Presence Of Aryl Hydrocarbon Receptor Research Articles

Aryl Hydrocarbon Receptor Regulates Muc2 Production Independently of IL-22 during Colitis.

We previously reported that an aryl hydrocarbon receptor (AhR) ligand, indole-3-carbinol (I3C), was effective at reducing colitis severity through immune cell-mediated interleukin-22 (IL-22) production. Intestinal epithelial cells (IECs) are also involved in regulating colitis, so we investigated their AhR-mediated mechanisms in the current report. A transcriptome analysis of IECs in wildtype (WT) mice revealed that during colitis, I3C regulated select mucin proteins, which could be attributed to goblet cell development. To address this, experiments under in vivo colitis (mice) or in vitro colon organoid conditions were undertaken to determine how select mucin proteins were altered in the absence or presence of AhR in IECs during I3C treatment. Comparing WT to IEC-specific AhR knockout mice (AhRΔIEC), the results showed that AhR expression was essential in IECs for I3C-mediated protection during colitis. AhR-deficiency also impaired mucin protein expression, particularly mucin 2 (Muc2), independently of IL-22. Collectively, this report highlights the important role of AhR in direct regulation of Muc2. These results provide justification for future studies aimed at determining how AhR might regulate select mucins through mechanisms such as direct transcription binding to enhance production.

Immune cell-intrinsic Ah receptor facilitates the expression of antimicrobial REG3G in the small intestine.

The intestinal epithelial layer is susceptible to damage by chemical, physiological and mechanical stress. While it is essential to maintain the integrity of epithelium, the biochemical pathways that contribute to the barrier function have not been completely investigated. Here we demonstrate an aryl hydrocarbon receptor (AHR)-dependent mechanism facilitating the production of the antimicrobial peptide AMP regenerating islet-derived protein 3 gamma (REG3G), which is essential for intestinal homeostasis. Genetic ablation of AHR in mice impairs pSTAT3-mediated REG3G expression and increases bacterial numbers of Segmented filamentous bacteria (SFB) and Akkermansia muciniphila in the small intestine. Studies with tissue-specific conditional knockout mice revealed that the presence of AHR in the epithelial cells of the small intestine is not required for the production of REG3G through the phosphorylated STAT3-mediated pathway. However, immune-cell-specific AHR activity is necessary for normal expression of REG3G in all regions of the small intestine. A diet rich in broccoli, capable of inducing AHR activity, increases REG3G production when compared to a semi-purified diet that is devoid of ligands that can potentially activate the AHR, thus highlighting the importance of AHR in antimicrobial function. Overall, these data suggest that homeostatic antimicrobial REG3G production is increased by an AHR pathway intrinsic to the immune cells in the small intestine.

ARYL HYDROCARBON RECEPTOR REGULATES GENES IN COLONIC EPITHELIAL CELLS TO MITIGATE COLITIS SEVERITY IN THE DSS-INDUCED MURINE MODEL

Abstract Current treatment of ulcerative colitis (UC) leaves a clear unmet need to target the colonic epithelial cells (CECs). In our previous study we found that aryl hydrocarbon receptor (AhR) expression on CECs has a vital role during indole-3-carbinol (I3C)-mediated protection against colitis. However, the precise mechanism behind this remains unclear. We investigated how intestinal regulatory components were altered in the absence or presence of AhR in CECs during I3C treatment under colitis (mouse model) or colitis-like conditions (colonic organoid). We generated conditional AhR-CEC knockout mice (AV) and induced colitis using the dextran sodium sulfate (DSS) model. Our results showed that the AV mice lost the protective effects of I3C against colitis. AV mice had higher disease scores and increased inflammation compared to controls. I3C treated AV mice that had DSS induced colitis were not able to regulate the colitis-associated parameters, even though flow cytometry analysis revealed AV mice were still capable of increasing protective interleukin-22 (IL-22) production in the colon. These results prompted us to study the CEC-specific mechanism involved in I3C-mediated protection against colitis. Whole transcriptome analysis of RNA isolated from enriched CECs of experimental mice was performed and results showed significant altered expression of several microRNAs, mucins (muc2 and muc13), and tight junction proteins (Cld3 and Cld4) in AV mice when compared to controls. RT-PCR data and direct effects of I3C on CECs (immunostaining) using organoids validated these results. This data concluded that AhR-dependent signaling orchestrates I3C-mediated protection of colitis. NIH 5P20GM103641

