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Ecotoxicology and Human Environmental Health

In Vitro and In Silico Evaluations of Binding Affinities of Perfluoroalkyl Substances to Baikal Seal and Human Peroxisome Proliferator-Activated Receptor # Hiroshi Ishibashi, Hirano Masashi, Eun-Young Kim, and Hisato Iwata Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b07273 • Publication Date (Web): 16 Jan 2019 Downloaded from http://pubs.acs.org on January 17, 2019

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In Vitro and In Silico Evaluations of Binding Affinities of Perfluoroalkyl

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Substances to Baikal Seal and Human Peroxisome Proliferator-Activated

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Receptor α

4 Hiroshi Ishibashi†,‡, Hirano Masashi§, Eun-Young Kimǁ, Hisato Iwata†,*

5 6 7



Center for Marine Environmental Studies (CMES), Ehime University, Bunkyo-cho 2-5,

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Matsuyama 790-8577, Japan ‡ Graduate

School of Agriculture, Ehime University, 3-5-7 Tarumi, Matsuyama 790-8566,

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Japan

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§ Department

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Technology, Kumamoto College, 2627 Hirayama-shinmachi, Yatsushiro, Kumamoto

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866-8501, Japan

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ǁ Department

of Biological and Chemical Systems Engineering, National Institute of

of Life and Nanopharmaceutical Science and Department of Biology, Kyung

Hee University, Hoegi-Dong, Dongdaemun-Gu, Seoul 130-701, Korea

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* Address

correspondence to:

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Hisato Iwata,

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Center for Marine Environmental Studies (CMES), Ehime University, Bunkyo-cho 2-5,

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Matsuyama 790-8577, Japan.

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Tel/Fax: +81-89-927-8172

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E-mail: [email protected]

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ABSTRACT: In this study, we assessed the binding affinities of perfluoroalkyl substances

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(PFASs), including perfluoroalkyl carboxylates (PFCAs) and perfluoroalkyl sulfonates

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(PFSAs), to the ligand-binding domains (LBDs) of Baikal seal (Pusa sibirica; bs) and human

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(h) peroxisome proliferator-activated receptor alpha (PPARα). An in vitro competitive

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binding assay showed that six PFCAs and two PFSAs could bind to recombinant bs and

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hPPARα LBD proteins in a dose-dependent manner. The relative binding affinities (RBAs)

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of PFASs to bsPPARα were as follows: PFOS > PFDA > PFNA > PFUnDA > PFOA >

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PFHxS > PFHpA > PFHxA. The RBAs to bsPPARα showed a significant positive correlation

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with those to hPPARα. In silico PPARα homology modeling predicted that there were two

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ligand-binding pockets (LBPs) in the bsPPARα and hPPARα LBDs. Structure-activity

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relationship analyses suggested that the binding potencies of PFASs to PPARα might depend

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on LBP binding cavity volume, hydrogen bond interactions, the number of perfluorinated

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carbons, and the hydrophobicity of PFASs. Interspecies comparison of the in vitro binding

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affinities revealed that bsPPARα had higher preference for PFASs with long carbon chains

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than that of hPPARα. The in silico docking simulations suggested that the 1st LBP of

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bsPPARα had higher affinities than that of hPPARα; however, the 2nd LBP of bsPPARα had

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lower affinities than that of hPPARα. To our knowledge, this is the first evidence showing

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interspecies differences in the binding of PFASs to PPARαs and their structure-activity

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relationships.

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INTRODUCTION

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Perfluoroalkyl substances (PFASs), such as perfluoroalkyl carboxylates (PFCAs) and

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perfluoroalkyl sulfonates (PFSAs), with different carbon chain length are man-made

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chemicals, which have been globally detected in the environment, humans and wildlife.1–8

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Owing to their high environmental persistence, bioaccumulation potential, and toxic

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properties, perfluorooctane sulfonate (PFOS) and its salts, as well as perfluorooctane sulfonyl

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fluoride have been globally regulated by the Stockholm Convention on Persistent Organic

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Pollutants.9,10 However, the toxic effects and risks of different PFASs in animals, particularly

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the non-model wildlife, are not fully understood.

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Previous studies have shown that PFASs, including PFOS and perfluorooctanoic acid

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(PFOA), exert a wide range of toxic effects, including reproductive and developmental

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toxicity, hepatotoxicity, carcinogenesis, neurotoxicity, immunotoxicity, and hormonal effects,

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in experimental animals.8,11–13 The results of these studies have suggested that adverse effects

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of PFASs are mostly mediated by peroxisome proliferator-activated receptors (PPARs).

