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We assessed the mechanism of human androgen receptor-mediated endocrine-disrupting effect by a triazole fungicide, metconazole in this study. The internationally validated stably transfected transactivation (STTA) in vitro assay, which was established for determination of a human androgen receptor (AR) agonist/antagonist by using 22Rv1/MMTV_GR-KO cell line, alongside an in vitro reporter-gene assay to confirm AR homodimerization was used. The STTA in vitro assay results showed that metconazole is a true AR antagonist. Furthermore, the results from the in vitro reporter-gene assay and western blotting showed that metconazole blocks the nuclear transfer of cytoplasmic AR proteins by suppressing the homodimerization of AR. These results suggest that metconazole can be considered to have an AR-mediated endocrine-disrupting effect. Additionally, the evidence from this study might help identify the endocrine-disrupting mechanism of triazole fungicides containing a phenyl ring., 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 © 2023 Elsevier B.V. All rights reserved.)

Per- and poly-fluoroalkyl substances (PFASs) are used extensively in a broad range of industrial applications and consumer products. While a few legacy PFASs have been voluntarily phased out, over 5000 PFASs have been produced as replacements for their predecessors. The potential endocrine disrupting hazards of most emerging PFASs have not been comprehensively investigated. In silico molecular docking to the human androgen receptor (hAR) combined with machine learning techniques were previously applied to 5206 PFASs and predicted 23 PFASs bind the hAR. Herein, the in silico results were validated in vitro for the five candidate AR ligands that were commercially available. Three manufactured PFASs namely (9-(nonafluorobutyl)- 2,3,6,7-tetrahydro-1 H,5 H,11 H-pyrano[2,3-f]pyrido[3,2,1-ij]quinolin-11-one (NON), 2-(heptafluoropropyl)- 3-phenylquinoxaline (HEP), and 2,2,3,3,4,4,5,5,5-nonafluoro-N-(4-nitrophenyl)pentanamide (NNN) elicited significant antiandrogenic effects at relatively low concentrations. We further investigated the mechanism of AR inhibition and found that all three PFASs inhibited AR transactivation induced by testosterone through a competitive binding mechanism. We then examined the antiandrogenic effects of these PFASs on AR expression and its responsive genes. Consistently, these PFASs significantly decreased the expression of PSA and FKBP5 and increased the expression of AR, similar to the effects elicited by a known competitive AR inhibitor, hydroxyflutamide. This suggests they are competitive antagonists of AR activity and western blot analysis revealed these PFASs decreased intracellular AR protein in androgen sensitive human prostate cancer cells. Hence, the findings presented here corroborate our published in silico approach and indicate these emerging PFASs may adversely affect the human endocrine system., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Under the Organisation for Economic Co-operation and Development (OECD), the Ministry of the Environment of Japan (MOE) added Japanese medaka (Oryzias latipes) to the test guideline fish short-term reproduction assay (FSTRA) developed by the United States Environmental Protection Agency (US EPA) using fathead minnow (Pimephales promelas). The FSTRA was designed to detect endocrine disrupting effects of chemicals interacting with the hypothalamic-pituitary-gonadal axis (HPG axis) such as agonists or antagonists on the estrogen receptor (Esr) and/or the androgen receptor (AR) and steroidogenesis inhibitors. We conducted the FSTRA with Japanese medaka, in accordance with OECD test guideline number 229 (TG229), for 16 chemicals including four Esr agonists, two Esr antagonists, three AR agonists, two AR antagonists, two steroidogenesis inhibitors, two progesterone receptor agonists, and a negative substance, and evaluated the usability and the validity of the FSTRA (TG229) protocol. In addition, in vitro reporter gene assays (RGAs) using Esr1 and ARβ of Japanese medaka were performed for the 16 chemicals, to support the interpretation of the in vivo effects observed in the FSTRA. In the present study, all the test chemicals, except an antiandrogenic chemical and a weak Esr agonist, significantly reduced the reproductive status of the test fish, that is, fecundity or fertility, at concentrations where no overt toxicity was observed. Moreover, vitellogenin (VTG) induction in males and formation of secondary sex characteristics (SSC), papillary processes on the anal fin, in females was sensitive endpoints to Esr and AR agonistic effects, respectively, and might be indicators of the effect concentrations in long-term exposure. Overall, it is suggested that the in vivo FSTRA supported by in vitro RGA data can adequately detect effects on the test fish, O. latipes, and probably identify the mode of action (MOA) of the chemicals tested., (© 2021 The Authors. Journal of Applied Toxicology published by John Wiley & Sons Ltd.)

