{"created":"2023-10-18T02:41:31.804875+00:00","id":2000049,"links":{},"metadata":{"_buckets":{"deposit":"9479efb6-2ff6-4f53-a627-5b5d547a5734"},"_deposit":{"created_by":12,"id":"2000049","owner":"12","owners":[12],"pid":{"revision_id":0,"type":"depid","value":"2000049"},"status":"published"},"_oai":{"id":"oai:obihiro.repo.nii.ac.jp:02000049","sets":["6:8"]},"author_link":[],"control_number":"2000049","item_3_alternative_title_1":{"attribute_name":"その他（別言語等）のタイトル","attribute_value_mlt":[{"subitem_alternative_title":"ゼブラフィッシュを用いた環境汚染物質の内分泌撹乱作用と発生毒性に関する研究","subitem_alternative_title_language":"ja"}]},"item_3_date_granted_12":{"attribute_name":"学位授与年月日","attribute_value_mlt":[{"subitem_dategranted":"2023-09-29"}]},"item_3_degree_grantor_10":{"attribute_name":"学位授与機関","attribute_value_mlt":[{"subitem_degreegrantor":[{"subitem_degreegrantor_language":"ja","subitem_degreegrantor_name":"帯広畜産大学"}],"subitem_degreegrantor_identifier":[{"subitem_degreegrantor_identifier_name":"10105","subitem_degreegrantor_identifier_scheme":"kakenhi"}]}]},"item_3_degree_name_9":{"attribute_name":"学位名","attribute_value_mlt":[{"subitem_degreename":"Doctor of Veterinary Medicine","subitem_degreename_language":"en"},{"subitem_degreename":"博士（獣医学）","subitem_degreename_language":"ja"}]},"item_3_description_11":{"attribute_name":"学位授与年度","attribute_value_mlt":[{"subitem_description":"2023","subitem_description_language":"ja","subitem_description_type":"Other"}]},"item_3_description_18":{"attribute_name":"フォーマット","attribute_value_mlt":[{"subitem_description":"application/pdf","subitem_description_language":"en","subitem_description_type":"Other"}]},"item_3_description_7":{"attribute_name":"抄録","attribute_value_mlt":[{"subitem_description":"A significant and varied array of endocrine-disrupting chemicals (EDCs) has been released into the environment since World War II (Colborn et al., 1993). EDCs have the capacity to interfere with the normal functioning of the endocrine and reproductive systems by mimicking or impeding the actions of endogenous hormones. Hence, there is concern regarding the impact of EDCs on ecosystems, leading to the conduction of numerous studies from this perspective. Studies have reported that exposure to anti-androgens and estrogens are associated with the feminization of wild fish. Considering the reproductive toxicity and endocrine system disruption to fish, it is crucial to identify environmental chemicals with anti-androgenic potential and to develop sensitive detection methods to assess anti-androgenic responses in fish. Although many evaluation systems have been established for detecting estrogenic activity of chemicals, few methods have been developed to identify anti-androgenic effects using fish species, such as three-spined stickleback (Gasterosteus aculeatus), Japanese medaka, and spiggin-gfp medaka. However, these testing methods require longer exposure durations and specific maintenance systems.