劉秉慧余豐益Biing-Hui LiuFeng-Yih Yu吳亭萱Ting-Shuan Wu醫學研究所2026-10-299999-12-312013https://handle.ncl.edu.tw/11296/9n8vg7https://scholars.csmu.edu.tw/handle/123456789/35943橘黴素(CTN)與棒曲毒素(PAT)都是泛存於食品或飼料中的黴菌次級代謝物，過去的研究指出這兩種黴菌毒素在多種動物模式中皆會造成器官毒性，尤其以腎臟為主要標的。目前CTN與PAT對於胚胎早期的影響仍屬未知，因此本研究利用斑馬魚胚胎具有澄清透明，發育快速而且生理反應與高等脊椎動物相似的特點來探討CTN及PAT對胚胎毒性的影響。在CTN的實驗中，利用CTN處理6-72 hpf或24-72 hpf胚胎所造成的一半致死濃度(LC50)分別為57.9μM及60.4 μM；雖然在50 μM CTN處理下並不會改變腎臟螢光魚Tg (wt1b: GFP)體內腎絲球和腎管的外觀型態，但是組織切片染色的結果顯示50 μM CTN會造成腎絲球及原腎小管的細胞排列混亂並且出現囊泡；進行腎功能評估時則觀察到只要7.5 μM 的CTN就會造成72 hpf胚胎的腎絲球過濾功能下降，若是同時處理可促進血流量的腎臟藥物penoxifylline則可以回復CTN所降低的腎絲球過濾功能。在分子層面上，以50 μM CTN處理24-72 hpf的胚胎會誘發發炎相關基因COX2、TNFα以及IL-1β的mRNA表現量，但是並不會改變腎絲球標記基因wt1a和原腎小管功能性蛋白質Na+/K+ ATPase的表達及分佈。在心臟發育方面，50 μM CTN會令24-72 hpf的胚胎心臟產生異常摺疊、腔室體積縮小、心包膜水腫以及紅血球堆積等現象，此外心跳和血液循環的速率都隨著CTN的濃度提升而顯著下降，但是整體紅血球的數量則不會受到影響。CTN也會令72 hpf胚胎中的tbx2a和junB mRNA表現量分別降低和上升，同時使用心臟藥物metoprolol則可以部分回復CTN調控之tbx2a與junB的基因表現、還原心跳速率以及拯救CTN所造成的胚胎死亡現象。CTN會在斑馬魚的心臟部位誘發aldh1a2及cspg2的mRNA訊號上升，已知這兩個基因在視甲酸 (RA)訊號傳遞途徑中扮演著重要的角色，並且皆為微型核醣核酸-138 (miR-138)所調控的標的基因。進一步研究則指出CTN會抑制心臟部位的miR-138表現量，若是利用顯微注射將miR-138模擬物注入受精卵中則可以顯著回復CTN所造成之心臟構型異常的比例。在PAT的實驗中，以PAT處理6 hpf胚胎到120 hpf時的LC50值為176.8 μM，即使高達200 μM 的PAT也不會影響24-72 hpf腎臟螢光魚的腎臟外觀型態，但是組織切片染色的結果則顯示腎絲球細胞的排列異常。 此外，50-100 μM PAT會造成胚胎腎絲球的過濾功能下降，若是利用可改變滲透壓的腎臟藥物mannitol與PAT共同處理則可以回復PAT所造成的腎功能損傷。但是PAT並不會影響斑馬魚胚胎的心臟外型，組織結構以及心跳功能，此外PAT也不會影響120 hpf胚胎的下顎軟骨發育。總結來說，CTN對斑馬魚胚胎具有心臟及腎臟毒性，推測其可能機轉是CTN藉由抑制miR-138的表現促使aldh1a2表現量上升，活化RA 傳遞途徑，進而抑制tbx2a的訊號並且影響心臟發育，受損的心臟功能可能誘發發炎反應，最後造成腎臟組織損傷並且導致腎功能下降。另一部分，PAT會造成斑馬魚胚胎腎臟組織及功能性的傷害，但是並不會影響其心臟結構及功能。本研究首次證實CTN及PAT所誘發的胚胎毒性並且探討相關的分子機制，所得到的數據和結論可以提供重要的資訊以維護大眾的公眾健康。Citrinin (CTN) and Patulin (PAT) are secondary metabolites secreted by fungi that frequently contaminate food and feed. The organotoxicity of CTN and PAT has been reported in several animal models, and kidney is the major target organ of these two toxins. However, their early developmental toxicities to embryos remain unclear. Since, zebrafish shows the characteristics of embryo transparency, rapid development, and similarity to human, the zebrafish embryo was used as a vertebrate model to study the developmental toxicity of CTN and PAT in the present study.In the CTN study, the LC 50 values of embryos at 6-72 hpf and 24-72 hpf were found to be 57.9 and 60.4 μM, respectively, after exposed to CTN. When the embryos of Tg (wt1b:GFP), a transgenic line with fluorescent nephrons, were exposed to 50 μM CTN, the gross morphology of glomeruli and pronephric tubules was not changed. Further histological examination of 50 μM CTN-treated embryos revealed the disorganization of cell arrangement and the presence of cysts in the glomeruli and tubule. The glomerulus filtration rate, an indicator of renal function, was significantly decreased in 7.5 μM CTN-treated group. The co-administration of pentoxifylline, a renal medicine improving blood flow, was able to restore the renal dysfunction induced by CTN. From the viewpoint of molecular level, 50 μM CTN induced the mRNA expression of proinflammatory genes, including COX2, TNFα, and