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An in vitro method to study the effects of thyroid hormone-disrupting chemicals on neuronal development
NeuroToxicology 33 (2012) 753–757
Contents lists available at SciVerse ScienceDirect
NeuroToxicology
An in vitro method to study the effects of thyroid hormone-disrupting chemicals on neuronal development Yu Xiong a,2, Kingsley Ibhazehiebo b,1,2, Toshiharu Iwasaki b,*, Noriyuki Koibuchi b a b
Department of Laboratory Sciences, Gunma University Graduate School of Health Sciences, 3-39-22 Showa-machi, Maebashi, Gunma 371-8511, Japan Department of Integrative Physiology, Gunma University Graduate School of Medicine, 3-39-22 Showa-machi, Maebashi, Gunma 371-8511, Japan
A R T I C L E I N F O
A B S T R A C T
Article history: Received 30 September 2011 Received in revised form 12 April 2012 Accepted 27 April 2012 Available online 9 May 2012
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. ß 2012 Elsevier Inc. All rights reserved.
Keywords: Primary culture Purkinje cell Thyroid hormone Thyroid hormone receptor Transcriptional regulation Brain development Screening system
1. Introduction Brain development and plasticity is involved in various epigenetic processes that activate specific genes at different time point (Evans, 1988; Iglesias et al., 1996; Legrand, 1986; Thompson and Bottcher, 1997). Neurological disorders can be caused by a number of environmental and genetic interference during brain developing periods as reviewed by Oppenheimer and Schwartz (1997). Environmental influences, such as stressor, endocrine disrupters, and malnutrition, may affect such processes. In adult brain, structural neuroplasticity is also greatly involved in functional adaptation to the changing condition of the external and internal environment. Circulating hormones mediate the effect of environment on neural plasticity to some extent (Koibuchi, 2008). Thyroid hormone (TH) is essential for proper mammalian development of various organs, including the central nervous system (Koibuchi and Chin, 2000; Koibuchi et al., 2003). TH plays particularly important role in the postnatal development of the rodent cerebellum (Hajos et al., 1973; Nicholson and Altman,
* Corresponding author. Tel.: +81 27 220 7923; fax: +81 27 220 7926. E-mail address: [email protected] (T. Iwasaki). 1 Present address: Department of Physiology, School of Basic Medical Sciences, College of Medical Sciences, University of Benin, P.M.B. 111, Benin City, Edo State, Nigeria. 2 These authors contributed equally. 0161-813X/$ – see front matter ß 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.neuro.2012.04.021
1972a,b; Rabie et al., 1979). TH deficiency causes specific defects in cell migration and differentiation (Legrand, 1986; Oppenheimer and Schwartz, 1997). Since TH deficiency results in multiple morphological alterations in neonatal rat brain (Schwartz, 1983; DeLong, 1996), particularly decreased dendritic arborization, synaptogenesis, number and size of dendritic spines of Purkinje cells (Heuer and Mason, 2003; Nicholson and Altman, 1972a,b; Legrand, 1979), the Purkinje cell is considered to be a good model to examine various TH actions in developing brain. Many studies suggested the role of TH in morphological development of cerebellum using rodent models (Legrand, 1979). Abnormal development seen in the perinatal hypothyroid rat cannot be rescued unless TH is replaced within the first 2 weeks of postnatal life in rodents (Koibuchi and Chin, 2000). Although neonatal TH deficiency severely impacts development, it is rarely fatal. Thus, the study of morphological, molecular and biochemical effects of TH on brain development have relied on the use of experimental animal models or cell culture models. TH exerts its major effect by binding to the nuclear TH receptor (TR), a ligand-regulated transcription factor (Chin and Yen, 1997), although TH actions at non-genomic sites such as mitochondria, plasma membrane, and cytoplasm have also been reported (Davis and Davis, 1996). Nuclear TRs are encoded by two genomic loci (a and b). TRb1 is expressed predominantly in the Purkinje cells, whereas TRa1 on other subset of neurons. Although these TRs are wildly expressed during the cerebellar development and their
