Potential Glucocorticoid and Mineralocorticoid Effects of Nine

Apr 21, 2017 - ... and Tissue-Specific Profiles of Polybrominated Diphenyl Ethers and Their Hydroxylated and Methoxylated Derivatives in Cats and Dogs...
0 downloads 0 Views 859KB Size
Subscriber access provided by ECU Libraries

Article

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

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Environmental Science & Technology is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 27

Environmental Science & Technology

1

Potential glucocorticoid and mineralocorticoid effects of

2

nine organophosphate flame retardants

3

Quan Zhang1,2, Jinghua Wang1, Jianqiang Zhu1, Jing Liu3, and Meirong Zhao1,2*

4

1. Key Laboratory of Microbial Technology for Industrial Pollution Control of

5

Zhejiang Province, College of Environment, Zhejiang University of Technology,

6

Hangzhou, Zhejiang, 310032, China

7

2. Department of Environmental Health, Harvard T.H. Chan School of Public Health,

8

Landmark Center West, Boston, MA, 02215, USA

9

3. College of Environmental and Resource Sciences, Zhejiang University, Hangzhou

10

310058, China

11 12 13 14 15 16 17

*To whom correspondence should be addressed. Phone: +86 571 8832 0265;

18

Fax: +86-571-88320265; E-mail: [email protected] (MR Zhao).

1

ACS Paragon Plus Environment

Environmental Science & Technology

19 20

TABLE OF CONTENT

21

2

ACS Paragon Plus Environment

Page 2 of 27

Page 3 of 27

Environmental Science & Technology

22 23

Abstract Organophosphate flame retardants (OPFRs), as alternatives of polybrominated

24

diphenyl ethers (PBDEs), have been frequently detected in the environment and biota,

25

and could pose adverse effects on organisms. However, information on the potential

26

endocrine disruption of OPFRs, especially their effects on steroid hormone receptors,

27

such as glucocorticoid and mineralocorticoid receptors (GR/MR), is limited. In this

28

study, the dual-luciferase reporter gene assay via GR/MR and a H295R

29

steroidogenesis assay were employed to evaluate the endocrine disruption of nine

30

OPFRs. We found TMPP, TPHP, and TDBPP exhibited both GR and MR antagonistic

31

activities, while TNBP and TDCIPP only showed MR antagonistic property within a

32

concentration range of 10-8 to 10-5 mol/L(M). In the H295R steroidogenesis assay, the

33

fold changes of eight steroidogenic genes in response to OPFRs were further studied.

34

We found CYP17,CYP21, and CYP11B1 expression were significantly

35

down-regulated following TMPP, TPHP, or TDBPP exposure at a concentration of

36

2×10-6 M. Meanwhile TMPP decreased the production of cortisol and TDBPP

37

down-regulated the secretion of aldosterone. Our results indicate that some OPFRs

38

can interact with GR and MR, and have the potential to disturb steroidogenesis. Data

39

provided here will be helpful to comprehensively understand the potential endocrine

40

disruption of OPFRs.

41

3

ACS Paragon Plus Environment

Environmental Science & Technology

42 43

Introduction Organophosphate flame retardants (OPFRs) have been the popular alternatives to

44

polybrominated diphenyl ethers (PBDEs) after their restrictive use in the United

45

States and Europe in the early 2000s.1With the increasing use of OPFRs, recent data

46

have shown that OPFRs are ubiquitous in air,2-5 water,6, 7 soil,8, 9 sediment,9, 10and

47

aquatic organisms,11-13 and they have also been detected in house dust,2, 4, 14-24

48

drinking water,25 and even in human milk13, 26 and urine.27-34 Studies have further

49

revealed that the OPFRs, especially tris(1,3-dichloro-2-propyl) phosphate (TDCIPP)

50

and triphenyl phosphate (TPHP), have been found in backpacking tents,35 hand

51

wipes,19, 21 silicone wristbands,36, 37and baby products,19, 21, 38, 39which have significant

52

implications on human exposures thru direct contact.

