Effects of Fe(II) and hydrogen peroxide interaction upon dissolving UO2 under geologic repository conditions.

Iron redox cycling is supposed to be one of the major mechanisms that control the geochemical boundary conditions in the near field of a geologic repository for UO2 spent nuclear fuel. This work investigates the impact of reactions between hydrogen peroxide (H2O2) and iron (Fe2+/Fe3+) on UO2 dissolution. The reaction partners were contacted with UO2 in oxygen-free batch reactor tests. The interaction in absence of UO2 gives a stoichiometric redox reaction of Fe2+ and H2O2 when the reactants are present in equal concentration. Predomination of H202 results in its delayed catalytic decomposition. With UO2 present, its dissolution is controlled by either a slow mechanism (as typical for anoxic environments) or uranium peroxide precipitation, depending strongly on the reactant ratio. Uranium peroxide (UO4 x nH2O, m-studtite), detected on UO2 surfaces after exposure to H2O2, was not found on the surfaces exposed to solutions with stoichometric Fe(II)/ H2O2 ratios. This suggests that H2O2 was deactivated in redox reactions before a formation of UO4 took place. ESR measurements employing the spin trapping technique revealed only the DMPO-OH adduct within the first minutes after the reaction start (high initial concentrations of the OH radical); however, in the case of Fe(II) and H2O2 reacting at 10(-4) mol/L with UO2, dissolved oxygen and Fe2+ concentrations indicate the participation of further Fe intermediates and, therefore, Fenton redox activities.

Enhanced gamma-glutamyl transpeptidase expression and superoxide production in Mpv17-/- glomerulosclerosis mice.

Recently, gamma-glutamyl transpeptidase, which initiates cleavage of extracellular glutathione, has been shown to promote oxidative damage to cells. Here we examined a murine disease model of glomerulosclerosis, involving loss of the Mpv17 gene coding for a peroxisomal protein. In Mpv17-/- cells, enzyme activity and mRNA expression (examined by quantitative RT-PCR) of membrane-bound gamma-glutamyl transpeptidase were increased, while plasma glutathione peroxidase and superoxide dismutase levels were lowered. Superoxide anion production in these cells was increased as documented by electron spin resonance spectroscopy. In the presence of Mn(III)tetrakis(4-benzoic acid)porphyrin, the activities of gamma-glutamyl transpeptidase and plasma glutathione peroxidase were unchanged, suggesting a relationship between enzyme expression and the amount of reactive oxygen species. Inhibition of gamma-glutamyl transpeptidase by acivicin reverted the lowered plasma glutathione peroxidase and superoxide dismutase activities, indicating reciprocal control of gene expression for these enzymes.

Chemical studies of proanthocyanidins and hydrolyzable tannins.

We investigated a number of natural polyphenols representing flavan-3-ols, gallotannins, and ellagitannins with regard to their antioxidant potential. For this purpose we used pulse radiolysis to determine scavenging rate constants with hydroxyl radicals and decay rates of the respective aroxyl radicals and EPR spectroscopy to identify the radicals after in situ oxidation. Using NMR spectroscopy, we could confirm phenolic coupling reactions of epigallocatechin gallate and pentagalloyl glucose after radical-induced oxidation.

Electron paramagnetic resonance studies of radical species of proanthocyanidins and gallate esters.

The polyphenols present in green tea or red wine comprise both regular flavon(ol)s and proanthocyanidins, i.e., derivatives of flavan-3-ols, whose distinct antioxidative potential is of great importance for explaining the beneficial effects of these nutrient beverages. Using EPR spectroscopy, we investigated radical structures obtained after oxidation of the parent compounds either by horseradish peroxidase/hydrogen peroxide or after autoxidation in moderately alkaline solutions. Both proanthocyanidins (monomers of condensed tannins, e.g., (+)-catechin, (-)-epicatechin, (-)-epicatechin gallate, (-)-epigallocatechin, (-)-epigallocatechin gallate, Pycnogenol) and gallate esters (hydrolyzable tannins, e.g., propylgallate, beta-glucogallin, pentagalloyl glucose and tannic acid) yielded predominantly semiquinone structures derived from the parent catechol or pyrogallol moieties. Evidence for a time-dependent oligomerization was obtained for (-)-epigallocatechin gallate, supporting our hypothesis that o-quinones formed from the initial semiquinone disproportionation are prone to nucleophilic addition reactions. Such phenolic coupling reactions would retain the numbers of reactive catechol/pyrogallol structures and thus the antioxidative potential. We therefore propose that proanthocyanidins are superior antioxidants as compared to flavon(ol)s proper, whose quinones are more likely to redox-cycle and act as prooxidants.

