Habitat Conservation of Aquatic Animals Including Dolphins Utilizing Estrogen Receptor Reporter Gene Assays

Abstract

Steroid hormones, including estrogen, play important roles in the reproduction, growth and development in all vertebrates. Estrogenic chemicals have been detected in the aquatic environment and estrogenic pharmaceuticals have been found to affect fish reproduction. Estrogenicity of chemicals can be screened by an in vitro reporter gene assay using estrogen receptors (ERs) from aquatic animal species. The goal of this research is to establish an in vitro dolphin ER reporter gene assay for screening of some environmental chemicals with estrogenic activity. To achieve this goal, dolphin ER gene was cloned. Due to low preservation quality of dolphin samples, only the ligand binding domain could be cloned. The ligand binding domain of dolphin ER was transfected into HEK293 cells (human embryonic kidney cell line lacking endogenous ERs expression) with reporter construct. Then, estrogenic potency of some environmental chemicals such as 4-nonylphenol (NP), bisphenol-A (BPA) and o,p’-DDT (DDT) and natural estrogen, 17-estradiol (E2) were analyzed using the reporter gene assay. An in silico docking model using amino acid sequence of dolphin ER ligand binding domain was applied to these chemicals to show potential docking stability to dolphin ER. The data obtained by the reporter gene assay matched with those of in silico docking model. Using these screening systems for chemicals with estrogenic activity, can be applied in the detection of environmental chemicals and used to minimize the release of estrogenic chemicals in habitats of dolphins and other aquatic mammals.