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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.

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.

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.

Funder: VUB Strategic Research Program, Exposure to volcanic ash is a long-standing health concern for people living near active volcanoes and in distal urban areas. During transport and deposition, ash is subjected to various physicochemical processes that may change its surface composition and, consequently, bioreactivity. One such process is the interaction with anthropogenic pollutants; however, the potential for adsorbed, deleterious organic compounds to directly impact human health is unknown. We use an in vitro bioanalytical approach to screen for the presence of organic compounds of toxicological concern on ash surfaces and assess their biological potency. These compounds include polycyclic aromatic hydrocarbons (PAHs), polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) and dioxin-like polychlorinated biphenyls (dlPCBs). Analysis of ash collected in or near urbanised areas at five active volcanoes across the world (Etna, Italy; Fuego, Guatemala; Kelud, Indonesia; Sakurajima, Japan; Tungurahua, Ecuador) using the bioassay inferred the presence of such compounds on all samples. A relatively low response to PCDD/Fs and the absence of a dlPCBs response in the bioassay suggest that the measured activity is dominated by PAHs and PAH-like compounds. This study is the first to demonstrate a biological potency of organic pollutants associated with volcanic ash particles. According to our estimations, they are present in quantities below recommended exposure limits and likely pose a low direct concern for human health.

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., SCOPUS: ar.j, info:eu-repo/semantics/published

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 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.

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.

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.

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-aenial 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-aenial 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.

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 min. 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 h Twisselmann extraction, the extraction of 1000 samples using MSE would result in annual savings of about 250 h or 32 working days. Our MSE procedure contributes to the European Commission's objective of harmonizing analytical results across the EU generated according to Commission Regulation (EU) 2017/644.

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.

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.

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-tetracholordibenzo-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 fromXenopus laevis(a frog; order Anura) andAmbystoma mexicanum(a salamander; order Urodela) identified three amino acid residues in the ligand-binding domain (LBD) that underlie low-affinity binding. InX. laevisAHR1β, these are A354, A370, and N325. Here we extend the analysis of amphibian AHRs to the caecilianGymnopis multiplicata, representing the remaining extant amphibian order, Apoda.G. multiplicataAHR 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 toX. laevisAhR1β (26.23 nM) andAmbystomaAHR (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 more ancient vertebrate groups (teleost fish) and those that appeared more recently (tetrapods). We suggest that AHRs with low TCDD affinity represent a basal characteristic that evolved in a common ancestor of all three extant amphibian groups.Research HighlightsA caecilian aryl hydrocarbon receptor exhibits low dioxin binding and sensitivity.The protein’s ligand-binding domain resembles frog and salamander AHRs in structure and function.AHR with low dioxin affinity likely evolved in a common ancestor of all three extant amphibian groups.Graphical Abstract

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.

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 H(2)O(2) 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 bi-substituted indirubins and tryptanthrins bearing halogens, alkyl or carbomethoxy groups. Afterwards, 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 ~4,000- 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.

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.

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 ± 4 ng E2-equivalents L−1), while the lowest level was found at the Belgian Oostende Harbor (0.05 ± 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. Keywords: Estrogenic activity, o-DGT, CALUX, DBL, Effluent, Sea water

BACKGROUND: Endocrine disrupting chemicals represent a broad class of compounds, are widespread in the environment and can pose severe health effects. OBJECTIVES: The objective of this study was to investigate and compare the overall estrogen and androgen activating potential of PM(10) air samples at an urban, rural and industrial location in Flanders, using a human in vitro cell bioassay. METHODS: PM(10) samples were collected on glass fiber filters every six days between April 2013 and January 2014 using a high-volume sampler. Extraction was executed with a hexane/acetone mixture before analysis using a recombinant estrogen- or androgen responsive human carcinoma cell line. Results were expressed as bioanalytical equivalents (BEQs) per cubic meter of air. RESULTS: High fluctuations in estrogenic activity were observed during the entire sampling period, with median BEQs of 32.1, 35.9 and 31.1 fg E2-Eq m(−3) in the industrial, urban and rural background area, respectively. Estrogenic activity was measured in 70% of the samples, while no androgenic activity was observed in any of the samples. The estrogenic activity in the industrial area was positively correlated with the airborne concentration of the sum of the non-carcinogenic PAHs pyrene and fluoranthene (rho = 0.48; p < 0.01) and the sum of the carcinogenic PAHs (rho = 0.36 p = 0.05). CONCLUSIONS: This study showed that no androgenic activity was present in PM(10) and that although the median estrogenic activity was rather low and comparable in the three locations, high fluctuations in estrogenic response exist over time. While atmospheric PAHs contributed to the observed estrogenic response, especially in the industrial area, the chemicals responsible for the majority of estrogenic activity remain to be identified.