IL-1β; however, the expression and distribution of glomerular marker gene wt1a and the pronephric tubule functional protein Na+/K+ATPase were not altered. On the other hand, CTN at 50 μM resulted in various cardiotoxic phenomena, including abnormal heart tube looping, decreased chamber volume, pericardium edema and erythrocyte accumulation. Additionally, the heartbeat and blood circulation of embryos were significantly decreased with the increasing CTN concentrations, but the gross number of erythrocytes was unaffected. CTN also down-regulated the mRNA levels of tbx2a and up-regulated that of junB in 72 hpf embryos. The co-presence of heart medicine metoprolol with CTN restored the mRNA levels of tbx2a and junB, recovered the heartbeat rate, and rescued the CTN-induced lethality. Further evidence demonstrated that CTN exposure increased aldh1a2 and cspg2 mRNA levels in the heart areas of 72 hpf embryos. Both aldh1a2 and cspg2 genes are known to play important roles in the retinoic acid (RA) signaling pathway and served as the target genes of microRNA-138 (miR-138). The expression of miR-138 was also significantly decreased in the CTN-treated heart. Microinjection of the miR-138 mimic into embryos at 1-2 cell stages was able to reduce the proportion of 72 hpf embryos with cardiac defects which were caused by CTN treatment.When 6 hpf zebrafish embryos were treated with nephrotoxin PAT till 120 hpf, the LC50 value was found to be 176.8 μM. The PAT concentration as high as 200 μM did not affect the kidney morphology of 24-72 hpf Tg (wt1b:GFP), but the disorganization of cell arrangement was found in the glomeruli of embryos by histological examination. In addition, the glomerular filtration rate of 72 hpf embryos was significantly decreased after 50-100 μM PAT treatments. Co-administration of mannitol, a clinical medicine with osmosis modulation ability, was able to repair the renal function damaged by PAT. However, PAT exposure did not cause any evident heart defect, no matter from the perspectives of morphology, histology and cardiac function. PAT treatment also showed no effect on the development of jaw cartilage in 120 hpf embryos.In conclusion, treatment of zebrafish embryos with CTN led to developmental cardiotoxicity and nephrotoxicity. CTN exerts part of its adverse effects on developing heart through suppressing miR-138 expression, subsequently promotes RA-related aldh1a2 and cspg2 activities and attenuates tbx2a levels. Moreover, heart defects and decreased blood circulation may be the cause of pro-inflammatory genes (COX2, TNFα and IL-1β) activation which results in decreasing renal function. On the other hand, PAT was able to cause developmental nephrotoxicity, but not cardiotoxicity, in zebrafish embryos. This study is the first time to demonstrate the developmental toxicity of CTN and PAT by applying a zebrafish model. Results collected herein could provide a solid base to evaluate the adverse effect of mycotoxin on the general public, including pregnant women and developing individuals.thesis