53

Recent studies have been conducted to evaluate the potential ecological and

54

health risks of OPFRs including ours in which we have found OPFRs induced anti/

55

estrogenic activity via estrogenic receptor α (ERα),40 and showed thyroid receptor β

56

(TRβ) antagonistic activity41 in the dual-luciferase reporter gene assays. Toxicological

57

studies have shown that OPFRs have potential endocrine disrupting effects via human

58

nuclear receptors,42 and could cause estrogen and thyroid disruption in zerbrafish.43, 44

59

Exposure to tris (2-butoxyethyl) phosphate (TBOEP) and TDCIPP may lead to

60

developmental malformations45 and neurotoxicity46 in zebrafish. Furthermore, Meeker

61

et al. revealed that bis(1,3-dichloro-2-propyl) phosphate (BDCPP) and diphenyl

62

phosphate (DPP), the urinary metabolites of TDCIPP and TPHP, were associated with 4

ACS Paragon Plus Environment

Page 4 of 27

Page 5 of 27

Environmental Science & Technology

63

male reproductive health to some degree.30 Thus, it is of vital importance to assess

64

the potential endocrine disrupting activity of OPFRs.

65

The estrogenic and thyroid hormones, which play essential roles in growth,

66

development and reproduction, have been reported to be disturbed by some OPFRs.

67

However, the effects of OPFRs on the glucocorticoid and mineralocorticoid hormones,

68

which are regulated by the hypothalamo–pituitary–adrenal (HPA) axis and are

69

extremely important in homeostasis, have not yet been elucidated.47 The HPA axis is a

70

central neuroendocrine system and its primary function is to mediate stress-associated

71

disorders such as chronic fatigue syndrome, melancholic depression and insomnia.48

72

Kojima et al. have reported that some OPFRs and their metabolites induced

73

antagonistic activities against human nuclear receptors including GR.42, 49 Several

74

studies have indicated that some OPFRs, such as TBOEP and TDCIPP, could change

75

the expression of genes involving with the GR and MR pathways in zebrafish

76

larvae.50, 51Further research manifests the alteration of cortisol in adult zebrafish under

77

long-term exposure to TPHP.52 However, neither MR agonistic/antagonistic effects

78

nor evaluating OPFRs in the H295R steroidogenesis assay for enzymatic activities of

79

steroidogenic genes,53 have been taken into account in those studies.

80

In this present study, we evaluated the glucocorticoid and mineralocorticoid

81

effects of nine OPFRs in dual-luciferase reporter gene assay. Furthermore, the levels

82

of cortisol and aldosterone, and the changes of genes in the pathways of

83

steroidogenesis synthesis were also examined in H295R cells. Data provided here will 5

ACS Paragon Plus Environment

Environmental Science & Technology

84

be helpful to evaluate the potential endocrine disruption effects of OPFRs.

85

Materials and methods

86

Chemicals and plasmids

87

Nine organophosphate flame retardants (OPFRs) were obtained from Dr.

88

Ehrenstorfer GmbH (Augsburg, Germany) and the details were listed in Table S1.

89

Hydrocortisone (HC; 98% pure) and aldosterone (AD; 97% pure) were both

90

purchased from J&K Scientific Ltd. (Beijing, China). All of the chemicals above were

91

dissolved in dimethyl sulfoxide (DMSO) obtained from Sigma-Aldrich (St. Louis,

92

MO, USA). The human glucocorticoid receptor α plasmid of pF25GFP-hGRα (GR)

93

and the response element plasmid of pMMTV-luc (MMTV) were kindly provided by

94

Dr. Evangelia Charmandari (Biomedical Research Foundation of the Academy in

95

Athens, Greece).The human mineralocorticoid receptor plasmid of EGFP-C1-hMR

96

(MR) was provided by Dr. Claudia Großmann (Julius Bernstein Institute for

97

Physiology, Martin Luther University, Germany). The internal control plasmid of

98

phRL-tk (TK) containing the Renilla luciferase gene was purchased from Promega

99

(Madison, WI, USA).

