Adipogenic Activity of Oligomeric Hexafluoropropylene Oxide

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Ecotoxicology and Human Environmental Health

Adipogenic Activity of Oligomeric Hexafluoropropylene Oxide (Perfluorooctanoic Acid Alternative) through Peroxisome Proliferator-Activated Receptor # Pathway Chuan-Hai Li, Xiao-Min Ren, and Liang-Hong Guo Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b06978 • Publication Date (Web): 20 Feb 2019 Downloaded from http://pubs.acs.org on February 21, 2019

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Environmental Science & Technology

1

Adipogenic Activity of Oligomeric Hexafluoropropylene Oxide

2

(Perfluorooctanoic

3

Proliferator-Activated Receptor γ Pathway

Acid

Alternative)

through

Peroxisome

4 5

Chuan-Hai Li1,2, Xiao-Min Ren1, Liang-Hong Guo1,2,3*

6 7

1State

8

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

9

Shuangqing Road, Beijing 100085, China

Key Laboratory of Environmental Chemistry and Eco-toxicology, Research

10

2College

11

Beijing 100039, China

12

3The

13

China

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

Third Affiliated Hospital of Guangzhou Medical University, Guangzhou 510150,

14 15

Corresponding authors:

16

Liang-Hong Guo, Email: [email protected]

17 18

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

19

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

20

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

21

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

1

ACS Paragon Plus Environment


23

ABSTRACT

24

Hexafluoropropylene oxide trimer acid (HFPO-TA) and hexafluoropropylene

25

oxide dimer acid (HFPO-DA) have been used as perfluorooctanoic acid (PFOA)

26

alternatives in the fluoropolymer industry for years. Their widespread environmental

27

distribution, high bioaccumulation capability and human exposure have caused great

28

concern. Nevertheless, their potential toxicity and health risk remain largely unknown.

29

In the present study, we compared potential disruption effects of HFPO-TA,

30

HFPO-DA and PFOA on peroxisome proliferator-activated receptor γ (PPARγ) via

31

the investigation of receptor binding, receptor activity and cell adipogenesis effects.

32

The receptor binding experiment showed HFPO-TA exhibited 4.8-7.5 folds higher

33

binding affinity with PPARγ than PFOA, whereas HFPO-DA exhibited weaker

34

binding affinity than PFOA. They also showed agonistic activity toward PPARγ

35

signaling pathway in HEK 293 cells in the order of HFPO-TA > PFOA > HFPO-DA.

36

Molecular docking simulation indicated HFPO-TA formed more hydrogen bonds than

37

PFOA, whereas HFPO-DA formed fewer hydrogen bonds than PFOA. HFPO-TA

38

promoted adipogenic differentiation and lipid accumulation in both mouse and human

39

preadipocytes with potency higher than PFOA. Adipogenesis in human preadipocytes

40

is a more sensitive end point than mouse preadipocytes. Collectively, HFPO-TA

41

exerts higher binding affinity, agonistic activity and adipogenesis activity than PFOA.

42

The potential health risk of HFPO-TA should be of concern.

43 44

KEY WORDS: PFOA alternative; HFPO-TA; HFPO-DA; PPARγ; binding potency; 2


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agonistic activity; adipogenesis activity

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3



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For Table of Contents Only

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50 51

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INTRODUCTION

54

Perfluorooctanoic acid (PFOA) is extensively used in industrial and consumer

55

products for many decades1. In recent years, however, strict regulations have been

56

issued to reduce the production and usage of PFOA2, 3. The phase-out of PFOA, due

57

to the concerns with its environmental occurrence and human health effects, has led to

58

an increase in the production and use of some novel replacement chemicals4-6.

59

Hexafluoropropylene oxide dimer acid (HFPO-DA) and hexafluoropropylene oxide

60

trimer acid (HFPO-TA), two oligomeric hexafluoropropylene oxides (HFPO, key

61

monomers in organofluorine products), are two novel PFOA alternatives that have

62

been used for fluoropolymer manufacture in recent years7.

63

HFPO-DA has subsequently been widely detected in river waters near

64

fluorochemical production plants in the Netherlands (with the highest concentration of

65

812 ng/L)8, Germany (with the highest concentration of 86.1 ng/L)9, USA (with the

66

mean concentration of 631 ng/L)5, and China (with the highest concentration of 3100

67

ng/L)9. HFPO-TA was also detected in surface waters in the United Kingdom,

68

Germany, Sweden, the Netherlands, the United States, China and Korea (with

69

concentrations range of 0.14 to 5.00 ng/L)10. Furthermore, the maximum

70

concentration of HFPO-TA reached 68.5 μg/L in river waters near a fluoropolymer

71

production plant in China, with concentrations comparable to PFOA11. These reports

72

suggest the potential of widespread environmental distribution of oligomeric HFPO

73

pollution. In addition, recent studies show that HFPO-TA was detected in wild

74

common carp (with the median blood concentration of 1510 ng/mL)11 and in black 5



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spotted frog (with the median liver concentration of 13.4 ng/g ww)12 near a

76

fluoropolymer production plant in China. Moreover, HFPO-TA was also detected in

77

the serum samples (~ 2.93 ng/ml) of the residents living near a fluoropolymer

78

production plant in China11.

