Tobi S E, Paul N, McMillan T J
Department of Biological Sciences, IENS, Lancaster University, UK.
J Photochem Photobiol B. 2000 Sep;57(2-3):102-12. doi: 10.1016/s1011-1344(00)00084-1.
We have developed an assay to detect reactive oxygen species (ROS) generated by UVA radiation utilising chemical probes which become fluorescent upon oxidation. Using a human bladder carcinoma cell line (MGH-U1) and spontaneously immortalised keratinocytes (HaCaT), we have shown a UVA (narrow band 365+/-5 nm) dose-dependent increase in fluorescence by flow cytometry following loading of the cells with either dihydrorhodamine 123 (DHR) or 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA). The UVA response of both DHR and DCFH was enhanced by elevation of intracellular levels of the photosensitiser protoporphyrin IX by incubation for 2.5 h with 5-aminolaevulinic acid. Depletion of the antioxidant glutathione (GSH) using the inhibitor D,L-buthionine-sulphoximine (BSO), resulted in an increase in the UVA-induced fluorescence of DCF but not of rhodamine 123. Conversely, raising intracellular GSH levels with N-acetyl cysteine (NAC) had relatively little protective effect in terms of degree of induced fluorescence.
我们开发了一种检测方法，利用氧化后会发荧光的化学探针来检测紫外线A（UVA）辐射产生的活性氧（ROS）。使用人膀胱癌细胞系（MGH-U1）和自发永生化角质形成细胞（HaCaT），我们发现，在用二氢罗丹明123（DHR）或2',7'-二氯二氢荧光素二乙酸酯（DCFH-DA）加载细胞后，通过流式细胞术检测到UVA（窄带365±5 nm）剂量依赖性荧光增加。通过与5-氨基乙酰丙酸孵育2.5小时来提高光敏剂原卟啉IX的细胞内水平，增强了DHR和DCFH对UVA的反应。使用抑制剂D,L-丁硫氨酸-亚砜亚胺（BSO）消耗抗氧化剂谷胱甘肽（GSH），导致DCF的UVA诱导荧光增加，但罗丹明123的荧光未增加。相反，用N-乙酰半胱氨酸（NAC）提高细胞内GSH水平，在诱导荧光程度方面的保护作用相对较小。
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