e ポスター

e ポスター 14. 細胞毒性、その他 e Poster 14. Cell toxicity, Others

2020/9/12　13:40～14:40　オンデマンドB-1 P3-22* 化学物質で誘発されたプルキンエ細胞の発達変化とGLAST分化の異常 Chemical-induced alteration of Purkinje cell development was related GLAST expression 岩永美咲¹、福島翠¹、松井紗羅沙¹、安達明理¹、諫田泰成²、吉田祥子¹ 1. 豊橋技術科学大学、2. 国立医薬品食品衛生研究所 Misaki Iwanaga¹, Midori Fukushima¹, Sarasa Matsui¹, Akari Adachi¹, Yasunari Kanda², Sachiko Yoshida¹ 1. Toyohashi University of Technology, 2. National Institute of Health Sciences Prenatal chemical exposure is a growing health concern as it can cause a lifelong lasting effect on the psychological and physiological health in both pregnant mother and offspring, which supports the Developmental Origins of Health and Disease (DOHaD) hypothesis. We have studied the neurodevelopmental toxicity to rat cerebellar cortex and observed the embryonic exposure to valproic acid (VPA), the antiepileptic drug, or Glyphosate (GLY), the herbicide, induced developmental alteration of the Purkinje cells on 2-weeks after birth. VPA is famous for both an antiepileptic agent and an ASD inducer, and a well-known HDAC inhibitor which causes wide- spread epigenetic change. GLY is widely-used less toxic herbicide but a candidate for Parkinson's disease inducer. In this study, we investigated the initial phenomena of the developmental neuronal toxicity of these chemicals. Even in postnatal day 5 (P5) cerebellum of VPA-administrated rat, this irregular folding appeared, and in the P7 cerebellum, the expression of the astrocyte-specific glutamate transporter, GLAST was increased in the external granular layer. In the cultured glial cells derived from VPA- or GLY-exposed neonatal rats, GLAST expression was increased in the astrocyte from VPA-exposed rats and decreased from GLY-exposed rats. Because GLAST on the astrocyte controls glutamate concentration around immature Purkinje cells (PC), we suggest increased GLAST expression in VPA-treated rats could protect the PC and decreased GLAST expression in GLY- treated rats could weaken the PC. We will compare these results to the GLAST expression in other HDAC inhibitor-treated rats, and clarify the role of GLAST in developmental neurotoxicity.		2020/9/12　13:40～14:40　オンデマンドB-1 P3-23 神経管障害のリスクファクターであるフモニシン B1 のエピジェネティックな作用 The epigenetic effects of Fumonisin B1, a risk factor for neural tube defects 佐藤薫¹、木下麻緖²、古沢博子²、本間正充²、杉山圭一² 1. 国立医薬品食品衛生研究所薬理部、2. 国立医薬品食品衛生研究所変異遺伝部 Kaoru Sato¹, Mawo Kinoshita², Hiroko Furusawa², Masamitsu Honma², Kei-ichi Sugiyama² 1. Div Pharmacol, NIHS, 2. Div Genetics Mutagenesis, NIHS Neural tube defects (NTDs) are the most serious and common birth defects in the clinic. It has been suggested that epigenetic mechanisms are affected in human NTDs. Mycotoxin Fumonisin B1 (FB1) is a secondary metabolite that is produced by certain Fusarium species. FB1 is found in corn-based foods and suggested to be a risk factor for NTDs. We therefore investigated the epigenetic effects of FB1 in this study. For this purpose, we first performed FLO assay, which detects epigenetic changes that affect the flocculation gene (FLO1) promote activity in budding yeast. 10-20 µM FB1 induced the increase in the reporter activities of the FLO1 promoter. 20 µM FB1 treatments promoted flocculation simultaneously. In vitro methylation assays using bacterial DNA methyltransferase (DNMT) showed that FB1 treatments increased DNMT activities. Furthermore, 100 µM FB1 caused significant increase in global DNA methylation in Human Embryonic Kidney cells 293 cells. Taken together, these results suggest that FB1 increases DNMT activities and leads to unique epigenetic alterations, which might be one of the underlying mechanisms for human NTDs.