79

Extensive studies have demonstrated that PFOA had hepatotoxicity, renal

80

toxicity, immune toxicity, neurotoxicity, genotoxicity, development and endocrine

81

disruption toxicity13. However, compared with PFOA, studies on the toxicological

82

effects of HFPO-TA and HFPO-DA are very scarce. In vivo studies suggest that

83

exposure to relatively low dose of HFPO-TA were more easily accumulated than

84

PFOA via quantified the HFPO-TA content in serum and liver samples and induced

85

more serious hepatotoxicity in mice than PFOA14. In wild common carp and black

86

spotted frog, HFPO-TA also exerted significantly higher bioconcentration factors than

87

PFOA, whereas HFPO-DA was less bioconcentration factors than PFOA11, 12. In vitro

88

studies also suggest that HFPO-TA showed greater toxic effects on cell viabilities of

89

HL-7702 human liver cells and exhibited higher binding potency to human liver fatty

90

acid binding protein (hl-FABP) than PFOA, whereas HFPO-DA showed weaker

91

cytotoxicity and binding potency than PFOA15. However, to the best of our

92

knowledge, the mechanisms of their toxicity are unknown.

93

The molecular mechanisms of PFOA-induced adipogenic toxicity have been

94

proposed to involve peroxisome proliferator-activated receptors γ (PPARγ) pathway13,

95

16, 17.

96

regulator of lipid metabolism, cell proliferation and differentiation18,

PPARγ, a subtype of peroxisome proliferator-activated receptors, is a master

6


19.

By using

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97

transient transfection assays, several in vitro studies investigated the activity of PFOA

98

toward PPARγ, showing that it had significant agonistic activity on PPARγ20, 21. In

99

our previous study using a competitive binding assay and transient transfection assay,

100

we also found PFOA bound to and activated human PPARγ22. Recently, PFOA has

101

been shown to bind to PPARγ, promote adipocyte differentiation in 3T3-L1

102

adipocytes16, and increase the expression level of PPARγ and adipogenesis related

103

genes17. As novel PFOA alternatives with chemical structures similar to PFOA,

104

HFPO-TA and HFPO-DA might have similar adipogenesis effects mediated by

105

PPARγ signaling pathways as PFOA.

106

In the present study, we determined the binding affinity of HFPO-TA and

107

HFPO-DA with human and mouse PPARγ and compared with PFOA. We further

108

tested their activities toward human and mouse PPARγ at the cellular level by using

109

luciferase reporter assays in HEK 293 embryonal kidney cells. Molecular docking

110

analysis was performed to analyze the structural characteristics of their binding and

111

activity to PPARγ. We also investigated their adipogenesis effects and evaluated the

112

expression level of adipogenic related genes both in human and mouse preadipocytes.

113

The primary goal of this study was to evaluate the potential health risks of HFPO-TA

114

and HFPO-DA as PFOA alternatives.

115 116

MATERIALS AND METHODS

117

Chemicals. The perfluorooctanoic acid (PFOA, 96%) and hexafluoropropylene oxide

118

dimer acid (HFPO-DA, 97%) were purchased from Alfa Aesar (Ward Hill, MA). 7



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119

Hexafluoropropylene oxide trimer acid (HFPO-TA, 97%) was purchased from

120

ChemEletronics, Inc. (Inglewood, California, USA) (SI Figure S1). The three

121

chemicals were dissolved in dimethyl sulfoxide (DMSO) to make stock solutions with

122

a concentration of 50 mM. The CMC values of PFOA, HFPO-DA and HFPO-TA

123

were measured23, with 3.1, 6.3 and 1.5 mM, respectively. There were not micelles

124

present at any of the test concentrations of the three studied chemicals in our

125

experiment system. Rosiglitazone (ROSI) was purchased from Sigma-Aldrich (St.

126

Louis, MO). 4,4-difluoro-5-methyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoic acid

127

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

128

Human and mouse PPARγ ligand binding domains (LBDs) were provided by

129

Genscript Biotechnology Co. Ltd. (Nanjing, China). The protein sequence (human

130

PPARγ-LBD:

131

NP_001295283.1, amino acids 207-474) were obtained from the NCBI GenBank (SI

132

Table S1).

133

Fluorescence Competitive Binding Assay. The binding affinity of HFPO-TA,

134

HFPO-DA and PFOA with human and mouse PPARγ-LBDs were measured by

135

fluorescence polarization (FP) competitive binding assay. The competitive binding

136

assay was the same as previously described24. Brieﬂy, 800 nM human and mouse

137

PPARγ-LBDs, 20 nM C1-BODIPY-C12 probe and different concentration of ligand

138

were mixed in Tris-HCl buffer (20 mM Tris-HCl, 100 mM NaCl, pH 7.4) in a total

139

volume of 20 μL. The content of DMSO in the final solution was kept below 1% to

140

avoid solvent effect. After incubation for 5 min at room temperature, the FP value was

NP_005028.4,

amino

acids

201-475;

8


mouse

PPARγ-LBD:

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141

measured and plotted as a function of the ligand concentration. The IC50 value of each

142

ligand was obtained from the competition curve processed by Origin 8.5 (OriginLab,

143

Northampton, MA, USA) using the sigmoidal model.

