Hypothalamic Monoaminergic Pathology in a Neurodevelopmental Rat Model Showing Prenatal 5-Bromo-2'-Deoxyuridine Treatment-Induced Hyperactivity and Hyporeproductivity.

OBJECTIVE: Prenatal treatment of rats with 5-bromo-2'-deoxyuridine (BrdU) is a neurodevelopmental model showing hyperactivity and impaired sexual activity. Human neurodevelopmental disorders, such as autism, exhibit sex-related pathology, but sex-related neurodevelopment has not been fully investigated in this model. We conducted this study to facilitate the understanding of the pathophysiology of neurodevelopmental disorders. METHODS: Pregnant rats received 50 mg/kg BrdU on gestational days 9-15. The tissue content of dopamine (DA), serotonin (5-HT), and their metabolites dihydroxyphenylacetic acid, homovanillic acid, and 5-hydroxyindoleacetic acid were measured in male and female offspring at 3 weeks (juveniles) and 10 weeks (adults) of age. RESULTS: Prenatally BrdU-treated rats had reduced DA metabolism or DA content in the hypothalamus from the juvenile through the adult period without sex differences, but sex-specific striatal DA abnormalities emerged after maturation. A reduction in 5-HT metabolism was measured in the hypothalamus without sex differences throughout development. Developmental alterations in the striatal 5-HT states were sex-dependent. Temporal changes in DA or 5-HT metabolism were found in the frontal cortex and midbrain. CONCLUSION: The sex-specific influence of a genotoxic factor on the development of the DA and 5-HT systems was clarified in the hypothalamus and striatum. The results suggest that the observed sex dependence and region specificity are related to the pathology of social dysfunction in neurodevelopmental disorders.

Stress-reactive rats (high-avoidance female rats) have a shorter lifespan than stress-nonreactive rats (low-avoidance female rats).

Although Hatano high-avoidance and low-avoidance rats (HAA and LAA, respectively) have been selectively bred for good versus poor avoidance learning, HAA rats are known to be more reactive to stress than LAA rats. In this study, HAA and LAA female rats were compared during reproductive aging by observing estrous cycles from 8 to 11 months of age. Furthermore, these rats were allowed to live out their natural lifespans, that is, until 24 months of age, in order to compare their survival and to clarify the relationship between reproductive aging and tumor development. At eight months of age, 2 of 35 HAA rats and 20 of 35 LAA rats had abnormal estrous cycles. The median lifespan of the HAA rats (673 days) was shorter than that of the LAA rats (733 days). The incidence of pituitary neoplasia was higher in the HAA rats than in the LAA rats. These results suggest that HAA female rats (i.e., stress-reactive rats) have a shorter lifespan than LAA female rats (i.e., stress-nonreactive rats) and develop pituitary neoplasia, which was one of the causal factors in their accelerated mortality. However, the onset of an age-matched abnormal cycle did not correspond with their lifespan.

Mother/offspring co-administration of the traditional herbal remedy yokukansan during the nursing period influences grooming and cerebellar serotonin levels in a rat model of neurodevelopmental disorders.

Neurodevelopmental impairment in the serotonergic system may be involved in autism spectrum disorder. Yokukansan is a traditional herbal remedy for restlessness and agitation in children, and mother-infant co-administration (MICA) to both the child and the nursing mother is one of the recommended treatment approaches. Recent studies have revealed the neuropharmacological properties of Yokukansan (YKS), including its 5-HT1A (serotonin) receptor agonistic effects. We investigated the influence of YKS treatment on behavior in a novel environment and on brain monoamine metabolism during the nursing period in an animal model of neurodevelopmental disorders, prenatally BrdU (5-bromo-2'-deoxyuridine)-treated rats (BrdU-rats). YKS treatment did not influence locomotor activity in BrdU-rats but reduced grooming in open-field tests. YKS treatment without MICA disrupted the correlation between locomotor behaviors and rearing and altered levels of serotonin and its metabolite in the cerebellum. These effects were not observed in the group receiving YKS treatment with MICA. These data indicate a direct pharmacological effect of YKS on the development of grooming behavior and profound effects on cerebellar serotonin metabolism, which is thought to be influenced by nursing conditions.

