2-ethoxycarbonyl-2-methyl-3,4-dihydro-2H-pyrrole-1-oxide: evaluation of the spin trapping properties

Olive, Gilles; Mercier, Anne; Moigne, F. Le; Rockenbauer, Antal; Tordo, Paul

doi:10.1016/s0891-5849(99)00254-3

Cited by 145 publications

(87 citation statements)

References 24 publications

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“…1E). The hyperfine splitting constants (Table 1) were identical to those of an EMPO hydroxyl radical adduct (Olive et al, 2000). As shown in Fig.…”

Section: Formation Of Empo Radical Adducts In Rat Redsupporting

confidence: 57%

“…EMPO was used instead of DMPO because the superoxide, hydroxyl, and thiyl radical adducts are more readily distinguished and because its superoxide radical adduct is more stable and does not spontaneously decay to a hydroxyl radical adduct signal (Olive et al, 2000;Zhang et al, 2000) as is observed with the DMPO-superoxide adduct (Thornalley, 1986;Makino et al, 1990;Hanna et al, 1992). We report that MAQ-NOH generated an EMPO-hydroxyl radical adduct (Fig.…”

Section: Discussionmentioning

confidence: 70%

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Primaquine-Induced Hemolytic Anemia: Formation of Free Radicals in Rat Erythrocytes Exposed to 6-Methoxy-8-hydroxylaminoquinoline

Bolchoz

Gelasco

Jollow³

et al. 2002

The Journal of Pharmacology and Experimental Therapeutics

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Primaquine is an important antimalarial drug that is often doselimited in therapy by the onset of hemolytic anemia. We have shown recently that an N-hydroxy metabolite of primaquine, 6-methoxy-8-hydroxylaminoquinoline (MAQ-NOH), is a directacting hemolytic agent in rat red cells and that the hemolytic activity of this metabolite is associated with GSH oxidation and oxidative damage to both membrane lipids and skeletal proteins. To determine whether the formation of free radicals may be involved in this process, rat red cells (40% suspensions) were incubated with hemolytic concentrations of MAQ-NOH (150 -750 M) and examined by EPR spectroscopy using 2-ethoxycarbonyl-2-methyl-3,4-dihydro-2H-pyrrole-1-oxide (EMPO) as a spin trap. Addition of MAQ-NOH to red cell suspensions containing 10 mM EMPO gave rise to an EPR spectrum with hyperfine constants consistent with those of an EMPO-hydroxyl radical adduct standard. Of interest, formation of EMPO-OH was constant for up to 20 min and dependent on the presence of erythrocytic GSH. Although no other radical adduct signals were detected in the cells by EPR, spectrophotometric analysis revealed the presence of ferrylhemoglobin, which indicates that hydrogen peroxide is generated under these experimental conditions. The data support the hypothesis that oxygen-derived and possibly other free radicals are involved in the mechanism underlying MAQ-NOH-induced hemolytic anemia.Hemolytic anemia and methemoglobinemia are well recognized dose-limiting side effects in the therapeutic use of arylamine drugs, such as primaquine and dapsone (Beutler, 1969). Because these compounds are not hemotoxic when incubated with red cells in vitro, it has long been appreciated that metabolites are responsible for the onset of the hemolytic response. In the cases of aniline (Harrison and Jollow, 1986), dapsone (Grossman and Jollow, 1988), and phenacetin (Jensen and Jollow, 1991), we have shown that the hemolytic metabolites are their N-hydroxy derivatives. Primaquine metabolism, on the other hand, is relatively more complex, and the metabolites that mediate the hemotoxic responses have not been identified. A variety of known and putative phenolic metabolites of primaquine are redox-active and therefore have the potential to mediate primaquine hemotoxicity (Strother et al., 1984;Baird et al., 1986;Fletcher et al., 1988;Agarwal et al., 1991); however, direct evidence for their hemolytic activity is lacking. We have recently explored an alternative hypothesis-that primaquine hemotoxicity is mediated by an N-hydroxylated metabolite, 6-methoxy-8-hydroxylaminoquinoline (MAQ-NOH).Although the mechanism underlying the damage and removal of red cells by hemolytic N-hydroxylamines remains unclear, oxidative stress has long been considered to play a prominent role in the process (for review, see Beutler, 1971). This concept is based on the well known association of hemotoxicity with oxidation of erythrocytic GSH (to glutathione disulfide and glutathione protein-mixed disulfides), with methemoglobin f...

