Effects of Flavored Nonalcoholic Beverages on Transcriptional

Article Cite This: J. Agric. Food Chem. 2018, 66, 12066−12078

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Effects of Flavored Nonalcoholic Beverages on Transcriptional Activities of Nuclear and Steroid Hormone Receptors: Proof of Concept for Novel Reporter Cell Line PAZ-PPARg Peter Illés,* Aneta Grycová, Kristýna Krasulová, and Zdeněk Dvořaḱ

J. Agric. Food Chem. 2018.66:12066-12078. Downloaded from pubs.acs.org by UNIV OF WINNIPEG on 11/14/18. For personal use only.

Regional Centre of Advanced Technologies and Materials, Faculty of Science, Palacky University, Slechtitelu 27, 783 71 Olomouc, Czech Republic ABSTRACT: We developed and characterized a novel human luciferase reporter cell line for the assessment of peroxisome proliferator-activated receptor γ (PPARγ) transcriptional activity, PAZ-PPARg. The luciferase activity induced by PPARγ endogenous agonist 15d-PGJ2 and prostaglandin PGD2 reached mean values of (87.9 ± 14.0)-fold and (89.6 ± 19.7)-fold after 24 h of exposure to 40 μM 15d-PGJ2 and 70 μM PGD2, respectively. A concentration-dependent inhibition of 15d-PGJ2- and PGD2-induced luciferase activity was observed after the application of T0070907, a selective antagonist of PPARγ, which confirms the specificity of response to both agonists. The PAZ-PPARg cell line, along with the reporter cell lines for the assessment of transcriptional activities of thyroid receptor (TR), vitamin D3 receptor (VDR), androgen receptor (AR), and glucocorticoid receptor (GR), were used for the screening of 27 commonly marketed flavored nonalcoholic beverages for their possible disrupting effects. Our findings indicate that some of the examined beverages have the potential to modulate the transcriptional activities of PPARγ, VDR, and AR. KEYWORDS: PPARγ, transcriptional activity, reporter cell line, nonalcoholic beverages

1. INTRODUCTION Peroxisome proliferator-activated receptors (PPARs) are ligandactivated transcription factors that belong to a superfamily of nuclear receptors. PPARs were described for the first time as receptors that are activated by a diverse group of agents causing extensive proliferation of peroxisomes and carcinogenesis in rodent liver.1 Three isotypes of PPARs have been identified up to now: PPARα (NR1C1), PPARβ/δ (NR1C2), and PPARγ (NR1C3). Each PPAR has its own, partially overlapping group of target genes (reviewed in ref 2). PPARs regulate gene expression by direct binding to specific peroxisome proliferatoractivated receptor response elements (PPREs) present in the regulatory regions of target genes in one or multiple copies. PPREs are composed of two hexameric half-sites, which consist of the AGGTCA consensus sequences, typically organized as direct repeats with a single nucleotide spacing between the repeats. Binding of PPARs to their response elements requires dimerization of receptors. Thus, formation of heterodimers with the retinoic X receptor (RXR) is essential for the PPARmediated regulation of gene expression. PPARγ plays a crucial role in differentiation of adipocytes and maintenance of their specific functions, including metabolism and storage of lipids.3−7 It is also involved in regulation of glucose metabolism and homeostasis through interactions with the insulin signaling pathway.8−10 In addition, PPARγ is implicated in the immune response of organisms and acts as an important factor in the control of inflammatory processes.11−15 PPARγ is predominantly expressed in brown and white adipose tissues; less extensive endogenous expression occurs in the large intestine, liver, spleen, kidney, heart, skeletal muscle, and some specific cells of the immune system.16−19 The transcriptional activity of PPARγ is facilitated by binding of specific ligands. Most of the PPARγ natural ligands belong to fatty acids or to a group of © 2018 American Chemical Society

eicosanoids that are generated in fatty acid metabolism. A wellknown endogenous ligand of PPARγ, 15-deoxy-δ12,14prostaglandin J2 (15d-PGJ2), is a derivative of prostaglandin D2 (PGD2) synthesized by cyclooxygenase-2 (COX-2) in a variety of tissues under inflammatory conditions.20,21 Among the synthetic ligands, thiazolidinedione compounds, a class of insulin-sensitizing drugs used for the treatment of type 2 diabetes in humans, represent a group of potent activators of PPARγ.22 Disruption of PPARγ function may lead to various pathophysiological disorders, and it is often associated with obesity, diabetes, atherosclerosis, or cardiovascular diseases (reviewed in refs 23−27). A variety of exogenous substances, generally termed endocrine disruptors (EDs), is capable of altering the function of the endocrine system and thus disturbing normal physiological processes in organisms, leading to undesirable health effects. It is widely accepted that one of the ways humans are exposed to EDs is through the diet. Food products contain a large number of compounds that can possibly affect the transcriptional activities of nuclear and steroid hormone receptors. These compounds can be either of natural character (e.g., anthocyanidins, carotenoids, isoflavonoids, polyphenols) or synthetic chemicals that are usually added to improve the quality and stability of food products (e.g., artificial flavors, colorants, sweeteners, antioxidants, preservatives). Recently, it has been reported that some commonly marketed flavored nonalcoholic beverages have the capability of inducing transcriptional activities of aryl hydrocarbon receptor (AhR) and Received: Revised: Accepted: Published: 12066

September 20, 2018 October 23, 2018 October 25, 2018 November 5, 2018 DOI: 10.1021/acs.jafc.8b05158 J. Agric. Food Chem. 2018, 66, 12066−12078

Article

Journal of Agricultural and Food Chemistry Table 1. List of Tested Nonalcoholic Beverages flavor

abbrev

brand

TEA-1

Dobra Voda

tea, lemon

TEA-2

Dobra Voda

tea, peach

TEA-3 TEA-4

Aquila Team Aquila Team

green tea, lemon black tea, lemon

TEA-5

Aquila Team

white tea, pomegranate

TEA-6

Aquila Team

black tea, peach

TEA-7

Nestea Vitao

red tea, pear

TEA-8 TEA-9 MW-1 MW-2 MW-3 MW-4 MW-5 MW-6 MW-7 MW-8 MW-9 MW-10 MW-11 MW-12 MW-13 MW-14 MW-15

Nestea Nestea Podebradka Podebradka Podebradka Podebradka Podebradka Podebradka Podebradka Podebradka Podebradka Dobra Voda Dobra Voda Dobra Voda Dobra Voda Dobra Voda Dobra Voda

