⥲⹰ࢅฺ⏕ࡊࡒ⎌ሾ᭯ᐐ∸㈻ࡡ⏍మᙫ㡢フ౮ࢨࢪࢷ࣑ࡡ
㛜Ⓠ
Development of novel bioassay system for evaluation of environmental toxic materials
using C. elegans
㇇ᶣᢇ⾙⛁ᏕኬᏕᕝᏕ㒂࢙࣬ࢤࣞࢩ࣭ᕝᏕ⣌ ຐᩅ᤭ ᾆ ಆᙢToshihiko Eki, Ph.D.
Associate Professor, Department of Ecological Engineering, Toyohashi University ofTechnology ₧ᅹⓏ࡞⎌ሾỗ᯹ࡡཋᅄ࡛࡝ࡾ᭯ᐐ∸㈻ࡡ᳠ฝ࣬フ౮ࢅ┘Ⓩ࡛ࡊ࡙ࠉ࣓ࢸࣜ⏍∸࡚࠵ࡾ⥲⹰ࢅ⏕࠷ࡒ◂✪ࢅ⾔ࡖࡒࠊ⥲⹰ࡢࣃࢹ࡛ྜྷᵕࡡኣ⣵⬂⏍∸࡚࠵ࡾ࡞ࡵ㛭ࢂࡼࡍࠉᐁ
㥺Ⓩ࡞ᐖ᪾࡞ᑋ࿤ࡡῼᏽ࠿࡚ࡀࡾ࡛࠷࠹ඁࡿࡒ≁㛏ࢅᣚࡖ࡙࠷ࡾࠊᮇ◂✪࡚ࡢࠉ⏍∸ࡡ⏍Ṓࢅᣞᵾ࡛ࡌࡾᚉᮮᆵࡡフ౮Ἢ࡞௥ࢂࡽ࠹ࡾࠉ⥲⹰ࡡⓆ⏍ࡷᑋ࿤࡫ࡡᙫ㡢ࢅᣞᵾ࡞ࡊࡒ᩺ぞ࡝᭯ᐐᛮフ౮Ἢࡡ☔❟ࢅ┘Ⓩ࡛ࡊࡒࠊ᭯ᐐ∸㈻ࢅྱࡳᐨኮᇰᆀ࡚㣣⫩ࡊࡒ⥲⹰㞗ᅆࡡ⏍Ꮛ᭜⥲࡞ᇱࡘ࠷࡙ᑋ࿤ࡡフ౮ࢅ⾔ࡖࡒ⤎ᯕࠉ㔔㔘ᒌ㸝㖙ࠉ࢜ࢺ࣐࣑ࢗ㸞ࡷὑ๠࡞ࡻࡾ㢟ⴥ࡝Ⓠ⏍㐔ᘇ࡛ᑋ࿤ࡡ▯⦨࠿ふᐳࡈࡿࠉᮇἪ࠿㔔㔘ᒌࡷ⏲㟻Ὡᛮ๠ࡡ᭯ᐐᛮフ౮࡞㐲⏕ྊ⬗࡚࠵ࡾࡆ࡛࠿♟ࡈࡿࡒࠊୌ᪁ࠉን␏ཋᛮ∸㈻㸝࢓ࣜ࢞ࣜ໩๠㸞࡞ᑊࡊ࡙ࡢࠉ఩୯⃨ᗐᇡ࡚ᑋ࿤࡫ࡡ▯⦨ຝᯕࡢ㍇᚜࡚࠵ࡖࡒࠊࡐࡆ࡚ን␏ཋᛮ∸㈻ࡡ᭯ᐐᛮ᳠ฝវᗐ
ࡡྡྷ୕ࢅ┘Ⓩ࡞ࠉIHHILQJ73& ᖱῦ 73&L Ἢ࡞ࡻࡾ )3& ಞᚗ㐿ఎᏄ⩄ࡡᶭ⬗ᢒโ࡞㛭ࡌࡾ᳠ゞࢅ⾔࠷ࠉ⥲⹰ᑋ࿤࡫ࡡᙫ㡢フ౮ࢅྜྷ᫤࡞㐅ࡴࡒࠊ௑ᚃࠉ73&L ࢅฺ⏕ࡊࡒᐁ㥺⣌ࡡᨭⰃࢅ㐅ࡴࡾࡆ࡛࡚᳠ฝវᗐ࡛≁␏ᛮࢅྡྷ୕࡚ࡀࡾ࡛⩻࠻ࡼࡿࡾࠊ
I studied on a novel assay system for detecting toxicity of reagents using nematode C.
elegans. The nematode is a representative multicellular organism like human, and we can easily
measure a lifespan of nematode to allow us to study experimentally any factors affecting alifespan of organism.

