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

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

Intestinal Fish Cell Barrier Model to Assess Transfer of Organic Chemicals in Vitro: An Experimental and Computational Study

Jenny Maner, Kristin Schirmer, Hannah Inka Schug, Anze Zupanic

We studied the role of the fish intestine as a barrier for organic chemicals using the epithelial barrier model built on the rainbow trout (Oncorhynchus mykiss) intestinal cell line, RTgutGC and the newly developed exposure chamber, TransFEr, specifically designed to work with hydrophobic and volatile chemicals. Testing 11 chemicals with a range of physicochemical properties (logK(ow): 2.2 to 6.3, logHLC: 6.1 to 2.3) and combining the data with a mechanistic kinetic model enabled the determination of dominant processes underlying the transfer experiments and the derivation of robust transfer rates. Against the current assumption in chemical uptake modeling, chemical transfer did not strictly depend on the logK(ow) but resulted from chemical-specific intracellular accumulation and biotransformation combined with paracellular and active transport. Modeling also identified that conducting elaborate measurements of the plastic parts, including the polystyrene insert and the PET filter, is unnecessary and that stirring in the TransFEr chamber reduced the stagnant water layers compared to theoretical predictions. Aside from providing insights into chemical uptake via the intestinal epithelium, this system can easily be transferred to other cell-based barrier systems, such as the fish gill or mammalian intestinal models and may improve in vitro-in vivo extrapolation and prediction of chemical bioaccumulation into organisms.

AMER CHEMICAL SOC2019