| 摘要: | 4-胺基聯苯（4-Aminobiphenyl；4-ABP）是人類致癌物質，許多流行病學研究指出4-胺基聯苯和膀胱癌之間有極大之相關性。4-胺基聯苯經由肝臟酵素系統代謝形成之衍生物可與DNA 形成鍵結物外，亦具有基因毒性及細胞毒性，然而4-胺基聯苯和DNA 氧化性傷害的相關性並不清楚。因此，本論文主要針對4-胺基聯苯及其同分異構物2-胺基聯苯（2-Aminobiphenyl；2-ABP）是否會造成Hep G2細胞氧化性傷害做探討。本研究利用流式細胞儀分析氧化性物質，發現Hep G2細胞經2-胺基聯苯或4-胺基聯苯作用一小時均會造成細胞產生H2O2，且具有濃度和時間的相關性。再進一步以彗星分析法（comet assay）發現2-或4-胺基聯苯均會直接造成細胞DNA斷裂；利用可修補氧化性傷害之酵素endonuclease III (Endo III) 和formamidopyrimidine DNA glycolase (Fpg) 作用發現2-或4-胺基聯苯分別會在DNA上形成氧化性purine和pyromidine傷害；另外，藉由5,5-dimethyl-pyrroline N-oxide (DMPO)和N-tert-butyl-α-phenylnitrone (PBN) 以及過氧化酶（Catalase）之作用，亦證實2或4-胺基聯苯會對Hep G2細胞產生氧化性DNA傷害。另一方面，添加鐵離子螯合劑（desferrioxamine ；DFO）、銅離子螯合劑（neocuproine；NC）則會降低2-或4-胺基聯苯對Hep G2細胞造成的傷害，鈣離子螯合劑（1,2-bis(2-aminophenoxy) ethane-N,N,N9,N9-tetraacetic acid acetoxymethyl ester；BAPTA/AM）會減少由4-胺基聯苯造成之DNA損傷。在氧化性修復基因及其蛋白表現方面，2-胺基聯苯會抑制OGG1和MTH1的mRNA的表現，而4-胺基聯苯則抑制OGG1蛋白質的表現。結果顯示2-或4-胺基聯苯雖為同分異構物，但在Hep G2細胞內所造成的DNA氧化性傷害及修復之機制不盡相同，且2-或4-胺基聯苯可能經由抑制細胞內DNA修補作用進而導致氧化性傷害之產生。; 4-Aminobiphenyl (4-ABP) can induce bladder and breast cancers in human. It has been proven 4-ABP could be metabolized into reactive products which form DNA adduct in liver cells. This adduct is known to cause DNA damage. However, whether the DNA damage caused by this chemical via the oxidative stress in Hep G2 cells remains to be evaluated. In this study, we determined the possibility that 4-ABP and its analogue, 2-aminobiphenyl (2-ABP), caused oxidative DNA damage in Hept G2 cells. By using flow cytometry assay, we showed that intracellular reactive oxygen species in these cells were detected in a does-dependent manner, after incubating cells with compounds for 60 min. The comet assay, a powerful method in detecting oxidative DNA damage in eukaryotic cells, was adopted to detect the oxidative DNA damage caused by 4-ABP and 2-ABP, respectively, in Hep G2 cells. Both compounds at 100 ?M of dose induced DNA strand breaks in these cells. This assay was also modified to permit the detection of oxidized bases by including a step in which DNA was digested with endonuclease III (Endo III) or amidopyrimidine-DNA glycosylase(Fpg) to assess oxidized pyrimidines and purines, respectively. Indeed, both compounds induced DNA strand breaks from excision of oxidative DNA adducts. This suggested that oxidative DNA damage occurs in Hep G2 cells treated with 2-ABP or 4-ABP. Pre-incubation of cells with iron and copper ions chelators decreased DNA damage, while calcium ion chelator only prevented DNA strands breaks in cells treated with 4-ABP but not in cells treated with 2-ABP(statistically not signfiant). The data suggested the participation of ions in 2-ABP and 4-ABP induced DNA strand breaks.Using RT-PCR technique, the expression OGG1 gene and MTH1 gene was decreased by 2-ABP. In addition, The production of OGG1 protein, was blocked by 4-ABP. That the amounts of OGG1 and MTH1, the DNA repair enzymes for 8-OH-Gua. Thus, we suggested the lower amounts of OGG1 and MTH1 in cells with both compounds would be one of mechanisms in inducing oxidative DNA damage. |
