Inhibition of the urea cycle by the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin increases serum ammonia levels in mice.

The aryl hydrocarbon receptor is a ligand-activated transcription factor known for mediating the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds. TCDD induces nonalcoholic fatty liver disease (NAFLD)-like pathologies including simple steatosis that can progress to steatohepatitis with fibrosis and bile duct proliferation in male mice. Dose-dependent progression of steatosis to steatohepatitis with fibrosis by TCDD has been associated with metabolic reprogramming, including the disruption of amino acid metabolism. Here, we used targeted metabolomic analysis to reveal dose-dependent changes in the level of ten serum and eleven hepatic amino acids in mice upon treatment with TCDD. Bulk RNA-seq and protein analysis showed TCDD repressed CPS1, OTS, ASS1, ASL, and GLUL, all of which are associated with the urea cycle and glutamine biosynthesis. Urea and glutamine are end products of the detoxification and excretion of ammonia, a toxic byproduct of amino acid catabolism. Furthermore, we found that the catalytic activity of OTC, a rate-limiting step in the urea cycle was also dose dependently repressed. These results are consistent with an increase in circulating ammonia. Collectively, the repression of the urea and glutamate-glutamine cycles increased circulating ammonia levels and the toxicity of TCDD.

Cell-specific AHR-driven differential gene expression in the mouse liver cell following acute TCDD exposure.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a persistent environmental contaminant that disrupts hepatic function leading to steatotic liver disease (SLD)-like pathologies, such as steatosis, steatohepatitis, and fibrosis. These effects are mediated by the aryl hydrocarbon receptor following changes in gene expression. Although diverse cell types are involved, initial cell-specific changes in gene expression have not been reported. In this study, differential gene expression in hepatic cell types was examined in male C57BL/6 mice gavaged with 30 µg/kg of TCDD using single-nuclei RNA-sequencing. Ten liver cell types were identified with the proportions of most cell types remaining unchanged, except for neutrophils which increased at 72 h. Gene expression suggests TCDD induced genes related to oxidative stress in hepatocytes as early as 2 h. Lipid homeostasis was disrupted in hepatocytes, macrophages, B cells, and T cells, characterized by the induction of genes associated with lipid transport, steroid hormone biosynthesis, and the suppression of ß-oxidation, while linoleic acid metabolism was altered in hepatic stellate cells (HSCs), B cells, portal fibroblasts, and plasmacytoid dendritic cells. Pro-fibrogenic processes were also enriched, including the induction retinol metabolism genes in HSCs and the early induction of anti-fibrolysis genes in hepatocytes, endothelial cells, HSCs, and macrophages. Hepatocytes also had gene expression changes consistent with hepatocellular carcinoma. Collectively, these findings underscore the effects of TCDD in initiating SLD-like phenotypes and identified cell-specific gene expression changes related to oxidative stress, steatosis, fibrosis, cell proliferation and the development of HCC.

Chorioallantoic membrane assay revealed the role of TIPARP (2,3,7,8-tetrachlorodibenzo-p-dioxin-inducible poly (ADP-ribose) polymerase) in lung adenocarcinoma-induced angiogenesis.

BACKGROUND: The chorioallantoic membrane (CAM) assay is a well-established technique to evaluate tumor invasion and angiogenesis and may overcome the shortcoming of the patient-derived xenograft (PDX) mouse model. Currently, few reports have described lung cancer invasion and angiogenesis in the CAM assay. We therefore used the CAM assay in the evaluation of lung cancer. METHOD: Lung cancer cell line-derived organoids or lung cancer cell lines were transplanted into the CAM on embryonic development day (EDD) 10, and an analysis was performed on EDD 15. Microscopic and macroscopic images and movies of the grafts on the CAM were captured and analyzed. The relationships between the graft and chick vessels were evaluated using immunohistochemistry. RESULTS: We transplanted lung cancer cell lines and cell line-derived organoid into a CAM to investigate angiogenesis and invasion. They engrafted on the CAM at a rate of 50-83%. A549-OKS cells showed enhanced cell invasion and angiogenesis on the CAM in comparison to A549-GFP cells as was reported in vitro. Next, we found that A549-TIPARP cells promoted angiogenesis on the CAM. RNA-seq identified 203 genes that were upregulated more than twofold in comparison to A549-GFP cells. A pathway analysis revealed many upregulated pathways related to degradation and synthesis of the extracellular matrix in A549-TIPARP cells. CONCLUSIONS: The CAM assay can be used to evaluate and research invasion and angiogenesis in lung cancer. The elevated expression of TIPARP in lung cancer may induce angiogenesis by remodeling the extracellular matrix.

