A Novel Dual-Color Luciferase Reporter Assay for Simultaneous

Article pubs.acs.org/crt

A Novel Dual-Color Luciferase Reporter Assay for Simultaneous Detection of Estrogen and Aryl Hydrocarbon Receptor Activation Patrick Tarnow,*,† Steffi Bross,† Lisa Wollenberg,† Yoshihiro Nakajima,‡ Yoshihiro Ohmiya,§ Tewes Tralau,† and Andreas Luch† †

German Federal Institute for Risk Assessment, Department of Chemical and Product Safety, Max-Dohrn-Strasse 8-10, 10589 Berlin, Germany ‡ Health Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 2217-14 Hayashi-cho, Takamatsu, Kagawa 761-0395, Japan § DAILAB, Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan ABSTRACT: Consumers are exposed to a plethora of anthropogenic and natural substances that can act as agonists or antagonists for various transcription factors. Depending on the exposure and potency, such interactions can potentially lead to adverse health effects, particularly for substances with multiple molecular targets. The early detection of such interactions is thus of high toxicological interest. Here, we report on the development of a new cellular dual-color reporter assay that allows for time-resolved and quantitative recording of estrogen receptor (ER) and aryl hydrocarbon receptor (AHR) activation in living cells. Both receptors are known for their ligand promiscuity. Moreover, both receptor signaling pathways are interconnected by direct protein−protein interactions as well as by shared protein factors and the competition for ligands. The assay is based on two rare beetle luciferases that emit light in the red (SLR) and green (ELuc) spectrum and that have been stably inserted into human T47D mammary carcinoma cells. The corresponding cell line is termed “XEER” and has been successfully subjected to proof-ofprinciple studies using prototypical ER and AHR ligands as well as various phytochemicals, xenobiotics, and extracts from various plastic products.

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INTRODUCTION

cofactor recruitment at the so-called estrogen-responsive elements (EREs) within the promoters of ER-regulated genes.4 Physiological reactions influenced by ERs comprise reproductive physiology as well as key developmental and metabolic processes. Meanwhile, the aryl hydrocarbon receptor (AHR) is one of the key regulators for xenobiotic phase I metabolism and tryptophan-mediated signaling.5−7 Apart from regulating CYP1 gene expression, AHR is involved in immune responses, autoimmunity, carcinogenesis, and estrogenic and host-microbiome signaling. AHR belongs to the class of basic helix-loophelix Per-ARNT-Sim domain (bHLH-PAS) proteins and is perceptible to a wide range of ligands, many of which are xenobiotics.8 In its unbound state, the receptor is part of a cytoplasmic complex together with HSP90, XAP2, and other proteins. Upon ligand binding, AHR translocates into the nucleus, where it heterodimerizes with its binding partner, the aryl hydrocarbon receptor nuclear translocator (ARNT) before binding at xenobiotic response elements (XREs) within the DNA. Cytochrome P450-dependent monooxygenases (CYPs) such as CYP1A1 and CYP1B1 are among the transcripts

Endocrine disruptors are substances or mixtures that are capable of interfering with the hormone system or with specific downstream events of hormone action to an extent that leads to adversity in an organism or a population.1,2 However, in contrast to classical toxicological end points that are usually based on observations of distinct pathophysiology or histopathology, the concept of endocrine disruption is a mechanistic one that subsumes a plethora of physiological effects. This not only poses an academic challenge but has far reaching consequences for the health assessment and regulation of chemicals with endocrine potential, many of which interfere with the endocrine system by direct binding to hormone receptors. Of particular interest in this context are the estrogen receptors (ERs) ERα and ERβ.3 As ligand-activated transcription factors, they are the target of a variety of anthropogenic or plant-derived substances, so-called xenoand phytoestrogens. In addition, ERα is subject to complicated crosstalk with the similarly promiscuous aryl hydrocarbon receptor (AHR) and its regulon. Binding of cognate ligands triggers ER dimerization followed by subsequent binding and © 2017 American Chemical Society

