研究者総覧

石橋 弘志 (イシバシ ヒロシ)

  • 大学院農学研究科 生物環境学専攻 准教授
Last Updated :2021/01/14

研究者情報

学位

  • 博士(学術)(長崎大学)

ホームページURL

科研費研究者番号

  • 90403857

J-Global ID

研究キーワード

  • 有機フッ素化合物   残留性有機汚染物質   内分泌かく乱物質   メダカ   ゼブラフィッシュ   海産甲殻類   核内受容体   ペルオキシソーム増殖剤応答性受容体   エストロゲン受容体   環境毒性学   生態系保全   食環境安全性学   リスク評価   

研究分野

  • 環境・農学 / 環境影響評価
  • 環境・農学 / ランドスケープ科学
  • 環境・農学 / 環境農学
  • ライフサイエンス / 薬系衛生、生物化学
  • ライフサイエンス / 分子生物学
  • 環境・農学 / 化学物質影響
  • 環境・農学 / 放射線影響

経歴

  • 2016年04月 - 現在  愛媛大学大学院農学研究科准教授
  • 2015年04月 - 2016年03月  愛媛大学農学部准教授
  • 2011年04月 - 2015年03月  尚絅大学短期大学部食物栄養学科准教授
  • 2008年07月 - 2011年03月  愛媛大学沿岸環境科学研究センター 化学汚染・毒性解析部門グローバルCOE准教授
  • 2006年10月 - 2008年06月  熊本県立大学環境共生学部 食環境安全性学講座
  • 2006年07月 - 2006年10月  長崎大学大学院医歯薬学総合研究科 生化学教室助手
  • 2005年04月 - 2006年07月  愛媛大学沿岸環境科学研究センター 生態毒性解析分野COE研究員
  • 2003年04月 - 2005年03月  熊本県立大学環境共生学部 食品安全性学講座博士研究員

学歴

  • 2000年 - 2003年   長崎大学大学院   生産科学研究科   海洋資源学専攻
  •         - 1999年   長崎大学   Graduate School, Division of Fisheries

所属学協会

  • 日本水環境学会   日本内分泌攪乱化学物質学会   日本食品衛生学会   生物化学的測定研究会   日本環境毒性学会   

研究活動情報

論文

  • Hiroshi Ishibashi, Masaya Uchida, Masashi Hirano, Taka Hayashi, Ryoko Yamamoto, Akira Kubota, Nobuhiro Ichikawa, Yasuhiro Ishibashi, Nobuaki Tominaga, Koji Arizono
    The Science of the Total Environment 767 144379 - 144379 2020年12月 研究論文(学術雑誌) 
    Equine estrogens (EEs) are widely used in hormone replacement therapy pharmaceuticals for postmenopausal women. Previous studies have shown that EEs occur in the aquatic environment; however, the potential estrogenicity and risk of EEs in aquatic organisms, including fish, have yet to be studied in detail. Therefore, we evaluated the estrogenic potential of major EEs, namely equilin (Eq), 17α-dihydroequilin (17α-Eq), 17β-dihydroequilin (17β-Eq), equilenin (Eqn), 17α-dihydroequilenin (17α-Eqn), and 17β-dihydroequilenin (17β-Eqn), on medaka (Oryzias latipes) using in vivo and in silico assays. Quantitative real-time RT-PCR analyses revealed that expression levels of choriogenin L (ChgL) and choriogenin H (ChgH) in medaka embryos responded to various types and concentrations of EEs in a concentration-dependent manner, whereas transcription levels of vitellogenin 1 were not significantly affected by any of the EEs in the concentration range tested. The order of the in vivo estrogenic potencies of EEs was as follows: 17β-Eq > Eq > 17β-Eqn > Eqn > 17α-Eqn > 17α-Eq. Additionally, the 50% effective concentrations (EC50) of 17β-Eq was lower than that of 17β-estradiol. We also investigated the interaction potential of EEs with medaka estrogen receptor (ER) subtypes in silico using a three-dimensional model of the ligand-binding domain (LBD) for each ER and docking simulations. All six EEs were found to interact with the LBDs of ERα, ERβ1, and ERβ2. The order of the in silico interaction potentials of EEs with each ER LBD was as follows: 17β-Eq > 17α-Eq > Eq > 17β-Eqn > 17α-Eqn > Eqn. Furthermore, we identified the key amino acids that interact with EEs in each ER LBD; our findings suggest that amino acids and/or their hydrogen bonding may be responsible for the ligand-specific interactions with each ER. This study is the first to comprehensively analyze the estrogenic potential of EEs in medaka both in vivo and in silico.
  • Rin Shitaoka, Hiroshi Ishibashi, Ichiro Takeuchi
    Marine Pollution Bulletin 111812 - 111812 2020年11月 研究論文(学術雑誌) 
    Increased seawater temperature has resulted in mass coral bleaching events globally. Acropora tenuis, the dominant hermatypic coral species in southern Japan, was exposed to four temperature treatments [28 °C, 30 °C, 32 °C, and >32 (=33.3 °C)] for 7 d. The coral colour was converted to R (red), G (green), and B (blue) values, each ranging from 0 (darkest) to 255 (brightest). RGB values exposed to 28 °C and 30 °C decreased slightly, whereas those exposed to 32 °C increased significantly after day 3-6, and those exposed to 33.3 °C changed to white within 2 d. Quantitative RT-PCR analysis revealed no significant changes in heat shock proteins in Acropora and symbiotic dinoflagellates at 28 °C and 30 °C after a 7 d exposure. Our findings revealed that 30 °C, higher than the mean temperature of the warmest month in southern Japan, was an inhabitable temperature for A. tenuis.
  • Masashi Hirano, Kenji Toyota, Hiroshi Ishibashi, Nobuaki Tominaga, Tomomi Sato, Norihisa Tatarazako, Taisen Iguchi
    Chemical Research in Toxicology 2020年10月 [査読有り]
     研究論文(学術雑誌) 
    Juvenile hormone (JH) is an important endocrine factor regulating many biological activities in arthropods. In daphnids, methoprene-tolerant (Met) belongs to a basic helix-loop-helix/Per-Arnt-Sim (bHLH/PAS) family protein which has recently been confirmed as a JH receptor and can bind and be activated by JHs and JH agonists. Although the activation of the JH signaling pathway causes many physiological effects, the molecular basis for the structural feature and ligand binding properties of Daphnia Met are not fully understood. To study the ligand preference in terms of structural features of Daphnia Met, we built in silico homology models of the PAS-B domain of Daphnia Mets from cladoceran crustaceans, Daphnia pulex and D. magna. Structural comparison of two Daphnia Met PAS-B domain models revealed that the volume in the main cavity of D. magna Met was larger than that of D. pulex Met. Compared with insect Met, Daphnia Met had a less hydrophobic cavity due to polar residues in the core-binding site. Molecular docking simulations of JH and its analogs with Daphnia Met indicated that the interaction energies were correlated with each of the experimental values of in vivo JH activities based on male induction and in vitro Met-mediated transactivation potencies. Furthermore, in silico site-directed mutagenesis supported experimental findings that Thr292 in D. pulex Met and Thr296 in D. magna Met substitution to valine contribute to JH selectivity and differential species response. This study demonstrates that in silico simulations of Daphnia Met and its ligands may be a tool for predicting the ligand profile and cross species sensitivity.
  • Ichiro Takeuchi, Daisuke Takaichi, Masakazu Katsumata, Hiroshi Ishibashi
    Marine Pollution Bulletin 154 2020年05月 [査読有り]
     研究論文(学術雑誌) 
    © 2020 Elsevier Ltd We investigated coral bleaching by monitoring colour changes and measuring the delayed fluorescence (DF) of symbiotic dinoflagellates in the hermatypic coral Acropora tenuis, exposed to 1.0 μg/L Irgarol 1051 (photosystem II herbicide) for 14 d. The Irgarol concentration corresponded to those from international port regions of the world. The coral colour and DFs under the control treatment were stable throughout the experiment, whereas under the Irgarol treatment the corals showed gradual bleaching. The Irgarol treatment caused a rapid decrease in the slow decay DF component (10.1–60.0 s), while the fast decay DF component (0.1–10.0 s) decreased significantly after 6 d. The significant correlation between the latter values and the coral colour indicates that if the electron accumulation function of quinones QA and QB is compromised, corals will bleach. The present study will contribute to the understanding of the mechanism involved in bleaching of coral exposed to herbicides.
  • Hiroshi Ishibashi, Masaya Uchida, Yuki Temma, Masashi Hirano, Nobuaki Tominaga, Koji Arizono
    Ecotoxicology and Environmental Safety 193 2020年04月 [査読有り]
     研究論文(学術雑誌) 
    © 2020 This study assessed the transcription levels of estrogen-responsive genes, such as vitellogenins (Vtg1 and Vtg2), choriogenins (ChgL, ChgH, and ChgHm), cytochrome P450 aromatase (CYP19a1b), and ER subtypes (ERα, ERβ1, and ERβ2), in 7 days-post-fertilization (dpf) embryos and 9 and 12 dpf larvae of medaka (Oryzias latipes) exposed to estrogenic endocrine-disrupting chemicals (EDCs). The <5 h-post-fertilization embryos were exposed to EDCs such as 17β-estradiol (E2), p-n-nonylphenol (NP), and bisphenol A (BPA). In E2 (0.10–222 nM)-treated 7 dpf embryos and 9 or 12 dpf larvae, ChgL, ChgH, and ChgHm expression was up-regulated in a concentration-dependent manner. By contrast, interestingly, Vtg1 and Vtg2 expression was not induced in E2-treated 7 dpf embryos but was significantly induced in 9 and 12 dpf larvae, suggesting a developmental-stage-specific regulatory mechanism underlying Vtg expression. The maximum concentrations of NP (0.09–1.5 μM) and BPA (1.8–30 μM) up-regulated Chg expression in 9 or 12 dpf larvae, and the relative estrogenic potencies (REPs) of E2, NP, and BPA were 1, 2.1 × 10−4, and 1.0 × 10−5, respectively. Chg messenger RNA (mRNA) in medaka embryos and larvae can be used as a sensitive biomarker for screening potential estrogenic EDCs. Our assay system using embryos and larvae can be used as an in vivo alternative model because independent feeding stages (e.g., embryonic and early larval stages) are suitable alternatives.
  • Akemi Yamaguchi, Masaya Uchida, Hiroshi Ishibashi, Masashi Hirano, Nobuhiro Ichikawa, Koji Arizono, Jiro Koyama, Nobuaki Tominaga
    Chemosphere 242 2020年03月 [査読有り]
     研究論文(学術雑誌) 
    © 2019 Elsevier Ltd Polycyclic aromatic hydrocarbons (PAHs), such as benzo[a]pyrene (BaP), are widely distributed in air, water, and sediments; however, limited data are available regarding their potential adverse effects on the early life stages of fish. In this study, we evaluated the embryonic teratogenicity and developmental toxicity of BaP in Japanese medaka (Oryzias latipes) using a nanosecond pulsed electric field (nsPEF) technique and predicted their molecular mechanisms via transcriptome analysis. The gas chromatography/mass spectrometry analyses revealed that the BaP was efficiently incorporated into the embryos by nsPEF treatment. The embryos incorporating BaP presented typical teratogenic and developmental effects, such as cardiovascular abnormalities, developmental abnormalities, and curvature of backbone. DNA microarray analysis revealed several unique upregulated genes, such as those involved in cardiovascular diseases, various cellular processes, and neural development. Furthermore, the gene set enrichment and network analyses found several genes and hub proteins involved in the developmental effects of BaP on the embryos. These findings suggest a potential mechanism of teratogenicity and developmental toxicity caused by exposure to BaP. The nsPEF and transcriptome analyses in combination can be effective for evaluating the potential effects of chemical substances on medaka embryos.
  • Akiko Takamoto, Hiroshi Ishibashi, Satoshi Fukushima, Hiroko Tomoyori, Koji Arizono
    Shokuhin eiseigaku zasshi. Journal of the Food Hygienic Society of Japan 61 5 192 - 199 2020年 研究論文(学術雑誌) 
    Herein, we quantified the concentrations of cadmium (Cd) and arsenic (As) in 63 milled rice (Oryza sativa L.) cultivated in Japan, Vietnam, and Indonesia. We estimated the daily intake of Cd and As by adults and children consuming this rice by inductively coupled plasma-mass spectrometer (ICP-MS). Cd and As were detected in all milled rice samples. No significant differences were observed in Cd concentrations between Japanese (50th percentile concentration: 0.036 mg/kg), Vietnamese (0.035 mg/kg), and Indonesian rice (0.022 mg/kg). However, As concentrations in Vietnamese rice (50th percentile concentration: 0.142 mg/kg) were significantly higher than those in Japanese (0.101 mg/kg, p<0.001) and Indonesian rice (0.038 mg/kg, p<0.0001). Target hazard quotients (THQs) were then calculated to evaluate the non-carcinogenic health risk from ingestion of individual heavy metals (Cd and As) by rice consumption. Results revealed that THQs of individual heavy metals for Japanese, Vietnamese, and Indonesian adults and children consuming this rice were all less than one, suggesting that no health risk is associated with the intake of a single heavy metal via rice consumption.
  • Misato Kamei, Kotaro Takayama, Hiroshi Ishibashi, Ichiro Takeuchi
    Marine Pollution Bulletin 150 2020年01月 [査読有り]
     研究論文(学術雑誌) 
    © 2019 Elsevier Ltd The effects of ecologically relevant concentrations of Irgarol 1051, a representative PSII herbicide, on hermatypic corals were studied in the laboratory. The colour and chlorophyll fluorescence of Acropora tenuis were examined following exposure to around ambient concentrations of Irgarol 1051 (20 ng/L and 200 ng/L) for 7 days. While the colour of corals was stable throughout the experiment at both concentrations, the maximum effective quantum yield (ΔF/Fm′) of symbiotic dinoflagellates decreased with increasing Irgarol 1051 concentration (day 7: 8%, 20 ng/L; 37%, 200 ng/L). The expression of heat shock protein (HSP) 70 and 90 in symbiotic dinoflagellates was upregulated after 7 days exposure to both Irgarol concentrations, whereas HSP90 in coral was not upregulated. The findings of the present study suggest that the threshold of chlorophyll fluorescence and HSP expression in symbiotic dinoflagellates is lower than 20 ng/L, which is around ecologically relevant concentrations in tropical to subtropical waters.
  • Tominaga Nobuaki, Shino Seiya, Uchida Masaya, Ishibashi Hiroshi, Iida Midori, Okobira Tadashi, Arizono Kayla, Yoshida Noriaki, Arizono Koji
    Fundamental Toxicological Sciences 6 2 31 - 36 2019年03月 [査読有り]
     
    ナノ秒パルス電場(nsPEF)技法とバイオインフォマティクス解析により、リチウム(Li)が、メダカ(Oryzias latipes)の胚発生毒性や催奇形性を及ぼす影響について基礎的検討を行い、その分子機序を明らかにした。その結果、1mg/LのLiCl処理により、胚発達遅滞、眼奇形、心肥大、血栓など重篤な奇形を及ぼす影響が認められた。また、nsPEFとLiCl処理した受精2日後胚のRNA-sq分析を行い、組織形成および器官形成関連の2483遺伝子における上方/下方調節遺伝子を同定した結果では、遺伝子オントロジーによるエンリッチ分析により、LiCl処理胚で、ヌクレオチド結合、ATP結合、RNA結合や核酸結合等の分子機能および細胞成分が有意にエンリッチされることが確認された。さらに、KEGGの遺伝子セットエンリッチ分析により、LiClのスプライセオソーム、細胞周期、セレン化合物代謝、TGF-βシグナリングおよびRNA分解への影響についても観察され、GSK3B、BAXおよびMAP3K8遺伝子の上方調節も認められた。
  • Akiko Takamoto, Yoshinori Ikenaka, Mayu Sugimoto, Manami Iwano, Satoshi Fukushima, Takahiro Ichise, Shouta M. M. Nakayama, Keisuke Takahashi, Keisuke Kato, Hiroshi Ishibashi, Mayumi Ishizuka, Koji Arizono
    Japanese Journal of Food Chemistry and Safety 26 1 17 - 27 日本食品化学学会 2019年 [査読有り]
     
