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.     

Antioxidant action of sodium diethyldithiocarbamate: Reaction with hydrogen peroxide and superoxide radical

The oxidation of sodium diethyldithiocarbamate (DDC) by hydrogen peroxide or superoxide radicals has been investigated. Hydrogen peroxide oxidizes DDC, leading to the formation of a hydrated form of disulfiram, a dimer of DDC having a disulfide group. In equimolar conditions, the overall process appears as a first-order reaction (k = 0.025±0.005 s⁻¹), the first step being a second-order reaction (k = 5.0±0.1mol⁻¹.1. s⁻¹). No radical intermediate was observed in this process. In the presence of an excess of any of the reagents, the hydrated form of disulfiram transforms into different products corresponding to the fixation of oxygen by sulfur atoms or replacement of C = S group by ketone function, in the presence of an excess of hydrogen peroxide. Superoxide anions (produced by steady-state ⁶⁰Co γ-radiolysis) oxidize DDC, yielding similar products to those obtained with hydrogen peroxide with a maximum oxidation G-value of 0.3 μmol.J⁻¹. The rate constant k(O₂^·− + DDC) is equal to 900 mol⁻¹. 1. s⁻¹.     

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 effects of reduced oxygen tension on swine granulosa cell

Follicular growth is characterized by an augmented vascularization, possibly driven by a fall in the oxygen supply. The present study was undertaken to investigate the effects of hypoxia on swine granulosa cells. At first, we quantified oxygen partial pressure (pO₂) in follicular fluid from different size follicles; the granulosa cells collected from large follicles (>5 mm) were subjected for 18 h to normoxia (19% O₂), partial (5% O₂) or total hypoxia (1% O₂). The effects of these conditions were tested on the main parameters of granulosa cell function, steroidogenesis and cell proliferation, and on vascular endothelial growth factor (VEGF), nitric oxide (NO) and superoxide anion (O₂⁻) production. Oxygen tension in follicular fluid was negatively related to follicular size, pointing out a gradual reduction during follicular growth. Severe hypoxic conditions determined a reduction of both 17β estradiol and progesterone production, while partial hypoxia did not seem to affect them. Hypoxia increased VEGF as well as O₂⁻ production in swine granulosa cells without impairing cell growth; in addition, it decreased NO output.

We may conclude that physiological hypoxia could play a pivotal role in the follicular angiogenic process stimulating VEGF synthesis by granulosa cells. ROS are possibly involved in hypoxic signalling.     

A comparative study of EPR spin trapping and cytochrome c reduction techniques for the measurement of superoxide anions

Superoxide anions (O₂^.−) generated by the reaction of xanthine with xanthine oxidase were measured by the reduction of cytochrome c and by electron paramagnetic resonance (EPR) spectroscopy using the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). Studies were performed to determine the relative sensitivities of these two techniques for the measurement of O₂^.−. Mixtures of xanthine, xanthine oxidase, DMPO generated two adducts, a transient DMPO-OOH and a smaller but longer-lived DMPO-OH. Both adducts were inhibited by superoxide dismutase (SOD), demonstrating they originated from O₂^.−, and were also significantly decreased when the experiments were performed using unchelated buffers, suggesting that metal ion impurities in unchelated buffers alter the formation or degradation of DMPO-adducts. O₂^.−, generated by concentrations of xanthine as low as 0.05 μM, were detectable using EPR spin trapping. In contrast, mixtures of xanthine, xanthine oxidase, and cytochrome c measured spectrophotometrically at 550 nm demonstrated that concentrations of xanthine above 1 μM were required to produce measurable levels of reduced cytochrome c. These studies demonstrate that spin trapping using DMPO was at least 20-fold more sensitive than the reduction of cytochrome c for the measurement of superoxide anions. However, at levels of superoxide generation where cytochrome c provides a linear measurement of production, EPR spin trapping may underestimate radical production, probably due to degradation of DMPO radical adducts.     

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.     

