(Ursus maritimus) Peroxisome Proliferator-Activated Receptor Gamma

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Environmental chemicals modulate polar bear (Ursus maritimus) peroxisome proliferator-activated receptor gamma (PPARG) and adipogenesis in vitro Heli Routti, Roger Lille-Langøy, Mari Katrine Berg, Trine Fink, Mikael Harju, Kurt Kristiansen, Pawel Rostkowski, Marte Rusten, Ingebrigt Sylte, Lene Øygarden, and Anders Goksøyr Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b03020 • Publication Date (Web): 07 Sep 2016 Downloaded from http://pubs.acs.org on September 13, 2016

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Environmental Science & Technology

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Environmental chemicals modulate polar bear

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(Ursus maritimus) peroxisome proliferator-activated

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receptor gamma (PPARG) and adipogenesis in vitro

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Heli Routti *,†, Roger Lille-Langøy ‡, Mari K. Berg †,‡, Trine Fink §, Mikael Harju ||, Kurt

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Kristiansen ┴, Pawel Rostkowski ╡, Marte Rusten ‡,∇, Ingebrigt Sylte ┴, Lene Øygarden †,‡,

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Anders Goksøyr ‡

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† Norwegian Polar Institute, Fram Centre, 9296 Tromsø, Norway

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‡ Department of Biology, University of Bergen, 5020 Bergen, Norway

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§ Department of Health Science and Technology, Aalborg University, 9220 Aalborg, Denmark

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||

Norwegian Institute for Air Research, Fram Centre, 9296 Tromsø, Norway

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┴ Department of Medical Biology, Faculty of Health Sciences, UiT - The Arctic University of

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Norway, 9037 Tromsø, Norway

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╡ Norwegian Institute for Air Research, 2007 Kjeller, Norway

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ACS Paragon Plus Environment

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ABSTRACT

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We studied interactions between polar bear peroxisome proliferator-activated receptor gamma

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(pbPPARG) and selected compounds using a luciferase reporter assay and predictions through

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molecular docking. Furthermore, we studied adipogenesis by liver and adipose tissue extracts

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from a polar bear and three synthetic mixtures of contaminants in murine 3T3-L1 preadipocytes

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and polar bear adipose tissue-derived stem cells (pbASCs). PCB153 and p,p’-DDE antagonized

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pbPPARG,

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tetrabromobisphenol A, and PCB170 had a weak agonistic effect on pbPPARG, while

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hexabromocyclododecane, bisphenol A, oxychlordane, and endosulfan were weak antagonists.

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pbPPARG-mediated luciferase activity was suppressed by synthetic contaminant mixtures

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reflecting levels measured in polar bear adipose tissue, as were transcript levels of PPARG and

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the PPARG target gene fatty acid binding protein 4 (FABP4) in pbASCs. Contaminant extracts

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from polar bear tissues enhanced triglyceride accumulation in murine 3T3-L1 cells and pbASCs,

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whereas triglyceride accumulation was not affected by the synthetic mixtures. Chemical

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characterization of extracts using non-target methods revealed presence of exogenous

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compounds that have previously been reported to induce adipogenesis. These compounds

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included phthalates, tonalide, and nonylphenol. In conclusion, major legacy contaminants in

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polar bear adipose tissue exert antagonistic effects on PPARG, but adipogenesis by a mixture

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containing emerging compounds may be enhanced through PPARG or other pathways.

although

their

predicted

receptor-ligand

affinity

was

weak.

