Mechanisms of Silver Nanoparticle Uptake by Embryonic Zebrafish Cells

Evaluation of the uptake pathways in cells during exposure to nanoparticles (NPs) is key for risk assessment and the development of safer nanomaterials, as the internalisation and fate of NPs is linked to their toxicity and mode of action. Here, we determined the uptake mechanisms activated during the internalisation of 10, 30, and 100 nm AgNPs by embryonic zebrafish cells (ZF4). The uptake results demonstrated an NP size- and time-dependent uptake, showing the highest total silver uptake for the smallest AgNP (10 nm) at the lowest exposure concentration (2.5 μg/mL) after 2 h, while after 24 h, the highest exposure concentration (10 μg/mL) of the 10 nm AgNPs revealed the highest cellular load at 8 pg/cell. Inhibition of the caveolae, clathrin, and macropinocytosis endocytic pathways by pharmaceutical inhibitors (genistein, chlorpromazine, and wortmannin respectively) revealed that uptake was mainly via macropinocytosis for the 10 nm AgNPs and via the caveolae-mediated pathway for the 30 and 100 nm AgNPs. The induction of autophagy was also strongly related to the NP size, showing the highest percentage of induction for the 10 nm (around 3%) compared to naive cells, suggesting that autophagy can be activated along with endocytosis to deal with exposure to NPs. TEM imaging revealed the distribution of NPs across the cytoplasm inside intracellular vesicles. An increase in Early Endosome formation (EE) was observed for the 30 and 100 nm sizes, whereas the 10 nm AgNPs disrupted the activity of EE. The data supports the establishment of adverse outcome pathways by increasing knowledge on the link between a molecular initiating event such as receptor-mediated endocytosis and an adverse outcome, as well as supporting the reduction of animal testing by using alternative testing models, such as fish cell lines.

Functionalized Nanoplastics (NPs) Increase the Toxicity of Metals in Fish Cell Lines

Nanoplastics (NPs) are one of the most abundant environment-threatening nanomaterials on the market. The objective of this study was to determine in vitro if functionalized NPs are cytotoxic by themselves or increase the toxicity of metals. For that, we used 50 nm polystyrene nanoparticles with distinct surface functionalization (pristine, PS-Plain; carboxylic, PS-COOH; and amino PS-NH2) alone or combined with the metals arsenic (As) and methylmercury (MeHg), which possess an environmental risk to marine life. As test model, we chose a brain-derived cell line (SaB-1) from gilthead seabream (Sparus aurata), one of the most commercial fish species in the Mediterranean. First, only the PS-NH2 NPs were toxic to SaB-1 cells. NPs seem to be internalized into the cells but they showed little alteration in the transcription of genes related to oxidative stress (nrf2, cat, gr, gsta), cellular protection against metals (mta) or apoptosis (bcl2, bax). However, NPs, mainly PS-COOH and PS-NH2, significantly increased the toxicity of both metals. Since the coexistence of NPs and other pollutants in the aquatic environment is inevitable, our results reveal that the combined effect of NPs with the rest of pollutants deserves more attention.

Isolation, Identification, and Genomic Analysis of a Novel Reovirus from Healthy Grass Carp and Its Dynamic Proliferation In Vitro and In Vivo

A new grass carp reovirus (GCRV), healthy grass carp reovirus (HGCRV), was isolated from grass carp in 2019. Its complete genome sequence was determined and contained 11 dsRNAs with a total size of 23,688 bp and 57.2 mol% G+C content, encoding 12 proteins. All segments had conserved 5' and 3' termini. Sequence comparisons showed that HGCRV was closely related to GCRV-873 (GCRV-I; 69.57–96.71% protein sequence identity) but shared only 22.65–45.85% and 23.37–43.39% identities with GCRV-HZ08 and Hubei grass carp disease reovirus (HGDRV), respectively. RNA-dependent RNA-polymerase (RdRp) protein-based phylogenetic analysis showed that HGCRV clustered with Aquareovirus-C (AqRV-C) prior to joining a branch common with other aquareoviruses. Further analysis using VP6 amino acid sequences from Chinese GCRV strains showed that HGCRV was in the same evolutionary cluster as GCRV-I. Thus, HGCRV could be a new GCRV isolate of GCRV-I but is distantly related to other known GCRVs. Grass carp infected with HGCRV did not exhibit signs of hemorrhage. Interestingly, the isolate induced a typical cytopathic effect in fish cell lines, such as infected cell shrank, apoptosis, and plague-like syncytia. Further analysis showed that HGCRV could proliferate in grass carp liver (L28824), gibel carp brain (GiCB), and other fish cell lines, reaching a titer of up to 7.5 × 104 copies/μL.

