Aryl Hydrocarbon Receptor Pathway Research Articles

Airborne particulate matter (PM10) induces cell invasion through Aryl Hydrocarbon Receptor and Activator Protein 1 (AP-1) pathway deregulation in A549 lung epithelial cells.

Particulate matter with an aerodynamic size ≤ 10μm (PM10) is a risk factor for lung cancer development, mainly because some components are highly toxic. Polycyclic aromatic hydrocarbons (PAHs) are present in PM10, such as benzo[a]pyrene (BaP), which is a well-known genotoxic and carcinogenic compound to humans, capable of activating AP-1 transcription factor family genes through the Aryl Hydrocarbon Receptor (AhR). Because effects of BaP include metalloprotease 9 (MMP-9) activation, cell invasion, and other pathways related to carcinogenesis, we aimed to demonstrate that PM10 (10µg/cm2) exposure induces the activation of AP-1 family members as well as cell invasion in lung epithelial cells, through AhR pathway. The role of the AhR gene in cells exposed to PM10 (10µg/cm2) and BaP (1µM) for 48h was evaluated using AhR-targeted interference siRNA. Then, the AP-1 family members (c-Jun, Jun B, Jun D, Fos B, C-Fos, and Fra-1), the levels/activity of MMP-9, and cell invasion were analyzed. We found that PM10 increased AhR levels and promoted its nuclear localization in A549 treated cells. Also, PM10 and BaP deregulated the activity of AP-1 family members. Moreover, PM10 upregulated the secretion and activity of MMP-9 through AhR, while BaP had no effect. Finally, we found that cell invasion in A549 cells exposed to PM10 and BaP is modulated by AhR. Our results demonstrated that PM10 exposure induces upregulation of the c-Jun, Jun B, and Fra-1 activity, the expression/activity of MMP-9, and the cell invasion in lung epithelial cells, effects mediated through the AhR. Also, the Fos B and C-Fos activity were downregulated. In addition, the effects induced by PM10 exposure were like those induced by BaP, which highlights the potentially toxic effects of the PM10 mixture in lung epithelial cells.

Crosstalk between aryl hydrocarbon receptor (AhR) and BCL-2 pathways suggests the use of AhR antagonist to maintain normal differentiation state of mammary epithelial cells during BCL-2 inhibition therapy

IntroductionActivating the aryl hydrocarbon receptor upon exposure to environmental pollutants promotes development of breast cancer stem cell (CSCs). BCL-2 family proteins protect cancer cells from the apoptotic effects of chemotherapeutic drugs. However, the crosstalk between AhR and the BCL-2 family in CSC development remains uninvestigated. ObjectivesThis study explored the interaction mechanisms between AhR and BCL-2 in CSC development and chemoresistance. MethodsA quantitative proteomic analysis study was performed as a tool for comparative expression analysis of breast cancer cells treated by AhR agonist. The basal and inducible levels of BCL-2, AhR, and CYP1A1 in vitro breast cancer and epithelial cell lines and in vivo mice animal models were determined by RT-PCR, Western blot analysis, immunofluorescence, flow cytometry, silencing of the target, and immunohistochemistry. In addition, an in silico toxicity study was conducted using DEREK software. ResultsActivation of the AhR/CYP1A1 pathway in mice increased EpCAMHigh/CD49fLow CD61+ luminal progenitor-like cells in early tumor formation but not in advanced tumors. In parallel, a chemoproteomic study on breast cancer MCF-7 cells revealed that the BCL-2 protein expression was the most upregulated upon AhR activation. The crosstalk between the AhR and BCL-2 pathways in vitro and in vivo modulated the CSCs features and chemoresistance. Interestingly, inhibition of BCL-2 in mice by venetoclax (VCX) increased EpCAMHigh/CD49fLow CD61+ luminal progenitor-like cells, causing inhibition of epithelial lineage markers, disruption of mammary gland branching and induced the epithelial-mesenchymal transition in mammary epithelial cells (MECs). The combined treatment of VCX and AhR antagonists in mice corrected the abnormal differentiation in MECs and protected mammary gland branching and cell identity. ConclusionsThis is the first study to report crosstalk between AhR and BCL-2 in breast CSCs and provides the rationale for using a combined treatment of BCL-2 inhibitor and AhR antagonist for more effective cancer prevention and treatment.

Aryl Hydrocarbon Receptor Agonism Antagonizes the Hypoxia-driven Inflammation in Cystic Fibrosis.