2020/9/12　13:40～14:40　オンデマンドB-1 P3-24* ヒト真皮線維芽細胞からのリアルタイムコラーゲン放出観察 Real time observation of collagen release from cultured human fibroblasts 仲田しずか¹、トーマス　ティオンクオン　スン¹、エドバグス　プラスティカ¹、小林和人²、穂積直裕¹、吉田祥子¹ 1. 豊橋技術科学大学、2. 本多電子株式会社 Shizuka Nakada¹, Thomas Tiong Kwong Soon¹, Edo Bagus Prastika¹, Kazuto Kobayashi², Naohiro Hozumi¹, Sachiko Yoshida¹ 1. Toyohashi Univ. of Technology, 2. Honda Electronics Co., Ltd. One of the main factors with skin aging is an attenuation of elasticity of skin due to a decrease in collagen released from skin fibroblasts, which construct extracellular matrices into the dermis. Traditionally, L-ascorbic acid is known as the collagen desmoplasia factor; however, releasing dynamism from living cells is less known because it is hard to observe it directly. Also, because L-ascorbic acid tends to be oxidized rapidly, it is important to determine which derivative or dosage is effective for collagen increase. In this research, we investigated real-time observation of collagen release from the fibroblast using acoustic microscopy, which visualizes the distribution of elastic materials as an acoustic impedance; actin bundles show high impedance, cytoplasm shows middle and culture medium shows low impedance.. Cultured human skin fibroblasts treated with phosphoric acid-L-ascorbic acid magnesium (MAP), an oxidation-resistant derivative, or dehydroascorbic acid (DHA), an oxidized derivative, were observed continuously. The results were compared with collagen assay in the culture medium by the ELISA system and immunohistological observation. The MAP treatment little changed in cell proliferation, while the DHA treatment slightly decreased it. Acoustic impedance around the cell was enhanced after MAP 0.2 mM treatment; however, it was decreased by DHA 1.0 mM treatment. The immunocytochemical observation showed the collagen distribution in the cells, whereas collagen released outside could not be observed. The quantitative of the collagen using the ELISA was consistent with the observation with acoustic microscopy. We suggest acoustic microscopy is useful for visualization and quantification of extracellular materials of living cells.		2020/9/12　13:40～14:40　オンデマンドB-1 P3-25 Glyphosate 神経毒性とHDACi神経毒性の腸内細菌叢から見た影響比較 Comparison of the effects of glyphosate neurotoxicity and HDACi neurotoxicity from the gut flora トーマスティオンクオンスン¹、浅井紀之²、二神健¹、稲川貴也¹、野村容子³、諫田泰成⁴、吉田祥子¹ 1. 豊橋技術科学大学、2. イチビキ株式会社、3. クイーンズカレッジニューヨーク市立大学、4. 国立衛生科学研究所 Kwong Soon Thomas Tiong¹, Noriyuki Asai², Ken Futagami¹, Takaya Inakawa¹, Yoko Nomura³, Yasunari Kanda⁴, Sachiko Yoshida¹ 1. Toyohashi University of Technology, 2. Ichibiki Co., Ltd, 3. Queens College, the City University of New York, 4. National Institute of Health Sciences Prenatal chemical exposure is proven to play a critical role in the brain development of offspring. Our previous studies revealed that exposure to histone deacetylase inhibitor (HDACi) such as sodium valproate (VPA) and herbicide such as glyphosate (GP) during pregnancy affects the cerebellar development in offspring using an animal model. This result suggested that both chemicals are neurotoxic to the cerebellar development of offspring. Interestingly, the gut flora of VPA- and GP-exposed pregnant dams were also altered, presenting different types of microbe composition. In this study, we compare the gut flora of the pregnant dam under different chemical exposures to better understand the connection between neurotoxicity and brain development in offspring. Pregnant dams were administrated with the following chemicals on gestation day 15, G15: 250 mg/kg BW of GP, 600 mg/kg BW of VPA, 400 mg/kg BW of sodium butyrate (BA), 50 mg/kg BW of Vorinostat (SAHA), 4 mg/kg BW of Entinostat (MS-275). The stool samples from the pregnant dams were collected on G6, G13, G15, G17, and G20 to record the gut flora alteration throughout pregnancy. The stool samples were purified and then subjected to 16S rRNA sequencing. Data analysis was done using QIIME2 with the DADA2 pipeline, as described by Callahan et al., 2016. The gut flora of GP exposed rats showed a significant decrease of butyrate-producing bacteria: Lachnospiraceae and Ruminococcaceae. These microbes are responsible for the metabolism of complex carbohydrates into short-chain fatty acid (SCFA), which plays an important role in the regulation of intestinal homeostasis. On the other hand, the HDACi groups showed a balance shift between the phylum Bacteroidetes and Firmicutes, which is an indication of gut inflammation.

