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Potential glucocorticoid and mineralocorticoid effects of nine organophosphate flame retardants Quan Zhang, Jinghua Wang, Jianqiang Zhu, Jing Liu, and Meirong Zhao Environ. Sci. Technol., Just Accepted Manuscript • Publication Date (Web): 21 Apr 2017 Downloaded from http://pubs.acs.org on April 22, 2017
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Potential glucocorticoid and mineralocorticoid effects of
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nine organophosphate flame retardants
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Quan Zhang1,2, Jinghua Wang1, Jianqiang Zhu1, Jing Liu3, and Meirong Zhao1,2*
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1. Key Laboratory of Microbial Technology for Industrial Pollution Control of
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Zhejiang Province, College of Environment, Zhejiang University of Technology,
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Hangzhou, Zhejiang, 310032, China
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2. Department of Environmental Health, Harvard T.H. Chan School of Public Health,
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Landmark Center West, Boston, MA, 02215, USA
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3. College of Environmental and Resource Sciences, Zhejiang University, Hangzhou
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310058, China
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*To whom correspondence should be addressed. Phone: +86 571 8832 0265;
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Fax: +86-571-88320265; E-mail:
[email protected] (MR Zhao).
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TABLE OF CONTENT
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Abstract Organophosphate flame retardants (OPFRs), as alternatives of polybrominated
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diphenyl ethers (PBDEs), have been frequently detected in the environment and biota,
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and could pose adverse effects on organisms. However, information on the potential
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endocrine disruption of OPFRs, especially their effects on steroid hormone receptors,
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such as glucocorticoid and mineralocorticoid receptors (GR/MR), is limited. In this
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study, the dual-luciferase reporter gene assay via GR/MR and a H295R
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steroidogenesis assay were employed to evaluate the endocrine disruption of nine
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OPFRs. We found TMPP, TPHP, and TDBPP exhibited both GR and MR antagonistic
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activities, while TNBP and TDCIPP only showed MR antagonistic property within a
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concentration range of 10-8 to 10-5 mol/L(M). In the H295R steroidogenesis assay, the
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fold changes of eight steroidogenic genes in response to OPFRs were further studied.
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We found CYP17,CYP21, and CYP11B1 expression were significantly
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down-regulated following TMPP, TPHP, or TDBPP exposure at a concentration of
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2×10-6 M. Meanwhile TMPP decreased the production of cortisol and TDBPP
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down-regulated the secretion of aldosterone. Our results indicate that some OPFRs
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can interact with GR and MR, and have the potential to disturb steroidogenesis. Data
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provided here will be helpful to comprehensively understand the potential endocrine
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disruption of OPFRs.
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Introduction Organophosphate flame retardants (OPFRs) have been the popular alternatives to
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polybrominated diphenyl ethers (PBDEs) after their restrictive use in the United
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States and Europe in the early 2000s.1With the increasing use of OPFRs, recent data
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have shown that OPFRs are ubiquitous in air,2-5 water,6, 7 soil,8, 9 sediment,9, 10and
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aquatic organisms,11-13 and they have also been detected in house dust,2, 4, 14-24
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drinking water,25 and even in human milk13, 26 and urine.27-34 Studies have further
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revealed that the OPFRs, especially tris(1,3-dichloro-2-propyl) phosphate (TDCIPP)
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and triphenyl phosphate (TPHP), have been found in backpacking tents,35 hand
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wipes,19, 21 silicone wristbands,36, 37and baby products,19, 21, 38, 39which have significant
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implications on human exposures thru direct contact.
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Recent studies have been conducted to evaluate the potential ecological and
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health risks of OPFRs including ours in which we have found OPFRs induced anti/
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estrogenic activity via estrogenic receptor α (ERα),40 and showed thyroid receptor β
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(TRβ) antagonistic activity41 in the dual-luciferase reporter gene assays. Toxicological
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studies have shown that OPFRs have potential endocrine disrupting effects via human
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nuclear receptors,42 and could cause estrogen and thyroid disruption in zerbrafish.43, 44
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Exposure to tris (2-butoxyethyl) phosphate (TBOEP) and TDCIPP may lead to
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developmental malformations45 and neurotoxicity46 in zebrafish. Furthermore, Meeker
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et al. revealed that bis(1,3-dichloro-2-propyl) phosphate (BDCPP) and diphenyl
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phosphate (DPP), the urinary metabolites of TDCIPP and TPHP, were associated with 4
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male reproductive health to some degree.30 Thus, it is of vital importance to assess
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the potential endocrine disrupting activity of OPFRs.
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The estrogenic and thyroid hormones, which play essential roles in growth,
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development and reproduction, have been reported to be disturbed by some OPFRs.
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However, the effects of OPFRs on the glucocorticoid and mineralocorticoid hormones,
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which are regulated by the hypothalamo–pituitary–adrenal (HPA) axis and are
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extremely important in homeostasis, have not yet been elucidated.47 The HPA axis is a
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central neuroendocrine system and its primary function is to mediate stress-associated
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disorders such as chronic fatigue syndrome, melancholic depression and insomnia.48
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Kojima et al. have reported that some OPFRs and their metabolites induced
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antagonistic activities against human nuclear receptors including GR.42, 49 Several
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studies have indicated that some OPFRs, such as TBOEP and TDCIPP, could change
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the expression of genes involving with the GR and MR pathways in zebrafish
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larvae.50, 51Further research manifests the alteration of cortisol in adult zebrafish under
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long-term exposure to TPHP.52 However, neither MR agonistic/antagonistic effects
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nor evaluating OPFRs in the H295R steroidogenesis assay for enzymatic activities of
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steroidogenic genes,53 have been taken into account in those studies.
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In this present study, we evaluated the glucocorticoid and mineralocorticoid
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effects of nine OPFRs in dual-luciferase reporter gene assay. Furthermore, the levels
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of cortisol and aldosterone, and the changes of genes in the pathways of
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steroidogenesis synthesis were also examined in H295R cells. Data provided here will 5
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be helpful to evaluate the potential endocrine disruption effects of OPFRs.
