Title 抗甲状腺剤を用いた一般毒性試験の枠組みでの発達神経毒性評価指標の確立に関する研究( 要約版(Digest) ) Author(s) 白木, 彩子 Report No.(Doctoral Degree) 博士(獣医学) 甲第454号 Issue Date 2016-03-14 Type 博士論文 Version none URL http://hdl.handle.net/20.500.12099/54527 ※この資料の著作権は、各資料の著者・学協会・出版社等に帰属します。
Ꮫ ㄽ ᩥ せ ⣙
Ặ ྡ ⓑ ᮌ ᙬ Ꮚ 㢟 ┠ ᢠ⏥≧⭢ࢆ⏝࠸ࡓ୍⯡ẘᛶヨ㦂ࡢᯟ⤌ࡳ࡛ࡢⓎ㐩⚄⤒ẘᛶホ౯ ᣦᶆࡢ☜❧㛵ࡍࡿ◊✲ ⫾ඣᮇ࠾ࡼࡧᑠඣᮇࡣ㸪Ꮫ≀㉁ᑐࡍࡿ᭚㟢≧ἣࡸឤཷᛶࡀ␗࡞ࡿࡇࡽ㸪㎰⸆ࡸ ୍㒊ࡢᏛ≀㉁࡛Ⓨ㐩⚄⤒ẘᛶヨ㦂ࡀᐇࡉࢀ࡚࠸ࡿࠋࡋࡋ㸪Ⓨ㐩ᮇ⚄⤒ẘᛶヨ㦂ࡢ⌧ ⾜ࡢ࢞ࢻࣛࣥ㸦OECD TG426㸪US EPA OPPTS 870.6300㸧࡛ࡣ, 㸯≀㉁ࡢホ౯ከࡃࡢ 㛫ື≀ࢆᚲせࡍࡿࡓࡵ, ࡼࡾຠ⋡ⓗ࡞ホ౯ࢩࢫࢸ࣒ࡢᵓ⠏ࡀồࡵࡽࢀ࡚࠸ࡿࠋᾏ㤿 ṑ≧ᅇࡢ㢛⢏⣽⬊ᒙୗᖏ㸦SGZ㸧࡛ࡣ㸪⏕ᚋጞࡲࡾ㸪ᡂ⇍ᚋࡶ⏕ᾭࢃࡓࡾ⥆ࡃࢽ࣮ࣗ ࣟࣥ᪂⏕ࡀ⾜ࢃࢀ࡚࠾ࡾ㸪ࡑࡢ㐣⛬࡛ࡣ⚄⤒ᖿ⣽⬊ࡢ⮬ᕫ」〇, ๓㥑⣽⬊ࡢቑṪࡸศ, ⚄ ⤒✺㉳ᙧᡂࡸࢩࢼࣉࢫᙧᡂ࡞ࡢ⌧㇟ࡀ⏕ࡌ࡚࠸ࡿࠋࡑࡢࡓࡵ㸪SGZ ࠾ࡅࡿࢽ࣮ࣗࣟࣥ ᪂⏕ࡣᵝࠎ࡞ẘᛶᶵᗎࡢ⚄⤒ẘᛶ≀㉁ࡢᶆⓗ࡞ࡿྍ⬟ᛶຍ࠼㸪ᡂ⇍ື≀ࡢⓎ㐩⚄⤒ ẘᛶ≀㉁ࡢ᭚㟢ࡼࡗ࡚ࡶ㞀ᐖࢆཷࡅࡿྍ⬟ᛶࡀ࠶ࡿࠋ࣐ࢡࣟࣞࢆ⏝࠸ࡓ㑇ఏᏊⓎ ⌧ࣉࣟࣇࣜࣥࢢࡣ, ᝈࡸᏛ≀㉁ࡢ᭚㟢ࡼࡿẘᛶⓎ⌧ࡢᶵᗎ㛵㐃ࡍࡿ⥙⨶ⓗ࡞ ሗ, ࡉࡽẘᛶホ౯㐺⏝࡛ࡁࡿ⣽⬊ᣦᶆࢆ⋓ᚓࡍࡿᡭẁ࡞ࡾ࠺ࡿࠋࡋࡋ㸪⌧≧࡛ ࡣࢽ࣮ࣗࣟࣥ᪂⏕㞀ᐖ㛵ࡋ࡚㸪Ⓨ㐩ᮇ࣭ᡂ⇍ᮇࡶ⥙⨶ⓗ࡞㑇ఏᏊⓎ⌧ゎᯒࢆྲྀࡾධ ࢀࡓ◊✲ࡀࢇ࡞ࡉࢀ࡚࠸࡞࠸ࠋࡲࡓ㸪⏥≧⭢࣍ࣝࣔࣥࡣ⬻ࡢⓎ㐩ࡸ⏕ᚋࡢᶵ⬟ᚲ せྍḞ࡛࠶ࡾ㸪ࣛࢵࢺ࠾࠸࡚㸪Ⓨ㐩ᮇ⏥≧⭢ᶵ⬟పୗࡼࡿ⬻ࡢⓎ㐩㞀ᐖࡀከᩘሗ࿌ ࡉࢀ࡚࠸ࡿࠋࡑࡇ࡛ᮏ◊✲ࡣ㸪⚄⤒Ⓨ㐩ࢆᵝࠎ࡞ᶵᗎ࡛㞀ᐖࡍࡿᢠ⏥≧⭢ࡢࣉࣟࣆࣝࢳ ࢜࢘ࣛࢩࣝ㸦6-propyl-2-thiouracil: