Preclinical Fluxomic Model of Drug-Induced Liver Injury

描述（由申请人提供）：新陈代谢是动态的。对乙酰氨基酚（APAP）过量是非稳态应激的典型例子，它会引发对毒性事件的动态级联反应，其严重程度取决于剂量。从I期和II期偶联途径到优越的生物能量代谢途径，许多代谢途径都可以防止APAP毒性。因此，本研究计划的目标是开发一种非稳态挑战测试，使用稳定标记的营养素，直接测量多种生物功能。量化多个通量率并利用它们来表征生物系统是“通量组学”，本文将结合四种生物医学工程技术来实现这一目标：(1)核磁共振兼容生物人工肝的组织工程，(2)体内13C核磁共振波谱，(3)代谢通量建模，以及多变量统计分析来识别生物标志物。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式。从真实实验体内13C NMR光谱时间过程中获得的额外通量组学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脏疾病的治疗方法非常重要。为了同时确定多个通量速率，从而进行通量组学，将建立一个最近开发的核磁共振兼容的人类生物人工肝脏来跟踪u- 13c -葡萄糖的过程。大量的代谢物将使用窄孔600兆赫核磁共振光谱仪进行分解。导师是一名体内核磁共振波谱学家/组织工程师（Macdonald博士），他与一名研究APAP毒性的世界肝病专家（Watkins博士）合作，创建了一个培训研究生的翻译团队。有两个具体目标：(1)第一年静电封装老鼠和人类肝细胞培养在最近3天建立NMR-compatible生物人工肝、文化和第二天的13 c NMR研究u-13C-glucose新陈代谢将会获得4人力资源注入,然后切换到12比较来确定利率的多重生化物质通量的衰减曲线,而吸收曲线将被用来识别有毒事件相比之下的级联控制;(2)与(1)中描述的相同的研究将在培养的第2天进行APAP挑战。将测试三种APAP剂量，分别是溶解在培养基中的1mm、10mm和20mm，并分析荧光组和代谢组，以确定APAP毒性的亚致死效应。实验结束后，提取细胞，用高分辨率1D 1H和2D 1H-{13C} HSQC NMR和共聚焦显微镜分析细胞活力。

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.