Are there protective enzymatic pathways to regulate high local nitric oxide (NO) concentrations in cells under stress conditions?

This paper examines, from a chemical perspective, the hypothesis of the existence of protective enzymes whose role would be to regulate the high local nitric oxide (NO) concentrations that are released in NO-generating cells in situations of response to oxidative stress. These enzymes should play the role, with respect to NO, either of a reductase or of a dismutase. The energetics of the intervening transformations is herein presented, along with a review of pertinent literature. An attempt is made in order to describe the physiognomy of such enzymes, in relation with the literature data. Experimental investigation is needed to further evaluate the validity of such a hypothesis.

Two-photon ionisation of the antitumor drug pazelliptine (BD40) by 355 nm laser photolysis.

The effect of 2 ns pulses of 355 nm laser light on aqueous solutions of pazelliptine (PZE) was investigated and biphotonic ionization was observed. The absorption spectrum corresponding to the pazelliptine radical cation (PZE+) and the hydrated electron simultaneously formed in this process was determined. In the absence of oxygen, eaq- reacted with unexcited PZE (k = 1.6 x 10(10) M-1 s-1) to give the pazelliptine radical anion (PZE-). This latter species was identified by separate pulse radiolysis experiments. The radicals cation and anion disappeared by recombination on the millisecond time range. In presence of oxygen, eaq- was scavenged by O2 leading to the formation of the superoxide radical (O2.-) in competition to the formation of the radical PZE.-.PZE+ reacted with O2.- to produce H2O2 (k = 9 x 10(9) M-1 s-1). The spectral analysis revealed that PZE triplet was also formed during the laser pulse. In the absence of oxygen, the triplet-triplet absorption decreased on the microsecond time scale (2k = 1.5 x 10(10) M-1 s-1). In oxygenated solutions, eaq- and the pazelliptine triplet decayed exponentially in the same time range.

[Peroxidation of human high density lipoproteins by oxygen-derived free radicals]. / Peroxydation des lipoprotéines de haute densité (HDL) humaines par des radicaux libres dérivés de l'oxygène.

The involvement of low density lipoprotein (LDL) peroxidation in atherogenesis is now admitted. The oxidation of high density lipoproteins (HDL) could contribute to the atherogenic process, by limiting their capacity to accept cholesterol from cell membranes. In this work, we studied the human HDL peroxidation initiated by OH. or OH./O2.- free radicals generated by gamma radiolysis. This method allows a quantitative and selective production of free radicals, and the resulting oxidation is less drastic than the chemical one. HDL oxidation was followed, as a function of the radiation dose, by the disappearance of endogenous vitamin E, the formation of thiobarbituric acid-reactive substances (TBARS) and the fluorescence at 440 nm. Human HDL turned out to be oxidizable by hydroxyl free radicals and oxygen potentiated this effect. The oxidative modification of HDL, leading to a rigidification of the HDL envelop, could contribute to reduce the ability of HDL to stimulate efflux of cholesterol from tissues.

Oxidation of low-density lipoproteins by OH. and OH./O2-. free radicals produced by gamma radiolysis.

The aim of this study was to analyze quantitatively the oxidative modification of low-density lipoproteins (LDLs) induced by OH. free radicals produced by gamma radiolysis, in the absence or in the presence of oxygen (action of OH. free radicals, or simultaneous action of OH. and O2-. free radicals, respectively). The effects of increasing radiation doses on aqueous LDL solutions have been monitored by several parameters: a decrease in endogenous vitamin E, the formation of thiobarbituric acid-reactive substances (TBARS) and conjugated dienes, the appearance of a differential fluorescence (excitation wavelength = 360 nm), and an increase in the relative electrophoretic mobility. Initial radiation yields (decrease in vitamin E, formation of TBARS) have been determined at pH 7 as a function of LDL concentration (from 0.75 to 9 g liter-1). From the comparison of these yields with those of OH. and O2-. free radicals produced by water radiolysis, we have deduced reaction mechanisms for the initiation of oxidation of LDLs by OH. and OH./O2-. free radicals.

Radioprotective effects of the immunostimulating lauroylpeptide LtriP (RP 56142).

The lipopeptide lauroyl-L-Ala-gamma-D-Glu-L,L-A2pm (LtriP) increased the resistance of mice to the lethal effect of gamma-ray irradiation. The radioprotective effect was dependent on the doses of LtriP and of radiation. Maximum survival was observed when the lipopeptide was injected on two successive days before irradiation. This activity seems to be related to immunostimulating functions, since the non-immunostimulating analog lauroyl-L-Ala-gamma-D-Glu-D,D-A2pm-Gly, containing D,D-diaminopimelic acid, was not radioprotective. The protective activity might result from an induction of cytokines, such as IL-1, TNF and M-CSF, since LtriP induced the mRNA expression and the secretion of these immunomodulators.

