Abstract
三氯化銻(SbCl3)和砷化鎵(GaAs)被應用於半導體工業已有相當年日。兩者在生物體及細胞方面的毒害情形有一些相關的報告，然而其所引發的遺傳毒性報告則相當少，目前僅有兩則數據：其一為1991年Kuroda等人發現: 以SbCl3處理V79細胞28小時，會提高姐妹染色体互換的頻率; 另一則為1997年Gebel等人也發現: 以SbCl3處理人類白血球細胞24小時，也有姐妹染色体互換頻率增加的結果。本研究將以HF和CHO-K1細胞處理上述金屬化合物，作遺傳及細胞毒性檢查，並探討其機制，所得有關於SbCl3與GaAs的結果分述如下。 0-400μM SbCl3處理哺乳類細胞CHO-K1及HF 4小時都可以引發劑量增加則微小核球形成及細胞毒性也增加的結果。Apoptosis的現象DNA fragmentation則發現於處理SbCl3的CHO-K1細胞，再經無藥環境培養至少16小時。所以，此為延遲式的程序性死亡現象。另外，CHO-K1和HF細胞，經0-400μM SbCl3 4小時處理後，或再經24小時以新鮮培養液作後續培養，以arsenazoIII作鈣離子染色，均可見細胞內所累積的鈣離子量，隨著處理劑量的增加而染色濃度增加。鈣離子通道阻斷劑verapamil 100μM和SbCl3共同處理，則可更進一步增加SbCl3所造成的HF和K1細胞內鈣離子染色。而SbCl3所造成的存活率下降情形，亦因verapamil的共同處理，而使細胞存活率更為下降(單獨處理verapamil，所造成的鈣離子堆積，並不會影響細胞存活率或造成遺傳毒性)；而SbCl3所造成的微小核球形成數目則會因verapamil的共同處理，而高於單獨處理SbCl3之實驗組。另一方面，SbCl3所引發之細胞微小核球形成數目，則因胞外鈣離子螯合劑(chelator)EGTA的共同處理，而明顯的低於單獨處理SbCl3之實驗組。因此，推測鈣離子在SbCl3導致細胞與遺傳毒性的過程中，可能扮演一個重要的調控角色。有關GaAs引發細胞與遺傳毒性方面，結果如下:處理大於40μM的GaAs 4小時，繼續用新鮮培養液培養24小時，在CHO-K1細胞中可觀察到DNA ladder現象。於HF細胞中，經由0-80μM GaAs處理4小時，會引發dose-dependent的微小核球形成數目(每500個雙核細胞有25-275個微小核球)；以Sulforhodamine B-staining(SRB)作存活率分析結果為：細胞存活率由100%降至22%。以上結果說明了哺乳類細胞短時間暴露在含有半導體金屬化合物SbCl3或GaAs的環境時，會發生遺傳和細胞毒性作用；並且這些金屬化合物皆可誘導CHO-K1細胞發生apoptosis。所以接觸這類金屬化合物的工作人員或許應做好完善的防護措施。Antimony trichloride (SbCl3) and Gallium arsenide (GaAs) havebeen used in semiconductor industrials for many years. Althoughthe relative adverse biological effects were reported fromanimals or cells after these metal exposure, not many availabledata on their genotoxic effects in treated cells were reportedpreviously. Recent studies indicated SbCl3 induced sisterchromatid exchange in V79 cells and human peripheral lymphocytesafter 24-28 h long time exposure. However, no available datawas found on the genotoxic and cytotoxic effects after a shortterm exposure of SbCl3 or GaAs in mammalian cells. In thisstudy, human fibroblasts (HF) and Chinese hamster ovary (CHO-K1)cells were used to examine the genotoxic and cytotoxic effectsafter 4 h treatment with SbCl3 or GaAs and to reveal thepossible mechanisms to cause cell damage. SbCl3-relatedresults are as follows. It was shown that apoptosis and DNAladder appeared in CHO-K1 cells immediately following 150 uMSbCl3 treatment for 4 h and post incubation for 16 or more h infresh medium. However, DNA ladder were not detected in CHO-K1cells after 4h SbCl3 treatment immediately. In addition, anincrease of calcium accumulation appeared in calcium specificarsenazo III staining in CHO-K1 cells and HF immediately after a4-h SbCl3 (0-400 uM) treatment, or after a 24-h post incubationin fresh medium. Dose-dependent cytotoxic effects andmicronuclei (MN) formation in CHO-K1 cells and HF after 4 hSbCl3 (0-400 uM) treatment. Co-treatment with calciummodulators such as verapamil (100 uM), EGTA (3 mM) and SbCl3 for4 h in HF showed that verapamil enhances SbCl3-induced MNformation and calcium accumulation. And, EGTA decreases MNformation. Cotreatment with 50-200 uM verapamil did notsignificantly enhance SbCl3-induced cytotoxicity in HF.GaAs-related results are as follows. Apoptosis and DNA ladderwere also appeared in CHO-K1 cells immediately following 40-160uM GaAs treatment for 4 h and post incubation for 24 h in freshmedium. In HF, 0-80 uM GaAs treatment for 4 h induced dose-dependent micronucleus (MN) formation, 25-275 MN per 500binuclei; and 0-78% cell killing examined by Sulforhodamine B-staining (SRB) survival assay. These results suggested that 4 hshort term exposure of semiconductor-used metal compounds suchas GaAs and SbCl3 induced genotoxic and cytotoxic effects inmammalian cells. Both metal compounds could induce apoptosis inCHO-K1 cells. The SbCl3-induced genotoxic effects could bemodulated with calcium modulators, such as VP and EGTA. Thecaution to protect workers using these metal compunds isrequired.