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. Author manuscript; available in PMC: 2018 Aug 15.
Published in final edited form as: Environ Sci Technol. 2017 Jul 28;51(16):9327–9333. doi: 10.1021/acs.est.7b02882

Ligand-Mediated Receptor Assembly As An Endpoint For High-Throughput Chemical Toxicity Screening

Elizabeth K Medlock Kakaley 1, Stephanie A Eytcheson 1, Gerald A LeBlanc 1,*
PMCID: PMC5831241  NIHMSID: NIHMS944762  PMID: 28708939

Abstract

The high throughput screening of chemicals for interaction with intracellular targets is gaining prominence in the toxicity evaluation of environmental chemicals. We describe ligand-mediated receptor assembly as an early event in receptor signaling and its application to the screening of chemicals for interaction with targeted receptors. We utilized bioluminescence resonance energy transfer (BRET) to detect and quantify assembly of the methyl farnesoate receptor (MfR) in response to various high-production volume and other chemicals. The hormone methyl farnesoate binds to the MfR to regulate various aspects of reproduction and development in crustaceans. The MfR protein subunits Met and SRC, cloned from Daphnia pulex, were fused to the fluorophore, mAmetrine and the photon generator, Rluc2, respectively. Ligand-mediated receptor assembly was measured by photon transfer from the photon donor to the fluorophore resulting in fluorescence emission. Overall, the BRET assay had comparable or greater sensitivity as compared to a traditional reporter gene assay. Further, chemicals that screened positive in the BRET assay also stimulated phenotypic outcomes in daphnids that result from MfR signaling. We concluded the BRET assay is an accurate, sensitive, and cost/time efficient alternative to traditional screening assays.

Introduction

Endocrine signaling pathways are crucial to survival and reproduction, however these pathways are often susceptible to disruption by environmental chemicals resulting in perturbations in normal physiology 1-4. Environmental exposure to endocrine disrupting chemicals has been associated with reproductive dysfunction 5, perturbations in reproductive development 6, 7, and population demise 8. As a result, significant effort has gone into the development of screening and testing methods for detecting endocrine-disrupting properties of chemicals, and hazards associated with the use of these chemicals9-13. Screening assays used to detect endocrine-disrupting activity of chemicals often consist of hormone-receptor binding assays or reporter gene transcription assays 9-11. Chemicals that screen positive in one or more of these assays then become a candidate for more definitive testing to assess whole organism consequences of this activity and the exposure concentrations at which effects occur 12. However, receptor-binding assays are relatively uninformative, because they provide no information on the consequence of binding (e.g., receptor activation, inhibition, or no consequence). Reporter gene transcription assays are more informative, however these assays rely upon reporter gene transcription and translation which increases the time required to provide a measurable endpoint.

Most hormone receptors consist of homo- or hetero-dimers 14. The first step in activation of many of these receptors is ligand-stimulated dimerization 14-16. This endpoint can serve as an initiating event in the adverse outcome pathway 17 for many endocrine signaling pathways. This study addresses the potential for protein dimerization to serve as an endpoint for the detection of chemical-induced receptor activation using the methyl farnesoate receptor as a model.

Methyl farnesoate has long been recognized as a hormone involved in reproduction and development in crustaceans 18-20. Methyl farnesoate stimulates male sex determination, in branchiopod crustaceans 18, 19 and male sex differentiation in some decapod crustaceans 21. Recently, we and others identified the protein receptor complex that mediates the actions of methyl farnesoate, the methyl farnesoate receptor (MfR) 22, 23. Further, we demonstrated methyl farnesoate stimulates the association of the protein methoprene tolerant (Met) with its partner, steroid receptor coactivator (SRC) 24. These assembled proteins comprise the active MfR 22-24.

