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Endpoint:
endocrine system modulation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2013
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
- Principle of test:

Examine the agonistic activity of bisphenol-AF through ERα and ERβ. Also investigated was the effects of bisphenol-AF on ER-mediated target genes.

- Short description of test conditions:

HepG2 and HeLa cells were used to determine the agonistic activity of bisphenol-AF 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 bisphenol-AF.

- Parameters analysed / observed:

As described above.
GLP compliance:
no
Type of method:
in vitro
Endpoint addressed:
toxicity to reproduction / fertility
Specific details on test material used for the study:
RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: n/a
- Specific activity: n/a
- Locations of the label: n/a
- Expiration date of radiochemical substance: n/a

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: not reported
- Stability under test conditions: not reported
- Solubility and stability of the test substance in the solvent/vehicle: test item disolved in ethanol
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not reported

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: test item dissolved in ethanol before being diluted in media
- Preliminary purification step (if any): n/a
- Final dilution of a dissolved solid, stock liquid or gel: test item dissolved in ethanol before being diluted in media- fianl concentration of solvent was 0.01 % v/v.
- Final preparation of a solid: n/a

FORM AS APPLIED IN THE TEST (if different from that of starting material): applied as a liquid

OTHER SPECIFICS: n/a
Species:
other: Human cell lines
Strain:
other: Human cell lines
Details on test animals or test system and environmental conditions:
n/a (in vitro test)
Details on analytical verification of doses or concentrations:
n/a (see "any other information on materials and methods" section)
Duration of treatment / exposure:
n/a (see "any other information on materials and methods" section)
Frequency of treatment:
n/a (see "any other information on materials and methods" section)
Post exposure period:
n/a (see "any other information on materials and methods" section)
No. of animals per sex per dose:
n/a (see "any other information on materials and methods" section)
Details on study design:
n/a (see "any other information on materials and methods" section)
Examinations:
n/a (see "any other information on materials and methods" section)
Positive control:
n/a (see "any other information on materials and methods" section)

ERE-mediated estrogenic activation of ERα and ERβ by bisphenol-AF

To evaluate the ERE-mediated transcriptional activity of ERα and ERβ, promoter activation in two ER-negative cell lines were examined (HepG2 and HeLa). The two luciferase reporters, 3×ERE (modified reporter) and pS2ERE (endogenous pS2 gene reporter) were used to determine the differential effects of bisphenol-AF. First, it was confirmed that there was no reporter activation in either of these ER-negative cell lines after stimulation with 10 nM E2.

HepG2 cells were highly responsive to E2, with up to 50-fold increases in 3×ERE-mediated transactivation. Bisphenol-AF strongly activated ERα3×ERE- mediated responses in HepG2 cells. In HeLa cells, bisphenol-AF significantly induced 3×ERE and pS2ERE mediated activity.

For ERβ ERE-mediated activation, both ERE reporters exhibited responses to E2 in HepG2 cells. BPAF exhibited strong activation of ERβ3×ERE and pS2ERE-mediated responses in HepG2 cells.

The effects of EDCs on expression of ER target genes

To characterize the ER-dependent response of bisphenol-AF, its effects on ERα target genes (PR, pS2, GREB1, SPUVE, WISP2, and SDF-1) using real-time PCR in Ishikawa/ERα stable cells was examined. Data were normalized to β-actin, relative to the vehicle control. Bisphenol-AF significantly induced the endogenous ERα target genes PR,pS2, GREB1, SPUVE, WISP2 and SDF-1. In contrast, expression of target genes in the Ishikawa/vector stable cells did not change with bisphenol-AF treatments, demonstrating that the changes in target gene expression are ER dependent.

Conclusions:
Bisphenol-AF strongly activated ERα estrogen responsive element (ERE)-mediated responses. Results of real-time polymerase chain reaction indicated that bisphenol AF consistently activated endogenous ER target genes.
Executive summary:

The agonistic activity of bisphenol-AF through ERα and ERβ was examined along with the effects of the test item on ER-mediated target genes.

HepG2 and HeLa cells were used to determine the agonistic activity of bisphenol-AF 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 the test item.

As shown by luciferase reporter analysis, bisphenol-AF acts as an ER agonists in a cell type (and promoter) specific manner. Bisphenol AF strongly activated ERα estrogen responsive element (ERE)-mediated responses. Results of real-time polymerase chain reaction indicated that bisphenol AF consistently activated endogenous ER target genes.

