Chlorinated Polyfluoroalkylether Sulfonates Exhibit Similar Binding

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Ecotoxicology and Human Environmental Health

Chlorinated Polyfluoroalkylether Sulfonates Exhibit Similar Binding Potency and Activity to Thyroid Hormone Transport Proteins and Nuclear Receptors as Perfluorooctane Sulfonate Yan Xin, Xiao-Min Ren, Ting Ruan, Chuanhai Li, Liang-Hong Guo, and Guibin Jiang Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b01494 • Publication Date (Web): 27 Jul 2018 Downloaded from http://pubs.acs.org on July 28, 2018

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Environmental Science & Technology

1

Chlorinated Polyfluoroalkylether Sulfonates Exhibit Similar Binding Potency

2

and Activity to Thyroid Hormone Transport Proteins and Nuclear Receptors as

3

Perfluorooctane Sulfonate

4

5

Yan Xin1,2, Xiao-Min Ren1, Ting Ruan1, Chuan-Hai Li1,2, Liang-Hong Guo1,2,3*,

6

Guibin Jiang1,2

7 8

1

9

Center for Eco-environmental Sciences, Chinese Academy of Sciences, 18

State Key Laboratory of Environmental Chemistry and Eco-toxicology, Research

10

Shuangqing Road, Beijing 100085, China

11

2

12

Beijing 100039, China

13

3

14

China

College of Resources and Environment, University of Chinese Academy of Sciences,

The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou 510150,

15 16 17 18

*Corresponding author: Liang-Hong Guo, Email: [email protected]

19

Address correspondence to: Prof. Liang-Hong Guo, State Key Laboratory of

20

Environmental

21

Eco-environmental Sciences, Chinese Academy of Sciences, 18 Shuangqing Road,

22

Beijing 100085, China. Telephone/Fax: 86 10 62849685, Email: [email protected].

Chemistry

and

Eco-toxicology,

1

ACS Paragon Plus Environment

Research

Center

for


23

ABSTRACT

24

Chlorinated polyfluoroalkylether sulfonates (Cl-PFAESs) have been used as

25

perfluorooctane sulfonate (PFOS) alternatives in the chrome plating industry for years.

26

Although Cl-PFAESs have become ubiquitous environmental contaminants,

27

knowledge on their toxicological mechanism remains very limited. We compared

28

potential thyroid hormone (TH) disruption effects of Cl-PFAESs and PFOS via the

29

mechanisms of competitive binding to TH transport proteins and activation of TH

30

receptors (TRs). Fluorescence binding assays revealed that 6:2 Cl-PFAES, 8:2

31

Cl-PFAES and F-53B (a mixture of 6:2 and 8:2 Cl-PFAES) all interacted with a TH

32

transport protein transthyretin (TTR), with 6:2 Cl-PFAES showing the highest affinity.

33

It was also found that the chemicals interacted with TRs, with the affinity following

34

the order of 6:2 Cl-PFAES > PFOS > 8:2 Cl-PFAES. In reporter gene assays the

35

chemicals exhibited agonistic activity toward TRs, with the potency of 6:2 Cl-PFAES

36

comparable to that of PFOS. The chemicals also promoted GH3 cell proliferation,

37

with 6:2 Cl-PFAES displaying the highest potency. Molecular docking and molecular

38

dynamic simulation revealed that both Cl-PFAESs fit into the ligand binding pockets

39

of TTR and TRs with the binding modes similar to PFOS. Collectively, our results

40

demonstrate that Cl-PFAESs might cause TH disruption effects through competitive

41

binding to transport proteins and activation of TRs.

42 43

Keywords: Chlorinated polyfluoroalkylether sulfonates; F-53B; PFOS alternative;

44

Thyroid hormone transport protein; Thyroid hormone receptor

45 2


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For Table of Contents Only

48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 3



82

INTRODUCTION

83

F-53B, a commercial mixture of chlorinated polyfluoroalkylether sulfonates

84

(Cl-PFAESs), has been widely used in the Chinese metal plating industry since 1970s

85

as the only available mist suppressant in place of PFOS1. Although it has been used

86

for such a long period of time, attention to its environmental occurrence and behavior

87

has been rare until recent years. Detection of 6:2 Cl-PFAES and 8:2 Cl-PFAES has

88

been reported in various environmental matrices, wildlifes as well as human bodies in

89

China, with the concentration levels slightly lower than or comparable to PFOS2-13.

90

Cl-PFAESs were also detected in East Greenland wildlifes, indicating Cl-PFAESs

91

may have the capability of long-distance transport14. More importantly, human

92

exposure levels may gradually increase with the increasing demand for PFOS

93

alternatives in other sectors2. Toxicological studies reported in recent years

94

demonstrated that 6:2 Cl-PFAES was moderately toxic based on its acute toxicity to

95

zebrafish. Its LC50 (96 h) was 15.5 mg/L, which is essentially the same as that of

96

PFOS (17 mg/L)2. In additon, Cl-PFAESs were found to induce cardiac and

97

developmental toxicity in zebrafish embryos, neurotoxicity in rats, and bind to fatty

98

acid binding proteins directly15-17. Our previous study also demonstrated that both 6:2

99

Cl-PFAES and 8:2 Cl-PFAES, like PFOS, could interact and activate all the three

100

peroxisome proliferator-activated receptors (PPARs) and futher promote adipogenesis

101

in 3T3-L1 cells18. Apparently, although all the above studies have shown some toxic

102

effects of Cl-PFAES both in vitro and in vivo, knowledge on their toxicities and

103

particularly toxicity mechanisms is still very limited. 4


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Previous studies have reported that PFOS exposure might impair thyroid hormone

105

homeostasis19-21. Epidemiological results indicated that high concentrations of PFOS

106

in human serum were positively correlated with thyroid disease22. Studies on the

107

molecular mechanisms have identified two possible routes for the disruption of

108

thyroid functions by PFOS. In one mechanism, PFOS was found to bind to TH

109

receptors (TRs), activate their transcriptional activity in cells, and subsequebtly

110

change the expression of the genes related to thyroid functions in Xenopus laevis at

111

low concentrations23. In another mechanism, PFOS was able to bind to transthyretin

112

(TTR), a TH transport protein with relatively high affinity. This binding interaction

113

could interfer with TH transport in vivo by competitively displacing TH from

114

TTR24,25. Since Cl-PFAESs are structurally similar to PFOS, it is reasonable to

115

assume that PFOS alternative, Cl-PFAESs, might also exert disruption effects on

116

thyroid hormones like PFOS.