    In this study, we determined the concentration of seven neonicotinoid insecticides and 13 of their metabolites in 46 bottled greenteabeverages, and estimated the daily intake of neonicotinoids in adults and children from these drinks. Liquid chromatographycoupled with electrospray ionization tandem mass spectrometry (LC–ESI/MS/MS) analysis revealed that six neonicotinoidsand two metabolites were detected in bottled green-tea beverages, and the 50th percentile concentration indicated dinotefuran > thiacloprid > clothianidin > imidacloprid > thiamethoxam > acetamiprid > N-desmethyl-acetamiprid > thiacloprid-amide. Theconcentration and composition profile of neonicotinoids in bottled green-tea beverages were different between brands; however,no significant differences were observed among manufacturing sites. The concentration of acetamiprid in bottled green-teabeverages was significantly correlated with that of N-desmethyl-acetamiprid. A significant relationship between concentrations ofthiamethoxam and clothianidin in bottled green-tea beverages was also observed, suggesting those neonicotinoids are metabolizedduring the tea leaves cultivation period. The daily intake for neonicotinoids in both adults and children from consumption ofbottled green-tea beverages was < 2.1% when compared with the acceptable daily intake (ADI) of neonicotinoids.
  • Hiroshi Ishibashi, Masashi Hirano, Eun Young Kim, Hisato Iwata
    Environmental Science and Technology 2019年 [査読有り]
     研究論文(学術雑誌) 
    © 2019 American Chemical Society. In this study, we assessed the binding affinities of perfluoroalkyl substances (PFASs), including perfluoroalkyl carboxylates (PFCAs) and perfluoroalkyl sulfonates (PFSAs), to the ligand-binding domains (LBDs) of Baikal seal (Pusa sibirica; bs) and human (h) peroxisome proliferator-activated receptor alpha (PPARα). An in vitro competitive binding assay showed that six PFCAs and two PFSAs could bind to recombinant bs and hPPARα LBD proteins in a dose-dependent manner. The relative binding affinities (RBAs) of PFASs to bsPPARα were as follows: PFOS > PFDA > PFNA > PFUnDA > PFOA > PFHxS > PFHpA > PFHxA. The RBAs to bsPPARα showed a significant positive correlation with those to hPPARα. In silico PPARα homology modeling predicted that there were two ligand-binding pockets (LBPs) in the bsPPARα and hPPARα LBDs. Structure-activity relationship analyses suggested that the binding potencies of PFASs to PPARα might depend on LBP binding cavity volume, hydrogen bond interactions, the number of perfluorinated carbons, and the hydrophobicity of PFASs. Interspecies comparison of the in vitro binding affinities revealed that bsPPARα had higher preference for PFASs with long carbon chains than hPPARα. The in silico docking simulations suggested that the first LBP of bsPPARα had higher affinities than that of hPPARα however, the second LBP of bsPPARα had lower affinities than that of hPPARα. To our knowledge, this is the first evidence showing interspecies differences in the binding of PFASs to PPARαs and their structure-activity relationships.
  • Hiroshi Ishibashi, Eun Young Kim, Koji Arizono, Hisato Iwata
    Environmental Science and Technology 52 20 11831 - 11837 2018年10月 [査読有り]
     研究論文(学術雑誌) 
    © 2018 American Chemical Society. We investigated the Baikal seal (Pusa sibirica) peroxisome proliferator-activated receptor α (bsPPARα) transactivation potencies of polybrominated diphenyl ethers (PBDEs) using an in vitro bsPPARα reporter gene assay. BDE47, BDE99, and BDE153 induced bsPPARα-mediated transcriptional activities in a dose-dependent manner. To compare bsPPARα transactivation potencies of PBDEs, perfluorooctanoic acid (PFOA)-based relative potencies (REPs), a ratio of 50% effective concentration of PFOA to the test chemical, were determined. The order of REPs of PBDEs was BDE153 (13) > BDE99 (8.1) > BDE47 (6.6) > PFOA (1.0) > BDE100, BDE154, and BDE183 (not activated). PBDEs with two bromine atoms at the ortho position showed higher bsPPARα transactivation potencies than those with three bromine atoms. Comparison of the lowest-observed-effect concentration in bsPPARα reporter gene assays revealed that BDE99 was 7-fold more potent than CB99, a polychlorinated biphenyl congener with the same IUPAC number, indicating that brominated congeners could more efficiently activate bsPPARα than chlorinated congeners. The REPs of PBDEs for bsPPARα transactivation were approximately 7- to 13-fold higher than those of perfluorochemicals (PFCs), suggesting that the effects of PBDEs on the bsPPARα signaling pathway may be superior to those of PFCs. This study provides the first evidence that PBDE congeners activate PPARα in vitro.
  • Hiroshi Ishibashi, Masaya Uchida, Keisuke Yoshimoto, Yuta Imamura, Ryoko Yamamoto, Yoshinori Ikenaka, Masahito Kawai, Nobuhiro Ichikawa, Yuji Takao, Nobuaki Tominaga, Yasuhiro Ishibashi, Koji Arizono
    Environmental Pollution 239 281 - 288 2018年08月 [査読有り]
     研究論文(学術雑誌) 
    © 2018 In this study, we determined the concentration of equine estrogens, such as equilin (Eq) and equilenin (Eqn), in the river water collected from nine research stations in Hokkaido, Japan. The LC–MS/MS analysis revealed that Eq concentrations were 2.7 ± 6.7, 0.22 ± 0.12, and 1.2 ± 0.64 ng/L in Sep 2015, Feb 2016, and Jul 2016, respectively. Eqn had concentration levels similar to those of Eq. Comparison of the concentrations at nine research stations showed that seasonal variation was observed in the detected Eq and Eqn concentration levels. This study was the first to show the occurrences and seasonal variation of Eq and Eqn in the river water of Japan. We further investigated the reproductive and transgenerational effects of Eq in Japanese medaka (Oryzias latipes) exposed to 10, 100, and 1000 ng/L for 21 days and assessed the transcriptional profiles of the estrogen-responsive genes in the livers of both sexes. The reproduction assay demonstrated that 1000 ng/L of Eq adversely affected the reproduction (i.e. fecundity) in the F0 generation and that the hatching of F1 generation fertilized eggs was reduced in the 100 and 1000 ng/L treatment groups. Our qRT-PCR assay revealed that the mRNA expression levels of hepatic vitellogenin 1 and 2, choriogenin L and H, and estrogen receptor α were significantly up-regulated in males exposed to 100 and/or 1000 ng/L of Eq. In contrast, the transcriptional levels of several genes, such as pregnane X receptor and cytochrome P450 3A, were down-regulated in the livers of males after the 21-d exposure. These results suggest that Eq has endocrine-disrupting potential such as reproductive and transgenerational effects by the modulation of hepatic estrogen-responsive genes expression on medaka. Occurrence, seasonal variation and endocrine-disrupting potentials of equine estrogens.
  • Hiroshi Ishibashi, Seigo Minamide, Ichiro Takeuchi
    Marine Pollution Bulletin 133 773 - 780 2018年08月 [査読有り]
     研究論文(学術雑誌) 
    © 2018 Elsevier Ltd To elucidate the effects of the herbicide Irgarol 1051 on the gene expression of heat shock protein 90 (HSP90) in hard corals, we isolated a full-length cDNA encoding HSP90 from Acropora tenuis, which has a deduced open reading frame of 732-amino acid residues with a predicted molecular mass of 84.5 kDa. The amino acid sequence of A. tenuis HSP90 showed a high degree of similarity with the hermatypic-coral HSP90 families. After a 7-d exposure to 1 or 10 μg/L of Irgarol, the body colours of corals in the 10 μg/L treatment group were significantly whiter (bleached), whereas no such effects were observed in the corals in the 1 μg/L treatment group. However, the expression level of coral HSP90 was significantly downregulated after exposure to both 1 and 10 μg/L Irgarol. These results suggest that A. tenuis HSP90 may be a useful molecular biomarker to predict bleaching caused by herbicides.
  • Shingo Nishiguchi, Naohisa Wada, Hideyuki Yamashiro, Hiroshi Ishibashi, Ichiro Takeuchi
    Marine Pollution Bulletin 131 422 - 427 2018年06月 [査読有り]
     研究論文(学術雑誌) 
    © 2018 Elsevier Ltd The colours of the hermatypic corals Porites sp. and Acropora cytherea at Sesoko Island, Okinawa, Japan, were photographed continuously, from 19 July to 6 September 2016, by an underwater camera equipped with a lens wiper. The average seawater temperature during the study period was 29.9 °C. The daily average seawater temperature (DAST) was >30.0 °C until 23 August 2016, and a maximum value of 31.2 °C was recorded on 2 August 2016. Red, green, and blue (RGB) values of these corals were analysed based on photographs taken at 14:00. The RGB values of Porites sp. were stable throughout the observation period, while those of A. cytherea gradually increased (i.e. moved toward the “white” end of the spectrum) until the beginning of September. The present study demonstrated the usefulness of RGB analysis of photographs taken by an underwater camera equipped with a lens wiper for monitoring coral beaching.
  • Akemi Yamaguchi, Hiroshi Ishibashi, Susumu Kono, Midori Iida, Masaya Uchida, Koji Arizono, Nobuaki Tominaga
    Journal of Applied Toxicology 38 5 714 - 723 2018年05月 [査読有り]
     研究論文(学術雑誌) 
    Copyright © 2017 John Wiley & Sons, Ltd. Herein, we propose using a nanosecond pulsed electric field (nsPEF) technique to assess teratogenicity and embryonic developmental toxicity of estradiol-17β (E 2 ) and predict the molecular mechanisms of teratogenicity and embryonic developmental defects caused by E 2 on medaka (Oryzias latipes). The 5 hour post-fertilization embryos were exposed to co-treatment with 10 μm E 2 and nsPEF for 2 hours and then continuously cultured under non-E 2 and nsPEF conditions until hatching. Results documented that the time to hatching of embryos was significantly delayed in comparison to the control group and that typical abnormal embryo development, such as the delay of blood vessel formation, was observed. For DNA microarray analysis, 6 day post-fertilization embryos that had been continuously cultured under the non-E 2 and nsPEF condition after 2 hour co-treatments were used. DNA microarray analysis identified 542 upregulated genes and one downregulated gene in the 6 day post-fertilization embryos. Furthermore, bioinformatic analyses using differentially expressed genes revealed that E 2 exposure affected various gene ontology terms, such as response to hormone stimulus. The network analysis also documented that the estrogen receptor α in the mitogen-activated protein kinase signaling pathway may be involved in regulating several transcription factors, such as FOX, AKT1 and epidermal growth factor receptor. These results suggest that our nsPEF technique is a powerful tool for assessing teratogenicity and embryonic developmental toxicity of E 2 and predict their molecular mechanisms in medaka embryos.
  • 内田 雅也, 平野 将司, 石橋 弘志, 冨永 伸明, 有薗 幸司
    環境毒性学会誌 20 2 59 - 68 日本環境毒性学会 2017年12月 [査読有り]
  • Keita Hirayama, Kotaro Takayama, Shinsuke Haruta, Hiroshi Ishibashi, Ichiro Takeuchi
    Marine Pollution Bulletin 124 2 678 - 686 2017年11月 [査読有り]
     研究論文(学術雑誌) 
    © 2017 Elsevier Ltd Colour change in Acropora tenuis, a representative species of Indo-Pacific hard coral, in response to low concentrations of Irgarol 1051 was examined in the laboratory. Branches of A. tenuis were exposed to 0, 1, and 10 μg Irgarol 1051/L for 14 days, and photographed daily using digital camera. These Irgarol 1051 concentrations were similar to those recorded at a number of sea ports. Red, green and blue (RGB) coral colour values were quantified from the photographs, with black represented by R = G = B = 0 and white as R = G = B = 255. Exposure to Irgarol 1051 caused RGB values to increase, moving towards the ‘white’ end of the spectrum as Irgarol 1051 concentration increased. These results suggest that the ambient levels of Irgarol 1051 recorded from port environments could be implicated in coral bleaching, if concentrations over nearby reef ecosystems are similar.
  • Hiroshi Ishibashi, Masaya Uchida, Akiko Koyanagi, Yoshihiro Kagami, Teruhiko Kusano, Ayami Nakao, Ryoko Yamamoto, Nobuhiro Ichikawa, Nobuaki Tominaga, Yasuhiro Ishibashi, Koji Arizono
    Journal of Applied Toxicology 36 11 1392 - 1400 2016年11月 [査読有り]
     研究論文(学術雑誌) 
    Copyright © 2016 John Wiley & Sons, Ltd. In the present study, we investigated transcriptional profiles of estrogen-responsive genes, such as vitellogenins (Vtg1 and Vtg2), choriogenins (ChgL and ChgH) and estrogen receptor subtypes (ERα, ERβ1, and ERβ2), in the liver of male medaka fish (Oryzias latipes) that were exposed to six equine estrogens (1–300 ng l−1) for 3 days. Our quantitative reverse transcription-polymerase chain reaction (RT-PCR) analyses revealed that the expression levels of hepatic Vtg, Chg and ERα genes in male medaka responded to various types and concentrations of equine estrogens. The estrogenic potentials of the tested chemicals were in the order of equilin > 17β-estradiol > equilenin > 17β-dihydroequilin > 17β-dihydroequilenin > 17α-dihydroequilin > 17α-dihydroequilenin, showing the higher estrogenic potential of equilin than that of 17β-estradiol. Our results also showed that the estrogenicities of 17β-dihydroequilin and 17β-dihydroequilenin were more potent than that of 17α-dihydroequilin and 17α-dihydroequilenin. Furthermore, in gene expression analyses of hepatic ER subtypes, observations were made to note that 17β-estradiol and equilin induced ERα transcription in male medaka, and the ERα transcription level had significantly positive correlations with the expression of Vtg and Chg genes. In contrast, in the same 17β-estradiol and equilin treatment groups, it was shown that the transcription levels of hepatic ERβ1 and/or ERβ2 had significantly negative correlations with the expression of Vtg and Chg genes. These results suggested some potential involvement of the ER subtypes in the regulation of Vtg and Chg gene expressions in the liver. This is the first report describing the comprehensive analyses of in vivo estrogenicity of the equine estrogens in male medaka. Copyright © 2016 John Wiley & Sons, Ltd.
  • Masaya Uchida, Masashi Hirano, Hiroshi Ishibashi, Jun Kobayashi, Yoshihiro Kagami, Akiko Koyanagi, Teruhiko Kusano, Minoru Koga, Koji Arizono
    Ecotoxicology and Environmental Safety 133 360 - 365 2016年11月 [査読有り]
     研究論文(学術雑誌) 
    © 2016 Elsevier Inc. Nonylphenol (NP) has been classified as an endocrine-disrupting chemical. In this study, we conducted mysid DNA microarray analysis with which has 2240 oligo DNA probes to observe differential gene expressions in mysid crustacean (Americamysis bahia) exposed to 1, 3, 10 and 30 μg/l of NP for 14 days. As a result, we found 31, 27, 39 and 68 genes were differentially expressed in the respective concentrations. Among these genes, the expressions of five particular genes were regulated in a similar manner at all concentrations of the NP exposure. So, we focused on one gene encoding cuticle protein, and another encoding cuticular protein analogous to peritrophins 1-H precursor. These genes were down-regulated by NP exposure in a dose-dependent manner, and it suggested that they were related in a reduction of the number of molting in mysids. Thus, they might become useful molecular biomarker candidates to evaluate molting inhibition in mysids.
  • Yukiko Ogino, Shigehiro Kuraku, Hiroshi Ishibashi, Hitoshi Miyakawa, Eri Sumiya, Shinichi Miyagawa, Hajime Matsubara, Gen Yamada, Michael E. Baker, Taisen Iguchi
    Molecular Biology and Evolution 33 1 228 - 244 2016年01月 [査読有り]
     研究論文(学術雑誌) 
    © The Author 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. Steroid hormone receptor family provides an example of evolution of diverse transcription factors through wholegenome duplication (WGD). However, little is known about how their functions have been evolved after the duplication. Teleosts present a good model to investigate an accurate evolutionary history of protein function after WGD, because a teleost-specific WGD (TSGD) resulted in a variety of duplicated genes in modern fishes. This study focused on the evolution of androgen receptor (AR) gene, as two distinct paralogs, ARa and ARb, have evolved in teleost lineage after TSGD. ARa showed a unique intracellular localization with a higher transactivation response than that of ARb. Using site-directed mutagenesis and computational prediction of protein-ligand interactions, we identified two key substitutions generating a new functionality of euteleost ARa. The substitution in the hinge region contributes to the unique intracellular localization of ARa. The substitution on helices 10/11 in the ligand-binding domain possibly modulates hydrogen bonds that stabilize the receptor-ligand complex leading to the higher transactivation response of ARa. These substitutions were conserved in Acanthomorpha (spiny-rayed fish) ARas, but not in an earlier branching lineage among teleosts, Japanese eel. Insertion of these substitutions into ARs from Japanese eel recapitulates the evolutionary novelty of euteleost ARa. These findings together indicate that the substitutions generating a new functionality of teleost ARa were fixed in teleost genome after the divergence of the Elopomorpha lineage. Our findings provide a molecular explanation for an adaptation process leading to generation of the hyperactive AR subtype after TSGD.
  • Akemi Yamaguchi, Hiroshi Ishibashi, Koji Arizono, Nobuaki Tominaga
    Ecotoxicology and Environmental Safety 120 198 - 205 2015年10月 [査読有り]
     研究論文(学術雑誌) 
    © 2015 . Various studies have demonstrated the estrogenic effect of bisphenol A (BPA), a member of bisphenol analogs (BPs), in in vitro and in vivo assays. However, limited data are available on the estrogenic potentials and risks of other BPs in aquatic organisms. In addition, the estrogenic effect of chemicals is known to have species-specific responses in teleost fish. The objective of this study was to evaluate the potential estrogenic effects of BPs on the medaka (Oryzias latipes) and common carp (Cyprinus carpio) using in vivo and in silico assays. Our quantitative real-time PCR analyses revealed that the expression levels of several hepatic estrogen-responsive biomarker genes in male medaka responded to various types and concentrations of BPs in a dose-response manner. The order of in vivo estrogenic potencies of BPs was as follows: BPC⋘BPAF>BPB>BPA⋙BPP. To further investigate the interaction potential of BPs with medaka estrogen receptor α (ERα) in silico, a three-dimensional model of the ERα ligand-binding domain (LBD) was built and docking simulations were performed. The docking simulation analysis revealed that BPC interaction potential for medaka ERα LBD was the most potent, followed by BPAF and BPA. Comparing this with carp ERα LBD revealed that the interaction potentials of these BPs to medaka ERα LBD were more stable than to carp ERα LBD. Furthermore, we identified key amino acid residues in medaka ERα LBD that interacted with BPC (Glu356, Arg397, and Cys533), BPAF (Thr350 and Glu356), and BPA (Glu356 and Met424), and found some differences in these key amino acid residues between medaka and carp ERα LBDs. These results of in vivo and in silico analyses showed potential estrogenic effects of BPs in teleost fish, and they also indicated that the differences in interaction potentials and key amino acid residues between medaka and carp ERα LBDs may be due to the differences between the species and estrogenic potencies of the selected BPs.
  • Masaya Uchida, Hiroshi Ishibashi, Ryoko Yamamoto, Akiko Koyanagi, Teruhiko Kusano, Nobuaki Tominaga, Yasuhiro Ishibashi, Koji Arizono
    Journal of Applied Toxicology 35 9 1040 - 1048 2015年09月 [査読有り]
     研究論文(学術雑誌) 
    © 2015 John Wiley & Sons, Ltd. Although several previous studies have demonstrated the presence of equine estrogens in the aquatic environment, limited data are currently available on the endocrine-disrupting potentials in fish and the risks they pose to aquatic organisms. To investigate the interactions of major equine estrogens equilin (Eq) and equilenin (Eqn), as well as their metabolites 17α-dihydroequilin, 17β-dihydroequilin, 17α-dihydroequilenin and 17β-dihydroequilenin, with the estrogen receptor α (ERα) of medaka (Oryzias latipes), a three-dimensional model of the ligand-binding domain (LBD) of ERα was built in silico, and docking simulations were performed. The docking simulation analysis indicated that the interaction of 17β-dihydroequilenin with the ERα LBD is the most potent, followed by those of 17α-dihydroequilin and 17β-dihydroequilin, whereas those of Eq and Eqn were least potent. We further analyzed gene expression profiles in the livers of male medaka exposed to Eq and Eqn. A DNA microarray representing 6000 genes revealed that 24-h exposure to Eq and Eqn (100ng/L) upregulated the expression of 6 and 34 genes in the livers of males, respectively. Genes upregulated by Eq included the estrogenic biomarker genes vitellogenins and choriogenins, suggesting the estrogenic potential of Eq. In contrast, Eqn exposure upregulated several cancer-related genes, such as mediator complex subunit 16 and RAS oncogene family members, suggesting a carcinogenic potential for Eqn. These results suggest that equine estrogens may have not only endocrine-disrupting potentials via the ERα signaling pathway but also carcinogenic potency in male medaka.
  • Ayako Inokuchi, Ryoko Yamamoto, Fumiyo Morita, Shota Takumi, Hiromi Matsusaki, Hiroshi Ishibashi, Nobuaki Tominaga, Koji Arizono
    Journal of Applied Toxicology 35 9 999 - 1006 2015年09月 [査読有り]
     研究論文(学術雑誌) 
    © 2015 John Wiley & Sons, Ltd. Lithium (Li) has been widely used to treat bipolar disorder, and industrial use of Li has been increasing; thus, environmental pollution and ecological impacts of Li have become a concern. This study was conducted to clarify the potential biological effects of LiCl and Li2CO3 on a nematode, Caenorhabditis elegans as a model system for evaluating soil contaminated with Li. Exposure of C. elegans to LiCl and Li2CO3 decreased growth/maturation and reproduction. The lowest observed effect concentrations for growth, maturation and reproduction were 1250, 313 and 10 000μm, respectively, for LiCl and 750, 750 and 3000μm, respectively, for Li2CO3. We also investigated the physiological function of LiCl and LiCO3 in C. elegans using DNA microarray analysis as an eco-toxicogenomic approach. Among approximately 300 unique genes, including metabolic genes, the exposure to 78μm LiCl downregulated the expression of 36 cytochrome P450, 16 ABC transporter, 10 glutathione S-transferase, 16 lipid metabolism and two vitellogenin genes. On the other hand, exposure to 375μm Li2CO3 downregulated the expression of 11 cytochrome P450, 13 ABC transporter, 13 lipid metabolism and one vitellogenin genes. No gene was upregulated by LiCl or Li2CO3. These results suggest that LiCl and Li2CO3 potentially affect the biological and physiological function in C. elegans associated with alteration of the gene expression such as metabolic genes. Our data also provide experimental support for the utility of toxicogenomics by integrating gene expression profiling into a toxicological study of an environmentally important organism such as C. elegans.
  • Shinichi Miyagawa, Ryohei Yatsu, Satomi Kohno, Brenna M. Doheny, Yukiko Ogino, Hiroshi Ishibashi, Yoshinao Katsu, Yasuhiko Ohta, Louis J. Guillette, Taisen Iguchi
    Endocrinology 156 8 2795 - 2806 2015年08月 [査読有り]
     研究論文(学術雑誌) 
    Androgens are essential for the development, reproduction, and health throughout the life span of vertebrates, particularly during the initiation and maintenance of male sexual characteristics. Androgen signaling is mediated by the androgen receptor (AR), a member of the steroid nuclear receptor superfamily. Mounting evidence suggests that environmental factors, such as exogenous hormones or contaminants that mimic hormones, can disrupt endocrine signaling and function. The American alligator (Alligator mississippiensis), a unique model for ecological research in that it exhibits environment-dependent sex determination, is oviparous and long lived. Alligators from a contaminated environment exhibit low reproductive success and morphological disorders of the testis and phallus in neonates and juveniles, both associated with androgen signaling; thus, the alterations are hypothesized to be related to disrupted androgen signaling. However, this line of research has been limited because of a lack of information on the alligator AR gene. Here, we isolated A mississippiensis AR homologs (AmAR) and evaluated receptor-hormone/chemical interactions using a transactivation assay.WeshowedthatAmARresponded to all natural androgens and their effects were inhibited by cotreatment with antiandrogens, such as flutamide, p,p-dichlorodiphenyldichloroethylene, and vinclozolin. Intriguingly,wefound a spliced form of theAR from alligator cDNA, which lacks seven amino acids within the ligand-binding domain that shows no response to androgens. Finally, we have initial data on a possible dominant-negative function of the spliced form of the AR against androgen-induced AmAR.
  • Saki Tohyama, Shinichi Miyagawa, Anke Lange, Yukiko Ogino, Takeshi Mizutani, Norihisa Tatarazako, Yoshinao Katsu, Masaru Ihara, Hiroaki Tanaka, Hiroshi Ishibashi, Tohru Kobayashi, Charles R. Tyler, Taisen Iguchi
    Environmental Science and Technology 49 12 7439 - 7447 2015年06月 [査読有り]
     研究論文(学術雑誌) 
    © 2015 American Chemical Society. Exposure to endocrine disrupting chemicals (EDCs) can elicit adverse effects on development, sexual differentiation, and reproduction in fish. Teleost species exhibit at least three subtypes of estrogen receptor (ESR), ESR1, ESR2a, and ESR2b; thus, estrogenic signaling pathways are complex. We applied in vitro reporter gene assays for ESRs in five fish species to investigate the ESR subtype-specificity for better understanding the signaling pathway of estrogenic EDCs. Responses to bisphenol A, 4-nonylphenol, and o,p′-DDT varied among ESR subtypes, and the response pattern of ESRs was basically common among the different fish species. Using a computational in silico docking model and through assays quantifying transactivation of the LBD (using GAL-LBD fusion proteins and chimera proteins for the ESR2s), we found that the LBD of the different ESR subtypes generally plays a key role in conferring responsiveness of the ESR subtypes to EDCs. These results also indicate that responses of ESR2s to EDCs cannot necessarily be predicted from the LBD sequence alone, and an additional region is required for full transactivation of these receptors. Our data thus provide advancing understanding on receptor functioning for both basic and applied research.
  • Jun Kobayashi, Yuki Imuta, Tomohiro Komorita, Katsumasa Yamada, Hiroshi Ishibashi, Fumitaka Ishihara, Naoya Nakashima, Jun Sakai, Koji Arizono, Minoru Koga
    Chemosphere 118 1 201 - 206 2015年 [査読有り]
     研究論文(学術雑誌) 
    © 2014 Elsevier Ltd. To evaluate trophic biomagnification of polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs) in an estuary of the Ariake Sea, Japan, we measured concentrations of 209 PCB congeners and 28 PBDE congeners, and nitrogen stable isotope (δ15N) levels in living aquatic organisms. The trophic magnification factor (TMF) for σPCBs (all 209 congeners) was 1.52, and TMFs for 58 PCB congeners ranged from 0.90 to 3.28. In contrast, TMF for σPBDEs was 1.17, and TMFs for 7 PBDE congeners ranged from 0.46 to 1.66. TMFs of PCB and PBDE congeners in this study were lower than those in marine food webs, and were similar to those in a lake food web. However, although negative relationships were observed between TMF and log octanol-water partition coefficient (KOW) values among PCB congeners in this study (log KOW up to 7), positive relationships have been reported in several other studies. In the present estuary, PCB concentrations in sea bass may not reach a steady state because sea bass are migratory species. Therefore, TMFs of highly chlorinated congeners with high log KOW values take longer to reach the steady state and may not increase with increasing log KOW.
  • Comparative study of the biological effects of antimicrobials, triclosan and trichlocarban, for C. elegans
    Ayako Inokuchi, Moritoshi Nihira, Midori Minakoshi, Ryoko Yamamoto, Hiroshi Ishibashi, Nobuaki Tominaga, Koji Arizono
    Journal of Environment and Safety 5 2 95 - 98 2014年 [査読有り]
  • Nakashima Shinya, Ji Hezhe, Ishitobi Sachiko, Kawazoe Sadahiro, Shinohara Ryota, Ishibashi Hiroshi, Tominaga Nobuyuki, Arizono Koji
    Japanese Journal of Food Chemistry and Safety 20 1 22 - 30 日本食品化学学会 2013年 [査読有り]
     
