The role of the aryl hydrocarbon receptor in the pathogenesis of congenital diaphragmatic hernia
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Date
2021-01-05
Authors
Falk, Landon
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Abstract
Environmental factors may contribute to 70% of congenital diaphragmatic hernia (CDH) cases. Patients with CDH are born with a hole in their diaphragm and abnormal lung development. A specific class of environmental chemicals can activate the transcription factor aryl hydrocarbon receptor (AHR) to change gene expression. We hypothesize that activation of AHR by these chemicals is involved in the pathogenesis of CDH.
Ethical approval was obtained prior to experiments (19-010 (AC11436)). We compared the response of AHR to nitrofen using known ligands - benzo[α]pyrene and resveratrol - in the BEAS-2B human epithelial cell line (n=3). AHR activity within a 24-hour exposure period was assessed with immunocytochemistry (ICC/IF). We further confirmed nitrofen as a ligand of AHR by performing induced-fit virtual docking using PyRx.8 software. We compared the protein abundance of AHR in nitrofen-treated rat pups (E21) and saccular lung sections (n=3) from human CDH patients (Week 39-40) to age-matched controls using immunofluorescence. AHR mRNA abundance was assessed using a novel in situ hybridization protocol called RNAScope in nitrofen-induced lung development and control E18, E21 (n=3) rat lung sections as well as end point gestation human CDH and control lung sections (n=3).
AHR activation was induced in BEAS-2B cells within six hours of treatment. We observed all ligands to induce the translocation of AHR fluorescence signal from the cytoplasm (inactive) to nucleus (active), suggesting nitrofen activates AHR. Induced-fit virtual docking confirmed this as we saw that nitrofen has a similar orientation to TCDD—a well-established ligand of AHR—within the AHR binding pocket and has a similar binding affinity and dissociation constant to TCDD. After 24 hours of treatment, the AHR signal was strictly cytoplasmic and diminished. CDH patients and nitrofen-induced rat lungs at E21 had increased AHR protein abundance in the mesenchyme and airways when compared to controls. AHR mRNA abundance was dynamic throughout development. At E21, we saw decreased AHR expression in the nitrofen treated lungs, but at E18, we saw increased AHR expression in nitrofen treated lungs. We also saw a similar trend with the human tissue where human control lungs had greater AHR mRNA expression relative to CDH lungs at end-point gestation.
We observed nuclear translocation of AHR suggesting activation of the receptor, which was corroborated by our virtual docking simulation results. We saw similar changes in AHR protein abundance in both nitrofen-induced hypoplastic rat lungs and human CDH lungs, suggesting that similar pathological mechanisms are involved. AHR mRNA expression was also altered throughout development. The dysregulated expression of AHR may contribute to abnormal lung development in babies born with CDH. The results suggest that environmental chemicals structurally similar to nitrofen may activate AHR to induce abnormal lung development in CDH.
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Keywords
congenital diaphragmatic hernia, AHR