Comparative study on estrogen receptor alpha dimerization and transcriptional activity of parabens.

Parabens are used as preservatives in various household products, including oral products, cosmetics, and hair/body washes. In recent years, the widespread use of parabens has raised concerns due to the potential health risks associated with their estrogenic effects. In the present study, we evaluated and compared the estrogenic activity of parabens using two cell-based in vitro tests: (1) bioluminescence resonance energy transfer (BRET)-based estrogen receptor alpha (ERα) dimerization using HEK293 cells that were stably transfected with ERα-fused NanoLuc luciferase (Nluc) and HaloTag (HT) expression vector, and (2) stably transfected transcriptional activation (STTA) assays using ERα-HeLa9903 cells. The following parabens were tested using the BRET-based ERα dimerization assay and showed estrogenic activity (PC20 values): methyl paraben (MP, 5.98 × 10-5 M), ethyl paraben (EP, 3.29 × 10-5 M), propylparaben (PP, 3.09 × 10-5 M), butyl paraben (BP, 2.58 × 10-5 M), isopropyl paraben (IsoPP, 1.37 × 10-5 M), and isobutyl paraben (IsoBP, 1.43 × 10-5 M). Except MP, all other parabens tested using the STTA assay also showed estrogenic activity: EP, 7.57 × 10-6 M; PP, 1.18 × 10-6 M; BP, 3.02 × 10-7 M; IsoPP, 3.58 × 10-7 M; and IsoBP, 1.80 × 10-7 M. Overall, EP, PP, BP, IsoPP, and IsoBP tested positive for estrogenic activity using both assays. These findings demonstrate that most parabens, albeit not all, induce ERα dimerization and possess estrogenic activity.

Comprehensive assessment of the estrogenic activity of resin composites.

Resin-based dental composites have been developed to restore decayed teeth or modify tooth color due to their excellent physical and chemical properties. Such composites may have intrinsic toxicity due to components released into the mouth during the early stage of polymerization, and afterward as a result of erosion or material decomposition. In addition, resin-based dental composites have potential environmental pollutant by elution of monomers and degradation. Since certain monomers of resin matrices are synthesized from bisphenol A (BPA), which acts as an estrogenic endocrine disruptor, these resin matrices may have estrogenic activity. Therefore, the estrogenic endocrine-disrupting activity of various dental composites should be evaluated. In this study, we evaluated the estrogenic endocrine-disrupting activity of 10 resin composites by using a BRET-based estrogen receptor (ER)α and ERß dimerization assays and ER transactivation assay. BPA, BisDMA, BisGMA, BisEMA, TEGDMA, HMBP, and DMPA mediated ERα dimerization, and BPA, BisDMA, and DMPA also mediated ERß dimerization. Except for UDMA and CQ, all the compounds were identified as estrogen agonists or antagonists. In-depth information for the safe use of dental composites was acquired, and it was confirmed how the component of dental composites acts in the ER signaling pathway. Further studies on the low-dose and long-term release of these compounds are needed to ensure the safe use of these resin-based dental composites.

Effects of gypenoside L-containing Gynostemma pentaphyllum extract on fatigue and physical performance: A double-blind, placebo-controlled, randomized trial.

This study was conducted to investigate the effect of Gynostemma pentaphyllum extract containing gypenoside L (GPE) on improving the cognitive aspects of fatigue and performance of the motor system. One hundred healthy Korean adults aged 19-60 years were randomized to the treatment (GPE for 12 weeks) and control groups, and efficacy and safety-related parameters were compared between the two groups. Maximal oxygen consumption (VO2 max) and O2 pulse were significantly higher in the treatment group than in the control group (p = 0.007 and p = 0.047, respectively). After 12 weeks, the treatment group showed significant changes such as decreases in the levels of free fatty acids (p = 0.042). In addition, there were significant differences in the rating of perceived exertion (RPE) (p < 0.05) and value of temporal fatigue between the treatment and control groups on the multidimensional fatigue scale (p < 0.05). Moreover, the level of endothelial nitric oxide synthase (eNOS) in the blood was significantly higher in the treatment group than in the control group (p = 0.047). In summary, oral administration of GPE has a positive effect on resistance to exercise-induced physical and mental fatigue.

Comprehensive analysis of cellular estrogen signaling in representative estrogen receptor ligands.

