プロトポルフィリノーゲン酸化酵素阻害剤が誘発するげっ歯類肝発がん機序における Constitutive androstane receptor の関与とヒトへの外挿性に関する研究
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(2) ࣉࣟࢺ࣏ࣝࣇࣜࣀ࣮ࢤࣥ㓟㓝⣲㜼ᐖࡀ ㄏⓎࡍࡿࡆࡗṑ㢮⫢Ⓨࡀࢇᶵᗎ࠾ࡅࡿ Constitutive androstane receptor ࡢ㛵 ࣄࢺࡢእᤄᛶ㛵ࡍࡿ◊✲. 2017 ᖺ. ᒱ㜧ᏛᏛ㝔㐃ྜ⋇་Ꮫ◊✲⛉. ᱓⏣ 1.
(3) ࠙┠ḟࠚ ᗎ ㄽ ........................................................................................................................................... 4 ➨ 1 ❶. ࢜࢟ࢧࢪࢰࣥࡀㄏⓎࡍࡿ࣐࢘ࢫ⫢Ⓨࡀࢇ࠾ࡅࡿ Constitutive androstane. receptor ࡢ㛵 ............................................................................................................................. 8 ⥴ ゝ ....................................................................................................................................... 9 ᮦᩱ࠾ࡼࡧ᪉ἲ ..................................................................................................................... 11 ⤖ ᯝ ..................................................................................................................................... 16 ⪃ ᐹ ..................................................................................................................................... 21 ᑠ ᣓ ..................................................................................................................................... 24. ➨ 2 ❶ ࢩࣇࣝ࢜ࣝࣇ࢙ࣥࡀㄏⓎࡍࡿ࣐࢘ࢫ⫢Ⓨࡀࢇ࠾ࡅࡿ Constitutive androstane receptor ࡢ㛵 .......................................................................................................................... 25 ⥴ ゝ ..................................................................................................................................... 26 ᮦᩱ࠾ࡼࡧ᪉ἲ ..................................................................................................................... 27 ⤖ ᯝ ..................................................................................................................................... 32 ⪃ ᐹ ..................................................................................................................................... 37 ᑠ ᣓ ..................................................................................................................................... 40. ➨ 3 ❶. ࣉࣟࢺ࣏ࣝࣇࣜࣀ࣮ࢤࣥ㓟㓝⣲㜼ᐖࡀㄏⓎࡍࡿ࣐࢘ࢫ⫢Ⓨࡀࢇࡢ ึ. ᮇẁ㝵࠾ࡅࡿ ȕ-catenin ኚ␗ࡢ㛵 .................................................................................... 41 ⥴ ゝ ..................................................................................................................................... 42 ᮦᩱ࠾ࡼࡧ᪉ἲ ..................................................................................................................... 44 ⤖ ᯝ ..................................................................................................................................... 47 ⪃ ᐹ ..................................................................................................................................... 50. 2.
(4) ᑠ ᣓ ..................................................................................................................................... 53. ⥲ྜ⪃ᐹ ..................................................................................................................................... 54 ⤖ ㄽ ......................................................................................................................................... 57 ㅰ ㎡ ......................................................................................................................................... 58. ᘬ⏝ᩥ⊩ ..................................................................................................................................... 59 せ ᪨ ......................................................................................................................................... 69 Abstract ....................................................................................................................................... 72 ᅗ ⾲ ......................................................................................................................................... 75. 3.
(5) ᗎ ㄽ ࣏ࣝࣇࣜࣥࡣ㸪࣒࣊ࡢྜᡂ㛵ࡍࡿ㓝⣲ࡢάᛶపୗࡀཎᅉ࡞ࡾ✀ࠎࡢែࢆ⏕ ࡌࡿᝈ࡛࠶ࡿ㸬࣒࣊ࡢྜᡂ⤒㊰ࡣ 8 ࡘࡢ㓝⣲ࡀᏑᅾࡋ㸪࠸ࡎࢀࡢ㓝⣲ᶵ⬟ࢆḞᦆࡋ ࡓࣄࢺ࡛ࡣ࣏ࣝࣇࣜࣥࢆⓎࡍࡿࡇࡀ▱ࡽࢀ࡚࠸ࡿࠋࡇࢀࡽ㓝⣲ࡢ୍ࡘ㸪ࣉࣟࢺ࣏ ࣝࣇࣜࣀ࣮ࢤࣥ㓟㓝⣲㸦PROTOX㸧ࡣ࣑ࢺࢥࣥࢻࣜෆ࡛ࣉࣟࢺ࣏ࣝࣇࣜࣀ࣮ࢤࣥࢆ ࣉࣟࢺ࣏ࣝࣇࣜࣥ IX㸦PPIX㸧㓟ࡉࡏࡿࠋࣄࢺࡢ࣏ࣝࣇࣜࣥ࠾࠸࡚㸪PROTOX ᶵ⬟ࡢḞᦆࢆ≉ᚩࡍࡿࡶࡢࡣ␗ᆺ࣏ࣝࣇࣜࣥࡤࢀࡿࠋPROTOX ᶵ⬟ࡀḞᦆࡍࡿ ࡇ࡛㸪⫢⮚୰ࡢࣉࣟࢺ࣏ࣝࣇࣜࣀ࣮ࢤࣥࡣ࣑ࢺࢥࣥࢻࣜࡽ⣽⬊㉁⛣⾜ࡋ㸪㓟 ࡉࢀ࡚ PPIX ࡞ࡿࠋ⏕ᡂࡉࢀࡓ PPIX ࡣ⫢⮚✚ࡋࡓᚋ㸪⫹Ồ࠾ࡼࡧ⣅୰ἥࡉࢀࡿ 㸦55㸧 ࠋ␗ᆺ࣏ࣝࣇࣜࣥࡢᝈ⪅࡛ࡣ㸪៏ᛶⓗ࡞⫢ᶵ⬟ࡢ␗ᖖࡸ㍍ᚤ࡞⫢ᶵ⬟㓝⣲ࡢቑຍ ࡀㄆࡵࡽࢀ㸪⫢Ⓨࡀࢇࣜࢫࢡࡀ㧗࠸ࡇࡀ▱ࡽࢀ࡚࠸ࡿ㸦53㸪55㸧 ࠋ ࡇࡢࡼ࠺࡞ PROTOX ࡼࡿ௦ㅰࡢ≉␗ⓗ࡞㜼ᐖࡣඛኳⓗ࡞ᝈࡢࡳ࡞ࡽࡎ㸪Ꮫ≀㉁ ࡼࡗ࡚ࡶㄏⓎࡉࢀࡿࡇࡀ▱ࡽࢀ࡚࠸ࡿࠋ࢜࢟ࢧࢪࢰࣥ㸦OX㸧࡞ࡢࢲࢪࣥ⣔㝖ⲡ ࡸࢩࣇࣝ࢜ࣝࣇ࢙ࣥ㸦ACI㸧࡞ࡢࢪࣇ࢙ࢽ࢚࣮ࣝࢸࣝ⣔㝖ⲡࡣ PROTOX 㜼ᐖ ࡤࢀ࡚࠾ࡾ㸪ᮏ᮶ࡢ㎰⸆ࡢస⏝ࡋ࡚᳜≀୰ࡢ PROTOX ࢆ㜼ᐖࡍࡿࡇ࡛㸪PPIX ࢆ✚ ࡉࡏࡿࠋ✚ࡋࡓ PPIX ࡣගᛂࡋ࡚άᛶ㓟⣲ࢆⓎ⏕ࡉࡏ㸪᳜≀ࢆᯤṚࡉࡏࡿ㸦13㸪22㸪 41㸧ࠋ PROTOX 㜼ᐖࡣ᳜≀ࡔࡅ࡛࡞ࡃࡆࡗṑ㢮ࡢ PROTOX ࢆ㜼ᐖࡍࡿࡇ࡛㸪 ⫢⮚୰ PPIX ࢆ✚ࡉࡏࡿ࡞␗ᆺ࣏ࣝࣇࣜࣥఝࡓែࢆㄏⓎࡍࡿ㸦29㸪41㸧 ࠋࡉࡽ㸪PROTOX 㜼ᐖࡋ࡚௦⾲ⓗ࡞ྜ≀࡛࠶ࡿ OX ACI ࡣ㑇ఏẘᛶࢆ♧ࡉ࡞࠸ࡀࠊࣛࢵࢺ࠾ࡼࡧ࣐ ࢘ࢫࢆ⏝࠸ࡓ៏ᛶẘᛶ㸭Ⓨࡀࢇᛶ」ྜヨ㦂࠾࠸࡚⫢⮚ࡢ⣽⬊യᐖ࠾ࡼࡧ⫢⭘⒆ࢆច㉳ࡍ ࡿࡇࡀࣜࢫࢡホ౯ᶵ㛵ࡢሗ࿌᭩グ㍕ࡉࢀ࡚࠸ࡿ㸦㎰⸆ホ౯᭩: http://www.fsc.go.jp/fsciis/evaluationDocument/show/kya20080111001㸹EPA Reregistration Eligibility Decision for Sodium Acifluorfen. Case No. 2605:. 4.
(6) http://archive.epa.gov/pesticides/reregistration/web/pdf/acifluorfen_red.pdf㸧 ࠋ ㏆ᖺࠊᏛ≀㉁ࡢẘᛶホ౯࠾ࡅࡿࣄࢺࡢእᤄᛶࡢ᭷↓ࡢ㔜せᛶࡽࠊࡆࡗṑ㢮⫢Ⓨ ࡀࢇࡢࣄࢺࡢእᤄᛶࡀẘᛶᏛⓗ◊✲ࡉࢀ࡚ࡁࡓࠋPeroxisome proliferator-activated receptor Į㸦33$5Į㸧ࡸ constitutive androstane/active receptor㸦CAR㸧࡞ࡢ᰾ෆཷᐜయࡢάᛶࡼ ࡿ⫢Ⓨࡀࢇࡀࣄࢺࡢእᤄᛶࡀ࡞࠸⪃࠼ࡽࢀ࡚࠸ࡿ୍᪉ࠊ⣽⬊യᐖࡀ㛵ࡍࡿ⤒㊰ࡣࣄ ࢺእᤄྍ⬟࡞స⏝ᶵᗎ⪃࠼ࡽࢀ࡚࠸ࡿ㸦20㸧 ࠋࡆࡗṑ㢮࠾ࡅࡿ࣏ࣝࣇࣜࣥࡣ㸪⫢ ⮚ࡢ⣽⬊യᐖࢆㄏⓎࡍࡿࡇ࡛⫢ⓎࡀࢇࢆㄏⓎࡍࡿ⪃࠼ࡽࢀ࡚ࡁࡓࡇࡽ㸪PROTOX 㜼ᐖࡀㄏⓎࡍࡿ⫢Ⓨࡀࢇᶵᗎࡣࣄࢺእᤄࡉࢀࡿࡶࡢࡋ࡚ศ㢮ࡉࢀࡁࡓ㸦58㸧 ࠋ ୍᪉࡛㸪OX ࡸ ACI ࢆጞࡵࡍࡿ PROTOX 㜼ᐖࡣ 33$5Į άᛶస⏝ࢆ᭷ࡍࡿࡇࡀ▱ ࡽࢀ࡚࠸ࡿ㸦51㸪61㸧 ࠋ33$5Į ࡣ᰾ෆཷᐜయࡢ 1 ✀࡛࠶ࡾ㸪33$5Į ࢦࢽࢫࢺࡣࡆࡗṑ㢮 ⫢⭘⒆ࢆច㉳ࡍࡿࡇࡀ▱ࡽࢀ࡚࠸ࡿ㸦5㸪26㸧 ࠋࡋࡓࡀࡗ࡚㸪PROTOX 㜼ᐖࡀㄏⓎࡍࡿ ࡆࡗṑ㢮⫢Ⓨࡀࢇࡣ 33$5Į ࡶ㛵ࡋ࡚࠸ࡿྍ⬟ᛶࡀ࠶ࡿࠋࣄࢺ࠾ࡅࡿ 33$5Į άᛶࡣ ⫢⣽⬊ቑṪࢆㄏⓎࡋ࡞࠸ࡇࡽ㸪33$5Į ࢦࢽࢫࢺࡀㄏⓎࡍࡿࡆࡗṑ㢮⫢Ⓨࡀࢇࡣࣄࢺ እᤄࡉࢀ࡞࠸⪃࠼ࡽࢀ࡚࠸ࡿ㸦33㸧 ࠋ 33$5Į ୪ࡧ㸪 CAR ࡣࡆࡗṑ㢮⫢Ⓨࡀࢇ㛵ࡍࡿ᰾ෆཷᐜయࡋ࡚ᗈࡃ▱ࡽࢀ࡚࠾ࡾ㸪 CAR άᛶ࡛࠶ࡿࣇ࢙ࣀࣂࣝࣅࢱ࣮ࣝ㸦PB㸧ࡣ CAR ࢆࡋ࡚ࡆࡗṑ㢮⫢⭘⒆ࢆច㉳ ࡍࡿࡇࡀ▱ࡽࢀ࡚࠸ࡿ㸦69㸧 ࠋPB ࡢ㸪ࣆ࣌ࣟࢽࣝࣈࢺ࢟ࢩࢻࡸࢺࣜࢰ࣮ࣝẅ⳦࡞ ከࡃࡢᏛ≀㉁ᢞࡀㄏⓎࡍࡿࡆࡗṑ㢮⫢Ⓨࡀࢇ㐣⛬࠾࠸࡚㸪CAR ࡀ㔜せ࡞ാࡁࢆࡋ ࡚࠸ࡿࡇࡀሗ࿌ࡉࢀ࡚࠸ࡿ㸦52㸪63㸧 ࠋ▩┪ࡋࡓሗ࿌ࡣ࠶ࡿࡶࡢࡢ㸦8㸧 㸪ࣄࢺ CAR ࣐࢘ ࢫ࡛ࡣ PB ࡼࡿ⫢⣽⬊ቑṪࡀㄆࡵࡽࢀࡎ㸪CAR ࢆࡋࡓࡆࡗṑ㢮⫢Ⓨࡀࢇࡣࣄࢺእᤄ ࡉࢀ࡞࠸⪃࠼ࡽࢀ࡚࠸ࡿ㸦17㸧ࠋࡑࡢࡓࡵ㸪ྜ≀ㄏⓎ⫢Ⓨࡀࢇ࠾ࡅࡿ CAR ࡢ㛵ࡣ ࣄࢺ⫢Ⓨࡀࢇࣜࢫࢡࢆホ౯ࡍࡿୖ࡛㔜せ࡞స⏝ᶵᗎ࡛࠶ࡿࠋ CAR ࢆࡋࡓࡆࡗṑ㢮⫢Ⓨࡀࢇࡢヲ⣽࡞ศᏊ⏕≀Ꮫⓗᶵᗎࡣ᫂ࡔࡀ㸪PB ࡀㄏⓎࡍࡿ⫢ Ⓨࡀࢇࡣ⫢⮚ࡢ ȕ-catenin ࢆࣀࢵࢡ࢘ࢺ㸦KO㸧ࡉࡏࡓ࣐࢘ࢫ࡛ᢚไࡉࢀࡿࡓࡵ㸪CAR ࢆ ࡋࡓࡆࡗṑ㢮⫢Ⓨࡀࢇࡣ ȕ-catenin ࡀᙉࡃ㛵ࢃࡿࡇࡀ♧၀ࡉࢀ࡚࠸ࡿ㸦57㸧 ࠋ 5.
(7) Diethylnitrosamine ࡼࡿࢽࢩ࢚࣮ࢩࣙࣥฎ⌮ᚋ㸪PB ࢆᢞࡋࡓ࣐࢘ࢫ࡛ㄆࡵࡽࢀࡓ⫢⭘ ⒆ࡢ࠺ࡕ㸪80%௨ୖ࡛ ȕ-catenin 㑇ఏᏊࡢኚ␗ࡀሗ࿌ࡉࢀ࡚࠸ࡿ㸦36㸧 ࠋȕ-catenin ࡢኚ␗ࡣ᰾ ࠾ࡅࡿ ȕ-catenin ⺮ⓑࡢ㞟✚ࢆច㉳ࡋ㸪ȕ-catenin ࡢᶆⓗศᏊ࡛࠶ࡿࢢࣝࢱ࣑ࣥྜᡂ㓝⣲ 㸦GS㸧ࡸ Axin2 ࡢⓎ⌧ࢆቑຍࡉࡏࡿ㸦44㸧 ࠋPB ࡼࡿㄏⓎࡉࢀࡓ࣐࢘ࢫ⫢⣽⬊⭢⭘࠾ࡼࡧ ⫢⣽⬊⒴࡛ࡣ㸪 ȕ-catenin ࡢኚ␗ࢆ♧ࡍ GS ࡢⓎ⌧ࢆ㢖⦾ㄆࡵࡿࡇࡀሗ࿌ࡉࢀ࡚࠸ࡿ 㸦14㸪 56㸧ࠋ ࡇࡢࡼ࠺Ꮫ≀㉁ࡼࡿࡆࡗṑ㢮ࡢ⫢⭘⒆Ⓨ⏕ CAR ࡢ㛵㐃ᛶࡀ᫂ࡽ࡞ࡾࡘࡘ ࠶ࡿࡀ㸪PROTOX 㜼ᐖࡼࡿ CAR ࡢᙳ㡪ࢆ᳨⣴ࡋࡓሗ࿌ࡣஈࡋ࠸ࠋ୍᪉࡛㸪ࢪࣇ࢙ࢽࣝ ࢚࣮ࢸࣝ⣔ࡢ PROTOX 㜼ᐖ࡛࠶ࡿ࢜࢟ࢩࣇࣝ࢜ࣝࣇ࢙ࣥࡣ㸪ࡆࡗṑ㢮 CAR ࢆάᛶࡉ ࡏࡿࡇࡀሗ࿌ࡉࢀ࡚࠾ࡾ㸦59㸧㸪PROTOX 㜼ᐖࡀㄏⓎࡍࡿࡆࡗṑ㢮⫢Ⓨࡀࢇࡣ CAR ࡀ㛵ࡋ࡚࠸ࡿྍ⬟ᛶࡶ⪃࠼ࡽࢀࡿࠋ ሗࡣ㝈ࡽࢀ࡚࠸ࡿࡶࡢࡢ๓㏙ࡢሗ࿌ࢆేࡏࡿ㸪PROTOX 㜼ᐖࡼࡿࡆࡗṑ㢮⫢Ⓨࡀ ࢇᶵᗎࡣ㸪ᚑ᮶ணࡉࢀ࡚ࡁࡓ࣏ࣝࣇࣜࣥ✚ࡼࡿ⣽⬊യᐖࡔࡅ࡛࡞ࡃ㸪᰾ෆᤵ య➼㸪」ᩘࡢస⏝ᶵᗎࡀᏑᅾࡍࡿࡇࡀᐃࡉࢀࡿࠋࡋࡋ࡞ࡀࡽ㸪ࡑࡢヲ⣽࡞⫢Ⓨࡀࢇ ᶵᗎࡑࡢࣄࢺࡢእᤄᛶࡣ᫂ࡽ࡞ࡗ࡚࠸࡞࠸ࠋPROTOX 㜼ᐖࡣ⌧ᅾࡶୡ⏺ⓗ ⏝ࡉࢀ࡚࠸ࡿ㎰⸆࡛࠶ࡾ㸪⫢Ⓨࡀࢇᶵᗎࢆ᫂ࡽࡋࣄࢺࡢእᤄᛶࢆ⪃ᐹࡍࡿࡇࡣࠊࣄ ࢺࡢᗣᙳ㡪ࢆ⪃࠼ࡿୖ࡛ᴟࡵ࡚㔜せ࡛࠶ࡿࠋࡑࡇ࡛ᮏ◊✲࡛ࡣ㸪ࣄࢺࡢእᤄᛶࢆ⪃ ࠼ࡿୖ࡛㔜せ࡞ CAR ╔┠ࡋ࡚㸪PROTOX 㜼ᐖ࡛࠶ࡿ OX ࠾ࡼࡧ ACI ࡀㄏⓎࡍࡿ⫢Ⓨࡀ ࢇࡢᶵᗎࢆ᳨⣴ࡋࡓࠋ ➨ 1 ❶࡛ࡣ㸪㔝⏕ᆺ㸦WT㸧࣐࢘ࢫ࠾ࡼࡧ CARKO ࣐࢘ࢫࢲࢪࣥ⣔ࡢ PROTOX 㜼 ᐖࡋ࡚▱ࡽࢀࡿ OX ࢆᢞࡋ㸪▷ᮇᢞࡼࡾㄏⓎࡉࢀࡿ⫢⮚ࡢኚ࠾ࡼࡧ㛗ᮇᢞ ࡼࡾᙧᡂࡉࢀࡿ⭘⒆ᑐࡍࡿ CAR ࡢᙳ㡪ࢆ☜ㄆࡋࡓࠋ➨ 2 ❶࡛ࡣ㸪ࢪࣇ࢙ࢽ࢚࣮ࣝࢸࣝ ⣔ࡢ PROTOX 㜼ᐖ࡛࠶ࡿ ACI ࢆ WT ࣐࢘ࢫ࠾ࡼࡧ CARKO ࣐࢘ࢫᢞࡍࡿࡇ࡛㸪 OX ㄏⓎ⫢Ⓨࡀࢇ࡛ㄆࡵࡓస⏝ᶵᗎࡀ PROTOX 㜼ᐖඹ㏻ࡋࡓࡶࡢ☜ࡵࡓࠋ ➨ 3 ❶࡛ࡣ㸪 OX ࠾ࡼࡧ ACI ࡀㄏⓎࡍࡿ⭘⒆ᛶኚ㸦⫢⣽⬊⭢⭘㸪⫢⣽⬊⒴㸧࠾ࡼࡧ⭘⒆ࡢ๓ẁ㝵⪃࠼ 6.
