Agonism, Estrogen receptor

Site of action: The molecular site of action is the estrogen receptor (ER). ERs are members of the steroid hormone receptor family which belongs to a group of nuclear receptors that are transcriptionally activated by ligands leading to downstream activation of many cellular processes. ERs are composed of three principal domains – N-terminal domain (NTD), DNA binding domain (DBD), and the ligand binding domain (LBD). ER binds to specific DNA sequences known as estrogen response elements (EREs); EREs are generally short sequences located in the promoter region but can also exist in introns or exons (Klinge, 2001). ER-mediated gene transcription is initiated by binding of the DBD to an ERE with two distinct transcriptional activation domains, AF1 and AF2, located on the NTD and LBD respectively (Kumar et al., 2011).

Responses at the macromolecular level: ER’s bind to endogenous and exogenous compounds and are activated by endogenous ligands such as estrone (E1), estradiol (E2) and estriol (E3) (Ng et al., 2014). There are numerous compounds (e.g., natural or pharmaceutical estrogens, alkylphenols, organochlorine pesticides, phthalates, etc.) that can act as estrogen agonists or antagonists, and effectively mimic or block the natural effects of estrogens on the ER (Pillon et al., 2005; Schmieder et al., 2014).

ER is part of a multi-protein complex consisting of HSP 90, HSP 70, and immunophilins (Stice & Knowlton, 2008). In this multi-protein complex HSP 90 is the dominant protein and its binding to ER is essential for ER conformational binding of 17β-estradiol (Segnitz & Gehring, 1997). When binding on the LBD receptor occurs ER dissociates from HSP 90 and leads to receptor dimerization which can either be homodimers from the same isoform (ERα-Erα) or heterodimers containing one unit from both isoforms (ERα-Erβ) (Fliss et al., 2000). The translocation of these dimers into the nucleus modulates gene transcription (Aranda & Pascual, 2001).

• OECD Test No. 455: Performance-based test guideline for stably transfected transactivation in vitro assays to detect estrogen receptor agonists and antagonists (OECD 2021).
• OECD Test No. 457: BG1Luc Estrogen Receptor Transactivation Test Method for Identifying Estrogen Receptor Agonists and Antagonists (OECD 2012).
• Standard Evaluation Procedure (SEP) for estrogen receptor transcriptional activation (Human Cell Line HeLa-9903) assay was developed by the U.S. Environmental Protection Agency (EPA).
• ER-based transactivation assays that have been used to detect ER agonists and antagonist using cell lines include T47D-Kbluc assay (Wehmas et al., 2011), the ERα CALUX assay (Van et al.); MELN assay (Berckmans et al., 2007); and the yeast estrogen screen (YES; (De Boever et al., 2001)). The T47D-Kbluc assay responds to both ERα and ERß agonists but support the assumption that ERα is inducing more reporter expression than ERß. Each of these assays have undergone some level of validation.
• Browne et al. (2015) integrated 18 ER ToxCast high-throughput screening (HTS) assays, measuring ER binding, dimerization, chromatin binding, transcriptional activation and ER-dependent cell proliferation, into the ToxCast ER pathway model. This mathematical model that in vitro assays to predict whether a chemical is an ER agonist or antagonist.

Taxonomic applicability: In mammals there are two ER subtypes, ER alpha (ERα) and ER beta (ERβ), which are located on chromosome 6 and 14 and encoded by two different genes (ESR1 and ESR2) (Ascenzi et al., 2006). ERs were conventionally identified as mammal specific, but most vertebrates contain functional ERs. However, although teleost fish have receptors homologous to mammilian ERα, ERβ is divided into ERβ1 and ERβ2 resulting in three distinct ERs (Asnake et al., 2019; Menuet et al., 2004; Menuet et al., 2002). The majority of invertebrates (i.e. mollusks) possess a gene that is the orthologue of the vertebrate ER but in many species it has been demonstrated to only have constitutive transcriptional activity, and is not activated by ligand binding (Balbi et al., 2019). However, ERs in annelids share functional characteristics with vertebrate ERs and its transcriptional activity can be disrupted by known endocrine-disrupting substances (Keay & Thornton, 2009).

This MIE would generally be viewed as relevant to vertebrates, but not invertebrates.

Life stage: This MIE is applicable to all life stages.

Sex: This MIE is applicable to both sexes.

