Chlorinated Polyfluorinated Ether Sulfonates Exhibit Higher Activity

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Chlorinated Polyfluorinated Ether Sulfonates Exhibit Higher Activity towards Peroxisome Proliferator-Activated Receptors Signaling Pathways than Perfluorooctane Sulfonate Chuan-Hai Li, Xiao-Min Ren, Ting Ruan, Lin-Ying Cao, Yan Xin, Liang-Hong Guo, and Guibin Jiang Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b06327 • Publication Date (Web): 01 Feb 2018 Downloaded from http://pubs.acs.org on February 2, 2018

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Environmental Science & Technology

1

Chlorinated Polyfluorinated Ether Sulfonates Exhibit Higher Activity towards

2

Peroxisome

3

Perfluorooctane Sulfonate

Proliferator-Activated

Receptors

Signaling

Pathways

than

4 5

Chuan-Hai Li1,2, Xiao-Min Ren1*, Ting Ruan1, Lin-Ying Cao1,2, Yan Xin1,2,

6

Liang-Hong Guo1,2*, 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, P. R. China

11

2

12

Beijing 100039, P. R. China

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

13 14 15

Corresponding authors:

16

Xiao-Min Ren, Email: [email protected]

17

Liang-Hong Guo, Email: [email protected]

18 19

Address correspondence to Liang-Hong Guo, State Key Laboratory of Environmental

20

Chemistry and Eco-toxicology, Research Center for Eco-environmental Sciences,

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Chinese Academy of Sciences, 18 Shuangqing Road, P.O. Box 2871, Beijing 100085,

22

P. R. China. Telephone/Fax: 86 010 62849685. E-mail: [email protected]

23

1

ACS Paragon Plus Environment


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ABSTRACT

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Chlorinated polyfluorinated ether sulfonates (Cl-PFAESs) are the alternative

26

products of perfluorooctane sulfonate (PFOS) in the metal plating industry in China.

27

The similarity in chemical structures between Cl-PFAESs and PFOS makes it

28

reasonable to assume they possess similar biological activities. In the present study,

29

we investigated whether Cl-PFAESs could induce cellular effects through peroxisome

30

proliferator-activated receptors (PPARs) signaling pathways like PFOS. By using

31

fluorescence competitive binding assay, we found two dominant Cl-PFAESs (6:2

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Cl-PFAES and 8:2 Cl-PFAES) bound to PPARs with affinity higher than PFOS. Based

33

on the luciferase reporter gene transcription assay, the two Cl-PFAESs also showed

34

agonistic activity towards PPARs signaling pathways with potency similar to (6:2

35

Cl-PFAES) or higher than (8:2 Cl-PFAES) PFOS. Molecular docking simulation

36

showed the two Cl-PFAESs fitted into the ligand binding pockets of PPARs with very

37

similar binding mode as PFOS. The cell function results showed Cl-PFAESs

38

promoted the process of adipogenesis in 3T3-L1 cells with potency higher than PFOS.

39

Taken together, we found for the first time that Cl-PFAESs have the ability to

40

interfere with PPARs signaling pathways, and current exposure level of 6:2 Cl-PFAES

41

in occupational workers has exceeded the margin of safety. Our study highlights the

42

potential health risks of Cl-PFAESs as PFOS alternatives.

43 44

KEY WORDS: PFOS alternative; F-53B; 6:2 Cl-PFAES; 8:2 Cl-PFAES; PPARs;

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agonistic activity; adipogenesis 2


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For Table of Contents Only

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49 50 51 52 53 54 55

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INTRODUCTION

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Perfluorooctane sulfonate (PFOS) has been widely used in industrial and

58

consumer products in the past few decades1. Due to environmental and human health

59

concerns, PFOS production was voluntarily phased out by major manufacturers in

60

2002, and has also been regulated under the Stockholm Convention on Persistent

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Organic Pollutants2. Consequently, some PFOS alternatives with similar structures

62

and physicochemical properties have been increasingly produced and extensively

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used3, 4. Chlorinated polyfluoroalkyl ether sulfonates (Cl-PFAESs), with a commercial

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name of F-53B, are one class of PFOS alternatives. The major component of F-53B is

65

6:2 Cl-PFAES, while 8:2 Cl-PFAES is produced as an impurity in the commercial

66

products (Figure 1).

67

With an annual production of 20−30 tons, the Cl-PFAESs have been widely used

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as a mist suppressant in the metal plating industry in China for decades. However,

69

their environmental occurrence, behavior and potential adverse effects have not been

70

investigated until recently. In 2013, Wang et al. first reported the environmental

71

occurrence of 6:2 Cl-PFAES in the wastewater from the chrome plating industry in

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China at concentrations ranging from 43 to 112 µg/L5. Later on, several studies

73

reported the detection of 6:2 Cl-PFAES in sewage sludge, river water and atmosphere

74

in China, with concentrations comparable to or higher than PFOS6-8. Furthermore, 6:2

75

Cl-PFAES was found in the bodies of wildlife such as freshwater fish and marine

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organisms9,10. 8:2 Cl-PFAES has also been found in sewage sludge and marine

77

organisms although with levels lower than 6:2 Cl-PFAES9. These environmental 4


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monitoring studies suggest the potential of widespread Cl-PFAESs pollution.

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Although China is the only known emission source, 6:2 Cl-PFAES has also been

80

detected in Arctic wildlife, suggesting the potential of long distance transport and

81

worldwide contamination of Cl-PFAESs11. More recently, increasing evidence has

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shown the widespread human exposure to Cl-PFAESs in China12. For example, 6:2

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Cl-PFAES was detected in occupationally exposed workers, high fish consuming

84

populations and general populations with median serum concentrations of 93.7, 51.5

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and 4.78 ng/mL respectively13. Moreover, 6:2 Cl-PFAES was also detected in the

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maternal serum (1.54 ng/mL), cord serum (0.6 ng/mL), and placenta (0.34 ng/mL),

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indicating that both general and perinatal exposure could occur14,15.

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PFOS toxicity has been extensively studied in the past few decades, showing

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hepatotoxicity, endocrine disruption effect, developmental and immune system

90

toxicity, hyperlipidemia, and even carcinogenic effect16-23. Compared with PFOS,

91

studies on the toxicological effects of Cl-PFAESs are very scarce. Acute toxicity

92

assays of 6:2 Cl-PFAES were executed on zebrafish, and the lethal concentration was

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determined at 15 mg/L during 96 h, indicating its moderate toxicity to zebrafish5.

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Besides, 6:2 Cl-PFAES was found to have embryo toxicity and cardiac development

95

toxicity in zebrafish24 and exert neurotoxicity in rats25. Recently, Sheng et al.

