Effect of propagators and inhibitors on the ultraweak luminescence from maize roots

This study has investigated the kinetics and mechanism of ultraweak luminescence in maize roots. Mannitol induced the second maximum and enhanced the main maximum of the relative intensity of luminescence from the roots. Hydroquinone and quinone enhanced the relative intensity of the luminescence. Catalase enhanced the maximum of the luminescence and changed the kinetics of the light emission. The effect of catalase on the kinetics was abolished by superoxide dismutase. Ascorbate in the presence of catalase reduced the luminescence maximum, but did not alter the kinetics. In the presence of catalase only, or in the combination with superoxide dismutase, or ascorbate, the luminescence intensity in the stationary phase was significantly lower compared to the control. The results support the participation of superoxide-radical, singlet oxygen, electron transfer and the role of peroxidase in the reactions generating ultraweak luminescence in the roots. Ascorbate, catalase and superoxide dismutase have a protective role in the luminescent reactions.     

Detection of activated O2 species in vitro and in rat lungs by chemiluminescence

This study used chemiluminescence, an "on-line" photon-counting technique, to detect and characterize activated O2 species in vitro and in isolated rat lungs. The sensitivity and specificity of enhanced chemiluminescence for superoxide anion (O2-.) and hydrogen peroxide (H2O2) was evaluated in vitro. The effect of media conditions (such as O2 tension, albumin concentration, and sulfhydryl group availability) on luminescence was assessed in vitro. Xanthine-xanthine oxidase (X-XO) primarily produced superoxide anion in vitro. Enhanced chemiluminescence varied directly with the dose of luminescent probe used and the quantity of activated O2 species administered. The strength of the luminescent signal was also dependent on the concentration of albumin and O2 in the media. Lucigenin was more sensitive than luminol to the presence of O2-. and, unlike luminol, lucigenin did not alter radical production by XO. However, neither luminescent probe was specific for O2-., as both detected H2O2 and O2 in vitro. H2O2-induced chemiluminescence was inhibited by catalase but not superoxide dismutase (SOD), while X-XO-induced luminescence was inhibited by SOD but not catalase. SOD-inhibitable chemiluminescence was a sensitive and specific marker for O2-. production in vitro. Once the sensitivity-specificity of enhanced chemiluminescence was defined in vitro, this technique was used to explore the mechanism by which exogenous X-XO reduced hypoxic vasoconstriction in isolated rat lungs. The vascular paresis, caused by administration of X-XO to the rat lung, resulted from a brief burst of O2-. production rather than a sustained alteration of lung radical levels.     

Reactions of pholasin with peroxidases and hypochlorous acid

The ability of myeloperoxidase (MPO) and horseradish peroxidase (HRP) to induce chemiluminescence (CL) in Pholasin (Knight Scientific, Plymouth, UK), the photoprotein of the Common Piddock Pholas dactylus, was studied. The oxidation of Pholasin by compound I or II of HRP induced an intense light emission, whereas native HRP showed only a small effect. The luminescence observed upon incubation of Pholasin with native MPO was diminished by preincubation with catalase. Considering the high instability of diluted MPO, it is concluded that traces of hydrogen peroxide in water converted MPO to its active forms, compound I and/or II, which are able to oxidize Pholasin. Indeed, the addition of hydrogen peroxide to a mixture of MPO and Pholasin induced an intense burst of light. This emission was enhanced in degree and duration in the absence of chloride. Hypochlorous acid, the reaction product of Cl(-) and compound I of MPO, was itself able to elicit a luminescent response in Pholasin and this luminescence was strongly inhibited by methionine and taurine. However, both of these HOCl scavengers only slightly reduced the light emission induced by MPO/H(2)O(2) in both the presence or absence of chloride. Thus, hypochlorous acid produced by the MPO/H(2)O(2)/Cl(-) system, under the conditions described in this study, did not contribute to Pholasin luminescence. The Pholasin luminescence elicited by formyl-leucyl-methionyl-phenylalanine (fMLP)-stimulated neutrophils depends both on superoxide anion radicals and higher oxidation states of myeloperoxidase (but not on hypochlorous acid). This is shown by the inhibition of luminescence with superoxide dismutase and potassium cyanide, together with the lack of effect of both methionine and taurine. The luminescence response is about eight times greater in cells stimulated with fMLP/cytochalasin B than with fMLP alone.     

