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 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. � 2016 hiraku et al.