Induced Aryl Hydrocarbon Receptor Activation Research Articles

PFOS and PFOSA induce oxidative stress-mediated cardiac defects in zebrafish via PPARγ and AHR pathways, respectively

Perfluorooctane sulfonate (PFOS) and its precursor, perfluorooctane sulfonamide (PFOSA), are widespread in the environment. Evidence suggests a strong link between maternal exposure to PFOS/PFOSA and congenital heart diseases in the offspring, but the underlying mechanisms remain unclear. We hypothesized that PFOS and PFOSA induce cardiac defects through the peroxisome proliferator-activated receptor gamma (PPARγ) and aryl hydrocarbon receptor (AHR) pathways, respectively. In this study, we demonstrated that exposing zebrafish embryos to either PFOSA or PFOS caused cardiac malformations and dysfunction. Both PFOS and PFOSA induced reactive oxygen species (ROS) overproduction, mitochondrial damage, and apoptosis in zebrafish larvae hearts. Blockade of PPARγ through either pharmaceutical inhibition or genetic knockdown only attenuated the changes caused by PFOS, but not those elicited by PFOSA. Conversely, inhibition of AHR alleviated the adverse effects induced by PFOSA but not by PFOS. Both PFOSA and PFOS exhibited similar binding affinities to AHR using molecular docking techniques. The varying ability of PFOS and PFOSA to induce AHR activity in zebrafish embryonic hearts can be attributed to their different capabilities for activating PPARγ. In summary, our findings indicate that PFOS and PFOSA induce excessive ROS production in zebrafish larvae via the PPARγ and AHR pathways, respectively. This oxidative stress in turn causes mitochondrial damage and apoptosis, leading to cardiac defects.

Molecular Docking of Natural Compounds for Potential Inhibition of AhR.

The aryl hydrocarbon receptor (AhR) is a highly conserved environmental sensor, historically known for mediating the toxicity of xenobiotics. It is involved in numerous cellular processes such as differentiation, proliferation, immunity, inflammation, homeostasis, and metabolism. It exerts a central role in several conditions such as cancer, inflammation, and aging, acting as a transcription factor belonging to the basic helix-loop-helix/Per-ARNT-Sim (bHLH-PAS) protein family. A key step in the canonical AhR activation is AhR-ARNT heterodimerization followed by the binding to the xenobiotic-responsive elements (XREs). The present work aims to investigate the potential AhR inhibitory activity of selected natural compounds. Due to the absence of a complete structure of human AhRs, a model consisting of the bHLH, the PAS A, and the PAS B domains was constructed. Blind and focused docking simulations revealed the presence of further binding pockets, different from the canonical one presented in the PAS B domain, which could be important for AhR inhibition due to the possibility to impede AhR:ARNT heterodimerization, either preventing conformational changes or masking crucial sites necessary for protein-protein interaction. Two of the compounds retrieved from the docking simulations, i.e., β-carotene and ellagic acid, confirmed their capacity of inhibiting benzo[a]pyrene (BaP)-induced AhR activation in in vitro tests on the human hepatoma cell line HepG2, validating the efficacy of the computational approach.

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.

Activation of AhR-NQO1 Signaling Pathway Protects Against Alcohol-Induced Liver Injury by Improving Redox Balance.