The estrogen receptor (ER)-mediated signaling pathway in physiological and biochemical aspects is very important in the environment, including food. The physiological action of estrogen is mediated by ER alpha (ERα) and beta (ERß), whose physiological action on estrogenic substances is complex because of the relatively low ligand-binding domain (LBD) similarity of the two ERs. In this study, the comprehensive activity of representative ER ligands was evaluated by using BRET-based ERα and ERß dimerization and ER transactivation assays to differentiate the specific binding and function of ERα and ERß from 12 representative natural and synthetic estrogenic substances. Results revealed that 11 chemicals mediated receptor ERα and ERß dimerization, 7 out of 12 chemicals were confirmed to be estrogen agonists, and 5 chemicals were antagonistic. Overall, this study demonstrated consistency between BRET dimerization and transactivation responses, supporting potential supplementary application of mechanism-based BRET assays as high-throughput screening methods for evaluation of potential endocrine-disrupting activity of environmental agents. This study also provided information about receptor specificity of ligand-mediated estrogenic activity via dimerization assays and elucidated cellular estrogen signaling pathways.

Human cell-based estrogen receptor beta dimerization assay.

Estrogen is not only responsible for important functions in the human body, such as cell growth, reproduction, differentiation, and development, but it is also deeply related to pathological processes, such as cancer, metabolic and cardiovascular diseases, and neurodegeneration. Estrogens and other estrogenic compounds have transcriptional activities through binding with the estrogen receptor (ER) to induce ER dimerization. The two estrogen receptor subtypes, estrogen receptor alpha (ERα) and estrogen receptor beta (ERß), show structural differences and have different expression ratios in specific cells and tissues. Currently, the methods for confirming the estrogenic properties of compounds are the binding (Test guideline no. 493) and transactivation (Test guideline no. 455) assays provided by the Organization for Economic Co-operation and Development (OECD). In a previous study, we developed an ERα dimerization assay based on the bioluminescence resonance energy transfer (BRET) system, but there are currently no available tests that can confirm the effect of estrogenic compounds on ERß. Therefore, in this study, we developed a BRET-based ERß dimerization assay to confirm the estrogenic prosperities of compounds. The BRET-based ERß dimerization assay was verified using nine representative ER ligands and the results were compared with the dimerization activity of ERα. In conclusion, our BRET-based ERß dimerization assay can provide information on the ERß dimerization potential of estrogenic compounds.

Effect of Annealing Temperature on the Structural and Electrochemical Properties of Hydrothermally Synthesized NiCo2O4 Electrodes.

In this study, a porous Ni-foam support was employed to enhance the capacitance of nickel cobaltite (NiCo2O4) electrodes designed for supercapacitors. The hydrothermal synthesis method was employed to grow NiCo2O4 as an active material on Ni-foam. The NiCo2O4 sample derived from hydrothermal synthesis underwent subsequent post-heat treatment at temperatures of 250 °C, 300 °C, and 350 °C. Thermogravimetric analysis of the NiCo2O4 showed that weight loss due to water evaporation occurs after 100 °C and enters the stabilization phase at temperatures above 400 °C. The XRD pattern indicated that NiCo2O4 grew into a spinel structure, and the TEM results demonstrated that the diffraction spots (DSs) on the (111) plane of the sample annealed at 350 °C were more pronounced than those of other samples. The specific capacitance of the NiCo2O4 electrodes exhibited a decrease with increasing current density across all samples, irrespective of the annealing temperature. The electrode annealed at 350 °C recorded the highest specific capacitance value. However, the capacity retention rate of the NiCo2O4 electrode revealed a deteriorating trend, declining to 88% at 250 °C, 75% at 300 °C, and 63% at 350 °C, as the annealing temperature increased.

Variations in the Physical Properties of RF-Sputtered CdS Thin Films Observed at Substrate Temperatures Ranging from 25 °C to 500 °C.

