Analysis of hematologic alterations, immune responses and metallothionein gene expression in Nile tilapia (Oreochromis niloticus) exposed to silver nanoparticles.

In this study, Nile tilapia (Oreochromis niloticus) fingerlings were used as a model to examine acute and chronic toxicity of silver nanoparticles (AgNP). Expression levels of metallothionein (MT) transcripts in fish exposed to 0, 1 or 100 mg AgNP/kg fish were investigated by quantitative real-time RT-PCR. The results showed MT expression levels were significantly decreased 0.3-0.7-fold in the liver and spleen of fish exposed to 1 or 100 mg AgNP/kg after 6-48 h. In contrast, during this period, MT mRNA expression levels were increased 2-3-fold in the head kidney of the fish exposed to either level of AgNP. Investigations of effects of AgNP on the fish immune responses and hematological parameters revealed that phagocytic activity, the amount of red blood cells (RBC) and the percent hematocrit (%Hct) in fish exposed to AgNP were decreased significantly 1 week after exposure, especially those exposed to 100 mg AgNP/kg. Fish immunized with Streptococcus agalactiae vaccine and simultaneously exposed to 100 mg AgNP/kg presented decreased antibody titers during the early phase. Lastly, a challenge test showed that vaccinated fish exposed to AgNP, regardless of concentration, remained protected against S. agalactiae infection, with a lower mortality (10-20%) compared to 70% in control fish. These findings indicated that expression patterns of the MT gene in the liver, spleen and head kidney at different timepoints could be used to assess acute and chronic exposure of Nile tilapia to AgNP. Additionally, changes in innate immune responses and hematological parameters in fish may prove useful for evaluation of AgNP toxicity. Data obtained in this study strongly support the use of Nile tilapia as an animal model to potentially serve as a bio-indicator of environment contamination caused by AgNP.

Molecular characterization and expression analyses of cDNAs encoding the thioredoxin-interacting protein and selenoprotein P genes and histological changes in Nile tilapia (Oreochromis niloticus) in response to silver nanoparticle exposure.

Herein, Nile tilapia thioredoxin-interacting protein (On-TXNIP) and selenoprotein P (On-SEPP) cDNAs were cloned and characterized. The full-length On-TXNIP cDNA contained 2 arrestin domains, 2 conserved cysteine residues that bind to thioredoxin to inhibit thioredoxin function, and 2 PPXY motifs, which negatively regulate the protein by stimulating binding to E3 ubiquitin ligase. The On-SEPP cDNA contained 17 selenocysteines (Sec) encoded by the TGA codon, which can be recognized as either a stop codon or a Sec codon. The On-SEPP cDNA also carried 2 typical SECIS elements located in the 3'UTR that are important for selenocysteine translation. Evolutionary analyses of both the On-TXNIP and On-SEPP genes revealed that these genes are closely related to the TXNIP and SEPP genes in zebrafish (Danio rerio), with amino acid similarities of 91.8% and 61.9%, respectively. A normal tissue distribution analysis indicated that the On-TXNIP and On-SEPP genes were ubiquitously expressed in all tissues examined, and the highest expression levels of these genes were observed in peripheral blood leukocytes (PBLs) and the trunk kidney, respectively. The expression levels of On-TXNIP and On-SEPP transcripts were acutely and chronically analyzed following the injection of fish with 1, 10 or 100mg/kg silver nanoparticles (Ag NPs). Significant up-regulation of On-TXNIP and On-SEPP transcripts was observed in the liver, spleen, and head kidney at the early phase of Ag NP exposure (hours 6 through 48). Down-regulation of On-SEPP transcripts was clearly observed in the liver at weeks 1 to 4. Histopathology analysis demonstrated that the fish livers exhibited a dramatic infiltration of Kupffer cells, elevated bi-nucleated cells, expanded sinusoidal blood congestion and severe necrosis in a dose-dependent manner. Based on these findings, coupling of the expression analysis of these two cellular stress response genes and histopathological observation of fish exposed to Ag NPs should be reliable for the assessment of Ag NP contamination in teleost fish.