3,5,6-Trichloro-2-pyridinol (TCP) is an important biomarker and one of the final metabolites of chlorpyrifos (CPF). TCP inhibits secretion of sex hormones. Similar to CPF, TCP can bind to sex steroid hormone receptors and decrease the secretion of sex hormones. However, little attention has been paid to the ability of TCP and CPF to interfere with androgen receptor (AR) in Sertoli cells. This study aimed to explain how TCP promotes the inhibitory effect of CPF on the paracrine function of Sertoli cells. Western blotting indicated that after 20 weeks of exposure, expression of AR in testes was significantly reduced by CPF. An in vitro assay measured the cytotoxicity of CPF, TCP and diethylphosphate (DEP) on viability of Sertoli cells by Cell Counting Kit-8. CPF cytotoxicity was greater than that of TCP, and TCP cytotoxicity was greater than that of DEP at concentrations of 1000 μmol/L. Western blotting indicated that TCP and CPF both decreased expression of AR and cAMP-response element binding protein phosphorylation, while DEP had no effect in Sertoli cells, which are important in regulating paracrine function of Sertoli cells. The fluorescence measurements and docking studies revealed that testosterone, CPF and TCP showed four types of intermolecular interactions with AR, highlighting alkyl bonds with some of the same amino acids. Compared with testosterone, CPF and TCP also showed significant synergistic interaction with AR. CPF interacted with more amino acids and interaction energy than TCP did. This research elucidates TCP in the antiandrogenic effect of CPF on the paracrine function and suggests that TCP or chemicals with a trichloropyridine structure must be considered during reproductive toxicity assessment of potential environmental pollutants., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Screening certain environmental chemicals for their ability to interact with endocrine targets, including the androgen receptor (AR), is an important global concern. We previously developed a model using a battery of eleven in vitro AR assays to predict in vivo AR activity. Here we describe a revised mathematical modeling approach that also incorporates data from newly available assays and demonstrate that subsets of assays can provide close to the same level of predictivity. These subset models are evaluated against the full model using 1820 chemicals, as well as in vitro and in vivo reference chemicals from the literature. Agonist batteries of as few as six assays and antagonist batteries of as few as five assays can yield balanced accuracies of 95% or better relative to the full model. Balanced accuracy for predicting reference chemicals is 100%. An approach is outlined for researchers to develop their own subset batteries to accurately detect AR activity using assays that map to the pathway of key molecular and cellular events involved in chemical-mediated AR activation and transcriptional activity. This work indicates in vitro bioactivity and in silico predictions that map to the AR pathway could be used in an integrated approach to testing and assessment for identifying chemicals that interact directly with the mammalian AR., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Many pesticides can antagonize the androgen receptor (AR) or inhibit androgen synthesis in vitro but their potential to cause reproductive toxicity related to disruption of androgen action during fetal life is difficult to predict. Currently no approaches for using in vitro data to anticipate such in vivo effects exist. Prioritization schemes that limit unnecessary in vivo testing are urgently needed., Objectives: The aim was to develop a quantitative in vitro to in vivo extrapolation (QIVIVE) approach for predicting in vivo anti-androgenicity arising from gestational exposures and manifesting as a shortened anogenital distance (AGD) in male rats., Methods: We built a physiologically based pharmacokinetic (PBK) model to simulate concentrations of chemicals in the fetus resulting from maternal dosing. The predicted fetal levels were compared with analytically determined concentrations, and these were judged against in vitro active concentrations for AR antagonism and androgen synthesis suppression., Results: We first evaluated our model by using in vitro and in vivo anti-androgenic data for procymidone, vinclozolin, and linuron. Our PBK model described the measured fetal concentrations of parent compounds and metabolites quite accurately (within a factor of five). We applied the model to nine current-use pesticides, all with in vitro evidence for anti-androgenicity but missing in vivo data. Seven pesticides (fludioxonil, cyprodinil, dimethomorph, imazalil, quinoxyfen, fenhexamid, o -phenylphenol) were predicted to produce a shortened AGD in male pups, whereas two ( λ -cyhalothrin , pyrimethanil) were anticipated to be inactive. We tested these expectations for fludioxonil, cyprodinil, and dimethomorph and observed shortened AGD in male pups after gestational exposure. The measured fetal concentrations agreed well with PBK-modeled predictions., Discussion: Our QIVIVE model newly identified fludioxonil, cyprodinil, and dimethomorph as in vivo anti-androgens. With the examples investigated, our approach shows great promise for predicting in vivo anti-androgenicity (i.e., AGD shortening) for chemicals with in vitro activity and for minimizing unnecessary in vivo testing. https://doi.org/10.1289/EHP6774.

