Multi-walled carbon nanotubes injure the plasma membrane of macrophages

Our plain-English summary of Multi-walled carbon nanotubes injure the plasma membrane of macrophages, by S. Hirano, S. Kanno, A. Furuyama (Toxicology and Applied Pharmacology, 2008).

The study

• Authors: S. Hirano, S. Kanno, A. Furuyama
• Journal: Toxicology and Applied Pharmacology
• Published: 2008;232(2):244-51
• Cited by: 138 articles, as counted by NIH iCite
• Read it: PubMed · Publisher

Abstract

Carbon nanotubes (CNTs) are emerging nanotechnology materials which are likely to be mass-produced in the near future. However, prior to mass-production, certain health-related concerns should first be addressed. For example, when inhaled, the thin-fibrous shape and the biopersistent characteristics of CNTs may cause pulmonary diseases, in a manner similar to asbestos. In the present study, mouse macrophages (J774.1) were exposed to highly-purified multi-walled CNTs (MWCNTs, 67 nm) or to UICC crocidolite in order to evaluate the toxicity of these nano-size fibers. The cytotoxicity of MWCNTs was found to be higher than that of crocidolite. The toxic effect of MWCNTs was not affected by N-acetylcysteine, an antioxidant, or buthionine sulfoximine, a glutathione synthesis inhibitor. cDNA microarray analyses suggested that the cytotoxicity of MWCNTs could not be explained satisfactorily by either an increase or decrease of gene expression, although mRNA levels of some cytokines were slightly increased by MWCNTs. Moreover, MWCNTs did not significantly activate either MAP kinases such as ERK, JNK and p38, nor common apoptosis pathways such as caspase 3 and PARP. Electron microscopic studies indicated that MWCNTs associate with the plasma membrane of macrophages and disrupt the integrity of the membrane. Several proteins were found to adsorb onto MWCNTs when MWCNT-exposed macrophages were gently lysed. One of these proteins was macrophage receptor with collagenous structure (MARCO). MARCO-transfected CHO-K1 cells associated with MWCNTs more rapidly than mock-transfected cells. These results indicate that MWCNTs probably trigger cytotoxic effects in phagocytotic cells by reacting with MARCO on the plasma membrane and rupturing the plasma membrane.

In plain English

This laboratory study exposed a mouse macrophage cell line (J774.1, a type of scavenging immune cell) to highly purified multi-walled carbon nanotubes (average diameter 67 nm) or to UICC crocidolite asbestos, which served as a reference fiber. No people or live animals were studied. Cell survival fell as nanotube dose and exposure time increased. The effect partly reversed above 100 μg/mL, which the authors speculated was due to the nanotubes clumping together. The concentration that killed half the cells (LC50) was about 1/25th as high for the nanotubes as for crocidolite. An antioxidant (N-acetylcysteine) and a glutathione blocker (buthionine sulfoximine) did not change this toxicity. Two common markers of programmed cell death, caspase-3 and PARP, were not activated. Electron microscopy showed nanotubes disrupting the cells' outer membrane after 8 hours. MARCO, a cell-surface receptor, was found bound to the nanotubes. Histone proteins were also recovered with them, but the authors said it was unclear whether histones bind directly or came along with DNA. The authors concluded that macrophages bind the nanotubes through MARCO and that the membrane is injured as it extends along the fibers.

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: In vitro (cell culture) laboratory experiment
• Who was studied: Mouse macrophage cell line J774.1, plus CHO-K1 cells used for experiments in which the MARCO receptor gene was introduced (transfection); Japan, National Institute for Environmental Studies
• Exposure: Multi-walled carbon nanotubes: average diameter 67 nm, surface area 26 m2/g, carbon purity 99.79 wt %, iron impurity about 2000 ppm. UICC crocidolite was the reference fiber. Doses ranged up to 100 μg/mL and above, for 1.5 to 32 hours depending on the experiment.
• Main results:
  • In J774.1 macrophages, the LC50 of the nanotubes was about 1/25th that of crocidolite.
  • The drop in cell viability partly reversed at nanotube concentrations over 100 μg/mL. Viability fell steadily with increasing crocidolite concentration.
  • Co-treatment with buthionine sulfoximine (BSO) or N-acetylcysteine (NAC) did not affect the loss of viability caused by the nanotubes.
  • The signaling proteins ERK44/42 and p38 were not activated by the nanotubes. JNK was slightly phosphorylated at higher concentrations.
  • The apoptosis markers caspase-3 and PARP were not activated by 50 μg/mL nanotubes.
  • In microarray tests, cytokine-related genes were only slightly upregulated after 8 hours of exposure to 10 μg/mL nanotubes.
  • Scanning electron microscopy after 8 hours of exposure to 20 μg/mL nanotubes showed the plasma membrane disrupted and infiltrated with nanotube fibers.
  • Among proteins recovered with the nanotubes, a 55 kDa protein was identified as the scavenger receptor MARCO, and lower-molecular-weight proteins were identified as histones.
  • CHO-K1 cells given the MARCO gene took up polystyrene beads rapidly (at 2 h) compared with cells given an empty vector. Most bead-associated MARCO cells were also associated with nanotubes.
• The authors' conclusion: The authors concluded that macrophages bind the nanotubes via MARCO, that the plasma membrane extends along the fibers and is injured in the process, and that nanotube cytotoxicity was about 25 times higher than that of crocidolite in these mouse macrophages.
• Limitations the authors state:
  • The nanotubes agglomerated (clumped) at higher concentrations despite ultrasonication. The authors speculated that this explains the unusual dose-response curve.
  • The precise length distribution of the nanotubes could not be determined.
  • It is unresolved whether histones bind the nanotubes directly or were extracted along with DNA.

Topics

Medical terms in this study, explained in our Lexicon: Macrophage, UICC, Crocidolite, Glutathione (GSH), Cytokine, Apoptosis.

Related studies

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• Macrophage receptor with collagenous structure (MARCO) is a dynamic adhesive molecule that enhances uptake of carbon nanotubes by CHO-K1 cells — S. Hirano, Y. Fujitani, et al. (2012)
• Multi-walled carbon nanotubes induce stronger migration of inflammatory cells in vitro than asbestos or granular particles but a similar pattern of inflammatory mediators — G.A. Westphal, N. Rosenkranz, et al. (2019)
• In vitro chromosomal aberrations induced by various shapes of multi-walled carbon nanotubes (MWCNTs) — T. Sasaki, M. Asakura, et al. (2016)
• Genotoxicity and cytotoxicity of multi-wall carbon nanotubes in cultured Chinese hamster lung cells in comparison with chrysotile A fibers — M. Asakura, T. Sasaki, et al. (2010)
• Carboxylated short single-walled carbon nanotubes but not plain and multi-walled short carbon nanotubes show in vitro genotoxicity — M. Mrakovcic, C. Meindl, et al. (2015)

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