(8) ࡽࢀ࡚࠸ࡿኚ␗⫢⣽⬊ᕢࡢศᏊ⏕≀Ꮫⓗ≉ᚩࢆ᳨ウࡋ㸪CAR άᛶ࡛࠶ࡿ PB ㄏⓎኚ ࡢ≉ᚩẚ㍑ࡋࡓࠋ. 7.
(9) ➨1❶. ࢜࢟ࢧࢪࢰࣥࡀㄏⓎࡍࡿ࣐࢘ࢫ⫢Ⓨࡀࢇ࠾ࡅࡿ Constitutive androstane receptor ࡢ㛵. 8.
(10) ⥴ ゝ ࣒࣊ࡢྜᡂ⤒㊰ࡣ 8 ࡘࡢ㓝⣲ࡀᏑᅾࡋ㸪࠸ࡎࢀࡢ㓝⣲ࡢᶵ⬟ࡀḞᦆࡋࡓࣄࢺ࡛ࡣ࣏ ࣝࣇࣜࣥࢆⓎࡍࡿࠋࡇࢀࡽ㓝⣲ࡢ୍ࡘ㸪ࣉࣟࢺ࣏ࣝࣇࣜࣀ࣮ࢤࣥ㓟㓝⣲ 㸦PROTOX㸧ࡣ࣑ࢺࢥࣥࢻࣜෆ࡛ࣉࣟࢺ࣏ࣝࣇࣜࣀ࣮ࢤࣥࢆࣉࣟࢺ࣏ࣝࣇࣜࣥ IX 㸦PPIX㸧㓟ࡉࡏࡿࠋPROTOX ᶵ⬟ࡢḞᦆࢆ≉ᚩࡋࡓࣄࢺࡢ㑇ఏᛶᕼᑡᝈࡣ␗ᆺ࣏ ࣝࣇࣜࣥࡤࢀࡿࠋPROTOX ᶵ⬟ࡀḞᦆࡍࡿࡇ࡛㸪⫢⮚୰ࡢࣉࣟࢺ࣏ࣝࣇࣜࣀ ࣮ࢤࣥࡣ࣑ࢺࢥࣥࢻࣜࡽ⣽⬊㉁⛣⾜ࡋ㸪㓟ࡉࢀ࡚ PPIX ࡞ࡿࠋ⏕ᡂࡉࢀࡓ PPIX ࡣ㸪⫹Ồ࠾ࡼࡧ⣅୰ἥࡉࢀࡿ㸦55㸧 ࠋ␗ᆺ࣏ࣝࣇࣜࣥࡢᝈ⪅࡛ࡣ㸪៏ᛶⓗ࡞⫢ᶵ⬟ ࡢ␗ᖖࡸ㍍ᚤ࡞⫢ᶵ⬟㓝⣲ࡢቑຍࡀㄆࡵࡽࢀ㸪⫢Ⓨࡀࢇࣜࢫࢡࡀ㧗࠸ࡇࡀ▱ࡽࢀ࡚࠸ࡿ 㸦53㸪55㸧 ࠋ ࣊ࢸࣟ⎔ᘧྜ≀࡛࠶ࡿ࢜࢟ࢧࢪࢰࣥ㸦OX㸧ࡣ㸪PROTOX 㜼ᐖࡤࢀࡿ㝖ⲡ࡛ ࠶ࡿࠋPROTOX 㜼ᐖࡼࡾ᳜≀୰✚ࡋࡓ PPIX ࡣ㸪ගᛂࡋ࡚ග㓟ࢆច㉳ࡍࡿࠋ OX ࡣࡆࡗṑ㢮⫢⮚ࡢ PROTOX ࢆ㜼ᐖࡍࡿࡇ࡛ PPIX ࢆ✚ࡉࡏࡿ㸦29㸪41㸧 ࠋ࣏ࣝࣇ ࣜࣥࡣࡆࡗṑ㢮⫢Ⓨࡀࢇ㛵ࡍࡿࡇࡀ▱ࡽࢀ࡚࠸ࡿ㸦58㸧 ࠋࡑࡢ⫢Ⓨࡀࢇᶵᗎࡣ⣽ ⬊യᐖࡀ㛵ࡋ࡚࠾ࡾ㸪ࣄࢺእᤄྍ⬟࡞స⏝ᶵᗎ⪃࠼ࡽࢀ࡚࠸ࡿ㸦20㸧 ࠋࣛࢵࢺ࠾ࡼࡧ ࣐࢘ࢫࢆ⏝࠸ࡓ៏ᛶẘᛶ㸭Ⓨࡀࢇᛶ」ྜヨ㦂࠾࠸࡚㸪OX ࡣ⫢⮚ࡢ⣽⬊യᐖ࠾ࡼࡧ⫢⭘ ⒆ࢆច㉳ࡋ࡚࠸ࡿ㸦㎰⸆ホ౯᭩㸸 http://www.fsc.go.jp/fsciis/evaluationDocument/show/kya20080111001㸧 ࠋ Peroxisome proliferator-activated receptor Į㸦33$5Į㸧ࡣ᰾ෆཷᐜయࡢ 1 ✀࡛࠶ࡾ㸪33$5Į ࢦࢽࢫࢺࡣࡆࡗṑ㢮࣌ࣝ࢜࢟ࢩࢰ࣮࣒ቑṪࢆక࠺⫢⫧㸪ࡑࡋ࡚⫢⭘⒆ࢆច㉳ࡍࡿࡇ ࡀ▱ࡽࢀ࡚࠸ࡿ㸦5㸪26㸧 ࠋOX ࡣ 33$5Į ࢆάᛶࡍࡿࡇࡀሗ࿌ࡉࢀ࡚࠾ࡾ㸦51㸧 㸪OX ࡀ ㄏⓎࡍࡿࡆࡗṑ㢮⫢Ⓨࡀࢇࡣ 33$5Į ࡀ㛵ࡋ࡚࠸ࡿྍ⬟ᛶࡀ࠶ࡿࠋࡲࡓ㸪ࣄࢺ࠾ࡅࡿ 33$5Į άᛶࡣ⫢⣽⬊ቑṪࢆㄏⓎࡏࡎ㸪33$5Į ࢦࢽࢫࢺࡀㄏⓎࡍࡿࡆࡗṑ㢮⫢Ⓨࡀࢇࡣ ࣄࢺእᤄࡉࢀ࡞࠸⪃࠼ࡽࢀ࡚࠸ࡿ㸦33㸧 ࠋ. 9.
(11) 33$5Į ࡢ constitutive androstane/active receptor㸦CAR㸧ࡀ㸪ࡆࡗṑ㢮⫢Ⓨࡀࢇ㛵 ࡍࡿ᰾ෆཷᐜయࡋ࡚▱ࡽࢀ࡚࠸ࡿࠋCAR άᛶ࡛࠶ࡿࣇ࢙ࣀࣂࣝࣅࢱ࣮ࣝ㸦PB㸧ࡣ㸪 CAR ࢆࡋ࡚ࡆࡗṑ㢮⫢⭘⒆ࢆច㉳ࡍࡿࡇࡀ▱ࡽࢀ࡚࠸ࡿ㸦69㸧 ࠋPB ࡢ㸪ࣆ࣌ࣟ ࢽࣝࣈࢺ࢟ࢩࢻࡸࢺࣜࢰ࣮ࣝẅ⳦࡞」ᩘࡢྜ≀ࡀㄏⓎࡍࡿࡆࡗṑ㢮⫢Ⓨࡀࢇ࠾ ࠸࡚㸪CAR ࡣ㔜せ࡞ാࡁࢆࡋ࡚࠸ࡿ㸦52㸪63㸧 ࠋOX CAR ࡢ㛵ಀࡣ᫂ࡔࡀ㸪OX ྠࡌ PROTOX 㜼ᐖࡋ࡚▱ࡽࢀࡿ࢜࢟ࢩࣇࣝ࢜ࣝࣇ࢙ࣥࡣ㸪ࡆࡗṑ㢮 CAR ࢆάᛶࡍࡿࡇ ࡀሗ࿌ࡉࢀ࡚࠸ࡿ㸦59㸧 ࠋࡋࡓࡀࡗ࡚㸪OX ࡀㄏⓎࡍࡿࡆࡗṑ㢮⫢Ⓨࡀࢇࡣ CAR ࡶ㛵ࡋ ࡚࠸ࡿྍ⬟ᛶࡀ࠶ࡿࠋ▩┪ࡋࡓሗ࿌ࡣ࠶ࡿࡶࡢࡢ㸦8㸧 㸪࣓࣐࢟ࣛ࢘ࢫࢆ⏝࠸ࡓᐇ㦂ࡽ㸪 CAR ࢆࡋࡓࡆࡗṑ㢮⫢Ⓨࡀࢇࡣࣄࢺእᤄࡉࢀ࡞࠸⪃࠼ࡽࢀ࡚࠸ࡿ㸦17㸧ࠋࡑࡢࡓࡵ㸪 ྜ≀ㄏⓎ⫢Ⓨࡀࢇ࠾ࡅࡿ CAR ࡢ㛵ࡢ☜ㄆࡣ㸪ࣄࢺ⫢Ⓨࡀࢇࣜࢫࢡࢆホ౯ࡍࡿୖ࡛㔜 せ࡛࠶ࡿࠋ ࡑࡇ࡛➨ 1 ❶࡛ࡣ㸪OX ࡀㄏⓎࡍࡿࡆࡗṑ㢮⫢Ⓨࡀࢇࡢᶵᗎࡑࡢࣄࢺࡢእᤄᛶࢆ᫂ࡽ ࡍࡿࡓࡵ㸪OX ⣽⬊യᐖ㸪33$5Į ࠾ࡼࡧ CAR ࡢ㛵ಀࢆ☜ࡵࡓࠋOX ࡼࡿ CAR ά ᛶࡢ᭷↓ࢆᤊ࠼ࡿࡓࡵ㸪C3H/HeNCrl ⣔⤫⏤᮶ࡢ CAR ࣀࢵࢡ࢘ࢺ㸦KO㸧࣐࢘ࢫࢆ⏝࠸ ࡓࠋ. 10.
(12) ᮦᩱ࠾ࡼࡧ᪉ἲ. Ꮫ≀㉁ OX㸦CAS No. 19666-30-9㸪⣧ᗘ 99.8%㸧ࡣග⣧⸆ᕤᴗᰴᘧ♫㸦㜰㸧 㸪diethylnitrosamine 㸦DEN㸪CAS No. 55-18-5㸪⣧ᗘ 99.9%㸧ࡣᮾிᡂᕤᴗᰴᘧ♫㸦ᮾி㸧ࡼࡾࡑࢀࡒࢀ㉎ධ ࡋࡓࠋ. ౪ヨື≀ ᰿ᓊ㞞ᙪඛ⏕㸦National Institute of Environmental Health Sciences㸧ࡽᥦ౪࠸ࡓࡔ࠸ࡓ C3H/HeNCrl ⣔⤫⏤᮶ࡢ CARKO ࣐࢘ࢫ㸦28㸪69㸧ࢆ㸪ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ࡚㣫⫱ ࡋ㸪ᐇ㦂౪ࡋࡓࠋ5 㐌㱋ࡢ C3H/HeNCrl ࣐࢘ࢫࢆ᪥ᮏࢳ࣮ࣕࣝࢫ࣭ࣜࣂ࣮ᰴᘧ♫㸦⚄ዉ ᕝ㸧ࡽ㉎ධࡋ㸪㔝⏕ᆺ㸦WT㸧࣐࢘ࢫࡋ࡚ᐇ㦂౪ࡋࡓࠋࡍ࡚ࡢື≀ᐇ㦂ィ⏬ࡣᐇ㦂 タ㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ㸧ࡢື≀ᐇ㦂ጤဨࡢᢎㄆࢆཷࡅ㸪ື≀ᐇ㦂᧯సࡣື≀ ࡢ⏝⟶⌮㛵ࡍࡿᣦ㔪ࢆ㑂Ᏺࡋ࡚⾜ࡗࡓࠋࡍ࡚ࡢᐇ㦂⪅ࡣ㑇ఏᏊ⤌࠼ᐇ㦂Ᏻጤ ဨࡽᩍ⫱ࢆཷࡅࡓࠋ. ᐇ㦂ࢹࢨࣥ ᐇ㦂 1 ࡛ࡣ㸪OX ࡢ▷ᮇᢞࡼࡿ⫢⮚ࡢᙳ㡪㸪ࡑࡢᙳ㡪ᑐࡍࡿ CAR ࡢ㛵ࢆ᳨ ウࡋࡓࠋ6 㐌㱋ࡢ㞝 WT ࣐࢘ࢫ࠾ࡼࡧ CARKO ࣐࢘ࢫࢆ㸪య㔜ᇶ࡙࠸࡚⩌ศࡅࡋ㸦5 ༉㸭 ⩌㸧㸪1000ppm ࡢ⃰ᗘ࡛ᇶ♏㣫ᩱ㸦CAF-1㸪࢚࢜ࣜࣥࢱࣝ㓝ẕᕤᴗᰴᘧ♫㸪ᮾி㸧ΰࡌ ࡓ OX ࢆ⮬⏤ᦤ䭱ࡉࡏࡓࠋᢞᮇ㛫ࡣ㐣ཤࡢヨ㦂ᇶ࡙࠸࡚ 1 㐌㸪4 㐌࠾ࡼࡧ 13 㐌㛫 ࡋࡓ㸦37㸧 ࠋᢞ⃰ᗘࡣ㐣ཤࡢ࣐࢘ࢫ៏ᛶẘᛶヨ㦂㸭ࡀࢇཎᛶ」ྜヨ㦂࡛⫢⣽⬊⭢⭘࠾ࡼࡧ ⫢⣽⬊⒴ࢆㄏⓎࡋࡓ 1000 ppm ࡋࡓ㸦㎰⸆ホ౯᭩㸸 http://www.fsc.go.jp/fsciis/evaluationDocument/show/kya20080111001㸧 ࠋᢞ⩌ྠᵝ㸪ᑐ↷ ⩌ࡣ⮬⏤ᦤ䭱࡚ᇶ♏㣫ᩱࢆ࠼ࡓࠋ⾑₢୰ PPIX ᐃ⏝ࡢ᥇⾑ࢆ⾜࠺ࡓࡵ㸪ู㏵ 6 㐌㱋ࡢ 11.
(13) 㞝 WT㸪CARKO ࣐࢘ࢫ 1000 ppm OX ࢆ 4 㐌㛫ΰ䭱ᢞࡋࡓࠋ ᐇ㦂 2 ࡛ࡣ㸪⫢⮚࠾ࡅࡿ⣽⬊യᐖ PPIX ࡢ⏝㔞┦㛵ࢆ☜ㄆࡋࡓࠋ6 㐌㱋ࡢ㞝 WT ࠾ࡼ ࡧ CARKO ࣐࢘ࢫ 0㸦ᑐ↷⩌㸧㸪300㸪1000 ࠾ࡼࡧ 3000 ppm OX ࢆ 4 㐌㛫ΰ䭱ᢞࡋࡓࠋ ᐇ㦂 1 ࡛ㄆࡵࡓ⣽⬊യᐖᑐࡋ࡚ࡼࡾ㍍ᗘ࠶ࡿ࠸ࡣࡼࡾ㔜ᗘ࡞യᐖࢆច㉳ࡉࡏࡿࡓࡵ㸪ᐇ 㦂 1 ࡛⏝࠸ࡓ 1000 ppm ࢆ୰㛫⏝㔞ࡋ㸪බẚ⣙ 3 ಸ࡛タᐃࡋࡓ 300 ࠾ࡼࡧ 3000 ppm OX ࢆ ࡑࢀࡒࢀప⏝㔞࠾ࡼࡧ㧗⏝㔞ࡋࡓࠋ ᐇ㦂 3 ࡛ࡣ㸪OX ㄏⓎ⫢Ⓨࡀࢇᑐࡍࡿ CAR ࡢ㛵ࢆ☜ㄆࡋࡓࠋ㐣ཤࡢሗ࿌ྠᵝ㸪6 㐌㱋ࡢࡍ࡚ࡢ WT㸪CARKO ࣐࢘ࢫ㸦50 ༉㸭㑇ఏᏊᆺ㸧㸪⫢Ⓨࡀࢇࢽࢩ࢚࣮ࢱ࣮࡛࠶ ࡿ DEN ࢆ 90 mg/kg ࡛༢ᅇ⭡⭍ෆᢞࡋࡓ㸦52㸧 ࠋDEN ᢞࡢ 2 㐌㛫ᚋ㸪ྛ㑇ఏᏊᆺ࣐࢘ࢫ 25 ༉ 1000ppm OX ࢆ 26 㐌㛫ΰ䭱ᢞࡋࡓࠋࡇࡢࣔࢹࣝࡣ㸪ྜ≀ࡢ⫢Ⓨࡀࢇࣉ࣮ࣟࣔࢩ ࣙࣥస⏝ࡢ᳨ฟ᭷⏝⪃࠼ࡽࢀ࡚࠸ࡿ㸦15㸧 ࠋ ᐇ㦂 1㸪2 ࠾ࡼࡧ 3 ࠾ࡅࡿࡍ࡚ࡢ࣐࢘ࢫࡣ㸪ᢞᮇ㛫୰ 1 ᪥ 1 ᅇࡢ୍⯡≧ែほᐹࢆ⾜ ࡗࡓࠋయ㔜ᦤ䭱㔞ࡣ㐌 1 ᗘ ᐃࡋࡓࠋゎ๗ࡣ㸪ࢯࣇࣝࣛࣥ㯞㓉ୗ࡛ࡍ࡚ࡢ࣐ ࢘ࢫࡢ㟼⬦ࡽ᥇⾑ࡋ㸪ᨺ⾑ࡼࡿᏳᴦṚࢆ⾜ࡗࡓࠋ⾑Ύࡣ ᐃ⏝࠸ࡿࡲ࡛-30Υ࡛ಖ ⟶ࡋ㸪ᰴᘧ♫࢚ࢫ࣮࢚ࣝࣝ㸦ᮾி㸧࡚⾑Ύ୰ࣛࢽ࣑ࣥࣀᇶ㌿⛣㓝⣲㸦ALT㸧ࢆ ᐃࡋࡓࠋᐇ㦂 1 ู࡛㏵㣫⫱ࡋࡓ࣐࢘ࢫࡢ⾑ᾮࡣ⾑₢୰ PPIX ᐃࡢࡳ౪ࡋࡓࠋ. ⌮⤌⧊Ꮫⓗゎᯒ ᐇ㦂 1 ࠾ࡼࡧ 2 ࡛ࡣ㸪⫢⮚㔜㔞 ᐃᚋ㸪ᕥⴥ࠾ࡼࡧ୰㛫ⴥࢆ 10㸣୰ᛶ⦆⾪࣐࣍ࣝࣜࣥᾮ ࡚ᅛᐃࡋࡓࠋᅛᐃࡋࡓ⫢⮚ࡣࣃࣛࣇࣥໟᇙฎ⌮ᚋⷧษࡋ㸪⌮⤌⧊᳨ᰝࡢࡓࡵ࣐࣊ࢺ ࢟ࢩ࣭࢚ࣜࣥ࢜ࢪࣥ㸦HE㸧ᰁⰍ㸪ࢩ࣮ࣗࣔࣝᛂ㸪࣌ࣝࢩࣥࣈ࣮ࣝᰁⰍ㸪࠾ࡼࡧ㐣ࣚ࢘ ⣲㓟ࢩࢵࣇ㸦PAS㸧ᰁⰍࢆ⾜ࡗࡓࠋ୍㒊ࡢ⫢⮚ࡣ OCT㸦ࢧࢡࣛࣇࣥࢸࢵࢡࢪࣕࣃࣥ㸪 ᮾி㸧⤖ໟᇙ㸪ⷧษࡋࡓᚋ㸪Oil red O ࡛ᰁⰍࡋࡓࠋ ᐇ㦂 3 ࡛ࡣ㸪10㸣୰ᛶ࣐࣍ࣝࣜࣥ⦆⾪ᾮᅛᐃࡋࡓࡍ࡚ࡢⴥࡢ⫢⮚ࢆ 13㹼18 ᩿㠃ษ ࡾฟࡋ㸦ᕥⴥ㸸4㹼6 ᩿㠃㸪୰㛫ⴥ㸸4㹼5 ᩿㠃㸪᪉ᙧⴥ࣭ᑿ≧ⴥ㸸5㹼8 ᩿㠃㸧 㸪⌮⤌⧊᳨ 12.
