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- Tissue responsiveness to estradiol and genistein in the sea bass liver and scalePublication . Estêvão, M. Dulce; Andrade, André; Santos, Soraia; Power, Deborah; Pinto, Patricia ISAs in mammals, estrogens in fish are essential for reproduction but also important regulators of mineral homeostasis. Fish scales are a non-conventional target tissue responsive to estradiol and constitute a good model to study mineralized tissues effects and mechanisms of action of estrogenic compounds, including phytoestrogens. The responsiveness to estradiol and the phytoestrogen genistein, was compared between the scales and the liver, a classical estrogenic target, in sea bass (Dicentrarchus labrax). Injection with estradiol and genistein significantly increased circulating vitellogenin (for both compounds) and mineral levels (estradiol only) and genistein also significantly increased scale enzymatic activities suggesting it increased mineral turnover. The repertoire, abundance and estrogenic regulation of nuclear estrogen receptors (ESR1, 2a and 2b) and membrane G-protein receptors (GPER and GPER-like) were different between liver and scales, which presumably explains the tissue-specific changes detected in estrogen-responsive gene expression. In scales changes in gene expression mainly consisted of small rapid increases, while in liver strong, sustained increases/decreases in gene expression occurred. Similar but not overlapping gene expression changes were observed in response to both estradiol and genistein. This study demonstrates for the first time the expression of membrane estrogen receptors in scales and that estrogens and phytoestrogens, to which fish may be exposed in the wild or in aquaculture, both affect liver and mineralized tissues in a tissue-specific manner. (C) 2015 Elsevier Ltd. All rights reserved.
- Efeitos de fitoestrogénios no metabolismo mineral em escamas de robalo e de tilápia moçambicanaPublication . Estêvão, Dulce; Pinto, Patricia IS; Santos, Soraia; Andrade, André; Power, DeborahO rápido desenvolvimento da aquacultura nas últimas décadas fez aumentar a procura por fontes proteicas adequadas para incluir nas rações dos peixes. A soja tem sido muito utilizada com fonte proteica de origem vegetal mas é particularmente rica em fitoestrogénios, incluindo a genisteína (GEN) e a daidzeína (DAI), que são as principais isoflavonas presentes na soja. Os peixes podem estar expostos aos fitoestrogénios no ambiente ou através das dietas que os contêm, como é o caso da soja. Estes compostos podem ter atividades estrogénicas e efeitos disruptivos na reprodução mas o seu impacto nos tecidos mineralizados continua a ser desconhecido. As escamas de peixe são um tecido mineralizado que, tal como o osso de mamíferos, é mantido por ciclos de formação e reabsorção, mediado por osteoblastos (OSB) e osteoclastos (OSC), respetivamente. As escamas são um tecido responsivo aos estrogénios e expressam os recetores de estrogénio nucleares (ERs). As atividades das enzimas fosfatase alcalina (ALP) e fosfatase ácida resistente ao tartrato (TRAP) são usadas como marcadores das atividades dos OSB e OSC, respetivamente, e são modificadas pelo estradiol (E2) nas escamas de várias espécies de peixe. Usando um ensaio in vitro, investigámos o possível impacto da exposição a GEN e a DAI no metabolismo mineral em escamas. O efeito destes compostos foi avaliado através da determinação das atividades de TRAP e ALP em escamas de robalo (Dicentrarchus labrax), uma espécie marinha, e de tilápia moçambicana (Oreochromis mossambicus), mantida em água salgada (AS) e em água doce (AD).