TCDD-Induced Allosteric Perturbation of the AhR:ARNT Binding to DNA.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates the biological and toxicological effects of structurally diverse chemicals, including halogenated aromatic hydrocarbons. In this work, we investigate the effects of the binding of the AhR prototypical ligand, TCDD, on the stability of the AhR:ARNT complex, as well as the mechanisms by which ligand-induced perturbations propagate to the DNA recognition site responsible for gene transcription. To this aim, a reliable structural model of the overall quaternary structure of the AhR:ARNT:DRE complex is proposed, based on homology modelling. The model shows very good agreement with a previous one and is supported by experimental evidence. Moreover, molecular dynamics simulations are performed to compare the dynamic behaviour of the AhR:ARNT heterodimer in the presence or absence of the TCDD. Analysis of the simulations, performed by an unsupervised machine learning method, shows that TCDD binding to the AhR PASB domain influences the stability of several inter-domain interactions, in particular at the PASA-PASB interface. The inter-domain communication network suggests a mechanism by which TCDD binding allosterically stabilizes the interactions at the DNA recognition site. These findings may have implications for the comprehension of the different toxic outcomes of AhR ligands and drug design.

Roles of the ubiquitin ligase CUL4B and ADP-ribosyltransferase TiPARP in TCDD-induced nuclear export and proteasomal degradation of the transcription factor AHR.

The aryl hydrocarbon receptor (AHR) is a transcription factor activated by exogenous halogenated polycyclic aromatic hydrocarbon compounds, including the environmental toxin TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin, and naturally occurring dietary and endogenous compounds. The activated AHR enhances transcription of specific genes including phase I and phase II metabolism enzymes and other targets genes such as the TCDD-inducible poly(ADP-ribose) polymerase (TiPARP). The regulation of AHR activation is a dynamic process: immediately after transcriptional activation of the AHR by TCDD, the AHR is exported from the nucleus to the cytoplasm where it is subjected to proteasomal degradation. However, the mechanisms regulating AHR degradation are not well understood. Here, we studied the role of two enzymes reported to enhance AHR breakdown: the cullin 4B (CUL4B)AHR complex, an E3 ubiquitin ligase that targets the AHR and other proteins for ubiquitination, and TiPARP, which targets proteins for ADP-ribosylation, a posttranslational modification that can increase susceptibility to degradation. Using a WT mouse embryonic fibroblast (MEF) cell line and an MEF cell line in which CUL4B has been deleted (MEFCul4b-null), we discovered that loss of CUL4B partially prevented AHR degradation after TCDD exposure, while knocking down TiPARP in MEFCul4b-null cells completely abolished AHR degradation upon TCDD treatment. Increased TCDD-activated AHR protein levels in MEFCul4b-null and MEFCul4b-null cells in which TiPARP was knocked down led to enhanced AHR transcriptional activity, indicating that CUL4B and TiPARP restrain AHR action. This study reveals a novel function of TiPARP in controlling TCDD-activated AHR nuclear export and subsequent proteasomal degradation.

