The oxidation of phenylhydrazine: superoxide and mechanism.

The oxidation of phenylhydrazine in buffered aqueous solutions is a complex process involving several intermediates. It can be initiated by metal cations, such as Cu2+; in which case EDTA acts as an inhibitor. It can also be intiated by oxyhemoglobin; in which case chelating agents do not interfere. Superoxide radical is both a product of this reaction and a chain propagator. The formation of O2- could be demonstrated in terms of a reduction of nitroblue tetrazolium, which was prevented by superoxide dismutase. The importance of O2- in carrying the reaction chains was shown by the inhibition of phenylhydrazine oxidation by superoxide dismutase. Hydrogen peroxide accumulated during the reaction and could be detected with catalase. The progress of this oxidation could be monitored in terms of oxygen consumption and by following increases in absorbance at 280 or 320 nm. The oxidation was markedly autocatalytic and superoxide dismutase had the effect of extending the lag period. The absorbance at 280 nm was due to an intermediate which first accumulated and was then consumed. This intermediate appears to be benzendiazonium ion. The absorbance at 320 nm was due to a stable product, which was not identified. The time course of oxygen consumption paralleled the increase in absorbance at 320 nm and lagged behind the changes at 280 nm. Exogenous benzenediazonium ion accelerated the oxidation of phenylhydrazine and eliminated the lag phase. Benzenediazonium ion must therefore react with phenylhydrazine to produce a very reactive intermediate, possibly phenyldiazene. A mechanism was proposed which is consistent with the data. The intermediates and products of the oxidation of phenylhydrazine include superoxide radical, hydrogen peroxide, phenylhydrazyl radical, phenyldiazene, and benzenediazonium ion. This is a minimal list: others remain to be detected and identified. It appears likely that the diverse biological effects of phenylhydrazine are largely due to the reactivities of these intermediates and products.     

The generation of O2-by the interaction of the hemolytic agent, phenylhydrazine, with human hemoglobin.

Phenylhydrazine reacts with adult oxy- or methemoglobin in the presence of molecular oxygen to generate O2-.The mechanism of the reaction apparently differs in each case. The generation of O2- is monitored by following the reduction of ferricytochrome c or the oxidation of epinephrine.     

Production of superoxide anion during the oxidation of hemoglobin by menadione.

Menadione in the presence of oxyhemoglobin will accelerate the formation of methemoglobin and result in the generation of superoxide anion. Menadione appears to oxidize slowly ferrohemoglobin to ferrihemoglobin, while forming menadione semiquinone in the process. Menadione semiquinone is known to react with molecular oxygen to yield superoxide anion. The superoxide anion appears to be the source of hydrogen peroxide which accounts for most of the observed methemoglobin formation when hemoglobin is reacted with menadione.     

The generation of superoxide anion in the reaction of tetrahydropteridines with molecular oxygen.

Cordycepin (100–200 μg/ml) blocked synthesis of all species of RNA separable by gel electrophoresis and by cellulose chromatography, similarly to actinomycin D, but more efficiently and rapidly. At low concentrations (40–80 μ/ml) cordycepin inhibited predominantly ribosomal RNA synthesis in Physarum, like toyocamycin, another adenosine analog.In nuclear preparations polyadenylylation of RNA was not affected by cordycepin. However, in the presence of cordycepin, no poly(A) RNA was found in the polysome fraction.     

Production of superoxide anion during the oxidation of hemoglobin by menadione

Menadione in the presence of oxyhemoglobin will accelerate the formation of methomoglobin and result in the generation of superoxide anion. Menadione appears to oxidize slowly ferrohemoglobin to ferrihemoglobin, while forming menadione semiquinone in the process. Menadione semiquinone is known to react with molecular oxygen to yield superoxide anion. The superoxide anion appears to be the source of hydrogen peroxide which accounts for most of the observed methemoglobin formation when hemoglobin is reacted with menadione.     

The sites of superoxide anion generation in higher plant mitochondria.