Received: March 20, 2017 Published: June 14, 2017 1436

DOI: 10.1021/acs.chemrestox.7b00076 Chem. Res. Toxicol. 2017, 30, 1436−1447

Article

Chemical Research in Toxicology

Cell Culture. Cell culture medium and serum were derived from PAN (Aidenbach, Germany) and supplemented with hygromycin B (Roth, Karlsruhe, Germany) as required. The human breast cancer cell line T-47D (ATCC HTB-133) was purchased from ATCC (LGC Standards GmbH, Wesel, Germany). For routine passaging, T47-D cells were cultivated in Dulbecco’s minimal essential medium (DMEM) supplemented with 10% (v/v) fetal calf serum (FCS), 0.2 U/mL insulin, and 1% (v/v) penicillin/streptomycin solution. The cells were cultivated in a CO2-enriched atmosphere (5%) at 37 °C and passaged twice per week. Plasmids and Clone Construction. EREs and XREs (5′CAGGTCACAGTGACCTG-3′ and 5′-TCTCACGCTAGCAGATT3′, respectively) were amplified as 6-fold repeats from two existing reporter plasmids, including the corresponding minimal promoter sequence using appropriate primers (Fwd: 5′-GCCTCGAGGGCCTAACTGGCCGGTACC-3′ and Rev: 5′-GCACTAGTTTACCAACAGTACCGGATTGCC-3′). The respective reporter constructs are based on the pGL4.26 luciferase reporter system (Promega) and were kindly provided by Dr. Peter J. Hofmann, Institute for Experimental Endocrinology, Charité, Berlin, Germany. Following PCR amplification, the promoter elements were purified using standard DNA purification kits (Qiagen, Hilden, Germany) and cloned into pELucPEST and pSLR using XhoI and SpeI.27,30 After verification by sequencing, the resulting constructs (pELuc-XRE and pSLR-ERE) were transfected into T-47D cells with successive transfections preventing nonhomologous end joining and coinsertion of the plasmids at the same genomic locus.31 Transfections were performed using Lipofectamine 2000 and Opti-MEM following the protocol of the manufacturer (Invitrogen) with subsequent clone selection from single colonies on 250 μg/mL of hygromycin B (pELuc-XRE) and 0.05 μg/mL of puromycin (pSLR-ERE) in conjunction with ligandinducible luminescence. Western Blotting. For Western blot analysis, cells (6 × 105) were seeded into 6-well plates using DMEM. The cells were allowed to attach for approximately 6 h before subjecting them to 65 h of hormone starvation in phenol red-free DMEM supplemented with 5% (v/v) CCD-FCS, followed by test substance stimulation as indicated. Subsequent to substance treatment, blotting was commenced as reported previously.32 Primary antibodies used were anti-AHR (H211), anti-CYP1A1 (H-70), anti-ERα (F-10) (all from Santa Cruz, Heidelberg, Germany), and anti-GAPDH (MAB374, Millipore, Darmstadt, Germany). The corresponding peroxidase-labeled secondary antibodies were obtained from Dianova (Hamburg, Germany). Dual-Color Luciferase Assay. Reporter cells (T-47D-XEER) were passaged using DMEM with 10% (v/v) FCS, 0.2 U/mL insulin, 1% (v/v) penicillin/streptomycin solution, 250 μg/mL of hygromycin, and 0.05 μg/mL of puromycin. Assays were performed in clearbottomed white 96-well tissue culture dishes (Corning) with an initial seed of 4.5 × 104 cells per 150 μL per well. The cells were allowed to attach for 6−8 h before the medium was replaced with HF-DMEM (DMEM without phenol red and supplemented with 5% (v/v) charcoal-stripped FCS and penicillin/streptomycin, hygromycin B, and puromycin as indicated previously). The cells were then subjected to hormone starvation for another 65 h. Subsequent to hormone starvation, the assay was commenced in fresh medium (NaHCO3-free DMEM supplemented with 25 mM HEPES, 5% (v/v) charcoalstripped FCS, and 0.2 U/mL insulin, pH 7.2; HF-DMEM-HEPES) containing 200 μM D-luciferin and test substances as required. DMSO concentrations were 0.1% (v/v) for stimulations with single substances and 0.2% (v/v) in coexposure experiments. Luminescence was measured in a SynergyHT microplate reader (BioTek, Bad Friedrichshall, Germany) at 37 °C under normal atmosphere in 1 h intervals or once after 20−24 h, respectively. Each well was measured twice using recording intervals of 20 s, thereby passing the signal of the second measurement through a 570 nm long-pass filter. Background corrected signals for ELuc (E) and SLR (R) were subsequently quantified based on the individual transmission coefficients (κE57 = 0.079 for ELuc and κR57 = 0.865 for SLR, Ltot = unfiltered luminescence, L570 = filtered luminescence) using eq 1

