Nitric oxide decreases the stability of DMPO spin adducts.

The effect nitric oxide (NO*) on the stability of 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) adducts has been investigated using EPR spectroscopy. We report that the DMPO/HO* adduct, generated by porcine pulmonary artery endothelial cells in the presence of H2O2 and DMPO, or by a Fenton system (Fe(II)+H2O2) is degraded in the presence of the NO*-donor, 2-(N,N-diethylamino)-diazenolate-2-oxide (DEANO) or by bolus addition of an aqueous solution of NO*. A similar effect of DEANO was observed on other DMPO adducts, such as DMPO/*CH3 and DMPO/*CH(CH3)OH, generated in cell-free systems. Measurements of the loss of DMPO/HO* in the presence of DEANO in aerated and oxygen-free buffers showed that in both of these settings the process obeys first-order kinetics and proceeds with similar efficacy. This indicates that direct interaction of the nitroxide with NO*, rather than with NO2* (formed from NO* and O2 in aerated media), is responsible for destruction of the spin adduct. These results suggest that the presence of NO* may substantially affect the quantitative determination of DMPO adducts. We also show that NO2* radicals, generated by a myeloperoxidase/H2O2/nitrite system, also degrade DMPO/HO*. Because DMPO is frequently used to study generation of superoxide and hydroxyl radicals in biological systems, these observations indicate that extra caution is required when studying generation of these species in the presence of NO* or NO2* radicals.     

Radiation-induced changes of structural and functional properties of human hemoglobin

Gel filtration and SDS-PAGE separation of hemoglobin (Hb) irradiated under argon or N2O show formation of covalent-aggregated Hb molecules. The production of covalent bonds is attributed mainly to the action of hydroxyl radicals, because addition of ethanol, a scavenger of these radicals, suppresses this reaction to a great extent. The oxidized heme iron forming metHb or hemichromes is found in all the separated fractions of irradiated Hb. It is also found that the radiation-modified Hb molecules exhibit a decrease of co-operative binding of oxygen.     

On the radiation chemistry of thymine in aqueous solution

The reactions of thymine in aqueous solution with radiation-induced radicals OH, H, and e-aq were studied under various conditions. Competition studies using scavengers of OH radicals (methanol, ethanol, iodide) or of e-aq and/or H atoms (N2O, H+, O2) led to the conclusion that OH and H radicals destroy the chromophoric group of thymine, but e-aq does not. A trace of O2 proved to be necessary to obtain maximal destruction. Removal of the last traces of O2 resulted in a decrease of the destruction yield, possibly through restitution reactions. It was found that (1) alcohol radicals destroy thymine, even in the presence of O2; (2) the rate constant, k(OH + thymine) = 4.3 X 10(9) M(-1) sec(-1) (from competition with iodide); and (3) k(H + thymine) = 8 X 10(8) M(-1) sec(-1) (from competition with O2 in acid solution).     

The influence of X-rays on human erythrocytes. Primary radicals

The effects on human erythrocytes of water-derived radicals generated by X-rays were studied under anaerobic conditions and in the presence of oxygen. Erythrocyte damage was estimated on the basis of the reduced GSH and MetHb content in the erythrocytes, the -SH group content in the membrane proteins and the amount of K(+)released from the erythrocytes. The results obtained show that the level of reduced GSH was the most sensitive indicator of erythrocyte damage by X-rays followed by the efflux of K(+). The processes of GSH oxidation took place most rapidly under air. At a dose of 100 Gy, the level of GSH fell to about 50%, whereas under argon and N(2)O to about 75% and 65%, respectively. A slight increase in the efflux of K(+)was observed in preparations irradiated under air. However, when erythrocytes were irradiated under argon and N(2)O, the loss of K(+)occurred at a dose 8-times higher. Changes in the remaining parameters occurred at considerably higher doses. On the basis of the results obtained one can say that oxygen is a factor increasing the toxicity of(.)OH radicals towards erythrocytes; however, e(-)(aq)present in the system can cause a decrease in damage to certain cellular components.     