Methyl-tert-butyl ether (MTBE) and its degradation by-product, tert-butyl alcohol (TBA), are widespread contaminants detected frequently in groundwater in California. Since MTBE was used as a fuel oxygenate for almost two decades, leaking underground fuel storage tanks are an important source of contamination. Gasoline components such as BTEX (benzene, toluene, ethylbenzene and xylenes) are often present in mixtures with MTBE and TBA. Investigations of interactions between BTEX and MTBE degradation have not yielded consistent trends, and the molecular mechanisms of BTEX compounds' impact on MTBE degradation are not well understood. We investigated trends in transcription of biodegradation genes in the MTBE-degrading bacterium, Methylibium petroleiphilum PM1 upon exposure to MTBE, TBA, ethylbenzene and benzene as individual compounds or in mixtures. We designed real-time quantitative PCR assays to target functional genes of strain PM1 and provide evidence for induction of genes mdpA (MTBE monooxygenase), mdpJ (TBA hydroxylase) and bmoA (benzene monooxygenase) in response to MTBE, TBA and benzene, respectively. Delayed induction of mdpA and mdpJ transcription occurred with mixtures of benzene and MTBE or TBA, respectively. bmoA transcription was similar in the presence of MTBE or TBA with benzene as in their absence. Our results also indicate that ethylbenzene, previously proposed as an inhibitor of MTBE degradation in some bacteria, inhibits transcription of mdpA, mdpJ and bmoAgenes in strain PM1.

© 2016, Springer-Verlag Berlin Heidelberg. The toxic effects of dioxins and related compounds (DRCs) are mediated by the aryl hydrocarbon receptor (AHR). Our previous study identified AHR1 and AHR2 genes from the red seabream (Pagrus major). Moreover, we found that AHR2 mRNA levels were notably elevated by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure in the early life stage of red seabream embryos, while AHR1 mRNA level was not altered. In this study, to investigate the regulatory mechanism of these AHR transcripts, we cloned and characterized 5′-flanking regions of AHR1 and AHR2 genes. Both of the 5′-flanking regions in these AHR genes contained three potential xenobiotic-responsive elements (XREs). To assess whether the 5′-flanking region is transactivated by rsAHR1 and rsAHR2 proteins, we measured the transactivation potency of the luciferase reporter plasmids containing the 5′-flanking regions by AHR1 and AHR2 proteins that were transiently co-expressed in COS-7. Only reporter plasmid (pGL4-rsAHR2-3XREs) that contained three putative XRE sites in the 5′-flanking region of AHR2 gene showed a clear TCDD dose-dependent transactivation by AHR1 and AHR2 proteins. TCDD-EC50 values for the rsAHR2-derived XRE transactivation were 1.3 and 1.4 nM for AHR1 and AHR2, respectively. These results suggest that the putative XREs of AHR2 gene have a function for AHR1- and AHR2-mediated transactivation, supporting our in ovo observation of an induction of AHR2 mRNA levels by TCDD exposure. Mutations in XREs of AHR2 gene led to a decrease in luciferase induction. Electrophoretic mobility shift assay showed that XRE1, the closest XRE from the start codon in AHR2 gene, is mainly responsible for the binding with TCDD-activated AHR. This suggests that TCDD-activated AHR1 and AHR2 up-regulate the AHR2 mRNA levels and this auto-induced AHR2 may amplify the signal transduction of its downstream targets including CYP1A in the red seabream.

