The evolutionary exploitation of vertebrate peroxisome proliferator

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TBT

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The evolutionary exploitation of vertebrate peroxisome proliferator-activated receptor γ by

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organotins

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Ana M. F. Capitão1,2●, Mónica S. Lopes-Marques1●, Yoichiro Ishii3, Raquel Ruivo1, Elza S. S.

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Fonseca1,2, Inês Páscoa1, Rodolfo P. Jorge1, Mélanie A. G. Barbosa1,2, Youhei Hiromori3,4,

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Takayuki Miyagi3, Tsuyoshi Nakanishi3, Miguel M. Santos1,2* and L. Filipe C. Castro1,2*

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1Interdisciplinary

Centre of Marine and Environmental Research (CIIMAR/CIMAR), University

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of Porto (U.Porto), 4450-208 Matosinhos, Portugal

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2Faculty

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Porto, Portugal

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3Laboratory

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1-25-4 Daigaku-nishi, Gifu, 501-1196, Japan

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4Faculty

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Minamitamagaki, Suzuka, Mie 513-8670, Japan.

of Sciences (FCUP), Department of Biology, University of Porto (U.Porto), 4169-007

of Hygienic Chemistry and Molecular Toxicology, Gifu Pharmaceutical University,

of Pharmaceutical Sciences, Suzuka University of Medical Science 3500-3,

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●Equal

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*Address

contribution correspondence to:

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Luís Filipe Costa Castro and Miguel Santos

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Interdisciplinary Centre for Marine and Environmental Research (CIIMAR)

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Av. General Norton de Matos s/n, 4450-208 Matosinhos, Portugal

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Tel.: +351223401800

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Fax: +351223390608

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E-mail: [email protected]; [email protected]

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Keywords: PPAR; chordates; evolutionary toxicology; obesogens

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Abstract

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Globally persistent man-made chemicals display ever-growing ecosystemic consequences, a

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hallmark of the Anthropocene. In this context, the assessment of how lineage-specific gene

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repertoires influence organism sensitivity towards endocrine disruptors is a central question in

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toxicology. A striking example highlights the role of a group of compounds known as obesogens.

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In mammals, most examples involve the modulation of the nuclear receptor, peroxisome

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proliferator-activated receptor  (PPAR). To address the structural and biological determinants

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of PPARγ exploitation by a model obesogen, tributyltin (TBT), in chordates, we employed

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comparative genomics, transactivation and ligand binding assays, homology modeling and site-

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directed-mutagenesis. We show that the emergence of multiple PPARs (,  and ) in vertebrate

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ancestry coincides with the acquisition of TBT agonist affinity, as can be deduced from the

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conserved transactivation and binding affinity of the chondrichthyan and mammalian PPAR. The

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amphioxus single copy PPAR is irresponsive to TBT; as well as the investigated teleosts, a

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probable consequence of a specific mutational remodeling of the ligand binding pocket. Our

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findings endorse the modulatory ability of man-made chemicals and suggest an evolutionary

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diverse setting, with impacts for environmental risk assessment.

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Introduction

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Over the past decades, various examples from disparate phylogenetic lineages have clearly

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established the central role of nuclear receptor (NR) exploitation by xenobiotics in the generation

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of endocrine imbalances. A critical question is to decipher how variations in NR gene function

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and repertoire underscore variable endocrine responses to xenobiotic exposure1. One of these

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examples has hypothesized that the exposure to an ample group of chemicals (termed obesogens)

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parallels the ongoing obesity epidemic

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extensive and includes a wide variety of compounds including phthalates, bisphenol A and

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organotins 5. Importantly, a string of studies using mice has documented that the obesity

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phenotype induced by obesogens such as tributyltin (TBT), results from the activation of a master

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gene regulator of adipogenesis: the peroxisome proliferator–activated receptor γ (PPARγ)

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In addition to mammals, this class of endocrine disruptors has also been shown to induce lipid

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alterations in other animal phyla 7-13, with several lipid metabolic modifications described upon

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obesogen exposure in teleosts and amphibians 7, 9, 10, 14-16. Yet, while the toxico-metabolic impact

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has been described in various taxa, is it mostly unknown whether these effects underpin conserved

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(or divergent) mechanisms of action, exploited by compounds such as TBT and acting via PPARs

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8, 17, 18.

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partial)

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revealed that TBT is a potent repressor of PPARα and β but inactive on PPARγ 22. In effect,

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animals exhibit conserved and divergent sensitivities to external stimuli yet, the role of PPARγ as

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a direct target of obesogens has been addressed in a minute number of species 3, 7, 23, 24. Thus, the

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extent to which genetic variation underlies such dissimilar responses is far from fully assessed.

