探討白藜蘆醇對過氧化氫傷害後酵母菌基因體穩定性的影響

The effect of resveratrol on genome stability in hydrogen peroxide damaged yeast cells

王如邦(Reu-Ben Wang)

指導教授 ： 羅翊禎

國立臺灣大學/生物資源暨農學院/食品科技研究所/碩士(2010年)

https://doi.org/10.6342/NTU.2010.00871

摘要

白藜蘆醇為植物用來抵禦外來侵害的抗菌物質，主要存在於葡萄皮、花生芽及藍莓。白藜蘆醇因有抗氧化、調節細胞週期、抗老化及對高熱量攝食老鼠之健康有正面功效而受各界矚目。為此，本實驗以酵母菌為模式，評估白藜蘆醇對經過氧化氫傷害後細胞基因體穩定性之影響及可能機制。實驗顯示，白藜蘆醇濃度於0.5~200 μM皆不影響酵母菌株BY4741 WT在八小時內生長曲線。同時於菌株BY4741 WT及RDKY3615 WT存活率測試中，白藜蘆醇濃度分別在5~100 μM及10~50 μM下，也不影響細胞存活率。另外，過氧化氫傷害效力測試中，以3 mM過氧化氫處理BY4741 WT 30及60分鐘後，存活率由70 %降為35 %；相同條件下，也可增加RDKY3615 WT之DNA受損修復訊息發送者磷酸化組蛋白2A (γ H2A)表現量。又不同濃度之白藜蘆醇 (10 μM及50 μM)，與相同過氧化氫 (3 mM，60分鐘)傷害條件處理細胞時，菌株存活率因白藜蘆醇濃度增高而有明顯下降；相同條件也增加RDKY3615 WT之DNA受損修復之訊息發送者γ H2A表現，顯示白藜蘆醇與過氧化氫對酵母菌磷酸化組蛋白2A表現量之調控具有加乘效應；此外，以白藜蘆醇 (50 μM)處理受損後細胞，會使細胞週期停滯在S期。為探討白藜蘆醇抗氧化之自由基捕捉能力，與過氧化氫所造成之氧化型DNA破壞基因體不穩定性關係，本實驗也測試白藜蘆醇對過氧化氫傷害後，酵母菌細胞內活性氧物質之含量。結果顯示，單獨使用白藜蘆醇時可降低RDKY3615 WT內生性過氧化物質；然而，細胞經過氧化氫 (3 mM，60分鐘)傷害後，並立即測定活性氧物質含量，無論過氧化氫傷害前有無添加白藜蘆醇 (10 μM或50 μM)，皆無法降低過氧化氫所造成活性氧物質含量增加的結果；但若提供酵母菌傷害後之修復時間 (1、2及3小時)並伴隨著修復時間中加入的50 μM的白藜蘆醇或不加入白藜蘆醇時，細胞中的活性氧含量皆會於修復一小時下降至最低。進一步以GCR ( gross chromosomal rearrangement )測試RDKY3615 WT之基因體穩定性發現，受損細胞持續培養在50 μM白藜蘆醇，可明顯降低過氧化氫所造成的基因不穩定的現象。綜合上述，白藜蘆醇可維持酵母菌於過氧化氫傷害後之基因穩定性，其機制可能是透過降低細胞內ROS含量，同時提高細胞受損後細胞週期S期的比例以延長修復時間，並藉由調節磷酸化組蛋白2A的表現量來達到總體促進細胞基因修復及維持基因穩定之效應。

關鍵字

白藜蘆醇 ； 酵母菌 ； 過氧化氫 ； 磷酸化組蛋白2A ； 細胞週期 ； 活性氧物質 ； 去氧核醣核酸修復

並列摘要

Resveratorl (trans-3,4,5-trihydorxystilbene), a phytoalexin found mainly in grape peels, peanuts, and berries, has numerous well known features, such as antioxidation, cell cycle regulation, and antiageing properties. Resveratrol has opposing effects on the health of high calorie diet mice. Recently, scientist have drawn attention to the biological functions of resveratrol. In this study, we investigate the effect and the mecahisms of resveratrol on the maintenance of genome stability in hydrogen peroxide damaged yeasts. Our results show that resveratrol (0~200 μM) does not affect the cell growth in yeast during eight hours of incubation. At the same time, the cell survival rate does not affected in the presence of resveratrol (10~50 μM). However, the survival rates were 70% and 35% when treated with 3 mM of hydrogen peroxide for 30 min and 60 min respectively. When cells are both treated with resveratrol and hydrogen peroxide, can lead to yeast H2A phosphorylation. The dose dependent phosphorylation levels of H2A with resveratrol (0~50 μM) also shows synergistic effect when combined with hydrogen peroxide treatment (3 mM, 60 min).Unexpectly, pre-incubation of resveratrol (50 μM) overnight before hydrogen peroxide damage (3 mM, 60 min) resulted in the decrease of cell viability. Resveratrol (50 μM) also enhances S phase arrest in cells treated with hydrogen peroxide. In order to elucidate the effects of resveratrol on the free radical scavenging properties and the maintenance of genome stability, we measure the intracellular ROS (reactive oxygen species) content and GCR (gross chromosomal rearragement) mutation rates of cells. Results show resveratrol (10 μM and 50 μM) treatment alone can reduce intracellular ROS contents. Nevertheless, the ROS contents remained unaffected when cells are both treated with resveratrol (50 μM) and hydrogen peroxide (3 mM, 60 min). Interestinly, cells that have been treated by long term incubation of resveratrol (50 μM) followed by hydrogen peroxide damage, exerts the lowest ROS levels at 1 hour during recovery. In addition, cells recover from resveratrol containing medium after hydrogen peroxide damage showed the lowest mutation rates. In conclusion, our results suggested that resveratrol can decrease intracellular ROS levels and possibly facilitate DNA repair through S phase arrest and increase H2A phosphoryation, which leads to the suppression on gene mutation as well as promoting genome stability in hydrogen peroxide damged yeast.

並列關鍵字

resveratrol ； yeast ； hydrogen peroxide ； histone 2A phosphorylation ； cell cycle ； reactive oxygen species ； DNA repair

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