An impermeable charged paramagnetic amphiphile 4-(dodecyl dimethyl ammonium)-1-oxyl-2,2,6,6-tetramethyl piperidine bromide can be used as a probe of membrane surface potentials. Upon energization of photosystem II or photosystems I + II in illuminated spinach chloroplast thylakoids, a decrease occurs in the potential of the outer surface of these membranes of up to 14 mV.     

Cytotoxicity and superoxide anion generation by some naturally occurring quinones.

Four naturally occurring quinones, mansonone-D (MD), mansonone-H (MH), thespone (TP) and thespesone (TPE), extracted from the heartwood of Thespesia populnea have been tested for their cytotoxic action by aerobic incubation with human breast adenocarcinoma (MCF-7) cells. Toxicity of the quinones follows the order MD > TP > MH approximately TPE. EPR spectrometric and Clark electrode oximetric studies indicate that redox cycling of these quinones produce superoxide anion radical (O2*-) and H2O2 on aerobic incubation with NADH:cytochrome c reductase. Generation of superoxide radical during enzymatic reduction of quinones, was confirmed by EPR spin trapping experiment using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as a spin trap. Cyclic voltammetric studies show reversible redox couples for MD and TP whereas TPE and MH show irreversible redox couple. The electrochemical results indicate that MH and TPE are more difficult to reduce than TP and MD.     

In vitro generation of superoxide anion by the hemocytes of Macrobrachium rosenbergii: possible mechanism and pathways

The hemocytes from the giant freshwater prawn Macrobrachium rosenbergii were examined for their ability to generate superoxide anion (O(2) (-)) in vitro upon exposure to various components derived from microbial cell wall components. Among the test molecules, laminarin (a polymer of beta-1, 3 glucans), mannan and LPS from five different bacterial species produced a differential response in terms of stimulated O(2) (-) production in prawn hemocytes, suggesting the ability of the hemocytes to differentiate non-self. This response was almost completely inhibited by superoxide dismutase (SOD) suggesting SOD-inhibitable O(2) (-) generation by prawn hemocytes. Phorbol 12-myristate 13-acetate (PMA) and Ca ionophore led to enhanced O(2) (-) generation by the hemocytes and this suggests the possible role of protein kinase C and Ca(2+) ions in such generation. Cytochemical analysis using nitroblue tetrazolium (NBT)-staining revealed the importance of granular hemocytes in O(2) (-) generation in these prawns. Inhibition of O(2) (-) generation by inhibitors of NADPH-oxidase and phenoloxidase pathways clearly reveal the involvement of two different pathways in non-self stimulated O(2) (-) generation by the prawn hemocytes. These findings demonstrate the importance of O(2) (-) generation and role of possible pathways in hemocyte mediated cellular immune response of a crustacean.     

Synthesis of nitric oxide from L-arginine by neutrophils. Release and interaction with superoxide anion.

Stimulated rat peritoneal neutrophils release a platelet inhibitory factor with the pharmacological properties of NO. This release is inhibited by NG-monomethyl-L-arginine and L-canavanine, indicating that it occurs through a mechanism similar to that in vascular endothelial cells and macrophages. As the degree of stimulation increases, the factor released is progressively inactivated by concomitant release of superoxide anions.     

The mechanism of interaction of ceruloplasmin with superoxide radicals

• 1.1. The mechanism of interaction of CP with O₂ radicals in chemical and enzymatic systems of Superoxide radical generation as well as in the pulse radiolysis technique was studied.
• 2.2. It is found that CP does not exert any kinetic influence on the decomposition of Superoxide radical and, unlike SOD, cannot catalyze the reaction of disproportionation of these radicals in systems with chemical and enzymatic generation of O₂⁻.
• 3.3. The data obtained confirm the suggestion that CP interacts with precursors of ₂⁻ radicals.
• 4.4. The irradiation of CP does not change its inhibiting activity in the reaction of the formation of Superoxide radicals in systems with enzymatic O₂⁻ generation, but decreases its oxidase activity.
• 5.5. The results obtained demonstrated that the increase in the radiation dose resulted in the decrease of the inhibiting activity of SOD, whereas the activity of CP did not change.

