Thyroid hormone- and estrogen receptor interactions with natural ligands and endocrine disruptors in the cerebellum

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Review article

Thyroid hormone- and estrogen receptor interactions with natural ligands and endocrine disruptors in the cerebellum ⁎

Attila Zsarnovszkya,b, , David Kissc, Gergely Jocsakc, Gabor Nemethd, Istvan Tothc, ⁎ Tamas L. Horvatha,b,e, a Department of Animal Physiology and Animal Health, Faculty of Agricultural and Environmental Sciences, Szent István University, Páter Károly u. 1, H-2100 Gödöllő, Hungary b Department of Comparative Medicine, Yale University School of Medicine, New Haven, CT, USA c Departments of Physiology and Biochemistry, University of Veterinary Medicine, Budapest 1078, Hungary d Department of Obstetrics and Gynecology, University of Szeged, School of Medicine, Szeged, Hungary e Departments of Anatomy and Histology, University of Veterinary Medicine, Budapest 1078, Hungary

A R T I C L E I N F O

A B S T R A C T

Classifications: 120: Hormones 150: Neurosteroids 60: Environmental factors

Although the effects of phytoestrogens on brain function is widely unknown, they are often regarded as “natural” and thus as harmless. However, the effects of phytoestrogens or environmental pollutants on brain function is underestimated. Estrogen (17beta-estradiol, E2) and thyroid hormones (THs) play pivotal roles in brain development. In the mature brain, these hormones regulate metabolism on cellular and organismal levels. Thus, E2 and THs do not only regulate the energy metabolism of the entire organism, but simultaneously also regulate important homeostatic parameters of neurons and glia in the CNS. It is, therefore, obvious that the mechanisms through which these hormones exert their effects are pleiotropic and include both intra- and intercellular actions. These hormonal mechanisms are versatile, and the experimental investigation of simultaneous hormoneinduced mechanisms is technically challenging. In addition, the normal physiological settings of metabolic parameters depend on a plethora of interactions of the steroid hormones. In this review, we discuss conceptual and experimental aspects of the gonadal and thyroid hormones as they relate to in vitro models of the cerebellum.

Keywords: Estradiol Thyrod hormone Cerebellum Neuronal and glial culture

1. Introduction Numerous studies provided evidence for the role of 17β-estradiol (estrogen, E2) (Ikeda, 2008; Fan et al., 2010) and thyroid hormones (THs), i.e., triiodothyronine, thyroxine (T3 and T4) (Koibuchi, 2008; Horn and Heuer, 2010) in the regulation of normal cerebellar development. Estrogen, as a traditionally known female reproductive hormone and THs, best known as regulators in energy homeostasis, are unique in that they play key roles in the regulation of several other physiological processes as well. Such E2-TH regulated processes are

neuronal/glial maturation (also involved in other somatic cells), cell migration (Kirby et al., 2004; Belcher et al., 2009), and the regulation of the intracellular metabolism, latter which significantly affects most intracellular events on its own. The listed hormonal regulatory effects are mediated by at least three known major mechanisms: 1. Specific (cognate) receptors (E2-, TH receptors, ERs and TRs) that function as transcription factors when activated by bound hormone ligands (generally considered as genomic effects) (Ikeda, 2008; Fan et al., 2010; Jakab et al., 2001; Belcher and Zsarnovszky, 2001); 2. Putative plasma membrane-bound/incorporated ligand-receptor complexes that

Abbreviations: AgRP, agouti-related protein; AMPK, adenosin monophosphate activated protein kinase; AN, arcuate nucleus; As, arsenic; AraC, cytosine β-D-arabinofuranoside; BDNF, brain derived neurotrophic factor; BPA, bisphenol A; Ca, calcium; cDNA, complementary deoxyribonucleic acid; CNS, central nervous system; D1-3, deiodinase type I-III; db, genetically diabetic mouse; DNA, deoxyribonucleic acid; E2, 17β-estradiol; EDTA, ethylenediaminetetraacetic acid; EGTA, ethylene glycol tetraacetic acid; ER, estrogen receptor; GDX, gonadectomy; GFAP, glial fibrillary acidic protein; Glia+, Glia containing; Glia−, Glia reduced; GLUT, glucose transporter; GnRH, gonadotropin releasing hormone; HRE, hormone response element; IGF, insulin-like growth factor; IgG, immunoglobulin G; LHN, lateral hypothalamic nucleus; MAPK, mitogen-activated protein kinase; MBC, 4-methylbenzylidene camphor; ME, median eminence; mRNA, messenger ribonucleic acid; NEB, negative energy balance; NPY, neuropeptide Y; ntC, non-treated controls; OT, oxytocin; P0-14, postnatal day 0-14; PMSF, phenylmethanesulfonylfluoride or phenylmethylsulfonyl fluoride; POMC, pro-opiomelanocortin; PPE, preproenkephalin; PVN, paraventricular nucleus; qPCR, quantitative Polymerase chain reaction; rT3, reverse triiodothyronine; SCN, suprachiasmatic nucleus; SON, suptraoptic nucleus; STAT, signal transducer and activator of transcription; T2, diiodothyronine; T3, triiodothyronine; T4, thyroxine; TG, thyroid gland; TH, thyroid hormone; TR, thyroid hormone receptor; TRH, thyreotrop releasing hormone; TSH, thyroid-stimulating hormone; UCP2, uncoupling protein 2; VMH, ventromedial hypothalamus; VMN, ventromedial nucleus ⁎ Corresponding authors at: Department of Animal Physiology and Animal Health, Faculty of Agricultural and Environmental Sciences, Szent István University, Páter Károly u. 1, H2100 Gödöllő, Hungary. E-mail addresses: [email protected] (A. Zsarnovszky), [email protected] (T.L. Horvath). http://dx.doi.org/10.1016/j.yfrne.2017.10.001 Received 11 May 2017; Received in revised form 6 September 2017; Accepted 4 October 2017 0091-3022/ © 2017 Elsevier Inc. All rights reserved.

Please cite this article as: Zsarnovszky, A., Frontiers in Neuroendocrinology (2017), http://dx.doi.org/10.1016/j.yfrne.2017.10.001

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through which E2 mediates its effects. For example, numerous studies indicate that changes in the expression or activity of E2-metabolizing enzymes or changes in the levels of E2-binding serum proteins can influence the rate of E2 metabolism resulting in alterations in the availability of free E2 at target tissues (Rosselli et al., 2000). This structural and mechanistic diversity of different estrogenic compounds suggests that E2 normally influences multiple mechanisms (i.e., genomic and rapid nongenomic signaling mechanisms) whose sum-effects result in a cell-type specific E2 responsive phenotype. EDs, such as phytoestrogens or environmental pollutants (also some mycotoxins, such as zearalenone) are selective ER and/or TR modulators and can act as agonists or antagonists of the hormones in question. During development, EDs can influence normal hormonal homeostasis and lead to immediate and/or life-long consequences (e.g., Zsarnovszky et al., 2007; Miodovnik et al., 2014). Here, we discuss effects of some frequently studied EDs, specifically bisphenol A (BPA), zearalenone (Zea), arsenic (As) and methyl-benzilidene-camphor (MBC) on TRα,β and ERβ in cells seeded into cell cultures and obtained from the cerebella of newborn rat pups. It should be noted at this point that this frequently deployed in vitro experimental system carries some advantages that make the primary cerebellar cell culture an excellent choice for the investigation of neurocellular effects: the absence of aromatase activity and serum-free medium allows full control over experimental estrogenic (or other steroidogenic) effects; the expression of both ERs and TRs allows for the investigation of substrate effects on these receptors, although effects on ERα cannot be reliably considered due to its involvement in brain reparation processes; inhibition of the growth of glia in this experimental system allows for the investigation of glial effects. Thus, results from this experimental model may not only apply for the cerebellum, but can also be suggestive to other basic celular processes in the CNS.

activate rapid, non-genomic intracellular signaling cascades (e.g., Pekary et al., 2006; Belcher, 2008; Leonard, 2008); and 3. Crosstalk on multiple levels of genomic and/or non-genomic E2- and TH-activated intracellular signaling pathways (Vasudevan et al., 2001a; Zhao et al., 2005), where hormone effects are evident but the exact role of the ligand alone or ligand-receptor complex is not yet clarified. Thus, the numerous trophic effects of E2 and THs that are mediated by ERα,β and TRα,β, are the result of the two hormone’s interactive effects on the expression level of each-other’s receptors, thereby modulating intracellular mechanisms that depend on the receptor’s signal-mediating functions. ERs (Shughrue et al., 1997) and TRs (Murray et al., 1988; Hodin et al., 1989) are widespread in the brain, however, their expression level depends on the brain region, age (Bernal, 2007; Al-Bader et al., 2008) and functional-hormonal status of the organism. Thus, it is not known, how ER-TR receptor expression levels correlate with realtime hormonal conditions and to what extent ER-TR gene transcriptional activity correlates to ER-TR protein synthesis in the developing cerebellum. Environmental estrogens are a large and structurally diverse group of compounds that can mimic, and in some cases antagonize the effects of endogenous estrogens and THs, and they are therefore often referred to as endocrine disruptors (EDs). As a result of their estrogen-like activities and the potential for some to block the normal actions of endogenous E2, there is currently much debate within the scientific community, and also considerable interest within the general public, regarding the relative benefits or threats associated with exposure to environmental estrogens and endocrine disruptors in general. Although most of the EDs mentioned herein are referred to as estrogenic compounds, it has recently been generally accepted that many of them can also disrupt thyroid actions as well. Compounds characterized as having estrogenic or thyroid properties are typically divided into three general categories, the xenoestrogens, the phytoestrogens and the mycoestrogens. Xenoestrogens are a diverse group of synthetic compounds that include pesticides, the widespread industrial pollutants poly-chlorinated biphenyls; bisphenol-A, the synthetic estrogen, diethylstilbesterol; and many others. As a result of their negative actions on reproductive tissues, xenoestrogens are a potential threat to wildlife and human populations and are therefore the subject of much active research. The phytoestrogens and mycoestrogens are a group of naturally occurring compounds with estrogenic (and thyroid) activity that are present in plants or that arise from bacterial or fungal metabolism of plant precursor compounds. To varying degrees, phytoestrogens (and mycoestrogens) can also act as agonists or antagonists of the normal actions of E2, and in adults they may have protective effects against certain forms of cancer, cardiovascular disease, and osteoporosis and may also prevent undesirable menopausal symptoms (Bingham et al., 1998). As a result of these potentially beneficial effects, phytoestrogens, especially soy isoflavones have increasingly gained widespread acceptance as safe and beneficial dietary components and as a “natural” alternative to estrogen-based hormone replacement therapies. This increased use of phytoestrogens has occurred even though their mechanisms of action and their effects (either positive or negative) on the developing and mature brain are not well understood. It is also of interest to note that the ready acceptance of the safety and the benefits associated with exposures to increased concentrations of the “natural” estrogenic compounds by the general public and the medical community is in sharp contrast to the common (and potentially accurate) perception that the actions of xenoestrogens are a threat to the health and well-being of human and wildlife populations. Some phytoestrogens have obvious structural similarity with E2 and are typically considered to act as E2 mimetics; however, many compounds characterized as having estrogen-like properties have few obvious structural similarities to E2 (this is especially true with regard to xenoestrogens and also arsenic, for example). It is also likely that some estrogenic compounds act through mechanisms unrelated to those

1.1. Bisphenol A (BPA) BPA is produced in large amounts for use as a monomer in the production of polycarbonate plastics and epoxide resins that are used as coatings for food cans and plastic packaging, dental sealants, and water pipes. As a result, there is extensive human exposure to BPA that has been estimated to range from 2 to 20 µg/kg/day (vom Saal et al., 1998). During pregnancy, BPA is detectable in maternal (0.3–18.9 ng/ml) and fetal (0.2–9.2 ng/ml) serum, demonstrating ready passage of BPA through the placenta (Schonfelder et al., 2002). Compared with other tissues, BPA is concentrated approximately 5-fold in amniotic fluid during early pregnancy, further indicating significant fetal exposure during important periods of human development (Ikezuki et al., 2002). The levels of BPA to which adult and fetal humans are exposed negatively impacts reproductive function, neurodevelopment, and hippocampal synapse formation in rodents (Palanza et al., 2002; Takai et al., 2000, 2001; Honma et al., 2002; MacLusky et al., 2005). With regard to ED effects in the cerebellum, it was previously shown that BPA can rapidly activate ERK1/2 in primary cerebellar granule cell cultures (Wong et al., 2003) and also, after injection of BPA into the cerebella of newborn rat pups (Zsarnovszky et al., 2005). These effects were dose-dependent, with a U-shaped dose-response curve, which could indicate compound actions of BPA. In support of these findings, Mathisen et al. (2013) described that perinatal BPA exposure increased Pax6 (transcription factor playing a role in granule cell development and migration) in newborn mice cerebella and in cerebellar cell cultures. In the hippocampus, BPA modulated dendritic morphogenesis via effects on ER. Likewise, BPA also promoted dendritic growth in maturing cerebellar Purkinje cells (Shikimi et al., 2004). This is consonant with previous results from our laboratory (Wong et al., 2003; Zsarnovszky et al., 2005). In addition to interactions between BPA and ERs, BPA can alter thyroid-specific gene expression (Gentilcore et al., 2013) and functions (Iwamuro et al., 2006; Delfosse et al., 2014). Our studies indicated that 2

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affected ERα, but not ERβ expression in breast carcinoma cells. Although information on As effects on ERβ is scarce, it appears that these As effects are tissue dependent.

the ratio of THs to E2 in the CNS is critical for the regulation of nuclear receptor expression (Scalise et al., 2012). While a growing body of evidence indicates that EDs, including BPA, interfere with CNS development, the exact mode of BPA action, and how it alters TR expression levels currently is not clear. Recently we showed that BPA alters TRα,β and ERβ mRNA and protein expression levels in primary cerebellar cell cultures. We also unmasled the outcome of combined treatments with the various hormones and revealed a role for glial cells in these processes.

