title: Multi-walled carbon nanotube induces nitrative DNA damage in human lung epithelial cells via HMGB1-RAGE interaction and Toll-like receptor 9 activation.
abstract: 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.
authors.affiliation: Department of Environmental and Molecular Medicine, Mie University Graduate School of Medicine, 2-174 Edobashi, Tsu, Mie, 514-8507, Japan. y-hiraku@doc.medic.mie-u.ac.jp.
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Hiraku
authors.affiliation: Department of Environmental and Molecular Medicine, Mie University Graduate School of Medicine, 2-174 Edobashi, Tsu, Mie, 514-8507, Japan.
authors.forename: Feiye
authors.initials: F
authors.lastname: Guo
authors.affiliation: Faculty of Nursing Science, Suzuka University of Medical Science, 3500-3 Minami-Tamagaki-cho, Suzuka, Mie, 513-8670, Japan.
authors.forename: Ning
authors.initials: N
authors.lastname: Ma
authors.affiliation: Department of Environmental and Molecular Medicine, Mie University Graduate School of Medicine, 2-174 Edobashi, Tsu, Mie, 514-8507, Japan.
authors.forename: Tatsuhiko
authors.initials: T
authors.lastname: Yamada
authors.affiliation: Department of Environmental and Molecular Medicine, Mie University Graduate School of Medicine, 2-174 Edobashi, Tsu, Mie, 514-8507, Japan.
authors.forename: Shumin
authors.initials: S
authors.lastname: Wang
authors.affiliation: Faculty of Pharmaceutical Sciences, Suzuka University of Medical Science, 3500-3 Minami-Tamagaki-cho, Suzuka, Mie, 513-8670, Japan.
authors.forename: Shosuke
authors.initials: S
authors.lastname: Kawanishi
authors.affiliation: Department of Environmental and Molecular Medicine, Mie University Graduate School of Medicine, 2-174 Edobashi, Tsu, Mie, 514-8507, Japan.
authors.forename: Mariko
authors.initials: M
authors.lastname: Murata
mesh_terms: D055371:Acute Lung Injury / Q000139:chemically induced* / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D000906:Antibodies / Q000494:pharmacology; D045744:Cell Line, Tumor; D018899:CpG Islands; D053903:DNA Breaks, Double-Stranded; D004305:Dose-Response Relationship, Drug; D004705:Endocytosis / Q000187:drug effects; D004847:Epithelial Cells / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D006147:Guanine / Q000031:analogs & derivatives / Q000378:metabolism; D024243:HMGB1 Protein / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D008168:Lung / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D008247:Lysosomes / Q000187:drug effects / Q000378:metabolism; D037742:Nanotubes, Carbon / Q000633:toxicity*; D009336:Necrosis; D009569:Nitric Oxide / Q000378:metabolism; D010316:Particle Size; D034622:RNA Interference; D026361:Reactive Nitrogen Species / Q000378:metabolism*; D000067759:Receptor for Advanced Glycation End Products / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism*; D018570:Risk Assessment; D015398:Signal Transduction / Q000187:drug effects; D013997:Time Factors; D051217:Toll-Like Receptor 9 / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
chemical_list: C095838:8-nitroguanine; C000593871:AGER protein, human; D000906:Antibodies; D024243:HMGB1 Protein; C585491:HMGB1 protein, human; D037742:Nanotubes, Carbon; D026361:Reactive Nitrogen Species; D000067759:Receptor for Advanced Glycation End Products; C495371:TLR9 protein, human; D051217:Toll-Like Receptor 9; D009569:Nitric Oxide; D006147:Guanine
keywords: 8-nitroguanine; Carbon nanotube; Carcinogenesis; Cytotoxicity; DNA damage; Endocytosis; High-mobility group box-1, Receptor for advanced glycation end-products, Toll-like receptor; Nitric oxide