Assessment of the permeation of the fragrance Tonalide across the fish intestinal barrier in vitro
This work focuses on Tonalide, a small fragrance molecule with an octanol-water partitioning coefficient of 5.7. Fragrances are an environmentally relevant substance class because they are produced in high amounts and strongly utilized for a multitude of products. The input into the aquatic environment is significant due to continuous use and low removal rates in waste water treatment plants. Their partly high hydrophobicity makes dietary uptake and bioaccumulation very likely. In this work, a cell line-based model for the intestinal barrier in fish and its application for compound permeability testing is presented and tested with Tonalide in a recently developed setup.
The model is based on intestinal epithelial cells from rainbow trout that were seeded into permeable supports. This creates a separation into an apical, representing the luminal side, and a basolateral, representing the blood-facing side, compartment, which generates a system in which permeation can be precisely studied. Complementary, an airtight stainless steel chamber was used for the compound exposure to reduce loss through evaporation and material binding. Furthermore, the system allowed the use of a passive dosing system for the exposure of hydrophobic substances as well as apical and basolateral stirring. For the basolateral compartment two setups were used: Medium without sink which leads to compound concentration gradient decrease over time and medium with sink used to maintain the concentration gradient. This setup is well suited for the assessment of fragrance permeation because it reduces compound loss, a common problem caused by their relative hydrophobicity and volatility.
It was found that experimental system and methodology are suitable to test the Tonalide permeation across the RTgut-GC cell layer. The accumulation in the cells and the permeation from the apical into the basolateral compartment was clearly detected through measurement of Tonalide mass in the different compartments. The permeation through permeable supports with and without cells was not significantly different. The same is true for the permeation with an without passive sink. The Tonalide permeability for experiments without sink is 4.95*10-6 ± 8.3*10-7 cm/s for cell-free and 6.595*10-6 ± 7.2*10-7 cm/s for cell-containing permeable supports, for experiments with sink the corresponding values are 6.667*10-6 ± 3.3*10-7 cm/s and 6.537*10-6 ± 1.6*10-6 cm/s. The accumulation in the cells increased twofold through the use of the passive sink. The measured permeation was compared to and evaluated with the help of permeabilities of other compounds. This revealed a dependency of substance lipophilicity and permeability across cell layers in permeable supports.