directly induced by AHR activation. As part of the eukaryotic phase I response, these two enzymes accept a wide range of (xenobiotic) substrates (e.g., polycyclic aromatic hydrocarbons (PAHs)), facilitating subsequent substrate conjugation and excretion. However, for some substrates, the monooxygenation triggers the formation of toxic metabolites (i.e., genotoxicants) rather than initiating substrate elimination.9 This is the case for benzo[a]pyrene (BaP) and other PAHs. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), the prototypic AHR activator, is also known to adversely affect estrous cyclicity and ovulation in rats.10,11 Likewise, CYP-generated catechol estrogen metabolites are known to form DNA adducts.12 Crosstalk of ER and AHR functions via several distinct mechanisms. Both receptors compete for the same rate-limiting cofactors,13 CYPs induced by AHR catalyze the conversion of endogenous estrogens,14 and most importantly, both receptors are able to physically interact with each other. This results in the recruitment of estrogen receptor alpha (ERα) to XREs and AHR/ARNT to ERE-containing promoters.15,16 Moreover, binding of activated AHR to ERα leads to rapid proteasomal degradation of the latter and thus to inhibition of ER-mediated signaling.17,18 Finally, both receptors have overlapping ligand ranges with some ER agonists binding to the AHR and vice versa.19,20 Although not fully understood, this crosstalk is likely to have far reaching biological and toxicological implications as chemical mixtures and environmental background exposures are likely to stimulate both receptors.21−23 For example, coplanar polychlorinated biphenyls (PCBs), which are persistent and ubiquitous contaminants, are known to activate AHR (so-called dioxin-like PCBs), whereas others have been shown to induce ER signaling24 or elicit antiestrogenic effects.25 Therefore, there is an urgent need for tools to investigate and screen AHR-ER costimulation in more detail. Transactivation assays are ideal for this purpose. They are reliable, robust, and fast and bring the benefit of being well-established tools for the screening of ligand-triggered receptor activation, be it for the discovery of new drugs or the identification of potential toxicants.26 However, most of the assays available are based on the firefly luciferase from Photinus pyralis. This luciferase features a low signal-to-noise ratio, a high dynamic range, and an emission predominantly comprising the yellow spectrum, thus making it unsuitable for monitoring more than one target gene. This study now reports on the creation of a new luciferase-based reporter system suitable for simultaneous detection of AHR and ER activation. The system makes use of two luciferases with distinct emission ranges, that is, the green-emitting emerald luciferase (ELuc) from the Brazilian click beetle Pyrearinus termitilluminans27 and the red-emitting “stable luciferase red” (SLR) luciferase from the railroad worm Phrixothrix hirtus.28,29 In addition, both luciferases conveniently use D-luciferin, which is cell permeable, nontoxic, and highly stable, thus allowing time-resolved quantitative measurements in living cells.

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EXPERIMENTAL PROCEDURES

Chemicals. TCDD was obtained as dissolved standard in dimethyl sulfoxide (DMSO) from LCG standards (Wesel, Germany), and Dluciferin was provided by PJK (Kleinbittersdorf, Germany). PCBs were obtained from LCG standards. All other chemicals were purchased from Sigma-Aldrich (Taufkirchen, Germany). All test substances were routinely dissolved in DMSO and stored in Teflon-capped glass vials at −20 °C until further use. 1437


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Figure 1. Measurement of ERα and AHR activation using prototypical ligands. (a) Schematic representation of the stably transfected reporter constructs. Expression of ELuc and SLR is under the transcriptional control of 6-fold tandem repeats of xenobiotic and estrogen response elements (XRE and ERE). In addition, ELuc was complemented with a C-terminal PEST sequence to enhance its proteolytic degradation for better timeresolved measurements as well as the assay’s sensitivity for the overlapping SLR-dependent signal. Note that the scale is arbitrary and does not correlate with the actual length of the DNA elements. (b) Time-resolved measurements following exposure to 17β-estradiol (E2, red curves), 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD, green curves), or coexposure to both substances (purple curves), respectively. The assay was performed in HFDMEM buffered with HEPES under normal atmosphere. Data shown represent the mean ± SEM from a representative experiment performed as a 6-fold replicate. (c) Luminescence of ELuc and SLR after 20 h of exposure to TCDD (green), E2 (red), or coexposure (purple) to both substances as indicated. The luminescence readings are normalized to 10 nM TCDD (ELuc) or 1 nM E2 (SLR), respectively. Data shown represent the mean ± SEM of three independent experiments each performed as a 6-fold replicate. (d) Western blot of AHR, ERα, and CYP1A1 in XEER reporter cells following stimulation with 10 nM TCDD, 1 nM E2, or both ligands together as indicated. One representative experiment is shown. *p < 0.05 compared to DMSO control determined by ANOVA followed by Dunett’s test.

⎛ Ltot ⎞ ⎛ 1 1 ⎞⎛ E ⎞ ⎟⎟ = ⎜ ⎜⎜ ⎟⎜ ⎟ κ κ ⎠⎝ R ⎠ ⎝ L E57 R57 ⎝ 570 ⎠

Dunnet’s test for two or more than two groups, respectively. Subsequent fitting of dose−response curves and calculation of EC50 and IC50 values were performed using GrapPad Prism (Graphpad Software, Inc., San Diego, CA, USA).

(1)

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Depending on the experiment, the luminescence was either reported as arbitrary units or relative to the corresponding positive control, that is, TCDD (10 nM) for ELuc and 17β-estradiol (E2, 1 nM) for SLR. Concomitantly, cell viability was assessed as a standard control for all experiments using an MTT viability assay as described previously with minor modifications.33 Preparation of Extracts. For the extraction of substances from plastic products, samples were cut into 2−5 mm pieces. After the addition of 1 mL of DMSO per gram matter, the samples were incubated at 37 °C for 72 h with mild agitation. For extraction of the thermal paper, 2 mL of DMSO/g was added. For the extraction procedure and subsequent storage, Teflon-capped glass vials were used. Data Evaluation and Statistics. All experiments were performed as biological triplicates at least. Plotted error bars refer to the standard error of the mean (SEM). Statistical group comparisons were performed using a Student’s t test or ANOVA with a posthoc

