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Characterization of Estrogenic and Androgenic Activities for Bisphenol A-like Chemicals (BPs): In Vitro Estrogen and Androgen Receptors Transcriptional Activation, Gene Regulation, and Binding Profiles

Katherine E. Pelch, Yin Li, Lalith Perera, Kristina A. Thayer, Kenneth S. Korach
DOI: https://doi.org/10.22427/NTP-DATA-002-00060-0001-0000-9

Publication

Abstract

Bisphenol A (BPA) is a high production volume chemical widely used in plastics, food packaging, and many other products. It is well known that endocrine-disrupting chemicals (EDC) might be harmful to human health due to interference with normal hormone actions. Recent studies report widespread usage and exposure to many BPA-like chemicals (BPs) that are structurally or functionally similar to BPA. However, the biological actions and toxicity of those BPs are still relatively unknown. To address this data gap, we used in vitro cell models to evaluate the ability of twenty-two BPs to induce or inhibit estrogenic and androgenic activity. BPA, Bisphenol AF (BPAF), bisphenol Z (BPZ), bisphenol C (BPC), tetramethyl bisphenol A (TMBPA), bisphenol S (BPS), bisphenol E (BPE), 4,4-bisphenol F (4,4-BPF), bisphenol AP (BPAP), bisphenol B (BPB), tetrachlorobisphenol A (TCBPA), and benzylparaben (PHBB) induced estrogen receptor (ER)α and/or ERβ-mediated activity. With the exception of BPS, TCBPA, and PHBB, these same BPs were also androgen receptor (AR) antagonists. Only three BPs were found to be ER antagonists. Bisphenol P (BPP) selectively inhibited ERβ-mediated activity and 4-(4-phenylmethoxyphenyl)sulfonylphenol (BPS-MPE) and 2,4-bisphenol S (2,4-BPS) selectively inhibited ERα-mediated activity. None of the BPs induced AR mediated activity. In addition, we identify that the BPs can bind to ER or AR with varying degrees by a molecular modeling analysis. Taken together, these findings help us to understand the molecular mechanism of BPs and further consideration of their usage in consumer products.

Figures

Figure 2A/Supplemental Figure 2

Induction of ERα/ERE mediated transcriptional activity in transiently transfected HepG2 cells. Each plot shows the vehicle control (blue down triangle), and the dose response curves for E2 (black open diamond), BPA (gray closed square) and the indicated BP (orange closed circle). A horizontal gray dotted line marks the 50% maximum response of E2. Data is presented as mean response normalized to vehicle control and the maximum E2 response ± SEM from three independent experiments. Only BPs inducing ≥20% of the activity of the positive control (E2) are considered active and shown in Figure 2. BPs considered inactive are shown in Supplemental Figure 2. *p < 0.05 versus vehicle control by ANOVA and Dunnett’s multiple comparison post hoc test. For improved clarity, only statistics for the indicated BP are shown in this figure.

Figure 2B/Supplemental Figure 3

Induction of ERβ/ERE mediated transcriptional activity in transiently transfected HepG2 cells. Each plot shows the vehicle control (blue down triangle), and the dose response curves for E2 (black open diamond), BPA (gray closed square) and the indicated BP (orange closed circle). A horizontal gray dotted line marks the 50% maximum response of E2. Data is presented as mean response normalized to vehicle control and the maximum E2 response ± SEM from three independent experiments. Only BPs inducing ≥20% of the activity of the positive control (E2) are considered active and shown in Figure 2. BPs considered inactive are shown in Supplemental Figure 4. *p < 0.05 versus vehicle control by ANOVA and Dunnett’s multiple comparison post hoc test. For improved clarity, only statistics for the indicated BP are shown in this figure.

Figure 3

Cells were treated with 10 nM E2 or 1 µM of an individual BP. Total RNA was extracted and used as a template for cDNA synthesis. Gene expression was measured by qPCR. Results are average fold change relative to vehicle control + SEM from three independent experiments. *p < 0.05 compared to vehicle control.

Figure 4A/Supplemental Figure 5

Each plot shows hormone free vehicle control (blue down triangle), 1.0 nM E2 (black open diamond), and the dose response curves for ICI (green X), BPA (gray closed square) and the indicated BP (orange closed circle). A horizontal gray dotted line marks 50% of the maximum response of 1.0 nM E2. Data is presented as mean response normalized to vehicle control and the 1.0 nM E2 response ± SEM from three independent experiments. BPs considered active are shown in Figure 4. BPs considered inactive are shown in Supplementary Figure 5. *p < 0.05 versus 1.0 nM E2 by ANOVA and Dunnett’s multiple comparison post hoc test. For improved clarity, only statistics for the indicated BP are shown in this figure. See Supplementary Figure 1 for statistics for ICI and BPA.