     

Effects of the generation of superoxide anion on permeability of liposomes.

Multilamellar liposomes released previously sequestered chromate ions when these artificial membrane structures were suspended in reaction mixtures containing hypoxanthine and xanthine oxidase. Release of chromate was reduced significantly if active superoxide dismutase and/or catalase were added to these reaction mixtures. These findings suggest that superoxide anion and/or related reactive molecules are capable of perturbing lipid bilayers sufficiently to cause leakage of relatively impermeant anions.     

Involvement of superoxide anion in the reaction mechanism of haemoglobin oxidation by nitrite.

The sigmoidal time course of haemoglobin oxidation by nitrite, involving an initial slow reaction accompanied by a subsequent rapid reaction, was extensively explored. The initial slow reaction was much prolonged by the addition of superoxide dismutase to the reaction mixture. On the other hand, in the presence of superoxide anion generated by xanthine oxidase systems, the slow phase disappeared and the reaction changed to first-order kinetics. The oxidation of intermediate haemoglobins [defined as haemoglobin tetramer in which different chains (alpha- or beta-) are in the ferric state and in the ferrous state] such as (alpha 2+ beta 3+)2 and (alpha 3+ beta 2+)2 also proceeded in a sigmoidal manner. Similar effects of superoxide anion on these reactions were observed. Since the intermediate haemoglobins such as (alpha 2+ beta 3+)2 and (alpha 3+ beta 2+)2 were found to be produced by the oxidation of haemoglobin by nitrite, the changes in oxyhaemoglobin, intermediate haemoglobins and methaemoglobin during the reaction were followed by isoelectric-focusing electrophoresis. The amounts of (alpha 2+ beta 3+)2 were larger than those of (alpha 3+ beta 2+)2 at the initial stages of the reaction, suggesting that there is a functional difference between alpha- and beta-chains in the oxyhaemoglobin tetramer. On the basis of these results, a reaction model of the haemoglobin oxidation by nitrite was tentatively proposed. The changes in oxyhaemoglobin, intermediate haemoglobins and methaemoglobin were well fitted to the simulation curves generated from the reaction model. Details of the derivation of the equations used for kinetic analysis have been deposited as Supplement SUP 50112 (5 pages) with the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K. from whom copies may be obtained on the terms indicated in Biochem. J. (1978) 169, 5.     

Osmotic stress stimulates generation of superoxide anion by spermatozoa in horses

The objective of this study was to examine the interplay between osmotic and oxidative stress as well as to determine mechanisms by which osmotic stress increases superoxide generation in spermatozoa of horses. Superoxide production, as measured by dihydroethidium (DHE), increased when spermatozoa of horses were incubated under either hyperosmotic or hyposmotic conditions. This increase in superoxide production was inhibited by the MAP kinase p38 inhibitor, SB203580, and by the superoxide scavenger, tiron. Incubation of spermatozoa under hyperosmotic conditions increased overall protein tyrosine phosphorylation as measured by western blotting techniques; however, a similar increase was not detected when spermatozoa were incubated under hyposmotic conditions. The general protein kinase C (PKC) and protein tyrosine kinase (PTK) inhibitor staurosporine inhibited (P < 0.05) tyrosine phosphorylation in samples from cells under hyperosmotic conditions. In addition, the NADPH oxidase inhibitor diphenyleneiodonium (DPI) also inhibited (P < 0.05) protein tyrosine phosphorylation in cells under hyperosmotic conditions. In summary, these data indicate that incubation of equine spermatozoa under both hyposmotic and hyperosmotic conditions can increase superoxide anion generation. Under hyperosmotic conditions, this increased generation of superoxide anion was accompanied by increased protein tyrosine phosphorylation.     