PBDEs,


2

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INTRODUCTION

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The polar bear (Ursus maritimus) is an apex predator of arctic marine ecosystems that uses sea

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ice as a platform to hunt its prey, namely ringed seals.1 During ringed seal pupping and the

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moulting period in spring, polar bears feed excessively and accumulate energy reserves to

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survive periods with limited access to food.2 In the absence of sea ice, polar bears go through

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extensive fasting periods, which may be prolonged up to eight months combined with subsequent

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denning by pregnant females.2,3 During fasting periods polar bears depend almost entirely on

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energy stored in white adipose tissue.2,4

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White adipose tissue consists mainly of adipocytes, whose primary function is to store lipids in

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periods of excess of energy and mobilize lipids during energy deprivation.5,6 The process where

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undifferentiated mesenchymal cells differentiate into preadipocytes and further into lipid-filled

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adipocytes is called adipogenesis.5 Adipogenesis and promotion of lipid stores proceeds in two

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waves, as studied extensively using the murine 3T3-L1 preadipocyte cell line as a model.7,8 The

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first wave of adipogenesis is induced by an adipogenic cocktail and involves several

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transcription factors such as CCAAT/enhancer-binding protein (C/EBP) β and –δ, the

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glucocorticoid receptor, and the cAMP-response element binding protein (CREB). Furthermore,

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the first wave induces the second wave, during which the cells go through terminal

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differentiation through activation of the nuclear receptor peroxisome proliferator-activated

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receptor gamma (PPARG, NR1C1). PPARG, that forms a heterodimer with retinoic X receptor,

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is the major regulator of adipogenesis and promotion of lipid stores, but it also plays a role in

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other cell types such as macrophages, and several kidney and bone cell types.7 Endogenous

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PPARG agonists include fatty acids and their derivatives, and oxidized polyunsaturated lipids.9,10


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Polar bears’ ability to store enough energy and to survive during sea ice free periods is

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challenged by global climate change and decline of Arctic sea ice, which has already been linked

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to negative impacts on survival and reproduction.11 Another anthropogenic factor, namely

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pollution, may also affect polar bear energy homeostasis negatively. The polar bear is among the

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most polluted arctic species due to its position at the top of the Arctic marine food web.12,13 In

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particular, persistent organic pollutants (POPs), including polychlorinated biphenyls (PCBs),

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polybrominated diphenyl ethers (PBDEs), and chlorinated pesticides, as well as their

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metabolites, are found at high concentrations in polar bears.14 Because over 90% of the body

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burden of POPs in polar bears is stored in white adipose tissue,15 adipose tissue is at special risk

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for toxic effects of pollutants. Recent in vitro studies suggest that several POPs found in polar

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bear adipose tissue may affect adipogenesis in mice.16-18 In addition, other synthetic chemicals

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including organotins, and phthalates can activate PPARG, and many of them also induce

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adipogenesis in vitro and in vivo.18-22 Furthermore, several studies suggest associations between

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exposure to POPs among other endocrine disrupting chemicals with common metabolic

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problems in humans, such as obesity and type 2 diabetes.23-25

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We hypothesize that contaminants in polar bears may target PPARG and disrupt adipose tissue

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functions. The aim of this study was to study interactions between polar bear PPARG

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(pbPPARG) and selected contaminants at concentrations reflecting levels measured in polar bear

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adipose tissue; the contaminants as single compounds and in mixtures. In order to construct a

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transactivation assay, we cloned and sequenced pbPPARG from polar bear adipose tissue

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mRNA. Transactivation of pbPPARG was examined using a luciferase reporter assay. Ligand

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binding affinity was predicted by a docking and scoring strategy. Furthermore, we investigated

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the effect of both extracts and synthetic mixtures of POPs on the first and second wave of


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adipogenesis using 3T3-L1 cells. POPs in the extracts were quantified using target analyses

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whereas the presence of emerging and endogenous compounds was characterized using

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suspect/non-target analytical screening methods. We also established a method using polar bear

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adipose tissue-derived stem cells (pbASCs) to study adipogenesis by exposure to extracts and

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synthetic mixtures in these cells.

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EXPERIMENTAL

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Preparation of polar bear tissue extracts and synthetic mixtures

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Preparation of polar bear tissue extracts, quantification of POPs and subsequent preparation of

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synthetic mixtures is described in detail in supporting information. Briefly, contaminants were

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extracted from liver and adipose tissue from a healthy 10-year-old male bear from Svalbard,

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Norway. The samples were homogenized in dry Na2SO4, extracted with acetone/cyclohexane

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and cleaned by adding the lipid extracts onto a 30 cm long semipermeable membrane device.

