Potential Glucocorticoid and Mineralocorticoid Effects of Nine

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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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Environmental Science & Technology

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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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ACS Paragon Plus Environment


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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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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.

23



Page 24 of 27

574 575

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

24


Page 25 of 27


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

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