Aryl Hydrocarbon Activity Research Articles

Effects of microplastic interaction with persistent organic pollutants on the activity of the aryl hydrocarbon and estrogen receptors

Environmental microplastics (MPs) are complex mixtures of plastic polymers and sorbed chemical pollutants with high degrees of heterogeneity, particularly in terms of particle size, morphology and degree of weathering. Currently, limitations exist in sampling sufficient amounts of environmental particles for laboratory studies to assess toxicity endpoints with statistical rigor and to examine chemical pollutant interactions. This study seeks to bridge this gap by investigating environmental plastic particle mimetics and pollutant-polymer interactions by mixing polymer particles with persistent organic pollutants (POPs) at set concentrations over time. Solutions containing combinations of polymers including polystyrene (PS), polypropylene (PP), polyethylene terephthalate (PET), and polyamide (PA) and POPs including 2,3,7,8 -Tetrachlorodibenzo-p-dioxin (TCDD), bisphenol A (BPA), and atrazine, were stirred for up to 19 weeks and monitored using assays to test for aryl hydrocarbon (AhR) and estrogen receptor (ER) activity which are cell signaling pathways impacted by environmental pollutants. TCDD induced AhR activity decreased over time in the presence of PS in a surface area dependent manner. BPA and atrazine also exhibited AhR antagonist activity in the presence of TCDD. The addition of BPA slowed the loss of activity but atrazine did not, suggesting that polymer chemistry impacts interactions with POPs. We also observed potential differences in TCDD sorption with different plastic polymers and that higher concentrations of PS particles may inhibit BPA-induced estrogen receptor activation. These results emphasize the need for additional understanding of how POPs and polymer chemistry impact their interaction and toxicity.

Performance Evaluation of a High Salinity Produced Water Treatment Train: Chemical Analysis and Aryl Hydrocarbon Activation

Performance Evaluation of a High Salinity Produced Water Treatment Train: Chemical Analysis and Aryl Hydrocarbon Activation

Effect-based monitoring of two rivers under urban and agricultural influence reveals a range of biological activities in sediment and water extracts

Chemical contaminants, such as pesticides, pharmaceuticals and industrial compounds are ubiquitous in surface water and sediment in areas subject to human activity. While targeted chemical analysis is typically used for water and sediment quality monitoring, there is growing interest in applying effect-based methods with in vitro bioassays to capture the effects of all active contaminants in a sample. The current study evaluated the biological effects in surface water and sediment from two contrasting catchments in Aotearoa New Zealand, the highly urbanised Whau River catchment in Tāmaki Makaurau (Auckland) and the urban and mixed agricultural Koreti (New River) Estuary catchment. Two complementary passive sampling devices, Chemcatcher for polar chemicals and polyethylene (PED) for non-polar chemicals, were applied to capture a wide range of contaminants in water, while composite sediment samples were collected at each sampling site. Bioassays indicative of induction of xenobiotic metabolism, receptor-mediated effects, genotoxicity, cytotoxicity and apical effects were applied to the water and sediment extracts. Most sediment extracts induced moderate to strong estrogenic and aryl hydrocarbon (AhR) activity, along with moderate toxicity to bacteria. The water extracts showed similar patterns to the sediment extracts, but with lower activity. Generally, the polar Chemcatcher extracts showed greater estrogenic activity, photosynthesis inhibition and algal growth inhibition than the non-polar PED extracts, though the PED extracts showed greater AhR activity. The observed effects in the water extracts were compared to available ecological effect-based trigger values (EBT) to evaluate the potential risk. For the polar extracts, most sites in both catchments exceeded the EBT for estrogenicity, with many sites exceeding the EBTs for AhR activity and photosynthesis inhibition. Of the wide range of endpoints considered, estrogenic activity, AhR activity and herbicidal activity appear to be the primary risk drivers in both the Whau and Koreti Estuary catchments.

Thrombolome and Its Emerging Role in Chronic Kidney Diseases.

