Extracellular superoxide dismutase in vessels and airways of humans and baboons

Extracellular superoxide dismutase (EC SOD) is generally the least abundant SOD isozyme in tissues, while the intracellular Cu,Zn SOD is usually the most abundant isozyme. The biological significance of EC SOD is unknown. Immunolocalization studies show that EC SOD is in the connective tissue surrounding smooth muscle in vessels and airways within the lung. Endothelium derived relaxing factor, thought to be a nitric oxide (NO^·) species, is a primary mediator of vascular relaxation. During NO^·′ diffusion between the endothelium and smooth muscle, extracellular superoxide would be the most efficient scavenger of NO^·. High levels of extracellualar superoxide dismutase in vessels could, therefore, be essential to enable NO' to modulate vascular tone. To evaluate the hypothesis that vessel walls are functionally rich in extracellular superoxide scavenging capacity, this study quantitates the EC SOD levels in pulmonary and systemic vessels and in airways. Both pulmonary and systemic arteries in humans and baboons were found to contain high activities of EC SOD. The level of EC SOD in all human and baboon arteries examined is greater than or equal to the level of intracellular Cu,Zn SOD, and EC SOD accounted for over 70% of the total SOD activity in some vessels examined. Immunolocalization of EC SOD in human and baboon vessels show similar distributions of this enzyme in pulmonary and systemic vessels. EC SOD is located beneath the endothelium, surrounding smooth muscle cells, and throughout the adventitia of vessels. The high level of EC SOD in vessels, and its localization between endothelial and smooth muscle cells, suggest that regulation of superoxide may be particularly important in this region, possibly in regulating vascular tone.     

The effects of superoxide dismutase on H2O2 formation

Numerous reports of the effects of overproduction of SODs have been explained on the basis of increased H2O2 production by the catalyzed dismutation of O2-. In this review we consider the effects of increasing [SOD] on H2O2 formation and question this explanation.     

Nitric oxide synthase inhibitors decrease human polymorphonuclear leukocyte luminol-dependent chemiluminescence

Nitric oxide synthase (NOS) inhibitors have been reported to modulate luminol-dependent chemiluminescence (CL) in rat macrophages, whereas the potent oxidant peroxynitrite (ONOO^-) was shown to react with luminol to yield CL in a cellfree system. We evaluated the role of the -arginine/NOS pathway in luminol CL by phorbol ester-activated human polymorpho-nuclear (PMN) leukocytes using the NOS inhibitors N^G-monomethyl- -arginine ( -NMMA) and N-iminoethyl- -omithine ( -NIO). Nitric oxide (·NO) release was determined by oxidation of oxymyoglobin. In addition, the effect of NOS inhibitors on superoxide anion O₂^-) production was measured. Luminol CL was notably diminished by -NMMA in a dose-dependent manner. Superoxide dismutase (SOD) also decreased luminol CL and -NMMA potentiated light emission decrease produced by SOD. Nitric oxide and O₂^·- production was significantly decreased by -NMMA; moreover, luminol-dependent CL but not O₂^·- production was attenuated by -NIO. These data suggest that products of catalytic activity of both ·NO synthase and NADPH oxidase are required to elicit maximal luminol CL in this system. These studies demonstrate that the NOS synthase pathway is involved in luminol CL by human PMN, and they suggest that ONOO would be an unrecognized mediator in this phenomenon.     

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.     

Target cell-derived superoxide anions cause efficiency and selectivity of intercellular induction of apoptosis