CdS films with a wide range of substrate temperatures as deposition parameters were fabricated on Corning Eagle 2000 glass substrates using RF magnetron sputtering. The crystallographic structure, microscopic surface texture, and stoichiometric and optical properties of each CdS film deposited at various substrate temperatures were observed to be highly temperature-dependent. The grown CdS thin films revealed a polycrystalline structure in which a cubic phase was mixed based on a hexagonal wurtzite phase. The relative intensity of the H(002)/C(111) peak, which represents the direction of the preferential growth plane, enhanced as the temperatures climbed from 25 °C to 350 °C. On the contrary, the intensity of the main growth peak at the higher temperatures of 450 °C and 500 °C was significantly reduced and exhibited amorphous-like behavior. The sharp absorption edge revealed in the transmission spectrum shifted from the long wavelength to the short wavelength region with the rise in the substrate temperature. The bandgap showed a tendency to widen from 2.38 eV to 2.97 eV when the temperatures increased from 25 °C to 350 °C. The CdS films grown at the temperatures of 450 °C and 500 °C exhibited glass-like transmittance with almost no interference fringes of light, which resulted in wide bandgap values of 3.09 eV and 4.19 eV, respectively.

Comparative study of estrogenic activities of phytoestrogens using OECD in vitro and in vivo testing methods.

With growing scientific interest in phytoestrogens, a number of studies have investigated the estrogenic potential of phytoestrogens in a wide variety of assay systems. However, evaluations of individual phytoestrogens with different assay systems make it difficult for predicting their relative estrogenic potency. The objective of this study was to compare estrogenic properties of fifteen known phytoestrogens using an estrogen receptor-α (ER-α) dimerization assay and Organization for Economic Cooperation and Development (OECD) standardized methods including in vitro estrogen receptor (ER) transactivation assay using VM7Luc4E2 cells and in vivo uterotrophic assay using an immature rat model. Human ER-α dimerization assay showed positive responses of eight test compounds and negative responses of seven compounds. These results were consistently found in luciferase reporter assay results for evaluating ER transactivation ability. Seven test compounds exhibiting relatively higher in vitro estrogenic activities were subjected to uterotrophic bioassays. Significant increases in uterine weights were only found after treatments with biochanin A, 8-prenylnaringenin, and coumestrol. Importantly, their uterotrophic effects were lost when animals were co-treated with antagonist of ER, indicating their ER-dependent effects in the uterus. In addition, analysis of estrogen responsive genes revealed that these phytoestrogens regulated uterine gene expressions differently compared to estrogens. Test methods used in this study provided a high consistency between in vitro and in vivo results. Thus, they could be used as effective screening tools for phytoestrogens, particularly focusing on their interactions with ER-α.

An In vitro dimerization assay for the adverse outcome pathway approach in risk assessment of human estrogen receptor α-mediated endocrine-disrupting chemicals.

The adverse outcome pathway (AOP) has been recently proposed as an effective framework for chemical risk assessment. The AOP framework offers the advantage of effectively integrating individual in vitro studies and in silico prediction models. Thus, the development of an effective testing method to measure key events caused by chemicals is essential for chemical risk assessment through a fully developed AOP framework. We developed a human cell-based estrogen receptor α (ERα) dimerization assay using the bioluminescence resonance energy transfer (BRET) technique and evaluated the ERα dimerization activities of 72 chemicals. Fifty-one chemicals were identified to mediate dimerization of ERα, and the BRET-based ERα dimerization assay could effectively measure the events that mediated dimerization of ERα by the estrogenic chemicals. These results were compared with the results of pre-existing assay to determine whether the BRET-based ERα dimerization assay could be employed as an in vitro test method to provide scientific information for explaining key events as a part of the AOP framework. Consequently, we propose that the BRET-based ERα dimerization assay is suitable for measuring the chemical-mediated dimerization of ERα, a key event in the AOP framework for cellular-level risk assessment of estrogenic chemicals.

Mechanistic insight into human androgen receptor-mediated endocrine-disrupting potentials by a stable bioluminescence resonance energy transfer-based dimerization assay.

To develop a novel cell-based assay to evaluate the androgenic endocrine-disrupting properties of chemical substances, we established a method to detect ligand-mediated androgen receptor (AR) dimerization in stably transfected human cell lines using a bioluminescence resonance energy transfer (BRET) system. Using stably transfected human embryonic kidney (HEK-293) cells, the BRET-based AR dimerization assay was optimized as a novel test method and was validated using test chemicals recommended by the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM). The BRET-based AR dimerization assay showed high accuracy, sensitivity, and specificity for the detection of androgenic endocrine-disrupting chemicals (EDCs), and the assay protocol is adequate for practical use. This dimerization assay is based on ligand-mediated hormone receptor dimerization and can provide accurate information about androgenic endocrine-disrupting properties at the cellular level, complementing conventional binding and transactivation assays.

