Reactive oxygen species and intracellular Ca2+, common signals for apoptosis induced by gallic acid

Gallic acid (3,4,5-trihydroxybenzoic acid), a naturally occurring plant phenol, induces cell death in apparently different manners, depending on cell lines. Flow cytometric analysis and agarose gel electrophoresis indicated that internucleosomal breakdown of chromatin DNA was observed in HL-60RG cells but not in dRLh-84, HeLa, and PLC/PRF/5 cells, and that the action of gallic acid was independent of cell cycle. A detailed study of signal transduction revealed that the gallic acid-induced cell death of all cells tested in this study was prevented by treatment with the intracellular thiol antioxidant N-acetyl-L-cysteine, catalase, and the intracellular calcium chelator bis-(o-aminophenoxy)-N,N,N,N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM). However, the effects of ascorbic acid, superoxide dismutase, EGTA, the endonuclease inhibitor zinc sulfate, the calmodulin inhibitor N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), and the NADPH oxidase inhibitor diphenyleneiodonium chloride on cell death were different depending on the cell type, suggesting that the death signal induced by gallic acid was diverse among different cell types, although the production of reactive oxygen species, such as H2O2, and the elevation of intracellular calcium concentration were required as common signals.     

The MGDS examination: a systematic approach. 3. Part II of the examination: diagnosis, treatment planning, execution of treatment, maintenance and appraisal, writing-up log diaries

The purpose of this study was to elucidate the differential contribution of catalase and glutathione peroxidase (GSH-Px) to H2O2 scavenging in cultured human dermal fibroblasts. Responses of the cells in terms of both enzyme activities were examined by using two sorts of inhibitors, 3-amino-1H-1,2,4-triazole (AT) for catalase and DL-buthionine-[S,R]-sulfoximine (BSO) for GSH-Px, under exposure to H2O2 or ultraviolet (UV) B radiation. AT treatment resulted in a decrease in H2O2 scavenging activity, while BSO treatment did not affect H2O2 scavenging. When fibroblasts were exposed to a low concentration of H2O2 (100 microM). AT treatment resulted in a significant decrease in cell survival, but BSO treatment did not affect survival. At higher concentrations of H2O2 ranging from 500 microM to 1 mM, BSO-treated fibroblasts showed reduced survival. In addition, AT treatment was much more cytotoxic in the presence of UVB than BSO treatment. The intracellular levels of H2O2 in fibroblasts treated with AT or BSO were also determined. BSO-treated cells showed similar H2O2 levels to control cells, but the intracellular H2O2 levels of AT-treated fibroblasts were 1.4-fold higher than found in control cells. These results with human dermal fibroblasts indicate that catalase acts as a primary defence against oxidative stress from exogenous or endogenous H2O2 at low concentrations. In contrast, GSH-Px helps protect the cell from damage during exposure to high concentrations of H2O2.     

Effects of hydroxyl radicals on KATP channels in guinea-pig ventricular myocytes

We studied the effects of oxygen free radicals on the ATP-sensitive potassium channel (KATP channel) of guinea-pig ventricular myocytes. Single KATP channel currents were recorded from inside-out patches in the presence of symmetrical K+ concentrations (140 mM in both bath and pipette solutions). Reaction of xanthine oxidase (0.1 U/ml) on hypoxanthine (0.5 mM) produced superoxide anions (.O2-) and hydrogen peroxide (H2O2). Exposure of the patch membrane to.O2- and H2O2 increased the opening of KATP channels, but this activation was prevented by adding 1 microM glibenclamide to the bath solution. In the presence of ferric iron (Fe3+: 0.1 mM), the same procedure produced hydroxyl radicals (.OH) via the iron-catalysed Haber-Weiss reaction.OH also activated KATP channels; however, this activation could not be prevented by, even very high concentrations of glibenclamide (10 microM). These different effects of glibenclamide suggest that the mode of action of these oxygen free radicals on KATP channels is different and that.OH is more potent than.O2-/H2O2 in activating KATP channels in the heart.     