2020/9/12　13:40～14:40　オンデマンドB-1 P3-26* 高用量のネオニコチノイド系殺虫剤アセタミプリドによるプルキンエ細胞奇形と行動異常の誘発 High-dose neonicotinoid insecticide, Acetamiprid induces Purkinje cell malformation and behavioral alteration Christine Lee Li Mei¹、大室拓平¹、Thomas Tiong Kwong Soon¹、Johnny Ademir Lopez²、Nomura Yoko²、諫田泰成³、吉田祥子¹ 1. 豊橋技術科学大学、2. ニューヨーク市立大学クインズカレッジ、3. 国立医薬品食品衛生研究所 Christine Lee Li Mei¹, TAKUHEI OMURO¹, Thomas Tiong Kwong Soon¹, Johnny Ademir Lopez², Yoko Nomura², Yasunari Kanda³, Sachiko Yoshida¹ 1. Toyohashi University of Technology, 2. Queens College, the City University of New York, 3. National Institute of Health Sciences Prenatal chemical exposure is a growing health concern as it can cause a lifelong lasting effect on the psychological and physiological health in both pregnant mother and offspring, which supports the Developmental Origins of Health and Disease (DOHaD) hypothesis. Neonicotinoids, a new class of insecticide, mimic the chemical structure of nicotine to bind to the nicotinic acetylcholine receptor (nAChR) to provide highly systemic insecticidal effects. Due to the difference of nAChR sensitivities between insects and mammal, neonicotinoids have been seen as safer insecticides than organophosphate compounds; however, recently, some reports suggest the developmental neurotoxicity of neonicotinoids. In this study, we investigate the effects of embryonic exposure of acetamiprid (ACE), a type of neonicotinoid, in different dosages (20 mg/kg-, 40 mg/kg-, or 60 mg/kg-ACE). The ACE-exposed pups showed Purkinje Cell (PC) misalignment and excess folding between lobule V and VI in the cerebellar vermis. In addition, we observed their behavioral alteration. The ACE-exposed pups showed ASD-like freezing behavior on 6-weeks after birth, and showed ADHD-like nervous behavior on 8-weeks after birth. Finally, we investigate the effects of ACE-exposure on the microbiome. The gut-brain axis is the focus of the immune network composed of the gut bacterial flora, the peripheral and central nervous system, and the immune system. ACE could perturb the peripheral nervous system and the microbiome environment. We will suggest the hypothesis on the relation between neuronal development and ACE exposure.		2020/9/12　13:40～14:40　オンデマンドB-1 P3-27 膀胱内腔ATPは炎症性変化を起こすことなく求心性神経を活性化し頻尿をもたらす Intravesical ATP instillation induces urinary frequency because of activation of bladder afferent nerves without inflammatory changes in mice 近藤誠¹、上田倫央^1,2、野々村祝夫²、島田昌一¹ 1. 大阪大学大学院医学系研究科神経細胞生物学講座、2. 大阪大学大学院医学系研究科泌尿器科学 Makoto Kondo¹, Norichika Ueda^1,2, Norio Nonomura², Shoichi Shimada¹ 1. Department of Neuroscience and Cell Biology, Graduate School of Medicine, Osaka University, 2. Department of Urology, Graduate School of Medicine, Osaka University ATP in the suburothelial layer is released from the bladder urothelium by mechanical stimuli. ATP directly activates purinergic receptors that are expressed on primary bladder afferent neurons and induces the micturition reflex. Although ATP is also released to the bladder lumen from the bladder urothelium, the role of ATP in the bladder lumen is unknown. Recently, clinical studies have reported that urinary ATP levels are much higher in patients with an overactive bladder than healthy controls. These results suggest that ATP in the bladder lumen is also involved in the micturition reflex. In this study, we performed intravesical ATP instillation in the mouse bladder. We evaluated urinary function with novel reliable methods using improved cystometry and ultrasonography, which we previously established. We found that intravesical ATP instillation induced urinary frequency because of activation of bladder afferent nerves without inflammatory changes in the bladder or an increase in post-void residual urine. These results suggest that not only ATP in the suburothelial layer, but also ATP in the bladder lumen, are involved in enhancement of the micturition reflex.