Usefulness of combined in vivo skin comet assay and in vivo skin micronucleus test.

We have already found that the in vivo skin comet assay is useful for the evaluation of primary DNA damage induced by genotoxic chemicals in epidermal skin cells. The aim of the present study was to evaluate the sensitivity and specificity of the combined in vivo skin comet assay and in vivo skin micronucleus (MN) test using the same animal to explore the usefulness of the new test method. The combined alkaline comet assay and MN test was carried out with three chemicals: 4-nitroquinoline-1-oxide (4NQO), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and benzo[a]pyrene (B[a]P). In the first experiment, we compared DNA- and chromosome-damaging effects of 3 [72, 24 and 3 hours (h) before sacrifice] and 4 applications (72, 48, 24 and 3h before sacrifice) of 4NQO, which induces dermal irritancy. The animals were euthanized and their skin was sampled for the combination test. As a result, the 4-application method was able to detect both DNA- and chromosome-damaging potential with a lower concentration; therefore, in the second experiment, MNNG and B[a]P were topically applied four times, respectively. The animals were euthanized, and then their skins were sampled for combination tests. In the alkaline comet assay, significant differences in the percent of DNA (%DNA) in the tail were observed in epidermal skin cells treated with MNNG and B[a]P. In the MN test, an increased frequency of MN cells (%MN) cells was observed by treatment with MNNG; however, there were no significant increases. In contrast, significant differences in %MN were observed by treatment with B[a]P. From these results, we conclude that the combined in vivo skin comet assay and in vivo MN test was useful because it can detect different genotoxicity with the same sampling time and reduce the number of animals used.

Current activities between the DevTox Berlin workshops and the Japanese Teratology Society Terminology Committee in harmonizing the terminology for classifying anomalies in laboratory animals in developmental toxicity studies: Report from the Satellite Workshop of the 60th Annual Meeting of the Japanese Teratology Society.

In recent years, the Japanese Teratology Society has worked with the DevTox Berlin Workshops project to provide internationally consistent terminology for teratogenic effects. This paper summarizes a satellite workshop of the 60th Annual Meeting of the Japanese Teratology Society, which was entitled "Current activities between DevTox Berlin Workshops to develop a harmonized terminology for classifying anomalies in laboratory animals in developmental toxicity studies." The Japanese Teratology Society - Laboratory Animal Terminology Project (JTS-LATP) reviewed "gray zone" anomalies and focused on developing criteria for reclassifying a large number of gray zone anomalies to clarify them and to make it easier to judge fetal categories. This effort will lead to international agreement, based on shared conceptions. The present article aimed to provide the reader with a summary of the issues discussed at the 2020 satellite meeting, which included discussions on open issues from the DevTox Berlin Workshops, ongoing work by the JTS-LATP on gray zone (GZ) anomalies, current industrial concerns, and future challenges.

Use of the in vivo skin comet assay to evaluate the DNA-damaging potential of chemicals applied to the skin.

The aim of the present study was to evaluate both sensitivity and specificity of an in vivo skin comet assay using chemically treated, hairless mouse dorsal skin as a model. N-methyl-N'-nitro-N-nitrosoguanidine (MNNG, 0.0125-0.2%), 4-nitroquinoline-1-oxide (4NQO, 0.01-0.25%), mitomycin C (MMC, 0.0125-0.05%), benzo[a]pyrene (B[a]P, 0.25-2%), and 7,12-dimethylbenz[a]anthracene (DMBA, 0.25-1%) were each applied once to the dorsal skin of hairless male mice; after 3h, epidermal skin cells were isolated, and the alkaline comet assay was performed. The assay was performed after 24h for only the B[a]P and DMBA. Furthermore, B[a]P and DMBA were evaluated by alkaline comet assay using liver cells after both 3 and 24h. The mean percent of DNA (%DNA) in tail in the 0.05-0.2% MNNG and 0.1-0.25% 4NQO treatment groups was markedly higher than in the control group at 3h post-application. Although the mean %DNA values in the tail in the B[a]P and DMBA groups were the same as the controls at 3h post-application, the 2% B[a]P and 1% DMBA groups showed significantly higher values versus controls 24h after application. No significant increases in the mean %DNA in the tail were observed in the MMC group. No clear increases in %DNA in the tail were observed in the B[a]P and DMBA groups at 3 or 24h after application in the liver. These results suggest that the in vivo skin comet assay is able to accurately identify DNA-damaging potential with a skin-specific response and is a useful method to detect the DNA-damaging potential of genotoxic chemicals on the skin.