1.4. Methyl-benzilidene-camphor (MBC) Camphor (4-methylbenzylidene camphor, MBC) is a natural compound in some tree species, rosemary leaves, etc., and it is also synthetically produced by the industry. It is used for its scent, as UV filters, as an ingredient in cooking, as an embalming fluid, for medicinal purposes, and in religious ceremonies. MBC, as an ED compound, affects the estrogenic and thyroid pathways as well (Schmutzler et al., 2004; Schlumpf et al., 2004a; Seidlová-Wuttke et al., 2006; Boas et al., 2012), but the exact mechanism of action is unknown. For example, a previous study demonstrated that MBC showed no binding to ERβ in human endometrial cells, nevertheless it affected ER mediated events (Mueller et al., 2003). Interestingly, however, Schlumpf et al. (2004a) found that MBC displaced 16-alpha-125-l-estradiol from ERβ, but not from ERα. Further studies also clarified that intrauterine exposure to MBC alters mRNA expression for both ERα and ERβ in uterine cells of the offspring (Durrer et al., 2005). A more detailed study by Schlumpf et al. (2004b) revealed that MBC exposure during the gestation affects ERα, ERβ, and progesterone receptor mRNA expression in various tissues, tissue-dependently, including the hypothalamus. MBC effects on the developing CNS is very little known; moreover, the relevant studies mostly focus on the hypothalamus-pituitary-gonad axis and the estrogen-related developmental changes during young ages (SeidlováWuttke et al., 2006; Maerkel et al., 2007; Carou et al., 2009).

1.2. Zearalenone (Zea) Zearalenone (Zea), also known as F-2 mycotoxin, is a product of fusarium molds; Zea is heat-stable and is found worldwide in a number of cereal crops, such as maize, barley, oats, wheat, rice, and sorghum, and also in bread. To the best of our knowledge, there is no available information on the effects of Zea on cerebellar cells. There are, however, several studies indicating that Zea stimulates cell proliferation in tumors of the female reproductive tract and breast malignancies in humans (Withanage et al., 2001; Pillay et al., 2002; Parveen et al., 2009). Neural cells are also obviously affected as shown in some sporadic reports. Zea clearly changes the expression of substances in the nerve fibers of the gastrointestinal tract such as vasoactive intestinal peptide, neuronal form of nitric oxide synthase, cocaine and amphetamine regulatory peptide, galanin, pituitary adenylate cyclase-activating peptide-27, and substance P (Gonkowski et al., 2015). Examining ovariectomized rats, Turcotte et al. (2005) also reported that 2 mg of Zea injected subcutaneously induced the expression of neuronal progestin receptors comparable to that of 10 µg E2, and altered their sexual behavior, stimulating sexual receptivity. Early studies suggested that the biological effects of Zea may be due to its ability to bind to intracellular estrogen binding sites (Greenman et al., 1979; Takemura et al., 2007) Indeed, reporter gene assays demonstrated that Zea and its metabolites α and β-zearalenol exhibit just slightly less strong estrogenic potency than 17-β estradiol itself (Frizzell et al., 2011). Further study indicated that in the liver, Zea could also bind to cellular proteins distinct from estrogen receptors (Mastri et al., 1985). Kuiper et al. (1998) found that Zea can bind to ERβ and can increase ERβ transcription.

1.5. The cerebellum in brief The cerebellum is the organ of coordination and learning of locomotion. The two main functions of the cerebellum are: 1. Correction and refinement of the movement plan comparing the central command and the actual peripheral movement; 2. Refining of movements during ontogenesis. Complex and complicated neural networks are formed supporting the complex tasks forwarded to the locomotor system. The cerebellum plays a crucial role in learning and storing complex movement plans. The rich afferentation is integrated by one cell type, namely the Purkinje cells and their efferents reach directly or indirectly the particular nuclei of the brain stem and the motor cortex. There is no direct efferent connection to the spinal cord, the cerebellum directs muscle function by indirectly influencing cortical movement patterns. The functional unit of the cerebellum – the cerebellar 'module' – is philogenetically very conservative, containing repetitive cytoarchitechtonical cell groups showing similarities in all three subdivisions of the cortex. The most important cell types involved in the module are: 1. GABAerg inhibitory cells – the Purkinje cell (the only cell type whose axons leave the cerebellum to the Deiters nucleus), stellate and basket cells. 2. glutamatergic excitatory cells and axons: granule cells, climbing and mossy fibers. The afferentation reaches the cortex in the form of climbing and mossy fibers after giving collaterals to the deep nuclei (nucleus Fastigii, interpositus [globosus et emboliformis] nuclei and dentate nucleus) transporting to them the same information. The climbing fibers (coming from the inferior oliva) directly connect the Purkinje cells that are of central importance in the cerebellum. The mossy fibers (from the spinal chord and the brain stem) are forming synapses with granule neurons that, in turn, are connected to the Purkinje cells in an up to several millimeter-wide zone parallelly with the surface. One of the two main afferents, the mossy fibers (continuing mainly the spinocerebellar tracts or afferents from the brain stem) give branches to the deep nuclei and the granule neurons. These cells are the most important cortical (glutamatergic) facilitating neurons. Their axons, running parallel with the surface of the cortex, ensure the connection between the cerebellar foliae. The parallel fibers of all the

1.3. Arsenic (As) Arsenic is present in ground water and food, however, its concentration as a ground water contaminant may considerably vary according to the geographical area examined. Arsenic exposure leads to a multitude of neurodevelopmental dysfunctions (Rodríguez-Barranco et al., 2013). In children, cognitive function could irreversibly decline (Rocha-Amador et al., 2007) and deleterious effects cause deficits in verbal and performance domains (Calderón et al., 2001; Wasserman et al., 2011). There are a number of reports describing neurotoxic effects of As on rodent pups resulting in severe decrease of locomotor activity and behavioral disorders (Rodríguez et al., 2001). Most studies, however, do not detail cellular and subcellular consequences that would manifest in the above phenomena. Arsenic effects include increased lipid peroxidation leading to damages in plasma membrane and the intracellular membrane system. Furthermore, As inhibits elimination of free radicals, interferes in methylation reactions and in coupling with thiol groups, inducing DNA and protein damage (Sundari et al., 1997; Zhong and Mass, 2001). It was Bodwell et al. (2004) who first reported that As can act as ED on receptors for progesterone, androgen and corticoids in a dose-dependent manner and enhance hormone-dependent gene transcription even at very low doses (Bodwell et al., 2006). ERs were involved in these examinations later on, however demonstrating highly varying effects of As on ER expression. For example, Cimino-Reale et al. (2008) reported that As increased ERβ expression in bone marrow cells. In contrast, Chen et al. (2002) found that As only 3

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observation suggests that loss of tissue integrity generates a need for more E2 and TH action in the cultured cells in order for the cells to adapt to their new environment and to maintain the highest possible cell vitality (Duenas et al., 1996; Zhu et al., 2004; Lamirand et al., 2008; Spence et al., 2009; Shulga et al., 2009; Mirzatoni et al., 2010). In addition, it should be considered that the cultured cells were obtained from immature, developing cerebella; therefore, maintenance of gene activation necessary for the completion of genetically programmed developmental processes may also require higher transcriptional activity of ligand bound TRs and ERs under the experimental conditions that we used. Interestingly, characteristic differences in the patterns of TRα,β mRNA[Glia+] vs TRα,β mRNA[Glia−] and protein[Glia+] vs. protein[Glia−] suggest that glial cells play key roles in the regulation of neuronal TRα,β protein biosynthesis, as higher than normal TRα,β mRNA expression levels accompanied lower than normal TRα,β protein levels in the absence of glia. One of the possible glial functions might be the mediation of molecular signals towards granule neurons that may be necessary links between the transcription and translation of TR genes. It should be noted, however, that the cell density in our cultures was set to minimize physical cellular contact. Therefore, all interactions in intercellular signaling observed could be accounted for by predominantly paracrine signaling, thereby supporting previous observations that paracrine signaling can activate gene expression in the brain of rodents or humans as well (Freitas et al., 2010). End-products, or socalled key-proteins, of protein biosyntheses usually play feedback roles to down-regulate their own biosynthesis. In this respect, it is also possible that glial cells play a role in the aforementioned negative feedback signaling, therefore, the lack of glia may result in flawed feedback in receptor biosyntheses and thus, imbalanced transcriptional and translational activities, leading to overt mRNA synthesis and insufficient protein production in glia-reduced neuronal populations. With regard to ERβ, increased mRNA[Glia+/−] levels were also accompanied by increased ERβ protein[Glia+/−] levels, regardless of the presence or absence of glia. This observation indicates that balancing the transcription and translation of ERβ gene is not, or is less dependent on glial contribution than that of the TR genes.

granule cells are stimulating a particular 'strip' of Purkinje neurons. The activation of these Purkinje cells means an increased inhibition for the deep nuclei. The neighboring Purkinje cells, which are not activated by the given granule cell fibers, are inhibited by the basket and stellate cells. The second afferent type of fibers reaching the cerebellum is called “climbing fibers” (the soma of these cells are located in the inferior oliva nucleus). These fibers convey the same information to the cerebellar deep nuclei and the Purkinje cells. Climbing fibers are directly connected to the Purkinje cells, facilitating them by glutamatergic mechanism. The first element of the cerebellar efferent system is given by the Purkinje cells that are inhibitory neurons, and they inhibit the firing of the deep nuclei. These nuclei can be released from this inhibition leading to increased mean discharge frequency of the efferents originating here. In essence, the incoming original afferent information and the same information after processing in the cortex are integrated in the deep nuclei. The main efferentation center of the cerebellum is the area of cerebellar deep nuclei: 1. nucl. Fastigii 2. interpositus (globulosus and emboliformis) nuclei 3. dentate nucleus. These nuclei receive the direct afferentation, and then the refined version of the movement pattern sent by the Purkinje cells, processed by the cerebellar cortex. The fibers originating here are not going to the spinal chord; instead they project to cerebral centers of the movement control: through the thalamus they reach the motor cortex, the nuclei of the brain stem and the nucleus Deiters. (The efferent information leaving the cerebellum in a small part is using axons of Purkinje cells directly reaching the Deiters nucleus.) Both the afferent and the efferent pathways depend on the approached cerebellar subdivision (vestibulo-, spino- or cerebrocerebellum), which the cerebellar “module” belongs to. 1.6. The primary cerebellar granule cell culture as model The primary cerebellar granule cell culture has been extensively used over the past decade for the study of cellular responses to various experimental cues. The methodology used for the discussion of the major data in this review was based on the work of Wong et al. (2001), however, minor modifications were made in order to minimize physical cellular contacts as described by Scalise et al. (2012). As a result, the model allowed for the investigation of cellular and intercellular actions (mostly via paracrine intercellular signaling), however, neither direct cell-to-cell adherence nor synaptic contacts were allowed to develop in the cultures. Therefore, while the in vitro model in the focus of the review is originated from the cerebellum, the model’s attributes allow for the formulation of more general conclusions that may apply for the entire neural system. Unfortunately, on the other hand, ED effects in the cerebellum and the entire CNS are extremely little known, despite of the large body of literature available on ED effects in the CNS. Thus, results from this or such an experimental model should be handled with care and it should be kept in mind that extrapolation of the results from any in vitro systems may not be directly relevant to the clinical practice, as a direct and clearcut conclusion from those (including the reviewed) experimental results would be misleading, and should be only taken as suggestive data. Selective inhibition of glial proliferation after cell seeding allowed for the investigation of processes mediated by the glia. We compared endocrine effects in glia-containing versus glia-reduced cultures. Differences between these groups was clearly the result of the presence or “absence” of glia, since in Glia− cultures granule neurons extremely outnumber sporadic and rudimentary glial cells and, therefore, it seems to be safe to interpret the treatment effects in Glia− as if they were only exerted by neurons. In the reference studies (E2 and TH treatments, please see Scalise et al., 2012), the applied experimental conditions lead to increases in mRNA expression levels compared to respective in situ samples. This

2. Reference studies: Hormonal effects on TRs and ERβ The hormone products of the thyroid gland have been known as thyroid hormones (THs). Today, the source and function of these hormones comprizes a great deal of knowledge, allowing for a remarkably complex view on the physiological roles of THs as reviewed by Somogyi et al. (2011). There are various types of THs: thyroxine (T4) containing four iodine atoms is generally considered to be a prohormone, while triiodothyronine (T3), containing three iodine atoms, is biologically more active than T4. These two hormones are inactivated by being converted into reverse triiodothyronine (rT3) or diiodothyronine (T2). Thyroid hormones act through binding to their cognate receptors (thyroid hormone receptors, TRs). TRs are nuclear receptors that can stimulate ligand-dependent transcription of certain genes (Sap et al., 1990; Brent et al., 1991). In mammals, there are four TR isoforms, TRα 1–2 and TRβ 1–2, encoded by two highly conserved genes (Thra and Thrb), respectively (Shi et al., 1992; Zhang and Lazar, 2000). TRα2 has not been found in non-mammalian species (Koenig et al., 1989). Unlike the other TRs, TRα2 acts as a week repressor instead of functioning as a receptor of T3 (Salto et al., 2001). TRs have both triiodothyronine (T3)-dependent and -independent functions. Interestingly, T3 can convert a T3-independent repressor into a T3-dependent activator (Chen and Evans, 1995). The distribution pattern of Thra and Thrb is age- and tissue-dependent. While TRα1 is widely expressed, TRβ 1–2 mRNA expression is restricted to specific ontogenetic states and is highly tissue specific (Bradley et al., 1994). Traditionally, estrogens (mostly 17β-estradiol, E2) have been considered as sex hormones, and indeed, many of their direct actions are 4