I3C-MEDIATED PROTECTION AGAINST COLITIS DEPENDENT ON AHR EXPRESSION ON VIL1-EXPRESSING COLONIC EPITHELIAL CELLS

Abstract Many current treatments for ulcerative colitis (UC) often causes side effects warranting safe therapeutic strategies to control the pathogenesis of colitis. Current study was aimed to ascertain the possible role of aryl hydrocarbon receptor (AhR) in vil1-expressing colonic epithelial cells (CECs) during I3C-mediated protection against colitis. We investigated how intestinal regulatory mediators were altered in the absence or presence of AhR in CECs during I3C treatment under colitis or colitis-like conditions. We generated conditional AhR knockout mice in vil1-expressing CECs using the cre-flox system and induced colitis using the dextran sodium sulfate (DSS) model. Results showed that the mice with AhR deficiency in CECs (AV mice) lost the protective effects of I3C treatment during colitis and had a higher disease score with increased inflammation in the colon compared to the controls. Also, after treatment with I3C during DSS-induced colitis, AV mice were not able to prevent colitis-associated gut microbial dysbiosis even though flow cytometry analysis revealed AV mice were still capable of increasing IL-22 production by ILC3s in the colon. IL-22-ILC3 immune cell response was not the only major mechanism involved in I3C-mediated protection against colitis and regulation of the gut microbiome. Transcriptome analysis of RNA isolated from enriched CECs of experimental mice showed significant altered expression of several microRNAs, mucins (muc3 and muc13), and tight junction proteins in AV mice compared to controls. PCR gene expression data and direct effects of I3C on CECs using colonic organoids validated these results. In summary, AhR expression in CECs play a critical role in I3C-mediated prevention of colitis.

AHR EXPRESSION ON VIL1-EXPRESSING COLONIC EPITHELIAL CELLS IS ESSENTIAL FOR I3C-MEDIATED PROTECTION AGAINST COLITIS

Abstract Ulcerative colitis (UC) is a chronic, idiopathic inflammatory disease that affects the colon and is characterized by localized tissue injury and disruption of the intestinal epithelial barrier. Pathophysiology of UC includes genetic predisposition, epithelial barrier defects, dysregulated immune responses, microbial dysbiosis, and environmental factors. Several therapeutic interventions for colitis have been identified, but many current treatments have various negative side effects, which is alarming given the incidence of UC is increasing around the world. Thus a search for safe therapeutic strategies to control the pathogenesis of colitis is still greatly needed. Previous studies from our laboratory reported that indole-3-carbinol (I3C), a natural plant product and ligand for the aryl hydrocarbon receptor (AhR), exhibited protective effects against colitis through innate lymphoid type 3 (ILC3) cells in an interleukin-22 (IL-22) dependent manner. The aim of this study was to ascertain the possible role of AhR in vil1-expressing colonic epithelial cells (CECs) during I3C-mediated protection against colitis. Using in vivo mouse studies and in vitro primary CEC organoid cultures, we investigated how key intestinal regulatory mediators (e.g. mucins and anti-microbial peptides, or AMPs) were altered in the absence or presence of AhR in CECs during I3C treatment under colitis or colitis-like conditions. We generated conditional AhR knockout mice in vil1-expressing CECs using the cre-flox system and induced colitis using the dextran sodium sulfate (DSS) model. Our results showed that the mice with AhR deficiency in CECs (AV mice) lost the protective effects of I3C treatment during colitis. Results showed that AV mice had a higher disease score and increased inflammation in the colon when compared to wild type (WT) or littermate (LM) controls. In addition, after treatment with I3C during DSS-induced colitis, AV mice were not able to prevent colitis-associated gut microbial dysbiosis even though flow cytometry analysis revealed AV mice were still capable of increasing IL-22 production by ILC3s in the colon. This suggested that the IL-22-ILC3 immune cell response was not the only major mechanism involved in I3C-mediated protection against colitis and regulation of the gut microbiome. Whole transcriptome analysis of RNA isolated from enriched CECs of experimental mice was performed and results showed significant altered expression of several microRNAs, mucins (muc3 and muc13), and tight junction proteins in AV mice when compared to wild type (WT) or littermate (LM) controls. PCR gene expression data and direct effects of I3C on CECs using colonic organoids from WT and AV mice validated these results. Collectively, these data showed that AhR expression in CECs play a critical role in I3C-mediated prevention of colitis.

