Oxygen toxicity in Streptococcus sanguis. The relative importance of superoxide and hydroxyl radicals

Streptococcus sanguis, whose growth appears to be independent of the availability of iron, makes no hemes, contains neither catalase nor peroxidase, and can accumulate millimolar concentration levels of H2O2 during aerobic growth. It possesses a single manganese-containing superoxide dismutase whose concentration can be varied over a 50-100-fold range by manipulating the availability of oxygen during growth. Cell extracts contain a soluble NADH-plumbagin diaphorase which mediates O2- production in vitro and presumably also in vivo. Plumbagin increased oxygen consumption by S. sanguis and imposed an oxygen-dependent toxicity. Cells grown aerobically and containing elevated levels of superoxide dismutase were resistant to this toxicity. Dimethyl sulfoxide, which was shown to permeate S. sanguis freely, was used as an indicating scavenger of OH. An in vitro enzymic source of O2- plus H2O2 generated formaldehyde from dimethyl sulfoxide, an indication of OH. production. Either superoxide dismutase or catalase inhibited this OH. production and iron salts augmented it. Intact, aerobic cells of S. sanguis also gave evidence of OH. production, in the presence of plumbagin, but all of it appeared to be generated outside the cells. In addition, 0.5 M dimethyl sulfoxide did not diminish the oxygen-dependent toxicity of plumbagin. We conclude that, in S. sanguis, O2- can exert a toxic effect independent of the production of OH..     

DNA breakage induced by 1,2,4-benzenetriol: relative contributions of oxygen-derived active species and transition metal ions

We report here the relative roles of metals and selected reactive oxygen species in DNA damage by the genotoxic benzene metabolite 1,2,4-benzenetriol, and the interactions of antioxidants in affording protection. 1,2,4-Benzenetriol induces scission in supercoiled phage DNA in neutral aqueous solution with an effective dose (ED(50)) of 6.7 microM for 50% cleavage of 2.05 microg/ml supercoiled PM2 DNA. In decreasing order of effectiveness: catalase (20 U/ml), formate (25 mM), superoxide dismutase (20 U/ml), and mannitol (50 mM) protected, from 85 to 28%. Evidently, H(2)O(2) is the dominant active species, with O(2)(*)(-) and *OH playing subordinate roles. Desferrioxamine or EDTA inhibited DNA breakage by 81-85%, despite accelerating 1,2,4-benzenetriol autoxidation. Consistent with this suggestion of a crucial role for metals, addition of cupric, cuprous, ferric, or ferrous ions enhanced DNA breakage, with copper being more active than iron. Combinations of scavengers protected more effectively than any single scavenger alone, with implications for antioxidants acting in concert in living cells. Synergistic combinations were superoxide dismutase with *OH scavengers, superoxide dismutase with desferrioxamine, and catalase with desferrioxamine. Antagonistic (preemptive) combinations were catalase with superoxide dismutase, desferrioxamine with *OH scavengers, and catalase with *OH scavengers. The most striking aspect of synergism was the extent to which metal chelation (desferrioxamine) acted synergistically with either catalase or superoxide dismutase to provide virtually complete protection. Concluding, 1,2,4-benzenetriol-induced DNA damage occurs mainly by site-specific, Fenton-type mechanisms, involving synergism between several reactive intermediates. Multiple antioxidant actions are needed for effective protection.     