\nThe susceptibility of organisms to chemical exposure is high during the earlier developmental stage, and chemical exposures during this period can cause irreversible outcomes such as malformation. Extensive studies have investigated the developmental effects of chemical exposure, but their connection to endocrine-disrupting effects remains unclear. Zebrafish, as a cost-effective and high fertility animal model, has been widely used in various research fields, including drug screening, gene function analyzing, developmental toxicity measuring, and endocrine-disrupting study. In recent years, molecular docking simulations have become essential in structure-based computational studies aimed at enhancing our understanding of receptor-ligand interactions at the atomic level. In this study, we used in vivo and in silico methods to develop a simple and rapid evaluation system to test anti-androgenic effects, which has been rarely reported so far. This study aimed to clarify the anti-androgenic effects of various environmental chemicals using the constructed evaluation system. Furthermore, we aimed to clarify the developmental toxicity of each environmental chemical and to evaluate the relationship between endocrine disrupting effects and developmental toxicity.\nChapter I established zebrafish-based in vivo and in silico assay systems to evaluate the anti-androgenic potential of environmental chemicals. Zebrafish embryos were exposed to 17α-methyltestosterone (TES) alone or in combination with the anti-androgen flutamide (FLU), as well as various pesticides known for their anti-androgenic activities, such as p,p′-DDE (DDE), vinclozolin (VIN), linuron (LIN), and fenitrothion (FEN). In order to explore the potential correlation between anti-androgenic potency and developmental toxicity, this chapter additionally conducted morphological assessments.\nThe expression of sulfotransferase family 2st3 (sult2st3), was measured as an indicator of anti-androgenic effects. The expression of sult2st3 mRNA was significantly induced by TES in the later developmental stages of embryos. However, the TES-induced expression of sult2st3 was inhibited by anti-androgen FLU in a concentration-dependent manner with IC50 of 5.7 μM, suggesting that the androgen receptor (AR) plays a role in sult2st3 induction. Similarly, DDE, VIN, and LIN repressed the TES-induced expression of sult2st3 with IC50 of 0.35, 3.90, and 52.00 μM, respectively. At the highest concentration tested (100 μM), FEN also suppressed sult2st3 expression almost completely, although its IC50 could not be calculated. Notably, DDE and LIN did not inhibit sult2st3 induction due to higher concentrations of TES; instead, they potentiated TES-induced sult2st3 expression. All tested pesticides affected the swim bladder inflation, especially FLU, VIN, LIN, and FEN impaired swim bladder inflation in a concentration-dependent manner. FLU, LIN, and FEN caused significant mortality at 30 uM, 300 uM, and 100 uM, respectively. LIN and FEN, which had relatively low anti-androgenic potentials in terms of sult2st3 inhibition, induced broader toxicities