Loss of heterozygosity on chromosome 16 in sporadic Wilms'' tumour

Activated cell-mediated immunity, associated for example with HIV infection, is accompanied by elevated concentrations of neopterin and 7,8-dihydroneopterin. Recent data have indicated a role of neopterin derivatives in virus activation and apoptotic cell death, processes likely to involve the action of oxygen free radicals. Because T cell death in AIDS is likely to involve the Fas/Fas ligand system and the action of oxygen free radicals and 7,8-dihydroneopterin, we compared the kinetics and sensitivity of apoptotic cell death of human leukemic Jurkat T cells to that of treatments with 7,8-dihydroneopterin, anti-Fas, and H2O2. Upon incubation with 5 mM 7,8-dihydroneopterin and 50 microM hydrogen peroxide over a period of 24 hr, bimodal kinetics were observed with peaks at 5.5 hr (7,8-dihydroneopterin, 13.1%; H2O2, 11.4%) and at 24 hr (7,8-dihydroneopterin, 11.2%; H2O2, 13.2%). In contrast, anti-Fas (20 ng/mL)-induced apoptosis increased steadily over time, peaking at 11 hr (43.2%). Interestingly, anti-Fas-induced apoptosis was suppressed upon co-incubation with 7,8-dihydroneopterin and H2O2 by 62% and 68%, respectively. We also compared the sensitivity to drug treatments of apoptosis induced by 7,8-dihydroneopterin, anti-Fas antibodies, and H2O2. 7,8-Dihydroneopterin-mediated, and similarly anti-Fas- and H2O2-mediated, apoptosis was not inhibited by a broad range of pharmacological inhibitors, such as actinomycin D, cycloheximide, cyclosporin A, and various protein kinase inhibitors. On the contrary, inhibitors with antioxidant abilities, such as pyrrolidinedithiocarbamate, significantly blocked 7,8-dihydroneopterin-, H2O2- as well as anti-Fas-mediated apoptosis. These results imply that 7,8-dihydroneopterin-, H2O2-, and anti-Fas-mediated cell death might involve related redox sensitive signal transduction pathways.     

Combined use of exhaled hydrogen peroxide and nitric oxide in monitoring asthma

Oxidative stress contributes to airway inflammation and exhaled hydrogen peroxide (H2O2) and nitric oxide (NO) are elevated in asthmatic patients. We determined the concentrations of expired H2O2 and NO in 116 asthmatic (72 stable steroid-naive, 30 stable steroid-treated, and 14 severe steroid-treated unstable patients) and in 35 healthy subjects, and studied the relation between exhaled H2O2, NO, FEV1, airway responsiveness, and eosinophils in induced sputum. Both exhaled H2O2 and NO levels were elevated in steroid-naive asthmatic patients compared with normal subjects (0.72 +/- 0.06 versus 0.27 +/- 0.04 microM and 29 +/- 1.9 versus 6.5 +/- 0. 32 ppb, respectively; p < 0.001) and were reduced in stable steroid-treated patients (0.43 +/- 0.08 microM, p < 0.05, and 9.9 +/- 0.97 ppb, p < 0.001). In unstable steroid-treated asthmatics, however, H2O2 levels were increased, but exhaled NO levels were low (0.78 +/- 0.16 microM and 6.7 +/- 1.0 ppb, respectively). There was a correlation between expired H2O2, sputum eosinophils and airway hyperresponsiveness (methacholine PC20). Exhaled NO also correlated with sputum eosinophils, but not with airway hyperresponsiveness. Our findings indicate that measurement of expired H2O2 and NO in asthmatic patients provides complementary data for monitoring of disease activity.     