    ペット飼料中に存在する植物エストロゲンと塩素系農薬は、共にバイオアッセイによりエストロゲン作用をもたらす可能性があると考えられる。我々は、様々な市販ペット飼料中の、植物エストロゲン、塩素系農薬とエストロゲン活性を調査し、エストロゲン活性への潜在的な寄与を、以前と比較し検討した。β-グルクロニダーゼによる加水分解後、genisteinとdaidzeinは検出された。これらの結果は以前の結果と同等であった。さらに、α-HCH、β-HCH、HCB、p,p'-DDE、dieldrinは、テストした15種の飼料中2つで検出された。全ての飼料で、イーストベース生物検定法では、α(ERα)より高いエストロゲン受容体β(ERβ)の活性化レベルを示した。これらの結果は、植物エストロゲン(たとえば、genisteinとdaidzein)がエストロゲン活性に寄与している主な物質であることを示している。さらに、5飼料の植物エストロゲン濃度は、重要なエストロゲン活性を生じるに十分な量と推定された。
  • Pham Thi Dau, Hiroki Sakai, Masashi Hirano, Hiroshi Ishibashi, Yuki Tanaka, Kenji Kameda, Takahiro Fujino, Eun Young Kim, Hisato Iwata
    Toxicological Sciences 131 1 116 - 127 2013年01月 [査読有り]
     研究論文(学術雑誌) 
    The constitutive androstane receptor (CAR) not only displays a high basal transcriptional activity but also acts as a ligand-dependent transcriptional factor. It is known that CAR exhibits different ligand profiles across species. However, the mechanisms underlying CAR activation by chemicals and the species-specific responses are not fully understood. The objectives of this study are to establish a high-throughput tool to screen CAR ligands and to clarify how CAR proteins from the Baikal seal (bsCAR) and the mouse (mCAR) interact with chemicals and steroid receptor coactivator 1 (SRC1). We developed the surface plasmon resonance (SPR) system to assess quantitatively the interaction of CAR with potential ligands and SRC1. The ligand-binding domain (LBD) of bsCAR and mCAR was synthesized in a wheat germ cell-free system. The purified CAR LBD was then immobilized on the sensor chip for the SPR assay, and the kinetics of direct interaction of CARs with ligand candidates was measured. Androstanol and androstenol, estrone, 17β-estradiol, TCPOBOP, and CITCO showed compound-specific but similar affinities for both CARs. The CAR-SRC1 interaction was ligand dependent but exhibited a different ligand profile between the seal and the mouse. The results of SRC1 interaction assay accounted for those of our previous in vitro CAR-mediated transactivation assay. In silico analyses also supported the results of CAR-SRC1 interaction; there is little structural difference in the ligand-binding pocket of bsCAR and mCAR, but there is a distinct discrimination in the helix 11 and 12 of these receptors, suggesting that the interaction of ligand-bound CAR and SRC1 is critical for determining species-specific and ligand-dependent transactivation over the basal activity. The SPR assays demonstrated a potential as a high-throughput screening tool of CAR ligands. © The Author 2012. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved.
  • Thuruthippallil Leena Mol, Eun Young Kim, Hiroshi Ishibashi, Hisato Iwata
    Environmental Science and Technology 46 1 525 - 533 2012年01月 [査読有り]
     研究論文(学術雑誌) 
    Our previous studies have detected high levels of dioxins and related compounds (DRCs) including polychlorinated dibenzo-p-dioxins (PCDDs), furans (PCDFs), and coplanar PCBs (Co-PCBs) in the black-footed albatross (BFA), Phoebastria nigripes, from the North Pacific region. We have also cloned two aryl hydrocarbon receptors, AHR1 and AHR2, of the BFA. To evaluate the sensitivity to DRCs in the BFA and to assess the status of cytochrome P450 1A (CYP1A) induction in the wild population, this study investigated the mRNA expression levels of BFA AHR1 and AHR2 and also the transactivation potencies of each AHR by 15 selected DRC congeners. Quantitative real-time PCR of BFA AHR mRNAs showed that hepatic AHR1 is more highly expressed than AHR2. Transactivation by graded concentrations of individual DRCs was measured in COS-7 cells, where BFA AHR1 or AHR2 was transiently transfected. For congeners that exhibited AHR-mediated dose-dependent activities, 50% effective concentration (EC 50) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) relative potencies (REPs) were estimated. Based on the estimates of the REPs, TCDD induction equivalency factors (IEFs) were determined. For BFA AHR1, PeCDF was equipotent to TCDD, but other congeners exhibited lower IEFs. For BFA AHR2, PCDD/F congeners except OCDD/F showed IEFs 1.0. Using BFA AHR1- or AHR2-IEFs and hepatic concentrations of DRCs in North Pacific BFAs, TCDD induction equivalents (IEQs) were calculated. We further constructed nonlinear regression models on the relationships between BFA AHR1- or AHR2-IEF derived total IEQ or WHO-TEF derived total TEQ and ethoxyresorufin-O-deethylase activity (EROD) in the liver of wild BFAs. The results indicated that the relationships of BFA AHR1- and AHR2-based IEQs and EROD were predictable from BFA AHR1- and AHR2-mediated transactivation by TCDD, respectively. Collectively, these results suggest that the in vitro assay incorporating the AHR of species of concern would be a useful tool to predict the sensitivity to DRCs in the species and CYP1A induction in the wild population. © 2011 American Chemical Society.
  • Hiroshi Ishibashi, Eun Young Kim, Hisato Iwata
    Environmental Science and Technology 45 7 3123 - 3130 2011年04月 [査読有り]
     研究論文(学術雑誌) 
    The present study assessed the transactivation potencies of the Baikal seal (Pusa sibirica) peroxisome proliferator-activated receptor α (BS PPARα) by perfluorochemicals (PFCs) having various carbon chain lengths (C4-C12) using an in vitro reporter gene assay. Among the twelve PFCs treated with a range of 7.8-250 μM concentration, eight perfluoroalkyl carboxylates (PFCAs) and two perfluoroalkyl sulfonates (PFSAs) induced BS PPARα-mediated transcriptional activities in a dose-dependent manner. To compare the BS PPARα transactivation potencies of PFCs, the present study estimated the PFOA induction equivalency factors (IEFs), a ratio of the 50% effective concentration of PFOA to the concentration of each compound that can induce the response corresponding to 50% of the maximal response of PFOA. The order of IEFs for the PFCs was as follows: PFOA (IEF: 1) > PFHpA (0.89) > PFNA (0.61) > PFPeA (0.50) > PFHxS (0.41) > PFHxA (0.38) ≈ PFDA (0.37) > PFBA (0.26) = PFOS (0.26) > PFUnDA (0.15) ≫ PFDoDA and PFBuS (not activated). The structure-activity relationship analysis showed that PFCAs having more than seven perfluorinated carbons had a negative correlation (r = -1.0, p = 0.017) between the number of perfluorinated carbons and the IEF of PFCAs, indicating that the number of perfluorinated carbon of PFCAs is one of the factors determining the transactivation potencies of the BS PPARα. The analysis also indicated that PFCAs were more potent than PFSAs with the same number of perfluorinated carbons. Treatment with a mixture of ten PFCs showed an additive action on the BS PPARα activation. Using IEFs of individual PFCs and hepatic concentrations of PFCs in the liver of wild Baikal seals, the PFOA induction equivalents (IEQs, 5.3-58 ng IEQ/g wet weight) were calculated. The correlation analysis revealed that the hepatic total IEQs showed a significant positive correlation with the hepatic expression levels of cytochrome P450 4A-like protein (r = 0.53, p = 0.036). This suggests that our approach may be useful for assessing the potential PPARα-mediated biological effects of complex mixtures of PFCs in wild Baikal seal population. © 2011 American Chemical Society.
  • Sadahiro Kawazoe, Kazuna Naka, Hiroshi Ishibashi, Tomomi Obara, Koji Arizono, Kaori Hashimoto, Yasuji Hojo, Takashi Suzuki
    Journal of the Food Hygienic Society of Japan 51 3 101 - 109 2010年06月 [査読有り]
     研究論文(学術雑誌) 
    We examined the combined estrogenic activity of soybean extract used in a dietary supplement and ethinyl estradiol (EE) contained in an oral contraceptive. Olive oil (control), soybean extract (0.0036 or 0.36 g/kg corresponding to doses of total isoflavone of 0.83 or 83 mg/kg respectively), EE (1 or 10 μg/kg), and soybean extract+EE were administered to ovariectomized CD-1 mice by oral gavage for 4 consecutive days. Soybean extract (0.0036 or 0.36 g/kg) and EE (1 μg/kg) did not increase the relative uterine weight. The relative uterine weight of the soybean extract (0.0036 or 0.36 g/kg)+EE (10 μg/kg) group was significantly higher than that of the control. The relative uterine weight of the soybean extract (0.36 g/kg)+ EE (10 μg/kg) group was also significantly higher than that of the EE (10 μg/kg) group. Soybean extract showed estrogenic activity for human estrogen receptor (hER)-α and-β. Coadministration of EE with soybean extract increased the estrogenic activity for hER-α and-β.
  • Contamination of pharmaceutical and personal care products in sewage treatment plants and surface waters in South Korea and their removal during activated sludge treatment
    Joon-Woo Kim, Hiroshi Ishibashi, Masashi Hirano, Hyo-Sang Jang, Jong-Gu Kim, Yuji Takao, Nobuhiro Ichikawa, Ryota Shinohara, Koji Arizono
    Japan Journal of Environmental Chemistry 20 2 127 - 136 2010年 [査読有り]
  • Hotaka Kai, Yasuhiro Ishibashi, Taiki Mori, Hiroshi Ishibashi, Isao Kawaguchi, Hiroki Ohwaki, Takehiro Takemasa, Koji Arizono
    Journal of Material Cycles and Waste Management 12 2 128 - 135 2010年 [査読有り]
     研究論文(学術雑誌) 
    Livestock wastewater is treated by activated sludge treatment. Untreated livestock wastewater has high estrogen activity because animal excreta contains estrogen. When activated sludge treatment is applied, the estrogen activity declines or is lost. However, the color of treated livestock wastewater is deep brownish-red because of the decomposition of organic compounds or the synthesis of metabolites. Discharging colored wastewater to the environment could cause some problems, so it is necessary to decolorize colored wastewater before it is discharged. It has been suggested that electrolysis decolorization technology is suitable for treating colored wastewater; however, the process produces volatile organic compounds (VOCs). In fact, little research has been conducted with reference to estrogen activity in wastewater that has undergone electrolysis, especially on the contribution of the electrolysis decolorization process to estrogen activity, i.e., the possibility of resynthesis of some substance with estrogen activity due to resolved and metabolized colored components. In this study, the concentration of VOC was measured for various electrolysis conditions, and estrogen activity was examined using a yeast two-hybrid assay. From the results, decolorization of colored livestock wastewater by electrolysis was possible, and the VOC generation during electrolysis could be controlled depending on the electrolysis conditions. Estrogen activity in colored livestock wastewater disappeared on electrolysis decolorization. © 2010 Springer Japan.
  • Akemi Yamaguchi, Hiroshi Ishibashi, Shinya Kohra, Koji Arizono, Keisuke Kato, Takayuki Nakahama, Yuichro Kanno, Yoshio Inouye, Nobuaki Tominaga
    Journal of Health Science 55 6 930 - 938 2009年12月 [査読有り]
     研究論文(学術雑誌) 
    Vitellogenin (VTG) is a useful biomarker for detecting the estrogenic activity of chemicals in aquatic environments. However, little information is available on the regulatory mechanisms of the expression of each VTG subtype, particularly the relationship between expression patterns of VTG1/2 and estrogen receptor (ER) subtypes, such as ERα and ERβ. In this paper, we measured VTG1 and VTG2 mRNA induction in male medaka liver, which was treated with ERα-selective ligand, (17α, 20E)-3-hydroxy-17,20-[(1- methoxyethylidene)bis(oxy)]-19-norpregna-1,3,5(10),20-tetraene-21-carboxylic acid, methyl ester or ERβ-selective ligand, 2-(4-hydroxyphenyl)-5-hydroxy- 1,3-benzoxazole and investigated the characteristics of ER subtype function in VTG1 and VTG2 inductions. Hepatic VTG1 mRNA was induced by ERα-selective ligands at even low concentration and maximum increases were the same as for E2. VTG2 mRNA was also increased, but its levels were very low. On the other hand, ERβ-selective ligands significantly increased VTG2 mRNA in the presence of ERα agonists. These results indicate that the expression of each VTG subtype is regulated by unique ER subtypes. VTG1 expression is only regulated by the action of ERα. In contrast, VTG2 expression is regulated by both ERα and ERβ, with ERα being essential for VTG2 gene expression and ERβ being essential for enhancement. ©2009 The Pharmaceutical Society of Japan.
  • Masashi Hirano, Hiroshi Ishibashi, Joon Woo Kim, Naomi Matsumura, Koji Arizono
    Comparative Biochemistry and Physiology - C Toxicology and Pharmacology 149 3 368 - 373 2009年04月 [査読有り]
     研究論文(学術雑誌) 
    We investigated the effects of environmentally relevant concentrations of nonylphenol (NP) on survival, growth, sexual development and molting of the mysid Americamysis bahia (Crustacea: Mysidacea), including assay of 20-hydroxyecdysone (20E) levels. Seven-day-old mysids were exposed to NP concentrations of 0.3, 1, 3, 10 and 30 μg/L for 14 days. Among growth-related traits we assayed, an effect on body length was the most prominent, with the effects observed at concentrations as low as 1 μg/L NP. In addition, the total number of molting exposed to NP for 14 days was significantly decreased in the treatment groups at 10 or 30 μg/L relative to the controls. These results clearly indicate that subchronic exposure to environmentally relevant concentrations of NP affects growth of mysids. Furthermore, we investigated the effect of NP on production of 20E in female mysids during the molting period. Production of 20E in pre-molting mysids exposed to 30 μg/L of NP was lower than those in control groups, and exposure of female mysids to NP disrupted molting cycles. Taken together, the results suggest that environmentally relevant concentrations of NP can disrupt growth of A. bahia via inhibition of 20E levels and an associated delay in molting. © 2008 Elsevier Inc. All rights reserved.
  • Joon Woo Kim, Hyo Sang Jang, Jong Gu Kim, Hiroshi Ishibashi, Masashi Hirano, Kazuaki Nasu, Nobuhiro Ichikawa, Yuji Takao, Ryota Shinohara, Koji Arizono
    Journal of Health Science 55 2 249 - 258 2009年04月 [査読有り]
     研究論文(学術雑誌) 
    The present study demonstrates the occurrence of pharmaceuticals and personal care products (PPCPs) in surface water from the Mankyung River, South Korea. Samples collected at 5 Sites along theMankyung River were assayed using a liquid chromatograph coupled with a tandem mass spectrometer (LC/MS/MS) for 13 PPCPs. Overall, 11 out of the 13 selected PPCPs, which span a range of therapeutic classes and one personal care product, were detected in surface water samples collected from the Mankyung River. The most prevalent contaminants were ibuprofen, with average concentrations ranging from not detected (ND) to 414 ng/l, followed by carbamazepine (ND- 595 ng/l), atenolol (ND-690 ng/l), clarithromycin (ND-443 ng/l), mefenamic acid (ND-326 ng/l), erythromycin (ND-137 ng/l), fluconazole (ND-111 ng/l), levofloxacin (ND-87.4 ng/l), indomethacin (ND-33.5 ng/l), propranolol (ND-40.1 ng/l), ifenprodil (ND-35.4 ng/l), disopyramide (ND) and triclosan (ND). PPCP concentrations were highest in surface water samples collected downstream from a sewage treatment plant (STP), implying possibly insuf- ficient removal efficiency of the PPCPs in the STP. Contamination by PPCPs such as ibuprofen, carbamazepine, atenolol, mefenamic acid and clarithromycin was high in the Mankyung River compared to concentrations in other countries.
  • Joon Woo Kim, Hiroshi Ishibashi, Ryoko Yamauchi, Nobuhiro Ichikawa, Yuji Takao, Masashi Hirano, Minoru Koga, Koji Arizono
    Journal of Toxicological Sciences 34 2 227 - 232 2009年04月 [査読有り]
     研究論文(学術雑誌) 
    Pharmaceutical and personal care products (PPCPs) enter aquatic environments via sewage treatment facilities and their potentially toxic effects on biota, particularly aquatic organisms, are of considerable concern. In this study, we investigated the acute toxicity of selected PPCPs on a freshwater crustacean (Thamnocephalus platyurus) and a fish species (Oryzias latipes). The 24-hr median lethal concentration (LC50) values of ibuprofen, mefenamic acid, indometacin, carbamazepine, propranolol, ifenprodil, clarithromycin and triclosan for T. platyurus were estimated to be 19.59, 3.95, 16.14, > 100, 10.31, 4.43, 94.23 and 0.47 mg/l respectively. Conversely, the 96-hr LC50 values for these PPCPs were estimated at > 100, 8.04, 81.92, 45.87, 11.40, 8.71, > 100 and 0.60 mg/l for O. latipes, respectively. The toxic sensitivity of T. platyurus to these PPCPs, except for carbamazepine, was therefore higher than for O. latipes. No acute toxicity effects were associated with PPCPs, such as atenolol, disopyramide, famotidine, fluconazole, erythromycin and levofloxacin, in the two aquatic organisms at the concentrations tested in this study (> 100 mg/l). These findings may help us to understand the potential biological effects and risks associated with PPCP exposure in aquatic organisms. Further long-term studies are required to fully assess the growth and reproduction of these compounds on aquatic biota.
  • Ryoko Yamauchi, Hiroshi Ishibashi, Masashi Hirano, Taiki Mori, Joon Woo Kim, Koji Arizono
    Aquatic Toxicology 90 4 261 - 268 2008年12月 [査読有り]
     研究論文(学術雑誌) 