Purification and partial characterization of Cu, Zn containing superoxide dismutase from entomogenous fungal species Cordyceps militaris

Cordyceps militaris mycelium produced mainly Cu, Zn containing superoxide dismutase (Cu, Zn-SOD). Cu, Zn-SOD activity was detectable in the culture filtrates, and intracellular Cu, Zn-SOD activity as a proportion protein was highest in early log phase culture. The effects of Cu²⁺, Zn²⁺, Mn²⁺ and Fe²⁺ on enzyme biosynthesis were studied. The Cu, Zn-SOD was isolated and purified to homogeneity from C. militaris mycelium and partially characterized. The purification was performed through four steps: (NH₄)₂SO₄ precipitation, DEAE-sepharose™ fast flow anion-exchange chromatography, CM-650 cation-exchange chromatography, and Sephadex G-100 gel filtration chromatography. The purified enzyme had a molecular weight of 35070 ± 400 Da and consisted of two equal-sized subunits each having a Cu and Zn element. Isoelectric point value of 7.0 was obtained for the purified enzyme. The N-terminal amino acid sequence of the purified enzyme was determined for 12 amino acid residues and the sequences was compared with other Cu, Zn-SODs. The optimum pH of the purified enzyme was obtained to be 8.2–8.8. The purified enzyme remained stable at pH 5.8–9.8, 25 °C and up to 50 °C at pH 7.8 for 1.5 h incubation. The purified enzyme was sensitive to H₂O₂, KCN. 2.5 mM NaN₃, PMSF, Triton X-100, β-mercaptoethanol and DTT showed no significant inhibition effect on the purified enzyme within 5 h incubation period.     

Altered nitric oxide production in mouse brain after administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydro-pyridin or methamphetamine

Several studies have demonstrated the involvement of reactive nitrogen and oxygen species (RNOS) in the neurotoxic effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydro-pyridin (MPTP) and methamphetamine (METH), so the contribution of altered nitric oxide synthase (NOS) enzyme function can be suspected. In this study, about 50% increase in nitric oxide (NO) production in the mouse striatum was found between 4 and 12 h after a single MPTP injection, allowing an increased peroxynitrite (ONOO⁻) formation in the target brain region. However, METH injection induced a rapid decrease of NO formation both in mouse striatum and hippocampus, reaching its minimum level at 2 h, and restored to the control value after 6 h in the striatum and 12 h in the hippocampus. The uncoupled function of NOS with increased superoxide (O₂⁻) production after METH injection is suggested.     

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.     

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.     

Nitric Oxide-Mediated Mitochondrial Damage in the Brain: Mechanisms and Implications for Neurodegenerative Diseases

Abstract: Within the CNS and under normal conditions, nitric oxide (^•NO) appears to be an important physiological signalling molecule. Its ability to increase cyclic GMP concentration suggests that ^•NO is implicated in the regulation of important metabolic pathways in the brain. Under certain circumstances ^•NO synthesis may be excessive and ^•NO may become neurotoxic. Excessive glutamate-receptor stimulation may lead to neuronal death through a mechanism implicating synthesis of both ^•NO and superoxide (O₂^•−) and hence peroxynitrite (ONOO⁻) formation. In response to lipopolysaccharide and cytokines, glial cells may also be induced to synthesize large amounts of ^•NO, which may be deleterious to the neighbouring neurones and oligodendrocytes. The precise mechanism of ^•NO neurotoxicity is not fully understood. One possibility is that it may involve neuronal energy deficiency. This may occur by ONOO⁻ interfering with key enzymes of the tricarboxylic acid cycle, the mitochondrial respiratory chain, mitochondrial calcium metabolism, or DNA damage with subsequent activation of the energy-consuming pathway involving poly(ADP-ribose) synthetase. Possible mechanisms whereby ONOO⁻ impairs the mitochondrial respiratory chain and the relevance for neurotoxicity are discussed. The intracellular content of reduced glutathione also appears important in determining the sensitivity of cells to ONOO⁻ production. It is concluded that neurotoxicity elicited by excessive ^•NO production may be mediated by mitochondrial dysfunction leading to an energy deficiency state.     

