Neonicotinoid Insecticides Alter the Gene Expression Profile of Neuron-Enriched Cultures from Neonatal Rat Cerebellum.

Neonicotinoids are considered safe because of their low affinities to mammalian nicotinic acetylcholine receptors (nAChRs) relative to insect nAChRs. However, because of importance of nAChRs in mammalian brain development, there remains a need to establish the safety of chronic neonicotinoid exposures with regards to children's health. Here we examined the effects of longterm (14 days) and low dose (1 µM) exposure of neuron-enriched cultures from neonatal rat cerebellum to nicotine and two neonicotinoids: acetamiprid and imidacloprid. Immunocytochemistry revealed no differences in the number or morphology of immature neurons or glial cells in any group versus untreated control cultures. However, a slight disturbance in Purkinje cell dendritic arborization was observed in the exposed cultures. Next we performed transcriptome analysis on total RNAs using microarrays, and identified significant differential expression (p < 0.05, q < 0.05, ≥1.5 fold) between control cultures versus nicotine-, acetamiprid-, or imidacloprid-exposed cultures in 34, 48, and 67 genes, respectively. Common to all exposed groups were nine genes essential for neurodevelopment, suggesting that chronic neonicotinoid exposure alters the transcriptome of the developing mammalian brain in a similar way to nicotine exposure. Our results highlight the need for further careful investigations into the effects of neonicotinoids in the developing mammalian brain.

Altered social interactions in male juvenile cynomolgus monkeys prenatally exposed to bisphenol A.

Bisphenol A (BPA) is a widespread environmental contaminant, and humans are routinely exposed to BPA. We investigated whether prenatal exposure to BPA influences behavioral development in juvenile cynomolgus monkeys (Macaca fascicularis). Pregnant cynomolgus monkeys were implanted with subcutaneous pumps and exposed to 10µg/kg/day BPA or vehicle (control) from gestational day 20 to 132. Both BPA-exposed and control juvenile monkeys (aged 1-2years) were assessed using the peer-encounter test that was conducted to evaluate behaviors in social interaction with a same-sex, same-treatment peer. In the encounter test, prenatal BPA exposure significantly reduced environmental exploration and presenting, a gesture related to sexual reproduction, and increased visual exploration, but only in males; furthermore, it significantly reduced the typical sexual dimorphism of the aforementioned behaviors normally observed between male and female juvenile cynomolgus monkeys. This study demonstrates that prenatal BPA exposure affects behavioral development during adolescence and results in the demasculinization of key sexually dimorphic behaviors in male juvenile monkeys.

Nicotine-like effects of the neonicotinoid insecticides acetamiprid and imidacloprid on cerebellar neurons from neonatal rats.

BACKGROUND: Acetamiprid (ACE) and imidacloprid (IMI) belong to a new, widely used class of pesticide, the neonicotinoids. With similar chemical structures to nicotine, neonicotinoids also share agonist activity at nicotinic acetylcholine receptors (nAChRs). Although their toxicities against insects are well established, their precise effects on mammalian nAChRs remain to be elucidated. Because of the importance of nAChRs for mammalian brain function, especially brain development, detailed investigation of the neonicotinoids is needed to protect the health of human children. We aimed to determine the effects of neonicotinoids on the nAChRs of developing mammalian neurons and compare their effects with nicotine, a neurotoxin of brain development. METHODOLOGY/PRINCIPAL FINDINGS: Primary cultures of cerebellar neurons from neonatal rats allow for examinations of the developmental neurotoxicity of chemicals because the various stages of neurodevelopment-including proliferation, migration, differentiation, and morphological and functional maturation-can be observed in vitro. Using these cultures, an excitatory Ca(2+)-influx assay was employed as an indicator of neural physiological activity. Significant excitatory Ca(2+) influxes were evoked by ACE, IMI, and nicotine at concentrations greater than 1 µM in small neurons in cerebellar cultures that expressed the mRNA of the α3, α4, and α7 nAChR subunits. The firing patterns, proportion of excited neurons, and peak excitatory Ca(2+) influxes induced by ACE and IMI showed differences from those induced by nicotine. However, ACE and IMI had greater effects on mammalian neurons than those previously reported in binding assay studies. Furthermore, the effects of the neonicotinoids were significantly inhibited by the nAChR antagonists mecamylamine, α-bungarotoxin, and dihydro-ß-erythroidine. CONCLUSIONS/SIGNIFICANCE: This study is the first to show that ACE, IMI, and nicotine exert similar excitatory effects on mammalian nAChRs at concentrations greater than 1 µM. Therefore, the neonicotinoids may adversely affect human health, especially the developing brain.

