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Direct evidence revealing structural elements essential for the high binding ability of bisphenol A to human estrogen-related receptor-gamma.

Okada H, Tokunaga T, Liu X, Takayanagi S, Matsushima A, Shimohigashi Y - Environ. Health Perspect. (2008)

Bottom Line: When we examined BPA derivatives to evaluate the structural essentials required for the binding of BPA to ERR-gamma , we found that only one of the two phenol-hydroxyl groups was essential for the full binding.The maximal activity was attained when one of the methyl groups was removed.These results indicate that the phenol derivatives are potent candidates for the endocrine disruptor that binds to ERR-gamma.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Structure-Function Biochemistry, Department of Chemistry, The Research-Education Centre of Risk Science, Faculty and Graduate School of Sciences, Kyushu University, Fukuoka, Japan.

ABSTRACT

Background: Various lines of evidence have shown that bisphenol A [BPA; HO-C6H4-C(CH3)2-C6H4-OH] acts as an endocrine disruptor when present in very low doses. We have recently demonstrated that BPA binds strongly to human estrogen-related receptor-gamma (ERR-gamma ) in a binding assay using [3H]4-hydroxytamoxifen ([3H]4-OHT). We also demonstrated that BPA inhibits the deactivation activity of 4-OHT.

Objectives: In the present study, we intended to obtain direct evidence that BPA interacts with ERR-gamma as a strong binder, and also to clarify the structural requirements of BPA for its binding to ERR-gamma.

Methods: We examined [3H]BPA in the saturation binding assay using the ligand binding domain of ERR-gamma and analyzed the result using Scatchard plot analysis. A number of BPA derivatives were tested in the competitive binding assay using [3H]BPA as a tracer and in the luciferase reporter gene assay.

Results: [3H]BPA showed a KD of 5.50 nM at a Bmax of 14.4 nmol/mg. When we examined BPA derivatives to evaluate the structural essentials required for the binding of BPA to ERR-gamma , we found that only one of the two phenol-hydroxyl groups was essential for the full binding. The maximal activity was attained when one of the methyl groups was removed. All of the potent BPA derivatives retained a high constitutive basal activity of ERR-gamma in the luciferase reporter gene assay and exhibited a distinct inhibitory activity against 4-OHT.

Conclusion: These results indicate that the phenol derivatives are potent candidates for the endocrine disruptor that binds to ERR-gamma.

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Related in: MedlinePlus

Luciferase-reporter gene assay of BPA and its derivatives for human ERR-γ . (A) Deactivation of the fully activated human ERR-γ by the inverse agonist 4-OHT and sustainment by BPA. (B) Reversing activity of BPA, bisphenol E, and bisphenol AF against the inverse agonist activity of 1.0 μM 4-OHT; 1.0 μM 4-OHT exhibited approximately 0.4-fold deactivation, and the inhibitory activities are shown by the percentage of relative activity. (C) Sustainment of the fully activated human ERR-γ by bisphenol E and bisphenol AF together with inverse agonist activity by 4-OHT. (D) Reversing activity of BPA, 4-α-cumylphenol, and 4-tert-butylphenol; the inverse agonist activity of 4-OHT was clearly reversed by all bisphenols tested in a dose-dependent manner. Data are from a single experiment performed in triplicate; two additional experiments gave similar results. High basal constitutive activity of ERR-γ was evaluated with the luciferase-reporter plasmid (pGL3/3 × ERRE), and the highest activity was estimated in a cell preparation of 1.0 × 105 HeLa cells/well.
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f6-ehp0116-000032: Luciferase-reporter gene assay of BPA and its derivatives for human ERR-γ . (A) Deactivation of the fully activated human ERR-γ by the inverse agonist 4-OHT and sustainment by BPA. (B) Reversing activity of BPA, bisphenol E, and bisphenol AF against the inverse agonist activity of 1.0 μM 4-OHT; 1.0 μM 4-OHT exhibited approximately 0.4-fold deactivation, and the inhibitory activities are shown by the percentage of relative activity. (C) Sustainment of the fully activated human ERR-γ by bisphenol E and bisphenol AF together with inverse agonist activity by 4-OHT. (D) Reversing activity of BPA, 4-α-cumylphenol, and 4-tert-butylphenol; the inverse agonist activity of 4-OHT was clearly reversed by all bisphenols tested in a dose-dependent manner. Data are from a single experiment performed in triplicate; two additional experiments gave similar results. High basal constitutive activity of ERR-γ was evaluated with the luciferase-reporter plasmid (pGL3/3 × ERRE), and the highest activity was estimated in a cell preparation of 1.0 × 105 HeLa cells/well.

Mentions: We found that BPA retained a high constitutive basal activity of ERR-γ in the luciferase reporter gene assay (Figure 6A). ERR-γ is in a full activation with no ligand; it is one of the self-activated NRs and is deactivated by the so-called “inverse agonists” such as 4-OHT (Greschik et al. 2004; Takayanagi et al. 2006). Although BPA shows no apparent effect on the high basal activity of ERR-γ , BPA evidently antagonizes or inhibits the deactivation activity of 4-OHT in a dose-dependent manner (Figure 6B), as reported by Takayanagi et al. (2006). This neutral antagonist is a distinct inhibitor or suppressor of the inverse agonist, reversing the deactivation conformation to the activation conformation.


