Zearalenone (ZEA), 6-(10-hydroxy-6-oxo-trans-1-undecenyl)-β-resorcyclic acid lactone, is an estrogenic mycotoxin produced by several Fusarium species and has been found to be a common and widespread contaminant in cereal grains and animal feedstuffs. Various health problems associated with this mycotoxin have been documented in farm animals. The most important toxic effect of ZEA is its estrogenic effect, which has recently led to identification of ZEA and zearalenols (ZOLs) as endocrine disrupters. ZEA can bind to estrogen receptors and cause diseases in the reproductive system, impaired fertility and abnormal fetal development in farm animals. In addition to these estrogen-related effects, hepatic and renal lesions including hepatocarcinogenesis, and hematotoxicity in rodents, immunotoxicity in mice and humans, the reduction of milk production in cows have been observed. The impact of ZEA on human and animal health is now increasingly recognized. Ruminants are less known for their sensitivity to the negative effects of ZEA than non-ruminants. In order to characterize the metabolism of ZEA in ruminants, a series of studies focusing were performed using adult goats.

In chapter 1, in order to clarify the features of ZEA metabolism in various organs, the in vitro zearalenone-reducing activity was investigated by postmitochondrial, microsome and cytosolic fractions of various organs of adult male and female goats. The results indicated that in the liver, α-ZOL was a major metabolite in cytosol, whereas β-ZOL was a predominant metabolite in microsomes. Post-mitochondrial fractions of the liver converted ZEA predominantly to more potent estrogenic compound, α-ZOL, indicating that the goat liver may function as an activation organ rather than as an inactivation organ. Such a feature of ZEA metabolism was confirmed by the Km and Vmax values from an enzyme kinetics experiment. In most other tissues including rumen tissue, the activity converting ZEA to α-ZOL was also higher than that to β-ZOL.

In chapter 2, in order to clarify whether 3α-hydroxysteroid dehydrogenase (HSD) and 3β-HSD are involved in ZEA metabolism, the effects of endogenous substrates for 3α-HSD and 3β-HSD were studied in vitro using liver. The results showed that the reduction of ZEA to α-ZOL and β-ZOL was competitively inhibited by the endogenous substrates, suggesting that 3α-HSD and 3β-HSD are involved in the ZEA to α-ZOL and to β-ZOL conversion. The expression of 3α-HSD mRNA in liver was increased, but that in the jejunum decreased, by intravenous injection of ZEA to goats, showing possible effects of ZEA on estrogen metabolism by these organs.

In chapter 3, in order to delineate the fate of ZEA in goats, the pharmacokinetics of ZEA were investigated by determining changes in blood plasma concentrations of ZEA and its metabolites in intravenously injected goats. Also the excretion of ZEA and its metabolites in urine and feces, and tissue residues of these metabolites in the liver were investigated. Kinetic evaluation of the plasma data revealed that the distribution half-life (t1/2α) and elimination half-life (t1/2β) of ZEA were 3.15 h and 28.58 h, respectively. The urine and feces samples revealed that ZEA is excreted as ZEA, α-ZOL and β-ZOL, β-ZOL being predominant metabolite. Urinary excretion showed that ZEA and its metabolites were mostly in glucuronide and /or sulfate conjugated forms. Faecal excretion showed that free forms of ZEA and ZOLs were major constituent. A large proportion of ZEA and its metabolites were excreted into urine during 24 h, and into feces during 48 h after administration of ZEA. In liver tissue samples, small amounts of α-ZOL and β-ZOL were identified, while ZEA was below detection limits.

In chapter 4, the effect of ZEA on expression of ERα and ERβ mRNA, and histopathological changes were investigated after intravenous administration. The expression of ERα mRNA was significantly increased in the liver, but no clear effect was found in the jejunum and rumen. The expression of ERβ mRNA was significantly increased in the jejunum, but not in the liver and rumen. These results suggest that ZEA can change the estrogenic response of the liver and jejunum. Histopathological changes such as lymphocytic infiltration were observed in the liver, kidney, uterus and epididymis, but not in gastrointestinal tract, ovary and testis. Although whether these ZEA-induced histopathological changes are caused via ERα and ERβ remains uncertain, the result shows that ZEA induces toxicity other than estrogenic effect in goats.

