Afri Michal, Frimer Aryeh A, Cohen Yael
The Department of Chemistry, Bar Ilan University, Ramat Gan 52900, Israel.
Chem Phys Lipids. 2004 Aug;131(1):123-33. doi: 10.1016/j.chemphyslip.2004.04.006.
2',7'-Dichlorodihydrofluorescein diacetate (DCFH-DA) is commonly used to detect the generation of reactive oxygen intermediates and for assessing the overall oxidative stress in toxicological phenomenon. It has been suggested that DCFH-DA crosses the cell membrane, subsequently undergoing deacetylation by intracellular esterases. The resulting 2',7'-dichlorodihydrofluorescein (DCFH) is proposed to react with intracellular hydrogen peroxide or other oxidizing ROS to give the fluorescent 2',7'-dichlorofluorescein (DCF). Using an NMR chemical shift-polarity correlation, we have determined that DCFH-DA and DCFH are located well within the lipid bilayer and certainly not at the interface. These results, therefore, put into serious question the proposed ability of DCFH to come in contact with the aqueous phase and thereby interact with aqueous intracellular ROS and components. However, H2O2 and superoxide can cross or at least penetrate the lipid bilayer and react with certain lipophilic substrates. This may well describe the mode of reaction of these and other ROS with DCFH.
2',7'-二氯二氢荧光素二乙酸酯（DCFH-DA）常用于检测活性氧中间体的产生，并用于评估毒理学现象中的整体氧化应激。有人提出，DCFH-DA可穿过细胞膜，随后被细胞内酯酶进行脱乙酰化。生成的2',7'-二氯二氢荧光素（DCFH）被认为会与细胞内的过氧化氢或其他氧化性活性氧反应，生成荧光性的2',7'-二氯荧光素（DCF）。通过核磁共振化学位移-极性相关性，我们已确定DCFH-DA和DCFH位于脂质双层内部，而非界面处。因此，这些结果严重质疑了DCFH与水相接触并进而与细胞内水性活性氧和成分相互作用的假设能力。然而，过氧化氢和超氧化物可以穿过或至少穿透脂质双层，并与某些亲脂性底物发生反应。这很可能描述了这些及其他活性氧与DCFH的反应模式。
