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.

Changes in biomarker levels and myofiber constitution in rat soleus muscle at different exercise intensities.

Although exercise affects the function and structure of skeletal muscle, our knowledge regarding the biomedical alterations induced by different intensities of exercise is incomplete. Here we report on the changes in biomarker levels and myofiber constitution in the rat soleus muscle induced by exercise intensity. Male adult rats at 7 weeks of age were divided into 3 groups by exercise intensity, which was set based on the accumulated lactate levels in the blood using a treadmill: stationary control (0 m/min), aerobic exercise (15 m/min), and anaerobic exercise (25 m/min). The rats underwent 30 min/day treadmill training at different exercise intensities for 14 days. Immediately after the last training session, the soleus muscle was dissected out in order to measure the muscle biomarker levels and evaluate the changes in the myofibers. The mRNA expression of citrate synthase, glucose-6-phosphate dehydrogenase, and Myo D increased with aerobic exercise, while the mRNA expression of myosin heavy-chain I and Myo D increased in anaerobic exercise. These results suggest that muscle biomarkers can be used as parameters for the muscle adaptation process in aerobic/anaerobic exercise. Interestingly, by 14 days after the anaerobic exercise, the number of type II (fast-twitch) myofibers had decreased by about 20%. Furthermore, many macrophages and regenerated fibers were observed in addition to the injured fibers 14 days after the anaerobic exercise. Constitutional changes in myofibers due to damage incurred during anaerobic exercise are necessary for at least about 2 weeks. These results indicate that the changes in the biomarker levels and myofiber constitution by exercise intensity are extremely important for understanding the metabolic adaptations of skeletal muscle during physical exercise.

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.

Effects of Gadolinium-Based Contrast Agents on Thyroid Hormone Receptor Action and Thyroid Hormone-Induced Cerebellar Purkinje Cell Morphogenesis.

Gadolinium (Gd)-based contrast agents (GBCAs) are used in diagnostic imaging to enhance the quality of magnetic resonance imaging or angiography. After intravenous injection, GBCAs can accumulate in the brain. Thyroid hormones (THs) are critical for the development and functional maintenance of the central nervous system. TH actions in brain are mainly exerted through nuclear TH receptors (TRs). We examined the effects of GBCAs on TR-mediated transcription in CV-1 cells using transient transfection-based reporter assay and TH-mediated cerebellar Purkinje cell morphogenesis in primary culture. We also measured the cellular accumulation and viability of Gd after representative GBCA treatments in cultured CV-1 cells. Both linear (Gd-diethylene triamine pentaacetic acid-bis methyl acid, Gd-DTPA-BMA) and macrocyclic (Gd-tetraazacyclododecane tetraacetic acid, Gd-DOTA) GBCAs were accumulated without inducing cell death in CV-1 cells. By contrast, Gd chloride (GdCl3) treatment induced approximately 100 times higher Gd accumulation and significantly reduced the number of cells. Low doses of Gd-DTPA-BMA (10(-8) to 10(-6)M) augmented TR-mediated transcription, but the transcription was suppressed at higher dose (10(-5) to 10(-4)M), with decreased ß-galactosidase activity indicating cellular toxicity. TR-mediated transcription was not altered by Gd-DOTA or GdCl3, but the latter induced a significant reduction in ß-galactosidase activity at high doses, indicating cellular toxicity. In cerebellar cultures, the dendrite arborization of Purkinje cells induced by 10(-9)M T4 was augmented by low-dose Gd-DTPA-BMA (10(-7)M) but was suppressed by higher dose (10(-5)M). Such augmentation by low-dose Gd-DTPA-BMA was not observed with 10(-9)M T3, probably because of the greater dendrite arborization by T3; however, the arborization by T3 was suppressed by a higher dose of Gd-DTPA-BMA (10(-5)M) as seen in T4 treatment. The effect of Gd-DOTA on dendrite arborization was much weaker than that of the other compounds. These results indicate that exposure to specific GBCAs may, at least in part, cause toxic effects in the brain by disrupting the action of THs on TRs. The toxic effects of GBCAs may depend on the chemical structure of GBCA and the dose. Thus, it is very important to choose appropriate GBCAs for imaging to prevent adverse side effects.

The change in thyroid hormone signaling by altered training intensity in male rat skeletal muscle.