(14) ᰝࡢࡓࡵ HE ᶆᮏࢆస〇ࡋࡓࠋቑṪᛶኚࡢⓎ⏕㢖ᗘ㸦ኚࢆㄆࡵࡿ࣐࢘ࢫࡢ༉ᩘ㸧࠾ࡼࡧ Ⓨ⏕ᩘ㸦࣐࢘ࢫẖࡢኚᩘ㸧ࢆ⟬ฟࡋࡓࠋ⫢⣽⬊⭢⭘ࡣࡑࡢࢧࢬࡽ 4 ࢡࣛࢫศ㢮ࡋ ࡓ㸦small: < 2 mm㸪middle: 2 - 3 mm㸪large: 3 - 4 mm㸪very large: > 4 mm㸧 ࠋ」ᩘࡢ HE ᶆᮏ࡛ ほᐹࡉࢀࡓᆺࡢ⤖⠇ࡣ୍ࡘࡢኚࡋ࡚ᩘ࠼ࡓࠋኚ␗⫢⣽⬊ᕢࡸ⫢⣽⬊⭢⭘࡞ቑṪᛶ ኚࡢデ᩿ࡣ International Harmonization of Nomenclature and Diagnostic Criteria of Lesions in Rats and Mice㸦65㸧ᚑࡗࡓࠋHE ᶆᮏ࠾ࡅࡿᙧែᏛⓗ≉ᚩࡽ㸪ኚ␗⫢⣽⬊ᕢ࠾ࡼࡧ⫢ ⣽⬊⭢⭘ࡣ㸪ዲ㓟ᛶ㸦ዲ㓟ᛶࢆ♧ࡍ⫢⣽⬊ࡼࡾᵓᡂ㸧㸪ዲሷᇶᛶ㸦ዲሷᇶᛶࢆ♧ࡍ⫢⣽⬊ࡼ ࡾᵓᡂ㸧㸪࠾ࡼࡧࡑࡢ㸦✵⬊ࢆከᩘ᭷ࡍࡿ⫢⣽⬊ࡼࡾᵓᡂ㸧ࡢ୕ࡘளศ㢮ࡋࡓࠋ. ㏱㐣㟁Ꮚ㢧ᚤ㙾ἲ ᐇ㦂 1 ࠾࠸࡚㸪OX ࢆ 4 㐌ᢞࡋࡓ࣐࢘ࢫࡢ⫢⮚ࢆ⏝࠸࡚㉸ᚤ⣽ᵓ㐀ほᐹࢆ⾜ࡗࡓࠋ⣽ ษࡋࡓᕥⴥࡢ⫢⮚ࢆ 2%ࢢࣝࢱࣝࣝࢹࣄࢻ㸦pH7.4㸧 4 㛫ᅛᐃࡋࡓᚋ㸪1%ᅄ㓟࢜ࢫ ࣑࣒࢘⦆⾪ᾮ 2 㛫ᅛᐃࡋࡓࠋ⫢⮚ࡣ࢚࣏ࣥ 812 ໟᇙࡋࡓࠋࢺࣝࢪࣥࣥࣈ࣮࡛ࣝᰁ ࡵࡓཌษࡾᶆᮏࢆཧ⪃ほᐹ㒊ࢆษࡾฟࡋ㸪㓑㓟࢘ࣛࢽࣝࢡ࢚ࣥ㓟㖄࡛ᾐࡋࡓ㉸ⷧษ ∦ࢆ㟁Ꮚ㢧ᚤ㙾㸦JEM-1400㸹᪥ᮏ㟁Ꮚᰴᘧ♫㸪ᮾி㸧ࢆ⏝࠸࡚ホ౯ࡋࡓࠋ. ච⤌⧊Ꮫⓗゎᯒ ᐇ㦂 1 ࠾࠸࡚㸪⫢⮚ࡢࣃࣛࣇࣥໟᇙษ∦ࢆస〇ࡋ㸪CYP1A ᢠయ㸦Abcam, Cambridge, UK; ab111868; 200 ಸᕼ㔘㸧 㸪CYP2B ᢠయ㸦Abcam; ab69652; 500 ಸᕼ㔘㸧 㸪CYP3A ᢠయ㸦Abcam; ab3572; 500 ಸᕼ㔘㸧㸪࠾ࡼࡧ CYP4A ᢠయ㸦Abcam; ab140635; 500 ಸᕼ㔘㸧 4°C ୍࡛ᬌ ᛂࡉࡏࡓࠋࡑࡢᚋ㸪࣌ࣝ࢜࢟ࢩࢲ࣮ࢮᶆ㆑ࢹ࢟ࢫࢺ࣏࣐࣮ࣛࣥࣜࢆ⤖ྜࡉࡏࡓḟᢠయ㸦ࣄ ࢫࢺࣇࣥࢩࣥࣉࣝࢫࢸࣥ MAX PO㸪ᰴᘧ♫ࢽࢳࣞࣂ࢜ࢧ࢚ࣥࢫ㸪ᮾி㸧 ᛂࡉࡏ㸪3-30-diaminobenzidine ᛂ㸦ᰴᘧ♫ྠோᏛ◊✲ᡤ㸪⇃ᮏ㸧ࡼࡾⓎⰍࡉࡏࡓࠋ ⫢⣽⬊ࡢቑṪάᛶࢆ☜ㄆࡍࡿࡓࡵ㸪ྠಶయࡢࣃࣛࣇࣥໟᇙษ∦ࢆ proliferating cell nuclear antigen㸦PCNA㸧ᢠయ㸦Dako, ி㒔; M0879; 100 ಸᕼ㔘㸧ᛂࡉࡏ㸪ୖグྠᵝࡢ 13.
(15) ᡭ㡰࡛ⓎⰍࡉࡏࡓࠋ㢧ᚤ㙾ࡢ 100 ಸど㔝࡚㸪ᑠⴥ㛫ᵓ㐀୰ᚰ㟼⬦㡿ᇦࢆྵࡴ㡿ᇦࢆ↓ సⅭ 5 ᡤ㑅ࢇࡔࠋ㑅ࢇࡔ㡿ᇦࡢ PCNA 㝧ᛶ⫢⣽⬊ᩘ࠾ࡼࡧ⥲⫢⣽⬊ᩘࢆ ᐃࡋࠊ⥲⫢ ⣽⬊ᩘᑐࡍࡿ PCNA 㝧ᛶ⫢⣽⬊ᩘࡢྜࢆ⟬ฟࡋࡓࠋ. Real-time RT-PCR ゎᯒ ᐇ㦂 1 ࠾࠸࡚㸪ᕥⴥࡢ⫢⮚⤌⧊ࢆ-80Υ࡛㎿㏿⤖ࡋࡓࠋ⫢⤌⧊ࡽ ISOGEN㸦Nippon Gene Co. Ltd., Tokyo, Japan㸧ࢆ⏝࠸࡚ RNA ࢆᢳฟࡋࡓࠋȝJ51$ ࡽ High Capacity Reverse Transcription kit㸦Applied Biosystems, Foster City, CA, USA㸧ࢆ⏝࠸࡚ cDNA ࢆྜᡂࡋࡓᚋ㸪 TaqMan Fast Universal PCR Master Mix㸦Applied Biosystems㸧 㸪TaqMan Gene Expression Assays 㸦Applied Biosystems㸧 㸪࠾ࡼࡧ 7900HT Fast Real-time PCR System㸦Applied Biosystems㸧ࢆ⏝ ࠸࡚ PCR ᛂࡉࡏࡓࠋ᧯సࡣྛ࢟ࢵࢺࡢࣉࣟࢺࢥ࣮ࣝᚑࡗࡓࠋPrimer-probe ࡣ Cyp1a1 㸦Mm00487218_m1㸧㸪Cyp1a2㸦Mm00487224_m1㸧 㸪Cyp2b10㸦Mm01972453_s1㸧 㸪Cyp3a11 㸦Mm00731567_m1㸧㸪Cyp4a10㸦Mm01188913_g1㸧㸪࠾ࡼࡧ P450 reductase㸦Mm00435876_m1㸧 ࢆ⏝࠸ࡓࠋ ྛ㑇ఏᏊࡢⓎ⌧ࡣ㸪┦ᑐ᳨㔞⥺ἲ࡚ィ⟬ࡋࡓᚋ㸪ෆᅉᛶ GAPDH㸦TaqMan Rodent GAPDH Control Reagent; Applied Biosystems㸧࡛⿵ṇࡋࡓࠋ. PPIX ᐃ ᐇ㦂 1 2 ࠾࠸࡚㸪⫢⮚୰ PPIX ࢆ㐣ཤࡢሗ࿌ᚑࡗ࡚ ᐃࡋࡓ㸦66㸧 ࠋᕥⴥࡢ⫢⮚⤌ ⧊ࢆ Tris HCl 10 mM㸦pH 7.0㸧࡛ 100mg/mL ㄪᩚࡋ㸪ᆒ㉁ࡋࡓࠋ⁐ゎᾮࢆ㓑㓟࢚ࢳ࣭ࣝ 㓑㓟㸦4:1㸧࡛ᢳฟࡋ㸪15 ศࡢ 1500 rpm 㐲ᚰศ㞳ࡼࡾỿẊࢱࣥࣃࢡࢆྲྀࡾ㝖࠸ࡓࠋPPIX ࢆ 1.5N ሷ㓟࡛ᢳฟࡋ㸪⺯ගගᗘィࢆ⏝࠸࡚ບ㉳Ἴ㛗 405 nm㸪⺯ගἼ㛗 590nm ࡚ ᐃࡋ ࡓࠋᶆ‽≀㉁ࡣ SigmaeAldrich㸦MO, USA㸧ࡽ㉎ධࡋࡓࠋᐇ㦂 1 ࠾࠸࡚ OX ࢆ 4 㐌㛫ᢞ ࡋࡓ࣐࢘ࢫࡢ⾑₢ࢆ⏝࠸࡚㸪PPIX ࢆ ᐃࡋࡓࠋ⾑₢୰ PPIX ࡣ㸪ᆒ㉁ฎ⌮ࢆ㝖࠸ࡓ⫢ ⮚୰ PPIX ࡢᡭἲྠᵝ ᐃࡋࡓࠋ. 14.
(16) ⤫ィゎᯒ ᐃ㔞್ࡣ⩌ࡢᖹᆒ್ᶆ‽೫ᕪ࡛♧ࡋࡓࠋOX ᢞ⩌ࡢ್ࡣᑐ↷⩌ࡢ್㸪CARKO ࣐࢘ ࢫࡢ್ࡣྠ⩌ࡢ WT ࣐࢘ࢫࡢ್ࡑࢀࡒࢀẚ㍑ࡋࡓࠋ್ࡢ➼ศᩓᛶࢆ F ᳨ᐃ࡛☜ㄆࡋ㸪➼ ศᩓࡢሙྜࡣ Student ࡢ t ᳨ᐃ㸪➼ศᩓࡢሙྜࡣ Welch ࡢ t ᳨ᐃࢆ⾜ࡗࡓࠋᐇ㦂 2 ࠾࠸ ࡚ OX ᢞ⩌ࡢ್ᑐ↷⩌ࡢ್ࢆẚ㍑ࡍࡿ㝿ࡣ㸪್ࡢ➼ศᩓᛶࢆ Bartlett ᳨ᐃ࡛☜ㄆࡋ㸪 ➼ศᩓࡢሙྜࡣ Dunnett ᳨ᐃ㸪➼ศᩓࡢሙྜࡣ Steel ᳨ᐃࢆ⾜ࡗࡓࠋᐇ㦂 3 ࠾ࡅࡿቑṪ ᛶኚࡢⓎ⏕㢖ᗘࡣ Fisher ἲࢆ⏝࠸᳨࡚ᐃࡋࡓࠋྛ᳨ᐃࡢ᭷ពỈ‽ࡣ 0.05 ࠾ࡼࡧ 0.01 ࡋ ࡓࠋ. 15.
(17) ⤖ ᯝ. OX ㉳ᅉࡋࡓṚஸ࠾ࡼࡧ୍⯡≧ࡣࡍ࡚ࡢ࣐࢘ࢫ࡛ㄆࡵࡽࢀ࡞ࡗࡓࠋゎ๗㸪⫢⮚ ࡢ⭘ࢆ㝖ࡁ⫗║ⓗኚࡣㄆࡵࡽࢀ࡞ࡗࡓࠋᦤ䭱㔞ࡣ㸪ᑐ↷⩌ OX ᢞ⩌ࡢ㛫᫂☜ ࡞ᕪࡣㄆࡵࡽࢀ࡞ࡗࡓࠋ. OX ▷ᮇ㛫ᢞࡼࡿ⫢⮚ࡢᙳ㡪㸦ᐇ㦂 1㸧 ᢞᮇ㛫୰ࡢ୧㑇ఏᏊᆺ࣐࢘ࢫࡢయ㔜ࡣ㸪ᑐ↷⩌ẚ㍑ࡋ࡚ OX ᢞ⩌࡛ప್ࢆ♧ࡋࡓࠋ ࡍ࡚ࡢ᳨ᰝᮇࡢ᭱⤊య㔜ࡣ㸪ᑐ↷⩌ẚ㍑ࡋ࡚ OX ᢞ⩌࡛᭷ព࡞ప್ࢆ♧ࡋࡓ㸦Table 1-1㸧ࠋᢞ 13 㐌┠࠾࠸࡚㸪WT ࣐࢘ࢫ࠾ࡼࡧ CARKO ࣐࢘ࢫࡢ᭱⤊య㔜ࡣ㸪ᑐ↷⩌ẚ ㍑ࡋ࡚ OX ᢞ⩌࡛ࡑࢀࡒࢀ 12.5%࠾ࡼࡧ 14.1%ῶᑡࡋࡓࠋࡍ࡚ࡢ᳨ᰝᮇࡢ୧㑇ఏᏊᆺ ࣐࢘ࢫࡢ┦ᑐ⫢㔜㔞ࡣ㸪ᑐ↷⩌ẚ㍑ࡋ࡚ OX ᢞ⩌࡛᭷ពቑຍࡋࡓ㸦Table 1-1㸧 ࠋᢞ 4 㐌┠ཬࡧ 13 㐌┠࠾ࡅࡿ OX ᢞ⩌ࡢ CARKO ࣐࢘ࢫࡢ┦ᑐ⫢㔜㔞ࡣ㸪ྠ⩌ࡢ WT ࣐࢘ࢫ ࡢ್ẚ㍑ࡋ࡚᭷ព࡞㧗್ࢆ♧ࡋࡓࠋ OX ࡢ▷ᮇ㛫ᢞࡼࡾ⫢⮚ࡘࡢせ࡞ኚࢆㄆࡵࡓࠋ୍ࡘ┠ࡢせ࡞ኚࡋ࡚㸪 ┦ᑐ⫢㔜㔞ࡢቑຍࢆక࠺⫢⣽⬊⫧ࢆㄆࡵࡓࠋࡍ࡚ࡢ᳨ᰝᮇࡢ୧㑇ఏᏊᆺ࣐࢘ࢫ࡛㸪 ୰ᚰᖏࡽ୰㛫ᖏࡅ࡚࠶ࡿ࠸ࡣࡧ៏ᛶ⫢⣽⬊⫧ࢆㄆࡵࡓ㸦Table 1-2㸧 ࠋ⫢⣽⬊⫧ ࡢ⛬ᗘࡣᢞ 4 㐌┠࡛᭱ࡶᙉࡃ㸪㑇ఏᏊᆺ㛫ࡢᕪࡣㄆࡵ࡞ࡗࡓ㸦Fig. 1-1㸧ࠋ㟁Ꮚ㢧ᚤ㙾ୗ ࡛ࡣ㸪1 㔜⭷ໟࡲࢀࡓ⌫≧ࡢ⣽⬊ᑠჾᐁ࡛࠶ࡿ࣐ࢡࣟ࣎ࢹ㸦࣌ࣝ࢜࢟ࢩࢰ࣮࣒㸧ࡢቑ ຍࢆㄆࡵࡓ㸦Fig. 1-2㸧 ࠋ ୧㑇ఏᏊᆺ࣐࢘ࢫࡢ⫧ࡋࡓ⫢⣽⬊࠾࠸࡚㸪 CYP2B ࠾ࡼࡧ CYP4A ࡢⓎ⌧ࢆㄆࡵࡓ 㸦Fig. 1-1㸧ࠋᢞ 13 㐌┠࡛ࡣ㸪୧㑇ఏᏊᆺ࣐࢘ࢫࡢ⫧ࡋࡓ⫢⣽⬊࠾࠸࡚ CYP3A ࡢⓎ⌧ࢆㄆ ࡵࡓࠋCARKO ࣐࢘ࢫ࠾ࡅࡿ CYP2B ࡢⓎ⌧ࡣ WT ࣐࢘ࢫࡢⓎ⌧ẚ㍑ࡋ࡚㍍ᗘࡔࡗࡓࠋ CYP4A ࠾ࡼࡧ CYP3A Ⓨ⌧㑇ఏᏊ㛫࡛᭷ព࡞ᕪࡣㄆࡵࡽࢀ࡞ࡗࡓࠋࡍ࡚ࡢ᳨ᰝᮇ ࡛㸪⫧ࡋࡓ⫢⣽⬊࠾ࡅࡿ CYP1A ࡢⓎ⌧ࡣㄆࡵࡽࢀ࡞ࡗࡓࠋ 16.
(18) ⸆≀௦ㅰ㓝⣲㑇ఏᏊࡢⓎ⌧㔞ࢆ Fig. 1-3 ♧ࡋࡓࠋ୧㑇ఏᏊᆺ࣐࢘ࢫࡢ Cyp4a10 Ⓨ⌧ࡣᑐ ↷⩌ẚ㍑ࡋ࡚ OX ᢞ⩌࡛᭷ពࡘ㢧ⴭቑຍࡋࡓ 㸦400㹼2500 ಸ㸧 ࠋ WT ࣐࢘ࢫࡢ Cyp2b10 Ⓨ⌧ࡣᑐ↷⩌ẚ㍑ࡋ࡚ OX ᢞ⩌࡛᭷ពቑຍࡋࡓ㸦29㹼97 ಸ㸧 ࠋࡓࡔࡋ㸪ᢞ 13 㐌┠ ࡢⓎ⌧⩌㛫ࡢ⤫ィᏛⓗ᭷ពᕪࡣㄆࡵ࡞ࡗࡓࠋࡍ࡚ࡢ᳨ᰝᮇ࡛ OX ࡣ୧㑇ఏᏊᆺ࣐࢘ ࢫࡢ Cyp3a11㸪P450 reductase Ⓨ⌧ࢆቑຍࡉࡏࡓࡀ㸪ᢞ 13 㐌┠࠾ࡅࡿ CARKO ࣐࢘ࢫࡢ P450 reductase Ⓨ⌧ࡣᑐ↷⩌ࡢⓎ⌧ྠ➼ࡔࡗࡓࠋࡲࡓ㸪ᢞ 1 㐌┠࠾࠸࡚㸪OX ࡣ୧㑇 ఏᏊᆺ࣐࢘ࢫࡢ Cyp1a ࠾ࡼࡧ Cyp1a2 Ⓨ⌧ࢆቑຍࡉࡏࡓࠋCyp2b10 ࢆ㝖ࡃ㑇ఏᏊⓎ⌧㑇ఏ Ꮚᆺ㛫࡛᭷ព࡞ᕪࡣㄆࡵࡽࢀ࡞ࡗࡓࠋ ࡘ┠ࡢせ࡞ኚࡋ࡚㸪⣽⬊യᐖࢆ OX ᢞ⩌ࡢ୧㑇ఏᏊᆺ࣐࢘ࢫㄆࡵࡓࠋ⣽⬊യ ᐖࢆ♧၀ࡍࡿᡤぢࡋ࡚⫢⣽⬊ࡢ༢⣽⬊ቯṚࢆㄆࡵࡽࢀࡓࠋ⫢⣽⬊ࡢ༢⣽⬊ቯṚࡣ୰ᚰ ࡽ୰㛫ᖏࡅ࡚ㄆࡵࡽࢀ㸪ࡑࡢᩘࡣᢞ 1 㐌┠ࡽ 13 㐌┠ࡅ࡚ቑຍࡋࡓࠋᢞ 4 㐌 ┠࠾ࡼࡧ 13 㐌┠࡛ࡣ㸪⫢⣽⬊ࡢ༢⣽⬊ቯṚ㝶ࡋ࡚ዲ୰⌫ᾐ₶ࡸࡧࡲࢇᛶ༢᰾⣽⬊ᾐ₶ ࡞ࡢ⅖ᛶኚࢆㄆࡵࡓ㸦Fig. 1-4A㸧 ࠋᢞ 1 㐌┠ࡽ 13 㐌┠ࡅ࡚⫢⣽⬊ࡢ᭷⣒ศ ീࡢቑຍࡀㄆࡵࡽࢀ㸪ᢞ 4 㐌┠࡛᭱ࡶ㧗㢖ᗘㄆࡵࡓࠋࡲࡓ㸪ᢞ 4 㐌┠࠾ࡼࡧ 13 㐌┠ ࡛ࡣࢡࢵࣃ࣮⣽⬊ࡢ〓ⰍⰍ⣲ỿ╔ࢆㄆࡵࡓ㸦Fig. 1-4B㸧 ࠋ〓ⰍⰍ⣲ࡣ PAS ᰁⰍ࠾ࡼࡧࢩࣗࣔ ࣮ࣝᛂ㝧ᛶ㸪࣌ࣝࢩࣥࣈ࣮ࣝᰁⰍ㝜ᛶ࡛࠶ࡾ㸪࣏ࣜࣇࢫࢳࣥࢆ♧၀ࡋ࡚࠸ࡓ㸦Fig. 1-4C㸧 ࠋ ࡍ࡚ࡢ᳨ᰝᮇ࠾࠸࡚㸪⫢⮚ࡢ⣽⬊യᐖࢆ♧၀ࡍࡿ⾑Ύ୰ ALT ್ࡣ㸪OX ᢞ⩌ࡢ ୧㑇ఏᏊᆺ࣐࢘ࢫ࡛㸪ᑐ↷⩌ẚ㍑ࡋ࡚ቑຍࡋࡓࠋᢞ 13 㐌┠ࡢ ALT ್⩌㛫࡛⤫ィᏛⓗ ᭷ពᕪࡣㄆࡵ࡞ࡗࡓ㸦Fig. 1-4D㸧ࠋ⌮⤌⧊Ꮫⓗ᳨ᰝ࡛ࡣ㑇ఏᏊᆺ㛫ᕪࢆㄆࡵ࡞ࡗࡓ ࡀ㸪ᢞ 4 㐌┠࠾ࡅࡿ CARKO ࣐࢘ࢫࡢ⾑Ύ୰ ALT ್ࡣ WT ࣐࢘ࢫࡢ್ᑐࡋ࡚ప್ࢆ ♧ࡋࡓࠋᢞ 1 㐌┠ࡽ 13 㐌┠࠾࠸࡚㸪୧㑇ఏᏊᆺ࣐࢘ࢫࡢ PCNA 㝧ᛶ⣽⬊⋡ࡣ㸪OX ᢞ⩌࡛ᑐ↷⩌ẚ㍑ࡋ࡚㧗್ࢆ♧ࡋࡓ㸦Fig. 1-4E㸧 ࠋPCNA 㝧ᛶ⣽⬊⋡㑇ఏᏊᆺ㛫࡛ᕪ ࡣㄆࡵ࡞ࡗࡓࠋ ࡍ࡚ࡢ᳨ᰝᮇ࠾࠸࡚㸪୧㑇ఏᏊᆺ࣐࢘ࢫࡢ⫢⮚୰ PPIX ್ࡣ㸪OX ᢞ⩌࡛ᑐ↷⩌ 17.