Pharmacokinetics of primary metabolites 5-hydroxythalidomide and 5'-hydroxythalidomide formed after oral administration of thalidomide in the rabbit, a thalidomide-sensitive species.

The teratogenicity of the chemotherapeutic drug thalidomide is species-specific and affects humans, non-human primates, and rabbits. The primary oxidation of thalidomide in previously investigated rodents predominantly resulted in the formation of deactivated 5'-hydroxythalidomide. In the current study, similar in vivo biotransformations to 5-hydroxythalidomide and 5'-hydroxythalidomide were confirmed by the analysis of blood plasma from male rabbits, a thalidomide-sensitive species, after oral administration of thalidomide (2.0 mg/kg). Similar levels of thalidomide in seminal plasma and in blood plasma were detected using liquid chromatography-tandem mass spectrometry at 4 hr and 7 hr after oral doses in male rabbits. Seminal plasma concentrations of 5-hydroxythalidomide and 5'-hydroxythalidomide were also seen in male rabbits in a roughly similar time-dependent manner to those in the blood plasma after oral doses of thalidomide (2.0 mg/kg). Furthermore, the values generated by a simplified physiologically based pharmacokinetic rabbit model were in agreement with the measured in vivo blood plasma data under metabolic ratios of 0.01 for the hepatic intrinsic clearance of thalidomide to both unconjugated 5-hydroxythalidomide and 5'-hydroxythalidomide. These results suggest that metabolic activation of thalidomide may be dependent on rabbit liver enzymes just it was for cytochrome P450 enzymes in humanized-liver mice; in contrast, rodent livers predominantly mediate biotransformation of thalidomide to 5'-hydroxythalidomide. A developmental toxicity test system with experimental animals that involves intravaginal exposures to the chemotherapeutic drug thalidomide via semen should be considered in the future.

Constructing a developmental and reproductive toxicity database of chemicals (DART NIHS DB) for integrated approaches to testing and assessment.

Developmental and reproductive toxicity (DART) is an important endpoint, and databases (DBs) are essential for evaluating the risk of untested substances using alternative methods. We have constructed a reliable and transparent DART DB, which we named DART NIHS DB, using the publicly available datasets of DART studies of industrial chemicals conducted by Japanese government ministries in accordance with the corresponding OECD test guidelines (OECD TG421 and TG422). This DB is unique because its dataset chemicals have little overlap with those of ToxRefDB, which compiles large-scale DART data, and it is reliable because the included datasets were created after reviewing the individual study reports. In DART NIHS DB, 171 of 404 substances exhibited signs of DART, which occurred during fertility and early embryonic development (49 substances), organogenesis (59 substances), and the perinatal period (161 substances). When the lowest-observed-adverse-effect level (LOAEL) of DART was compared with that of repeated-dose toxicity (RDT), 15 substances (12%) had a lower LOAEL for DART than for RDT. Of these, five substances displayed significant DART at doses of ≤ 50 mg/kg bw/day. The chemical and toxicity information in this DB will be useful for the development of stage-specific adverse outcome pathways (AOPs) via integration with mechanistic information. The whole datasets of the DB can be implemented in read-across support tools such as the OECD QSAR Toolbox, which will further lead to future integrated approaches to testing and assessment based on AOPs.

Visualizing the spatial localization of ciclesonide and its metabolites in rat lungs after inhalation of 1-µm aerosol of ciclesonide by desorption electrospray ionization-time of flight mass spectrometry imaging.

