Peroxide-metabolizing systems of the crystalline lens

The ability of transparent and cataractous human, rabbit and mice lenses to metabolize hydrogen peroxide in the surrounding medium was evaluated. Using a chemiluminescence method in a system of luminol-horseradish peroxidase and a photometric technique, the temperature-dependent kinetics of H₂O₂ decomposition by lenses were measured. The ability of opaque human lenses to catalyze the decomposition of 10^?4 M H₂O₂ was significantly decreased. However, this was reserved by the addition of GSH to the incubation medium. Incubation of the mice lenses with the initial concentration H₂O₂ 10^?4 M led to partial depletion of GSH in normal and cataractous lenses. Human cataractous lenses showed decreased activities of glutathione reductase, glutathione peroxidase (catalyzing reduction of organic hydroperoxides including hydroperoxides of lipids), superoxide dismutase, but no signs of depletion in activities of catalase or glutathione peroxidase (utilizing H₂O₂). The findings indicated an impairment in peroxide metabolism of the mature cataractous lenses compared to normal lenses to be resulted from a deficiency of GSH. An oxidative stress induced by accumulation of lipid peroxidation products in the lens membranes during cataract progression could be considered as a primary cause of GSH deficiency and disturbance of the redox balance in the lens.     

Aldehyde oxidase generates deoxyribonucleic acid single strand nicks in vitro

SUMMARY

We purified aldehyde oxidase (AO) from rabbit livers and found that AO produced deoxyribonucleic acid (DNA) single strand nicks in vitro. Acetaldehyde, benzaldehyde, and certain purine bases were effective substrates for AO catalyzed DNA strand nicking. DNA strand nicking did not occur with the reducing substrates nicotinamide-adenine dinucleotide or dithionite that produce superoxide anion (O_2′^?). Inclusion of electron transport inhibitors, potassium cyanide, ferricyanide or menadione, prevented AO catalyzed nicking. AO induced DNA strand nicking was dependent upon hydrogen peroxide (H₂O₂) formation and most likely generation of hydroxyl radical (HO'). The present observations may be pertinent to the recently proposed involvement of AO in inherited juvenile familial amyotrophic lateral sclerosis (JFALS) and other oxygen radical mediated diseases.     

Hyperbaric oxygen enhances apoptosis in hematopoietic cells

Hyperbaric oxygen (HBO) is 100% oxygen administered at elevated atmospheric pressure. In this study, we examined the effect of HBO on hematopoietic cell apoptosis. Cells exposed to HBO were incubated in a chamber containing 97.9% O₂ and 2.1% CO₂ at 2.4 atmospheres absolute (ATA). HBO enhanced spontaneous HL-60 cell apoptosis in a time-dependent manner; a 12 h exposure increased apoptosis by 42%. Exposing these cells to hyperoxia at standard atmospheric pressure (95% O₂, 5% CO₂ at 1 ATA) or increased pressure alone (8.75% O₂, 2.1% CO₂ at 2.4 ATA) had minimal effect on apoptosis. HBO also enhanced stimulus-induced apoptosis. HL-60 cells stimulated to die using radiation underwent 33% more apoptosis than cells exposed to radiation alone. HBO enhanced melphalan, camptothecin, and chlorambucil-induced apoptosis by 22%, 13%, and 8%, respectively. Jurkat cells stimulated to die with anti-Fas antibody underwent 44% more apoptosis when exposed to HBO. Spontaneous apoptosis was increased by 15% in HBO-exposed murine thymocytes. HBO's effect on apoptosis did not require new protein synthesis. As expected, HBO exposure increased the intracellular concentration of H₂O₂. Incubating HL-60 cells in the presence of dehydroascorbic acid partially abrogated HBO-induced increases in intracellular H₂O₂ and apoptosis. In summary, HBO enhances spontaneous and stimulus-induced apoptosis in hematopoietic cells, at least in part, by enhancing the intracellular accumulation of H₂O₂.     

