Patterns of destruction of mouse neuroblastoma cells by extracellular hydrogen peroxide formed by 6-hydroxydopamine and ascorbate

Summary The patterns of the cytolytic effects of 6-hydroxydopamine (6-OHDA), with/without ascorbate, on C-1300 and three other cloned mouse neuroblastoma cell lines (N1E-115, NS-20, N-18) were studied in vitro. The sensitivity to 6-OHDA differed and the three cloned cell lines were more sensitive than the wild type C-1300 cell line. Ascorbate synergistically potentiated the cytolytic effect of 6-OHDA to all four cell lines. The 6-OHDA cytotoxicity was eliminated by the addition of exogenous catalase but not by addition of other oxygen free radical scavengers, thereby suggesting that the hydrogen peroxide formed might influence the cells, extracellularly. In addition, the critical time for tumor cell lysis was the first 60 min of the reaction. The cytotoxicity induced by the unmasked cyclophosphamide, 4-hydroperoxycyclophosphamide, was synergistically enhanced in the presence of a nontoxic concentration of 6-OHDA and ascorbate. These data suggest that reactive oxygen intermediates may prove to be a good tool for destroying neuroblastoma cells.     

Exhaled nitric oxide and hydrogen peroxide concentrations in asthmatic smokers

BACKGROUND: Cigarette smoking is associated with decreased nitric oxide (NO) production and increased oxidative stress in the airways. Exhaled NO levels are not higher in asthmatic smokers than in healthy non-smokers, and the value of exhaled NO for diagnosing asthma in smokers has been questioned. OBJECTIVES: To compare exhaled NO concentrations between healthy and steroid-naive and steroid-treated asthmatic smokers and non-smokers. To also assess the acute effect of cigarette smoking on exhaled NO and hydrogen peroxide (H(2)O(2)) levels in asthmatic smokers. METHODS: Exhaled NO was measured by chemiluminescence and exhaled H(2)O(2) spectrophotometrically. In 7 steroid-naive asthmatic smokers exhaled NO and H(2)O(2) was measured both before and 15 min after smoking one cigarette. Data are given as median (range). RESULTS: Exhaled NO level was significantly higher in steroid-naive asthmatic smokers than in healthy smokers [7.7 (3.4-32.5) ppb vs. 3.2 (2.0-7.2) ppb, p < 0.001]. Exhaled NO values were lower in smokers than in non-smokers both in healthy subjects and in steroid-naive asthmatic patients. Steroid-treated asthmatic smokers had a tendency for lower exhaled NO values [5.4 (1.7-12.0) ppb] compared to steroid-naive asthmatic smokers. Cigarette smoking caused an acute increase in exhaled H(2)O(2) concentrations together with a decrease in exhaled NO concentration. CONCLUSIONS: Our data suggest that an elevation in exhaled NO concentration is associated with asthma in smokers. This difference may be useful for diagnosing the disease in smokers, but its clinical value needs further evaluation. Acute increase in exhaled H(2)O(2) concentrations suggests that smoking increases the oxidative stress in the asthmatic airways.     

Elevated concentrations of exhaled hydrogen peroxide in asthmatic patients

BACKGROUND: Airway inflammation is important in the development and progression of asthma. Activation of inflammatory cells induces a respiratory burst resulting in the production of reactive oxygen species, such as H(2)O(2). The aim of this study was to measure the concentration of H(2)O(2) in exhaled breath condensate and its correlation with airway obstruction, airway hyperresponsiveness, and concentration of eosinophil cationic protein (ECP) in serum in 70 steroid-naive, atopic patients with unstable asthma (20 men; age range, 18 to 62 years) and 17 normal subjects (7 men; age range, 19 to 34 years). METHODS: Exhaled H(2)O(2) was measured using a colorimetric assay, and the concentration of ECP in serum was measured using radioimmunoassay. Airway hyperresponsiveness was expressed as the provocative concentration of inhaled histamine causing a 20% fall in FEV(1) (PC(20)). RESULTS: In patients with asthma, the mean H(2)O(2) concentration was significantly elevated compared to values in normal subjects: 0.127 +/- 0.083 mol/L vs 0.024 +/- 0.016 mol/L (p < 0.001). There was a significant correlation among H(2)O(2) concentration, FEV(1), PC(20), and ECP in serum. CONCLUSION: We conclude that exhaled H(2)O(2) is significantly elevated in asthmatic patients. This is correlated with disease severity and indirect markers of airway inflammation. Measurement of exhaled H(2)O(2) may be useful to assess airway inflammation and oxidative stress in asthmatic patients.     

