Examining Molecular Mechanisms of Endocrine Disruption from Chemicals Used in Hydraulic Fracturing
Presentation Number: SAT 261
Date of Presentation: April 1st, 2017
Rana Kennedy*1, Jennifer N Cornelius-Green2 and Susan Carol Nagel1
1University of Missouri, Columbia, MO, 2University of Missouri
Hydraulic fracturing as a method for extracting oil and natural gas has become a widespread practice. The hydraulic fracturing industry utilizes over 1000 chemicals combined with water to fracture the shale or coal bed layer and release trapped natural gas and oil. More than 100 of the chemicals used in this process are known or suspected endocrine disruptors. Using a reporter gene assay in Ishikawa human endometrial carcinoma cells we have previously characterized 23 of these chemicals and found them to be endocrine disrupting chemicals (EDCs). These chemicals disrupted the activities of estrogen (ER), androgen, glucocorticoid, thyroid and/or progesterone receptors in this reporter gene assay. EDCs can disrupt normal receptor function by direct binding to the receptor or through indirect interactions. The current study aims to elucidate the mechanisms behind this disruption for these 23 chemicals. A mammalian two-hybrid assay was used to assess the interaction of two fusion proteins; VP16-ERα ligand binding domain and a Gal4 activation domain – ER interacting peptide. In this assay, when an ER ligand binds to the ERα ligand binding domain, it induces a conformational change in the protein and as a result can alter binding of ER interacting peptides. The interaction of the VP16-ERα ligand binding domain and Gal4-ER interacting peptide allows for the interaction of VP16 and Gal4. This interaction allows these proteins to activate a Gal4 luciferase reporter gene. The resulting luciferase activity was used as a marker for direct ligand interaction with ERα. Using this method it was determined that thirteen of these EDCs bind directly to ERα and ten likely do not directly bind ERα, suggesting that the mechanism of ERα disruption is a result of indirect interactions. To further investigate the effects and mechanisms of endocrine disruption from these EDCs, gene expression for hormone responsive genes, receptors and coregulators will be evaluated.
Nothing to Disclose: RK, JNC, SCN