Involvement of Molecular Oxygen in the Enzyme-Catalyzed NADH Oxidation and Ferric Leghemoglobin Reduction

Ferric leghemoglobin reductase (FLbR) from soybean (Glycine max [L.] Merr) nodules catalyzed oxidation of NADH, reduction of ferric leghemoglobin (Lb⁺³), and reduction of dichloroindophenol (diaphorase activity). None of these reactions was detectable when O₂ was removed from the reaction system, but all were restored upon readdition of O₂. In the absence of exogenous electron carriers and in the presence of O₂ and excess NADH, FLbR catalyzed NADH oxidation with the generation of H₂O₂ functioning as an NADH oxidase. The possible involvement of peroxide-like intermediates in the FLbR-catalyzed reactions was analyzed by measuring the effects of peroxidase and catalase on FLbR activities; both enzymes at low concentrations (about 2 μg/mL) stimulated the FLbR-catalyzed NADH oxidation and Lb⁺³ reduction. The formation of H₂O₂ during the FLbR-catalyzed NADH oxidation was confirmed using a sensitive assay based on the fluorescence emitted by dichlorofluorescin upon reaction with H₂O₂. The stoichiometry ratios between the FLbR-catalyzed NADH oxidation and Lb⁺³ reduction were not constant but changed with time and with concentrations of NADH and O₂ in the reaction solution, indicating that the reactions were not directly coupled and electrons from NADH oxidation were transferred to Lb⁺³ by reaction intermediates. A study of the affinity of FLbR for O₂ showed that the enzyme required at least micromolar levels of dissolved O₂ for optimal activities. A mechanism for the FLbR-catalyzed reactions is proposed by analogy with related oxidoreductase systems.     

Electron acceptor function of O2 in radical N-demethylation reactions catalyzed by hemeproteins

In aerobic solutions, O₂ consumption correlated well with N-demethylation of N,N-dimethyl-$\text{p}$-toluidine catalyzed by horseradish peroxidase, in the presence or absence of H₂O₂. In the absence of added H₂O₂, superoxide dismutase stimulated, and catalase inhibited, both reactions; in the presence of H₂O₂, argon inhibition of formaldehyde production increased with increasing concentration of horseradish peroxidase. These results provide evidence for competing reactions of the enzymatically-generated substrate radical: oxidation by O₂ increases formaldehyde production, while radical dimerization decreases the yield of this product. Implications of these findings for similar reactions catalyzed by microsomal cytochrome P-450 are suggested.     

Hydrogen peroxide formation and stoichiometry of hydroxylation reactions catalyzed by highly purified liver microsomal cytochrome P-450.

The stoichiometry of hydroxylation reactions catalyzed by cytochrome P-450 was studied in a reconstituted enzyme system containing the highly purified cytochrome from phenobarbital-induced rabbit liver microsomes. Hydrogen peroxide was shown to be formed in the reconstituted system in the presence of NADPH and oxygen; the amount of peroxide produced varied with the substrated added. NADPH oxidation, oxygen consumption, and total product formation (sum of hydroxylated compound and hydrogen peroxide) were shown to be equimolar when cyclohexane, benzphetamine, or dimethylaniline served as the substrate. The stoichiometry observed represents the sum of two activities associated with cytochrome P-450. These are (1) hydroxylase activity: NADPH + H⁺ + O₂ + RH → NADP⁺ + H₂O + ROH; and (2) oxidase activity: NADPH + H⁺ + O₂ → NADP⁺ + H₂O₂. Benzylamphetamine (desmethylbenzphetamine) acts as a pseudosubstrate in that it stimulates peroxide formation to the same extent as the parent compound (benzphetamine), but does not undergo hydroxylation. Accordingly, when benzylamphetamine alone is added in control experiments to correct for the NADPH and O₂ consumption not associated with benzphetamine hydroxylation, the expected 1:1:1 stoichiometry for NADPH oxidation, O₂ consumption, and formaldehyde formation in the hydroxylation reaction is observed.     

