Concetta Calabrò

Immunohistochemical localization of nNOS and VIP in the mantle integument of the mussel, Mytilus galloprovincialis

The phylogeny and functional roles of many bioactive compounds in the invertebrate integument are still unclear. In order to deal with this issue, we performed an immunohistochemical investigation of the integument of the mussel, Mytilus galloprovincialis, to demonstrate the presence of nNOS- and VIP-positive nerve fibers in subepidermal connective tissue of the mantle. Positive nerve cell bodies were detected in this tissue as well as in cortex of sperm follicles, and adjacent to maturating oocytes and spermatocytes located in the thickness of the mantle. These results indicate involvement of a local inhibitory non-adrenergic-non-cholinergic (NANC) regulatory mechanism of epidermal functions, such as mucous secretion and ciliary beating. At the gonadic level, this mechanism probably regulates the cycle of maturation and release of the gametes in both sexes.

Exposure of sea bream (Sparus aurata) to toxic concentrations of benzo[a]pyrene: possible human health effect.

Polycyclic aromatic hydrocarbons (PAHs) can accumulate in the food chain, due to their lipophilic properties. Fish can accumulate contaminants including PAHs and frequent consumption of such contaminated fish can pose risk to human health. The aim of this study was to clarify if acute exposure of sea bream (Sparus aurata, a fish species of great economic importance in the Atlantic and Mediterranean areas) to a PAH, benzo[a]pyrene (B[a]P), at a dose that can induce CYP1A and pathological changes in fish gills, liver and muscle, can induce accumulation in muscle. We investigated the cytotoxic effects (as changes in cell viability, DNA laddering and glutathione content) of in vitro exposure of human peripheral blood mononuclear cells (PBMCs) to organic extracts obtained from muscle of sea breams previously exposed via water to B[a]P (2mg/l, for 12, 24 and 72 h). At this level of exposure, B[a]P caused morphological changes, inflammatory response and CYP1A induction not only in sea bream gills and liver but also in muscle; furthermore, in fish muscle we observed a substantial B[a]P accumulation, which may be associated with the increased CYP1A activity in liver and especially in muscle. However, when PBMCs were exposed to organic extracts obtained from sea bream muscle contaminated with B[a]P, a toxic, although modest effect was revealed, consisting in a significant decrease in cell glutathione levels without alterations in cell viability and DNA laddering. This suggests that consumption of sea breams from B[a]P contaminated waters might represent a risk for human health.

Histological, histochemical and immunohistochemical study on the growing oocytes of the abyssal teleost Hoplostethus mediterraneus (V).

The oocytes of the abyssal Teleost, Hoplostethus mediterraneus were studied. Four stages of growth were observed and the oocytes of all the stages were surrounded by follicular cells and had several nucleoli in the nucleus. In the oocytes of the II degrees stage, vacuoles without contents, in oocytes of the III degrees stage several vacuoles with a basophilic contents and small yolk globules were identified. General and basic proteins, ribonucleoproteins, acid proteoglycans with -COOH groups were recognized in the cytoplasm, in the nucleoli of oocytes in the II degrees stage and in the vacuolar contents of oocytes in the III degrees stage. In the follicular cells, in the pellucid zone, in the yolk globules, from their beginning, glycoproteins were present. Positivity, for all lectins used, was revealed in the follicular cells and in the four stages of oocytes growth. alpha-D-glucose and alpha-D-mannose binding sites were in the pellucid zone and in the initial yolk globules. In the lather galactose and beta-N-acetyl glucosamine were present too. nNOS and VIP immunopositivity revealed at the periphery of the cytoplasm and at network of nerve fibres between oocytes, suggests NO is involved in a mechanism of regulation of the gametogenesis and of the spawning.

