The possibility of N-Nitrosation in the absence of nitrosating agents was studied on model solutions and film coated tablets containing metformin. N-nitrosodimethylamine (NDMA) and N-nitrosation precursors (dimethylamine and nitrites) were determined using previously published fully validated analytical methods. Alternative routes to N-nitrosation were found. Dimethylamine can undergo an oxidation to nitrite in the presence of strong oxidants (e.g., H2O2), as was observed during wastewater treatment in several published works. The resulting nitrite can consecutively act as a nitrosating agent. We proved that the described reaction indeed leads to N-nitrosation (NDMA formation in case of dimethylamine precursor) in model solutions made of dimethylamine and H2O2. An experiment was designed in order to prove those reactions take place in dosage forms. Film coated tablets present a highly heterogenous system with several solid phases and low water activity, which is in stark contrast to the liquid wastewater, where this reaction was originally studied. Despite that, the described reaction took place even in the tablets, but only to a small degree. The amount formed via this alternative route corresponds to less than 10 % of the total formed NDMA. The pH optimum of this alternative route lies in the alkaline range which was confirmed by the determined NDMA concentration in model solutions. The solid phase system (i.e., tablets) was found to behave differently. The addition of Na2CO3 into the tablets during manufacture resulted in tablets without NDMA (cNDMA < LOQ) even in batches spiked with both dimethylamine and H2O2. Thus, adjusting the pH of the solid dosage forms remains a sufficient measure of controlling N-nitrosamines in the product, even in product with limit amounts of oxidating agent (H2O2) and N-nitrosation precursor (dimethylamine).
- MeSH
- dimethylaminy MeSH
- dimethylnitrosamin MeSH
- dusitany * MeSH
- léčivé přípravky MeSH
- nitrosace MeSH
- peroxid vodíku * MeSH
- Publikační typ
- časopisecké články MeSH
The ultra-performance liquid chromatography (UPLC) method, which involves pre-column derivatization of nitrite with 2,3-diaminonaphthalene (DAN) to form 2,3-naphthotriazole (NAT), offers the advantages of easy sample preparation, simple derivatization, stable derivatives, rapid analysis, high sensitivity and specificity and lack of interferences for determining nitrite in pharmaceutical samples. Determination of NAT was performed on a an Acquity UPLC HSS T3 column using a gradient elution of 0.1% formic acid with acetonitrile at flow rate of 0.4 mL/min and temperature at 45 °C. The single-quadrupole mass detector was operated in the positive ion mode. Quadrupole mass analyser was employed in selected ion monitoring mode using a target ion at m/z = 170 as [M+H]+. The UPLC-MS method was validated as per International Council on Harmonization (ICH) guidelines in terms of linearity, limit of detection, limit of quantification, selectivity, accuracy, precision, intermediate precision and stability. The UPLC-MS method was demonstrated to be applicable for the determination of nitrite in various pharmaceutical samples. The proposed UPLC-MS method was used to study the effect of nitrite content in pharmaceutical products on the formation of N-nitrosamines. The high importance of nitrites in relation to the N-nitrosation reaction was discussed. As deduced from theory and justified by the presented results, reducing the nitrite concentration could definitely solve the N-nitrosamine contamination. Nitrites, unlike secondary and tertiary amines, are universal precursors to any N-nitrosamine, so this solution is easily transferable to any relevant pharmaceutical product.
S-nitrosation as a redox-based posttranslational modification of protein cysteine has emerged as an integral part of signaling pathways of nitric oxide across all types of organisms. Protein S-nitrosation status is controlled by two key mechanisms: by direct denitrosation performed by the thioredoxin/thioredoxin reductase system, and in an indirect way mediated by S-nitrosoglutathione reductase (GSNOR). GSNOR, which has been identified as a key component of S-nitrosothiols catabolism, catalyzes an irreversible decomposition of abundant intracellular S-nitrosothiol, S-nitrosoglutathione (GSNO) to oxidized glutathione using reduced NADH cofactor. In plants, GSNOR has been shown to play important roles in plant growth and development and plant responses to abiotic and biotic stress stimuli. In this chapter, optimized protocols of spectrophotometric measurement of GSNOR enzymatic activity and activity staining in native polyacrylamide gels in plant GSNOR are presented.