Molecular characterization of the aryl hydrocarbon receptor 2 gene in black rockfish, Sebastes schlegelii, and its expression patterns upon exposure to benzo[a]pyrene, 2,3,7,8-tetrachlorodibenzo-p-dioxin, and ß-naphthoflavone.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates the toxicity of halogenated and polycyclic aromatic hydrocarbons in vertebrates. Thus, increased knowledge of AhR-mediated responses to xenobiotics is imperative. Sebastes schlegelii is increasingly being used as a model for studying environmental toxicology; hence, in this study, the presence of AhR2 was evaluated in S. schlegelii. The results showed that the predicted AhR2 amino acid sequence contained regions characteristic of other vertebrate AhRs, including the basic helix-loop-helix and PER-ARNT-SIM domains in the N-terminal half, but it had minor similarity with other vertebrate AhRs across the C-terminal half; it did not contain the distinct glutamine-rich domains found in mammalian AhR2. Phylogenetic analysis demonstrated that S. schlegelii AhR2 was clustered within the teleost AhR2 branch. Additionally, AhR2 mRNA was detectable in all 11 tissues tested, with the highest mRNA levels in the heart, pyloric ceca, and liver. Furthermore, exposure to the AhR agonists showed that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, 1 µg/g body weight) induced a significantly higher increases in AhR2 expression in the gills, liver, kidneys, and spleen in 48 h than benzo[a]pyrene (2 µg/g body weight), and ß-naphthoflavone (50-µg/g body weight); AhR2 mRNA levels upon TCDD exposure were up-regulated by 16- and 10-fold in the gills and liver, respectively. These findings indicated that AhR was a highly sensitive receptor against TCDD. Thus, investigating AhR2 expression in the presence of other xenobiotics might offer further information for the elucidation of its crucial role in mediating toxicant metabolism in S. schlegelii.

Liver-specific decrease in Tff3 gene expression in infant mice perinatally exposed to 2,3,7,8-tetrabromodibenzofuran or 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Polybrominated dibenzo-p-dioxins and dibenzofurans (PBDD/DFs) are byproducts of brominated flame retardants and can cause adverse health effects. Although exposure to polychlorinated (PC) DD/DFs induces toxic effects, including liver injury and neurobehavioral disorder, little is known about toxicities associated with PBDD/DF exposure. Thus, we examined effects of perinatal exposure to brominated congener on the infant mouse. Gene expression in several organs, such as the liver and brain, was analyzed in mouse offspring born to dams administered 2,3,7,8-tetrabromodibenzofuran (TBDF; 9 or 45 µg/kg body weight) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 3 µg/kg body weight) on gestational day 12.5. An increase in liver size was observed in TBDF- or TCDD-exposed offspring in infancy. Gene microarray analysis revealed that 163 and 36 genes were markedly upregulated and downregulated, respectively, in the liver of TBDF-exposed mice compared with those in vehicle-treated mice on postnatal day (PND) 5. Significant increases in Cyp1a1, Cyp1a2, Fmo3, and Pnliprp1 and decreases in Tff3, Ocstamp, Kcnk16, and Lgals2 mRNA levels in TBDF-exposed offspring on PNDs 5 and 12 were confirmed by quantitative PCR. In particular, a significant reduction in Tff3 mRNA in the liver, but not in the brain, small intestine, colon, and kidney, was observed in offspring perinatally exposed to TBDF or TCDD. Ultrasonic calls of TBDF- or TCDD-exposed offspring on PNDs 3-5 were impaired. Taken together, perinatal exposure to polyhalogenated dioxin/furan congeners disrupts gene expression patterns in the liver and ultrasonic calling during infancy. These results suggest that liver injury may contribute to neurobehavioral disorder.

