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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 [ 3 H]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.

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., (Copyright © 2021 The Academy of Dental Materials. Published by Elsevier Inc. All rights reserved.)

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., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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.

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., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

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.

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., Competing Interests: Competing interestsThe authors declare that they have no competing interests., (© The Author(s) 2021.)

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 AHR1β, 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 AhR1β (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., (Copyright © 2020 Elsevier Inc. All rights reserved.)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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.

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., Competing Interests: Declarations Competing interests The authors declare that they have no competing interests.

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., (© 2019 EAACI and John Wiley and Sons A/S. Published by John Wiley and Sons Ltd.)

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 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.

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., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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.

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., (Copyright © 2017 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

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., (© 2017 SETAC.)

A novel type of diffusive gradients in thin-film (DGT) was combined with a chemically activated luciferase gene expression bioassay (CALUX) to measure estrogens in aquatic systems. The performance of this novel method was assessed with 17β-estradiol (E2) as the model steroid hormone, XAD 18 resin gel as the binding phase in the DGT method and VM7Luc4E2 cells (formerly BG1Luc4E2) for the Estrogen Responsive Element (ERE)-CALUX bioassay. The measured effective diffusion coefficient of E2 in agarose diffusive gel was 4.65 ± 0.37 × 10 -6 cm 2 s -1 at 25 °C. The detection limit of this combined DGT/ERE-CALUX method for 1 day of sampling (0.026 ± 0.003 ng L -1 of E2) is significantly lower than that obtained by spot sampling combined with GC-MS/MS or LC-MS/MS analysis (0.1-7.0 ng L -1 ). The method is independent of pH (5-8), ionic strength (0.001-0.5 M), and dissolved organic matter (DOM; concentrations up to 30 mg L -1 ). Field applications of this novel DGT in effluents of three sewage treatment plants in Beijing city (China) showed comparable results to conventional spot (grab) sampling. This study demonstrates that the combined DGT/ERE-CALUX approach is an effective and sensitive tool for in situ monitoring of estrogenic activity in waters and wastewaters.

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., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Laboratory safety requires protecting personnel from chemical exposures. Working with stock solutions of polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/PCDFs) in routine analysis of feed and food with bioanalytical or physicochemical methods raises some concerns. Since PCDD/PCDFs are considered as possibly acutely toxic, the potential risks were evaluated to determine whether supervision of their use is necessary. Based on LD 50 -data for oral or dermal intake, hazard classification of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) as a substance (category 1) and in commercially available TCDD standard solutions (category 4) is different. As worst case exposure scenario during routine laboratory work it was assumed that a dose of 100 ng TCDD gets onto the skin and is absorbed. This would result in the total body burden of a 70 kg person with 15 kg fat increasing from 10 (upper range of current background levels) to ∼17 pg of toxic equivalents (TEQs) of PCDD/PCDFs per g lipid, a level commonly observed over past decades. Chloracne, the main acute effect occurring weeks after exposure, is observed at much higher blood concentrations than estimated from accidental laboratory exposure. Immunotoxicity, developmental effects and other toxic effects may occur at lower blood levels, but require longer periods to develop. Since acute toxic symptoms don't occur within an "8 h acute time window", no supervision is necessary when working with standard solutions in routine analysis. Nevertheless, precautionary measures are needed regarding long-term adverse health effects and appropriate workplace conditions must exist to ensure that additional occupational exposure to PCDD/PCDFs by laboratory personnel is negligible., (Copyright © 2017 Elsevier Ltd. 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 O 2 consumption 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 O 2 consumption [half maximal inhibitory concentration (IC 50 ): 3.8μM] and ATP synthesis (IC 50 : 0.9μM), 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 [(EC 50 ): 4.5μM)]. 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. https://doi.org/10.1289/EHP1404.

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., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Elucidation of the dimerization process of the aryl hydrocarbon receptor (AhR) with the AhR nuclear translocator (ARNT) is crucial for understanding the mechanisms underlying the functional activity of AhR, including mediation of the toxicity of environmental contaminants. In this work, for the first time a structural model of the AhR:ARNT dimer encompassing the entire bHLH-PASA-PASB domain region is proposed. It is developed by using a template-based modeling approach, relying on the recently available crystallographic structures of two dimers of homologous systems in the bHLH-PAS family of proteins: the CLOCK:BMAL1 and the HIF2α:ARNT heterodimers. The structural and energetic characteristics of the modeled AhR:ARNT protein-protein interface are determined by evaluating the variations in solvent accessible surface area, the total binding free energy and the per-residue free energy contributions obtained by the MM-GBSA method and the Energy Decomposition Analysis. The analyses of the intricate network of inter-domain interactions at the dimerization interfaces provide insights into the key determinants of dimerization. These are confirmed by comparison of the computational findings with the available experimental mutagenesis and functional analysis data. The results presented here on the AhR:ARNT dimer structure and interactions provide a framework to start analyzing the mechanism of AhR transformation into its functional DNA binding form.

