Effects of Perfluorooctane Sulfonate on Cerebellar Cells via Inhibition of Type 2 Iodothyronine Deiodinase Activity.

Perfluorooctane sulfonate (PFOS) has been used in a wide variety of industrial and commercial products. The adverse effects of PFOS on the developing brain are becoming of a great concern. However, the molecular mechanisms of PFOS on brain development have not yet been clarified. We investigated the effect of early-life exposure to PFOS on brain development and the mechanism involved. We investigated the change in thyroid hormone (TH)-induced dendrite arborization of Purkinje cells in the primary culture of newborn rat cerebellum. We further examined the mechanism of PFOS on TH signaling by reporter gene assay, quantitative RT-PCR, and type 2 iodothyronine deiodinase (D2) assay. As low as 10-7 M PFOS suppressed thyroxine (T4)-, but not triiodothyronine (T3)-induced dendrite arborization of Purkinje cells. Reporter gene assay showed that PFOS did not affect TRα1- and TRß1-mediated transcription in CV-1 cells. RT-PCR showed that PFOS suppressed D2 mRNA expression in the absence of T4 in primary cerebellar cells. D2 activity was also suppressed by PFOS in C6 glioma-derived cells. These results indicate that early-life exposure of PFOS disrupts TH-mediated cerebellar development possibly through the disruption of D2 activity and/or mRNA expression, which may cause cerebellar dysfunction.

A Novel Mechanism of S-equol Action in Neurons and Astrocytes: The Possible Involvement of GPR30/GPER1.

S-equol is a major bacterial metabolite of the soy isoflavone daidzein. It is known to be a phytoestrogen that acts by binding to the nuclear estrogen receptors (ERs) that are expressed in various brain regions, including the cerebellum. However, the effects of S-equol on cerebellar development and function have not yet been extensively studied. In this study, the effects of S-equol were evaluated using a mouse primary cerebellar culture, Neuro-2A clonal cells, and an astrocyte-enriched culture. S-equol augmented the dendrite arborization of Purkinje cells induced by triiodothyronine (T3) and the neurite growth of Neuro-2A cell differentiation. Such augmentation was suppressed by G15, a selective G-protein coupled ER (GPR30) antagonist, and ICI 182,780, an antagonist for ERs in both cultures. On the other hand, in astrocytes, S-equol induced cell proliferation and cell migration with an increase in the phosphorylated extracellular-signal-regulated kinase 1/2 and F-actin rearrangements. Such effects were suppressed by G15, but not by ICI. These findings indicated that S-equol may enhanced cerebellar development by affecting both neurons and astrocytes through several signaling pathways, including GPR30 and ERs. We here report a novel mechanism of S-equol in cerebellar development that may provide a novel possibility to use S-equol supplementation during development.

Prenatal exposure to bisphenol A impacts neuronal morphology in the hippocampal CA1 region in developing and aged mice.

Bisphenol A (BPA), a widely used raw component of polycarbonate plastics and epoxy resins, has been reported to induce developmental neurotoxicity in offspring born to dams exposed to low doses of BPA; however, the toxicity mechanism remains elusive. To study the effects of in utero BPA exposure on neuronal morphology, we studied spine density and dendritic growth in the hippocampal CA1 of aged mice and developing mice prenatally exposed to low doses of BPA. Pregnant mice were orally administered BPA at a low dose of 0, 40, or 400 µg/kg body weight/day on gestational days 8.5-17.5/18.5. Mouse progenies were euthanized at 3 weeks or 14 months, and their brains were analyzed for dendritic arborization of GFP-expressing neurons or spine densities of Golgi-stained neurons in the hippocampal CA1. Regardless of the dose, in utero BPA exposure reduced spine densities in the hippocampal CA1 of the 14-month-old mice. In the developing brain from the 3-week-old mice born to dams exposed to BPA at a dose of 400 µg/kg body weight/day, overall length and branching number of basal dendrites but not apical dendrites were decreased. In utero low doses of BPA exposure disrupts hippocampal CA1 neuronal morphology during development, and this disruption is believed to persist in adulthood.

Associations between estrogen receptor genetic polymorphisms, smoking status, and prostate cancer risk: a case-control study in Japanese men.

