Endocrine disrupting chemicals (EDCs) are exogenous compounds that affect the endocrine system, disrupt the hormonal balance and consequently cause the adverse health effects in humans. These compounds persist and accumulate in the environment and can easily gain entry to the food chain. Peroxisome proliferator-activated receptor gamma (PPARγ) and its heterodimeric partner retinoid X receptor alpha (RXRα) are very often targets of EDCs. PPARγ play crucial role in lipid and glucose metabolism and disturbing of PPARγ signaling leads to serious diseases such as obesity, diabetes, insulin resistance and cardiovascular diseases. Therefore, it is of topical interest to develop reliable, high throughput in vitro system allowing screening and identification of compounds affecting PPARγ activity. In the current work, we developed the unique stably transfected human reporter cell line T24/83-PPARgamma for the assessment of PPARγ transcriptional activity. Reporter cell line was prepared by transfecting the human bladder carcinoma cell line T24/83 with reporter plasmid pNL2.1[Nluc/Hygro] containing three copies of PPARγ response element, coupled with minimal promoter. Design of response element was based on the sequences of promoter region of human acyl-CoA-oxidase. In the cells treated with PPARγ ligand 15-deoxy-δ12,14-prostaglandin J2 and prostaglandin D2 for 24 h, luciferase activity ranged from 40-fold to 50-fold and from 70-fold to 80-fold, respectively (RLU 104105). Application of PPARγ selective antagonist T0070907 resulted in inhibition of luciferase activity, indicating specific response of T24/83-PPARgamma cell line. The inducibility of luciferase activity remained unaffected after cryopreservation and even after prolonged cultivation for more than 2 months. Based on these results, the novel human reporter cell line T24/83-PPARgamma can be considered as a potential tool for screening of compounds affecting PPARγ activity, applicable in various toxicological and environmental studies.
20 May 2017 - 23 May 2017