Maternal plasma polychlorinated biphenyl levels in cynomolgus monkeys (Macaca fascicularis) affect infant social skills in mother-infant interaction.

Polychlorinated biphenyls (PCBs) are endocrine disrupting chemicals that disturb normal development of embryonic brains. In the present study, we evaluated the relationship between maternal plasma PCB concentration and infant behavioral characteristics in mother-infant interactions. We grouped 20 pregnant cynomolgus monkeys (Macaca fascicularis) into higher and lower PCB exposure groups; monkeys in the higher PCB group had PCB concentrations above 15 pg/g, which is representative of natural exposure levels. Maternal PCB concentration correlated negatively with infant behaviors (approach, look, proximity, locomotion) at the age of 6 months (p < .05), when an increase in these behaviors should normally occur. These results suggest that maternal PCB exposure may affect the development of infant social behavior in cynomolgus monkeys. Furthermore, this study provides primate evidence to support observations of associations between behavioral and learning disabilities and prenatal exposure to PCBs in humans.

Alterations in male infant behaviors towards its mother by prenatal exposure to bisphenol A in cynomolgus monkeys (Macaca fascicularis) during early suckling period.

Bisphenol A (BPA) is an environmental chemical with physiological potencies that cause adverse effects, even at environmentally relevant exposures, on the basis of a number of studies in experimental rodents. Thus, there is an increasing concern about environmental exposure of humans to BPA. In the present study, we used experimentally controlled cynomolgus monkeys (Macaca fascicularis) to assess the influence of prenatal exposure to BPA (10 microg/(kg day)) via subcutaneously implanted pumps and examined social behaviors between infants and their mothers during the suckling period. Mother-infant interactions in cynomolgus monkeys had behavioral sexual dimorphism associated with sex of infant from early suckling period. Prenatal exposure to BPA altered the behaviors of male infants significantly; BPA-exposed male infants behaved as female infants. And it also affected some of female infant behaviors. Consequently, gestational BPA exposure altered some behaviors of their mothers, mainly in male-nursing mothers. These results suggest that BPA exposure affects behavioral sexual differentiation in male monkeys, which promotes the understanding of risk of BPA exposure in human.

Alterations in gene expression of glutamate receptors and exocytosis-related factors by a hydroxylated-polychlorinated biphenyl in the developing rat brain.

Polychlorinated biphenyls (PCBs), major environmental hormonally active agents, are metabolized into hydroxylated PCBs in the liver to facilitate excretion. Some of hydroxylated PCBs also have potencies disturbing endogenous hormonal activities at least in vitro. Hormonal activities of hydroxylated PCBs raise a possibility of their interfering with normal brain development which is strictly regulated by endogenous hormones. We investigated whether and how prenatal exposure to a congener of hydroxylated PCBs (4-OH-2',3,3',4',5'-penta CB; 4-OH-PCB106) having activities to disrupt thyroid hormone-dependent signals in vitro could perturb normal gene expression in the developing brain in vivo. Pregnant rats were exposed to 4-OH-PCB106 subcutaneously at the dose of 1.0mg/(kgday) from day 7 of gestation to postnatal day 1. Then three brain regions (cerebral cortex, hippocampus and striatum) were obtained from offspring on postnatal day 1 and subjected to further gene expression analyses. Comprehensive analyses of mRNA expression by oligo DNA microarrays and subsequent validations by quantitative RT-PCR revealed that prenatal exposure to 4-OH-PCB106 affected mRNA expression of glutamate receptors as well as that of thyroid hormone-responsive genes in region-specific manners. Concomitantly 4-OH-PCB106 exposure increased mRNA expression of genes related to exocytosis in the three brain regions. These results raise the possibility that prenatal exposure to some hydroxylated PCBs with thyroid hormone-disrupting potencies leads to abnormal brain development via perturbations on the expression of genes involved in glutamatergic neurotransmission.

Estrogen and environmental estrogenic chemicals exert developmental effects on rat hypothalamic neurons and glias.