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PPARs are members of the ligand-activated nuclear receptor superfamily, and three different 4 ACS Paragon Plus Environment

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PPAR subtypes, PPARα, PPARβ/δ, and PPARγ, have been identified in various animal

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species.14–16 The ligand-PPAR complex heterodimerizes with retinoid X receptor α (RXRα),

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interacts with the peroxisome proliferator-responsive element (PPRE) in the promoter regions

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of target genes, such as cytochrome P450 (CYP) 4A, and consequently regulates their

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transcriptional activity.11 Studies in PPARα gene knockout mice clearly showed that PPARα

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regulated the expression of CYP4A, which can catalyze the oxidation of fatty acids.17,18

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Exposure to PFOA, as well as fatty acids, can also upregulate hepatic CYP4A mRNA and

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protein expression in rats.19 Previous studies using in vitro reporter gene assays have

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indicated differences in the transactivation potencies of PFASs with different carbon chain

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length, including PFOS and PFOA, for human, mouse, and rat PPARα,20–23 suggesting

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significant interspecies differences among PPARα in their ligand preference. However,

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limited data are available on the interspecies differences in PPARα activation by PFASs.

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The Baikal seal (Pusa sibirica), a top predator in Lake Baikal, Russia, accumulates

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high levels of environmental pollutants.24–26 Our research group has previously determined

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the residual levels of various PFASs in the liver and serum of wild Baikal seals, which were

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particularly high for PFOS, perfluorononanoic acid (PFNA), and perfluorodecanoic acid

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(PFDA).27 We have further obtained and sequenced the full-length cDNA of Baikal seal

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PPARα (bsPPARα), showing that PPARα mRNA and a CYP4A-like protein expression were

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upregulated by high levels of PFASs, such as PFNA and PFDA in the liver of wild seals.28

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Our in vitro reporter gene assay, using a bsPPARα expression vector and a PPRE-containing

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reporter vector, has shown that certain PFASs, such as PFOS, PFOA, PFNA, PFDA, and

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PFUnDA, had transactivation capabilities for bsPPARα.29 These results indicated that

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bsPPARα-mediated response might be useful for assessing potential biological/toxicological

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effects of PFASs on the wild Baikal seal population. However, it has not been fully

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investigated whether a molecular event, such as the binding of PFASs to PPARα triggers its

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downstream signaling. Although various studies have reported the in vitro transactivation

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potencies of certain PFASs for PPARα,20–23 it is not known yet whether any of these

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chemicals directly interacts with bsPPARα. Additionally, a previous study identified another

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new ligand-binding pocket (2nd LBP) in the crystal structure of human PPARα (hPPARα),

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besides the classical LBP of hPPARα, 1st LBP.30 However, the binding potencies of PFASs to

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these two LBPs of PPARα remain unclear.

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In this study, we aimed to evaluate the direct binding affinities of PFCAs and PFSAs

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with various carbon chain lengths (C4-C11) to bsPPARα and hPPARα, as well as their

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structure-activity relationships and interspecies differences. We expressed and purified a

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recombinant bsPPARα ligand-binding domain (LBD) protein using an in vitro wheat germ

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cell-free protein synthesis system and measured the direct binding affinities of PFASs to

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bsPPARα and hPPARα LBDs using a competitive binding assay. We further performed in

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silico docking simulation analyses to predict the molecular interactions of PFASs with the 1st

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and 2nd LBPs of bsPPARα and hPPARα LBDs. Finally, we compared the structure-activity

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relationships among PFASs with respect to their binding to bsPPARα and hPPARα LBDs

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and evaluated the interspecies differences.

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MATERIALS AND METHODS Chemicals. Eleven PFASs (eight PFCAs and three PFSAs) were used as test

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compounds, and their full names, providers, and purities are shown in the Supporting

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Information (Table S1). A PPARα agonist,

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[4-[[[2-[3-fluoro-4-(trifluoromethyl)phenyl]-4-methyl-5-thiazolyl]methyl]thio]-2-methylphen

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oxy]acetic acid (GW0742),31,32 used as a positive control ligand, was obtained from Cayman

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Chemical (Ann Arbor, MI, USA). For all PFASs, 250 mM stock solutions were prepared by

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dissolving each chemical in dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO,

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USA), and the final DMSO concentration was adjusted to 1% (v/v). A 1.85 mM stock

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solution of GW0742 was prepared in a similar manner. For the PPARα competitive binding

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assay, test solutions of PFASs (final concentrations: 1.14-2,500 µM) and GW0742

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(0.31-18,500 nM) were prepared by serially diluting the stock solutions with the binding

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assay buffer included in the PPARα binding assay kit (Invitrogen, Tokyo, Japan).

