OXYGEN DEPENDENCE OF CELLULAR UPTAKE OF EF5 [2-(2-NITRO-1H-IMIDAZOL-1-YL)-N-(2,2,3,3,3-PENTAFLUOROPROPYL)ACETAMIDE] - ANALYSIS OF DRUG ADDUCTS BY FLUORESCENT-ANTIBODIES VS BOUND RADIOACTIVITY
OXYGEN DEPENDENCE OF CELLULAR UPTAKE OF EF5 [2-(2-NITRO-1H-IMIDAZOL-1-YL)-N-(2,2,3,3,3-PENTAFLUOROPROPYL)ACETAMIDE] - ANALYSIS OF DRUG ADDUCTS BY FLUORESCENT-ANTIBODIES VS BOUND RADIOACTIVITY
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DOI：
10.1038/bjc.1995.426
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发表时间：
1995-10-01
影响因子：
8.8
通讯作者：
LORD, EM
中科院分区：
文献类型：
--
作者：
KOCH, CJ;EVANS, SM;LORD, EM
目前的研究开始定量的氧依赖性的生物还原代谢诱导的结合EF 5，五氟化衍生物的2-硝基咪唑，依他硝唑。比较了两种不同的测定法：第一，放射性药物掺入细胞裂解物中，这提供了药物代谢或摄取的直接测量;第二，全细胞透化和固定后EF 5的细胞大分子加合物的单克隆抗体检测。将抗体（命名为ELK 3 -51的单个克隆）与荧光染料Cy 3缀合，通过荧光显微镜和流式细胞术测定荧光。对于测试的两种细胞系（V79中国仓鼠成纤维细胞和9 L大鼠神经胶质瘤），发现两种技术的结合的氧依赖性相同。使用抗体结合技术，荧光信号在实验之间是高度可重复的，耐光或化学漂白，并且在细胞或组织染色后随时间稳定。来自用EF 5在体内或体外处理的大鼠9 L肿瘤的细胞的流式细胞术分析显示荧光信号的分布，其在相对和绝对基础上与体外结果非常相容。我们的研究结果表明，免疫荧光技术提供了一个定量测定生物还原性药物加合物，因此可能能够测量的绝对氧浓度分布在细胞群和组织的利益。
The present studies were initiated to quantitate the oxygen dependence of bioreductive metabolism-induced binding of EF5, a pentafluorinated derivative of the 2-nitroimidazole, etanidazole. Two different assays were compared: first, radioactive drug incorporation into cell lysates, which provides a direct measure of drug metabolism or uptake; second, monoclonal antibody detection of cellular macromolecular adducts of EF5 after whole cell permeabilisation and fixing. The antibodies (a single clone designated ELK3-51) were conjugated with the fluorescent dye Cy3, with fluorescence determined by fluorescence microscopy and flow cytometry. For the two cell lines tested (V79 Chinese hamster fibroblasts and 9L rat glioma), the oxygen dependence of binding was found to be the same for the two techniques. Using the antibody binding technique, the fluorescence signal was highly reproducible between experiments, resistant to light or chemical bleaching and stable over time following cell or tissue staining. Flow cytometric analysis of cells from rat 9L tumours treated with EF5 in vivo or in vitro showed a distribution of fluorescent signal which was very compatible, on both a relative and absolute basis, with the in vitro results. Our results indicate that immunofluorescent techniques provide a quantitative assay for bioreductive drug adducts, and therefore may be able to measure the absolute oxygen concentration distribution in cell populations and tissues of interest.