Impaired lysosomal activity mediated autophagic flux disruption by graphite carbon nanofibers induce apoptosis in human lung epithelial cells through oxidative stress and energetic impairment
Authors: Alok Kumar Pandey
Raja Gopal Rayavarapu
Lalit Kumar Singh Chauhan
Pradeep Kumar Sharma
Sandeep Mittal
Ratnakar Tiwari
Jai Shankar
Journal: Particle and Fibre Toxicology (2017)
Discipline: PHARMACOLOGY & TOXICOLOGY
Keywords: ATP loss; Apoptosis; Autophagy; Destabilization of lysosomes; Genotoxicity
Affiliations: Academy of Scientific and Innovative Research (AcSIR), CSIR-IITR Campus, Lucknow, India., Nanomaterials Toxicology Laboratory, Nanotherapeutics and Nanomaterial Toxicology Group, CSIR - Indian Institute of Toxicology Research (CSIR - IITR), Vishvigyan Bhawan, 31, Mahatma Gandhi Marg, Lucknow, 226001, Uttar Pradesh, India., Environmental Carcinogenesis Laboratory, Food, Drug and Chemical Toxicology Group, CSIR - Indian Institute of Toxicology Research (CSIR - IITR), Vishvigyan Bhawan, 31, Mahatma Gandhi Marg, Lucknow, 226001, Uttar Pradesh, India., Developmental Toxicology Laboratory, System Toxicology and Health Risk Assessment Group, CSIR - Indian Institute of Toxicology Research (CSIR - IITR), Vishvigyan Bhawan, 31, Mahatma Gandhi Marg, Lucknow, 226001, Uttar Pradesh, India., Electron Microscopy Laboratory, CSIR - Indian Institute of Toxicology Research (CSIR - IITR), Vishvigyan Bhawan 31, Mahatma Gandhi Marg, Lucknow, 226001, Uttar Pradesh, India., Academy of Scientific and Innovative Research (AcSIR), CSIR-IITR Campus, Lucknow, India. ., Nanomaterials Toxicology Laboratory, Nanotherapeutics and Nanomaterial Toxicology Group, CSIR - Indian Institute of Toxicology Research (CSIR - IITR), Vishvigyan Bhawan, 31, Mahatma Gandhi Marg, Lucknow, 226001, Uttar Pradesh, India. .

Abstract:  Background: 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. Methods: 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. Results: 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. Conclusion: 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. 

Retracted: No

PubPeer comments:
 Indicated blots of this paper have been taken from a previous paper of this group. ![file] , 
 The first 2 panels in Figure 6a (control vs. 1ug ml) look very similar to each other. The red graphs appear to show the exact same shape, yet the count percentages are very different. ![file] , 
 ![file] Same loading control used for entirely different treatment conditions. ,