MW-16

Dobra Voda

MW-17 MW-18

Rajec Rajec

peach lemon lemon grapefruit plum, elderberry sour cherry pomelo, cranberry lime orange plum passion flower mango lemon orange grapefruit pear lemon, green tea, passion flower orange, violet, hawthorn, blueberry dandelion chestnut

composition sugar, tea extract 2.1 g/L, natural aroma, citric acid, sodium citrate, sodium benzoate, potassium sorbate, dimethyl dicarbonate sugar, tea extract 2.1 g/L, natural aroma, citric acid, sodium citrate, sodium benzoate, potassium sorbate, dimethyl dicarbonate sugar, green tea extract, natural green tea and lemon aromas, natural lemon juice, sodium benzoate sugar, citric acid, caramel dye, black tea extract, phosphoric acid, sodium benzoate, natural lemon aroma, vitamin C sugar, white tea extract, citric acid, ascorbic acid, natural pomegranate aroma, pomegranate juice, sodium citrate, sodium benzoate sugar, citric acid, caramel dye, black tea extract, phosphoric acid, sodium benzoate, natural peach aroma, vitamin C sugar, citric acid, sodium citrate, rooibos extract 0.1%, pear juice 0.1% of concentrate, aroma, ascorbic acid, polyphenols 440 mg/L sugar, citric acid, sodium citrate, tea extract 0.1%, aroma, peach juice 0.1% of concentrate sugar, citric acid, sodium citrate, tea extract 0.1%, aroma, lemon juice 0.1% of concentrate, ascorbic acid citric acid, aroma, acesulfame K, aspartame citric acid, aroma, acesulfame K, aspartame citric acid, sodium citrate, L-carnitine 100 mg, acesulfame K, aspartame, fiber 1 g/L citric acid, aroma, acesulfame K, aspartame citric acid, sodium citrate, L-carnitine 100 mg, acesulfame K, aspartame, fiber 1 g/L citric acid, aroma, acesulfame K, aspartame citric acid, aroma, acesulfame K, aspartame citric acid, sodium benzoate, aroma, glucose-fructose syrup citric acid, sodium benzoate, aroma, glucose-fructose syrup vitamin C 92 mg/L, sugar, citric acid, aroma vitamin C 92 mg/L, sugar, citric acid, aroma vitamin C 92 mg/L, sugar, citric acid, aroma vitamin C 92 mg/L, sugar, citric acid, aroma vitamin C 92 mg/L, sodium benzoate, sugar, citric acid, dimethyl dicarbonate, aroma, preservative sugar, aroma, herb extract 2.5 g/L, sodium benzoate, aspartame, acesulfame K, citric acid, ascorbic acid sugar, aroma, herb extract 4 g/L, sodium benzoate, aspartame, acesulfame K, citric acid, ascorbic acid glucose syrup, citric acid, dandelion extract 0.1 g/L, apple juice, aroma, potassium sorbate, sodium benzoate fructose, citric acid, chestnut extract 0.1 g/L, aroma, sodium benzoate

flavored nonalcoholic beverages, which can be applied particularly in research of food−drug interactions.

pregnane X receptor (PXR),28,29 which play an important role in the metabolism of xenobiotics. The main aim of our work was to develop a human-based, stable luciferase reporter system that can serve as a reliable tool for the assessment of the transcriptional activity of PPARγ in various applications. As a proof of concept, the novel PAZ-PPARg cell line was used for screening of 27 flavored nonalcoholic beverages for their potential disrupting effects on the transcriptional activity of PPARγ. Nuclear and steroid hormone receptors represent the most important end points in endocrine regulation of fundamental physiological processes. Due to the complexity and possible interconnection of mechanisms involved in regulation of transcriptional activities of these receptors, the effect of one particular endocrine disruptor may lead to a simultaneous disturbance of activities of different receptors.30 To the best of our knowledge, no comprehensive study comparing the effects of flavored nonalcoholic beverages on the function of nuclear and steroid hormone receptors has yet been published. Therefore, 27 flavored nonalcoholic beverages were examined for their possible disrupting effects on transcriptional activities of thyroid receptor (TR), vitamin D3 receptor (VDR), androgen receptor (AR), and glucocorticoid receptor (GR), using the luciferase reporter cell lines developed previously in our laboratory. Our current study brings new information about potential endocrine disrupting effects of

2. MATERIALS AND METHODS 2.1. Chemicals. Prostaglandin D2 (PGD2), 15-deoxy-δ12,14prostaglandin J2 (15d-PGJ2), cholecalciferol (VD3), 3,3′,5-triiodo-Lthyronine (T3), 9-cis-retinoic acid (9-cis-RA), all-trans-retinoic acid (all-trans-RA), WY 14643 (pirinixic acid), fenofibrate, and 2-chloro-5nitro-N-4-pyridinylbenzamide (T0070907) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Aldosterone, dexamethasone (DEX), 5α-dihydrotestosterone (DHT), 17β-estradiol, progesterone, thiazolyl blue tetrazolium bromide (MTT), McCoy’s 5A medium, Dulbecco’s modified Eagle’s medium (DMEM), RPMI-1640 medium, fetal bovine serum, and charcoal-stripped bovine serum were acquired from Sigma-Aldrich (Prague, Czech Republic). FuGENE HD Transfection Reagent, Luciferase Assay System, Nano-Glo Luciferase Assay System, and Reporter Lysis 5× Buffer were purchased from Promega (Madison, WI, USA). All other chemicals were of the highest commercially available quality. 2.2. Cell Lines. Human bladder carcinoma cell line T24/83 (ECACC 85061107) obtained from the European Collection of Authenticated Cell Cultures was cultivated in McCoy’s 5A medium supplemented with 10% of fetal bovine serum and 4 mM L-glutamine. The stably transfected reporter cell line AIZ-AR (derived from human prostate carcinoma cell line 22RV1)31 was cultivated in RPMI-1640 medium supplemented with 10% of fetal bovine serum and 4 mM Lglutamine. The stably transfected reporter cell lines AZ-GR (derived from human cervix carcinoma cell line HeLa),32 IZ-VDRE (derived from human colon adenocarcinoma cell line LS180),33 and PZ-TR 12067

