Isolation, Identification and Characterization of a Toxin Causing Biliary Atresia

引起胆道闭锁的毒素的分离、鉴定和表征

• 批准号: 9131852
• 负责人: MICHAEL A PACK
• 金额: $ 44.8万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-04-01 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9131852
• 关键词: 3-Dimensional; Adult; Affect; Age; Animal Disease Models; Antioxidants; Apical; Australia; Bile fluid; Biliary; Biliary Atresia; Binding; Biological; Biological Assay; Candidate Disease Gene; Cattle; Cell Polarity; Cells; Child; Childhood; Cholestasis; Chromosomes, Human, Pair 10; Clinical; Complex; Congenital atresia of extrahepatic bile duct; Coupled; Cysteine; Data; Diagnosis; Disease; Disease Outbreaks; Duct (organ) structure; Ductal; Employee Strikes; Environmental Exposure; Epidemiology; Etiology; Extrahepatic; Extrahepatic Cholestasis; Fibrosis; Fishes; Funding; Genetic; Goals; Health; Histidine; Human; Human Chromosomes; Immune system; In Vitro; Ingestion; Libraries; Link; Liver; Livestock; Maps; Mediating; Messenger RNA; Microtubules; Modeling; Molecular; Mothers; Mus; Neonatal; Newborn Infant; Obstruction; Oxidation-Reduction; Oxidative Stress; Pathogenesis; Plants; Poison; Polyribosomes; Population; Predisposition; Pregnancy; Reduced Glutathione; Reporting; Research Personnel; Resolution; Role; Sheep; Signal Pathway; Stress; Structure; Structure-Activity Relationship; Testing; Time; Toxic effect; Toxin; Zebrafish; base; bile duct; biological adaptation to stress; chemical genetics; cholangiocyte; epidemiologic data; genetic risk factor; insight; liver transplantation; macrophage; mutant; neonate; novel; pregnant; research study; transcription factor

 DESCRIPTION (provided by applicant): Biliary atresia (BA) is a fibrotic disorder that is the leading cause of neonatal cholestasis and the most common indication for liver transplant in the pediatric population. Although epidemiologic data suggest that BA arises from the interplay of genetic risk factors coupled with environmental exposures, the etiology is unknown. Insight into the pathogenesis of BA comes from the study of a naturally-occurring animal model of the disease. Over the last 40 years, there have been four outbreaks of BA in newborn livestock in Australia associated with ingestion of plants from the genus Dysphania by pregnant sheep and cows. Clinical and pathological findings from the affected lambs and calves show striking similarities with human BA, in particular marked fibrosis at the time of diagnosis. In our original proposal, we proposed to isolate the Dysphania biliary toxin. We now report that we have identified a selective extrahepatic biliary toxin, a previously undescribed isoflavonoid termed biliatresone, and have developed a new model of BA in larval zebrafish. Additionally, we have 1) identified structural features of biliatresone responsible for biliary toxicity, 2) demonstrated tht it binds reduced glutathione, cysteine and histidine in vitro and shown that the binding may be important for toxicity in zebrafish and mammalian cholangiocyte models, 3) identified a genetic link between toxin susceptibility in zebrafish and BA in humans, 4) demonstrated that biliatresone destabilizes mammalian cholangiocyte microtubules and alters cholangiocyte polarity, and 5) shown that biliatresone causes changes in Sox17 in mammalian cholangiocytes that are paralleled by changes observed in human BA livers. We hypothesize that biliatresone-induced toxicity is mechanistically relevant to human BA, and that signaling pathways related to susceptibility loci on human chromosome 10, oxidative stress, cell polarity, and Sox17 are critical to extrahepatic ductal atresia. Our overall goal is to employ a combined chemical, genetic, and cell biological approach to further understand the mechanism of extrahepatic duct obstruction and atresia. There are four specific aims: 1) to determine the effects of biliatresone on redox stress in the neonatal liver and bile ducts; 2) to determine the mechanism of biliatresone-mediated bile duct disruption at a cellular level, specifically to determine the relationship between oxidative stress, microtubules, and polarity in BA, and to identify signaling pathways downstream of Sox17 that could cause BA; 3) to identify genetic modifiers of biliatresone toxicity and determine their relevance to human BA; and 4) to synthesize and conduct structure-function studies of biliatresone and related compounds in order to identify critical structural features underlying toxicity. The proposed experiments will yield novel information about the mechanism of biliatresone-mediated BA; more importantly, they will provide insight into general mechanisms of extrahepatic ductal damage and potential therapies that are highly relevant to human BA.

 描述（由申请方提供）：胆道闭锁（BA）是一种纤维化疾病，是新生儿胆汁淤积的主要原因，也是儿科人群中最常见的肝移植适应症。虽然流行病学数据表明，BA是由遗传风险因素与环境暴露的相互作用引起的，但病因尚不清楚。对BA发病机制的了解来自对该疾病的自然发生的动物模型的研究。在过去的40年里，澳大利亚新生牲畜中发生了四次BA爆发，与怀孕的绵羊和奶牛摄入Dysphania属植物有关。受影响的羔羊和小牛的临床和病理学结果显示与人BA惊人的相似性，特别是在诊断时明显的纤维化。在我们最初的 在此基础上，我们提出了分离Dysphania胆毒素的方法。我们现在报告，我们已经确定了一种选择性肝外胆汁毒素，以前未描述的胆甾体称为biliatresone，并已开发出一种新的模式，BA在斑马鱼幼虫。此外，我们已经1）鉴定了导致胆汁毒性的胆甾烯松的结构特征，2）证明了其在体外结合还原型谷胱甘肽、半胱氨酸和组氨酸，并且显示该结合可能对斑马鱼和哺乳动物胆管细胞模型中的毒性很重要，3）鉴定了斑马鱼中的毒素易感性和人类中的BA之间的遗传联系，4）证明了胆甾酯松使哺乳动物胆管细胞微管不稳定并改变胆管细胞极性，和5）显示了胆甾酯松引起哺乳动物胆管细胞中Sox 17的变化，这被在人BA肝脏中观察到的变化所证实。我们假设胆固醇共振诱导的毒性机制与人类BA相关，并且与人类10号染色体上的易感基因座、氧化应激、细胞极性和Sox 17相关的信号传导途径对肝外胆管闭锁至关重要。我们的总体目标是采用化学，遗传学和细胞生物学相结合的方法，以进一步了解肝外胆管梗阻和闭锁的机制。有四个具体目标：2）在细胞水平上确定胆脂松介导的胆管破坏的机制，特别是确定氧化应激、微管和BA极性之间的关系，并鉴定可能导致BA的Sox 17下游的信号通路; 3）鉴定胆甾烯松毒性的遗传修饰剂并确定它们与人BA的相关性;和4）合成胆甾烯松和相关化合物并进行结构-功能研究，以鉴定毒性潜在的关键结构特征。拟议的实验将产生新的信息的机制，胆汁淤积介导的BA;更重要的是，他们将提供洞察肝外胆管损伤的一般机制和潜在的治疗，是高度相关的人类BA。