超氧化物岐化酶及過氧化氫酶過量表現對於氧化劑引起之細胞毒性及蛋白質羰基化的影響之探討

Effect of overexpression superoxide dismutase or catalase on oxidants induced cytoxicity and protein carbonylation

張永聰(Yung-Tsong Chang)

指導教授 ： 王祖興

中山醫學大學/醫學科技學院/生物醫學科學學系碩士班/碩士(2007年)

https://doi.org/10.6834/CSMU.2007.00097

摘要

砷化物和香煙皆為已知的致癌物，研究指出它們透過產生活性氧的方式傷害DNA，但是砷化物和香煙在哺乳動物細胞中造成蛋白質羰基化的部份仍然少被討論。蛋白質羰基化是一種氧化傷害，會使蛋白質失去正常的結構和功能，所以偵測蛋白質羰基化的程度可以視為蛋白質受到氧化傷害的指標。實驗中我們使用中國倉鼠卵巢細胞(Chinese hamster ovary cells，CHO-K1)探討亞砷酸鈉(sodium arsenite)及香煙萃取液(Cigarette smoke extract，CSE)造成的蛋白質氧化傷害與超氧化物歧化酶(superoxide dismutase)過度表現(CHO- SOD)及過氧化氫酶(catalase)過度表現(CHO- CAT)之間的關係。我們用點轉漬技術(Dot blot)，加入2,4-dinitrophenyl hydrazine 和被氧化的蛋白質鍵結，將蛋白質羰基化的程度表示出來。用細胞毒性測試抗氧化酵素不同表現細胞對氧化劑的耐受性。處理20% CSE 1小時後，CHO-CAT在羰基化程度上較CHO-K1低26.3%；處理過氧化氫5 mM 1小時後，CHO-CAT在羰基化程度上較CHO-K1低26%；以半數致死劑量處理48小時，在62.5 μM 亞砷酸鈉處理下，CHO-CAT蛋白質羰基化的程度比CHO-K1下降 35.2%，在 5% CSE處理下，CHO-CAT蛋白質羰基化的程度比CHO-K1下降 49.2%。此外CHO-CAT暴露在過氧化氫下的存活率也比CHO-K1高，但是暴露在CSE下則沒有明顯差異。這些結果顯示 catalase 與亞砷酸鈉及CSE誘發的活性氧有密切關係。

關鍵字

蛋白質羰基化 ； 亞砷酸鈉 ； 香煙萃取液 ； 過氧化氫酶超氧化物歧化酶

並列摘要

The arsenics and cigarette smoke are well known as carcinogens. They significantly damage DNA through a reactive oxygen species-dependent pathway. However, there is still little known about arsenics and cigarette smoke induced protein carbonylation in mammalian cells. Carbonylation of proteins is an irreversible oxidative damage that often leads to a loss of protein function, it is considered as a widespread indicator of severe oxidative damage and disease-derived protein dysfunction. Oxidative stress carbonylates some specific protein targets and is related to arsenics and cigarette smoke induced apoptosis. In this study we investigated the effect of catalase (CAT) and Cu/Zn-superoxide dismutase (SOD) overexpression on protein carbonylation induced by sodium arsenite and cigarette extract on Chinese hamster ovary cells (CHO-K1). Oxidative damage was assessed using dot blot analysis of DNPH-derivatized carbonyl groups on oxidized proteins. The protein carbonyl levels and cytotoxicity were increased by sodium arsenite and cigarette smoke extract (CSE) treatment in a dose-dependent manner. Catalase overexpression cells (CHO-CAT) significantly decreased protein carbonylation by 35.2% for sodium arsenite (62.5 μM 48h), 26.3% and 49.2% for cigarette smoke extract (20% 1h and 5% 48h), and 26% for H2O2 (5 mM 1h). In addition, CHO-CAT cells showed higher survival percentage when exposed to hydrogen peroxide, but not showes resistant to CSE exposure. These findings suggest that CAT may involve on sodium arsenite and CSE -induced reactive oxygen species.

並列關鍵字

Protein carbonylation ； sodium arsenite ； cigarette smoke extract ； catalase ； superodie dismutase

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