100 101

Cell Culture and Exposure medium Chinese hamster ovary K1 (CHO) cells were purchased from the Cell Bank of

102

Chinese Academy of Sciences (Shanghai, China). The cells were cultured in modified

103

RPMI medium (HyClone; Logan, UT, USA) with 10% fetal bovine serum (FBS;

104

HyClone) in a humidified atmosphere of 5% CO2 at 37 °C. Before transfection, the 6

ACS Paragon Plus Environment

Page 6 of 27

Page 7 of 27

Environmental Science & Technology

105

cells were cultured for 24 h in phenol red-free RPMI 1640 medium (Gibco, Grand

106

Island, NY, USA) supplemented with 5% charcoal−dextran stripped FBS (CD-FBS;

107

Gemini, USA).

108

The human adrenocortical carcinoma (H295R) cells were kindly provided by

109

Professor Qunfang Zhou (Research Center for Eco-Environmental Sciences, Chinese

110

Academy of Sciences). The cells were cultured in Dulbecco’s modified Eagle’s

111

medium/F12 (DMEM/F12; Hyclone) supplemented with 1% Ultroser G (Pall

112

Corporation, Port Washington, NY, USA), a serum substitute for animal cell culture,

113

1% insulin-transferrin-selenium (Gibco), 1% L-glutamine (Gibco), and 1%

114

penicillin-streptomycin (Gibco). For real-time polymerase chain reaction (RT-PCR)

115

and hormone measurement assays, the cells were incubated with phenol red-free

116

DMEM/F12 medium (Gibco) substituted with phenol red DMEM/F12 medium. Cells

117

were seeded in 6-well culture plates, and starved in exposure medium for 24 h and

118

then treated with the test chemicals.

119

MTS Assay

120

The cytotoxicity of nine OPFRs in CHO and H295R cells were assessed by Cell

121

Proliferation Assay (MTS assay; Promega, USA) as previously described.40 The cells

122

were grown in 96-well plates (Corning, NY, USA) containing 100 µl exposure

123

medium. The confluence of cells was approximately 80% and the density was

124

approximately 5000 cells/well. The cells were then treated with various

125

concentrations of test chemicals and the DMSO set (≤ 0.1% v/v) as the negative 7

ACS Paragon Plus Environment

Environmental Science & Technology

126

control for 24 h. The absorbance was measured at 490 nm by a microplate reader

127

(Infinite M200, Tecan, Switzerland). Only the non-cytotoxic concentrations were used

128

for following experiments.

129

Dual-Luciferase Reporter Gene Assays for GR and MR

130

The CHO cells were grown in a 96-well plate at a density of 20000 cells/well.

131

For GR activity, each well was transfected with a DNA mixture containing 10 ng of

132

GR, 120 ng of MMTV, and 10 ng of TK with 0.5 µl of transfection reagent

133

(Lipofectamine 2000; Invitrogen, MD, USA). For MR activity, 20 ng of MR, 160 ng

134

of MMTV, and 10 ng of TK were added to each well with 0.5 µl of transfection

135

reagent. After 4 h of transfection, the cells were changed with fresh exposure medium

136

overnight. Cells were then treated with various concentrations of test chemicals for

137

GR- or MR-mediated agonistic activities. For antagonistic activities, cells were

138

exposed to different concentrations of test chemicals in the presence of 50 nM HC or

139

0.1 nM AD.The activities of firefly luciferase and renilla luciferase were measured

140

witha fluorescence microplate reader (Infinite M200) following the protocol specified

141

in the dual-luciferase reporter assay kit (Promega, WI, USA). The results were

142

normalized to the renilla luciferase.