in zebrafish embryos. Thus, the relationship between developmental toxicities and anti-androgenic potency was unclear. Additionally, an in silico docking simulation showed that all five chemicals interact with the zebrafish AR at relatively low interaction energies and with Arg702 as a key amino acid in ligand binding, whereas several chemicals classified into other groups (e.g., potassium permanganate, 4-hydroxytamoxifen, and 20-hydroxyecdysone) have higher interaction energies. This indicates that anti-androgenic compounds exhibit a higher stability in bonding with zfAR compared to compounds not classified into anti-androgens.\nThe in vivo and in vitro anti-androgenic and anti-estrogenic effects of bisphenol A (BPA) have been reported. Still, there is limited research on its analogs about these aspects. Therefore, Chapter II aimed to measure the anti-androgenic potency of BPA and its various analogs applied the established method in Chapter I, and to test their anti-estrogenic potentials both in vivo and in silico. To better understand the involvement of estrogen receptor (ER) signaling in the developmental toxicity of BPA and its analogs, this chapter also performed morphological assessments of cardiovascular toxicity, such as pericardial edema and blood flow reduction.\nFor gene expression analysis, zebrafish embryos were exposed to TES or 17β-estradiol (E2) alone or in combination with BPA or each of its analogs, such as bisphenol AF (BPAF), bisphenol E (BPE), bisphenol F (BPF), bisphenol B (BPB), bisphenol C2 (BP C2), 2,2′-bisphenol F (2,2′-BPF), 4,4′-(1,3-dimethylbutylidene)diphenol (Bis-MP), bisphenol Z (BPZ), and bisphenol S (BPS), at 72 h postfertilization. After 24 h of exposure, all samples were collected to measure the mRNA expression levels of sult2st3 and CYP19A1b, which were used to assess the anti-androgenic and estrogenic potential of the tested compounds, respectively. To better understand the ligand binding affinity, we analyzed the binding mode of bisphenols to the zebrafish AR and estrogen receptor subtypes (ERα, ERβ1, and ERβ2). For morphological assessment, embryos were exposed to BPA or its analogs in the presence or absence of ER antagonist fulvestrant (ICI) at 72 hpf and were observed at 96 hpf.\nBPAF, BPE, BPA, BPF, and BPB inhibited the expression of TES-induced sult2st3 with IC50 values of 0.53, 3.7, 4.7, 12, and 87 μM, respectively. BP C2, 2,2′-BPF, and Bis-MP showed an inhibitory effect on TES-induced sult2st3 at the higher tested concentrations. However, BPZ and BPS did not exhibit inhibitory effects on TES-induced sult2st3. These results indicate that the anti-androgenic effect of BPA and its analogs follows this order: BPAF > BPE > BPA > BPF > BPB > BP C2 > 2,2′-BPF > Bis-MP >> BPZ ≈ BPS. In AR ligand binding domain, BPA formed a hydrogen bond with Met695, while most of analogs hydrogen-bonded to Asn655 and/or Gln661, indicating that these amino acid residues are essential for exhibiting anti-androgenic activity of bisphenols. Furthermore, BPAF, BPA, BPF, BPB, BP