Pharmacologic ascorbic acid concentrations selectively kill cancer cells: action as a pro-drug to deliver hydrogen peroxide to tissues

Human pharmacokinetics data indicate that i.v. ascorbic acid (ascorbate) in pharmacologic concentrations could have an unanticipated role in cancer treatment. Our goals here were to test whether ascorbate killed cancer cells selectively, and if so, to determine mechanisms, using clinically relevant conditions. Cell death in 10 cancer and 4 normal cell types was measured by using 1-h exposures. Normal cells were unaffected by 20 mM ascorbate, whereas 5 cancer lines had EC(50) values of <4 mM, a concentration easily achievable i.v. Human lymphoma cells were studied in detail because of their sensitivity to ascorbate (EC(50) of 0.5 mM) and suitability for addressing mechanisms. Extracellular but not intracellular ascorbate mediated cell death, which occurred by apoptosis and pyknosis/necrosis. Cell death was independent of metal chelators and absolutely dependent on H(2)O(2) formation. Cell death from H(2)O(2) added to cells was identical to that found when H(2)O(2) was generated by ascorbate treatment. H(2)O(2) generation was dependent on ascorbate concentration, incubation time, and the presence of 0.5-10% serum, and displayed a linear relationship with ascorbate radical formation. Although ascorbate addition to medium generated H(2)O(2), ascorbate addition to blood generated no detectable H(2)O(2) and only trace detectable ascorbate radical. Taken together, these data indicate that ascorbate at concentrations achieved only by i.v. administration may be a pro-drug for formation of H(2)O(2), and that blood can be a delivery system of the pro-drug to tissues. These findings give plausibility to i.v. ascorbic acid in cancer treatment, and have unexpected implications for treatment of infections where H(2)O(2) may be beneficial.     