Background & AimsAryl hydrocarbon receptor (AhR) is a liver-enriched xenobiotic receptor that plays important role in detoxification response in liver. This study aimed to investigate how AhR signaling may impact the pathogenesis of alcohol-related liver disease (ALD).MethodsChronic alcohol feeding animal studies were conducted with mouse models of hepatocyte-specific AhR knockout (AhRΔhep) and NAD(P)H quinone dehydrogenase 1 (NQO1) overexpression, and dietary supplementation of the AhR ligand indole-3-carbinol. Cell studies were conducted to define the causal role of AhR and NQO1 in regulation of redox balance and apoptosis.ResultsChronic alcohol consumption induced AhR activation and nuclear enrichment of NQO1 in hepatocytes of both alcoholic hepatitis patients and ALD mice. AhR deficiency exacerbated alcohol-induced liver injury, along with reduction of NQO1. Consistently, in vitro studies demonstrated that NQO1 expression was dependent on AhR. However, alcohol-induced NQO1 nuclear translocation was triggered by decreased cellular oxidized nicotinamide adenine dinucleotide (NAD+)-to-NADH ratio, rather than by AhR activation. Furthermore, both in vitro and in vivo overexpression NQO1 prevented alcohol-induced hepatic NAD+ depletion, thereby enhancing activities of NAD+-dependent enzymes and reversing alcohol-induced liver injury. In addition, therapeutic targeting of AhR in the liver with dietary indole-3-carbinol supplementation efficiently reversed alcoholic liver injury by AhR-NQO1 signaling activation.ConclusionsThis study demonstrated that AhR activation is a protective response to counteract alcohol-induced hepatic NAD+ depletion through induction of NQO1, and targeting the hepatic AhR-NQO1 pathway may serve as a novel therapeutic approach for ALD.

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.

6-Formylindolo(3,2-b)carbazole induced aryl hydrocarbon receptor activation prevents intestinal barrier dysfunction through regulation of claudin-2 expression

6-Formylindolo(3,2-b)carbazole (FICZ), a high-affinity aryl hydrocarbon receptor (AhR) ligand, plays a protective role in inflammatory bowel disease (IBD) through activation of AhR. Interleukin-6 (IL-6) induced intestinal epithelial barrier dysfunction is involved in the pathological process of IBD. In this study, we investigated the protective effects of FICZ on IL-6 induced intestinal epithelial barrier injury. Our data show that AhR activation by FICZ ameliorated colonic inflammation, decreased IL-6 and claudin-2 expression, and maintained intestinal barrier function in a mouse model of dextran sulphate sodium (DSS)-induced colitis. In Caco-2 and T84 intestinal epithelial cells, FICZ also prevented the increase of intestinal epithelial permeability and claudin-2 expression induced by IL-6. Depletion of AhR expression by small interfering (si)RNA reversed FICZ induced decrease of claudin-2. Furthermore, IL-6 induced upregulation of claudin-2 was required for increased caudal-related homeobox 2 (CDX-2) and hepatocyte-nuclear factor (HNF)-1α. However, FICZ repressed the increase of CDX-2 and HNF-1α expression induced by IL-6. These results reveal the protective effects of FICZ on IL-6 induced disruption of intestinal epithelial barrier function through suppressing the expression of claudin-2. In addition, CDX-2 and HNF-1α are involved in the regulation of claudin-2 after IL-6 and FICZ treatment. Therefore compounds related to AhR ligands may be potential pharmaceutical agents to treat IBD.

Malassezia pachydermatis up-regulates AhR related CYP1A1 gene and epidermal barrier markers in human keratinocytes.

Cytochrome P450 CYP1A1 and CYP1B1 enzymes are regulated by the aryl hydrocarbon receptor (AhR), a transcription factor activated by a variety of ligands among which Malassezia metabolites. In this study, we analyzed the modulation of CYP1A1, CYP1B1, and AhR in human keratinocytes infected with different strains of Malassezia pachydermatis, as well as the upregulation of some genes involved in the epidermal homeostasis. We demonstrated that all the strains induced AhR activation and its nuclear translocation in HaCaT cells infected for 24h, compared to untreated cells. The expression of CYP1A1 and CYP1B1, prototypical markers of the AhR signaling pathway, were upregulated with the level of CYP1A1 mRNA approximately 100-fold greater than that for CYP1B1. Filaggrin, involucrin, and TGaseI, proteins involved in epidermal differentiation, were all modulated by Malassezia pachydermatis strains, with the strongest induction observed for filaggrin. By contrast, quinone oxidoreductase 1 (NQO1), which is part of the antioxidant defense system involved in detoxification, was not modulated in our experimental model. In conclusions, our findings suggest that Malassezia pachydermatis infection of human keratinocytes induces activation of the AhR, and increases the expression of its responsive genes and markers of epidermal differentiation, paving the way for occurrence/exacerbation of pathological skin conditions.

