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A teleost in vitro reporter gene assay to screen for agonists of the peroxisome proliferator‐activated receptors
Author(s) -
Liu Gele,
Moon Thomas W.,
Metcalfe Chris D.,
Lee Lucy E. J.,
Trudeau Vance L.
Publication year - 2005
Publication title -
environmental toxicology and chemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.1
H-Index - 171
eISSN - 1552-8618
pISSN - 0730-7268
DOI - 10.1897/04-405r.1
Subject(s) - bezafibrate , reporter gene , luciferase , peroxisome proliferator activated receptor , retinoid x receptor , biology , microbiology and biotechnology , receptor , transfection , biochemistry , chemistry , nuclear receptor , gene expression , gene , transcription factor
Several contaminants detected in aquatic ecosystems are agonists of peroxisome proliferator‐activated receptors (PPARs). Peroxisome proliferator‐activated receptors interact with the retinoid × receptor (RXR) to activate the transcription of genes that control a variety of physiological functions. We cloned and sequenced partial cDNA fragments of rainbow trout ( Oncorhynchus mykiss ) PPARα and PPARβ from rainbow trout (rt) gill‐W1 cells, a cell line derived from rainbow trout gills; predicted amino acid identities are 77% and 82% compared with their respective human homologs and 83 to 88% and 91 to 98% identical to fish homologs. A reporter gene assay was developed by transfecting rt‐gill‐W1 cells with a reporter gene construct containing the peroxisome proliferator response element (PPRE) of the rat liver 3‐ketoacyl‐CoA thiolase B (TB) gene, which drives luciferase expression. Agonists of both PPARα (WY14,643 and gemfibrozil) and PPARβ (bezafibrate) induced luciferase activity, while rosiglitazone, a PPARγ agonist, was not effective. The fibrate drug, bezafibrate increased luciferase activity in a dose‐dependent manner, but addition of 50 nM 9‐ cis ‐retinoic acid to the transfected rt‐gill‐W1 cell culture maximized the sensitivity of the assay so that bezafibrate could be detected at concentrations as low as 6 nM. Extracts from treated domestic wastewater containing fibrate drugs induced luciferase activity in the transfected gill cells. This in vitro reporter gene assay shows promise as a rapid and sensitive technique for screening environmental samples for PPAR‐active substances.