Dihydroartemisinin alleviates AngII-induced vascular smooth muscle cell proliferation and inflammatory response by blocking the FTO/NR4A3 axis.

OBJECTIVE: Inflammation and proliferation of vascular smooth muscle cells (VSMCs), induced by angiotensin II (AngII) and other growth factors, play important roles in the pathogenesis of hypertension, restenosis, and atherosclerosis. Dihydroartemisinin (DHA) exhibits broad protective effects. However, the effects of DHA on AngII-induced inflammation and proliferation of VSMCs remain unknown. MATERIALS AND METHODS: AngII was used to construct VSMCs and vascular inflammation model in vitro and in vivo. The protective roles of DHA in inflammatory response and proliferation were evaluated through CCK-8, BrdU assay and immunofluorescence staining. The level of mRNA N6-methyladenosine was measured by m6A-RNA immunoprecipitation (MeRIP) assay. Western blot and quantitative real-time PCR were used to investigate the relationship between FTO and its potential downstream signaling molecules. RESULTS: In the present study, we found that DHA significantly suppressed AngII-induced proliferation of VSMCs and the expression of IL-6 and Ccl2 in a dose-dependent manner. Additionally, we confirmed that fat mass and obesity-associated (FTO) plays a critical role in AngII-induced VSMC proliferation and inflammation. FTO knockdown increased the methylation level of NR4A3 mRNA, whereas FTO, but not mutated FTO overexpression, reduced the methylation level of NR4A3 mRNA. These results suggest that DHA plays a protective role in AngII-induced VSMC proliferation and the associated inflammation by inhibiting the FTO/NR4A3 axis. CONCLUSION: Our findings provide new insight into the mechanisms of DHA and its critical role in the pathogenesis of hypertension-related vascular complications.

Use of miR­145 and testicular nuclear receptor 4 inhibition to reduce chemoresistance to docetaxel in prostate cancer.

The human testicular nuclear receptor 4 (TR4) is a critical regulatory gene for the progression of prostate cancer (PCa). Although it has been revealed that TR4 causes chemoresistance in PCa via the activation of octamer­binding transcription factor 4 (OCT4), the detailed mechanism remains unexplored. In the present study, it was revealed that inhibition of TR4 by shRNA in PCa enhanced the sensitivity to docetaxel in vitro and in vivo. TR4 induced the downregulation of miR­145 by directly binding it to the promoter of miR­145, which was confirmed by chromatin immunoprecipitation analysis and luciferase assay. The overexpression of miR­145 suppressed both the chemoresistance and the expression of OCT4 mRNA and protein. Additionally, the TR4 shRNA mediated re­sensitization to docetaxel, along with the downregulated expression of OCT4, were reversed by the concurrent inhibition of miR­145. The luciferase assay revealed that the activity of the wild­type OCT4 3' untranslated region reporter was suppressed. This suppression diminished when the miR­145 response element mutated. These findings suggest an undescribed regulatory pathway in PCa, by which TR4 directly suppressed the expression of miR­145, thereby inhibiting its direct target OCT4, leading to the promotion of chemoresistance in PCa.

Investigation of NH3 as a selective thyroid hormone receptor modulator in larval zebrafish (Danio rerio).

