Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/51779
標題: Homocysteine and DNA damage in cultured cells: protection by resveratrol and potentiation by s-adenosylhomocysteine
同半胱胺酸與細胞DNA傷害: 白藜蘆醇的保護作用及腺甘同半胱胺酸的協同作用
作者: 林佩縈
Lin, Pei-ying
關鍵字: Homocysteine;同半胱胺酸;resveratrol;s-adenosylhomocysteine;DNA damage;白藜蘆醇;腺甘同半胱胺酸;DNA傷害
出版社: 食品科學系
摘要: 
許多臨床研究顯示,當血漿中同半胱胺酸濃度 (Homocysteine, Hcy) 不正常累積時,可能增加罹患心血管疾病的機率; 同時也是退化性腦部病變如帕金森氏症及阿茲海默氏症等的可能致病因子。故有許多研究學者紛紛探討Hcy對心血管疾病及退化性腦部病變可能的作用機制,其中一項假說認為因為Hcy可造成細胞內氧化壓力上升,主要因為Hcy具有一個游離的硫醇基 (-SH) 可發生自氧化的現象,同時陸續也有許多文獻提出當試驗系統中存在銅離子 (Cu2+) 時,可加速Hcy的自氧化而造成明顯的氧化傷害。在單碳循環中,s-adenosylhomocysteine (SAH) 和Hcy互為可逆的代謝前趨物及產物。研究指出Hcy造成內皮細胞傷害可能與腺苷同半胱胺酸 (SAH) 造成細胞的低甲基化有關。 由於多數有關Hcy會造成氧化傷害的報導來自體外試驗的結果,並且使用極高的濃度(1-10 mM),使得Hcy經由氧化壓力而造成內皮傷害的理論受到質疑。 因此本論文研究乃採用較低濃度的Hcy探討在細胞模式中的氧化傷害作用。 同時,本研究並探討一種富含於紅酒中的多酚類─白藜蘆醇 (resveratrol, RSV,被認為是所謂的 “The French paradox” 現象中的一個保護因子) 對Hcy和Cu+2所造成的內皮細胞氧化傷害。 然後,本研究又探討了Hcy與SAH之間的相互作用。 本論文研究共分成二部分。
(1)第一部份為探討RSV是否可抑制Hcy和Cu+2所造成的內皮細胞氧化傷害。 結果顯示:SVEC 4-10細胞 (株化的老鼠血管內皮細胞) 以500μM Hcy/100 μM Cu2+處理後,細胞脂質過氧化及DNA傷害均有明顯的增加。 以RSV預培養後,再以Hcy/Cu2+處理時可發現:當RSV濃度低於系統中Cu2+離子濃度時,可有效抑制Hcy/Cu2+造成的DNA傷害及細胞內活性氧 (ROS) 的產生,但對於脂質過氧化並沒有抑制效果; 但當RSV濃度等於或大於系統中Cu2+離子濃度時,抑制效果不如低濃度的RSV為佳。以上結果顯示:RSV可降低胞內活性氧含量而抑制Hcy/Cu2+造成的DNA傷害,然而此效果似乎與RSV和系統中Cu2+間的比率有關。
( II ) 第二部分為探討Hcy與SAH的相互作用;作法為利用BV-2細胞 (株化的老鼠微神經膠細胞) 單獨培養或共同培養Hcy及SAH。 結果顯示:細胞以100 μM Hcy或4 μM SAH單獨培養不會造成明顯的DNA傷害,但Hcy加上SAH則會以協同(synergistic)方式促進DNA傷害以及DNA低甲基化的程度,並呈現SAH的濃度效應以及對SAH的專一性(DNA低基化的程度對Hcy同樣專一性,但對SAH的專一性較強)。 我們也發現細胞以Hcy/SAH同時培養時並不會造成BV-2細胞中iNOS表現量及NO含量增加。加入抗氧化酶 (SOD或catalase) 或一種離鐵子螫合劑 (desferal) 可強烈抑制Hcy/SAH造成的DNA傷害。 經由相關性分析發現Hcy/SAH所造成的DNA傷害與胞內過氧化氫含量以及低甲基化程度呈現顯著正相關。因此BV-2細胞中氧化壓力上升及低甲基化程度的增加可能是Hcy/SAH造成DNA傷害的共同作用因子。
綜合上述結果,本論文研究顯示RSV具有抑制Hcy/Cu2+對內皮細胞傷害的能力,其作用與減少胞內活性氧有關;然而RSV的作用以較低的濃度為佳。同時本研究也發現,在不會對微神經細胞的DNA造成傷害的Hcy濃度下,加入少量的SAH卻會對DNA造成加成性的傷害以及DNA的低甲基化,而且SAH對這兩種作用具有專一性。 有關SAH與Hcy相互作用的機制有待更深入的探討,但本研究結果提示SAH的升高可能是人體某些疾病的一個危險因子。

Plasma homocysteine (Hcy) is a risk factor for cardiovascular disease (CVD) and perhaps, neurodegenerative diseases. Much work has been done on the possible mechanisms underlying the effects of Hcy. One theory holds that Hcy, which contains a free sulfhydryl group, may undergo autoxidation, particularly in the presence of copper ions, with concomitant production of hydrogen peroxide, resulting in injury to endothelial cells. Another theory holds that plasma S-adenosylhomocysteine (SAH), which rises as a result of chronic Hcy-related diseases, may mediate Hcy-related vascular changes through hypomethylating effects of SAH. However, whether the effect of Hcy in vivo is related to generation of ROS is still in dispute because the evidence comes mainly from in vitro studies in which very high Hcy levels (1 to 10 mM) have been used. In this thesis study, we therefore employed relatively low levels of Hcy to study its oxidative effects in cultured cells. Meanwhile, we tested the effect of resveratrol, a polyphenol present in red wine that has been implicated in “The French paradox” as a protective factor against CVD, on oxidative damage induced by a system related to Hcy. In addition, we studied the interaction between Hcy and SAH. The study is divided into two parts.