Effects of 1,3,7-tribromodibenzo-p-dioxin, a natural dioxin on chicken embryos: Comparison with effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Several naturally occurring dioxins, including 1,3,7-tribromodibenzo-p-dioxin (1,3,7-TriBDD), synthesized by red algae, have been detected in the marine environment. As 1,3,7-TriBDD is accumulated in mussels and fish, predators, such as marine birds, are exposed to this congener, similar to anthropogenic dioxins (including 2,3,7,8-tetrachlorodibenzo-p-dioxin TCDD). However, little is known about the impact of 1,3,7-TriBDD exposure on the bird health. To understand the effects of 1,3,7-TriBDD on birds, the phenotypic effects and hepatic transcriptome were investigated in chicken (Gallus gallus) embryos treated with 27 µM (2.9 ng/g egg) and 137 µM (14.4 ng/g egg) 1,3,7-TriBDD. The blood glucose levels in the 1,3,7-TriBDD-treated groups were lower than those in the control group. The transcriptome analysis of 6520 sequences in the 27 and 137 µM 1,3,7-TriBDD-treated groups identified 733 and 596 differentially expressed genes (DEGs). Cytochrome P450 1A4 and 1A5 were also identified as DEGs, suggesting that the aryl hydrocarbon receptor is activated by this congener. Pathway and network analyses with DEGs suggested that 1,3,7-TriBDD may induce carcinogenic effects and metabolic alterations. These results were similar to the effects on TCDD-treated embryos. Nevertheless, the overall transcriptome results suggested that compared with TCDD, 1,3,7-TriBDD has a unique impact on insulin- and peroxisome-signaling pathways in chicken embryos. Differences in altered transcriptome profiles between 1,3,7-TriBDD- and TCDD-treated embryos may lead to different phenotypic effects: less severe effects of 1,3,7-TriBDD and more fatal effects of TCDD. Collectively, these findings warrant the further assessment of the hazard and risk of 1,3,7-TriBDD on marine animals, considering increased exposure due to climate change.

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 h after oral gavage with 30 µg/kg TCDD were integrated with bulk RNA-sequencing (RNAseq) time-course (2-72 h) and dose-response (0.01-30 µg/kg) datasets to assess putative AhR, HNF4α and COUP-TFII interactions associated with differential gene expression. Functional enrichment analysis of differentially expressed genes (DEGs) identified differential binding enrichment for AhR, COUP-TFII, and HNF4α 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.

The role of MEG3 in the proliferation of palatal mesenchymal cells is related to the TGFß/Smad pathway in TCDD inducing cleft palate.

Cleft palate (CP) is a common birth defect with a high incidence of occurrence in humans. The 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) is a highly toxic halogenated aromatic hydrocarbon, with a strong CP effect on mice. Increasing recent evidences have shown that long-noncoding RNAs (lncRNAs) play an important role in several diseases, including CP. However, there is a paucity of studies on the role of lncRNA MEG3 in the occurrence and development of TCDD-induced CP. In this study, the relationship between MEG3 and the proliferation of palatal mesenchymal cells and the underlying molecular mechanism were studied by establishing fetal CP with TCDD (64 µg/kg) in C57BL/6N mice. The results revealed that MEG3 was highly expressed during the critical period of CP formation and that the fetal mesenchymal proliferation was significantly inhibited at certain critical periods in the mice receiving TCDD. In addition, we noted a possibility of a crosstalk between MEG3 and the TGF-ß/Smad pathway, such that the inhibition of the TGF-ß/Smad pathway was induced by TCDD. Cumulatively, our study suggests that TCDD-induced CP may be caused by MEG3 inhibition of the proliferation of palatal mesenchymal cells involving the TGFß/Smad pathway, which may provide a novel perspective to understand the pathogenesis of CP.

2,3,7,8-Tetrachlorodibenzo-p-dioxin prompted differentiation to CD4+CD8-CD25+ and CD4+CD8+CD25+ Tregs and altered expression of immune-related genes in the thymus of chicken embryos.