Patients with chronic kidney disease (CKD) are at an increased risk of thromboembolic complications, including myocardial infarction, stroke, deep vein thrombosis, and pulmonary embolism. These complications lead to increased mortality. Evidence points to the key role of CKD-associated dysbiosis and its effect via the generation of gut microbial metabolites in inducing the prothrombotic phenotype. This phenomenon is known as thrombolome, a panel of intestinal bacteria-derived uremic toxins that enhance thrombosis via increased tissue factor expression, platelet hyperactivity, microparticles release, and endothelial dysfunction. This review discusses the role of uremic toxins derived from gut-microbiota metabolism of dietary tryptophan (indoxyl sulfate (IS), indole-3-acetic acid (IAA), kynurenine (KYN)), phenylalanine/tyrosine (p-cresol sulfate (PCS), p-cresol glucuronide (PCG), phenylacetylglutamine (PAGln)) and choline/phosphatidylcholine (trimethylamine N-oxide (TMAO)) in spontaneously induced thrombosis. The increase in the generation of gut microbial uremic toxins, the activation of aryl hydrocarbon (AhRs) and platelet adrenergic (ARs) receptors, and the nuclear factor kappa B (NF-κB) signaling pathway can serve as potential targets during the prevention of thromboembolic events. They can also help create a new therapeutic approach in the CKD population.

Mapping multiple endocrine disrupting activities in Virginia rivers using effect-based assays

Water sources are frequently contaminated with natural and anthropogenic substances having known or suspected endocrine disrupting activities; however, these activities are not routinely measured and monitored. Phenotypic bioassays are a promising new approach for detection and quantitation of endocrine disrupting chemicals (EDCs). We developed cell lines expressing fluorescent chimeric constructs capable of detecting environmental contaminants which interact with multiple nuclear receptors. Using these assays, we tested water samples collected in the summers of 2016, 2017 and 2018 from two major Virginia rivers. Samples were concentrated 200× and screened for contaminants interacting with the androgen (AR), glucocorticoid (GR), aryl hydrocarbon (AhR) and thyroid receptors. Among 45 tested sites, over 70% had AR activity and 60% had AhR activity. Many sites were also positive for GR and TRβ activation (22% and 42%, respectively). Multiple sites were positive for more than one type of contaminants, indicating presence of complex mixtures. These activities may negatively impact river ecosystems and consequently human health.

Assessment of source and treated water quality in seven drinking water treatment plants by in vitro bioassays – Oxidative stress and antiandrogenic effects after artificial infiltration

Drinking water quality and treatment efficacy was investigated in seven drinking water treatment plants (DWTPs), using water from the river Göta Älv, which also is a recipient of treated sewage water. A panel of cell-based bioassays was used, including measurements of receptor activity of aryl hydrocarbon (AhR), estrogen (ER), androgen (AR), peroxisome proliferator-activated receptor alpha (PPARα) as well as induction of oxidative stress (Nrf2) and micronuclei formation. Grab water samples were concentrated by solid phase extraction (SPE) and water samples were analyzed at a relative enrichment factor of 50. High activities of AhR, ER and AR antagonism were present in WWTP outlets along the river. Inlet water from the river exhibited AhR and AR antagonistic activities. AhR activity was removed by DWTPs using granulated activated carbon (GAC) and artificial infiltration. AR antagonistic activity was removed by the treatment plants, except the artificial infiltration plant, which actually increased the activity. Furthermore, treated drinking water from the DWTP using artificial infiltration exhibited high Nrf2 activity, which was not found in any of the other water samples. Nrf2 activity was found in water from eight of the 13 abstraction wells, collecting water from the artificial infiltration. No genotoxic activity was detected at non-cytotoxic concentrations. No Nrf2 or AR antagonistic activities were detected in the inlet or outlet water after the DWTP had been replaced by a new plant, using membrane ultrafiltration and GAC. Neither target chemical analysis, nor chemical analysis according to the drinking water regulation, detected any presence of chemicals, which could be responsible of the prominent effects on oxidative stress and AR antagonistic activity in the drinking water samples. Thus, bioanalysis is a useful tool for detection of unknown hazards in drinking water and for assessment of drinking water treatments.

