Live-cell detection of estrogenic endocrine disruptors

This article and associated images are based on a poster originally authored by Gyuho Choi, Joko Logis, Martin Mangold, and Tae-Jin Kim and presented at ELRIG Drug Discovery 2026 in affiliation with Pusan National University and BMG LABTECH.

This poster is being hosted on this website in its raw form, without modifications. It has not undergone peer review but has been reviewed to meet AZoNetwork's editorial quality standards. The information contained is for informational purposes only and should not be considered validated by independent peer assessment.

This study presents a novel BRET platform that enables screening and classification of eEDCs. It utilizes a BRET-based biosensor to assess all three types of endogen receptor (ER) dimerization, delivering BRET screening in a robust, high-throughput format on the CLARIOstar^{® Plus}.

Introduction

Estrogenic endocrine-disrupting chemicals (eEDCs) are a significant public health concern due to their ability to mimic or interfere with the function of the estrogen receptor (ER).

The binding of a ligand to the ER triggers a critical step, receptor dimerization, in which the ER can form three distinct functional subtypes: two homodimers (ERα/α, ERβ/β) and a heterodimer (ERα/β). ERα homodimers are known to drive breast cancer growth, while ERβ homodimers exhibit anti-proliferative effects.¹ 

The heterodimers, consisting of both ER­ and ERβ, provide a unique balance in regulating gene expression and cellular functions.² Current regulatory guidelines (e.g., OECD Test Guidelines) comprehensively cover ligand binding and transcriptional activation but do not include an assay to assess ER dimerization, leaving a crucial gap in eEDC hazard evaluation.

To address this, a unified platform of highly sensitive ER dimerization-detecting biosensors (ERDDBs) based on bioluminescence resonance energy transfer (BRET) was developed.³ This system enables rapid, real-time assessment and subtype-specific classification of eEDCs, providing necessary mechanistic data for risk assessment.

Assay principle

The ERDDB platform is engineered to detect the physical interaction (dimerization) of two ER monomers in live cells. Each ER monomer is tagged with a BRET component: the donor (Nano-Luciferase) is fused to one monomer, and the acceptor (HaloTag 618) is fused to the other.

Upon binding of estradiol (E2) or eEDC, the receptor dimerizes, bringing the donor and acceptor within 10 nm of each other, resulting in an efficient BRET energy transfer and increased BRET ratio (Figure 1).

Fig. 1. Schematic of the nanoBRET activation mechanism of ERDDB. Image Credit: Gyuho, C et al.

Materials and methods

• White flat-bottom 96-well half-area plate (#675083, Greiner Bio-One)
• CLARIOstar Plus (BMG LABTECH)
• 17β-estradiol (E2) (#S1709, Selleck Chem)
• Seventy-two estrogen analogs or EAs (MFDS, Rep. of Korea)
• HaloTag^® NanoBRET^® 618 Ligand, NanoBRET Nano-Glo^® Substrate, Nano-Glo Endurazine Substrate (#G980A, #N157A, #N257B) Promega
• α/α, α/β and β/β ERDDB stable cell lines (according to 3)
• Assay media (according to 3)
• Chemicals and drugs from various manufacturers (according to 3)

eEDCs screening

For the eEDCs screening assay, the assay media consisted of clear DMEM (GenDEPOT, CM004-310) supplemented with 0.5% FBS, 1% penicillin (100 U/mL), and 100 μg/mL streptomycin.

Stable cells were cultured in 75T flasks (SPL, 70075) and harvested prior to the assay. The cells were resuspended in assay media, and 50,000 cells were added to a white solid-bottom 96-well half-area plate and incubated overnight. The endurazine substrate (final dilution 1:100) was injected into the wells two hours before the measurement.

After two hours, luminescence was measured to obtain data before treatment, and then the wells were treated with estrogen hormone E2 and various EAs at a 1 μM final concentration. Plates were measured at six, 12, and 24 hours after treatment.

The sigmoidal dose-response curves and logEC values were generated by plotting log(agonist) values against the response-variable slope3. The BRET ratio and nBRs were calculated using the following formula:

Classification of dimerization was performed using an E2 standard graph based on the criteria shown in the range below.

Fig. 2. Classification of dimerization using E2 standards. Dose-response curves (A) and BRET ratios (B) were established using E2 to classify the ERDDB biosensors. Image Credit: Image courtesy of Gyuho Choi et al., in partnership with ELRIG (UK) Ltd.

Instrument settings

 Source: BMG LABTECH GmbH

Results and discussion

The optimized ERDDB stable cell line demonstrated highly sensitive, dose-dependent responses to E2 across all three dimer types, as shown by the sigmoidal dose-response curves for all three dimer types (Figures 3A–C).

Fig. 3. The assay was further validated using known ER-modulating drugs (e.g., Fulvestrant, 4-Hydroxytamoxifen), which elicited the expected dose-dependent increases in the BRET ratio across all three dimer types, confirming that the biosensors specifically respond to ER dimerization-inducing agents (results not shown here).

Next, a total of 72 estrogen analogs (EAs) were screened using the ERDDB stable cell line to identify their specific dimerization profiles. The Normalized BRET Ratio (nBR) of each EA was compared against classification criteria mentioned above (Data not shown, available as Figure 5 in 3). Image Credit: Image courtesy of Gyuho Choi et al., in partnership with ELRIG (UK) Ltd.

Progesterone and Hydroxyflutamide were identified as selectively inducing only the ERα/α homodimer. Phytoestrogens like Coumestrol, Daidzein, and Genistein were classified as preferentially affecting ERβ-containing dimers.

Strong inducers like 17β-Estradiol (E2), Fulvestrant, and Tamoxifen were confirmed to promote dimerization across all three subtypes (α/α, α/β, and β/β). Overall, this cross-comparison method enabled the classification of 26 estrogen analogs based on their induction profiles across the three ER dimer types.

Conclusion

The optimized nanoBRET-based ERDDB platform provides a robust, reproducible, and sensitive method for the real-time detection of estrogen receptor dimerization in live cells.

The performance and sensitivity of the CLARIOstar Plus microplate reader enable this assay to successfully classify eEDCs by subtype, fulfilling a critical need for comprehensive assessment based on ER dimerization. This is a promising screening tool for eEDCs that is likely to be incorporated into the OECD test guidelines.

Download the original poster

References

1. Song, D., et al. (2022). ERα and ERβ Homodimers in the Same Cellular Context Regulate Distinct Transcriptomes and Functions. Frontiers in endocrinology, 13. DOI:10.3389/fendo.2022.930227. https://www.frontiersin.org/journals/endocrinology/articles/10.3389/fendo.2022.930227/full
2. MMonroe, D.G., et al. (2005). Estrogen Receptor α and β Heterodimers Exert Unique Effects on Estrogen- and Tamoxifen-Dependent Gene Expression in Human U2OS Osteosarcoma Cells. Molecular Endocrinology, 19(6), pp.1555–1568. DOI:10.1210/me.2004-0381. https://academic.oup.com/mend/article/19/6/1555/2738062
3. Choi, G., et al. (2024). Novel Estrogen Receptor Dimerization BRET-Based Biosensors for Screening Estrogenic Endocrine-Disrupting Chemicals. Biomaterials Research, 28. DOI:10.34133/bmr.0010. https://spj.science.org/doi/full/10.34133/bmr.0010

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Last Updated: Oct 12, 2026