In this study, I tried to establish a novel assay system based onmonitoring influence of toxic materials in nematode lifespan and development, which can bereplaced to current live-or-death-based toxicity tests. The lifespan of nematodes cultured withheavy metals (copper, cadmium), or detergent was significantly reduced and development oflava was inhibited in a concentration dependent manner, suggesting that this system may be
applied for detection of contaminated heavy metals and detergents.

- 1 -On the other hand,reduction of lifespan of nematode treated by alkylating reagent (i.e., mutagen) was not sosignificant. Thus, I am applying the feeding RNA interference (RNAi) technology to reduce
DNA repair activity, which could make the nematode more sensitive to DNA damages and thesystem more sensitive and specific to mutagen.

࠘◂✪⤊㐛࠙࠘◂✪┘Ⓩ࠙⏐༵᭿࡞࠵ࡾ⥲⹰㸝㔕⏍ᆵ Bristol N2 ᰬ㸞 ⎌ሾỗ᯹ࢅ㜭Ḿࡌࡾࡒࡴࡡ㔔さ࡝஢㜭ࢅ࢓ࣜ࢜ࣛ࣬ࣇ࣭ࣛࢲἪ࡞ࡻࡽྜྷㄢࡊࡒⓏᡥṹࡡୌࡗ࡛ࡊ࡙ࠉຝ⋙Ⓩ࡝໩ྙ∸ࡡ⥲⹰ ࡡ ᖺ ⹰㞗 ᅆ ࢅᚋ ࡒ ࠊ ⥲⹰ ࡡ ᇰ 㣬ࡢ᭯ᐐᛮフ౮Ἢࡡᑙථ࠿᭿ᙽࡈࡿ࡙࠷ࡾࠊ

NGM ᐨኮᇰᆀࢅྱࡳ 6cm ᙼࡡࢸ࢔ࢴࢨࡱࡒ⏐ᴏ⏲࡞࠽࠷࡙ࡢḚᕗࢅ୯ᚨ࡞⿿ဗࣖࢅ ࣂ ࣚ ࣆ࢔ ࣜ ࣑࡚ ᑌ ࡋ ࡙౐ ⏕ ࡊ ࡒࠊ࡞⏕࠷ࡼࡿࡾ໩Ꮥ∸㈻ࡡ᭯ᐐᛮࢅཚᐠ࡞

2xYT ᇰᆀ࡚ᇰ㣬ࡊࡒኬ⭘Ⳟ OP50 ᰬࢅ㐪ၡ࠹ᶭ㐘࠿㧏ࡱࡖ࡙࠷ࡾࠊ∸㈻ࡡ᭯ᐐᛮ

ᚨ࡚⃨⦨ࡊࠉᇰᆀࡡ୯ᚨ௛㎾࡞ළ≟࡞⁪フ౮࡞ࡢࠉࡆࡿࡱ࡚ࣁࢠࢷࣛ࢓ࡷ࣐ࢩࣤ

ୖࡊࡒࡵࡡࢅ⥲⹰ࡡ㣭࡛ࡊ࡙୙࠻ࠉ20 ᗐࢤ࡝࡜ỀᲿ⏍∸ࡡ⏍Ṓࢅᇱ‵࡛ࡊࡒフ౮࡚↋ⳞⓏ࡞㣣⫩ࢅ⾔ࡖࡒࠊモⷾࢅ῟ຊࡌἪ࠿ 㐲 ⏕ ࡈࡿ ࠉ ࡐࡆ ࠾ ࡼ ᑙ࠾ ࡿ ࡒ ᩐೋࡾሔྙࡢࠉᐨኮᇰᆀ࡞⤂⃨ᗐ࡞࡝ࡾࡻ࠹LD50㸝50%⮬Ṓ㔖㸞࡝࡜࠿᭯ᐐᛮࡡᣞᵾ࡞࠵ࡼ࠾ࡋࡴຊ࠻ࡒࠊ᭯ᐐモⷾࡡ⃨ᗐ࠿࡛ࡊฺ࡙⏕ࡈࡿ࡙ࡀࡒࠊࡊ࠾ࡊ࡝࠿ࡼࡆ㧏ࡂ࡝ࡾ࡛㣣⫩୯ࡡ⥲⹰࠿ࢸ࢔ࢴࢨࣖአࡡࡻ࠹࡝᛬ᛮẐᛮ࡞ࡻࡾ⏍Ṓึᏽモ㥺ࡡ

