Activation of Peroxisome Proliferator-Activated Receptor Gamma and

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Activation of Peroxisome Proliferator-activated Receptor Gamma and Disruption of Progesterone Synthesis of 2Ethylhexyl Diphenyl Phosphate in Human Placental Choriocarcinoma Cells: Comparison with Triphenyl Phosphate Wenxin Hu, Fumei Gao, Hong Zhang, Youhei Hiromori, Shuhei Arakawa, Hisamitsu Nagase, Tsuyoshi NAKANISHI, and Jianying Hu Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b00872 • Publication Date (Web): 10 Mar 2017 Downloaded from http://pubs.acs.org on March 12, 2017

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Environmental Science & Technology

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Activation of Peroxisome Proliferator-activated Receptor Gamma and Disruption of

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Progesterone Synthesis of 2-Ethylhexyl Diphenyl Phosphate in Human Placental

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Choriocarcinoma Cells: Comparison with Triphenyl Phosphate

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Wenxin Hu1, Fumei Gao1, Hong Zhang1, Youhei Hiromori2,3, Shuhei Arakawa2, Hisamitsu

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Nagase2, Tsuyoshi Nakanishi2, and Jianying Hu1*

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1

8

Peking University, Beijing 100871, China

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2

MOE Laboratory for Earth Surface Processes, College of Urban and Environmental Sciences,

Laboratory of Hygienic Chemistry and Molecular Toxicology, Gifu Pharmaceutical

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University, 1-25-4 Daigaku-nishi, Gifu, Gifu, 501-1196, Japan

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3

12

Minamitamagaki, Suzuka, Mie 513-8670, Japan

Faculty of Pharmaceutical Sciences, Suzuka University of Medical Science 3500-3,

13 14 15 16

*Address for correspondence

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Dr. Jianying Hu

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College of Urban and Environmental Sciences

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Peking University

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Beijing 100871, China.

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TEL & FAX: 86-10-62765520;

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Email: [email protected]

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ACS Paragon Plus Environment


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Abstract

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2-Ethylhexyl diphenyl phosphate (EHDPP), an organophosphate flame retardants

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(OPFRs), is frequently detected in human blood. In this study, sensitive dual-luciferase

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reporter gene assay and molecular docking were used to investigate the activation of EHDPP

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to human peroxisome proliferator-activated receptor gamma (PPARG). Results show that

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EHDPP exhibited stronger PPARG activation (EC20: 2.04 µM) than triphenyl phosphate

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(TPhP) (EC20: 2.78 µM). EHDPP upregulated the gene expression of 3β-hydroxysteroid

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dehydrogenase type 1 (3β-HSD1) in human placental choriocarcinoma cells in a

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dose-dependent manner, and the lowest observable effective concentration was 10 µM, lower

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than that of TPhP (20 µM). EHDPP significantly altered progesterone secretion at a lower

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concentration (10 µM) than that of TPhP (20 µM), and both EHDPP and TPhP significantly

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promoted human chorionic gonadotropin (hCG) production at 20 µM. Furthermore,

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inactivation of PPARG by either pharmacological inhibitor (GW9662) or small interfering

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RNA (siRNA) abolished the change in progesterone secretion and gene expression in the cells

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exposed to EHDPP, suggesting that the PPARG signaling pathway plays a role in the

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upregulation of progesterone by the two OPFRs. This is the first report to show that OPFRs

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can alter the biosynthesis of progesterone in the placenta, which could affect female

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reproduction and fetal development.

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Keywords: Organophosphate flame retardants; PPARG; Human placenta; Human chorionic

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gonadotropin.

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Introduction

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There is growing concern about the possible health threat posed by anthropogenic

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compounds. Increasing evidence has shown that substances found in the environment, food,

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and consumer products can interfere with hormone biosynthesis, metabolism, and activity,

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resulting in deviation from normal homeostatic control or reproduction.1 The wide variety of

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pollutants reported to disrupt endocrine function includes pesticides, polycyclic aromatic

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hydrocarbons, phthalate plasticizers, polychlorinated biphenyls, dioxins, furans, alkylphenols,

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and synthetic steroids.2

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Of all environmental chemicals showing reproductive toxicity, organophosphate flame

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retardants (OPFRs) are of particular concern. OPFRs are used as replacements for brominated

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flame retardants (BFRs) such as polybrominated diphenyl ethers (PBDEs), thus their use has

