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) × 10(5) M(-1) s(-1), (8.6 ± 0.5) × 10(3) 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.

Nitroxyl (HNO) reacts with molecular oxygen and forms peroxynitrite at physiological pH. Biological Implications.

Nitroxyl (HNO), the protonated one-electron reduction product of NO, remains an enigmatic reactive nitrogen species. Its chemical reactivity and biological activity are still not completely understood. HNO donors show biological effects different from NO donors. Although HNO reactivity with molecular oxygen is described in the literature, the product of this reaction has not yet been unambiguously identified. Here we report that the decomposition of HNO donors under aerobic conditions in aqueous solutions at physiological pH leads to the formation of peroxynitrite (ONOO(-)) as a major intermediate. We have specifically detected and quantified ONOO(-) with the aid of boronate probes, e.g. coumarin-7-boronic acid or 4-boronobenzyl derivative of fluorescein methyl ester. In addition to the major phenolic products, peroxynitrite-specific minor products of oxidation of boronate probes were detected under these conditions. Using the competition kinetics method and a set of HNO scavengers, the value of the second order rate constant of the HNO reaction with oxygen (k = 1.8 × 10(4) m(-1) s(-1)) was determined. The rate constant (k = 2 × 10(4) m(-1) s(-1)) was also determined using kinetic simulations. The kinetic parameters of the reactions of HNO with selected thiols, including cysteine, dithiothreitol, N-acetylcysteine, captopril, bovine and human serum albumins, and hydrogen sulfide, are reported. Biological and cardiovascular implications of nitroxyl reactions are discussed.

Reaction between peroxynitrite and triphenylphosphonium-substituted arylboronic acid isomers: identification of diagnostic marker products and biological implications.

Aromatic boronic acids react rapidly with peroxynitrite (ONOO(-)) to yield phenols as major products. This reaction was used to monitor ONOO(-) formation in cellular systems. Previously, we proposed that the reaction between ONOO(-) and arylboronates (PhB(OH)2) yields a phenolic product (major pathway) and a radical pair PhB(OH)2O(•-)···(•)NO2 (minor pathway). [Sikora, A. et al. (2011) Chem. Res. Toxicol. 24, 687-697]. In this study, we investigated the influence of a bulky triphenylphosphonium (TPP) group on the reaction between ONOO(-) and mitochondria-targeted arylboronate isomers (o-, m-, and p-MitoPhB(OH)2). Results from the electron paramagnetic resonance (EPR) spin-trapping experiments unequivocally showed the presence of a phenyl radical intermediate from meta and para isomers, and not from the ortho isomer. The yield of o-MitoPhNO2 formed from the reaction between o-MitoPhB(OH)2 and ONOO(-) was not diminished by phenyl radical scavengers, suggesting a rapid fragmentation of the o-MitoPhB(OH)2O(•-) radical anion with subsequent reaction of the resulting phenyl radical with (•)NO2 in the solvent cage. The DFT quantum mechanical calculations showed that the energy barrier for the dissociation of the o-MitoPhB(OH)2O(•-) radical anion is significantly lower than that of m-MitoPhB(OH)2O(•-) and p-MitoPhB(OH)2O(•-) radical anions. The nitrated product, o-MitoPhNO2, is not formed by the nitrogen dioxide radical generated by myeloperoxidase in the presence of the nitrite anion and hydrogen peroxide, indicating that this specific nitrated product may be used as a diagnostic marker product for ONOO(-). Incubation of o-MitoPhB(OH)2 with RAW 264.7 macrophages activated to produce ONOO(-) yielded the corresponding phenol o-MitoPhOH as well as the diagnostic nitrated product, o-MitoPhNO2. We conclude that the ortho isomer probe reported here is most suitable for specific detection of ONOO(-) in biological systems.

Construction Products between Testing Laboratory and Market Surveillance: Case study of Cementitious Ceramic Tile Adhesives.

This article presents the results of the interlaboratory comparison (ILC) study of the following four characteristics of ceramic tile adhesives (CTAs): initial tensile adhesion strength, tensile adhesion strength after heat ageing, tensile adhesion strength after immersion in water, and tensile adhesion strength after freeze-thaw cycles. The results showed that the objective of the ILC was achieved-the z-score analysis carried out following ISO 13528 allowed for classifying all results obtained by 23 laboratories out of 27 as satisfactory. The results of the remaining four laboratories were rated worse. Despite the achieved goal, the ILC notes high heterogeneity of the results in terms of failure patterns, as well as significant differences between the lowest and the highest values of tensile adhesion strength for various measurement conditions. The results of the ILC were discussed in terms of the possibility of including them in the risk analysis conducted by the manufacturer. The results of the ILC are also valuable information for market surveillance authorities, who, in the authors' opinion, should be more cautious about results on samples taken from the market. The ILC results for CTAs are also a valuable recommendation for a possible revision of EN 12004.

Mechanism of oxidative conversion of Amplex® Red to resorufin: Pulse radiolysis and enzymatic studies.

Amplex® Red (10-acetyl-3,7-dihydroxyphenoxazine) is a fluorogenic probe widely used to detect and quantify hydrogen peroxide in biological systems. Detection of hydrogen peroxide is based on peroxidase-catalyzed oxidation of Amplex® Red to resorufin. In this study we investigated the mechanism of one-electron oxidation of Amplex® Red and we present the spectroscopic characterization of transient species formed upon the oxidation. Oxidation process has been studied by a pulse radiolysis technique with one-electron oxidants (N3(•), CO3(•-),(•)NO2 and GS(•)). The rate constants for the Amplex® Red oxidation by N3(•) ((2)k=2.1·10(9)M(-1)s(-1), at pH=7.2) and CO3(•-) ((2)k=7.6·10(8)M(-1)s(-1), at pH=10.3) were determined. Two intermediates formed during the conversion of Amplex® Red into resorufin have been characterized. Based on the results obtained, the mechanism of transformation of Amplex® Red into resorufin, involving disproportionation of the Amplex® Red-derived radical species, has been proposed. The results indicate that peroxynitrite-derived radicals, but not peroxynitrite itself, are capable to oxidize Amplex® Red to resorufin. We also demonstrate that horseradish peroxidase can catalyze oxidation of Amplex® Red not only by hydrogen peroxide, but also by peroxynitrite, which needs to be considered when employing the probe for hydrogen peroxide detection.

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

Over the last 40 years, there has been tremendous progress in understanding the biological reactions of reactive oxygen species (ROS) and reactive nitrogen species (RNS). It is widely accepted that the generation of ROS and RNS is involved in physiological and pathophysiological processes. To understand the role of ROS and 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, we have focused on the mechanistic and quantitative aspects of their detection with the use of selected fluorogenic probes. The challenges, limitations and perspectives of these methods are discussed.