Thyroid hormones (THs) are essential for normal vertebrate development and diverse environmental chemicals are hypothesized to cause developmental toxicity by disrupting TH-mediated signaling. The larval zebrafish (Danio rerio) is an emerging in vivo model of developmental TH disruption; however, the effects of TR antagonism have not yet been studied in zebrafish. NH3, generally considered a potent and specific thyroid hormone receptor (TR) antagonist, has been used in rodents and Xenopus laevis to characterize phenotypes of TR antagonism. The objective of this study is to determine the effects of NH3 on endpoints previously determined to be TH-sensitive in larval zebrafish, specifically teratology and mortality, photomotor behavior, and mRNA expression of TH signaling genes. Zebrafish embryos were exposed to NH3 via static waterborne exposure at concentrations ranging from 0.001 to 10 µM beginning at 6 h post-fertilization (hpf) through 5 days post fertilization (dpf). Significant mortality and teratogenesis was observed at 3, 4, and 5 dpf in zebrafish exposed to NH3 at 10 µM. At concentrations that did not cause significant mortality, NH3 did not exert a consistent antagonistic effect on photomotor behavior assays or mRNA expression when administered alone or in the presence of exogenous T4. Rather, depending on the NH3 concentration and larval age NH3 decreased or increased swimming triggered by transition from light to dark. Similarly, inconsistent antagonistic and agonistic effects on mRNA expression of TH signaling genes were noted following treatment with NH3 alone. NH3 did inhibit T4 (30 nM)-induced gene expression; however, this was only consistently observed at a concentration of NH3 (10 µM) that also caused significant mortality. Collectively, these results suggest that NH3 does not act solely as a TR antagonist in larval zebrafish, but instead exhibits complex modulatory effects on TR activity. These data support the hypothesis that NH3 is a selective thyroid hormone receptor modulator. Further studies of NH3 interactions with the zebrafish thyroid hormone receptor are required to characterize the activity of NH3 in target tissues of the larval zebrafish at the molecular level, highlighting the importance of characterizing NH3 effects in specific models of TH-disruption to better interpret its actions in mechanistic screens of environmental chemicals for TH action.

Thyroid Hormone Signaling in Embryonic Stem Cells: Crosstalk with the Retinoic Acid Pathway.

While the role of thyroid hormones (THs) during fetal and postnatal life is well-established, their role at preimplantation and during blastocyst development remains unclear. In this study, we used an embryonic stem cell line isolated from rat (RESC) to study the effects of THs and retinoic acid (RA) on early embryonic development during the pre-implantation stage. The results showed that THs play an important role in the differentiation/maturation processes of cells obtained from embryoid bodies (EB), with thyroid hormone nuclear receptors (TR) (TRα and TRß), metabolic enzymes (deiodinases 1, 2, 3) and membrane transporters (Monocarboxylate transporters -MCT- 8 and 10) being expressed throughout in vitro differentiation until the Embryoid body (EB) stage. Moreover, thyroid hormone receptor antagonist TR (1-850) impaired RA-induced neuroectodermal lineage specification. This effect was significantly higher when cells were treated with retinoic acid (RA) to induce neuroectodermal lineage, studied through the gene and protein expression of nestin, an undifferentiated progenitor marker from the neuroectoderm lineage, as established by nestin mRNA and protein regulation. These results demonstrate the contribution of the two nuclear receptors, TR and RA, to the process of neuroectoderm maturation of the in vitro model embryonic stem cells obtained from rat.

Targeting the radiation-induced TR4 nuclear receptor-mediated QKI/circZEB1/miR-141-3p/ZEB1 signaling increases prostate cancer radiosensitivity.

Early studies indicated that the testicular nuclear receptor 4 (TR4) might play key roles in altering prostate cancer (PCa) progression; however, its ability to alter PCa radiosensitivity remains unclear. Here, we found that suppressing TR4 expression promoted radiosensitivity and better suppressed PCa by modulating the protein quaking (QKI)/circZEB1/miR-141-3p/ZEB1 signaling pathway. Mechanism dissection studies revealed that TR4 could transcriptionally increase the RNA-binding protein QKI to increase circZEB1 levels, which then sponges the miR-141-3p to increase the expression of its host gene ZEB1. Preclinical studies with an in vivo mouse model further proved that combining radiation therapy (RT) with metformin promoted radiosensitivity to suppress PCa progression. Together, these results suggest that TR4 may play key roles in altering PCa radiosensitivity and show that targeting this newly identified TR4-mediated QKI/circZEB1/miR-141-3p/ZEB1 signaling pathway may help in the development of a novel RT to better suppress the progression of PCa.

Targeting TR4 nuclear receptor with antagonist bexarotene increases docetaxel sensitivity to better suppress the metastatic castration-resistant prostate cancer progression.

