Rainbow trout | EPFL Graph Search

The rainbow trout (Oncorhynchus mykiss) is a species of trout native to cold-water tributaries of the Pacific Ocean in Asia and North America. The steelhead (sometimes called steelhead trout) is an anadromous (sea-run) form of the coastal rainbow trout (O. m. irideus) or Columbia River redband trout (O. m. gairdneri) that usually returns to freshwater to spawn after living two to three years in the ocean. Freshwater forms that have been introduced into the Great Lakes and migrate into tributaries to spawn are also called steelhead. Adult freshwater stream rainbow trout average between , while lake-dwelling and anadromous forms may reach . Coloration varies widely based on subspecies, forms, and habitat. Adult fish are distinguished by a broad reddish stripe along the lateral line, from gills to the tail, which is most vivid in breeding males. Wild-caught and hatchery-reared forms of the species have been transplanted and intro

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

Yang Yue

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.

EPFL2015

Extending the Concept of Predicting Fish Acute Toxicity In Vitro to the Intestinal Cell Line RTgutGC

Jenny Maner, Kristin Schirmer, Hannah Inka Schug

Testing chemicals for fish acute toxicity is a legal requirement in many countries as part of environmental risk assessment. To reduce the number of fish used, substantial efforts have been focused on alternative approaches. Prominently, the cell viability assay with the rainbow trout (Oncorhynchus mykiss) gill cell line, RTgill-W1, has proven to be highly predictive and robust. Like the gills, the intestine is considered a major site of chemical uptake and biotransformation, but, in contrast to the gills, it is expected to be exposed to more hydrophobic chemicals, which enter the fish via food. In the present study, we therefore aimed to extend the cell bioassay to the rainbow trout epithelial cell line from intestine, RTgutGC. Using 16 hydrophobic and volatile chemicals from the fragrance palette, we show that also the RTgutGC cell line can be used to predict fish acute toxicity of chemicals and yields intra-laboratory variability in line with other bioassays. By comparing the RTgutGC toxicity to a study employing the RTgill-W1 assay on the same group of chemicals, a fragrance-specific relationship was established that reflects an almost perfect 1:1 relationship between in vitro and in vivo toxicity results. Thus, both cell lines can be used to predict fish acute toxicity, either by extrapolating based on the in vivo-in vitro relationship or by taking the in vitro results at face value. We moreover demonstrate the derivation of non-toxic concentrations for downstream applications that rely on a healthy cell state, such as the assessment of biotransformation or chemical transfer.

SPEKTRUM AKADEMISCHER VERLAG-SPRINGER-VERLAG GMBH2020

A fish intestinal barrier model to assess the interaction with chemicals in vitro

Hannah Inka Schug

The intestinal epithelium acts as vital gate keeper between the fish and its surrounding environment. A single layer of connected epithelial cells forms a selective barrier, which, among other essential functions, is important for nutrient uptake and defence against pathogens and putatively toxic chemicals. Despite its recognised significance, large knowledge gaps exist regarding the functionality of the fish intestine in general and the epithelium in specific, in large part due to the unavailability of suitable model systems. This thesis exploits a recently established in vitro fish intestinal cell barrier model from rainbow trout (Oncorhynchus mykiss), which is based on the so far only available fish intestinal epithelial cell line, RTgutGC. Motivated by the fact that the RTgutGC-based barrier model allows unprecedented opportunities for experimentation, this thesis engaged in embedding this cell line model into an in vitro testing strategy for assessing chemical transfer into fish. The focus was set on generally difficult-to-test chemicals, i.e. chemicals that are hydrophobic and/or volatile. For this purpose fragrances were selected, which combine chemicals in regular use with the described properties. Moreover, the response of the model epithelium to pathogenic stimulation was explored, in order to lay the groundwork for extending the in vitro testing strategy to different states of immune challenge in the future. To initiate this research, fragrance cellular toxicity was assessed to derive non-toxic concentrations as prerequisite for transfer experiments and for in vitro to in vivo toxicity extrapolation of fish acute toxicity, which was both successfully achieved. Chemical transfer experiments were subsequently carried out with a set of fragrance chemicals using a purposefully designed transfer chamber, which reduces losses due to binding and evaporation of chemicals. The obtained transfer data were combined with in silico kinetic models to distinguish mechanisms of transfer and enable reliable result interpretation. Based on the mechanistic model, transfer processes were identified to involve intracellular accumulation and biotransformation, combined with paracellular transport and active transport processes. This outcome is contradictory to the current assumptions used in bioaccumulation models that chemical uptake is logKOW-dependent. While chemical transfer assessment was conducted under optimal cellular conditions, the in vivo intestine is likely exposed to pathogens activating the immune system and potentially impairing barrier tightness. Indeed, also the RTgutGC cell barrier model was found to respond to bacterial and viral stimulation in a manner previously shown in vivo. Toll-like receptor-mediated regulation of cytokines and interferon were recorded within hours of exposure while long-term exposure resulted in an, albeit small, loss of epithelial tightness. To conclude, this thesis comprises a detailed exploration of the RTgutGC-based fish intestinal cell model in terms of its physical and immunological barrier properties and demonstrates the value of this in vitro model for fish gut physiology, immunology and toxicology. This unique barrier model has the potential to successfully span mechanistic knowledge on gut functionality to practical application, such as fish feed development and chemical hazard assessment, while contributing to the refinement, reduction and even the potential replacement of animal testing

EPFL2019