Transformed fibroblasts are specifically eliminated by their nontransformed neighbors through intercellular induction of apoptosis. This process depends on the number of nontransformed effector cells and on the local density of transformed target cells. Intercellular signalling is inhibited by SOD (a scavenger of superoxide anions), taurine (a scavenger of HOCl), 4-aminobenzoyl hydrazide (a mechanism-based inhibitor of peroxidase), DMSO (a hydroxyl radical scavenger), and two inhibitors of NO synthase. Therefore, selective apoptosis induction seems to be based on superoxide anion production by transformed cells, their spontaneous dismutation to hydrogen peroxide, and HOCl generation by a novel effector cell-derived peroxidase. HOCl then interacts with target cell–derived superoxide anions to yield hydroxyl radicals. Due to the short diffusion pathway of superoxide anions, hydroxyl radical generation is confined to the intimate vicinity of transformed cells. In parallel, NO derived from effector cells interacts with superoxide anions of target cells to yield the apoptosis inducer peroxynitrite. Reconstitution experiments using transformed or nontransformed cells in conjunction with myeloperoxidase, HOCl, or an NO donor demonstrated that superoxide anions generated extracellularly by transformed cells participate in intercellular signalling and at the same time determine transformed cells as selective targets for intercellular induction of apoptosis.     

Efficient nitrosation of glutathione by nitric oxide

Nitrosothiols are increasingly regarded as important participants in a range of physiological processes, yet little is known about their biological generation. Nitrosothiols can be formed from the corresponding thiols by nitric oxide in a reaction that requires the presence of oxygen and is mediated by reactive intermediates (NO₂ or N₂O₃) formed in the course of NO autoxidation. Because the autoxidation of NO is second order in NO, it is extremely slow at submicromolar NO concentrations, casting doubt on its physiological relevance. In this paper we present evidence that at submicromolar NO concentrations the aerobic nitrosation of glutathione does not involve NO autoxidation but a reaction that is first order in NO. We show that this reaction produces nitrosoglutathione efficiently in a reaction that is strongly stimulated by physiological concentrations of Mg²⁺. These observations suggest that direct aerobic nitrosation may represent a physiologically relevant pathway of nitrosothiol formation.     

The role of the reactions of NO with superoxide and oxygen in biological systems: A kinetic approach

In this study we calculate the half-life of ^·NO in its reactions with superoxide and with oxygen under various conditions using the known rate constants for these reactions. The measured half-life of ^·NO in biological systems is 3–5 s, which agrees well with the calculated value for intracellular ^·NO, but not for extracellular ^·NO under normal physiological conditions. The autoxidation of ^·NO to yield NO₂ as a final product cannot be responsible for such a short measured half-life under normal as well as pathologic conditions. Therefore, if there is direct evidence for the occurrence of the reaction of ^·NO with O₂ in the medium, one has to assume that the steady state concentrations of free ^·NO are much lower than those measured. The very low concentrations of free ^·NO in biological systems may result from its reversible strong binding to biological molecules. Simulation of the mechanism of the autoxidation of ^·NO indicates that the binding constants of ^·NO to O₂ or to another ^·NO are too small to account for the very low concentration of free ^·NO in biological systems. Nevertheless, the reaction of ^·NO with oxygen cannot be neglected in biological systems if the intermediate ONOO· reacts rapidly with a biological target. The biological damage caused by ONOO′ is expected to be due to the radical itself and to peroxynitrite, which is most probably formed via the reaction of ONOO· with the biological molecule.     

Hemodynamic effects of metalloporphyrin catalytic antioxidants: structure-activity relationships and species specificity

Superoxide plays a role in blood pressure regulation in certain vascular diseases, however, its involvement in regulating basal blood pressure is uncertain. Vascular superoxide concentrations are limited by extracellular superoxide dismutase (EC-SOD), which is highly expressed in the vasculature of most animal species. Metalloporphyrins are low molecular weight, synthetic, redox-active, catalytic antioxidants that act as SOD mimetics. We evaluated the effects of metalloporphyrins on blood pressure in different animal species. The metalloporphyrin AEOL10113 (5–10 μg/kg iv), but not native or polyethylene glycol-CuZnSOD, caused a dose-dependent reduction in blood pressure in anesthetized rats. AEOL10113 had no effect on blood pressure in mice (wild-type or EC-SOD knockouts), guinea pigs, dogs, or baboons at doses up to 5 mg/kg iv Structure-activity studies indicated that metalloporphyrins with high SOD activity were more effective in lowering rat blood pressure than low-activity analogs. The blood pressure effect of AEOL10113 was not attributable to the release of manganese, nor was it affected by inhibitors of nitric oxide synthase (L-NAME) and guanylate cyclase (ODQ, 8-bromo-cGMP, and methylene blue) or nitric oxide scavengers (HbA_o). Chlorpheniramine attenuated the effect, suggesting that the blood pressure response in rats is related to histamine release rather than the protection of nitric oxide.     