Prostate cancer (PCa) is the second leading cause of cancer death in men in America, and there are no curative options for metastatic castration-resistant prostate cancer (mCRPC). Docetaxel (DTX) has been used as a standard chemotherapy for the mCRPC. However, resistance to DTX is a significant clinical problem as half of patients fail to respond to therapy. The TR4 nuclear receptor has been reported to play an important role in PCa progression, however, its linkage to the DTX resistance remains unclear. Here we found that TR4 was upregulated after DTX chemotherapy in the mCRPC cells and patients, and TR4 expression is correlated with DTX sensitivity with a higher level conferring chemo-resistance. Targeting TR4 with an antagonist bexarotene (Bex, a derivative of retinoid) suppressed the TR4 transactivation with increased DTX chemo-sensitivity. Mechanism dissection studies revealed that TR4 might alter the DTX chemo-sensitivity via modulating the TR4/lincRNA-p21/HIF-1α/VEGF-A signaling. Together, these results suggest that targeting this newly identified TR4/lincRNA-p21/HIF-1α/VEGF-A signaling with Bex, an FDA-approved drug, may increase the DTX chemo-sensitivity to better suppress the mCRPC progression.

Bisphenols and Thyroid Hormone.

In recent decades, attention has been directed toward the effects of bisphenol A (BPA) on human health. BPA has estrogenic activity and is regarded as a representative endocrine disruptor. In addition, mounting evidence indicates that BPA can disrupt thyroid hormone and its action. This review examined human epidemiological studies to investigate the association between BPA exposure and thyroid hormone levels, and analyzed in vivo and in vitro experiments to identify the causal relationship and its mechanism of action. BPA is involved in thyroid hormone action not only as a thyroid hormone receptor antagonist, but also through several other mechanisms. Since the use of bisphenols other than BPA has recently increased, we also reviewed the effects of other bisphenols on thyroid hormone action.

The molecular mechanism of the antagonistic activity of hydroxylated polybrominated biphenyl (OH-BB80) toward thyroid receptor ß.

Polybrominated biphenyls (PBBs) were widely used as additive brominated flame retardants. Their hydroxylated products (OH-PBBs) have been detected frequently in various marine mammals, causing an increased health risk. Till now, there lacks information on the potential disruption of OH-PBBs toward thyroid hormone receptor (TR) and the molecular characteristics of their interactions remain largely unknown. We herein in vitro and in silico evaluated the disrupting effect of 3,3',5,5'-tetrabromobiphenyl (BB80) and its metabolite 2,2'-dihydroxy- 3,3',5,5'-tetrabromobiphenyl (OH-BB80) toward human TR. The recombinant human TRß two-hybrid yeast assay reveals the moderate antagonistic activity of OH-BB80 with IC20 at 2 µmol/L, while BB80 shows no agonistic or antagonistic activity. OH-BB80 binds at the binding cavity of TRß ligand binding domain (LBD) and forms one hydrogen bond with Phe272. Electrostatic interactions and hydrophobic interactions contribute much to their interactions. The binding of OH-BB80 quenches the intrinsic fluorescence of TRß LBD at static quenching mode. Our study extends knowledge on the endocrine disrupting effect of OH-PBBs and suggests the full consideration of the biotransformation for further health risk assessment of PBBs and related structurally similar emerging contaminants.

A comparison of endocrine disruption potential of nonylphenol ethoxylate, vanillin ethoxylate, 4-n-nonylphenol and vanillin in vitro.