(19) ẚ㍑ࡋ࡚᭷ពቑຍࡋࡓ 㸦Fig. 1-4F㸧 ࠋ ᢞ 4 㐌┠࠾ࡅࡿ WT ࣐࢘ࢫࡢ PPIX ್ࡣ㸪CARKO ࣐࢘ࢫࡢ್ᑐࡋ࡚᭷ពపࡗࡓࡀ㸪ᢞ 1 㐌┠࠾ࡼࡧ 13 㐌┠ࡢ PPIX ್ࡣ㑇ఏᏊ㛫࡛ ᭷ព࡞ᕪࡣㄆࡵࡽࢀ࡞ࡗࡓࠋ OX ࢆ 4 㐌㛫ᢞࡋࡓ୧㑇ఏᏊᆺ࣐࢘ࢫࡢ⾑₢୰ PPIX ್ࡣ㸪 ᑐ↷⩌ࡢ್ẚ㍑ࡋ࡚᭷ពቑຍࡋࡓ㸦Fig. 1-4G㸧 ࠋ ᑐ↷⩌ẚ㍑ࡋ࡚㸪OX ᢞ⩌࡛ࡣ⫢⮚࠾ࡼࡧ⾑₢୰ࡢ PPIX ್ࡀ᭷ពቑຍࡋࡓࡀ㸪࣏ ࣝࣇࣜࣥࢆ♧၀ࡍࡿ⌮⤌⧊Ꮫⓗኚࡣ⫢⮚ㄆࡵࡽࢀ࡞ࡗࡓࠋᢞ 4 㐌┠࠾ࡼࡧ 13 㐌┠㸪OX ᢞ⩌ࡢ୧㑇ఏᏊᆺ࣐࢘ࢫࡢ⫢⮚࡛୰ᚰࡽ୰㛫ᖏࡅ࡚⫢⣽⬊ࡢ✵⬊ࢆ ㄆࡵࡓࠋ✵⬊ࡣࢢࣜࢥࣥࢤࣥ㝧ᛶࢆ♧ࡍ PAS ᰁⰍ࠾ࡼࡧ⬡⫫㝧ᛶࢆ♧ࡍ Oil red O ᰁⰍ ࡶ㝧ᛶࡔࡗࡓࠋࡑࡢ㸪ᢞ 13 㐌┠࠾ࡅࡿ OX ᢞ⩌ࡢ୧㑇ఏᏊᆺ࣐࢘ࢫ࡛ࡣ⫢ ⣽⬊ࡢᕧ᰾ࢆ㢖⦾ㄆࡵࡓࠋ. ⫢⮚࠾ࡅࡿ⣽⬊യᐖ PPIX ✚ࡢ⏝㔞┦㛵㸦ᐇ㦂 2㸧 ヨ㦂ᮇ㛫୰ࡢ୧㑇ఏᏊᆺ࣐࢘ࢫࡢయ㔜ࡣ㸪1000 ࠾ࡼࡧ 3000 ppm OX ᢞ⩌࡛ᑐ↷⩌ẚ ㍑ࡋ࡚᭷ពῶᑡࡋࡓࠋࡍ࡚ࡢ OX ᢞ⩌ࡢ᭱⤊య㔜ࡣᑐ㇟⩌ࡢ್ẚ㍑ࡋ࡚᭷ព࡞ప್ ࢆ♧ࡋࡓ㸦Table1-3㸧ࠋ1000 ppm ࠾ࡼࡧ 3000 ppm OX ᢞ⩌࠾ࡅࡿ CARKO ࣐࢘ࢫࡢ┦ᑐ ⫢㔜㔞ࡣ㸪ྠ⩌ࡢ WT ࣐࢘ࢫࡢ್ᑐࡋ࡚ࡑࢀࡒࢀప್ࢆ♧ࡋࡓࠋ ⫢㔜㔞ࡢቑຍక࠸㸪⌮⤌⧊Ꮫⓗ⫢⣽⬊ࡢ⫧ࢆㄆࡵࡓࠋ⌮⤌⧊Ꮫⓗ᳨ᰝࡢ⤖ᯝ㸪 ࡍ࡚ࡢ OX ᢞ⩌࡛⫢⣽⬊⫧ࢆ㸪1000 ppm ࠾ࡼࡧ 3000ppm OX ᢞ⩌࡛⫢⣽⬊ࡢ༢⣽ ⬊ቯṚࡸࡧ៏ᛶ༢᰾⣽⬊ᾐ₶࡞ࡢ⣽⬊യᐖᛶࡢኚࢆㄆࡵࡓ㸦Table 1-4㸪Fig. 1-5A㸧 ࠋᐇ 㦂 2 ࡛ㄆࡵࡓ⌮⤌⧊Ꮫⓗኚࡣᐇ㦂 1 ࡛ㄆࡵࡓኚ㢮ఝࡋ࡚࠸ࡓࠋࡓࡔࡋ㸪⫢⣽⬊ࡢ ༢⣽⬊ቯṚࡣ 3000 ppm OX ⩌࡛ 1000 ppm OX ⩌ẚ㍑ࡋ࡚ࡼࡾ㢖⦾ㄆࡵࡽࢀࡓࠋ 3000 ppm OX ⩌ࡢ WT ࣐࢘ࢫ࡛ࡣዲ㓟ᛶ㢛⢏ࢆ⫧ࡋࡓ⫢⣽⬊୰ㄆࡵࡓࡀ㸪ྠ⩌ࡢ CARKO ࣐࢘ࢫ ࡛ࡣㄆࡵࡽࢀ࡞ࡗࡓࠋ WT ࣐࢘ࢫࡢ⾑Ύ୰ ALT ್ࡣࡍ࡚ࡢ OX ᢞ⩌࡛㸪CARKO ࣐࢘ࢫࡢ⾑Ύ୰ ALT ್ࡣ 1000 ࠾ࡼࡧ 3000 ppm OX ᢞ⩌࡛㸪 ᑐ↷⩌ࡢ್ẚ㍑ࡋ࡚ࡑࢀࡒࢀ᭷ព࡞㧗್ࢆ♧ࡋࡓ 㸦Fig. 18.
(20) 1-5B㸧ࠋ300 ࠾ࡼࡧ 1000 ppm OX ᢞ⩌࠾ࡅࡿ CARKO ࣐࢘ࢫࡢ⾑Ύ୰ ALT ್ࡣ㸪WT ࣐ ࢘ࢫࡢ್ẚ㍑ࡋ࡚᭷ព࡞ప್ࢆ♧ࡋࡓࠋ୧㑇ఏᏊᆺ࣐࢘ࢫࡢ ALT ್ࡣ⏝㔞┦㛵ࡋ࡚ቑ ຍࡋࡓࠋ ୧㑇ఏᏊᆺ࣐࢘ࢫࡢ⫢⮚୰ PPIX ್ࡀ㸪OX ᢞ⩌࡛ᑐ↷⩌ẚ㍑ࡋ࡚᭷ព࡞㧗࠸್ࢆ♧ ࡋࡓ㸦Fig. 1-5C㸧 ࠋ300 ࠾ࡼࡧ 1000 ppm OX ᢞ⩌࠾࠸࡚㸪CARKO ࣐࢘ࢫࡢ⫢⮚୰ PPIX ್ࡣ WT ࣐࢘ࢫࡢ್ẚ㍑ࡋ࡚᭷ពపࡗࡓࠋ WT ࣐࢘ࢫࡢ⫢⮚୰ PPIX ್ࡣ 1000 ppm OX ⩌࡛᭱ࡶ㧗࠸್ࢆ♧ࡋ࡚࠾ࡾ㸪⏝㔞┦㛵ᛶࢆ♧ࡉ࡞ࡗࡓࠋ. ⭘⒆ᙧᡂᑐࡍࡿ OX ࡢᙳ㡪㸦ᐇ㦂 3㸧 ኚ␗⫢⣽⬊ᕢ࠾ࡼࡧ⫢⣽⬊⭢⭘ࡢⓎ⏕㢖ᗘࢆ Table 1-5 ♧ࡋࡓࠋ୧㑇ఏᏊᆺ࣐࢘ࢫࡢዲ 㓟ᛶ࠾ࡼࡧዲሷᇶᛶኚ␗⫢⣽⬊ᕢࡢⓎ⏕㢖ᗘࡣ㸪OX ᢞ⩌࡛ᑐ↷⩌ẚ㍑ࡋ࡚㧗್ࢆ♧ࡋ ࡓࠋࡓࡔࡋ㸪WT ࣐࢘ࢫࡢࡇࢀࡽࡢኚࡢⓎ⏕㢖ᗘ⩌㛫࡛᭷ព࡞ᕪࡣㄆࡵࡽࢀ࡞ࡗࡓࠋ ୧㑇ఏᏊᆺ࣐࢘ࢫࡢዲ㓟ᛶ⫢⣽⬊⭢⭘ࡢⓎ⏕㢖ᗘࡣ㸪OX ᢞ⩌࡛ᑐ↷⩌ẚ㍑ࡋ࡚㧗್ࢆ ♧ࡋ㸪 ࡑࡢ CARKO ࣐࢘ࢫࡢⓎ⏕㢖ᗘࡣ WT ࣐࢘ࢫࡢⓎ⏕㢖ᗘẚ㍑ࡋ࡚᭷ពῶᑡࡋࡓࠋ ᑐ↷⩌ẚ㍑ࡋ࡚ OX ᢞ⩌࡛ࡣ㸪CARKO ࣐࢘ࢫࡢዲሷᇶᛶ⫢⣽⬊⭢⭘ࡢⓎ⏕㢖ᗘࡣῶᑡ ࡋ㸪୧㑇ఏᏊᆺ࣐࢘ࢫࡢࡑࡢኚ␗⫢⣽⬊ᕢࡢⓎ⏕㢖ᗘࡣቑຍࡋࡓࠋ ኚ␗⫢⣽⬊ᕢ࠾ࡼࡧ⫢⣽⬊⭢⭘ࡢⓎ⏕ᩘࢆ Fig. 1-6A ♧ࡋࡓࠋ୧㑇ఏᏊᆺ࣐࢘ࢫࡢዲ㓟 ᛶኚ␗⫢⣽⬊ᕢ࠾ࡼࡧ⫢⣽⬊⭢⭘ࡢⓎ⏕ᩘࡣ㸪OX ᢞ⩌࡛ᑐ↷⩌ẚ㍑ࡋ࡚᭷ពቑຍࡋ ࡓࠋᆺⓗ࡞ዲ㓟ᛶቑṪᛶኚ࡛ࡣࡍࡾ◪Ꮚ≧ࡢ⣽⬊㉁ࢆㄆࡵࡿࡀ㸪OX ᢞ⩌ࡢኚ࡛ࡣ ㄆࡵࡽࢀ࡞ࡗࡓ㸦Fig. 1-6B㸧ࠋCARKO ࣐࢘ࢫࡢዲ㓟ᛶኚ␗⫢⣽⬊ᕢ࠾ࡼࡧ⫢⣽⬊⭢⭘ࡢ Ⓨ⏕ᩘࡣ WT ࣐࢘ࢫࡢⓎ⏕ᩘẚ㍑ࡋ࡚᭷ព࡞ప್ࢆ♧ࡋࡓࠋᑐ↷⩌ẚ㍑ࡋ࡚ OX ⩌࡛ ࡣ㸪୧㑇ఏᏊᆺ࣐࢘ࢫࡢዲሷᇶᛶኚ␗⫢⣽⬊ᕢࡢⓎ⏕ᩘࡀ᭷ពቑຍࡋࡓࡀ㸪ዲሷᇶᛶ⫢ ⣽⬊⭢⭘ࡢⓎ⏕ᩘ᭷ព࡞ᕪࡣ࡞ࡗࡓࠋᑐ↷⩌ࡢ WT ࣐࢘ࢫ࠾ࡅࡿዲ㓟ᛶኚ␗⫢⣽⬊ ᕢ࠾ࡼࡧ⫢⣽⬊⭢⭘㸪ዲሷᇶᛶ⫢⣽⬊⭢⭘㸪࡞ࡽࡧࡑࡢኚ␗⫢⣽⬊ᕢࡢⓎ⏕ᩘࡣ㸪ྠ ⩌ࡢ CARKO ࣐࢘ࢫࡢⓎ⏕ᩘẚ㍑ࡋ࡚᭷ព࡞ప್ࢆ♧ࡋࡓࠋ 19.
(21) ⫢⣽⬊⭢⭘ࢆ┤ᚄࢧࢬ࡛ศ㢮ࡋ㸪ࡑࡢศᕸࢆ Fig. 1-6C ♧ࡋࡓࠋᑐ↷⩌ẚ㍑ࡋ࡚㸪 OX ᢞ⩌ࡢ WT ࣐࢘ࢫ࡛ࡣࡍ࡚ࡢࢧࢬࡢ⫢⣽⬊⭢⭘ࡀቑຍࡋࡓࠋ OX ᢞ⩌ࡢ CARKO ࣐࢘ࢫ࡛ࡣ small㸪middle ࠾ࡼࡧ large ศ㢮ࡉࢀࡓ⫢⣽⬊⭢⭘ࡀቑຍࡋࡓࡀ㸪very large ศ㢮ࡉࢀࡓ⫢⣽⬊⭢⭘ࡣቑຍࡋ࡞ࡗࡓࠋ OX ᢞ⩌ࡢ୧㑇ఏᏊᆺ࣐࢘ࢫ࡛ࡣ㸪⫢⣽⬊ࡢ༢⣽⬊ቯṚࡸࡧࡲࢇᛶ༢᰾⣽⬊ᾐ₶࡞ࡢ ⣽⬊യᐖᛶኚࡀᐇ㦂 1 ࠾ࡼࡧ 2 ྠᵝㄆࡵࡽࢀࡓࠋ. 20.
(22) ⪃ ᐹ. ᮏᐇ㦂࠾࠸࡚㸪OX ᢞ⩌ࡢ CARKO ࣐࢘ࢫ࠾ࡅࡿቑṪᛶኚࡢⓎ⏕㢖ᗘ࠾ࡼࡧⓎ⏕ᩘ ࡣ㸪ྠ⩌ࡢ WT ࣐࢘ࢫࡢ್ẚ㍑ࡋ࡚ῶᑡࡋࡓࡀ㸪ᑐ↷⩌ࡢ CARKO ࣐࢘ࢫࡢ್ẚ㍑ࡋ ࡚ቑຍࡋࡓࠋࡇࡢ⤖ᯝࡣ㸪CAR ࡀ OX ㄏⓎ⫢Ⓨࡀࢇ୍㒊㛵ࢃࡗ࡚࠸ࡿࡶࡢࡢ㸪CAR ௨እ ࡢᅉᏊࡀ⫢Ⓨࡀࢇ㔜せ࡞ാࡁࢆࡋ࡚࠸ࡿࡇࢆ♧၀ࡋ࡚࠸ࡓࠋࡆࡗṑ㢮ࡢ⫢Ⓨࡀࢇึᮇ ࠾࠸࡚㔜せ࡞㑇ఏᏊ࠶ࡿ࠸ࡣ⣽⬊ࣞ࣋ࣝࡢᶵᗎࡣ࡚ࡀ᫂ࡽ࡞ࡗ࡚࠸࡞࠸ࡀ㸪PB ࠾ ࡼࡧ PB 㢮ఝࡍࡿ CAR άᛶࡀㄏⓎࡍࡿ⫢Ⓨࡀࢇࡣ CAR ࡀ㔜せ࡞ാࡁࢆࡍࡿࡇࡀ ሗ࿌ࡉࢀ࡚࠸ࡿ㸦37㸪52㸪67㸪69㸧 ࠋᮏ❶࡛ OX ࡣ CAR ࡢࢱ࣮ࢤࢵࢺ࡛࠶ࡿ Cyp2b10 㑇ఏ Ꮚ࠾ࡼࡧ CYP2B ⺮ⓑࡢⓎ⌧ࢆቑຍࡉࡏࡓࡀ㸪OX ࡢᢞᮇ㛫ࡀᘏࡧࡿࡘࢀ࡚ Cyp2b10 Ⓨ ⌧ࡢ⛬ᗘࡣᙅࡲࡗࡓࠋCAR ࡢάᛶࡣ⅖ࡼࡾᢚไࡉࢀࡿࡇࡀሗ࿌ࡉࢀ࡚࠾ࡾ㸦30㸧 㸪ᮏ ❶࠾ࡅࡿ Cyp2b10 Ⓨ⌧ࡢᢚไࡣ㸪OX ࡼࡿ⫢⮚࠾ࡅࡿ⅖ࡀ୍ᅉࡋ࡚᥎ᐹࡉࢀࡓࠋ ࡲࡓ㸪PB ࠾ࡼࡧࡑࡢ CAR άᛶࢆᢞࡋࡓ࣐࢘ࢫࡢ⫢⮚࡛ࡣ㸪ග㢧ୗ࡛ࡍࡾ◪Ꮚ≧ ࡢዲ㓟ᛶ⣽⬊㉁ࢆ♧ࡍ⫢⣽⬊⫧㸪㟁Ꮚ㢧ᚤ㙾ୗ࡛⫢⣽⬊ෆࡢᑠ⬊యቑ⏕ࢆඹ㏻ࡢᙧែീ ࡋ࡚ㄆࡵࡿࡀ㸦33㸧 㸪ᮏ❶࡛ࡇࢀࡽࡢኚࡣㄆࡵࡽࢀ࡞ࡗࡓࠋࡇࢀࡽࡢ⤖ᯝࡣ OX ㄏⓎ ⫢Ⓨࡀࢇᑐࡍࡿ CAR ࡢ㛵ࡀ㝈ᐃⓗ࡛࠶ࡿࡇࢆᨭᣢࡍࡿࡶࡢ࡛࠶ࡗࡓࠋ OX ㄏⓎ⫢Ⓨࡀࢇ㛵ࡍࡿ CAR ௨እࡢᅉᏊࡋ࡚㸪⣽⬊യᐖ࠾ࡼࡧ 33$5Į ࡢᙳ㡪ࢆ᳨ ウࡋࡓࠋ⣽⬊യᐖᛶኚࡋ࡚㸪ᮏ❶࠾࠸࡚ OX ࡣ⫢⣽⬊༢⣽⬊ቯṚ㸪⅖㸪ALT ቑຍ ࡞ࢆច㉳ࡋࡓࠋࡇ࠺ࡋࡓ⣽⬊Ṛࡸ⅖ࢆึᮇࡽక࠺യᐖᛶኚࡣ㸪PB ᢞࡢ⫢⮚࡛ࡣ ច㉳ࡉࢀ࡞࠸㸦52㸪68㸧ࠋInternational Programme on Chemical Safety ࡼࡿࡆࡗṑ㢮⫢⭘⒆Ⓨ ⏕ᶵᗎࡢศ㢮࠾࠸࡚㸪⣽⬊യᐖࡑࢀ⥆Ⓨࡍࡿᣢ⥆ⓗ࡞⣽⬊ቑṪᛂࡣ⫢Ⓨࡀࢇ㔜 せ࡞స⏝ᶵᗎ࡛࠶ࡿࡇࡀཷࡅධࢀࡽࢀ࡚࠸ࡿ㸦20㸧 ࠋᮏ❶࡛ࡣ⣽⬊ቑṪάᛶࢆ♧ࡍ PCNA 㝧ᛶ⫢⣽⬊⋡ࡀᣢ⥆ⓗቑຍࡋ࡚࠾ࡾ㸪OX ㄏⓎ⫢Ⓨࡀࢇࡣ⣽⬊യᐖࡀ㔜せ࡞ാࡁࢆࡋ࡚ ࠸ࡿ⪃࠼ࡽࢀࡓࠋ ⫢⣽⬊࠾ࡅࡿ࣏ࣝࣇࣜࣥỿ╔ࡣ㸪ᢠ⳦ࢢࣜࢭ࢜ࣇࣝࣅࣥࡢሗ࿌ࡽ⣽⬊യᐖࢆㄏ 21.
