Abstract
砷化物是一已知的人類致癌物，本研究使用人類纖維母細胞（HF）當材料，探討一種無機砷化物、亞砷酸鈉（SA），處理HF細胞後，所引起的細胞和遺傳毒性之機轉。當80 μM SA處理HF 4 小時後，處理組有鈣離子累積的情形。 在培養液中加入45Ca，再以不同劑量 SA 處理 HF 後，結果為：細胞內鈣離子累積量，與 SA 劑量有正相關。進一步地，用鈣離子modifiers ，鈣離子通道的阻斷劑 -verapamil和鈣離子ionophore-A23187，除可增加鈣離子胞內累積量，且使SA所誘導的微小核球數（MN）和細胞毒性亦增加. 反之，以細胞內鈣離子移除劑-quin-2預先處理HF，或細胞外鈣離子移除劑- EGTA處理，均明顯地抑制SA所引起的MN的產生，然而，在減少細胞毒性方面，作用不明顯。鈣離子為蛋白質激活酵素-C（PKC）的一個輔助因子，SA處理細胞引起之鈣離子不平衡，是否亦影響PKC？ 本研究應用西方點墨法及抗 PKC 抗體，檢查 SA 處理後的細胞PKC 含量，結果顯示：PKC 含量隨 SA 劑量的增加而增加。以 PKC 抑制劑-H7 則可以使 SA 所誘導的微小核球及細胞毒性數目減少，反之PKC的活化劑-TPA則增加SA所引起的 MN 數目。而一般所認為與砷化物解毒有關的胞內分子或酵素（GSH，GST and catalase)，在本研究中，不論是由SA處理細胞（4小時）後立刻測量,或是再以新鮮培養液培養0-48 小時後再測量，都沒有明顯的變化。以上結果結論：鈣離子增加以及與鈣離子相關的PKC增加，均是SA處理細胞後引發的細胞和遺傳毒性原因之一。Arsenic is the first metal to be identified as a humancarcinogen. In this study, arsenite (inorganic arsenic) wasused to study the mechanisms of arsenic-induced cytotoxicityand genotoxicity (micronuclei assay) in human fibroblasts.Surprisingly, calcium accumulation was observed in cells afterarsenite treatment. In addition, cells treated with variousconcentrations of arsenite for 4 h in the presence of 1 μCi/ml45Ca showed that the calcium influx was dose- dependent. Inorder to examine the effects of calcium modifiers on arsenitetoxicity, it showed calcium channel blocker, verapamil, andcalcium ionophore, A23187, could increase micronuclei inductionand cytotoxic effects of arsenite. Moreover, pretreated cellswith intracellular calcium chelator, quin-2, or cotreated cellswith extracellular calcium chelator, EGTA, significantlydecreased micronuclei induction of arsenite. However, quin-2and EGTA could not protect cells against arsenite-inducedcytotoxicity. Since arsenite treatment could increase cellularcalcium accumulation, one would expect that intracellularcalcium-dependent protein kinase (PKC) was involved in arsenite-induced induced geno- and cytotoxicity in HF. Western blot wasapplied to detect PKC expression and found that dose-dependentincrease of PKC protein accompanied arsenite treatment. Inaddition, H7 (PKC inhibitor) could inhibit and TPA (PKCactivator) could slightly increase arsenite-induced micronucleiformation and cytotoxicity. These results indicated thatcalcium ion and calcium-dependent PKC were involved in arsenite-induced cytotoxicity and genotoxicity in human fibroblasts.