In vitro and in vivo genotoxic effects of straight versus tangled multi-walled carbon nanotubes

Our plain-English summary of In vitro and in vivo genotoxic effects of straight versus tangled multi-walled carbon nanotubes, by J. Catalán, K.M. Siivola, P. Nymark, et al (Nanotoxicology, 2016).

The study

• Authors: J. Catalán, K.M. Siivola, P. Nymark, H. Lindberg, S. Suhonen, H. Järventaus, A.J. Koivisto, C. Moreno, E. Vanhala, H. Wolff, K.I. Kling, K.A. Jensen, K. Savolainen, H. Norppa
• Journal: Nanotoxicology
• Published: 2016;10(6):794-806
• Cited by: 43 articles, as counted by NIH iCite
• Read it: PubMed · Publisher

Abstract

Some multi-walled carbon nanotubes (MWCNTs) induce mesothelioma in rodents, straight MWCNTs showing a more pronounced effect than tangled MWCNTs. As primary and secondary genotoxicity may play a role in MWCNT carcinogenesis, we used a battery of assays for DNA damage and micronuclei to compare the genotoxicity of straight (MWCNT-S) and tangled MWCNTs (MWCNT-T) in vitro (primary genotoxicity) and in vivo (primary or secondary genotoxicity). C57Bl/6 mice showed a dose-dependent increase in DNA strand breaks, as measured by the comet assay, in lung cells 24 h after a single pharyngeal aspiration of MWCNT-S (1-200 μg/mouse). An increase was also observed for DNA strand breaks in lung and bronchoalveolar lavage (BAL) cells and for micronucleated alveolar type II cells in mice exposed to aerosolized MWCNT-S (8.2-10.8 mg/m(3)) for 4 d, 4 h/d. No systemic genotoxic effects, assessed by the γ-H2AX assay in blood mononuclear leukocytes or by micronucleated polychromatic erythrocytes (MNPCEs) in bone marrow or blood, were observed for MWCNT-S by either exposure technique. MWCNT-T showed a dose-related decrease in DNA damage in BAL and lung cells of mice after a single pharyngeal aspiration (1-200 μg/mouse) and in MNPCEs after inhalation exposure (17.5 mg/m(3)). In vitro in human bronchial epithelial BEAS-2B cells, MWCNT-S induced DNA strand breaks at low doses (5 and 10 μg/cm(2)), while MWCNT-T increased strand breakage only at 200 μg/cm(2). Neither of the MWCNTs was able to induce micronuclei in vitro. Our findings suggest that both primary and secondary mechanisms may be involved in the genotoxicity of straight MWCNTs.

In plain English

This laboratory study compared straight (MWCNT-S, Mitsui MWCNT-XNRI-7) and tangled (MWCNT-T, CheapTubes) multi-walled carbon nanotubes. Asbestos itself was not tested. The authors exposed human bronchial epithelial BEAS-2B cells in culture. They also exposed female C57Bl/6 mice by a single pharyngeal aspiration or by inhaling aerosols 4 hours a day for 4 days. They then measured DNA damage and micronuclei, a sign of chromosome damage. In cells, the straight nanotubes increased DNA strand breaks at 5 and 10 µg/cm2, reaching 2.2-fold the control level. The tangled type did so only at the highest dose, and neither type caused micronuclei. In mice, the straight type increased lung-cell DNA damage by both methods, and increased micronucleated alveolar type II lung cells after inhalation. The blood and bone marrow tests used showed no genotoxic effects of the straight type. The tangled type did not increase DNA damage in mice; after aspiration, DNA damage in lung and lung-wash cells fell significantly as the dose rose. The authors concluded that both primary and secondary (inflammation-related) mechanisms may be involved in the genotoxicity of straight MWCNTs.

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 study combining in vitro experiments in human cells and in vivo animal experiments in mice (pharyngeal aspiration and inhalation), using a battery of genotoxicity assays (comet assay, gamma-H2AX assay, micronucleus assays)

• Who was studied: Human bronchial epithelial BEAS-2B cells in culture, and female C57Bl/6 mice aged 7-8 weeks. For aspiration, six mice per group received one dose. For inhalation, eight mice per group were exposed, plus a further experiment with six mice per group for the lung micronucleus test. Work was done at the Finnish Institute of Occupational Health with approval from Finnish animal experiment authorities. Dates of the experiments are not given.

• Exposure: Straight multi-walled carbon nanotubes (MWCNT-S: Mitsui MWCNT-XNRI-7) and tangled multi-walled carbon nanotubes (MWCNT-T: CheapTubes, 8-15 nm outer diameter).