RESULTS Performance with Prototypical Ligands. The assay is based on luciferases ELuc and SLR put under the transcriptional control of a minimal promoter (MP), which is preceded by 6-fold tandem repeats of XREs and EREs, respectively (Figure 1a). The corresponding reporter constructs (XRE6ELuc and ERE6-SLR) were stably inserted into human mammary carcinoma T-47D cells, creating the reporter cell line XEER (XRE-ELuc/ERE-SLR). Cell line T-47D was chosen as host for the initial proof of concept as it is well-established, robust and, most importantly, expresses both receptors (i.e., AHR and ERα) endogenously. Challenged with single substances such as the prototypical agonist TCDD for AHR or E2 for the ER, the reporter system responds with specific 1438


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Figure 2. Performance of XEER with various mono- and bifunctional ligands for AHR and ERα in HEPES- (a) and CO2-buffered medium (b and c). Cells were stimulated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 10 nM), benzo[a]pyrene (BaP; 3 μM), 3-methylcholanthrene (3MC; 3 μM), β-naphthoflavone (bNF; 3 μM), bisphenol A (BPA; 10 μM), ICI-182,780/fulvestrant (ICI; 100 nM), 4-hydroxytamoxifen (4-OHT; 1 μM), resveratrol (Res; 10 μM), and 3,3′-diindolylmethane (DIM; 10 μM) with and without the presence of 17β-estradiol (E2; 1 nM). Assays were performed in HF-DMEM buffered with HEPES under normal atmosphere (a) or in HF-DMEM under 5% CO2 (b). The luminescence readings are normalized to 10 nM TCDD (ELuc) or 1 nM E2 (SLR), respectively. Data shown represent the mean ± SEM of at least three independent experiments each performed as a 3-fold replicate. (c) Exemplary dynamic measurements of XEER following stimulation with 1 nM E2 or 10 nM TCDD. Data shown represent the mean ± SEM of one representative experiment performed as a 3-fold replicate. *p < 0.05 compared to controls with DMSO or E2 by ANOVA followed by Dunett’s test. #p < 0.05 for DMSO vs E2 cotreatment by t test.

pyrene (BaP) and 3-methylcholanthrene (3MC) as well as the synthetic flavonoid β-naphthoflavone (bNF). The ER/SLR was tested with bisphenol A (BPA) as xenoestrogenic agonist and ICI 182,780 (also known as ICI or fulvestrant) and 4hydroxytamoxifen (4-OHT) as antagonist and selective estrogen receptor modulator, respectively. Additional substances included resveratrol (3,4′,5-trihydroxystilbene or Res) and 3,3′diindolylmethane (DIM). Both are plant-derived substances that have previously been shown to modulate the signaling pathways of AHR and ERα alike. Found in grape skin and red wine, Res is an agonist for ER but acts as antagonist on AHR.36−38 The metabolite DIM on the other hand is a condensation product of indol-3-carbinol found in the Brassica genus and has likewise been reported to be an AHR antagonist as well an indirect activator of ERα.39−41 It should be noted that the effect of DIM on AHR appears to be concentrationdependent with higher concentrations reportedly leading to induction of CYP1A1.42 All AHR agonists, that is TCDD (10 nM), BaP (3 μM), 3MC (3 μM), and bNF (3 μM), were able to selectively induce XRE-driven ELuc (Figure 2a, for clarity, results shown refer to 20 h although measurements were recorded continuously as described before). Likewise, luciferase activity of ER-driven SLR was induced by the ERα agonist BPA and repressed by ICI (100 nM) and 4-OHT (1 μM). However, although ELuc activation appeared to be unaffected in the presence of E2, TCDD, BaP, 3MC, and bNF exerted an inhibitory effect on SLR luminescence. The bifunctional substrates Res (10 μM) and DIM (10 μM) also mostly featured as expected with both inducing SLR and Res showing no activity for ELuc. However,

and dose-dependent signals as expected. In the case of TCDD, XEER featured a half maximal effective concentration (EC50) of ∼380 pM (Figure 1b and c) with the ELuc signal peaking at approximately 23 h, whereas E2 specifically induced SLR with an EC50 of ∼13 pM and a maximal induction at 18 h (Figure 1b and c). Effects of the single substances on the respective concomitant reporter were only minor (Figure 1b and c). For addressing the crosstalk between AHR and ER, XEER cells were subsequently stimulated with TCDD and E2 simultaneously (Figure 1a and b, purple curves and bars). Co-stimulation with mixtures of both ligands showed only minor effects on ELuc but corroborated a strong repression of E2-mediated SLR luminescence when compared to E2 stimulation alone (Figure 1b and c). The most likely reason is an underlying TCDD-mediated proteasomal degradation of ERα,18 which was also confirmed by Western blot (Figure 1d). As reported before,34,35 both ligands induced the degradation of their cognate receptors as early as 3.5 h. However, although this did not affect the induction of AHR targets such as CYP1A1 after 24 h, it had a marked inhibitory effect on the expression of ERα. This repression was found to be even more pronounced following coexposure to TCDD and E2 (Figure 1d). Extended Proof of Concept with Specific and Bifunctional Ligands. Following the establishment of the XEER cell line, the basic functionality tests were extended to include other well-established ligands of AHR and ER as well as substances reported to activate both systems. This comprised single substance stimulation as well as costimulation in the presence of E2 to establish noninterference of the two luciferase systems. For AHR/ELuc, the ligands included the PAHs benzo[a]1439


