臺灣博碩士論文加值系統

摘 要
砷化鎵 (GaAs) 是一種砷和鎵的化合物，在半導體工業上已被廣泛使用。 雖然個別對砷離子或鎵離子的毒性報導有很多，但直接研究砷化鎵化合物的細胞毒性的報導卻非常少。 根據本實驗室最近的研究指出，在砷化鎵四小時的短暫暴露後，會分別對人類纖維母細胞引發微小球核之形成，以及對中國倉鼠卵巢細胞在細胞週期上有影響。 因此，本研究主要是進一步檢測在短時間處理下，GaAs的細胞毒性與遺傳毒性，並對其參與的機制作一探討。
已知細胞毒性中，快速形成的氧化逆境，常是金屬類刺激的第一線反應，經由中介路徑reactive nitrogen species (RNS) 和reactive oxygen species (ROS)之產生，在耗損細胞內抗氧化蛋白glutathione後，細胞之DNA、蛋白質、lipid 會被氧化，造成細胞損壞或凋亡。 為了瞭解砷化鎵的細胞毒性可能參與的機制，中國倉鼠卵巢細胞，在砷化鎵處理一段短時間後，細胞內以DAN螢光監測的NO2.的含量會增加，且與砷化鎵的處理劑量和處理時間呈正相關，表示氧化逆境為砷化鎵的細胞毒性參與的機制之一。 為了區分其中介氧化逆境路徑，藉使用reactive nitrogen species (RNS) 抑制劑，N-nitro-L-arginine methyl ester (NAME) 或S-methyl-L-thiocitrullin (MTC)， 發現會降低砷化鎵處理細胞，所引起的DAN螢光強度，表示RNS為砷化鎵的氧化逆境的必須中介路徑。 在使用reactive oxygen species (ROS) 抑制劑，6-hyroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) 也可有效抑制一氧化氮的產生，表示ROS亦為砷化鎵的氧化逆境的必須中介路徑。 另外，以Dichlorofluorescin (DCF) 螢光監測的結果亦顯示，與砷化鎵處理之正相關性，以及Trolox可有效降低DCF的螢光強度，而再肯定ROS為砷化鎵的氧化逆境的必須中介路徑。 從遺傳毒性方面來看，砷化鎵會增加細胞內微小球核的形成。 共同處理砷化鎵和NAME 或 MTC，會減少砷化鎵處理所引起的微小球核形成，表示RNS也是砷化鎵造成微小球核的形成的必須中介路徑。 在細胞存活率方面，用Sulforhodamine B (SRB) 方法測量，在砷化鎵處理四小時後，零到百分之八十的中國倉鼠卵巢細胞會死亡。 綜合以上所述，砷化鎵會造成細胞毒性和遺傳毒性，而且這個機制和氧化逆境中的ROS及RNS皆有關。因此，在使用砷化鎵時，應注意防護的工作。

Abstract
Gallium arsenide (GaAs), a chemical compound of gallium and arsenic, is widely used material in various semiconductor products. While cytotoxic effects of individual gallium and arsenic were well documented, the direct investigation of cytotoxic effects of mere gallium arsenide compound is largely unexplored. We have previously shown in our laboratory that short-term exposure of human fibroblast cells to gallium arsenide exhibits genotoxic micronuclei (MN) formation while of Chinese hamster oocyte (CHO) cells impacts on regulation of cell cycle. In this study, I used CHO-K1 cells to investigate both the cytotoxic and genotoxic mechanisms of the short-term effect of gallium arsenide treatment and to delineate the possible inductive pathway(s) involved.
Oxidative stress is the prompt cellular response for many metal stimuli. The oxidative process contributes to glutathione depletion, DNA and/or protein damage, and lipid peroxidation through production of intermediate reactive nitrogen species (RNS) and/or reactive oxygen species (ROS) and finally leads to cell damage or apoptosis. To investigate the involvement of oxidative stress in gallium arsenide treatment, the DAN assay was used to detect a stable product of nitric oxide (NO) in GaAs-treated CHO-K1 cells. The induction of nitrite level was concentration- and time-dependent suggesting the induction is GaAs-specific. The DAN assay was then challenged against pathway-specific inhibitors. Treatment with NO synthase (NOS) inhibitor, either N-nitro-L-arginine methyl ester (NAME) or S-methyl-L-thiocitrullin (MTC), inhibits the GaAs-induced nitric oxide (NO) production, suggesting the involvement and the necessity of RNS intermediates in GaAs induction. Likewise, 6-hyroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) scavenging hydroxyl radical —OH. and perooxynitrite ONOO- also suppressed the increase of nitrite level, suggesting the involvement and the necessity of ROS intermediates in GaAs induction. Consistent results were also obtained independently by the DCF assay with those of the DAN assay in a concomitantly increase of DCF fluorescence intensity and in the pathway-specific inhibitor experiments using Trolox and either NAME or MTC. In the genotoxic aspect, GaAs induced micronuclei (MN) formation and it is also mediated by ROS and RNS. As cell viability was tested, 0-80 % of cells was killed as examined by Sulforhodamine B (SRB) assay. These results indicated that GaAs possesses cytotoxic and genotoxic effects and both RNS and ROS are involved and required for GaAs stimulation in micronuclei (MN) formation.
Page
Chinese Abstract……………..………………………………………01
English Abstract……………………………………………………...02
Introduction…………………………………………………………..03
Material and Methods..……………………………………………….07
Results………………………………………………………………..10
Discussion……………………………………………………………14
References...………………………………………………………….17
Figures………………………………………………………………..21

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