Peroxidase mediated hydrogen peroxide production in barley roots grown under stress conditions

All applied metals (Co, Al, Cu, Cd) and NaCl inhibited barley root growth. No root growth inhibition was caused by drought exposure, in contrast to cold treatment. 0.01 mM H₂O₂ stimulated root growth and GA application did not affect root growth at all. Other activators and inhibitors of H₂O₂ production (SHAM, DTT, 10 mM H₂O₂, 2,4-D) inhibited root growth. Loss of cell viability was most significant after Al treatment, followed by Cd and Cu, but no cell death was induced by Co. Drought led to slight increase in Evans blue uptake, whereas neither NaCl nor cold influenced this parameter. DTT treatment caused slight increase in Evans blue uptake and significant increases were detected after 2,4-D and 10 mM H₂O₂ treatment, but were not induced by others stressors. Metal exposure increased guaiacol-POD activity, which was correlated with oxidation of NADH and production of H₂O₂. Exposure to drought caused a minor change in NADH oxidation, but neither H₂O₂ production nor guaiacol-POD activity was increased. Cold and NaCl application decreased all monitored activities. Increase in NADH oxidation and guaiacol-POD activity was caused by 10 mM H₂O₂ and 0.01 mM 2,4-D treatment, which also caused enhancement of H₂O₂ production. Slight inhibition of all activities was caused by 0.01 mM H₂O₂, GA, DTT; more pronounced inhibition was detected after SHAM treatment. The role of H₂O₂ production mediated by POD activity in relation to root growth and cell viability under exposure to some abiotic stress factors is discussed.     

Inactivation of Rabbit Muscle Creatine Kinase by Hydrogen Peroxide

The effects of xanthine + xanthine oxidase-generated reactive oxygen species (ROS) on rabbit muscle creatine kinase (CK) were studied. Xanthine (0.1 mM) + xanthine oxidase (30 mU/ml) inhibited activity of rabbit muscle CK (1.2mU/ml). Catalase (100/ml), but not SOD (100 U/ml), deferoxamine (100μM) or mannitol (20 mM), protected CK from inactivation; suggesting that H₂O₂ was responsible for inactivation. These results were different from previously reported findings on bovine heart CK that superoxide radicals inactivate the enzyme. Thus, enzymes with homologous structures may have different reactivities to different ROS. H₂O₂-induced inactivation of rabbit muscle CK was accompanied by a decrease in its thiol group content, whereas no significant changes in the protein structure were detected by SDS-PAGE or carbonyl content. These results suggest that oxidation of -SH groups by H₂O₂ seems to be a major mechanism of activation of rabbit muscle CK by xanthine + xanthine oxidase. Such inactivation of CK by H₂O₂ may be important in ROS-induced pathology.     

Scuba diving enhances endogenous antioxidant defenses in lymphocytes and neutrophils

The aim was to study the effects of a scuba diving session on the lymphocyte antioxidant system, NO synthesis, the capability to produce reactive oxygen species and the antioxidant response in neutrophils. For that purpose seven male divers performed an immersion at a depth of 40 m for 25 min. The same parameters were measured after an hyperbaric oxygen (HBO) treatment at resting conditions in a hyperbaric chamber. Lymphocyte H₂O₂ production rose after diving and after HBO treatment. Glutathione peroxidase (GPx) and catalase activities increased after diving in lymphocytes, while after HBO exposure only increased GPx activity. Lymphocyte HO-1 mRNA expression increased after diving and after HBO exposure, while iNOS levels and nitrite levels significantly increased after diving. The hyperoxia associated to scuba diving leads to a condition of oxidative stress with increased lymphocyte H₂O₂ production, HO-1 expression, NO synthesis and antioxidant enzyme adaptations in order to avoid oxidative damage.     

Effects of putrescine on oxidative stress induced by hydrogen peroxide in Salvinia natans L.

Salvinia natans L. response to hydrogen peroxide (H₂O₂) induced oxidative stress through physiological activities was evaluated. The plants were incubated with varying concentrations (0, 50, 100 µM) of H₂O₂ and 100 µM of H₂O₂ supplemented with 1 mM putrescine (Put) in hydroponic culture. This is observed with the decline in proline content and its biosynthetic enzymes viz. γ-glutamyl kinase and γ-glutamyl phosphate reductase activity. Protein carbamylated derivative by protein oxidation was another trait for oxidative damages by H₂O₂. The antioxidative enzymes like guaiacol peroxidase (GPX), glutathione reductase (GR), and catalase (CAT) recorded to express through in-gel staining with the H₂O₂ exposure. On nuclear level, plants were sensitive to H₂O₂ where the DNA disintegration was studied with comet assay and maximum comet tail observed at 100 µM H₂O₂ treatment. Application of Put reduced the generation of protein oxidation and comet tail length as well as moderated the enzyme activity as revealed through in-gel staining.     