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Figure 3. Effects of AHR antagonists. Cells were stimulated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 1 and 10 nM) or 17β-estradiol (E2; 0.1 and 1 nM) with and without the presence of α-naphthoflavone (aNF; 10 μM), CH-223191 (CH; 1 or 10 μM) (a) or resveratrol (Res; 10 μM) and 3,3′-diindolylmethane (DIM; 10 μM) (b). The luminescence readings were normalized to 10 nM TCDD (ELuc) or 1 nM E2 (SLR), respectively. Assays were performed in HF-DMEM buffered with HEPES under normal atmosphere. Data shown represent the mean ± SEM of at least three independent experiments each performed as a 3-fold replicate. *p < 0.05 compared to DMSO, E2, or TCDD cotreatment by ANOVA followed by Dunett’s test.

AHR antagonist and CYP inhibitor, although it can also act as a partial agonist at high concentrations.45,46 Cells were stimulated with two doses of E2 and TCDD with and without the presence of the corresponding AHR inhibitors (Figure 3a). As expected, CH inhibited TCDD-mediated induction of the ELuc reporter, as did aNF at the 10 μM dose. However, when administered alone, the latter slightly elevated basal ELuc luminescence and also inhibited E2mediated SLR induction. Also tested for their antagonistic activity were the aforementioned bifunctional compounds Res and DIM (Figure 3b). Both substances partially antagonized TCDD-induced Eluc luminescence. Correspondingly, they activated ER (compare to Figure 2), an effect slightly reduced in the presence of TCDD. Application of XEER for Assessing the ER and AHR Activating Potential of Various Polychlorinated Biphenyls. Polychlorinated biphenyls (PCBs) are environmental contaminants that are known to exert estrogenic properties as well as binding to the AHR. Toxicologically, they are of particular interest due to their ubiquitous occurrence and the fact that their activation potential for either of the two receptors can vary depending on their individual structure. The XEER assay was thus used to assess six different PCBs, namely 138, 110, 44, 49, 77, and 126. All PCBs were tested at 10 μM except for PCB 126, which was tested at 1 μM due to its lower solubility (Figure 4). Although PCB 138 showed no activity,

DIM was also identified to be a weak AHR agonist and a competitive E2 antagonist (Figure 2a). Assays were routinely performed under normal atmosphere using HEPES-buffered medium to ensure convenient assaying using a multimode plate reader. Besides the obvious ease of use, this warranted continuous luminescence recording in situ. Nevertheless, these conditions deviate from those of routine cell culture performed in a CO2-enriched atmosphere with carbonate buffering. All experiments were thus re-evaluated under standard cell culture conditions with the plate reader placed in a CO2 incubator and carbonate-buffered medium. The differences observed were minor and restricted to an occasional slight increase in signal strength for some of the substances (i.e., enhanced readings for BaP, DIM, and Res) (Figure 2b). Similarly, peaking of luminescence was unaltered (Figure 2c). However, luminescence readings were detectable for a longer time period with ERE-driven SLR luminescence above baseline still being recorded after 40 h. Effects of Specific and Nonspecific AHR Antagonists. After proving the general reliability of the XEER reporter system, further testing addressed different types of AHR antagonists. The ligand CH-223191 (CH) selectively antagonizes the effects of TCDD without interfering with PAH- or flavonoid-mediated AHR activation or estrogen signaling.43,44 In contrast, α-naphthoflavone (aNF) is an effective general 1440


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Figure 4. Estrogenic and AHR activating activities of selected PCBs. Cells were stimulated with polychlorinated biphenyls (PCBs), 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD; 10 nM), and 17β-estradiol (E2; 1 nM) as indicated. The luminescence readings were normalized to 10 nM TCDD (ELuc) or 1 nM E2 (SLR), respectively. Assays were performed in HF-DMEM buffered with HEPES under normal atmosphere. Dark red bars represent PCBs known to be estrogenic, and dark green bars represent PCBs known to be dioxin-like. PCB 138 is known to be neither estrogennor dioxin-like and is thus shown in blue. Data shown represent the mean ± SEM of at least three independent experiments each performed as a 6fold replicate. *p < 0.05 compared to DMSO by ANOVA followed by Dunett’s test. #p < 0.05 according to student’s t test.