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“…1E). The hyperfine splitting constants (Table 1) were identical to those of an EMPO hydroxyl radical adduct (Olive et al, 2000). As shown in Fig.…”

Section: Formation Of Empo Radical Adducts In Rat Redsupporting

confidence: 57%

Section: Discussionmentioning

confidence: 70%

Primaquine-Induced Hemolytic Anemia: Formation of Free Radicals in Rat Erythrocytes Exposed to 6-Methoxy-8-hydroxylaminoquinoline

Bolchoz

Gelasco

Jollow³

et al. 2002

The Journal of Pharmacology and Experimental Therapeutics

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“…For example, human neutrophils upon stimulation generates considerable flux of O 2 •-which can be easily detected using DMPO but caution should be made when dealing with other types of cells where radical production is significantly less robust (e.g., endothelial or epithelial cells). Several nitrones have been developed to increase the half-life of the of O 2 •-adduct such as the use of EMPO and BMPO with half-lives of 7-8 min 12,13 , and DEPMPO which gives the longest half-life of 14 min 21 . However, the rates of spin trapping with O 2 •-of these spin traps still remain slow compared to DMPO, and therefore still requires the use of high concentration of the spin traps.…”

Section: Discussionmentioning

confidence: 99%

Detection of Nitric Oxide and Superoxide Radical Anion by Electron Paramagnetic Resonance Spectroscopy from Cells using Spin Traps

Gopalakrishnan¹,

Nash²,

Murugesan³

et al. 2012

JoVE

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Reactive nitrogen/oxygen species (ROS/RNS) at low concentrations play an important role in regulating cell function, signaling, and immune response but in unregulated concentrations are detrimental to cell viability 1,2 . While living systems have evolved with endogenous and dietary antioxidant defense mechanisms to regulate ROS generation, ROS are produced continuously as natural by-products of normal metabolism of oxygen and can cause oxidative damage to biomolecules resulting in loss of protein function, DNA cleavage, or lipid peroxidation Nitric oxide (NO) synthase which comes in three isoforms, as inducible-, neuronal-and endothelial-NOS, or iNOS, nNOS or eNOS, respectively, catalyzes the conversion of L-arginine to L-citrulline, using NADPH to produce NO 6 . Here, we generated NO from endothelial cells. Under oxidative stress conditions, eNOS for example can switch from producing NO to O 2 •-in a process called uncoupling, which is believed to be caused by oxidation of heme 7 or the co-factor, tetrahydrobiopterin (BH 4 ) 8 .There are only few reliable methods for the detection of free radicals in biological systems but are limited by specificity and sensitivity. Spin trapping is commonly used for the identification of free radicals and involves the addition reaction of a radical to a spin trap forming a persistent spin adduct which can be detected by electron paramagnetic resonance (EPR) spectroscopy. The various radical adducts exhibit distinctive spectrum which can be used to identify the radicals being generated and can provide a wealth of information about the nature and kinetics of radical production Video LinkThe video component of this article can be found at

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“…Because CP-H is poorly transported across the cell membrane (9 -11, 17, 36), we used the more cell-permeable spin trap, EMPO, to investigate the identity of the intracellular ROS observed in the FLIPR fluorescence experiments. EMPO reacts with both O 2 Ϫ ⅐ and HO⅐ radicals and exhibits EPR spectral characteristics that distinguish the two radical adducts (35,40,42). Figure 7A shows an EPR spectrum that indicates the presence of EMPO spintrapped radical species in indoxyl sulfate-treated mesangial cells.…”

Section: Indoxyl Sulfate Alters Reducing Rates In Rat Renal Mesangialmentioning

confidence: 99%

Indoxyl sulfate induces complex redox alterations in mesangial cells

Gelasco¹,

Raymond²

2006

American Journal of Physiology-Renal Physiology

110

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Indoxyl sulfate is a protein metabolite that is concentrated in the serum of patients with chronic renal insufficiency. It also is a uremic toxin that has been implicated in the progression of chronic renal disease in rodent models. We have shown previously that mesangial cell redox status is related to activation of mitogen-activated protein kinases and cell proliferation, which are factors related to glomerular damage. We used three methods to examine the ability of indoxyl sulfate to alter mesangial cell redox as a possible mechanism for its toxicity. Indoxyl sulfate increases mesangial cell reduction rate in a concentration-dependent manner as demonstrated by redox microphysiometry. Alterations occurred at concentrations as low as 100 M, with more marked alterations occurring at higher concentrations associated with human renal failure. We demonstrated that indoxyl sulfate induces the production of intracellular reactive oxygen species (ROS) in mesangial cells (EC50 ϭ 550 M) by using the ROS-sensitive fluorescent dye CM-DCF. ROS generation was only partially (ϳ50%) inhibited by the NADPH oxidase inhibitor diphenylene iodinium at low (Յ300 M) indoxyl sulfate concentrations. Diphenylene iodinium was without effect at higher concentrations of indoxyl sulfate. We also used electron paramagnetic spin resonance spectroscopy with extracellular and intracellular spin traps to show that indoxyl sulfate increases extracellular SOD-sensitive O 2

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2-ethoxycarbonyl-2-methyl-3,4-dihydro-2H-pyrrole-1-oxide: evaluation of the spin trapping properties

Cited by 145 publications

References 24 publications

Primaquine-Induced Hemolytic Anemia: Formation of Free Radicals in Rat Erythrocytes Exposed to 6-Methoxy-8-hydroxylaminoquinoline

Primaquine-Induced Hemolytic Anemia: Formation of Free Radicals in Rat Erythrocytes Exposed to 6-Methoxy-8-hydroxylaminoquinoline

Detection of Nitric Oxide and Superoxide Radical Anion by Electron Paramagnetic Resonance Spectroscopy from Cells using Spin Traps

Indoxyl sulfate induces complex redox alterations in mesangial cells

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