96

demonstrated the cytotoxicity of 6:2 Cl-PFAES to HL-7702 human liver cells and the

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direct binding of 6:2 Cl-PFAES to the liver fatty acid binding protein, suggesting the

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potential hepatotoxicity of 6:2 Cl-PFAES26. Although Cl-PFAESs have been

99

demonstrated to induce some toxicity effects, the current toxicological information is 5



100

insufficient for comprehensive assessment of their health risk. Meanwhile,

101

mechanistic studies of Cl-PFAESs are very limited and the mechanisms of their

102

toxicity are unknown.

103

Peroxisome proliferator-activated receptors (PPARs, including PPARα, PPARβ

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and PPARγ subtypes) mediated signaling pathways play key roles in the regulation of

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many biological functions such as lipid metabolism, cellular growth, differentiation

106

and inflammation27. The structural similarity of PFOS to fatty acids, the natural

107

ligands of PPARs, made the PPARs receptors to be the preferred targets of PFOS in

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toxicological studies28. Actually, many mechanistic studies have demonstrated the

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PPARs signaling pathways are important adverse outcome pathways for the toxicity

110

of PFOS16,29,30. For example, a number of mammalian toxicology studies have shown

111

that PFOS induced hepatotoxicity such as hepatomegaly, hepatic lipid accumulation,

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hepatic steatosis and hepatic carcinoma was associated with PPARs signaling

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pathways28,31-33. A mode of action through PPARα signaling pathway has been

114

proposed and elucidated in a number of cases for the induction of liver cancer in mice

115

and rats by PFOS28-30,34. Besides, direct binding of PFOS to PPARs and activation the

116

subsequent signaling pathways have been well demonstrated by many in vitro

117

studies28,34. The Cl-PFAESs, with very similar chemical structures as PFOS, might

118

have similar effects as PFOS. Here, we hypothesize that Cl-PFAESs might have

119

similar effects on PPARs signaling pathways as PFOS, which may lead to similar

120

adverse effects.

121

In the present study, we measured the binding affinity of 6:2 Cl-PFAES and 8:2 6


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Cl-PFAES to three subtypes of PPARs by fluorescence competitive binding assay.

123

Then, their activity on PPARs signaling pathways were investigated by PPARs

124

mediated luciferase reporter gene assay. By assessing the lipid content and expression

125

of four adipogenic related genes in 3T3-L1 pre-adipocyte cells, we further

126

investigated their effects on adipogenesis as an example to illustrate the potential

127

adverse effects of Cl-PFAESs mediated by PPARs signaling pathways. Molecular

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docking analysis was performed to investigate the structural characteristics of their

129

binding and activity towards PPARs.

130 131

MATERIALS AND METHODS

132

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

133

MA, USA) (Figure 1). 6:2 Cl-PFAES (with purity > 95%) and 8:2 Cl-PFAES (with

134

purity > 95%) standards were purified from the commercial F-53B mist suppressant

135

product (with 6:2 Cl-PFAES and 8:2 Cl-PFAES content of 77.6% and 6.0%,

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respectively) by preparative liquid chromatography7. The purity of the 6:2 Cl-PFAES

137

and 8:2 Cl-PFAES standards was determined by high performance liquid

138

chromatography coupled with an evaporative light-scattering detector according to the

139

procedure described previously7. The three chemicals were dissolved in dimethyl

140

sulfoxide (DMSO) to make stock solutions with a concentration of 50 mM. Linoleic

141

acid (LA), WY14643, GW501516 and rosiglitazone were purchased from

142

Sigma-Aldrich (St. Louis, MO, USA). Human PPARα, PPARβ and PPARγ ligand

143

binding domains (LBDs) were provided by Zhongding Biotechnology Co. Ltd. 7



144

(Nanjing, China). 4,4-Difluoro-5-methyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoic

145

acid (C1-BODIPY-C12) probe was purchased from Life Technologies (Carlsbad, CA,

146

USA).

147

Cell culture. Human HEK 293 embryonal kidney cells and mouse 3T3-L1

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pre-adipocytes cells were purchased from American Type Culture Collection

149

(Manassas, VA, USA). Cells were cultured in culture medium: high-glucose

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Dulbecco’s minimal essential medium (DMEM) supplemented with 10% fetal bovine

151

serum (Life Technologies), 100 units/mL penicillin and 100 µg/mL streptomycin (Life

152

Technologies) at 37 ℃ in a humidified 5% CO2 atmosphere. All cells used in our

153

experiments were at the exponential growth phase.

154

Fluorescence competitive binding assay. A fluorescence polarization (FP) based

155

competitive binding assay was employed to determine the binding affinity of the two

156

Cl-PFAESs and PFOS with PPAR ligand-binding domains (PPAR-LBDs). The

157

detailed procedure for competitive binding assay is provided in the supporting

158

information.

159

PPAR mediated luciferase reporter gene assay. The pBIND-PPARs vectors,

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pGL4.35 [luc2P/9×GAL4UAS/Hygro] vector and PRL-TK vector were transiently

161

transfected into HEK 293 cells to characterize and compare the activity of chemicals

162

on the PPARs signaling pathways. The detailed procedures for vector construction and

163

transient transfection are provided in the supporting information.

164

3T3-L1 adipogenesis assay. Mouse 3T3-L1 adipogenesis assay was performed

165

according to the protocol described previously35,36. The lipid content of cells was 8


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determined by Oil Red O staining assay. The detailed procedure for 3T3-L1

167

adipogenesis assay and Oil Red O staining assay is provided in the supporting

168

information. The concentrations of chemicals used in the adipogenesis assay had no

169

cytotoxicity on 3T3-L1 cells (Figure S4).

170

RNA extraction and quantitative real-time PCR. Total RNA was extracted from

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3T3-L1 cells by Trizol reagent (Invitrogen, Carlsbad, CA, USA) following the

172

manufacturer’s instruction. First-strand cDNAs were synthesized with RevertAid First

173

Strand cDNA Synthesis Kit (ThermoFisher Scientific, Waltham, MA, USA). The

174

transcriptional

175

CCAAT/enhancer-binding protein alpha (C/EBPα), adipocyte Protein 2 (aP2),

176

adiponectin (Adip) and leptin (Lep), were analyzed by quantitative real-time PCR

177

using GoTaq® qPCR Master Mix (Promega, Madison, WI) with MX Real-time

178

Polymerase Chain Reaction system (Light Cycler 480, Roche, Basel, Switzerland).

179

β-Actin was used as the reference gene. Specific primers for these genes were

180

described in the Table S1. The fold change of gene transcription related to the

181

reference was determined by 2-∆∆Ct method37.