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Further cleanup was achieved using a high performance liquid chromatography (HPLC) system

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utilizing gel permeation chromatography and a column packed with activated Florisil,

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fractionating the sample into three fractions. The first fraction contained neutral compounds such

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as PCBs, PBDEs and organochlorine pesticides, while the second and third fraction contained

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MeSO2-PCBs/DDE and hydroxylated (OH) PCBs/phenols, respectively. The volume of the final

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solvent, dimethyl sulphoxide (DMSO) was adjusted until the concentrations of the extracts were

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equivalent to 100 times the concentrations measured in polar bear tissues. Three separate

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synthetic mixtures including 1) 44 neutral POPs, 2) 10 neutral POPs, and 3) 16 MeSO2-

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metabolites of POPs were composed according to the concentrations and composition found in


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polar bear adipose tissue and dissolved in DMSO (Table S1). The mixture of 10 neutral POPs

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contained only compounds present at more than 0.1µM in the 44 neutral POP mixture.

104 105

Suspect/Non-target screening

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Extracts of liver and adipose tissue were prepared for suspect/non-target screening and

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analyses were conducted by an ultra-high performance liquid chromatography system coupled to

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a high resolution quadrupole time-of flight mass spectrometer (Agilent 6550) as described in

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supporting information. The data was processed with various modules part of the Agilent Mass

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Hunter data processing platform (ver. B.07). The levels of confidence were set as previously

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proposed.26,27 Briefly, confidence level 1 means that the structure is confirmed by a reference

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standard. Level 2 refers to probable structure based on a library match and diagnostic evidence,

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and level 3 means that the structure is tentative. At confidence level 4 the molecular formula is

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unequivocal, whereas at confidence level 5 the compounds mass is of interest but there is no

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match with a reference standard or with a tentative candidate. We performed a systematic

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literature search to find out which compounds identified in the polar bear extracts have

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previously been reported to enhance adipogenesis. These compounds or closely related

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compounds were classified as “compounds of special interest” and we made a particular effort

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for improving their confidence level.

120 121

Identification, cloning, and sequencing of polar bear PPARG

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Tissue sampling, RNA extraction, cDNA synthesis, and amplification of two distinct isoforms

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of pbPPARG cloned from liver and adipose tissue is described in supporting information. To


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evaluate the phylogeny of pbPPARG2 cloned from adipose tissue, nuclear receptor amino acid

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sequences were aligned using Clustal Omega.28

126 127

Luciferase reporter assay (LRA)

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Construction of a LRA plasmid encoding a fusion protein of the GAL4 DNA-binding domain

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(DBD) and the ligand-binding domain (LBD) of pbPPARG2 is described in detail in supporting

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information. Briefly, COS7 cells were transiently co-transfected with pCMX-GAL4-pbPPARG,

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tk(MH100)x4-luciferase, and pCMV-β-galactosidase at a mass ratio of 1:2:2 using the

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TransIT®-LT1 Transfection Reagent (Mirus Bio, Madison, WI, USA). The cells were exposed

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to test chemicals diluted in exposure medium for 24 hours. After exposure, the cells were lysed,

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and enzymatic activity was measured. Test compounds for agonistic effects included BDE28,

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BDE47, BDE99, BDE100, and BDE153, hexabromocyclododecane (HBCDD), bisphenol A

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(BPA), tetrabromobisphenol A (TBBPA), PCB118, PCB138, PCB153, PCB170, p,p’-DDE, and

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endosulfan. We assessed antagonistic effects of BPA, HBCDD, PCB-153, p,p’-DDE,

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oxychlordane, and endosulfan by exposing the cells to the test compounds in the presence of 0.5

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µM of a known PPARG ligand, rosiglitazone (Cayman Chemical, Tallinn, Estonia). We also

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tested agonistic and antagonistic effects of the mixture of neutral POPs, and the mixture of

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MeSO2-metabolites of POPs. Functionality of the LRA was established by exposure to

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rosiglitazone, and antagonists, bisphenol A diglycidylether (BADGE), and GW-9662 (Sigma

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Aldrich).29-31 All compounds for agonistic effects were tested in the range of (5 x 10-3) – 25 µM.