Energy-coupling mechanisms under aerobic and anaerobic conditions in autotrophically grown Pseudomonas saccharophila

The cell-free preparations from autotrophieally grown Pseudomonas saccharophila catalyzed the process of electron transport from H₂ or various other organic electron donors to either O₂ or NO₃^? with concomitant ATP generation. The respective $\text{P}\text{O}$ ratios with H₂ and NADH were 0.63 and 0.73, the respective $\text{P}\text{NO}{}_3{}^{\mathrm{?}}$ ratios were 0.57 and 0.54. In contrast, the $\text{P}\text{O}$ and $\text{P}\text{NO}{}_3{}^{\mathrm{?}}$ ratios with succinate were 0.18 and 0.11, respectively. ATP formation coupled to the oxidation of ascorbate, in the absence or presence of added N,N,N′,N′-tetramethyl-p-phenylenediamine or cytochrome c, could not be detected. Various uncouplers inhibited phosphorylation with either O₂ or NO₃^? as terminal electron acceptors without affecting the oxidation of H₂ or other substrates. The NADH oxidation at the expense of O₂ or NO₃^? reduction as well as the associated phosphorylation were inhibited by rotenone and amytal. The aerobic and anaerobic H₂ oxidation and coupled ATP synthesis, on the other hand, was unaffected by the flavoprotein inhibitors as well as by the NADH trapping system. The NADH, H₂, and succinate-linked electron transport to O₂ or NO₃^? and the associated phosphorylations were sensitive, however, to antimycin A or 2-n-nonyl-4-hydroxyquino-line-N-oxide, and cyanide or azide. The data indicated that although the phosphorylation sites 1 and II were associated with NADH oxidation by O₂ or NO₃^?, the energy conservation coupled to H₂ oxidation under aerobic or anaerobic conditions appeared to involve site II only.     

Water Oxidation by a Cytochrome P450: Mechanism and Function of the Reaction

P450_cam (CYP101A1) is a bacterial monooxygenase that is known to catalyze the oxidation of camphor, the first committed step in camphor degradation, with simultaneous reduction of oxygen (O₂). We report that P450_cam catalysis is controlled by oxygen levels: at high O₂ concentration, P450_cam catalyzes the known oxidation reaction, whereas at low O₂ concentration the enzyme catalyzes the reduction of camphor to borneol. We confirmed, using ¹⁷O and ²H NMR, that the hydrogen atom added to camphor comes from water, which is oxidized to hydrogen peroxide (H₂O₂). This is the first time a cytochrome P450 has been observed to catalyze oxidation of water to H₂O₂, a difficult reaction to catalyze due to its high barrier. The reduction of camphor and simultaneous oxidation of water are likely catalyzed by the iron-oxo intermediate of P450_cam, and we present a plausible mechanism that accounts for the 1∶1 borneol:H₂O₂ stoichiometry we observed. This reaction has an adaptive value to bacteria that express this camphor catabolism pathway, which requires O₂, for two reasons: 1) the borneol and H₂O₂ mixture generated is toxic to other bacteria and 2) borneol down-regulates the expression of P450_cam and its electron transfer partners. Since the reaction described here only occurs under low O₂ conditions, the down-regulation only occurs when O₂ is scarce.     

Reduced nicotinamide adenine dinucleotide phosphate (NADPH)-dependent formation and breakdown of hydrogen peroxide during mixed function oxidation reactions in liver microsomes.

