Pulse-radiolytic investigations of catechols and catecholamines. II. Reactions of Tiron with oxygen radical species.

Transient spectra and kinetic data of Tiron (1,2-dihydroxybenzene-3,5-disulphonic acid) are reported, obtained after pulse-radiolytic oxidation by hydroxyl radicals (.OH), superoxide anions (O-2) or a combination of both oxygen radicals. The rate constant with .OH radicals was determined at 1.0.10(9) M-1.s-1. Contrary to a previous report (Greenstock, C.L. and Miller, R.W. (1975) Biochim. Biophys. Acta 396, 11--16), the rate constant with O-2 of 1.0.10(7) M-1.s-1 is lower by one order of magnitude; also the semiquinone absorbs at 300 nm rather than at 400 nm. The ratio of the rate constants with .OH and O-2 of 100 again demonstrates that any oxidation reaction by the latter radical is unspecific due to the more efficient reaction of .OH radicals, leading to the same products with catechol compounds.

The involvement of oxygen radicals during the autoxidation of adrenalin.

1. In unbuffered alkaline solutions, autoxidizing adrenalin generates superoxide anions: both the scavenging by adrenalin itself, leading to adrenochrome, and the formation of nitrite from hydroxylamine are inhibited by superoxide dismutase. No hydroxyl radical could be detected. 2. The yield of hydrogen peroxide increases with pH in a way similar to that of adrenochrome and nitrite. The dissociated form of adrenalin (pK = 8.5) is proposed as the source of superoxide anions. 3. Superoxide dismutase delays rather than inhibits the reaction. In addition to the diminished formation of adrenochrome due to the scavenging of superoxide anions and re-reduction of the semiquinone by hydrogen peroxide, respectively, adrenochrome is further removed by hydrogen peroxide, with final products absorbing at 310 nm. 4. The diminished inhibitory effect of superoxide dismutase above pH 10 is due to superoxide-independent reactions. This effect is masked by the alkaline conversion of adrenochrome to indole compounds. 5. It is concluded that monitoring the absorption of adrenochrome in alkaline solutions does not produce reliable evidence for superoxide anions.

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.

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.

A critical reevaluation of some assay methods for superoxide dismutase activity.

We have compared the direct method of pulse radiolysis to the indirect methods of cytochrome c and nitroblue tetrazolium for assaying the superoxide dismutase activity of a compound. We have shown that with pulse radiolysis, where high concentrations of O2- are generated, the "turnover" rate constant, kcat, can be determined directly, while with the indirect methods, where relatively low steady state concentrations of O2- are formed, the value of kcat determined by these methods, can be orders of magnitude lower than that determined directly. The main reason for the lower values obtained with the indirect methods is due to the fast reoxidation of the reduced compound by molecular oxygen. Additional problems which arise with the use of indirect methods for determining superoxide dismutase catalytic activity are discussed.

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.

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.

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)

Characterization of Cat-2t, a radiation-induced dominant cataract mutation in mice.

A dominant cataract mutation was detected recently among the offspring of x-ray-irradiated male mice. The mutation, which causes total lens opacity, has provisionally been designated by the gene symbol Cat-2t. In the lenses of heterozygous and homozygous Cat-2t mutants, the epithelial and fiber cells were swollen and the lens capsule was ruptured. The histologic analysis demonstrated a complete destruction of the cellular organization of the lens, which might be caused by its altered developmental processes. The data derived from biochemical investigations indicate that biochemistry of the cataractous Cat-2t lenses is affected: the osmotic state as indicated by the increased water content and increased Na(+)-K(+)-adenosinetriphosphatase (ATPase) activity; the energy state as indicated by the decreased adenosine triphosphate (ATP) concentration; and the redox state as indicated by the enhanced content of oxidized glutathione. Additionally, the lenticular protein composition is altered because of the presence of vimentin in the water-soluble fraction. This cannot be explained by the enhanced crosslinking activity of transglutaminase. The changes of the osmotic, energy, and redox states are considered to be secondary in relation to the altered lenticular development. In contrast, the variations concerning vimentin and transglutaminase might be a biochemical indication of the changed development. Possible similarities to other dominantly expressed murine cataract mutants are discussed.

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.

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.

On the cytotoxicity of irradiated media. To what extent are stable products of radial chain reactions in physiological saline responsible for cell death?

In a previous publication (Czapski et al. 1992) we reported that HOCl accounts for the toxicity of irradiated phosphate-buffered saline towards Escherichia coli bacterial cells. We have now investigated the respective toxicities towards lambda phage and mammalian cells. For phage, as with bacteria, cytotoxicity of the irradiated media seems to derive from HOCl without detectable contribution of H2O2. Mammalian cells (V79 CHO), in contrast, are more sensitive to H2O2 than to HOCl. Both agents, however, are not able to account quantitatively for the toxicity of irradiated solutions towards V79 cells; a hitherto unidentified chlorine/oxygen derivative--being formed in the sub-micromolar concentration range--is suggested to be responsible for toxicity in the case of eukaryotes.

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.

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.

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.