A chemiluminescent probe with a Cypridina luciferin analog, 2-methyl-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-3-one, specific and sensitive for O2- production in phagocytizing macrophages

When a Cypridina luciferin analog (the title compound) was added to a macrophage suspension in Hank's balanced salt solution (control), the system emitted a weak, but detectable light, which was not altered in the presence of superoxide dismutase. The same system, however, emitted a much stronger light, just after the addition of a trigger, opsonized zymosan. The luminescence was suppressed to the control level in the presence of superoxide dismutase, while it was only slightly influenced, if at all, by NaN3, a scavenger of singlet oxygen and an inhibitor of myeloperoxidase. Some other results obtained also indicate the participation of O2- in the luciferin analog-dependent luminescence in macrophages during phagocytosis.     

Superoxide, hydrogen peroxide, and oxygen toxicity in two free-living nematode species

Two species of free-living nematodes, Turbatrix aceti and Caenorhabditis elegans, exhibited a marked sensitivity to 3 atm of 100% O2. Environmental changes in pH and temperature, which altered nematode respiration, resulted in alterations in the survival of these organisms under high pO2. Levels of defensive enzymes such as superoxide dismutase, catalase, glutathione peroxidase, and dianisidine peroxidase were measured in the two species. No changes in the level of superoxide dismutase or catalase activity were induced by exposure of the nematodes to high pO2. Manipulation of these two enzymes was however achieved using the inhibitors 3-amino-1,2,4-triazole and diethyldithiocarbamate. 3-Amino-1,2,4-triazole (20 mM) eliminated greater than or equal to 80% of the catalase activity in vivo and diethyldithiocarbamate (5 mM) decreased the level of CuZn superoxide dismutase by greater than or equal to 70%. Both of these compounds increased the sensitivity of C. elegans to high pO2 toxicity. Compounds capable of intracellular redox-cycling with O2- -production, such as plumbagin, increased CN- -resistant respiration in the nematodes and imposed an O2-dependent toxicity. These experiments demonstrate the toxicity of intracellular O2- and H2O2 in nematodes and the importance of superoxide dismutase and catalase in providing a defense against these toxic molecules in vivo.     

Ultraweak light emission is a common response of bacterial cells to chemico-physical stress

Escherichia coli JM101 cells were subjected to pore-forming electric fields, irradiation with ultraviolet light or oxidative stress by either the lipoxygenase products 9- and 13-hydroperoxyoctadecadienoic acids (9- and 13-HPOD) or hydrogen peroxide. It was found that all chemico-physical stresses enhanced ultraweak light emission from the bacterial cells, the most effective treatment being electroporation (up to 20-fold increase in luminescence compared to the control value), followed by oxidative stress with 9- or 13-HPOD (up to 4-fold increase) and irradiation with UV light (up to 2.8-fold increase). Bacterial luminescence was always in the red edge of the spectrum and was paralleled by changes in membrane oxidative index and specific activity of catalase and superoxide dismutase. © 1998 John Wiley & Sons, Ltd.     

Spontaneous luminescence from soybean Rhizobium bacteroids

Abstract: A remarkable spontaneous photon emission was observed in isolated bacteroids of three strains of soybean rhizobia from different genera, but not for the same rhizobia when cultured in liquid medium. The photon emission is oxygen-dependent and can be inhibited by desferal or dipyridyl (both good iron-chelating agents), superoxide dismutase or β-carotene. It is enhanced by catalase. The emission spectrum indicates that singlet oxygen is partly responsible for the luminescence.     