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Materials and methods
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Chemicals and plasmids
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Nine organophosphate flame retardants (OPFRs) were obtained from Dr.
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Ehrenstorfer GmbH (Augsburg, Germany) and the details were listed in Table S1.
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Hydrocortisone (HC; 98% pure) and aldosterone (AD; 97% pure) were both
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purchased from J&K Scientific Ltd. (Beijing, China). All of the chemicals above were
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dissolved in dimethyl sulfoxide (DMSO) obtained from Sigma-Aldrich (St. Louis,
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MO, USA). The human glucocorticoid receptor α plasmid of pF25GFP-hGRα (GR)
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and the response element plasmid of pMMTV-luc (MMTV) were kindly provided by
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Dr. Evangelia Charmandari (Biomedical Research Foundation of the Academy in
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Athens, Greece).The human mineralocorticoid receptor plasmid of EGFP-C1-hMR
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(MR) was provided by Dr. Claudia Großmann (Julius Bernstein Institute for
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Physiology, Martin Luther University, Germany). The internal control plasmid of
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phRL-tk (TK) containing the Renilla luciferase gene was purchased from Promega
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(Madison, WI, USA).
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Cell Culture and Exposure medium Chinese hamster ovary K1 (CHO) cells were purchased from the Cell Bank of
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Chinese Academy of Sciences (Shanghai, China). The cells were cultured in modified
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RPMI medium (HyClone; Logan, UT, USA) with 10% fetal bovine serum (FBS;
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HyClone) in a humidified atmosphere of 5% CO2 at 37 °C. Before transfection, the 6
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cells were cultured for 24 h in phenol red-free RPMI 1640 medium (Gibco, Grand
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Island, NY, USA) supplemented with 5% charcoal−dextran stripped FBS (CD-FBS;
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Gemini, USA).
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The human adrenocortical carcinoma (H295R) cells were kindly provided by
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Professor Qunfang Zhou (Research Center for Eco-Environmental Sciences, Chinese
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Academy of Sciences). The cells were cultured in Dulbecco’s modified Eagle’s
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medium/F12 (DMEM/F12; Hyclone) supplemented with 1% Ultroser G (Pall
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Corporation, Port Washington, NY, USA), a serum substitute for animal cell culture,
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1% insulin-transferrin-selenium (Gibco), 1% L-glutamine (Gibco), and 1%
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penicillin-streptomycin (Gibco). For real-time polymerase chain reaction (RT-PCR)
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and hormone measurement assays, the cells were incubated with phenol red-free
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DMEM/F12 medium (Gibco) substituted with phenol red DMEM/F12 medium. Cells
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were seeded in 6-well culture plates, and starved in exposure medium for 24 h and
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then treated with the test chemicals.
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MTS Assay
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The cytotoxicity of nine OPFRs in CHO and H295R cells were assessed by Cell
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Proliferation Assay (MTS assay; Promega, USA) as previously described.40 The cells
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were grown in 96-well plates (Corning, NY, USA) containing 100 µl exposure
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medium. The confluence of cells was approximately 80% and the density was
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approximately 5000 cells/well. The cells were then treated with various
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concentrations of test chemicals and the DMSO set (≤ 0.1% v/v) as the negative 7
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control for 24 h. The absorbance was measured at 490 nm by a microplate reader
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(Infinite M200, Tecan, Switzerland). Only the non-cytotoxic concentrations were used
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for following experiments.
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Dual-Luciferase Reporter Gene Assays for GR and MR
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The CHO cells were grown in a 96-well plate at a density of 20000 cells/well.
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For GR activity, each well was transfected with a DNA mixture containing 10 ng of
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GR, 120 ng of MMTV, and 10 ng of TK with 0.5 µl of transfection reagent
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(Lipofectamine 2000; Invitrogen, MD, USA). For MR activity, 20 ng of MR, 160 ng
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of MMTV, and 10 ng of TK were added to each well with 0.5 µl of transfection
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reagent. After 4 h of transfection, the cells were changed with fresh exposure medium
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overnight. Cells were then treated with various concentrations of test chemicals for
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GR- or MR-mediated agonistic activities. For antagonistic activities, cells were
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exposed to different concentrations of test chemicals in the presence of 50 nM HC or
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0.1 nM AD.The activities of firefly luciferase and renilla luciferase were measured
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witha fluorescence microplate reader (Infinite M200) following the protocol specified
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in the dual-luciferase reporter assay kit (Promega, WI, USA). The results were
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normalized to the renilla luciferase.
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RNA Isolation and RT-PCR
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H295R cells were seeded in a 6-well plate with 2 mL of exposure medium at a
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density of 6×105 cells/well. The cells were then exposed to vehicle (≤ 0.1% v/v
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DMSO), 0.1 µM, 1 µM or 2 µM of each chemical for24 h. After removal of the 8
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medium, the total RNA of the H295R cells was extracted using TRIzol reagent
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(Takara, Otsu, Japan) following the manufacturer’s instructions. The RNA, in a
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A260/280 ratio measured with a K5500 spectrophotometer (Kaiao, Beijing, China) in
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the range of 1.8 to 2.0, was used for cDNA synthesis immediately or stored at -80 °C
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until needed. The synthesis of cDNA was performed by a ReverTra Ace qPCR RT Kit
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(Toyobo, Osaka, Japan) using a 2720 thermal cycler (Applied Biosystems, CA, USA).
