Department of Environmental and Molecular Medicine, Mie University Graduate School of Medicine, 2-174 Edobashi, Tsu, Mie 514-8507, Japan.
Toxicol Appl Pharmacol. 2012 Apr 15;260(2):183-92. doi: 10.1016/j.taap.2012.02.010. Epub 2012 Feb 21.
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.
碳纳米管（CNT）由于其独特的物理化学性质，在工业用途的材料科学领域具有广阔的应用前景。然而，已有研究报道腹腔内给予 CNT 可导致实验动物发生间皮瘤。慢性炎症可能与纤维材料诱导的致癌作用有关。8-硝基鸟嘌呤是炎症过程中形成的诱变 DNA 损伤，可能在 CNT 诱导的致癌作用中发挥作用。在这项研究中，我们通过荧光免疫细胞化学法检测了经多壁 CNT（MWCNT）处理的 A549 人肺泡上皮细胞中 8-硝基鸟嘌呤的形成。直径为 20-30nm（CNT20）和 40-70nm（CNT40）的 MWCNT 均能显著诱导 5μg/ml 和 10μg/ml 浓度下的 8-硝基鸟嘌呤形成（p<0.05），这种诱导作用可持续 24 小时，尽管这些 MWCNT 的 DNA 损伤能力无显著差异。MWCNT 能显著诱导诱导型一氧化氮合酶（iNOS）在 24 小时内的表达（p<0.05）。MWCNT 还能显著增加培养上清液中氧化型 NO 的水解产物亚硝酸盐在 4 小时和 8 小时时的水平（p<0.05）。iNOS 抑制剂（1400 W）、核因子-κB（Bay11-7082）、肌动蛋白聚合抑制剂（细胞松弛素 D）、小窝蛋白介导的内吞抑制剂（甲基-β-环糊精，MBCD）和网格蛋白介导的内吞抑制剂（单丹磺酰尸胺，MDC）可显著抑制 MWCNT 诱导的 8-硝基鸟嘌呤形成和 iNOS 表达。电子显微镜显示，MWCNT 主要位于细胞质中的囊泡结构中，MBCD 和 MDC 可减少其细胞内化。这些结果表明，MWCNT 通过网格蛋白和小窝蛋白介导的内吞作用进入细胞，引起包括 iNOS 表达在内的炎症反应，导致硝化 DNA 损伤，这可能与致癌作用有关。