Effect of pH on low-density lipoprotein oxidation by O2-./HO2. free radicals produced by gamma radiolysis.

This study aimed to analyze quantitatively the initiation and the consequences of low-density lipoprotein (LDL) peroxidation by O2-./HO2. free radicals produced by gamma radiolysis. The action of increasing radiation doses on aqueous LDL solutions has been monitored simultaneously by several parameters: a decrease in endogenous vitamin E, the formation of thiobarbituric acid-reactive substances (TBARS) and conjugated dienes, the appearance of a differential fluorescence (excitation wavelength = 360 nm), and an increase of the relative electrophoretic mobility. Initial radiation yields (decrease in vitamin E, formation of TBARS) have been determined at pH 7 and pH 5.7 as a function of LDL concentration (from 0.75 to 9 g liter-1). From the comparison of these yields with those of O2-. radicals produced by water radiolysis, we have deduced reaction mechanisms for LDL peroxidation initiated by O2-./HO2. free radicals.

[Biological roles of free radicals: introduction]. / Rôles biologiques des radicaux libres: introduction.

Free radicals are produced during cellular oxidations. The primary ones are superoxide anions (O2-) and hydroxyl radicals (OH). They generate in vivo various secondary radicals. Radical mechanisms are invoked in a number of biological processes including, phagocytosis, ischemia or ageing.

Intramolecular semiquinone disproportionation in DNA. Pulse radiolysis study of the one-electron reduction of daunorubicin intercalated in DNA.

The one-electron reduction of daunorubicin, a quinonic antitumor antibiotic, intercalated in DNA was studied by pulse radiolysis using carboxyl radicals as reductants. The reaction's first stage is the daunorubicin semiquinone formation (k = 1.9 x 10(8) mol-1.dm3.s-1) in a way entirely consistent with a simple competition between .COO- disproportionation and the drug reduction. The semiquinone drug disappears by a first-order reaction (k = 1340 s-1) producing the hydroquinone form. This reaction leads to an equilibrium similar to the one without DNA and the equilibrium constant is very close to its value free in water (Kc approximately 25). In addition, the stoichiometry of the first-order reaction is the one of a dismutation process. Therefore, it appears that the disproportionation occurs along an intramolecular path across DNA. This migration takes place under our experimental conditions, over a distance of ca. 100 base pairs, with a mobility of ca. 4.4 X 10(-11) m2.V-1.s-1, similar in magnitude to an excess electron mobility in doped organic polymers.

Reduction of daunorubicin in the presence of sulfur-containing peptides.

Daunorubicin, an anthracycline antitumor antibiotic, was reduced in the presence of reduced (GSH) or oxidized (GSSG) glutathione to evaluate the possibilities of detoxification or of potentiation of the drug by these compounds. The reductants were .COO- free radicals produced by gamma radiolysis. In both cases, the final product is 7-deoxydaunomycinone, i.e., the same as without glutathione. The reduction yield is also the same as without GSH or GSSG (0.23 mumol.J-1). No glutathione depletion was observed. Limits for the rate constants of some possible nonenzymatic detoxification reactions are given. To evaluate the possible interactions of daunorubicin with sulfur-containing proteins, the reduction of this drug by .COO- free radicals was also studied in the presence of a polypeptide containing two disulfide bridges, aponeocarzinostatine. The final product is also 7-deoxydaunomycinone. The yields of reduction of the drug and of a protein disulfide bridge are, respectively, 0.23 mumol.J-1 and less than or equal to 6 nmol.J-1. These values indicate that disulfide radical anions of the protein can reduce the drug, giving back the disulfide bridge, but that the drug transients neither oxidize nor reduce the protein.

Spectra and structure of alpha-tocopherol radicals produced in anoxic media.

Electron spin resonance and flash photolysis studies have been combined to determine, amid conflicting reports from radiolysis studies, the spectrum for the phenoxyl radical of alpha-tocopherol. Triplet state ketones were used to abstract the alpha-tocopherol phenolic hydrogen in order to obtain both the transient ESR spectrum and optical spectrum. Analysis of the ESR data yields g = 2.00469, a(H, 5-CCH3) = 6.04G, a(H,7-CCH3) = 4.51G, a(H, CH2) = 1.38G, a(H', CH2) = 1.49G, and a(H,8-CCH3) = 0.89G. The g factor shows large spin population on oxygen in this radical and demonstrates conclusively involvement of the phenoxyl radical. Both spectra were in agreement with those produced by radiolysis of alpha-tocopherol in N2 saturated EtOH. While the spectral characteristics of the phenoxyl radical now appear to be clarified, uncertainties remain concerning optical spectra for radiolysis of alpha-tocopherol in air- and oxygen-containing systems.

