Control of singlet oxygen-induced oxidative damage in Escherichia coli

Singlet oxygen ((1)O(2)) is a highly reactive form of molecular oxygen that may harm living systems by oxidizing critical cellular macromolecules. The oxyR gene product regulates the expression of the enzymes and proteins that are needed for cellular protection against oxidative stress. In this study, the role of oxyR in cellular defense against a singlet oxygen was investigated using Escherichia coli oxyR mutant strains. Upon exposure to methylene blue and visible light, which generates singlet oxygen, the oxyR overexpression mutant was much more resistant to singlet oxygen-mediated cellular damage when compared to the oxyR deletion mutant in regard to growth kinetics, viability and protein oxidation. Induction and inactivation of major antioxidant enzymes, such as superoxide dismutase and catalase, were observed after their exposure to a singlet oxygen generating system in both oxyR strains. However, the oxyR overexpression mutant maintained significantly higher activities of antioxidant enzymes than did the oxyR deletion mutant. These results suggest that the oxyR regulon plays an important protective role in singlet oxygen-mediated cellular damage, presumably through the protection of antioxidant enzymes.     

Inactivation of biologically active DNA by gamma-ray-induced superoxide radicals and their dismutation products singlet molecular oxygen and hydrogen peroxide.

Since superoxide radicals are involved in many metabolically important as well as in some other, detrimental cellular processes, the reactivity of gamma-ray-induced superoxide radicals and its dismutation products singlet molecular oxygen and hydrogen peroxide with DNA have been studied. Superoxide dismutase which removes superoxide radicals and inhibits the formation of singlet oxygen in the solution protects the biologically active replicative form of DNA (from bacteriophage theta X174) against inactivation by ionizing radiation. Catalase which removes hydrogen peroxide also protects the DNA. Attempts with various chemical sources of singlet oxygen to determine whether this species inactivates DNA did not give an unequivocal answer. It is concluded from the presented experiments that a combination of the protonated form of the superoxide radical (HO-2) and H2O2 do inactivate DNA.     

Prooxidant Action of Maltol: Role of Transition Metals in the Generation of Reactive Oxygen Species and Enhanced Formation of 8-hydroxy-2′-deoxyguanosine Formation in DNA

Maltol (3-hydroxy-2-methyl-4-pyrone) produced reactive oxygen species as a complex with transition metals. Maltol/iron complex inactivated aconitase the most sensitive enzyme to oxidative stress. The inactivation of aconitase was iron-dependent, and prevented by TEMPOL, a scavenger of reactive oxygen species, suggesting that the maltol/iron-mediated generation of superoxide anion is responsible for the inactivation of aconitase. Addition of maltol effectively enhanced the ascorbate/copper-mediated formation of 8-hydroxy-2′-deoxyguanosine in DNA. Oxidation of ascorbic acid by CuSO₄ was effectively stimulated by addition of maltol, and the enhanced oxidation rate was markedly inhibited by the addition of catalase and superoxide dismutase. These results suggest that maltol can stimulate the copper reduction coupled with the oxidation of ascorbate, resulting in the production of superoxide radical which in turn converts to hydrogen peroxide and hydroxyl radical. Cytotoxic effect of maltol can be explained by its prooxidant properties: maltol/transition metal complex generates reactive oxygen species causing the inactivation of aconitase and the production of hydroxyl radical causing the formation of DNA base adduct.     

Photoactivation of the nematicidal compound alpha-terthienyl from roots of marigolds (Tagetes species). A possible singlet oxygen role.

The nematicidal compound alpha-terthienyl from roots of Tagetes species generates upon irradiation with near ultraviolet light reactive oxygen species on which the in vitro nematicidal activity depends. This system was studied by following the inhibition of glucose-6-phosphate dehydrogenase by photoactivated alpha-terthienyl and protection of the enzyme activity in the absence of oxygen and by various additions. Addition of mannitol, benzoate, superoxide dismutase or catalase did not have any effect nor did H2O2. This suggests that OH., O-.2, and H2O2 are not the reactive oxygen species involved. The enzyme was protected against photoactivated alpha-terthienyl in air-saturated solutions by singlet oxygen quenchers such as histidine, methionine, tryptophan, bovine serum albumin, and NaN3. Furthermore, inactivation of the enzyme was about 3.5 times faster in D2O than in H2O. When alpha-terthienyl in CH2Cl2 was irradiated in the presence of the olefin adamantylideneadamantane, a stable dioxetane was formed which decomposed to adamantanone when heated above its melting point. These results indicate a singlet oxygen-mediated process.     

