The aryl hydrocarbon receptor maintains antitumor activity of liver resident natural killer cells after partial hepatectomy in C57BL/6J mice.

BACKGROUND: Liver-resident natural killer (lr-NK) cells are distinct from conventional NK cells and exhibit higher cytotoxicity against hepatoma via tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). However, the mechanism by which partial hepatectomy (PH) significantly suppresses TRAIL expression in lr-NK cells remains unclear. METHODS: This study aimed to investigate the PH influence on the function and characteristics of liver-resident NK (lr-NK) cells using a PH mouse model. RESULTS: Here, we report that PH alters the differentiation pattern of NK cells in the liver, and an aryl hydrocarbon receptor (AhR) molecule is involved in these changes. Treatment with the AhR agonist 6-formylindolo[3,2-b]carbazole (FICZ) inhibited the maturation of NK cells. FICZ increased the immature subtype proportion of NK cells with high TRAIL activity and decreased the mature subtype of NK cells with low TRAIL activity. Consequently, FICZ increased the expression of TRAIL and cytotoxic activity of NK cells in the liver, and this effect was confirmed even after hepatectomy. The participation of AhR promoted FoxO1 expression in the mTOR signaling pathway involved in the maturation of NK cells, resulting in TRAIL expression. CONCLUSION: Our findings provide direct in-vivo evidence that partial hepatectomy affects lrNK cell activity through NK cell differentiation in the liver. Perioperative therapies using an AhR agonist to improve NK cell function may reduce the recurrence of hepatocellular carcinoma after hepatectomy.

AhR activation attenuates calcium oxalate nephrocalcinosis by diminishing M1 macrophage polarization and promoting M2 macrophage polarization.

Calcium oxalate (CaOx) crystal can trigger kidney injury, which contributes to the pathogenesis of nephrocalcinosis. The phenotypes of infiltrating macrophage may impact CaOx-mediated kidney inflammatory injury as well as crystal deposition. How aryl hydrocarbon receptor (AhR) regulates inflammation and macrophage polarization is well understood; however, how it modulates CaOx nephrocalcinosis remains unclear. Methods: Mice were intraperitoneally injected with glyoxylate to establish CaOx nephrocalcinosis model with or without the treatment of AhR activator 6-formylindolo(3,2-b)carbazole (FICZ). Positron emission tomography computed tomography (PET-CT) imaging, Periodic acid-Schiff (PAS) staining, and polarized light optical microscopy were used to evaluate kidney injury and crystal deposition in mice kidney. Western blotting, immunofluorescence, chromatin immunoprecipitation, microRNA-fluorescence in situ hybridization, and luciferase reporter assays were applied to analyze polarization state and regulation mechanism of macrophage. Results: AhR expression was significantly upregulated and negatively correlated with interferon-regulatory factor 1 (IRF1) and hypoxia inducible factor 1-alpha (HIF-1α) levels in a murine CaOx nephrocalcinosis model following administration of FICZ. Moreover, AhR activation suppressed IRF1 and HIF-1α levels and decreased M1 macrophage polarization in vitro. In terms of the mechanism, bioinformatics analysis and chromatin immunoprecipitation assay confirmed that AhR could bind to miR-142a promoter to transcriptionally activate miR-142a. In addition, luciferase reporter assays validated that miR-142a inhibited IRF1 and HIF-1α expression by directly targeting their 3'-untranslated regions. Conclusions: Our results indicated that AhR activation could diminish M1 macrophage polarization and promote M2 macrophage polarization to suppress CaOx nephrocalcinosis via the AhR-miR-142a-IRF1/HIF-1α pathway.

Cinnamtannin D1 attenuates autoimmune arthritis by regulating the balance of Th17 and treg cells through inhibition of aryl hydrocarbon receptor expression.

