Environmental Chemicals Modulate Polar Bear (Ursus maritimus) Peroxisome Proliferator-Activated Receptor Gamma (PPARG) and Adipogenesis in Vitro.

We studied interactions between polar bear peroxisome proliferator-activated receptor gamma (pbPPARG) and selected compounds using a luciferase reporter assay and predictions through molecular docking. Furthermore, we studied adipogenesis by liver and adipose tissue extracts from a polar bear and three synthetic mixtures of contaminants in murine 3T3-L1 preadipocytes and polar bear adipose tissue-derived stem cells (pbASCs). PCB153 and p,p'-DDE antagonized pbPPARG, although their predicted receptor-ligand affinity was weak. PBDEs, tetrabromobisphenol A, and PCB170 had a weak agonistic effect on pbPPARG, while hexabromocyclododecane, bisphenol A, oxychlordane, and endosulfan were weak antagonists. pbPPARG-mediated luciferase activity was suppressed by synthetic contaminant mixtures reflecting levels measured in polar bear adipose tissue, as were transcript levels of PPARG and the PPARG target gene fatty acid binding protein 4 (FABP4) in pbASCs. Contaminant extracts from polar bear tissues enhanced triglyceride accumulation in murine 3T3-L1 cells and pbASCs, whereas triglyceride accumulation was not affected by the synthetic mixtures. Chemical characterization of extracts using nontarget methods revealed presence of exogenous compounds that have previously been reported to induce adipogenesis. These compounds included phthalates, tonalide, and nonylphenol. In conclusion, major legacy contaminants in polar bear adipose tissue exert antagonistic effects on PPARG, but adipogenesis by a mixture containing emerging compounds may be enhanced through PPARG or other pathways.

Environmental contaminants activate human and polar bear (Ursus maritimus) pregnane X receptors (PXR, NR1I2) differently.

BACKGROUND: Many persistent organic pollutants (POPs) accumulate readily in polar bears because of their position as apex predators in Arctic food webs. The pregnane X receptor (PXR, formally NR1I2, here proposed to be named promiscuous xenobiotic receptor) is a xenobiotic sensor that is directly involved in metabolizing pathways of a wide range of environmental contaminants. OBJECTIVES: In the present study, we comparably assess the ability of 51 selected pharmaceuticals, pesticides and emerging contaminants to activate PXRs from polar bears and humans using an in vitro luciferase reporter gene assay. RESULTS: We found that polar bear PXR is activated by a wide range of our test compounds (68%) but has a slightly more narrow ligand specificity than human PXR that was activated by 86% of the 51 test compounds. The majority of the agonists identified (70%) produces a stronger induction of the reporter gene via human PXR than via polar bear PXR, however with some notable and environmentally relevant exceptions. CONCLUSIONS: Due to the observed differences in activation of polar bear and human PXRs, exposure of each species to environmental agents is likely to induce biotransformation differently in the two species. Bioinformatics analyses and structural modeling studies suggest that amino acids that are not part of the ligand-binding domain and do not interact with the ligand can modulate receptor activation.

Designing green oxidation catalysts for purifying environmental waters.

We describe the synthesis, characterization, aqueous behavior, and catalytic activity of a new generation of Fe(III)-TAML (tetraamido macrocycle ligand) activators of peroxides (2), variants of [Fe{(OC)(2)(o,o'-NC(6)H(4)NCO)(2)CMe(2)}(OH(2))(-)] (2d), which have been designed to be especially suitable for purifying water of recalcitrant oxidizable pollutants. Activation of H(2)O(2) by 2 (k(I)) as a function of pH was analyzed via kinetic studies of Orange II bleaching. This was compared with the known behavior of the first generation of Fe(III)-TAMLs (1). Novel reactivity features impact the potential for oxidant activation for water purification by 2d and its aromatic ring-substituted dinitro (2e) and tetrachloro (2f) derivatives. Thus, the maximum activity for 2e occurs at pH 9, the closest yet to the EPA guidelines for drinking water (6.5-8.5), allowing 2e to rapidly activate H(2)O(2) at pH 7.7. In water, 2e has two axial water ligands with pK(a)'s of 8.4 and 10.0 (25 degrees C). The former is the lowest for all Fe(III)-TAMLs developed to date and is key to 2e's exceptional catalytic activity in neutral and slightly basic solutions. Below pH 7, 2d was found to be quite sensitive to demetalation in phosphate buffers. This was overcome by iterative design to give 2e (hydrolysis rate 2d > 100 x 2e). Mechanistic studies highlight 2e's increased stability by establishing that to demetalate 2e at a comparable rate to which H(2)PO(4)(-) demetalates 2d, H(3)PO(4) is required. A critical criterion for green catalysts for water purification is the avoidance of endocrine disruptors, which can impair aquatic life. Fe(III)-TAMLs do not alter transcription mediated by mammalian thyroid, androgen, or estrogen hormone receptors, suggesting that 2 do not bind to the receptors and reducing concerns that the catalysts might have endocrine disrupting activity.