Reactivity of semiquinones with ascorbate and the ascorbate radical as studied by pulse radiolysis.

Free-radical interactions between hydroquinones (QH2) and ascorbate (AscH-) have a profound impact in many biological situations. Despite the obvious biological significance, not much is known about the kinetics of reactions of QH2 and AscH- with their corresponding free radicals, i.e., semiquinones, Q1.-, and the ascorbate radical, Asc.-. Furthermore, a general approach to reliably measure rate constants for the above reactions is fraught with complications. In this work, the kinetic behavior of Q.- and Asc.-, after pulse radiolytic oxidation of mixtures of a series of alkyl- and methoxysubstituted hydroquinones and ascorbate by azide radicals in aqueous buffer, pH 7.40, was monitored in submillisecond range by time-resolved UV spectroscopy. Rate constants for reactions of Q.- with AscH-(reaction [1]) and Asc.- (reaction [2]) were directly determined by using new kinetic procedures which distinguished between reactions [1] and [2]. The results show that the rate constants for reaction [2] vary only within a narrow range from 1.2 x 10(8) to 2.5 x 10(8) M(-1) s(-1) and do not display any pronounced correlation with Q.- structures. In contrast, the value of k1 for nonsubstituted Q.- was found to be (1.8 +/- 0.2) x 10(5) M(-1) s(-1) and decreases with the number of alkyl and methoxy substituents as well as with the decrease of the one-electron reduction potential E(Q.-/QH2).

Arguments against the significance of the Fenton reaction contributing to signal pathways under in vivo conditions.

One of the common explanations for oxidative stress in the physiological milieu is based on the Fenton reaction, i.e. the assumption that radical chain reactions are initiated by metal-catalyzed electron transfer to hydrogen peroxide yielding hydroxyl radicals. On the other hand - especially in the context of so-called "iron switches" - it is postulated that cellular signaling pathways originate from the interaction of reduced iron with hydrogen peroxide. Using fluorescence detection and EPR for identification of radical intermediates, we determined the rate of iron complexation by physiological buffer together with the reaction rate of concomitant hydroxylations of aromatic compounds under aerobic and anaerobic conditions. With the obtained overall reaction rate of 1,700 M(-1)s(-1) for the buffer-dependent reactions and the known rates for Fenton reactions, we derive estimates for the relative reaction probabilities of both processes. As a consequence we suggest that under in vivo conditions initiation of chain reactions by hydroxyl radicals generated by the Fenton reaction is of minor importance and hence metal-dependent oxidative stress must be rather independent of the so-called "peroxide tone". Furthermore, it is proposed that - in the low (subtoxic) concentration range - hydroxylated compounds derived from reactions of "non-free" (crypto) OH radicals are better candidates for iron-dependent sensing of redox-states and for explaining the origin of cellular signals than the generation of "free" hydroxyl radicals.

Studies on radical intermediates in the early stage of the nonenzymatic browning reaction of carbohydrates and amino acids.

Determination of the color intensity of heated mixtures of L-alanine and carbohydrate degradation products revealed furan-2-carboxaldehyde and glycolaldehyde as by far the most effective color precursors. EPR studies demonstrated that furan-2-carboxaldehyde generated colored compounds exclusively via ionic mechanisms, whereas glycolaldehyde led to color development accompanied by intense radical formation. In agreement with literature data, these radicals were also detected in heated mixtures of L-alanine and pentoses or hexoses, respectively, and were identified as 1,4-dialkylpyrazinium radical cations by EPR as well as LC/MS measurements. Studies on the mechanisms of radical formation revealed that under the reaction conditions applied, glyoxal is formed as an early product in hexose/L-alanine mixtures prior to radical formation. Reductones then initiate radical formation upon reduction of glyoxal and/or glyoxal imines, formed upon reaction with the amino acid, into glycolaldehyde, which was found as the most effective radical precursor. LC/MS measurements gave evidence that these pyrazinium radicals cations are not stable but are easily transformed into hydroxylated 1,4-dialkyl-1, 4-dihydropyrazines upon oxidation and hydrolysis of intermediate diquarternary pyrazinium ions. Besides other types of color precursors, these intermediates might be involved in the formation of colored compounds in the Maillard reaction.

