Hatano Research Institute

Hadano, Japan

Hatano Research Institute

Hadano, Japan
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Ohta R.,Hatano Research Institute | Takagi A.,Japan National Institute of Health Sciences | Ohmukai H.,Hatano Research Institute | Marumo H.,Hatano Research Institute | And 5 more authors.
Journal of Toxicological Sciences | Year: 2012

The concern over endocrine disruptors prompted international establishment of a strategic framework for the identification of the estrogenic compounds. OECD has launched the Conceptual Framework tool box containing various screening and testing methods including the uterotrophic assay. The (anti)estrogenicity of 36 chemicals suspected to be estrogen-receptor interactive by in silico and/or in vitro screening in the Extended Scheme for Endocrine Disruptor Screening and Testing of the Ministry of Health, Labour and Welfare, Japan, were monitored by the uterotrophic assay using C57BL/6J ovariectomized adult female mice after a 7-day exposure by oral gavage (po) and subcutaneous injection (sc). Ethynyl estradiol was used as reference for agonist and antagonist detection. In addition, Bisphenol A (sc) and Genistein (po) were tested for the comparison to rat assays. Among the 36, 2-[Bis(4-hydroxyphenyl) methyl]benzylalcohol, 2,2',4,4'-Tetrahydroxybenzophenone, 2,4-Dihydroxybenzophenone, 3,3',5-Triiodothyroacetic acid, New fuchsin and alpha-Naphtholbenzein, showed both estrogenic agonistic and antagonistic activities; first two showed U-shaped dose-response in antagonistic studies. N,N-Diphenyl- p-phenylenediamine, 2,2'-Dihydroxy-4,4'-dimethoxybenzophenone, n-Butyl 4-hydroxybenzoate, and Reserpine were agonistic by sc. Benzo [a] pyrene, Benz [a] anthracene, Dibenz [a,h] anthracene, 2-(2H-Benzotriazol-2-yl)-4,6-di(t-pentyl)phenol, Rosemarinic acid, meta-Thymol, 6-Gingerol, Colchicine, Malachite green base, Fenbuconazole, and Lead acetate were antagonistic. The rest, i.e. n-Heptyl 4-hydroxybenzoate, Tetrazolium violet, Pravastatin sodium salt, Physostigmine, salicylate (1:1), Nordihydroguaiaretic acid, o-Cresolphthalein, 1,3-Dinitrobenzene, C.I. Pigment orange, Tetrabromobisphenol- A, 2-Hydroxy-4-methoxybenzophenone, Ethylparaben, Propyl p-hydroxybenzoate, Kaempferol, 2-(2-Benzotriazolyl)-p-cresol and Phenolphthalein were negative for both effects. Taking together with in silico/ in vitro screening, the result suggested that the ovariectomized mouse uterotrophic bioassay has sufficient performance comparable to rat for the screening of (anti)estrogenicity of various chemicals.


Kakiuchi H.,Azabu University | Kawarai-Shimamura A.,Anicom Holdings Inc. | Kuwagata M.,Hatano Research Institute | Orito K.,Azabu University
European Journal of Pharmacology | Year: 2014

Tranexamic acid suppresses post-partum haemorrhage and idiopathic menorrhagia through its anti-fibrinolytic action. Although it is clinically useful, it is associated with high risks of side effects such as emesis. Understanding the mechanisms underlying tranexamic acid-induced emesis is very important to explore appropriate anti-emetic drugs for the prevention and/or suppression of emesis. In this study, we examined the receptors involved in tranexamic acid-induced kaolin intake in rats, which reflects the drug's clinical emetogenic potential in humans. Further, we examined the brain regions activated by administration of tranexamic acid and elucidated pivotal pathways of tranexamic acid-induced kaolin intake. We examined the effects of ondansetron, a 5-hydroxytryptamine 3 receptor antagonist, domperidone, a dopamine 2 receptor antagonist, and aprepitant, a tachykinin neurokinin 1 (NK1) receptor antagonist, on tranexamic acid-induced kaolin intake in rats. Then, we determined the brain regions that showed increased numbers of c-Fos immunoreactive cells. Finally, we examined the effects of an antagonist(s) that reduced tranexamic acid-induced kaolin intake on the increase in c-Fos immunoreactive cells. Aprepitant significantly decreased tranexamic acid-induced kaolin intake. However, neither ondansetron nor domperidone decreased kaolin intake. Tranexamic acid significantly increased c-Fos immunoreactive cells by approximately 5.5-fold and 22-fold in the area postrema and nucleus of solitary tract, respectively. Aprepitant decreased the number of c-Fos immunoreactive cells in both areas. Tranexamic acid induced kaolin intake possibly via stimulation of tachykinin NK1 receptors in rats. The tachykinin NK1 receptor could be targeted to prevent and/or suppress emesis in patients receiving tranexamic acid. © 2013 Published by Elsevier B.V. Behavioural Pharmacology.


