Cytotoxic and genotoxic effects of tert-butyl hydroperoxide on Chinese hamster B14 cells

The organic hydroperoxide, tert-butyl hydroperoxide (t-BHP), is a useful model compound to study mechanisms of oxidative cell injury. In the present work, we examined the features of the interactions of this oxidant with Chinese hamster B14 cells. The aim of our study was to reveal a possible role of structural modifications in membranes and loss of DNA integrity in t-BHP-induced cell injury and death. The tert-butyl hydroperoxide treatment (100-1000 microM, 37 degrees C for 1h) did not decrease cell viability (as measured by cell-specific functional activity with an MTT test), but completely prevented cell growth. We observed intracellular reduced glutathione (GSH) oxidation and total glutathione (GSH+GSSG) depletion, a slight increase in the level of lipid-peroxidation products, an enhancement of membrane fluidity, intracellular potassium leakage and a significant decrease of membrane potential. At oxidant concentrations of 100-1500 microM, a significant damage to DNA integrity was observed as revealed by the Comet assay. The inhibition of cell proliferation (cell-growth arrest) may be explained by genotoxicity of t-BHP, by disturbance of the cellular redox-equilibrium (GSH oxidation) and by structural membrane modifications, which result in ion-non-selective pore formation. The disturbance in passive membrane permeability and the DNA damage may be the most dramatic cell impairments induced by t-BHP treatment. The presence of another oxidant, hypochlorous acid (HOCl), completely prevented t-BHP-induced DNA strand breaks, perhaps due to extracellular oxidation of t-BHP by HOCl.     

Rat lung glutathione release: response to oxidative stress and selenium deficiency

We performed experiments to characterize the glutathione-dependent metabolism occurring during tert-butyl hydroperoxide infusion in isolated perfused rat lungs and to examine the effect of selenium deficiency on this metabolism. Selenium deficiency resulted in decreased lung glutathione peroxidase activity but normal glutathione reductase activity and glutathione content. Infusion of the hydroperoxide into control lungs caused a proportional increase in tissue glutathione disulfide (GSSG) concentration and release of GSSG into the perfusate up to an infusion rate of 250 nmol of tert-butyl hydroperoxide X min-1 X 100 g body wt-1. Infusion rates greater than this resulted in continued rise of tissue GSSG concentrations but GSSG release into the perfusate plateaued. Infusion of tert-butyl hydroperoxide into selenium-deficient rat lungs resulted in much lower concentrations of tissue GSSG and GSSG release into the perfusate; however, release in the selenium-deficient rat lung was also found to be saturable at infusion rates of 450 nmol of tert-butyl hydroperoxide X min-1 X 100 g of body wt-1. Selenium deficiency in the rat decreases the rate of reduction of infused tert-butyl hydroperoxide by glutathione and may predispose the lung to free radical damage.     

Oxidation of active center cysteine of bovine 1-Cys peroxiredoxin to the cysteine sulfenic acid form by peroxide and peroxynitrite.

Peroxiredoxins are antioxidant enzymes whose peroxidase activity depends on a redox-sensitive cysteine residue at the active center. In this study we investigated properties of the active center cysteine of bovine 1-Cys peroxiredoxin using a recombinant protein (BRPrx). The only cysteine residue of the BRPrx molecule was oxidized rapidly by an equimolar peroxide or peroxynitrite to the cysteine sulfenic acid. Approximate rates of oxidation of BRPrx by different peroxides were estimated using selenium glutathione peroxidase as a competitor. Oxidation of the active center cysteine of BRPrx by H2O2 proceeded only several times slowly than that of the selenocysteine of glutathione peroxidase. The rate of oxidation varied depending on peroxides tested, with H2O2 being about 7 and 80 times faster than tert-butyl hydroperoxide and cumene hydroperoxide, respectively. Peroxynitrite oxidized BRPrx slower than H2O2 but faster than tert-butyl hydroperoxide. Further oxidation of the cysteine sulfenic acid of BRPrx to higher oxidation states proceeded slowly. Oxidized BRPrx was reduced by dithiothreitol, dihydrolipoic acid, and hydrogen sulfide, and demonstrated peroxidase activity (about 30 nmol/mg/min) with these reductants as electron donors. beta-Mercaptoethanol formed a mixed disulfide and did not support peroxidase activity. Oxidized BRPrx did not react with glutathione, cysteine, homocysteine, N-acetyl-cysteine, and mercaptosuccinic acid.     