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Antagonistic effects were tested in the range of (9.6 x 10-3) – 50 µM. All compounds were

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dissolved in DMSO, and DMSO percentages were kept at 1% or below. In PPARG agonism and

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antagonism assays general cell viability was monitored through stability of β-galactosidase


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activities. To assess cell viability for antagonistic testing, we repeated the experiments in the

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presence of 10 µM of rosiglitazone. Potential toxicity of rosiglitazone was tested by Cytotoxicity

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detection kit LDH (LDH; Roche, Oslo, Norway). Effects of solvent (up to 2.5% DMSO) on the

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metabolic activity (CDFA-AM) and membrane integrity (resazurin reduction) of COS7 cells was

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perfomed.32

152 153

Modeling of PPARG-ligand interactions

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Polar bear PPARG ligand binding potential of environmental contaminants was studied using

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protein-ligand docking and scoring methods described in supporting information. Briefly, 3D X-

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ray structures of human PPARG (LBD 100% identical) in complex with structurally diverse

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ligands were used for docking into the LBD of pbPPARG. A molecular mechanical approach

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was used to calculate the potential for receptor binding of the test compounds to h/pbPPARG

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LBD. The virtual ligand screening scoring function was used to predict binding of test

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compounds to PPARG relative to data achieved for the ligands in the X-ray crystal structures.

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Test compounds with binding scores below -31 were considered as compounds with high

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putative binding affinities towards pbPPARG.

163 164 165

3T3-L1 differentiation assay

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Detailed methods for 3T3-L1 assay are described in supporting information. Briefly, to study

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how extracts and synthetic mixtures affect adipogenesis in 3T3-L1 cells through the first wave of

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the differentiation process,7 adipogenesis was carried out without the first-wave adipogenic

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mixture (isobutylmethylxanthine [IBMX], dexamethazone [DEX], and insulin). Effect of tissue


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extracts and synthetic mixtures on differentiation of 3T3-L1 cells through the first wave was

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determined after exposure to culture medium including 1 µg/ml insulin and test mixtures,

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DMSO, or rosiglitazone throughout a 10-day growth period. Contaminant extracts and synthetic

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mixtures dissolved in DMSO were diluted 1:100 (v/v) to Dulbecco´s Modified Eagle′s Medium

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(DMEM) to give a final concentration of contaminants in the culture medium equivalent to the

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concentrations found in polar bear tissues, and a solvent concentration of 1% (v/v). To

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investigate a dose-response effect, the contaminant mixtures were further diluted 1:3, 1:9, and

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1:27. The DMSO used for negative control in experiments with tissue extracts was subjected to

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the same extraction and clean-up steps as the polar bear tissue extracts. Rosiglitazone (1 µM in

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DMSO) was used as positive control. For our maximum adipogenic differentiation control, the

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culture medium given at day 0 contained 1 µg/ml insulin, 1 µM rosiglitazone, 500 µM IBMX

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and 1 µM DEX. The content of triglycerides in the 3T3-L1 cells was quantified

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spectrophotometrically (OD570nm, Adipogenesis Detection Kit Abcam, Cambridge, UK).

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Cytotoxicity was assessed by measuring the amount of lactate dehydrogenase released into the

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culture medium using Cytotoxicity Detection Kit Plus (LDH; Roche).