࡞㏠ࡅ࡙ࡊࡱ࠹ࡒࡴࠉ㣭ࡡ࿔ᅑࢅ⤎ᬏ໩ࡲ࡚ࡢࠉ⌟ᅹࠉ♣ఌⓏ࡞ᠩᛍࡈࡿ࡙࠷ࡾࡊࡒࣂ࣐ࣜࢲࣤ㓗࡚ᅑࡳࡆ࡛࡚⥲⹰ࡡ㏠々ྙⓏ࡚㛏᭿㸝ᖳ༟న㸞࡞ῳࡾ⏍∸ᙫ㡢ஷࢅᢒโࡊࡒࠊ࡝࠽ࣂ࣐ࣜࢲࣤ㓗࡞ࡻࡾࢅᮇᙔ࡞フ౮࡚ࡀ࡙࠷ࡾ࠾␪ၡ࠿ṟࡾࠊ⥲⹰ᑋ࿤࡫ࡡᙫ㡢ࡢ࡝࠷ࡆ࡛ࡢ☔ヾῥࡲࡐࡡࡒࡴ࡞ࡢ࣏ࢗࢪ࡝࡜ࢅ⏕࠷ࡒフ౮モ࡚࠵ࡾࠊᑋ࿤ῼᏽࡡࡒࡴࡡ㣣⫩࡞ࡢኬ⭘㥺࠿⩻࠻ࡼࡿࡾ࠿ࠉᖳ༟నࡡ᫤㛣࡛ኣኬⳞࢅሧᕱࡊࠉ25µM 5-fluoro-2’-deoxyuridine࡝ࢤࢪࢹ࠿ᚪさ࡚࠵ࡽࠉࢷࢪࢹ࡚ࡀࡾ໩(FUdR)ࢅ῟ຊࡊࡒ NGM ᇰᆀࢅ౐⏕ࡊࡒࠊྙ∸ࡡᩐࡢ㝀ᏽࡈࡿࡾࠊࡱࡒິ∸យ㆜ࡡ

FUdR ࡢ⥲⹰ࡡ⏐༵ࢅᢒโࡊࠉᑋ࿤ῼᏽࡡ

ふⅤ࠾ࡼࡵၡ㢗࠿࠵ࡾࠊࡐࡆ࡚ᮇ◂✪࡚ጄᐐ࡛࡝ࡾᏄᏘ⥲⹰ࡡΊථࢅᢒ࠻ࡾࡒࡴࡢᑋ࿤ࡡῼᏽ࠿ᐖ᪾࡚㐿ఎᏄ᧧షࡵྊ⬗࡞౐⏕ࡊࡒ㸝FUdR ࡞ࡻࡾᑋ࿤࡫ࡡᙫ㡢ࡢ࡝ኣ⣵⬂⏍∸࡚࠵ࡾ⥲⹰(C. elegans)ࢅᮞ

