The effects of bisphenol A, benzyl butyl phthalate, and di(2-ethylhexyl) phthalate on estrogen receptor alpha in estrogen receptor-positive cells under hypoxia

Environmental Pollution 248 (2019) 774e781

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The effects of bisphenol A, benzyl butyl phthalate, and di(2-ethylhexyl) phthalate on estrogen receptor alpha in estrogen receptor-positive cells under hypoxia* Choa Park, Jeonggeun Lee, Byounguk Kong, Joonwoo Park, Heewon Song, KeunOh Choi, Taeeun Guon, YoungJoo Lee* Department of Integrative Bioscience and Biotechnology, College of Life Science, Sejong University, Seoul, 05006, Republic of Korea

a r t i c l e i n f o

a b s t r a c t

Article history: Received 12 September 2018 Received in revised form 12 February 2019 Accepted 21 February 2019 Available online 21 February 2019

Endocrine-disrupting chemicals (EDCs) are widely used in various consumer goods. Consequently, humans are constantly exposed to EDCs, which is associated with a variety of endocrine-related diseases. In this study, we demonstrated the effects of bisphenol A (BPA), benzyl butyl phthalate (BBP), and di(2ethylhexyl) phthalate (DEHP) on estrogen receptor alpha (ERa) expression under normoxia and hypoxia. First, we confirmed the effects of EDCs on ER activity using OECD Test Guideline 455. Compared to the 100% activity induced by 1 nM 17-b-estradiol (positive control), BPA and BBP exhibited 50% ERa activation at concentrations of 1.31 mM and 4.8 mM, respectively. In contrast, and consistent with previous reports, DEHP did not activate ERa. ERa is activated and degraded by hypoxia in breast cancer cells. BPA, BBP, and DEHP enhanced ERa-mediated transcriptional activity under hypoxia. All three EDCs decreased ERa protein levels under hypoxia in MCF-7 cells. The transcriptional activity of hypoxia-inducible factor-1 was decreased and secretion of vascular endothelial growth factor (VEGF) was increased by BPA and BBP under hypoxia in MCF-7 cells, but not by DEHP. All three EDCs decreased the ERa protein expression level in Ishikawa human endometrial adenocarcinoma cells, and DEHP caused a weak decrease in VEGF secretion under hypoxia. These results demonstrate down-regulation of ERa by EDCs may influence the pathological state associated with hypoxia. © 2019 Elsevier Ltd. All rights reserved.

Keywords: EDC ER alpha Hypoxia

1. Introduction People are constantly exposed to endocrine-disrupting chemicals (EDCs), such as the phthalates, benzyl butyl phthalate (BBP) and di(2-ethylhexyl) phthalate (DEHP), and bisphenol A (BPA) (Morgan et al., 2017), which are used in diverse consumer products and have been detected in soils, sediments, surface waters, and groundwater. BPA has been detected in surface waters in Southeast Asia, and the maximum BPA concentration in 14 selected rivers was 1950 ng/L (Yamazaki et al., 2015). The U.S. Environmental Protection Agency classifies BBP and DEHP as environmental pollutants.

Abbreviations: ERa, estrogen receptor alpha; EDC, endocrine disrupting chemical; BPA, bisphenol A; BBP, benzyl butyl phthalate; DEHP, di(2-ethylhexyl) phthalate. * This paper has been recommended for acceptance by Charles Wong. * Corresponding author. Department of Bioscience and Biotechnology, Sejong University, Kwang-Jin-Gu, Seoul, Republic of Korea. E-mail address: [email protected] (Y. Lee). https://doi.org/10.1016/j.envpol.2019.02.069 0269-7491/© 2019 Elsevier Ltd. All rights reserved.

Phthalates can be released into the air or evaporate and become particles in dust and air. The BBP concentration in indoor air ranges between 18 ng/m3 and 3.97 mg/m3, while the DEHP concentration ranges between 82 ng/m3 and 2.43 mg/m3 (Pei et al., 2013). BBP has been detected in perfumes with a maximum concentration of 201.724 ppm (Al-Saleh and Elkhatib, 2016). Other exposure pathways are contaminated foodstuffs, such as pasta, fruit, fish, and meat, and bottled water and juice contaminated with phthalates (Ceretti et al., 2010; Lee et al., 2014; Liu et al., 2015). DEHP has been detected at concentrations of 1e220 mg/L in surface water, 7.5e2045 mg/kg in the sediments at sites where DEHP was produced (Facts, 2008), and 1085 mg/L in a wastewater treatment facility (Olujimi et al., 2012). EDCs mimic the structure of the endogenous ligand, interfering with the action of the hormone and causing a physiological process that can cause hormone-related diseases (Kelce et al., 1998; Kelce and Wilson, 1997; Sonnenschein and Soto, 1998). They exert endocrine effects by affecting the activities of the androgen