(23) Ⓨࡍࡿࡇࡀ▱ࡽࢀ࡚࠸ࡿࠋࢢࣜࢭ࢜ࣇࣝࣅࣥࡣ࣒࣊ྜᡂ⤒㊰ࡢ᭱⤊㓝⣲࡛࠶ࡿ㕲ຍ㓝 ⣲ࢆ㜼ᐖࡋ㸪⾑₢࠾ࡼࡧ⫢⮚୰ࡢ PPIX ್ࡢቑຍ㸪⫢⣽⬊ࡢ࣏ࣝࣇࣜࣥỿ╔㸪ࡑࡋ࡚⣽⬊ യᐖࢆㄏⓎࡍࡿ㸦19㸪27㸪64㸧 ࠋࡋࡋ㸪ᮏ❶ࡢ OX ᢞ⩌࡛ࡣ⫢⣽⬊ࡢ࣏ࣝࣇࣜࣥỿ╔ ࡣㄆࡵࡽࢀ࡞ࡗࡓࠋ࣏ࣝࣇࣜࣥ⤖ᬗࡣ೫ග㢧ᚤ㙾ୗ࡛ᒅᢡᛶࢆ♧ࡍ≀㉁ࡋ࡚㟁Ꮚ㢧 ᚤ㙾ୗ࡛⤖ᬗᵓ㐀ࡋ࡚ㄆࡵࡽࢀࡿࡀ㸦25㸧 㸪ᮏ❶࡛ࡣ࠸ࡎࢀࡶᤊ࠼ࡽࢀ࡞ࡗࡓࠋ ࡉࡽ㸪ᮏ❶࠾ࡅࡿ 1000 ppm OX ࢆ 4 㐌㛫ᢞࡋࡓ WT ࣐࢘ࢫࡢ⫢⮚୰࠾ࡼࡧ⾑₢୰ PPIX ࡢ್㸦29.5 ± 2.0 ȝg/g ࠾ࡼࡧ 15.1 ± 1.5 ȝg/100 mL㸧 ࡣ㸪Poh-Fitzpatrick ࡽࡀሗ࿌ࡋࡓ 20000 ppm ࢢࣜࢭ࢜ࣇࣝࣅࣥࢆ 4 㐌㛫ᢞࡋࡓ࣐࢘ࢫࡢ್㸦1167 ± 1023 ȝg/g ࠾ࡼࡧ 209 ± 274 ȝg/100 mL㸧ẚ㍑ࡋ࡚㢧ⴭపࡗࡓ㸦50㸧 ࠋࡇࢀࡽࡢ㐪࠸ࡽ㸪࣏ࣝࣇࣜࣥࡣ OX ࡀㄏ Ⓨࡍࡿ⣽⬊യᐖࡢᅉ࡛ࡣ࡞࠸⪃࠼ࡽࢀࡓࠋOX ᢞ⩌࡛ࡣ⫢⣽⬊ࡢ࣏ࣝࣇࣜࣥ✚ࡸ ⫢⮚୰ PPIX ್ࡢ⤒ⓗ࠾ࡼࡧ⏝㔞┦㛵ⓗ࡞ቑຍࡣㄆࡵࡽࢀ࡞ࡗࡓࠋቑຍࡀㄆࡵࡽࢀ࡞ࡗ ࡓ୍ᅉࡋ࡚㸪PPIX ࡀ⫢⮚ࡽ⫹Ồ୰࠶ࡿ࠸ࡣ⾑୰ἥࡉࢀࡓࡇࡀ⪃࠼ࡽࢀࡓࠋ ᮏ❶࡛ࡣ OX ᢞࡼࡿ 33$5Į ࡢ㛵㐃ᛶࡘ࠸࡚ࡶ᳨ウࡋࡓࠋᮏ❶ࡢ OX ᢞ⩌࡛ㄆࡵ ࡓ Cyp4a10 㑇ఏᏊ࠾ࡼࡧ CYP4A ⺮ⓑⓎ⌧ࡢቑຍ㸪࡞ࡽࡧ⫢⣽⬊୰ࡢ࣌ࣝ࢜࢟ࢩࢰ࣮࣒ቑ ⏕ࡣ 33$5Į άᛶࢆ♧ࡍኚ࡛࠶ࡗࡓ㸦2㸪11㸪48㸧 ࠋࡇࢀࡽࡢ⤖ᯝࡣ㐣ཤࡢሗ࿌୍⮴ࡋ ࡚࠸ࡓ㸦51㸧 ࠋ33$5Į ࡢάᛶࡣቑṪไᚚ㑇ఏᏊࡢⓎ⌧ㄏᑟ㸪⣽⬊ቑṪάᛶࡢቑຍ㸪ࡑࡋ࡚ ࠋࡋࡓࡀࡗ࡚㸪PPARĮ ࡢάᛶࡣ㸪CAR ࠾ࡼࡧ⣽⬊യᐖ ⭘⒆ᛶኚࡢᙧᡂࢆಁࡍ㸦6㸪26㸧 ྠᵝ OX ㄏⓎ⫢Ⓨࡀࢇᐤࡋ࡚࠸ࡿ⪃࠼ࡽࢀࡓࠋ OX ࡣ Cyp3a11 㑇ఏᏊ࠾ࡼࡧ CYP3A ⺮ⓑࡢⓎ⌧ࢆቑຍࡉࡏ࡚࠾ࡾ㸪᰾ෆཷᐜయࡢ୍ࡘ࡛ ࠶ࡿ pregnane X Receptor 㸦PXR㸧ࢆάᛶࡉࡏࡿ⪃࠼ࡽࢀࡓࠋOX ࡣࣄࢺ PXR ࢆάᛶࡉ ࡏࡿࡇࡀሗ࿌ࡉࢀ࡚࠸ࡿ 㸦35㸧 ࠋࡋࡋ㸪PXR ࡢάᛶࡣ࣐࢘ࢫࡢ⫢⣽⬊ቑṪࢆច㉳ࡋ ࡞࠸ࡇࡀ▱ࡽࢀ࡚࠸ࡿ㸦54㸧 ࠋࡲࡓ㸪OX ࡣ AhR ࡢάᛶࢆ♧ࡍ Cyp1a1 ࠾ࡼࡧ Cyp1a2 㑇. ఏᏊࡢⓎ⌧㔞ࢆቑຍࡉࡏࡓࡀ㸪CYP1A ⺮ⓑࡢⓎ⌧ࡣቑຍࡉࡏ࡞ࡗࡓࠋࡋࡓࡀࡗ࡚㸪OX ㄏⓎ⫢Ⓨࡀࢇᑐࡍࡿ PXR ࠾ࡼࡧ AhR ࡢ᫂☜࡞㛵ࡣㄆࡵࡽࢀ࡞ࡗࡓࠋ ᮏ❶࡛ࡣ⣽⬊യᐖ㸪CAR㸪࠾ࡼࡧ 33$5Į ࡀ OX ㄏⓎ⫢Ⓨࡀࢇ㛵ࡋ࡚࠸ࡿࡇࡀ♧၀ 22.
(24) ࡉࢀࡓࠋࡋࡋ㸪ࡇࢀࡽ୕ࡘࡢᅉᏊࡢ┦㛵ಀࡣ࡛᫂࠶ࡗࡓࠋ⣽⬊യᐖࢆࡋࡓࡆࡗṑ 㢮⫢Ⓨࡀࢇస⏝ᶵᗎࡣࣄࢺእᤄྍ⬟⪃࠼ࡽࢀ࡚࠾ࡾ㸪OX ࡀㄏⓎࡋࡓ⣽⬊യᐖࡣࣄࢺࡢ ⫢Ⓨࡀࢇࣜࢫࢡࢆホ౯ࡍࡿୖ࡛≉㔜せ⪃࠼ࡽࢀࡓࠋ. 23.
(25) ᑠ ᣓ. ➨ 1 ❶࡛ࡣ㸪 CARKO ࣐࢘ࢫࢆ⏝࠸࡚ OX ㄏⓎ⫢Ⓨࡀࢇᑐࡍࡿ CAR ࡢ㛵ࢆ᳨⣴ࡋࡓࠋ 1000 ppm OX ࢆ 1, 4 ࠾ࡼࡧ 13 㐌㛫ΰ䭱ᢞࡋࡓ WT ࣐࢘ࢫࡢ⫢⮚࡛ࡣ Cyp2b10 㑇ఏᏊࡢⓎ ⌧ࡀቑຍࡋࡓࡀ㸪CARKO ࣐࢘ࢫࡢ⫢⮚࡛ࡣ᭷ព࡞ቑຍࡣㄆࡵࡽࢀ࡞ࡗࡓࠋྠ㸪OX ࡣ PPARĮ άᛶ࠾ࡼࡧ⫢⣽⬊࠾ࡅࡿയᐖᛶኚࢆច㉳ࡋࡓࡀ㸪ࡑࡢ⛬ᗘ㑇ఏᏊᆺ㛫ࡢ ᕪࡣㄆࡵࡽࢀ࡞ࡗࡓࠋ⫢⣽⬊ࡢ࣏ࣝࣇࣜࣥỿ╔ࡣࡍ࡚ࡢ࣐࢘ࢫ࡛ㄆࡵࡽࢀ࡞ࡗࡓࠋ DEN ࡼࡿࢽࢩ࢚࣮ࢩࣙࣥฎ⌮ᚋ OX ࢆ 26 㐌㛫ΰ䭱ᢞࡋࡓ⤖ᯝ㸪ኚ␗⫢⣽⬊ᕢ࠾ࡼࡧ ⫢⣽⬊⭢⭘ࡀㄏⓎࡉࢀࡓࠋOX ᢞ⩌ࡢ CARKO ࣐࢘ࢫ࠾ࡅࡿኚ␗⫢⣽⬊ᕢ㸪⫢⣽⬊⭢⭘ ࡢⓎ⏕㢖ᗘ࠾ࡼࡧⓎ⏕ᩘࡣྠ⩌ࡢ WT ࣐࢘ࢫẚ㍑ࡋ࡚ప್ࢆ♧ࡋࡓࡀ㸪ᑐ↷⩌ࡢ CARKO ࣐࢘ࢫẚ㍑ࡋ࡚ప್ࢆ♧ࡋࡓࠋ ௨ୖࡼࡾ㸪CAR㸪33$5Į㸪࠾ࡼࡧ⣽⬊യᐖࡀ OX ㄏⓎ⫢Ⓨࡀࢇ」ྜⓗ㛵ࡋ࡚࠸ࡿࡇ ࡀ♧ࡉࢀࡓࠋணࡍࡿ⤖ᯝࡋ࡚㸪࣏ࣝࣇࣜࣥࡣ OX ㄏⓎ⣽⬊യᐖࡢせᅉ࡛ࡣ࡞ ࠸⪃࠼ࡽࢀࡓࠋ. 24.
(26) ➨2❶ ࢩࣇࣝ࢜ࣝࣇ࢙ࣥࡀㄏⓎࡍࡿ࣐࢘ࢫ⫢Ⓨࡀࢇ࠾ࡅࡿ Constitutive androstane receptor ࡢ㛵. 25.
(27) ⥴ ゝ. ➨ 1 ❶࡛ࡣ㸪࢜࢟ࢧࢪࢰࣥ㸦OX㸧ㄏⓎ⫢Ⓨࡀࢇ㸪⣽⬊യᐖ㸪Constitutive androstane receptor㸦CAR㸧 㸪࠾ࡼࡧ peroxisome proliferator-DFWLYDWHGUHFHSWRUĮ㸦33$5Į㸧ࡀ」ྜⓗ㛵 ࡋ࡚࠸ࡿࡇࡀ♧ࡉࢀࡓࠋ OX ࡣࣉࣟࢺ࣏ࣝࣇࣜࣀ࣮ࢤࣥ㓟㓝⣲㸦PROTOX㸧㜼ᐖࡼࡤࢀࡿࢲࢪࣥ⣔㝖 ⲡ࡛࠶ࡿࡀ㸪ࡑࡢ⣔⤫ࡢ PROTOX 㜼ᐖࡋ࡚ࢩࣇࣝ࢜ࣝࣇ࢙ࣥ㸦Acifluorfen, ACI㸧 ࡞ࡢࢪࣇ࢙ࢽ࢚࣮ࣝࢸࣝ⣔㝖ⲡࡀ▱ࡽࢀ࡚࠸ࡿ㸦41㸧ࠋOX ྠᵝ ACI ࡣࣛࢵࢺ࠾ࡼ ࡧ࣐࢘ࢫࢆ⏝࠸ࡓ៏ᛶẘᛶ㸭Ⓨࡀࢇᛶ」ྜヨ㦂࠾࠸࡚⫢⮚ࡢ⣽⬊യᐖ࠾ࡼࡧ⫢⭘⒆ࢆច ㉳ࡍࡿࡇࡀሗ࿌ࡉࢀ࡚࠸ࡿࡀ㸪ࡑࡢヲ⣽࡞ᶵᗎࡣ࡛᫂࠶ࡿ㸦EPA Reregistration Eligibility Decision for Sodium Acifluorfen. Case No. 2605, available at: http://archive.epa.gov/pesticides/reregistration/web/pdf/acifluorfen_red.pdf㸧 ࠋ ACI ࡣ 33$5Į άᛶస⏝ࡀሗ࿌ࡉࢀ࡚࠸ࡿ㸦61㸧 ࠋACI CAR ࡢ㛵㐃ᛶࡘ࠸࡚ࡣሗ࿌ ࡀ࡞࠸ࡀ㸪ACI ྠࡌࢪࣇ࢙ࢽ࢚࣮ࣝࢸࣝ⣔㝖ⲡ࡛࠶ࡿ࢜࢟ࢩࣇࣝ࢜ࣝࣇ࢙ࣥࡣ࣐࢘ࢫ⫢ ⮚ࡢ 33$5Į ࠾ࡼࡧ CAR ࢆάᛶࡉࡏࡿࡇࡀሗ࿌ࡉࢀ࡚࠸ࡿ㸦60㸧 ࠋࡋࡓࡀࡗ࡚㸪࣐࢘ࢫ ࠾ࡅࡿ ACI ㄏⓎ⫢Ⓨࡀࢇࡣ㸪OX ㄏⓎ⫢Ⓨࡀࢇྠᵝ⣽⬊യᐖ㸪33$5Į㸪CAR ࡞ ࡢస⏝ᶵᗎࡀ」ྜⓗ㛵ࡋ࡚࠸ࡿྍ⬟ᛶࡀ࠶ࡿࠋ ➨ 2 ❶࡛ࡣ㸪OX ࡛ㄆࡵࡽࢀࡓ⫢Ⓨࡀࢇᑐࡍࡿ」ྜⓗ࡞స⏝ᶵᗎࡢ㛵ࡀ PROTOX 㜼 ᐖඹ㏻ࡋࡓኚ࡛࠶ࡿ☜ࡵࡿࡓࡵ㸪ACI ㄏⓎ⫢Ⓨࡀࢇࡢస⏝ᶵᗎࢆ᳨⣴ࡋࡓࠋᮏ❶ ࡛ࡣ≉ CAR ╔┠ࡋ᳨࡚ウࡍࡿࡓࡵ㸪CAR άᛶࡋ࡚ᗈࡃ▱ࡽࢀࡿࣇ࢙ࣀࣂࣝࣅࢱ ࣮ࣝ㸦PB㸧ࢆྠᐇ㦂⏝࠸ࡓࠋ. 26.
(28) ᮦᩱ࠾ࡼࡧ᪉ἲ. Ꮫ≀㉁ ACI㸦CAS No. 50594-66-6㸪⣧ᗘ 99.6%㸧࠾ࡼࡧ PB ࢼࢺ࣒ࣜ࢘㸦CASNo. 57-30-7㸪⣧ᗘ 98%㸧 ࡣ WAKO Pure Chemicals Industries, Ltd.㸦㜰㸧ࡼࡾ㸪diethylnitrosamine㸦DEN㸪CAS No. 55-18-5㸪 ⣧ᗘ 99.9%㸧ࡣ Tokyo Chemical Industry Co., Ltd.㸦ᮾி㸧ࡼࡾࡑࢀࡒࢀ㉎ධࡋࡓࠋ. ౪ヨື≀ ᰿ᓊ㞞ᙪඛ⏕㸦National Institute of Environmental Health Sciences㸧ࡽᥦ౪࠸ࡓࡔ࠸ࡓ C3H/HeNCrl ⣔⤫ࡢ CAR ࣀࢵࢡ࢘ࢺ㸦KO㸧࣐࢘ࢫ㸦28㸪69㸧ࢆᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ ᡤ࡚㣫⫱ࡋ㸪ᐇ㦂౪ࡋࡓࠋ5 㐌㱋ࡢ C3H/HeNCrl ࣐࢘ࢫࢆ᪥ᮏࢳ࣮ࣕࣝࢫ࣭ࣜࣂ࣮ᰴᘧ ♫㸦⚄ዉᕝ㸧ࡽ㉎ධࡋ࡚㔝⏕ᆺ㸦WT㸧࣐࢘ࢫࡋ࡚ᐇ㦂౪ࡋࡓࠋC3H/HeNCrl ⣔⤫ ࡣ㸪B6C3F1 ࡸ C57BL/6 ࡞ࡑࡢ⣔⤫ẚ࡚㸪⫢⭘⒆ዲⓎ࡞⣔⤫࡛࠶ࡿ㸦39㸧 ࠋࡍ ࡚ࡢື≀ᐇ㦂ィ⏬ࡣᐇ㦂タ㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ㸧ࡢື≀ᐇ㦂ጤဨࡢᢎㄆࢆཷ ࡅ㸪ື≀ᐇ㦂᧯సࡣື≀ࡢ⏝⟶⌮㛵ࡍࡿᣦ㔪ࢆ㑂Ᏺࡋ࡚⾜ࡗࡓࠋ. ᐇ㦂ࢹࢨࣥ ᐇ㦂 1 ࡛ࡣ ACI ࠾ࡼࡧ PB ࡢ▷ᮇᢞࡼࡿ⫢⮚ࡢᙳ㡪ࢆ᳨ウࡋࡓࠋ6 㐌㱋ࡢ㞝 WT ࣐࢘ ࢫ࠾ࡼࡧ CARKO ࣐࢘ࢫࢆ 1㸪4㸬࠾ࡼࡧ 13 㐌ᢞ⩌㸦5 ༉㸭⩌㸧ࡑࢀࡒࢀ⩌ศࡅࡋࡓࠋ 2500 ppm ACI ࠾ࡼࡧ 500 ppm PB ࢆᇶ♏㣫ᩱ㸦CAF-1㸪࢚࢜ࣜࣥࢱࣝ㓝ẕᕤᴗᰴᘧ♫㸪ᮾ ி㸧ࡑࢀࡒࢀΰࡌ㸪6 㐌㱋ࡽ⮬⏤ᦤ䭱ࡉࡏࡓࠋᢞᮇ㛫ࡣ㐣ཤࡢヨ㦂ᇶ࡙࠸࡚タᐃ ࡋࡓ㸦37㸧 ࠋᮏ❶࠾ࡅࡿ ACI ࡢᢞ⃰ᗘࡣ㸪ணഛᐇ㦂㸦625㸪1250㸪࠾ࡼࡧ 2500 ppm ACI ࡢ 4 㐌㛫ΰ䭱ᢞ㸧࡛᫂ࡽ࡞⫢⮚ࡢ⣽⬊യᐖࢆ♧ࡋࡓ 2500ppm ࡋࡓࠋPB ࡢᢞ⃰ᗘࡣ 㛗ᮇᢞ࠾࠸࡚⫢⭘⒆ࡀሗ࿌ࡉࢀ࡚࠸ࡿ 500ppm ࡋࡓ㸦24㸪52㸧 ࠋACI ࠾ࡼࡧ PB ᢞ⩌ ྠᵝ㸪ᑐ↷⩌ࡣᇶ♏㣫ᩱࢆ⮬⏤ᦤ䭱ࡉࡏࡓࠋ 27.