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.

© 2018 by the authors. Licensee MDPI, Basel, Switzerland. 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.

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.

Estrogenic endocrine-disrupting chemicals are found in environmental and biological samples, commercial and consumer products, food, and numerous other sources. Given their ubiquitous nature and potential for adverse effects, a critical need exists for rapidly detecting these chemicals. The authors developed an estrogen-responsive recombinant human ovarian (BG1Luc4E2) cell line recently accepted by the US Environmental Protection Agency (USEPA) and Organisation for Economic Co-operation and Development (OECD) as a bioanalytical method to detect estrogen receptor (ER) agonists/antagonists. Unfortunately, these cells appear to contain only 1 of the 2 known ER isoforms, ERα but not ERβ, and the differential ligand selectivity of these ERs indicates that the currently accepted screening method only detects a subset of total estrogenic chemicals. To improve the estrogen screening bioassay, BG1Luc4E2 cells were stably transfected with an ERβ expression plasmid and positive clones identified using ERβ-selective ligands (genistein and Br-ERβ-041). A highly responsive clone (BG1LucERβc9) was identified that exhibited greater sensitivity and responsiveness to ERβ-selective ligands than BG1Luc4E2 cells, and quantitative reverse-transcription polymerase chain reaction confirmed the presence of ERβ expression in these cells. Screening of pesticides and industrial chemicals identified chemicals that preferentially stimulated ERβ-dependent reporter gene expression. Together, these results not only demonstrate the utility of this dual-ER recombinant cell line for detecting a broader range of estrogenic chemicals than the current BG1Luc4E2 cell line, but screening with both cell lines allows identification of ERα- and ERβ-selective chemicals.

When compared to the European guidelines, PM10 (particulate matter up to 10-μm size) concentrations in Algeria are often exceeding the maximum limits, and in general, no information exists on the compounds bound on its surface. The objective of this study was to measure the dioxin-like activity of polychlorinated dibenzodioxines and dibenzofurans (PCDD/Fs) and dioxin-like polychlorinated biphenyls (PCBs) in the PM10 fraction at the Sour El Ghozlane cement plant in Algeria. PM10 samples (n = 23) were taken between 24 March and 15 April 2013, using a medium volume sampler and 47-mm PTFE filters. The 24-h samples were dried to determine the PM10 content and afterward extracted, cleaned up, and analyzed with the dioxin-responsive element–chemical-activated luciferase gene expression (DRE-CALUX) bioassay. Our results showed that the measured bioanalytical equivalents (BEQs) were similar to those in other international industrial sites worldwide. The PCDD/Fs and dioxin-like PCBs (dl-PCBs) were positively correlated (rho = 0.6, p = 0.002), indicating that they have similar sources. Furthermore, samples from March showed higher PCDD/F and dl-PCB BEQs and humidity but lower temperatures compared to samples from April, while there was no difference in the PM10 concentrations between the two months. These results reveal that PM10 alone is not a good proxy and that meteorological conditions are an important factor in assessing dioxin-like pollution in the atmosphere. It seems that, at present, there is no health hazard through direct airborne human exposure to dioxin-like pollutants in PM10 from this site. However, it is important to monitor these POPs for a longer period of time and also to gain more insight in their distribution between the particulate and gas phase in relation to meteorological conditions.

