Estrogen receptor-mediated transcriptional activities of spent coffee

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Food Safety and Toxicology

Estrogen receptor-mediated transcriptional activities of spent coffee grounds and spent coffee grounds compost, and their phenolic acid constituents Byoung Ha An, Hyesoo Jeong, Jin-Hee Kim, Sujeong Park, Jin-Hyun Jeong, Min Jung Kim, and Minsun Chang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b02452 • Publication Date (Web): 08 Jul 2019 Downloaded from pubs.acs.org on July 19, 2019

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Journal of Agricultural and Food Chemistry

Estrogen receptor-mediated transcriptional activities of spent coffee grounds and spent coffee grounds compost, and their phenolic acid constituents

Byoung Ha An†,‡, Hyesoo Jeong†,‡, Jin-Hee Kim†,§, Sujeong Park‡, Jin-Hyun Jeong§, Min Jung Kimǀǀ,⊥, and Minsun Changǀǀ,⊥,*

‡Graduate

School of Biological Sciences, Sookmyung Women’s University, 100 Chungparo

47-gil, Seoul 04310, Republic of Korea § College

of Pharmacy, Yonsei Institute of Pharmaceutical Sciences, Yonsei University, 85

Songdogwahak-ro, Yeonsu-gu, Incheon 21983, Republic of Korea ǀǀDepartment

of Biological Science, College of Science, Sookmyung Women’s University,

100 Chungparo 47-gil, Seoul 04310, Republic of Korea ⊥ Research

Institute of Women’s Health, Sookmyung Women’s University, 100 Chungparo

47-gil, Seoul 04310, Republic of Korea

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ABSTRACT: Spent coffee grounds (SCG) are the most abundant coffee by-product and are generally discarded as waste. The horticultural use of SCG and SCG compost (SCGC) has become popular due to a growing interest in environmentally friendly measures for waste disposal. Estrogen-like endocrine disrupting chemicals in the soil can be absorbed by plants and subsequently by humans who consume these plants. The objectives of this study are to determine the phytochemical profiles of extracts of SCG and SCGC and to evaluate the estrogen-like activities of SCG, SCGC, and the major coffee phenolic acids, specifically, 5O-caffeoylquinic acid (CQA), caffeic acid, and ferulic acid. Their inductive effects on estrogen receptor (ER)-mediated gene transcription have been examined in cultured cell lines. CQA was the most abundant phenolic acid in SCG and SCGC and was further examined for its ER-mediated estrogen-like activity using various assays. This is the first study to report the estrogen-like signaling activities of coffee by-products and their major constituents.

KEYWORDS: spent coffee grounds, spent coffee grounds compost, phenolic acids, 5-Ocaffeoylquinic acid, estrogen receptors, endocrine disrupting chemical


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INTRODUCTION

Coffee is one of the most popular beverages and is the second only to petroleum as a globally traded commodity.1 Coffee is made by grinding and aqueous heat extraction or brewing of roasted coffee beans. Processes involved in the preparation of coffee beverages result in the production of a solid residue known as spent coffee grounds (SCG), which comprise the most abundant coffee by-product (55~67%).2 The International Coffee Organization has reported the increased production of SCG as coffee consumption increases world-wide, with an annual global production of approximately 8 million tons of SCG.3 The vast majority of SCG are discarded as waste and SCG may represent a pollution hazard. Therefore, the use of SCG for various applications is becoming an important environmental issue. SCG have been investigated for the removal of lead from water, biodiesel production, composting enhancer, agricultural medium, and food ingredients.4 In particular, the use of SCG in horticulture as compost has become popular with the growing interest in developing environmentally friendly horticulture techniques and recycling waste management.5 SCG contain polysaccharides such as hemicelluloses and sugars, as the major constituents (40–53%), as well as caffeine (0.02%) and polyphenols (2.5%).4 Polyphenols of SCG are represented mainly by several highly bioavailable and bioactive phenolic acids such as caffeic acid (CA), ferulic acid (FA), and chlorogenic acids. The latter are diester derivatives of caffeoyl acids and quinic acids. Among the coffee chlorogenic acids, 5-Ocaffeoylquinic acid (CQA) is the most abundant constituent.6 CQA is metabolized to CA and further to FA in humans by colonic microbiota and catechol O-methyltransferase.7 Extracts of SCG have antioxidant, genoprotective, and ultraviolet (UV) ray-protective activities.8-9 Studies have shown that CQA exerts anti-inflammatory, antibacterial, antitumor, cancer preventive, antidiabetic, and antilipidemic activities,10 suggesting that CQA is a valuable phytochemical that may be useful as a nutraceutical or food additive. 3 ACS Paragon Plus Environment


Estrogen receptors (ERs) are ligand-dependent transcription factors that are involved in the induction of estrogen-responsive genes. ER signal transduction is mediated by two types of ERs: ER and ER.11 Even though the exact physiological roles of the two subtypes remain unknown, they clearly have unique biological roles and tissue distribution patterns. In particular, ER plays a more prominent role in mammary gland and uterine tissues, as well as in the preservation of bone homeostasis and the regulation of metabolism, whereas ER has more profound effects on the central nervous and immune systems, as well as antiproliferative effects in breast and uterine tissues.12 Therefore, it is thought that ERα-mediated signal transduction plays a major role in the disruption of both reproductive function and normal development in humans and other animals by estrogen-mimicking endocrine disrupting chemicals (EDCs).13 Exposure of humans and other animals to EDCs occurs mainly through inhalation, ingestion, and dermal contact of EDC-contaminated personal products, soil, air, water, and sewage. Plants may absorb various types of EDCs or pharmaceuticals present in sewage sludge or from soils in contact with treated sewage waters.14 Ingestion of plants grown in contaminated soil can be considered as one of the bioaccumulation pathways of EDCs in humans, although the quantity of chemicals that plants absorb from these soil may be very minor. Considering the ongoing and increasing generation of SCG and the interest in horticultural use for SCG and SCGC, it is important to evaluate the potential harmful effects of SCG, SCGC, and their constituents. In the present study, SCG and SCGC were identified for the first time as estrogen-like active coffee by-product. The ER-mediated biological activity of hot water extracts of SCG and SCGC was evaluated in cultured cell lines to assess whether SCG or SCGC can play a role in ER-mediated signaling activation. Phenolic acids such as CQA, CA, and FA were investigated for their roles in ER-mediated gene transcription. CQA was further investigated as a main phenolic acid responsible for MCF-7 cell 4 ACS Paragon Plus Environment