Aryl Hydrocarbon Receptor Activation Modulates Intestinal Epithelial Barrier Function by Maintaining Tight Junction Integrity.

Activation of Aryl hydrocarbon receptor (AhR) is involved in the control of intestinal mucosal homeostasis. Intestinal barrier dysfunction contributes to the development of many intestinal diseases, such as inflammatory bowel disease (IBD). In this study, we investigated the mechanisms of AhR activation in the maintenance of intestinal barrier function. Adult C57BL/6 mice were treated with dextran sulphate sodium (DSS) for 7 days, with or without 6-Formylindolo(3,2-b)carbazole (FICZ), a ligand of AhR. We found that AhR activation by FICZ attenuated the decreased TJ protein expression in the colonic mucosa of the DSS-induced mice. Further, the increase of both MLC phosphorylation and MLCK expression in the mice with DSS-induced colitis was also significantly inhibited by FICZ induced AhR activation. For in vitro experiments, Caco-2 cells were treated with tumour necrosis factor alpha (TNF-α)/interferon gamma (IFN-γ) for 48 h, with or without FICZ. AhR activation prevented TNF-α/IFN-γ-induced decrease in TER and morphological disruption of the TJs in Caco-2 monolayers. It also inhibited TNF-α/IFN-γ-induced increase in MLCK expression and MLC phosphorylation by suppression of NF-κB p65 signaling pathway. Thus, AhR-activating factors might have potential as therapeutic agents for the treatment of patients with IBD.

Effects of the aryl hydrocarbon receptor agonist 3-methylcholanthrene on the 17β-estradiol regulated mRNA transcriptome of the rat uterus

Polycyclic aromatic hydrocarbons (PAHs) are products of incomplete combustion of organic compounds, abundant in exhaust fumes and cigarette smoke. They act by binding to the aryl hydrocarbon receptor (AHR) which induces expression of phase 1 and phase 2 enzymes in the liver. PAH induced AHR activation may also lead to adverse effects by modulating other pathways, for example estrogen receptor (ER) signaling in the female reproductive tract. We have investigated the effects of the PAH 3-methylcholanthrene (3-MC) on 17β-estradiol (E2) dependent signaling in the uterus of ovariectomized rats to characterize the cross talk between AHR and ER on an mRNA transcriptome wide scale. A standard three day uterotrophic assay was performed in young adult Lewis rats. Treatment induced effects were analyzed using histology, immunohistochemistry and gene expression analysis by microarray and qPCR. 3-MC shows broad E2 antagonistic effects on uterine mRNA transcription of the vast majority of E2 regulated genes, significantly altering prostaglandin biosynthesis, complement activation, coagulation pathways and other inflammatory response pathways. The regulation of ER expression in the uterus, but not the regulation of E2 metabolism in the liver, was identified as a potentially important factor in mediating this general antiestrogenic effect. The regulation of prostaglandin biosynthesis by E2 is important for inflammation-like events during pregnancy including the initiation of birth. Our results suggest that adverse effects of PAHs on prostaglandin related pathways are likely caused by the interference with E2 signaling, specifically by inhibiting the E2 mediated downregulation of PGF2α. Characterization of the generalized antagonistic effect of 3-MC on E2 dependent signaling in the rat uterus thus contributes to a better understanding of molecular mechanisms of the toxicity of PAHs in female reproductive organs.

Detection of Aryl Hydrocarbon Receptor Activation by Some Chemicals in Food Using a Reporter Gene Assay.