We investigated effects of 17beta-estradiol (E(2)) and endocrine disrupters, nonylphenol (NP) and bisphenol-A (BPA), focusing on the neuronal development in cultures of fetal rat hypothalamic cells. We applied different concentrations of E(2), NP or BPA to the cultured hypothalamic cells and observed their effects on dendritic and synaptic development by immunocytochemistry using anti-microtubule associated protein-2 (MAP2) and anti-synapsin I antibodies, respectively. Administration of E(2) for 7 days affected MAP2-positive area as well as synapsin I-positive area. NP and BPA also influenced neuronal developments. The significant increase both in MAP2- and synapsin I-positive areas was observed at 10 and/or 100 nM of them, while 1 microM of them reduced the positive areas. Synaptic densities calculated from synapsin I-positive area/MAP2-positive area were not constant among different doses of three chemicals, but increased at 10 and/or 100 nM and decreased at 1 microM. Furthermore, immunostaining of NP-treated cells with the antibody against glial fibrillary acidic protein (GFAP) revealed that glial development was similarly influenced by NP. Therefore, the present results demonstrated that not only E(2) but also the environmental estrogenic chemicals, NP and BPA, affect development of fetal rat hypothalamic cells in vitro.

Disrupting effects of hydroxy-polychlorinated biphenyl (PCB) congeners on neuronal development of cerebellar Purkinje cells: a possible causal factor for developmental brain disorders?

Polychlorinated biphenyls (PCBs) and hydroxy-PCB (OH-PCB) metabolites are widely distributed bioaccumulative environmental chemicals and have similar chemical structures to those of thyroid hormones (THs). Previously, we reported that THs are essential for neuronal development and the low doses of two OH-PCBs, namely, 4-OH-2',3,3',4',5'-pentachlorobiphenyl (4'-OH-PeCB-106) and 4-OH-2',3,3',4',5,5'-hexachlorobiphenyl (4'-OH-HxCB-159), inhibited the TH-dependent dendritic development of Purkinje cells in mouse cerebellar cultures using serum-free defined medium. To determine which type of OH-PCBs affect neuronal development, we further examined several OH-PCBs and other estrogenic chemicals using this simple and sensitive assay system. Two-way ANOVA was used to assess the effects of OH-PCBs and other chemicals on both factors of their concentrations and with/without T4 in the assay of TH-dependent dendritic development of Purkinje cells. Aside from the two OH-PCBs, 4-OH-2',3,4',5,6'-pentachlorobiphenyl (4'-OH-PeCB-121) and bisphenol A significantly inhibited the TH-dependent dendritic development of Purkinje cells, whereas 4-OH-2',3,3',5',6'-pentachlorobiphenyl (4'-OH-PeCB-112), 4-OH-2',3,3',5,5',6'-hexachlorobiphenyl (4'-OH-HxCB-165), 4-OH-2,2',3,4',5,5',6-heptachlorobiphenyl (4-OH-HpCB-187), progesterone and nonylphenol did not induce any inhibition, but significantly promoted the dendritic extension of Purkinje cells in the absence of THs. Other estrogenic chemicals, including beta-estradiol, diethyl stilbestrol and p-octylphenol did not show significant inhibitory or promoting effects. From these results, it is suggested that exposure to OH-PCBs and other environmental chemicals may disrupt normal neuronal development and cause some developmental brain disorders, such as LD, ADHD, and autism.

Gestational and lactational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin affects social behaviors between developing rhesus monkeys (Macaca mulatta).

Pregnant rhesus monkeys (Macaca mulatta) were exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) at 30 and 300 ng/kg by subcutaneous injection at gestational day 20, followed by additional injections of TCDD (1.5 and 15 ng/kg, respectively) every 30 days till 90 days after parturition. The offspring delivered from these experimentally TCDD-exposed mothers were subjected to a series of behavioral tests after the weaning at 12-14 months old (MO): a finger maze learning task (12-15 MO), encounter tests between two monkeys (at 12-15 and 24-27 MO), and an eye-contact test (23-26 MO) to estimate learning ability, social interaction with a peer subject, and interest or hostility to a human observer, respectively. TCDD exposure had no significant effect on learning ability or interest/hostility to an observer. It did, however, significantly affect behavioral characteristics in the encounter tests. In the first encounter test, monkeys exposed to TCDD showed more visual exploration and mutual proximity but less stereotypy behavior compared to control monkeys. In the second encounter test, these differences seemed to disappear, suggesting that the behavioral effects of TCDD exposure in the encounter tests might disappear as the monkey develops. This study produced evidence of the behavioral toxicity of TCDD in social interactions using non-human primates.