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proliferation and ER subtype-dependent transcriptional activities. Molecular docking analysis was utilized to predict the binding mode of CQA to the ligand binding domain (LBD) of ERs. Finally, the estrogen-like activity of CQA was examined in immature female rats by determining the effects of CQA on endometrial proliferation. The results of this study provide a rationale for further evaluation of novel biological activities of SCG and SCGC, and their constituents as endocrine disrupting products/chemicals.

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MATERIALS AND METHODS Chemicals. All chemicals and reagents were purchased from Sigma-Aldrich (St.

Louis, MO) unless stated otherwise. Solvents for liquid chromatography experiments were purchased from Burdick & Jackson (Morristown, NJ). All cell culture reagents were purchased from Gibco (Grand Island, NY) unless stated otherwise. ICI 182,780 (ICI) was obtained from Tocris (Ellisville, MO). 17-Estradiol (E2), ICI, CQA, CA, and FA were dissolved in dimethylsulfoxide (DMSO), and stock solutions were stored at −20 °C for further cell experiments. Preparation of Extracts of SCG and SCGC. SCG were obtained from a local coffee shop in Kwangmyoung, South Korea. SCGC was purchased from KT Co., Ltd (Gwangju, Gyeonggi, South Korea; SCGC-KT). Another SCGC product was purchased from Danong Co., LTD (Namyangju, Gyeonggi, South Korea; SCGC-GC). SCG or SCGC samples were dried in an oven at 40 ºC for 12 h and then stored in an air tight container at −20 ºC. Aqueous extracts of SCG or SCGC were prepared based on a previously described method with minor modifications.15 Briefly, dried SCG or SCGC powder (2 g) was dissolved in 100 mL of deionized water. The aqueous solution of SCG or SCGC was boiled for 5 min while stirring, and the resulting solution was cooled for 20 min to 20 ºC. After centrifugation 5 ACS Paragon Plus Environment


(12,000 × g, 4 °C, 15 min) of SCG or SCGC solutions, the supernatant was filtered through a 0.45 m-polytetrafluoroethylene syringe filter, and the filtrate was injected into high performance liquid chromatography (HPLC) and LC-electrospray ionization-tandem mass spectrometry (ESI-MS/MS) systems for analysis of phenolic acids. Analytical Instrumentation. The HPLC system (Agilent, Santa Clara, CA) consisted of an Agilent 1200 binary pump, a diode array detector (DAD), and an Agilent 1260 autosampler. A Kinetex® C18 column (5 m, 4.6 × 150 mm; Phenomenex, Torrance, CA) protected by a KrudKatcher Ultra HPLC in-line filter (Phenomenex) was used for the separation. Mobile phase A consisted of water with 0.1% (v/v) formic acid, while mobile phase B was acetonitrile. The solvent flow rate was 0.8 mL/min, and the column temperature was set at 30 °C. The gradient began with 10% B and this was maintained for 7 min and was increased to 20% B over 1 min, and followed by a return to the initial condition over a period of 22 min. The DAD was set at a wavelength of 330 nm. LC-ESI-MS/MS analysis was performed using an LTQ XL linear ion trap mass spectrometer (Thermo Fisher Scientific, San Jose, CA) linked to the same HPLC instrument components described above, with the exception of the use of a Kinetex® C18 column (2.6 m, 4.6 × 100 mm, Phenomenex) for separation. The flow rate was 0.2 mL/min, and the column temperature was set at 30 °C. The same mobile phase A and B that were used for HPLC-DAD analysis were also used for MS analysis. The gradient began with 10% B and was maintained for 10 min, after which it was increased to 20% B over 2 min, and finally increased to 25% B over 6 min. This was followed by return to the initial condition over a period of 12 min. A mass spectrometer was operated in the negative ion electrospray mode at an ion spray voltage of 4.2 kV and a source temperature of 275 °C. The sheath gas flow rate was 35 arb, the aux gas flow rate was 5 arb, the capillary voltage was set to −24 V, and the 6 ACS Paragon Plus Environment

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tube lens was set to −62.48 V. A data acquisition was performed using Xcalibur software version 2.2 (Thermo Fisher Scientific) with a full-scan MS analysis from m/z 170 to 360. MS2 analysis was used to monitor precursor to product ion transitions of m/z 353 → 191 for CQA, m/z 179 → 135 for CA, and m/z 193 →134 for FA). Cell Culture. The MCF-7 human breast cancer cell line MCF-7 and the human embryo kidney (HEK) cell lines were obtained from the American Type Culture Collection (Manassas, VA). The cells were maintained in Dulbecco’s modified Eagle’s essential medium containing 10% fetal bovine serum (FBS) and 1% antibiotics/antimycotics at 37 °C in a 5% CO2 atmosphere. Estrogen-free medium was prepared by supplementing phenol redfree medium with 3×dextran-coated charcoal-treated FBS while the other components remained the same. Cell Proliferation Assay. MCF-7 cells were seeded at 1.5 × 104 cells/well in 24-well plates after culture in estrogen-free medium for 2 days. The next day, the medium was replaced with medium containing either test sample, E2, ICI, or an appropriate combination of samples. The cells were replenished each day with medium-containing test compounds. Cell growth was determined by measuring the amount of DNA as described previously.16 Briefly, the cell lysates were incubated with 200 L of 2 g/mL of Hoechst 33258 (Bio-Rad Laboratories, Hercules, CA) for 1 h and DNA-dye conjugate was detected using a 96-well fluorescence SpectraMax i3x plate reader (Molecular Devices, San Jose, CA) with an excitation filter of 360 to 390 nm and an emission filter of 450 to 470 nm. Plasmid Transfection and Luciferase Reporter Assay. An estrogen response element (ERE)-luciferase plasmid containing three copies of the Xenopus laevis vitellogenin A2 ERE upstream of the firefly luciferase gene was a gift from Dr. V. C. Jordan (MD Anderson Cancer Center, Houston, TX). The cells were cultured in estrogen-free media for 2 7 ACS Paragon Plus Environment


