Novel NIR fluorescent probe for hypochlorite ion detection in biological systems.

This study presents the synthesis and application of a novel fluorescent probe, NR-ClO, for the detection of hypochlorite ion (ClO-) in biological systems. The probe was synthesized through a nucleophilic substitution reaction between Nile red and dimethylcarbamothioic chloride. The synthesized probe had high sensitivity and selectivity towards ClO-, with a detection limit of 75 nM and a linear range of 0.1-200 µM. The probe's efficacy was validated through in vitro studies using HepG2 cells and in vivo experiments using a mouse model of rheumatoid arthritis. The findings demonstrate that the NR-ClO probe is a promisingly reliable tool for real-time monitoring of ClO- in complex biological environments.

One-Pot Hydrothermal Synthesis of Carbon Dots as Fluorescent Probes for the Determination of Mercuric and Hypochlorite Ions.

Nitrogen and sulfur codoped carbon dots (NSCDs) were synthesized via a one-pot hydrothermal method, and citric acid, ethylenediamine, and methyl blue were used as precursors. The obtained NSCDs were spherical with an average size of 1.86 nm. The fluorescence emission spectra of the NSCDs were excitation independent and emitted blue fluorescence at 440 nm with an excitation wavelength at 350 nm. The quantum yield of the NSCDs was calculated to be 68.0%. The NSCDs could be constructed as fluorescent probes for highly selective and sensitive sensing mercuric (Hg2+) and hypochlorite (ClO-) ions. As the addition of Hg2+ or ClO- ions to the NSCDs, the fluorescence intensity was effectively quenched due to dynamic quenching. Under the optimal conditions, the linear response of the fluorescence intensity ranged from 0.7 µM to 15 µM with a detection limit of 0.54 µM and from 0.3 µM to 5.0 µM with a limit of detection of 0.29 µM for Hg2+ and ClO- ions, respectively. Finally, the proposed method was successfully used for quantifying Hg2+ and ClO- ions in spiked tap water samples.

High quantum yield aminophenylboronic acid-functionalized N-doped carbon dots for highly selective hypochlorite ion detection.

High quantum yield 3-aminophenylboronic acid-functionalized nitrogen-doped carbon dots (GAAP-CDs) were fabricated using a simple hydrothermal route and used as a sensing probe for toxic hypochlorite (ClO-). The as-synthesized GAAP-CDs showed absorption peaks at 252, 297, and 370 nm and an emission peak at 375 nm with an excitation wavelength of 310 nm. The quantum yield of GAAP-CDs reached 58.28%, with no noticeable fluorescence change observed under high ionic strength conditions and a three-month long-term test. GAAP-CDs-based ClO- sensing was carried out by UV-vis absorbance and fluorescence spectroscopy; the detection limit was as low as 0.77 µM (linear range of 0-100 µM), and 0.50 µM (linear range of 0.1-100 µM), respectively. In addition, the as-synthesized GAAP-CDs showed excellent selectivity towards ClO- ions in the presence of various interfering chemicals. The satisfactory results from the proposed method of ClO- detection in tap water and drinking water samples, suggesting promising application of GAAP-CDs for ClO- detection.

Radiation activates myeloperoxidase (MPO) to generate active chlorine species (ACS) via a dephosphorylation mechanism - inhibitory effect of LGM2605.

BACKGROUND: Radiation exposure of tissues is associated with inflammatory cell influx. Myeloperoxidase (MPO) is an enzyme expressed in granulocytes, such as neutrophils (PMN) and macrophages, responsible for active chlorine species (ACS) generation. The present study aimed to: 1) determine whether exposure to γ-irradiation induces MPO-dependent ACS generation in murine PMN; 2) elucidate the mechanism of radiation-induced ACS generation; and 3) evaluate the effect of the synthetic lignan LGM2605, known for ACS scavenging properties. METHODS: MPO-dependent ACS generation was determined by using hypochlorite-specific 3'-(p-aminophenyl) fluorescein (APF) and a highly potent MPO inhibitor, 4-aminobenzoic acid hydrazide (ABAH), and confirmed in PMN derived from MPO-/- mice. Radiation-induced MPO activation was determined by EPR spectroscopy and computational analysis identified tyrosine, serine, and threonine residues near MPO's active site. RESULTS: γ-radiation increased MPO-dependent ACS generation dose-dependently in human MPO and in wild-type murine PMN, but not in PMN from MPO-/- mice. LGM2605 decreased radiation-induced, MPO-dependent ACS. Protein tyrosine phosphatase (PTP) and protein serine/threonine phosphatase (PSTP) inhibitors decreased the radiation-induced increase in ACS. Peroxidase cycle results demonstrate that tyrosine phosphorylation blocks MPO Compound I formation by preventing catalysis on H2O2 in the active site of MPO. EPR data demonstrate that γ-radiation increased tyrosyl radical species formation in a dose-dependent manner. CONCLUSIONS: We demonstrate that γ-radiation induces MPO-dependent generation of ACS, which is dependent, at least in part, by protein tyrosine and Ser/Thr dephosphorylation and is reduced by LGM2605. This study identified for the first time a novel protein dephosphorylation-dependent mechanism of radiation-induced MPO activation.

A coumarin-based fluorescent probe for hypochlorite ion detection in environmental water samples and living cells.

In this study, we developed a new fluorescent probe (CMM) based on coumarin dye and malononitrile, for highly sensitive and selective detection of hypochlorite ion (ClO-). CMM showed a 45-fold fluorescence enhancement at 459 nm in the presence of ClO- and displayed an excellent selectivity over other competing species. The probe featured a fast response time (<15 s), which could be in favor of the real-time detection towards ClO-. Meanwhile, probe CMM could effectively monitor ClO- in physiological pH condition and the detection limit was estimated to be as low as 5.7 nM. Furthermore, its preeminent recognition properties made the successful application for monitoring ClO- in environmental water samples and labeling ClO- in living biological cells.