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Further, we studied the effect of synthetic mixtures on terminal differentiation, a process

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primarily driven by PPARG.7 Effect of synthetic mixtures of contaminants on terminal

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differentiation of 3T3-L1 cells was determined by OilRed O33 staining in cells given culture

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medium added adipogenic cocktail (500 µM IBMX, 250 nM DEX and 5 µg/ml insulin) at day 0,

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culture medium added 5 µg/ml insulin and the tested mixtures (or controls) at day 2 and culture

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medium during days 4-10. Synthetic mixtures gave a final concentration equivalent to

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concentrations in polar bear tissues, and a solvent concentration of 1% (v/v). To study dose-

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response relationships, the mixtures were further diluted 1:2 and 1:10.


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Adipogenic differentiation of polar bear adipose tissue-derived stem cells (pbASCs)

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Isolation, growth and establishment of suitable conditions for adipogenic differentiation of

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pbASCs are described in supporting information. pbASCs were seeded in 96-well plates at a

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density of 6400 cells/well (20,000 cells/cm2) and cultured to 100 % confluency (day -2). After

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two days adipogenic differentiation was induced in adipogenic medium (culture medium

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supplemented with 1 µg/mL insulin, 0.45 mM IBMX, 0.1 µM DEX), also containing the tested

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extract, synthetic mixture, or controls. The adipogenic media was changed day 3, 7 and 10. For

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exposures, the adipose tissue extract and the mixture of 10 neutral POPs in DMSO were diluted

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1:100 (v/v) in the adipogenic medium and the highest concentration in the medium was thus

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equivalent to the concentrations quantified in the polar bear adipose tissue (Table S1).

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Differentiation control cells were given 1 µM rosiglitazone while solvent control cells were

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given 1% (v/v) DMSO in addition to adipogenic medium. Undifferentiated control cells were

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maintained in basal medium (culture medium supplemented with 1 µg/mL insulin and 1%

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DMSO) without adipogenic medium. Experiments with synthetic mixtures at three different

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concentration levels were conducted using pbASCs originating from the two polar bears. They

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were seeded in triplicates, and the experiments were repeated twice. Due to a limited amount of

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extract, experiments with extract from polar bear adipose tissue were conducted at one

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concentration level using pbASCs from the 10 year male polar bear, and repeated three times.

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Controls were seeded in triplicates, while cells receiving the extracts were seeded in duplicates.

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At day 14, the accumulation of triglycerides in pbASCs was quantified spectrophotometically

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(OD570nm, Adipogenesis Detection Kit, Abcam).


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Transcriptional activity of PPARG and its target gene FABP4 was studied in pbASCs

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following exposure to the mixture of 10 neutral POPs for 14 days. Adipogenic differentiation of

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pbASCs was performed in 12-well plates and otherwise conducted as described above. Detailed

218

methods are given in supporting information.

219 220

Data analyses

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Statistical analysis was carried out using R version 3.2.2.34 Agonistic luciferase data were

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normalized over (divided by) solvent control (DMSO) and antagonistic data over positive control

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(0.5 µM rosiglitazone). Triglyceride levels in 3T3-L1 and pbASCs and transcript levels of

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PPARG and FABP4 were normalized over levels in solvent control.

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Effects of contaminants and their mixtures on transactivation of pbPPARG, transcriptional

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activity of PPARG and FABP4 in pbASCs and triglyceride accumulation in 3T3-L1 cells and

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pbASCs was investigated using linear models. Linear models were run separately for each

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compound/mixture. Exposure concentration was used as a categorical explanatory variable and

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each exposure concentration was compared to the control using treatment contrasts. The null

230

hypothesis was rejected at α=0.05. Diagnostic plots of residuals were used to verify that the

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model assumptions were met (most importantly constant variance between residuals).35

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In one model for agonistic effects (BDE153) on pbPPARG transactivation the diagnostic plots

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of residuals revealed an outlier, which was omitted for further statistical analysis. The omittance

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of the one extreme value of BDE153 led to a significant positive effect at the 25 µM

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concentration, but the estimate was likely more robust without this outlier. Relative pbPPARG-

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mediated luciferase activity for agonistic and antagonistic effects were calculated from β(Cmax test

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compound)/

β(rosiglitazone) *100 and β(Cmax test compound)/ β(GW9662) *100, respectively. Relative triglyceride


11


test compound)/

β(max

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accumulation was calculated from β(Cmax

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difference in fold change between the maximal concentration and the control based on linear

240

models.

change)

*100.

