檳榔子萃取物對中國倉鼠卵巢細胞之細胞毒性與基因毒性的探討

Abstract

嚼食檳榔是受歡迎的口腔嗜好之一，估計全球約有二到六億的嚼食人口。流行病學的研究發現嚼食檳榔與口腔癌間具有強烈的關連性。在台灣，檳榔嚼塊通常是由新鮮的檳榔子，紅灰或白灰，再加上荖花或荖葉所組成。許多的研究都指出檳榔子具有致癌性與基因毒性；然而，截至目前為止，對於檳榔子的致癌機轉仍是不完全清楚。從本篇論文中發現隨著濃度與時間的遞增，檳榔子萃取物會抑制CHO-K1 細胞的生長與分裂。經過二十四小時的處理後，濃度高於400μg/ml 的檳榔子萃取物會增加細胞內過氧化氫的含量。從細胞質分裂阻斷之微核分析法中發現檳榔子萃取物會誘導微核的生成且伴隨著細胞內過氧化氫濃度的提升。當以400~800μg/ml 的濃度作用二十四小時，發現會造成細胞週期停滯於G2/M phase；當作用時間拉長，發現檳榔子萃取物會造成細胞死亡。有趣的是，我們還觀察到檳榔子萃取物作用二十四小時後的細胞會出現細胞質分裂缺損，且雙核細胞的比例有明顯增多的現象。相似的濃度下，發現檳榔子萃取物會擾亂 actin filament 的分佈。而抗氧化劑Catalase (2000U/ml)與檳榔子萃取物(800μg/ml)共同作用下的細胞，雙核細胞的生成會受到明顯的抑制，這也意味著檳榔子萃取物所造成的細胞質分裂缺損與活性氧的生成有關。另外，長時間的作用下多倍體，多核或微核細胞之數量有明顯的增多，同時也發現數量異常之中心體。除此之外，我們還發現2mM 之咖啡因會加成檳榔子萃取物之細胞毒性，且可有效的減少多倍體細胞的數量。故從本實驗的結果，我們認為檳榔子萃取物會增加微核頻率，誘導細胞週期停滯於G2/M phase，造成細胞質分裂缺損與多倍體細胞的增多；而這些現象可能與活性氧的生成，actin filament 的異常與中心體的變異有關。

Betel quid (BQ) chewing is the fourth most popular oral habit. There are about 200 - 600 million BQ chewers in the world. Epidemiological evidences have found the strong association between BQ chewing and oral cancer. In Taiwan, BQ consists of fresh areca nut (AN) and lime with or without inflorescence piper betle or betle leaf. Many studies have demonstrated that AN exerts mutagenicity and genotoxicity. However, the precise toxic mechanisms responsible for AN-induced carcinogenesis are not fully clear. In this thesis, we found that AN extract (ANE) inhibited the growth and proliferation of CHO-K1 cells in a concentration- and time-dependent manner. After 24-h of exposure, ANE increased the intracellular H2O2 production at concentrations higher than 400 µg/ml. By using cytokinesis-block micronucleus assay, ANE induced micronuclei formation in CHO-K1 cells, accompanied by an increase in intracellular H2O2. Incubation of CHO-K1 cells with ANE (400~800 µg/ml) for 24-h caused G2/M cell cycle arrest and prolonged exposure to 800 µg/ml of ANE induced cell death. Interestingly, we observed that ANE itself caused cytokinesis failure and increased the frequency of binucleated cells following 24-h exposure. At similar concentrations, ANE induced actin filament disorganization. Co-incubation of cells to catalase (2000 U/ml) and ANE (800 µg/ml) reduced the binucleated cells generation, indicating that ANE-induced cytokinesis failure was associated with ROS production. After prolonged exposure to ANE, the accumulation of hyperploid cells, micronucleated or multinucleated cells were noted concomitantly with centrosome amplification. In addition, we found that co-exposure of cells to caffeine (2 mM) and ANE (800 µg/ml) resulted in additive or synergistic cytotoxicity, accompanied by a decrease in hyperploid cells. In summary, our results suggest that ANE increases micronuclei frequency, induces cell cycle arrest at G2/M phase, causes cytokinesis failure and accumulates hyperploid cells. The events are possibly associated with ROS production, actin filament disorganization and centrosome aberration.