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increased significantly in recent years, with annual global production currently reaching

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approximately 2,000,000 tons.3 OPFRs are widely used as plasticizers in various consumer

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products, building materials, and baby products,4-6 and are therefore ubiquitous in various

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environmental media such as house dust, sediment, sludge, river water, biota, and even in

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human blood and placenta.7-13 There is increasing evidence that OPRFs exposure can disrupt

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hormonal metabolism and biosynthesis in animals and humans. Tris(1,3-dichloro-2-propyl)

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phosphate (TDCIPP) and triphenyl phosphate (TPhP) have been shown to reduce fecundity

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by significantly increasing plasma estradiol levels and inhibiting androgen levels in

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zebrafish.14 A similar phenomenon has also been observed in stably transfected human breast

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cancer cell line MVLN and human adrenocortical carcinoma cell line (H295R).15 One recent

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epidemiological study showed that OPFRs in house dust were associated with serum free 3



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thyroxin (T4), prolactin, and decreased semen quality in men.16 In addition to alteration of

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hormonal metabolism and biosynthesis, OPFRs can also interact with various nuclear

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receptors (NRs) such as constitutive androstane receptor (CAR), pregnane X receptor (PXR),

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estrogen receptor (ER), androgen receptor (AR), glucocorticoid receptor (GR), and

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peroxisome proliferator-activated receptor gamma (PPARG).17,18 Of these NRs, PPARG is

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abundantly expressed in human placenta and serves as an essential regulator of endocrine

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functions that play a vital role in maintaining pregnancy.19-22 Thus, it would be interesting to

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determine whether OPFRs can disrupt endocrine functions in human placenta. Among

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various OPFRs, only TPhP and tributyl phosphate (TBP) have been identified as agonists of

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PPARG,17 for a broad range of OPFRs with diverse structures the activity to PPARG have not

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been evaluated. Particularly, 2-ethylhexyl diphenyl phosphate (EHDPP), tris-(2-chloroethyl)

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phosphate (TCEP), and tris-2-chloroisopropyl phosphate (TCPP) have been detected in the

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blood of the Chinese population, with median concentrations of EHDPP (1.22 ng/ml) and

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TCPP (0.71 ng/ml) found to be higher than that of TPhP (0.43 ng/ml).13 To date, however,

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their activation to PPARG remain to be elucidated.

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In this study, TCEP, TCPP and EHDPP PPARG activation were determined using

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dual-luciferase reporter assay. The TBP and TPhP PPARG activation were also determined to

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enable a direct comparison of the unknown PPARG activation by TCEP, TCPP and EHDPP

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based on the same assay. We also investigated their effects on the synthesis of progesterone

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and human chorionic gonadotropin (hCG) in human placental choriocarcinoma cell line

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JEG-3, which retains characteristics of normal pregnancy trophoblast cells. Finally, the

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potential role in promotion of progesterone production was also investigated by co-exposure 4


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with a pharmacological inhibitor of PPARG (GW9662) and small interfering RNA (siRNA)

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targeting PPARG in JEG-3 cells.

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Materials and Methods

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Chemicals and Reagents. The standards of TCEP, TCPP, TBP, EHDPP, and TPhP were

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purchased from Tokyo Chemical Industry Co (Tokyo, Japan). Rosiglitazone, GW9662,

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3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), and dimethyl sulfoxide

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(DMSO) were purchased from Sigma-Aldrich (St. Louis, MO, USA). TRIzol was obtained

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from Invitrogen (Carlsbad, CA, USA).

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Cell Cultures and Treatments. The human placental choriocarcinoma cell line JEG-3

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was cultured in DMEM-F12 medium (Hyclone, Logan, UT, USA) supplemented with 10%

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fetal bovine serum (FBS, Gibco, Grand Island, NY, USA), and incubated at 37°C in a

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humidified, 5% CO2 atmosphere. The JEG-3 cells were plated in 24-well plates in

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DMEM-F12 medium and allowed to become confluent (2–3 d). To determine the effects of

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EHDPP, TPhP, TCEP, TBP, and TCPP on synthesis-related gene expression and production of

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progesterone and hCG, the JEG-3 cells were seeded, pre-cultured for 24 h, and then treated

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with various concentrations of EHDPP, TPhP, TCEP, TBP, or TCPP in DMSO or with

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vehicle-control (0.1% DMSO).