When compared to the European guidelines, PM 10 (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 PM 10 fraction at the Sour El Ghozlane cement plant in Algeria. PM 10 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 PM 10 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 PM 10 concentrations between the two months. These results reveal that PM 10 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 PM 10 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.

Inorganic arsenic is a human toxicant and carcinogen that has been extensively studied over decades; however, no definitive understanding of the underlying mechanisms has been established yet. Arsenic is capable of modulating the expression of aryl hydrocarbon receptor (AhR)-regulated genes, nevertheless, whether its trivalent organic metabolites have similar effects or not need to be investigated. Therefore, in this study we examined the effects of monomethylarsonous acid (MMA(III)) as compared to its parent compound sodium arsenite (As(III)) on the expression of CYP1A1 in HepG2 cells. HepG2 cells were treated with MMA(III) (5μM) or its parents compound, As(III) (5μM), in the absence and presence of the prototypical AhR ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 1nM). Experiments were conducted at 6h for gene expression; 24h for XRE-driven luciferase activity, protein expression, and EROD activity. Our results showed that both MMA(III) and As(III) decreased CYP1A1 mRNA, protein, and catalytic activity levels; and inhibit the TCDD-mediated induction of CYP1A1 mRNA, protein, and catalytic activity levels. MMA(III) and As(III) significantly inhibited XRE-driven luciferase activity and it inhibited the TCDD-mediated induction of XRE-driven luciferase reporter gene expression. Although MMA(III) and As(III) were not shown to be AhR ligands, both compounds showed inhibition of nuclear accumulation of AhR transcription factor as evidenced by immunocytochemical analysis. MMA(III) and As(III) had no effect on CYP1A1 mRNA stability; however MMA(III), but not As(III), decreased the protein stability of CYP1A1. As(III), but not MMA(III), induced HO-1 mRNA levels. Both MMA(III) and As(III) increased ROS production. Our results demonstrate for the first time that, MMA(III) down-regulates CYP1A1 mainly through transcriptional and post-translational mechanisms. This modulation of CYP1A1 proves that trivalent metabolites of arsenic are highly reactive and could participate in arsenic toxicity., (Copyright © 2017 Elsevier B.V. All rights reserved.)

1,4-Dihydroxy-2-naphthoic acid (1,4-DHNA) is a bacterial-derived metabolite that binds the aryl hydrocarbon receptor (AhR) and exhibits anti-inflammatory activity in the gut. The structure-dependent AhR activity of hydroxyl/carboxy-substituted naphthoic acids (NAs) was determined in young adult mouse colonic (YAMC) cells and human Caco2 colon cancer cells using CYP1A1/CYP1B1 mRNAs as Ah-responsive genes. Compounds used in this study include 1,4-, 3,5-, and 3,7-DHNA, 1,4-dimethoxy-2-naphthoic acid (1,4-DMNA), 1- and 4-hydroxy-2-naphthoic acid (1-HNA, 4-HNA), 1- and 2-naphthoic acid (1-NA, 2-NA), and 1- and 2-naphthol (1-NOH, 2-NOH). 1,4-DHNA was the most potent compound among hydroxyl/carboxy naphthalene derivatives, and the fold induction response for CYP1A1 and CYP1B1 was similar to that observed for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in YAMC and Caco2 cells. 1- and 4-HNA were less potent than 1,4-DHNA but induced maximal (TCDD-like) response for CYP1B1 (both cell lines) and CYP1A1 (Caco2 cells). With the exception of 1- and 2-NA, all compounds significantly induced Cyp1b1 in YAMC cells and these responses were not observed in AhR-deficient YAMC cells generated using CRISPR/Cas9 technology. In addition, we also observed that 1- and 2-NOH (and 1,4-DHNA) were weak AhR agonists, and 1- and 2-NOH also exhibited partial AhR antagonist activity. Structure-activity relationship studies for CYP1A1 but not CYP1B1 were similar in both cell lines, and CYP1A1 induction required one or both 1,4-dihydroxy substituents and activity was significantly enhanced by the 2-carboxyl group. We also used computational analysis to show that 1,4-DHNA and TCDD share similar interactions within the AhR binding pocket and differ primarily due to the negatively charged group of 1,4-DHNA., (© The Author 2016. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

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 reponse.