    This study demonstrates the effects of synthetic polycyclic musks such as Galaxolide (HHCB), Tonalide (AHTN), Traseolide (ATII), Celestolide (ADBI), Phantolide (AHMI) and Cashmeran (DPMI), both on the early life stage and on gene expression in the livers of male medaka (Oryzias latipes). The toxicity ranking (the 96-h median lethal concentration) of the chemicals tested on 24-h-old medaka larvae descended in the order HHCB (0.95 mg/L) = ATII (0.95 mg/L) > AHTN (1.0 mg/L) > AHMI (1.2 mg/L) > ADBI (2.0 mg/L) ≫ DPMI (12 mg/L), indicating high acute toxicity of these compounds on the early life stages of medaka. Expression analysis of hepatic vitellogenin (VTG) protein showed potential estrogenic effects upon the addition of AHTN and HHCB, indicative of the induction of VTG synthesis in the livers of male medaka. We also investigated mRNA expression levels of two estrogen receptor (ER) subtypes (ERα and β), two VTGs (VTG I and II), pregnane X receptor (PXR), and two cytochromes P450 (CYP) 3As (CYP3A38 and 3A40) in the livers of male medaka treated with AHTN and HHCB at 5, 50 and 500 μg/L. Quantitative real-time PCR analyses revealed that hepatic ERα, VTG I, VTG II, and CYP3A40 mRNA responded to 500 μg/L of AHTN and/or HHCB after 3 days exposure, whereas no effects of these compounds on ERβ, PXR, and CYP3A38 mRNA transcription were observed. These results suggest that certain polycyclic musks, including AHTN and HHCB, induce the expression levels of hepatic ERα and VTG mRNA/protein and modulate expression levels of CYP3A40 mRNA in the livers of male medaka. © 2008 Elsevier B.V. All rights reserved.
  • Akemi Yamaguchi, Shinya Kohra, Hiroshi Ishibashi, Koji Arizono, Nobuaki Tominaga
    Journal of Health Science 54 5 596 - 601 2008年10月 [査読有り]
     研究論文(学術雑誌) 
    Menadione, a synthetic vitamin K3, exhibits anti-estrogenic activity on in vitro assay. However, the in vivo anti-estrogenic effects of menadione have not been determined, while correlations between biological effects and structural changes are unclear. Thus, we investigated the in vivo anti-estrogenic activity of menadione under fluorescent light and dark conditions. Suppression of the hepatic estrogen response genes vitellogenin1 (VTG1), VTG2 and estrogen receptor-α (ER-α) was used as an index of antiestrogenic activity. Male medaka (Oryzias latipes) were treated with nominal concentrations of menadione in the presence or absence of 17β-estradiol (E2), and hepatic VTG1, VTG2 and ER-α mRNA levels were determined by quantitative real-time PCR. In the presence of E2 under dark conditions, expression of hepatic VTG2 and ER-α genes was suppressed by menadione treatment. On the other hand, menadione activity was lost under fluorescent light conditions. These results suggest that menadione has antiestrogenic activity in vivo, and that this activity is diminished under fluorescent light, probably due to a structural change in menadione.
  • Hisato Iwata, Hiroshi Ishibashi, Eun-Young Kim, Lin Tao, Kurunthachalam Kannan, Shinsuke Tanabe, Valeriy B. Batoev, Evgeny A. Petrov
    MARINE ENVIRONMENTAL RESEARCH 66 1 72 - 73 2008年07月
  • Hiroshi Ishibashi, Ryoko Yamauchi, Munekazu Matsuoka, Joon Woo Kim, Masashi Hirano, Akemi Yamaguchi, Nobuaki Tominaga, Koji Arizono
    Chemosphere 71 10 1853 - 1859 2008年05月 [査読有り]
     研究論文(学術雑誌) 
    Here we report on the in vivo estrogenic effects of two fluorotelomer alcohols, such as 1H,1H,2H,2H-perfluorooctan-1-ol (6:2 FTOH) and 1H,1H,2H,2H-perfluorodecan-1-ol (8:2 FTOH), in male medaka (Oryzias latipes). An in vitro yeast two-hybrid assay indicated a significant, dose-dependent interaction between medaka estrogen receptor α (ERα) and coactivator TIF2 upon treatment with 6:2 FTOH, 8:2 FTOH or 2,2,3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-nonadecafluoro-1-decanol (NFDH). The relative ranks of tested chemicals on the estrogenic effects for medaka ERα descended in the order of estradiol-17β (100) ≫ 6:2 FTOH (0.16) > NFDH (0.016) > 8:2 FTOH (0.0044). In contrast, no interaction with the ERα was observed upon treatment with perfluorooctane sulfonate (PFOS), perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), perfluorododecanoic acid (PFDA) or perfluoroundecanoic acid (PFUnDA). Expression analysis of hepatic vitellogenin (VTG) protein showed estrogenic potentials with, 6:2 FTOH and 8:2 FTOH, indicative of the induction of VTG synthesis in the livers of male medaka. We also investigated mRNA expression levels of two ER subtypes (ERα and β) and two VTGs (VTG I and VTG II) in the livers of male medaka following exposure to FTOHs. Quantitative real-time polymerase chain reaction analyses revealed that hepatic ERα, VTG I, and VTG II mRNA responded rapidly to FTOHs such as 6:2 FTOH and 8:2 FTOH after 8-h exposure, whereas no effects of these compounds on ERβ mRNA transcription were observed. These results from both in vitro and in vivo assays strongly suggest that certain FTOHs, such as 6:2 FTOH and 8:2 FTOH, induce hepatic VTG through activation of ERα in male medaka. © 2008 Elsevier Ltd. All rights reserved.
  • Hiroshi Ishibashi, Hisato Iwata, Eun Young Kim, Lin Tao, Kurunthachalam Kannan, Masao Amano, Nobuyuki Miyazaki, Shinsuke Tanabe, Valeriy B. Batoev, Evgeny A. Petrov
    Environmental Science and Technology 42 7 2295 - 2301 2008年04月 [査読有り]
     研究論文(学術雑誌) 
    Concentrations of perfluorochemicals (PFCs) including perfluoroalkylsulfonates (PFSAs) and perfluoroalkylcarboxylates (PFCAs) were determined in liver and serum of Baikal seals (Pusa sibirica) collected from Lake Baikal, Russia in 2005. Among the 10 PFC compounds measured, perfluorononanoic acid (PFNA, 3.3-72 ng/g wet wt) concentrations were the highest in liver, followed by perfluorooctanesulfonate (PFOS, 2.6-38 ng/g). The accumulation profile of long-chain (C7-C12) PFCAs in particular, the predominance of PFNA, indicated that 8:2 fluorotelomer alcohol or commercially manufactured PFNA is a major local source of PFCs in Lake Baikal. No gender-related differences in the concentrations of individual PFCs or total PFCs were found. Tissues from pups and juveniles contained relatively higher concentrations of PFCs than tissues from subadults and adults, suggesting that maternal transfer of PFCs is of critical importance. Comparison of concentrations of PFCs in livers and sera collected from the same individuals of Baikal seals revealed that residue levels of PFOS, PFNA, perfluorodecanoic acid (PFDA), and perfluoroundecanoic acid (PFUnDA) were significantly higher in liver than in serum. The concentration ratios of PFNA and PFDA between liver and serum were calculated to be 14 and 15, respectively, whereas the ratio of PFDS was 2.4. This suggests preferential retention of both PFNA and PFDA in liver. Concentrations of PFDS, PFNA, and PFDA in liver were significantly correlated with those in serum, whereas concentrations of PFUnDA were not correlated in between the two tissues, suggesting differences in pharmacokinetics among these PFCs. Temporal comparisons of hepatic PFC concentrations in seals collected between 1992 and 2005 showed that the concentrations of PFDS (p = 0.0006), PFNA (p = 0.061) and PFDA (p = 0.017) were higher in animals collected in recent years, indicating ongoing sources of PFC contamination in Lake Baikal. © 2008 American Chemical Society.
  • Yuji Takao, Miki Shimazu, Mayumi Fukuda, Hiroshi Ishibashi, Masaki Nagae, Shinya Kohra, Yasuhiro Tabira, Yasuhiro Ishibashi, Koji Arizono
    Journal of Health Science 54 2 240 - 243 2008年04月 [査読有り]
     研究論文(学術雑誌) 
    Seasonal and diurnal fluctuations in pharmaceuticals and personal care products (PPCPs) concentrations in residential sewage water were ascertained in an area with no businesses industry (e.g., plants or offices) upstream. PPCPs with high detection rates included ibuprofen, acetaminophen and indomethacin (antipyretic analgesics), atenolol and disopyramide (antiarrhythmics), clarithromycin (antibiotic), levofloxacin (synthetic antimicrobial agent) and triclosan (disinfectant). In summer, the concentration of triclosan was the highest, while in winter, the concentrations of ibuprofen and acetaminophen were higher than the others. Moreover, three types of diurnal fluctuations were observed: no marked diurnal changes (triclosan), high daytime concentrations (disopyramide) and high nighttime concentrations (acetaminophen).
  • Hiroshi Ishibashi, Hisato Iwata, Eun Young Kim, Lin Tao, Kurunthachalam Kannan, Shinsuke Tanabe, Valeriy B. Batoev, Evgeny A. Petrov
    Environmental Science and Technology 42 7 2302 - 2308 2008年04月 [査読有り]
     研究論文(学術雑誌) 
    To investigate the biological effects of perfluorochemicals (PFCs) and to identify biomarkers of exposure to PFCs, this study focused on the effects mediated by peroxisome proliferator-activated receptor α (PPARα) in Baikal seals (Pusa sibirica). We cloned a full-length cDNA, encoding PPARα from the liver of Baikal seal, which has a deduced open reading frame of 468-amino acid residues with a predicted molecular mass of 52.2 kDa. Comparison of the amino-acid sequence of Baikal seal PPARα with that of other mammalian PPARα showed considerable similarities with PPARα of dog (97%), human (95%), rat (92%), and mouse (91%). The quantitative real-time RT-PCR analyses of tissues from Baikal seals revealed that PPARα mRNAs were primarily expressed in the liver, kidney, heart, and muscle. The hepatic expression levels of PPARα mRNA showed a positive correlation with the expression levels of immunochemically detected cytochrome P450 (CYP) 4A-like protein, indicating that the PPARα-CYP4A signaling pathway in Baikal seal is likely conserved. This study also developed an in vitro PPARα reporter gene assay using African green monkey kidney CV-1 cells transiently transfected with Baikal seal PPARα cDNA expression vector and a reporter vector containing a peroxisome proliferator-responsive element. The in vitro reporter gene assay displayed significant response to clofibrate, which is a known PPARα agonist in humans and rodents. Treatment with perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), perfluorodecanoic acid (PFDA), perfluoroundecanoic acid (PFUnDA), or perfluorooctane sulfonate (PFOS) induced PPARα-mediated transcriptional activity in a dose-dependent manner, showing the lowest-observed-effect concentrations of 62.5, 125, 125, 62.5, and 125 μM, respectively. In the livers of wild Baikal seals, expression levels of PPARα mRNA showed a significant positive correlation with PFNA levels. Moreover, expression of hepatic CYP4A-like protein was significantly correlated with the hepatic concentrations of PFNA and PFDA. These results suggest modulation of the PPARα-CYP4A signaling pathway by PFCs in the wild Baikal seals. Our study demonstrates that the PPARα-mediated response may be a useful biomarker to evaluate potential biological effects of PFCs in wildlife. © 2008 American Chemical Society.
  • Toxicity to early life stages on medaka (Oryzias latipes) and in vitro estrogen intensity of bisphenol compounds
    Keiko Yokota, Chihiro Kato, Masashi Hirano, Hiroshi Ishibashi, Hideki Shiratsuchi, Katsuyasu Tachibana, Koji Arizono
    Japan Journal of Environmental Toxicology 11 2 133 - 142 2008年 [査読有り]
  • Hiroshi Ishibashi, Haruna Ishida, Munekazu Matsuoka, Nobuaki Tominaga, Koji Arizono
    Biological and Pharmaceutical Bulletin 30 7 1358 - 1359 2007年07月 [査読有り]
     研究論文(学術雑誌) 
    The present study demonstrates the estrogenic effects of fluorotelomer alcohols (FTOHs). In a yeast two-hybrid assay, treatment with 1H,1H,2H,2H-perfluorooctan-1-ol (6:2 FTOH), 1H,1H,2H,2H-perfluoro-decan-1-ol (8:2 FTOH) and 2,2,3,3,4,-4,5,5,6,6,7,7,8,8,9,9,10,10,10-nonadecafluoro-1- decanol (NFDH) showed a dose-dependent interaction between the human estrogen receptor (hER) isoforms hERα or hERβ ligand-binding domain and coactivator TIF2, whereas there were no estrogenic effects of perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) for these hERs. The estrogenic effects of FTOHs on hERα were higher than those on hERβ, indicating a differential responsiveness of hERs to FTOHs. The relative ranks of tested chemicals on the estrogenic effects for hERα and hERβ descended in the order of estradiol-17β⋙6:2 FTOH> NFDH>8:2 FTOH. These results suggest that certain FTOHs including 6:2 FTOH, 8:2 FTOH and NFDH interact with hER isoforms α and β in vitro. Further studies are necessary to investigate contamination levels, potential biological effects and the risks of these compounds on human health. © 2007 Pharmaceutical Society of Japan.
  • Manabu Sumi, Yukio Kawashima, Tatsunori Fukumaki, Hiroshi Ishibashi, Koji Arizono, Taisen Iguchi, Makoto Shimizu
    Environmental sciences : an international journal of environmental physiology and toxicology 14 1 41 - 54 2007年01月 [査読有り]
     研究論文(学術雑誌) 
    To investigate endocrine disruption and chemical contaminant levels in the aquatic environment, serum vitellogenin (Vtg) induction, steroid hormone synthesis, gonad histopathology and nonylphenol (NP) bioaccumulation were measured in common carp (Cyprinus carpio) in three sites (two rivers and one lake, in which high, medium, and low NP concentrations were detected in a previous study) in Japan from November 2001 to February 2002. The average gonadosomatic indexes (GSIs) for males were 3.9% in the Ishizu River, 5.6% in the Imba discharge channel, and 4.2% in Teganuma. No testis-ova in the gonads were found in any of the captured carp in this study. Abnormalities in testis were observed in individuals (n = 2/15) captured in the Imba discharge channel, and retraction and unusual cell proliferation were observed in the testis of the same fish. Serum Vtg was detected in 40% of the male samples captured in the Ishizu River and in 53% of the male samples captured in the Imba discharge channel. The levels of alkylphenols and bisphenol A, as well as estrogenic activity, were determined in the aquatic environment. In additional caging experiments in the Nikko River (medium NP concentration), male carp showed NP bioconcentration after the exposure to the river water.
  • Taiki Mori, Mitsuru Iida, Hiroshi Ishibashi, Shinya Kohra, Yuji Takao, Takehiro Takemasa, Koji Arizono
    Environmental sciences : an international journal of environmental physiology and toxicology 14 4 195 - 202 2007年 [査読有り]
     研究論文(学術雑誌) 
    Synthetic musk fragrance compounds, such as polycyclic musks (PCMs), are a group of chemicals used extensively as personal care products, and can be found in the environment and the human body. PCMs, such as 1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexa-methylcyclopenta-gamma-2-benzopyran (HHCB) and 7-acetyl-1,1,3,4,4,6-hexamethyltetralin (AHTN), are known to have agonistic activities toward human estrogen receptor alpha (hERalpha) and hERbeta, and have antagonistic activity toward the human androgen receptor (hAR), as shown in several reporter gene assays. However, little is known about the interaction of PCMs with the human thyroid hormone receptor (hTR), and the hormonal effects of other PCMs except for HHCB and AHTN. In this study, we focus on the interactions of six PCMs, namely, HHCB, AHTN, 4-acetyl-1,1-dimethyl-6-tert-butyl-indan (ADBI), 6-acetyl-1,1,2,3,3,5-hexamethylindan (AHMI), 6,7-dihydro-1,1,2,3,3-pentamethyl-4(5H)-indanone (DPMI), and 5-acetyl-1,1,2,6-tetramethyl-3-isopropy-lindan (ATII) with hERalpha, hAR, and hTRbeta by in vitro reporter gene assay using Chinese hamster ovary cells. All the samples were found to be agonists toward hERalpha, whereas no agonistic activities of these PCMs for hAR and hTRbeta were observed. No antagonistic activities for hERalpha and hTRbeta were observed at the concentrations tested. However, several PCMs, namely, HHCB, AHTN, ATII, ADBI, and AHMI, showed dose-dependent antagonistic activities for hAR, and the IC50 values of these compounds were estimated to be 1.0 x 10(-7), 1.5 x 10(-7), 1.4 x 10(-7), 9.8 x 10(-6), and 1.4 x 10(-7) M, respectively. The results suggest that these PCMs interact with hERalpha and hAR but have no hormonal effect on hTRbeta. This is the first report on the agonistic and antagonistic activities of ATII, ADBI, AHMI, and DPMI for hERalpha and hAR as determined by in vitro reporter gene assay using stably transfected Chinese hamster ovary cells.
  • Hiroshi Ishibashi, Masashi Hirano, Naomi Matsumura, Naoko Watanabe, Yuji Takao, Koji Arizono
    Chemosphere 65 6 1019 - 1026 2006年11月 [査読有り]
     研究論文(学術雑誌) 
    The objective of this study was to investigate the reproductive success (fertility and fecundity) of medaka (Oryzias latipes) exposed to 4-nonylphenol (4-NP) in water and the viability of their F1 offspring. In addition, we measured the bioconcentration of 4-NP in eggs. After a 21-d exposure to 100 μg/l 4-NP, medaka showed reduced egg production and fertility. Hepatic vitellogenin levels were increased significantly in males treated with 10, 50 and 100 μg/l of 4-NP. In the F1 generations, the hatchability and time to hatching of embryos in the 100 μg/l treatment group were adversely affected, and 2-7 μg 4-NP/g egg was found in spawned eggs (the bioconcentration factor: 30-100). These results indicate that (1) 4-NP adversely affected the reproduction (fecundity and fertility) of adult medaka, (2) 4-NP accumulated in eggs through maternal transfer and (3) these levels of 4-NP were associated with adverse effects in the F1 offspring. © 2006 Elsevier Ltd. All rights reserved.
  • Makito Kobayashi, Hiroshi Ishibashi, Toshihisa Moriwaki, Tomohiko Koshiishi, Satoshi Ogawa, Takeru Matsumoto, Koji Arizono, Shugo Watabe
    Environmental sciences : an international journal of environmental physiology and toxicology 13 2 125 - 136 2006年 [査読有り]
     研究論文(学術雑誌) 
    A low-estrogenic diet for goldfish Carassius auatus was produced for an in vivo estrogen activity test, because commercial fish feed has estrogenic activity and may affect the results of estrogen assays. The newly produced diet (FD5) was formulated with defatted rice bran and casein, and did not contain any soybean meal or fish meal. Phytoestrogen contents (genistein, daidzein, equol, and coumestrol) of FD5 were measured by liquid chromatography-mass spectroscopy/mass spectroscopy (LC-MS/MS) and compared with those of the commercial trout diet (TD) and carp diet (CD). The genistein, daidzein, and coumestrol contents of TD and CD were much higher (5-2000 times) than those of FD5, but equol was detected only in FD5. Estrogenic activity of the fish diets was estimated in vitro by the yeast estrogen-screen assay (YES assay). The estrogenic activity was detected in TD and CD, but not in FD5. The in vivo estrogenic activity of the diets was examined by determining the production of vitellogenin in male goldfish. When male goldfish were fed TD or CD, plasma vitellogenin levels increased, but fish that were fed FD5 maintained low vitellogenin levels. These results indicate that FD5 produced in the present study has a low estrogenic activity, and FD5 would be suitable for the in vivo estrogen activity test using goldfish.
