Strategic Project - LA 15 - 2011-2012

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Publications

A fish scale in vitro bioassay to screen for endocrine disrupting compounds

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

A 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.

2016Other

Open Access

Development of an in vitro cell system from zebrafish suitable to study bone cell differentiation and extracellular matrix mineralization

Publication . Vijayakumar, Parameswaran; Laizé, Vincent; Cardeira Da Silva, João; Trindade, Marlene; Cancela, M. Leonor

Mechanisms of bone formation and skeletal development have been successfully investigated in zebrafish using a variety of in vivo approaches, but in vitro studies have been hindered due to a lack of homologous cell lines capable of producing an extracellular matrix (ECM) suitable for mineral deposition. Here we describe the development and characterization of a new cell line termed ZFB1, derived from zebrafish calcified tissues. ZFB1 cells have an epithelium-like phenotype, grow at 28 degrees C in a regular L-15 medium supplemented with 15% of fetal bovine serum, and are maintained and manipulated using standard methods (e.g., trypsinization, cryopreservation, and transfection). They can therefore be propagated and maintained easily in most cell culture facilities. ZFB1 cells show aneuploidy with 2n=78 chromosomes, indicative of cell transformation. Furthermore, because DNA can be efficiently delivered into their intracellular space by nucleofection, ZFB1 cells are suitable for gene targeting approaches and for assessing gene promoter activity. ZFB1 cells can also differentiate toward osteoblast or chondroblast lineages, as demonstrated by expression of osteoblast- and chondrocyte-specific markers, they exhibit an alkaline phosphatase activity, a marker of bone formation in vivo, and they can mineralize their ECM. Therefore, they represent a valuable zebrafish-derived in vitro system for investigating bone cell differentiation and extracellular matrix mineralization.

2013-12Journal article

Open Access

Central role of betaine-homocysteine S-methyltransferase 3 in chondral ossification and evidence for sub-functionalization in neoteleost fish

Publication . Rosa, Joana; Tiago, Daniel; Marques, Cátia L.; Vijayakumar, Parameswaran; Fonseca, Luís; Cancela, Leonor; Laizé, Vincent

Background: To better understand the complex mechanisms of bone formation it is fundamental that genes central to signaling/regulatory pathways and matrix formation are identified. Cell systems were used to analyze genes differentially expressed during extracellular matrix mineralization and bhmt3, coding for a betaine-homocysteine S-methyltransferase, was shown to be down-regulated in mineralizing gilthead seabream cells.Methods: Levels and sites of bhmt3 expression were determined by qPCR and in situ hybridization throughout seabream development and in adult tissues. Transcriptional regulation of bhmt3 was assessed from the activity of promoter constructs controlling luciferase gene expression. Molecular phylogeny of vertebrate BHMT was determined from maximum likelihood analysis of available sequences.Results: bhmt3 transcript is abundant in calcified tissues and localized in cartilaginous structures undergoing endo/perichondral ossification. Promoter activity is regulated by transcription factors involved in bone and cartilage development, further demonstrating the central role of Bhmt3 in chondrogenesis and/or osteogenesis. Molecular phylogeny revealed the explosive diversity of bhmt genes in neoteleost fish, while tissue distribution of bhmt genes in seabream suggested that neoteleostean Bhmt may have undergone several steps of sub-functionalization.Conclusions: Data on bhmt3 gene expression and promoter activity evidences a novel function for betaine-homocysteine S-methyltransferase in bone and cartilage development, while phylogenetic analysis provides new insights into the evolution of vertebrate BHMTs and suggests that multiple gene duplication events occurred in neoteleost fish lineage.General significance: High and specific expression of Bhmt3 in gilthead seabream calcified tissues suggests that bone-specific betaine-homocysteine S-methyltransferases could represent a suitable marker of chondral ossification.

2016-07Journal article

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Matrix Gla protein repression by miR-155 promotes oncogenic signals in breast cancer MCF-7 cells

Publication . Tiago, Daniel; Conceição, Natércia; Caiado, Helena; Laizé, Vincent; Cancela, Leonor

MGP is a protein that was initially associated with the inhibition of calcification in skeleton, soft tissues, and arteries, but more recently also implicated in cancer. In breast cancer, higher levels of MGP mRNA were associated with poor prognosis, but since this deregulation was never demonstrated at the protein level, we postulated the involvement of a post-transcriptional regulatory mechanism. In this work we show that MGP is significantly repressed by miR-155 in breast cancer MCF-7 cells, and concomitantly there is a stimulation of cell proliferation and cell invasiveness. This study brings new insights into the putative involvement of MGP and oncomiR-155 in breast cancer, and may contribute to develop new therapeutic strategies.

2016-04Journal article

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