Impaired lysosomal activity mediated autophagic flux disruption by graphite carbon nanofibers induce apoptosis in human lung epithelial cells through oxidative stress and energetic impairment.
Impaired lysosomal activity mediated autophagic flux disruption by graphite carbon nanofibers induce apoptosis in human lung epithelial cells through oxidative stress and energetic impairment.
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DOI：
10.1186/s12989-017-0194-4
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发表时间：
2017-04-28
影响因子：
10
通讯作者：
Pandey AK
中科院分区：
文献类型：
--
作者：
Mittal S;Sharma PK;Tiwari R;Rayavarapu RG;Shankar J;Chauhan LKS;Pandey AK
石墨碳纳米纤维（GCNF）由于其优异的上级物理化学性能，已成为碳纳米管（CNT）在各种生物医学应用中的潜在替代品。因此，深入了解GCNF在生物系统中诱导的毒性效应和潜在机制具有重要意义。目前，纳米材料的自噬激活被认为是一种新兴的毒性机制。然而，GCNF诱导的毒性与这种形式的细胞死亡的关联在很大程度上是未知的。在这项研究中，我们评估了可能的机制，特别是自噬的作用，潜在的GCNF诱导的毒性。将人肺腺癌（A549）细胞暴露于一系列GCNF浓度，并分析各种细胞参数（长达48 h）。透射电镜、免疫荧光染色、western blot和真实的实时定量PCR检测GCNF处理后细胞的凋亡、自噬诱导、溶酶体不稳定和细胞骨架破坏。DCFDA法检测细胞内活性氧（ROS）的产生。用N-乙酰-L-半胱氨酸（NAC）、3-甲基腺嘌呤（3-MA）和LC 3 siRNA进行实验以证实氧化应激和自噬参与GCNF诱导的细胞死亡。进行彗星试验和微核（MN）试验以评估遗传毒性潜力。在本研究中，发现GCNF通过自噬体积累诱导人肺细胞中的纳米毒性，随后通过细胞内ROS的产生诱导细胞凋亡。从机制上讲，溶酶体功能受损和细胞骨架破坏介导的自噬通量阻断被认为是累积的主要原因，而不是自噬诱导，其进一步激活细胞凋亡。整个过程与增加的ROS水平一致，并且它们的药理学抑制导致GCNF诱导的细胞死亡的减轻。此外，自噬的抑制减弱细胞凋亡，表明自噬作为细胞死亡过程的作用。还发现GCNF诱导基因组不稳定性。我们的研究表明，GCNF干扰了各种相关的信号通路，并揭示了GCNF通过靶向ROS-自噬-凋亡轴的潜在纳米毒性机制。目前的研究对于评估纤维状碳纳米材料在其潜在用途之前的安全性和风险评估具有重要意义，并建议谨慎将其用于生物医学研究。本文的在线版本（doi：10.1186/s12989-017-0194-4）包含补充材料，可供授权用户使用。
Graphite carbon nanofibers (GCNF) have emerged as a potential alternative of carbon nanotubes (CNT) for various biomedical applications due to their superior physico-chemical properties. Therefore in-depth understanding of the GCNF induced toxic effects and underlying mechanisms in biological systems is of great interest. Currently, autophagy activation by nanomaterials is recognized as an emerging toxicity mechanism. However, the association of GCNF induced toxicity with this form of cell death is largely unknown. In this study, we have assessed the possible mechanism; especially the role of autophagy, underlying the GCNF induced toxicity. Human lung adenocarcinoma (A549) cells were exposed to a range of GCNF concentrations and various cellular parameters were analyzed (up to 48 h). Transmission electron microscopy, immunofluorescent staining, western blot and quantitative real time PCR were performed to detect apoptosis, autophagy induction, lysosomal destabilization and cytoskeleton disruption in GCNF exposed cells. DCFDA assay was used to evaluate the reactive oxygen species (ROS) production. Experiments with N-acetyl-L-cysteine (NAC), 3-methyladenine (3-MA) and LC3 siRNA was carried out to confirm the involvement of oxidative stress and autophagy in GCNF induced cell death. Comet assay and micronucleus (MN) assay was performed to assess the genotoxicity potential. In the present study, GCNF was found to induce nanotoxicity in human lung cells through autophagosomes accumulation followed by apoptosis via intracellular ROS generation. Mechanistically, impaired lysosomal function and cytoskeleton disruption mediated autophagic flux blockade was found to be the major cause of accumulation rather than autophagy induction which further activates apoptosis. The whole process was in line with the increased ROS level and their pharmacological inhibition leads to mitigation of GCNF induced cell death. Moreover the inhibition of autophagy attenuates apoptosis indicating the role of autophagy as cell death process. GCNF was also found to induce genomic instability. Our present study demonstrates that GCNF perturbs various interrelated signaling pathway and unveils the potential nanotoxicity mechanism of GCNF through targeting ROS-autophagy-apoptosis axis. The current study is significant to evaluate the safety and risk assessment of fibrous carbon nanomaterials prior to their potential use and suggests caution on their utilization for biomedical research. The online version of this article (doi:10.1186/s12989-017-0194-4) contains supplementary material, which is available to authorized users.