This proposal comprised 4 work packages (WP) describing the experimental part and was complemented with training strategies to enhance the career path of the fellow and defined communication/dissemination/exploitation objectives.
WP1 involved the collection of information from in vivo and human clinical samples. These experiments started immediately after submission of the project proposal as some of the samples were re-used from previous projects (in alignment with the 3Rs) and the fellow was already working with the host group. The work was performed with the help of 2 PhD students of the group and published (Van Campenhout et al. 2019, Gijbels et al. 2020). Some work concerning this WP is currently being concluded by 2 other PhD students and will be published in 2 forthcoming research papers, 1 including the remaining in vivo data and another 1 concerning clinical data.
WP2 concerned the collection of data using the selected human liver-based in vitro model. For that, the fellow did her secondment at the Karolinska Institutet in Sweden where she spent 2.5 months acquiring skills in the generation, maintenance and handling of spheroids of primary human hepatocytes. This knowledge was brought and transferred to the host laboratory. For this project, the spheroids were incubated with selected cholestatic drugs under a 28-day repeated exposure protocol, with assessment of results through total adenosine triphosphate (ATP) quantification. Initially, a dose-response curve was performed for each drug, time-point and donor, to select the concentrations to proceed with the experiment. Then, the selected concentrations were tested in the presence and in the absence of a concentrated bile acid mixture containing the most abundant bile acids in humans. This allowed to calculate the cholestatic index (CIx), a ratio between the viability in the presence and in the absence of bile acid mixture, for each drug, time-point and donor. A CIx below 0.8 was taken as an indication of the cholestatic potential of that drug and selected for transcriptomic analysis, which is currently being finalized with the help of a lab technician. The results collected from WP2 will be included in a research article that is currently in preparation and waiting for the results from the transcriptomic analysis.
WP3 comprised the development of the AOP network, which started with the data collected from an in vivo model of extrahepatic cholestasis (Gijbels et al. 2020). The remaining data that is being collected from WP1 and 2 will also be integrated in the AOP network, which will be updated in the research paper mentioned for WP2.
In WP4, the selected in vitro model was exposed to selected chemicals from distinct categories: paraquat, a biocide; tartrazine, a food additive; and triclosan, a cosmetic ingredient. These chemicals have been associated with cholestatic injury, though information is scarce and not yet fully understood. The literature search included in this WP resulted in 3 review papers addressing the different categories of chemicals and what is known about their ability to cause cholestasis/ hepatotoxicity (Vilas-Boas et al. 2019, Vilas-Boas et al. 2020, Vilas-Boas and Vinken 2020). The review paper addressing the hepatotoxic effects of nanomaterials (Vilas-Boas and Vinken 2020) has been highlighted by the Animal Free Safety Assessment (AFSA) collaboration and has caught the attention of stakeholders with regulatory influence, such as the Scientific Committee on Consumer Safety.
During the timeframe of this project, the fellow had the chance to strengthen her network and build up her CV by attending specific training courses, such as “Safety assessment of cosmetics in the EU – Intensive course 2020”, “Applied In Vitro Toxicology Course” and “Leadership for postdocs”, among others. The participation in conferences was restricted by the COVID-19 outbreak, as the ESTIV conference 2020 and the World Congress on Alternatives and Animal Use in Life Sciences 2020 were postponed.