PTU㸧ࢆ⏝࠸࡚ࣛࢵࢺⓎ㐩ᮇ᭚㟢ᐇ㦂࠾ࡼࡧ 28 ᪥㛫 ᢞᐇ㦂ࢆ⾜࠸㸪ᾏ㤿ṑ≧ᅇ࠾ࡅࡿࢽ࣮ࣗࣟࣥ᪂⏕㞀ᐖᙳ㡪ࡶ㸪ᾏ㤿ṑ≧ᅇࢆྵ ࡴ」ᩘࡢ⬻㒊࡛ࡢ㑇ఏᏊⓎ⌧ࣉࣟࣇࣝࢆẚ㍑ࡋ㸪ᡂ⇍ᮇື≀ࢆ⏝࠸ࡓ୍⯡ẘᛶヨ㦂 ࡢᯟ⤌ࡳ࡛ࡢⓎ㐩⚄⤒ẘᛶホ౯ἲࢆ☜❧ࡍࡿࡇࢆ┠ᶆࡋ࡚⥙⨶ⓗ࡞ᣦᶆ㛤Ⓨࢆ⾜ࡗࡓࠋ ➨1 ❶࡛ࡣ㸪ࣛࢵࢺᑐࡋ⫾㱋 6 ᪥┠ࡽ⏕ᚋ 21 ᪥┠ࡢ㞳ஙࡲ࡛ PTU ࡢ 0㸪1㸪3㸪 10 ppm ࡢ⃰ᗘ࡛ࡢẕື≀ࡢ㣧ỈᢞࡼࡿⓎ㐩ᮇ᭚㟢ᐇ㦂ࢆ⾜࠸㸪㞳ஙཬࡧᡂ⇍࡛ ࡢSGZ ࠾ࡅࡿࢽ࣮ࣗࣟࣥ᪂⏕࠼ࡿᙳ㡪ࢆ᳨ウࡋࡓࠋࡑࡢ⤖ᯝ㸪⏕ᚋ 21 ᪥┠ࡢ㞝Ꮚ ື≀࠾࠸࡚㸪10 ppm ⩌࡛ glial fibrillary acidic protein㸦GFAP㸧㝧ᛶࡢ type-1 ᖿ⣽⬊㸪3 ppm ௨ୖ⩌࡛paired box 6㸦PAX6㸧㝧ᛶࡢ type-1 ࡽ type-2 ๓㥑⣽⬊࠾ࡼࡧ doublecortin㸦DCX㸧 㝧ᛶࡢtype-2b ࡽᮍ⇍㢛⢏⣽⬊ࡀῶᑡࡋࡓࠋࡉࡽṑ≧ᅇ㛛࠾ࡅࡿ γ-aminobutyric acid 㸦GABA㸧ᛶᅾࢽ࣮ࣗࣟࣥ㛵ࡋ࡚㸪10 ppm ⩌࡛ somatostatin㸦SST㸧㝧ᛶ⣽⬊㸪3 ppm ௨ୖ⩌࡛reelin㸦RELN㸧࠾ࡼࡧ calbindin-D-29K㸦CALB2㸧ࡀቑຍ㸪parvalbumin㸦PVALB㸧 㝧ᛶ⣽⬊ࡀῶᑡࡋࡓࠋPAX6 㝧ᛶ⣽⬊ᩘࡢῶᑡ࠾ࡼࡧ CALB2㸪SST 㝧ᛶ⣽⬊ᩘࡢቑຍࡣ⏕ ᚋ77 ᪥┠࡛ࡶᣢ⥆ࡋ࡚࠸ࡓࠋࡇࢀࡽࡢ⤖ᯝࡼࡾ㸪PTU ࡢࣛࢵࢺⓎ㐩ᮇ᭚㟢ࡀ SGZ ࠾ࡅ ࡿศึᮇẁ㝵ࡽࡢྍ㏫ⓗ࡞ࢽ࣮ࣗࣟࣥ᪂⏕㞀ᐖࢆㄏⓎࡍࡿࡇࡀ᥎ᐹࡉࢀࡓࠋ ➨2 ❶࡛ࡣ㸪ᡂ⇍ᮇື≀ࡢᾏ㤿ṑ≧ᅇ࠾ࡅࡿࢽ࣮ࣗࣟࣥ᪂⏕㞀ᐖࡢ᳨ฟᛶࢆ᳨ウࡍࡿ ┠ⓗ࡛㸪⏕ᚋ5 㐌㱋ࡢࣛࢵࢺᑐࡍࡿ PTU ࡢ 0㸪0.1 ࠾ࡼࡧ 10 mg/kg ࡢ⤒ཱྀ⏝㔞࡛ࡢ 28 ᪥㛫ᢞࢆ⾜࠸㸪ᾏ㤿ṑ≧ᅇ࠾ࡅࡿࢽ࣮ࣗࣟࣥ᪂⏕࠼ࡿᙳ㡪ࢆ᳨ウࡋࡓࠋࡑࡢ ⤖ᯝ㸪SGZ ࠾࠸࡚ 10 mg/kg ⩌࡛ DCX 㝧ᛶ⣽⬊ᩘࡀῶᑡࡋ㸪ṑ≧ᅇ㛛࠾࠸࡚ 10 mg/kg ⩌࡛RELN 㝧ᛶ⣽⬊ᩘ㸪0.1 ࠾ࡼࡧ 10 mg/kg ⩌࡛ SST 