An efficient vector-based CRISPR/Cas9 system in an Oreochromis mossambicus cell line using endogenous promoters

CRISPR/Cas9 gene editing is effective in manipulating genetic loci in mammalian cell cultures and whole fish but efficient platforms applicable to fish cell lines are currently limited. Our initial attempts to employ this technology in fish cell lines using heterologous promoters or a ribonucleoprotein approach failed to indicate genomic alteration at targeted sites in a tilapia brain cell line (OmB). For potential use in a DNA vector approach, endogenous tilapia beta Actin (OmBAct), EF1 alpha (OmEF1a), and U6 (TU6) promoters were isolated. The strongest candidate promoter determined by EGFP reporter assay, OmEF1a, was used to drive constitutive Cas9 expression in a modified OmB cell line (Cas9-OmB1). Cas9-OmB1 cell transfection with vectors expressing gRNAs driven by the TU6 promoter achieved mutational efficiencies as high as 81% following hygromycin selection. Mutations were not detected using human and zebrafish U6 promoters demonstrating the phylogenetic proximity of U6 promoters as critical when used for gRNA expression. Sequence alteration to TU6 improved mutation rate and cloning efficiency. In conclusion, we report new tools for ectopic expression and a highly efficient, economical system for manipulation of genomic loci and evaluation of their causal relationship with adaptive cellular phenotypes by CRISPR/Cas9 gene editing in fish cells.

Development of a Liquid Chromatography/Mass Spectrometry-Based Inhibition Assay for the Screening of Steroid 5-α Reductase in Human and Fish Cell Lines

Steroid 5-α reductase (5AR) is responsible for the reduction of steroids to 5-α reduced metabolites, such as the reduction of testosterone to 5-α dihydrotestosterone (DHT). A new adverse outcome pathway (AOP) for 5AR inhibition to reduce female reproduction in fish (AOP 289) is under development to clarify the antiestrogenic effects of 5AR inhibitors in female fish. A sensitive method for the DHT analysis using chemical derivatization and liquid chromatography–tandem mass spectrometry was developed. A cell-based 5AR inhibition assay that utilizes human cell lines, a transient overexpression system, and fish cell lines was developed. The measured IC50 values of two well-known 5AR inhibitors, finasteride and dutasteride, were comparable in the different systems. However, the IC50 of dutasteride in the fish cell lines was lower than that in the human cell lines. Finasteride showed a higher IC50 against the RTG-2 cell line. These results demonstrated that 5ARs inhibition could differ in terms of structural characteristics among species. The assay has high sensitivity and reproducibility and is suitable for the application in 5AR inhibition screening for various endocrine disruption chemicals (EDCs). Future studies will continue to evaluate the quantitative inhibition of 5AR by EDCs to compare the endocrine-disrupting pathway in different species.

Assessment of engineered nanosilver as an alternative nano-antibiotic in marine water pollution using biomarker of fish cell line

A large volume of antibiotics is used in fish farms to treat diseases because the farmed fish are fully affected by diseases and parasites in the aquaculture and particularly in the ocean environment where disease pathogens multiply quickly. The frequent use of these antibiotics in aquaculture has resulted in animal; stress, infection, and their dissemination in the form of antibiotic resistant genes to other bacteria including human and animal pathogens. The problems arising with antibiotics can be overcome by using silver nanoparticles (AgNPs) due to their physiochemical properties and low toxicity. So AgNPs could be combined with antibiotics to induce infections in fish cell lines and to protect dissemination of antibiotics in the form of antibiotics resistant genes. We expose AgNPs on fish cell lines as a new nano-antibacterial agent to investigate and obtain findings in terms of the cell viability and toxicity. The experimental data is analyzed to examine the antibacterial effects of nanosilver as a replacement agent and discuss the complex scenario, drawbacks, techniques, methods, main mechanisms, and procedures to perform antibacterial tests of exposed AgNPs. There would be an attempt to deal with the AgNPs antibacterial therapies for the fish cell lines.

Screening of Fish Cell Lines for Piscine Orthoreovirus-1 (PRV-1) Amplification: Identification of the Non-Supportive PRV-1 Invitrome

Piscine reovirus (PRV) is the causative agent of heart and skeletal muscle inflammation (HSMI), which is detrimental to Atlantic Salmon (AS) aquaculture, but so far has not been cultivatable, which impedes studying the disease and developing a vaccine. Homogenates of head kidney and red blood cells (RBC) from AS in which PRV-1 had been detected were applied to fish cell lines. The cell lines were from embryos, and from brain, blood, fin, gill, gonads, gut, heart, kidney, liver, skin, and spleen, and had the shapes of endothelial, epithelial, fibroblast, and macrophage cells. Most cell lines were derived from the Neopterygii subclass of fish, but one was from subclass Chondrostei. Cultures were examined by phase contrast microscopy for appearance, and by quantitative polymerase chain reaction (qPCR) for PRV-1 RNA amplification and for the capacity to transfer any changes to new cultures. No changes in appearance and Ct values were observed consistently or transferable to new cultures. Therefore, 31 cell lines examined were unable to support PRV-1 amplification and are described as belonging to the non-supportive PRV-1 invitrome. However, these investigations and cell lines can contribute to understanding PRV-1 cellular and host tropism, and the interactions between virus-infected and bystander cells.