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

• Main Authors: Yuan-Jen Huang, 黃苑甄
• Other Authors: Miao-Lin Hu
• Format: Others
• Language: zh-TW
• Published: 2005
• Online Access: http://ndltd.ncl.edu.tw/handle/95044845669537298464

碩士 === 國立中興大學 === 食品科學系 === 93 === 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.