Development of a liquid chromatography electrospray ionization tandem mass spectrometry method for analysis of stable 4-hydroxyequilenin-DNA adducts in human breast cancer cells.
Development of a liquid chromatography electrospray ionization tandem mass spectrometry method for analysis of stable 4-hydroxyequilenin-DNA adducts in human breast cancer cells.
复制标题
开发用于分析人乳腺癌细胞中稳定的 4-羟基马萘醌-DNA 加合物的液相色谱电喷雾电离串联质谱方法。
DOI：
10.1021/tx900063g
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发表时间：
2009
影响因子：
4.1
通讯作者：
Bolton,JudyL
中科院分区：
文献类型：
--
作者：
Wang,Zhican;Edirisinghe,Praneeth;Sohn,Johann;Qin,Zhihui;Geacintov,NicholasE;Thatcher,GregoryRJ;Bolton,JudyL
雌激素- DNA加合物是评估雌激素依赖性癌症风险和进展的潜在生物标志物。4-羟基喹啉（4-OHEN）是激素替代疗法配方中马雌激素的主要儿茶酚代谢物，它会自氧化成一种反应性醌，随后会导致DNA损伤。在体外和体内已经报道了稳定的立体异构体环4-OHEN - DNA加合物的形成，但它们在细胞内DNA修复过程中的去除尚未确定。当用4-OHEN处理培养细胞时，这种研究受到环加合物产量低和可重复性差的阻碍。这些问题部分归因于4-OHEN在好氧水介质中的不稳定性。我们在此表明，4-OHEN双乙酸酯作为一种新型的、细胞可渗透的4-OHEN前体，结合用于检测人乳腺癌细胞中加合物的灵敏LC-MS/MS方法，可以克服低产量和重复性。该方法包括分离细胞DNA，将DNA消化成脱氧核苷，然后在LC-MS/MS分析之前添加同位素标记的内标（4-OHEN−15N5-dG加合物）。暴露于4-OHEN二乙酸酯后，MCF-7细胞中加合物水平呈浓度依赖性增加。还研究了加合物的化学稳定性，以证实加合物在测定条件下是稳定的。综上所述，新建立的LC-MS/MS方法可以对人乳腺癌细胞中的4-OHEN−DNA加合物进行检测和相对定量，适用于人类样品中的加合物检测。
Estrogen−DNA adducts are potential biomarkers for assessing cancer risk and progression in estrogen-dependent cancer. 4-Hydroxyequilenin (4-OHEN), the major catechol metabolite of equine estrogens present in hormone replacement therapy formulations, autoxidizes to a reactiveo-quinone that subsequently causes DNA damage. The formation of stable stereoisomeric cyclic 4-OHEN−DNA adducts has been reported in vitro and in vivo, but their removal by DNA repair processes in cells has not been determined. Such studies have been hampered by low yields of cyclic adducts and poor reproducibility when treating cells in culture with 4-OHEN. These problems are attributed in part to the instability of 4-OHEN in aerobic, aqueous media. We show herein that low yields and reproducibility can be overcome by 4-OHEN diacetate as a novel, cell-permeable 4-OHEN precursor, in combination with a sensitive LC-MS/MS method developed for detecting adducts in human breast cancer cells. This method involves isolation of cellular DNA, DNA digestion to deoxynucleosides, followed by the addition of an isotope-labeled internal standard (4-OHEN−15N5-dG adduct) prior to analysis by LC-MS/MS. A concentration-dependent increase in adduct levels was observed in MCF-7 cells after exposure to 4-OHEN diacetate. The chemical stabilities of the adducts were also investigated to confirm that adducts were stable under assay conditions. In conclusion, this newly developed LC-MS/MS method allows detection and relative quantification of 4-OHEN−DNA adducts in human breast cancer cells, which could be adapted for adduct detection in human samples.