The mixture effects of bisphenol derivatives on estrogen receptor and androgen receptor

Bisphenol A (BPA) is a well-known for endocrine-disrupting chemical (EDC) and is one of the highest amounts of chemicals produced worldwide. Some countries restrict the use of BPA, which is widely used in the production of a variety products. Considering the toxicity and limitations on use of BPA, efforts are needed to find safer alternatives. Increasingly, bisphenol F (BPF) and bisphenol S (BPS) are alternatives of BPA, which is increasing their exposure levels in various environments. There are many ways to assess whether a chemical is an EDC. Here, we evaluated the endocrine-disrupting risks of the bisphenols by investigating their agonist and antagonist activities with the estrogen (ER), androgen (AR), and aryl hydrocarbon (AhR) receptors. Our results showed that BPA, BPS, and BPF (BPs) have estrogen agonist and androgen antagonist activities and decrease the ERα protein level. Interestingly, a mixture of the BPs had ER and anti-AR activity at lower concentrations than BPs alone. The activation of AhR was not a concentration-dependent effect of BPs, although it was increased significantly. In conclusion, BPs have estrogen agonist and androgen antagonist activities, and the effect of exposure to a BPs mixture differs from that of BPs alone.

Abstract 4361: Kynurenine - Aryl hydrocarbon receptor axis: A crucial modulator of immunometabolism in cisplatin resistant lung cancer

Abstract The effect of tumor metabolism on the tumor microenvironment is not well established. Our previous data have shown that cisplatin resistant (CR) lung cancer cells increased secretion of thioredoxin-1 (TRX1) and kynurenine (KYN). Interestingly, high TRX1 and KYN levels in tumor microenvironment can enhance immunosuppressive environment. We have demonstrated that CR cells possessed lower levels of hypoxia-inducible factor-1α (HIF1α) due to metabolic re-programming. ARNT or HIF1β is a known binding partner of both HIF1α and aryl hydrocarbon (AHR). Importantly, recent study indicates that KYN can serve as an endogenous ligand for AHR in cancer cells. Thus, we hypothesize that in the absence of HIF1α, ARNT is now available to bind and form a new partner with KYN/AHR and initiate the transcription of genes which favor survival/proliferation of CR cells. Four pairs of NSCLC cell lines and their CR variants (ALC, FC, H460R, A549R) were used. Using immunofluorescence technique, our result showed that AHR localized primary in the nucleus of CR cells when compared with parental cell counterparts. We further determined that KYN was specific to AHR activation in CR cells by inhibiting AHR translocation using 10µM of dimethoxyflavone (DMF; an AHR antagonist). AHR was less accumulated inside nuclease after treatment. Importantly, treatment of DMF also resulted in suppression of Indoleamine 2,3-Dioxygenase-1 (IDO1) activities. To further verify these findings, we assayed the expression of AHR-target gene (LAT1 and CYP1B1) with and without adding KYN. Both genes expressions were higher in CR cells and were further augmented upon an addition of KYN. In contrast, we did not find an increase in LAT1 after exposure to KYN in parental cells. Using flow cytometer, we found that CR cells possessed higher surface-LAT1 levels when compared to parental cell counterparts, and treatment of KYN resulted in significant tryptophan uptake in CR cells. Importantly, treatment with IDO1 inhibitor (5.5uM) significantly suppressed LAT1 and CYP1B1 expression in CR cells and reverse cisplatin resistance in CR from 2.5 to 0.75 μg/ml (3.3 fold). Thus, our data strongly indicate that KYN/AHR/ARNT axis plays a unique modulator role in CR cells metabolism. Overall these results will have potential impact on (i) how one can effectively exploit the KP pathway to treat CR tumors, and (ii) improving understanding on how CR cells evade immune surveillance. Supported by Department of Veterans Affairs (BLR&D Merit review and CDA2) Citation Format: Medhi Wangpaichitr, Dan JM Nguyen, Ying-Ying Li, Chunjing Wu, Lynn G. Feun, Niramol Savaraj. Kynurenine - Aryl hydrocarbon receptor axis: A crucial modulator of immunometabolism in cisplatin resistant lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4361.

Biological activities associated with the volatile compound 2,5-bis(1-methylethyl)-pyrazine.