Monitoring synaptogenesis in the developing mouse cerebellum with an original oligonucleotide microarray.

Use of DNA microarrays in neuroscience have been limited to rough screening purposes, mainly because the reliability and sensitivity of available arrays are not high enough. Because only a few hundred to one thousand genes are usually found to change expression levels in most experiments, we attempted to develop a more quantitative array by the following strategies: 1) limit the genes to those relevant to the experimental system, 2) design oligonucleotide probes of specified molecular properties so that more stringent hybridization conditions can be employed, 3) place six spots per probe on one slide and use multiple normalization genes, and 4) use a new type of gold-coated slide with higher S/N ratio. Genes involved in the construction and functioning of the synapse were selected from the literature as well as experimentally by comparing cerebella from hypothyroid and control mice at postnatal day 15 (P15). Although the number of genes covered was not yet large (172 genes), the custom array "Synaptoarray" thus constructed was capable of detecting +/-20% difference in gene expression ratios. Analysis of the postnatal development of the mouse cerebellum by using Synaptoarray demonstrated a general expression pattern with a peak at P7, followed by a decline at P15 and a partial recovery after P21. P10 clearly marked the end of the initial growth stage and a major transcriptional turning point in this system. This result suggests that such a custom array should be useful in monitoring perturbations to the normal developmental program.

Attention-deficit and hyperactive neurobehavioural characteristics induced by perinatal hypothyroidism in rats.

Thyroid hormone is essential for the proper development of the mammalian central nervous system (CNS). In the present study, we examined behavioural alterations caused by transient perinatal hypothyroidism induced by an anti-thyroid drug, propylthiouracil (PTU). This drug produces perinatal disruption of the thyroid system and subsequent behavioural changes, which we investigated using a series of behavioural tests and focusing particularly on attention-deficit/hyperactivity disorder (ADHD)-like behaviours. In the open field test, both male and female rats that had experienced perinatal hypothyroidism (HT rats) showed an increased percent of locomotion behaviour and reduced grooming behaviour, suggesting that HT rats may be hyperactive and show fewer anxiety characteristics. Neither male nor female HT rats showed retention in the passive avoidance test. Male HT rats showed a significantly lower rate of correct avoidance responses than control rats in earlier sessions in the active avoidance test. In addition, we observed significant increases in the number of times that rats crossed the partition during inter-trial intervals and the percent of failure of avoidance during 5 s electrical stimuli in HT rats, suggesting that HT rats are restless, have a shortened attention span and panic easily. In measuring spontaneous motor activity during a period of darkness, male HT rats appeared to plunge into active phase with short, quick steps, while male control rats showed only long active phases during a stress-free period of darkness. These abnormal behavioural characteristics in HT rats might coincide with those found in some cases of ADHD.

Hydroxylated metabolites of polychlorinated biphenyls inhibit thyroid-hormone-dependent extension of cerebellar Purkinje cell dendrites.

Thyroid hormones (THs) are important for brain development, and polychlorinated biphenyl (PCB) accumulation in humans is a serious problem because PCBs may affect TH functions. To determine the effects of hydroxylated metabolites of PCBs (OH-PCBs) on brain development, we performed mouse cerebellar culture assays. 4-OH-2',3,3',4',5'-pentachlorobiphenyl and 4-OH-2',3,3',4',5,5'-hexachlorobiphenyl significantly inhibited the TH-dependent extension of Purkinje cell dendrites even at 5 x 10(-11) M and 5 x 10(-12) M, respectively. OH-PCBs may disturb TH-dependent brain development.

Differences in development and cellular composition between neuronal cultures of rat accessory and main olfactory bulbs.

We previously established a primary culture system of the accessory olfactory bulb (AOB) to investigate the functional roles of individual types of neuron in pheromonal signal processing. However, the detailed characteristics of cultured AOB neurons were not yet apparent. In the present study, we address the cytological aspects of cultured AOB neurons using immunocytochemical staining methods. Cultured AOB neurons were compared with cultured main olfactory bulb (MOB) neurons in neuronal composition, maturational time course, and cell size. The number of total neurons, measured by microtubule-associated protein 2 (MAP2) immunostaining, progressively decreased, and glutamic acid decarboxylase positive (GAD+) interneurons were scarcely changed in their number in both AOB and MOB cultures over the culture periods. In contrast, the number of tyrosine hydroxylase positive (TH+) neurons in AOB cultures showed a slight, but significant, increase over time in culture, while those in MOB cultures remarkably decreased. The numbers of total neurons and GAD+ neurons were significantly greater in AOB cultures than in MOB cultures at all investigated time points. However, the numbers of TH+ neurons were lower at 7 days in vitro (DIV) and greater at 21 DIV in AOB cultures than in MOB cultures. The somatic sizes of all types of neurons at 14 DIV were significantly larger in AOB cultures than in MOB cultures. Furthermore, the frequency distributions of somatic sizes of total, GAD+, and TH+ neurons were significantly different between AOB and MOB cultures. These subtle differences in vitro may reflect in vivo differences between the AOB and MOB.