࡝࠷㸞ࠊ㣣⫩୯ࡡ㞗ᅆ࡞ࡗ࠷࡙ࠉ⤽᫤Ⓩ㸝ୌᩩ࡞㐽ࡦࠉࡐࡡ಴మᑋ࿤࡞ཀྵ࡯ࡌᙫ㡢㸝ᑋ᪝࠽ࡀ㸞࡞ᐁమ㢟᚜㙶࡞ࡻࡽふᐳࡌࡾࡆ࿤ࡡ▯⦨㸞ࢅ᩺ࡒ࡝ᣞᵾ࡛ࡌࡾ᭯ᐐᛮフ࡛࡚྘⥲⹰಴మࡡ⏍Ṓࢅึᏽࡊࠉ⥲⹰㞗౮Ἢࡡ㛜Ⓠࢅ┘ᣞࡊࡒࠊᅆࡡ⏍Ꮛ᭜⥲ࢅᥝࡂࡆ࡛࡚⾔ࡖࡒࠊ྘⃨ᗐࢡ࣭ࣜࣈ࠵ࡒࡽ 30 ༆࠾ࡼ 70 ༆ࡡ⥲⹰- 2 -ࢅ⏕࠷ࡒ㸝ࢸ࢔ࢴࢨࣖ࠵ࡒࡽ⣑ 10 ༆㸞ࠊ⏍Ṓࡢ㐘ິᛮࡡ႕኶࡞ࡻࡽึ᩷ࡊࡒ࠿ࠉᙟឺⓏ࡞ࡵᐖ᪾࡞ึืྊ⬗࡚࠵ࡾ㸝Ṓ࡟
࡛㏩᪺࡞࡝ࡾ㸞ࠊ Ⓠ⏍࡫ࡡᙫ㡢フ౮࡞ࡗ࠷࡙ࡢ௧ୖࡡᡥ㡨࡚⾔ࡖࡒࠊࡌ࡝ࢂࡔࠉ⏐༵᭿࡞ථࡖࡒ
⥲⹰ࢅ 24 ᫤㛣ࠉ྘⃨ᗐࡡモ࡛ⷾኬ⭘Ⳟࢅྱࡳ NGM ᐨኮᇰᆀ࡞࡙㣣⫩ࡊࡒᚃࠉ᩺ࡊ࠷ᐨኮᇰᆀ࡞⛛ࡊ࡙ 8 ᫤㛣㣣⫩ࡊࠉུ⢥ 㖙࢕࢛ࣤ㸝◪㓗㖙㸞࡞ࡻࡾ⥲⹰ࡡᑋ࿤༵ࢅ⏐༵ࡈࡎࡒࠊࡐࡡᚃࠉུ⢥༵࠾ࡼⓆ▯⦨࠽ࡻࡦᠺ㛏㜴ᐐ࠿ P2 ௧୕ࡡ⃨ᗐ⏍ࡊࡒᏄᏘ⥲⹰⩄ࡡమ㛏ࢅ⤽᫤Ⓩ࡞ῼᏽᇡ࡚ふᐳࡈࡿࡒ㸝ᅒ 㸞ࠊ㜴ᐐࡡ⛤ᗐࡢࡌࡾࡆ࡛࡚ࠉ⥲⹰ࡡⓆ⏍࣬ᠺ㛏ࡡ࣓ࢼࢰ⃨ᗐ౪ᏋⓏ࡚࠵ࡽࠉ஦⛸㢦ࡡ࢓ࢴࢬ࢕⣌࣭ࢅ⾔ࡖࡒࠊᮇ◂✪࡚ࡢࠉ⎌ሾỗ᯹࠽ࡻ࡞࠽࠷࡙ࣂ࡚ࣚࣝࣜ࠵ࡾࡆ࡛࠿ึ᪺ࡊࡒࠊࡦ⏍∸ᏕⓏᙫ㡢ࡡふⅤ࠾ࡼ㔔さ࡛⩻࠻ࡼࢸ࣭ࢰࡢ♟ࡈ࡝࠷࠿ࠉ࢜ࢺ࣐࣑ࢗ࡞࠽࠷ࡿࡾ㔔㔘ᒌ㢦ࠉ⏲㟻Ὡᛮ๠࠽ࡻࡦን␏ཋ࡙ࡵྜྷᵕࡡ⤎ᯕࢅᚋࡒࠊࡱࡒ୹さ࡝Ề⎌ᛮ∸㈻ࢅࢷࢪࢹ∸㈻࡛ࡊ࡙フ౮ࢅ⾔ࡖࡒࠊ ሾỗ᯹ࡡཋᅄ࡚࠵ࡾὑ๠࡚ࡵྜྷᵕࡡ㢟ⴥ㔔㔘ᒌ࡛ࡊ࡙㖙࡛࢜ࢺ࣐࣑ࢗࢅࠉ⏲㟻Ὡ࡝ᑋ࿤▯⦨ຝᯕ࠿ヾࡴࡼࡿࡒ㸝ᅒ 㸞ࠊᛮ๠࡛ࡊ࡙ᕰ㈅ࡡὑ๠ࠉࡱࡒን␏ཋᛮ∸㈻࡛ ࡊ ࡙ ௥⾪ Ⓩ ࡝࢓ ࣜ ࢞ ࣜ໩ ๠ ࡚ ࠵ࡾ
methyl methanesulfonate (MMS)ࢅ⏕࠷ࡒࠊୌ᪁ࠉ࢓ࣜ࢞ࣜ໩๠ 228 ࡞ࡻࡾᑋ࿤▯⦨ຝᯕࡢ  \[ ௧୕ࡡ⃨ᗐᇡ࡚ヾࡴࡼࡿࡒ㸝ᅒ 㸞ࠊ఩୯⃨ᗐᇡ࡞࠽࠷࡙ࡢࠉᠺ㛏㜴ᐐຝᯕࢅྱࡴṜ࡜ᙫ㡢ࡢヾࡴࡼࡿ ࡝࠾ࡖࡒࠊ- 3 -ࢨࣘࢴࢠ࡝࡜㸞࡞ࡻࡾⓆ⌟ㄇᑙࡢ᳠ฝࡈࡿࡒ࠿ࠉᚺ➽ࡡ≁␏ᛮࡢ఩ࡂࠉࣈ࣓࣭ࣞࢰࡡ⛸㢦ࡡ᳠ゞ࡝࡜࠿ᚪさ࡛⩻࠻ࡼࡿࡒࠊ࠘◂✪ᠺᯕ࠙ ⥲⹰಴మࡡᑋ࿤࠽ࡻࡦⓆ⏍࣬ᠺ㛏࡞୙࠻ࡾᙫ㡢ࢅᣞᵾ࡛ࡊࡒ᭯ᐐ∸㈻ࡡフ౮Ἢࡡ᳠ゞࢅ⾔ࡖࡒ⤎ᯕࠉᮇἪ࠿㔔㔘ᒌࡷ⏲㟻Ὡᛮ๠ࡡ᭯ᐐᛮフ౮࡞㐲⏕࡚ࡀࡾࡆ࡛
࠿ᐁッࡈࡿࡒࠊࡱࡒ feeding RNAi Ἢࢅె ⌟ᅹࠉን␏ཋᛮ∸㈻ࡡ᳠ฝវᗐࢅྡྷ୕