㝧ᛶ⣽⬊ᩘࡀቑຍࡋࡓࠋࡇࢀࡽࡢ⤖ᯝࡼࡾ㸪Ⓨ㐩ᮇ᭚㟢ẚࡿᙳ㡪ࡣᑠࡉࡗࡓࡶࡢࡢ㸪ᡂ⇍ᮇື≀ࡢ᭚㟢ࡼࡗ࡚ࡶ ᾏ㤿ṑ≧ᅇ SGZ ࠾ࡅࡿࢽ࣮ࣗࣟࣥ᪂⏕ࡀ㞀ᐖࢆཷࡅ㸪㢛⢏⣽⬊⣔㆕࠾ࡼࡧ GABA ᛶ ᅾࢽ࣮ࣗࣟࣥࡢᣦᶆࢆ⏝࠸ࡓච⤌⧊ᏛᰁⰍࡼࡾ㸪㞀ᐖࢆ᳨ฟ࡛ࡁࡿྍ⬟ᛶࡀ♧ࡉࢀ ࡓࠋ ➨3 ❶࡛ࡣ㸪Ⓨ㐩⚄⤒ẘᛶࡢ⌮Ꮫⓗ࣓࢝ࢽࢬ࣒ࢆᫎࡍࡿᶆⓗ㑇ఏᏊࣉࣟࣇࣝࢆ ≉ᐃࡍࡿ┠ⓗ࡛㸪PTU ࡢⓎ㐩ᮇ᭚㟢ᐇ㦂࠾ࡼࡧ 28 ᪥㛫ᢞᐇ㦂࡛ᚓࡽࢀࡓ⬻ࢧࣥࣉ ࣝࢆ⏝࠸㸪ᵓ㐀ࡸᶵ⬟ࡢ␗࡞ࡿᾏ㤿ṑ≧ᅇ㸪⬻⓶㉁㸪⬻ᱱ㸪ᑠ⬻ࡢ4 㒊ࡘ࠸࡚㸪࣐ ࢡࣟࣞࡼࡿ⬻㒊≉␗ⓗ࡞⥙⨶ⓗ㑇ఏᏊⓎ⌧ゎᯒࢆ⾜ࡗࡓࠋ⬻㒊ࡈ㸪࠾ࡼࡧ ᭚㟢ᮇࡈࡢ㑇ఏᏊⓎ⌧ࣉࣟࣇࣝࡢẚ㍑ࡼࡾ㸪Ⓨ㐩⚄⤒ẘᛶࢆ㗦ᩄᫎࡍࡿ⬻ 㒊࠾ࡼࡧ㸪㞀ᐖᣦᶆೃ⿵㑇ఏᏊࡢ⋓ᚓࢆ┠ᣦࡋࡓࠋࡑࡢ⤖ᯝ㸪Ⓨ㐩ᮇ᭚㟢࡛ࡣ᳨⣴ࡋࡓ ࡚ࡢ⬻㒊࡛⚄⤒ࡢⓎ㐩ࡸศ㸪⣽⬊⛣ື㸪ࢩࢼࣉࢫࡢᶵ⬟࠾ࡼࡧ㍈⣴ᙧᡂᑐࡍࡿ㞀 ᐖࢆ♧၀ࡍࡿ㑇ఏᏊ⩌ࡀኚືࡋࡓࠋ≉㸪࢚ࣇࣜࣥࢩࢢࢼࣜࣥࢢ⣔ࡸGABA ᛶᅾࢽ࣮ࣗ ࣟࣥᣦᶆࡢ㑇ఏᏊࡣ」ᩘࡢ㒊࡛ඹ㏻ࡋ࡚ኚືࡋࡓࠋࡲࡓ㸪࣑࢚ࣜࣥᙧᡂ㞀ᐖࡸࢢࣜࡢ Ⓨ㐩㞀ᐖࢆ♧၀ࡍࡿ㑇ఏᏊ⩌ࡀ㸪⬻ᱱ࠾ࡼࡧ⬻⓶㉁㝈ᒁࡋ࡚ኚືࡋࡓࠋ୍᪉㸪28 ᪥㛫 ᢞ࡛ࡣ㸪࢚ࣇࣜࣥࢩࢢࢼࣜࣥࢢ⣔ࢆྵࡴ⚄⤒Ⓨ㐩㛵ࢃࡿ㑇ఏᏊ⩌ࡣᾏ㤿ṑ≧ᅇ ⬻⓶㉁㸪࣑࢚ࣜࣥᙧᡂ㛵ࢃࡿ㑇ఏᏊ⩌ࡣᾏ㤿ṑ≧ᅇ࡛Ⓨ⌧ኚືࢆ♧ࡋ㸪≉Ⓨ㐩ᮇ᭚ 㟢ඹ㏻ࡢኚື㑇ఏᏊࡣᾏ㤿ṑ≧ᅇ࡛ከࡃㄆࡵࡽࢀࡓࠋࡇࢀࡽࡢ⤖ᯝࡼࡾ㸪⏥≧⭢ᶵ⬟ప ୗࡼࡿ㞀ᐖᶆⓗೃ⿵㑇ఏᏊࡋ࡚㸪ࢽ࣮ࣗࣟࣥࡢ⛣ືࡸศ㛵ࢃࡿEph family gene ࡸ Robo3㸪GABA ᛶᅾࢽ࣮ࣗࣟࣥᣦᶆࡢ Reln㸪Pvalb ࠾ࡼࡧ Calb2㸪࣑࢚ࣜࣥᙧᡂ㛵ࢃࡿ Mbp ࠾ࡼࡧ Plp1㸪ࢢࣜⓎ㐩㛵ࢃࡿ Vim㸪Gfap ࠾ࡼࡧ Ret ࢆ⋓ᚓࡋࡓࠋࡲࡓ㸪28 ᪥㛫 ᢞヨ㦂ࡢᯟ⤌ࡳ࡛㑇ఏᏊⓎ⌧ࡼࡾⓎ㐩⚄⤒ẘᛶࢆホ౯ࡍࡿୖ࡛ࡣ㸪ᾏ㤿ṑ≧ᅇࡢឤ ཷᛶࡀ㧗࠸ࡇࡀ♧၀ࡉࢀࡓࠋ ➨ 4 ❶࡛ࡣ㸪➨ 3 ❶࡛ᚓࡽࢀࡓⓎ㐩⚄⤒ẘᛶࢆᫎࡍࡿ㑇ఏᏊⓎ⌧ࣉࣟࣇࣝࡽ㸪 ච⤌⧊Ꮫⓗゎᯒࡼࡾ⡆౽ホ౯ྍ⬟࡞㞀ᐖᣦᶆศᏊࡢ᥈⣴ࢆ⾜ࡗࡓࠋࡑࡢ⤖ᯝ㸪Ⓨ 㐩ᮇ᭚㟢ᐇ㦂ࡢ㞝Ꮚື≀࠾࠸࡚㸪ࢩࢼࣉࢫࡢྍረᛶ㛵ࢃࡿศᏊࡋ࡚ࡣ㸪3 