392Identification of androgen disruptors using a cell-based androgen receptor dimerization assay

Barry Whyte, Scott Klayner and Carl Peters
BMG LABTECH, Cary, NC, USA

  • Androgen receptor dimerization assay (AR2) enables screening of agonists, antagonists and xenobiotic metabolites

  • AR2 receptor dimerization assay is a viable alternative to existing toxicology assays for androgen disruptors

  • Detection using NanoLuc® Binary Technology (NanoBiT®) was performed on the CLARIOstar® Plus microplate reader

Introduction

Androgen-disrupting chemicals are molecules found in the environment that interfere with male fertility by disrupting hormone signaling.1 Current screening assays for agonists and antagonists of androgen receptors are limited to time-consuming and resource-intensive in vitro tests and in vivo animal assays. Viable alternatives are therefore needed that offer high throughput capabilities for screening of androgen-disrupting chemicals and their metabolites (xenobiotics).

One type of assay that is amenable to scale and predictive value for the identification of androgen disruptors is the androgen receptor dimerization assay. Androgen receptor dimers control the expression of key genes involved in hormonal signaling and the different events linked to normal male development. Androgen receptor dimerization assays can be used to study agonists that activate the dimeric androgen receptor or antagonists which block the normal function of the active receptor.

In this study, a novel cell-based receptor dimerization assay (AR2) was developed that measures ligand-dependent androgen receptor homodimerization using the NanoLuc® Binary Technology (NanoBiT®).2 When the Large BiT and Small BiT components come together, they form a functional NanoLuc luciferase enzyme suitable for determination of protein-protein interactions for the homodimer receptor (Fig.1). This system can be used to study the effects of different agonists and antagonists.

Fig. 1: Assay principle for androgen receptor dimerization assay using NanoBit technology.For antagonists, fluorescent cell viability measurements were performed in parallel. These measurements permit assessment of any loss of luminescence due to antagonists impacting cell viability as opposed to androgen receptor activity. In addition, the AR2 receptor dimerization method was extended to include measurements related to cytochrome P450 (CYP) enzymes and to determine the impact of xenobiotic compounds on receptor activity.

 

Materials & methods
  • Solid white 384-well microplates, Greiner Bio-One
  • OptiMEM medium, Life Technologies
  • Human hepatocellular carcinoma cells (HepG2), American Type Culture Collection
  • LgBiT and SmBiT (NanoBiT), Promega
  • Furimazine, AOBIOUS
  • Reazurin sodium, Thermo Fisher
  • CLARIOstar Plus microplate reader, BMG LABTECH

Experimental procedure
AR2 receptor dimerization assays were performed to measure responses to different agonists and antagonists. Assessment of cell viability was added to antagonist mode tests. The complete experimental details have been previously reported.2

Instrument settings for cell viability assays

Optic settings
 
 

 
 
Fluorescence, top optic
Monochromator
settings

Excitation: 530
Dichroic: 558.8
Emission: 590
Gain

860
Focal height

7.8 mm
General settings
 Number of flashes 35
Cycle time 20 sec

 

 

Instrument settings for AR2 receptor dimerization assays

Optic settings
 
Luminescence, endpoint
Gain

EDR
General settings
 Integration time 0.08 sec

Results & Discussion

In this study, more than 100 compounds were tested in a novel androgen receptor dimerization assay for activity as agonist or antagonists. An example of agonist mode testing with AR2 receptor dimerization assays is shown in Fig.2. R1881 (metribolone) elicited a >50-fold response at concentrations above 10 nM and a half-maximal activity concentration (AC50) of 319 pM. Testosterone was not as effective as R1881, evoking only a 23-fold response. In subsequent experiments, R1881 was selected as the positive control for agonist mode experiments where test chemicals were evaluated for their ability to promote AR
receptor dimerization.

AN392-Fig2Fig. 2: Agonist mode testing in AR2 receptor dimerization assays. AR2 cells were treated with agonist R1881 (purple squares) and testosterone (grey circles) at 100 pM to 1 μM. NanoLuc luciferase activity was measured after 18 h of incubation. Data represent duplicate wells for each concentration tested in two independent experiments.

Figure 3 shows an example of antagonist mode testing using AR2 receptor dimerization assays. R1881 was co-administered with a titrated series of five known AR antagonists and a negative control for 18 h. Cyproterone acetate was the most potent AR antagonist with an AC50 of 240 nM. Amitrole was used as a negative control and did not significantly inhibit AR dimerization. Collectively the data show that the AR2 receptor dimerization assay responds predictably to both agonists and antagonists.

Fig. 3: Antagonist mode testing in AR2 receptor dimerization assays. Samples were cotreated with 10 nM R1881 and selected antagonists ranging from 10 nM to 100 μM for 18 h before measuring nanoluciferase activity. Data represent individual wells from fi ve independent experiments. Amitrole (yellow squares); cyproterone acetate (green circles).

The 128 chemicals used in the study were re-tested in the presence of individual human CYP enzymes using an mRNA transfection method.3 This reevaluation changed the activity calls for five compounds (as an example, the dose-response curve for flutamide is shown in Fig.4) and shifted the estimated potencies for several others. The AR2 receptor dimerization assay was thus capable of being retrofitted to look at androgen-disrupting metabolites generated by human cytochrome P450 (CYP) enzymes.

Fig. 4: Bioactivation of fl utamide by CYP1A2. AR2 cells were transfected with mRNAs encoding beta-galactosidase (black), CYP1A2 (red) or no RNA (grey) for 6 h then exposed to fl utamide for 18 h in antagonist mode. Data represent individual wells from three independent experiments.

Conclusion

The AR2 receptor dimerization assay is a robust, fast, and reliable way to screen for agonists, antagonists and xenobiotic metabolites. With robust Z’ factors of 0.94 and 0.85 for agonists and antagonists, respectively, this assay is well suited to high-throughput screening. The AR2 receptor dimerization assay successfully classified known agonists and antagonists as well as or better than legacy assays with equal or higher estimated potencies. The BMG LABTECH CLARIOstar Plus microplate reader was the ideal platform to measure luminescence changes for the NanoBiT in these cell-based assays which was crucial to the measurement of androgen disruptor activity.

References

  1. Luccio-Camelo DC and Prins GS. Disruption of androgen receptor signaling in males by environmental chemicals. Journal of Steroid Biochemistry and Molecular Biology (2011) 127(1-2):74-82. doi: 10.1016/j.jsbmb.2011.04.004.

  2. Brown EC et al., High-throughput AR dimerization assay identifies androgen disrupting chemicals and metabolites. Frontiers in Toxicology (2023) 5:1134783.doi:10.3389/ftox.2023.1134783.

  3. DeGroot DE et al., mRNA transfection retrofi ts cellbased assays with xenobiotic metabolism. Journal of Pharmacological and Toxicological Methods (2018) 92:77-94. doi: 10.1016/j.vascn.2018.03.002.
Newsletter Sign-up