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levels are greater in adult, the expression of many TH-responsive genes is altered by TH status only during early postnatal ‘critical period’. TR is bound to specific DNA sequences called TH-response elements (TREs). When TR binds to TRE, it interacts with retinoid X receptor (RXR) to form heterodimers, which, in turn, binds to a number of cofactors such as coreprssors and coactivators. In the absence of ligand, the TR/RXR complex binds to a corepressor complex that may contain NCoR (nuclear receptor corepressor) or SMRT (silencing mediator of retinod and thyroid hormone receptor), and shows histone deacetylase (HDAC) activities. These factors mediate repression of transcription probably by restricting the accessibility of basal transcription factors to targeted promoters. In the presence of T3, such corepressors are replaced by a coactivator complex that includes the p160 coactivator family, which contains histone acetylase (HAT) activity for chromatin remodeling. Besides these cofactors, many cofactors interact with TR to regulate the expression of target gene precisely (Wu and Koenig, 2000; Xu et al., 1999). Polybrominated diphenyl ethers (PBDEs) have been commonly used as flame retardants, and are becoming ubiquitous environmental contaminants. PBDEs can cross the blood–brain barrier (BBB)
to accumulate in the central nervous system (Naert et al., 2007). PBDEs and hydroxylated PBDE (OH-PBDE) metabolites are antiandrogenic in vitro by acting on, or interfering with, a number of potential targets within the hypothalamic-pituitary-gonadal axis (Stoker et al., 2005; Canto´n et al., 2008, 2007a,b, 2006; Harju et al., 2007; He et al., 2008; Legler, 2008). Furthermore, PBDEs, that include 209 congeners, or their metabolites may affect TH-related function by altering TH or TR function, by binding to TR or other nuclear hormone receptors, or through direct effects on thyroid gland tissue (Costa and Giordano, 2007; Darnerud, 2008; Legler, 2008; Kitamura et al., 2008; Kojima et al., 2009; Li et al., 2010). Previously, we reported that some environmental compounds suppress the TRmediated transcription and this suppression was due to dissociation of TR from TH-response element (TRE). Based on this observation, we developed a screening system, liquid chemiluminescent DNA-pull down assay (LCDPA), to examine the effect of chemicals on TR-TRE binding (Iwasaki et al., 2008). However, since LCDPA is in vitro protein–DNA binding assay, a screening system to examine the effect of developing brain was required. Thus, we developed a primary culture system to examine the effect of environmental chemicals using rat primary cerebellar culture (Ibhazehiebo et al., 2011a,b).
Fig. 1. The effect of T4 on Purkinje cell dendritic arborization (17 days in culture). (A–D) Photomicrographs showing the effect of T4 on Purkinje cell morphology. The control without T4 (A), and +T4 (10 10 M; B), +T4 (10 9 M; C), +T4 (10 8 M; D) were added to the primary cultured cerebellar cells that contain the whole subset of cerebellar cells. T4 was converted to T3, physiologically active form of TH, by deiodenase 2 in astrocyte. Immunocytochemistry was performed using anti-calbindin antibody to visualize Purkinje cells. Bars = 50 mm. (E) Change in dendritic areas of Purkinje cells by T4. Data are expressed as mean SE (n = 10 determinations) from one experiment and represent at least three indepentdent experiments. *p < 0.01 by ANOVA, for T4 (+) compared with T4 ( ).
Y. Xiong et al. / NeuroToxicology 33 (2012) 753–757
2. Materials and methods 2.1. Chemicals Tri-iodothyronine (T3) and thyroxine (T4) were purchased from Sigma Chemical Co. (St. Louis, MO). BDE209 was purchased from AccuStandard Chemicals (New Haven, CT), and was >98% pure. BDE209 was dissolved in absolute ethanol at concentration less than 10 4 M, while T3 and T4 were dissolved in dimethylsulfoxide (DMSO) at 100 mM concentration. Dilutions using culture medium were made from stock solutions immediately before use. Repeated freezing and thawing were avoided. 2.2. Method of screening system based on primary cerebellar culture In this assay, newborn rats were decapitated under direct ether anesthesia on postnatal day 1. The culture protocol has been described by Kimura-Kuroda et al. (2007). Briefly, cerebella were digested and plated in poly-L-lysine-coated wells of chamber slides (8-mm-diameter wells), and cultured overnight in a 5% CO2 incubator. Then, T4 and/or environmental chemicals were added to the culture medium and cultured for 17 days. For TH treatment, T4 was used in primary culture because, physiologically, it is preferentially transported to brain through the BBB, and primary culture contains the whole subset of cerebellar cells, including astrocytes. Thus, T4 was converted to T3, physiologically active form of TH, by deiodenase 2 in astrocyte. Purkinje cells were immunostained with a mouse monoclonal anti-calbindin-28K antibody. The morphological changes were examined under a laser confocal scanning microscope and finally quantified dendritic arborization using NIH Image software (Vischer, 2006).