⏕ࡌࡾࡆ࡛࡚ࠉᮇ⣌࡚Ẓ㍉Ⓩ᳠ฝࡡᅏ㞬ࡈࡎࡾ┘Ⓩ࡚ࠉ73&L Ἢࢅ⏕࠷ࡒᮇ࢓ࢴࢬ࡝᭯ᐐ∸㈻࡞ᑊࡊ࡙ࡵ᳠ฝវᗐ࡛≁␏ᛮ࢕⣌ࡡᨭⰃࢅ㐅ࡴ࡙࠷ࡾࠊ73&L Ἢ࡞ࡻࡽࢅྡྷ୕࡚ࡀࡾྊ⬗ᛮ࠿♟ࡈࡿࡒࠊ)3& ಞᚗ㐿ఎᏄᶭ⬗ࢅᢒโࡌࡾࡆ࡛࡚ࠉን␏ཋᛮ∸㈻࡞ᑊࡌࡾវུᛮࡷ≁␏ᛮࢅ㧏࠘௑ᚃࡡㄚ㢗࡛Ⓠᒈ࠙ࡴࡾ ࡆ ࡛ ࠿᭿ ᙽ ࡚ࡀ ࡾ ࠊ ⤽⤾ Ⓩ ࡞ ⤝㣭  ᮇ⣌ࢅᵕࠍ࡝᭯ᐐ∸㈻㸝㎨ⷾࠉ᭯ᶭ

