Oxidative cellular damage associated with transformation of Helicobacter pylori from a bacillary to a coccoid form

Exposure to unfavorable conditions results in the transformation of Helicobacter pylori, a gastric pathogen, from a bacillary form to a coccoid form. The mechanism and pathophysiological significance of this transformation remain unclear. The generation of the superoxide radical by H. pylori has previously been shown to inhibit the bactericidal action of nitric oxide, the concentration of which is relatively high in gastric juice. With the use of chemiluminescence probes, both the quality and quantity of reactive oxygen species generated by H. pylori have now been shown to change markedly during the transformation from the bacillary form to the coccoid form. The transformation of H. pylori was associated with oxidative modification of cellular proteins, including urease, an enzyme required for the survival of this bacterium in acidic gastric juice. Although the cellular abundance of urease protein increased during the transformation, the specific activity of the enzyme decreased and it underwent aggregation. Specific activities of both superoxide dismutase and catalase in H. pylori also decreased markedly during the transformation. The transformation of H. pylori was also associated with oxidative modification of DNA, as revealed by the generation of 8-hydroxyguanine, and subsequent DNA fragment. These observations indicate that oxidative stress elicited by endogenously generated reactive oxygen species might play an important role in the transformation of H. pylori from the bacillary form to the coccoid form.     

Colony formation by Helicobacter pylori after long-term incubation under anaerobic conditions

To evaluate the viability of Helicobacter pylori cultured under anaerobic conditions, H. pylori strain TK1029 was grown on blood agar in a microaerophilic environment at 37 degrees C for 4 days, and subsequently cultured under anaerobic conditions for 1 to 35 days. Colony formation by bacteria on blood agar plates cultured under anaerobic conditions was observed only for up to 4 days of microaerophilic incubation. By Gram staining, the morphological form of the bacteria was shown to be predominantly coccoid. However, bacteria cultured under anaerobic conditions for 15 to 35 days formed colonies on blood agar after pre-incubation of bacteria with PBS, but not without pre-incubation. These results suggest that H. pylori survives long-term culture under anaerobic conditions and that both pre-incubation in non-nutrient solution and high density of bacterial concentration might be important for recovery of H. pylori cultured for a prolonged time under anaerobic conditions.     

Morphological changes and outer membrane protein patterns in Helicobacter pylori during conversion from bacillary to coccoid form

Conversion from bacillary to fully coccoid form via an intermediate U-and V-shaped form has been described in prolonged cultures of H. pylori. This morphological transformation may be the expression of transitory adaptation to a particular environment and may play an important role in antibiotic resistance and the difficulty to eradicate the pathogen. The aim of this study was to evaluate morphological and outer membrane protein changes in H. pylori during ageing-induced conversion to coccoid morphology. We used two H. pylori strains (the reference NCTC 11639 and a fresh clinical isolate) cultivated in microaerophilic environment at 37 degrees C, monitoring their morphological and biochemical evolutions for 11 days. Microscopic examination revealed the passage from spiral to U- and V-shaped form after 5-8 days of incubation, the conversion to coccoid form and the entry into viable but non-culturable state (VBNC) between days 9 and 11. Protein pattern difference appeared at 97.4 to 45 and 30 kDa molecular weight. Biochemical tests demonstrated not only a modification of outer membrane protein profiles, but also an intra-specific variability by comparison between the two analysed strains. Our findings suggest that structural and outer membrane changes associated with coccoid transformation represent a typical response in H. pylori and may constitute a survival strategy in adverse environmental conditions.     

Mechanisms of generation of oxygen radicals and reductive mobilization of ferritin iron by lipoamide dehydrogenase.

