N-甲基-N-亚硝基脲在哺乳动物细胞中诱导 3'-谷胱甘肽化 DNA 切割产物,Analytical Chemistry

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N-甲基-N-亚硝基脲在哺乳动物细胞中诱导 3'-谷胱甘肽化 DNA 切割产物
Analytical Chemistry ( IF 6.7 ) Pub Date : 2022-11-04 , DOI: 10.1021/acs.analchem.2c02003
Jiekai Yin ₁ , Kent S Gates _{2,

3} , Yinsheng Wang _{1,

4}

Affiliation

无嘌呤/无嘧啶（AP）位点，即无碱基位点，是最常引起的 DNA 损伤之一。自发或 DNA 糖基酶介导的 3'-磷酰基 β-消除可导致 AP 位点链断裂，产生高反应性、亲电子的 3'-磷酸-α,β-不饱和醛 (3'-PUA) 残余物。后者可以与生物小分子、DNA和蛋白质的胺基或硫醇基反应，产生各种受损的3'端产物。考虑到其细胞内浓度较高，谷胱甘肽 (GSH) 可能与 3'-PUA 结合产生 3-谷胱甘肽-2,3-二脱氧核糖 (GS-ddR)，这可能构成一个重要的、但以前未被识别的内源性病变。在这里，我们开发了一种液相色谱串联质谱方法，结合使用稳定同位素标记的内标，来定量培养的人类细胞基因组 DNA 中的 GS-ddR。我们的结果揭示了未处理细胞的 DNA 中存在 GS-ddR，并且在暴露于烷化剂N-甲基-N-亚硝基脲 (MNU) 后，其水平在细胞中增加。此外，抑制 AP 核酸内切酶 (APE1) 会导致 MNU 处理的细胞 DNA 中 GS-ddR 水平升高。我们在这里首次共同报道了细胞 DNA 中 GS-ddR 水平的存在、DNA 烷化剂诱导 GS-ddR 的作用，以及 APE1 在调节人类细胞中其水平的作用。

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N-Methyl-N-nitrosourea Induced 3′-Glutathionylated DNA-Cleavage Products in Mammalian Cells

Apurinic/apyrimidinic (AP) sites, that is, abasic sites, are among the most frequently induced DNA lesions. Spontaneous or DNA glycosylase-mediated β-elimination of the 3′-phosphoryl group can lead to strand cleavages at AP sites to yield a highly reactive, electrophilic 3′-phospho-α,β-unsaturated aldehyde (3′-PUA) remnant. The latter can react with amine or thiol groups of biological small molecules, DNA, and proteins to yield various damaged 3′-end products. Considering its high intracellular concentration, glutathione (GSH) may conjugate with 3′-PUA to yield 3-glutathionyl-2,3-dideoxyribose (GS-ddR), which may constitute a significant, yet previously unrecognized endogenous lesion. Here, we developed a liquid chromatography tandem mass spectroscopy method, in combination with the use of a stable isotope-labeled internal standard, to quantify GS-ddR in genomic DNA of cultured human cells. Our results revealed the presence of GS-ddR in the DNA of untreated cells, and its level was augmented in cells upon exposure to an alkylating agent, N-methyl-N-nitrosourea (MNU). In addition, inhibition of AP endonuclease (APE1) led to an elevated level of GS-ddR in the DNA of MNU-treated cells. Together, we reported here, for the first time, the presence of appreciable levels of GS-ddR in cellular DNA, the induction of GS-ddR by a DNA alkylating agent, and the role of APE1 in modulating its level in human cells.

更新日期：2022-11-04

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