days and plated (1.5 × 105 cells/well) in triplicate in a 24-well plate. The cells were transiently transfected with the ERE-luciferase plasmid (0.25 μg/well) and Renilla luciferase plasmid (0.125 μg/well) using Lipofectamine 2000 Reagent (Invitrogen) as described previously.17 In some cases, cells were co-transfected with either an ERα- or ERβ-expressing plasmid (0.25 μg/well) in addition to the ERE-luciferase plasmid. At 24 h post transfection, the cells were treated with various concentrations of SCG, SCGC, CQA, CA, FA, and E2 (1 nM), ICI (1 μM), or appropriate combinations, and they were incubated for an additional 24 h. The cells were then harvested with Passive Lysis Buffer (Promega). Luciferase activity in the cell lysates was measured using the Dual Luciferase Assay kit (Promega) with a SpectraMax i3x plate reader (Molecular Devices) in a 96-well plate format. Data were reported as relative luciferase activity (firefly luciferase reading divided by the Renilla luciferase reading). Animals and Ethics. All animal studies were performed in accordance with the guidelines of the Korea Ministry of Food and Drug Safety. The study protocol was reviewed and approved by the Institutional Animal Care and Use Committee of Sookmyung Women’s University (IACUC-1806-013). Immature female Sprague Dawley (SD) rats (13−14 days old, 30–35 g) were obtained from Orient Bio (Sungnam-si, Kyeonggi, South Korea) and were housed under standard animal laboratory conditions (21 ± 2 °C; 50–80% relative humidity; 12-h light/dark cycle) with free access to water and a phytoestrogen-free diet (2020X, Harlan, Host, the Netherlands). Forty-nine SD rats (20-21 days old) were randomly divided into seven groups (n=7 per group) and subcutaneously injected once each day for 3 days with either E2 (5 μg/kg/day) or CQA (25 and 50 mg/kg/day) dissolved in 1% carboxymethyl cellulose (CMC) solution. One percent CMC solution was used as a vehicle control. Twentyfour hours after the last administration, the animals were sacrificed and the wet weight of each uterus was measured as described previously.18 8 ACS Paragon Plus Environment

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Quantitative Polymerase Chain Reaction (qPCR) Analysis. MCF-7 cells grown in estrogen-free media were treated with the appropriate test compounds or with vehicle control. Total RNA was extracted using a Qiagen RNeasy kit (Hilden, Germany) and cDNA was synthesized from total RNA (2 g) by oligo (dT) and M-MLV reverse transcriptase (Promega). A qPCR analysis was performed to determine the mRNA levels of each gene of interest on an ABI Prism 7500 Sequence Detection System (Applied Biosystems, Foster City, CA) using SYBR Green PCR Master Mix (Toyobo, Osaka, Japan) and a gene-specific primer set (Table S1 for primer sequences in experiments with RNA from MCF-7 cells and Table S2 for those from rat uterus tissue). ABI 7500 Software (v2.0.6) was used to estimate the threshold cycle (Ct). The 2-ΔΔCt method was applied for quantification of the relative amount of target genes. The data were normalized against the expression level of the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene. Results were expressed as fold change when the gene expression level in the vehicle control-treated sample was set to 1. Western Blots. Cells were trypsinized, pelleted, washed in PBS, resuspended in lysis buffer (50 mM HEPES, 150 mM NaCl, 1 mM EDTA, 2.5 mM EGTA, 10 mM βglycerophosphate, 10% glycerol, and 0.5% NP-40, pH 8.0) containing cOmpleteTM protease inhibitor cocktail (Roche Biochem, Indianapolis, IN), mixed, and centrifuged at 12,000 × g for 10 min at 4 °C. Protein concentrations in the supernatant were measured by Bradford assay. Equal amounts of protein were electrophoresed on a 10% SDS-polyacrylamide gel and transferred to a PVDF membrane using a Trans-Blot Turbo transfer system (Bio-Rad, Hercules, CA). The membrane was blocked in TBS-Tween 20 (TBST) containing 10% nonfat dry milk at 4 °C overnight, and then incubated for 2 h with mouse anti-ERα (D8H8) antibody (1:1000; Cell Signaling Technology, Danvers, MA) or mouse anti-β-actin (AC-15) antibody (1:10,000; Sigma-Aldrich) in TBST containing 2.5% nonfat dry milk. The membranes were washed in TBST containing 2.5% nonfat milk and incubated with the 9 ACS Paragon Plus Environment