The suppression of the abnormal systemic immune response constitutes a primary strategy for treatment of rheumatoid arthritis (RA); toward this end, the identification of natural compounds with immunosuppressive activity represents a promising strategy for RA drug discovery. Cinnamtannin D1 (CTD-1), a polyphenolic compound isolated from Cinnamomum tamala, was previously reported to possess good immunosuppressive activity. However, the beneficial effect of CTD-1 on RA is currently unknown. The aim of this study was to evaluate the anti-arthritic effect of CTD-1 in collagen-induced arthritis (CIA) mice and clarify the underlying mechanisms. CTD-1 treatment significantly alleviated the severity of CIA mice, affording reduced clinical scores and paw swelling, along with reduced inflammatory cell infiltration and cartilage damage in the joints; in addition, the serum levels of IL-17, IL-6, and IL-1ß were decreased whereas those of TGF-ß and IL-10 were increased. CTD-1-treated mice exhibited lower frequency of Th17 cells and higher frequency of Treg cells compared to those in untreated mice, indicating that the balance of Th17/Treg cells may serve as the target for CTD-1. Consistent with this, in ex vivo assays, CTD-1 inhibited Th17 cell differentiation through the downregulation of phospho-STAT3/RORγt, whereas it promoted Treg differentiation by upregulating phospho-STAT5/Foxp3 in response to the stimulation of collagen type II. Moreover, in an in vitro naïve CD4+ T cell differentiation assay, CTD-1 directly inhibited Th17 cell differentiation and promoted Treg differentiation, suggesting that CTD-1 regulated the balance of Th17 and Treg cells to inhibit excessive immune response. Furthermore, the regulation effect of CTD-1 on Th17 and Treg cells was dependent on Ahr expression, as this effect was abolished when Ahr was knocked down and was impaired when Ahr was overexpressed. Together, our results indicated that CTD-1 treatment benefits CIA mice by regulating Th17 and Treg differentiation through the inhibition of AHR expression, and suggested a potential application of CTD-1 toward RA treatment.

Effects of 1,25-dihydroxyvitamin D3 on experimental periodontitis and AhR/NF-κB/NLRP3 inflammasome pathway in a mouse model.

Vitamin D has been known to have important regulatory functions in inflammation and immune response and shows inhibitory effects on experimental periodontitis in animal models. However, the potential mechanism has yet to be clarified. Recent studies have highlighted Aryl hydrocarbon receptor (AhR) and its downstream signaling as a crucial regulator of immune homeostasis and inflammatory regulation. OBJECTIVE: This study aimed to clarify the effect of 1,25-dihydroxyvitamin D3 (VD3) on experimental periodontitis and AhR/nuclear factor-κB (NF-κB)/NLR pyrin domain-containing 3 (NLRP3) inflammasome pathway in the gingival epithelium in a murine model. METHODOLOGY: We induced periodontitis in male C57BL/6 wild-type mice by oral inoculation of Porphyromonas gingivalis (P. gingivalis), and subsequently gave intraperitoneal VD3 injection to the mice every other day for 8 weeks. Afterwards, we examined the alveolar bone using scanning electron microscopy (SEM) and detected the gingival epithelial protein using western blot analysis and immunohistochemical staining. RESULTS: SEM images demonstrated that alveolar bone loss was reduced in the periodontitis mouse model after VD3 supplementation. Western blot analyses and immunohistochemical staining of the gingival epithelium showed that the expression of vitamin D receptor, AhR and its downstream cytochrome P450 1A1 were enhanced upon VD3 application. Additionally, VD3 decreased NF-κB p65 phosphorylation, and NLRP3, apoptosis-associated speck-like protein, caspase-1, interleukin-1ß (IL-1ß) and IL-6 protein expression. CONCLUSIONS: These results implicate the alleviation of periodontitis and the alteration of AhR/NF-κB/NLRP3 inflammasome pathway by VD3 in the mouse model. The attenuation of this periodontal disease may correlate with the regulation of AhR/NF-κB/NLRP3 inflammasome pathway by VD3.

