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).

Radical mechanisms of cephalosporins: a pulse radiolysis study.

Radiosterilization induces radicals, and it is very important to describe radical mechanisms before the possible use of cephalosporins gamma sterilization. Moreover, physiological or radiotherapeutically induced free radicals also initiate radical mechanisms. For this study, pulse radiolysis was used. This method permits to avoid in vivo direct study difficulties of bioradical processes and gives quantitative data. Reactions of solvated electron (eaq-), hydroxyl radical (.OH), azide radical (N3.), dibromine radical anions (Br2.-), oxygen, and superoxide radical (O2.-) with three cephalosporins have been studied. Absorption spectra and rate constants have been determined. It has been found that both eaq- and .OH quickly react (k congruent to 10(10) mol-1 L s-1) with the molecules to give radicals with similar absorption spectra. N3. gives an absorption spectra that has been attributed to an electron transfer, whereas a part of .OH and Br2.- could add themselves to an unsaturated bond.

Kinetic studies of four types of nitroheterocyclic radicals by pulse radiolysis. Correlation of pharmacological properties to decay rates.

The chemical properties of the nitro radical of four types of nitroheterocyclic compounds, nitrofurans, 2-nitroimidazoles, 4(5)-nitroimidazoles, 5-nitroimidazoles, having radiosensitizing and cytotoxic properties, have been studied by pulse radiolysis. The acid-base equilibria involving the nitro radical, the imidazole ring and some residues on the heterocycle have been determined. The pH-dependence of the rate of the disproportionation reaction of the nitro radical have been extensively studied. While the nitro radical derived from nitrofurans, 4- and 5-nitroimidazoles had a second-order decay, those of the 2-nitroimidazoles were found to decay through simultaneous first-order and second-order processes. Intrinsic second-order rate constants of the decay of the radical species in its various acidic and basic forms, could be determined. The intrinsic rate constants that determine the overall decay rates in the physiologically important 6 to 7.5 pH-range could be related to the one-electron redox potential E7(1). The implication of such chemical properties to enzyme-catalyzed reduction processes and to the mechanisms of radiosensitization and cytotoxicity of nitroheterocyclic compounds are briefly discussed. Pharmacological properties such as in vitro radiosensitization efficiency or metabolic reduction rates could be related to two of the nitro radical intrinsic disproportionation rates.

Astrocytes enhance radical defence in capillary endothelial cells constituting the blood-brain barrier.

Astrocytes (AC) induce blood-brain barrier (BBB) properties in brain endothelial cells (EC). As antioxidative activity (AOA) is assumed to be a BBB characteristic, we tested whether AC improve AOA of EC. Monocultivated AC showed higher AOA [manganese superoxide dismutase (SOD), catalase (Cat), glutathione peroxidase (GPx)] than EC. Cocultivation elevated AOA in EC (MnSOD, CuZnSOD, Cat, GPx), and AC (MnSOD, CuZnSOD, GPx). Hypoxia increased radical-induced membrane lipid peroxidation in monocultivated, but not in cocultivated EC. Thus, EC/AC cocultivation intensifies AOA in both cell types, protects the EC, and therefore, the BBB against oxidative stress. The high AOA is regarded as an essential property of the BBB, which is induced by AC.

Pulse radiolysis study of vitamin K1 reduction in ethanolic solution.

A pulse radiolysis study of vitamin K1 reduction was carried out in argon-saturated ethanolic solutions. The alpha-hydroxyethyl radicals CH3C.HOH (R.) and the solvated electrons (e-solv) reduce vitamin K1, leading to the semiquinone transient K1H.. This species, characterized by its absorption spectrum, decays by disproportionation and leads to the formation of the hydroquinone K1H2. The rate constants of the monoelectronic exchanges involved in this reduction have been determined.

Direct observation of HO2/O2- free radicals generated in water by a high-linear energy transfer pulsed heavy-ion beam.