143

RNA Isolation and RT-PCR

144

H295R cells were seeded in a 6-well plate with 2 mL of exposure medium at a

145

density of 6×105 cells/well. The cells were then exposed to vehicle (≤ 0.1% v/v

146

DMSO), 0.1 µM, 1 µM or 2 µM of each chemical for24 h. After removal of the 8

ACS Paragon Plus Environment

Page 8 of 27

Page 9 of 27

Environmental Science & Technology

147

medium, the total RNA of the H295R cells was extracted using TRIzol reagent

148

(Takara, Otsu, Japan) following the manufacturer’s instructions. The RNA, in a

149

A260/280 ratio measured with a K5500 spectrophotometer (Kaiao, Beijing, China) in

150

the range of 1.8 to 2.0, was used for cDNA synthesis immediately or stored at -80 °C

151

until needed. The synthesis of cDNA was performed by a ReverTra Ace qPCR RT Kit

152

(Toyobo, Osaka, Japan) using a 2720 thermal cycler (Applied Biosystems, CA, USA).

153

The final cDNA samples were used immediately or stored at -20 °C. RT-PCR was

154

undertaken with a 7300 Real Time PCR System (Applied Biosystems, CA, USA)

155

using a SYBR Green Real-time PCR Master Mix Kit (Toyobo, Osaka, Japan). The

156

primer sequences of GAPDH, StAR, HMGR, 3βHSD2, CYP11A1, CYP11B1, CYP11B2,

157

CYP17, and CYP21 purchased from Sangon Biotech Co., Ltd. (Shanghai, China) are

158

shown in Table S2. The thermal cycle was as follows: 1 min at 95 °C, followed

159

closely by 40 cycles of 15 s at 95 °C, and 60 s at 60 °C. The expression of

160

steroidogenesis gene was normalized to the housekeeping gene

161

glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Fold change was calculated

162

with the 2-∆∆Ct method, as described by Livak andSchmittgen.54

163

Hormone Measurements

164

H295R cells were plated in 6-well plates at a density of 106 cells/well with 2.5

165

mL of medium. The medium was changed and the cells were exposed to vehicle (≤

166

0.1% v/v DMSO), or 5 µM of each chemical for48 h after the cells were attached to

167

the plate overnight. Then the medium was collected and stored at -80 °C for detection 9

ACS Paragon Plus Environment

Environmental Science & Technology

168

of cortisol and aldosterone. A radioimmunoassay kit (Beijing North Institute of

169

Biological Technology) was used for measuring the cortisol and aldosterone contents

170

according to the manufacturer’s protocol and three replicated samples were measured.

171

The limits of detection were 10 ng/mL for cortisol and 50 pg/mL for aldosterone. The

172

intra-assay coefficients of variation were below 10%.

173

Statistical Analysis

174

All results were analyzed using Microsoft excel and Origin 9.0 (OriginLab,

175

Northampton, MA). Data were presented as mean ± SD (standard deviation) for at

176

least three independent experiments with triplicates. Statistical significance was

177

determined using the Student’s t-test following a one-way ANOVA and the significant

178

difference was set at p< 0.05.

179

Results

180

Cytotoxicity of OPFRs on MTS assay

181

The cytotoxicity of the nine OPFRs against CHO cells was described in a

182

previous study,40 and here we tested the cell viability of H295R cells with various

183

concentrations of nine OPFRs (Figures S1). The concentrations (≤5×10-6 M for

184

TMPP, TPHP, TDBPP and ≤10-5 M for TNBP, TBOEP, TCEP, TDCIPP, TCIPP and

185

TEHP) that did not induce cytotoxicity were used for the further experiments.

186

Agonistic and antagonistic activities of the OPFRs against GR and MR

187

We fitted the dose response curve of HC or AD at various concentrations using

188

logistic model in order to calculate the EC20 from the fitting equation. As previously 10

ACS Paragon Plus Environment

Page 10 of 27

Page 11 of 27

Environmental Science & Technology

189

described, the EC20 value of HC for GR was 4.2 × 10−9 M from the dose response

190

curve.55 The EC20 value of AD for MR was estimated to be 3.1×10-11 M from the dose

191

response curve ranging from 10-13 M to 10-7 M, as shown in Figure S2. The

192

concentration from the curve, which reached the upper plateau was used for agonistic

193

activity, while the approximate EC80 value was used for antagonistic activity, as

194

previously reported.56 When the effect value of individual OPFRs in the presence with

195

positive control is below than 80% of effect value of positive control alone, that

196

specific OPFRs was considered as an antagonist in our study.