Inhibition of mitochondria-dependent apoptosis by 635-nm irradiation in sodium nitroprusside-treated SH-SY5Y cells

Nitric oxide (NO) is a major factor contributing to the loss of neurons in ischemic stroke, demyelinating diseases, and other neurodegenerative disorders. NO not only functions as a direct neurotoxin, but also combines with superoxide (O₂⁻) by a diffusion-controlled reaction to form peroxynitrite (ONOO⁻), a species that contributes to oxidative signaling and cellular apoptosis. However, the mechanism by which ONOO⁻ induces apoptosis remains unclear, although subsequent formation of reactive oxygen species (ROS) has been suggested. The aim of this study was to further investigate the triggers of the apoptotic pathway using O₂⁻ scavenging with light irradiation to block ONOO⁻ production. Antiapoptotic effects of light irradiation in sodium nitroprusside (SNP)-treated SH-SY5Y cells were assayed by reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, DNA fragmentation, flow cytometry, Western blot, and caspase activity assays. In addition, NO, total ROS, O₂⁻, and ONOO⁻ levels were measured to observe changes in NO and its possible involvement in radical induction. Cell survival was reduced to approximately 40% of control levels by SNP treatment, and this reduction was increased to 60% by low-level light irradiation. Apoptotic cells were observed in the SNP-treated group, but the frequency of these was reduced in the irradiation group. NO, O₂⁻, total ROS, and ONOO⁻ levels were increased after SNP treatment, but O₂⁻, total ROS, and ONOO⁻ levels were decreased after irradiation, despite the high NO concentration induced by SNP treatment. Cytochrome c was released from mitochondria of SNP-treated SH-SY5Y cells, but not of irradiated cells, resulting in a decrease in caspase-3 and -9 activity in SNP-treated cells. Finally, these results show that 635-nm irradiation, by promoting the scavenging of O₂⁻, protected against neuronal death through blocking the mitochondrial apoptotic pathway induced by ONOO⁻ synthesis.     

Direct chemiluminescence detection of nitric oxide in aqueous solutions using the natural nitric oxide target soluble guanylyl cyclase

Nitric oxide (NO) is a free radical involved in many physiological processes including regulation of blood pressure, immune response, and neurotransmission. However, the measurement of extremely low, in some cases subnanomolar, physiological concentrations of nitric oxide presents an analytical challenge. The purpose of this methods article is to introduce a new highly sensitive chemiluminescence approach to direct NO detection in aqueous solutions using a natural nitric oxide target, soluble guanylyl cyclase (sGC), which catalyzes the conversion of guanosine triphosphate to guanosine 3′,5′-cyclic monophosphate and inorganic pyrophosphate. The suggested enzymatic assay uses the fact that the rate of the reaction increases by about 200 times when NO binds with sGC and, in so doing, provides a sensor for nitric oxide. Luminescence detection of the above reaction is accomplished by converting inorganic pyrophosphate into ATP with the help of ATP sulfurylase followed by light emission from the ATP-dependent luciferin–luciferase reaction. Detailed protocols for NO quantification in aqueous samples are provided. The examples of applications include measurement of NO generated by a nitric oxide donor (PAPA-NONOate), nitric oxide synthase, and NO gas dissolved in buffer. The method allows for the measurement of NO concentrations in the nanomolar range and NO generation rates as low as 100 pM/min.     