117

In the present study, we first measured the binding affinity of PFOS alternatives

118

(6:2 Cl-PFAES, 8:2 Cl-PFAES and F-53B) with TTR and TBG, and compared with

119

PFOS in their ability to interfering with TH transport. We then assessed the agonistic

120

activity of Cl-PFAESs on TRs and compared with PFOS by measuring their binding

121

affinity with TRs, their transcriptional activity in cells, and their effects on cell

122

proliferation. Furthermore, molecular docking and molecular dynamic (MD)

123

simulation were performed to compare the binding interactions of Cl-PFAESs and

124

PFOS with TH transport proteins and TRs.

125 5



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126

MATERIALS AND METHODS

127

Chemicals. PFOS (with purity > 95%) was purchased from Alfa Aesar (Ward Hill,

128

MA, USA). Commercial product F-53B (77.6% 6:2 Cl-PFAES, 6.0% 8:2 Cl-PFAES)

129

was purchased from Shanghai Synica Co., Ltd. The structures of Cl-PFAESs are

130

shown in the Supporting Information, Table S1. The molar concentration of F-53B

131

was calculated from its mass concentration by using the percentage-weighted

132

molecular weight of 6:2 Cl-PFAES and 8:2 Cl-PFAES in the commercial mixture.

133

The standards of 6:2 Cl-PFAES and 8:2 Cl-PFAES (with purity > 95%) were

134

laboratory-purified from F-53B according to the published procedure. All the stock

135

solutions of these chemicals were prepared in DMSO at a concentration of 50 mM.

136

Due to the hydrophobic nature of perfluorinated chemicals, aggregation into micelles

137

occurs in water beyond the critical micelle concentration (CMC) and could confound

138

exposures in aqueous medium. PFOS has been reported with a CMC value of 570

139

mg/L in pure water (USEPA)26. In the system containing 1% DMSO, the CMC values

140

of PFOS, 6:2 Cl-PFAES, 8:2 Cl-PFAES and F-53B were measured27, with 2.9, 5.6,

141

0.82 and 2.7 mM, respectively. In our study, laboratory-grade distilled water was used

142

for all solutions with the maximum concentrations derived from stock solutions no

143

greater than their CMC threshold. Occurrence of self-aggregation was not evident at

144

any

145

fluorescein-triiodothyronine (F-T3), were prepared in our laboratory according to a

146

method reported previously28. Human TH transport proteins TTR and TBG were

147

purchased from Calbiochem (San Diego, California, USA). Human TRα and TRβ

of

the

test

concentrations.

Fluorescein-thyroxine

6


(F-T4)

and

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148

ligand binding domains (LBDs) were prepared by Zhongding Biotechnology Co. Ltd.

149

(Nanjing, Jiangsu, China). All other reagents are of the highest purity available.

150

Cell culture. Human HEK 293 embryonal cells and GH3 rat pituitary cancer cells

151

were purchased from Cell Resource Center, IBMS, CAMS/PUMC (Beijing, China).

152

Dulbecco's modified eagle medium (DMEM) supplemented with 10% (v/v)

153

heat-inactivated fetal bovine serum (FBS, Gibco) and antibiotics (100 U/mL penicillin

154

and 100 µg/mL streptomycin, Gibco) was used to culture HEK 293 cells. Dulbecco's

155

Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12) supplemented with

156

10% (v/v) heat-inactivated FBS and antibiotics was used to culture GH3 cells. All

157

cells were cultured in a humidified atmosphere composed of 95% air and 5% CO2 at

158

37 °C.

159

Competitive fluorescence binding assay. A fluorescence polarization (FP) based

160

competitive binding assay was used to measure the binding affinities of 6:2

161

Cl-PFAES, 8:2 Cl-PFAES, F-53B and PFOS with TH transport proteins (TTR and

162

TBG) and TRs (TRα-LBD and TRβ-LBD) according to our previous study25,29. The

163

detailed procedure is provided in the Supporting Information.

164

TRs mediated luciferase reporter gene assay. The agonistic activity to TRs at the

165

cellular level was quantitatively determined using luciferase reporter gene assay as

166

described in our previous study18, with some modifications. The detailed procedure is

167

provided in the Supporting Information.

7



168

T-screen assay. The effects of Cl-PFAESs and PFOS on cell proliferation were

169

detected using the T-screen assay according to Ren et al29. The detailed experimental

170

procedures for T-screen assay are described in the Supporting Information.

171

Molecular docking analysis. We used AutoDock 4.2 (La Jolla, California, USA) to

172

determine the binding modes of PFOS and Cl-PFAESs to TTR, TRα and TRβ. The

173

detail of molecular docking is same as the description in a previous study25,29. The

174

detailed procedures and related parameters are described in the Supporting

175

Information.

176

Molecular dynamic simulation. The conformations of TTR, TRα and TRβ with

177

PFOS and Cl-PFAESs were further optimized using the molecular dynamic (MD)

178

simulation. Details of the MD simulation, binding pattern analysis and binding free

179

energy calculation are described in the Supporting Information.

180

Statistical analysis. All data in experiments are expressed as the mean ± standard

181

deviation (S.D.) (n = 3). Comparison of the mean values among experimental groups

182

was performed with one-way ANOVA tests using SPSS 17.0 software (Chicago,

183

Illinois, USA), with signiﬁcance level set at * p < 0.01.

