Patricia Morcillo

Heavy metals produce toxicity, oxidative stress and apoptosis in the marine teleost fish SAF-1 cell line.

The use of cell lines to test the toxicity of aquatic pollutants is a valuable alternative to fish bioassays. In this study, fibroblast SAF-1 cells from the marine gilthead seabream (Sparus aurata L.) were exposed for 24 h to the heavy metals Cd, Hg, MeHg (Methylmercury), As or Pb and the resulting cytotoxicity was assessed. Neutral red (NR), MTT-tetrazolio (MTT), crystal violet (CV) and lactate dehydrogenase (LDH) viability tests showed that SAF-1 cells exposed to the above heavy metals produced a dose-dependent reduction in the number of viable cells. Methylmercury showed the highest toxicity (EC50 = 0.01 mM) followed by As, Cd, Hg and Pb. NR was the most sensitive method followed by MTT, CV and LDH. SAF-1 cells incubated with each of the heavy metals also exhibited an increase in the production of reactive oxygen species and apoptosis cell death. Moreover, the corresponding gene expression profiles pointed to the induction of the metallothionein protective system, cellular and oxidative stress and apoptosis after heavy metal exposure for 24 h. This report describes and compares tools for evaluating the potential effects of marine contamination using the SAF-1 cell line.

Differential proteome profile of skin mucus of gilthead seabream (Sparus aurata) after probiotic intake and/or overcrowding stress

UNLABELLED Gilthead seabream (Sparus aurata L.) is the major cultured fish species in the Mediterranean area. High density stocking causes stress and increases the impact of diseases leading to economic losses. Probiotics could represent a solution to prevent diseases through several mechanisms such as improving the immune status and/or mucosal microbiota or competing with pathogens. The probiotic Shewanella putrefaciens, also known as Pdp11, was firstly isolated from the skin of healthy gilthead seabream. Our study focuses on the skin mucus proteome after dietary probiotic Pdp11 intake in fish maintained under normal or overcrowding conditions. 2-DE of skin mucus followed by LC-MS/MS analysis was done for each experimental group and differentially expressed proteins were identified. The results showed differentially expressed proteins especially involved in immune processes, such as lysozyme, complement C3, natural killer cell enhancing factor and nonspecific cytotoxic cell receptor protein 1, whose transcript profiles were studied by qPCR. A consistency between lysozyme protein levels in the mucus and lysozyme mRNA levels in skin was found. Further research is necessary to unravel the implications of skin mucosal immunity on fish welfare and disease. BIOLOGICAL SIGNIFICANCE The present work reveals the proteomic changes, which are taking place in the skin mucus of stressed and non-stressed gilthead seabream after Pdp11 probiotic intake. The study contributes to improving the knowledge on skin mucosal immunology of this relevant farmed fish species. Furthermore, the paper shows for the first time how a suitable proteomic methodology, in this case 2-DE followed by LC-MS/MS is useful to perform a comparative study with a non-invasive technique of skin mucus of gilthead seabream.

Characterization of the IFN pathway in the teleost fish gonad against vertically transmitted viral nervous necrosis virus.

One of the most powerful innate immune responses against viruses is mediated by type I IFN. In teleost fish, it is known that virus infection triggers the expression of ifn and many IFN-stimulated genes, but the viral RNA sensors and mediators leading to IFN production are scarcely known. Thus, we have searched for the presence of these genes in gilt-head sea bream (Sparus aurata) and European sea bass (Dicentrarchus labrax), and evaluated their expression after infection with viral nervous necrosis virus (VNNV) in the brain, the main viral target tissue, and the gonad, used to transmit the virus vertically. In sea bream, a fish species resistant to the VNNV strain used, we found an upregulation of the genes encoding MDA5 (melanoma differentiation-associated gene 5), TBK1 (TANK-binding kinase 1), IRF3 (IFN regulatory factor 3), IFN, Mx [myxovirus (influenza) resistance protein] and PKR (dsRNA-dependent protein kinase receptor) proteins in the brain, which were unaltered in the gonad and could favour the dissemination by gonad fluids or gametes. Strikingly, in European sea bass, a very susceptible species, we also identified, transcripts coding for LGP2 (Laboratory of Genetics and Physiology 2), MAVS (mitochondrial antiviral signalling), TRAF3 (TNF receptor-associated factor 3), TANK (TRAF family member-associated NFκB activator) and IRF7 (IFN regulatory factor 7), and found that all the genes analysed were upregulated in the gonad, but only mda5, lgp2, irf3, mx and pkr were upregulated in the brain. These findings supported the notion that the European sea bass brain innate immune response is unable to clear the virus and pointed to the importance of gonad immunity to control the dissemination of VNNV to the progeny--an aspect that is worth investigating in aquatic animals.

