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

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