Ohmukai H.,Hatano Research Institute | Negura T.,Hatano Research Institute | Tachibana S.,Hatano Research Institute | Ohta R.,Hatano Research Institute
Reproductive Toxicology | Year: 2017

To confirm genetic variation in low-dose effects of diethylstilbestrol (DES), two inbred strains of rats, which have been selectively bred for high- and low-avoidance learning (HAA and LAA, respectively), were used in this study. LAA rats characteristically show later sexual maturation, earlier reproductive senescence, and lower body weight as compared to HAA rats. Female neonates of each strain were daily administered DES by oral gavage at doses of 0 (vehicle only), 0.05 and 0.5. μg/kg for the first 5. days after birth. As a result, early onset of abnormal estrous cycles was observed during the same period in HAA and LAA rats treated with 0.5. μg/kg. However, accelerated puberty and excessive body weight gains were observed only in LAA rats treated with 0.05 and 0.5. μg/kg. These results suggest that the effects of neonatal DES exposure vary with the genetic background of the female rats used. © 2017 Elsevier Inc.


Ohta R.,Hatano Research Institute | Ohmukai H.,Hatano Research Institute | Marumo H.,Hatano Research Institute | Shindo T.,Hatano Research Institute | And 2 more authors.
Reproductive Toxicology | Year: 2012

A one-lifespan test was carried out to establish a test protocol for endocrine-disrupting chemicals (EDCs). Diethylstilbestrol was administered by oral gavage to neonatal rats at doses of 0.05, 0.5 and 5 μg/kg/day for 5 days after birth. Abnormal estrous cycles were observed throughout the study in all females from the 5 μg/kg group, and in 40% from the 0.5 μg/kg group from 24 weeks of age. The conception rate of 12-week-old females in the 5 μg/kg group was 0%, and that of the 23-week-old females in the 0.5 μg/kg group was 33.3%. No effect of DES was observed at the first parturition in any group, except for the 5 μg/kg group. However, litter size was significantly reduced in the 0.5 μg/kg group at the second parturition. These results indicated that a prolonged period of observation of reproductive function is necessary to determine EDCs reliably. © 2012 Elsevier Inc.


Nakagomi M.,Hatano Research Institute | Suzuki E.,Hatano Research Institute
Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences | Year: 2017

15α-Hydroxyestrogens (15α-OHEs) are metabolites of the female hormone estradiol. In this study, to discover physiological markers that can be utilized for monitoring fetal conditions and estrogen-induced cancers, we established a method for quantifying 15α-OHEs in rat urine via immunoaffinity column extraction and HPLC-electrochemical detection, and detected 15α-OHEs in urine obtained male rats treated with estradiol. Notably, the standard curves for quantification obtained using the column were linear over a range of 0.5–50 ng 15α-OHEs. The accuracy of the analytical method with cleanup was 97–109% for the three kinds of 15α-OHEs examined, and the intra-assay precision of the measured values had a coefficient of variation of ≤20.6%. Therefore, the theoretical limit of quantification was 0.5 ng. However, the actual measured values obtained from the urine of male rats indicated that the detection limits were 0.425, 0.103, and 0.047 ng for estetrol, 15α-hydroxyestradiol, and 15α-hydroxyestrone, respectively. Our newly established method for measuring 15α-OHE concentrations in urine could facilitate characterization of the in vivo metabolic profile of 15α-OHEs in mammals under various physiological conditions, which could comprise clinical markers for monitoring human fetal health conditions in mammals. © 2017 Elsevier B.V.


