Rosmarinus officinalis L. Leaf Extracts and Their Metabolites Inhibit the Aryl Hydrocarbon Receptor (AhR) Activation In Vitro and in Human Keratinocytes: Potential Impact on Inflammatory Skin Diseases and Skin Cancer.

Aryl hydrocarbon receptor (AhR) activation by environmental agents and microbial metabolites is potentially implicated in a series of skin diseases. Hence, it would be very important to identify natural compounds that could inhibit the AhR activation by ligands of microbial origin as 6-formylindolo[3,2-b]carbazole (FICZ), indirubin (IND) and pityriazepin (PZ) or the prototype ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Five different dry Rosmarinus officinalis L. extracts (ROEs) were assayed for their activities as antagonists of AhR ligand binding with guinea pig cytosol in the presence of [3H]TCDD. The methanolic ROE was further assayed towards CYP1A1 mRNA induction using RT-PCR in human keratinocytes against TCDD, FICZ, PZ, and IND. The isolated metabolites, carnosic acid, carnosol, 7-O-methyl-epi-rosmanol, 4',7-O-dimethylapigenin, and betulinic acid, were assayed for their agonist and antagonist activity in the presence and absence of TCDD using the gel retardation assay (GRA). All assayed ROE extracts showed similar dose-dependent activities with almost complete inhibition of AhR activation by TCDD at 100 ppm. The methanol ROE at 10 ppm showed 99%, 50%, 90%, and 85% inhibition against TCDD, FICZ, IND, and PZ, respectively, in human keratinocytes. Most assayed metabolites exhibited dose-dependent antagonist activity. ROEs inhibit AhR activation by TCDD and by the Malassezia metabolites FICZ, PZ, and IND. Hence, ROE could be useful for the prevention or treatment of skin diseases mediated by activation of AhR.

Assessing the estrogenic activity of chemicals present in resin based dental composites and in leachates of commercially available composites using the ERα-CALUX bioassay.

OBJECTIVE: The biocompatibility of resin based dental composites has not yet been fully characterized even though certain monomers used in these composites are synthesized from Bisphenol A (BPA), a well-known estrogenic endocrine disruptor. As a result, they show structural relationship to BPA and can contain it as an impurity. Therefore, the estrogenic activity of 9 monomers, 2 photoinitiators, one photostabilizer and leachates of 4 commercially available composites was determined. METHODS: The ERα-CALUX bioassay was used to determine both agonistic and antagonistic estrogenic activities of the pure compounds (BPA, BisDMA, BisGMA, BisEMA(3), BisEMA(6), BisEMA(10), TEGDMA, TCD-DI-HEA, BADGE, UDMA, HMBP, DMPA, CQ) and the leachates of cured composite disks. The leachates of 4 commercially available composites (Solitaire 2, Ceram.x Spectra ST, G-ænial Posterior and Filtek Supreme XTE) in water and 0.1 M NaOH (pH = 13, 'worst-case scenario') were tested for estrogenic activity (pooled leachates from 10 cured composite disks). RESULTS: Agonistic estrogenic activity was found for the monomer BisDMA, the photostabilizer HMBP and photoinitiator DMPA. All leachates from the 4 tested composites showed significant agonistic estrogenic activity higher than the DMSO control, and the highest activity (potency and efficacy) was found for Solitaire 2, followed by Ceram.x Spectra ST. Furthermore, antagonistic estrogenic activity was found in the leachates from G-ænial Posterior. SIGNIFICANCE: These results show that significant estrogenic activity was found in all leachates of the cured composite disks, and that this estrogenicity is most likely due to a mixture effect of multiple estrogenic compounds (including BPA, HMBP and DMPA). This indicates that further research into the endocrine activity of all the compounds that are present in these composites (even at low quantities) and their possible mixture effect is warranted to guarantee their safe use.

Woodsmoke Extracts Cross-Link Proteins and Induce Cornified Envelope Formation without Stimulating Keratinocyte Terminal Differentiation.