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receptor (AR) and estrogen receptor (ER) (Harris et al., 1997; Takeuchi et al., 2005), which in turn results in adverse consequences, such as developmental and reproductive effects (e.g., deregulated sex steroid hormone levels, impaired sperm production and functionality, and gonad malformations) (Fisher, 2004; Lyche et al., 2009). Accumulating data show that, rather than being confined to sex hormone receptors, the mechanisms of action of EDCs are diverse and can include asexual reproduction, ion channels, inflammation, and epigenetic changes. For example, BPA is primarily considered to be an ERa and ERb agonist, but it can also act as an AR or aryl hydrocarbon receptor (AhR) antagonist, via its cross-talk with ERs, affecting other endocrine pathways (Rubin, 2011). The phthalates dibutyl phthalate (DBP) and BBP both bind to ERa in MCF-7 cells and increase the ER activity and cell proliferation (Harris et al., 1997; Jobling et al., 1995; Takeuchi et al., 2005; Zacharewski et al., 1998). Phthalates not only interfere with sexhormone activity, they also promote adipogenesis in obesity via the peroxisome proliferator-activated receptor (PPAR)g and CCAAT/ enhancer-binding protein alpha (Pereira-Fernandes et al., 2014; Yin et al., 2016). The recent second Scientific Statement from the Endocrine Society concluded that long-term permanent physiological changes due to early or chronic low-dose EDC exposure may alter susceptibility to cardiovascular diseases, diabetes, chronic respiratory diseases, and cancers (Gore et al., 2015). The analysis of the 1999e2004 National Health and Nutrition Examination Survey data indicated that polychlorinated biphenyls (PCBs), BPA, and phthalates are alter the expression of several estrogen-responsive genes that are linked to breast cancer, although only PCBs showed a significant relationship with breast cancer (Statistics, 2007). Nonetheless, the results suggest that the risk of breast cancer from these EDCs may be enhanced due to disturbance of estrogen-responsive genes through the ER signaling pathway (Roy et al., 2015). The ER is a ligand-activated nuclear transcription factor (Huang et al., 2010), through which estrogen signaling occurs by regulating target gene expression. The disruption of the ERa signaling pathway by EDCs can lead to an increased risk of abnormal fetal growth and development, impaired fertility, and hormone-dependent cancers (Morgan et al., 2017). ERa signaling occurs via both genomic and non-genomic pathways in response to ligands (Hammes and Davis, 2015). In the genomic pathway, ERa regulates the expression of target genes by directly binding to them (Hall et al., 2001). The non-genomic pathway activates different signaling pathways, such as mitogen-activated protein kinase or phosphoinositide 3-kinase (PI3K) signaling pathways (Hall et al., 2001; Mendelsohn, 2000). In addition, the non-genomic pathway of ER signaling includes crosstalk with growth factor receptors and G-protein-coupled receptors in the cytosol within seconds to minutes after exposure to estrogens (Tanos et al., 2012). The ER is activated by ligands, but can be activated even in the absence of a ligand. Unliganded ERs can be activated by growth factors, such as insulin and the epidermal growth factor, or by intracellular signaling pathways and hypoxic conditions (Smith, 1998; Yi et al., 2009). Hypoxia plays a major role in tumor development and physiological processes and development (Koh et al., 2010). Moreover, studies have demonstrated associations between hypoxia and malignant breast cancer (Lundgren et al., 2007). For instance, the physical interaction between the ER and hypoxia-inducible factor (HIF)-1 was reported by our group (Cho et al., 2006), where ERs were degraded within 6e12 h under hypoxia (Stoner et al., 2002). The estrogen response depends on and is proportional to the initial ER concentration (Chen et al., 2001). Therefore, hypoxia may play an important role in the development of acquired hormone resistance in breast cancer by modulating ERa levels (Kurebayashi, 2003; Kurebayashi et al., 2001; Stoner et al., 2002). Only a few

