Induction and recovery of CpG site specific methylation changes in human bronchial cells after long-term exposure to carbon nanotubes and asbestos

Our plain-English summary of Induction and recovery of CpG site specific methylation changes in human bronchial cells after long-term exposure to carbon nanotubes and asbestos, by D. Öner, M. Ghosh, R. Coorens, et al (Environment International, 2020).

The study

• Authors: D. Öner, M. Ghosh, R. Coorens, H. Bové, M. Moisse, D. Lambrechts, M. Ameloot, L. Godderis, P.H.M. Hoet
• Journal: Environment International
• Published: 2020;137:105530
• Cited by: 28 articles, as counted by NIH iCite
• Read it: PubMed · Publisher

Abstract

Introduction: Inhalation of asbestos induces lung cancer via different cellular mechanisms. Together with the increased production of carbon nanotubes (CNTs) grows the concern about adverse effects on the lungs given the similarities with asbestos. While it has been established that CNT and asbestos induce epigenetic alterations, it is currently not known whether alterations at epigenetic level remain stable after withdrawal of the exposure. Identification of DNA methylation changes after a low dose of CNT and asbestos exposure and recovery can be useful to determine the fibre/particle toxicity and adverse outcome.

Methods: Human bronchial epithelial cells (16HBE) were treated with a low and non-cytotoxic dose (0.25 µg/ml) of multi-walled carbon nanotubes (MWCNTs-NM400) or single-walled carbon nanotubes (SWCNTs-SRM2483) and 0.05 µg/ml amosite (brown) asbestos for the course of four weeks (sub-chronic exposure). After this treatment, the cells were further incubated (without particle/fibre) for two weeks, allowing recovery from the exposure (recovery period). Nuclear depositions of the CNTs were assessed using femtosecond pulsed laser microscopy in a label-free manner. DNA methylation alterations were analysed using microarrays that assess more than 850 thousand CpG sites in the whole genome.

Results: At non-cytotoxic doses, CNTs were noted to be incorporated with in the nucleus after a four weeks period. Exposure to MWCNTs induced a single hypomethylation at a CpG site and gene promoter region. No change in DNA methylation was observed after the recovery period for MWCNTs. Exposure to SWCNTs or amosite induced hypermethylation at CpG sites after sub-chronic exposure which may involve in 'transcription factor activity' and 'sequence-specific DNA binding' gene ontologies. After the recovery period, hypermethylation and hypomethylation were noted for both SWCNTs and amosite. Hippocalcinlike 1 (HPCAL1), protease serine 3 (PRSS3), kallikrein-related peptidase 3 (KLK3), kruppel like factor 3 (KLF3) genes were hypermethylated at different time points in either SWCNT-exposed or amosite-exposed cells.

Conclusion: These results suggest that the specific SWCNT (SRM2483) and amosite fibres studied induce hypo- or hypermethylation on CpG sites in DNA after very low-dose exposure and recovery period. This effect was not seen for the studied MWCNT (NM400).

Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.

In plain English

This was a laboratory experiment on a human bronchial (airway lining) cell line called 16HBE, not a study of people. The authors exposed the cells for four weeks to low doses of amosite asbestos (0.05 µg/ml) or to one of two carbon nanotube types: multi-walled NM400 or single-walled SRM2483, each at 0.25 µg/ml. The cells then grew for two weeks without exposure, as a recovery period. The authors measured DNA methylation, a chemical tag on DNA, at more than 850 thousand sites. No cell toxicity was seen. After four weeks, amosite-exposed cells had nine sites with increased methylation. After recovery, six sites differed: two decreased and four increased. Single-walled nanotubes changed 11 sites, and 21 after recovery. Multi-walled nanotubes changed one site and one gene promoter, and none after recovery. Some genes, including PRSS3, KLF3 and KLK3, showed changes after both amosite and single-walled nanotubes. The authors concluded that these specific amosite and single-walled nanotube fibres induced methylation changes after very low-dose exposure and recovery. They noted they tested only one material of each nanotube type and only four replicates.