Antioxidant activity of hemocyanin; a pulse radiolysis study.

In order to determine the reactivity on hemocyanin from Androctonus australis, the reaction of superoxide anion has been investigated using pulse radiolysis. The kinetics of O2- decays have been studied in aqueous buffered media at various basic pH (8, 8.5 and 9), first in the absence and then in the presence of hemocyanin (in oxygenated solutions containing formate anion 0.16 mol.l-1). We have shown that, in the presence of hemocyanin, O2- decay is a first-order process whose apparent rate constant is proportional to protein concentration (10(-7) to 10(-6) mol.l-1) and pH independent between 8 to 9. A second-order rate constant of 3.5 +/- 0.1.10(7) mol-1.l.s-1, has been deduced for the catalytic rate constant of hemocyanin with O2-. Meanwhile, this activity is smaller than that described for free copper, eukaryotic Cu-Zn-SOD or some copper chelates. We have verified that apohemocyanin--the copper deprived protein--does not exhibit such an activity vs. SOD (superoxide dismutase).

One-electron reduction of daunorubicin intercalated in DNA or in a protein: a gamma radiolysis study.

The one-electron reduction of daunorubicin, an anthracycline antibiotic, intercalated in DNA or in the apoprotein of the riboflavin binding protein, was studied by gamma radiolysis. The two reduction mechanisms appear very similar to the one found for the non-intercalated drug. Hydrogen peroxide, which oxidizes non-intercalated hydroquinone daunorubicin with two electrons in one step (C. Houée-Levin, M. Gardès-Albert and C. Ferradin, FEBS lett., 173, 27-30, (1984], reacts with daunorubicin hydroquinone in DNA but not in the protein. It appears thus that the site accessibility to hydrogen peroxide in DNA is better than in the protein. Biological consequences are discussed.

One-electron reduction of daunorubicin intercalated in DNA or in a protein: a gamma radiolysis study.

The one-electron reduction of daunorubicin, an anthracycline antibiotic, intercalated in DNA or in the apoprotein of the riboflavin binding protein, was studied by gamma radiolysis. The two reduction mechanisms appear very similar to the one found for the non-intercalated drug. Hydrogen peroxide, which oxidizes non-intercalated hydroquinone daunorubicin with two electrons in one step (C. Houée-Levin, M. Gardès-Albert and C. Ferradin, FEBS lett., 173, 27-30, (1984)), reacts with daunorubicin hydroquinone in DNA but not in the protein. It appears thus that the site accessibility to hydrogen peroxide in DNA is better than in the protein. Biological consequences are discussed.

Vitamin E and correlated antioxidants: a gamma radiolysis study.

gamma irradiations of Vit.E-Vit.C aerated ethanolic solutions have been performed for several ratios (Vit.E)/(Vit.C) between 0.1 and 50. The obtained results show that Vit.C is able to regenerate Vit.E from its oxidized radical, this regeneration being total for a ratio (Vit.E)/(Vit.C) greater than or equal to 27 in our conditions of irradiation. The ratio (Vit.E)/(Vit.C) seems to be the main factor of this synergestic effect towards peroxyl radicals scavenging.

Gamma and pulse radiolysis investigation of the reaction of desferrioxamine with superoxide anions.

The kinetic scheme of the reaction of desferrioxamine (DFO) with O2-. was studied using pulse and gamma-radiolysis. The rate constant k(O2-. + DFO) is equal to 1.3 +/- 0.1 x 10(6) dm3 mol-1s-1 at pH 7.4. Studying the competition between DFO and ferricytochrome-c for O2-. generated by gamma-radiolysis, we observed that the nitroxide free radical resulting from the reaction of O2-. with DFO and the product(s) resulting from the decay of this nitroxide radical act inversely towards the cytochrome-c-Fe3+/cytochrome-c-Fe2+ redox couple. This explains the discrepancy between our value of k(O2-. + DFO) and the one measured previously using ferricytochrome-c for the detection of O2-. The reported results show that DFO acts as a powerful O2-. scavenger, and that the products resulting from the reaction of DFO with O2-. can initiate oxidative and/or reductive reactions that should be taken into account in interpreting the effects of DFO in vitro and in vivo.

Intramolecular electron transfer in proteins. Radiolysis study of the reductive activation of daunorubicin complexed in egg white apo-riboflavin binding protein.

