Mutations by near-ultraviolet radiation in Escherichia coli strains lacking superoxide dismutase

Om wild-type Escherichia coli, near-ultraviolet radiation (NUV) was only weakly mutagenic. However, in an allelic mutant strain (sodA sodB) that lacks both Mn- and Fe-superoxide dismutase (SOD) and assumed to have excess superoxide anion (O₂⁻), NUV induced a 9-fold increase in mutation above the level that normally occurs in this double mutant. When a sodA sodB double mutant contained a plasmid carrying katG⁺ HP-I catalase), mutation by NUV was reduced to wild-type (sodA⁺sodB⁺) levels. Also, in the sodA sodB xthA triple mutant, which lacks exonuclease III (exoIII) in addition to SOD, the mutations frequency by NUV was reduced to wild-type levels. This synergistic action of NUV and O₂⁻ suggested that pre-mutational lesions occur, with exoIII converting these lesions to stable mutants. Exposure to H₂O₂ induced a 2.8 fold increase in mutations in sodA sodB double mutants, but was reduced to control levels when a plasmid carrying katG⁺ was introduced. These results suggest that NUV, in addition to its other effects on cells, increases mutations indirectly by increasing the flux of OH^. radicals, possibly by generating excess H₂O₂.     

Mechanisms of H2O2-induced oxidative stress in endothelial cells

Hydrogen peroxide, produced by inflammatory and vascular cells, induces oxidative stress that may contribute to endothelial dysfunction. In smooth muscle cells, H₂O₂ induces production of O₂⁻ by activating NADPH oxidase. However, the mechanisms whereby H₂O₂ induces oxidative stress in endothelial cells are poorly understood. We examined the effects of H₂O₂ on O₂⁻ levels on porcine aortic endothelial cells (PAEC). Treatment with 60 μmol/L H₂O₂ markedly increased intracellular O₂⁻ levels (determined by conversion of dihydroethidium to hydroxyethidium) and produced cytotoxicity (determined by propidium iodide staining) in PAEC. Overexpression of human manganese superoxide dismutase in PAEC reduced O₂⁻ levels and attenuated cytotoxicity resulting from treatment with H₂O₂. L-NAME, an inhibitor of nitric oxide synthase (NOS), and apocynin, an inhibitor of NADPH oxidase, reduced O₂⁻ levels in PAEC treated with H₂O₂, suggesting that both NOS and NADPH oxidase contribute to H₂O₂-induced O₂⁻ in PAEC. Inhibition of NADPH oxidase using apocynin and NOS rescue with L-sepiapterin together reduced O₂⁻ levels in PAEC treated with H₂O₂ to control levels. This suggests interaction-distinct NOS and NADPH oxidase pathways to superoxide. We conclude that H₂O₂ produces oxidative stress in endothelial cells by increasing intracellular O₂⁻ levels through NOS and NADPH oxidase. These findings suggest a complex interaction between H₂O₂ and oxidant-generating enzymes that may contribute to endothelial dysfunction.     

Ovotransferrin possesses SOD-like superoxide anion scavenging activity that is promoted by copper and manganese binding

The embryo of oviparous species is confronted by a highly oxidative stress generating as it grows and must rely on effective antioxidant system for protection. Proteins of avian egg albumin have been suggested to play the major redox-modulatory role during embryo development. Recently, we found that ovotransferrin (OTf) undergoes distinct thiol-linked self-cleavage in a redox-dependent process. In this study, we explore that OTf is SOD mimic protein with a potent superoxide anion (O₂⁻) scavenging activity. The O₂⁻ scavenging activity was investigated using the natural xanthine/xanthine oxidase (X/XOD) coupling system. OTf exhibited O₂⁻ scavenging activity in a dose-dependent manner and showed remarkably higher scavenging activity than the known antioxidants, ascorbate or serum albumin. The isolated half-molecules of OTf exhibited higher scavenging activity than the intact molecule, whereas the N-lobe showed much greater activity. OTf dramatically quenched the O₂⁻ flux but had no effect on the urate production in the X/XOD system, indicating its unique specificity to scavenge O₂⁻ but not oxidase inhibition. Strikingly, metal-bound OTf exhibited greater O₂⁻ dismutation capacity than the apo-protein, ranging from moderate (Zn²⁺-OTf and Fe²⁺-OTf) to high (Mn²⁺-OTf and Cu²⁺-OTf) activity with the Cu²⁺-OTf being the most potent scavenger. In a highly sensitive fluorogenic assay, the metal-bound OTf exhibited significant increase in the rate of H₂O₂ production in the X/XOD reaction than the apo-OTf, providing evidence that Zn²⁺-, Mn²⁺- and Cu²⁺-OTf possess SOD mimic activity. This finding is the first to describe that OTf is an O₂⁻ scavenging molecule, providing insight into its novel SOD-like biological function, and heralding a fascinating opportunity for its potential candidacy as antioxidant drug.     