Hydroxyl radicals is not a significant intermediate in the vanadate-stimulated oxidation of NAD(P)H by O2

The vanadate-stimulated oxidation of NADH by an enzymatic flux of O2- is inhibited by superoxide dismutase, but not by catalase. Keller et al. (1989, Free Radical Biol. Med. 6, 15-22) observed inhibition by catalase presumably because they used a commercial preparation contaminated with superoxide dismutase. Their proposal, that H2O2 and hydroxyl radical play significant roles in vanadate-stimulation of NAD(P)H oxidation, may be discounted on the basis of these and of previously reported results.     

Superoxide dismutase of the eye: relative functions of superoxide dismutase and catalase in protecting the ocular lens from oxidative damage.

1. Activities of superoxide dismutase (superoxide: superoxide oxidoreductase, EC 1.15.1.1) have been estimated in eye tissues. In rabbit eye, superoxide dismutase is present in corneal epithelium, corneal endothelium, lens, iris, ciliary body and retina. In lens the activity is in capsule epithelium. 2. Copper chelator diethyldithiocarbamate inhibited lens superoxide dismutase in vitro and in vivo in rabbit. 3. H2O2 caused inhibition of superoxide dismutase activity of lens extract, and this inhibition was potentiated by the catalase inhibitor 3-amino-1H-1,2,4-triazole (3-aminotriazole) or NaN3. 3-Aminotriazole or NaN3 had no effect on lens superoxide dismutase. Thus endogenous catalase of lens affords protection to the lens superoxide dismutase from inactivation by H2O2. 4. In rabbit having early cataract (vacuolar stage) induced by feeding-3-aminotriazole, there was a decrease in superoxide dismutase of lens, a fall in ascorbic acid of ocular humors and lens, and a 2--3-Fold increase in H2O2 of aqueous humor and vitreous humor. We conclude that catalase of eye affords protection to the lens from H2O2 and it also protects superoxide dismutase of lens from inactivation by H2O2. Superoxide dismutase, in turn, protects the lens from the superoxide radical, O2.-. It is likely that inhibition of these enzymes may lead to production of the highly reactive oxidant, the hydroxyl radical, under pathological conditions when H2O2 concentration in vivo exceeds physiological limits as in cataract induced by 3-aminotriazole. A scheme of reaction mechanism has been proposed to explain the relative functions of ocular catalase and superoxide dismutase. Such a mechanism may be involved in cataractogenic process in the human.     

A sensitive and specific assay for superoxide anion released by neutrophils or macrophages based on bioluminescence of polynoidin

Using the luminescent protein polynoidin, present in the bioluminescent system isolated from the marine annelid Harmothoe lunulata, we have developed a new method to measure, specifically, superoxide anion (O2-) released by macrophages or neutrophils. A small quantity of an aqueous crude extract of polynoidin is used to detect O2- released by stimulated cells. Light emission is linearly dependent on the number of cells over a wide range (10(3) to 10(7) cells), and the assay is thus more sensitive than either luminol or ferricytochrome c reduction. Luminescence is enhanced 20% by mannitol, 80% by catalase, and is totally quenched by superoxide dismutase. For the same number of cells, neutrophils showed a threefold higher release of O2- and a twofold faster first-order light decay than stimulated macrophages, in accordance with data obtained by other methods.     