Air pollution poses a serious risk to human health. To help understand the contribution of smoke from wood burning to the harmfulness of air pollution toward the skin, we studied the effects of liquid smoke, aqueous extracts of wood smoke condensate, a commercially available food flavor additive, in cultured keratinocytes. We report that liquid smoke can react with and cross-link keratinocyte cellular proteins, leading to abnormal cross-linked envelope formation. Instead of inducing genes ordinarily involved in terminal differentiation, liquid smoke induced expression of genes associated with stress responses. When transglutaminase activity was inhibited, liquid smoke still promoted protein cross-linking and envelope formation in keratinocytes. This phenomenon likely results from oxidative stress and protein adducts from aldehydes as either preloading the cells with N-acetylcysteine or reducing the aldehyde content of liquid smoke decreased its ability to promote protein cross-linking and envelope formation. Finally, liquid smoke-induced envelopes were found to have elevated protein content, suggesting oxidative cross-linking and formation of protein adducts might impair barrier function by inducing abnormal incorporation of cellular proteins into envelopes. Since the cross-linked protein envelope provides structural stability to the stratum corneum and serves as a scaffold for the organization of the corneocyte lipid envelope (hydrophobic barrier to the environment), these findings provide new insight into the mechanism by which pro-oxidative air pollutants can impair epidermal function.

Bioanalytical screening of low levels of dioxins and dioxin-like PCBs in pig meat (pork) for checking compliance with EU maximum and action levels using highly sensitive "third generation" recombinant H4L7.5c2 rat hepatoma cells.

BACKGROUND: Low maximum and action levels set by the European Union for polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) and dioxin-like polychlorinated biphenyls (DL-PCBs) in pig meat (pork) have led to a demand for reliable and cost-effective bioanalytical screening methods implemented upstream of gas chromatography/high-resolution mass spectrometry confirmatory technology, that can detect low levels of contamination in EU-regulated foods with quick turn-around times. RESULTS: Based on the Chemically Activated LUciferase gene eXpression (CALUX) bioassay, extraction and clean-up steps were optimized for recovery and reproducibility within working ranges significantly lower than in current bioassays. A highly sensitive "3rd generation" recombinant rat hepatoma cell line (H4L7.5c2) containing 20 dioxin responsive elements was exposed to pork sample extracts, and their PCDD/Fs and DL-PCBs levels were evaluated by measuring luciferase activity. The method was validated according to the provisions of Commission Regulation (EU) 2017/644 of 5 April 2017 with spiking experiments performed selectively for PCDD/Fs and DL-PCBs and individual calibration for PCDD/Fs, DL-PCBs and the calculated sum of PCDD/Fs and DL-PCBs. The resulting performance parameters met all legal specifications as confirmed by re-calibration using authentic samples. Cut-off concentrations for assessing compliance with low maximum levels and action levels set for PCDD/Fs and DL-PCBs within a range of 0.50-1.25 pg WHO-TEQ/g fat were derived, ensuring low rates of false-compliant results (ß-error < 1%) and keeping the rate of false-noncompliant results well under control (α-error < 12%). CONCLUSIONS: We present a fast and efficient bioanalytical routine method validated according to the European Union's legal requirements on the basis of authentic samples, allowing the analyst to reliably identify pork samples and any other EU-regulated foods of animal origin suspected to be noncompliant with a high level of performance and turn-around times of 52 h. This was facilitated in particular by a quick and efficient extraction step followed by selective clean-up, use of a highly sensitive "3rd generation" H4L7.5c2 recombinant rat hepatoma cell CALUX bioassay, and optimized assay performance with improved calibrator precision and reduced lack-of-fit errors. New restrictions are proposed for the calibrator bias and the unspecific background contribution to reportable results. The procedure can utilize comparably small sample amounts and allows an annual throughput of 840-1000 samples per lab technician. The described bioanalytical method contributes to the European Commission's objective of generating accurate and reproducible analytical results according to Commission Regulation (EU) 2017/644 across the European Union.

Using Estrogenic Activity and Nontargeted Chemical Analysis to Identify Contaminants in Sewage Sludge.