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Therefore, we have carried out an extensive examination of PPAR gene diversity in the main

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chordate lineages, including cephalochordates (amphioxus), chondrichthyans (sharks, skates and

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chimaeras), ray-finned fish and amphibians. We surveyed a phylogenetic blueprint to address the

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impact of a model obesogen, TBT, acting via PPARγ, a critical regulator of adipogenesis. By

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investigating representative species of the chordate phylum (Figure S1), we provide a detailed

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The list of confirmed and suspected obesogens is

3, 6, 7.

For instance, while in mammals TBT was shown to act as a PPARγ agonist 7, 19 (although 20, 21,

in the teleost fish Pleuronectes platessa (European plaice) a reporter gene assay

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map of TBT activation/repression of PPARγ, as a proxy for potential obesogenic disorder. Our

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study illustrates the central importance of comparative approaches to unravel the full ecosystem

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impact of endocrine disruptors and contributes for the informed selection of model animal systems

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in human health risk assessments.

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Materials and Methods

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Sequence investigation

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Tblastn and blastp searches using human PPARα (Q07869), PPARβ (Q03181) and

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PPARγ (P37231) as query were performed in the available databases NCBI Ensembl and JGI.

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Ppar sequences were retrieved for the major vertebrate lineages: mammals (Homo sapiens, Mus

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musculus); birds (Gallus gallus) and reptiles (Anolis carolinesis); amphibians (Xenopus

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tropicalis); coelacanths (Latimeria chalumnae); Lepisosteiformes (Lepisosteus oculatus),

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Osteoglossomorpha (Pantodon buchholzi) and Cypriniforme (Danio rerio), Chondrichthyes

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(Callorhinchus milii and Leucoraja erinacea) and for the following invertebrates, tunicates

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(Ciona intestinalis) cephalochordate (Branchiostoma floridae, Branchiostoma lanceolatum,

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Branchiostoma belcheri) (Figure S1 and Table S1 for accession numbers).

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Phylogenetic analysis

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Phylogenetic analysis was performed using the set of sequences through database search

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as well as the isolated sequences. A total of 36 sequences were aligned in MAFFT 25, 26 with L-

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INS-I method. The resulting sequence alignment was stripped of all columns containing 90% gaps

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leaving a total of 515 positions for phylogenetic analysis. Maximum likelihood phylogenetic

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analysis was performed in PhyML V3.0 27 and the evolutionary model was determined using the

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smart model selection (SMS) option resulting in a JTT +G. The branch support was calculated

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using aBayes. The resulting tree was analyzed in Fig Tree V1.3.1 available at

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http://tree.bio.ed.ac.uk/software/figtree/ and rooted with the invertebrate sequences.

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Synteny maps

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Comparative synteny maps were constructed using NCBI Gene database, using as

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reference the latest available genome assemblies for the following species: H. sapiens

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(GCF_000001405.33), L. oculatus (GCF_000242695.1), C. milii (GCF_000165045.1), B.

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floridae (GG666565.1) and B. belcheri (AYSS01004225.)1. For each species, with the exception

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of the cephalochordates that present a sole PPAR, we analysed the genomic location of PPARα,

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PPARβ and PPARγ gene. The genomic locus of each PPAR gene was retrieved, as well as, the

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five contiguous flanking genes to each side, when possible. Following the assembly of the synteny

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maps, we proceeded to identify and localize the corresponding human orthologues of non-

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conserved neighboring genes in the B. floridae and B belcheri. Orthology was determined through

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the Ensembl orthologue-paralogue pipeline and our own phylogenetic analysis (not shown).

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Finally, synteny maps and annotated orthologues were then used to infer the localization of the

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ancestral PPAR gene in the reconstructed genome of the vertebrate ancestor using as reference

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the reconstruction presented by Nakatani and colleagues

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Putnam and colleagues 29.

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Gene isolation and cloning

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and the reconstruction presented by

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Hinge and ligand binding domain (LBD) of PPARγ genes from Homo sapiens, X.

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tropicalis, P. buchholzi, D. rerio, L. oculatus, L. erinacea and PPAR from B. lanceolatum (Table

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S1) were predicted using NCBI's conserved domain database30 and isolated using a polymerase

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chain reaction (PCR) approach (Table S2 for primer sequence). The hinge and LBD were then

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cloned into the pBIND vector to produce a nuclear receptor (NR) LBD-Gal4 hybrid protein. This

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hybrid protein, contain the DNA binding domain (DBD) of Gal4 and acts on an upstream

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activation sequence (UAS) response element. The hinge and LBD of PPARγ from L. erinacea

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was then cloned into the pCold-TF vector (Takara bio) to produce a PPARγ LBD-His6-tagged

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trigger factor hybrid protein (HisTF-LePPARγ). Plasmid sequences were confirmed using Sanger

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sequencing (GATC).