secondary antibody (1:3000) for 2 h. β-Actin was used as a loading control. The specific proteins were detected using an enhanced chemiluminescence western blotting analysis system (GE Healthcare Life Sciences, Pittsburgh, PA) and photographed using a LAS-3000 Image analyzer (Fujifilm Life Science, Tokyo, Japan). Molecular Docking Study. The structures of CQA as a ligand bound to the LBD of human ERs were constructed using the SYBYL-X2.1.1 molecular modeling software (Tripos St. Louis, MO) and were energy minimized by the Powell method using Gasteiger−Marsili charge and the Tripos force field.19 The crystal structures of agonist-induced conformations of human ERα and ERβ were obtained from the Protein Data Bank (PDB code 1A5220 for ERα and PDB code 3OLS21 for ERβ). All the water molecules from the crystal structures were removed while the missing hydrogen atoms were added to the structures. The protein and ligand preparations were done according to the respective protocols in the program. Docking studies were carried out using Surflex-Dock in SYBYL-X2.1.1. Both bound E2 and CQA in 1A52 and 3OLS were subjected to a redocking process and the best docked conformation of CGA-bound LBDs was superimposed to those of E2-bound LBDs. The protein and ligand preparations were done according to the respective protocols in the program. The Surplex-Dock scoring function is the sum of the hydrophobic, polar, repulsive, and entropic terms including crash and solvation over the appropriate atom pairs. Statistical Analysis. In vitro experiments were performed in triplicate. The results are representative of at least three independent experiments. All results are presented as mean ± SD values. An F-test was used to determine if the variances among groups were equal. If sample variances were homogenous, analysis of variance (ANOVA) was used followed by Bonferroni post hoc tests using Prism Version 4.0 (GraphPad Software, San Diego, CA). Measurement of EC50 values was performed using Prism 4.0. 10 ACS Paragon Plus Environment

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RESULTS Phenolic Acid Contents in SCG and SCGC Extracts. The presence and quantity of

the CQA, CA, and FA phenolic acids (the chemical structures are provided in Figure 1D), present in hot water extracts of a SCG sample and the SCGC-KT and SCGC-GC samples were determined using HPLC-DAD and LC-ESI-MS/MS analyses. CQA, CA, and FA are known to be the major phenolic acid derivatives found in coffee beans, coffee drinks, and SCG.8 CQA, CA, and FA were eluted at retention times of 7.0, 9.5, and 23.5 min, respectively, under our analytical conditions (Figure 1A-C). The peak corresponding to CQA was observed as a major peak in the HPLC chromatograms of the SCG and SCGC-KT samples, while no CQA peak was found in SCGC-GC. CA was eluted from both SCGC samples and FA was found only in minor quantities in SCGC-GC. The amount of CQA present in SCG (2.44 mg/g dry weight) was approximately 100-fold to that observed in SCGC-KT (Table 1). SCGC-GC contained the highest quantity of CA among the samples tested. The differential presence of each phenolic acid among the test samples was further confirmed by LC-ESI -MS/MS analysis (Figure 2A-C and Figure S1 for MS/MS spectra of each phenolic acid). The identity of the peaks corresponding to each phenolic acid was validated using commercially available reference chemicals and MS/MS spectra in the literature.22 ER-Mediated Transcriptional Activity of SCG and SCGC Extracts in MCF-7 Cells. The signaling pathway involved in the interaction of ligand-bound ERs and EREs is a classical molecular mechanism underlying the actions of estrogen-like xenobiotics. Thus, ERE-driven reporter activity was examined in ER-positive MCF-7 cells. ERE-luciferase activities increased in a dose-dependent manner in cells treated with extracts derived from 11 ACS Paragon Plus Environment


SCG and the SCGC acquired from the two sources (Figure 3). The luciferase activity induced by exposure to SCG extracts (5000 g/mL) was as efficacious as that induced by E2 (1 nM). The two SCGC induced approximately 25% of maximal luciferase activity obtained by E2 treatment. Co-treatment of either SCG or SCGC with E2 did not affect the activities induced by E2 alone (Figure 3), implying that SCG or SCGC did not antagonize or synergize E2induced reporter activity. Co-treatment with ICI, a full ER antagonist, and either SCG or SCGC completely abolished the induction of luciferase activity (Figure 3), suggesting that modulation of the ERE-luciferase gene by SCG and SCGC involves ER-mediated mechanisms. Concentration and ER Subtype-Dependent Transcriptional Activity of Coffee Phenolic Acids. Given that the relative luciferase activities of SCG and the two SCGC extracts were positively correlated with the presence and amount of total phenolic acids (Table 1 and Figure 3), the ability of each phenolic acid constituent to induce ER-mediated transcription was further investigated in a concentration and ER subtype-dependent manner using an ERE-reporter gene assay. Treatments with 50 M CQA, CA, and FA induced reporter gene transcription in MCF-7 cells where EMax values of CQA, CA, and FA were 89.0, 82.1, and 32.9%, respectively (Table 2). ER-mediated mechanisms associated with phenolic acid-induced ERE reporter activity were confirmed by co-incubation of cells with ICI and each phenolic acid (data not shown). ER subtype-dependent ERE-mediated activity was investigated in HEK cells transfected with plasmids expressing either human ER or ER. The EC50 of CQA was 8.16, 7.54, and 11.4 M for induction of ER/ERE-associated transcription in MCF-7, HEK/ER, and HEK/ER cells, respectively (Table 2). The EMax values of CQA were 100% and 61% for transcriptional activities through ER and ER, respectively. The values of EC50 and EMax 12 ACS Paragon Plus Environment

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suggested that CQA may act as an agonist of both ERs and CQA may exert more potent and efficacious estrogen-like activity through ER than through ER CA exhibited partial agonistic activities for both ER and ER with a higher preference for ER compared to that of CQA, while FA acted only as an ER agonist with negligible reporter activity in HEK/ER cells. CQA was the most potent agonist for both ER and ER and FA was the least potent agonist among the phenolic acids tested in our study. Analysis of Expression of Endogenous Estrogen-Responsive Genes in MCF-7 Cells. The observation that CQA was the most abundant of the phenolic acids examined, with the best ER-mediated signaling activity prompted further study of the estrogen-like activities of CQA in MCF-7 cells at the molecular level and in rodent models. To investigate the effects of CQA on the activation of ER target genes in situ, the levels of the endogenous estrogen-responsive gene in CQA-treated MCF-7 cells were determined using RT-qPCR. The genes encoding the progesterone receptor (PGR) and the gene regulated in breast cancer 1 (GREB1) were monitored, as it is well documented that their mRNA expression is upregulated upon exposure to estrogenic or anti-estrogenic compounds.23-24 Treatment of MCF-7 cells with E2 resulted in 27- and 2.9-fold induction of PGR and GREB1, respectively, compared to the gene expression levels in samples obtained DMSO-treated cells (Figure 4A and 4B). PGR expression was induced in a CQA concentration-dependent manner, with 2.1and 6.7-fold induction observed in cells treated with 10 and 25 M CQA, respectively, compared to that of DMSO-treated cells (Figure 4A). The GREB1 gene expression was also induced by CQA (25 M) exposure and was found to be 1.9-fold higher than that of the control (Figure 4B). CQA-mediated induction of PGR and GREB1 was completely blocked when cells were co-incubated with ICI (Figure 4A and 4B), suggesting that ER-associated signal transduction is involved in the modulation of gene expression by CQA. 13 ACS Paragon Plus Environment