Diverse organic compounds, many derived from consumer products, are found in sewage sludge worldwide. Understanding which of these poses the most significant environmental threat following land application can be investigated through a variety of predictive and cell-based toxicological techniques. Nontargeted analysis using high-resolution mass spectrometry with predictive estrogenic activity modeling was performed on sewage sludge samples from 12 wastewater treatment plants in California. Diisobutyl phthalate and dextrorphan were predicted to exhibit estrogenic activity and identified in >75% of sludge samples, signifying their universal presence and persistence. Additionally, the application of an estrogen-responsive cell bioassay revealed reductions in agonistic activity during mesophilic and thermophilic treatment but significant increases in antagonism during thermophilic treatment, which warrants further research. Ten nontarget features were identified (metoprolol, fenofibric acid, erythrohydrobupropion, oleic acid, mestranol, 4'-chlorobiphenyl-2,3-diol, medrysone, scillarenin, sudan I, and N,O-didesmethyltramadol) in treatment set samples and are considered to have influenced the in vitro estrogenic activity observed. The combination of predictive and in vitro estrogenicity with nontargeted analysis has led to confirmation of 12 estrogen-active contaminants in California sewage sludge and has highlighted the importance of evaluating both agonistic and antagonistic responses when evaluating the bioactivity of complex samples.

Identifying Toxicologically Significant Compounds in Urban Wildfire Ash Using In Vitro Bioassays and High-Resolution Mass Spectrometry.

Urban wildfires may generate numerous unidentified chemicals of toxicity concern. Ash samples were collected from burned residences and from an undeveloped upwind reference site, following the Tubbs fire in Sonoma County, California. The solvent extracts of ash samples were analyzed using GC- and LC-high-resolution mass spectrometry (HRMS) and using a suite of in vitro bioassays for their bioactivity toward nuclear receptors [aryl hydrocarbon receptor (AhR), estrogen receptor (ER), and androgen receptor (AR)], their influence on the expression of genetic markers of stress and inflammation [interleukin-8 (IL-8) and cyclooxygenase-2 (COX-2)], and xenobiotic metabolism [cytochrome P4501A1 (CYP1A1)]. Genetic markers (CYP1A1, IL-8, and COX-2) and AhR activity were significantly higher with wildfire samples than in solvent controls, whereas AR and ER activities generally were unaffected or reduced. The bioassay responses of samples from residential areas were not significantly different from the samples from the reference site despite differing chemical compositions. Suspect and nontarget screening was conducted to identify the chemicals responsible for elevated bioactivity using the multiple streams of HRMS data and open-source data analysis workflows. For the bioassay endpoint with the largest available database of pure compound results (AhR), nontarget features statistically related to whole sample bioassay response using Spearman's rank-order correlation coefficients or elastic net regression were significantly more likely (by 10 and 15 times, respectively) to be known AhR agonists than the overall population of compounds tentatively identified by nontarget analysis. The findings suggest that a combination of nontarget analysis, in vitro bioassays, and statistical analysis can identify bioactive compounds in complex mixtures.

Evaluation of the dioxin-like toxicity in soil samples from Thua Thien Hue province using the AhR-CALUX bioassay - An update of Agent Orange contamination in Vietnam.

In this study, an AhR-responsive reporter-gene cell-based bioassay CALUX was used to assess the biological potency of dioxins and dioxin-like PCBs (dl-PCBs) in top soil samples collected from a former airbase (A-So) and remote regions from urban and agricultural areas in Thua Thien Hue, Vietnam. In top soil collected from A-So airbase, Bioanalytical EQuivalent (BEQ) concentrations of up to 2700 pg 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) per g dry weight (pg BEQ-TCDD g-1 dw) were assessed. Interestingly, while BEQ values for dl-PCBs were found to be up to 13 pg BEQ-TCDD g-1 dw, the dl-PCB activity was not detected in all the hotspot sample extracts. In contrasts, BEQ values for dioxin like compounds from remote regions were much lower and occasionally below the quantification limits of the method. The BEQ activities obtained in this study have a similar trend to the WHO-TEQ results for the samples collected in the A-So airbase. However, BEQ values were higher than those of TEQ, probably reflecting the presence of additional AhR ligands and/or possible non-additive interactions in the sample mixture. This study confirms that after more than 60 years, a strong residual pollution of PCDD/Fs remains on this former air base following the use and storage of Agent Orange during the Vietnam War, raising a health risk for populations exposed in this area because livestock animals graze there.

An aryl hydrocarbon receptor from the caecilian Gymnopis multiplicata suggests low dioxin affinity in the ancestor of all three amphibian orders.

