Targeting the Aryl Hydrocarbon Receptor with Microbial Metabolite Mimics Alleviates Experimental Colitis in Mice.

Targeting the aryl hydrocarbon receptor (AhR) is an emerging therapeutic strategy for multiple diseases (e.g., inflammatory bowel disease). Thermosporothrix hazakensis microbial metabolite 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE) is a putative AhR endogenous ligand. To improve the chemical stability, we synthesized a series of ITE chemical mimics. Using a series of in vitro assays, we identified 2-(1H-indole-3-carbonyl)-N-methyl thiazole-4-carboxamide (ITE-CONHCH3) as a highly potent (EC50 = 1.6 nM) AhR agonist with high affinity (Ki = 88 nM). ITE-CONHCH3 triggered AhR nuclear translocation and dimerization of AhR-ARNT, enhanced AhR binding in the CYP1A1 promoter, and induced AhR-regulated genes in an AhR-dependent manner. The metabolic stability of ITE-CONHCH3 in a cell culture was 10 times higher than that of ITE. Finally, we observed protective effects of ITE-CONHCH3 in mice with DSS-induced colitis. Overall, we demonstrate and validate a concept of microbial metabolite mimicry in the therapeutic targeting of AhR.

Effects of Flavored Nonalcoholic Beverages on Transcriptional Activities of Nuclear and Steroid Hormone Receptors: Proof of Concept for Novel Reporter Cell Line PAZ-PPARg.

We developed and characterized a novel human luciferase reporter cell line for the assessment of peroxisome proliferator-activated receptor γ (PPARγ) transcriptional activity, PAZ-PPARg. The luciferase activity induced by PPARγ endogenous agonist 15d-PGJ2 and prostaglandin PGD2 reached mean values of (87.9 ± 14.0)-fold and (89.6 ± 19.7)-fold after 24 h of exposure to 40 µM 15d-PGJ2 and 70 µM PGD2, respectively. A concentration-dependent inhibition of 15d-PGJ2- and PGD2-induced luciferase activity was observed after the application of T0070907, a selective antagonist of PPARγ, which confirms the specificity of response to both agonists. The PAZ-PPARg cell line, along with the reporter cell lines for the assessment of transcriptional activities of thyroid receptor (TR), vitamin D3 receptor (VDR), androgen receptor (AR), and glucocorticoid receptor (GR), were used for the screening of 27 commonly marketed flavored nonalcoholic beverages for their possible disrupting effects. Our findings indicate that some of the examined beverages have the potential to modulate the transcriptional activities of PPARγ, VDR, and AR.

Activated thyroid hormone receptor modulates dioxin-inducible aryl hydrocarbon receptor-mediated CYP1A1 induction in human hepatocytes but not in human hepatocarcinoma HepG2 cells.

Aryl hydrocarbon receptor (AhR) is a transcription factor, the activity of which is modulated by hormones including glucocorticoids and estrogens. In this study, we examined the effects of triiodothyronine (T3), a ligand and activator of thyroid hormone receptor (TR), on transcriptional activity of AhR and the expression of its target gene CYP1A1. Study was carried out in human hepatocellular carcinoma cells HepG2 and primary cultures of human hepatocytes (HH). Gene reporter assay in stably transfected AZ-AhR cells revealed that T3 dose-dependently augmented 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-inducible AhR-dependent luciferase activity. In contrast, T3 had no effect on TCDD-inducible expression of CYP1A1 mRNA, protein and catalytic activity. Incubation of human hepatocytes with T3 had modulatory and inter-individual (7 cell cultures from 7 different liver donors) effects on both basal and dioxin-inducible CYP1A1/2. Since there was no correlation between T3 effects on CYP1A expression and T3-dependent expression of Spot14 mRNA, the involvement of additional factors besides TR is supposed. Overall, the co-incubation of normal and cancer human hepatic cells with TCDD and T3 suggested transcriptional cross-talk between AhR and TR, which may have physiological and toxicological implications.

In vitro modulatory effects of functionalized pyrimidines and piperidine derivatives on Aryl hydrocarbon receptor (AhR) and glucocorticoid receptor (GR) activities.

The development of biologically active molecules based on molecular recognition is an attractive and challenging task in medicinal chemistry and the molecules that can activate/deactivate certain receptors are of great medical interest. In this contribution, selected pyrimidine/piperidine derivatives were synthesized and tested for the ability to activate/deactivate Aryl hydrocarbon receptor (AhR) and Glucocorticoid receptor (GR). Tested compounds are shown to activate the receptors but to much lesser extent than positive controls, dioxin and dexamethasone for Ahr and GR, respectively. However, some of them antagonized the positive controls action. Although further in vivo studies are needed to fully characterize the bioactivities of these compounds, the reported in vitro evidences demonstrate that they might be used as the modulators of AhR and GR activities.

Assessment of the aryl hydrocarbon receptor-mediated activities of polycyclic aromatic hydrocarbons in a human cell-based reporter gene assay.