Developmental changes of GABAergic synapses formed between primary cultured cortical neurons.

The characteristics of functional changes of GABAergic synapses between cultured rat cortical neurons were observed by monitoring intracellular calcium level ([Ca2+]in) during development in vitro. After 5 days in vitro (DIV), cultured cortical neurons spontaneously exhibited synchronous oscillatory changes in [Ca2+]in, which were derived from synaptic activity. Exposure to bicuculline, antagonist of gamma-aminobutyric acid (GABA)(A) receptors, caused a marked decrease in the frequency of [Ca2+]in oscillations at 7-20 DIV. Although the frequency of spontaneous oscillations increased during this culture period, the ratio of the decrease in the frequency following bicuculline treatment did not significantly change. Thereafter, to investigate the detailed morphological changes of GABAergic synapses during development in vitro, the cultured neurons were immunostained with antibodies to glutamic acid decarboxylase (GAD), synaptophysin and GABA(A) receptor and were observed under a confocal laser microscope. Most of the GAD-positive puncta colocalized with synaptophysin-positive puncta and were opposed to GABA(A) receptor-positive structures. The images of GAD-positive puncta were reconstructed from the confocal three-dimensional data to analyze their number, volume, and surface area. The number of these puncta increased with culture time at 7-20 DIV. Although the volume of individual GAD-positive puncta did not significantly change, the surface area decreased in a time-dependent manner over the culture period. This system that we developed enabled us to investigate in detail the morphological and functional changes of GABAergic synapses during neuronal development.

Behavioral alterations in response to fear-provoking stimuli and tranylcypromine induced by perinatal exposure to bisphenol A and nonylphenol in male rats.

The purpose of this study was to examine whether perinatal exposure to two major environmental endocrine-disrupting chemicals, bisphenol A (BPA; 0.1 mg/kg/day orally) and nonylphenol [NP; 0.1 mg/kg/day (low dose) and 10 mg/kg/day (high dose) orally] daily from gestational day 3 to postnatal day 20 (transplacental and lactational exposures) would lead to behavioral alterations in the male offspring of F344 rats. Neither BPA nor NP exposure affected behavioral characteristics in an open-field test (8 weeks of age), in a measurement of spontaneous motor activity (12 weeks of age), or in an elevated plus-maze test (14 weeks of age). A passive avoidance test (13 weeks of age) showed that both BPA- and NP-treated offspring tended to delay entry into a dark compartment. An active avoidance test at 15 weeks of age revealed that BPA-treated offspring showed significantly fewer avoidance responses and low-dose NP-treated offspring exhibited slightly fewer avoidance responses. Furthermore, BPA-treated offspring significantly increased the number of failures to avoid electrical unconditioned stimuli within 5-sec electrical shock presentation compared with the control offspring. In a monoamine-disruption test using 5 mg/kg (intraperitoneal) tranylcypromine (Tcy), a monoamine oxidase inhibitor, both BPA-treated and low-dose NP-treated offspring at 22-24 weeks of age failed to show a significant increment in locomotion in response to Tcy, whereas control and high-dose NP-treated offspring significantly increased locomotion behavior after Tcy injection. In addition, when only saline was injected during a monoamine-disruption test, low-dose NP-treated offspring showed frequent rearing compared with the control offspring. The present results indicate that perinatal low-dose BPA or NP exposure irreversibly influenced the reception of fear-provoking stimuli (e.g., electrical shock), as well as monoaminergic neural pathways. Key words: behavior, bisphenol A, fear, learning, monoamine, nonylphenol.

Comparative study on toxicokinetics of bisphenol A in F344 rats, monkeys (Macaca fascicularis), and chimpanzees (Pan troglodytes).

