Posters

      In Vitro Exposure Of Organotypical 3D Epithelial Tissues To Cigarette Smoke As A Potential Alternative To Rodent Inhalation Studies

      Weisensee, D.; Kurkowsky, B.; Hebestreit, M.; Wagner, S.; Schueller, J.
      Conference date
      Mar 6, 2011
      Conference name
      Society of Toxicology (SOT) 2011
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      Topic
      Summary

      Airway epithelium is the initial barrier affected by toxic gases or particles in the atmosphere, such as cigarette smoke (CS), which induces, among other things, inflammatory processes that can lead to COPD and tissue remodeling. Here we describe an in vitro test system which examines the toxicological effects of repeated CS exposure on organotypical 3D epithelial tissues derived from differentiated human airway epithelial cells. Tissues were exposed at the air liquid interface to either fresh air or to mainstream smoke from the reference cigarette 3R4F in the Vitrocell® system for 1-3 cycles of 20 min each to a CS concentration of 20% (12-well format) or for 7, 14, 21, and 28 min to a CS concentration of 10%, 15%, or 20% (24-well format). Cell viability, as determined by resazurin reduction 24h after CS exposure, was not adversely affected. For morphological characterization of the reconstituted tissue H&E, AB-PAS, and immunohistochemical p63 (basal cells) stainings were performed. These stainings showed intact basal and pseudostratified epithelium with mucociliary phenotype similar to in vivo proximal airway epithelium. Gene expression of hemoxygenase-1 (HO-1), cytochrome p450 1A1 (CYP1A1), heat shock 70kDa protein A6 (HPSA6), and Transforming Growth Factor ß1 (TGF ß1) was strongly up-regulated (QRT-PCR) under all conditions. Matrix metalloproteinase-1 (mmp-1), an enzyme possibly involved in the pathogenesis of COPD, was dose-dependently increased (ELISA measurement). Results for HO-1 and CYP1A1 and MMP-1 are comparable to in vivo data from previous rodent inhalation studies which showed increased HO-1 and CYP1A gene expression in the lung and increased mmp-1 secretion in the lavage fluid of rats exposed to CS for up to 1 month. The in vitro study design presented here serves as a potential alternative to rodent inhalation studies for the investigation of the toxicology of CS and supports the 3R strategy of refinement, reduction, and replacement of animal experimentation. 

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