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studies have shown effects of EDCs on hypoxic pathways (Sen et al., 2015). For example, dibutyl phthalate reduced follicle stimulating hormone-induced expression of HIF-1a (Wang et al., 2016), and BPA inhibited the hypoxic response via degradation of HIF-1a (Kubo et al., 2004). In this study, we determined the effects of BPA, BBP, and DEHP on ERa expression under normoxia and hypoxia. All three EDCs decreased ERa protein levels under hypoxia in both MCF-7 breast cancer and Ishikawa human endometrial adenocarcinoma cells, suggesting that not only BPA and BBP, which exhibit estrogenic activity, but even DEHP, which lacks estrogenic activity, influence the ERa signaling pathway via an indirect mechanism. 2. Material and methods 2.1. Chemicals BBP (CAS # 85-68-7,
98%), DEHP (CAS # 117-81-7,
99.5%), BPA (CAS # 80-05-7,
99%), and 17-b-estradiol (E2; CAS # 50-28-2,
98%) were obtained from Sigma-Aldrich (St. Louis, MO, USA). BPA was dissolved in dimethyl sulfoxide (DMSO; CAS # 67-68-5,
99%, Sigma-Aldrich), while the other chemicals were dissolved in ethanol (Duksan Pure Chemicals, Duksan, Gyunggi-do, Korea). 2.2. Cell culture and hypoxic conditions The human breast cancer MCF-7 cell line (Korean Cell Line Bank, Seoul, Korea) was cultured in Dulbecco's modified Eagle's medium (DMEM; WelGENE, Daegu, South Korea) containing 10% fetal bovine serum (FBS; WelGENE) and penicillin/streptomycin (Gibco, NY, USA). The Ishikawa cell line (Korean Cell Line Bank) was cultured in DMEM/F12 (WelGENE) containing 10% FBS. MCF-7 and Ishikawa cells were ERa positive cell lines (Al-Bader et al., 2011; Hou et al., 2014). The human ERa-expressing HeLa-9903 cell line (HeLa9903; JCRB Cell Bank, Osaka, Japan) was cultured in DMEM containing 10% charcoal-dextran stripped FBS. All of the media used in this study lacked phenol red. All treatments were done in media containing 5% charcoaledextran stripped FBS (CD-FBS). All three cell lines were cultured stably at 37  C in a humidified 5% CO2 atmosphere. For the hypoxic microenvironment, cells were incubated in a hypoxic chamber (Thermo Fisher Scientific, Waltham, MA, USA) at 37  C containing 5% CO2/1% O2/94% N2 atmosphere. 2.3. Transfection and luciferase assays MCF-7 cells were transiently transfected with the hypoxia response element (HRE)-luciferase reporter plasmid (Park and Lee, 2014) or the estrogen response element (ERE)-luciferase reporter plasmid (plasmid number 11354; Addgene, Cambridge, MA, USA) using polyethylenimine (Polyscience, Warrington, PA, USA) and Tom media (WelGENE). The following day, the cells were isolated from a 100-Ø dish, seeded at a density of 5  104 cells/well in a 24well plate using a hemocytometer, and then stabilized in the assay medium. Each plate was treated with the reagents for 24 h (final concentration ethanol 0.1% and DMSO 0.1%). Luciferase activity was measured using the luciferase assay kit (Promega Corp., Madison, WI, USA) and detected with an AutoLumat LB9507 luminometer (EG & Berthold, Bad Wildbad, Germany), according to the manufacturer's manual. HeLa9903 cells were treated with the serially diluted EDCs, followed by incubation for 20e24 h. Luciferase activity was investigated according to the OECD Test Guideline (TG) 455 method (OECD, 2016) using the Steady-Glo Luciferase assay system (Promega Corp.). EDCs were tested for cytotoxicity using cell counting kit-8 (CCK-8; Enzo Life Sciences, Lausen, Switzerland).

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2.4. ToxCast data mining

3. Results

Publicly available data in the ToxCast program were analyzed using the iCSS ToxCast Dashboard (http://actor.epa.gov/dashboard/ ; last accessed 1 January 2018). In the ToxCast program, two ER agonist assays yielded results comparable to our ER agonist OECD TG 455 assay. ToxCast data measured reporter protein levels in the HEK293T (Tox21_ERa_BLA_Agonist_Ratio) cell line. The Tox_21_ERa_LUC_BG1_Agonist assay assessed the reporter protein level in the BG1 cell line (Brennan et al., 2016). Not all assays were performed on each EDC.

3.1. Effects of EDCs on ERa transactivation

2.5. Quantitative real-time polymerase chain reaction Total RNA was extracted and the first-strand cDNA was synthesized according to a previously reported method (Song et al., 2017). Quantitative real-time polymerase chain reaction (PCR) was performed using Power SYBR® Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA). Estrogen receptor gene (ESR)1 and b-actin primers were used as reported previously (Park and Lee, 2017). The final reaction volume was 20 mL. Real-time PCR was conducted using the StepOnePlus Real-Time PCR System (Life Technologies). Data were analyzed using the software program provided by Applied Biosystems and the results were calculated following the manufacturer's instructions (Guide).