GPX2, encoding a phospholipid hydroperoxide glutathione peroxidase homologue, codes for an atypical 2-Cys peroxiredoxin in Saccharomyces cerevisiae

We have previously reported that Saccharomyces cerevisiae has three glutathione peroxidase homologues (GPX1, GPX2, and GPX3) (Inoue, Y., Matsuda, T., Sugiyama, K., Izawa, S., and Kimura, A. (1999) J. Biol. Chem. 274, 27002-27009). Of these, the GPX2 gene product (Gpx2) shows the greatest similarity to phospholipid hydroperoxide glutathione peroxidase. Here we show that GPX2 encodes an atypical 2-Cys peroxiredoxin which uses thioredoxin as an electron donor. Gpx2 was essentially in a reduced form even in mutants defective in glutathione reductase or glutaredoxin under oxidative stressed conditions. On the other hand, Gpx2 was partially oxidized in a mutant defective in cytosolic thioredoxin (trx1Deltatrx2Delta) under non-stressed conditions and completely oxidized in tert-butyl hydroperoxide-treated cells of trx1Deltatrx2Delta and thioredoxin reductase-deficient mutant cells. Alanine scanning of cysteine residues of Gpx2 revealed that an intramolecular disulfide bond was formed between Cys37 and Cys83 in vivo. Gpx2 was purified to determine whether it functions as a peroxidase that uses thioredoxin as an electron donor in vitro. Gpx2 reduced H2O2 and tert-butyl hydroperoxide in the presence of thioredoxin, thioredoxin reductase, and NADPH (for H2O2, Km= 20 microm, kcat = 9.57 x 10(2) s(-1); for tert-butyl hydroperoxide, Km= 62.5 microm, kcat = 3.68 x 10(2) s(-1)); however, it showed remarkably less activity toward these peroxides in the presence of glutathione, glutathione reductase, and NADPH. The sensitivity of yeast cells to tert-butyl hydroperoxide was found to be exacerbated by the co-existence of Ca2+, a tendency that was most obvious in gpx2Delta cells. Although the redox state of Gpx2 was not affected by Ca2+, the Gpx2 level was markedly increased in the presence of both tert-butyl hydroperoxide and Ca2+. Gpx2 is likely to play an important role in the protection of cells from oxidative stress in the presence of Ca2+.     

Modulating effect of thiol-disulfide status on [14C]aminopyrine accumulation in the isolated parietal cell

Thiol-oxidizing agents were found to stimulate [14C] aminopyrine accumulation, a reliable index of acid secretory function of isolated canine parietal cells. Glutathione is the predominant intracellular free thiol; thus, its oxidation status largely determines the thiol-disulfide status of the cell by thiol-disulfide interchange reactions. Three agents which alter glutathione oxidation status by different mechanisms were applied to parietal cells in vitro to investigate whether enhanced formation of GSSG alters acid secretory function. The agents studied were diamide (which nonenzymatically oxidizes GSH to GSSG), tert-butyl hydroperoxide (an organic peroxide specifically reduced by glutathione peroxidase, thereby generating GSSG for GSH), and 1,3-bis(2-chloroethyl)-1-nitrosourea (an inhibitor of NADPH:GSSG reductase, which presumably allows the accumulation of GSSG). Each of these agents stimulated aminopyrine accumulation in a dose-dependent fashion. Simple depletion of GSH by diethyl maleate or 2-cyclohexene-1-one did not stimulate aminopyrine accumulation. Likewise, enhanced aminopyrine accumulation occurred at diamide concentrations which did not cause significant depletion of total cellular glutathione. The thiol-reducing agent, dithiothreitol, prevented enhanced aminopyrine accumulation by 1,3-bis(2-chloroethyl)-1-nitrosourea and tert-butyl hydroperoxide. These observations support the hypothesis that thiol-disulfide interchange reactions involving GSSG modulate the acid secretory function of the isolated parietal cell.     