AHR EXPRESSION ON VIL1-EXPRESSING COLONIC EPITHELIAL CELLS IS ESSENTIAL FOR I3C-MEDIATED PROTECTION AGAINST COLITIS

Ulcerative colitis (UC) is a chronic, idiopathic inflammatory disease that affects the colon and is characterized by localized tissue injury and disruption of the intestinal epithelial barrier. Pathophysiology of UC includes genetic predisposition, epithelial barrier defects, dysregulated immune responses, microbial dysbiosis, and environmental factors. Several therapeutic interventions for colitis have been identified, but many current treatments have various negative side effects, which is alarming given the incidence of UC is increasing around the world. Thus a search for safe therapeutic strategies to control the pathogenesis of colitis is still greatly needed. Previous studies from our laboratory reported that indole-3-carbinol (I3C), a natural plant product and ligand for the aryl hydrocarbon receptor (AhR), exhibited protective effects against colitis through innate lymphoid type 3 (ILC3) cells in an interleukin-22 (IL-22) dependent manner. The aim of this study was to ascertain the possible role of AhR in vil1-expressing colonic epithelial cells (CECs) during I3C-mediated protection against colitis. Using in vivo mouse studies and in vitro primary CEC organoid cultures, we investigated how key intestinal regulatory mediators (e.g. mucins and anti-microbial peptides, or AMPs) were altered in the absence or presence of AhR in CECs during I3C treatment under colitis or colitis-like conditions. We generated conditional AhR knockout mice in vil1-expressing CECs using the cre-flox system and induced colitis using the dextran sodium sulfate (DSS) model. Our results showed that the mice with AhR deficiency in CECs (AV mice) lost the protective effects of I3C treatment during colitis. Results showed that AV mice had a higher disease score and increased inflammation in the colon when compared to wild type (WT) or littermate (LM) controls. In addition, after treatment with I3C during DSS-induced colitis, AV mice were not able to prevent colitis-associated gut microbial dysbiosis even though flow cytometry analysis revealed AV mice were still capable of increasing IL-22 production by ILC3s in the colon. This suggested that the IL-22-ILC3 immune cell response was not the only major mechanism involved in I3C-mediated protection against colitis and regulation of the gut microbiome. Whole transcriptome analysis of RNA isolated from enriched CECs of experimental mice was performed and results showed significant altered expression of several microRNAs, mucins (muc3 and muc13), and tight junction proteins in AV mice when compared to wild type (WT) or littermate (LM) controls. PCR gene expression data and direct effects of I3C on CECs using colonic organoids from WT and AV mice validated these results. Collectively, these data showed that AhR expression in CECs play a critical role in I3C-mediated prevention of colitis.

Glass-bottled drinking water: a time capsule to study the historic presence of hazardous chemicals using effect-based methods

BackgroundContamination of drinking water by hazardous chemicals can be associated with human health risks. Recent studies using effect-based in vitro methods have demonstrated that a large part of the observed toxic effects are caused by unknown chemicals. In this study, we have used a panel of effect-based methods to study the presence of chemical contaminants in a unique material; glass-bottled Swedish tap water collected during the 1990s. These water samples were compared to drinking water from the same source waters and drinking water facilities, yet collected about 25 years later, in 2020.ResultsSamples were concentrated by solid phase extraction and evaluated for the following activities; estrogen receptor activity, androgen receptor activity, antiandrogenic activity, aryl hydrocarbon receptor activity, and oxidative stress response. We observed aryl hydrocarbon receptor activities in almost all studied samples and estrogen receptor activity in three out of ten studied samples. No activities were observed for androgen receptor activity, antiandrogenic activity or oxidative stress response. In general, observed activities were more frequent and higher in the water samples collected during the 1990s as compared to the corresponding samples collected in 2020.ConclusionsThis study demonstrates that it is possible to conduct an effect-based evaluation of the presence of hazardous chemicals in drinking water, with as small starting volume as 330 mL, by using miniaturized bioassays. Further, by comparing the glass-bottled water samples with newly collected water samples from the same drinking water treatment facilities, our results indicate that the presence of aryl hydrocarbon receptor and estrogen receptor activating compounds in the drinking water has decreased over the approximately quarter of a century that is separating the two sampling occasions. This difference could be due to improved raw water quality and/or improved treatment efficiency in the treatment plants.