2.6. Western blot analysis Protein isolation and western blot analysis were performed according to previously reported methods (Park and Lee, 2014). After sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the membrane from which the protein was transferred was blocked with 5% skim milk (Sigma) and probed with primary antibodies. Anti-ERa (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was diluted to 1:1000, and blots were incubated overnight. Anti-b-actin (Sigma) was diluted to 1:5000, and blots were incubated for 1 h. The membrane was washed and then incubated with a 1:5000 dilution of the secondary antibody. Horseradish peroxidase conjugated-secondary antibody goat anti-rabbit IgG (H þ L) and goat anti-mouse IgG (H þ L) were obtained from Invitrogen (Grand Island, NY, USA). The blots were detected using enhanced chemiluminescence (Amersham Pharmacia Biotech, Buckinghamshire, UK). The band strength of ERa bands was quantified using Quantity One software (Bio-Rad).

2.7. Vascular endothelial growth factor ELISA After the desired cell culture time had elapsed, cells were removed from the culture medium using centrifugation. The supernatant was stored at 80  C until use in ELISA. The ELISA kit (R&D Systems, Minneapolis, MN, USA) was used to investigate the secreted vascular endothelial growth factor (VEGF) concentration according to the manufacturer's protocol.

2.8. Statistical analysis Data were analyzed and expressed as the means ± standard deviation. The mean values among the experimental groups were statistically compared by analysis of variance using GraphPad PRISM software (GraphPad Software Inc., La Jolla, Ca, USA). Values of P < 0.05 were considered statistically significant.

We first measured the estrogenic activity of BPA, BBP, and DEHP using luciferase assays. Stably transfected cells from the ERaHeLa9903 cell line were used to monitor the activation of human ERa by BPA, BBP, or DEHP. HeLa9903 cells were treated with BPA, BBP, or DEHP for 24 h. None of the EDCs were cytotoxic to the cells at any of the concentrations tested in this experiment, as indicated by the results of the CCK-8 assay (data not shown). CCK-8 assay was used to test the cytotoxicity. BPA and BBP markedly activated ERa, with respective half-maximal effective concentration (EC50) values of 1.15 and 13.84 mM (Fig. 1A and B). In contrast, none of the DEHP concentrations activated ERa (Fig. 1C). We compared the EC50 values by mining data from ToxCast, the program with the aim of developing methods for utilizing various in vitro high throughput screening (HTS) technologies to quickly screen for potential toxicity (Judson et al., 2009). The half-maximal activation concentration (AC50) values of BPA were 1.01 and 0.363 mM in the TOX21_ERa_BLA_Agonist_ratio and TOX21_ERa_LUC_BG1_Agonist assays, respectively. According to our TG455 assay (OECD, 2016) for detecting ER ligands, the EC50 value of BPA was 1.15 mM. There were some discrepancies between the ToxCast results and our TG 455 assay, ERa transcriptional activation assay, for BBP (Table 1). BBP had AC50 values of 24.8 and 20.8 mM in the TOX21_ERa_BLA_Agonist_ratio and TOX21_ERa_LUC_BG1_Agonist assays, respectively. According to our TG 455 assay, BBP had an EC50 of 13.84 mM. This discrepancy was likely due to differences in the experimental setups. DEHP was not tested in either the ERa_BLA_Agonist_ratio or ERa_Luc_BG1_Agonist assays. 3.2. Effect of EDCs on ERa under hypoxia in MCF-7 cells Studies have shown that hypoxic conditions impact ERa-mediated transcriptional activity (Cho et al., 2006; Yang et al., 2015). In addition, concurrent treatment of hypoxia with E2 synergistically enhanced ERE-driven luciferase activity (Cho et al., 2006). We used the ERE-driven reporter gene to investigate the effect of ERamediated gene transcriptional activity of BPA, BBP, and DEHP in the hypoxic state using MCF-7 cells transfected with an ERE-luciferase plasmid. BPA and BBP significantly increased ERE-mediated transcriptional activity under normoxic and hypoxic conditions, similar to E2 (Fig. 2A). Interestingly, DEHP did not activate ERE-driven luciferase activity under normoxia. However, DEHP induced ERa transcriptional activation under hypoxia, although to a lesser extent than E2. Then, we determined the effects of EDCs on ESR1 mRNA levels in the hypoxic state. E2 treatment, as a positive control, decreased ESR1 mRNA expression under normoxia and hypoxia. ESR1 mRNA levels were reduced by BPA, BBP, and DEHP to levels similar to those by E2 under normoxic and hypoxic conditions (Fig. 2B). Previous reports demonstrated that ESR1 mRNA expression was downregulated in response to DEHP treatment in generations of wild-type mice compared to PPARa-knockout mice. This reduction of ESR1 mRNA expression by DEHP was mediated by the PPAR-dependent pathway (Kawano et al., 2014). Under hypoxic conditions, ESR1 mRNA levels are reduced and the ERa protein is degraded by proteasome-mediated pathways in human breast cancer cells (Cho et al., 2005; Ryu et al., 2011; Stoner et al., 2002). Therefore, we examined whether BPA, BBP, and DEHP regulated ERa protein levels under a hypoxic microenvironment in MCF-7 cells. The E2 positive control reduced ERa protein levels under both normoxia and hypoxia (Fig. 3). We confirmed that ERa protein down-regulation was similar to that of E2 under both normoxic and hypoxic conditions after treatment with BPA and BBP