Effects of CQA on ER Expression in MCF-7 Cells. The effects of CQA on the expression of both ERα mRNA and proteins in MCF-7 cells were determined, as estrogenic compounds downregulate ERαs at both the mRNA and protein levels, whereas pure ER antagonists suppress the protein levels but fail to affect mRNA levels.25 Treatment of MCF-7 cells with E2 (1 nM) resulted in greater than 90% reduction in the ER protein levels (Figure 5A and 5B) and 70% reduction in its gene level (Figure 5C). ER protein levels were reduced by 21% and 40% as a result of treatment of cells with 10 and 25 M CQA, respectively (Figure 5A). Similarly, the ER gene was slightly downregulated in cells treated with 10 and 25 M of CQA (Figure 5C). On the other hand, ICI had no effect on ERα gene expression. The combination of ICI with either E2 or CQA resulted in >90% decrease in the ERα protein (Figure 5A and 5B). Our data demonstrate that CQA may share similar mechanism with E2 in regard to modulation of ERα at the transcription and translational levels in MCF-7 cells. Cell Proliferation Activity of CQA in MCF-7 Cells. Cell proliferation assays were performed to investigate the effects of CQA on the estrogen-dependent growth of MCF-7 cells. The MCF-7 cell proliferation assay is a well-established method that provides data in a rapid and straightforward manner for test compounds with even weak estrogenicity. A dosedependent increase in cell numbers in response to test compounds is considered as evidence of estrogenicity.26 In our study, the cell numbers (measured by DNA amount) increased in CQA-treated cells in a concentration-dependent manner with an EC50 of 18.5 M (Figure 6). The maximum efficacy (EMax) of cell proliferation was achieved using 50 M CQA, which was 50% of E2 (1 nM)-induced cell growth. The proliferative activity by CQA or E2 was completely abolished in the presence of ICI (1 M). The results suggested that ER-mediated signaling pathways are associated with cell proliferation activity observed after CQA 14 ACS Paragon Plus Environment

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treatment. Molecular docking analysis of CQA bound to the LBD of ERs. Molecular docking analysis was performed to provide molecular insights into the binding modes of CQA with the LBD of ERα and ERβ. The docking study results demonstrated that the molecular interactions between CQA and the amino acid residues within the ER-LBD included hydrogen bonding networks in addition to hydrophobic contributions resulting from electron-rich and electron-deficient groups present in CQA. The modeling data indicated that CQA tends to adopt the favorable binding modes of CQA within the ER-LBD, and this involves multiple hydrogen bonds. CQA engages in hydrogen bonding interactions with Glu353, Arg394, Gly521, and His524 in the LBD of ER (Figure 7A). Dihydroxyl groups at the C-3 position within the caffeic acid moiety and the C-3 position within the quinic acid moiety are analogous to those located at C-3 and C-17 positions within E2 that allow for agonistic molecular interactions with Glu353 and His524 (Figure 7A). Dock scores were calculated as 6.8201 and 8.0694 for E2 and CQA, respectively, indicating that CQA possesses comparable binding affinity to that of E2 toward the ER-LBD. CQA in the LBD of ER also displayed hydrogen bonding interactions with five amino acids (Glu305, Arg346, Leu339, Gly472, and Leu476) (Figure 7B). Dihydroxyl groups at the C-3 and C-17 positions in E2 associated with three amino acids, Glu305, Arg346, and His475, through hydrogen bonding interactions (Figure 7B). Three hydroxyl groups at the C3 and C-4 positions within the caffeic acid moiety and the C-4 position within the quinic acid moiety were attributed to hydrogen bonding interactions. The docking score of CQA to the ER-LBD was 3.778, which was only half of that in the CQA-ER-LBD (8.0694) or E2ER-LBD (6.8201), implying that CQA may be positioned in a less stabilized conformation 15 ACS Paragon Plus Environment


when bound to the ER-LBD compared to the ER-LBD. These differences found in the docking analyses between ER isoforms corroborated the ERE-luciferase reporter assay data that demonstrated the preference of CQA toward ER-mediated transcription activity compared to the ER-mediated pathway (Table 2). Effects of CQA on Uterus Weights and Gene Expression in Female Immature Rats. The effects of CQA on uterus weights were investigated in female immature rats, since the endometrium tends to proliferate and its weight increases in response to estrogen via ERdependent signaling pathways in estrogen-deprived rodents.27 E2 treatment resulted in a 1.7fold increase in the uterine weight to body weight ratio. In contrast, CQA administration did not result in an increase in this ratio, with a 1.1- and 1.0-fold induction in the 25 mg/kg and 50 mg/kg dose groups, respectively, compared to that in the control group (Figure 8A). The changes in gene expression levels in the rat uterine tissues were analyzed to determine the effects of CQA treatment on the molecular pathways involved in proliferation of the endometrium. The estrogen-responsive genes and two subtypes of ERs were analyzed by RT-qPCR. PGR and estrogen-enhanced transcript-1 (EET-1) were selected as the estrogen-responsive marker genes.28 PGR was upregulated by approximately 2.0-fold by both doses of CQA (25 and 50 mg/kg) and 1.6-fold in the E2 group compared to levels observed in the vehicle-treated group (Figure 8B). EET-1 was also slightly increased in groups treated with E2 and CQA, although the values were not statistically significantly different among groups. The gene levels of both ER subtypes also increased upon administration with both E2 and CQA. ER gene expression was induced up to 3.8-fold and 2.4-fold in the CQA 25 mg/kg-treated and CQA 50 mg/kg-treated groups, respectively (Figure 8B). The relative gene expression of ER increased by 2.1-fold and 1.7-fold in low dose (CQA, 25 mg/kg) and high dose (CQA, 50 mg/kg) groups, respectively (Figure 8B). 16 ACS Paragon Plus Environment

