Excited Oxygen Species Research Articles

Lack of association between MnSOD gene polymorphism and sporadic Alzheimer’s Disease

Substantial evidence supports the hypothesis that impairment of mitochondrial function and increased oxidative damage are involved in the pathogenesis of several neurodegenerative disorders including Alzheimer's disease (AD). Manganese superoxide dismutase (MnSOD) plays a major role in protecting the mitochondrion from oxidative damage due to superoxide radicals and other excited oxygen species. Recent studies have indicated that MnSOD mRNA levels are significantly increased in the lymphocytes of AD patients, supporting the role of oxidative alterations in the pathogenetic mechanism underlying this neurodegeneration. A potentially functional amino acid polymorphism (Ala-9Val) has been described in the signal sequence of enzymes associated with decreased defense capacity against oxidative stress. The object of this study was to investigate the association between this polymorphism of the MnSOD gene and AD in the Italian population. The Ala-9Val polymorphism was genotyped by PCR amplification and SSCP analysis in 227 AD patients and 198 healthy controls. No significant differences in genotype or allele frequencies between cases and controls, even after stratification for APOE carrier status, were observed. Our data suggest that the Ala-9Val polymorphism in the MnSOD gene is not associated with genetic susceptibility in AD patients.

Poly (ethylene terephthalate) decomposition process in oxygen plasma; emission spectroscopic and surface analysis for oxygen–plasma reaction

Emission spectroscopy was applied to observe the reaction process of poly (ethylene terephthalate) (PET) in an oxygen (O2) plasma generated by a microwave discharge. As the PET was exposed in the O2 plasma flow, light emitted from the PET surface was monitored. In the diagnosis measurement, several emission peaks assigned to the Hα atomic line at 652 nm, Hβ at 486 nm, OH (2Σ→2Π) transition near 244–343 nm and CO (b3 Σ→a3 Σ) near 283–370 nm were observed and measured at various discharge times. These results indicated that after the plasma etching, the PET sample was decomposed by the oxygen plasma reaction, and then, hydrogen abstraction and carbon oxidation processes. We also observed the time profile of oxygen atom, as the atom-emission intensity at 777 nm was monitored. As Hβ atomic and OH molecule lines appeared in the presence of PET, the O atom intensity was significantly reduced. In the surface analysis on Fourier transform infrared and x-ray photoelectron spectroscopy measurements, it was found that for the PET surface treated by O2 plasma containing excited atomic oxygen species, ester bands were broken and carbonization formed on the PET surface.

On the antioxidant properties of therapeutic drugs: quenching of singlet molecular oxygen by aminosalicylic acids

The ability of the widely employed therapeutic drugs 4-aminosalicylic acid and 5-aminosalicylic acid to act as singlet molecular oxygen (O2(1Δg)) scavengers was investigated at pH 7 and pH 12. The isomer 3-aminosalicylic acid was also included in the study for comparative purposes. All three compounds quench photochemically generated O2(1Δg) with rate constants in the range of 107-108 M-1s-1, depending on the experimental conditions. No chemical reaction (oxidation of the aminosalicylic acids) was detected at the neutral pH, whereas at pH 12 both chemical and physical interactions with O2(1Δg) operated. The physical process implies the de-activation of the oxidant species without destruction of the aminosalicylic acid. The quotients between the overall and reactive rate constants for O2(1Δg) quenching at pH 12 (kr/kt ratios), which account for the actual effectiveness of photodegradation, were relatively low (0.22, 0.04, and 0.06 for 3-, 4- and 5-aminosalicylic acids, respectively). This indicates that the drugs, particularly the 4- and 5-amino derivatives, de-activate the excited oxygen species, at both pH values studied, mainly in a physical fashion, preventing its photodegradation and providing an antioxidative protection for possible photo-oxidizable biological targets in the surroundings.