Hypoxia contributes to the exaggerated yet ineffective airway inflammation that fails to oppose infections in cystic fibrosis (CF). However, the potential for impairment of essential immune functions by HIF-1α (hypoxia-inducible factor 1α) inhibition demands a better comprehension of downstream hypoxia-dependent pathways that are amenable for manipulation. We assessed here whether hypoxia may interfere with the activity of AhR (aryl hydrocarbon receptor), a versatile environmental sensor highly expressed in the lungs, where it plays a homeostatic role. We used murine models of Aspergillus fumigatus infection invivo and human cells invitro to define the functional role of AhR in CF, evaluate the impact of hypoxia on AhR expression and activity, and assess whether AhR agonism may antagonize hypoxia-driven inflammation. We demonstrated that there is an important interferential cross-talk between the AhR and HIF-1α signaling pathways in murine and human CF, in that HIF-1α induction squelched the normal AhR response through an impaired formation of the AhR:ARNT (aryl hydrocarbon receptor nuclear translocator)/HIF-1β heterodimer. However, functional studies and analysis of the AhR genetic variability in patients with CF proved that AhR agonism could prevent hypoxia-driven inflammation, restore immune homeostasis, and improve lung function. This study emphasizes the contribution of environmental factors, such as infections, in CF disease progression and suggests the exploitation of hypoxia:xenobiotic receptor cross-talk for antiinflammatory therapy in CF.

Activation of the kynurenine-aryl hydrocarbon receptor axis impairs the chondrogenic and chondroprotective effects of human umbilical cord-derived mesenchymal stromal cells in osteoarthritis rats.

It has been proven that intra-articular injection of mesenchymal stromal cells (MSCs) can alleviate cartilage damage in osteoarthritis (OA) by differentiating into chondrocytes and protecting inherent cartilage. However, the mechanism by which the OA articular microenvironment affects MSCs' therapeutic efficiency is yet to be fully elucidated. The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor involved in various cellular processes, such as osteogenesis and immune regulation. Tryptophan (Trp) metabolites, most of which are endogenous ligand for AHR, are abnormally increased in synovial fluid (SF) of OA and rheumatoid arthritis (RA) patients. In this study, the effects of kynurenine (KYN), one of the most important metabolites of Trp, were evaluated on the chondrogenic and chondroprotective effects of human umbilical cord-derived mesenchymal stromal cells (hUC-MSCs). hUC-MSCs were cultured in conditioned medium containing different proportions of OA/RA SF, or stimulated with KYN directly, and then, AHR activation, proliferation, and chondrogenesis of hUC-MSCs were measured. Moreover, the chondroprotective efficiency of short hairpin-AHR-UC-MSC (shAHR-UC-MSC) was determined in a rat surgical OA model (right hind joint). OA SF could activate AHR signaling in hUC-MSCs in a concentration-dependent manner and inhibit the chondrogenic differentiation and proliferation ability of hUC-MSCs. Similar results were observed in hUC-MSCs stimulated with KYN in vitro. Notably, shAHR-UC-MSC exhibited superior therapeutic efficiency in OA rat upon intra-articular injection. Taken together, this study indicates that OA articular microenvironment is not conducive to the therapeutic effect of hUC-MSCs, which is related to the activation of the AHR pathway by tryptophan metabolites, and thus impairs the chondrogenic and chondroprotective effects of hUC-MSCs. AHR might be a promising modification target for further improving the therapeutic efficacy of hUC-MSCs on treatment of cartilage-related diseases such as OA.

Ameliorative effect of the probiotic peptide against benzo(α)pyrene-induced inflammatory damages in enterocytes

Probiotics are living bacteria that provide health benefits to the host when consumed in sufficient quantities. However, the protective effect of the bioactive peptides isolated from the probiotics against benzo(α)pyrene (BaP) induced gastrointestinal injury has never been investigated. The current work used a bio-assay guided technique to identify-four new cyclic peptides in BaP-induced Caco-2 cell culture and mouse colitis model. Lactobacillus rhamnosus cycle (Thr-His-Ala-Trp) peptide-1 (LRCP-1) effectively inhibited BaP-induced epithelial cytokine over-release and intracellular ROS over-production. Simultaneously, LRCP-1 attenuated BaP-induced NAD (P)H: oxidases (NOXs), Matrix metalloproteinases (MMPs) over-expression, respectively. Furthermore, increased NAD (P)H: quinone oxidoreductase 1 (NQO1)/heme oxygenase-1 (HO-1)/nuclear factor E2-related factor 2 (Nrf2) expression and aryl hydrocarbon receptor (AhR) pathway activation induced by the BaP-exposure were also inhibited after the LRCP-1 treatment. Notably, LRCP-1 is a promising agent protecting gastrointestinal epithelial cells from BaP-induced inflammatory and oxidative damages.

Gut Microbiota Regulation of AHR Signaling in Liver Disease.