Aerobic (sub lactate threshold; sub-LT) exercise training facilitates oxidative phosphorylation and glycolysis of skeletal muscle. Thyroid hormone (TH) also facilitates such metabolic events. Thus, we studied whether TH signaling pathway is activated by treadmill training. Male adult rats received 30 min/day treadmill training with different exercise intensity for 12 days. Then plasma lactate and thyrotropin (TSH) levels were measured. By lactate levels, rats were divided into stationary control (SC, 0 m/min), sub-LT (15 m/min) and supra lactate threshold (supra-LT; 25 m/min) training groups. Immediately after the last training, the soleus muscles were dissected out to measure TH receptor (TR) mRNA and protein expressions. Other rats received intraperitoneal injection of T3, 24 h after the last training and sacrificed 6 h after the injection to measure TH target gene expression. TSH level was suppressed in both sub-LT and supra-LT groups during the exercise. TRß1 mRNA and protein levels were increased in sub-LT group. Sensitivity to T3 was altered in several TH-target genes by training. Particularly, induction of Na(+)/K(+)-ATPase ß1 expression by T3 was significantly augmented in sub-LT group. These results indicate that sub-LT training alters TH signaling at least in part by increasing TRß1 expression. Such TH signaling alteration may contribute metabolic adaptation in skeletal muscle during physical training.

Aberrant Cerebellar Development in Mice Lacking Dual Oxidase Maturation Factors.

BACKGROUND: Thyroid hormone (TH) plays a key role in the developing brain, including the cerebellum. TH deficiency induces organizational changes of the cerebellum, causing cerebellar ataxia. However, the mechanisms causing these abnormalities are poorly understood. Various animal models have been used to study the mechanism. Lacking dual oxidase (DUOX) and its maturation factor (DUOXA) are major inducers of congenital hypothyroidism. Thus, this study examined the organizational changes of the cerebellum using knockout mice of the Duoxa gene (Duoxa-/-). METHODS: The morphological, behavioral, and electrophysiological changes were analyzed in wild type (Wt) and Duoxa-deficient (Duoxa-/-) mice from postnatal day (P) 10 to P30. To detect the changes in the expression levels of presynaptic proteins, Western blot analysis was performed. RESULTS: The proliferation and migration of granule cells was delayed after P15 in Duoxa-/- mice. However, these changes disappeared by P25. Although the cerebellar structure of Duoxa-/- mice was not significantly different from that of Wt mice at P25, motor coordination was impaired. It was also found that the amplitude of paired-pulse facilitation at parallel fiber-Purkinje cell synapses decreased in Duoxa-/- mice, particularly at P15. There were no differences between expression levels of presynaptic proteins regulating neurotransmitter release at P25. CONCLUSIONS: These results indicate that the anatomical catch-up growth of the cerebellum did not normalize its function because of the disturbance of neuronal circuits by the combined effect of hypothyroidism and functional disruption of the DUOX/DUOXA complex.

Aberrant cerebellar development of transgenic mice expressing dominant-negative thyroid hormone receptor in cerebellar Purkinje cells.

To study the role of the thyroid hormone (TH) in cerebellar development, we generated transgenic mice expressing a dominant-negative TH receptor (TR) in cerebellar Purkinje cells. A mutant human TRß1 (G345R), which binds to the TH-response element but cannot bind to T3, was subcloned into exon 4 of the full-length L7/Pcp-2 gene, which is specifically expressed in Purkinje and retinal rod bipolar cells. The transgene was specifically expressed in Purkinje cells in the postnatal cerebellum. Purkinje cell dendrite arborization was significantly delayed in the transgenic mice. Surprisingly, granule cell migration was also significantly delayed. In the primary cerebellar culture, TH-induced Purkinje cell dendrite arborization was also suppressed. In quantitative real-time RT-PCR analysis, the expression levels of several TH-responsive genes were altered. The expression levels of inositol trisphosphate receptor type 1 and retinoic acid receptor-related orphan receptorα mRNAs, which are mainly expressed in Purkinje cells, and brain-derived neurotrophic factor mRNA, which is expressed in both Purkinje and granule cells, were significantly decreased. The expression levels of neurotrophin-3 and hairless mRNAs, which are mainly expressed in granule cells, and myelin basic protein mRNA, which is mainly expressed in oligodendrocytes, were also decreased. The motor coordination of transgenic mice was significantly disrupted. These results indicate that TH action through its binding to TR in Purkinje cells is required for the normal cerebellar development. TH action through TR in Purkinje cells is also important for the development of other subsets of cerebellar cells such as granule cells and oligodendrocytes.

