E2SCALE: Scaling-down estrogenic responses in fish

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Effects of estrogens and estrogenic disrupting compounds on fish mineralized tissues

Publication . Pinto, Patricia IS; Estevao, Maria D.; Power, Deborah M.

Estrogens play well-recognized roles in reproduction across vertebrates, but also intervene in a wide range of other physiological processes, including mineral homeostasis. Classical actions are triggered when estrogens bind and activate intracellular estrogen receptors (ERs), regulating the transcription of responsive genes, but rapid non-genomic actions initiated by binding to plasma membrane receptors were recently described. A wide range of structurally diverse compounds from natural and anthropogenic sources have been shown to interact with and disrupt the normal functions of the estrogen system, and fish are particularly vulnerable to endocrine disruption, as these compounds are frequently discharged or run-off into waterways. The effect of estrogen disruptors in fish has mainly been assessed in relation to reproductive endpoints, and relatively little attention has been given to other disruptive actions. This review will overview the actions of estrogens in fish, including ER isoforms, their expression, structure and mechanisms of action. The estrogen functions will be considered in relation to mineral homeostasis and actions on mineralized tissues. The impact of estrogenic endocrine disrupting compounds on fish mineralized tissues will be reviewed, and the potential adverse outcomes of exposure to such compounds will be discussed. Current lacunae in knowledge are highlighted along with future research priorities.

2014-08Journal article

Open Access

Duplicated membrane estrogen receptors in the European sea bass (Dicentrarchus labrax): Phylogeny, expression and regulation throughout the reproductive cycle

Publication . Pinto, Patricia IS; Andrade, André; Estêvão, M. Dulce; Alvarado, M. Victoria; Felip, Alicia; Power, Deborah

The numerous estrogen functions reported across vertebrates have been classically explained by their binding to specific transcription factors, the nuclear estrogen receptors (ERs). Rapid non-genomic estrogenic responses have also been recently identified in vertebrates including fish, which can be mediated by membrane receptors such as the G protein-coupled estrogen receptor (Gper). In this study, two genes for Gper, namely gpera and gperb, were identified in the genome of a teleost fish, the European sea bass. Phylogenetic analysis indicated they were most likely retained after the 3R teleost-specific whole genome duplication and raises questions about their function in male and female sea bass. Gpera expression was mainly restricted to brain and pituitary in both sexes while gperb had a widespread tissue distribution with higher expression levels in gill filaments, kidney and head kidney. Both receptors were detected in the hypothalamus and pituitary of both sexes and significant changes in gpers expression were observed throughout the annual reproductive season. In female pituitaries, gpera showed an overall increase in expression throughout the reproductive season while gperb levels remained constant. In the hypothalamus, gpera had a higher expression during vitellogenesis and decreased in fish entering the ovary maturation and ovulation stage, while gperb expression increased at the final atresia stage. In males, gpers expression was constant in the hypothalamus and pituitary throughout the reproductive cycle apart from the mid- to late testicular development stage transition when a significant up-regulation of gpera occurred in the pituitary. The differential sex, seasonal and subtype-specific expression patterns detected for the two novel gper genes in sea bass suggests they may have acquired different and/or complementary roles in mediating estrogens actions in fish, namely on the neuroendocrine control of reproduction.

2018Journal article

Open Access

Proteomics of sea bass skin-scales exposed to the emerging pollutant fluoxetine compared to estradiol

Publication . Pinto, Patricia; Anjos, L.; Estêvão, Dulce; Santos, S.; Santa, C.; Manadas, B.; Monsinjon, T.; Canario, Adelino; Power, Deborah

Teleost fish skin-scales are essential for protection and homeostasis and the largest tissue in direct contact with the environment, but their potential as early indicators of pollutant exposure are hampered by limited knowledge about this model. This study evaluated multi-level impacts of in vivo exposure of European sea bass to fluoxetine (FLX, a selective serotonin-reuptake inhibitor and an emerging pollutant) and 17 beta-estradiol (E2, a natural hormone and representative of diverse estrogenic endocrine-disrupting pollutants). Exposed fish had significantly increased circulating levels of FLX and its active metabolite nor-FLX that, in contrast to E2, did not have estrogenic effects on most fish plasma and scale indicators. Quantitative proteomics using SWATH-MS identified 985 proteins in the scale total proteome. 213 proteins were significantly modified 5 days after exposure to E2 or FLX and 31 were common to both treatments and responded in the same way. Common biological processes significantly affected by both treatments were protein turnover and cytoskeleton reorganization. E2 specifically up-regulated proteins related to protein production and degradation and down-regulated the cytoskeleton/extracellular matrix and innate immune proteins. FLX caused both up- and down-regulation of protein synthesis and energy metabolism. Multiple estrogen and serotonin receptor and transporter transcripts were altered in sea bass scales after E2 and/or FLX exposure, revealing complex disruptive effects in estrogen/serotonin responsiveness, which may account for the partially overlapping effects of E2 and FLX on the proteome. A large number (103) of FLX-specifically regulated proteins indicated numerous actions independent of estrogen signalling. This study provides the first quantitative proteome of the fish skin-scale barrier, elucidates routes of action and biochemical and molecular signatures of E2 or FLX-exposure and identifies potential physiological consequences and candidate biomarkers of pollutant exposure, for monitoring and risk assessment.

2022Journal article

Restricted

Estradiol and genistein effects on the sea bass (Dicentrarchus labrax) scales: transcriptome dataset

Publication . Pinto, Patricia IS; Andrade, André; Thorne, Michael A.S.; Estêvão, M. Dulce; Canario, Adelino; Power, Deborah

Fish scales are mineralized structures that play important roles in protection and mineral homeostasis. This tissue expresses multiple estrogen receptor subtypes and can be targeted by estrogens or estrogenic endocrine-disrupting compounds, but their effects are poorly explored. The transcriptome data here presented support the findings reported in the research article "Genistein and estradiol have common and specific impacts on the sea bass (Dicentrarchus labrax) skin-scale barrier" [1]. Juvenile sea bass were exposed to estradiol and the phytoestrogen genistein for 1 and 5 days, by intraperitoneal injections, and the effects on scale transcript expression were analysed by RNA-seq using an Illumina Hi-seq 1500. The raw reads of the 30 libraries produced have been deposited in the NCBI-SRA database with the project accession number SRP102504. Mapping of RNA-seq reads against the sea bass reference genome using the Cufflinks/TopHat package identified 371 genes that had significant (FDR<0.05) differential expression with the estradiol or genistein treatments in relation to the control scales at each exposure time, 254 of which presented more than a 2-fold change in expression. The identity of the differentially expressed genes was obtained using both automatic and manual annotations against multiple public sequence databases and they were grouped according to their patterns of expression using hierarchical clustering and heat-maps. The biological processes and KEGG pathways most significantly affected by the estradiol and/or genistein treatments were identified using Cytoscape/ClueGO enrichment analyses.

2019-12Journal article

Open Access

The effects of di-n-butyl phthalate and 4-tert-octylphenol in osteoclastic and osteoblastic activities in teleost fish scales

Publication . Pinto, Patricia IS; Estêvão, Dulce; Santos, Soraia; Andrade, André; Power, Deborah

Di-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.

2015Conference object

Open Access