The metal-mediated formation of hydroxyl radical by aqueous extracts of cigarette tar

Aqueous extracts of cigarette tar produce hydroxyl radicals that are spin trapped by 5,5-dimethyl-1-pyrroline-N-oxide. The addition of catalase almost completely inhibits and superoxide dismutase partially inhibits spin adduct formation. The addition of ethylenediamine tetraacetic acid greatly increases the amount of hydroxyl radical adduct observed; in contrast, diethylenetriamine pentaacetic acid causes complete inhibition of spin adduct formation. We suggest that the hydroxyl radical arises from the metal-mediated decomposition of hydrogen peroxide, and that hydrogen peroxide is formed from the reduction of dioxygen by the semiquinones present in the cigarette tar.     

Activation and inactivation of cyclo-oxygenase in rat alveolar macrophages by aqueous cigarette tar extracts.

Cyclo-oxygenase (COX) activity and its level of expression, the release of arachidonic acid (AA), and the accumulation of prostaglandins (PGs) were determined in isolated rat pulmonary alveolar macrophages (PAM) exposed to aqueous cigarette tar (ACT) extracts. COX activity increased 3-fold above the initial activity within 2 h of incubation with ACT extracts and gradually decreased below the initial activity after 8 h of incubation. The increased COX activity after 2 h of incubation did not lead to increased accumulation of PGE2. Accumulated levels of PGE2 increased dramatically after 12 h of incubation despite decreased COX activity in cells incubated with ACT extracts. This increased accumulation of PGE2 was greater in cells derived from vitamin E deficient rats compared with control rats. Release of AA from cells was dramatically increased in cells incubated with ACT extracts in parallel to PG accumulation. Thus increased accumulation of PGE2 despite decreased COX activity after 12 h of incubation is likely the result of increased substrate availability. These results suggest that, contrary to earlier reports, cigarette smoke stimulates the formation of PGs in alveolar macrophages. Increased PG production may lead to suppressed immune response and enhanced risk of tumorigenesis in smokers' lungs.     

Fluorescence detection of enzymatically formed hydrogen peroxide in aqueous solution and in reversed micelles

A sensitive enzymatic assay for oxidase reactions both in aqueous solution and in hexadecyltrimethylammoniumbromide (CTAB) reversed micelles has been developed. The assay is based on the fluorescence detection of dichlorofluorescein, which is formed by hydrogen peroxide oxidation of the nonfluorescent precursor dichlorofluorescin. Hydrogen peroxide as product of the reaction catalyzed by glucose oxidase served to select the reaction conditions. The reaction rate is distinctly enhanced in CTAB reversed micelles as compared to the rate in aqueous solution. This effect, combined with the high sensitivity owing to the strong fluorescence of dichlorofluorescein, makes the assay attractive for the detection of low enzyme, substrate, or peroxide concentrations.     

Aqueous cigarette tar extracts damage human alpha-1-proteinase inhibitor

The elastase inhibitory capacity (EIC) of human alpha-1-proteinase inhibitor (alpha 1PI) is severely compromised by aqueous cigarette tar extract (ACTE). An aqueous extract of the tar from two cigarettes causes a loss of EIC of at least 60% in 24 h at 37 degrees C (pH 7.4) and the damaging capability of the ACTE is retained for many hours. Hydrogen peroxide appears to be an essential component of the mechanism by which ACTE damages alpha 1 PI, since catalase substantially protects alpha 1PI from ACTE-mediated damage. Only mild protection is offered by 10 mM diethylenetriamine pentaacetic acid, indicating only a minor role for transition metal ions in the alpha 1PI-damaging process. Hydroxyl radicals are unlikely agents of alpha 1PI damage in the ACTE system, as judged from hydroxyl radical scavenger studies. Ascorbate and various thiols offer protection to different degrees, dependent on the incubation conditions. Of several amino acids tested, cysteine and methionine (but not methionine sulfoxide) are the only two that protect alpha 1PI. We suggest that components of cigarette smoke particulate matter extracted into the aqueous lung fluid environment may cause local deficiencies in alpha 1PI in smokers' lungs.     

Quantitative analysis of hydrogen peroxide with special emphasis on biosensors

Bioprocess and Biosystems Engineering - Determination of hydrogen peroxide (H2O2) has become essential in pharmaceutical, biological, clinical and environmental studies. The conventional detection...     

