以次細胞分佈及組織病理學評估不同粒徑之水域奈米銅對草魚之影響

Subcellular partitioning and histopathological effects of size-dependent waterborne nanocopper on grass carp (Ctenopharyngodon idella)

蔡育霖(Yu-Ling Tsai)

指導教授 ： 陳韋妤

高雄醫學大學/生命科學院/生物醫學暨環境生物學系碩士班/碩士(2017年)

摘要

奈米銅目前廣泛應用之奈米材料，其大部分之應用為半導體產業及積體電路板之製程。若奈米銅釋放至環境，可能懸浮於水體中、沉積於底泥及累積於生物體內。過去研究中發現可將細胞之區塊分為代謝活化區(Metabolically active pool, MAP)及代謝解毒區(Metabolically detoxified pool, MDP)並以此作為金屬產生毒性及解毒作用之指標。然而，過去研究較少比較不同粒徑之奈米銅對可食用魚所造成之影響。因此，本研究將對草魚(Ctenopharyngodon idella)暴露25、40-60及60-80 nm之奈米銅，觀察銅於鰓、肝臟、小腸及腦之次細胞累積情況，並以代謝活化區及代謝解毒區評估其產生之毒性，最後以組織病理學切片觀察其組織暴露於奈米銅後所出現之病理形態以及受損程度。結果顯示累積之程度以鰓為最高，次高為肝臟及小腸，而腦並無累積之情況產生，且銅濃度有下降之趨勢。次細胞之累積情況中，三種不同粒徑之奈米銅於鰓及小腸之代謝解毒區(MDP)主要累積區塊皆為金屬富集區塊(Metal-rich granules, MRG)，而肝臟及腦於代謝解毒區(MDP)主要累積區塊為熱穩定蛋白(Heat-stable protein, HSP)。鰓之累積情況中，以25 nm奈米銅累積最為明顯，並顯著高於40-60及60-80 nm奈米銅，但細胞中代謝活化區(MAP)及代謝解毒區(MDP)分布之結果指出三種不同粒徑奈米銅於代謝活化區(MAP)皆上升，而60-80 nm高於另外兩粒徑奈米銅，表示60-80 nm對鰓為毒性最高之奈米銅。肝臟中，以60-80 nm奈米銅累積最為明顯，而代謝活化區(MAP)及代謝解毒區(MDP)分布之結果僅有60-80 nm於肝臟出現代謝活化區(MAP)上升之情形，表示60-80 nm之奈米銅亦對肝臟產生毒性。而小腸雖然於60-80 nm有顯著之累積，但代謝活化區(MAP)中之分布並無上升，但代謝解毒區(MDP)出現下降之情況，表示奈米銅分布可能轉移至細胞碎片(Cellular debris, CD)。此結果表示粒徑間存在不同之累積程度及毒性，而此差異可能為奈米顆粒之大小所造成，25及40-60 nm可以粒子形態進入細胞，而60-80 nm被阻擋於細胞外且持續釋放銅離子進入細胞中。而在組織切片之結果，發現奈米銅於所有器官皆出現毒性之反應，而鰓之組織病理切片及次細胞之結果較為吻合。本研究以次細胞之分布探討不同粒徑之奈米銅對草魚之影響，並且發現其粒徑之大小可能所造成之累積及產生毒性機制之不同，對奈米物質之毒性進行之探討提供不同粒徑間對生物所造成之影響可能之不同，期望對生態環境之毒性評估有所貢獻。

關鍵字

奈米銅 ； 因粒徑而異 ； 淡水魚 ； 次細胞 ； 組織病理 ； 生態毒理

並列摘要

Copper oxide (CuO) nanoparticles (NPs) is one of the most frequently used nanomaterials, especially used in integrated circuits and semiconductor. Once CuNPs release into aquatic environment, it may accumulate and induce toxic effect in aquatic organism. Some studies have showed that the metal toxicity and detoxification mechanism can determined by metabolically active pool (MAP) and metabolically detoxified pool (MDP) of subcellular distribution. Most studies have concentrated on health risk that by one particle size of CuNPs. However, there is a lack of size-dependent waterborne CuNPs and subcellular-related mechanism in the edible freshwater fish. Therefore, this study aimed to understand the size-dependent CuNPs toxic effect and its mechanism in gill, liver, intestine and brain of grass carp (Ctenopharyngodon idella) by observing CuNPs accumulation in subcellular distribution and histopathological effects on tissue morphology. This study carried out CuNPs exposure bioassays with grass carp varying with nanoscales of 25, 40-60 and 60-80 nm and concentrations to determine metabolically active and metabolically detoxified capacities, and also to observe the tissue morphology. Results showed that the highest to lowest accumulation organ was gill > intestine > liver >brain. In gill, the accumulation of 25 nm CuNPs was significantly higher than that of 40-60 and 60-80 nm CuNPs. In liver and intestine, the accumulation of 60-80 nm CuNPs was significantly higher than that of 25 and 40-60 nm CuNPs. In brain, the accumulation of 40-60 nm CuNPs was the lowest while 25 and 60-80 nm had no different. Subcellular distribution of copper of 25 and 40-60 nm CuNPs were mainly in matal-rich granules (MRG), 60-80 nm CuNPs was mainly in heat-denature protein (HDP). The metabolically pool distribution of 25 and 40-60 nm CuNPs increased significantly in both pool, but the distribution in metabolically detoxified pool (MDP) was higher than that in metabolically active pool (MAP). The metabolically pool distribution of 60-80 nm CuNPs was increased significantly in metabolically active pool (MAP), but decreased significantly in metabolically detoxified pool (MDP). These results showed that the accumulation and the toxicity mechanism were different size of CuNPs.This study could provide the toxic mechanism of size-dependent CuNPs in grass carp and could applied in ecological risk assessment.

並列關鍵字

Copper nanoparticle ； Size-dependent ； Freshwater fish ； Subcellular partitioning ； Histopathology ； Ecotoxicology

參考文獻

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