A fish intestinal epithelial barrier model established from the rainbow trout (Oncorhynchus mykiss) cell line, RTgutGC

The intestine of fish is a multifunctional organ: lined by only a single layer of specialized epithelial cells, it has various physiological roles including nutrient absorption and ion regulation. It moreover comprises an important barrier for environmental toxicants, including metals. Thus far, knowledge of the fish intestine is limited largely to in vivo or ex vivo investigations. Recently, however, the first fish intestinal cell line, RTgutGC, was established, originating from a rainbow trout (Oncorhynchus mykiss). In order to exploit the opportunities arising from RTgutGC cells for exploring fish intestinal physiology and toxicology, we present here the establishment of cells on commercially available permeable membrane supports and evaluate its suitability as a model of polarized intestinal epithelia. Within 3 weeks of culture, RTgutGC cells show epithelial features by forming tight junctions and desmosomes between adjacent cells. Cells develop a transepithelial electrical resistance comparable to in vivo measured values, reflecting the leaky nature of the fish intestine. Immunocytochemistry reveals evidence of polarization, such as basolateral localization of Na+/K+-ATPase (NKA) and apical localization of the tight junction protein ZO-1. NKA mRNA abundance was induced as physiological response toward a saltwater buffer, mimicking the migration of rainbow trout from fresh to seawater. Permeation of fluorescent molecules proved the barrier function of the cells, with permeation coefficients being comparable to those reported in fish. Finally, we demonstrate that cells on permeable supports are more resistant to the toxicity elicited by silver ions than cells grown the conventional way, likely due to improved cellular silver excretion

Effect of TiO2 Nanoparticles and UV Radiation on Extracellular Enzyme Activity of Intact Heterotrophic Biofilms

When introduced into the aquatic environment, TiO2 NP are likely to settle from the water column, which results in increased exposure of benthic communities. Here, we show that the activity of two extracellular enzymes of intact heterotrophic biofilms, beta-glucosidase (carbon-cycling) and l-leucin aminopeptidase (nitrogen-cycling), was reduced following exposure to surface functionalized TiO2 NP and UV radiation, depending on the particles' coating. This reduction was partially linked to ROS production. Alkaline phosphatase (phosphorus-cycling) activity was not affected, however in contrast, an alkaline phosphatase isolated from E. coli was strongly inhibited at lower concentrations of TiO2 NP than the intact biofilms. These results indicate that enzymes present in the biofilm matrix are partly protected against exposure to TiO2 NP and UV radiation. Impairment of extracellular enzymes which mediate the uptake of nutrients from water may affect ecosystem function

TransFEr: a new device to measure the transfer of volatile and hydrophobic organic chemicals across an in vitro intestinal fish cell barrier

Transfer of compounds across cellular barriers is a critical step of compound uptake into organisms. Using in vitro barrier systems to evaluate such transfer is attractive because of the higher throughput and reduced resource needs compared to animal studies. Thus far, however, studying the transfer of hydrophobic and volatile compounds was hampered by the unavailability of in vitro exposure systems that allow for stable and predictable chemical exposure concentrations. To overcome this limitation, we constructed a novel exposure chamber, TransFEr, and tested it with an in vitro epithelial barrier model using the rainbow trout (Oncorhynchus mykiss) intestinal cell line, RTgutGC. Key features of the chamber are its closed design and rotatable silicon segments, which can serve for chemical dosing and sampling. Using the fragrance damascone beta (log K-OW: 3.7, log HLC: -3.9) as a pilot chemical, we were able to demonstrate that our exposure chamber provides for stable chemical exposure concentrations and full mass balance. The RTgutGC epithelium served as barrier for damascone beta transfer, which we attribute to chemical retention and biotransformation in the intestinal cells. Nevertheless, substantial transfer of the chemical across the epithelium occurred. When a chemical sink, i.e. a silicon segment, was included in the basolateral chamber to mimic blood constituents binding in vivo, transfer was about three-fold enhanced. We suggest that the presented methodology can help to obtain insights into chemical uptake mechanisms via the intestinal or other epithelia of fish and other animals for hydrophobic and volatile chemicals

Extending the concept of predicting fish acute toxicity in vitro to the intestinal cell line rtgutgc

© The Authors, 2019. 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.ISSN:1868-8551ISSN:0946-7785ISSN:1868-596

Improving a fish intestinal barrier model by combining two rainbow trout cell lines: epithelial RTgutGC and fibroblastic RTgutF

An in vitro model of the fish intestine is of interest for research and application in diverse fields such as fish physiology, aquaculture and chemical risk assessment. The recently developed epithelial barrier model of the fish intestine relies on the RTgutGC cell line from rainbow trout (Oncorhynchus mykiss), cultured in inserts on permeable membranes. Our aim was to extend the current system by introducing intestinal fibroblasts as supportive layer in order to reconstruct the epithelial-mesenchymal interface as found in vivo. We therefore initiated and characterized the first fibroblast cell line from the intestine of rainbow trout, which has been termed RTgutF. Co-culture studies of RTgutGC and RTgutF were performed on commercially available electric cell substrate for impedance sensing (ECIS) and on newly developed ultrathin, highly porous alumina membranes to imitate the cellular interaction with the basement membrane. Cellular events were examined with non-invasive impedance spectroscopy to distinguish between barrier tightness and cell density in the ECIS system and to determine transepithelial electrical resistance for cells cultured on the alumina membranes. We highlight the relevance of the piscine intestinal fibroblasts for an advanced intestinal barrier model, particularly on ultrathin alumina membranes. These membranes enable rapid crosstalk of cells cultured on opposite sides, which led to increased barrier tightening in the fish cell line-based epithelial-mesenchymal model

Effect of TiO₂ Nanoparticles and UV Radiation on Extracellular Enzyme Activity of Intact Heterotrophic Biofilms

When introduced into the aquatic environment, TiO₂ NP are likely to settle from the water column, which results in increased exposure of benthic communities. Here, we show that the activity of two extracellular enzymes of intact heterotrophic biofilms, β-glucosidase (carbon-cycling) and l-leucin aminopeptidase (nitrogen-cycling), was reduced following exposure to surface functionalized TiO₂ NP and UV radiation, depending on the particles’ coating. This reduction was partially linked to ROS production. Alkaline phosphatase (phosphorus-cycling) activity was not affected, however in contrast, an alkaline phosphatase isolated from <i>E. coli</i> was strongly inhibited at lower concentrations of TiO₂ NP than the intact biofilms. These results indicate that enzymes present in the biofilm matrix are partly protected against exposure to TiO₂ NP and UV radiation. Impairment of extracellular enzymes which mediate the uptake of nutrients from water may affect ecosystem function

Chemical Aspects of Nanoparticle Ecotoxicology

Nanoecotoxicology strives to understand the processes and mechanisms by which engineered nanoparticles (ENP) may exert toxic effects on aquatic organisms. Detailed knowledge of the chemical reactions of nanoparticles in the media and of their interactions with organisms is required to understand these effects. The processes of agglomeration of nanoparticles, of dissolution and release of toxic metal ions, and of production of reactive oxygen species (ROS) are considered in this article. Important questions concern the role of uptake of nanoparticles in various organisms, in contrast to uptake of ions released from nanoparticles and to nanoparticle attachment to organism surfaces. These interactions are illustrated for effects of silver nanoparticles (AgNP), cerium oxide (CeO2 NP) and titanium dioxide (TiO2 NP), on aquatic organisms, including algae, biofilms, fish cells and fish embryos