Postersot2013_niehof

Cytochrome P450 monooxygenases expression in human
epithelial lung cell lines
Abbreviation
Concentration
Receptor
Calu-3 cells, CYP gene expression
Fraunhofer Institute for Toxicology and Experimental Medicine, Hannover, Germany Table 1: Various CYP inducers used for the treatment of Calu-3 and A549 cells. INTRODUCTION
2C(8-19)
The pulmonary epithelium is the first barrier for airborne Basal expression of CYP1B1, CYP1A1, CYP2J2, CYP2E1, monooxygenases (CYP) participate in metabolic inacti- CYP3A7 was detected in both cell lines (Fig. 1, Fig, 2), vation of xenobiotics. Beyond that some compounds which is consistent with CYP expression in the human Ct-values (threshold cycles), real time RT-qPCR
2C(8-19)
require enzymatic activation to exert their toxic effects or lung. Furthermore, potential CYP inducers were analyzed their desirable functions. The bronchiale epithelial cell line (Table 1). Omeprazole acts on aryl hydrocarbon receptor Figure 1: CYP expression in untreated Calu-3 cells 48 h after seeding Calu-3 and the type II-like pulmonary epithelial cell line (AhR) activation and induced CYP1A1 and CYP1B1 in (n = 3). Gene expression was analysed by real time RT-qPCR. Ct A549 serve as cell culture models for the human both cell lines (Table 2, 3). Rifampicin acts on pregnane x (threshold cycle) reflects the cycle number at which the fluorescence generated within a reaction crosses the threshold.
respiratory epithelium. So far, there is little information receptor (PXR) and phenobarbitale acts predominantly on regarding their metabolic properties especially for Calu-3 constitutive androstane receptor (CAR), however, both Table 2: Fold change in CYP gene expression in Calu-3 cells 24 h after cells. In this study we want to further characterize A549 receptors are not expressed in the lung. Accordingly, both treatment with different inducers. Fold changes > 2.5 with p<0.05 (n = 3) are A549 cells, CYP gene expression
and Calu-3 cells regarding their basal and inducible CYP agents did not induce any CYP in these cells (Table 2, indicated in bold (green boxes). Analysis was performed with the opensource software REST 2009 (TU Munich/Qiagen) using multiple different reference genes, respectively. nd = not detected.
members of the CYP3A family, mainly CYP3A7 in Calu-3 cells (Table 2), and CYP3A5 and CYP3A7 in A549 cells MATERIAL AND METHODS
(Table 3). CITCO is known to act as a CAR agonist and is normally used to induce CYP2B6/7. However, it is a potent inducer of CYP1B1 in Calu-3 cells (Table 2), and of Cells were cultured according to ATCC conditions. After CYP1A1 and CYP1B1 in A549 cells (Table 3).
2C(8-19)
24 h cells were treated with the respective inducers or 2C(8-19)
left untreated. Again after 24 h cells were harvested and RNA was isolated. Subsequently, CYP expression was CONCLUSION
quantitative polymerase chain reaction (RT-qPCR) with Ct-values (threshold cycles), real time RT-qPCR
the Light Cycler SystemTM (Roche) or ABI Prism® 7500 Calu-3 cells and A549 cells express a broad range of Table 3: Fold change in CYP gene expression in A549 CYPs with preserved inducibility and are valuable models Figure 2: CYP expression in untreated A549 cells 48 h after seeding treatment with different inducers. Fold changes > 2.5 with p<0.05 (n = 3) are (n = 3). Gene expression was analysed by real time RT-qPCR. Ct indicated in bold (green boxes). Analysis was performed with the open of the airway epithelial barrier for metabolic in vitro (threshold cycle) reflects the cycle number at which the fluorescence source software REST 2009 (TU Munich/Qiagen) using multiple different generated within a reaction crosses the threshold.
reference genes, respectively. nd = not detected.
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Source: http://www.item.fraunhofer.de/content/dam/item/de/neuedokumente/Veranstaltungen/SOT13/HandoutSOT2013_Niehof.pdf