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The final cDNA samples were used immediately or stored at -20 °C. RT-PCR was
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undertaken with a 7300 Real Time PCR System (Applied Biosystems, CA, USA)
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using a SYBR Green Real-time PCR Master Mix Kit (Toyobo, Osaka, Japan). The
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primer sequences of GAPDH, StAR, HMGR, 3βHSD2, CYP11A1, CYP11B1, CYP11B2,
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CYP17, and CYP21 purchased from Sangon Biotech Co., Ltd. (Shanghai, China) are
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shown in Table S2. The thermal cycle was as follows: 1 min at 95 °C, followed
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closely by 40 cycles of 15 s at 95 °C, and 60 s at 60 °C. The expression of
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steroidogenesis gene was normalized to the housekeeping gene
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glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Fold change was calculated
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with the 2-∆∆Ct method, as described by Livak andSchmittgen.54
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Hormone Measurements
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H295R cells were plated in 6-well plates at a density of 106 cells/well with 2.5
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mL of medium. The medium was changed and the cells were exposed to vehicle (≤
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0.1% v/v DMSO), or 5 µM of each chemical for48 h after the cells were attached to
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the plate overnight. Then the medium was collected and stored at -80 °C for detection 9
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of cortisol and aldosterone. A radioimmunoassay kit (Beijing North Institute of
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Biological Technology) was used for measuring the cortisol and aldosterone contents
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according to the manufacturer’s protocol and three replicated samples were measured.
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The limits of detection were 10 ng/mL for cortisol and 50 pg/mL for aldosterone. The
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intra-assay coefficients of variation were below 10%.
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Statistical Analysis
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All results were analyzed using Microsoft excel and Origin 9.0 (OriginLab,
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Northampton, MA). Data were presented as mean ± SD (standard deviation) for at
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least three independent experiments with triplicates. Statistical significance was
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determined using the Student’s t-test following a one-way ANOVA and the significant
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difference was set at p< 0.05.
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Results
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Cytotoxicity of OPFRs on MTS assay
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The cytotoxicity of the nine OPFRs against CHO cells was described in a
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previous study,40 and here we tested the cell viability of H295R cells with various
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concentrations of nine OPFRs (Figures S1). The concentrations (≤5×10-6 M for
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TMPP, TPHP, TDBPP and ≤10-5 M for TNBP, TBOEP, TCEP, TDCIPP, TCIPP and
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TEHP) that did not induce cytotoxicity were used for the further experiments.
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Agonistic and antagonistic activities of the OPFRs against GR and MR
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We fitted the dose response curve of HC or AD at various concentrations using
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logistic model in order to calculate the EC20 from the fitting equation. As previously 10
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described, the EC20 value of HC for GR was 4.2 × 10−9 M from the dose response
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curve.55 The EC20 value of AD for MR was estimated to be 3.1×10-11 M from the dose
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response curve ranging from 10-13 M to 10-7 M, as shown in Figure S2. The
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concentration from the curve, which reached the upper plateau was used for agonistic
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activity, while the approximate EC80 value was used for antagonistic activity, as
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previously reported.56 When the effect value of individual OPFRs in the presence with
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positive control is below than 80% of effect value of positive control alone, that
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specific OPFRs was considered as an antagonist in our study.
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Although none of the nine OPFRs induced any agonistic activity against GR or
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MR receptors (Figures S3 and S4), we found three and five OPFRs that have
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antagonistic activities in the GR and MR transactivation assay, respectively (Figure
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1). The relative effective concentrations, RIC20 and RIC50, levels of OPFRs inhibiting
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20% or 50% of luciferase activities induced by 50 nM of HC and 0.1 nM of AD,
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respectively, were both used for evaluating the antagonism of OPFRs via GR and MR
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(Table 1). For GR antagonism (Figure 1A-1C and S5), TMPP, TPHP, and TDBPP
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exhibited potent antagonistic activities in the following order, TDBPP > TMPP >
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TPHP, with RIC20 values of 1.1×10-6, 1.2×10-6, and 2.6×10-6 M, respectively. In the
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MR assay (Figure 1D-1H and S6), TNBP, TMPP, TPHP, TDCIPP, and TDBPP
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showed the antagonistic properties and the corresponding RIC20 values were listed in
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Table 1. Among those five OPFRs, TMPP, TPHP, and TDBPP induced significant
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antagonistic activities with RIC20 values lower than 10-6M, and TNBP and TDCIPP 11
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showed weak antagonistic effects with RIC20 values higher than 5×10-6 M.
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Steroidogenic genes expression profile of treated H295R cells
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To further investigate the effects of OPFRs on corticosteroid homeostasis, eight
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steroidogenic genes, StAR, HMGR, 3βHSD2, CYP11A1, CYP11B1, CYP11B2, CYP17,
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and CYP21, involving in the principal pathways for synthesis of aldosterone and
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cortisol in H295R cells were determined (Table S2). As shown in Figure 2, most of
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the OPFRs treatments led to a significant decrease in the expression of genes. More
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than three genes’ expression was modified by TMPP, TPHP, and TDBPP (Table S3
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and Table S4). CYP17, CYP21, and CYP11B1 expression were down-regulated by
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~34-97% following 2×10-6 M of TMPP, TPHP, or TDBPP exposure. When exposed to
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2×10-6 M, TMPP up-regulated the HMGR expression, TPHP inhibited the expression
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of StAR, 3βHSD2 andCYP11A1, and TDBPP decreased the expression of 3βHSD2,
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CYP11A1, and CYP11B2. In addition, TDCIPP and TEHP both affected the expression
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of three genes. TDCIPP induced a significant increase in CYP17 and CYP21, but
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decreased the expression of CYP11B1 at 2×10-6 M. TEHP significantly up-regulated
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3βHSD2 and CYP21, and maintained a repressive effect on StAR exposure to 2×10-6
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M. TNBP, TBOEP, and TCEP showed a dominant inhibition in CYP17, CYP17, and
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StAR, respectively, while TCIPP manifested great up-regulation in 3βHSD2
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expression at the 2×10-6 M.
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Effects on the production of cortisol and aldosterone
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The levels of cortisol and aldosterone were measured to assess the effects of nine 12
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OPFRs on the production of steroid hormones in H295R cells when exposing to
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5×10-6 M. Table 2 showed that TPHP and TDCIPP significantly increased the
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production of cortisol by 2.27 and 1.76 folds, meanwhile TNBP, TPHP, and TDCIPP
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increased the levels of aldosterone by 1.88, 2.55, and 2.36 folds, respectively.
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Exposure to TMPP and TDBPP caused a decrease in cortisol by 0.68 fold and
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aldosterone by 0.72 fold, respectively. Other OPFRs, however, did not change the
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production of these two steroid hormones compared with control (≤ 0.1% v/v
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DMSO).
