The Echinodermata PPAR: Functional characterization and exploitation by the model lipid homeostasis regulator tributyltinā˜†

https://doi.org/10.1016/j.envpol.2020.114467Get rights and content

Highlights

  • ā€¢

    TBT is a known modulator of lipid homeostasis in mammals.

  • ā€¢

    The previous studies in the field are mainly focus in vertebrates.

  • ā€¢

    First time an Echinodermata gene orthologue of PPAR characterized.

  • ā€¢

    TBT modulates PPAR/RXR and alter gene expression and lipid profile in P.Ā lividus.

Abstract

The wide ecological relevance of lipid homeostasis modulators in the environment has been increasingly acknowledged. Tributyltin (TBT), for instance, was shown to cause lipid modulation, not only in mammals, but also in fish, molluscs, arthropods and rotifers. In vertebrates, TBT is known to interact with a nuclear receptor heterodimer module, formed by the retinoid X receptor (RXR) and the peroxisome proliferator-activated receptor (PPAR). These modulate the expression of genes involved in lipid homeostasis. In the present work, we isolated for the first time the complete coding region of the Echinodermata (Paracentrotus lividus) gene orthologues of PPAR and RXR and evaluated the ability of a model lipid homeostasis modulator, TBT, to interfere with the lipid metabolism in this species. Our results demonstrate that TBT alters the gonadal fatty acid composition and gene expression patterns: yielding sex-specific responses in fatty acid levels, including the decrease of eicosapentaenoic acid (C20:5 n-3, EPA) in males, and increase of arachidonic acid (20:4n-6, ARA) in females, and upregulation of long-chain acyl-CoA synthetase (acsl), ppar and rxr. Furthermore, an inĀ vitro test using COS-1Ā cells as host and chimeric receptors with the ligand binding domain (LBD) of P.Ā lividus PPAR and RXR shows that organotins (TBT and TPT (Triphenyltin)) suppressed activity of the heterodimer PPAR/RXR in a concentration-dependent manner. Together, these results suggest that TBT acts as a lipid homeostasis modulator at environmentally relevant concentrations in Echinodermata and highlight a possible conserved mode of action via the PPAR/RXR heterodimer.

Introduction

Sea urchins, members of the Echinodermata phylum, play an important ecological role in ecosystem functioning through their grazing activity that controls the algae biomass (GonzĆ”lez-Irusta etĀ al., 2010, Ribeiro etĀ al., 2015, Romero etĀ al., 2016). From an economic standpoint, species such as the herbivorous Paracentrotus lividus, widely distributed in the Atlantic and Mediterranean coasts (Arafa etĀ al., 2012, Carboni etĀ al., 2012, Kabeya etĀ al., 2017), also hold a high commercial value, as their gonads are considered a gastronomic delicacy (Arafa etĀ al., 2012, Guidetti, 2004, Guidetti etĀ al., 2004, Shpigel etĀ al., 2005a).

The sea urchin gonads are rich in lipids, carbohydrates and proteins (Archana and Babu, 2016). Among lipids, fatty acids display essential roles in gonad maturation and larvae development (Carboni etĀ al., 2013). Before reaching the feeding stage, sea urchin embryos use nutrients provided by the egg. This makes maternal provisions, including essential fatty acids, crucial for embryo development and offspring success (Carboni etĀ al., 2013). In agreement, previous studies found that total lipid levels were maintained constant prior to hatching; yet, they decreased after the digestion of the envelope, between free-swimming blastula and the first feeding stages (Sewell, 2005, Smith etĀ al., 2008). Among fatty acids, several long-chain are known to play an important role in larvae development: docosahexaenoic acid (C22:6 n-3, DHA), eicosapentaenoic acid (C20:5 n-3, EPA), and arachidonic acid (20:4n-6, ARA) (Carboni etĀ al., 2013). The sea urchin P.Ā lividus is able to synthetize ARA and EPA from the precursors linoleic acid (18: 2n-6, LA) and Ī±-linolenic acid (18: 3n-3, ALA) (Kabeya etĀ al., 2017). Moreover, the equilibrium between n-6 and n-3 fatty acids is important from the economic standpoint since the imbalance between n-6 and n-3 consumption in humans can lead to health disorders, such as cardiovascular diseases (GonzĆ”lez-MaƱƔn etĀ al., 2012, RincĆ³n-Cervera etĀ al., 2016).