The purpose of this study was to examine whether a simple bioassay used for the detection of dioxins (DXNs) could be applied to detect trace amounts of harmful DXN-like substances in food products. To identify substances with possible DXN-like activity, we assessed the ability of various compounds in the environment to bind the aryl hydrocarbon receptor (AhR) that binds specifically to DXNs. The compounds tested included 19 polycyclic aromatic hydrocarbons (PAHs), 20 PAH derivatives (nitrated, halogenated, and aminated derivatives), 23 pesticides, six amino acids, and eight amino acid metabolites. The AhR binding activities (AhR activity) of these compounds were measured using the chemical activated luciferase gene expression (CALUX) reporter gene assay system. The majority of the PAHs exhibited marked AhR activity that increased in a concentration-dependent manner. Furthermore, there was a positive link between AhR activity and the number of aromatic rings in the PAH derivatives. Conversely, there appeared to be a negative correlation between AhR activity and the number of chlorine residues present on halogenated PAH derivatives. However, there was no correlation between AhR activity and the number and position of substituents among nitrated and aminated derivatives. Among the pesticides tested, the indole-type compounds carbendazim and thiabendazole showed high levels of activity. Similarly, the indole compound tryptamine was the only amino acid metabolite to induce AhR activity. The results are useful in understanding the identification and characterization of AhR ligands in the CALUX assay.

Activation of aryl hydrocarbon receptor mediates suppression of hypoxia-inducible factor-dependent erythropoietin expression by indoxyl sulfate.

Indoxyl sulfate (IS) is a representative uremic toxin that accumulates in the blood of patients with chronic kidney disease (CKD). In addition to the involvement in the progression of CKD, a recent report indicates that IS suppresses hypoxia-inducible factor (HIF)-dependent erythropoietin (EPO) production, suggesting that IS may also contribute to the progression of renal anemia. In this report, we provide evidence that aryl hydrocarbon receptor (AhR) mediates IS-induced suppression of HIF activation and subsequent EPO production. In HepG2 cells, IS at concentrations similar to the blood levels in CKD patients suppressed hypoxia- or cobalt chloride-induced EPO mRNA expression and transcriptional activation of HIF. IS also induced AhR activation, and AhR blockade resulted in abolishment of IS-induced suppression of HIF activation. The HIF transcription factor is a heterodimeric complex composed of HIF-α subunits (HIF-1α and HIF-2α) and AhR nuclear translocator (ARNT). IS suppressed nuclear accumulation of the HIF-α-ARNT complex accompanied by an increase of the AhR-ARNT complex in the nucleus, implying the involvement of interactions among AhR, HIF-α, and ARNT in the suppression mechanism. In rats, oral administration of indole, a metabolic precursor of IS, inhibited bleeding-induced elevation of renal EPO mRNA expression and plasma EPO concentration and strongly induced AhR activation in the liver and renal cortex tissues. Collectively, this study is the first to elucidate the detailed mechanism by which AhR plays an indispensable role in the suppression of HIF activation by IS. Hence, IS-induced activation of AhR may be a potential therapeutic target for treating renal anemia.

Ex Vivo Induced Regulatory Human/Murine Mesenchymal Stem Cells as Immune Modulators.

Over the past decade there has been a growing interest in using mesenchymal stem cells (MSCs) as an immune-regulatory agent for prevention and treatment of various immune disorders including graft-versus-host disease (GVHD), transplanted organ rejection, and autoimmune diseases. However, the high diversity in the results from clinical trials using MSCs for such disorders emphasizes the need for MSCs to be "professionalized" ex vivo to a more defined regulatory phenotype before administering to patients. To this aim, we have established an ex vivo immunomodulatory triple combination treatment (TCT) for MSCs, using IFNγ, TGFβ, and kynurenine. We show that pretreated MSCs acquire an immunomodulatory phenotype, have improved regulatory functions, and upregulate the expression of inducible nitric oxide synthase, indoleamine 2,3-dioxygenase, cyclooxygenase-2 (COX2), heme oxygenase 1, leukemia inhibitory factor (LIF), and programmed death ligand 1. We define the pathway of kynurenine induced aryl hydrocarbon receptor activation in MSCs and how it contributes to the upregulation of COX2 expression and IL-6 downregulation. The combination of reduced IL-6 secretion with enhanced LIF expression leads to the inhibition of Th17 differentiation in coculture of TCT MSCs and lymphocytes. To test the immunomodulatory function of TCT MSCs in vivo, we used the cells as GVHD prophylaxis in a GVHD mouse model. TCT MSCs administration significantly decreased GVHD score and improved mouse survival. Importantly, single administration could attenuate disease symptoms for more than 3 weeks. Based on these results, we suggest considering TCT MSCs as an improved cell therapy for systemic diseases with an underlying inflammatory and immunologic etiology. Stem Cells 2015;33:2256-2267.