184 185

RESULTS AND DISCUSSION

186

Binding potency of Cl-PFAESs and PFOS with TH transport proteins and

187

receptors. Binding potency of Cl-PFAESs and PFOS with TH transport proteins and

188

TH receptors was assessed and compared quantitatively using the FP based

189

competitive binding assay. The competition curves obtained from the assays are 8


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shown in Figure 1 and SI, Figure S1-S3. The calculated IC50 values of these chemicals

191

are listed in Table 1.

192

It can be seen from the data that PFOS could bind to TTR with an IC50 of 2.1 µM,

193

but did not show any appreciable binding to TBG (Table 1), which are consistent with

194

previous results24,25. The binding potency of 6:2 Cl-PFAES (IC50 4.8 µM) was slightly

195

weaker than PFOS, and the IC50 value of 8:2 Cl-PFAES exceeded 500 µM, suggesting

196

its binding potency is much weaker than PFOS. In addition, F-53B (IC50 7.3 µM)

197

showed weaker binding potency than 6:2 Cl-PFAES (SI, Figure S1). Like PFOS,

198

neither the two Cl-PFAES standards nor F-53B binds to TBG (SI, Figure S2), which

199

are similar to the reported results for the hydroxylated polychlorinated biphenyls

200

(PCBs)30.

201

In the TRα-LBD binding assay, PFOS displaced the binding of the F-T3 probe from

202

the protein in a dose-dependent manner (Figure 1), with an IC50 value of 16.0 µM

203

(Table 1). This is consistent with a previous report29. Compared with PFOS, 6:2

204

Cl-PFAES showed slightly higher binding potency with an IC50 value of 10.3 µM,

205

whereas 8:2 Cl-PFAES binds to TRα-LBD with much weaker potency (IC50 232.7

206

µM) (Table 1). For F-53B, its IC50 (8.4 µM) is even smaller than 6:2 Cl-PFAES.

207

Therefore, the order of the binding potency for these four compounds is F-53B > 6:2

208

Cl-PFAES > PFOS > 8:2 Cl-PFAES (Table 1). Similarly, all the four compounds

209

could also bind to TRβ-LBD (SI, Figure S3). But the order of binding potency is 6:2

210

Cl-PFAES > F-53B > PFOS > 8:2 Cl-PFAES (Table 1). After comparison, the

211

binding affinity of F-53B to TRα-LBD indicated that there may be a synergistic effect 9



212

between 6:2 Cl-PFAES and 8:2 Cl-PFAES. But, for such synergistic effect, we could

213

not give a definitive conclusion based on the current results. After all, there are other

214

unknown ingredients in F-53B except 6:2 Cl-PFAES and 8:2 Cl-PFAES. Using the

215

same method, our previous study demonstrated that both 6:2 Cl-PFAES and 8:2

216

Cl-PFAES exhibited stronger binding potencies toward PPARs than PFOS18. The

217

difference in binding potency of Cl-PFAESs toward TRs and PPARs may be due to

218

the specificity of binding pockets of different nuclear receptors. Besides, by

219

comparing IC50 values, we found that 6:2 Cl-PFAES may be more likely to bind to

220

TRs while 8:2 Cl-PFAES may prefer to bind to PPARs. To our knowledge, this is the

221

first study demonstrating that Cl-PFAESs could bind to TTR and TRs directly.

222

Activity of Cl-PFAESs and PFOS towards TRs. TRs-mediated luciferase reporter

223

assay was used to investigate the activity of Cl-PFAESs and PFOS on TRs. The

224

results are shown in Figure 2 and SI, Figure S4-S6. Triiodothyronine (T3), as a

225

positive control, effectively enhanced the TRs-mediated luciferase transcriptional

226

activity (SI, Figure S7). For the tested compounds, 6:2 Cl-PFAES, 8:2 Cl-PFAES,

227

F-53B and PFOS all enhanced the luciferase transcriptional activity in a

228

dose-dependent manner, suggesting they all have agonistic activity on TRα and TRβ.

229

More quantitatively, PFOS exposure induced approximately 1.6-fold enhancement of

230

TRα-mediated transcriptional activity at 25.0 µM, which is similar to previous result29.

231

Similar to PFOS, exposure to 6:2 Cl-PFAES and 8:2 Cl-PFAES exhibited

232

approximately 2.8 fold and 1.4 fold enhancement of transcriptional activity at 25 µM,

233

respectively (Figure 2). In addition, F-53B showed 2.7 fold activity enhancement at 10


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the same mass concentration as 6:2 Cl-PFAES, with 13.3 mg/L (equivalent to 25 µM

235

for 6:2 Cl-PFAES) (SI, Figure S5). As such, agonistic activity of 6:2 Cl-PFAES on

236

TRα was higher than PFOS, whereas 8:2 Cl-PFAES was the weakest among these

237

chemicals. Similarly, PFOS treatment also enhanced TRβ-mediated transcriptional

238

activity, with 1.7 fold increase at 25 µM (SI, Figure S4). In comparison, 6:2

239

Cl-PFAES showed similar agonistic effect while 8:2 Cl-PFAES showed weaker

240

agonistic effect for TRβ than PFOS (SI, Figure S4), and F-53B showed 2.2 fold

241

agonistic effect at the same mass concentration as 6:2 Cl-PFAES, with 13.3 mg/L

242

(equivalent to 25 µM for 6:2 Cl-PFAES) (SI, Figure S6). Compared with the results of

243

TR binding assays, there seems to exist a good correlation between TR binding

244

affinity and TR agonistic activity for the three purified standards. But, for the mixture

245

F-53B, the TR agonistic activity of F-53B is not exactly consistent with its binding

246

potency. We speculate that it may be related to the uptake of F-53B during cell

247

exposure. As a mixture, the uptake process of F-53B is extremely complicated, and

248

may be different from the standards. Nevertheless, we can confirm that F-53B

249

exhibited the TR agonistic activity, indicating its potential THs disruption effect.