Development of a Human Estrogen Receptor Dimerization Assay for the Estrogenic Endocrine-Disrupting Chemicals Using Bioluminescence Resonance Energy Transfer.

Endocrine-disrupting chemicals (EDCs) are found in food and various other substances, including pesticides and plastics. EDCs are easily absorbed into the body and have the ability to mimic or block hormone function. The radioligand binding assay based on the estrogen receptors binding affinity is widely used to detect estrogenic EDCs but is limited to radioactive substances and requires specific conditions. As an alternative, we developed a human cell-based dimerization assay for detecting EDC-mediated ER-alpha (ERα) dimerization using bioluminescence resonance energy transfer (BRET). The resultant novel BRET-based on the ERα dimerization assay was used to identify the binding affinity of 17ß-estradiol (E2), 17α-estradiol, corticosterone, diethylhexyl phthalate, bisphenol A, and 4-nonylphenol with ERα by measuring the corresponding BRET signals. Consequently, the BRET signals from five chemicals except corticosterone showed a dose-dependent sigmoidal curve for ERα, and these chemicals were suggested as positive chemicals for ERα. In contrast, corticosterone, which induced a BRET signal comparable to that of the vehicle control, was suggested as a negative chemical for ERα. Therefore, these results were consistent with the results of the existing binding assay for ERα and suggested that a novel BRET system can provide information about EDCs-mediated dimerization to ERα.

(-)-Epicatechin-Enriched Extract from Camellia sinensis Improves Regulation of Muscle Mass and Function: Results from a Randomized Controlled Trial.

Loss of skeletal muscle mass and function with age represents an important source of frailty and functional decline in the elderly. Antioxidants from botanical extracts have been shown to enhance the development, mass, and strength of skeletal muscle by influencing age-related cellular and molecular processes. Tannase-treated green tea extract contains high levels of the antioxidants (-)-epicatechin (EC) and gallic acid that may have therapeutic benefits for age-related muscle decline. The aim of this study was to investigate the effect of tannase-treated green tea extract on various muscle-related parameters, without concomitant exercise, in a single-center, randomized, double-blind, placebo-controlled study. Administration of tannase-treated green tea extract (600 mg/day) for 12 weeks significantly increased isokinetic flexor muscle and handgrip strength in the treatment group compared with those in the placebo (control) group. In addition, the control group showed a significant decrease in arm muscle mass after 12 weeks, whereas no significant change was observed in the treatment group. Blood serum levels of follistatin, myostatin, high-sensitivity C-reactive protein (hs-CRP), interleukin (IL)-6, IL-8, insulin-like growth factor-1 (IGF-1), and cortisol were analyzed, and the decrease in myostatin resulting from the administration of tannase-treated green tea extract was found to be related to the change in muscle mass and strength. In summary, oral administration of tannase-treated green tea extract containing antioxidants without concomitant exercise can improve muscle mass and strength and may have therapeutic benefits in age-related muscle function decline.

Shape- and Size-Controlled Palladium Nanocrystals and Their Electrocatalytic Properties in the Oxidation of Ethanol.

Pd nanoparticles (PdNPs) were synthesized in an aqueous environment via the reduction of K2PdCl4 by a surfactant under a high temperature. Highly monodisperse spherical PdNPs and multi-pod PdNPs with a controlled size ranging from 18 to 50 nm were prepared in high yields by varying the concentration of cetyltrimethylammonium chloride. The structural and optical properties of the synthesized Pd NPs were characterized by transmission electron microscopy, X-ray diffraction and UV-vis spectroscopy. The spherical and multi-pod PdNPs exhibited catalytic properties that were unique to their size and shape and presented efficient electrocatalytic activities toward the ethanol oxidation reaction.

Nano-sized Zinc in Broiler Chickens: Effects on Growth Performance, Zinc Concentration in Organs, and Intestinal Morphology.