(29) ᐇ㦂 2 ࡛ࡣ ACI ࠾ࡼࡧ PB ࡀㄏⓎࡍࡿ⫢Ⓨࡀࢇᑐࡍࡿ CAR ࡢᙳ㡪ࢆ᳨ウࡋࡓࠋ㐣ཤࡢ ሗ࿌ྠᵝ㸪6 㐌㱋ࡢࡍ࡚ࡢ WT ࠾ࡼࡧ CARKO ࣐࢘ࢫ㸪⫢Ⓨࡀࢇࢽࢩ࢚࣮ࢱ࣮࡛࠶ ࡿ DEN ࢆ 90 mg/kg ࡛༢ᅇ⭡⭍ෆᢞࡋࡓ㸦52㸧 ࠋDEN ᢞࡢ 2 㐌㛫ᚋ㸪2500 ppm ACI ࠶ࡿ ࠸ࡣ 500 ppm PB ࢆྛ⩌ࡢ࣐࢘ࢫ㸦24㹼25 ༉㸭㑇ఏᏊᆺ㸧 26 㐌㛫ΰ䭱ᢞࡋࡓࠋ ᐇ㦂 1 ࠾ࡼࡧ 2 ࡢᢞᮇ㛫୰㸪ࡍ࡚ࡢ࣐࢘ࢫࡢ୍⯡≧ែࢆᑡ࡞ࡃࡶ 1 ᪥ 1 ᅇほᐹࡋ ࡓࠋయ㔜ᦤ䭱㔞ࢆ㐌 1 ᗘ ᐃࡋ㸪ᦤ䭱㔞ࡽ ACI ࠾ࡼࡧ PB ࡢ 1 ᪥ᙜࡓࡾᦤྲྀ㔞 㸦mg/kg/day㸧ࢆ⟬ฟࡋࡓࠋゎ๗㸪ࢯࣇࣝࣛࣥ㯞㓉ୗ࡛ࡍ࡚ࡢ࣐࢘ࢫࡢ㟼⬦ࡽ᥇ ⾑ࡋ㸪ᨺ⾑ࡼࡿᏳᴦṚࢆ⾜ࡗࡓࠋ⾑Ύࡣ-30Υ࡛ಖ⟶ࡋ㸪ᰴᘧ♫࢚ࢫ࣮࢚ࣝࣝ㸦ᮾி㸧 ࡚⾑Ύ୰ࣛࢽ࣑ࣥࣀᇶ㌿⛣㓝⣲㸦ALT㸧ࢆ ᐃࡋࡓࠋ. ⌮⤌⧊Ꮫⓗゎᯒ ᐇ㦂 1 ࡛ࡣ㸪⫢⮚㔜㔞 ᐃᚋ㸪ᕥⴥ࠾ࡼࡧ୰㛫ⴥࢆ 10㸣୰ᛶ⦆⾪࣐࣍ࣝࣜࣥᾮ࡚ᅛᐃ ࡋࡓࠋᅛᐃࡋࡓ⫢⮚ࡣᐃᆺࡢฎ⌮ࢆ⾜ࡗࡓᚋⷧษࡋ㸪࣐࣊ࢺ࢟ࢩ࣭࢚ࣜࣥ࢜ࢪࣥ㸦HE㸧ᰁ Ⰽ㸪ࢩ࣮ࣗࣔࣝᛂ㸪࣌ࣝࢩࣥࣈ࣮ࣝᰁⰍ㸪࠾ࡼࡧ㐣ࣚ࢘⣲㓟ࢩࢵࣇ㸦PAS㸧ᰁⰍࢆ⾜ࡗ ࡓࠋගᏛ㢧ᚤ㙾࠶ࡿ࠸ࡣ೫ග㢧ᚤ㙾ࢆ⏝࠸࡚స〇ࡋࡓᶆᮏࢆほᐹࡋࡓࠋᢞ 4 㐌┠ࡢ࣐࢘ ࢫࡢ⫢⮚ࢆ⣽ษࡋ㸪2%ࢢࣝࢱࣝࣝࢹࣄࢻ㸦pH7.4㸧࡛ 4 㛫ᅛᐃࡋࡓࠋ⥆࠸࡚ 1%ᅄ㓟 ࢜ࢫ࣑࣒࢘⦆⾪ᾮ 2 㛫ᅛᐃࡋࡓᚋ㸪࢚࣏ࣥ 812 ໟᇙࡋࡓࠋ㓑㓟࢘ࣛࢽࣝ࠾ࡼࡧࢡ࢚ ࣥ㓟㖄࡛ᾐࡋࡓ㉸ⷧษ∦ࢆ㟁Ꮚ㢧ᚤ㙾㸦JEM-1400㸹᪥ᮏ㟁Ꮚᰴᘧ♫㸪ᮾி㸧ࢆ⏝࠸࡚ホ ౯ࡋࡓࠋ ᐇ㦂 2 ࡛ࡣ㸪࣐࣍ࣝࣜࣥᅛᐃࡋࡓࡍ࡚ࡢⴥࡢ⫢⮚ࢆ 14㹼16 ᩿㠃ษࡾฟࡋ㸪స〇ࡋࡓ HE ᶆᮏࢆ⏝࠸࡚⌮⤌⧊Ꮫⓗ᳨ᰝࢆ⾜ࡗࡓࠋቑṪᛶኚࡢⓎ⏕㢖ᗘ㸦ྛኚࢆㄆࡵࡿ࣐࢘ ࢫࡢ༉ᩘ㸧Ⓨ⏕ᩘ㸦࣐࢘ࢫẖࡢኚᩘ㸧ࢆ⟬ฟࡋࡓࠋ⫢ቑṪᛶኚࡢデ᩿ࡣ International Harmonization of Nomenclature and Diagnostic Criteria of Lesions in Rats and Mice㸦65㸧ᚑࡗ ࡓࠋࡉࡽ㸪ኚ␗⫢⣽⬊ᕢ࠾ࡼࡧ⫢⣽⬊⭢⭘ࡣዲ㓟ᛶ㸪ዲሷᇶᛶ㸪࠾ࡼࡧࡑࡢ㸦✵⬊ࢆ ከᩘ᭷ࡍࡿኚ㸧ࡢ୕ࡘศ㢮ࡋࡓࠋࡲࡓ㸪⤖ᯝᚋ㏙ࡍࡿ࠾ࡾ㸪ᮏ❶࡛ࡣᢞ㉳ᅉࡢ 28.
(30) ኚࡋ࡚⏕ᛶ⫢⣽⬊㐣ᙧᡂࡀㄆࡵࡽࢀࡓࠋ⏕ᛶ⫢⣽⬊㐣ᙧᡂࡣᣢ⥆ⓗ࡞⫢⮚ࡢ⣽⬊ യᐖᑐࡍࡿ⏕ᛂࡼࡗ࡚ᙧᡂࡉࢀࡿሗ࿌ࡉࢀ㸪ᙧែᏛⓗࡣ୰ᚰ㟼⬦㸪⫹⟶㸪࠶ ࡿ࠸ࡣ࣮࢜ࣂࣝ⣽⬊࡞ࢆㄆࡵ㸪ᑠⴥᏑᅾࡍࡿ᪤Ꮡࡢᵓ㐀ࡀಖࡓࢀ࡚࠸ࡿࡇࢆ≉ᚩ ࡍࡿ㸦65㸧 ࠋࡋࡋ࡞ࡀࡽ㸪࣐࢘ࢫࡢ⫢⣽⬊⭢⭘ࡣ␗ᆺᛶࡀஈࡋ࠸ࡶࡢࡶከࡃ㸪⭘⒆㛫 ᪤Ꮡ⤌⧊ࢆㄆࡵࡿሙྜࡶ࠶ࡾ㸪⏕ᛶ⫢⣽⬊㐣ᙧᡂࡣ⫢⣽⬊⭢⭘ࡢ㚷ูࡀᅔ㞴࡛࠶ࡗࡓࠋ ࡑࡇ࡛㸪ᮏ❶࡛ࡣ୰ᚰ㟼⬦㸪⫹⟶ୖ⓶㸪࠶ࡿ࠸ࡣ࣮࢜ࣂࣝ⣽⬊࡞᪤Ꮡࡢ⫢⮚Ꮡᅾࡍࡿ ᡂศࢆ᫂ࡽㄆࡵࡿኚࢆ⏕ᛶ⫢⣽⬊㐣ᙧᡂศ㢮ࡋࡓࠋ. ච⤌⧊Ꮫⓗゎᯒ ᐇ㦂 1 ࡢ⫢⮚ࡽࣃࣛࣇࣥໟᇙษ∦ࢆస〇ࡋ㸪CYP1A ᢠయ㸦Abcam, Cambridge, UK; ab111868; 200 ಸᕼ㔘㸧㸪CYP2B ᢠయ㸦Abcam; ab69652; 500 ಸᕼ㔘㸧 㸪CYP3A ᢠయ㸦Abcam; ab3572; 500 ಸᕼ㔘㸧㸪࠾ࡼࡧ CYP4A ᢠయ㸦Abcam; ab140635; 500 ಸᕼ㔘㸧 4°C ୍࡛ᬌ ᛂࡉࡏࡓࠋࡑࡢᚋ㸪࣌ࣝ࢜࢟ࢩࢲ࣮ࢮᶆ㆑ࢹ࢟ࢫࢺ࣏࣐࣮ࣛࣥࣜࢆ⤖ྜࡉࡏࡓḟᢠయ㸦ࣄ ࢫࢺࣇࣥࢩࣥࣉࣝࢫࢸࣥ MAX PO㸪ᰴᘧ♫ࢽࢳࣞࣂ࢜ࢧ࢚ࣥࢫ㸪ᮾி㸧 ᛂࡉࡏ㸪3-30-diaminobenzidine ᛂ㸦ᰴᘧ♫ྠோᏛ◊✲ᡤ㸪⇃ᮏ㸧ࡼࡾⓎⰍࡉࡏࡓࠋ ୰ᚰ㟼⬦࿘ᅖ࠶ࡿ࠸ࡣ୰ᚰ㟼⬦ࡽ୰㛫ᖏࡢ⫢⣽⬊࠾ࡅࡿⓎ⌧ࡣs㸪⫧ࡋࡓ⫢⣽⬊ ࠾ࡅࡿ㍍ᗘ࡞Ⓨ⌧ࡣ㸩㸪⫧ࡋࡓ⫢⣽⬊࠾ࡅࡿ୰⛬ᗘ௨ୖࡢⓎ⌧ࡣ㸩㸩ศ㢮ࡋࡓࠋ ⫢⣽⬊ࡢቑṪάᛶࢆ☜ㄆࡍࡿࡓࡵ㸪ྠಶయ࠾ࡅࡿ⫢⮚ࡢࣃࣛࣇࣥໟᇙษ∦ࢆ proliferating cell nuclear antigen㸦PCNA㸧ᢠయ㸦Dako, ி㒔, M0879; 100 ಸᕼ㔘㸧ᛂࡉࡏ㸪 ୖグྠᵝࡢᡭ㡰࡛ⓎⰍࡉࡏࡓࠋ㢧ᚤ㙾ࡢ 100 ಸど㔝࡚㸪ᑠⴥ㛫ᵓ㐀୰ᚰ㟼⬦㡿ᇦࢆ ྵࡴ㡿ᇦࢆ↓సⅭ 5 ᡤ㑅ࢇࡔࠋ㑅ࢇࡔ㡿ᇦࡢ PCNA 㝧ᛶ⫢⣽⬊ᩘ࠾ࡼࡧ⥲⫢⣽⬊ᩘࢆ ᐃࡋࠊ⥲⫢⣽⬊ᩘᑐࡍࡿ PCNA 㝧ᛶ⫢⣽⬊ᩘࡢྜࢆ⟬ฟࡋࡓࠋ. Real-time RT-PCR ゎᯒ ᐇ㦂 1 ࡢᕥⴥࡢ⫢⮚⤌⧊ࢆ-80Υ࡛㎿㏿⤖ࡋࡓࠋ⫢⤌⧊ࡽ ISOGEN㸦Nippon Gene Co. 29.
(31) Ltd., Tokyo, Japan㸧ࢆ⏝࠸࡚ Total RNA ࢆᢳฟࡋࡓࠋȝJ51$ ࡽ High Capacity Reverse Transcription kit㸦Applied Biosystems, Foster City, CA, USA㸧ࢆ⏝࠸࡚ cDNA ࢆྜᡂࡋࡓᚋ㸪 TaqMan Fast Universal PCR Master Mix㸦Applied Biosystems㸧 㸪TaqMan Gene Expression Assays 㸦Applied Biosystems㸧 㸪࠾ࡼࡧ 7900HT Fast Real-time PCR System㸦Applied Biosystems㸧ࢆ⏝ ࠸࡚ PCR ᛂࡉࡏࡓࠋ᧯సࡣྛ࢟ࢵࢺࡢࣉࣟࢺࢥ࣮ࣝᚑࡗࡓࠋPrimer-probe ࡣ Cyp1a2 㸦Mm00487224_m1㸧㸪Cyp2b10㸦Mm01972453_s1㸧 㸪Cyp3a11㸦Mm00731567_m1㸧 㸪Cyp4a10 㸦Mm01188913_g1㸧 㸪࠾ࡼࡧ Tnfa㸦Mm00443258_m1㸧ࢆ⏝࠸ࡓࠋྛ㑇ఏᏊࡢⓎ⌧ࡣ㸪┦ᑐ ᳨㔞⥺ἲ࡚ィ⟬ࡋࡓᚋ㸪ෆᅉᛶ GAPDH㸦TaqMan Rodent GAPDH Control Reagent; Applied Biosystems㸧࡛⿵ṇࡋࡓࠋ. ࣉࣟࢺ࣏ࣝࣇࣜࣥ IX㸦PPIX㸧 ᐃ ᐇ㦂 1 ࠾࠸࡚㸪⫢⮚୰ PPIX ࢆ㐣ཤࡢሗ࿌ᚑࡗ࡚ ᐃࡋࡓ㸦66㸧 ࠋᕥⴥࡢ⫢⮚⤌⧊ࢆ pH 7.0 ࡢ 10 mM Tris HCl ࡛ 100mg/mL ㄪᩚࡋ㸪ᆒ㉁ࡉࡏࡓࠋ⁐ゎᾮࢆ㓑㓟࢚ࢳ࣭ࣝ㓑 㓟㸦4:1㸧࡛ᢳฟࡋ㸪15 ศࡢ 1500 rpm 㐲ᚰศ㞳ࡼࡾỿẊࢱࣥࣃࢡࢆྲྀࡾ㝖࠸ࡓࠋPPIX ࢆ 1.5N ሷ㓟࡛ᢳฟࡋ㸪⺯ගගᗘィࢆ⏝࠸࡚ບ㉳Ἴ㛗 405 nm㸪⺯ගἼ㛗 590nm ࡚ ᐃࡋࡓࠋ ᳨㔞⥺ࡣ SigmaeAldrich㸦MO, USA㸧ࡼࡾ㉎ධࡋࡓ PPIX ࢆ⏝࠸࡚ 0.14 ࡽ 3.69 mM ࡢ⠊ᅖ ࡛タᐃࡋ㸪⫢⮚୰ PPIX ್ࡣ nmol/g ࡋ࡚♧ࡋࡓࠋ. ࢳ࢜ࣂࣝࣅࢶ࣮ࣝ㓟ᛂᛶ≀㉁㸦TBARS㸧 ᐃ ᐇ㦂 1 ࡢ⫢⮚୰ࢳ࢜ࣂࣝࣅࢶ࣮ࣝ㓟ᛂᛶ≀㉁㸦TBARS㸧ࢆ㐣ཤࡢሗ࿌ᚑࡗ࡚ ᐃࡋ ࡓ㸦47㸧 ࠋTBARS ࡢయࡣ⬡㉁㐣㓟ᛂࡢ⏘≀࡛࠶ࡿ࣐ࣟࣥࢪࣝࢹࣄࢻ㸦MDA㸧࡛࠶ ࡿࡇࡀ▱ࡽࢀ࡚࠸ࡿࠋᕥⴥࡢ⫢⮚⤌⧊ࢆ 1.15% KCL ࡛ᆒ㉁ࡋ㸪3000 rpm ࡛ 10 ศ㛫㐲ᚰ ศ㞳ࡋࡓࠋࡑࡢୖΎ 10 mL ࢆ㸪8.1%ࢻࢹࢩࣝ◲㓟ࢼࢺ࣒ࣜ࢘ 40mL㸪0.4%ࢳ࢜ࣂࣝࣅࢶ࣮ࣝ 㓟ࢆྵࡴ 10%㓑㓟㸦pH3.5㸧600 mL㸪࠾ࡼࡧ␃Ỉ 150 mL ΰࡌ㸪95Υ࡛ 60 ศ ࡵ㸪෭༷ ࡋࡓࠋ⥆࠸࡚㸪ΰྜ≀ n-ࣈࢱࣀ࣮ࣝ࠾ࡼࡧࣆࣜࢪࣥ㸦15:1 v/v㸧ࢆ 500 mL ຍ࠼㸪3000 rpm 30.
(32) ࡛ 10 ศ㛫㐲ᚰศ㞳ࡋࡓࠋࣈࢱࣀ࣮ࣝᒙࡢ TBARS ࢆ⺯ගගᗘィ㸦Thermo Scientific㸧ࢆ⏝࠸ ࡚ບ㉳Ἴ㛗 405 nm ࠾ࡼࡧ⺯ගἼ㛗 590nm ࡚ ᐃࡋࡓࠋTBARS ್ࡣ Pierce BCA protein assay kit㸦Thermo Scientific㸧࡛ ᐃࡋࡓ⺮ⓑ㔞࡛⿵ṇࡋࡓࠋMDA ࡢ᳨㔞⥺ࡣຍỈศゎࡉࡏ ࡓ 1,1,3,3,-tetramethoxypropane㸦Wako㸧ࢆ⏝࠸࡚タᐃࡋࡓࠋ. ࢢࣝࢱࢳ࢜ࣥ ᐃ ᐇ㦂 1 ࠾࠸࡚㸪GSH-Glo Glutathione Assay㸦Promega, Madison Wisconsin㸧ࢆ⏝࠸࡚ࢢࣝ ࢱࢳ࢜ࣥࡢᯤῬࢆホ౯ࡋࡓࠋ ᕥⴥࡢ⫢⮚⤌⧊ࢆ EDTA2mM ྵࡴࣜࣥ㓟ሷࣂࢵࣇ࣮ 㸦pH 7.4㸧 ࡛ᆒ㉁ࡋ㸪3000 rpm ࡛ 10 ศ㐲ᚰศ㞳ࡋࡓࠋࡑࡢୖΎ 10μL ࢆ㸪ࣝࢩࣇ࢙ࣜࣥㄏᑟయ࠾ࡼ ࡧࢢࣝࢱࢳ࢜ࣥ S-ࢺࣛࣥࢫࣇ࢙࣮ࣛࢮࢆྵࡴ GSH-Glo Reagent 100 μL ຍ࠼㸪㐽ගୗࡘ ᐊ ࡛ 30 ศ㛫ᛂࡉࡏࡓࠋࡑࡢᚋ㸪ࢧࣥࣉࣝ luciferin detection 100μL ࢆຍ࠼㸪15 ศ㛫 ᛂࡉࡏ㸪GloMax 20/20 Luminometer㸦Promega㸧ࢆ⏝࠸࡚⺯ගࢆ ᐃࡋࡓࠋGSH ್ࡣ Pierce BCA protein assay kit㸦Thermo Scientific㸧ࢆ⏝࠸࡚ ᐃࡋࡓ⺮ⓑ⃰ᗘ࡛⿵ṇࡋࡓࠋ. ⤫ィゎᯒ ᐃ㔞್ࡣ⩌ࡢᖹᆒ್ᶆ‽೫ᕪ࡛♧ࡋࡓࠋᢞ⩌ࡢ್ࡣᑐ↷⩌ࡢ್㸪ྛ⩌ࡢ CARKO ࣐࢘ࢫࡢ್ࡣྠ⩌ࡢ WT ࣐࢘ࢫࡢ್ࡑࢀࡒࢀẚ㍑ࡋࡓࠋ ್ࡢ➼ศᩓᛶࢆ F ᳨ᐃ࡛☜ㄆࡋ㸪 ➼ศᩓࡢሙྜࡣ Student ࡢ t ᳨ᐃ㸪➼ศᩓࡢሙྜࡣ Welch ࡢ t ᳨ᐃࢆ⾜ࡗࡓࠋStudent ࠾ࡼ ࡧ Welch ࡢ t ᳨ᐃࡣ୧ഃ࡛ᐇࡋࡓࠋᐇ㦂 2 ࠾ࡅࡿቑṪᛶኚࡢⓎ⏕㢖ᗘࡣ Fisher ἲࢆ⏝ ࠸᳨࡚ᐃࡋࡓࠋྛ᳨ᐃࡢ᭷ពỈ‽ࡣ 0.05 ࠾ࡼࡧ 0.01 ࡋࡓࠋ. 31.
(33) ⤖ ᯝ. ACI ࠾ࡼࡧ PB ᢞ⩌࠾࠸࡚ᢞ㉳ᅉࡍࡿ୍⯡≧࠾ࡼࡧṚஸࡣㄆࡵࡽࢀ࡞ࡗࡓࠋ ࡍ࡚ࡢ᳨⣴ᮇ࠾࠸࡚㸪୧㑇ఏᏊᆺ࣐࢘ࢫࡢ᭱⤊య㔜࠾ࡼࡧయ㔜ቑຍ㔞ࡣ㸪ᑐ↷⩌ ẚ㍑ࡋ࡚ ACI ᢞ⩌࡛᭷ព࡞ప್ࢆ♧ࡋࡓ㸦Table 2-1㸧ࠋᢞ 13 㐌┠࠾ࡼࡧ 26 㐌┠࠾࠸ ࡚㸪ᑐ↷⩌ࡢ CARKO ࣐࢘ࢫࡢయ㔜ࡣྠ⩌ࡢ WT ࣐࢘ࢫࡢ್ẚ㍑ࡋ࡚᭷ព࡞ቑຍᢚไࢆ ♧ࡋࡓ㸦Table 2-1㸧ࠋᢞ 4 㐌┠ࡽ 26 㐌┠ࡅ࡚㸪ᑐ↷⩌ࡢ CARKO ࡢయ㔜ቑຍ㔞ࡣ ྠ⩌ࡢ WT ࣐࢘ࢫࡢ್ẚ㍑ࡋ࡚᭷ព࡞ప್ࢆ♧ࡋࡓ㸦Table 2-1㸧ࠋ୧㑇ఏᏊᆺ࣐࢘ࢫࡢᦤ 䭱㔞ࡣ㸪ᑐ↷⩌ẚ㍑ࡋ࡚ ACI ᢞ⩌࡛ప್ࢆ♧ࡋࡓࠋᢞ 13 㐌┠࠾ࡅࡿ WT ࠾ࡼࡧ CARKO ࣐࢘ࢫࡢ ACI ᦤྲྀ㔞ࡣࡑࢀࡒࢀ 387 ࠾ࡼࡧ 461 mg/kg/᪥㸪 PB ᦤྲྀ㔞ࡣࡑࢀࡒࢀ 427 ࠾ࡼࡧ 528 mg/kg/᪥⟬ฟࡉࢀࡓࠋ ᐇ㦂 1 ࡢࡍ࡚ࡢ᳨ᰝᮇ࡛㸪ACI ᢞ⩌ࡢ୧㑇ఏᏊᆺ࣐࢘ࢫ࡛⫗║ⓗ࡞⫢⮚ࡢ⭘࠾ࡼ ࡧ⫢㔜㔞ࡢ㧗್ࢆㄆࡵࡓ㸦Table 2-1㸧ࠋྠ᳨ᰝᮇࡢ PB ᢞ⩌ࡢ WT ࣐࢘ࢫ࡛ࡣ⫢⮚ࡢ⫧ ࠾ࡼࡧ⫢㔜㔞ࡢ㧗್ࢆㄆࡵࡓࡀ㸪ྠ⩌ࡢ CARKO ࣐࢘ࢫ࡛ࡣㄆࡵ࡞ࡗࡓࠋACI ࡣ⫢⮚ ࡘࡢせ࡞⌮⤌⧊Ꮫⓗኚ㸦⫢⣽⬊⫧࠾ࡼࡧ⣽⬊യᐖ㸧ࢆច㉳ࡋࡓ㸦Table 2-2㸧ࠋ. ⫢⣽⬊⫧㛵㐃ࡍࡿኚ ࡍ࡚ࡢ᳨ᰝᮇ࠾࠸࡚㸪ACI ᢞ⩌ࡢ୧㑇ఏᏊᆺ࣐࢘ࢫ࡛ࡣ⫢⣽⬊⫧ࢆㄆࡵࡓࠋ⫢ ⣽⬊⫧ࡣᑠⴥ୰ᚰࡽ୰㛫ᖏࡅ࡚㸪࠶ࡿ࠸ࡣࡧࡲࢇᛶㄆࡵࡽࢀ㸪ࡑࡢ⛬ᗘ㑇ఏ Ꮚᆺ㛫࡛᭷ព࡞ᕪࡣㄆࡵࡽࢀ࡞ࡗࡓ㸦Fig. 2-1㸪Table 2-2㸧 ࠋࡍ࡚ࡢ᳨ᰝᮇ࠾࠸࡚㸪 ACI ᢞ⩌ࡢ⫧ࡋࡓ⫢⣽⬊ࡣ CYP2B㸪CYP3A㸪࠾ࡼࡧ CYP4A ࢆⓎ⌧ࡋ࡚࠸ࡓ㸦Fig. 2-1㸧 ࠋ ⫧ࡋࡓ⫢⣽⬊࠾ࡅࡿ CYP2B Ⓨ⌧ࡣ㸪WT ࣐࢘ࢫẚ㍑ࡋ࡚ CARKO ࣐࢘ࢫ࡛㍍ᗘ࡛࠶ ࡾ㸪CYP3A ࠾ࡼࡧ CYP4A Ⓨ⌧㑇ఏᏊᆺ㛫࡛᭷ព࡞ᕪࡣㄆࡵࡽࢀ࡞ࡗࡓࠋࡍ࡚ࡢ᳨ ᰝᮇ࡛⫧ࡋࡓ⫢⣽⬊࠾ࡅࡿ CYP1A Ⓨ⌧ࡣㄆࡵࡽࢀ࡞ࡗࡓࠋ㟁Ꮚ㢧ᚤ㙾ୗ࡛ࡣ㸪⫧ ࡋࡓ⫢⣽⬊ࡢ⣽⬊㉁࠾࠸࡚ 1 㔜⭷ໟࡲࢀࡓ⌫≧ࡢ⣽⬊ᑠჾᐁ࡛࠶ࡿ࣐ࢡࣟ࣎ࢹ 32.