DOI: 10.1021/acs.jafc.8b05158 J. Agric. Food Chem. 2018, 66, 12066−12078

Article

Journal of Agricultural and Food Chemistry

Figure 1. Response of the stably transfected clones to 15d-PGJ2 and PGD2. The cells seeded into 96-well plates were stabilized for 16 h and treated with 50 μM 15d-PGJ2, 50 μM PGD2, or vehicle (UT; 0.1% DMSO v/v). Luciferase activity was measured in cell lysate after 24 h of exposure. The data are the means ± SD of quadruplicate measurements and are the representatives of three independent experiments. The values are expressed as a fold induction over the DMSO-treated cells (A) and the corresponding relative light units (B). (derived from human hepatocellular carcinoma cell line HepG2)34 were cultivated in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% of fetal bovine serum, 4 mM L-glutamine, and 1% of nonessential amino acids. Cells were maintained at 37 °C and 5% CO2 in a humidified incubator. 2.3. Reporter Plasmid. The pNL2.1-PPREγ reporter plasmid was prepared by cloning of a synthesized DNA fragment into the multiple cloning site of the pNL2.1[Nluc/Hygro] vector (Cat. No. N1061, Promega, Madison, WI, USA). The synthesized DNA fragment consisted of three copies of the PPARγ response element derived from the promoter region of ACOX gene20 and the minimal promoter that was attached downstream of the PPARγ response elements. For insertion of the DNA fragment into the reporter vector, XhoI/HindIII restriction cloning was used. 2.4. Stable Transfection and Selection. Human bladder carcinoma cell line T24/83, characterized by endogenous expression of both PPARγ and RXRα and functional PPARγ signaling pathway,35,36 was used for stable transfection. The T24/83 cells at a density of 1.5 × 106 cells in 5 mL of McCoy’s 5A culture medium were transfected with 3 μg of the pNL2.1-PPREγ reporter plasmid using the FuGENE HD transfection reagent. Transfection was performed according to the manufacturer’s standard protocol at a ratio of 3/1 (reagent/DNA), and the transfected cells were seeded into 60 mm cell

culture dishes. After 24 h of incubation, the culture medium containing transfection reagent was replaced by selection medium supplemented with hygromycin B (60 μg/mL). Medium was exchanged every 4−5 days for a period of 6 weeks, until a polyclonal population of transfected cells was selected. The cells were subsequently transferred into 10 mm cell culture dishes at a density of 100−1000 cells in 10 mL of selection medium supplemented with hygromycin B (60 μg/mL) and were cultivated for an additional 4 weeks, until small colonies appeared. Thereafter, 24 individual colonies were transferred into 24-well tissue culture plates and cultivated for an additional 2 weeks in selection medium to obtain monoclonal populations of stably transfected, hygromycin B resistant clones. All resulting clones were tested for their responsiveness to 50 μM 15d-PGJ2 and 50 μM PGD2, and the most susceptible clone termed PAZ-PPARg was used for further characterization. For long-term cultivation of the stably transfected PAZ-PPARg cells, the culture medium supplemented with hygromycin B (60 μg/ mL) was used every fourth passage. The use of GMO at the Faculty of Science, Palacky University Olomouc, was approved by the Ministry of the Environment of the Czech Republic (ref. 91997/ENV/10). 2.5. Luciferase Reporter Assay. The reporter cells were seeded into 96-well plates at a density of 4 × 104 (PAZ-PPARg), 2.5 × 104 (IZVDRE), 5 × 104 (AIZ-AR), 2 × 104 (AZ-GR), and 5 × 104 (PZ-TR) cells in 0.2 mL of appropriate culture medium supplemented with 10% 12068


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Figure 2. Dose−response of the PAZ-PPARg cells to 15d-PGJ2 and PGD2. The cells seeded into 96-well plates were stabilized for 16 h and treated with 15d-PGJ2 or PGD2 (1−100 μM) or vehicle (UT; 0.1% DMSO v/v) for 24 h. After the treatments, the cells were lysed and the luciferase activity was measured (A, B). The data are the means ± SD of quadruplicate measurements and are the representatives of six independent experiments. The values are expressed as a fold induction over the DMSO-treated cells. In parallel experiments, the cytotoxicity assay was performed (C, D). The data represent the means ± SD of eight independent experiments and are expressed as a percentage of viability of the DMSO-treated cells. charcoal-stripped fetal bovine serum instead of normal fetal bovine serum. Following 16 h of stabilization, the cells were treated with examined compounds or extracts of beverages as described in the figure captions for the indicated period of time. The vehicle DMSO (0.1% v/ v) or/and ethanol (0.1% v/v) was used as a negative control (specified in the figure captions). After the treatments, the cells were lysed in properly diluted Reporter Lysis 5× Buffer and the luciferase activity was measured in a 96-well plate format using the Nano-Glo Luciferase Assay System for PAZ-PPARg and IZ-VDRE cell lines and Luciferase Assay System for AIZ-AR, AZ-GR, and PZ-TR cell lines. The measurement of luciferase activity was performed by a Tecan Infinite M200 plate reader (Tecan, Männedorf, Switzerland). 2.6. Cytotoxicity Assay. The PAZ-PPARg reporter cells were seeded into 96-well plates at a density of 4 × 104 cells in 0.2 mL of McCoy’s 5A culture medium supplemented with 10% charcoal-stripped fetal bovine serum. After 16 h of stabilization, the cells were treated with studied compounds as described in the figure captions for 24 h. The vehicle DMSO (0.1% v/v) was used as a negative control. Subsequently, the medium was replaced by culture medium supplemented with 0.3 mg/mL MTT and the cells were incubated for an additional 30 min. The MTT assay was measured spectrophotometrically at λ 540 nm using a Tecan Infinite M200 plate reader (Tecan, Männedorf, Switzerland). 2.7. Extracts of Nonalcoholic Beverages and Treatment of Cells. Nine commonly marketed ready-to-drink teas (TEA-1−TEA-9) and 18 flavored mineral waters (MW-1−MW-18) were purchased in various supermarkets in Olomouc City, Czech Republic (Table 1). The extracts of nonalcoholic beverages were prepared as previously described.28,29 The dried extracts obtained from 1000 mL of beverages were dissolved in 1 mL of ethanol, resulting in stock solutions of 1000× concentrated ethanol extracts of the tested TEAs and MWs. For

