Crow J P
Department of Anesthesiology, University of Alabama at Birmingham, 35233, USA.
Nitric Oxide. 1997 Apr;1(2):145-57. doi: 10.1006/niox.1996.0113.
2,7-Dichlorodihydrofluorescein (DCDHF), commonly known as dichlorofluorescin, and dihydrorhodamine 123 (DHR) are often used to detect the production of reactive nitrogen and oxygen species in cells via oxidation to their respective fluorescent products. To determine which biological oxidants might be involved, DCDHF and DHR were exposed to a number of oxidants in vitro to determine which are capable of oxidizing these compounds. Formation of dichlorofluorescein (DCF) and rhodamine is typically monitored by measuring their intrinsic fluorescence, however, absorbance can also be utilized (epsilon500 nm = 59,500 and 78,800 M(-1) cm(-1) for DCF and rhodamine, respectively). Peroxynitrite (ONOO-) readily oxidized both compounds with an efficiency equal to 38% of added ONOO- for DCDHF and 44% for DHR. Addition of nitric oxide (NO) to a superoxide-generating system resulted in DCDHF and DHR oxidation which was inhibitable by superoxide dismutase (SOD). SIN-1-mediated oxidation of DCDHF and DHR was also SOD-inhibitable, suggesting that peroxynitrite is the primary oxidant formed from SIN-1 decomposition. Aerobic addition of NO resulted in DCDHF oxidation in a manner consistent with nitrogen dioxide (.NO2) formation. NO did not oxidize DHR and actually inhibited UV-light-induced DHR oxidation. Simultaneous addition of NO and ONOO- resulted in an apparent inhibition of indicator oxidation; however, subsequent addition of ONOO- alone 20 s later produced a higher than average amount of oxidized indicator. Addition of indicator after NO + ONOO- followed by subsequent ONOO- addition gave similar results, suggesting the formation of a relatively stable, oxidant-activated NO/ONOO- adduct. At pH 7.4, hypochlorous acid was 66% efficient at oxidizing DHR but only 9% with DCDHF. Neither H2O2 (1 mM) nor superoxide flux alone produced significant indicator oxidation. Oxidation of DCDHF by horseradish peroxidase (HRP) plus H2O2 was considerably less efficient than oxidation of DHR. At 20-fold higher concentrations, HRP alone oxidized DHR but the rate was much lower than when H2O2 was present. Catalase largely inhibited HRP-mediated oxidation of DHR but not DCDHF, suggesting a direct effect of the peroxidase on DCDHF. These results reveal that peroxynitrite, hypochlorous acid, and H2O2 plus peroxidase all oxidize DCDHF and DHR to varying degrees but that neither superoxide, H2O2 alone, nor physiological levels of nitric oxide are capable of indicator oxidation. Thus, DCDHF or DHR oxidation in any given cell type may involve more than one oxidant. In cell systems where nitric oxide production occurs, oxidation of either DCDHF or DHR is likely to include a peroxynitrite component. Identification of relevant oxidants will best be achieved with a combined experimental approach which exploits the differential reactivities of DCDHF and DHR and the judicious use of inhibitors and oxidant scavengers.
2,7 - 二氯二氢荧光素（DCDHF），通常称为二氯荧光素，以及二氢罗丹明123（DHR），常被用于通过氧化成各自的荧光产物来检测细胞中活性氮和氧物种的产生。为了确定可能涉及哪些生物氧化剂，将DCDHF和DHR在体外暴露于多种氧化剂，以确定哪些能够氧化这些化合物。二氯荧光素（DCF）和罗丹明的形成通常通过测量它们的固有荧光来监测，不过，吸光度也可被利用（DCF和罗丹明在500 nm处的摩尔吸光系数分别为59,500和78,800 M⁻¹ cm⁻¹）。过氧亚硝酸根（ONOO⁻）能轻易氧化这两种化合物，对于DCDHF，氧化效率相当于所加ONOO⁻的38%，对于DHR则为44%。向超氧化物生成系统中添加一氧化氮（NO）会导致DCDHF和DHR氧化，这可被超氧化物歧化酶（SOD）抑制。SIN - 1介导的DCDHF和DHR氧化也可被SOD抑制，表明过氧亚硝酸根是由SIN - 1分解形成的主要氧化剂。有氧条件下添加NO会导致DCDHF氧化，其方式与二氧化氮（·NO₂）的形成一致。NO不会氧化DHR，实际上还会抑制紫外线诱导的DHR氧化。同时添加NO和ONOO⁻会导致指示剂氧化明显受到抑制；然而，20秒后单独添加ONOO⁻会产生高于平均量的氧化指示剂。在NO + ONOO⁻之后添加指示剂，随后再添加ONOO⁻会得到类似结果，表明形成了一种相对稳定的、被氧化剂激活的NO/ONOO⁻加合物。在pH 7.4时，次氯酸氧化DHR的效率为66%，但氧化DCDHF的效率仅为9%。单独的过氧化氢（1 mM）或超氧化物通量都不会产生显著的指示剂氧化。辣根过氧化物酶（HRP）加过氧化氢对DCDHF的氧化效率远低于对DHR的氧化效率。在浓度高20倍时，单独的HRP能氧化DHR，但速率远低于有过氧化氢存在时。过氧化氢酶在很大程度上抑制了HRP介导的DHR氧化，但对DCDHF没有抑制作用，这表明过氧化物酶对DCDHF有直接作用。这些结果表明，过氧亚硝酸根、次氯酸以及过氧化氢加过氧化物酶都能不同程度地氧化DCDHF和DHR，但超氧化物、单独的过氧化氢以及生理水平的一氧化氮都不能氧化指示剂。因此，在任何给定的细胞类型中，DCDHF或DHR氧化可能涉及不止一种氧化剂。在发生一氧化氮产生的细胞系统中，DCDHF或DHR的氧化很可能包括过氧亚硝酸根成分。通过结合利用DCDHF和DHR的不同反应性以及明智地使用抑制剂和氧化剂清除剂的实验方法，将最有助于鉴定相关的氧化剂。