Pyrazines are 1,4-diazabenzene-based volatile organic compounds and known for their broad-spectrum antimicrobial activity. In the present study, we assessed the antimicrobial activity of 2,5-bis(1-methylethyl)-pyrazine, produced by Paenibacillus sp. AD87 during co-culture with Burkholderia sp. AD24. In addition, we were using transcriptional reporter assays in E. coli and mammalian cells to decipher the possible mode of action. Bacterial and mammalian luciferase reporter strains were deployed to elucidate antimicrobial and toxicological effects of 2,5-bis(1-methylethyl)-pyrazine. At high levels of exposure, 2,5-bis(1-methylethyl)-pyrazine exerted strong DNA damage response. At lower concentrations, cell-wall damage response was observed. The activity was corroborated by a general toxicity reporter assay in E. coli ΔampD, defective in peptidoglycan turnover. The maximum E. coli cell-wall stress activity was measured at a concentration close to the onset of the mammalian cytotoxicity, while other adverse outcome pathways, such as the activation of aryl hydrocarbon and estrogenic receptor, the p53 tumour suppressor and the oxidative stress-related Nrf2 transcription factor, were induced at elevated concentrations compared to the response of mammalian cells. Because of its broad-spectrum antimicrobial activity at lower concentrations and the relatively low mammalian toxicity, 2,5-bis(1-methylethyl)-pyrazine is a potential bio-based fumigant with possible applications in food industry, agriculture or logistics.

Assessment of endocrine disruption and oxidative potential of bisphenol-A, triclosan, nonylphenol, diethylhexyl phthalate, galaxolide, and carbamazepine, common contaminants of municipal biosolids

The use of biosolids as a soil conditioner and fertiliser is hindered by the limited knowledge on the risks of micro-contaminants they contain. This study investigated the binding of six organic contaminants commonly found in biosolids, to the estrogen (ER), androgen (AR), aryl hydrocarbon (AhR), and transthyretin (TTR) receptors and their redox activity. Triclosan (TCS), bisphenol-A (BPA), and technical nonylphenol (TNP) had affinity for the TTR with relative potencies of 0.3, 0.03, and 0.076 respectively. Further, binding to TTR was the only toxicological response observed for carbamazepine, which induced sub-maximal response and relative potency of 0.0017. Estrogenic activity was induced by BPA, galaxolide (HHCB), diethylhexyl phthalate (DEHP) and TNP with BPA having the strongest potency of 5.1 × 10−6 relative to estradiol. Only BPA showed androgenic activity but it was not quantifiable. BPA also showed anti-androgenic activity along with TCS, HHCB, and TNP in the order of TNP > HHCB > TCS ~ BPA (relative potencies 0.126, 0.042, 0.032, 0.03). No compounds exhibited anti-estrogenic or AhR activity, or were redox-active in the dithiothreitol assay. The results highlight the multiple modes of action through which these compounds may impact exposed organisms, and the concentrations at which effects may occur. This allows assessment of the likelihood of effects being observed at environmental concentrations, and the potential contribution of these compounds.

Adaptation of the human aryl hydrocarbon receptor to sense microbiota-derived indoles.

Ligand activation of the aryl hydrocarbon (AHR) has profound effects upon the immunological status of the gastrointestinal tract, establishing and maintaining signaling networks, which facilitate host-microbe homeostasis at the mucosal interface. However, the identity of the ligand(s) responsible for such AHR-mediated activation within the gut remains to be firmly established. Here, we combine in vitro ligand binding, quantitative gene expression, protein-DNA interaction and ligand structure activity analyses together with in silico modeling of the AHR ligand binding domain to identify indole, a microbial tryptophan metabolite, as a human-AHR selective agonist. Human AHR, acting as a host indole receptor may exhibit a unique bimolecular (2:1) binding stoichiometry not observed with typical AHR ligands. Such bimolecular indole-mediated activation of the human AHR within the gastrointestinal tract may provide a foundation for inter-kingdom signaling between the enteric microflora and the immune system to promote commensalism within the gut.