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levels were significantly increased compared to those in situ. This is consistent with previous findings that hypothyroidism leads to an increase in TR expression levels (Chattopadhyay et al., 1995; Gereben et al., 1998), although in our experimental model this was only reflected in TRα,β mRNA expression, but not in receptor protein levels. The increase in TRα mRNA expression might also be the result of compensatory mechanism(s) to ameliorate the reduction of nutrient substrates in the culture compared to in situ conditions. Increased TRα levels in CNS cells after ischemic conditions support this idea (Zhu et al., 2004). Additionally/alternatively, the observed increase in transcriptional activity may also reflect a regenerative action on the part of explanted cells (Panaite and Barakat-Walter, 2010). When cultured cells were deprived of THs (E2 treatment only), not even increased transcriptional activity could maintain in situ levels of TRα protein production. Comparison of similarly treated subgroups of Glia+/Glia− samples revealed that non-treated controls do not differ significantly from each other in their TRα protein levels. This phenomenon, however, was backed by significantly different respective ntC[Glia−] vs. ntC[Glia+] mRNA expression values. Therefore, it is reasonable to conclude that granule neurons possess a high degree of adaptability on (TRα) transcriptional level to compensate for the lack of glia and resulting lack of possible neuron-glia interactions. These findings suggest that the clear overall glial effect on TRα protein production is dependent on the presence of the hormones used. The overall glial effect was not seen with respect to TH-deprivation (E2[Glia+]), as TRα protein expression values for both E2[Glia+/−] subgroups were equally and significantly lower compared to in situ levels. This observation implies that with respect to TRα protein expression, effects of THs, but not those of E2, are conveyed by glial cells. In turn, this phenomenon may also indicate that when the glia is present, cerebellar TRα protein expression depends on the presence of either of the used THs, but not on E2. Cerebellar glial cells are able to present T3 to granule neurons due to their T4 to T3 conversion by type 2 deiodinase activity (Guadano-Ferraz et al., 1997). Further, excess amounts or unnecessary T3 in neurons is deactivated by type 3 deiodinase (Escamez et al., 1999; Peeters et al., 2001). In order to prevent neuronal T3 deactivation before its physiological role is accomplished and to protect neurons, glial cells can inhibit neuronal type 3 deiodinase activity (Lamirand et al., 2008). This interactive mechanism between the glia and neurons might be the reason why TRα,β[Glia+/−] protein expressions tended to be at near similar levels. It is also possible, however, that the glial contribution to the maintenance of normal levels of TRα protein levels is not dependent on glial type 2 deiodinase activity, but rather, on some other glial signaling mechanism. Deprivation of cultured neurons from both E2 and THs or from E2 alone (ntC[Glia+]) does not lead to a significant change in TRα protein expression, although mean values were decreased. If glia growth was blocked (ntC[Glia−]), the latter decrease reached significance. This significant decrease in TRα protein levels observed in all Glia− samples was accompanied by remarkably high mRNA expression levels (compared to respective in situ samples), where there was a significant heterogeneity between TRα mRNA[Glia−] groups. The aforementioned, treatment- (ligand-) dependent heterogeneity in mRNA levels might demonstrate how granule neurons attempt to maintain a subnormal but steady expression level of TRα proteins with the lack of glial contribution. Specifically, results from TRα mRNA[Glia−] samples suggest that the most TRα mRNA is required when no receptor ligand is present (ntC[Glia−]), while, considering the mean values, deprivation of neurons from either T4 or E2 (T3[Glia−] group) resulted in a state with the second highest TRα transcriptional activity. In contrast, mean values of E2/T4/E2 + T3/E2 + T4[Glia−] samples were closer to those in situ, suggesting that the potency of T3 to induce TRα mRNA synthesis differs from that of the other hormones used. Altogether, mean values of TRα mRNA[Glia−] groups may indicate that in the absence of glia, TRα transcriptional activity may depend more on E2 than on THs. Additionally, the highly variable mean and SEM values also suggest that

17 1 -estradiol

Triiodothyronine T

TThyroxine

b bisphenol A

zzearalenone

4-methylbenzylidene camphor 4 Fig. 1. Molecular structure of 17β-estradiol (E2), bisphenol A (BPA), zearalenone (Zea) and 4-methylbenzylidene camphor (MBC).

exerted on reproductive organs and tissues. Estrogens are primarily synthesized within the developing follicles of the ovaries and exert an influence on the female reproductive system; however, other sites of biosynthesis are present throughout the body, including the adipose tissue and the brain itself. The functions of E2 were originally thought to be mediated through E2′s classical intracellular/nuclear receptors (ERs), ERα and ERβ, which act as transcription factors, binding to the estrogen responsive elements (ERE) of respective genes. An interesting mechanism of estrogenic effects is the synaptogenic influence of E2 on neuronal connectivity. The goal of the reference studies discussed below was to assess the possible regulatory effects of THs and E2 on the expression (both on transcription and translation) of their own and each other’s receptors in order to establish the basis of comparison to further ED effects on these physiological mechanisms (see Figs. 1 and 2).

2.1. Effects of E2 and THs on TRα Results from the Glia+ subgroup show that loss of tissue integrity, on its own, does not alter the expression level of TRα proteins, with the only exception of the E2[Glia+] group. The maintenance of normal levels of TRα protein, however, seems to require higher than normal transcriptional activity, as relevant TRα mRNA[Glia+] expression 5

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Fig. 2. Summarizes the experimental groups and treatments that gave the basis of this review.

mRNA and protein expression, regardless of the presence or absence of glia. It is speculated that such an increase in ERβ (mRNA and protein) might be a reparative/regenerative response on the part of cerebellar cells, and highlights the potential role of ERβ in the regulation of neuronal viability (Lee and McEwen, 2001; Wong et al., 2003). With regard to ERβ mRNA expression pattern, there are many similarities between ligand-induced TR and ERβ transcriptional activities. For example, Glia+ values were closer to each other than those in Glia− samples. There were clear differences in both the magnitude and ligand-dependent variances between respective Glia+ and Glia− groups, which indicates the apparent effect of glia in the regulation of neuronal ERβ gene transcription. On the other hand, granule neurons seem to possess a great degree of adaptability in their ERβ transcriptional activity so as to set a final ERβ protein level adequate to the experimental conditions applied. An interesting observation is that, while Glia+ vs. Glia− ERβ mRNA values displayed a considerably different pattern and therefore suggest obvious glial effects in the regulation of ERβ gene transcription, Glia+ protein expression values were much less different from those found in Glia− ERβ protein groups. This finding may suggest that the neuronal adaptability mentioned above could compensate for the absence of glial contribution. This idea raises the question of what is the exact role of glia in the regulation of neuronal ERβ (and TRα,β) expression, if ERβ (and TRα,β) protein expression values do not show (when comparing treatment groups within given Glia+ and/or Glia− goups) such an apparent regulatory role for the glia. ERβ has been identified in both neurons and glia of the developing cerebellum (Jakab et al., 2001; Price and Handa, 2000). Therefore, it is reasonable to assume that mechanisms that set ERβ expression levels and that are adequate to a given environment are available in both cell types. This assumption does not rule out the possibility of glia-neuron interaction (s), when the glia is present, to synchronize their ERβ biosynthetic activity for optimal adjustment to conditions of this present experimentation.

neurons that lack the glial contribution, possess a high degree of adaptability in TRα,β transcriptional levels to maintain a realtively steady (and lower than normal) level of TRα,β protein expression. 2.2. Effects of E2 and THs on TRβ In Glia+/− non-treated controls, TRβ protein expression levels fell significantly as compared to in situ values. Based on this observation, one may speculate that loss of tissue integrity leads to a decrease in TRβ protein expression. However, in the presence of glia (Glia+), loss of tissue integrity alone does not lead to a decrease in TRβ protein expression, as removal of both E2 and THs was necessary to reach the aforementioned significant decrease; addition of any or both of these hormones prevented a loss in TRβ protein expression. In contrast, all subgroups deprived from glia (Glia−) displayed TRβ protein levels significantly lower than those detected in situ, regardless of the presence or absence of physiological amounts of E2 and/or THs. Therefore, it appears that the hormonal effects on the maintenance of normal TRβ protein expression levels are mediated by glial cells. This idea is supported by the finding that analogously treated counterparts (all Glia− groups) displayed significantly lower levels of TRβ protein. Interestingly, in the presence of glia (Glia+ groups), only THs maintained TRβ protein expression values at in situ levels so as to significantly differ from the non-treated control of the Glia+ group; nevertheless, all other treatment subgroups showed higher TRβ protein expression values than their Glia+ non-treated control. Comparison of E2[Glia−/+] subgroups indicated that the glia mediates both E2 and TH effects: deprivation of cultures from THs resulted in lower TRβ protein expression when the glia is absent, however, a glia-mediated E2 effect was also detected, as E2, T3, T4, E2 + T3[Glia+] values were significantly different from those in the glia-reduced samples. As in the case of TRα, measured TRβ protein levels were backed by significantly higher than normal mRNAs, suggesting that there may be compensatory mechanism(s) in cultured cells on a transcriptional level to maintain the TRβ protein expressions observed. Although there is no distinction between TRα and TRβ isoforms here, other researchers do make a distinction between TRα and TRβ isoforms since TRα and TRβ are derived from separate genes (Lazar, 1993; Oppenheimer et al., 1995; Vasudevan and Pfaff, 2007); this may explain the relatively minor differences detected between TRα and TRβ expression patterns (both transciptional and translational levels). It is perplexing that T3 in the TRα and TRβ and T4 in the ERβ mRNA expression would show increased fold changes in Glia− groups. However, previous work may offer some explanation (Zhao et al., 2005; Vasudevan and Pfaff, 2007), proposing that E2 and THs (T3, T4) may act synergistically and are subject to both genomic and nongenomic mechanisms characteristic of members of the nuclear family of steroid receptors (Evans, 1988).

3. Possible mechanisms of ligand-dependent ER-TR interactions Although early reports are controversial about the presence of TRs in astrocytes of the rat brain (Carlson et al., 1994), the majority of the relevant studies reported that both ERs and TRs are expressed in cerebellar glia (Kolodny et al., 1985; Ortiz-Caro et al., 1986; Hubank et al., 1990; Lebel et al., 1993; Leonard et al., 1994; Carlson et al., 1996) and neurons (Wallis et al., 2010) as well. Thus, both cell types are direct targets of both E2 and THs. Therefore, hormonal and receptorial interactions are possibly intracellular, as well as intercellular via interactions between glial cells and neurons. ERs and TRs bind to hormone response elements (EREs and THREs), present in the promoter region of certain genes, as either homo- or heterodimers. Lee et al. (1998) suggested that ERβ can bind to TRs and form ER-TR heterodimers in yeast and mammalian two-hybrid tests. In addition, Zhu et al. (1996) pointed to the existence of an identical half-site at the hormone response elements, possibly shared by ERs and TRs. The latter observation suggests that ERs and TRs may compete for binding to their promoter binding sites and, although both Lee’s and Zhu’s results were obtained from

2.3. Effects of E2 and THs on ERβ The most salient observation was the overall increase in ERβ mRNA and protein expression levels in cultures compared to in situ values. This finding suggests that loss of tissue integrity induces an increase in ERβ 6

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cultured and transfected cells, these findings altogether implicate the possibility of a high degree of co-operation between certain functions of E2 and THs. Such a possibility is supported by a number of other studies as well (Vasudevan et al., 2001a, 2001b, 2001c; Vasudevan et al., 2002a, 2002b; Vasudevan and Pfaff, 2005). As mentioned above, one of the likely mechanisms of hormonalreceptorial interactions involve hormonal signaling through specific receptors, i.e., ERs and TRs, that are transcription factors and, thus, are able to activate genes that possess estrogen- and/or thyroid hormone responsive elements (EREs, THREs). Since TH actions through TRs can modulate E2-induced transcription from EREs in neuroblastoma cells (Zhao et al., 2005), it is possible that such mechanisms also function in neurons and glia of the developing cerebellum. Moreover, the findings of Zhao et al. (2005) also suggest a more complex interplay between the two hormones, as the aforementioned genomic interaction was mediated by mitogen-activated protein kinase (MAPK) activation, and other studies report that both E2- and THs can activate the MAPK pathway (Wong et al., 2003; Zsarnovszky et al., 2005; Ghosh et al., 2005; Lin et al., 2009). Interestingly, TH-dependent MAPK-activation can also lead to ER phosphorylation (Tang et al., 2004). The MAPK pathway is but one of the rapid, non-genomic intracellular signaling mechanisms that represent potential crosstalk between E2 and TH signaling (a broad review of the molecular mechanisms of crosstalk between E2 and THS was provided by Vasudevan and Pfaff, 2005). For example, brain derived neurotrophic factor (BDNF) can also be activated by both hormones (Koibuchi et al., 1999; Sasahara et al., 2007). The cited literature also indicates that E2- and/or TH-induced MAPK- or BDNF activation mediates developmental signals. Since activation of genes involved in the regulation of developmental processes is sequential and follows a well defined temporal pattern (Wechsler-Reya, 2003), the exact roles of the two hormones in the regulation of neurodevelopment seem to be even more inter-related, making distinctions more difficult to identify, as inadequate/imbalanced hormonal signaling is likely to affect a longer period of neurodevelopment. As part of this complexity of the discussed hormone interactions, it is worth mentioning that, especially in the cerebellum, the glia plays an important role in the mediation of developmental signals (maturational, migrational) towards neurons (Morest and Silver, 2003). Considering that we used a relatively diluted cell suspension to minimize physical cell-cell contact, all glial effects observed (a form of glia-neuron interaction) could mostly be mediated by a paracrine way of intercellular signaling. Since previous study indicated that neuronastroglia interactions in the cerebellum involve both cell contact and soluble factors (Martinez and Gomes, 2005), it is possible that one of the reasons for detecting differences between in situ and in vitro results is the lack of cell contact-based signaling mechanisms in our experimental model.