Activation of cyclic electron flow by hydrogen peroxide in vivo

Cyclic electron flow (CEF) around photosystem I is thought to balance the ATP/NADPH energy budget of photosynthesis, requiring that its rate be finely regulated. The mechanisms of this regulation are not well understood. We observed that mutants that exhibited constitutively high rates of CEF also showed elevated production of H₂O₂. We thus tested the hypothesis that CEF can be activated by H₂O₂ in vivo. CEF was strongly increased by H₂O₂ both by infiltration or in situ production by chloroplast-localized glycolate oxidase, implying that H₂O₂ can activate CEF either directly by redox modulation of key enzymes, or indirectly by affecting other photosynthetic processes. CEF appeared with a half time of about 20 min after exposure to H₂O₂, suggesting activation of previously expressed CEF-related machinery. H₂O₂-dependent CEF was not sensitive to antimycin A or loss of PGR5, indicating that increased CEF probably does not involve the PGR5-PGRL1 associated pathway. In contrast, the rise in CEF was not observed in a mutant deficient in the chloroplast NADPH:PQ reductase (NDH), supporting the involvement of this complex in CEF activated by H₂O₂. We propose that H₂O₂ is a missing link between environmental stress, metabolism, and redox regulation of CEF in higher plants.In oxygenic photosynthesis, linear electron flow (LEF) is the process by which light energy is captured to drive the extraction of electrons and protons from water and transfer them through a system of electron carriers to reduce NADPH. LEF is coupled to proton translocation into the thylakoid lumen, generating an electrochemical gradient of protons (Δμ~H+) or proton motive force (pmf). The pmf drives the synthesis of ATP to power the reactions of the Calvin–Benson–Bassham (CBB) cycle and other essential metabolic processes in the chloroplast. The pmf is also a key regulator of photosynthesis in that it activates the photoprotective q_E response to dissipate excess light energy and down-regulates electron transfer by controlling the rate of oxidation of plastoquinol at the cytochrome b₆f complex (b₆f), thus preventing the buildup of reduced intermediates (1, 2).LEF results in the transfer or deposition into the lumen of three protons for each electron transferred through PSII, plastoquinone (PQ), b₆f, plastocyanin, and photosystem I (PSI) to ferredoxin (Fd). The synthesis of one ATP is thought to require the passage of 4.67 protons through the ATP synthase, so that LEF should produce a ratio of ATP/NADPH of about 1.33; this ratio is too low to sustain the CBB cycle or supply ATP required for translation, protein transport, or other ATP-dependent processes (3). In addition, the relative demands for ATP and NADPH can change dramatically depending on environmental, developmental, and other factors, leading to rapid energy imbalances that require dynamical regulation of ATP/NADPH balance.Several alternative electron flow pathways in the chloroplast have been proposed to augment ATP production, thus balancing the ATP/NADPH budget of the chloroplast (2, 3). Perhaps the most important and complicated of these pathways is cyclic electron flow around photosystem I (CEF), in which electron flow from the acceptor side of PSI is shunted back into the PQ pool, generating additional pmf that can power ATP production without net NADPH production. There are several proposed CEF pathways that may operate under different conditions or in different species (reviewed in refs. 2 and 3). In higher-plant chloroplasts, the most studied routes of CEF are the antimycin A-sensitive pathway, which involves a complex of two CEF-related proteins, PGR5 (Proton Gradient Regulation 5) and PGRL1 (PGR5-like 1), directly reducing the quinone pool (4–7), the respiratory complex I analog, the NADPH dehydrogenase (NDH) complex (8–10), which oxidizes Fd or NAD(P)H to reduce plastoquinone (8, 11), and through the Q_i site of b₆f (12, 13). Different CEF mechanisms seem to operate in other species. In Chlamydomonas, for example, CEF seems to be conducted by a supercomplex of PSI, b₆f, and the PGRL1 protein (14, 15), and the involvement of PGR5 has recently been described as important for CEF under hypoxia (16, 17).Regardless of the mechanism of CEF, the overall process must be well regulated to properly balance the production of ATP to match the demands of metabolism. The mechanism of this regulation is not known, but many general models have been proposed. Perhaps the most widely cited regulatory model is the antenna state transition, which was previously shown to be correlated with activation of CEF in Chlamydomonas reinhardtii (14, 18) and favor the formation of the PSI–b₆f supercomplex (14). However, it was recently shown that state transitions are not required for CEF activation, supporting models that include redox control (15–17, 19–22). Other possible regulatory mechanisms include sensing of ATP/ADP ratios (23, 24), the redox status of NAD(P)H or Fd (25), various CBB metabolic intermediates (reviewed in ref. 26), calcium signaling (15, 27), phosphorylation of CEF-related proteins (27), and the reactive oxygen species H₂O₂ (26–29).One possibility is that CEF may be at least partly regulated by H₂O₂ (26), which is produced by the light reactions of photosynthesis and already known to regulate other cellular processes such as plant growth, development, and defense (30–32). Based on in vitro studies, it was previously proposed that H₂O₂ could activate CEF or chlororespiration by modifying the NDH complex (27). It has also been shown that H₂O₂ can increase the expression of the NDH complex (29) and may further affect the accumulation of photosynthetic metabolites, indirectly activating CEF (26). Consistent with this possibility, H₂O₂ is a well-documented signaling molecule (33), possibly through its ability to oxidize thiols (34, 35). Furthermore, H₂O₂ is expected to be produced under many conditions that initiate CEF [e.g., under a deficit of ATP, when electrons should accumulate in the PSI acceptor pools, leading to superoxide production that can be converted to H₂O₂ by superoxide dismutase (36)].This study aims to test the hypothesis that CEF can be initiated in vivo by H₂O₂ using a combination of in vivo spectroscopy and genetic modifications to selectively and rapidly initiate H₂O₂ production in the chloroplast.     