GA strategy for variable selection in QSAR studies: GA-based PLS analysis of calcium channel antagonists

To clarify a possible role of advanced glycation end-products (AGE) on photoaging of human skin, the interaction between AGE and ultraviolet A light (UVA) was examined from both a biological and chemical perspective. Human dermal fibroblasts that were exposed to UVA in the presence of AGE bound with bovine serum albumin (AGE-BSA) exhibited a significant decrease of cell viability as compared to control cells, which were exposed to UVA in the absence of AGE-BSA. Further, UVA-irradiated AGE-BSA reduced nitroblue tetrazolium to its formazan, but the reaction was inhibited by addition of superoxide dismutase in the system. UVA dose-dependent formation of H2O2 in AGE-BSA was also observed. An ESR spin-trapping study revealed the generation of unstable free radicals in AGE-BSA under UVA irradiation. After addition of Fe2+ in the system, an ESR spectrum due to the formation of hydroxyl radicals was observed. On the basis of these results, the authors propose that AGE is an important factor for promoting photoaging in the skin via generation of active oxygen species involving .O2-, H2O2, and .OH.     

Caveolae: from a morphological point of view

The protective effect of quercetin against oxidant-induced cell injury (hypoxanthine/xanthine oxidase system) was studied in the renal tubular epithelial cell line LLC-PK1. Pretreatment with quercetin provided protection from structural and functional cell damage in a concentration-dependent manner (10-100 microM). Comparison with structural variants revealed that the protective property of quercetin depends on the number of hydroxyl substituents in the B-ring, the presence of an extended C-ring chromophore, 3-D-planarity and lipophilicity, indicating that membrane affinity is essential for protection. The hypothesis that quercetin exerts its protective effects via inhibition of lipid peroxidation was further examined. Protection by quercetin was found when lipid peroxidation, assessed by the release of malondialdehyde, was initiated by H2O2 or by the combination of 1-chloro-2,4-dinitrobenzene and aminotriazole. In contrast, the bioflavonoid was not protective when oxidative cell damage was induced by menadione and occurred in the absence of lipid peroxidation. These data suggest that cytoprotective effects of quercetin are related to membrane affinity and may be explained by interruption of membrane lipid peroxidation rather than by intracellular scavenging of oxygen free radicals.     

Prostate cancer in the African American: is this a different disease?

Reactive oxygen species such as superoxides, hydrogen peroxide (H2O2) and hydroxyl radicals have been suggested to be involved in the catalytic action of nitric oxide synthase (NOS) to produce NO from L-arginine. An examination was conducted on the effects of oxygen radical scavengers and oxygen radical-generating systems on the activity of neuronal NOS and guanylate cyclase (GC) in rat brains and NOS from the activated murine macrophage cell line J774. Catalase and superoxide dismutase (SOD) showed no significant effects on NOS or GC activity. Nitroblue tetrazolium (NBT, known as a superoxide radical scavenger) and peroxidase (POD) inhibited NOS, but their inhibitory actions were removed by increasing the concentration of arginine or NADPH respectively, in the reaction mixture. NOS and NO-dependent GC were inactivated by ascorbate/FeSO4 (a metal-catalyzed oxidation system), 2'2'-azobis-amidinopropane (a peroxy radical producer), and xanthine/xanthine oxidase (a superoxide generating system). The effects of oxygen radicals or antioxidants on the two isoforms of NOS were almost similar. However, H2O2 activated GC in a dose-dependent manner from 100 microM to 1 mM without significant effects on NOS. H2O2-induced GC activation was blocked by catalase. These results suggested that oxygen radicals inhibited NOS and GC, but H2O2 could activate GC directly.     

Inhibition of vasoactive amine induced contractions of vascular smooth muscle by hydrogen peroxide in rabbit aorta

