Uptake and cytotoxic effects of multi-walled carbon nanotubes in human bronchial epithelial cells

Our plain-English summary of Uptake and cytotoxic effects of multi-walled carbon nanotubes in human bronchial epithelial cells, by S. Hirano, Y. Fujitani, A. Furuyama, et al (Toxicology and Applied Pharmacology, 2010).

The study

• Authors: S. Hirano, Y. Fujitani, A. Furuyama, S. Kanno
• Journal: Toxicology and Applied Pharmacology
• Published: 2010;249(1):8-15
• Cited by: 69 articles, as counted by NIH iCite
• Read it: PubMed · Publisher

Abstract

Carbon nanotubes (CNT) are cytotoxic to several cell types. However, the mechanism of CNT toxicity has not been fully studied, and dosimetric analyses of CNT in the cell culture system are lacking. Here, we describe a novel, high throughput method to measure cellular uptake of CNT using turbimetry. BEAS-2B, a human bronchial epithelial cell line, was used to investigate cellular uptake, cytotoxicity, and inflammatory effects of multi-walled CNT (MWCNT). The cytotoxicity of MWCNT was higher than that of crocidolite asbestos in BEAS-2B cells. The IC(50) of MWCNT was 12 μg/ml, whereas that of asbestos (crocidolite) was 678 μg/ml. Over the course of 5 to 8 h, BEAS-2B cells took up 17-18% of the MWCNT when they were added to the culture medium at a concentration of 10 μg/ml. BEAS-2B cells were exposed to 2, 5, or 10 μg/ml of MWCNT, and total RNA was extracted for cytokine cDNA primer array assays. The culture supernatant was collected for cytokine antibody array assays. Cytokines IL-6 and IL-8 increased in a dose dependent manner at both the mRNA and protein levels. Migration inhibitory factor (MIF) also increased in the culture supernatant in response to MWCNT. A phosphokinase array study using lysates from BEAS-2B cells exposed to MWCNT indicated that phosphorylation of p38, ERK1, and HSP27 increased significantly in response to MWCNT. Results from a reporter gene assays using the NF-κB or AP-1 promoter linked to the luciferase gene in transiently transfected CHO-KI cells revealed that NF-κB was activated following MWCNT exposure, while AP-1 was not changed. Collectively, MWCNT activated NF-κB, enhanced phosphorylation of MAP kinase pathway components, and increased production of proinflammatory cytokines in human bronchial epithelial cells.

Copyright © 2010 Elsevier Inc. All rights reserved.

In plain English

This laboratory study, done in cell cultures in Japan, tested multi-walled carbon nanotubes (MWCNT) on a human bronchial (airway lining) cell line called BEAS-2B. Crocidolite asbestos was used as a comparison fiber. No people or animals were studied. The authors described a new way to measure how much nanotube material became associated with the cells, counting both engulfed and membrane-bound material. The cells took up 12.5% of the added MWCNT within 5 hours. After 24 hours, the concentration that lowered measured cell viability to half the control level (the IC50) was 12 μg/ml for MWCNT and 678 μg/ml for crocidolite, so MWCNT were much more toxic to these cells. That toxicity depended on how densely the cells were grown. MWCNT raised the cells' messenger RNA for several inflammation signals, including IL-6 and IL-8. IL-6, IL-8 and MIF also rose in the culture fluid, but those increases were statistically significant only for IL-6 and MIF at 10 μg/ml. The authors concluded that MWCNT were highly cytotoxic in human bronchial epithelial cells and triggered inflammatory signals through p38 MAP kinase and NF-κB activation.

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, including development of a turbidity-based method to measure cellular uptake of carbon nanotubes
• Who was studied: No human or animal subjects. Two cell lines were used: BEAS-2B human bronchial epithelial cells, and CHO-K1 Chinese hamster ovary cells (mainly for the reporter gene assay). The work was done at the National Institute for Environmental Studies, Japan.
• Exposure: Multi-walled carbon nanotubes (MWCNT, mean diameter 67 nm, length 1.0 to 13.5 μm, median 2.7 μm), compared with UICC crocidolite as a reference biopersistent fiber. Concentrations of 2, 5 and 10 μg/ml were used for cytokine tests, and various concentrations over 24 hours for cytotoxicity.
• Main results:
  • In BEAS-2B cells, the IC50 was 12 μg/ml for MWCNT. This is the concentration that reduced viability, measured by the WST-8 assay after 24 h, to half the control level. MWCNT were much more cytotoxic than crocidolite.
  • The crocidolite IC50 in BEAS-2B cells was 678 μg/ml (Fig. 3, WST-8 viability assay).
  • BEAS-2B cells exposed to 10 μg/ml MWCNT took up 12.5% of the MWCNT (2.5 μg) within 5 h. Uptake was slow in the first 2 h. 'Uptake' included both engulfed and membrane-bound MWCNT.
  • Toxicity in BEAS-2B cells depended on cell density. At 10 μg/ml for 24 h, essentially no low-density cells survived, while most high-density cells survived. This density difference was not seen in CHO-K1 cells.
  • Messenger RNA for IL-1α, IL-6, IL-8 and CXCL2 rose with MWCNT dose in BEAS-2B cells. IL-8 mRNA was 9.56-fold the control level at 10 μg/ml.
  • IL-6, IL-8 and MIF in the culture fluid rose with MWCNT dose. The increases were statistically significant only for IL-6 and MIF at 10 μg/ml. Apparent rises in IL-13 and sICAM-1 were not statistically significant.
  • In CHO-K1 reporter cells, NF-κB was activated by MWCNT while AP-1 was unchanged.
  • Of 46 kinases tested in BEAS-2B cells exposed to 10 μg/ml MWCNT for 8 h, phosphorylation of p38α, ERK1/2 and HSP27 increased significantly. AKT phosphorylation did not change.
• The authors' conclusion: The authors concluded that MWCNT were highly cytotoxic in human bronchial epithelial cells, which produced proinflammatory cytokines via p38 MAP kinase and NF-κB activation. They stated that more studies are needed on how the cell membrane associates with MWCNT.

Topics

Medical terms in this study, explained in our Lexicon: Crocidolite, Cytokine.

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