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Plasmid Construction and Recombinant Protein Synthesis. The bsPPARα LBD

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cDNA (National Center for Biotechnology Information accession number: LC072268) was

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amplified by polymerase chain reaction using specific primers, containing XhoI and NotI

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restriction enzyme recognition sites. The amplicon of bsPPARα LBD cDNA was then

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digested with the restriction enzymes and subcloned into the XhoI/NotI site of the

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pEU-E01-GST-TEV-MCS-N2 expression vector (CellFree Sciences Co., Ltd., Ehime,

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Japan), and the subcloned bsPPARα LBD cDNA was sequenced to ensure that no mutations

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were introduced by PCR, as previously reported.28 bsPPARα LBD mRNA was prepared by in

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vitro transcription using SP6 RNA polymerase. A glutathione-S-transferase (GST)-tagged

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bsPPARα LBD protein was synthesized using an in vitro wheat germ cell-free protein

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synthesis system, with some modifications.33 The GST-tagged bsPPARα LBD protein was

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purified on glutathione Sepharose™ 4B (GE Healthcare, Tokyo, Japan), according to the

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manufacturer’s instructions, and the purified yield was confirmed by sodium dodecyl sulfate

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polyacrylamide gel electrophoresis (SDS-PAGE). A recombinant hPPARα LBD protein was

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purchased from Invitrogen.

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PPARα Competitive Binding Assay. The competitive binding of the test chemicals to

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bsPPARα and hPPARα LBD proteins was determined using the LanthaScreen™

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time-resolved fluorescence resonance energy transfer (TR-FRET) PPARα competitive

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binding assay kit, according to the manufacturer's instructions (Invitrogen). Briefly, serially

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diluted solutions of the test chemicals (10 µL) were prepared in a 384-well polypropylene

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assay plate. Then, 5 µL of 4× Fluormone™ Pan-PPAR Green was added into each well,

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followed by the addition of 5 µL of 4× GST-tagged PPARα LBD protein/terbium-labeled

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anti-GST antibody in the TR-FRET assay buffer. The plate was gently mixed and then

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incubated at room temperature for 2 h. TR-FRET was measured at emission wavelengths of

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520 and 495 nm using an Infinite™ F500 fluorescence plate reader (Tecan Japan Co., Ltd.,

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Kanagawa, Japan) with the recommended settings. The results are presented as the means ±

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standard deviations from three independent experiments, in which each consisted of two

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technical replicates for each concentration tested. A binding curve was generated by plotting

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the 520/495 nm ratio versus the log concentration, and the median inhibitory concentration

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(IC50) was determined as the competitor concentration that caused 50% displacement of

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Fluormone™ Pan-PPAR Green using GraphPad Prism™ version 7.0 (GraphPad Software,

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San Diego, CA, USA). Regarding PFBA, PFPeA, and PFBS, which showed weak binding

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potencies to both PPARαs, a binding curve over 50% inhibition was not obtained; thus, the

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IC50 value for each chemical was predicted from a regression line using plots from the

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highest three test concentrations. The relative binding affinity (RBA) of PFASs to bsPPARα

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and hPPARα LBDs was estimated as the ratio of IC50 of PFOA to that of the test chemicals,

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and PFOA was selected as a reference to compare the RBAs among PFASs.

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Homology Modeling and Docking Simulation. Homology modeling of bsPPARα

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LBD protein and docking simulation analysis were performed using the Molecular Operation

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Environment (MOE) program (Chemical Computing Group, Inc., Montreal, QB, Canada), as

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previously described.34,35 The homology model of bsPPARα LBD was constructed based on

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the X-ray crystal structure of the hPPARα LBD bound with WY14643 (Protein Data Bank

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ID: 4BCR), because both of 1st and 2nd LBPs were identified only in this structure.30 Detailed

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procedures are described in the Supporting Information.

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RESULTS AND DISCUSSION Expression of bsPPARα LBD Protein and Binding Affinity of GW0742. An X-ray

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crystallographic structure analysis and in vitro binding assays have shown the utility of

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human and rodent recombinant PPARα LBD proteins expressed in Escherichia coli.36,37

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However, the in vitro wheat germ cell-free protein synthesis technology, developed by Endo

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and Sawasaki,33,38 is advantageous in terms of the quality and quantity of various synthesized

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recombinant proteins. In the present study, a GST-tagged bsPPARα LBD protein was

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synthesized using the in vitro wheat germ cell-free protein synthesis system. SDS-PAGE of

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the purified protein showed a single band of bsPPARα LBD of the expected molecular

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weight (approximately 52 kDa) (Figure S1, lane 4). This result suggested that recombinant

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GST-tagged bsPPARα LBD protein was successfully synthesized using this in vitro system.

