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Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/48806

Title: 二價汞藉由干擾 DNA 傷害移除步驟進而抑制斑馬魚胚胎中核酸切割修補作用
Mercury (II) impairs nucleotide excision repair (NER) in zebrafish (Danio rerio) embryos by targeting primarily at the stage of DNA incision
Authors: Chang, Yung
張永
Contributors: 國立臺灣海洋大學:生命科學暨生物科技學系
Keywords: 胚胎;;核酸切割修復;紫外線;斑馬魚
embryo;mercury;nucleotide excision repair;UV;zebrafish
Date: 2017
Issue Date: 2018-08-22T06:31:29Z
Abstract: 二價汞離子(Hg2 +)是污染水生環境中最常見的無機汞形式。已有文獻指出在水生生物與哺乳類生物中二價汞離子會透過抑制 DNA 修復進而導致遺傳毒性,本研究探討二價汞離子對斑馬魚(Danio rerio)胚胎中核酸剪切修復各步驟之效應。透過體外轉錄為基礎的 DNA 修復實驗,將紫外線照射誘導損傷的 DNA 與斑馬魚胚胎萃取液混和進行修復,以轉錄出的RNA為判斷依據,發現0.1µM 至 2.5µM氯化汞處理斑馬魚胚胎9小時後NER修復活性呈現濃度關聯性的抑制。汞暴露對斑馬魚胚胎 NER 辨識與切割蛋白基因表現僅有微弱的抑制或刺激作用。透過凝膠電泳阻滯分析結果得知斑馬魚胚胎暴露在汞 1µM 與 2.5µM 後對6-PP光產物之辨識活性並無抑制作用,顯示NER的辨識步驟未受汞影響。由於真核生物 NER 切除的小片段 DNA 為24-32個寡核甘酸,平均長度為26個寡核甘酸的切割產物大量出現在直接照射紫外線1 kJ/m2的斑馬魚胚胎中,顯示紫外線直接照射斑馬魚胚胎會誘導NER的產生。藉由體內切割分析實驗結果顯示,先暴露在汞 0.1µM 至 2.5µM 後的斑馬魚胚胎再照射紫外線, NER 切割步驟幾乎喪失功能,因此得知汞會明顯抑制 NER 的切割步驟。本研究結果發現環境中的二價汞會透過干擾 NER 切割活性進而造成 NER 系統喪失修復活性。
Mercuric ion (Hg2+) is the most prevalent form of inorganic Hg found in polluted aquatic environment. As inhibition of DNA damage repair has been proposed as one of the mechanisms of Hg2+-induced genotoxicity in aquatic animals and mammalian cells, this study explored the susceptibility of different stages of nucleotide excision repair (NER) in zebrafish (Danio rerio) embryos to Hg2+ using UV-damaged DNA as the repair substrate. Exposure of embryos at 1 h post fertilization (hpf) to HgCl2 at 0.1 to 2.5 μM for 9 h caused a concentration-dependent inhibition of NER capacity monitored by a transcription-based DNA repair assay. The extracts of embryos exposed to 2.5 μM Hg2+ failed to up-regulate UV-suppressed marker cDNA transcription. Hg2+ exposure imposed either weak inhibitory or stimulating effects on the gene expression of NER factors.Band shift assay showed no inhibition of photolesion binding activities in embryos treated with 1 to 2.5 μM Hg2+. The damage incision stage of NER in zebrafish embryos was found to be more sensitive to Hg2+ than photolesion binding capacity due to the complete loss of damage incision activity in the extracts of embryos exposed to 1 to 2.5 μM Hg2+. NER-related DNA incision was induced in UV-irradiated embryos based on the production of short DNA fragments matching the sizes of excision products generated by eukaryotic NER. Pre-exposure of embryos to Hg2+ at 0.1 to 2.5 μM all suppressed DNA incision in UV-irradiated embryos, reflecting a high sensitivity of DNA damage incision to Hg2+. Our results showed the potential of Hg2+ at environmental relevant levels to disturb NER in zebrafish embryos by targeting primarily at the stage of DNA incision.
URI: http://ethesys.lib.ntou.edu.tw/cgi-bin/gs32/gsweb.cgi?o=dstdcdr&s=G001043B006.id
http://ntour.ntou.edu.tw:8080/ir/handle/987654321/48806
Appears in Collections:[生命科學暨生物科技學系] 博碩士論文

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