A teleost in vitro reporter gene assay to screen for agonists of the peroxisome proliferator-activated receptors

Abstract

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.

Transcriptional activities of medaka Oryzias latipes peroxisome proliferator-activated receptors and their gene expression profiles at different temperatures

“…It has been claimed that PPARs in mammals and amphibians form heterodimers with retinoid X receptor, which is activated by retinoic acid [8]. Furthermore, ligand-dependent transcriptional activities of rainbow trout PPAR are enhanced in the presence of 9-cis-retinoic acid [24]. However, further investigation is required to confirm whether medaka PPARb and c also require another type of receptor to exert their activities.…”

“…Furthermore, full-length nucleotide sequences have been reported for PPARa in thicklip grey mullet Chelon labrosus [20] and PPARb in goldfish Carassius auratus [21], along with partial nucleotide sequences of PPARc in Atlantic salmon Salmo salar [22,23] and PPARa and b in rainbow trout Onchorhyncus mykiss [24]. Interestingly, torafugu contains 2 types of PPARa [15,16], and zebrafish contains 2 types of PPARb [14].…”

Cloning and transcription of nuclear receptors and other toxicologically relevant genes, and exposure biomarkers in European hake (Merluccius merluccius) after the Prestige oil spill

“…These amino acid residues include the P-and D-boxes that interact directly with the response elements of regulated genes. Teleost PPARαs have been proved to heterodimerize with RXR to bind PPREs of mammalian and piscine genes (Boukouvala et al, 2004;Leaver et al, 2005;Liu et al, 2005;Kondo et al, 2007). This suggests that M. merluccius PPARα may bind with similar affinity to the same type of PPRE-driven genes.…”

“…The retinoid X receptor (RXR) is the obligate heterodimerization partner of PPAR, allowing binding to peroxisome proliferator response elements (PPRE) that regulate target genes involved in peroxisome proliferation (Bardot et al, 1993;Liu et al, 2005). Three different PPAR subtypes (α, β and γ) encoded by separate genes and showing different tissue distribution patterns have been described in different vertebrate species including fish Salmo salar (Andersen et al, 2000;Leaver et al, 2007), Sparus aurata and Pleuronectes platessa (Leaver et al, 2005), Danio rerio (RobinsonRechavi et al, 2001), Fugu rubripes (Maglich et al, 2003;Kondo et al, 2007), Dicentrarchus labrax (Boukouvala et al, 2004), Salmo trutta (Batista-Pinto et al, 2005), Chelon labrosus (Raingeard et al, 2006(Raingeard et al, , 2009, Carassius auratus (Mimeault et al, 2006), and Tetraodon nigroviridis (Metpally et al, 2007).…”

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“…This could result in binding to specific peroxisome proliferator response elements (PPRE) in the promoter regions of numerous target genes (Liu, Moon, Metcalfe, Lee, & Trudeau, 2005). Three kinds of PPAR isotypes PPARα, PPARβ and PPARγ have been identified in fish.…”