Effectiveness of the in vitro Comet assay in the determination of cigarette smoke effects in human lung epithelial cells in conjunction with an air-liquid interface exposure system


Authored by  S Weber, M Hebestreit, T Wilms, B Kurkowsky, T Meisgen

Presented at International Comet Assay Workshop (ICAW)     

Abstract

The alkaline single cell gel electrophoresis assay (comet assay) is a quick and elegant tool to determine the genotoxic activity of different agents. To demonstrate the effectiveness of the comet assay as a tool for in vitro assessment, cigarette-smoke-induced DNA damage was determined in human lung epithelial cells (BEAS-2B and A549). To quantify the effectiveness of the comet assay, reproducibility and repeatability were determined. To improve the exposure system and to achieve more realistic exposure conditions, the novel air-liquid interface exposure system Vitrocell 24® was adapted to simulate exposure conditions in inhalation studies. Vitrocell 24® consists of a dilution system, an exposure chamber with 24 Microwell fittings for higher throughput, and a smoking robot. By growing the human lung epithelial cells on the Microwell fittings, a more organotypical differentiation was induced. BEAS-2B and A549 cells were exposed to different flows of freshly generated, diluted mainstream smoke from the reference cigarette 3R4F. A clear dose-dependent increase in DNA-damage, expressed as tail intensity, was obtained in all experiments for both cell lines (p ≤ 0.001), with A549 cells demonstrating a higher resistance to genotoxic insults than BEAS-2B cells. Reproducibility and repeatability were acceptable, with a relative standard deviation of approximately 25%. The study shows that the in vitro comet assay in combination with the Vitrocell 24® air-liquid exposure system is able to detect cigarette-smoke-induced DNA damage in a reproducible and repeatable manner in human lung epithelial cells. 

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