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DISCUSSION

Coffee is the second most popular commodity world-wide.1 Meeting this demand produces considerable organic waste in the coffee industry. Many gardening and horticultural "grey literatures" have encouraged the use of SCG as a direct soil amendment and SCGC as a nutrient-rich fertilizer.29 For example, Starbucks Korea in cooperation with the South Korea Ministry of Environment donated 3.6 tons of SCG-derived compost to local farmers in 2018.30 The effects of SCG and SCGC and their use as fertilizer have been described.31-32 However, few studies have addressed the safety issues underlying the application of SCG and SCGC to soil. Particularly, no scientific reports have addressed the potential endocrine disrupting properties of these by-products. The results of the present study suggest that extracts of SCG and SCGC, and their phenolic acids, have ER-mediated signaling activity in cultured cancer cells. Based on our dose- and ER subtype-dependent activity studies, CQA, the major phenolic acid constituent in SCG, exerts the estrogen-like ER transactivation property. In turn, treatment of MCF-7 cells with CQA resulted in estrogen-responsive genes such as PGR and GREB-1 since the liganded ERs act as transcription factors leading to induction of target genes. PGR is a nuclear transcription factor that is involved in differentiation of the endometrium, control of implantation, maturation of the mammary epithelium, and modulation of gonadotropinreleasing hormone pulsatility,33 whereas GREB1 is an early estrogen-responsive gene and a critical co-factor for ER-mediated transcription leading to E2-stimulated growth of breast cancer cells.24 Induction of both PGR and GREB1 in response to CQA in MCF-7 cells suggests that CQA functions as an agonistic ER-signaling molecule. CQA-mediated ER degradation also suggests that CQA mirrors the behavior of E2 17 ACS Paragon Plus Environment


in MCF-7 cells (Figure 5). Binding of either E2 or ICI to ER causes proteosomal degradation of ER via different mechanisms.25 Estrogens downregulate ER mRNA by inhibiting ER gene transcription at early stages, whereas ICI does not affect ER transcription and, instead, blocks agonist-dependent ER downregulation.34 In the present study, both the mRNA and protein levels of ER in MCF-7 cells were reduced by CQA and E2 treatment, whereas the combination of ICI with either E2 or CQA resulted in ER degradation but had no significant effect on the levels of ER mRNA. These data suggest that CQA functions as an estrogen-like phytochemical in MCF-7 cells. Our study suggests that CQA may be classified as a phytoestrogen. Phytoestrogens possess endocrine disrupting properties in vertebrates, including humans, although they can be beneficial in terms of nutritional values and favorable cardiovascular and bone health profiles.35-36 In particular, genistein, a major soy isoflavone, is a well-known phytoestrogen with health-promoting effects that are especially prominent in post-menopausal women. Genistein is also the most studied natural endocrine disrupting phytochemical due to its potent ER-mediated signaling activity. Genistein was nominated for the endocrine disrupter project in 1995 by the National Toxicology Program of the United States National Institute of Environmental Health Sciences. Since then, various in vitro, in vivo, and alternative model studies have been performed to determine the endocrine disrupting properties, carcinogenicity, and toxicological effects of genistein.37 The results of the present study on CQA and previous reports on genistein suggest that CQA has unique biological and pharmacokinetic behaviors that are comparable to those of genistein. Both genistein and CQA displayed ER-mediated hormone action in cell culture models. Genistein exhibited ER preference over ER, suggesting that it can be characterized as a beneficial phytoestrogen in terms of cell proliferation and metabolic 18 ACS Paragon Plus Environment

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profile.38 In contrast, our data demonstrated that CQA transmits signals preferentially via ER (Table 2), and its metabolites CA and FA also displayed similar effects on ERmediated action. Both the potency and efficacy of ER-mediated signaling activation by CQA were lower than those of genistein in cell-based assays (unpublished data). However, the lower estrogen-like activity of CQA does not exclude the possibility of endocrine disruption property because most of the negative biological effects of phytoestrogens are derived from ER-mediated signaling pathways. In addition, phytoestrogens with hormonal activities that are lower than those of genistein have been reported to interfere with reproductive functions in vertebrates.39 Finally, as the amount, exposure route, race, health status, and duration of exposure could collectively affect the long-term effects of environmental estrogens,40 it is worthwhile to further evaluate the ER-mediated endocrine disrupting activity of CQA. Microflora biotransformation appears to be common for both genistein and CQA. The biotransformation of genistein leads to the formation of 5-hydroxyequol.41 This microflora metabolite is reported to possess more potent hormonal activity than its parent compound. The pharmacokinetic fate of CQA in humans also involves its biotransformation into CA and FA by colonic bacteria, including Escherichia coli, Bifidobacterium lactis, and Lactobacillus gasseri, which express cinnamoyl esterase, and unidentified microflora which are able to catalyze O-methylation.7, 42 The present study demonstrates that CA and FA are weak ER agonists. Therefore, it is reasonable to conclude that the consumption of CQA would result in a pharmacodynamically longer half-life through the generation of bioactive metabolites in a similar pattern to genistein. There is a lack of evidence concerning the uterotrophic properties of genistein and CQA. Several studies have demonstrated no significant increase in endometrial thickness as a result of genistein consumption, although this remains equivocal in some human studies.43 19 ACS Paragon Plus Environment