The chicken (Gallus gallus), which has three aryl hydrocarbon receptor (AHR) isoforms (ckAHR1, ckAHR2, and ckAHR1ß) and two AHR nuclear translocator (ARNT) isoforms (ckARNT1 and ckARNT2), is highly sensitive to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and can serve as an avian model to gain an understanding of the mechanism underlying dioxin toxicity. To elucidate the mechanism of TCDD-induced immunotoxicity in avian species, we treated chicken embryos in ovo with graded concentrations of TCDD (1.5, 2.5, 3.0, 3.3, 3.5, and 4.0 µM). Initially, we measured mRNA expression levels of ckAHR and ckARNT isoforms and analyzed the T cell populations and transcriptome in the thymuses of TCDD-treated chicken embryos. Quantitative polymerase chain reaction analysis revealed that mRNA expressions of ckAHR1 and ckARNT2 were dominant in the thymus. Severe weight loss and thymus atrophy were observed in the TCDD-treated embryos. Immunophenotyping analyses demonstrated significant increases in CD4+CD8-CD25+ and CD4+CD8+CD25+ regulatory T cells (Tregs) populations following TCDD exposure, suggesting that TCDD suppresses T cell-mediated immune responses in chicken embryos. In addition, thymic transcriptome analyses intimated that alteration of the signaling pathways related to erb-b2 receptor tyrosine kinase 4 (ERBB4) and wnt family member 5A (WNT5A), and bone morphogenetic protein (BMP) may be associated with the TCDD-induced thymus atrophy. We also observed significantly altered expression levels of genes including interleukine 13 receptor subunit alpha 2 (IL13RA2), transforming growth factor beta 1 (TGFß1), collagen type III alpha 1 chain (COL3A1), and collagen type IX alpha 3 chain (COL9A3), implying immunosuppression, fibrosis development, and collagen deposition. Collectively, these findings suggest that TCDD exposure activates the ckAHR1-ckARNT2 signaling pathway and suppresses immune responses through the prompted differentiation to CD4+CD8-CD25+ and CD4+CD8+CD25+ Tregs and altered expressions of immune-related genes in the thymus of chicken embryos.

Rapamycin Alleviates 2,3,7,8-Tetrachlorodibenzo-p-dioxin-Induced Aggravated Dermatitis in Mice with Imiquimod-Induced Psoriasis-Like Dermatitis by Inducing Autophagy.

Recently, the mTOR signaling has emerged as an important player in the pathogenesis of psoriasis. We previously found that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced psoriatic skin inflammation was related to the inhibition of autophagy in keratinocytes. However, the effects and detailed molecular mechanisms of the mTOR inhibitor rapamycin and TCDD on psoriasis in vivo remain to be elucidated. In this study, we aimed to evaluate the effects of rapamycin and TCDD on skin lesions in imiquimod (IMQ)-induced psoriasis using a mouse model. TCDD aggravated skin inflammation in an IMQ-induced psoriatic mouse model. Furthermore, TCDD increased the expression of aryl hydrocarbon receptor (AHR), CYP1A1, proinflammatory cytokines, oxidative stress markers (NADPH oxidase (Nox) 2, Nox4), and phosphorylated P65NF-ĸB, whereas the expression of autophagy-related factors and the antioxidant marker nuclear factor-erythroid 2-related factor 2 (NRF2) decreased. Rapamycin reduced the aggravated skin inflammation induced by TCDD and restored TCDD-induced autophagy suppression and the increase of AHR expression, oxidative stress, and inflammatory response in the skin lesions of a psoriatic mouse model. In conclusion, we demonstrated that rapamycin alleviates TCDD-induced aggravated dermatitis in mice with imiquimod-induced psoriasis-like dermatitis through AHR and autophagy modulation.

Effects of Acute 2,3,7,8-Tetrachlorodibenzo-p-Dioxin Exposure on the Circulating and Cecal Metabolome Profile.