The oxidase reaction of lipoamide dehydrogenase with NADH generates superoxide radicals and hydrogen peroxide under aerobic conditions. ESR spin trapping using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) was applied to characterize the oxygen radical species generated by lipoamide dehydrogenase and the mechanism of their generation. During the oxidase reaction of lipoamide dehydrogenase, DMPO-OOH and DMPO-OH signals were observed. The DMPO-OOH signal disappeared on addition of superoxide dismutase. These results demonstrate that the DMPO-OOH adduct was produced from the superoxide radical generated by lipoamide dehydrogenase. In the presence of dimethyl sulfoxide, a DMPO-CH3 signal appeared at the expense of the DMPO-OH signal, indicating that the DMPO-OH adduct was produced directly from the hydroxyl radical rather than by decomposition of the DMPO-OOH adduct. The DMPO-OH signal decreased on addition of superoxide dismutase, catalase, or diethylenetriaminepentaacetic acid, indicating that the hydroxyl radical was generated via the metal-catalyzed Haber-Weiss reaction from the superoxide radical and hydrogen peroxide. Addition of ferritin to the NADH-lipoamide dehydrogenase system resulted in a decrease of the DMPO-OOH signal, indicating that the superoxide radical interacted with ferritin iron.     

Extracellular superoxide production by Enterococcus faecalis requires demethylmenaquinone and is attenuated by functional terminal quinol oxidases

The intestinal commensal bacterium, Enterococcus faecalis, is unusual among prokaryotic organisms in its ability to produce substantial extracellular superoxide. Transposon mutagenesis, allelic replacement, and electron spin resonance (ESR)-spin trapping showed that superoxide production and generation of derivative hydroxyl radical were dependent on membrane-associated demethylmenaquinone. Extracellular superoxide was generated through univalent reduction of oxygen by reduced demethylmenaquinone. Moreover, extracellular superoxide production was inhibited by exogenous haematin, an essential cofactor for cytochrome bd, and by fumarate, a substrate for fumarate reductase. As integral membrane quinol oxidases, cytochrome bd and fumarate reductase redox cycle demethylmenaquinone, and are necessary for aerobic and anaerobic respiration respectively. A rat model of intestinal colonization demonstrated that conditions exist in the mammalian intestinal tract that permit a mode of respiration for E. faecalis that results in the formation of hydroxyl radical. These results identify and characterize the mechanism by which E. faecalis generates extracellular free radicals.     

Electron paramagnetic resonance evidence that cellular oxygen toxicity is caused by the generation of superoxide and hydroxyl free radicals

Cells require molecular oxygen for the generation of energy through mitochondrial oxidative phosphorylation; however, high concentrations of oxygen are toxic and can cause cell death. A number of different mechanisms have been proposed to cause cellular oxygen toxicity. One hypothesis is that reactive oxygen free radicals may be generated; however free radical generation in hyperoxic cells has never been directly measured and the mechanism of this radical generation is unknown. In order to determine if cellular oxygen toxicity is free radical mediated, we applied electron paramagnetic resonance, EPR, spectroscopy using the spin trap 5,5'-dimethyl-1-pyrroline-N-oxide, DMPO, to measure free radical generation in hyperoxic pulmonary endothelial cells. Cells in air did not give rise to any detectable signal. However, cells exposed to 100% O2 for 30 min exhibited a prominent signal of trapped hydroxyl radical, DMPO-OH, while cell free buffer did not give rise to any detectable radical generation. This cellular radical generation was demonstrated to be derived from the superoxide radical since the observed signal was totally quenched by superoxide dismutase, but not by equal concentrations of the denatured enzyme. It was confirmed that the hydroxyl radical was generated since in the presence of ethanol the CH3 CH(OH) radical was formed. Loss of cell viability as measured by uptake of trypan blue dye was observed paralleling the measured free radical generation. Thus, superoxide and hydroxyl radicals are generated in hyperoxic pulmonary endothelial cells and this appears to be an important mechanism of cellular oxygen toxicity.     

