Abstract
本論文探討鉛化物誘發核酸損傷機制. 首先利用質體DNA 斷裂測定,證實醋酸鉛在Hepes (pH 6.8)緩衝液中會造成DNA 斷裂,且隨著濃度與反應時間增加而加強;相較於鉛,醋酸鋅則不顯著地造成DNA 斷裂.鉛所造成的 DNA 斷裂可明顯地被單線態氧清除劑 NaN3及 TEMP 所抑制,再者單線態氧的加強劑 D2O, GSH 及 2-mercaptoethanol 加強鉛造成DNA 斷裂的現象,而且以電子順磁共振光譜 (EPR) 也偵測出鉛在 Hepes (pH 6.8) 緩衝液中有單線態氧的訊號出現,相較地,氫氧自由基訊號則不明顯;以上結果均支持單現態氧是造成鉛的 DNA 斷裂性的要素.維生素C 顯著地加強鉛造成 DNA 斷裂,且斷裂的程度隨著維生素 C 濃度的增加而增加.過氧化酵素,NaN3 及 EDTA 可明顯地降低鉛/維生素C 造成的 DNA斷裂,顯示此反應中產生過氧化氫,並且單線態氧及鉛均參與此反應,此結果推測鉛促使維生素 C 自動氧化,而將 O2 還原成 H2O2,並加速催化 Haber-Weiss 反應.此外,過氧化氫也顯著地加強鉛造成 DNA 斷裂,且 NaN3,TEMP,GSH,BSA,EDTA及過氧化氫酵素均有抑制鉛/過氧化氫的 DNA 斷裂性,顯示單線態氧,鉛與過氧化氫均是在此系統中造成 DNA 斷裂不可缺少的反應物.另外利用HPLC-UV-ECD 測定 8-OHdG 核酸鍵結物,發現鉛略使 8-OHdG 含量上升,且在過氧化氫存在情況下,DNA 上 8-OHdG 含量隨著鉛濃度的增加成正相關地增加.單線態氧清除劑比氫氧自由基清除劑更有效地抑制鉛及過氧化氫造成之 8-OHdG.在鉛/過氧化氫反應中,EPR 偵測出單線態氧的訊號強度比鉛或過氧化氫單獨下的訊號強度高;相反地,隨反應時間加長,鉛明顯地抑制過氧化氫造成的氫氧自由基訊號.本研究發現鉛誘發 DNA 損傷與單線態氧的生成成正相關.此結果也印證鉛誘發基因突變特殊圖譜與單線態氧及8-OHdG 有關(Yang et al., 1996).本研究指出鉛雖然不屬於過渡金屬族,卻能如過渡金屬銅或鐵離子之用作用,催化自由基之產生.The mechanism of DNA damage induced by lead was explored inthis study. Lead acetate could induce DNA strand breakage inHepes buffer, pH 6.8, in a dose- and time-dependent manner asdetermined using the plasmid relaxationassay. When compared withlead, zinc acetate did not significantly induced DNA breakage.Singlet oxygen scavengers NaN3 and 2,2,6,6-tetramethyl-4-piperidone (TEMP) markedly inhibited lead-induced DNA breakage.D2O, low dosesof glutathione and 2-mercaptoethanol (singletoxygen enhancers) could enhancelead-induced DNA breakage.Electron paramagnetic resonance (EPR) spectra showedthat singletoxygen but not hydroxyl radical signal was detected in leadsolution. Furthermore, lead-induced DNA breakage was markedlyenhanced by ascorbate in a dose-dependent manner. Catalase,NaN3, and EDTA significantlyinhibited lead/ascorbate-induced DNAbreakage, indicating the participation ofH2O2, singlet oxygen,and lead in this reaction. This result suggests thatleadpromotes ascorbate autooxidation, enhances the reduction ofO2 to H2O2 simultaneously, and catalyzes Haber-Weiss reaction.Additionally, lead-inducedDNA breakage was markedly enganced byH2O2, and this induction was inhibited byNaN3, TEMP,glutathione, BSA, EDTA, and catalase. The results have indicatedthat singlet oxygen, lead, and H2O2 are all involved in thereaction system.Lead could cause a small amount of8-hydrodeoxyguanosine (8-OHdG) formation in calf thymus DNA, anddose-dependent induced the formation of this adduct in thepresence of H2O2 using HPLC-UV-ECDmethod. Singlet oxygenscavengers were more effective than hydroxyl radical scavengersin protection from lead/H2O2-induced 8-OHdG adducts. EPRspectrashowed that singlet oxygen signal was additively inducedby lead/H2O2, whereaslead inhibited the hydroxylll radicalsignal induced by H2O2 in a prolongedincubation time. Takentogether, singlet oxygen is an important factor for leadinducedDNA breakage. The results were well correlated to that theobservation of lead -induced specific mutational spectrum maydue to singlet oxygen and 8-OHdG (Yang et al., MolCarcinogenesis: 17:181-191, 1996). This study showed that leadcould function as transition metao ions Cu2+ and Fe3+ tocatalyzefree radical generation.