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Browsing ESS4-Vários by Author "Andrade, André"
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- 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).
- 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.
- Membrane and nuclear estrogen receptors in sea bass provide insight to explore genomic and non-genomic estrogenic actions: the mineralized scale examplePublication . Pinto, Patricia IS; Andrade, André; Félix, Rute; Cardoso, João CR; D Estêvão, M; Power, DeborahThe numerous estrogen functions across vertebrates have been classically explained by binding to nuclear estrogen receptors (ERs) regulating the transcription of responsive genes. It is now known that estrogenic compounds can also produce rapid non-genomic actions initiated by binding to plasma estrogen membrane receptors, such as the recently identified G protein-coupled estrogen receptor1 (GPER). Sea bass (Dicentrarchus labrax) express three ER subtype genes, one esr1 and two esr2 genes that appear to have been originated from the original esr2 gene in the teleost-specific whole genome duplication. We have recently identified two genes for GPER in the sea bass genome and phylogenetic analyses also suggest they are teleost-specific gene duplicates. Quantitative PCR revealed a wide tissue distribution for the five receptors in both male and female sea bass and expression throughout the reproductive cycle in brain and pituitary, although with subtype-specific and seasonal differences. When analyzing the sea bass scales, mineralized structures previously shown to be estrogen-responsive, a different receptor repertoire and regulation was detected compared to liver, a classical target gene. In juvenile sea bass scales, the main forms expressed were esr2a and gperb, which were also up regulated after injection with the natural estrogen estradiol (E2) and the phytoestrogen genistein (Gen). Both rapid (30 min) and slow (1 day or more) changes in the activities of enzymes related to mineral turnover were detected in fish scales in response to E2, Gen and xenoestrogens and the gene networks activated 1-5 days after injection of E2 and Gen are being characterized by transcriptomics, revealing both common and compound-specific effects at the transcriptional level. Functional characterization of the three sea bass ER subtypes and two GPERs is underway in mammalian cells, to allow to compare their signaling to different estrogenic compounds. These studies will help to understand the normal estrogen regulation of fish scale functions as well as its possible disruption by phytoestrogens and other xenoestrogens and the relative importance of genomic and non-genomic mechanisms of action of the five receptors.
- 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.