Endocytosis of Multiwalled Carbon Nanotubes in Bronchial Epithelial and Mesothelial Cells

Our plain-English summary of Endocytosis of Multiwalled Carbon Nanotubes in Bronchial Epithelial and Mesothelial Cells, by K. Maruyama, H. Haniu, N. Saito, et al (BioMed Research International, 2015).

The study

• Authors: K. Maruyama, H. Haniu, N. Saito, Y. Matsuda, T. Tsukahara, S. Kobayashi, M. Tanaka, K. Aoki, S. Takanashi, M. Okamoto, H. Kato
• Journal: BioMed Research International
• Published: 2015;2015:793186
• Cited by: 41 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Bronchial epithelial cells and mesothelial cells are crucial targets for the safety assessment of inhalation of carbon nanotubes (CNTs), which resemble asbestos particles in shape. Intrinsic properties of multiwalled CNTs (MWCNTs) are known to cause potentially hazardous effects on intracellular and extracellular pathways. These interactions alter cellular signaling and affect major cell functions, resulting in cell death, lysosome injury, reactive oxygen species production, apoptosis, and cytokine release. Furthermore, CNTs are emerging as a novel class of autophagy inducers. Thus, in this study, we focused on the mechanisms of MWCNT uptake into the human bronchial epithelial cells (HBECs) and human mesothelial cells (HMCs). We verified that MWCNTs are actively internalized into HBECs and HMCs and were accumulated in the lysosomes of the cells after 24-hour treatment. Next, we determined which endocytosis pathways (clathrin-mediated, caveolae-mediated, and macropinocytosis) were associated with MWCNT internalization by using corresponding endocytosis inhibitors, in two nonphagocytic cell lines derived from bronchial epithelial cells and mesothelioma cells. Clathrin-mediated endocytosis inhibitors significantly suppressed MWCNT uptake, whereas caveolae-mediated endocytosis and macropinocytosis were also found to be involved in MWCNT uptake. Thus, MWCNTs were positively taken up by nonphagocytic cells, and their cytotoxicity was closely related to these three endocytosis pathways.

In plain English

This laboratory study examined how multiwalled carbon nanotubes, which the authors describe as resembling asbestos in shape, enter human cells. No asbestos was tested. Normal human bronchial epithelial cells and normal human mesothelial cells were exposed to a nanotube product called MWNT-7. Most of the bronchial cells had taken up nanotubes within 6 hours, and at 24 hours the nanotubes appeared to collect in lysosomes. At 10 micrograms per millilitre for 24 hours, bronchial cell viability was about 85.5% in one dispersing liquid (FBS) and 64.7% in another (gelatin). Mesothelial cell viability was higher than that of the bronchial cells. The authors then used drugs that block different uptake routes in a bronchial cell line and in a malignant pleural mesothelioma cell line. Two blockers of clathrin-mediated uptake reduced uptake in both lines. Caveolae blockers gave mixed results. A macropinocytosis blocker was statistically significant only at its highest dose in the mesothelioma cells. The authors concluded that clathrin-mediated uptake played the main role, with other routes possibly involved. In the blocker experiments they measured uptake only at 2 hours, because high blocker doses were toxic.

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: microscopy of nanotube uptake, cell viability assay, and flow cytometry with endocytosis inhibitors
• Who was studied: Purchased human cells: normal human bronchial epithelial cells (HBECs, from Lonza), normal human mesothelial cells (HMCs, from Zen-Bio), the BEAS-2B human bronchial epithelial cell line, and the ACC-MESO-1 human malignant pleural mesothelioma cell line
• Exposure: Multiwalled carbon nanotubes (MWNT-7, Hodogaya Chemical) dispersed in 0.1% gelatin or 2% fetal bovine serum. Viability was tested at 1, 10 and 100 micrograms per millilitre for 24 hours, and imaging and uptake were tested at 10 micrograms per millilitre. No asbestos was used.
• Main results:
  • Viability of normal bronchial epithelial cells (HBECs) at 10 micrograms per millilitre MWNT-7 for 24 hours was approximately 85.5% in FBS and 64.7% in gelatin
  • HBEC viability decreased further at 100 micrograms per millilitre in both dispersants (69.9% versus 47.7%)
  • In HBECs, uptake of MWNT-7 was seen in most cells within 6 hours, and at 24 hours MWNT-7 appeared to accumulate in lysosomes
  • Viability of normal mesothelial cells (HMCs) exposed to MWNT-7 in gelatin fell with concentration but stayed higher than that of HBECs
  • Side-scatter (SSC) uptake ratios in control cells not pretreated with inhibitors at 2 hours were 1.355 to 1.426 in BEAS-2B cells and 1.137 to 1.258 in MESO-1 cells (baseline for cells not exposed to nanotubes: 1.000)
  • Clathrin-pathway inhibitors in BEAS-2B cells: 50 micromolar chlorpromazine lowered the SSC ratio to 1.039, and 2 micromolar phenylarsine oxide gave 1.040
  • Clathrin-pathway inhibitors in MESO-1 cells: the lowest SSC ratios were 1.032 with 50 micromolar chlorpromazine and 1.025 with 5 micromolar phenylarsine oxide
  • Caveolae-pathway inhibitors: nystatin significantly decreased the SSC ratio in both cell lines except at 1 micromolar in MESO-1 cells
  • Caveolae-pathway inhibitors: indomethacin tended to inhibit uptake in a concentration-dependent way in both cell lines, but the differences were not statistically significant
  • Caveolae inhibition rates in BEAS-2B cells: nystatin 30.7%, and indomethacin 27.9% at 100 micromolar
  • Caveolae inhibition rates in MESO-1 cells: indomethacin 35.2%, nystatin 23.9%
  • Macropinocytosis inhibitor 5-(N-ethyl-N-isopropyl)amiloride: differences were not significant except at 80 micromolar in MESO-1 cells. Inhibition rates at 80 micromolar were 42.7% in BEAS-2B cells and 56.6% in MESO-1 cells.
• The authors' conclusion: The authors concluded that normal human bronchial epithelial and mesothelial cells took up MWCNTs by a combination of clathrin-mediated endocytosis, caveolae-mediated endocytosis and macropinocytosis. In their view clathrin-mediated endocytosis played the most important role, and the other pathways may be involved to varying degrees.
• Limitations the authors state:
  • Variability (SD) of SSC ratios tended to increase with cell passage number, so few statistically significant differences were found for nystatin and the macropinocytosis inhibitor
  • Uptake in the inhibitor experiments was measured only at an early time point, 2 hours, because high inhibitor concentrations were cytotoxic
  • The role of the nanotubes' degree of aggregation could not be clarified
  • Other uptake pathways not tested may exist

Topics

The National Library of Medicine indexes this study under subjects our site covers: Mesothelioma.

Medical terms in this study, explained in our Lexicon: Mesothelial Cells, Reactive Oxygen Species (ROS), Apoptosis, Cytokine, Mesothelioma.

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