ppm ௨ୖ ⩌࡛ᾏ㤿ṑ≧ᅇࡢcyclooxygenase-2 ࠾ࡼࡧ Eph receptor A4㸦EPHA4㸧㝧ᛶ㢛⢏⣽⬊ᩘ㸪10 ppm ⩌࡛activity-regulated cytoskeleton-associated protein㸦ARC㸧㝧ᛶ㢛⢏⣽⬊ᩘࡀῶᑡࡋࡓࠋ⏕ ᚋ77 ᪥㱋࡛ࡣ㸪EPHA4 ࠾ࡼࡧ ARC 㝧ᛶ㢛⢏⣽⬊ᩘࡣቑຍࡋࡓࠋEPHA4 㝧ᛶࢽ࣮ࣗࣟࣥ ࡣ㸪⏕ᚋ21 ᪥┠࠾ࡼࡧ 77 ᪥┠ࡢ⬻⓶㉁࡛ࡶ 3 ppm ௨ୖ⩌࡛ῶᑡࡋࡓࠋࡲࡓ㸪GABA ᛶ ᅾࢽ࣮ࣗࣟࣥᣦᶆࡘ࠸࡚㸪⏕ᚋ21 ᪥┠ࡢ⬻⓶㉁࡛㸪10 ppm ⩌࡛ RELN ࠾ࡼࡧ CALB2 㝧ᛶ⣽⬊ᩘࡀቑຍࡋ㸪3 ppm ௨ୖ⩌࡛ PVALB ࠾ࡼࡧ NPY 㝧ᛶ⣽⬊ᩘࡀῶᑡࡋࡓࠋᑠ⬻ ࠾࠸࡚ࡶ㸪10 ppm ⩌࡛ RELN ࠾ࡼࡧ SST 㝧ᛶ⣽⬊ᩘࡀቑຍࡋ㸪3 ppm ௨ୖ⩌࡛ PVALB 㝧 ᛶ⣽⬊ᩘࡀῶᑡࡋࡓࠋࡇࢀࡽࡢኚືࡢከࡃࡣ⏕ᚋ77 ᪥┠࡛ࡶᣢ⥆ࡋ࡚࠸ࡓࠋ࣑࢚ࣜࣥᙧᡂ 㛵㐃ศᏊࡋ࡚ࡣ㸪⏕ᚋ21 ᪥┠ࡢ 3 ppm ௨ୖ⩌࡛㸪⬻ᱱ࠾ࡼࡧ⬻⓶㉁࠾ࡅࡿ myelin basic protein ࡢᰁⰍᛶࡀపୗࡋࡓࠋࢢࣜⓎ㐩㛵㐃ศᏊࡋ࡚ࡣ㸪⏕ᚋ 21 ᪥┠ࡢ⬻ᱱ࠾ ࠸࡚㸪1 ppm ௨ୖ⩌࡛ vimentin㸪10 ppm ⩌࡛ GFAP 㝧ᛶࡢᮍ⇍࡞ࢫࢺࣟࢧࢺࡀቑຍࡋ㸪 1 ppm ௨ୖ⩌࡛ oligodendrocyte transcription factor 2 㝧ᛶࢢࣜ⣽⬊ࡀῶᑡࡋࡓࠋࡋࡋ㸪28 ᪥㛫ᢞᐇ㦂࠾࠸࡚ࡣ㸪ࡇࢀࡽࡢศᏊࡣ࠸ࡎࢀࡶචᰁⰍࡼࡿᕪࡣぢฟࡏ࡞ࡗ ࡓࠋࡇࢀࡽࡢ⤖ᯝࡼࡾ㸪Ⓨ㐩ᮇ᭚㟢ᐇ㦂࡛ࡣ㸪ࢩࢼࣉࢫྍረᛶ㸪GABA ᛶᅾࢽ࣮ࣗࣟࣥ㸪 ࣑࢚ࣜࣥᙧᡂ࠾ࡼࡧࢢࣜⓎ㐩ᑐࡍࡿ㞀ᐖᛶࡘ࠸࡚㸪ච⤌⧊Ꮫⓗⓗゎᯒࡼࡾホ ౯ྍ⬟࡞ศᏊࢆぢฟࡋࡓࡶࡢࡢ㸪ᾏ㤿ṑ≧ᅇࡢࢽ࣮ࣗࣟࣥ᪂⏕㞀ᐖ㛵㐃ࡋࡓGABA ᛶ ᅾࢽ࣮ࣗࣟࣥࡢኚື௨እ㸪ᡂ⇍ື≀ࡢ᭚㟢ࡼࡿ㞀ᐖᛶࢆホ౯ࡍࡿୖ࡛ࡣ᭷⏝࡛࡞࠸ ⪃࠼ࡽࢀࡓࠋ ௨ୖࡼࡾ㸪୍⯡ẘᛶヨ㦂ࡢᯟ⤌ࡳ࡛Ⓨ㐩⚄⤒ẘᛶࢆホ౯ࡍࡿ࠶ࡓࡗ࡚ࡣ㸪ᾏ㤿ṑ≧ᅇ ࡀホ౯㒊ࡋ࡚㧗ឤཷᛶ࡛࠶ࡿࡇࡀ⥙⨶ⓗ࡞㑇ఏᏊⓎ⌧ゎᯒࡽ♧၀ࡉࢀࡓࠋࡉࡽ㸪 SGZ ࠾ࡅࡿ㢛⢏⣽⬊⣔㆕࠾ࡼࡧṑ≧ᅇ㛛࠾ࡅࡿ GABA ᛶᅾࢽ࣮ࣗࣟࣥᣦᶆࡢච
