Formation of TEMPOL-hydroxylamine during reaction between TEMPOL and hydroxyl radical: HPLC/ECD study

Nitroxyl radicals are important antioxidants that have been used to protect animal tissues from oxidative damage. Their reaction with hydroxyl radical (^?OH) is generally accepted to be the mechanism of antioxidant function. However, the direct interaction of nitroxyl radicals with ^?OH does not always provide a satisfactory explanation in various pH, because the concentration of hydrogen ion may affect the generation of secondary ^?OH-derived radicals. In the present study, it was confirmed that the reaction between 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPOL) and ^?OH generated TEMPOL-hydroxylamine, 4-oxo-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPON) and TEMPON-hydroxylamine using HPLC coupled with electrochemical detection. In the absence of NADH, TEMPOL-H may be generated by the reaction with secondary ^?OH-derived radicals in acidic condition. In the presence of NADH, a large proportion of the non-paramagnetic products was TEMPOL-H. Finally, it was clarified that TEMPOL-H was generated during dopamine metabolism, which is believed to be one of the ^?OH sources in pathological processes such as Parkinson's disease.     

Biologically active oxidized lipids (phytoprostanes) in the plant diet and parenteral lipid nutrition

Both ⁷⁹Br and ⁸¹Br nuclei have very large magnetic moments and hence can give rise to large hyperfine splittings. However, for the title compound, dibromonitroso-p-benzene sulphonate (DBNBS), which is a widely used spin-trap, liquid-phase spectra of radical adducts are characterised by narrow EPR features with no bromine splitting, despite the expected delocalisation of spin-density onto bromine. I find that for such radicals in the solid-state the ¹⁴N parallel features are very broad, although the seven expected hyperfine features from the ⁷⁹Br and ⁸¹Br nuclei were not clearly defined. I use these results to offer an explanation for the complete absence of any hyperfine splitting from bromine in the isotropic spectra.     

Nitroxyl exacerbates ischemic cerebral injury and oxidative neurotoxicity

Nitroxyl (HNO) donor compounds function as potent vasorelaxants, improve myocardial contractility and reduce ischemia-reperfusion injury in the cardiovascular system. With respect to the nervous system, HNO donors have been shown to attenuate NMDA receptor activity and neuronal injury, suggesting that its production may be protective against cerebral ischemic damage. Hence, we studied the effect of the classical HNO-donor, Angeli's salt (AS), on a cerebral ischemia/reperfusion injury in a mouse model of experimental stroke and on related in vitro paradigms of neurotoxicity. I.p. injection of AS (40 μmol/kg) in mice prior to middle cerebral artery occlusion exacerbated cortical infarct size and worsened the persistent neurological deficit. AS not only decreased systolic blood pressure, but also induced systemic oxidative stress in vivo indicated by increased isoprostane levels in urine and serum. In vitro , neuronal damage induced by oxygen-glucose-deprivation of mature neuronal cultures was exacerbated by AS, although there was no direct effect on glutamate excitotoxicity. Finally, AS exacerbated oxidative glutamate toxicity – that is, cell death propagated via oxidative stress in immature neurons devoid of ionotropic glutamate receptors. Taken together, our data indicate that HNO might worsen cerebral ischemia-reperfusion injury by increasing oxidative stress and decreasing brain perfusion at concentrations shown to be cardioprotective in vivo .     

The nitric oxide–iron interplay in mammalian cells: Transport and storage of dinitrosyl iron complexes

Nitrogen monoxide (NO) is a vital effector and messenger molecule that plays roles in a variety of biological processes. Many of the functions of NO are mediated by its high affinity for iron (Fe) in the active centres of proteins. Indeed, NO possesses a rich coordination chemistry with this metal and the formation of dinitrosyl–dithiolato–Fe complexes (DNICs) is well known to occur intracellularly. In mammals, NO produced by activated macrophages acts as a cytotoxic effector against tumour cells by binding and releasing cancer cell Fe that is vital for proliferation. Glucose metabolism and the subsequent generation of glutathione (GSH) are critical for NO-mediated Fe efflux and this process occurs by active transport. Our previous studies showed that GSH is required for Fe mobilisation from tumour cells and we hypothesized it was effluxed with Fe as a dinitrosyl–diglutathionyl–Fe complex (DNDGIC). It is well known that Fe and GSH release from cells induces apoptosis, a crucial property for a cytotoxic effector like NO. Furthermore, NO-mediated Fe release is mediated from cells expressing the GSH transporter, multi-drug resistance protein 1 (MRP1). Interestingly, the glutathione-S-transferase (GST) enzymes act to bind DNDGICs with high affinity and some members of the GST family act as storage intermediates for these complexes. Since the GST enzymes and MRP1 form a coordinated system for removing toxic substances from cells, it is possible to hypothesize these molecules regulate NO levels by binding and transporting DNDGICs.     

Cysteine reactivity in Thermoanaerobacter brockii alcohol dehydrogenase.