144

PPARγ Mediated Luciferase Reporter Gene Assay. The activity of HFPO-TA,

145

HFPO-DA and PFOA toward human and mouse PPARγ were determined by

146

PPARγ-mediated luciferase reporter gene assay. The luciferase reporter gene assay

147

was performed according to our previous study24. Brieﬂy, the HEK 293 cells (2 × 105

148

cells/well) were seeded in 24-well plates for 24 h. Then, the cells were transiently

149

transfected

150

pGL4.35[luc2P/9XGAL4UAS/Hygro] vector using Lipofectamine 2000 (Invitrogen).

151

After 24 hours, the wells were replaced with fresh medium containing tested

152

compounds with various concentrations for another 24 hours. The concentrations of

153

HFPO-TA, HFPO-DA and PFOA used in the experiment were noncytotoxic

154

determined by WST-1 assay. The cells were then harvested and measured their

155

luciferase activity using a dual-luciferase reporter assay kit (Promega) and normalized

156

to the renilla luciferase activity.

157

Molecular Docking Analysis. 3D crystal structure of human PPARγ (hPPARγ, PDB

158

ID: 3U9Q) was obtained from the RCSB Protein Data Bank [RCSB (Research

159

Collaboratory for Structural Bioinformatics); PDB (http://www.rcsb.org/pdb)]25. Due

160

to the crystallographic structure of mouse PPARγ (mPPARγ) is not available to date,

161

the crystal structure of mPPARγ was built by homology modeling using Modeller 9v8

162

software26. Structures of the ligands were prepared by ChemBioDraw Ultra and

with

300

ng

of

pBIND-PPARγ

vector

9


and

300

ng

of


163

PRODRG server27. The tested chemicals were docked into PPARγ using Lamarckian

164

genetic algorithm provided by Auto Dock 4.2. The detailed procedures for homology

165

modeling and molecular docking are provided in the SI.

166

Differentiation of Human and Mouse Preadipocytes. The culture and

167

differentiation of mouse 3T3-L1 preadipocyte cells were the same as described in our

168

previous work24. Human preadipocytes-subcutaneous (HPA-s) (ScienCell Research

169

Laboratories, CA, USA) from human subcutaneous fat tissue were cultured in

170

preadipocyte medium (PAM) supplemented with 5% fetal bovine serum (FBS) at 37

171

℃ in a humidified 5% CO2 atmosphere. For differentiation, HPA-s cells were seeded

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in six-well dishes in PAM, containing 5% FBS. After cells reached confluence, they

173

were treated with MDI medium containing 1 μM dexamethasone, 0.5 mM

174

3-isobutyl-1-methylxan-thine (IBMX), and 10 μg/ml insulin in PAM supplemented

175

with 5% FBS. After 2 days, the medium was changed with 10 μg/ml insulin in PAM

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supplemented with 5% FBS. From days 4 to 10, cells were cultured with PAM

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supplemented with 5% FBS and replenished every 2 days. Cell treatments were

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performed on day 0 treated with a range of concentrations for each compound.

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Oil Red O Staining and Analysis. After 10 days of adipocyte differentiation, cells

180

were washed twice with phosphate-buffered saline (PBS) buffer [137 mM sodium

181

chloride (NaCl), 2.7 mM potassium chloride (KCl), 10 mM disodium phosphate

182

(Na2HPO4), and 1.8 mM monopotassium phosphate (KH2PO4), pH 7.4], and fixed

183

using 4% paraformaldehyde for 30 min at 4 ℃ and stained with Oil Red O for 15 min

184

as previously described24. The images of Oil Red O staining cells were taken using an 10


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Olympus CKX41 inverted microscope. The stained oil droplets were extracted in

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isopropanol and quantified at 520 nm by using SpectraMax i3x Multi-mode detection

187

platform (Molecular Devices, CA, USA).

188

RNA Extraction and Quantitative Real-Time PCR. Total RNA was extracted from

189

differentiating human and mouse preadipocytes by using Trizol reagent (Invitrogen,

190

Carlsbad, CA). RNA was reverse transcribed using RevertAid First Strand cDNA

191

Synthesis Kit (ThermoFisher Scientific, Waltham, MA). Quantitative real-time PCR

192

was performed by using the Roche Light Cycler 480 System (Roche Applied Science,

193

Basel, Switzerland) with GoTaq qPCR Master Mix (Promega, Madison, WI). The

194

primers of adipogenic related genes including adipocyte Protein 2 (aP2),

195

CCAAT/enhancer-binding protein alpha (Cebpα), lipoprotein lipase (LPL), leptin

196

(Lep), perilipin (PLIN), adiponectin (Adip) and PPARγ, were listed in the SI Table S2.

197

The relative mRNA level was normalized to β-Actin.

198

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

199

are presented as mean ± SEM. Comparison of mean values among experimental

200

groups were performed with one-way analysis of variance (ANOVA) and Duncan’s

201

multiple range test using SPSS 17.0 software (Chicago, IL, USA), with significance

202

level set at *p < 0.05.

