Nitrative DNA damage in human lung epithelial cells treated with multi-walled carbon nanotube

Abstract

Carbon nanotube (CNT) has the potential for applications in industry because of its unique physicochemical property. However, intraperitoneal administration of CNT was reported to cause mesothelioma in experimental animals. Therefore, there is a concern that nanomaterials may induce carcinogenesis in humans. 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 alveolar epithelial cell line treated with multi-walled CNT (MWCNT) by fluorescent immunocytochemistry. Both MWCNTs with diameter of 20–30 nm (CNT20) and 40–70 nm (CNT40) induced 8-nitroguanine formation in a dose-dependent manner, which persisted for 24 h. MWCNTs significantly induced the expression of inducible nitric oxide synthase (iNOS) for 24 h ( p < 0.05). MWCNTs also significantly increased the level of NO 2 - , 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 suppressed by inhibitors of iNOS (1400 W), nuclear factor-kB (Bay11-7082), actin polymerization (cytochalasin D) and caveola- (methyl-b-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 largely reduced by MBCD and MDC. These results suggest that MWCNT is internalized into cells via endocytosis, and that internalized particles play the key role in inflammatory reactions, including iNOS expression, and resulting nitrative DNA damage, which may contribute to carcinogenesis.