Catechins in tea suppress the activity of cytochrome P450 1A1 through the aryl hydrocarbon receptor activation pathway in rat livers

Polycyclic aromatic hydrocarbons (PAHs) and halogenated aromatic hydrocarbons (HAHs) develop various adverse effects through activation of an aryl hydrocarbon receptor (AhR). The suppressive effects of brewed green tea and black tea on 3-methylcholanthrene (MC)-induced AhR activation and its downstream events were examined in the liver of rats. Ad-libitum drinking of green tea and black tea suppressed MC-induced AhR activation and elevation of ethoxyresorufin O-deethylase activity in the liver, whereas the teas themselves did not induce them. Tea showed a suppressive fashion on the expression of cytochrome P450 1A1 (CYP1A1). Tea suppressed the AhR activation induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) ex vivo. A part of catechins and theaflavins was present in plasma and liver as conjugated and intact forms. The results of this study suggested that active component(s) of tea are incorporated in the liver and suppress the activity of CYP1As through the AhR activation pathway.

Water exposure assessment of aryl hydrocarbon receptor agonists in Three Gorges Reservoir, China using SPMD-based virtual organisms

SPMD-based virtual organisms (VOs) were deployed at five to eight sites in the Three Gorges Reservoir (TGR), China for five periods in 2008, 2009 and 2011. The water exposure of aryl hydrocarbon receptor (AhR) agonists was assessed by the VOs. The chosen bioassay response for the extracts of the VOs, the induction of 7-ethoxyresorufin-O-deethylase (EROD) was assayed using a rat hepatoma cell line (H4IIE). The results show that the extracts from the VOs could induce AhR activity significantly, whereas the chemically derived 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) equivalent (TEQcal) accounted for <11% of the observed AhR responses (TEQbio). Unidentified AhR-active compounds represented a greater proportion of the TCDD equivalent in VOs from TGR. High TEQbio value in diluted extract and low TEQbio in concentrated extract of the same sample was observed suggesting potential non-additive effects in the mixture. The levels of AhR agonists in VOs from upstream TGR were in general higher than those from downstream reservoir, indicating urbanization effect on AhR agonist pollution. The temporal variation showed that levels of AhR agonists in 2009 and 2011 were higher than those in 2008, and the potential non-additive effects in the area close to the dam were also obviously higher in 2009 and 2011 than in 2008, indicating big changes in the composition of pollutants in the area after water level reached a maximum of 175m. Although the aqueous concentration of AhR agonists of 0.8–4.8pgTCDDL−1 in TGR was not alarming, the tendency of accumulating high concentration of AhR agonists in VO lipid and existence of possible synergism or antagonism in the water may exhibit a potential hazard to local biota being exposed to AhR agonists.