  • Y. Nomura, W. Teshima, T. Kawahara, N. Tanaka, H. Ishibashi, M. Okazaki, K. Arizono
    Journal of Materials Science: Materials in Medicine 17 1 29 - 32 2006年01月 [査読有り]
     研究論文(学術雑誌) 
    The polymerization initiators for resins cured using visible light usually consist of a photosensitizer, primarily camphorquinone (CQ), and a reducing agent, which is often a tertiary amine (DMPT, DMAEMA), while the initiator used for self-curing resins consists of benzoyl peroxide (BPO) and a tertiary amine (DMPT). The genotoxicities of camphorquinone (CQ), benzoyl peroxide (BPO), dimethyl-para-toluidine (DMPT), 2-dimethylamino-ethyl-methacrylate (DMAEMA), and 1-allyl-2-thiourea (ATU) were examined using the bioluminescent bacterial genotoxicity test. 4-Nitroquinoline-N-oxide (4NQO) was prepared for comparison with these chemicals. Acetone solutions of the five polymerization initiators and 4NQO were prepared. Benzoyl peroxide (BPO), dimethyl-para-toluidine (DMPT), and 1-allyl-2-thiourea (ATU) showed significant genotoxic activity at 24 h in the bioluminescent bacterial genotoxicity test, at concentrations of approximately 5 μM, 4 mM, and 1 mM, respectively. 2-Dimethyloamino-ethyl- methacrylate (DMAEMA) did not have genotoxic activity and CQ had questionable genotoxic activity. In comparison, 4NQO had strong genotoxicity, at 4 μM, roughly the same as that of BPO. Therefore, BPO should be used carefully in clinical dentistry. © 2006 Springer Science + Business Media, Inc.
  • Kei Nomiyama, Teiji Tanizaki, Hiroshi Ishibashi, Koji Arizono, Ryota Shinohara
    Environmental Science and Technology 39 22 8762 - 8769 2005年11月 [査読有り]
     研究論文(学術雑誌) 
    The oxidative photodegradation behaviors of selected polychlorinated biphenyls (PCBs) [2,3,3′,4′-tetraCB (BZ number: CB56), 2,3′,4′,5-tetraCB (CB70), and 3,4,4′,5-tetraCB (CB81: coplanar PCB)] using titanium dioxide (TiO2) in water were investigated. The main purposes were to clarify the structural relation between the original PCBs and the intermediates derived by TiO2 oxidation and to evaluate the estrogenic activity in the treated PCBs during the oxidative reactions. Approximately 90% of the three tetraCBs decomposed within 120 min. Intermediates by decomposition of three tetraCBs, such as some OH-tetraCBs and OH-triCBs, carboxylic intermediates, phenolic intermediates, and other intermediates produced by the cleavage of a benzene ring were identified and quantified. In the degradation pathways, the produced amounts of OH-tetraCB and OH-triCB increased within 60 min of irradiation time. Estrogenic activities of the intermediates from the three tetraCBs in water were assessed by using a yeast two-hybrid assay system for human estrogen receptor α (hERa). The maximal estrogenic activities were induced by the solutions of decomposed CB81 with irradiation time at 60 min. We found that the solutions at an irradiation time of 60-120 min contained several 4-OH-tetraCBs and 4-OH-triCBs substituted with OH and CI at para- and para′-positions. It is presumed that the chemical structures of the 4-OH-PCBs are similar to that of 17β-estradiol (β-E2); these intermediates present strong estrogenic activities. Moreover, we learned that there is a high possibility of conversion from some low toxic PCBs congeners to strong estrogenic OH-PCBs. © 2005 American Chemical Society.
  • Hiroshi Ishibashi, Naoko Watanabe, Naomi Matsumura, Masashi Hirano, Yukiko Nagao, Hideki Shiratsuchi, Shinya Kohra, Shin Ichi Yoshihara, Koji Arizono
    Life Sciences 77 21 2643 - 2655 2005年10月 [査読有り]
     研究論文(学術雑誌) 
    In a recent study, it was reported that 4-methyl-2,4-bis(4-hydroxyphenyl) pent-1-ene (MBP), a metabolite of bisphenol A (BPA; 2,2-bis(4-hydroxyphenyl) propane), showed estrogenic activity in several in vitro assays, and the estrogenic activity of MBP was higher than that of BPA. In this study, we have investigated the early life stage toxicity and estrogenic effect of MBP on medaka (Oryzias latipes). The 96-h median lethal concentration value of MBP and BPA with 24-h-old larvae was estimated to be 1640 and 13,900 μg/l, respectively. The hatchability of fertilized eggs exposed to MBP and BPA over 14 days was significantly decreased at doses of 2500 μg/l and 12,500 μg/l, respectively. Moreover, to compare the potency of estrogenic activity in vivo, male medaka were exposed to various concentrations of MBP and BPA for 21 days. The lowest-observed-effect concentrations of MBP and BPA for hepatic vitellogenin induction in male medaka were estimated to be 4.1 and 1000 μg/l, respectively. These results suggest that MBP has high toxicity for early life stages of the medaka, and that the estrogenic activity of MBP was about 250-fold higher than that of BPA to male medaka. © 2005 Elsevier Inc. All rights reserved.
  • Naomi Matsumura, Hiroshi Ishibashi, Masashi Hirano, Yukiko Nagao, Naoko Watanabe, Hideki Shiratsuchi, Toshinori Kai, Tetsuji Nishimura, Akihiko Kashiwagi, Koji Arizono
    Biological and Pharmaceutical Bulletin 28 9 1748 - 1751 2005年09月 [査読有り]
     研究論文(学術雑誌) 
    We investigated the effects of nonylphenol (NP) and triclosan (TCS) on production of vitellogenin (Vg), testosterone (T), and hepatic cytochrome P450 1A and 2B activities in male South African clawed frogs (Xenopus laevis). In a 14-d waterborne exposure test, no significant differences in the level of plasma Vg synthesis in male frogs were observed among the control, 10, 50, and 100 μg/l NP and 20, 100, and 200 μg/l TCS treatment groups. Intraperitoneal injection of male frogs with 2, 20, and 200 μg/g body weight NP resulted in no significant differences in plasma Vg levels among the control and all treatment groups. However, the levels of plasma Vg in all TCS treatment groups (intraperitoneal injection of 4, 40, and 400 μg/g body weight) were lower than that in the solvent control group, and male frogs injected with high doses of NP or TCS had lower T levels than the control group. No significant differences in hepatic cytochrome P450 1A and 2B activities were observed among the all treatment groups. Male frogs injected with 20 μg/g body weight of estradiol-17β had significantly higher plasma Vg levels than the control group. These results suggest that profiles of plasma Vg and T production in male Xenopus laevis could be useful biomarkers for detecting hormonally active agents. © 2005 Pharmaceutical Society of Japan.
  • Akemi Yamaguchi, Hiroshi Ishibashi, Shinya Kohra, Koji Arizono, Nobuaki Tominaga
    Aquatic Toxicology 72 3 239 - 249 2005年04月 [査読有り]
     研究論文(学術雑誌) 
    To evaluate the estrogenic activities of selected estrogenic compounds such as estradiol-17β (E2), nonylphenol (NP), 4-(1-adamantyl)phenol (AdP), bisphenol A (BPA), BPA metabolite 4-methyl-2,4-bis(4-hydroxyphenyl)pent-1-ene (MBP) and 4,4′-dihydroxy-α-methylstilbene (DHMS) in the shortest possible time, we investigated the expression of estrogen-responsive genes such as vitellogenin I, vitellogenin II and α-type estrogen receptor genes in the liver of male medaka (Oryzias latipes) using reverse transcription- polymerase chain reaction (RT-PCR) techniques. These estrogen-responsive genes responded rapidly to selected estrogenic compounds after 8 h exposure, and the expression of hepatic vitellogenin II and estrogen receptor α mRNA was found to be more responsive than that of vitellogenin I mRNA. As a result, the relative estrogenic potencies of tested chemicals descended in the order of E2 (100) > MBP (0.38) > AdP (0.25) > DHMS (0.05) > NP (0.02) > BPA (0.001). Moreover, this preliminary study indicates that AdP and DHMS should be considered as candidate estrogenic compounds with the potential to induce hepatic estrogen-responsive genes in male medaka. These results suggest that vitellogenin I, vitellogenin II and estrogen receptor α gene expression patterns alter in male medaka treated with selected estrogenic compounds, and that these genes may be useful molecular biomarkers for screening estrogenic endocrine-disrupting chemicals in the shortest possible time. © 2005 Elsevier B.V. All rights reserved.
  • Toshikazu Ayaki, Yoshiharu Kawauchino, Chihiro Nishimura, Hiroshi Ishibashi, Koji Arizono
    Integrative and Comparative Biology 45 1 39 - 42 2005年02月 [査読有り]
     研究論文(国際会議プロシーディングス) 
    To clarify the effect of environmental endocrine disruptors on the induction of secondary sexual characteristics in wild animals, we observed 1,527 male and 1,847 female specimens of sexually mature Japanese freshwater crabs, Geothelphusa dehaani, collected at 12 sample points from 10 rivers in Sasebo City, Nagasaki Prefecture, Japan. The results obtained were as follows: 1. Abnormal males, with external appearance of a complete male, but having female genital openings, were observed at frequencies of 8-32% of males collected at all sample points. In contrast to this, all females collected were normal. 2. The frequency of abnormal males increased significantly with the increase in individual carapace width. This species inhabits mountain streams in Japan, which are free from tributyltin that is known to induce the sexual mosaic phenomenon in mollusks. The streams are most likely polluted by some agricultural chemicals and chemicals present in waste gases from cars and especially particulate materials from diesel engines. The clear relationship of the frequencies of abnormal males to size (or age) suggests that the sexual mosaic phenomenon is induced not only at early stages of male development but after sexual maturation, in response to long-term exposure to chemical(s) during the whole life cycle of males. The definitive causal factors and mechanisms inducing sexual mosaics in this species are still unclear.
  • Acute and subchronic toxicity of a bisphenol A metabolite, 4-metyl-2,4-bis(4-hydroxyphenyl)pent-1-ene on the invertebrates
    Masashi Hirano, Munekazu Matsuoka, Naomi Matsumura, Takashi Nakamoto, Shinya Kohra, Shin-ichi Yoshihara, Nobuaki Tominaga, Hiroshi Ishibashi, Koji Arizono
    Japan Journal of Environmental Toxicology 8 2 65 - 76 2005年 [査読有り]
  • Munekazu Matsuoka, Hiroshi Ishibashi, Marie Ushijima, Makiko Inudo, Eiko Honda, Masayoshi Iwahara, Hyeon Seo Cho, Yasuhiro Ishibashi, Koji Arizono
    Journal of Health Science 51 2 212 - 219 2005年 [査読有り]
     研究論文(学術雑誌) 
    Phytoestrogens and organochlorine pesticides in the diet of laboratory animals are a possible source of interference in bioassays that assess estrogenic activity. In the present study, we investigated the levels of dietary phytoestrogens, organochlorine pesticides and the estrogenic activity of various diets for an experimental fish and discuss the potential contribution of these substances to estrogenic activity, in comparison with those used in previous studies. After hydrolysis with β-glucuronidase, genistein and daidzein were detected in all of the diets, and there were no significant differences in the contents of these substances among present and previous investigations. In addition, organochlorine pesticides, such as hexachlorobenzene (HCB), β-benzene hexachloride (β-BHC), γ-BHC, trans-nonachlor, and/or endrin, were detected in most fish diets. All of these diets exhibited higher levels of activation of β estrogen receptors than with α estrogen receptors in an in vitro yeast-based bioassay. These results indicate that phytoestrogens, such as genistein and daidzein, were the main substances contributing to the estrogenic activity of the diet. Moreover, some diets may exert estrogenic activity in in vivo tests, indicating the necessity for more careful selection of the feeding diet and measurement of estrogenic substances when performing routine screening assays for endocrine-disrupting chemicals.
  • Makiko Inudo, Hiroshi Ishibashi, Naomi Matsumura, Munekazu Matsuoka, Taiki Mori, Shigeto Taniyama, Kiwao Kadokami, Minoru Koga, Ryota Shinohara, T. H. Hutchinson, Taisen Iguchi, Koji Arizono
    Comparative Medicine 54 6 673 - 680 2004年12月 [査読有り]
     研究論文(学術雑誌) 
    Endocrine-disrupting chemicals (EDCs) are giving rise to serious concerns for humans and wildlife. Phytoestrogens, such as daidzein and genistein in plants, and organochlorine pesticides are suspected EDCs, because their chemical structure is similar to that of natural or synthetic estrogens and they have estrogenic activity in vitro and in vivo. We assessed estrogenic activity and dietary phytoestrogen and organochlorine pesticide contents of various fish diets made in the United Kingdom, and compared them with those features of diets made in Japan that were tested in a previous study. Genistein and daidzein were detected in all of the diets. Using an in vitro bioassay, many of these diets had higher activation of estrogen β-receptors than estrogen α-receptors. Organochlorine pesticides such as hexachlorobenzene, β-benzene hexachloride (BHC), and γ-BHC were detected in all fish diets. On the basis of these data, we investigated the effect of differing dietary phytoestrogen content in Japanese fish diets on hepatic vitellogenin production and reproduction (fecundity and fertility) in medaka (Oryzias latipes). Assessment of the effects of a 28-day feeding period on reproduction of paired medaka did not indicate significant differences in the number of eggs produced and fertility among all feeding groups. However, hepatic vitellogenin values were significantly higher for male medaka fed diet C (genistein, 58.5 ± 0.6 μg/g; daidzein, 37.3 ± 0.2 μg/g) for 28 days compared with those fed diet A (genistein, < 0.8 μg/g; daidzein, < 0.8 μg/g) or diet B (genistein, 1.4 ± 0.1 μg/g; daidzein, 2.0 ± 0.1 μg/g). Our findings indicate that fish diets containing high amounts of phytoestrogens, such as diet C, have the potential to induce hepatic vitellogenin production in male medaka, even if reproductive parameters are unaffected. Therefore, some diets, by affecting vitellogenin production in males, may alter estrogenic activity of in vivo tests designed to determine activity of test compounds added to the diet. Copyright 2004 by the American Association for Laboratory Animal Science.
  • Yuji Takao, Miki Shimazu, Shinya Kohra, Masaki Nagae, Yasuhiro Ishibashi, Nobuaki Tominaga, Hiroshi Ishibashi, Shinich Yoshihara, Koji Arizono
    Journal of Health Science 50 6 576 - 580 2004年12月 [査読有り]
     研究論文(学術雑誌) 
    Exposure experiments in medaka and photodecomposition tests were performed using a metabolite of bisphenol A [4-methyl-2,4-bis(p-hydroxyphenyl)-pent-1-ene; MBP], the solubility limit of which is 42 mg/l of water. Three adult medaka were kept in a 21 glass beaker at 25 ± 1°C for 4 days. The LC 50 for 96 hr was > 1000 ppb. The measured average MBP concentration in the breeding water (nominal concentration of 100 ppb) was 49.2 ppb. The average concentration in the whole bodies of medaka after 4 days was 1.92 mg/g-wet body, and the bioconcentration factor (BCF) of MBP was calculated to be 39.0. MBP in water and acetone was decomposed very easily, with about 98% of the MBP being decomposed after several hours under sunlight. MBP was also decomposed after 48 hr of illumination under a white fluorescent lamp.
  • Hiroshi Ishibashi, Makito Kobayashi, Yuki Tomiyasu, Maki Miyahara, Katsuyasu Tachibana, Mutsuyosi Tsuchimoto, Koji Arizono
    Journal of Health Science 50 2 169 - 173 2004年04月 [査読有り]
     研究論文(学術雑誌) 
    Plasma vitellogenin (VTG) assay was developed using ovariectomized goldfish (Carassius auratus) for determining the estrogenic effects of endocrine-disrupting chemicals. In a laboratory study, we assessed the estrogenic activity of commercial fish diets by using a diet for ornamental carp (CD) and a casein-based formulated fish diet (FD), which was shown to not contain soybean or fish meal in a previous study. In ovariectomized fish, plasma VTG concentrations were significantly higher in the CD-fed group than in the FD-fed group. These results indicate that the estrogen activity of CD may be high enough to cause induction of plasma VTG in ovariectomized goldfish as previously observed in male goldfish. Moreover, the effect of estrogen on plasma VTG induction was confirmed by significant plasma VTG production following the exposure of FD-fed ovariectomized goldfish to a nominal estradiol-17β concentration of 100 μg/l for 31 days. Our data suggest that induction of plasma VTG using ovariectomized goldfish is a good tool for evaluating the estrogenic effects of endocrine-disrupting chemicals.
  • Hiroshi Ishibashi, Naomi Matsumura, Masashi Hirano, Munekazu Matsuoka, Hideki Shiratsuchi, Yasuhiro Ishibashi, Yuji Takao, Koji Arizono
    Aquatic Toxicology 67 2 167 - 179 2004年04月 [査読有り]
     研究論文(学術雑誌) 
