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

Vietti G, Ibouraadaten S, Palmai-Pallag M, Yakoub Y, Bailly C, Fenoglio I, Marbaix E, Lison D, van den Brule S - Part Fibre Toxicol (2013)

Bottom Line: 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.Results obtained by WST1 cell activity were confirmed with cell counting and cell cycle (PI staining) assays.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Louvain centre for Toxicology and Applied Pharmacology, Université catholique de Louvain, Avenue E, Mounier, 52 - bte B1,52,12, 1200 Brussels, Belgium. giulia.vietti@uclouvain.be.

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.

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In vitro effect of MWCNT on proliferation of several fibroblast types assessed by cell counting. Primary murine lung fibroblasts and human and mouse fibroblast cell lines (HFL-1, BALB/3T3 and MLg) were treated for 24 h with different doses of NM400 dispersed in 1.4 mg BSA /ml (final concentration 0.14 mg BSA /ml). PDGF-BB (50 ng/ml) was used as a positive control. Results are expressed relative to non-treated cell number (Ctl). * p < 0.05, ** p < 0.01, *** p < 0.001 vs non-treated cells (n = 3, t-test or Dunnet multiple comparison test as appropriate).
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Figure 4: In vitro effect of MWCNT on proliferation of several fibroblast types assessed by cell counting. Primary murine lung fibroblasts and human and mouse fibroblast cell lines (HFL-1, BALB/3T3 and MLg) were treated for 24 h with different doses of NM400 dispersed in 1.4 mg BSA /ml (final concentration 0.14 mg BSA /ml). PDGF-BB (50 ng/ml) was used as a positive control. Results are expressed relative to non-treated cell number (Ctl). * p < 0.05, ** p < 0.01, *** p < 0.001 vs non-treated cells (n = 3, t-test or Dunnet multiple comparison test as appropriate).

Mentions: To examine the response of different fibroblast cell types, the effect of NM400 on human fetal lung fibroblasts (HFL-1), mouse embryonic fibroblasts (BALB-3T3) and mouse lung fibroblasts (MLg) was evaluated. At the concentrations tested, NM400 had a similar and significant stimulating effect on the proliferation of all fibroblasts (Figure 4). MLg were chosen for subsequent experiments since the propagation of this cell in culture is more convenient than primary cells and furthermore, the in vitro results obtained with these cells can be directly compared to in vivo experiments performed in mice.


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

Vietti G, Ibouraadaten S, Palmai-Pallag M, Yakoub Y, Bailly C, Fenoglio I, Marbaix E, Lison D, van den Brule S - Part Fibre Toxicol (2013)

In vitro effect of MWCNT on proliferation of several fibroblast types assessed by cell counting. Primary murine lung fibroblasts and human and mouse fibroblast cell lines (HFL-1, BALB/3T3 and MLg) were treated for 24 h with different doses of NM400 dispersed in 1.4 mg BSA /ml (final concentration 0.14 mg BSA /ml). PDGF-BB (50 ng/ml) was used as a positive control. Results are expressed relative to non-treated cell number (Ctl). * p < 0.05, ** p < 0.01, *** p < 0.001 vs non-treated cells (n = 3, t-test or Dunnet multiple comparison test as appropriate).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3852297&req=5

Figure 4: In vitro effect of MWCNT on proliferation of several fibroblast types assessed by cell counting. Primary murine lung fibroblasts and human and mouse fibroblast cell lines (HFL-1, BALB/3T3 and MLg) were treated for 24 h with different doses of NM400 dispersed in 1.4 mg BSA /ml (final concentration 0.14 mg BSA /ml). PDGF-BB (50 ng/ml) was used as a positive control. Results are expressed relative to non-treated cell number (Ctl). * p < 0.05, ** p < 0.01, *** p < 0.001 vs non-treated cells (n = 3, t-test or Dunnet multiple comparison test as appropriate).
Mentions: To examine the response of different fibroblast cell types, the effect of NM400 on human fetal lung fibroblasts (HFL-1), mouse embryonic fibroblasts (BALB-3T3) and mouse lung fibroblasts (MLg) was evaluated. At the concentrations tested, NM400 had a similar and significant stimulating effect on the proliferation of all fibroblasts (Figure 4). MLg were chosen for subsequent experiments since the propagation of this cell in culture is more convenient than primary cells and furthermore, the in vitro results obtained with these cells can be directly compared to in vivo experiments performed in mice.

Bottom Line: 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.Results obtained by WST1 cell activity were confirmed with cell counting and cell cycle (PI staining) assays.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Louvain centre for Toxicology and Applied Pharmacology, Université catholique de Louvain, Avenue E, Mounier, 52 - bte B1,52,12, 1200 Brussels, Belgium. giulia.vietti@uclouvain.be.

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.

Show MeSH
Related in: MedlinePlus