Inhaled ciclesonide (CIC), a corticosteroid used to treat asthma that is also being investigated for the treatment of corona virus disease 2019, hydrolyzes to desisobutyryl-ciclesonide (des-CIC) followed by reversible esterification when exposed to fatty acids in lungs. While previous studies have described the distribution and metabolism of the compounds after inhalation, spatial localization in the lungs remains unclear. We visualized two-dimensional spatial localization of CIC and its metabolites in rat lungs after administration of a single dose of a CIC aerosol (with the mass median aerodynamic diameter of 0.918-1.168 µm) using desorption electrospray ionization-time of flight mass spectrometry imaging (DESI-MSI). In the analysis, CIC, des-CIC, and des-CIC-oleate were imaged in frozen lung sections at high spatial and mass resolutions in negative-ion mode. MSI revealed the coexistence of CIC, des-CIC, and des-CIC-oleate on the airway epithelium, and the distribution of des-CIC and des-CIC-oleate in peripheral lung regions. In addition, a part of CIC independently localized on the airway epithelium. These results suggest that distribution of CIC and its metabolites in lungs is related to both the intended delivery of aerosols to pulmonary alveoli and peripheral regions, and the potential deposition of CIC particles on the airway epithelium.

25th anniversary of the Berlin workshop on developmental toxicology: DevTox database update, challenges in risk assessment of developmental neurotoxicity and alternative methodologies in bone development and growth.

25 years after the first Berlin Workshop on Developmental Toxicity this 10th Berlin Workshop aimed to bring together international experts from authorities, academia and industry to consider scientific, methodologic and regulatory aspects in risk assessment of developmental toxicity and to debate alternative strategies in testing developmental effects in the future. Proposals for improvement of the categorization of developmental effects were discussed as well as the update of the DevTox database as valuable tool for harmonization. The development of adverse outcome pathways relevant to developmental neurotoxicity (DNT) was debated as a fundamental improvement to guide the screening and testing for DNT using alternatives to animal methods. A further focus was the implementation of an in vitro mechanism-based battery, which can support various regulatory applications associated with the assessment of chemicals and mixtures. More interdisciplinary and translation research should be initiated to accelerate the development of new technologies to test developmental toxicity. Technologies in the pipeline are (i) high throughput imaging techniques, (ii) models for DNT screening tests, (iii) use of computer tomography for assessment of thoracolumbar supernumerary ribs in animal models, and (iv) 3D biofabrication of bone development and regeneration tissue models. In addition, increased collaboration with the medical community was suggested to improve the relevance of test results to humans and identify more clinically relevant endpoints. Finally, the participants agreed that this conference facilitated better understanding innovative approaches that can be useful for the identification of developmental health risks due to exposure to chemical substances.

Novel hepatotoxicity biomarkers of extracellular vesicle (EV)-associated miRNAs induced by CCl4.

Recent findings have revealed that extracellular vesicles (EVs) are secreted from cells and circulate in the blood. EVs are classified as exosomes (40-100 nm), microvesicles (50-1,000 nm) or apoptotic bodies (500-2,000 nm). EVs contain mRNAs, microRNAs, and DNAs and have the ability to transfer them from cell to cell. Recently, especially in humans, the diagnostic accuracy of tumor cell type-specific EV-associated miRNAs as biomarkers has been found to be more than 90 %. In addition, microRNAs contained in EVs in blood are being identified as specific biomarkers of chemical-induced inflammation and organ damage. Therefore, microRNAs contained in the EVs released into the blood from tissues and organs in response to adverse events such as exposure to chemical substances and drugs are expected to be useful as novel biomarkers for toxicity assessment. In this study, C57BL/6 J male mice orally dosed with carbon tetrachloride (CCl4) were used as a hepatotoxicity animal model. Here, we report that not only the known hepatotoxicity biomarkers miR-122 and miR-192 but also 42 novel EV-associated biomarkers were upregulated in mice dosed with CCl4. Some of these novel biomarkers may be expected to be able to use for better understanding the mechanism of toxicity. These results suggest that our newly developed protocol using EV-associated miRNAs as a biomarker would accelerate the rapid evaluation of toxicity caused by chemical substances and/or drugs.