Conditions for the recovery of H₂O₂ from microsomes and for determination of the rate and extent of H₂O₂ formation during oxidation of NADPH by liver microsomes have been investigated. H₂O₂ was determined by two methods that are applicable to conditions existing during microsomal mixed function oxidation reactions, provided that contaminating catalase activity is inhibited by azide and that interference by other mixed function oxidation reactions can be excluded. To estimate the formation of H₂O₂ in absence of azide, H₂O₂ was determined indirectly by the production of HCHO during oxidation of cold and ¹⁴C-labeled methanol and an excess of exogenous catalase. As additional catalase-independent decomposition of H₂O₂ also occurs during oxidation of NADPH, the kinetics of H₂O₂ formation in microsomes is influenced by two independent processes. H₂O₂ will be produced under optimal conditions i.e., at V when O₂ and NADPH are in excess. Addition or formation of increasing amounts of H₂O₂ raises the substrate (H₂O₂) concentration and will enhance the rate of breakdown of H₂O₂.     

Oxidative stress response in eukaryotes: effect of glutathione,superoxide dismutase and catalase on adaptation to peroxide and menadione stresses in Saccharomyces cerevisiae

Abstract

Aiming to clarify the mechanisms by which eukaryotes acquire tolerance to oxidative stress, adaptive and cross-protection responses to oxidants were investigated in Saccharomyces cerevisiae. Cells treated with sub-lethal concentrations of menadione (a source of superoxide anions) exhibited cross-protection against lethal doses of peroxide; however, cells treated with H₂O₂ did not acquire tolerance to a menadione stress, indicating that menadione response encompasses H₂O₂ adaptation. Although, deficiency in cytoplasmic superoxide dismutase (Sod1) had not interfered with response to superoxide, cells deficient in glutathione (GSH) synthesis were not able to acquire tolerance to H₂O₂ when pretreated with menadione. These results suggest that GSH is an inducible part of the superoxide adaptive stress response, which correlates with a decrease in the levels of intracellular oxidation. On the other hand, neither the deficiency of Sod1 nor in GSH impaired the process of acquisition of tolerance to H₂O₂ achieved by a mild pretreatment with peroxide. Using a strain deficient in the cytosolic catalase, we were able to conclude that the reduction in lipid peroxidation levels produced by the adaptive treatment with H₂O₂ was dependent on this enzyme. Corroborating these results, the pretreatment with low concentrations of H₂O₂ promoted an increase in catalase activity.     

Lead-hemoglobin interaction as a possible source of reactive oxygen species—A chemiluminescent study

Pb²⁺-hemoglobin interaction as a possible source of reactive oxygen species was investigated. It was found that the products of this reaction are able to promote peroxidase catalyzed luminol oxidation with light emission. Superoxide dismutase and catalase strongly inhibited this effect. A conclusion was done that the interaction between Pb²⁺ and oxyhemoglobin yields reactive oxygen species, possibly O₂^? and H₂O₂.     

Catecholamines Enhance Dihydrolipoamide Dehydrogenase Inactivation by the Copper Fenton System. Enzyme Protection by Copper Chelators

Catecholamines (CAs: epinephrine, norepinephrine, dopamine, L-DOPA, 6-hydroxydopamine) and o-diphenols (DOPAC and catechol) enhanced dihydrolipoamide dehydrogenase (LADH) inactivation by Cu(II) /H₂O₂ (Cu-Fenton system). The inhibition of LADH activity correlated with Cu(II), H₂O₂ and CA concentrations. Similar inhibitions were obtained wit! the assayed CAs and o-diphenols. CAs enhanced HO radical production by Cu(II) /H₂O₂, as demonstrated by benzoate hydroxylation and deoxyribose oxidation; LADH counteracted the pro-oxidant effect of CAs by scavenging hydroxyl radicals. Captopril, dihydrolipo amide, dihydrolipoic acid, DL-dithiothreitol, GSSG, try-panothione and histidine effectively preserved LADH from oxidative damage, whereas N-acetylcysteine, N-(2-mercaptopropionylglycine) and lipoamide were less effective protectors. Catalase (though neither bovine serum albumin nor superoxide dismutase) protected LADH against the Cu(II)/H₂O₂/CAs systems. Dena tured catalase protected less than the native enzyme, its action possibly depending on Cu-binding. LADH in creased and Captopril inhibited epinephrine oxidation by Cu(II)/H₂O₂ and Cu(II). The summarized evidence supports the following steps for LADH inactivation: (1) reduction of LADH linked-Cu(II) to Cu(I) by CAs; (2) production of HO^* from H₂O₂ by LADH-linked Cu(I) (Haber-Weiss reaction) and (3) oxidation of aminoacid residues at the: enzyme active site by site-specifically generated HO^* radicals. Hydrogen peroxide formation from CAs autoxidation may contribute to LADH inactivation.     

