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The miniaturized enzyme-modified comet assay for genotoxicity testing of nanomaterials

The in vitro comet assay is a widely applied method for investigating genotoxicity of chemicals including engineered nanomaterials (NMs). A big challenge in hazard assessment of NMs is possible interference between the NMs and reagents or read-out of the test assay, leading to a risk of biased resul...

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Autores principales: El Yamani, N., Rundén-Pran, E., Collins, A. R., Longhin, E. M., Elje, E., Hoet, P., Vinković Vrček, I., Doak, S. H., Fessard, V., Dusinska, M.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9597874/
https://www.ncbi.nlm.nih.gov/pubmed/36310692
http://dx.doi.org/10.3389/ftox.2022.986318
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author El Yamani, N.
Rundén-Pran, E.
Collins, A. R.
Longhin, E. M.
Elje, E.
Hoet, P.
Vinković Vrček, I.
Doak, S. H.
Fessard, V.
Dusinska, M.
author_facet El Yamani, N.
Rundén-Pran, E.
Collins, A. R.
Longhin, E. M.
Elje, E.
Hoet, P.
Vinković Vrček, I.
Doak, S. H.
Fessard, V.
Dusinska, M.
author_sort El Yamani, N.
collection PubMed
description The in vitro comet assay is a widely applied method for investigating genotoxicity of chemicals including engineered nanomaterials (NMs). A big challenge in hazard assessment of NMs is possible interference between the NMs and reagents or read-out of the test assay, leading to a risk of biased results. Here, we describe both the standard alkaline version of the in vitro comet assay with 12 mini-gels per slide for detection of DNA strand breaks and the enzyme-modified version that allows detection of oxidized DNA bases by applying lesion-specific endonucleases (e.g., formamidopyrimidine DNA glycosylase or endonuclease III). We highlight critical points that need to be taken into consideration when assessing the genotoxicity of NMs, as well as basic methodological considerations, such as the importance of carrying out physicochemical characterization of the NMs and investigating uptake and cytotoxicity. Also, experimental design—including treatment conditions, cell number, cell culture, format and volume of medium on the plate—is crucial and can have an impact on the results, especially when testing NMs. Toxicity of NMs depends upon physicochemical properties that change depending on the environment. To facilitate testing of numerous NMs with distinct modifications, the higher throughput miniaturized version of the comet assay is essential.
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spelling pubmed-95978742022-10-27 The miniaturized enzyme-modified comet assay for genotoxicity testing of nanomaterials El Yamani, N. Rundén-Pran, E. Collins, A. R. Longhin, E. M. Elje, E. Hoet, P. Vinković Vrček, I. Doak, S. H. Fessard, V. Dusinska, M. Front Toxicol Toxicology The in vitro comet assay is a widely applied method for investigating genotoxicity of chemicals including engineered nanomaterials (NMs). A big challenge in hazard assessment of NMs is possible interference between the NMs and reagents or read-out of the test assay, leading to a risk of biased results. Here, we describe both the standard alkaline version of the in vitro comet assay with 12 mini-gels per slide for detection of DNA strand breaks and the enzyme-modified version that allows detection of oxidized DNA bases by applying lesion-specific endonucleases (e.g., formamidopyrimidine DNA glycosylase or endonuclease III). We highlight critical points that need to be taken into consideration when assessing the genotoxicity of NMs, as well as basic methodological considerations, such as the importance of carrying out physicochemical characterization of the NMs and investigating uptake and cytotoxicity. Also, experimental design—including treatment conditions, cell number, cell culture, format and volume of medium on the plate—is crucial and can have an impact on the results, especially when testing NMs. Toxicity of NMs depends upon physicochemical properties that change depending on the environment. To facilitate testing of numerous NMs with distinct modifications, the higher throughput miniaturized version of the comet assay is essential. Frontiers Media S.A. 2022-10-12 /pmc/articles/PMC9597874/ /pubmed/36310692 http://dx.doi.org/10.3389/ftox.2022.986318 Text en Copyright © 2022 El Yamani, Rundén-Pran, Collins, Longhin, Elje, Hoet, Vinković Vrček, Doak, Fessard and Dusinska. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Toxicology
El Yamani, N.
Rundén-Pran, E.
Collins, A. R.
Longhin, E. M.
Elje, E.
Hoet, P.
Vinković Vrček, I.
Doak, S. H.
Fessard, V.
Dusinska, M.
The miniaturized enzyme-modified comet assay for genotoxicity testing of nanomaterials
title The miniaturized enzyme-modified comet assay for genotoxicity testing of nanomaterials
title_full The miniaturized enzyme-modified comet assay for genotoxicity testing of nanomaterials
title_fullStr The miniaturized enzyme-modified comet assay for genotoxicity testing of nanomaterials
title_full_unstemmed The miniaturized enzyme-modified comet assay for genotoxicity testing of nanomaterials
title_short The miniaturized enzyme-modified comet assay for genotoxicity testing of nanomaterials
title_sort miniaturized enzyme-modified comet assay for genotoxicity testing of nanomaterials
topic Toxicology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9597874/
https://www.ncbi.nlm.nih.gov/pubmed/36310692
http://dx.doi.org/10.3389/ftox.2022.986318
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