C2, and Bis-MP showed inhibitory effects on E2-induced CYP19A1b expression in a concentration-independent manner, indicating their anti-estrogenic effects. Compared to our previous study, the binding mode of BPA and its analogs differed between their antagonistic and agonistic effects on ER subtypes. Specifically, ERβ2 exhibited completely distinct binding modes for bisphenols in the agonistic and antagonistic modes, suggesting the potential role of ERβ2 in distinguishing agonistic and antagonistic activities of bisphenols. BP C2, Bis-MP, and BPAF showed more potent cardiovascular toxicity, as their lowest observed effect concentrations are much lower than those of BPA, BPE, and BPF. The incidence of pericardial edema and blood flow reduction induced by BP C2, Bis-MP, and BPAF was large/completely reduced by ICI, whereas this effect was not observed with BPA, BPE, and BPF. This suggests that ER signaling may not mediate the cardiovascular toxicity induced by BPA, BPE, or BPF.\nIn summary, the present study provided the faster and simpler in vivo methods for assessing anti-androgenic potentials of a wider range of environmental chemicals using zebrafish embryos. The majority of tested bisphenols exhibited both anti-androgenic and anti-estrogenic effects, while BPZ and BPS did not demonstrate either. No correlation was observed between the concentration-dependent effects of anti-androgenic or anti-estrogenic activities and their cardiovascular toxicity. Some BPA alternatives, such as BP C2, Bis-MP, and BPAF, showed stronger cardiovascular toxicity than BPA, BPE, and BPF. The cardiovascular toxicity caused by BPA, BPE, and BPF may not be mediated by ER signaling, but further investigations are needed to confirm the (non)involvement of ER in cardiovascular toxicity induced by these bisphenols. Based on our findings, utilizing zebrafish-based in vivo and in silico assessments appears to be a promising approach for evaluating the anti-androgenic or anti-estrogenic potentials of environmental chemicals. Careful consideration should be given to the toxicities of alternative itself when selecting BPA replacement.","subitem_description_language":"en","subitem_description_type":"Abstract"},{"subitem_description":"第二次世界大戦以降、多種多様な化学物質が生産・使用され人類に利便性をもたらしてきた。一方、生産・使用された化学物質が環境中に放出され、中には極微量で生物の内分泌系を撹乱する作用を示すいわゆる内分泌撹乱物質（環境ホルモン）が含まれていることも明らかにされてきた。とくに、あらゆる環境化学物質の到達点である水圏において、内分泌撹乱物質の生態系に対する影響が懸念されたため、こうした視点の研究がこれまで数多く実施されてきた。魚類を用いた研究では、環境化学物質がエストロゲン作用や抗アンドロゲン作用を示し、メス化と関連することが報告されている。したがって、多様な化学物質についてエストロゲン作用や抗アンドロゲン作用を魚類で評価することは重要である。これまでエストロゲン作用の評価系については多くの報告例があるものの、抗アンドロゲン作用の評価系については報告例が少なく、また評価にかかる時間やコストなどの面で課題もあった。\n一般に化学物質に対する感受性は発達期で高く、その時期における曝露は奇形等の不可逆的かつ重大な影響を引き起こす。これまでに化学物質の発達期での影響を調べた研究は多いが、内分泌撹乱作用との関連性については不明な点が多い。\n本研究では、環境毒性学研究ですでに重要な地位を築いているゼブラフィッシュをモデルとして、インビボ試験およびインシリコ手法を用いて、これまでに報告例の少ない抗アンドロゲン作用の簡便迅速な評価系を構築すること、ならびに構築した評価系を用いて多様な環境化学物質の抗アンドロゲン作用を明らかにすることを目的とした。さらに、各環境化学物質の発生毒性を明らかにするとともに、抗アンドロゲン作用と発生毒性の関連性について評価することも目的とした。\n第Ⅰ章では、抗アンドロゲン作用の評価系の構築について検討した。