Radical-assisted melanoidin formation during thermal processing of foods as well as under physiological conditions.

Color-generating reactions of protein-bound lysine with carbohydrates were studied under thermal as well as under physiological conditions to gain insights into the role of protein/carbohydrate reactions in the formation of food melanoidins as well as nonenzymatic browning products in vivo. EPR spectroscopy of orange-brown melanoidins, which were isolated from heated aqueous solutions of bovine serum albumin and glycolaldehyde, revealed the protein-bound 1,4-bis(5-amino-5-carboxy-1-pentyl)pyrazinium radical cation (CROSSPY) as a previously unknown type of cross-linking amino acid leading to protein dimerization. To verify their formation in foods, wheat bread crust and roasted cocoa as well as coffee beans, showing elevated nonenzymatic browning, were investigated by EPR spectroscopy. An intense radical was detected, which, by comparison with the radical formed upon reaction bovine serum albumin with glycolaldehyde, was identified as the protein-bound CROSSPY. The radical-assisted protein oligomerization as well as the browning of bovine serum albumin in the presence of glycolaldehyde occurred also rapidly under physiological conditions, thereby suggesting CROSSPY formation to be probably involved also in nonenzymatic glycation reactions in vivo.

Antioxidant capacity of flavanols and gallate esters: pulse radiolysis studies.

Reactivities of several proanthocyanidins (monomers of condensed tannins) and gallate esters (representing hydrolyzable tannins) with hydroxyl radicals, azide radicals, and superoxide anions were investigated using pulse radiolysis combined with kinetic spectroscopy. We determined the scavenging rate constants and the decay kinetics of the aroxyl radicals both at the wavelength of the semiquinone absorption (275 nm) and the absorption band of the gallate ester ketyl radical (400-420 nm). For most compounds second-order decay kinetics were observed, which reflect disproportionation of the semiquinones. In the case of the oligomeric hydrolysable tannins, pentagalloyl glucose and tannic acid, the decay kinetics were more complex involving sequential first-order and second-order reactions, which could only be resolved by kinetic modeling. A correlation of the reaction rates with hydroxyl radicals (k*OH) with the number of adjacent aromatic hydroxyl groups (i.e., representing catechol and/or pyrogallol structures) was obtained for both condensed and hydrolyzable tannins. Similar correlation for the reactions with azide radicals and superoxide anions are less obvious, but exist as well. We consider proanthocyanidins superior radical scavenging agents as compared with the monomeric flavonols and flavones and propose that these substances rather than the flavonoids proper represent the antioxidative principle in red wine and green tea.

Radical functions in vivo: a critical review of current concepts and hypotheses.

Most of the basic knowledge about radical reactions comes from radiation chemical studies in vitro. In view of the rapidly increasing knowledge on radical reaction in vivo, it is important to reconcile the fundamental physico-chemical reaction characteristics of radicals with the need to explain their alleged biological effects. Severe problems in the understanding of their in vivo action remain unsolved. An example is phagocytosis, which seems to be a paradigm of a 'deleterious' radical process. The exact mechanism is not clear; so it is an open question whether the intruder is eventually killed by radicals (like OH) or by endproducts of radical reactions (like H2O2 and/or HOCl). It is even more difficult to understand signalling by radicals: owing to their chemical nature they are 'unspecifically' reacting species--they withdraw or add electrons--and thus their reactions are governed by redox-properties. Since all radicals have different redox characteristics and different molecular shapes, the usual key-and-keyhole picture for molecular interaction does not apply, as there, is no reactive site conceivable which has the property of reacting with radicals 'specifically. Our intent in this article is: (i) to briefly review some fundamental characteristics of in vitro radical reactions, (ii) to extrapolate from this to the conditions in vivo, and (iii) to discuss current hypotheses concerning the redox-regulation of cellular signalling. This leads us to the tentative conclusion that radicals per se must be tolerated by the cell and do not threaten its life, if they stay below a certain concentration limit. The main biological implication of radical-reactions seems to be that the cell derives signals from the balance of oxidative versus reductive processes and that radicals may interact with pathways of intra- and intercellular communication.

