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Effects of Benzo(e)pyrene on Reactive Oxygen/Nitrogen Species and Inflammatory Cytokines Induction in Human RPE Cells and Attenuation by Mitochondrial-involved Mechanism

PURPOSE: To identify inhibitors that could effectively lower reactive oxygen/nitrogen species (ROS/RNS), complement and inflammatory cytokine levels induced by Benzo(e)pyrene [B(e)p], an element of cigarette smoke, in human retinal pigment epithelial cells (ARPE-19) in vitro. METHODS: ARPE-19 cells...

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Autores principales: Estrago-Franco, M. Fernanda, Moustafa, M. Tarek, Riazi-Esfahani, Mohammad, Sapkal, Ashish U., Piche-Lopez, Rhina, Patil, A. Jayaprakash, Sharma, Ashish, Falatoonzadeh, Payam, Chwa, Marilyn, Luczy-Bachman, Georgia, Kuppermann, Baruch D., Kenney, M. Cristina
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Medknow Publications & Media Pvt Ltd 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5139551/
https://www.ncbi.nlm.nih.gov/pubmed/27994808
http://dx.doi.org/10.4103/2008-322X.194091
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author Estrago-Franco, M. Fernanda
Moustafa, M. Tarek
Riazi-Esfahani, Mohammad
Sapkal, Ashish U.
Piche-Lopez, Rhina
Patil, A. Jayaprakash
Sharma, Ashish
Falatoonzadeh, Payam
Chwa, Marilyn
Luczy-Bachman, Georgia
Kuppermann, Baruch D.
Kenney, M. Cristina
author_facet Estrago-Franco, M. Fernanda
Moustafa, M. Tarek
Riazi-Esfahani, Mohammad
Sapkal, Ashish U.
Piche-Lopez, Rhina
Patil, A. Jayaprakash
Sharma, Ashish
Falatoonzadeh, Payam
Chwa, Marilyn
Luczy-Bachman, Georgia
Kuppermann, Baruch D.
Kenney, M. Cristina
author_sort Estrago-Franco, M. Fernanda
collection PubMed
description PURPOSE: To identify inhibitors that could effectively lower reactive oxygen/nitrogen species (ROS/RNS), complement and inflammatory cytokine levels induced by Benzo(e)pyrene [B(e)p], an element of cigarette smoke, in human retinal pigment epithelial cells (ARPE-19) in vitro. METHODS: ARPE-19 cells were treated for 24 hours with 200 μM, 100 μM, and 50 μM B(e)p or DMSO (dimethyl sulfoxide)-equivalent concentrations. Some cultures were pre-treated with ROS/RNS inhibitors (NG nitro-L-arginine, inhibits nitric oxide synthase; Apocynin, inhibits NADPH oxidase; Rotenone, inhibits mitochondrial complex I; Antimycin A, inhibits mitochondria complex III) and ROS/RNS levels were measured with a fluorescent H(2) DCFDA assay. Multiplex bead arrays were used to measure levels of Interleukin-6 (IL-6), Interleukin-8 (IL-8), Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF), Transforming Growth Factor alpha (TGF-α) and Vascular Endothelial Growth Factor (VEGF). IL-6 levels were also measured by an enzyme-linked immunosorbent assay. Real-time qPCR analyses were performed with primers for C3 (component 3), CFH (inhibits complement activation), CD59 (inhibitor of the complement membrane attack complex (MAC)) and CD55/DAF (accelerates decay of target complement target proteins). RESULTS: The ARPE-19 cultures treated with B(e)p showed significantly increased ROS/RNS levels (P < 0.001), which were then partially reversed by 6 μM Antimycin A (19%, P = 0.03), but not affected by the other ROS/RNS inhibitors. The B(e)p treated cultures demonstrated increased levels of IL-6 (33%; P = 0.016) and GM-CSF (29%; P = 0.0001) compared to DMSO-equivalent controls, while the expression levels for components of the complement pathway (C3, CFH, CD59 and CD55/DAF) were not changed. CONCLUSION: The cytotoxic effects of B(e)p include elevated ROS/RNS levels along with pro-inflammatory IL-6 and GM-CSF proteins. Blocking the Qi site of cytochrome c reductase (complex III) with Antimycin A led to partial reduction in B(e)p induced ROS production. Our findings suggest that inhibitors for multiple pathways would be necessary to protect the retinal cells from B(e)p induced toxicity.
