Amaya Azqueta

Universidad de Navarra

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Adela López de Ceráin

Universidad de Navarra

Andrew Collins

University of Oslo

Sabine A. S. Langie

Maastricht University

Peter Møller

University of Copenhagen

Ariane Vettorazzi

Universidad de Navarra

Gunnar Brunborg

Norwegian Institute of Public Health

Hugo Cerecetto

Universidad de la República

Solange Costa

National Institute of Health Dr. Ricardo Jorge

Maria Dusinšká

Norwegian Institute for Air Research

Sergey Shaposhnikov

Oslo Cancer Cluster

Cooperative Institutions

Universidad de Navarra

Centre National de la Recherche Scientifique

Consejo Superior de Investigaciones Científicas

Inserm

Universidad de Granada

Universidad de la República

Instituto de Salud Carlos III

Navarre Institute of Health Research

University of Oslo

Universitat de Barcelona

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Measuring DNA modifications with the comet assay: a compendium of protocols

Nature Protocols (2023)

Andrew Collins Peter Møller Goran Gajski Soňa Vodenková Abdulhadi Abdulwahed

Comet Assay

10.1038/s41596-022-00754-y

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Citations (193)

In vitro comet assay for DNA repair: a warning concerning application to cultured cells

Mutagenesis (2009)

Amaya Azqueta Yolanda Lorenzo Andrew Collins

The comet assay (single-cell gel electrophoresis) is a sensitive and simple method for measuring DNA damage. An early modification of the assay involved the application of specific repair endonucleases to convert lesions to breaks; thus, for example, endonuclease III was used to measure oxidized pyrimidines. This concept has now been extended to produce an in vitro assay for DNA repair activity in a cell-free extract, for example from lymphocytes. The extract is incubated with substrate DNA containing specific base damage, and repair incision is detected as breaks in this DNA. We have recently been studying effects of phytochemicals in cultured cells, whether as antioxidants or as potential modulators of DNA repair. We realized that there is a need to check that observed effects that appear as an enhancement of repair (i.e. increased breaks in substrate DNA) are not simply due to a direct damaging effect of the phytochemical or to induction of non-specific nucleases. Here, we describe a rigorous approach to testing for this possibility, which we recommend to anyone carrying out similar experiments.

Comet Assay

10.1093/mutage/gep009

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Citations (28)

Corrigendum to “Novel approach for the detection of alkylated bases using the enzyme-modified comet assay” [Toxicol. Lett. 330 (2020) 108–117]

Toxicology Letters (2020)

Damián Muruzabal Julen Sanz-Serrano Sylvie Sauvaigo Kristine B. Gützkow Adela López de Ceráin

10.1016/j.toxlet.2020.10.011

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In vitro genotoxicity assessment of French fries from mass catering companies: a preliminary study

Mutagenesis (2022)

Julen Sanz-Serrano Roncesvalles Garayoa Ana Isabel Vitas Adela López de Ceráin Amaya Azqueta

It is generally assumed that French fries are likely to have weak in vitro mutagenic activity, but most studies thereof have only assessed gene mutations. In this article, the genotoxicity of 10 extracts of French fries was assessed using the in vitro micronucleus test (following the principles of the OECD 487 guidelines). Each sample was obtained from a different mass catering company in Navarra (Spain). This assay, together with the Ames test, is recommended in the basic in vitro phase included in the European Food Safety Authority Opinion on Genotoxicity Testing Strategies Applicable to Food and Feed Safety Assessment. Eight of 10 samples from mass catering companies induced chromosomal aberrations in the in vitro micronucleus test. Moreover, French fries deep-fried in the laboratory for different periods of time (0, 3, 5, 10, 20, 30 min) were assessed using the in vitro micronucleus test. Genotoxicity was observed in all time periods from 3 min on. The biological relevance of these results must be further explored.

Ames test

10.1093/mutage/geac021

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Citations (4)

Does the duration of lysis affect the sensitivity of the in vitro alkaline comet assay?

Mutagenesis (2014)

Juan Enciso Olga Sánchez Adela López de Ceráin Amaya Azqueta

The alkaline comet assay is now the method of choice for measuring different kinds of DNA damage in cells. Several attempts have been made to identify and evaluate the critical points affecting the comet assay outcome, highlighting the requirement of arriving at a standardised protocol in order to be able to compare the results obtained in different laboratories. However, reports on the effect of modifying the time of lysis are lacking. Here we tested different times of lysis (from no lysis to 1 week) in control HeLa cells and HeLa cells treated with different concentrations of methyl methanesulfonate (MMS) or H2O2. We also tested different times of lysis in the comet assay combined with formamidopyrimidine DNA glycosylase (FPG) in untreated and Ro 19-8022 plus light-treated HeLa cells. The same DNA damage levels were detected in the absence of lysis or after 1h of lysis when the standard comet assay was used to detect the MMS- and H2O2-induced lesions; the response increased when longer lysis was used, up to at least 1 week. When FPG was used, a minimum lysis period of 5 min was necessary to allow the enzyme to reach the DNA; the same DNA damage levels were detected after 5 min or 1h of lysis and the response increased up to 24h. In conclusion, the time of lysis can be varied depending on the sensitivity needed in both versions of the assay, and a constant time of lysis should be used if results from different experiments or laboratories are to be compared.

