DOI: 10.5061/dryad.m63xsj48x
title: Low-dose biliatresone treatment of pregnant mice causes subclinical biliary disease in their offspring: Evidence for a spectrum of neonatal injury

description: Biliary atresia is a neonatal disease characterized by damage,
 inflammation, and fibrosis of the liver and bile ducts and by abnormal
 bile metabolism. It likely results from a prenatal environmental exposure
 that spares the mother and affects the fetus. Our aim was to develop a
 model of fetal injury by exposing pregnant mice to low-dose biliatresone,
 a plant toxin implicated in biliary atresia in livestock, and then to
 determine whether there was a hepatobiliary phenotype in their pups.
 Pregnant mice were treated orally with 15 mg/kg/d biliatresone for 2 days.
 Histology of the liver and bile ducts, serum bile acids, and liver immune
 cells of pups from treated mothers were analyzed at P5 and P21. Pups had
 no evidence of histological liver or bile duct injury or fibrosis at
 either timepoint. In addition, growth was normal. However, serum levels of
 glycocholic acid were elevated at  P5, suggesting altered bile
 metabolism, and the serum bile acid profile became increasingly abnormal
 through P21, with enhanced glycine conjugation of bile acids. There was
 also immune cell activation observed in the liver at P21. These results
 suggest that prenatal exposure to low doses of an environmental toxin can
 cause subclinical disease including liver inflammation and aberrant bile
 metabolism even in the absence of histological changes. This finding
 suggests a wide potential spectrum of disease after fetal biliary injury.
description: <strong>Materials</strong>
   The bile acids ursodeoxycholic acid
 (UDCA), hyodeoxycholic acid (HDCA), taurodeoxycholic acid (TDCA),
 taurocholic acid (TCA), cholic acid (CA), taurochenodeoxycholic acid
 (TCDCA), glycocholic acid (GCA), β-muricholic acid (BMA),
 tauro-β-muricholic acid (TBMCA), α-muricholic acid (AMA), deoxycholic acid
 (DCA), glycoursodeoxycholic acid (GUDCA), glycohyodeoxycholic acid
 (GHDCA), glycochenodeoxycholic acid (GCDCA) and glycine-β-muricholic acid
 (GBMCA) as well as an internal standard stock solution containing a
 mixture of CA-d4, GCA-d4 and DCA-d4 were obtained from Cayman Chemical
 (Ann Arbor, Michigan, USA).  Stock solutions of 1 mg/mL in 100% methanol
 were diluted to obtain calibration curves for concentrations ranging from
 15.6 to 1000 nM. Percoll was obtained from GE Healthcare Life Sciences
 (Chicago, Illinois, USA). Antibodies against CD4, Ly6G and CD90.2 were
 obtained from BD Pharmingen (San Diego, CA, USA) and antibodies against
 CD8α, NK1.1, B220 and Ly6C from BioLegend (San Diego, CA, USA). Goat
 anti-collagen I antibody for staining was obtained from Southern Biotech
 (Birmingham, AL, USA). Rabbit anti-Cytokeratin 19 (KRT 19) antibody was
 obtained from Abcam (Boston, MA, USA). Biotinylated Goat Anti-Rabbit IgG
 Antibody was obtained from Vector Laboratories, Inc. (Burlington, CA,
 USA). Biotinylated HA-binding protein for staining HA was obtained from
 EMD Millipore  (Burlington, MA, USA). LIVE/DEAD fixable viability dye was
 from Life Technologies (Carlsbad, CA, USA). Biliatresone was synthesized
 as previously described [31]. Stock solutions were made in DMSO and
 diluted in 1X PBS for gavage.  
 <strong>Animal experiments</strong>
   We used BALB/c mice obtained from
 Jackson Laboratories as animal model. All animal experiments were
 conducted following the National Institutes of Health policy, and the
 study was approved by the Institutional Animal Care and Use Committee at
 the University of Pennsylvania under protocol #804862, ensuring that all
 procedures were performed ethically and with minimal harm to the
 animals.   To investigate
 the effects of biliatresone, pregnant female mice were administered either
 15 mg/kg of biliatresone or vehicle (containing an equivalent
 concentration of DMSO, at 0.3 ml/kg) via gavage on days 14 and 15 post
 mating. All gavaging procedures were carried out with utmost consideration
 to ensure humane and ethical treatment throughout the experiment. Animals
 were closely monitored post-gavaging for any signs of distress, and no
 instances of stressed animals were recorded during the observation period.