Response of the antioxidant defense system to tert-butyl hydroperoxide and hydrogen peroxide in a human hepatoma cell line (HepG2)

The aim of this work was to investigate the response of the antioxidant defense system to two oxidative stressors, hydrogen peroxide and tert-butyl hydroperoxide, in HepG2 cells in culture. The parameters evaluated included enzyme activity and gene expression of superoxide dismutase, catalase, glutathione peroxidase, and activity of glutathione reductase. Besides, markers of the cell damage and oxidative stress evoked by the stressors such as cell viability, intracellular reactive oxygen species generation, malondialdehyde levels, and reduced glutathione concentration were evaluated. Both stressors, hydrogen peroxide and tert-butyl hydroperoxide, enhanced cell damage and reactive oxygen species generation at doses above 50 microM. The concentration of reduced glutathione decreased, and levels of malondialdehyde and activity of the antioxidant enzymes consistently increased only when HepG2 cells were treated with tert-butyl hydroperoxide but not when hydrogen peroxide was used. A slight increase in the gene expression of Cu/Zn superoxide dismutase and catalase with 500 microM tert-butyl hydroperoxide and of catalase with 200 microM hydrogen peroxide was observed. The response of the components of the antioxidant defense system evaluated in this study indicates that tert-butyl hydroperoxide evokes a consistent cellular stress in HepG2.     

Free radical involvement in the oxidative phenomena induced by tert-butyl hydroperoxide in erythrocytes

Free radical involvement in the oxidative events induced by tert-butyl hydroperoxide in erythrocytes has been demonstrated by the use of the electron spin resonance technique of spin trapping with the spin trap 5.5-dimethyl-1-pyrroline-N-oxide (DMPO). The reactions of tert-butyl hydroperoxide with haemoglobins and intact cell systems were studied. Oxyhaemoglobin-containing system showed exclusive production of the t-butyloxy radical spin adduct of DMPO (DMPO-OBut), indicating t-butyloxy radical production. Methaemoglobin-containing systems showed the production of an oxidised derivative of DMPO, 5,5-dimethyl-2-ketopyrrolidino-1-oxyl (DMPOX)-previously associated with the generation of highly oxidised haem-iron. Carbon monoxyhaemoglobin-containing systems show the production of both DMPO-OBut and DMPOX but markedly slower than in either of the other haemoglobin systems. Generally, free radical production in haemoglobin systems was faster than in intact cell systems, indicating a membrane transport rate-limiting step for the tert-butyl hydroperoxide-mediated effects. Data from the use of free radical scavengers to inhibit DMPO-OBut production was consistent with the known reactivities of the scavengers toward t-butyloxy radicals. These and previously reported results (Trotta, R. J., Sullivan, S. G. and Stern, A. (1981) Biochim. Biophys. Acta 679, 230-237 and (1982) Biochem. J. 204, 405-415) implicate important roles for t-butyloxy radicals and haem intermediates in tert-butyl hydroperoxide-induced lipid peroxidation and haemoglobin oxidation in erythrocytes, respectively.     

Pro-oxidative activity of nitroxides in their reactions with glutathione

Nitroxides are unreactive towards glutathione in vitro. Interaction of nitroxides with peroxynitrite does not lead to a significant loss of their electron paramagnetic resonance (EPR) signal. However, addition of peroxynitrite to a solution containing glutathione and nitroxides induces an irreversible disappearance of EPR signal of nitroxides and augmentation of glutathione oxidation which is a pro-oxidant effect of these compounds. Nitroxide loss leading to the formation of amine derivatives is initiated by products of glutathione oxidation by peroxynitrite. The pro-oxidant action of nitroxides at micromolar concentrations may be important in view of the proposed use of these compounds as antioxidants.     

