CCM4-Vários
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Browsing CCM4-Vários by Author "Andrade, André"
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- 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.