Immunological identification of the heart myoglobin radical formed by hydrogen peroxide

This study reports the detection of protein free radicals using the specific free radical reactivity of nitrone spin traps in conjunction with nitrone-antibody specificity. Polyclonal antibodies were developed that bind to protein adducts of the nitrone spin-trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO). The antibodies were used to detect DMPO protein adducts produced on horse myoglobin resulting from self-peroxidation. Western blot analysis demonstrates that myoglobin forms the predominant radical-derived nitrone adduct in rat heart supernatant.     

Spectrofluorometric analysis of hydrogen peroxide

The pH of maximum fluorescence (above pH 7) and the optimal excitation and emission wavelengths (468 nm and 519 nm, respectively) were determined for 2′,7′-dichlorofluorescein (DCF). The stoichiometry after hydrolysis of the oxidation of the stable nonfluorescent compound 2′,7′-dichlorofluorescin diacetate (LDADCF) was determined and found to be 2 moles of DCF produced per mole of hydrogen peroxide used.     

Quantification of hydrogen peroxide in plant extracts by the chemiluminescence reaction with luminol

The chemiluminescence of luminol (3-aminophthalhydrazide) with H₂O₂ has been used to quantify endogenous amounts of H₂O₂ in plant tissues. The reaction is linear over at least three orders of magnitude between 10^?5 and 10^?2M H₂O₂. Interference by coloured compounds in the crude extract is calibrated by a purification step with Dowex AG 1-X8. The extract is calibrated with an internal H₂O₂ standard, and the specificity verified by H₂O₂ purging with catalase. The minimum delectability for H₂O₂ of this assay is at least 1 ng, corresponding to 0.1–1 g fresh material. Data are presented for the levels of H₂O₂ in potatoes after treatment with oxygen and ethylene, in tomatoes before and after ripening and in untreated germinating castor beans as well as in beans treated with aminotriazol to inhibit catalase activity. Though data using the titanium test are generally confirmed, the method presented here has the advantage of higher sensitivity and specificity.     

Scavenging of hydrogen peroxide and inhibition of ultraviolet light-induced oxidative DNA damage by aqueous extracts from green and black teas

Aqueous extracts of green and black teas have been shown to inhibit a variety of experimentally induced animal tumors, particularly ultraviolet (UV) B light-induced skin carcinogenesis. In the present study, we compared the effects of different extractable fractions of green and black teas on scavenging hydrogen peroxide (H2O2), and UV irradiation-induced formation of 8-hydroxy 2'-deoxyguanosine (8-OHdG) in vitro. Green and black teas have been extracted by serial chloroform, ethyl acetate and n-butanol, and divided into four subfractions designated as GT1-4 for green tea and BT1-4 for black tea, respectively. The total extracts from green and black teas exhibited a potent scavenging capacity of exogenous H2O2 in a dose-dependent manner. It appeared that the total extracts from black tea scavenged H2O2 more potently than those from green tea. When tested individually, the potency of scavenging H2O2 by green tea subfractions was: GT2 > GT3 > GT1 > GT4, whereas the order of efficacy for black tea was: BT2 > BT3 > BT4 > BT1. In addition, we demonstrated that total fractions of green and black teas substantially inhibited the induction of 8-OHdG in calf thymus by all three portions of UV spectrum (UVA, B and C). Consistent with the capacity of scavenging H2O2, the subfractions from black tea showed a greater inhibition of UV-induced 8-OHdG than those from green tea. At low concentrations, the order of potency of quenching of 8-OHdG by green tea subfractions was: GT2 > GT3 > GT4 > GT1 and the efficacy of all subfractions became similar at high concentrations. All subfractions of the black tea except BT1 strongly inhibited UV-induced 8-OHdG and the order of potency was: BT2 > BT3 > BT4 > BT1. Addition of (-)-epigallocatechin gallate (EGCG), an ingredient of green tea extract, to low concentration of green and black tea extracts substantially enhanced the scavenging of H2O2 and quenching of 8-OHdG, suggesting the important role of EGCG in the antioxidant activities of tea extracts. The potent scavenging of oxygen species and blocking of UV-induced oxidative DNA damage may, at least in part, explain the mechanism(s) by which green/black teas inhibit photocarcinogenesis.     