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-EC 50 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.

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 PM10 air samples at an urban, rural and industrial location in Flanders, using a human in vitro cell bioassay., Methods: PM10 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 PM10 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., (Copyright © 2016 Elsevier Inc. All rights reserved.)

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.

Background: Association of particulate matter with adverse health effects has been established in epidemiological studies and animal experiments. Epidemiological studies are difficult to undertake while animal studies are impractical for high-throughput toxicity testing. The ease and rapidity of in vitro tests emphasizes their potential for use in risk assessment of chemicals and particles. We examined the association between in vitro and in vivo responses to ambient particles, to determine the potential of cell-based assays as standalone toxicity screening tools., Methods: Assays of cytotoxicity and key inflammatory mediators were applied to determine the in vitro biological potency of a panel of urban and mineral particles in J774A.1 macrophages and A549 lung epithelial cells. The particles were also screened for the presence of AhR agonists using the Ah receptor-dependent gene induction assay and for endotoxin using the Limulus amebocyte lysate assay. A subset of the particles with a contrasting in vitro toxicity profile was delivered intratracheally in BALB/c mice to assess their in vivo biological potency. Results from various bioassays were combined within the in vitro and in vivo models. The combined potency measures were examined for associations., Results: Overall, J774A.1 cells were more sensitive to particle effects than A549 cells. Whereas the combined cytotoxicity estimates were highly correlated between the two cell lines, the combined in vitro inflammatory potency estimates were not, emphasizing functional differences of the two cell types. Secretion of inflammatory markers by J774A.1 cells was correlated with AhR ligand binding profile and endotoxin levels of particles. Particle instillation led to an acute toxicity response in BALB/c mice, with neutrophilia and release of inflammatory mediators. While the combined toxicity estimates were not correlated between in vitro and in vivo models, the combined inflammatory and integrated potency estimates (toxicity and inflammation) approached the threshold for significance (p = 0.052) in a correlation within in vitro and in vivo models, with a ranking of fine particle (DWR1), minerals (TiO2, CRI) and coarse particles (SRM-, EHC-type) from low to high potency., Conclusion: Integration of in vitro endpoints shows promise in determining adverse outcomes of particle exposures in vivo. The devised data reduction and computational approach will prove useful in the development of models for assessment of hazard potential of particles; however, distinct models may be needed for particles of different type, such as urban particles vs. mineral particles, nanomaterials.

Fishes in estuarine waters are frequently exposed to treated wastewater effluent, among numerous other sources of contaminants, yet the impacts of these anthropogenic chemicals are not well understood in these dynamic and important waterways. Inland silversides (Menidia beryllina) at an early stage of development [12 days posthatch (dph)] were exposed to waters from two estuarine wastewater-treatment outfall locations in a tidal estuary, the Sacramento/San Joaquin Delta (California, USA) that had varied hydrology and input volumes. The genomic response caused by endocrine-disrupting compounds (EDCs) in these waters was determined using quantitative polymerase chain reaction on a suite of hormonally regulated genes. Relative androgenic and estrogenic activities of the waters were measured using CALUX reporter bioassays. The presence of bifenthrin, a pyrethroid pesticide and known EDC, as well as caffeine and the anti-inflammatory pharmaceutical ibuprofen, which were used as markers of wastewater effluent input, were determined using instrumental analysis. Detectable levels of bifenthrin (2.89 ng L(-1)) were found on one of the sampling dates, and caffeine was found on all sampling dates, in water from the Boynton Slough. Neither compound was detected at the Carquinez Strait site, which has a much smaller effluent discharge input volume relative to the receiving water body size compared with Boynton Slough. Water samples from both sites incubated in the CALUX cell line induced estrogenic and androgenic activity in almost all instances, though the estrogenicity was relatively higher than the androgenicity. Changes in the abundance of mRNA transcripts of endocrine-responsive genes and indicators of general chemical stress were observed after a 96-h exposure to waters from both locations. The relative levels of endocrine response, changes in gene transcript abundance, and contaminant concentrations were greater in water from the Boynton Slough site despite those effluents undergoing a more advanced treatment process. The availability of a widely geographically distributed estuarine model species (M. beryllina) now allows for improved assessment of treated effluent impacts across brackish, estuarine, and marine environments.

