- 學位論文
咖啡因在斑馬魚早期胚胎發育時期引發之畸形
Induced zebrafish larvae malformation by caffeine
摘要
咖啡因是一種廣泛的存在於許多食品中，包括咖啡、茶葉、可可。然而，在我們食用或飲用這些日常食品的時候，常常不自覺得過量食用咖啡因。咖啡因的結構為一種生物鹼，這種物質對人類而言是一種植物性的中樞神經興奮劑 (O'Connell and Zurzola, 1984)，對於研究咖啡因在人體中的作用已經有許多的報導。在服用50-200mg的咖啡因後，會產生潮紅、寒顫、煩亂、興奮、沒有胃口、虛弱、顫抖，另外還有高血壓、低血壓、心跳過速、嘔吐、發熱、妄想症、幻覺、疾病發作、心律不整、心臟停止跳動、昏迷和死亡(Kerrigan and Lindsey, 2005)；若是口服大量的劑量（服用超過5 g的咖啡因）是一種極度有毒的藥劑，會造成人體的心律不整，心跳過快、嘔吐、抽搐、昏睡和死亡 (Kerrigan and Lindsey, 2005)。但是我們仍然對咖啡因在胚胎發育時期的影響並不明瞭，所以我們接著選擇斑馬魚作為我們的模式物種，以斑馬魚為模式物種的優點有：個體數高和胚胎透明容易觀察。這兩個優點讓我們能夠更細微地觀察咖啡因在斑馬魚早期發育所造成的影響。因此我們找到咖啡因對斑馬魚胚胎的半數致死濃度，LC50為250 ppm。然而，我們為了收集更多的觀察樣本，於是將濃度調降為150 ppm，並觀察其外觀變異，發現胚胎的體長變短。接著在能動性 (motility) 的測驗下，咖啡因濃度越高，能動性越低。於是，我們以F59、Znp1、aat螢光抗體染色做更細微的觀察，結果發現肌纖維排列混亂，運動神經軸索（axon）無法正常延伸發育、感覺神經數量減少、腦部的發育延遲、軀體彎曲以及心臟和血管發育不良的現象。由此，我們認為咖啡因對斑馬魚胚胎對神經發育、肌肉發育、以及心臟發育具有相當的毒性。
並列摘要
Caffeine is a commonly existing in many kinds of food, including coffee, tea, and coca. However, little is known about the caffeine-induced toxicity and teratogenicity during early embryonic development. Here, we used zebrafish as a model to test the toxicity and teratogenicity because of their transparent eggs; therefore, the organogenesis of zebrafish embryos is easy to observe. After low dosages of caffeine (1-100 ppm) treatment, the zebrafish embryos exhibited a 100 % survival rate. As the exposure dosages increased, the survival rates decreased. No embryos survived after treatment with 1000 ppm caffeine. The 50% lethal dose (LD50) of zebrafish is found to be in the range of 200-275 ppm. Shortened and curved body were observed after treatment with caffeine. Compared to untreated littermates (vehicle-treated control), caffeine-treated embryos exhibited significantly reduced tactile sensitivity frequencies of touch-induced movement (vehicle-treated control: 10±3 v.s. 150 ppm caffeine: 0±0; N=30). Subtle changes are easily observed morphology by staining with specific monoclonal antibodies F59, Znp1 and aat to detect changes in muscle fibers, motor axons, sensory neurons, Rb cell and brain, respectively. Our data showed that the treatment of caffeine led to misalignment of muscle fibers, motor neuron innervations, less acetyl-choline receptor cluster and Rohon-beard cell. On the basis of these observations, we suggest that caffeiene is able to induce neurotoxicity and nephrotoxicity of zebrafish larvae.