The presence of the cag pathogenicity island is associated with increased superoxide anion radical scavenging activity by Helicobacter pylori

Reactive oxygen species (ROS) generated by Helicobacter pylori infection have been suggested to be important factors in induction of gastric malignancies. Utilizing electron spin resonance spectrometry, H. pylori-dependent radical formation and hydroxyl- and superoxide-anion radical scavenging activity was investigated. In contrast to previous reports, we found that H. pylori does not produce ROS, but displays superoxide scavenging activity. This scavenging activity was increased in cag-positive H. pylori strains when compared to strains lacking an intact cag pathogenicity island, and was dependent on enzyme activity. We hypothesize that the increased scavenging activity of cag-positive H. pylori strains is an adaptation to the increased inflammatory response associated with the cag-positive genotype of H. pylori.     

Hydroxyl radical formation as a result of the interaction between primaquine and reduced pyridine nucleotides. Catalysis by hemoglobin and microsomes

Kinetic, circular dichroism, and NADH and NADPH fluorescence quenching studies indicate that these compounds interact with the antimalarial drug primaquine (PQ). The affinity of both pyridine nucleotides for PQ is similar. The data are in contrast with a previous report (Thornalley et al. (1983) Biochem. Pharmacol. 32, 3571-3575) suggesting specificity for the interaction with NADPH. The complex was seen to facilitate electron transfer from NAD(P)H to oxygen, generating oxygen-free radicals which were detected by the spin-trapping technique and to flavin nucleotides, giving rise to flavin semiquinone radicals which were demonstrated by direct ESR spectroscopy under anaerobic conditions. A twofold increase in oxygen uptake and hydroxyl radical generation by the NAD(P)H-PQ complex was observed in the presence of hemoglobin. This effect was independent of heme concentration (in the range 1 X 10(-5)-1 X 10(-4) M) and oxidation state of the iron. Under anaerobic conditions, the NAD(P)H-PQ complex reduces Fe-III to Fe-II hemoglobin, and under aerobic conditions about 65% of the heme chromophore is irreversibly destroyed. Superoxide dismutase inhibits hydroxyl radical generation by the NAD(P)H-PQ pair; this effect is not observed in the presence of hemoglobin. In the presence of microsomes there is a 10-fold increase in both oxygen consumption and hydroxyl radical generation by the NAD(P)H-PQ pair. The fact that both pyridine nucleotides are active, and the inability of SKF 525A in decreasing hydroxyl radical generation, suggests that microsomal reductases are involved in the catalysis.     

Hydroxyl Radical Generation Depending on O2 or H2O by a Photocatalyzed Reaction in an Aqueous Suspension of Titanium Dioxide

Photocatalytic production of the electron (e^-) and positive hole (h⁺) in an aqueous suspension of TiO₂ (anatase form) under illumination by near-UV light (295-390 nm) generated the superoxide (O₂ ^-) and hydroxyl radical (?OH), which both proceeded linearly with reaction time, while H₂O₂ accumulated non-linearly. Under anaerobic conditions (introduced Ar gas), the yields of three active species of oxygen were decreased to 10-20% of those detected in the air-saturated reaction. The electron spin resonance (ESR) signal characteristics of ?OH were obtained when a spin trap of 5,5-dimthyl-1-pyrroline-N-oxide (DMPO) was included in the illuminating mixture. The intensity of the ESR signal was increased by Cu/Zn superoxide dismutase, and decreased under anaerobic conditions, amounting to only 20% of the intensity detected in the aerobic reaction. The addition of H₂O₂ to the reaction mixture resulted in about an 8-fold increase of ?OH production in the anaerobic reaction, but only about 1.5-fold in the aerobic reaction, indicating that e^- generated by the photocatalytic reaction reduced H₂O₂ to produce ?OH plus OH^-. On the other hand, D₂O lowered the yield of ?OH generation to 18% under air and 40% under Ar conditions, indicating the oxidation of H₂O by h⁺. The addition of Fe(III)-EDTA as an electron acceptor effectively increased ?OH generation, 2.3-fold in the aerobic reaction and 8.4-fold in the anaerobic reaction, the yield in the latter exceeding that in the air-saturated reaction.     