Singlet molecular oxygen-mediated photo-oxidation of tetracyclines: kinetics, mechanism and microbiological implications

Members of the biologically active series tetracyclines (TCs) suffer visible-light-promoted photodynamic degradation to different extents, depending on their respective chemical structures and reaction conditions (solvent polarity and pH). The photo-oxidation is accompanied by a partial loss of the antimicrobial power. The photodamage is very fast in the alkaline pH range and less aggressive, although not negligible in kinetic terms, in the physiological pH region. Photo-oxidation quantum efficiencies, evaluated for eight TC derivatives, through singlet molecular oxygen [O 2( 1Δ g)] phosphorescence detection, spectrophotometric and polarographic methods, range from 0.12 to 0.65 as upper limits in alkaline medium. The photo-oxidation essentially proceeds via a O 2( 1Δ g)-mediated process, with rose bengal or eosine as dyesensitizers. Nevertheless, as a minor reactive pathway, the excited triplet state of the dye sensitizers interacts with TCs in a competitive process with O 2( 1Δ g) generation. The O 2( 1Δ g)-mediated photo-oxidation of TCs appears to be a plausible mechanism to account for their phototransformations in biological media, in the presence of visible-absorbing pigments. In both highly and moderately polar media, the quenching of the excited oxygen species is mainly represented by a reactive interaction. It is exerted by the TC molecule through a cooperative effect from the different contributions of several nuclear and extranuclear O 2( 1Δ g)-sensitive substituents, as discussed in detail in this paper. The TC phenolic function plays the driving role as a O 2( 1Δ g)-quencher only at pH values higher than 9. The quantum yields for O 2( 1Δ g) generation ( Φ Δ) by TCs, upon direct photo-irradiation, have also been investigated. This family of compounds possesses extremely poor ( Φ Δ) values, lower than 0.03 in the most favourable cases. Nevertheless, the TC photoproduct, formed through direct irradiation, efficiently generates O 2( 1Δ g) with ( Φ Δ) = 0.24. This important finding constitutes the first direct evidence of Type II sensitization by TC photoproducts, and could contribute to the elucidation of the mechanism of TC phototoxicity.

Quinones and hydroxyquinones as generators and quenchers of singlet molecular oxygen

The role of quinones and hydroxyquinones as sensitizers and as quenchers in Type II photooxygenations has been examined. The second aspect is discussed here, through a systematic study, for the first time in the open literature. Quinonic compounds are excellent generators of O2(1Δg) in aprotic solvents (excluding those quinones possessing substituents in positions adjacent to the carbonyl groups, in the case of anthraquinone derivatives). Benzoquinones, anthraquinones, and hydroxy derivatives are good O2(1Δg) quenchers upon dye-sensitized photoirradiation. The excited oxygen species is deactivated with rate constants in the range 106–107 M−1 s−1 depending on the solvent employed. The quenching process deactivates O2(1Δg) without further destruction of the quinone. The main interaction with O2(1Δg) is driven by the quinone moiety, in spite of the presence of potentially active nuclear substituents. The quenching mechanism could involve a reversible charge transfer intermediate, with the quinonic compound acting as an electron donor. Keywords: photooxidation, quenching, quinones, rose bengal, singlet oxygen.

Deriving “skin notations” for occupational exposure limits; a method validated by ECETOC