250

Similar to TRs, our previous study found that Cl-PFAESs also exhibited obviously

251

agonistic effects toward PPARs signaling pathways.

252

Effects of Cl-PFAESs and PFOS on GH3 cell proliferation. Previous studies have

253

shown that GH3 cell proliferation is regulated by TRs31. We therefore investigated the

254

effects of Cl-PFAESs and PFOS on GH3 cell proliferation to further verify their

255

potential health risks. T3, as positive control, effectively enhanced the GH3 cell 11



256

proliferation in a dose-dependent manner (SI, Figure S8). Similarly, PFOS exposure

257

also increased GH3 cell proliferation in a dose-dependent manner (Figure 3), with

258

maximum enhancement of 1.5 fold at 12.5 µM, suggesting PFOS exposure

259

significantly affected cell function. All the three Cl-PFAESs also induced cell

260

proliferation effects in a dose-dependent manner, with 1.7 fold increase for 6:2

261

Cl-PFAES, 1.2 fold increase for 8:2 Cl-PFAES (Figure 3), and 1.4 fold enhancement

262

for F-53B (SI, Figure S9). Therefore, all the compounds promoted the GH3 cell

263

proliferation in the order of 6:2 Cl-PFAES > PFOS > F-53B > 8:2 Cl-PFAES.

264

Similarly, Deng and coworkers also reported that GH3 cell proliferation was

265

significantly enhanced by F-53B (refers to 6:2 Cl-PFAES in their study) in a

266

dose-dependent manner32.

267

Molecular docking of Cl-PFAESs with TTR and TRs. Molecular docking was

268

carried out for the binding interactions of Cl-PFAESs with TTR, TRα, and TRβ, and

269

comparison with PFOS was made to explore the structural basis for the similar

270

binding potency and activity between Cl-PFAESs and PFOS. Natural ligands T3 and

271

T4 were also included in the docking analysis for comparison. The structures of the

272

ligand-protein complexes obtained from the molecular docking analysis are shown in

273

Figure 4, and the amino acid residues of the protein participated in the binding

274

interactions with the ligand are listed in SI, Table S2.

275

As shown in SI, Figure S10, T4 was docked into the interior of the ligand binding

276

pocket in TTR with an orientation that is very similar to the configuration illustrated

277

in their crystal structure33. The hydrogen bonding interaction between T4 and LYS15 12


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of TTR was also observed (SI, Table S2). In the docked structures, 6:2 Cl-PFAES, 8:2

279

Cl-PFAES and PFOS could all fit into the TTR ligand-binding pocket and the sulfonic

280

acid groups form a hydrogen bond with LYS15, with the fluorinated carbon tail

281

adopting an extended conformation (Figure 4A). The structures are very similar with

282

each other, and also resemble the one with T4. Zhang’s study showed that PFOS

283

interacts with human TTR with the hydrophobic tail deep in the pocket and the head

284

forming salt bridge with LYS1534. Actually, their results are consistent with our

285

observation because the salt bridge is a type of hydrogen bond interaction between

286

pairs of oppositely charged groups35.

287

T3 was docked into TRα and TRβ with its polar substituent towards the inner part

288

of a binding pocket in TRs, forming hydrogen bonds with the residues of the receptors

289

(SI, Table S2) (ARG 228 in TRα, ARG 282, ARG 320, and HIS 435 in TRβ). The

290

docking results are in good agreement with the crystallographic structures36.

291

For the tested chemicals, 6:2 Cl-PFAES, 8:2 Cl-PFAES and PFOS all fit into the

292

binding pocket of TRα and TRβ. The negative charged end group is located near the

293

entrance of the ligand-binding pocket, and the hydrophobic part towards the interior

294

of the binding site (Figure 4B and 4C). In addition, all formed hydrogen bonds with

295

residues in the binding pocket region (SI, Table S2).

296

Molecular docking analysis revealed similar structural characteristics between

297

Cl-PFAESs and PFOS in their binding interactions with transport proteins and

298

hormone receptors. The docking results have provided additional evidence for the

299

relatively high affinity interactions of the compounds with TTR and TRs. 13



300

Furthermore, the docked structures can also illustrate why Cl-PFAESs act as TR

301

agonists. For a TR agonist, hydrogen bonding interactions with ARG 228 of TRα or

302

ARG 282 and ARG 320 of TRβ are critical for receptor activation36,37.

303

MD Simulations. The molecular interactions were further investigated by performing

304

MD simulations which could well describe the formed halogen bonds. The results of

305

root-mean-squared deviation (RMSD) indicated that the systems reached the

306

equilibrium state in the MD process, suggesting the conformations could be used to

307

perform the binding pattern analysis (SI, Figure S11-S13). The conformations and the

308

calculated binding free energies are list in SI, Figure S14 and Table S3.

309

Similar to T3 and T4, PFOS and Cl-PFAESs could insert into the binding pocket of

310

TTR and TRs with obviously similar patterns (SI, Figure S15). These conformations

311

are consistent with the results of molecular docking. From SI, Figure S14, we can

312

further find that 8:2 Cl-PFAES could not bind to the binding pocket as deeper as

313

PFOS or 6:2 Cl-PFAES. It may be related to the fact that the longer carbon chain of

314

8:2 Cl-PFAES caused larger steric hindrance. As fluorine atoms in PFOS and

315

Cl-PFAESs are likely to behave as hydrogen bond acceptors, they could form halogen

316

(fluorine) bonds. Taking these non-bond interactions together, among the three

317

compounds, 8:2 Cl-PFAES formed fewer non-bond interactions, resulting in a high

318

binding free energy of 8:2 Cl-PFAES (SI, Table S3). The weaker binding free energy

319

may explain why 8:2 Cl-PFAES exhibited the weakest binding potency in competitive

320

fluorescence binding assays.