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Prior to investigating the binding potential of PFASs to bsPPARα LBD, the binding

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affinity of GW0742, a PPARα agonist, was measured using the LanthaScreen™ TR-FRET

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assay system. In this assay, ligand binding to PPARα LBD was assessed by displacement of a

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fluorescent PPARα ligand (Fluormone™ Pan-PPAR Green) from the receptor, leading to a

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decrease in the FRET signal derived from the interaction of the fluorescent ligand and a

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terbium-labeled antibody with the GST-tag on PPARα.39 Results showed that GW0742

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displaced Fluormone™ Pan-PPAR Green from bsPPARα LBD in a concentration- and

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time-dependent manner (Figure S2A). The IC50 values of GW0742 were 217 nM (95%

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confidence interval: 112-421 nM) at 0.5 h, 260 nM (131-515 nM) at 1 h, 339 nM (162-709

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nM) at 2 h, and 371 nM (168-818 nM) at 3 h. Similarly, GW0742 also showed a binding

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affinity to hPPARα LBD in a concentration- and time-dependent manner (IC50 values: 140,

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184, 207, and 221 nM at 0.5, 1, 2, and 3 h, respectively; Figure S2B), and these values were

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lower than the IC50 value (680 nM) of GW0742.39 Comparison of the IC50 values of GW0742

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for bsPPARα and hPPARα LBDs showed that they were similar at 2 and 3 h, suggesting that

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the reactions reached a steady state after 2-h exposure. These results suggested that both

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bsPPARα LBD, synthesized in vitro in this study, and hPPARα LBD proteins were functional

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and able to bind to the PPARα agonist, GW0742.

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Competitive Binding of PFASs to bsPPARα and hPPARα. Several previous studies

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have shown that PFOS and PFOA can directly bind to a liver fatty acid-binding protein and

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serum binding protein in several animal species.40–48 However, to our knowledge, there are

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limited data on the direct binding of PFASs to the PPARα protein, although a recent study

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using an in vitro binding assay reported the direct binding of PFOS to hPPARα LBD

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protein.40 Thus, in the present study, we assessed the direct binding abilities of PFCAs and

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PFSAs with various numbers of perfluorinated carbons for bsPPARα and hPPARα LBDs

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using the LanthaScreen™ TR-FRET assay. Among the 11 PFASs tested, six PFCAs

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(PFHxA, PFHpA, PFOA, PFNA, PFDA, and PFUnDA) and two PFSAs (PFHxS and PFOS)

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could directly bind to bsPPARα (Figures S3 and S4) and hPPARα LBDs (Figures S5 and S6)

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in a dose-dependent manner. For hPPARα LBD, the IC50 value of PFOS was 237 µM (Table

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1), which is in line with the results of a previous study (IC50: 247.7 µM).40 However, PFASs

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with shorter carbon chains, such as PFBA (number of perfluorinated carbons: 3), PFPeA (4),

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and PFBS (4), exhibited weak binding responses (˂ 50% inhibition) only at high

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concentrations for both PPARα LBDs (Figures S3-S6, A and/or B). To our knowledge, this is

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the first evidence on the direct binding of PFCAs and PFSAs with various numbers of

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perfluorinated carbons to bsPPARα and hPPARα LBDs.

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Structure-Activity Relationships of PFASs and Their Binding to bsPPARα and

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hPPARα. An in vitro competitive binding assay was employed to determine the binding

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affinities of short/long-chain fatty acids to hPPARα LBD and their structure-activity

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relationships.49,50 However, the relationship between the structure of short/long-chain PFASs

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and their PPARα-binding affinities has not been investigated yet. To compare the

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bsPPARα-binding potencies of PFASs, we initially calculated the RBA of each PFAS to

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bsPPARα LBD relative to that of PFOA (Table 1). PFOS showed the highest binding affinity

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to bsPPARα LBD. The order of RBAs for the 11 PFASs was as follows: PFOS (3.75) >

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PFDA (2.24) > PFNA (1.71) > PFUnDA (1.36) > PFOA (1) > PFHxS (0.65) > PFHpA (0.53)

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> PFHxA (0.11) >> PFBA (0.067), PFPeA (0.10), and PFBS (0.043) (Table 1). Our previous

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study has suggested, based on the data obtained from an in vitro reporter gene assay, that the

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number of perfluorinated carbons in PFCAs is one of the factors determining their

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transactivation potencies for bsPPARα.29 Therefore, the current study evaluated the

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relationship between the number of perfluorinated carbons in PFASs and their RBAs.

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Pearson correlation test revealed a positive correlation (p = 0.0001, r = 0.818) between the

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number of perfluorinated carbons and RBAs of PFASs with 3-10 perfluorinated carbons to

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bsPPARα LBD (Figure 1A). For hPPARα LBD, the RBAs of PFASs also increased with the

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increase in the number of perfluorinated carbons (p = 0.0072, Figure 1B), although the RBA

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of PFUnDA (number of perfluorinated carbons: 10) appeared to be an outlier. These results

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suggested that the number of perfluorinated carbons in PFASs, besides the fatty acid chain

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length, might contribute to their binding potencies for bsPPARα and hPPARα LBDs.49

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On the other hand, our previous bsPPARα transactivation assay has indicated that

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PFCAs with more than seven perfluorinated carbons showed a negative correlation with

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PFOA induction equivalent factors (IEFs).29 Comparison of RBAs and IEFs of PFASs that

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were obtained from the bsPPARα-binding and transactivation assays, respectively, revealed

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that IEFs were lower than RBAs for PFASs with more than eight perfluorinated carbons,

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such as PFNA (IEF: 0.61), PFDA (0.37), PFUnDA (0.15), and PFOS (0.26).29 The data from

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the present and previous studies suggest that bsPPARα is activated through direct binding of

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PFASs. However, the high binding affinity but low transactivation potency of PFNA, PFDA,

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PFUnDA, and PFOS suggest that these compounds may be partial antagonists for bsPPARα

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transactivation. Further study is necessary to assess the antagonist potencies of these PFASs

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for bsPPARα transactivation.

