Endocrine-Disrupting Chemicals (EDCs): In Vitro Mechanism of Estrogenic Activation and Differential Effects on ER Target Genes

Abstract

Background: Endocrine-disrupting chemicals (EDCs) influence the activity of estrogen receptors (ERs) and alter the function of the endocrine system. However, the diversity of EDC effects and mechanisms of action are poorly understood.

Objectives: We examined the agonistic activity of EDCs through ERα and ERβ. We also investigated the effects of EDCs on ER-mediated target genes.

Methods: HepG2 and HeLa cells were used to determine the agonistic activity of EDCs on ERα and ERβ via the luciferase reporter assay. Ishikawa cells stably expressing ERα were used to determine changes in endogenous ER target gene expression by EDCs.

Results: Twelve EDCs were categorized into three groups on the basis of product class and similarity of chemical structure. As shown by luciferase reporter analysis, the EDCs act as ER agonists in a cell type- and promoter-specific manner. Bisphenol A, bisphenol AF, and 2-2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane (group 1) strongly activated ERα estrogen responsive element (ERE)-mediated responses. Daidzein, genistein, kaempferol, and coumestrol (group 2) activated both ERα and ERβ ERE-mediated activities. Endosulfan and kepone (group 3) weakly activated ERα. Only a few EDCs significantly activated the "tethered" mechanism via ERα or ERβ. Results of real-time polymerase chain reaction indicated that bisphenol A and bisphenol AF consistently activated endogenous ER target genes, but the activities of other EDCs on changes of ER target gene expression were compound specific.

Conclusion: Although EDCs with similar chemical structures (in the same group) tended to have comparable ERα and ERβ ERE-mediated activities, similar chemical structure did not correlate with previously reported ligand binding affinities of the EDCs. Using ERα-stable cells, we observed that EDCs differentially induced activity of endogenous ER target genes.

Figure 1

The chemical structures of EDCs tested in this study.

Figure 2

EDCs act as agonists on ERα (A,B) and ERβ (C,D) to activate the classical mechanism (ERE) in HepG2 and HeLa cells. (A,B) Activation of ERα in HepG2 (A) and HeLa (B) cells transfected with ERE-luc (3×ERE or pS2 ERE), pRL-TK, and pcDNA/WT-ERα or pcDNA/WT-ERβ plasmids overnight, and then treated with vehicle (control), 10 nM E₂, 100 nM ICI, or EDCs for 18 hr; ERα ERE-mediated activation was detected by luciferase reporter assays. (C,D) Activation of ERβ in HepG2 (C) and HeLa (B) cells transfected with ERE-luc (3×ERE or pS2 ERE), pRL-TK, and pcDNA/WT-ERβ plasmids overnight and then treated with vehicle (control), 10 nM E₂, 100 nM ICI, or EDCs for 18 hr; ERβ ERE-mediated activation was detected by luciferase reporter assays. See “Materials and Methods” for details of the experiments. Data shown represent mean fold change (± SE) relative to the control. *p < 0.05, **p < 0.01, and ^#p < 0.001, compared with control.

Figure 3

EDCs act as agonists on ERα and ERβ to activate the tethered mechanism (AP-1). (A) Effects of EDCs on ERα 7×AP-1 Luc reporter activity in HepG2 (top) and HeLa (bottom) cells transfected with 7×AP-1 Luc, pRL-TK, vehicle (control), 10 nM E₂, 100 nM ICI, or EDCs for 18 hr; ER AP-1–mediated activation was detected by luciferase reporter assays. (B) Effects of EDCs on ERα‑ and ERβ-73Col AP-1 Luc reporter activity in HeLa cells transfected with -73Col AP-1 Luc, pRL-TK, and pcDNA/WT-ERα or -ERβ plasmids overnight and then treated with vehicle (control), 10 nM E₂, 100 nM ICI, or EDCs for 18 hr; ER -73Col AP-1–mediated activation was detected by luciferase reporter assays. See “Materials and Methods” for details of the experiments. Data shown represent mean fold change (± SE) relative to the control. *p < 0.05, **p < 0.01, and ^#p < 0.001, compared with control.

Figure 4

Effects of EDCs on expression of the ER target genes of PR, pS2, GREB1, SPUVE, WISP2, and SDF-1 in Ishikawa/vector and Ishikawa/ERα cells. Total RNA was extracted from Ishikawa/vec or Ishikawa/ERα cells after treatment with vehicle (control), 10 nM E₂, 100 nM ICI, or EDCs for 18 hr; mRNA levels of PR, pS2, GREB1, SPUVE, WISP2, and SDF-1 were quantified by real-time PCR. Data were normalized to β‑actin and represent mean fold change (± SE) relative to control Ishikawa/vec cells. *p < 0.05, **p < 0.01, and ^#p < 0.001, compared with control Ishikawa/vec cells.