Flow Cytometric Analysis of Oxidative Stress in Escherichia coli B Strains Deficient in Genes of the Antioxidant Defence.

The detection of reactive oxygen species (ROS) and the analysis of oxidative stress are frequent applications of functional flow cytometry. Identifying and quantifying the ROS species generated during oxidative stress are crucial steps for the investigation of molecular mechanisms underlying stress...

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Autores: Jávega B, Herrera G, O'Connor JE
Formato: artículo
Estado:Versión publicada
Fecha de publicación:2022
País:España
Recursos:Centro de Investigación Principe Felipe (CIPF)
Repositorio:r-CIPF. Repositorio Institucional Producción Científica del Centro de Investigación Principe Felipe (CIPF)
OAI Identifier:oai:cipf.fundanetsuite.com:p4036
Acesso em linha:https://cipf.fundanetsuite.com/Publicaciones/ProdCientif/PublicacionFrw.aspx?id=4036
Access Level:acceso abierto
Palavra-chave:OxyR, SodA, SodB, catalase, fluorescence, hydrogen peroxide, organic hydroperoxides, superoxide dismutase
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spelling Flow Cytometric Analysis of Oxidative Stress in Escherichia coli B Strains Deficient in Genes of the Antioxidant Defence.Jávega BHerrera GO'Connor JEOxyR, SodA, SodB, catalase, fluorescence, hydrogen peroxide, organic hydroperoxides, superoxide dismutaseThe detection of reactive oxygen species (ROS) and the analysis of oxidative stress are frequent applications of functional flow cytometry. Identifying and quantifying the ROS species generated during oxidative stress are crucial steps for the investigation of molecular mechanisms underlying stress responses. Currently, there is a wide availability of fluorogenic substrates for such purposes, but limitations in their specificity and sensitivity may affect the accuracy of the analysis. The aim of our work was to validate a new experimental model based in different strains of Escherichia coli B deficient in key genes for antioxidant defense, namely oxyR , sodA and s odB . We applied this model to systematically assess issues of specificity in fluorescent probes and the involvement of different ROS in a bacterial model of oxidative stress, as the probes can react with a variety of oxidants and free radical species. Our results confirm the higher sensitivity and specificity of the fluorescent probe mitochondrial peroxy yellow 1 (MitoPY1) for the detection of H 2 O 2 , and its very low capacity for organic hydroperoxides, thus extending MitoPY1's specificity for H 2 O 2 in mammalian cells to a bacterial model. On the contrary, the fluorescent probe 2',7'-dichlorodihydrofluorescein diacetate (H 2 DCF-DA) is more sensitive to organic peroxides than to H 2 O 2 , confirming the lack of selectivity of H 2 DCF-DA to H 2 O 2 . Treatment with organic peroxides and H 2 O 2 suggests a superoxide-independent oxidation of the fluorescent probe Hydroethidine (HE). We found a positive correlation between the lipophilicity of the peroxides and their toxicity to E. coli , suggesting greater quantitative importance of the peroxidative effects on the bacterial membrane and/or greater efficiency of the protection systems against the intracellular effects of H 2 O 2 than against the membrane oxidative stress induced by organic peroxides. Altogether, our results may aid in preventing or minimizing experimental errors and providing recommendations for the proper design of cytometric studies of oxidative stress, in accordance with current recommendations and guidelines.MDPI2022info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttps://cipf.fundanetsuite.com/Publicaciones/ProdCientif/PublicacionFrw.aspx?id=4036INTERNATIONAL JOURNAL OF MOLECULAR SCIENCESISSN: 14220067reponame:r-CIPF. Repositorio Institucional Producción Científica del Centro de Investigación Principe Felipe (CIPF)instname:Centro de Investigación Principe Felipe (CIPF)Inglésinfo:eu-repo/semantics/openAccessoai:cipf.fundanetsuite.com:p40362026-06-17T11:19:47Z
dc.title.none.fl_str_mv Flow Cytometric Analysis of Oxidative Stress in Escherichia coli B Strains Deficient in Genes of the Antioxidant Defence.
title Flow Cytometric Analysis of Oxidative Stress in Escherichia coli B Strains Deficient in Genes of the Antioxidant Defence.
spellingShingle Flow Cytometric Analysis of Oxidative Stress in Escherichia coli B Strains Deficient in Genes of the Antioxidant Defence.