Possible involvement of IGF-1 signaling on compensatory growth of the infraspinatus muscle induced by the supraspinatus tendon detachment of rat shoulder.

A rotator cuff tear (RCT) is a common musculoskeletal disorder among elderly people. RCT is often treated conservatively for functional compensation by the remaining muscles. However, the mode of such compensation after RCT has not yet been fully understood. Here, we used the RCT rat model to investigate the compensatory process in the remaining muscles. The involvement of insulin-like growth factor 1 (IGF-1)/Akt signaling which potentially contributes to the muscle growth was also examined. The RCT made by transecting the supraspinatus (SSP) tendon resulted in atrophy of the SSP muscle. The remaining infraspinatus (ISP) muscle weight increased rapidly after a transient decrease (3 days), which could be induced by posttraumatic immobilization. The IGF-1 mRNA levels increased transiently at 7 days followed by a gradual increase thereafter in the ISP muscle, and those of IGF-1 receptor mRNA significantly increased after 3 days. IGF-1 protein levels biphasically increased (3 and 14 days), then gradually decreased thereafter. The IGF-1 protein levels tended to show a negative correlation with IGF-1 mRNA levels. These levels also showed a negative correlation with the ISP muscle weight, indicating that the increase in IGF-1 secretion may contribute to the ISP muscle growth. The pAkt/Akt protein ratio decreased transiently by 14 days, but recovered later. The IGF-1 protein levels were negatively correlated with the pAkt/Akt ratio. These results indicate that transection of the SSP tendon activates IGF-1/Akt signaling in the remaining ISP muscle for structural compensation. Thus, the remaining muscles after RCT can be a target for rehabilitation through the activation of IGF-1/Akt signaling.

Spikar, a novel drebrin-binding protein, regulates the formation and stabilization of dendritic spines.

Dendritic spines are small, actin-rich protrusions on dendrites, the development of which is fundamental for the formation of neural circuits. The actin cytoskeleton is central to dendritic spine morphogenesis. Drebrin is an actin-binding protein that is thought to initiate spine formation through a unique drebrin-actin complex at postsynaptic sites. However drebrin overexpression in neurons does not increase the final density of dendritic spines. In this study, we have identified and characterized a novel drebrin-binding protein, spikar. Spikar is localized in cell nuclei and dendritic spines, and accumulation of spikar in dendritic spines directly correlates with spine density. A reporter gene assay demonstrated that spikar acts as a transcriptional co-activator for nuclear receptors. We found that dendritic spine, but not nuclear, localization of spikar requires drebrin. RNA-interference knockdown and overexpression experiments demonstrated that extranuclear spikar regulates dendritic spine density by modulating de novo spine formation and retraction of existing spines. Unlike drebrin, spikar does not affect either the morphology or function of dendritic spines. These findings indicate that drebrin-mediated postsynaptic accumulation of spikar regulates spine density, but is not involved in regulation of spine morphology.

Lactational exposure to hydroxylated polychlorinated biphenyl (OH-PCB 106) causes hyperactivity in male rat pups by aberrant increase in dopamine and its receptor.

Polychlorinated biphenyls (PCBs) are recognized as persistent environmental pollutants that may cause adverse health problems. Despite extensive investigations of PCB in neural function, little is known about behavioral traits by PCB exposure and its neurochemical mechanism. Here, we report the behavioral study of a rat pup that was exposed to hydroxylated-PCB 106 (OH-PCB 106; 4-hydroxy-2',3,3',4',5'-pentachlorobiphenyl) through maternal milk. The different groups of mothers received via gavage corn oil vehicle, 0.5, 5, or 50 mg/kg body weight of OH-PCB 106 every second day from day 3 to 13 after delivery. The exposure did not affect the body weight of the dams or the physical development of the newborn pups in both sexes. Male rats exposed to OH-PCB 106 showed hyperactivity that was characterized by increased locomotor activity in novel environment and circadian period. Interestingly, OH-PCB 106-exposed rat pups displayed abnormally high levels of dopamine and D2 dopamine receptor (D2DR), but not D1DR and D5DR, in the striatum, an important center for the coordination of behavior. These findings indicate that OH-PCB 106 has a significant neurotoxic effect on rat behavior, which may be associated with increased D2DR mediated signals.

The effect of low dose lipopolysaccharide on thyroid hormone-regulated actin cytoskeleton modulation and type 2 iodothyronine deiodinase activity in astrocytes.

