黃麴毒素B1對斑馬魚胚胎發育的影響

Effects of Aflatoxin B1 on the Development of Zebrafish Embryos

鄭雅之(Ya-Chih Cheng)

指導教授 ： 劉秉慧

國立臺灣大學/醫學院/毒理學研究所/碩士(2017年)

https://doi.org/10.6342/NTU201704094

摘要

黃麴毒素B1（Aflatoxins B1, AFB1）為黃麴黴 (Aspergillus flavus) 等黴菌產生之二級代謝產物，已知其對人類及動物具有肝毒性及致肝癌性。目前對於AFB1在胚胎發育上的影響及毒性機制尚不明瞭，因此本研究將利用斑馬魚胚胎作為動物模式來探討AFB1對於胚胎發育的影響。實驗結果發現，AFB1處理斑馬魚胚胎達120 hpf (hours post fertilization) 後，雖然導致胚胎的高死亡率，卻不會對胚胎造成明顯的不良型態變化。以0.25及0.5 μM的AFB1處理轉殖肝螢光魚胚胎Tg（lfabp：eGFP）以觀察AFB1對肝臟發育的影響 (6-96 hpf)，結果顯示肝臟螢光強度隨毒素劑量上升而顯著下降，組織切片分析也顯示肝臟有縮小現象。末端脫氧核苷酸轉移酶脫氧尿苷三磷酸切口末端標記螢光染色 (Terminal deoxynucleotidyl transferase dUTP nick end labeling, TUNEL)的結果指出AFB1會促進肝臟細胞凋亡，磷酸化組蛋白H3免疫染色 (phospho-histone H3 immunostaining) 的結果則顯示AFB1可能可以抑制肝臟細胞增生。以prox1和hhex探針進行全胚體原位雜交法 (Whole-mount in situ hybridization) ，發現胚胎肝臟在特化 (specification) 階段（24hpf）就會受到AFB1的傷害，進一步在肝臟發育不同的不同時期以AFB1處理轉殖肝螢光魚胚胎Tg（lfabp：eGFP）觀察，得知肝臟在出芽/分化 (budding/ differentiation) 及增長 (outgrowth) 階段都有可能受AFB1影響而造成肝臟縮小的情形。AFB1的暴露亦會導致肝臟特異性的miR-122表現下降，但不會影響胚胎中凝血因子基因（f7、f3a、f3a）和肝臟特異性基因（vtna、cp）的表現。此外AFB1會誘發斑馬魚肝細胞（ZFL）中的氧化壓力上升，而抗氧化劑N-乙醯半胱氨酸（N-acetylcysteine , NAC）和維生素C (Vitamin C, Vit C) 的使用可以降低AFB1造成的細胞傷害、胚胎死亡率，而維生素C更可能可以改善肝臟縮小的情形。
過去研究指出AFB1也會造成中樞神經的損傷。我們由行為測試觀察到AFB1的暴露造成斑馬魚胚胎異常的游泳模式，同時也使胚胎移動的距離下降，因此推測AFB1可能會影響胚胎發育階段的神經生長。早期神經元的標記（HuC）和神經毒性的生物指標（gfap）的mRNA變化證實了AFB1會影響胚胎神經發育而產生神經毒性。乙醯化微管蛋白免疫染色 (Acetyl-alpha Tubulin staining, AcTub staining) 的結果也顯示，0.25及0.5 μM的AFB1明顯干擾了24 hpf胚胎神經元的發育。將 6 hpf 斑馬魚胚胎以 0.5 μM AFB1 處理至 48 hpf 後進行微陣列技術分析(microarray)，發現多個和神經發育相關基因的表現皆受到AFB1的影響。以半定量及定量聚合酶鏈鎖反應 (PCR) 確認在處理AFB1達24h及48h的胚胎中，ngfa 及atp1b1b mRNA的表現量顯著下降prtga mRNA的表現量則上升，上述結果表示AFB1可能可以干擾神經元的能量來源並抑制斑馬魚胚胎神經元的早期分化。
本篇研究證實AFB1會干擾早期胚胎肝臟和神經的發育，而相關的發育毒性的機制仍需要進一步的實驗來確認。

關鍵字

黃麴毒素 ； 斑馬魚 ； 發育 ； 肝臟 ； 神經

並列摘要

Aflatoxins B1 (AFB1) is a naturally occurring mycotoxin produced by the Aspergillus flavus group of fungi. It is also a well-known hepatocarcinogen that contributes significantly to the high incidence of hepatocellular carcinoma. Since there are few studies examining the developmental toxicity of AFB1, we used the zebrafish embryo as a vertebrate model herein. Treatment of zebrafish embryos from 6 hours post-fertilization (hpf) to 120 hpf resulted in a high mortality rate, but the morphology of embryos remained unchanged. To observe the effect of AFB1 on liver development, the embryos of transgenic zebrafish Tg(lfabp:eGFP) were treated with AFB1 ranging from 0.25 to 0.5 μM. We found that the liver fluorescence was decreased in a dose-dependent manner and histological analysis also indicated a shrunk liver size. TUNEL assay revealed that AFB1 promoted liver apoptosis and pH3 immunostaining implied that AFB1 might also inhibited liver proliferation. Whole-mount in situ hybridization with prox1 and hhex probes and observation of Tg(lfabp:eGFP) showed that the embryonic liver was affected by AFB1 at the specification and the budding/ differentiation stages (24-72hpf). Also, AFB1 treatment led to the down-regulation of liver-specific miR-122. However, AFB1 did not affect the gene expression of coagulation factors (f7, f3a, and f3a) and liver-specific genes (vtna and cp) in 6-72 hpf embryos. As AFB1 was found to increase the oxidative stress in zebrafish liver cell line (ZFL), ZFL cell death as well as embryo mortality mediated by AFB1 was restored in the presence of antioxidants N-acetylcysteine (NAC) and Vitamin C (Vit C). In addition, Vit C is likely to rescue the liver shrinkage that caused by AFB1.
Previous studies indicate that AFB1 also leads to brain damage. From our results of behavior test, AFB1 exposure decreased the total distance moved of embryos which swam in an abnormal shaking patterns. Therefore, we hypothesized that AFB1 might affect the neurodevelopment during developmental stage of embryos. The marker of early born neurons (HuC) and the biomarker of neurotoxicity (gfap) were both modulated by AFB1. Additionally, acetylated alpha-tubulin (AcTub) staining indicated that AFB1 disrupted the development of embryonic neurons. Microarray profiles of embryos after 48 hr AFB1 treatment showed an alteration of several genes which were related to neurodevelopment, including nerve growth factor a (ngfa), atp1b1b, and protogenin homolog a (prtga). PCR analysis further confirmed that AFB1 significantly decreased the expression of both ngfa and atp1b1b, and increased that of prtga gene in 6-48 hpf embryos, which suggest that AFB1 might have the ability to suppress neuronal differentiation and reduce the source of energy demand in neurons.
In conclusion, AFB1 might interfere with the embryonic liver and neural development. Further experiments are required to confirm the mechanism of developmental hepatotoxicity and neurotoxicity associated with AFB1.

並列關鍵字

Aflatoxin B1 ； zebrafish ； development ； liver ； neuron

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