The G Protein-Coupled Estrogen Receptor Agonist G-1 Inhibits Nuclear Estrogen Receptor Activity and Stimulates Novel Phosphoproteomic Signatures.

Estrogen exerts cellular effects through both nuclear (ESR1 and ESR2) and membrane-bound estrogen receptors (G-protein coupled estrogen receptor, GPER); however, it is unclear if they act independently or engage in crosstalk to influence hormonal responses. To investigate each receptor's role in proliferation, transcriptional activation, and protein phosphorylation in breast cancer cells (MCF-7), we employed selective agonists for ESR1 propyl-pyrazole-triol (PPT), ESR2 diarylpropionitrile (DPN), and GPER (G-1) and also determined the impact of xenoestrogens bisphenol-A (BPA) and genistein on these effects. As anticipated, 17ß-estradiol (E2), PPT, DPN, BPA, and genistein each enhanced proliferation and activation of an ERE-driven reporter gene whereas G-1 had no significant impact. However, G-1 significantly reduced E2-, PPT-, DPN-, BPA-, and genistein-induced proliferation and ERE activation at doses greater than 500 nM indicating that G-1 mediated inhibition is not ESR isotype specific. As membrane receptors initiate cascades of phosphorylation events, we performed a global phosphoproteomic analysis on cells exposed to E2 or G-1 to identify potential targets of receptor crosstalk via downstream protein phosphorylation targets. Of the 211 phosphorylated proteins identified, 40 and 13 phosphoproteins were specifically modified by E2 and G-1, respectively. Subnetwork enrichment analysis revealed several processes related to cell cycle were specifically enriched by G-1 compared with E2. Further there existed a number of newly identified proteins that were specifically phosphorylated by G-1. These phosphorylation networks highlight specific proteins that may modulate the inhibitory effects of G-1 and suggest a novel role for interference with nuclear receptor activity driven by E2 and xenoestrogens.

Proteomics in aquatic amphipods: can it be used to determine mechanisms of toxicity and interspecies responses after exposure to atrazine?

Proteomics has gained popularity in the field of ecotoxicology as a holistic tool for unraveling novel mechanisms of toxicity and elucidating subtle effects of contaminant exposure. The holoarctic amphipod Diporeia spp. is declining at precipitous rates in the Great Lakes, and we are evaluating the use of the well-studied amphipod model Hyalella azteca as a surrogate for Diporeia spp. This article presents proteomics data from both amphipod species exposed to atrazine (ATZ) and one of its metabolites, desethylatrazine (DEA; 3 and 30 µg/L for 21 and 42 d). We used a proteomics approach to determine whether these two species of amphipods responded similarly to the same chemicals and to understand better the mechanisms of toxicity of ATZ and DEA in aquatic invertebrates. We observed disruption in energy production and mitochondrial function as well as hormesis in exposed organisms. In addition, we identified a two proteins (GAPDH and HSP 90 kDa) that have been linked to hormonal disruptions, suggesting potential endocrine disruption. Finally, we found that H. azteca and Diporeia spp. responded with similar proteomic profiles after ATZ and DEA exposure, suggesting that H. azteca may be used as a surrogate model organism for Diporeia spp.

Use of GC × GC/TOF-MS and LC/TOF-MS for metabolomic analysis of Hyalella azteca chronically exposed to atrazine and its primary metabolite, desethylatrazine.

Atrazine is one of the most commonly detected contaminants in the U.S. Little information is available on one of atrazine's metabolites, desethylatrazine (DEA). Two-dimensional gas chromatography and liquid chromatography coupled with time of flight- mass spectrometry were used to examine metabolite profiles of Hyalella azteca chronically exposed to 30 µg/L atrazine and DEA. The majority of identified metabolites were by-products of ß-oxidation of fatty acids suggesting possible disruption in energy metabolism. Eicosanoids increased in exposed females suggesting possible perturbations in neuropeptide hormonal systems. Overall, this research demonstrates the feasibility of utilizing metabolomic profiling of invertebrate species exposed to environmental contaminants as a way to determine mechanisms of toxicity.

Use of gene expression, biochemical and metabolite profiles to enhance exposure and effects assessment of the model androgen 17ß-trenbolone in fish.

The impact of exposure by water to a model androgen, 17ß-trenbolone (TRB), was assessed in fathead minnows using an integrated molecular approach. This included classical measures of endocrine exposure such as impacts on testosterone (T), 17ß-estradiol (E2), and vitellogenin (VTG) concentrations in plasma, as well as determination of effects on the hepatic metabolome using proton nuclear magnetic resonance spectroscopy. In addition, the rates of production of T and E2 in ovary explants were measured, as were changes in a number of ovarian gene transcripts hypothesized to be relevant to androgen exposure. A temporally intensive 16-d test design was used to assess responses both during and after the TRB exposure (i.e., depuration/recovery). This strategy revealed time-dependent responses in females (little impact was seen in the males), in which changes in T and E2 production in the ovary, as well as levels in plasma, declined rapidly (within 1 d), followed shortly by a return to control levels. Gene expression measurements revealed dynamic control of transcript levels in the ovary and suggested potential mechanisms for compensation during the exposure phase of the test. Proton nuclear magnetic resonance spectroscopy revealed a number of hepatic metabolite changes that exhibited strong time and dose dependence. Furthermore, TRB appeared to induce the hepatic metabolome of females to become more like that of males at both high test concentrations of TRB (472 ng/L) and more environmentally relevant levels (33 ng/L).

Liver proteome response of largemouth bass (Micropterus salmoides) exposed to several environmental contaminants: potential insights into biomarker development.

Liver proteome response of largemouth bass (Micropterus salmoides) exposed to environmental contaminants was analyzed to identify novel biomarkers of exposure. Adult male bass were exposed to cadmium chloride (CdCl(2)), atrazine, PCB 126, phenanthrene, or toxaphene via intraperitoneal injection with target body burdens of 0.00067, 3.0, 2.5, 50, and 100 microg/g, respectively. After a 96 h exposure, hepatic proteins were separated with two-dimensional gel electrophoresis and differentially expressed proteins (vs. controls) recognized and identified with MALDI-TOF/TOF mass spectrometry. We identified, 30, 18, eight, 19, and five proteins as differentially expressed within the CdCl(2), atrazine, PCB 126, phenanthrene, and toxaphene treatments, respectively. Alterations were observed in the expression of proteins associated with cellular ion homeostasis (toxaphene), oxidative stress (phenanthrene, PCB 126), and energy production including glycolysis (CdCl(2), atrazine) and ATP synthesis (atrazine). This work supports the further evaluation of several of these proteins as biomarkers of contaminant exposure in fish.