Liver health plays a vital role in human health and disease. Emerging evidence has shown the importance of the aryl hydrocarbon receptor (AHR) in liver diseases such as alcoholic liver disease, fatty liver disease, and liver failure. As a ligand-activated transcription factor, AHR can be activated by endogenous ligands of microbial metabolites such as tryptophan (Trp), kynurenine (Kyn) or indole derivatives locally or distantly. However, the therapeutic effects of the gut microbiota-regulated AHR pathway remain to be clarified. In this review, we summarize recent progress and examine the role of AHR signaling as a target for gut microbiota intervention in liver diseases. The focus on AHR signaling will identify a promising target in the gut microbiota for better understanding and therapeutic opportunities in liver diseases.

Polychlorinated biphenyl congeners PCB 118 and PCB 126 induce upregulation PD-L1 expression in human thyrocytes through aryl hydrocarbon receptor (AHR) pathways

Polychlorinated biphenyl congeners PCB 118 and PCB 126 induce upregulation PD-L1 expression in human thyrocytes through aryl hydrocarbon receptor (AHR) pathways

Changes in uridine 5'-diphospho-glucuronosyltransferase 1A6 expression by histone deacetylase inhibitor valproic acid.

Valproic acid (VPA) is well-known as a histone deacetylase (HDAC) inhibitor. It has been reported that HDAC inhibitors enhance basal and aryl hydrocarbon receptor (AhR) ligand-induced aryl hydrocarbon receptor-responsive gene expression. Other studies suggested that HDAC inhibition might significantly activate the NF-E2-related factor-2 (Nrf2). Moreover, VPA activates mitogen-activated protein kinases (MAPKs). MAPK pathways regulate Nrf2 transactivation domain activity. Uridine 5'-diphospho-glucuronosyltransferase (UGT) 1A6 is one of the important isoforms to affect drug pharmacokinetics. UGT1A6 gene is regulated transcriptionally by AhR and Nrf2. The present study aimed to investigate whether UGT1A6 expression was changed by VPA and to elucidate the mechanism of the alteration. Following VPA treatment for 72h in Caco-2 cells, UGT1A6 mRNA was increased by 7.9-fold. Moreover, UGT1A6 mRNA was increased by other HDAC inhibitors, suggesting that HDAC inhibition caused the UGT1A6 mRNA induction. AhR and Nrf2 proteins in the nucleus of Caco-2 cells were increased by 1.5- and 1.7-fold, respectively, following the VPA treatment. However, VPA treatment did not activate the extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) pathways in Caco-2 cells. In conclusion, we observed that VPA induced UGT1A6 mRNA expression via AhR and Nrf2 pathways, but not via the ERK or JNK pathways.

Concentration-response gene expression analysis in zebrafish reveals phenotypically-anchored transcriptional responses to retene.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants and are associated with human disease. Canonically, many PAHs induce toxicity via activation of the aryl hydrocarbon receptor (AHR) pathway. While the interaction between PAHs and the AHR is well-established, understanding which AHR-regulated transcriptional effects directly result in observable phenotypes and which are adaptive or benign is important to better understand PAH toxicity. Retene is a frequently detected PAH in environmental sampling and has been associated with AHR2-dependent developmental toxicity in zebrafish, though its mechanism of toxicity has not been fully elucidated. To interrogate transcriptional changes causally associated with retene toxicity, we conducted whole-animal RNA sequencing at 48 h post-fertilization after exposure to eight retene concentrations. We aimed to identify the most sensitive transcriptomic responses and to determine whether this approach could uncover gene sets uniquely differentially expressed at concentrations which induce a phenotype. We identified a concentration-response relationship for differential gene expression in both number of differentially expressed genes (DEGs) and magnitude of expression change. Elevated expression of cyp1a at retene concentrations below the threshold for teratogenicity suggested that while cyp1a expression is a sensitive biomarker of AHR activation, it may be too sensitive to serve as a biomarker of teratogenicity. Genes differentially expressed at only non-teratogenic concentrations were enriched for transforming growth factor-β (TGF-β) signaling pathway disruption while DEGs identified at only teratogenic concentrations were significantly enriched for response to xenobiotic stimulus and reduction-oxidation reaction activity. DEGs which spanned both non-teratogenic and teratogenic concentrations showed similar disrupted biological processes to those unique to teratogenic concentrations, indicating these processes were disrupted at low exposure concentrations. Gene co-expression network analysis identified several gene modules, including those associated with PAHs and AHR2 activation. One, Module 7, was strongly enriched for AHR2-associated genes and contained the strongest responses to retene. Benchmark concentration (BMC) of Module seven genes identified a median BMC of 7.5 µM, nearly the highest retene concentration with no associated teratogenicity, supporting the hypothesis that Module seven genes are largely responsible for retene toxicity.

