNA in each Comet in the agarose, fits the distribution of Comets in the gel, and conveys the technical measurement of Comets as a descriptor that other researchers can understand. It should be emphasized that we do not embark on a mission to promote only one Comet Assay descriptor.
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The Comet Assay as a tool for human biomonitoring studies: The ComNet Project
Mutation Research - Reviews in Mutation Research, 2014Co-Authors: Andrew Collins, Marek Kruszewski, Iris Benzie, Vanessa Valdiglesias, Alok Dhawan, Maria Dusinska, Emilio Rojas, Gudrun Koppen, Peter Moller, Erdem CoskunAbstract:The Comet Assay is widely used in human biomonitoring to measure DNA damage as a marker of exposure to genotoxic agents or to investigate genoprotective effects. Studies often involve small numbers of subjects, and design may be sub-optimal in other respects. In addition, Comet Assay protocols in use in different laboratories vary significantly. In spite of these difficulties, it is appropriate to carry out a pooled analysis of all available Comet Assay biomonitoring data, in order to establish baseline parameters of DNA damage, and to investigate associations between Comet Assay measurements and factors such as sex, age, smoking status, nutrition, lifestyle, etc. With this as its major objective, the ComNet project has recruited almost 100 research groups willing to share datasets. Here we provide a background to this project, discussing the history of the Comet Assay and practical issues that can critically affect its performance. We survey its diverse applications in biomonitoring studies, including environmental and occupational exposure to genotoxic agents, genoprotection by dietary and other factors, DNA damage associated with various diseases, and intrinsic factors that affect DNA damage levels in humans. We examine in depth the quality of data from a random selection of studies, from an epidemiological and statistical point of view. © 2013 Elsevier B.V.
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the Comet Assay as a tool for human biomonitoring studies the comnet project
Mutation Research-reviews in Mutation Research, 2014Co-Authors: Andrew Collins, Vanessa Valdiglesias, Alok Dhawan, Maria Dusinska, Iris F. F. Benzie, Emilio Rojas, Gudrun Koppen, Peter Moller, Marcin Kruszewski, Erdem CoskunAbstract:The Comet Assay is widely used in human biomonitoring to measure DNA damage as a marker of exposure to genotoxic agents or to investigate genoprotective effects. Studies often involve small numbers of subjects, and design may be sub-optimal in other respects. In addition, Comet Assay protocols in use in different laboratories vary significantly. In spite of these difficulties, it is appropriate to carry out a pooled analysis of all available Comet Assay biomonitoring data, in order to establish baseline parameters of DNA damage, and to investigate associations between Comet Assay measurements and factors such as sex, age, smoking status, nutrition, lifestyle, etc. With this as its major objective, the ComNet project has recruited almost 100 research groups willing to share datasets. Here we provide a background to this project, discussing the history of the Comet Assay and practical issues that can critically affect its performance. We survey its diverse applications in biomonitoring studies, including environmental and occupational exposure to genotoxic agents, genoprotection by dietary and other factors, DNA damage associated with various diseases, and intrinsic factors that affect DNA damage levels in humans. We examine in depth the quality of data from a random selection of studies, from an epidemiological and statistical point of view.
R Hauser - One of the best experts on this subject based on the ideXlab platform.
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reliability of the Comet Assay in cryopreserved human sperm
Human Reproduction, 2002Co-Authors: Susan M Duty, Z Chen, Tian Huang, Narendra P. Singh, Louise Ryan, Cora E Lewis, R HauserAbstract:BACKGROUND: Although the Comet Assay has potential value for measuring DNA damage in large epidemiological human sperm studies, it is impractical to perform the Assay daily on fresh semen samples. Therefore, before its use in epidemiological studies, the reliability of the Comet Assay in measuring DNA damage in cryopreserved sperm should be compared with that in fresh human sperm. METHODS: Semen samples from 16 men were cryopreserved in liquid nitrogen (LN) using four methods: flash freezing with and without cryopreservative, and programmable freezing with and without cryopreservative. Neutral microgel electrophoresis was performed and Comets were stained with YOYO-1. Comet length was measured using an eyepiece micrometer at x400 magnification. RESULTS: The highest correlation was between Comet Assay results obtained from fresh human semen compared with semen flash frozen without cryopreservative (R = 0.88). However, the method of cryopreservation, as compared with other sources of variability, accounted for only 6% of the variability. Inter-individual variability accounted for 20%, and individual sperm-to-sperm variability within an ejaculate accounted for 65%. CONCLUSIONS: Flash-freezing in LN without cryopreservative most closely reproduced the results obtained using fresh human semen samples, and thereby represents the most appropriate cryopreservation method for human semen in epidemiological studies utilizing the neutral Comet Assay.
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Reliability of the Comet Assay in cryopreserved human sperm
Human Reproduction, 2002Co-Authors: Susan M Duty, C Lewis, Z Chen, Tian Huang, Narendra P. Singh, Louise Ryan, R HauserAbstract:Background: Although the Comet Assay has potential value for measuring DNA damage in large epidemiological human sperm studies, it is impractical to perform the Assay daily on fresh semen samples. Therefore, before its use in epidemiological studies, the reliability of the Comet Assay in measuring DNA damage in cryopreserved sperm should be compared with that in fresh human sperm. Methods: Semen samples from 16 men were cryopreserved in liquid nitrogen (LN) using four methods: flash freezing with and without cryopreservative, and programmable freezing with and without cryopreservative. Neutral microgel electrophoresis was performed and Comets were stained with YOYO-1. Comet length was measured using an eyepiece micrometer at × 400 magnification. Results: The highest correlation was between Comet Assay results obtained from fresh human semen compared with semen flash frozen without cryopreservative (R = 0.88). However, the method of cryopreservation, as compared with other sources of variability, accounted for only 6% of the variability. Inter-individual variability accounted for 20%, and individual sperm-to-sperm variability within an ejaculate accounted for 65%. Conclusions: Flash-freezing in LN without cryopreservative most closely reproduced the results obtained using fresh human semen samples, and thereby represents the most appropriate cryopreservation method for human semen in epidemiological studies utilizing the neutral Comet Assay.