- The effects of di-n-butyl phthalate and 4-tert-octylphenol in osteoclastic and osteoblastic activities in teleost fish scalesPublication . Pinto, Patricia IS; Estêvão, Dulce; Santos, Soraia; Andrade, André; Power, DeborahDi-n-butyl phtalate (DBP) and 4-tert-octylphenol (OP) are environmental pollutants with estrogenic activity that have been shown to have endocrine disruptive actions in reproduction of several fish species. However, their impact in bone and scale metabolism, which are estrogen-responsive tissues, remains unknown. In this study, we evaluated the impact of these compounds on mineral metabolism in fish scales that, like bone, are a dynamic tissue maintained by continuous cycles of formation and resorption mediated, respectively, by osteoblasts (OSB) and osteoclasts (OSC). Using an in vitro bioassay, Atlantic sea bass (a marine species) and Mozambique tilapia (a freshwater species) scales were incubated with a range of concentrations of OP and DBP in culture media for a short (30 minutes) or long (24 hours) incubation time. Effects on the activity of tartrate resistant acid phosphatase (TRAP) and alkaline phosphatase (ALP), markers for OSC and OSB activities, respectively, were assessed using a colorimetric enzymatic assay. DBP (10-6 M) affected TRAP activity in both species. While in sea bass, TRAP activity increased with DBP after 30 min incubation but was unaffected after 24 h, in tilapia no alterations were observed at the short term but a significant decrease was observed after 24 h incubation with this compound. None of the tested concentrations (10-10 to 10-6 M) affected ALP activity in both species. On the contrary, OP effects were only observed on the activity of ALP, which was significantly decreased after a 24 h incubation with 10-8 M of OP in the scales of both species. These results suggest that the exposure to these compounds may have disruptive effects on the metabolism of mineralized tissues in both marine and freshwater species. Future studies will investigate the mechanisms involved in these responses and the consequences for fish health.
- Genistein and estradiol have common and specific impacts on the sea bass (Dicentrarchus labrax) skin-scale barrierPublication . Pinto, Patricia IS; Andrade, André; Moreira, Catarina; Zapater, Cinta; Thorne, Michael A.S.; Santos, Soraia; Estêvão, M. Dulce; Gomez, Ana; Canario, Adelino; Power, DeborahTeleost fish scales play important roles in animal protection and homeostasis. They can be targeted by endogenous estrogens and by environmental estrogenic endocrine disruptors. The phytoestrogen genistein is ubiquitous in the environment and in aquaculture feeds and is a disruptor of estrogenic processes in vertebrates. To test genistein disrupting actions in teleost fish we used a minimally invasive approach by analysing scales plucked from the skin of sea bass (Dicentrarchus labrax). Genistein transactivated all three fish nuclear estrogen receptors and was most potent with the Esr2, had the highest efficacy with Esr1, but reached, in all cases, transactivation levels lower than those of estradiol. RNA-seq revealed 254 responsive genes in the sea bass scales transcriptome with an FDR < 0.05 and more than 2-fold change in expression, 1 or 5 days after acute exposure to estradiol or to genistein. 65 genes were specifically responsive to estradiol and 106 by genistein while 83 genes were responsive to both compounds. Estradiol specifically regulated genes of protein/matrix turnover and genistein affected sterol biosynthesis and regeneration, while innate immune responses were affected by both compounds. This comprehensive study revealed the impact on the fish scale transcriptome of estradiol and genistein, providing a solid background to further develop fish scales as a practical screening tool for endocrine disrupting chemicals in teleosts.
- Responsiveness of pituitary to galanin throughout the reproductive cycle of male European sea bass (Dicentrarchus labrax)Publication . Pinto, P.; Velez, Zélia; Sousa, Carmen; Santos, Soraia; Andrade, André; Alvarado, M. V.; Felip, A.; Zanuy, S.; Canario, A.V.M.The neuropeptide galanin (Gal) is a putative factor regulating puberty onset and reproduction through its actions on the pituitary. The present study investigated the pituitary responsiveness to galanin and the patterns of galanin receptors (Galrs) expression throughout the reproductive cycle of two years old male European sea bass (Dicentrarchus labrax), an important aquaculture species. Quantitative analysis of pituitary and hypothalamus transcript expression of four galr subtypes revealed differential regulation according to the testicular developmental stage, with an overall decrease in expression from the immature stage to the mid-recrudescence stage. Incubation of pituitary cells with mammalian 1-29 Gal peptide induced significant changes in cAMP concentration, with sensitivities that varied according to the testicular development stages. Furthermore 1-29 Gal was able to stimulate both follicle stimulating hormone (Fsh) and luteinizing hormone (Lh) release from pituitary cell suspensions. The magnitude of the effects and effective concentrations varied according to reproductive stage, with generalized induction of Fsh and Lh release in animals sampled in January (full spermiation). The differential expression of galrs in pituitary and hypothalamus across the reproductive season, together with the differential effects of Gal on gonadotropins release in vitro strongly suggests the involvement of the galaninergic system in the regulation the hypothalamus-pituitary-gonad axis of male sea bass. This is to our knowledge the first clear evidence for the involvement of galanin in the regulation of reproduction in non-mammalian vertebrates. (C) 2017 Elsevier Inc. All rights reserved.