Finally, the system was challenged with a xenoestrogenic model mixture consisting of BPA, BuPA, BP2, and DIM to assess its capability of mimicking the established dose additivity for such scenarios.48 Indeed, the respective xenoestrogens showed a strong dose-dependent effect when mixed at concentrations below their individual EC50 values (Figure 5e). ER and AHR Activation by Substances Extracted from Plastics. Imminent and foreseeable applications of the XEER assay are large substance screens and potential hazard prioritization. The assay was therefore tested with DMSO extracts from various plastic materials to evaluate its suitability for detecting estrogenic and AHR coactivation.49,50 Test items comprised a tarpaulin cover (extract 1), rubber rings (extract 2), a cell scraper (extract 4), a rubber hose (extract 5), a tube rack (extract 6), and earphone plugs (extract 7). Also included was an extract from standard thermal paper (extract 3) as it is known to contain high amounts of estrogenic BPA or bisphenol S.51 The corresponding extracts were tested using serial dilutions ranging from 10−3 to 10−6 (Figure 6). Extracts 1−4 showed statistically significant dose-dependent estrogenic activity. Although the data of a subsequent MTT assay clearly indicated dose-dependent cytotoxicity at higher doses for extracts 2, 5, and 6, none of the extracts induced AHR-dependent ELuc luminescence. At first sight, this is at odds with the expectation of AHR agonists being present in many plastic materials. However, many AHR ligands are subject to rapid degradation by AHRregulated CYP enzymes.49 Luminescence recordings for ELuc were hence repeated as time-resolved measurements, revealing a transient increase in AHR-triggered luminescence for extract 4−10 h after stimulation (Figure 7b and data not shown). Subsequent measurements showed the corresponding activity to originate from the yellow polystyrene handle (PE4y) with the white polyethylene scraper as such (PE4w) being tested negative (Figure 7c and d).

PCB 77 and 126 were found to activate the AHR and the remaining three (PCB 110, 44 and 49) showed signs of modest estrogenic activity. Dose Dependency of the XEER Reporter Assay. Finally, the assay was tested for its capability to assess dose-dependent effects. This was first tested using several phyto- and xenoestrogens comprising the aforementioned BPA, Res, and DIM as well as four additional substances. These were genistein (Gen), 2,2′,4,4′-tetrahydroxybenzophenone (BP2), 4-methylbenzylidene camphor (4MBC), and butylparaben (BuPa). The first is a well-known phytoestrogen found in soy, whereas the xenoestrogens BP2 and 4MBC are used as UV filters in sunscreen lotions and BuPa might serve as a preservative in cosmetics. All substances induced the ERE-driven SLR reporter in a dose-dependent manner (Figure 5a) with EC50 values of 72, 232, and 742 nM and 2.2, 3.1, 3.5, and 3.7 μM for Gen, BPA, 4-MBC, BP2, BuPa, DIM, and Res, respectively. Three substances (i.e., Gen, BuPa, and Res) acted as superagonists with the maximal induction exceeding the positive control (1 nM E2). It should be noted though that, at least for Gen, this phenomenon has already been previously reported and is attributed to a substrate-induced stabilization of luciferase.47 The assay also performed well for the ER antagonists ICI and 4-OHT, yielding half maximal inhibitory concentration (IC50) values of 4 and 20 nM in the presence of 1 nM E2 (Figure 5b). Upon treatment with high concentrations of both ER antagonists, the SLR signal droped beyond the basal level. The reason for this effect is the unavoidable low levels of residual estrogen that induce some basal ER activity. Likewise, stimulation with AHR agonists induced a dosedependent increase in ELuc luminescence. For 3MC and bNF, the signal reached saturation at a maximal effect (Emax) of ∼40% of 10 nM TCDD and PCB126 showing an Emax at ∼65% for 10 nM TCDD (Figure 5c). The corresponding EC50 values were 0.03, 0.94, and 1.9 μM for PCB126, 3MC, and bNF, respectively. Exposure to BaP only induced up to 25% of the maximal luminescence at 3 μM with higher concentrations seeing the signal drop again. For the AHR antagonists CH and aNF, the IC50 values were 0.857 and ∼8 μM, respectively (Figure 5d). The latter value represents only an estimate as aNF did not completely inhibit TCDD-induced ELuc luminescence.

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DISCUSSION ERα and AHR are two ligand-activated transcription factors that are functionally interlinked, allowing a fine-tuned and balanced regulation of estrogenic responses,52 hormone synthesis,53 immune modulation,54,55 and phase I metabo1441


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Figure 5. Dose dependency of XEER following stimulation with agonists and antagonists of ERα (a and b), AHR (c and d), or xenoestrogenic mixtures (e). Cells were stimulated with substances as indicated. For antagonist testing, cells were costimulated with either 1 nM 17β-estradiol (E2) (b) or 10 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (d). Dose-dependent estrogenic activity following exposure to a mix of four xenoestrogens at concentrations below their individual EC50 is shown in (e). Doses of the mix refer to the sum of the individual substances. Assays were performed in HF-DMEM buffered with HEPES under normal atmosphere. The luminescence readings are normalized to 1 nM E2 (SLR) or 10 nM TCDD (ELuc). Data shown represent the mean ± SEM of at least three independent experiments each performed as a 3-fold replicate. Abbreviations used include benzo[a]pyrene (BaP), bisphenol A (BPA), 2,2′,4,4′-tetrahydroxybenzophenone (BP2), butylparaben (BuPa), CH223191 (CH), 3,3′-diindolylmethane (DIM), genistein (Gen), ICI-182,780/fulvestrant (ICI), 4-methylbenzylidene camphor (4MBC), 3methylcholanthrene (3MC), α-naphthoflavone (aNF), β-naphthoflavone (bNF), 4-hydroxytamoxifen (4-OHT), polychlorinated biphenyl (PCB), and resveratrol (RES). *p < 0.05 compared to DMSO by ANOVA followed by Dunett’s test.