The Aryl Hydrocarbon Receptor Attenuates Acute Cigarette Smoke-Induced Airway Neutrophilia Independent of the Dioxin Response Element.

Cigarette smoke is a prevalent respiratory toxicant that remains a leading cause of death worldwide. Cigarette smoke induces inflammation in the lungs and airways that contributes to the development of diseases such as lung cancer and chronic obstructive pulmonary disease (COPD). Due to the presence of aryl hydrocarbon receptor (AhR) ligands in cigarette smoke, activation of the AhR has been implicated in driving this inflammatory response. However, we have previously shown that the AhR suppresses cigarette smoke-induced pulmonary inflammation, but the mechanism by which the AhR achieves its anti-inflammatory function is unknown. In this study, we use the AhR antagonist CH-223191 to inhibit AhR activity in mice. After an acute (3-day) cigarette smoke exposure, AhR inhibition was associated with significantly enhanced neutrophilia in the airways in response to cigarette smoke, mimicking the phenotype of AhR-deficient mice. We then used genetically-modified mouse strains which express an AhR that can bind ligand but either cannot translocate to the nucleus or bind its cognate response element, to show that these features of the AhR pathway are not required for the AhR to suppress pulmonary neutrophilia. Finally, using the non-toxic endogenous AhR ligand FICZ, we provide proof-of-concept that activation of pulmonary AhR attenuates smoke-induced inflammation. Collectively, these results support the importance of AhR activity in mediating its anti-inflammatory function in response to cigarette smoke. Further investigation of the precise mechanisms by which the AhR exerts is protective functions may lead to the development of therapeutic agents to treat people with chronic lung diseases that have an inflammatory etiology, but for which few therapeutic options exist.

Aryl Hydrocarbon Receptor Agonists in European Herring Gull (Larus argentatus) Eggs From Norway

Herring gull eggs from two locations in Norway, an island situated in the north (Musvær, 69.88° N, 18.55° E) and an island in the southeast (Reiaren, 59.15° N, 10.46° E) of the country, were analyzed for the presence of aryl hydrocarbon receptor (AhR) agonists. AhR agonist activity was determined using the dioxin-responsive chemically activated luciferase expression (DR-CALUX) assay to calculate the toxic equivalent quotient (TEQ)CALUX. TEQCALUX ranged from 16 to 401 pg TEQ/g lipid in the samples from the north (n = 11) and between 6 and 360 pg TEQ/g lipid (n = 12) in the southeastern samples. The large variance between the individual samples is postulated to be due to different feeding habits of individual birds. The levels of AhR agonists detected might lead to adverse effects for the developing embryo or to a significant increase of contaminant load for human consumers of eggs.

Chemometrics-Assisted Effect-Directed Analysis of Crude and Refined Oil Using Comprehensive Two-Dimensional Gas Chromatography–Time-of-Flight Mass Spectrometry