The effects of dietary supplementation of zinc (Zn) sources and concentrations were investigated on growth performance, absorption into tissues, fecal excretion, nutrient retention, and intestinal morphology in broilers fed a corn-soybean meal basal diet. A total of 525 one-day-old chicks (Ross 308) were assigned based on body weight to seven dietary treatments. There were five replicate pens for each treatment and 15 broilers per replicate pen. The dietary treatments included a basal diet (control, without supplementing Zn), and basal diet supplemented with Zn, as inorganic zinc sulfate (ZnS; 110 mg/kg); organic Zn-methionine (ZnM; 110 mg/kg); hot-melt extruded (HME) 25 zinc sulfate (27.5 mg/kg); HME50 zinc sulfate (55 mg/kg); HME75 zinc sulfate (82.5 mg/kg); or HME100 zinc sulfate (110 mg/kg) for 35 days in two phases (d 1-21, phase I and d 22-35, phase II). Bodyweight and feed efficiency of broiler chicks fed diets supplemented with increasing dietary concentrations of HME-Zn improved linearly during the study period (P<0.05). Compared to the control treatment, the ZnS, ZnM, and HME diets increased Zn concentrations in the serum and liver. Inorganic ZnS supply resulted in the highest Zn concentration in excreta. Increasing supplemented Zn content in diets as HME linearly increased Zn concentration in the excreta, serum, liver, and tibia. Broiler chicks fed diets supplemented with increasing concentrations of HME increased villus height (VH; linear and quadratic) of the jejunum and VH of the ileum (linear). Increasing concentrations of dietary Zn supplied as HME resulted in linearly enhanced dry matter, gross energy, and nitrogen retention of broilers on day 21. These results suggest that dietary HME-Zn at a lower level (55 ppm) shows the same growth performance as common ZnSO4 at 110 ppm.

3-MCPD (3-monochloro-1,2-propanediol) inhibit myogenic differentiation in murine skeletal myoblasts.

3-Monochloro-1,2-propanediol (3-MCPD) is a chemical compound that is unintentionally produced during food processing such as acid hydrolysis. There has been reports regarding the role of this chemical compound in reproductive toxicity, as well as genotoxicity, neurotoxicity, and kidney toxicity. In this study, the in vitro muscle toxicity of 3-MCPD was assessed using C2C12 myoblast cells. The reduction in muscle regulatory factors (MRFs), which is related to muscle differentiation, was identified as significant with the increase concentration of 3-MCPD. Also, significantly decreased protein expression in mTOR and p70S6 kinase, which are the downstream targets of the pathway associated with muscle synthesis, was also confirmed. Therefore, the inhibitory effect of 3-MCPD on muscle differentiation is considered to be the cause of suppressing mTOR and p70S6 kinase expression. In conclusion, it was confirmed that 3-MCPD inhibits muscle differentiation in C2C12 myoblasts through suppressing the expression of several genetic factors involving muscle differentiation.

Development and characterization of a human cell line-based transactivation assay to assess thyroid EDCs.

Thyroid hormones (THs) are one of the most important hormones, playing key roles in the regulation of various physiological functions. Although THs have important function in human, in vitro test methods based on human cells are currently insufficient to effectively screen and test TH-related endocrine disrupting chemicals (EDCs). We established a TH agonist TA assay using the adenocarcinomic human alveolar basal epithelial cell line A549 to test and screen potential TH agonists. To establish the TH agonist TA assay, a TRE-secNluc-IRES-EGFP reporter cassette was constructed and transfected into the A549 cell line using a retrovirus. We evaluated the TH agonistic properties of several chemicals which were tested by existing thyroid agonists testing method (OECD GD 207). Comparing the results of the TH agonist TA assay with the OECD GD 207, T3, T4, tiratricol, and tetrac (natural TH and 3,3',5,5'-tetraiodothyroacetic acid derivatives), which are TH agonists according to the OECD GD 207, also tested positive in the TH agonist TA assay using the A549 cell line. These results suggested that the TH agonist TA assay developed in this study using a human cell line can provide the information, such as accuracy and specificity to TH agonistic properties of chemicals.

Biological Evaluation of Hot-Melt Extruded Nano-selenium and the Role of Selenium on the Expression Profiles of Selenium-Dependent Antioxidant Enzymes in Chickens.