 We used a dosage of 15 mg/kg of biliatresone in order to avoid pregnancy
 loss, which was reported by Yang et al. [1] with higher doses. The average
 litter size in the biliatresone-treated group was 6, comparable to the
 control group. We chose E14 and 15 for biliatresone treatment in alignment
 with the time of hepatoblast differentiation and early biliary network
 formation [2].   Half of the
 pups from each litter (chosen randomly) were euthanized at P5, and the
 other half were euthanized at P21. All animals were handled with humane
 care. Carbon dioxide was utilized as the primary method for euthanasia.
 Additionally, P5 pups underwent a secondary physical method of euthanasia
 through decapitation, while other animals underwent cervical dislocation
 as the secondary method..We did not determine sex given that anogenital
 distance measurements at P5 lack accuracy, but our random selection of
 pups for euthanasia at a specific day typically yields a balanced
 male-female distribution. Mother mice and P21 pups were euthanized on the
 same day, and blood, EHBDs, and liver samples were collected from all the
 animals. Placental tissue was not collected.  Although in utero exposure
 is the most likely route of biliatresone toxicity in pups, we could not
 rule out transfer in milk [3] and therefore kept nursing mothers with pups
 until P21, then euthanized both.  
 Serum samples were analyzed for albumin, alkaline phosphatase
 (ALP) and aspartate transaminase (AST) levels, as well as bile acid
 concentrations. Liver samples were analyzed for bile acids and immune
 cells. EHBD and liver samples were fixed and stained for further analysis.
 There were 13-15 total pups in each group; however, due to limited serum
 sample volumes, especially in P5 pups, not all analyses could be performed
 on all samples.  
 <strong>Histochemistry and
 immunostaining</strong>  
 After collection, EHBDs and liver samples were fixed in 10%
 formalin and embedded in paraffin. The embedded samples were then
 sectioned at 5 µm thickness, and slides were stained for Hematoxylin and
 Eosin (H&E). Standard protocols were followed for processing the
 slides.   For antibody
 staining, EHBD sections were deparaffinized with xylene and rehydrated
 through a graded series of alcohols and distilled water. Antigen retrieval
 was performed in 10 mM citric acid buffer (pH 6.0). Sections were blocked
 with 5% bovine serum albumin and permeabilized with 0.4% Triton X-100
 prior to antibody incubation. Sections were stained for collagen I, HA and
 DAPI as described in [4]. For collagen cy3 anti-goat antibodies and for
 HA-binding protein, Cy2-streptavidin secondary antibodies were used
 (1:500, Vector Laboratories).  
 Liver sections were stained for KRT 19 and labelled with
 diaminobenzidine. Sections were incubated with 3% H2O2 to quench
 endogenous peroxidases and blocked with StartingBlock™ T20/PBS Blocking
 Buffer (Thermo Fisher Scientific, Waltham, MA, USA) and Avidin D and
 Biotin Blocking Reagents, prior to incubation with primary KRT 19
 antibodies (1:500) overnight at 4oC. The next day, sections were incubated
 with secondary antibodies (1:500) for 30 minutes at 37oC and visualized
 using an Avidin-Biotin Complex detection system (Vector Elite Kit, Vector
 Laboratories, Burlingame, CA, USA). Signals were developed by a
 diaminobenzidine substrate kit for peroxidases (Vector Laboratories) and
 counterstained with hematoxylin.  
 <strong>Histology assessment</strong>
   For bile duct H&E-stained
 slides, a qualitative assessment of damage was performed by grading the
 slides as normal or abnormal based on several features. These features
 included the presence of luminal debris, marked inflammation, detachment
 of surface epithelium, and signs of regeneration (including multi-layered
 surface epithelium and peribiliary gland expansion). Similarly, liver
 H&E-stained slides were graded as normal or abnormal based on the
 presence of bile duct damage, ductular reaction and the presence of bile
 plugs. The grading was performed independently by two researchers, IDJ and
 NDT, with more than eight animals per group being analyzed.