An effect-directed analysis (EDA) of fresh and artificially weathered (evaporated, photooxidized) samples of North Sea crude oil and residual heavy fuel oil is presented. Aliphatic, aromatic, and polar oil fractions were tested for the presence of aryl hydrocarbon receptor (AhR) agonist and androgen receptor (AR) antagonist, demonstrating for the first time the AR antagonist effects in the aromatic and, to a lesser extent, polar fractions. An extension of the typical EDA strategy to include an N-way partial least-squares (N-PLS) model capable of relating the comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry (GC × GC-TOFMS) data set to the bioassay data obtained from normal-phase LC fractions is proposed. The predicted AhR binding effects in the fresh and artificially weathered aromatic oil fractions facilitated the identification of alkyl-substituted three- and four-ring aromatic systems in the active fractions through the weighting of their contributions to the observed effects. A N-PLS chemometric model is demonstrated as a potentially useful strategy for future EDA studies that can streamline the compound identification process and provide additional reduction of samples' complexity. The AhR binding effects of the suspected compounds predicted by N-PLS and identified by GC × GC-TOFMS were confirmed using quantitative structure-activity relationship (QSAR) estimates.

Occurrence of aryl hydrocarbon receptor agonists and genotoxic compounds in the river systems in Southern Taiwan

Water and sediment samples from river systems located in Southern Taiwan were investigated for the presence of aryl hydrocarbon receptor (AhR) agonists and genotoxicants by a combination of recombinant cell assays and gas chromatography–mass spectrometry analysis. AhR agonist activity and genotoxic response were frequently detected in samples collected during different seasons. In particular, dry-season water and sediment samples from Erren River showed strong AhR agonist activity (201–1423ngL−1 and 1374–5631ngg−1 β-naphthoflavone equivalents) and high genotoxic potential. Although no significant correlation was found between AhR agonist activity and genotoxicity, potential genotoxicants in sample extracts were suggested to be causative agents for yeast growth inhibition in the AhR-responsive reporter gene assay. After high performance liquid chromatography fractionation, AhR agonist candidates were detected in several fractions of Erren River water and sediment extracts, while possible genotoxicants were only found in water extracts. In addition, polycyclic aromatic hydrocarbons, the typical contaminants showing high AhR binding affinity, were only minor contributors to the AhR agonist activity detected in Erren River sediment extracts. Our findings displayed the usefulness of bioassays in evaluating the extent of environmental contamination, which may be helpful in reducing the chances of false-negative results obtained from chemical analysis of conventional contaminants. Further research will be undertaken to identify major candidates for xenobiotic AhR agonists and genotoxicants to better protect the aquatic environments in Taiwan.

The aryl hydrocarbon receptor and estrogen receptor alpha differentially modulate nuclear factor erythroid-2-related factor 2 transactivation in MCF-7 breast cancer cells

Nuclear factor erythroid-2-related factor 2 (NRF2; NFE2L2) plays an important role in mediating cellular protection against reactive oxygen species. NRF2 signaling is positively modulated by the aryl hydrocarbon receptor (AHR) but inhibited by estrogen receptor alpha (ERα). In this study we investigated the crosstalk among NRF2, AHR and ERα in MCF-7 breast cancer cells treated with the NRF2 activator sulforaphane (SFN), a dual AHR and ERα activator, 3,3′-diindolylmethane (DIM), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or 17β-estradiol (E2). SFN-dependent increases in NADPH-dependent oxidoreductase 1 (NQO1) and heme oxygenase I (HMOX1) mRNA levels were significantly reduced after co-treatment with E2. E2-dependent repression of NQO1 and HMOX1 was associated with increased ERα but reduced p300 recruitment and reduced histone H3 acetylation at both genes. In contrast, DIM+SFN or TCDD+SFN induced NQO1 and HMOX1 mRNA expression to levels higher than SFN alone, which was prevented by RNAi-mediated knockdown of AHR. DIM+SFN but not TCDD+SFN also induced recruitment of ERα to NQO1 and HMOX1. However, the presence of AHR at NQO1 and HMOX1 restored p300 recruitment and histone H3 acetylation, thereby reversing the ERα-dependent repression of NRF2. Taken together, our study provides further evidence of functional interplay among NRF2, AHR and ERα signaling pathways through altered p300 recruitment to NRF2-regulated target genes.

