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Multi-walled carbon nanotube induces nitrative DNA damage in human lung epithelial cells via HMGB1-RAGE interaction and Toll-like receptor 9 activation

BACKGROUND: Carbon nanotube (CNT) is used for various industrial purposes, but exhibits carcinogenic effects in experimental animals. Chronic inflammation in the respiratory system may participate in CNT-induced carcinogenesis. 8-Nitroguanine (8-nitroG) is a mutagenic DNA lesion formed during inflam...

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Autores principales: Hiraku, Yusuke, Guo, Feiye, Ma, Ning, Yamada, Tatsuhiko, Wang, Shumin, Kawanishi, Shosuke, Murata, Mariko
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4812657/
https://www.ncbi.nlm.nih.gov/pubmed/27026438
http://dx.doi.org/10.1186/s12989-016-0127-7
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author Hiraku, Yusuke
Guo, Feiye
Ma, Ning
Yamada, Tatsuhiko
Wang, Shumin
Kawanishi, Shosuke
Murata, Mariko
author_facet Hiraku, Yusuke
Guo, Feiye
Ma, Ning
Yamada, Tatsuhiko
Wang, Shumin
Kawanishi, Shosuke
Murata, Mariko
author_sort Hiraku, Yusuke
collection PubMed
description BACKGROUND: Carbon nanotube (CNT) is used for various industrial purposes, but exhibits carcinogenic effects in experimental animals. Chronic inflammation in the respiratory system may participate in CNT-induced carcinogenesis. 8-Nitroguanine (8-nitroG) is a mutagenic DNA lesion formed during inflammation. We have previously reported that multi-walled CNT (MWCNT) induced 8-nitroG formation in lung epithelial cells and this process involved endocytosis. To clarify the mechanism of CNT-induced carcinogenesis, we examined the role of Toll-like receptor (TLR) 9, which resides in endosomes and lysosomes, in 8-nitroG formation in human lung epithelial cell lines. METHODS: We performed immunocytochemistry to examine 8-nitroG formation in A549 and HBEpC cells treated with MWCNT with a length of 1-2 μm (CNT-S) or 5-15 μm (CNT-L) and a diameter of 20-40 nm. We examined inhibitory effects of endocytosis inhibitors, small interfering RNA (siRNA) for TLR9, and antibodies against high-mobility group box-1 (HMGB1) and receptor for advanced glycation end-products (RAGE) on 8-nitroG formation. The release of HMGB1 and double-stranded DNA (dsDNA) into the culture supernatant from MWCNT-treated cells was examined by ELISA and fluorometric analysis, respectively. The association of these molecules was examined by double immunofluorescent staining and co-immunoprecipitation. RESULTS: CNT-L significantly increased 8-nitroG formation at 0.05 μg/ml in A549 cells and its intensity reached a maximum at 1 μg/ml. CNT-L tended to induce stronger cytotoxicity and 8-nitroG formation than CNT-S. Endocytosis inhibitors, TLR9 siRNA and antibodies against HMGB1 and RAGE largely reduced MWCNT-induced 8-nitroG formation. MWCNT increased the release of HMGB1 and dsDNA from A549 cells into culture supernatant. The culture supernatant of MWCNT-exposed cells induced 8-nitroG formation in fresh A549 cells. Double immunofluorescent staining and co-immunoprecipitation showed that TLR9 was associated with HMGB1 and RAGE in lysosomes of MWCNT-treated cells. CONCLUSIONS: MWCNT induces injury or necrosis of lung epithelial cells, which release HMGB1 and DNA into the extracellular space. The HMGB1-DNA complex binds to RAGE on neighboring cells and then CpG DNA is recognized by TLR9 in lysosomes, leading to generation of nitric oxide and 8-nitroG formation. This is the first study demonstrating that TLR9 and related molecules participate in MWCNT-induced genotoxicity and may contribute to carcinogenesis. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12989-016-0127-7) contains supplementary material, which is available to authorized users.