Crystal and microparticle effects on MDCK cell superoxide production: oxalate-specific mitochondrial membrane potential changes

We have previously shown that crystals of calcium oxalate (COM) elicit a superoxide (O₂⁻) response from mitochondria. We have now investigated: (i) if other microparticles can elicit the same response, (ii) if processing of crystals is involved, and (iii) at what level of mitochondrial function oxalate acts. O₂⁻ was measured in digitonin-permeabilized MDCK cells by lucigenin (10 μM) chemiluminescence. [¹⁴C]-COM dissociation was examined with or without EDTA and employing alternative chelators. Whereas mitochondrial O₂⁻ in COM-treated cells was three- to fourfold enhanced compared to controls, other particulates (uric acid, zymosan, and latex beads) either did not increase O₂⁻ or were much less effective (hydroxyapatite +50%, p < 0.01), with all at 28 μg/cm². Free oxalate (750 μM), at the level released from COM with EDTA (1 mM), increased O₂⁻ (+50%, p < 0.01). Omitting EDTA abrogated this signal, which was restored completely by EGTA and partially by ascorbate, but not by desferrioxamine or citrate. Omission of phosphate abrogated O₂⁻, implicating phosphate-dependent mitochondrial dicarboxylate transport. COM caused a time-related increase in the mitochondrial membrane potential (Δψ_m) measured using TMRM fluorescence and confocal microscopy. Application of COM to Fura 2-loaded cells induced rapid, large-amplitude cytosolic Ca²⁺ transients, which were inhibited by thapsigargin, indicating that COM induces release of Ca²⁺ from internal stores. Thus, COM-induced mitochondrial O₂⁻ requires the release of free oxalate and contributes to a synergistic response. Intracellular dissociation of COM and the mitochondrial dicarboxylate transporter are important in O₂⁻ production, which is probably regulated by Δψ_m.     

Trehalose: Protector of antioxidant enzymes or reactive oxygen species scavenger under heat stress?

Trehalose is known to protect membranes and macromolecules. Its accumulation has been implicated in allowing plants to tolerate stress, including heat-shock. However, under heat-shock, it is not clear whether trehalose eliminates reactive oxygen species (ROS) directly or indirectly by protecting antioxidant enzymes. In this study, we initially examined the effects of trehalose on the activities of key antioxidant enzymes, including superoxide dismutases (SODs), ascorbate catalases (CATs), and ascorbate peroxidases (APX) from wheat (Triticum aestivum L.), and then measured the ability of trehalose to scavenge hydrogen peroxide (H₂O₂) and superoxide anions (O₂⁻). Our results indicated that trehalose protected SOD activity slightly. However, it inhibited CAT and APX activities under heat stress, with a little protection of CAT activity (only about 7% promotion) at 22 °C. Moreover, trehalose scavenged H₂O₂ and O₂⁻ greatly in a concentration-dependent manner, reaching the maximal scavenging H₂O₂ rate of 95% and O₂⁻ rate of 78%, respectively, at 50 mM trehalose. These results suggest that trehalose plays a direct role in eliminating H₂O₂ and O₂⁻ in wheat under heat stress.     

Biosynthesis and regulation of superoxide dismutases

The past two decades have witnessed an explosion in our understanding of oxygen toxicity. The discovery of superoxide dismutases (SODs) (EC.1.15.1.1), which specifically catalyze the dismutation of superoxide radicals (O₂⁻) to hydrogen peroxide (H₂O₂) and oxygen, has indicated that O₂⁻ is a normal and common byproduct of oxygen metabolism. There is an increasing evidence to support the conclusion that superoxide radicals play a major role in cellular injury, mutagenesis, and many diseases. In all cases SODs have been shown to protect the cells against these deleterious effects. Recent advances in molecular biology and the isolation of different SOD genes and SOD c-DNAs have been useful in proving beyond doubt the physiological function of the enzyme. The biosynthesis of SODs, in most biological systems, is under rigorous controls. In general, exposure to increased pO₂, increased intracellular fluxes of O₂⁻, metal ions perturbation, and exposures to several environmental oxidants have been shown to influence the rate of SOD synthesis in both prokaryotic and eukaryotic organisms. Recent developments in the mechanism of regulation of the manganese-containing superoxide dismutase of Escherichia coli will certainly open new research avenues to better understand the regulation of SODs in other organisms.     