Ethnopharmacological Relevance: Many prostate cancer (PCa) patients in Mainland China and other Asian countries often use Chinese herbal medicines as an adjuvant treatment while receiving Western medicines. However, concerns have been raised about the potential herb-drug interaction when using herbal medicines containing phytoandrogens., Aim of the Study: This study aimed to investigate the effects of the selected 21 Chinese herbal medicines on the proliferation and tumor growth using the relevant in vitro and in vivo models of PCa., Materials and Methods: After treatment of LNCaP and 22Rv1 cells with different concentrations of 70% ethanol extracts of the 21 selected herbal medicines for 48 h, the proliferative activity, the effects on androgen receptor (AR) and prostate specific antigen (PSA) were determined. The anti-tumor effects of the 21 herbs on PCa growth were also investigated on a subcutaneous mouse model of PCa., Results: The results showed that Epimedii Folium (EF) and Codonopsis Radix (CNR) could significantly increase the cell viability in LNCaP cells (p < 0.05 for both) and 22Rv1 cells (p < 0.05 for both), protein expressions of AR in LNCaP cells (p < 0.05 for both) and 22Rv1 cells (p < 0.05 for both), and PSA (p < 0.05 for both) in LNCaP cells. EF, CNR, and Cistanches Herba (CCH) markedly accentuated the tumor growth (p < 0.05 for three drugs) and AR expression (p < 0.05 for three herbs) in tumor tissues. On the other hand, treatment with Astragali Radix (AGR), Chuanxiong Rhizoma (CXR) and Bruceae Fructus (BF) significantly inhibited the cell viability in LNCaP cells (p < 0.05, p < 0.05 and p < 0.001, respectively) and in 22Rv1 cells (p < 0.05, p < 0.05 and p < 0.001, respectively), and the protein expression of AR in LNCaP cells (p < 0.05 for three herbs) and 22Rv1 cells (p < 0.05, p < 0.05 and p < 0.001, respectively), and the protein expression of PSA (p < 0.05 for three herbs) in LNCaP cells, as well as tumor growth (p < 0.05 for three herbs) and the AR expression (p < 0.05 for AGR and CXR, p < 0.001 for BF) in tumor tissues., Conclusion: Our results revealed that AGR, CXR and BF suppressed the PCa development via inhibition of AR expression, while EF, CNR and CCH promoted the development and progression of PCa via enhancement of AR expression. The results strongly suggest that caution should be exercised when using androgenic Chinese herbal medicines in PCa patients., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Multilayer materials used in food packaging are commonly manufactured with a polyurethane adhesive layer in its structure that may contain cyclic esters oligomers as potential migrants. However, little is known about their toxicity. In this work, two cyclic esters of polyurethane are evaluated in migration from 20 multilayer packaging samples. They were composed by adipic acid (AA), diethylene glycol (DEG) and isophthalic acid (IPA) and their structure was AA-DEG and AA-DEG-IPA-DEG. The concentration of these compounds in migration exceeded the maximum level established by Regulation EU/10/2011 (10 ng g -1 ). Bioaccessibility of both compounds was evaluated by studying gastric and intestinal digestion. The studies showed that the concentration of the compounds decreased during digestion and that their hydrolysed molecules increased. Furthermore, endocrine activity in vitro assays were performed. A weak androgen receptor antagonism was identified, whereas no arylhydrocarbon receptor activity or binding to the thyroid hormone transport protein was found., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Introduction: Human exposure to persistent organic pollutants (POPs) has been linked to genitourinary health-related conditions such as decreased sperm quality, hypospadias, and prostate cancer (PCa). Conventional risk assessment of POPs focuses on individual compounds. However, in real life, individuals are exposed to many compounds simultaneously. This might lead to combinatorial effects whereby the global effect of the mixture is different from the effect of the single elements or subgroups. POP mixtures may act as endocrine disruptors via the androgen receptor (AR) and potentially contribute to PCa development., Aim: To determine the endocrine disrupting activity of a POP mixture and sub-mixtures based upon exposure levels detected in a human Scandinavian population, on AR transactivation and translocation in vitro., Materials and Methods: The Total POP mixture combined 29 chemicals modelled on the exposure profile of a Scandinavian population and 6 sub-mixtures: brominated (Br), chlorinated (Cl), Cl + Br, perfluorinated (PFAA), PFAA + Br, PFAA + Cl, ranging from 1/10× to 500× relative to what is found in human blood. Transactivation was measured by reporter gene assay (RGA) and translocation activity was measured by high content analysis (HCA), each using stably transfected AR model cell lines., Results: No agonist activity in terms of transactivation and translocation was detected for any POP mixtures. In the presence of testosterone the Cl + Br mixture at 100× and 500× blood level antagonised AR transactivation, whereas the PFAA mixture at blood level increased AR transactivation (P < 0.05). In the presence of testosterone the Cl and PFAA + Br mixtures at 1/10×, 1×, and 50× blood level antagonised AR translocation (P < 0.05)., Conclusion: Taken together, some combinations of POP mixtures can interfere with AR translocation. However, in the transactivation assay, these combinations did not affect gene transactivation. Other POP combinations were identified here as modulators of AR-induced gene transactivation without affecting AR translocation. Thus, to fully evaluate the effect of environmental toxins on AR signalling, both types of assays need to be applied., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Chemicals that disrupt androgen receptor (AR) function in utero induce a cascade of adverse effects in male rats including reduced anogenital distance, retained nipples, and reproductive tract malformations. The objective of this study was to compare the in vitro and in utero activities of two novel AR antagonists, bisphenol C (BPC) and pyrifluquinazon (PFQ). In vitro, BPC was as potent an AR antagonist as hydroxyflutamide. Furthermore, BPC inhibited fetal testis testosterone production and testis gene expression ex vivo. However, when BPC was administered at 100 and 200 mg/kg/d in utero, the reproductive tract of the male offspring was minimally affected. None of the males displayed reproductive malformations. For comparison, in utero administration of flutamide has been shown to induce malformations in 100% of males at 6 mg/kg/d. In vitro, PFQ was several orders of magnitude less potent than BPC, vinclozolin, or procymidone. However, in utero administration of 12.5, 25, 50, and 100 mg PFQ/kg/d on GD 14-18 induced antiandrogenic effects at all dosage levels and 91% of the males displayed reproductive malformation in the high dose group. Overall, BPC was ∼380-fold more potent than PFQ in vitro, whereas PFQ was far more potent than BPC in utero. Incorporating toxicokinetic and toxicodynamic data into in vitro to in vivo extrapolations would reduce the discordance between the in vitro and in utero effects of PFQ and BPC and combining in vitro results with a short-term Hershberger assay would reduce the uncertainty in predicting the in utero effects of antiandrogenic chemicals., (Published by Oxford University Press on behalf of the Society of Toxicology 2019.)