experimental use, the stock solutions were diluted 1000 times in the appropriate culture medium supplemented with 10% charcoal-stripped fetal bovine serum. Hence, the concentrations of constituents of examined beverages in the medium used for the treatments were identical with those in the original beverages. For detailed analyses, a concentration scale of MW-9 extract was prepared. The extract was diluted with ethanol to final concentrations ranging from 10 to 100% of the original concentration. Prepared samples were consequently diluted 1000 times in the appropriate culture medium supplemented with 10% charcoal-stripped fetal bovine serum. The effects of TEAs and MWs on transcriptional activities of three nuclear receptors, peroxisome proliferator-activated receptor γ (PPARγ), vitamin D3 receptor (VDR), and thyroid receptor (TR), and two steroid hormone receptors, androgen receptor (AR) and glucocorticoid receptor (GR), were examined. Transcriptional activities of selected receptors were assessed in the stably transfected reporter cell lines PAZ-PPARg (PPARγ), IZ-VDRE (VDR), PZ-TR (TR), AIZ-AR (AR), and AZ-GR (GR), respectively. The analyses were performed in two different experimental layouts. In the agonist mode, the cells were treated with the extracts of examined beverages diluted 1:1000 for 24 h. In the antagonist mode, the cells were incubated with the extracts of examined beverages diluted 1:1000 in the presence of model agonists of particular receptors for 24 h. To induce the luciferase activity in the reporter cells, 15-deoxy-δ12,14-prostaglandin J2 (15d-PGJ2; 40 μM), cholecalciferol (VD3; 50 nM), 3,3′,5-triiodo-L-thyronine (T3; 50 nM), 5αdihydrotestosterone (DHT; 100 nM), and dexamethasone (DEX; 100 nM) were used as model agonists of PPARγ, VDR, TR, AR, and GR, respectively. For calculation of fold induction of the luciferase activity, the appropriate culture medium containing 0.1% (v/v) of ethanol was used as a negative control in the agonist experimental layout. In the antagonist experimental layout, the appropriate culture 12069


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Journal of Agricultural and Food Chemistry medium containing both 0.1% (v/v) of ethanol and 0.1% (v/v) of DMSO was used as a negative control. The culture medium containing an adequate concentration of the particular ligand supplemented with 0.1% (v/v) of ethanol was used as a positive control in antagonist experimental layout. 2.8. Data and Statistical Analyses. The results were obtained from at least three independent experiments (indicated in the figure captions), and for each experiment quadruplicate measurements were performed. Statistical significance was tested by Student’s t test, and the differences were considered significant at p < 0.05. IC50 values and curve fittings were determined using GraphPad Prism 5 software (GraphPad Software, La Jolla, CA, USA).

the luciferase activity was measurable as soon as after 12 h of application of PPARγ agonists. The values of luciferase activity corresponded to 32% (40 μM 15d-PGJ2) and 15% (70 μM PGD2) of the activity obtained after 24 h of exposure, respectively (Figure 3).

3. RESULTS 3.1. Construction of Stably Transfected PAZ-PPARg Reporter Cell Line. Human bladder carcinoma cell line T24/ 83 was transfected with the pNL2.1-PPREγ reporter plasmid to obtain the stably transfected reporter cell line for the assessment of PPARγ transcriptional activity. After the cultivation of transfected cells under the selection pressure of hygromycin B, we gained 24 hygromycin B resistant, stably transfected clones. All resulting clones were tested for their responsiveness to 50 μM 15d-PGJ2 (natural ligand of PPARγ) and 50 μM PGD2 (precursor of 15d-PGJ2). After 24 h of treatment, the highest induction of luciferase activity was measured in clones 1 and 15, reaching 23-fold for 15d-PGJ2 and 50-fold for PGD2 in clone 1 and 15-fold for 15d-PGJ2 and 75-fold for PGD2 in clone 15, respectively (Figure 1A). However, the values of the relative light units (RLU) in clone 15 were significantly lower (103−104 RLU) than those in clone 1 (105 RLU; Figure 1B). Therefore, clone 1 (termed PAZ-PPARg) was selected as the most reliable candidate and was used for further detailed characterization. 3.2. Characterization of PAZ-PPARg Reporter Cell Line. In the next series of experiments, we characterized the dose-dependent response of the PAZ-PPARg cell line to 15dPGJ2 and PGD2. The PAZ-PPARg cells were treated with increasing concentrations of 15d-PGJ2 and PGD2 (1−100 μM) for 24 h, which resulted in the induction of luciferase activity with the maximum ranging from 60- to 120-fold for both compounds. The 15d-PGJ2-treated cells reached the maximum of fold induction at a concentration of 40 μM and the PGD2treated cells at a concentration of 70 μM, respectively (Figure 2A,B). These concentrations were set as reference values. Acquired findings are in accordance with the results of previous experiments, in which a relatively low level of fold induction was measured in the PAZ-PPARg cells treated with 50 μM 15dPGJ2, in comparison to the cells treated with 50 μM PGD2 (Figure 1A). In parallel experiments, the cytotoxic effect of PPARγ agonists on the PPAZ-PPARg cells was tested. After 24 h of exposure to 15d-PGJ2 and PGD2 at concentrations of 1−100 μM, we observed a decrease in viability of the PAZ-PPARg cells (Figure 2C,D). However, any substantial cytotoxic effect of 15dPGJ2 and PGD2 at their reference concentrations was not observed. The values of cell viability reached 74% (40 μM 15dPGJ2) and 86% (70 μM PGD2) of the viability of control cells, respectively. In order to determine a minimal incubation period for the reliable identification of PPARγ agonists, the time-course analyses of luciferase induction were performed. The PAZPPARg cells were treated with 40 μM 15d-PGJ2 and 70 μM PGD2 for 6, 12, 18, and 24 h. After the treatments, the luciferase activity was measured and the time course of induction of luciferase activity was estimated as a percentage of the maximum fold induction obtained after 24 h of treatment. We found that

Figure 3. Time-course analyses of luciferase activity in the PAZ-PPARg cells. The cells seeded into 96-well plates were stabilized for 16 h and treated with 40 μM 15d-PGJ2, 70 μM PGD2, or vehicle (0.1% DMSO v/v) for 6, 12, 18, and 24 h. After the treatments, the cells were lysed and the luciferase activity was measured. The data are the means ± SD of three independent experiments and are expressed as a percentage of the maximum fold induction obtained after 24 h of treatment.

As an important parameter, the PAZ-PPARg cell line was tested for its survivability and the maintenance of functionality after the cryopreservation. The cells were frozen according to the standard procedure, using the fetal bovine serum and DMSO as a cryoprotectant (9:1), and stored for 2 weeks at −80 °C. The fresh cells and the cells after the freeze−thaw cycle at the third and fifth passage were treated with 40 μM 15d-PGJ2 and 70 μM PGD2 for 24 h, and the luciferase activity was measured. We found that, after the freeze−thaw cycle, the induction of luciferase activity in the cryopreserved cells decreased rapidly, but it was fully recovered within the five subsequent passages (Figure 4). The maintenance of ability of the PAZ-PPARg cells to respond to PPARγ agonists over a long period is another important feature of the newly prepared reporter cell line required for its practical use. Therefore, the PAZ-PPARg cells were treated with 40 μM 15d-PGJ2 and 70 μM PGD2 for 24 h after every fifth passage, and the luciferase activity was measured. We showed that the induction of luciferase activity remained stable for at least 57 days, which corresponded to 25 passages (Table 2). Finally, we compared the morphology of the PAZ-PPARg cells and the parental T24/83 cells. We did not find any significant differences in the morphology of either cell line, even after the cells were maintained in culture for 10 passages (Figure 5). 3.3. Specificity of PAZ-PPARg Cell Line Response. The ability of the PAZ-PPARg cell line to respond specifically to agonists of PPARγ was determined using T0070907, a selective antagonist of PPARγ. For this purpose, the PAZ-PPARg cells were treated with T0070907 at concentrations of 0.01−30 μM in the presence of 40 μM 15d-PGJ2 or 70 μM PGD2 for 24 h. The application of T0070907 resulted in a significant decrease in 15d-PGJ2- and PGD2-induced luciferase activity in the PAZ12070


