Preclinical Fluxomic Model of Drug-Induced Liver Injury

药物性肝损伤的临床前 Fluxomic 模型

• 批准号: 7694390
• 负责人: Rex E. Jeffries
• 金额: $ 2.92万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-01 至 2010-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7694390
• 关键词: Acetaminophen; Affect; Artificial Liver; Arts; Biochemical; Bioenergetics; Biological Markers; Biological Process; Biomedical Engineering; Biotechnology; Cell Death; Cells; Collaborations; Confocal Microscopy; Consumption; Culture Media; Data Set; Disease; Dose; Encapsulated; Equation; Equilibrium; Event; Glucose; Goals; Hepatocyte; Human; Infusion procedures; Injury; Label; Liver; Liver diseases; Measurement; Measures; Mentors; Metabolic; Metabolic Pathway; Metabolism; Methods; Modeling; NMR Spectroscopy; Nutrient; Pathway interactions; Pharmaceutical Preparations; Phase; Phosphorylation; Rattus; Reaction; Research Proposals; Resolution; Severities; Signal Transduction; Stress; Technology; Testing; Time; Tissue Engineering; Toxic Actions; Toxic effect; Training; Xenobiotics; acetaminophen overdose; biological systems; detector; glucose metabolism; graduate student; in vivo; inhibitor/antagonist; metabolomics; pre-clinical; research study; response; therapy development; toxicant; uptake

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.

描述（由申请人提供）：代谢是动态的。对乙酰氨基酚（APAP）过量是非稳态应力的一个经典例子，它启动了对毒性事件的动态级联反应，因为剂量变化而变化变化。许多代谢途径可以预防APAP毒性，从I期和II期结合途径到上级生物能代谢。因此，该研究建议的目的是使用稳定标记的营养素开发出非稳定的国家挑战测试，该养分直接测量了多种生物学功能。多个通量率及其用它们表征生物系统的定量是“通量学”，此处将结合使用四种生物医学工程技术以实现这一目标：（1）NMR与生物人工肝的组织工程，（2）在Vivo 13C NMR谱图中（2）体内13C NMR光谱谱，（3）识别生物剂量统计的模型和多数分析。这些是最新的体内方法，将用于验证APAP药物损伤机制的现有范式。从实际实验13C NMR光谱时间课程中获得的添加的通量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很重要。为了同时确定多重通量速率并执行通量，将建立最近发展的NMR兼容的人工肝脏以跟踪U-13C-葡萄糖的过程。使用窄孔600 MHz NMR光谱仪将解决多种代谢物。这些导师是一名体内NMR光谱学家/组织工程师（MacDonald博士），他与一位有关APAP毒性（沃特金斯博士）的世界专家肝病学家合作，创建了一个转化团队，以培训研究生。 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与对照相比，衰减曲线中的生化曲线的生化曲线将使用摄取曲线来识别一系列有毒事件。 （2）（1）中描述的同一项研究将在培养第2天进行APAP挑战。将测试三种APAP剂量，其中1,10和20 mM溶解在培养基中，并分析了通量组和代谢组，以确定APAP毒性的亚致死作用。在实验结束时，将通过高分辨率1d 1h和2d 1H- {13C} HSQC NMR光谱和共聚焦显微镜来提取和分析细胞。