Catalase, superoxide dismutase, and the production of O2-sensitive mutants of Bacillus coagulans

A number of facultatively anaerobic members of the genus Bacillus were screened for their catalase, diaminobenzidine peroxidase, and superoxide dismutase activities. A strain of Bacillus coagulans (7050) lacking peroxidatic activity and containing single catalatic and superoxide dismutase activities was selected. Responses of the superoxide dismutase activity and catalase level to the partial pressure of oxygen, and Fe and Mn levels, as well as to aerobic and fermentative metabolism, were determined. There appeared to be a relationship between high endogenous catalase levels and the high H2O2 evolution and KCN insensitivity of B. coagulans respiration. Bacillus coagulans 7050 was mutagenized with N-methyl-N'-nitro-N-nitrosoguanidine and screened for the expression of oxygen intolerance. All of the 38 stable oxygen sensitive mutants obtained had very low or completely absent catalatic activity and catalase protein. No mutant lacked superoxide dismutase, although five showed significantly lowered levels of the enzyme. Exogenous bovine liver catalase restored aerotolerance and reduced cell pleomorphism in the mutants.     

The effects of "oxygen radicals" generated in the medium on lenses in organ culture: inhibition of damage by chelated iron

Rat lenses in organ culture were exposed to activated species of oxygen generated in the culture medium either by xanthine oxidase and hypoxanthine or by riboflavin and visible light, two systems which have been shown to produce superoxide and H2O2. In each case there was marked damage to carrier-mediated transport systems of the lens. Under standard culture conditions this damage was strongly inhibited by catalase, but not by superoxide dismutase (SOD). By the addition to the medium of chelated iron, hydroxyl radicals were produced in a Fenton reaction with a concomitant decrease in H2O2 levels. With both oxygen radical-generating systems, the addition of chelated iron strongly inhibited lens damage. This inhibitory effect could be reversed by the addition of SOD with the chelated iron. Under such conditions SOD converts superoxide anion to H2O2, thereby preventing reduction of the chelated iron and thus stopping the generation of hydroxyl radicals. Increased lens damage following addition of SOD to the iron-containing systems correlated with higher H2O2 concentrations, and was inhibited by catalase. These findings suggest that, when generated in the fluids surrounding the lens, H2O2 poses a much greater oxidative stress for the lens than do the superoxide or hydroxyl free radicals.     

Superoxide radical inhibits catalase

Catalase was inhibited by a flux of O2- generated in situ by the aerobic xanthine oxidase reaction. Two distinct types of inhibition could be distinguished. One of these was rapidly established and could be as rapidly reversed by the addition of superoxide dismutase. The second developed slowly and was reversed by ethanol, but not by superoxide dismutase. The rapid inhibition was probably due to conversion of catalase to the ferrooxy state (compound III), while the slow inhibition was due to conversion to the ferryl state (compound II). Since neither compound III nor compound II occurs in the catalatic reaction pathway, they are inactive. This inhibition of catalase by O2- provides the basis for a synergism between superoxide dismutase and catalase. Such synergisms have been observed in vitro and may be significant in vivo.     

Characteristics of chemiluminescence observed in the horseradish peroxidase-hydrogen peroxide-tyrosine system.

Electrolysis or horseradish peroxidase (HRP)-catalyzed oxidation of tyrosine and bityrosine in aqueous solution at pH 7.4 resulted in light emission in the visible region. Electrolysis of tyrosine emitted light which peaked at 490 nm and was almost completely quenched by superoxide dismutase (SOD), while emission by bityrosine peaked at 530 nm. In the HRP-H(2)O(2)-tyrosine system the oxidation-reduction of tyrosine emitted light with two prominent peaks, 490 and 530 nm, and was not quenched by SOD. The phenoxyl neutral radical of the tyrosine in HRP-H(2)O(2)-tyrosine system was detected by electron spin resonance (ESR) spectrometry using tert-nitrosobutane as a spin trap; the spin adduct was found to adhere to the HRP molecule during the enzymatic reaction. Further, bityrosine was detected in the HRP-H(2)O(2)-tyrosine reaction system. Changes in absorption spectra of HRP and chemiluminescence intensities during HRP-catalyzed oxidation of tyrosine suggest that for photon emission compound III is a candidate superoxide donor to the phenoxyl cation radical of tyrosine on the enzyme molecule. The luminescence observed in this study might be originated from at least two exciplexes involved with the tyrosine cation radical (Tyr(*+)) and the bityrosine cation radical (BT(*+))     