( I ) Chapter 1 focused on the effect of resveratrol on oxidative damage to SVEC 4-10 cells (an endothelial cell line) induced by 500 μM Hcy/100 μM Cu2+. We found that incubation of cells with Hcy/Cu2+ markedly induced oxidative damage and elevated intracellular reactive oxygen species (ROS). Pre-incubation of cells with RSV reduced Hcy/Cu2+-induced DNA damage (but not lipid peroxidation) when the ratio of RSV to Cu2+ was less than 1; whereas the protective effect of RSV weakened, when the ratio of RSV to Cu2+ was greater than 1. The same change was found for intracellular ROS. Thus, RSV is able to prevent Hcy/Cu2+-induced DNA damage, possibly by inhibiting ROS production, but the effect of RSV appears to depend on the ratio of RSV to copper ions.
( II ) Chapter 2 focused on the oxidative interaction of Hcy and SAH in BV-2 cells (immortalized mouse microglia cell line). We found that Hcy at 100 μM and 4 μM SAH alone resulted in little or no oxidative damage. However, a combination of SAH and Hcy (Hcy/SAH) led to marked and synergistic DNA strand breaks that were completely inhibited by addition of superoxide dismutase, catalase and an iron chelator, suggesting the involvement of ROS. However, reactive nitrogen species are probably not involved because Hcy/SAH did not increase the expression of inducible NO synthase (iNOS) or nitric oxide levels. SAH or Hcy alone induced DNA hypomethylation but the effect of the former was much stronger than that of the latter. Moreover, SAH/Hcy also synergistically potentiated DNA hypomethylation. The effects of Hcy/SAH on DNA damage and DNA hypomethylation were specific to SAH, but not to Hcy, because the effect of SAH was not or only partially reproduced by adenosine (the intracellular indicator of adenine), whereas the effect of Hcy was completely reproduced by a similar SH-containing compound, cysteine.
Overall, the results of this thesis study demonstrate that RSV can prevent Hcy/Cu2+-induced DNA damage, possibly by inhibiting ROS production and that the effect of RSV appears to depend on the ratio of RSV to copper ions. By contrast, we showed that Hcy combined with SAH, but not individually, induced DNA damage, possibly by increasing generation of ROS and hypomethylation of DNA. The results suggest a possible detrimental role for elevated SAH levels. The in vivo relevance of our finding is unclear, but the protection by RSV and potentiation by SAH are an interesting area awaiting further exploration.
URI: http://hdl.handle.net/11455/51779
Appears in Collections:食品暨應用生物科技學系

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