The roles of degranulation and superoxide anion generation in neutrophil aggregation

Human neutrophils when exposed to appropriate stimuli aggregate, generate O(2) and secrete lysosomal constituents. To determine whether a causal relationship may exist between these responses neutrophils were exposed to either N-formyl-methionyl-leucyl-phenylalanine, phorbol myristate acetate, or the two calcium ionophores, A23187 and prostaglandin Bx. Each agent elicited all of the above responses. The concentrations required to elicit the aggregation of 30 . 10(6) neutrophils/ml were comparable to that required for O(2) generation or lysozyme release. In a series of experiments designed to dissociate these responses, cells were suspended in a concentration too dilute (3 . 10(6) neutrophils/ml) to permit aggregation to occur. O(2) generation and lysozyme release was measurable and varied in a dose-dependent fashion to the concentration of stimulus. In a second series of experiments, neutrophils were treated with 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid to inhibit degranulation without affecting O(2) generation. Aggregation was inhibited in a parallel fashion with lysozyme release. When detectable O(2) was removed from the medium by superoxide dismutase and catalase, aggregation and lysozyme release unaffected showing that aggregation can not be due to the presence of O(2) or its products in the extracellular medium. Neither aggregation of resting cells nor augmentation of fMet-Leu-Phe-induced aggregation was observed when cells were exposed to either supernatants of degranulated neutrophils or constituents of specific granules (lysozyme, lactoferrin). Kinetic analysis showed that in the absence of cytochalasin B degranulation preceded aggregation, while in its presence aggregation preceded degranulation.     

Role of the epithelial layer in the generation of superoxide anion by the guinea-pig isolated trachea.

The lucigenin-dependent chemiluminescence generation by guinea-pig isolated tracheal two rings preparations was studied. Tracheal preparations stimulated with phorbol myristate acetate (PMA) or opsonized zymosan generated chemiluminescence. The total amount of chemiluminescence generated in 120 min was 754+/-63 mV x min for PMA and 4832+/-396 mV x min for zymosan. Generation of chemiluminescence was decreased by more than 50% when the tissues were co-incubated with superoxide dismutase (100 U/ml). Also, addition of direct donors of nitric oxide diminished chemiluminescence generation by zymosan-activated tracheal rings significantly by about 50%. However, the presence of the precursor or of inhibitors of nitric oxide synthase did not influence zymosan-induced chemiluminescence. Removal of the epithelial layer from tracheal rings caused an approximately 90% decrease in chemiluminescence response. However, isolated epithelial cell suspensions did not generate chemiluminescence. Histologic examination showed that the number of eosinophils in the tracheal tissue was reduced from 56+/-7 to 18+/-8 per mm basal membrane when the epithelial layer was removed. These results indicated that (1) superoxide anion formation can take place in the guinea-pig trachea, (2) eosinophils in the epithelial and submucosal layers of guinea-pig trachea are likely candidates for superoxide generation although other cell types can also be involved, and (3) besides relaxing airway smooth muscle, nitric oxide donors may also affect superoxide in the airways.     

Oxidative damage by phenylhydrazine diminishes erythrocyte anion transport

Human erythrocytes were exposed to oxidative stress by treatment with the slowly hemolytic drug phenylhydrazine. Phenylhydrazine has been previously shown to trigger the production of toxic oxygen metabolites including O-2 and H2O2 and the formation of Heinz bodies. The concentration-dependent formation of Heinz bodies was confirmed using optical microscopy. Heinz body formation was accompanied by surface protuberances as shown by scanning electron microscopy. The formation of Heinz bodies was accompanied by inhibition of anion translocation. Anion translocation was measured using the anionic fluorescent substrate analog N-(2-aminoethylsulfonate)-7-nitrobenz-2-oxa-1,3-diazole (NBD-taurine). The efflux of NBD-taurine was measured by continuous monitoring of transport by fluorescence (CMTF). The mean value of the kinetic rate constant for transport, k, was found to be -0.090 +/- 0.017 min-1. Phenylhydrazine was found to decrease k to less than one-half of control values in a dose-dependent fashion. The disruption of anion translocation may be related to the oxidative effects of phenylhydrazine and to the generation of Heinz bodies, which bind to the N-terminal domain of band 3.     