Presently, uterine weight change was not induced by CQA in the rat model (Figure 8A). The rodent uterotrophic assay is an established way to evaluate the endocrine disrupting property of test chemicals, and has revealed the milestone outcome of molecular disruption of signaling pathways mediated by the estrogen-like EDCs.44-45 To investigate the possible mechanisms underlying the lack of uterotrophic activity by CQA, the transcription of ERs and several genes regulated by estrogens were measured in uterine tissues. In rat uterus, PGR expression is been reported to respond rapidly to E2 and to increase significantly, after which it decreases to control levels,46 and is upregulated by two-fold upon ethinylestradiol treatment in a similar type of assay.47 Uterine PGR expression was also upregulated by the oral administration of E2 and soy isoflavone concentrate (by 2.8 and 3.7-fold, respectively) in a 4wk study on ovariectomized rats.48 Therefore, PGR can be classified as a uterine gene that is upregulated by estrogen-like chemicals, depending on the potency of the chemicals and the animal study design. The slight increase in PGR expression after E2 treatment under the experimental conditions of the present study is consistent with previously reported results.46 Meanwhile, the two-fold upregulation of PGR after CQA treatment in the present study can likely be attributed to the transcriptional responses to CQA, as an estrogen-like chemical that modulates PGR expression at rates that are different from those of responses to E2. The estrogen-induced upregulation of EET-1 was reported to occur rapidly and was affected by both chemical type and study design.28 The absence of significant of EET-1 induction by either E2 or CQA in the present study suggests that CQA shares the regulatory mechanism of EET-1 expression with that of E2. It is possible that genistein, an ER-selective phytoestrogen, exerts no significant uterotrophic activity, despite its potent activities toward both ER and ER, as the ERmediated signaling pathway is thought to play a role in anti-proliferation.49 The present study demonstrated that CQA is ER-selective but still possesses ER-mediated signaling activity 20 ACS Paragon Plus Environment

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(up to 61% that of E2; Table 2), suggesting that ER-mediated transactivation by CQA may negatively regulate ER-mediated proliferative effects, at least in part. Other studies have suggested that genistein is also involved in various anti-proliferative mechanisms, such as caspase-3 activation, inhibition of tyrosine kinase, and repression of telomerase activity.50 In this regard, the molecular mechanisms by which CQA failed to induce endometrium proliferation warrants further research. It should be noted that the negligible or weak uterotrophic activity of certain chemicals does not rule out their endocrine disrupting potentials, as evidenced by genistein. Given this, further reproductive toxicology studies are warranted for estrogen-like signaling molecules, such as CQA, that exert weak or no effects on endometrial thickness. In summary, to our knowledge this is the first study demonstrating the ER-mediated transcriptional activities of SCG, SCGC, and their major phenolic acid constituents, specifically, CQA, CA, and FA. In particular, CQA, one of the major chlorogenic acids present in coffee beans, coffee drinks, and SCG, was the most estrogenic phytochemical in cell culture models without in vivo uterotrophic activity among the coffee phenolic acids tested in our study. CQA can be either directly ingested via coffee consumption or indirectly ingested through edible plants grown in SCG or SCGC-containing soil, implying that the amount of CQA ingested can vary. The impact of EDCs on organisms is dependent on the route of administration, exposure frequency, pharmacokinetic profiles of chemicals, amounts, and health/medical conditions of the subjects. Therefore, even the apparently small amount of some EDCs still requires further evaluation in the context of their endocrine disrupting effects. Based on our data and the trends in the horticultural use of SCG and SCGC, further studies are warranted to elucidate the EDC effects of these compounds on the plants and wildlife, and their chemical fates in the ecosystem.

21 ACS Paragon Plus Environment




ASSOCIATED CONTENT

Supplementary data The supplementary data associated with this article is available free of charge on the ACS Publications website at DOI:



AUTHOR INFORMATION

Corresponding Author *Telephone: +82 2 2077 7626. E-mail: [email protected] Author Contributions †Byoung

Ha An, Hyesoo Jeong, and Jin-Hee Kim contributed equally to this work.

Funding NRF-2016K1A1A8A01939090 , NRF-2016R1A2B4012963, and NRF-2019R1A2C1003497 (National Research Foundation, Republic of Korea). Notes The authors declare no competing financial interest.



REFERENCES

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Figure Legends Figure 1.

HPLC-DAD chromatograms showing the presence of CQA, CA, and FA in aqueous extracts of spent coffee grounds (SCG) (A), SCG compost (SCGC)-KT (B), and SCGC-GC (C). Each peak in the chromatogram was identified, and the quantity was calculated based on results from reference chemicals. Chemical structures (D) of 5-O-caffeoylquinic acid (CQA), caffeic acid (CA), and ferulic acid (FA) present in aqueous extracts of spent coffee grounds (SCG) and SCG compost (SCGC). The IUPAC numbering system51 was used for CQA.

Figure 2.

Liquid Chromatography-electrospray-tandem mass spectrometry chromatograms showing the presence of CQA, CA, and FA in aqueous extracts of spent coffee grounds (SCG) (A), SCG compost (SCGC)-KT (B), and SCGCGC (C). Each peak was validated by mass fragmentation patterns using tandem MS analysis.

Figure 3.

ER-mediated activation of ERE-luciferase activity by extracts of SCG and two types of SCGC (SCGC-KT and SCGC-GC) in MCF-7 cells. Cells were transiently transfected with the ERE-luciferase reporter plasmid and Renilla luciferase plasmid. Twenty-four hours after transfection, cells were treated with vehicle control, E2 (1 nM), various concentrations (g/mL) of SCG and SCGC extracts, ICI (1 M), or appropriate combinations for 24 h. Luciferase activities in the cell lysates were determined by measuring the luminescence intensity. Data are shows mean ± SD. An asterisk indicates p < 0.001 compared to the vehicle control.

Figure 4.

