DNA instability (strand breakage, uracil misincorporation, and defective repair) is increased by folic acid depletion in human lymphocytes in vitro
DNA instability (strand breakage, uracil misincorporation, and defective repair) is increased by folic acid depletion in human lymphocytes in vitro
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DOI：
10.1096/fasebj.12.14.1491
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发表时间：
1998-11-01
期刊：
影响因子：
4.8
通讯作者：
Hawdon, A
中科院分区：
文献类型：
--
作者：
Duthie, SJ;Hawdon, A
叶酸对DNA的合成和修复是必不可少的。我们报道了叶酸耗竭对体外培养的正常人淋巴细胞DNA稳定性的影响。采用彗星试验(单细胞凝胶电泳法)检测DNA链断裂、尿嘧啶掺入错误、DNA氧化碱基损伤和DNA修复能力，以淋巴细胞增殖作为正常复制的指标。从人静脉血中分离的淋巴细胞在含叶酸(1 ng/ml~2µg/ml)的完全培养液和缺乏叶酸的培养液中刺激生长10天。彗星分析细胞用细菌DNA修复酶Ⅲ内切酶检测淋巴细胞DNA中氧化嘧啶的水平，或用尿嘧啶DNA糖基酶检测尿嘧啶的错误结合，测量细胞数和存活率。正常的人淋巴细胞DNA中含有可检测到的尿嘧啶错误结合量(估计每个细胞约为1000)。淋巴细胞培养后，随着叶酸浓度的降低，DNA链断裂和尿嘧啶掺入错误增加，呈时间和浓度依赖关系。在人血浆中常规观察到的叶酸浓度(1-10 ng/ml)可引起DNA损伤，在叶酸缺乏条件下培养的淋巴细胞与对照细胞相比不能正常生长。然而，通过台盼蓝拒染法检测，所有淋巴细胞仍然活着。缺乏叶酸的细胞不能有效地修复过氧化氢引起的DNA氧化损伤。修复抑制作用在培养8d后达到最大。叶酸供应对淋巴细胞DNA中氧化嘧啶的水平没有影响，即使在培养10d后也是如此，这表明叶酸缺乏相对特异性地增加了尿嘧啶的误掺入。这些体外实验结果有助于确定叶酸维持脱氧核糖核酸稳定性的机制(S)。
Folic acid is essential for the synthesis and repair of DNA. We report the effects of folate depletion on DNA stability in normal human lymphocytes in vitro. DNA strand breakage, uracil misincorporation, oxidative DNA base damage, and DNA repair capability were determined using variants of the comet assay (single cell gel electrophoresis), Lymphocyte proliferation was measured as an indicator of normal replication. Lymphocytes isolated from human venous blood were stimulated to grow in either complete medium containing folic acid (1 ng/ml-2 mu g/ml) or medium deficient in folic acid for up to 10 days. Cells prepared for comet analysis were treated either with the bacterial DNA repair enzyme endonuclease III to determine the level of oxidized pyrimidines in lymphocyte DNA or with uracil DNA glycosylase, which detects misincorporated uracil, Cell number and viability were measured. Normal human lymphocyte DNA contained detectable amounts of misincorporated uracil (estimated as approximately 1000 per cell). DNA strand breakage and uracil misincorporation increased in a time- and concentration-dependent manner after lymphocytes were cultured with decreasing amounts: of folic acid. DNA damage was induced at folic acid concentrations routinely observed in plasma from the human population (1-10 ng/ml), Lymphocytes cultured under folate-deficient conditions failed to grow normally compared with control cells. However, all lymphocytes remained viable as measured by Trypan blue exclusion. Cells deprived of folate were unable to efficiently repair oxidative DNA damage induced by hydrogen peroxide. Inhibition of repair was maximal after 8 days in culture. Folate supply had no effect on the level of oxidized pyrimidines in lymphocyte DNA, even after 10 days in culture, suggesting that folate deficiency increases uracil misincorporation relatively specifically. These in vitro results: help to determine the mechanism(s) through which folic acid maintains DNA stability.