Comet Assay

HeLa

Alkaline lysis

Lysis buffer

Ethyl methanesulfonate

10.1093/mutage/geu047

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Citations (29)

Controlling variation in the comet assay

Frontiers in Genetics (2014)

Andrew Collins Naouale El Yamani Yolanda Lorenzo Sergey Shaposhnikov Gunnar Brunborg

Variability of the comet assay is a serious issue, whether it occurs from experiment to experiment in the same laboratory, or between different laboratories analysing identical samples. Do we have to live with high variability, just because the comet assay is a biological assay rather than analytical chemistry? Numerous attempts have been made to limit variability by standardizing the assay protocol, and the critical steps in the assay have been identified; agarose concentration, duration of alkaline incubation, and electrophoresis conditions (time, temperature, and voltage gradient) are particularly important. Even when these are controlled, variation seems to be inevitable. It is helpful to include in experiments reference standards, i.e., cells with a known amount of specific damage to the DNA. They can be aliquots frozen from a single large batch of cells, either untreated (negative controls) or treated with, for example, H2O2 or X-rays to induce strand breaks (positive control for the basic assay), or photosensitiser plus light to oxidize guanine (positive control for Fpg- or OGG1-sensitive sites). Reference standards are especially valuable when performing a series of experiments over a long period-for example, analysing samples of white blood cells from a large human biomonitoring trial-to check that the assay is performing consistently, and to identify anomalous results necessitating a repeat experiment. The reference values of tail intensity can also be used to iron out small variations occurring from day to day. We present examples of the use of reference standards in human trials, both within one laboratory and between different laboratories, and describe procedures that can be used to control variation.

Comet Assay

Agarose

10.3389/fgene.2014.00359

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Citations (100)

1,2,4‐Triazine N‐Oxide Derivatives: Studies as Potential Hypoxic Cytotoxins. Part 3.

ChemInform (2004)

Hugo Cerecetto Mercedes González Mariela Risso Patricia Saenz‐Méndez Claudio Olea‐Azar

Abstract For Abstract see ChemInform Abstract in Full Text.

Triazine

10.1002/chin.200435155

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Citations (0)

An optimized comet-based in vitro DNA repair assay to assess base and nucleotide excision repair activity

Nature Protocols (2020)

Soňa Vodenková Amaya Azqueta Andrew Collins Maria Dusinšká Isabel Gaivão

Comet Assay

Agarose

Agarose gel electrophoresis

10.1038/s41596-020-0401-x

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Citations (38)

An ECVAG inter-laboratory validation study of the comet assay: inter-laboratory and intra-laboratory variations of DNA strand breaks and FPG-sensitive sites in human mononuclear cells

Mutagenesis (2013)

Clara Ersson Peter Møller Lykke Forchhammer Steffen Loft Amaya Azqueta

The alkaline comet assay is an established, sensitive method extensively used in biomonitoring studies. This method can be modified to measure a range of different types of DNA damage. However, considerable differences in the protocols used by different research groups affect the inter-laboratory comparisons of results. The aim of this study was to assess the inter-laboratory, intra-laboratory, sample and residual (unexplained) variations in DNA strand breaks and formamidopyrimidine DNA glycosylase (FPG)-sensitive sites measured by the comet assay by using a balanced Latin square design. Fourteen participating laboratories used their own comet assay protocols to measure the level of DNA strand breaks and FPG-sensitive sites in coded samples containing peripheral blood mononuclear cells (PBMC) and the level of DNA strand breaks in coded calibration curve samples (cells exposed to different doses of ionising radiation) on three different days of analysis. Eleven laboratories found dose–response relationships in the coded calibration curve samples on two or three days of analysis, whereas three laboratories had technical problems in their assay. In the coded calibration curve samples, the dose of ionising radiation, inter-laboratory variation, intra-laboratory variation and residual variation contributed to 60.9, 19.4, 0.1 and 19.5%, respectively, of the total variation. In the coded PBMC samples, the inter-laboratory variation explained the largest fraction of the overall variation of DNA strand breaks (79.2%) and the residual variation (19.9%) was much larger than the intra-laboratory (0.3%) and inter-subject (0.5%) variation. The same partitioning of the overall variation of FPG-sensitive sites in the PBMC samples indicated that the inter-laboratory variation was the strongest contributor (56.7%), whereas the residual (42.9%), intra-laboratory (0.2%) and inter-subject (0.3%) variations again contributed less to the overall variation. The results suggest that the variation in DNA damage, measured by comet assay, in PBMC from healthy subjects is assay variation rather than variation between subjects.

Comet Assay

Coefficient of variation

10.1093/mutage/get001

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Citations (84)

The enzyme-modified comet assay: Past, present and future

Food and Chemical Toxicology (2020)

Damián Muruzabal Andrew Collins Amaya Azqueta

Comet Assay

Uracil

10.1016/j.fct.2020.111865

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Citations (56)