Département Toxicologie de l’environnement

In this study we present the first fish-gut-on-chip model. This model is based on the reconstruction of the intestinal barrier by culturing two intestinal cell lines from rainbow trout, namely epithelial RTgutGC and fibroblastic RTgutF, in an artificial microenvironment. For a realistic mimicry of the interface between the intestinal lumen and the interior of the organism we i) developed ultrathin and highly porous silicon nitride membranes that serve as basement membrane analogues and provide a culture interface for the fish cells; ii) constructed a unique micro-well plate-based microfluidic bioreactor that enables parallelization of experiments and creates realistic fluid flow exposure scenarios for the cells; iii) integrated electrodes in the reactor for non-invasive impedance sensing of cellular well-being. In a first approach, we used this reactor to investigate the response of epithelial fish cells to in vivo-like shear stress rates of 0.002–0.06 dyne per cm², resulting from fluid flow within the intestinal lumen. Moreover, we investigated the interplay of epithelial and fibroblast cells under optimal flow conditions to carefully evaluate the benefits and drawbacks of the more complex reconstruction of the intestinal architecture. With our fish-gut-on-chip model we open up new strategies for a better understanding of basic fish physiology, for the refinement of fish feed in aquaculture and for predicting chemical uptake and bioaccumulation in fish for environmental risk assessment. The basic principles of our reactor prototype, including the use of ultrathin membranes, an open microfluidic circuit for perfusion and the micro-well plate-based format for simplified handling and avoidance of air-bubbles, will as well be of great value for other barrier-on-chip models.

Titanium dioxide nanoparticles (TiO₂ NPs) are widely incorporated in various consumer products such as cosmetics and food. Despite known human exposure, the potential risks of TiO₂ NPs during pregnancy are not fully understood, but several studies in mice elucidated toxic effects on fetal development. It has also been shown that modifying NPs with positive or negative surface charge alters cellular uptake and abolishes fetotoxicity of silicon dioxide (SiO₂) NPs in mice.
Here, we investigated accumulation and translocation of positively charged TiO₂-NH₂ and negatively charged TiO₂-COOH NPs at the placental barrier, to clarify whether surface charge provides a means to control TiO₂ NP distribution at the placental barrier. To ensure outcome relevant for humans, the recently developed in vitro human placental co-culture model and the gold standard amongst placental translocation models – the ex vivo perfusion of human term placental tissue - were employed during this study. Sector field-ICP-MS analysis of maternal and fetal supernatants as well as placental cells/tissues revealed a substantial accumulation of both TiO₂ NP types while no considerable placental translocation was apparent in both models. Characterization of agglomeration behavior demonstrated a strong and fast agglomeration of TiO₂-NH₂ and TiO₂-COOH NPs in the different culture media.
Overall, our results indicate that surface charge is not a key factor to steer placental uptake and transfer of TiO₂. Moreover, the negligible placental transfer but high accumulation of TiO₂ NPs in placental tissue suggests that potential effects on fetal health may occur indirectly, which calls for further studies elucidating the impact of TiO₂ NPs on placental tissue functionality and signaling.

Testing chemicals for fish acute toxicity is a legal requirement in many countries as part of environmental risk assessment. To reduce the numbers of fish used, substantial efforts have been focussed on alternative approaches. Prominently, the cell viability assay with the rainbow trout (Oncorhynchus mykiss) gill cell line, RTgill-W1, has been recognized, owing to its high predictive power and robustness. Like gills, the intestine is considered a major site of chemical uptake and biotransformation but, in contrast to gills, is expected to be exposed to rather 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 showed 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 which 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 using the obtained 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 which rely on a healthy cell state, such as the assessment of biotransformation or chemical transfer.

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.

Out of the >107 million chemicals already registered with the Chemical Abstracts Services, less than 0.5% are being regulated, and even fewer are evaluated for their safety. Consequently, a new paradigm in risk assessment is urgently needed. It should encompass faster and less costly methods and reduce the number of animals needed for testing. One proposal is to combine computational modeling with small-scale bioassay methods. This chapter describes the methods that link in vitro bioassays using fish cells with physiologically based toxicokinetic (PBTK) modeling in order to predict the acute toxicity, bioaccumulation, and impact of chemicals on fish growth. The main focus is on PBTK modeling; thus all the model equations and parameters available for eight fish species as well as suggestions for possible software implementation will be provided. The PBTK model described here can account for respiratory and dietary uptake routes and for chemical biotransformation processes.