Increased levels of superoxide in brains from old female rats

Hypertension, aging and a range of neurodegenerative diseases are associated with increased oxidative damage. The present study examined whether superoxide (O₂^•-) levels in brain are increased during aging in female rats, and the role of superoxide dismutase (SOD) and oestrogen in regulating O₂^•- levels.

Young adult (3 month) and old (11 month) female spontaneously hypertensive stroke prone rats (SHRSP) and normotensive Wistar-Kyoto rats (WKY) were studied. O₂^•- levels were measured in brain homogenates by lucigenin chemiluminescence and SOD expression by Western blotting. Ageing significantly increased brain O₂^•- levels in WKY (cortex +216%, hippocampus +320%, striatum +225%) and to a greater extent in SHRSP (cortex +540%, hippocampus +580%, striatum +533%). Older SHRSP showed a decline in cortical Cu/Zn SOD expression compared to young adult SHRSP. Oestrogen did not attenuate O₂^•- levels.

The results show a significant age-dependent increase in brain O₂^•- levels which is exaggerated in SHRSP. The excess cortical O₂^•- levels in the SHRSP may be associated with a down-regulation of Cu/Zn SOD but are not related to a decrease in oestrogen.     

Direct measurement of oscillatory generation of superoxide anions by single phagocytes

Phagocytic cells such as neutrophils generate superoxide anions (O₂⁻) within phagocytic vacuoles for killing and digesting microorganisms. Here we report the simultaneous observation of morphological changes and O₂⁻ generation in single phagocytic cells during phagocytosis. Point stimulation of a cell by contact with an opsonized microelectrode at the cell surface induced significant deformation to engulf the electrode, and also induced the O₂⁻ generation which was measured by the electrode. Periodic fluctuations in the magnitude of the O₂⁻ generation were observed in the time course. These oscillations may be caused by metabolic regulation of the formation of NADPH, which is the substrate for the O₂⁻ generation.     

Bipyridylium quaternary salts and related compounds. V. Pulse radiolysis studies of the reaction of paraquat radical with oxygen. Implications for the mode of action of bipyridyl herbicides

1. Rate constants for reduction of paraquat ion (1,1′-dimethyl-4,4′-bipyridy-lium, PQ²⁺) to paraquat radical (PQ⁺·) by e⁻_aq and CO₂⁻· have been measured by pulse radiolysis. Reduction by e⁻_aq is diffusion controlled (k = 8.4·10¹⁰ M⁻¹·s⁻¹) and reduction by CO₂⁻· is also very fast k = 1.5·10¹⁰ M⁻¹·s⁻¹).

2. The reaction of paraquat radical with oxygen has been analysed to give rate constants of 7.7·10⁸ M⁻¹·s⁻¹ and 6.5·10⁸ M⁻¹·s⁻¹ for the reactions of paraquat radical with O₂ and O₂⁻·, respectively. The similarity in these rate constants is in marked contrast to the difference in redox potentials of O₂ and O₂⁻· (− 0.59 V and + 1.12 V, respectively).

3. These rate constants, together with that for the self-reaction of O₂⁻·, have been used to calculate the steady-state concentration of O₂⁻· under conditions thought to apply at the site of reduction of paraquat in the plant cell. On the basis of these calculations the decay of O₂⁻· appears to be governed almost entirely by its self-reaction, and the concentration 5 μm away from the thylakoid is still 90% of that at the thylakoid itself. Thus, O₂⁻· persists long enough to diffuse as far as the chloroplast envelope and tonoplast, which are the first structures to be damaged by paraquat treatment. O₂⁻· is therefore sufficiently long-lived to be a candidate for the phytotoxic product formed by paraquat in plants.     