DNA photosensitization by several furocoumarins (including 3-carbethoxypsoralen (3-CPs), 8-methoxypsoralen (8-MOP), 5-methoxypsoralen (5-MOP) and angelicin was investigated by using DNA sequencing methodology. 3-CPs induces photo-oxidation of guanine residues leading to alkali-labile sites in DNA (revealed by hot piperidine), whereas 8-MOP, 5-MOP and angelicin do not. There is a preferential photo-oxidation of G when located on the 5′ side of GG doublets, likely to reflect a better accessibility of the G moiety in such a context. Mechanisms operating via both radicals (type I) and singlet oxygen (type II) are involved in the photo-oxidation of G residues by 3-CPs. Photo-oxidized G residues are produced independently of the formation of photoadducts, and scavengers of singlet oxygen or radicals do not inhibit photobinding of 3-CPs to DNA. This leads us to propose that covalent photoadducts arise from the intercalated excited sensitizer molecules, whereas G photo-oxidations are produced either by electron transfer reactions involving bound 3-CPs or by energy transfer to molecular oxygen, thereby producing singlet oxygen that subsequently reacts with guanine bases. Quantification of both types of DNA lesions indicated that in vitro photo-oxidized G residues are produced in DNA by 3-CPs plus ultraviolet light at least to the same extent as photoadducts, under our conditions. A calf thymus redoxyendonuclease, equivalent to the endonuclease III of Escherichia coli, specific for oxidative DNA damages, recognizes and cleaves DNA at sites of photo-oxidized G residues. The extent of the cleavage by this enzyme was close to that observed by hot piperidine and followed the amount of photo-oxidized G residues produced when the lifetime of excited oxygen species is modified. The redoxyendonuclease did not incise DNA treated with 8-MOP, 5-MOP or angelicin plus ultraviolet light. The exonuclease III and endonuclease IV of E. coli also involved in the repair of oxidative DNA damage, convert the replicative form I of 3-CPs-treated DNA to replicative form II. This suggests that the lesions recognized by these enzymes are apurinic-like lesions. In view of the low toxicity and mutagenicity of 3-CPs, DNA photo-oxidation products induced by the photodynamic effect of 3-CPs are likely to be efficiently taken care of by the DNA repair system(s). It is clear that 3-CPs photo-induces several classes of DNA damage, including oxidative damage. This latter type of damage was not detected in vitro in the case of other furocoumarins, such as 8-MOP and 5-MOP, whose genotoxicity is much higher than that of 3-CPs.

Excited oxygen beam by R.F. type multicapillary ion source

We have developed a compact ion source for ion-assisted film formation. This ion source has two specific features. The first is the ability to feed the discharge gas through a multicapillary nozzle that consists of a few hundred bundled capillaries. Consequently, the gaseous flow becomes intense and collimated. The second feature is producing the plasma by inductive coupled R.F. discharge. Since the R.F. coil is not exposed in the plasma, this ion source can operate with various reactive gases. Moreover, since ions can be extracted into the chamber without extraction grids, the ion energy can be low. It is found that the excited oxygen beam produced by this method includes oxygen ions and atomic oxygen. The flux of excited oxygen species is calculated from the increasing mass of an oxidized silver thin film bombarded by the excited oxygen beam. In the pressure range from 10 −1 to 10 −2 Pa, a flux density of excited oxygen species above 10 16 atoms cm −2 s −1 is obtained.

Oxidative stress in rodent closed duodenal loop pancreatitis.

Production of excited oxygen species is earlier in the liver than in the pancreas and could contribute to damage in a reflux model. Treatment with SOD could attenuate 59% light emission in pancreas, but did not modify serum enzyme levels or pancreatic edema, resulting as an insufficient isolated therapy. Unexpectedly, it was found an increased plasma antioxidant capacity that was related to total bilirubin levels, and declined at late stages probably denoting other circulating antioxidant consumption. Oxidative stress has been shown to play a role in different models of acute pancreatitis, although it has not been studied in the severe necrohemorrhagic model produced by closed duodenal loop pancreatitis. We studied Sprague Dawley female rats in two groups: a closed duodenal loop pancreatitis group and a control, sham-operated group. In order to evidence the oxygen excited species production, in situ spontaneous chemiluminescence from living and naturally perfused pancreas and liver was measured at 0, 0.5, 1.5, 3, 6, 12, and 24 h after the duodenal ligature. Blood pancreatic amylase and aminotransferases levels were determined as expression of tissue damage in pancreas and liver. At the same time, plasma antioxidant capacity was measured by the peroxyl radical trapping capability of plasma samples compared to that of Trolox (synthetic analog of vitamin E), and results are expressed as Trolox equivalence. Bovine superoxide dismutase (SOD) was administered to attenuate oxygen free radicals activity at the beginning of the peroxidation chain and also as a therapeutic tool. The experimental procedure induced a severe pancreatitis, as evidenced by pancreatic enzymes that rose markedly in the early hours of disease and remained heightened throughout the experiment. The results show early light emission from the liver at 3 h and peak levels at 12 h, whereas in the pancreas, luminescence increased at 6 h and doubled later at 12 h, both returning to control levels at 24 h.