Kinetics and mechanism of the reduction of ferricytochrome c by the superoxide anion

The temperature and pH dependence of the reaction of the superoxide radical anion with ferricytochrome c have been measured using the pulse-radiolysis technique. The temperature dependence of the reaction at low ionic strength yields an activation energy of 31 +/- 5 kJ/mol as compared to 14 +/- 3 kJ/mol for the reaction of CO2.(-) under the same conditions. The pH dependence fits the single pK'a of ferricytochrome c of 9.1. The bimolecular rate constant for the reaction of the superoxide anion with ferricytochrome c at pH 7.8, 21 +/- 2 degrees C, in the presence of 50 mM phosphate and 0.1 mM EDTA is (2.6 +/- 0.1) X 10(5) M-1 s-1. Using this value, 1 unit of superoxide dismutase activity (McCord, J. M., and Fridovich, I. (1969) J. Biol. Chem. 244, 6049-6055) is calculated to be 3.6 +/- 0.3 pmol of enzyme if the assay is performed in a total volume of 3.0 ml. Copper ions reduce the yield of the reaction of ferricytochrome c with CO2.(-). The reactivities of native and singly modified 4-carboxy-2,4-dinitrophenyllysine cytochromes c towards the superoxide anion radical are in the order native greater than 4-carboxy-2,4-dinitrophenyllysine 60 greater than lysine 13 greater than lysine 87 greater than lysine 27 greater than lysine 86 greater than lysine 72, indicating that electron transfer takes place at or close to the solvent accessible heme edge. The mechanism of the reaction is discussed in terms of the approach of superoxide anion radicals to the heme edge and the available molecular orbitals of both heme and free radicals.     

Effect of alkylated and intercalated DNA on the generation of superoxide anion by riboflavin

Superoxide anion (O ₂ ^.– ) was photogenerated upon illumination of riboflavin in fluorescent light. The rate of O ₂ ^.– formation was stimulated by double stranded DNA but not by denatured DNA or RNA. Depurinated DNA, which was predominantly depleted in guanine residues, did not exhibit the stimulatory effect, indicating an interaction of riboflavin, or active oxygen species derived from it, with guanine bases. Also, the stimulation of O ₂ ^.– photogeneration was not observed with ethidium bromide but was seen with proflavin-intercalated DNA. Since ethidium bromide intercalates preferentially between purines and pyrimidines, and proflavin prefers dA-dT rich sites, these results were interpreted to suggest that the interaction of riboflavin with DNA is mainly with GC or CG base pairs.     

Nitric oxide decreases superoxide anion generation by microsomes from soybean embryonic axes

Experiments were carried out to evaluate the effects of exposure to nitric oxide on the ability by NADPH‐dependent microsomal electron transfer to generate oxygen radicals. Such interactions could play a role in the potential antioxidant action of nitric oxide (NO). Isolated microsomes from soybean ( Glycine max [L.] Merr. cv. Hood) embryonic axes were exposed to an exogenously added source of nitric oxide (NO) (S‐nitrosoglutathione + dithiothreitol). The O₂⁻ generation rate by microsomes exposed to NO decreased significantly as compared to the rate measured in microsomes incubated in the absence of NO. The exposure of the microsomes to the NO donor did not alter the microsomal rate of hydroxyl radical generation. Preincubation of the microsomes with the NO donor affected neither iron reduction rate nor activity of cytochrome c reductase. However, cytochrome P₄₅₀ activity was significantly inhibited after exposure to NO. This inhibition was completely prevented by hemoglobin. The data are consistent with the hypothesis that NO exhibits a potential antioxidant role in the plant cell by decreasing the rate of generation of superoxide anion. Since endogenous NO was detected in homogenates of soybean embryonic axes by EPR studies, this interaction between NO and cytochrome P₄₅₀ in soybean embryonic axes could be a factor of relevance for the control of oxidative stress in vivo.