Vitamin D receptor activation enhances Benzo[a]pyrene metabolism via CYP1A1 expression in macrophages

Benzo[a]pyrene (BaP) activates the aryl hydrocarbon (AHR) and induces the expression of xenobiotic metabolic genes, including cytochrome P450 (CYP) 1A1. CYP1A1 is involved not only in BaP detoxification but also in metabolic activation, which results in DNA adduct formation. Vitamin D receptor (VDR) also regulates expression of other xenobiotic metabolism genes, including CYP3A. We investigated the cross‐talk between AHR and VDR signaling pathways and found that 1α,25‐ dihydroxyvitamin D3 (1,25(OH)2D3) enhanced BaP‐induced transcription of CYP1A1 in human monocytic U937 cells and THP‐1 cells. 1,25(OH)2D3 alone did not induce CYP1A1 and 1,25(OH)2D3 plus BaP did not increase other CYP1 family (CYP1A2 or CYP1B1) mRNA expression in U937 cells. Combination of 1,25(OH)2D3 and BaP increased CYP1A1 protein levels, BaP hydroxylation activity and BaP‐DNA adduct formation in U937 cells and THP‐1 cells more effectively than BaP alone. The combined effect of 1,25(OH)2D3 and BaP on CYP1A1 mRNA expression in U937 cells and/or THP‐1 cells was inhibited by VDR knockdown and VDR or AHR antagonists. Electrophoretic mobility shift assays and chromatin immunoprecipitation assays showed that VDR directly bound to an everted repeat 8 motif in the human CYP1A1 promoter. Thus, induction of CYP1A1 by the activation of VDR and AHR may contribute to BaP‐mediated toxicity and the physiological function of this enzyme.

Screening for potential effects of endocrine-disrupting chemicals in peri-urban creeks and rivers in Melbourne, Australia using mosquitofish and recombinant receptor–reporter gene assays

Sexually mature male mosquitofish (Gambusia holbrooki) were collected from various sites around Melbourne in 2009 to evaluate the performance of gonopodial indices as a biomarker for endocrine disruption in Melbourne's waterways. The mosquitofish indices assessed were body length (BL), gonopodial length (GL)/BL ratio, ray 4:6 ratio and the absence or presence of hooks and serrae, and these varied between sites. The study was complemented by measurements of estrogenic, retinoid, thyroid and aryl hydrocarbon (AhR) receptor activities of the water. Male mosquitofish were 16.3-21.5 mm in length, and although there was a statistically significant positive relationship showing that bigger fish had longer gonopodia than small fish (r2 = 0.52, p < 0.001), there were few significant differences in GL/BL ratio of fish between sites. Measured estrogenic activity was mostly in the range 0.1-1.7 ng/L EEQ, with one site having much higher levels (~12 ng/L EEQ). Aryl hydrocarbon (AhR) receptor activity was observed in all water samples (7-180 ng/L βNF EQ), although there was no consistent pattern in the level of AhR activity observed, i.e., 'clean' sites were as likely to return a high AhR activity response as urban or wastewater treatment plant (WWTP)-impacted sites. There was no correlation between measurements of receptor actvity and gonopodial length (GL):BL ratio and BL. We conclude that the mosquitofish gonopodia only fulfills part of the criteria for biomarker selection for screening. The mosquitofish indices assessed were cheap and easy-to-perform procedures; however, there is no baseline data from the selected sites to evaluate whether differences in the morpholical indices observed at a site were a result of natural selection in the population or due to estrogenic exposure.

Vitamin D Receptor Activation Enhances Benzo[a]pyrene Metabolism via CYP1A1 Expression in Macrophages

Benzo[a]pyrene (BaP) activates the aryl hydrocarbon (AHR) and induces the expression of genes involved in xenobiotic metabolism, including CYP1A1. CYP1A1 is involved not only in BaP detoxification but also in metabolic activation, which results in DNA adduct formation. Vitamin D receptor (VDR) belongs to the NR1I subfamily of the nuclear receptor superfamily, which also regulates expression of xenobiotic metabolism genes. We investigated the cross-talk between AHR and VDR signaling pathways and found that 1α,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], a potent physiological VDR agonist, enhanced BaP-induced transcription of CYP1A1 in human monocytic U937 cells and THP-1 cells, breast cancer cells, and kidney epithelium-derived cells. 1,25(OH)(2)D(3) alone did not induce CYP1A1, and 1,25(OH)(2)D(3) plus BaP did not increase CYP1A2 or CYP1B1 mRNA expression in U937 cells. The combination of 1,25(OH)(2)D(3) and BaP increased CYP1A1 protein levels, BaP hydroxylation activity, and BaP-DNA adduct formation in U937 cells and THP-1 cells more effectively than BaP alone. The combined effect of 1,25(OH)(2)D(3) and BaP on CYP1A1 mRNA expression in U937 cells and/or THP-1 cells was inhibited by VDR knockdown, VDR antagonists, and α-naphthoflavone, an AHR antagonist. Electrophoretic mobility shift assays and chromatin immunoprecipitation assays showed that VDR directly bound to an everted repeat (ER) 8 motif in the human CYP1A1 promoter. Thus, CYP1A1 is a novel VDR target gene involved in xenobiotic metabolism. Induction of CYP1A1 by the activation of VDR and AHR may contribute to BaP-mediated toxicity and the physiological function of this enzyme.

