Wang Zhican, Edirisinghe Praneeth, Sohn Johann, Qin Zhihui, Geacintov Nicholas E, Thatcher Gregory R J, Bolton Judy L
Department of Medicinal Chemistry and Pharmacognosy (M/C 781), College of Pharmacy, University of Illinois at Chicago, 833 South Wood Street, Chicago, Illinois 60612-7231, USA.
Chem Res Toxicol. 2009 Jun;22(6):1129-36. doi: 10.1021/tx900063g.
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 reactive o-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-(15)N(5)-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.
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 - 羟基马萘雌酮（4 - OHEN）是激素替代疗法制剂中马雌激素的主要儿茶酚代谢产物，可自动氧化为具有反应性的邻醌，随后导致DNA损伤。体外和体内均已报道稳定的立体异构环状4 - OHEN - DNA加合物的形成，但尚未确定细胞中DNA修复过程对其的清除情况。在用4 - OHEN处理培养细胞时，环状加合物产量低和重现性差阻碍了此类研究。这些问题部分归因于4 - OHEN在有氧水性介质中的不稳定性。我们在此表明，通过使用4 - 羟基马萘雌酮二乙酸酯作为一种新型的、可穿透细胞的4 - 羟基马萘雌酮前体，并结合开发用于检测人乳腺癌细胞中加合物的灵敏液相色谱 - 串联质谱法（LC - MS/MS），可以克服产量低和重现性差的问题。该方法包括分离细胞DNA，将DNA消化为脱氧核苷，然后在通过LC - MS/MS分析之前添加同位素标记的内标（4 - OHEN - （15）N（5） - dG加合物）。在MCF - 7细胞暴露于4 - 羟基马萘雌酮二乙酸酯后，观察到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，以确认加合物在测定条件下是稳定的。总之，这种新开发的LC - MS/MS方法能够检测和相对定量人乳腺癌细胞中的4 - OHEN - DNA加合物，该方法可适用于人样品中加合物的检测。