Effects of 1, 25-dihydroxyvitamin D3 on experimental periodontitis and AhR/NF-κB/NLRP3 inflammasome pathway in a mouse model

Abstract Vitamin D has been known to have important regulatory functions in inflammation and immune response and shows inhibitory effects on experimental periodontitis in animal models. However, the potential mechanism has yet to be clarified. Recent studies have highlighted Aryl hydrocarbon receptor (AhR) and its downstream signaling as a crucial regulator of immune homeostasis and inflammatory regulation. Objective: This study aimed to clarify the effect of 1,25-dihydroxyvitamin D3 (VD3) on experimental periodontitis and AhR/nuclear factor-κB (NF-κB)/NLR pyrin domain-containing 3 (NLRP3) inflammasome pathway in the gingival epithelium in a murine model. Methodology: We induced periodontitis in male C57BL/6 wild-type mice by oral inoculation of Porphyromonas gingivalis (P. gingivalis), and subsequently gave intraperitoneal VD3 injection to the mice every other day for 8 weeks. Afterwards, we examined the alveolar bone using scanning electron microscopy (SEM) and detected the gingival epithelial protein using western blot analysis and immunohistochemical staining. Results: SEM images demonstrated that alveolar bone loss was reduced in the periodontitis mouse model after VD3 supplementation. Western blot analyses and immunohistochemical staining of the gingival epithelium showed that the expression of vitamin D receptor, AhR and its downstream cytochrome P450 1A1 were enhanced upon VD3 application. Additionally, VD3 decreased NF-κB p65 phosphorylation, and NLRP3, apoptosis-associated speck-like protein, caspase-1, interleukin-1β (IL-1β) and IL-6 protein expression. Conclusions: These results implicate the alleviation of periodontitis and the alteration of AhR/NF-κB/NLRP3 inflammasome pathway by VD3 in the mouse model. The attenuation of this periodontal disease may correlate with the regulation of AhR/NF-κB/NLRP3 inflammasome pathway by VD3.

Activation and overexpression of the aryl hydrocarbon receptor contribute to cutaneous squamous cell carcinomas: an immunohistochemical study.

BACKGROUND: In vitro studies showed that the aryl hydrocarbon receptor (AHR) contributed to the development of cutaneous squamous cell carcinomas, but supporting clinical data are lacking. METHODS: Immunohistochemical analysis was used to detect the expression of AHR, CYP1A1, EGFR, and Ki-67 in 10 actinic keratosis (AK) cases, 10 Bowen disease (BD) cases, 20 cutaneous squamous cell carcinoma (cSCC) cases and 20 normal skin samples. H-scores were used to assess the immunoreactivity. RESULTS: Weak positive AHR immunoreactivity was found in all normal skin samples, while strong positive AHR immunoreactivity was found in atypical squamous proliferation (AK, BD and cSCC) cases. H-scores and the rate of strong immunostaining of the atypical squamous proliferation cases were higher than those of normal controls (p < 0.01). Nuclear expression of AHR was higher in atypical squamous proliferation cases than in normal controls (p < 0.01). H-scores and the nuclear expression rate of AHR were significantly higher in AK and BD cases than cSCC cases (p < 0.01). CYP1A1 expression was low and showed no differences among the four studied groups (p > 0.05). The H-score of AHR was positively correlated with EGFR expression (r = 0.54, p < 0.01) in atypical squamous proliferation cases but was not correlated with CYP1A1 (r = - 0.17, p = 0.295) and Ki-67 (r = - 0.48, p = 0.222) expression. CONCLUSION: AHR plays a vital role in cSCC pathogenesis. The overexpression and activation of AHR are involved in the early development of skin cancers. AHR expression correlates with EGFR expression and may influence cell proliferation. AHR is a valuable therapeutic target for skin cancers.

ITE inhibits growth of human pulmonary artery endothelial cells.

