Chemical nature of attention deficit hyperactivity disorder (ADHD) - related chemical subfamily.

Endocrine disruptors have been subjected to health risk assessments. Bioassays and chemoinformatics are very useful tools to characterize their chemical nature. By performing rat hyperactivity assays, we screened some endocrine disruptors, resulting in the classification of two groups: hyperactivity-associated and hyperactivity-negative chemicals. Moreover, many epidemiological studies have reported the correlation between most of the hyperactivity-associated chemicals identified in our bioassay and patients with attention deficit hyperactivity disorder (ADHD); thus, these chemicals are emerging as a subfamily of hyperactivity-associated chemicals among endocrine disruptors. Using RDKit, chemoinformatic analyses revealed no significant differences in the distribution of molecular weight between the two groups, but significant differences in "Fraction CSP3" (number of sp3-hybridized carbons/total carbon count) and the Tanimoto coefficient were observed. Additionally, hyperactivity-associated chemicals were distinguished from two known classes of dopaminergic toxins by the Tanimoto coefficient. Machine learning methods were also applied for classification, regression analyses, and prediction. A neural network model classified the two groups. Random forest methods also showed good prediction (R = 0.9, MAE (mean absolute error) = 0.06). Using a junction tree variational autoencoder, the core structure was interpolated between phthalate and phenol in the hyperactivity-associated group. Thus, I describe the chemical nature of a new chemical family that might promote the development of ADHD in humans.

Neonatal rotenone lesions cause onset of hyperactivity during juvenile and adulthood in the rat.

Attention deficit hyperactivity disorder (ADHD) is characterized by behavioral and cognitive symptoms. Longitudinal studies demonstrated that the symptoms remains clinically significant for the majority of ADHD children into adulthood. Furthermore, a population-based birth cohort provided the initial evidence of adult ADHD that lacks a history of childhood ADHD. We previously demonstrated that neonatal exposure to bisphenol A, an environmental chemical caused hyperactivity in the juvenile. Here, we extend to examine other chemical such as rotenone, a dopaminergic toxins. Oral administration of rotenone (3mg/kg) into 5-day-old male Wistar rats significantly caused hyperactivity at adulthood (8∼11 weeks old; p<0.05). It was about 1.3∼1.4-fold more active in the nocturnal phase after administration of rotenone than control rats. Higher dose (16mg/kg) or repeated lower dose of rotenone (1mg/kg/day for 4days) caused hyperactivity in the juvenile. Furthermore, DNA array analyses showed that neonatal exposure to rotenone altered the levels of gene expression of several molecules related to apoptosis/cell cycle, ATPase, skeletal molecule, and glioma. Bivariate normal distribution analysis indicates no correlation in gene expression between a hyperactivity disorder model and a Parkinson's disease model by rotenone. Thus, we demonstrate a rotenone models of ADHD whose onset varies during juvenile and adulthood.

Repulsive Apoptosis During Exposure of Mesencephalic Neural Stem Cells to Silver Nanoparticles in a Neurosphere Assay In Vitro.

Neurodevelopmental toxicity of silver nanoparticles (AgNPs) remains largely unknown. In this study, we applied a neurosphere assay for neurodevelopmental effects of AgNPs. The neural stem cells were isolated from rat mesencephalon. They were cultured as a sphere. In an assay with coated plates, cells appeared by anchoraging on the dish and then started to migrate along the radial axis from the neurosphere. AgNPs inhibited cell migration in a dose-dependent manner. There was a linear correlation between the inhibition of migration and the logarithm of the particle concentration (1.25-10 µg/ml); the half-maximal inhibitory concentration was 0.41 µg/ml for 16-h exposure. Preceding migrated cells were retarded and/or collapsed by exposure to AgNPs: lower doses of AgNPs (0.31-1.2 µg/ml) caused a 42% retardation for 48 h, while higher doses of AgNPs (2.5-10 µg/ml) collasped migrating cells. Furthermore, collapsed cells were TUNEL-positive and showed a defect in the mitochondrial membrane potential. Thus, we showed the neurodevelopmental toxicity of AgNPs using an in vitro neurosphere assay system.