Animal lipid composition is not static and can be altered by diet, life-cycle stage or external stimuli (Arafa etĀ al., 2012). Recently, several environmental chemicals were shown to modulate lipid homeostasis (Castro and Santos, 2014, De Cock and Van de Bor, 2014, Diamanti-Kandarakis etĀ al., 2009, GrĆ¼n and Blumberg, 2009, JordĆ£o etĀ al., 2016b, Lyssimachou etĀ al., 2015, Ouadah-Boussouf and Babin, 2016, Santos etĀ al., 2012). Those compounds, able to interfere with lipid homeostasis in favour of lipid storage are commonly known as obesogens and were primarily found to modulate lipid homeostasis in mammals; yet, recent studies suggested that their scope of action transcends mammals, or even vertebrates (CapitĆ£o etĀ al., 2017, Janer etĀ al., 2007, JordĆ£o etĀ al., 2015, Lyssimachou etĀ al., 2009). Tributyltin (TBT), an endocrine disrupting chemical (EDC), is a well-recognised model of lipid homeostasis modulator. Although TBT is no longer used as biocide in anti-fouling paint for boats, its levels are still high in some areas: reaching 241,8Ā Ī¼g Sn/Kg, as reported in biological samples from one of the largest harbour regions in China (Chen etĀ al., 2017). TBT was found to disrupt mammalian lipid homeostasis through the interaction with the nuclear receptors peroxisome proliferator-activated receptor Ī³ (PPARĪ³) and retinoid X receptor (RXR) (CapitĆ£o etĀ al., 2018, Harada etĀ al., 2015, Hiromori etĀ al., 2009, le Maire etĀ al., 2009). PPARĪ³ and RXR cooperate in the form of a permissive heterodimer (PPARĪ³/RXR) that is considered a master regulator of lipid homeostasis in vertebrates (Ahmadian etĀ al., 2013, Berkenstam and Gustafsson, 2005, Hiromori etĀ al., 2015, Janesick and Blumberg, 2011, Ouadah-Boussouf and Babin, 2016, Santos etĀ al., 2012). PPARĪ³ is member of a nuclear receptor superfamily that in vertebrates include also peroxisome proliferator-activated receptor Ī± and Ī² (PPARĪ± and PPARĪ²). PPARs act as ligand-activated transcription factors by the binding of specific ligands, such as fatty acids, inducing a conformational change that cause the replacement of corepressors with coactivators triggering the transcription of specific genes from pathways involved in lipid homeostasis (EcheverrĆ­a etĀ al., 2016, Lodhi and Semenkovich, 2014, Tyagi and Gupta, 2011).

TBT was identified and shown to modulate RXR transactivation in several invertebrate groups, including annelids (AndrĆ© etĀ al., 2017), gastropods (Nishikawa etĀ al., 2004), crustaceans (Wang and LeBlanc, 2009) and rotifers (Lee etĀ al., 2019). In vertebrates, TBT, and the related organotin, triphenyltin (TPT), were also shown to interact with PPARĪ³ exhibiting a similar binding mode as that described for RXR: through the interaction of the tin atom and a cysteine residue (Harada etĀ al., 2015, Hiromori etĀ al., 2009, le Maire etĀ al., 2009). Outside deuterostomes, PPAR was only reported in the mollusk Crassostrea gigas; yet, its characterization was limited to in silico analysis (Vogeler etĀ al., 2014, Vogeler etĀ al., 2017). Although nuclear receptors (NRs) are widespread in metazoans (Bridgham etĀ al., 2010, Santos etĀ al., 2018), the available functional data has limited taxonomic scope (Castro and Santos, 2014, Santos etĀ al., 2018; Tan and Palli, 2008, Thornton, 2003, Vogeler etĀ al., 2017). Since echinoderms are deuterostomes (Lavado etĀ al., 2006), the characterization of their NRs involved in lipid homeostasis is essential to better understand the evolutionary response to lipid modulating chemicals.

Echinoderms are deuterostomes and, together with the sister group of hemichordates, share a closer ancestry with chordates than any other invertebrate phyla (Lavado etĀ al., 2006, Rottinger and Lowe, 2012). In this work, we aimed to get additional insights into the adverse outcomes of a model lipid homeostasis modulator in early diverging deuterostomes. To achieve this aim, we isolated and functionally characterized inĀ vitro, and for the first time, the PPAR orthologue of a non-chordate, the echinoderm P.Ā lividus, using transactivation assays. We also evaluated the ability of the model lipid modulator, TBT, to alter lipid homeostasis following 3 weeks of exposure to environmentally relevant concentrations (100 and 250Ā ng Sn/L). In parallel, screening of the fatty acid profile and key gene expression levels was performed.