Bioluminescence resonance energy transfer (BRET) technology has gained prominence as a means of measuring protein-protein interactions in cells and in real time25, 26. The method involves the construction of fusion proteins whereby one protein of interest is fused to a luciferase protein. The other protein of interest is fused to a fluorophore. When the proteins associate, photons generated by the luciferase can excite the fluorophore resulting in fluorescence emission. BRET has been extensively used in the study of G-protein coupled receptors 26-28, but more recently has been used in the study of homo- and hetero-dimerization of hormone nuclear receptors 15, 16, 29.

Herein, we describe the construction and optimization of a novel approach to screen chemicals for hormone receptor activation using the MfR cloned from daphnids (Daphnia pulex). We propose that this methodology could be applied to all receptor complexes whose subunit dimerization is agonist driven. Further, we screened several compounds for comparison of specificity and sensitivity to the more traditional luciferase reporter gene transcription assay. Finally, compounds that screened positive in the BRET assay were evaluated in vivo to determine whether results from the cell-based assay accurately predicted phenotypic outcomes in the whole organism.

Material and Methods

Methyl farnesoate (Echelon Biosciences Inc., Salt Lake City, Utah), and all other chemicals (Sigma-Aldrich Corp., St. Louis, MO) screened in BRET and luciferase reporter gene assays, were dissolved in DMSO for delivery to assay solutions. Final DMSO concentrations were 0.001% v/v in the BRET assays and 0.0005% v/v in the reporter gene assays. Hydroprene and diofenolan were dissolved in ethanol for in vivo experiments, where the final concentration of ethanol was 0.0003% v/v.

Fusion Protein Construction

Four fusion proteins were constructed to identify those constructs that provided the optimum BRET signal. The daphnid met open reading frame, described previously 22, was fused to the fluorophore mAmetrine open reading frame (mAme) (excitation: 510 nm, emission: 535nm) (Addgene, Cambridge MA) at either the 5′ or 3′end of met. The daphnid SRC open reading frame, described previously 24, was fused to the Renilla luciferase 2 open reading frame (RLuc2) (substrate: coelenterazine 400A, emission: 410 nm) (Dr. Sanjiv Gambhir, Stanford University, School of Medicine, Stanford, CA) at either the 5′ or 3′ end of SRC.

The met gene was amplified using primers harboring NotHFI (forward containing linker sequence, ATAGCGGAAGTGGTAGCGGAAGTGGT) and ApaI (reverse) restriction enzyme sites, and sub-cloned into the pMT-B vector (ThermoFischer Scientific). mAme was amplified from the pBad cloning vector using primers with KpnI (forward) and NotHFI (reverse) sites, and subcloned at the 5′-terminus of the pMT-met, to create pMT-mAme-linker-met (mAme-Met). A similar procedure was used to construct the pMT-met-linker-mAme (Met-mAme), with some exceptions. Primers harboring KpnI (forward) and NotHFI (reverse) sites were used to amplify met, while primers with NotHFI (forward containing linker sequence) and BstBI (reverse) sites amplified mAme.

SRC was amplified from the TOPO cloning vector using primers harboring BstB1 (forward) and AgeI (reverse) restriction enzyme sites, and was sub-cloned into the pMT-B vector. Rluc2 was amplified from the pcDNA storage vector using primers harboring XhoI (forward) and BstBI (reverse) sites. The reverse primer also contained a nucleotide “linker” sequence (AGCGGAAGTGGTAGCGGAAGTGGC) to lengthen the distance between the two proteins and decrease probability of incorrect folding. The Rluc2-linker sequence was sub-cloned at the 5′-terminus of the pMT-SRC plasmid, to create pMT- Rluc2-linker-SRC (Rluc2-SRC). A similar procedure was used to construct the pMT-SRC-linker-Rluc2 (SRC-Rluc2), with some exceptions. Primers harboring Xho1 (forward) and BstB1 (reverse) sites was used to amplify SRC, while primers with BstB1 (forward containing linker sequence) and Age1 (reverse) sites were used to amplify Rluc2. All four fusion proteins, Rluc2-SRC, SRC-Rluc2, mAme-Met, and Met-mAme, were sequenced (Eton Bioscience, San Diego, CA) to ensure the fluorescent/luminescent proteins were in frame with the respective MfR subunit protein. All fusion constructs were successfully sub-cloned without amino acid substitutions.