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Reporter Assay. The 293T cells were seeded in 24-well plates in 500 µL of DMEM-F12

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containing 10% FBS. After incubation for 24 h, the cells showed 70–80% confluence and

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were then transiently transfected with pBIND-PPARG-LBD, GAL4-pGL4-luc (provided by

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Shuyi Si, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and

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Peking Union Medical College) and pGL4.74 (Promega, Madison, WI, USA) or GAL4-RXR, 5



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pG5-luc and pGL4.74 using Lipofectamine 2000 Transfection Reagent (Invitrogen, Carlsbad,

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CA, USA) according to the manufacturer’s instructions.23 Following 24 h of incubation, the

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medium was replaced and the cultures were treated with DMSO (0.1%), EHDPP, TPhP, TCEP,

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TBP, or TCPP (0.05–25 µM) for 24 h. The extracts were prepared and assayed for firefly

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luciferase (LUC) activity using the Dual-Luciferase Reporter Assay System (Promega,

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Madison, WI, USA) and a LB 941 TriStar Multimode Microplate Reader (Berthold

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Technologies, Bad Wildbad, Germany) according to the manufacturer’s instructions. In brief,

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the cells were collected with phosphate buffered saline (PBS), and then passive lysis buffer

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was added. The supernatant was collected to test the firefly LUC and Renilla LUC activities.

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Results were expressed as the average relative firefly LUC activity of at least triplicated

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samples.

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Molecular Docking. Molecular simulation software Scigress (Ultra Version 3.0.0,

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Fujitsu, USA) was used to dock flexible ligands into a rigid protein active site. In brief, the

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PPARG ligand binding domain (PDB ID code 2PRG) was obtained from the Protein Data

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Bank (PDB, http://www.rcsb.org/). Explicit hydrogen atoms were created and heteroatoms

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such as water, ions, and cofactors were removed. Docking calculations were evaluated using

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a genetic algorithm with a 15 × 15 × 15 Å grid box with 0.3 Å grid spacing, which contained

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the active site for the original ligand. The procedure was set to run for 20000 generations with

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an initial population size of 100, elitism of 8, crossover rate of 0.8, mutation rate of 0.5, and

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convergence of 1.0. Other parameters were set at their default values. The Potential of Mean

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Force (PMF) scores, a knowledge-based approach that extracts pairwise atomic potentials

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from the structure information on known protein−ligand complexes contained in the Protein 6


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Data Bank, was determined and used to score the binding activity of a chemical to the active

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site of human PPARG. The PMF of rosiglitazone, the native ligand in the complex, was

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−91.15 kcal/mol when docked into the binding site. The root-mean-square error between the

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predicted conformation and the actual conformation from the crystal structure was 1.78 Å,

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smaller than the X-ray crystallography resolution (2.20 Å).24 Thus the parameter set used for

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the docking simulation was considered to be reasonable.

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Plasmid construction. pBIND-PPARG-LBD mutant construct, carrying a Ser342 to Ala

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mutation, was generated by site-directed mutagenesis of pBIND-PPARG-LBD. The

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sequences

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5’-GGTTCTCATAGCAGAGGGCCAAGGCTTCATGAC-3’

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CTTGGCCCTCTGCTATGAGAACCCCATCTTTAT-3’.

of

the

mutagenic

primers

are

and

5’-

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Cell Viability Assays. Confluent JEG-3 cultures were treated with vehicle-control

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(DMSO, 0.1%), EHDPP, TPhP, TCEP, TBP, or TCPP (5–40 µM) for 48 h. Cytotoxicity was

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measured using MTT assay. Briefly, JEG-3 cells were seeded in 96-cell plates for 24 h, and

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then treated with different concentrations of OPFRs for 48 h. At the end of treatment, 20 µL

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of MTT was added and the solution was incubated for an additional 4 h. The formazan

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crystals were then dissolved in 200 µL of DMSO. After shaking the plate for 10 min, cell

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viability was assessed by measuring the absorbance at 490 nm using a spectrophotometer

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(Bio-Rad Model 550; Bio-Rad Laboratories, Inc., Hercules, CA, USA). Cell viability was

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also assessed by CellTiter-Glo Reagent (Promega, Madison, WI, USA) according to the

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manufacturer’s instructions. In brief, cells were seeded, pre-cultured for 24 h, and then treated

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with various concentrations of EHDPP, TPhP, TCEP, TBP, or TCPP in 0.1% DMSO or with 7



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vehicle-control (0.1% DMSO) for 48 h. After 100 µl of CellTiter-Glo Reagent was added to

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each well, the plate and its contents were equilibrated at room temperature for approximately

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30 min. The luminescence was determined using the LB 941 TriStar Multimode Microplate

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Reader. Absorbance or luminescence in the experimental wells were normalized by dividing

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the absorbance or luminescence in untreated cultures and reported as “Fold Change from

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Medium”. Results were expressed as the average relative firefly LUC activity of at least

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triplicated samples.