Systemic infection/inflammation can severely interfere with brain development. Lipopolysaccharide (LPS) is a major cell wall component of gram-negative bacteria and commonly used to model the response by infections. Since perinatal exposure to LPS shows neurodevelopmental defects partly similar to those seen in perinatal hypothyroidism, we examined the effect of LPS on thyroxin (T4)-mediated signalings in astrocytes. Initially, C6 rat glioma-derived clonal cells were used, whose biological nature is similar to that of astrocytes. To measure the effects of LPS and T4, actin polymerization and D2 activity assays were carried out. LPS treatment (10 ng/mL) markedly induced actin depolymerization, whereas 10 nM T4 promoted actin polymerization. Furthermore, T4 partly rescued LPS-induced actin depolymerization. LPS treatment (10 ng/mL) increased D2 activity, whereas T4 (10 nM) suppressed this activity. T4 restored LPS-increased D2 activity at 10 nM. LPS-induced actin depolymerization and D2 activity were blocked by p38 MAP kinase inhibitor. Such effects were not seen in T4-mediated changes. Furthermore, similar results were found in the cerebellar primary astrocyte. These results indicate that, although LPS affects T4-regulated cellular events such as actin polymerization and D2 activity, which may induce neurodevelopmental defects similar to those in perinatal hypothyroidism, LPS signaling pathways are independent of T4 signaling pathways.

Interaction of silencing mediator for retinoid and thyroid receptors with steroid and xenobiotic receptor on multidrug resistance 1 promoter.

AIMS: The steroid and xenobiotic receptor (SXR) regulates the transcription of its target genes by interacting with various nuclear receptor cofactors. We have previously shown that silencing mediator for retinoid and thyroid receptors (SMRT) interacts with SXR even in the presence of rifampicin on cytochrome P450 monooxygenase 3A4 (CYP3A4) promoter in HepG2 cells. To examine the specificity of such interaction, the involvement of SMRT on SXR-mediated transcription through multidrug resistance (MDR) 1 gene promoter was examined using LS174T intestine-derived clonal cells. MAIN METHODS: Transient transfection-based reporter gene assay was carried out to examine the effect of SMRT or nuclear receptor corepressor (NCoR) on SXR-mediated transcription in LS174T cells. Semi-quantitative RT-PCR was performed to confirm the expression of MDR1 mRNA in LS174T cells. To examine the interaction of SMRT with SXR, we carried out mammalian one-hybrid assay in CV-1 cells and immunoprecipitation study in HEK-293 cells. KEY FINDINGS: SMRT, but not NCoR suppressed rifampicin-induced SXR-mediated transcription. The SXR-mediated MDR1 mRNA expression was augmented in the presence of rifampicin, whereas it suppressed the expression following the overexpression of SMRT. In mammalian one-hybrid assay, only SMRT but not NCoR interacted with SXR on MDR1 promoter in the presence of rifampicin. In immunoprecipitation study, SMRT bound to SXR regardless of the presence or absence of rifampicin. SIGNIFICANCE: SMRT may be recruited in the SXR-cofactor complex even in the presence of ligand. SMRT may be involved not only in SXR-mediated suppression without ligand, but also in ligand-activated transcription to suppress the overactivation of transcription.

Aberrant cerebellar neurotrophin-3 expression induced by lipopolysaccharide exposure during brain development.

Autism is a developmental disorder affecting communication, social interaction, motor skills, and cerebellar structure and functions. Recent studies have indicated that maternal infection during brain development may be one of the risk factors for autism. We have previously demonstrated the abnormal overexpression of neurotrophin-3 (NT-3) in autistic cerebellum. To examine further the potential link between autism and maternal infection, and specifically NT-3 expression in the cerebellum, we used maternal lipopolysaccharide (LPS)-exposed rat model of infection. In group 1, pregnant female rats were exposed to 200 µg/kg body weight LPS delivered subcutaneously from gestational days (G) 10 to G15, and pups were exposed to LPS from postnatal days (P) 5 to P10, whereas in group 2, pups were exposed to the same dose of LPS from P5 to P10. There was no change in body mass of pups and mothers following LPS treatment. Cerebellar NT-3 levels were examined by enzyme-linked immunosorbent assay on P6, P12, and P21. We report here that cerebellar NT-3 levels were elevated in pups of both LPS groups as compared to the controls on P21. Our results suggest that altered neurotrophin levels may affect normal brain development and contribute to autistic pathology.

Alterations of biochemical marker levels and myonuclear numbers in rat skeletal muscle after ischemia-reperfusion.