Oxygen-based free radical generation by ferrous ions and deferoxamine

Deferoxamine accelerates the autooxidation of iron as measured by the rapid disappearance of Fe2+, the associated appearance of Fe3+, and the uptake of oxygen. Protons are released in the reaction. The formation of H2O2 was detected by the horseradish peroxidase-catalyzed oxidation of scopoletin, and the formation of hydroxyl radicals (OH.) was suggested by the formation of the OH. spin trap adduct (DMPO/OH). with the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and the generation of the methyl radical adduct on the further addition of dimethyl sulfoxide. (DMPO/OH). adduct formation was inhibited by catalase but not by superoxide dismutase. The oxidant formed converted iodide to a trichloroacetic acid-precipitable form (iodination) and was bactericidal to logarithmic phase Escherichia coli. Both iodination and bactericidal activity was inhibited by catalase and by OH. scavengers, but not by superoxide dismutase. Iodination was optimal in 5 x 10(-4) M acetate buffer, pH 5.0, and when the Fe2+ and deferoxamine concentrations were equimolar at 10(-4) M. Fe2+ could not be replaced by Fe3+, Co2+, Zn2+, Ca2+, Mg2+, or Mn2+, or deferoxamine by EDTA, diethylenetriaminepentaacetic acid, or bathophenanthroline. These findings indicate that Fe2+ and deferoxamine can act as an oxygen radical generating system, which may contribute to its biological effects in vitro and in vivo.     

Characterization of free radical generation by xanthine oxidase. Evidence for hydroxyl radical generation

Xanthine oxidase has been hypothesized to be an important source of biological free radical generation. The enzyme generates the superoxide radical, .O2- and has been widely applied as a .O2- generating system; however, the enzyme may also generate other forms of reduced oxygen. We have applied electron paramagnetic resonance (EPR) spectroscopy using the spin trap 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO) to characterize the different radical species generated by xanthine oxidase along with the mechanisms of their generation. Upon reaction of xanthine with xanthine oxidase equilibrated with air, both DMPO-OOH and DMPO-OH radicals are observed. In the presence of ethanol or dimethyl sulfoxide, alpha-hydroxyethyl or methyl radicals are generated, respectively, indicating that significant DMPO-OH generation occurred directly from OH rather than simply from the breakdown of DMPO-OOH. Superoxide dismutase totally scavenged the DMPO-OOH signal but not the DMPO-OH signal suggesting that .O2- was not required for .OH generation. Catalase markedly decreased the DMPO-OH signal, while superoxide dismutase + catalase totally scavenged all radical generation. Thus, xanthine oxidase generates .OH via the reduction of O2 to H2O2, which in turn is reduced to .OH. In anaerobic preparations, the enzyme reduces H2O2 to .OH as evidenced by the appearance of a pure DMPO-OH signal. The presence of the flavin in the enzyme is required for both .O2- and .OH generation confirming that the flavin is the site of O2 reduction. The ratio of .O2- and .OH generation was affected by the relative concentrations of dissolved O2 and H2O2. Thus, xanthine oxidase can generate the highly reactive .OH radical as well as the less reactive .O2- radical. The direct production of .OH by xanthine oxidase in cells and tissues containing this enzyme could explain the presence of oxidative cellular damage which is not prevented by superoxide dismutase.     

Catalase and superoxide dismutase in Escherichia coli

We assessed the roles of intrabacterial catalase and superoxide dismutase in the resistance of Escherichia coli to killing by neutrophils. E. coli in which the synthesis of superoxide dismutase and catalase were induced by paraquat 10-fold and 5-fold, respectively, did not resist killing by neutrophils. When bacteria were allowed to recover from the toxicity of paraquat for 1 h on ice and for 30 min at 37 degrees C, they still failed to resist killing by neutrophils. Induction of the synthesis of catalase 9-fold by growth in the presence of phenazine methosulfate did not render E. coli resistant to killing by either neutrophils or by H2O2 itself. The lack of protection by intrabacterial catalase from killing by neutrophils could not be attributed to an impermeable bacterial membrane; the evolution of O2 from H2O2 was no less rapid in suspensions of E. coli than in lysates. The failure of intrabacterial catalase or superoxide dismutase to protect bacteria from killing by neutrophils might indicate either that the flux of O-2 and H2O2 in the phagosome is too great for the intrabacterial enzymes to alter or that the site of injury is at the bacterial surface.     

Formation of a TBA reaction product from crown ether in the presence of KO2.