The aryl hydrocarbon receptor (AHR) plays pleiotropic roles in the development and physiology of vertebrates in conjunction with xenobiotic and endogenous ligands. It is best known for mediating the toxic effects of dioxin-like pollutants such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). While most vertebrates possess at least one AHR that binds TCDD tightly, amphibian AHRs bind TCDD with very low affinity. Previous analyses of AHRs from Xenopus laevis (a frog; order Anura) and Ambystoma mexicanum (a salamander; order Caudata) identified three amino acid residues in the ligand-binding domain (LBD) that underlie low-affinity binding. In X. laevis AHR1ß, these are A354, A370, and N325. Here we extend the analysis of amphibian AHRs to the caecilian Gymnopis multiplicata, representing the remaining extant amphibian order, Gymnophiona. G. multiplicata AHR groups with the monophyletic vertebrate AHR/AHR1 clade. The LBD includes all three signature residues of low TCDD affinity, and a structural homology model suggests that its architecture closely resembles those of other amphibians. In transactivation assays, the EC50 for reporter gene induction by TCDD was 17.17 nM, comparable to X. laevis AhR1ß (26.23 nM) and Ambystoma AHR (34.09 nM) and dramatically higher than mouse AhR (0.13 nM), a trend generally reflected in direct measures of TCDD binding. These shared properties distinguish amphibian AHRs from the high-affinity proteins typical of both vertebrate groups that diverged earlier (teleost fish) and those that appeared more recently (other tetrapods). These findings suggest the hypothesis that AHRs with low TCDD affinity represent a characteristic that evolved in a common ancestor of all three extant amphibian groups.

The Cellular and Molecular Determinants of Naphthoquinone-Dependent Activation of the Aryl Hydrocarbon Receptor.

1,2-naphthoquinone (1,2-NQ) and 1,4-naphthoquinone (1,4-NQ) are clinically promising biologically active chemicals that have been shown to stimulate the aryl hydrocarbon receptor (AhR) signaling pathway, but whether they are direct or indirect ligands or activate the AhR in a ligand-independent manner is unknown. Given the structural diversity of AhR ligands, multiple mechanisms of AhR activation of gene expression, and species differences in AhR ligand binding and response, we examined the ability of 1,2-NQ and 1,4-NQ to bind to and activate the mouse and human AhRs using a series of in vitro AhR-specific bioassays and in silico modeling techniques. Both NQs induced AhR-dependent gene expression in mouse and human hepatoma cells, but were more potent and efficacious in human cells. 1,2-NQ and 1,4-NQ stimulated AhR transformation and DNA binding in vitro and was inhibited by AhR antagonists. Ligand binding analysis confirmed the ability of 1,2-NQ and 1,4-NQ to competitively bind to the AhR ligand binding cavity and the molecular determinants for interactions were predicted by molecular modeling methods. NQs were shown to bind distinctly differently from that of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and differences were also observed between species. Mutation of amino acid residues (F289, M334, and M342) involved in critical NQ:AhR binding interactions, decreased NQ- and AhR-dependent gene expression, consistent with a role for these residues in binding and activation of the AhR by NQs. These studies provide insights into the molecular mechanism of action of NQs and contribute to the development of emerging NQ-based therapeutics.

Development of a novel recombinant cell line for detection and characterization of Ah receptor nuclear translocation in intact cells.

The Ah receptor (AhR) is a ligand-dependent transcriptional factor that mediates the effects of structurally diverse chemicals. Ligand binding stimulates nuclear translocation of the AhR and leads to AhR DNA binding and increased gene expression. Studies of the molecular mechanisms by which ligands bind to and activate the AhR and AhR-dependent signal transduction require methods to easily examine each step of the AhR signaling pathway. While current assays can measure ligand and DNA binding in vitro and gene expression in cells, there is no simple method to monitor AhR nuclear translocation. We developed a stably transfected mouse hepatoma cell line (yAHAYc6) that expresses yellow fluorescent protein-tagged AhR (yAhR) for use in qualitative or semiquantitative assessment of nuclear/cytoplasmic distribution of yAhR in living cells by fluorescent microscopy. yAhR nuclear translocation was stimulated in a concentration- and time-dependent manner by AhR agonists and inhibited by antagonists. Inhibition of nuclear export channels by leptomycin B, resulted in increased nuclear accumulation of yAhR in the absence of added ligand, indicating endogenous nucleocytoplasmic shuttling of unliganded AhR and demonstrating the utility of these cells. This novel cell line can be used to detect and characterize AhR ligands and will facilitate mechanistic studies of AhR signaling.