4.1. Effects of EDs on TRα Under in vitro circumstances it is especially interesting that BPA and Zea alone suppress TRα mRNA expression. This finding is supported by the findings of Sheng et al. (2012), however, earlier data also suggest that the suppressing effect of BPA (or other EDs) on TRs may only be temporary, and symptoms that emerge later in life resulting from perinatal exposure to BPA may develop on the grounds of BPA-linked mechanisms indirectly. Arsenic and MBC did not affect TRα mRNA expression in Glia+, yet, AllEDs seemed to increase TRα mRNA substancialy, and with a high variability (therefore, this effect did not reach significance, but is still alarming). The observation that ntC values in Glia− cultures superseded those measured in ntC[Glia+] suggests that the glia plays a role in the regulation of transcription, regardless of the type of receptor examined; even when cells were only treated with the EDs alone, consequential decrease in TRα transcription was more remarkable if the glia was absent in the culture. Therefore, it is surprising that when all EDs were given to the Glia− cultures, the degree of TRα reduction was less and did not reach significance when compared to the individual ED effects. While these observations are in concordance with previous results (Fauquier et al., 2014), our data also show that ED effects on TR transcription is multifactorial and, in addition, differ depending on the presence or the absence of glia. In contrast to the suppressing effect of BPA and Zea on TRα mRNA expression, the combination of EDs with any of the hormones provoked remarkably high versatility in the evoked transcriptional activity, regardless of the hormone used or the receptor examined. Such a robust activational effect by EDs has been reported earlier (Zhang et al., 2013), yet, this finding should still be alarming. When comparing the effects of the various EDs on TRα mRNA expression, it becomes apparent that there are similarities between EDs either or in Glia+/Glia− conditions. In Glia+, BPA and MBC effects are rather similar, while Zea, in general, exerts an opposite effect and As does not seem to affect TRα mRNA expression. Since results in the AllEDs experimental groups are reminiscent to those found in BPA-MBC groups, it is suggested that the latter two EDs dominate in the shaping of TRα mRNA levels, however, the mechanism(s) behind this phenomenon is/are not known. One of the possible causes might lie on the grounds of the competitive binding of the used EDs to TRα. To our knowledge, currently there is no explanation for this additive effect, although it is likely that the ability of BPA and/or MBC to act on TRs plays a role in the potentiation of transcription (Zoeller, 2005). It was generally observed that effects of EDs or EDs in combination with E2 and/or THs on translation (receptor protein expression) were less prominent than those found with regard to mRNA expression. At this point the distinct ED effects should be pointed out. Overall patterns of effects of BPA, Zea and As are rather similar, and in Glia− cultures, ED effects seemed to be uniform. It is interesting that when all EDs were co-administered, effects were as in the case of the individual EDs, but no further additive effects was found. In Glia+, however, MBC seemed to act differently from BPA, Zea and As, yet, simultaneous exposure to all EDs mimicked MBC effects. At the present time we do not know the explanation of this phenomenon, however, it seems to be safe to say that MBC might act differently on glia than the other EDs used. Since the specific cellular effects of hormones are mostly mediated by their cognate receptors, this observation can explain why the biological effects of ED-exposure could have remained masked or even unrecognized for a long time in spite of the dramatically increased or decreased transcriptional activity. Several studies suggested that exposure to EDs early in life leads to altered CNS development and functional deficiencies later in adulthood (Mathisen et al., 2013). The present results suggest that the conventional receptorial mediation of the ligand effects in the final, altered outcome of hormonal signaling during ED exposure may only partly account for those anomalies; this also means that linked and/or alternative mechanisms should receive special attention in future ED effect studies. For example, increased

4. Experiments on EDs Environmental and plant estrogens have been identified as compounds that when ingested, disrupt the physiological pathways of endogenous estrogen actions and thus, act as agonists or antagonists of estrogen. However, recent studies have made it clear that many of the exogenous compounds with estrogenic effects also influence thyroid actions and vice versa. In light of the relevant literature and our reference studies, this is not so surprizing since, as shown above, there is a complex interplay between the estrogenic and thyroid system in the CNS, but also on the organismal level. Below we discuss the effects of some well-known EDs one by one, followed by their combined effects on the hormonal regulation of the estrogenic-thyroid system. The latter may be of particular importance considering that humans and animals are, in general, exposed to a number of EDs simultaneously, and the possible consequences of this multiple exposure is extremely little determined yet. 7

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shaping of TRα receptor protein levels in the case of BPA, Zea and As, but not MBC (compared to respective ntCs). Since in AllED treatment groups the four afore-mentioned EDs were co-administered, it was surprising that results in Glia+ groups mimicked MBC effects. This phenomenon indicates that combined effects of EDs may not reflect the mathematical average of the individual ED effects, and that exposure to yet more EDs may result in unpredictable consequences. Differences were found between groups treated with the hormones only and those exposed to BPA as well. This observation suggests that in the presence of glia, THs must be present for the maintenance of the afore-mentioned double levels of TRα protein expression (compared to ntC[Glia+]), and that under such circumstances BPA does not further increase the TH-regulated TRα expression. The finding that in Glia− cultures these twofold ntC values were only detected if BPA was present alone or in combination with the hormones indicates that in neurons of Glia− cultures BPA determined the actual TRα protein expression values, regardless of whether the hormones were present. Finally, the smaller variances in Glia− cultures may be due to the more unified response of the homogeneous cell populations compared to those with mixed cell types. In Glia+, TRα receptor protein expression nearly doubled when the cultures were exposed to any of the used ligands, with the exception of E2. E2 treatment alone did not cause a change in receptor protein level when compared to the ntC. Thus, the potency of BPA and the other EDs used, as known estrogenic chemicals, to influence TRα expression more than E2 underlines the importance of these EDs to be considered as general nuclear receptor modulators, rather than just chemicals with estrogenic or thyroid effects.

material and energy consumption by CNS cells in the process of enhanced transcription could also lead to energy-deficient intracellular conditions that could be, at least in part, responsible for developmental deficiencies. This idea is consonant with the report of Nakagawa and Tayama (2000) that BPA toxicity caused a decrease in cellular ATP levels in hepatocytes. The unproportional ED effects in transcription versus translation also indicate that regulatory mechanisms that are interposed between transcription and translation, such as microRNA regulation, may also be affected by-, or could be specific targets of EDs, as indicated by AvissarWhiting et al. (2010) and Tilghman et al. (2012). These mechanisms apparently play a crucial role in blunting-buffering ED effects downstream of transcription. This idea signifies the importance of further research of these mechanisms, since the potential vulnerability of such interposed regulatory mechanisms may well determine the severity of ED effects. 4.2. TRα mRNA: Glia+ vs Glia− While differences between Glia+ and Glia− groups show comparable trends after BPA treatment, treatment with other EDs resulted in distinct and characteristic differences. For example, combining hormones with BPA leads to increased TRα mRNA expression in both Glia + and Glia−; Arsenic combined with hormones did not result in significant differences in Glia+, but in general, decreased TRα mRNA mean values in Glia−. It is noteworthy that in Glia+, BPA treatment alone lead to lower TRα mRNA expression than E2 treatment alone, in contrast to the opposite findings in Glia− cultures. Whether or not differences between treatment groups compared to their respective ntC followed similar trends (changes relative to each other), results indicate that the differences are due to glial effects. One possible mechanism underlying the afore-mentioned idea is that the glia may mediate BPA effects on the level of transcription in a T3-dependent manner, which is likely due to the astroglia’s ability to convert T4 to T3 through deiodination (Leonard, 1988). When cultured cells in Glia+ were treated with all four EDs, ED exposure resulted in the elevation of mean TRα mRNA values, especially without hormone exposure or co-exposure with either E2 or T3, and to a much lesser extent in other Glia+ groups. Thus, considering the effects of the different EDs alone or in combination with E2 and/or THs, we tend to conclude that a mixed-type effect could be detected: on the one hand, mean values show a characteristic increase in TRα mRNA, on the other hand, however, SEM values were also high, therefore these differences did not reach significance. In contrast, in Glia− groups, mean values were lower after AllED exposure compared to the respective ntC. This phenomenon indicates that in general, overall ED effects on neurons are mediated and shaped by the glia, and therefore, clinical conditions with glial deficiencies may be more affected by multiple exposure to EDs on transcriptional level.

4.4. TRα mRNA vs TRα protein Comparison of TRα mRNA and protein expression patterns suggest that the seemingly ligand-independent receptor protein expression levels were based on highly ligand-dependent transcriptional activity, especially in cultures treated with BPA or BPA in combination with any of the hormones. In Glia+ cultures, with the exception of the ntC and the E2 treated group, TRα protein expression levels were comparable (no significant differences), but were two fold compared to ntC. This phenomenon means that these cell populations could either not produce more TRα protein (because of their limited capacity or the activation of some down-regulating mechanism), or the detected increased TRα expression levels after treatments were necessary for the maintenance of survival under the applied experimental conditions. Although robust increases in transcription (e.g., in Glia+: E2 + BPA, T3 + BPA, T4 + BPA and E2 + T3/T4 + BPA) may potentially exhaust cellular energy sources, it is more likely that the comparison of transcription and translation reveals one of the cellular adaptation mechanisms, in the form of a high degree of plasticity on transcriptional level to maintain a relatively steady receptor protein expression. In fact, this idea or concept may apply to our TRβ results as well. Unproportional reactions in transcription vs translation, of the cells to BPA treatment alone also suggests that BPA could act to influence receptor protein expression levels in a receptor-independent manner, i.e., independently of potentially binding to TRα.

4.3. TRα protein: Glia+ vs Glia− In Glia− cultures, hormone treatments did not cause significant changes in TRα receptor protein levels when compared to ntCs. This steady level of TRα receptor protein expression is in contrast with the ligand dependent changes seen in TRα mRNA levels. Therefore, it is concluded that in neurons, there is a high level of adaptability to hormonal (i.e., E2 and THs) conditions, and without the glial mediation of hormone effects, neurons are able to maintain normal TRα receptor protein expression (or, in other words, neurons may be insensitive to these specific hormone conditions). Interestingly, when EDs were added to the cultures, a nearly uniform increase was detected in TRα receptor protein levels, regardless of the EDs used. In this respect, MBC did not seem to act differently in the presence of glia, while BPA, Zea and As effects in Glia+ apparently differed from MBC effects. Thus, it is suggested that in the presence of glia, there is a glia-dependence in the

4.5. TRβ mRNA: Glia+ vs. Glia− Differences between the members of Glia+ and Glia− groups resemble, in many respects, to those found with regard to TRα mRNA. Differences between respective treatment groups in Glia+ vs Glia− suggest that the mediating role of glia in the regulation of neuronal TRβ receptor expression is ligand dependent, and also indicates that this mediating activity, in addition to the mere glial presentation of T3 (after conversion of T4 to T3) to neurons, contains additional functional element(s) as well, whose identification warrants further experiments. 8

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4.6. TRβ protein: Glia+ vs. Glia−, mRNA vs. TRβ protein

5. Effects of endocrine disruptors

The overall pattern of TRβ protein expression values may suggest that there is no glial contribution in the determination of the actual TRβ protein expression levels. It is, therefore, important to consider the role of glia in the regulation of TRβ transcription, since the simple examination of potential glia effects on TRβ protein expression would be misleading. It is also interesting that exposure to BPA, alone or in combination with any of the hormones used, leads to significantly elevated TRβ protein expression. This finding suggests that BPA could influence TRβ protein in a hormone ligand-independent manner, as also indicated with regard to TRα. Again, this conclusion would be misleading regarding the effects of BPA on TRβ protein expression, therefore we conclude that BPA effects on transcription and translation should be comparatively evaluated to understand the complexity of BPA, and maybe other ED effects on the regulation of TR expression.

5.1. Cellular mechanisms of action When reaching their target cells, EDs permeate the plasma membrane and follow the intracellular cascades normally activated by E2. The major intracellular mechanisms of E2 actions are genomic, via their cognate receptors ERα and ERβ. Estrogen receptors (ERs) bind to E2responsive elements within the promoter regions of E2-responsive genes and modify transcription through protein-protein interactions or influence transcription through interactions with the AP1 transcription factors jun and fos. There is a growing number of studies reporting the ability of different EDs to bind to rat (Kuiper et al., 1998; Casanova et al., 1999) and human ERs (Nikov et al., 2000). Further, some industrial chemicals, the dioxins, have been reported to modulate ER mRNA expression in rats (Chaffin et al., 1996). These results indicate that certain EDs are capable of acting via rodent and human ER-dependent mechanisms and raise such a possibility with regard to untested EDs as well. Recent research conducted on non-neural (Wehling, 1997; Falkenstein et al., 2000) and neural tissues (Wong et al., 2003; Zsarnovszky et al., 2005) revealed that the cellular effects of E2 can be non-genomic as well. These rapid actions can increase intracellular calcium (Kuiper et al., 1998), generate inositol 1,4,5-triphosphate and diacylglycerol, can activate adenylate cyclase, phospholipase C, and the ERK1/2 mitogen activated protein kinase (MAPK) pathway in cancer cells, as well as in nervous tissues (reviewed by Belcher and Zsarnovszky, 2001). Some of these mechanisms are initiated by the binding of E2 at membrane-associated ERs that are closely related to the ‘classical’ intracellular receptors, but also, some evidence exists that rapid cellular E2 effects may not always require ERs (Falkenstein et al., 2000; Schmidt et al., 2000). The idea of non-genomic E2 signaling has evolved rapidly over the past years and, in particular, the nervous system has been found to be a unique tissue for rapid E2 effects (McEwen, 1991; Moss et al., 1997; Wong et al., 2003; Zsarnovszky et al., 2005). Kato et al. (1995) were the first to show that the phosphorylation of Ser 118 in AF1 region of human ERα functionally activated AF1 (an activation domain) and this was accomplished specifically by MAPK. This established for the first time a cross-talk between membrane-type growth factors and nuclear receptors. Indeed, since Kato’s report, a plethora of studies have confirmed and further detailed the relationship of ERs and neurotrophins in the activation/phosphorylation of the MAPK pathway in the CNS. Other studies suggest that E2 activation of membrane-bound neurotrophin receptors and ERs may be more ‘intimate’ than previously thought. For example, distinct regions in the developing forebrain are not only targets for E2 and the neurotrophins, but are sites of E2- and neurotrophin synthesis as well (Toran-Allerand et al., 1992). Estrogen is able to regulate the expression of several growth factors and their receptors, such as NGF, trkA and p75 in cholinergic neurons (Toran-Allerand, 1996), TGF-α in the hypothalamus (Ma et al., 1992), and BDNF in the cortex (Sohrabji et al., 1995), respectively. In turn, NGF regulates ER action in the forebrain post-translationally. Altogether, neurotrophins and E2 may act in concert, as well as reciprocally, to regulate the differentiation of their target neurons. Notwithstanding, the activation of the MAPK pathway by both neurotrophins and ERs has become the subject of scientific debate. Recently, Singh et al. (2000) demonstrated that, unlike in wildtype mice, the MAPK/MEK-kinase-dependent ERK phosphorylation in the cerebral cortex of ERα knock-out mice could not be inhibited by ICI 182,780, a specific ER blocker, and using genistein as a specific ligand for ERβ and 16α-iodo-17β-oestradiol as a specific ERα ligand did not induce ERK phosphorylation. Therefore, they concluded that E2-induced activation of this pathway is not ER dependent. Even though the above-mentioned substances are structurally selective ligands for the two ERs, their functional properties are not well characterized. Further, recent studies show that ERK phosphorylation can

