隨科技進步，奈米物質使用日益增加，其中奈米銀和奈米銅是其中最被廣泛使用的奈米材料，多應用於治療劑、抗菌劑、化妝品或工業之用。而曾被限用於局部治療之高腎毒性抗生素colistin，隨著革蘭氏陰性細菌之抗藥性出現，以及後線有效抗生素之短缺，全球使用量亦隨之上升。近年來，許多研究開始發現各種藥品、抗生素和個人護理產品存在於各式水域系統，被公認為屬於新興的環境污染物。隨著奈米顆粒廣泛使用以及colistin後線抗生素全球用量的不斷攀升，並以各種方式進入環境及水域系統。此類新興環境汙染物暴露之可能性，及其對生物或環境的毒性影響也是我們亟需思考的問題。然而目前並無適當的毒理研究證實此類新興環境汙染物對水生動物的負面影響，特別是耳毒性方面之研究。
由於魚類側線系統位於皮膚，會直接暴露外來物質，且其側線毛細胞於表面易於觀察，加上側線神經丘中之毛細胞的結構及功能與人類內耳的機械感官性毛細胞極為相似，故其側線系統之毛細胞應可作為測試潛在有毒物質之耳毒性的合理模式。在本研究中，我們使用斑馬魚胚胎之側線毛細胞，以研究早期暴露此類新興環境汙染物對斑馬魚胚胎本身及其側線毛細胞之影響。我們將斑馬魚胚胎自受孕後連續暴露96小時於不同濃度之特定環境污染物（包括抗生素colistin、奈米銀、奈米銅），觀察其存活率、胚胎孵化率、外觀及側線毛細胞的數量及功能影響。
抗生素colistin之暴露明顯降低了胚胎存活率，此降低與其濃度具明顯相關性；暴露於抗生素colistin 96小時後50％致死之濃度（LC50）值為3.0 μM或3.5 mg/L，但並未改變其孵化率和體長。同時，經由觀測FM1-43標記之毛細胞數量下降，及使用掃描式離子選擇電極技術偵測側線毛細胞處之鈣離子流減少，證實抗生素colistin於濃度≥ 0.1 μM時，已影響其側線毛細胞之功能。以此斑馬魚胚胎側線毛細胞模式，亦發現常見的奈米銀、奈米銅造成斑馬魚胚胎之存活率下降，此降低亦與暴露濃度具明顯相關性；暴露於奈米銀、奈米銅96小時之LC50分別為6.1 ppm（56.5 μM）和2.61 ppm（41.1 μM）。當奈米銀濃度≥ 1 ppm（9.3 μM）或奈米銅濃度≥ 0.01 ppm（0.16 μM），FM1-43標記的毛細胞數量會開始減少，並出現微觀結構改變；當奈米銀濃度≥ 0.1 ppm（0.9 μM）或奈米銅濃度≥ 0.01 ppm（0.16 μM），雖毛細胞數量無顯著下降，但掃描式離子選擇電極技術測得之毛細胞鈣離子流已經開始明顯下降。
此研究證明，藉助以掃描式電子顯微鏡觀測FM1-43標記之斑馬魚側線毛細胞數量，合併掃描式離子選擇電極技術測量之毛細胞功能，來作為研究耳毒性物質之斑馬魚模型之可行性。

With nanotechnology advances, nanomaterials are widely applied in tissue engineering, imaging, surface texturing, and preservatives in cosmetics. Among all nanomaterials, the most common nanoparticles are silver (AgNPs) and copper nanoparticles (CuNPs). In addition, colistin, one of the few cationic antimicrobial peptides, was mostly used in local treatment due to its renal toxicity related to systemic use in the past few decades. Due to the emergence of multi-resistant gram-negative bacterial infections, and a shortage of new-generation antimicrobial agents, colistin gains attention as a last-line antibiotic. Its global consumption rate increases greatly in the last decade. With the presence antibiotics, chemotherapeutic agents, personal care products in the aquatic environment system, those agents were recognized as emerging environmental contaminants in recent years. But their toxicological effects on aquatic animals have not been properly investigated, especially the hair cell toxicity of lateral line, which involves a wide range of behaviors on aquatic animals. The impairment of lateral line may furtherly impact their survival.
Due to neuromasts of lateral line system in the fish being located in the skin and directly contacting environmental exposure, and the structure and functions of hair cells in lateral line neuromasts mimicking those of mechanosensory hair cells in the inner ear of humans, lateral line system in zebrafish is suitable for investigating potential hair cell damage of the emerging environmental contaminants. In this study, we examined the survival, hatching, morphological changes and hair cell toxicity of selected contaminants, including colistin, AgNPs or CuNPs, on zebrafish embryos, which were incubated in different concentrations of these selective agents during 0~96 hour post fertilization.
Colistin decreased the survival rate in a dose-dependent manner (LC50 was 3.0 μM or 3.5 mg/L), but it did not change the hatching rate, and body length of embryos. However, colistin impaired lateral-line hair cells in the skin of embryos. Colistin (at ≥ 0.1 μM) decreased the number of FM1-43-labeled hair cells. Ca2+ influxes at hair bundles of hair cells were measured with a scanning ion-selective microelectrode technique (SIET) to evaluate the function of hair cells. The reduced mechanotransduction-mediated Ca2+ influx at hair bundles suggests that sublethal concentrations of colistin can impair lateral line function. Both AgNPs and CuNPs were found to also cause toxic effects in zebrafish embryos in a dose-dependent manner. Values of the 96-h 50% lethal concentration (LC50) of AgNPs and CuNPs were 6.1 ppm (56.5 μM) and 2.61 ppm (41.1 μM), respectively. The number of FM1-43-labeled hair cells and the microstructure of hair bundles were significantly impaired by AgNPs [≥ 1 ppm (9.3 μM)] and CuNPs [≥ 0.01 ppm (0.16 μM)]. AgNPs [≥ 0.1 ppm (0.9 μM)] and CuNPs [≥ 0.01 ppm (0.16 μM)] were both found to significantly reduce Ca2+ influxes.
This study revealed that lateral-line hair cells of zebrafish are susceptible to colistin, AgNPs and CuNPs functionally and morphologically at different specific concentration. By combining SIET to detect Ca2+ influxes through mechanoelectrical transducer channel and FM1-43 staining with scanning electron microscopy, lateral line hair cells in zebrafish could be used as a model animal for investigating ototoxins.

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