OBJECTIVE: The aims were to investigate the effects of H2O2 on arterial contractions induced by vasoactive amine agonists and a high concentration of potassium ions (high K+) in vitro and to explore the possible underlying mechanism(s) involved. METHODS: Isometric tension of rabbit isolated aortic strips was measured and the effects of pretreatment with H2O2 on contractions induced by phenylephrine and high K+ were compared. The effects of H2O2 on precontracted strips were determined in the presence and absence of the aortic endothelium and compared with those of acetylcholine. RESULTS: The tension developed in response to an agonist was expressed as a percentage of the contraction induced by high K+ (64.7 mM) superfusion. Pretreatment with 300 microM H2O2 reduced the mean phenylephrine (0.3 microM) induced contraction from 96.2(SEM 1.4) to 61.8(2.8)%; the effect was stable and reversed by washing out the H2O2. Hydrogen peroxide relaxed phenylphrine precontracted strips with and without endothelium but it showed no relaxant effect when the strips were precontracted by high K+, whereas acetylcholine (1 microM) induced transient relaxation of high K+ precontracted strips by 27.8(2.9)%. The relaxant effect of H2O2 was not affected by pretreatment with indomethacin (a cyclo-oxygenase inhibitor), desferrioxamine (a hydroxyl radical scavenger), or diphenylphenylenediamine (a lipophilic antioxidant). CONCLUSIONS: H2O2 inhibits vasoactive amine induced contractions of the vascular smooth muscle of rabbit aorta in vitro without affecting voltage dependent Ca2+ influx or contractile machinery. The mechanism responsible for its inhibitory effects may be related to impairments of the cellular signalling reactions initiated by the agonists.     

Superoxide: a two-edged sword

Superoxide (O2-) is the compound obtained when oxygen is reduced by one electron. For a molecule with an unpaired electron, O2- is surprisingly inert, its chief reaction being a dismutation in which it reacts with itself to form H2O2 and oxygen. The involvement of O2- in biological systems was first revealed by the discovery in 1969 of superoxide dismutase, an enzyme that catalyzes the dismutation of O2-. Since then it has been found that biological systems produce a bewildering variety of reactive oxidants, all but a few arising ultimately from O2-. These oxidants include O2- itself, H2O2 and alkyl peroxides, hydroxyl radical and other reactive oxidizing radicals, oxidized halogens and halamines, singlet oxygen, and peroxynitrite. These various oxidants are able to damage molecules in their environment, and are therefore very dangerous. They are thought to participate in the pathogenesis of a number of common diseases, including among others malignancy, by their ability to mutate the genome, and atherosclerosis, by their capacity for oxidizing lipoproteins. Their properties are put to good use, however, in host defense, where they serve as microbicidal and parasiticidal agents, and in biological signalling, where their liberation in small quantities results in redox-mediated changes in the functions of enzymes and other proteins.     

The role of oxidative stress in rhinovirus induced elaboration of IL-8 by respiratory epithelial cells

A direct correlation has been reported between the severity of symptoms associated with rhinovirus infection and the concentration of interleukin-8 in nasal secretions. The purpose of these studies was to examine the mechanism of rhinovirus-induced IL-8 elaboration. Rhinovirus infection induced oxidative stress in Beas-2b cells and the concentration of H2O2 in supernatant media from rhinovirus challenged cells was 12.5 +/- 6.1 microM 1 h after challenge compared to 0.7 +/- 0.3 microM in supernatant from control cells. N-acetyl cysteine inhibited RV-induced NF-kappaB activation and IL-8 elaboration. IL-8 concentrations were 36 +/- 2 pg/ml and 10 +/- 1 pg/ml 6 h after virus challenge in untreated and NAC-treated (30 mM NAC) cells, respectively. Despite the effects of NAC on IL-8 elaboration and NF-kappaB activation, RV stimulated increases in supernatant H2O2 were not altered by NAC. These data suggest that RV stimulation of IL-8 in respiratory epithelium is mediated through production of oxidative species and the subsequent activation of NF-kappaB.     

Cytotoxic activity of BCG-activated macrophages against L929 tumor cells is nitric oxide-dependent