    Triclosan (2,4,4′-trichloro-2′-hydroxydiphenyl ether) is widely used as antibacterial agent in various industrial products, such as textile goods, soap, shampoo, liquid toothpaste and cosmetics, and often detected in wastewater effluent. In this study, the effects of TCS on the early life stages and reproduction of medaka (Oryzias latipes) were investigated. The 96-h median lethal concentration value of TCS for 24-h-old larvae was 602μg/l. The hatchability and time to hatching in fertilized eggs exposed to 313μg/l TCS for 14 days were significantly decreased and delayed, respectively. An assessment of the effects of a TCS 21-day exposure period on the reproduction of paired medaka showed no significant differences in the number of eggs produced and fertility among the control and 20, 100 and 200μg/l TCS treatment groups. However, concentrations of hepatic vitellogenin were increased significantly in males treated with TCS at 20 and 100μg/l. In the F1 generations, although the hatching of embryos in the 20μg/l treatment showed adverse effects, there was no dose-response relationship between hatchability and TCS treatment levels. These results suggest that TCS has high toxicity on the early life stages of medaka, and that the metabolite of TCS may be a weak estrogenic compound with the potential to induce vitellogenin in male medaka but with no adverse effect on reproductive success and offspring. © 2003 Elsevier B.V. All rights reserved.
  • Hiroshi Ishibashi, Katsuyasu Tachibana, Mutsuyosi Tsuchimoto, Kiyoshi Soyano, Norihisa Tatarazako, Naomi Matsumura, Yuki Tomiyasu, Nobuaki Tominaga, Koji Arizono
    Comparative Medicine 54 1 54 - 62 2004年02月 [査読有り]
     研究論文(学術雑誌) 
    The effects of nonylphenol (NP) on plasma vitellogenin (VTG) and steroid hormone values, as well as hepatic cytochrome P450 1A (CYP1A) and glutathione-S-transferase (GST) activities, were measured in goldfish (Carassius auratus) fed a diet with a low (formulated diet, FD) or high (commercial diet, CD) content of phytoestrogens, including genistein and daidzein. Male goldfish with secondary sexual characteristics were exposed to nominal NP concentrations of 0.1, 1.0, 10, and 100 μg/L in the water for 28 days while being fed either the FD or CD diet at 1.0% of body weight daily. Plasma VTG concentration in male goldfish exposed to 100 μg of NP/L and fed FD was significantly higher than that in the FD-fed control fish at seven, 21, and 28 days. However, fish of the CD-fed group exposed to 100 μg of NP/L had significantly higher plasma VTG concentration than did fish of the CD-fed control group at 28 days only. Moreover, plasma VTG concentration in fish of the CD-fed control group was about 100-fold higher than that in fish of the FD-fed control group. Although the estrogenic effects of a phytoestrogen-enriched diet caused a decrease in testosterone and/or 11-ketotestosterone values in the CD-fed fish, there was no dose-response relationship between androgen and amount of NP to which the FD-fed fish were exposed. Nonylphenol does not have appreciable effects on hepatic CYP1A and GST activities in male goldfish at concentrations as low as 100 μg/L. These results suggest that NP has estrogenic activity in male goldfish at the nominal concentration of 100 μg/L, and that phytoestrogens, such as genistein and daidzein, in the CD inhibit an aspect(s) of steroid release and/or synthesis common to testosterone and 11-ketotestosterone. However, results of in vivo screening assays for endocrine-disrupting chemicals may be seriously affected by phytoestrogens in the diet, depending on content or potency of estrogenic activity; therefore, we recommend use in research of a standardized, open-formula diet in which estrogenic substances have been reduced to amounts that do not alter the results of studies that are influenced by exogenous estrogens.
  • Masashi Hirano, Hiroshi Ishibashi, Naomi Matsumura, Yukiko Nagao, Naoko Watanabe, Akiko Watanabe, Norio Onikura, Katsuyuki Kishi, Koji Arizono
    Journal of Health Science 50 1 97 - 100 2004年02月 [査読有り]
     研究論文(学術雑誌) 
    The acute toxicity of endocrine disrupters in two crustaceans, Americamysis bahia (A. bahia) and Daphnia magna (D. magna), was investigated and the toxicological responses compared. Bisphenol A had the lowest toxicity to D. magna, the 48 hr median lethal concentrations (LC50) values was 12.8 mg/l. However, the toxic sensitivity of A. bahia to bisphenol A was approximately 10-fold higher than for D. magna (48 hr LC50; 1.34 mg/l). The 48 hr LC50 of estradiol-17β was 2.97 and 1.69 mg/l in D. magna and A. bahia, respectively. Nonylphenol had the highest lethal toxicity against both A. bahia (48 hr LC50; 0.051 mg/l) and D. magna (48 hr LC50; 0.18 mg/l). However, ecdysteroids, and ecdysteroidal activity insecticides, such as tebufenozide, and juvenile hormone analog, had no toxic effects against either A. bahia or D. magna at the concentrations tested in this study. These results suggest that A. bahia is a more suitable toxicity test organism for endocrine disrupters than D. magna because of the higher sensitivity of A. bahia to the toxicological effect.
  • Masashi Hirano, Hiroshi Ishibashi, Naoko Watanabe, Naomi Matsumura, Akiko Watanabe, Norio Onikura, Katsuyuki Kishi, Hideki Shiratsuchi, Koji Arizono
    Environmental sciences : an international journal of environmental physiology and toxicology 11 5 259 - 268 2004年 [査読有り]
     研究論文(学術雑誌) 
    A 14-day partial life-cycle test was performed to assess the effects of 17beta-estradiol (E2) on the survival, growth, sexual development and molting cycles of a marine crustacean mysid shrimp (Americamysis bahia). Seven-day-old mysids were exposed to the nominal E2 concentrations of 31.3, 62.5, 125, 250 and 500 microg/l for 14 days. The total length and the body weight of mysids significantly decreased relative to the controls when exposed to 62.5, 250 and 500 microg/l E2 for 14 days. Moreover, the carapace length significantly decreased in the 500 microg/l E2 treatment groups. No significant differences were observed in sex ratio with the appearance of secondary sex characteristics in the all treatment groups including the control and solvent control groups. However, the percentage of females with eggs in the oviduct or brood sac decreased significantly in mysids treated with E2 at 62.5, 125, 250 and 500 microg/l. The cumulative total number of molting cycles when exposed to E2 for 14 days significantly decreased in the treatment groups at 500 microg/l relative to the controls. These results suggest that concentrations of E2 over 62.5 microg/l may cause growth suppression in mysid shrimp, and that the disruption of molting cycles may result in alterations in growth due to a sublethal response to toxicant exposure.
  • Norihisa Tatarazako, Hiroshi Ishibashi, Kenji Teshima, Katsuyuki Kishi, Koji Arizono
    Environmental sciences : an international journal of environmental physiology and toxicology 11 2 133 - 140 2004年 [査読有り]
     研究論文(学術雑誌) 
    Triclosan (2,4,4'-trichloro-2'-hydroxydiphenyl ether) is widely used as an antibacterial agent in various industrial products, such as textile goods, soap, shampoo, liquid toothpaste and cosmetics, and often detected in wastewater effluent. However, there is a paucity of data on the toxicity of triclosan and its effects on aquatic organisms. In this study, the acute toxicity of triclosan to the Microtox bacterium (Vibrio fischeri), a microalga (Selenastrum capricornutum), a crustacean (Ceriodaphnia dubia) and fish (Danio rerio and Oryzias latipes) was examined. As a result, the MicrotoxR bacterium, crustacean and fish had similar sensitivities towards triclosan toxicity (i.e., IC25 from 0.07 to 0.29 mg/L triclosan). In contrast, the microalga was about 30-80-fold (IC25 = 0.0034 mg/L triclosan) more sensitive to triclosan toxicity than the bacterium and fish. Therefore, triclosan is quite highly toxic to aquatic animals, and is particularly highly toxic to the green alga used as a test organism in this study. This result indicates that triclosan exerts a marked influence on algae, which are important organisms being the first-step producers in the ecosystem; therefore, the possible destruction of the balance of the ecosystem is expected if triclosan is discharged into the environment at high levels.
  • H. Shimada, N. Tominaga, S. Kohra, H. Ishibashi, Y. Mitsui, K. Ura, K. Arizono
    Trace Elements and Electrolytes 20 4 240 - 243 2003年12月 [査読有り]
     研究論文(学術雑誌) 
    Previously, we reported that the induction of vitellogenin mRNA in the larvae of Caenorhabditis elegans (C. elegans) can be used as a biomarker for short-term screening of environmental endocrine disrupters. Therefore, the present study was designed to determine if the induction of metallothionein (MT) mRNA in the larvae of C. elegans would be a biomarker for short-term screening of heavy metals. The larvae were exposed to various concentrations of cadmium (Cd), mercury (Hg), zinc chloride (Zn), cupper chloride (Cu) or lead acetate (Pb) for 3 h. Cd (1, 10 and 100 μM) and Hg (0.01, 0.1, 1 and 10 μM) exposures resulted in the marked induction of MT-I and MT-II mRNAs in the larvae of C. elegans as measured by reverse transcription polymerase chain reaction. The Cd-and Hg induction of MT-II mRNA was higher than that of MT-I mRNA, and concentration-dependent increase was observed in MT-II but not in MT-I. Time course analyses for MT-I and MT-II mRNA expressions with Cd and Hg were also determined. Cd induction of MT-I and MT-II mRNAs reached a peak at 2 h after the exposure (10 μM), and the levels of MT-II were higher than that of MT-I. For Hg, an initial peak of induction of MT-II mRNA occurred 15 min after the exposure (0.1 μM), and the levels reached maximum by 2 h. The initial peak of induction of MT-I mRNA occurred much later (∼ 2 h after Hg exposure) than MT-II mRNA. These results indicated that the induction of MT-II mRNA in the larvae of C. elegans can be used as a potential biomarker for short-term exposure to Cd and Hg.
  • Maki Miyahara, Hiroshi Ishibashi, Makiko Inudo, Haruka Nishijima, Taisen Iguchi, Louis Joseph Guillette, Koji Arizono
    Journal of Health Science 49 6 481 - 491 2003年12月 [査読有り]
     研究論文(学術雑誌) 
    Phytoestrogens, such as daidzein and genistein in plants are suspected as endocrine-disrupting chemicals (EDCs), because their chemical structures are similar to natural or synthetic estrogens, and have estrogenic activity in vitro and in vivo. An EDC study was carried out on the diets of various animals in vivo. However, many of these diets include phytoestrogens and may already possess estrogenic activity. In this study, we evaluated the estrogenic activity of phytoestrogens (such as daidzin, genistin, daidzein, genistein, coumestrol and equol) and the feed diets for experimental animals, such as fish, amphibians and reptiles, towards human estrogen receptors α (hER-α) and β (hER-β), and the genistein and daidzein content in these diets from HPLC analysis. Coumestrol showed the highest estrogenic activity for hER-α and -β in the -S9 test. Equol showed the highest estrogenic activity for hER-α in the +S9 test. The estrogenic activities of coumestrol, equol and genistein were approximately one hundred to two thousand times higher than that of daidzein. Many of these compounds showed higher compatibility with hER-β than with hER-α. A diet for fish from soybean was indicated to contain the highest amounts of genistein and daidzein. Moreover, this fish diet had the highest estrogenic activity for hER-α and -β. The estrogenic activity was found with hydrolysis by β-glucuronidase, showing higher compatibility with hER-β than with hER-α. In addition, correlation between the contents of genistein and estrogenic activities in the diets was found, with the exception of part of the diet. Therefore, this indicates that the genistein content contributes to the estrogenic activity of the diets. These results suggest that in vivo estrogenic activity might be caused by the diet provided to an experimental animal, indicating the necessity for more careful selection of the feeding diet and measurement of estrogenic substances when performing an in vivo screening assay for EDCs.
  • Y. Nomura, H. Ishibashi, M. Miyahara, R. Shinohara, F. Shiraishi, K. Arizono
    Journal of Materials Science: Materials in Medicine 14 4 307 - 310 2003年04月 [査読有り]
     研究論文(学術雑誌) 
    Three monomers (Bis-GMA, UDMA, and TEGDMA) and five polymerization initiators (CQ, BPO, DMPT, DMAEMA, and ATU) commonly used in dental composite resins were tested for estrogenic activity using a reporter gene assay (yeast two-hybrid system) in vitro, and compared with bisphenol-A (BPA). Estrogenic activity was indicated by agonist and antagonist activity, with (+S9) and without (-S9) metabolic activation using rat liver cells. No estrogenic agonist activity was seen for each monomer and polymerization initiator in either the -S9 and +S9 tests in the concentration ranges examined in this study. On the other hand, estrogen antagonist activity was found with BPO and DMPT. BPO showed antagonist activity at a concentration of ∼ 1800 nM with the -S9 test, but not with the +S9 test. With DMPT, antagonist activity was not seen with the -S9 test, but it was seen at a concentration of ∼ 610 nM using the +S9 test. With BPA, the +S9 test indicated antagonist activity at a concentration of ∼ 780 nM. The estrogen antagonist activities of DMPT and BPA appeared to be similar. CQ, DMAEMA, ATU, and the three monomers did not show antagonist activity as demonstrated by the -S9 or +S9 tests within the concentration range tested in this study. © 2003 Kluwer Academic Publishers.
  • Monitoring of environmental pollutants by a combination of biomarkers in Minamata river water using goldfish (Carassius auratus)
    Hiroshi Ishibashi, Katsuyasu Tachibana, Mutsuyosi Tsuchimoto, Yuki Tomiyasu, Akiko Urakabe, Koremoto Morishita, Masahiro Tachibana, Norihisa Tatarazako, Koji Arizono
    Environmental Sciences 10 3 175 - 186 2003年 [査読有り]
  • Hiroshi Ishibashi, Makito Kobayashi, Tomohiko Koshiishi, Toshihisa Moriwaki, Katsuyasu Tachibana, Mutsuyosi Tsuchimoto, Kiyoshi Soyano, Taisen Iguchi, Chisato Mori, Koji Arizono
    Journal of Health Science 48 5 427 - 434 2002年10月 [査読有り]
     研究論文(学術雑誌) 
    In this study, we analyzed contents of phytoestrogens (genistein, daidzein, equol, and coumestrol) in two commercial fish diets [a diet for trout (TD) and a diet for ornamental carp (CD)] using Liquid Chromatography-Mass Spectroscopy/Mass Spectroscopy (LC-MS/MS), and these contents were compared with that of a casein-based formulated fish diet (FD) which does not contain soya bean or fish meal. We also analyzed phytoestrogen contents in commercial infant casein- and soya bean-based diets. The contents of phytoestorogens were generally high in CD, TD, and soya milk, and low or non-detectable in FD and casein-based milks. Among these samples, CD showed the highest phytoestrogen contents: genistein, 390800 ng/g; daidzein, 416800 ng/g; coumestrol, 1325 ng/g; equol, 6.4 ng/g. We also determined the estrogenic activity of the fish diets using male goldfish by measuring plasma vitellogenin (VTG) levels as a biomarker of estrogen exposure. When male goldfish were fed one of these diets for 31 days, plasma VTG was detected in CD-fed fish (78.01 ± 48.18 μg/ml) and TD-fed fish (3.51 ± 3.83 μg/ml), whereas plasma VTG was not detected in FD-fed fish (less than 0.040 μg/ml). These results indicate that the commercial fish diets examined contain a large amount of phytoestrogens and showed estrogenic activity that were strong enough to induce VTG production in male goldfish. It is necessary to eliminate estrogenic substances other than test chemicals in the screening test system for estrogenic endocrine-disrupting chemicals (EDCs). Since the formulated diet developed in the present study contain less phytoestrogens than the commercial fish diets and has low estrogenic activity, it is suggested that VTG production using male goldfish in combination with the low estrogen fish diet is a good in vivo system for evaluation of estrogenic effects of EDCs.
  • 石橋 弘志, 鑪迫 典久, 有薗 幸司
    環境技術 = Environmental conservation engineering 31 8 604 - 609 環境技術学会 2002年08月 [査読有り]
  • Evaluation of freshwater environment by combination biomarkers in goldfish (Carassius auratus)
    Hiroshi Ishibashi, Katsuyasu Tachibana, Mutsuyosi Tsuchimoto, Kiyoshi Soyano, Yasuhiro Ishibashi, Shinya Kohra, Nobuaki Tominaga, Norihisa Tatarazako, Koji Arizono
    Environmental Sciences 8 1 103 - 113 2001年 [査読有り]
  • Hiroshi Ishibashi, Katsuyasu Tachibana, Mutsuyosi Tsuchimoto, Kiyoshi Soyano, Yasuhiro Ishibashi, Masaki Nagae, Shinya Kohra, Yuji Takao, Nobuaki Tominaga, Koji Arizono
    Journal of Health Science 47 2 213 - 218 2001年 [査読有り]
     研究論文(学術雑誌) 
    Vitellogenin (VTG) is believed to be an effective and sensitive biomarker to detect the effects of endocrine-disrupting chemicals (EDCs) on fish. An enzyme-linked immunosorbent assay (ELISA) was developed in this study for the quantification of VTG in goldfish (Carassius auratus) blood plasma using a monoclonal antibody against carp lipovitellin which is known to cross-react with goldfish VTG, and a working range of 7.8 to 500 ng VTG/ml was established. A laboratory study involving the dosing of male goldfish for 28 days with a range of bisphenol-A (BPA) concentrations was conducted, and the established ELISA was used. There was no significant induction of VTG at the concentrations of 1 and 10 μg/l. Exposure to 100 μg/l and 1000 μg/l BPA for 28 days significantly elevated plasma VTG concentrations to 201 ± 90 μg/ml and 104552 ± 24920 μg/ml, respectively. To confirm the effect of temperature on VTG induction, male goldfish were exposed to the nominal concentration of 17β-estradiol (E2) 100 μg/l at 10° and 30°C for 10 days. Plasma VTG concentration in male goldfish increased more quickly in the fish exposed at 30°C than in those at 10°C. In addition, the plasma VTG concentration in the 30°C group (10463 ± 3268 μg/ml) was higher than that of the 10°C group (1.42 ± 1.6 μg/ml) after 1 day of exposure. Thus goldfish are useful for the investigation of EDCs under various conditions since they are relatively small in size, and the ELISA established in the present study will contribute to various types of research.

