Abstract
Drug-induced liver injury, including cholestasis, is an important clinical issue and economic burden for pharmaceutical industry and healthcare systems. However, human-relevant in vitro information on the ability of other types of chemicals to induce cholestatic hepatotoxicity is lacking. This work aimed at investigating the cholestatic potential of non-pharmaceutical chemicals using primary human hepatocytes cultured in 3D spheroids. Spheroid cultures were repeatedly (co-) exposed to drugs (cyclosporine-A, bosentan, macitentan) or non-pharmaceutical chemicals (paraquat, tartrazine, triclosan) and a concentrated mixture of bile acids for 4 weeks. Cell viability (adenosine triphosphate content) was checked every week and used to calculate the cholestatic index, an indicator of cholestatic liability. Microarray analysis was performed at specific time-points to verify the deregulation of genes related to cholestasis, steatosis and fibrosis. Despite the evident inter-donor variability, shorter exposures to cyclosporine-A consistently produced cholestatic index values below 0.80 with transcriptomic data partially supporting its cholestatic burden. Bosentan confirmed to be hepatotoxic, while macitentan was not toxic in the tested concentrations. Prolonged exposure to paraquat suggested fibrotic potential, while triclosan markedly deregulated genes involved in different types of hepatotoxicity. These results support the applicability of primary human hepatocyte spheroids to study hepatotoxicity of non-pharmaceutical chemicals in vitro.
Highlights
Cholestasis denotes the disruption of bile synthesis or flow leading to the accumulation of toxic levels of bile acids (BAs) in hepatocytes or systemic circulation [1]
Cholestasis often results from the direct inhibition of hepatocellular canalicular transporters involved in the efflux of BAs, such as the bile salt export pump (BSEP, ABCB11) and multidrug resistance-associated protein (MRP) 2 (ABCC2) [2]
Considering the acknowledged applicability of the PHH spheroids for depicting drug-induced liver injury (DILI) in a sensitive and human-relevant manner, this in vitro model was used, for the first time, to assess the hepatotoxic potential of non-pharmaceutical chemicals, which have been linked to the manifestation of cholestatic effects
Summary
Cholestasis denotes the disruption of bile synthesis or flow leading to the accumulation of toxic levels of bile acids (BAs) in hepatocytes or systemic circulation [1]. In a recent study performed by our group, a genetic signature partially based on this AOP was employed to depict non-pharmaceutical chemicals with assumed cholestatic characteristics but failed to identify genes relevant for cholestasis for all the treatments This outcome could be attributed to a number of reasons, one of them being the need for a longer-term exposure screening method, which is not feasible in the human hepatoma HepaRG model, resulting in a relative lower sensitivity towards testing non-pharmaceutical compounds [20]. Considering the acknowledged applicability of the PHH spheroids for depicting DILI in a sensitive and human-relevant manner, this in vitro model was used, for the first time, to assess the hepatotoxic potential of non-pharmaceutical chemicals, which have been linked to the manifestation of cholestatic effects For this purpose, spheroids from three different PHH donors were generated in the present study and used to investigate the ability of PQ, TAR and TRI to induce (cholestatic) hepatotoxicity. Hepatotoxic drugs, including CyA, BOS and macitentan (MAC), were used as benchmark chemicals
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