Sahyouni, Fatima
Szarka, Szabolcs
Nguyen, Vien
Prokai-Tatrai, Katalin
Prokai, Laszlo


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Endocrine disrupting chemicals (EDCs) are a class of chemicals that interfere with the biological actions of hormones, and there has been significant public concern about EDCs in the environment adversely affecting both wildlife and human health. They can alter processes regardless of whether they are related to reproduction. The mammalian uterus is one of the most sensitive organs for estrogenicity, but the widely used uterotrophic rat assay to assess known and potential EDCs merely considers the uterine weight gain endpoint. In this presentation, we focus on the quantitative proteomic investigation of estrogenic endocrine disruption in the rat uterus utilizing a comprehensive systems biology approach. Using 17β-estradiol (E2), an endogenous estrogen, will serve as a reference for subsequent studies of agents with estrogenic potential. Purpose (a): To validate potential markers of estrogenicity of discovery identified differentially expressed estrogen-induced proteins in rat uterine tissue using quantitative proteomics. Methods (b): Ovariectomized rats were treated short-term with subcutaneous E2 injections using corn oil as a vehicle. Approximately 10 mg of tissue were dissected from the uterus of vehicle-treated control and E2-treated animals for proteomic analyses. Uterine proteins were extracted with 8M urea for 30 minutes and subsequently processed by reduction, alkylation and digestion for mass spectrometry analysis. The samples were analyzed using a hybrid linear ion trap–Fourier transform ion cyclotron mass spectrometer equipped with an electrospray ionization source and connected to a nanoflow liquid chromatography system. MS/MS data was searched against a composite UniProt rat protein database using the Mascot software. Quantitation was performed using an MS-based total precursor intensity approach using the Scaffold software. Additionally, the differentially expressed proteins were mapped to signaling networks and biological processes using Ingenuity Pathway Analysis (IPA). Results (c): The mammalian uterus increases its weight due to fluid imbibition and cell proliferation by exogenously administered estrogenic compounds. With the observation of weight gain in the treated uterus compared to non-treated control rats, we confirmed E2’s uterotrophic effects for our subsequent proteomics study. Estrogen-regulated proteins were identified using an MS-based label-free quantitative approach. With p<0.05 considered statistically significant and >2-fold change as threshold, 135 proteins were differentially regulated by the hormone. Of these significantly differentially regulated proteins, 97 were up-regulated in E2-treated uteri and 38 were down-regulated in E2-treated uteri. When these 135 proteins were submitted for bioinformatic pathway analysis, 131 proteins were mapped into 14 networks that merged into E2-regulated pathways. Major molecular processes involve metabolic pathways, steroid signaling, and inflammatory signaling. Top networks include post-translational modification, protein folding, carbohydrate metabolism, cell death and survival, cancer, and endocrine system disorders. Implicated diseases include endocrine system and metabolic disorders. Proteotypic peptides from proteins that were strongly influenced by E2 administration have been selected for targeted validation studies. Conclusions (d): In addition to confirming the expected increase in wet uterine weights, we have derived interaction networks that mechanistically dissect E2’s uterotrophic effect at the proteome level. We have selected proteotypic peptides of strongly regulated proteins for future targeted validation as a potential biomarker panel for estrogenicity. (Supported by the Robert A. Welch Foundation, BK-0031, and the NIH grant AG031535).


Research Appreciation Day Award Winner - 2014 For HER, Translational Research Poster Award