T-2 toxin is one of type A trichothecerie mycotoxins, belonging to 12,13 epoxytrichothecenes. It has a double bond at position C-9;10, a 12-13-epoxide ring, and a variable number of hydroxyl and acetoxy groups.T-2 toxin is produced by various species of Fusarium. The most important fungus is F. sporotrichioides , a saprophyte (i.e. not pathogenic to plants) which grows between -2°C and 35°C and only at high water activities (above 0.88). In consequence, T-2 toxin is normally found in grains at harvest, resulting from water damage to grains, which may occur when they remain in the field for extended periods during or after harvest, especially in cold weather, or when become wet during storage. Therefore, T-2 toxin has been found to contaminate foods, animal feeds and agricultural products, and has been found in wide area of the world.

T-2 toxin, possesses the highest toxic in animals among type A trichothecene. The toxin exerts such toxic effects in farm animals and human as reduced consumption of feed, skin irritation, diarrhoea, multiple haemorrhage and immunosuppressive effects. Actively dividing cells are particularly sensitive to the toxin, and therefore hematopoietic organs such as bone marrow, spleen and thymus are primary target organs to the toxin. The outbreaks of alimentary toxic aleukia have occurred in Russia in 1913 and 1944 in association with ingestion of moldy wheat grains contaminated mainly with T-2 toxin.

The metabolic fate of T-2 toxin in farm animals has long been of great interest. The presence of T-2 toxin in grains and feedstuffs causes not only animal health problems, but also a concern about the transmission of potentially toxic residues into animal-derived products intended for human consumption. T-2 toxin metabolism occurs in the liver but also in the digestive tract and more particularly in the rumen of ruminants. Four basic metabolic biotransformations, namely hydrolysis, oxidation, reduction and conjugation, occur during the metabolism of T-2 toxin by different tissues or organs. Various metabolites of T-2 toxin have been isolated and identified in animals, but most of them are non-toxic or weakly toxic compared with T-2 toxin. In most of studies, researchers have given T-2 toxin to animals via oral, intramuscular, intravenous or intraperitoneal route rather than subcutaneous (s.c.) route to animals. In vitro experiment using animal tissues have shown that the liver of various animal species can metabolize T-2 toxin mainly to HT-2 toxin. Formation of other metabolites by liver tissues has been noted, but their quantity is very little compared with that of HT-2. Pfeiffer et al (1988) have reported that the metabolic profile of T-2 toxin in rats is largely affected by the route of its administration. However, the fate of subcutaneously administrated T-2 toxin in pigs is unknown. Although many toxicological studies have already been done, little is known about the effect of T-2 toxin and the effect of the toxin on liver drug metabolizing enzymes in pigs.

In order to gain insight into the fate and the effect on liver drug metabolizing enzymes of T-2 toxin in piglets, a series of experiments were performed.

In Chapter 1, the author investigated the distribution of T-2 and its metabolites in piglets received a single s.c. injection of T-2 toxin. In order to monitor the distribution of T-2 toxin in piglets, a GC/MS method was employed. The GC/MS was chosen in this experiment because it is used widely for analysis of trichothecenes. Mass spectrometric analysis increases the sensitivity of the method considerably compared to other spectrometry-based detection techniques, because selected ion monitoring (SIM) can be used to detect only desired ions produced by the analytes. As metabolites, HT-2 toxin was detected in serum, urine and faeces, but not in bile at both 24hr and 48hr after subcutaneous injection at dosage of 0.3mg/kg bwt; Relating high levels of T-2 triol and Neosolaniol were found in urine and feces only at 48hr. No any parent toxin and its metabolites were detected in bile samples at any time.