The Expression of Aryl Hydrocarbon Receptor in Porcine Ovarian Cells

The aryl hydrocarbon receptor (AHR) is an intracellular transcription factor best known for mediating the toxicity of dioxins. The AHR is phylogenetically highly conserved among invertebrates and vertebrates and may play a significant role in the regulation of physiological processes including female reproduction. This study was performed to determine the partial nucleotide sequence of porcine AHR and to evaluate the AHR mRNA (real-time PCR) and AHR protein (Western blot) expression in granulosa and theca interna cells harvested from medium (3-6mm) and pre-ovulatory (≥8mm) follicles as well as in luteal cells obtained from corpora lutea collected during the mid-luteal phase (days: 8-10) of the porcine oestrous cycle. In the study, the partial nucleotide sequence of porcine AHR containing 1021bp (GenBank accession no: HM488957.1) was determined. The AHR transcript and protein were found in all ovarian cells obtained during both phases of the porcine oestrous cycle. The highest AHR transcript level was detected in theca interna cells isolated from pre-ovulatory follicles as well as in luteal cells. Higher AHR protein expression was found in granulosa cells isolated from pre-ovulatory follicles in comparison with all remaining cell types. The presence of AHR in the examined ovarian cells may account for their sensitivity to some environmental pollutants. Moreover, the differences found in AHR mRNA expression between granulosa and theca cells as well as between cells originating from follicles of different size suggest the involvement of AHR in the modulation of reproductive processes in the porcine ovary.

Disruption of CLOCK-BMAL1 Transcriptional Activity Is Responsible for Aryl Hydrocarbon Receptor–Mediated Regulation of Period1 Gene

The aryl hydrocarbon receptor (AhR) is a period-aryl hydrocarbon receptor nuclear transporter-simple minded domain transcription factor that shares structural similarity with circadian clock genes and readily interacts with components of the molecular clock. Activation of AhR by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) alters behavioral circadian rhythms and represses the Period1 (Per1) gene in murine hematopoietic stem and progenitor cells. Per1 expression is driven by circadian locomotor activity cycles kaput-brain muscle ARNT-like (CLOCK-BMAL1)-dependent activation of Eboxes in the Per1 promoter. We hypothesized that the effects of AhR activation on the circadian clock are mediated by disruption of CLOCK-BMAL1 function and subsequent Per1 gene suppression. Effects of AhR activation on rhythmic Per1 transcripts were examined in livers of mice after treatment with the AhR agonist, TCDD; the molecular mechanisms of Per1 repression by AhR were determined in hepatoma cells using TCDD and beta-napthoflavone as AhR activators. This study reports, for the first time, that AhR activation by TCDD alters the Per1 rhythm in the mouse liver and that Per1 gene suppression depends upon the presence of AhR. Furthermore, AhR interaction with BMAL1 attenuates CLOCK-BMAL1 activity and decreases CLOCK binding at Ebox1 and Ebox3 in the Per1 promoter. Taken together, these data suggest that AhR activation represses Per1 through disrupting CLOCK-BMAL1 activity, producing dysregulation of rhythmic Per1 gene expression. These data define alteration of the Per1 rhythm as novel signaling events downstream of AhR activation. Downregulation of Per1 could contribute to metabolic disease, cancer, and other detrimental effects resulting from exposure to certain environmental pollutants.

Aryl Hydrocarbon Receptor Functions as a Potent Coactivator of E2F1-Dependent Trascription Activity

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates a spectrum of toxic and biological effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin and related compounds. Several reports have shown that the AhR plays an important role in the control of cell-cycle progression, and this function is thought to be partly associated with the tumor promotion activity of dioxin. However, the underling mechanisms are not fully understood. We have previously shown that overexpression of AhR, as well as AhR ligand treatment, stimulates cell proliferation of human lung cancer A549 cells. In AhR-activated cells, the expression levels of DNA synthesis-related genes such as proliferating cell nuclear antigen (PCNA) and RFC38 are notably increased. Expression of these genes is mainly regulated by E2F1, a transcription factor that is crucial for transition of the cell cycle from G1 to S phase. We show here that the transcriptional activity of E2F1 is increased by the AhR agonist treatment and that this effect depends on the presence of AhR. Functional mapping of AhR showed that the Per-Arnt-Sim (PAS) B domain is required for promotion of E2F1 activity. The mechanism involves formation of a complex of AhR and E2F1 on the regulatory region in the E2F1 target gene, followed by recruitment of coactivator activator for thyroid hormone and retinoid receptors (ACTR). Consequently, the results in this study indicate the physiological function of AhR as a potent transcriptional coactivator of E2F1-dependent transcription and implicate the AhR-E2F1 interaction as a part of the mechanism by which AhR/Arnt promotes cell proliferation.