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a polyhalogenated planar hydrocarbon belonging to a group of highly toxic and persistent environmental contaminants known as "dioxins". TCDD is an animal teratogen and carcinogen that is well characterized for causing immunosuppression through activation of aryl hydrocarbon receptor (AHR). In this study, we investigated the effect of exposure of mice to an acute dose of TCDD on the metabolic profile within the serum and cecal contents to better define the effects of TCDD on host physiology. Our findings demonstrated that within the circulating metabolome following acute TCDD exposure, there was significant dysregulation in the metabolism of bioactive lipids, amino acids, and carbohydrates when compared with the vehicle (VEH)-treated mice. These widespread changes in metabolite abundance were identified to regulate host immunity via modulating nuclear factor-kappa B (NF-κB) and extracellular signal-regulated protein kinase (ERK1/2) activity and work as biomarkers for a variety of organ injuries and dysfunctions that follow TCDD exposure. Within the cecal content of mice exposed to TCDD, we were able to detect changes in inflammatory markers that regulate NF-κB, markers of injury-related inflammation, and changes in lysine degradation, nicotinamide metabolism, and butanoate metabolism, which collectively suggested an immediate suppression of broad-scale metabolic processes in the gastrointestinal tract. Collectively, these results demonstrate that acute TCDD exposure results in immediate irregularities in the circulating and intestinal metabolome, which likely contribute to TCDD toxicity and can be used as biomarkers for the early detection of individual exposure.

Aryl Hydrocarbon Receptor Repressor Is Hypomethylated in Psoriasis and Promotes Psoriasis-like Inflammation in HaCaT Cells.

It is known that DNA hypomethylation of aryl hydrocarbon receptor repressor (AhRR), one of the epigenetic markers of environmental pollutants, causes skin diseases. However, the function and mechanisms are still unknown. We aimed to determine whether AhRR is hypomethylated in PBMC of psoriasis patients, as well as to examine the expression of psoriasis-related inflammatory cytokines and antimicrobial peptides after 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) treatment in HaCaT cells overexpressing or silencing AhRR. AhRR was determined by qPCR, Western blot, immunohistochemistry, and immunocytochemistry in skin tissue and HaCaT cells. DNA methylation of AhRR was performed by Infinium Human Methylation450 BeadChip in PBMC of psoriasis patients and methylation-specific PCR (MSP) in HaCaT cells. NF-κB pp50 translocation and activity were performed by immunocytochemistry and luciferase reporter assay, respectively. We verified AhRR gene expression in the epidermis from psoriasis patients and healthy controls. AhRR hypomethylation in PBMC of psoriasis patients and pAhRR-HaCaT cells was confirmed. The expression level of AhRR was increased in both TCDD-treated HaCaT cells and pAhRR-HaCaT cells. NF-κB pp50 translocation and activity increased with TCDD. Our results showed that AhRR was hypomethylated and overexpressed in the lesional skin of patients with psoriasis, thereby increasing AhRR gene expression and regulating pro-inflammatory cytokines through the NF-κB signaling pathway in TCDD-treated HaCaT cells.

Trajectory Shifts in Interdisciplinary Research of the Aryl Hydrocarbon Receptor-A Personal Perspective on Thymus and Skin.

Identifying historical trajectories is a useful exercise in research, as it helps clarify important, perhaps even "paradigmatic", shifts in thinking and moving forward in science. In this review, the development of research regarding the role of the transcription factor "aryl hydrocarbon receptor" (AHR) as a mediator of the toxicity of environmental pollution towards a link between the environment and a healthy adaptive response of the immune system and the skin is discussed. From this fascinating development, the opportunities for targeting the AHR in the therapy of many diseases become clear.

Aryl Hydrocarbon Receptor (AhR) Activation by 2,3,7,8-Tetrachlorodibenzo-p-Dioxin (TCDD) Dose-Dependently Shifts the Gut Microbiome Consistent with the Progression of Non-Alcoholic Fatty Liver Disease.