The tumoricidal activity of activated macrophages has been attributed largely to the release of tumor necrosis factor (TNF), or to the production of reactive oxygen or nitrogen intermediates. The L929 tumor cell line (a murine fibroblast-like cell) when treated with actinomycin D (ActD) has been used to measure TNF alpha cytotoxicity. In the present study, we determined the cytotoxic activity of BCG-activated peritoneal macrophages against ActD-untreated L929 tumor cells. Furthermore, we measured the production of hydrogen peroxide (H2O2), nitric oxide (NO) and TNF by macrophages cultured in the presence or absence of L929 cells. As expected, BCG-activated macrophages produced significant amounts of H2O2 (16.0 +/- 3.0 microM), TNF (512 U/ml) and NO (71.5 +/- 3.2 microM). TNF (256 U/ml) and NO (78.9 +/- 9.7 microM) production was unchanged in co-cultures of L929 cells with BCG-activated macrophages but H2O2 production was totally inhibited. The cytotoxic activity was dependent on NO release since L-NAME (2.5, 5.0 and 10 mM), which blocks NO synthase, inhibited the killing of L929 cells. Addition of anti-TNF (20 micrograms/ml) antibodies to the cultures did not affect the tumoricidal activity of macrophages. Our results indicate that macrophage-mediated killing of L929 cells is largely dependent on NO production but independent of H2O2 or TNF release.     

Acylated ascorbate stimulates collagen synthesis in cultured human foreskin fibroblasts at lower doses than does ascorbic acid

Acylated derivatives of ascorbic acid were found to be active in a number of biochemical and physiological processes. In the present study we investigated the effects of 6-O-palmitoyl ascorbate on collagen synthesis by cultured foreskin human fibroblasts. Our observations indicate a marked stimulatory effect on collagen synthesis by 6-O-palmitoyl ascorbate in the concentration range of 5-20 microM, while the synthesis stimulated by ascorbic acid was maximal at concentrations of 20-100 microM. Cells treated with 10 microM palmitoyl ascorbate for 36 h exhibited a production of collagen threefold greater than those in the presence of 10 microM ascorbic acid, and it was about the same as in cells treated with 100 microM ascorbic acid. By 48 h differences were not significant. Acylated ascorbate impaired vitality of the treated fibroblasts at concentrations exceeding 20 microM in media supplemented with 0.5% FCS. However, most of the cytotoxic effect was neutralized by FCS at a concentration of 10%. The resistance of acylated ascorbate against oxidative degradation as well as the role of free radicals in the modulation of collagen synthesis by ascorbic acid and by its derivatives is discussed.     

The origin of the hydroxyl radical oxygen in the Fenton reaction

There is an ongoing discussion in the chemical literature regarding the nature of the highly reactive hydroxyl radical formed from the reaction between ferrous iron and hydrogen peroxide (the Fenton reaction). However, the fundamental experiment of directly determining the source of the hydroxyl radicals formed in the reaction has not yet been carried out. In this study, we have used both hydrogen peroxide and water labeled with 17O, together with ESR spin trapping, to detect the hydroxyl radicals formed in the reaction. ESR experiments were run in phosphate buffer with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as a spin trap, and either H2O2 or H2O labeled with 17O. The hydroxyl radical was generated by addition of Fe2+ ion to H2O2, or as a control, by photolysis of H2O2 in the ESR cavity. Observed ESR spectra were the sum of DMPO/.16OH and DMPO/.17OH radical adduct spectra. Within experimental accuracy, the percentage of 17O-labeled hydroxyl radical trapped by the DMPO was the same as in the original hydrogen peroxide, for either method of hydroxyl radical generation, indicating that the trapped hydroxyl radical was derived exclusively from hydrogen peroxide and that there was no exchange of oxygen atoms between H2O2 and solvent water. Likewise, the complementary reaction with ordinary H2O2 and 17O-labeled water also showed that none of the hydroxyl radical was derived from water. Our results do not preclude the ferryl intermediate, [Fe = O]2+ reacting with DMPO to form DMPO/.OH if the ferryl oxygen is derived from H2O2 rather than from a water ligand.     

