Genome-wide ChIPseq analysis of AhR, COUP-TF, and HNF4 enrichment in TCDD-treated mouse liver

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor known for mediating the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds. Although the canonical mechanism of AhR activation involves heterodimerization with the aryl hydrocarbon receptor nuclear translocator, other transcriptional regulators that interact with AhR have been identified. Enrichment analysis of motifs in AhR-bound genomic regions implicated co-operation with COUP transcription factor (COUP-TF) and hepatocyte nuclear factor 4 (HNF4). The present study investigated AhR, HNF4α and COUP-TFII genomic binding and effects on gene expression associated with liver-specific function and cell differentiation in response to TCDD. Hepatic ChIPseq data from male C57BL/6 mice at 2 hrs after oral gavage with 30 μg/kg TCDD were integrated with bulk RNA-sequencing (RNAseq) time-course (2 - 72 hrs) and dose-response (0.01 - 30 μg/kg) datasets to assess putative AhR, HNF4α and COUP-TFII interactions associated with differential gene expression. TCDD treatment resulted in the genomic enrichment of 23,701, 11,688, and 9,547 binding regions for AhR, COUP-TFII and HNF4α, respectively, throughout the genome. Functional enrichment analysis of differentially expressed genes (DEGs) identified differential binding enrichment for AhR, COUP-TFII, and HNF4a to regions within liver-specific genes suggesting intersections associated with the loss of liver-specific functions and hepatocyte differentiation. Analysis found that the repression of liver-specific, HNF4α target and hepatocyte differentiation genes, involved increased AhR and HNF4α binding with decreased COUP-TFII binding. Collectively, these results suggested TCDD-elicited loss of liver-specific functions and markers of hepatocyte differentiation involved interactions between AhR, COUP-TFII and HNF4α.

Epigenetically Mediated Pathogenic Effects of Phenanthrene on Regulatory T Cells

Phenanthrene (Phe), a polycyclic aromatic hydrocarbon (PAH), is a major constituent of urban air pollution. There have been conflicting results regarding the role of other AhR ligands 2,3,7,8- tetrachlorodibenzo-p-dioxin (TCDD) and 6-formylindolo [3,2-b]carbazole (FICZ) in modifying regulatory T cell populations (Treg) or T helper (Th)17 differentiation, and the effects of Phe have been understudied. We hypothesized that different chemical entities of PAH induce Treg to become either Th2 or Th17 effector T cells through epigenetic modification of FOXP3. To determine specific effects on T cell populations by phenanthrene, primary human Treg were treated with Phe, TCDD, or FICZ and assessed for function, gene expression, and phenotype. Methylation of CpG sites within theFOXP3locus reducedFOXP3expression, leading to impaired Treg function and conversion of Treg into a CD4+CD25loTh2 phenotype in Phe-treated cells. Conversely, TCDD treatment led to epigenetic modification of IL-17A and conversion of Treg to Th17 T cells. These findings present a mechanism by which exposure to AhR-ligands mediates human T cell responses and begins to elucidate the relationship between environmental exposures, immune modulation, and initiation of human disease.

Effect of restraint stress on 2,3,7,8 tetrachloro dibenzo-p-dioxin induced testicular and epididymal toxicity in rats

Dioxins like 2,3,7,8 tetrachlorodibenzo-p—dioxin (TCDD) impair male reproductive system by increasing the generation of reactive oxygen species (ROS). Glucocorticoids have been found to suppress male reproductive function and also influence TCDD pathway. As stress is characterized by an increase in the level and activity of glucocorticoids, the present experiments were conducted to evaluate the effect of restraint stress on TCDDinduced testicular and epididymal toxicity. Adult male Wistar rats were subjected to either restraint stress (5 hours/day) or TCDD treatment (100 ng/kg b.w./day) or both for 15 days. Restraint stress or TCDD treatment raised the serum level of corticosterone and suppressed the testicular level of steroidogenic acute regulatory (StAR) protein and serum level of testosterone significantly. In the testis and epididymis, restraint stress or TCDD treatment raised the levels of lipid peroxidation and hydrogen peroxide and suppressed the activities of antioxidant enzymes significantly. In rats subjected to both restraint stress and TCDD treatment, a significant increase in the serum level of corticosterone and a significant decrease in the testicular level of StAR protein and serum level of testosterone were observed as compared to rats treated with TCDD alone. A significant increase in the levels of lipid peroxidation and hydrogen peroxide and a significant decrease in the activities of antioxidant enzymes were observed in the testis and epididymis of rats subjected to both restraint stress and TCDD treatment as compared to TCDD alone treated rats. Thus, restraint stress potentiates the adverse effects of TCDD on male reproductive organs.