Gut dysbiosis with disrupted enterohepatic bile acid metabolism is commonly associated with non-alcoholic fatty liver disease (NAFLD) and recapitulated in a NAFLD-phenotype elicited by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in mice. TCDD induces hepatic fat accumulation and increases levels of secondary bile acids, including taurolithocholic acid and deoxycholic acid (microbial modified bile acids involved in host bile acid regulation signaling pathways). To investigate the effects of TCDD on the gut microbiota, the cecum contents of male C57BL/6 mice orally gavaged with sesame oil vehicle or 0.3, 3, or 30 µg/kg TCDD were examined using shotgun metagenomic sequencing. Taxonomic analysis identified dose-dependent increases in Lactobacillus species (i.e., Lactobacillus reuteri). Increased species were also associated with dose-dependent increases in bile salt hydrolase sequences, responsible for deconjugation reactions in secondary bile acid metabolism. Increased L. reuteri levels were further associated with mevalonate-dependent isopentenyl diphosphate (IPP) biosynthesis and o-succinylbenzoate synthase, a menaquinone biosynthesis associated gene. Analysis of the gut microbiomes from cirrhosis patients identified an increased abundance of genes from the mevalonate-dependent IPP biosynthesis as well as several other menaquinone biosynthesis genes, including o-succinylbenzoate synthase. These results extend the association of lactobacilli with the AhR/intestinal axis in NAFLD progression and highlight the similarities between TCDD-elicited phenotypes in mice to human NAFLD.

An aryl hydrocarbon receptor agonist suppresses the growth of human umbilical vein endothelial cells in vitro: Potent effect with polyunsaturated fatty acids.

Human umbilical vein endothelial cells (HUVECs) are a pivotal component of the hematopoietic microenvironment linked to the modulation of the immune response, inflammation and carcinogenesis. HUVEC expresses the aryl hydrocarbon receptor (AHR), which regulates gene expression by binding to the xenobiotic-responsive element. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a potent agonist for AHR signalling. Treatment with TCDD (0.1-100 nmol/L) was found to suppress the proliferation and to stimulate the death of HUVEC. TCDD's effects were abolished by culturing with CH223191, an inhibitor of AHR signalling. Mechanistically, TCDD treatment increased the protein levels of cell growth suppressors, including p53, Rb, p21 and regucalcin, and caspase-3 implicated in apoptotic cell death, and decreased the levels of Stat3, mitogen-activated protein kinase (MAPK/Erk1/2) and phospho-MAPK/Erk1/2. Treatment with polyunsaturated fatty acids (PUFAs), including docosahexaenoic acid, eicosapentaenoic acid and arachidonic acid, suppressed the proliferation and stimulated the death of HUVEC in vitro, and decreased the levels of Stat3, MAPK/Erk1/2 and phospho-MAPK/Erk1/2 and increased caspase-3. Notably, the effects of TCDD in suppressing proliferation and stimulating death of HUVEC were modulated by coculturing with PUFAs. These effects were reversed by treatment with CH223191, an inhibitor of AHR. Treatment with both TCDD and PUFAs collaboratively enhanced the levels of AHR, CYP1A1, p53, p21, Rb and regucalcin. Moreover, TCDD suppressed migration with wound healing of HUVEC. Notably, the combination of TCDD and PUFAs revealed potent suppressive effects on angiogenesis of HUVEC, potentially related to disorders of the stromal microenvironment.

Serum microRNA profiles among dioxin exposed veterans with monoclonal gammopathy of undetermined significance.