Apocynin decreases hydrogen peroxide and nirtate concentrations in exhaled breath in healthy subjects

The imbalance between reactive oxygen species (ROS) synthesis and antioxidants might be involved in the pathogenesis of many inflammatory diseases. NADPH oxidase, an enzyme responsible for ROS production, may represent an attractive therapeutic target to inhibit, for the treatment of these diseases.Apocynin is an inhibitor of activation of NADPH oxidase complex present in the inflammatory cells.In double blind, placebo-controlled, cross-over study, we investigated the effect of nebulized apocynin on ROS synthesis in 10 nonsmoking healthy volunteers. Apocynin (6 ml of 0.5 mg/ml) was administered by nebulization and its effects on H₂O₂, NO₂^? and NO₃^? generation were assessed after 30, 60 and 120 min by collecting exhaled breath condensate (EBC) samples using an EcoScreen analyzer. Additionally, respiratory parameters have been evaluated, utilizing spirometry and DLCO. We also analyzed peripheral blood differential counts and NO₂^? serum level, cough scale control and blood pressure as safety parameters.Apocynin caused reduction of H₂O₂ concentration in EBC as compared to placebo, after 60 min. of inhalation (0.18 μM vs. 0.31 μM, p < 0.05) as well as after 120 min. (0.2 μM vs. 0.31 μM, p < 0.05). Similarly, apocynin significantly decreased concentration of NO₃^? as compared to placebo, after 60 and 120 min. (6.8 μM vs. 14.4 μM and 6.5 μM vs. 14.9 μM respectively, p < 0.05). Apocynin was well tolerated and no adverse events have been observed throughout the study.Thus, as apocynin significantly influence ROS concentration, it might have also antiinflammatory properties. As it is safe, it may have a potential to become a drug in airway inflammatory diseases treatment.     

Protective factors against oxygen free radicals and hydrogen peroxide in rheumatoid arthritis synovial fluid

Oxygen free radicals are probably involved in the pathogenesis of rheumatoid arthritis (RA). The enzymes involved in protection against oxygen free radicals and H2O2 (superoxide dismutase, catalase, and glutathione peroxidase) were measured. Superoxide dismutase was not increased, glutathione peroxidase was slightly and catalase was strongly elevated in RA synovial fluid (SF) compared with control SF. Although these enzymes are present in SF, the activities are insufficient to protect against oxygen free radicals and H2O2. In contrast to transferrin, ferritin was increased in RA synovial fluid. Ceruloplasmin was also elevated. When rat liver microsomes were used as a target for oxygen free radicals, serum and SF were both protective. Gel filtration experiments showed that the fraction pattern in which there was maximal protective potential against lipid peroxidation corresponded closely to the level of ceruloplasmin. After removal of ceruloplasmin from serum or SF, about 70% of the protective capacity disappeared. It is concluded that ceruloplasmin is an important protector against oxygen free radicals.     

Ascorbate radical and ascorbic acid level in human serum and age

Free radical in human serum was observed by an electron spin resonance (ESR) technique and was assigned to ascorbate radical. Quantitative estimation revealed that the ESR intensity could be used as an expedient mean. The ESR intensity of ascorbate radical, the concentrations of total ascorbic acid and oxidized form of ascorbic acid were determined on sera of 200 healthy individuals whose ages ranged from 12 to 96 years. The ESR intensity as well as the concentrations of total and oxidized form of ascorbic acid declined with age. There were significant correlations between the ESR intensity and the concentrations of total and oxidized form of ascorbic acid. From these results, the clinical significance of the concentrations of total ascorbic acid, ascorbate radical and the oxidized form was discussed in relation to age.     

Quantitative measurement of nitric oxide and hydrogen peroxide in Helicobacter pylori-infected Mongolian gerbils in vivo

OBJECTIVE: Peroxynitrite formation, as reflected by nitrotyrosine expression, is low in Helicobacter pylori-infected Mongolian gerbils despite pronounced expression of radical-forming enzymes. The aim of the present study was to investigate in vivo whether H. pylori inhibits either one or both of the nitro- and oxyradical formation pathways. MATERIAL AND METHODS: Male Mongolian gerbils were infected with two different H. pylori strains, TN2GF4 and SS1. Six months after inoculation, direct measurement of NO and H2O2 was performed in vivo using electrochemical microsensors positioned in close proximity to the gastric mucosa. RESULTS: In the TN2GF4-infected animals the level of NO was significantly lower than that in controls. No significant difference in NO levels was detected between the SS1-infected group and the controls. H2O2 was significantly increased in the SS1 animals compared with that in controls after 6 months. The H2O2 level in the TN2GF4 group did not differ from that in controls. CONCLUSIONS: The results indicate that H. pylori infection is associated with strain-dependent functional inhibition of both the NO and oxyradical formation pathways in the gastric mucosa.     