Abstract 4653: 5F203-induced reactive oxidative species, DNA damage and apoptosis involves aryl hydrocarbon activation in ovarian cancer cells

Abstract Ovarian cancer is the 2nd cause of death by gynecological malignancies after breast cancer. These tumors have poor prognoses because they are detected at advanced stages of disease and show acquired or intrinsic resistance to therapy.Benzothiazoles, agents with preclinical antitumor activity against ovarian and breast cancer models, belong to a novel mechanistic class which bind to the arylhydrocarbon receptor (AhR). Phortress the lysylamide prodrug of 2-(4-amino-3-methylphenyl)-5- fluorobenzothiazole (5F203) is currently under phase I clinical evaluation. We previously demonstrated that these compounds activate AhR signaling in sensitive breast cancer cells, the goal of this project was to evaluate the role of AhR in 5F203 activity in two human ovarian cell lines: IGROV-1 sensitive to 5F203 and SKOV-3 resistant to this agent. We observed by MTS assay IGROV-1 cell growth inhibition of 31.9±12.2% by 0.01 μM 5F203 and almost total growth inhibition (91.9±3.4%) after treatment of IGROV-1 cells with 1 μM 5F203. Pre-incubation with the AhR inhibitor ΔNF (1 μM), partially blocked the effect of 5F203 (inhibition of 21.6±4.7% and 27.7±5.3% respectively). In contrast, 5F203 failed to inhibit growth of SKOV-3 cells.5F203 (1 μM) induced AhR translocation from cytosolic to nuclear fractions evaluated by Western blot and confocal microscopy in IGROV-1 cells only. Moreover, activation of CYP1A1 related promoter sequences and EROD activity further indicated activation of AhR signaling. 5F203 treatment for 1h increased ROS and induced NFκB nuclear translocation only in IGROV-1 cells, effects which were attenuated by pre-treatment with ΔNF. Detection of γH2AX revealed DNA double strand breaks 2h post 5F203 treatment of IGROV-1 cells only. After 24h exposure, 5F203 resulted in increased IGROV-1 cell apoptosis, evaluated by DAPI staining of apoptotic bodies, increased pP53, caspase 3 and PARP cleavage. We also observed G1 cell cycle arrest, increased P21 expression and decreased cyclin D1 expression induced by 5F 203. We conclude that the antitumor/pro-apoptotic effects of 5F203 observed in IGROV-1 cells is mediated by AhR which may be a new molecular target in the treatment of ovarian tumors and that this agent may represent a potential novel treatment for ovarian cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4653. doi:1538-7445.AM2012-4653

Biological Analysis of Endocrine-Disrupting Compounds in Tunisian Sewage Treatment Plants