Endpoint:
endocrine system modulation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
- Principle of test:

Determine the relative preference of bisphenol AF for the human nuclear estrogenic receptors ERα and ERβ and the bisphenol A–specific estrogen-related receptor ERRγ, and to clarify structural characteristics of receptors that influence bisphenol AF binding.

- Short description of test conditions:

Receptor-binding activities of bisphenol AF relative to [3H]17β‑estradiol (for ERα and ERβ) and [3H]bisphenol A (for ERRγ) were examined. Functional luciferase reporter gene assays were performed to assess receptor activation in HeLa cells.

- Parameters analysed / observed:

Receptor binding selectivity (ERα vs. ERβ, ERα vs. ERRγ, ERβ vs. ERRγ)
GLP compliance:
no
Type of method:
in vitro
Endpoint addressed:
toxicity to reproduction / fertility
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Not reported
- Stability under test conditions: Not reported
- Solubility and stability of the test substance in the solvent/vehicle: Not reported
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Not reported

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Bisphenol AF was dissolved in 0.3 % DMSO in 1% bovine serum albumin
- Preliminary purification step (if any):Not reported
- Final dilution of a dissolved solid, stock liquid or gel: Not reported
- Final preparation of a solid: Not reported

FORM AS APPLIED IN THE TEST (if different from that of starting material) Liquid

OTHER SPECIFICS: n/a

Table 1. Receptor‐binding characteristics of BPA and bisphenol AF for ERα, ERβ, and ERRγ.

Compound

IC50(nM)


ERα

ERβ

ERRγ

17β-estradiol

0.88 ± 0.04

2.17 ± 0.12

NB

4-OHT


2.88 ± 0.15

3.17 ± 0.24

10.3 ± 0.8

BPA

1,030 ± 70

900 ± 70

9.70 ± 0.59

Bisphenol AF

53.4 ± 3.1

18.9 ± 0.84

358 ± 3.1

HPTE

59.1 ± 1.5

18.1 ± 1.9

36.4 ± 4.4

Abbreviations: HPTE, 2,2‐bis(p‐hydroxyphenyl)‐1,1,1‐trichloroethane; NB, not bound (no significant receptor binding at 10 μM, the highest concentration tested).

Table 2. Receptor‐binding selectivity of BPA and AF for ERα, ERβ, and ERRγ

Compound

 

Receptor-binding selectivity

 

ERα vs. ERβ

ERα vs. ERRγ

ERβ vs. ERRγ

Preferred receptor(s)

17β-estradiol

2.47 ERα

(ERα)a

(ERβ)a

ERα

4-OHT


1.10 ERα

3.58 ERα

3.25 ERβ

ERα ~ ERβ

BPA

1.14 ERβ

106.18 ERRγ

92.78 ERRγ

ERα ~ ERβ

Bisphenol AF

2.83 ERβ

6.70 ERα

18.94 ERβ

ERβ

HPTE

3.27 ERβ

1.63 ERRγ

2.01 ERβ

ERβ

HPTE, 2,2‐bis(p‐hydroxyphenyl)‐1,1,1‐trichloroethane. Data aren‐fold strength of the preferred receptor compared with the non-preferred receptor; for example, ”2.47 ERα” means that 17β‐estradiol binds to ERα 2.47 times more strongly than to ERβ.aBecause of inactivity of 17β‐estradiol in ERRγ, 17β‐estradiol is active exclusively in ERα and ERβ.

Conclusions:
Bisphenol AF could function as an endocrine-disrupting chemical by acting as an agonist or antagonist to perturb physiological processes mediated through ERα and/or ERβ.
Executive summary:

Receptor binding activity of Bisphenol AF to estrogen receptors (ERα and ERβ ) was assessed using tritium-labeled ligand [3H]17β-estradiol (5.96 TBq/mmol in a receptor binding assay based on Scatchard plot analysis of Saturation-binding assays. Receptor populations with the appropriate dissociation constant (Kd) and receptor density (Bmax) were used for each radioligand receptor-binding assay.  Receptor protein GST-ERα-LBD or GST-ERβ-LBD (0.3 nM) was incubated with increasing concentrations of [3H]17β-estradiol (0.1–30 nM) in a final volume of 100 μL binding bu er (10 mM Tris, 1 mM EDTA, 1 mM EGTA, 1 mM sodium vanadate(V), 0.5 mM phenylmethylsulfonyl uoride, 0.2 mM leupeptin, 10% glycerol; pH 7.4. Nonspecific binding was determined in a parallel set of incubations that included 10 μM nonradiolabeled 17β-estradiol. After incubation for 2 hr at 20°C, free radioligand was removed by incubation with 0.4% dextran- coated charcoal (Sigma-Aldrich Inc.) in phosphate buffered saline (PBS; pH 7.4) for 10 min on ice and then centrifuged for 10 min at 15,000 rpm. 