Reactions of the ferri-ferrocytochrome-c system with superoxide/oxygen and CO2/CO2 studied by fast pulse radiolysis

The reduction of ferricytochrome c by O₂⁻ and CO₂⁻ was studied in the pH range 6.6–9.2 and Arrhenius as well as Eyring parameters were derived from the rate constants and their temperature dependence. Ionic effects on the rate indicate that the redox process proceeds through a multiply-positively charged interaction site on cytochrome c. It is shown that the reaction with O₂⁻ and correspondingly with O₂ of ferrocytochrome c) is by a factor of approx. 10³ slower than warranted by factors such as redox potential. Evidence is adduced to support the view that this slowness is connected with the role of water in the interaction between O₂⁻/O₂ and ferri-ferrocytochrome c in the positively charged interaction site on cytochrome c in which water molecules are specifically involved in maintaining the local structure of cytochrome c and participate in the process of electron equivalent transfer.     

Protection of Chinese hamster ovary cells from paraquat-mediated cytotoxicity by a low molecular weight mimic of superoxide dismutase (DF-Mn)

Paraquat exerts a cytotoxic effect of Chinese hamster ovary cells in culture via the superoxide radical (O₂⁻. We have described a superoxide dismutase (SOD) mimic based on manganese (DF-Mn) which consists of a one-to-one complex between desferrioxamine B (Desferal) and MnO₂. It is a small molecular weight molecule, easy to prepare and possesses considerable stability. It is now shown to protect mammalian cells from paraquat toxicity. Thus, 20 μM DF-Mn affords up to complete protection against the cytotoxicity of 200 μM paraquat in Chinese hamster ovary cells. Desferrioxamine B or MnO₂ alone gave no protection. MnCl₂ or catalase provided little or no protection against the paraquat, respectively. Equivalent amounts of human Cu-Zn SOD in terms of activity, also provided no protection. Copper diisopropylsalicylate (CuDIPS) provided limited, yet significant, protection, but this is explained in terms other than SOD activity. Finally, at higher concentrations, purified human SOD, exerts a limited toxicity as well as a protective ability against paraquat (similar to DF-Mn) both of which are eliminated upon heat denaturation of the enzyme. It appears that the SOD mimic, DF-Mn, can enter mammalian cells and can protect against the cytotoxic effects of O₂⁻.     

The reaction between the superoxide anion radical and cytochrome c

1. 1. The superoxide anion radical (O₂⁻) reacts with ferricytochrome c to form ferrocytochrome c. No intermediate complexes are observable. No reaction could be detected between O₂⁻ and ferrocytochrome c.

2. 2. At 20 °C the rate constant for the reaction at pH 4.7 to 6.7 is 1.4 · 10⁶ M⁻¹ · s⁻¹ and as the pH increases above 6.7 the rate constant steadily decreases. The dependence on pH is the same for tuna heart and horse heart cytochrome c. No reaction could be demonstrated between O₂⁻ and the form of cytochrome c which exists above pH ≈ 9.2. The dependence of the rate constant on pH can be explained if cytochrome c has pKs of 7.45 and 9.2, and O₂⁻ reacts with the form present below pH 7.45 with k = 1.4 · 10⁶ M⁻¹ · s⁻¹, the form above pH 7.45 with k = 3.0 · 10⁵ M⁻¹ · s⁻¹, and the form present above pH 9.2 with k = 0.

3. 3. The reaction has an activation energy of 20 kJ mol⁻¹ and an enthalpy of activation at 25 °C of 18 kJ mol⁻¹ both above and below pH 7.45. It is suggested that O₂⁻ may reduce cytochrome c through a track composed of aromatic amino acids, and that little protein rearrangement is required for the formation of the activated complex.

4. 4. No reduction of ferricytochrome c by HO₂ radicals could be demonstrated at pH 1.2–6.2 but at pH 5.3, HO₂ radicals oxidize ferrocytochrome c with a rate constant of about 5 · 10⁵–5 · 10⁶ M⁻¹ · s⁻¹

.     

Superoxide anion burst and taxol production induced by Ce in suspension cultures of Taxus cuspidata