203 204

RESULTS AND DISCUSSION

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Binding Potency of HFPO-TA, HFPO-DA and PFOA with Human and Mouse

206

PPARγ-LBDs. We determined the binding affinity of HFPO-TA, HFPO-DA as well 11



207

as PFOA with human and mouse PPARγ-LBDs by using the FP competitive binding

208

assay. As shown in Figure 1A, PFOA competed the binding of the fluorescence probe

209

to human PPARγ-LBD in a dose-dependent manner with IC50 value of 370.2 μM.

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Like PFOA, HFPO-TA and HFPO-DA bound to human PPARγ-LBD with IC50 value

211

of 49.3 μM and beyond detection, respectively (Figure 1A, Table 1). HFPO-TA

212

exhibited approximately 7.5-fold stronger binding potency than PFOA with human

213

PPARγ-LBD (Table 1). For mouse PPARγ-LBD, PFOA (with IC50 value of 396.2

214

μM), HFPO-TA (with IC50 value of 82.7 μM) and HFPO-DA (with IC50 value of

215

beyond detection) bound to mouse PPARγ-LBD in a dose-dependent manner (Figure

216

1B, Table 1). HFPO-TA showed higher binding potency than PFOA with the RP

217

value of 4.8-fold (Table 1). From the above results, the order of these chemicals in

218

terms of binding potency to both human and mouse PPARγ-LBDs was HFPO-TA >

219

PFOA > HFPO-DA.

220

Several in vitro studies have reported the binding potency of PFOA with human

221

PPARγ22, 28. The binding potency of PFOA with mouse PPARγ was also measured,

222

which was lower or comparable to human PPARγ. As PFOA alternatives with similar

223

chemical structure, HFPO-TA exhibited higher binding affinity with PPARγ than

224

PFOA, whereas HFPO-DA exhibited weaker binding affinity than PFOA, which are

225

similar to the reported results for hl-FABP. By using a fluorescence displacement

226

assay, Sheng et al. found HFPO-TA, HFPO-DA and PFOA could bind to hl-FABP,

227

with the order of binding potency is HFPO-TA > PFOA > HFPO-DA15.

228

Activity of HFPO-TA, HFPO-DA and PFOA toward Human and Mouse PPARγ. 12


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To explore the activity of HFPO-TA, HFPO-DA and PFOA toward PPARγ, human

230

and mouse PPARγ-mediated luciferase reporter gene assays were performed. As

231

shown in SI Figure S2A, rosiglitazone (ROSI), a known PPARγ agonist, strongly

232

enhanced the human and mouse PPARγ-mediated luciferase transcriptional activity in

233

a dose-dependent manner, indicating the accuracy of the method. For human PPARγ

234

transcriptional activity, HFPO-TA, HFPO-DA and PFOA enhanced the transcriptional

235

activity in a dose-dependent manner, with the lowest effective concentrations (LOECs)

236

of 12, 50 and 25 μM and the highest transcriptional activity of 2.7-, 1.2- and 1.4-fold

237

at 50 µM respectively (Figure 2, Figure S2), suggesting they had agonistic activity

238

toward human PPARγ signaling pathway. For mouse PPARγ transcriptional activity,

239

they also showed agonistic activity in a dose-dependent manner (Figure 2). By

240

comparing the activity of human and mouse PPARγ, we found the agonistic effects on

241

the human PPARγ transcriptional activity were higher than those on mouse PPARγ

242

transcriptional activity (Figure S2), which is consistent with the results of the receptor

243

binding assays.

244

As reported previously, PFOA also exhibited human and mouse PPARγ

245

transcriptional activity20-22. By using a luciferase reporter assay based on Hep G2 liver

246

cancer cells, Zhang et al. showed that PFOA had agonistic activity to human PPARγ22.

247

In addition, Buhrke et al. found that PFOA activated human PPARγ approximately

248

1.5-fold at 50 µM in HEK 293 embryonal kidney cells28. Moreover, by using a

249

transient transfection assay in COS-1 cells, Takacs and Abbott found PFOA could

250

activate human PPARγ but not the mouse PPARγ20. Similarly, in the present study, 13



251

we found the agonistic activity on human PPARγ was significantly higher than that on

252

mouse PPARγ. However, by using luciferase reporter assays based on 3T3-L1

253

preadipocyte cells, Vanden Heuvel et al. found that PFOA could activate both human

254

and mouse PPARγ21. This inconsistent effect may be dependent on cell types and

255

reporter system. Our study also showed that HFPO-TA exhibited stronger agonistic

256

activity than PFOA both on human and mouse PPARγ, which is consistent with the

257

result that it had higher binding affinity to PPARγ than PFOA, indicating that the

258

potency for PPARγ activation is dependent on their backbone length, and correlates

259

well with their binding affinity. Like PFOA, HFPO-TA and HFPO-DA showed

260

agonistic effects on human PPARγ transcriptional activity, but weaker agonistic

261

effects on mouse PPARγ, which is consistent with the binding results that they had

262

higher binding affinity to human PPARγ than mouse PPARγ. These results suggest

263

that human PPARγ is probably more responsive than mouse PPARγ. Combining the

264

results of receptor binding and receptor activation assays, it is clear that HFPO-TA

265

exerts higher binding affinity and agonistic activity with PPARγ than PFOA, and

266

human PPARγ is more responsive than the mouse receptor.