Background The Scheldt estuary is historically a highly polluted river system. While several studies have focused on contamination with metals, pesticides, Polycyclic Aromatic Hydrocarbons (PAHs) and marker PolyChlorinated Biphenyls (PCBs), no data are available concerning past contamination by dioxin-like compounds. Objectives The objective of this study is to determine spatial and time trends of PolyChlorinated Dibenzo-p-Dioxins and DibenzoFurans (PCDD/Fs) and dioxin-like PCBs (dl-PCBs) in sediment samples and Suspended Particulate Matter (SPM) from the Scheldt River basin and the North Sea Coast. Methods Dioxin-like compounds (PCDD/F and dl-PCB fractions) were measured with the CALUX-bioassay. Bioanalytical EQuivalent concentrations (BEQs) and Total Organic Carbon (TOC) content of historical (1982–1984) and recent (2011–2015) sediment and SPM samples from different locations in the coastal area and the estuary, were evaluated. Results A decrease in dioxin-like compound concentrations was found at all stations over time, especially for the PCDD/Fs. Dl-PCBs were relatively low in all samples. The Scheldt mouth and the Antwerp harbor yielded the highest BEQs and levels were higher in SPM than in sediment due to the higher organic carbon content in this fraction. Conclusions Current PCDD/F and dl-PCB levels in the Belgian Coastal Zone and Scheldt estuary are much lower than their levels 30 years ago and pose a relatively low risk to the aquatic system. This is the result of a strong decrease in emissions, however, large local variabilities in sediment concentration levels can still exist because of local variability in sedimentation, erosion rates and in organic carbon content.

© 2017, Public Health Services, US Dept of Health and Human Services. All rights reserved. Background: Quaternary ammonium salts (QUATS), such as cetylpyridinium chloride (CPC) and benzalkonium chloride (BAK), are frequently used in antiseptic formulations, including toothpastes, mouthwashes, lozenges, throat and nasal sprays, and as biocides. Although in a recent ruling, the U.S. Food and Drug Administration (FDA) banned CPC from certain products and requested more data on BAK’s efficacy and safety profile, QUATS, in general, and CPC and BAK, in particular, continue to be used in personal health care, food, and pharmaceutical and cleaning industries. Objectives: We aimed to assess CPC's effects on mitochondrial toxicity and endocrine disruption in vitro. METHOD: Mitochondrial O2consumption and adenosine triphosphate (ATP) synthesis rates of osteosarcoma cybrid cells were measured before and after CPC and BAK treatment. Antiestrogenic effects of the compounds were measured by a luciferase-based assay using recombinant human breast carcinoma cells (VM7Luc4E2, ERalpha-positive). Results: CPC inhibited both mitochondrial O2consumption [half maximal inhibitory concentration (IC50): 3.8 μM] and ATP synthesis (IC50: 0.9 μM), and additional findings supported inhibition of mitochondrial complex 1 as the underlying mechanism for these effects. In addition, CPC showed concentration-dependent antiestrogenic activity half maximal effective concentration [(EC50): 4.5 μM)]. BAK, another antimicrobial QUATS that is structurally similar to CPC, and the pesticide rotenone, a known complex 1 inhibitor, also showed mitochondrial inhibitory and antiestrogenic effects. In all three cases, there was overlap of the antiestrogenic activity with the mitochondrial inhibitory activity. Conclusions: Mitochondrial inhibition in vitro occurred at a CPC concentration that may be relevant to human exposures. The antiestrogenic activity of CPC, BAK, rotenone, and triclosan may be related to their mitochondrial inhibitory activity. Our findings support the need for additional research on the mitochondrial inhibitory and antiestrogenic effects of QUATS, including CPC and BaK.