• In cells: 5 to 200 µg/cm2 for the comet assay, and 2.5 to 100 µg/cm2 for the micronucleus assay.

• In mice by single pharyngeal aspiration: MWCNT-S at 1 to 200 µg/mouse; MWCNT-T at 10 to 200 µg/mouse according to the methods.

• In mice by inhalation, 4 hours a day for 4 days: MWCNT-S at mean 8.2 and 10.8 mg/m3; MWCNT-T at mean 17.5 mg/m3.

A tungsten carbide-cobalt mixture was used as a positive control.

• Main results:
  • In BEAS-2B cells, MWCNT-S increased DNA strand breaks at 5 and 10 µg/cm2 (p<0.005), reaching 2.2-fold the control level. At 50 and 100 µg/cm2 the increase was not statistically significant, and no dose-dependence was detected.
  • In BEAS-2B cells, MWCNT-T induced DNA damage only at the highest dose, 200 µg/cm2 (p<0.003).
  • Neither nanotube type induced micronuclei in BEAS-2B cells.
  • After single pharyngeal aspiration of MWCNT-S, DNA damage in mouse lung cells was significantly increased only at the highest dose, 200 µg/mouse (p=0.0021), with a significant positive dose-response.
  • After aspiration, MWCNT-T induced no significant increase in DNA damage in mice. Instead, it showed a significant decreasing dose-response in both BAL (lung wash) cells and lung cells.
  • After 4 days of inhalation, MWCNT-S significantly increased DNA damage in mouse BAL and lung cells (p<0.001). The level in BAL cells was 6 times that of the negative control group.
  • After inhalation, MWCNT-T did not increase DNA damage in mouse BAL or lung cells.
  • MWCNT-S induced no significant systemic genotoxic effect in mice, either in blood (gamma-H2AX assay) or in bone marrow (micronucleus assay).
  • After inhalation, MWCNT-T caused a significant decrease (p=0.0024) in micronucleated polychromatic erythrocytes in mouse bone marrow.
  • Inhalation of MWCNT-S (mean 10.8 mg/m3) significantly increased micronucleated alveolar type II cells in mouse lungs (p=0.009). The authors described these cells as supposed target cells of lung carcinogenesis in mice.
  • After inhalation, lung tissue from MWCNT-S-exposed mice, but not MWCNT-T-exposed mice, showed infiltration of eosinophils and activated macrophages, which the authors took to indicate lung inflammation.
• The authors' conclusion: The authors concluded that straight MWCNTs induced DNA damage in cells and both DNA damage and micronuclei in mouse lungs, while tangled MWCNTs showed only a slight increase in DNA damage in cells. They suggested that both primary and secondary mechanisms are involved in the genotoxicity of straight MWCNTs. They also suggested that the erythrocyte micronucleus assay may not be appropriate for detecting the genotoxicity of nanomaterials.
• Limitations the authors state:
  • By volume and mass, the aspiration exposures were dominated by agglomerates and aggregates in the 10 to over 100 µm size range, representing more the inhalable than the respirable fraction.
  • The authors noted that better-dispersed suspensions might have been obtained with another approach. They used PBS with bovine serum albumin to allow comparison with the in vitro studies.
  • The negative dose-response for MWCNT-S in the in vitro micronucleus assay should be taken with caution. At the highest dose tested, cytostasis was over 60%, above the OECD upper testing limit.

Topics

Medical terms in this study, explained in our Lexicon: Mesothelioma, Genotoxicity, Carcinogenesis, Micronuclei, Comet Assay, Bronchoalveolar Lavage (BAL).

Related studies

• Comparative carcinogenicity study of a thick, straight-type and a thin, tangled-type multi-walled carbon nanotube administered by intra-tracheal instillation in the rat — D.M. Saleh, W.T. Alexander, et al. (2020)
• Role of p53 in the chronic pulmonary immune response to tangled or rod-like multi-walled carbon nanotubes — K.S. Duke, E.A. Thompson, et al. (2018)
• 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)
• Free radical scavenging and formation by multi-walled carbon nanotubes in cell free conditions and in human bronchial epithelial cells — P. Nymark, K.A. Jensen, et al. (2014)
• Osteopontin mRNA expression by rat mesothelial cells exposed to multi-walled carbon nanotubes as a potential biomarker of chronic neoplastic transformation in vitro — S. Sridharan, A. Taylor-Just, et al. (2021)
• Effect of surface functionalizations of multi-walled carbon nanotubes on neoplastic transformation potential in primary human lung epithelial cells — T.A. Stueckle, D.C. Davidson, et al. (2017)

Browse more research in Nanotoxicology, or return to medical research.