Daunorubicin, an anthracycline antitumor antibiotic, can be complexed in egg white apo-riboflavin binding protein. The reduction of this complex was studied by gamma-radiolysis and pulse radiolysis using COO.- free radicals as reductants. The final products are 7-deoxydaunomycinone intercalated in the protein and thiol groups coming from the reduction of disulfide bonds of the protein, in the respective proportions of 90% and 10%. One-electron reduction of the complex gives daunorubicin semiquinone radical and a disulfide anion. The rate constants of the reactions of COO.- ions with the complex and with the disulfide bond in the protein alone are respectively equal to 2.4 x 10(8) mol-1.L.s-1 and 6.4 x 10(7) mol-1.L.s-1. Daunorubicin semiquinone decays by a first-order process, the rate constant of which is independent of the initial protein and radical concentrations. Without protein, daunorubicin semiquinone undergoes a disproportionation-comproportionation equilibrium [Houée-Levin, C., Gardès-Albert, M., Ferradini, C., Faraggi, M., & Klapper, M. (1985) FEBS Lett. 179, 46-50]. We propose that a protein residue reduces semiquinone by an intramolecular path. This creates an electron hole in the protein which may alter its function. This reduction process is very different from the reduction mechanism of riboflavin binding protein by the same reductant [Faraggi, M., Steiner, J.P., & Klapper, M.H. (1985) Biochemistry 24, 3273-3279]. These results suggest a new deleterious pathway to explain the antitumor and/or cytotoxic effect of this drug.

Role of the superoxide anion in the oxidative activation of the new antitumor drug BD40: a radiolysis study.

BD40, a new antitumor drug derived from 9-azaellipticine, is thought to have an oxygen-dependent metabolism in vivo. We have investigated the one-electron oxidation of this drug by gamma radiolysis using OH. free radicals as oxidants and the reaction of O2-. with the BD40 oxidized transient(s). The absorption spectrum of the one-electron oxidized free radical was determined by pulse radiolysis using OH. or N.3 as reactant. In the absence of O2 and O2-., the initial yield of disappearance of the drug is equal to 2.5 x 10(-7) mol J-1 independently of the initial concentration of the drug and of the dose rate. When BD40 is oxidized by OH. radicals in the presence of O2 and O2-., the yield is the same. This yield is halved if superoxide dismutase is present during irradiation. Superoxide anions do not react directly with the drug. Thus it is suggested that these radicals oxidize the BD40 free radical produced by oxidation with OH. Biological implications are discussed.

gamma-radiolysis study of the reduction by COO- free radicals of daunorubicin intercalated in DNA.

The reduction of daunorubicin intercalated in DNA was studied using COO- free radicals produced by gamma-radiolysis as reductants. The reduction process of the drug intercalated in DNA was found to be very similar to the one of daunorubicin in aqueous solution without DNA. (a) the final product is the same (7-deoxy daunomycinone); (b) the reduction yield is the same [2.6 +/- 0.2) x 10(-7) mol.J-1); (c) H2O2 reacts with hydroquinone daunorubicin giving back the drug in a one-step reaction. However 7-deoxy daunomycinone precipitation was so slow that this aglycone could be reduced by COO- free radicals giving its hydroquinone form, which cannot be observed without DNA. This shows that the whole 4-electron reduction process takes place in DNA. The implications of these findings are discussed.

Parameters controlling the kinetics of ferric and ferrous hemeproteins reduction by hydrated electrons.

To clarify the processes of hemeproteins reduction, three classes of these proteins (ferric, ferrous and desFe) were reduced by hydrated electrons generated by pulse radiolysis. Spectral and kinetic investigations were made on alpha hemoglobin chain and myoglobin. Human alpha chain has been chosen to avoid all ferric contaminations and horse ferric myoglobin to eliminate all ferrous protein fractions. We have successively studied the influences of: the iron presence, its oxidation state (II and III), the protein charge and the iron-ligand nature (H2O, OH-, N3- and CN-). For alpha human hemoglobin chain without metallic ion or with ferrous iron, the reduction rates are the same: 1.1 +/- 0.2.10(10) M-1.s-1. In the case of horse ferric myoglobin, the reduction rates depend principally on the protein charge (from pH 6.3 to pH 9.5, the reduction rate of Mb(FeIII)N3- decreases from 2.5 +/- 0.5.10(10) M-1.s-1 to 1.2 +/- 0.2.10(10) M-1.s-1) and are also modulated by the equilibrium constant of the hemeprotein-ligand association (1.2 +/- 0.2.10(10) M-1.s-1 for Mb(FeIII)N3- and 0.8 +/- 0.2.10(10) M-1.s-1 for Mb(FeIII)CN-, at pH 9.8).