In June 2005, a World Health Organization (WHO)-International Programme on Chemical Safety expert meeting was held in Geneva during which the toxic equivalency factors (TEFs) for dioxin-like compounds, including some polychlorinated biphenyls (PCBs), were reevaluated. For this reevaluation process, the refined TEF database recently published by Haws et al. (2006, Toxicol. Sci. 89, 4-30) was used as a starting point. Decisions about a TEF value were made based on a combination of unweighted relative effect potency (REP) distributions from this database, expert judgment, and point estimates. Previous TEFs were assigned in increments of 0.01, 0.05, 0.1, etc., but for this reevaluation, it was decided to use half order of magnitude increments on a logarithmic scale of 0.03, 0.1, 0.3, etc. Changes were decided by the expert panel for 2,3,4,7,8-pentachlorodibenzofuran (PeCDF) (TEF = 0.3), 1,2,3,7,8-pentachlorodibenzofuran (PeCDF) (TEF = 0.03), octachlorodibenzo-p-dioxin and octachlorodibenzofuran (TEFs = 0.0003), 3,4,4',5-tetrachlorbiphenyl (PCB 81) (TEF = 0.0003), 3,3',4,4',5,5'-hexachlorobiphenyl (PCB 169) (TEF = 0.03), and a single TEF value (0.00003) for all relevant mono-ortho-substituted PCBs. Additivity, an important prerequisite of the TEF concept was again confirmed by results from recent in vivo mixture studies. Some experimental evidence shows that non-dioxin-like aryl hydrocarbon receptor agonists/antagonists are able to impact the overall toxic potency of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds, and this needs to be investigated further. Certain individual and groups of compounds were identified for possible future inclusion in the TEF concept, including 3,4,4'-TCB (PCB 37), polybrominated dibenzo-p-dioxins and dibenzofurans, mixed polyhalogenated dibenzo-p-dioxins and dibenzofurans, polyhalogenated naphthalenes, and polybrominated biphenyls. Concern was expressed about direct application of the TEF/total toxic equivalency (TEQ) approach to abiotic matrices, such as soil, sediment, etc., for direct application in human risk assessment. This is problematic as the present TEF scheme and TEQ methodology are primarily intended for estimating exposure and risks via oral ingestion (e.g., by dietary intake). A number of future approaches to determine alternative or additional TEFs were also identified. These included the use of a probabilistic methodology to determine TEFs that better describe the associated levels of uncertainty and "systemic" TEFs for blood and adipose tissue and TEQ for body burden.

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

Surface waters can contain a diverse range of organic pollutants, including pesticides, pharmaceuticals and industrial compounds. While bioassays have been used for water quality monitoring, there is limited knowledge regarding the effects of individual micropollutants and their relationship to the overall mixture effect in water samples. In this study, a battery of in vitro bioassays based on human and fish cell lines and whole organism assays using bacteria, algae, daphnids and fish embryos was assembled for use in water quality monitoring. The selection of bioassays was guided by the principles of adverse outcome pathways in order to cover relevant steps in toxicity pathways known to be triggered by environmental water samples. The effects of 34 water pollutants, which were selected based on hazard quotients, available environmental quality standards and mode of action information, were fingerprinted in the bioassay test battery. There was a relatively good agreement between the experimental results and available literature effect data. The majority of the chemicals were active in the assays indicative of apical effects, while fewer chemicals had a response in the specific reporter gene assays, but these effects were typically triggered at lower concentrations. The single chemical effect data were used to improve published mixture toxicity modeling of water samples from the Danube River. While there was a slight increase in the fraction of the bioanalytical equivalents explained for the Danube River samples, for some endpoints less than 1% of the observed effect could be explained by the studied chemicals. The new mixture models essentially confirmed previous findings from many studies monitoring water quality using both chemical analysis and bioanalytical tools. In short, our results indicate that many more chemicals contribute to the biological effect than those that are typically quantified by chemical monitoring programs or those regulated by environmental quality standards. This study not only demonstrates the utility of fingerprinting single chemicals for an improved understanding of the biological effect of pollutants, but also highlights the need to apply bioassays for water quality monitoring in order to prevent underestimation of the overall biological effect.

© 2017 Ligand-dependent activation of the Ah receptor (AhR) can result in an extremely diverse spectrum of biological and toxic effects that occur in a ligand-, species- and tissue-specific manner. While the classical mechanism of AhR-dependent signal transduction is directly related to its ability to modulate gene expression, the dramatic diversity in responses observed following AhR activation or inhibition is inconsistent with a single molecular mechanism of AhR action. Recent studies have revealed that key molecular events underlying the AhR signaling pathway are significantly more varied and complex than previously established, and the specificity and diversity in AhR response can be selectively modulated by a variety of factors. Here we describe new insights into the mechanistic diversity in AhR signal transduction that can contribute to ligand-, species- and tissue-specific differences in AhR response.

