title: Characterization of Fluorescein-Based Monoboronate Probe and Its Application to the Detection of Peroxynitrite in Endothelial Cells Treated with Doxorubicin.
abstract: 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.
authors.affiliation: Institute of Applied Radiation Chemistry, Lodz University of Technology , Lodz, Poland.
authors.forename: Karolina
authors.initials: K
authors.lastname: Dębowska
authors.affiliation: Institute of Applied Radiation Chemistry, Lodz University of Technology , Lodz, Poland.
authors.forename: Dawid
authors.initials: D
authors.lastname: Dębski
authors.affiliation: Institute of Applied Radiation Chemistry, Lodz University of Technology , Lodz, Poland.
authors.forename: Bartosz
authors.initials: B
authors.lastname: Michałowski
authors.affiliation: Institute of Applied Radiation Chemistry, Lodz University of Technology , Lodz, Poland.
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Dybala-Defratyka
authors.affiliation: Jagiellonian Centre for Experimental Therapeutics (JCET), Jagiellonian University , Kraków, Poland.
authors.forename: Tomasz
authors.initials: T
authors.lastname: Wójcik
authors.affiliation: Institute of Applied Radiation Chemistry, Lodz University of Technology , Lodz, Poland.
authors.forename: Radosław
authors.initials: R
authors.lastname: Michalski
authors.affiliation: Institute of Applied Radiation Chemistry, Lodz University of Technology , Lodz, Poland.
authors.forename: Małgorzata
authors.initials: M
authors.lastname: Jakubowska
authors.affiliation: Jagiellonian Centre for Experimental Therapeutics (JCET), Jagiellonian University , Kraków, Poland.
authors.forename: Anna
authors.initials: A
authors.lastname: Selmi
authors.affiliation: Institute of Applied Radiation Chemistry, Lodz University of Technology , Lodz, Poland.
authors.forename: Renata
authors.initials: R
authors.lastname: Smulik
authors.affiliation: Institute of Applied Radiation Chemistry, Lodz University of Technology , Lodz, Poland.
authors.forename: Łukasz
authors.initials: Ł
authors.lastname: Piotrowski
authors.affiliation: Institute of Applied Radiation Chemistry, Lodz University of Technology , Lodz, Poland.
authors.forename: Jan
authors.initials: J
authors.lastname: Adamus
authors.affiliation: Institute of Applied Radiation Chemistry, Lodz University of Technology , Lodz, Poland.
authors.forename: Andrzej
authors.initials: A
authors.lastname: Marcinek
authors.affiliation: Jagiellonian Centre for Experimental Therapeutics (JCET), Jagiellonian University , Kraków, Poland.|Chair of Pharmacology, Jagiellonian University Medical College , Kraków, Poland.
authors.forename: Stefan
authors.initials: S
authors.lastname: Chlopicki
authors.affiliation: Institute of Applied Radiation Chemistry, Lodz University of Technology , Lodz, Poland.
authors.forename: Adam
authors.initials: A
authors.lastname: Sikora
mesh_terms: D001897:Boronic Acids / Q000737:chemistry*; D004317:Doxorubicin / Q000494:pharmacology*; D042783:Endothelial Cells / Q000187:drug effects* / Q000378:metabolism; D019793:Fluorescein / Q000737:chemistry*; D006801:Humans; D015335:Molecular Probes; D030421:Peroxynitrous Acid / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
chemical_list: D001897:Boronic Acids; D015335:Molecular Probes; D030421:Peroxynitrous Acid; D004317:Doxorubicin; D019793:Fluorescein