Lysine-derived cross-links in the egg shell membrane

Egg shell membrane protein contains significant quantities of the lysine-derived aldehyde, allysine, and its aldol condensation product. NaB³H₄ reduction followed by alkaline hydrolysis of purified protein revealed that there were six residues/1000 of both allysine and the reduced aldol while only traces of desmosine and isodesmosine were detected. The amino acid composition of the membrane protein did not resemble that of mammalian elastin.     

Superoxide and hydrogen peroxide production and NADPH oxidation stimulated by nitrofurantoin in lung microsomes: possible implications for toxicity.

The addition of nitrofurantoin to aerobic incubation mixtures containing rat lung microsomes strongly enhanced the generation of adrenochrome from epinephrine. Adrenochrome formation in this system was blocked by superoxide dismutase, but not by catalase. Hydrogen peroxide production was also strongly enhanced by nitrofurantoin in these preparations; superoxide dismutase did not significantly alter the amount of H₂O₂ measured, but no H₂O₂ was detected in incubation mixtures in the presence of catalase. Nitrofurantoin enhanced the oxidation of NADPH in lung microsomal suspensions under aerobic conditions; the enhancement was unaffected by catalase but was partially prevented by superoxide dismutase. Neither adrenochrome formation nor H₂O₂ production were enhanced by nitrofurantoin under anaerobic (N₂) conditions, but NADPH oxidation in the presence of nitrofurantoin was greater under anaerobic conditions than under aerobic conditions. These results are consistent with the view that the redox cycling of nitrofurantoin in lung microsomes in the presence of oxygen results in the consumption of NADPH and the production of activated oxygen species, emphasizing some $\text{in vitro}$ metabolic similarities with the lung-toxic herbicide, paraquat.     

Observations on the use of guaiacol and 2,2′-azino-di(3-ethylbenzthiazoline-6-sulfonic acid) as peroxidase substrates

Aging of aqueous guaiacol (o-methoxyphenol) solutions over a period of several months led to the spontaneous formation of peroxidatic compound(s) and other unidentified oxidation products of guaiacol. This accelerated the oxidation of guaiacol catalyzed by lactoperoxidase (LPO) severalfold depending on the pH of the reaction mixture. The peroxide(s) acted like H₂O₂ while the aromatic oxidation products may be more reactive than guaiacol. Five- to 12-month-old 20 mm stock solutions contained even 0.05-0.3% of H₂O₂ equivalents. The formation of the peroxidatic compound(s) was found to be a photochemical process which progressed in a few hours at 254 nm and slowly (detectable in 2-week-old solutions) in regular glass bottles kept under normal laboratory illumination. The kinetics and pH dependence of the oxidation of aged guaiacol solutions by LPO were distinctly different from those found with fresh substrate. The spontaneously formed peroxidatic compound is possibly a better oxygen donor in LPO assays than H₂O₂. The spontaneously formed aromatic oxidation products of guaiacol may include compounds that contain diphenoquinone groups. The complexity of the oxidation of guaiacol and the multitude of reaction products formed require special consideration in kinetic studies of LPO. The use of 2,2′-azino-di(3-ethylbenzthiazoline-6-sulfonic acid) as a LPO substrate was studied. The published method utilizing this substrate was modified into a more sensitive procedure by readjusting some of the reaction conditions.     

Oxidation of 4-chloroaniline catalyzed by human myeloperoxidase

The incubation of 4-chloroaniline with H₂O₂ and myeloperoxidase results in the formation of at least 10 products. Possibly some structures with high complexity, like 4,4′-dichloroazobenzene, are present; however, no 4-chloronitrosobenzene is detectable. This result contrasts with the oxidation of 4-chloroaniline catalyzed by chloroperoxidase, which only yields 4-chloronitrosobenzene.