The Aryl hydrocarbon Receptor (AhR) is a transcription factor that mediates the biochemical response to xenobiotics and the toxic effects of a number of environmental contaminants, including dioxins. Recently, endogenous regulatory roles for the AhR in normal physiology and development have also been reported, thus extending the interest in understanding its molecular mechanisms of activation. Since dimerization with the AhR Nuclear Translocator (ARNT) protein, occurring through the Helix-Loop-Helix (HLH) and PER-ARNT-SIM (PAS) domains, is needed to convert the AhR into its transcriptionally active form, deciphering the AhR:ARNT dimerization mode would provide insights into the mechanisms of AhR transformation. Here we present homology models of the murine AhR:ARNT PAS domain dimer developed using recently available X-ray structures of other bHLH-PAS protein dimers. Due to the different reciprocal orientation and interaction surfaces in the different template dimers, two alternative models were developed for both the PAS-A and PAS-B dimers and they were characterized by combining a number of computational evaluations. Both well-established hot spot prediction methods and new approaches to analyze individual residue and residue-pairwise contributions to the MM-GBSA binding free energies were adopted to predict residues critical for dimer stabilization. On this basis, a mutagenesis strategy for both the murine AhR and ARNT proteins was designed and ligand-dependent DNA binding ability of the AhR:ARNT heterodimer mutants was evaluated. While functional analysis disfavored the HIF2α:ARNT heterodimer-based PAS-B model, most mutants derived from the CLOCK:BMAL1-based AhR:ARNT dimer models of both the PAS-A and the PAS-B dramatically decreased the levels of DNA binding, suggesting this latter model as the most suitable for describing AhR:ARNT dimerization. These novel results open new research directions focused at elucidating basic molecular mechanisms underlying the functional activity of the AhR.

Endocrine disrupting chemicals represent a broad class of compounds, are widespread in the environment and can pose severe health effects. The objective of this study was to investigate the overall estrogen activating potential of PM10 air samples at an urban location with high traffic incidence in Flanders, using a human in vitro cell bioassay. PM10 samples (n = 36) 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-responsive human ovarian carcinoma (BG1Luc4E2) cell line. In addition, several samples and procedural blanks were extracted with ultra-pure ethanol or acetonitrile to compare extraction efficiencies. Results were expressed as bioanalytical equivalents (BEQs) in femtogram 17β-estradiol equivalent (fg E2-Eq) per cubic meter of air. High fluctuations in estrogenic activity were observed during the entire sampling period, with mean and median BEQs of 50.7 and 35.9 fg E2-Eq m(-)(3), respectively. Estrogenic activity was measured in more than 70% of the samples and several sample extracts showed both high BEQs and high cytotoxicity, which could not be related to black carbon, PM10 or heavy metal concentrations. At this moment, it remains unclear which substances cause this toxicity, but comparison of results obtained with different extraction solvents indicated that acetone/hexane extracts contained more compounds that were cytotoxic and suppressive of responses than those extracted using ultra-pure ethanol. Although more research is needed, the use of a more polar extraction solvent seems to be advisable., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

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., (© 2015 SETAC.)

