Development of a proteomic tool to predict hepatic toxicity

药物肝毒性是药物开发中的主要问题之一。事实上，这是新候选药物在临床试验中失败和商业化后退出市场的主要原因之一。此外，尽管进行了全面的临床前慢性毒性研究，但药物性肝损伤仍然是工业国家急性肝衰竭最常见的原因之一。开发新工具来更准确地预测肝毒性对于加速药物开发至关重要。蛋白质组学是一种有趣的工具，因为它可以监测蛋白质的表达、相互作用和翻译后修饰。此外，蛋白质组学分析可以提供比较体外和体内相似终点的机会。然而，传统的单层细胞培养模型在检测毒性方面受到很大限制。这种限制主要是由于培养细胞和器官的表型不同。在过去的几年里，越来越多的证据表明，3D细胞培养，如球体，可以诱导各种细胞系表型的急剧改善。在这个项目中，我们的目标是利用HepG2开发和表征一个球体3D模型来研究肝毒性。球体3D模型将使用极化分化肝细胞的标记物，如糖蛋白-1、白蛋白和外源代谢酶来表征。然后用对乙酰氨基酚、胺碘酮、双氯芬酸、二甲双胍、苯双胍和丙戊酸对HepG2球体和单层细胞诱导肝毒性。细胞活力将根据球体产生ATP的能力来测量。LC50将用于体外模型和与文献中发现的LD50值进行比较。每种药物的毒性谱也将由蛋白质组学确定，以确定预测肝毒性的特定标记物。为了验证这些潜在的新的体外生物标志物，Balb/c小鼠将接受400mg /kg的对乙酰氨基酚治疗。收集血清和肝脏组织进行蛋白质组学分析，并通过肝脏组织病理学分析和血清中AST和ALT的定量分析来确认肝毒性

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