Classification of phthalates based on an in vitro neurosphere assay using rat mesencephalic neural stem cells.

Exposure to environmental neurotoxic chemicals both in utero and during the early postnatal period can cause neurodevelopmental disorders. To evaluate the disruption of neurodevelopmental programming, we previously established an in vitro neurosphere assay system using rat mesencephalic neural stem cells that can be used to evaluate. Here, we extended the assay system to examine the neurodevelopmental toxicity of the endocrine disruptors butyl benzyl phthalate, di-n-butyl phthalate, dicyclohexyl phthalate, diethyl phthalate, di(2-ethyl hexyl) phthalate, di-n-pentyl phthalate, and dihexyl phthalate at a range of concentrations (0-100 µM). All phthalates tested inhibited cell migration with a linear or non-linear range of concentrations when comparing migration distance to the logarithm of the phthalate concentrations. On the other hand, some, but not all, phthalates decreased the number of proliferating cells. Apoptotic cells were not observed upon phthalate exposure under any of the conditions tested, whereas the dopaminergic toxin rotenone induced significant apoptosis. Thus, we were able to classify phthalate toxicity based on cell migration and cell proliferation using the in vitro neurosphere assay.

Rat hyperactivity by bisphenol A, but not by its derivatives, 3-hydroxybisphenol A or bisphenol A 3,4-quinone.

Detoxification in the central nervous system is largely unknown. The mechanism of neurotoxicity of bisphenol A, a toxic environmental chemical remains obscure. We examined the effects of bisphenol A, and its derivatives, 3-hydroxybisphenol A and bisphenol A 3,4-quinone on rat behavior as possible metabolites of bisphenol A. A single intracisternal administration of bisphenol A (20 µg equivalent to 87 nmol) into 5-day-old male Wistar rats caused significant hyperactivity at 4-5 weeks of age. It was about 1.3 fold more active in the nocturnal phase than control rats. However, neither 3-hydroxybisphenol A nor bisphenol A 3,4-quinone at the same amount (87 nmol) increased the spontaneous motor activity. Gas chromatographic-mass spectrometric (GC-MS) analyses of the treated brain revealed that 7% of the parent chemical resided in the brain at 8 weeks of age, but its derivatives were not found. This suggested a difference in metabolic turnover of these compounds or a difference in their stabilities. We conclude that bisphenol A per se caused hyperactivity in the rat, eliminating the possibility that possible metabolic forms of bisphenol A, 3-hydroxybisphenol A and bisphenol A 3,4-quinone have the ability to elicit rat hyperactivity, probably because of longer-lasting residence of the parent compound in the brain.

Neurotoxicity of endocrine disruptors: possible involvement in brain development and neurodegeneration.

Environmental chemicals that act as endocrine disruptors do not appear to pose a risk to human reproduction; however, their effects on the central nervous systems are less well understood. Animal studies suggested that maternal exposure to endocrine-disrupting chemicals (EDC) produced changes in rearing behavior, locomotion, anxiety, and learning/memory in offspring, as well as neuronal abnormalities. Some investigations suggested that EDC exert effects on central monoaminergic neurons, especially dopaminergic neurons. Our data demonstrated that EDC attenuate the development of dopaminergic neurons, which might be involved in developmental disorders. Perinatal exposure to EDC might affect neuronal plasticity in the hippocampus, thereby potentially modulating neuronal development, leading to impaired cognitive and memory functions. Endocrine disruptors also attenuate gender differences in brain development. For example, the locus ceruleus is larger in female rats than in males, but treatments with bisphenol-A (BPA) enlarge this region in males. Some reports indicated that EDC induce hypothyroidism, which might be evidenced as abnormal brain development. Endocrine disruptors might also affect mature neurons, resulting in neurodegenerative disorders such as Parkinson's disease. The current review focused on alterations in the brain induced by EDC, specifically on the possible involvement of EDC in brain development and neurodegeneration.