14


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321

Toxicological and health risk implication of Cl-PFAESs. Previous studies have

322

proposed two potential mechanisms for the disruption effects of PFOS on THs, one by

323

interfering with TH transport24, and the other by activating TR-mediated

324

transcriptional pathways23. As two major TH transport proteins, TTR and TBG are

325

responsible for the primary transport of the THs including triiodothyronine (T3) and

326

thyroxine (T4)25. It has been reported that in humans, TBG distributes about 75% of

327

serum THs while TTR binds and transports 15-20% of serum THs38. In our previous

328

study, with a fluorescence-based competitive binding assay, we found that the

329

dissociation constants (Kd) values for T4 and T3 with TBG are 29 and 59 nM,

330

respectively, whereas Kd values for T4 and T3 with TTR are 239 and 1472 nM,

331

respectively, suggesting TBG has a stronger binding potency to THs than TTR39. It is

332

for these reasons that TBG is sometimes referred to as the “most important” TH

333

distributor in human blood-stream40. Due to species difference, in rodents it is TTR

334

that is mostly responsible for TH transport in blood41. If a chemical can bind to TTR

335

or TBG with appreciable affinity, it might be able to displace TH from the protein in

336

plasma by competitive binding, thereby interfering with TH transport. Our results

337

demonstrated that Cl-PFAESs as well as PFOS are more likely to bind to TTR than to

338

TBG. Bearing this limitation in mind, we made a simplified estimation for the

339

likelihood of TH displacement by Cl-PFAESs in humans. In a previous study, it was

340

estimated that for occupationally exposed workers almost all of TTR are occupied by

341

PFOS, and competitive displacement of T4 from TTR by PFOS could not be neglected.

342

In the current study, we found that the compound showing the highest protein binding 15



343

affinity was 6:2 Cl-PFAES, which could bind to TTR with an IC50 of 4.8 µM,

344

although approximately 2-fold weaker than PFOS (IC50 2.1 µM). According to

345

previous reports, the median concentrations of PFOS and 6:2 Cl-PFAES

346

concentrations in occupationally exposed workers were 4.0 µM42,43 and 0.1 µM11,

347

respectively. Using the same estimation method25, it can be said that competitive

348

displacement of T4 from TTR by Cl-PFAESs is unlikely to occur at current exposure

349

levels even for the highly exposed populations, and Cl-PFAESs would not interfere

350

with TH transport. However, due to the persistent and bioaccumulative properties of

351

the perfluorinated compounds, Cl-PFAESs might interfere with TH transport if their

352

plasma levels increase as a result of long-term exposure.

353

As described in the introduction, previous studies indicated that 6:2 Cl-PFAES

354

exhibited higher developmental toxicity, hepatic cytotoxicity and neurotoxicity than

355

PFOS15-18. Similarly, after investigating the possibility of Cl-PFAESs disruption

356

effect on THs, we found that 6:2 Cl-PFAES exhibits clearly higher activity on

357

TR-mediated pathways than PFOS. F-53B, as a commercial mixture of 6:2 Cl-PFAES

358

and 8:2 Cl-PFAES with the former as the major component, showed TR agonistic

359

activity similar to PFOS. In Deng’s study32, using zebrafish larvae as a model species,

360

the changes in thyroid hormone level and expression of THs related genes were

361

studied after exposure to F-53B (which refers to 6:2 Cl-PFAES). The results showed

362

that 6:2 Cl-PFAES exposure induced significant developmental inhibition and

363

increased thyroxine (T4) level accompanied by an increase in TTR protein and

364

transcript levels of most genes involved in the hypothalamic-pituitary-thyroid (HPT) 16


Page 16 of 30

Page 17 of 30


365

axis. These in vivo data strongly support our results.

366

In conclusion, in this study we investigated the possibility of Cl-PFAESs disruption

367

effects on THs by the mechanism of competitive binding with TH transport proteins

368

and by the mechanism of activating TR-mediated transcriptional pathways. We found

369

that even though 6:2 Cl-PFAES has significantly higher binding affinity for TTR than

370

8:2 Cl-PFAES, its interference on TH transport in vivo is unlikely because of the

371

relatively low exposure levels at present. However, if the exposure increases to the

372

level of PFOS, its effects on TH transport would become significant. We also found

373

that Cl-PFAESs bind to TRs with relatively high affinity, exhibited agonistic activity

374

toward TRs pathways, and promoted GH3 cell proliferation. The results suggest that

375

these compounds might be able to disrupt TH functions by acting on TR-mediated

376

signaling pathways. Actually, in the commercial product F-53B, there are close to

377

16.0% unknown components except 6:2 Cl-PFAES and 8:2 Cl-PFAES. It is still

378

unclear whether these unknown components would also cause thyroid hormone

379

disruption effect. Nevertheless, the major component 6:2 Cl-PFAES has been shown

380

to have a higher agonistic activity on TRs than PFOS in our study, suggesting that the

381

Cl-PFAESs might not be safe alternatives of PFOS and deserve further evaluation.

382 383 384 385 386 17



387 388

Notes The authors declare that there are no conflicts of interest.

389 390

ACKNOWLEDGMENTS

391

This work was supported by the Chinese Academy of Sciences (XDB14040100,

392

QYZDJ-SSW-DQC020), the National Natural Science Foundation of China

393

(91543203, 21621064, 21577151 and 21777187) and the Royal Society International

394

Collaboration Award for Research Professors (IC160121).

395 396

ASSOCIATED CONTENT

397

Supporting Information. The Supporting Information is available free of charge on

398

the ACS Publications website.

399

Details of competitive fluorescence binding assay, luciferase reporter gene assay,

400

T-screen assay, molecular docking and molecular dynamic simulation, and additional

401

figures and tables.