In Chapter 2, the author investigated changes of T-2 and HT-2 toxins in blood plasma after s.c. injection of T-2 toxin in a piglet. The author investigated the distributions of T-2 toxin in plasma of piglet for 48 hours continuously. In order to collect blood samples continuously, an indwelling catheter was surgically implanted in the left jugular vein. A liquid chromatography/time-of flight mass spectrometric method was employed to monitor the distribution of T- toxin and HT-2 toxin precisely, one of its metabolites in plasma of the piglet. The limits of detection of T-2 toxin and HT-2 toxin were 0.2 and 0.6 ppb respectively. T-2 toxin dissolved in DMSO (1 mg/ml) was administrated to the animal at a dose of 0.3mg/Kg BW by the subcutaneous route into the right lower half of the ventral abdomen and care was taken to avoid injections into any wrong positions. Blood samples were taken from jugular vein catheter before and after T-2 toxin administration at time 0.0min, 15min, 30min, 60min, 90min, 2hr, 4hr, 8hr,20hr and 48hr. The highest peak concentrations in plasma appeared at about 30 and 90minute for T-2 and HT-2 toxin was observed after administration of T-2 toxin to piglet by subcutaneous route. The results of this study show that the speed of biotransformation T-2 toxin in piglet was delayed greatly. The reasons for the time delay may be mainly caused by the administration method.

From all over the results of Chapter 1 and 2, the fate of T-2 toxin injected s.c. to piglets was summarized as follows: T-2 toxin is absorbed rapidly and metabolized to HT-2 toxin in the liver, and then both the toxins may be excreted into the intestine via bile and metabolized to less toxic neosolaniol and T-2 triol.

In Chapter 3, the effect of T-2 toxin on hepatic drug metabolizing enzymes in piglets was investigated. In order to investigate the effects of subcutaneous administration of T-2 toxin on hepatic drug metabolizing enzymes in piglets, the activities of phase I and phase II toward corresponding substrates in livers of piglets were measured. Piglets were administrated 0.3 mg T-2 toxin/Kg Bwt dissolved in DMSO by single s.c. injection. Control animals received only vehicle (DMSO). The activities of Phase I and Phase II enzymes were determined at 24h and 48h after the last s.c. injection. The activities of cytochrome P450 (CYP) 1A2 and 2E1 increased slightly at 24h and 48h (P<0.05). The CYP3A4 activity increased at 24h (P<0.01), and tended to decrease at 48h, but not significantly (P>0.05). The glutathione S-transferase activity towards cumene hydroperoxide increased slightly at 24h (P<0.05), but decreased slightly at 48h (P<0.05). No significant changes were observed in the glutathione s-transferase activity toward CDNB either at 24hr or 48hr. Western blot analysis of liver fractions revealed increased levels of 1 A2, 2E1, 3A4, GSTa , GST M1-1 at 24h, and that of CYP2E1 at 48h.The results suggest that T-2 toxin causes modulation of Phase I and Phase II drug metabolizing enzymes in piglets.

Thus all over the results of this study presented new and valuable findings on the fate of T-2 toxin and the effect of this toxin on drug metabolizing enzymes in piglets.

T-2トキシンはフザリウム属のカビにより産生されるカビ毒であり、収穫時の穀物を汚染することが多く、その結果、食品や飼料を含め農産物を広く汚染することが認められている。T2-トキシンは類似化学構造をもつA型トリコテセン化合物の中では最強の毒性を有し、家畜や人に対して免疫能低下、出血、下痢、食欲低下、皮膚炎等を招来する。とくに細胞分裂の盛んな骨髄、脾臓、胸腺等の造血組織はこの毒素に感受性が高く、第一の標的臓器となる。飼料原料である穀物のT-2トキシン汚染はそれ自体が動物や人の健康を障害するだけでなく、人が摂取する動物由来の食品への残留による問題も引き起こす。摂取されたT-2トキシンは肝臓で代謝されるとともに、反芻動物の第一胃を含め、消化管においても代謝されることが認められている。動物体内において見出されるそれらの代謝物の大部分は、無毒性またはT2-トキシンに比して低毒性であることが認められている。以上、T-2トキシンの動物体内代謝と毒性に関する知見は多数得られているが、ブタの体内におけるT-2トキシンの代謝運命とブタの肝臓の薬物代謝酵素に及ぼすT-2トキシンの影響についての知見は少ない。著者は、著者は、カビ毒によるブタの被害が大きいこと、また、ヒトのモデル動物としてのブタの有用性を考慮し、ブタを用いて以下の一連の研究を行った。