⤌⧊Ꮫⓗゎᯒࡼࡾ㸪ࢽ࣮ࣗࣟࣥ᪂⏕ᑐࡍࡿᙳ㡪ࡀホ౯ྍ⬟࡛࠶ࡿࡇࡀ♧၀ࡉࢀࡓࠋ ࡲࡓ㸪Ⓨ㐩⚄⤒ẘᛶᐇ㦂ࡼࡗ࡚㸪⥙⨶ⓗ㑇ఏᏊⓎ⌧ゎᯒࡼࡾࢩࢼࣉࢫྍረᛶ㸪࣑࢚ࣜࣥ ᙧᡂ࠾ࡼࡧࢢࣜⓎ㐩㞀ᐖࡢᣦᶆೃ⿵ศᏊࡀᚓࡽࢀ㸪ࡇࢀࡽࡢศᏊࡢච⤌⧊Ꮫⓗホ౯ ࡀ⌧⾜ࡢヨ㦂ࡢ⢭ᗘྥୖ㈉⊩࡛ࡁࡿࡇࡀᮇᚅࡉࢀࡿࠋࡉࡽ㸪GABA ᛶᅾࢽ࣮ࣗࣟ ࣥᣦᶆࡀ」ᩘࡢ⬻㒊࡛Ⓨ㐩⚄⤒ẘᛶ≀㉁ࡢྍ㏫ⓗ࡞ᙳ㡪ࢆホ౯ࡍࡿୖ࡛᭷⏝࡛࠶ࡿࡇ ࡀ♧၀ࡉࢀࡓࠋ
Ꮫ ㄽ ᩥ せ ⣙
Ặ ྡ SHIRAKI, Ayako
㢟 ┠ Studies on the Development of Developmental Neurotoxicity Markers in the Framework of General Toxicity Study Using Anti-thyroid
Agents
㸦ᢠ⏥≧⭢ࢆ⏝࠸ࡓ୍⯡ẘᛶヨ㦂ࡢᯟ⤌ࡳ࡛ࡢⓎ㐩⚄⤒ẘᛶ ᣦᶆࡢ☜❧㛵ࡍࡿ◊✲㸧
Developmental neurotoxicity testing is a field that is in need of a rapid screening system because testing one chemical with the current guidelines, i.e., OECD Test Guideline 426 and US EPA Health Effects Test Guidelines OPPTS 870.6300, is time consuming and requires hundreds of animals to conduct one study. Therefore, more efficient screening system is necessary to be established. In the subgranular zone (SGZ) of the hippocampal dentate gyrus, adult neurogenesis of granular cells starts postnatally and continues throughout life. The process of the hippocampal neurogenesis consists of self-renewal of stem cells and production of progenitor cells, proliferation and differentiation of progenitor cells, and maturation involving neuritogenesis and synaptogenesis of granule cell lineages. Therefore, hippocampal neurogenesis is a possible target of many neurotoxicants targeting these processes by both developmental and adult-stage exposure. On the other hand, gene expression profiling using microarrays provides global view of tissue-specific changes on the mechanisms underlying diseases or toxicity following chemical exposure and also offers opportunity to obtain new cellular markers applicable for toxicity testing. However, there have been only a few studies dealing with neurotoxic changes in the adult neurogenesis employing global gene expression profiling tool at both developmental and adult stages.