Potassium superoxide (KO2), which can be dissolved in dimethyl sulfoxide containing crown ether, has been used as a source of O2-. for superoxide reaction systems. We have found that crown ether reacts with thiobarbituric acid (TBA) in the presence of KO2 to form a red pigment, which is a well-known reaction product of lipoperoxide.     

Effects of superoxide dismutase, dithiothreitol and formate ion on the inactivation of papain by hydroxyl and superoxide radicals in aerated solutions.

Losses in enzyme activity and sulphydryl content have been studied in aerated papain solutions containing formate, superoxide dismutase and dithiothreitol. Both formate and dithiothreitol converted .OH to .O2-, whereas superoxide dismutase completely suppressed the inactivation by .O2-. Using results from all three systems, the fraction of .O2- reactions with papain that caused inactivation of the enzyme was 0.33 +/- 0.07. The results also showed that the fraction of .OH reactions, which cause inactivation of papain, is significantly higher in aerated than in oxygen-free solutions.     

Spin trapping study on the kinetics of Fe2+ autoxidation: formation of spin adducts and their destruction by superoxide.

The oxidation of Fe2+ was investigated by electron spin resonance spin trapping techniques with N-t-butyl-alpha-phenylnitrone (PBN) and dimethyl sulfoxide. Under pure oxygen, the spin adduct PBN/.OCH3 was rapidly generated by the addition of Fe2+ (0.2-1.2 mM) into phosphate buffer containing ethylenediaminetetraacetate (EDTA), dimethyl sulfoxide, and PBN at pH 7.4, but it decayed. The decay process of PBN/.OCH3 consists of two components. The fast decay was dependent on Fe2+ concentration. Another was due to destruction of the spin adduct by superoxide anion (.O2-), because superoxide dismutase (SOD) markedly prevented the decay. Catalase decreased the yield of PBN/.OCH3. When EDTA was replaced by diethylenetriaminepentaacetic acid (DTPA), both the generation and decay process of PBN/.OCH3 were slow. SOD and catalase effects were similar to those in EDTA. Fe2+ produced PBN/.OCH3 even in the absence of chelators. We could estimate the kinetic parameters by computer simulation, comparing the Fe2+ oxidation in EDTA with that in DTPA. These results demonstrate that Fe2+ reacts with O2 to generate .O2- and then H2O2, which produces .CH3 by reaction with Fe2+ and dimethyl sulfoxide.(.)OCH3 results from the reaction between .CH3 and O2. The adduct PBN/.OCH3 decays by reaction with Fe2+ and .O2-.     

Clear evidence for the participation of OH in lambda DNA breakage induced by the enzymatic reduction of adriamycin in the presence of iron-ADP. Importance of local OH concentration for DNA strand cleavage

lambda DNA (a double-stranded DNA) was exposed to several adriamycin-mediated active oxygen generating systems (O2- and H2O2 generating, OH generating, and perferryl ion complex generating), extracted, and analyzed by gel electrophoresis on agarose gel. Only the DNA exposed to and subsequently isolated from the adriamycin-mediated OH generating system contained many DNA fragments of low molecular weight, indicating the breakage of DNA strands. Such a breakage was strikingly inhibited by catalase or 50 mM sodium benzoate, but not by superoxide dismutase. The local OH concentration near the DNA strand was considered to be important for DNA strand cleavage.     

Glycolate formation catalyzed by spinach leaf transketolase utilizing the superoxide radical