The free cysteine residues in the extremely thermophilic Thermoanaerobacter brockii alcohol dehydrogenase (TBADH) were characterized using selective chemical modification with the stable nitroxyl biradical bis(1-oxy-2,2,5,5-tetramethyl-3-imidazoline-4-yl)disulfide, via a thiol-disulfide exchange reaction and with 2[14C]iodoacetic acid, via S-alkylation. The respective reactions were monitored by electron paramagenetic resonance (EPR) and by the incorporation of the radioactive label. In native TBADH, the rapid modification of one cysteine residue per subunit by the biradical and the concomitant loss of catalytic activity was reversed by DTT. NADP protected the enzyme from both modification and inactivation by the biradical. RPLC fingerprint analysis of reduced and S-carboxymethylated lysyl peptides from the radioactive alkylated enzyme identified Cys 203 as the readily modified residue. A second cysteine residue was rapidly modified with both modification reagents when the catalytic zinc was removed from the enzyme by o-phenanthroline. This cysteine residue, which could serve as a putative ligand to the active-site zinc atom, was identified as Cys 37 in RPLC. The EPR data suggested a distance of < or 10 A between Cys 37 and Cys 203. Although Cys 283 and Cys 295 were buried within the protein core and were not accessible for chemical modification, the two residues were oxidized to cystine when TBADH was heated at 75 degrees C, forming a disulfide bridge that was not present in the native enzyme, without affecting either enzymatic activity or thermal stability. The status of these cysteine residues was verified by site directed mutagenesis.     

DNA Single Strand Breaks by Aromatic Nitroso Compounds in the Presence of Thiols

Aromatic nitroso compounds, nitrosobenzene (NB), N, N-dimethyl-4-nitrosoaniline (DMNA) and 3,5-dibromo-4-nitrosobenzene sulfonate (DBNBS), caused DNA single strand breaks in the presence of thiol compounds. The strand breaking was inhibited completely by free radical scavenger ethanol. Electron spin resonance (ESR) studies showed that hydronitroxyl (or sulfur-substituted nitroxyl) radicals were generated in the early stage of the interactions. Formation of these radicals was not inhibited by ethanol, indicating that these radicals did not directly contribute to the strand breaking. The DNA strand breaking was inhibited partially by superoxide dismutase and catalase under the limited conditions, but not by removal of oxygen from or addition of metal chelators to the reaction mixture. By ESR-spin trapping technique using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), the DMPO-OH spin adduct was detected. Formation of the spin adduct was inhibited by superoxide dismutase and catalase. The hydronitroxyl (or the sulfur-substituted nitroxyl) radicals may reduce oxygen into active oxygen species and also transformed by themselves into other unidentified free radical species to cause the DNA strand breaks.     

An ESR contrast agent is transported to rat liver through organic anion transporter

Carboxy PROXYL is a useful extracellular paramagnetic contrast reagent in electron spin resonance (ESR) and magnetic resonance imaging (MRI). Active transfer of the probe was investigated using an in situ liver model in rats. Carboxy PROXYL, a nitroxyl spin probe, was perfused into in situ liver perfusion system from Wistar rats. Concentration of nitroxyl form of the spin probe in effluent increased gradually after introducing perfusate with the spin probe and reached a plateau. The disappearance of Carboxy PROXYL from the perfusate was 40%, which could not be explained with its partition coefficient. Administration of non-selective inhibitors of organic anion transporters, p-aminohippuric acid and penicillin G, inhibited competitively and in a dose dependent manner the transfer of Carboxy PROXYL into rat liver in situ, resulting in increases of Carboxy PROXYL in the effluent. The results demonstrate that there is an active transfer system of an ESR contrast reagent into in situ rat liver through organic anion transporters.     

Enhanced intraarticular free radical reactions in adjuvant arthritis rats

One of the reasons of rheumatoid arthritis (RA) development is widely recognized the relation of free radical reactions in tissue injuries. The aim of this study was to evaluate the location where in vivo free radical reactions was enhanced in adjuvant arthritis (AA) model rats using in vivo electron spin resonance (ESR)/nitroxyl spin probe technique. The signal decay after intravenous injection of spin probe was enhanced in AA than that in control and suppressed by the pre-treatment of dexamethasone (DXT). Interestingly, the decay in joint cavity occurred prior to paw swelling of AA and suppressed by a simultaneous injection of free radical scavengers, indicating that the enhancement of free radical reactions in joint cavity of AA rats. This technique would be useful tool to determine the location of the enhanced free radical reactions and evaluate the activity of antioxidant medicine with non-invasive real-time measurement.     

Triarylmethyl-based biradical as a superoxide probe

Abstract

Superoxide radical represents one of the most biologically relevant reactive oxygen species involved in numerous physiological and pathophysiological processes. Superoxide measurement through the decay of an electron paramagnetic resonance (EPR) signal of a triarylmethyl (TAM) radical possesses the advantage of a high selectivity and relatively high rate constant of TAM reaction with the superoxide. Hereby we report a straightforward synthesis and characterization of a TAM–TAM biradical showing a high reactivity with superoxide (second-order rate constant, (6.7?±?0.2)?×?10³ M^?1 s^?1) enabling the measurement of superoxide radical by following the increase of a sharp EPR signal associated with the formation of a TAM-quinone-methide monoradical product.     

Hydrophobic Nitroxyl Radicals Inhibit Linoleyl Alcohol Oxidation by 5-Lipoxygenase

The linoleyl alcohol oxidation catalyzed by potato tuber 5-lipoxygenase was found to be efficiently inhibited by stable nitroxyl radicals: 1-oxyl-2,2,6,6-tetramethylpiperidin-4-yl bicyclo[2,2,2]octane-1-carboxylate, 1-adamantylacetate, dodecanoate, and octadecanoate. The dependence of apparent IC₅₀ values on the rotational correlation times of 4-hydroxy-1-oxyl-2,2,6,6-tetramethylpiperidine and its derivatives in model micellar systems was analyzed. An inhibition mechanism was proposed; it involves the interaction of a hydrophobic nitroxyl radical with the intermediate radical enzyme–substrate complex.