Transitional States in Ligand-Dependent Transformation of the Aryl Hydrocarbon Receptor into Its DNA-Binding Form.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates the biological and toxicological effects of an AhR lacking the entire PASB structurally diverse chemicals, including halogenated aromatic hydrocarbons. Ligand-dependent transformation of the AhR into its DNA binding form involves a ligand-dependent conformational change, heat shock protein 90 (hsp90), dissociation from the AhR complex and AhR dimerization with the AhR nuclear translocator (ARNT) protein. The mechanism of AhR transformation was examined using mutational approaches and stabilization of the AhR:hsp90 complex with sodium molybdate. Insertion of a single mutation (F281A) in the hsp90-binding region of the AhR resulted in its constitutive (ligand-independent) transformation/DNA binding in vitro. Mutations of AhR residues within the Arg-Cys-rich region (R212A, R217A, R219A) and Asp371 (D371A) impaired AhR transformation without a significant effect on ligand binding. Stabilization of AhR:hsp90 binding with sodium molybdate decreased transformation/DNA binding of the wild type AhR but had no effect on constitutively active AhR mutants. Interestingly, transformation of the AhR in the presence of molybdate allowed detection of an intermediate transformation ternary complex containing hsp90, AhR, and ARNT. These results are consistent with a stepwise transformation mechanism in which binding of ARNT to the liganded AhR:hsp90 complex results in a progressive displacement of hsp90 and conversion of the AhR into its high affinity DNA binding form. The available molecular insights into the signaling mechanism of other Per-ARNT-Sim (PAS) domains and structural information on hsp90 association with other client proteins are consistent with the proposed transformation mechanism of the AhR.

Rapid extraction of total lipids and lipophilic POPs from all EU-regulated foods of animal origin: Smedes' method revisited and enhanced.

BACKGROUND: Persistent organic pollutants (POPs) such as dioxins, dioxin-like chemicals and non-dioxin-like PCBs causing adverse effects to human health bio-accumulate through the food web due to their affinity for adipose tissues. Foods of animal origin are therefore the main contributors to human dietary exposure. The European Union's (EU) food safety policy requires checking of a wide range of samples for compliance with legal limits on a regular basis. Several methods of varying efficiency are applied by official control laboratories for extraction of the different classes of lipids and associated POPs, bound to animal tissue and animal products in varying degrees, sometimes leading to discrepancies especially in fresh weight based analytical results. RESULTS: Starting from Smedes' lipid extraction from marine tissue, we optimized the extraction efficiency for both lipids and lipophilic pollutants, abandoning the time-consuming centrifugation step. The resulting modified Smedes extraction (MSE) method was validated based on multiple analyses of a large number of real world samples, matrix calibration and performance assessment in proficiency testing utilizing both instrumental and bioanalytical methodologies. Intermediate precision in 12 different foods was below 3% in chicken eggs, egg powder, animal fat, fish, fish oil, poultry, whole milk, milk fat and milk powder, and below 5% in bovine meat, liver, and infant food. In comparison to Twisselmann hot extraction, results presented here show an increased efficiency of MSE by +25% for bovine liver, +14% for chicken eggs, +13% for poultry meat, +12% for fish, 8% for bovine meat, and 6% for infant food. CONCLUSIONS: For the first time, a fast and reliable routine method is available that enables the analyst to reproducibly extract "total" lipids from any EU-regulated food sample of animal origin within 6 to 8 minutes. Increased efficiency translates into a considerable increase in both lipid and wet weight-based analytical results measured for associated POPs, reducing the risk of false non-compliant results. Compared to a 4 hour Twisselmann extraction, the extraction of 1000 samples using MSE would result in annual savings of about 250 hours or 32 working days. Our MSE procedure contributes to the European Commission's objective of harmonising analytical results across the EU generated according to Commission Regulation (EU) 2017/644.

