Bioactivity screening and mass spectrometric confirmation for the detection of PPAR-delta agonists that increase type 1 muscle fibres
| Autor: | Marco Blokland, Martien L. Essers, Astrid R. M. Hamers, Toine F.H. Bovee, Sander Kersten, Michel W. F. Nielen, H.H. Heskamp, Leendert A. van Ginkel |
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| Jazyk: | angličtina |
| Rok vydání: | 2014 |
| Předmět: |
Novel Foods & Agrochains
receptors Novel Foods & Agroketens Biochemistry Phenoxyacetates Polymerase Chain Reaction Mass Spectrometry BU Veterinary Drugs Analytical Chemistry Voeding Metabolisme en Genomica Limit of Detection fat Bioassay BU Toxicology Novel Foods & Agrochains PPAR delta Directie Receptor Expression vector Chemistry BU Toxicology Transfection Hep G2 Cells Organische Chemie Metabolism and Genomics macrophages Dose–response relationship Muscle Fibers Slow-Twitch BU Toxicologie Novel Foods & Agroketens Metabolisme en Genomica Nutrition Metabolism and Genomics Biological Assay acid BU Toxicologie human skeletal-muscle BU Dierbehandelingsmiddelen GW501516 Voeding Cell Line Tumor expression medicine Angiopoietin-Like Protein 4 Humans Luciferase RNA Messenger gene VLAG Nutrition Dose-Response Relationship Drug Organic Chemistry medicine.disease Molecular biology Actins Thiazoles Cell culture cells gamma Angiopoietins |
| Zdroj: | Analytical and Bioanalytical Chemistry 406 (2014) Analytical and Bioanalytical Chemistry, 406, 705-713 |
| ISSN: | 1618-2642 |
| Popis: | Sensitive and robust bioassays able to detect nuclear receptor activation are very useful for veterinary and doping control, pharmaceutical industry and environmental scientists. Here, we used bioassays based on human leukemic monocyte lymphoma U937 and human liver hepatocellular carcinoma HepG2 cell lines to detect the ligand-induced activation of the peroxisome proliferator-activated receptor delta (PPARδ). Exposure of U937 cells to the PPARδ agonist GW501516 resulted in a marked increase in mRNA expression of the PPARδ target gene Angptl4 which was quantified by qRT-PCR analysis. Exposure of HepG2 cells transiently transfected with a PPARδ expression plasmid and a PPAR-response element-driven luciferase reporter plasmid to PPARδ agonists GW501516, GW610742 and L-165041 resulted in clear dose–response curves. Although the qRT-PCR resulted in higher fold inductions, the luciferase assay with transfected HepG2 cells is cheaper and quicker and about ten times more sensitive to GW501516 compared to analysis of Angptl4 mRNA expression in U937 cells by qRT-PCR. The HepG2-based luciferase assay was therefore used to screen GW501516-spiked supplements and feed and water samples. After liquid extraction and clean-up by solid phase extraction using a weak anion exchange column, extracts were screened in the HepG2 bioassay followed by confirmation with a newly developed UPLC-MS/MS method, using two transitions for each compound, i.e., for GW501516, 454.07>188.15 (collision energy (CE) 46 V) and 454.07>257.08 (CE 30 V); for GW610742, 472.07>206.2 (CE 48 V) and 472.07>275.08 (CE 30 V); and for L-165041, 401.2>193.15 (CE 26 V) and 401.2>343.2 (CE 20 V). |
| Databáze: | OpenAIRE |
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