水中鉀離子干擾一價鉈對青鱂魚之神經毒性效應探討

Abstract

鉈 (Thallium, Tl) 常用於電子、光學及半導體產業中，隨著科技快速發展，鉈可能隨產業製程及廢水進入環境中，因此在世界各地皆有監測到鉈於水域環境中流布。目前認為一價Tl+ 是環境中主要存在形式且美國環保署認定為具有高毒性之汙染物。然而關於Tl+ 對水生生物毒性機制仍不清楚。由於Tl+ 離子半徑與K+ 相似， Tl+ 可能與K+ 相關離子通道競爭 (e.g. Na+/K+-ATPase, NKA)，並干擾其生物有效性及毒性。本研究目的為使用青鱂魚作為模式生物，探討水體環境中Tl+ 及K+ 之生物及化學作用，包括死亡率、游泳行為、生物累積量、神經相關基因表現量、神經傳遞物質含量及氧化壓力等分析，以評估Tl+ 之急毒及非致死效應。結果顯示，Tl+ 對青鱂魚苗之96-h急毒性大小依序為Tl+ -LK (低鉀) > Tl+-MK (中鉀) > Tl+-HK (高鉀)。生物累積性大小為Tl+ -LK > Tl+-MK，同時Tl+ -LK (25 μg/L) 處理之魚體內鈣離子的濃度顯著上升。青鱂魚苗暴露於非致死濃度 (Tl+ = 5-25 μg/L) 七日後，Tl+ –LK (15-25 μg/L) 處理組對魚苗之平均游泳速度及活動百分比顯著下降，絕對旋轉角度則顯著上升；Tl+ -MK僅在Tl+ (25 μg/L) 時上述參數具顯著差異。研究續以即時聚合酶連鎖反應進行基因表現量分析，滲透壓調節相關基因鉀鈉幫浦 (atp1a1) 表現量受Tl+ -MK誘導；神經發育相關基因如 tuba1c 及 mbp 表現量受Tl+ -LK抑制；Tl+ -MK 則不受Tl+ 抑制；神經傳導相關基因多巴胺受體 (drd2) 及代謝相關基因 (comt) 表現量受兩種處理組抑制；多巴胺合成相關基因 (ddc) 僅受到Tl+ -MK抑制；ache表現量受兩種處理組抑制。其他相關生理指標也呈現類似的結果: 在Tl+ -MK (15 μg/L)中，滲透壓調節指標鉀鈉幫浦活性 (NKA) 亦顯著被誘導；Tl+ -LK則無顯著差異。神經傳遞物質多巴胺含量在Tl+ -LK無顯著差異；Tl+ -MK (25 μg/L) 顯著下降；乙醯膽鹼酯酶 (AChE) 活性在兩處理組均無顯著差異。此外，氧化壓力指標顯示Tl+ -LK會抑制抗氧化酵素如CAT、SOD、及GST活性；Tl+ -MK則無顯著差異。本研究以生物累積量作為評估終點進一步探討Tl+ 及K+ 競爭離子通道之相關證據。初步結果顯示魚體內鉈濃度在加入NKA抑制劑 (Ouabain) 與未加抑制劑相比些微上升，但無顯著差異；魚體內K+、Na+ 及Ca2+ 離子濃度未顯著改變。綜合上述結果，水體中K+ 濃度是影響Tl+ 對青鱂魚苗毒性的重要因子: K+ 濃度較低時，青鱂魚死亡率顯著增加且魚體內Tl+ 累積含量顯著增加。此外，Tl+ 的非致死性毒理機制應與改變神經發育及傳導相關基因表現量、干擾滲透壓調節、降低神經傳遞物質含量及抑制抗氧化酵素活性有關，進而導致青鱂魚游泳行為異常，另外關於Tl+ 及K+ 競爭離子通道之相關證據仍需要更多研究去證實。

Thallium (Tl) is a trace element commonly used in the electronic, optical and semi-conductive manufactures etc. With the booming of these high-tech industries, Tl usage is rapidly increased, leading to frequent occurrence of Tl being detected in the aquatic environment via various routes of human activities. Tl mainly exists in the environment as monovalent Tl+ and it is a priority pollutant with high toxicity suggested by the US Environmental Protection Agency and the European Water Framework Directive. However, information regarding toxic mechanisms of Tl+ is currently limited, so the associated risk of Tl+ exposure and the toxicity to the aquatic ecosystem is currently unclear. Since ionic radius of Tl+ is similar to that of potassium (K+), Tl+ may interact with K+ ion channels (e.g. Na+/K+-ATPase) and interfere with its bioavailability and metabolic processes as well as toxicity of aquatic organisms. The objective of this study is to use larvae of medaka (Oryzias latipes) fish as a model organism to investigate the chemical and biological interaction of Tl+ versus K+ in aquatic organisms. The mortality, behavioral analysis, bioaccumulation, gene expression, neurontransmitter and oxidative stress were used to assess the acute and sublethal neurotoxic effects. Moreover, NKA inhibitor was used to find out the evidence that Tl+ may compete with K+ and interacted with K+ ion channels. Our results showed that Tl+ induced higher mortality to larvae in dosing solutions containing lower K+ concentrations than those with higher K+ concentrations. After 7-day exposure, the trend of Tl bioaccumulation in fish was similar to acute mortality. The average velocity and percent time active of larvae exposed to Tl+ -LK (15-25 μg/L) significantly decreased, while the absolute turn angle of treated larvae significantly increased. Only Tl+ -MK (25 μg/L) significantly altered the parameters of locomotion mentioned above. The mRNA expression of genes for osmotic regulation gene atp1a1 was induced by Tl+ -MK. And the mRNA expression of genes for nerve function and development including tuba1c and mbp were significantly decreased by Tl+ -LK. Genes related to neurotransmitter and dopaminergic pathway including ache, drd2 and comt were suppressed by Tl+ -LK and Tl+ -MK, ddc was only suppressed by Tl+ -MK. Physiological biomarker also showed a similar trend. The osmoregulation biomarker NKA was induced by Tl+ -MK. Dopamine content was decrease by Tl+ -MK. Additionally, the activities of antioxidant enzyme such as CAT, SOD and GST were inhibited by Tl+-LK. The concentration of metals (Tl+, K+, Na+ and Ca2+) in fish did not significantly different between no inhibitor and NKA inhibitor treatments. In summary, the concentration of K+ was an important factor affecting Tl+ toxicity in aquatic systems. Besides, Tl+ accumulated in fish, interfered the osmoregulation, altered the transcription of nervous system genes, decreased dopamine content, reduced the catalase activities, and resulted in decreased locomotor behavior in medaka larvae.