Phosphorylation of steroidogenic factor 1 is mediated by cyclin-dependent kinase 7.

The nuclear receptor steroidogenic factor-1 (SF1) is critical for development and function of steroidogenic tissues. Posttranslational modifications are known to influence the transcriptional capacity of SF1, and it was previously demonstrated that serine 203 is phosphorylated. In this paper we report that serine 203 is phosphorylated by a cyclin-dependent kinase 7 (CDK7)-mediated process. As part of the CDK-activating kinase complex, CDK7 is a component of the basal transcription factor TFIIH, and phosphorylation of SF1 as well as SF1-dependent transcription was clearly reduced in cells carrying a mutation that renders the CDK-activating kinase complex unable to interact with the TFIIH core. Coimmunoprecipitation analyses revealed that SF1 and CDK7 reside in the same complex, and kinase assays demonstrated that immunoprecipitated CDK7 and purified TFIIH phosphorylate SF1 in vitro. The CDK inhibitor roscovitine blocked phosphorylation of SF1, and an inactive form of CDK7 repressed the phosphorylation level and the transactivation capacity of SF1. Structural studies have identified phosphoinositides as potential ligands for SF1. Interestingly, we found that mutations designed to block phospholipid binding dramatically decreased the level of SF1 phosphorylation. Together our results suggest a connection between ligand occupation and phosphorylation and association with the basic transcriptional machinery, indicating an intricate regulation of SF1 transactivation.

MRNA expression of genes regulating lipid metabolism in ringed seals (Pusa hispida) from differently polluted areas.

There is a growing concern about the ability of persistent organic pollutants (POPs) to influence lipid metabolism. Although POPs are found at high concentrations in some populations of marine mammals, for example in the ringed seal (Pusa hispida) from the Baltic Sea, little is known about the effects of POPs on their lipid metabolism. An optimal regulation of lipid metabolism is crucial for ringed seals during the fasting/molting season. This is a physiologically stressful period, during which they rely on the energy stored in their fat reserves. The mRNA expression levels for seven genes involved in lipid metabolism were analyzed in liver and/or blubber tissue from molting ringed seals from the polluted Baltic Sea and a less polluted reference location, Svalbard (Norway). mRNA expression of genes encoding peroxisome proliferator-activated receptors (PPAR) α and γ and their target genes acyl-coenzyme A oxidase 1 (ACOX1) and cluster of differentiation 36 (CD36) were analyzed in liver. mRNA expression level of genes encoding PPARß, PPARγ and their target genes encoding fatty acid binding protein 4 (FABP4) and adiponectin (ADIPOQ) were measured in inner and middle blubber layers. In addition, we evaluated the influence of molting status on hepatic mRNA expression of genes encoding PPARs and their target genes in ringed seals from Svalbard. Our results show higher mRNA expression of genes encoding hepatic PPARγ and adipose PPARß, FABP4, and ADIPOQ in the Baltic seals compared to the Svalbard seals. A positive relationship between mRNA expressions of genes encoding hepatic PPARγ, adipose FABP4, adipose ADIPOQ and ΣPOP concentrations was observed. These findings suggest that lipid metabolism may be affected by contaminant exposure in the Baltic population. mRNA expression of genes encoding PPARß, PPARγ, FABP4 and ADIPOQ were similar between the mid and inner adipose layer. Hepatic mRNA expression of genes encoding PPARα and PPARγ was higher in the pre-molting individuals compared to the molting ones highlighting differential regulation of these metabolic sensors through the molting period.

A characterization of the ZFL cell line and primary hepatocytes as in vitro liver cell models for the zebrafish (Danio rerio).