Synthetic secoisolariciresinol diglucoside (LGM2605) inhibits myeloperoxidase activity in inflammatory cells.

BACKGROUND: Myeloperoxidase (MPO) generates hypochlorous acid (HOCl) during inflammation and infection. We showed that secoisolariciresinol diglucoside (SDG) scavenges radiation-induced HOCl in physiological solutions. However, the action of SDG and its synthetic version, LGM2605, on MPO-catalyzed generation of HOCl is unknown. The present study evaluated the effect of LGM2605 on human MPO, and murine MPO from macrophages and neutrophils. METHODS: MPO activity was determined fluorometrically using hypochlorite-specific 3'-(p-aminophenyl) fluorescein (APF). The effect of LGM2605 on (a) the peroxidase cycle of MPO was determined using Amplex Red while the effect on (b) the chlorination cycle was determined using a taurine chloramine assay. Using electron paramagnetic resonance (EPR) spectroscopy we determined the effect of LGM2605 on the EPR signals of MPO. Finally, computational docking of SDG was used to identify energetically favorable docking poses to enzyme's active site. RESULTS: LGM2605 inhibited human and murine MPO activity. MPO inhibition was observed in the absence and presence of Cl-. EPR confirmed that LGM2605 suppressed the formation of Compound I, an oxoiron (IV) intermediate [Fe(IV)O] containing a porphyrin π-radical of MPO's catalytic cycle. Computational docking revealed that SDG can act as an inhibitor by binding to the enzyme's active site. CONCLUSIONS: We conclude that LGM2605 inhibits MPO activity by suppressing both the peroxidase and chlorination cycles. EPR analysis demonstrated that LGM2605 inhibits MPO by decreasing the formation of the highly oxidative Compound I. This study identifies a novel mechanism of LGM2605 action as an inhibitor of MPO and indicates that LGM2605 may be a promising attenuator of oxidant-dependent inflammatory tissue damage.

Designing antioxidant peptides based on the antioxidant properties of the amino acid side-chains.

Amino acids exert characteristic antioxidant activities depending on the properties of their side residues. The hydrophobic residues were effective against peroxyl radical, while acidic residues and their analogs were effective against peroxynitrite. Peptides containing tyrosine showed different activities against different reactive oxygen species (ROS) and/or reactive nitrogen species (RNS). The number and position of tyrosine did not affect the antioxidant activity against hypochlorite ion. Against the peroxyl radical, the number of tyrosine residues affected the antioxidant activity, while its position did not have a significant effect. The tyrosine position was an important factor for the antioxidant activity against peroxynitrite. The peptide GWWW showed higher antioxidant activity against peroxyl radical than tryptophan at concentrations below 25 µM, and high activity against peroxynitrite at 250 µM. Our results suggest that antioxidant peptides against a specific target ROS or RNS can be designed based on the characteristics of the amino acid side chains.

Gamma-irradiation produces active chlorine species (ACS) in physiological solutions: Secoisolariciresinol diglucoside (SDG) scavenges ACS - A novel mechanism of DNA radioprotection.

BACKGROUND: Secoisolariciresinol diglucoside (SDG), the main lignan in whole grain flaxseed, is a potent antioxidant and free radical scavenger with known radioprotective properties. However, the exact mechanism of SDG radioprotection is not well understood. The current study identified a novel mechanism of DNA radioprotection by SDG in physiological solutions by scavenging active chlorine species (ACS) and reducing chlorinated nucleobases. METHODS: The ACS scavenging activity of SDG was determined using two highly specific fluoroprobes: hypochlorite-specific 3'-(p-aminophenyl) fluorescein (APF) and hydroxyl radical-sensitive 3'-(p-hydroxyphenyl) fluorescein (HPF). Dopamine, an SDG structural analog, was used for proton (1)H NMR studies to trap primary ACS radicals. Taurine N-chlorination was determined to demonstrate radiation-induced generation of hypochlorite, a secondary ACS. DNA protection was assessed by determining the extent of DNA fragmentation and plasmid DNA relaxation following exposure to ClO(-) and radiation. Purine base chlorination by ClO(-) and γ-radiation was determined by using 2-aminopurine (2-AP), a fluorescent analog of 6-aminopurine. RESULTS: Chloride anions (Cl(-)) consumed >90% of hydroxyl radicals in physiological solutions produced by γ-radiation resulting in ACS formation, which was detected by (1)H NMR. Importantly, SDG scavenged hypochlorite- and γ-radiation-induced ACS. In addition, SDG blunted ACS-induced fragmentation of calf thymus DNA and plasmid DNA relaxation. SDG treatment before or after ACS exposure decreased the ClO(-) or γ-radiation-induced chlorination of 2-AP. Exposure to γ-radiation resulted in increased taurine chlorination, indicative of ClO(-) generation. NMR studies revealed formation of primary ACS radicals (chlorine atoms (Cl) and dichloro radical anions (Cl2¯)), which were trapped by SDG and its structural analog dopamine. CONCLUSION: We demonstrate that γ-radiation induces the generation of ACS in physiological solutions. SDG treatment scavenged ACS and prevented ACS-induced DNA damage and chlorination of 2-aminopurine. This study identified a novel and unique mechanism of SDG radioprotection, through ACS scavenging, and supports the potential usefulness of SDG as a radioprotector and mitigator for radiation exposure as part of cancer therapy or accidental exposure.