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spelling pubmed-48126572016-03-31 Multi-walled carbon nanotube induces nitrative DNA damage in human lung epithelial cells via HMGB1-RAGE interaction and Toll-like receptor 9 activation Hiraku, Yusuke Guo, Feiye Ma, Ning Yamada, Tatsuhiko Wang, Shumin Kawanishi, Shosuke Murata, Mariko Part Fibre Toxicol Research BACKGROUND: Carbon nanotube (CNT) is used for various industrial purposes, but exhibits carcinogenic effects in experimental animals. Chronic inflammation in the respiratory system may participate in CNT-induced carcinogenesis. 8-Nitroguanine (8-nitroG) is a mutagenic DNA lesion formed during inflammation. We have previously reported that multi-walled CNT (MWCNT) induced 8-nitroG formation in lung epithelial cells and this process involved endocytosis. To clarify the mechanism of CNT-induced carcinogenesis, we examined the role of Toll-like receptor (TLR) 9, which resides in endosomes and lysosomes, in 8-nitroG formation in human lung epithelial cell lines. METHODS: We performed immunocytochemistry to examine 8-nitroG formation in A549 and HBEpC cells treated with MWCNT with a length of 1-2 μm (CNT-S) or 5-15 μm (CNT-L) and a diameter of 20-40 nm. We examined inhibitory effects of endocytosis inhibitors, small interfering RNA (siRNA) for TLR9, and antibodies against high-mobility group box-1 (HMGB1) and receptor for advanced glycation end-products (RAGE) on 8-nitroG formation. The release of HMGB1 and double-stranded DNA (dsDNA) into the culture supernatant from MWCNT-treated cells was examined by ELISA and fluorometric analysis, respectively. The association of these molecules was examined by double immunofluorescent staining and co-immunoprecipitation. RESULTS: CNT-L significantly increased 8-nitroG formation at 0.05 μg/ml in A549 cells and its intensity reached a maximum at 1 μg/ml. CNT-L tended to induce stronger cytotoxicity and 8-nitroG formation than CNT-S. Endocytosis inhibitors, TLR9 siRNA and antibodies against HMGB1 and RAGE largely reduced MWCNT-induced 8-nitroG formation. MWCNT increased the release of HMGB1 and dsDNA from A549 cells into culture supernatant. The culture supernatant of MWCNT-exposed cells induced 8-nitroG formation in fresh A549 cells. Double immunofluorescent staining and co-immunoprecipitation showed that TLR9 was associated with HMGB1 and RAGE in lysosomes of MWCNT-treated cells. CONCLUSIONS: MWCNT induces injury or necrosis of lung epithelial cells, which release HMGB1 and DNA into the extracellular space. The HMGB1-DNA complex binds to RAGE on neighboring cells and then CpG DNA is recognized by TLR9 in lysosomes, leading to generation of nitric oxide and 8-nitroG formation. This is the first study demonstrating that TLR9 and related molecules participate in MWCNT-induced genotoxicity and may contribute to carcinogenesis. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12989-016-0127-7) contains supplementary material, which is available to authorized users. BioMed Central 2016-03-29 /pmc/articles/PMC4812657/ /pubmed/27026438 http://dx.doi.org/10.1186/s12989-016-0127-7 Text en © Hiraku et al. 2016 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Research
Hiraku, Yusuke
Guo, Feiye
Ma, Ning
Yamada, Tatsuhiko
Wang, Shumin
Kawanishi, Shosuke
Murata, Mariko
Multi-walled carbon nanotube induces nitrative DNA damage in human lung epithelial cells via HMGB1-RAGE interaction and Toll-like receptor 9 activation
title Multi-walled carbon nanotube induces nitrative DNA damage in human lung epithelial cells via HMGB1-RAGE interaction and Toll-like receptor 9 activation
title_full Multi-walled carbon nanotube induces nitrative DNA damage in human lung epithelial cells via HMGB1-RAGE interaction and Toll-like receptor 9 activation
title_fullStr Multi-walled carbon nanotube induces nitrative DNA damage in human lung epithelial cells via HMGB1-RAGE interaction and Toll-like receptor 9 activation
title_full_unstemmed Multi-walled carbon nanotube induces nitrative DNA damage in human lung epithelial cells via HMGB1-RAGE interaction and Toll-like receptor 9 activation
title_short Multi-walled carbon nanotube induces nitrative DNA damage in human lung epithelial cells via HMGB1-RAGE interaction and Toll-like receptor 9 activation
title_sort multi-walled carbon nanotube induces nitrative dna damage in human lung epithelial cells via hmgb1-rage interaction and toll-like receptor 9 activation
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4812657/
https://www.ncbi.nlm.nih.gov/pubmed/27026438
http://dx.doi.org/10.1186/s12989-016-0127-7
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