Microbiota-derived metabolite Indoles induced aryl hydrocarbon receptor activation and inhibited neuroinflammation in APP/PS1 mice

Gut microbiota alterations might affect the development of Alzheimer’s disease (AD) through microbiota-derived metabolites. For example, microbiota-derived Indoles via tryptophan metabolism prevented Aβ accumulation and Tau hyperphosphorylation, restored synaptic plasticity, and then promoted the cognitive and behavioral ability of APP/PS1 mice. The imbalanced compositions of Indoles-producing bacteria with tryptophan deficiency were found in male APP/PS1 mice, but the molecular mechanisms remained unclear. Our current study revealed that Indoles (including indole, indole-3-acetic acid and indole-3-propionic acid) upregulated the production of aryl hydrocarbon receptor (AhR), inhibited the activation of the NF-κB signal pathway as well as the formation of the NLRP3 inflammasome, reduced the release of inflammatory cytokines, including TNF-α, IL-6, IL-1β and IL-18, alleviating the inflammatory response of APP/PS1 mice. These findings demonstrated the roles of Indoles-producing bacteria in activating the AhR pathway to regulate neuroinflammation of AD through gut microbiota-derived Indoles, which implied a novel way for AD treatment.

Aryl Hydrocarbon Receptor (AhR)-Mediated Signaling in iPSC-Derived Human Motor Neurons.

Exposure to environmental pollutants and endogenous metabolites that induce aryl hydrocarbon receptor (AhR) expression has been suggested to affect cognitive development and, particularly in boys, also motor function. As current knowledge is based on epidemiological and animal studies, in vitro models are needed to better understand the effects of these compounds in the human nervous system at the molecular level. Here, we investigated expression of AhR pathway components and how they are regulated by AhR ligands in human motor neurons. Motor neurons generated from human induced pluripotent stem cells (hiPSCs) were characterized at the molecular level and by electrophysiology. mRNA levels of AhR target genes, CYP1A1 and CYP1B1 (cytochromes P450 1A1/1B1), and AhR signaling components were monitored in hiPSCs and in differentiated neurons following treatment with AhR ligands, 2,3,7,8,-tetrachlodibenzo-p-dioxin (TCDD), L-kynurenine (L-Kyn), and kynurenic acid (KA), by RT-qPCR. Changes in AhR cellular localization and CYP1A1 activity in neurons treated with AhR ligands were also assessed. The neurons we generated express motor neuron-specific markers and are functional. Transcript levels of CYP1B1, AhR nuclear translocators (ARNT1 and ARNT2) and the AhR repressor (AhRR) change with neuronal differentiation, being significantly higher in neurons than hiPSCs. In contrast, CYP1A1 and AhR transcript levels are slightly lower in neurons than in hiPSCs. The response to TCDD treatment differs in hiPSCs and neurons, with only the latter showing significant CYP1A1 up-regulation. In contrast, TCDD slightly up-regulates CYP1B1 mRNA in hiPSCs, but downregulates it in neurons. Comparison of the effects of different AhR ligands on AhR and some of its target genes in neurons shows that L-Kyn and KA, but not TCDD, regulate AhR expression and differently affect CYP1A1 and CYP1B1 expression. Finally, although TCDD does not significantly affect AhR transcript levels, it induces AhR protein translocation to the nucleus and increases CYP1A1 activity. This is in contrast to L-Kyn and KA, which either do not affect or reduce, respectively, CYP1A1 activity. Expression of components of the AhR signaling pathway are regulated with neuronal differentiation and are differently affected by TCDD, suggesting that pluripotent stem cells might be less sensitive to this toxin than neurons. Crucially, AhR signaling is affected differently by TCDD and other AhR ligands in human motor neurons, suggesting that they can provide a valuable tool for assessing the impact of environmental pollutants.

Combinatory Exposure to Urolithin A, Alternariol, and Deoxynivalenol Affects Colon Cancer Metabolism and Epithelial Barrier Integrity in vitro