Low-density lipoprotein modification by normal,myeloperoxidase-deficient and NADPH oxidase-deficient granulocytes and the impact of redox active transition metal ions

The modification of low-density lipoprotein (LDL) by normal, myeloperoxidase (MPO)-deficient and NADPH oxidase-deficient granulocytes was investigated using the monoclonal antibody (mAb) OB/04, which was originally generated against copper-oxidized LDL. Incubation of LDL with normal granulocytes increased the reactivity of LDL with mAb OB/04. These effects were even more pronounced using MPO-deficient granulocytes. Inhibitors of oxidative reactions (the NADPH oxidase inhibitor diphenyleneiodonium chloride [DPI], catalase, superoxide dismutase [SOD]) did not significantly reduce LDL oxidation by normal granulocytes. Furthermore, granulocytes of a patient with NADPH oxidase deficiency were almost equally effective as normal granulocytes, indicating that oxidative burst-derived reactive oxygen species are of only minor importance in the generation of mAb OB/04-detectable new epitopes on LDL in vitro. In contrast, incubation of LDL with iron and copper prior to and during incubation with normal granulocytes markedly enhanced the generation of OB/04-detectable epitopes. It is supposed that, besides superoxide (in normal and MPO-deficient granulocytes) or instead of superoxide (in NADPH oxidase-deficient granulocytes), lytic enzymes released by activated granulocytes may enhance the availability of transition metals for oxidation of LDL. Our results support the concept that transition-metal-dependent pathways of LDL oxidation in combination with degranulation products of granulocytes are important.     

Ferrous ion-mediated cytochrome P-450 degradation and lipid peroxidation in adrenal cortex mitochondria.

The relationship between the degradation reaction of cytochrome P-450 and lipid peroxidation was studied utilizing bovine adrenal cortex mitochondria. The two reactions were found to be closely correlated in terms of their response to storage of the mitochondrial preparation, stimulation by Fe2+, inhibition by EDTA and their initiation by cumene hydroperoxide. Both reactions were also found not to be inhibited by catalase, superoxide dismutase, 1,4-diazabicyclo-(2,2,2)-octane and alcohols, indicating that H2O2, superoxide, singlet oxygen and hydroxyl radicals do not participate in these reactions. Yet, diphenylamine proved to be a powerful inhibitor for both reactions, suggesting the involvement of a radical species. Cumene hydroperoxide could induce these two reactions at below 0.1 mM concentrations in the presence of molecular oxygen. The chemiluminescence observed during the Fe2+-mediated lipid peroxidation reaction which was not inhibited by either superoxide dismutase or 1,4-diazabicyclo-(2,2,2)-octane, was biphasic: one was a rapid burst; and the other was a slowly increasing emission. The latter portion of the emission of light coincided with the formation of malondialdehyde. These results indicate that in adrenal cortex mitochondria the degradation of cytochrome P-450 is closely related to lipid peroxidation.     

Superoxide, hydrogen peroxide and singlet oxygen in hematoporphyrin derivative-cysteine, -NADH and -light systems

Hematoporphyrin derivative and light in the presence of cysteine or glutathione were found to convert oxygen to superoxide and hydrogen peroxide at pH less than approx. 6.5, while at pH greater than 6.5 no superoxide or hydrogen peroxide production was observed. However, at pH values greater than 6.5 the rate of oxygen consumption increased. This rate paralleled the acid dissociation curve of the cysteine thiol group and is consistent with the chemical quenching of 1O2 by cysteine. The superoxide and hydrogen peroxide formation observed below pH 6.5 appeared not to be related to the singlet oxygen production of hematoporphyrin derivative. In addition, superoxide and hydrogen peroxide production was observed with hematoporphyrin derivative and light in the presence of NADH, both above and below pH 6.5. Direct detection of singlet oxygen luminescence at 1268 nm in the hematoporphyrin derivative-light system (2H2O as solvent) revealed an apparent linear increase in the singlet oxygen emission intensity as the p2H was raised from 7.0 to 10.0. Azide efficiently quenched this observed emission. In addition, at p2H 7.4, 1 mM cysteine resulted in a 40% reduction of the singlet oxygen luminescence, while at p2H 9.4 the signal was quenched by over 95% (under the experimental conditions employed). In total, we interpret these results as consistent with the chemical quenching of 1O2 by the ionized thiol group of cysteine.     

