Chlorinated Polyfluorinated Ether Sulfonates Exhibit Higher Activity

Feb 1, 2018 - Chlorinated polyfluorinated ether sulfonates (Cl-PFAESs) are the alternative products of perfluorooctanesulfonate (PFOS) in the metal pl...
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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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Chlorinated Polyfluorinated Ether Sulfonates Exhibit Higher Activity towards

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Peroxisome

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Perfluorooctane Sulfonate

Proliferator-Activated

Receptors

Signaling

Pathways

than

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

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Liang-Hong Guo1,2*, Guibin Jiang1,2

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1

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

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

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Shuangqing Road, Beijing 100085, P. R. China

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2

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Beijing 100039, P. R. China

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

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Corresponding authors:

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Xiao-Min Ren, Email: xmren@rcees.ac.cn

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Liang-Hong Guo, Email: LHGuo@rcees.ac.cn

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

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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,

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P. R. China. Telephone/Fax: 86 010 62849685. E-mail: LHGuo@rcees.ac.cn

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ABSTRACT

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

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

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

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

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

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

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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

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

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

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

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

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

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

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

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

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

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potential health risks of Cl-PFAESs as PFOS alternatives.

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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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INTRODUCTION

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

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

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

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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

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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

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

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products (Figure 1).

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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,

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

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

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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

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

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

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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

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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

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

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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

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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

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

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

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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

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

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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

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

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insufficient for comprehensive assessment of their health risk. Meanwhile,

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

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toxicity are unknown.

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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

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

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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

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

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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

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

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

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subsequent signaling pathways have been well demonstrated by many in vitro

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

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

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

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adverse effects.

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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.

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

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

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

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

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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

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binding and activity towards PPARs.

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MATERIALS AND METHODS

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

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

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

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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

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

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

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procedure described previously7. The three chemicals were dissolved in dimethyl

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

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

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

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

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

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

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USA).

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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

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(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

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

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

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experiments were at the exponential growth phase.

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

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

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

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detailed procedure for competitive binding assay is provided in the supporting

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information.

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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

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

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

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transient transfection are provided in the supporting information.

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

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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

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

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

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

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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

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

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

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transcriptional

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

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

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

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

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

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

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reference was determined by 2-∆∆Ct method37.

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

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

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RCSB (Research Collaboratory for Structural Bioinformatics) Protein Data Bank

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

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

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

levels

of

four

adipogenesis

related

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

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in the supporting information.

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

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

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

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

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

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

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set at *p < 0.05.

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RESULTS AND DISCUSSION

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

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

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

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

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

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

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

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

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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

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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

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

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

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

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

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

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

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

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

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

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PPAR-LBDs directly.

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

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

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

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

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

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

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

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

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

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

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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

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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

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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

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

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

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Cl-PFAESs could activate the PPARs signaling pathways.

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

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

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

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

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

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

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

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

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

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

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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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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towards PPARs.

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Effects

of

Cl-PFAESs

on

adipogenesis

in

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pre-adipocytes.

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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.

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

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

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

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

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

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

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

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

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

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

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

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

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

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WY14643 and GW501516 significantly enhanced adipogenesis by about 2.39- and

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

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potential contribution of PPARα and PPARβ pathways in adipocyte differentiation,

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

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

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

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

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

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

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

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

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

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

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

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

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higher agonistic activity towards PPARs signaling pathways than PFOS. By

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

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

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

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

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

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adipogenesis by activating the PPARs signaling pathways with PPARγ pathway

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playing the predominant role.

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

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

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

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

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

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

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

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

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

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

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

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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

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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

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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

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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

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

565 566 567

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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

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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

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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

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(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

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

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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)

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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)

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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)

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Environmental Science & Technology

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)

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