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nonexistent; whereas those with T4 showed elaborate dendrites characterized by the presence of a main thick primary shaft and several other secondary shafts with bifurcating branches (Fig. 1A– D). T4 augments the Purkinje cell dendritic arborization in a dosedependent manner. Although the dendrite development showed high sensitivity and dependence of the T4 concentration, but the number of Purkinje cells was not always in parallel with the dendritic arborization. 3.2. Effect of PBDEs on Purkinje cell dendritic arborization Using Purkinje cell screening system, we examined the effect of PBDEs. Purkinje cells cultured with BDE209 showed abnormally shaped dendrite, very poor growth and the especially small secondary branches (Fig. 2A); the area of these Purkinje cell dendrites was significantly reduced (Fig. 2B). (Reproduced with permission from Environmental Health Perspectives.) 3.3. Effect of BDE209 on TR-mediated transcription We observed the greatest magnitude of suppression (45%) of TRb1-mediated transcription thought the F2-TRE-LUC by 10 9 M BDE209 (Fig. 3A). BDE209 also suppressed the transcription
2.3. Plasmids Expression vector for TRb1 has been previously described (Iwasaki et al., 2001). The luciferase (LUC) reporter constructs containing TRE, the chick lysozyme (F2)- and artificial direct repeat (DR4)-thymidine kinase (TK)-LUC in the PT109 vector have been described elsewhere (Koibuchi et al., 1999). 2.4. Clonal cell culture and transient transfection-based reporter gene assay CV-1 cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% small lipophilic hormonedeprived fetal bovine serum at 37 8C, 5% CO2. Cells were plated in 24-well plates 48 h before transfection using calcium-phosphate coprecipitation method (Iwasaki et al., 2002). Cytomegalovirus-b-galactosidase plasmid was used as internal control. 16–24 h after transfection, wells were refilled with fresh medium containing the indicated concentrations of ligand for 24 h. Cells were then harvested to measure the LUC activity as described previously (Iwasaki et al., 2002). Total amounts of DNA per well were balanced by adding pcDNA3 plasmids (Invitrogen, San Diego, CA). The LUC activities were normalized to b-galactosidase activity and then calculated as relative LUC activities. All transfection studies were repeated at least three times in triplicate. Data shown represent mean SEM of one experiment. 3. Results 3.1. Rat Purkinje dendritic arborization was induced by thyroid hormone in a dose-dependent manner As shown in Fig. 1, Purkinje cells in the control medium ( T4) showed that the growth of dendrites was poor or almost
Fig. 2. The effect of BDE209 on Purkinje cell dendritic arborization (17 days in culture). (A) Photomicrographs showing the effect of BDE209 on Purkinje cell morphology. BDE209 (10 10 M) was added to the culture in the absence or presence of T4 (10 8 M) and immunocytochemisty was performed using anti-calbindin antibody to visualize Purkinje cells. Bars = 50 mm. (B) Change in dendritic areas of Purkinje cells by T4. Data are expressed as mean SE (n = 10 determinations) from one experiment and represent at least three indepentdent experiments. *p < 0.01 by ANOVA, for T4 (+)/BDE209 ( ) compared with T4 ( )/BDE209 ( ). #p < 0.01 by ANOVA, for T4 (+)/BDE209 (+) compared with T4 (+)/BDE209 ( ).
