Nature and nurture: Environmental toxins and biliary atresia
Authors: Chad Walesky, Wolfram Goessling
Journal: Hepatology (2016)

Potential conflict of interest: Dr. Goessling consults and holds intellectual property rights with Fate Therapeutics. See Articles on pages 880 and 894 Biliary atresia (BA) is one of the most severe liver diseases affecting children. As the most common cause of infantile cholestasis and pediatric liver transplantation, it is characterized by progressive scarring and occlusion of the extrahepatic bile ducts. The etiology of BA has remained elusive in many cases, in contrast to those patients with Alagille syndrome, an inherited paucity of bile ducts, where the Notch signaling pathway has been demonstrated to be a genetic cause in the pathogenesis of the disease. Familial cases of BA are rare, suggesting a lack of primary genetic factors in disease development. However, recent evidence suggests that exposure to select toxins and toxicants may contribute to the pathogenesis of BA. Reports of BA in the veterinary literature reveal an epidemic clustering of timing and geographic location, suggesting an environmental insult as a major contributing factor in disease pathogenesis. A classic example of this phenomenon is highlighted by outbreaks of BA in herds of grazing animals in New South Wales, Australia, affecting large percentages of newborn animals with no effect on their mothers or other adult animals.1 An elegant, collaborative study from the laboratories of Dr. Michael Pack and Dr. Rebecca Wells led to the discovery of a novel biliary toxin as the causative agent in these cases3: the investigators were able to identify flora ingested by pregnant animals that were unique to each occurrence of a BA outbreak, leading them to examine particular chenopods in the genus Dysphania. Sequential extraction and fractionation of these plants, along with in vivo zebrafish bioassays, identified a compound, named biliatresone, as the causative toxic molecule. These groups have continued the investigation of this exciting story and are presenting two articles in the current issue of hepatology, examining the mechanisms surrounding the biliary insult caused by biliatresone. The study by Zhao et al. explores the mechanism of biliatresone‐induced bile duct injury using the zebrafish model.4 Transcriptomic analysis by RNA sequencing of larval zebrafish following exposure to biliatresone revealed activation of genes involved in phase 2 metabolism and, in particular, genes required for glutathione (GSH) biosynthesis and metabolism. Further, up‐regulation of target genes of the nuclear factor erythroid 2‐related factor 2/Kelch ECH associating protein 1 (Nrf2/Keap1) pathway was observed, which is known to play an important role in the induction of phase 2 metabolism. These findings were corroborated by the observation that biliatresone exposure led to hepatic GSH depletion, which was found to be most severe in extrahepatic cholangiocytes. GSH depletion appeared to sensitize extrahepatic cholangiocytes to the toxic effects of biliatresone, which could be blocked by replenishing GSH levels by treatment with N‐acetylcysteine or stimulation of the Nrf2/Keap1 pathway. In a concurrent study by Waisbourd‐Zinman et al., the focus is switched to a mammalian in vitro model system for investigation of BA pathology in response to biliatresone exposure5: biliatresone induced lumen obstruction, loss of polarity, and increased epithelial permeability in mouse cholangiocytes cultured as spheroids as well as in bile duct explants. These changes were found to be in response to GSH depletion and, as in the Zhao et al. study, could be prevented by treatment with N‐acetylcysteine or by stimulation of the Nrf2/Keap1 pathway. Further, biliatresone caused a reduction in SRY (sex‐determining region Y)‐box 17 (Sox17). Loss of Sox17 caused similar changes in cholangiocyte integrity as observed with biliatresone exposure. In contrast, overexpression of Sox17 did not have a protective effect and, surprisingly, caused disruption of the epithelial monolayer in the absence of biliatresone, suggesting a delicate balance of Sox17 activity required for optimal cholangiocyte maintenance. Taken together, these studies highlight a potential critical role for environmental insults in the pathogenesis of BA. The authors emphasize that there is no current evidence that pregnant women are exposed to biliatresone itself, but there is a high likelihood that other xenobiotics exist that may function in a similar manner. The articles by Zhao et al. and Waisbourd‐Zinman