A Biomimetic, One-Step Transformation of Simple Indolic Compounds to Malassezia-Related Alkaloids with High AhR Potency and Efficacy.

Malassezia furfur isolates from diseased skin preferentially biosynthesize compounds which are among the most active known aryl-hydrocarbon receptor (AhR) inducers, such as indirubin, tryptanthrin, indolo[3,2-b]carbazole, and 6-formylindolo[3,2-b]carbazole. In our effort to study their production from Malassezia spp., we investigated the role of indole-3-carbaldehyde (I3A), the most abundant metabolite of Malassezia when grown on tryptophan agar, as a possible starting material for the biosynthesis of the alkaloids. Treatment of I3A with H2O2 and use of catalysts like diphenyldiselenide resulted in the simultaneous one-step transformation of I3A to indirubin and tryptanthrin in good yields. The same reaction was first applied on simple indole and then on substituted indoles and indole-3-carbaldehydes, leading to a series of mono- and bisubstituted indirubins and tryptanthrins bearing halogens, alkyl, or carbomethoxy groups. Afterward, they were evaluated for their AhR agonist activity in recombinant human and mouse hepatoma cell lines containing a stably transfected AhR-response luciferase reporter gene. Among them, 3,9-dibromotryptanthrin was found to be equipotent to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) as an AhR agonist, and 3-bromotryptanthrin was 10-times more potent than TCDD in the human HG2L7.5c1 cell line. In contrast, 3,9-dibromotryptanthrin and 3-bromotryptanthrin were ∼4000 and >10,000 times less potent than TCDD in the mouse H1L7.5c3 cell line, respectively, demonstrating that they are species-specific AhR agonists. Involvement of the AhR in the action of 3-bromotryptanthrin was confirmed by the ability of the AhR antagonists CH223191 and SR1 to inhibit 3-bromotryptanthrin-dependent reporter gene induction in human HG2L7.5c1 cells. In conclusion, I3A can be the starting material used by Malassezia for the production of both indirubin and tryptanthrin through an oxidation mechanism, and modification of these compounds can produce some highly potent, efficacious and species-selective AhR agonists.

Modeling the binding of diverse ligands within the Ah receptor ligand binding domain.

The Ah receptor (AhR) is a ligand-dependent transcription factor belonging to the basic helix-loop-helix Per-Arnt-Sim (bHLH-PAS) superfamily. Binding to and activation of the AhR by a variety of chemicals results in the induction of expression of diverse genes and production of a broad spectrum of biological and toxic effects. The AhR also plays important roles in several physiological responses, which has led it to become a novel target for the development of therapeutic drugs. Differences in the interactions of various ligands within the AhR ligand binding domain (LBD) may contribute to differential modulation of AhR functionality. We combined computational and experimental analyses to investigate the binding modes of a group of chemicals representative of major classes of AhR ligands. On the basis of a novel computational approach for molecular docking to the homology model of the AhR LBD that includes the receptor flexibility, we predicted specific residues within the AhR binding cavity that play a critical role in binding of three distinct groups of chemicals. The prediction was validated by site-directed mutagenesis and evaluation of the relative ligand binding affinities for the mutant AhRs. These results provide an avenue for understanding ligand modulation of the AhR functionality and for rational drug design.

Vemurafenib acts as an aryl hydrocarbon receptor antagonist: Implications for inflammatory cutaneous adverse events.

BACKGROUND: In recent years, the BRAF inhibitor vemurafenib has been successfully established in the therapy of advanced melanoma. Despite its superior efficacy, the use of vemurafenib is limited by frequent inflammatory cutaneous adverse events that affect patients' quality of life and may lead to dose reduction or even cessation of anti-tumor therapy. To date, the molecular and cellular mechanisms of vemurafenib-induced rashes have remained largely elusive. METHODS: In this study, we deployed immunohistochemistry, RT-qPCR, flow cytometry, lymphocyte activation tests, and different cell-free protein-interaction assays. RESULTS: We here demonstrate that vemurafenib inhibits the downstream signaling of the canonical pathway of aryl hydrocarbon receptor (AhR) in vitro, thereby inducing the expression of proinflammatory cytokines (eg, TNF) and chemokines (eg, CCL5). In line with these results, we observed an impaired expression of AhR-regulated genes (eg, CYP1A1) and an upregulation of the corresponding proinflammatory genes in vivo. Moreover, results of lymphocyte activation tests showed the absence of drug-specific T cells in respective patients. CONCLUSION: Taken together, we obtained no hint of an underlying sensitization against vemurafenib but found evidence suggesting that vemurafenib enhances proinflammatory responses by inhibition of canonical AhR signaling. Our findings contribute to our understanding of the central role of the AhR in skin inflammation and may point toward a potential role for topical AhR agonists in supportive cancer care.