Prolonged ischemia-reperfusion results in various damages in skeletal muscle. Following reperfusion, although the damaged muscles undergo regeneration, the precise process and mechanism of regeneration have not yet been fully understood. Here, we show the altered levels of plasma biochemical markers of muscle damage, and the change in myonuclear numbers in adult rat skeletal muscle by ischemia-reperfusion. Male Wistar rats were subjected to unilateral hindlimb ischemia by clamping the anterior tibial artery for 2 h before reperfusion. Both plasma creatine kinase activity and C-reactive protein levels in plasma were increased significantly at 0.5 h of reperfusion and returned to the control level at 24 h. The transverse sectional area of muscle belly of the anterior tibial muscles in ischemic side was significantly decreased by 20 % compared with those in sham-ischemic (control) side at 2 days, and returned to the control level at 5 days of reperfusion. Moreover, the number of interstitial nuclei in the ischemic side were significantly increased at 5-14 days and returned to the control level at 21 days of reperfusion. Central nuclei that are specifically observed in regenerating muscle, appeared at 5 days, reached a peak at 14 days, and disappeared at 28 days of reperfusion. Furthermore, MyoD, a regulatory factor for myogenesis, showed a transient expression at 5 days of reperfusion. These results indicate that, although the size of muscle seems to be recovered by 5 days of reperfusion, the most active muscle regeneration occurs much later, as shown by the increase in central nuclei.

An in vitro method to study the effects of thyroid hormone-disrupting chemicals on neuronal development.

Thyroid hormones (THs) play critical roles for normal cerebellar development. It has been reported that several environmental chemicals may affect cerebellar development through TH system. One such example is the suppression of TH receptor (TR)-mediated transcription by polybrominated diphenyl ethers (PBDEs). To determine the effect of these chemicals on brain development, we established a primary culture system of rat cerebellar Purkinje cells. Using this system, as low as 10(-10)M TH induced Purkinje cell dendritic arborization and such effect was dose-dependent. We examined the effect of decabromodiphenyl ether (BDE209) using this system. Dendritic development of the Purkinje cells was suppressed by 10(-10)M BDE209, that was compatible to the result of the suppression of TR-mediated transcription by using reporter gene assay. These results suggest that TH plays a pivotal role in the development of the Purkinje cell dendrites. Together with in vitro assay system such as reporter gene assay and liquid chemiluminescent DNA-pull down assay, an in vitro protein-DNA binding assay, these assay systems provide us with precise information about environmental chemicals on brain development.

Ligand-induced downregulation of TrkA is partly regulated through ubiquitination by Cbl.

Nerve growth factor (NGF) binding to its receptor TrkA, which belongs to the family of receptor tyrosine kinases (RTKs), is known to induce its internalization, endosomal trafficking and subsequent lysosomal degradation. The Cbl family of ubiquitin ligases plays a major role in mediating ubiquitination and degradation of RTKs. However, it is not known whether Cbl participates in mediating ubiquitination of TrkA. Here we report that c-Cbl mediates ligand-induced ubiquitination and degradation of TrkA. TrkA ubiquitination and degradation required direct interactions between c-Cbl and phosphorylated TrkA. c-Cbl and ubiquitinated TrkA are found in a complex after NGF stimulation and are degraded in lysosomes. Taken together, our data demonstrate that c-Cbl can induce downregulation of NGF-TrkA complexes through ubiquitination and degradation of TrkA.

Suppression of thyroid hormone receptor-mediated transcription and disruption of thyroid hormone-induced cerebellar morphogenesis by the polybrominated biphenyl mixture, BP-6.

Polybrominated biphenyls (PBBs) are polyhalogenated, bioaccumulative flame retardant chemicals, which have been used in a variety of consumer and household products. They were accidentally introduced into the food chain in Michigan in 1973 and have remained a source of health concern. Studies have shown that exposure to PBB may cause adverse neurotoxic effects. We therefore examined the effects of BP-6, a PBB mixture, on thyroid hormone (TH) receptor (TR)-mediated transcription, on TH-induced Purkinje cell dendritogenesis, and on TH-induced cerebellar granule cell neurite extension. Our study shows that BP-6 suppressed TR-mediated transcription in CV-1 cells. Mammalian two-hybrid studies revealed that BP-6 did not inhibit coactivator binding to TR nor did it recruit corepressors to TR. Further examination using the liquid chemiluminescent DNA pull down assay revealed partial dissociation of TR from TH response element (TRE). In primary rat cerebellar culture, BP-6 significantly suppressed TH-induced dendrite arborization of Purkinje cells, and in reaggregate rat granule cell culture, impaired TH-induced neurite extension of granule cells. Taken together, our results indicate that BP-6 may disrupt TH homeostasis and consequently impair normal neuronal development.