β is the estimated

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Dose-response curves for transactivation of pbPPARG were calculated using R library drc

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version 2.5.12.36 The curves were fitted using a four parameter log-logistic model.36,37 To choose

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a best-fitting model for compounds showing >20% relative increase/decrease in luciferase

244

activity compared to rosiglitazone/GW9662, we compared three models using a lack-of-fit F-

245

test.38 These models included 1) a four parameter model, 2) a three parameter model where the

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upper plateau was fixed to the upper asymptote of rosiglitazone (agonistic effects) or the lower

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plateau to lower asymptote of GW9662 (antagonistic effects), and 3) a two parameter model

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where the lower and upper asymptotes were fixed to lower and upper plateaus of

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rosiglitazone/GW9662

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corresponding to a response midway between the estimates of the lower and upper plateaus

251

(EC50 for agonist, IC50 for antagonist) were determined based on nonlinear least-squares

252

estimates of the parameters of these models. EC/IC50 was calculated only for compounds

253

showing

254

rosiglitazone/GW9662. Dose-response models were also used to determine 20% of minimum

255

response induced by known ligands.

>20%

for

relative

agonistic/antagonistic

increase/decrease

in

effects.

In

luciferase

addition,

activity

concentrations

compared

to

256


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RESULTS AND DISCUSSION

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Two PPARG isoforms in polar bears

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PPARG was cloned from liver and adipose tissue, and encoded to 475 and 505 amino acid

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(AA) proteins respectively (Figure S1). The shorter PPARG cloned from liver tissue (equivalent

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to hPPARG1), lacks the initial 30 AAs when compared to adipose tissue PPARG (equivalent to

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hPPARG2). This corresponds well to human PPARG, where the hPPARG1 is 28 AAs shorter

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than PPARG2. In many vertebrates, PPARG exists as at least two isoforms called PPARG1 and

264

PPARG2. PPARG2 is expressed predominantly in adipose tissue as well as in macrophages,

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whereas PPARG1 is more widely expressed throughout the body.39,40 Of the two isoforms, the

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longer PPARG2 is expressed only at very low levels in liver.41 Compared to the PPARG coding

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sequence predicted from a polar bear genome sequencing (GenBank: XM_008697869)42, the

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longer adipose tissue PPARG lacked the initial 24 AA.

269

Comparison of PPARG in polar bear to PPARG in other mammals revealed strong

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conservation, with overall AA identities of 98.2% (human), 99.6% (dog) and 97% (mouse)

271

(Figure S2). The comparison of these PPARG orthologs confirms the variability of the N-

272

terminal A/B-region, and the high degree of conservation (100%) of the DBD (C-region) and

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hinge (D-region). While the LBD of PPARG from human, dog, and polar bear was identical,

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three amino acids were substituted in the LBD of mouse PPARG. The high degree of

275

conservation underlines the importance of this receptor and implies that PPARG orthologs would

276

be expected to have similar activation patterns.

277 278

Validation of the transactivation assay


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For the ligand transactivation assay, we used the hinge and LBD domain of PPARG. Thus, the

280

results from the assay should represent both the short and the long pbPPARG forms. In the

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GAL4-construct system, the risk of cross-reactivity by other cellular pathways is highly

282

reduced,43 and we can thus assume that any activity we saw was due to the activation of

283

pbPPARG-LBD and not nuclear receptors found endogenously in the cell. This assumption is

284

supported by the results of the agonistic and antagonistic effects of known ligands on pbPPARG

285

activation (Table 1, Figure S3). In the transiently transfected COS7 cells, luciferase activities

