Research ArticleBiliary Atresia

Identification of a plant isoflavonoid that causes biliary atresia

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Science Translational Medicine  06 May 2015:
Vol. 7, Issue 286, pp. 286ra67
DOI: 10.1126/scitranslmed.aaa1652
  • Fig. 1. Biliatresone identification.

    (A and B) Lateral fluorescent image of a live 8 dpf wild-type (wt) control larva (A) and a larva treated with biliatresone (B) for 48 hours beginning at 5 dpf (1.0 μg/ml). Both larvae were soaked in Bodipy-C16 for 4 hours before imaging. Gallbladder fluorescence (arrow) is absent, and intestinal fluorescence (arrowheads) is markedly reduced in the toxin-treated larva. (C) Chemical structures of biliatresone and related isoflavones noted in the text. Arrow points to site of C-ring cleavage during possible metabolism of betavulgarin to biliatresone. (D to G) Confocal projections of the gallbladder and extrahepatic bile ducts of 8 dpf immunostained control (D) and toxin-treated (E to G) larvae. Insets show thin (1 μm) confocal sections of gallbladders. Increased doses of the toxin caused progressive changes in morphology of the gallbladder and extrahepatic ducts with preservation of the intrahepatic ducts. The variation seen in intrahepatic duct morphology is within normal limits. g, gallbladder; cd, cystic duct; cbd, common bile duct; ihd, intrahepatic bile ducts; hd, hepatic duct. Scale bars, 100 μm (A and B); 20 μm (D to G).

  • Fig. 2. Biliatresone tissue specificity.

    (A and B) Histological cross sections showing severe morphological defects of the gallbladder (asterisks) and cystic duct (arrows) in an 8 dpf biliatresone–treated larva (B) compared with a control larva (A). (C and D) Normal histological appearance of hepatocytes and liver sinusoids in an 8 dpf toxin-treated larva (D) compared with a control larva (C). Sinusoids (containing nucleated red blood cells; arrows) are the principal vascular channel in the liver of zebrafish larvae; thus, branches of the portal vein and artery are not seen in these histological sections. Intrahepatic bile ducts are too small to be seen. (E to H) Bright-field images of live 8 dpf control (E) and toxin-treated (F) Tg(lfabp-RFP) larvae with corresponding fluorescent images of the liver (G and H). Arrows, liver (E and F). Scale bars, 10 μm (A to D); 200 μm (E to H).

  • Fig. 3. Innate immune response in biliatresone-treated larvae.

    Confocal projections through the liver of Tg(mpx:GFP) and Tg(mpeg-1:GFP) larvae immunostained with the 2F11 monoclonal antibody (green) and anti-GFP antibody (red). (A and D) Control larvae (5 dpf) show a well-formed gallbladder. (B, C, E, and F) Biliatresone-treated larvae show progressive accumulation of neutrophils (B and C) and macrophages (E and F) with increasing duration of treatment. Twelve-hour biliatresone treatment (B and E) causes milder changes in gallbladder morphology compared with 48-hour treatment (C and F). Biliatresone treatment was initiated at 5 dpf in all larvae. Scale bars, 20 μm.

  • Fig. 4. Zebrafish ductbend sensitization.

    (A to D) Confocal projections through the liver and extrahepatic biliary system of control and toxin-treated wt and ductbend (dtb) larvae. Insets show thin sections through the gallbladder (A and B) or gallbladder and extrahepatic duct (C and D). Compared to the control larvae, extrahepatic BA (A) is evident in the treated wt larva (B), because the three principal components of the extrahepatic system (gallbladder, common bile duct, and cystic duct) distal to the common hepatic duct (chd) cannot be identified. (C) The control ductbend larva lacks intrahepatic bile ducts and has a hypoplastic gallbladder. (D) Toxin-induced damage to the extrahepatic system is more pronounced in the treated ductbend larva compared with wt (C). wt and ductbend larvae were treated with biliatresone (1 μg/ml) from 5 to 7 dpf. (E) Simplified genetic synteny map of the ductbend locus indicating the position of genes relative to their human orthologs at the BA susceptibility loci (16p13.3 and 10q25.1). Numbers refer to chromosomal location in Mb. Blue asterisks, positions of SNPs in BA susceptibility loci; red asterisk, position of SNP 27434434, ~1 centimorgan (cM) (0.6 Mb) from the ductbend locus. ihd, intrahepatic bile ducts; g, gallbladder; cbd, common bile duct; cd, cystic duct; chd, common hepatic duct; pd, pancreatic duct. Scale bars, 20 μm.

  • Fig. 5. Biliatresone-induced mouse cholangiocyte injury.

