Royall J A, Ischiropoulos H
Department of Pediatrics, University of Alabama, Birmingham 35233.
Arch Biochem Biophys. 1993 May;302(2):348-55. doi: 10.1006/abbi.1993.1222.
2',7'-Dichlorofluorescein and dihydrorhodamine 123 were evaluated as probes for detecting changes in intracellular H2O2 in cultured endothelial cells. Stable intracellular levels of these probes were established within 15 min of exposure to the probe in culture medium. With continued presence of the probe in the medium, intracellular levels were unchanged for 1 h. However, if medium without the probes was used after intracellular loading had occurred, there was a greater than 90% loss of intracellular dichlorofluorescin, dichlorofluorescein, and dihydrorhodamine 123 while intracellular rhodamine 123 decreased by only 15%. Exposure of endothelial cells to exogenous 100 microM H2O2 for 1 h increased intracellular rhodamine 123 by 83%, but there was a reproducible decrease of 53% in intracellular dichlorofluorescein. Exposure to 0.05 mM BCNU plus 10 mM aminotriazole for 2 h increased intracellular rhodamine 123 by 111%. In vitro studies of dihydrorhodamine 123 oxidation were similar to previous reports of dichlorofluorescin oxidation. Oxidation of dihydrorhodamine 123 does not occur with H2O2 alone, but is mediated by a variety of secondary H2O2-dependent intracellular reactions including H2O2-cytochrome c and H2O2-Fe2+. Our results suggest that detection of increased oxidation of these probes in endothelial cells is most useful as a marker of a change in general cellular oxidant production.
2',7'-二氯荧光素和二氢罗丹明123被评估为检测培养的内皮细胞内过氧化氢变化的探针。在培养基中暴露于探针15分钟内可建立这些探针稳定的细胞内水平。随着探针持续存在于培养基中，细胞内水平在1小时内保持不变。然而，如果在细胞内加载后使用不含探针的培养基，细胞内二氯荧光素、二氯荧光素和二氢罗丹明123损失超过90%，而细胞内罗丹明123仅下降15%。将内皮细胞暴露于外源性100微摩尔过氧化氢1小时，细胞内罗丹明123增加83%，但细胞内二氯荧光素可重复性下降53%。暴露于0.05毫摩尔卡莫司汀加10毫摩尔氨基三唑2小时，细胞内罗丹明123增加111%。二氢罗丹明123氧化的体外研究与先前关于二氯荧光素氧化的报道相似。二氢罗丹明123的氧化不会单独由过氧化氢发生，而是由多种依赖过氧化氢的细胞内次级反应介导，包括过氧化氢-细胞色素c和过氧化氢-Fe2+。我们的结果表明，检测这些探针在内皮细胞中氧化增加最有助于作为一般细胞氧化剂产生变化的标志物。
