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

Yves Guichard,Binet Stéphane,Cristina Langlais,Schmit Julien,Marie-Claire Bottin,Laurent Gaté,Christian Darne,Michèle Goutet,Jean-Claude Micillino

doi:10.1155/2010/323828

Yves Guichard, Binet Stéphane + Show 7 more

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https://doi.org/10.1155/2010/323828

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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.

Highlights

Asbestos forms a group of naturally occurring mineral fibres that are associated with the development of both malignant and nonmalignant diseases of the lung and pleura [1]
Big Blue Rat2 embryonic fibroblast (Stratagene, La jolla, CA) and alveolar macrophage-derived NR8383 (American Type Culture Collection, Manassas, VA) cell lines were cultured at 37◦C, 5% CO2, in a complete medium (Dulbecco’s modified Eagle’s medium, Invitrogen, France) containing 10% foetal bovine serum (Dutscher, France), 2 mM L-glutamine (Invitrogen), and antibiotics (50 units/mL Penicillin, 50 μg/mL Steptomycin, Invitrogen)
When NR8383 cells were exposed to zymosan concentrations of 7.5, 15, and 30 μg/cm2, a dosedependant increase in luminescence was observed, indicating superoxide production in stimulated cells

Summary

Introduction

Asbestos forms a group of naturally occurring mineral fibres (defined as having a ≥3 : 1 length to diameter ratio) that are associated with the development of both malignant (cancer, mesothelioma) and nonmalignant (asbestosis) diseases of the lung and pleura [1]. Mutagenic assays suitable for detecting either large deletion or homologous recombination have been used to show the mutagenic potential of various fibre types They suggest that direct exposure of cells to asbestos induces major deletions rather point mutations [5, 6]. These genotoxicity data may not reflect every possible effect of in vivo exposure, indirect genotoxicity, which is related to the production of DNA reactive radicals (ROS or RNS) via secondary mechanisms [7]. Several studies using transgenic mutational assays (detecting in vivo point mutation but not clastogenic effects) in fibre genotoxicity testing have reported gene mutations induced by asbestos fibres [8,9,10,11].

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