182

Molecular docking analysis. 3D crystal structures of the three PPARs (PPARα, PDB

183

ID: 4BCR; PPARβ, PDB ID: 3OZ0; PPARγ, PDB ID: 3U9Q) were obtained from the

184

RCSB (Research Collaboratory for Structural Bioinformatics) Protein Data Bank

185

(http://www.rcsb.org/pdb). Structures of the ligands were drawn with ChemBioDraw

186

Ultra and transformed into PDB format through the PRODRG server38. The binding

187

mode of a ligand to PPARs was determined using Lamarckian genetic algorithm

levels

of

four

adipogenesis

related

9


genes

including


188

provided by Auto Dock 4.2. The detailed procedure for Molecular docking is provided

189

in the supporting information.

190

Statistical analysis. All the experiments were conducted in triplicates and the data

191

were expressed in terms of mean ± SEM (n = 3). Comparison of the mean values

192

among experimental groups was analyzed using SPSS 17.0 software (Chicago, IL,

193

USA). The normality and homogeneity of variance of all data were analyzed by

194

one-way analysis of variance (ANOVA), and the multiple comparison for differences

195

in means was then performed by Duncan’s multiple range test, with signiﬁcance level

196

set at *p < 0.05.

197 198

RESULTS AND DISCUSSION

199

Binding potency of Cl-PFAESs to PPAR-LBDs. Using the FP based competitive

200

binding assay, we studied the binding potency of 6:2 Cl-PFAES, 8:2 Cl-PFAES as

201

well as PFOS with three PPAR-LBDs. The competition curves are shown in Figure 2,

202

and the obtained IC50 values are listed in Table 1. As shown in Figure S1D, linoleic

203

acid (used as a positive control) competed the binding of the fluorescence probe to

204

PPAR-LBDs in a dose-dependent manner, suggesting the validity of the method.

205

Based on the competitive binding results, we found PFOS could bind with all the

206

three PPAR-LBDs (Figure 2). Several previous studies reported PFOS could bind

207

with PPARα and PPARγ34. Consistent with these studies, we also demonstrated PFOS

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bound with PPARα-LBD and PPARγ-LBD. However, the direct binding of PFOS to

209

PPARβ has never been studied before. Here, we found PFOS could also bind to 10


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PPARβ-LBD directly.

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Like PFOS, 6:2 Cl-PFAES and 8:2 Cl-PFAES could also bind to all the three

212

PPAR-LBDs (Figure 2). By comparing the IC50 values of Cl-PFAESs with that of

213

PFOS, we found they even showed much higher binding potency than PFOS (Table 1).

214

6:2 Cl-PFAES exhibited approximately 2.4-, 4.0- and 1.9-fold stronger binding

215

potency than PFOS for PPARα-LBD, PPARβ-LBD and PPARγ-LBD, respectively

216

(Table 1). 8:2 Cl-PFAES exhibited approximately 2.9-, 2.0- and 1.7-fold stronger

217

binding potency than PFOS for PPARα-LBD, PPARβ-LBD and PPARγ-LBD,

218

respectively (Table 1). The replacement of fluorine by chlorine increases the

219

hydrophobicity of Cl-PFAESs, which might lead to stronger binding potency. Here,

220

we provided the first evidence that, like PFOS, the Cl-PFAESs could bind to

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PPAR-LBDs directly.

222

Activity of Cl-PFAESs towards PPARs transactivation. We further studied the

223

activity of the two Cl-PFAESs and PFOS towards the three PPARs signaling

224

pathways by PPARs-mediated luciferase reporter gene assay. As shown in Figure S2,

225

WY14643, GW501516 and rosiglitazone (known specific agonists for PPARα,

226

PPARβ and PPARγ respectively) enhanced the luciferase transcriptional activity in a

227

dose-dependent manner, suggesting the validity of the assay. For PFOS, it also

228

enhanced the PPARs-mediated luciferase transcriptional activity in a dose-dependent

229

manner (Figure 3), suggesting its agonistic activity towards the three PPARs signaling

230

pathways. Multiple previous studies have demonstrated the agonistic activity of PFOS

231

towards human PPARs signaling pathways which our results are in agreement 11



232

with30,34, although there are also a few studies showing PFOS had no effect28,39,40.

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Similar to PFOS, 6:2 Cl-PFAES and 8:2 Cl-PFAES also enhanced the luciferase

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transcriptional activity in a dose-dependent manner (Figure 3), suggesting they also

235

had agonistic activity towards the three PPARs signaling pathways. By comparing the

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activity of the two Cl-PFAESs with PFOS at three high concentrations (50 µM, 75 µM

237

and 100 µM), we found 8:2 Cl-PFAES even showed higher agonistic activity than

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PFOS (Figure 3), which is consistent with the result that it had higher binding potency

239

to PPARs than PFOS. For the 6:2 Cl-PFAES, it showed similar activity as PFOS

240

(Figure 3). Here, for the first time, we demonstrated that like PFOS, binding of the

241

Cl-PFAESs could activate the PPARs signaling pathways.

242

Molecular docking of Cl-PFAESs with PPARs. Molecular docking analysis

243

between Cl-PFAESs and PPARs was performed so as to provide some explanation

244

about the structural characteristics of their binding and activity with PPARs. Decanoic

245

acid (DA, an endogenous ligand for PPARγ) and PFOS were also docked into PPARs

246

for comparison. The binding geometries are illustrated in Figure 4, while the

247

hydrogen bond interactions obtained from the docking analysis are listed in Table 2.

248

As shown in Figure 4, DA docked into all the three PPARs with its hydrophobic chain

249

residing towards the inner part and its polar carboxylic acid end group towards the

250

entrance of the binding pocket. As shown in Table 2, DA formed hydrogen bonds with

251

the residues in PPARs with its carboxylic acid substituent (residues of SER280,

252

HIS440 and TYR464 in PPARα, THR289, HIS323, HIS449 and TYR473 in PPARβ,

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SER289, HIS323, HIS449 and TYR473 in PPARγ). It should be noted that the 12


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residues that formed hydrogen bonds with DA are in the AF-2 region of the PPARs.

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The docking results of DA with PPARγ are in good agreement with the

256

crystallographic results48-50, suggesting the accuracy of the docking method.