AIM: Pulmonary arterial hypertension (PAH), a deadly disorder is associated with excessive growth of human pulmonary artery endothelial (HPAECs) and smooth muscle (HPASMCs) cells. Current therapies primarily aim at promoting vasodilation, which only ameliorates clinical symptoms without a cure. 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE) is an endogenous aryl hydrocarbon receptor (AhR) ligand, and mediates many cellular function including cell growth. However, the roles of ITE in human lung endothelial cells remain elusive. Herein, we tested a hypothesis that ITE inhibits growth of human pulmonary artery endothelial cells via AhR. MATERIALS AND METHODS: Immunohistochemistry was performed to localize AhR expression in human lung tissues. The crystal violet method and MTT assay were used to determine ITE's effects on growth of HPAECs. The AhR activation in HPAECs was confirmed using Western blotting and RT-qPCR. The role of AhR in ITE-affected proliferation of HPAECs was assessed using siRNA knockdown method followed by the crystal violet method. RESULTS: Immunohistochemistry revealed that AhR was present in human lung tissues, primarily in endothelial and smooth muscle cells of pulmonary veins and arteries, as well as in bronchial and alveolar sac epithelia. We also found that ITE dose- and time-dependently inhibited proliferation of HPAECs with a maximum inhibition of 83% at 20 µM after 6 days of treatment. ITE rapidly decreased AhR protein levels, while it increased mRNA levels of cytochrome P450 (CYP), family 1, member A1 (CYP1A1) and B1 (CYP1B1), indicating activation of the AhR/CYP1A1 and AhR/CYP1B1 pathways in HPAECs. The AhR siRNA significantly suppressed AhR protein expression, whereas it did not significantly alter ITE-inhibited growth of HPAECs. CONCLUSIONS: ITE suppresses growth of HPAECs independent of AhR, suggesting that ITE may play an important role in preventing excessive growth of lung endothelial cells.

A Novel Dual-Color Luciferase Reporter Assay for Simultaneous Detection of Estrogen and Aryl Hydrocarbon Receptor Activation.

Consumers are exposed to a plethora of anthropogenic and natural substances that can act as agonists or antagonists for various transcription factors. Depending on the exposure and potency, such interactions can potentially lead to adverse health effects, particularly for substances with multiple molecular targets. The early detection of such interactions is thus of high toxicological interest. Here, we report on the development of a new cellular dual-color reporter assay that allows for time-resolved and quantitative recording of estrogen receptor (ER) and aryl hydrocarbon receptor (AHR) activation in living cells. Both receptors are known for their ligand promiscuity. Moreover, both receptor signaling pathways are interconnected by direct protein-protein interactions as well as by shared protein factors and the competition for ligands. The assay is based on two rare beetle luciferases that emit light in the red (SLR) and green (ELuc) spectrum and that have been stably inserted into human T-47D mammary carcinoma cells. The corresponding cell line is termed "XEER" and has been successfully subjected to proof-of-principle studies using prototypical ER and AHR ligands as well as various phytochemicals, xenobiotics, and extracts from various plastic products.

Impact of untreated wastewater on a major European river evaluated with a combination of in vitro bioassays and chemical analysis.

Complex mixtures of micropollutants, including pesticides, pharmaceuticals and industrial chemicals emitted by wastewater effluents to European rivers may compromise the quality of these water resources and may pose a risk to ecosystem health and abstraction of drinking water. In the present study, an integrated analytical and bioanalytical approach was applied to investigate the impact of untreated wastewater effluents from the city of Novi Sad, Serbia, into the River Danube. The study was based on three on-site large volume solid phase extracted water samples collected upstream and downstream of the untreated wastewater discharge. Chemical screening with liquid chromatography high resolution mass spectrometry (LC-HRMS) was applied together with a battery of in vitro cell-based bioassays covering important steps of the cellular toxicity pathway to evaluate effects on the activation of metabolism (arylhydrocarbon receptor AhR, peroxisome proliferator activated receptor gamma PPARγ), specific modes of action (estrogen receptor ERα, androgen receptor AR) and adaptive stress responses (oxidative stress, inflammation). Increased effects, significantly changed contamination patterns and higher chemical concentrations were observed downstream of the wastewater discharge. A mass balance approach showed that enhanced endocrine disruption was in good agreement with concentrations of detected hormones, while only a smaller fraction of the effects on xenobiotic metabolism (<1%) and adaptive stress responses (0-12%) could be explained by the detected chemicals. The chemical and effects patterns observed upstream of the discharge point were fairly re-established at about 7 km downstream, demonstrating the enormous dilution capacity of this large river.

Genistein decreases basal hepatic cytochrome P450 1A1 protein expression and activity in Swiss Webster mice.

