林瑞生Ruey-Hseng, Lin李寶珍Pao-Chen, LeeLeePao-Chen醫學研究所2026-10-289999-12-311999https://handle.ncl.edu.tw/11296/b8f5n5https://scholars.csmu.edu.tw/handle/123456789/34866中文摘要 (Chinese abstract) 銅(Copper)、錳(Manganese)、鎳(Nickel)和鉛(Lead)四種重金屬廣泛分佈於自然界中，所以可由地殼和海洋中釋出。環境之污染亦可能來自半導體工業、電子工業、汽車工業、儀器工業、航空零件和日用品工業。雖然銅和錳亦是人體內微量的元素，但已知含量過高或過低對健康都有所危害。由於半導體的製造在這些年相當盛行，其製造過程中需使用許多化學物質，這些化學物質之毒性除強酸、強鹼溶液、有機溶媒和各種光源被較詳細的研究外，金屬部份則很少有毒性的探討。由於此四種重金屬化合物不僅可用在半導體之連線，亦可用於複合鍍層，例如鎳則常用於印刷電路板及BGA之墊層。半導體工業表面看來相當安全和安靜，其實其製造過程中充滿危險性，其廢棄物如隨地棄置亦會造成環境的污染，因此吾人以培養之動物肺細胞為標本來探討四種重金屬鹽之細胞-遺傳毒性。 本研究為體外實驗(In vitro)，將V79中國頰鼠肺細胞暴露在適量的銅（Cu2+）、錳（Mn2+）、鎳（Ni2+）和鉛（Pb2+）鹽溶液中，首先利用微小核法(Micronuclei method) 探討其遺傳毒性(Genotoxicity)和以MTT法(MTT assay)探討其細胞毒性(Cytotoxicity)，並且以核分裂指數(NDI)判別其對細胞分裂的影響。然後藉由流式細胞技術(Flow cytometry)觀察其對細胞週期(Cell cycle)之分布比例的影響，以及探討其是否造成V79肺細胞進行細胞凋亡（Apoptosis）？另外，這四種重金屬分別和半導體晶片常用的銻(Antimony)和銦(Indium)二種元素共同作用，探討是否相互加強細胞毒性和遺傳毒性？並以提高細胞外鈣(Calcium)或鎂(Magnesium)離子的濃度，來研究此四種重金屬之細胞毒性和遺傳毒性的機轉。本研究亦以金屬中毒之解毒劑，D-penicillamine(D-PA)和Dimercaprol(BAL)，來探討其對銅、錳、鎳和鉛之細胞毒性之影響，以期提供適當的解毒劑。 遺傳毒性之研究結果發現高濃度的Cu2+(1mM)、Mn2+ (200-300μM)和Ni2+ (0.3-1mM)才具有統計意義增加V79中國頰鼠肺細胞之微小核頻率，Pb2+則在低濃度(0.1-3μM)皆能增加微小核數，但不呈劑量-相關性。遺傳毒性之結果顯示Pb2+之遺傳毒性較具特異性。當此四種重金屬離子分別加上In3+或Sb3+時，In3+之遺傳毒性可被Ni2+所降低，Sb3+則被Cu2+所加強而其它金屬離子之交互遺傳毒性則不具統計意義，顯示出半導體重金屬之交互作用十分複雜。於培養液中增加Ca2+或Mg2+的濃度，Ca2+能夠降低Pb2+所增加之微小核數，而Mg2+能夠反轉Mn2+和Pb2+所造成的遺傳毒性，由此顯示Mn2+和Pb2+對染色體DNA的傷害，和干擾Ca2+和Mg2+之功能有關。 細胞毒性之研究結果顯示，高濃度的Cu2+（1mM）、Mn2+（200-300μM）和Ni2+（1mM）均抑制NDI也就是延長細胞分裂時間。MTT法之結果亦顯示Cu2+、Mn2+和Ni2+都具有統計意義地降低細胞存活率。另外，Pb2+在研究濃度範圍內不影響NDI，但3μM的Pb2+具有明顯的細胞毒性。此四種重金屬與In3+或Sb3+之交互細胞毒性方面，只有Pb2+和Sb3+共同作用會加強細胞毒性。3mM和10mM Ca2+可反轉Mn2+或Pb2+之細胞毒性，3mM Mg2+對四種金屬離子之細胞毒性均可逆轉，但高濃度時（10mM）則只能反轉Cu2+或Mn2+之細胞毒性。金屬解毒劑之研究結果顯示3μM D-PA會加強四種金屬之細胞毒性； 30μM BAL可對抗Pb2+所造成的細胞毒性。 以FACScan分析V79肺細胞之DNA content，檢視Cu2+、Mn2+、Ni2+和Pb2+是否造成細胞凋亡？結果並沒有sub-G1出現，所以此四種重金屬離子在本研究條件下不造成V79肺細胞之細胞凋亡。此外，發現很有趣的現象是Cu2+、Ni2+和正對照組之Adriamycin，均使含G2/M phase之細胞數目增加，進一步以cell cycle distribution analysis證實Cu2+、Ni2+和Adriamycin確實會在V79肺細胞造成G2/M phase block。 在細胞型態上，四種重金屬化合物除了Mn2+外，其它與對照組比較則無明顯差異。Mn2+造成V79肺細胞凝縮，形成圓形亮點，另外，可能會與培養液或血清產生作用，在細胞表面及培養皿底面呈現黑色沈澱物。 以上結果顯示V79肺細胞短時間暴露在含有半導體重金屬Cu2+、Mn2+、Ni2+或Pb2+的環境時，會發生細胞-遺傳毒性，由於Ca2+和Mg2+可降低V79肺細胞之細胞-遺傳毒性所以推測此四種重金屬離子至少部分干擾鈣和鎂離子之功能而導致V79產生細胞-遺傳毒性。此外，Pb2+之細胞毒性可以BAL當解毒劑。半導體金屬間之交互細胞-遺傳毒性呈現十分複雜。Cu2+和Ni2+可誘導V79肺細胞發生G2/M phase block，而Mn2+明顯改變細胞型態，顯示四種重金屬之細胞-遺傳毒性具有差異性。既然此四種重金屬離子會在V79肺細胞造成細胞-遺傳毒性，所以在工作環境有必要做好妥善的防護措施，以避免危及工作人員之健康及造成環境污染。English abstract Copper, manganese, nickel and lead are widely distributed in the biosphere, being released into the atmosphere by degreasing from the earth crusts and the oceans. In addition, these metals are released into the environment by human activities, such as combustion of fossil fuels and other industrial release. Recent studies suggest that semiconductor workers have an increased incidence of worker-related illness. Semiconductor manufacturing is a chemically intensive industry involving many potentially hazardous operations, the relatively high toxicity of compounds routinely handled by employees. The important health effects of major solvents, acids, caustics, reactive gases, metals, energy and dopands were evaluated by many authors, whereas there are few studies on metals-induced cytogenotoxicities in cultured mammalian cells. In this study, the frequency of micronuclei (MN) was assayed to evaluate the genotoxic effects of Cu2+, Mn2+, Ni2+ and Pb2+ in V79 Chinese hamster lung (V79) cells. The cytotoxic effects of the four heavy metals were conducted by using MTT assay, and nuclear division index (NDI) index. In addition, effects of the four heavy metals on cell cycle distribution and apoptosis in V79 lung cells were assayed by flow cytometry. We also studied the cytogenotoxic interactions of the