MISC

  • In silico試験系を用いたパラオキシ安息香酸エチルのエストロゲン作用の評価
    広田 菜々, 平野 将司, 石橋 弘志, 山元 涼子, 高本 亜希子, 有薗 幸司 環境ホルモン学会研究発表会要旨集 20回 83 -83 2017年12月
  • 有薗 幸司, 石橋 弘志, 内田 雅也, 吉本 圭佑, 山元 涼子, 冨永 伸明, 石橋 康弘 日本毒性学会学術年会 44 (0) P -118 2017年06月 
    【目的】更年期障害のホルモン補充用医薬品プレマリンは、牝馬の尿中に存在するエクイリン(Eq)などエクインエストロゲン類(EQs)を成分とする。これまでに我々は北海道の河川から複数のEQsを初めて検出し、存在実態を明らかにした。本研究では、魚類に対するEqの有害性とそのメカニズムを明らかにするため、雌雄ヒメダカ(<i>Oryzias latipes</i>)の繁殖及び次世代に対する影響を評価し、肝臓中の遺伝子発現解析を行った。<br>【方法】成熟雌雄ペア(5ペア/濃度区)を助剤対照区、Eq-10, 100及び1000 ng/Lに21日間曝露した。曝露期間中、各ペアの産卵数、受精率及び生死と産卵後のF<sub>1</sub>胚の孵化率を算出した。曝露後は全長、体重、肝臓(HSI)及び生殖腺対指数(GSI)を算出した。遺伝子発現解析は、肝臓中<i>vitellogenin</i>s (<i>Vtg1</i>, <i>2</i>)、<i>choriogenin</i>s (<i>ChgL</i>, <i>H</i>)、<i>estrogen receptor</i>s (<i>ERα</i>, <i>β1</i>, <i>β2</i>)、<i>pregnane X receptor</i> (<i>PXR</i>)、<i>cytochrome P4501A</i>及び<i>3A</i> (<i>CYP1A</i>, <i>3A</i>)と内部標準遺伝子をリアルタイムPCRで相対定量した。<br>【結果及び考察】繁殖試験の結果、雌雄の全長、体重、HSI及びGSIに変化はみられなかったが、1000 ng/L区における21日間の平均産卵数は有意に減少した。また、F<sub>1</sub>胚の孵化率は100及び1000 ng/L区で有意に減少し、1000 ng/L区の孵化仔魚では背骨の湾曲など奇形が観察された。遺伝子発現解析の結果、雄では<i>Vtg</i>s、<i>Chg</i>s、<i>ERα</i>/<i>β1</i>及び<i>PXR</i>遺伝子の濃度依存的な発現誘導がみられたが、雌では測定対象とした遺伝子の発現に変化はなかった。これらのことから、Eqはヒメダカの繁殖及び次世代に影響し(最小作用濃度100 ng/L)、肝臓中の遺伝子発現応答の性差が示唆された。<br>【謝辞】本研究は、環境省「EXTEND2010基盤的研究」の助成を受けて実施した。
  • 医薬品プレマリン成分エクイリン曝露によるヒメダカ繁殖阻害メカニズムの解明
    有薗 幸司, 石橋 弘志, 内田 雅也, 吉本 圭佑, 山元 涼子, 冨永 伸明, 石橋 康弘 The Journal of Toxicological Sciences 42 (Suppl.) S268 -S268 2017年06月
  • 線虫DNAマイクロアレイによるリチウムの影響評価
    井口 綾子, 内匠 正太, 山元 涼子, 石橋 弘志, 冨永 伸明, 有薗 幸司 The Journal of Toxicological Sciences 34 (Suppl.) S95 -S95 2009年06月
  • 立花 賢浩, 鑪迫 典久, 石橋 弘志, 有薗 幸司 環境技術 32 (3) 244 -251 2003年
  • 石橋 弘志, 鑪迫 典久, 有薗 幸司 資源環境対策 38 (12) 39 -44 2002年

受賞

  • 2020年05月 日本食品化学学会 島津製作所論文賞
  • 2012年07月 日本毒性学会 第2回ファイザー賞
  • 2011年07月 日本環境化学会 第18回環境化学論文賞
  • 2008年03月 Best Poster Award, International Symposium on Biological Responses to Chemical Pollutants:Toward Establishing an Asian Network of Environmental Toxicology
  • 2001年01月 日本水環境学会九州支部 学術奨励賞
     
    受賞者: 石橋 弘志

共同研究・競争的資金等の研究課題

  • 次世代型有機フッ素化合物による環境汚染・生物蓄積の実態解明と毒性影響評価
    日本学術振興会:科学研究費助成事業 基盤研究(A)
    研究期間 : 2020年04月 -2025年03月 
    代表者 : 石橋 弘志, 高橋 真, 川嶋 文人, 有薗 幸司, 加藤 恵介, 日下部 太一, 冨永 伸明, 平野 将司, 久保田 彰
  • 化学物質による水棲哺乳類細胞内受容体シグナル撹乱と感受性を規定する分子機構の解明
    日本学術振興会:科学研究費助成事業 基盤研究(A)
    研究期間 : 2019年04月 -2024年03月 
    代表者 : 岩田 久人, 国末 達也, 石橋 弘志
  • マイクロカプセルを介した化学物質の新たな環境動態の解明と評価
    日本学術振興会:科学研究費助成事業 基盤研究(A)
    研究期間 : 2019年04月 -2024年03月 
    代表者 : 鑪迫 典久, 石橋 弘志, 寺崎 正紀, 堀江 好文, 山岸 隆博, 山室 真澄, 山本 裕史
  • 臭素化ダイオキシンを蓄積する海産無脊椎動物を対象とした毒性リスク評価
    日本学術振興会:科学研究費助成事業 基盤研究(B)
    研究期間 : 2019年04月 -2022年03月 
    代表者 : 平野 将司, 石橋 弘志, 内田 雅也, 小林 淳
  • 先端的分子生物学的手法によるサンゴ白化に及ぼす高水温と化学物質の複合影響の解明
    日本学術振興会:科学研究費助成事業 基盤研究(B)
    研究期間 : 2018年04月 -2021年03月 
    代表者 : 竹内 一郎, 高山 弘太郎, 山城 秀之, 石橋 弘志
     