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Fig. 1. Activation of ERa by BPA, BBP, and DEHP. ERa-HeLa9903 cells were treated with E2 (1 nM) or (a) BPA (1.00  1010 to 1.00  104 M), (b) BBP (1.00  109 to 1.00  103 M), and (c) DEHP (1.00  109 to 1.00  103 M) for 24 h, as indicated. Each value represents the mean of three replicates. *P < 0.05 vs. vehicle control.

Table 1 Comparison to ToxCast data. CAS No.

NAME

TOX21_ERa_BLA _Agonist_ratio

TOX21_ERa_LUC-BG1_Agonist

TG455_Hela 9903_ this study

80-05-07 85-68-7 117-81-7

BPA BBP DEHP

1.01 24.8 Not tested

0.363 20.8 Not tested

1.15 13.84 ####

The iCSS ToxCast dashboard was used to analyze publicly available data in the ToxCast program (http://actor.epa.gov/dashboard/; last accessed January 30, 2018). EC50 values (mM) for ToxCast assays relative to ERa activation are compared with the results presented here. ####: Negative, Not-tested show assays in ESR1 have not been tested.

Fig. 2. Effect of EDC on ESR1 gene transcription in MCF-7 cells. (a) MCF-7 cells were transiently transfected with an ERE-luciferase reporter gene. MCF-7 cells were treated with E2 (10 nM), BPA, BBP, or DEHP for 24 h under normoxic or hypoxic conditions. The treatment concentration of EDCs was 10 mM. After incubation, luciferase expression was determined. (b) MCF-7 cells were treated with E2 (10 nM), BPA, BBP, or DEHP for 24 h under normoxic or hypoxic conditions. Total RNA was extracted, and expression of ESR1 mRNA was analyzed by real-time PCR. *P < 0.05 vs. normoxia control; #P < 0.05 vs. hypoxia control.

(Fig. 3A and B). ERa protein levels were not decreased by DEHP under normoxic conditions, but were slightly decreased under hypoxic conditions (Fig. 3C). These data indicate that DEHP modulates ERa expression in the hypoxic state. 3.3. Regulation of HIF-1a-mediated gene transcription by EDCs in MCF-7 cells One of the important transcription factors activated in hypoxia is HIF-1 (Dengler et al., 2014). HIF-1 can recognize and bind to HREs in hypoxia-inducible promoters. Studies have shown that HIF-la protein levels are decreased by BPA under hypoxic conditions (Kubo et al., 2004). To determine whether elevated ER activity caused by EDCs under hypoxia is associated with HIF-1, we conducted HREluciferase reporter assays in MCF-7 cells (Fig. 4A). Although E2 did not inhibit HRE activity, BPA, BBP and DEHP slightly inhibited HRE activity in MCF-7 cells transfected with an HRE-luciferase

reporter plasmid. VEGF is a well-known angiogenesis-related growth factor that is secreted under various conditions, including hypoxia (Morfoisse et al., 2015). We evaluated the relationship between EDCs and the levels of secreted VEGF protein in the supernatant of MCF-7 cells under hypoxia. BPA and BBP increased the levels of secreted VEGF under hypoxia, but to a lesser extent compared to E2. DEHP did not increase the level of hypoxiainduced VEGF secretion (Fig. 4B). BPA and BBP increased the levels of VEGF secretion despite decreased HRE transactivation in MCF-7 cells under hypoxia. Although DEHP decreased HRE transactivation, it did not affect the VEGF levels in MCF-7 cells under hypoxic conditions. 3.4. Effects of EDCs on ERa and VEGF in Ishikawa cells Ishikawa cells are ERa-positive cells similar to MCF-7 cells, but they differ in terms of the influence of E2 and hypoxia on ERa