2020/9/12　13:40～14:40　オンデマンドB-1 P3-28* 穿刺脳損傷の修復における環状ホスファチジン酸誘導体2ccPAの効果 The effect of cyclic phosphatidic acid derivative 2-carba-cPA on repair of brain stab wound 遠藤美沙紀¹、中島麻里¹、池島（片岡）宏子²、後藤真里¹、室伏きみ子¹、宮本泰則¹ 1. お茶の水女子大学、2. 早稲田大学 Misaki Endo¹, Mari Nakashima¹, Hiroko Ikeshima-Kataoka², Mari Gotoh¹, Kimiko Murakami-Murofushi¹, Yasunori Miyamoto¹ 1. Ochanomizu Univ., 2. Waseda Univ. Traumatic brain injury (TBI) is caused by physical damage to the brain and induces blood-brain barrier (BBB) breakdown and hemorrhage. To diminish the sequelae of TBI, the suppression of hemorrhage is important. We previously revealed that stable derivative of cyclic phosphatidic acid (cPA) : 2-carbacyclic phosphatidic acid (2ccPA) contributes to the repair of TBI. cPA is a naturally occurring phospholipid mediator with a unique cyclic phosphate ring at the sn-2 and sn-3 positions of its glycerol. 2ccPA suppresses hemorrhage after a stab wound injury as a murine TBI model. However, the molecular mechanisms how 2ccPA suppresses the hemorrhage by TBI remain unexplained. In this study, we aimed to investigate the effect of 2ccPA on the hemorrhage and BBB-related mRNA expression level by 2ccPA after the injury of mouse cerebral cortices. First, we examined the effect of 2ccPA on the mRNA expression levels of proteolysis-related genes in the stab-wound cerebral cortices. Realtime RT-PCR analysis found that 2ccPA down-regulated the mRNA expression level of plasminogen activator inhibitor-1, but did not affect that of tissue-type plasminogen activator and urokinase-type plasminogen activator on days 3 after injury. Next, we examined the effect of the mRNA expression levels of BBB integrity and tight junction-related genes in the stab wounded regions. The results showed that 2ccPA down-regulated the TBI-induced increase of the mRNA expression of transferrin receptor (TFRC), which is involved in the integrity of BBB, on days 3 and 5. Taken together, these data suggest that 2ccPA promotes the proteolysis in the repairing process of wounded regions, and suppresses the mRNA expression of TFRC as a result of reduction of BBB breakdown by 2ccPA.		2020/9/12　13:40～14:40　オンデマンドB-1 P3-29 順遺伝学スクリーニングによる新規疼痛候補遺伝子の解析 Analysis of candidates of pain-related genes by forward genetic screening 田中達英¹、奥田洋明²、寺田雄紀¹、新城武明¹、西村和也¹、石西綾美¹、三谷早希³、竹村晶子¹、辰巳晃子¹、和中明生¹ 1. 奈良県立医科大学医学部解剖学第２、2. 金沢大学医学部機能解剖学、3. 大阪歯科大学歯科麻酔学 Tatsuhide Tanaka¹, Hiroaki Okuda², Yuki Terada¹, Takeaki Shinjo¹, Kazuya Nishimura¹, Ayami Isonishi¹, Saki Mitani³, Shoko takemura¹, kouko Tatsumi¹, Akio Wanaka¹ 1. Nara Med. Univ., 2. Kanazawa Univ., 3. Osaka Dental Univ. Pain significantly reduces quality of life (QOL) in various diseases. Although pain research has revealed pain-modulating cells, molecules and neural pathways, the cellular and molecular mechanisms of pain remain largely unknown. We noticed that pain responses to mechanical and chemical stimuli were extremely reduced in a bacterial artificial chromosome-transgenic (BAC-TG, Mlc1-tTA #Rhn) mice. Swelling and redness hardly occurred when chemical substances were injected into the hind paw of the mice. Since this TG mice harbors a BAC transgene (clone RP23-114I6), we first speculated that exogenous genes in the BAC may modulate pain behaviors. The exogenous gene expressions, however, were indistinguishable from those in naive mice. Based on a next hypothesis that the large transgene might affect endogenous gene expression and thereby influence pain behaviors, we performed forward genetic screening using next generation sequencing and cDNA microarray analysis. The BAC transgene was inserted into chromosome 8 and three genes in the vicinity of the insertion site were almost knocked out. None of the three genes have been so far identified as pain-related factors. Whether these genes are involved in pain-sensing mechanisms is an open question. To elucidate the roles of the candidates in pain behaviors, we are investigating knock out (KO) and conditional KO mice.