The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that can be activated by structurally diverse chemicals. To examine the mechanisms responsible for the promiscuity in AhR ligand binding, we determined the effects of mutations within the AhR ligand-binding domain (LBD) on the activity of diverse AhR ligands. Site-directed mutagenesis identified Ile319 of the mouse AhR and, to a lesser extent, Phe318 as residues involved in ligand-selective modulation of AhR transformation using a panel of 12 AhR ligands. These ligands could be categorized into four distinct structurally related groups based on their ability to activate AhR mutants at position 319 in vitro. The mutation I319K was selectively activated by FICZ and not by other examined ligands in vitro and in cell culture. F318L and F318A mutations resulted in the conversion of AhR agonists β-naphthoflavone and 3-methylcholanthrene, respectively, into partial agonists/ antagonists. Hsp90 binding to the AhR was decreased with several mutations and was inversely correlated with AhR ligand-binding promiscuity. Together, these data define overlapping amino acid residues within the AhR LBD involved in the selectivity of ligand binding, the agonist or antagonist mode of ligand binding, and hsp90 binding and provide insights into the ligand diversity of AhR activators. © 2014, American Society for Microbiology.

The aryl hydrocarbon receptor (AhR) is involved in the regulation of immune responses, T-cell differentiation, and immunity. Here, we show that inflammatory stimuli such as LPS induce the expression of AhR in human dendritic cells (DC) associated with an AhR-dependent increase of CYP1A1 (cytochrome P4501A1). In vivo data confirmed the elevated expression of AhR by LPS and the LPS-enhanced 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-mediated induction of CYP1A1 in thymus of B6 mice. Inhibition of nuclear factor-κB (NF-κB) repressed both normal and LPS-enhanced, TCDD-inducible, AhR-dependent gene expression and canonical pathway control of RelA-regulated AhR-responsive gene expression. LPS-mediated induction of AhR was NF-κB-dependent, as shown in mouse embryonic fibroblasts (MEFs) derived from Rel null mice. AhR expression and TCDD-mediated induction of CYP1A1 was significantly reduced in RelA-deficient MEF compared with wild type MEF cells and ectopic expression of RelA restored the expression of AhR and induction of CYP1A1 in MEF RelA null cells. Promoter analysis of the human AhR gene identified three putative NF-κB-binding elements upstream of the transcription start site. Mutation analysis of the AhR promoter identified one NF-κB site as responsible for mediating the induction of AhR expression by LPS and electrophoretic shift assays demonstrated that this NF-κB motif is recognized by the RelA/p50 heterodimer. Our results show for the first time that NF-κB RelA is a critical component regulating the expression of AhR and the induction of AhR-dependent gene expression in immune cells illustrating the interaction of AhR and NF-κB signaling.

Malassezia yeasts are commensal microorganisms, which under insufficiently understood conditions can become pathogenic. We have previously shown that specific strains isolated from diseased human skin can preferentially produce agonists of the aryl hydrocarbon receptor (AhR), whose activation has been linked to certain skin diseases. Investigation of skin scale extracts from patients with Malassezia-associated diseases demonstrated 10- to 1,000-fold higher AhR-activating capacity than control skin extracts. Liquid chromatography-tandem mass spectrometry analysis of the patients' extracts revealed the presence of indirubin, 6-formylindolo[3,2-b]carbazole (FICZ), indolo[3,2-b]carbazole (ICZ), malassezin, and pityriacitrin. The same compounds were also identified in 9 out of 12 Malassezia species culture extracts tested, connecting their presence in skin scales with this yeast. Studying the activity of the Malassezia culture extracts and pure metabolites in HaCaT cells by reverse transcriptase real-time PCR revealed significant alterations in mRNA levels of the endogenous AhR-responsive genes Cyp1A1, Cyp1B1, and AhRR. Indirubin- and FICZ-activated AhR in HaCaT and human HepG2 cells with significantly higher, yet transient, potency as compared with the prototypical AhR ligand, dioxin. In loco synthesis of these highly potent AhR inducers by Malassezia yeasts could have a significant impact on skin homeostatic mechanisms and disease development.

