Self-assembling protein nanoparticles are used as a novel vaccine design platform to improve the stability and immunogenicity of safe subunit vaccines, while providing broader protection against ...viral infections. Infectious Hematopoietic Necrosis virus (IHNV) is the causative agent of the WOAH-listed IHN diseases for which there are currently no therapeutic treatments and no globally available commercial vaccine. In this study, by genetically fusing the virus glycoprotein to the H. pylori ferritin as a scaffold, we constructed a self-assembling IHNV nanovaccine (FerritVac). Despite the introduction of an exogenous fragment, the FerritVac NPs show excellent stability same as Ferritin NPs under different storage, pH, and temperature conditions, mimicking the harsh gastrointestinal condition of the virus main host (trout). MTT viability assays showed no cytotoxicity of FerritVac or Ferritin NPs in zebrafish cell culture (ZFL cells) incubated with different doses of up to 100 µg/mL for 14 hours. FerritVac NPs also upregulated expression of innate antiviral immunity, IHNV, and other fish rhabdovirus infection gene markers (mx, vig1, ifit5, and isg-15) in the macrophage cells of the host. In this study, we demonstrate the development of a soluble recombinant glycoprotein of IHNV in the E. coli system using the ferritin self-assembling nanoplatform, as a biocompatible, stable, and effective foundation to rescue and produce soluble protein and enable oral administration and antiviral induction for development of a complete IHNV vaccine. This self-assembling protein nanocages as novel vaccine approach offers significant commercial potential for non-mammalian and enveloped viruses.
Environmental pollution caused by pesticides is a growing concern. Pyridaben, a widely used organochlorine insecticide, is a representative water pollutant. Owing to its extensive usage, it has been ...detected in various aquatic ecosystems, including rivers and oceans. Pyridaben is highly toxic to aquatic organisms; however, the mechanism of its toxicity in the liver, which is important in toxicant metabolism, has not been studied. Therefore, we employed zebrafish and its well-characterized liver cell line, ZFL to assess pyridaben hepatotoxicity and explore its potential mechanisms of action. Pyridaben led to reduction of the liver size and fluorescence intensity of dsRed-labeled Tg (fabp10a:dsRed) zebrafish. It reduced the viability and proliferation of ZFL cells in vitro by inducing apoptosis and cell cycle arrest. These changes might be primarily linked to uncontrolled intracellular calcium flow in ZFL cells exposed to pyridaben. Additionally, it also downregulates the PI3K/Akt signaling cascade, leading to the inactivation of Gsk3β and nuclear translocation of β-catenin. Taken together, our findings suggest that pyridaben could have hepatotoxic effects on aquatic organisms. This study is the first to provide insight into the hepatotoxic mechanism of pyridaben using both in vivo and in vitro models.
•Cytotoxicity occurred in ZFL-cells exposed to trabectedin.•Trabectedin caused morphological alterations in ZFL-cells.•Trabectedin caused mortality and malformations in Danio rerio ...embryo-larvae.•Locomotor impairments in Danio rerio larvae were induced by trabectedin.•The zebrafish larvae-cell line system can be a suitable tool for AAs ecotoxicology.
Anticancer agents pose a great environmental risk due to their high toxicity. The aim of the current study is to assess the toxicity of trabectedin, a cytotoxic but atypical DNA binder, to liver cell line (ZFL) and embryo-larvae of the zebrafish Danio rerio employing an innovative approach. In ZFL cells, trabectedin cytotoxicity was measured using MTT and Trypan blue exclusion assay, and cell morphology was evaluated by fluorescence-activated cell sorting (FACS) and by immunofluorescence analysis. Trabectedin was 60-fold more toxic to ZFL cells than to zebrafish embryo-larvae in terms of mortality/cell viability, with mortality being observed in 42.7 µg.L−1 for embryo-larvae and non-viability in 0.04 µg.L−1 for cultured cells. Immunofluorescence staining showed morphology alterations of ZFL-cells exposed to trabectedin in a dose-dependent manner, from 0.04 to 0.15 µg.L−1. Furthermore, trabectedin induced morphological abnormalities to zebrafish embryo-larvae, such as tail malformations, pericardial edema and lack of equilibrium at concentrations lower than 50.3 µg.L−1. Regarding larvae behavior analysis, trabectedin increased velocity and total distance covered by zebrafish exposed to 42.7 µg.L−1 under dark conditions. These results reveal trabectedin to be toxic in both in vitro and in vivo zebrafish models, and thus the occurrence and persistence of this anticancer agent in the environment may represent a potential risk factor to the biota.