lism.56,57 Accordingly, screening their activation and activity is of high toxicological relevance, be it for potential hazard identification or a better molecular understanding, even more so as both receptors are targeted by various industrial chemicals as well as environmental contaminants. However, the available molecular reporter systems are usually restricted to single receptor measurements only. Here, we present a novel reporter cell line that allows simultaneous measurement of the activation of both receptors in a time-resolved quantitative manner. The respective cell line, XEER, passed the proof of concept showing stable and comparable reactions to prototypical ligands like E2 and TCDD. An EC50 of 13 pM for E2 corresponds well to values of

other reporter cell lines such as the stable ERα-Hela9903 reporter system previously used in our laboratory (EC50 = 2.9 pM E2).58 Other mammalian systems report EC50 values of 1.5−50 pM E2.59 Likewise, the EC50 of 0.38 nM for TCDD matches the range of 0.1−1 nM reported in human epithelial cells or hepatocytes.60 Although other reporter cell lines might feature a slightly increased sensitivity for TCDD, it should be kept in mind that absolute readouts will not only depend on reporter expression levels but can also be subject to differences in the host cell system or reporter species. For example, nonhuman AHR has a 10-fold higher affinity to TCDD due to specific amino acid changes.61 Comparisons between different systems will thus inherently be limited to qualitative aspects. It 1442


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Figure 6. ER-/AHR-activating potential of extracts from plastic commodities and thermal paper. XEER cells were stimulated with diluted extracts as indicated and recorded after 20−22 h. Assays were performed in HF-DMEM buffered with HEPES under normal atmosphere followed by subsequent cytotoxicity measurements using the MTT assay. Shown are relative luminescence readings for ELuc (green) and SLR (red) signals as well as the MTT (purple) results. The luminescence readings are normalized to 1 nM E2 (SLR) or 10 nM TCDD (ELuc); MTT values refer to the DMSO control. All data shown represent the mean ± SEM of at least three independent experiments each performed as a 3-fold replicate. *p < 0.05 compared to DMSO control determined by ANOVA followed by Dunett’s test.

activation by TCDD induces proteasomal degradation of ERα,17,18 a mechanism that is also functional in the XEER cell line. Third, competition for shared transcriptional cofactors can also potentially shape receptor cross talk. Accordingly, sequestration of shared cofactors by AHR could hence theoretically limit any concomitant (xeno)estrogenic response. However, although the data clearly show such a limitation of the ER response during AHR activation, they fail to support the opposite scenario, that is, inhibition of the AHR during ER

should also be noted that, similarly to other reporters, the E2 signal will be primarily mediated by ERα because T-47D cells express 9-fold more ERα than ERβ.62 The observed inhibition of ERα during costimulation with TCDD is in line with well-known antiestrogenic actions of TCDD in vivo and in vitro.63,64 Several mechanisms might converge to explain these observations. First of all, AHR activation induces CYP expression, which is also responsible for the metabolism of natural estrogens such as E2.14 Second, AHR 1443


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Figure 7. Transient stimulation of AHR by extracts from a cell scraper made out of polystyrene/polyethylene. (a) Photograph of the cell scraper indicating the parts investigated: the whole tool (PE4), its handle (PE4y), and the scraper as such (PE4w). (b) Time-resolved activation of AHRdependent ELuc after stimulation with extracts from a cell scraper. The assay was performed in HF-DMEM buffered with HEPES under normal atmosphere. Shown is the mean ± SEM luminescence of one representative measurement conducted in 6-fold determinations. Arrows in (b) indicate the time points shown in (c) and (d). Relative readings of ELuc after 10 h (c) or 24 h (d) are normalized to 10 nM TCDD. The dilution (1:1000−1:10000) of the corresponding extracts is indicated. Data shown represent the mean ± SEM from three independent experiments each performed as a 6-fold replicate. *p < 0.05 compared to DMSO control determined by ANOVA followed by Dunett’s test.

expression and breast development, suggesting a rather antiestrogenic action of 3MC in vivo.70 Meanwhile, all tested AHR ligands reliably induced ELuc expression, although the substrates BaP, 3MC, and bNF failed to reach the maximal signal intensity recorded for the positive control. In contrast to TCDD, these compounds are readily subjected to CYP-mediated degradation. This phenomenon has previously been observed in other AHR-responsive cell lines, too. Particularly unstable AHR agonists are hence better detected with fast responding reporter constructs.71 Conversely to the aforementioned effect of AHR ligands on SLR, ELuc was hardly affected by (co)exposure to E2 except for a slight decrease in signal intensity, which most likely results from concomitantly induced cell proliferation. Furthermore, XEER performed as expected with the bifunctional substrates DIM and Res, identifying both correctly as AHR antagonist and ER agonists,36−42 which included recapitulation of the previously noted partial antagonism for DIM under conditions of cotreatment.72 Similarly, all antagonists reliably inhibited the activation of their corresponding receptor with the ER-specific ones even decreasing the signal to sub-basal levels as a consequence of blocking the activity of residual estrogens in the cell culture medium. Interestingly, the AHR antagonists not only inhibited activation of the AHR by TCDD but also targeted the ER, at least at higher concentrations (10 μM). Antagonist-liganded AHR might thus potentially be able to inhibit ER signaling. Depending on their concentration, ligands such as aNF act either as antagonists or as partial agonists on AHR. It appears well conceivable that such substances might leave the AHR in a bifunctional conformation capable of