Role of water and ethanol radicals in the radiation destruction of human deoxyhemoglobin

In this study destruction of human deoxyhemoglobin (deoxy-Hb) induced by the action of primary products of water radiolysis, mainly .OH, eaq-, H, and of secondary ethanol radicals was investigated. The extent of destruction was estimated on the basis of absorbance ratio A505/A563 after conversion of hemoglobin into methemoglobin (MetHb) and the changes in the parameters of hemoglobin oxygenation. Deoxy-Hb solutions were irradiated under the atmosphere of argon and N2O, in the absence and presence of ethanol. In each case the radiation-chemical yield for hemoglobin destruction Gd = sigma i fi gi was determined (where: fi--fractional efficiency of destruction by the individual radicals, gi-fraction of radicals reacting with deoxy-Hb). Results were elaborated statistically estimating, by the method of least squares, the parameters of a multidimensional regression function which for each experiment with deoxy-Hb conc. 5 mg ml-1 was of the form: Gd = 0.09 gOH + 0.018 geaq- -0.099 gH + 0.03 g R.Et. The destruction efficiencies f.OH and fR.Et were statistically significant while feaq- and fH were not.     

ESR studies on reaction of saccharide with the free radicals generated from the xanthine oxidase/hypoxanthine system containing iron

The free radicals generated from the iron containing system of xanthine oxidase and hypoxanthine (Fe-XO/HX) were directly detected by using spin trapping. It was found that not only superoxide anion (O(2)*-) and hydroxyl radical (OH*), but also alkyl or alkoxyl radicals (R*) were formed when saccharides such as glucose, fructose and sucrose were added into the Fe-XO/HX system. The generated amount of R* was dependent on the kind and concentration of saccharides added into the Fe-XO/HX system and no R* were detected in the absence of saccharides, indicating that there is an interaction between the saccharide molecules and the free radicals generated from the Fe-XO/HX system and saccharide molecules are essential for generating R* in the Fe-XO/HX system. It is expected that the toxicity of R* would be greater than of hydrophilic O(2)*- and OH* because they are liposoluble and their lives are longer and the active sites of biomolecules are closely related with lipophilic phase, thus they can damage cells more seriously than O(2)*- and OH*. The R* generated from the saccharide containing Fe-XO/HX can be effectively scavenged by selenium containing abzyme (Se-abzyme), indicating Se-abzyme is a promising antioxidant.     

Intra- and intermolecular oxidation of oxymyoglobin and oxyhemoglobin induced by hydroxyl and carbonate radicals

The mechanism of the reactions of myoglobin and hemoglobin with *OH and CO3*- in the presence of oxygen was studied using pulse and gamma-radiolysis. Unlike *NO2, which adds to the porphyrin iron, *OH and CO3*- form globin radicals. These secondary radicals oxidize the Fe(II) center through both intra- and intermolecular processes. The intermolecular pathway was further demonstrated when BSA radicals derived from *OH or CO3*- oxidized oxyhemoglobin and oxymyoglobin to their respective ferric states. The oxidation yields obtained by pulse radiolysis were lower compared to gamma-radiolysis, where the contribution of radical-radical reactions is negligible. Full oxidation yields by *OH-derived globin radicals could be achieved only at relatively high concentrations of the heme protein mainly via an intermolecular pathway. It is suggested that CO3*- reaction with the protein yields Tyr and/or Trp-derived phenoxyl radicals, which solely oxidize the porphyrin iron under gamma-radiolysis conditions. The *OH particularly adds to aromatic residues, which can undergo elimination of H2O forming the phenoxyl radical, and/or react rapidly with O2 yielding peroxyl radicals. The peroxyl radical can oxidize a neighboring porphyrin iron and/or give rise to superoxide, which neither oxidize nor reduce the porphyrin iron. The potential physiological implications of this chemistry are that hemoglobin and myoglobin, being present at relatively high concentrations, can detoxify highly oxidizing radicals yielding the respective ferric states, which are not toxic.     