Characteristic behavioral anomalies in rats prenatally exposed to 5-bromo-2'-deoxyuridine.

The aim of the present study was to characterize behavioral anomalies in rats prenatally exposed to 5-bromo-2'-deoxyuridine, a useful model of hyperactive disorder. Rats were treated with BrdU at 50mg/kg IP or carboxymethylcellulose, its vehicle, on gestational Days 9 through 15, and their offsprings were subjected to behavioral tests. Rats prenatally exposed to 5-bromo-2'-deoxyuridine showed higher locomotor activity levels when the lights were turned off, and these levels kept increasing throughout the dark cycle. In an elevated plus maze, the rats prenatally exposed to 5-bromo-2'-deoxyuridine exhibited decreased anxiety-related behavior, including higher open arm entries and a longer time spent per one open arm entry when compared with rats prenatally exposed to carboxymethylcellulose. Methylphenidate, a psychostimulant that suppresses hyperactivity in humans with attention-deficit hyperactivity disorder, increased locomotor activity in both rats, with a greater sensitivity in rats prenatally exposed to 5-bromo-2'-deoxyuridine. Desipramine, a specific noradrenaline uptake inhibitor, normalized the hyperactivity of rats prenatally exposed to 5-bromo-2'-deoxyuridine. Paroxetine, a selective serotonin reuptake inhibitor, also normalized the hyperactivity and the low anxiety-related behavior in the elevated plus maze. These results suggest that rats prenatally exposed to 5-bromo-2'-deoxyuridine are hyperactive and exhibit a lower anxiety level. Dysfunctional monoaminergic neurons may be, at least in part, the cause of the behavioral anomalies.

Induction of a thoracolumbar supernumerary rib in rat developmental toxicity studies: A short discussion on the critical window.

Thoracolumbar supernumerary ribs (TSRs) are classified as less severe skeletal anomalies in rat developmental toxicity studies, although their incidence is relatively high in rodent studies. To investigate the characteristics of the critical window for chemically-induced TSR, in this study, rats were administered 5-fluorocytocine (5-FC) or sodium salicylate (SAL) at one of three time periods on gestational day (GD) 9, early morning (7:00 am), midday (12:00 pm to 1:00 pm), or late afternoon (4:00 pm or 7:00 pm). The incidence of TSR and other anomalies were assessed in GD20 fetuses. A single treatment with both chemicals on GD9-induced TSR, with the incidence highest when administered at 7:00 Am, decreasing gradually when administered later. This trajectory was clearer in rats treated with 5-FC than with SAL. The critical period of TSR induction is shorter in rats administered 5-FC than SAL. The characteristics of the critical window may cause variability in the incidence of TSR observed in developmental toxicity studies.

Update of the DevTox data database for harmonized risk assessment and alternative methodologies in developmental toxicology: Report of the 9th Berlin Workshop on Developmental Toxicity.

Representatives of applied science (e.g. governmental organizations, academia, and industry) met to discuss the progress towards a harmonized human health risk assessment in developmental toxicology of plant protection products, biocidal products, and other environmental chemicals at the 9th Berlin Workshop on Developmental Toxicity held in September 2018. Within the focus of the scientific discussion were the future of in-vitro methods for developmental and reproductive toxicology, the potential relevance of alternative species in testing of developmental effects, and risk and hazard assessment of developmental and endocrine effects. Furthermore, the need for a harmonized terminology for classification of anomalies in laboratory animals in developmental toxicity studies aiming for human health risk assessment was determined. Here, the DevTox database was identified as an extremely valuable tool. Overall, the participants agreed that still one of the biggest challenges for testing developmental toxicity in the 21st century is the development of animal-free test strategies and alternatives to animal testing that could provide human-relevant information in a rapid, efficient, and mechanistically informative manner.

Historical control data on developmental toxicity studies in rats.