OBJECTIVE: Prostate cancer (PCa) is one of the major causes of death among men. Our study investigated the association of ESR1 and ESR2 genotypes with susceptibility to PCa in relation to smoking status in Japanese. METHOD: A case-control study was performed with 750 Japanese prostate cancer patients and 870 healthy controls. After age-matching in case-controls, 352 controls and 352 cases were enrolled in this study. By using logistic regression analysis, the different genotypes from ESR1 and ESR2 were analyzed according to case/control status. RESULT: ESR2 rs4986938 AG and AG + AA genotypes were associated with significantly decreased risk of PCa (AG: OR = 0.68, 95 % CI 0.47-0.97, P < 0.05 and AG + AA: OR = 0.67, 95 % CI 0.47-0.94, P < 0.05). However, there was no significant association between ESR1 rs2234693 and PCa risk. When patients were grouped according to smoking status, the ESR2 rs1256049 AA genotype (OR = 0.48, 95 % CI 0.25-0.95, P < 0.05) and ESR2 rs4986938 AG + AA genotype (OR = 0.64, 95 % CI 0.41-1.00, P < 0.05) showed significantly decreased PCa risk in the ever-smoker group. CONCLUSION: Our results suggest that the estrogen receptor ESR2 has a very important function to predict PCa and that different SNPs have different predictive values. Smoking may influence estrogenic activity and may influence PCa together with the estrogen receptor.

Disruption of paired-associate learning in rat offspring perinatally exposed to dioxins.

The prevalence of cognitive abnormalities in children has partly been ascribed to environmental chemical exposure. Appropriate animal models and tools for evaluating higher brain function are required to examine this problem. A recently developed behavioral test in which rats learn six unique flavor-location pairs in a test arena was used to evaluate paired-associate learning, a hallmark of the higher cognitive function that is essential to language learning in humans. Pregnant Long-Evans rats were dosed by gavage with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or 2,3,7,8-tetrabromodibenzo-p-dioxin (TBDD) at a dose of 0, 200, or 800 ng/kg (referred as Control, TCDD-200, TCDD-800, TBDD-200, or TBDD-800, hereafter) on gestational day 15, and the offspring was tested during adulthood. Paired-associate learning was found to be impaired in the TCDD-200 and TBDD-200 groups, but not in either group exposed to 800 ng/kg, the observations of which were ensured by non-cued trials. As for the emotional aspect, during habituation, the TCDD-200 and TBDD-200 groups showed significantly longer latencies to enter the test arena from a start box than the Control, TCDD-800, and TBDD-800 groups, suggesting that the TCDD-200 and TBDD-200 groups manifested anxiety-like behavior. Thus, both the chlorinated dioxin and its brominated congener affected higher brain function to a similar extent in a nearly identical manner. Use of the behavioral test that can evaluate paired-associate learning in rats demonstrated that in utero and lactational exposure to not only TCDD but also TBDD perturbed higher brain function in rat offspring in a nonmonotonic manner.

Prevalence and interannual changes in multiple chemical sensitivity in Japanese workers.

OBJECTIVE: We aimed to evaluate the prevalence rates and interannual fluctuations in multiple chemical sensitivity (MCS) in Japanese workers. METHODS: We assessed MCS using the Quick Environmental Exposure and Sensitivity Inventory, employing both Miller and Japanese criteria. Workers of two manufacturing companies located in Kyushu, Japan, were assessed, with company A surveyed in 2003, 2006 and 2011, and company B in 2003 and 2011. RESULTS: In company A, the Miller criteria-based MCS prevalence rate was higher in 2011 than in 2003, and according to the Japanese criteria, it was higher in 2011 than 2006. In company B, the Miller criteria-based MCS prevalence rate was lower in 2011 than in 2003. CONCLUSION: The results indicated that MCS exists among industrial workers in Japan. We found no statistically significant interannual changes in MCS rates.

[Awareness of issues in research and educational activities and expectations for young researchers' activities and supporting].