Display omitted
The potential of chitosan-based nanoencapsulation as a tool for delivering ascorbic acid (AA) to marine and freshwater organisms was investigated. Polymeric non-loaded and loaded vitamin C ...nanoparticles (NPs) were made by ionic gelation and the particles were characterized. In vitro performance of nanoparticles was evaluated in a zebrafish liver cell-line (ZFL) and in vivo studies were carried out in fish (post-metamorphic larvae of Solea senegalensis) and rotifers (Brachionus plicatilis) to assess the potential use of these NPs to be used as a tool in nutritional aquaculture studies. The results showed that NPs are suitable to trap hydrosoluble compounds such as AA by forming positively charged complexes (30–35mV), in a nanosize range (<300nm), with encapsulation efficiency (EE) higher than 15% and high stability (>90% of loaded AA remained within nanoparticles after 2h in seawater). The potential cytotoxicity of the NPs was evaluated in ZFL cells and no decrease in cell viability was noted up to 2.5mg/ml of nanoparticle concentration. The NP uptake was analyzed in ZFL cells by FACS cytometry and confocal laser scanning microscopy (CLSM). Time course and dose–response experiments were performed using fluorescein isothiocyanate labeled NPs (FITC-NPs). The in vitro endocytosis assays with ZFL cells showed a maximum uptake after 6h of incubation and a dose-dependent increase of fluorescence intensity directly proportional to the FITC-NP concentration. The antioxidant properties of vitamin C nanoparticles (AA-NPs) were also analyzed in ZFL cell extracts. Lipopolysaccharide (LPS) was added to ZFL cells to induce oxidative stress. The total antioxidant capacity of the AA-NP-treated cells showed a statistically significant increase with respect to the control with non-loaded nanoparticles (71.00±9.6 and 25.36±3.96μM Trolox equivalent; p<0.05 respectively). The NPs' ability to penetrate fish intestinal epithelium was also evaluated. After 2h, NPs were able to penetrate through intestinal epithelium in post-metamorphic larvae of S. senegalensis as detected by CLSM. The potential use of NPs as additive to rotifers was analyzed using AA-loaded or non-loaded NPs in rotifer enrichment for 2h. Rotifers fed with AA-NPs increased up to 2-fold of their ascorbic acid levels in comparison to control groups. As a whole, results show that these polymeric NPs might represent an interesting vehicle for oral administration of AA and other active compounds in aquaculture.
•In vitro assays show that nanoparticles (NPs) did not produce any toxic effect.•These NPs entered cells and increased total antioxidant capacity.•Ascorbic acid (AA) was loaded into NPs and formed a stable complex.•After two hours in seawater at 20ºC, at least 90 % of AA remains within NPs.•Rotifers enriched with AA-NPs showed optimal AA content to be used as larvae prey.
The number of environmental chemical contaminants suspected to act as endocrine disruptor compounds by interacting with estrogen receptor (ER) signaling pathway has been continuously increasing. To ...study such interaction, the use of stable reporter gene assays is relevant, but species-specific in vitro screening assays are still lacking to address hazard assessment of estrogenic chemicals in aquatic vertebrates. Here, we describe the development of stable reporter gene assays based on stable expression of subtypes of zebrafish ER (zfERα, zfERβ1, and zfERβ2) coupled to estrogen response element-driven luciferase in a zebrafish liver (ZFL) cell line. The three established cell models, named ZELH-zfERα, ZELH-zfERβ1, and ZELH-zfERβ2, expressed stable and significant basal luciferase signal, which was induced by 17β-estradiol (E2) in a sensitive and dose-response manner at EC50s of 0.2, 0.03, and 0.05nM, respectively. In addition, E2 significantly altered cell proliferation in ZELH-zfERα and ZELH-zfERβ2 cells, but not in parental ZFL and ZELH-zfERβ1 cells, suggesting a functionality of these two receptors to modulate endogenous gene expression in the transfected clones. The screening of various xenoestrogens from different classes in the three models resulted in different luciferase response patterns. Natural and synthetic estrogens and 1,1,1-trichloro-2-(2 chlorophenyl)-2-(4-chlorophenyl)ethane were active at lower concentrations in ZELH-zfERβ1 and ZELH-zfERβ2 than in ZELH-zfERα cells, whereas genistein and zearalenone metabolites as well as three benzophenone derivatives preferentially activated zfERα. Altogether, the newly established models provide specific and convenient in vitro tool for comparative assessment of zfERs selective activation by chemicals within ZFL cell context.