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Fig. 3. Reduction of ERa protein levels by EDC in MCF-7 cells. MCF-7 cells were treated with E2 (10 nM) or (a) BPA, (b) BBP, and (c) DEHP for 24 h under normoxic or hypoxic conditions. The treatment concentration of EDCs was 10 mM. Equal amounts of total protein extracts were immunoblotted as directed. *P < 0.05 vs. normoxia control; #P < 0.05 vs. hypoxia control.

Fig. 4. Regulation of HIF-1a-mediated gene transcription by EDCs. (a) MCF-7 cells were used to analyze HRE-luciferase activity and secreted VEGF protein levels. MCF-7 cells were treated with E2 (10 nM), BPA, BBP, or DEHP for 24 h under hypoxic conditions. The treatment concentration of EDCs was 10 mM. After incubation, luciferase activity was measured. (b) Secreted VEGF protein levels in MCF-7 cell supernatant were analyzed by ELISA. *P < 0.05 vs. normoxia control; #P < 0.05 vs. hypoxia control.

expression. Studies have shown that E2 decreases ERa protein levels without decreasing ESR1 mRNA levels in Ishikawa endometrial cancer cells (Amita et al., 2016; Park et al., 2012; Vivacqua et al., 2006). In addition, Ishikawa cells show decreased ERa protein levels under hypoxic conditions but do not repress ESR1 mRNA levels (Ryu et al., 2011). We determined the effects of BBP and DEHP on ERa protein levels in Ishikawa cells to confirm whether ERa was reduced by phthalates in other ERa-positive cells. When treated with BPA, ERa was reduced to a level similar to that by E2 in the normoxic and hypoxic states (Fig. 5A). Compared to the control group, the decrease in ERa expression by BBP was similar to that by E2 under normoxic and hypoxic conditions (Fig. 5B). In contrast, DEHP did not reduce ERa under normoxia but did under hypoxia, albeit to a lesser extent than BPA and BBP (Fig. 5C). We evaluated the levels of VEGF protein secreted by Ishikawa cells under hypoxia. Unlike MCF-7 cells, E2, BPA, and BBP had no effect on the level of secreted VEGF protein in Ishikawa cells under hypoxia. The level of secreted VEGF protein tended to decrease slightly with DEHP under hypoxia (Fig. 5D). 4. Discussion Hundreds of EDCs interfere with hormone action, but in general, they affect estrogen, androgen, and thyroid hormone activities and

steroidogenesis, as reflected in the level 2 OECD TGs for evaluating EDCs. However, considering the complex cellular signaling pathways activated by ERs, ARs, and thyroid hormone receptors, as well as additional newly discovered cellular targets, it is becoming increasingly clear that EDCs can contribute to numerous adverse outcomes, such as hormone-sensitive cancers, obesity, and cardiovascular and inflammatory diseases (Desvergne et al., 2009; Gore et al., 2015; Kim and Park, 2014; Morgan et al., 2017; Teitelbaum et al., 2012). Studies have suggested that the inexplicably rising rates of metabolic disorders are caused by exposure to chemicals (Heindel et al., 2017). In particular, early exposure is suspected to alter susceptibility to common non-communicable diseases, including cancers, cardiovascular diseases, chronic respiratory diseases, and diabetes mellitus (Organization and Unit, 2014). ERa expression is important in the treatment of tamoxifen resistance and endocrine therapy during breast cancer and brain development (Gutierrez et al., 2005; Yang et al., 2015). In this study, BPA and BBP downregulated ERa under both normoxia and hypoxia. Surprisingly, DEHP, which does not exhibit any estrogenic activity, still decreased ERa, implying that DEHP influences ERa pathways by as-yet unknown indirect mechanisms. Consistent with our results, other studies have implied that, although DEHP exhibits no apparent estrogenic activity, it is still capable of

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Fig. 5. Effects of EDCs on ERa and VEGF in Ishikawa cells. Ishikawa cells were treated with E2 (10 nM) or (a) BPA, (b) BBP, and (c) DEHP and incubated under normoxic or hypoxic conditions for 24 h, followed by western blot analysis. The treatment concentration of the three EDCs was 10 mM. (d) After incubation, secreted VEGF protein levels in Ishikawa cell supernatant were analyzed by ELISA. *P < 0.05 vs. normoxia control; #P < 0.05 vs. hypoxia control.