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Table 1. In Vitro Binding Potencies of Perfluoroalkyl Carboxylates (PFCAs) and Perfluoroalkyl

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Sulfonates (PFSAs) for the Baikal Seal and Human PPARα Ligand-Binding Domains and

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Interspecies Differences

IC50 (µM)a

RBAb

abbreviation

IC50 ratioc bsPPARα

hPPARα

bsPPARα

hPPARα

PFCAs

PFBA

4,637

3,224

0.067

0.12

1.44

PFPeA

3,036

3,279

0.10

0.11

0.93

PFHxA

2,790 ± 1.31

904 ± 2.13

0.11

0.41

3.08

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PFHpA

586 ± 1.14

275 ± 1.54

0.53

1.35

2.13

PFOA

311 ± 1.40

371 ± 1.62

1

1

0.84

PFNA

182 ± 2.16

277 ± 2.48

1.71

1.34

0.66

PFDA

139 ± 2.41

366 ± 4.57

2.24

1.01

0.38

PFUnDA

228 ± 2.85

3,265 ± 6.38

1.36

0.11

0.070

7,231

7,745

0.043

0.048

0.93

PFHxS

480 ± 1.55

140 ± 1.71

0.65

2.65

3.44

PFOS

83 ± 2.18

237 ± 2.55

3.75

1.57

0.35

PFSAs

PFBS

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aMedian

inhibitory concentration ± standard error: the competitor concentration that causes 50%

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displacement of FluormoneTM Pan-PPAR Green. bRelative binding affinity: IC50 of PFOA/IC50 of

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competitor. cRatio of IC50 for the binding of bsPPARα LBD to IC50 for the binding of hPPARα LBD.

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The IC50 values for PFBA, PFPeA, and PFBS were predicted from a regression line using plots from

244

the highest three test concentrations, because these chemicals showed weak binding potencies to both

245

PPARαs and a binding curve over 50% inhibition was not obtained.

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A

y = 0.271x − 2.06 p = 0.0001, r = 0.818

B

y = 0.233x − 1.67 p = 0.0072, r = 0.615

Log [relative binding affinity (RBA)]

1 0 -1 -2 2 1

PFBA (3) PFPeA (4) PFHxA (5) PFHpA (6) PFOA (7) PFNA (8) PFDA (9) PFUnDA (10) PFUdA (10) PFBS (4) PFBuS (4) PFHxS (6) PFOS (8)

0 -1 -2

3

4

5

6

7

8

9

10

247

Perfluorinated carbon number

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Figure 1. Relationships between the numbers of perfluorinated carbons in perfluoroalkyl

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substances (PFASs) and their relative binding affinities (RBAs) to the (A) Baikal seal PPARα

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ligand-binding domain (LBD) and (B) human PPARα LBD. The number in parentheses next

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to the PFAS name represents the respective number of perfluorinated carbons in the

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molecule.

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The present study also estimated the RBAs of PFASs to hPPARα LBD (Table 1).

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Results showed that PFHxS had the highest RBA value (2.65) for hPPARα LBD, followed by

256

PFOS (1.57), PFHpA (1.35), and PFNA (1.34). Similarly, the hPPARα LBD-binding abilities

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of PFASs also increased with the increase in the number of perfluorinated carbons, except for 17 ACS Paragon Plus Environment

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PFUnDA (Figure 1B). Using an in vitro reporter gene assay, a previous study showed that

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PFCAs with a medium chain length, such as PFHpA (number of perfluorinated carbons: 6)

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and PFOA (7), had a high potential for hPPARα activation, whereas PFCAs with short or

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long carbon chain lengths showed a low potential.20 These results suggested that the

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hPPARα-binding and transactivation profiles of PFCAs were similar and the transactivation

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of hPPARα by PFCAs directly reflected their binding.

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Our previous studies have shown that in silico analysis is a useful tool to understand the

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molecular basis of the binding of environmental chemicals to nuclear receptors.34,35

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Moreover, a previous study reported a new ligand-binding pocket (2nd LBP) interacting with

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WY14643, a typical hPPARα ligand in the crystal structure of hPPARα LBD.30 In this study,

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we calculated the in silico binding scores (S-score, kcal/mol) for the interaction of each PFAS

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with the 1st LBP, the classical LBP,30 and 2nd LBP of bsPPARα and hPPARα LBDs and then

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examined the relationships between these scores and the number of perfluorinated carbons of

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PFASs. To initially construct a homology model for bsPPARα LBD, we used the X-ray

272

crystal structure of hPPARα LBD (PDB code: 4BCR) as a template. Results revealed that the

273

bsPPARα LBD homology model closely resembled the X-ray crystal structure of hPPARα

274

LBD (Figure S7A), showing a low root-mean-square distance (RMSD) value (0.622 Å3).