Interaction of Oxoferrylmyoglobin and Dinitrosyl-Iron Complexes

It is shown that dinitrosyl-iron complexes (DNIC) with glutathione can reduce oxoferrylmyoglobin forming on interaction of tert-butyl hydroperoxide and metmyoglobin. A rapid decrease in the DNIC concentration was observed under the conditions of production of tert-butyl free radicals; however, destruction of DNIC in the presence of oxoferrylmyoglobin alone was negligible. It is demonstrated that DNIC reduces oxoferrylmyoglobin more than an order more efficiently than S-nitrosoglutathione and glutathione. DNIC also inhibits formation of the thiyl radicals of glutathione in a medium containing metmyoglobin and tert-butyl hydroperoxide. A mechanism of the antioxidant action of DNIC based on regeneration of the nitrosyl complexes from the products of their interaction with oxoferrylheme is proposed.     

Mechanism of cell death induction by nitroxide and hyperthermia

Heat stress and nitroxides induce reactive oxygen species (ROS) and proapoptotic effects. The underlying mechanisms remain largely elusive. Here we report that Tempo (2,2,6,6-tetramethylpiperidine-N-oxyl) is a potent thermosensitizer for promoting cell death in human leukemia U937 cells. Treatment with Tempo (10 mM, 37 degrees C/30 min) and hyperthermia (44 degrees C/30 min) induced 30 and 70-80% apoptosis, respectively, through Bax-mediated cytochrome c release and DEVDase activation. The Tempo/heat combination also caused Bax-mediated cytochrome c release, but switched heat-induced apoptosis to the particular pyknotic cell death, resulting in the irreparable inhibition of proliferation. Tempo and heat stress, but not the combination, caused an early transient elevation of H2O2/O2*- and a late induction of only O2*-, respectively. Mitochondrial Ca2+ overloads were indistinguishable after any treatment. Heat stress induced the pan-caspase inhibitor zVAD-fmk-suppressible low-Deltapsi (mitochondrial membrane potential) in 75% of cells as a result of DEVDase activation. In contrast, Tempo yielded low-Deltapsi by deprivation of the mitochondrial H+ gradient. The combined treatment induced 97% zVAD-resistant low-Deltapsi cells through irreversible mitochondrial dysfunction. Together, thus, Tempo or heat stress induced Bax-mediated mitochondrial apoptosis with the possible help of ROS or mitochondrial Ca2+, and Tempo when combined with hyperthermia acts a sensitizer by inducing irreparable pyknotic cell death through irreversible mitochondrial dysfunction.     

The oxidation of chiral alcohols catalyzed by catalase in organic solvents

The catalytic properties of bovine liver catalase have been investigated in organic solvents. In tetrahydrofuran, dioxane, and acetone (all containing 1% to 3% of water), the enzyme breaks down tert-butyl hydroperoxide several fold faster than in pure water. Furthermore, the rate of catalase-catalyzed production of tert-butanol from tert-butyl hydroperoxide increases more than 400-fold upon transition from aqueous buffer to ethanol as the reaction medium. The mechanistic rationale for this striking effect is that in aqueous buffer the rate-limiting step of the enzymatic process involves the reduction of catalase's compound I by tert-butyl hydroperoxide. In ethanol, and additional step in the reaction scheme becomes available in which ethanol, greatly outcompeting the hydroperoxide, is oxidized by compound I regenerating the free enzyme. In solvents, such as acetonitrile or tetrahydrofuran, which themselves are not oxidizable by compound I, catalase catalyzes the oxidation of numerous primary and secondary alcohols with tert-butyl hydroperoxide to the corresponding aldehydes or ketones. The enzymatic oxidation of some chiral alcohols (2,3-butanediol, citronellol, and menthol) under these conditions occurs enantioselectively. Examination of the enantioselectivity for the oxidation of 2,3-butanediol in a series of organic solvents reveals a considerable solvent dependence. (c) 1995 John Wiley & Sons, Inc.     