Participation of peroxynitrite in oxidative modification of LDL by aqueous extracts of cigarette smoke

Aqueous extracts of cigarette smoke (CSE) can oxidatively modify plasma low-density lipoprotein (LDL). The aim of the present study was to elucidate the participation of peroxynitrite in LDL oxidation. When LDL was incubated with CSE, its oxidative modification was dependent on time and concentration. It could be effectively prevented by vitamin E, partially by superoxide dismutase, but hardly by catalase, mannitol and metal chelators. CSE also increased the 3-nitrotyrosine content in LDL. A similar increase of 3-nitrotyrosine occurred after incubation of LDL with a peroxynitrite generating agent, 3-morpholinosydnonimine, thus suggesting that prominent pro-oxidants in CSE are peroxynitrite-generating species.     

Glutathione peroxidase in lens and a source of hydrogen peroxide in aqueous humour

1. Glutathione peroxidase has been demonstrated in cattle, rabbit and guineapig lenses. 2. The enzyme will oxidize GSH either with hydrogen peroxide added at the start of the reaction or with hydrogen peroxide generated enzymically with glucose oxidase. 3. No product other than GSSG was detected. 4. Oxidation of GSH can be coupled with oxidation of malate through the intermediate reaction of glutathione reductase and NADPH₂. 5. Traces of hydrogen peroxide are present in aqueous humour: it is formed when the ascorbic acid of aqueous humour is oxidized. 6. Hydrogen peroxide will diffuse into the explanted intact lens and oxidize the contained GSH. The addition of glucose to the medium together with hydrogen peroxide maintains the concentration of lens GSH. 7. Glutathione peroxidase in lens extracts will couple with the oxidation of ascorbic acid. 8. It is suggested that, as there is only weak catalase activity in lens, glutathione peroxidase may act as one link between the oxygen of the aqueous humour and NADPH₂.     

The inhibitory effect of extracts of cigarette tar on electron transport of mitochondria and submitochondrial particles.

Acetonitrile extracts of cigarette tar inhibit state 3 and state 4 respiration of intact mitochondria. Exposure of respiring submitochondrial particles to acetonitrile extracts of cigarette tar results in a dose-dependent inhibition of oxygen consumption and reduced nicotinamide adenine dinucleotide (NADH) oxidation. This inhibition was not due to a solvent effect since acetonitrile alone did not alter oxygen consumption or NADH oxidation. Intact mitochondria are less sensitive to extracts of tar than submitochondrial particles. The NADH-ubiquinone (Q) reductase complex is more sensitive to inhibition by tar extract than the succinate-Q reductase and cytochrome complexes. Nicotine or catechol did not inhibit respiration of intact mitochondria. Treatment of submitochondrial particles with cigarette tar results in the formation of hydroxyl radicals, detected by electron spin resonance (ESR) spin trapping. The ESR signal attributable to the hydroxyl radical spin adduct requires the presence of NADH and is completely abolished by catalase and to a lesser extent superoxide dismutase (SOD). Catalase and SOD did not protect the mitochondrial respiratory chain from inhibition by tar extract, indicating that the radicals detected by ESR spin trapping are not responsible for the inhibition of the electron transport. We propose that tar causes at least two effects: (1) Tar components interact with the electron transport chain and inhibit electron flow, and (2) tar components interact with the electron transport chain, ultimately to form hydroxyl radicals.     

Quantitative analysis of hemin-induced neutrophil extracellular trap formation and effects of hydrogen peroxide on this phenomenon

Formation of neutrophil extracellular traps (NETs) can perpetuate sterile inflammation; thus, it is important to clarify their pathophysiological characteristics. Free heme, derived via hemolysis, is a major contributor to organ damage, and reportedly induces neutrophil activation as well as reactive oxygen species (ROS) production and NET formation. For this study, we examined hemin (Fe³⁺ -protoporphyrin IX)-induced NET formation quantitatively in vitro as well as the effects of oxidative stress.NETs formed in vitro from cultured neutrophils were quantitatively detected by using nuclease treatment and Sytox Green, a nucleic acid stain. Hemin-induced NET production was found to be in a dose-dependent manner, NADPH oxidase-dependent and toll-like receptor (TLR)-4 independent. Additionally, the iron molecule in the porphyrin ring was considered essential for the formation of NETs. In the presence of low concentrations of hydrogen peroxide, low concentrations of hemin-induced NETs were enhanced, unlike those of phorbol myristate acetate (PMA)-induced NETs.Quantitative analysis of NET formation may prove to be a useful tool for investigating NET physiology, and hemin could function as a possible therapeutic target for hemolysis-related events.     