Transcriptome of tributyltin-induced apoptosis of the cultured rat mesencephalic neural stem cells.

Exposure to environmental neurotoxic chemicals both in utero and during the early postnatal period can cause neurodevelopmental disorders. To evaluate the disruption of neurodevelopmental programming, we previously established an in vitro neurosphere assay system, using rat mesencephalic neural stem cells (mNSC). Here, we examined the developmental neurotoxicity of tributyltin (TBT) in an in vitro neurosphere assay. A neurosphere was driven from rat E16 mesencephalon and seeded in a poly-l-ornithine/laminin-coated plate. Exposure to TBT increased the number of terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL)-positive cells in time-dependent and dose-dependent manner: it was significantly detectable with treatment of 100pM TBT for 90min, or of 1µM TBT for 30min. Disruption of mitochondrial membrane potential and activation of caspase-3/7 were concomitantly observed. Furthermore, DNA microarray analyses using Affymetrix GeneChip revealed that as early as 0.5h after exposure to the metal (1µM), the expression levels of 71 genes were increased by more than 2-fold, whereas those of 8 genes were decreased by 2-fold or less: it was notably altered in expression of Ca(2+)-mobilizing-related genes, and retinoic-acid signal-related genes, as well as bifunctional apoptosis-related genes. The levels of gene expression of Wnt family were also significantly changed. Thus, we established transcriptome of TBT-induced apoptosis of mNSC. This would help to evaluate developmental neurotoxicity of TBT in vivo, contributing to the risk assessment methods based on infant physiology.

Activation of STAT3 by pituitary adenylate cyclase-activating polypeptide (PACAP) during PACAP-promoted neurite outgrowth of PC12 cells.

Addition of pituitary adenylate cyclase-activating polypeptide (PACAP) into the cultured PC12 cells promoted neurite outgrowth of the cells, indicating cell differentiation. Using DNA macroarray techniques, we have characterized the PC12 cell transcriptome, revealing that several genes were regulated by PACAP. Among many, we focused to investigate whether STAT3 molecule, whose message was up-regulated, might be involved in PACAP signaling. Reverse transcription polymerase chain reaction supported the results obtained by DNA macroarray; PACAP increased gene expression of STAT3, at least up to 24 h, being a maximum 9.5-fold with 3 h-treatment of 1 nM PACAP. Reporter assay analyses demonstrated that PACAP activated STAT3-response promoter, but neither GAS- nor ISRE-response promoters. Enzyme-linked immunosorbent assay revealed that PACAP increased the amount of STAT3 proteins about 30%. Concurrently, phospho-STAT3 Tyr705 was increased in the nuclei by the neuropeptide. Ectopic expression of dominant negative forms of STAT3, which had Tyr705 to phenylalanine substitution, effectively decreased PACAP-promoted neurite extension (55%). During the activation of STAT3, the secretion of interleukin-6 (IL-6) was stimulated by PACAP in a dose-dependent manner. Treatment with IL-6-neutralizing antibody significantly diminished PACAP-activated STAT3 promoter activity and PACAP-induced neurite outgrowth. Thus, our findings have demonstrated that STAT3 is involved in PACAP signaling and function during PC12 cell differentiation by the neuropeptide.

Inhibition by rotenone of mesencephalic neural stem-cell migration in a neurosphere assay in vitro.

Parkinson disease is an age-related neurodegenerative disorder. Although the underlying pathophysiological mechanisms are incompletely understood, it has been suggested that environmental origins of sporadic Parkinson disease occur early in life. Here we examined the in vitro effects of the environmental dopaminergic toxin rotenone on neural stem cells, derived from the rat E16 mesencephalon. The neurospheres were cultured on the uncoated glass dishes. Cells emerged from the neurospheres and migrated along the radial axis. The migrating populations comprised cells that were positive for nestin, microtubule-associated proteins, and glial fibrillary acidic protein. Exposure to rotenone inhibited cell migration, decreased proliferative cells in a dose-dependent manner, and increased the number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells. Quantitative analysis revealed a linear function between the inhibition of migration and the rotenone concentration. Thus, we showed for the first time that rotenone exerted inhibitory effects on the migration as well as the proliferation of neural stem cells in vitro.