257

For 6:2 Cl-PFAES, 8:2 Cl-PFAES and PFOS, they showed very similar binding

258

geometry between each other (Figure 4). It is obvious that the three chemicals have

259

very similar structures (Figure 1). Like DA, they all fit into the ligand binding pockets

260

of the three PPARs with their hydrophobic chain residing toward the inner part and

261

their polar sulfonic acid end group toward the entrance of the binding pocket (Figure

262

4). Besides, these three chemicals formed hydrogen bonds with the same residues of

263

each PPAR through their sulfonic acid groups. They all formed hydrogen bond

264

interactions with TYR464 in PPARα, with THR289 and HIS449 in PPARβ and with

265

SER289, HIS449 and TYR473 in PPARγ (Table 2). It is worth noting that these

266

residues are the ones on the AF-2 region of the PPARs which form hydrogen bonds

267

with DA. Previous studies have revealed that the hydrogen bond interactions with

268

these residues play important roles in the ligand binding and activation of PPARs50-52.

269

Hydrogen bond interactions of a ligand with the residues in AF-2 region could lead to

270

the packing of AF-2 helix to the binding pocket of PPAR, which results in the

271

coactivator binding and PPAR mediated gene transcription51. Therefore, we infer that

272

these three chemicals probably activate PPARs in the same manner as fatty acids by

273

forming hydrogen bonds with the residues in AF-2 region through their acid groups,

274

which could result in the packing of AF-2 helix to the binding pocket of PPAR.

275

Overall, the docking results could explain the observed Cl-PFAESs and PFOS activity 13



276

towards PPARs.

277

Effects

of

Cl-PFAESs

on

adipogenesis

in

3T3-L1

Page 14 of 36

pre-adipocytes.

278

Adipogenesis is an important process of cell differentiation regulated by PPARs

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signaling pathways with PPARγ pathway playing the predominant role36,41,42.

280

Numerous in vitro and in vivo studies have showed PFOS could promote adipogenesis

281

by activating the PPARs signaling pathways35,36,41-43. Here, we studied the effect of

282

Cl-PFAESs on adipogenesis as an example to illustrate the role of PPARs mediated

283

adverse effects. The change of lipid content and expression of four adipogenic related

284

genes were assessed on the process of Cl-PFAESs mediate adipogenesis in 3T3-L1

285

pre-adipocyte cells. It is well known that PPARγ is the predominant receptor

286

regulating adipogenesis, while PPARα and PPARβ may play a leading role in some

287

other functions such as differentiation of certain adipose depots44,45. As shown in

288

Figure S5, 100 nM rosiglitazone (specific agonistic for PPARγ, used as a positive

289

control) enhanced the lipid content of 3T3-L1 by 7.2-fold compared to the vehicle,

290

suggesting the promotion of adipocyte differentiation41,46,47. Furthermore, we also

291

investigated the effects of WY14643 and GW501516 (specific agonists of PPARα and

292

PPARβ respectively) on adipogenesis in 3T3-L1 preadipocytes. The results showed

293

WY14643 and GW501516 significantly enhanced adipogenesis by about 2.39- and

294

2.26-fold at 10 µM and 100 nM (Figure S5) respectively. These results indicate the

295

potential contribution of PPARα and PPARβ pathways in adipocyte differentiation,

296

although with less potency than PPARγ, which is consistent with previous results36.

297

The cytotoxicity of each chemical on 3T3-L1 cells was tested. As shown in Figure S4, 14


Page 15 of 36


298

the lowest concentrations of 6:2 Cl-PFAES and 8:2 Cl-PFAES for cytotoxicity were

299

lower than that of PFOS, indicating Cl-PFAESs had stronger cytotoxicity than PFOS.

300

The adipogenesis assay was performed at the levels with no cytotoxicity. For PFOS, it

301

enhanced the lipid content in a dose-dependent manner with 2.1 folds enhancement at

302

100 µM (Figure 5A). Our result was in line with a previous study which demonstrated

303

the promotion of adipocyte differentiation of PFOS to 3T3-L1 pre-adipocyte cells35,36.

304

Similar to PFOS, 6:2 Cl-PFAES and 8:2 Cl-PFAES also promoted adipogenesis in a

305

dose-dependent manner with 2.3 folds and 2.6 folds enhancement at 100 µM,

306

respectively (Figure 5A and 5B). These results are consistent with the results that

307

Cl-PFAESs showed higher binding potency to PPARs than PFOS, and comparable or

308

higher agonistic activity towards PPARs signaling pathways than PFOS. By

309

comparing the activities of these three chemicals at the three tested concentrations (10

310

µM, 50 µM and 100 µM), we found 6:2 Cl-PFAES and 8:2 Cl-PFAES even showed

311

higher activity than PFOS. Combining the results of receptor binding and luciferase

312

assays, which showed PFOS and Cl-PFAESs bound to or activated different PPARs

313

subtypes with similar potency, we speculate PFOS and Cl-PFAESs might promote

314

adipogenesis by activating the PPARs signaling pathways with PPARγ pathway

315

playing the predominant role.

316

We also investigated the effect of Cl-PFAESs on adipogenesis by determining

317

the expression level of four adipogenic related genes (C/EBPα, aP2, Adip and Lep) in

318

3T3-L1 cells. As shown in Figure S6, 100 nM rosiglitazone enhanced the expression

319

level of C/EBPα, aP2, Adip and Lep by 10.8, 4692, 1.9 and 4.0-folds compared to day 15



320

0 (Figure S6), which is in line with the results of previous studies41,44,45. Similarly,

321

PFOS, 6:2 Cl-PFAES and 8:2 Cl-PFAES also enhanced the expression level of the

322

four genes although with potency much weaker than rosiglitazone. For PFOS, it

323

enhanced the expression level of C/EBPα, aP2, Adip and Lep for 6.8, 966.3, 2.6 and

324

2.6-folds at 100 µM, respectively (Figure 5C), which is in line with the previous

325

literature35. 6:2 Cl-PFAES (8:2 Cl-PFAES) also enhanced the expression level of

326

these four genes, with 5.6 (6.2), 610 (645), 3.0 (2.7) and 3.7 (3.5)-folds for C/EBPα,

327

aP2, Adip and Lep at 100 µM, respectively (Figure 5C). Compared with the

328

differential responses among PFOS and Cl-PEAESs in the TG assay, we found the

329

differential responses of the four adipogenic related genes are less consistent. We

330

speculate that this is because the transcriptional responses of the adipogenic related

331

genes might have reached a ceiling effect. By combing the results of Oil Red O

332

staining assay and gene transcription assay, we found the Cl-PFAESs even showed

333

higher adipogenesis activity than PFOS (Figure 5). Based on the above results, we

334

found that PFOS and Cl-PFAESs could promote adipogenesis by activating the

335

PPARs signaling pathways, probably dominated by the PPARγ pathway. Here, using

336

adipogenesis as a representative effect mediated by PPARs, we demonstrated the

337

potential toxicity induced by Cl-PFAESs through PPARs signaling pathways.

