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.

Enzyme-Treated Zizania latifolia Ethanol Extract Improves Liver-Related Outcomes and Fatigability.

Long-term hepatic damage is associated with human morbidity and mortality owing to numerous pathogenic factors. A variety of studies have focused on improving liver health using natural products and herbal medicines. We aimed to investigate the effect of enzyme-treated Zizania latifolia ethanol extract (ETZL), which increases the content of tricin via enzymatic hydrolysis, for 8 weeks on liver-related outcomes, lipid metabolism, antioxidant activity, and fatigue compared to a placebo. Healthy Korean adult males aged 19-60 years were randomized into ETZL treatment and placebo groups, and alcohol consumption was 24.96 and 28.64 units/week, respectively. Alanine transaminase, a blood marker associated with liver cell injury, significantly decreased after 8 weeks compared to the baseline in the ETZL treatment group (p = 0.004). After 8 weeks, the treatment group showed significant changes in the levels of high-density lipoprotein and hepatic steatosis index compared to the baseline (p = 0.028 and p = 0.004, respectively). ETZL treatment tended to reduce antioxidant-activity-related factors, total antioxidant status, and malondialdehyde, but there was no significant difference. In the multidimensional fatigue scale, ETZL treatment showed a significant reduction in general fatigue and total-fatigue-related values after 8 weeks compared to the baseline (p = 0.012 and p = 0.032, respectively). Taken together, the 8-week treatment of enzyme-treated Zizania latifolia ethanol extract demonstrated positive effects on liver-related outcomes, lipid metabolism, and mental fatigue without adverse effects on safety-related parameters.

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.

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.

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.

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 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.