four heavy metals with Sb3+, In3+, Ca2+ or Mg2+, respectively. Since D-PA and BAL are metal chelators and have been widely used as antidotes in cases of heavy metal poisoning, we also studied the effects of D-PA and BAL on metals-induced cytotoxicity in V79 cells. All of Cu2+, Mn2+ and Ni2+ at higher concentrations (1mM Cu2+; 200-300μM Mn2+; 0.3-1mM Ni2+) significantly increased MN frequencies in V79 cells in this study. However, Pb2+ at lower concentrations of 0.1-3μM significantly increased MN frequencies, but Pb2+-induced genotoxicity was not in a concentration-dependent manner. The results suggested that Pb2+ induced genotoxicity in V79 cells with more specific effect. The results of genotoxic interactions of the four metals with In3+ or Sb3+ were complication: Ni2+ decreased In3+-induced genotoxicity, Cu2+ increased Sb3+-induced genotoxicity, but genotoxic interactions between other metals were not significant. In addition, Ca2+ decreased Pb2+-induced genotoxicity. Mg2+ reversed the genotoxicity of Mn2+ or Pb2+ to the control level, respectively. It appears that both Mn2+- and Pb2+-induced genotoxicity might relate to interfere the biological functions of Mg2+ and Ca2+. Cu2+ (1mM), Mn2+ (200-300μM), and Ni2+ (1mM) significantly decreased NDI, but Pb2+ at all concentra-tions treated didn't significantly decreased NDI. The results of MTT assay also showed that all of Cu2+, Mn2+, and Ni2+ at higher concentrations significantly decreased cell viability, but Pb2+ at lower concentration of 3μM significantly induced cytotoxicity. In addition, just only Pb2+ significantly increased Sb3+-induced cytotoxicity. Ca2+ at concentrations of 3mM and 10mM significantly antagonized the cytotoxicity of Mn2+ or Pb2+, respectively. Mg2+ at concentration of 3mM significantly reversed cytotoxicity of the four metals, but 10mM Mg2+ only reversed cytotoxicity of Cu2+ or Mn2+. The data of metal chelators study showed that 3μM D-PA significantly increased all of the four heavy metals induced cytotoxicity, but 30μM BAL significantly reduced Pb2+-induced cytotoxicity. The results of hypoploidy analysis showed that all of Cu2+, Mn2+, Ni2+ and Pb2+ didn't induce sub-G1 V79 cells, the results indicated all of the four metal ions didn't cause apoptosis in V79 cells. However, both Cu2+ and Ni2+ as adriamycin (positive control) caused G2/M phase block and increased population of V79 cells with higher granularity. Cell cycle distribution analysis further demonstrated that both Cu2+ and Ni2+ as adriamycin increased population of V79 cells in G2/M phase. There is only Mn2+ among the four metal ions as adriamycin markedly changed morphology of V79 lung cells. In addition, Mn2+ caused black precipitation, which might react with ingredients of medium or serum. These results showed that all of Cu2+, Mn2+, Ni2+ and Pb2+ induced cytogenotoxicities in V79 cells after two hours exposure. However, both Ca2+ and Mg2+ significantly decreased their cytogenotoxicity. Accordingly, we suggested that the mechanism of the four metals induced cytogenotoxicity might be partly related interfere with the biological functions of Mg2+ and Ca2+. In addition, BAL decreased Pb2+-induced cytotoxicity. Cytogenotoxic interactions between semiconductor metals were complication. Both Cu2+ and Ni2+ caused G2/M phase block. Mn2+ markedly changed morphology of V79 cells. These results showed that the four heavy metal ions induced cytogenotoxicities in V79 cells is in different properties.硝酸銅硝酸錳硝酸鎳硝酸鉛遺傳毒性細胞毒性微小核法流式細胞分析法G2/M期阻斷細胞凋亡金屬中毒之解毒劑copper nitratemanganese nitratenickel nitratelead nitrateGenotoxicityCytotoxicityMicronuclei methodFlow cytometryG2/M phase blockApoptosisAntidote - D-PA (D-penicillamine) and BAL (Dimercaprol)thesis