OBJECTIVES: To understand the actual situation and needs of young researchers and to provide reference for the management of Young Researchers Association (YRA) and the Japanese Society for Hygiene activities in the future. METHODS: An Internet survey was conducted on 67 members registered in YRA of the Japanese Society for Hygiene. The questions included those on basic information, research content and impressions about the activities of the society. RESULTS: Although members of YRA differ in backgrounds, research method used, and years of research experience, the respondents rated the organization as highly useful and participated continuously. In particular, they considered that participation in the planning of academic conferences and summer gatherings of YRA not only helped improve interpersonal relationships and expertise, but also provided opportunities to consult regarding educational activities and collect information. Regarding the format of conferences, it was shown that the majority of requests were for a hybrid format. It was also shown that most of the respondents expected opportunities for collaboration and joint research through participation in YRA. CONCLUSION: Through YRA, we would like to contribute to the further revitalization of young researchers and the Japanese Society for Hygiene by understanding and responding to the needs of diverse young researchers.

Association of genotypes of carcinogen-metabolizing enzymes and smoking status with bladder cancer in a Japanese population.

OBJECTIVES: Arylamines are considered to be the primary causative agent of bladder cancer in tobacco smokers. To test the hypothesis that variation in the genes that metabolize tobacco carcinogens contribute to bladder cancer, we examined the effects of single nucleotide polymorphisms in the genes of four key enzymes: cytochrome P450 1A2, N-acetyltransferase (NAT) 2, sulfotransferase 1A1, and UDP-glucuronosyltransferase (UGT) 2B7. METHODS: In this study, 282 Japanese patients with transitional cell carcinoma, the most common bladder cancer, and 257 healthy controls were surveyed and compared for frequencies of the genotypes of the four enzymes. Genotypes were determined using PCR-restriction fragment length polymorphism and TaqMan assays. Smoking information was collected by personal interview. Logistic regression analysis and the chi-square test were employed as statistical methods. RESULTS: The NAT2 slow genotype was significantly associated with the risk of bladder cancer [odds ratio (OR) 3.41, 95 % confidence interval (95 % CI) 1.68-6.87; p < 0.05). The NAT2 slow genotype also significantly increased the risk of bladder cancer in heavy smokers (OR 8.57, 95 % CI 1.82-40.25; p < 0.05). Among the different combinations of the four enzyme genotypes, the highest OR (4.20; 95 % CI 1.34-13.14; p < 0.05) was obtained with the NAT2 slow genotype when present in combination with the UGT2B7 *2/*2 or *1/*2 genotype. CONCLUSIONS: Our results suggest that individuals with different genotypes for the enzymes involved in metabolizing carcinogenic arylamines have a different risk of developing bladder cancer. In particularly, the combination of the NAT2 slow genotype with UGT2B7 *1/*2 or *2/*2 genotype is a high risk factor for bladder cancer.

Gadolinium-based contrast agent accelerates the migration of astrocyte via integrin αvß3 signaling pathway.

Gadolinium (Gd)-based contrast agents (GBCAs) are chemicals injected intravenously during magnetic resonance imaging to enhance the diagnostic yield. Repeated use of GBCAs causes their deposition in the brain. Such deposition may affect various neuronal cells, including astrocytes. In this study, we examined the effect of GBCAs (Omniscan, Magnescope, Magnevist, and Gadovist) on astrocyte migration, which is critical for formation of neurons during development and maintaining brain homeostasis. All GBCAs increased cell migration and adhesion with increased actin remodelling. Knockdown of integrin αvß3 by RNAi or exposure to integrin αvß3 inhibitor reduced astrocyte migration. GBCAs increased phosphorylation of downstream factors of αvß3, such as FAK, ERK1/2, and Akt. The phosphorylation of all these factors were reduced by RNAi or integrin αvß3 inhibitor. GBCAs also increased the phosphorylation of their downstream factor, Rac1/cdc42, belonging to the RhoGTPases family. Coexposure to the selective RhoGTPases inhibitors, decreased the effects of GBCAs on cell migration. These findings indicate that GBCAs exert their action via integrin αvß3 to activate the signaling pathway, resulting in increased astrocyte migration. Thus, the findings of the study suggest that it is important to avoid the repeated use of GBCAs to prevent adverse side effects in the brain, particularly during development.

Involvement of integrin αvß3 in thyroid hormone-induced dendritogenesis.

