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.

Morcillo P ，Cordero H ，Meseguer J ，Esteban MÁ ，Cuesta A ... -  《-》

In vitro effects of metals on isolated head-kidney and blood leucocytes of the teleost fish Sparus aurata L. and Dicentrarchus labrax L.

The in vitro use of fish leucocytes to test the toxicity of aquatic pollutants, and particularly the immutoxicological effects, could be a valuable alternative to fish bioassays but has received little attention. In this study, head-kidney and peripheral blood leucocytes (HKLs and PBLs, respectively) from gilthead seabream (Sparus aurata L.) and European sea bass (Dicentrarchus labrax L.) specimens were exposed to Cd, MeHg (methylmercury), Pb or As for 24 h being evaluated the resulting cytotoxicity. Exposure to metals produced a dose-dependent reduction in the viability, and MeHg showed the highest toxicity followed by Cd, As and Pb. Interestingly, leucocytes from European sea bass are more resistant to metal exposure than those from gilthead seabream. Similarly, HKLs are always more sensitive than those isolated from blood from the same fish species. Moreover, fish leucocytes incubated with metals exhibited alterations in gene expression profiles that were more pronounced in the HKLs in general, being Pb the metal provoking less effects. Concretely, genes related to cellular protection (metallothionein), stress (heat shock protein 70) and oxidative stress (superoxide dismutase, catalase and glutathione reductase) were, in general, down-regulated in seabream HKLs but up-regulated in seabream PBLs and sea bass HKLs and PBLs. In addition, this profile leads to the increase of expression in genes related to apoptosis (Bcl2 associated X protein and caspase 3). Finally, transcription of genes involved in immunity (interleukin-1β and immunoglobulin M) was down-regulated, mainly in seabream leucocytes. This study points to the benefits for evaluating the toxicological mechanisms of marine pollution using fish leucocytes in vitro and insight into the mechanisms at gene level.

Morcillo P ，Meseguer J ，Esteban MÁ ，Cuesta A ... -  《-》

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.

Morcillo P ，Cordero H ，Meseguer J ，Esteban MÁ ，Cuesta A ... -  《-》

In vitro effects of virgin microplastics on fish head-kidney leucocyte activities.

Microplastics are well-documented pollutants in the marine environment that result from production or fragmentation of larger plastic items. The knowledge about the direct effects of microplastics on immunity, including fish, is still very limited. We investigated the in vitro effects of microplastics [polyvinylchloride (PVC) and polyethylene (PE)] on gilthead seabream (Sparus aurata) and European sea bass (Dicentrarchus labrax) head-kidney leucocytes (HKLs). After 1 and 24 h of exposure of HKLs with 0 (control), 1, 10 and 100 mg mL-1 MPs in a rotatory system, cell viability, innate immune parameters (phagocytic, respiratory burst and peroxidase activities) and the expression of genes related to inflammation (il1b), oxidative stress (nrf2, prdx3), metabolism of xenobiotics (cyp1a1, mta) and cell apoptosis (casp3) were studied. Microplastics failed to affect the cell viability of HKLs. In addition, they provoke very few significant effects on the main cellular innate immune activities, as decrease on phagocytosis or increase in the respiratory burst of HKLs with the highest dose of microplastics tested. Furthermore, microplastics failed to affect the expression of the selected genes on sea bass or seabream, except the nrf2 which was up-regulated in seabream HKLs incubated with the highest doses. Present results seem to suggest that continue exposure of fish to PVC or PE microplastics could impair fish immune parameters probably due to the oxidative stress produced in the fish leucocytes.

Espinosa C ，García Beltrán JM ，Esteban MA ，Cuesta A ... -  《-》

Cytokine expression in head-kidney leucocytes of European sea bass (Dicentrarchus labrax L.) after incubation with the probiotic Vagococcus fluvialis L-21.

The European sea bass (Dicentrarchus labrax L.) is one of the most extensively farmed marine fish in the Mediterranean sea. Under the high-density condition, common in aquaculture, the infectious diseases can cause significant economic losses. Probiotics are presented as an alternative to antibiotics for the control of aquaculture diseases. This study used real-time PCR to investigate in vitro the dynamic of expression of immune-related genes in sea bass after incubation with live and inactivated (heat and Uv-light) probiotic Vagoccus fluvialis L-21 at different times (T1, T12, T24, T48). The immune associated genes, interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin 10 (IL-10), TumourTumour necrosis factor- (TNF-), ciclo-oxigenase-2 (COX-2), caspase-3 (Casp-3) and Mx were studied in head-kidney (HK) leucocytes of sea bass after incubation with the probiotic strain. Transcript of proinflammatory cytokines (IL-1, TNF-, COX-2) was highly up-regulated after 1 h of incubation with the probiotic strain V. fluvialis L-21. We found statistically significant difference in pick of expression of TNF-, after 1 h of incubation with Uv-light inactivated probiotic strain. The COX-2 expression was highly up-regulated at all times studied, with the exception of 12 and 24 h post incubation for the Uv-light inactivated bacteria. Transcript of IL-10 and Casp-3 showed the higher statistically significant differences of expression after 48 h post incubation with live bacteria. In the contrast, sea bass HK leucocytes expressed Mx at 12 and 48 h without statistically differences among treatments. Our results suggest that V. fluvialis L-21 is able to stimulate in vitro some immune-related genes associated with the early inflammatory response. Future studies in vivo are necessary to clarify this process in sea bass.

Román L ，Real F ，Padilla D ，El Aamri F ，Déniz S ，Grasso V ，Acosta F ... -  《-》