Historical control data from prenatal developmental toxicity studies in rats have been used to evaluate whether toxicology outcomes were induced by exposure to a chemical or were within the range of spontaneous variation. These data are also important for monitoring animal characteristics. As a follow-up to historical control data from 1998 to 2010, this study analyzed control data from prenatal developmental studies performed in rats from 2011 to 2015. Data were collected from studies performed by 24 Japanese laboratories, including 15 pharmaceutical and chemical companies and nine contract research organizations, in Sprague-Dawley and two-sub-strains of Wistar Hannover rats. The data included maternal reproductive findings at terminal cesarean section and fetal findings, including incidences of spontaneous external, visceral, and skeletal anomalies. No noticeable differences in maternal reproductive data were observed among laboratories. The inter-laboratory variations in the incidences of fetal anomalies seemed to be due to differences in the selection of observation parameters, observation criteria, and classification of the findings, as well as to differences in terminology of fetal alterations. These historical control data may be helpful for adequate interpretation of experimental results and for evaluating the reproductive and developmental toxicities of various chemicals.

Different effects of 26-week dietary intake of rapeseed oil and soybean oil on plasma lipid levels, glucose-6-phosphate dehydrogenase activity and cyclooxygenase-2 expression in spontaneously hypertensive rats.

We intended to determine whether or not dietary canola oil (CO) elevates plasma lipids and oxidative stress, since both of these are, possibly, related to the CO-induced life shortening through exacerbation of hypertension-associated vascular lesions found in stroke-prone spontaneously hypertensive rats (SHRSP). Spontaneously hypertensive rats (SHR) were used in this study to avoid a potential bias in the results due to the irregular death by stroke seen in SHRSP. SHR were fed for 26 weeks on a chow containing either, 10 wt/wt% of CO or soybean oil (SO), i.e., the control. Elevated plasma lipids and glucose-6-phosphate dehydrogenase (G6PD) activation in the liver and erythrocyte were found in SHR fed CO compared to that fed SO, while anti-oxidative enzymes other than G6PD were not activated. The CO diet brought about significant vascular lesions in the kidney, in which abundant cyclooxygenase-2 (COX-2) positive foci were immunochemically located in the juxtaglomerular apparatus. These results suggest that dietary CO induces a hyperlipidemic condition, in which G6PD may serve as an NADPH provider, and aggravates genetic diseases in SHR (also, probably, in SHRSP). The increased COX-2 expression indicates a role of renin-angiotensin-aldosterone system activation in the increased vascular lesions, whereas the effects of oxidative stress remain unclear.

The evaluation of early embryonic neurogenesis after exposure to the genotoxic agent 5-bromo-2'-deoxyuridine in mice.

Developmental neurotoxicity (DNT) is an important issue in children's health. Neurogenesis occurs throughout the early fetal to the postnatal period. The proliferation of embryonic stem cells can be a target for toxicants, especially genotoxic compounds. 5-Bromo-2'-deoxyuridine (BrdU), a thymidine analogue, has been used as a marker for proliferating cells. However, we reported that prenatal BrdU exposure induced behavioral abnormalities such as hyperactivity in rat and mouse offspring. In this study, to further clarify the toxic effect of BrdU on the early neurogenesis and to examine the usefulness of the evaluation of this process in DNT, C57BL/6 mice were exposed to 100 mg/kg of BrdU once on gestational day (GD) 9 or 11, and serial sections from a wide variety of areas of the embryonic brains 24 h after the exposure were examined. BrdU exposure on GD11 induced cell death in some specific areas, such as the neocortex and striatum, but not in the substantia nigra, raphe and pons, even though BrdU was incorporated into those cells. BrdU decreased the number of cells positive for phosphorylated histone 3 (phospho-histone 3), a marker for proliferating cells at metaphase of mitosis, in the cortex, mammillary body and cerebellum, suggesting that BrdU affected the proliferation of neural stem cells. Exposure on GD9 did not induce cell death in the fetal brain. These results indicate that BrdU actually impaired the early neurogenesis, supporting the postnatal results, and demonstrated that embryonic neurogenesis has heterogeneous sensitivity to the genotoxic agents BrdU that differs according to the area and developmental stage. The evaluation of events in early neurogenesis such as the proliferation of neural stem cells shortly after chemical exposure will be one of the valuable endpoints for studying postnatal neurodevelopmental disorders.