286

increased approximately 8-fold when exposed to rosiglitazone, whereas rosiglitazone-induced

287

(0.5 µM) luciferase production was inhibited by the known PPARG-antagonists GW9662 and

288

BADGE (Table 1). EC50 and IC50 for rosiglitazone and GW9662 (Table 1) were comparable to

289

previous studies using GAL4 systems.30,44,45 BADGE exposure led to a similar decrease of

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luciferase activity as GW9662, although at higher exposure concentrations (Table 1B, Figure

291

S3B). This is in accordance with previous studies that have characterized BADGE as a low-

292

affinity antagonist.29,46 Thus, the pbPPARG LRA appears to produce results comparable to those

293

of previous studies.

294

The relatively constant levels of β-galactosidase activities observed in cells exposed to

295

different concentrations of test compounds (not shown), indicated that the rate of transcription

296

and translation in the COS7 cells was unaffected by the exposures. At 1% and lower DMSO

297

concentration, no significant reduction in metabolic activity (CDFA-AM) or membrane integrity

298

(Resazurin reduction) was seen. Furthermore, antagonistic effects of test compounds were less

299

pronounced in the presence of a higher concentration (10 µM) of rosiglitazone (results not

300

shown). Thus, the viability of the cells appeared not to be severely affected by exposure of test

301

compounds.


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302 303

Activation and predicted binding of polar bear PPARG by brominated compounds

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With the exception of bisphenol A and HBCDD, all the tested individual brominated

305

compounds had a weak agonistic effect on pbPPARG (Table 1A, Figure S3A).. The most potent

306

pbPPARG agonist in the test panel was TBBPA, which induced reporter gene transcription 2.5-

307

fold at the highest exposure concentration, which was only about one fifth of the maximal

308

activity induced by rosiglitazone (Table 1A). Of the other brominated flame retardants, all five

309

PBDEs had weak agonistic activity on pbPPARG in our system and the most potent PBDE was

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BDE-47 (Table 1A). The measured agonistic effects of BDE47 and TBBPA are supported by

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previous studies which report induction of hPPARG by BDE47 and/or TBBPA in a similar

312

system to ours and HEK293H cells stably expressing GAL4-PPARG-LBD.18,20 Furthermore,

313

TBBPA had agonistic activity in stably-transfected HGELN-cells with full-length hPPARG.47

314

Induction of hPPARG by BDE47 and TBBPA, but not BDE99 and BDE100 as shown in this

315

study, has also been shown by using hPPARG-CALUX assay based on human osteosarcoma

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(U2OS) cell lines.48 Our results, as well as previous in vitro studies,18,20,48 refutes the hypothesis

317

by Luthe et al.,49 which suggests that PBDEs would not interact with PPARs based on a

318

theoretical structure-activity assessment.

319

In contrast to the other brominated compounds tested in our study, BPA and HBCDD

320

decreased pbPPARG-mediated luciferase activity induced by rosiglitazone (Table 1B, Figure

321

S3B). In a previous study, no BPA-mediated human PPARG antagonism was observed.50 The

322

discrepancy between the results may be related to detailed differences in experimental set-up.

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Docking of brominated compounds predicted weak receptor-ligand affinity for the brominated

324

compound in this study (binding score < -31). This corresponds to previous in vitro and


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modeling studies which show that brominated compounds have weak binding potency to

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hPPARG LBD compared to rosiglitazone.47,51 Moreover, the results from the docking analysis

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were in agreement with the weak agonistic potential of brominated compounds demonstrated in

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the luciferase reporter gene assays.