Figure 4B/Supplemental Figure 6

Each plot shows hormone free vehicle control (blue down triangle), 1.0 nM E2 (black open diamond), and the dose response curves for ICI (green X), BPA (gray closed square) and the indicated BP (orange closed circle). A horizontal gray dotted line marks 50% of the maximum response of 1.0 nM E2. Data is presented as mean response normalized to vehicle control and the 1.0 nM E2 response ± SEM from three independent experiments. BPs considered active are shown in Figure 4. BPs considered inactive are shown in Supplementary Figure 5. *p < 0.05 versus 1.0 nM E2 by ANOVA and Dunnett’s multiple comparison post hoc test. For improved clarity, only statistics for the indicated BP are shown in this figure. See Supplementary Figure 1 for statistics for ICI and BPA.

Figure 5/Supplemental Figure 8

Antagonism of AR/MMTV-mediated activity in stably transfected MDA-kb2 cells treated concurrently with 1.0 nM T. Each plot shows hormone free vehicle control (blue down triangle), 1.0 nM T (black open diamond), and the dose response curves for Flut (green X), BPA (gray closed square) and the indicated BP (orange closed circle). A horizontal gray dotted line marks 50% of the maximum response of 1.0 nM T. Data is presented as mean response normalized to vehicle control and the 1.0 nM T response ± SEM from three independent experiments. BPs considered active are shown in Figure 5. BPs considered inactive are shown in Supplemental Figure 8. *p < 0.05 versus 1.0 nM T by ANOVA and Dunnett’s multiple comparison post hoc test. For improved clarity, only statistics for the indicated BP are shown in this figure. See Supplementary Figure 1 for statistics for Flut and BPA.

Supplemental Figure 1

Response curves for controls used in the luciferase reporter gene assays are shown. Hormone free vehicle control (blue down triangle), positive control (E2 or T; black open diamond), BPA (gray closed square) are shown for assays run in agonist mode for ERα (A) or ERβ (B) or AR (C). Hormone free vehicle control (blue down triangle), 1.0 nM E2 or T control (black open diamond), positive control (ICI or Flut; black open diamond), BPA (gray closed square) are shown for assays run in antagonist mode for ERα (D) or ERβ (E) or AR (F). A horizontal gray dotted line marks the 50% maximum response of E2 or T in all figures. Data is presented as mean response normalized to vehicle control and the maximum E2 response ± SEM from n=3 independent experiments. *p ≤ 0.05 compared to hormone free vehicle control in agonist assays or 1.0 nM E2 or T control in antagonist assays.

Supplemental Figure 4

Transiently transfected HepG2 cells were treated with E2, BPA or the BPs at 1 µM in the absence (gray) or presence (black) of 10 µM ICI. Data is average fold induction relative to vehicle treated cells in the absence of ICI ± SEM from three independent experiments. A dashed gray line is shown at relative fold induction = 1 for improved clarity. *p≤0.05 compared to vehicle control in the absence of ICI by ANOVA and Dunnet’s multiple comparison post hoc test.

Supplemental Figure 7

BPs did not induce AR/MMTV-mediated transcriptional activity ≥ 20% in stably transfected MDA-kb2 cells and were not statistically different from 1.0 nM T so were considered to be inactive in the AR agonism assays. Each plot shows the dose response curve for the AR agonists T (black open diamond), and DHT (blue triangle), BPA (gray closed square) and the indicated BP (orange closed circle). A horizontal gray dotted line marks the 50% maximum response of T. Data is presented as mean response normalized to vehicle control and the maximum T response ± SEM from three technical replicates. This assay was only completed once since none of the BPs showed AR agonist activity.

Supplemental Information

Cytotoxicity

Cytotoxicity was determined for HepG2 and MDA-kb2 cells used in the luciferase reporter gene assays. Fluorescence was normalized by determining the percentage difference from the concurrent vehicle control. One-way ANOVA followed by Dunnett’s post hoc test was used to determine if each data point was significantly different than vehicle control. BPs with a statistically significant increase in fluorescence signal ≥15% compared to vehicle were considered cytotoxic.