disrupting ERa activity. Exposure to DEHP increases serum estradiol levels (Barakat et al., 2017; Somasundaram et al., 2017) and promotes the proliferation of ERa-positive MCF-7 cells (Blom et al., 1998; Chen and Chien, 2014; Chou et al., 2017; Okubo et al., 2003). For example, male rats exposed to DEHP for 4 weeks or more exhibited dramatically reduced ERa activity, and ER expression affected ceruloplasmin protein levels (Eagon et al., 1994). The exact mechanism by which DEHP degrades ERa is unknown, but one possibility is that DEHP activates coregulators of HIF-1 via proteasome activity or affects other indirect targets that can degrade ERa. The several known targets of DEHP, such as ERa, AR, and aryl hydrocarbon receptor, suggest that the adverse effects triggered by DEHP may be due to interactions among multi-molecular pathways rather than by a single pathway (Chou et al., 2017). Hypoxia is an important regulator of tumorigenesis and chronic inflammatory diseases, and induces adaptive mechanisms in cancer cells that contribute to the selection of more aggressive cells (Michiels et al., 2016). This process leads to therapeutic resistance and causes poor prognoses. The transcriptional activity of glucocorticoid receptors (Kodama et al., 2003), ERs (Cho et al., 2006; Cho et al., 2005; Lim et al., 2009; Stoner et al., 2002; Yi et al., 2009), ARs (Park et al., 2006), and PPARs (Belanger et al., 2007; Huss et al., 2001; Li et al., 2007; Narravula and Colgan, 2001) were regulated in the hypoxic state in our experiment. These results suggest that hypoxia may influence not only tumorigenesis, but also other pathophysiological phenomena such as inflammation and metabolic processes. In previous studies, we and others have shown that ERa under hypoxic conditions is activated and destabilizes via the proteasome pathway (Cho et al., 2005; Park and Lee, 2017; Stoner et al., 2002), which

depends on the physical interaction between ERa and HIF-1a and the expression of HIF-1a. A recent study demonstrated the suppression of HIF-1a-dependent ERa in breast cancer (Wolff et al., 2017). Hypoxia affects estrogen pathways, and estrogen activates HIF-1a and induces vascular endothelial growth factor gene expression via the PI3K/AKT pathway in rat uteri (Kazi and Koos, 2007; Kazi et al., 2009). Because hypoxia can affect hormonal pathways, it is important to investigate the behavior changes of EDCs under hypoxic conditions. The results of our reporter gene assays demonstrated that estrogenic activity was enhanced under hypoxia with BPA, BBP, and DEHP. In contrast, HIF-1-dependent reporter gene activity was reduced by EDCs. Other studies have shown that BPA inhibits the hypoxic response via degradation of HIF-1a (Kubo et al., 2004), while BPA can promote HIF-1a and VEGF under hypoxia via the G-coupled estrogen receptor pathway, depending on the model system used (Xu et al., 2017). Our results showed that E2, BPA, and BBP, but not DEHP, stimulated VEGF secretion in MCF-7 cells in the hypoxic state. The pattern of secreted VEGF expression with EDC treatment under hypoxic conditions in Ishikawa cells differed from that of MCF-7 cells. Ishikawa cells are also an ERa-positive cell line, but with different ESR1 expression by ERa ligands relative to MCF-7 cells. Under a hypoxic microenvironment, VEGF expression is mediated by both HIF-1 and interdependent gene expression pathways involving a variety of factors. To our knowledge, this study provides the first evidence that phthalates affect VEGF secretion. The mechanism of cross-talk between hypoxia and estrogen receptors is not well understood, but it appears that EDCs are also influenced by hypoxia, suggesting that the behavior of EDCs under hypoxia may be augmented in hypoxic microenvironments.