Anthocyanins are plant pigments occurring in flowers and berry fruits. Since a phenomenon of food-drug interactions is increasingly emerging, we examined the effects of 21 major anthocyanins and the extracts from 3 food supplements containing anthocyanins on the aryl hydrocarbon receptor (AhR) – cytochrome P450 CYP1A1 signaling pathway in human hepatocytes and human hepatic HepG2 and intestinal LS174T cancer cells. Pelargonidin-3-O-rutinoside (PEL-2) and cyanidin-3,5-O-diglucoside (CYA-3) dose-dependently activated AhR, as revealed by gene reporter assay. PEL-2 and CYA-3 induced CYP1A1 mRNA but not protein in HepG2 and LS174T cells. Neither compound induced CYP1A1 mRNA and protein in four different primary human hepatocytes cultures. The effects of PEL-2 and CYA-3 on AhR occurred by ligand-dependent and ligand-independent mechanisms, respectively, as demonstrated by ligand binding assay. In a direct enzyme inhibition assay, none of the antocyanins tested inhibited the CYP1A1 marker activity to less than 50% even at 100 µM concentration. PEL-2 and CYA-3 at 100 µM inhibited CYP1A1 to 79% and 65%, respectively. In conclusion, with exception of PEL-2 and CYA-3, there were no effects of 19 major anthocyanins and 3 food supplements containing anthocyanins on AhR-CYP1A1 signaling, implying zero potential of these compounds for food-drug interactions with respect to AhR-CYP1A1 pathway.

We examined the effects of anthocyanidins (cyanidin, delphinidin, malvidin, peonidin, petunidin, pelargonidin) on the aryl hydrocarbon receptor (AhR)-CYP1A1 signaling pathway in human hepatocytes, hepatic HepG2 and intestinal LS174T cancer cells. AhR-dependent reporter gene expression in transfected HepG2 cells was increased by pelargonidin in a concentration-dependent manner at 24h. Similarly, pelargonidin induced the expression of CYP1A1 mRNA up to 5-fold in HepG2 and LS174T cells relative to the induction by 5 nM 2,3,7,8-tetrachlorodibenzodioxin (TCDD), the most potent activator of AhR. CYP1A1 and CYP1A2 mRNAs were also increased by pelargonidin in three primary human hepatocytes cultures (approximately 5% of TCDD potency) and the increase in CYP1A1 protein in HepG2 and LS174T cells was comparable to the increase in catalytic activity of CYP1A1 enzyme. Ligand binding analysis demonstrated that pelargonidin was a weak ligand of AhR. Enzyme kinetic analyses using human liver microsomes revealed inhibition of CYP1A1 activity by delphinidin (IC50 78 μM) and pelargonidin (IC50 33 μM). Overall, although most anthocyanidins had no effects on AhR-CYP1A1 signaling, pelargonidin can bind to and activate the AhR and AhR-dependent gene expression, and pelargonidin and delphinidin inhibit the CYP1A1 catalytic activity.

International audience; Biosolids are a potentially valuable source of carbon and nutrients for agricultural soils; however, potential unintended impacts on human health and the environment must be considered. Virtually all biosolids contain trace amounts endocrine-disrupting chemicals derived from human use of pharmaceuticals and personal care products (PPCPs). One potential way to reduce the bioavailability of PPCPs is to co-apply biosolids with biochar to soil, because biochar's chemical (e.g., aromaticity) and physical properties (e.g., surface area) give it a high affinity to bind many organic chemicals in the environment. We developed a soil-specific extraction method and utilized a luciferase-based bioassay (CALUX) to detect endocrine active chemicals in a biosolids-biochar co-amendment soil greenhouse study. Both biochar (walnut shell, 900 °C) and biosolids had positive impacts on carrot and lettuce biomass accumulation over our study period. However, the walnut shell biochar stimulated aryl hydrocarbon receptor activity, suggesting the presence of potential endocrine active chemicals in the biochar. Since the biochar rate tested (100 t ha-1) is above the average agronomic rate (10-20 t ha-1), endocrine effects would not be expected in most environmental applications. The effect of high temperature biochars on endocrine system pathways must be explored further, using both quantitative analytical tools to identify potential endocrine active chemicals and highly sensitive bioanalytical assays such as CALUX to measure the resulting biological activity of such compounds.