The enzyme aromatase (CYP19; cytochrome P450 19) in humans undergoes highly tissue- and promoter-specific regulation. In hormone-dependent breast cancer, aromatase is over-expressed via several normally inactive promoters (PII, I.3, I.7). Aromatase biosynthesizes estrogens, which stimulate breast cancer cell proliferation. The placenta produces estrogens required for healthy pregnancy and the major placental CYP19 promoter is I.1. Exposure to certain pesticides, such as atrazine, is associated with increased CYP19 expression, but little is known about the effects of neonicotinoid insecticides on CYP19. We developed sensitive and robust RT-qPCR methods to detect the promoter-specific expression of CYP19 in human adrenocortical carcinoma (H295R) and primary umbilical vein endothelial (HUVEC) cells, and determined the potential promoter-specific disruption of CYP19 expression by atrazine and the commonly used neonicotinoids imidacloprid, thiacloprid, and thiamethoxam. In H295R cells, atrazine concentration-dependently increased PII- and I.3-mediated CYP19 expression and aromatase catalytic activity. Thiacloprid and thiamethoxam induced PII- and I.3-mediated CYP19 expression and aromatase activity at relatively low concentrations (0.1-1.0 µM), exhibiting non-monotonic concentration-response curves with a decline in gene induction and catalytic activity at higher concentrations. In HUVEC cells, atrazine slightly induced overall (promoter-indistinct) CYP19 expression (30 µM) and aromatase activity (≥ 3 µM), without increasing I.1 promoter activity. None of the neonicotinoids increased CYP19 expression or aromatase activity in HUVEC cells. Considering the importance of promoter-specific (over)expression of CYP19 in disease (breast cancer) or during sensitive developmental periods (pregnancy), our newly developed RT-qPCR methods will be helpful tools in assessing the risk that neonicotinoids and other chemicals may pose to exposed women., (© The Author 2015. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Surface water can contain countless organic micropollutants, and targeted chemical analysis alone may only detect a small fraction of the chemicals present. Consequently, bioanalytical tools can be applied complementary to chemical analysis to detect the effects of complex chemical mixtures. In this study, bioassays indicative of activation of the aryl hydrocarbon receptor (AhR), activation of the pregnane X receptor (PXR), activation of the estrogen receptor (ER), adaptive stress responses to oxidative stress (Nrf2), genotoxicity (p53) and inflammation (NF-κB) and the fish embryo toxicity test were applied along with chemical analysis to water extracts from the Danube River. Mixture-toxicity modeling was applied to determine the contribution of detected chemicals to the biological effect. Effect concentrations for between 0 to 13 detected chemicals could be found in the literature for the different bioassays. Detected chemicals explained less than 0.2% of the biological effect in the PXR activation, adaptive stress response, and fish embryo toxicity assays, while five chemicals explained up to 80% of ER activation, and three chemicals explained up to 71% of AhR activation. This study highlights the importance of fingerprinting the effects of detected chemicals.

The Ah receptor (AhR)-responsive CALUX (chemically activated luciferase expression) cell bioassay is commonly used for rapid screening of samples for the presence of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin), dioxin-like compounds, and AhR agonists/antagonists. By increasing the number of AhR DNA recognition sites (dioxin responsive elements), we previously generated a novel third generation (G3) recombinant AhR-responsive mouse CALUX cell line (H1L7.5c3) with a significantly enhanced response to DLCs compared to existing AhR-CALUX cell bioassays. However, the elevated background luciferase activity of these cells and the absence of comparable G3 cell lines derived from other species have limited their utility for screening purposes. Here, we describe the development and characterization of species-specific G3 recombinant AhR-responsive CALUX cell lines (rat, human, and guinea pig) that exhibit significantly improved limit of detection and dramatically increased TCDD induction response. The low background luciferase activity, low minimal detection limit (0.1 pM TCDD) and enhanced induction response of the rat G3 cell line (H4L7.5c2) over the H1L7.5c3 mouse G3 cells, identifies them as a more optimal cell line for screening purposes. The utility of the new G3 CALUX cell lines were demonstrated by screening sediment extracts and a small chemical compound library for the presence of AhR agonists. The improved limit of detection and increased response of these new G3 CALUX cell lines will facilitate species-specific analysis of DLCs and AhR agonists in samples with low levels of contamination and/or in small sample volumes.