Mesencephalic neurodegeneration in the orally administered bisphenol A-caused hyperactive rats.

Since an emerging body of evidence is accumulating that endocrine disruptors exert their effects on the central nervous system, their neuronal risk assessment is now required. A previous study showed that a single intracisternal administration of bisphenol A, an endocrine disruptor, into neonatal rats caused hyperactivity. To evaluate the neural risk assessment of bisphenol A, it is very important to test the potential of the chemical via an environmental exposure route. In this study, we tested the hypothesis that oral exposure to bisphenol A would exhibit effects observed previously with direct instillation. Oral administration of 600mug/pup/day bisphenol A into male Wistar rats aged 5 days-3 weeks caused significant hyperactivity at 4-5 weeks of age. Treated rats were about 1.3 times as active in the nocturnal phase as were vehicle-treated control rats (p<0.005). The long-term effects of the chemical resulted in a large reduction of immunoreactivity for tyrosine hydroxylase in the midbrain at 7 weeks of age, where terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL)-positive cells were detected. Furthermore, bisphenol A decreased gene expression levels of dopamine transporter in adult rats.

Behavioural characteristics and gene expression in the hyperactive wiggling (Wig) rat.

Recently, congenic wiggling (Wig) rats were described as a good model for attention-deficit hyperactivity disorder; 12- to 14-week-old animals demonstrated hyperactivity, impulsive behaviour and an impaired working memory. Here, we show that 4- to 5-week-old Wig rats displayed significantly greater spontaneous motor activity than control rats during a period of darkness. Subcutaneous injection of 4 mg/kg methamphetamine exacerbated hyperactivity, the reverse of its effect in rats with neonatally induced 6-hydroxydopamine lesions. Immunohistochemistry showed low levels of tyrosine hydroxylase in the ventral midbrain, similar to 6-hydroxydopamine-treated rats. In cDNA macroarrays, 4-week-old Wig rats showed increased expression of the adenosine A2a receptor in the dorsal striatum, macrophage migration inhibitory factor in the frontal cortex, ventral striatum and midbrain, and calbindin 2 in the dorsal and ventral midbrain. Expression of the gamma-aminobutyric acid (GABA) transporter and sterol carrier protein 2 genes was reduced in all regions. Dopamine transporter gene expression was increased in the dorsal midbrain but decreased in the ventral midbrain, a pattern distinct from that induced by 6-hydroxydopamine. Although abnormal development of dopaminergic neurons may underlie motor hyperactivity, other mechanisms may control responsiveness to methamphetamine. Wig rats may provide a model of attention-deficit hyperactivity disorder in which treatment with psychostimulants accelerate the hyperactivity.

Melatonin inhibits maneb-induced aggregation of alpha-synuclein in rat pheochromocytoma cells.

Melatonin, a secretory product of the pineal gland, is involved in the regulation of circadian and seasonal rhythms, in oncostasis, and in inducing osteoblast differentiation. Furthermore, melatonin is a scavenger of a number of reactive oxygen and reactive nitrogen species both in vitro and in vivo. In this study, the antioxidant nature of melatonin was shown to prevent cultured neural cells from apoptosis induced by endocrine-disrupting chemical, maneb. The neurotoxicity of the fungicide, maneb (1 microg/mL), on the PC12 cells was elicited through apoptotic cell death, concomitant with aggregation of alpha-synuclein, a feature of Parkinson's disease. Activation of caspase-3/7 was associated with this process. A fluorescence rationing technique using a mitochondrial dye revealed that maneb altered the mitochondrial membrane potential of the neural cells. However, melatonin (1 nm) largely prevented the neural cells from the neural toxicant by inhibition of both caspase-3/7 activation and disruption of the mitochondrial transmembrane potential. Furthermore, aggregation of alpha-synuclein by maneb was also inhibited by melatonin. Thus, melatonin prevents maneb-induced neurodegeneration at a nighttime physiological blood concentration, most likely by inhibiting the aggregation of alpha-synuclein as well as preventing mitochondrial dysfunction in PC 12 cells.

