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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...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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BioMed Central
2016
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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. |
format | Online Article Text |
id | pubmed-4812657 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2016 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
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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