4.7. Further considerations To the best of our knowledge, no clear definition of the differences between the functional roles of TRα and TRβ is available, most probably because of the usual co-activation of these receptors by their common ligands, as reviewed earlier (Somogyi et al., 2011). There are, however, studies reporting on physiological functions partly regulated by either of these receptors. Based on knockout and knockin experiments, TRα was found to influence cardiac functions, thermogenesis, hemopoiesis, and the maturation of intestines and bones (O'Shea et al., 2005; Plateroti et al., 2006). TRβ is crucial for normal, physiological endocrine and sensory functions such as those regulated by the hypothalamic-pituitary-thyroid axis, hepatic reactions to T3, behavior, audition, color sensation, and tactile senses (Amma et al., 2001; Ng et al., 2001; Flores-Morales et al., 2002; Abel et al., 2003; Esaki et al., 2003; Siesser et al., 2005). TRα and TRβ, however, can be co-expressed in the same tissues and can substitute for each other’s function to a certain extent (Gothe et al., 1999). Considering the latter potency of TRs together with the similarities and differences between TRα-TRβ receptor protein expression patterns in our experiments in Glia− vs Glia+ cultures allow us to reasonably suggest that one or more regulatory mechanisms interposed between transcription and translation is/are affected by BPA exposure. This idea is supported by reported alterations in microRNA expression in human placenta cells (AvissarWhiting et al., 2010), MCF-7 breast cancer cells (Tilghman et al., 2012) and ovine fetal ovary (Veiga-Lopez et al., 2013) after BPA-exposure. In our experimental model, one or more of these mechanisms may function in the glia and/or neurons. We are aware that the in vitro conditions, in general may, by themselves, substantially modify physiological parameters. Yet, it is more than likely that the observed BPA effects, combined with numerous biologically linked mechanisms, also occur in vivo, with the notion that in vivo, TRβ expression is restricted to specific ontogenetic states and is highly tissue specific (Bradley et al., 1994). Altogether, this idea is consonant with results from animal models that have shown that hypothyroidism during critical periods of development causes a variety of abnormalities in the central nervous system (Pasquini and Adamo, 1994; Martinez-Galan et al., 1997; Simorangkir et al., 1997), and that TRα and TRβ can compensate for each other’s hypofunction. Finally, it should be noted that a growing body of evidence exists to show that BPA and other EDs increase intracellular reactive oxygen species, generate oxidative stress conditions in mitochondria and endoplasmic reticulum (Huc et al., 2012; Babu et al., 2013), and activate apoptotic processes, such as the caspase 3 and caspase 9 apoptotic pathways, altogether leading to cell death and poor tissue development. The intracellular mediators of these ED effects towards influencing TR expression are currently studied. 9

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5.2. Interneuronal mechanisms

be rapidly induced by E2, as well as BPA, in the developing and mature cerebellum both in vivo (Zsarnovszky et al., 2005) and in vitro (Wong et al., 2003). Interestingly, both ICI 182,780 and bisphenol A can evoke E2-like effects on the activation of the ERK MAPK pathway. According to these studies, E2 can activate the ERK signaling cascade by acting on the plasma membrane. Therefore, the aforementioned estrogenic effects of ICI 182,780 and bisphenol A are believed to be initiated at membrane sites that are also responsive to E2. Noteworthy is among the novel actions of estrogens the activation/ phosphorylation of the transcription regulator CREB. It is well established by now that E2 can regulate-promote neurite growth and dendritic arborisation, and this mechanism involves E2 binding to membrane sites that activate cAMP/PKA and consequentially the CREB signaling cascade. Because this process can be blocked with cAMP/PKA antagonist or calcium depletion, but not with ICI 182,780, it has been concluded that the activation of the CREB cascade is not dependent on ERs (Beyer and Karolczak, 2000). On the other hand, however, CBP/ p300, which was identified as a co-factor of the CREB signaling pathway may be involved with both genomic and non-genomic E2 actions, and this also means that both ERα and ERβ may be capable of CREB activation/phosphorylation. The ability of EDs to bind to, and act through ERs is rather disturbing. Firstly, it has been shown that brain areas other than the neuroendocrine hypothalamus do also contain ERs. For example, limbic cortical structures that play a role in learning, memory maintenance and the regulation of behavior are also responsive to E2 and may be affected by EDs. The reported changes in sex-specific behavioral patterns after ED exposure may result from the intervention of these chemicals in the function of the above-mentioned higher brain regions. Secondly, recent experiments have identified ERs in brain areas where previously they were not detected, in both the developing and the adult brain. During the perinatal period, for example, ERs are expressed in virtually every region of the rat brain and probably in that of other mammalian species as well. It has been demonstrated that changes in ER- or ER mRNA-expression in certain brain regions, such as the cerebellum (Belcher, 1999; Jakab et al., 2001) and the cerebral cortex (Zsarnovszky and Belcher, 2001) occur at the time of important neurodevelopmental events. These observations suggest that ED exposure may not only affect neuroendocrine, but also cerebrocortical and cerebellar development. This, in turn, may impact later exploratory behavior, learning, cognitive functions and cerebellar regulation of motor functions (Zsarnovszky et al., 2005). Steroidogenic compounds can modulate both E2- and androgen dependent mechanisms. Androgen action is essential for the normal masculinization of the XY embryo during development. Testosterone is the major circulating androgen in males; however, within certain tissues, testosterone is enzymatically reduced to the more potent androgen, 5-alpha-dihydrotestosterone by 5-alphareductase. Little evidence exists about the exact effects of 5-alpha-reductase inhibition in the developing male brain. Nevertheless, it is suspected that androgen inhibition leads to failure in the masculinisation of dimorphic brain regions. Androgen receptors (ARs), as well as ERs, being present in the brains of rodents, primates and humans, are major mediators of androgen effects and can be activated by both estrogens and androgens. In fact, a recent study demonstrated that the immediate AR mRNA activators in the developing male rat brain are estrogens rather than androgens (McAbee and Doncarlos, 1999). Androgens dynamically activate nuclear ARs in rodents, which, during a certain period of brain development, is necessary for the activation of brain aromatase and promotes neuronal differentiation. This, in turn, can be blocked by the AR inhibitors imidazole, fadrozole and some triazoles, respectively. Connoly et al. (1994) demonstrated that exogenous androgens can activate ARs in the fetal primate brain, too and therefore, it is reasonable to assume that androgenic or antiandrogenic compounds may affect human fetal brain development as well.

There is evidence for neuronal membrane-mediated effects of E2 in the hypothalamic arcuate nucleus (AN). Postsynaptic membrane recognition is critical in the establishment and maintenance of synaptic contacts. Freezefracture analysis of AN neurons demonstrated sex differences in plasma membrane components that could play a role in the recognition of pre- and postsynaptic terminals. Significant sex differences were found in perikaryal and dendritic membranes in both the developing and the adult rat. Postsynaptic neuronal membranes of female rats contain significantly greater numbers of intramembranous protein particles (IMPs) than do those of male rats, but IMPs of males were found to be larger. Estrogen treatment causes aggregation of small IMPs (characteristic of females) and reduces the number of small IMPs. Thus, it appears that E2 causes the defeminisation of neuronal membranes in the AN. Although IMP numbers revert to the usual female levels after the E2 surge during the cycle, they remain fixed at male levels when the animal is put into constant estrus by E2 treatment and is in senescent constant estrus. Such E2-induced membrane effects and sexual dimorphism have also been described in primates. While these studies suggest that estrogen can have a dynamic modulatory effect on neuronal plasma membrane, further evidence indicates that the membrane effects of E2 may be of particular importance during brain development. It has been described that estrogen induces rapid increase in the number of exo- and endocytotic images in the AN both in vitro (Garcia-Segura et al., 1987) and in vivo (Garcia-Segura et al., 1988). These findings indicate that E2 may regulate both the insertion of proteins in the membranes and the internalisation of membrane components, and plays an important role in the genderisation of the neuronal plasma membrane. This may be, at least in part, an initiator of sex differences in synaptic plasticity later in life. In the hypothalamus, biphasic plasma E2 levels result in conspicuous changes in the number of both excitatory and inhibitory synapses in rats (Parducz et al., 2003), as well as in primates (Zsarnovszky et al., 2001). This, in turn, was found to correlate with consequential alterations in GnRH and subsequent LH release. Thus, it appears that a considerable means of E2 action regulating female reproductive functions is the determination of sex differences in plasma/synaptic membrane composition and later regulation of neuronal connectivity by the induction of synaptic plasticity. This idea is further supported by the observations mentioned above, that estrogen regulates neurite growth and dendritic arborisation, and influences the number of dendritic spines. There are few data available on the possible effects of EDs on synaptic activity (MacLusky et al., 2005). However, more forms of estrogens (17β-estradiol, estradiol benzoate, estradiol valerate, etc.) were found to be capable of inducing synaptic retraction/synaptic reapplication in rodents and primates, and to result in changes in neuroendocrine functions. Thus, it is reasonable to assume that some forms of EDs may be able to produce similar effects when reaching considerable concentrations in the blood. 6. Conclusions Results of recent studies reveal that in the developing cerebellum, there is a highly complex interplay between E2 and THs in the maintenance of normal levels of each-other’s cognate receptors, and that the hormone effects are most probably mediated by the glia. Our results explain at least some, and raise even more, questions regarding the role and mechanisms of E2 and THs in neurodevelopment, and underscore the importance of the optimal/physiological ratio of E2/THs in the precise orchestration of cerebellar development, memory formation and neuroprotection when necessary. On the other hand, our observations implicate that abnormalities in glial and/or thyroid functions or in tissue E2/TH levels impact, on multiple levels, cerebellar development, cerebellar functions later in life, and the regenerative capability of the cerebellar tissue in case of injury, all of which should be considered in 10