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.


Senuma M.,Hatano Research Institute | Mori C.,Chiba University | Ogawa T.,Saitama University | Kuwagata M.,Hatano Research Institute
International Journal of Developmental Neuroscience | Year: 2014

Prenatal arsenite exposure has been associated with developmental disorders in children, including reduced IQ and language abnormalities. Animal experiments have also shown that exposure to arsenite during development induced developmental neurotoxicity after birth. However, the evidence is not enough, and the mechanism is poorly understood, especially on the exposure during early brain development. This study assessed effects of sodium (meta) arsenite shortly after exposure on early developing fetal rat brains.Pregnant rats were administered 50. mg/L arsenite in their drinking water or 20. mg/kg arsenite orally using a gastric tube, on gestational days (GD) 9-15. Fetal brains were examined on GD16.Pregnant rats administered 20. mg/kg arsenite showed reductions in maternal body weight gain and food consumption during treatment, but not with 50. mg/L arsenite. Arsenite did not affect fetal development, as determined by body weight, mortality and brain size. Arsenite also did not induce excessive cell death or affect neural cell division in any region of the fetal neuroepithelium. Thyrosine hydroxylase immunohistochemistry revealed no difference in the distribution of catecholaminergic neurons between fetuses of arsenite treated and control rats. However, reductions in the number of serotonin positive cells in the fetal median and dorsal raphe nuclei were observed following maternal treatment with 20. mg/kg arsenite. Image analysis showed that the serotonin positive areas decreased in all fetal mid- and hind-brain areas without altering distribution patterns. Maternal stress induced by arsenite toxicity did not alter fetal development. These results suggest that arsenite-induced neurodevelopmental toxicity involves defects in the early development of the serotonin nervous system. © 2014 ISDN.Published by Elsevier Ltd.


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.


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

The repeated-dose liver micronucleus (RDLMN) assay has the potential to detect liver carcinogens and can be integrated into general toxicological studies. In this study, thioacetamide (TAA) was tested in 14- and 28-day RDLMN assays to assess the performance of the assay.The test substance, TAA, was administered orally to 6-week-old male Crl:CD (SD) rats once daily for 14 or 28 days at a dosage of 5, 10 or 20. mg/kg/day. Hepatocytes were collected approximately 24. h after the last TAA administration, and the incidence of micronuclei was assessed. In this study, bone marrow micronucleus assays were also conducted in the same animals. The 14- and 28-day RDLMN assays indicated that none of the TAA dosages significantly increased the proportion of micronucleated hepatocytes. Bone marrow micronucleus assays with TAA also provided negative results. It is known that TAA is a liver carcinogen in mice and rats. In the previous genotoxic studies, the Ames test and the chromosomal aberration test using CHL/IU cells have yielded negative results [1-4]. The liver micronucleus assay using young adult rats singly dosed with TAA (75 and 150. mg/kg) also produced negative results [5]. TAA gave positive results only in the mouse bone marrow micronucleus assays [6,7]. © 2014 Elsevier B.V.


Ohta R.,Hatano Research Institute | Ohmukai H.,Hatano Research Institute | Toyoizumi T.,Hatano Research Institute | Shindo T.,Hatano Research Institute | And 2 more authors.
Reproductive Toxicology | Year: 2014

In a previous study, we found that early life exposure to low-dose diethylstilbestrol (DES) induced early onset of spontaneous abnormalities in estrus cycle and shortened survival in female Sprague-Dawley rats. In order to confirm the repeatability of the previous study, neonates of C57BL/6J mice were orally administered DES at doses of 0.005, 0.05, 0.5 and 5. μg/kg/day, and the aging of their reproductive function was observed. As a result, delayed toxicity on ovarian function was found in females treated with 0.5. μg/kg/day of DES. Concomitantly, the females in the 0.05. μg/kg/day of DES, or greater, groups, had increased body weights and, in the 0.5. μg/kg/day of DES, or greater, groups, had developed pituitary tumors, which were causal factors in their accelerated mortality. Thus, we found that early life exposure to low-dose DES induced early onset of spontaneous abnormalities in estrus cycle not only in female rats but also in female mice. © 2014 Elsevier Inc.

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