We compared the toxicokinetics of bisphenol A (BPA) among three animal species: rats, cynomolgus monkeys and chimpanzees. Rats and monkeys were administered BPA orally or subcutaneously at 10 or 100 mg/kg body weight, while chimpanzees were administered only 10 mg/kg of BPA. BPA in serum was measured by ELISA. In oral administration of BPA at 10 mg/kg, both C(max) and AUC were rats < chimpanzee < monkeys. In oral administration of BPA at 100 mg/kg, both C(max) and AUC were rats < monkeys. Subcutaneous BPA administrations also revealed similar results, although the values of toxicokinetic parameters in subcutaneous administration were higher than those in oral administration. These results suggest that orally or subcutaneously administered BPA in primates is more easily absorbed than that in rats. We conclude that there are considerable differences in distribution, metabolism, and excretion of BPA between rodents and primates.

[Assay system for synapse formation between primary cultured neurons].

Synapse plasticity, in particular, formation of new synapses, plays crucial roles in learning and memory. We have developed a convenient assay system for measuring the number of newly formed synapses between cultured rat cerebrocortical neurons using the multisite fluorometry system of intracellular calcium. We found that cultured neurons exhibited spontaneous oscillatory changes in intracellular calcium levels and that the frequency of the oscillation was strongly correlated with synaptic density. Combined with immunohistochemical studies, this assay system enables us to study the molecular mechanism of synapse formation, in particular, the involvement of ecto-protein kinase. Other applications of the assay system are discussed here.

Endogenous dopamine maintains synchronous oscillation of intracellular calcium in primary cultured-mouse midbrain neurons.

We demonstrated synchronous oscillation of intracellular Ca2+ in cultured-mouse mid-brain neurons. This synchronous oscillation was thought to result from spontaneous and synchronous neural bursts in a synaptic neural network. We also examined the role of endogenous dopamine in neural networks showing synchronous oscillation. Immunocytochemical study revealed a few tyrosine hydroxylase (TH)-positive dopaminergic neurons, and that cultured neurons expressed synaptophysin and synapsin I. Western blot analyses comfirmed synaptophysin, TH, and 2 types of dopamine receptor (DR), D1R and D2R expression. The synchronous oscillation in midbrain neurons was abolished by the application of R(-)-2-amino-5-phosphonopentanoic acid (AP-5) as an N-methyl-D-aspartate receptor (NMDAR) antagonist. This result suggests that the synchronous oscillation in midbrain neurons requires glutamatergic transmissions, as was the case in previously reported cortical neurons. SCH-12679, a D1R antagonist, inhibited synchronous oscillation in midbrain neurons, while raclopride, a D2R antagonist, induced a transient increase of intracellular Ca2+ and inhibited synchronous oscillation. We consider that endogenous dopamine maintains synchronous oscillation of intracellular Ca2+ through D1R and D2R, and that these DRs regulate intracellular Ca2+in distinctly different ways. Synchronous oscillation of midbrain neurons would be a useful tool for in vitro researches into various neural disorders directly or indirectly caused by dopaminergic neurons.

Polychlorinated biphenyls suppress thyroid hormone receptor-mediated transcription through a novel mechanism.

Polychlorinated biphenyls (PCBs) are environmental compounds that disrupt the endocrine system, and exposure to low doses causes abnormalities, particularly in the developing central nervous system. Because they are structurally similar to thyroid hormone (TH), PCBs might affect systems involving this hormone. We previously found, using reporter assays, that hydroxylated-PCB at doses as low as 10(-10) m suppress TH-induced transcriptional activation of TH receptor (TR). To understand the molecular mechanisms of this process, we examined whether PCBs alter coactivator or corepressor interaction with TR. Polychlorinated biphenyls suppressed steroid receptor coactivator-1 enhanced TR-mediated transcription, but did not reduce TR/steroid receptor coactivator-1 interaction in mammalian two-hybrid and glutathione S-transferase pull-down studies. Thus, the suppression was probably not caused by coactivator dissociation. Nuclear receptor co-repressor was not recruited to TR by PCBs either in vivo or in vitro, indicating that TR corepressor binding did not induce the suppression. We then examined whether PCB influences TR binding to the thyroid hormone-response element (TRE). Electrophoretic mobility shift assays revealed that the TR/retinoid X receptor heterodimer complex was partially dissociated from TRE in the presence of PCB. These results indicate that partial dissociation of TR/retinoid X receptor heterodimer complex from the TRE is involved in the suppression of transcription induced by PCB.