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Our in silico docking simulation analyses, using bsPPARα (Figure S8A) and hPPARα

276

(Figure S8B) 1st LBP models, showed negative correlations between the number of

277

perfluorinated carbons and S-scores of PFASs. All PFASs fitted into PPARα 1st LBPs of both

278

species (Figures S10 and S11). Moreover, the S-scores of PFHxDA (number of

279

perfluorinated carbons: 15) and PFODA (17) were relatively high and outliers from the

280

regression line (Figure S8A). Detailed evaluation of the S-scores showed that the E_refine

281

values (the sum of van der Waals interaction, electrostatic interaction, and solvation effect

282

energies) in the S-score of PFHxDA (E_refine value: 25.5 kcal/mol) and PFODA (37.8

283

kcal/mol) were much higher than those of the other 14 PFASs (-15.8 to -2.73 kcal/mol).

284

Thus, these energies might be involved in the low binding potentials of PFHxDA and

285

PFODA to bsPPARα 1st LBP. Taken together, these results suggested that the number of

286

perfluorinated carbons (i.e., molecular size) and E_refine values of PFASs might contribute

287

to their binding potencies for bsPPARα and hPPARα 1st LBPs.

288

For bsPPARα 2nd LBP, a significant negative correlation was observed between the

289

number of perfluorinated carbons and S-scores of PFASs (Figure S8C). However, most of

290

PFASs did not fit into the LBP (Figure S12), and their S-scores (-4.90 to -4.05 kcal/mol)

291

were higher than those of bsPPARα 1st LBP (-8.16 to -4.73 kcal/mol) and hPPARα 2nd LBP

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(-6.00 to -4.39 kcal/mol) (Table S2). This might be attributable to the tight space of bsPPARα

293

2nd LBP (244.0 Å3, Figure S9C). Thus, the binding to bsPPARα 2nd LBP might be weaker

294

than that to bsPPARα 1st LBP. hPPARα 2nd LBP model also revealed a significant negative

295

correlation between the number of perfluorinated carbons and S-scores of PFASs (Figure

296

S8D). Although most of PFASs fitted into hPPARα 2nd LBP owing to its greater cavity

297

volume (511.3 Å3, Figure S9C) than that of bsPPARα 2nd LBP, long-chain PFASs, such as

298

PFTeDA (13), PFHxDA (15), and PFODA (17), did not fit into hPPARα 2nd LBP (Figures

299

S13K-S13M). These PFASs had no amino acid residues involved in hydrogen bond

300

interactions (Table S2). The low binding potencies of these long-chain PFASs may be

301

attributable to no-fitting in the cavity and no hydrogen bond formation with the amino acids

302

in hPPARα 2nd LBP.

303

We also examined the relationships between LogKow values and RBAs of PFASs (data

304

not shown). Results showed a similar relationship to that between the number of

305

perfluorinated carbons and RBAs of PFASs (Figure 1). Thus, the hydrophobicity of PFASs,

306

i.e. LogKow values, might also affect the binding capabilities of PFASs to bsPPARα and

307

hPPARα LBDs. Taken together, these results suggested that the binding potencies of PFASs

308

to PPARα might depend on LBP binding cavity volume, hydrogen bond interactions, the

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number of perfluorinated carbons, and hydrophobicity of PFASs. Species Differences in Binding of PFASs to bsPPARα and hPPARα. Several studies

311

have suggested species differences in the transactivation potencies of PFASs for PPARα

312

among rats, mice, and humans.21–23 However, no data are available on the species differences

313

in direct binding of PFASs to PPARα. To compare the binding affinities of PFASs among

314

species, this study estimated the ratio of IC50 values for binding to bsPPARα and hPPARα for

315

each PFAS (Table 1). Results revealed that the ratios for PFASs with five or six

316

perfluorinated carbons were more than 1, whereas the ratios for PFASs with more than seven

317

perfluorinated carbons were ˂ 1. These results suggested that bsPPARα showed preference

318

for PFASs with longer carbon chains, compared to that of hPPARα. We have previously

319

found that the amino-acid sequence of bsPPARα LBD was 96% identical to that of hPPARα

320

LBD.28 A previous study identified the key amino acids involved in hydrogen bond

321

interactions for hPPARα 1st and 2nd LBPs based on their X-ray crystal structure (WY14643,

322

Ser280, Tyr314, His440, and Tyr464 for hPPARα 1st LBP and Glu251, Lys266, His274, and

323

Asp453 for hPPARα 2nd LBP).30 The amino acids of hPPARα 1st and 2nd LBPs were similar

324

to those of bsPPARα 1st and 2nd LBPs (Figure S7). Therefore, these amino acids of PPARα

325

1st and 2nd LBPs did not account for the interspecies differences in the binding of PFASs.