- MeSH
- aldehydoxidoreduktasy metabolismus MeSH
- barvení a značení metody MeSH
- enzymatické testy metody MeSH
- fluorescence MeSH
- NAD chemie MeSH
- nativní elektroforéza na polyakrylamidovém gelu MeSH
- nitrosace MeSH
- oxid dusnatý metabolismus MeSH
- průběh práce MeSH
- rostlinné extrakty izolace a purifikace metabolismus MeSH
- rostliny enzymologie MeSH
- S-nitrosoglutathion chemická syntéza chemie MeSH
- S-nitrosothioly metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
S-nitrosoglutathione reductase (GSNOR) exerts crucial roles in the homeostasis of nitric oxide (NO) and reactive nitrogen species (RNS) in plant cells through indirect control of S-nitrosation, an important protein post-translational modification in signaling pathways of NO. Using cultivated and wild tomato species, we studied GSNOR function in interactions of key enzymes of reactive oxygen species (ROS) metabolism with RNS mediated by protein S-nitrosation during tomato root growth and responses to salinity and cadmium. Application of a GSNOR inhibitor N6022 increased both NO and S-nitrosothiol levels and stimulated root growth in both genotypes. Moreover, N6022 treatment, as well as S-nitrosoglutathione (GSNO) application, caused intensive S-nitrosation of important enzymes of ROS metabolism, NADPH oxidase (NADPHox) and ascorbate peroxidase (APX). Under abiotic stress, activities of APX and NADPHox were modulated by S-nitrosation. Increased production of H2O2 and subsequent oxidative stress were observed in wild Solanumhabrochaites, together with increased GSNOR activity and reduced S-nitrosothiols. An opposite effect occurred in cultivated S. lycopersicum, where reduced GSNOR activity and intensive S-nitrosation resulted in reduced ROS levels by abiotic stress. These data suggest stress-triggered disruption of ROS homeostasis, mediated by modulation of RNS and S-nitrosation of NADPHox and APX, underlies tomato root growth inhibition by salinity and cadmium stress.
- MeSH
- aldehydoxidoreduktasy metabolismus MeSH
- askorbátperoxidasa metabolismus MeSH
- benzamidy chemie metabolismus farmakologie MeSH
- chlorid sodný farmakologie MeSH
- fyziologický stres MeSH
- kadmium toxicita MeSH
- kořeny rostlin účinky léků růst a vývoj metabolismus MeSH
- NADPH-oxidasy metabolismus MeSH
- nitrosace MeSH
- oxid dusnatý metabolismus MeSH
- oxidační stres účinky léků MeSH
- peroxid vodíku metabolismus MeSH
- pyrroly chemie metabolismus farmakologie MeSH
- reaktivní formy dusíku chemie metabolismus MeSH
- reaktivní formy kyslíku chemie metabolismus MeSH
- regulace genové exprese u rostlin účinky léků MeSH
- rostlinné proteiny metabolismus MeSH
- S-nitrosoglutathion farmakologie MeSH
- S-nitrosothioly metabolismus MeSH
- Solanum lycopersicum účinky léků růst a vývoj metabolismus MeSH
- Solanum růst a vývoj metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Nitric oxide (NO) is considered as a signalling molecule involved in a variety of important physiological and pathological processes in plant and animal systems. The major pathway of NO reactions in vivo represents S-nitrosation of thiols to form S-nitrosothiols. S-nitrosoglutathione reductase (GSNOR) is the key enzyme in the degradation pathway of S-nitrosoglutathione (GSNO), a low-molecular weight adduct of NO and glutathione. GSNOR indirectly regulates the level of protein S-nitrosothiol in the cells. This study was focused on the dynamic regulation of the activity of plant GSNORs through reversible S-nitrosation and/or oxidative modifications of target cysteine residues. Pre-incubation with NO/NO- donors or hydrogen peroxide resulted in a decreased reductase and dehydrogenase activity of all studied plant GSNORs. Incubation with thiol reducing agent completely reversed inhibitory effects of nitrosative modifications and partially also oxidative inhibition. In biotin-labelled samples, S-nitrosation of plant GSNORs was confirmed after immunodetection and using mass spectrometry S-nitrosation of conserved Cys271 was identified in tomato GSNOR. Negative regulation of constitutive GSNOR activity in vivo by nitrosative or oxidative modifications might present an important mechanism to control GSNO levels, a critical mediator of the downstream signalling effects of NO, as well as for formaldehyde detoxification in dehydrogenase reaction mode.
