Adverse effects of industrial multiwalled carbon nanotubes on human pulmonary cells

Our plain-English summary of Adverse effects of industrial multiwalled carbon nanotubes on human pulmonary cells, by L. Tabet, C. Bussy, N. Amara, et al (Journal of Toxicology and Environmental Health. Part a, 2009).

The study

• Authors: L. Tabet, C. Bussy, N. Amara, A. Setyan, A. Grodet, M.J. Rossi, J.C. Pairon, J. Boczkowski, S. Lanone
• Journal: Journal of Toxicology and Environmental Health. Part a
• Published: 2009;72(2):60-73
• Cited by: 96 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

The aim of this study was to evaluate adverse effects of multiwalled carbon nanotubes (MWCNT), produced for industrial purposes, on the human epithelial cell line A549. MWCNT were dispersed in dipalmitoyl lecithin (DPL), a component of pulmonary surfactant, and the effects of dispersion in DPL were compared to those in two other media: ethanol (EtOH) and phosphate-buffered saline (PBS). Effects of MWCNT were also compared to those of two asbestos fibers (chrysotile and crocidolite) and carbon black (CB) nanoparticles, not only in A549 cells but also in mesothelial cells (MeT5A human cell line), used as an asbestos-sensitive cell type. MWCNT formed agglomerates on top of both cell lines (surface area 15-35 microm(2)) that were significantly larger and more numerous in PBS than in EtOH and DPL. Whatever the dispersion media, incubation with 100 microg/ml MWCNT induced a similar decrease in metabolic activity without changing cell membrane permeability or apoptosis. Neither MWCNT cellular internalization nor oxidative stress was observed. In contrast, asbestos fibers penetrated into the cells, decreased metabolic activity but not cell membrane permeability, and increased apoptosis, without decreasing cell number. CB was internalized without any adverse effects. In conclusion, this study demonstrates that MWCNT produced for industrial purposes exert adverse effects without being internalized by human epithelial and mesothelial pulmonary cell lines.

In plain English

This laboratory study used human cells grown in dishes. The authors tested industrial multiwalled carbon nanotubes from France on two human cell lines: A549 lung epithelial cells and MeT5A mesothelial cells. They compared nanotubes at 0.1 to 100 μg/ml with chrysotile and crocidolite asbestos and carbon black, each at 100 μg/ml. A549 cells received nanotubes in three suspension liquids; MeT5A cells, only in dipalmitoyl lecithin. In A549 cells, 100 μg/ml nanotubes lowered a measure of cell metabolism to 60% of control after 48 h. Chrysotile and crocidolite caused similar decreases. Neither nanotubes nor asbestos significantly reduced viability on a membrane-integrity test; asbestos increased dye uptake. Nanotubes reduced A549 cell number by 15 to 20% without increasing apoptosis (a form of cell death). Crocidolite raised apoptosis to about 5% of cells after 72 h. Asbestos fibers and carbon black were seen inside cells; nanotubes were not. In nanotube-exposed cells, four oxidative stress genes showed no significant change at 6 or 24 h. The authors concluded that the nanotubes had adverse effects without entering cells and differed in effect from asbestos, though not definitively.

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) experiment on human cell lines
• Who was studied: Two human cell lines grown in culture: A549 alveolar epithelial cells (used as a model of alveolar type II cells) and MeT5A mesothelial cells (used as an asbestos-sensitive cell type)
• Exposure: Industrial multiwalled carbon nanotubes (Graphistrength C100, ARKEMA, France, made by chemical vapor deposition) at 0.1 to 100 μg/ml, suspended in dipalmitoyl lecithin, ethanol or phosphate-buffered saline. These were compared with UICC chrysotile and crocidolite asbestos (suspended in culture medium) and with carbon black nanoparticles (suspended in phosphate-buffered saline), each at 100 μg/ml, for 6 to 72 hours. MeT5A cells were exposed only to nanotubes suspended in dipalmitoyl lecithin.
• Main results:
  • In A549 cells, nanotubes reduced metabolic activity (MTT assay) similarly in all three suspension media. At 100 μg/ml after 48 h, activity reached 60% of control values. Similar results were seen in MeT5A cells exposed to nanotubes suspended in dipalmitoyl lecithin.
  • In both cell types, chrysotile and crocidolite at 100 μg/ml caused decreases in metabolic activity that were not different from those caused by 100 μg/ml nanotubes after 24 h.
  • On the neutral red assay, a test of plasma membrane permeability, nanotubes caused no significant alteration of A549 viability in any medium at any time point, with similar results in MeT5A cells.
  • On the same neutral red assay, neither asbestos fibers nor carbon black significantly reduced viability in either cell type.
  • Asbestos fibers increased neutral red dye incorporation in both cell types, as did carbon black in MeT5A cells at 6 or 24 h.
  • Nanotubes at 100 μg/ml significantly reduced A549 cell number by 15 to 20% of control at 24, 48 and 72 h, whatever the suspension medium. No such effect was seen in MeT5A cells.
  • Asbestos fibers and carbon black did not significantly reduce total DNA content (cell number). The one exception was carbon black in MeT5A cells at 24 h.
  • Apoptosis did not change significantly in cells exposed to nanotubes (control cells: 1 to 2% apoptotic nuclei). Crocidolite significantly increased apoptosis after 72 h, to about 5% of total cells, with similar results in MeT5A cells.
  • A second apoptosis test (DNA laddering) agreed with these results, except that it showed no change after asbestos exposure.
  • Electron microscopy found no evidence that nanotubes entered A549 cells in any medium. Carbon black particles and chrysotile and crocidolite fibers were seen inside cells.
  • In cells exposed to nanotubes for 6 or 24 h, mRNA expression of the oxidative stress genes HO-1, SOD2 and GPx showed no significant change; NOX4 mRNA also did not change.
  • Nanotubes formed clumps (agglomerates) on top of A549 cells, ranging from 15 to 30 μm2. The clumps were significantly larger when the nanotubes were suspended in phosphate-buffered saline than in dipalmitoyl lecithin or ethanol.
• The authors' conclusion: The authors concluded that these industrially produced nanotubes had adverse biological effects on human epithelial and mesothelial lung cells without being taken up by the cells. They also stated that the effects of the nanotubes and of asbestos fibers were different in nature, though the study could not give definitive answers on that point.
• Limitations the authors state:
  • Small individual nanotubes might have entered cells without being visible by light or electron microscopy.
  • The lack of oxidative stress response may relate to A549 being a cancer cell line, since cancer cell lines resist oxidant stress.
  • The mechanisms behind the reduced cell metabolism and cell number were not identified.
  • The study could not give definitive answers on whether nanotube and asbestos effects differ in nature.
  • The light-scattering instrument was not sensitive enough to detect individual nanotubes in some suspensions.

Topics

The National Library of Medicine indexes this study under subjects our site covers: Chrysotile, Crocidolite.

Medical terms in this study, explained in our Lexicon: Chrysotile, Crocidolite, A549 Cells, Mesothelial Cells, Apoptosis, Oxidative Stress.

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