Effect of free-radical scavengers on enumeration of thermally stressed cells of Staphylococcus aureus MF-31.

Exposure of crude cell lysates of Staphylococcus aureus MF-31 to 5 or 10 mM hydrogen peroxide resulted in a linear decrease in superoxide dismutase activity. Approximately 13% of the superoxide dismutase activity was lost after 16 min. Thermally stressed and nonstressed cells were exposed to a photochemically generated exogenous flux of superoxide radicals (O2.-). The death of thermally stressed cells was linear with time. Addition of superoxide dismutase or catalase to the O2.- generating system resulted in protection of thermally stressed and nonstressed cells, with the protective effect being greater for thermally stressed cells. Incorporation of O2-, hydroxyl radical, or singlet oxygen scavengers or antioxidants to tryptic soy agar containing 7.5% NaCl did not increase the enumeration of thermally stressed cells.     

The interaction of bovine erythrocyte superoxide dismutase with hydrogen peroxide: chemiluminescence and peroxidation.

Reaction of bovine erythrocyte superoxide dismutase with H2O2 was accompanied by a luminescence whose intensity was a function of the concentration of H2O2 and whose duration was coincident with the inactivation of the enzyme by this reagent. Oxygen, which protected against inactivation, also diminished the luminescence. Several other compounds which prevented the inactivation by H2O2 also modified the luminescence. Thus urate, formate, and triethylamine inhibited luminescence whereas imidazole and xanthine augmented it. These seemingly contrary effects can be explained by assuming that the compounds which protected the enzyme were peroxidized in competition with the sensitive group on the enzyme. The luminescence arises because that group on the enzyme was oxidized to a product in an electronically excited state, which could return to the ground state by emitting light. Imidazole and xanthine gave electronically excited products whose quantum efficiency was greater than that of the group on the enzyme, whereas urate, formate, and triethylamine gave products with much lower luminescent efficiencies. This superoxide dismutase could catalyze the peroxidation of a wide range of compounds, including ferrocytochrome c, luminol, diphenylisobenzofuran, dianisidine, and linoleic acid. In control experiments, boiled enzyme was inactive. This peroxidative activity can lead to unexpected effects when superoxide dismutase is added to H2O2-producing systems, as a probe for the involvement of O2-. Several examples from the literature are cited to illustrate the misinterpretations which this previously unrecognized peroxidative activity can generate.     

Sustained Photoproduction of Ammonia from Nitrate by Anacystis nidulans

Exposure of crude cell lysates of Staphylococcus aureus MF-31 to 5 or 10 mM hydrogen peroxide resulted in a linear decrease in superoxide dismutase activity. Approximately 13% of the superoxide dismutase activity was lost after 16 min. Thermally stressed and nonstressed cells were exposed to a photochemically generated exogenous flux of superoxide radicals (O2.-). The death of thermally stressed cells was linear with time. Addition of superoxide dismutase or catalase to the O2.- generating system resulted in protection of thermally stressed and nonstressed cells, with the protective effect being greater for thermally stressed cells. Incorporation of O2-, hydroxyl radical, or singlet oxygen scavengers or antioxidants to tryptic soy agar containing 7.5% NaCl did not increase the enumeration of thermally stressed cells.