267

Molecular Docking of HFPO-TA, HFPO-DA and PFOA Interactions with

268

Human and Mouse PPARγ. To provide some explanation about the structural

269

characteristics of the binding and activity with human and mouse PPARγ, we

270

performed molecular docking analysis on the binding interactions of HFPO-TA,

271

HFPO-DA and PFOA with human and mouse PPARγ. For PPARγ, the ligand binding

272

pocket is a “Y”-shaped ligand-binding cavity and consists of three-arm binding 14


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cavity30, 31. Arm Ⅰ, where the entrance of the ligand binding pocket is located, is a

274

substantially polar cavity which could form hydrogen bonds with the polar acid end

275

group of ligands. Arm Ⅱ and arm Ⅲ, which are located in the inner part of the ligand

276

binding pocket, are two hydrophobic cavities32. The homology modeling structures of

277

mouse PPARγ also showed a similar ligand binding pocket with three-arm binding

278

cavity. The docked structures of decanoic acid (DA) with human and mouse PPARγ

279

were used as the reference for comparison because DA is an endogenous ligand of

280

PPARγ. As shown in Figure 3, DA was docked into human and mouse PPARγ with

281

its hydrophobic chain residing toward the inner part and its polar carboxylic acid end

282

group toward the AF-2 region of the protein. It formed hydrogen bond interactions

283

with residues Ser289 (Helix 3), His323 (Helix 5), His449 (Helix 10) and Tyr473

284

(AF-2) of human PPARγ, and Ser287 (Helix 3) and Tyr471 (AF-2) of mouse PPARγ

285

(Figure 3 and Table 1). The results of endogenous ligand (DA) are in good agreement

286

with the previously published results obtained from crystallographic experiment and

287

molecular docking22, 33.

288

As shown in Figure 3A, for human PPARγ, HFPO-TA, HFPO-DA and PFOA fit

289

into the ligand binding pocket of the receptor with a binding geometry similar to DA,

290

with their hydrophobic fluorinated alkyl chain residing toward the inner part and polar

291

end group residing toward the AF-2 region. HFPO-TA formed hydrogen bonds with

292

residues Ser289, His323, His449 and Tyr473, HFPO-DA formed hydrogen bonds

293

with residues His449 and Tyr473, and PFOA formed hydrogen bonds with residues

294

Ser289, His449 and Tyr473 (Table 1). For mouse PPARγ (Figure 3B), all the three 15



295

compounds fit into the ligand binding pocket of mouse PPARγ with similar binding

296

geometry as DA. They formed hydrogen bonds with the same residues (Ser287 and

297

Tyr471) of mouse PPARγ through their acid end groups.

298

Compared with PFOA, HFPO-TA formed more hydrogen bonds with human

299

PPARγ, and HFPO-DA formed less hydrogen bonds with human PPARγ. This

300

finding might explain the distinct binding affinities among HFPO-TA, HFPO-DA and

301

PFOA. Furthermore, HFPO-TA had higher hydrophobicity (LogKow) than PFOA, and

302

HFPO-DA had lower hydrophobicity than PFOA. Previous studies have shown that

303

hydrophobic interactions play an important role in the PPARγ receptor binding34.

304

HFPO-TA displayed a stronger hydrophobic interaction when fitted into the inner part

305

of the ligand binding pocket, which might help to stabilize the binding conformation.

306

Previous crystal structure studies revealed that the stabilization of the AF-2

307

region plays an important role in the PPARγ receptor activation, which caps the

308

ligand binding pocket closely when agonists are bound30, 35-37. Hydrogen bonds with

309

residues in Helix 3, Helix 5 and Helix 10 are reported to be the key to the ligand

310

binding and activation of PPARγ35, 36. In our study, PFOA, HFPO-DA and HFPO-TA

311

formed hydrogen bonds with these residues, which might result in the agonistic

312

conformation of PPARγ. With human PPARγ, HFPO-TA formed hydrogen bonds

313

with residues in Helix 3, Helix 5, Helix 10 and AF-2 region; HFPO-DA formed

314

hydrogen bonds with residues only in Helix 10 and AF-2 region; PFOA formed

315

hydrogen bonds with residues in Helix 3, Helix 10 and AF-2 region. The number and

316

position of hydrogen bond interactions tend to result in the distinct agonistic activity 16


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with human PPARγ. In addition, these three compounds only formed two hydrogen

318

bonds in Helix 3 and AF-2 region with mouse PPARγ, and formed more hydrogen

319

bonds with human PPARγ (Table 1), which might explain our experimental results

320

that the compounds displayed stronger potency in receptor binding and activation

321

with human PPARγ than the mouse counterpart.

