- 學位論文
檳榔素及其氧化物對肝細胞之細胞毒性、遺傳毒性及致突變性之探討
Cytotoxic, Genotoxic and Mutagenic Effects of Arecoline and Its Oxides on Liver Cells
摘要
背景：在亞洲，檳榔經常被人們拿來食用，主要是為了放鬆緊張 壓力、提神、分泌唾液與體溫升高等效果。檳榔中含有許多的生 物鹼，研究證實在嚼食檳榔者的唾液中以檳榔素 (arecoline, AR ) 的含量最多。從國內醫療院所數年來的研究顯示，唾液中含大量 檳榔素會使口腔黏膜細胞發生變異，進而導致口腔癌的發生。檳 榔素不只導致口腔癌，有研究指出它也具有遺傳及細胞毒性，且 與肝硬化的形成有關。而肝臟是人體重要代謝器官，檳榔素進入 人體經氧化作用後，產物為檳榔素氧化物 (arecoline oxides, AROs)。 目的：本研究想了解當檳榔素轉變成檳榔素氧化物後，對正常大 鼠肝臟細胞 (clone 9 cell) 的細胞毒性、遺傳毒性、致突變效果及 細胞內氧化壓力的變化與造成肝臟損傷的關連性，並以斑馬魚來 確定在細胞模式下之發現。方法：檳榔素及檳榔素氧化物對細胞 毒殺能力、DNA 損傷、致突變及細胞內氧化壓力等之影響，分別 以結晶紫染色法、慧星分析法、體外細菌回復突變分析法 (Ames test) 及 DCHF-DA 染色法進行測量。結果：正常大鼠肝細胞經過A 檳榔素氧化物及檳榔素處理後，檳榔素氧化物的細胞毒性IC50 為 100 μM，而檳榔素的細胞毒性IC50 為500 μM，其間差異達5 倍， 可知在細胞毒性方面檳榔素氧化物的毒性遠大於檳榔素的毒性。 彗星分析測量結果顯示，檳榔素氧化物對DNA 造成的股斷裂傷害 也明顯大於檳榔素造成之DNA 股斷裂傷害。另外以250-500 μM 檳榔素氧化物處理也造成DNA crosslinks (DCs) 形成的情況，細胞 處理檳榔素則不會有明顯DCs 的形成。在致突變效果方面，以體 外細菌回復突變分析法分析結果發現隨著加入檳榔素氧化物的劑 量越高，使沙門氏突變菌TA100 產生回復突變的菌落數也越多， 而處理檳榔素則無明顯致突變效果。在細胞內氧化壓力方面，檳 榔素氧化物可明顯誘導細胞內活性氧物種(reactive oxygen species, ROS)含量增加，檳榔素的效果較不明顯。以0-500 μM 檳榔素或檳 榔素氧化物處理斑馬魚胚胎及以31.25-62.5 μM 檳榔素氧化物處 理幼蟲，發現檳榔素氧化物對胚胎具有明顯毒殺性，對幼蟲可明 顯誘發體內活性氧物種的生成；而處理檳榔素後，胚胎的存活率 無明顯改變，且輕微誘發幼蟲體內活性氧物種生成。結論：綜合 上述結果，檳榔素氧化物對正常大鼠肝細胞的毒殺性、遺傳毒性、 致突變性及誘導細胞內氧化壓力提高的能力皆強過於檳榔素。檳 榔素氧化物有可能是一種對肝臟造成病變，導致肝癌發生的重要 致癌因子。
並列摘要
The International Association of Research on Cancer has classified the areca nut as a human environmental carcinogen. Previous studies have shown that arecoline (AR) is the major areca nut alkaloid present in the saliva of areca nut chewers. Domestic medical institutions over the years studies have shown that saliva contains a large number of arecoline make the oral mucosa cells mutate, leading to oral cancer. But, arecoline also induces liver toxicity, and depresses the antioxidant system. There is accumulating evidence that implicates areca nut in the development of other astrointestinal malignancies, including liver carcinoma. However, the active compound accounting for arecoline-induced damage in normal liver cells is still uncharacterized. Therefore, the purpose of this study was to determine cytotoxic, genotoxic and mutagenic effects of arecoline and its oxidative metabolites, arecoline oxides (AROs) in normal rat liver cells. The cytotoxic effects were detected by crystal violet staining. The genotoxic effects were detected by the alkaline comet assay. The mutagenic effects were detected by the Salmonella typhimurium mutagenicity test. The results showed that the half maximal inhibitory concentration of AROs was 100μM, but of AR was greater than 500 μM for clone 9 cells. The difference was 5 folds. In genotoxic effects, AROs could lead to DNA strand breaks significantly and dose-depended. When we treated cells with H2O2 after AROs, DNA crosslink (DCs) situation was significant and dose-depended. But when treating cells with H2O2 after AR, DPC was not significant. In mutagenic effects, AROs cause mutagenicity in Salmonella typhimurium tester strains TA 100 and TA 98 than AR. The possible role of arecoline oxides in the induction of liver carcinoma by areca nut chewing can be discussed.
參考文獻
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