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Determination of hCG and Progesterone Production. The JEG-3 cells were plated in

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24-well plates. After 24 h of culture, the JEG-3 cells were treated with various concentrations

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of EHDPP, TPhP, TCEP, TBP, or TCPP for a further 48 h. After incubation, the culture

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supernatants were collected, and the concentrations of hCG and progesterone were

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determined by radioimmunoassay (RIA) with a Progesterone Direct RIA Kit and hCG Direct

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RIA Kit (ICN Biomedicals, Irvin, CA, USA) according to the manufacturer’s protocols.

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Quantitative RT-PCR. Total RNA was extracted using TRIzol reagent (Invitrogen,

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Carlsbad, CA, USA) according to the manufacturer’s protocols, and digested by DNaseI

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(TaKaRa Biotechnology, Dalian, China) in case of genomic DNA contamination. The

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concentration and quality of RNA was analyzed using a Nanovue Plus spectrophotometer

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(GE Healthcare Life Science, Little Chalfont, Buckinghamshire, England). The purity of each

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sample was between 1.8 and 2.0 (A260/A280 nm ratio). Total RNA was reverse transcribed

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with MMLV reverse transcriptase in the presence of oligo (dT) and dNTP (TaKaRa

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Biotechnology, Dalian, China). The complete reaction mixture was incubated at 37°C for 50

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min, followed by 95°C for 5 min to stop the reaction. SYBR Green PCR Kits (TOYOBO, 8


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Osaka, Japan) were used for Q-PCR analysis. A real-time PCR profile was used: first,

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enzyme activation at 95°C (10 min), 40 cycles at 95°C (30 s per cycle), and 60 s at 58–62°C,

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depending on the target transcript, followed by post-run melt curve analysis (65°C).

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Real-time fluorescence detection was carried out using the STEP ONE PLUS sequence

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detection system (Applied Biosystems, Foster City, CA. USA). Relative gene expression was

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evaluated by the 2-∆∆Ct method, as suggested by Applied Biosystems. The primers

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sequences used are shown in Table S1.

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RNA Interference Assay. Sequence-specific small interfering RNA (siRNA) targeting

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PPARG (siPPARG) shown in Table S2 was purchased from Dharmacon/GE Healthcare

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(Little Chalfont, Buckinghamshire, England). AllStars Negative Control siRNA purchased

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from Qiagen (Valencia, CA) was used for the negative control siRNA. JEG-3 cells (3 × 105

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cells/well) were seeded in 6-well plates and precultured at 37 °C for 24 h. The cells then were

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transfected with siRNA duplexes (20 nM/well) by using Lipofectamine RNAiMAX

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(Invitrogen/Thermo Fisher Scientific, Grand Island, NY) in accordance with the

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manufacturer’s instructions.

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Statistical Analysis. Results were presented as means ± standard errors of the mean

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(SEM) and tested for statistical significance by analysis of variance (ANOVA) followed by

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post hoc Dunnett’s test using SPSS 16.0 (SPSS Inc., Chicago, IL, USA). The number of

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replicates is indicated in each figure legend. Dose response curves were performed with

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Prism 5 (Graphpad Inc., La Jolla, CA). A p-value < 0.05 was considered statistically

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significant.