第一章では、1ヶ月齢ブタにおけるT-2トキシンの体内動態を明らかにするために同毒素(0.3mg/kg)を皮下投与し、24および48時間後に、血清、尿、糞、胆汁を採取し、各試料中の同毒素とその代謝物をガスクロマトグラフ質量分析計で分析した。その結果、代謝物として、T-2トキシンと同等の毒性を持つことが知られているHT-2トキシンが血清、尿、糞に見出された。毒性が極めて低いことが知られているT-2Triolとネオソラニオールは、48時間目にのみ尿と糞に認められた。胆汁中にはいずれの時間にもT-2トキシンもその代謝物も認められなかった。

第二章では、第一章において血清中に認められたT-2トキシンとHT-2トキシンについて、投与後の血中濃度変化の推移を明らかにするために、1ヶ月齢ブタにT-2トキシン(0.3mg/kg)を皮下投与した後に、頚静脈に挿着したカニューレより経時的に投与48時間後まで採血し、血漿中のT-2トキシンとHT-2トキシンを液体クロマトグラフ質量分析計で分析した。その結果、血漿中T-2トキシン濃度の最高値は投与30分後に、HT-2トキシン濃度の最高値は90分後に、それぞれ認められ、その後24時間以内に両毒素とも速やかに減少するが、その減少はHT-2トキシンの方がT-2トキシンよりも遅れることが見出された。

以上、第一章と第二章の結果から、仔豚におけるT-2トキシンの体内運命の重要な側面を明らかにすることができた。すなわちこれまでの知見と合わせ、本研究の結果より、T-2トキシンは速やかに投与部位から吸収され、肝臓においてHT-2トキシンに代謝されること、両毒素は一部、24時間以内に胆汁を介して、または血液循環より直接に、腸管腔内に排泄され、腸管腔内で低毒性代謝物であるネオソラニオール等に代謝変換され、一部は再吸収されてから尿中に、残余は糞中に排泄されることが明らかにされた。

第三章では、T-2トキシンの肝薬物代謝機能に及ぼす影響を究明するために、ブタ(1-1.5ヶ月齢)にT-2トキシン(0.3mg/kg)を皮下投与し、24および48時間後に肝臓を摘出し、ミクロゾームとサイトゾール画分を得た。ミクロゾーム画分については、3-cyano-7-ethoxycoumarin、7-methoxy-4-trifluoromethylcoumarin、dibenzylfluorescin等を基質としてチトクロームP450(CYP)活性を、サイトゾール画分については、cumene hydroperoxide等を基質としてグルタチオンS-トランスフェラーゼ(GST)活性を、それぞれin vitro試験系を用いて測定した。その結果、CYP1A2とCYP2E1活性は24および48時間後に若干増加すること、CYP3A4活性は24時間後に増加し、48時間後に減少することが認められた。また、GST活性についてはcumene hydroperoxideに対する活性が24時間後に増加し、48時間後に減少することが見出された。また、各酵素たんぱくの発現をウェスタンブロット法により測定した結果、CYP1A2、CYP2E1, CYP3A4、GSTα、GSTM1-1は24時間後に増加すること、CYP2E1は48時間後にも増加することが認められた。代謝運命の結果とあわせ以上の結果から、T-2トキシンはブタにおいて、解毒代謝される前の投与24時間以内に肝薬物代謝機能に顕著な影響を招来すること、これにより種々の化学物質の生体影響を修飾するであろうことが示唆された。

以上、本研究により、仔豚におけるT-2トキシンの生体内運命および肝薬物代謝機能に及ぼす影響について今まで不明であった重要な側面が明らかにされた。よって審査員一同は本論文が博士(獣医学)の学位を授与するのに値するものと認めた。