In the radioligand binding assays for competitive binding, Bisphenol AF, BPA, 17β-estradiol, and 4-OHT were dissolved in 0.3% DMSO in 1% bovine serum albumin (BSA; a blocker of nonspecific adsorption to the reaction vessels). HPTE was tested as a reference com- pound that acted as an ERα agonist and an ERβ antagonist. The reaction mixtures were incubated overnight at 4°C, and free radioligand was removed with 1% dextran-coated charcoal by ltration. Radioactivity was determined on a liquid scintillation counter.  To measure the antagonistic activity of bisphenol AF for ERβ, we examined four concentrations (0.01, 0.1, 1.0, and 10 μM) of bisphenol AF for a serial concentration of 17β-estradiol (10–12 to 10–5 M in the final solution). Also, a serial concentration of bisphenol AF (10–12 to 10–5 M in the final solution) was assayed in the presence of 10 or 100 nM concentrations of 17β-estradiol, which normally elicit full activation of ERβ. 

Reporter gene activity after bisphenol AF exposure in HeLa cells transiently cotransfected with an ERα or ERβ expression plasmid and an estrogen-response element (ERE)-luciferase reporter plasmid. La cells were seeded at 5 × 105 cells per 6-cm dish for 24 hr and then transfected with 4 μg reporter gene (pGL3/3xERE) and 3 μg of ERα or ERβ expression plasmid (pcDNA3/ERs) by Lipofectamine Plus reagent (Invitrogen Japan, Tokyo, Japan) according to the manufacturer’s protocol. 

The results of this study indicated that Bisphenol AF strongly and selectively binds to ERs over ERRγ. Furthermore, Bisphenol AF receptor-binding activity was three times stronger for ERβ [IC50 (median inhibitory concentration) = 18.9 nM] than for ERα. When examined using a reporter gene assay, bisphenol AF was a full agonist for ERα. In contrast, it was almost completely inactive in stimulating the basal constitutive activity of ERβ. Surprisingly, bisphenol AF acted as a distinct and strong antagonist against the activity of the endogenous ERβ agonist 17β-estradiol. 

Under the condition of the study, Bisphenol AF could function as an endocrine-disrupting chemical by acting as an agonist or antagonist to perturb physiological processes mediated through ERα and/or ERβ. 

 

Endpoint:
endocrine system modulation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2012
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
In vitro models were used to evaluate the mechanistic actions of BPAF on estrogen receptor (ER) α and ERβ.
GLP compliance:
no
Type of method:
in vitro
Endpoint addressed:
toxicity to reproduction / fertility
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Not reported
- Stability under test conditions: Not reported
- Solubility and stability of the test substance in the solvent/vehicle: Not reported
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Not reported

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Not reported
- Preliminary purification step (if any): Not reported
- Final dilution of a dissolved solid, stock liquid or gel: Not reported
- Final preparation of a solid: n/a

FORM AS APPLIED IN THE TEST (if different from that of starting material): Not reported

OTHER SPECIFICS: n/a

Estrogenic activation of ERα and ERβ by BPAF

To evaluate ERE-mediated transcriptional activity of ERα and ERβ, promoter activation in Ishikawa, HeLa, and HepG2 cells were investigated. The luciferase reporter assay system was used to determine the treatment effects of BPAF on ERα or ERβ in cells derived from different tissues. Reporter activation of ERE‑luc in response to BPAF was observed only with both ERα and ERβ expression plasmids but not with pcDNA control plasmid.

For ERα activation in Ishikawa cells, estradiol activated the ERE‑luc reporter at a concentration of 1 nM (4‑fold induction). At low concentrations (≤ 10 nM), BPAF had weak estrogenic activity compared with estradiol, but stronger activation was observed at higher concentrations (1,000 nM). ERα activation in response to BPAF did not occur except with higher concentrations (≥ 100 nM). HepG2 cells were highly responsive to estradiol, with 10‑to 20‑fold increases in ERE-mediated transactivation at 10–1,000 nM estradiol.