Activation and/or modulation of the membrane-associated receptors plays a critical role in brain development. Thyroid hormone (TH) acts on both nuclear receptors (thyroid hormone receptor, TR) and membrane-associated receptors, particularly integrin αvß3 in neurons and glia. Integrin αvß3-mediated signal transduction mediates various cellular events during development including morphogenesis, migration, synaptogenesis, and intracellular metabolism. However, the involvement of integrin αvß3-mediated TH action during brain development remains poorly understood. Thus, we examined the integrin αvß3-mediated effects of TH (T3, T4, and rT3) in the neurons and astrocytes using primary cerebellar culture, astrocyte-enriched culture, Neuro-2A clonal cells, and co-culture of neurons and astrocytes. We found that TH augments dendrite arborization of cerebellar Purkinje cells. This augmentation was suppressed by knockdown of integrin αvß3, as well as TRα and TRß. A selective integrin αvß3 antagonist, LM609, was also found to suppress TH-induced arborization. However, whether this effect was a direct action of TH on Purkinje cells or due to indirect actions of other cells subset such as astrocytes was not clarified. To further study neuron-specific molecular mechanisms, we used Neuro-2A clonal cells and found TH also induces neurite growth. TH-induced neurite growth was reduced by co-exposure with LM609 or knockdown of TRα, but not TRß. Moreover, co-culture of Neuro-2A and astrocytes also increased TH-induced neurite growth, indicating astrocytes may be involved in neuritogenesis. TH increased the localization of synapsin-1 and F-actin in filopodia tips. TH exposure also increased phosphorylation of FAK, Akt, and ERK1/2. Phosphorylation was suppressed by co-exposure with LM609 and TRα knockdown. These results indicate that TRs and integrin αvß3 play essential roles in TH-induced dendritogenesis and neuritogenesis. Furthermore, astrocytes-neuron communication via TR-dependent and TR-independent signaling through membrane receptors and F-actin are required for TH-induced neuritogenesis.

Absence of Thyroid Hormone Induced Delayed Dendritic Arborization in Mouse Primary Hippocampal Neurons Through Insufficient Expression of Brain-Derived Neurotrophic Factor.

Thyroid hormone (TH) plays important roles in the developing brain. TH deficiency in early life leads to severe developmental impairment in the hippocampus. However, the mechanisms of TH action in the developing hippocampus are still largely unknown. In this study, we generated 3,5,3'-tri-iodo-l-thyronine (T3)-free neuronal supplement, based on the composition of neuronal supplement 21 (NS21), to examine the effect of TH in the developing hippocampus using primary cultured neurons. Effects of TH on neurons were compared between cultures in this T3-free culture medium (-T3 group) and a medium in which T3 was added (+T3 group). Morphometric analysis and RT-qPCR were performed on 7, 10, and 14 days in vitro (DIV). On 10 DIV, a decreased dendrite arborization in -T3 group was observed. Such difference was not observed on 7 and 14 DIV. Brain-derived neurotrophic factor (Bdnf) mRNA levels also decreased significantly in -T3 group on 10 DIV. We then confirmed protein levels of phosphorylated neurotrophic tyrosine kinase type 2 (NTRK2, TRKB), which is a receptor for BDNF, on 10 DIV by immunocytochemistry and Western blot analysis. Phosphorylated NTRK2 levels significantly decreased in -T3 group compared to +T3 group on 10 DIV. Considering the role of BDNF on neurodevelopment, we examined its involvement by adding BDNF on 8 and 9 DIV. Addition of 10 ng/ml BDNF recovered the suppressed dendrite arborization induced by T3 deficiency on 10 DIV. We show that the lack of TH induces a developmental delay in primary hippocampal neurons, likely caused through a decreased Bdnf expression. Thus, BDNF may play a role in TH-regulated dendritogenesis.

Hydroxylated PCB induces Ca2+ oscillations and alterations of membrane potential in cultured cortical cells.