activation. This indicates the influence of competition for shared cofactors in XEER to be minor. However, this might be different in other cells or tissues. Finally, antiestrogenic effects of TCDD are partially convened by inhibitory XREs that are located in the promoters of ER-regulated genes.65,66 However, for the observed inhibition in XEER, this is a rather unlikely mechanism because the transgenic reporter constructs contain consensus EREs and XREs only. Compared to other ERspecific reporter systems, the AHR-mediated attenuation of the ER signal during mixture testing might at first sight appear disadvantageous because it will impede sensitivity. However, because the AHR is expressed ubiquitously, this situation resembles human cellular physiology more closely, particularly when compared to other cell systems that do not express AHR, such as yeast. In addition, this behavior of XEER allows the clear discrimination of real ER antagonists from AHR agonists, something that is hardly possible in other ER reporter cell lines. Other AHR agonists also inhibited E2-mediated SLR luminescence. Namely, these were the PAHs BaP and 3MC as well as bNF, although the latter appeared to be less effective. Although this could partly be attributed to mechanisms similar to TCDD, this result is to some extent surprising as some reports associate 3MC and BaP with activation of ERα possibly as a consequence of CYP-catalyzed hydroxylation of these compounds.67−69 However, even with prolonged continuous assaying, XEER failed to record signs of an intermediate induction of SLR (data not shown). Likely explanations for this are cell-line specific differences regarding receptor content and CYP expression. Indeed, a recent rat study found 3MC to alter estrogen-responsive gene expression and formation of terminal end buds only marginally but to inhibit E2-induced gene 1444



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directly affecting ER function. However, this hypothesis would require further confirmation by orthogonal assays. The proof of concept was concluded by testing a defined set of PCB congeners, all of which were correctly identified for their respective potential to activate the AHR (i.e., PCB 77 and 126), ER (i.e., PCB 110, 44 and 49),63,73−75 or neither of the receptors (i.e., PCB 138).63,74 In addition, several DMSO extracts from commodities were tested as an example of a practical application for the screening of mixtures. As expected, the extract from the thermal paper receipt showed strong estrogenic effects most likely due to the presence of BPA or its substitutes. However, estrogenic activity could also be observed in most of the other extracts with several also inducing cytotoxic effects at higher doses. Activation of the AHR was recorded for one of the extracts from a cell scraper handle made of polystyrene. The transient occurrence of the signal is in line with other literature reports, indicating metabolism of the corresponding AHR ligand(s).49 Potential candidates for these ligands are styrene oligomers, which have recently been shown to be AHR activators.76 For such ligands, the option of timeresolved measurements is a clear advantage over other assays, although longer incubation times will put obvious limits on the assay’s high-throughput compatibility and require the consideration of possible false negative results due to potential test substance metabolism. Altogether we present a novel reporter system termed XEER that is suitable for the simultaneous and time-resolved measurement of AHR and ERα activation in living cells. Thanks to slightly altered culture conditions, the system is suitable to perform continuous readings over extended periods of time, thus providing a simple, cost-effective, and powerful tool for investigating AHR/ER signaling crosstalk as well as for the screening of single substances and mixtures for their receptor activating potential.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +49 30 184123885. ORCID

Patrick Tarnow: 0000-0002-9250-6775 Funding

We acknowledge intramural funding at the German Federal Institute for Risk Assessment (BfR) Grants 1322-548 and 1322665. Notes

The authors declare no competing financial interest.

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ABBREVIATIONS 3MC, 3-methylcholanthrene; 4MBC, 4-methylbenzylidene camphor; 4-OHT, 4-hydroxytamoxifen; AHR, aryl hydrocarbon receptor; BaP, benzo[a]pyrene; BPA, bisphenol A; CH, CH223191; CYP, cytochrome P450-dependent monooxygenase; DIM, 3,3′-diindolylmethane; DMSO, dimethyl sulfoxide; E2, 17β-estradiol; ELuc, emerald luciferase; ER, estrogen receptor; ERE, estrogen response element; ICI, ICI-182,780/fulvestrant; aNF, α-naphthoflavone; bNF, β-naphthoflavone; PAH, polycyclic aromatic hydrocarbon; PE, plastic extract; PCB, polychlorinated biphenyl; Res, resveratrol; SLR, stable luciferase red; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; XEER, XRE-ELuc/ERE-SLR; XRE, xenobiotic response element 1445


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