利用斑馬魚胚胎模式研究烯酸酯類殺菌劑pyraclostrobin 之毒性

Abstract

百克敏(pyraclostrobin)是一種被大量使用的農業除真菌劑，透過地表徑流等途徑汙染環境，在世界各地的水域已被檢出。其對於水生生物包括魚類以及兩棲類，具有極高的非專一毒性。先前研究證實百克敏對於斑馬魚具有粒線體毒性、心毒性並造成發育延遲、增加氧化壓力、降低活動力。但對魚類其他生理功能的影響仍不清楚。本研究利用斑馬魚胚胎作爲毒理模式，將其曝露於100-1000 ppb的百克敏96小時後，觀察其對於基因表現、離子調節、側線感覺功能的變化；曝露百克敏120小時後，觀察聽覺與視覺行爲的改變。並利用轉基因螢光魚觀察中樞神經系統的影響。結果顯示百克敏96小時半致死濃度為204.6 ppb。濃度≥100 ppb 造成心率下降、側線毛細胞損傷、觸碰逃跑速度下降；濃度≥150 ppb造成體長降低，眼睛、耳石囊、耳石面積變小，卵黃消耗減少，體内Ca2+、K+離子含量下降，離子細胞數目減少；濃度200 ppb造成圍心腔水腫、皮膚幹細胞減少以及側線毛細胞減少。利用RNA定序分析基因表現發現許多異常包括:離子調節(atp1a1a, atp2b,atp6v, kcnj1a, rhcg)、側線感覺(cib2, myo6b, s100)、粒線體電子傳遞鏈(cytb, cox3、cox19)、內耳感覺(otomp, cep290, oc90)、視覺(cabp5a, clcc1)、細胞凋亡(casp3, casp8, casp17)等相關基因。在行為實驗發現，百克敏會降低聲音驚嚇反應之敏感度，並延長視覺運動反應時間。螢光魚的結果顯示，百克敏會造成脊髓運動神經元減少。綜上所述，本研究提出百克敏可能會造成魚類離子調節、周邊感覺(側線、視覺、聽覺)以及中樞神經異常，對魚類生存造成威脅，需更審慎評估百克敏的使用。Pyraclostrobin (PCB) is a commonly used agricultural fungicide that contaminates the environment through surface run-off and has been detected in aquatic environments globally. PCB exhibits high non-specific toxicity to aquatic organisms. Previous studies demonstrated that PCB caused mitochondria toxicity, cardiotoxicity, developmental retardation, oxidative stress, and hypoactivity in zebrafish. However, the effects of PCB on other physiological functions of fish are still not clear. In this study, zebrafish embryos were exposed to PCB at 100-1000 ppb for 96 h to investigate changes in gene expression, ion regulation, and lateral line sensory function. After 120 h of exposure to PCB, changes in auditory and visual behavior were also examined. In addition, the effects on the central nervous system were assessed using transgenic fish. Results showed that 96-h-LC50 of PCB was 204.6 ppb. PCB concentrations ≥100 ppb caused a decrease in heart rate, damage to lateral line hair cells, and a decrease in Vmax of touch-response. PCB concentrations ≥150 ppb caused decreases in body length, size of the eye, otolith vesicle, and sagitta otolith, yolk consumption, the ion contents (Ca2+ and K+), and the number of ionocytes. PCB at 200 ppb caused pericardial edema and decreased the number of skin p63+ stem cells and lateral line hair cells. RNA sequencing analysis revealed changes in multiple genes involved in ion regulation (atp1a1a, atp2b,atp6v, kcnj1a, rhcg), lateral line (cib2, myo6b, s100), the mitochondrial electron transport chain (cytb, cox3、cox19), audition (otomp, cep290, oc90), vision (cabp5a, clcc1) and apoptosis (casp3, casp8, casp17). In conclusion, the present study suggests that PCB can cause toxic effects on multiple physiological functions of fish, and the use of PCB in agriculture should be carefully evaluated.