Thyroid hormones are crucial for both normal brain development and adult brain function. In rat developmental hypothyroidism model, aberrant brain growth and impairment in brain structures and functions involving both neuronal and glial differentiation have been reported. The present study was undertaken to establish an efficient system to detect developmental neurotoxicity in the framework of a 28-day regular toxicity study using rat hypothyroidism models. To this end, I used propylthiouracil (PTU), an anti-thyroid agent, to induce hypothyroidism and compared the pattern in the aberration in neurogenesis in the hippocampal SGZ and the gene expression profiles in multiple brain regions including the dentate gyrus between the developmental and adult-stage hypothyroidism.
In chapter 1, I investigated the effect of developmental hypothyroidism on the hippocampal neurogenesis at the weaning and adult stage. Pregnant rats were given drinking water containing PTU at 0 (control), 1, 3, and 10 ppm from gestational day 6 until weaning on postnatal day (PND) 21. For examination of developmental neurotoxicity, I analyzed the distribution of granule cell lineages in the SGZ and γ-aminobutyric acid (GABA) ergic interneurons in the hilus of the hippocampal dentate gyrus in offspring on PND 21 and PND 77. On PND 21, there were fewer glial fibrillary acidic protein (GFAP)+ type-1 stem cells, paired box 6 (PAX6)+ type-1 to type-2 progenitor cells, and doublecortin (DCX)+ type-2b to immature granule cells in the SGZ at ≥ 3 or 10 ppm. Regarding GABAergic interneuron subpopulations, there were more reelin (RELN)+ cells
and calbindin-D-29K (CALB2)+ cells at ≥ 3 ppm and somatostatin (SST)+ cells at 10 ppm, and fewer parvalbumin (PVALB)+ cells at ≥ 3 ppm in the dentate hilus of offspring. Decrease in number of PAX6+ cells in the SGZ and increase in number of CALB2+ and SST+ cells were sustained until PND 77. These results suggested that developmental exposure to PTU induces irreversible impairment in hippocampal neurogenesis from the early stage of granule cell lineage.
In chapter 2, I investigated the effect of adult-stage hypothyroidism on the hippocampal neurogenesis in the framework of 28-day repeated oral dosing study. For this purpose, PTU was orally administrated to 5-week-old male rats at 0, 0.1 and 10 mg/kg by gavage for 28 days. The distribution of granule cell lineages in the SGZ and GABAergic interneurons in the hilus of the hippocampal dentate gyrus were analyzed. As a result, there were fewer DCX+ cells in the SGZ, but more RELN+ and SST+ cells in the dentate hilus at 10 mg/kg. These results suggested that hippocampal neurogenesis is sensitive to adult-onset hypothyroidism and may be useful for detection of developmental neurotoxicity in the framework of 28-day repeated dosing study.