Radiation chemistry of physiological saline reinvestigated: evidence that chloride-derived intermediates play a key role in cytotoxicity.

Contrary to common belief, hydrogen peroxide (H2O2) and hypochlorite (HOCl) are not produced continuously and independently during the irradiation of buffer solution containing chloride. Different buildup and decay reactions are involved in a complex interaction of these substances during irradiation. Which of the species predominates is determined by the parameters of the solution. The amount of either compound detectable after irradiation depends on the dissolved gas (O2, N2O or N2), on the pH value and to some extent on the presence of catalytic metals: Under slightly acidic conditions, low oxygen content and high generation rates of OH radicals, the only detectable species is hypochlorite; at high oxygen content and at pH values in the physiological range, hydrogen peroxide is the main detectable product. However, H2O2 and HOCl react with each other in a pH-dependent way, yielding the stable products O2 and Cl-. This reaction limits the expected lifetime of both species in aqueous solution to some tens of seconds. Therefore, analysis of the sample solution after irradiation determines only the substance that was present in greater relative concentration at the termination of irradiation. Such analysis, however, does not allow conclusions about the processes that occurred during irradiation. We have investigated the decay and formation reactions of H2O2 and HOCl under all relevant irradiation conditions and found evidence that the formation and further reaction of HOCl-, the precursor of HOCl, is of central importance even in cases where no significant amounts of H2O2 or HOCl are detectable after irradiation. We discuss the consequences of these results for the cytotoxicity observed after irradiation of cells suspended in physiological saline and conclude that analogous processes must also be relevant for irradiations under in vivo conditions.

Radiation-induced cell killing is highly dependent upon buffer treatment (filtration compared to autoclaving) due to metal-catalyzed formation of hypochlorite: a cautionary note.

Buffer solutions used in experiments in radiation biology may be sterilized by either autoclaving or filtration. We show here that for phosphate-buffered saline such differences in buffer treatment may result in widely differing dose-effect curves for cell killing. The temperature-dependent transformation of monophosphate ions into di- or polyphosphate evidently proceeds to an appreciable extent upon autoclaving the buffers at 120 degrees C for 10 to 20 min. This increases the capability of the buffer to chelate spurious metal contaminations and, as a consequence, to reduce the amount of cytotoxic hypochlorite being produced. Depending on conditions of buffer treatment we have observed dose modification factors for the colony-forming ability of yeast cells up to the order of 3. Thus effects due to buffer treatment might easily outweigh the effect which the experiment was originally designed to determine. We strongly advise, therefore, that results of parallel sets of experiments in which different methods of buffer sterilization have been used should not be compared directly.

Marchantins and related polyphenols from liverwort: physico-chemical studies of their radical-scavenging properties.

Structurally unique macrocyclic phenols from liverwort, i.e., marchantins and related substances, were studied for their antioxidative potential using pulse-radiolytic and EPR-spectroscopic techniques. The generally diffusion-controlled rate constants for scavenging of azide radicals as a model electrophilic species and the sufficiently slow bimolecular decay confirm their antioxidative potential. Transient spectra after pulse radiolysis and the EPR spectra both demonstrate the internal strain of the macrocyclic ring. One compound, Perrottetin D, furthermore gave proof to the hitherto only kinetically verifyable superior radical-scavenging capability of the aroxyl radical derived from a phenolic antioxidant.

Interaction of flavonoids with ascorbate and determination of their univalent redox potentials: a pulse radiolysis study.

Concurrent pulse-radiolytic generation of flavonoid aroxyl radicals and ascorbyl radicals causes a complex kinetic interplay of competing and parallel reactions. Evaluation by "kinetic modelling," that is, taking into account all possible reactions by a set of differential equations, allowed us to determine equilibria constants for the univalent steps by a novel method. From these kinetic data we were able to calculate the redox potentials for dihydroquercetin, quercetin, rutin (a quercetin 3-glycoside), kaempferol, fisetin, and luteolin. Despite the limited number of substances, two structural criteria became apparent: all substances containing the B-ring catechol group and the 2,3-double bond have a higher redox potential than ascorbate and are consequently able to oxidize it to the ascorbyl radical. With fisetin and kaempferol having values very similar to ascorbate, only the flavanone dihydro-quercetin was capable of reducing the ascorbyl radical, thus fulfilling the so-called "ascorbate-protective" function, originally proposed by Szent-Györgyi. While flavonoids are effective radical scavengers, these rather high redox potentials for most flavonols may explain their occasional prooxidative behavior.