The widely used surfactant nonylphenol ethoxylate (NPEO) and its raw material 4-n-nonylphenol (4-n-NP), as well as its degradation products, are recognized as endocrine disrupting chemicals. The USA Environmental Protection Agency (EPA) released an assessment that looked for safe alternatives to NPEO. Vanillin ethoxylate (VAEO) is a novel substitute for NPEO and is quite similar to NPEO in structure; there is a risk that it has similar endocrine disrupting effects to NPEO. However, their effects on various nuclear hormone receptors have not been thoroughly examined. In this study, the effects of NPEO, VAEO, 4-n-NP and Vanillin on the estrogen receptor α (ERα), androgen receptor (AR), thyroid hormone receptor (TR), retinoic X receptor ß (RXRß) and estrogen-related receptor γ (ERRγ) were determined and compared using a battery of recombined yeast strains expressing ß-galactosidase. The results showed that NPEO and 4-n-NP acted as significant antagonists of ER, AR, TR and ERRγ. In addition, 4-n-NP also had antagonistic activity toward RXRß. Moreover, VAEO was shown to be a very weak antagonist of TR and ERRγ, and Vanillin had no interaction with any nuclear receptors. For the first time, it was found that NPEO had AR, TR and ERRγ antagonistic effects and that 4-n-NP was an antagonist of RXRß. The in vitro data indicated that NPEO, 4-n-NP and VAEO have the potential to act as endocrine disruptors involving more than one nuclear hormone receptor, but VAEO has much lower endocrine disrupting potential than NPEO. Thus, it is critical to find safe substitutes for NPEO and a substitute of NPEO with structural analogues should be carried out with caution. Furthermore, to look for preferable alternatives for NPEO, more in vivo and in vitro studies of the alternatives concerning endocrine disruption are needed, especially in vitro studies need to involve various target points, not only focus on their effects on ER but also take other nuclear hormone receptor pathways into consideration.

Classification of thyroid hormone receptor agonists and antagonists using statistical learning approaches.

In silico models are presented for modeling and predicting thyroid hormone receptor (TR) agonists and antagonists. A data set consisting of 258 compounds is used in the present work. The C4.5, random forest (RF) and support vector machine (SVM) statistical methods were used for evaluation. The performance of the quantitative structure-activity relationships was further validated with fivefold cross-validation and an independent external test set. The C4.5 model is slightly weak, and the prediction accuracies of the agonists and antagonists are 93.2 and 57.8% for cross-validation, respectively, averaging 83.1% of correctly classified compounds in the test set. The RF model possesses an average prediction accuracy of 84.0 and 87.1% for the cross-validation and external validation, respectively. Furthermore, the overall prediction accuracy and the external prediction accuracy are 96.6 and 97.2%, respectively, for the SVM model. The results would validate the reliability of the derived models, further demonstrating that RF and SVM models are useful tools capable of classifying TR-binding ligands as agonists or antagonists.

GLP-1 Receptor Agonist Exendin-4 Attenuates NR4A Orphan Nuclear Receptor NOR1 Expression in Vascular Smooth Muscle Cells.

AIMS: Recently, incretin therapy has attracted increasing attention because of its potential use in tissue-protective therapy. Neuron-derived orphan receptor 1 (NOR1) is a nuclear orphan receptor that regulates vascular smooth muscle cell (VSMC) proliferation. In the present study, we investigated the vascular-protective effect of Exendin-4 (Ex-4), a glucagon-like peptide-1 receptor agonist, by inhibiting NOR1 expression in VSMCs. METHODS: We classified 7-week-old male 129X1/SvJ mice into control group and Ex-4 low- and high-dose-treated groups fed normal or high-fat diets, respectively. Endothelial denudation injuries were induced in the femoral artery at 8 weeks of age, followed by the evaluation of neointima formation at 12 weeks of age. To evaluate VSMC proliferation, bromodeoxyuridine incorporation assay and cell cycle distribution analysis were performed. NOR1 and cell cycle regulators were detected using immunohistochemistry, western blotting, quantitative reverse-transcription polymerase chain reaction, and luciferase assays. RESULTS: Ex-4 treatment reduced vascular injury-induced neointima formation compared with controls. In terms of VSMCs occupying the neointima area, VSMC numbers and NOR1-expressing proliferative cells were significantly decreased by Ex-4 in a dose-dependent manner in both diabetic and non-diabetic mice. In vitro experiments using primary cultured VSMCs revealed that Ex-4 attenuated NOR1 expression by reducing extracellular signal-regulated kinase-mitogen-activated protein kinase and cAMP-responsive element-binding protein phosphorylations. Furthermore, in the cell cycle distribution analysis, serum-induced G1-S phase entry was significantly attenuated by Ex-4 treatment of VSMCs by inhibiting the induction of S-phase kinase-associated protein 2. CONCLUSION: Ex-4 attenuates neointima formation after vascular injury and VSMC proliferation possibly by inhibiting NOR1 expression.

Endocrine disrupting activities and immunomodulatory effects in lymphoblastoid cell lines of diclofenac, 4-hydroxydiclofenac and paracetamol.

