Interaction of Silver Nanoparticles (AgNP) with Rainbow Trout Gill Cell Lines

Owing to their unique antimicrobial properties, silver nanoparticles (AgNP) are among the most widely used engineered nanoparticles in a variety of consumer products and medical applications. Their resulting release into the aquatic environment raises concern about potential adverse effects in aquatic organisms. This thesis therefore focused on the interaction of AgNP with cells derived from fish gill, specifically a fish gill cell line from rainbow trout (Oncorhynchus mykiss), in an integrative way, starting from AgNP behavior in exposure media and upon contact with cells, following the formation of a protein corona around the AgNP during trafficking in intact cells and quantifying short- and long-term impact on cell viability. The composition of cell exposure media was found to have a major effect on the AgNP behaviour and toxicity to the rainbow trout gill cell line, RTgill-W1. Three different exposure media were used with varying ionic strength and chloride content, which had a dominant effect on the AgNP agglomeration, deposition and dissolved silver species in exposure media. Comparing the behaviour and toxicity of AgNP in the different media, stronger agglomeration of AgNP correlated with higher toxicity. The newly developed low ionic strength medium, d-L15/ex, which can stabilize AgNP and better mimic the freshwater environment, offers an excellent exposure solution to study cellular and molecular effects of nanoparticles to gill cells. Furthermore, the lysosomal membrane integrity was significantly more sensitive to AgNP exposure than cellular metabolic activity or cell membrane integrity and showed the weakest protection by silver ligands. This revealed that AgNP seem to particularly affect RTgill-W1 cell lysosomes. To shed light on the path of AgNP in cells leading to toxicity, AgNP uptake by the cells was explored on exposure in d-L15/ex medium. Electron microscope imaging not only suggested that RTgill-W1 cells take up AgNP via an energy depend pathway but also that particles are stored in endocytic compartments, which include lysosomes and endosomes. With subcellular fractionation, the AgNP-protein corona was recovered from intact subcellular compartments with high acid phosphatase activity and/or high silver content. 383 proteins were identified in this way and broadly classified as belonging to cell membrane functions as well as endocytosis and vesicle-mediated transport pathways. This is the first study that focuses on the interaction of industrial nanoparticles with proteins in living cells and an initial mechanism of AgNP uptake and toxicity was derived from it. Long-term exposures of cells in culture is thus far restricted to media containing a non-defined serum component. Therefore, in order to allow long-term studies on the impact of nanoparticles with fish gill cells, a new in vitro cell model, based on the RTgill-W1 cell line, was developed and renamed as RTgill-W1-pf (protein free). This cell line was obtained by means of selection in a commercial, protein-free culture medium. Initial results with this new cell line demonstrated that AgNP inhibited RTgill-W1-pf cell proliferation in a 12 day exposure test. To conclude, this thesis offers insights into the mechanisms of interaction of AgNP with fish gill cells. It provides fundamental information about AgNP uptake and interaction with proteins in these cells, which is useful for understanding AgNP toxicity and sustainable nanoparticle development.

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