Photosensitization by the trypanocidal agent crystal violet. Type I versus type II reactions

The photoreduction of crystal violet to a carbon-centered radical was detected directly by electron spin resonance (ESR) spectroscopy under anaerobic conditions. The linewidth (0.9 G) of this radical was less broad than the linewidth (11.0 G) of the free radical obtained in Trypanosoma cruzi incubations. No crystal violet radical could be detected under aerobic conditions. However, crystal violet was found to convert oxygen to superoxide anion and hydrogen peroxide in the presence of light. This superoxide anion and hydrogen peroxide formation was greatly enhanced by reducing agents such as NAD(P)H. In addition, irradiation of crystal violet did not generate detectable amounts of singlet oxygen.     

Plasma membrane-generated reactive oxygen intermediates and their role in cell growth of plants

Reactive oxygen species (ROS) produced as intermediates in the reduction of O2 to H2O (superoxide radical, hydrogen peroxide, hydroxyl radical), are generally regarded as harmful products of oxygenic metabolism causing cell damage in plants, animals and microorganisms. However, oxygen radical chemistry can also play useful roles if it takes place outside of the protoplast. In plants, the production of these ROS initiated by the plasma membrane NAD(P)H oxidase can be used for controlled polymer breakdown leading to wall loosening during extension growth. Backbone cleavage of cell wall polysaccharides can be accomplished by hydroxyl radicals produced from hydrogen peroxide and superoxide in a reaction catalyzed by cell wall peroxidase. Growing plant organs such as coleoptiles or roots of maize seedlings produce these ROS specifically in the apoplast of actively growing tissues, e.g. in the epidermis of the coleoptile and the growing zone of the root. Auxin promotes the release of hydroxyl radicals when inducing elongation growth. Experimental generation of hydroxyl radicals in the wall causes an increase in wall extensibility in vitro and replaces auxin in inducing growth. Auxin-induced growth can be inhibited by scavengers of ROS or inhibitors interfering with the formation of these molecules in the cell wall. These results provide the experimental background for a novel hypothesis on the mechanism of plant cell growth in which the generation of hydroxyl radicals, initiated by the plasma membrane NAD(P)H oxidase, plays a central role.     

Superoxide and peroxyl radical generation from the reduction of polyunsaturated fatty acid hydroperoxides by soybean lipoxygenase.

Soybean lipoxygenase is shown to catalyze the breakdown of polyunsaturated fatty acid hydroperoxides to produce superoxide radical anion as detected by spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). In addition to the DMPO/superoxide radical adduct, the adducts of peroxyl, acyl, carbon-centered, and hydroxyl radicals were identified in incubations containing linoleic acid and lipoxygenase. These DMPO radical adducts were observed just prior to the system becoming anaerobic. Only a carbon-centered radical adduct was observed under anaerobic conditions. The superoxide radical production required the presence of fatty acid substrates, fatty acid hydroperoxides, active lipoxygenase, and molecular oxygen. Superoxide radical production was inhibited when nordihydroguaiaretic acid, butylated hydroxytoluene, or butylated hydroxyanisole was added to the incubation mixtures. We propose that polyunsaturated fatty acid hydroperoxides are reduced to form alkoxyl radicals and that after an intramolecular rearrangement, the resulting hydroxyalkyl radical reacts with oxygen, forming a peroxyl radical which subsequently eliminates superoxide radical anion.     

One-electron reduction of carcinogen chromate by microsomes, mitochondria, and Escherichia coli: identification of Cr(V) and .OH radical.

Earlier studies have shown that a long-lived Cr(V) species is produced during the reduction of chromate (Cr(VI] by microsomes/NADPH, mitochondria, and other cellular constituents and that this Cr(V) species plays a significant role in the mechanism of Cr(VI) toxicity. The present work indicates that this species is a Cr(V) complex involving the diol moieties of NADPH as the ligand. Additionally, ESR spin trapping investigations show that the hydroxyl (.OH) radical is also generated in the reduction process. Hydrogen peroxide (H2O2) enhances the .OH generation but suppresses the Cr(V)-NADPH complex formation. Catalase decreases the .OH radical generation and enhances the Cr(V)-NADPH formation. Measurements under anaerobic atmosphere show decreased .OH radical generation, indicating that during the cellular Cr(VI) reduction process molecular oxygen is reduced to H2O2, which reacts with the Cr(V)-NADPH complex to generate the .OH radical via a Fenton-like mechanism.     