Polychlorinated biphenyls (PCBs) are known as environmental pollutants that may cause adverse health effects. Although some congeners have been shown to affect brain development or function, the molecular mechanisms mediating their toxicity are not yet fully understood. Since signal transduction via intracellular Ca(2+) is crucial for neuronal development and plasticity, we investigated the effect of PCBs on Ca(2+) homeostasis and membrane potential in cultured mouse cortical cells. Acute exposure to hydroxylated PCB 106 [4(OH)-2',3,3',4',5'-pentachlorobiphenyl, OH-PCB 106, 0.1 microM] caused recurring Ca(2+) oscillations that were classified into three prototypes. Although extracellular Ca(2+) deprivation significantly reduced the oscillations, 54% of the cells still showed different patterns of oscillations or gradual increase in the intracellular Ca(2+) concentration, indicating possible involvement of multiple Ca(2+) channels in a cell-specific manner. Such a possibility was further confirmed by differential responses to several channel/receptor blockers, including nifedipine, ryanodine, xestospongine and tetrodotoxin. Although all chemicals had partial inhibition action in different subsets of neurons, nifedipine blocked the OH-PCB 106 action in the largest subpopulation of cells and with the greatest magnitude. Ryanodine also blocked the action with a similar magnitude, but in a smaller subpopulation of cells. Moreover, OH-PCB 106 induced depolarization of the plasma membrane in all the recorded cells. Taken together, our results indicate that OH-PCB 106 alters membrane potential as well as Ca(2+) dynamics in part by inducing extracellular influx and/or intracellular release of Ca(2+). These mechanisms may be responsible for their neurotoxicity.

Soy Isoflavones Accelerate Glial Cell Migration via GPER-Mediated Signal Transduction Pathway.

Soybean isoflavones, such as genistein, daidzein, and its metabolite, S-equol, are widely known as phytoestrogens. Their biological actions are thought to be exerted via the estrogen signal transduction pathway. Estrogens, such as 17ß-estradiol (E2), play a crucial role in the development and functional maintenance of the central nervous system. E2 bind to the nuclear estrogen receptor (ER) and regulates morphogenesis, migration, functional maturation, and intracellular metabolism of neurons and glial cells. In addition to binding to nuclear ER, E2 also binds to the G-protein-coupled estrogen receptor (GPER) and activates the nongenomic estrogen signaling pathway. Soybean isoflavones also bind to the ER and GPER. However, the effect of soybean isoflavone on brain development, particularly glial cell function, remains unclear. We examined the effects of soybean isoflavones using an astrocyte-enriched culture and astrocyte-derived C6 clonal cells. Isoflavones increased glial cell migration. This augmentation was suppressed by co-exposure with G15, a selective GPER antagonist, or knockdown of GPER expression using RNA interference. Isoflavones also activated actin cytoskeleton arrangement via increased actin polymerization and cortical actin, resulting in an increased number and length of filopodia. Isoflavones exposure increased the phosphorylation levels of FAK (Tyr397 and Tyr576/577), ERK1/2 (Thr202/Tyr204), Akt (Ser473), and Rac1/cdc42 (Ser71), and the expression levels of cortactin, paxillin and ERα. These effects were suppressed by knockdown of the GPER. Co-exposure of isoflavones to the selective RhoA inhibitor, rhosin, selective Cdc42 inhibitor, casin, or Rac1/Cdc42 inhibitor, ML-141, decreased the effects of isoflavones on cell migration. These findings indicate that soybean isoflavones exert their action via the GPER to activate the PI3K/FAK/Akt/RhoA/Rac1/Cdc42 signaling pathway, resulting in increased glial cell migration. Furthermore, in silico molecular docking studies to examine the binding mode of isoflavones to the GPER revealed the possibility that isoflavones bind directly to the GPER at the same position as E2, further confirming that the effects of the isoflavones are at least in part exerted via the GPER signal transduction pathway. The findings of the present study indicate that isoflavones may be an effective supplement to promote astrocyte migration in developing and/or injured adult brains.

Neurotoxic effects of lactational exposure to perfluorooctane sulfonate on learning and memory in adult male mouse.

The present study aims to examine the effect of early lactational perfluorooctane sulfonate (PFOS) exposures on learning and memory in male mice and reveal the underlying mechanisms involved. PFOS solution was orally administered to dams from the postpartum days 1-14, so that pups would be exposed through breast milk. At 8-10 weeks of age, we performed object location test (OLT), object recognition test (ORT), and pairwise visual discrimination (VD) task. We also performed in vivo microdialysis, and mRNA and protein analysis of genes responsible for hippocampal development and function. In both OLT and ORT, the performance of mice in the PFOS-exposed group was significantly lower than those in the control group. In the VD task, the PFOS-exposed group learned significantly slower than the control group. Concentrations of glutamate and gamma-aminobutyric acid in the dorsal hippocampus were significantly higher in the PFOS-exposed group than in the control group. No notable differences were shown in our mRNA and protein studies. The present study showed that lactational PFOS exposure has a profound, long-lasting neurotoxic effect in the hippocampus and consequently leads to learning and memory deficits.