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.     

Is the activity-linked electrostatic gradient of bovine Cu, Zn superoxide dismutases conserved in homologous enzymes irrespective of the number and distribution of charges?

Electrostatic potential calculations have been performed on three different Cu, Zn superoxide dismutases (superoxide: superoxide oxidoreductase, EC 1.15 1.1 ), in order to evaluate the degree of conservation of the pattern of the electrostatic interactions between O₂⁻ and the active site recently pointed out in bovine Cu Zn SOD. The three Cu, Zn SODs that have been selected for this study, namely the bovine, ovine, and porcine enzymes, are highly homologous as to reasonably assume identical three-dimensional structure but display large differences in their net charge, as shown by their pI's which span over a wide range pHrange: 8.0 (sheep), 6.5(pig), 5.2(ox). Despite such a large difference in the net protein charge and in the spatial arrangement of electrostatic charges, electrostatic potential calculations show that the electrostatic channel directing the negatively charged substrate toward the positive catalytic site is strictly preserved with the same features for the three proteins. This suggests that the electrostatic funnel for conducting small anions into the active site is a highly conservative property in the evolution of Cu, Zn SOD.     

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.     

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.     

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.     

Actual and potential dark respiration rates and different electron transport pathways in freshwater aquatic plants

The rates of respiratory O₂ uptake have been studied in leaves, stems and whole shoots of several freshwater plants: 6 angiosperms, 2 bryophytes and one alga. For angiosperm leaves, rates varied widely with species (30–142 μmol O₂ (gDW)⁻¹ h⁻¹), were correlated with chlorophyll content and were higher than those of the stems (13–71 μmol O₂ (gDQ)⁻¹ h⁻¹). The rates for the shoots of bryophytes (53–66 μmol O₂ (gDW)⁻¹ h⁻¹) and for the alga Cladophora glomerata (L.) Kütz. (96 μmol O₂ (gDW)⁻¹ h⁻¹) were slightly higher than those of most angiosperm stems, but lower than those for most leaves.

These plants had a significant cyanide-resistant respiration, suggesting the existence of an alternative pathway to the “classic” cytochrome system. This pathway was found to be active in all the species studied, as judged by responses to a specific inhibitor, SHAM (salicylhydroxamic acid). Measurement of electron-transport system (ETS) activity showed that there is a large electron-transport capacity which is not normally used by respiration in vivo.     

Nitric oxide stimulates prostaglandin synthesis in cultured rabbit gastric cells

Both prostaglandins (PGs) and nitric oxide (NO) have cytoprotective and hyperemic effects in the stomach. However, the effect of NO on PG synthesis in gastric mucosal cells is unclear. We examined whether sodium nitroprusside (SNP), a releaser of NO, stimulates PG synthesis in cultured rabbit gastric mucus-producing cells. These cells did not release NO themselves. Co-incubation with SNP (2 × 10⁻⁴, 5 × 10⁻⁴, 10⁻³ M) increased PGE₂ synthesis, and SNP (10⁻³ M) increased PGI₂ synthesis in these cells. Hemoglobin, a scavenger of NO, (10⁻⁵ M) eliminated the increase in PGE₂ synthesis by SNP, but methylene blue, an inhibitor of soluble guanylate cyclase, (5 × 10⁻⁵ M) did not affect the increase in PGE₂ synthesis by SNP. 8-bromo guanosine 3′ : 5′-cyclic monophosphate (8-bromo cGMP), a cGMP analogue, (10⁻⁶, 10⁻⁵, 10⁻⁴, 10⁻³ M) did not affect PGE₂ synthesis. These findings suggest that NO increased PGE₂ and PGI₂ synthesis via a cGMP-independent pathway in cultured rabbit gastric cells.     

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.