Yeast thioredoxin peroxidase expression enhances the resistance of Escherichia coli to oxidative stress induced by singlet oxygen

Singlet oxygen ((1)O(2)) is a highly reactive form of molecular oxygen that may harm living systems by oxidizing critical cellular macromolecules. A soluble protein from Saccharomyces cerevisiae specifically provides protection against a thiol-containing metal-catalyzed oxidation system (thiol/Fe(3+)/O(2)) but not against an oxidation system without thiol. This 25 kDa protein acts as a peroxidase but requires the NADPH-dependent thioredoxin system or a thiol-containing intermediate, and was named thioredoxin peroxidase (TPx). The role of TPx in the cellular defense against oxidative stress induced by singlet oxygen was investigated in Escherichia coli containing an expression vector with a yeast genomic DNA fragment that encodes TPx and mutant in which the catalytically essential amino acid cysteine (Cys-47) has been replaced with alanine by a site-directed mutagenesis. Upon exposure to methylene blue and visible light, which generates singlet oxygen, there was a distinct difference between the two strains in regard to growth kinetics, viability, the accumulation of oxidized proteins and lipids, and modulation of activities of superoxide dismutase and catalase. The results suggest that TPx may play an important protective role in a singlet oxygen-mediated cellular damage.     

Antioxidant enzyme inhibitors enhance singlet oxygen-induced cell death in HL-60 cells

Singlet oxygen is a highly reactive form of molecular oxygen that may harm living systems by oxidizing critical cellular macromolecules and it also promotes deleterious processes such as cell death. The protective role of antioxidant enzymes against singlet oxygen-induced oxidative damage in HL-60 cells was investigated in control and cells pre-treated with diethyldithiocarbamic acid, aminotriazole and oxlalomalate, specific inhibitors of superoxide dismutase, catalase and NADP⁺-dependent isocitrate dehydrogenase, respectively. Upon exposure to rose bengal (20 μM)/light (15 min), which generates singlet oxygen, to HL-60 cells, the viability was lower and the lipid peroxidation and oxidative DNA damage were higher in inhibitor-treated cells as compared to control cells. We also observed the significant increase in the endogenous production of reactive oxygen species as well as the significant decrease in the intracellular GSH level in inhibitor-treated HL-60 cells exposed to singlet oxygen. Upon exposure to rose bengal (3 μM)/light (15 min), which induced apoptotic cell death, a clear inverse relationship was observed between the control and inhibitor-treated HL-60 cells in their susceptibility to apoptosis. These results suggest that antioxidant enzymes play an important role in cellular defense against singlet oxygen-induced cell death including necrosis and apoptosis.     

Antioxidant enzyme inhibitors enhance singlet oxygen-induced cell death in HL-60 cells

Singlet oxygen is a highly reactive form of molecular oxygen that may harm living systems by oxidizing critical cellular macromolecules and it also promotes deleterious processes such as cell death. The protective role of antioxidant enzymes against singlet oxygen-induced oxidative damage in HL-60 cells was investigated in control and cells pre-treated with diethyldithiocarbamic acid, aminotriazole and oxlalomalate, specific inhibitors of superoxide dismutase, catalase and NADP⁺-dependent isocitrate dehydrogenase, respectively. Upon exposure to rose bengal (20 μM)/light (15 min), which generates singlet oxygen, to HL-60 cells, the viability was lower and the lipid peroxidation and oxidative DNA damage were higher in inhibitor-treated cells as compared to control cells. We also observed the significant increase in the endogenous production of reactive oxygen species as well as the significant decrease in the intracellular GSH level in inhibitor-treated HL-60 cells exposed to singlet oxygen. Upon exposure to rose bengal (3 μM)/light (15 min), which induced apoptotic cell death, a clear inverse relationship was observed between the control and inhibitor-treated HL-60 cells in their susceptibility to apoptosis. These results suggest that antioxidant enzymes play an important role in cellular defense against singlet oxygen-induced cell death including necrosis and apoptosis.     

Formation of lipid peroxides in isolated rat liver microsomes by singlet molecular oxygen.