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Carlson, D.J., Strait, K.A., Schwartz, H.L., Oppenheimer, J.H., 1996. Thyroid hormone receptor isoform content in cultured type 1 and type 2 astrocytes. Endocrinology 137, 911–917. Carou, M.E., Deguiz, M.L., Reynoso, R., Szwarcfarb, B., Carbone, S., Moguilevsky, J.A., Scacchi, P., Ponzo, O.J., 2009. Impact of the UV-B filter 4-(Methylbenzylidene)camphor (4-MBC) during prenatal development in the neuroendocrine regulation of gonadal axis in male and female adult rats. Environ. Toxicol. Pharmacol. 27, 410–414. Casanova, M., You, L., Gaido, K.W., Archibeque-Engle, S., Janszen, D.B., Heck, H.A., 1999. Developmental effects of dietary phytoestrogens in Sprague-Dawley rats and inter actions of genistein and daidzein with rat estrogen receptors alpha and beta in vitro. Toxicol. Sci. 51, 236–244. Chaffin, C.L., Peterson, R.E., Hutz, R.J., 1996. In utero and lactational exposure of female Holtzman rats to 2,3,7,8-tetrachlorodibenzo-p-dioxin: modulation of the estrogen signal. Biol. Reprod. 55, 62–67. Chattopadhyay, N., Kher, R., Virmani, J., Godbole, M.M., 1995. Differential expression of alpha- and beta-thyroid hormone receptor genes in the developing rat brain under hypothyroidism. Biol. Neonate 67, 64–71. Chen, J.D., Evans, R.M., 1995. A transcriptional co-repressor that interacts with nuclear hormone receptors. Nature 377, 454–457. Chen, G.C., Guan, L.S., Hu, W.L., Wang, Z.Y., 2002. Functional repression of estrogen receptor a by arsenic trioxide in human breast cancer cells. Anticancer Res. 22, 633–638. Cimino-Reale, G., Ferrario, D., Casati, B., Brustio, R., Diodovich, C., Collotta, A., Vahter, M., Gribaldo, L., 2008. Combined in utero and juvenile exposure of mice to arsenate and atrazine in drinking water modulates gene expression and clonogenicity of myeloid progenitors. Toxicol. Lett. 180, 59–66. Connoly, P.B., Choate, J.V., Resko, J.A., 1994. Effects of exogenous androgen on brain androgen receptors of the fetal rhesus monkey. Neuroendocrinology 59, 271–276. Delfosse, V., Grimaldi, M., Maire, A., Bourguet, W., Balaguer, P., 2014. Nuclear receptor profiling of bisphenol-A and its halogenated analogues. Vitam. Horm. 94, 229–251. Duenas, M., Torres-Aleman, I., Naftolin, F., Garcia-Segura, L.M., 1996. Interaction of insulin-like growth factor-I and estradiol signaling pathways on hypothalamic neuronal differentiation. Neuroscience 74, 531–539. Durrer, S., Maerkel, K., Schlumpf, M., Lichtensteiger, W., 2005. Estrogen target gene regulation and coactivator expression in rat uterus after developmental exposure to the ultraviolet filter 4-methylbenzylidene camphor. Endocrinology 146, 2130–2139. Esaki, T., Suzuki, H., Cook, M., Shimoji, K., Cheng, S.Y., Sokoloff, L., Nunez, J., 2003. Functional activation of cerebral metabolism in mice with mutated thyroid hormone nuclear receptors. Endocrinology 144, 4117–4122. Escamez, M.J., Guadano-Ferraz, A., Cuadrado, A., Bernal, J., 1999. Type 3 iodothyronine deiodinase is selectively expressed in areas related to sexual differentiation in the newborn rat brain. Endocrinology 140, 5443–5446. Evans, R.M., 1988. The steroid and thyroid hormone receptor superfamily. Science 240, 889–895. Falkenstein, E., Tillman, H.C., Christ, M., Feuring, M., Wehling, M., 2000. Multiple actions of steroid hormones. A focus on rapid, nongenomic effects. Pharmacol. Rev. 52, 513–556. Fan, X., Xu, H., Warner, M., Gustafsson, J.A., 2010. ERbeta in CNS: new roles in development and function. Prog. Brain Res. 181, 233–250. Fauquier, T., Chatonnet, F., Picou, F., Richard, S., Fossat, N., Aguilera, N., Lamonerie, T., Flamant, F., 2014. Purkinje cells and Bergmann glia are primary targets of the TRα1 thyroid hormone receptor during mouse cerebellum postnatal development. Development 141, 166–175. Flores-Morales, A., Gullberg, H., Fernandez, L., Stahlberg, N., Lee, N.H., Vennstrom, B., Norstedt, G., 2002. Patterns of liver gene expression governed by TRbeta. Mol. Endocrinol. 16, 1257–1268. Freitas, B.C., Gereben, B., Castillo, M., Kallo, I., Zeold, A., Egri, P., Liposits, Z., Zavacki, A.M., Maciel, R.M., Jo, S., Singru, P., Sanchez, E., Lechan, R.M., Bianco, A.C., 2010. Paracrine signaling by glial cell–derived triiodothyronine activates neuronal gene expression in the rodent brain and human cell. J. Clin. Invest. 120, 2206–2217. Frizzell, C., Ndossi, D., Verhaegen, S., Dahl, E., Eriksen, G., Sørlie, M., Ropstad, E., Muller, M., Elliott, C.T., Connolly, L., 2011. Endocrine disrupting effects of zearalenone, alpha- and beta-zearalenol at the level of nuclear receptor binding and steroidogenesis. Toxicol. Lett. 206, 210–217. Garcia-Segura, L.M., Olmos, G., Tranque, P., Naftolin, F., 1987. Rapid effects of gonadal steroids upon hypothalamic neuronal membrane ultrastructure. J. Steroid Biochem. 27, 615–627. Garcia-Segura, L.M., Perez, J., Tranque, P.A., Olmos, G., Naftolin, F., 1988. Sexual differentiation of the neuronal plasma membrane: neonatal levels of sex steroids modulate the number of exo-endocytotic images in the developing rat arcuate neurons. Neurosci. Lett. 91, 19–23. Gentilcore, D., Porreca, I., Rizzo, F., Ganbaatar, E., Carchia, E., Mallardo, M., De Felice, M., Ambrosino, C., 2013. Bisphenol A interferes with thyroid specific gene expression. Toxicology 304, 21–31. Gereben, B., Kollar, A., Bartha, T., Buys, N., Decuypere, E., Rudas, P., 1998. 3,3',5Triiodothyronine (T3) uptake and expression of thyroid hormone receptors during the adaptation to hypothyroidism of the brain of chicken. Acta Vet. Hung. 46, 473–485. Ghosh, M., Gharami, K., Paul, S., Das, S., 2005. Thyroid hormone-induced morphological differentiation and maturation of astrocytes involves activation of protein kinase A and ERK signalling pathway. Eur. J. Neurosci. 22, 1609–1617. Gonkowski, S., Obremski, K., Calka, J., 2015. The influence of low doses of zearalenone on distribution of selected active substances in nerve fibers within the circular muscle layer of porcine ileum. J. Mol. Neurosci. 56, 878–886. Gothe, S., Wang, Z., Ng, L., Kindblom, J.M., Barros, A.C., Ohlsson, C., Vennstrom, B.,

the diagnostics and treatment of relevant clinical conditions. Considering the extreme complexity of interactions between E2 and TH signaling, both intracellularly and intercellularly, it is reasonable to hypothesize that the failure of the integrated ER-TR signaling at one or more points may account, at least in part, for the formation of neuronor glia-based tumours. The ED effects discussed in this review seem to be of more importance in humans health than in animals. At the same time, analogous experiments can certainly not be carried out on humans for ethical reasons. Therefore, it is also concluded that there is an immense need for further clinical determination of the relationship of ED effects on the cerebellum and the entire CNS and the relevant clinical conditions. Acknowledgement Research discussed in this review was supported by OTKA 104982; OTKA K-115613; Research Centre of Excellence Fund 8526-5/2015/ TUDPOL (Hungary); NKB 15711, 15984, NKB JG 15912; KK-PhD 15257 and KK-PhD 15263 and the 11475-4/2016/FEKUT grant of the Hungarian Ministry of Human Resources. References Abel, E.D., Moura, E.G., Ahima, R.S., Campos-Barros, A., Pazos-Moura, C.C., Boers, M.E., Kaulbach, H.C., Forrest, D., Wondisford, F.E., 2003. Dominant inhibition of thyroid hormone action selectively in the pituitary of thyroid hormone receptor-beta null mice abolishes the regulation of thyrotropin by thyroid hormone. Mol. Endocrinol. 17, 1767–1776. Al-Bader, M.D., El-Abdallah, A.A., Redzic, Z.B., 2008. Ontogenic profile of estrogen receptor alpha and beta mRNA and protein expression in fetal rat brain. Neurosci. Lett. 440, 222–226. Amma, L.L., Campos-Barros, A., Wang, Z., Vennstrom, B., Forrest, D., 2001. Distinct tissue-specific roles for thyroid hormone receptors beta and alpha1 in regulation of type 1 deiodinase expression. Mol. Endocrinol. 15, 467–475. Avissar-Whiting, M., Veiga, K.R., Uhl, K.M., Maccani, M.A., Gagne, L.A., Moen, E.L., Marsit, C.J., 2010. Bisphenol A exposure leads to specific microRNA alterations in placental cells. Reprod. Toxicol. 29, 401–406. Babu, S., Uppu, S., Claville, M.O., Uppu, R.M., 2013. Prooxidant actions of bisphenol A (BPA) phenoxyl radicals: implications to BPA-related oxidative stress and toxicity. Toxicol. Mech. Methods 23, 273–280. Belcher, S.M., 1999. Regulated expression of estrogen receptor alpha and beta mRNA in granule cells during development of the rat cerebellum. Brain Res. Dev. Brain Res. 115, 57–69. Belcher, S.M., 2008. Rapid signaling mechanisms of estrogens in the developing cerebellum. Brain Res. Rev. 57, 481–492. Belcher, S.M., Ma, X., Le, H.H., 2009. Blockade of estrogen receptor signaling inhibits growth and migration of medulloblastoma. Endocrinology 150, 1112–1121. Belcher, S.M., Zsarnovszky, A., 2001. Estrogenic actions in the brain: estrogen, phytoestrogens, and rapid intracellular signaling mechanisms. J. Pharmacol. Exp. Ther. 299, 408–414. Bernal, J., 2007. Thyroid hormone receptors in brain development and function. Nat. Clin. Pract. Endocrinol. Metab. 3, 249–259. Beyer, C., Karolczak, M., 2000. Estrogenic stimulation of neurite growth in midbrain dopaminergic neurons depends on cAMP/protein kinase A signaling. J. Neurosci. Res. 59, 107–116. Bingham, S.A., Atkinson, C., Liggins, J., Bluck, L., Coward, A., 1998. Phyto-oestrogens: where are we now? Br. J. Nutr. 79, 393–406. Boas, M., Feldt-Rasmussen, U., Main, K.M., 2012. Thyroid effects of endocrine disrupting chemicals. Mol. Cell. Endocrinol. 355, 240–248. Bodwell, J.E., Gosse, J.A., Nomikos, A.P., Hamilton, J.W., 2006. Arsenic disruption of steroid receptor gene activation: complex dose-response effects are shared by several steroid receptors. Chem. Res. Toxicol. 19, 1619–1629. Bodwell, J.E., Kingsley, L.A., Hamilton, J.W., 2004. Arsenic at very low concentrations alters glucocorticoid receptor (GR)-mediated gene activation but not GR-mediated gene repression: complex dose-response effects are closely correlated with levels of activated GR and require a functional GR DNA binding. Chem. Res. Toxicol. 17, 1064–1076. Bradley, D.J., Towle, H.C., Young 3rd, W.S., 1994. Alpha and beta thyroid hormone receptor (TR) gene expression during auditory neurogenesis: evidence for TR isoformspecific transcriptional regulation in vivo. Proc. Natl. Acad. Sci. USA 91, 439–443. Brent, G.A., Moore, D.D., Larsen, P.R., 1991. Thyroid hormone regulation of gene expression. Annu. Rev. Physiol. 53, 17–35. Calderón, J., Navarro, M.E., Jimenez-Capdeville, M.E., Santos-Diaz, M.A., Golden, A., Rodriguez-Leyva, I., Borja-Aburto, V., Díaz-Barriga, F., 2001. Exposure to arsenic and lead and neuropsychological development in mexican children. Environ. Res. 85, 69–76. Carlson, D.J., Strait, K.A., Schwartz, H.L., Oppenheimer, J.H., 1994. Immunofluorescent localization of thyroid hormone receptor isoforms in glial cells of rat brain. Endocrinology 135, 1831–1836.