Humans and other organisms are exposed to multi-chemical mixtures including commonly found carcinogens such as polycyclic aromatic hydrocarbons (PAHs) and heavy metal/loids. The joint effects of these chemicals as beyond the binary mixtures have not been well characterised. In this study, we evaluated the combined genotoxicity of mixtures of PAHs and heavy metal/loids containing benzo(a)pyrene (B[a]P), naphthalene (Nap), phenanthrene (Phe), pyrene (Pyr), arsenic (As), cadmium (Cd) and chromium (Cr) using in vitro micronucleus (MN) test in HepG2 cells. The induction of aryl hydrocarbon receptor (AhR) by single and mixed PAHs was also measured. The results indicated that individual and mixed Nap, Phe and Pyr did not induce significant MN frequencies. PAHs mixture containing B[a]P and B[a]P alone caused significant but similar level of MN frequencies. The same pattern was found in their AhR induction. Individual metal/loids induced significant cytostasis and MN formation of which Cd was found the most potent inducer. Mixture of metal/loids caused higher frequency of MN suggesting a possible additive effect among metal/loids. In addition, binary mixture of metal/loids and B[a]P, namely As/B[a]P, Cd/B[a]P and Cr/B[a]P, increased MN formation. Mixture of Cd and B[a]P induced the highest level of MN. Exposure of cells to the mixture containing B[a]P and Cd/Cr/As at lower concentration (0.25 µM) resulted in significant MN frequency, the level of which was equal to that by Cd/B[a]P at 1.0 µM. The results of the study suggested that an additive effect may exist between PAHs and heavy metal/loids in a compound- and concentration-dependent manner. The compounds with highest potencies of genotoxicity in the mixture seem dominant as driving sources in the final combined genotoxicity of PAHs and heavy metal/loids., (© The Author 2015. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Small molecular weight protein kinase inhibitors are frequently used tools to unravel the complex network of cellular signal transduction under certain physiological and pathophysiological conditions. 4-amino-5-(4-chlorophenyl)-7-(dimethylethyl)pyrazolo[3,4-d]pyrimidine (PP2) is a widely used compound to block the activity of Src family kinases, the major group of non-receptor tyrosine kinases, which trigger multiple cellular signaling pathways. Here, we show that PP2 induces cytochrome P450 1A1 mRNA expression and enzyme activity in a dose-dependent manner in human HepG2 hepatoma cells and NCTC 2544 keratinocytes. By means of reporter gene assays, RNA interference, electrophoretic mobility shift assay, and competitive ligand-binding assay, we further demonstrate that PP2 is a ligand for the aryl hydrocarbon receptor (AHR), an intracellular chemosensor that regulates xenobiotic metabolism, environmental stress responses, and immune functions. Upon ligand-dependent activation, the AHR translocates into the nucleus and dimerizes with the AHR nuclear translocator (ARNT) to modulate the expression of its target genes. In addition, AHR activation is frequently accompanied by an activation of the tyrosine kinase c-Src, resulting in stimulation of cell-surface receptors and downstream signal transduction. As PP2 activates the AHR/ARNT pathway by simultaneously blocking c-Src-mediated alternative signaling routes, this compound may be a suitable tool to study the contribution of the different AHR-dependent signaling pathways to biological processes and adverse outcomes. On the other hand, the unexpected property of PP2 to stimulate AHR/ARNT signaling should be carefully taken into account in future investigations in order to avoid a false interpretation of experimental results and molecular interrelations.

The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that mediates the toxic and biological effects of structurally diverse chemicals, including the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). As part of a larger effort to identify the full spectrum of chemicals that can bind to and activate the AhR, we have examined the ability of several naturally occurring marine-derived brominated indoles and brominated (methylthio)indoles (collectively referred to as brominated indoles) to bind to the AhR and stimulate AhR-dependent gene expression. Incubation of mouse, rat, and guinea pig recombinant cell lines containing a stably transfected AhR-responsive luciferase reporter gene with eight brominated indoles revealed that all compounds stimulated luciferase reporter gene activity, although some species-specific differences were observed. All compounds induced significantly more luciferase activity when incubated with cells for 4 h as compared to 24 h, demonstrating that these compounds are transient activators of the AhR signaling pathway. Three of the brominated indoles induced CYP1A1 mRNA in human HepG2 cells in vitro and Cyp1a mRNA in zebrafish embryos in vivo. The identification of the brominated indoles as direct ligands and activators/agonists of the AhR was confirmed by their ability to compete with [(3)H]TCDD for binding to the AhR and to stimulate AhR transformation and DNA binding in vitro. Taken together, these results indicate that marine-derived brominated indoles are members of a new class of naturally occurring AhR agonists.

Structural features of the aryl hydrocarbon receptor (AHR) can underlie species- and population-specific differences in its affinity for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). These differences often explain variations in TCDD toxicity. Frogs are relatively insensitive to dioxin, and Xenopus AHRs bind TCDD with low affinity. Weak TCDD binding results from the combination of three residues in the ligand-binding domain: A354 and A370, and N325. Here we sought to determine whether this mechanism of weak TCDD binding is shared by other amphibian AHRs. We isolated an AHR cDNA from the Mexican axolotl (Ambystoma mexicanum). The encoded polypeptide contains identical residues at positions that confer low TCDD affinity to X. laevis AHRs (A364, A380, and N335), and homology modeling predicts they protrude into the binding cavity. Axolotl AHR bound one-tenth the TCDD of mouse AHR in velocity sedimentation analysis, and in transactivation assays, the EC50 for TCDD was 23 nM, similar to X. laevis AHR1β (27 nM) and greater than AHR containing the mouse ligand-binding domain (0.08 nM). Sequence, modeled structure, and function indicate that axolotl AHR binds TCDD weakly, predicting that A. mexicanum lacks sensitivity toTCDD toxicity. We hypothesize that this characteristic of axolotl and Xenopus AHRs arose in a common ancestor of the Caudata and Anura.