Region-dependent differences and alterations of protective thiol compound levels in cultured astrocytes and brain tissues.

We examined region-dependent differences and alterations in the levels of protective thiol compounds, glutathione (GSH) and metallothionein (MT)-I and -II, in cultured rat astrocytes under several culture conditions and in brain tissues of rats at postnatal and weaning periods. Regardless of culture conditions, both protein concentrations and mRNA expressions of MT-I and -II were much higher in the cerebral hemisphere than in cerebellar astrocytes, whereas no difference was observed in GSH concentration. In both astrocytes, the GSH concentrations did not change within 12 h but significantly increased 24 h after being maintained in a serum-free defined medium. At 24 h, protein concentrations and mRNA expressions of MT-I and -II also increased in the respective astrocytes, and were further enhanced when maintained in the presence of 50 microM Zn(2+). In the brain tissues, the MT-I/-II protein concentrations were significantly higher in the cerebral cortex (a part of the cerebral hemisphere) than in the cerebellum, whereas the GSH concentration was similar at both postnatal day (P)1 and P35. In addition, the concentrations in the respective regions were significantly higher at P35 than at P1. These results suggest that region-dependent differences in the cellular levels of GSH and MTs in cultured astrocytes might reflect the in vivo differences, and that the levels of the respective thiol compounds in cultured astrocytes increase after serum elimination along with the region-dependent differences.

Alteration of gene expression of G protein-coupled receptors in endocrine disruptors-caused hyperactive rats.

We examined the effects of endocrine disruptors on rat behavioral and cellular responses. Single intracisternal administration of bisphenol A, p-octylphenol, nonylphenol, dibutylphthalate (DBP), dicyclohexylphthalate (DCHP), or diethylhexylphthalate (DEHP) into 5-day-old male Wistar rats caused significant hyperactivity at 4-5 weeks of age. It was about 1.3- to 1.6-fold more active in the nocturnal phase than control rats. Based on DNA macroarray analyses of the midbrain at 8 weeks of age, the endocrine disruptors altered the levels of gene expression of G protein-coupled receptors that were involved in not only dopaminergic neurotransduction but also many peptidergic neurotransduction. The gene expression of dopamine receptor D1A was decreased by nonylphenol, DBP, or DEHP by 0.23- to 0.4-fold, whereas that of dopamine D2 was increased by nonylphenol or DBP by 2- to 2.8-fold. It was notable that four of six endocrine disruptors tested, i.e. nonylphenol, DBP, DCHP, and DEHP largely downregulated the levels of gene expression of galanin receptor 2 by 0.11- to 0.28-fold. Bisphenol A, DBP or DCHP significantly decreased the levels of gene expression of dopamine transporter at 8 weeks more than 0.5-fold. Immunohistochemical analyses revealed that p-octylphenol impaired the immunoreactivity for tyrosine hydroxylase in substantia nigra pars compacta. Thus, endocrine disruptors caused hyperactivity in the rat, probably regulating the levels not only of gene expression but also of proteins of both G-protein-coupled receptors systems and dopaminergic neurotransduction system.

Overexpression of Bcl-2 inhibits nuclear localization of annexin I during tumor necrosis factor-alpha-mediated apoptosis in porcine renal LLC-PK1 cells.

