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Muneoka K.,Showa University | Kuwagata M.,Hatano Research Institute | Ogawa T.,Showa University | Shioda S.,Showa University
Cerebellum | Year: 2015

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. © 2014, Springer Science+Business Media New York.

Muneoka K.,Showa University | Muneoka K.,Chiba University | Kuwagata M.,Hatano Research Institute | Ogawa T.,Showa University | Shioda S.,Showa University
Life Sciences | Year: 2010

Aims: The early neonatal period is critical for the development of the rodent brain. Neurosteroid levels in the brain decline from the late gestation to the neonatal period. Previous studies indicate effects of neurosteroid treatment during the neonatal period on the development of the dopaminergic system. In this study, we investigated the sex-specific effects of neonatal treatment with the neurosteroid progesterone on monoamine metabolism. Separately, we examined the contribution of pre-pubertal castration on the effect of neonatal treatment of pregnenolone (a neurosteroid precursor). Main methods: Progesterone (Experiment 1) or pregnenolone (Experiment 2) treatments in Sprague-Dawley rats were performed from postnatal days 3 through 7. Castration in experiment 2 was performed in male rats at postnatal day 21. We measured the brain tissue contents of dopamine, serotonin (5-HT), and their metabolites in rats at age 10. weeks. Key findings: Results showed that neonatal progesterone treatment altered striatal 5-hydroxy-3-indolacetic acid/5-HT ratios in males and females in opposite directions, in addition to dopaminergic effects. The treatment also influenced dopamine and 5-HT metabolism without sex-specificity in the frontal cortex. In addition, there was no significant difference in striatal monoamine metabolism between sham-operated, castrated and castrated pregnenolone-treated group. Significance: The present result indicates a sex-specific influence of progesterone during the early neonatal period on the development of the serotonergic system, depending on brain region in addition to of the dopaminergic system. © 2010 Elsevier Inc.

Tsuji S.,Bio Frontier Project Promotion Section | Ohbayashi T.,Tottori University | Yamakage K.,Hatano Research Institute | Oshimura M.,Tottori University | Tada M.,Tottori University
PLoS ONE | Year: 2016

The elimination of unfavorable chemicals from our environment and commercial products requires a sensitive and high-throughput in vitro assay system for drug-induced hepatotoxicity. Some previous methods for evaluating hepatotoxicity measure the amounts of cytoplasmic enzymes secreted from damaged cells into the peripheral blood or culture medium. However, most of these enzymes are proteolytically digested in the extracellular milieu, dramatically reducing the sensitivity and reliability of such assays. Other methods measure the decrease in cell viability following exposure to a compound, but such endpoint assays are often confounded by proliferation of surviving cells that replace dead or damaged cells. In this study, with the goal of preventing false-negative diagnoses, we developed a sensitive luminometric cytotoxicity test using a stable form of luciferase. Specifically, we converted Gaussia luciferase (G-Luc) from an actively secreted form to a cytoplasmic form by adding an ER-retention signal composed of the four amino acids KDEL. The bioluminescent signal was >30-fold higher in transgenic HepG2 human hepatoblastoma cells expressing G-Luc+KDEL than in cells expressing wild-type G-Luc. Moreover, G-Luc+KDEL secreted from damaged cells was stable in culture medium after 24 hr at 37°C. We evaluated the accuracy of our cytotoxicity test by subjecting identical samples obtained from chemically treated transgenic HepG2 cells to the G-Luc+KDEL assay and luminometric analyses based on secretion of endogenous adenylate kinase or cellular ATP level. Time-dependent accumulation of G-Luc+KDEL in the medium increased the sensitivity of our assay above those of existing tests. Our findings demonstrate that strong and stable luminescence of G-Luc+KDEL in human hepatocyte-like cells, which have high levels of metabolic activity, make it suitable for use in a high-throughput screening system for monitoring time-dependent cytotoxicity in a limited number of cells. © 2016 Tsuji et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Shimada Y.,Hokko Chemical Industry Co. | Sui H.,Hatano Research Institute | Wako Y.,LSI Corporation | Kawasako K.,LSI Corporation
Mutation Research - Genetic Toxicology and Environmental Mutagenesis | Year: 2015

The micronucleus induction by p-dimethylaminoazobenzene (DAB), a genotoxic rat liver carcinogen, was assessed in the liver and bone marrow of young adult rats after the repeated administration of DAB for 14 (Lab. 1) and 28 (Lab. 2) days. Three dose levels, 25, 50 and 100. mg/kg/day, were used for the investigations in both labs.The frequency of micronucleated hepatocytes was significantly increased in a dose-dependent manner after the repeated administration of DAB at 50. mg/kg/day or more for 14 and 28 days. Similarly, the frequency of micronucleated immature erythrocytes in the bone marrow was increased after the repeated administration of DAB at 100. mg/kg/day for 14 and 28 days.These results indicate that the repeated-dose liver micronucleus assay allowed for the detection of micronucleus induction by DAB, and that the lowest detectable dose for micronucleus induction in the liver was lower than in the bone marrow. Thus, the repeated-dose liver micronucleus assay using young adult rats is considered suitable for the detection of micronucleus induction by liver carcinogens, such as DAB. © 2014 Elsevier B.V.

Yakabe T.,Kagome Co. | Takashima H.,Hatano Research Institute | Kuwagata M.,Hatano Research Institute | Fukao M.,Kagome Co. | And 2 more authors.
Food and Chemical Toxicology | Year: 2011

Lactobacillus brevis KB290 (KB290), a plant-derived probiotic lactic acid bacterium, improves gut health and stimulates immune function. Here we extensively investigated the teratogenicity of KB290 in rats and rabbits. We observed no adverse maternal or fetal effects and concluded that the no observable adverse effect level for maternal general toxicity, maintenance of pregnancy, and teratogenicity should be ≥1010cfu/kg/day. Our results suggest that KB290 would be safe for pregnant females and their offspring. © 2010 Elsevier Ltd.

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