Identification of nonregulated pollutants in north sea‐produced water discharges

Produced water collected from oil and gas platforms on the United Kingdom Continental Shelf was characterized for nonregulated pollutants through an effects-directed analysis procedure. Produced water samples were characterized for the presence of aryl hydrocarbon receptor (AhR) and estrogen receptor (ER) agonists using the dioxin-responsive, chemical-activated luciferase gene expression assay (DR-CALUX) and yeast estrogen screen (YES) bioassays. The AhR and ER agonists were then isolated by normal-phase, high-performance liquid chromatography and identified using gas chromatography coupled to mass spectrometry in a number of formats. The identified compounds were cross-referenced with those compounds routinely analyzed and regarded by the Oslo and Paris (OSPAR) Commission for the North East Atlantic as priority hazardous substances. The occurrence in produced water of a number of nonregulated compounds with demonstrable potential environmental effects is presented, to our knowledge for the first time. These include persistent organic contaminants, such as hexachlorobenzene, decachlorobiphenyl, and octachlorodibenzofuran.

Oxidative stress in rainbow trout ( Oncorhynchus mykiss) exposed to sewage treatment plant effluent

Effluents from sewage treatment plants (STPs) can be regarded as “hot spots” of discharge releasing large amounts of chemicals into the aquatic environment. Many of these compounds are toxic to organisms due to their ability to form reactive oxygen species (ROS) and cause oxidative stress. In order to investigate if STP effluents contain compounds that may cause oxidative stress, rainbow trout ( Oncorhynchus mykiss) were exposed to effluent from a Swedish STP at different dilutions in a flow-through system. Antioxidant enzymes analyzed were glutathione reductase (GR), catalase (CAT) and DT-diaphorase (DTD). Catalytic activities of CYP1A (EROD) and the conjugating enzyme glutathione- S transferase (GST) were also analyzed. Results indicate that the effluent contains prooxidants since the activities of the antioxidant enzymes GR, CAT, and DTD were all elevated after 5 days of exposure. A prolonged exposure resulted in an inhibition of DT diaphorse activity, suggesting a depleted cellular ROS defence. EROD activities increased in a dose- and time-dependent manner, which suggests the presence of aryl hydrocarbon receptor (AHR) ligands such as polycyclic aromatic hydrocarbons (PAHs) in the effluent. These results indicate that STPs do not have the capacity to biodegrade harmful chemicals sufficiently to protect the aquatic environment. However, STPs are designed to remove nutrients and not persistent pollutants from the sewage and effort should be made to diminish the amount of chemicals entering the sewage in the first place.

2,3,7,8-Tetrachlorodibenzo-p-dioxin Alters the Differentiation of Alloreactive CD8+ T Cells Toward a Regulatory T Cell Phenotype by a Mechanism that is Dependent on Aryl Hydrocarbon Receptor in CD4+ T Cells

Activation of aryl hydrocarbon receptor (AhR) by 2,3,7,8-tetracholordibenzo- p-dioxin (TCDD) during an acute graft-versus-host response induces a population of alloreactive donor CD4+CD25+ regulatory T (Treg)-like cells that have potent suppressive activity in vitro. In the present studies, we show that TCDD induced a similar population of donor CD8+CD25+ T-cells with suppressive activity in vitro. Like the CD4+ Treg cells, donor CD8+CD25+ T-cells also expressed higher levels of CD28, glucocorticoid-induced TNFR (GITR) and CTLA-4 along with low levels of CD62L. These TCDD-induced phenotypic changes were not observed if donor T-cells were obtained from AhR-KO mice. When CD4+ and CD8+ donor T-cells from AhR-WT and AhR-KO mice were injected in various combinations into F1 mice, the enhanced expression of CD25 on CD8+ T-cells required AhR in donor CD4+ T-cells, while down-regulation of CD62L required AhR in the donor CD8+ T-cells themselves. Changes in GITR and CTLA-4 on donor CD8+ T-cells were partially mediated by AhR in both T-cells subsets. In contrast, all phenotypic changes in donor CD4+ T-cells were dependent on the presence of AhR in the CD4+ T-cells themselves. These findings suggest that the direct effects of AhR-mediated signaling in CD8+ T-cells are more limited than the direct effects in CD4+ T-cells, and that AhR signaling in CD4+ T-cells may be a unique pathway for the induction of both CD4+ and CD8+ adaptive Treg.

