2--7--dichlorodihydrofluorescein and dihydrorhodamine-123

Topics: Animals; Fluoresceins; Humans; Nitric Oxide; Nitrogen Compounds; Rhodamines

The aim of this study was to investigate the oxidation of two common fluorescent probes, dichlorodihydrofluorescein (DCFH2) and dihydrorhodamine (DHR), and their oxidized forms, dichlorofluorescein and rhodamine, by the radical products of peroxynitrite chemistry, *OH, NO2*, and CO3*-. At pH 8.0-8.2, rate constants for the interaction of carbonate radical with probes were estimated to be 2.6 x 10(8) x M(-1) s(-1) for DCFH2 and 6.7 x 10(8) M(-1) s(-1) for DHR. Nitrogen dioxide interacted more slowly than carbonate radical with these probes: the rate constant for the interaction between NO2* and DCFH2 was estimated as 1.3 x 10(7) M(-1) s(-1). Oxidation of DHR by nitrogen dioxide led to the production of rhodamine, but the kinetics of these reactions were complex. Hydroxyl radical interacted with both probes with rate constants close to the diffusion-controlled limit. We also found that oxidized forms of these fluorescent probes reacted rapidly with carbonate, nitrogen dioxide, and hydroxyl radicals. These data suggest that probe oxidation may often be in competition with reaction of the radicals with cellular antioxidants.

Topics: Antioxidants; Carbonates; Diffusion; Fluoresceins; Free Radicals; Hydrogen-Ion Concentration; Hydroxyl Radical; Kinetics; Models, Chemical; Nitrogen Dioxide; Oxygen; Rhodamines; Spectrometry, Fluorescence; Spectrophotometry; Time Factors

The oxidations of dichlorodihydrofluorescein and dihydrorhodamine by peroxynitrite are zero-order in the indicator between pH 3 and 10. The yield of the oxidized products, dichlorofluorescein and rhodamine, significantly increased at pH values>7, and the maximal molar yields were 0.47 +/- 0.04 mol rhodamine and 0.54 +/- 0.06 mol, dichlorofluorescein per mol peroxynitrite at pH 8.5. The increase in yield of oxidized products as a function of pH indicates that the peroxynitrite anion may form an adduct with the indicator, followed by protonation and oxidation of the indicator. Carbon dioxide decreased the yield of fluorescent products to about 5%, relative to peroxynitrite, and the rate of product formation is again zero-order in the indicator. Given this yield, it is proposed that nitrogen dioxide and trioxocarbonate (*1-) are the reactive species that oxidize the indicators.

Topics: Fluoresceins; Fluorescent Dyes; Hydrogen-Ion Concentration; Kinetics; Molecular Structure; Oxidation-Reduction; Peroxynitrous Acid; Rhodamines

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 DH

Topics: Fluoresceins; Indicators and Reagents; Nitrates; Nitrogen; Oxidation-Reduction; Reactive Oxygen Species; Rhodamines; Spectrophotometry, Ultraviolet

It is well established that the fluorescent probes dichlorodihydrofluorescein diacetate (H2DCF-DA) and dihydrorhodamine 123 (H2R123) can be used to detect the respiratory burst response of mature myeloid cells. We describe a simple, fast and quantitative assay for myeloid differentiation based on the oxidation of these probes, which can be performed from start to finish in 96-well dishes. A bis(acetoxymethyl) ester of H2DCF-DA, 5-(and-6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate (CODCF-DA) is also capable of detecting the respiratory burst, but is less suitable than H2DCF-DA or H2R123 in our system. The amount of fluorescence produced can be quantified using a calibration curve, and values can be normalised to cell numbers using the 3-(4,5-dimethylthiazol-2-yl)2,5-diphenylte-trazolium bromide (MTT) cell proliferation assay. Results are expressed as 'equivalents of soluble fluorescein' (ESF) produced per cell under the defined reaction conditions. The extent to which HL60 cells reduce MTT is unaffected by differentiation induced by retinoic acid or 1 alpha,25-dihydroxyvitamin D3, and normalisation of fluorescence values using the MTT assay appears to be valid for a wide range of myeloid cell lines and differentiation inducers or cytokines.

Topics: Calibration; Cell Differentiation; Fluoresceins; Fluorescent Dyes; Fluorometry; Granulocytes; HL-60 Cells; Humans; Monocytes; Oxidation-Reduction; Respiratory Burst; Rhodamines; Tetrazolium Salts; Thiazoles