Liquid chemiluminescent DNA pull-down assay to measure nuclear receptor-DNA binding in solution.

Electrophoretic mobility shift assays (EMSAs) are commonly used to investigate protein-DNA binding in vitro. However, EMSA can generate considerable amounts of undesirable waste, particularly when toxic compounds are examined. We therefore developed a novel in vitro protein-DNA binding assay called liquid chemiluminescent DNA pull-down assay, which is based on solution hybridization between digoxigenin-labeled DNA and glutathione S-transferase (GST)-fused DNA binding protein bound to glutathione-Sepharose beads.

Identification of the functional domain of thyroid hormone receptor responsible for polychlorinated biphenyl-mediated suppression of its action in vitro.

BACKGROUND: Polychlorinated biphenyls (PCBs), polychlorinated dibenzo-p-dioxins, and poly-chlorinated dibenzofurans adversely affect the health of humans and various animals. Such effects might be partially exerted through the thyroid hormone (TH) system. We previously reported that one of the hydroxylated PCB congeners suppresses TH receptor (TR)-mediated transcription by dissociating TR from the TH response element (TRE). However, the binding site of PCB within TR has not yet been identified. OBJECTIVES: We aimed to identify the functional TR domain responsible for the PCB-mediated suppression of TR action by comparing the magnitude of suppression using several representative PCB/dioxin congeners. MATERIALS AND METHODS: We generated chimeric receptors by combining TR and glucocorticoid receptor (GR) and determined receptor-mediated transcription using transient transfection-based reporter gene assays, and TR-TRE binding using electrophoretic mobility shift assays. RESULTS: Although several PCB congeners, including the hydroxylated forms, suppressed TR-mediated transcription to various degrees, 2,3,7,8-tetrachlorodibenzo-p-dioxin did not alter TR action, but 2,3,4,7,8-pentachlorodibenzofuran weakly suppressed it. The magnitude of suppression correlated with that of TR-TRE dissociation. The suppression by PCB congeners was evident from experiments using chimeric receptors containing a TR DNA-binding domain (DBD) but not a GR-DBD. CONCLUSIONS: Several nondioxin-like PCB congeners and hydroxylated PCB compounds suppress TR action by dissociating TR from TRE through interaction with TR-DBD.

The Effects of Low-Dose Bisphenol A and Bisphenol F on Neural Differentiation of a Fetal Brain-Derived Neural Progenitor Cell Line.

Environmental chemicals are known to disrupt the endocrine system in humans and to have adverse effects on several organs including the developing brain. Recent studies indicate that exposure to environmental chemicals during gestation can interfere with neuronal differentiation, subsequently affecting normal brain development in newborns. Xenoestrogen, bisphenol A (BPA), which is widely used in plastic products, is one such chemical. Adverse effects of exposure to BPA during pre- and postnatal periods include the disruption of brain function. However, the effect of BPA on neural differentiation remains unclear. In this study, we explored the effects of BPA or bisphenol F (BPF), an alternative compound for BPA, on neural differentiation using ReNcell, a human fetus-derived neural progenitor cell line. Maintenance in growth factor-free medium initiated the differentiation of ReNcell to neuronal cells including neurons, astrocytes, and oligodendrocytes. We exposed the cells to BPA or BPF for 3 days from the period of initiation and performed real-time PCR for neural markers such as ß III-tubulin and glial fibrillary acidic protein (GFAP), and Olig2. The ß III-tubulin mRNA level decreased in response to BPA, but not BPF, exposure. We also observed that the number of ß III-tubulin-positive cells in the BPA-exposed group was less than that of the control group. On the other hand, there were no changes in the MAP2 mRNA level. These results indicate that BPA disrupts neural differentiation in human-derived neural progenitor cells, potentially disrupting brain development.

A Possible Novel Mechanism of Action of Genistein and Daidzein for Activating Thyroid Hormone Receptor-Mediated Transcription.