197

Although none of the nine OPFRs induced any agonistic activity against GR or

198

MR receptors (Figures S3 and S4), we found three and five OPFRs that have

199

antagonistic activities in the GR and MR transactivation assay, respectively (Figure

200

1). The relative effective concentrations, RIC20 and RIC50, levels of OPFRs inhibiting

201

20% or 50% of luciferase activities induced by 50 nM of HC and 0.1 nM of AD,

202

respectively, were both used for evaluating the antagonism of OPFRs via GR and MR

203

(Table 1). For GR antagonism (Figure 1A-1C and S5), TMPP, TPHP, and TDBPP

204

exhibited potent antagonistic activities in the following order, TDBPP > TMPP >

205

TPHP, with RIC20 values of 1.1×10-6, 1.2×10-6, and 2.6×10-6 M, respectively. In the

206

MR assay (Figure 1D-1H and S6), TNBP, TMPP, TPHP, TDCIPP, and TDBPP

207

showed the antagonistic properties and the corresponding RIC20 values were listed in

208

Table 1. Among those five OPFRs, TMPP, TPHP, and TDBPP induced significant

209

antagonistic activities with RIC20 values lower than 10-6M, and TNBP and TDCIPP 11

ACS Paragon Plus Environment

Environmental Science & Technology

210

showed weak antagonistic effects with RIC20 values higher than 5×10-6 M.

211

Steroidogenic genes expression profile of treated H295R cells

212

To further investigate the effects of OPFRs on corticosteroid homeostasis, eight

213

steroidogenic genes, StAR, HMGR, 3βHSD2, CYP11A1, CYP11B1, CYP11B2, CYP17,

214

and CYP21, involving in the principal pathways for synthesis of aldosterone and

215

cortisol in H295R cells were determined (Table S2). As shown in Figure 2, most of

216

the OPFRs treatments led to a significant decrease in the expression of genes. More

217

than three genes’ expression was modified by TMPP, TPHP, and TDBPP (Table S3

218

and Table S4). CYP17, CYP21, and CYP11B1 expression were down-regulated by

219

~34-97% following 2×10-6 M of TMPP, TPHP, or TDBPP exposure. When exposed to

220

2×10-6 M, TMPP up-regulated the HMGR expression, TPHP inhibited the expression

221

of StAR, 3βHSD2 andCYP11A1, and TDBPP decreased the expression of 3βHSD2,

222

CYP11A1, and CYP11B2. In addition, TDCIPP and TEHP both affected the expression

223

of three genes. TDCIPP induced a significant increase in CYP17 and CYP21, but

224

decreased the expression of CYP11B1 at 2×10-6 M. TEHP significantly up-regulated

225

3βHSD2 and CYP21, and maintained a repressive effect on StAR exposure to 2×10-6

226

M. TNBP, TBOEP, and TCEP showed a dominant inhibition in CYP17, CYP17, and

227

StAR, respectively, while TCIPP manifested great up-regulation in 3βHSD2

228

expression at the 2×10-6 M.

229

Effects on the production of cortisol and aldosterone

230

The levels of cortisol and aldosterone were measured to assess the effects of nine 12

ACS Paragon Plus Environment

Page 12 of 27

Page 13 of 27

Environmental Science & Technology

231

OPFRs on the production of steroid hormones in H295R cells when exposing to

232

5×10-6 M. Table 2 showed that TPHP and TDCIPP significantly increased the

233

production of cortisol by 2.27 and 1.76 folds, meanwhile TNBP, TPHP, and TDCIPP

234

increased the levels of aldosterone by 1.88, 2.55, and 2.36 folds, respectively.