The zebrafish (Danio rerio) is a widely used model species in biomedical research. The ZFL cell line, established from zebrafish liver, and freshly isolated primary hepatocytes from zebrafish have been used in several toxicological studies. However, no previous report has compared and characterized these two systems at the level of gene expression. The aim of this study was to evaluate the ZFL cell line in comparison to primary hepatocytes as in vitro models for studying effects of environmental contaminants in zebrafish liver. Using quantitative real-time PCR, the basal level and transcriptional induction potential of key genes involved in toxic responses in the ZFL cell line, primary hepatocytes and whole liver from zebrafish were compared. The study showed that the ZFL cells have lower levels of mRNA of most selected genes compared to zebrafish liver. The induced gene transcription following exposure to ligand was much lower in ZFL cells compared to zebrafish primary hepatocytes at the doses tested. Importantly, oestrogen receptor and vitellogenin genes showed low basal transcription and no induction response in the ZFL cell line. In conclusion, it appears that primary hepatocytes are well suited for studying environmental contaminants including xenoestrogens, but may show large sex-dependent differences in gene transcription. The ZFL cell line shows potential in toxicological studies involving the aryl hydrocarbon receptor pathway. However, low potential for transcriptional induction of genes in general should be expected, especially notable when studying estrogenic responses.

Functional roles of protein kinase A (PKA) and exchange protein directly activated by 3',5'-cyclic adenosine 5'-monophosphate (cAMP) 2 (EPAC2) in cAMP-mediated actions in adrenocortical cells.

In the adrenal cortex, the biosynthesis of steroid hormones is controlled by the pituitary-derived hormone ACTH. The functions of ACTH are principally relayed by activating cAMP-dependent signaling pathways leading to the induction of genes encoding enzymes involved in the conversion of cholesterol to steroid hormones. Previously, protein kinase A (PKA) was thought to be the only direct effector of cAMP. However, the discovery of the cAMP sensors, exchange proteins directly activated by cAMP (EPAC1 and 2), has led to a reevaluation of this assumption. In the present study, we demonstrate the occurrence of the EPAC2 splicing variant EPAC2B in adrenocortical cancer cells. Immunocytochemistry demonstrated that EPAC2B is localized predominantly in the nucleus. EPAC2B is functional because it activates Rap1 in these cells. Using the cAMP analogs 8-p-chlorophenylthio-2'-O-methyl-cAMP and N6-benzoyl-cAMP, which specifically activate EPAC1/2 and PKA, respectively, we evaluated the contribution of these factors in steroid hormone production, cell morphology, actin reorganization, and migration. We demonstrate that the expression of cAMP-inducible factors involved in steroidogenesis (steroidogenic acute regulatory protein, cytochrome P450 11A1 and 17, and nerve growth factor-induced clone B) and the cAMP-induced biosynthesis of steroid hormones (cortisol and aldosterone) are mediated by PKA and not by EPAC2B. In contrast, both PKA- and EPAC-specific cAMP analogs induced cell rounding, loss of stress fibers, and blocked migration. Taken together, the presented data confirm PKA as the central cAMP mediator in steroid hormone production and reveal the involvement of EPAC2B in cAMP-induced effects on cytoskeleton integrity and cell migration.

Effects of water regime on archaeal community composition in Arctic soils.

Effects of water regime on archaeal communities in Arctic soils from Spitsbergen were studied using denaturing gradient gel electrophoresis (DGGE) of amplified 16S rRNA genes, with subsequent sequencing of amplicons and ordination analysis of binary DGGE data. Samples with major differences in soil water regime showed significant differences in their archaeal community profiles. Methanomicrobiales, Methanobacteriaceae and Methanosaeta were detectable only in environments that were wet during most of the growth season, while a novel euryarchaeotal cluster was detected only in less reduced solifluction material. Group 1.3b of Crenarchaeota had a high relative abundance within the archaeal community in a wide range of wet soils. Along a natural soil moisture gradient, changes in archaeal community composition were observed only in upper soil layers. The results indicated that members of Methanomicrobiales were relatively tolerant to soil aeration. Differences in archaeal community composition associated with soil water regime were predominant over regional and seasonal variation, and over differences between individual wetlands. The results suggest that the observed 'on-off switch' mechanism of soil hydrology for large-scale variations in methane emissions from northern wetlands is at least partly caused by differences in the community structure of organisms involved in methane production.