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decrease in cell viability was observed after the treatment of the cells with T0070907 at the highest concentrations (10−30 μM). These findings indicate that the decrease in luciferase activity was a result of specific inhibition of luciferase induction by the selective antagonist of PPARγ T0070907 but was not a consequence of cytotoxicity. In addition, the selectivity of the PAZ-PPARg cell line toward PPARγ agonists was confirmed using different model agonists of nuclear and steroid hormone receptors. The PAZ-PPARg cells were treated with agonists of thyroid receptor (3,3′,5-triiodo-Lthyronine; T3), vitamin D3 receptor (cholecalciferol; VD3), retinoic X receptor (9-cis-retinoic acid; 9-cis-RA), retinoic acid receptor (all-trans-retinoic acid; all-trans-RA), mineralocorticoid receptor (aldosterone), glucocorticoid receptor (dexamethasone), estrogen receptor (17β-estradiol), progesterone receptor (progesterone), and androgen receptor (5α-dihydrotestosterone) and two agonists of peroxisome proliferatoractivated receptor α (pirinixic acid and fenofibrate) at concentrations of 0.001−50 μM for 24 h. We did not observe any significant induction of luciferase activity after the application of the examined agonists, indicating that the responsivity of the PPAZ-PPARg cells to the agonists of PPARγ was highly selective (Figure 7). 3.4. Effect of Flavored Nonalcoholic Beverages on Transcriptional Activities of Nuclear and Steroid Hormone Receptors. Nine commonly marketed ready-todrink teas (TEA-1−TEA-9) and 18 flavored mineral waters (MW-1−MW-18) were screened for their potential effects on transcriptional activities of PPARγ, VDR, TR, A,R and GR. After the treatments, we did not observe any substantial increase in luciferase activity in any reporter cell line, in the agonist mode of experiments (Figures 8A−C and 9A,B). The only exceptions were the PZ-TR and AIZ-AR cells, in which the application of TEA-7 slightly induced luciferase activity (Figures 8C and 9A). The inductions of luciferase activity reached 1.50-fold in the PZTR cells and 2.23-fold in the AIZ-AR cells. Even though the increase in luciferase activity was statistically significant, the level of luciferase induction represented only a small portion of the luciferase activity induced by model agonists in the PZ-TR (2.59-fold) and AIZ-AR cells (13.85-fold), respectively. In contrast, in the PAZ-PPARg cells treated with MW-9, a significant drop in basal luciferase activity was observed (Figure 8A). Similarly to previous experiments, no significant changes in the induction of luciferase activity were observed in the PZ-TR and AZ-GR reporter cell lines, in the antagonist mode of

Figure 4. Effect of cryopreservation on responsivity of the PAZ-PPARg cells to 15d-PGJ2 and PGD2. The fresh cells and the cells after the freeze−thaw cycle at the third and fifth passage were seeded into 96well plates and stabilized for 16 h. After 24 h of treatment with 40 μM 15d-PGJ2, 70 μM PGD2, or vehicle (0.1% DMSO v/v), the cells were lysed and the luciferase activity was measured. The data correspond to the means ± SD of quadruplicate measurements and are expressed as a fold induction over the DMSO-treated cells. Similar results were obtained from three independent experiments.

Table 2. Long-Term Maintenance of the PAZ-PPARg Cell Line Responsivity to PPARγ Agonists passage number

days in culture

40 μM 15d-PGJ2 (fold ± SD)

70 μM PGD2 (fold ± SD)

5 10 15 20 25

9 19 32 44 57

74 ± 3.9 106 ± 5.6 80 ± 11.2 119 ± 9.3 78 ± 10.9

84 ± 1.2 93 ± 6.4 86 ± 7.7 94 ± 6.7 82 ± 8.8

PPARg cells (Figure 6A,B). The inhibition of luciferase activity was dose-dependent, with IC50 values representing 117.15 ± 51.27 nM for 40 μM 15d-PGJ2 and 259.52 ± 97.00 nM for 70 μM PGD2. To exclude the loss of cell viability due to the cytotoxic effect of compounds used for the treatments as a reason for decreased luciferase activity, a cytotoxicity assay was performed. In parallel experiments, the PAZ-PPARg cells were exposed to T0070907 (0.01−30 μM) in the presence of 40 μM 15d-PGJ2 or 70 μM PGD2 for 24 h and after that period the viability of cells was measured. We found that the application of T0070907 in combination with PPARγ agonists did not affect the viability of cells markedly (Figure 6C,D). Only a slight

Figure 5. Morphology of the T24/83 and PAZ-PPARg cell lines. Micrographs of the parental T24/83 cells (A) and the PAZ-PPARg reporter cells (B), both at the 10th passage. 12071


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Figure 6. Inhibition of luciferase activity in the PAZ-PPARg cells by T0070907. The cells seeded into 96-well plates were stabilized for 16 h and treated with a selective antagonist of PPARγ T0070907 (0.01−30 μM) in the presence of 40 μM 15d-PGJ2 or 70 μM PGD2. A vehicle (0.2% DMSO v/v) was used as a control. After 24 h of exposure, the cells were lysed and the luciferase activity was measured (A, B). The data are the means ± SD of five independent experiments and are expressed as a percentage of the maximum 15d-PGJ2 or PGD2 fold induction. In parallel experiments, the cytotoxicity assay was performed (C, D). The data represent the means ± SD of four independent experiments and are expressed as a percentage of viability of the 15d-PGJ2- treated or PGD2-treated cells.