402 403 404 405 406 407 408 409 410 18


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REFERENCES

412

(1) Yan, X.; Liu, X.; Qi, C.; Wang, D.; Lin, C. Mechanochemical destruction of a chlorinated

413

polyfluorinated ether sulfonate (F-53B, a PFOS alternative) assisted by sodium persulfate. RSC

414

Adv. 2015, 5 (104), 85785-85790.

415

(2) Wang, S.; Huang, J.; Yang, Y.; Hui, Y.; Ge, Y.; Larssen, T.; Yu, G.; Deng, S.; Wang, B.; Harman, C.

416

First report of a Chinese PFOS alternative overlooked for 30 years: its toxicity, persistence, and

417

presence in the environment. Environ. Sci. Technol. 2013, 47 (18), 10163-10170.

418

(3) Lin, Y.; Liu, R.; Hu, F.; Liu, R.; Ruan, T.; Jiang, G. Simultaneous qualitative and quantitative

419

analysis of fluoroalkyl sulfonates in riverine water by liquid chromatography coupled with Orbitrap

420

high resolution mass spectrometry. J. Chromatogr. A 2016, 1435, 66-74.

421

(4) Wang, T.; Vestergren, R.; Herzke, D.; Yu, J.; Cousins, I. T. Levels, isomer profiles, and estimated

422

riverine mass discharges of perfluoroalkyl acids and fluorinated alternatives at the mouths of Chinese

423

rivers. Environ. Sci. Technol. 2016, 50 (21), 11584-11592.

424

(5) Ruan, T.; Lin, Y.; Wang, T.; Liu, R.; Jiang, G. Identification of novel polyfluorinated ether

425

sulfonates as PFOS alternatives in municipal sewage sludge in China. Environ. Sci. Technol. 2015, 49

426

(11), 6519-6527.

427

(6) Gomis, M. I.; Wang, Z.; Scheringer, M.; Cousins, I. T. A modeling assessment of the

428

physicochemical properties and environmental fate of emerging and novel per-and polyfluoroalkyl

429

substances. Sci. Total. Environ. 2015, 505, 981-991.

430

(7) Zhang, Y.; Wang, B.; Wang, W.; Li, W.; Huang, J.; Deng, S.; Wang, Y.; Yu, G. Occurrence and

431

source apportionment of Per-and poly-fluorinated compounds (PFCs) in North Canal Basin, Beijing.

432

Sci. Rep. 2016, 6, 36683.

19



433

(8) Liu, W.; Qin, H.; Li, J.; Zhang, Q.; Zhang, H.; Wang, Z.; He, X. Atmospheric chlorinated

434

polyfluorinated ether sulfonate and ionic perfluoroalkyl acids in 2006 to 2014 in Dalian, China.

435

Environ. Toxicol. Chem. 2017, 36 (10), 2581-2586.

436

(9) Shi, Y.; Vestergren, R.; Zhou, Z.; Song, X.; Xu, L.; Liang, Y.; Cai, Y. Tissue distribution and whole

437

body burden of the chlorinated polyfluoroalkyl ether sulfonic acid F-53B in crucian carp (Carassius

438

carassius): Evidence for a highly bioaccumulative contaminant of emerging concern. Environ. Sci.

439

Technol. 2015, 49 (24), 14156-14165.

440

(10) Liu, Y.; Ruan, T.; Lin, Y.; Liu, A.; Yu, M.; Liu, R.; Meng, M.; Wang, Y.; Liu, J.; Jiang, G.

441

Chlorinated polyfluoroalkyl ether sulfonic acids in marine organisms from Bohai sea, China:

442

occurrence, temporal variations, and trophic transfer behavior. Environ. Sci. Technol. 2017, 51 (8),

443

4407-4414.

444

(11) Shi, Y.; Vestergren, R.; Xu, L.; Zhou, Z.; Li, C.; Liang, Y.; Cai, Y. Human exposure and

445

elimination kinetics of chlorinated polyfluoroalkyl ether sulfonic acids (Cl-PFESAs). Environ. Sci.

446

Technol. 2016, 50 (5), 2396-2404.

447

(12) Gao, K.; Gao, Y.; Li, Y.; Fu, J.; Zhang, A. A rapid and fully automatic method for the accurate

448

determination of a wide carbon-chain range of per-and polyfluoroalkyl substances (C4–C18) in human

449

serum. J. Chromatogr. A 2016, 1471, 1-10.

450

(13) Chen, F.; Yin, S.; Kelly, B. C.; Liu, W. Chlorinated polyfluoroalkyl ether sulfonic acids in

451

matched maternal, cord, and placenta samples: A study of transplacental transfer. Environ. Sci. Technol.

452

2017, 51, 6387−6394.

20


Page 20 of 30

Page 21 of 30


453

(14) Gebbink, W. A.; Bossi, R.; Rigét, F. F.; Rosing-Asvid, A.; Sonne, C.; Dietz, R. Observation of

454

emerging per-and polyfluoroalkyl substances (PFASs) in Greenland marine mammals. Chemosphere

455

2016, 144, 2384-2391.

456

(15) Shi, G.; Cui, Q.; Pan, Y.; Sheng, N.; Sun, S.; Guo, Y.; Dai, J. 6:2 Chlorinated polyfluorinated ether

457

sulfonate, a PFOS alternative, induces embryotoxicity and disrupts cardiac development in zebrafish

458

embryos. Aquat. Toxicol. 2017, 185, 67-75.

459

(16) Zhang, Q.; Liu, W.; Niu, Q.; Wang, Y.; Zhao, H.; Zhang, H.; Song, J.; Tsuda, S.; Saito, N. Effects

460

of perfluorooctane sulfonate and its alternatives on long-term potentiation in the hippocampus CA1

461

region of adult rats in vivo. Toxicol. Res. 2016, 5 (2), 539-546.