まずインビボ試験では、アンドロゲン活性のマーカー遺伝子として知られるsulfotransferase 2st3（sult2st3）を指標として、テストステロン（TES）誘導性のsult2st3発現に対する化学物質の抑制能を評価した。このため、受精後72時間のゼブラフィッシュ胚にメチルテストステロンならびにこれまで抗アンドロゲン作用が報告されているフルタミド（FUL）と4種の農薬（p,p'-DDE（DDE）、ビンクロゾリン（VIN）、リニュロン（LIN）、フェニトロチオン（FEN））をそれぞれ共曝露した。受精後96時間で発生毒性を評価した後、曝露胚を採材した。採材した曝露胚は、全RNA抽出をした後、cDNA合成を行い、リアルタイムPCR法でsult2st3の発現量を測定した。曝露試験の結果、TES誘導性のsult2st3発現量は抗アンドロゲン剤FLUによって濃度依存的に抑制され、その用量効果（IC50）は5.7 μMであった。したがって、TESによるsult2st3発現誘導にはアンドロゲン受容体（AR）が関与していると考えられた。同様に、DDE、VIN、LINについても、それぞれTES誘導性のsult2st3発現量は濃度依存的に抑制され、IC50はそれぞれ、0.35μM、3.9μM、52μMであった。FENについては、本曝露試験の最高濃度である100μMでのみ、TES誘導性のsult2st3発現の抑制が認められた。また、TES誘導性のsult2st3発現の抑制に関する用量効果が相対的に低かったFENとLINにおいて、高濃度側で循環障害や浮袋の膨張不全など様々な発生毒性が認められた。これらの結果より、発生毒性と抗アンドロゲン作用の間に明瞭な相関性は認められなかった。\n次いで、インシリコ解析では、ヒトARと水酸化フルタミド（hydroxyflutamide）の結晶構造をテンプレートとして、ホモロジーモデリングによりゼブラフィッシュAR（zfAR）の3次元構造を構築した。構築したzfARと、上記のインビボ試験で抗アンドロゲン作用を評価した化合物についてドッキングシミュレーションを行い、リガンド結合に重要な役割を果たしているアミノ酸を推定するとともに、相互作用エネルギーを算出した。インシリコ解析の結果、FUL、DDE、VIN、LIN、FENの5化合物は、いずれもzfARのリガンド結合ポケットに位置するArg702と水素結合を形成し、その相互作用エネルギーは抗アンドロゲン作用をもたない他の化合物群と比べて相対的に低い値をもつことが示された。以上より、インビボ試験で抗アンドロゲン作用を示した化合物は、他の化合物群と比べてzfARとより安定的に結合することが示唆された。\n第II章では、第Ⅰ章で構築した抗アンドロゲン作用のインビボ・インシリコ評価系を用いて、ビスフェノールA（BPA）とその関連化合物（ビスフェノール類）の抗アンドロゲン作用を評価した。加えて、エストロゲン受容体（ER）の標的遺伝子であるCYP19A1bを指標として、17β-エストラジオール（E2）誘導性のCYP19A1b発現に対する各ビスフェノール類の抑制能（抗エストロゲン作用）を評価した。このためインビボ試験では、受精後72時間のゼブラフィッシュ胚にTESまたはE2と、各ビスフェノール類をそれぞれ共曝露した。いずれも受精後96時間で曝露胚を採材し、全RNA抽出をした後、cDNA合成を行い、リアルタイムPCR法でsult2st3またはCYP19A1bの発現量を測定した。さらに、BPAおよび一部のビスフェノール類（BPE、BPF、BP C2、Bis-MP、BPAF）については、同様に曝露試験を行い、心臓周囲浮腫や体幹血流の低下を評価した。また、ER拮抗薬であるフルベストラント（ICI）との共曝露試験を行い、ビスフェノール類の発生毒性に対するERの関与を検討した。\n抗アンドロゲン作用に関する曝露試験の結果、TES誘導性のsult2st3発現量は多くのビスフェノール類によって濃度依存的に抑制され、その用量効果（IC50）はBPAF（0.53μM）、BPE（3.7μM）、BPA（4.7μM）、BPF（12μM）、およびBPB（87 μM）の順であった。また、BP C2、2,2′-BPF、およびBis-MPは、試験した最高濃度でのみTES誘導性のsult2st3発現を抑制した。一方、BPZおよびBPSはTES誘導性のsult2st3発現を抑制しなかった。次いで、インシリコ解析では、BPAはzfARのリガンド結合ポケットに位置するMet695と水素結合を形成し、その他のビスフェノール類の多くはAsn655もしくはGln661と水素結合を形成した。したがって、これらアミノ酸残基がビスフェノール類の抗アンドロゲン作用で重要な役割を担うと推察された。\n抗エストロゲン作用に関する曝露試験の結果、E2誘導性のCYP19A1b発現は、試験した高濃度のBPAF、BPA、BPF、BPB、BP C2、およびBis-MPによって抑制されたものの、明瞭な濃度依存性は認められなかった。したがって、これらビスフェノール類は、用量効果は低いものの抗エストロゲン作用を有することが示唆された。次いで、インシリコ解析では、多くのビスフェノール類は各ERサブタイプ（zfERα、zfERβ1、zfERβ2）のリガンド結合ポケットに位置するアミノ酸残基と水素結合を形成した。さらに、本研究で明らかにしたERアンタゴニスト作用に関する結合モードは、当研究グループのこれまでの研究で明らかにしたERアゴニスト作用に関する結合モードと異なることが示された。とくに、ERβ2ではアゴニスト作用とアンタゴニスト作用でまったく異なる結合モードを示したことから、ERβ2がビスフェノールのアゴニスト作用とアンタゴニスト作用を区別する重要な役割をもつことが示唆された。\nビスフェノール類の曝露胚における心血管毒性の評価では、試験したいずれのビスフェノール類（BPA、BPE、BPF、BP C2、Bis-MP、BPAF）についても、心臓周囲浮腫や体幹血流の低下が認められた。その用量効果は、BP C2、Bis-MP、およびBPAF で高く、逆にBPA、BPE、およびBPFでは低かった。また、BP C2、Bis-MP、およびBPAFで誘発された心臓周囲浮腫および体幹血流の低下はICIによって大幅に改善したが、同様の改善効果はBPA、BPE、およびBPF誘発性の心血管毒性では観察されなかった。したがって、BPA、BPE、BPF誘発性の心血管毒性はERシグナル介在性ではないと考えられた。\n以上の結果より、多くのビスフェノール類は、抗アンドロゲン作用と抗エストロゲン作用の両方を有することが明らかとなった。一方、試験したビスフェノール類では、抗アンドロゲン作用や抗エストロゲン作用の用量効果と心血管毒性の用量効果の間に相関関係は認められなかったことから、ビスフェノール類誘発性の心血管毒性はERやAR介在性ではないと推察された。