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Establishment of pathway-specific gene expression analysis using the example of the paraquat action on human lung epithelial cells

: Niehof, Monika; Augustin, Christian; Hansen, Tanja

Naunyn-Schmiedebergs archives of pharmacology 387 (2014), Supplement 1, S.S71, A284
ISSN: 0028-1298
ISSN: 1432-1912
Deutsche Gesellschaft für Experimentelle und Klinische Pharmakologie und Toxikologie (Annual Meeting) <80, 2014, Hannover>
Zeitschriftenaufsatz, Konferenzbeitrag
Fraunhofer ITEM ()

The aim of the present study was the establishment of "Pathway Arrays" (RT2 Profiler PCR Arrays, SABiosciences) with which the expression of a variety of genes can be assessed simultaneously. These arrays combine real-time PCR performance and the ability of microarrays to detect the expression of many genes simultaneously. Paraquat, an herbicide, was used as test substance. It evolves its toxic effects by forming reactive oxygen species (ROS). ROS can lead to the activation of transcription factors, which regulate the transcription of genes relevant for inflammatory response, cell growth, differentiation or apoptosis. Paraquat is preferably accumulated in alveolar epithelial cells type II. Therefore, the human alveolar epithelial cell line A549 was used in this study. Due to the effect of Paraquat on inflammatory response an array for the NFkB-Signaling-Pathway was used for establishment (84 genes). About 40% of the tested genes were significantly regulated. A classification of the genes into functions showed that, as expected, a great number of cytokines, being involved in inflammatory response, were regulated (e.g. IL8, IL1A, IL1R1, TNF, TLR4, CSF2). Furthermore, it could be shown that Paraquat regulate different transcription factors (e.g. EGR1, STAT1, JUN, FOS), which could take part in the regulation of cytokine expression. To validate the array results 15% of the genes (13 genes, "up", "down" and "unregulated") were chosen. The expression of these genes was completely confirmed via real-time RTqPCR with the Lightcycler™ (Roche). The results of this study show that the establishment of these arrays was successful and that they yield a high precision and quality. The "Pathway-Arrays" take much less time compared to the analysis of individual genes and represent a fast knowledge-based alternative to genome-wide gene expression analysis in toxicogenomic studies.