BRET Assay

BRET assays were performed as we described previously 24. Assays were performed in Drosophila Schneider (S2) cells (Invitrogen). Cells were grown in Schneider's medium, containing 10% heat inactivated fetal bovine serum (Gibco, Carlsbad, CA, USA), 50 mg/ml penicillin G and 50 mg/ml streptomycin sulfate (Fisher Scientific, Pittsburgh, PA), and incubated at 23°C under ambient air atmosphere. Cells were seeded at a density of 3 × 106 in 35 mm dishes in 6-well plates and transfected 24 hours after plating.

The relevant plasmids were transiently transfected into cells by calcium phosphate DNA precipitation. Total DNA concentration remained constant across all experiments at 2.8 ng/μL, while the photon donor: protein acceptor ratio was held at an optimized 1: 6 ratio (produced greatest energy transfer). Transcription was induced by exposing cells to 500 μM CuSO4 for 24 hours. Cells were treated with test chemical or vehicle control for 1 hour in 1× phosphate-buffered saline medium. The Rluc2 substrate, coelenterazine 400A (Biotium, Inc.), was then added to each well (5.0 μM), and light emission was measured immediately at 410 ± 40 nm (emission produced by Rluc2) and 535 ± 15 nm (emission produced by mAme) using a FluoroStar fluorimeter (BMG Labtech). The ratio of light emitted at 535 nm/410 nm (corrected for basal level donor emission of Rluc2 15, 25) is termed the BRET ratio. The BRET ratio provided a quantitative measure of the degree of MfR protein association. All treatments were replicated 3 times.

Luciferase Reporter Gene Assay

Luciferase-based transcription reporter gene assays were conducted for comparison to BRET with respect to specificity and sensitivity. S2 cells were transiently transfected with daphnid met full open reading frame fused to the Gal4 DNA binding domain22, daphnid SRC, Renilla Luciferase (pRL-CMV, internal transfection control, Promega) and the firefly luciferase reporter gene vector (pGL5-Luc, Promega) that contained five upstream GAL4 binding sites. Following transfection, transcription of Met-Gal4 and SRC was induced with CuSO4 (100 μM for 24 hours). Cells then were treated with test chemical in Ex-cellTM 420 insect serum-free medium with L-glutamine (SAFC Biosciences, Sigma, St. Louis, MO). Cells were harvested after 24 hours incubation. Emissions from the firefly luciferase and Renilla luciferases were measured using the Dual-Glo® luciferase system (Promega). Firefly luciferase emission was normalized to Renilla luciferase emission, and each chemical treatment group was normalized to DMSO control treated cells. All treatments were replicated 3times.

Male Sex Determination

D. magna were cultured under parthenogenetic rearing conditions where all offspring produced are female29,30. Hydroprene and diofenolan were used in in vivo exposure assays to determine their potency in stimulating male sex determination. Gravid adult female daphnids of the same age were selected from cultures, and placed individually in 50 mL beakers containing 40 mL daphnid media. The daphnids were exposed to serial dilutions of the evaluated chemicals, where each treatment consisted of ten individual daphnids. Solutions were renewed daily. Animals were fed 0.20 mg (dry wt) fish food and 7 × 106 algae cells (Pseudokirchneriella subcapitata), prepared as described elsewhere 4, daily and daphnids were assessed for survival, brood release. Survival of maternal organisms was ≥90% in all treatments. The percentage of males in the second brood was used to determine chemical potency. Experiments were terminated after the second brood release. Methods used are described in greater detail elsewhere 30,31.