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Relative luciferase activity
A 5
-T3 +T3
4
*
3
*
*
2 1
F2-TRE CV-1
0 TRβ1
-
+
BDE209
-
-
+
10 -12
+
+
10 -11
+
10 -10
10 -9 (M)
Relative luciferase activity
B 5
-T3 +T3
4 3
*
*
*
2 1
DR4-TRE CV-1
0 TRβ1
-
+
BDE209
-
-
+
10 -12
+
10 -11
+
10 -10
+
are regulated by TH in laboratory animal (Koibuchi et al., 1999; Koibuchi and Chin, 2000). Nuclear TH action is mediated through TR, which increases or decreases the expression of target genes. TRa and b are produced during the development of both Purkinje and granule cells in the cerebellum (Bradley et al., 1989; Strait et al., 1991). Although these TRs are wildly expressed during the cerebellar development and their levels are greater in adult, the expression of many TH-responsive genes is altered by TH status only during early postnatal critical period. Not only the expression levels of TRs but also those of cofactors and other nuclear hormone receptors may play a role in regulating TH sensitivity in the developing cerebellum. To examine the effect of mutant TR in Purkinje cells, we are now investigating the transgenic mouse that specifically expresses mutant TRb1 in Purkinje cells (data not shown). In summary, we established a convenient assay system to screen the effect of neurotoxic chemicals on TH-related function in developing brain. Together with in vitro screening system, these assays provide us with precise information about chemicals on developing brain. We hope our study will provide significant clarifications on the mechanisms of TH-related action in the developing brain. Conflict of interest
10 -9 (M) All authors declare no conflict of interest.
Fig. 3. Suppression of TR-mediated transcription by BDE209. (A and B) Expression plasmids encoding TRb1 (10 ng) were cotransfected with F2-TRE-LUC (100 ng) (A), DR4-TRE-LUC (100 ng) (B) into CV-1 cells. Cells were incubated with or without 10 7 M T3 and indicated amounts of BDE209. Total amounts of DNA for each well were balanced by adding vector pcDNA3. Data are mean SE of experiments performed in triplicate. *p < 0.01 by ANOVA, compared with TRb1 (+), T3 (+), and BDE209 ( ).
Acknowledgments
through the DR4-TRE (45%) at 10 9 M (Fig. 3B). These results are compatible to the result of primary Purkinje cell dendritic arborization.
˜ o, T. Aoki, K. Takata, We thank Drs. J. Kimura-Kuroda, M. London K. Hanamura, Y. Lu, N. Shimokawa and T. Shirao for helpful assistance, and Dr. A. Takeshita for kindly providing plasmids. This project was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT) (17510039, T.I, 17390060, N.K.), and a grant from Ministry of the Environment of Japan (to N.K.).
4. Discussion
References
Through our study, we confirm that the essential role of TH on the TH-dependent development of Purkinje cells using the primary culture system of rat cerebellar cells. It has been reported that TH induces the dendritic development of Purkinje cells via TR (Heuer and Mason, 2003). In the cerebellum, TR is expressed in most types of neuron, including Purkinje and granule cells (Bradley et al., 1989; Strait et al., 1991). TH also activates several astrocyte functions, which are important in Purkinje cell development (Siegrist-Kaiser et al., 1990; Farwell et al., 1995; Gomes et al., 1999). TH may also affect Purkinje cell through the hormones’ interactions with granule cells, which are major neuronal cells in terms of cell number in the culture system (Kimura-Kuroda et al., 2007). Thus, our system may be useful to examine the effect of toxic chemicals since our primary culture contains all sets of cerebellar cells. In addition, we used rat cerebellum and efficiency is much better than mice. Thus, our system is good for screening