    (A) Confocal sections through cholangiocyte spheroids treated with vehicle, betavulgarin (2 μg/ml), or biliatresone (2 μg/ml) for 24 hours and stained with antibodies against F-actin or the β1-integrin subunit, or with DAPI. The biliatresone-treated spheroid shows disruption of the spheroid lumen and abnormal cholangiocyte polarity, as evidenced by altered distribution of F-actin and by cholangiocyte stratification. Most betavulgarin-treated spheroids demonstrated decreased lumen size, but there was no loss of polarity or disruption of the monolayer (see fig. S17). Representative images from five independent experiments are shown. DAPI, 4′,6-diamidino-2-phenylindole. (B) Dose-response experiment showing progressive increase in the percentage of abnormal biliatresone-treated spheroids beginning at 0.5 μg/ml. Scale bars, 7 μm. n = 48 to 89 spheroids counted per condition.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/7/286/286ra67/DC1

    Fig. S1. Swallowing function of biliatresone-treated larvae.

    Fig. S2. Scheme for biliatresone isolation.

    Fig. S3. Biliatresone-mediated biliary secretion defect.

    Fig. S4. Biliatresone dose response in zebrafish.

    Fig. S5. Biliatresone biliary secretion dose response.

    Fig. S6. Macrophages not required for biliatresone toxicity.

    Fig. S7. Biliatresone treatment during embryogenesis.

    Fig. S8. No effect of biliatresone on digestive epithelia.

    Fig. S9. Bile flow requirement for biliatresone toxicity.

    Fig. S10. Zebrafish ductbend mutants.

    Fig. S11. ductbend biliatresone sensitivity.

    Fig. S12. ductbend candidate gene expression.

    Fig. S13. Biliatresone-induced defects of cholangiocyte cilia and microtubules.

    Fig. S14. Dose-dependent loss of cholangiocyte microtubules with biliatresone treatment.

    Fig. S15. Effects of biliatresone on hepatocytes.

    Fig. S16. Lack of effect of isoflavanone-3 on mammalian cholangiocyte spheroids.

    Fig. S17. Effects of betavulgarin on mammalian cholangiocyte spheroids.

    Table S1. Biliatresone toxicity in DAPT-treated larvae.

    Movie S1. Biliary anatomy of a wild-type larva.

    Movie S2. Biliary anatomy of a toxin-treated wild-type larva.

  • Supplementary Material for:

    Identification of a plant isoflavonoid that causes biliary atresia

    Kristin Lorent, Weilong Gong, Kyung A. Koo, Orith Waisbourd-Zinman, Sara Karjoo, Xiao Zhao, Ian Sealy, Ross N. Kettleborough, Derek L. Stemple, Peter A. Windsor, Stephen J. Whittaker, John R. Porter, Rebecca G. Wells,* Michael Pack*

    *Corresponding author. E-mail: rgwells{at}mail.med.upenn.edu (R.G.W.); mpack{at}mail.med.upenn.edu (M.P.)

    Published 6 May 2015, Sci. Transl. Med. 7, 286ra67 (2015)
    DOI: 10.1126/scitranslmed.aaa1652

    This PDF file includes:

    • Fig. S1. Swallowing function of biliatresone-treated larvae.
    • Fig. S2. Scheme for biliatresone isolation.
    • Fig. S3. Biliatresone-mediated biliary secretion defect.
    • Fig. S4. Biliatresone dose response in zebrafish.
    • Fig. S5. Biliatresone biliary secretion dose response.
    • Fig. S6. Macrophages not required for biliatresone toxicity.
    • Fig. S7. Biliatresone treatment during embryogenesis.
    • Fig. S8. No effect of biliatresone on digestive epithelia.
    • Fig. S9. Bile flow requirement for biliatresone toxicity.
    • Fig. S10. Zebrafish ductbend mutants.
    • Fig. S11. ductbend biliatresone sensitivity.
    • Fig. S12. ductbend candidate gene expression.
    • Fig. S13. Biliatresone-induced defects of cholangiocyte cilia and microtubules.
    • Fig. S14. Dose-dependent loss of cholangiocyte microtubules with biliatresone treatment.
    • Fig. S15. Effects of biliatresone on hepatocytes.
    • Fig. S16. Lack of effect of isoflavanone-3 on mammalian cholangiocyte spheroids.
    • Fig. S17. Effects of betavulgarin on mammalian cholangiocyte spheroids.
    • Table S1. Biliatresone toxicity in DAPT-treated larvae.
    • Legends for movies S1 and S2

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.mov format). Biliary anatomy of a wild-type larva.
    • Movie S2 (.mov format). Biliary anatomy of a toxin-treated wild-type larva.

    [Download Movie S1]

    [Download Movie S2]

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