- A fish scale in vitro bioassay to screen for endocrine disrupting compoundsPublication . Pinto, Patricia IS; Estêvão, Dulce; Santos, Soraia; Andrade, André; Power, DeborahA wide range of natural and anthropogenic compounds are accumulating in the aquatic environment, many of which can interact with and disrupt the endocrine system. Estrogenic endocrine disruptors (EDCs) are a particular problem with impact on humans, ecosystems and wildlife and are particularly relevant in aquatic organisms like fish that may experience life-long exposures. The effects of EDCs in fish have mainly been assessed using reproductive endpoints and in vivo animal experiments. We propose that using other potential endpoints, such as the effect of estrogens on mineralized tissue, would allow development of a simple non invasive assay using scales. Fish scales are mineralized tissues that express both membrane and nuclear estrogen receptors, and are targets for natural estrogens and EDCs. The in vitro bioassay optimized in this work includes sampling of fish scales, incubation in culture media containing the tested compounds and measurement of enzymatic activities related to calcium turnover (TRAP, tartrate-resistant acid phosphatase and ALP, alkaline phosphatase). Several variables were optimized including culture media, compounds concentrations and incubation conditions (e.g. temperature, time), using both sea bass (Dicentrarchus labrax) and tilapia (Oreochromis mossambicus) scales. Significant effects of E2 and EDCs were detected, including both rapid (30 minutes) or slow (1day) changes in scale TRAP or ALP activities, but the responses were of low magnitude and varied with the individual, age, time of year, species and culture conditions. The in vitro fish scale assay is a promising non-invasive screening tool for E2 and EDCs effects, complying with the 3Rs of animal welfare. However, current technical limitations are its limited sensitivity for some parameters eg. TRAP/ALP activity and alternative, sensitive, robust and easy to measure endpoints are under investigation.
- In vitro screening for estrogenic endocrine disrupting compounds using Mozambique tilapia and sea bass scalesPublication . Pinto, Patricia; Estêvão, M. Dulce; Santos, Soraia; Andrade, André; Power, DeborahA wide range of estrogenic endocrine disruptors (EDCs) are accumulating in the environment and may disrupt the physiology of aquatic organisms. The effects of EDCs on fish have mainly been assessed using reproductive endpoints and in vivo animal experiments. We used a simple non-invasive assay to evaluate the impact of estrogens and EDCs on sea bass (Dicentrarchus labrax) and tilapia (Oreochromis mossambicus) scales. These were exposed to estradiol (E2), two phytoestrogens and six anthropogenic estrogenic/anti-estrogenic EDCs and activities of enzymes related to mineralized tissue turnover (TRAP, tartrate-resistant acid phosphatase and ALP, alkaline phosphatase) were measured. Semi-quantitative RT-PCR detected the expression of both membrane and nuclear estrogen receptors in the scales of both species, confirming scales as a target for E2 and EDCs through different mechanisms. Changes in TRAP or ALP activities after 30 minute and 24 h exposure were detected in sea bass and tilapia scales treated with E2 and three EDCs, although compound-, time- and dose-specific responses were observed for the two species. These results support again that the mineralized tissue turnover of fish is regulated by estrogens and reveals that the scales are a mineralized estrogen-responsive tissue that may be affected by some EDCs. The significance of these effects for whole animal physiology needs to be further explored. The in vitro fish scale bioassay is a promising non-invasive screening tool for E2 and EDCs effects, although the low sensitivity of TRAP/ALP quantification limits their utility and indicates that alternative endpoints are required.
- High pressure processing of European sea bass (Dicentrarchus labrax) fillets and tools for flesh quality and shelf life monitoringPublication . Tsironi, Theofania; Anjos, Liliana; Pinto, Patricia IS; Dimopoulos, George; Santos, Soraia; Santa, Cátia; Manadas, Bruno; Canario, Adelino; Taoukis, Petros; Power, DeborahThe effects of high pressure (HP:600 MPa, 5 min, 25 °C) on European sea bass fillets were investigated using microbiological, physicochemical and sensory indices, and “omics” technologies. HPP led to more than a 5 log(cfu/g) reduction in initial bacterial total viable counts and altered the bacterial microbiome, reducing the proportion of food spoilage genera. Lightness and hardness of the fish flesh significantly increased after HPP and were associated with modified muscle tissue histology, with fibers appearing fused and more compact in comparison to the unprocessed control. Sensory evaluation (based on a lower limit of 5 for overall acceptability scoring) indicated a shelf life of 11 days for untreated control samples and 2 months for the HP-treated fillets. Quantitative SWATH proteomics revealed 281 proteins that had modified levels between control and HP-processed fish flesh. The metagenomics and proteomics provided detailed insight into how the change in HP-processed sea bass fillets is linked to the modifications in the microbiome and proteome.