Dithiothreitol induces the sacrificial antioxidant property of human serum albumin in a metal-catalyzed oxidation and gamma-irradiation system

The species *OH or H2O2 are produced by both metal-catalyzed oxidation (MCO) of reducing equivalents and gamma-irradiation. Intact or Cys-34-modified human serum albumin (HSA) was significantly degraded in the MCO system containing dithiothreitol (DTT) as electron donor, but as long as it lasted, HSA prohibited *OH or H2O2 from initiating molecular damage of DNA. However, in the GSH and ascorbate (nonthiol) MCO system, HSA was not sacrificially degraded, and indeed accelerated the formation of DNA strand breaks. In the y-irradiation system producing *OH from H2O, only DTT attenuated the generation of DNA strand breaks by HSA. It did not degrade more H2O2 in the presence of reduced GSH (thiol-linked peroxidase) than in its absence. Therefore it would seem that in an MCO system, the antioxidant activity of HSA depends on the effectiveness of reducing equivalents to induce exposure of a functional group scavenging the *OH or H2O2 species, by reduction of its disulfide-bonds. In the presence of DTT, disulfide bonds in HSA were quantitatively reduced to cysteinyl residues but not significantly reduced by ascorbate or GSH. In conclusion, the antioxidant activity of HSA in the D     

Allopurinol-insensitive oxygen radical formation by milk xanthine oxidase systems.

Oxygen radical generation in the xanthine- and NADH-oxygen reductase reactions by xanthine oxidase, was demonstrated using the ESR spin trap 5,5'-dimethyl-1- pyrroline-N-oxide. No xanthine-dependent oxygen radical formation was observed when allopurinol-treated xanthine oxidase was used. The significant superoxide generation in the NADH-oxygen reductase reaction by the enzyme was increased by the addition of menadione and adriamycin. The NADH-menadione and -adriamycin reductase activities of xanthine oxidase were assessed in terms of NADH oxidation. From Lineweaver-Burk plots, the Km and Vmax of xanthine oxidase were estimated to be respectively 51 microM and 5.5 s-1 for menadione and 12 microM and 0.4 s-1 for adriamycin. Allopurinol-inactivated xanthine oxidase generates superoxide and OH.radicals in the presence of NADH and menadione or adriamycin to the same extent as the native enzyme. Adriamycin radicals were observed when the reactions were carried out under an atmosphere of argon. The effects of superoxide dismutase and catalase revealed that OH.radicals were mainly generated through the direct reaction of H2O2 with semiquinoid forms of menadione and adriamycin.     

The interaction of alcohol radicals with human hemoglobin

Aqueous deoxyhemoglobin solutions (2 mg/ml) were gamma-irradiated by a⁶⁰Co source in the presence of methanol, ethanol, 1-butanol andt-butanol under N₂O or argon. The effects of the interaction of the particular alcohol radical species with hemoglobin were determined according to the detected spectral alterations in the visible range. The amounts of stable final products in the form of methemoglobin (MetHb) and the sum of hemichromes and cholehemichromes (Hemichr) were estimated in irradiated preparations. For preparations irradiated under N₂O, the radiation yield for MetHb formation was threefold lower in the presence of ethanol and 1-butanol [G(MetHb)=0.33] compared with preparations irradiated in the presence oft-butanol or without alcohol [G(MetHb)=1.00]. The yield of hemichromes and cholehemichromes in preparations irradiated under N2O increased in the order: ethanol (G=0.38), 1-butanol (G=0.52),t-butanol (G=0.59), and in the absence of alcohol (G=0.72). The high effectivity oft-butanol radicals for iron oxidation and Hb destruction is apparently due to their oxidative properties, compared with the other radicals. It was also shown that ethanol radicals reduce MetHb 10 times more effectively [G(Fe(II)) = 2.5] compared witht-butanol radicals [G(Fe(II)) = 0.24]. For samples irradiated under argon all the observed changes were similar, regardless of the presence of alcohols. This effect can be attributed to reconstruction reactions of Hb molecules in the presence of both oxidizing (OH ort-but·) and reducing agents (e _aq ^/– ). The following sequence of effectivities of water radiolysis products and secondary alcohol radicals for hemoglobin destruction has been identified: meth·, eth·1-but·e _aq ^/– t-but··OH.This work was supported by State Committee for Scientific Research (Poland), grant no. 44509203     