Ozone decomposition on glass and silica

Abstract Ozone decomposition on glass and silica surfaces is studied by means of non‐porous particles in a fixed bed (sand, glass bead, crushed glass bead, silica). Influences of gas velocity, particle diameter and reactor volume are investigated. Ozone is produced by a silent discharge generator. A model considering the geometric characteristics of material is used so that the ozone decomposition rate per unit of surface area is identified. The decomposition reaction is represented by means of an apparent first‐order kinetic constant. The high dependency of the apparent kinetic constant on the surface area, for given flow rates, suggests that the ozone decomposition mechanism is not simple. Due to the values of the apparent first order kinetic constant, the existence of catalytic species produced by the ozone generator, as well as excited ozone and/or oxygen species, are considered, by means of several models, to explain the experimental results.

Structure and DNA sequence of the mouse MnSOD gene

Oxidative damage to the cell has been implicated in the pathogenesis of a number of disorders, including chronic inflammation, aging, and cancer. Manganese superoxide dismutase (Mn-SOD) plays a major role in the protection of the mitochondrion from oxidative damage due to superoxide radicals and other excited oxygen species. In this report we describe the genomic organization and DNA sequence of the murine MnSOD gene. This gene is interrupted by four introns. The coding sequence of this gene was examined in C57BL/6J and C3H/HeJ mice that are SUSCEPTIBLE AND RESISTANT, respectively, to the pulmonary injuries induced by the inhaled oxidants, ozone, and hyperoxia. Since the predicted amino acid sequence for MnSOD does not differ for these strains, nor does the size or steady-state level of this transcript, biologic variability in the pulmonary inflammatory response to ozone and hyperoxia does not arise from an altered gene structure. Examination of the noncoding sequence revealed a dC.dA polymorphism in intron 2 and a StyI RFLV in intron 4 of the MnSOD gene. These sequence and mapping data provide the basis for continued study of biologic variability in the MnSOD gene as a cause of disease.

Multiband analysis of photoluminescence spectra from electronically excited gas-phase species produced during laser ablation of lead oxide, zirconium oxide, titanium oxide, and lead zirconate titanate targets

The analysis of multiband/line photoluminescence spectra produced by electronically excited gas-phase species in the plasma plume, generated by impact of a KrF excimer laser beam (248 nm) on various metal oxide targets, has been carried out, and the results are reported. It was established that, at 1.1 J/cm{sup 2} in 900 mTorr of O{sub 2}, (i) Pb(I), Pb(II), and O{sub 2}{sup +} are generated from PbO(s) targets; (ii) Zr(I), Zr(II), ZrO, O{sub 2}, and O{sub 2}{sup +} from ZrO{sub 2}(s) targets; (iii) Ti(I), Ti(II), TiO, TiO{sub 2}, and O{sub 2}{sup +} from TiO{sub 2}(s) targets; and (iv) Pb(I), Pb(II), PbO, Zr(I), Zr(II), ZrO, Ti(I), Ti(II), TiO, TiO{sub 2}, O(I), O(II), O{sub 2}, and O{sub 2}{sup +} from a solid Pb(Zr{sub x}Ti{sub 1-x})O{sub 3}(PZT) target. The observation of excited oxygen species may be attributed to the presence of ambient molecular O{sub 2} and/or the oxygen-rich metal oxide and PZT targets. The much lower standard enthalpy of atomization, {Delta}H{sub a}{sup 0}, for PbO(s) and the much higher {Delta}H{sub a}{sup 0} values for ZrO{sub 2}(s) and TiO{sub 2}(s) are consistent with observing molecular metal oxide species, MO{sub n}(g), in the ablation plume from ZrO{sub 2}(s) and TiO{sub 2}(s) but observing almost exclusively atomicmore » Pb(I) and Pb(II) from the PbO(s) target. Additionally, a detailed explanation regarding the observation of lower-charged atomic metal species and not the higher-charged (formally ionic) species is presented. Particle ejection is briefly discussed as also is the relative influence of the observed species on thin-film deposition. 30 refs., 4 figs., 8 tabs.« less