322

Adipogenesis of HFPO-TA, HFPO-DA and PFOA in Human and Mouse

323

Preadipocytes. The activation of PPARγ pathway has been demonstrated to modulate

324

the progression of adipogenesis38. Based on the results of binding assay and

325

transactivation activity assay, we used primary human preadipocytes (HPA-s) and

326

mouse 3T3-L1 preadipocytes (3T3-L1) to study the effects of HFPO-TA, HFPO-DA

327

and PFOA on adipogenesis, which has been shown to be regulated by PPARγ

328

pathways39-41. With HPA-s cells, HFPO-TA, HFPO-DA and PFOA significantly

329

increased lipid accumulation after 10 days of exposure (Figure 4A and B). HFPO-TA

330

showed higher activity of adipogenesis than PFOA, but HFPO-DA showed

331

comparable or weaker adipogenesis activity than PFOA (Figure 4B). HFPO-TA,

332

HFPO-DA and PFOA enhanced the lipid content with the lowest effective

333

concentrations (LOECs) of 1, 6 and 6 μM and the highest adipogenesis activity of 3.9-,

334

1.5- and 1.9-fold at 25 μM, respectively (Figure 4B). With 3T3-L1 cells, all the three

335

compounds enhanced the lipid content with the order of HFPO-TA > PFOA >

336

HFPO-DA (Figure 5A and B). HFPO-TA, HFPO-DA and PFOA had LOECs of 12,

337

25 and 25 μM and maximum adipogenesis activity of 3.7-, 1.4- and 1.8-fold at 50 μM,

338

respectively (Figure 5B). Combining the results of human and mouse preadipocytes, 17



339

HPA-s cells were more responsive to HFPO-TA than 3T3-L1 cells in lipid

340

accumulation.

341

We then measured the mRNA levels of key adipogenic factors after 10 days of

342

exposure in both HPA-s and 3T3-L1 cells. HFPO-TA, HFPO-DA and PFOA

343

significantly increased the expression of adipogenic markers in both HPA-s and

344

3T3-L1 cells (Figure 4C and Figure 5C). With HPA-s cells, HFPO-TA enhanced the

345

expression level of PPARγ, Cebpα, aP2, LPL, Lep and PLIN by 5.2-, 26-, 11-, 10-,

346

2.9-, and 1.9- fold at 25 μM, respectively (Figure 4C). The rank order of the

347

expression level of adipogenic genes is as follows: HFPO-TA > PFOA > HFPO-DA.

348

With 3T3-L1 cells, a similar trend was also observed for the three compounds.

349

Comparing the results of HPA-s and 3T3-L1 cells with HFPO-TA at similar

350

concentration, HPA-s cells showed higher effect than 3T3-L1 cells. By combing the

351

results of Oil Red O staining assay and gene transcription assay, we found HFPO-TA

352

exerts higher adipogenic activity than PFOA, whereas HFPO-DA exerts weaker

353

adipogenic activity than PFOA, which is in line with the results of receptor binding

354

and activation assays. Taken together, our study suggests strongly that HFPO-TA also

355

lead to disruption effects on PPARγ through direct binding and activation of PPARγ,

356

with more potency than PFOA. Recent studies also reported that HFPO-TA showed

357

higher cytotoxicity than PFOA in human liver cells15 and higher bioaccumulation

358

potential and hepatotoxicity in mice14. HFPO-TA exhibited greater toxic effects on

359

human liver HL-7702 cell viabilities and higher binding affinity to hl-FABP than

360

PFOA, whereas HFPO-DA exhibited weaker toxic effects than PFOA15. In our study, 18


Page 18 of 37

Page 19 of 37


361

the cytotoxicity of HFPO-TA on HEK 293 cells, 3T3-L1 cells and HPA-s cells were

362

also higher than PFOA, HFPO-DA exhibited weaker cytotoxicity than PFOA (SI

363

Figure S3-S5), which was also related to their backbone length42. HFPO-TA exerted

364

higher hepatotoxic effects than PFOA on mice after 28 days of exposure via oral

365

gavage, significantly affected lipid metabolism in mouse liver with LOEC of 0.2

366

mg/kg/d (~ 10 μM in serum)14. By comparing the activities of HFPO-TA in human

367

and mouse preadipocytes, we also found human (with LOEC of 1 μM) showed higher

368

adipogenesis activities than mouse (with LOEC of 10 μM), indicating that the effects

369

of adipogenesis are more likely to occur in human bodies. Combining the results of

370

previous studies and our present study, the effect of HFPO-TA on adipogenesis in

371

HPA-s cells might be a more sensitive end point.

372

Toxicological and Health Risk Assessment of HFPO-TA. Toxicological studies

373

have demonstrated HFPO-TA exhibited higher cytotoxicity and hepatotoxicity than

374

PFOA. For example, HFPO-TA had higher bioaccumulation potential than PFOA in

375

common carp11 and black-spotted frog12, had greater cytotoxicity on human liver

376

HL-7702 cells15 and hepatotoxic effects on mice14 than PFOA. We also found that

377

HFPO-TA is more potent than PFOA at binding to and activating mouse and human

378

PPARγ directly and inducing adipocyte differentiation in mouse and primary human

379

preadipocytes. According to the results of previous studies14, 15 and our present study,

380

HFPO-TA has higher activities than PFOA, and PPARγ is the potential target

381

molecule of HFPO-TA. Therefore, we speculate that HFPO-TA might lead to higher

382

potential adverse effects mediated by PPARγ signaling pathways than PFOA in the 19



383

human body. HFPO-TA might not be suitable as a substitute for PFOA and deserve

384

further studies for risk assessment.