Effect of marchantins and related compounds on 5-lipoxygenase and cyclooxygenase and their antioxidant properties: a structure activity relationship study.

Marchantins and related compounds, isolated from different species of liverworts, were investigated for their inhibitory potential on cyclooxygenase (COX) and 5-lipoxygenase 5-(LOX), the key enzymes of the arachidonic acid cascade, and additionally in a cell-free lipid-peroxidation system. The results were compared with rate constants obtained in pulse-radiolytic studies. All substances tested showed significant inhibitory COX and 5-LOX-activity. Marchantin B with two catechol moieties, as in NDGA, exerted an enhanced inhibitory effect in all test systems. The most active compound in the 5-LOX-test was Perrottetin D (IC(50) = 0.66 µM), which was also effective in the COX and lipid-peroxidation assay. While the rate constant determined by pulse radiolysis was rather low, EPR studies at elevated temperatures demonstrated for perrottetin D the presence of a pyrogallol-type radical, generated by a furan-ring opening reaction. In conclusion, all compounds from liverworts investigated possess significant antiinflammatory activities. The results let us suggest that all these phenolic compounds can be considered as promising leads due to their strong radical scavenger potential.

Signalling by O2-. and NO.: how far can either radical, or any specific reaction product, transmit a message under in vivo conditions?

With regard to the stability of the NO. radical as a chemical entity, it is without doubt able to serve as an intra- as well as an intercellular messenger. The radical O2-., in contrast, does not seem to be suited to far-range signalling in the vascular system. Its short chemical half-life, which is limited by the presence of various reactive blood constituents to below 50 ms, results in a free diffusion path length of less than 40 microns, i.e. only the distance between just a few cells. While accelerated 'downstream' transport by arterial blood may help to extend the action sphere, there is no possibility for O2-. to serve as a signal in an upstream direction. The estimates presented, however, do not invalidate arguments for a possible role of superoxide anions in intra- or pericellular signalling phenomena. Cross-talk between NO.- and O2-.-dependent signal routes, e.g. by peroxynitrite formation, is unlikely to be a relevant process under the conditions which prevail in the vascular system.

Lack of an association between serum vitamin E and myocardial infarction in a population with high vitamin E levels.

The antioxidant effects of vitamin E may protect low density lipoproteins from peroxidation and thus inhibit the development of arteriosclerosis. Inverse associations between vitamin E levels and coronary heart disease have been reported from cross-sectional and ecologic studies. In the population-based MONICA Augsburg cohort (2023 men, 1999 women, age 25-64 years at baseline in 1984, 93% of whom were reexamined in 1987/88) we investigated the relationship between serum vitamin E concentrations and the risk of subsequent myocardial infarction (MI). Between 1984 and 1991, 46 cases of fatal and non-fatal myocardial infarction from this cohort were recruited for a nested case-control study. Four controls were sampled from the cohort for each case of MI with matching for age, sex, and total cholesterol. There were no marked differences between cases and their matched controls in the means of vitamin E concentrations (33.9 mumol/l vs. 32.8 mumol/l, P = 0.37) or in the mean vitamin E/total cholesterol ratios (4.89 mumol/mmol vs. 4.82 mumol/mmol, P = 0.75). The covariate adjusted relative risk (RR) for fatal plus non-fatal MI in the lowest tertile of vitamin E relative to the upper two tertiles was 0.72 (90% confidence interval: 0.33-1.57). Likewise, for the lowest tertile of the ratio (vitamin E/total cholesterol) the RR was 0.81 (0.42-1.56). The association was not modified by history of previous coronary heart disease, fatality of MI, temporal distance of MI onset from vitamin E determinations, or season.(ABSTRACT TRUNCATED AT 250 WORDS)