Isolation, Identification and Characterization of a Toxin Causing Biliary Atresia

 描述(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，也是儿科人群中肝移植最常见的适应症。虽然流行病学数据表明BA是由遗传风险因素和环境暴露相互作用引起的，但其病因尚不清楚。对BA发病机制的洞察来自于对这种疾病的自然发生的动物模型的研究。在过去的40年里，澳大利亚发生了四起与怀孕的绵羊和奶牛摄入Dysphania属植物有关的新生家畜BA疫情。受影响的羔羊和小牛的临床和病理结果与人类BA有惊人的相似之处，特别是在诊断时明显的纤维化。在我们的原版中 建议，我们提出了分离胆汁困难毒素的建议。我们现在报告，我们已经确定了一种选择性的肝外胆汁毒素，一种以前未被描述的被称为胆红素的异黄酮类化合物，并在斑马鱼幼体中开发了一种新的BA模型。此外，我们已1)证实了胆汁酮可导致胆汁毒性的结构特征，2)在体外证明了它能与还原型谷胱甘肽、半胱氨酸和组氨酸结合，并证明这种结合可能在斑马鱼和哺乳动物胆管细胞模型中的毒性中起重要作用，3)已确定斑马鱼的毒素敏感性与人类BA之间的遗传联系，4)证明了胆固醇酮可破坏哺乳动物胆管细胞微管的稳定，并改变了胆管细胞的极性，5)表明胆固醇酮可引起哺乳动物胆管细胞中Sox17的变化，这一变化与人类BA肝脏的变化相平行。我们假设胆红素诱导的毒性与人BA有机械联系，与人类10号染色体易感基因、氧化应激、细胞极性和Sox17相关的信号通路在肝外胆管闭锁中起关键作用。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来进一步了解肝外胆管阻塞和闭锁的机制。有四个特定的目标：1)确定胆红素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红素介导的胆管破坏的机制，特别是确定氧化应激、微管和BA的极性之间的关系，并确定Sox17下游可能导致BA的信号通路；3)确定胆红素毒性的遗传修饰因素，并确定它们与人类BA的相关性；以及4)合成并进行胆红素及其相关化合物的结构-功能研究，以确定潜在的毒性关键结构特征。这些拟议的实验将产生关于胆红素介导的BA机制的新信息；更重要的是，它们将提供对肝外胆管损伤的一般机制的洞察，以及与人类BA高度相关的潜在治疗方法。

 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.