Figure 7. Responsivity of the PAZ-PPARg cells to agonists of selected nuclear and steroid hormone receptors. The cells seeded into 96-well plates were stabilized for 16 h and treated with different agonists of nuclear and steroid hormone receptors (0.001−50 μM) or vehicle (UT; 0.1% DMSO v/v) for 24 h. After the treatments, the cells were lysed and the luciferase activity was measured. The data are the means ± SD of three independent experiments and are expressed as a fold induction over the DMSO-treated cells.

experiments (Figures 8F and 9D). However, exposure of the AIZ-AR cells to TEA-7 in the presence of 100 nM DHT resulted

in a significant increase in luciferase activity, in comparison to the DHT-treated cells (Figure 9C). The level of luciferase 12072


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Figure 8. Effects of nonalcoholic beverages on transcriptional activity of nuclear receptors. The PAZ-PPARg, IZ-VDRE, and PZ-TR cells were seeded into 96-well plates and stabilized for 16 h. Subsequently, the cells were treated with extracts of ready-to-drink teas (TEA-1−TEA-7) and flavored mineral waters (MW-1−MW-18) in the absence (A−C; agonist mode) or presence (D−F; antagonist mode) of model agonists of PPARγ (40 μM 15dPGJ2), VDR (50 nM VD3), or TR (50 nM T3). A vehicle (UT; 0.1% ethanol or/and DMSO v/v) was used as a control. After 24 h, the cells were lysed and the luciferase activity was measured. The data represent the means ± SD of three independent experiments and are expressed either as a fold induction over the vehicle-treated cells in the agonist mode (A−C) or as a percentage of the maximum fold induction by model agonists in the antagonist mode (D−F). An asterisk (*) indicates a significant difference (p < 0.05) in comparison to the vehicle-treated cells (A−C) or to the model agonist-treated cells (D−F).

PAZ-PPARg cells to TEA-3 and TEA-7 in the presence of 40 μM 15d-PGJ2 resulted in a substantial decrease in luciferase activity (Figure 8D). The values of luciferase induction corresponded to 49% (TEA-3) and 63% (TEA-7) of the induction attained after the treatment with 40 μM 15d-PGJ2 alone. Similar, a statistically significant inhibition of luciferase activity was observed after the treatment of the PAZ-PPARg cells with 40 μM 15d-PGJ2 in combination with MW-5, MW-8, MW-11, or MW-18 (Figure 8D). The values of luciferase induction ranged from 53 to 72% of the level of induction obtained after the treatment with 40 μM 15d-PGJ2. Finally, we found that MW-9, which inhibited basal luciferase activity in the PAZ-PPARg cells in the agonist mode, completely abolished the 15d-PGJ2-induced luciferase activity (Figure 8D). To analyze the effects of MW-9 on PPARγ transcriptional activity in more detail, an extract of MW-9 diluted to a scale of concentrations was used for the treatments of the PAZ-PPARg cells. The application of MW-9 at relative concentrations corresponding to 10−100% of the original

induction reached 160% of the induction attained after the treatment with 100 nM DHT alone. The same sample slightly induced the luciferase activity in the AIZ-AR cells also in the agonist mode. The application of TEA-7 in the presence of 50 nM VD3 induced a significant increase in luciferase activity in the IZ-VDRE reporter cells, as well (Figure 8E). Moreover, we observed the enhanced induction of luciferase activity in the IZVDRE cells after the treatment with 50 nM VD3 in combination with TEA-3, MW-15, MW-16, or MW-17 (Figure 8E). The levels of luciferase induction corresponded to 127% (TEA-3), 134% (TEA-7), 120% (MW-15), 119% (MW-16), and 124% (MW-17) of the induction attained after the treatment with 50 nM VD3 alone. Interestingly, the extracts of ready-to-drink teas TEA-3, which increased the VD3-induced luciferase activity in the IZ-VDRE cell line, and TEA-7, which increased the model agonist-induced luciferase activity in both the IZ-VDRE and AIZ-AR cell lines, had an opposite effect on the transcriptional activity of PPARγ in the PAZ-PPARg cell line. Exposure of the 12073


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Figure 9. Effects of nonalcoholic beverages on transcriptional activity of steroid hormone receptors. The AIZ-AR and AZ-GR cells were seeded into 96-well plates and stabilized for 16 h. Subsequently, the cells were treated with extracts of ready-to-drink teas (TEA-1−TEA-7) and flavored mineral waters (MW-1−MW-18) in the absence (A, B; agonist mode) or presence (C, D; antagonist mode) of model agonists of AR (100 nM DHT) or GR (100 nM DEX). A vehicle (UT; 0.1% ethanol or/and DMSO v/v) was used as a control. After 24 h, the cells were lysed and the luciferase activity was measured. The data represent the means ± SD of three independent experiments and are expressed either as a fold induction over the vehicle-treated cells in the agonist mode (A, B) or as a percentage of the maximum fold induction by model agonists in the antagonist mode (C, D). An asterisk (*) indicates a significant difference (p < 0.05) in comparison to the vehicle-treated cells (A, B) or to the model agonist-treated cells (C, D).

of a large number of substances for their potential agonist or antagonist activity. The construction of such systems is based on either transient or stable transfection of cells with a reporter plasmid carrying luciferase gene driven by a specific receptor response element. Although the generation of stably transfected reporter cell line is a material- and time-consuming process, the prospective benefits of this type of reporter system are undoubted. The main advantages of a stable reporter assay are high sensitivity and good reproducibility of the results due to the homogeneous transfection of all cells with reporter plasmid. There is also no need for additional normalization of data with respect to the efficiency of transfection. Moreover, an expensive and time-consuming transfection procedure at the initial phase of each experiment is not required. An important parameter for development of a reliable reporter system is the selection of an appropriate cell line. Generally, a low level or complete absence of endogenous receptors in the cells, which are used for the transfection, is compensated by their cotransfection with an expression plasmid carrying a gene encoding for this receptor. The fact that introduction of expression plasmid into the reporter system usually results in overexpression of receptor is often omitted. This may lead to alterations in the stoichiometric ratio between the receptor and other transcriptional regulators and does not reflect the natural situation. In the current article, we present a novel stably transfected luciferase reporter cell line for the assessment of PPARγ transcriptional activity, PAZ-PPARg. To develop an exclusively human reporter system, which overcomes possible effects resulting from overexpression of PPARγ, three main criteria for selection of the cell line were defined: (1) human origin of the cell line, (2) sufficient endogenous expression of PPARγ and

concentration revealed that the inhibition of basal luciferase activity occurred only after the exposure of the cells to an undiluted extract of MW-9 in the agonist mode (Figure 10A). In parallel experiments, the cytotoxic effect of MW-9 on the PPAZPPARg cells was examined. A significant decrease in cell viability was observed only in the cells exposed to an undiluted extract of MW-9 (Figure 10B). The value of cell viability reached 63% of the viability of control cells. These findings indicate that the cytotoxic effect of MW-9 can participate in the inhibition of basal luciferase activity in the PAZ-PPARg cells exposed to an undiluted extract of MW-9. In the antagonist mode of experiments, the treatment of the PAZ-PPARg cells with a concentration scale of MW-9 in the presence of 40 μM 15dPGJ2 resulted in a concentration-dependent inhibition of luciferase activity (Figure 10C). A statistically significant decrease in luciferase activity was measured in the cells exposed to MW-9 at relative concentrations ranging from 30 to 100% of the original concentration. The values of luciferase induction corresponded to 43−4% of the induction obtained after the treatment with 40 μM 15d-PGJ2. Although the application of MW-9 at relative concentrations of 40−100% in the presence of 40 μM 15d-PGJ2 slightly decreased the viability of cells, no statistically significant cytotoxic effect was observed (Figure 10D). Therefore, the inhibition of 15d-PGJ2-induced luciferase activity was a specific effect of MW-9, rather than a consequence of cytotoxicity.