Experimental exposure of blue mussels (Mytilus galloprovincialis) to high levels of benzo[a]pyrene and possible implications for human health

Polycyclic aromatic hydrocarbons (PAHs) are lipophilic compounds able to accumulate in the food chain. Mussels showed to bioaccumulate contaminants, such as PAHs, so that recurrent consumption of such contaminated food represents a risk for human health. This study was aimed to elucidate if acute exposure of Mediterranean blue mussel (Mytilus galloprovincialis), a bivalve of great economic importance in several countries, to a PAH, benzo[a]pyrene (B[a]P), at doses able to induce cytochrome P450 1A (CYP1A) and pathological changes in mussel gills, can produce accumulation in soft tissue. We explored the cytotoxic effects (cell viability, DNA laddering, and glutathione levels) of in vitro exposure of human peripheral blood mononuclear cells (PBMCs) to organic extracts obtained from blue mussels previously exposed for 12 and 72h via water to B[a]P (0.5-1mg/L). In our experimental conditions, B[a]P induced CYP1A induction and morphological changes in mussel gills and a significant B[a]P accumulation in soft tissue. Conversely, exposing PBMCs to organic extracts obtained from contaminated mussels, resulted in a significant reduction of cell viability and cell glutathione content, and in an increase in DNA laddering. This confirms that consumption of mussels from B[a]P polluted waters might affect human health. Our data lead us to suggest that CYP1A activity in mussel gills may be useful (more than the amount of detected PAHs in the mussel edible tissue) as a marker in assessment of risk for health of consumers exposed to PAHs through ingestion of shellfish.

Effects of fasting and refeeding on the digestive tract of zebrafish (Danio rerio) fed with Spirulina (Arthrospira platensis), a high protein feed source

Abstract In the present work, morphological and molecular effects of short-term feed deprivation and refeeding with Spirulina (Arthrospira platensis) on zebrafish digestive tract were determined. Once elucidated the proximate composition of Spirulina feed, immunohistochemical and western blot analyses of peptide transporter (PepT1) and cholecystokinin (CCK8) were carried out in the gastrointestinal tract of zebrafish, previously morphologically investigated. Two and five fasting days caused not only morphostructural alterations, but also the downregulation of PepT1 and CCK8 proteins. Conversely, the recovery of normal morphological conditions, along with an increased PepT1 and CCK8 expression, were observed after refeeding with Spirulina. The increase of PepT1 expression in zebrafish may be responsible for the enhanced CCK8 secretion, so that both proteins may contribute to an improved digestion process during refeeding. These observations could be supported not only by compensatory mechanisms induced by fasting and refeeding but also by an higher protein quality of Spirulina-based diet.

Immunohistochemical Characterization of PepT1 and Ghrelin in Gastrointestinal Tract of Zebrafish: Effects of Spirulina Vegetarian Diet on the Neuroendocrine System Cells After Alimentary Stress

Gastrointestinal function in vertebrates is influenced by stressors, such as fasting and refeeding, different types of diet and hormonal factors. The aim of this paper was to analyze the effect of a Spirulina (Arthrospira platensis) diet, a microalga known for its nutraceutical properties, on the gastrointestinal tract of zebrafish (Danio rerio) regarding expression of oligopeptide transporter 1 (PepT1) and ghrelin (GHR). Food deprivation and refeeding was investigated to elucidate expression of PepT1 and GHR at a gastrointestinal level and the zebrafish compensatory mechanism. PepT1 is responsible for absorbing di- and tripeptides through a brush border membrane of intestinal mucosa. GHR is a brain-gut peptide in fish and mammals, stimulating growth hormone secretion and regulating appetite. Samples were taken after 2 and 5 days of specimen fasting, and 2 and 5 days of refeeding with Sera Spirulina tabs, in which the major constituent is Spirulina sp. (50.2% protein). Morphological and immunohistochemical analysis of PepT1 and GHR were carried out. Control specimen intestinal tract showed normal morphology of the digestive tract. Fasting caused fold structural changes and intestinal lumen constriction. Immunohistochemical analysis showed a PepT1 level reduction after fasting and an increase after refeeding, reaching very high levels after 5 days, compared to controls. GHR levels increased after food deprivation and gradually decreased after refeeding. Increased expression of PepT1 in refeeding fish suggests a compensatory physiological mechanism, as does the increase in GHR levels in fasting fish followed by a reduction after refeeding. A compensatory mechanism may be induced by fasting and refeeding and by a higher protein Spirulina diet. The microalga, for its nutraceutical properties, is an excellent candidate for animal breeding and human diet.