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Discussion
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We have identified the GR and MR antagonistic activities of nine OPFRs in
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dual-luciferase reporter gene assay and the modification of gene expression in the
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pathways for the synthesis of cortisol and aldosterone using H295R cells. The results
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indicated that TMPP, TPHP, and TDBPP could behave as GR and MR antagonists,
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while TNBP and TDCIPP only exhibited MR antagonistic effects. The data from
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RT-PCR showed that most of the genes involving in the synthesis of cortisol and
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aldosterone were down-regulated under the exposure conditions. The productions of
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steroid hormones including cortisol and aldosterone were also affected after exposure
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to TNBP, TMPP, TPHP, TDCIPP, and TDBPP at 5×10-6 M. The results presented here
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have enriched the evidence that OPFRs as a class of potent endocrine disrupting
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chemicals (EDCs), and will be helpful for us to better understanding the ecological
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and health risks of those emerging contaminates at a new sight. 13
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Abundant evidence from recent studies has shown that OPFRs are widely
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detected in various environmental matrixes, consumer products, organisms, and
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biological samples collected from individuals. Studies have demonstrated that human
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has been exposed to OPFRs through dust ingestion57, dermal absorption58 and dietary
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intake59. Hoffman et al. reported that the geometric mean of TPHP in house dust
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collected from North Carolina was 1020 ppb (≈ng/g).21 TDCIPP and TCIPP, were
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detected in children’s car seats and mattress.60 TPHP was also detected in perch at
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levels of 21-180 ppb, and TNBP was found in human breast milk.13 Those residue
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data not only have revealed the high levels of some OPFRs in the environment, but
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should have aroused the concern due to the fact that some of the levels have exceeded
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the lowest observed effect level (LOEL) as well. As defined in our reporter gene
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assays, the value of LOEL is the lowest concentration leading to the inhibition of 20%
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of luciferase activities and OPFR’s molecular weight. Although most of the OPFRs
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residues reported by recent studies were still one to two orders of magnitude lower
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than our LOEL as calculated here, it does not directly imply that those levels would
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not impose the potential ecological and health risks. Two important factors should be
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considered in assessing the environmental relevancy of the potential risks of those
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emerging OPFRs. First of all, OPFRs had been found in our everyday environment,
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including in the drinking water in which the total concentrations of OPFRs could be
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as high as 1660 ng/L (median level at 48.7) ng/L reported in Korea61 and 325 ng/L in
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China62. TPHP, as one of the most commonly found OPFRs in drinking water, had 14
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been identified as GR/MR mediated disruptor in our study. The ubiquity of OPFRs in
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water indicated human especially susceptible sub-population has been exposed to
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OPFRs more frequently than previously thought. Secondly, because several OPFRs
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have relative high octanol-water partition coefficients (Kow) such as TNBP, TMPP,
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TPHP, TDBPP and TEHP,63-67 OPFRs as a group are considered persistent, and will be
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bio-accumulated in organisms as the result of prolong exposure. As being the
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emerging contaminates, the usage of OPFRs will be increasing in the foreseeable
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future in which will facilitate the increasing body burden of OPFRs in biota that will
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trigger the potential adverse effects, such as the endocrine disrupting effects reported
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here.
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GR and MR, as two potential targets for EDCs, play vital roles in glucose
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metabolism and electrolyte homeostasis.68-70 Several diseases, such as neurological
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diseases and metabolic disorders, are linked to the perturbation of glucocorticoid and
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mineralocorticoid activities.71A recent study has shown that OPFRs could be
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transported to tissues, even in the brain of adult fish,72 suggesting the transportation
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and bio-accumulation of OPFRs from the environment to organisms. Herein, we
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reported the RIC20 of OPFRs via GR/MR, and data provided here would be used as
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reference values for further study regard of biological effects and risks associated with
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OPFRs exposure. Moreover, besides the GR and MR mediated effects, our previous
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studies have shown that TPHP and TDCIPP could induce estrogenic and thyroid
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antagonistic activities.40, 41 Considering multi-endocrine disrupting effects and the 15
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ubiquity of OPFRs in the environment, the potential ecological and health risks
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associated with OPFRs exposure should be carefully examined without any further
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delay.
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Other than the dual-luciferase reporter gene assay, we used RT-PCR to assess the
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change in the expression of related genes and to measure the corresponding steroid
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hormones containing cortisol and aldosterone. In the H295R steroidogenesis pathway,
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genes such as StAR, HMGR, CYP11A1, 3βHSD2, CYP21, CYP11B1 and CYP11B2,
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involve in the synthesis of aldosterone, and CYP17 is associated with the synthesis of
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cortisol.CYP17, CYP21, and CYP11B1 encodes members of the cytochrome P450
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superfamily of enzymes.CYP17plays a key role in the conversion of pregnenolone and
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progesterone to their 17-α-hydroxylated products, while CYP21 is related to carbon
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metabolism, and cannot function without cortisol and aldosterone. As the last step in
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the synthesis of steroids, CYP11B1 is responsible for catalyzing the transformation of
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11-deoxycorticosterone and 11-deoxycortisol to corticosterone and cortisol,
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respectively. In this study, we have shown that TDCIPP could activate the synthesis of
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cortisol and aldosterone by up-regulating the expression of CYP17 and CYP21, which
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play a role in the intermediate steps. This study also showed that reduction of
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transcription of CYP17, CYP21 and CYP11B1 would probably decrease the
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production of cortisol when treated with TMPP. Similarly, exposure to TDBPP
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down-regulated the expression of CYP11A1, 3βHSD2, CYP21, CYP11B1 and
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CYP11B2, so aldosterone production was decreased significantly. However, the 16
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hormones production increased when treated with TNBP and TPHP even though none
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of genes were up-regulated significantly. Such discrepancies between the gene
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expression and hormone production could be a consequence of the chemical
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structures and properties or the other signaling pathways that they involve in H295R
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cells.73 The underlying mechanism for this finding should be further elucidated in the
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future studies.