Rat liver microsomes were incubated in neutral aqueous solution of potassium peroxychromate, a system which generates singlet molecular oxygen. Such incubation resulted both in a rapid decline in NADPH-cytochrome c reductase activity, and in an increase in formation of lipid peroxides. These reactions were not inhibited by either superoxide dismutase (SOD) or mannitol, nor were they entirely duplicated by incubating microsomes with hydrogen peroxide. However, a high concentration of 1,4-diazabicyclo-[2,2,2]octane (DABCO), a known scavenger of singlet oxygen, prevented both decline in reductase activity and formation of lipid peroxides. These results suggest that the observed effects are, in fact, attributable to singlet oxygen, and not to hydrogen peroxide, superoxide radical, or hydroxyl radical.     

Genotoxicity of singlet oxygen

Singlet oxygen, ¹O₂(¹Δ_g), fulfills essential prerequisites for a genotoxic substance, like hydroxyl radicals and other oxygen radicals: it can react efficiently with DNA and it can be generated inside cells, e.g. by photosensitization and enzymatic oxidation. As might be anticipated from the non-radical character of singlet oxygen, the pattern of DNA modifications it produces is very different from that caused by hydroxyl radicals. While hydroxyl radicals produce DNA strand breaks and sites of base loss (AP sites) in high yield and react with all four bases of DNA, singlet oxygen generates predominantly modified guanine residues and few strand breaks and AP sites. There is now convincing evidence that a major product of base modification caused by singlet oxygen is 8-hydroxyguanine (7,8-dihydro-8-oxoguanine). Indeed, the recently reported miscoding properties of 8-hydroxyguanine can explain the predominant type of mutations observed when DNA modified by singlet oxygen is replicated in cells. There are also strong indications that singlet oxygen generated by photosensitization can act as an ultimate DNA modifying species inside cells. However, indirect genotoxic mechanisms involving other reactive oxygen species produced from singlet oxygen are also possible and appear to predominate in some cases. The cellular defense system against oxidants consists of effective singlet oxygen scavengers such as carotenoids. The observation that carotenoids can inhibit neoplastic cell transformation when administered not only together with but also after the application of chemical or physical carcinogens might indicate a role of singlet oxygen in tumor promotion that could be independent of the direct or indirect DNA damaging properties.     

Revealing mechanisms of selective,concentration-dependent potentials of 4-hydroxy-2-nonenal to induce apoptosis in cancer cells through inactivation of membrane-associated catalase

Tumor cells generate extracellular superoxide anions and are protected against superoxide anion-mediated intercellular apoptosis-inducing signaling by the expression of membrane-associated catalase. 4-Hydroxy-2-nonenal (4-HNE), a versatile second messenger generated during lipid peroxidation, has been shown to induce apoptosis selectively in malignant cells. The findings described in this paper reveal the strong, concentration-dependent potential of 4-HNE to specifically inactivate extracellular catalase of tumor cells both indirectly and directly and to consequently trigger apoptosis in malignant cells through superoxide anion-mediated intercellular apoptosis-inducing signaling. Namely, 4-HNE caused apoptosis selectively in NOX1-expressing tumor cells through inactivation of their membrane-associated catalase, thus reactivating subsequent intercellular signaling through the NO/peroxynitrite and HOCl pathways, followed by the mitochondrial pathway of apoptosis. Concentrations of 4-HNE of 1.2 µM and higher directly inactivated membrane-associated catalase of tumor cells, whereas at lower concentrations, 4-HNE triggered a complex amplificatory pathway based on initial singlet oxygen formation through H₂O₂ and peroxynitrite interaction. Singlet-oxygen-dependent activation of the FAS receptor and caspase-8 increased superoxide anion generation by NOX1 and amplification of singlet oxygen generation, which allowed singlet-oxygen-dependent inactivation of catalase. 4-HNE and singlet oxygen cooperate in complex autoamplificatory loops during this process. The finding of these novel anticancer pathways may be useful for understanding the role of 4-HNE in the control of malignant cells and for the optimization of ROS-dependent therapeutic approaches including antioxidant treatments.     

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.     

Generation of reactive oxygen species by photolysis of the ruthenium(II) complex Ru(NH3)5(pyrazine)2+ in oxygenated solution.

Metal-to-ligand charge transfer photolysis of the ruthenium(II) pyrazine complex Ru(NH3)5pz2+ (I) in pH 7.4 oxygenated phosphate buffer solution generates the Ru(III) analog Ru(NH3)5pz3+ plus the reactive oxygen species singlet oxygen and superoxide. Based on the very short MLCT lifetime (re-measured as approximately 250 ps in D2O) of I* and the quantum yield for singlet oxygen formation (0.01 for aerated D2O) the rate constant for oxygen quenching of I* was calculated to be approximately (3+/-1)x10(10) M-1 s-1.     