- MeSH
- aldehydoxidoreduktasy antagonisté a inhibitory chemie metabolismus MeSH
- cystein chemie metabolismus MeSH
- donory oxidu dusnatého farmakologie MeSH
- nitrosace MeSH
- oxid dusnatý metabolismus MeSH
- oxidace-redukce MeSH
- peroxid vodíku farmakologie MeSH
- posttranslační úpravy proteinů MeSH
- rekombinantní proteiny chemie genetika metabolismus MeSH
- rostlinné proteiny antagonisté a inhibitory chemie metabolismus MeSH
- S-nitrosoglutathion metabolismus MeSH
- S-nitrosothioly metabolismus MeSH
- signální transdukce MeSH
- Solanum lycopersicum genetika růst a vývoj metabolismus MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
This review deals with the mechanism of the formation of carcinogenic N-nitrosoamines in the production of amine oxides by the oxidation of tertiary amines with hydrogen peroxide. The crucial point is the nature of the nitrogen substrate and nitrosating species as they are not added to the process directly. As the nitrogen substrates, we consider particularly primary and secondary amines. Various structures of the nitrosating agents are discussed, for example nitrogen oxides from the ambient air, corrosion inhibitors in the hydrogen peroxide (e.g. NaNO2) or other side nitroso compounds.
Stress-activated plant mitogen-activated protein (MAP) kinase pathways play roles in growth adaptation to the environment by modulating cell division through cytoskeletal regulation, but the mechanisms are poorly understood. We performed protein interaction and phosphorylation experiments with cytoskeletal proteins, mass spectrometric identification of MPK6 complexes and immunofluorescence analyses of the microtubular cytoskeleton of mitotic cells using wild-type, mpk6-2 mutant and plants overexpressing the MAP kinase-inactivating phosphatase, AP2C3. We showed that MPK6 interacted with γ-tubulin and co-sedimented with plant microtubules polymerized in vitro. It was the active form of MAP kinase that was enriched with microtubules and followed similar dynamics to γ-tubulin, moving from poles to midzone during the anaphase-to-telophase transition. We found a novel substrate for MPK6, the microtubule plus end protein, EB1c. The mpk6-2 mutant was sensitive to 3-nitro-l-tyrosine (NO2 -Tyr) treatment with respect to mitotic abnormalities, and root cells overexpressing AP2C3 showed defects in chromosome segregation and spindle orientation. Our data suggest that the active form of MAP kinase interacts with γ-tubulin on specific subsets of mitotic microtubules during late mitosis. MPK6 phosphorylates EB1c, but not EB1a, and has a role in maintaining regular planes of cell division under stress conditions.
- MeSH
- anafáze účinky léků MeSH
- aparát dělícího vřeténka účinky léků metabolismus MeSH
- Arabidopsis cytologie účinky léků enzymologie MeSH
- butadieny farmakologie MeSH
- cytokineze účinky léků MeSH
- extracelulárním signálem regulované MAP kinasy metabolismus MeSH
- fosforylace účinky léků MeSH
- fyziologický stres * účinky léků MeSH
- kinetochory účinky léků metabolismus MeSH
- meristém cytologie účinky léků metabolismus MeSH
- mikrotubuly účinky léků metabolismus MeSH
- mitogenem aktivované proteinkinasy metabolismus MeSH
- nitrily farmakologie MeSH
- nitrosace účinky léků MeSH
- proliferace buněk účinky léků MeSH
- proteiny asociované s mikrotubuly metabolismus MeSH
- proteiny huseníčku metabolismus MeSH
- rostlinné buňky účinky léků metabolismus MeSH
- segregace chromozomů účinky léků MeSH
- telofáze účinky léků MeSH
- tubulin metabolismus MeSH
- tyrosin analogy a deriváty farmakologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
S-Nitrosation is a new type of protein posttranslational modification. S-nitrosothiols, which are stable and mobile reservoirs of nitric oxide, are considered convergence points of signalling pathways of reactive oxygen and nitrogen species. This review summarizes the current knowledge of the origin, properties and biological functions of both low-molecular-weight and protein S-nitrosothiols. The biological functions of the former nitrosothiols are derived from their ability to decompose yielding NO or to trans-nitrosate protein thiols. Altered levels of the S-nitrosothiols and S-nitrosation of proteins are associated with nitrosation stress, resulting in a series of pathological processes. Although considerable advances in the research on S-nitrosothiols and protein S-nitrosation have been recently achieved, understanding the exact role and functions of S-nitrosation in signalling pathways of reactive nitrogen species is still very limited.
- MeSH
- dusitany MeSH
- lidé MeSH
- morfoliny metabolismus škodlivé účinky toxicita MeSH
- nitrosace MeSH
- nitrososloučeniny metabolismus toxicita MeSH
- rozpouštědla metabolismus škodlivé účinky toxicita MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- přehledy MeSH
