Intracellular levels of homocysteine and S-adenosylhomocysteine in relation to DNA damage in a mouse endothelial cell line during folate deficiency

Abstract

許多研究皆指出，葉酸缺乏會導致染色體的不穩定，但機制至今尚未完全清楚，大多研究著重在葉酸缺乏造成核苷酸imbalance和misincorporation而導致 DNA傷害，較少文獻探討葉酸缺乏時，methionine cycle中間代謝產物濃度改變與DNA傷害之相關性與機制。前人研究認為，葉酸缺乏時是由於同半胱胺酸(Homocysteine, Hcy)上升、氧化壓力增加，進而造成DNA傷害或細胞凋亡，但有關Hcy之研究文獻，大多使用相當高的濃度，與生理濃度相差甚遠。故葉酸缺乏時，Hcy對細胞DNA傷害之機制仍待進ㄧ步釐清。此外，與Hcy相關之許多疾病研究中皆發現methionine cycle的另一中間代謝產物- 腺核苷同半胱胺酸(S-adenosylhomocysteine, SAH)-亦扮演重要之調控角色。故本研究利用小鼠內皮細胞(SVEC4-10 cells)，以葉酸缺乏以及外加Hcy或SAH兩種模式，探討SAH及Hcy與細胞DNA傷害之協同作用。 本研究將內皮細胞培養於葉酸缺乏條件下四至六天，會顯著抑制細胞生長，並造成細胞DNA傷害。葉酸缺乏造成細胞內SAM濃度下降、細胞內Hcy、SAH濃度則顯著上升。進ㄧ步探討Hcy、SAH與DNA傷害之機制發現，葉酸缺乏造成細胞內外SAH濃度上升時，細胞內SAM- dependent methyltransferase活性受到抑制，且導致SAM/SAH ratio及5-mdc含量(為細胞內兩種甲基化指標)顯著下降。另一方面，葉酸缺乏導致細胞內外Hcy上升時，其細胞內ROS亦明顯上升；兩者呈現高度正相關。以上結果暗示，葉酸缺乏所導致之DNA傷害中，細胞內外上升之SAH與Hcy，可能扮演相當重要之角色。 我們亦探討細胞培養於葉酸充足或缺乏條件下，外加Hcy與SAH誘發DNA傷害之能力與其可能機制。細胞於葉酸充足之條件下，以SAH或Hcy培養48hr誘發DNA傷害時，細胞內SAH及Hcy之濃度，與葉酸缺乏時細胞內濃度變化比較時發現，葉酸缺乏時細胞內SAH濃度可以上升至足以傷害DNA之濃度(葉酸缺乏第四天細胞內SAH濃度上升至492 pmole/106 cells；葉酸充足以50 μM SAH誘發DNA傷害時，細胞內SAH上升至432 pmole/106 cells)。但葉酸缺乏時，細胞內Hcy上升程度略低於足以傷害DNA之濃度，但其濃度均達到nmole/106 cells(葉酸缺乏時，細胞內Hcy最高上升至7.1 nmole/106 cells；葉酸充足以2.5 mM Hcy誘發DNA傷害時，細胞內Hcy上升至29 nmole/106 cells)。結果亦顯示，無論將細胞培養於葉酸缺乏或正常之條件下，SAH誘發DNA傷害之能力皆大於Hcy。我們並進ㄧ步發現，在葉酸缺乏之條件下，以低濃度之SAH或高濃度之Hcy培養48hr造成DNA傷害時，細胞內SAH及Hcy濃度皆會顯著上升。在葉酸缺乏之模式下，亦發現低濃度SAH與Hcy共同培養會顯著促進細胞之DNA傷害。由上述結果得知葉酸缺乏時所導致之DNA傷害應是由SAH及Hcy共同上升所導致。另外，以高濃度Hcy培養造成DNA傷害時，會造成細胞內 SAM濃度下降，及導致SAH及Hcy及Cys濃度顯著上升。由此推測，外加Hcy所造成之DNA傷害不單是由Hcy所導致，而可能是影響了整個methionine cycle之結果。 由以上結果推測，葉酸缺乏時所導致之DNA傷害，一部分是藉由Hcy上升，導致氧化壓力增加而造成DNA傷害；另外，此時細胞內SAH濃度亦上升，抑制DNA甲基轉移酵素活性，導致DNA hypomethylation而擴大DNA 傷害。總之，我們認為在葉酸缺乏之老鼠內皮細胞中，除了Hcy之外, SAH亦為相當重要之調控因子。