Production of superoxide/H2O2 by dihydroorotate dehydrogenase in rat skeletal muscle mitochondria

Dehydrogenases that use ubiquinone as an electron acceptor, including complex I of the respiratory chain, complex II, and glycerol-3-phosphate dehydrogenase, are known to be direct generators of superoxide and/or H₂O₂. Dihydroorotate dehydrogenase oxidizes dihydroorotate to orotate and reduces ubiquinone to ubiquinol during pyrimidine metabolism, but it is unclear whether it produces superoxide and/or H₂O₂ directly or does so only indirectly from other sites in the electron transport chain. Using mitochondria isolated from rat skeletal muscle we establish that dihydroorotate oxidation leads to superoxide/H₂O₂ production at a fairly high rate of about 300 pmol H₂O₂·min⁻¹·mg protein⁻¹ when oxidation of ubiquinol is prevented and complex II is uninhibited. This H₂O₂ production is abolished by brequinar or leflunomide, known inhibitors of dihydroorotate dehydrogenase. Eighty percent of this rate is indirect, originating from site II_F of complex II, because it can be prevented by malonate or atpenin A5, inhibitors of complex II. In the presence of inhibitors of all known sites of superoxide/H₂O₂ production (rotenone to inhibit sites in complex I (site I_Q and, indirectly, site I_F), myxothiazol to inhibit site III_Qo in complex III, and malonate plus atpenin A5 to inhibit site II_F in complex II), dihydroorotate dehydrogenase generates superoxide/H₂O₂, at a small but significant rate (23 pmol H₂O₂·min⁻¹·mg protein⁻¹), from the ubiquinone-binding site. We conclude that dihydroorotate dehydrogenase can generate superoxide and/or H₂O₂ directly at low rates and is also capable of indirect production at higher rates from other sites through its ability to reduce the ubiquinone pool.     

NADPH oxidase- and mitochondrion-derived superoxide at rostral ventrolateral medulla in endotoxin-induced cardiovascular depression

We evaluated the contribution of superoxide anion (O2*-) generated by NADPH oxidase or mitochondria in the rostral ventrolateral medulla (RVLM), where sympathetic premotor neurons for arterial pressure maintenance are located, on cardiovascular depression induced by inducible nitric oxide synthase-derived NO after Escherichia coli lipopolysaccharide (LPS) treatment. In Sprague-Dawley rats maintained under propofol anesthesia, microinjection of LPS bilaterally into the RVLM induced progressive hypotension, bradycardia, and reduction in sympathetic vasomotor outflow over our 240-min observation period. This was accompanied by an increase in O2*- production (60-240 min) in the RVLM, alongside phosphorylation of p47(phox) or p67(phox), upregulation of gp91(phox) or p47(phox) protein, and increase in Rac-1 or NADPH oxidase activity (60-120 min), and a depression of mitochondrial respiratory enzyme activity (120-240 min). Whereas inhibition of NADPH oxidase or knockdown of the gp91(phox) or p47(phox) gene blunted the early phase (60-150 min), coenzyme Q10 or mitochondrial K(ATP) channel inhibitor antagonized the delayed phase (120-240 min) of LPS-induced increase in O2*- production in RVLM and cardiovascular depression. We conclude that, whereas NADPH oxidase-derived O2*- in RVLM participates predominantly in the early phase, O2*- generated by depression in mitochondrial respiratory enzyme activity or opening of mitoK(ATP) channels mediates the delayed phase of LPS-induced cardiovascular depression.     

Nitric oxide diffusion to red blood cells limits extracellular,but not intraphagosomal,peroxynitrite formation by macrophages