   <strong>Image
 analysis</strong>  
 Image analysis of stained sections was performed with Fiji
 ImageJ and QuPath v0.2.0 software. The QuPath selection tool was used to
 calculate the area of the biliary submucosa that was occupied by HA (based
 on HA-binding protein staining) relative to the entire submucosal area. As
 a second measure for the thickness of the HA layer, the width between the
 lumen and the HA-collagen interface was measured in at least 5 different
 places, and was adjusted relative to the entire thickness of the bile duct
 wall.   For KRT 19 stained
 samples, the QuPath pixel classification tool was utilized to measure the
 KRT 19 positive area relative to the field area. The number of KRT
 19-positive foci per portal triad was counted manually.
   <strong>Liver
 immunology</strong>  
 Intrahepatic leukocytes were isolated by Percoll density
 gradient centrifugation and stained with LIVE/DEAD fixable viability dye,
 or with antibodies against CD4, CD8α, NK1.1, B220, Ly6C, Ly6G, and CD90.2.
 All samples were separated on a MACSQuant flow cytometer (Miltenyi Biotec,
 Gaithersburg, MD, USA) and analyzed using FlowJo software version 10.6
 (Tree Star) (Fig 4A).  
 <strong>Sample processing for
 HPLC</strong>   Bile
 acids were extracted from homogenized liver and serum samples as described
 [5,6].  Separations were performed on a Waters BEH C18 Column (2.1 mm x 50
 mm 1.7 μm). Mobile phase A was water with 0.1% formic acid, and mobile
 phase B was methanol with 0.1% formic acid at 0.4 mL/min flow. The
 gradient started at 5% B and was changed to 40% B over 2 min, then to 99%
 B over 2 min, held constant for 3 minutes then back to the initial
 composition for equilibration of the column, for a total chromatographic
 separation time of 12 min. Analysis was conducted on a Thermo Q Exactive
 HF coupled to an Ultimate 3000 UHPLC interfaced with a heated electrospray
 ionization (HESI-II) source. The instrument was operated in negative ion
 mode alternating between full scan from 250-800 m/z at a resolution of
 120,000 and parallel reaction monitoring at 60,000 resolution with a
 precursor isolation window of 0.7 m/z. Since sample amounts were limited,
 not all analyses were performed on all samples. Bile acid values were
 normalized to average values obtained for control pups in each set of
 experiments. Some bile acids could not be detected in all samples; for
 purposes of the analysis, only those bile acids detected in at least 5
 samples were considered.  
 <strong>Statistical analysis</strong>
   Statistical significance was
 calculated by one and two-tailed Student’s t-tests. Differences in
 variance were tested using the F test [7]. The number of samples tested
 for each experiment is given in parentheses in the graphs. All data are
 shown as boxplots.  
 <strong>References</strong>
   1. Yang Y, Wang J, Zhan Y, Chen G,
 Shen Z, Zheng S, et al. The synthetic toxin biliatresone causes biliary
 atresia in mice. Lab Investig. 2020;100: 1425–1435.
 doi:10.1038/s41374-020-0467-7  
 2. Su X, Shi Y, Zou X, Lu ZN, Xie G, Yang JYH, et al.
 Single-cell RNA-Seq analysis reveals dynamic trajectories during mouse
 liver development. BMC Genomics. 2017;18: 946.
 doi:10.1186/s12864-017-4342-x  
 3. Kotb MA, Kotb A, Talaat S, Shehata SM, El Dessouki N,
 Elhaddad AA, et al. Congenital aflatoxicosis, mal-detoxification genomics
 & ontogeny trigger immune-mediated Kotb disease biliary atresia
 variant: SANRA compliant review. Med (United States). 2022;101: E30368.
 doi:10.1097/MD.0000000000030368  
 4.de Jong IEM, Hunt ML, Chen D, Du Y, Llewellyn J, Gupta K, et
 al. A fetal wound healing program after intrauterine bile duct injury may
 contribute to biliary atresia. J Hepatol. 2023;79: 1396–1407.
 doi:10.1016/j.jhep.2023.08.010  
 5. Huang J, Bathena SPR, Csanaky IL, Alnouti Y. Simultaneous
 characterization of bile acids and their sulfate metabolites in mouse
 liver, plasma, bile, and urine using LC-MS/MS. J Pharm Biomed Anal.
