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

Guichard Y, Gaté L, Darne C, Bottin MC, Langlais C, Micillino JC, Goutet M, Julien S, Stéphane B - J Toxicol (2010)

Bottom Line: Asbestos-induced mutagenicity in the lung may involve reactive oxygen/nitrogen species (ROS/RNS) released by alveolar macrophages.In particular, both particles induced up-regulation of iNOS expression in Big Blue, suggesting the formation of potentially genotoxic nitrogen species.NR8383 activation by crocidolite is probably insufficient to induce in vitro mutagenic events.

View Article: PubMed Central - PubMed

Affiliation: Institut National de Recherche et de Sécurité, rue du Morvan CS 60027, 54519 Vandoeuvre-Les-Nancy Cedex, France.

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.

No MeSH data available.


Related in: MedlinePlus

ROS production in NR8383 analysed by flow cytometry. NR8383 cells were preincubated with H2DCFDA (25 μm) for 30 minutes and then exposed to 10 μg/cm2 of crocidolite or zymosan for 3 hours. Representative histograms plot the relative green DCF fluorescence (FL1) within 15000 live cells (counts: numbers of events).
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fig2: ROS production in NR8383 analysed by flow cytometry. NR8383 cells were preincubated with H2DCFDA (25 μm) for 30 minutes and then exposed to 10 μg/cm2 of crocidolite or zymosan for 3 hours. Representative histograms plot the relative green DCF fluorescence (FL1) within 15000 live cells (counts: numbers of events).

Mentions: In the fluorescence assay, NR8383 cells preincubated with H2DCFDA were exposed to crocidolite (1, 5, and 10 μg/cm2) or zymosan (10 μg/cm2) for a period of 3 hours. Higher concentrations of fibres or particles disrupted the flow cytometry analysis. Figure 2 shows typical histograms of DCF fluorescence in NR8383 cells exposed to 10 μg/cm2 of crocidolite or zymosan, and Table 2 details the corresponding mean fluorescence values. Compared to the control cells, an approximately 3-fold increase in fluorescence was detected in the zymosan-treated cells, but no significant change was detected in the crocidolite-treated cells (at all concentrations tested), indicating that only zymosan particles stimulated ROS production in NR8383 cells.


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

Guichard Y, Gaté L, Darne C, Bottin MC, Langlais C, Micillino JC, Goutet M, Julien S, Stéphane B - J Toxicol (2010)

ROS production in NR8383 analysed by flow cytometry. NR8383 cells were preincubated with H2DCFDA (25 μm) for 30 minutes and then exposed to 10 μg/cm2 of crocidolite or zymosan for 3 hours. Representative histograms plot the relative green DCF fluorescence (FL1) within 15000 live cells (counts: numbers of events).
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2901601&req=5

fig2: ROS production in NR8383 analysed by flow cytometry. NR8383 cells were preincubated with H2DCFDA (25 μm) for 30 minutes and then exposed to 10 μg/cm2 of crocidolite or zymosan for 3 hours. Representative histograms plot the relative green DCF fluorescence (FL1) within 15000 live cells (counts: numbers of events).
Mentions: In the fluorescence assay, NR8383 cells preincubated with H2DCFDA were exposed to crocidolite (1, 5, and 10 μg/cm2) or zymosan (10 μg/cm2) for a period of 3 hours. Higher concentrations of fibres or particles disrupted the flow cytometry analysis. Figure 2 shows typical histograms of DCF fluorescence in NR8383 cells exposed to 10 μg/cm2 of crocidolite or zymosan, and Table 2 details the corresponding mean fluorescence values. Compared to the control cells, an approximately 3-fold increase in fluorescence was detected in the zymosan-treated cells, but no significant change was detected in the crocidolite-treated cells (at all concentrations tested), indicating that only zymosan particles stimulated ROS production in NR8383 cells.

Bottom Line: Asbestos-induced mutagenicity in the lung may involve reactive oxygen/nitrogen species (ROS/RNS) released by alveolar macrophages.In particular, both particles induced up-regulation of iNOS expression in Big Blue, suggesting the formation of potentially genotoxic nitrogen species.NR8383 activation by crocidolite is probably insufficient to induce in vitro mutagenic events.

View Article: PubMed Central - PubMed

Affiliation: Institut National de Recherche et de Sécurité, rue du Morvan CS 60027, 54519 Vandoeuvre-Les-Nancy Cedex, France.

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.

No MeSH data available.


Related in: MedlinePlus