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In summary, we evaluated the potential endocrine disrupting effects mediated by
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GR and MR for nine OPFRs in in vitro and in silico models for the first time. Among
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those nine OPFRs, TMPP, TPHP, and TDBPP showed potential glucocorticoid
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antagonistic activities and TNBP, TMPP, TPHP, TDCIPP, and TDBPP exhibited
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potential mineralocorticoid antagonistic effects. The data provided in this paper is
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significant from the perspective of comprehensive evaluation of the biological and
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ecological risks of OPFRs.
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Acknowledgements This study was funded by the National Natural Science Foundation of China (21377119, 21307109, and 21577129).
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Reference
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(1) Darnerud, P. O.; Eriksen, G. S.; Jóhannesson, T.; Larsen, P. B.; Viluksela, M. Polybrominated Diphenyl Ethers: Occurrence, Dietary Exposure, and Toxicology. Environ. Health Perspect. 2001,109 (s1), 49-68. (2) Takigami, H.; Suzuki, G.; Hirai, Y.; Ishikawa, Y.; Sunami, M.; Sakai, S. Flame retardants in indoor dust and air of a hotel in Japan. Environ. Int. 2009,35 (4), 688-693. (3) Bergh, C.; Aberg, K. M.; Svartengren, M.; Emenius, G.; Ostman, C. Organophosphate and phthalate esters in indoor air: a comparison between multi-storey buildings with high and low prevalence of sick building symptoms. J. Environ. Monit. 2011,13 (7), 2001-2009. (4) Fromme, H.; Lahrz, T.; Kraft, M.; Fembacher, L.; Mach, C.; Dietrich, S.; Burkardt, R.; Volkel, W.; Goen, T. Organophosphate flame retardants and plasticizers in the air and dust in German daycare centers and human biomonitoring in visiting children (LUPE 3). Environ. Int. 2014,71, 158-163. (5) Shoeib, M.; Ahrens, L.; Jantunen, L.; Harner, T. Concentrations in air of organobromine, organochlorine and organophosphate flame retardants in Toronto, Canada. Atmos. Environ. 2014,99, 140-147. (6) Andresen, J. A.; Grundmann, A.; Bester, K. Organophosphorus flame retardants and plasticisers in surface waters. Sci. Total Environ. 2004,332 (1-3), 155-166. (7) Venier, M.; Salamova, A.; Hites, R. A. Halogenated Flame Retardants in the Great Lakes Environment. Acc. Chem. Res. 2015,48 (7), 1853-1861. (8) David, M. D.; Seiber, J. N. Analysis of organophosphate hydraulic fluids in US Air Force base soils. Arch. Environ. Contam. Toxicol. 1999,36 (3), 235-241. (9) Lu, J. X.; Ji, W.; Ma, S. T.; Yu, Z. Q.; Wang, Z.; Li, H.; Ren, G. F.; Fu, J. M. Analysis of Organophosphate Esters in Dust, Soil and Sediment Samples Using Gas Chromatography Coupled with Mass Spectrometry. Chinese J. Anal. Chem. 2014,42 (6), 859-865. (10) Cao, S. X.; Zeng, X. Y.; Song, H.; Li, H. R.; Yu, Z. Q.; Sheng, G. Y.; Fu, J. M. Levels and distributions of organophosphate flame retardants and plasticizers in sediment from Taihu Lake, China. Environ. Toxicol. Chem. 2012,31 (7), 1478-1484. (11) Ma, Y. Q.; Cui, K. Y.; Zeng, F.; Wen, J. X.; Liu, H.; Zhu, F.; Ouyang, G. F.; Luan, T. G.; Zeng, Z. X. Microwave-assisted extraction combined with gel permeation chromatography and silica gel cleanup followed by gas chromatography-mass spectrometry for the determination of organophosphorus flame retardants and plasticizers in biological samples. Anal. Chim. Acta 2013,786, 47-53. (12) McGoldrick, D. J.; Letcher, R. J.; Barresi, E.; Keir, M. J.; Small, J.; Clark, M. G.; Sverko, E.; Backus, S. M. Organophosphate flame retardants and organosiloxanes in predatory freshwater fish from locations across Canada. Environ. Pollut. 2014,193, 254-261. 18
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Page 19 of 27
Environmental Science & Technology
375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416
(13) Sundkvist, A. M.; Olofsson, U.; Haglund, P. Organophosphorus flame retardants and plasticizers in marine and fresh water biota and in human milk. J. Environ. Monit. 2010,12 (4), 943-951. (14) Stapleton, H. M.; Eagle, S.; Fuh, J.; Blum, A.; Webster, T. F. Detection of Organophosphate Flame Retardants in Furniture Foam and U.S. House Dust. Environ. Sci. Technol. 2009,43, 7490–7495. (15) Mercier, F.; Glorennec, P.; Thomas, O.; Le Bot, B. Organic Contamination of Settled House Dust, A Review for Exposure Assessment Purposes. Environ. Sci. Technol. 2011,45 (16), 6716-6727. (16) Dodson, R. E.; Perovich, L. J.; Covaci, A.; Van den Eede, N.; Ionas, A. C.; Dirtu, A. C.; Brody, J. G.; Rudel, R. A. After the PBDE Phase-Out: A Broad Suite of Flame Retardants in Repeat House Dust Samples from California. Environ. Sci. Technol. 