In situ measurement of estrogenic activity in various aquatic systems using organic diffusive gradients in thin-film coupled with ERE-CALUX bioassay.

Organic-diffusive gradients in thin-film samplers (o-DGT), were developed and applied for accumulation of estrogen and estrogen-like compounds on a XAD18 resin and deployed in situ in the effluents of Beijing Gaobeidian Wastewater Treatment Plant (GWWTP) and Brussels North Wastewater Treatment Plant as well as in several aquatic systems in Belgium, including the Zenne River, the Belgian Oostende Harbor and the North Sea. Estrogenic compounds accumulate on the XAD18 resin and the estrogenic activity of the resin extract was measured with the Estrogen Responsive Elements-Chemically Activated LUciferase gene eXpression (ERE-CALUX) bioassay. With this result and by applying Fick's diffusion law, it is possible to calculate the estrogenic activity in the aquatic system, if the diffusion boundary layer (DBL) is known or negligible compared to the hydrogel diffusive layer thickness. The DBL thickness in our study varied from 0.010 to 0.023 cm and ignoring the DBL thickness would for instance, underestimate the estrogenic activity by 10-20%. Estrogenic activities in the secondary effluent of GWWTP were the highest (29 ±â€¯4 ng E2-equivalents L-1), while the lowest level was found at the Belgian Oostende Harbor (0.05 ±â€¯0.01 ng E2-equivalents L-1). Comparable estrogenic activities in water samples measured by o-DGT and grab sampling were obtained, confirming that o-DGT can be efficiently used in various aquatic systems. The advantage of our sampling and measuring method is that very low, time averaged estrogenic activities can be determined, with a minimum of sample treatment. The risk of sample contamination is very low as well as the cost of the whole analytical procedure.

Sustained activation of the Aryl hydrocarbon Receptor transcription factor promotes resistance to BRAF-inhibitors in melanoma.

BRAF inhibitors target the BRAF-V600E/K mutated kinase, the driver mutation found in 50% of cutaneous melanoma. They give unprecedented anti-tumor responses but acquisition of resistance ultimately limits their clinical benefit. The master regulators driving the expression of resistance-genes remain poorly understood. Here, we demonstrate that the Aryl hydrocarbon Receptor (AhR) transcription factor is constitutively activated in a subset of melanoma cells, promoting the dedifferentiation of melanoma cells and the expression of BRAFi-resistance genes. Typically, under BRAFi pressure, death of BRAFi-sensitive cells leads to an enrichment of a small subpopulation of AhR-activated and BRAFi-persister cells, responsible for relapse. Also, differentiated and BRAFi-sensitive cells can be redirected towards an AhR-dependent resistant program using AhR agonists. We thus identify Resveratrol, a clinically compatible AhR-antagonist that abrogates deleterious AhR sustained-activation. Combined with BRAFi, Resveratrol reduces the number of BRAFi-resistant cells and delays tumor growth. We thus propose AhR-impairment as a strategy to overcome melanoma resistance.

Comparative In Vitro and In Silico Analysis of the Selectivity of Indirubin as a Human Ah Receptor Agonist.