Activation of the aryl hydrocarbon receptor (AhR)-mediated activity is one of key events in toxicity of polycyclic aromatic hydrocarbons (PAHs). Although various classes of AhR ligands may differentially activate human and rodent AhR, there is presently a lack of data on the human AhR-inducing relative potencies (REPs) of PAHs. Here, we focused on estimation of the AhR-mediated activities of a large set of environmental PAHs in human gene reporter AZ-AhR cell line, with an aim to develop the human AhR-based REP values with potential implications for risk assessment of PAHs. The previously identified weakly active PAHs mostly failed to activate the AhR in human cells. The order for REPs of individual PAHs in human cells largely corresponded with the available data from rodent-based experimental systems; nevertheless, we identified differences up to one order of magnitude in REP values of PAHs between human and rodent cells. Higher REP values were found in human cells for some important environmental contaminants or suspected carcinogens, such as indeno[1,2,3-cd]pyrene, benz[a]anthracene or benzo[b]fluoranthene, while lower REP values were determined for methyl-substituted PAHs. Our results also indicate that a different rate of metabolism for individual PAHs in human vs. rodent cells may affect estimation of REP values in human cell-based assay, and potentially alter toxicity of some compounds, such as benzofluoranthenes, in humans. We applied the AZ-AhR assay to evaluation of the AhR-mediated activity of complex mixtures of organic compounds associated with diesel exhaust particles, and we identified the polar compounds present in these mixtures as being particularly highly active in human cells, as compared with rodent cells. The present data suggest that differences may exist between the AhR-mediated potencies of PAHs in human and rodent cells, and that the AhR-mediated effects of polar PAH derivatives and metabolites in human cell models deserve further attention.

Profiling of Vitamin D Metabolic Intermediates toward VDR Using Novel Stable Gene Reporter Cell Lines IZ-VDRE and IZ-CYP24.

Variety of xenobiotics, including therapeutically used vitamin D analogues or environmental and alimentary endocrine disruptors, may interfere with vitamin D receptor (VDR) signaling, with serious physiological or pathophysiological consequences. Therefore, it is of topical interest to have reliable and efficient in vitro screening tools for the identification of agonists and activators of human VDR. We present here two novel stably transfected human reporter cell lines allowing rapid, high-throughput, and selective identification of VDR agonists and activators. Human colon adenocarcinoma cells LS180 were stably transfected with reporter plasmids CYP24_minP-pNL2.1[Nluc/Hygro] (IZ-CYP24 cells contain the -326/-46 sequence from the human CYP24A1 promoter) or VDREI3_SV40-pNL2.1[Nluc/Hygro] (IZ-VDRE cells contain three copies of vitamin D response elements VDRE-I from the human CYP24A1 promoter). Both cell lines remained fully functional for over two months in the culture and also after cryopreservation. Luciferase inductions ranged from 10-fold to 25-fold (RLU 10(6)-10(7)) and from 30-fold to 80-fold (RLU 10(3)-10(4)) in IZ-VDRE and IZ-CYP24 cells, respectively. Time-course analyses revealed that detection of VDR activators is possible as soon as after 8 h of incubation. Cell lines were highly selective toward VDR agonists, displaying no cross-activation by retinoids, thyroids, and steroids. As a proof of concept, we used IZ-VDRE and IZ-CYP24 cells for profiling analogues of vitamin D, and intermediates in vitamin D2 and vitamin D3 metabolic pathways against VDR transcriptional activity. The data obtained revealed significant activation of VDR not only by obligatory ligands calcitriol and ergocalcitriol but also by their precursors and degradation products.

Impurities contained in antifungal drug ketoconazole are potent activators of human aryl hydrocarbon receptor.

Antifungal drug ketoconazole is a mixture of (+)/(-) cis-enantiomers, which also contains several impurities. Ketoconazole was identified as an activator of aryl hydrocarbon receptor AhR by three independent research teams. In the current paper we demonstrate that impurities contained in ketoconazole preparations are strong activators of human AhR and inducers of CYP1A1. Impurity IMP-C had similar potency (EC50), but 10-15 times higher efficacy (magnitude of induction) towards AhR, comparing to (+)-ketoconazole, as revealed by gene reporter assay in AZ-AHR stably transfected cells. Impurities IMP-B and IMP-C, and in lesser extent IMP-E, induced a formation of AhR-DNA complex, as demonstrated by electromobility shift assay EMSA. Impurities IMP-C and IMP-E dose-dependently induced CYP1A1 mRNA after 24 h, and their effects were comparable to those by (+)-ketoconazole. The level of CYP1A1 protein in HepG2 cells was strongly increased by IMP-C after 48h. In conclusion, our data further elucidated molecular effects of ketoconazole towards AhR signaling pathway, with possible implications in ketoconazole role in skin chemoprevention and/or damage, involving AhR.