Kingsley Ibhazehiebo

Low-dose exposure to bisphenol A and replacement bisphenol S induces precocious hypothalamic neurogenesis in embryonic zebrafish

Significance Here we demonstrate that bisphenol A (BPA) exposure during a time point analogous to the second trimester in humans has real and measurable effects on brain development and behavior. Furthermore, our study is the first, to our knowledge, to show that bisphenol S, a replacement used in BPA-free products, equally affects neurodevelopment. These findings suggest that BPA-free products are not necessarily safe and support a societal push to remove all structurally similar bisphenol analogues and other compounds with endocrine-disruptive activity from consumer goods. Our data here, combined with over a dozen physiological and behavioral human studies that begin to point to the prenatal period as a BPA window of vulnerability, suggest that pregnant mothers limit exposure to plastics and receipts. Bisphenol A (BPA), a ubiquitous endocrine disruptor that is present in many household products, has been linked to obesity, cancer, and, most relevant here, childhood neurological disorders such as anxiety and hyperactivity. However, how BPA exposure translates into these neurodevelopmental disorders remains poorly understood. Here, we used zebrafish to link BPA mechanistically to disease etiology. Strikingly, treatment of embryonic zebrafish with very low-dose BPA (0.0068 μM, 1,000-fold lower than the accepted human daily exposure) and bisphenol S (BPS), a common analog used in BPA-free products, resulted in 180% and 240% increases, respectively, in neuronal birth (neurogenesis) within the hypothalamus, a highly conserved brain region involved in hyperactivity. Furthermore, restricted BPA/BPS exposure specifically during the neurogenic window caused later hyperactive behaviors in zebrafish larvae. Unexpectedly, we show that BPA-mediated precocious neurogenesis and the concomitant behavioral phenotype were not dependent on predicted estrogen receptors but relied on androgen receptor-mediated up-regulation of aromatase. Although human epidemiological results are still emerging, an association between high maternal urinary BPA during gestation and hyperactivity and other behavioral disturbances in the child has been suggested. Our studies here provide mechanistic support that the neurogenic period indeed may be a window of vulnerability and uncovers previously unexplored avenues of research into how endocrine disruptors might perturb early brain development. Furthermore, our results show that BPA-free products are not necessarily safer and support the removal of all bisphenols from consumer merchandise.

Disruption of thyroid hormone receptor-mediated transcription and thyroid hormone-induced purkinje cell dendrite arborization by Polybrominated diphenyl ethers

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

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

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

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

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

Retinoic Acid Receptor–Related Orphan Receptor Alpha–Enhanced Thyroid Hormone Receptor–Mediated Transcription Requires Its Ligand Binding Domain Which Is Not, by Itself, Sufficient: Possible Direct Interaction of Two Receptors

BACKGROUND Natural mutant staggerer (sg) mice harbor a mutated retinoic acid receptor-related orphan receptor alpha (RORalpha). A genetic deletion corresponding to the ligand-binding domain (LBD) of RORalpha results in aberrant cerebellar development in the sg mice. These mice show similar neurotrophin expression to that seen in perinatal hypothyroid animals. RORalpha augments thyroid hormone receptor (TR)-mediated transcription, which may be partly responsible for the similar cerebellar abnormalities between sg and hypothyroid animals. The objective of this study is to examine further the mechanisms of augmentation of TR action by RORalpha. We examined whether TR directly binds to ROR and which regions of TR or ROR are required for the TR-ROR interaction. METHODS A transient transfection-based reporter gene assay was performed to measure the activity of TR-mediated transcription in CV-1 cells. To examine TR-RORalpha binding mammalian two-hybrid and glutathione-S-transferase (GST) pull-down assays were carried out. RESULTS Although full-length RORalpha augmented TRalpha1- or beta1-mediated transcription, such augmentation was not observed with sg-type mutant RORalpha (RORsg) that contained the RORalpha N-terminal and DNA-binding domain (DBD) and a part of the LBD. On the other hand, the transcription of Gal4-DBD-fused TRbeta1-LBD was suppressed by RORalpha, indicating that RORalpha does not interact with TR-LBD. Full-length TRbeta1 bound to RORalpha or RORsg in GST pull-down assays; however, RORalpha-LBD did not bind to TRalpha1 or beta1. CONCLUSION The full-length forms of both RORalpha and TR are essential for the augmentation of TR-mediated transcription by RORalpha.

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

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

Impact of endocrine-disrupting chemicals on thyroid function and brain development

Endocrine disrupting chemicals (EDCs) are synthetic or natural substances in the environment. EDCs have been shown to disrupt reproductive, developmental and other homeostatic systems by interfering with the synthesis, secretion, transport, metabolism and action of endogenous hormones including the thyroid hormone (TH) system. Since TH plays a critical role in brain development, the exposure to TH-system disrupting EDCs during development may have serious consequences. In this article, representative previous studies showing the effect of representative EDCs on the TH system are summarized. Then, the molecular mechanisms of action of polychlorinated biphenyls and polybrominated diphenyl ethers on the TH system are discussed further. Particularly, the effect of polychlorinated biphenyls and polybrominated diphenyl ethers on TH-mediated brain development is discussed. Our recent studies may provide a novel idea regarding the effect of EDCs on the TH system.

A novel metabolism-based phenotypic drug discovery platform in zebrafish uncovers HDACs 1 and 3 as a potential combined anti-seizure drug target

Drug discovery in epilepsy has historically been biased towards transmembrane channels and receptors. Ibhazehiebo et al. describe a novel, metabolism-based drug discovery platform that assays for bioenergetic changes in zebrafish models, and demonstrate that histone deacetylases 1 and 3 decrease mitochondrial hyperexcitability and represent a combined target for anti-seizure drug development.

Brominated Organohalogens and Neurodevelopment: Different Mechanisms, Same Consequence

Brominated organohalogens including polybrominated diphenyl ethers (PBDEs), hexabromocyclododecane (HBCD), tetrabromobisphenol A (TBBPA), and polybrominated biphenyl mixture (BP-6) are ubiquitous industrial chemicals used extensively as flame retardants in a wide range of consumer and household items including furnitures and electronics. Their ability to persist in the environment and bioaccumulate in humans and wildlife is currently of great health concern. These brominated organohalogens have been implicated in developmental neurotoxicity in numerous in vitro and in vivo models, acting through multiple mechanisms. In this chapter, we will examine different pathways and mechanisms of brominated organohalogen actions and their consequences for neurodevelopment. Although many mechanisms and pathways have been postulated for brominated organohalogen actions, they all converge at same consequences of impaired brain development.