Reactions of reactive oxygen species (ROS) with curcumin analogues: Structure-activity relationship

Three curcumin analogues viz., bisdemethoxy curcumin, monodemethoxy curcumin, and dimethoxycurcumin that differ at the phenolic substitution were synthesized. These compounds have been subjected for free radical reactions with DPPH radicals, superoxide radicals (O(2)(?-)), singlet oxygen ((1)O(2)) and peroxyl radicals (CCl(3)O(2)(?)) and the bimolecular rate constants were determined. The DPPH radical reactions were followed by stopped-flow spectrometer, (1)O(2) reactions by transient luminescence spectrometer, and CCl(3)O(2)(?) reactions using pulse radiolysis technique. The rate constants indicate that the presence of o-methoxy phenolic OH increases its reactivity with DPPH and CCl(3)O(2)(?), while for molecules lacking phenolic OH, this reaction is very sluggish. Reaction of O(2)(?-) and (1)O(2) with curcumin analogues takes place preferably at β-diketone moiety. The studies thus suggested that both phenolic OH and the β-diketone moiety of curcumin are involved in neutralizing the free radicals and their relative scavenging ability depends on the nature of the free radicals.     

Polysaccharide degradation by Fenton reaction--or peroxidase-generated hydroxyl radicals in isolated plant cell walls

The formation of hydroxyl radicals (OH*) by peroxidase was confirmed by EPR spectroscopy using ethanol/alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone as a spin-trapping system specific of OH*. The effect of OH*, generated either non-enzymatically with the Fenton reaction (H(2)O(2) + Fe(2+)) or with horseradish peroxidase in the presence of O(2) and NADH, on cell walls isolated from maize (Zea mays) coleoptiles or soybean (Glycine max) hypocotyls was investigated. OH* produced by these reactions attack polysaccharides in the wall, demonstrated by the release of a heterogeneous mixture of polymeric breakdown products into the incubation medium. The peroxidase-catalyzed degradation of cell-wall polysaccharides can be inhibited by KCN and superoxide radical (O(2)*) or OH* scavengers. These data support the hypothesis that OH*, produced by cell-wall peroxidases in vivo, act as wall-loosening agents in plant extension growth.     

Decomposition of hydroxylamine by hemoglobin

The reaction between hydroxylamine (NH2OH) and human hemoglobin (Hb) at pH 6-8 and the reaction between NH2OH and methemoglobin (Hb+) chiefly at pH 7 were studied under anaerobic conditions at 25 degrees C. In presence of cyanide, which was used to trap Hb+, Hb was oxidized by NH2OH to methemoglobin cyanide with production of about 0.5 mol NH+4/mol of heme oxidized at pH 7. The conversion of Hb to Hb+ was first order in [Hb] (or nearly so) but the pseudo-first-order rate constant was not strictly proportional to [NH2OH]. Thus, the apparent second-order rate constant at pH 7 decreased from about 30 M-1 X s-1 to a limiting value of 11.3 M-1 X s-1 with increasing [NH2OH]. The rate of Hb oxidation was not much affected by cyanide, whereas there was no reaction between NH2OH and carbonmonoxyhemoglobin (HbCO). The pseudo-first-order rate constant for Hb oxidation at 500 microM NH2OH increased from about 0.008 s-1 at pH 6 to 0.02 s-1 at pH 8. The oxidation of Hb by NH2OH terminated prematurely at 75-90% completion at pH 7 and at 30-35% completion at pH 8. Data on the premature termination of reaction fit the titration curve for a group with pK = 7.5-7.7. NH2OH was decomposed by Hb+ to N2, NH+4, and a small amount of N2O in what appears to be a dismutation reaction. Nitrite and hydrazine were not detected, and N2 and NH+4 were produced in nearly equimolar amounts. The dismutation reaction was first order in [Hb+] and [NH2OH] only at low concentrations of reactants and was cleanly inhibited by cyanide. The spectrum of Hb+ remained unchanged during the reaction, except for the gradual formation of some choleglobin-like (green) pigment, whereas in the presence of CO, HbCO was formed. Kinetics are consistent with the view advanced previously by J. S. Colter and J. H. Quastel [1950) Arch. Biochem. 27, 368-389) that the decomposition of NH2OH proceeds by a mechanism involving a Hb/Hb+ cycle (reactions [1] and [2]) in which Hb is oxidized to Hb+ by NH2OH.     