Combined chemical and toxicological long-term monitoring for AhR agonists with SPMD-based virtual organisms in drinking water Danjiangkou Reservoir, China

SPMD-based virtual organisms (VOs) were employed for time-integrating, long-term sampling combined biological and chemical analyses for exposure assessment of hydrophobic organic pollutants (HOPs) in a drinking water reservoir, China. The SPMDs were deployed at four and five sites in the Danjiangkou (DJK) reservoir over two periods of 26 and 31d to sequester the hydrophobic contaminants in water. The chosen bioassay response for the extracts of the SPMDs, the induction of 7-ethoxyresorufin-o-deethylase (EROD) was assayed using a rat hepatoma cell line (H4IIE). The known aryl hydrocarbon receptor (AhR) agonists PAHs and PCBs were analyzed by HRGC/HRMS instrument. The cause-effect relationship between the observed AhR activities and chemical concentrations of detected AhR agonists was examined. The results show that the extracts from the SPMD samples could induce AhR activity significantly, whereas the chemically derived 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) equivalent (TEQcal) was not correlated with the bioassay-derived TCDD equivalent (TEQbio). The known AhR agonists could only account for 2–10% of the observed AhR responses among which the contribution of PCBs could almost be neglected. Unidentified AhR-active compounds represented a greater proportion of the TCDD equivalent (TCDD-EQ) in SPMD samples from DJK. Based on the first assessment, the VO followed by the combination of chemical and biological analyses emerges as a resource efficient water monitoring device in ecotoxicological assessment for toxicologically relevant compounds which are readily available for uptake by resident aquatic biota in drinking water resources.

Generation of IL-8 and IL-9 Producing CD4+T Cells Is Affected by Th17 Polarizing Conditions and AHR Ligands

The T helper cell subsets Th1, Th2, Th17, and Treg play an important role in immune cell homeostasis, in host defense, and in immunological disorders. Recently, much attention has been paid to Th17 cells which seem to play an important role in the early phase of the adoptive immune response and autoimmune disease. When generating Th17 cells under in vitro conditions the amount of IL-17A producing cells hardly exceeds 20% while the nature of the remaining T cells is poorly characterized. As engagement of the aryl hydrocarbon receptor (AHR) has also been postulated to modulate the differentiation of T helper cells into Th17 cells with regard to the IL-17A expression we ask how far do Th17 polarizing conditions in combination with ligand induced AHR activation have an effect on the production of other T helper cell cytokines. We found that a high proportion of T helper cells cultured under Th17 polarizing conditions are IL-8 and IL-9 single producing cells and that AHR activation results in an upregulation of IL-8 and a downregulation of IL-9 production. Thus, we have identified IL-8 and IL-9 producing T helper cells which are subject to regulation by the engagement of the AHR.

Aryl Hydrocarbon Receptor Deficient Donor T Cells Display Decreased Migration and Cause Reduced Or Enhanced Graft-Versus-Host Diseases In Different Transplant Models