(34) 㸦࣌ࣝ࢜࢟ࢩࢰ࣮࣒㸧ࡢቑຍࢆㄆࡵࡓ㸦Fig. 2-1㸧 ࠋ ࡍ࡚ࡢ᳨ᰝᮇ࠾࠸࡚㸪ACI ᢞ⩌ࡢ WT ࣐࢘ࢫ࡛ࡣᑠⴥ୰ᚰࡽ୰㛫ᖏࡅ࡚ ⫢⣽⬊⫧ࢆㄆࡵࡓࡀ㸪 ྠ⩌ࡢ CARKO ࣐࢘ࢫ࡛ࡣ⫢⣽⬊⫧ࡣㄆࡵࡽࢀ࡞ࡗࡓ 㸦Fig. 2-2㸧 ࠋ PB ᢞ⩌࡛ㄆࡵࡓ⫧ࡋࡓ⫢⣽⬊ࡣ CYP2B ࠾ࡼࡧ CYP3A ࢆⓎ⌧ࡋ࡚࠸ࡓࡀ㸪CYP1A ࠾ ࡼࡧ CYP4A ࡣⓎ⌧ࡋ࡚࠸࡞ࡗࡓࠋ㟁Ꮚ㢧ᚤ㙾ୗ࡛ࡣ㸪⫧ࡋࡓ⫢⣽⬊࠾࠸࡚㠃ᑠ⬊ యࡢቑຍࢆㄆࡵࡓ㸦Fig. 2-2㸧ࠋ ⫢⮚࠾ࡅࡿ⸆≀௦ㅰ㓝⣲㑇ఏᏊࡢⓎ⌧㔞ࢆ Fig. 2-3 ♧ࡋࡓࠋࡍ࡚ࡢ᳨ᰝᮇ࠾࠸ ࡚㸪୧㑇ఏᏊᆺ࣐࢘ࢫࡢ Cyp2b10 Ⓨ⌧ࡣ㸪ᑐ↷⩌ẚ㍑ࡋ࡚ ACI ᢞ⩌࡛᭷ពቑຍࡋࡓ 㸦24㹼70 ಸ㸧ࠋACI ᢞ⩌࠾ࡼࡧᑐ↷⩌࠾ࡅࡿ CARKO ࣐࢘ࢫࡢ Cyp2b10 Ⓨ⌧ࡣ㸪ྠ⩌ࡢ WT ࣐࢘ࢫࡢⓎ⌧ẚ㍑ࡋ࡚᭷ពῶᑡࡋࡓࠋࡍ࡚ࡢ᳨ᰝᮇ࠾࠸࡚㸪୧㑇ఏᏊᆺ࣐࢘ ࢫࡢ Cyp4a10 Ⓨ⌧ࡣ㸪ᑐ↷⩌ẚ㍑ࡋ࡚ ACI ᢞ⩌࡛㢧ⴭቑຍࡋࡓ㸦4570㹼12656 ಸ㸧ࠋ ᢞ 1 㐌┠࠾ࡼࡧ 4 㐌┠ࡢ WT ࣐࢘ࢫ࡞ࡽࡧᢞ 1 㐌┠ࡢ CARKO ࠾࠸࡚㸪ACI ࡣ Cyp3a11 Ⓨ⌧ࢆ᭷ពቑຍࡉࡏࡓࠋࡍ࡚ࡢ᳨ᰝᮇ࠾࠸࡚㸪୧㑇ఏᏊᆺ࣐࢘ࢫࡢ Cyp1a2 Ⓨ⌧ᑐ↷⩌ ACI ᢞ⩌㛫࡛᭷ព࡞ᕪࡣㄆࡵࡽࢀ࡞ࡗࡓࠋ ࡍ࡚ࡢ᳨ᰝᮇ࠾࠸࡚㸪ᑐ↷⩌ẚ㍑ࡋ࡚ PB ⩌࡛ࡣ㸪WT ࣐࢘ࢫࡢ Cyp2b10 Ⓨ⌧ࡀ ቑຍࡋࡓࡀ㸪CARKO ࣐࢘ࢫࡢⓎ⌧ࡣኚືࡋ࡞ࡗࡓࠋࡍ࡚ࡢ᳨ᰝᮇ࠾࠸࡚㸪୧㑇ఏ Ꮚᆺ࣐࢘ࢫࡢ Cyp4a10 Ⓨ⌧ᑐ↷⩌ PB ᢞ⩌㛫࡛᭷ព࡞ᕪࡣㄆࡵࡽࢀ࡞ࡗࡓࠋᑐ↷⩌ ẚ㍑ࡋ࡚ PB ᢞ⩌࡛ࡣ㸪୧㑇ఏᏊᆺ࣐࢘ࢫࡢ Cyp3a11 ࠾ࡼࡧ Cyp1a2 Ⓨ⌧ࡀᩘᅇࡢ᳨ᰝ ᮇ࡛ቑຍࡋࡓࠋCARKO ࣐࢘ࢫࡢ Cyp3a11 ࠾ࡼࡧ Cyp1a2 Ⓨ⌧ࡣ WT ࣐࢘ࢫࡢⓎ⌧ẚ㍑ ࡋ࡚ప࠸ഴྥࢆ♧ࡋࡓࠋ. ⫢⮚ࡢ⣽⬊യᐖ㛵㐃ࡍࡿኚ ACI ᢞ⩌ࡢ୧㑇ఏᏊᆺ࣐࢘ࢫ࡛ࡣ㸪⣽⬊യᐖ㛵㐃ࡍࡿኚࡀㄆࡵࡽࢀࡓࠋᢞ 1 㐌┠ ࡽ 13 㐌┠ࡅ࡚୧㑇ఏᏊᆺ࣐࢘ࢫࡢ⾑Ύ ALT ್ࡀ⤒ⓗቑຍࡋࡓࠋࡋࡋ࡞ࡀࡽ㸪 CARKO ࣐࢘ࢫࡢ ALT ್ࡣ WT ࣐࢘ࢫࡢ್ẚ㍑ࡋ࡚᭷ពప್࡛࠶ࡗࡓ㸦Fig. 2-4㸧 ࠋᢞ 33.
(35) 1 㐌┠ࡽ 13 㐌┠ࡅ࡚㸪⅖ᛶࢧࢺ࢝ࣥ TNF-Į ࢆࢥ࣮ࢻࡍࡿ 7QIĮ 㑇ఏᏊࡢⓎ⌧ࡀ WT ࣐࢘ࢫ࡛⤒ⓗቑຍࡋࡓࡀ㸪CARKO ࣐࢘ࢫ࡛ࡣኚືࡣㄆࡵࡽࢀ࡞ࡗࡓࠋ ⌮⤌⧊Ꮫ᳨ᰝࡢ⤖ᯝ㸪ACI ᢞ⩌ࡢ WT ࣐࢘ࢫ࡛⫢⣽⬊ࡢ༢⣽⬊ቯṚࡀᢞ 4 㐌┠ ࡽㄆࡵࡽࢀ㸪ࡑࡢ⛬ᗘࡣᢞ 13 㐌┠࡛ࡼࡾቑᙉࡋ࡚ほᐹࡉࢀࡓ㸦Fig. 2-5㸪Table 2-2㸧ࠋྠ⩌ ࡢ WT ࣐࢘ࢫ࡛ࡣ㸪ቯṚ⫢⣽⬊࿘ᅖࡢዲ୰⌫ᾐ₶ࡸࡧࡲࢇᛶ༢᰾⣽⬊ᾐ₶࡞ࡢ⅖ᛂ ࢆྠㄆࡵࡓ㸦Fig. 2-5A㸧 ࠋࡇࢀࡽࡢኚࡣ CARKO ࣐࢘ࢫ࡛ࡶㄆࡵࡽࢀࡓࡀ㸪ࡑࡢ⛬ᗘ ࡣ WT ࣐࢘ࢫࡢ⛬ᗘẚ㍑ࡋ࡚᫂ࡽపࡗࡓ㸦Fig. 2-5B, Table 2-2㸧 ࠋᢞ 4 㐌┠࠾ࡼࡧ 13 㐌┠ࡢ୧㑇ఏᏊᆺ࣐࢘ࢫ࡛ࢡࢵࣃ࣮⣽⬊࠾ࡅࡿⰍ⣲ỿ╔ࡀㄆࡵࡽࢀࡓ㸦Fig. 2-5C㸧 ࠋⰍ ⣲ỿ╔ࡢ⛬ᗘࡣ WT ࣐࢘ࢫẚ㍑ࡋ࡚ CARKO ࣐࢘ࢫ࡛㍍ᗘࡔࡗࡓࠋỿ╔ࡋࡓⰍ⣲ࡣ PAS ᰁⰍ࠾ࡼࡧࢩ࣮ࣗࣔࣝᛂ㝧ᛶ㸪࣌ࣝࢩࣥࣈ࣮ࣝ㝜ᛶࢆ♧ࡋࡓࡇࡽ㸪࣏ࣜࣇࢫ ࢳࣥࢆ♧၀ࡋ࡚࠸ࡓ㸦Fig. 2-5D㸪PAS ࠾ࡼࡧ࣌ࣝࢩࣥࣈ࣮ࣝᰁⰍࡢ⤖ᯝឡ㸧 ࠋࡲࡓ㸪ᢞ 13 㐌┠ࡢ୧㑇ఏᏊᆺ࣐࢘ࢫ࡛ࡣ⫢⣽⬊ࡢ᭷⣒ศീ࠾ࡼࡧᕧ᰾ࡀㄆࡵࡽࢀ㸪ࡇࢀࡽ ኚࡢⓎ⏕㢖ᗘࡣ CARKO ࣐࢘ࢫᑐࡋ࡚ WT ࣐࢘ࢫ࡛㧗ࡗࡓࠋ. ⣽⬊ቑṪάᛶ ࡍ࡚ࡢ᳨ᰝᮇ࠾࠸࡚㸪୧㑇ఏᏊᆺ࣐࢘ࢫࡢ PCNA 㝧ᛶ⣽⬊⋡ࡀ㸪ᑐ↷⩌ẚ㍑ࡋ ࡚ ACI ᢞ⩌࡛᭷ពቑຍࡋࡓ㸦Fig. 2-4C㸧 ࠋᢞᮇ㛫┦㛵ࡋࡓ㝧ᛶ⣽⬊⋡ࡢ㧗್ࡀ WT ࣐࢘ࢫ࡛ㄆࡵࡽࢀࡓࡶࡢࡢ㸪CARKO ࣐࢘ࢫ࡛ࡣㄆࡵࡽࢀ࡞ࡗࡓࠋACI ᢞ⩌ࡢ CARKO ࣐࢘ࢫࡢ㝧ᛶ⣽⬊⋡ࡣᑐ↷⩌ࡢ CARKO ࣐࢘ࢫࡢ್ẚ㍑ࡋ࡚㧗್ࢆ♧ࡋࡓࡀ㸪ACI ᢞ⩌ ࡢ WT ࣐࢘ࢫࡢ್ẚ࡚ప್ࢆ♧ࡋ࡚࠸ࡓࠋPB ᢞ⩌࡛ࡣ WT ࣐࢘ࢫࡢ PCNA 㝧ᛶ⣽⬊ ⋡ࡀᢞ 1 㐌┠୍࡛ⓗቑຍࡋࡓࡀ㸪CARKO ࣐࢘ࢫࡢ್ࡣኚືࡋ࡞ࡗࡓࠋ. ⫢⮚ࡢ࣏ࣝࣇࣜࣥ✚࠾ࡼࡧ㓟ࢫࢺࣞࢫ ACI ᢞࡣ୧㑇ఏᏊᆺ࣐࢘ࢫࡢ⫢⮚ PPIX ࡢ್ࡣ㸪ᑐ↷⩌ẚ㍑ࡋ࡚ ACI ᢞ⩌࡛ቑຍࡋ ࡓ㸦Fig. 2-4D㸧 ࠋᢞ 4 㐌┠࠾ࡅࡿ CARKO ࣐࢘ࢫࡢ್ࡣ WT ࣐࢘ࢫࡢ್ẚ㍑ࡋ࡚᭷ព 34.
(36) ࡞ప್ࢆ♧ࡋࡓࡀ㸪ᢞ 1 㐌┠࠾ࡼࡧ 13 㐌┠ࡢ್㑇ఏᏊᆺ㛫ࡢ᭷ព࡞ᕪࡣㄆࡵࡽࢀ࡞ ࡗࡓࠋග㢧ୗ࠾ࡼࡧ㟁㢧ୗ࠾࠸࡚࣏ࣝࣇࣜࣥࢆ♧၀ࡍࡿኚࡣ⫢⮚ㄆࡵࡽࢀࡎ㸪೫ ග㢧ᚤ㙾ୗ࠾࠸࡚࣏ࣝࣇࣜࣥࡢ≉ᚩ࡛࠶ࡿ೫ගࢆ♧ࡍᵓ㐀≀ࡣㄆࡵࡽࢀ࡞ࡗࡓࠋ ᢞ 4 㐌┠࠾ࡼࡧ 13 㐌┠࠾ࡅࡿ WT ࣐࢘ࢫࡢ TBARS ್ࡀ㸪ᑐ↷⩌ẚ㍑ࡋ࡚ ACI ᢞ ⩌࡛ቑຍࡋࡓ㸦Fig. 2- 4E㸧ࠋACI ᢞ⩌ࡢ CARKO ࣐࢘ࢫࡢ TBARS ್ࡣ㸪ᑐ↷⩌ࡢྠ࣐ ࢘ࢫࡢ್ẚ㍑ࡋ࡚ᢞ 4 㐌┠࡛᭷ពቑຍࡋࡓࡀ㸪ᢞ 13 㐌┠࡛᭷ពῶᑡࡋࡓࠋᢞ 13 㐌┠ࡢ ACI ᢞ⩌࠾ࡅࡿ CARKO ࣐࢘ࢫࡢ TBARS ್ࡣ㸪ྠ⩌ࡢ WT ࣐࢘ࢫࡢ್ẚ ㍑ࡋ࡚᭷ពప࠸್ࢆ♧ࡋࡓࠋPB ᢞ⩌࡛ࡣ㸪ᑐ↷⩌ẚ㍑ࡋ࡚ WT ࣐࢘ࢫࡢ TBARS ್ ࡀᢞ 1 㐌࠾ࡼࡧ 4 㐌┠࡛㸪CARKO ࣐࢘ࢫࡢ್ࡀᢞ 1 㐌┠࡛᭷ពቑຍࡋࡓࠋ ࡍ࡚ࡢ᳨ᰝᮇ࠾࠸࡚㸪୧㑇ఏᏊᆺ࣐࢘ࢫࡢ⫢⮚ GSH ࡣᑐ↷⩌ẚ㍑ࡋ࡚ ACI ᢞ ⩌࡛᭷ពቑຍࡋࡓ㸦Fig. 2-4F㸧ࠋPB ᢞ⩌࡛ࡣ GSH ್ࡢኚືࢆㄆࡵ࡞ࡗࡓࠋ. ⫢⭘⒆ᙧᡂ ᐇ㦂 2 ࡢゎ๗㸪ACI ࠾ࡼࡧ PB ᢞ⩌ࡢ WT ࣐࢘ࢫ࡛ࡣ⫢⮚」ᩘࡢᆺ⤖⠇ࡀ⫗║ⓗ ㄆࡵࡽࢀࡓࠋቑṪᛶኚࡢⓎ⏕㢖ᗘࢆ Table 2-3 ♧ࡋࡓࠋᑐ↷⩌ྵࡴࡍ࡚ࡢ⩌ࡢ WT ࣐࢘ࢫ࡛ࡣዲ㓟ᛶ࠾ࡼࡧዲሷᇶᛶኚ␗⫢⣽⬊ᕢࡀ㢖ᗘ㧗ࡃㄆࡵࡽࢀࡓࠋᑐ↷⩌࠾ࡅࡿ CARKO ࣐࢘ࢫࡢ⣙༙ᩘ࡛ࡣྛࢱࣉࡢኚ␗⫢⣽⬊ᕢࡀㄆࡵࡽࢀ㸪ACI ᢞ⩌ࡢ CARKO ࣐ ࢘ࢫ࡛ࡣࡑࢀࡽኚࡀࡼࡾ㧗㢖ᗘㄆࡵࡽࢀࡓࠋ୧㑇ఏᏊᆺ࣐࢘ࢫࡢዲ㓟ᛶ⫢⣽⬊⭢⭘ࡢ Ⓨ⏕㢖ᗘࡣ㸪ᑐ↷⩌ẚ㍑ࡋ࡚ ACI ᢞ⩌࡛㧗㢖ᗘㄆࡵࡽࢀࡓࠋ⤫ィᏛⓗ᭷ពᕪࡣㄆࡵ ࡽࢀ࡞ࡗࡓࡀ㸪WT ࣐࢘ࢫࡢዲሷᇶᛶ⫢⣽⬊⭢⭘ࡢⓎ⏕㢖ᗘࡣ㸪ᑐ↷⩌ẚ㍑ࡋ࡚ ACI ᢞ⩌࡛㧗್ࢆ♧ࡋࡓࠋWT ࣐࢘ࢫ࠾ࡅࡿዲ㓟ᛶ࠾ࡼࡧዲሷᇶᛶ⫢⣽⬊⭢⭘ࡢⓎ⏕㢖ᗘࡣ㸪 CARKO ࣐࢘ࢫࡢ㢖ᗘẚ㍑ࡋ࡚᭷ព࡞ప್ࢆ♧ࡋࡓࠋCARKO ࣐࢘ࢫ࠾ࡅࡿࡑࡢኚ␗ ⫢⣽⬊ᕢࡢⓎ⏕㢖ᗘࡣ㸪ᑐ↷⩌ẚ㍑ࡋ࡚ ACI ᢞ⩌࡛᭷ពቑຍࡋࡓࠋ⏕ᛶ⫢⣽⬊㐣 ᙧᡂ࠾ࡼࡧ⫢⣽⬊⒴ࡣ ACI ᢞ⩌ࡢ WT ࣐࢘ࢫࡢࡳㄆࡵࡽࢀ㸪⫢⣽⬊⒴ࡢከࡃࡣ⏕ᛶ ⫢⣽⬊㐣ᙧᡂྠㄆࡵࡽࢀࡓࠋ 35.
