Biliary atresia (BA) is a rapidly progressive and destructive fibrotic disorder of unknown etiology affecting the extrahepatic biliary tree of neonates. Epidemiological studies suggest that an environmental factor, such as a virus or toxin, is the cause of the disease, although none have been definitively established. Several naturally occurring outbreaks of BA in Australian livestock have been associated with the ingestion of unusual plants by pregnant animals during drought conditions. We used a biliary secretion assay in zebrafish to isolate a previously undescribed isoflavonoid, biliatresone, from Dysphania species implicated in a recent BA outbreak. This compound caused selective destruction of the extrahepatic, but not intrahepatic, biliary system of larval zebrafish. A mutation that enhanced biliatresone toxicity mapped to a region of the zebrafish genome that has conserved synteny with an established human BA susceptibility locus. The toxin also caused loss of cilia in neonatal mouse extrahepatic cholangiocytes in culture and disrupted cell polarity and monolayer integrity in cholangiocyte spheroids. Together, these findings provide direct evidence that BA could be initiated by perinatal exposure to an environmental toxin.
Biliary atresia, the most common indication for pediatric liver transplantation, is a fibrotic disease of unknown etiology affecting the extrahepatic bile ducts of newborns. The recently-described toxin biliatresone causes lumen obstruction in mouse cholangiocyte spheroids and represents a new model of biliary atresia. Our aim was to determine the cellular changes caused by biliatresone in mammalian cells that ultimately lead to biliary atresia and extra-hepatic fibrosis. We treated mouse cholangiocytes in 3D spheroid culture and neonatal extra-hepatic duct explants with biliatresone and compounds that regulate glutathione. We examined the effects of biliatresone on SOX17 levels, and determined the effects of Sox17 knockdown on cholangiocytes in 3D culture. We found that biliatresone caused disruption of cholangiocyte apical polarity and loss of monolayer integrity. Spheroids treated with biliatresone had increased permeability as shown by rhodamine efflux within 5 hours compared to untreated spheroids, which retained rhodamine for longer than 12 hours. Neonatal bile duct explants treated with the toxin showed lumen obstruction with increased subepithelial staining for α-smooth muscle actin and collagen, consistent with fibrosis. Biliatresone caused a rapid and transient decrease in glutathione, which was both necessary and sufficient to mediate its effects in cholangiocyte spheroid and bile duct explant systems. It also caused a significant decrease in in cholangiocyte levels of SOX17, and Sox17 knockdown in cholangiocyte spheroids mimicked the effects of biliatresone. Conclusion Biliatresone decreases glutathione and SOX17 in mouse cholangiocytes. In 3D cell systems, this leads to cholangiocyte monolayer damage and increased permeability and in extrahepatic bile duct explants it leads to disruption of the extra-hepatic biliary tree and subepithelial fibrosis. This mechanism may be important in understanding human biliary atresia.
A methanolic extract of dried Schisandra fruit (Schisandra chinensis Baill.; Schisandraceae) significantly attenuated the neurotoxicity induced by L-glutamate in primary cultures of rat cortical cells. Five dibenzocyclooctadiene lignans (deoxyschisandrin, gomisin N, gomisin A, schisandrin, and wuweizisu C) were isolated from the methanolic extract; their protective effects against glutamate-induced neurotoxicity were then evaluated. Among the five lignans, deoxyschisandrin, gomisin N, and wuweizisu C significantly attenuated glutamate-induced neurotoxicity as measured by 1). an inhibition in the increase of intracellular [Ca(2+)]; 2). an improvement in the glutathione defense system, the level of glutathione, and the activity of glutathione peroxidase; and 3). an inhibition in the formation of cellular peroxide. These results suggest that dibenzocyclooctadiene lignans from Schisandra chinensis may possess therapeutic potential against oxidative neuronal damage induced by excitotoxin.
Biliatresone is an electrophilic isoflavone isolated from Dysphania species plants that has been causatively linked to naturally occurring outbreaks of a biliary atresia (BA)-like disease in livestock. Biliatresone has selective toxicity for extrahepatic cholangiocytes (EHC) in zebrafish larvae. To better understand its mechanism of toxicity, we performed transcriptional profiling of liver cells isolated from zebrafish larvae at the earliest stage of biliatresone-mediated biliary injury, with subsequent comparison of biliary and hepatocyte gene expression profiles. Transcripts encoded by genes involved in redox stress response, particularly those involved in glutathione (GSH) metabolism, were among the most prominently upregulated in both cholangiocytes and hepatocytes of biliatresone-treated larvae. Consistent with these findings, hepatic GSH was depleted at the onset of biliary injury, and in situ mapping of the hepatic GSH redox potential using a redox-sensitive GFP (roGFP) biosensor showed that it was significantly more oxidized in EHC both before and after treatment with biliatresone. Pharmacological and genetic manipulation of GSH redox homeostasis confirmed the importance of GSH in modulating biliatresone-induced injury as GSH depletion sensitized both EHC and the otherwise resistant intrahepatic cholangiocytes (IHC) to the toxin, whereas replenishing GSH level via N-acetylcysteine administration or activation of nuclear factor, erythroid 2-like 2 (Nrf2), a transcriptional regulator of GSH synthesis, inhibited EHC injury. Conclusion: These findings strongly support redox stress as a critical contributing factor in biliatresone-induced cholangiocyte injury, and suggest variations in intrinsic stress responses underlie the susceptibility profile. Insufficient antioxidant capacity of EHC may be critical to the early pathogenesis of human BA.
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