4. DISCUSSION Cell-based reporter gene systems represent a valuable and widely used instrument in nuclear receptor research. This experimental approach enables simultaneous in vitro screening 12074


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Figure 10. Effects of MW-9 on the transcriptional activity of PPARγ. The PAZ-PPARg cells were seeded into 96-well plates and stabilized for 16 h. Subsequently, the cells were treated with an extract of MW-9 at relative concentrations corresponding to 10−100% of the original concentration in the absence (A, B; agonist mode) or presence (C, D; antagonist mode) of the model agonist of PPARγ (40 μM 15d-PGJ2). A vehicle (UT; 0.1% ethanol or/and DMSO v/v) was used as a control. After 24 h, the cells were lysed and the luciferase activity was measured (A, C). The data represent the means ± SD of three independent experiments and are expressed either as a fold induction over the vehicle-treated cells in the agonist mode (A) or as a percentage of the maximum fold induction by 15d-PGJ2 in the antagonist mode (C). In parallel experiments, the cytotoxicity assay was performed (B, D). The data represent the means ± SD of three independent experiments and are expressed either as a percentage of viability of the vehicle-treated cells (B) or as a percentage of viability of the 15d-PGJ2-treated cells (D). An asterisk (*) indicates a significant difference (p < 0.05) in comparison to the vehicle-treated cells (A, B) or to the 15d-PGJ2-treated cells (C, D).

reporter cell line was constructed on the same principle.38 In contrast to the previous cell line, a chimeric plasmid used for transfection of the HeLa cells carried the ligand binding domain of zebrafish PPARγ, instead of human PPARγ. The presence of the zebrafish PPARγ ligand binding domain resulted in the inability to induce luciferase activity by either endogenous or synthetic model agonists of human PPARγ. This has limited the use of the HG5LN-GAL4-zfPPARγ reporter cell line to study of PPARγ transcriptional activity exclusively in the zebrafish model organism. Recently, two stable luciferase reporter cell lines for the assessment of transcriptional activity of PPARγ, PPARγ1 CALUX and PPARγ2 CALUX, were developed.39 The PPARγ1 CALUX cell line was prepared by transfection of the U2OS cells with pGL3-3xPPRE-tata-luc reporter plasmid and expression vector for PPARγ1. To generate the PPARγ2 CALUX cell line, the U2OS cells were transfected with pGL4-3xPPRE-tata-luc reporter plasmid and expression vector for PPARγ2. Reporter plasmids contained luciferase gene driven by three copies of specific PPARγ response element and minimal promoter, similarly to a reporter plasmid used for construction of the PAZ-PPARg cell line. However, cotransfection of U2OS cells with PPARγ expression plasmids leading to a possible overexpression of PPARγ in both CALUX reporter cell lines

its heterodimeric partner RXRα, and (3) functional mechanism responsible for PPARγ-mediated regulation of gene expression. On the basis of available data, the T24/83 human bladder carcinoma cell line was selected as the most suitable candidate that meets all required criteria.35,36 The transfection of the T24/ 83 cells was performed using a reporter plasmid carrying luciferase gene driven by the specific PPREγ derived from the promoter region of ACOX gene.20 No necessity of additional cotransfection with a PPARγ expression plasmid enabled the preservation of the stoichiometric ratio between the PPARγ and other transcriptional regulators, reflecting the natural situation in human cells. Several stable37−39 and transient40−42 luciferase reporter systems for monitoring PPARγ transcriptional activity have been constructed over the past decade. The stably transfected HG5LN GAL4 PPARγ reporter cell line was developed by introducing a chimeric plasmid containing the ligand binding domain of human PPARγ fused to the yeast GAL4 DNA binding domain and a reporter plasmid carrying luciferase gene driven by a pentamer of the yeast activator GAL4 binding sites into the HeLa cells.37 This reporter cell line was specifically designed for identification of compounds that are capable to bind and activate human PPARγ. Later, the HG5LN-GAL4-zfPPARγ stable 12075


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which biotransformation of tested substances is not considered. If the activities of compounds should be examined following metabolic transformations, the incubation with a competent system such as human liver microsomes or human hepatocytes should precede, in general, the application of compounds to the reporter cell line. Alternatively, appropriately designed microphysiological systems or organoids could be used.45 The reliability of the PAZ-PPARg cell line was demonstrated by screening of 27 flavored nonalcoholic beverages for their potential disrupting effects on transcriptional activity of PPARγ. To obtain more complex information, all tested beverages were examined also for their ability to modulate transcriptional activities of TR, VDR, AR, and GR. Using the cell-based in vitro model systems, it has been shown that some flavored mineral waters are able to activate AhR and induce expression of CYP1A1 gene.28 Similar effects have been reported for some ready-to-drink teas.29 The authors observed that the exposure of cells to ready-to-drink teas can lead to an increase in transcriptional activities of AhR and PXR and induction of CYP3A4 gene expression. In the current study, we revealed that none of the examined nonalcoholic beverages remarkably affected transcriptional activities of nuclear and steroid hormone receptors in agonist mode of experiments. The only exception was one sample of ready-to-drink tea that slightly increased activities of TR and AR. On the other hand, several tested readyto-drink teas and mineral waters had the capability of potentiating transcriptional activities of VDR and AR induced by their model agonists. Interestingly, we found that some of the examined nonalcoholic beverages inhibited or completely abolished model agonist induced activity of PPARγ. To propose a possible mechanism for how beverages can affect transcriptional activities of nuclear and steroid hormone receptors, it is necessary to know the complete compositions of all examined beverages. Since our information on compositions of beverages is based solely on the limited lists of constituents provided by the producers (Table 1), we can only speculate which components are responsible for these effects. The main components of the majority of mineral waters used in our experiments were artificial sweeteners. It has been shown that artificial sweeteners such as aspartame, acesulfame, saccharin, and cyclamate do not affect transcriptional activities of AhR and GR.46 It is likely that these compounds did not induce changes in transcriptional activities of receptors in our experiments either. This assumption was supported by the findings that some of the mineral waters containing artificial sweeteners altered activities of PPARγ and VDR, whereas the rest of the mineral waters with contents of artificial sweeteners did not affect activities of these receptors (Figure 8D,E). Similarly, alterations in activities of nuclear and steroid hormone receptors did not correlate with the presence of most other known constituents such as citric acid, sodium citrate, and sodium benzoate in tested mineral waters and readyto-drink teas. Finally, the only variable components specifically occurring in each examined mineral water were artificial aromas. Unfortunately, any relevant data on chemical structures or characteristics of these substances were not provided by the producers. Nevertheless, we can speculate that artificial aromas might be involved in modulations of transcriptional activities of PPARγ and VDR, with respect to the particular effects of all tested mineral waters. Another group of substances which could play a possible role in modulations of transcriptional activities of nuclear and steroid hormone receptors are compounds present in tea extracts. Extracts of teas, the main components of all examined ready-to-drink teas, contain a variety of bioactive