Although taurine has been shown to play multiple important physiological roles in teleosts, little is known about the molecular mechanisms underlying dietary requirements. Cell lines can provide ...useful tools for deciphering biosynthetic pathways and their regulation. However, culture media and sera contain variable taurine levels. To provide a useful cell line for the investigation of taurine homeostasis, an adult zebrafish liver cell line (ZFL) has been adapted to a taurine-free medium by gradual accommodation to a commercially available synthetic medium, UltraMEM™-ITES. Here we show that ZFL cells are able to synthesize taurine and be maintained in medium without taurine. This has allowed for the investigation of the effects of taurine supplementation on cell growth, cellular amino acid pools, as well as the expression of the taurine biosynthetic pathway and taurine transporter genes in a defined fish cell type. After taurine supplementation, cellular taurine levels increase but hypotaurine levels stay constant, suggesting little suppression of taurine biosynthesis. Cellular methionine levels do not change after taurine addition, consistent with maintenance of taurine biosynthesis. The addition of taurine to cells grown in taurine-free medium has little effect on transcript levels of the biosynthetic pathway genes for cysteine dioxygenase (CDO), cysteine sulfinate decarboxylase (CSAD), or cysteamine dioxygenase (ADO). In contrast, supplementation with taurine causes a 30% reduction in transcript levels of the taurine transporter, TauT. This experimental approach can be tailored for the development of cell lines from aquaculture species for the elucidation of their taurine biosynthetic capacity.
Microcystin-LR (MC-LR) is a highly toxic hepatotoxin that poses great hazards to aquatic organisms and even human health. The zebrafish liver cell line (ZFL) is a valuable model for investigating ...toxicity and metabolism of toxicants. However, the toxicity of MC-LR and its effects on gene transcription of ZFL cells remains to be characterized. In this study, we determined the toxicity of MC-LR for ZFL cells and investigated the effects of MC-LR on cellular transcriptome dynamics. The EC
of MC-LR for ZFL cells was 80.123 μg/mL. The ZFL cells were exposed to 10 μg/mL MC-LR for 0, 1, 3, 6, 12 or 24 h, and RNA-sequencing was performed to analyze gene transcription. A total of 10,209 genes were found to be regulated by MC-LR. The numbers of up- and down-regulated genes at different time points ranged from 2179 to 3202 and from 1501 to 2597, respectively. Furthermore, 1543 genes underwent differential splicing (AS) upon MC-LR exposure, of which 620 were not identified as differentially expressed gene (DEG). The effects of MC-LR on cellular functions were highly time-dependent. MAPK (mitogen-activated protein kinase) and FoxO (forkhead box O) signaling pathways were the most prominent pathways activated by MC-LR. Steroid biosynthesis and terpenoid backbone biosynthesis were the most enriched for the down-regulated genes. A gene regulatory network was constructed from the expression profile datasets and the candidate master transcription factors were identified. Our results shed light on the molecular mechanisms of MC-LR cellular toxicity and the transcriptome landscapes of ZFL cells upon MC-LR toxicity.