 2011;55: 1111–1119. doi:10.1016/j.jpba.2011.03.035
   6. Ghaffarzadegan T, Essén S,
 Verbrugghe P, Marungruang N, Hållenius FF, Nyman M, et al. Determination
 of free and conjugated bile acids in serum of Apoe(−/−) mice fed different
 lingonberry fractions by UHPLC-MS. Sci Rep. 2019;9: 3800.
 doi:10.1038/s41598-019-40272-8  
 7. Mohr DL, Wilson WJ, Freund RJ. Statistical Methods.
 Statistical Methods. New Delhi: Wiley Blackwell; 2021.
 doi:10.1016/B978-0-12-823043-5.00015-1
description: # Low-dose biliatresone treatment of pregnant mice causes subclinical
 biliary disease in their offspring: evidence for a spectrum of neonatal
 injury This dataset
 comprises data collected from pups born to mothers administered either
 biliatresone or a vehicle control via gavage. Analysis was conducted on
 pups at postnatal days 5 and 21, with mothers analyzed alongside pups at
 postnatal day 5. Physical measurements, liver, bile duct, and serum
 samples were collected. Serum samples were utilized for assessing serum
 biochemistry relevant to liver diseases and bile acid content. Liver
 samples underwent analysis for cytokeratin-19 (KRT19) staining, immune
 profiling, and bile acid measurements. Bile duct samples were subjected to
 hyaluronic acid staining. ## Description of the data and file structure
 The data are structured in accordance with the organization of figures in
 our manuscript, with two comparison groups designated: "D"
 representing the vehicle control group and "B" representing the
 biliatresone-treated group. Each cell value within the groups represents
 data obtained from individual pups. High-performance liquid chromatography
 (HPLC) measurements are presented as normalized area ratios for specific
 bile acids. The first cell in each sheet provides a brief background on
 the experiment and the specific measurement presented in the sheet. ##
 Sheet 1 Sheet title: Fig 1B.P5 serum biochemistry Description: Pregnant
 mothers (E14 and E15) were treated with 15 mg/kg of biliatresone or
 vehicle control (DMSO), and P5 pups were analyzed. Serum biochemistry and
 physical parameters for P5 pups. The number of pups is shown in
 parentheses. D denotes DMSO treated, B denotes Biliatresone treated. Each
 cell denote value from individual pup. ALP (U/L), AST (U/L), Albumin
 (g/dL), and Weight (gm) were analyzed and are reported. ## Sheet 2 Sheet
 title: Fig 1D.P5 KRT19 quantification Description: Pregnant mothers (E14
 and E15) were treated with 15 mg/kg of biliatresone or vehicle control
 (DMSO), and P5 pups were analyzed. Quantification of the number of
 cytokeratin 19 (KRT19) positive foci per portal triad and KRT19 positive
 area per field in P5 pups, with the number of pups indicated in
 parentheses. D denotes DMSO treated, B denotes Biliatresone treated. Each
 cell denote value from individual pup. KRT 19 +ve Area per field (%) and
 KRT 19 +ve duct/PT were analyzed and are reported. PT: Portal traid. ##
 Sheet 3 Sheet title: Fig 1F.P5 hyaluronic acid Description: Pregnant
 mothers (E14 and E15) were treated with 15 mg/kg of biliatresone or
 vehicle control (DMSO), and P5 pups were analyzed. Quantification of
 submucosal area stained by hyaluronic acid (HA)-binding protein. The
 number of P5 pups is shown in parentheses. D denotes DMSO treated, B
 denotes Biliatresone treated. Each cell denote value from individual
 pup.HA content (%) is reported in this sheet. ## Sheet 4 Sheet title: Fig
 2B.P21 serum biochemistry Description: Pregnant mothers (E14 and E15) were
 treated with 15 mg/kg of biliatresone or vehicle control (DMSO), and P21
 pups were analyzed. Serum biochemistry and physical parameters for P21
 pups. The number of pups is shown in parentheses. D denotes DMSO treated,
 B denotes Biliatresone treated. Each cell denote value from individual
 pup. ALP (U/L), AST (U/L), Albumin (g/dL), and Weight (gm) were analyzed
 and are reported. ## Sheet 5 Sheet title: Fig 2D.P21 KRT19 quantification
 Description: Pregnant mothers (E14 and E15) were treated with 15 mg/kg of
 biliatresone or vehicle control (DMSO), and P21 pups were analyzed.