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Activation and predicted binding of polar bear PPARG by chlorinated compounds

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The chlorinated compounds included in this study had very weak or no agonistic potential on

332

pbPPARG, however, many of them had antagonistic properties towards pbPPARG (Table 1,

333

Figure S3). PCB153 seems to be a full antagonist with similar efficacy but lower potency

334

compared to that of GW9662 (Table 1B). Furthermore, PCB153 reduced the basal luciferase

335

activity in the absence of rosiglitazone (Table 1A) which suggests that PCB153 is an inverse

336

pbPPARG agonist

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luciferase reporter gene by PCB153 has also been observed in NIH-3T3 cells transfected with

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murine PPARG LBD coupled with GAL4.17 Exposure to PCB170 led to a weak increase in

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pbPPARG-induced luciferase activity (Table 1A). A similar tendency was also observed

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following PCB138 exposure. The different effects of PCB170 and PCB138 compared to PCB153

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may be due to their different chlorine-substitution patterns. PCB153 has no vicinal H-atoms,

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while PCB170 and PCB138 possess vicinal H-atoms in ortho-meta positions. Exposure to

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PCB118, the only tested PCB that easily acquires a planar configuration, did not lead to any

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significant changes in luciferase activity.

52

in the luciferase reporter gene assay. Reduction of basal expression of the

345

The chlorinated pesticides p,p’-DDE, oxychlordane and endosulfan showed antagonistic

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properties towards PPARG (Table 1B, Figure S3B,). p,p’-DDE appears to be a full antagonist

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with similar efficacy but lesser activity per dosing equivalent than GW9662 (Table 1B).


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Furthermore, and in contrast to PCB153, p,p’-DDE acted as a silent antagonist, as it did not

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reduce basal luciferase activity in absence of rosiglitazone (Table 1A). Oxychlordane partially

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decreased rosiglitazone-induced luciferase activity, the efficacy corresponding to one fourth of

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the maximal GW9662-induced inhibition (Table 1B). Endosulfan seemed to be a partial agonist

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(Table 1A) that also acted as a competitive antagonist in the presence of the full agonist

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rosiglitazone (Table 1B).

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Docking revealed that the chlorinated compounds which showed antagonistic activity did not

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show strong binding affinity towards pbPPARG LBD (score < -31). PPARG antagonism, as

356

observed in our in vitro results, may be acquired through both covalent and weaker non-covalent

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modification of LBD.30,53,54 It is likely that most ligands interact with LBD through non-covalent

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interactions only. A molecule with a rigid and bulky distal phenyl, benzyl, or phenethyl

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substituent is important for non-covalent PPARG antagonist activity, which is strengthened to

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full antagonism by a chlorine substituent at the distal ring.54 These rigid molecules prevent helix

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12 to adopt an active state conformation53,55 which is needed for stable binding of ligand and

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recruitment of coactivators of PPARG.56 The full antagonists identified in our study, PCB153

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and p,p’-DDE, are both rigid bulky chlorine substituted molecules. This could explain their

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antagonistic potential towards pbPPARG in vitro, although they were not predicted as strong

365

binders.

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Synthetic POP mixtures suppress polar bear PPARG

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Exposure to synthetic mixtures of neutral and MeSO2-POPs suppressed the activity of

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pbPPARG (Table 1B, Figure S3B). Relative inhibition at highest exposure concentration,

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corresponding to those identified in polar bear adipose tissue, was 30-50% of the maximal


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inhibition induced by GW9662 (Table 1B). Interestingly, the IC50 for the neutral 10 POP

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mixture was ~10 µM (concentration of summed compounds), which was slightly lower than the

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concentrations in adipose tissue of the male polar bear from Svalbard in which the mixture was

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based on (Table S1). However, 10 µM is within the range of average POP concentrations found

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in adipose tissue of polar bears from circumpolar subpopulations.14

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The inhibition at highest exposure concentrations was identical following exposure to the

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mixture of 44 and 10 neutral POPs. However, the inhibitory effect occurred at a lower

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concentration with the 44 compound mixture than with the 10 compound mixture (Figure S3B).

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The latter mixture contains only compounds present at >0.1 µM concentration in the 44 neutral

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POP mixture. This suggests that the compounds found at

(Ursus maritimus) Peroxisome Proliferator-Activated Receptor Gamma

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