Jávega B
OxyR, SodA, SodB, catalase, fluorescence, hydrogen peroxide, organic hydroperoxides, superoxide dismutase
title_short Flow Cytometric Analysis of Oxidative Stress in Escherichia coli B Strains Deficient in Genes of the Antioxidant Defence.
title_full Flow Cytometric Analysis of Oxidative Stress in Escherichia coli B Strains Deficient in Genes of the Antioxidant Defence.
title_fullStr Flow Cytometric Analysis of Oxidative Stress in Escherichia coli B Strains Deficient in Genes of the Antioxidant Defence.
title_full_unstemmed Flow Cytometric Analysis of Oxidative Stress in Escherichia coli B Strains Deficient in Genes of the Antioxidant Defence.
title_sort Flow Cytometric Analysis of Oxidative Stress in Escherichia coli B Strains Deficient in Genes of the Antioxidant Defence.
dc.creator.none.fl_str_mv Jávega B
Herrera G
O'Connor JE
author Jávega B
author_facet Jávega B
Herrera G
O'Connor JE
author_role author
author2 Herrera G
O'Connor JE
author2_role author
author
dc.subject.none.fl_str_mv OxyR, SodA, SodB, catalase, fluorescence, hydrogen peroxide, organic hydroperoxides, superoxide dismutase
topic OxyR, SodA, SodB, catalase, fluorescence, hydrogen peroxide, organic hydroperoxides, superoxide dismutase
description The detection of reactive oxygen species (ROS) and the analysis of oxidative stress are frequent applications of functional flow cytometry. Identifying and quantifying the ROS species generated during oxidative stress are crucial steps for the investigation of molecular mechanisms underlying stress responses. Currently, there is a wide availability of fluorogenic substrates for such purposes, but limitations in their specificity and sensitivity may affect the accuracy of the analysis. The aim of our work was to validate a new experimental model based in different strains of Escherichia coli B deficient in key genes for antioxidant defense, namely oxyR , sodA and s odB . We applied this model to systematically assess issues of specificity in fluorescent probes and the involvement of different ROS in a bacterial model of oxidative stress, as the probes can react with a variety of oxidants and free radical species. Our results confirm the higher sensitivity and specificity of the fluorescent probe mitochondrial peroxy yellow 1 (MitoPY1) for the detection of H 2 O 2 , and its very low capacity for organic hydroperoxides, thus extending MitoPY1's specificity for H 2 O 2 in mammalian cells to a bacterial model. On the contrary, the fluorescent probe 2',7'-dichlorodihydrofluorescein diacetate (H 2 DCF-DA) is more sensitive to organic peroxides than to H 2 O 2 , confirming the lack of selectivity of H 2 DCF-DA to H 2 O 2 . Treatment with organic peroxides and H 2 O 2 suggests a superoxide-independent oxidation of the fluorescent probe Hydroethidine (HE). We found a positive correlation between the lipophilicity of the peroxides and their toxicity to E. coli , suggesting greater quantitative importance of the peroxidative effects on the bacterial membrane and/or greater efficiency of the protection systems against the intracellular effects of H 2 O 2 than against the membrane oxidative stress induced by organic peroxides. Altogether, our results may aid in preventing or minimizing experimental errors and providing recommendations for the proper design of cytometric studies of oxidative stress, in accordance with current recommendations and guidelines.
publishDate 2022
dc.date.none.fl_str_mv 2022
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv https://cipf.fundanetsuite.com/Publicaciones/ProdCientif/PublicacionFrw.aspx?id=4036
url https://cipf.fundanetsuite.com/Publicaciones/ProdCientif/PublicacionFrw.aspx?id=4036
dc.language.none.fl_str_mv Inglés
language_invalid_str_mv Inglés
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.publisher.none.fl_str_mv MDPI
publisher.none.fl_str_mv MDPI
dc.source.none.fl_str_mv INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
ISSN: 14220067
reponame:r-CIPF. Repositorio Institucional Producción Científica del Centro de Investigación Principe Felipe (CIPF)
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