Soy consumption has been associated with risk reduction for chronic diseases such as cancer. One proposed mechanism for cancer prevention by soy is through decreasing cytochrome P450 1A1 (Cyp1a1) activity. However, it is not known with certainty which soy components modulate Cyp1a1, or the characteristics or mechanisms involved in the responses after short-term (<20 days) dietary treatment without concomitant carcinogen-mediated induction. Therefore, the objective was to test the hypothesis that physiologic concentrations of dietary genistein and/or daidzein will decrease basal hepatic Cyp1a1 protein expression and activity in male and female Swiss Webster mice via inhibiting the bindings of aryl hydrocarbon receptor (AhR)-AhR nuclear translocator (ARNT) and estrogen receptor-α to the Cyp1a1 promoter region xenobiotic response element. The mice were fed the AIN-93G diet supplemented with 1500 mg/kg of genistein or daidzein for up to 1 week. Genistein, but not daidzein, significantly decreased basal hepatic microsomal Cyp1a1 protein expression and activity. AhR protein expression was not altered. Molecular mechanisms were investigated in Hepa-1c1c7 cells treated with 5 µmol/L purified aglycones genistein, daidzein, or equol. Cells treated with genistein exhibited inhibitions in ARNT and estrogen receptor-α bindings to the Cyp1a1 promoter region. This study demonstrated that genistein consumption reduced constitutive hepatic Cyp1a1 protein expression and activity, thereby contributing to the understanding of how soy isoflavone aglycones modulate cytochrome P450 biotransformation enzymes.

Expression of interleukin-22 and its significance in the pathogenesis of chronic rhinosinusitis.

In order to explore the role of IL-22 in the pathogenesis of CRS, we observed the expression of IL-22 and associated factors in chronic rhinosinusitis with nasal polyps (CRSwNP) and chronic rhinosinusitis without nasal polyps (CRSsNP). Immunohistochemical staining was applied to detect the expression of IL-22, IL-22R, STAT3, retinoic acid orphan receptor C (RORC) and aryl hydrocarbon recptor (AhR). There was significantly higher expression of IL-22 in CRSsNP than in controls (P<0.05). But the expression of IL-22 had no significant difference when comparing CRSwNP with CRSsNP and controls. The expression of IL-22R was significantly lower in CRSwNP compared to controls and CRSsNP (P<0.05). The expression of AhR was lower in CRSwNP than in CRSsNP (P<0.05). There was no significant difference of RORC and STAT3 among CRSwNP, CRSsNP and controls. IL-22 plays the important role in the pathogenesis of CRS, and further research is needed to understand the complex interactions with other cytokines and the exact mechanism of transcriptional regulation for IL-22.

Expression and functional regulation of the nuclear receptors AHR, PXR, and CAR, and the transcription factor Nrf2 in rat parotid gland.

Nuclear receptors and transcription factors regulate the functions of many genes involved in cellular physiology and pathology (e.g. tumorigenesis and autoimmune diseases). The present study was performed to define the expression and the regulation of aryl hydrocarbon receptor (AhR), pregnane X receptor (PXR), constitutive androstane receptor (CAR), and nuclear factor E2-related factor 2 (Nrf2) in the rat parotid gland. Constitutive expression, as well as expression after stimulation with specific inducers for AhR [2,3,7,8-tetrachloro-dibenzylo-p-dioxin (TCDD)], Nrf2(oltipraz), PXR (dexamethasone), and CAR (phenobarbital), was evaluated using the quantitative PCR. Cellular localization of the nuclear receptors and the transcription factor was visualized by immunohistochemical staining. The study revealed constitutive expression of AhR as well as Nrf2, and their induction by TCDD andoltipraz, respectively. Immunohistochemical analysis revealed constitutive, predominantly cytoplasmic, expression of the AhR receptor, especially in interlobular striated duct cells, with nuclear shift upon exposure to TCDD. Inducible expression of Nfr2 was found mainly in the cytoplasm of intralobular striated duct cells. Constitutive expression of PXR and CAR was not found. Bearing in mind the involvement of AhR and Nrf2 in the regulation of many genes, it seems that these factors may play also a role in salivary gland physiology and pathology.

