Towards predicting the lung fibrogenic activity of nanomaterials: experimental validation of an in vitro fibroblast proliferation assay

Our plain-English summary of Towards predicting the lung fibrogenic activity of nanomaterials: experimental validation of an in vitro fibroblast proliferation assay, by G. Vietti, S. Ibouraadaten, M. Palmai-Pallag, et al (Particle and Fibre Toxicology, 2013).

The study

• Authors: G. Vietti, S. Ibouraadaten, M. Palmai-Pallag, Y. Yakoub, C. Bailly, I. Fenoglio, E. Marbaix, D. Lison, S. van den Brule
• Journal: Particle and Fibre Toxicology
• Published: 2013;10:52
• Cited by: 67 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Background: Carbon nanotubes (CNT) can induce lung inflammation and fibrosis in rodents. Several studies have identified the capacity of CNT to stimulate the proliferation of fibroblasts. We developed and validated experimentally here a simple and rapid in vitro assay to evaluate the capacity of a nanomaterial to exert a direct pro-fibrotic effect on fibroblasts.

Methods: The activity of several multi-wall (MW)CNT samples (NM400, the crushed form of NM400 named NM400c, NM402 and MWCNTg 2400) and asbestos (crocidolite) was investigated in vitro and in vivo. The proliferative response to MWCNT was assessed on mouse primary lung fibroblasts, human fetal lung fibroblasts (HFL-1), mouse embryonic fibroblasts (BALB-3T3) and mouse lung fibroblasts (MLg) by using different assays (cell counting, WST-1 assay and propidium iodide PI staining) and dispersion media (fetal bovine serum, FBS and bovine serum albumin, BSA). C57BL/6 mice were pharyngeally aspirated with the same materials and lung fibrosis was assessed after 2 months by histopathology, quantification of total collagen lung content and pro-fibrotic cytokines in broncho-alveolar lavage fluid (BALF).

Results: MWCNT (NM400 and NM402) directly stimulated fibroblast proliferation in vitro in a dose-dependent manner and induced lung fibrosis in vivo. NM400 stimulated the proliferation of all tested fibroblast types, independently of FBS- or BSA- dispersion. Results obtained by WST1 cell activity were confirmed with cell counting and cell cycle (PI staining) assays. Crocidolite also stimulated fibroblast proliferation and induced pulmonary fibrosis, although to a lesser extent than NM400 and NM402. In contrast, shorter CNT (NM400c and MWCNTg 2400) did not induce any fibroblast proliferation or collagen accumulation in vivo, supporting the idea that CNT structure is an important parameter for inducing lung fibrosis.

Conclusions: In this study, an optimized proliferation assay using BSA as a dispersant, MLg cells as targets and an adaptation of WST-1 as readout was developed. The activity of MWCNT in this test strongly reflects their fibrotic activity in vivo, supporting the predictive value of this in vitro assay in terms of lung fibrosis potential.

In plain English

This laboratory study combined cell-culture experiments with mouse experiments to test whether a fibroblast proliferation assay can predict lung fibrosis. Four multi-wall carbon nanotube (MWCNT) samples (NM400, crushed NM400 called NM400c, NM402 and MWCNTg 2400), crocidolite asbestos and Min-U-Sil crystalline silica were studied. Fibroblasts (mouse primary lung fibroblasts, HFL-1, BALB-3T3 and MLg) were exposed for 24 hours to doses of 1 to 37.5 micrograms per square centimetre, with proliferation read by cell counting, an adapted WST-1 assay and propidium iodide staining. C57BL/6 mice received pharyngeal aspiration of NM400 or NM402 at 12.5 to 100 micrograms per mouse, of NM400c, MWCNTg 2400 or crocidolite at 100 micrograms, or of 2.5 mg Min-U-Sil as positive control, and were examined 2 months later. The authors reported that NM400 and NM402 increased fibroblast proliferation dose-dependently and increased lung collagen (hydroxyproline), while NM400c and MWCNTg 2400 did neither. Crocidolite also stimulated fibroblast proliferation and induced pulmonary fibrosis, but to a lesser extent than NM400. The authors concluded that the in vitro proliferative activity reflected the in vivo fibrosis findings, and that nanotube structure/length is an important determinant, while noting the assay needs wider confirmation.

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: Experimental laboratory study: in vitro fibroblast proliferation assays combined with in vivo mouse instillation experiments
• Who was studied: Cultured fibroblasts (mouse primary lung fibroblasts, human fetal lung fibroblasts HFL-1, mouse embryonic BALB-3T3, mouse lung fibroblasts MLg) and C57BL/6 female mice (at least five per group), Belgium
• Exposure: MWCNT samples NM400, NM400c (crushed), NM402, MWCNTg 2400; crocidolite asbestos; Min-U-Sil crystalline silica as positive control. In vitro 1 to 37.5 micrograms per square centimetre for 24 h; in vivo NM400 and NM402 at 100, 50, 25 and 12.5 micrograms per mouse, NM400c, MWCNTg 2400 and crocidolite at 100 micrograms per mouse, Min-U-Sil at 2.5 mg per mouse, all by pharyngeal aspiration
• Main results:
  • In cultured primary mouse lung fibroblasts, both NM400 and NM402 increased cell number dose-dependently, the highest concentration producing an effect similar to the PDGF positive control
  • In mice, NM400 and NM402 produced dose-dependent lung collagen accumulation at 2 months; at 50-100 micrograms per mouse this was similar in extent to 2.5 mg Min-U-Sil
  • In the MLg fibroblast WST-1 assay, intact NM400 and crocidolite stimulated proliferation whereas NM400c and MWCNTg 2400 did not; NM400 had a stronger proliferative effect than crocidolite
  • In mice given 100 micrograms, lung collagen content rose significantly with intact NM400, silica and crocidolite, but not with NM400c or MWCNTg 2400
  • Sirius red histology showed collagen accumulation and greater collagen fibre thickness only in lungs of mice given NM400 or crocidolite, with NM400 producing a stronger fibrotic response than crocidolite
  • In bronchoalveolar lavage fluid, total TGF-beta1, PDGF-BB and osteopontin were significantly increased in mice exposed to intact NM400, more so than with silica and crocidolite
• The authors' conclusion: The authors concluded that the in vitro proliferative activity of CNT reflected the in vivo fibrosis findings, supporting the predictive value of their assay, and that CNT structure/length is an important determinant of lung fibrosis capacity
• Limitations the authors state:
  • The authors stated the assay must still be tested with a wider range of CNT and in an inter-laboratory comparison for reproducibility and transferability
  • The authors noted the doses used in vitro and in vivo were higher than those used by other investigators
  • The authors noted a discrepancy with their own earlier study in which MWCNTg 2400 did induce lung fibrosis, attributed to differences in protocol
  • The authors noted that carbon nanotubes can interfere with conventional in vitro assays, several of which gave unstable or biased results
  • The authors noted that in vivo CNT are not uniformly distributed and that CNT do not entirely sediment onto cells in vitro, complicating dose comparison

Topics

The National Library of Medicine indexes this study under subjects our site covers: Crocidolite.

Medical terms in this study, explained in our Lexicon: Fibrogenic, Fibroblast, Inflammation, Fibrosis, Crocidolite, Histopathology, Collagen, Cytokine.

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