The human gastrointestinal tract is an important site of nutrient absorption and a crucial barrier against xenobiotics. It regularly faces “chemical cocktails” composed of food constituents, their human and microbial metabolites, and foodborne contaminants, such as mycotoxins. Hence, the colonic epithelium adapts to dietary molecules tuning its immune response, structural integrity, and metabolism to maintain intestinal homeostasis. While gut microbiota metabolites of berry ellagitannins, such as urolithin A (Uro A) might contribute to physiological epithelial barrier integrity, foodborne co-contaminating mycotoxins like alternariol (AOH) and deoxynivalenol (DON) could hamper epithelial function. Hence, we investigated the response of differentiated Caco-2 cells (clone C2BBe1) in vitro to the three compounds alone or in binary mixtures. In virtue of the possible interactions of Uro A, AOH, and DON with the aryl hydrocarbon receptor (AhR) pathway, potential effects on phase-I-metabolism enzymes and epithelial structural integrity were taken as endpoints for the evaluation. Finally, Liquid chromatography tandem mass spectrometry measurements elucidated the absorption, secretion, and metabolic capacity of the cells under single and combinatory exposure scenarios. Uro A and AOH as single compounds, and as a binary mixture, were capable to induce CYP1A1/1A2/1B1 enzymes triggered by the AhR pathway. In light of its ribosome inhibiting capacity, the trichothecene suppressed the effects of both dibenzo-α-pyrones. In turn, cellular responsiveness to Uro A and AOH could be sustained when co-exposed to DON-3-sulfate, instead of DON. Colonic epithelial structural integrity was rather maintained after incubation with Uro A and AOH: this was reinforced in the combinatory exposure scenario and disrupted by DON, an effect, opposed in combination. Passage through the cells as well as the metabolism of Uro A and AOH were rather influenced by co-exposure to DON, than by interaction with each other. Therefore, we conclude that although single foodborne bioactive substances individually could either support or disrupt the epithelial structure and metabolic capacity of colon cancer, exposure to chemical mixtures changes the experimental outcome and calls for the need of combinatory investigations for proper risk assessment.

P438: ARYL HYDROCARBON RECEPTOR SIGNALLING IN NORMAL HAEMATOPOIETIC (HSC) AND LEUKEMIA STEM CELLS (LSC): DIFFERENTIAL EFFECT OF AHR ANTAGONIST ON LSC COMPARED TO HSC

Background: Acute myeloid leukaemia (AML) is a devastating disease with poor prognosis. It is driven by leukemia stem cells (LSCs), which are able to survive treatment and drive relapse. This highlights the importance of characterizing LSCs. LSC-targeted treatment strategies are limited because there are no known surface markers that are specific for LSCs in every leukaemia. Targeting a signalling pathway that is altered in all LSCs could be a novel, promising treatment strategy. The aryl hydrocarbon receptor (AHR) pathway was shown to be critical for the self-renewal properties of normal haematopoietic stem cells (HSCs). Aims: We aimed to study the consequences of manipulating the AHR pathway in vivo in a murine leukaemia model. Methods: We used the following methods: murine bone marrow transplantation leukemia model (MBMTLM) - CALM-AF10 with limiting dilution assay (LDA); competitive repopulation assay (CRA) with LDA (CD45.2 donors into CD45.1 recipient mice), transcriptome analysis (sequencing with the Illumina platform and analisys with the DESeq2). Treatment of the cells was performed at various experiments with the AHR agonist, ITE, and an AHR antagonist, CH-223191 (a vehicle control is DMSO). Results: We used a murine retroviral transduction bone marrow transplantation leukemia model (MBMTLM)) with the CALM-AF10 minimal fusion gene (CAMF) as the oncogenic driver, to study the effect of AHR agonist (ITE) and an AHR antagonist (CH-223191) treatment on LSCs. Interestingly, both AHR agonist and antagonist treatment affected LSC frequency and survival of mice transplanted with treated cells. The LSC frequency as measured by limiting dilution transplantation assays (LDA) was 3.7 times lower after agonist treatment (1:163, blue survival curve in fig. 1, A) compared to a vehicle control (1:44, DMSO, black survival curve)). Very interestingly, after antagonist treatment the LSC frequency was so low that could not be measured as none of the mice transplanted with antagonist-treated cells in the LDAs developed leukaemia (n=9, CH-223191 (red curve in fig. 1, A)). Of note, our CAMF MBMTLM has a very high LSC frequency of about 1:4 in cells that were not cultured after thawing. We performed RNA-Seq on 90 samples to determine changes in gene expression patterns after treatment of healthy bone marrow cells and CAMF leukemia cells with the AHR agonist and antagonist. Preliminary differential expression analysis with DESeq2 revealed differential expression of genes involved in apoptosis and proliferation in leukaemia cells treated with CH-223191. As leukemia did not develop in recipients of cells treated with the antagonist CH-223191, genes differentially regulated in these cells might be critical and specific to LSC function and will be candidate targets for LSC-specific treatments. To confirm that AHR antagonist treatment affects LSCs differently than normal HCS, we performed competitive repopulation assay (CRA). Our preliminary data (n = 12) show that treatment of HSCs with CH-223191 results in a steady increase in the engraftment over a 47-week period following transplantation (coral bars, fig. 1, B), as opposed to the grafts from the ITE-treated arm (blue bars), vehicle or untreated HSCs (black and green bars, respectively). Image:Summary/Conclusion: In summary, our data show that the LSCs react differently than normal HSCs to changes in AHR signalling. These differences suggest it might be possible to specifically inhibit LSCs while sparing normal HSCs, thus paving the way to develop strategies to treat leukemia based on manipulating AHR signalling.

