Development of a Human Estrogen Receptor Dimerization Assay for the Estrogenic Endocrine-Disrupting Chemicals Using Bioluminescence Resonance Energy Transfer.

Hyeyeong Seo,Kwang Suk Ko,Seok-Hee Lee,Hye Mi Kim,Yooheon Park,Hee-Seok Lee

doi:10.3390/ijerph18168875

Abstract

Endocrine-disrupting chemicals (EDCs) are found in food and various other substances, including pesticides and plastics. EDCs are easily absorbed into the body and have the ability to mimic or block hormone function. The radioligand binding assay based on the estrogen receptors binding affinity is widely used to detect estrogenic EDCs but is limited to radioactive substances and requires specific conditions. As an alternative, we developed a human cell-based dimerization assay for detecting EDC-mediated ER-alpha (ERα) dimerization using bioluminescence resonance energy transfer (BRET). The resultant novel BRET-based on the ERα dimerization assay was used to identify the binding affinity of 17β-estradiol (E2), 17α-estradiol, corticosterone, diethylhexyl phthalate, bisphenol A, and 4-nonylphenol with ERα by measuring the corresponding BRET signals. Consequently, the BRET signals from five chemicals except corticosterone showed a dose-dependent sigmoidal curve for ERα, and these chemicals were suggested as positive chemicals for ERα. In contrast, corticosterone, which induced a BRET signal comparable to that of the vehicle control, was suggested as a negative chemical for ERα. Therefore, these results were consistent with the results of the existing binding assay for ERα and suggested that a novel BRET system can provide information about EDCs-mediated dimerization to ERα.

Highlights

Endocrine-disrupting chemicals (EDCs) interfere with the functions of the endocrine system by mimicking or blocking hormone function in humans and animals
Vectors encoding all possible combinations of N- (Nluc–ERα, HT vector combined at N-terminal of ERα (HT–ERα)) and C-terminal (ERα–NanoLuc luciferase (Nluc), ERα–HT) fusion between Nluc and HT with ERα were constructed for testing using the bioluminescence resonance energy transfer (BRET)-based dimerization assay
By establishing the optimal condition for the ERα dimerization assay based on the NanoBRET system, it enhances the performance of the dimerization assay for detecting estrogenic EDCs using the in vitro model

Summary

Introduction

Endocrine-disrupting chemicals (EDCs) interfere with the functions of the endocrine system by mimicking or blocking hormone function in humans and animals. A considerable number of EDCs are widely found in everyday products, such as industrial solvents/byproducts, agricultural pesticides, plastics, and other manufactured products, and include natural chemicals, such as phytoestrogen [3,4]. These contaminants may enter the food chain by direct contact with food components [5,6]. Phenolic EDCs from industrial sewage plant flow in the aquatic environment and bisphenol A, another kind of EDC, are contained in plastics used for food packing materials or beverage containers. Exposure and accumulation of bisphenol A in the body can cause

Objectives

Methods

Results

Discussion

Conclusion