AHR Activation Affects Enteric Neuronal Survival by Inducing Apoptosis
Open Access
- Author:
- Kumar, Supriya
- Area of Honors:
- Biochemistry and Molecular Biology
- Degree:
- Bachelor of Arts
- Document Type:
- Thesis
- Thesis Supervisors:
- Andrew Patterson, Thesis Supervisor
Joseph C. Reese, Thesis Honors Advisor - Keywords:
- Aryl Hydrocarbon Receptor
TCDD
Enteric Nervous System
Persistant Organic Pollutant
Ceramide Synthesizing Genes
Apoptosis
Longitudinal Muscle Myenteric Plexus
Gut Motility
IM-FEN Cells
Neuronal Marker Genes
Nitrergic Neurons
Cholinergic Neurons
GI Tract - Abstract:
- 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a persistent organic pollutant (POP) and a potent activator of the aryl hydrocarbon receptor (AHR), a ligand-dependent transcription factor. There has been considerable progress in understanding how AHR activation by TCDD affects the central nervous system (CNS) while TCDD effects on the enteric nervous system (ENS) remains relatively underexplored. This study sought to better understand the effects of TCDD toxicity via AHR activation in the ENS with both in vivo and in vitro methods. The upregulation of ceramide synthesizing genes and apoptosis were explored as possible routes of TCDD toxicity in the ENS. In both in vivo and in vitro studies, AHR activation was confirmed with elevated cytochrome P4501A1 (Cyp1a1) gene expression levels with TCDD treatment through quantitative polymerase chain reaction (qPCR). Cyp1a1 is a known AHR target gene involved in xenobiotic metabolism. For the in vivo study, the longitudinal muscle myenteric plexus (LMMP) was isolated from male C57BL/6J mice and used for immunostaining and qPCR analysis. A stool frequency test was done before sacrificing the mice for gut motility observations. For the in vitro study, immorto fetal enteric neuronal (IM-FEN) cells were used for qPCR analysis (neuronal marker genes and ceramide genes), LDH cytotoxicity assay, Caspase-Glo 3/7 assay, cleaved caspase-3 western blot analysis, and neutral sphingomyelinase (N-Smase) activity assay. Results showed that TCDD reduced the total number of neurons (TUJ1), specifically nitrergic neurons (nNOS) with no difference in cholinergic neurons (ChAT) in TCDD-treated IM-FEN cells and primary cells isolated from WT mice but not AHR global knockout (Ahr-/-). Mice treated with TCDD exhibited a reduced interstitial motility (decreased stool frequency). TCDD also elicited cytotoxicity in IM-FEN cells with increased caspase 3/7 levels and activity indicating apoptosis as a consequence of TCDD cytotoxicity. Increased expression of ceramide synthesizing genes and increased N-Smase activity suggests that ceramides may have a significant role in the intrinsic apoptotic process of TCDD-induced cytotoxicity. The results indicate that AHR signaling within the ENS could potentially have an impact on the gastrointestinal pathophysiology caused by TCDD and protective measures should be taken with regard to the GI tract in industrial places prone to TCDD exposure.