In Vivo Kinetic Biomarkers of Hepatic Toxicity

描述(由申请人提供)： 大量的候选药物增加了对测量肝脏毒性的新检测方法的需求。我们已经开发了利用2H2O标记来定量体内多种代谢通量的方法。在这里，我们建议验证这种方法作为毒性筛查。我们将测试14种不同作用机制的肝毒素，以检测它们改变肝细胞周转(肝细胞、内皮细胞、总肝细胞)、线粒体生物合成和胶原合成的能力，作为肝损伤和体内平衡的标志。我们将把这些测量分别与细胞分裂的静态标志物、窦内皮细胞的形态、线粒体细胞色素c和胶原沉积的变化相关联。我们假设，已知的影响后一种表型的药物的毒性浓度将强烈调节潜在代谢途径中的通量。我们将比较静态标记物和2H标记在限制剂量/暴露时间内检测药物引起的亚临床变化和揭示新的毒性活性的能力。最后，我们将筛选通过我们的测试检测到的预测最终器官损伤的基因表达模式。这项工作将为监管部门批准我们的毒性筛查、将我们的工作扩展到其他终端器官以及用于人类奠定坚实的基础。

DESCRIPTION (provided by applicant): A flood of drug candidates increases the need for novel assays to measure liver toxicity. We have developed methods for quantifying multiple metabolic fluxes in vivo using 2H2O labeling. Here we propose to validate this approach as a toxicity screen. We will test 14 hepatotoxins with diverse mechanisms of action for their ability to alter liver cell turnover (hepatocytes, endothelial cells, total liver cells), mitochondrial biogenesis, and collagen synthesis as markers of liver damage and homeostasis. We will correlate these measurements with changes in static markers of cell division; morphology of sinusoidal endothelial cells; mitochondrial cytochrome c; and collagen deposition, respectively. We hypothesize that toxic concentrations of drugs known to affect the latter phenotypes will strongly modulate fluxes in the underlying metabolic pathways. We will compare static markers and 2H labeling for their ability to detect drug-induced subclinical changes at limiting doses/exposure times and to reveal novel toxic activities. Finally, we will screen for gene expression patterns that predict end organ damage as detected by our test. This work will provide a solid foundation for regulatory approval of our toxicity screen, for extension of our work to other end organs, and for use in humans.