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spelling pubmed-51395512016-12-19 Effects of Benzo(e)pyrene on Reactive Oxygen/Nitrogen Species and Inflammatory Cytokines Induction in Human RPE Cells and Attenuation by Mitochondrial-involved Mechanism Estrago-Franco, M. Fernanda Moustafa, M. Tarek Riazi-Esfahani, Mohammad Sapkal, Ashish U. Piche-Lopez, Rhina Patil, A. Jayaprakash Sharma, Ashish Falatoonzadeh, Payam Chwa, Marilyn Luczy-Bachman, Georgia Kuppermann, Baruch D. Kenney, M. Cristina J Ophthalmic Vis Res Original Article PURPOSE: To identify inhibitors that could effectively lower reactive oxygen/nitrogen species (ROS/RNS), complement and inflammatory cytokine levels induced by Benzo(e)pyrene [B(e)p], an element of cigarette smoke, in human retinal pigment epithelial cells (ARPE-19) in vitro. METHODS: ARPE-19 cells were treated for 24 hours with 200 μM, 100 μM, and 50 μM B(e)p or DMSO (dimethyl sulfoxide)-equivalent concentrations. Some cultures were pre-treated with ROS/RNS inhibitors (NG nitro-L-arginine, inhibits nitric oxide synthase; Apocynin, inhibits NADPH oxidase; Rotenone, inhibits mitochondrial complex I; Antimycin A, inhibits mitochondria complex III) and ROS/RNS levels were measured with a fluorescent H(2) DCFDA assay. Multiplex bead arrays were used to measure levels of Interleukin-6 (IL-6), Interleukin-8 (IL-8), Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF), Transforming Growth Factor alpha (TGF-α) and Vascular Endothelial Growth Factor (VEGF). IL-6 levels were also measured by an enzyme-linked immunosorbent assay. Real-time qPCR analyses were performed with primers for C3 (component 3), CFH (inhibits complement activation), CD59 (inhibitor of the complement membrane attack complex (MAC)) and CD55/DAF (accelerates decay of target complement target proteins). RESULTS: The ARPE-19 cultures treated with B(e)p showed significantly increased ROS/RNS levels (P < 0.001), which were then partially reversed by 6 μM Antimycin A (19%, P = 0.03), but not affected by the other ROS/RNS inhibitors. The B(e)p treated cultures demonstrated increased levels of IL-6 (33%; P = 0.016) and GM-CSF (29%; P = 0.0001) compared to DMSO-equivalent controls, while the expression levels for components of the complement pathway (C3, CFH, CD59 and CD55/DAF) were not changed. CONCLUSION: The cytotoxic effects of B(e)p include elevated ROS/RNS levels along with pro-inflammatory IL-6 and GM-CSF proteins. Blocking the Qi site of cytochrome c reductase (complex III) with Antimycin A led to partial reduction in B(e)p induced ROS production. Our findings suggest that inhibitors for multiple pathways would be necessary to protect the retinal cells from B(e)p induced toxicity. Medknow Publications & Media Pvt Ltd 2016 /pmc/articles/PMC5139551/ /pubmed/27994808 http://dx.doi.org/10.4103/2008-322X.194091 Text en Copyright: © 2016 Journal of Ophthalmic and Vision Research http://creativecommons.org/licenses/by-nc-sa/3.0 This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as the author is credited and the new creations are licensed under the identical terms.
spellingShingle Original Article
Estrago-Franco, M. Fernanda
Moustafa, M. Tarek
Riazi-Esfahani, Mohammad
Sapkal, Ashish U.
Piche-Lopez, Rhina
Patil, A. Jayaprakash
Sharma, Ashish
Falatoonzadeh, Payam
Chwa, Marilyn
Luczy-Bachman, Georgia
Kuppermann, Baruch D.
Kenney, M. Cristina
Effects of Benzo(e)pyrene on Reactive Oxygen/Nitrogen Species and Inflammatory Cytokines Induction in Human RPE Cells and Attenuation by Mitochondrial-involved Mechanism
title Effects of Benzo(e)pyrene on Reactive Oxygen/Nitrogen Species and Inflammatory Cytokines Induction in Human RPE Cells and Attenuation by Mitochondrial-involved Mechanism
title_full Effects of Benzo(e)pyrene on Reactive Oxygen/Nitrogen Species and Inflammatory Cytokines Induction in Human RPE Cells and Attenuation by Mitochondrial-involved Mechanism
title_fullStr Effects of Benzo(e)pyrene on Reactive Oxygen/Nitrogen Species and Inflammatory Cytokines Induction in Human RPE Cells and Attenuation by Mitochondrial-involved Mechanism
title_full_unstemmed Effects of Benzo(e)pyrene on Reactive Oxygen/Nitrogen Species and Inflammatory Cytokines Induction in Human RPE Cells and Attenuation by Mitochondrial-involved Mechanism
title_short Effects of Benzo(e)pyrene on Reactive Oxygen/Nitrogen Species and Inflammatory Cytokines Induction in Human RPE Cells and Attenuation by Mitochondrial-involved Mechanism
title_sort effects of benzo(e)pyrene on reactive oxygen/nitrogen species and inflammatory cytokines induction in human rpe cells and attenuation by mitochondrial-involved mechanism
topic Original Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5139551/
https://www.ncbi.nlm.nih.gov/pubmed/27994808
http://dx.doi.org/10.4103/2008-322X.194091
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