Aryl hydrocarbon receptor activation-mediated vascular toxicity of ambient fine particulate matter: contribution of polycyclic aromatic hydrocarbons and osteopontin as a biomarker

BackgroundExposure to ambient fine particulate matter (PM2.5) is associated with vascular diseases. Polycyclic aromatic hydrocarbons (PAHs) in PM2.5 are highly hazardous; however, the contribution of PM2.5-bound PAHs to PM2.5-associated vascular diseases remains unclear. The ToxCast high-throughput in vitro screening database indicates that some PM2.5-bound PAHs activate the aryl hydrocarbon receptor (AhR). The present study investigated whether the AhR pathway is involved in the mechanism of PM2.5-induced vascular toxicity, identified the PAH in PM2.5 that was the major contributor of AhR activation, and identified a biomarker for vascular toxicity of PM2.5-bound PAHs.ResultsTreatment of vascular smooth muscle cells (VMSCs) with an AhR antagonist inhibited the PM2.5-induced increase in the cell migration ability; NF-κB activity; and expression of cytochrome P450 1A1 (CYP1A1), 1B1 (CYP1B1), interleukin-6 (IL-6), and osteopontin (OPN). Most PM2.5-bound PAHs were extracted into the organic fraction, which drastically enhanced VSMC migration and increased mRNA levels of CYP1A1, CYP1B1, IL-6, and OPN. However, the inorganic fraction of PM2.5 moderately enhanced VSMC migration and only increased IL-6 mRNA levels. PM2.5 increased IL-6 secretion through NF-κB activation; however, PM2.5 and its organic extract increased OPN secretion in a CYP1B1-dependent manner. Inhibiting CYP1B1 activity and silencing OPN expression prevented the increase in VSMC migration ability caused by PM2.5 and its organic extract. The AhR activation potencies of seven PM2.5-bound PAHs, reported in the ToxCast database, were strongly correlated with their capabilities of enhancing the migration ability of VSMCs. Benzo(k)fluoranthene (BkF) contributed the most to the AhR agonistic activity of ambient PM2.5-bound PAHs. The association between PM2.5-induced vascular toxicity, AhR activity, and OPN secretion was further verified in mice; PM2.5-induced intimal hyperplasia in pulmonary small arteries and OPN secretion were alleviated in mice with low AhR affinity. Finally, urinary concentrations of 1-hydroxypyrene, a major PAH metabolite, were positively correlated with plasma OPN levels in healthy humans.ConclusionsThe present study offers in vitro, animal, and human evidences supporting the importance of AhR activation for PM2.5-induced vascular toxicities and that BkF was the major contributor of AhR activation. OPN is an AhR-dependent biomarker of PM2.5-induced vascular toxicity. The AhR activation potency may be applied in the risk assessment of vascular toxicity in PAH mixtures.

Staphylococcus epidermidis WF2R11 Suppresses PM2.5-Mediated Activation of the Aryl Hydrocarbon Receptor in HaCaT Keratinocytes

The skin supports a diverse microbiome whose imbalance is related to skin inflammation and diseases. Exposure to fine particulate matter (PM2.5), a major air pollutant, can adversely affect the skin microbiota equilibrium. In this study, the effect and mechanism of PM2.5 exposure in HaCaT keratinocytes were investigated. PM2.5 stimulated the aryl hydrocarbon receptor (AhR) to produce reactive oxygen species (ROS) in HaCaT cells, leading to mitochondrial dysfunction and intrinsic mitochondrial apoptosis. We observed that the culture medium derived from a particular skin microbe, Staphylococcus epidermidis WF2R11, remarkably reduced oxidative stress in HaCaT cells caused by PM2.5-mediated activation of the AhR pathway. Staphylococcus epidermidis WF2R11 also exhibited inhibition of ROS-induced inflammatory cytokine secretion. Herein, we demonstrated that S. epidermidis WF2R11 could act as a suppressor of AhRs, affect cell proliferation, and inhibit apoptosis. Our results highlight the importance of the clinical application of skin microbiome interventions in the treatment of inflammatory skin diseases.

Nanovesicles From Lactobacillus johnsonii N6.2 Reduce Apoptosis in Human Beta Cells by Promoting AHR Translocation and IL10 Secretion.