Brain-derived neurotrophic factor (BDNF) ameliorates the suppression of thyroid hormone-induced granule cell neurite extension by hexabromocyclododecane (HBCD).

Thyroid hormone (TH) plays an essential role in growth and differentiation of the central nervous system. Deficiency of TH during perinatal period results in abnormal brain development known as cretinism in human. We recently reported that an environmental chemical 1,2,5,6,9,10-α-hexabromocyclododecane (HBCD) suppressed TH receptor (TR)-mediated transcription. To examine the effect of HBCD on cerebellar granule cells, we used purified rat cerebellar granule cells in reaggregate culture. Low dose HBCD (10(-10)M) significantly suppressed TH-induced neurite extension of granule cell aggregate. To clarify further the mechanisms of such suppression, we added brain-derived neurotrophic factor (BDNF) into culture medium, since BDNF plays a critical role in promoting granule cell development and is regulated by TH. BDNF completely rescued HBCD-induced suppression of granule cell neurite extension in the presence of T3. These results indicate that HBCD may disrupt TH-mediated brain development at least in part due to a disruption of the T3 stimulated increase in BDNF and BDNF may possess ability to ameliorate the effect of HBCD in granule cells.

1,2,5,6,9,10-αHexabromocyclododecane (HBCD) impairs thyroid hormone-induced dendrite arborization of Purkinje cells and suppresses thyroid hormone receptor-mediated transcription.

1,2,5,6,9,10-αHexabromocyclododecane (HBCD) is a nonaromatic, brominated cyclic alkane used as an additive flame retardant. It bioaccumulates, persists in the environment, and has been detected in humans and wildlife. Its developmental neurotoxicity is of great concern. We investigated the effect of HBCD on thyroid hormone (TH) receptor (TR)-mediated transcription using transient transfection-based reporter gene assays and found that a low-dose (10(-10) M) HBCD suppressed TR-mediated transcription. We further examined the effect of HBCD on interaction of TR with TH response element (TRE) and found a partial dissociation of TR from TRE. HBCD did not dissociate steroid receptor coactivator-1 from TR in the presence of TH; neither did it recruit corepressors (N-CoR and SMRT) to TR in the absence of TH. Furthermore, low-dose HBCD (10(-10) M) significantly suppressed TH-induced dendrite arborization of Purkinje cells in primary cerebellar culture derived from newborn rat. These results show that low-dose HBCD can potentially disrupt TR-mediated transactivation and impairs Purkinje cell dendritogenesis, suggesting that HBCD can interfere with TH action in target organs, including the developing brain.

Disruption of thyroid hormone receptor-mediated transcription and thyroid hormone-induced Purkinje cell dendrite arborization by polybrominated diphenyl ethers.

BACKGROUND: Polybrominated diphenyl ethers (PBDEs) have been used as flame retardants and are becoming a ubiquitous environmental contaminant. Adverse effects in the developing brain are of great health concern. OBJECTIVE: We investigated the effect of PBDEs/hydroxylated PBDEs (OH-PBDEs) on thyroid hormone (TH) receptor (TR)-mediated transcription and on TH-induced dendrite arborization of cerebellar Purkinje cells. METHODS: We examined the effect of PBDEs/OH-PBDEs on TR action using a transient transfection-based reporter gene assay. TR-cofactor binding was studied by the mammalian two-hybrid assay, and TR-DNA [TH response element (TRE)] binding was examined by the liquid chemiluminescent DNA pull-down assay. Chimeric receptors generated from TR and glucocorticoid receptor (GR) were used to identify the functional domain of TR responsible for PBDE action. The change in dendrite arborization of the Purkinje cell in primary culture of newborn rat cerebellum was also examined. RESULTS: Several PBDE congeners suppressed TR-mediated transcription. The magnitude of suppression correlated with that of TR-TRE dissociation. PBDEs suppressed transcription of chimeric receptors containing the TR DNA binding domain (TR-DBD). We observed no such suppression with chimeras containing GR-DBD. In the cerebellar culture, PBDE significantly suppressed TH-induced Purkinje cell dendrite arborization. CONCLUSIONS: Several PBDE congeners may disrupt the TH system by partial dissociation of TR from TRE acting through TR-DBD and, consequently, may disrupt normal brain development.