The addition of tumor necrosis factor (TNF)-alpha into the cultured porcine kidney LLC-PK1 cells caused apoptosis concomitantly with caspase-3 activation and the inductions of an endogenous Bcl-2 protein. An SDS-polyacrylamide electrophoretic analysis revealed that a 37-kDa protein in a nuclear fraction was increased during TNF-alpha-induced apoptosis. Partial amino acid sequence of the protein was A-L-T-G-H-L-E-E-V, perfectly matching that of annexin I. Immunocytochemistry revealed that annexin I migrated to the nucleus and/or peri-nucleus region upon exposure to TNF-alpha. Overexpression of Bcl-2 proteins inhibited the nuclear localization of annexin I during TNF-alpha-induced apoptosis. Antisense oligodeoxynucleotides complementary to annexin I-inhibited TUNEL (terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labelling) staining in TNF-alpha-treated cells, suggesting that annexin I expression is a possible prerequisite for the induction of apoptosis by the cytokine. Thus, it is first time to show that annexin I is regulated by an anti-apoptotic Bcl-2 protein in TNF-alpha-induced renal apoptotic events.

Transcriptome of pituitary adenylate cyclase-activating polypeptide-differentiated PC12 cells.

Addition of pituitary adenylate cyclase-activating polypeptide (PACAP) into the cultured PC12 cells secreted dopamine and promoted neurite outgrowth of the cells, indicating cell differentiation. To characterize the PACAP-differentiated PC12 cell transcriptome, we applied DNA macroarray techniques, using Atlas Rat 1.2 Array membranes (BD Biosciences Clontech) that have 1176 cDNA. RNA samples were harvested from PC12 cells before and at a time of 6 h treatment with 1 nM PACAP, when neuritogenesis was remarkably observed under the condition used. Several genes regulated by PACAP have been associated with neuritogenesis (i.e. villin 2 and tissue plasminogen activator) or cell growth/differentiation (i.e. cyclin or ornitine decarboxylase). Also, cytoskeleton proteins such as actin or tubulin were up-regulated for cell morphology remodeling. A message of vehicle trafficking molecule (synaptotagmin IV) was more remarkably increased (3.95-6.85-fold). Signaling molecules such as small G proteins (rab12, rab16, or ral), IkappaB, or STAT3 were altered by PACAP. It is noteworthy that PACAP inhibited the expression of galanin receptor 2, whose ligand was shown to inhibit tyrosine hydroxylase activity. Thus, in this study the transcriptome of PACAP-differentiated PC12 was established, leading to the elucidation of the molecular mechanism of neuritogenesis by the neuropeptide.

Motor hyperactivity caused by a deficit in dopaminergic neurons and the effects of endocrine disruptors: a study inspired by the physiological roles of PACAP in the brain.

Recent studies have revealed that the pituitary adenylate cyclase-activating polypeptide (PACAP) might act as a psychostimulant. Here we investigated the mechanisms underlying motor hyperactivity in patients with pervasive developmental disorders, such as autism, and attention-deficit hyperactivity disorder (ADHD). We studied the effects of intracisternal administration of 6-hydroxydopamine (6-OHDA) or endocrine disruptors (EDs) on spontaneous motor activity (SMA) and multiple gene expression in neonatal rats. Treatment with 6-OHDA caused significant hyperactivity during the dark phase in rats aged 4-5 weeks. Motor hyperactivities also were observed after treatment with endocrine disruptors, such as bisphenol A, nonylphenol, diethylhexyl phthalate and dibutyl phthalate, during both dark and light phases. Gene-expression profiles produced using cDNA macroarrays of 8-week-old rats with 6-OHDA lesions revealed the altered expression of several classes of gene, including the N-methyl-D-aspartate (NMDA) receptor 1, glutamate/aspartate transporter, gamma-aminobutyric-acid transporter, dopamine transporter 1, D4 receptor, and peptidergic elements such as the galanin receptor, arginine vasopressin receptor, neuropeptide Y and tachykinin 2. The changes in gene expression caused by treatment with endocrine disruptors differed from those induced by 6-OHDA. These results suggest that the mechanisms underlying the induction of motor hyperactivity and/or compensatory changes in young adult rats might differ between 6-OHDA and endocrine disruptors.

Motor activity and gene expression in rats with neonatal 6-hydroxydopamine lesions.