L. johnsonii N6.2 releases nano-sized vesicles (NVs) with distinct protein and lipid contents. We hypothesized that these NVs play a central role in the delivery of bioactive molecules that may act as mechanistic effectors in immune modulation. In this report, we observed that addition of NVs to the human pancreatic cell line βlox5 reduced cytokine-induced apoptosis. Through RNAseq analyses, increased expression of CYP1A1, CYP1B1, AHRR, and TIPARP genes in the aryl hydrocarbon receptor (AHR) pathways were found to be significantly induced in presence of NVs. AHR nuclear translocation was confirmed by confocal microscopy. The role of NVs on beta cell function was further evaluated using primary human pancreatic islets. It was found that NVs significantly increased insulin secretion in presence of high glucose concentrations. These increases positively correlated with increased GLUT6 and SREBF1 mRNA and coincided with reduced oxidative stress markers. Furthermore, incubation of NVs with THP-1 macrophages promoted the M2 tolerogenic phenotype through STAT3 activation, expression of AHR-dependent genes and secretion of IL10. Altogether, our findings indicate that bacterial NVs have the potential to modulate glucose homeostasis in the host by directly affecting insulin secretion by islets and through the induction of a tolerogenic immune phenotype.

The involvement of CYP1A2 in biodegradation of dioxins in pigs.

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is one of the most harmful chemicals showing resistance to biodegradation. The majority of TCDD effects is mediated by the aryl hydrocarbon receptor (AhR) pathway. TCDD binding to AhR results in the activation of cytochrome P450 enzymes (CYP1A1, CYP1A2, CYP1B1) involved in dioxin biodegradation. The goal of the study was to explore the potential role of CYP1A2 in the metabolism of TCDD. We investigated a molecular structure of CYP1A2 and the binding selectivity and affinity between the pig CYP1A2 and: 1/ DiCDD or TCDD (dioxins differing in toxicity and biodegradability) or 2/ their selected metabolites. pCYP1A2 demonstrated higher affinity towards DiCDD and TCDD than other pCYP1 enzymes. All dioxin-pCYP1A2 complexes were found to be stabilized by hydrophobic interactions. The calculated distances between the heme oxygen and the dioxin carbon nearest to the oxygen, reflecting the hydroxylating potential of CYP1A2, were higher than in other pCYP1 enzymes. The distances between the heme iron and the nearest dioxin carbon exceeded 5 Å, a distance sufficient to allow the metabolites to leave the active site. However, the molecular dynamics simulations revealed that two access channels of CYP1A2 were closed upon binding the majority of the examined dioxins. Moreover, the binding of dioxin metabolites did not promote opening of channel S–an exit for hydroxylated products. It appears that the undesired changes in the behavior of access channels prevail over the hydroxylating potential of CYP1A2 towards TCDD and the favorable distances, ultimately trapping the metabolites at the enzyme’s active site.

Time-course of host cell transcription during the HTLV-1 transcriptional burst.

The human T-cell leukemia virus type 1 (HTLV-1) transactivator protein Tax has pleiotropic functions in the host cell affecting cell-cycle regulation, DNA damage response pathways and apoptosis. These actions of Tax have been implicated in the persistence and pathogenesis of HTLV-1-infected cells. It is now known that tax expression occurs in transcriptional bursts of the proviral plus-strand, but the effects of the burst on host transcription are not fully understood. We carried out RNA sequencing of two naturally-infected T-cell clones transduced with a Tax-responsive Timer protein, which undergoes a time-dependent shift in fluorescence emission, to study transcriptional changes during successive phases of the HTLV-1 plus-strand burst. We found that the transcriptional regulation of genes involved in the NF-κB pathway, cell-cycle regulation, DNA damage response and apoptosis inhibition were immediate effects accompanying the plus-strand burst, and are limited to the duration of the burst. The results distinguish between the immediate and delayed effects of HTLV-1 reactivation on host transcription, and between clone-specific effects and those observed in both clones. The major transcriptional changes in the infected host T-cells observed here, including NF-κB, are transient, suggesting that these pathways are not persistently activated at high levels in HTLV-1-infected cells. The two clones diverged strongly in their expression of genes regulating the cell cycle. Up-regulation of senescence markers was a delayed effect of the proviral plus-strand burst and the up-regulation of some pro-apoptotic genes outlasted the burst. We found that activation of the aryl hydrocarbon receptor (AhR) pathway enhanced and prolonged the proviral burst, but did not increase the rate of reactivation. Our results also suggest that sustained plus-strand expression is detrimental to the survival of infected cells.