A critical literature review reveals that knowledge of side effects of pharmaceuticals diclofenac and paracetamol is extremely important because of their widespread use and occurrence in the environment. In order to delineate whether these compounds have endocrine activity and influence on the immune system, we assessed the potential endocrine disrupting and immunomodulatory activities of: diclofenac (DIC), its metabolite 4-hydroxydiclofenac (4-HD) and paracetamol (PAR). Herein, we report on their impact on estrogen receptor (ER), androgen receptor (AR), glucocorticoid receptor (GR) and thyroid hormone receptor (TR). The endocrine disrupting effects were assessed in vitro in MDA-kb2 and GH3.TRE-Luc cell lines and by the XenoScreen YES/YAS assay. Moreover, binding affinity to nuclear receptors (GR and AR) was also measured. Immunomodulatory properties of the compounds were evaluated in lymphoblastoid cell lines. All the tested compounds showed endocrine disrupting and immunomodulatory activities. The results revealed that both DIC and its metabolite 4-HD exhibited significant estrogenic, anti-androgenic (in YAS assay), (anti)-androgenic, (anti)-glucocorticoid and anti-thyroid hormonal activities (in luciferase reporter gene assays). DIC showed direct binding to the GR, while its metabolite 4-HD to the GR and AR. Only metabolite 4-HD showed estrogenic, androgenic (in YAS assay) and thyroid-hormonal activities. PAR had anti-androgenic activity and anti-thyroid hormonal activity. PAR displayed GR agonist activity with competition to its receptor and agonistic activity to AR. All of the compounds significantly modulated pro-inflammatory and immunoregulatory cytokine production in lymphoblastoid cell lines and were thus proven immunomodulatory. The study is useful in determining toxicological effects and contributes to the knowledge of possible side effects of diclofenac, its metabolite and paracetamol.

Thyroid Hormone Receptor Antagonists: From Environmental Pollution to Novel Small Molecules.

Thyroid hormone receptors (TRs) are nuclear receptors which control transcription, and thereby have effects in all cells within the body. TRs are an important regulator in many basic physiological processes including development, growth, metabolism, and cardiac function. The hyperthyroid condition results from an over production of thyroid hormones resulting in a continual stimulation of thyroid receptors which is detrimental for the patient. Therapies for hyperthyroidism are available, but there is a need for new small molecules that act as TR antagonists to treat hyperthyroidism. Many compounds exhibit TR antagonism and are considered detrimental to health. Some drugs in the clinic (most importantly, amiodarone) and environmental pollution exhibit TR antagonist properties and thus have the potential to induce hypothyroidism in some people. This chapter provides an overview of novel small molecules that have been specifically designed or screened for their TR antagonist activity as novel treatments for hyperthyroidism. While novel compounds have been identified, to date none have been developed sufficiently to enter clinical trials. Furthermore, a discussion on other sources of TR antagonists is discussed in terms of side effects of current drugs in the clinic as well as environmental pollution.

Risk assessment of the endocrine-disrupting effects of nine chiral pesticides.

The increased release of chiral pesticides into the environment has generated interest in the role of enantioselectivity in the environmental fate and ecotoxicological effects of these compounds. However, the information on the endocrine disrupting effects (EDEs) of chiral pesticides is still limited and discrepancies are also usually observed among different assays. In this study, we investigated the enantioselectivity of EDEs via estrogen and thyroid hormone receptors for nine chiral pesticides using in vitro and in silico approaches. The results of the luciferase reporter gene assays showed 7 chiral pesticides possessed enantioselective estrogenic activities and 2 chiral pesticides exerted thyroid hormone antagonistic effects. Proliferation assays in MCF-7 and GH3 cells were also used to verify the results of the dual-luciferase reporter gene assays. At last, the molecular docking results indicated that the enantioselective EDEs of chiral pesticides were partially due to enantiospecific binding affinities with receptors. Our data not only show enantioselective EDEs of nine chiral pesticides, but also would be helpful to better understanding the molecular biological mechanisms of enantioselectivity in EDEs of chiral pesticides.

