Toward selective detection of reactive oxygen and nitrogen species with the use of fluorogenic probes – Limitations, progress, and perspectives

International audienceOver the last 40 years, there has been tremendous progress in understanding the biological reactions ofreactive oxygen species (ROS) and reactive nitrogen species (RNS). It is widely accepted that the generationof ROS and RNS is involved in physiological and pathophysiological processes. To understand the role of ROSand RNS in a variety of pathologies, the specific detection of ROS and RNS is fundamental. Unfortunately,the intracellular detection and quantitation of ROS and RNS remains a challenge. In this short review, wehave focused on the mechanistic and quantitative aspects of their detection with the use of selectedfluorogenic probes. The challenges, limitations and perspectives of these methods are discussed

Oxidation of ethidium-based probes by biological radicals: mechanism, kinetics and implications for the detection of superoxide

International audienceHydroethidine (HE) and hydropropidine (HPr +) are fluorogenic probes used for the detection of the intra-and extracellular superoxide radical anion (O •− 2). In this study, we provide evidence that HE and HPr + react rapidly with the biologically relevant radicals, including the hydroxyl radical, peroxyl radicals, the trioxidocarbonate radical anion, nitrogen dioxide, and the glutathionyl radical, via one-electron oxidation, forming the corresponding radical cations. At physiological pH, the radical cations of the probes react rapidly with O •− 2 , leading to the specific 2-hydroxylated cationic products. We determined the rate constants of the reaction between O •− 2 and the radical cations of the probes. We also synthesized N-methylated analogs of HPr + and HE which were used in mechanistic studies. Methylation of the amine groups was not found to prevent the reaction between the radical cation of the probe and the superoxide, but it significantly increased the lifetime of the radical cation and had a substantial effect on the profiles of the oxidation products by inhibiting the formation of dimeric products. We conclude that the N-methylated analogs of HE and HPr + may be used as a scaffold for the design of a new generation of probes for intra-and extracellular superoxide

Characterization of fluorescein-based monoboronate probe and its application to the detection of peroxynitrite in endothelial cells treated with doxorubicin

Boronate probes have emerged recently as a versatile tool for the detection of reactive oxygen and nitrogen species. Here, we present the characterization of a fluorescein-based monoboronate probe, a 4-(pinacol boronate)benzyl derivative of fluorescein methyl ester (FBBE), that proved to be useful to detect peroxynitrite in cell culture experiments. The reactivity of FBBE toward peroxynitrite as well hypochlorite, hydrogen peroxide, and tyrosyl hydroperoxide was determined. Second-order rate constants of the reactions of FBBE with peroxynitrite, HOCl, and H2O2 at pH 7.4 were equal to (2.8 ± 0.2) × 105 M-1 s-1, (8.6 ± 0.5) × 103 M-1 s-1, and (0.96 ± 0.03) M-1 s-1, respectively. The presence of glutathione completely blocked the oxidation of the probe by HOCl and significantly inhibited its oxidation by H2O2 and tyrosyl hydroperoxide but not by peroxynitrite. The oxidative conversion of the probe was also studied in the systems generating singlet oxygen, superoxide radical anion, and nitric oxide in the presence and absence of glutathione. Spectroscopic characterization of FBBE and its oxidation product has been also performed. The differences in the reactivity pattern were supported by DFT quantum mechanical calculations. Finally, the FBBE probe was used to study the oxidative stress in endothelial cells (Ea.hy926) incubated with doxorubicin, a quinone anthracycline antibiotic. In endothelial cells pretreated with doxorubicin, FBBE was oxidized, and this effect was reversed by PEG-SOD and L-NAME but not by catalase