Abstract
砷化合物是第一個被定義為人類的致癌物質，然而其致癌機轉仍尚未清楚。在最近的研究發現，四小時亞砷酸鈉處理會造成中國倉卵巢細胞內鈣離子堆積及微小核球形成。另外EGTA (細胞外鈣離子螯合劑)或高濃度的蛋白質激活酵素C (PKC)的抑制劑H7 (500μM) 與亞砷酸鈉共同處理四小時，可以減少由亞砷酸鈉所誘導的微小核球形成。微小核球的形成是一種基因毒性的指標，它來自無中節或完整的染色體在有絲分裂時無法正常包入子核的遺傳物質。在本研究裡，以人類纖維母細胞作為實驗細胞株得到以下結果。隨著TPA (PKC的活化劑) 與亞砷酸鈉共同處理的濃度增加(0~10 ng/ml)，其亞砷酸鈉所誘導的微小核球形成隨之增加，並且TPA單獨處理四小時亦可促使微小核球形成隨TPA濃度增加而增加。而以低濃度H7 (50μM) 與亞砷酸鈉共同處理，即可抑制亞砷酸鈉所誘導的微小核球行形，且隨H7與亞砷酸鈉共同處理的濃度增加 (0~400μM)，其抑制效果愈顯著。以西方轉漬法 (Western blotting) 分析亞砷酸鈉處理的細胞蛋白質中的不同種類的PKC發現; 只有跟鈣有關的cPKC會受到亞砷酸鈉誘導而表現量增加，而此種cPKC在核內的含量，亦隨亞砷酸鈉處理的濃度增加而增加。另外以激活酵素分析 ( kinase assay) 法發現;使細胞核內的PKC活化。但低濃度的H7 (50μM) 無法有效抑制亞砷酸鈉所誘導的PKC活化。 另外利用免疫沉澱法 (immunoprecipitation) 及一種跟鈣/鎂有關的內切核甘酸脢的活性分析 (Ca2+/Mg2+-dependent endonucleaseactivity assay) 法發現，亞砷酸鈉可活化鈣/鎂有關的內切核甘酸脢，且H7(50μM )可抑制此亞砷酸鈉所誘導內切核甘酸脢的活化; 而且cPKC與此種內切核甘酸脢是有相關的。綜合以上的結果，亞砷酸鈉所誘導cPKC在細胞中的表現增加、cPKC出現在細胞核的量及活性增加、鈣/鎂有關的內切核甘酸脢的活化均與亞砷酸鈉所誘導的微小核球行成有相當程度的關係。Arsenic is the first identified human carcinogen. However,the carcinogenic mechanism of arsenic is not yet defined.Recently, it was showed that 4-h arsenite treatment induced Ca2+accumulation, micronuclei (MN) formation in CHO-K1 cells. AndEGTA (Ca2+ chelator) and H7 (500mM, PKC inhibitor) could reducearsenite-induced MN formation in CHO-K1 cells. MN formation isan index of genotoxicity. In this study, the data showed that4-h arsenite treatment cotreated with TPA ( 0~10 ng/ml, proteinkinase C, PKC activator) could potentate arsenite-inducedmicronuclei formation in human fibroblasts (HF) and TPAtreatment alone also induced MN formation in a dose-dependentmanner. Arsenite treatment combined with H7 (50mM) coulddecrease arsenite-induced MN formation. Applied Westernblotting in whole cell extracts from arsenite-treated humanfibroblasts, the result showed that a dose-dependent increase ofclassical PKC (cPKC) expression, but the expression of the otherthree PKC isoforms, e (nPKC), h (nPKC) and z, (aPKC) could notbe affected in arsenite-treated HF. According to previousresults indicated that Ca2+ ion is involved in the arsenite-induced MN formation in HF. Assayed with Western blotting andkinase assay, the expression and activity levels of cPKC wereincreased in nuclear fractions after 4-h arsenite treatment.Examined with endonuclease activity assay afterimmunoprecipitation, preliminary result indicated that thenuclear immunoprecipitated complex probed with anti-cPKCantibody had Ca2+/Mg2+-dependent endonuclease activity in 4-harsenite-treated HF. This result indicated that cPKC mayassociate with this Ca2+/Mg2+-dependent endonuclease in HF. Onthe basis of above findings, it was suggested that cPKCexpression in whole cell extract, the increased content andactivity of cPKC in nuclear fraction, and Ca2+/Mg2+-dependentendonuclease activation may involve in arsenite-inducedmicronuclei formation.