A teleost in vitro reporter gene assay to screen for agonists of the peroxisome proliferator-activated receptors.
Several contaminants detected in aquatic ecosystems are agonists of peroxisome proliferator-activated receptors (PPARs). Peroxisome proliferator-activated receptors interact with the retinoid X 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) PPARalpha and PPARbeta 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 PPARalpha (WY14,643 and gemfibrozil) and PPARbeta (bezafibrate) induced luciferase activity, while rosiglitazone, a PPARgamma 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.
Liu
Gele
G
Centre for Advanced Research in Environmental Genomics and Department of Biology, University of Ottawa, Ottawa, Ontario K1N 6N5, Canada.
Moon
Thomas W
TW
Metcalfe
Chris D
CD
Lee
Lucy E J
LE
Trudeau
Vance L
VL
D000102:Acetyl-CoA C-Acyltransferase / Q000235:genetics; D000077556:Alitretinoin; D020533:Angiogenesis Inhibitors / Q000494:pharmacology; D000818:Animals; D001629:Bezafibrate / Q000494:pharmacology; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004784:Environmental Monitoring / Q000379:methods*; D015248:Gemfibrozil / Q000494:pharmacology; D017930:Genes, Reporter; D006801:Humans; D000960:Hypolipidemic Agents / Q000494:pharmacology; D066298:In Vitro Techniques; D008099:Liver / Q000201:enzymology; D008156:Luciferases / Q000378:metabolism; D017686:Oncorhynchus mykiss; D047493:PPAR alpha / Q000235:genetics; D048068:PPAR-beta / Q000235:genetics; D047492:Peroxisome Proliferator-Activated Receptors / Q000819:agonists*; D020025:Peroxisome Proliferators / Q000494:pharmacology; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011743:Pyrimidines / Q000494:pharmacology; D051381:Rats; D047488:Retinoid X Receptors / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection; D014212:Tretinoin / Q000494:pharmacology; D014874:Water Pollutants, Chemical / Q000032:analysis
D020533:Angiogenesis Inhibitors; D018076:DNA, Complementary; D000960:Hypolipidemic Agents; D047493:PPAR alpha; D048068:PPAR-beta; D047492:Peroxisome Proliferator-Activated Receptors; D020025:Peroxisome Proliferators; D011743:Pyrimidines; D047488:Retinoid X Receptors; D014874:Water Pollutants, Chemical; D000077556:Alitretinoin; D014212:Tretinoin; C006253:pirinixic acid; D004247:DNA; D008156:Luciferases; D000102:Acetyl-CoA C-Acyltransferase; D015248:Gemfibrozil; D001629:Bezafibrate