Statistical Analysis

Comparisons of two means were evaluated for significance (p<0.05) using Student's t test. Equal variance between multiple treatment groups was confirmed with Brown-Forsythe's test. One-way analysis of variance (ANOVA), followed by Tukey's multiple comparison procedure, was used to evaluate significant differences between the control and multiple treatments. All statistics were performed using Origin software (OriginLab Corp., Northhampton, MA).

Results

BRET Assay

Fusion proteins were constructed (Fig. 1) in different configurations and used in BRET assays to determine which configuration provided the strongest BRET ratio. Significant increases in BRET ratio upon the addition of methyl farnesoate occurred with all fusion protein configurations (Fig. 2). However, the greatest increase in BRET ratio between the control and hormone treatment occurred in cells expressing both subunit proteins fused at the N-terminus with the fluorophore or luciferase protein: mAme-Met and Rluc2-SRC (Fig. 2). All subsequent BRET assays were performed with these two protein constructs.

Figure 1. The detection of MfR agonists using BRET.

Figure 1

A) SRC is fused to Rluc2, which metabolizes coelenterazine 400A to emit light at 410 nm. B) In the presence of an agonist (Ag), dimerization with Met is stimulated and the light emitted at 410 nm excites the proximal yellow fluorescent protein, mAmetrine (mAme), that is fused to Met to emit a secondary light at 535 nm.

Figure 2. BRET signaling using MfR subunits with different bioluminescent/fluorescent protein configurations.

Figure 2

Cells were transfected with four different sets of fusion proteins, where Met was fused with a mAmetrine and SRC was fused with Renilla luciferase 2 at either the N- or C- terminus. White bars indicate cells treated with vehicle (DMSO) control and grey bars indicate cells treated with 100 μM methyl farnesoate. Data are presented as mean with standard deviation (n = 3) and an asterisk denotes a significant increase in Met: SRC binding compared to control (p<0.05).

Twenty-nine environmental chemicals (Table S1) were screened at 100 μM for their ability to stimulate MfR subunit association. Chemicals were selected based upon propensity for environmental exposure or structural similarity to methyl farnesoate. All compounds appeared to be non-toxic at 100 μM except for TBBPA which caused a significant (p<0.05, ANOVA) reduction in Rluc2 luciferase. The positive control, methyl farnesoate, and six of the test compounds significantly (p<0.05) stimulated MfR subunit association compared to controls (Fig. 3). These compounds, methoprene, kinoprene, pyriproxyfen, fenoxycarb, diofenoaln, and hydroprene, were all insect growth regulating (IGR) insecticides.

Figure 3. Chemical screen for MfR activation using BRET assay.

Figure 3

White bar indicates DMSO vehicle control, light grey bar indicates the positive positive control (methyl farnesoate), and dark grey bars indicate screened chemicals. Data are presented as means with standard deviations (n = 3) and an asterisk denotes a significant increase in Met-SRC dimerization compared to DMSO control (p<0.05).

The compounds that screened positive were assayed over a range of chemical concentrations to determine relative potency in stimulating MfR association. Chemical potency or assay sensitivity were judged by the lowest observed effect concentration (LOEC). The positive control, methyl farnesoate, significantly activated subunit association with an LOEC of 3.0 μM (Fig. 4A). Methoprene (LOEC = 10 μM) (Fig. 4B) and kinoprene (LOEC = 100 μM) (Fig. 4C) were both less potent at stimulating subunit assembly compared to methyl farnesoate. The remaining compounds, pyriproxyfen (LOEC = 1.0 μM) (Fig. 4D), hydroprene (LOEC = 0.3 μM) (Fig. 4E), diofenolan (LOEC = ≤0.0030 μM) (Fig. 4F), and fenoxycarb (LOEC = 0.0030 μM) (Fig. 4G) were all more potent than the hormone at stimulating MfR subunit association.

Figure 4. MfR BRET assay: concentration-response analysis for MfR activators identified in the initial BRET screen.