Copper is an essential metal to aquatic animals, but it can be toxic when in elevated concentrations in water. The objective of the present study was to analyze copper effects in zebrafish ...hepatocytes (ZFL cell-line). The number of viable cells and copper accumulation were determined in hepatocytes exposed
in vitro to different copper concentrations (5–30
mg
Cu/L). Intracellular reactive oxygen species (ROS) formation, total antioxidant capacity against peroxyl radicals, and expression of genes related do DNA repair system were also measured in hepatocytes exposed to 5 and 20
mg
Cu/L. After 24
h of exposure, hepatocytes showed an exponential kinetics of copper accumulation. Copper exposure (24 and 48
h) significantly reduced hepatocyte number in all concentrations tested, except at the lowest one (5
mg
Cu/L). Exposure to 20
mg
Cu/L for 6, 12 and 24
h significantly increased intracellular ROS formation. However, no significant change in total antioxidant capacity was observed. After 12 and 24
h of exposure to 20
mg
Cu/L, a significant decrease in expression of
p53 and
CDKI genes was observed. Conversely, expression of
Gadd45α,
CyclinG1 and
Bax genes was significantly induced after 24
h of exposure to 20
mg
Cu/L. In hepatocytes exposed to 5
mg
Cu/L, any significant alteration in expression of these genes was observed. In a broad view, most of genes encoding for DNA repair proteins were inhibited after copper exposure, especially in hepatocytes exposed to 20
mg
Cu/L. Taken all together, results obtained suggest that the increased intracellular ROS formation induced by copper exposure would be responsible for the alteration in gene expression pattern observed.
► Nine differentially expressed membrane proteins were revealed by proteomics in ZFL cells exposed to methyl parathion. ► The differential expression of these membrane proteins were further validated ...by real-time PCR and Western blotting. ► MP exerted significant effects on cytoskeleton system, signal transduction, metabolism, transport and endocrine in ZFL cell. ► These differential proteins have the potential to be used as biomarkers for monitoring MP contamination in environment.
Methyl parathion (MP) is an extensively used organophosphorus pesticide, which has been associated with a wide spectrum of toxic effects on environmental organisms. The aim of this study is to investigate the alterations of membrane protein profiles in zebrafish liver (ZFL) cell line exposed to MP for 24h using proteomic approaches. Two-dimensional gel electrophoresis revealed a total of 13 protein spots, whose expression levels were significantly altered by MP. These differential proteins were subjected to matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry analysis, and nine proteins were identified to be membrane proteins, among which seven were up-regulated, while two were down-regulated. In addition, the mRNA levels corresponding to these differential membrane proteins were further analyzed by quantitative real-time PCR. And the differential expression of arginase-2 was specially validated via Western blotting. Regarding the physiological functions, these proteins are involved in molecular chaperon, cytoskeleton system, cell metabolism, signal transduction, transport and hormone receptor respectively, suggesting the complexity of MP-mediated toxicity to ZFL cell. These data could provide useful insights for better understanding the hepatotoxic mechanisms of MP and develop novel protein biomarkers for effectively monitoring MP contamination level in aquatic environment.
The basic-helix-loop-helix/PAS (bHLH/PAS) family of proteins is a group of transcription factors that regulate key pathways during normal development and in the response to stress. The aryl ...hydrocarbon receptor (AHR) is a member of this family. Recently,
Danio rerio (zebrafish) has become an important model system in the study of the signal transduction pathway and complements the results seen in mammalian models. However, studies of the AHR protein have been limited by the lack of antibody reagents and thus, little is known concerning the localization and degradation of the zebrafish AHR (zfAHR). In this report, we describe the production and characterization of specific polyclonal antibodies to the zfAHR2 protein and the analysis of AHR-mediated signal transduction in the zebrafish liver cell line (ZFL). The results show that the zfAHR2 is degraded via the 26S proteasome following exposure of cells to β-naphthoflavone (BNF). Interestingly, the time course is slower and the magnitude of zfAHR2 degradation is not as great as seen for the mammalian AHR. Studies also show that the zfAHR2 is rapidly degraded in a ligand-independent manner by exposure of cells to geldanamycin (GA) to levels consistent with mammalian AHR. Finally, immunohistochemical staining of the ZFL cells suggest that the unliganded AHR resides in both the cytoplasm and nucleus and undergoes active nucleocytoplasmic shuttling in the absence of ligand. These results suggest that there is conservation of function between fish and mammals with respect to ligand-dependent and -independent degradation of the AHR and that the zfAHR2 is degraded via the 26S proteasome.
PDF