many toxic chemicals at one time. However, it takes 17 days to obtain results. To shorten the period of culture for screening systems, we recently developed primary culture system using granule cells and astrocytes. The relationship among these assays is now under investigation including genomic and non-genomic actions. It has been reported that the development of cerebellum including Purkinje cells is affected by many kinds of neurotrophic factors (e.g., neurotrophin-3; NT-3, brain-derived neurotrophic factor; BDNF), and extracellular matrix proteins (e.g., laminin), that
Bradley DJ, Young WS III, Weinberger C. Differential expression of alpha and beta thyroid hormone receptor genes in rat brain and pituitary. Proc Natl Acad Sci U S A 1989;86:7250–4. Canto´n RF, Bovee T, Bergman A, Daamen FEJ, van den Berg M, van Duursen MBM. In vitro anti-androgenicity of PBDEs, HBCD, TBP and hydroxylated and methoxylated PBDEs based on a yeast bioassay. Organohal Comp 2007a;69:682–5. Canto´n RF, Nijmeijer S, Daamen FEJ, van den Ven LTM, van den Berg M. In vivo steroidogenic effects of several brominated flame retardants in Wistar rats. Organohal Comp 2007b;69:2647–50. Canto´n RF, Sanderson JT, Nijmeijer S, Bergman A, Letcher RJ, van den Berg M. In vitro effects of brominated flame retardants and metabolites on CYP17 catalytic activity: a novel mechanism of action. Toxicol Appl Pharmacol 2006;216:274–81. Canto´n RF, Scholten DE, Marsh G, de Jong PC, van den Berg M. Inhibition of human placental aromatase activity by hydroxylated polybrominated diphenyl ethers (OH-PBDEs). Toxicol Appl Pharmacol 2008;227:68–75. Chin WW, Yen PM. Molecular mechanisms of nuclear thyroid hormone action. In: Braverman LE, editor. Diseases of the thyroid. Totowa: Humana Press Inc.; 1997, pp. 1–15. Costa LG, Giordano G. Developmental neurotoxicity of polybrominated diphenylether (PBDE) flame retardants. Neurotoxicology 2007;28:1047–67. Darnerud PO. Brominated flame retardants as possible endocrine disrupters. Int J Androl 2008;31:152–60. Davis PJ, Davis FB. Nongenomic actions of thyroid hormone. Thyroid 1996;6:497–504. DeLong GR. The neuromuscular system and brain in hypothyroidism. 7th ed. In: Braverman LE, Utiger RD, editors. Werner and Ingbar’s the thyroid a fundamental and clinical text, vol. 67. Philadelphia: JB Lippincott-Raven Co; 1996. p. 826–35. Evans RM. The steroid and thyroid hormone receptor superfamily. Science 1988;240: 889–95. Farwell AP, Tranter MP, Leonard JL. Thyroxine-dependent regulation of integrin– laminin interactions in astrocytes. Endocrinology 1995;136:3909–15. Gomes FC, Maia CG, de Menezes JR, Neto VM. Cerebellar astrocytes treated by thyroid hormone modulate neuronal proliferation. Glia 1999;25:247–55. Hajos F, Patel AJ, Balazs R. Effect of thyroid deficiency on the synaptic organization of the rat cerebellar cortex. Brain Res 1973;50:387–401.
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Contents lists available at SciVerse ScienceDirect
NeuroToxicology
An in vitro method to study the effects of thyroid hormone-disrupting chemicals on neuronal development Yu Xiong a,2, Kingsley Ibhazehiebo b,1,2, Toshiharu Iwasaki b,*, Noriyuki Koibuchi b a b
Department of Laboratory Sciences, Gunma University Graduate School of Health Sciences, 3-39-22 Showa-machi, Maebashi, Gunma 371-8511, Japan Department of Integrative Physiology, Gunma University Graduate School of Medicine, 3-39-22 Showa-machi, Maebashi, Gunma 371-8511, Japan
A R T I C L E I N F O
A B S T R A C T
Article history: Received 30 September 2011 Received in revised form 12 April 2012 Accepted 27 April 2012 Available online 9 May 2012
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. ß 2012 Elsevier Inc. All rights reserved.