Gamma radiolysis as a tool to study lipoprotein oxidation mechanisms

Well-defined quantities of *OH, O2*-,HO2* or RO2*)radicals (reactive oxygen species) can be specifically produced by radiolysis of water or ethanol. Such radical species can initiate one-electron oxidation or one-electron reduction reactions on numerous biological systems. The oxidative hypothesis of atherosclerosis classically admits the involvement of the oxidation of low density lipoproteins (LDLs) but also of high density lipoproteins (HDLs) in the development of the atherosclerotic process. The initiation mechanisms of this oxidation are still incompletely defined, although free radicals are likely involved. Therefore, gamma-radiolysis appears as a method of choice for the in vitro study of the mechanisms of oxidation of LDLs and HDLs by oxygen-centred free radicals (*OH, O2*-,HO2* and RO2*). Radiolytically oxidized lipoproteins exhibited a very well defined oxidation status (radiation dose-dependent quantification of vitamin E, beta-carotene, lipid peroxidation, protein carbonylation ...). gamma-Radiolysis is a less drastic method than other oxidation procedures such as for example copper ions. Moreover, gamma-radiolysis is also especially suitable for studying the reducing properties of antioxidant compounds with regard to their scavenging capacity.     

Copper-catalyzed protein oxidation and its modulation by carbon dioxide: enhancement of protein radicals in cells

It is well known that hydrogen peroxide (H2O2)-induced copper-catalyzed fragmentation of proteins follows a site-specific oxidative mechanism mediated by hydroxyl radical-like species (i.e. Cu(I)O, Cu(II)/*OH or Cu(III)) that ends in increased carbonyl formation and protein fragmentation. We have found that the nitrone spin trap DMPO (5,5-dimethyl-1-pyrroline N-oxide) prevented such processes by trapping human serum albumin (HSA)-centered radicals, in situ and in real time, before they reacted with oxygen. When (bi)carbonate (CO2, H2CO3, HCO3- and CO3(-2)) was added to the reaction mixture, it blocked fragmentation mediated by hydroxyl radical-like species but enhanced DMPO-trappable radical sites in HSA. In the past, this effect would have been explained by oxidation of (bi)carbonate to a carbonate radical anion (CO3*) by a bound hydroxyl radical-like species. We now propose that the CO3* radical is formed by the reduction of HOOCO2- (a complex of H2O2 with CO2) by the protein-Cu(I) complex. CO3* diffuses and produces more DMPO-trappable radical sites but does not fragment HSA. We were also able, for the first time, to detect discrete but highly specific H2O2-induced copper-catalyzed CO3*-mediated induction of DMPO-trappable protein radicals in functioning RAW 264.7 macrophages. We conclude that carbon dioxide modulates H2O2-induced copper-catalyzed oxidative damage to proteins by preventing site-specific fragmentation and enhancing DMPO-trappable protein radicals in functioning cells. The pathophysiological significance of our findings is discussed.     

