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Sensitive CometChip assay for screening potentially carcinogenic DNA adducts by trapping DNA repair intermediates

Genotoxicity testing is critical for predicting adverse effects of pharmaceutical, industrial, and environmental chemicals. The alkaline comet assay is an established method for detecting DNA strand breaks, however, the assay does not detect potentially carcinogenic bulky adducts that can arise when...

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Autores principales: Ngo, Le P, Owiti, Norah A, Swartz, Carol, Winters, John, Su, Yang, Ge, Jing, Xiong, Aoli, Han, Jongyoon, Recio, Leslie, Samson, Leona D, Engelward, Bevin P
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7026589/
https://www.ncbi.nlm.nih.gov/pubmed/31822921
http://dx.doi.org/10.1093/nar/gkz1077
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author Ngo, Le P
Owiti, Norah A
Swartz, Carol
Winters, John
Su, Yang
Ge, Jing
Xiong, Aoli
Han, Jongyoon
Recio, Leslie
Samson, Leona D
Engelward, Bevin P
author_facet Ngo, Le P
Owiti, Norah A
Swartz, Carol
Winters, John
Su, Yang
Ge, Jing
Xiong, Aoli
Han, Jongyoon
Recio, Leslie
Samson, Leona D
Engelward, Bevin P
author_sort Ngo, Le P
collection PubMed
description Genotoxicity testing is critical for predicting adverse effects of pharmaceutical, industrial, and environmental chemicals. The alkaline comet assay is an established method for detecting DNA strand breaks, however, the assay does not detect potentially carcinogenic bulky adducts that can arise when metabolic enzymes convert pro-carcinogens into a highly DNA reactive products. To overcome this, we use DNA synthesis inhibitors (hydroxyurea and 1-β-d-arabinofuranosyl cytosine) to trap single strand breaks that are formed during nucleotide excision repair, which primarily removes bulky lesions. In this way, comet-undetectable bulky lesions are converted into comet-detectable single strand breaks. Moreover, we use HepaRG™ cells to recapitulate in vivo metabolic capacity, and leverage the CometChip platform (a higher throughput more sensitive comet assay) to create the ‘HepaCometChip’, enabling the detection of bulky genotoxic lesions that are missed by current genotoxicity screens. The HepaCometChip thus provides a broadly effective approach for detection of bulky DNA adducts.
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spelling pubmed-70265892020-02-25 Sensitive CometChip assay for screening potentially carcinogenic DNA adducts by trapping DNA repair intermediates Ngo, Le P Owiti, Norah A Swartz, Carol Winters, John Su, Yang Ge, Jing Xiong, Aoli Han, Jongyoon Recio, Leslie Samson, Leona D Engelward, Bevin P Nucleic Acids Res Methods Online Genotoxicity testing is critical for predicting adverse effects of pharmaceutical, industrial, and environmental chemicals. The alkaline comet assay is an established method for detecting DNA strand breaks, however, the assay does not detect potentially carcinogenic bulky adducts that can arise when metabolic enzymes convert pro-carcinogens into a highly DNA reactive products. To overcome this, we use DNA synthesis inhibitors (hydroxyurea and 1-β-d-arabinofuranosyl cytosine) to trap single strand breaks that are formed during nucleotide excision repair, which primarily removes bulky lesions. In this way, comet-undetectable bulky lesions are converted into comet-detectable single strand breaks. Moreover, we use HepaRG™ cells to recapitulate in vivo metabolic capacity, and leverage the CometChip platform (a higher throughput more sensitive comet assay) to create the ‘HepaCometChip’, enabling the detection of bulky genotoxic lesions that are missed by current genotoxicity screens. The HepaCometChip thus provides a broadly effective approach for detection of bulky DNA adducts. Oxford University Press 2020-02-20 2019-12-11 /pmc/articles/PMC7026589/ /pubmed/31822921 http://dx.doi.org/10.1093/nar/gkz1077 Text en © The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research. http://creativecommons.org/licenses/by/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Methods Online
Ngo, Le P
Owiti, Norah A
Swartz, Carol
Winters, John
Su, Yang
Ge, Jing
Xiong, Aoli
Han, Jongyoon
Recio, Leslie
Samson, Leona D
Engelward, Bevin P
Sensitive CometChip assay for screening potentially carcinogenic DNA adducts by trapping DNA repair intermediates
title Sensitive CometChip assay for screening potentially carcinogenic DNA adducts by trapping DNA repair intermediates
title_full Sensitive CometChip assay for screening potentially carcinogenic DNA adducts by trapping DNA repair intermediates
title_fullStr Sensitive CometChip assay for screening potentially carcinogenic DNA adducts by trapping DNA repair intermediates
title_full_unstemmed Sensitive CometChip assay for screening potentially carcinogenic DNA adducts by trapping DNA repair intermediates
title_short Sensitive CometChip assay for screening potentially carcinogenic DNA adducts by trapping DNA repair intermediates
title_sort sensitive cometchip assay for screening potentially carcinogenic dna adducts by trapping dna repair intermediates
topic Methods Online
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7026589/
https://www.ncbi.nlm.nih.gov/pubmed/31822921
http://dx.doi.org/10.1093/nar/gkz1077
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