Ascorbate- and dehydroascorbic acid-mediated reduction of free radicals in the human erythrocyte

Nitroxides were used as models of persistent free radicals to study the antioxidant function of ascorbic acid in the human erythrocyte. It was concluded that: 1) ascorbate and other reductant(s) derived from dehydroascorbic acid (DHA) in the presence of thiols are the only significant reducing agents for nitroxides, 2) glutathione and DHA reduce nitroxides by a process that cannot be inhibited by ascorbic acid oxidase, 3) erythrocytes can be depleted of ascorbic acid by exhaustive washing in the presence of membrane-permeable cationic nitroxides such as N,N-dimethylamino-Tempo, 4) ascorbate-depleted cells do not reduce nitroxides; however, nitroxide reduction is restored when the cells are incubated with DHA, 5) reduction of nitroxides in ascorbate-depleted, DHA-treated cells is significantly faster than in buffered solutions of DHA and glutathione, 6) several equivalents of nitroxide are reduced relative to the intracellular ascorbate pool, 7) sustained nitroxide reduction is observed even when most of the intracellular ascorbate is oxidized, 8) spin trapping of oxyradicals in tert-butyl hydroperoxide-treated cells is accelerated with ascorbate depletion and inhibited with ascorbate loading, 9) ascorbate can be quantified within intact cells by analyzing the initial reduction rates of membrane-permeable cationic nitroxides, and 10) DHA-stimulated reduction of cationic nitroxides is slower and less extensive in erythrocytes deficient in glucose-6-phosphate dehydrogenase than in normal erythrocytes.     

Contribution of peroxidized cardiolipin to inactivation of bovine heart cytochrome c oxidase

The lipid-soluble peroxides, tert-butyl hydroperoxide and peroxidized cardiolipin, each react with bovine cytochrome c oxidase and cause a loss of electron-transport activity. Coinciding with loss of activity is oxidation of Trp19 and Trp48 within subunits VIIc and IV, and partial dissociation of subunits VIa and VIIa. tert-Butyl hydroperoxide initiates these structural and functional changes of cytochrome c oxidase by three mechanisms: (1) radical generation at the binuclear center; (2) direct oxidation of Trp19 and Trp48; and (3) peroxidation of bound cardiolipin. All three mechanisms contribute to inactivation since blocking a single mechanism only partially prevents oxidative damage. The first mechanism is similar to that described for hydrogen peroxide [Biochemistry43:1003-1009; 2004], while the second and third mechanism are unique to organic hydroperoxides. Peroxidized cardiolipin inactivates cytochrome c oxidase in the absence of tert-butyl hydroperoxide and oxidizes the same tryptophans within the nuclear-encoded subunits. Peroxidized cardiolipin also inactivates cardiolipin-free cytochrome c oxidase rather than restoring full activity. Cardiolipin-free cytochrome c oxidase, although it does not contain cardiolipin, is still inactivated by tert-butyl hydroperoxide, indicating that the other oxidation products contribute to the inactivation of cytochrome c oxidase. We conclude that both peroxidized cardiolipin and tert-butyl hydroperoxide react with and triggers a cascade of structural alterations within cytochrome c oxidase. The summation of these events leads to cytochrome c oxidase inactivation.     

Different effectiveness of piperidine nitroxides against oxidative stress induced by doxorubicin and hydrogen peroxide

The piperidine nitroxides Tempamine and Tempace have been studied for their effect on doxorubicin (DOX) and hydrogen peroxide (H₂O₂) cytotoxicity in immortalized B14 cells, a model for neoplastic phenotype. The significance for nitroxide performance of the substituent in position 4 of the piperidine ring was evaluated. The cells were exposed to DOX/H₂O₂ alone or in combination with the nitroxides Tempamine or Tempace. Two other piperidine nitroxides, Tempo and Tempol, were used for comparison. All the nitroxides except Tempamine modestly reduced DOX cytotoxicity. Tempamine evoked a biphasic response: at concentrations lower than 200 μmol/L the nitroxide decreased DOX cytotoxicity, while at concentrations higher than 200 μmol/L, it enhanced DOX cytotoxicity. In contrast to Tempo and Tempol, Tempamine and Tempace ameliorated hydrogen peroxide cytotoxicity, but none of the nitroxides influenced TBARS stimulated by hydrogen peroxide. The cytoprotective effect of Tempace, Tempo and Tempol in DOX-treated cells correlated with the inhibition of DOX-induced lipid peroxidation. The bioreduction rates of the investigated nitroxides differed significantly and were variously affected by DOX depending on the nitroxide substituent. In combination with DOX, Tempo and Tempol were reduced significantly more slowly, while no influence of DOX on Tempamine and Tempace bioreduction was observed. Our results suggest that the structure of the 4-position substituent is an important factor for biological activity of piperidine nitroxides. Among the investigated nitroxides, Tempace displayed the best protective properties in vitro but Tempamine was the only nitroxide that potentiated cytotoxicity of DOX and did not influence DOX-induced lipid peroxidation. However, this nitroxide showed different performance depending on its concentration and conditions of oxidative stress.     