Direct evidence revealing structural elements essential for the high binding ability of bisphenol A to human estrogen-related receptor-gamma.

Okada H, Tokunaga T, Liu X, Takayanagi S, Matsushima A, Shimohigashi Y - Environ. Health Perspect. (2008)

Luciferase-reporter gene assay of BPA and its derivatives for human ERR-γ . (A) Deactivation of the fully activated human ERR-γ by the inverse agonist 4-OHT and sustainment by BPA. (B) Reversing activity of BPA, bisphenol E, and bisphenol AF against the inverse agonist activity of 1.0 μM 4-OHT; 1.0 μM 4-OHT exhibited approximately 0.4-fold deactivation, and the inhibitory activities are shown by the percentage of relative activity. (C) Sustainment of the fully activated human ERR-γ by bisphenol E and bisphenol AF together with inverse agonist activity by 4-OHT. (D) Reversing activity of BPA, 4-α-cumylphenol, and 4-tert-butylphenol; the inverse agonist activity of 4-OHT was clearly reversed by all bisphenols tested in a dose-dependent manner. Data are from a single experiment performed in triplicate; two additional experiments gave similar results. High basal constitutive activity of ERR-γ was evaluated with the luciferase-reporter plasmid (pGL3/3 × ERRE), and the highest activity was estimated in a cell preparation of 1.0 × 105 HeLa cells/well.
© Copyright Policy - public-domain
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2199305&req=5

f6-ehp0116-000032: Luciferase-reporter gene assay of BPA and its derivatives for human ERR-γ . (A) Deactivation of the fully activated human ERR-γ by the inverse agonist 4-OHT and sustainment by BPA. (B) Reversing activity of BPA, bisphenol E, and bisphenol AF against the inverse agonist activity of 1.0 μM 4-OHT; 1.0 μM 4-OHT exhibited approximately 0.4-fold deactivation, and the inhibitory activities are shown by the percentage of relative activity. (C) Sustainment of the fully activated human ERR-γ by bisphenol E and bisphenol AF together with inverse agonist activity by 4-OHT. (D) Reversing activity of BPA, 4-α-cumylphenol, and 4-tert-butylphenol; the inverse agonist activity of 4-OHT was clearly reversed by all bisphenols tested in a dose-dependent manner. Data are from a single experiment performed in triplicate; two additional experiments gave similar results. High basal constitutive activity of ERR-γ was evaluated with the luciferase-reporter plasmid (pGL3/3 × ERRE), and the highest activity was estimated in a cell preparation of 1.0 × 105 HeLa cells/well.
Mentions: We found that BPA retained a high constitutive basal activity of ERR-γ in the luciferase reporter gene assay (Figure 6A). ERR-γ is in a full activation with no ligand; it is one of the self-activated NRs and is deactivated by the so-called “inverse agonists” such as 4-OHT (Greschik et al. 2004; Takayanagi et al. 2006). Although BPA shows no apparent effect on the high basal activity of ERR-γ , BPA evidently antagonizes or inhibits the deactivation activity of 4-OHT in a dose-dependent manner (Figure 6B), as reported by Takayanagi et al. (2006). This neutral antagonist is a distinct inhibitor or suppressor of the inverse agonist, reversing the deactivation conformation to the activation conformation.

Bottom Line: When we examined BPA derivatives to evaluate the structural essentials required for the binding of BPA to ERR-gamma , we found that only one of the two phenol-hydroxyl groups was essential for the full binding.The maximal activity was attained when one of the methyl groups was removed.These results indicate that the phenol derivatives are potent candidates for the endocrine disruptor that binds to ERR-gamma.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Structure-Function Biochemistry, Department of Chemistry, The Research-Education Centre of Risk Science, Faculty and Graduate School of Sciences, Kyushu University, Fukuoka, Japan.

ABSTRACT

Background: Various lines of evidence have shown that bisphenol A [BPA; HO-C6H4-C(CH3)2-C6H4-OH] acts as an endocrine disruptor when present in very low doses. We have recently demonstrated that BPA binds strongly to human estrogen-related receptor-gamma (ERR-gamma ) in a binding assay using [3H]4-hydroxytamoxifen ([3H]4-OHT). We also demonstrated that BPA inhibits the deactivation activity of 4-OHT.

Objectives: In the present study, we intended to obtain direct evidence that BPA interacts with ERR-gamma as a strong binder, and also to clarify the structural requirements of BPA for its binding to ERR-gamma.

Methods: We examined [3H]BPA in the saturation binding assay using the ligand binding domain of ERR-gamma and analyzed the result using Scatchard plot analysis. A number of BPA derivatives were tested in the competitive binding assay using [3H]BPA as a tracer and in the luciferase reporter gene assay.

Results: [3H]BPA showed a KD of 5.50 nM at a Bmax of 14.4 nmol/mg. When we examined BPA derivatives to evaluate the structural essentials required for the binding of BPA to ERR-gamma , we found that only one of the two phenol-hydroxyl groups was essential for the full binding. The maximal activity was attained when one of the methyl groups was removed. All of the potent BPA derivatives retained a high constitutive basal activity of ERR-gamma in the luciferase reporter gene assay and exhibited a distinct inhibitory activity against 4-OHT.

Conclusion: These results indicate that the phenol derivatives are potent candidates for the endocrine disruptor that binds to ERR-gamma.

Show MeSH
Related in: MedlinePlus