Effect of Flavonoids on the Photooxidation of Fats ‐A Study on their Activity as Singlet Molecular Oxygen [O2(1δ)] Generators and Quenchers

AbstractThe protective effect of the polyhydroxyflavones Fisetin and Baicalein on the sensitized [O2(1δg] ‐ mediated photooxidation of fats was investigated through a kinetic study. These flavonoids at concentrations in the order of a few ppm efficiently inhibit the photoperoxidation of linoleic acid, which was chosen as an example of photooxidizable fat. This property was attributed to the ability of Fisetin and Baicalein to quench photochemically generated O2(1δg). The rate constants for such a process of quenching were 1.9 and 1.4 × 108 M−1S−1 for Fisetin and Baicalein respectively. The ratio between the overall and the chemical rate constants were in the order of 0.01, indicating that the flavones are practically not consumed in the process of O2(1δg) quenching. The presence of the ‐OH groups in the aromatic rings of the hydroxyflavones confer to these compounds the ability as O2(1δg) quenchers. The parent compound Flavone does not quench that oxygen excited species. Only Flavone generates O2(1δg) upon direct irradiation, at 337 nm, in the absence of added sensitizers, with a quantum yield of 0.16. Fisetin and Baicalein were totally inefficient for such a process. This fact constitutes a very convenient property from the point of view of the protective (anti oxidative) activity of the hydroxyflavones.

Influence of the nuclear and extranuclear substitution on the singlet molecular oxygen [O 2(1?g)]-mediated photooxidation of tyrosine derivatives: A kinetic study

The effect of the substitution pattern on the kinetics of the Type II (O2((1)Δg)-mediated) dye-sensitized photooxidation of a series of nine tyrosine derivatives was investigated. Overall (kt) and reactive (kr) rate constants for the interaction of the excited oxygen species with the amino acid derivatives were determined. A parallel study on solvent and pH effects was carried out.The presence of different substituents in nuclear positions or in the amino acid side chain greatly affect the photooxidation rates.An upper limit for photooxidation quantum yield, calculated from the kinetic data, varies from 0.03 to 0.25, being the higher for halogenated tyrosines and the lower for esterified tyrosines and for the nitro-derivative.The variation of solvent polarity and pH of the reaction medium confirm that the presence of the ionized phenolate group in tyrosine, clearly dominates the quenching process. As already postulated for generic phenolic derivatives, it proceeds through a polar intermediate complex which posses some component of charge-transfer character.Esterification of the carboxilic acid of tyrosine selectively decreases the contribution of the reactive step to the overall process of O2((1)Δg) quenching. An amide group in the same position does not produce noticiable changes in this sense. The presence of a highly deactivating nitro group in nuclear positions greatly diminishes the magnitude of both overall and reactive interactions.For all three, o-, m- and p-tyrosine the values of photooxidation quantum yields show an excellent parallelism with the rates of consumption of the - NH2 group of the amino acid chain, upon sensitized irradiation. It could react, in the cases of 0- and m-tyrosine in a secondary, non photochemical, step.