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces expression of p27(kip¹) and FoxO3a in female rat cerebral cortex and PC12 cells.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a potent toxin that alters normal brain development, producing cognitive disability and motor dysfunction. Previous studies in rats have proved that female rats are more sensitive to TCDD lethality than male ones. Recent studies have shown that TCDD induces cell cycle arrest and apoptosis, but the regulatory proteins involved in these processes have yet to be elucidated. In this study, we constructed an acute TCDD injury female rat model, and investigated the effects of TCDD on apoptosis and expression of cell cycle regulators, forkhead box class O 3a (FoxO3a) and p27(kip1), in the central nervous system (CNS). Increased levels of active caspase-3 were observed in the cerebral cortex of female rats treated with TCDD, suggesting that TCDD-induced apoptosis occurs in the CNS. The terminal deoxynucleotidyl transferase-mediated biotinylated-dUTP nick-end labeling assay showed that apoptosis primarily occurred in neurons. Furthermore, Western blot analysis, reverse transcription-polymerase chain reaction, and immunohistochemistry showed a significant up-regulation of FoxO3a and p27(kip1) in the cerebral cortex. Immunofluorescent labeling indicated that FoxO3a and p27(kip1) were predominantly localized in apoptotic neurons, but not in astrocytes. In vitro experiments using PC12, a rat neuron-like pheochromocytoma cell line, also revealed that TCDD induced apoptosis and an increase in FoxO3a and p27(kip1) expression. Furthermore, knockdown of FoxO3a expression inhibited p27(kip1) transcription and TCDD-induced apoptosis. Based on our data, induction of FoxO3a may play an important role in TCDD-induced neurotoxicity.

The levels of PAHs and aryl hydrocarbon receptor effects in sediments of Taihu Lake, China.

A total of 16 priority polycyclic aromatic hydrocarbons (PAHs) in sediment samples from Taihu Lake were analyzed by instruments, and sediment extracts were assayed for aryl hydrocarbon receptor (AhR)-mediated ethoxyresorufin-o-deethylase (EROD) induction using a rat hepatoma cell line (H4IIE). The cause-effect relationship between the observed EROD activity and chemical concentrations of PAHs was examined. Our results showed that sediment extracts could induce significant AhR effects, and the bioassay-derived 2,3,7,8-tetrachlorodibenzo-p-dioxin equivalents of raw extracts (TEQ(bio)s) ranged from 2.7 to 39.8 pg g(-1) dw. Chemical analysis showed that 16 PAHs were all detected in all samples, and their total concentrations (Σ16PAHs) ranged from 179.8 to 1,669.4 ng g(-1) dw. The abundance of sedimentary PAHs in the three regions (Meiliang Bay, Gonghu Bay, and Xukou Bay) showed a decreasing trend from the inflow region to the outflow region. Chemical analysis-derived TEQs (TEQ(cal)s) contributed by PAHs ranged from 1.6 to 20.7 pg g(-1) dw. The mean contribution rates (CRs) of PAHs to TEQ(bio)s were 48.9%. In Meiliang Bay, EROD effects of 60% samples were caused by PAHs whose CRs were more than 60%, while in most sampling sites of Gonghu Bay and Xukou Bay, the CRs of PAHs to TEQ(bio)s were basically below 40%. In addition, preliminary ecological risk assessment found that PAHs in sediments have very low ecological impact based on the chemical data of PAHs, while the sediments might pose an unacceptable risk to aquatic organisms and their predators based on the data of TEQ(bio). These findings showed that EROD effects of sediment extracts from Taihu Lake were also caused by other compounds, such as dioxins, polychlorinated biphenyls, etc., together.