Recent studies have established that metformin (MET), an oral anti-diabetic drug, possesses antioxidant activity and is effective against different types of cancer in several carcinogen-induced animal models and cell lines. However, whether MET can protect against breast cancer has not been reported before. Therefore, the overall objectives of the present study are to elucidate the potential chemopreventive effect of MET in non-cancerous human breast MCF10A cells and explore the underlying mechanism involved, specifically the role of cytochrome P4501A1 (CYP1A1)/aryl hydrocarbon receptor (AhR) pathway. Transformation of the MCF10A cells into initiated breast cancer cells with DNA adduct formation was conducted using 7,12-dimethylbenz[a]anthracene (DMBA), an AhR ligand. The chemopreventive effect of MET against DMBA-induced breast carcinogenesis was evidenced by the capability of MET to restore the induction of the mRNA levels of basic excision repair genes, 8-oxoguanine DNA glycosylase (OGG1) and apurinic/apyrimidinic endonuclease1 (APE1), and the level of 8-hydroxy-2-deoxyguanosine (8-OHdG). Interestingly, the inhibition of DMBA-induced DNA adduct formation was associated with proportional decrease in CYP1A1 and in, Nad(p)h: quinone oxidoreductase 1 (NQO1) gene expression. Mechanistically, the involvements of AhR and nuclear factor erythroid 2-related factor-2 (Nrf2) in the MET-mediated inhibition of DMBA-induced CYP1A1 and NQO1 gene expression were evidenced by the ability of MET to inhibit DMBA-induced xenobiotic responsive element and antioxidant responsive element luciferase reporter gene expression which suggests an AhR- and Nrf2-dependent transcriptional control. However, the inability of MET to bind to AhR suggests that MET is not an AhR ligand. In conclusion, the present work shows a strong evidence that MET inhibits the DMBA-mediated carcinogenicity and adduct formation by inhibiting the expression of CYP1A1 through an AhR ligand-independent mechanism., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Among the few bacteria known to utilize methyl tert-butyl ether (MTBE) as a sole carbon source, Methylibium petroleiphilum PM1 is a well-characterized organism with a sequenced genome; however, knowledge of the genetic regulation of its MTBE degradation pathway is limited. We investigated the role of a putative transcriptional activator gene, mdpC, in the induction of MTBE-degradation genes mdpA (encoding MTBE monooxygenase) and mdpJ (encoding tert-butyl alcohol hydroxylase) of strain PM1 in a gene-knockout mutant mdpC(-). We also utilized quantitative reverse transcriptase PCR assays targeting genes mdpA, mdpJ and mdpC to determine the effects of the mutation on transcription of these genes. Our results indicate that gene mdpC is involved in the induction of both mdpA and mdpJ in response to MTBE and tert-butyl alcohol (TBA) exposure in PM1. An additional independent mechanism may be involved in the induction of mdpJ in the presence of TBA., (© FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Malassezia furfur yeast strains isolated from diseased human skin preferentially biosynthesize indole alkaloids which can be detected in the human skin and are highly potent activators of the aryl hydrocarbon receptor (AhR) and AhR-dependent gene expression. Chemical analysis of an EtOAc extract of a M. furfur strain obtained from diseased human skin and grown on l-tryptophan agar revealed several known AhR active tryptophan metabolites along with a previously unidentified compound, pityriazepin. While its structure resembled that of the known alkaloid pityriacitrin, the comprised pyridine ring had been transformed into an azepinone. The indoloazepinone scaffold of pityriazepin is extremely rare in nature and has only been reported once previously. Pityriazepin, like the other isolated compounds, was found to be a potent activator of the AhR-dependent reporter gene assay in recombinant cell lines derived from four different species, although significant species differences in relative potency were observed. The ability of pityriazepin to competitively bind to the AhR and directly stimulate AhR DNA binding classified it as a new naturally-occurring potent AhR agonist. M. furfur produces an expanded collection of extremely potent naturally occurring AhR agonists, which produce their biological effects in a species-specific manner., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: Chemicals that have estrogenic activity (EA) can potentially cause adverse health effects in mammals including humans, sometimes at low doses in fetal through juvenile stages with effects detected in adults. Polycarbonate (PC) thermoplastic resins made from bisphenol A (BPA), a chemical that has EA, are now often avoided in products used by babies. Other BPA-free thermoplastic resins, some hypothesized or advertised to be EA-free, are replacing PC resins used to make reusable hard and clear thermoplastic products such as baby bottles., Methods: We used two very sensitive and accurate in vitro assays (MCF-7 and BG1Luc human cell lines) to quantify the EA of chemicals leached into ethanol or water/saline extracts of fourteen unstressed or stressed (autoclaving, microwaving, UV radiation) thermoplastic resins. Estrogen receptor (ER)-dependent agonist responses were confirmed by their inhibition with the ER antagonist ICI 182,780., Results: Our data showed that some (4/14) unstressed and stressed BPA-free thermoplastic resins leached chemicals having significant levels of EA, including one polystyrene (PS), and three Tritan™ resins, the latter reportedly EA-free. Exposure to UV radiation in natural sunlight resulted in an increased release of EA from Tritan™ resins. Triphenyl-phosphate (TPP), an additive used to manufacture some thermoplastic resins such as Tritan™, exhibited EA in both MCF-7 and BG1Luc assays. Ten unstressed or stressed glycol-modified polyethylene terephthalate (PETG), cyclic olefin polymer (COP) or copolymer (COC) thermoplastic resins did not release chemicals with detectable EA under any test condition., Conclusions: This hazard survey study assessed the release of chemicals exhibiting EA as detected by two sensitive, widely used and accepted, human cell line in vitro assays. Four PC replacement resins (Tritan™ and PS) released chemicals having EA. However, ten other PC-replacement resins did not leach chemicals having EA (EA-free-resins). These results indicate that PC-replacement plastic products could be made from EA-free resins (if appropriate EA-free additives are chosen) that maintain advantages of re-usable plastic items (price, weight, shatter resistance) without releasing chemicals having EA that potentially produce adverse health effects on current or future generations.

