Low-dose biliatresone treatment of pregnant mice causes subclinical biliary disease in their offspring: Evidence for a spectrum of neonatal injury

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.

 

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.

 

 

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.

 

 

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.

 

 

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.

 

 

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.

 

 

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).

 

 

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.

 

 

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.

 

 

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