Preclinical Fluxomic Model of Drug-Induced Liver Injury

描述(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经典例子，它会根据剂量的不同而对不同严重程度的中毒事件启动动态级联反应。许多代谢途径可以预防APAP的毒性，从I相和II相结合途径到优越的生物能量代谢。因此，这项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物质，直接测量多种生物功能。对多个通量速率的量化并利用它们来表征生物系统是“通量组学”，这里将结合四种生物医学工程技术来实现这一点：(1)组织工程的核磁共振兼容生物人工肝，(2)活体13C核磁共振波谱，(3)代谢通量建模，以及用于识别生物标志物的多元统计分析。这些都是最先进的体内方法，将被用来验证APAP药物损伤机制(S)的现有范式。从活体13C核磁共振实验光谱时间过程中获得的额外流体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的治疗方法很重要。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与核磁共振兼容的人类生物人工肝来跟踪u-13C-葡萄糖的过程。多种代谢物将使用窄口径600 MHz核磁共振波谱仪进行解析。导师是体内核磁共振光谱学家/组织工程师(麦克唐纳博士饰)，与APAP毒性方面的世界肝病专家(沃特金斯博士)合作，创建了一个翻译团队来培训研究生。该中心有两个具体目标：(1)在第一年，静电包裹的大鼠和人肝细胞将在最近建立的核磁共振兼容生物人工肝中培养3天，在培养的第2天，将获得u-13C-葡萄糖代谢的13CNMR研究，持续4小时，然后切换到12C-葡萄糖，以从衰减曲线确定多种生物化学物质的通量速率，同时摄取曲线将用于通过与对照相比确定一连串的毒性事件；(2)将进行(1)中所述的相同研究，并在培养第2天进行APAP挑战。将测试三个APAP剂量，1，10和20 mM溶解在培养基中，并分析流动体和代谢组，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1D1H和2D1H-13C HSQC核磁共振波谱和共聚焦显微镜分析细胞的活力。

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.