Biphasic effect of hydrogen peroxide on field potentials in rat hippocampal slices

In the CA1 region of rat hippocampal slices, H2O2 (0.294-2.94 mM) caused initial augmentation, and subsequent long-lasting depression, of population spikes and excitatory postsynaptic potentials. The effect of H2O2 may not be mediated by its degradation product, hydroxyl radicals, because an iron chelator deferoxamine did not block the effect. A catalase inhibitor 3-amino-1,2,4-triazole only modestly attenuated the initial augmentation, suggesting that the effect of H2O2 is not attributable to catalase-dependent O2 generation, either. An N-methyl-D-aspartate receptor antagonist DL-2-amino-5-phosphonovaleric acid had no influence on the effect of H2O2, whereas a gamma-aminobutyric acid type A receptor channel blocker picrotoxin attenuated long-lasting depression, indicating that gamma-aminobutyric acid-mediated inhibition is altered during the depression phase. The initial augmentation but not subsequent depression was attenuated by a phospholipase A2/C inhibitor 4-bromophenacyl bromide, suggesting the involvement of lipid signaling molecule(s) in the enhancement of excitatory synaptic transmission. These results suggest that H2O2 regulates hippocampal synaptic transmission via multiple mechanisms.     

Fluorophotometric quantitation of oxidative stress in the retina in vivo

PURPOSE: To establish a new fluorophotometric method to quantitate oxidative stress in the retina in vivo with a hydrogen peroxide (H2O2)-sensitive fluorescent dye. METHODS: For in vitro fluorophotometric study, nonfluorescent 2',7'-dichlorofluorescein (DCFH) was incubated with H2O2 (10 pM to 100 nM), and the production of fluorescent 2',7'-dichlorofluorescein (DCF) was measured with fluorophotometric analysis. The inhibitory effect of catalase was also examined. For in vivo fluorophotometric study, rabbit eyes received vitrectomy and were perfused with 5 microM 2',7'-dichlorofluorescein diacetate (DCF-DA) or 2',7'-dichlorofluorescein diacetate (DCFH-DA). For oxidative stress, 300 microM H2O2 was infused after perfusion of DCFH-DA. Fluorophotometric measurements of the chorioretinal peak were performed. The eyes were enucleated for fluorescent microscopic examination to determine the localization of DCF fluorescence. RESULTS: H2O2 converted DCFH to DCF in a dose-dependent manner, which was inhibited by catalase dose dependently. In vivo fluorophotometric study showed DCF-DA and DCFH-DA caused production of 2006 +/- 274 picomole/ml (mean +/- SD, n = 5) and 8.35 +/- 1.11 picomole/ml (n = 5), respectively, in the chorioretinal peak. DCFH-DA with stimulation by H2O2 induced 30.7 +/- 13.1 (n = 4) picomole/ml DCF. Fluorescent microscopy showed DCF production in the retina was significant in the eye treated with DCF-DA and minimal in the eye treated with DCFH-DA. Moderate DCF production in the nerve fiber layer was observed in the eye treated with DCFH-DA and H2O2. CONCLUSIONS: This new fluorophotometric method with DCFH-DA may be useful in quantitatively evaluating oxidative stress in the retina in vivo.     

Purification and characterization of ryanotoxin, a peptide with actions similar to those of ryanodine

We purified and characterized ryanotoxin, an approximately 11.4-kDa peptide from the venom of the scorpion Buthotus judiacus that induces changes in ryanodine receptors of rabbit skeletal muscle sarcoplasmic reticulum analogous to those induced by the alkaloid ryanodine. Ryanotoxin stimulated Ca2+ release from sarcoplasmic reticulum vesicles and induced a state of reduce unit conductance with a mean duration longer than that of unmodified ryanodine receptor channels. With Cs+ as the current carrier, the slope conductance of the state induced by 1 microM ryanotoxin was 163 +/- 12 pS, that of the state induced by 1 microM ryanodine was 173 +/- 26 pS, and that of control channels was 2.3-fold larger (396 +/- 25 pS). The distribution of substate events induced by 1 microM RyTx was biexponential and was fitted with time constants approximately 10 times shorter than those fitted to the distribution of substates induced by 1 microM ryanodine. Bath-applied 5 microM ryanotoxin had no effect on the excitability of mouse myotubes in culture. When 5 microM ryanotoxin was dialyzed into the cell through the patch pipette in the whole-cell configuration, there was a voltage-dependent increase in the amplitude of intracellular Ca2+ transients elicited by depolarizing potentials in the range of -30 to +50 mV. Ryanotoxin increased the binding affinity of [3H]ryanodine in a reversible manner with a 50% effective dose (ED50) of 0.16 microM without altering the maximum number (Bmax) of [3H]ryanodine-binding sites. This result suggested that binding sites for ryanotoxin and ryanodine were different. Ryanotoxin should prove useful in identifying domains coupling the ryanodine receptor to the voltage sensor, or domains affecting the gating and conductance of the ryanodine receptor channel.     