A study was conducted to determine the effect of dietary selenium (Se) concentration and source for broiler chickens on performance, nutrient digestibility, plasma Se, glutathione peroxidase (GPx) activity, and thiobarbituric acid reactive substances (TBARS). A total of 700 1-day-old broiler chicks were assigned to 7 diets with 20 birds per cage and 5 replicates per treatment. The experimental diets were fed for 32 days in 2 phases (phase 1, day 0 to 14 and phase 2, day 15 to 32). Treatments were as follows: control (without Se supplementation), sodium selenite (SeS; 0.15, 0.30, or 0.45 ppm), and hot-melt extruded sodium selenite (SeHME; 0.15, 0.30, or 0.45 ppm). There were significant linear responses (P < 0.01) for higher plasma Se concentration in SeS and SeHME treatments. Moreover, an increased (P < 0.01) Se concentration of plasma occurred in SeHME treatment compared with that in SeS treatment. The serum GPx analyses revealed that supplemental SeS and SeHME increased significantly the activity of GPx in the plasma in phase 1 (P < 0.05) and phase 2 (P < 0.05). There were significant linear (P < 0.01) responses of SeS and SeHME treatments for the expression of SelW, GPx1, GPx3, and GPx4 in the livers and spleens. In addition, SeHME showed an upregulated expression of GPx-4 in the livers (P < 0.01) and SelW in the spleens (P < 0.05) compared with SeS treatment. SeHME showed a lower TBARS on day 9. Moreover, a decreased (P < 0.01) TBARS occurred in SeS treatment compared with that in control treatment. In conclusion, SeHME can increase antioxidant activity and Se absorption, consequently being a more suitable source of Se than regular sodium selenite.

Creation of two-dimensional layered Zintl phase by dimensional manipulation of crystal structure.

The discovery of new families, beyond graphene, of two-dimensional (2D) layered materials has always attracted great attention. However, it has been challenging to artificially develop layered materials with honeycomb atomic lattice structure composed of multicomponents such as hexagonal boron nitride. Here, through the dimensional manipulation of a crystal structure from sp3-hybridized 3D-ZnSb, we create an unprecedented layered structure of Zintl phase, which is constructed by the staking of sp2-hybridized honeycomb ZnSb layers. Using structural analysis combined with theoretical calculation, it is found that the 2D-ZnSb has a stable and robust layered structure. The bidimensional polymorphism is a previously unobserved phenomenon at ambient pressure in Zintl families and can be a common feature of transition metal pnictides. This dimensional manipulation of a crystal structure thus provides a rational design strategy to search for new 2D layered materials in various compounds, enabling unlimited expansion of 2D libraries and corresponding physical properties.

Enhancement of androgen transcriptional activation assay based on genome edited glucocorticoid knock out human prostate cancer cell line.

Endocrine-disrupting chemicals (EDCs) interfere with the biological activity of hormones. Among EDC's, (anti-)androgenic compounds potentially cause several androgen-related diseases. To improve the accuracy of an in vitro transactivation assay (TA) for detection of (anti-)androgenic compounds, We established the glucocorticoid receptor (GR) knockout 22Rv1/MMTV cell line by using an RNA-guided engineered nuclease (RGEN)-derived CRISPR/Cas system. The 22Rv1/MMTV GRKO cell line was characterized and validated by androgen receptor (AR)-mediated TA assay compared with the AR-TA assay using 22Rv1/MMTV. In conclusion, the AR-TA assay with the 22Rv1/MMTV GRKO cell line was more accurate, excluding the misleading signals derived from glucocorticoids or equivalent chemicals, and might be an effective method for screening potential (anti-)androgenic compounds.

Plant-derived compounds regulate formation of the insect juvenile hormone receptor complex.

Insect growth regulators (IGRs) are attractive pest control agents due to their high target specificity and relative safety to the environment. Recently, plants have been shown to synthesize IGRs that affect the insect juvenile hormone (JH) as a part of their defense mechanisms. Using a yeast two-hybrid system transformed with the Aedes aegypti JH receptor as a reporter system, we identified several JH agonists (JHAs) and antagonists (JHANs) causing retardation in the ovarian development of female Asian tiger mosquito, Aedes albopictus, from plant essential oil compounds. While the JHAs increased the expression of a JH-induced gene, the JHANs caused a reduction in the expression of the same gene. The compounds identified in this study could provide insights into plant-insect interactions and may be useful for the development of novel IGR insecticides.