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Glutathione antioxidant pathway activity and reserve determine toxicity and specificity of the biliary toxin biliatresone in zebrafish.

ABSTRACT: 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 (EHCs) 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 up-regulated 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 green fluorescent protein biosensor showed that it was significantly more oxidized in EHCs 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 given that GSH depletion sensitized both EHCs and the otherwise resistant intrahepatic cholangiocytes to the toxin, whereas replenishing GSH level by N-acetylcysteine administration or activation of nuclear factor erythroid 2-like 2 (Nrf2), a transcriptional regulator of GSH synthesis, inhibited EHC injury.These findings strongly support redox stress as a critical contributing factor in biliatresone-induced cholangiocyte injury, and suggest that variations in intrinsic stress responses underlie the susceptibility profile. Insufficient antioxidant capacity of EHCs may be critical to early pathogenesis of human BA. (Hepatology 2016;64:894-907).

SUBMITTER: Zhao X

PROVIDER: S-EPMC5251204 | biostudies-literature | 2016 Sep

REPOSITORIES: biostudies-literature

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Glutathione antioxidant pathway activity and reserve determine toxicity and specificity of the biliary toxin biliatresone in zebrafish.

Zhao Xiao X Lorent Kristin K Wilkins Benjamin J BJ Marchione Dylan M DM Gillespie Kevin K Waisbourd-Zinman Orith O So Juhoon J Koo Kyung Ah KA Shin Donghun D Porter John R JR Wells Rebecca G RG Blair Ian I Pack Michael M

Hepatology (Baltimore, Md.) 20160611 3

<h4>Unlabelled</h4>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 (EHCs) 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 bil ...[more]

PMID: 27102575

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Project description:In our previous work, we identified a natural toxin, biliatresone, from Dysphania glomulifera and D. littoralis, endemic plants associated with outbreaks of biliary atresia in Australian neonatal livestock. Biliatresone is a very rare isoflavonoid with an ?-methylene ketone between two phenyls, 1,2-diaryl-2-propenone, along with methylenedioxy, dimethoxyl, and hydroxyl functional groups, that causes extrahepatic biliary toxicity in zebrafish. The toxic core of biliatresone is a methylene in the ?-position relative to the ketone of 1,2-diaryl-2-propenone that serves as an electrophilic Michael acceptor. The ?-methylene of biliatresone spontaneously conjugated with water and methanol (MeOH), respectively, via Michael addition in a reverse phase high-performance liquid chromatography (RP-HPLC) analysis. We here report the reactivity of biliatresone toward glutathione (GSH), several amino acids, and other thiol- or imidazole-containing biomolecules. LC-MS and HPLC analysis of the conjugation reaction showed the reactivity of biliatresone to be in the order histidine > N-acetyl-d-cysteine (D-NAC) = N-acetyl-l-cysteine (L-NAC) > histamine > glutathione ? cysteine ? glycine > glutamate > phenylalanine, while serine and adenine had no reactivity due to intramolecular hydrogen bonding in the protic solvents. The reactivity of ethyl vinyl ketone (EVK, 1-penten-3-one), an example of a highly reactive ?,ß-unsaturated ketone, toward GSH gave a 6.7-fold lower reaction rate constant than that of biliatresone. The reaction rate constant of synthetic 1,2-diaryl-2-propen-1-one (DP), a core structure of the toxic molecule, was 10-fold and 1.5-fold weaker in potency compared to the reaction rate constants of biliatresone and EVK, respectively. These results demostrated that the methylenedioxy, dimethoxyl, and hydroxyl functional groups of biliatresone contribute to the stronger reactivity of the Michael acceptor ?-methylene ketone toward nucleophiles compared to that of DP and EVK.

Project description:UnlabelledBiliary 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. The goal of this study was to determine the cellular changes caused by biliatresone in mammalian cells that ultimately lead to biliary atresia and extrahepatic fibrosis. We treated mouse cholangiocytes in three-dimensional (3D) spheroid culture and neonatal extrahepatic duct explants with biliatresone and compounds that regulate glutathione (GSH). 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 with 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 GSH, 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 cholangiocyte levels of SOX17, and Sox17 knockdown in cholangiocyte spheroids mimicked the effects of biliatresone.ConclusionBiliatresone decreases GSH and SOX17 in mouse cholangiocytes. In 3D cell systems, this leads to cholangiocyte monolayer damage and increased permeability; in extrahepatic bile duct explants, it leads to disruption of the extrahepatic biliary tree and subepithelial fibrosis. This mechanism may be important in understanding human biliary atresia. (Hepatology 2016;64:880-893).

Dataset Information

Glutathione antioxidant pathway activity and reserve determine toxicity and specificity of the biliary toxin biliatresone in zebrafish.

Publications

Glutathione antioxidant pathway activity and reserve determine toxicity and specificity of the biliary toxin biliatresone in zebrafish.

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