Distinct autophagy-apoptosis related pathways activated by Multi-walled (NM 400) and Single-walled carbon nanotubes (NIST-SRM2483) in human bronchial epithelial (16HBE14o-) cells

Our plain-English summary of Distinct autophagy-apoptosis related pathways activated by Multi-walled (NM 400) and Single-walled carbon nanotubes (NIST-SRM2483) in human bronchial epithelial (16HBE14o-) cells, by M. Ghosh, S. Murugadoss, L. Janssen, et al (Journal of Hazardous Materials, 2020).

The study

• Authors: M. Ghosh, S. Murugadoss, L. Janssen, S. Cokic, C. Mathyssen, K. Van Landuyt, W. Janssens, S. Carpentier, L. Godderis, P. Hoet
• Journal: Journal of Hazardous Materials
• Published: 2020;387:121691
• Cited by: 15 articles, as counted by NIH iCite
• Read it: PubMed · Publisher

Abstract

Given the recent development in the field of particle and fibre toxicology, parallels have been drawn between Carbon nanotubes (CNTs) and asbestos. It is now established that both multi-walled (MWCNTs) and single-walled (SWCNTs) carbon nanotubes might contribute to pulmonary disease. Although multiple mechanisms might be involved in CNT induced pathogenesis, systematic understanding of the relationship between different CNT exposure (MWCNT vs SWCNT) and autophagy/ apoptosis/ necrosis, in human lung epithelial cells remains limited. In this study, we demonstrate that exposure to MWCNT (NM-400), but not SWCNT (NIST-SRM2483), leads to an autophagic response after acute exposure (24 h). MWCNT exposure was characterized by an increase in anti-apoptotic BCL2, downregulation of executor Caspase-3/7 and increase in expression of genes from the autophagy machinery. For SWCNT exposure however, we observed an overexpression of executor Caspase-3/7 and upregulation of pro-apoptotic BAX; enrichment for processes like cornification, apoptotic process, cell differentiation from proteomic analysis. These results clearly indicate a major difference in the pathways initiated by the CNTs, in vitro. While the present study design provides mechanistic understanding after an acute exposure for the tested CNTs, we believe that the information obtained here would have relevance in better understanding of CNT toxicity and pathogenesis in general.

Copyright © 2019 Elsevier B.V. All rights reserved.

In plain English

This was a laboratory study of human bronchial epithelial cells (16HBE14o-), not of people. The authors exposed the cells for 24 hours to one multi-walled carbon nanotube (NM-400) and one single-walled carbon nanotube (NIST-SRM2483), at 2.5 and 25 micrograms per ml. They looked for signs of cell death and of autophagy, a process in which cells break down their own components. With the single-walled nanotubes, staining for caspase-3/7 (enzymes linked to apoptotic cell death) rose significantly at both concentrations, and the pro-apoptotic gene BAX increased significantly at both concentrations. With the multi-walled nanotubes, caspase-3/7 changed little, the anti-apoptotic gene BCL2 was higher, and the autophagy genes ATG5, ATG7 and ATG12 increased significantly. Protein analysis linked the single-walled nanotubes to cornification and apoptotic processes. For the multi-walled nanotubes it showed no direct enrichment of autophagy. The authors concluded that the multi-walled nanotubes led to an autophagic response and the single-walled nanotubes to apoptosis. They cautioned that the results may be specific to this cell type and these nanotubes. Asbestos itself was not tested.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Laboratory (in vitro) cell-culture experiment comparing two reference carbon nanotube materials, using staining, gene-expression (mRNA), proteomic and extracellular-vesicle measurements
• Who was studied: Cultured human bronchial epithelial cells of the 16HBE14o- cell line. The proteomic experiment used three samples each of control, multi-walled and single-walled nanotube-exposed cells at 25 micrograms per ml. No human subjects were studied.
• Exposure: Multi-walled carbon nanotubes (NM-400) and single-walled carbon nanotubes (NIST-SRM2483), each at 2.5 and 25 micrograms per ml for 24 hours (acute exposure). No asbestos was tested.
• Main results:
  • Propidium iodide staining increased for both nanotube types and was significantly higher for the single-walled nanotubes. The authors read this as indicating late apoptosis and/or necrosis.
  • BCL2 (anti-apoptotic) gene expression was reduced in single-walled nanotube-exposed cells and higher in multi-walled nanotube-exposed cells.
  • BAX (pro-apoptotic) gene expression increased significantly at both single-walled nanotube concentrations and was lower for the multi-walled nanotubes.
  • Caspase-3/7 staining showed negligible change for multi-walled nanotube-exposed cells compared with the negative control, and a significant increase at both single-walled nanotube concentrations.
  • mRNA expression of the autophagy genes ATG5, ATG7 and ATG12 increased significantly with the multi-walled nanotubes and was downregulated with the single-walled nanotubes.
  • At the lower concentration (2.5 micrograms per ml), both nanotube types decreased mitochondrial membrane potential. At the higher concentration, mitochondrial membrane potential increased significantly.
  • The proteomic analysis identified 2790 proteins. Only 5 genes were shared between the multi-walled and single-walled nanotube results.
  • For multi-walled nanotube-exposed cells, pathway analysis did not show direct enrichment of autophagy, though it did show related processes such as necroptosis, Rab signalling and regulation of chaperone-mediated autophagy.
  • Changes in single-walled nanotube-exposed cells were enriched for cornification, apoptotic process and cell differentiation.
  • There was no dose-response relation for IL-6 and IL-8 levels in the culture medium. mRNA levels of IL-6 and IL-8 were significantly increased for the multi-walled nanotubes and similar to the negative control for the single-walled nanotubes.
• The authors' conclusion: The authors concluded that, after 24-hour exposure, the multi-walled nanotube NM-400 led to an autophagic response and the single-walled nanotube NIST-SRM2483 led to signs suggesting late apoptosis.
• Limitations the authors state:
  • Only one representative material was used for each nanotube type.
  • The results may be specific to this cell type and these nanotubes and need validation in other conditions.
  • Some conventional cell-death methods (Annexin V-FITC flow cytometry, cell cycle analysis) could not be used because the nanotubes interfered with them.
  • Steady-state measurements of apoptosis and autophagy markers may be inconclusive, because these processes are dynamic.
  • Extracellular vesicles could not be characterized quantitatively, and interpretation of them awaits further method optimization.

Topics

Medical terms in this study, explained in our Lexicon: Apoptosis, Necrosis.

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