2,3,7,8-Tetrachlorodibenzo-p-dioxin poly(ADP-ribose) polymerase (TiPARP, ARTD14) is a mono-ADP-ribosyltransferase and repressor of aryl hydrocarbon receptor transactivation.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD)-inducible poly(ADP-ribose) polymerase (TiPARP/ARTD14) is a member of the PARP family and is regulated by the aryl hydrocarbon receptor (AHR); however, little is known about TiPARP function. In this study, we examined the catalytic function of TiPARP and determined its role in AHR transactivation. We observed that TiPARP exhibited auto-mono-ADP-ribosyltransferase activity and ribosylated core histones. RNAi-mediated knockdown of TiPARP in T-47D breast cancer and HuH-7 hepatoma cells increased TCDD-dependent cytochrome P450 1A1 (CYP1A1) and CYP1B1 messenger RNA (mRNA) expression levels and recruitment of AHR to both genes. Overexpression of TiPARP reduced AHR-dependent increases in CYP1A1-reporter gene activity, which was restored by overexpression of AHR, but not aryl hydrocarbon receptor nuclear translocator. Deletion and mutagenesis studies showed that TiPARP-mediated inhibition of AHR required the zinc-finger and catalytic domains. TiPARP and AHR co-localized in the nucleus, directly interacted and both were recruited to CYP1A1 in response to TCDD. Overexpression of Tiparp enhanced, whereas RNAi-mediated knockdown of TiPARP reduced TCDD-dependent AHR proteolytic degradation. TCDD-dependent induction of AHR target genes was enhanced in Tiparp(-/-) mouse embryonic fibroblasts compared with wildtype controls. Our findings show that TiPARP is a mono-ADP-ribosyltransferase and a transcriptional repressor of AHR, revealing a novel negative feedback loop in AHR signalling.

Circulating Th22 and Th9 levels in patients with acute coronary syndrome.

BACKGROUND: CD4+ T helper (Th) cells play critical roles in the development and progression of atherosclerosis and the onset of acute coronary syndromes (ACS, including acute myocardial infarction (AMI) and unstable angina pectoris (UAP)). In addition to Th1, Th2, and Th17 cells, Th22 and Th9 subsets have been identified in humans. In the present study, we investigated whether Th22 cells and Th9 cells are involved in the onset of ACS. METHODS: The frequencies of Th22 and Th9 cells were detected using a flow cytometric analysis and their related cytokine and transcription factor were measured in the AMI, UAP, stable angina pectoris (SAP), and control groups. RESULTS: The results revealed a significant increase in the peripheral Th22 number, AHR expression, and IL-22 levels in patients with ACS compared with those in the SAP and control groups. Although there was no difference in the peripheral Th9 number among the four groups, the PU.1 expression and IL-9 levels were significantly increased in patients with ACS compared with the SAP and control groups. CONCLUSIONS: Circulating Th22 and Th9 type responses may play a potential role in the onset of ACS symptom.

The aryl hydrocarbon receptor contributes to the proliferation of human medulloblastoma cells.

The aryl hydrocarbon receptor (AhR), a ligand-activated member of the basic helix-loop-helix (bHLH)/PER-ARNT-SIM (PAS) transcription superfamily, is known to regulate the toxicity of polyaromatic halogenated hydrocarbon environmental chemicals, most notably dioxin. However, the AhR has also been implicated in multiple stages of tumorigenesis. Medulloblastoma (MB), a primary cerebellar brain tumor arising in infants and children, is thought to originate from abnormally proliferating cerebellar granule neuron precursors (GNPs). GNPs express high levels of the AhR in the external germinal layer of the developing cerebellum. Moreover, our laboratory has previously reported that either abnormal activation or deletion of the AhR leads to dysregulation of GNP cell cycle activity and maturation. These observations led to the hypothesis that the AhR promotes the growth of MB. Therefore, this study evaluated whether the AhR serves a pro-proliferative role in an immortalized MB tumor cell line (DAOY). We produced a stable AhR knockdown DAOY cell line [AhR short hairpin RNA (shRNA)], which exhibited a 70% reduction in AhR protein levels. Compared with wild-type DAOY cells, AhR shRNA DAOY cells displayed an impaired G(1)-to-S cell cycle transition, decreased DNA synthesis, and reduced proliferation. Furthermore, these cell cycle perturbations were correlated with decreased levels of the pro-proliferative gene Hes1 and increased levels of the cell cycle inhibitor p27(kip1). Supplementation experiments with human AhR restored the proliferative activity in AhR shRNA DAOY cells. Taken together, our data show that the AhR promotes proliferation of MB cells, suggesting that this pathway should be considered as a potential therapeutic target for MB treatment.

Integrated decision support for assessing chemical liabilities.