11

Frontiers in Neuroendocrinology xxx (xxxx) xxx–xxx

A. Zsarnovszky et al.

dimorphic gene regulation in brain as a target for endocrine disrupters: developmental exposure of rats to 4-methylbenzylidene camphor. Toxicol. Appl. Pharmacol. 218, 152–165. Martinez, R., Gomes, F.C., 2005. Proliferation of cerebellar neurons induced by astrocytes treated with thyroid hormone is mediated by a cooperation between cell contact and soluble factors and involves the epidermal growth factor-protein kinase a pathway. J. Neurosci. Res. 80, 341–349. Martinez-Galan, J.R., Pedraza, P., Santacana, M., Escobar del Ray, F., Morreale de Escobar, G., Ruiz-Marcos, A., 1997. Early effects of iodine deficiency on radial glial cells of the hippocampus of the rat fetus. A model of neurological cretinism. J. Clin. Invest. 99, 2701–2709. Mastri, C., Mistry, P., Lucier, G.W., 1985. In vivo oestrogenicity and binding characteristics of alpha-zearalanol (P-1496) to different classes of oestrogen binding proteins in rat liver. J. Steroid Biochem. 23, 279–289. Mathisen, G.H., Yazdani, M., Rakkestad, K.E., Aden, P.K., Bodin, J., Samuelsen, M., Nygaard, U.C., Goverud, I.L., Gaarder, M., Loberg, E.M., Bolling, A.K., Becher, R., Paulsen, R.E., 2013. Prenatal exposure to bisphenol A interferes with the development of cerebellar granule neurons in mice and chicken. Int. J. Dev. Neurosci. 31, 762–769. McAbee, M.D., Doncarlos, L.L., 1999. Estrogen, but not androgens, regulates androgen receptor messenger ribonucleic acid expression in the developing male rat forebrain. Endocrinology 140, 3674–3681. McEwen, B.S., 1991. Non-genomic and genomic effects of steroids on neural activity. Trends Pharmacol. Sci. 12, 141–147. Miodovnik, A., Edwards, A., Bellinger, D.C., Hauser, R., 2014. Developmental neurotoxicity of ortho-phthalate diesters: review of human and experimental evidence. Neurotoxicology 41, 112–122. Mirzatoni, A., Spence, R.D., Naranjo, K.C., Saldanha, C.J., Schlinger, B.A., 2010. Injuryinduced regulation of steroidogenic gene expression in the cerebellum. J. Neurotrauma 27, 1875–1882. Morest, D.K., Silver, J., 2003. Precursors of neurons, neuroglia, and ependymal cells in the CNS: what are they? Where are they from? How do they get where they are going? Glia 43, 6–18. Moss, R.L., Gu, Q., Wong, M., 1997. Estrogen: nontranscriptional signaling pathway. Rec. Progr. Horm. Res. 52, 33–69. Mueller, S.O., Kling, M., Firzani, P.A., Mecky, A., Duranti, E., Shields-Botella, J., Delansorne, R., Broschard, T., Kramer, P.J., 2003. Activation of estrogen receptor alpha and ER beta by 4-methylbenzylidene-camphor in human and rat cells: comparison with phyto- and xenoestrogens. Toxicol. Lett. 142, 89–101. Murray, M.B., Zilz, N.D., McCreary, N.L., MacDonald, M.J., Towle, H.C., 1988. Isolation and characterization of rat cDNA clones for two distinct thyroid hormone receptors. J. Biol. Chem. 263, 12770–12777. Nakagawa, Y., Tayama, S., 2000. Metabolism and cytotoxicity of bisphenol A and other bisphenols in isolated rat hepatocytes. Arch. Toxicol. 74, 99–105. Ng, L., Pedraza, P.E., Faris, J.S., Vennstrom, B., Curran, T., Morreale de Escobar, G., Forrest, D., 2001. Audiogenic seizure susceptibility in thyroid hormone receptor betadeficient mice. NeuroReport 12, 2359–2362. Nikov, G.N., Hopkins, N.E., Boue, S., Alworth, W.L., 2000. Interactions of dietary estrogens with human estrogen receptors and the effect on estrogen receptor-estrogen response element complex formation. Environ. Health Perspect. 108, 867–872. Oppenheimer, J.H., Schwartz, H.L., Strait, K.A., 1995. In: Weintraub, B., Raven (Eds.), Basic Concepts and Clinical Correlations. MolecularEndocrinology, New York, pp. 249–268. Ortiz-Caro, J., Yusta, B., Montiel, F., Villa, A., Aranda, A., Pascual, A., 1986. Identification and characterization of l-triiodothyronine receptors in cells of glial and neuronal origin. Endocrinology 119, 2163–2167. O'Shea, P.J., Bassett, J.H., Sriskantharajah, S., Ying, H., Cheng, S.Y., Williams, G.R., 2005. Contrasting skeletal phenotypes in mice with an identical mutation targeted to thyroid hormone receptor alpha1 or beta. Mol. Endocrinol. 19, 3045–3059. Palanza, P.L., Howdeshell, K.L., Parmigiani, S., Vom Saal, F.S., 2002. Exposure to a low dose of bisphenol A during fetal life or in adulthood alters maternal behavior in mice. Environ. Health Perspect. 110 (Suppl 3), 415–422. Panaite, P.A., Barakat-Walter, I., 2010. Thyroid hormone enhances transected axonal regeneration and muscle reinnervation following rat sciatic nerve injury. J. Neurosci. Res. 88, 1751–1763. Parducz, A., Zsarnovszky, A., Naftolin, F., Horvath, T.L., 2003. Estradiol affects axo-somatic contacts of neuroendocrine cells in the arcuate nucleus of adult rats. Neuroscience 117, 791–794. Parveen, M., Zhu, Y., Kiyama, R., 2009. Expression profiling of the genes responding to zearalenone and its analogues using estrogen-responsive genes. FEBS Lett. 583, 2377–2384. Pasquini, J.M., Adamo, A.M., 1994. Thyroid hormones and the central nervous system. Dev. Neurosci. 16, 1–8. Peeters, R., Fekete, C., Goncalves, C., Legradi, G., Tu, H.M., Harney, J.W., Bianco, A.C., Lechan, R.M., Larsen, P.R., 2001. Regional physiological adaptation of the central nervous system deiodinases to iodine deficiency. Am. J. Physiol. Endocrinol. Metab. 281, E54–61. Pekary, A.E., Sattin, A., Stevens, S.A., 2006. Rapid modulation of TRH-like peptides in rat brain by thyroid hormones. Peptides 27, 1577–1588. Pillay, D., Churturgoon, A.A., Nevines, E., Manickum, T., Deppe, W., Dutton, M.F., 2002. The quantitative analysis of zearalenone and its derivatives in plasma of patients with breast and cervical cancer. Clin. Chem. Lab. Med. 40, 946–951. Plateroti, M., Kress, E., Mori, J.I., Samarut, J., 2006. Thyroid hormone receptor alpha1 directly controls transcription of the beta-catenin gene in intestinal epithelial cells. Mol. Cell. Biol. 26, 3204–3214. Price Jr., R.H., Handa, R.J., 2000. Expression of estrogen receptor-beta protein and mRNA

Forrest, D., 1999. Mice devoid of all known thyroid hormone receptors are viable but exhibit disorders of the pituitary-thyroid axis, growth, and bone maturation. Genes Dev. 13, 1329–1341. Greenman, D.L., Mehta, R.G., Wittliff, J.L., 1979. Nuclear interaction of Fusarium mycotoxins with estradiol bindingsites in the mouse uterus. J. Toxicol. Environ. Health 5, 593–598. Guadano-Ferraz, A., Obregon, M.J., St Germain, D.L., Bernal, J., 1997. The type 2 iodothyronine deiodinase is expressed primarily in glial cells in the neonatal rat brain. Proc. Natl. Acad. Sci. USA 94, 10391–10396. Hodin, R.A., Lazar, M.A., Wintman, B.I., Darling, D.S., Koenig, R.J., Larsen, P.R., Moore, D.D., Chin, W.W., 1989. Identification of a thyroid hormone receptor that is pituitaryspecific. Science 244, 76–79. Honma, S., Suzuki, A., Buchanan, D.L., Katsu, Y., Watanabe, H., Iguchi, T., 2002. Low dose effect of in utero exposure to bisphenol A and diethylstilbestrol on female mouse reproduction. Reprod. Toxicol. 16, 117–122. Horn, S., Heuer, H., 2010. Thyroid hormone action during brain development: more questions than answers. Mol. Cell. Endocrinol. 315, 19–26. Hubank, M., Sinha, A.K., Gullo, D., Ekins, R.P., 1990. Nuclear tri-iodothyronine (T3) binding in neonatal rat brain suggests a direct glial requirement for T3 during development. J. Endocrinol. 126, 409–415. Huc, L., Lemarie, A., Gueraud, F., Helies-Toussaint, C., 2012. Low concentrations of bisphenol A induce lipid accumulation mediated by the production of reactive oxygen species in the mitochondria of HepG2 cells. Toxicol. In Vitro 26, 709–717. Ikeda, Y., 2008. Expression of the two estrogen receptor (ER) subtypes, ERalpha and ERbeta, during postnatal development of the rat cerebellum. Cerebellum 7, 501–502. Ikezuki, Y., Tsutsumi, O., Takai, Y., Kamei, Y., Taketani, Y., 2002. Determination of bisphenol A concentrations in human biological fluids reveals significant early prenatal exposure. Hum. Reprod. 17, 2839–2841. Iwamuro, S., Yamada, M., Kato, M., Kikuyama, S., 2006. Effects of bisphenol A on thyroid hormone-dependent up-regulation of thyroid hormone receptor alpha and beta and down-regulation of retinoid X receptor gamma in Xenopus tail culture. Life Sci. 79, 2165–2171. Jakab, R.L., Wong, J.K., Belcher, S.M., 2001. Estrogen receptor beta immunoreactivity in differentiating cells of the developing rat cerebellum. J. Comp. Neurol. 430, 396–409. Kato, S., Endoh, H., Masuhiro, Y., Kitamoto, T., Uchiyama, S., Sasaki, H., Masushige, S., Goto, Y., Nishida, E., Kawashima, H., Metzger, D., Chambon, P., 1995. Activation of the estrogen receptor through phosphorylation by mitogen-activated protein kinase. Science 270, 1491–1494. Kirby, M., Zsarnovszky, A., Belcher, S.M., 2004. Estrogen receptor expression in a human primitive neuroectodermal tumor cell line from the cerebral cortex: estrogen stimulates rapid ERK1/2 activation and receptor-dependent cell migration. Biochem. Biophys. Res. Commun. 319, 753–758. Koenig, R.J., Lazar, M.A., Hodin, R.A., Brent, G.A., Larsen, P.R., Chin, W.W., Moore, D.D., 1989. Inhibition of thyroid hormone action by a non-hormone binding c-erbA protein generated by alternative mRNA splicing. Nature 337, 659–661. Koibuchi, N., 2008. The role of thyroid hormone on cerebellar development. Cerebellum 7, 530–533. Koibuchi, N., Fukuda, H., Chin, W.W., 1999. Promoter-specific regulation of the brainderived neurotropic factor gene by thyroid hormone in the developing rat cerebellum. Endocrinology 140, 3955–3961. Kolodny, J.M., Leonard, J.L., Larsen, P.R., Silva, J.E., 1985. Studies of nuclear 3,5,3′triiodothyronine binding in primary cultures of rat brain. Endocrinology 117, 1848–1857. Kuiper, G.G.J.M., Lemmen, J.G., Carlsson, B., Corton, J.C., Safe, S.H., van der Saag, P.T., van der Burg, B., Gustafsson, J.Å., 1998. Interaction of estrogenic chemicals and phytoestrogens with estrogen receptor. Endocrinology 139, 4252–4263. Lamirand, A., Pallud-Mothre, S., Ramauge, M., Pierre, M., Courtin, F., 2008. Oxidative stress regulates type 3 deiodinase and type 2 deiodinase in cultured rat astrocytes. Endocrinology 149, 3713–3721. Lazar, M.A., 1993. Thyroid hormone receptors: multiple forms, multiple possibilities. Endocr. Rev. 14, 184–193. Lebel, J.M., L’Herault, S., Dussault, J.H., Puymirat, J., 1993. Thyroid hormone upregulates thyroid hormone receptor β gene expression in rat cerebral hemisphere astrocyte cultures. Glia 9, 105–112. Lee, S.J., McEwen, B.S., 2001. Neurotrophic and neuroprotective actions of estrogens and their therapeutic implications. Annu. Rev. Pharmacol. Toxicol. 41, 569–591. Lee, S.K., Choi, H.S., Song, M.R., Lee, M.O., Lee, J.W., 1998. Estrogen receptor, a common interaction partner for a subset of nuclear receptors. Mol. Endocrinol. 12, 1184–1192. Leonard, J.L., 1988. Dibutyryl cAMP induction of type II 5'deiodinase activity in rat brain astrocytes in culture. Biochem. Biophys. Res. Commun. 151, 1164–1172. Leonard, J.L., Farwell, A.P., Yen, P.M., Chin, W.W., Stula, M., 1994. Differential expression of thyroid hormone receptor isoforms in neurons and astroglial cells. Endocrinology 135, 548–555. Leonard, J.L., 2008. Non-genomic actions of thyroid hormone in brain development. Steroids 73, 1008–1012. Lin, H.Y., Sun, M., Tang, H.Y., Lin, C., Luidens, M.K., Mousa, S.A., Incerpi, S., Drusano, G.L., Davis, F.B., Davis, P.J., 2009. L-Thyroxine vs. 3,5,3′-triiodo-L-thyronine and cell proliferation: activation of mitogen-activated protein kinase and phosphatidylinositol 3-kinase. Am. J. Physiol. Cell Physiol. 296, C980–991. Ma, Y.J., Junier, M.P., Costa, M.E., Ojeda, S.R., 1992. Transforming growth factor-β gene expression in the hypothalamus is developmentally regulated and linked to sexual maturation. Neuron 9, 657–670. MacLusky, N.J., Hajszan, T., Leranth, C., 2005. The environmental estrogen bisphenol A inhibits estradiol-induced hippocampal synaptogenesis. Environ. Health Perspect. 113, 675–679. Maerkel, K., Durrer, S., Henseler, M., Schlumpf, M., Lichtensteiger, W., 2007. Sexually