 Quantification of the number of cytokeratin 19 (KRT19) positive foci per
 portal triad and KRT19 positive area per field in P21 pups, with the
 number of pups indicated in parentheses. D denotes DMSO treated, B denotes
 Biliatresone treated. Each cell denote value from individual pup. KRT 19
 +ve Area per field (%) and KRT 19 +ve duct/PT were analyzed and are
 reported. PT: Portal traid. ## Sheet 6 Sheet title: Fig 3A.P5 liver bile
 acid Pregnant mothers (E14 and E15) were treated with 15 mg/kg of
 biliatresone or vehicle control (DMSO). Liver from P5 pups were analyzed.
 Relative amounts of various bile acids from P5 liver are reported here.
 Individual bile acids were normalized to the corresponding mean from
 control pups from each experiment. Each column denote value from
 individual pup. D denotes DMSO treated. B denotes Biliatresone treated.
 Empty cells represent the values that could not be determined. Each row
 denotes individual bile acid that were determined. The abbreviations for
 bile acid could be found in method section. ## Sheet 7 Sheet title: Fig
 3B.P5 serum bile acid Pregnant mothers (E14 and E15) were treated with 15
 mg/kg of biliatresone or vehicle control (DMSO). Serum from P5 pups were
 analyzed. Relative amounts of various bile acids from P5 Serum are
 reported here. Individual bile acids were normalized to the corresponding
 mean from control pups from each experiment. Each Column denote value from
 individual pup. D denotes DMSO treated. B denotes Biliatresone treated.
 Empty cells represent the values that could not be determined. Each row
 denotes individual bile acid that were determined. The abbreviations for
 bile acid could be found in method section. ## Sheet 8 Sheet title: Fig
 3C.P21 liver bile acid Pregnant mothers (E14 and E15) were treated with 15
 mg/kg of biliatresone or vehicle control (DMSO). Liver from P21 pups were
 analyzed. Relative amounts of various bile acids from P21 liver are
 reported here. Individual bile acids were normalized to the corresponding
 mean from control pups from each experiment. Each Column denote value from
 individual pup. D denotes DMSO treated. B denotes Biliatresone treated.
 Empty cells represent the values that could not be determined. Each row
 denotes individual bile acid that were determined. The abbreviations for
 bile acid could be found in method section. ## Sheet 9 Sheet title: Fig
 3D.P21 serum bile acid Pregnant mothers (E14 and E15) were treated with 15
 mg/kg of biliatresone or vehicle control (DMSO). Serum from P21 pups were
 analyzed. Relative amounts of various bile acids from P21 Serum are
 reported here. Individual bile acids were normalized to the corresponding
 mean from control pups from each experiment. Each Column denote value from
 individual pup. D denotes DMSO treated. B denotes Biliatresone treated.
 Empty cells represent the values that could not be determined. Each row
 denotes individual bile acid that were determined. The abbreviations for
 bile acid could be found in method section. ## Sheet 10 Sheet title: Fig
 4B.P21 Immune cells Pregnant mothers (E14 and E15) were treated with 15
 mg/kg of biliatresone or vehicle control (DMSO). Liver from P21 pups were
 analyzed. Quantification showing numbers of T-cells, CD4 cells, CD8 cells,
 B-cells, monocytes and neutrophils in livers isolated from P21 pups. The
 number of pups is shown in parentheses. Each cell denote value from
 individual pup. D denotes DMSO treated. B denotes Biliatresone treated. ##
 Sheet 11 Sheet title: Fig 5BMother serum biochemistry Pregnant mothers
 (E14 and E15) were treated with 15 mg/kg of biliatresone or vehicle
 control (DMSO). Mothers were euthanized along with P21 pups, and serum
 biochemistry and physical parameters were measured for both control and
 biliatresone-treated mothers.Each cell denote value from individual pup. D
 denotes DMSO treated. B denotes Biliatresone treated. ## Sheet 12 Sheet
 title: Fig Fig 5C.Mother immune cells Pregnant mothers (E14 and E15) were
 treated with 15 mg/kg of biliatresone or vehicle control (DMSO). Mothers
 were euthanized along with P21 pups, and the numbers of immune cells in
 livers isolated from both control and biliatresone-treated mothers were
 quantified. Quantification showing numbers of T-cells, CD4 cells, CD8
 cells, B-cells, monocytes and neutrophils in livers isolated from mothers.