385

Previous environmental and human biomonitoring study has shown that the

386

maximum concentrations of PFOA, HFPO-TA and HFPO-DA were 197 μg/L, 68.5

387

μg/L and 2.1 μg/L in river waters near a fluoropolymer production plant in China11.

388

The median concentrations of PFOA, HFPO-TA and HFPO-DA in human serum

389

samples near the fluoropolymer production were 126 ng/mL (~ 304 nM), 2.93 ng/mL

390

(~ 6 nM), and not detected, respectively11. In our human preadipocytes adipogenesis

391

assay, PFOA, HFPO-TA and HFPO-DA enhanced the lipid content with LOECs of 6,

392

1 and 6 μM. Based on the available human biomonitoring data11 and our results, we

393

evaluated the potential health risk of HFPO-TA, HFPO-DA and PFOA by using the

394

hazard quotient (HQ) value43,

395

lowest observed adverse effect level (LOAEL) derived from the differentiation of

396

HPA-s cells with the available human exposure concentrations of the compound.

397

Details of the calculation are provided in the SI. The HQ value of HFPO-TA was 0.59

398

(< 1) for the residents residing near this fluoropolymer production plant, suggesting

399

that the current exposure level might be safe. The HQ value of PFOA was 5.7 (> 1)

400

for the residents, suggesting such serum level in the residents might be of concern.

401

The HQ value of HFPO-DA was far smaller than 1 for the residents, suggesting

402

current exposure concentrations of HFPO-DA might be safe. By comparing the HQ

403

values among HFPO-DA, HFPO-TA and PFOA, currently the potential health risk of

404

HFPO-DA and HFPO-TA are lower than that of PFOA. However, for 20% of these

44.

The HQ value was calculated by comparing the

20


Page 20 of 37

Page 21 of 37


405

residents, their exposure concentration of HFPO-TA was 36.8 ng/mL (~ 88 nM), and

406

the HQ value was 7.4 (> 1), indicating that these people might be at risk. Furthermore,

407

with the regulations on PFOA, the use of HFPO-TA as a PFOA alternative is

408

expected to increase, which would likely result in higher environmental

409

contamination and human exposure levels. Over time, the human exposure level of

410

HFPO-TA might reach a point when the HQ value exceeds 1 and its potential health

411

risk cannot be neglected.

412

In conclusion, to the best of our knowledge, this is the first report for the

413

disruption effects of HFPO-TA on the PPARγ mediated pathway. It exerts higher

414

binding affinity, agonistic activity and adipogenesis activity than PFOA. The

415

combined data from our study strongly suggest that HFPO-TA has more potential

416

adverse effects than PFOA. Its potential health risk should be studied further and

417

should not be overlooked.

418 419

Supporting Information

420

Details of the cell viability assay for HEK 293, 3T3-L1 and HPA-s cells; homology

421

modeling and molecular docking; hazard quotients (HQs). Structures of PFOA,

422

HFPO-DA and HFPO-TA (Figure S1); effects of rosiglitazone, PFOA, HFPO-DA and

423

HFPO-TA on human and mouse PPAR γ mediated luciferase reporter gene

424

transcription activity (Figure S2); the cytotoxicity of PFOA, HFPO-DA and

425

HFPO-TA on HEK 293 cells (Figure S3), 3T3-L1 cells (Figure S4) and HPA-s cells

426

(Figure S5) determined by WST-1 assay. List of protein sequence used for human and 21



427

mouse PPARγ ligand binding domains protein synthesis (Table S1). List of primer

428

pairs used for quantitative real-time PCR (Table S2).

429 430

Notes

431

The authors declare that there are no conflicts of interest.

432 433

Acknowledgments

434

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

435

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

436

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

437

Collaboration Awards for Research Professors.

438

22


Page 22 of 37

Page 23 of 37


440

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10-carbon fatty acid as modulating ligand of peroxisome proliferator-activated

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A. Crystal structure of the ligand binding domain of the human nuclear receptor

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PPARgamma. J. Biol. Chem. 1998, 273 (47), 31108.

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receptors. J. Biol. Chem. 2012, 287 (1), 183-195.

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receptor structures: ligand specificity, molecular switch and interactions with 27



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regulators. Biochim Biophys Acta. 2007, 1771 (8), 915-925.

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Y.; Nettles, K. W.; Griffin, P. R. Partial agonists activate PPAR gamma using a helix

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domain of peroxisome proliferator-activated receptor gamma. J. Biol. Chem. 2006,

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Ligand Binding Domain of PPARγ and Functional Implications. J. Phys. Chem. B.

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2015, 119, 15418–15429.

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

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murine 3T3-L1 preadipocytes. J. Steroid Biochem. Mol. Biol. 2011, 127 (1-2), 9-15.