In vitro immunotoxicological effects of heavy metals on European sea bass (Dicentrarchus labrax L.) head-kidney leucocytes

The knowledge about the direct effects of heavy metals on fish leucocytes is still limited. We investigate the in vitro effects of heavy metals (Cd, Hg, Pb or As) on oxidative stress, viability and innate immune parameters of head-kidney leucocytes (HKLs) from European sea bass (Dicentrarchus labrax). Production of free oxygen radicals was induced by Cd, Hg and As, mainly after 30 min of exposure. Cd and Hg promoted both apoptosis and necrosis cell death while Pb and As did only apoptosis, in all cases in a concentration-dependent manner. Moreover, expression of genes related to oxidative stress and apoptosis was significantly induced by Hg and Pb but down-regulated by As. In addition, the expression of the metallothionein A gene was up-regulated by Cd and Pb exposure though this transcript, as well as the heat shock protein 70, was down-regulated by Hg. Cd, methylmercury (MeHg) and As reduced the phagocytic ability, whereas Hg and Pb increased it. Interestingly, all the heavy metals decreased the phagocytic capacity (the number of ingested particles per cell). Leucocyte respiratory burst changed depending on the metal exposure, usually in a time- and dose-manner. Interestingly, the expression of immune-related genes was slightly affected by Cd, MeHg, As or Pb being Hg the form producing the greatest alterations, which included down-regulation of immunoglobulin M and hepcidin, as well as the up-regulation of interleukin-1 beta mRNA levels. This study provides an in vitro approach for elucidating the heavy metals toxicity, and particularly the immunotoxicity, in fish leucocytes.

Toxicological in vitro effects of heavy metals on gilthead seabream (Sparus aurata L.) head–kidney leucocytes

Heavy metals provoke toxicological effects on aquatic animal species, including fish, though their effects on fish leucocytes and immunotoxicology are still limited. In the present work the effects of heavy metals (Cd, Hg, Pb or As) on viability, oxidative stress and innate immune parameters of isolated head-kidney leucocytes from gilthead seabream (Sparus aurata) are studied. Cytotoxicity results indicated that short exposures (30 min or 2h) to Hg promoted both apoptosis and necrosis cell death of leucocytes whilst Cd, Pb and As did only by apoptosis, in all cases in a concentration- and time-dependent manner. In addition, production of free oxygen radicals was induced by Cd, Hg and As heavy metals. Cd failed to change phagocytosis but Hg and As increased the percentage of phagocytic cells but decreased the number of ingested particles per cell whilst Pb increased both phagocytic parameters. On the other hand, respiratory burst activity was significantly reduced by incubation with Cd, Hg and As but increased with Pb. Furthermore, the gene expression profiles partly support the functional finding of this work. This study provides an in vitro approach for elucidating the heavy metals toxicity, and particularly the immunotoxicity, in fish leucocytes.

In vitro characterization of 6-Coumarin loaded solid lipid nanoparticles and their uptake by immunocompetent fish cells.

The primary aim of the present work was to evaluate the in vitro uptake of 6-Coumarin (6COUM) loaded solid lipid nanoparticles (SLN) by two gilthead seabream (Sparus aurata L.) cell types: an established cell line (SAF-1 cells) and the primary cultures of head-kidney (HK)-the main haemopoietic organ in fish, equivalent to mammalian bone marrow-leucocytes. For this purpose, after the physicochemical characterization of SLN, the uptake by those immunocompetent fish cells was evaluated using flow cytometry and confocal microscopy. Concomitantly, the uptake of 6-COUM loaded SLN was compared with that achieved with 6-COUM loaded pectin microparticles (MPs), which were selected as a competitor of the delivery carriers. After SLN and MP physicochemical characterization, the results demonstrated that SAF-1 cells were able to internalize high percentages of 6-COUM SLNs when incubated for 4, 8 and 24h, with the highest SLN concentration tested (10 μg/ml). The ability of HK leucocytes to internalize SLN was also found to vary depending on both incubation time and SLN concentration. The highest values of HK leucocytes internalizing SLN particles (around 16%) were detected at the maximum SLN concentration (20 μg/ml) at incubation times of 4 or 8h. Conversely, HK leucocytes were unable to internalize MPs at any tested concentration and incubation time. A possible mechanism explaining the uptake into cells is proposed. The present work constitutes the first approximation to consider SLN as nanocarriers for delivering biologically active substances to fish.