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326

Among the 11 PFASs tested in this study, the IC50 value (228 µM) of PFUnDA for

327

bsPPARα was much lower than that for hPPARα (3,265 µM) (Table 1), suggesting that

328

PFUnDA had the most striking interspecies difference in the binding to PPARα (Figure S14).

329

To understand the molecular basis of interspecies differences in the binding of PFASs to

330

PPARα, we compared the in silico binding scores (S-score) for the interaction of each PFAS

331

with bsPPARα and hPPARα 1st and 2nd LBPs. Results revealed that the S-scores of PFASs

332

for bsPPARα and hPPARα 1st LBPs were plotted to the left side from the 1:1 linear

333

regression line (y = x, Figure S15A); however, those for bsPPARα and hPPARα 2nd LBPs

334

were plotted to the right side from the 1:1 linear regression line (Figure S15B). Among the

335

S-scores of PFASs for PPARα 2nd LBPs, PFUnDA showed the most striking outlier from the

336

1:1 linear regression line (Figure S15B), indicating its stronger binding to hPPARα 2nd LBP

337

than that to bsPPARα 2nd LBP. For most of PFASs, the S-scores for hPPARα 1st LBP were

338

lower than or comparable to those for hPPARα 2nd LBP (Table S2). In contrast, the S-score

339

of PFUnDA for the 2nd LBP was the lowest among those of PFASs, and was lower than that

340

for the 1st LBP (Table S2). These results indicated that PFUnDA preferably bound to

341

hPPARα 2nd LBP. We further identified the key amino acids interacting with PFUnDA in

342

hPPARα 2nd LBP. A previous study examining the X-ray crystal structure of hPPARα

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343

showed that key amino acids, such as Glu251, Lys266, His274, and Asp453 (Figure S7B,

344

green boxes), were involved in hydrogen bond interactions between hPPARα 2nd LBP and

345

WY14643; in addition, polar residues participating in hydrogen bonds were important for

346

stabilizing the ligand-receptor interaction.30 Our results showed that PFUnDA formed a

347

hydrogen bond with a polar residue (Thr279); however, the other PFASs mainly formed a

348

hydrogen bond with a non-polar residue (Cys275) (Table S2). Moreover, the E_refine value

349

of PFUnDA (E_refine value: -21.1 kcal/mol) was lower than those of other PFASs (-20.2 to

350

-10.0 kcal/mol). Thus, the higher binding potency of PFUnDA for hPPARα 2nd LBP might be

351

attributable to the formation of a hydrogen bond with a polar residue and the low E_refine

352

value in the 2nd LBP. Since the binding affinity in this study was measured based on the

353

competition between the tested PFASs and Fluormone™ Pan-PPAR Green, PFUnDA might

354

not have fully competed with the fluorescent probe in the 1st LBP, leading to an apparently

355

weak binding of PFUnDA to hPPARα. Taken together, these results suggested that there

356

were little interspecies differences in the relative binding potencies of PFASs with various

357

carbon chain lengths (C4-C11) for the bsPPARα and hPPARα LBD proteins (Figure S14),

358

although there might be interspecies differences in their absolute binding potencies to each

359

LBP (Figure S15).

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360

We finally compared between the S-scores of PFASs determined using in silico

361

docking simulations (PPARα 1st and 2nd LBPs) and their RBAs determined using in vitro

362

PPARα binding assays. Results revealed a significant negative correlation between the

363

S-scores of PFASs for bsPPARα 1st and 2nd LBPs and their RBAs determined using in vitro

364

bsPPARα-binding assays (Figures 2A and 2C). In addition, the RBAs of PFASs determined

365

using in vitro hPPARα-binding assays also negatively correlated with the in silico S-scores of

366

PFASs (Figures 2B and 2D), although PFUnDA unexpectedly showed a low RBA value.

367

These results suggested that in silico docking simulation may be a useful tool for screening

368

potential ligands for bsPPARα and hPPARα 1st and 2nd LBPs.

369

Overall, our study provided the first evidence of interspecies differences in the binding

370

of PFASs to PPARαs and their structure-activity relationships. However, the toxicological

371

implications from the binding of PFASs to the PPARα remain unknown. In a previous study,

372

we have measured the hepatic concentrations of total 10 PFASs (14-143 ng/g wet weight) in

373

wild Baikal seals.27 For example, the hepatic concentrations of PFOS (mean 11.4 ng/g wet

374

weight) in the seals27 were much lower than the IC50 value of PFOS (83 µM = 42 µg/g) for

375

bsPPARα-binding affinity (Table 1). However, the wild Baikal seals are chronically exposed

376

to a mixture of PFASs.27 The mixture may additively and/or synergistically affect the

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377

bsPPARα signaling pathway even at the low concentration of each PFAS, since the mixture

378

concentration including unknown ligands may reach the effective level at which bsPPARα is

379

activated.28 Exploring how the binding of PFASs to PPARα is transferred into their

380

downstream responses will be the next challenge for understanding the toxic effects of these

381

compounds.

