Preclinical Fluxomic Model of Drug-Induced Liver Injury

描述（由申请人提供）：代谢是动态的。对乙酰氨基酚（APAP）过量是非稳态应激的典型示例，其启动对毒性事件的动态级联反应，其严重程度取决于剂量。许多代谢途径可以防止APAP毒性，从I相和II相缀合途径到上级生物能代谢。因此，本研究提案的目标是使用稳定标记的营养素开发一种非稳态挑战测试，直接测量多种生物功能。多个通量率的量化并使用它们表征生物系统是“通量组学”，并且本文将结合四种生物医学工程技术来实现这一点：（1）NMR兼容的生物人工肝的组织工程，（2）体内13 C NMR光谱，（3）代谢通量建模，和多变量统计分析以识别生物标志物。这些是最先进的体内方法，将用于验证APAP药物损伤机制的现有范例。从体内真实的实验13 C NMR光谱时间过程中获得的通量组数据集将对代谢组学做出相当大的贡献。这种新的生物技术对于理解和开发肝病疗法非常重要。为了同时测定多个通量率，从而执行通量组学，最近开发的核磁共振兼容的人类生物人工肝将被建立以跟踪u-13 C-葡萄糖的过程。将使用窄孔600 MHz NMR光谱仪解析大量代谢物。导师是体内NMR光谱学家/组织工程师（Macdonald博士），与APAP毒性世界专家肝病学家（Watkins博士）合作，为研究生的培训创建了一个翻译团队。有两个具体目标：（1）在第一年，将静电封装的大鼠和人肝细胞在最近建立的NMR相容的生物人工肝中培养3天，并且在培养的第2天，将获得输注4小时的13 C-葡萄糖代谢的13 C NMR研究，然后转换为12 C-葡萄糖以从衰变曲线确定多种生化物质的通量率，而摄取曲线将用于通过与对照比较来鉴定毒性事件的级联;和（2）将进行（1）中所述的相同研究，随后在培养的第2天进行APAP攻击。将测试三种APAP剂量，1、10和20 mM溶解在培养基中，并分析通量组和代谢组以确定APAP毒性的亚致死效应。在实验结束时，将提取细胞并通过高分辨率1D 1H和2D 1H-{13 C} HSQC NMR光谱和共聚焦显微镜进行分析，以确定活力。

DESCRIPTION (provided by applicant): Metabolism is dynamic. Acetaminophen (APAP) overdose is a classic example of non-steady state stress that initiates a dynamic cascade of responses to toxic events occurring to varying severity depending on dose. Many metabolic pathways can protect against APAP toxicity, ranging from phase I and phase II conjugation pathways to superior bioenergetic metabolism. Therefore, the goal of this research proposal is to developed a nonsteady-state challenge test using stable-labeled nutrients that directly measures a multitude of biological functions. The quantification of multiple flux rates and using them characterize a biological system is "fluxomics", and herein four biomedical engineering technologies will be combined to achieve this: (1) tissue engineering of NMR-compatible bioartificial liver, (2) in vivo 13C NMR spectroscopy, (3) metabolic flux modeling, and multivariate statistical analysis to identify biomarkers. These are state-of- the-art in vivo methods will be used to verify the existing paradigm of the mechanism(s) of APAP drug injury. A considerable contribution to metabolomics will be made by the added fluxomic dataset acquired from real experimental in vivo 13C NMR spectral time courses. This new biotechnology is important for understanding and developing therapies for liver disease. In order to simultaneously determine multiple flux rates and thus perform fluxomics, a recently develop NMR-compatible human bioartificial liver will be established to track the course of u-13C-glucose. A multitude of metabolites will be resolved using a narrow- bore 600 MHz NMR spectrometer. The mentors are an in vivo NMR spectroscopist/tissue engineer (Dr. Macdonald), in a collaboration with a world-expert hepatologist on APAP toxicity (Dr. Watkins), creating a translational team for the training of the graduate student. There are two specific aims: (1) In the first year electrostatically encapsulated rat and human hepatocytes will be cultured for 3 days in a recently established NMR-compatible bioartificial liver, and on day 2 of culture 13C NMR studies of u-13C-glucose metabolism will will be obtained for 4 hr of infusion and then switched to 12C-glucose to determine flux rates of mutliple biochemicals from the decay curve, while the uptake curve will be used to identify the cascade of toxic events by comparison to control; and (2) the same study described in (1) will be performed followed by a APAP challenge on day 2 of culture. Three APAP doses will tested, 1,10, and 20 mM dissolved in the culture media, and the the fluxome and metabolome analyzed to determine the sub-lethal effects of APAP toxicity. At the end of the experiment, the cells will be extracted and analyzed by high resolution 1D 1H and 2D 1H-{13C} HSQC NMR spectroscopy and confocal microscopy for determining viability.