Organochlorine compounds in liver and concentrations of vitellogenin and 17β-estradiol in plasma of sea bass fed with a commercial or with a natural diet

Results from previous experiments directed to determine the effect of different nutritional factors or the effect of xenobiotics on hormonal control of reproduction, lead to the hypothesis that hormonal perturbations repeatedly observed in sea bass ( Dicentrarchus labrax) broodstock feeding commercial diets could have been caused by the presence of aryl hydrocarbon receptor (AhR) ligands, such as dioxins, furans and polychlorinated biphenyls (PCBs) in the diet. To evaluate this hypothesis, dioxins and related compounds were analysed in liver of female sea bass fed with a commercial or with a natural diet consisting of trash fish (bogue, Boops boops), and concentrations of vitellogenin (VTG) and 17β-estradiol (E2) were determined in plasma obtained previously in monthly samplings of these animals. As observed in other experiments, females fed with a commercial diet exhibited lower VTG and higher E2 plasma levels than females fed with the natural diet. In liver, sea bass fed with the commercial diet exhibited a profile clearly dominated by high-chlorinated dioxins while in fish fed with the natural diet this profile was dominated by low chlorinated furans. However, typical AhR ligands, such as 2,3,7,8-tetrachlorodibenzo- p-dioxin showed no differences between groups or, as is the case of planar PCBs, showed higher concentrations in the liver of fish fed with the natural diet. These results do not permit to explain the observed hormonal alterations by a possible antiestrogenic effect caused by dioxins and related compounds.

Induction of murine NAD(P)H:quinone oxidoreductase by 2,3,7,8-tetrachlorodibenzo-p-dioxin requires the CNC (cap 'n' collar) basic leucine zipper transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2): cross-interaction between AhR (aryl hydrocarbon receptor) and Nrf2 signal transduction.

TCDD (2,3,7,8-tetrachlorodibenzo- p -dixoin) induces phase II drug-metabolizing enzyme NQO1 [NAD(P)H:quinone oxidoreductase; EC 1.6.99.2; DT-diaphorase] in a wide range of mammalian tissues and cells. Here, we analysed the molecular pathway mediating NQO1 induction by TCDD in mouse hepatoma cells. Inhibition of protein synthesis with CHX (cycloheximide) completely blocks induction of NQO1 by TCDD as well as the basal expression and induction by phenolic antioxidant tBHQ (2-t-butylbenzene-1,4-diol), implicating a labile factor in NQO1 mRNA expression. The inhibition is both time- and concentration-dependent, requires inhibition of protein synthesis, and occurs at a transcriptional level. Inhibition of NQO1 transcription by CHX correlates with a rapid reduction of the CNC bZip (cap 'n' collar basic leucine zipper) transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2) through the 26 S proteasome pathway. Moreover, blocking Nrf2 degradation with proteasome inhibitor MG132 increases the amount of Nrf2 and superinduces NQO1 in the presence of TCDD or tBHQ. Finally, genetic experiments using AhR (aryl hydrocarbon receptor)-, Arnt (aryl hydrocarbon receptor nuclear translocator)- or Nrf2-deficient cells reveal that, while induction of NQO1 by TCDD depends on the presence of AhR and Arnt, the basal and inducible expression of NQO1 by either TCDD or tBHQ requires functional Nrf2. The findings demonstrate a novel role of Nrf2 in the induction of NQO1 by TCDD and provide new insights into the mechanism by which Nrf2 regulates the induction of phase II enzymes by both phenolic antioxidants and AhR ligands.