Isolation, Identification and Characterization of a Toxin Causing Biliary Atresia



 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.