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Homology modeling

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Homology models were calculated in SWISS-MODEL homology modelling workspace

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in alignment mode

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submitted to SWISS-MODEL for homology modelling. Homology models were calculated using

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ProMod3 and human PPARγ crystal structure 3WJ4 was selected as template. All models were

31, 32.

For each species PPARγ sequence corresponding to the LBD was

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evaluated for the following parameters: sequence identity, GQME (Global model Quality

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estimation), QMEAN 33, 34 and rendered as reliable (Table S3). Finally, models were visualized,

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inspected and aligned to human crystal structure 3WJ4 in PyMOL v1.74 35.

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Chemicals and solutions

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All chemicals and reagents were obtained from Sigma-Aldrich unless stated otherwise in

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the text. Three TBT-Cl (tributyltin chloride) and three TPT-Cl (triphenyltin chloride) (250μM,

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100μM and 10μM), three ARA (Arachidonic acid), three EPA (cis-5,8,11,14,17-

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Eicosapentaenoic acid) and three erucic acid (200mM, 100mM and 50mM), one Rosiglitazone

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(Cayman) (1mM) and one T0070907 (Cayman) (1mM) solutions were prepared in DMSO

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(dimethyl sulfoxide).

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Cell culture and in vitro assays

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Cell culture and transactivation assays were performed as described in André et al. 36.

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Briefly, Cos-1 cells were seeded in 24-well culture plates. After 24 hours cells

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were transfected using lipofectamine 2000 reagent (Invitrogen), in Opti-MEM reduced

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serum medium (Gibco, Thermo Fisher), according manufacturer’s indications and 0.5 μg

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of pBIND PPAR LBD-Gal4 or RXR LBD-Gal4 and 1μg of pGL4.31 luciferase reporter

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vector, containing five UAS elements upstream the firefly luciferase reporter gene. For

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heterodimer transfection assays 0.5 μg of pBind PPAR LBD-Gal4, 0.5 μg of pACT and

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1μg of pGL4.31, were used. After 5 hours of incubation the medium was replaced with medium

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containing the test compounds (TBT – 10nM, 100nM and 250nM; TPT– 10nM, 100nM and

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250nM; ARA - 50µM, 100µM and 200µM; EPA - 50µM, 100µM and 200µM; Erucic acid -

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50µM, 100µM and 200µM; Rosiglitazone (Rosi) - 1µM and T0070907 - 1µM) dissolved in

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DMSO (0.1%). DMSO (0.1%) was used as solvent control. 24 hours later cells were lysed and

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the luminescent activities of Firefly luciferase (pGL4.31) and Renilla luciferase (pBIND) were

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determined. All assays were performed, independently, five times and each time two technical

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replicates per condition were performed to validate the results. Two different batches of Cos-1

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cells were used; three replicates per batch were included per assay.

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Ligand Binding Assay

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The L. erinacea PPARγ LBD-His6-tagged trigger factor hybrid protein were expressed

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in Escherichia coli BL21 (DE3) cells and purified by using His-select nickel affinity gel (Sigma

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Aldrich). Ligand binding assay was assessed as previously described (39-41). In brief, the purified

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protein (100, 200, 400 or 800 µg/ml) were incubated with 200 nM of tri[U-14C]phenyltin

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hydroxide ([14C]TPT; radiochemical purity > 96.6 %, 2.04 GBq/mmol; Amersham Biosciences)

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or 50 nM of [3H]rosiglitazone ([3H]rosiglitazone; radiochemical purity > 97 %, 2.168 TBq/mmol;

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PerkinElmer). After incubation at 4 ◦C for 1 h, hydroxyapatite was added to precipitate the

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receptor protein and bound radioactive compounds. The hydroxyapatite pellets was washed and

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then radioactivity in the pellet was determined by liquid scintillation. Binding in the presence of

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a 100-fold molar excess of unlabeled compound was defined as nonspecific binding; specific

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binding was defined as total binding minus nonspecific binding. For competition binding assay,

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the purified protein (800 µg/ml) were incubated with 200 nM of [14C]TPT and increasing

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concentrations of test compounds.

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Statistical analysis

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Transactivation data was normalized dividing by the solvent control (DMSO) and the

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mean of the technical replicates was used in the statistical analysis. One-way ANOVA followed

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by Tukey post hoc test was used to analyze differences when parametric criteria was achieved,

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otherwise a non-parametric Kruskal-wallis ANOVA followed by a Games-Howel test was used.