The transcription factor aryl hydrocarbon receptor (AhR) is a cytosolic sensor of numerous small synthetic compounds (xenobiotics) and natural chemicals. Ligand binding of AhR causes a conformational change of the receptor, allowing its translocation to nucleus to regulate an array of genes. Different ligands could induce different sets of genes. AhR was first discovered as the mediator of dioxin toxicity. Its role in immunity was recently extensively investigated and expanded exponentially. Here we investigated the role of AhR in donor T cell-induced graft-versus-host diseases (GvHD) after bone marrow transplant in two MHC-mismatched models. B6D2 or Balb/c recipients were irradiated with one dose (950 rads for B6D2 and 800 rads for Balb/c) one day before transplant. C57BL/6 WT or AhR deficient (AhR KO) T cells were transplanted with T cell-depleted bone marrow to B6D2 or Balb/c, at a dose of three million T cells or five hundred thousand T cells, respectively. At different time points, recipient lymphoid organs or target organs were harvested and used for flow analysis or ELISA. A group of recipient mice were observed for an extended time to establish GvHD scores and survival curves. In B6 to B6D2 model, recipient mice transplanted with AhR KO T cells displayed lower GvHD scores and survived longer than mice transplanted with WT T cells (Figure 1C). We found that AhR KO donor T cells failed to accumulate in lymph nodes and spleen as rapidly as WT T cells as evident on day 1 and day 3 post-transplant (Figure 1A). Further investigation showed that the proliferation and cell death were similar between WT and AhR donor T cells, suggesting decreased migration of AhR KO T cells to lymphoid organs. It has been shown that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induced AhR activation leads to the up-regulation of CCR4 and CCR5 in T cells, supporting our data that AhR affects cell migration. We also observed that lower percentage of AhR KO T cells produced IFN-g and IL-17 in lymph nodes and spleen early after transplant, but the differences disappeared at later time points (day 7). Colonic homogenates of AhR KO transplants contained less IFN-g, TNF-a, and IL-1a than WT transplants, which indicated that AhR KO T cells caused less inflammation in the gut (Figure 1B). Gut-associated injury due to inflammation is the major cause of death in this model. However, in B6 to Balb/c model, recipient mice transplanted with AhR KO T cells displayed the opposite survival curve. They had diminished survival compared to mice receiving WT T cells with most mice succumbing to GvHD around day 10 to day 15 before the onset of diarrhea. AhR KO T cells in this model still accumulated less in lymph nodes at early time points, which suggested that AhR played a role in cell migration in both models. It was noteworthy that after day 6, the number of AhR KO T cells in the spleen was actually similar to or higher than WT T cells in both models, suggesting cell proliferation was not affected by AhR deficiency. In conclusion, AhR KO T cells demonstrate decreased migration to lymphoid organs after bone marrow transplant compared to WT T cells. This decrease in migration may be the reason why mice transplanted with AhR KO T cells exhibit less inflammation in the gut and subsequently survive better from GvHD in the B6 to B6D2 model. However, mice transplanted with AhR KO T cells have diminished survival in B6 to Balb/c model despite of lower T cell numbers in lymph nodes at early time points. Thus, certain function of AhR in donor T cells is model dependent while other function is conserved among different models. These data suggest that investigators should be cautious regarding the supplementation of AhR ligands to diminish GvHD in clinical studies. Disclosures: No relevant conflicts of interest to declare.

Identification of Ah Receptor Agonists in Soil of E-waste Recycling Sites from Taizhou Area in China

In recent years, increasing concern has surrounded the consequences of improper electric and electronic waste (e-waste) disposal. In order to mitigate or remediate the potentially severe toxic effects of e-waste recycling on the environment, organisms, and humans, many contaminated sites must first be well-characterized. In this study, soil samples were taken from Taizhou city, one of the largest e-waste disposal centers in China, which was involved in recycling for nearly 30 years. The extracts of the samples were assayed for aryl hydrocarbon receptor (AhR)-mediated ethoxyresorufin-O-deethylase (EROD) induction in the rat hepatoma cell line H4IIE. Some of the target AhR agonists, including polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs), polychlorinated biphenyls (PCBs), and polycyclic aromatic hydrocarbons (PAHs), were instrumentally analyzed as well. The cause-effect relationship and dose-response relationship between the chemical concentrations of AhR agonists and observed EROD activity were examined. The results showed that soil extracts could induce AhR activity significantly, and the chemically derived 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) equivalents (TEQcal) were perfectly correlated to bioassay-derived TCDD equivalents (TEQbio; R = 0.96, P < 0.001), which indicated that the known AhR agonists could account for the observed responses. Among different contributors, PCBs accounted for 87.2-98.2% and PCDD/Fs contributed 1.7-11.6% of TEQcal, while the contribution of PAHs could almost be neglected. Under these conditions, a quantitative dose-effect relationship between TEQ(PCB) and EROD activity could be evaluated, suggesting that the observed AhR effect was mainly caused by PCBs. Further source identification by congener profiles analysis showed that the crude dismantling of electric power devices and open burning of electric wires and printed circuit boards may be the main sources of these dioxin-like compounds. This study suggests that the combination of in vitro bioassay and chemical analysis is useful to screen, identify, and prioritize AhR agonists in soil from e-waste recycling areas.