Induction of estrogen-responsive genes by CQA in MCF-7 cells. Total RNA was isolated from cells treated with vehicle, E2 (1 nM), CQA, or in combination 29 ACS Paragon Plus Environment


with ICI (1 M) for 24 h. RNA was subjected to cDNA synthesis and real-time PCR quantitation of progesterone receptor (PGR) (A) and gene regulated in breast cancer 1 (GREB-1) genes (B). CQA10 and CQA25 refer to the sample treated with 10 or 25 M of CQA, respectively. The data shown are a representative of three independent experiments. The error bars represent SD, and significant (*, p < 0.001 and #, p < 0.01) induction compared with vehicle control is indicated above each bar. Figure 5.

Modulation of estrogen receptor (ER)  protein (A and B) and mRNA (C) by CQA in MCF-7 cells. ER was detected by SDS-PAGE and immunoblotting of CQA-treated cell lysates (A) and the quantitation of blot results are shown in (B). mRNA level of ER (C) was quantitated using real-time qPCR. CQA10 and CQA25 refer to the sample treated with 10 or 25 M of CQA, respectively. The data shown are a representative of three independent experiments. The error bars represent SD, and significant (*, p < 0.001 and #, p < 0.01) reduction compared with vehicle control is indicated above each bar.

Figure 6.

Concentration-dependent MCF-7 cell proliferation by CQA. Cells were treated with vehicle control (DMSO), E2 (1 nM), various concentration (1~50 M) of CQA, and appropriate combinations with ICI (1 M) for 3 days. Cell growth was quantified by measuring DNA amount. Each sample was performed in triplicate and the data represent mean ± SD. An asterisk indicates p < 0.001 compared to the vehicle control.

Figure 7.

Binding pockets and potential binding modes of CGA and E2 (green) in the ligand binding domain (LBD) of ERα (PDB code: 1A52, A) and ERβ (PDB code: 3OLS, B). The key amino acid residues that showed hydrogen bonding 30 ACS Paragon Plus Environment

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interaction with CGA (magenta in A and blue in B) are displayed as stick model representations by atom type, with oxygen atoms in red and proton in white. The proposed hydrogen bonding interactions of CQA with the amino acid residues in the ligand binding pocket of ERs are shown as yellow dashed lines. The predicted distances (Å) between atoms involved in the hydrogen bonding interactions are also shown. Figure 8.

Uterus weight gain in immature Sprague Dawley rats after SC administration of CQA. Rats were administered with 1% CMC (control group), estradiol (E2) (0.5 mg/kg/d), and CQA (two groups; 25 and 50 mg/kg/d). The uterotrophic activity of CQA was determined by measuring the ratio of uterus wet weight to body weight (A). The mRNA levels of progesterone receptor (PGR), estrogenenhanced transcript-1 (EET-1), estrogen receptor (ER), and ER were measured by quantitative polymerase chain reaction (B). Values are expressed as mean ± SD. ANOVA was performed to estimate differences between corresponding vehicle control and sample-treated groups. An asterisk indicates significant differences from the corresponding control (p < 0.01) and a sharp (#) indicate p < 0.001.



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Table 1. Quantity of three phenolic acids present in spent coffee grounds (SCG) and SCG compost (SCGC) extracts determined by HPLC-DAD analysisa

Sample

5-O-Caffeoylquinic acid (CQA)

Caffeic acid (CA)

Ferulic acid (FA)

2440b

N.D.c

N.D.

27.2

4.11

N.D.

N.D.

22.9

14.2

Spent coffee ground (SCG) Spent coffee ground compost (SCGC)-KT Spent coffee ground compost (SCGC)-GC aPhenolic

acids were separated on HPLC-DAD analysis and were validated based on

analytical results of reference chemicals. The quantity was calculated based on the standard curve obtained by reference analysis. HPLC-DAD analytical conditions are described in Materials and Methods. bUnit

of quantity is g per g of dried sample weight.

cThe

presence of the corresponding phenolic acid was not detected under our analytical

conditions.


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Table 2. ERE-reporter gene transcription activities induced by phenolic acids present in SCG/SCGC extracts through estrogen receptor (ER)  and  in various cell lines

Cell line

EC50 (M)a

EMax (%)b

/ EC50selectivity ratiog

MCF-7

8.16 ± 1.01

89.0

-

-

HEK/ERd

7.54 ± 0.871

100 0.661

1.64

-

-

0.061

1.24

-

-

N.D.

3.27

Phenolic acid

CQAc

CA

FA

HEK/ERe

11.4 ± 1.04

61.0

MCF-7

9.46 ± 1.44

92.1

HEK/ER

7.63 ± 0.911

43.2

HEK/ER

124 ± 11.9

34.7

MCF-7

18.2 ± 2.12

32.9

HEK/ER

13.3 ± 1.21

37.9

HEK/ER

N.D.f

aHalf-maximal

bMaximal

cCQA,

dHEK

RTE (/)h

11.6

effective concentration.

effect normalized to the activity with 1 nM E2, which was set as 100%.

CA, and FA refer 5-O-caffeoylquinic acid, caffeic acid, and ferulic acid, respectively.

cells were transfected with a mammalian expression plasmid for ER in combination

with an (ERE)3-luciferase reporter plasmid and treated with various concentrations of the phenolic acids and luciferase activity was measured. Values are means of at least three experiments. eHEK

cells were transfected with a mammalian expression plasmid for ER in combination 33 ACS Paragon Plus Environment


with an (ERE)3-luciferase reporter plasmid and treated with various concentrations of the phenolic acids, and luciferase activity was measured. Values are means of at least three experiments. fEC 50

value was not determined due to low activity values among concentrations of FA under

our assay conditions. g/

EC50-selectivity ratio was calculated by dividing the EC50 value for ER activation by

that for ER. A greater ratio indicates increased ER selectivity of the test compound. hRelative

transcriptional efficacy (RTE) was obtained by dividing the EMax for ER by that

for ER. Greater values indicate that the compound is more efficacious in an ER-selective manner.


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Estrogen receptor-mediated transcriptional activities of spent coffee

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