The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that modulates gene expression following its binding and activation by structurally diverse chemicals. Species differences in AhR functionality have been observed, with the mouse AhR (mAhR) and human AhR (hAhR) exhibiting significant differences in ligand binding, coactivator recruitment, gene expression and response. While the AhR agonist indirubin (IR) is a more potent activator of hAhR-dependent gene expression than the prototypical ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), it is a significantly less potent activator of the mAhR. DNA binding analysis confirmed the greater potency/efficacy of IR in stimulating transformation/DNA binding of the hAhR in vitro and domain-swapping experiments demonstrated that the enhanced response to IR was primarily due to the hAhR ligand binding domain (LBD). Site-directed mutagenesis and functional analysis studies revealed that mutation of H326 and A349 in the mAhR LBD to the corresponding residues in the hAhR LBD significantly increased the potency of IR. Since these mutations had no significant effect on ligand binding, these residues likely contribute to an enhanced efficiency of transformation/DNA binding by IR-bound hAhR. Molecular docking to mAhR LBD homology models further elucidated the different roles of the A375V mutation in TCDD and IR binding, as revealed by [³H]TCDD competitive binding results. These results demonstrate the differential binding of structurally diverse ligands within the LBD of a given AhR and confirm that amino acid differences within the LBD of AhRs contribute to significant species differences in ligand response.

Aryl hydrocarbon receptor is activated in patients and mice with chronic kidney disease.

Patients with chronic kidney disease (CKD) are exposed to uremic toxins and have an increased risk of cardiovascular disease. Some uremic toxins, like indoxyl sulfate, are agonists of the transcription factor aryl hydrocarbon receptor (AHR). These toxins induce a vascular procoagulant phenotype. Here we investigated AHR activation in patients with CKD and in a murine model of CKD. We performed a prospective study in 116 patients with CKD stage 3 to 5D and measured the AHR-Activating Potential of serum by bioassay. Compared to sera from healthy controls, sera from CKD patients displayed a strong AHR-Activating Potential; strongly correlated with eGFR and with the indoxyl sulfate concentration. The expression of the AHR target genes Cyp1A1 and AHRR was up-regulated in whole blood from patients with CKD. Survival analyses revealed that cardiovascular events were more frequent in CKD patients with an AHR-Activating Potential above the median. In mice with 5/6 nephrectomy, there was an increased serum AHR-Activating Potential, and an induction of Cyp1a1 mRNA in the aorta and heart, absent in AhR-/- CKD mice. After serial indoxyl sulfate injections, we observed an increase in serum AHR-AP and in expression of Cyp1a1 mRNA in aorta and heart in WT mice, but not in AhR-/- mice. Thus, the AHR pathway is activated both in patients and mice with CKD. Hence, AHR activation could be a key mechanism involved in the deleterious cardiovascular effects observed in CKD.

Methylated polycyclic aromatic hydrocarbons and/or their metabolites are important contributors to the overall estrogenic activity of polycyclic aromatic hydrocarbon-contaminated soils.

In the present study 42 polycyclic aromatic compounds (PACs) were investigated for their estrogenic potential using the VM7Luc4E2 transactivation assay. Relative potencies were determined for mass-balance analysis. In addition, compounds were tested in combination with the estrogen receptor (ER) antagonist ICI182,780 (ICI) and the aryl hydrocarbon receptor antagonist/CYP1A1 inhibitor α-naphthoflavone. Luciferase induction and CYP1A1-dependent ethoxyresorufin-O-deethylase (EROD) activity were measured to assess whether the estrogenic activity was elicited by the compound itself and/or by its metabolites. Relative potencies ranged between 10-7 and 10-4 . The ability of ICI to decrease luciferase activity stimulated by all compounds indicated that the induction responses were ER-dependent. The aryl hydrocarbon receptor antagonist/CYP1A1 inhibitor α-naphthoflavone decreased luciferase induction and EROD activity by several compounds, including the methylated chrysenes, suggesting that metabolites of these chemicals contributed to ER activation. Several PACs, such as acridine and its derivatives, appear to directly activate the ER. Furthermore, extracts of soils from industrial areas were examined using this bioassay, and estrogenic activity was detected in all soil samples. Mass-balance analysis using a combination of relative potencies and chemical analysis of the samples suggested that polycyclic aromatic hydrocarbons (PAHs) and alkylated PAHs, such as 1- and 3-methylchrysene, are important contributors to the overall estrogenic activity. However, these results revealed that a considerable proportion of the estrogenic activity in the soil remained unexplained, indicating the presence of other significant estrogenic compounds. Environ Toxicol Chem 2018;37:385-397. © 2017 SETAC.