title: Nitrative DNA damage induced by multi-walled carbon nanotube via endocytosis in human lung epithelial cells.
abstract: Carbon nanotube (CNT) has a promising usage in the field of material science for industrial purposes because of its unique physicochemical property. However, intraperitoneal administration of CNT was reported to cause mesothelioma in experimental animals. Chronic inflammation may contribute to carcinogenesis induced by fibrous materials. 8-Nitroguanine is a mutagenic DNA lesion formed during inflammation and may play a role in CNT-induced carcinogenesis. In this study, we examined 8-nitroguanine formation in A549 human lung alveolar epithelial cells treated with multi-walled CNT (MWCNT) by fluorescent immunocytochemistry. Both MWCNTs with diameter of 20-30 nm (CNT20) and 40-70 nm (CNT40) significantly induced 8-nitroguanine formation at 5 and 10 μg/ml (p<0.05), which persisted for 24h, although there was no significant difference in DNA-damaging abilities of these MWCNTs. MWCNTs significantly induced the expression of inducible nitric oxide synthase (iNOS) for 24 h (p<0.05). MWCNTs also significantly increased the level of nitrite, a hydrolysis product of oxidized NO, in the culture supernatant at 4 and 8 h (p<0.05). MWCNT-induced 8-nitroguanine formation and iNOS expression were largely suppressed by inhibitors of iNOS (1400 W), nuclear factor-κB (Bay11-7082), actin polymerization (cytochalasin D), caveolae-mediated endocytosis (methyl-β-cyclodextrin, MBCD) and clathrin-mediated endocytosis (monodansylcadaverine, MDC). Electron microscopy revealed that MWCNT was mainly located in vesicular structures in the cytoplasm, and its cellular internalization was reduced by MBCD and MDC. These results suggest that MWCNT is internalized into cells via clathrin- and caveolae-mediated endocytosis, leading to inflammatory reactions including iNOS expression and resulting nitrative DNA damage, which 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.
authors.forename: Feiye
authors.initials: F
authors.lastname: Guo
authors.forename: Ning
authors.initials: N
authors.lastname: Ma
authors.forename: Yoshiteru
authors.initials: Y
authors.lastname: Horibe
authors.forename: Shosuke
authors.initials: S
authors.lastname: Kawanishi
authors.forename: Mariko
authors.initials: M
authors.lastname: Murata
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Hiraku
mesh_terms: D021941:Caveolae / Q000378:metabolism; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D002966:Clathrin / Q000037:antagonists & inhibitors / Q000378:metabolism; D004249:DNA Damage; D004705:Endocytosis / Q000187:drug effects*; D004847:Epithelial Cells / Q000187:drug effects; D005434:Flow Cytometry; D006147:Guanine / Q000031:analogs & derivatives / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D007249:Inflammation / Q000139:chemically induced* / Q000378:metabolism; D008168:Lung / Q000187:drug effects* / Q000378:metabolism; D046529:Microscopy, Electron, Transmission; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000378:metabolism; D037742:Nanotubes, Carbon / Q000633:toxicity*; D052247:Nitric Oxide Synthase Type II / Q000037:antagonists & inhibitors / Q000378:metabolism
chemical_list: C095838:8-nitroguanine; D002966:Clathrin; D016328:NF-kappa B; D037742:Nanotubes, Carbon; D006147:Guanine; C496319:NOS2 protein, human; D052247:Nitric Oxide Synthase Type II