微囊藻毒Microcystin-LR抑制斑馬魚胚發育之研究

Abstract

微囊藻毒（Microcystin-LR, MC-LR）是一種由淡水藍綠藻產生的毒素，其主要作用在於抑制絲氨酸-酥氨酸蛋白質去磷酸酶（serine/threonine protein phosphatase）1（PP1）及2A（PP2A）之活性。MC-LR可藉由抑制蛋白質去磷酸化而使細胞內功能蛋白過度磷酸化導致細胞骨架變形甚致細胞死亡。微囊藻毒對魚類之危害雖有報導但其毒性卻因魚種類及試驗方法不同而有極大差異，而其對幼魚或早期魚胚之影響亦是如此。推測其因，可能與微囊藻毒本身之細胞膜通透性低有關，因此在本實驗中我們利用顯微注射來進行MC-LR的處理並觀察其對胚發育之影響，結果發現，當MC-LR處理的濃度越高，斑馬魚胚的死亡率畸型率相對增高，在最高濃度（900 nM）MC-LR處理的斑馬魚胚會有細胞堆疊、細胞移位、分裂不正常的現象；除此之外，利用免疫化學染色法並在共軛對焦顯微鏡下觀察，我們發現MC-LR可使得協助胚細胞間連結的cadherin和β-catenin分布明顯地受到干擾，肌動蛋白（actin）也有不正常分布與堆聚的情況，而存活之斑馬仔魚也會受到MC-LR影響而發生各種不同畸型。綜合以上結果，我們證明微囊藻毒在進入斑馬魚胚內會對其分裂及發育有毒害，而其毒理作用機制則可能是經由對細胞骨架及其調控因子磷酸化之調節，但此推測仍須進一步探討其細胞內相關訊息傳導機制始可證實。

Microcystin-LR（MC-LR）, a cyanobacterial toxin, is a potent inhibitor of protein phosphatase 1（PP1）and protein phosphatase 2A（PP2A）. PP1 and PP2A are critical regulators in embryonic development. However, the effects of MC-LR in embryonic development are controversial. MC-LR has been demonstrated to be highly toxic in medaka, but not in zebrafish or rabbit embryos. The causes of difference may be due to membrane impermeability that impaired the delivery of MC-LR into cytoplasm of zebrafish and rabbit embryos. Therefore, we microinjected MC-LR directly into the developing zebrafish embryos and investigated the effects of MC-LR on embryonic development. We demonstrated that MC-LR induced the lethality of zebrafish embryos in a dose- and time-dependent manner. MC-LR also induced the loss of blastomere coherence via the interference of β-catenin and cadherins distributions. Furthermore, the MC-LR treated fry revealed various developmental defects. These results suggested that MC-LR might affect the phosphorylation equilibrium of signaling molecules, including β-catenin and cadherins, required early in zebrafish embryonic development.