Use of dihydrorhodamine 123 for detecting intracellular generation of peroxides upon UV irradiation in epidermal keratinocytes
Use of dihydrorhodamine 123 for detecting intracellular generation of peroxides upon UV irradiation in epidermal keratinocytes
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DOI：
10.1007/bf00373382
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发表时间：
2004
影响因子：
3
通讯作者：
H. Sakurada;H. Koizumi;A. Ohkawara;T. Ueda;N. Kamo
中科院分区：
文献类型：
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作者：
H. Sakurada;H. Koizumi;A. Ohkawara;T. Ueda;N. Kamo
来自太阳和人工紫外线源的紫外线（UV）辐射是一种环境危害，会在人体皮肤中产生炎症、色素沉着和致癌作用。近年来，超氧阴离子、羟基自由基、过氧化氢等过氧化物被认为是主要有害物质[7,11,14]。然而，一种直接检测紫外线照射后活角质形成细胞中过氧化氢的方法尚未开发。目前，过氧化物的检测方法有两种，一种是使用自旋捕获试剂的ESR光谱法[6,14]，另一种是使用荧光染料如二氯荧光素（DCF）的流式细胞术[1,3,8,10,20]。当这些方法应用于辐照研究时，我们遇到了严重的问题。所用的试剂会吸收紫外线，从而使结果的解释复杂化。在生理条件下测量过氧化物的生成是可取的，但自旋捕获试剂只能在细胞均浆后才能使用，并且一些染料如DCF在细胞中不稳定。最近，一种新的染料二氢hodamine 123 （DHR 123）被开发出来，并成功地应用于化学刺激下白细胞过氧化氢生成的研究[16,17]。在这里，我们扩展了这种染料在辐射研究中的应用，并首次报道了紫外线照射下完整表皮角质形成细胞中产生过氧化物。
Ultraviolet (UV) radiation from the sun and artificial UV sources is an environmental hazard that produces inflammation, pigmentation and carcinogenesis in human skin [15]. Recently, peroxides such as superoxide anion, hydroxyradical and hydrogen peroxide have been suggested as primary hazardous substances [7, 11, 14]. However, a direct method to detect peroxide in living keratinocytes after UV irradiation has not yet been developed. At present, peroxides are detected either by ESR spectroscopy using spin trapping reagents [6, 14], or by flow cytometry using fluorescent dyes such as dichlorofluorescein (DCF)[1, 3, 8, 10, 20]. When these methods were applied to irradiation studies, we encountered serious problems. The reagents used absorb UV, and so complicate the interpretation of the results. It is preferable to measure the generation of peroxides under physiological conditions, but spin trapping reagents can be used only after homogenizing the cells, and some dyes such as DCF are not stable in the cell. Recently a new dye, dihydrorhodamine 123 (DHR 123), has been developed, and applied successfully to the study of peroxide generation in leucocytes upon chemical stimulation [16, 17]. Here we extend the use of this dye for radiation studies, and report for the first time that peroxides are generated in intact epidermal keratinocytes upon UV irradiation.