Inactivation of cytochrome P-450 by hydrogen peroxide formed in the catalytic cycle during peroxy-complex degradation

It was shown that the crucial role in the inactivation of microsomal cytochrome P-450 in reactions of hydroxylation of type I (DMA, AP, BPh, p-NA) and type II (AN) substrates belongs to H2O2 directly formed in the enzyme active center during the decomposition of the peroxy complex. Hydrogen peroxide formed via an indirect pathway during the dismutation of superoxide radicals does not play a role in the hemoprotein inactivation.     

Oxidation of p-coumaroylagmatine in barley seedling extracts in the presence of hydrogen peroxide or thiols

When p-coumaroylagmatine is oxidized in the presence of hydrogen peroxide in a crude extract of barley seedlings, among several products, hordatine A is formed. However, unlike the natural isomer, this is optically inactive. In the absence of hydrogen peroxide and when a thiol (glutathione, cysteine, mercaptoethanol or dithiothreitol) is added to the incubation medium, p-coumaroylagmatine is rapidly transformed to a thiol adduct, probably through a peroxidase-dependent co-oxidation reaction. The reactions with hydrogen peroxide or with a thiol are completely inhibited by 1 mM ascorbate.     

Peroxyoxalate chemiluminescent assay for oxidase activities based on detecting enzymatically formed hydrogen peroxide

A sensitive peroxyoxalate chemiluminescent (PO-CL) assay for activities of oxidases (uricase, choline oxidase, cholesterol oxidase and xanthine oxidase) which catalyse a formation of hydrogen peroxide was developed using 4,4′-oxalyl-bis[(trifluoromethylsulphonyl)imino]trimethylene-bis(4-methylmorpholinium)trifluoromethanesulphonate as a chemiluminogenic reagent and 2,4,6,8-tetramorpholinopyrimido[5,4-d]pyrimidine as a fluorophore. The standard curve for hydrogen peroxide was linear over the range 1 × 10^?7-1 × 10^?4 mol/L. Relative standard deviations for oxidase assays were 5.1–12.7% (n = 10). Detection limits were 1 × 10^?3 U/mL for uricase, 5 × 10^?4 U/mL for choline oxidase, 5 × 10^?3 U/mL for cholesterol oxidase and 5 × 10^?4 U/mL xanthine oxidase (sample to blank ratio, 3).     

Effect of a weak electromagnetic field on the rate of hydrogen peroxide production in aqueous solutions

A theoretical model of the mechanism of action of weak electromagnetic fields on water solutions has been constructed. The model predicts the redistribution of protons on spatial inhomogeneities in water medium. It is shown that an external field leads to the phasing of ions on the standing wave, which is considered as an inhomogeneity. As a result of an inhomogeneous distribution of hydrogen ions, local regions with a higher and lower acidity arise. The acidity of medium substantially affects the rate of chemical reactions; therefore, the exposure to a weak external field can change this parameter. The effect of local changes in acidity on the rate of hydrogen peroxide production was considered. It was predicted that the exposure to a weak electromagnetic field with particular parameters can increase the rate and, as a consequence, the concentration of hydrogen peroxide in solution.     

Formation of hydrogen peroxide by VUV-photolysis of water and aqueous solutions with methanol

The hydrogen peroxide production upon vacuum ultraviolet (VUV) irradiation of water is reviewed, because published results from the last 10 years lead to conflicting mechanistic interpretations. This work confirms that in pure water, hydrogen peroxide is only produced in the presence of molecular oxygen. Mechanistic schemes explain these findings and confirm earlier statements that recombination of hydroxyl radicals is kinetically disfavoured. In agreement with other recent publications, this work confirms that enhanced hydrogen peroxide production takes place upon VUV irradiation of aqueous solutions of organic compounds. For these investigations, methanol was chosen as an organic model compound. During photolyses, hydrogen peroxide, dissolved molecular oxygen, pH-value of the reaction system, methanol and its products of oxidative degradation were analyzed, and kinetic studies were undertaken to explain the evolution of the concentrations of these components.