Keywords: Primary culture Purkinje cell Thyroid hormone Thyroid hormone receptor Transcriptional regulation Brain development Screening system
1. Introduction Brain development and plasticity is involved in various epigenetic processes that activate specific genes at different time point (Evans, 1988; Iglesias et al., 1996; Legrand, 1986; Thompson and Bottcher, 1997). Neurological disorders can be caused by a number of environmental and genetic interference during brain developing periods as reviewed by Oppenheimer and Schwartz (1997). Environmental influences, such as stressor, endocrine disrupters, and malnutrition, may affect such processes. In adult brain, structural neuroplasticity is also greatly involved in functional adaptation to the changing condition of the external and internal environment. Circulating hormones mediate the effect of environment on neural plasticity to some extent (Koibuchi, 2008). Thyroid hormone (TH) is essential for proper mammalian development of various organs, including the central nervous system (Koibuchi and Chin, 2000; Koibuchi et al., 2003). TH plays particularly important role in the postnatal development of the rodent cerebellum (Hajos et al., 1973; Nicholson and Altman,
* Corresponding author. Tel.: +81 27 220 7923; fax: +81 27 220 7926. E-mail address: [email protected] (T. Iwasaki). 1 Present address: Department of Physiology, School of Basic Medical Sciences, College of Medical Sciences, University of Benin, P.M.B. 111, Benin City, Edo State, Nigeria. 2 These authors contributed equally. 0161-813X/$ – see front matter ß 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.neuro.2012.04.021
1972a,b; Rabie et al., 1979). TH deficiency causes specific defects in cell migration and differentiation (Legrand, 1986; Oppenheimer and Schwartz, 1997). Since TH deficiency results in multiple morphological alterations in neonatal rat brain (Schwartz, 1983; DeLong, 1996), particularly decreased dendritic arborization, synaptogenesis, number and size of dendritic spines of Purkinje cells (Heuer and Mason, 2003; Nicholson and Altman, 1972a,b; Legrand, 1979), the Purkinje cell is considered to be a good model to examine various TH actions in developing brain. Many studies suggested the role of TH in morphological development of cerebellum using rodent models (Legrand, 1979). Abnormal development seen in the perinatal hypothyroid rat cannot be rescued unless TH is replaced within the first 2 weeks of postnatal life in rodents (Koibuchi and Chin, 2000). Although neonatal TH deficiency severely impacts development, it is rarely fatal. Thus, the study of morphological, molecular and biochemical effects of TH on brain development have relied on the use of experimental animal models or cell culture models. TH exerts its major effect by binding to the nuclear TH receptor (TR), a ligand-regulated transcription factor (Chin and Yen, 1997), although TH actions at non-genomic sites such as mitochondria, plasma membrane, and cytoplasm have also been reported (Davis and Davis, 1996). Nuclear TRs are encoded by two genomic loci (a and b). TRb1 is expressed predominantly in the Purkinje cells, whereas TRa1 on other subset of neurons. Although these TRs are wildly expressed during the cerebellar development and their
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Y. Xiong et al. / NeuroToxicology 33 (2012) 753–757
levels are greater in adult, the expression of many TH-responsive genes is altered by TH status only during early postnatal ‘critical period’. TR is bound to specific DNA sequences called TH-response elements (TREs). When TR binds to TRE, it interacts with retinoid X receptor (RXR) to form heterodimers, which, in turn, binds to a number of cofactors such as coreprssors and coactivators. In the absence of ligand, the TR/RXR complex binds to a corepressor complex that may contain NCoR (nuclear receptor corepressor) or SMRT (silencing mediator of retinod and thyroid hormone receptor), and shows histone deacetylase (HDAC) activities. These factors mediate repression of transcription probably by restricting the accessibility of basal transcription factors to targeted promoters. In the presence of T3, such corepressors are replaced by a coactivator complex that includes the p160 coactivator family, which contains histone acetylase (HAT) activity for chromatin remodeling. Besides these cofactors, many cofactors interact with TR to regulate the expression of target gene precisely (Wu and Koenig, 2000; Xu et al., 1999). Polybrominated diphenyl ethers (PBDEs) have been commonly used as flame retardants, and are becoming ubiquitous environmental contaminants. PBDEs can cross the blood–brain barrier (BBB)
to accumulate in the central nervous system (Naert et al., 2007). PBDEs and hydroxylated PBDE (OH-PBDE) metabolites are antiandrogenic in vitro by acting on, or interfering with, a number of potential targets within the hypothalamic-pituitary-gonadal axis (Stoker et al., 2005; Canto´n et al., 2008, 2007a,b, 2006; Harju et al., 2007; He et al., 2008; Legler, 2008). Furthermore, PBDEs, that include 209 congeners, or their metabolites may affect TH-related function by altering TH or TR function, by binding to TR or other nuclear hormone receptors, or through direct effects on thyroid gland tissue (Costa and Giordano, 2007; Darnerud, 2008; Legler, 2008; Kitamura et al., 2008; Kojima et al., 2009; Li et al., 2010). Previously, we reported that some environmental compounds suppress the TRmediated transcription and this suppression was due to dissociation of TR from TH-response element (TRE). Based on this observation, we developed a screening system, liquid chemiluminescent DNA-pull down assay (LCDPA), to examine the effect of chemicals on TR-TRE binding (Iwasaki et al., 2008). However, since LCDPA is in vitro protein–DNA binding assay, a screening system to examine the effect of developing brain was required. Thus, we developed a primary culture system to examine the effect of environmental chemicals using rat primary cerebellar culture (Ibhazehiebo et al., 2011a,b).