Valproate-induced developmental neurotoxicity is affected by maternal conditions including shipping stress and environmental change during early pregnancy.

Prenatal stress is known to affect the development of the brain, and exaggerate the developmental toxicity of chemicals. Many studies of developmental neurotoxicity (DNT) use pregnant rodents mated at the supplier, which consequently suffer from the stress of shipping and of environmental changes. Here, we demonstrated differences in the developmental neurotoxicity induced by valproate (VPA) between pregnant rats mated at our own animal facility (in-house group) and rats purchased pregnant (supplier group). Rats were treated with VPA (800mg/kg) orally on gestation day (GD) 9 or 11 (VPAE9 or VPAE11), and the fetal brain was examined at embryonic day 14 using immunohistochemistry for TuJ1 (a marker for immature neurons). The size of the fetal brain was also measured. The treatment decreased fetal live viability and fetal body weight only in the supplier group. VPA treatment on either day impaired the development of TuJ1-positive neurons in the cerebral cortex. The size of the forebrain was also affected by VPA. The supplier group was much more sensitive to these toxic effects. Therefore, difference in mating place (one's own animal facility or supplier) takes part in reproducibility of valproate-induced DNT.

Dopamine transporter density and behavioral response to methylphenidate in a hyperlocomotor rat model.

Rats exposed prenatally to 5-bromo-2'-deoxyuridine (BrdU-rats) display hyperlocomotive activity, making them a possibly useful animal model for the study of attention deficit hyperactivity disorder (ADHD). Using this model, we investigated dopamine transporter (DAT) density and behavioral outcomes in BrdU-rats, some of which were also administered methylphenidate, a psychostimulant that is widely used for the treatment of ADHD. Pregnant rats were exposed to BrdU from gestational day 9 through 15. In male offspring, DAT densities in different regions of the striatum were quantified at three weeks of age. At seven weeks of age, locomotor, rearing and grooming behaviors were evaluated in an open-field setting, with or without methylphenidate treatment (1 mg/kg or 4 mg/kg). The results revealed no significant changes in striatal DAT densities in BrdU-rats compared with controls. Extreme hyperlocomotion of BrdU-rats was detected in the open-field environment, an effect that was exacerbated following treatment with the lower and higher dose of methylphenidate. Such increase in locomotor activity was observed only with the higher dose in control animals. In summary, degeneration of dopaminergic neurons in the terminal field was not detected in juvenile BrdU-rats, although adult animals displayed hyperactive behavior in a mildly stressful environment as well as hypersensitivity to a psychostimulant that facilitates dopaminergic neurotransmission.

Neuropathological examination of fetal rat brain in the 5-bromo-2'-deoxyuridine-induced neurodevelopmental disorder model.

The majority of prior developmental neurotoxicity studies focused on postnatal subjects rather than on the fetus. In the present paper, we demonstrate the use of histological examination of fetal rat (embryonic day 16.5) brain serial sections, employing Nissl staining and microtubule-associated protein 2 (MAP2) immunohistochemistry, in evaluating a chemical-induced neurodevelopmental disorder. Since prenatal treatment with 5-bromo-2'-deoxyuridine (BrdU) is known to induce behavioral abnormalities such as locomotor hyperactivity in offspring, pregnant rats were administered 50 mg/kg on gestation days 9.5 through 15.5. The fetal brains at embryonic day 16.5 were collected and processed for neuropathological study. Cell death, including DNA strand breaks, was observed in specific areas of the fetal brain such as the neuroepithelium, intermediate zone and/or differentiating zones (e.g. neocortex and striatum) in exposed fetuses. In addition, the neocortex had an abnormal appearance cortical plate, which was also detected by MAP2 immunohistochemistry. The abnormal cortical plate was observed consistently, while the grade of cell death was generally very mild and variable. No significant alteration was detected in the brainstem. The present study reveals that histological observation of the fetal brain includes sensitive endpoints in developmental neurotoxicity, and that BrdU, at a dose generally administered to label proliferating cells, affects the development of the fetal neocortex.