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（由申请人提供）：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，也是儿科人群肝移植的最常见适应症。尽管流行病学数据表明 BA 是由遗传风险因素与环境暴露相互作用引起的，但其病因尚不清楚。对 BA 发病机制的深入了解来自对该疾病自然发生的动物模型的研究。过去 40 年来，澳大利亚新生牲畜爆发了四次 BA 疫情，与怀孕的羊和牛摄入 Dysphania 属植物有关。受影响羔羊和犊牛的临床和病理结果与人类 BA 具有惊人的相似性，特别是在诊断时出现明显的纤维化。在我们原来的 提案中，我们建议分离Dysphania胆汁毒素。我们现在报告说，我们已经鉴定出一种选择性肝外胆汁毒素，一种以前未被描述的异黄酮类化合物，称为胆汁松，并在斑马鱼幼虫中开发了一种新的 BA 模型。此外，我们还 1) 确定了导致胆道毒性的胆阿曲松的结构特征，2) 证明它在体外结合还原型谷胱甘肽、半胱氨酸和组氨酸，并表明这种结合可能对斑马鱼和哺乳动物胆管细胞模型的毒性很重要，3) 确定了斑马鱼毒素敏感性和人类 BA 之间的遗传联系， 4) 证明胆管细胞微管不稳定并改变胆管细胞极性，5) 表明胆管细胞中胆管细胞 Sox17 发生变化，这与人类 BA 肝脏中观察到的变化是平行的。我们假设胆阿曲松诱导的毒性在机制上与人类 BA 相关，并且与人类 10 号染色体上的易感位点、氧化应激、细胞极性和 Sox17 相关的信号通路对于肝外导管闭锁至关重要。我们的总体目标是采用化学、遗传和细胞生物学相结合的方法来进一步了解肝外导管阻塞和闭锁的机制。有四个具体目标：1）确定胆阿曲松对新生儿肝脏和胆管氧化还原应激的影响； 2）在细胞水平上确定胆管破坏的胆管破坏机制，特别是确定BA中氧化应激、微管和极性之间的关系，并确定Sox17下游可能导致BA的信号通路； 3) 鉴定胆阿曲松毒性的遗传修饰因子并确定其与人类 BA 的相关性； 4) 合成胆阿曲松及相关化合物并进行结构功能研究，以确定潜在毒性的关键结构特征。所提出的实验将产生有关双阿曲松介导的 BA 机制的新信息；更重要的是，他们将深入了解肝外导管损伤的一般机制以及与人类 BA 高度相关的潜在疗法。