2012,46 (24), 13056-13066. (17) Suzuki, G.; Tue, N. M.; Malarvannan, G.; Sudaryanto, A.; Takahashi, S.; Tanabe, S.; Sakai, S.; Brouwer, A.; Uramaru, N.; Kitamura, S.; Takigami, H. Similarities in the endocrine-disrupting potencies of indoor dust and flame retardants by using human osteosarcoma (U2OS) cell-based reporter gene assays. Environ. Sci. Technol. 2013,47 (6), 2898-2908. (18) Fang, M. L.; Stapleton, H. M. Evaluating the Bioaccessibility of Flame Retardants in House Dust Using an In Vitro Tenax Bead-Assisted Sorptive Physiologically Based Method. Environ. Sci. Technol. 2014,48 (22), 13323-13330. (19) Stapleton, H. M.; Misenheimer, J.; Hoffman, K.; Webster, T. F. Flame retardant associations between children's handwipes and house dust. Chemosphere 2014,116, 54-60. (20) Takeuchi, S.; Kojima, H.; Saito, I.; Jin, K.; Kobayashi, S.; Tanaka-Kagawa, T.; Jinno, H. Detection of 34 plasticizers and 25 flame retardants in indoor air from houses in Sapporo, Japan. Sci. Total Environ. 2014,491, 28-33. (21) Hoffman, K.; Garantziotis, S.; Birnbaum, L. S.; Stapleton, H. M. Monitoring indoor exposure to organophosphate flame retardants: hand wipes and house dust. Environ. Health Perspect. 2015,123 (2), 160-165. (22) Cristale, J.; Hurtado, A.; Gomez-Canela, C.; Lacorte, S. Occurrence and sources of brominated and organophosphorus flame retardants in dust from different indoor environments in Barcelona, Spain. Environ. Res. 2016,149, 66-76. (23) Langer, S.; Fredricsson, M.; Weschler, C. J.; Beko, G.; Strandberg, B.; Remberger, M.; Toftum, J.; Clausen, G. Organophosphate esters in dust samples collected from Danish homes and daycare centers. Chemosphere 2016,154, 559-566. (24) Wu, M.; Yu, G.; Cao, Z. G.; Wu, D. K.; Liu, K.; Deng, S. B.; Huang, J.; Wang, B.; Wang, Y. J. Characterization and human exposure assessment of organophosphate flame retardants in indoor dust from several microenvironments of Beijing, China. Chemosphere 2016,150, 465-471. (25) Khan, M. U.; Li, J.; Zhang, G.; Malik, R. N. First insight into the levels and distribution of flame retardants in potable water in Pakistan: An underestimated 19
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417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458
problem with an associated health risk diagnosis. Sci. Total Environ. 2016,565, 346-359. (26) Kim, J. W.; Isobe, T.; Muto, M.; Tue, N. M.; Katsura, K.; Malarvannan, G.; Sudaryanto, A.; Chang, K. H.; Prudente, M.; Viet, P. H.; Takahashi, S.; Tanabe, S. Organophosphorus flame retardants (PFRs) in human breast milk from several Asian countries. Chemosphere 2014,116, 91-97. (27) Moller, K.; Crescenzi, C.; Nilsson, U. Determination of a flame retardant hydrolysis product in human urine by SPE and LC-MS. Comparison of molecularly imprinted solid-phase extraction with a mixed-mode anion exchanger. Anal. Bioanal. Chem. 2004,378 (1), 197-204. (28) Schindler, B. K.; Foerster, K.; Angerer, J. Determination of human urinary organophosphate flame retardant metabolites by solid-phase extraction and gas chromatography-tandem mass spectrometry. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2009,877 (4), 375-381. (29) Cooper, E. M.; Covaci, A.; van Nuijs, A. L. N.; Webster, T. F.; Stapleton, H. M. Analysis of the flame retardant metabolites bis(1,3-dichloro-2-propyl) phosphate (BDCPP) and diphenyl phosphate (DPP) in urine using liquid chromatography-tandem mass spectrometry. Anal. Bioanal. Chem. 2011,401 (7), 2123-2132. (30) Meeker, J. D.; Cooper, E. M.; Stapleton, H. M.; Hauser, R. Exploratory analysis of urinary metabolites of phosphorus-containing flame retardants in relation to markers of male reproductive health. Endocr. Disruptors (Austin) 2013,1 (1), e26306. (31) Van den Eede, N.; Neels, H.; Jorens, P. G.; Covaci, A. Analysis of organophosphate flame retardant diester metabolites in human urine by liquid chromatography electrospray ionisation tandem mass spectrometry. J. Chromatogr. A 2013,1303, 48-53. (32) Butt, C. M.; Congleton, J.; Hoffman, K.; Fang, M.; Stapleton, H. M. Metabolites of organophosphate flame retardants and 2-ethylhexyl tetrabromobenzoate in urine from paired mothers and toddlers. Environ. Sci. Technol. 2014,48 (17), 10432-10438. (33) Dodson, R. E.; Van den Eede, N.; Covaci, A.; Perovich, L. J.; Brody, J. G.; Rudel, R. A. Urinary biomonitoring of phosphate flame retardants: levels in California adults and recommendations for future studies. Environ. Sci. Technol. 2014,48 (23), 13625-13633. (34) Hoffman, K.; Daniels, J. L.; Stapleton, H. M. Urinary metabolites of organophosphate flame retardants and their variability in pregnant women. Environ. Int. 2014,63, 169-172. (35) Gomes, G.; Ward, P.; Lorenzo, A.; Hoffman, K.; Stapleton, H. M. Characterizing Flame Retardant Applications and Potential Human Exposure in Backpacking Tents. Environ. Sci. Technol. 2016,50 (10), 5338-5345. (36) Kile, M. L.; Scott, R. P.; O'Connell, S. G.; Lipscomb, S.; MacDonald, M.; McClelland, M.; Anderson, K. A. Using silicone wristbands to evaluate preschool children's exposure to flame retardants. Environ. Res. 2016,147, 365-372. 20