• Aranda, A., & Pascual, A. (2001). Nuclear hormone receptors and gene expression. Physiological reviews, 81(3), 1269-1304.
• Ascenzi, P., Bocedi, A., & Marino, M. (2006). Structure–function relationship of estrogen receptor α and β: Impact on human health. Molecular aspects of medicine, 27(4), 299-402.
• Asnake, S., Modig, C., & Olsson, P.-E. (2019). Species differences in ligand interaction and activation of estrogen receptors in fish and human. The Journal of steroid biochemistry and molecular biology, 195, 105450.
• Balbi, T., Ciacci, C., & Canesi, L. (2019). Estrogenic compounds as exogenous modulators of physiological functions in molluscs: Signaling pathways and biological responses. Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology, 222, 135-144.
• Berckmans, P., Leppens, H., Vangenechten, C., & Witters, H. (2007). Screening of endocrine disrupting chemicals with MELN cells, an ER-transactivation assay combined with cytotoxicity assessment. Toxicology in vitro, 21(7), 1262-1267.
• Browne, P., Judson, R. S., Casey, W. M., Kleinstreuer, N. C., & Thomas, R. S. (2015). Screening Chemicals for Estrogen Receptor Bioactivity Using a Computational Model. Environmental Science & Technology, 49(14), 8804-8814. https://doi.org/10.1021/acs.est.5b02641
• De Boever, P., Demaré, W., Vanderperren, E., Cooreman, K., Bossier, P., & Verstraete, W. (2001). Optimization of a yeast estrogen screen and its applicability to study the release of estrogenic isoflavones from a soygerm powder. Environmental Health Perspectives, 109(7), 691-697.
• Fliss, A. E., Benzeno, S., Rao, J., & Caplan, A. J. (2000). Control of estrogen receptor ligand binding by Hsp90. The Journal of steroid biochemistry and molecular biology, 72(5), 223-230.
• Keay, J., & Thornton, J. W. (2009). Hormone-activated estrogen receptors in annelid invertebrates: implications for evolution and endocrine disruption. Endocrinology, 150(4), 1731-1738.
• Klinge, C. M. (2001). Estrogen receptor interaction with estrogen response elements. Nucleic Acids Res, 29(14), 2905-2919. https://doi.org/10.1093/nar/29.14.2905
• Kumar, R., Zakharov, M. N., Khan, S. H., Miki, R., Jang, H., Toraldo, G., Singh, R., Bhasin, S., & Jasuja, R. (2011). The dynamic structure of the estrogen receptor. Journal of amino acids, 2011.
• Menuet, A., Le Page, Y., Torres, O., Kern, L., Kah, O., & Pakdel, F. (2004). Analysis of the estrogen regulation of the zebrafish estrogen receptor (ER) reveals distinct effects of ERalpha, ERbeta1 and ERbeta2. Journal of Molecular Endocrinology, 32(3), 975-986.
• Menuet, A., Pellegrini, E., Anglade, I., Blaise, O., Laudet, V., Kah, O., & Pakdel, F. (2002). Molecular characterization of three estrogen receptor forms in zebrafish: binding characteristics, transactivation properties, and tissue distributions. Biology of reproduction, 66(6), 1881-1892.
• Ng, H. W., Perkins, R., Tong, W., & Hong, H. (2014). Versatility or Promiscuity: The Estrogen Receptors, Control of Ligand Selectivity and an Update on Subtype Selective Ligands. International Journal of Environmental Research and Public Health, 11(9), 8709-8742. https://www.mdpi.com/1660-4601/11/9/8709
• Pillon, A., Boussioux, A.-M., Escande, A., Aït-Aïssa, S., Gomez, E., Fenet, H., Ruff, M., Moras, D., Vignon, F., & Duchesne, M.-J. (2005). Binding of estrogenic compounds to recombinant estrogen receptor-α: application to environmental analysis. Environmental Health Perspectives, 113(3), 278-284.
• Schmieder, P. K., Kolanczyk, R. C., Hornung, M. W., Tapper, M. A., Denny, J. S., Sheedy, B. R., & Aladjov, H. (2014). A rule-based expert system for chemical prioritization using effects-based chemical categories. SAR and QSAR in Environmental Research, 25(4), 253-287. https://doi.org/10.1080/1062936X.2014.898691
• Segnitz, B., & Gehring, U. (1997). The function of steroid hormone receptors is inhibited by the hsp90-specific compound geldanamycin. Journal of Biological Chemistry, 272(30), 18694-18701.
• Stice, J. P., & Knowlton, A. A. (2008). Estrogen, NFκB, and the heat shock response. Molecular Medicine, 14, 517-527.
• Van, d., Winter, R., Weimer, M., Beckmanns, P., Suzuki, G., Gijsberg, L., Jonas, A., Van, d. W., Hilda, & Aarts, J. Optimization and Prevalidation of the in Vitro ER CALUX Method to Test Estrogenic and Antiestrogenic Activity of Compounds.
• Wehmas, L. C., Cavallin, J. E., Durhan, E. J., Kahl, M. D., Martinovic, D., Mayasich, J., Tuominen, T., Villeneuve, D. L., & Ankley, G. T. (2011). Screening complex effluents for estrogenic activity with the T47D‐KBluc cell bioassay: Assay optimization and comparison with in vivo responses in fish. Environmental toxicology and chemistry, 30(2), 439-445.