The yeast glutaredoxins are active as glutathione peroxidases

The yeast Saccharomyces cerevisiae contains two glutaredoxins, encoded by GRX1 and GRX2, which are active as glutathione-dependent oxidoreductases. Our studies show that changes in the levels of glutaredoxins affect the resistance of yeast cells to oxidative stress induced by hydroperoxides. Elevating the gene dosage of GRX1 or GRX2 increases resistance to hydroperoxides including hydrogen peroxide, tert-butyl hydroperoxide and cumene hydroperoxide. The glutaredoxin-mediated resistance to hydroperoxides is dependent on the presence of an intact glutathione system, but does not require the activity of phospholipid hydroperoxide glutathione peroxidases (GPX1-3). Rather, the mechanism appears to be mediated via glutathione conjugation and removal from the cell because it is absent in strains lacking glutathione-S-transferases (GTT1, GTT2) or the GS-X pump (YCF1). We show that the yeast glutaredoxins can directly reduce hydroperoxides in a catalytic manner, using reducing power provided by NADPH, GSH, and glutathione reductase. With cumene hydroperoxide, high pressure liquid chromatography analysis confirmed the formation of the corresponding cumyl alcohol. We propose a model in which the glutathione peroxidase activity of glutaredoxins converts hydroperoxides to their corresponding alcohols; these can then be conjugated to GSH by glutathione-S-transferases and transported into the vacuole by Ycf1.     

A bis-cyclodextrin diselenide with glutathione peroxidase-like activity

A diselenide, 2,2'-diseleno-bis-beta-cyclodextrin (2-SeCD), was synthesized to imitate the antioxidant enzyme glutathione peroxidase (GPX). The GPX mimic accepts a variety of hydroperoxides as substrates. The GPX activities, reduction of H(2)O(2), tert-butyl hydroperoxide and cumenyl hydroperoxide by glutathione, are 7.4, 4.5 and 10.2 U/micromol, respectively. In contrast to ebselen (PZ51), the diselenide displays high GPX-like activity. The reduction of hydroperoxide by glutathione in the presence of a radical trap shows that the mimic catalyzes the reaction via a non-radical mechanism. A ping-pong mechanism was observed in the steady-state kinetic studies of the 2-SeCD-catalyzed reaction.     

The interaction of ferritin and myoglobin as inductors of lipid peroxidation. The role of reactive oxygen and nitrogen species

The effect of iron dinitrosyl complexes, S-nitrosoglutathione, and glutathione on free radical oxidation of rat heart mitochondria induced by tert-butyl hydroperoxide and metmyoglobin or their combination with ferritin was studied. It was shown that iron dinitrosyl complexes or the combination of S-nitrosoglutathione and glutathione inhibited most effectively the peroxidation of mitochondrial membranes. It was found that ferritin stimulated the prooxidant action of metmyoglobin. Using EPR spectroscopy, it was established that, in conditions of O2*- generation, the destruction of iron dinitrosyl complexes took place. Iron dinitrosyl complexes also inhibited the formation of thiyl radicals, which appeared during O2*- generation in the system containing glutathione and S-nitrosoglutathione. It is essential that the formation of iron dinitrosyl complexes in this reaction system took place with the involvement of ferritin. It was proposed that the prooxidant action of ferritin and myoglobin could be inverted to the antioxidant one.     

