Multi-walled carbon nanotube induces nitrative DNA damage in human lung epithelial cells via HMGB1-RAGE interaction and Toll-like receptor 9 activation.
Multi-walled carbon nanotube induces nitrative DNA damage in human lung epithelial cells via HMGB1-RAGE interaction and Toll-like receptor 9 activation.
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DOI：
10.1186/s12989-016-0127-7
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发表时间：
2016-03-29
影响因子：
10
通讯作者：
Murata M
中科院分区：
文献类型：
--
作者：
Hiraku Y;Guo F;Ma N;Yamada T;Wang S;Kawanishi S;Murata M
碳纳米管（CNT）用于各种工业目的，但在实验动物中表现出致癌作用。呼吸系统的慢性炎症可能参与CNT诱导的致癌作用。8-硝基鸟嘌呤（8-硝基G）是炎症期间形成的一种致突变DNA损伤。我们以前曾报道，多壁碳纳米管（MWCNT）诱导肺上皮细胞中8-硝基G的形成，这一过程涉及内吞。为了阐明CNT诱导的致癌作用的机制，我们研究了Toll样受体（TLR）9的作用，它驻留在核内体和溶酶体，在8-硝基G形成在人肺上皮细胞系。我们用免疫细胞化学方法检测了长1-2 μm（CNT-S）或5-15 μm（CNT-L）、直径20-40 nm的MWCNT处理A549和HBEpC细胞后8-nitroG的形成。我们研究了内吞抑制剂、TLR 9的小干扰RNA（siRNA）以及针对高迁移率族蛋白1（HMGB 1）和晚期糖基化终产物受体（RECEPTOR）的抗体对8-硝基G形成的抑制作用。通过ELISA和荧光分析分别检测了MWCNT处理的细胞释放到培养上清液中的HMGB 1和双链DNA（dsDNA）。这些分子的协会进行了检查，通过双重免疫荧光染色和免疫共沉淀。CNT-L浓度为0.05 μg/ml时能显著增加A549细胞8-硝基G的生成，浓度为1 μg/ml时达到最大值。CNT-L比CNT-S诱导更强的细胞毒性和8-硝基G的形成。内吞作用抑制剂、TLR 9 siRNA和针对HMGB 1和HMGB 2的抗体在很大程度上减少了MWCNT诱导的8-硝基G形成。多壁碳纳米管增加了A549细胞向培养上清中HMGB 1和dsDNA的释放。MWCNT暴露的细胞的培养上清液诱导新鲜A549细胞中8-硝基G的形成。免疫荧光双染和免疫共沉淀显示，TLR 9与多壁碳纳米管处理的细胞溶酶体中的HMGB 1和HMGB 2结合。多壁碳纳米管诱导肺上皮细胞损伤或坏死，从而将HMGB 1和DNA释放到细胞外空间。HMGB 1-DNA复合物与邻近细胞上的CpG结合，然后CpG DNA被溶酶体中的TLR 9识别，导致产生一氧化氮和8-硝基G形成。这是第一项研究表明TLR 9和相关分子参与MWCNT诱导的遗传毒性，并可能导致致癌作用。本文的在线版本（doi：10.1186/s12989-016-0127-7）包含补充材料，可供授权用户使用。
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. 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. 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. 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. The online version of this article (doi:10.1186/s12989-016-0127-7) contains supplementary material, which is available to authorized users.