In chapter 3, brain region-specific global gene expression profiling was performed to obtain target gene profile related to pathological mechanisms of developmental neurotoxicity. Brain samples were those obtained in chapters 1 and 2, and four brain regions were selected to cover cerebral and cerebellar tissues, i.e., the parietal cortex (cerebral cortex), corpus callosum, hippocampal dentate gyrus and cerebellar vermis. As a result, developmental PTU exposure caused gene expression alterations related to neuronal development, cell migration, synaptic function and axonogenesis in all brain regions examined. Characteristically, gene expression profiles suggestive of affection of ephrin signaling and glutamate transmission were obtained in multiple brain regions. Gene clusters suggestive of suppression of myelination and glial development were specifically detected in the corpus callosum and cerebral cortex. On the other hand, adult-stage PTU exposure caused expression alterations in genes related to neural development in the cerebral cortex and hippocampal dentate gyrus, and in genes related to myelination in the hippocampal dentate gyrus. In the corpus callosum and cerebellar vermis, only a few gene populations in these gene clusters showed expression changes. From these results, genes related to neuronal development involving
Eph family genes, Robo3 and GABAergic interneuron markers, glial cell differentiation involving Vim, Gfap and Ret, and myelination involving Mbp and Plp1, were obtained as possible targets of
hypothyroidism-induced developmental neurotoxicity. In addition, in the framework of 28-day repeated dosing study, hippocampal dentate gyrus was suggested to show high sensitivity to detect developmental neurotoxicity.
In chapter 4, I searched the sensitive immunohistochemical parameters of developmental neurotoxicity following developmental PTU-exposure, as well as those in the 28-day toxicity study of adult animals. Candidate target molecules were selected from gene expression profiles obtained in chapter 3. Regarding molecules related to synaptic plasticity, decrease in number of cyclooxygenase-2+ cells and Eph receptor A4 (EPHA4)+ cells at ≥ 3 ppm and activity-regulated cytoskeleton-associated protein (ARC)+ cells at 10 ppm were observed in the hippocampal dentate gyrus of offspring on PND 21. On PND 77, number of EPHA4+ cells and ARC+ cells were increased at ≥ 3 ppm and 10 ppm, respectively. Number of EPHA4+ cells was also decreased in the cerebral cortex of offspring on PND 21 and PND 77 at ≥ 3 ppm. Regarding GABAergic interneuron markers in developmental exposure study, increase in number of RELN+ cells and CALB2+ cells at 10 ppm and decrease in number of PVALB+ cells and NPY+ cells at ≥ 3 ppm were observed in the cerebral cortex of offspring on PND 21. Increase in number of RELN+ cells and SST+ cells at 10 ppm and decrease in number of PVALB+ cells at ≥ 3 ppm were also observed in the cerebellum. Most of these distribution changes of GABAergic interneuron subpopulations
observed in the cerebral cortex and cerebellum were sustained on PND 77. Regarding molecules related to myelination, immunoreactive intensity of myelin basic protein was decreased in the corpus callosum and cerebral cortex of offspring on PND 21 at ≥ 3 ppm. Regarding molecules related to glial cell development, immature astrocytes immunoreactive for vimentin at ≥ 1 ppm and GFAP at 10 ppm were increased, and oligodendrocytes immunoreactive for oligodendrocyte lineage transcription factor 2 at ≥ 1 ppm were decreased in the corpus callosum of offspring on PND 21. On the other hand, distributions of these molecules were not changed in animals administrated PTU at adult-stage. In summary, sensitive immunohistochemical parameters suggestive of aberration in synaptic plasticity, GABAergic interneuron subpopulations, myelinations and glial cell development were obtained by developmental PTU exposure study, while these parameters were not sensitive for detecting developmental neurotoxicity in adult-stage exposure study, except for GABAergic interneuron subpopulations in association with hippocampal neurogenesis.
In the present study, results of global gene expression profiling after adult-stage PTU exposure study suggested the hippocampal dentate gyrus to be the most sensitive region for evaluation of developmental neurotoxicity in the framework of general toxicity study. In addition, immunohistochemistry against granule cell lineage at the SGZ and GABAergic interneuron markers in the hilus of hippocampal dentate gyrus were useful for evaluation of effects of developmental neurotoxicants on hippocampal neurogenesis after adult-stage exposure. On the other hand, in the developmental PTU exposure study, global gene expression profiling revealed candidate target molecules, which reflect affection of synaptic plasticity, myelination and glial cell development. Immunohistochemistry of these molecules may contribute to improve sensitivity of current developmental neurotoxicity testing. In addition, immunohistochemical analysis of GABAergic interneuron markers was suggested to be sensitive to evaluate irreversible effect of developmental neurotoxicants in the multiple brain regions.