For a better understanding of species-specific relative effect potencies (REPs), responses of dioxin-like compounds (DLCs) were assessed. REPs were calculated using chemical-activated luciferase gene expression assays (CALUX) derived from guinea pig, rat, and mouse cell lines. Almost all 20 congeners tested in the rodent cell lines were partial agonists and less efficacious than 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). For this reason, REPs were calculated for each congener using concentrations at which 20% of the maximal TCDD response was reached (REP20TCDD). REP20TCDD values obtained for PCDD/Fs were comparable with their toxic equivalency factors assigned by the World Health Organization (WHO-TEF), while those for PCBs were in general lower than the WHO-TEF values. Moreover, the guinea pig cell line was the most sensitive as indicated by the 20% effect concentrations of TCDD of 1.5, 5.6, and 11.0 pM for guinea pig, rat, and mouse cells, respectively. A similar response pattern was observed using multivariate statistical analysis between the three CALUX assays and the WHO-TEFs. The mouse assay showed minor deviation due to higher relative induction potential for 2,3,7,8-tetrachlorodibenzofuran and 2,3,4,6,7,8-hexachlorodibenzofuran and lower for 1,2,3,4,6,7,8-heptachlorodibenzofuran and 3,3',4,4',5-pentachlorobiphenyl (PCB126). 2,3,7,8-Tetrachlorodibenzofuran was more than two times more potent in the mouse assay as compared with that of rat and guinea pig cells, while measured REP20TCDD for PCB126 was lower in mouse cells (0.05) as compared with that of the guinea pig (0.2) and rat (0.07). In order to provide REP20TCDD values for all WHO-TEF assigned compounds, quantitative structure-activity relationship (QSAR) models were developed. The QSAR models showed that specific electronic properties and molecular surface characteristics play important roles in the AhR-mediated response. In silico derived REP20TCDD values were generally consistent with the WHO-TEFs with a few exceptions. The QSAR models indicated that, e.g., 1,2,3,7,8-pentachlorodibenzofuran and 1,2,3,7,8,9-hexachlorodibenzofuran were more potent than given by their assigned WHO-TEF values, and the non-ortho PCB 81 was predicted, based on the guinea-pig model, to be 1 order of magnitude above its WHO-TEF value. By combining in vitro and in silico approaches, REPs were established for all WHO-TEF assigned compounds (except OCDD), which will provide future guidance in testing AhR-mediated responses of DLCs and to increase our understanding of species variation in AhR-mediated effects.