(37) ቑṪᛶኚࡢⓎ⏕ᩘࢆ Fig. 2-6 ♧ࡋࡓࠋᑐ↷⩌ẚ㍑ࡋ࡚ ACI ᢞ⩌࡛ࡣ㸪WT ࣐࢘ࢫ ࠾ࡅࡿዲ㓟ᛶኚ␗⫢⣽⬊ᕢ࠾ࡼࡧ⫢⣽⬊⭢⭘㸪࡞ࡽࡧዲሷᇶᛶኚ␗⫢⣽⬊ᕢ࠾ࡼࡧ⫢ ⣽⬊⭢⭘ࡢⓎ⏕ᩘࡀቑຍࡋࡓࡀ㸪CARKO ࣐࢘ࢫࡢⓎ⏕ᩘࡣኚືࡋ࡞ࡗࡓࠋ௦⾲ⓗ࡞ዲ 㓟ᛶ⫢⣽⬊⭢⭘ࢆ Fig. 2-7A ♧ࡋࡓࠋ⏕ᛶ⫢⣽⬊㐣ᙧᡂ࠾ࡼࡧ⫢⣽⬊⒴ࡣ ACI ᢞ⩌ ࡢ WT ࣐࢘ࢫࡢࡳㄆࡵࡽࢀࡓࠋ⫢⣽⬊⒴ࡢከࡃࡣ⏕ᛶ⫢⣽⬊㐣ᙧᡂ୰ㄆࡵࡽࢀࡓ 㸦Fig. 2-7㸧 ࠋCARKO ࣐࢘ࢫ࠾ࡅࡿࡑࡢኚ␗⫢⣽⬊ᕢ࠾ࡼࡧ⫢⣽⬊⭢⭘ࡢⓎ⏕ᩘࡣ㸪ᑐ ↷⩌ẚ㍑ࡋ࡚ ACI ᢞ⩌࡛㍍ᗘቑຍࡋࡓࠋ ᑐ↷⩌ẚ㍑ࡋ࡚ PB ᢞ⩌࡛ࡣ㸪WT ࣐࢘ࢫࡢዲ㓟ᛶ⫢⣽⬊⭢⭘ࡢⓎ⏕㢖ᗘࡀ᭷ពቑ ຍࡋࡓࡀ㸪CARKO ࣐࢘ࢫࡢⓎ⏕㢖ᗘࡣኚືࡋ࡞ࡗࡓࠋCARKO ࣐࢘ࢫ࠾ࡅࡿࡍ࡚ࡢ ࢱࣉࡢኚ␗⫢⣽⬊ᕢ࠾ࡼࡧ⫢⣽⬊⭢⭘ࡢⓎ⏕㢖ᗘࡣ㸪ᑐ↷⩌ẚ㍑ࡋ࡚ PB ᢞ⩌࡛ྠ⛬ ᗘࡢ್࠶ࡿ࠸ࡣప್ࢆ♧ࡋࡓ㸦Table 2-3㸧 ࠋᑐ↷⩌ẚ㍑ࡋ࡚ PB ᢞ⩌࡛ࡣ㸪WT ࣐࢘ࢫࡢ ዲ㓟ᛶኚ␗⫢⣽⬊ᕢ࠾ࡼࡧ⫢⣽⬊⭢⭘࡞ࡽࡧዲሷᇶᛶኚ␗⫢⣽⬊ᕢࡢⓎ⏕ᩘࡀቑຍࡋࡓ ࡀ㸪CARKO ࣐࢘ࢫࡢⓎ⏕ᩘࡣኚືࡋ࡞ࡗࡓࠋ ᐇ㦂 2 ࠾ࡅࡿ ACI ᢞ⩌ࡢ୧㑇ఏᏊᆺ࣐࢘ࢫ࡛ࡣ㸪⫢⣽⬊ࡢ༢⣽⬊ቯṚࡸࡧࡲࢇᛶ༢ ᰾⣽⬊ᾐ₶࡞ࡢ⣽⬊യᐖ㛵㐃ࡋࡓኚ࠾ࡼࡧ⫢⣽⬊⫧ࢆㄆࡵࡓ㸦Table 2-2㸧 ࠋCARKO ࣐࢘ࢫ࠾ࡅࡿ⣽⬊യᐖᛶኚࡢⓎ⏕㢖ᗘ࠾ࡼࡧ⛬ᗘࡣ㸪WT ࣐࢘ࢫࡢ㢖ᗘ࠾ࡼࡧ⛬ᗘẚ ㍑ࡋ࡚ప್࡛࠶ࡗࡓࠋPB ᢞ⩌ࡢ WT ࣐࢘ࢫ࡛ࡣ⫢⣽⬊⫧࠾ࡼࡧᕧ᰾ࡀ㧗࠸㢖ᗘ࡛ㄆ ࡵࡽࢀࡓࡀ㸪ྠ⩌ࡢ CARKO ࣐࢘ࢫ࡛ࡣࡇࢀࡽࡢኚࡣㄆࡵࡽࢀ࡞ࡗࡓࠋ. 36.
(38) ⪃ ᐹ. ᐇ㦂 2 ࠾ࡅࡿቑṪᛶኚࡢⓎ⏕㢖ᗘ࠾ࡼࡧⓎ⏕ᩘࡢ⤖ᯝࡽ㸪CAR ࡀ ACI ㄏⓎ⫢Ⓨࡀ ࢇ㛵ࡋ࡚࠸ࡿࡇࡀ♧ࡉࢀࡓࠋCAR ࢆࡋࡓ⫢Ⓨࡀࢇࡢヲ⣽࡞ศᏊᶵᗎࡣ᫂ࡔࡀ㸪 CAR άᛶࡣ⣽⬊࿘ᮇ㸪࣏ࢺ࣮ࢩࢫ㸪Wnt/ȕ-catenin ⤒㊰࡞ከࡃࡢ㑇ఏᏊࡢⓎ⌧ࢆㄏᑟ ࡍࡿࡇࡀ▱ࡽࢀ࡚࠸ࡿ㸦49㸧 ࠋACI ࡣ WT ࣐࢘ࢫ࡛ CAR ࡢᶆⓗ࡛࠶ࡿ Cyp2b10 㑇ఏᏊ࠾ ࡼࡧ CYP2B ⺮ⓑࡢⓎ⌧ࢆቑຍࡉࡏࡓࡀ㸪CARKO ࣐࢘ࢫ࡛ࡇࢀࡽࡢኚࡣప್࡛࠶ࡗࡓࠋ CAR άᛶ࡛࠶ࡿ PB ࡣ CAR ౫Ꮡࡋ࡚ Cyp2b10 㑇ఏᏊ࠾ࡼࡧ CYP2B ⺮ⓑࡢⓎ⌧ࢆㄏ ᑟࡋࡓࠋࡇࢀࡽࡢ⤖ᯝࡣ㸪ACI ࡀ CAR ࢆάᛶࡉࡏ࡚࠸ࡿࡇࢆ♧၀ࡋ࡚࠸ࡓࠋ ➨ 2 ❶ࡢ◊✲࡛ࡣ㸪➨ 1 ❶ࡢ OX ࡣ␗࡞ࡾ㸪CAR ࡀ ACI ㄏⓎ⣽⬊യᐖ㔜せ࡞ാࡁࢆ ࡋ࡚࠸ࡓࠋ୍⯡ⓗࡆࡗṑ㢮⫢Ⓨࡀࢇࡢస⏝ᶵᗎࡢ୍ࡘࡋ࡚㸪⣽⬊യᐖ⥆Ⓨࡍࡿᣢ⥆ ⓗ࡞⣽⬊ቑṪάᛶ่⃭ࡀ࠶ࡆࡽࢀ࡚࠸ࡿ㸦17㸪20㸧 ࠋᮏ❶ࡢ◊✲࡛ࡣ ACI ࡀ⫢⣽⬊ࡢ༢⣽⬊ ቯṚ㸪⅖㸪ALT ್ቑຍ࠸ࡗࡓ⣽⬊യᐖࢆច㉳ࡋ࡚࠾ࡾ㸪ࡑࢀ┦㛵ࡋ࡚ PCNA 㝧ᛶ⣽ ⬊⋡ࡢᣢ⥆ⓗ࡞ቑຍࡀㄆࡵࡽࢀࡓࠋࡇࢀࡽࡢ⣽⬊യᐖ㛵㐃ࡍࡿኚࡣ WT ࣐࢘ࢫẚ㍑ ࡋ࡚ CARKO ࣐࢘ࢫ࡛ᙅࡗࡓࠋACI ᢞ⩌ࡢ WT ࣐࢘ࢫ࡛≉ᚩⓗ࡞ኚࡋ࡚㸪ᣢ⥆ⓗ ࡞⣽⬊യᐖᑐࡍࡿ⏕ᛂࡼࡾᙧᡂࡉࢀࡿ⏕ᛶ⫢⣽⬊㐣ᙧᡂࢆ㢖⦾ㄆࡵࡓࠋࡋ ࡋ㸪ࡇࡢኚࡣྠ⩌ࡢ CARKO ࣐࢘ࢫ࡛ࡣㄆࡵࡽࢀ࡞ࡗࡓࠋࡉࡽ ACI ᢞ⩌ࡢ≉ᚩ ࡋ࡚㸪⫢⣽⬊⒴ࡀ WT ࣐࢘ࢫࡢࡳㄆࡵࡽࢀ㸪ࡑࡢከࡃࡣ⏕ᛶ⫢⣽⬊㐣ᙧᡂ୰Ꮡᅾࡋ ࡚࠸ࡓࠋᮏ❶ࡢᐇ㦂⤖ᯝࡽᶵᗎࢆ᫂ࡽࡍࡿࡇࡣ࡛ࡁ࡞ࡗࡓࡀ㸪⫢⣽⬊⒴ࡢⓎ⌧ 㒊➼ࢆ⪃៖ࡍࡿ㸪ᣢ⥆ࡋࡓ⣽⬊യᐖࡀ ACI ㄏⓎ⫢Ⓨࡀࢇ㛵ࡋ࡚࠸ࡿྍ⬟ᛶࡀ⪃࠼ ࡽࢀࡓࠋ ࣏ࣝࣇࣜࣥࡑࢀ⥆Ⓨࡍࡿ⣽⬊യᐖࡣ㸪ࣄࢺእᤄᛶࡢ࠶ࡿࡆࡗṑ㢮⫢Ⓨࡀࢇࡢస ࠋᢠ⳦ࢢࣜࢭ࢜ࣇࣝࣅࣥࡣ࣒࣊ྜᡂ⤒㊰ࡢࣇ࢙ ⏝ᶵᗎࡢ୍ࡘ⪃࠼ࡽࢀ࡚࠸ࡿ㸦17㸪20㸧 ࣟࢣࣛࢱ࣮ࢮࢆ㜼ᐖࡋ㸪ᙉ࠸࣏ࣝࣇࣜࣥỿ╔ࢆక࠺⣽⬊യᐖࢆ⫢⮚ច㉳ࡍࡿ㸦19㸪27㸪 64㸧ࠋẅ⳦࣊࢟ࢧࢡࣟࣟ࣋ࣥࢮࣥࡣ࣒࣊ྜᡂ⤒㊰ࡢ࣏࢘ࣟࣝࣇࣜࣀ࣮ࢤࣥ III ࢹ࢝ࣝ࣎ 37.
(39) ࢟ࢩ࣮ࣛࢮࢆ㜼ᐖࡋ㸪㕲ࡢ㐣㈇Ⲵࢆࡋࡓ⣽⬊യᐖࢆច㉳ࡍࡿ㸦10㸧 ࠋᮏ❶ࡢ ACI ᢞ⩌ࡢ ⫢⮚࡛ࡣ࣏ࣝࣇࣜࣥ࠶ࡿ࠸ࡣ㕲ࡢỿ╔ࡣㄆࡵࡽࢀ࡞ࡗࡓࠋࡉࡽ㸪ACI ᢞ⩌࠾ࡅࡿ ⫢⮚ PPIX ್ࡣ WT ࣐࢘ࢫ CARKO ࣐࢘ࢫ࡛ྠ⛬ᗘ࡛࠶ࡾ㸪⣽⬊യᐖࡢ⛬ᗘࡣ┦㛵ࡋ࡞ ࡗࡓࠋࡇࢀࡽࡢ⤖ᯝࡣ㸪࣏ࣝࣇࣜࣥ✚ࡀ ACI ㄏⓎ⣽⬊യᐖࡢせᅉ࡛ࡣ࡞࠸࠸࠺ ᪂ࡓ࡞⤖ᯝࢆ♧၀ࡋ࡚࠸ࡓࠋ ⣽⬊യᐖࢆច㉳ࡍࡿせᅉࡢ୍ࡘࡋ࡚㓟ࢫࢺࣞࢫࡀ▱ࡽࢀ࡚࠸ࡿࠋᮏ❶࡛ࡢ◊✲⤖ᯝࡼ ࡾ㸪ACI ᢞ⩌࠾࠸࡚⫢⮚࠾ࡼࡧࢡࢵࣃ࣮⣽⬊ࡢ࣏ࣜࣇࢫࢳࣥỿ╔ࡸ TBARS ್ࡢቑຍ࡞ 㓟ࢫࢺࣞࢫࢆ♧၀ࡍࡿኚࡀㄆࡵࡽࢀࡓࠋࡋࡋ㸪୧㑇ఏᏊᆺ࣐࢘ࢫ࡛ GSH ࡢᾲῬࡣ ㄆࡵࡽࢀ࡞ࡗࡓࠋGSH ࡣ⣽⬊ෆ࠾ࡅࡿᢠ㓟≀㉁࡛࠶ࡾ㸪ࢭࢺ࣑ࣀࣇ࢙ࣥࡸࣆࣞ ࣥ࡞ࡢྜ≀ࡣ GSH ࢆᾲῬࡉࡏࡿࡇ࡛⣽⬊യᐖࢆច㉳ࡍࡿ㸦70㸪71㸧 ࠋCARKO ࣐࢘ࢫ ࡛ࡣࣆࣞࣥࡼࡿ GSH ࡢᯤῬࡀᢚไࡉࢀ㸪⤖ᯝ⣽⬊യᐖࡀᢚไࡉࢀࡓ࠸࠺ሗ࿌ࡀ࠶ࡿ 㸦71㸧 ࠋᮏ❶࠾ࡅࡿ GSH ࡢ⤖ᯝࡣ㸪ACI ࡀㄏⓎࡍࡿ⣽⬊യᐖࡢᶵᗎࡀࢭࢺ࣑ࣀࣇ࢙ࣥ ࡸࣆࣞࣥࡣ␗࡞ࡿࡇࢆ♧၀ࡋ࡚࠸ࡓࠋࡲࡓ㸪33$5Į άᛶ⸆࡛࠶ࡿࣇ࢙ࣀࣇࣈ࣮ࣛ ࢺࡣ 33$5Į ࢆࡋ࡚࣐࢘ࢫࡢ⫢⮚୰ GSH ࢆቑຍࡉࡏࡿࡇࡀ▱ࡽࢀ࡚࠸ࡿ㸦1㸪11㸪48㸧ࠋ ACI ࡣ 33$5Į άᛶస⏝ࢆ᭷ࡋ࡚࠾ࡾ㸪 33$5Į ࡢάᛶࡀ ACI ᢞ⩌࠾ࡅࡿ⫢⮚୰ GSH ࡢቑຍࡘ࡞ࡀࡗࡓྍ⬟ᛶࡀ♧၀ࡉࢀࡓࠋ ➨ 1 ❶࡛グ㍕ࡋࡓ࠾ࡾ㸪ACI ྠᵝࡢ PROTOX 㜼ᐖ࡛࠶ࡿ OX ࡣ CAR 㛵ࡏࡎ ⣽⬊യᐖࢆច㉳ࡋ㸪᭱⤊ⓗ⫢ⓎࡀࢇࢆㄏⓎࡍࡿ㸦32㸧 ࠋCAR ࡣ ACI ࡀㄏⓎࡍࡿ⣽⬊യᐖ ≉␗ⓗ࡞ᙺࢆᯝࡓࡋࡓࠋࡋࡋ୍᪉࡛㸪⣽⬊യᐖࡑࡢࡶࡢࡣ ACI ࠾ࡼࡧ OX ࡀㄏⓎࡍ ࡿ⫢Ⓨࡀࢇඹ㏻ࡋࡓస⏝ᶵᗎ࡛࠶ࡿ⪃࠼ࡽࢀࡓࠋ PPARĮ άᛶࡣቑṪไᚚ㑇ఏᏊ➼ࡢⓎ⌧ࢆㄏᑟࡋ㸪⣽⬊ቑṪάᛶࢆ㧗ࡵࡿࡇ࡛⫢⭘⒆ ࢆᙧᡂࡍࡿ㸦6㸪12㸪26㸧 ࠋCAR 33$5Į ࡢඹάᛶࡀ㸪ࣉࣟࢼ࣑ࢻㄏⓎࡆࡗṑ㢮⫢Ⓨࡀࢇ ࡢస⏝ᶵᗎࡋ࡚ሗ࿌ࡉࢀ࡚࠸ࡿ㸦34㸧 ࠋᮏ❶࡛㸪ACI ࡣ୧㑇ఏᏊᆺ࣐࢘ࢫ࠾࠸࡚⫢⣽⬊ ⫧ࢆక࠺ Cyp4a10 㑇ఏᏊ࠾ࡼࡧ CYP4A Ⓨ⌧ࡢቑຍ㸪 ࣌ࣝ࢜࢟ࢩࢰ࣮࣒ࡢቑṪࢆច㉳ࡋࡓࠋ ࡇࢀࡽኚࡣ 33$5Į άᛶࢆ♧ࡋ࡚࠸ࡓࠋPB ᢞ⩌࠾࠸࡚ CAR άᛶࡀ㠃ᑠ⬊యቑ 38.
(40) Ṫࢆక࠺⫢⣽⬊⫧ࢆច㉳ࡋࡓࡇࢆ⪃៖ࡍࡿ㸪ACI ᢞ⩌࠾ࡅࡿ⫢⣽⬊⫧ࡢᅉࡣ CAR ࡼࡾࡣࡴࡋࢁ PPARa άᛶ࡛࠶ࡿྍ⬟ᛶࡀ࠶ࡿࠋࡋࡋ୍᪉㸪ACI ᢞ⩌ࡢ CARKO ࣐࢘ࢫ࠾ࡅࡿቑṪᛶኚࡢⓎ⏕㢖ᗘ࠾ࡼࡧⓎ⏕ᩘࡣ 33$5Į άᛶࡣ↓㛵ಀ㢧ⴭῶ ᑡࡋ࡚࠾ࡾ㸪PPA5Į άᛶࡣ ACI ㄏⓎ⫢Ⓨࡀࢇࡢせᅉ࡛ࡣ࡞࠸⪃࠼ࡽࢀࡓࠋACI ㄏⓎ ⫢Ⓨࡀࢇࡢ 33$5Į ࡢ㛵ࢆ᫂ࡽࡍࡿࡓࡵࡣ 33$5Į KO ࣐࢘ࢫ➼ࢆ⏝࠸ࡓࡉࡽ࡞ࡿ ᳨ウࡀᚲせ⪃࠼ࡽࢀࡓࠋ ACI ࡣ Cyp3a11 㑇ఏᏊ࠾ࡼࡧ CYP3A ⺮ⓑࡢⓎ⌧ࢆቑຍࡉࡏ࡚࠾ࡾ㸪᰾ෆཷᐜయࡢ୍ࡘ࡛ ࠶ࡿ pregnane X Receptor㸦PXR㸧ࢆάᛶࡉࡏࡿ⪃࠼ࡽࢀࡓࠋPB ࢆጞࡵከࡃࡢྜ≀ࡣ CAR PXB ୧᪉ࢆάᛶࡉࡏࡿ㸦43㸧 ࠋPXB ࡣ CAR ࢡࣟࢫࢺ࣮ࢡࡍࡿࡇ࡛ CAR ඹ ㏻ࡍࡿከࡃࡢ㑇ఏᏊࡢⓎ⌧ࢆไᚚࡍࡿ㸦38㸪42㸪43㸧 ࠋࡋࡋ㸪PXR ࡢάᛶࡣ࣐࢘ࢫࡢ⫢ ⣽⬊ࡢቑṪࢆច㉳ࡋ࡞࠸ࡇࡀ▱ࡽࢀ࡚࠾ࡾ㸦54㸧 㸪PXR ࡣ ACI ㄏⓎ⫢Ⓨࡀࢇࡢ࣮࢟࣋ࣥ ࢺ࡛ࡣ࡞࠸⪃࠼ࡽࢀࡓࠋ ᮏ❶ࡢ◊✲⤖ᯝࡽ㸪CAR ࠾ࡼࡧ CAR ࢆࡋࡓ⣽⬊യᐖࡀ ACI ㄏⓎ⫢Ⓨࡀࢇ㔜せ࡞ స⏝ᶵᗎ࡛࠶ࡿࡇࡀ♧၀ࡉࢀࡓࠋࡲࡓ㸪⫢⮚୰ࡢ PPIX ್㑇ఏᏊᆺ㛫࡛ᕪࡣㄆࡵࡽࢀ࡞ ࡗࡓࡇࡽ㸪࣏ࣝࣇࣜࣥࡣ ACI ㄏⓎ⫢Ⓨࡀࢇࡢ࣮࢟࣋ࣥࢺ࡛ࡣ࡞࠸⪃࠼ࡽࢀࡓࠋ 2500 ppm ACI ࡢΰ䭱ᢞࡣ࣐࢘ࢫ࡛㢧ⴭ࡞య㔜ቑຍᢚไ⣽⬊യᐖࢆ♧ࡋࡓࡀ㸪ᢞ㉳ᅉ ࡞ࡿṚஸࡣច㉳ࡋ࡞ࡗࡓࠋᮏ❶࡛㑅ᢥࡋࡓ⃰ᗘ 2500 ppm ࡣ ACI ࡀㄏⓎࡍࡿ⣽⬊യᐖཬ ࡧ⫢Ⓨࡀࢇࢆ᳨⣴ࡍࡿ࠺࠼࡛㐺ษ⪃࠼ࡽࢀࡓࠋ⣽⬊യᐖࢆࡋࡓࡆࡗṑ㢮⫢Ⓨࡀࢇࡢస ⏝ᶵᗎࡣࣄࢺእᤄྍ⬟⪃࠼ࡽࢀࡿ୍᪉㸪ࣄࢺ⫢⮚࠾࠸࡚ ACI ࡀ CAR ࢆࡋࡓ⣽⬊യ ᐖࢆㄏⓎࡍࡿࡣ࡛᫂࠶ࡗࡓࠋ. 39.
図
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