338

Compared with the zebrafish non-lethal concentration of 6:2 Cl-PFAES at 5 mg/L

339

(~10µM)5, developmental toxicity of zebrafish embryo at 6 mg/L (~11µM)21 and

340

cytotoxicity of HL-7702 human liver cells at 25µM26, the effect of 6:2 Cl-PFAES on

341

adipogenesis might be the most sensitive endpoint (with LOEC of 10 µM) reported so 16


Page 16 of 36

Page 17 of 36


342

far.

343

Toxicological and health risk implication of Cl-PFAESs. At present, the occurrence

344

of Cl-PFAESs in humans has been documented5,10. Biomonitoring of the Chinese

345

general population has shown the median concentration of 6:2 Cl-PFAES with 4.7

346

ng/mL13,15. Based on our study, Cl-PFAESs could activate the PPARs signaling

347

pathways and induce 3T3-L1 cell adipogenesis at the LOEC of 10 µM (~ 5 µg/mL),

348

which is much higher than the reported exposure levels of the general population. The

349

effects of Cl-PFAESs on PPARs mediated physiological functions might not occur at

350

the current human exposure level. However, although current usage of Cl-PFAESs is

351

limited to the chrome plating industry, the increasing demand for PFOS alternatives in

352

other sectors may result in expanded usage of Cl-PFAESs5. The environmental and

353

human levels of Cl-PFAESs are expected to increase in China over the coming years.

354

Therefore, we think Cl-PFAESs deserve more attention in the future. Moreover, a

355

study reported that the highest 6:2 Cl-PFAES serum concentration of the

356

plating workers in China

357

activating the PPAR signaling pathway detected in our experiments. To further

358

evaluate the potential health risk of 6:2 Cl-PFAES, we estimated the margin of safety

359

for both the general and occupational populations. Details of the calculation are

360

provided in SI. The results showed the hazard quotient (HQ) values of 6:2 Cl-PFAES

361

were 0.26 (< 1) for the general population and 5.3 (> 1) for the occupational

362

population, suggesting that the current exposure level in the occupational population

363

might be of concern.

metal

was 5.1 µg/mL13, which is close to the LOEC for

17



364

Several studies have compared the environmental behavior and toxicological

365

effect of Cl-PFAESs with PFOS10,13,15,24,26. For example, Shi et al. found the

366

bioaccumulation potential of 6:2 Cl-PFAES was higher than that of PFOS in crucian

367

carp10. More recently, Shi et al showed 6:2 Cl-PFAES and 8:2 Cl-PFAES had slower

368

elimination kinetics than that of PFOS13. Chen et al. found the placental transfer

369

efficiencies of 8:2 Cl-PFAES and 6:2 Cl-PFAES were higher than that of PFOS13,15. In

370

addition, the Cl-PFAESs have relatively higher trophic transfer behavior than PFOS9.

371

All these findings indicated that Cl-PFAESs might exhibit higher health risk than

372

PFOS. Besides, some recent toxicological studies have indicated that 6:2 Cl-PFAES

373

exhibited higher developmental toxicity, hepatic cytotoxicity and neurotoxicity than

374

PFOS24-26. Combining the results of these previous studies and our present study, we

375

speculate that Cl-PFAESs might have higher toxic effects than PFOS, and may not be

376

suitable substitutes for PFOS.

377

To conclude, we found in the present study that Cl-PFAESs interacted with

378

PPARs directly, showed agonistic activity towards PPARs signaling pathways, and

379

promoted the process of adipogenesis in 3T3-L1 cells, all similar to PFOS but with

380

higher potency. Molecular docking analysis revealed similar binding modes and

381

hydrogen bond interactions in the PPARs ligand binding pockets and suggests that

382

Cl-PFAESs bind to PPARs in a manner similar to PFOS. Our results indicate potential

383

toxicity of Cl-PFAESs mediated by PPARs signaling pathways.

384 385

Notes 18


Page 18 of 36

Page 19 of 36


386

The authors declare that there are no conflicts of interest.

387 388

Acknowledgments

389

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

390

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

391

(91543203, 21621064, 21622705, and 21777187), and the Royal Society International

392

Collaboration Awards for Research Professors.

393 394

19



395

References

396

(1) Kissa, E. Fluorinated Surfactants and Repellents, 2nd ed.; Marcel Dekker Inc.:

397

New York, 2001.

398

(2) Stockholm Convention on persistent organic pollutants adoption of amendments

399

to annexes B. Treaties-4; United Nations Environment Programme (UNEP), 2009;

400

http://chm.pops.int/Implementation/NewPOPs/TheNewPOPs/tabid/672/Default.aspx.

401

(3) Heydebreck, F.; Tang, J. H.; Xie, Z. Y.; Ebinghaus, R. Alternative and Legacy

402

Perfluoroalkyl Substances: Differences between European and Chinese River/Estuary

403

Systems. Environ. Sci. Technol. 2015, 49 (14), 8386-8395.

404

(4) Wang, T.; Vestergren, R.; Herzke, D.; Yu, J.; Cousins, I. T. Levels, Isomer Profiles,

405

and Estimated Riverine Mass Discharges of Perfluoroalkyl Acids and Fluorinated

406

Alternatives at the Mouths of Chinese Rivers. Environ. Sci. Technol. 2016, 50 (21),

407

11584-11592.

408

(5) Wang, S.; Huang, J.; Yang, Y.; Hui, Y.; Ge, Y.; Larssen, T.; Yu, G.; Deng, S.;

409

Wang, B.; Harman, C. First report of a Chinese PFOS alternative overlooked for 30

410

years: its toxicity, persistence, and presence in the environment. Environ. Sci. Technol.

411

2013, 47 (18), 10163-10170.

412

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

413

chlorinated polyfluorinated ether sulfonate and ionic perfluoroalkyl acids in 2006 to

414

2014 in Dalian, China. Environ. Toxicol. Chem. 2017, 36 (10), 2581-2586.

415

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

416

polyfluorinated ether sulfonates as PFOS alternatives in municipal sewage sludge in 20


Page 20 of 36

Page 21 of 36


417

China. Environ. Sci. Technol. 2015, 49 (11), 6519-6527.

418

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

419

quantitative analysis of fluoroalkyl sulfonates in riverine water by liquid

420

chromatography coupled with Orbitrap high resolution mass spectrometry. J.

421

Chromatogr. A 2016, 1435, 66-74.

422

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

423

Jiang, G. Chlorinated Polyfluoroalkyl Ether Sulfonic Acids in Marine Organisms from

424

Bohai Sea, China: Occurrence, Temporal Variations, and Trophic Transfer Behavior.