Singlet Oxygen Generated in PS￠ò Particles Promotes the Superoxide Generation

The relationship between superoxide and singlet oxygen in PSⅡ particle of spinach under strong illumination was studied with spin-trap ESR technique. The generation of superoxide was increased under D2 environment which prolonged the half life of singlet oxygen. The generation of superoxide was decreased when histidine existed as a scavenger of singlet oxygen. It is highly possible that the superoxide generated in PSⅡ particle originates from singlet oxygen.     

Effect of ascorbic acid on the production of singlet oxygen by purified human myeloperoxidase

We have previously studied purified human myeloperoxidase-hydrogen peroxide-halide ion systems as models of possible singlet oxygen production by granulocytes. While myeloperoxidase could efficiently produce singlet oxygen, the yield of singlet oxygen at a physiological pH with Cl- was very small due to enzyme inactivation. In that Bolscher et al. [(1984) Biochim. Biophys. Acta 784, 189-191] observed that micromolar concentrations of ascorbic acid prevented inactivation of myeloperoxidase and increased the production of hypochlorous acid, we examined whether ascorbic acid would augment singlet oxygen production by the myeloperoxidase-hydrogen peroxide-halide ion systems. Ascorbic acid, however, fails to increase the singlet oxygen yield, suggesting that it does not augment singlet oxygen production in the intact granulocyte by a myeloperoxidase-dependent mechanism.     

Yeast thioredoxin peroxidase expression enhances the resistance of Escherichia coli to oxidative stress induced by singlet oxygen

Abstract

Singlet oxygen (¹O₂) is a highly reactive form of molecular oxygen that may harm living systems by oxidizing critical cellular macromolecules. A soluble protein from Saccharomyces cerevisiae specifically provides protection against a thiol-containing metal-catalyzed oxidation system (thiol/Fe³⁺/O₂) but not against an oxidation system without thiol. This 25 kDa protein acts as a peroxidase but requires the NADPH-dependent thioredoxin system or a thiol-containing intermediate, and was named thioredoxin peroxidase (TPx). The role of TPx in the cellular defense against oxidative stress induced by singlet oxygen was investigated in Escherichia coli containing an expression vector with a yeast genomic DNA fragment that encodes TPx and mutant in which the catalytically essential amino acid cysteine (Cys-47) has been replaced with alanine by a site-directed mutagenesis. Upon exposure to methylene blue and visible light, which generates singlet oxygen, there was a distinct difference between the two strains in regard to growth kinetics, viability, the accumulation of oxidized proteins and lipids, and modulation of activities of superoxide dismutase and catalase. The results suggest that TPx may play an important protective role in a singlet oxygen-mediated cellular damage.     

DNA damage by oxygen radicals and excited state species: a comparative study using enzymatic probes in vitro

Repair enzyme-containing extracts from a variety of cell types are used to analyse and compare DNA damage induced by oxygen radicals and excited molecules. The differing potentials of these extracts for recognising DNA damage leads to characteristic DNA damage profiles after treatment with superoxide (xanthine/xanthine oxidase), gamma-rays, chemically generated singlet oxygen, photosensitizers (rose bengal, methylene blue), UV254 and a 1,2-dioxetane. Three different types of damage profiles are distinguished and assigned to the predominant action of hydroxyl radicals, singlet oxygen or to the photoexcitation of thymine residues. The method applied in this study allows the analysis of DNA damage and the identification or exclusion of the participation of different ultimate reactive species without chemical identification of the lesions.     

Photoilluminated riboflavin/riboflavin-Cu(II) inactivates trypsin: Cu(II) tilts the balance

Riboflavin (RF) upon irradiation with fluorescent light generates reactive oxygen species like superoxide anion, singlet and triplet oxygen, flavin radicals and substantial amounts of hydrogen peroxide (H2O2). H2O2 can freely penetrate cell membrane and react with a transition metal ion like Cu(ll), generating hydroxyl radical via the modified metal-catalyzed Haber-Weiss reaction. Earlier, it was reported that trypsin-chymotrypsin mixture served as an indirect antioxidant and decreased free radical generation. Thus, in the present study, we used photoilluminated RF as a source of ROS to investigate the effect of free radicals on the activity of trypsin. We also compared the damaging effect of photoilluminated RF and RF-Cu(ll) system using trypsin as a target molecule. RF caused fragmentation of trypsin and the effect was further enhanced, when Cu(II) was added to the reaction. Results obtained with various ROS scavengers suggested that superoxide radical, singlet and triplet oxygen were predominantly responsible for trypsin damage caused by photoilluminated RF. On the other hand, when Cu(ll) was added to the reaction, hydroxyl radical was mainly responsible for trypsin damage. A mechanism of generation of various ROS in the reaction is also proposed. Trypsin did not show any antioxidant effect with RF alone or with RF-Cu(II) combination.     