Polygonum tinctorium leaves suppress sodium dextran sulfate-induced colitis through interleukin-10-related pathway

Indigo naturalis, a herbal medicine purified from indigo-containing plants, such as Strobilanthes cusia, Isatis tinctoria, and Polygonum tinctorium, has been reported to be useful in the treatment of ulcerative colitis by activating the aryl hydrocarbon receptor. However, the aryl hydrocarbon receptor pathway causes crucial side effects, such as pulmonary arterial hypertension. Although P. tinctorium is one of the plant derivatives of indigo naturalis, it is not identical to it. To date, the pure leaves of P. tinctorium have not been reported to ameliorate ulcerative colitis. Therefore, we investigated the effect of pure P. tinctorium leaves, which are consumed in some regions, on experimental colitis induced in mice using sodium dextran sulfate. We found that P. tinctorium leaves ameliorated weight loss (P < 0.01) and pathological inflammatory changes in the colon (P < 0.05), enhanced mRNA expression of interleukin-10 (P < 0.05), and decreased expression of tumor necrosis factor-in colonic tissues (P < 0.05), as determined using quantitative real-time reverse transcription polymerase chain reaction. The intraperitoneal administration of an aryl hydrocarbon receptor antagonist did not antagonize the inhibition of mucosal destruction, whereas an anti-interleukin-10 receptor antibody did. These results suggest that P. tinctorium ameliorate sodium dextran sulfate-induced intestinal inflammation via interleukin-10-related pathway, independent of the aryl hydrocarbon receptor pathway. P. tinctorium leaves have the potential to be a new, safe treatment for ulcerative colitis.

Aryl hydrocarbon Receptor (AhR) Promotes Cell Growth, Induces Stemness Like Characteristics and Metastasis in Ovarian Cancer Cells via Activation of Akt, β‐Catenin and EMT

BackgroundOvarian cancer is the most lethal form of all gynecological malignancies with highpitched tumor relapse and chemoresistance. Cancer stem cells (CSCs), a small sub population of cancer cells driving tumor initiation, progression, and metastasis, are known to be a major contributor of therapy resistance. Recent studies have depicted a link between CSC development and exposure to environmental pollutants like 2,3,7,8‐Tetrachlorodibenzo‐p‐dioxin (TCDD). Reports suggest that one of the major pathways that mediate the metabolism of these environmental chemicals is the aryl hydrocarbon receptor (AhR) pathway, whose role in several CSCs is lately demonstrated. However, its role in ovarian cancer development and chemoresistance remains uninvestigated. We hypothesize that AhR and its regulatory genes CYP1A1 and CYP1B1 are involved in the ovarian cancer progression and chemoresistance. The objectives of the present study were a) to examine the role and involvement of AhR in ovarian cancer progression and chemoresistance and b) to explore the underlying mechanisms.MethodsTo test our hypothesis, seven ovarian cancer cell lines; A2780, A2780cis (cisplatin resistant), OVCAR‐3, SKOV‐3, CAOV‐3, and SW‐626 were utilized in this study. Ovarian cancer cells were treated for 24 h with a potent AhR activator, tetrachlorodibenzo‐p‐dioxin (TCDD, 10 nM), thereafter protein expression of AhR, CYP1A1, CYP1B1, pro‐ and anti‐apoptotic, and stemness markers were determined by immunoblotting.Resultsour study showed that SKOV‐3 cells showed highest basal expression of AhR, whereas A2780cis and A2780 expressed the highest basal expression of CYP1A1 and CYP1B1 proteins, respectively. At the inducible levels, treatment of selected cells (OVCAR‐3, SKOV‐3 and A2780) with TCDD significantly induced the protein level of AhR, CYP1A1 in OVCAR‐3 and SKOV‐3, and CYP1B1 in only SKOV‐3 and A2780 cells. To investigate the mechanism involved, we utilized A2780 cells as a model to explore the effect of TCDD on pro‐ and anti‐apototic, Epithelial to Mesenchymal Transition (EMT), and stemness markers. Our results showed that activation of AhR by TCDD in A2780 cells induced phosphorylation of Akt protein with a concomitant increase in the expression of anti‐apoptotic proteins BCL‐2, BCL‐xl and MCL‐1. In addition, a significant increase of aldehyde dehydrogenase (ALDH1), a functional CSC‐specific marker exhibiting important role in cancer progression and chemoresistance was also observed. This effect was also associated with an accumulation of βCatenin, a Wnt transcription factor. Moreover, we found a decrease in E‐cadherin, an epithelial marker, followed by a concurrent increase in Vimentin, a mesenchymal marker, upon AhR activation, confirming the induction of EMT.ConclusionThe results from our study indicate that AhR regulates ovarian cancer progression, exerts stemness characteristics and induces metastasis via activation of Akt, Wnt/β‐Catenin and EMT. This study provides a better understanding of the regulation of ovarian cancer cells in relation to AhR that could help in devising novel therapeutics for better management of ovarian cancer and prevention.