In Ishikawa and HepG2 cells, ERβ was weak in response to BPAF compared with estradiol. In HeLa cells, ERβ responses to 100 or 1,000 nM BPAF were similar to those of estradiol. Taken together, these results demonstrate that BPAF can activate ERE-mediated transcription in different cell types via ERα and ERβ, and that the estrogenic activity of each compound is cell type and concentration dependent.

Activity of ERα and ERβ is antagonized by low doses of BPAF

BPAF showed weak activity in certain cell types at low doses. The antagonistic effects of BPAF on ERα and ERβ using the ERE-mediated reporter assay system was investigated. Cells were transiently transfected with ERα or ERβ expression plasmids and then treated with 1 or 10 nM BPAF with or without 10 nM estradiol co‑treatment. BPAF did not inhibit estradiol activation. estradiol-mediated activation was inhibited with estradiol co-treatment. No antagonistic effects of BPAF on ERα in HeLa or HepG2 cells was observed.

For ERβ, estradiol-ERE–mediated reporter activity was reduced in HeLa cells treated with BPAF. The test item did not have an antagonistic effect on ERβ in Ishikawa or HepG2 cells. Data demonstrate that low doses of BPAF (≤ 10 nM) antagonized ERα activity and ERβ activity, respectively; however, these effects were cell-type specific.

Effects of BPAF on ERα functionality

To link BPAF-mediated activation to a specific ERα functionality, we used four ERα mutants: H1‑ERα_(ERE-mediated activation, but no tethered-mediated activation), AA‑ERα (tethered-mediated activation, but no ERE-mediated activation), E1‑ERα (AF‑1 inactive), and AF‑2-ERα (AF‑2 inactive). Estrogenic effects of BPAF at 100 nM with WT‑ERα in Ishikawa cells was observed. Therefore, this concentration was used in the mutant experiments. Cells were transiently transfected with an ERE‑luc reporter plasmid and ERα mutant expression plasmids. Reporter activity was calculated for each mutant relative to activity in the vehicle control. Responses to BPAF were similar with H1‑ERα and WT‑ERα; however, BPAF did not show activation of AA‑ERα; this is consistent with ERE‑mediated activity, in which ERE reporter activity in response to BPAF with E1‑ERα (AF‑1 inactive) was similar to activity with WT‑ERα. Overall, these data indicate that ERE‑mediated activation by BPAF was via the AF‑2 function on ERα.

In determining a role for ERα and activation with coactivators, we found that both SRC2 and p300 coactivated ERα- ERE–mediated activity with BPAF. These data indicated that SRC2 and p300 act as ERα coactivators to increase transactivation in the presence of BPAF, consistent with its AF‑2 functionality.

BPAF activate genes via p44/42 MAPK and src tyrosine kinase pathways.

To examine phosphorylation events, we investigated the involvement of rapid action responses by BPAF-mediated signaling pathways in Ishikawa cells stably expressing the vector control or WT‑ERα. First, ERα expression was confirmed in Ishikawa/ERα cells by Western blot  and then confirmed ERE‑mediated activation by estradiol, BPAF using the reporter assay system. estradiol and BPAF induced phospho-p44/42 MAPK in Ishikawa/ERα cells but not in Ishikawa/vec cells, suggesting ERα-dependent and ligand-dependent activation. In addition, phospho-GSK-3β expression was only weakly induced by estradiol and BPAF, and no increase in phospho-Akt was observed in response to estradiol or BPAF in this model system.

The effect of two specific kinase inhibitors were examined—PD 98059 (MAPK inhibitor) and PP2 (src family tyrosine kinase inhibitor) on BPAF-mediated expression of progesterone receptor (PR), a classic ER target gene, in Ishikawa/ ERα cells. Both inhibitors blocked BPAF‑mediated endogenous PR gene expression, suggesting that BPAF is involved in the p44/42 MAPK and tyrosine kinase src pathways. In contrast, induction of PR expression by estradiol was inhibited by PD 98059 but not by PP2. Thus, while BPAF appeared to activate the p44/42 MAPK pathway in an ER-dependent manner, other kinase signaling pathways, such as the tyrosine kinase src, may also involve BPAF activation.