Section snippets

Gene isolation and cloning

The full sequence of the gene rxr and ppar, and the partial sequences of the genes acc (acetyl-CoA carboxylase) and acsl (long-chain acyl-CoA synthetase) were obtained using a combination of PCR-based approaches. Briefly, degenerate PCR primers were designed from conserved regions using CODEHOP (Rose etĀ al., 2003) (COnsensus-DEgenerate Hybrid Oligonucleotide Primer) program to obtain the initial fragment or fragments. For rxr, 5ā€² and 3ā€² ends were further extended using the SMARTerā„¢ RACE cDNA

Results and discussion

It is well established that different endocrine disrupting chemicals (EDCs) are able to interfere with the lipid metabolism of mammals; yet, data on the effect of those compounds in other lineages is still very limited. Nonetheless, recent research supports the hypothesis that lipid homeostasis and obesogenic outcomes transcend mammals (CapitĆ£o etĀ al., 2017, Janer etĀ al., 2007, JordĆ£o etĀ al., 2015, JordĆ£o etĀ al., 2016b, Lyssimachou etĀ al., 2009, Lyssimachou etĀ al., 2015, Maradonna etĀ al., 2015

Conclusions

The present study is, to our knowledge, the first to isolate and functionally characterize the NRs PPAR and RXR from an echinoderm. Additionally, the inĀ vivo observations support the hypothesis that TBT acts as a lipid homeostasis modulator in this group. The present work provides robust evidence for the ability of TBT to interfere with sea urchin lipid metabolism and reveal the modulation of PPAR/RXR as a potential mechanism. Future developmental and full life cycle studies should further

Funding

This work was supported by the project 031544 cofinanced by COMPETE 2020, Portugal 2020, and the European Union through the ERDF, and by FundaĆ§Ć£o para a CiĆŖncia e a Tecnologia through national funds and the support to A.M.F.C (SFRH/BD/90664/2012).

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

We acknowledge Ana AndrƩ, Tiago Torres and Ricardo Capela for their help in the sampling process.

References (99)

  • E.J. Cook et al.

    Effect of variation in the protein value of the red macroalga Palmaria palmata on the feeding, growth and gonad composition of the sea urchins Psammechinus miliaris and Paracentrotus lividus (Echinodermata)

    Aquaculture

    (2007)
  • M. De Cock et al.

    Obesogenic effects of endocrine disruptors, what do we know from animal and human studies?

    Environ. Int.

    (2014)
  • F. EcheverrĆ­a et al.

    Long-chain polyunsaturated fatty acids regulation of PPARs, signaling: relationship to tissue development and aging

    Prostaglandins Leukot. Essent. Fat. Acids

    (2016)
  • F. EcheverrĆ­a et al.

    Reduction of high-fat diet-induced liver proinflammatory state by eicosapentaenoic acid plus hydroxytyrosol supplementation: involvement of resolvins RvE1/2 and RvD1/2

    J.Ā Nutr. Biochem.

    (2019)
  • J. Folch et al.

    AĀ simple method for the isolation and purification of total lipides from animal tissues

    J.Ā Biol. Chem.

    (1957)
  • A. Georgiadi et al.

    Mechanisms of gene regulation by fatty acids

    Adv. Nutr.

    (2012)
  • J.V. Goldstone et al.

    The chemical defensome: environmental sensing and response genes in the Strongylocentrotus purpuratus genome

    Dev. Biol.

    (2006)
  • F. GrĆ¼n et al.

    Endocrine disrupters as obesogens

    Mol. Cell. Endocrinol.

    (2009)
  • A.C. Guedes et al.

    Fatty acid composition of several wild microalgae and cyanobacteria, with a focus on eicosapentaenoic, docosahexaenoic and Ī±-linolenic acids for eventual dietary uses

    Food Res. Int.

    (2011)
  • P. Guidetti et al.

    Effects of the edible sea urchin, Paracentrotus lividus, fishery along the Apulian rocky coast (SE Italy, Mediterranean Sea)

    Fish. Res.

    (2004)
  • H. Harino et al.

    Temporal trends of organotin compounds in the aquatic environment of the Port of Osaka

    Japan

    (1999)
  • Y. Hiromori et al.

    Structure-dependent activation of peroxisome proliferator-activated receptor (PPAR) gamma by organotin compounds

    Chem. Biol. Interact.

    (2009)
  • E. His et al.

    AĀ comparison between oyster (Crassostrea gigas) and sea urchin (Paracentrotus lividus) larval bioassays for toxicological studies

    Water Res.

    (1999)
  • A.D. Hughes et al.

    The transformation of long chain polyunsaturated fatty acids in benthic food webs: the role of sea urchins

    J.Ā Exp. Mar. Bio. Ecol.

    (2011)
  • G. Janer et al.

    Exposure to TBT increases accumulation of lipids and alters fatty acid homeostasis in the ramshorn snail Marisa cornuarietis

    Comp. Biochem. Physiol. C Toxicol. Pharmacol.

    (2007)
  • A. Janesick et al.

    Minireview: PPARĪ³ as the target of obesogens

    J.Ā Steroid Biochem. Mol. Biol.

