Cargando…

Skin Immuno-CometChip in 3D vs. 2D Cultures to Screen Topical Toxins and Skin-Specific Cytochrome Inducers

The targets of topical genotoxic agents are basal and stem cells of the skin. These cells may misrepair DNA lesions, resulting in deleterious mutations of tumor suppressors or oncogenes. However, the genotoxicity of many compounds has not as yet been determined and needs to be tested using a relevan...

Descripción completa

Detalles Bibliográficos
Autores principales: Rosenthal, Dean S., Kuo, Li-Wei, Seagrave, Sarah L., Soni, Vikas, Islam, Nusrat, Minsky, Geetanjali, Dussan-Cuellar, Lucia, Ell, Brian, Simbulan-Rosenthal, Cynthia M., Sykora, Peter
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10048716/
https://www.ncbi.nlm.nih.gov/pubmed/36980902
http://dx.doi.org/10.3390/genes14030630
_version_ 1785014265050038272
author Rosenthal, Dean S.
Kuo, Li-Wei
Seagrave, Sarah L.
Soni, Vikas
Islam, Nusrat
Minsky, Geetanjali
Dussan-Cuellar, Lucia
Ell, Brian
Simbulan-Rosenthal, Cynthia M.
Sykora, Peter
author_facet Rosenthal, Dean S.
Kuo, Li-Wei
Seagrave, Sarah L.
Soni, Vikas
Islam, Nusrat
Minsky, Geetanjali
Dussan-Cuellar, Lucia
Ell, Brian
Simbulan-Rosenthal, Cynthia M.
Sykora, Peter
author_sort Rosenthal, Dean S.
collection PubMed
description The targets of topical genotoxic agents are basal and stem cells of the skin. These cells may misrepair DNA lesions, resulting in deleterious mutations of tumor suppressors or oncogenes. However, the genotoxicity of many compounds has not as yet been determined and needs to be tested using a relevant skin model. To this end, we designed a new high-throughput assay for the detection of agents that create DNA damage in epidermal stem and basal cells and used it to test known DNA-damaging agents. We utilized either 2D epidermal cells or 3D skin equivalents and topically exposed them to different compounds. The Skin Immuno-CometChip assay uses arrays of microwells formed in a collagen/agarose mixture to capture single basal cells in each microwell by virtue of collagen binding to α2β1 integrin, which is present only on basal and stem cells. The presence of β1 integrin was verified by immunofluorescent labeling cells that were then subjected to an electrical field, allowing for the migration of nicked DNA out of the nucleoid in alkali, with the resulting DNA comets stained and imaged. Furthermore, using improved comet detection software allowed for the automated and rapid quantification of DNA damage. Our study indicates that we can accurately predict genotoxicity by using 3D skin cultures, as well as keratinocytes grown in 2D monolayers.
format Online
Article
Text
id pubmed-10048716
institution National Center for Biotechnology Information
language English
publishDate 2023
publisher MDPI
record_format MEDLINE/PubMed
spelling pubmed-100487162023-03-29 Skin Immuno-CometChip in 3D vs. 2D Cultures to Screen Topical Toxins and Skin-Specific Cytochrome Inducers Rosenthal, Dean S. Kuo, Li-Wei Seagrave, Sarah L. Soni, Vikas Islam, Nusrat Minsky, Geetanjali Dussan-Cuellar, Lucia Ell, Brian Simbulan-Rosenthal, Cynthia M. Sykora, Peter Genes (Basel) Article The targets of topical genotoxic agents are basal and stem cells of the skin. These cells may misrepair DNA lesions, resulting in deleterious mutations of tumor suppressors or oncogenes. However, the genotoxicity of many compounds has not as yet been determined and needs to be tested using a relevant skin model. To this end, we designed a new high-throughput assay for the detection of agents that create DNA damage in epidermal stem and basal cells and used it to test known DNA-damaging agents. We utilized either 2D epidermal cells or 3D skin equivalents and topically exposed them to different compounds. The Skin Immuno-CometChip assay uses arrays of microwells formed in a collagen/agarose mixture to capture single basal cells in each microwell by virtue of collagen binding to α2β1 integrin, which is present only on basal and stem cells. The presence of β1 integrin was verified by immunofluorescent labeling cells that were then subjected to an electrical field, allowing for the migration of nicked DNA out of the nucleoid in alkali, with the resulting DNA comets stained and imaged. Furthermore, using improved comet detection software allowed for the automated and rapid quantification of DNA damage. Our study indicates that we can accurately predict genotoxicity by using 3D skin cultures, as well as keratinocytes grown in 2D monolayers. MDPI 2023-03-02 /pmc/articles/PMC10048716/ /pubmed/36980902 http://dx.doi.org/10.3390/genes14030630 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Rosenthal, Dean S.
Kuo, Li-Wei
Seagrave, Sarah L.
Soni, Vikas
Islam, Nusrat
Minsky, Geetanjali
Dussan-Cuellar, Lucia
Ell, Brian
Simbulan-Rosenthal, Cynthia M.
Sykora, Peter
Skin Immuno-CometChip in 3D vs. 2D Cultures to Screen Topical Toxins and Skin-Specific Cytochrome Inducers
title Skin Immuno-CometChip in 3D vs. 2D Cultures to Screen Topical Toxins and Skin-Specific Cytochrome Inducers
title_full Skin Immuno-CometChip in 3D vs. 2D Cultures to Screen Topical Toxins and Skin-Specific Cytochrome Inducers
title_fullStr Skin Immuno-CometChip in 3D vs. 2D Cultures to Screen Topical Toxins and Skin-Specific Cytochrome Inducers
title_full_unstemmed Skin Immuno-CometChip in 3D vs. 2D Cultures to Screen Topical Toxins and Skin-Specific Cytochrome Inducers
title_short Skin Immuno-CometChip in 3D vs. 2D Cultures to Screen Topical Toxins and Skin-Specific Cytochrome Inducers
title_sort skin immuno-cometchip in 3d vs. 2d cultures to screen topical toxins and skin-specific cytochrome inducers
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10048716/
https://www.ncbi.nlm.nih.gov/pubmed/36980902
http://dx.doi.org/10.3390/genes14030630
work_keys_str_mv AT rosenthaldeans skinimmunocometchipin3dvs2dculturestoscreentopicaltoxinsandskinspecificcytochromeinducers
AT kuoliwei skinimmunocometchipin3dvs2dculturestoscreentopicaltoxinsandskinspecificcytochromeinducers
AT seagravesarahl skinimmunocometchipin3dvs2dculturestoscreentopicaltoxinsandskinspecificcytochromeinducers
AT sonivikas skinimmunocometchipin3dvs2dculturestoscreentopicaltoxinsandskinspecificcytochromeinducers
AT islamnusrat skinimmunocometchipin3dvs2dculturestoscreentopicaltoxinsandskinspecificcytochromeinducers
AT minskygeetanjali skinimmunocometchipin3dvs2dculturestoscreentopicaltoxinsandskinspecificcytochromeinducers
AT dussancuellarlucia skinimmunocometchipin3dvs2dculturestoscreentopicaltoxinsandskinspecificcytochromeinducers
AT ellbrian skinimmunocometchipin3dvs2dculturestoscreentopicaltoxinsandskinspecificcytochromeinducers
AT simbulanrosenthalcynthiam skinimmunocometchipin3dvs2dculturestoscreentopicaltoxinsandskinspecificcytochromeinducers
AT sykorapeter skinimmunocometchipin3dvs2dculturestoscreentopicaltoxinsandskinspecificcytochromeinducers