Generation of reactive oxygen species (ROS) induced by Ce⁴⁺ in suspension cultures of Taxus cuspidata was investigated. The burst of superoxide anions (O₂√⁻) occurred rapidly after the addition of Ce⁴⁺ and reached maximum at 4.3 h, while the total level of the cellular reactive oxygen species maintained unchanged. The intracellular superoxide dismutase (SOD) and catalase (CAT) were activated while the intra/extracellular peroxidases (PODs) were inhibited accompanying the O₂√⁻ burst. The pretreatment of the suspension cultures with diphenylene iodonium (DPI), a suicide inhibitor of the NADPH oxidase, blocked the O₂√⁻ burst, inhibiting the cell apoptosis and taxol production induced by Ce⁴⁺. These results show that NADPH oxidase played a key role in O₂√⁻ burst and O₂√⁻ served as a mediator of Ce⁴⁺ for cell apoptosis and taxol production. The pretreatments of the suspension cultures with anthracene-9-carboxylate, an ion-channel blocker, nifedipine, a Ca²⁺-channel blocker, neomycin, a phospholipase C (PLC) inhibitor, or suramin, a G-protein inhibitor, decreased O₂√⁻ burst induced by Ce⁴⁺. It is thus inferred that Ce⁴⁺-induced O₂√⁻ burst, which mediated cell apoptosis and taxol production by activating the ion-channels, PLC, G-proteins and NADPH oxidase.     

The antioxidant action of N-acetylcysteine: Its reaction with hydrogen peroxide, hydroxyl radical, superoxide, and hypochlorous acid

N-acetylcysteine has been widely used as an antioxidant in vivo and in vitro. Its reaction with four oxidant species has therefore been examined. N-acetylcysteine is a powerful scavenger of hypochlorous acid (H---OCl); low concentrations are able to protect ₁-antiproteinase against inactivation by HOCl. N-acetylcysteine also reacts with hydroxyl radical with a rate constant of 1.36 × 10¹⁰ M⁻¹s⁻¹, as determined by pulse radiolysis. It also reacts slowly with H₂O₂, but no reaction of N-acetylcysteine with superoxide (O₂⁻) could be detected within the limits of our assay procedures.     

Mitochondrial superoxide production during oxalate-mediated oxidative stress in renal epithelial cells

Crystals of calcium oxalate monohydrate (COM) in the renal tubule form the basis of most kidney stones. Tubular dysfunction resulting from COM-cell interactions occurs by mechanism(s) that are incompletely understood. We examined the production of reactive oxygen intermediates (ROI) by proximal (LLC-PK1) and distal (MDCK) tubular epithelial cells after treatment with COM (25–250 μg/ml) to determine whether ROI, specifically superoxide (O₂^•−), production was activated, and whether it was sufficient to induce oxidative stress. Employing inhibitors of cytosolic and mitochondrial systems, the source of ROI production was investigated. In addition, intracellular glutathione (total and oxidized), energy status (ATP), and NADH were measured. COM treatment for 1–24 h increased O₂^•− production 3–6-fold as measured by both lucigenin chemiluminescence in permeabilized cells and dihydrorhodamine fluorescence in intact cells. Using selective inhibitors we found no evidence of cytosolic production. The use of mitochondrial probes, substrates, and inhibitors indicated that increased O₂^•− production originated from mitochondria. Treatment with COM decreased glutathione (total and redox state), indicating a sustained oxidative insult. An increase in NADH in COM-treated cells suggested this cofactor could be responsible for elevating O₂^•− generation. In conclusion, COM increased mitochondrial O₂^•− production by epithelial cells, with a subsequent depletion of antioxidant status. These changes may contribute to the reported cellular transformations during the development of renal calculi.     

Evidence that de novo protein synthesis participates in a time-dependent augmentation of the chemotactic peptide-induced respiratory burst in neutrophils: Effects of recombinant human colony stimulating factors and dihydrocytochalasin B

We examined the effects of the recombinant human colony stimulating factors GM-CSF and G-CSF, cycloheximide (a protein synthesis inhibitor) and dihydrocytochalasin B (a microfilament disrupting agent) upon FMLP (N-formyl-methionyl-leucylphenylalanine)-stimulated O₂ ⁻ production by neutrophils. We confirmed a time dependent augmentation of O₂ ⁻ production following preincubation of neutrophils either alone or with colony stimulating factors. Furthermore, we found that GM-CSF, but not G-CSF, increased O₂ ⁻ production at some concentrations of the stimulus. Preincubation of neutrophils with cycloheximide in the absence of CSF caused a marked fall in O₂⁻-production that was first evident at 2 hours. The fall in O₂⁻-forming capacity caused by cycloheximide was much less pronounced if dihydrocytochalasin B was also included in the preincubation buffer. These findings suggest a previously unrecognized role for de novo protein synthesis in maintaining the ability of neutrophils to manufacture O₂⁻, and support earlier studies indicating that the cycling of FMLP receptors between the cell membrane and an intracellular compartment is important in determining the magnitude of the respiratory burst in FMLP-stimulated neutrophils.     