Macrophage-derived nitric oxide (^•NO) participates in cytotoxic mechanisms against diverse microorganisms and tumor cells. These effects can be mediated by ^•NO itself or ^•NO-derived species such as peroxynitrite formed by its diffusion-controlled reaction with NADPH oxidase-derived superoxide radical anion (O₂^•−). In vivo, the facile extracellular diffusion of ^•NO as well as different competing consumption routes limit its bioavailability for the reaction with O₂^•− and, hence, peroxynitrite formation. In this work, we evaluated the extent by which ^•NO diffusion to red blood cells (RBC) can compete with activated macrophages-derived O₂^•− and affect peroxynitrite formation yields. Macrophage-dependent peroxynitrite production was determined by boron-based probes that react directly with peroxynitrite, namely, coumarin-7-boronic acid (CBA) and fluorescein-boronate (Fl-B). The influence of ^•NO diffusion to RBC on peroxynitrite formation was experimentally analyzed in co-incubations of ^•NO and O₂^•−-forming macrophages with erythrocytes. Additionally, we evaluated the permeation of ^•NO to RBC by measuring the intracellular oxidation of oxyhemoglobin to methemoglobin. Our results indicate that diluted RBC suspensions dose-dependently inhibit peroxynitrite formation, outcompeting the O₂^•− reaction. Computer-assisted kinetic studies evaluating peroxynitrite formation by its precursor radicals in the presence of RBC are in accordance with experimental results. Moreover, the presence of erythrocytes in the proximity of ^•NO and O₂^•--forming macrophages prevented intracellular Fl-B oxidation pre-loaded in L1210 cells co-cultured with activated macrophages. On the other hand, Fl-B-coated latex beads incorporated in the macrophage phagocytic vacuole indicated that intraphagosomal probe oxidation by peroxynitrite was not affected by nearby RBC. Our data support that in the proximity of a blood vessel, ^•NO consumption by RBC will limit the extracellular formation (and subsequent cytotoxic effects) of peroxynitrite by activated macrophages, while the intraphagosomal yield of peroxynitrite will remain unaffected.     

Thiourea and Dimethylthiourea Inhibit Peroxynitrite-Dependent Damage: Nonspecificity as Hydroxyl Radical Scavengers

Thiourea and, more recently, dimethylthiourea, have been used as hydroxyl radical (OH√) scavengers in experiments both in vitro and in vivo. We show that both compounds can inhibit nitration of the amino acid tyrosine on addition of peroxynitrite, and also the inactivation of -antiproteinase by peroxynitrite. Hence, protective effects of (dimethyl) thiourea could be due to inhibition of peroxynitrite-dependent damage as well as to OH√ scavenging, and these compounds must not be regarded as specific OH√ scavengers. © 1997 Elsevier Science Inc.     

Thiyl radicals react with nitric oxide to form S-nitrosothiols with rate constants near the diffusion-controlled limit

A possible route to S-nitrosothiols in biology is the reaction between thiyl radicals and nitric oxide. D. Hofstetter et al. (Biochem. Biophys. Res. Commun.360:146-148; 2007) claimed an upper limit of (2.8+/-0.6)x10(7) M(-1)s(-1) for the rate constant between thiyl radicals derived from glutathione and nitric oxide, and it was suggested that under physiological conditions S-nitrosation via this route is negligible. In the present study, thiyl radicals were generated by pulse radiolysis, and the rate constants of their reactions with nitric oxide were determined by kinetic competition with the oxidizable dyes 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) and a phenothiazine. The rate constants for the reaction of nitric oxide with thiyl radicals derived from glutathione, cysteine, and penicillamine were all in the range (2-3) x10(9) M(-1)s(-1), two orders of magnitude higher than the previously reported estimate in the case of glutathione. Absorbance changes on reaction of thiyl radicals with nitric oxide were consistent with such high reactivity and showed the formation of S-nitrosothiols, which was also confirmed in the case of glutathione by HPLC/MS. These rate constants imply that formation of S-nitrosothiols in biological systems from the combination of thiyl radicals with nitric oxide is much more likely than claimed by Hofstetter et al.     

