Microplastics are ubiquitous environmental pollutants frequently detected in aquatic environments. Here we used the Atlantic salmon epithelial gill cell line (ASG-10) to investigate the uptake and effects of polystyrene (PS) microplastic. The ASG-10 cell line has phagocytotic/endocytic capacities and can take up clear PS particles at 0.2 and 1.0 µm, while PS at 10 µm was not taken up. As a response to the uptake, the ASG-10 cells increased their lysosomal activity. Furthermore, no effects on the mitochondria were found, neither on the mitochondrial membrane potential nor the mitochondria morphology (branch length and diameter). Interestingly, even a very high concentration of PS (200 µg/ml) with all tested particle sizes had no effects on cell viability or cell cycle. The environmental toxin Benzo(a)pyrene (B(a)P), a known inducer of CYP1A, is highly hydrophobic and thus sticks to the PS particles. However, co-exposure of B(a)P and PS the particles did not increase the induction of CYP1A activity compared to B(a)P alone. Our study contributes to the understanding of the cellular effects of PS particles using a highly relevant Atlantic salmon gill epithelium in vitro model.

Here we evaluated the gill epithelial cell line ASG-10 from Atlantic salmon, as an in vitro model for research on known water quality challenges in aquaculture. Ammonia/ammonium (NH3/NH4+), a recognized challenge in water-intensive recirculating aquaculture systems (RAS), induced lysosomal vacuolization, reduced protein degradation and cell migration of the ASG-10 cells. Aluminium (Aln+), another challenge in freshwater aquaculture facilities had only minor effects. Next, we investigated the tolerance for direct water exposure of ASG-10. The cells tolerated water with osmolarity between 169 and 419 mOsmol/kg for 24 h. However, cells exposed for 3 h to water at 863 mOsmol/kg changed cellular morphology and induced gene expression related to stress (gpx1, casp3, hsp70), and after 24 h exposure cellular viability was severely reduced. Nevertheless, when the cells were grown in transwell inserts, they tolerated 863 mOsmol/kg for 3 h and induction of stress response associated genes was considerably reduced. Lastly, the ASG-10 cells were exposed to water samples, with no known quality issues, from different aquaculture facilities. The cells showed no differences in viability or morphology compared to their representative control. In conclusion, the ASG-10 cell line is a promising in vitro model to study water quality challenges and whole water samples.

Fish gills are not only the respiratory organ, but also essential for ion-regulation, acid-base control, detoxification, waste excretion and host defense. Multifactorial gill diseases are common in farmed Atlantic salmon, and still poorly understood. Understanding gill pathophysiology is of paramount importance, but the sacrifice of large numbers of experimental animals for this purpose should be avoided. Therefore, in vitro models, such as cell lines, are urgently required to replace fish trials. An Atlantic salmon gill epithelial cell line, ASG-10, was established at the Norwegian Veterinary institute in 2018. This cell line forms a monolayer expressing cytokeratin, e-cadherin and desmosomes, hallmarks of a functional epithelial barrier. To determine the value of ASG-10 for comparative studies of gill functions, the characterization of ASG-10 was taken one step further by performing functional assays and comparing the cell proteome and transcriptome with those of gills from juvenile freshwater Atlantic salmon. The ASG-10 cell line appear to be a homogenous cell line consisting of epithelial cells, which express tight junction proteins. We demonstrated that ASG-10 forms a barrier, both alone and in co-culture with the Atlantic salmon gill fibroblast cell line ASG-13. ASG-10 cells can phagocytose and express several ATP-binding cassette transport proteins. Additionally, ASG-10 expresses genes involved in biotransformation of xenobiotics and immune responses. Taken together, this study provides an overview of functions that can be studied using ASG-10, which will be an important contribution to in vitro gill epithelial research of Atlantic salmon.

In order to ensure the proper use and interpretation of results from laboratory test systems, it is important to know the characteristics of your test system. Here we compare mitochondria and the handling of reactive oxygen species (ROS) in two gill epithelial cell lines, the well-known RTgill-W1 cell line from Rainbow trout and the newly established ASG-10 cell line from Atlantic salmon. Rotenone was used to trigger ROS production. Rotenone reduced metabolic activity and induced cell death in both cell lines, with RTgill-W1 far more sensitive than ASG-10. In untreated cells, the mitochondria appear to be more fragmented in RTgill-W1 cells compared to ASG-10 cells. Furthermore, rotenone induced mitochondrial fragmentation, reduced mitochondria membrane potential (Δψm) and increased ROS generation in both cell lines. Glutathione (GSH) and catalase is important to maintain the cellular oxidative balance by eliminating hydrogen peroxide (H2O2). In response to rotenone, both GSH and catalase depletion were observed in the RTgill-W1 cells. In contrast, no changes were found in the GSH levels in ASG-10, while the catalase activity was increased. In summary, the two salmonid gill cell lines have different tolerance towards ROS, probably caused by differences in mitochondrial status as well as in GSH and catalase activities. This should be taken into consideration with the selection of experimental model and interpretation of results.
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UNASSIGNED: The online version contains supplementary material available at 10.1007/s10616-022-00560-0.