    平成30年度は、下記の1.~3.の実験等を実施した。 1. ミドリイシ属サンゴの毒性実験方法の開発 ミドリイシ属サンゴ等の枝状サンゴに適した毒性実験方法の開発等を行った。すなわち、ミドリイシ属サンゴの同一の箇所へのクロロフィル蛍光測定装置による光合成活性の測定方法を専用のホルダー等を用いて確立したほか、紫外線(UV)硬化性樹脂やUVライト等を使用したサンゴのポリカーボネート製ボルトへの新たな接着方法を開発した。 2. ミドリイシ属サンゴ主要種の温度耐性の解明 沖縄県瀬底島周辺の沿岸域からミドリイシ属サンゴを採集し、琉球大学熱帯生物圏研究センター瀬底研究施設の陸上水槽にて馴致飼育後、愛媛県松山市の愛媛大学大学院農学研究科の実験室に輸送し、実験に供した。インキュベータ、LEDライト、小型シャーレ等を用い29から32℃の間で4温度区を設定し、1週間の飼育実験を実施し、その間のサンゴの体色や光合成活性を測定した。その結果、コユビミドリイシ等は、32℃区では白化するものの、29℃~30℃では体色および光合成収率は変動しないこと等が明らかになった。 3. サンゴと共生藻の熱ショックタンパク質遺伝子の塩基配列の決定 ウスエダミドリイシからtotal RNAおよびmRNA試料を抽出・精製し、逆転写酵素によりcDNAを合成後、各生物種のHSP cDNA配列の保存領域から設計・合成したプライマーとポリメラーゼ連鎖反応(PCR)によりDNA増幅およびシーケンスを行った。その結果、ウスエダミドリイシのHSP70 cDNAの部分配列を得ることができた。共生藻については、既報と同じHSP90およびHSP70 cDNAの配列があることが確認された。また、これらHSP遺伝子に特異的なプライマーとSYBR Greenを用いた定量的リアルタイムPCR測定系を構築した。
  • 魚類脳神経系に対するネオニコチノイド系農薬環境変化体の影響評価
    日本学術振興会:科学研究費助成事業 基盤研究(C)
    研究期間 : 2018年04月 -2021年03月 
    代表者 : 甲斐 穂高, 山口 雅裕, 石橋 弘志, 石橋 康弘
     
    ネオニコチノイド(NNI)系農薬は、ミツバチなどの大量死など有害性を示すことから、欧州をはじめとする諸外国で、NNIの使用や販売が禁止され始めている。また、NNIは農薬としてだけでなく、動物用医薬品や建材などにも使用されるため、最終的に水系に排出されることで水生生物に影響を及ぼす可能性がある。NNIは、自然環境中において代謝されその構造が変化していくが、その代謝物(環境変化体)は代謝前のNNI(親化合物)と同等以上の毒性があることが懸念されている。しかし、その詳細は明らかにされていない。加えて、一部のNNI環境変化体は、同親化合物と比較して、ニコチン性アセチルコリン受容体(nAChR)に強い結合親和性を示すことが明らかにされており、NNI環境変化体がnAChRを介して脳神経系に何らかの影響を及ぼす可能性も考えられる。本申請課題では、バイオアッセイ試験のモデル生物の一つであるヒメダカ(Oryzias latipes)を対象として、NNI環境変化体がヒメダカ脳神経系へ与える影響について、1)異なる成長段階に対する毒性試験、2)抗体染色による脳神経系観察、3)発現変動遺伝子解析、4)対象物質の体内蓄積量の測定から、個体―組織―細胞レベルにおいて包括的にNNI環境変化体の影響を解析することを目的としている。 当該年度は、NNIの親化合物、環境変化体、最終代謝物を1種類ずつ選択し、ヒメダカ胚に対する毒性試験の暴露条件、ヒメダカ脳神経系の観察方法、LC-MS-MSによる水試料の分析条件について検討した。その結果、ヒメダカ胚の発生に与える影響が対象化合物毎に異なっていることが示唆された。また、脳神経系観察のための最適な暴露条件を見出すことができた。加えて、抗体を用いた蛍光免疫染色による脳神経系の観察方法についてもおおよそ確立できた。水試料を対象としたNNIsのLC-MS-MS分析方法も一部確立できた。
  • メダカ非アルコール性脂肪肝炎(NASH)モデルを用いた食品由来PPARリガンド成分と治療薬の同時摂取による効果の評価
    公益財団法人日本食品科学研究振興財団:
    研究期間 : 2020年04月 -2021年03月 
    代表者 : 内田雅也、平野将司、山元涼子、石橋弘志
  • AOPによる環境毒性学発展のための基盤形成研究
    日本学術振興会:科学研究費助成事業 挑戦的研究(萌芽)
    研究期間 : 2018年06月 -2020年03月 
    代表者 : 冨永 伸明, 平野 将司, 石橋 弘志
     
    本研究課題では,環境毒性学にAdverse Outcome Pathway(AOP)の概念によるリスク評価・管理システムを導入するためのモデル生物による環境毒性AOPの基盤構築を行うことを目的とした.本年度は,以下の研究成果を得た. 環境化学物質の有害影響と初期応答のキーイベントを直接つなぐ経路を整備・構築するため,PPARsに注目し,モデル化合物の選別を分子シミレーションソフトによるインシリコ解析にて行った.ヒトPPARaの結晶構造を基にメダカPPARaの立体構造を予測した.得られたモデルはRMSD値が0.78 Aと算出され,良好なモデルが構築できた.そこで,PPARa作動性の環境化学物質として有機フッ素化合物(PFCs)に着目し,PFCs17種についてドッキングシミュレーションを実施し,相互作用および親和性について検討したところ,炭素鎖長が長いPFODA,PFOSはメダカPPARaに対して既知の哺乳動物PPARαへの結合と同様に安定した強い相互作用を示し,炭素鎖長が短いPFBA,PFBSは弱いことが分かった.これら結合には,ヒトPPARaの重要残基であるTyr314,His440に対応するHis327,His453がメダカPPARaでは関与していることを見出した.これらことから,メダカにおける結果は哺乳動物における影響評価に外挿できる可能性が高いことが考えられた.AOPモデル構築における最初のターゲットとなる分子レベルのキーイベントについての情報が得られた.また,結合性の高いフルアゴニストおよびパーシャルアゴニストの選定を行った.さらに,PPARsレポーターアッセイを立ち上げ,アゴニスト活性の比較をはじめた.メダカ卵への取り込み暴露試験については,発生影響評価およびトランスクリプトーム解析を開始した.
  • AOP研究基盤の高度化を目指した魚類を用いた化学物質の影響評価
    日本学術振興会:科学研究費助成事業 基盤研究(B)
    研究期間 : 2017年04月 -2020年03月 
    代表者 : 有薗 幸司, 冨永 伸明, 石橋 弘志, 吉田 佳督
     
    エクインエストロゲン(EQs)は、妊馬の尿中から検出されるステロイドホルモンであり、医薬品の原料としても使用されている。欧米では、下水処理水などからEQsが検出されており、水生生物への影響が危惧されている。昨年度まで、北海道河川水からEQsの一種であるエクイリン(Eq)やエクイレニン(Eqn)を検出し、日本の水環境におけるEQsの存在実態を初めて明らかにし、Eqを3週間曝露したメダカにおいて、産卵数の減少や次世代胚の孵化率の低下を明らかにした(Ishibashi et al., 2018)。本年度は胚の孵化率の低下のメカニズムを明らかにすべく高電界パルス印加法を用いてメダカ受精卵にEq及びEqnを導入し、発生毒性と胚トランスクリプトームに及ぼす影響を調査した。結果としてEq及びEqn共通して主に神経発達や細胞接着などの発生影響に関与するパスウェイが同定された。また、Eq特異的にレチノール代謝の撹乱による胚の奇形に関与するMAPK signaling, retinol metabolismパスウェイ、Eqn特異的に細胞の分化や増殖などへの影響するcytokine-cytokine receptor interactionパスウェイが確認された。さらにゼブラフィシュ胚を用いてエクイン類のエストロゲン作用のED50はエクイン類の相互作用スコア(S-Score)との間で有意な正の相関をもつことと、Eq、Eqnは17位がカルボニル基からα、β体のヒドロキシ基になることでエストロゲンレセプター結合性が強化される事実が明らかになった。以上平成30年度はEQsは発生初期における代謝経路の攪乱を惹起、多くの代謝経路に関わるネットワーク系に影響を及ぼし、代謝されさらに強いエストロゲン活性を持つ可能性を明らかにできた。
  • New POPsによるPPARαシグナル伝達撹乱の比較生物学的リスク評価
    日本学術振興会:科学研究費助成事業 基盤研究(B)
    研究期間 : 2016年04月 -2020年03月 
    代表者 : 石橋 弘志, 有薗 幸司
     
    残留性有機汚染物質(Persistent Organic Pollutants: POPs)に関するストックホルム条約において、新たなPOPsとして追加された臭素系難燃剤(Brominated Flame Retardants: BFRs)や有機フッ素化合物(Perfluoroalkyl Substances: PFASs)に対する学術的・社会的関心が高まっている。これらnew POPsによる環境汚染の実態や毒性影響については依然として不明な点が多い。また、実験動物では、new POPsなどの環境汚染物質による毒性影響(作用濃度)において、系統差・種間差などが存在することが知られている。本申請課題では、実験動物に加え、生態学的に重要視されている野生生物を対象として、脂質代謝に関与するペルオキシソーム増殖剤応答性受容体(PPARα)シグナル伝達経路に対するnew POPsの比較生物学的リスク評価を行うことを目的としている。 平成30年度は、PPARα-リガンド分子間相互作用において、より精度の高いインシリコアッセイ系を構築するため、従来から知られているPPARαリガンド結合ポケット(PPARα 1st LBP)に加え、近年発見された第2のPPARαリガンド結合ポケット(PPARα 2nd LBP)について、PFASsとのドッキングシュミレーション解析を行い、PPARαリガンド結合領域(LBD)の組換えタンパク質に対するPFASsのインビトロ競合結合試験の結果と比較した。その結果、ヒト(h)およびバイカルアザラシ(bs)のPPARα 1st/2nd LBPsに対するPFASsの結合親和性が初めて明らかになった。また、hおよびbsPPARα 1st/2nd LBPsに対するPFASsの異なる結合親和性には、PPARα 1st/2nd LBPsの体積や水素結合相互作用に関与するアミノ酸、PFASsのフッ素化された炭素数(分子量)や疎水性などの関与が示唆された。さらに、インビトロ競合結合試験におけるPFASs結合強度とインシリコ評価系の結果との間で有意な相関関係がみられ、インシリコ評価系の有用性が示された。
  • 日本学術振興会:科学研究費助成事業 基盤研究(B)
    研究期間 : 2016年04月 -2019年03月 
    代表者 : 冨永 伸明, 高橋 圭介, 大河平 紀司, 有薗 幸司, 石橋 弘志
     
    本研究は,メダカ受精卵を用いた高感度な化学物質の生物影響評価法の開発を行った.高電界パルスを用いた化学物質導入法を用いることで,メダカ受精卵に高効率かつ安定的に取り込ませることができた.また,ネオニコチノイド系農薬をモデル化合物として評価したところ,急性毒性は低いものの発生異常が生じることが分かった.また,その影響は代謝物においても同様に引き起こされた.また,顕著な形態的な影響が現れていない時期においても遺伝子発現が変化していることが分かった.
  • 日本学術振興会:科学研究費助成事業 基盤研究(B)
    研究期間 : 2014年04月 -2017年03月 
    代表者 : 竹内 一郎, 冨永 伸明, 高橋 真, 高山 弘太郎, 治多 伸介, 石橋 弘志
     
    実験室内でLED照明装置、人工海水等によるサンゴやサボテングサ用の飼育システムを構築した。本システムにより、低濃度のイルガロール 1051およびジウロン(1および10 μg/L)のミドリイシ属のサンゴ等への毒性実験を実施したが、RGB値(R、赤:G、緑:B、青)によるサンゴ等の体色解析、高感度ルミノメーターによる遅延発光の解析ともに、イルガロール 1051の毒性影響がジウロンより強い傾向が認められた。イルガロール 1051では、これらの濃度下で、2週間以内の曝露でサンゴが白化した。また、次世代シーケンス解析により、これらの除草剤曝露によって特異的に発現変動するサンゴの遺伝子群が同定された。
  • 日本学術振興会:科学研究費助成事業 挑戦的萌芽研究
    研究期間 : 2013年04月 -2015年03月 
    代表者 : 石橋 弘志
     
    水環境汚染が問題視されている医薬品類や化粧品等のパーソナルケア製品(PPCPs)と魚類エストロゲン受容体(ER)サブタイプの分子間相互作用を評価するため、in silicoスクリーニング系を開発した。鎮痛剤/抗炎症剤、脂質調節剤、β遮断薬、抗鬱剤などのPPCPsは、一部の魚類ERαに対して比較的強く相互作用し、相互作用に関与したERsのアミノ酸の種類や数、水素結合の有無やリガンド-ERs間の距離は異なり、生物種間差が明らかになった。開発したin silico試験系は、PPCPs-ERs分子間相互作用のスクリーニング手法として極めて有用であり、生態リスク評価ツールとしての活用が期待される。
  • 日本学術振興会:科学研究費助成事業 基盤研究(B)
    研究期間 : 2012年04月 -2015年03月 
    代表者 : 石橋 弘志, 有薗 幸司
     
    新たな残留性有機汚染物質 (POPs) であるポリ臭素化ジフェニルエーテルなど臭素系難燃剤に着目し、核内受容体ペルオキシソーム増殖剤応答性受容体α (PPARα) を介した生体影響・作用機序の解明とリスク評価することを目的とした。系統学的・生態学的に重要な野生高等動物種のPPARαシグナル伝達系に影響する臭素系難燃剤のハイスループットスクリーニングシステムの開発に成功し、本研究で初めて「臭素系難燃剤によるPPARαシグナル伝達撹乱」という新たなシグナル毒性の作用機序の一端を明らかにした。
  • 日本学術振興会:科学研究費助成事業 基盤研究(S)
    研究期間 : 2009年05月 -2014年03月 
    代表者 : 岩田 久人, 阿草 哲郎, 宮崎 龍彦, 鈴木 賢一, 石橋 弘志, 能勢 眞人, 平野 将司
     
    成果の概要は以下のように要約できる。 1)有害な環境汚染物質をスクリーニングするため、野生動物の細胞内受容体蛋白質を発現させたin vitroアッセイ法を構築した。2)環境汚染物質曝露に対して細胞内受容体は種・アイソフォーム特異的な感受性を有すること。3)in vitroアッセイ系を利用することにより、種特異的感受性を考慮した野生個体群のリスクが評価できること。4)細胞内受容体およびシトクロムP450(CYP)の特異的なリガンド結合能には、進化の過程で変異した特定のアミノ酸残基が寄与していること。5)細胞内受容体・CYP以外にも感受性を規定する因子が存在すること。
  • 日本学術振興会:科学研究費助成事業 基盤研究(B)
    研究期間 : 2010年 -2012年 
    代表者 : 有薗 幸司, 渡辺 知歩, 石橋 弘志, 吉田 佳督, 石橋 康弘
     
    中国「癌村」において、対象地域と癌村の複数場所で環境試料(遺伝毒物を含有すると思われる生活環境水)を得て、癌村周辺の工業地域の情報をもとに多様な化学物質の濃度を分析したところ。o,p'-DDTが環境水中に検出された。そこでその生態影響評価をメダカのDNAマイクロアレイを用いて行った。一方で、カドミウムに特化したイムノクロマトグラフィを活用した環境水や食品汚染調査(農作物および水産物)を中国海南省CDCと協力して実施した。地域住民の健康調査も重金属分析とともに行った。その結果、海南省水産物の鉛汚染は0.005~0.717mg/kg、カドミウム河川汚染は0.100×10-3~0.648 mg/kgが検出された。
  • バイカルアザラシPPAR-CYP4を介した有機フッ素化合物の毒性メカニズム解明
    日本学術振興会:科学研究費助成事業 若手研究(B)
    研究期間 : 2006年 -2007年 
    代表者 : 石橋 弘志
     
    本研究の目的は,野生生物における有機フッ素化合物(PFCs)の汚染実態および蓄積特性を明らかにし,peroxisome proliferator-activated receptor(PPAR)-cytochrome P450 4A(CYP4A)シグナル伝達系を介した毒性影響メカニズムを解明することである。LC-MS/MSによる分析から,ロシア・バイカル湖の固有種バイカルアザラシ(Pusa sibirica)の肝臓および血清中のPFCs蓄積濃度を明らかにした。これら蓄積濃度は,水棲哺乳類を対象とした既報値と比較して同程度あるいは低値であったが,PFNAおよびPFDAの肝臓への特異的な集積がみられた。また,幼獣のPFCs蓄積濃度は成獣と比較して高く,幼獣の低いPFCs代謝能あるいは母子間移行の可能性が考えられた。さらに,2005年に採取したバイカルアザラシ肝臓中のPFOS・PFNAおよびPFDA蓄積濃度は,1992年のそれらと比較して高く,バイカル湖におけるPFCs汚染の顕在化が明らかとなった。そこで,バイカルアザラシPPARα cDNAの単離を試みたところ,水棲哺乳類で初めて完全長PPARα cDNAの単離に成功した。バイカルアザラシPPARα発現プラスミドを導入したin vitroレポーター遺伝子アッセイ系を構築し,転写活性化能を測定したところ,PFOA・PFNA・PFDA・PFUnDAおよびPFOSによりPPARαは活性化された。また,PFCsの蓄積したバイカルアザラシ肝臓中では,PFNAとPPARαmRNA発現量の間に,PFNAあるいはPFDAとCYP4Aタンパク発現量の間に正の相関関係が認められた。これらことから,バイカルアザラシの肝臓中に蓄積したPFCsは,PPARα-CYP4Aシグナル伝達系に影響していることが強く示唆された。

愛媛大学教員活動実績

教育活動(B)

担当授業科目(B01)

  • 2019, 前期, 学部, 環境生態毒性学
  • 2019, 前期, 修士, 環境保全学特論


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