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Page 20 of 27
Page 21 of 27
Environmental Science & Technology
459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500
(37) Hammel, S. C.; Hoffman, K.; Webster, T. F.; Anderson, K. A.; Stapleton, H. M. Measuring Personal Exposure to Organophosphate Flame Retardants Using Silicone Wristbands and Hand Wipes. Environ. Sci. Technol. 2016,50 (8), 4483-4491. (38) Stapleton, H. M.; Klosterhaus, S.; Keller, A.; Ferguson, P. L.; van Bergen, S.; Cooper, E.; Webster, T. F.; Blum, A. Identification of Flame Retardants in Polyurethane Foam Collected from Baby Products. Environ. Sci. Technol. 2011,45 (12), 5323-5331. (39) Hoffman, K.; Butt, C. M.; Chen, A.; Limkakeng, A. T.; Stapleton, H. M. High Exposure to Organophosphate Flame Retardants in Infants: Associations with Baby Products. Environ. Sci. Technol. 2015,49 (24), 14554-14559. (40) Zhang, Q.; Lu, M. Y.; Dong, X. W.; Wang, C.; Zhang, C. L.; Liu, W. P.; Zhao, M. R. Potential estrogenic effects of phosphorus-containing flame retardants. Environ. Sci. Technol. 2014,48 (12), 6995-7001. (41) Zhang, Q.; Ji, C. Y.; Yin, X. H.; Yan, L.; Lu, M. Y.; Zhao, M. R. Thyroid hormone-disrupting activity and ecological risk assessment of phosphorus-containing flame retardants by in vitro, in vivo and in silico approaches. Environ. Pollut. 2016,210, 27-33. (42) Kojima, H.; Takeuchi, S.; Itoh, T.; Iida, M.; Kobayashi, S.; Yoshida, T. In vitro endocrine disruption potential of organophosphate flame retardants via human nuclear receptors. Toxicology 2013,314 (1), 76-83. (43) Wang, Q. W.; Lam, J. C. W.; Han, J.; Wang, X. F.; Guo, Y. Y.; Lam, P. K. S.; Zhou, B. S. Developmental exposure to the organophosphorus flame retardant tris(1,3-dichloro-2-propyl) phosphate: Estrogenic activity, endocrine disruption and reproductive effects on zebrafish. Aquat. Toxicol. 2015,160, 163-171. (44) Liu, X. S.; Ji, K.; Jo, A.; Moon, H. B.; Choi, K. Effects of TDCPP or TPP on gene transcriptions and hormones of HPG axis, and their consequences on reproduction in adult zebrafish (Danio rerio). Aquat. Toxicol. 2013,134, 104-111. (45) Ma, Z. Y.; Song, T.; Su, G. Y.; Miao, Y. Q.; Liu, H. L.; Xie, Y. W.; Giesy, J. P.; Saunders, D. M. V.; Hecker, M.; Yu, H. X. Effects of tris (2-butoxyethyl) phosphate (TBOEP) on endocrine axes during development of early life stages of zebrafish ( Danio rerio ). Chemosphere 2016,144, 1920-1927. (46) Wang, Q. W.; Lam, J. C. W.; Man, Y. C.; Lai, N. L. S.; Kwok, K. Y.; Guo, Y. Y.; Lam, P. K. S.; Zhou, B. S. Bioconcentration, metabolism and neurotoxicity of the organophorous flame retardant 1,3-dichloro 2-propyl phosphate (TDCPP) to zebrafish. Aquat. Toxicol. 2015,158, 108-115. (47) Hosseinichimeh, N.; Rahmandad, H.; Wittenborn, A. K. Modeling the hypothalamus-pituitary-adrenal axis: A review and extension. Math. Biosci. 2015,268, 52-65. (48) Makino, S.; Hashimoto, K.; Gold, P. W. Multiple feedback mechanisms activating corticotropin-releasing hormone system in the brain during stress. Pharmacology Biochemistry & Behavior 2002,73 (1), 147-158. (49) Kojima, H.; Takeuchi, S.; Van den Eede, N.; Covaci, A. Effects of primary 21
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Environmental Science & Technology
501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542
metabolites of organophosphate flame retardants on transcriptional activity via human nuclear receptors. Toxicol. Lett. 2016,245, 31-39. (50) Liu, C. S.; Wang, Q. W.; Liang, K.; Liu, J. F.; Zhou, B. S.; Zhang, X. W.; Liu, H. L.; Giesy, J. P.; Yu, H. X. Effects of tris(1,3-dichloro-2-propyl) phosphate and triphenyl phosphate on receptor-associated mRNA expression in zebrafish embryos/larvae. Aquat. Toxicol. 2013,128-129, 147-157. (51) Ma, Z. Y.; Yu, Y. J.; Tang, S.; Liu, H. L.; Su, G. Y.; Xie, Y. W.; Giesy, J. P.; Hecker, M.; Yu, H. X. Differential modulation of expression of nuclear receptor mediated genes by tris(2-butoxyethyl) phosphate (TBOEP) on early life stages of zebrafish (Danio rerio). Aquat. Toxicol. 2015,169, 196-203. (52) Liu, X. S.; Jung, D.; Jo, A.; Ji, K.; Moon, H. B.; Choi, K. Long-term exposure to triphenylphosphate alters hormone balance and HPG, HPI, and HPT gene expression in zebrafish (Danio rerio). Environ. Toxicol. Chem. 2016,35 (9), 2288-2296. (53) USEPA, Steroidogenesis (Human Cell Line - H295R) OCSPP Guideline 890.1550. In Washington, DC 20460, 2011. (54) Livak, K. J.; Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(T)(-Delta Delta C) method. Methods 2001,25 (4), 402-408. (55) Zhang, Q.; Wang, J. H.; Zhu, J. Q.; Liu, J.; Zhang, J. Y.; Zhao, M. R. Assessment of the endocrine-disrupting effects of short-chain chlorinated paraffins in in vitro models. Environ. Int. 2016,94, 43-50. (56) Kojima, H.; Katsura, E.; Takeuchi, S.; Niiyama, K.; Kobayashi, K. Screening for Estrogen and Androgen Receptor Activities in 200 Pesticides by In Vitro Reporter Gene Assays Using Chinese Hamster Ovary Cells. Environ. Health Perspect. 