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BPA is excreted after binding with uridine 50 -diphosphoglucuronic acid (UDPGA), while the phthalates BBP and DEHP are first hydrolyzed in vivo. The toxicity of phthalates is believed to be partly due to their metabolites. BBP is hydrolyzed to mono-n-butyl phthalate and mono-benzylphthalate, while the hydrolysate of DEHP is mono(2-ethylhexyl) phthalate. The hydrolysis of BBP and DEHP differs with each enzyme in each organ. For example, bovine pancreas lipase hydrolyzed DEHP within 15 min and BBP within 30 min (Saito et al., 2010). Rat pancreatic and small intestine microsomes showed high activity for DEHP, while liver microsomes show marginal activity toward DEHP (Ozaki et al., 2017). Our results suggest that DEHP without estrogen receptor (ER) activity in normoxic conditions increases the ER activity in hypoxic conditions, so that the in vitro and in vivo activities of metabolic products and their maternal chemicals may differ. In the present study, we demonstrated BPA-, BBP-, and DEHPinduced ERa downregulation and activation under hypoxia in both MCF-7 breast cancer and Ishikawa human endometrial adenocarcinoma cells. Our results suggest that not only BPA and BBP, which have estrogenic activity, but also DEHP, which lacks estrogenic activity, can influence ERa pathways via modulation of ERa levels under hypoxic conditions. Considering the enhancement of the adverse effects of EDCs in chronic metabolic and degenerative diseases, the potential amplifying behavior of EDCs in hypoxic microenvironments should be researched further. Acknowledgements This research was supported by Research Program To Solve Social Issues of the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT. (No. NRF2017M3C8A6091777) References Al-Bader, M., Ford, C., Al-Ayadhy, B., Francis, I., 2011. Analysis of estrogen receptor isoforms and variants in breast cancer cell lines. Exp. Therapeut. Med. 2, 537e544. Al-Saleh, I., Elkhatib, R., 2016. Screening of phthalate esters in 47 branded perfumes. Environ. Sci. Pollut. Control Ser. 23, 455e468. Amita, M., Takahashi, T., Igarashi, H., Nagase, S., 2016. Clomiphene citrate downregulates estrogen receptor-a through the ubiquitin-proteasome pathway in a human endometrial cancer cell line. Mol. Cell. Endocrinol. 428, 142e147. Barakat, R., Lin, P.-C.P., Rattan, S., Brehm, E., Canisso, I.F., Abosalum, M.E., Flaws, J.A., Hess, R., Ko, C., 2017. Prenatal exposure to DEHP induces premature reproductive senescence in male mice. Toxicol. Sci. 156, 96e108. Belanger, A.J., Luo, Z., Vincent, K.A., Akita, G.Y., Cheng, S.H., Gregory, R.J., Jiang, C., 2007. Hypoxia-inducible factor 1 mediates hypoxia-induced cardiomyocyte lipid accumulation by reducing the DNA binding activity of peroxisome proliferator-activated receptor a/retinoid X receptor. Biochem. Biophys. Res. Commun. 364, 567e572. Blom, A., Ekman, E., Johannisson, A., Norrgren, L., Pesonen, M., 1998. Effects of xenoestrogenic environmental pollutants on the proliferation of a human breast cancer cell line (MCF-7). Arch. Environ. Contam. Toxicol. 34, 306e310. Brennan, J.C., Bassal, A., He, G., Denison, M.S., 2016. Development of a recombinant human ovarian (BG1) cell line containing estrogen receptor a and b for improved detection of estrogenic/antiestrogenic chemicals. Environ. Toxicol. Chem. 35, 91e100. Ceretti, E., Zani, C., Zerbini, I., Guzzella, L., Scaglia, M., Berna, V., Donato, F., Monarca, S., Feretti, D., 2010. Comparative assessment of genotoxicity of mineral water packed in polyethylene terephthalate (PET) and glass bottles. Water Res. 44, 1462e1470. Chen, F.-P., Chien, M.-H., 2014. Lower concentrations of phthalates induce proliferation in human breast cancer cells. Climacteric 17, 377e384. Chen, Z., Zheng, H., Dong, K.-W., 2001. Identification of negative and positive estrogen response elements in human GnRH upstream promoter in the placental JEG-3 cells. Mol. Cell. Endocrinol. 184, 125e134. Cho, J., Bahn, J.-J., Park, M., Ahn, W., Lee, Y.J., 2006. Hypoxic activation of unoccupied estrogen-receptor-alpha is mediated by hypoxia-inducible factor-1 alpha. J. Steroid Biochem. Mol. Biol. 100, 18e23. Cho, J., Kim, D., Lee, S., Lee, Y., 2005. Cobalt chloride-induced estrogen receptor a down-regulation involves hypoxia-inducible factor-1a in MCF-7 human breast cancer cells. Mol. Endocrinol. 19, 1191e1199. Chou, C.-K., Yang, Y.-T., Yang, H.-C., Liang, S.-S., Wang, T.-N., Kuo, P.-L., Wang, H.-

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