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All analyses were performed using IBM SPSS Statistics 24. P Thr and Phe363>Met. Additionally, Cys285 is replaced by a tyrosine in

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butterfly fish, though conserved in the spotted gar (Figure 5A). The butterfly fish homology model

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reveals that the aromatic ring of tyrosine projects into the LBP, altering the pocket organization

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as observed in human PPARγ (Figures 5A, D and E). Also, the replacement of Ile281 by a Phe in

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Helix 3 in both spotted gar and butterfly fish results in a second aromatic ring protruding into the

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LBP (Figures 5A, 5D and 5E). We next constructed an homology model of the LBP of zebrafish

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PPARγ, a popular comparative model in lipid endocrine disruption studies

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interacting residues of the PPARγ-LBP in zebrafish are conserved with those of the butterfly fish,

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in line with the incapacity of TBT to bind and transactivate PPARγ (Figures 5A and 5E). Finally,

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B. lanceolatum presents the lowest number of conserved TBT-interacting residues; aside from the

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replacement of the Cys285 by a leucine, we find six additional substitutions, including amino

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acids that were found to be totally conserved in vertebrates such as Phe282>Leu and Phe360>Leu

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(Figure 5G). Overall, our analysis strongly suggests that, Cys285 is essential for PPARγ

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transactivation in the presence of TBT.

9, 10.

The TBT-

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Conserved and derived molecular features in PPARγ exploitation by organotins

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Animal endocrine systems encompass an intricate network of signaling cascades, that

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when perturbated lead to episodes of disruption with developmental and physiological

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impairment 48. In this context, a critical and complex issue is that of how man-made chemicals

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differently exploit the genetic and physiological make-up of animal species and influence

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homeostasis. This question assumes a key importance, since animal model-derived studies are

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essential to evaluate the risk of man-made chemicals in human populations. In recent years, the

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concept of obesogens has gathered increased attention as the obesity epidemic gained pace in

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western societies. The overall feature of these compounds is their capacity to activate the

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mammalian adipogenesis master control gene PPAR; yet, PPAR is largely uncharacterized in

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most vertebrate lineages and in some species seems irresponsive to a model obesogen, TBT 22, 23,

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47.

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heterodimer emerged in the ancestor of vertebrates concomitant with the appearance of PPAR

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(Fig.7). This might result in potential obesogenic impacts at environmental conditions which

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require further investigations. The conversion of activation to a neutral/negative profile observed

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in the tested teleost represents a secondary event dictated by changes in the ligand pocket of

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PPAR. These observations raise a paradoxical scenario. While both mammals and teleosts

Considering this set-up, we show that the TBT agonist capacity towards PPARγ/RXR

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experience obesogenic outcomes upon organotin exposure

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mechanisms of such disruption are possibly different, since the tested teleost PPAR does not

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activate transcription in the presence of TBT

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apparent absence of obesogenic impacts in fish. Yet, TBT was suggested to inhibit teleost

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PPAR/

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accumulation 9. Other hypothesis to consider includes the activation of the heterodimer

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PPARγ/RXR by TBT through the RXR monomer 10, although our in vitro results are contradictory

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with this scenario. Overall, our results pave the way for future functional and in vivo studies to

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investigate the diversity of physiological landscapes resulting from the exposure to obesogens.

22,

22

7, 9, 10, 49

(Fig. 7), the molecular

(this work) (Fig. 7). This would involve an

eventually suppressing lipid oxidation in vivo, which could potentially cause fat

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Fig. 7 – The chordate taxonomic scope of PPARγ exploitation by TBT. PPARs transactivation results in the presence of TBT.

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Acknowledgments

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This work was supported by Norte2020 and FEDER (Coral - Sustainable Ocean Exploitation -

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Norte-01-0145-FEDER-000036), Project No. 031342 co-financed by COMPETE 2020, Portugal

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2020 and the European Union through the ERDF, and by FCT through national funds. We

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acknowledge the Branchiostoma lanceolatum genome consortium for providing access to the

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genome sequence and Fundação para a Ciência e a Tecnologia for the support to AC

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(SFRH/BD/90664/2012), RR (SFRH/BPD/72519/2010) and to EF (SFRH/BD/100262/2014). We

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acknowledge Ana André and Ricardo Capela for their help in the sampling process.

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The authors declare no competing financial interest.

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Supporting Information.

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Additional results and experimental details regarding gene isolation, homology modeling,

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transactivation assays and synteny maps.

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