High-throughput transcriptomic (HTTr) technologies are increasingly being used to screen environmental chemicals in vitro to identify molecular targets and provide mechanistic context for regulatory testing. Here, we describe the development and validation of a novel gene expression biomarker to identify androgen receptor (AR)-modulating chemicals using a pattern matching method. Androgen receptor biomarker genes were identified by their consistent expression after exposure to 4 AR agonists and 4 AR antagonists and included only those genes that were regulated by AR. The 51 gene biomarker was evaluated as a predictive tool using the fold-change, rank-based Running Fisher algorithm. Using 158 comparisons from cells treated with 95 chemicals, the biomarker gave balanced accuracies for prediction of AR activation or AR suppression of 97% or 98%, respectively. The biomarker correctly classified 16 out of the 17 AR reference antagonists including those that are "weak" and "very weak". Predictions based on microarray profiles from AR-positive LAPC-4 cells treated with 28 chemicals in antagonist mode were compared with those from an AR pathway model which used 11 in vitro HT assays. The balanced accuracy for suppression was 93%. Using our approach, we identified conditions in which AR was modulated in a large collection of microarray profiles from prostate cancer cell lines including (1) constitutively active mutants or knockdown of AR, (2) decreases in availability of androgens by castration or removal from media, and (3) exposure to chemical modulators that work through indirect mechanisms including suppression of AR expression. These results demonstrate that the AR gene expression biomarker could be a useful tool in HTTr to identify AR modulators.

Mild analgesics have been associated with antiandrogenic effects, but there are no such studies on dipyrone, despite its high prevalence of use in many countries. We examined the production of steroid hormones in human H295R cells after exposure to dipyrone and two metabolites, 4-Methylaminoantipyrine (MAA) and 4-Aminoantipyrine (AA), as well as fetal testicular testosterone production in rats following maternal dipyrone exposure. Androgen agonistic/antagonistic effects were examined in vitro for dipyrone and its metabolites in the Yeast Androgen Screen (YAS) assay and in vivo for dipyrone through the Hershberger assay. In vitro we tested dipyrone, MAA, and AA (0.1-1000 μM) while in vivo we used dipyrone (50, 100, 200 mg/kg/day). In the H295R assay, dipyrone, MAA and AA reduced the production of androgens and corticosteroids. Testosterone was reduced at concentrations 4-13 times higher than the maximum plasma concentrations reported in humans for MAA and AA. No effects were observed in the fetal testosterone production assay. In the YAS and Hershberger assays, no androgen agonistic/antagonistic activities were observed. These results indicate that dipyrone and its metabolites do not interact with the androgen receptor, but have the potential to inhibit steroidogenesis, however only at concentrations that are not relevant under normal medical use., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Personal care products are used increasingly, resulting in growing concern concerning their potential disruption of normal hormonal functions. Recent results on the bioaccumulation of cosmetic ingredients in wildlife and humans point to the need for an in-depth analysis for endocrine activity, in particular with respect to their influence on the androgen (AR), glucocorticoid (GR), and thyroid hormone receptors (TRs). Furthermore, humans are commonly exposed simultaneously to complex mixtures of endocrine active compounds. We have therefore examined 3 frequently used cosmetic ingredients: 2-methylresorcinol (2MR), butylated hydroxyanisole (BHA) and avobenzone (AVB), for (anti)-androgen-, (anti)-glucocorticoid-, and (anti)-thyroid hormone-like activities. Their binary and ternary mixtures at EC50 or IC50 concentrations have also been examined for anti-androgen-, glucocorticoid-, and thyroid hormone-like activities. In the MDA-kb2 reporter cell line, compounds possessed anti-androgen-, glucocorticoid-, and anti-glucocorticoid-like activities (except AVB). A new cell line, GH3.TRE-Luc, was used to evaluate anti-thyroid and thyroid hormone-like activities. The combinations 2MR + BHA and 2MR + BHA + AVB have glucocorticoid-like activity: only 2MR + AVB has anti-androgen-like activity. On the other hand, binary and ternary mixtures of compounds showed no thyroid hormone-like activity. Thus, in addition to identifying new endocrine disrupting compounds, it is also necessary to determine the effects of their mixtures in order to assess fully their risk to human health., (© The Author 2017. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Endocrine active compounds (EACs) remain an important group of chemicals that require additional evaluation to determine their environmental impacts. While estrogens and androgens were previously demonstrated to impact organisms during environmental exposures, progestagens have recently been shown to have strong impacts on aquatic organisms. To gain an understanding of the impacts of these types of chemicals on aquatic species, experiments evaluating the mechanisms of action of progestagen exposure were conducted with the Eastern Mosquitofish (Gambusia holbrooki). The objective of this study was to conduct hepatic microarray analysis of male and female G. holbrooki exposed to progestins and anti-progestagens. In addition, we evaluated the ability of levonorgestrel, a synthetic progesterone (progestin), to induce anal fin elongation and to determine how anal fin growth is modulated during co-exposures with progesterone and androgen receptor antagonists. Gene expression analyses were conducted on male and female G. holbrooki exposed for 48h to the agonist levonorgestrel, the antagonist mifepristone, or a mixture of the two chemicals. Microarray analysis revealed that mifepristone does not act as an anti-progestagen in G. holbrooki in liver tissues, and that levonorgestrel elicits strong effects on the processes of embryo development and lipid transport. Levonorgestrel was also demonstrated to induce male secondary sexual characteristic formation in females, and co-exposure of either an androgen or levonorgestrel in the presence of the anti-androgen flutamide prevented anal fin elongation. These results provide indications as to the potential impacts of progestins, including non-target effects such as secondary sexual characteristic formation, and demonstrate the importance of this class of chemicals on aquatic organisms., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The incidence of immune-related diseases increased over the last years in industrialized countries, suggesting a contribution of environmental factors. Impaired glucocorticoid action has been associated with immune disorders. Thus, there is an increasing interest to identify chemicals disrupting glucocorticoid action. The widely used flame retardant tetrabromobisphenol A (TBBPA) was reported earlier to potently inhibit glucocorticoid receptor (GR) and moderately androgen receptor (AR) activity in yeast-based reporter gene assays. To further characterize possible GR disrupting effects of TBBPA, transactivation experiments using a human HEK-293 cell-based reporter gene assay and cell-free receptor binding experiments were performed in the present study. Both, transactivation and GR binding experiments failed to detect any activity of TBBPA on GR function. Molecular docking calculations supported this observation. Additionally, the current study could confirm the antiandrogenic activity of TBBPA seen in the yeast assay, although the effect was an order of magnitude less pronounced in the HEK-293 cell-based system. In conclusion, TBBPA does not directly affect GR function and, considering its rapid metabolism and low concentrations found in humans, it is unlikely to cause adverse effects by acting through AR. This study emphasizes the use of cell-free assays in combination with cell-based assays for the in vitro evaluation of endocrine disrupting chemicals., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

The compound p-nitrophenol, which shows the anti-androgenic activity, can easily become anthropogenic pollutants and pose a threat to the environment and human health. Previous work indicates that the anti-androgenic mechanism of p-nitrophenol is complex and may involve several components in the AR signaling pathway, but the molecular details of how p-nitrophenol inhibits AR signaling are still not quite clear. Here, we characterized p-nitrophenol binds to the FK1 domain of an AR positive regulator FKBP51 with micromolar affinity and structural analysis of FK1 domain in complex with p-nitrophenol revealed that p-nitrophenol occupies a hydrophobic FK1 pocket that is vital for AR activity enhancement. Molecular dynamics simulation indicated that p-nitrophenol is stably bound to the FK1 pocket and the hotspot residues that involved p-nitrophenol binding are mainly hydrophobic and overlap with the AR interaction site. Furthermore, we showed that p-nitrophenol inhibits the androgen-dependent growth of human prostate cancer cells, possibly through down-regulating the expression levels of AR activated downstream genes. Taken together, our data suggests that p-nitrophenol suppresses the AR signaling pathway at least in part by blocking the interaction between AR and its positive regulator FKBP51. We believe that our findings could provide new guidelines for assessing the potential health effects of p-nitrophenol., (Copyright © 2015 Elsevier B.V. All rights reserved.)

There is a great need for alternative testing methods for reproductive toxicants that are practical, fast, cost-effective and easy to interpret. Previously we followed a pragmatic approach using readily available tests, which was successful in predicting reproductive toxicity of chemicals [13]. This initial battery still contained apical tests and is fairly complex and low in its throughput. The current study aimed to simplify this screening battery using a mechanistic approach and a panel of high throughput CALUX reporter gene assays. A mechanistic approach was taken to validate this high throughput test battery. To this end it was challenged with two preselected sets of chemicals addressing two major apical effect classes relevant in reproductive toxicity. We found selectivity in this battery in that 82% of the compounds inducing reproductive organ deformities were predicted correctly, while for compounds inducing neural tube defects this was the case in 47% only. This is consistent with the mechanisms of toxicity covered in the battery. The most informative assays in the battery were ERalpha CALUX to measure estrogenicity and the AR-anti CALUX assay to measure androgen receptor antagonism., (Copyright © 2014 Elsevier Inc. All rights reserved.)

