Abstract
砷和鎘是人類的致癌物,但其致癌機轉仍不清楚.最近的研究報告指出,含氧自由基(ROS)及含氮自由基(RNS)與砷和鎘,所誘發染色體異常以及微小核球形成有關.雖然染色體異常與微小核球主要來自於核酸鏈損傷,但核酸鏈受損是需一些細胞內的步驟才變成染色體異常,所以本論文問一個比較簡單的問題:砷和鎘如何造成核酸鏈斷裂?本實驗的目的在找出砷和鎘如何引發核酸鏈斷裂?含氧自由基(ROS)和/或含氮自由基(RNS)是否參與其過程?經砷或鎘處理的牛主動脈內皮細胞,其核酸鏈斷裂由單細胞鹼性電泳法測得.一氧化氮合成酵素抑制劑,Nw-nitro-L-arginine methyl ester, S-methyl-L-thiocitrulline及氫氧自由基清除劑,dimethyl sulfoxide, D-mannitol,可以降低砷誘發之核酸鏈斷裂,但不能降低鎘誘發之核酸鏈斷裂.相反的過氧化氫酵素可以降低鎘誘發之核酸鏈斷裂,但不能降低砷誘發之核酸鏈斷裂.然而superoixde dismutase,超氧離子清除劑及uric aicd和Trolox,peroxyn-itrite清除劑,都可以降低鎘和砷誘發之核酸鏈斷裂.這些結果顯示,砷誘發之核酸斷裂是經由nitric oxide,peroxynitrite;而鎘是經由超氧離子,過氧化氫,氫氧自由基.然而,鈣似乎也參與砷和鎘所早造成核酸鏈斷裂的過程.因為鈣離子螯合劑,EGTA,Quin 2,BAPTA,都可以降低砷和鎘所造成核酸鏈斷裂.而鈣離子通透劑,A23187,也可以增加砷所造成之核酸鏈斷裂.一般認為三價無機砷化物較其代謝物,甲基砷化物毒性高.然而最近研究發現,亞砷酸鈉在人類肺上皮細胞,並不會誘發核酸鏈斷裂,但是其代謝物,雙甲基砷酸則可以誘發核酸鏈的斷裂.我們以單細胞鹼性電泳法,比較亞砷酸鈉及雙甲基砷酸在人類胚胎肺細胞株 MRC-5及牛主動脈內皮細胞所誘發核酸鏈斷裂的情形.結果顯示,無論有無hydroxyurea(Hu)及arabinofuranosyl cytosine(AraC)這兩種核酸聚合抑制劑的協助,皆可以偵測到亞砷酸鈉誘發的核酸鏈斷裂;然而雙甲基砷酸則需要在有Hu及AraC的協助下,才可以偵測到核酸鏈的斷裂.在誘發核酸鏈斷裂的時程上,亞砷酸鈉與Hu及AraC所誘發的核酸鏈斷裂約在作用後六小時左右達到高峰,而雙甲基砷酸與Hu及AraC所誘發的核酸鏈斷裂則於作用之後的十小時之內,皆隨著作用的時間呈持續性的增加.亞砷酸鈉所誘發的核酸鏈斷裂的修復速度也比雙甲基砷酸所誘發核酸鏈斷裂的修復快.此外,亞砷酸鈉經由一氧化氮的產生而誘發核酸鏈的斷裂,此過程會受胞內鈣離子濃度的影響;相反地,雙甲基砷酸在誘發核酸鏈斷裂過程中,則與一氧化氮的產生及胞內鈣離子濃度變化沒有明顯的關聯性.這些結果顯示,亞砷酸鈉與雙甲基砷酸是經由不同的機轉和路徑誘發細胞產生核酸鏈斷裂.Arsenic and cadmium are well-reconized human carcinogens, buttheir mechanismsare not entirely clear. Recent studiessuggested that reactive oxygen species(ROS) and/or reactivenitrogen species (RNS) are involved in the clastogenici-ty ofarsenic and cadmium. Since chromosome aberrations mainly comefrom DNA damage, and DNA damages may require several cellularsteps to become chromoso-me aberrations, a more simple questionto be asked is: how DNA damages are in-duced by arsenic andcadmium? The aim of this invesgation is to find out whatkindsof ROS and RNS are involved in arsenic- and cadmium-induced DNAstrand breaks. DNA strand breaks in sodium arsenite (SA) andcadmium chloride (Cd) treated bovine aortic endothelialcellswere analyzed by single-cell alkaline electrophoresis. Theresults indicate that inhibitors of nitric oxide syntha-se, Nw-nitro-L-arginine methyl ester (NAME) and S-methyl-L-thiocitrulline (MTC),scavengers of hydroxyl radicals, dimethylsulfoxide and D-mannitol, could decrease DNA strand breaks inSA- but not in Cd-treated cells. On the contray, catalase, ascavenger of hydrogen peroxide, could decrease DNA strand breaksin Cd- but not in SA-treated cells. However, superoxidedimutase,a scavenger of superoxide, uric acid and Trolox,scavengers of peroxynitrite, could decrea-sse DNA strand breaksbreaks in both SA- and in Cd-treated cells. These rsultssuggest that SA may induce DNAstrand breaks via nitric oxideandperoxynitrite; and Cd induce DNA strand breaks via superoxide,hydrogenperoxide,and hydroxyl radicals. Moreover, calcium seemsto be involved in both SA- and Cd-induced DNA breaks, sincecalcium chelators, ethylene glycol-bis (beta-ami-ethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), Quin 2, 1,2-Bis(2-aminophe-noxy)ethane-N,N,N',N'-tetra-acetic acid (BAPTA), supressed SA-as well as Cd- induced DNA strand breaks. calcium ionophore,A23187, increased SA- but not Cd-induced comet. Inorganictrivalent arsenic compounds are generally recognized to be moretoxic than their metabolite, methylated arsenic compounds.However, SA has recently been shown not to induce DNA strandbreaks in human diploid pulmonary epithelial cells, but itsmetabolite, dimethylarsinic acid (DMA) does. We have comparedthe induction of DNA strand breaks by SA and DMA in a humanembryo lung cell line, MRC-5, and in bovine aortic endothelialcells by single-cell alkaline electrophoresis. The resultsindicate that SA could induce DNA strand breaks in both celllines with or wihtout the blockage of DNA repair synthesis byhydroxyurea (Hu) plus cytosine-beta-D-arabinofuranoside (AraC).Whereas, DMA did not induce DNA strand breaks without Hu/AraC.Induction of DNA strand breaks by SA plus Hu/AraC, reached aplateau by 6 h of treatment, wheras, DNA strand breaks inducedby DMA plus Hu/AraC contiously increased beyound 10 h. The SA-induced DNA strand breaks were also repaired in a faster ratethan that of DMA. Moreover, while the SA-induced DNA strandbreaks were mediated by the nitric oxide production and weresensitive to calcium modulation, the DMA-induced DNA strandbreaks were insensitive to nitric oxide synthase inhibitors andcalcium modulators. These results indicate that SA and DMAinduce DNA strand breaks through distinct different mechanisms.