Chemical liabilities, such as adverse effects and toxicity, have a major impact on today's drug discovery process. In silico prediction of chemical liabilities is an important approach which can reduce costs and animal testing by complementing or replacing in vitro and in vivo liability models. There is a lack of integrated, extensible decision support systems for chemical liability assessment which run quickly and have easily interpretable results. Here we present a method which integrates similarity searches, structural alerts, and QSAR models which all are available from the Bioclipse workbench. Emphasis has been placed on interpretation of results, and substructures which are important for predictions are highlighted in the original chemical structures. This allows for interactively changing chemical structures with instant visual feedback and can be used for hypothesis testing of single chemical structures as well as compound collections. The system has a clear separation between methods and data, and the extensible architecture enables straightforward extension via addition of more plugins (such as new data sets and computational models). We demonstrate our method on three important safety end points: mutagenicity, carcinogenicity, and aryl hydrocarbon receptor (AhR) activation. Bioclipse and the decision support implementation are free, open source, and available from http://www.bioclipse.net/decision-support .

Effects-directed analysis of sediments from polluted marine sites in Norway.

The environmental status of two polluted marine sites in Norway was investigated by a combination of target chemical analysis and effect-directed analysis (EDA). The two selected sites, the Grenland area and Oslo harbor, in addition to two reference sites, were classified according to the Norwegian environmental classification system based upon results of the target chemical analyses. The polluted sites were characterized by high levels of metals, polycyclic aromatic hydrocarbons (PAH), and polychlorinated biphenyls (PCB). High levels of organotin compounds were also detected in Oslo harbor. The aryl hydrocarbon receptor (AhR) agonist activity in extracts of sediments from marine sites close to Oslo, Oslo harbor, and Grenland were investigated using the CALUX (chemical-activated luciferase expression) assay, which showed elevated levels of activity. As expected from the history of dioxin release into the Grenland area, the results were highest in this area. The presence of estrogen receptor (ER) and androgen receptor (AR) antagonists was also detected in the sediment extracts. Following fractionation of the sediment extracts, EDA was used to tentatively identify the AhR agonists. The compounds responsible for AhR agonist activity in samples from Oslo harbor were isolated in fraction 13, and to a lesser extent in fractions 9-11. In Grenland, the main activity was found in the more polar fractions, namely fractions 14-18. The AhR agonists identified in Oslo harbor were mainly PAH, while in the Grenland area the compounds identified were mainly nitrogen/oxygen-containing polyaromatic compounds (N/O-PAC).

Polar compounds dominate in vitro effects of sediment extracts.

Sediment extracts from three polluted sites of the river Elbe basin were fractionated using a novel online fractionation procedure. Resulting fractions were screened for mutagenic, aryl hydrocarbon receptor (AhR)-mediated, transthyretin (TTR)-binding, and estrogenic activities and their potency to inhibit gap junctional intercellular communication (GJIC) to compare toxicity patterns and identify priority fractions. Additionally, more than 200 compounds and compound classes were identified using GC-MS/MS, LC-MS/MS, and HPLC-DAD methods. For all investigated end points, major activities were found in polar fractions, which are defined here as fractions containing dominantly compounds with at least one polar functional group. Nonpolar PAH fractions contributed to mutagenic and AhR-mediated activities while inhibition of GJIC and estrogenic and TTR-binding activities were exclusively observed in the polar fractions. Known mutagens in polar fractions included nitro- and dinitro-PAHs, azaarenes, and keto-PAHs, while parent and monomethylated PAHs such as benzo[a]pyrene and benzofluoranthenes were identified in nonpolar fractions. Additionally, for one sample, high AhR-mediated activities were determined in one fraction characterized by PCDD/Fs, PCBs, and PCNs. Estrone, 17ß-estradiol, 9H-benz[de]anthracen-7-one, and 4-nonylphenol were identified as possible estrogenic and TTR-binding compounds. Thus, not only nonpolar compounds such as PAHs, PCBs, and PCDD/Fs but also the less characterized and investigated more polar substances should be considered as potent mutagenic, estrogenic, AhR-inducing, TTR-binding, and GJIC-inhibiting components for future studies.