425

Environ. Sci. Technol. 2017, 51 (8), 4407-4414.

426

(10) Shi, Y.; Vestergren, R.; Zhou, Z.; Song, X.; Xu, L.; Liang, Y.; Cai, Y. Tissue

427

distribution and whole body burden of the chlorinated polyfluoroalkyl ether sulfonic

428

acid F-53B in crucian carp (Carassius carassius): Evidence for a highly

429

bioaccumulative contaminant of emerging concern. Environ. Sci. Technol. 2015, 49

430

(24), 14156-14165.

431

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

432

Observation of emerging per-and polyfluoroalkyl substances (PFASs) in Greenland

433

marine mammals. Chemosphere 2016, 144, 2384-2391.

434

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

435

the accurate determination of a wide carbon-chain range of per-and polyfluoroalkyl

436

substances (C4–C18) in human serum. J. Chromatogr. A 2016, 1471, 1-10.

437

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

438

exposure and elimination kinetics of chlorinated polyfluoroalkyl ether sulfonic acids 21



439

(Cl-PFESAs). Environ. Sci. Technol. 2016, 50 (5), 2396-2404.

440

(14) Pan, Y.; Zhu, Y.; Zheng, T.; Cui, Q.; Buka, S. L.; Zhang, B.; Guo, Y.; Xia, W.;

441

Yeung, L. W.; Li, Y. Novel chlorinated polyfluorinated ether sulfonates and legacy

442

per-/polyfluoroalkyl substances: placental transfer and relationship with serum

443

albumin and glomerular filtration rate. Environ. Sci. Technol. 2016, 51 (1), 634-644.

444

(15) Chen, F.; Yin, S.; Kelly, B. C.; Liu, W. Chlorinated Polyfluoroalkyl Ether

445

Sulfonic Acids in Matched Maternal, Cord, and Placenta Samples: A Study of

446

Transplacental Transfer. Environ. Sci. Technol. 2017, 51, 6387-6394

447

(16) Wan, H.; Zhao, Y.; Wei, X.; Hui, K.; Giesy, J.; Wong, C. K. PFOS-induced

448

hepatic steatosis, the mechanistic actions on β-oxidation and lipid transport. Biochim.

449

Biophys. Acta 2012, 1820 (7) 1092-1101.

450

(17) Lau, C.; Butenhoff, J. L.; Rogers, J. M. The developmental toxicity of

451

perfluoroalkyl acids and their derivatives. Toxicol. Appl. Pharmacol. 2004, 198 (2),

452

231-241.

453

(18) DeWitt, J. C.; Peden-Adams, M. M.; Keller, J. M.; Germolec, D. R.

454

Immunotoxicity of perfluorinated compounds: recent developments. Toxicol. Pathol.

455

2012, 40 (2), 300-311.

456

(19) Johansson, N.; Fredriksson, A.; Eriksson, P., Neonatal exposure to

457

perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) causes

458

neurobehavioural defects in adult mice. Neurotoxicology 2008, 29 (1), 160-169.

459

(20) Jensen, A. A.; Leffers, H., Emerging endocrine disrupters: perfluoroalkylated

460

substances. Int. J. Androl. 2008, 31 (2), 161-169. 22


Page 22 of 36

Page 23 of 36


461

(21) Jacquet, N.; Maire, M.; Landkocz, Y.; Vasseur, P. Carcinogenic potency of

462

perfluorooctane sulfonate (PFOS) on Syrian hamster embryo (SHE) cells. Arch.

463

Toxicol. 2012, 86 (2), 305-314.

464

(22) Chang, E. T.; Adami, H.-O.; Boffetta, P.; Cole, P.; Starr, T. B.; Mandel, J. S. A

465

critical review of perfluorooctanoate and perfluorooctanesulfonate exposure and

466

cancer risk in humans. Crit. Rev. Toxicol. 2014, 44 (Suppl 1), 1-81.

467

(23) Lau, C.; Anitole, K.; Hodes, C.; Lai, D.; Pfahles-Hutchens, A.; Seed, J.

468

Perfluoroalkyl acids: a review of monitoring and toxicological findings. Toxicol. Sci.

469

2007, 99 (2), 366-394.

470

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

471

polyfluorinated ether sulfonate, a PFOS alternative, induces embryotoxicity and

472

disrupts cardiac development in zebrafish embryos. Aqua. Toxicol. 2017, 185, 67-75.

473

(25) Zhang, Q.; Liu, W.; Niu, Q.; Wang, Y.; Zhao, H.; Zhang, H.; Song, J.; Tsuda, S.;

474

Saito, N. Effects of perfluorooctane sulfonate and its alternatives on long-term

475

potentiation in the hippocampus CA1 region of adult rats in vivo. Toxicol. Res. 2016,

476

5 (2), 539-546.

477

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

478

fluorinated alternatives to long-chain perfluoroalkyl substances to human liver cell

479

line and their binding capacity to human liver fatty acid binding protein. Arch. Toxicol.

480

2017, 51 (17), 9553-9560.

481

(27) Grygiel-Górniak, B. Peroxisome proliferator-activated receptors and their ligands:

482

nutritional and clinical implications-a review. Nutr. J. 2014, 13 (1), 17. 23



483

(28) Vanden Heuvel, J. P.; Thompson, J. T.; Frame, S. R.; Gillies, P. J. Differential

484

activation of nuclear receptors by perfluorinated fatty acid analogs and natural fatty

485

acids: a comparison of human, mouse, and rat peroxisome proliferator-activated

486

receptor-α,-β, and-γ, liver X receptor-β, and retinoid X receptor-α. Toxicol. Sci. 2006,

487

92 (2), 476-489.

488

(29) Corton, J. C.; Cunningham, M. L.; Hummer, B. T.; Lau, C.; Meek, B.; Peters, J.

489

M.; Popp, J. A.; Rhomberg, L.; Seed, J.; Klaunig, J. E. Mode of action framework

490

analysis for receptor-mediated toxicity: The peroxisome proliferator-activated

491

receptor alpha (PPAR α) as a case study. Crit. Rev. Toxicol. 2014, 44 (1), 1-49.

492

(30) Wolf, C. J.; Takacs, M. L.; Schmid, J. E.; Lau, C.; Abbott, B. D. Activation of

493

mouse and human peroxisome proliferator− activated receptor alpha by perfluoroalkyl

494

acids of different functional groups and chain lengths. Toxicol. Sci. 2008, 106 (1),

495

162-171.