Stable Nitroxide Radicals Protect Lipid Acyl Chains From Radiation Damage

The present study focused on protective activity of two six-membered-ring nitroxide radicals, 2,2,6,6-tetramethylpiperidine-1-oxyl (Tempo) and 4-hydroxy-Tempo (Tempol), against radiation damage to acyl chain residues of egg phosphatidylcholine (EPC) of small unilamellar vesicles (SUV). SUV were -irradiated (10–12 kGy) under air at ambient temperature in the absence and presence of nitroxides. Acyl chain composition of the phospholipids before and after irradiation was determined by gas chromatography. Both Tempo and Tempol effectively and similarly protected the acyl chains of EPC SUV, including the highly sensitive polyunsaturated acyl chains, C20:4, C22:5, and C22:6. The conclusions of the study are: (a) The higher the degree of unsaturation in the acyl chain, the greater is the degradation caused by irradiation. (b) The fully saturated fatty acids palmitic acid (C16) and stearic acid (C18) showed no significant change in their levels. (c) Both Tempo and Tempol provided similar protection to acyl chain residues. (d) Nitroxides' lipid-bilayer/aqueous distribution is not validly represented by their n-octanol/saline partition coefficient. (e) The lipid-bilayer/aqueous partition coefficient of Tempo and Tempol cannot be correlated with their protective effect. (f) The nitroxides appear to protect via a catalytic mode. Unlike common antioxidants, such as -tocopherol, which are consumed under irradiation and are, therefore, less effective against high radiation dose, nitroxide radicals are restored and terminate radical chain reactions in a catalytic manner. Furthermore, nitroxides neither yield secondary radicals upon their reaction with radicals nor act as prooxidants. Not only are nitroxides self-replenished, but also their reduction products are effective antioxidants. Therefore, the use of nitroxides offers a powerful strategy to protect liposomes, membranes, and other lipid-based assemblies from radiation damage. © 1997 Elsevier Science Inc.     

In vivo thiyl free radical formation from hemoglobin following administration of hydroperoxides

Although free radical formation due to the reaction between red blood cells and organic hydroperoxides in vitro has been well documented, the analogous in vivo ESR spectroscopic evidence for free radical formation has yet to be reported. We successfully employed ESR to detect the formation of the 5,5-dimethyl-1-pyrroline-N-oxide (DMPO)/hemoglobin thiyl free radical adduct in the blood of rats dosed with DMPO and tert-butyl hydroperoxide, cumene hydroperoxide, ethyl hydrogen peroxide, 2-butanone hydroperoxide, 15(S)-hydroperoxy-5,8,11,13-eicosatetraenoic acid, or hydrogen peroxide. We found that pretreating the rats with either buthionine sulfoximine or diethylmaleate prior to dosing with tert-butyl hydroperoxide decreased the concentration of nonprotein thiols within the red blood cells and significantly enhanced the DMPO/hemoglobin thiyl radical adduct concentration. Finally, we found that pretreating rats with the glutathione reductase inhibitor 1,3-bis(2-chloroethyl)-1-nitrosourea prior to dosing with tert-butyl hydroperoxide enhanced the DMPO/hemoglobin thiyl radical adduct concentration and induced the greatest decrease in nonprotein thiol concentration within the red blood cells.     

Carmustine augments the effects of tert-butyl hydroperoxide on calcium signaling in cultured pulmonary artery endothelial cells

The effects of oxidant stress and inhibition of glutathione reductase on the bradykinin-stimulated changes in cytosolic free Ca2+ concentration ([Ca2+]i) of calf pulmonary artery endothelial cells were determined using the intracellular fluorescent probe, fura-2. Changes in [Ca2+]i upon stimulation with bradykinin were measured after incubation of cells with the chemical oxidant tert-butyl hydroperoxide (0.4 mM) for various times. After 60 min, bradykinin-stimulated Ca2+ influx was significantly decreased. With more prolonged incubations with the peroxide, bradykinin had little effect on cytosolic calcium concentration. Preincubation of cells with the glutathione reductase inhibitor, carmustine, led to elevated basal [Ca2+]i, yet the cells remained responsive to bradykinin. However, incubation of carmustine-treated cells with tert-butyl hydroperoxide for 30 min dramatically reduced both bradykinin-stimulated release of Ca2+ from internal stores and influx of Ca2+ from the extracellular space. These results suggest that inhibition of glutathione reductase alters cytosolic Ca2+ homeostasis and enhances the effects of oxidative stress on signal transduction in vascular endothelial cells.