BPAF induced expression of ER target genes

We confirmed ERα- dependent responses to BPAF by detecting endogenous gene expression of PR, GREB1 (gene regulation by estrogen in breast cancer 1), MCM3 (minichromosome maintenance complex component 3), and SPUVE (also known as PRSS23; a member of the trypsin family of serine proteases) by real time-PCR. In Ishikawa/ERα cells, estradiol and BPAF induced endogenous PR and GREB expression. MCM3 and SPUVE expression was not induced by BPAF. In addition, it was confirmed that expression of PR, GREB, MCM3, and SPUVE was not induced in Ishikawa/vec cells treated with BPAF.

Conclusions:
BPAF can function as an endocrine disrupting chemical by acting as an agonist (at higher concentrations; ≥ 10 nM) and as an antagonists (at lower concentrations; ≤ 10 nM) for ERα and ERβ. These actions were found to be cell-type specific. BPAF activated the AF‑2 function of ERα in addition to inducing the p44/42 MAPK pathway, suggesting that it can mediate rapid action responses involved in endogenous ER signaling events as well as genomic responses. Taken together, the data demonstrate the mechanistic importance of cell type specificity in evaluation of the potential activities of BPAF.
Executive summary:

In vitro models were used to evaluate the mechanistic actions of BPAF on estrogen receptor (ER) α and ERβ.

Three human cell lines (Ishikawa, HeLa, and HepG2) representing three cell types to evaluate the estrogen promoter activity of BPAF 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 indicated that BPAF showed strong estrogenic activity as agonists for ERα in a dose-dependent manner. At lower concentrations, BPAF acted as an antagonist for ERβ in HeLa cells. ERE-mediated activation by BPAF was via the AF‑2 function of ERα. Endogenous ERα target genes and rapid signaling via the p44/42 MAPK pathway were activated by BPAF.

In conclusion, BPAF can function as an endocrine disrupting chemical by acting as cell type–specific agonists (≥ 10 nM) or antagonists (≤ 10 nM) for ERα and ERβ. In addition, BPAF induced the rapid action-mediated response for ERα.

Description of key information

Bisphenol AF may function as an endocrine-disrupting chemical by acting as an agonist or antagonist to perturb physiological processes mediated through ERα and/or ERβ. Bisphenol AF action is considered to be cell-type specific and involve the activation of AF‑2 function of ERα therefore inducing the p44/42 MAPK pathway, which is involved in the regulation of meiosis, mitosis and post mitotic function in differentiated cells.

Additional information

Matsushima 2010) - Receptor binding activity of Bisphenol AF to estrogen receptors (ERα and ERβ) was assessed. The result indicated that Bisphenol AF strongly and selectively binds to ERs over ERRγ. Furthermore, Bisphenol AF receptor-binding activity was three times stronger for ERβ [IC50 (median inhibitory concentration) = 18.9 nM] than for ERα. When examined using a reporter gene assay, bisphenol AF was a full agonist for ERα. In contrast, it was almost completely inactive in stimulating the basal constitutive activity of ERβ. Surprisingly, bisphenol AF acted as a distinct and strong antagonist against the activity of the endogenous ERβ agonist 17β-estradiol. Bisphenol AF could function as   an endocrine-disrupting chemical by acting as an agonist or antagonist to perturb physiological processes mediated through ERα and/or ERβ. 

Li et al 2013: agonistic activity of bisphenol-AF through ERα and ERβ was examined along with the effects of the test item on ER-mediated target genes. Bisphenol-AF acts as an ER agonists in a cell type (and promoter) specific manner. Bisphenol AF strongly activated ERα estrogen responsive element (ERE)-mediated responses. Furthermore, Bisphenol AF also activated endogenous ER target genes.  

In another study (Li 2012) - In vitro models were used to evaluate the mechanistic actions of BPAF on estrogen receptor (ER) α and ERβ using three human cell lines (Ishikawa, HeLa, and HepG2). BPAF showed strong estrogenic activity as agonists for ERα in a dose-dependent manner. At lower concentrations, BPAF acted as an antagonist for ERβ in HeLa cells. ERE-mediated activation by BPAF was via the AF‑2 function of ERα. Endogenous ERα target genes and rapid signaling via the p44/42 MAPK pathway were activated by BPAF.