一方で、BPAよりも抗アンドロゲン作用、抗エストロゲン作用、心血管毒性のいずれも高いビスフェノール類が複数あることが明らかとなったことから、構造的に類似の代替物質を利用する際にはそれらの毒性を明らかにする必要があると考えられた。","subitem_description_language":"ja","subitem_description_type":"Abstract"}]},"item_3_description_8":{"attribute_name":"内容記述","attribute_value_mlt":[{"subitem_description":"博士学位論文","subitem_description_language":"ja","subitem_description_type":"Other"},{"subitem_description":"大学院畜産学研究科 獣医学専攻","subitem_description_language":"ja","subitem_description_type":"Other"},{"subitem_description":"Doctoral Program of Veterinary Science","subitem_description_language":"en","subitem_description_type":"Other"}]},"item_3_dissertation_number_13":{"attribute_name":"学位授与番号","attribute_value_mlt":[{"subitem_dissertationnumber":"甲第138号"}]},"item_3_publisher_5":{"attribute_name":"公開者","attribute_value_mlt":[{"subitem_publisher":"帯広畜産大学","subitem_publisher_language":"ja"}]},"item_3_rights_14":{"attribute_name":"権利","attribute_value_mlt":[{"subitem_rights":"CHEN Xing","subitem_rights_language":"en"}]},"item_3_subject_20":{"attribute_name":"日本十進分類法","attribute_value_mlt":[{"subitem_subject":"491","subitem_subject_language":"ja","subitem_subject_scheme":"NDC"}]},"item_3_version_type_19":{"attribute_name":"著者版フラグ","attribute_value_mlt":[{"subitem_version_resource":"http://purl.org/coar/version/c_970fb48d4fbd8a85","subitem_version_type":"VoR"}]},"item_access_right":{"attribute_name":"アクセス権","attribute_value_mlt":[{"subitem_access_right":"open access","subitem_access_right_uri":"http://purl.org/coar/access_right/c_abf2"}]},"item_creator":{"attribute_name":"著者","attribute_type":"creator","attribute_value_mlt":[{"creatorNames":[{"creatorName":"CHEN, Xing","creatorNameLang":"en"},{"creatorName":"チャン, シン","creatorNameLang":"ja"}]}]},"item_files":{"attribute_name":"ファイル情報","attribute_type":"file","attribute_value_mlt":[{"accessrole":"open_access","date":[{"dateType":"Available","dateValue":"2023-10-18"}],"filename":"CHEN.pdf","filesize":[{"value":"3.8 MB"}],"format":"application/pdf","mimetype":"application/pdf","url":{"label":"全文　Full text","objectType":"fulltext","url":"https://obihiro.repo.nii.ac.jp/record/2000049/files/CHEN.pdf"},"version_id":"3e98ccd2-b425-43cf-8a2f-ddc31a82aa06"},{"accessrole":"open_access","date":[{"dateType":"Available","dateValue":"2023-10-18"}],"filename":"Abstract(CHEN).pdf","filesize":[{"value":"243 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and their developmental toxicity using zebrafish","item_titles":{"attribute_name":"タイトル","attribute_value_mlt":[{"subitem_title":"Research on the endocrine disrupting potential of environmental contaminants and their developmental toxicity using zebrafish","subitem_title_language":"en"}]},"item_type_id":"3","owner":"12","path":["8"],"pubdate":{"attribute_name":"PubDate","attribute_value":"2023-10-18"},"publish_date":"2023-10-18","publish_status":"0","recid":"2000049","relation_version_is_last":true,"title":["Research on the endocrine disrupting potential of environmental contaminants and their developmental toxicity using zebrafish"],"weko_creator_id":"12","weko_shared_id":-1},"updated":"2023-10-30T00:16:21.028699+00:00"}