Chemiluminescence as a method for oxidative rancidity assessment in autoxidized marine oils

Chemiluminescence based on light emission from excited oxygen species,e.g. singlet oxygen and triplet carbonyls, has been used to measure the oxidative rencidity of fish oils. The luminescence was recorded after sodium hypochlorite addition to the oils dissolved intert‐butanol. In addition to high sensitivity and ability to detect small changes in rancidity, the method is fast and may be used as a supplement to the standard chemical methods for quality assessment and antioxidant evaluation. However, care has to be taken in interpretation of the chemiluminescence data of different fish oils, as the light emission depends on both the composition and the rancidity level of the oil. The autoxidized oils were also characterized by peroxide value, thiobarbituric acid value, anisidine value, Kreis rancidity index, iodine value, ultraviolet measurements, capillary gas chromatography, size‐exclusion chromatography and sensoric evaluation.

Investigation on Leakage Current Reduction of Photo‐CVD Tantalum Oxide Films Accomplished by Active Oxygen Annealing

Previously, the authors demonstrated that photo‐CVD films formed on a Si substrate using and acquire very low leakage current by active oxygen annealing. This paper investigates in detail the mechanism of the leakage current reduction. Low‐leakage‐current films are produced provided there is a sufficient UV‐activation of molecules during CVD, an abundant supply of electronically excited oxygen, O(1D) and , during annealing, and Si migration from the substrate into the films during annealing, or CVD. A phenomenological model of the leakage current reduction is proposed. In the model, a certain void in the Ta‐O network is compensated by chemically incorporating a Si atom and several excited oxygen species; O(1D) or . This model can consistently interpret a series of phenomena on leakage current reduction reported in this paper and in our previous papers.

Nonradical oxidants of the phagocyte type induce the activation of plasmatic single chain- urokinase

Single chain- urokinase (scu-PA) is the proenzyme of the plasminogen activator urokinase (tcu-PA). In human blood scu-PA is of great stability. Activated phagocytes generate large amounts of single chain- urokinase and of reactive oxidants (chloramines and HOCl). Since these cells participate in physiologic fibrinolysis, we were interested in the interaction between plasmatic scu-PA and chloramines. The oxidants dose dependently induce the activation of plasmatic scu-PA. Optimal activation of scu-PA occurs at about 3 – 5 mmol/l of chloramine-T. The findings suggest a control mechanism of scu-PA stability/ activity by oxidatively modifiable plasma proteins, such as a-2-antiplasmin. The oxidation mechanism seems to be mediated by singlet molecular oxygen, an excited oxygen species. Basing on this scu-PA/ oxidant synergism a sensitive and fast functional assay of scu-PA in human plasma is presented. Plasmatic inhibitors normally interfering with functional scu-PA measurements are inactivated by addition of chloramine-T, imitating the physiological oxidants generated by activated phagocytes. The scu-PA concentration in plasma of n= 36 healthy individuals has been determined to be 5.8 ± 1.6 ng/ml. The lower detection limit of plasma scu-PA by the procedure described is about 1.5 ng/ ml of plasma. By means of this technique scu-PA concentration during thrombolytic therapy can be measured within minutes in undiluted (direct) plasma samples, allowing adjustments of the scu-PA dosage. The present study gives further credence for a role of singlet molecular oxygen, possibly a new type of locally acting hormones (autacoid), in the regulation of the fibrinolytic pathway.