A range of 1,4-substituted-1,2,3-N-phenyltriazoles were synthesized and evaluated as non-steroidal androgen receptor (AR) antagonists. The motivation for this study was to replace the N-phenyl amide portion of small molecule antiandrogens with a 1,2,3-triazole and determine effects, if any, on biological activity. The synthetic methodology presented herein is robust, high yielding and extremely rapid. Using this methodology a series of 17 N-aryl triazoles were synthesized from commercially available starting materials in less than 3h. After preliminary biological screening at 20 and 40 μM, the most promising three compounds were found to display IC50 values of 40-50 μM against androgen dependent (LNCaP) cells and serve as a starting point for further structure-activity investigations. All compounds in this work were the focus of an in silico study to dock the compounds into the human androgen receptor ligand binding domain (hARLBD) and compare their predicted binding affinity with known antiandrogens. A comparison of receptor-ligand interactions for the wild type and T877A mutant AR revealed two novel polar interactions. One with Q738 of the wild type site and the second with the mutated A877 residue., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Hydroxylated polychlorinated biphenyls (HO-PCBs), major metabolites of PCBs, may have the potential to disrupt androgen hormone homeostasis. However, there is a lack of systematic investigation into the intermolecular interaction mechanism between HO-PCBs and the androgen receptor (AR). In this study, the combination of three-dimensional quantitative structure-activity relationship (3D-QSAR), molecular docking, and molecular dynamics (MD) simulations was performed to elucidate structural characteristics that influence the anti-androgen activity of HO-PCBs, and to provide a better understanding of the binding modes between HO-PCBs and AR. A predictive comparative molecular field analysis (CoMFA) model was developed with good robustness and predictive ability. Graphical interpretation of the model provided some insights into the structural features that affect the anti-androgen activity of HO-PCBs. The hydrogen bond interaction with Gln711, and hydrophobic interactions with residues in the hydrophobic pocket played important roles in the binding of ligand with receptor. These results are expected to be beneficial to predict anti-androgen activities of other HO-PCBs and provided possible clues for further elucidation of the binding mechanism of HO-PCBs with AR., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The management of hormone-refractory prostate cancer represents a major challenge in the therapy of this tumor, and identification of novel androgen receptor antagonists is needed to render treatment more effective. We analyzed the activity of two novel androgen receptor antagonists, (S)-11 and (R)-9, in in vitro and in vivo experimental models of hormone-sensitive or castration-resistant prostate cancer (CRPC). In vitro experiments were performed on LNCaP, LNCaP-AR, LNCaP-Rbic and VCaP human prostate cancer cells. Cytotoxic activity was assessed by SRB and BrdU uptake, AR transactivation by luciferase reporter assay and PSA levels by Real Time RT-PCR and ELISA assays. Cell cycle progression-related markers were evaluated by western blot. In vivo experiments were performed on SCID mice xenografted with cells with different sensitivity to hormonal treatment. In hormone-sensitive LNCaP and LNCaP-AR cells, the latter expressing high androgen receptor levels, (R)-9 and (S)-11 exhibited a higher cytotoxic effect compared to that of the reference compound ((R)-bicalutamide), also in the presence of the synthetic androgen R1881. Furthermore, the cytotoxic effect produced by (R)-9 was higher than that of (S)-11 in the two hormone-resistant LNCaP-AR and VCaP cells. A significant reduction in PSA levels was observed after exposure to both molecules. Moreover, (S)-11 and (R)-9 inhibited DNA synthesis by blocking the androgen-induced increase in cyclin D1 protein levels. In vivo studies on the toxicological profile of (R)-9 did not reveal the presence of adverse events. Furthermore, (R)-9 inhibited tumor growth in various in vivo models, especially LNCaP-Rbic xenografts, representative of recurrent disease. Our in vitro results highlight the antitumor activity of the two novel molecules (R)-9 and (S)-11, making them a potentially attractive option for the treatment of CRPC.

Androgen receptor (AR) is an essential component to activate AR dependent gene transcriptions. Despite wide acceptance of pharmacological controls on transcriptional pathway depending on competitive inhibitions of ligand binding, only a few examples, AR antagonism via ligand-independent mechanisms, have been recognized. Pyrifluquinazon(PFQ), a newly developed pesticide, induced representative AR antagonism against rats in in vivo and in vitro. Intriguingly, this AR antagonism was not based on inhibition of ligand binding. Instead, the evidence suggested that the AR antagonism was induced as a consequence of decline of cellular AR protein level. This study demonstrated that AR N-terminal region could be an essential element for a ligand-independent mechanism underling the AR antagonism by PFQ. Our findings should provide a novel insight into the regulation of AR-mediated transcription., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background: The androgen receptor plays a critical role throughout the progression of prostate cancer and is an important drug target for this disease. While chromatin immunoprecipitation coupled with massively parallel sequencing (ChIP-Seq) is becoming an essential tool for studying transcription and chromatin modification factors, it has rarely been employed in the context of drug discovery., Results: Here we report changes in the genome-wide AR binding landscape due to dose-dependent inhibition by drug-like small molecules using ChIP-Seq. Integration of sequence analysis, transcriptome profiling, cell viability assays and xenograft tumor growth inhibition studies enabled us to establish a direct cistrome-activity relationship for two novel potent AR antagonists. By selectively occupying the strongest binding sites, AR signaling remains active even when androgen levels are low, as is characteristic of first-line androgen ablation therapy. Coupled cistrome and transcriptome profiling upon small molecule antagonism led to the identification of a core set of AR direct effector genes that are most likely to mediate the activities of targeted agents: unbiased pathway mapping revealed that AR is a key modulator of steroid metabolism by forming a tightly controlled feedback loop with other nuclear receptor family members and this oncogenic effect can be relieved by antagonist treatment. Furthermore, we found that AR also has an extensive role in negative gene regulation, with estrogen (related) receptor likely mediating its function as a transcriptional repressor., Conclusions: Our study provides a global and dynamic view of AR's regulatory program upon antagonism, which may serve as a molecular basis for deciphering and developing AR therapeutics.