Antioxidant properties of melatonin: a pulse radiolysis study

Various one-electron oxidants such as OH*, tert-BuO*, CCl3OO*, Br2*- and N3*, generated pulse radiolytically in aqueous solutions at pH 7, were scavenged by melatonin to form two main absorption bands with lambda(max) = 335 nm and 500 nm. The assignment of the spectra and determination of extinction coefficients of the transients have been reported. Rate constants for the formation of these species ranged from 0.6-12.5x10(9) dm3 mol(-1) s(-1). These transients decayed by second order, as observed in the case of Br2*- and N3* radical reactions. Both the NO2* and NO* radicals react with the substrate with k = 0.37x10(7) and 3x10(7) dm3 mol(-1) s(-1), respectively. At pH approximately 2.5, the protonated form of the transient is formed due to the reaction of Br2*- radical with melatonin, pKa ( MelH* <=> Mel* + H+) = 4.7+/-0.1. Reduction potential of the couple (Mel*/MelH), determined both by cyclic voltammetric and pulse-radiolytic techniques, gave a value E(1)7 = 0.95+/-0.02 V vs. NHE. Repair of guanosine radical and regeneration of melatonin radicals by ascorbate and urate ions at pH 7 have been reported. Reactions of the reducing radicals e(aq)- and H* atoms with melatonin have been shown to occur at near diffusion rates.     

Hydroxyl radical-induced apoptosis in human tumor cells is associated with telomere shortening but not telomerase inhibition and caspase activation

Reactive oxygen species (ROS) have been found to trigger apoptosis in tumor cells. At the same time, telomerase is found to be associated with malignancy and reduced apoptosis. However little is known about the linkage between ROS such as *OH and telomerase/telomere. To address the interrelations between *OH and telomerase/telomere in tumor cell killing, HeLa, 293 and MW451 cells were induced to undergo apoptosis with *OH radicals generated via Fe(2+)-mediated Fenton reactions (0.1 mM FeSO(4) plus 0.3-0.9 mM H2O2) and telomerase activity, telomere length were measured during apoptosis. We found that during *OH-induced apoptosis, telomere shortening took place while no changes in telomerase activity were observed. Our results suggest that *OH-induced telomere shortening is not through telomerase inhibition but possibly a direct effect of *OH on telomeres themselves indicating that telomere shortening but not telomerase inhibition is the primary event during *OH-induced apoptosis. Strikingly, we also found that *OH-induced apoptosis in HeLa cells is caspase-3-independent but is associated with reduction of mitochondrial transmembrane potential. Our results indicate that *OH triggers apoptotic tumor cell death through a telomere-related, caspase-independent pathway.     

Salivary thiocyanate/nitrite inhibits hydroxylation of 2-hydroxybenzoic acid induced by hydrogen peroxide/Fe(II) systems under acidic conditions: possibility of thiocyanate/nitrite-dependent scavenging of hydroxyl radical in the stomach

Formation of OH radicals in the stomach is possible by Fenton-type reactions, as gastric juice contains ascorbic acid (AA), iron ions and H2O2. An objective of the present study is to elucidate the effects of salivary SCN- and NO2- on the hydroxylation of salicylic acid which was induced by H2O2/Fe(II) and AA/H2O2/Fe(II) systems. Thiocyanate ion inhibited the hydroxylation of salicylic acid by the above systems in acidic buffer solutions and in acidified saliva. The inhibition by SCN- was deduced to be due to SCN- -dependent scavenging of OH radicals. Nitrite ion could enhance the SCN- -dependent inhibition of the hydroxylation induced by AA/H2O2/Fe(II) systems. The enhancement was suggested to be due to scavenging of OH radicals by NO which was formed by the reactions among AA, HNO2 and SCN- contained in the reaction mixture. The concentrations of SCN- and NO2-, which were effective for the inhibition, were in ranges of their normal salivary concentrations. These results suggest that salivary SCN- can cooperate with NO2- to protect stomach from OH radicals formed by AA/H2O2/Fe(II) systems under acidic conditions.