A rat model of a hyperkinetic disorder was used to investigate the mechanisms underlying motor hyperactivity. Rats received an intracisternal injection of 6-hydroxydopamine on post-natal day 5. At 4 weeks of age, the animals showed significant motor hyperactivity during the dark phase, which was attenuated by methamphetamine injection. Gene expression profiling was carried out in the striatum and midbrain using a DNA macroarray. In the striatum at 4 weeks, there was increased gene expression of the NMDA receptor 1 and tachykinins, and decreased expression of a GABA transporter. At 8 weeks, expression of the NMDA receptor 1 in the striatum was attenuated, with enhanced expression of the glial glutamate/aspartate transporter. In the midbrain, a number of genes, including the GABA transporter gene, showed decreased expression at 4 weeks. At 8 weeks, gene expression was augmented for the dopamine transporter, D4 receptor, and several genes encoding peptides, such as tachykinins and their receptors. These results suggest that in the striatum the neurotransmitters glutamate, GABA and tachykinin may play crucial roles in motor hyperactivity during the juvenile period. Several classes of neurotransmitters, including dopamine and peptides, may be involved in compensatory mechanisms during early adulthood. These data may prompt further neurochemical investigations in hyperkinetic disorders.

Dicyclohexylphthalate causes hyperactivity in the rat concomitantly with impairment of tyrosine hydroxylase immunoreactivity.

Endocrine disruptors possibly exert effects on neuronal functions leading, in particular, to behavioural alterations. In this study, we examined the effects of dicyclohexylphthalate (DCHP), an endocrine disruptor, on rat behavioural and cellular responses. Single intracisternal administration of DCHP (0.87-87 nmol) into 5-day-old male Wistar rats caused significant hyperactivity at 4-5 weeks of age. It was about 1.4-fold more active in the nocturnal phase after administration of 87 nmol of DCHP than control rats (p < 0.001). The response had a tendency to be dose-dependent. Based on DNA macoarray analyses, DCHP down-regulated the levels of gene expression of the dopamine D4 receptor at 4 weeks old in both the midbrain and the striatum, and the dopamine transporter in the midbrain at 8 weeks old 1.7- to 2-fold. The gene expression of several subtypes of glutamate receptors was facilitated in the striatum at 4 weeks old and in the midbrain at 8 weeks old. Some normalization and/or compensatory changes seemed to occur in gene expression of GABA or glycine transmission. Furthermore, DCHP abolished immunoreactivity of tyrosine hydroxylase in the substantia nigra at 8 weeks of age, where TUNEL-positive cells were seen. We conclude that DCHP affected the developing rat brain, resulting in hyperactivity, probably as a result of degeneration of mesencephalic tyrosine hydroxylase rather than alteration of the level of gene expression.

Effects of neonatal treatment with 6-hydroxydopamine and endocrine disruptors on motor activity and gene expression in rats.

To investigate the mechanisms underlying motor hyperactivity, we performed intracisternal injection of 6-hydroxydopamine or endocrine disruptors in rats on postnatal day 5. 6-Hydroxydopamine (100 microg, 488 nmol) caused a significant increase in spontaneous motor activities at 4 weeks of age. Gene-expression profiling using a cDNA membrane array revealed alterations in several classes of gene at 8 weeks of age. In the midbrain, gene expression was enhanced in dopamine transporter 1; a platelet-derived growth factor receptor; dopamine receptor D4; galanin receptor 2; arginine vasopressin receptor 2; neuropeptide Y; tachykinin 2; and fibroblast growth factor 10. Expression was also enhanced in the glutamate/aspartate transporter gene in the striatum. Rats received an endocrine disruptor (87 nmol), such as bisphenol A, nonylphenol, p-octylphenol, or diethylhexylphthalate, which also caused motor hyperactivity at 4 weeks. The effects of bisphenol A on motor activity were dose-dependent from 0.87 to 87 nmol. The phenols caused a deficit in dopamine neurons, similarly to the deficit caused by 6-hydroxydopamine. Gene-expression profiles after treatment with endocrine disruptors showed variation and differed from those of 6-hydroxydopamine. The results suggest that neonatal treatment with environmental chemicals can generate an animal model of attention-deficit hyperactivity disorder, in which clinical symptoms are pervasive.