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(40) Ewy, T.; Peshdary, V.; Gagné, R.; Rowan-Carroll, A.; Yauk, C. L.; Boudreau, A.;

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Atlas, E. Adipogenic Effects and Gene Expression Profiling of Firemaster® 550

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Components in Human Primary Preadipocytes. Environ. Health Perspect. 2017, 125

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A β-D-Glucuronide (BPA-G) on Adipogenesis in Human and Murine Preadipocytes.

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

576

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

577

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

578

2018, 92 (1), 359-369.

579

(43) Ludwicki, J. K.; Góralczyk, K.; Struciński, P.; Wojtyniak, B.; Rabczenko, D.;

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582

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584

polyfluoroalkyl substances in cord blood of newborns in Shanghai, China:

585

Implications for risk assessment. Environ. Int. 2016, 97, 7-14.

29



Page 30 of 37

587

Table 1. LogKow, IC50, RP (relative potency to PFOA) values and hydrogen bond

588

interactions of the tested chemicals for human and mouse PPARγ-LBDs by the

589

competitive binding assay and molecular docking analysis.

590 Human PPARγ

Molecular Chemicals

Mouse PPARγ

LogKow formula

IC50 (μM)

RP

hydrogen bonds

IC50 (μM)

RP

hydrogen bonds

-

-

Ser287, Tyr471

396.2

1

Ser287, Tyr471

NA

NA

Ser287, Tyr471

82.7

4.8

Ser287, Tyr471

Ser289, His323, DA

C10H20O2

-

-

His449, Tyr473 Ser289, His449,

PFOA

C8HF15O2

4.46

370.2

1 Tyr473

HFPO-DA

C6HF11O3

3.93

NA

NA

His449, Tyr473 Ser289, His323,

HFPO-TA

C9HF17O4

6.43

49.3

7.5 His449, Tyr473

591

Note: DA, decanoic acid, an endogenous ligand of PPARγ, was used as the reference

592

for comparison in docking results; -, no information was collected at that particular

593

examination point; NA, means not available (a compound could displace the

594

fluorescence probe from the PPARγ LBD but could not reveal the IC50 value). The

595

LogKow values for the chemicals were determined with ChemBioDraw.

596

30


Page 31 of 37

598


Figure legends:

599 600

Figure 1. Competitive binding curves of PFOA, HFPO-DA and HFPO-TA to human

601

and mouse PPARγ-LBDs.

602 603

Figure 2. Effects of PFOA, HFPO-DA and HFPO-TA on human and mouse PPARγ

604

mediated luciferase reporter gene transcription activity. The relative luciferase activity

605

was determined by setting 0.1% DMSO (Veh) treated cells as 1.

606 607

Figure 3. Molecular docking results of Decanoic acid (DA), PFOA, HFPO-DA and

608

HFPO-TA with human (A) and mouse (B) PPARγ. PPARγ is represented in blue, and

609

the chemicals are colored by atom type (carbon in gray, oxygen in red, fluorine in

610

green and sulfur in yellow).

611 612

Figure 4. Effects of PFOA, HFPO-DA and HFPO-TA on adipogenesis in HPA-s cells.

613

(A) Oil Red O staining of HPA-s cells after treated with 25 µM PFOA, HFPO-DA

614

and HFPO-TA for 10 days. (B) Comparison of lipid contents of PFOA, HFPO-DA

615

and HFPO-TA treated HPA-s cells by Oil Red O staining assay. The relative Oil Red

616

O density was determined by setting MDI medium with 0.1% DMSO (Veh) treated

617

cells as 1. *p < 0.05, compared with Veh. (C) Effects of PFOA, HFPO-DA and

618

HFPO-TA on expression of aP2, Cebpα, PLIN, Lep, LPL and PPARγ genes in HPA-s

619

cells. The relative mRNA levels were normalized to β-Actin mRNA level. *p < 0.05 31



620

compared with Veh (day 10).

621 622

Figure 5. Effects of PFOA, HFPO-DA and HFPO-TA on adipogenesis in 3T3-L1

623

cells. (A) Oil Red O staining of 3T3-L1 cells after treated with 50 µM PFOA,

624

HFPO-DA and HFPO-TA for 10 days. (B) Comparison of lipid contents of PFOA,

625

HFPO-DA and HFPO-TA treated 3T3-L1 cells by Oil Red O staining assay. The

626

relative Oil Red O density was determined by setting MDI medium with 0.1% DMSO

627

(Veh) treated cells as 1. *p < 0.05, compared with Veh. (C) Effects of PFOA,

628

HFPO-DA and HFPO-TA on expression of aP2, Cebpα, Adip, Lep, LPL and PPARγ

629

genes in 3T3-L1 cells. The relative mRNA levels were normalized to β-Actin mRNA

630

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

631

3).

632

32


Page 32 of 37

Page 33 of 37


634 635

Figure 1

636

33



638 639

Figure 2

640

34


Page 34 of 37

Page 35 of 37


642 643

Figure 3

644

35



645 646

Figure 4

36


Page 36 of 37

Page 37 of 37


647 648

Figure 5

649

37


Adipogenic Activity of Oligomeric Hexafluoropropylene Oxide

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