496

(31) Klaunig, J. E.; Babich, M. A.; Baetcke, K. P.; Cook, J. C.; Corton, J. C.; David, R.

497

M.; DeLuca, J. G.; Lai, D. Y.; McKee, R. H.; Peters, J. M. PPARα agonist-induced

498

rodent tumors: modes of action and human relevance. Crit. Rev. Toxicol. 2003, 33 (6),

499

655-780.

500

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

501

Elcombe, C. R.; Butenhoff, J. L. Sub-chronic dietary toxicity of potassium

502

perfluorooctanesulfonate in rats. Toxicol. 2003, 183 (1), 117-131.

503

(33) Cwinn, M. A.; Jones, S. P.; Kennedy, S. W. Exposure to perfluorooctane

504

sulfonate or fenofibrate causes PPAR-α dependent transcriptional responses in 24


Page 24 of 36

Page 25 of 36


505

chicken embryo hepatocytes. Comp. Biochem. Phys. C 2008, 148 (2), 165-171.

506

(34) Zhang, L.; Ren, X. M.; Wan, B.; Guo, L. H. Structure-dependent binding and

507

activation of perfluorinated compounds on human peroxisome proliferator-activated

508

receptor γ. Toxicol. Appl. Pharmacol. 2014, 279 (3), 275-283.

509

(35) Xu, J.; Shimpi, P.; Armstrong, L.; Salter, D.; Slitt, A. L. PFOS induces

510

adipogenesis and glucose uptake in association with activation of Nrf2 signaling

511

pathway. Toxicol. Appl. Pharmacol. 2016, 290, 21-30.

512

(36) Watkins, A. M.; Wood, C. R.; Lin, M. T.; Abbott, B. D. The effects of

513

perfluorinated chemicals on adipocyte differentiation in vitro. Mol. Cell. Endocrinol.

514

2015, 400, 90-101.

515

(37) Livak, K. J.; Schmittgen, T. D. Analysis of relative gene expression data using

516

real-time quantitative PCR and the 2− ∆∆CT method. Methods 2001, 25 (4), 402-408.

517

(38) SchuÈttelkopf, A. W.; Van Aalten, D. M. PRODRG: a tool for high-throughput

518

crystallography of protein–ligand complexes. Acta Crystallogr. D 2004, 60 (8),

519

1355-1363.

520

(39) Takacs, M. L.; Abbott, B. D. Activation of mouse and human peroxisome

521

proliferator–activated receptors (α, β/δ, γ) by perfluorooctanoic acid and

522

perfluorooctane sulfonate. Toxicol. Sci. 2006, 95 (1), 108-117.

523

(40) Maloney, E. K.; Waxman, D. J. trans-Activation of PPARα and PPARγ by

524

structurally diverse environmental chemicals. Toxicol. Appl. Pharmacol. 1999, 161 (2),

525

209-218.

526

(41) Tontonoz, P.; Hu, E.; Spiegelman, B. M. Stimulation of adipogenesis in 25



527

fibroblasts by PPARγ2, a lipid-activated transcription factor. Cell 1994, 79 (7),

528

1147-1156.

529

(42) Li, X.; Ycaza, J.; Blumberg, B. The environmental obesogen tributyltin chloride

530

acts via peroxisome proliferator activated receptor gamma to induce adipogenesis in

531

murine 3T3-L1 preadipocytes. J. Steroid Biochem. Mol. Biol. 2011, 127 (1), 9-15.

532

(43) Jones, J. R.; Barrick, C.; Kim, K.-A.; Lindner, J.; Blondeau, B.; Fujimoto, Y.;

533

Shiota, M.; Kesterson, R. A.; Kahn, B. B.; Magnuson, M. A. Deletion of PPARγ in

534

adipose tissues of mice protects against high fat diet-induced obesity and insulin

535

resistance. Proc. Natl. Acad. Sci. USA, 2005, 102 (17), 6207-6212.

536

(44) Neels, J. G.,; Grimaldi, P. A. Physiological functions of peroxisome

537

proliferator-activated receptor β. Phys. Rev. 2014, 94 (3), 795-858.

538

(45) Kersten, S.; Desvergne, B.; Wahli, W. Roles of PPARs in health and disease.

539

Nature, 2000, 405(6785), 421-424.

540

(46) Bouaboula, M.; Hilairet, S.; Marchand, J.; Fajas, L.; Le Fur, G.; Casellas, P.

541

Anandamide induced PPARγ transcriptional activation and 3T3-L1 preadipocyte

542

differentiation. Eur. J. Pharmacol. 2005, 517 (3), 174-181.

543

(47) Manaharan, T.; Ming, C. H.; Palanisamy, U. D. Syzygium aqueum leaf extract

544

and its bioactive compounds enhances pre-adipocyte differentiation and 2-NBDG

545

uptake in 3T3-L1 cells. Food Chem. 2013, 136 (2), 354-363.

546

(48) Sierra, M. L.; Beneton, V.; Boullay, A.-B.; Boyer, T.; Brewster, A. G.; Donche, F.;

547

Forest, M.-C.; Fouchet, M.-H.; Gellibert, F. J.; Grillot, D. A. Substituted

548

2-[(4-aminomethyl) phenoxy]-2-methylpropionic acid PPARα agonists. 1. Discovery 26


Page 26 of 36

Page 27 of 36


549

of a novel series of potent HDLc raising agents. J. Med. Chem. 2007, 50 (4), 685-695.

550

(49) Liu, T.; Zhang, H.; Liu, F.; Wu, Q.; Shen, X.; Yang, Q. Structural determinants of

551

an insect β-N-acetyl-D-hexosaminidase specialized as a chitinolytic enzyme. J. Biol.

552

Chem. 2011, 286 (6), 4049-4058.

553

(50) Malapaka, R. R.; Khoo, S.; Zhang, J.; Choi, J. H.; Zhou, X. E.; Xu, Y.; Gong, Y.;

554

Li, J.; Yong, E.-L.; Chalmers, M. J. Identification and mechanism of 10-carbon fatty

555

acid as modulating ligand of peroxisome proliferator-activated receptors. J. Biol.

556

Chem. 2012, 287 (1), 183-195.

557

(51) Bernardes, A.; Souza, P. C.; Muniz, J. R.; Ricci, C. G.; Ayers, S. D.; Parekh, N.

558

M.; Godoy, A. S.; Trivella, D. B.; Reinach, P.; Webb, P. Molecular mechanism of

559

peroxisome proliferator-activated receptor α activation by WY14643: a new mode of

560

ligand recognition and receptor stabilization. J. Mol. Biol. 2013, 425 (16), 2878-2893.