2003,112 (5), 524-531. (57) Schreder, E. D.; Uding, N.; La Guardia, M. J. Inhalation a significant exposure route for chlorinated organophosphate flame retardants. Chemosphere 2016,150, 499-504. (58) Liu, X. T.; Yu, G.; Cao, Z. G.; Wang, B.; Huang, J.; Deng, S. B.; Wang, Y. J. Occurrence of organophosphorus flame retardants on skin wipes: Insight into human exposure from dermal absorption. Environ. Int. 2017,98, 113-119. (59) Poma, G.; Glynn, A.; Malarvannan, G.; Covaci, A.; Darnerud, P. O. Dietary intake of phosphorus flame retardants (PFRs) using Swedish food market basket estimations. Food Chem. Toxicol. 2016,100, 1-7. (60) Cooper, E. M.; Kroeger, G.; Davis, K.; Clark, C. R.; Ferguson, P. L.; Stapleton, H. M. Results from Screening Polyurethane Foam Based Consumer Products for Flame Retardant Chemicals: Assessing Impacts on the Change in the Furniture Flammability Standards. Environ. Sci. Technol. 2016,50 (19), 10653-10660. (61) Lee, S.; Jeong, W.; Kannan, K.; Moon, H. B. Occurrence and exposure assessment of organophosphate flame retardants (OPFRs) through the consumption of drinking water in Korea. Water Res 2016,103, 182-188. (62) Li, J.; Yu, N. Y.; Zhang, B. B.; Jin, L.; Li, M. Y.; Hu, M. Y.; Zhang, X. W.; Wei, 22
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Environmental Science & Technology
543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573
S.; Yu, H. X. Occurrence of organophosphate flame retardants in drinking water from China. Water Research 2014,54, 53-61. (63) NCBI. Pubchem-Tributyl phosphate https://pubchem.ncbi.nlm.nih.gov/compound/31357#section=Vapor-Pressure (64) NCBI. Pubchem-Tri-o-cresyl phosphate https://pubchem.ncbi.nlm.nih.gov/compound/6527#section=Top (65) NCBI. Pubchem-Triphenyl phosphate https://pubchem.ncbi.nlm.nih.gov/compound/8289#section=Top (66) NCBI. Pubchem-Tris(2,3-dibromopropyl) phosphate https://pubchem.ncbi.nlm.nih.gov/compound/31356#section=Top (67) NCBI. Pubchem-Tris(2-ethylhexyl) phosphate https://pubchem.ncbi.nlm.nih.gov/compound/6537#section=Top (68) Herman, J. P.; Cullinan, W. E. Neurocircuitry of stress: Central control of the hypothalamo-pituitary-adrenocortical axis. Trends Neurosci. 1997,20 (2), 78-84. (69) Baker, M. E.; Hardiman, G. Transcriptional analysis of endocrine disruption using zebrafish and massively parallel sequencing. J. Mol. Endocrinol. 2014,52 (3), R241-R256. (70) Zhang, J. Y.; Zhang, J.; Liu, R.; Gan, J.; Liu, J.; Liu, W. P. Endocrine-Disrupting Effects of Pesticides through Interference with Human Glucocorticoid Receptor. Environ. Sci. Technol. 2016,50 (1), 435-443. (71) Odermatt, A.; Gumy, C. Glucocorticoid and mineralocorticoid action: why should we consider influences by environmental chemicals? Biochem. Pharmacol. 2008,76 (10), 1184-1193. (72) Wang, G. W.; Du, Z. K.; Chen, H. Y.; Su, Y.; Gao, S. X.; Mao, L. Tissue-Specific Accumulation, Depuration, and Transformation of Triphenyl Phosphate (TPHP) in Adult Zebrafish (Danio rerio). Environ. Sci. Technol. 2016,50 (24), 13555-13564. (73) Wang, C.; Ruan, T.; Liu, J. Y.; He, B.; Zhou, Q. F.; Jiang, G. B. Perfluorooctyl Iodide Stimulates Steroidogenesis in H295R Cells via a Cyclic Adenosine Monophosphate Signaling Pathway. Chem. Res. Toxicol. 2015,28 (5), 848-854.
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Figures and Tables
576
Table 1 Antagonistic activities of OPFRs via GR and MR in the dual-luciferase reporter gene
577
assay MR
GR Chemicals
TNBP
RLA RIC20(mol/l)
RIC50(mol/l)
ND
ND
1.2×10
-6
TPHP
2.6×10
-6
TBOEP TCEP
TMPP
RLA LOEL(ppb)
(%) /
-6
4.4×10
45
/ 737
RIC20(mol/l)
RIC50(mol/l)
5.1×10-6
ND
9.5×10
-7 -7
ND
63
1631
7.9×10
ND
ND
/
/
ND
ND
ND
/
/
ND -6
LOEL(ppb) (%) 68
2663
38
368
ND
54
326
ND
/
/
ND
/
/
3.7×10
-6
TDCIPP
ND
ND
/
/
5.2×10
ND
76
4309
TDBPP
1.1×10-6
3.7×10-6
46
1395
7.5×10-7
2.2×10-6
32
698
TCIPP
ND
ND
/
/
ND
ND
/
/
TEHP
ND
ND
/
/
ND
ND
/
/
578
ND: not detected in the experimental concentrations.
579
RIC20/50: relative effective concentration, which means the concentrations of OPFRs inhibiting
580
20% or 50% of luciferase activities induced by hydrocortisone (HC, 50 nM) and aldosterone (AD,
581
0.1 nM) respectively.
582
RLA: relative luciferase activity; the highest inhibition effects of OPFRs in the experimental
583
concentrations compared with HC and AD which defined as 100%.
584
LOEL: The lowest observed effective level.
585
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586 587
Table 2 The production of cortisol and aldosterone in H295R cells treated with 5 µM of OPFRs
588
for 48 h Chemicals (5 µM)
Cortisol (ng/mL)
Aldosterone (ng/mL)
Control
19.67±1.53
0.75±0.08
TNBP
19.33±1.53
1.41±0.24* (↑)
TMPP
13.33±2.08* (↓)
0.69±0.10
TPHP
44.67±5.69* (↑)
1.91±0.08* (↑)
TBOEP
19.67±2.08
0.72±0.09
TCEP
19.67±1.53
0.74±0.04
TDCIPP
34.67±3.21* (↑)
1.77±0.09* (↑)
TDBPP
19.33±2.52
0.54±0.07* (↓)
TCIPP
20.33±3.06
0.71±0.06
TEHP
19.33±3.21
0.75±0.08
589
Results were presented as mean ± SD from three replicated samples. * indicated p