Congrès annuel de la SFT - Brest, 13 et 14 octobre 2005
"Allergies et Toxiques " Macrophage activation for assaying fiber biopersistence. Hermine DIKA NGUEA (1), Aymon de REYDELLET (2), Patrice LEHUEDE (3), (1) Laboratoire de Bactériologie Virologie, Faculté de Médecine, Université Henri Poincaré -Nancy 1, 54501 Vandoeuvre-lès-Nancy, France. Toxicity of man made mineral fibers (MMMF) is usually evaluated by testing biopersistence in rodents. A cellular model would be convenient to reduce, refine and replace animal models. As a matter of fact, macrophages are the cells of choice as MMMF toxicity is the consequence of fibers interactions with alveolar macrophages. Indeed, we developed an in vitro assay to investigate MMMF biodegradation. We activated U-937 monocytes with several factors such as Escherichia coli and Curtobacterium sp extracts, interleukine 6 (IL6), lipopolysaccharide (LPS), and tumor necrosis factor a (TNFa). Fiber surface integrity was checked by scanning electron microscopy. We assessed MMMF chemical biodegradation by performing induction coupled plasma atomic emission spectrometry to measure silicon release in extracellular medium. RNA extraction from E. coli -activated U-937 monocytes and native U-937 monocytes in presence of MMMF allowed us to measure gene expression with 20 K-microarrays. E. coli supernatant induced U-937 monocytes differentiation into active macrophages as shown by light microscopy. Tested high alumina containing fibers displayed surface degradation as evidenced by surface erosion and holes formation when incubated with E. coli - or Curtobacterium sp - activated U-937 monocytes, whereas natural mineral fibers, namely crocidolite, did not show any surface erosion. Physical degradation of fibers was dramatically increased when a combination of Curtobacterium sp and IL-6 or TNF-a was used to activate U-937 monocytes. In our assay, silicon release was dose and time dependent. The comparison of gene expression showed some relevant genes with differential expression. One of them was related to oxidative stress and involved in hydrogen peroxide formation. Indeed, tested fiber shown surface erosion when incubated with hydrogen peroxide. Thus, using various bacterial extracts as well as various cytokines, we mimic physiological activation of cultured macrophages leading to physical and chemical degradation of MMMF.
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