et al. can focus our attention on molecules with similar structure and functional groups that could be contributing to BA pathogenesis in humans. Additionally, these studies provide a novel view of BA pathogenesis mechanistically, which will aid in further elucidating and preventing the causes of the disease. The authors postulate that the differential concentration of GSH in various liver cell types and the observed activation of hepatic redox stress response pathways may explain the selective toxicity of biliatresone. This is highlighted by the fact that biliatresone appears to be selectively toxic to extrahepatic bile ducts, whereas the intrahepatic bile ducts are most likely protected by an increased level of GSH due to their their close proximity to GSH‐producing hepatocytes (Fig. 1). Decreases in GSH have been implicated in microtubule disruption, and data presented in the current studies suggest that this is occurring within cholangiocytes exposed to biliatresone. Further, Sox17 has been shown to play an important role in maintaining the epithelium of extrahepatic bile ducts. Changes associated with GSH depletion and reduction of Sox17 could lead to loss of cholangiocyte integrity. This may contribute to disease formation by allowing bile, as well as toxic compounds within the bile, to leak into the submucosa, causing inflammation and a secondary fibrotic response (Fig. 1). These findings enhance our understanding of classic toxicologic principles and highlight the possibility that susceptibility to toxins and toxicants is also determined by the (micro)environment, or niche, the specific cell lies within.Figure 1: Biliatresone is selectively toxic to extrahepatic bile ducts. Biliatresone, a novel biliary toxin, is eliminated through biliary excretion following GSH conjugation. Extrahepatic bile ducts, which have low levels of GSH, appear to be susceptible to biliatresone toxicity. Biliatresone exposure results in GSH depletion, as well as reduced Sox17, in extrahepatic bile ducts, leading to redox stress, loss of cholangiocyte integrity, and a fibrotic response. However, intrahepatic bile ducts appear to be protected due to their close association with GSH‐rich hepatocytes.Much remains to be learned about the factors that contribute to xenobiotic‐induced toxicity seen in BA. Understanding all of the toxicodynamics and kinetics surrounding biliatresone is of great importance. The more we know about biliatresone, the more tools we will have at our disposal to find a true cause of BA in children afflicted with this devastating disease. Identification of candidate molecules that induce biliary injury using a similar mechanism as biliatresone, as well as defining factors that may increase susceptibility to these given molecules, such as genetic and environmental factors, are of great importance. For example, polymorphisms in glutathione S‐transferase and related enzymes have been implicated in a number of pathologies, including liver diseases, such as hepatocellular carcinoma.6 It is highly possible that select polymorphisms affecting glutathione S‐transferase production or activity may influence susceptibility to molecules like biliatresone. Additionally, polymorphisms affecting activity or response of the Nrf2/Keap1 pathway may play a role, as well as variations that influence production and uptake of cysteine, which is the rate‐limiting substrate in GSH synthesis. Importantly, it is well established that the gut microbiome can directly biotransform molecules.7 Therefore, the microbiome, which can be highly variable between individuals, can directly modulate xenobiotic‐induced toxicity. Analysis of biliatresone reveals that it is a 1,2‐diaryl‐2‐propenone structure.8 Similar metabolites have been shown to be produced by intestinal clostridia in humans who ingested dietary isoflavonoids, suggesting that the gut microbiome may play an important role in the presence of molecules that could contribute to BA pathogenesis.9 In conclusion, the current studies highlight the role of a select biliary toxin (biliatresone) in the pathogenesis of BA. In combination, they elegantly demonstrate the synergistic strengths of innovative in vitro techniques with the unique opportunities in the zebrafish model. We can now apply this knowledge to further understand BA pathogenesis, discover other environmental insults contributing to disease incidence in humans, and possibly explore modulation of redox signaling pathways and GSH content as preventive measures in patients at risk. Author names in bold designate shared co‐first authorship.