In Vitro Study of Mutagenesis Induced by Crocidolite-Exposed Alveolar Macrophages NR8383 in Cocultured Big Blue Rat2 Embryonic Fibroblasts

Our plain-English summary of In Vitro Study of Mutagenesis Induced by Crocidolite-Exposed Alveolar Macrophages NR8383 in Cocultured Big Blue Rat2 Embryonic Fibroblasts, by Y. Guichard, L. Gaté, C. Darne, et al (Journal of Toxicology, 2010).

The study

• Authors: Y. Guichard, L. Gaté, C. Darne, M.C. Bottin, C. Langlais, J.C. Micillino, M. Goutet, S. Julien, B. Stéphane
• Journal: Journal of Toxicology
• Published: 2010;2010:323828
• Cited by: 5 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Asbestos-induced mutagenicity in the lung may involve reactive oxygen/nitrogen species (ROS/RNS) released by alveolar macrophages. With the aim of proposing an alternative in vitro mutagenesis test, a coculture system of rat alveolar macrophages (NR8383) and transgenic Big Blue Rat2 embryonic fibroblasts was developed and tested with a crocidolite sample. Crocidolite exposure induced no detectable increase in ROS production from NR8383, contrasting with the oxidative burst that occurred following a brief exposure (1 hour) to zymosan, a known macrophage activator. In separated cocultures, crocidolite and zymosan induced different changes in the gene expressions involved in cellular inflammation in NR8383 and Big Blue. In particular, both particles induced up-regulation of iNOS expression in Big Blue, suggesting the formation of potentially genotoxic nitrogen species. However, crocidolite exposure in separated or mixed cocultures induced no mutagenic effects whereas an increase in Big Blue mutants was detected after exposure to zymosan in mixed cocultures. NR8383 activation by crocidolite is probably insufficient to induce in vitro mutagenic events. The mutagenesis assay based on the coculture of NR8383 and Big Blue cannot be used as an alternative in vitro method to assess the mutagenic properties of asbestos fibres.

In plain English

This was a laboratory study on cells grown in dishes, not on people or animals. The authors tested whether a rat lung immune cell line (alveolar macrophages called NR8383), grown together with genetically engineered rat fibroblast cells (Big Blue), could serve as a test of whether asbestos fibres cause gene mutations. They exposed the cells to a crocidolite sample. For comparison they used zymosan, a known stimulant of macrophages. Crocidolite did not measurably increase the macrophages' release of reactive oxygen species (damaging oxygen-containing molecules), while zymosan did. Crocidolite raised the activity of some inflammation-related genes in both cell types. One was iNOS in the fibroblasts: after 3 hours at 60 μg/cm2 it rose 1.9-fold with crocidolite, compared with 5.2-fold with zymosan. Crocidolite caused no significant change in the fibroblasts' mutation frequency, whether the two cell types were separated by a membrane or mixed together. Zymosan increased mutation frequency in mixed cultures, by up to 2.4-fold. The authors concluded that this coculture test cannot be used as an alternative laboratory method to assess the mutagenic properties of asbestos fibres.

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) experiment testing a proposed mutagenesis assay
• Who was studied: Two commercially obtained cell lines: rat alveolar macrophage-derived NR8383 cells and transgenic Big Blue Rat2 rat embryonic fibroblasts. They were grown either separated by a membrane insert or mixed together, and each experiment was performed at least three times.
• Exposure: A crocidolite sample, in which 83.1% of fibres were shorter than 5 μm and 16.9% longer than 5 μm, all under 3 μm in diameter. It was tested at concentrations between 1 and 60 μg/cm2 for 3, 24 or 72 hours depending on the assay. Zymosan was used as a macrophage stimulant for comparison, and ENU as a known mutagen to validate the mutation assay.
• Main results:
  • In the chemiluminescence assay, crocidolite at 15, 30 and 60 μg/cm2 produced no increase in superoxide from NR8383 macrophages compared with unexposed cultures.
  • In the fluorescence assay, mean fluorescence was 236.5 in zymosan-treated (10 μg/cm2) NR8383 cells versus 77.1 in controls, an approximately 3-fold increase. Crocidolite-treated cells (1, 5 and 10 μg/cm2) showed no significant change.
  • In NR8383 macrophages, 3-hour crocidolite exposure significantly increased iNOS (2.9-fold) and TNF-α (2.2-fold) gene expression but had no significant effect on IL1-β.
  • In NR8383 macrophages, 24-hour crocidolite exposure at 15 μg/cm2 increased iNOS, IL1-β and TNF-α expression by 26.1-fold, 2.5-fold and 3.4-fold respectively.
  • In cocultured Big Blue fibroblasts, iNOS expression after 3 hours at 60 μg/cm2 rose 5.2-fold with zymosan and 1.9-fold with crocidolite.
  • Crocidolite caused no significant change in Big Blue mutation frequency, whether in separated cocultures (3 and 15 μg/cm2 for 72 hours) or mixed cocultures (10 μg/cm2 for 24 hours).
  • In mixed cocultures, 3-hour zymosan exposure significantly increased Big Blue mutation frequency. At 60 μg/cm2 the increase was 2.4-fold: 30.8 per 100,000 versus 13.0 per 100,000 in control cocultures.
  • The known mutagen ENU at 500 μg/mL raised mutation frequency in Big Blue monocultures 8.6-fold: 83.3 per 100,000 versus 9.6 per 100,000 in controls.
• The authors' conclusion: The authors concluded that the NR8383/Big Blue coculture mutagenesis assay cannot be used as an alternative in vitro method to assess the mutagenic properties of asbestos fibres. They also concluded that activation of NR8383 cells by crocidolite is probably insufficient to induce mutations in neighbouring cells in vitro.
• Limitations the authors state:
  • Higher concentrations of fibres or particles could not be tested in the fluorescence assay because they disrupted the flow cytometry analysis.
  • Because of cytotoxicity, crocidolite exposure of mixed cocultures was limited to 24 hours.
  • The authors noted that human blood monocytes, which the literature indicates generate reactive oxygen species on contact with mineral fibres, would have been of interest. They were not used because they are difficult to obtain routinely.

Topics

Medical terms in this study, explained in our Lexicon: Crocidolite, Alveolar Macrophage, Fibroblast, Macrophage, Inflammation.

Related studies

• Lung mesothelial cell and fibroblast responses to pleural and alveolar macrophage supernatants and to lavage fluids from crocidolite-exposed rats — I.Y. Adamson, H. Prieditis, et al. (1997)
• Differential proliferation of rat lung fibroblasts induced by the platelet-derived growth factor-AA, -AB, and -BB isoforms secreted by rat alveolar macrophages — J.C. Bonner, A.R. Osornio-Vargas, et al. (1991)
• Prevention of asbestos-induced cell death in rat lung fibroblasts and alveolar macrophages by scavengers of active oxygen species — M.A. Shatos, J.M. Doherty, et al. (1987)
• Alveolar macrophage stimulation of lung fibroblast growth in asbestos-induced pulmonary fibrosis — I. Lemaire, H. Beaudoin, et al. (1986)
• Alveolar macrophage cytokine and growth factor production in a rat model of crocidolite-induced pulmonary inflammation and fibrosis — K.E. Driscoll, J.K. Maurer, et al. (1995)
• Mechanisms of asbestos-induced nitric oxide production by rat alveolar macrophages in inhalation and in vitro models — T.R. Quinlan, K.A. BeruBe, et al. (1998)

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