should be considered. In comparison to the existing transient cell-based luciferase reporter systems,40−42 using the stably transfected PAZ-PPARg cell line provides several advantages. The main benefits of the PAZ-PPARg cell line are high sensitivity and good reproducibility of the results. In the transient reporter systems, the efficiency of transfection can significantly affect acquired data; thus, an additional normalization of the results is required. Moreover, the level of luciferase induction in transiently transfected cells is usually too low; maximum values of luciferase activity range from 3- to 5fold.40,42 In addition, other important attributes representing the advantages of the PAZ-PPARg cell line in comparison to the existing transient luciferase reporter systems are no need for any transfection procedures and the possibility of long-term storing and maintenance of stable luciferase induction. The maintenance of functionality of the PAZ-PPARg cell line after cryopreservation was evaluated as an important parameter for its practical use. It is generally accepted that, after the freeze− thaw cycle, the cells undergo at least three subsequent passages until they propagate to a sufficient amount to perform the experiment. During this period, the cells recover from the stress caused by the freeze−thaw procedure, long-term storage at low temperatures (−80 and −196 °C), and presence of cryoprotectant (5−10% DMSO). A period of three passages is usually sufficient for the complete recovery of the cells, which means that the induction of luciferase activity in recovered cells is equivalent to the luciferase induction in the cells before the cryopreservation.31,34 However, the measurement of luciferase activity in the IZ-VDRE reporter cell line showed that the maximum fold induction dropped after the freeze−thaw cycle, in comparison to the values obtained in the fresh cells.33 On the other hand, an increased induction of luciferase activity was observed after cryopreservation in the AZ-AHR reporter cell line.43 Subsequent passaging of the cells resulted in the stabilization of luciferase induction in both reporter cell lines.33,43 In the PAZ-PPARg reporter cell line, a substantial decrease in induction of luciferase activity was observed within the first three passages after the freeze−thaw cycle. However, the induction of luciferase activity increased to the level of the luciferase induction in the fresh cells after two additional passages (Figure 4). Taken together, the PAZ-PPARg cells recovered from the stress caused by cryopreservation within the first five subsequent passages and they could be considered as fully functional. This fact was confirmed by the findings that the induction of luciferase activity remained consistent over the next 20 successive passages (Table 2). According to available data, the existing reporter cell lines are able to maintain stable luciferase induction during the period ranging from 12 passages (PZ-TR)34 to 32 passages (IZ-VDRE),33 which is comparable to our obtained results. The newly developed PAZ-PPARg reporter cell line represents a model system that can be used as an effective tool for in vitro monitoring of PPARγ transcriptional activity in various food safety, environmental, toxicological and pharmacological applications. Despite the versatility of such reporter gene systems, there are some limitations in their use. These limitations result from the fact that metabolic transformations, as well as pharmacokinetic and pharmacodynamic interactions, which occur in vivo, are lacking in reporter gene models. As a consequence, the results obtained by in vitro models can not always be directly extrapolated to in vivo effects.44 Since the xenobiotic-metabolizing apparatus of the PAZ-PPARg cells is very limited, this model is suitable preferentially for studies, in 12076


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Journal of Agricultural and Food Chemistry organic molecules such as alkaloids, flavonoids, polyphenols, and terpenes. In particular, flavonoids and polyphenols found in extracts of green tea and rooibos (red tea) have been reported for their endocrine disrupting effects.47,48 Since the changes in activities of receptors were observed after the exposure to TEA-3 and TEA-7 (containing green tea and rooibos extracts), we can suppose that these organic compounds might participate on alterations in transcriptional activities of AR, PPARγ, TR, and VDR (Figures 8C−E and 9A,C). Our findings indicate that constituents of some commonly marketed flavored nonalcoholic beverages can disrupt normal activities of nuclear and steroid hormone receptors. Such modulations of transcriptional activities may consequently result in alterations in expression of target genes.

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AUTHOR INFORMATION

Corresponding Author

*P.I.: tel, +420 585 634 904; e-mail, [email protected]. ORCID

Peter Illés: 0000-0003-3909-1491 Funding

This work was supported by the grant from the Czech Science Foundation GACR 16−07544S (to P.I., A.G,. and K.K.), a student grant from Palacky University PrF-2018−005 (to Z.D.), and the Operational Programme Research, Development and Education-European Regional Development Fund project no. CZ.02.1.01/0.0/0.0/16_019/0000754 of the Ministry of Education, Youth and Sports of the Czech Republic (to Z.D.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Alzbeta Srovnalova, Michaela Svecarova, and Hana Prichystalova for their assistance in performing the experiments.

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ABBREVIATIONS USED 15d-PGJ2, 15-deoxy-δ12,14-prostaglandin J2; 9-cis-RA, 9-cisretinoic acid; all-trans-RA, all-trans-retinoic acid; AhR, aryl hydrocarbon receptor; AR, androgen receptor; COX-2, cyclooxygenase-2; DEX, dexamethasone; DHT, 5α-dihydrotestosterone; ED, endocrine disruptor; GR, glucocorticoid receptor; MW, flavored mineral water; PGD2, prostaglandin D2; PPAR, peroxisome proliferator-activated receptor; PPRE, peroxisome proliferator-activated receptor response element; PXR, pregnane X receptor; RXR, retinoic X receptor; T3, 3,3′,5-triiodo-Lthyronine; TEA, ready-to-drink tea; TR, thyroid receptor; VD3, cholecalciferol (vitamin D3); VDR, vitamin D3 receptor

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