Biological Reduction of Aromatic Nitroso Compounds: Evidence for the Involvement of Superoxide Anions

The in vitro formation of phenylhydronitroxide and 2-methylphenylhydronitroxide free radicals from nitrosobenzene (NB) and 2-nitrosotoluene (NT), respectively, in either red blood cells (RBC) or RBC hemolysates, was confirmed by electron spin resonance spectroscopy (ESR). Free radicals were generated nonenzymatically from reaction of the respective nitroso compounds with a number of biological reducing agents as corroborated by model studies of NB or NT with NAD(P)H. Under aerobic conditions, phenylhydronitroxide and 2-methylphenylhydronitroxide underwent a subsequent one-electron transfer to oxygen, which then resulted in the formation of superoxide anion (O₂^-). The latter product was confirmed by the superoxide dismutase (SOD)-inhibitable reduction of cytochrome c (cyt c). Apparently, oxygen is needed for continuous formation of the hydronitroxide radical derivatives. On the other hand, under anaerobic conditions, no phenylhydronitroxide radical was generated from NB in the presence of NADH, but the formation of phenylhydroxylamine from NB was detected by the absorption spectrometry. These results suggest that oxygen is a preferential electron acceptor for hydronitroxide radical derivatives.     

Chemiluminescent detection and imaging of reactive oxygen species in live mouse skin exposed to UVA

The recent increase of ultraviolet (UV) rays on Earth due to the increasing size of the ozone hole is suggested to be harmful to life and to accelerate premature photoaging of the skin. The detrimental effects of UV radiation on the skin are associated with the generation of reactive oxygen species (ROS) such as superoxide anion radical (*O(-)(2)), hydrogen peroxide (H(2)O(2)), hydroxyl radical (*OH), and singlet oxygen ((1)O(2)). However, direct proof of such ROS produced in the skin under UV irradiation has been elusive. In this study, we report first in vivo detection and imaging of the generated ROS in the skin of live mice following UVA irradiation, in which both a sensitive and specific chemiluminescence probe (CLA) and an ultralow-light-imaging apparatus with a CCD camera were used. In addition, we found that *O(-)(2) is formed spontaneously and (1)O(2) is generated in the UVA-irradiated skin. This method should be useful not only for noninvasive investigation of the spatial distribution and quantitative determination of ROS in the skin of live animals, but also for in vivo evaluation of the protective ability of free radical scavengers and antioxidants.     

Photosensitized damage to calf thymus DNA by a hypocrellin derivative: mechanisms under aerobic and anaerobic conditions

The di-cysteine substituted hypocrellin B (DCHB) derivative has been found to be a potential phototherapeutic agent and exhibit photosensitized damage to DNA. Electronic paramagnetic resonance (EPR) and spectrophotometry demonstrate that one-electron transfer from calf thymus DNA to triplet DCHB induces the generation of the reduced form of DCHB (DCHB*- radical), followed by the second electron transfer from DNA to DCHB*- or the disproportionation of DCHB*- to form the hydroquinone of DCHB (DCHBH2) in anaerobic conditions. This electron transfer process induces the direct damage to DNA in oxygen-free media and contributes partly to the damage of DNA in aerobic media. Superoxide radical and hydroxyl radical are formed with enhanced efficiencies while singlet oxygen is generated with a reduced efficiency from irradiation of DCHB and DNA solution under aerobic conditions as compared with the case in the absence of DNA. All of three reactive oxygen species play an evident role in the photosensitized damage to DNA in aerobic system in addition to the direct electron-transfer damage.