Elevated levels of expired breath hydrogen peroxide in bronchiectasis

Airway inflammation is important in the development and progression of many lung diseases, including bronchiectasis. Activation of inflammatory cells such as neutrophils, eosinophils, and macrophages induces a respiratory burst resulting in the production of reactive oxygen species such as hydrogen peroxide (H2O2). We have measured exhaled H2O2 in patients with documented bronchiectasis and investigated whether the concentration of H2O2 is related to the disease severity, as defined by lung function. We also investigated whether the concentrations of expired H2O2 were different in bronchiectatic patients who received inhaled corticosteroids compared with steroid-na?ve patients. In 37 patients with bronchiectasis (mean age, 45 +/- 2.5 yr; FEV1, 59 +/- 3% pred), mean H2O2 concentration in exhaled breath condensate was significantly elevated as compared with the values in 25 age-matched (mean age, 42 +/- 2 yr) normal subjects (0.87 +/- 0.01 versus 0.26 +/- 0.04 microM, p < 0.001). There was a significant negative correlation between H2O2 and FEV1 (r = -0.76, p < 0.0001). Patients treated with inhaled corticosteroids had values of H2O2 similar to those of steroid-na?ve patients (0.8 +/- 0.1 versus 0.9 +/- 0.1, p > 0.05). We conclude that H2O2 is elevated in exhaled air condensate of patients with bronchiectasis and is correlated with disease severity. Measurement of H2O2 may be used as a simple noninvasive method to monitor airway inflammation and oxidative stress.     

Hydrogen atom abstraction of 3,5-disubstituted analogues of paracetamol by horseradish peroxidase and cytochrome P450

1. The formation of free radicals during enzyme catalysed oxidation of eight 3,5-disubstituted analogues of paracetamol (PAR) has been studied. A simple peroxidase system as well as cytochrome P450-containing systems were used. Radicals were detected by electron spin resonance (ESR) on incubation of PAR and 3,5-diCH3-, 3,5-diC2H5-, 3,5-ditC4H9-, 3,5-diOCH3-, 3,5-diSCH3-, 3,5-diF-, 3,5-diCl- and 3,5-diBr-substituted analogues of PAR with horseradish peroxidase in the presence of hydrogen peroxide (H2O2). Initial analysis of the observed ESR spectra revealed all radical species to be phenoxy radicals, based on the absence of dominant nitrogen hyperfine splittings. No radicals were detected in rat liver cytochrome P450-containing microsomal or reconstituted systems. 2. To rationalize the observed ESR spectra, hydrogen atom abstraction of PAR and four of the 3,5-disubstituted analogues (3,5-diCH3-, 3,5-diOCH3-, 3,5-diF- and 3,5-diCl-PAR) was calculated using ab initio calculations, and a singlet oxygen atom was used as the oxidizing species. The calculations indicated that for all compounds studied an initial hydrogen atom abstraction from the phenolic hydroxyl group is favoured by approximately 125 kJ/mol over an initial hydrogen atom abstraction from the acetylamino nitrogen atom, and that after hydrogen abstraction from the phenolic hydroxyl group, the unpaired electron remains predominantly localised at the phenoxy oxygen atom (+/-85%). 3. The experimental finding of phenoxy radicals in horseradish peroxidase/H2O2 incubations paralleled these theoretical findings. The failure to detect experimentally phenoxy radicals in cytochrome P450-catalysed oxidation of any of the eight 3,5-disubstituted PAR analogues is more likely due to the reducing effects that agents like NADPH and protein thiol groups have on phenoxy radicals rather than on the physical instability of the respective substrate radicals.