Figure 4

Cells containing the MfR BRET fusion proteins were treated with increasing concentrations of compounds that screened positive in the BRET assay: (A) Methyl Farnesoate, (B) Methoprene, (C) Kinoprene, (D) Pyriproxyfen, (E) Hydroprene, (F) Diofenolan, (G) Fenoxycarb. The red circle on the y-axis represents the control group where cells were treated with the DMSO vehicle control. Data are presented as mean with standard deviation (n = 3) and an asterisk denotes significant increase in Met: SRC dimerization compared to control (p<0.01, ANOVA, Tukey's Multiple Comparison test).

MfR Reporter Gene Activation

The same suite of twenty-nine environmental chemicals (Table S1) also were screened for their ability to stimulate MfR-initiated gene transcription (Fig. 5). Only the positive control and five of the six compounds that were active in the BRET assay significantly stimulated transcription of the reporter gene at 100 μM. Kinoprene, the weak agonist in the BRET assay (Fig. 3) failed to activate transcription in the reporter gene assay (Fig. 5).

Figure 5. Chemical screen for MfR activation using luciferase reporter gene assay.

Figure 5

White bar indicates DMSO vehicle control, light grey bar indicates the positive control (methyl farnesoate), and dark grey bars indicate screened chemicals. Data are presented as means with standard deviations (n = 3) and an asterisk denotes significant increase in Met-SRC dimerization compared to DMSO control (p<0.05).

Concentration-response analyses were performed with respect to reporter gene transcriptional activation using methyl farnesoate and the same suite of compounds that were active in the BRET assay. Methyl farnesoate significantly stimulated transcription with an LOEC of 30 μM (Fig. 6A). Methoprene was comparable to the hormone in potency using the reporter gene assay (LOEC = 30 μM, Fig. 6B). Kinoprene, again did not activate transcription in the reporter gene assay (Fig. 6C). Pyriproxyfen (LOEC = 1.0 μM, Fig. 6D), hydroprene (LOEC = 0.30 μM, Fig. 6E), diofenolan (LOEC = 0.30 μM, Fig. 6F), and fenoxycarb (LOEC = 0.010 μM, Fig. 6G) were all more potent than methyl farnesoate in activating gene transcription.

Figure 6. Reporter gene assay: concentration response analysis for MfR activators identified in the initial BRET screen.

Figure 6

Cells expressing MfR subunits and a luciferase reporter gene were treated with increasing concentration compounds that screened positive in the BRET assay:(A) Methyl Farnesoate, (B) Methoprene, (C) Kinoprene, (D) Pyriproxyfen, (E) Hydroprene, (F) Diofenolan, (G) Fenoxycarb. The red circle on the y-axis represents the control group where cells were treated with the DMSO vehicle control. Data are presented as means with standard deviations (n = 3) and an asterisk denotes significant increase in transcription of the luciferase reporter gene compared to control (p<0.05, ANOVA, Tukey's Multiple Comparison test).

Male Sex Determination

Lastly, we evaluated the ability of the BRET MfR subunit association assay to predict male sex determination in D. magna. Two of the most potent chemicals, hydroprene and diofenolan, that generated a BRET signal also stimulated the production of male offspring at low exposure concentrations. Daphnids exposed to 0.001 nM diofenolan (Fig. 7A), and 0.03 nM hydroprene (Fig. 7B) produced all male offspring. All other compounds that screened positive in the BRET assay have been previously shown to stimulate male sex determination in vivo 31, 32, although kinoprene did not stimulate male sex determination in our previous in vivo assay31.

Figure 7. Male offspring production in response to diofenolan and hydroprene exposure.

Figure 7

Adult female daphnids were exposed to diofenolan (A) and hydroprene (B). Data are presented as the percentage of females that produced a male-containing brood at each treatment concentration (n = 9-10).

Discussion

Screening assays that detect the potential for a chemical to elicit toxicity typically utilize an early event in a relevant toxicity pathway33, e.g. ligand-mediated gene transcription. In fact, transcriptional activation assays quantified by reporter genes are extensively used to detect nuclear receptor-chemical interactions in toxicity screening formats34-36. We have demonstrated the use of an endpoint upstream of gene transcription that is well-suited for the evaluation of chemical interactions with many different nuclear receptors.