The pyrethroid insecticide, cypermethrin has been demonstrated to be an environmental anti-androgen in the androgen receptor (AR) reporter gene assay. The amino- and carboxyl-terminal (N/C) interaction is required for transcription potential of the AR. In order to characterize the anti-androgen effects of cypermethrin involved in the N/C interaction of AR, the mammalian two-hybrid assay has been developed in the study. The fusion vectors pVP16-ARNTD, pM-ARLBD and the pG5CAT Reporter Vector were cotransfected into the CV-1 cells. The assay displayed appropriate response to the potent, classical AR agonist 5α-dihydrotestosterone (DHT) and known AR antagonist nilutamide. The N/C interaction was induced by DHT from 10(-11)M to 10(-5)M in a dose-dependent manner. Nilutamide did not activate N/C interaction, while inhibited DHT-induced AR N/C interaction at the concentrations from 10(-7)M to 10(-5)M. Treatment of CV-1 cells with cypermethrin alone did not activate the reporter CAT. Cypermethrin significantly decreased the DHT-induced reporter CAT expression at the higher concentration of 10(-5)M. The mammalian two-hybrid assay provides a promising tool both for defining mechanism involved in AR N/C interaction of EDCs and for screening of chemicals with androgen agonistic and antagonistic activities. Cypermethrin exhibits inhibitory effects on the DHT-induced AR N/C interaction, while the potency is weaker than that of nilutamide., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

We evaluated the effect of the methanol extract of Basella alba (MEBa) on testosterone level and fecundity/fertility in male rats exposed in utero to flutamide - an androgen receptor antagonist. For this purpose, 1.5- and 2.5 -month-old male rats exposed in utero to flutamide were treated with the MEBa (1 mg kg(-1) ) for 2 and 1 month respectively. Five days before the end of treatment, rats were housed with females to assess their fecundity/fertility. Thereafter, rats were sacrificed and blood collected for the quantification of testosterone. Flutamide-exposed male rats showed a decrease in their ano-genital distance (AGD, P < 0.05) and were infertile. In normal (methylcellulose-exposed) animals, MEBa provoked an increase in testosterone level in 1.5- (P < 0.008) and 2.5 -month-old rats (P < 0.01) concomitantly with the improvement in their fecundity by 25%. In flutamide-exposed male rats, MEBa increased testosterone level in 1.5 -month-old rats (P < 0.001) without any effect on their fecundity; while in 2.5- month-old rats, MEBa did not affect the testosterone level but improved fecundity (by 25%) and fertility (P < 0.001). This study demonstrated the positive effect of MEBa to enhance fecundity/fertility in normal male rats and in rats exposed to the antiandrogen flutamide during their foetal life., (© 2011 Blackwell Verlag GmbH.)

A series of 4-phenylpyrrole derivatives D were designed, synthesized, and evaluated for their potential as novel orally available androgen receptor antagonists therapeutically effective against castration-resistant prostate cancers. 4-Phenylpyrrole compound 1 exhibited androgen receptor (AR) antagonistic activity against T877A and W741C mutant-type ARs as well as wild-type AR. An arylmethyl group incorporated into compound 1 contributed to enhancement of antagonistic activity. Compound 4n, 1-{[6-chloro-5-(hydroxymethyl)pyridin-3-yl]methyl}-4-(4-cyanophenyl)-2,5-dimethyl-1H-pyrrole-3-carbonitrile exhibited inhibitory effects on tumor cell growth against the bicalutamide-resistant LNCaP-cxD2 cell line as well as the androgen receptor-dependent JDCaP cell line in a mouse xenograft model. These results demonstrate that this series of pyrrole compounds are novel androgen receptor antagonists with efficacy against prostate cancer cells, including castration-resistant prostate cancers such as bicalutamide-resistant prostate cancer., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Methoxylated brominated diphenyl ethers (MeO-BDEs) in aquatic environments have been found to be primarily of natural origin in the marine environment and not from biotransformation of synthetic PBDEs. Two of the eight MeO-PBDEs (2'-MeO-BDE-68 and 6-MeO-BDE-47) that were detected in anchovy from the Yangtze River Delta, were natural products from marine organisms. So 2'-MeO-BDE-68 and 6-MeO-BDE-47 were chosen to study the potential to modulate androgen, estrogen, or thyroid hormone receptor- (AR, ER, ThR) mediated responses by use of reporter gene assays. 2'-MeO-BDE-68 was antiandrogenic at 50 μM, estrogenic at 10 μM and antiestrogenic at 10 and 50 μM (IC50=4.88 μM). 2'-MeO-BDE-68 enhanced luciferase expression by 5 nM T3 at 50 μM. 6-MeO-BDE-47 exhibited potent antiandrogenicity at 1 μM and greater (IC50=41.8 μM) and possessed estrogenic activity at 10 μM and antiestrogenic activity at 10 and 50 μM (IC50=6.02 μM)., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Few data exist on the possible effects of pulp and paper effluent discharge on native fish populations in the Southern Hemisphere, relative to the research done in the Northern Hemisphere. The present research examined two native fish species (Trichomycterus areolatus and Percilia gillissi) for effects at both the molecular and individual level due to the discharge of effluent from a tertiary treated elemental chlorine-free pulp mill into a fluvial system in Central Chile over three seasons (February 2007, October 2007, January 2008). Different responses were observed between species and between sexes. There was an increase in the production of gonadal 17β-estradiol in the females of both species but a drop in 11-ketotestosterone production in P. gillissi males. Female gonadal size was increased, especially during the summer period, with corresponding increases the frequency of advanced oocyte development, and in the oocyte diameter in both species. Hepatic ethoxyresorufin-O-deethylase (EROD) activity was elevated for both species downstream of the discharge point, although overall it was higher in P. gillissi than T. areolatus. Decreases in the frequency of smaller-sized fish for both species, as well as a drop in the size of the adults downstream of the discharge point, were observed. The present study is the first evidence of endocrine disruption in native freshwater fish associated with modern pulp mills in South America. This study establishes possible links in the reproductive alterations observed at the subindividual and individual levels that could explain the changes observed at the population level., (Copyright © 2011 SETAC.)