235

Exposure to TMPP and TDBPP caused a decrease in cortisol by 0.68 fold and

236

aldosterone by 0.72 fold, respectively. Other OPFRs, however, did not change the

237

production of these two steroid hormones compared with control (≤ 0.1% v/v

238

DMSO).

239

Discussion

240

We have identified the GR and MR antagonistic activities of nine OPFRs in

241

dual-luciferase reporter gene assay and the modification of gene expression in the

242

pathways for the synthesis of cortisol and aldosterone using H295R cells. The results

243

indicated that TMPP, TPHP, and TDBPP could behave as GR and MR antagonists,

244

while TNBP and TDCIPP only exhibited MR antagonistic effects. The data from

245

RT-PCR showed that most of the genes involving in the synthesis of cortisol and

246

aldosterone were down-regulated under the exposure conditions. The productions of

247

steroid hormones including cortisol and aldosterone were also affected after exposure

248

to TNBP, TMPP, TPHP, TDCIPP, and TDBPP at 5×10-6 M. The results presented here

249

have enriched the evidence that OPFRs as a class of potent endocrine disrupting

250

chemicals (EDCs), and will be helpful for us to better understanding the ecological

251

and health risks of those emerging contaminates at a new sight. 13

ACS Paragon Plus Environment

Environmental Science & Technology

252

Abundant evidence from recent studies has shown that OPFRs are widely

253

detected in various environmental matrixes, consumer products, organisms, and

254

biological samples collected from individuals. Studies have demonstrated that human

255

has been exposed to OPFRs through dust ingestion57, dermal absorption58 and dietary

256

intake59. Hoffman et al. reported that the geometric mean of TPHP in house dust

257

collected from North Carolina was 1020 ppb (≈ng/g).21 TDCIPP and TCIPP, were

258

detected in children’s car seats and mattress.60 TPHP was also detected in perch at

259

levels of 21-180 ppb, and TNBP was found in human breast milk.13 Those residue

260

data not only have revealed the high levels of some OPFRs in the environment, but

261

should have aroused the concern due to the fact that some of the levels have exceeded

262

the lowest observed effect level (LOEL) as well. As defined in our reporter gene

263

assays, the value of LOEL is the lowest concentration leading to the inhibition of 20%

264

of luciferase activities and OPFR’s molecular weight. Although most of the OPFRs

265

residues reported by recent studies were still one to two orders of magnitude lower

266

than our LOEL as calculated here, it does not directly imply that those levels would

267

not impose the potential ecological and health risks. Two important factors should be

268

considered in assessing the environmental relevancy of the potential risks of those

269

emerging OPFRs. First of all, OPFRs had been found in our everyday environment,

270

including in the drinking water in which the total concentrations of OPFRs could be

271

as high as 1660 ng/L (median level at 48.7) ng/L reported in Korea61 and 325 ng/L in

272

China62. TPHP, as one of the most commonly found OPFRs in drinking water, had 14

ACS Paragon Plus Environment

Page 14 of 27

Page 15 of 27

Environmental Science & Technology

273

been identified as GR/MR mediated disruptor in our study. The ubiquity of OPFRs in

274

water indicated human especially susceptible sub-population has been exposed to

275

OPFRs more frequently than previously thought. Secondly, because several OPFRs

276

have relative high octanol-water partition coefficients (Kow) such as TNBP, TMPP,

277

TPHP, TDBPP and TEHP,63-67 OPFRs as a group are considered persistent, and will be

278

bio-accumulated in organisms as the result of prolong exposure. As being the

279

emerging contaminates, the usage of OPFRs will be increasing in the foreseeable

280

future in which will facilitate the increasing body burden of OPFRs in biota that will

281

trigger the potential adverse effects, such as the endocrine disrupting effects reported

282

here.

