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2014•Toxicology Mechanisms and MethodsRequires access

A novel ERα-mediated reporter gene assay for screening estrogenic/antiestrogenic chemicals based on LLC-MK2 cells

Xiaoming Huang, Jie Huang, Lishi Zhang, Yanfeng Zhu, Yun Li

Open publisher page 4 citations

Abstract

Low concentration of endocrine-disrupting chemicals (EDCs) may lead to serious consequences in animals and human, so it is essential to develop an effective assay for EDCs detection. In this study, we developed a novel ERα-mediated reporter gene assay based on the LLC-MK2 cells by co-transfecting pERE-sv40-Luc, hERα-pcDNA3.1, and pRL-tk. Then we determined 17β-estradiol (E2) and some estrogenic/antiestrogenic chemicals to verify the validity of this assay. Data showed that the assay possesses a concentration-dependent responses to E2 and diethylstilbestrol (DES) from 10(-12 )M to 10(-8 )M with EC(50) 3.4 × 10(-10 )M and 5.9 × 10(-10 )M, and ICI 182,780 completely blocks the luciferase activity induced by 10(-9 )M E2. Bisphenol A (BPA), nonylphenol (NP), genistein (GS), and tamoxifen (TAM) also showed corresponding estrogenic or antiestrogenic activity at test concentrations. All evidences proved that the LLC-MK2 reporter gene assay was specific and sensitive to estrogen receptor (ER) agonistic and antagonistic chemicals.

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What this paper is about

Low concentration of endocrine-disrupting chemicals (EDCs) may lead to serious consequences in animals and human, so it is essential to develop an effective assay for EDCs detection. In this study, we developed a novel ERα-mediated reporter gene assay based on the LLC-MK2 cells by co-transfecting pERE-sv40-Luc, hERα-pcDNA3.1, and pRL-tk. Then we determined 17β-estradiol (E2) and some estrogenic/antiestrogenic chemicals to verify the validity of this assay. Data showed that the assay possesses a concentration-dependent responses to E2 and diethylstilbestrol (DES) from 10(-12 )M to 10(-8 )M with EC(50) 3.4 × 10(-10 )M and 5.9 × 10(-10 )M, and ICI 182,780 completely blocks the luciferase activity induced by 10(-9 )M E2. Bisphenol A (BPA), nonylphenol (NP), genistein (GS), and tamoxifen (TAM) also showed corresponding estrogenic or antiestrogenic activity at test concentrations. All evidences proved that the LLC-MK2 reporter gene assay was specific and sensitive to estrogen receptor (ER) agonistic and antagonistic chemicals.

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Available abstract

Low concentration of endocrine-disrupting chemicals (EDCs) may lead to serious consequences in animals and human, so it is essential to develop an effective assay for EDCs detection. In this study, we developed a novel ERα-mediated reporter gene assay based on the LLC-MK2 cells by co-transfecting pERE-sv40-Luc, hERα-pcDNA3.1, and pRL-tk. Then we determined 17β-estradiol (E2) and some estrogenic/antiestrogenic chemicals to verify the validity of this assay. Data showed that the assay possesses a concentration-dependent responses to E2 and diethylstilbestrol (DES) from 10(-12 )M to 10(-8 )M with EC(50) 3.4 × 10(-10 )M and 5.9 × 10(-10 )M, and ICI 182,780 completely blocks the luciferase activity induced by 10(-9 )M E2. Bisphenol A (BPA), nonylphenol (NP), genistein (GS), and tamoxifen (TAM) also showed corresponding estrogenic or antiestrogenic activity at test concentrations. All evidences proved that the LLC-MK2 reporter gene assay was specific and sensitive to estrogen receptor (ER) agonistic and antagonistic chemicals.

Key concepts: Reporter gene, Estrogen receptor, Nonylphenol, Diethylstilbestrol, Estrogen receptor alpha, Endocrine disruptor, Chemistry, Estrogen

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