A homogeneous preparation of transketolase was obtained from spinach leaf; the specific enzyme activity was 9.5 mumolo of glyceraldehyde-3-P formed (mg of protein)-1 min-1, when xylulose-5-P and ribose-5-P were used as the donor and acceptor, respectively, of the ketol residue. Transketolase catalyzed the formation of glycolate from fructose-6-P coupled with the O2- -generating system of xanthine-xanthine oxidase. The addition of superoxide dismutase (145 units) or 1,2-dihydroxybenzene-3,5-disulfonic acid (Tiron) (5 mM), both O2- scavengers, to the reaction system inhibited glycolate formation 72 and 58%, respectively. The reacton was not inhibited by catalase. Mannitol, an .OH scavenger, and beta-carotene and 1,4-diazobicyclo[2.2.2]octane, 1O2 scavengers, showed little or no inhibitory effects. The rate of glycolate formation catalyzed by the transketolase system was measured in a coupled reaction with a continuous supply of KO2 dissolved in dimethyl sulfoxide, used as an O2- -generating system. The optimum pH of the reaction was above pH 8.5. The second-order rate constant for the reaction between transketolase and O2-, determined by the competition for O2- between nitroblue tetrazolium (NBT) and transketolase, was 1.0 X 10(6) M-1 s-1. Transketolase showed an inhibitory effect on the O2- -dependent reduction of NBT only if the reaction mixture was previously incubated with ketol donors such as fructose-6-P, xylulose-5-P, or glycolaldehyde. The results suggest the possibility that transketolase catalyzes O2- -dependent glycolate formation under increased steady-state levels of O2- in the chloroplast stroma.     

In vitro production of free oxygen radicals induced by pulsed ultrasounds in whole blood exposed to diagnostical frequencies and intensities.

DNA alkalinization experiments on lymphocytes from sonicated whole blood and on in vitro cultured lymphocytes in presence of free radical scavengers (superoxide dismutase, catalase and mannitol) showed that lesions inflicted upon DNA by pulsed ultrasounds could be ascribed to production of free radicals (O2-, OH.) and H2O2, which could mediate the production of still unidentified organic radicals, likely to be responsible for DNA damage.     

An electron spin resonance study of oxyradical generation in superoxide dismutase- and catalase-deficient mutants of Escherichia coli K-12

The response of superoxide dismutase- and catalase-deficient strains of Escherichia coli to redox active compounds was examined by electron spin resonance. Levels of radicals formed in response to pyocyanine in situ were extremely low and were found to be predominantly extracellular, even in a strain completely deficient in both superoxide dismutase and catalase. In cell-free extracts of superoxide dismutase-minus strains incubated with NADPH and pyocyanine, the primary accumulating radical was the superoxide anion (O2-), although low levels of the hydroxyl radical (.OH) were also detected. In contrast, extracts from strains lacking catalase were found to accumulate higher levels of hydroxyl radicals.     

Involvement of oxidative stress in hydroquinone-induced cytotoxicity in catalase-deficient Escherichia coli mutants

Hydroquinone is a benzene-derived metabolite. To clarify whether the reactive oxygen species (ROS) are involved in hydroquinone-induced cytotoxicity, we constructed transformants of Escherichia coli (E. coli) strains that express mammalian catalase gene derived from catalase mutant mice (Cs(b), Cs(c)) and the wild-type (Cs(a)) using a catalase-deficient E. coli UM255 as a recipient. Specific catalase activities of these tester strains were in order of Cs(a) > Cs(c) > Cs(b) > UM255, and their susceptibility to hydrogen peroxide (H2O2) showed UM255 > Cs(b) > Cs(c) > Cs(a). We found that hydroquinone exposure reduced the survival of catalase-deficient E. coli mutants in a dose-dependent manner significantly, especially in the strains with lower catalase activities. Hydroquinone toxicity was also confirmed using zone of inhibition test, in which UM255 was the most susceptible, showing the largest zone of growth inhibition, followed by Cs(b), Cs(c) and Cs(a). Furthermore, we found that hydroquinone-induced cell damage was inhibited by the pretreatment of catalase, ascorbic acid, dimethyl sulfoxide (DMSO), and ethylenediaminetetraacetic acid (EDTA), and augmented by superoxide dismutase (both CuZnSOD and MnSOD). The present results suggest that H2O2 is probably involved in hydroquinone-induced cytotoxicity in catalase-deficient E. coli mutants and catalase plays an important role in protection of the cells against hydroquinone toxicity.     