561

(52) Wu, C.-C.; Baiga, T. J.; Downes, M.; La Clair, J. J.; Atkins, A. R.; Richard, S. B.;

562

Fan, W.; Stockley-Noel, T. A.; Bowman, M. E.; Noel, J. P. Structural basis for specific

563

ligation of the peroxisome proliferator-activated receptor δ. Proc. Natl. Acad. Sci.

564

USA, 2017, 114 (13), E2563-E2570.

565 566 567

27



Page 28 of 36

568

Table 1. Results of IC50 and RP (relative potency to Linoleic acid (LA)) values of the

569

tested chemicals for PPARα-LBD, PPARβ-LBD and PPARγ-LBD by the competitive

570

binding assay.

571

PPARα

PPARβ

PPARγ

Chemicals IC50 (µM)

RP

IC50 (µM)

RP

IC50 (µM)

RP

LA

233.6

1.00

83.9

1.00

66.4

1.00

PFOS

247.7

0.94

456.5

0.18

189.5

0.35

6:2 Cl-PFAES

105.3

2.22

114.0

0.96

98.5

0.67

8:2 Cl-PFAES

84.7

2.76

232.6

0.36

109.4

0.61

572 573 574

28


Page 29 of 36


575

Table 2. Results of hydrogen bond interactions of Decanoic acid (DA), PFOS 6:2

576

Cl-PFAES and 8:2 Cl-PFAES with PPAR-LBDs by molecular docking analysis.

577

Chemicals

PPARα

PPARβ

PPARγ

SER280, HIS440,

SER289, HIS323,

SER289, HIS323,

TYR464

HIS449, TYR473

HIS449, TYR473

SER280, TYR464

THR289, HIS449

DA

SER289, HIS323, PFOS

HIS449, TYR473 SER280, HIS440,

THR289, HIS323,

SER289, HIS449,

TYR464

HIS449, TYR473

HIS449,TYR473

6:2 Cl-PFAES

SER289, HIS323, 8:2 Cl-PFAES

HIS440, TYR464

THR289, HIS449 HIS449, TYR473

578 579

29



580

Figure legends:

581 582

Figure 1. Structures of 6:2 Cl-PFAES, 8:2 Cl-PFAES and PFOS.

583 584

Figure 2. Competitive binding curves of PFOS, 6:2 Cl-PFAES and 8:2 Cl-PFAES to

585

PPARα-LBD (A), PPARβ-LBD (B) and PPARγ-LBD (C).

586 587

Figure 3. Effects of PFOS, 6:2 Cl-PFAES and 8:2 Cl-PFAES on PPARα (A), PPARβ

588

(B) and PPARγ (C) mediated luciferase reporter gene transcription activity. The

589

relative luciferase activity was determined by setting 0.1% DMSO (Veh) treated cells

590

as 1.

591 592

Figure 4. Molecular docking results of Decanoic acid (DA), PFOS, 6:2 Cl-PFAES and

593

8:2 Cl-PFAES with PPARα (A), PPARβ (B) and PPARγ (C). PPAR (α, β, γ) are

594

represented in blue and brown (AF-2 helix), and the chemicals are colored by atom

595

type (carbon in gray, oxygen in red, fluorine in green and sulfur in yellow).

596 597

Figure 5. Effects of PFOS, 6:2 Cl-PFAES and 8:2 Cl-PFAES on adipogenesis in

598

3T3-L1 cells. (A) Oil Red O staining of 3T3-L1 cells after treated with 100 µM PFOS,

599

6:2 Cl-PFAES or 8:2 Cl-PFAES for 10 days. (B) Comparison of lipid contents of

600

PFOS, 6:2 Cl-PFAES and 8:2 Cl-PFAES treated 3T3-L1 cells. The relative

601

triglyceride (TG) content was determined by setting MDI medium with 0.1% DMSO 30


Page 30 of 36

Page 31 of 36


602

(Veh) treated cells as 1. *p < 0.05, compared with Veh. (C) Effects of PFOS, 6:2

603

Cl-PFAES and 8:2 Cl-PFAES on expression of Cebpα, aP2, Adip and Lep genes in

604

3T3-L1 cells. The relative mRNA levels were normalized to β-actin mRNA level. *p

605

< 0.05 compared with Veh (day 10). Data are presented as means ± SE (n = 3).

606 607

31



Figure 1. Structures of 6:2 Cl-PFAES, 8:2 Cl-PFAES and PFOS. 77x40mm (300 x 300 DPI)


Page 32 of 36

Page 33 of 36


Figure 2. Competitive binding curves of PFOS, 6:2 Cl-PFAES and 8:2 Cl-PFAES to PPARα-LBD (A), PPARβLBD (B) and PPARγ-LBD (C). 41x11mm (600 x 600 DPI)



Figure 3. Effects of PFOS, 6:2 Cl-PFAES and 8:2 Cl-PFAES on PPARα (A), PPARβ (B) and PPARγ (C) mediated luciferase reporter gene transcription activity. The relative luciferase activity was determined by setting 0.1% DMSO (Veh) treated cells as 1. 40x10mm (600 x 600 DPI)


Page 34 of 36

Page 35 of 36


Figure 4. Molecular docking results of Decanoic acid (DA), PFOS, 6:2 Cl-PFAES and 8:2 Cl-PFAES with PPARα (A), PPARβ (B) and PPARγ (C). PPAR (α, β, γ) are represented in blue and brown (AF-2 helix), and the chemicals are colored by atom type (carbon in gray, oxygen in red, fluorine in green and sulfur in yellow). 256x439mm (300 x 300 DPI)



Figure 5. Effects of PFOS, 6:2 Cl-PFAES and 8:2 Cl-PFAES on adipogenesis in 3T3-L1 cells. (A) Oil Red O staining of 3T3-L1 cells after treated with 100 µM PFOS, 6:2 Cl-PFAES or 8:2 Cl-PFAES for 10 days. (B) Comparison of lipid contents of PFOS, 6:2 Cl-PFAES and 8:2 Cl-PFAES treated 3T3-L1 cells. The relative triglyceride (TG) content was determined by setting MDI medium with 0.1% DMSO (Veh) treated cells as 1. *p < 0.05, compared with Veh. (C) Effects of PFOS, 6:2 Cl-PFAES and 8:2 Cl-PFAES on expression of Cebpα, aP2, Adip and Lep genes in 3T3-L1 cells. The relative mRNA levels were normalized to β-actin mRNA level. *p < 0.05 compared with Veh (day 10). Data are presented as means ± SE (n = 3). 167x187mm (300 x 300 DPI)


Page 36 of 36

Chlorinated Polyfluorinated Ether Sulfonates Exhibit Higher Activity

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