Figure 2

382 383 2

22

A Log [relative binding affinity (RBA)]

1

00

-1

-1-1

-2

-8

-7

-6

-5

y = −0.980x − 5.34 p = 0.0032, r = 0.683

11

0

-4

-2-2 -6.5 -6.5

-6.0 -6.0

-5.5 -5.5

-5.0 -5.0

-4.5 -4.5

-4.0 -4.0

2

2

y = −2.63x − 11.6 p = 0.0117, r = 0.525

C 1

D 0

-1

-1

-4.8

-4.6

-4.4

-4.2

-4.0

-3.8

-2 -6.5

PFBA PFBA(3) (3) PFPeA PFPeA(4) (4) PFHxA PFHxA(5) (5) PFHpA PFHpA(6) (6) PFOA PFOA(7) (7) PFNA PFNA(8) (8) PFDA PFDA(9) (9) PFUnDA (10) PFUdA (10) PFBS PFBuS (4)(4) PFHxS PFHxS(6) (6) PFOS PFOS(8) (8)

y = −1.26x − 6.52 p = 0.0322, r = 0.456

1

0

-2 -5.0

384

B

y = −0.923x − 5.76 p < 0.0001, r = 0.825

-6.0

-5.5

-5.0

-4.5

-4.0

Docking simulation (S-score, kcal/mol)

385

Figure 2. Relationship between the S-scores obtained by in silico docking simulation and the

386

relative binding affinities (RBAs) of perfluoroalkyl substances (PFASs), obtained using an in

387

vitro Baikal seal (bs) PPARα- and human (h) PPARα-binding assay. (A) bsPPARα 1st ligand

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388

binding pocket (LBP), (B) hPPARα 1st LBP, (C) bsPPARα 2nd LBP, and (D) hPPARα 2nd

389

LBP. The number in parentheses next to the PFAS name represents the respective number of

390

perfluorinated carbons in the molecule.

391 392 393

ACKNOWLEDGMENTS This study was supported by Grant-in-Aid for Scientific Research (S) (no. 21221004 &

394

26220103) and (B) (no. 24380179 & 16H05057) from Japan Society for the Promotion of

395

Science (JSPS), and “Joint Usage/Research Center – Leading Academia in Marine and

396

Environment Pollution Research (LaMer)” from the Ministry of Education, Culture, Sports,

397

Science and Technology (MEXT), Japan. Financial assistance was also provided in part by

398

Basic Research in ExTEND2005 (Enhanced Tack on Endocrine Disruption) from the

399

Ministry of Environment, Japan. We would like to thank Editage (www.editage.jp) for

400

English language editing.

401 402

ASSOCIATED CONTENT

403

Supporting Information

404

Table S1: PFASs used in this study. Table S2: Interaction energies and amino acids predicted

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405

to participate in hydrogen bonding. Figure S1: Expression of the GST-tagged bsPPARα LBD

406

protein. Figure S2: Binding affinities of GW0742 to the bsPPARα and hPPARα LBDs.

407

Figure S3: Binding affinities of PFCAs to the bsPPARα LBD. Figure S4: Binding affinities

408

of PFSAs to the bsPPARα LBD. Figure S5: Binding affinities of PFCAs to the hPPARα

409

LBD. Figure S6: Binding affinities of PFSAs to the hPPARα LBD. Figure S7: Structural

410

comparison of bsPPARα and hPPARα LBDs. Figure S8: Relationships between the

411

perfluorinated carbon number of PFASs and the S-score. Figure 9S: Predicted cavity volume

412

of bsPPARα and hPPARα 1st and 2nd LBPs. Figure S10: Docking poses of PFASs to

413

bsPPARα 1st LBP. Figure S11: Docking poses of PFASs to hPPARα 1st LBP. Figure S12:

414

Docking poses of PFASs to bsPPARα 2nd LBP. Figure S13: Docking poses of PFASs to

415

hPPARα 2nd LBP. Figure S14: Relationship between the IC50 values of PFASs for the

416

binding to bsPPARα and hPPARα LBDs. Figure S15: Relationships between the S-scores of

417

the in silico docking of PFASs to bsPPARα and hPPARα. This material is available free of

418

charge via the Internet at http://pubs.acs.org.

419 420

AUTHOR INFORMATION

421

Corresponding Author

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422

*Phone: +81-89-927-8172; e-mail: [email protected]

423

Notes

424

The authors declare no competing financial interest.

425 426

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