DNA strand scission by enzymatically reduced mitomycin C: evidence for participation of the hydroxyl radical in the DNA damage

Phage DNA, as well as plasmid and mammalian DNA's, were exposed to a superoxide and hydroxyl radical-generating system containing NADPH-cytochrome P-450 reductase and mitomycin C, both with and without added Fe3+-ADP, in phosphate buffer at pH 7.5. The generation of superoxide (O2-.) and hydroxyl (.OH) radicals in the system was demonstrated by using ESR spectrometry with N-tert -butyl-alpha-phenylnitrone (PBN) as a spin trapping agent. Only the lambda DNA isolated after exposure to the O2-./.OH-generating system containing many lower molecular weight DNA fragments indicating DNA strand breaks. This breakage was completely inhibited by a .OH radical scavenger (sodium benzoate) and by catalase, but only slightly by superoxide dismutase. Thyroid and plasmid DNA's were both cleaved when exposed to the O2-./.OH-generating systems. It is suggested that the mechanism of DNA scission by mitomycin C described here closely resembles that induced by the anthracycline drugs.     

Free-radical chain oxidation of 2-nitropropane initiated and propagated by superoxide.

The superoxide radical O2.-, whether produced by the xanthine/xanthine oxidase reaction or infused as KO2, solubilized by a crown ether in dry dimethyl sulphoxide, initiated a free-radical chain oxidation of anionic 2-nitropropane. Superoxide dismutase, but not catalase, inhibited oxidation of the nitroalkane. Xanthine oxidase suffered a syncatalytic inactivation, during the co-oxidation of 2-nitropropane, which was reversed by dialysis. Cyanide exacerbated this syncatalytic inactivation and rendered it irreversible. The frequently observed oxidations of nitroalkanes by flavoenzymes now need to be re-examined to clarify the extent to which O2.--initiated free-radical chain oxidation contributed to the overall nitroalkane oxidation.     

Superoxide dismutase-rich bacteria. Paradoxical increase in oxidant toxicity

Superoxide dismutase is considered important in protection of aerobes against oxidant damage, and increased tolerance to oxidant stress is associated with induction of this enzyme. However, the importance of superoxide dismutase in this tolerance is not clear because conditions which promote the synthesis of superoxide dismutase likewise affect other antioxidant enzymes and substances. To clarify the role of superoxide dismutase per se in organismal defense against oxidant-generating drugs, we employed Escherichia coli transformed with multiple copies of the gene for bacterial iron superoxide dismutase. These bacteria have greater than ten times the superoxide dismutase activity of wild-type E. coli but, importantly, are normal in other oxidant defense parameters including catalase, peroxidases, glutathione, and glutathione reductase. High superoxide dismutase and control bacteria were exposed to the O2- -generating drug paraquat and to elevated pO2. We find; high superoxide dismutase E. coli are more readily killed by paraquat under aerobic, but not anaerobic, conditions. During exposure to paraquat, high superoxide dismutase E. coli accumulate more H2O2. Coincidentally, the reduced glutathione content of high superoxide dismutase E. coli declines more than in control E. coli. E. coli with high superoxide dismutase activity are also more readily killed by hyperoxia. Interestingly, the susceptibility of the parental and high superoxide dismutase E. coli to killing by exogenous H2O2 is not significantly different. Thus, under these experimental conditions, greatly enhanced superoxide dismutase activity accelerates H2O2 formation. The increased H2O2 probably accounts for the exaggerated sensitivity of high superoxide dismutase bacteria to oxidant-generating drugs. These results support the concept that the product of superoxide dismutase, H2O2, is at least as hazardous as the substrate, O2-. We conclude that effective organismal defense against reactive oxygen species may require balanced increments in antioxidant enzymes and cannot necessarily be improved by increases in the activity of single enzymes.     

