β‑Actin
Control
CYPIAlandCYPIA2mRNAgeneexpressionintheliverofp53(+/‑)and(+/+)with
andwithoutAPNHtreatment･OneqmicrogramaliquotsofRNAextractedfromliverof
･り?
((竃ぷ∈n百葛む)彗叫冨トd宏‑5hトU
つム一'一
l 5
0
5
5
∧U
5
0 3 10 30 100
田:Male(‑/‑) 8:Male(+/一) 由:Male(+/+)
0 3 10 30 100
APNH(PPm)
Sigi丘cantlydiffbrencefromrespectivecontrol;☆:P<0.05,**:P<0.00l,☆**;P<0.0001
Figure18‑A
RelativequantutationofCYPIAlnormalizedwithrespecttoARPinliversofthreegenotypesmaleandfbmalemice
treatedwithAPNHfbr7days(A)andbothsexesofthreegenotypesmicetreatedwith30ppmofAPNHfbr7days(B).
RelativequantutationofCYPIA2normalizedwithrespecttoARPinliversofbothsexesthreegenotypesmicetreated
･箪† ((司巴Oq∈n白むちむ)已○叫芸2d蟹‑く一丸トU
5
(‑/‑) (+/‑) (+/+) (‑/‑) (+/‑)
Male
(+/+)
･り甲
((d巴遥∈n白むちむ)彗叫芸2d宏云葡一丸トU
5
0
5
10
APNH(PPm)
30 100
ロ:Male(‑/‑) 臼:Male(+/‑)
□:Female(‑/一)
臼:Female(+/‑)
T⊥■｢‑⊥‑
･り?
(卜.≡且一む>む〓Unppイ
JTDNAadductlevel
T̲■‑⊥ T̲†⊥
T†
*勺J 10 30 100
APNH(PPm)
【結論】
本研究では,新規ヘテロサイクリックアミンaminophenylnorharman(APNH)
を用いイニシエーション作用に基づいた肝臓における中期がん原性試験(血v血 nve憎eekinitiationassay)および遺伝子改変動物を用いた発がん性試験を行い,
APNHの発がん性について検討し,さらにAPNHの代謝と発がんとの関連性に
ついて解析した｡本研究は大きく分けて,以下の3つの研究からなる(Figure20)｡
第1に,イニシエーション作用に基づいた肝臓における中期がん原性試験(f〝
Vivo丘ve珊eekinitiationassay)によりAPNHの発がん過程におけるイニシエーシ
ヨン活性の検索を行った｡
その結果,肝臓のglutathioneSlransftraseplacentalform(GSTゼ)陽性巣面積の 検索において,APNHを投与していない対照群と比較して,10mgkgb.w.投与群,
3mgkgb･W･投与群および1mgkgb.w.投与群に用量相関性の増加が認められた｡
GSTゼ陽性巣数においては,APNH投与群,APNHを投与しない対照群に比較し て有意な増加がみられた｡これらのことより,APNHが発がん過程においてイ ニシエーション活性を有することが示唆された0さらに,APNHlOmgkgb.w.投 与後2AAF,CC14処置を行わない群では,GSTゼ陽性巣面積がAPNHを投与し ない対照群に比較して有意な増加を示したことから,APNHの肝臓に対する標
的性も強く示唆された｡
第2に,APNHの発がん性と〆jがん抑制遺伝子の関係について,〆jが
ん抑制遺伝子を欠損した〆jノックアウト(EO)マウスを用いて検索を行った｡
その結果,APNH混餌投与により肝臓にovalcellhyperplasia(OCH),altered
Cellfoci(Foci),肝細胞腺腫(HCA),肝細胞癌(HCC)等の多彩な病変が観察され たo APNH投与15週後では肝臓のOCHがAPNH30ppm投与群のp53(イう,P53
(+/うおよび〆j(+/+)マウスにそれぞれ2′14(14･3恥14/23(60.9%)および2/10 (20%)観察されたo APNH投与40週後の安楽殺例では,HCCがAPNH30ppm
投与群の雌〆j(+/うマウス16′46(34･8%)および雌〆j(+′+)マウス10/27(37.0%)