Fig. 1. The effect of T4 on Purkinje cell dendritic arborization (17 days in culture). (A–D) Photomicrographs showing the effect of T4 on Purkinje cell morphology. The control without T4 (A), and +T4 (10 10 M; B), +T4 (10 9 M; C), +T4 (10 8 M; D) were added to the primary cultured cerebellar cells that contain the whole subset of cerebellar cells. T4 was converted to T3, physiologically active form of TH, by deiodenase 2 in astrocyte. Immunocytochemistry was performed using anti-calbindin antibody to visualize Purkinje cells. Bars = 50 mm. (E) Change in dendritic areas of Purkinje cells by T4. Data are expressed as mean SE (n = 10 determinations) from one experiment and represent at least three indepentdent experiments. *p < 0.01 by ANOVA, for T4 (+) compared with T4 ( ).
Y. Xiong et al. / NeuroToxicology 33 (2012) 753–757
2. Materials and methods 2.1. Chemicals Tri-iodothyronine (T3) and thyroxine (T4) were purchased from Sigma Chemical Co. (St. Louis, MO). BDE209 was purchased from AccuStandard Chemicals (New Haven, CT), and was >98% pure. BDE209 was dissolved in absolute ethanol at concentration less than 10 4 M, while T3 and T4 were dissolved in dimethylsulfoxide (DMSO) at 100 mM concentration. Dilutions using culture medium were made from stock solutions immediately before use. Repeated freezing and thawing were avoided. 2.2. Method of screening system based on primary cerebellar culture In this assay, newborn rats were decapitated under direct ether anesthesia on postnatal day 1. The culture protocol has been described by Kimura-Kuroda et al. (2007). Briefly, cerebella were digested and plated in poly-L-lysine-coated wells of chamber slides (8-mm-diameter wells), and cultured overnight in a 5% CO2 incubator. Then, T4 and/or environmental chemicals were added to the culture medium and cultured for 17 days. For TH treatment, T4 was used in primary culture because, physiologically, it is preferentially transported to brain through the BBB, and primary culture contains the whole subset of cerebellar cells, including astrocytes. Thus, T4 was converted to T3, physiologically active form of TH, by deiodenase 2 in astrocyte. Purkinje cells were immunostained with a mouse monoclonal anti-calbindin-28K antibody. The morphological changes were examined under a laser confocal scanning microscope and finally quantified dendritic arborization using NIH Image software (Vischer, 2006).
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nonexistent; whereas those with T4 showed elaborate dendrites characterized by the presence of a main thick primary shaft and several other secondary shafts with bifurcating branches (Fig. 1A– D). T4 augments the Purkinje cell dendritic arborization in a dosedependent manner. Although the dendrite development showed high sensitivity and dependence of the T4 concentration, but the number of Purkinje cells was not always in parallel with the dendritic arborization. 3.2. Effect of PBDEs on Purkinje cell dendritic arborization Using Purkinje cell screening system, we examined the effect of PBDEs. Purkinje cells cultured with BDE209 showed abnormally shaped dendrite, very poor growth and the especially small secondary branches (Fig. 2A); the area of these Purkinje cell dendrites was significantly reduced (Fig. 2B). (Reproduced with permission from Environmental Health Perspectives.) 3.3. Effect of BDE209 on TR-mediated transcription We observed the greatest magnitude of suppression (45%) of TRb1-mediated transcription thought the F2-TRE-LUC by 10 9 M BDE209 (Fig. 3A). BDE209 also suppressed the transcription
2.3. Plasmids Expression vector for TRb1 has been previously described (Iwasaki et al., 2001). The luciferase (LUC) reporter constructs containing TRE, the chick lysozyme (F2)- and artificial direct repeat (DR4)-thymidine kinase (TK)-LUC in the PT109 vector have been described elsewhere (Koibuchi et al., 1999). 2.4. Clonal cell culture and transient transfection-based reporter gene assay CV-1 cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% small lipophilic hormonedeprived fetal bovine serum at 37 8C, 5% CO2. Cells were plated in 24-well plates 48 h before transfection using calcium-phosphate coprecipitation method (Iwasaki et al., 2002). Cytomegalovirus-b-galactosidase plasmid was used as internal control. 