Thyroid hormone receptors (TRs) are members of the nuclear receptor superfamily that regulate their target genes for controlling organ development and functional maintenance. Soybean isoflavones, especially genistein and daidzein, modulate various hormone-mediated pathways. However, their effects on TRs have not yet been extensively studied. In this study, the effects of these isoflavones on TR action were evaluated using transient transfection-based reporter gene assays and molecular docking studies. Genistein and daidzein augmented T3-liganded TR-mediated transcription in a concentration-dependent manner. In the mammalian 2-hybrid study, these isoflavones augmented the recruitment of steroid receptor coactivator-1 and nuclear corepressor to liganded or unliganded TRs. Using a series of mutant TRs, we also showed that the activation function-2 domain of TRs was responsible for the augmentation by these isoflavones. CV-1 cells had expressed TRα, TRß1, and ERα mRNAs. However, neither the overexpression nor the knocking down of ERα altered the augmentation of TR action by isoflavones, indicating that the effects of isoflavones are exerted through their direct action on TRs. In silico molecular docking studies showed that genistein and daidzein can directly bind to the TR-ligand-binding domain. These findings indicate that the augmentation of the TR-mediated transcription by genistein and daidzein is due to their direct binding to TR-ligand-binding domain to induce the recruitment of steroid receptor coactivator-1. Our study reports a novel mode of action of soybean isoflavones on TR function. The biological effects and the relevance of these isoflavones to human health may be partially attributable to the activation of thyroid hormone signaling.

In Utero and Postnatal Propylthiouracil-Induced Mild Hypothyroidism Impairs Maternal Behavior in Mice.

Thyroid hormones (THs) play crucial roles in general and brain development. Even if the hypothyroidism is mild, it may alter brain function, resulting in irreversible behavioral alterations. Although various behavioral analyses have been conducted, the effects of propylthiouracil (PTU) treatment during in utero and postnatal periods on maternal behavior have not yet been studied. The present study examined in mice whether THs insufficiency during development induce behavioral changes. Pregnant C57BL/6j mice were divided into three groups, and each group was administered different dosages of PTU (0, 5, or 50 ppm) in drinking water during in utero and postnatal periods (from gestational day 14 to postnatal day 21). First, locomotor activity and cognitive function were assessed in the offspring at 10 weeks. Next, female offspring were mated with normal mice and they and their offspring were used to assess several aspects of maternal behavior (identifying first pup, returning all pups to nest, time spent nursing, and licking pups). As expected, locomotor and cognitive functions in these mice were disrupted in a PTU dose-dependent manner. On postpartum day 2, dams who had been exposed 50 ppm PTU during in utero and postnatal periods displayed a significantly longer time identifying the first pup and returning all three pups back to the nest, less time nursing, and decreased licking behavior. The decrease in maternal behavior was significantly correlated with a decrease in cognition. These results indicate that insufficiency of THs during in utero and postnatal periods impairs maternal behavior, which may be partly induced by impaired cognitive function.

The interaction of TRbeta1-N terminus with steroid receptor coactivator-1 (SRC-1) serves a full transcriptional activation function of SRC-1.

Steroid receptor coactivator-1 (SRC-1) plays a crucial role in nuclear receptor-mediated transcription including thyroid hormone receptor (TR)-dependent gene expression. Interaction of the TR-ligand binding domain and SRC-1 through LXXLL motifs is required for this action. However, potential interactions between the TRbeta1-N terminus (N) and SRC-1 have not been explored and thus are examined in this manuscript. Far-Western studies showed that protein construct containing TRbeta1-N + DNA binding domain (DBD) bound to nuclear receptor binding domain (NBD)-1 (amino acid residue, aa 595-780) of SRC-1 without ligand. Mammalian two-hybrid studies showed that NBD-1, as well as SRC-1 (aa 595-1440), bound to TRbeta1-N+DBD in the absence of ligand in CV-1 cells. However, NBD-2 (aa 1237-1440) did not bind to this protein. Glutathione-S-transferase pull-down studies showed that TRbeta1-N (aa 1-105) bound to the broad region of SRC-1-C terminus. Expression vectors encoding a series of truncations and/or point mutations of TRbeta1 were used in transient transfection-based reporter assays in CV-1 cells. N-terminal truncated TRbeta1 (DeltaN-TRbeta1) showed lower activity than that of wild-type in both artificial F2-thyroid hormone response element and native malic enzyme response element. These results suggest that there is the interaction between N terminus of TRbeta1 and SRC-1, which may serve a full activation of SRC-1, together with activation function-2 on TRbeta1-mediated transcription.