462

(17) Sheng, N.; Cui, R.; Wang, J.; Guo, Y.; Wang, J.; Dai, J. Cytotoxicity of novel fluorinated

463

alternatives to long-chain perfluoroalkyl substances to human liver cell line and their binding capacity

464

to human liver fatty acid binding protein. Arch. Toxicol. 2017, 1-11.

465

(18) Li, C.; Ren, X.; Ruan, T.; Cao, L.; Xin, Y.; Guo, L.; Jiang, G. Chlorinated polyfluorinated ether

466

sulfonates exhibit higher activity toward peroxisome proliferator-activated receptors signaling

467

pathways than perfluorooctanesulfonate. Environ. Sci. Technol. 2018, 52 (5), 3232-3239.

468

(19) Lau, C.; Thibodeaux, J. R.; Hanson, R. G.; Rogers, J. M.; Grey, B. E.; Stanton, M. E.; Butenhoff, J.

469

L.; Stevenson, L. A. Exposure to perfluorooctane sulfonate during pregnancy in rat and mouse. II:

470

postnatal evaluation. Toxicol. Sci. 2003, 74 (2), 382-392.

471

(20) Luebker, D. J.; Case, M. T.; York, R. G.; Moore, J. A.; Hansen, K. J.; Butenhoff, J. L.

472

Two-generation reproduction and cross-foster studies of perfluorooctanesulfonate (PFOS) in rats.

473

Toxicology 2005, 215 (1), 126-148.

21



474

(21) Seacat, A. M.; Thomford, P. J.; Hansen, K. J.; Clemen, L. A.; Eldridge, S. R.; Elcombe, C. R.;

475

Butenhoff, J. L. Sub-chronic dietary toxicity of potassium perfluorooctanesulfonate in rats. Toxicology

476

2003, 183 (1), 117-131.

477

(22) Melzer, D.; Rice, N.; Depledge, M. H.; Henley, W. E.; Galloway, T. S. Association between serum

478

perfluorooctanoic acid (PFOA) and thyroid disease in the US National Health and Nutrition

479

Examination Survey. Environ. Health Persp. 2010, 118 (5), 686.

480

(23) Cheng, Y.; Cui, Y.; Chen, H.; Xie, W. Thyroid disruption effects of environmental level

481

perfluorooctane sulfonates (PFOS) in Xenopus laevis. Ecotoxicology 2011, 20 (8), 2069.

482

(24) Weiss, J. M.; Andersson, P. L.; Lamoree, M. H.; Leonards, P. E.; van Leeuwen, S. P.; Hamers, T.

483

Competitive binding of poly-and perfluorinated compounds to the thyroid hormone transport protein

484

transthyretin. Toxicol. Sci. 2009, 109 (2), 206-216.

485

(25) Ren, X.; Qin, W.; Cao, L.; Zhang, J.; Yang, Y.; Wan, B.; Guo, L. Binding interactions of

486

perfluoroalkyl substances with thyroid hormone transport proteins and potential toxicological

487

implications. Toxicology 2016, 366, 32-42.

488

(26) U.S. Environmental Protection Agency. Perfluorooctyl sulfonates; proposed significant new use

489

rule. Fed Reg 65(202). 2000, 40 CFR Part 721, 62319–62333.

490

(27) Domínguez, A.; Fernández, A.; González, N.; Iglesias, E.; Montenegro, L. Determination of

491

critical micelle concentration of some surfactants by three techniques. J. Chem. Educ. 1997, 74 (10),

492

1227-1231.

493

(28) Smith, D. Enhancement fluoroimmunoassay of thyroxine. FEBS Lett. 1977, 77 (1), 25-27.

22


Page 22 of 30

Page 23 of 30


494

(29) Ren, X.; Zhang, Y.; Guo, L.; Qin, Z.; Lv, Q.; Zhang, L. Structure–activity relations in binding of

495

perfluoroalkyl compounds to human thyroid hormone T3 receptor. Arch. Toxicol. 2015, 89 (2),

496

233-242.

497

(30) Hallgren, S.; Darnerud, P. O. Polybrominated diphenyl ethers (PBDEs), polychlorinated biphenyls

498

(PCBs) and chlorinated paraffins (CPs) in rats—testing interactions and mechanisms for thyroid

499

hormone effects. Toxicology 2002, 177 (2-3), 227-243.

500

(31) Ghisari, M.; Bonefeld-Jorgensen, E. C. Impact of environmental chemicals on the thyroid

501

hormone function in pituitary rat GH3 cells. Mol. Cell. Endocrinol. 2005, 244 (1), 31-41.

502

(32) Deng, M.; Wu, Y.; Xu, C.; Jin, Y.; He, X.; Wan, J.; Yu, X.; Rao, H.; Tu, W. Multiple approaches to

503

assess the effects of F-53B, a Chinese PFOS alternative, on thyroid endocrine disruption at

504

environmentally relevant concentrations. Sci. Total Environ. 2018, 624, 215-224.

505

(33) Wojtczak, A.; Neumann, P.; Cody, V. Structure of a new polymorphic monoclinic form of human

506

transthyretin at 3 Å resolution reveals a mixed complex between unliganded and T4-bound tetramers of

507

TTR. Acta Crystallogr. 2001, 57 (7), 957-967.

508

(34) Zhang, J.; Begum, A.; Brännström, K.; Grundström, C.; Iakovleva, I.; Olofsson, A.;

509

Sauer-Eriksson, A. E.; Andersson, P. L. Structure-based virtual screening protocol for in silico

510

identification of potential thyroid disrupting chemicals targeting transthyretin. Environ. Sci. Technol.

511

2016, 50 (21), 11984-11993.

512

(35) Bissantz, C.; Kuhn, B.; Stahl, M. A medicinal chemist’s guide to molecular interactions. J. Med.

513

Chem. 2010, 53 (14), 5061-5084.