Inhibition of thyroid hormone receptor locally in the retina is a therapeutic strategy for retinal degeneration.

Thyroid hormone (TH) signaling regulates cell proliferation, differentiation, and metabolism. Recent studies have implicated TH signaling in cone photoreceptor viability. Using mouse models of retinal degeneration, we demonstrated that antithyroid drug treatment and targeting iodothyronine deiodinases (DIOs) to suppress cellular tri-iodothyronine (T3) production or increase T3 degradation preserves cones. In this work, we investigated the effectiveness of inhibition of the TH receptor (TR). Two genes, THRA and THRB, encode TRs; THRB2 has been associated with cone viability. Using TR antagonists and Thrb2 deletion, we examined the effects of TR inhibition. Systemic and ocular treatment with the TR antagonists NH-3 and 1-850 increased cone density by 30-40% in the Rpe65-/- mouse model of Leber congenital amaurosis and reduced the number of TUNEL+ cells. Cone survival was significantly improved in Rpe65-/- and Cpfl1 (a model of achromatopsia with Pde6c defect) mice with Thrb2 deletion. Ventral cone density in Cpfl1/Thrb2-/- and Rpe65-/- /Thrb2-/- mice was increased by 1- to 4-fold, compared with age-matched controls. Moreover, the expression levels of TR were significantly higher in the cone-degeneration retinas, suggesting locally elevated TR signaling. This work shows that the effects of antithyroid treatment or targeting DIOs were likely mediated by TRs and that suppressing TR protects cones. Our findings support the view that inhibition of TR locally in the retina is a therapeutic strategy for retinal degeneration management.-Ma, H., Yang, F., Butler, M. R., Belcher, J., Redmond, T. M., Placzek, A. T., Scanlan, T. S., Ding, X.-Q. Inhibition of thyroid hormone receptor locally in the retina is a therapeutic strategy for retinal degeneration.

Triiodothyronine promotes the proliferation of epicardial progenitor cells through the MAPK/ERK pathway.

Thyroid hormone has important functions in the development and physiological function of the heart. The aim of this study was to determine whether 3,5,3'-Triiodothyronine (T3) can promote the proliferation of epicardial progenitor cells (EPCs) and to investigate the potential underlying mechanism. Our results showed that T3 significantly promoted the proliferation of EPCs in a concentration- and time-dependent manner. The thyroid hormone nuclear receptor inhibitor bisphenol A (100 µmol/L) did not affect T3's ability to induce proliferation. Further studies showed that the mRNA expression levels of mitogen-activated protein kinase 1 (MAPK1), MAPK3, and Ki67 in EPCs in the T3 group (10 nmol/L) increased 2.9-, 3-, and 4.1-fold, respectively, compared with those in the control group (P < 0.05). In addition, the mRNA expression of the cell cycle protein cyclin D1 in the T3 group increased approximately 2-fold compared with the control group (P < 0.05), and there were more EPCs in the S phase of the cell cycle (20.6% vs. 12.0%, P < 0.05). The mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling pathway inhibitor U0126 (10 µmol/L) significantly inhibited the ability of T3 to promote the proliferation of EPCs and to alter cell cycle progression. This study suggested that T3 significantly promotes the proliferation of EPCs, and this effect may be achieved through activation of the MAPK/ERK signaling pathway.

Endocrine Activity of AVB, 2MR, BHA, and Their Mixtures.

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.

[Effect of Dissolved Humic Acid on Thyroid Receptor Antagonistic Activity of Zinc in Aquatic Environment].