Cell culture studies have shown that folate deficiency causes DNA instability. These effects can be attributed to impaired homocysteine (Hcy) metabolism which can increase generation of reactive oxygen species (ROS). However, this proposed mechanism is still in dispute because the evidence comes mainly from in vitro studies using very high Hcy concentration. It is noteworthy that a preponderance of evidence has shown that S-adenosyl-homocysteine (SAH), the immediate precursor for Hcy biosynthesis, is also associated with Hcy-related diseases. The aims of this study were to investigate the interaction of SAH and Hcy on DNA damage in a mouse endothelial cell line (SVEC4-10 cells) and the possible mechanisms. To better understand the role of Hcy and SAH in cellular physiology and pathology during folate deficiency, SVEC4-10 cells were incubated with folate deficiency medium for 4-6 days. As expected, folate deficiency significantly inhibited cell proliferation and increased DNA damage. In comparison, the concentrations of intracellular and extracellular Hcy were dramatically increased in cells grown in folate deficient medium at the end of a 1-week cultivation period. We also found a high correlation between the levels of intracellular and extracellular Hcy and ROS. Additionally, folate deficiency increased the levels of intracellular and extracellular SAH. SAH has been reported to be a competitive inhibitor of DNA methyltransferase. We also found the activity of cellular methyltransferase was significantly inhibited during folate deficiency, and the intracellular methylation markers (both SAM/SAH ratio and 5-mdc content) also were significantly decreased. We also investigated the DNA damage induced by added SAH and Hcy in SVEC4-10 cells during folate deficiency in comparison with normal folate status. During folate deficiency, intracellular SAH but not Hcy increased to a concentration that could lead to DNA damage. The addition of 50μM SAH in the folate complete medium effectively resulted in DNA damage, with the intracellular SAH concentrations increased to 432 pmole/106 cells, which was comparable to the levels of SAH at 4th day during folate deficiency (492 pmole/106 cells). However, addition of 2.5mM Hcy in the folate complete medium caused DNA damage, with the intracellular Hcy concentrations increased to 29 nmole/106 cells. These levels were higher than the highest level (7.1 nmole/106 cells) of intracellular Hcy during folate deficiency. Additionally, when cells were incubated in folate-complete medium for 48 hr, SAH significantly enhanced DNA damage in a dose-dependent manner, whereas Hcy had a much weaker effect. A similar trend occurred in folate deficiency. After cultivation in folate deficiency for 48 hr, administrations of SAH or Hcy at concentration sufficient to evoke DNA damage markedly increased the levels of intracellular SAH and Hcy. Finally, we found that the addition of 200μM Hcy or 2μM SAH alone only slightly enhanced DNA damage. However, the combination of SAH and Hcy (Hcy/SAH) led to marked and synergistic DNA strand breaks. In summary, our results support a pivotal role for both of SAH and Hcy as mediators of DNA damage in folate- deficient endothelial cells. Additionally, when cells were treated with high concentrations of Hcy, which caused DNA damage, the levels of intracellular SAM decreased but the concentrations of Hcy, SAH, and Cys increased. The result suggests that DNA damage induced by administration of high concentrations of Hcy may not be resulted only from the increased levels of intracellular Hcy, but may also be associated with the change in levels of other intermediates of the methionine cycle. In summary, our results demonstrate cellular DNA strand breakage induced by folate deficiency is preceded by the enhanced intracellular ROS. Additionally, our results demonstrate that folate deficiency increases the levels of cellular SAH, which may impair DNA methyation by inhinibtion of DNA methyltransferase activity. The results support a pivotal role for both of SAH and Hcy as a mediator of DNA damage in folate-deficient endothelial cells.