Radiosensitization and radioprotection of E. coli by thiourea in nitrous oxide saturated suspensions

N2O did not modify the radiosensitivity of E. coli BH and H/r-30 strains as to colony-forming ability and DNA single-strand breaks. In N2O-saturated suspensions of E. coli, thiourea and thiosemicarbasite sensitized at low concentrations, while cysteamine and cysteine protected at all concentrations. Protection by thiourea in N2O-saturated suspensions was observed only in the frozen state. These results suggest that the conversion of e-aq to OH radicals may be responsible for sensitization and this sensitization is probably due to the thiourea and thiosemicarbasite radicals produced extracellularly.     

Oxygen free radical generation and regulation of proliferative activity of human mononuclear cells responding to different mitogens.

We have compared various mitogenic stimuli for their ability to induce hydrogen peroxide (H2O2) and superoxide anion (O2-) production by PBMC and the effect of these reactive oxygen species and hydroxyl radical (OH.) has been assessed on proliferation. Our results show that pokeweed mitogen (PWM) stimulated PBMC to release H2O2 which interfered with proliferation since inclusion of catalase enhanced PBMC thymidine uptake. In contrast, phytohemagglutinin (PHA) and monoclonal antibody to CD3 (alpha CD3) did not induce PBMC to generate H2O2. O2- release by PBMC, which is readily induced by phorbol myristate acetate (PMA), did not occur when the cells were stimulated with PWM, PHA, or alpha CD3. In correlation, the O2- scavenger enzyme superoxide dismutase (SOD) had no effect on the proliferative response of the cells to the same mitogens, whereas it impaired the thymidine uptake of PMA-stimulated PBMC. A regulatory role for OH. was implied by studies using a battery of OH scavengers known to inhibit PMA-stimulated PBMC proliferation. OH. scavengers markedly inhibited the lymphoblastic transformation of alpha CD3-stimulated cells but had little or no effect on PHA- and PWM-stimulated PBMC. Thus, one manner by which PBMC proliferation is regulated is through oxygen free radical production which varies depending on the type of mitogenic stimulus.     

Superoxide Dismutase and Oxygen Toxicity in a Eukaryote

Saccharomyces cerevisiae var. ellipsoideus contained 6.5 times more superoxide dismutase and 2.3 times more catalase when grown under 100% O(2) than when grown anaerobically. Growth under oxygen caused equal increases in both the cyanide-sensitive and the cyanide-insensitive superoxide dismutases of this organism. Experience with other eukaryotes has shown that cyanide sensitivity is a property of the cupro-zinc superoxide dismutase of the cytosol, whereas cyanide insensitivity is a property of the corresponding mangani-enzyme found in mitochondria. Cu(2+), which has been shown to increase the radioresistance of yeast, also caused an increase of both of the superoxide dismutases of S. cerevisiae. Yeast which had been grown under 1 atm of O(2) were more resistant toward the lethal effects of 20 atm of O(2) than were yeast which had been grown in the absence of O(2). Escherichia coli K-12 his(-) responded to growth under 1 atm of O(2) by increasing its content of catalase and of peroxidase, but not of superoxide dismutase. This contrasts with E. coli B, which was previously shown to respond to O(2) by a striking increase in superoxide dismutase. E. coli K-12 his(-) did not gain resistance toward 20 atm of O(2) because of having been grown under 1 atm of O(2). Once again, this contrasts with the behavior of E. coli B. These data indicate that, in both prokaryotes and in eukaryotes, superoxide dismutase is an important component of the defenses against oxygen toxicity.     

Mechanism of NADH-sensitized formation of DNA breaks during irradiation with near UV light]

NADH-photosensitized in vitro formation of single-stranded breaks in plasmid DNA pBR322 depends on both the concentration of the sensitizer and the influence of near-UV radiation (320-400 nm). Scavengers and inhibitors of different activated oxygen species (sodium azide, sodium benzoate, catalase and superoxide dismutase) prevent the formation of breaks in full or partly. The data obtained show that hydroxyl radical (.OH) and singlet oxygen (1O2) are directly involved in the induction of breaks. In this process hydrogen peroxide (H2O2) plays the role of an intermediate in the reaction of .OH formation from superoxide anion-radical (O2-.) which is the first NAD.H-photogenerated product.