Allover the results of these studies delineated firstly a characteristic feature of ZEA metabolism in goats, i.e., ZEA is metabolized to more potent α-ZOL and less potent β-ZOL not only in the liver but also in the other organs, β-ZOL being more rapidly excreted in feces and urine. Toxic effects other than estrogenic effects of ZEA were also observed in the liver and other organs. Thus this study may provide a better understanding on ZEA metabolism in relation to its toxic effects in goats, and background information for other ruminant species.

ゼアラレノン(以下ZEA)はフザリウム属のカビにより産生されるエストロゲン作用を有するカビ毒であり、動物飼料を含め穀物を広く汚染することが認められている。

ZEAはエストロゲンレセプターに結合し、家畜において繁殖障害や胎児発育障害を引き起こすことが知られている。また、エストロゲン様作用による障害以外にも、げっ歯類動物において肝臓がんや造血障害、マウスとヒトにおいて免疫毒性、ウシにおいて搾乳量減少等を招来することが認められている。ヒトや動物の健康に及ぼす影響への懸念が増大しているにもかかわらず、とくに反芻動物における知見が限られていることから、この分野の研究の推進が望まれている。そこで反芻動物体内におけるZEAの代謝に関する知見を得るために、本研究においては成熟ヤギを用い、一連の研究を行った。

第一章では、ヤギの諸臓器におけるZEA代謝像を究明するために、成熟雌雄ヤギの臓器組織の細胞分画のZEA代謝活性を調べた。その結果、肝臓の代謝活性が最も高く、サイドゾーム分画により、エストロゲン作用の強いα-ゼアラレノール(以下ZOL)へ、ミクロゾール分画によりエストロゲン作用の弱いβ-ZOLへ、また、ポストミトコンドリア分画により主にα-ZOLへの各変換が認められたことから、ヤギ肝臓はZEAの不活化よりも活性化の機能をもつものと考えられた。こうしたZEA代謝の特徴は、Km値とVmax値においても認められた。第一胃を含め、他の組織においても、α-ZOLへの変換活性の方がβ-ZOLへの変換活性よりも高かった。

第二章では、ZEA代謝への3α-Hydroxysteroid dehydrogenase(以下HSD)と3β-HSDの関与を究明するために、肝臓組織を用いて両酵素の生体内基質であるステロイドホルモンのZEA代謝に及ぼす影響をin vitroで調べた。その結果、これらステロイドホルモンは、ZEAのα-ZOLとβ-ZOLへの変換を抑制することが認められたことから、ZEAの代謝に3α-HSDと3β-HSDが関与するものと考えられた。さらに、この点を確認するために、ヤギの肝臓等の組織における両酵素のmRNA発現を測定したところ、肝臓や消化管等にその発現が認められ、ZEAによって肝臓中3α-HSDのmRNA発現量が増加したことから、ZEAが肝臓のステロイド代謝に影響を及ぼすことが考えられた。

第三章では、ヤギ個体におけるZEAの生体内運命を究明するために、ZEAを静脈内投与後に、血漿中のZEAとその代謝物の濃度変化を追跡すると共に、糞尿中への排泄を調べた。ZEAの血漿濃度の変化から、分布半減期と消失半減期はそれぞれ3.15および28.58時間であること、また、糞尿中にはZEA、α-ZOL、β-ZOLが排泄されること、尿中へは抱合型のみで糞中へは抱合型と遊離型で排泄されることが認められた。これらの成績から、ZEAのα-ZOLとβ-ZOLへの代謝変換がin vivoでも確認されるとともに、ZEAの反芻動物における生体内運命が始めて明らかにされた。

第四章では、ヤギの肝臓や子宮等の組織中のエストロゲンレセプター(以下ER)αとERβのmRNA発現を測定比較するとともに、ZEAの静脈内投与によるこれらmRNA発現に及ぼす影響を調べた。その結果、両ERのmRNA発現は肝と子宮でとくに高いことが見出され、これら組織ではZEAによりERの発現が上昇すること、空腸においてはERβの発現が低下することが観察されるなど、ZEAのER発現に及ぼす影響が始めて明らかにされた。また、ZEAを静脈内投与されたヤギにおける病理組織学的変化を調べたところ、肝臓と子宮にリンパ球浸潤等の病理変化が認められなど、ER発現の変化とともにZEAの毒性影響が誘起されることが認められた。

以上、本研究は、ヤギにおけるZEAの代謝活性と生体内運命に関して、毒性発現機序の理解促進に役立つ重要な側面を明らかにし、関連獣医学分野に貢献するところ大であると判断された。よって審査委員一同は、本論文が博士(獣医学)の学位を授与するに値するものと認めた。