16–24 h after transfection, wells were refilled with fresh medium containing the indicated concentrations of ligand for 24 h. Cells were then harvested to measure the LUC activity as described previously (Iwasaki et al., 2002). Total amounts of DNA per well were balanced by adding pcDNA3 plasmids (Invitrogen, San Diego, CA). The LUC activities were normalized to b-galactosidase activity and then calculated as relative LUC activities. All transfection studies were repeated at least three times in triplicate. Data shown represent mean SEM of one experiment. 3. Results 3.1. Rat Purkinje dendritic arborization was induced by thyroid hormone in a dose-dependent manner As shown in Fig. 1, Purkinje cells in the control medium ( T4) showed that the growth of dendrites was poor or almost
Fig. 2. The effect of BDE209 on Purkinje cell dendritic arborization (17 days in culture). (A) Photomicrographs showing the effect of BDE209 on Purkinje cell morphology. BDE209 (10 10 M) was added to the culture in the absence or presence of T4 (10 8 M) and immunocytochemisty was performed using anti-calbindin antibody to visualize Purkinje cells. Bars = 50 mm. (B) Change in dendritic areas of Purkinje cells by T4. Data are expressed as mean SE (n = 10 determinations) from one experiment and represent at least three indepentdent experiments. *p < 0.01 by ANOVA, for T4 (+)/BDE209 ( ) compared with T4 ( )/BDE209 ( ). #p < 0.01 by ANOVA, for T4 (+)/BDE209 (+) compared with T4 (+)/BDE209 ( ).
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Relative luciferase activity
A 5
-T3 +T3
4
*
3
*
*
2 1
F2-TRE CV-1
0 TRβ1
-
+
BDE209
-
-
+
10 -12
+
+
10 -11
+
10 -10
10 -9 (M)
Relative luciferase activity
B 5
-T3 +T3
4 3
*
*
*
2 1
DR4-TRE CV-1
0 TRβ1
-
+
BDE209
-
-
+
10 -12
+
10 -11
+
10 -10
+
are regulated by TH in laboratory animal (Koibuchi et al., 1999; Koibuchi and Chin, 2000). Nuclear TH action is mediated through TR, which increases or decreases the expression of target genes. TRa and b are produced during the development of both Purkinje and granule cells in the cerebellum (Bradley et al., 1989; Strait et al., 1991). Although these TRs are wildly expressed during the cerebellar development and their levels are greater in adult, the expression of many TH-responsive genes is altered by TH status only during early postnatal critical period. Not only the expression levels of TRs but also those of cofactors and other nuclear hormone receptors may play a role in regulating TH sensitivity in the developing cerebellum. To examine the effect of mutant TR in Purkinje cells, we are now investigating the transgenic mouse that specifically expresses mutant TRb1 in Purkinje cells (data not shown). In summary, we established a convenient assay system to screen the effect of neurotoxic chemicals on TH-related function in developing brain. Together with in vitro screening system, these assays provide us with precise information about chemicals on developing brain. We hope our study will provide significant clarifications on the mechanisms of TH-related action in the developing brain. Conflict of interest
10 -9 (M) All authors declare no conflict of interest.
Fig. 3. Suppression of TR-mediated transcription by BDE209. (A and B) Expression plasmids encoding TRb1 (10 ng) were cotransfected with F2-TRE-LUC (100 ng) (A), DR4-TRE-LUC (100 ng) (B) into CV-1 cells. Cells were incubated with or without 10 7 M T3 and indicated amounts of BDE209. Total amounts of DNA for each well were balanced by adding vector pcDNA3. Data are mean SE of experiments performed in triplicate. *p < 0.01 by ANOVA, compared with TRb1 (+), T3 (+), and BDE209 ( ).
Acknowledgments
through the DR4-TRE (45%) at 10 9 M (Fig. 3B). These results are compatible to the result of primary Purkinje cell dendritic arborization.
˜ o, T. Aoki, K. Takata, We thank Drs. J. Kimura-Kuroda, M. London K. Hanamura, Y. Lu, N. Shimokawa and T. Shirao for helpful assistance, and Dr. A. Takeshita for kindly providing plasmids. This project was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT) (17510039, T.I, 17390060, N.K.), and a grant from Ministry of the Environment of Japan (to N.K.).
4. Discussion
References
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