- Proteome dataset of sea bass (Dicentrarchus labrax) skin-scales exposed to fluoxetine and estradiolPublication . L, Anjos; PI Pinto, PPinto; Santos, Soraia; Estêvão, M. Dulce; Santa, Cátia; Manadas, Bruno; Canario, A.V.M.; Power, DeborahContamination of aquatic ecosystems with anthropogenic pollutants, including pharmaceutical drugs, is a major concern worldwide. Aquatic organisms such as fish are particularly at risk of exposure to pollutants. The surface of fish is the first point of contact with pollutants, but few studies have considered the impact of pollutants on the skin-scale barrier. The present proteome data are the basis of the findings discussed in the associated research article "Proteomics of sea bass skin-scales exposed to the emerging pollutant fluoxetine compared to estradiol" [1]. Juvenile sea bass were exposed by intraperitoneal injections to: a) the antidepressant fluoxetine (FLX), a widely prescribed psychotropic drug and an emerging pollutant; b) the natural estrogen 17 beta-estradiol (E2) and c) the vehicle, coconut oil (control). The scale proteome of fish exposed to these compounds for 5 days was analysed using quantitative label-free proteomics technology SWATH-MS (sequential windowed data-independent a cquisition of the total high-resolution-mass spectra). The proteome data generated was submitted to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD020983. LC-MS data from pooled protein extracts from the scales of all experimental groups was acquired using information-dependent acquisition (IDA) and 1,254 proteins were identified by searching against the sea bass genome database. 715 proteins were quantified by SWATH acquisition, and 213 proteins had modified levels (p < 0.05) between the E2- or FLX-exposed fish compared to the control. The main biological processes and KEGG pathways affected by E2 or FLX treatments were identified using Cytoscape/ClueGO enrichment analyses. (c) 2022 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)
- Experimental data from flesh quality assessment and shelf life monitoring of high pressure processed European sea bass (Dicentrarchus labrax) filletsPublication . Anjos, Liliana; Pinto, Patricia IS; Tsironi, Theofania; Dimopoulos, George; Santos, Soraia; Santa, Cátia; Manadas, Bruno; Canario, Adelino; Taoukis, Petros; Power, DeborahFresh fish are highly perishable food products and their short shelf-life limits their commercial exploitation and leads to waste, which has a negative impact on aquaculture sustainability. New non-thermal food processing methods, such as high pressure (HP) processing, prolong shelf-life while assuring high food quality. The effect of HP processing (600MPa, 25 °C, 5min) on European sea bass (Dicentrarchus labrax) fillet quality and shelf life was investigated. The data presented comprises microbiome and proteome profiles of control and HP-processed sea bass fillets from 1 to 67 days of isothermal storage at 2 °C. Bacterial diversity was analysed by Illumina high-throughput sequencing of the 16S rRNA gene in pooled DNAs from control or HP-processed fillets after 1, 11 or 67 days and the raw reads were deposited in the NCBI-SRA database with accession number PRJNA517618. Yeast and fungi diversity were analysed by high-throughput sequencing of the internal transcribed spacer (ITS) region for control and HP-processed fillets at the end of storage (11 or 67 days, respectively) and have the SRA accession number PRJNA517779. Quantitative label-free proteomics profiles were analysed by SWATH-MS (Sequential Windowed data independent Acquisition of the Total High-resolution-Mass Spectra) in myofibrillar or sarcoplasmic enriched protein extracts pooled for control or HP-processed fillets after 1, 11 and 67 days of storage. Proteome data was deposited in the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifiers PXD012737. These data support the findings reported in the associated manuscript "High pressure processing of European sea bass (Dicentrarchus labrax) fillets and tools for flesh quality and shelf life monitoring", Tsironi et al., 2019, JFE 262:83-91, doi.org/10.1016/j.jfoodeng.2019.05.010.