514

(36) Wagner, R. L.; Apriletti, J. W.; McGrath, M. E.; West, B. L.; Baxter, J. D.; Fletterick, R. J. A

515

structural role for hormone in the thyroid hormone receptor. Nature 1995, 378 (6558), 690-697. 23



516

(37) Apriletti, J. W.; Ribeiro, R. C.; Wagner, R. L.; Feng, W.; Webb, P.; Kushner, P. J.; West, B. L.;

517

Nilsson, S.; Scanlan, T. S.; Fletterick, R. J. Molecular and structural biology of thyroid hormone

518

receptors. Clin. Exp. Pharmacol. P. 1998, 25 (S1), S2-S11.

519

(38) Hagen, G. A.; Elliott, W. J. Transport of thyroid hormones in serum and cerebrospinal fluid. J.

520

Clin. Endocr. Metab. 1973, 37 (3), 415-422.

521

(39) Ren, X.; Guo, L. Assessment of the binding of hydroxylated polybrominated diphenyl ethers to

522

thyroid hormone transport proteins using a site-specific fluorescence probe. Environ. Sci. Technol. 2012,

523

46 (8), 4633-4640.

524

(40) Richardson, S. J.; Wijayagunaratne, R. C.; D'Souza, D. G.; Darras, V. M.; Van Herck, S. L.

525

Transport of thyroid hormones via the choroid plexus into the brain: the roles of transthyretin and

526

thyroid hormone transmembrane transporters. Front. Neurosci. 2015, 9, 66.

527

(41) Cheek, A. O.; Kow, K.; Chen, J.; McLachlan, J. A. Potential mechanisms of thyroid disruption in

528

humans: interaction of organochlorine compounds with thyroid receptor, transthyretin, and

529

thyroid-binding globulin. Environ. Health Persp. 1999, 107 (4), 273.

530

(42) Chang, E. T.; Adami, H. O.; Boffetta, P.; Cole, P.; Starr, T. B.; Mandel, J. S. A critical review of

531

perfluorooctanoate and perfluorooctanesulfonate exposure and cancer risk in humans. Crit. Rev. Toxicol.

532

2014, 44 (S1), 1-81.

533

(43) Fromme, H.; Tittlemier, S. A.; Völkel, W.; Wilhelm, M.; Twardella, D. Perfluorinated

534

compounds–exposure assessment for the general population in Western countries. Int. J. Hyg. Envir.

535

Heal. 2009, 212 (3), 239-270.

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Figure Legends

539

Figure 1. Competitive fluorescence binding curves of PFOS, 6:2 Cl-PFAES, 8:2

540

Cl-PFAES and F-53B to TRα-LBD. The error bar represents the standard deviation

541

of three independent measurements.

542 543

Figure 2. Activities of PFOS, 6:2 Cl-PFAES and 8:2 Cl-PFAES toward TRα. The

544

error bar represents the standard deviation of three independent measurements.

545 546

Figure 3. Effects of PFOS, 6:2 Cl-PFAES and 8:2 Cl-PFAES on GH3 cell

547

proliferation. The error bar represents the standard deviation of three independent

548

measurements.

549 550

Figure 4. Molecular docking results of PFOS, 6:2 Cl-PFAES and 8:2 Cl-PFAES with

551

TH transport protein (TTR) and TH receptors (TRα, TRβ). (A) PFOS, 6:2 Cl-PFAES

552

and 8:2 Cl-PFAES with TTR. (B) PFOS, 6:2 Cl-PFAES and 8:2 Cl-PFAES with TRα.

553

(C) PFOS, 6:2 Cl-PFAES and 8:2 Cl-PFAES with TRβ. In the docking results, TTR,

554

TRα and TRβ are represented in blue whereas ligands are represented in other colors.

555 556 557 558 559 25



Page 26 of 30

560

Table 1. IC50 values of Cl-PFAESs and PFOS with TH transport proteins (TTR,

561

TBG) and TH receptors (TRα-LBD, TRβ-LBD) by competitive fluorescence binding

562

assays. TRα

TRβ

TTR

TBG

PFOS (µM)

16.0±0.4

20.5±0.6

2.1±0.2

ND

6:2 Cl-PFAES (µM)

10.3±0.3

7.1±0.4

4.8±0.7

ND

8:2 Cl-PFAES (µM)

232.7±2.6

>500

>500

ND

F-53B (µM)

8.4±1.8

12.9±1.3

7.3±0.9

ND

563

26


Page 27 of 30


Figure 1. Competitive fluorescence binding curves of PFOS, 6:2 Cl-PFAES, 8:2 Cl-PFAES and F-53B to TRαLBD. The error bar represents the standard deviation of three independent measurements. 47x36mm (600 x 600 DPI)



Figure 2. Activities of PFOS, 6:2 Cl-PFAES and 8:2 Cl-PFAES toward TRα. The error bar represents the standard deviation of three independent measurements. 45x36mm (600 x 600 DPI)


Page 28 of 30

Page 29 of 30


Figure 3. Effects of PFOS, 6:2 Cl-PFAES and 8:2 Cl-PFAES on GH3 cell proliferation. The error bar represents the standard deviation of three independent measurements. 45x36mm (600 x 600 DPI)



Figure 4. Molecular docking results of PFOS, 6:2 Cl-PFAES and 8:2 Cl-PFAES with TH transport protein (TTR) and TH receptors (TRα, TRβ). (A) PFOS, 6:2 Cl-PFAES and 8:2 Cl-PFAES with TTR. (B) PFOS, 6:2 Cl-PFAES and 8:2 Cl-PFAES with TRα. (C) PFOS, 6:2 Cl-PFAES and 8:2 Cl-PFAES with TRβ. In the docking results, TTR, TRα and TRβ are represented in blue whereas ligands are represented in other colors. 38x26mm (600 x 600 DPI)


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Chlorinated Polyfluoroalkylether Sulfonates Exhibit Similar Binding

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