Endocrin-disrupting compounds (EDCs) are frequently found in wastewater treatment plants (WWTPs). So far, research has been mainly focused on the detection of estrogenic compounds and very little work has been carried out on other receptors activators. In this study, we used reporter cell lines, which allow detecting the activity of estrogen (ERalpha), androgen (AR), pregnane X (PXR), glucocorticoid (GR), progesterone (PR), mineralocorticoid (MR), and aryl hydrocarbon (AhR) receptors, to characterise the endocrine-disrupting profile of the aqueous, suspended particulate matter, and sludge fractions from three Tunisian WWTPs. The aqueous fraction exhibited estrogenic and androgenic activities. Suspended particulate matter and sludge extracts showed estrogenic, aryl hydrocarbon and pregnane X receptor activities. No GR, MR, or PR (ant) agonistic activity was detected in the samples, suggesting that environmental compounds present in sewage might have a limited spectrum of activity. By performing competition experiments with recombinant ERalpha, we demonstrated that the estrogenic activity detected in the aqueous fraction was due to EDCs with a strong affinity for ERalpha. Conversely, in the sludge fraction, it was linked to the presence of EDCs with weak affinity. Moreover, by using different incubation times, we determined that the EDCs present in suspended particulate matter and sludge, which can activate AhR, are metabolically labile compounds. Finally, we showed in this study that environmental compounds are mainly ER, AR, PXR, and AhR activators. Concerning AR and PXR ligands, we do not to know the nature of the molecules. Concerning ER and AhR compounds, competition experiments with recombinant receptor and analysis at different times of exposure of the AhR activation gave some indications of the compound's nature that need to be confirmed by chemical analysis.

In Vitro Biologic Activities of the Antimicrobials Triclocarban, Its Analogs, and Triclosan in Bioassay Screens: Receptor-Based Bioassay Screens

BackgroundConcerns have been raised about the biological and toxicologic effects of the antimicrobials triclocarban (TCC) and triclosan (TCS) in personal care products. Few studies have evaluated their biological activities in mammalian cells to assess their potential for adverse effects.ObjectivesIn this study, we assessed the activity of TCC, its analogs, and TCS in in vitro nuclear-receptor–responsive and calcium signaling bioassays.Materials and methodsWe determined the biological activities of the compounds in in vitro, cell-based, and nuclear-receptor–responsive bioassays for receptors for aryl hydrocarbon (AhR), estrogen (ER), androgen (AR), and ryanodine (RyR1).ResultsSome carbanilide compounds, including TCC (1–10 μM), enhanced estradiol (E2)-dependent or testosterone-dependent activation of ER- and AR-responsive gene expression up to 2.5-fold but exhibited little or no agonistic activity alone. Some carbanilides and TCS exhibited weak agonistic and/or antagonistic activity in the AhR-responsive bioassay. TCS exhibited antagonistic activity in both ER- and AR-responsive bioassays. TCS (0.1–10 μM) significantly enhanced the binding of [3H]ryanodine to RyR1 and caused elevation of resting cytosolic [Ca2+] in primary skeletal myotubes, but carbanilides had no effect.ConclusionsCarbanilides, including TCC, enhanced hormone-dependent induction of ER- and AR-dependent gene expression but had little agonist activity, suggesting a new mechanism of action of endocrine-disrupting compounds. TCS, structurally similar to noncoplanar ortho-substituted poly-chlorinated biphenyls, exhibited weak AhR activity but interacted with RyR1 and stimulated Ca2+ mobilization. These observations have potential implications for human and animal health. Further investigations are needed into the biological and toxicologic effects of TCC, its analogs, and TCS.

Lack of antagonism of 2,3,7,8-tetrachlorodibenzo- p-dioxin's (TCDDs) induction of cytochrome P4501A1 ( CYP1A1) by the putative selective aryl hydrocarbon receptor modulator 6-alkyl-1,3,8-trichlorodibenzofuran (6-MCDF) in the mouse hepatoma cell line Hepa-1c1c7

Regulation of gene expression by the aryl hydrocarbon (AHR) receptor is a much-studied pathway of molecular toxicology. Activation of AHR by the xenobiotic ligand 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) is hypothesized as the mechanism by which TCDD exerts its toxic and carcinogenic effects. Paradoxically, some studies have shown that TCDD acts as an antiestrogen. This has led to the hypothesis that so-called selective aryl hydrocarbon receptor modulators (SAhRMs), AHR ligands that retain the antiestrogenic effects but lack the transcriptional effects of TCDD associated with toxicity, may be utilized as cancer chemotherapeutics in conjunction with other antiestrogenic compounds such as tamoxifen. The present study attempts to further define the molecular mechanism of action of the putative SAhRMs, 6-alkyl-1,3,8-trichlorodibenzofuran (6-MCDF), and diindolylmethane (DIM), focusing particularly on the former. We tested 6-MCDF and DIM for the recruitment of AHR and RNA polymerase II (pol II) to the regulatory region of the AHR responsive gene, cytochrome P4501A1 ( CYP1A1), using the chromatin immunoprecipitation (ChIP) assay in the mouse hepatoma cell line Hepa-1c1c7 (Hepa-1). We also tested the level of CYP1A1 induction in Hepa-1 cells using quantitative real-time PCR. We show no difference in the recruitment of AHR or pol II to the regulatory region of CYP1A1 in response to TCDD, 6-MCDF, or co-treatment with both TCDD and 6-MCDF. Our results also show no antagonism of CYP1A1 induction with co-treatment of Hepa-1 cells with TCDD and 6-MCDF. These data suggest that 6-MCDF exhibits agonist activity with respect to induction of CYP1A1 in the Hepa-1 cell line.