Previously an increased risk for monoclonal gammopathy of undetermined significance (MGUS), a precursor of multiple myeloma (MM), was reported among Vietnam veterans exposed to Agent Orange and its contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Dysregulated expression of certain microRNAs (miRNAs) was demonstrated in MGUS and MM. Given the important role of miRNAs in cellular homeostasis, the aim of this study was to determine if there was an association between serum levels of selected miRNAs and TCDD in 47 MGUS cases identified in our previous investigation using serum specimens and exposure data archived by the Air Force Health Study (AFHS). A total of 13 miRNA levels (let-7a, let-7i, miR-16, miR-20a, miR-21, miR-34a, miR-106b, miR-146a, miR-181a, miR-192, miR-205, miR-335, and miR-361) was measured in serum stored during the 2002 AFHS follow-up and the relationship to lipid-adjusted serum TCDD levels in 1987 was determined. miR-34a showed the strongest relationship with TCDD; after age-adjustment, this positive association was more pronounced. In contrast, the other 12 miRNAs displayed absolute values of age adjusted coefficient estimates below 1.16 and non-significant p-values. The observed strong positive association between high body burdens of TCDD and miR-34a, a tumor suppressor regulated by p53, in this MGUS population warrants clarification of the TCDD-miR-34a relationship and its role in the pathogenesis of MGUS and risk for MM.

The AHR1-ARNT1 dimerization pair is a major regulator of the response to natural ligands, but not to TCDD, in the chicken.

The activation of the aryl hydrocarbon receptor (AHR) occurs through the binding of dioxin-like compounds (DLCs) or natural ligands. In this pathway, the AHR-ARNT (AHR nuclear translocator) heterodimer serves to regulate critical physiological functions, such as immune responses and the metabolism of xenobiotics. Birds have three AHR isoforms (AHR1, AHR1ß, and AHR2) and two ARNT isoforms (ARNT1 and ARNT2). However, how AHR and ARNT dimerization pair in birds regulates the AHR signaling pathway in an isoform-specific manner remains unknown. In this study, we initially sought to clarify the major chicken AHR-ARNT (ckAHR-ckARNT) pairs by estimating the mRNA tissue distributions of various ckAHR and ckARNT isoforms. Our results indicated that the ckAHR1-ckARNT1 represented the major dimerization pair in most tissues except the brain. We then measured the transactivation potencies of various ckAHR-ckARNT pairs by natural ligands and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), in in vitro reporter gene assays using COS-7 and LMH cell lines. Our results from the in vitro assays demonstrated that the ckAHR1-ckARNT1 pair was strongly activated by the five natural ligands, namely, 6-formylindolo [3,2-b]carbazole, L-kynurenin, kynurenic acid, indoxyl-3-sulfate, and 1,3,7-tribromodibenzo-p-dioxin, but not by TCDD. In in silico ligand docking simulations with ckAHR1 homology models, all the natural ligands showed a interaction pattern that was distinct from that observed with anthropogenic DLCs, including TCDD. In conclusion, our findings indicate that the ckAHR1-ckARNT1 may be the most important dimerization pair in most tissues for regulating the physiological functions driven by natural ligands, although it was less reactive to TCDD.

Role of Aryl Hydrocarbon Receptor Activation and Autophagy in Psoriasis-Related Inflammation.

Aryl hydrocarbon receptor (AhR) and autophagy reportedly regulate immune responses in the skin. This study explored the effects of AhR activation on autophagy in human keratinocytes, and the relevance of AhR and autophagy in psoriasis pathogenesis. AhR activation by 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) repressed autophagy, while autophagy inhibition induced AhR activation in HaCaT cells and normal human epidermal keratinocytes (NHEKs). A particularly strong interaction between AhR and autophagy was observed in proinflammatory cytokines-stimulated keratinocytes, an in vitro model of psoriasis. In skin biopsies from psoriasis patients, a similar impact of AhR on autophagy and inflammation was observed. AhR inhibition blocked TCDD- and chloroquine-induced p65NF-κB and p38MAPK phosphorylation in proinflammatory cytokines-stimulated HaCaT cells. Moreover, higher expression of AhR and CYP1A1, and lower expression of LC3, were detected in psoriatic skin tissues, compared to the controls. These data demonstrated that AhR modulated autophagy leads to skin inflammation in human keratinocytes via the p65NF-κB/p38MAPK signaling pathways, suggesting that AhR signaling and autophagy might be involved in the pathogenesis of chronic inflammatory disorders such as psoriasis.