Nonradical excited oxygen species induce selective thrombolysis in vivo

All the thrombolytic agents currently in clinical use act as plasminogen activators. In this study evidence is presented that also oxidants of the phagocyte type are of fibrinolytic efficiency in vivo. Activated phagocytes participate in physiologic fibrinolysis. The cells generate plasminogen activators and reactive oxidants of the nitrogen- chlorine type. Experimental mimicry of this oxidative inflammatory response induces selective thrombolysis in a rabbit jugular vein model. Intravenous bolus administration of sub-millimolar blood concentrations of chloramine-T resulted in thrombolysis after about 30 min without notable systemic toxicity; the coagulation parameters activated partial thromboplastin time (aPTT), thrombin time, fibrinogen, and a-2-antiplasmin were not influenced. Control experiments with 2000 IU of urokinase/ kg induced thrombolysis after about 90 min with systemic changes of the hemostatic system. The fibrinolysis promoting effect of the oxidants of the phagocyte type could be inhibited by quenchers of singlet molecular oxygen and was not affected at all by inhibitors of oxygen radicals. The data gives evidence that nonradical excited oxygen species (NEOS) act as powerful pro-fibrinolytic and anti-coagulant agents in vivo. It might be suggested that NEOS could represent a novel class of regulators of the fibrinolytic system. The long lived and hydrophilic chloramine derivatives can either accumulate or diffuse far from their site of generation. Therefore, on the one hand oxidants in high (local) concentrations might be considered as direct pro- fibrinolytic agents due to their powerful protein modulating efficacy. On the other hand, oxidants at low concentrations may act as indirect pro- fibrinolytic compounds, i.e. as chemoattractants to concentrate phagocytes to the site of a thrombus. In this case the oxidants would play the role of signal elements faraway from the thrombus,a self amplifying mechanism possibly mediated by oxidation of blood arachidonat/ lipid metabolites.

Influence of water vapor in the chemically excited oxygen flow generated from the reaction of Cl2 with alkaline H2O2

The estimation for the water vapor included in chemically generated excited oxygen flow and the influence of the water vapor on the excited oxygen species are presented. The concentration of the water vapor in the flow was changed by altering the temperatures of the solution and the cold trap and then, it was estimated by measuring the the pressure change and by measuring the concentration of the excited oxygen molecules. The fraction of O2(1Δ) to the total oxygen did not change with the water vapor pressure, but the concentration of O2(1Σ) changed significantly. Applying the high sensitivity of O2(1Σ) concentration to H2O, the estimation of a small quantity of water vapor concentration was available in the system.

Flow cytometric study of injuries in cultured endothelial cells by neutrophils of the inherited cataract rats

We examined the mechanism of endothelial injuries in the inherited cataract rats (ICR), which have a number of age-associated spontaneous injuries in the aortic endothelium. Cell cycle traverse rate of endothelial cells of ICR was shorter than that of Wistar rats. The rate was estimated from bromodeoxyuridine (BrdU) incorporation into cell nuclei measured periodically after BrdU pulse labeling. Next we established the method for measurement of cultured endothelial cell injury by neutrophils with flow cytometry by assessing the regeneration of injured endothelial cells. By the use of the gate analysis method, contaminated neutrophils were excluded from the analysis. Endothelial cell injury by neutrophils of Wistar rats was detectable at 1 × 10 5 neutrophils (1 neutrophil to 1 endothelial cell) when stimulated with 10 ng/ml phorbol myristate acetate (PMA). Extent of injury increased with an increasing number of neutrophils and the concentration of a stimulator, PMA. We detected endothelial cell injury by ICR neutrophils not only when they were stimulated but also in a resting condition, and ICR neutrophils yielded more injury than Wistar rat neutrophils. Number of adhered neutrophils to endothelial cells and effects of plasma or lymphocytes were the same between two strain rats. Scavengers of hydrogen peroxide and singlet oxygen inhibited the ICR neutrophil-induced endothelial cell injury. These findings indicate that an increase of generation of excited oxygen species from neutrophils, particularly of singlet oxygen, may cause injury of endothelial cells in this specific strain of rats.

Oxidative damage to bacterial DNA and evidence for its repair

Oxidative damage to DNA can be caused by excited oxygen species, which are produced by radiation or are by-products of aerobic metabolism. Endogenous levels of 8-hydroxy-2′-deoxyguanosine (8-OH-dG), an adduct that results from the damage of DNA caused by hydroxyl radical, have been detected inE. coli andS. typhimurium. Treatment of bacterial cells with various concentrations of hydrogen peroxide caused a moderate increase in the 8-OH-dG content. The enzymatic release of 8-OH-dG from asocorbate/Cu(II)-treated DNA was effected by an extract ofE. coli cells. These results indicate that 8-OH-dG is formedin vivo in bacterial DNA through endogenous oxidative mechanisms and on treatment with an oxygen radical-producing agent and that it is repairable.