Effects of Shewanella putrefaciens on innate immunity and cytokine expression profile upon high stocking density of gilthead seabream specimens

High stocking density increases the number of emerging diseases triggering economic losses worldwide. Probiotics provide an effective and natural solution for preventing some diseases through an improvement of innate immune system among others. In the present work dietary administration of the probiotic Shewanella putrefaciens (known as Pdp11) was evaluated under stress by high stocking density after 2 and 4 weeks of administration to gilthead seabream (Sparus aurata) specimens. Results showed an increase in cellular peroxidase and respiratory burst activity as well as a modulation of cytokine profile when Pdp11 was administered to fish reared at high stocking density. Overall, our results showed how Pdp11 is not only able to improve to some extent the cellular and humoral immunity but also to increase the gene expression profile of pro-inflammatory cytokines such as il1b or il6 in response to high stocking density in gilthead seabream. These findings may support the potential use of this probiotic as functional feed against stress in fish farms.

Establishment of a new teleost brain cell line (DLB-1) from the European sea bass and its use to study metal toxicology

In teleost fish, there are no commercial cell lines for the European sea bass (Dicentrarchus labrax). Thus, we have established the sea bass brain (DLB-1) cell line, using a fish retrovirus for immortalization, which resemble epithelial cells and express glial cells markers. Exposure to metals [Cd, methylmercury (MeHg), Pb or As] produces cytotoxicity and induction of reactive oxygen species (ROS) production. Interestingly, cell cycle analysis of DLB-1 cells shows that exposure to metals alters it significantly. Moreover, all the metals induce apoptosis as indicated by sub-Go/G1 population and annexin V binding. Finally, exposure of DLB-1 cells to metals also produces significant alterations at gene expression level, which confirm the above functional results. This is the first study in which metal cytotoxicity has been evaluated in a fish brain cell line and results seem to support that DLB-1 cells are suitable for toxicological studies.

Molecular oxidative stress markers in olive ridley turtles (Lepidochelys olivacea) and their relation to metal concentrations in wild populations

Due to their longevity and extensive migration areas, marine turtles are able to accumulate diverse contaminants over many years and as a consequence they represent an interesting bioindicator species for marine ecosystem pollution. Metals provoke toxicological effects in many aquatic animal species, but marine turtles have been under-investigated in this area. Thus, we have determined the presence of certain inorganic elements (As, Cd, Cu, Ni, Pb, Se and Zn) in olive ridley turtles (Lepidochelys olivacea) and related them to metallothionein (MT), superoxide dismutase (SOD), catalase (CAT) and glutathione reductase (GR) transcription and/or enzymatic activities. Gene expression of sod, cat and gr was found to be higher in blood than liver or kidney but most of the significant relationships were found in liver, not only for gene expression but also for enzyme activities. This must be related to the role the liver has as the first filter organ. Several positive relationships of sod, cat and gr gene expression in the different tissues were found in this population, as well as very high Cd concentrations. This could mean that these turtles are adapting to the metals-production of ROS and damage through a high transcription of these antioxidants. Multiple positive relationships with GR seem to be part of its compensatory effect due to the decrease of SOD production against the high and chronic exposure to certain xenobiotics. CAT, on the other hand, seems not to be used much, and glutathione detoxification of H2O2 may be more important in this species. Finally, despite the very high Cd concentrations found in this population, no significant relationship was found in any tissue with metallothionein gene expression. These results, along with very high Cd concentrations and a negative relationship with Cu, lead us to consider some kind of disruption in mt gene expression in these turtles.

Adhesion, invasion, cytotoxic effect and cytokine production in response to atypical Salmonella Typhimurium infection

Pathogenic bacteria such as Salmonella have the ability to respond to a wide variety of environmental stimuli. These responses allow them to survive and withstand insults both of an external location as well as within the host. The aim of this study was to investigate the effect of preadaptation in stressful conditions encountered in seawater microcosms for different periods of time on Salmonella Typhimurium survival, antibiotic susceptibility and interactions with Caco-2 cells. These results showed that the number of bacterial cells depends from the periods of stress in culture medium, highlighting the importance of using the right culture medium for the enumeration of stressed bacteria. The antibiotic resistance of starved cells was modified and their exposure to stressful conditions in seawater during 12 months significantly increased adhesion, invasion and cytotoxic activities on Caco-2 cells. Moreover, cellular cytokines IL-6 and IL-8 secretions were up-regulated. Present results seem to suggest that the preadaptation of S. Typhimurium in seawater microcosms affect the cultural characters by the appearance of the atypical cells that may play a critical role in the intestinal infection and in the systemic spread of the disease. These findings are very important to understand bacterial responses to changing conditions and explain the persistence of these atypical in eukaryotic cells.