Review of aerosolized hydrogen peroxide,vaporized hydrogen peroxide,and hydrogen peroxide gas plasma in the decontamination of filtering facepiece respirators

BackgroundCoronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to over 170?million cases worldwide with over 33.2?million cases and 594,000 deaths in the US alone as of May 31st, 2021. The pandemic has also created severe shortages of personal protective equipment, particularly of filtering facepiece respirators (FFRs). The Centers for Disease Control and Prevention (CDC) has issued recommendations to help conserve FFRs, as well as crisis standards, including four criteria required for decontamination of the traditionally single use respirators. This review is designed to provide an overview of the current literature on vaporized hydrogen peroxide (vHP), hydrogen peroxide gas plasma (HPGP), and aerosolized hydrogen peroxide (aHP) with respect to each of the four CDC decontamination criteria.MethodsPubMed and Medrxiv were queried for relevant articles. All articles underwent a title and abstract screen as well as subsequent full text screen by two blinded reviewers if indicated.ResultsSearches yielded 195 papers, of which, 79 were found to be relevant. Of those, 23 papers presented unique findings and 8 additional articles and technical papers were added to provide a comprehensive review. Overall, while there are potential concerns for all 3 decontamination methods, we found that vHP has the most evidence supporting its use in FFR decontamination consistent with CDC recommendation.ConclusionsFuture research is recommended to evaluate biological inactivation and real world fit failures after FFR reuse.     

In vivo generation of hydrogen peroxide in the rat corpus luteum during luteolysis

The hypothesis that hydrogen peroxide generation occurs in the corpora lutea of superovulated rats during luteolysis was tested using a peroxide-dependent inhibitor of catalase, 3-amino-1,2,4-triazole (AT). Luteal regression was induced during midpseudopregnancy by injection of 500 micrograms prostaglandin F2 alpha (PGF2 alpha) 1 h before administration of AT (0.1 g/kg, ip) and was confirmed by progesterone analysis of peripheral blood serum. Within groups of both PGF2 alpha-treated and untreated control rats, other rats also received ethanol (0.2 g/kg, ip), which prevents hydrogen peroxide-mediated inhibition of catalase by AT. Diluted homogenates of ovaries removed 1 h after AT administration were assayed for catalase activity by measuring the decrease in absorbance at 240 nm for 30 sec after the addition of hydrogen peroxide (10 mM). Ethanol-sensitive catalase inhibition by AT was significantly higher (47.9 +/- 3.38%) in samples from PGF2 alpha-treated groups than in controls (23.1 +/- 4.82%; P less than 0.01; n = 9). Similar increases in catalase inhibition by AT were found in luteal tissue of rats treated with PGF2 alpha 24 h earlier and in rats in which luteolysis was allowed to occur spontaneously in late pseudopregnancy. Hemoglobin an AT assays revealed that the changes in catalase activity were not the result of altered blood contamination or AT concentration in the luteal homogenates. Since catalase inhibition by AT is only seen in the presence of hydrogen peroxide, these results support the conclusion that an early and sustained component of corpus luteum regression is the generation of hydrogen peroxide in luteal tissue.     

Quantitative measurement of nitric oxide and hydrogen peroxide in Helicobacter pylori-infected Mongolian gerbils in vivo

Objective. Peroxynitrite formation, as reflected by nitrotyrosine expression, is low in Helicobacter pylori-infected Mongolian gerbils despite pronounced expression of radical-forming enzymes. The aim of the present study was to investigate in vivo whether H. pylori inhibits either one or both of the nitro- and oxyradical formation pathways. Material and methods. Male Mongolian gerbils were infected with two different H. pylori strains, TN2GF4 and SS1. Six months after inoculation, direct measurement of NO and H₂O₂ was performed in vivo using electrochemical microsensors positioned in close proximity to the gastric mucosa. Results. In the TN2GF4-infected animals the level of NO was significantly lower than that in controls. No significant difference in NO levels was detected between the SS1-infected group and the controls. H₂O₂ was significantly increased in the SS1 animals compared with that in controls after 6 months. The H₂O₂ level in the TN2GF4 group did not differ from that in controls. Conclusions. The results indicate that H. pylori infection is associated with strain-dependent functional inhibition of both the NO and oxyradical formation pathways in the gastric mucosa.     

