Nitrative DNA damage induced by carbon-black nanoparticles in macrophages and lung epithelial cells
Nitrative DNA damage induced by carbon-black nanoparticles in macrophages and lung epithelial cells
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DOI：
10.1016/j.mrgentox.2017.04.002
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发表时间：
2017-06-01
影响因子：
1.9
通讯作者：
Murata, Mariko
中科院分区：
文献类型：
--
作者：
Hiraku, Yusuke;Nishikawa, Yoshihiro;Murata, Mariko
炭黑（CB）是一种主要用于橡胶制品的纳米材料。在实验动物中，通过吸入接触CB可引起恶性肺肿瘤。吸入黑炭黑可引起呼吸系统慢性炎症，导致癌变，但其机制尚不清楚。炎症和上皮细胞在炎症条件下产生活性氧和活性氮，由此导致的DNA损伤可能有助于致癌。在这项研究中，我们通过免疫细胞化学来确定CB暴露是否会诱导RAW 264.7巨噬细胞和A549肺上皮细胞中8-硝基鸟嘌呤（8-nitroG）的形成，8-硝基鸟嘌呤是炎症条件下形成的一种硝化DNA病变。我们比较了原始直径56 nm （CB56）和95 nm （CB95）的CB颗粒对dna的损伤作用。两种CB均诱导8-nitroG的形成，主要在RAW 264.7和A549细胞的细胞核中，CB95诱导的8-nitroG的形成倾向于比CB56更多。流式细胞术显示CB95比CB56在RAW 264.7细胞中产生更多的活性氧。Griess法显示CB95产生的一氧化氮（NO）明显多于CB56。流式细胞术显示CB95比CB56更有效地内化到细胞中。单丹糖cadaverine（一种网格蛋白介导的内吞作用抑制剂）和Ctic（网格蛋白重链）基因的siRNA可以减少RAW 264.7细胞对CB的细胞摄取和8-硝基的形成。CB诱导培养细胞的硝化DNA损伤，至少部分参与了网格蛋白介导的内吞作用。
Carbon black (CB) is a nanomaterial used mainly in rubber products. Exposure to CB by inhalation causes malignant lung tumors in experimental animals. CB inhalation may cause chronic inflammation in the respiratory system, leading to carcinogenesis, but the mechanism remains unclear. Reactive oxygen and nitrogen species are generated from inflammatory and epithelial cells under inflammatory conditions, and resulting DNA damage may contribute to carcinogenesis. In this study, we performed immunocytochemistry to determine whether CB exposure induces formation of 8-nitroguanine (8-nitroG), a nitrative DNA lesion formed under inflammatory conditions, in RAW 264.7 macrophage and A549 lung epithelial cells. We compared the DNA-damaging effects of CB particles with primary diameter 56 nm (CB56) and 95 nm (CB95). Both types of CB induced 8-nitroG formation, mainly in the nucleus of RAW 264.7 and A549 cells, and CB95 tended to induce more 8-nitroG formation than did CB56. Flow cytometry revealed that CB95 generated larger amount of reactive oxygen species than did CB56 in RAW 264.7 cells. The Griess method showed that CB95 produced significantly larger amount of nitric oxide (NO) than did CB56. Flow cytometry showed that CB95 was more efficiently internalized into the cells than was CB56. The cellular uptake of CB and 8-nitroG formation in RAW 264.7 cells were reduced by monodansylcadaverine, an inhibitor of clathrin-mediated endocytosis, and by siRNA for Ctic (clathrin heavy chain) gene. CB induces nitrative DNA damage in cultured cells, and clathrin-mediated endocytosis is involved, at least in part.