In continuation of our previous work, a novel series of steroid derivatives were synthesized and their androgen receptor (AR) antagonist activities and in vivo antiandrogenic properties were evaluated. Twenty-one heterocyclic derivatives containing a cyanopyrane ring fused to a steroidal moiety were conveniently synthesized and screened for their antagonistic, antiandrogen and prostate anticancer activities comparable to that of bicalutamide as the reference control. Some of the compounds exhibited better antagonistic, antiandrogen and prostate anticancer activities than the reference controls. Initially the acute toxicity of the compounds was assayed via the determination of their LD(50). Synthetic steroidal structures fused to a substituted cyanopyrane ring seem to be a promising approach in the search for novel leads for potent antagonistic, antiandrogen and prostate anticancer agents.

Widely used conazole antifungals were tested for endocrine disruptive effects using a panel of in vitro assays. They all showed endocrine disrupting potential and ability to act via several different mechanisms. Overall the imidazoles (econazole, ketoconazole, miconazole, prochloraz) were more potent than the triazoles (epoxiconazole, propiconazole, tebuconazole). The critical mechanism seems to be disturbance of steroid biosynthesis. In the H295R cell assay, the conazoles decreased the formation of estradiol and testosterone, and increased the concentration of progesterone, indicating inhibition of enzymes involved in the conversion of progesterone to testosterone. Prochloraz was most potent followed by econazole~miconazole>ketoconazole>tebuconazole>epoxiconazole>propiconazole. In the MCF-7 cell proliferation assay, the conazoles showed anti-estrogenic effect, including aromatase inhibition, since they inhibited the response induced by both 17β-estradiol (miconazole>econazole~ketoconazole>prochloraz>tebuconazole>epoxiconazole>propiconazole) and testosterone (econazole>miconazole>prochloraz>ketoconazole>tebuconazole>epoxiconazole>propiconazole). The triazoles were anti-androgenic in an androgen receptor reporter gene assay (epoxiconazole∼tebuconazole>propiconazole). This effect could not be evaluated for the pharmaceutical imidazoles due to cytotoxicity., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Pesticides have been suspected to act as endocrine disruptive compounds (EDCs) through several mechanisms of action, however data are still needed for a number of currently used pesticides. In the present study, 30 environmental pesticides selected from different chemical classes (azole, carbamate, dicarboximide, organochlorine, organophosphorus, oxadiazole, phenylureas, pyrazole, pyrimidine, pyrethroid and sulfonylureas) were tested for their ability to alter in vitro the transcriptional activity of the androgen receptor in the MDA-kb2 reporter cell line. The responsiveness of the system was checked by using a panel of reference ligands of androgen and glucocorticoid receptors. When tested alone at concentrations up to 10 μM, none of the studied pesticides were able to induce the reporter gene after a 18 h exposure. Conversely, co-exposure experiments with 0.1 nM dihydrotestosterone (DHT) allowed identifying 15 active pesticides with IC(50) ranging from 0.2 μM for vinclozolin to 12 μM for fenarimol. Fipronil and bupirimate were here newly described for their AR antagonistic activity., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Besides other modes of action, endocrine disruptors may interact with hormone receptors thereby modifying the physiological function of endogenous hormones. In the present study, we report the results obtained with yeast based assays to detect the (anti-)estrogenic potential (YES) and the (anti-)androgenic potential (YAS) of 105 substances. The results show very high reproducibility and good concordance with literature data of in vivo and/or in vitro studies: the overall true positive rate, true negative rate and accuracy of the assays were 78%, 95%, and 87% (estrogen agonism), and 70%, 97%, and 90% (estrogen antagonism), 88%, 96%, and 95% (androgen agonism) and 81%, 88%, and 85% (androgen antagonism). Furthermore, the performance of the YES assay has been compared to the HeLa based transcriptional activation assay using 20 compounds. The overall true positive rate, true negative rate, and accuracy obtained for the 20 compounds were 100%, 88%, and 95% (mammalian cell based HeLa assay) and 92%, 86%, and 90% (yeast based YES assay). Taken together, the YES and YAS are robust systems, easy to handle and satisfying the requirements for screening systems that can be applied in programs including the US Environmental Protection Agency's Endocrine Disruptor Screening Program., (Copyright © 2010 Elsevier Ltd. All rights reserved.)