Toxicity of hydroxylated and quinoid PCB metabolites: inhibition of gap junctional intercellular communication and activation of aryl hydrocarbon and estrogen receptors in hepatic and mammary cells.

In the present study, a series of 32 hydroxy- and dihydroxy-polychlorinated biphenyls (OH-PCBs) and PCB-derived quinones were prepared and evaluated for their in vitro potencies to downregulate gap junctional intercellular communication (GJIC) and to activate the aryl hydrocarbon receptor (AhR) and the estrogen receptor alpha (ER) in well-established liver and mammary cell models. The rat liver epithelial cell line WB-F344 was used for in vitro determination of GJIC inhibition; the AhR-inducing activity was determined in the rat hepatoma H4IIE.Luc cells stably transfected with a luciferase reporter gene; ER-mediated activity was measured in two breast carcinoma cell lines, MVLN and T47D.Luc, stably transfected with luciferase under the control of estrogen responsive element. Acute inhibition of GJIC, potentially associated with tumor promotion, was detected after treatment with all OH-PCBs under study, with the persistent OH-PCBs being the strongest ones. Several compounds were found to significantly induce the AhR-mediated activity, including 4'-OH-PCB 79, a metabolite of PCB 77, and 2-(4'-chloro)- and 2-(3',4'-dichloro)-1,4-benzoquinones and 1,4-hydroquinones. Low molecular weight OH-PCBs, such as 3'-hydroxy, 4'-, and 3',4'-dihydroxy-4-chlorobiphenyl, elicited significant estrogenic activity and potentiated effect of 17beta-estradiol. Antiestrogenic potencies, determined in the presence of 17beta-estradiol, were found for persistent 4-OH-PCB 187, 4-OH-PCB 146, and some low chlorinated PCB derivatives. However, no apparent association between induction of AhR activity and antiestrogenicity was observed. The majority of the OH-PCBs suppressed the 17beta-estradiol response only at cytotoxic concentrations. Spearman's rank correlations were calculated for these biological data and the physicochemical descriptors, hydrophobicity (log P), molar volume, pKa, log D, and dihedral angle. Significant correlations were found between potency to downregulate GJIC and log P and molar volume (R = -0.7, p < 0.0001). Antiestrogenic effects were also negatively correlated with hydrophobicity and molar volume. No significant correlations among other biological end points and the physicochemical descriptors were observed for the entire set of compounds. These results show that oxygenated PCB metabolites are capable of multiple adverse effects, including gap junction inhibition, AhR-mediated activity, and (anti)estrogenicity. The inhibition of GJIC by OH-PCBs represents a novel mode of action of both the lower chlorinated and the persisting high molecular weight OH-PCBs.

Effect of Dioxin on the Gene Promoters

Effect of and other polychlorinated polyaromatic compounds on mammalian gene's expression is reviewed. The attention is focused on molecular mechanisms of gene promoter activation. We describe first the classical activation pathway: activation of aryl hydrocarbon (AhR) by agonists, the association of activated AhR with Aromatic hydrocarbon nuclear translocator (Arnt) and the activation by this dimer of the response elements of several genes. We then discuss the interaction of proteins implicated in AhR activation such as hsp90 and Arnt in the cross talk with other cellular regulatory pathways. Action of polychlorinated polyaromatic compounds on genes by mechanisms not involving dioxin receptor is also examined.