A rapid recombinant human thyroid (hTR) gene yeast bioassay was used to evaluate the effect of dissolved humic acid on thyroid receptor antagonistic activity of ZnCl2.The concentration of bio-available zinc after its reaction with dissolved humic acids was measured by anodic stripping voltammetry (ASV).Furthermore,the reaction mechanism of humic acid and zinc was investigated by three-dimensional excitation-emission matrix fluorescence spectroscopy (3DEEM).The results revealed that ZnCl2 demonstrated strong thyroid receptor antagonistic activity,and the concentration inhibiting 20% of the maximum effect of ZnCl2 was 1.70×10-5 mol·L-1.The thyroid receptor antagonistic activity of ZnCl2 was reduced by 30%-50% after the reaction of dissolved humic acids.The results of ASV showed that the concentration of bio-available zinc was decreased after the reaction of dissolved humic acids,the result was similar to that of bioassay test.The thyroid receptor antagonistic activity of the mixed solution of humic acid and ZnCl2 was increased after UV radiation treatment,however it was still lower than the antagonistic activity induced by ZnCl2.The results of 3DEEM showed that ZnCl2 could reduce the fluorescence peak intensity of humic acid,which could intuitively characterize the interaction between humic acid and ZnCl2.The above results can provide basic data and theoretical support for zinc toxicity study in aquatic environment and the establishment of water quality criteria for znic.

Influence of metabolism on endocrine activities of bisphenol S.

Bisphenol S (BPS; bis[4-hydroxyphenyl]sulfone) is commonly used as a replacement for bisphenol A in numerous consumer products. The main goal of this study was to examine the influence of different metabolic reactions that BPS undergoes on the endocrine activity. We demonstrate that hydroxylation of the aromatic ring of BPS, catalyzed mainly by the cytochrome P450 enzymes CYP3A4 and CYP2C9, is its major in-vitro phase I biotransformation. Nevertheless, coupled oxidative-conjugative reactions analyses revealed that glucuronidation and formation of BPS glucuronide is the predominant BPS metabolic pathway. BPS reactive metabolites that can be tracked as glutathione conjugates were not detected in the present study. Two in-vitro systems were used to evaluate the endocrine activity of BPS and its two main metabolites, BPS glucuronide and hydroxylated BPS 4-(4-hydroxy-benzenesulfonyl)-benzene-1,2-diol (BPSM1). In addition, we have tested two structural analogs of BPS, bis[4-(2-hydroxyetoxy)phenyl]sulfone (BHEPS) and 4,4-sulfonylbis(2-methylphenol) (dBPS). The test systems were yeast cells, for evaluating estrogenic and androgenic activities, and the GH3.TRE-Luc reporter cell line for measuring thyroid hormone activity. BPS and BPSM1 were weak agonists of the estrogen receptor, EC50 values of 8.4 × 10(-5) M and 6.7 × 10(-4) M, respectively. Additionally, BPSM1 exhibited weak antagonistic activity toward the thyroid hormone receptor, with an IC50 of 4.3 × 10(-5) M. In contrast to BPSM1, BPS glucuronide was inactive in these assays, inhibiting neither the estrogen nor the thyroid hormone receptors. Hence, glucuronidation appears to be the most important pathway for both BPS metabolism and detoxification.

T-screen and yeast assay for the detection of the thyroid-disrupting activities of cadmium, mercury, and zinc.

In the present study, a two-hybrid yeast bioassay and a T-screen were used to screen for the thyroid receptor (TR)-disrupting activity of select metallic compounds (CdCl2, ZnCl2, HgCl2, CuSO4, MnSO4, and MgSO4). The results reveal that none of the tested metallic compounds showed TR-agonistic activity, whereas ZnCl2, HgCl2, and CdCl2 demonstrated TR antagonism. For the yeast assay, the dose-response relationship of these metallic compounds was established, and the concentrations producing 20 % of the maximum effect of ZnCl2, HgCl2, and CdCl2 were 9.1 × 10(-5), 3.2 × 10(-6), and 1.2 × 10(-6) mol/L, respectively. The T-screen also supported the finding that ZnCl2, HgCl2, and CdCl2 decreased the cell proliferation at concentrations ranging from 10(-6) to 10(-4) mol/L. Furthermore, the thyroid-disrupting activity of metallic compounds in environmental water samples collected from the Guanting Reservoir, Beijing, China was evaluated. Solid-phase extraction was used to separate the organic extracts, and a modified two-hybrid yeast bioassay revealed that the metallic compounds in the water samples could affect thyroid hormone-induced signaling by decreasing the binding of the thyroid hormone. The addition of ethylenediaminetetraacetic acid (30 mg/L) could eliminate the effects. Thus, the cause(s) of the thyroid toxicity in the water samples appeared to be partly related to the metallic compounds.