283

GR and MR, as two potential targets for EDCs, play vital roles in glucose

284

metabolism and electrolyte homeostasis.68-70 Several diseases, such as neurological

285

diseases and metabolic disorders, are linked to the perturbation of glucocorticoid and

286

mineralocorticoid activities.71A recent study has shown that OPFRs could be

287

transported to tissues, even in the brain of adult fish,72 suggesting the transportation

288

and bio-accumulation of OPFRs from the environment to organisms. Herein, we

289

reported the RIC20 of OPFRs via GR/MR, and data provided here would be used as

290

reference values for further study regard of biological effects and risks associated with

291

OPFRs exposure. Moreover, besides the GR and MR mediated effects, our previous

292

studies have shown that TPHP and TDCIPP could induce estrogenic and thyroid

293

antagonistic activities.40, 41 Considering multi-endocrine disrupting effects and the 15

ACS Paragon Plus Environment

Environmental Science & Technology

294

ubiquity of OPFRs in the environment, the potential ecological and health risks

295

associated with OPFRs exposure should be carefully examined without any further

296

delay.

297

Other than the dual-luciferase reporter gene assay, we used RT-PCR to assess the

298

change in the expression of related genes and to measure the corresponding steroid

299

hormones containing cortisol and aldosterone. In the H295R steroidogenesis pathway,

300

genes such as StAR, HMGR, CYP11A1, 3βHSD2, CYP21, CYP11B1 and CYP11B2,

301

involve in the synthesis of aldosterone, and CYP17 is associated with the synthesis of

302

cortisol.CYP17, CYP21, and CYP11B1 encodes members of the cytochrome P450

303

superfamily of enzymes.CYP17plays a key role in the conversion of pregnenolone and

304

progesterone to their 17-α-hydroxylated products, while CYP21 is related to carbon

305

metabolism, and cannot function without cortisol and aldosterone. As the last step in

306

the synthesis of steroids, CYP11B1 is responsible for catalyzing the transformation of

307

11-deoxycorticosterone and 11-deoxycortisol to corticosterone and cortisol,

308

respectively. In this study, we have shown that TDCIPP could activate the synthesis of

309

cortisol and aldosterone by up-regulating the expression of CYP17 and CYP21, which

310

play a role in the intermediate steps. This study also showed that reduction of

311

transcription of CYP17, CYP21 and CYP11B1 would probably decrease the

312

production of cortisol when treated with TMPP. Similarly, exposure to TDBPP

313

down-regulated the expression of CYP11A1, 3βHSD2, CYP21, CYP11B1 and

314

CYP11B2, so aldosterone production was decreased significantly. However, the 16

ACS Paragon Plus Environment

Page 16 of 27

Page 17 of 27

Environmental Science & Technology

315

hormones production increased when treated with TNBP and TPHP even though none

316

of genes were up-regulated significantly. Such discrepancies between the gene

317

expression and hormone production could be a consequence of the chemical

318

structures and properties or the other signaling pathways that they involve in H295R

319

cells.73 The underlying mechanism for this finding should be further elucidated in the

320

future studies.

321

In summary, we evaluated the potential endocrine disrupting effects mediated by

322

GR and MR for nine OPFRs in in vitro and in silico models for the first time. Among

323

those nine OPFRs, TMPP, TPHP, and TDBPP showed potential glucocorticoid

324

antagonistic activities and TNBP, TMPP, TPHP, TDCIPP, and TDBPP exhibited

325

potential mineralocorticoid antagonistic effects. The data provided in this paper is

326

significant from the perspective of comprehensive evaluation of the biological and

327

ecological risks of OPFRs.

328 329 330 331

Acknowledgements This study was funded by the National Natural Science Foundation of China (21377119, 21307109, and 21577129).

332 333

17

ACS Paragon Plus Environment

Environmental Science & Technology

334 335

Reference

336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374

(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

ACS Paragon Plus Environment

Page 18 of 27

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

ACS Paragon Plus Environment

Environmental Science & Technology

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

ACS Paragon Plus Environment

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

ACS Paragon Plus Environment

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

ACS Paragon Plus Environment

Page 22 of 27

Page 23 of 27

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.

23

ACS Paragon Plus Environment

Environmental Science & Technology

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

ACS Paragon Plus Environment

Page 25 of 27

Environmental Science & Technology

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