| Direct Evidence Revealing Structural
Elements Essential for the High
Binding Ability of
Bisphenol A to Human Estrogen-Related Receptor-γ Hiroyuki Okada, Takatoshi Tokunaga,
Xiaohui Liu, Sayaka Takayanagi, Ayami Matsushima, and Yasuyuki
Shimohigashi 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-γ (ERR-γ) 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-γ as a strong binder, and also to clarify the structural requirements of BPA for its binding to ERR-γ. Methods: We examined [3H]BPA in the saturation binding assay using the ligand binding domain of ERR-γ 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-γ, 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-γ 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-γ. Key words: bisphenol A, constitutive activity, endocrine disruptor, estrogen receptor, estrogen-related receptor-γ, inverse agonist, nuclear receptor. Environ Health Perspect 116:32–38 (2008) . doi:10.1289/ehp.10587 available via http://dx.doi.org/ [Online 5 October 2007]
Address correspondence to Y. Shimohigashi, Laboratory of Structure-Function Biochemistry, Department of Chemistry, The Research-Education Centre of Risk Science, Faculty of Sciences, Kyushu University, Fukuoka 812-8581, Japan. Telephone: 81-92-642-2584. Fax: 81-92-642-2584. E-mail: shimoscc@mbox.nc.kyushu-u.ac.jp This study was supported by Health and Labour Sciences Research Grants for Research on the Risk of Chemical Substances from the Ministry of Health, Labor and Welfare of Japan. The authors declare they have no competing financial interests. Received 22 June 2007 ; accepted 4 October 2007.
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