The Interaction of Environmentally Relevant Pollutants with Nuclear Hormone Receptors of European Flounder (Platichthys flesus)

Abstract

Nuclear hormone receptors (NHRs) are ligand-activated transcriptions factors which transduce the effects of various hormones as well as nutritional and other environmental signals. They thus function to maintain physiological homeostasis by integrating the tissue expression of specific target genes to regulate a wealth of biological processes including reproduction, development, metabolism and environmental adaptation. Mounting evidence indicates NHRs are the target of endocrine disrupting compounds (EDCs), exogenous chemicals, often of anthropogenic origin, which disrupt NHRs and thus the processes under their control. EDCs can interfere with NHR signalling by activating receptors (agonists), by inhibiting the actions of the receptor (antagonists), or by disrupting endogenous hormone synthesis, secretion, transport or metabolism. Much of the focus to date has been on the risk of EDCs to reproductive functions, via estrogen and androgen NHRs in humans, and also in aquatic organisms. However environmental pollutants also have the potential to interact with other NHRs, particularly in aquatic environments, and cause dysregulation of other critical physiological processes, including energy homeostasis, immune functions and the stress response. To address this possibility a reporter gene assay was developed, allowing the high-throughput screening of pollutants for their interactions with piscine NHRs with critical roles in energy homeostasis, stress reponse and immune functions, namely the peroxisome proliferator-activated receptors (PPARs) and corticosteroid receptors (CRs) from European plaice (Pleuronectes platessa) and European flounder (Platichthys flesus), respectively. Complementary DNA (cDNA) sequences encoding the ligand-binding domains of PPARs and CRs, critical for receptor-ligand interactions and receptor activation, were ligated to the DNA-binding domain (DBD) of the yeast Gal4 transcription activator protein to create experimental expression plasmid constructs. Co-transfection of these expression plasmids into the fathead minnow (FHM) cell line with an upstream-activating sequence (UAS)-firefly luciferase reporter gene plasmid increased luciferase expression in the presence of known PPAR and CR ligands. Several aquatic pollutants including pharmaceuticals, industrial by-products and biocides were tested for their potential to disrupt PPAR and CR functions by interacting with these receptors in an agonistic or antagonistic manner. Several fibrates, a group of pharmaceutical compounds used to treat dyslipidemia in humans by targeting the PPARs, were able to activate plaice Gal4-PPARα and Gal4-PPARβ in the reporter gene assay, indicative of an interaction with PPAR receptors in non-target species. Fibrates which did not activate Gal4-PPARα were able to inhibit the activation of Gal4-PPARα by the PPARα-specific agonist, Wy14643, suggesting differential effects of fibrates on human and flounder PPARs. In addition some metabolites of widespread phthalate ester pollutants were also agonists of the Gal4-PPARα and Gal4-PPARβ constructs. The Gal4-PPARγ construct was unresponsive to almost all the compounds tested, including the mammalian PPARγ agonist, rosiglitazone. The exception to this was the phthalate metabolite monobenzylphthalate, which induced a small increase in firefly luciferase in Gal4-PPARγ transfected cells. All of the above effects required concentrations of at least 10 µM, which are unlikely to be encountered in the aquatic environment. In contrast bis(tributyltin) oxide (TBTO), a notorious environmental pollutant, inhibited Gal4-PPARα and Gal4-CR constructs at concentrations as low as 1 nM and 100 nM, respectively. These concentrations are lower than those reported in aquatic environments, or in fish tissues, making TBTO a candidate endocrine disruptor in fish by inhibiting PPARα and CR signalling. A European flounder cDNA microarray was used to investigate the trasnscriptional responses of flounder hepatocytes to TBTO (10 nM) exposure. Exposure to TBTO and Wy14643, both alone and in combination, indicated a TBTO-driven downregulation of several potential PPARα-target genes with functions in the immune system, the proteasome, and lipid metabolism, although, based on mammalian comparisons, some potential PPARα-target genes were also upregulated, indicating differences in mammalian and fish PPAR-target genes or reflecting the complexity of organisms at a higher organisational level than cell-based assay systems. However, the microarray-based approach was useful in formulating further hypotheses about the effects of TBTO on PPARα signalling. Overall, these results indicate that exogenous chemicals entering the aquatic environment can interfere with NHRs with functions in energy homeostasis, immune functions and stress, in non-target organisms. The cell-based reporter gene assay is a useful tool for identifying potential endocrine disruptors which target PPARs and CRs and would be a useful method in a first tier testing approach, limiting the use of live animal models and enabling investigation into specific receptors which are targets of endocrine disrupting compounds. Although more work is required to confirm the physiological consequences of TBTO inhibition of PPARα, the results presented here indicate that organisms inhabiting TBTO-polluted environments may experience suppression of the immune system, an increase in non-functional or misfolded proteins through suppression of genes involved in the ubiquitin/proteasome system and a disruption in lipid homeostasis.