199 9



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Results and Discussion

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Human PPARG Activation. In this study, we determined the activation of TCEP, TCPP,

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and EHDPP to PPARG using a sensitive dual-luciferase reporter assay, as shown in Figure

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S1 (half effective concentration of rosiglitazone is 0.01µM). Although TPhP and TBP

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PPARG activation have been reported previously,25 we show their PPARG activation in

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Figure 1. The 20% effective concentrations (EC20) were 2.78 µM and 5.96 µM for TPhP and

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TBP, respectively, slightly lower than those for TPhP (3.27 µM) and TBP (13.35 µM)

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reported previously.25 Among the three OPFRs newly determined in this study, TCEP and

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TCPP showed no significant PPARG activation under the testing concentrations, whereas

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EHDPP showed significant PPARG activation compared with the vehicle-control, with a

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EC20 value of 2.46 µM, showing stronger PPARG activation than that of TPhP. The EC20

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value of EHDPP was similar to that of mono(2-ethylhexyl) phthalate (2.12 µM) and

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3-hydroxide-2,2’,4,4’-tetrabromodiphendyl ether (3-OH-BDE47) (2.99 µM), but higher than

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that of tetrabromobisphenol A (TBBPA) (0.41 µM) and 3,5,3′,5′-tetrachlorobisphenol A

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(TCBPA) (0.47 µM).25 Although used as a replacement for PBDE, it should be noted that the

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activation of EHDPP to PPARG was stronger than that of BDE47 (7.00 µM).25

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Molecular docking was performed to understand the structural basis for the observed

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activation of OPFRs to PPARG using Scigress. The PMF values of EHDPP, TPhP and TBP

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were calculated to be −54.134, −44.288, and −32.534 kcal/mol, respectively, consistent with

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their PPARG activation. As shown in Figure 2, hydrophobicity of TBP, EHDPP, and TPhP

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plays an important role in human PPARG activation, as the nine important amino acid

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residues (Leu333, Phe347, Ile341, Met348, Phe352, Leu270, Phe287, Ile281, and Phe363) 10


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present in the inner surface of the ligand-binding domain (LBD) of human PPARG form the

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hydrophobic agonist pocket. EHDPP and TPhP both showed π-π interaction between their

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benzene rings and Phe287 of PPARG-LBD (face to face). In contrast to TPhP, an important

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feature of the EHDPP interaction was the formation of hydrogen bonds between EHDPP and

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the residue Ser342 of PPARG-LBD (2.217 Å), which might contribute to the relatively high

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PPARG activation by EHDPP. Such differences in binding mode might also explain the

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differences in PPARG activation of the three chemicals. To verify the involvement of the

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hydrogen bonds in the activation of EHDPP to PPARG, we substituted Ser342 of PPARG

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with alanine and then carried out PPARG activation of EHDPP using the dual-luciferase

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reporter assay. The activation of EHDPP to the PPARG mutant was found to be about 2.5 fold

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lower than that of wild-type (Figure 3), demonstrating the importance of hydrogen bond in

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PPARG activation by EHDPP.

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To further demonstrate the PPARG activity of OPFRs, three main downstream genes of

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PPARG, hCG, pregnancy-associated plasma protein-A (PAPP-A), and mucin gene Muc1

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were also quantified in human placental choriocarcinoma JEG-3 cells.26,27 Confluent JEG-3

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cells showed no change in cell viability or cell proliferation after treatment with EHDPP,

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TPhP, TCEP, TBP, or TCPP at 5–40 µM for 48 h, and therefore the concentrations of 5, 10, 20,

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and 40 µM were used in the following experiments (Figure S2). EHDPP and TPhP

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significantly upregulated Muc1 and hCG expression but significantly down-regulated

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PAPP-A gene expression compared with the control (Figure 4), further demonstrating

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PPARG activation of TPhP and EHDPP in human placenta.

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243

Disruption of Progesterone Secretion. Since hCG governs the production of

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progesterone in the corpus luteum during the first trimester,26,28 and significantly upregulated

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hCG alterations may disrupt progesterone secretion. In addition to the gene expression of

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hCG, its production alterations were also determined. Both TPhP and EHDPP significantly

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increased hCG levels in the 20 and 40 µM groups compared with the control (Figure 5).

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To investigate whether the five target OPFRs could disrupt progesterone biosynthesis,

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JEG-3 cells were grown to confluence and treated with OPFRs at different concentrations,

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and concentration of progesterone was determined. Among the OPFRs tested, EHDPP and

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TPhP induced progesterone in a dose-dependent manner, while no changes in progesterone

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level were observed in cells exposed to TBP, TCEP, or TCPP (Figure 5). The progesterone

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concentration was significantly upregulated in the 20 and 40 µM TPhP exposure groups, with

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1.29 ± 0.09 and 1.28 ± 0.07-fold increases, respectively, relative to that of the control (p

Activation of Peroxisome Proliferator-Activated Receptor Gamma and

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