Nuclear receptors typically function as homo- or hetero-dimers, with dimerization initiated by the binding of an activating ligand37. The BRET assay, as described herein, can be exploited to detect this initial receptor assembly response to agonist binding in living cells and in real-time with no requirement for transcription, translation, and accumulation of a reporter gene product.

We used the BRET assay to screen a battery of 29 chemicals for their ability to activate the methyl farnesoate receptor (MfR). Several of these compounds are designed for environmental use (e.g., pesticides) or designated as high production volume compounds (compounds produced or imported into the United States in quantities equal to or greater than one million pounds per year)38, 39 and thus have potential environmental exposure. Others were selected because they are known to stimulate male sex determination in daphnids or are structurally related to such compounds. In comparison to the reporter gene assay, the BRET assay was as or more efficient in detecting MfR agonists with no false negatives detected. There was one positive result from the BRET assay that was not detected using the reporter gene assay, kinoprene. Kinoprene is a member of the insect growth regulating class of insecticides, akin to all other assayed members of this chemical class which yielded positive weresults in both the BRET and reporter gene assays. This positive result likely represented greater sensitivity of the BRET assay. Furthermore, kinoprene yielded positive results in an in vivo assay for activity towards the MfR40. Based upon this limited dataset, the BRET assay appears to have comparable or greater sensitivity than the reporter gene assay (Table S2).

The MfR mediates the action of the hormone methyl farnesoate in crustaceans22 and the MfR signaling pathway is involved in larval development, metamorphosis, reproductive maturation, and sex determination of offspring21. The methyl farnesoate hormone is the unepoxidated form of an insect hormone ortholog, juvenile hormone III41. Results of the present study suggest that the MfR exhibits a high degree of ligand specificity, as the only compounds that stimulated dimerization, reporter gene activation, and in vivo responses were insect growth-regulating insecticides. These compounds are considered to elicit insecticidal activity by mimicking the action of juvenile hormone42. Further investigation is warranted to establish the extent of this apparent specificity.

All compounds that screened positive in the BRET assay have been shown to stimulate male sex determination in daphnids22, 31, 32, 43. We had not previously evaluated hydroprene and diofenolan in vivo, while, others had reported high potency of these compounds with respect to stimulating male sex determination40, 44. Therefore, we evaluated these compounds in the present study. Consistent with previous results, both compounds stimulate male sex determination at low nM concentrations.

A negative aspect of the BRET assay as compared to reporter gene assays is the lower signal intensity. Maximum BRET ratios detected in the present study were less than 10× those measured in controls. While, maximum reporter gene responses approached 50× control values. Despite the lower signal intensity, BRET results were sufficient to derive statistically discernible activation along with concentration-response relationships. Major positive attributes to the BRET assay are its rapidity, its conduciveness to real-time measurements in intact cells, and reduced cost due to reduced time commitment.

The BRET assay has demonstrated utility with other nuclear receptors such as the estrogen receptor dimer15, the peroxisome proliferator-activated receptor:retinoid × receptor heterodimer45, and the thyroid hormone receptor:retinoid × receptor heterodimer16. The assay typically has been used to assess receptor interaction with its endogenous ligand. Clearly, the methodology can be applied to the screening of environmental chemicals for activity with nuclear receptors. With proper validation46 such assays could significantly increase the capacity of screening programs47 to evaluate receptors and chemicals for interaction.

Supplementary Material

supporting Information

Acknowledgments

This research was supported by US EPA grant RD-835165 and NIEHS grant T32 ES007046.

Footnotes

Supporting Information: Table of the thirty compounds used in screening experiments, along with corresponding IUPAC name, commercial use and/or activity and the screening rationale.

Notes: The authors declare no competing financial interest.

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