In vivo real-time measurement of superoxide anion radical with a novel electrochemical sensor

The dynamics of superoxide anion (O₂⁻) in vivo remain to be clarified because no appropriate method exists to directly and continuously monitor and evaluate O₂⁻ in vivo. Here, we establish an in vivo method using a novel electrochemical O₂⁻ sensor. O₂⁻ generated is measured as a current and evaluated as a quantified partial value of electricity (Q_part), which is calculated by integration of the difference between the baseline and the actual reacted current. The accuracy and efficacy of this method were confirmed by dose-dependent O₂⁻ generation in xanthine–xanthine oxidase in vitro in phosphate-buffered saline and human blood. It was then applied to endotoxemic rats in vivo. O₂⁻ current began to increase 1 h after lipopolysaccharide, and Q_part increased significantly for 6 h in endotoxemic rats, in comparison to sham-treated rats. These values were attenuated by superoxide dismutase. The generation and attenuation of O₂⁻ were indirectly confirmed by plasma lipid peroxidation with malondialdehyde, endothelial injury with soluble intercellular adhesion molecule-1, and microcirculatory dysfunction. This is a novel method for measuring O₂⁻ in vivo and could be used to monitor and treat the pathophysiology caused by excessive O₂⁻ generation in animals and humans.     

Membrane peroxidation: Inhibiting effects of water- soluble antioxidants on phospholipids of different charge types

Quantitative kinetic methods of autoxidation are used to determine the antioxidant activities of two water-soluble antioxidants of the chromanol type, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) and 6-hydroxy-2,5,7,8- tetramethyl-2-N,N,N-trimethylethanaminium methylbenzene-sulfonate (MDL 73404), during free radical peroxidation of phospholipid membranes of different charge types. The stoichiometric factor (n) for peroxyl radical trapping for both Trolox and MDL 73404 was found to be 2. Trolox was found to partition partially, approximately 20%, into the lipid phase of liposomes. The antioxidant activity of Trolox during peroxidation of membranes determined by measurements of the absolute rate constant for inhibition of oxygen uptake,k_inh, was found to vary with the membrane surface charge that is controlled by variation in pH. When peroxidation is initiated in the lipid phase by azo-bis-2,4-dimethylvaleronitrile (ADVN), using a typical zwitterionic liposome, dilinoleoylphosphatidyl choline (DLPC), the k_inh was found to be 2.98 × 10³ M⁻¹s⁻¹. The k_inh of Trolox increased approximately 2-fold for membranes that have positive surface, including DLPC at pH 4, DLPC containing stearylamine at pH 7, and for a membrane of dimyristoylphosphatidic acid containing linoleic acid (DMPA/LA). Conversely, Trolox does not inhibit peroxidation of negatively charged dilinoleoylphosphatidyl glycerol (DLPG) at pH 7–11. Studies made of the positively charged MDL 73404 show that its antioxidant activity using DLPC and DLPG is pH dependent. Trolox inhibits the peroxidations of DLPC initiated in the aqueous phase by azo-bis(2-amidinopropane·HCl)(ABAP) at pH 4 or 7. However, Trolox does not inhibit the peroxidation of DLPG at pH 7. The different antioxidant activities of Trolox and MDL 73404 are rationalized in terms of a peroxyl-radical diffusion model and specific charge interactions between antioxidants and membrane surface.     

Why does SOD overexpression sometimes enhance,sometimes decrease,hydrogen peroxide production? A minimalist explanation

Toxic effects of superoxide dismutase (SOD) overexpression are commonly attributed to increased hydrogen peroxide (H₂O₂) production. Still, published experiments yield contradictory evidence on whether SOD overexpression increases or decreases H₂O₂ production. We analyzed this issue using a minimal mathematical model. The most relevant mechanisms of superoxide consumption are treated as pseudo first-order processes, and both superoxide production and the activity of enzymes other than SOD were considered constant. Even within this simple framework, SOD overexpression may increase, hold constant, or decrease H₂O₂ production. At normal SOD levels, the outcome depends on the ratio between the rate of processes that consume superoxide without forming H₂O₂ and the rate of processes that consume superoxide with high (≥ 1) H₂O₂ yield. In cells or cellular compartments where this ratio is exceptionally low (< 1), a modest decrease in H₂O₂ production upon SOD overexpression is expected. Where the ratio is higher than unity, H₂O₂ production should increase, but at most linearly, with SOD activity. The results are consistent with the available experimental observations. According to the minimal model, only where most superoxide is eliminated through H₂O₂-free processes does SOD activity have the moderately large influence on H₂O₂ production observed in some experiments.