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: long-term, low-dose exposure followed by a recovery period, with genome-wide DNA methylation microarray analysis
• Who was studied: Human bronchial epithelial cell line 16HBE (16HBE14o-), obtained from the University of California, San Francisco; 4 replicates per exposure condition (3 for controls)
• Exposure: UICC reference amosite (South African) asbestos at 0.05 µg/ml; multi-walled carbon nanotubes (NM400) and single-walled carbon nanotubes (SRM2483) each at 0.25 µg/ml. Cells were exposed repeatedly every 2 to 3 days for 4 weeks, followed by 2 weeks without exposure (recovery period)
• Main results:
  • No cellular toxicity was observed for any of the three materials
  • Amosite-exposed cells: after 4 weeks of exposure, 9 CpG sites were differentially methylated, all hypermethylated (increased methylation)
  • Amosite-exposed cells: after the recovery period, 6 CpG sites were differentially methylated, 2 hypomethylated and 4 hypermethylated
  • Single-walled nanotube-exposed cells: 11 hypermethylated CpG sites after 4 weeks of exposure
  • Single-walled nanotube-exposed cells: after recovery, 21 differentially methylated CpG sites, 3 hypomethylated and 18 hypermethylated. Only hypermethylation of HPCAL1 was present at both time points
  • Multi-walled nanotube-exposed cells: 1 CpG site and 1 gene promoter were hypomethylated after 4 weeks, and there was no differential methylation after recovery
  • PRSS3 was hypermethylated after the 4-week exposure in both single-walled nanotube- and amosite-exposed cells
  • KLK3 was hypermethylated after the recovery period in both amosite- and single-walled nanotube-exposed cells. KLF3 was hypermethylated after the 4-week exposure in amosite-exposed cells and after recovery in single-walled nanotube-exposed cells
  • Both nanotube types were found inside cell nuclei after 4 weeks of exposure and were not detectable after the recovery period. Amosite fibres were seen piercing some cells
• The authors' conclusion: The authors concluded that the specific single-walled nanotube (SRM2483) and amosite fibres studied induced hypo- or hypermethylation at CpG sites after very low-dose exposure and a recovery period. This effect was not seen for the multi-walled nanotube (NM400). They emphasized the potential toxicity of single-walled nanotubes based on similarities with amosite.
• Limitations the authors state:
  • Only one representative material was used for each nanotube type, so the results may not apply to all carbon nanotubes
  • Using four replicates may have limited statistical power and underestimated differential methylation
  • At these low doses the nanotubes did not contact all cells, which may have reduced the observed effects
  • The study did not directly examine the link between methylation changes and disease

Topics

Medical terms in this study, explained in our Lexicon: Lung Cancer, Asbestos Exposure, Amosite, Deposition.

Related studies

• Global and gene-specific DNA methylation effects of different asbestos fibres on human bronchial epithelial cells — D. Öner, M. Ghosh, et al. (2018)
• Distinct autophagy-apoptosis related pathways activated by Multi-walled (NM 400) and Single-walled carbon nanotubes (NIST-SRM2483) in human bronchial epithelial (16HBE14o-) cells — M. Ghosh, S. Murugadoss, et al. (2020)
• A comparison of acute and long-term effects of industrial multiwalled carbon nanotubes on human lung and immune cells in vitro — T. Thurnherr, C. Brandenberger, et al. (2011)
• 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)
• Different Cellular Response of Human Mesothelial Cell MeT-5A to Short-Term and Long-Term Multiwalled Carbon Nanotubes Exposure — L. Ju, W. Wu, et al. (2017)
• Uptake and cytotoxic effects of multi-walled carbon nanotubes in human bronchial epithelial cells — S. Hirano, Y. Fujitani, et al. (2010)

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