IHHILQJ ࢅ௒ࡊ࡙⥲⹰࡞ 73&L ࢅㄇᑙ࡚⁈๠ࠉᤴ࢝ࢪᠺฦ࡝࡜㸞࡞㐲⏕࡚ࡀࡾ࠾ࠉ

ࡀࡾ IHHILQJ73&L Ἢࢅ㐲⏕ࡌࡾࡆ࡛࡚ࠉ࠵ࡾ࠷ࡢ々ྙⓏ࡝᭯ᐐᛮࡡフ౮࡞㐲⏕ྊ㛏᭿㛣࡞ࢂࡒࡽ㐿ఎᏄᶭ⬗ᢒโ≟ឺࢅ㐡⬗࠾᳠ゞࡌࡾࡆ࡛ࠉ  73&L Ἢ࡛⤄ࡲྙࢂᠺ࡚ࡀࡾ࡛⩻࠻ࡼࡿࡒࠊࡐࡆ࡚ᑋ࿤ᨥ㒼ࡎࡒᐁ㥺⣌ࡡវᗐ࡛≁␏ᛮࡡྡྷ୕ࢅ㐅ࡴ㐿ఎᏄ࡚࠵ࡾ IFI 㐿ఎᏄ࡛ IFI 㐿ఎࡾࡆ࡛ࠉ௧୕ࡡ஦Ⅴ࠿௑ᚃࡡㄚ㢗࡚࠵ࡾࠊ

Ꮔ࡞ᑊࡌࡾ IHHILQJ73&L ฌ⌦⥲⹰ࡡᑋ࿤ῼᏽᐁ㥺ࢅ⾔ࡖࡒ⤎ᯕࠉ྘ࠍᑋ࿤࠿༖ฦ࠘Ⓠ⾪ㄵᩝࣛࢪࢹ࠙࡞▯⦨࠵ࡾ࠷ࡢ  ಶ࡞ᘇ㛏ࡊࡒࡆ࡛࠾㸝ᅗහᏕఌⓆ⾪㸞ࡼࠉᮇᐁ㥺⣌࡞ࡻࡽ᭿ᙽ㏳ࡽࡡ㐿ఎᏄᶭ୯ᮟḿᬍኬᠩಆᗛᏭ⸠⍘⨶࿰⏛⬗ᢒโຝᯕࢅ㐡ᠺ࡚ࡀࡾࡆ࡛࠿ᐁッࡈࡿⱹᯐ ⌦ᜠⰴᒱᩝ㞕ᾆ ಆᙢࠔ⥲⹰ࡒࠊ⌟ᅹࠉ)3& ಞᚗ㐿ఎᏄ⩄ࡡᶭ⬗ᢒโ⥲ࡡࢣࢿ࣑Ꮽᏽ໩࡞㛭୙ࡌࡾ᩺ぞࣉ࣭ࣛ࢜⹰ࢅ⏕࠷࡙ᑋ࿤ῼᏽᐁ㥺ࢅ㐅ࡴ࡙࠷ࡾࠊࢭᵕ㐿ఎᏄࡡᶭ⬗よᯊࠕ᪝ᮇฦᏄ⏍∸Ꮥᮇ◂✪࡚ࡢࠉᑋ࿤ῼᏽᐁ㥺࡛ࡢืࡡཋ⌦ఌ➠  ᅂᖳఌ㸝♼ᡖ㸞 ᖳ  ᭮࡞ᇱࡘࡂフ౮Ἢ࡛ࡊ࡙ࠉࢪࢹࣝࢪᚺ➽ࣈཋ⏛ᾀ᪺ᯐ ⌦ᜠᕰᕖᗛᖲᾆ ಆ࣓࣭ࣞࢰࡡᨥ㒼ୖ࡞࠵ࡾ ,+5 Ⓠ⌟⥲⹰ࢅᙢࠔࢣࢿ࣑ഭᐐ࡞ࡻࡾ⥲⹰ࡡ಴మᑋ࿤࡞⏕࠷ࡒ᭯ᐐᛮフ౮Ἢࡡ᳠ゞࡵ୩⾔ࡊ࡙⾔ཀྵ࡯ࡌᙫ㡢࡞㛭ࡌࡾ◂✪ࠕ᪝ᮇⷾᏕఌ➠ࡖࡒࠊࡊ࠾ࡊࠉ᭿ᙽ㏳ࡽࡡࢪࢹࣝࢪ㸝⇍ ᅂᖳఌ㸝᮶ா㸞 ᖳ  ᭮- 4 -