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. 2012 Jul;120(7):1029-35.
doi: 10.1289/ehp.1104689. Epub 2012 Apr 11.

Differential estrogenic actions of endocrine-disrupting chemicals bisphenol A, bisphenol AF, and zearalenone through estrogen receptor α and β in vitro

Yin Li  1 , Katherine A Burns, Yukitomo Arao, Colin J Luh, Kenneth S Korach
Affiliations

Differential estrogenic actions of endocrine-disrupting chemicals bisphenol A, bisphenol AF, and zearalenone through estrogen receptor α and β in vitro

Yin Li et al. Environ Health Perspect. 2012 Jul.

Erratum in

  • Environ Health Perspect. 2012 Dec;120(12):A455

Abstract

Background: Endocrine-disrupting chemicals (EDCs) are widely found in the environment. Estrogen-like activity is attributed to EDCs, such as bisphenol A (BPA), bisphenol AF (BPAF), and zearalenone (Zea), but mechanisms of action and diversity of effects are poorly understood.

Objectives: We used in vitro models to evaluate the mechanistic actions of BPA, BPAF, and Zea on estrogen receptor (ER) α and ERβ.

Methods: We used three human cell lines (Ishikawa, HeLa, and HepG2) representing three cell types to evaluate the estrogen promoter activity of BPA, BPAF, and Zea on ERα and ERβ. Ishikawa/ERα stable cells were used to determine changes in estrogen response element (ERE)-mediated target gene expression or rapid action-mediated effects.

Results: The three EDCs showed strong estrogenic activity as agonists for ERα in a dose-dependent manner. At lower concentrations, BPA acted as an antagonist for ERα in Ishikawa cells and BPAF acted as an antagonist for ERβ in HeLa cells, whereas Zea was only a partial antagonist for ERα. ERE-mediated activation by BPA and BPAF was via the AF-2 function of ERα, but Zea activated via both the AF-1 and AF-2 functions. Endogenous ERα target genes and rapid signaling via the p44/42 MAPK pathway were activated by BPA, BPAF, and Zea.

Conclusion: BPA and BPAF can function as EDCs by acting as cell type-specific agonists (≥ 10 nM) or antagonists (≤ 10 nM) for ERα and ERβ. Zea had strong estrogenic activity and activated both the AF-1 and AF-2 functions of ERα. In addition, all three compounds induced the rapid action-mediated response for ERα.

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Conflict of interest statement

The authors declare they have no actual or potential competing financial interests.

Figures

Figure 1
Figure 1
BPA, BPAF, and Zea act as agonists or antagonists for ERα and ERβ. (A–F) Dose–response curves for ERα (A–C) or ERβ (D–F) in Ishikawa (A,D), HeLa (B,E), and HepG2 (C,F) cells transfected with ERE-luc, pRL‑TK, and either pcDNA/WT‑ERα or pcDNA/WT‑ERβ plasmids and treated with vehicle (control), 1, 10, 100, or 1,000 nM E2, BPA, BPAF, or Zea for 18 hr; ERE-mediated ERα and ERβ activation was detected by luciferase reporter assays. Data shown are mean ± SEM fold change relative to control for three independent experiments. (G,H) BPA, BPAF, and Zea antagonize E2-mediated ERα activation. Cells transfected with ERE-luc, pRL‑TK, and pcDNA/WT ERα (Ishikawa cells) or pcDNA/WT ERβ (HeLa cells) plasmids were treated with vehicle (control), 10 nM E2, or BPA, BPAF, or Zea (1 or 10 nM alone or with E2 for 18 hr, and ERE-mediated ERα and ERβ activation was detected by luciferase reporter assay. Data shown are mean ± SEM fold change relative to control for three independent experiments. *p < 0.05 compared with control. **p < 0.05 compared with 10 nM E2 treatment.
Figure 2
Figure 2
Functional analysis of BPA, BPAF, and Zea on WT and ERα mutants and coactivation of ERα by SRC2 or p300 in Ishikawa cells. (A) For functional analysis, cells transfected with ERE-luc, pRL‑TK, and pcDNA/WT ERα, pcDNA/H1 ERα, pcDNA/AA ERα, pcDNA/E1 ERα, or pcDNA/AF2 ERα plasmid were treated with vehicle, 10 nM E2, 100 nM BPA, 100 nM BPAF, 100 nM Zea, or 100 nM ICI for 18 hr, and ERα-ERE–mediated activity was detected by luciferase reporter assay. Data shown are mean ± SE fold change relative to control for three independent experiments relative to control. (B,C) Coactivation of ERα by SRC2 (B) or p300 (C) in cells transfected with ERE-luc, pRL‑TK, and pcDNA/SRC2 or p300, pcDNA/WT ERα, or pcDNA/SRC2 or p300 plus pcDNA/WT ERα plasmids and treated with the vehicle, 10 nM E2, or 100 nM BPA, BPAF, or Zea for 18 hr. ERE-mediated activation was detected by luciferase reporter assay. Data shown are mean ± SE fold change relative to control for three independent experiments relative to control. *p < 0.05 compared with control. **p < 0.05 compared with the vehicle for co-transfections.
Figure 3
Figure 3
BPA, BPAF, and Zea affect p44/42 MAPK and src tyrosine kinase pathways in Ishikawa/ERα–stable cells. (A) Detection of ERα protein expression by Western blot in whole cell lysates prepared from Ishikawa/vec or Ishkawa/ERα cells. (B) ERE-mediated activity in cells transiently transfected with ERE-luc and pRL‑TK plasmids and treated with vehicle, 10 nM E2 or 100 nM BPA, BPAF, or Zea. Activity was detected by luciferase reporter assays, and data are mean ± SEM fold change relative to control for three independent experiments. (C) Western blot detection of phospho-p44/42 MAPK, phospho-GSK-3β, and phospho-Akt activation by 100 nM E2, 1,000 nM BPA, 1,000 nM BPAF, or 1,000 nM Zea. (D) Effect of PD 98059 and PP2 on the induction of PR gene expression by vehicle (control), 10 nM E2 or 100 nM BPA, BPAF, or Zea. PR transcripts were quantified by real time-PCR, and results are presented as mean ± SEM fold change relative to control for three independent experiments. *p < 0.05 compared with control.
Figure 4
Figure 4
BPA, BPAF, and Zea regulate expression of the ER target genes PR, GREB, MCM3, and SPUVE in Ishikawa/ERα cells. Ishikawa/ERα cells were treated with vehicle (control), 10 nM E2, or 100 nM BPA, BPAF, or Zea for 18 hr and total RNA was extracted; mRNA levels of PR, GREB, MCM3 and SPUVE were quantified by real-time PCR. Results are presented as fold change (mean ± SEM) relative to the control for three independent experiments. *p < 0.05 compared with control.
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