A new type of O(2)(-)-generating tool for oxidative stress studies by remodeling neutrophil NADPH oxidase

The effects of reactive oxygen species on cells have attracted much attention in relation to redox regulation and oxidative stress-related diseases. Superoxide (O₂⁻) is the reactive oxygen species primarily formed in biological systems. However, no convenient O₂⁻-generating device has been available for use in cell or tissue culture. The neutrophil NADPH oxidase, a professional enzyme for killing bacteria, has a high ability to produce O₂⁻. However, the cell-free activation process requires several protein factors and an anionic amphiphile, and moreover, the activation is transient. To utilize the enzyme as an O₂⁻ generator, we improved the cell-free activation method by remodeling regulatory components, optimizing lipid composition, and modifying the mixing conditions. We established a new method to produce an active enzyme that is stable, efficient, and preservable. As an application, we examined the effect of the device on cultured HEK293 cells and observed that it caused cell death. This system has several advantages over the xanthine oxidase system often used. The new device will be useful for studies of oxidative stress and related diseases.     

Up-expression of NapA and other oxidative stress proteins is a compensatory response to loss of major Helicobacter pylori stress resistance factors

Twenty-six Helicobacter pylori targeted mutant strains with deficiencies in oxidative stress combating proteins, including 12 double mutant strains were analyzed via physiological and proteomic approaches to distinguish the major expression changes caused by the mutations. Mutations were introduced into both a Mtz^S and a Mtz^R strain background. Most of the mutations caused increased growth sensitivity of the strains to oxygen, and they all exhibited clear compensatory up-expression of oxidative stress resistance proteins enabling survival of the bacterium. The most frequent up-expressed oxidative stress resistance factor (observed in 16 of the mutants) was the iron-sequestering protein NapA, linking iron sequestration with oxidative stress resistance. The up-expression of individual proteins in mutants ranged from 2 to 10 fold that of the wild type strain, even when incubated in a low O₂ environment. For example, a considerably higher level of catalase expression (4 fold of that in the wild-type strain) was observed in ahpC napA and ahpC sodB double mutants. A Fur mutant up-expressed ferritin (Pfr) protein 20-fold. In some mutant strains the bacterial DNA is protected from oxidative stress damage apparently via overexpression of oxidative stress-combating proteins such as NapA, catalase or MdaB (an NADPH quinone reductase). Our results show that H. pylori has a variety of ways to compensate for loss of major oxidative stress combating factors.     

Oxidative phosphorylation coupled to oxygen uptake and nitrate reduction in Micrococcus denitrificans

A procedure is described for preparing particles from cells of Micrococcus denitrificans which were broken osmotically after treatment with lysozyme.

1. 1. The preparations catalysed ATP synthesis coupled to O₂ uptake or NO₃⁻ reduction. With NADH or succinate as the electron donors the P:O ratios were about 1.5 and 0.5, respectively; and the P:NO₃⁻ ratios were about 0.9 and 0.06, respectively.

2. 2. Addition of ADP or P_i to the reaction mixture increased the rates of NADH-dependent O₂ uptake and NO₃⁻ reduction. Addition of 1 mM 2,4-dinitrophenol, which inhibited phosphorylation by 50–60%, increased the basal rates of electron transport.

3. 3. Evidence derived from spectrophotometry and from the differential inhibition by antimycin A of O₂ and NO₃⁻ reduction leads to the conclusion that the nitrate reductase interacted with the respiratory chain in the region of the b-type cytochrome, and that the c-type cytochrome present was not involved in the reduction of NO₃⁻ to NO₂⁻.

Vanadate-stimulated oxidation of NAD(P)H

Vanadate stimulates the oxidation of NAD(P)H by biological membranes because such membranes contain NAD(P)H oxidases which are capable of reducing dioxygen to O₂⁻ and because vanadate catalyzes the oxidation of NAD(P)H by O₂⁻, by a free radical chain mechanism. Dihydropyridines, such as reduced nicotinamide mononucleotide (NMNH), which are not substrates for membrane-associated NAD(P)H oxidases, are not oxidized by membranes plus vanadate unless NAD(P)H is present to serve as a source of O₂⁻. When [NMNH] greatly exceeds [NAD(P)H], in such reaction mixtures, one can observe the oxidation of many molecules of NMNH per NAD(P)H consumed. This reflects the chain length of the free radical chain mechanism. We have discussed the mechanism and significance of this process and have tried to clarify the pertinent but confusing literature.