    (2011)
  • R. JordĆ£o et al.

    Compounds altering fat storage in Daphnia magna

    Sci. Total Environ.

    (2016)
  • R. Lavado et al.

    Triphenyltin alters androgen metabolism in the sea urchin Paracentrotus lividus

    Aquat. Toxicol.

    (2006)
  • J. Lengqvist et al.

    Polyunsaturated fatty acids including docosahexaenoic and arachidonic acid bind to the retinoid X receptor alpha ligand-binding domain

    Mol. Cell. Proteomics

    (2004)
  • D. Lima et al.

    Tributyltin-induced imposex in marine gastropods involves tissue-specific modulation of the retinoid X receptor

    Aquat. Toxicol.

    (2011)
  • K.J. Livak et al.

    Analysis of relative gene expression data using real-time quantitative PCR and the 2āˆ’Ī”Ī”CT method

    Methods

    (2001)
  • I.J. Lodhi et al.

    Peroxisomes: a nexus for lipid metabolism and cellular signaling

    Cell Metabol.

    (2014)
  • A. Lyssimachou et al.

    Triphenyltin alters lipid homeostasis in females of the ramshorn snail Marisa cornuarietis

    Environ. Pollut.

    (2009)
  • F. Maradonna et al.

    Xenobiotic-contaminated diets affect hepatic lipid metabolism: implications for liver steatosis in Sparus aurata juveniles

    Aquat. Toxicol.

    (2015)
  • M.G. Marin et al.

    Embryotoxicity of butyltin compounds to the sea urchin Paracentrotus lividus

    Mar. Environ. Res.

    (2000)
  • N. Ouadah-Boussouf et al.

    Pharmacological evaluation of the mechanisms involved in increased adiposity in zebrafish triggered by the environmental contaminant tributyltin

    Toxicol. Appl. Pharmacol.

    (2016)
  • T.F.B.R. Repolho et al.

    Broodstock diet effect on sea urchin Paracentrotus lividus (Lamarck, 1816) endotrophic larvae development: potential for their year-round use in environmental toxicology assessment

    Ecotoxicol. Environ. Saf.

    (2011)
  • S. Ribeiro et al.

    Toxicity screening of diclofenac, propranolol, sertraline and simvastatin using danio rerio and paracentrotus lividus embryo bioassays

    Ecotoxicol. Environ. Saf.

    (2015)
  • M.Ɓ. RincĆ³n-Cervera et al.

    Vegetable oils rich in alpha linolenic acid increment hepatic n-3 LCPUFA, modulating the fatty acid metabolism and antioxidant response in rats

    Prostaglandins Leukot. Essent. Fat. Acids

    (2016)
  • A. Romero et al.

    Cell mediated immune response of the Mediterranean sea urchin Paracentrotus lividus after PAMPs stimulation

    Dev. Comp. Immunol.

    (2016)
  • M.M. Santos et al.

    Identifying the gaps: Resources and perspectives on the use of nuclear receptor based-assays to improve hazard assessment of emerging contaminants

    J.Ā Hazard. Mater.

    (2018)
  • M. Shpigel et al.

    Improving gonad colour and somatic index in the European sea urchin Paracentrotus lividus

    Aquaculture

    (2005)
  • M. Shpigel et al.

    Improving gonad colour and somatic index in the European sea urchin Paracentrotus lividus

    Aquaculture

    (2005)
  • M. Shpigel et al.

    Effects of dietary carotenoid on the gut and the gonad of the sea urchin Paracentrotus lividus

    Aquaculture

    (2006)
  • H.H. Steineger et al.

    Gene transcription of the retinoid X receptor alpha (RXRalpha) is regulated by fatty acids and hormones in rat hepatic cells

    J.Ā Lipid Res.

    (1998)
  • S. Takeuchi et al.

    InĀ vitro screening of 200 pesticides for agonistic activity via mouse peroxisome proliferator-activated receptor (PPAR)Ī± and PPARĪ³ and quantitative analysis of inĀ vivo induction pathway

    Toxicol. Appl. Pharmacol.

    (2006)
  • A. Tan et al.

    Identification and characterization of nuclear receptors from the red flour beetle , Tribolium castaneum

    Insect Biochem. Mol. Biol.

    (2008)
  • Y.H. Wang et al.

    Interactions of methyl farnesoate and related compounds with a crustacean retinoid X receptor

    Mol. Cell. Endocrinol.

    (2009)
  • M. Ahmadian et al.

    PPAR Ī³ signaling and metabolism: the good , the bad and the future

    Nat. Med.

    (2013)
  • Cited by (0)

    ā˜†

    This paper has been recommended for acceptance by Christian Sonne.

    View full text