に認められた0しかし,雄〆j(+′〕およびp5j(+/+)マウスに観察されたHCCは, それぞれ1/45(2･2%)および2′12(16･7%)であった040週屠殺例〆j(+′うおよび〆j
(+/+)マウスに観察された腫瘍の〆jがん抑制遺伝子exon5から8について, PCR･づinglestrandconformationpolymorphismanalysis(PCRづSCP法)により検索
したところ,〆jがん抑制遺伝子の変異は認められなかった｡
これらの結果からAPNHのp53KOマウスに対する標的臓器は肝臓であり, APNHの肝発がん性が示唆されたoAPNHの肝発がん性は,P53がん抑制遺伝子
の変異との関連性は低く,雌でより多くの腫瘍発生が認められることから代謝 あるいはホルモン環境との関連性が考えられた｡
第3に,肝臓でのチトクロームP450(CYP)による代謝とAPNHによる肝臓腫
瘍形成との関連性について検討し,さらに,APNHの肝臓におけるDNA付加体
の意義についても検討した｡APNHによる〆∬KOマウスにおける肝臓腫瘍形成 はp53がん抑制遺伝子の有無に影響されないが,APNH投与により肝臓のCYP
1AlmRNAが誘導されることが明らかになった｡さらに,肝臓において,APNH 投与濃度依存性にDNA付加体が増加した｡以上のことから,肝臓腫瘍形成に
CYPIAlによるAPNHの代謝活性化が関与することが示唆された｡
1.Initiationef鮎ctofAPNH OmCarCi血OgemeSis
2･CarCinogemiepotentialof
､IPヽll
3.Rela也0皿5hipbetweenmetabolism andcarcinogenicpotentialofAPNII
【謝辞】
本研究を終えるにあたり,終始御指導と御高閲を賜った岐阜大学大学院連合 獣医学研究科の柵木利昭教授,松井高峯教授,岡田幸助教授,三森国敏教授, 丸尾幸嗣教授に謹んで感謝の意を表します｡
また,本研究を進めるにあたり懇篤な御指導と御助言を頂きました愛知県が んセンター研究所立松正衛副所長,および腫瘍病理学部の塚本徹哉室長,山本
昌美技師,岐阜大学応用生物科学部家畜病理学教室柳井徳磨助教授および酒井 洋樹助手に深甚なる謝辞を表します｡
本研究の遂行に際し,終始暖かい御理解御協力を頂いたファイザー株式会社
中央研究所堀井郁夫博士,浜田悦昌博士,橘正克博士および関係者各位に深く 感謝いたします｡
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【要旨】
新 規 に 認 め られた 含 窒 素 芳香族 異 項環化 合物,
9‑(4'‑aminophenyl)‑9H‑pyrido[3,4‑b]indole(Aminophenylnorharman,APNH)は,S9
mix存在下でnorharmanとanilineの反応によって生成され,5b血one肋Typhimurium TÅ98とYGlO24に変異原性を発現する｡その遺伝子変異の強度は既知のヘテロサ イクリックアミンに匹敵する｡
No血aⅢnanは,タバコの煙,食品中など広く環境中に存在し,普通の食事を 摂取している健康なヒトの尿中にも,常に存在している｡また,anilineは,タ バコの煙,ある種の果物,野菜中に存在し,同様にヒトの尿中および乳汁中に も検出されている｡これらのことから,ヒトは日常的にnorharmanとanilineに 暴露されることは避けがたく,APNHは常に体内で生成,暴露されていると考 えられる｡そのためAPNHの発がん性の詳細なデータが必要と考えられる｡
本研究において,APNHの発がん性を検索するにあたり,イニシエーショ ン作用に基づいた肝臓における中期発がん性試験(In vivofive‑Weekinitiation
assay)および遺伝子改変動物を用いたがん原性試験を行い,APNHの発がん性 について検討した(第一,二章)｡さらにAPNHの代謝と発がんとの関連性につ いて解析した(第三章)｡
第1に,イニシエーション作用に基づいた肝臓における中期がん原性試験 によりAPNHの発がん過程におけるイニシエーション活性の検索を行った｡そ
の結果,肝臓のglutathioneS‑tranSferaseplacentalform(GSTJP)陽性巣面積の検索に おいて,APNHを投与していない対照群と比較して,APNHlO,3および1mg/kg
の各体重投与群に用量相関性の増加が認められた｡GSTしP陽性巣数においては,