Activation of aryl hydrocarbon receptor by TCDD prevents diabetes in NOD mice and increases Foxp3+ T cells in pancreatic lymph nodes

The ligand-activated transcription factor, aryl hydrocarbon receptor (AHR), is a novel inducer of adaptive Tregs. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), the most potent AHR ligand, induces adaptive CD4+CD25+ Tregs during an acute graft-versus-host (GvH) response and prevents the generation of allospecific cytotoxic T lymphocytes. TCDD also suppresses the induction of experimental autoimmune encephalitis in association with an expanded population of Foxp3+ Tregs. In this study, we show that chronic treatment of NOD mice with TCDD potently suppresses the development of autoimmune Type 1 diabetes in parallel with greatly reduced pancreatic islet insulitis and an expanded population of CD4+CD25+Foxp3+ cells in the pancreatic lymph nodes. When treatment with TCDD was terminated after 15 weeks (23 weeks of age), mice developed diabetes over the next 8 weeks in association with lower numbers of Tregs and decreased activation of AHR. Analysis of the expression levels of several genes associated with inflammation, T-cell activation and/or Treg function in pancreatic lymph node cells failed to reveal any differences associated with TCDD treatment. Taken together, the data suggest that AHR activation by TCDD-like ligands may represent a novel avenue for treatment of immune-mediated diseases.

Role of aryl hydrocarbon receptor in modulation of the expression of the hypoxia marker carbonic anhydrase IX

Tumour-associated expression of CA IX (carbonic anhydrase IX) is to a major extent regulated by HIF-1 (hypoxia-inducible factor-1) which is important for transcriptional activation and consists of the oxygen-regulated subunit HIF-1α and the partner factor ARNT [AhR (aryl hydrocarbon receptor) nuclear translocator]. We have previously observed that HIF-1α competes with the AhR for interaction with ARNT under conditions when both conditionally regulated factors are activated. We have therefore investigated whether TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin)-induced activation of the AhR pathway might interfere with CA IX expression. The results from the present study suggest that TCDD treatment reduces hypoxic induction of both CA IX mRNA and protein expression. Moreover, the transcriptional activity of the CA9 promoter was significantly reduced by expression of CAAhR (constitutively active AhR), which activates transcription in a ligand-independent manner. Finally, we found that ARNT is critical for both hypoxic induction and the TCDD-mediated inhibition of CA9 expression.

Dioxin Affects Glucose Transport via the Arylhydrocarbon Receptor Signal Cascade in Pluripotent Embryonic Carcinoma Cells

Intoxication by dioxins such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) leads, among other damages, to early embryo loss, fetal malformations, and cardiovascular toxicity. Apart from binding to the arylhydrocarbon receptor (AhR), the mechanism of TCDD-mediated embryo toxicity is still unclear. We investigated possible modes of a TCDD-mediated toxicity, particularly in glucose metabolism, in pluripotent P19 mouse embryonic carcinoma cells. Undifferentiated P19 cells were exposed to 1–100 nm TCDD and characterized for AhR signaling. For studying cell differentiation, P19 cells were exposed to 10 nm TCDD at stage of embryoid body formation, and analyzed on glucose metabolism and cardiac differentiation during the next 3 wk. TCDD treatment activated the AhR-signaling cascade within 1 h, confirmed by AhR translocation, induction of cytochrome P450 1A1 expression, and activation of the xenobiotic response element. Although cell viability and transcription of the cardiac marker protein α-myosin heavy chain were affected, TCDD did not inhibit the differentiation of P19 cells to pulsating cardiomyocytes. TCDD significantly down-regulated the expression levels of the glucose transporter (GLUT) isoforms 1 and 3. After 24-h TCDD treatment, GLUT1 was no longer localized in the plasma membrane of P19 cells. The impaired GLUT expression correlated with a lower glucose uptake in 5-d-old embryoid bodies. The TCDD effects were mediated by AhR, as shown by preculture with the AhR antagonist α-naphthoflavone. Our data demonstrate that an AhR-mediated disturbance in GLUT expression and insufficient glucose uptake may be major mechanisms in TCDD embryo toxicity.