Development of a proteomic tool to predict hepatic toxicity

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Drug hepatotoxicity is one of the major concerns in drug development. Indeed, it is one of the primary reasons for failure of new candidates in clinical trials and drug withdrawal from the market after commercialization. Moreover, despite thorough preclinical chronic toxicity studies drug-induced liver injury is still one of the mostfrequent causes of acute liver failure in industrial countries. Development of new tools to predict moreaccurately liver toxicity is crucial to accelerate drug development. Proteomics is an interesting tool since it can monitor protein expression, interaction and post-translational modifications. Furthermore, proteomic analysis could provide the opportunity compare similar end points in vitro and in vivo. However, conventionalmonolayer cell culture models are highly limited to detect toxicity. The limitations are mostly due to thedifferences between the phenotype of cultured cells and an organ. In the past years, mounting evidence hassuggested that 3D cell culture, such as spheroids, could induce a drastic improvement in the phenotypes ofvarious cell lines. In this project, we aim to develop and characterized a spheroid 3D model using HepG2 to study hepatotoxicity. The spheroid 3D model will be characterized using markers of polarized differentiated hepatocytes such as glycoprotein-1, albumin and xenobiotic-metabolizing enzymes. Afterwards, hepatotoxicity will be induced on HepG2 spheroids and monolayer cells using acetaminophen, amiodarone, diclofenac,metformin, phenformin and valproic acid. Cells viability will be measured based on the ability of spheroids to produce ATP. LC50 will be determined for both in vitro models and compared to LD50 values found in theliterature. A toxicity profile of each drug will also be determined by proteomic to identify specific markers to predict hepatotoxicity. To validate those potential new in vitro biomarkers, Balb/c mice will be treated with acetaminophen at 400 mg/kg. Serum and liver tissue will be collected for proteomic analysis and hepatotoxicitywill be confirmed by histopathological analysis of the liver and quantification of AST and ALT in the serum