Cervicovaginal fluid and semen block the microbicidal activity of hydrogen peroxide produced by vaginal lactobacilli

Background  

H₂O₂ produced by vaginal lactobacilli is believed to protect against infection, and H₂O₂-producing lactobacilli inactivate pathogens in vitro in protein-free salt solution. However, cervicovaginal fluid (CVF) and semen have significant H₂O₂-blocking activity.     

Important role of endogenous hydrogen peroxide in pacing-induced metabolic coronary vasodilation in dogs in vivo.

OBJECTIVES: We examined whether endogenous hydrogen peroxide (H2O2) is involved in pacing-induced metabolic vasodilation in vivo. BACKGROUND: We have previously demonstrated that endothelium-derived H2O2 is an endothelium-derived hyperpolarizing factor in canine coronary microcirculation in vivo. However, the role of endogenous H2O2 in metabolic coronary vasodilation in vivo remains to be examined. METHODS: Canine subepicardial small coronary arteries (> or =100 microm) and arterioles (<100 microm) were continuously observed by a microscope under cyclooxygenase blockade (ibuprofen, 12.5 mg/kg intravenous [IV]) (n = 60). Experiments were performed during paired right ventricular pacing under the following 7 conditions: control, nitric oxide (NO) synthase inhibitor (N(G)-monomethyl-L-arginine [L-NMMA], 2 micromol/min for 20 min intracoronary [IC]), catalase (a decomposer of H2O2, 40,000 U/kg IV and 240,000 U/kg/min for 10 min IC), 8-sulfophenyltheophylline (SPT) (an adenosine receptor blocker, 25 mug/kg/min for 5 min IC), L-NMMA+catalase, L-NMMA+tetraethylammonium (TEA) (K(Ca)-channel blocker, 10 microg/kg/min for 10 min IC), and L-NMMA+catalase+8-SPT. RESULTS: Cardiac tachypacing (60 to 120 beats/min) caused coronary vasodilation in both-sized arteries under control conditions in response to the increase in myocardial oxygen consumption. The metabolic coronary vasodilation was decreased after L-NMMA in subepicardial small arteries with an increased fluorescent H2O2 production compared with catalase group, whereas catalase decreased the vasodilation of arterioles with an increased fluorescent NO production compared with the L-NMMA group, and 8-SPT also decreased the vasodilation of arterioles. Furthermore, the metabolic coronary vasodilation was markedly attenuated after L-NMMA+catalase, L-NMMA+TEA, and L-NMMA+catalase+8-SPT in both-sized arteries. CONCLUSIONS: These results indicate that endogenous H2O2 plays an important role in pacing-induced metabolic coronary vasodilation in vivo.     

Absence of a role for superoxide anion, hydrogen peroxide and hydroxyl radical in endothelium-mediated relaxation of rabbit aorta

We tested the hypothesis that the endothelium-dependent relaxation of rabbit thoracic aorta in vitro is mediated by reduced metabolites of oxygen. Helical vascular strips were contracted with either norepinephrine or phenylephrine. Oxygen metabolites, generated by the xanthine oxidase reaction, completely relaxed norepinephrine-induced contractile tone but not tone induced by phenylephrine. A mixture of oxygen metabolite scavengers (superoxide dismutase, catalase and mannitol) eliminated the relaxation induced by the xanthine oxidase products. Acetylcholine caused a dose-dependent and endothelium-dependent relaxation of the strips; this was not inhibited by the presence of the scavengers. We conclude that reduced oxygen metabolites have little direct effect on rabbit aortic smooth muscle in vitro, although they indirectly but specifically relax norepinephrine-induced tone, presumably by oxidation of norepinephrine. Oxygen metabolites do not appear to mediate the endothelium-dependent relaxation response of this tissue to acetylcholine.