The role of compound III in reversible and irreversible inactivation of lactoperoxidase

In the presence of iodide (I-, 10 mM) and hydrogen peroxide in a large excess (H2O2, 0.1-10 mM) catalytic amounts of lactoperoxidase (2 nM) are very rapidly irreversibly inactivated without forming compound III (cpd III). In contrast, in the absence of I- cpd III is formed and inactivation proceeds very slowly. Increasing the enzyme concentration up to the micromolar range significantly accelerates the rate of inactivation. The present data reveal that irreversible inactivation of the enzyme involves cleavage of the prosthetic group and liberation of heme iron. The rate of enzyme destruction is well correlated with the production of molecular oxygen (O2), which originates from the oxidation of excess H2O2. Since H2O2 and O2 per se do not affect the heme moiety of the peroxidase, we suggest that the damaging species may be a primary intermediate of the H2O2 oxidation, such as oxygen in its excited singlet state (1 delta gO2), superoxide radicals (O-.2), or consequently formed hydroxyl radicals (OH.).     

Effects of Oxygen Radical Scavengers on the Inactivation of SS ΦX174 DNA by the Semi-Quinone Free Radical of the Antitumor Agent Etoposide

We have studied the effects of oxygen radical scavengers on the inactivation of ss ΦX174 DNA by the semi-quinone free radical of the antitumor agent etoposide (VP 16-213), which was generated from the ortho-quinone of etoposide at pH ≥ 7.4. A semi-quinone free radical of etoposide is thought to play a role in the inactivation of ss ΦDX174 DNA by its precursors 3',4'-ortho-quinone and 3',4'-ortho-dihydroxy-derivative. The possible role of oxygen radicals formed secondary to semi-quinone formation in the inactivation of DNA by the semi-quinone free radical was investigated using the hydroxyl radical scavengers t-butanol and DMSO. the spin trap DMPO, the enzymes catalase and superoxide dismutase, the iron chelator EDTA and potassium superoxide. Hydroxyl radicals seem not important in the process of inactivation of DNA by the semi-quinone free radical, since t-butanol, DMSO, catalase and EDTA had no inhibitory effect on DNA inactivation. The spin trapping agent DMPO strongly inhibited DNA inactivation and semi-quinone formation from the ortho-quinone of etoposide at pH ≥ 7.4 with the concomitant formation of a DMPO-OH adduct. This adduct probably did not arise from OH· trapping but from trapping of O₂^-. DMSO increased both the semi-quinone formation from and the DNA inactivation by the ortho-quinone of etoposide at pH ≥ 7.4. Potassium superoxide also stimulated ΦDX174 DNA inactivation by the ortho-quinone at pH ≤ 7. From the present study, it is also concluded that superoxide anion radicals probably play an important role in the formation of the semi-quinone free radical from the orthoquinone of etoposide, thus indirectly influencing DNA inactivation.     

Cytotoxicity, redox cycling and photodynamic action of two naturally occurring quinones

Two naturally occurring anthraquinones, barleriaquinone-I (BQ-I) and barleriaquinone-II (BQ-II), extracted from Barleria buxifolia, are tested for their cytotoxic action by aerobic incubation with human breast adenocarcinoma cells (MCF7). Cytotoxicities, measured as LD(50) (50% inhibition of colony formation) values, show BQ-II to be more active than BQ-I. Electron paramagnetic resonance studies confirm that BQ-II is reductively activated by NADH:cytochrome c reductase to superoxide anion radical. Cyclic voltammetric studies show one quasi-reversible redox couple for both BQ-I and BQ-II. Also, aerobic solutions of both BQ-I and BQ-II on visible illumination generate reactive oxygen species. Formation of O*-2 is studied by both EPR spin trapping and SOD-inhibitable cytochrome c reduction techniques. BQ-I generates more singlet oxygen as evidenced from the photobleaching of N,N-dimethyl-4-nitrosoaniline.