12

Frontiers in Neuroendocrinology xxx (xxxx) xxx–xxx

A. Zsarnovszky et al.

Biochem. Biophys. Acta. 1362, 169–176. Takai, Y., Tsutsumi, O., Ikezuki, Y., Hiroi, H., Osuga, Y., Momoeda, M., Yano, T., Taketani, Y., 2000. Estrogen receptor-mediated effects of a xenoestrogen, bisphenol A, on preimplantation mouse embryos. Biochem. Biophys. Res. Commun. 270, 918–921. Takai, Y., Tsutsumi, O., Ikezuki, Y., Kamei, Y., Osuga, Y., Yano, T., Taketan, Y., 2001. 2001 Preimplantation exposure to bisphenol A advances postnatal development. Reprod. Toxicol. 15, 71–74. Takemura, H., Shim, J.Y., Sayama, K., Tsubura, A., Zhu, B.T., Shimoi, K., 2007. Characterization of the estrogenic activities of zearalenone and zeranol in vivo and in vitro. J. Steroid Biochem. Mol. Biol. 103, 170–177. Tang, H.Y., Lin, H.Y., Zhang, S., Davis, F.B., Davis, P.J., 2004. Thyroid hormone causes mitogen-activated protein kinase-dependent phosphorylation of the nuclear estrogen receptor. Endocrinology 145, 3265–3272. Tilghman, S.L., Bratton, M.R., Segar, H.C., Martin, E.C., Rhodes, L.V., Li, M., McLachlan, J.A., Wiese, T.E., Nephew, K.P., Burow, M.E., 2012. Endocrine disruptor regulation of microRNA expression in breast carcinoma cells. PLoS ONE 7, e32754. Toran-Allerand, C.D., 1996. The estrogen/neurotrophin connection during neural development: is co-localization of estrogen receptors with the neurotrophins and their receptors biologically relevant? Dev. Neurosci. 18, 36–48. Toran-Allerand, C.D., Miranda, R.C., Bentham, W., Sohrabji, F., Brown, T.J., Hochberg, R.B., MacLusky, N.J., 1992. Estrogen receptors colocalize with low-affinity nerve growth factor receptors in cholinergic neurons of the basal forebrain. Proc. Natl. Acad. Sci. USA 89, 4668–4672. Turcotte, J.C., Hunt, P.J., Blaustein, J.D., 2005. Estrogenic effects of zearalenone on the expression of progestin receptors and sexual behavior in female rats. Horm. Behav. 47, 178–184. Vasudevan, N., Davidkova, G., Zhu, Y.S., Koibuchi, N., Chin, W.W., Pfaff, D., 2001a. Differential interaction of estrogen receptor and thyroid hormone receptor isoforms on the rat oxytocin receptor promoter leads to differences in transcriptional regulation. Neuroendocrinology 74, 309–324. Vasudevan, N., Kia, H.K., Inoue, S., Muramatsu, M., Pfaff, D., 2002a. Isoform specificity for oestrogen receptor and thyroid hormone receptor genes and their interactions on the NR2D gene promoter. J. Neuroendocrinol. 14, 836–842. Vasudevan, N., Koibuchi, N., Chin, W.W., Pfaff, D.W., 2001b. Differential crosstalk between estrogen receptor (ER)alpha and ERbeta and the thyroid hormone receptor isoforms results in flexible regulation of the consensus ERE. Brain Res. Mol. Brain Res. 95, 9–17. Vasudevan, N., Kow, L.M., Pfaff, D.W., 2001c. Early membrane estrogenic effects required for full expression of slower genomic actions in a nerve cell line. Proc. Natl. Acad. Sci. USA 98, 12267–12271. Vasudevan, N., Ogawa, S., Pfaff, D., 2002b. Estrogen and thyroid hormone receptor interactions: physiological flexibility by molecular specificity. Physiol. Rev. 82, 923–944. Vasudevan, N., Pfaff, D.W., 2005. Molecular mechanisms of crosstalk between thyroid hormones and estrogens. Curr. Opin. Endocrinol. Diabetes 12, 381–388. Vasudevan, N., Pfaff, D.W., 2007. Membrane-initiated actions of estrogens in neuroendocrinology: emerging principles. Endocr. Rev. 28, 1–19. Veiga-Lopez, A., Luense, L.J., Christenson, L.K., Padmanabhan, V., 2013. Developmental programming: gestational bisphenol-A treatment alters trajectory of fetal ovarian gene expression. Endocrinology 154, 1873–1884. vom Saal, F.S., Cooke, P.S., Buchanan, D.L., Palanza, P., Thayer, K.A., Nagel, S.C., Parmigiani, S., Welshons, W.V., 1998. A physiologically based approach to the study of bisphenol A and other estrogenic chemicals on the size of reproductive organs, daily sperm production, and behavior. Toxicol. Ind. Health 14, 239–260. Wallis, K., Dudazy, S., van Hogerlinden, M., Nordstrom, K., Mittag, J., Vennstrom, B., 2010. The thyroid hormone receptor alpha1 protein is expressed in embryonic postmitotic neurons and persists in most adult neurons. Mol. Endocrinol. 24, 1904–1916. Wasserman, G.A., Liu, X., Parvez, F., Factor-Litvak, P., Ahsan, H., Levy, D., Kline, J., van Geen, A., Mey, J., Slavkovich, V., Siddique, A.B., Islam, T., Graziano, J.H., 2011. Arsenic and manganese exposure and children’s intellectual function. Neurotoxicology 32, 450–457. Wehling, M., 1997. Specific, nongenomic actions of steroid hormones. Annu. Rev. Physiol. 59, 365–393. Wechsler-Reya, R.J., 2003. Analysis of gene expression in the normal and malignant cerebellum. Recent Prog. Horm. Res. 58, 227–248. Withanage, G., Murata, H., Koyama, T., Ishiwata, I., 2001. Agonistic and antagonistic effects of zearalenone, an etrogenic mycotoxin, on SKN, HHUA, HepG2 human cancer cell lines. Vet. Hum. Toxicol. 43, 6–10. Wong, J.K., Kennedy, P.R., Belcher, S.M., 2001. Simplified serum- and steroid-free culture conditions for the high-throughput viability analysis of primary cultures of cerebellar neurons. J. Neurosci. Methods 110, 45–55. Wong, J.K., Le, H.H., Zsarnovszky, A., Belcher, S.M., 2003. Estrogens and ICI182,780 (Faslodex) modulate mitosis and cell death in immature cerebellar neurons via rapid activation of p44/p42 mitogen-activated protein kinase. J. Neurosci. 23, 4984–4995. Zhang, J., Lazar, M.A., 2000. The mechanism of action of thyroid hormones. Annu. Rev. Physiol. 62, 439–466. Zhang, W.Z., Yong, L., Jia, X.D., Li, N., Fan, Y.X., 2013. Combined subchronic toxicity of bisphenol A and dibutyl phthalate on male rats. Biomed. Environ. Sci. 26, 63–69. Zhao, X., Lorenc, H., Stephenson, H., Wang, Y.J., Witherspoon, D., Katzenellenbogen, B., Pfaff, D., Vasudevan, N., 2005. Thyroid hormone can increase estrogen-mediated transcription from a consensus estrogen response element in neuroblastoma cells. Proc. Natl. Acad. Sci. USA 102, 4890–4895. Zhong, C.X., Mass, M.J., 2001. Both hypomethylation and hypermethylation of DNA associated with arsenite exposure in cultures of human cells identified by methylation-

in the cerebellum of the rat. Neurosci. Lett. 288, 115–118. Rocha-Amador, D., Navarro, M.E., Carrizales, L., Morales, R., Calderón, J., 2007. Decreased intelligence in children and exposure to fluoride and arsenic in drinking water. Cad. Saude Pub. 23, S579–S587. Rodríguez, V., Carrizales, L., Jiménez-Capdeville, M., Dufour, L., Giordano, M., 2001. The effects of sodium arsenite exposure on behavioral parameters in the rat. Brain Res. Bull. 55, 301–308. Rodríguez-Barranco, M., Lacasaña, M., Aguilar-Garduño, C., Alguacil, J., Gil, F., González-Alzaga, B., Rojas-García, A., 2013. Association of arsenic, cadmium and manganese exposure with neurodevelopment and behavioural disorders in children: a systematic review and meta-analysis. Sci. Total Environ. 454, 562–577. Rosselli, M., Reinhart, K., Imthurn, B., Keller, P.J., Dubey, R.K., 2000. Cellular and biochemical mechanisms by which environmental oestrogens influence reproductive function. Hum. Reprod. Update 6, 332–350. Sap, J., de Magistris, L., Stunnenberg, H., Vennström, B., 1990. A major thyroid hormone response element in the third intron of the rat growth hormone gene. EMBO J. 9, 887–896. Sasahara, K., Shikimi, H., Haraguchi, S., Sakamoto, H., Honda, S., Harada, N., Tsutsui, K., 2007. Mode of action and functional significance of estrogen-inducing dendritic growth, spinogenesis, and synaptogenesis in the developing Purkinje cell. J. Neurosci. 27, 7408–7417. Salto, C., Kindblom, J.M., Johansson, C., Wang, Z., Gullberg, H., Nordström, K., Mansén, A., Ohlsson, C., Thorén, P., Forrest, D., Vennström, B., 2001. Ablation of TRa2 and a concomitant overexpression of a1 yields a mixed hypo- and hyperthyroid phenotype in mice. Mol. Endocrinol. 15, 2115–2128. Scalise, T.J., Gyorffy, A., Toth, I., Kiss, D.S., Somogyi, V., Goszleth, G., Bartha, T., Frenyo, L.V., Zsarnovszky, A., 2012. Ligand-induced changes in oestrogen and thyroid hormone receptor expression in the developing rat cerebellum: a comparative quantitative PCR and Western blot study. Acta Vet. Hung. 60, 263–284. Schlumpf, M., Jarry, H., Wuttke, W., Ma, R., Lichtensteiger, W., 2004a. Estrogenic activity and estrogen receptor beta binding of the UV filter 3-benzylidene camphor. Comparison with 4-methylbenzylidene camphor. Toxicology 199, 109–120. Schlumpf, M., Schmid, P., Durrer, S., Conscience, M., Maerkel, K., Henseler, M., Gruetter, M., Herzog, I., Reolon, S., Ceccatelli, R., Faass, O., Stutz, E., Jarry, H., Wuttke, W., Lichtensteiger, W., 2004b. Endocrine activity and developmental toxicity of cosmetic UV filters—an update. Toxicology 205, 113–122. Schmidt, B.M., Gerdes, D., Feuring, M., Falkenstein, E., Christ, M., Wehling, M., 2000. Rapid, nongenomic steroid actions: a new age? Front. Neuroendocrinol. 21, 57–94. Schmutzler, C., Hamann, I., Hofmann, P.J., Kovacs, G., Stemmler, L., Mentrup, B., Schomburg, L., Ambrugger, P., Grüters, A., Seidlova-Wuttke, D., Jarry, H., Wuttke, W., Köhrle, J., 2004. Endocrine active compounds affect thyrotropin and thyroid hormone levels in serum as well as endpoints of thyroid hormone action in liver, heart and kidney. Toxicology 205, 95–102. Schonfelder, G., Wittfoht, W., Hopp, H., Talsness, C.E., Paul, M., Chahoud, I., 2002. Parent bisphenol A accumulation in the human maternal-fetal-placental unit. Environ. Health Perspect. 110, A703–A707. Seidlová-Wuttke, D., Christoffel, J., Rimoldi, G., Jarry, H., Wuttke, W., 2006. Comparison of effects of estradiol with those of octylmethoxycinnamate and 4-methylbenzylidene camphor on fat tissue, lipids and pituitary hormones. Toxicol. Appl. Pharmacol. 214, 1–7. Sheng, Z.G., Tang, Y., Liu, Y.X., Yuan, Y., Zhao, B.Q., Chao, X.J., Zhu, B.Z., 2012. Low concentrations of bisphenol a suppress thyroid hormone receptor transcription through a nongenomic mechanism. Toxicol. Appl. Pharmacol. 259, 133–142. Shi, Y.B., Yaoita, Y., Brown, D.D., 1992. Genomic organization and alternative promoter usage of the two thyroid hormone receptor b genes in Xenopus laevis. J. Biol. Chem. 267, 733–738. Shikimi, H., Sakamoto, H., Mezaki, Y., Ukena, K., Tsutsui, K., 2004. Dendritic growth in response to environmental estrogens in the developing Purkinje cell in rats. Neurosci. Lett. 364, 114–118. Shughrue, P.J., Lane, M.V., Merchenthaler, I., 1997. Comparative distribution of estrogen receptor-alpha and -beta mRNA in the rat central nervous system. J. Comp. Neurol. 388, 507–525. Shulga, A., Blaesse, A., Kysenius, K., Huttunen, H.J., Tanhuanpaa, K., Saarma, M., Rivera, C., 2009. Thyroxin regulates BDNF expression to promote survival of injured neurons. Mol. Cell. Neurosci. 42, 408–418. Siesser, W.B., Cheng, S.Y., McDonald, M.P., 2005. Hyperactivity, impaired learning on a vigilance task, and a differential response to methylphenidate in the TRbetaPV knockin mouse. Psychopharmacology 181, 653–663. Simorangkir, D.R., Wreford, N.G., De Kretser, D.M., 1997. Impaired germ cell development in the testes of immature rats with neonatal hypothyroidism. J. Androl. 18, 186–193. Singh, M., Setalo Jr., G., Guan, X., Frail, D.E., Toran-Allerand, D.C., 2000. Estrogen induced activation of the mitogen-activated protein kinase cascade in the cerebral cortex of estrogen receptor-α knock-out mice. J. Neurosci. 20, 1694–1700. Sohrabji, F., Miranda, R.C., Toran-Allerand, C.D., 1995. Identification of a putative estrogen response element in the gene encoding brain-derived neurotropic factor. Proc. Natl. Acad. Sci. USA 92, 11110–11114. Somogyi, V., Gyorffy, A., Scalise, T.J., Kiss, D.S., Goszleth, G., Bartha, T., Frenyo, V.L., Zsarnovszky, A., 2011. Endocrine factors in the hypothalamic regulation of food intake in females: a review of the physiological roles and interactions of ghrelin, leptin, thyroid hormones, oestrogen and insulin. Nutr. Res. Rev. 24, 132–154. Spence, R.D., Zhen, Y., White, S., Schlinger, B.A., Day, L.B., 2009. Recovery of motor and cognitive function after cerebellar lesions in a songbird: role of estrogens. Eur. J. Neurosci. 29, 1225–1234. Sundari, P.N., Wilfred, G., Ramakrishna, B., 1997. Does oxidative protein damage play a role in the pathogenesis of carbon tetrachloride-induced liver injury in the rat?

13

Frontiers in Neuroendocrinology xxx (xxxx) xxx–xxx

A. Zsarnovszky et al.

female rat primary somatosensory cortex. Brain Res. Dev. Brain Res. 129, 39–46. Zsarnovszky, A., Foldvari, E.G., Ronai, Z., Bartha, T., Frenyo, L.V., 2007. Oestrogens in the mammalian brain: from conception to adulthood – a review. Acta Vet. Hung. 55, 333–347. Zsarnovszky, A., Horvath, T.L., Garcia-Segura, L.M., Horvath, B., Naftolin, F., 2001. Oestrogen-induced changes in the synaptology of the monkey (Cercopithecus aethiops) arcuate nucleus during gonadotropin feedback. J. Neuroendocrinol. 13, 22–28. Zsarnovszky, A., Le, H.H., Wang, H.S., Belcher, S.M., 2005. Ontogeny of rapid estrogenmediated extracellular signal-regulated kinase signaling in the rat cerebellar cortex: potent nongenomic agonist and endocrine disrupting activity of the xenoestrogen bisphenol A. Endocrinology 146, 5388–5396.

sensitive arbitrarily-primed PCR. Toxicol. Lett. 122, 223–234. Zhu, Z., Zhao, B., Wang, X., Zhu, S., Zhang, Q., Xu, Y., Hui, R., Tepel, M., 2004. Differentially expressed genes in hypertensive rats developing cerebral ischemia. Life Sci. 74, 1899–1909. Zhu, Y.S., Yen, P.M., Chin, W.W., Pfaff, D.W., 1996. Estrogen and thyroid hormone interaction on regulation of gene expression. Proc. Natl. Acad. Sci. USA 93, 12587–12592. Zoeller, R.T., 2005. Environmental chemicals as thyroid hormone analogues: new studies indicate that thyroid hormone receptors are targets of industrial chemicals. Mol. Cell. Endocrinol. 242, 10–15. Zsarnovszky, A., Belcher, S.M., 2001. Identification of a developmental gradient of estrogen receptor expression and cellular localization in the developing and adult

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