The formation and decay of HO2/O2- radical from the radiolysis of water by heavy 36S16+ ions (2.7 GeV) have been observed by time-resolved absorption spectroscopy at 260 nm. The experiment was performed at the Grand Accélérateur National d'Ions Lourds (Caen, France). In deaerated water, for a linear energy transfer (LET) of 250 eV/nm, the yield of HO2/O2- is (6 +/- 2) x 10(-9) mol J-1. In aerated solution, an additional formation of O2- is observed due to the reaction of hydrogen atom and e(aq)- with oxygen. The experimental G values are compared to those obtained with light ions for the same LET. The importance of the initial velocity is discussed briefly.

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.

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.

Pulse radiolysis study of the reactivity of chloramphenicol, diazepam and clonazepam with the electron and the hydroxyl radical.

Chloramphenicol (CAP), diazepam (DIAZ) and clonazepam (CLONA) have been irradiated in aqueous and methanolic solutions by pulsed electrons (Febetron 707). Spectra of the species formed by the attack of the solvated electron and of OH radical have been determined. The kinetics of reaction of CAP, DIAZ, and CLONA with the solvated electron have been measured. Reactions of radical-radical recombination have also been studied. All these reactions are limited by the diffusion of the reactive species.

Radicals of nitroimidazole derivatives: pH dependence of rates of formation and decay related to acid-base equilibria.

Three analogous 5-nitroimidazoles, having radiosensitizing and cytotoxic properties, have been studied by pulse-radiolysis in N2O-saturated aqueous formate solutions. Rates of formation of the radicals ImNO2-. are found to have little pH dependence. Decay of the radicals always follows second-order kinetics. The observed rates of decay decrease by three to four orders of magnitude over the pH range 0-12. A pK at 2.3 has been observed kinetically for metronidazole. The pK assigned to the radical couple (ImH)NO2H./(ImH)-NO2-., or alternatively (ImH2+)-NO2-./(ImH)-NO2-., varies from 4.7 to 6.1, depending on the substituents on the imidazole ring. Intrinsic second-order rate constants for decay of the acidic form of the radical, of the anionic form and of the mixed reactions were determined. While the anionic radical reacts slowly with itself, both the acidic radical self-reaction and the mixed reaction proceed at fast rates. The implications of these chemical properties to the mechanisms of radiosensitization and cytotoxicity of the nitroaryl compounds are briefly discussed.

[Detection of hydroxyl and superoxide radicals in radiolysis of water with time-resolved chemiluminescence]. / Détection des radicaux OH et O2- issus de la radiolyse de l'eau par chimiluminescence résolue en temps.

A new method for the detection of low concentrations of hydroxyl and superoxide radicals, formed by water radiolysis, is described in this article. The method used is the time resolved chemiluminescence. It has been performed with an electron beam delivered by a Febetron 707 accelerator. This method allows to measure hydroxyl and superoxide radical concentrations in a large range of concentrations, between 10(-5) and 10(-8) M.

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.

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.

[Production of superoxide radicals with pulse radiolysis of water with high linear energy transfer]. / Production de radicaux superoxydes par radiolyse pulsée de l'eau à transfert d'énergie linéique (TEL) élevé.

The radiolysis of water with heavy ions of high linear energy transfer (LET) (-dE/dx) is characterized, in deaerated medium, by the production of superoxide anions, the radiolytic yields of which increase with the LET. Radiobiological interest in such radical species comes from the oxidative stress which may be generated by their dismutation in O2 and H2O2 in anoxic medium (radiotherapy with heavy ions). A brief review of the measurements of superoxide free radicals in aqueous solution by indirect or direct methods is presented. Moreover, some experimental results obtained by pulse radiolysis with Ar18+ ions (TEL = 290 keV x microm(-1)), are described. The interpretation of the kinetics takes into account the superoxide absorbance and that of hydrogen peroxide, which is present at the millisecond time scale.