Involvement of cytochrome P450 and the flavin-containing monooxygenase(s) in the sulphoxidation of simple sulphides in human liver microsomes

This study was conducted to examine the involvement of cytochrome P450 (CYP450) and the flavin-containing monooxygenase (FMO) in the sulphoxidation of ethyl methyl sulphide (EMS), 4-chlorophenyl methyl sulphide (CPMS) and diphenyl sulphide (DPS) in human liver microsomes from a phenotypic CYP2D6 extensive metabolizer. Human liver microsomes catalyzed the sulphoxidation of EMS, CPMS and DPS to their corresponding sulphoxides. Lineweaver-Burk plots for the sulphoxidation of EMS in human liver microsomes indicated that the apparent K(m) and V(max) were 1.53 +/- 0.07 mM and 1.11 +/- 0.25 nmoles/mg protein/min, respectively. The apparent K(m) and V(max) for the sulphoxidation of CPMS were 0.17 +/- 0.05 mM and 1.41 +/- 0.16 nmoles/mg protein/min, respectively. The apparent K(m) and V(max) for the sulphoxidation of DPS were 0.10 +/- 0.01 mM and 1.08 +/- 0.05 nmoles/mg protein/min, respectively. Methimazole noncompetitively inhibited the sulphoxidation of EMS, CPMS and DPS by human liver microsomes with K(i) values of 8.6 +/- 0.6, 5.7 +/- 0.4 and 6.6 +/- 0.5 mM, respectively. SKF525A noncompetitively inhibited the sulphoxidation of CPMS and DPS by human liver microsomes with K(i) values of 6.6 +/- 0.4 and 0.40 +/- 0.1 mM, respectively. The results suggest that FMO is involved in the sulphoxidation of EMS, CPMS and DPS while CYP450 is involved in the sulphoxidation of CPMS and DPS in human liver microsomes.     

An oxygenase from guinea-pig liver which catalyses sulphoxidation

A mono-oxygenase catalysing the conversion of 2-ethyl-4-thioisonicotinamide (ethionamide) into its sulphoxide was purified from guinea-pig liver homogenates. The enzyme required stoicheiometric amounts of oxygen and NADPH for the sulphoxidation reaction. The purified protein is homogeneous by electrophoretic, antigenic and chromatographic criteria. The enzyme has mol.wt. 85000 and it contains 1g-atom of iron and 1mol of FAD per mol, but not cytochrome P-450. The enzyme shows maximal activity at pH7.4 in a number of different buffer systems and the K(m) values calculated for the substrate and NADPH are 6.5x10(-5)m and 2.8x10(-5)m respectively. The activation energy of the reaction was calculated to be 36kJ/mol. Under optimal conditions, the molecular activity of the enzyme (mol of substrate oxidized/min per mol of enzyme) is calculated to be 2.1. The oxygenase belongs to the class of general drug-metabolizing enzymes and it may act on different compounds which can undergo sulphoxidation. The mechanism of sulphoxidation was shown to be mediated by superoxide anions.     

Comparison of the effects of superoxide dismutase and cytochrome c on luminol chemiluminescence produced by xanthine oxidase-catalyzed reactions

Superoxide dismutase (superoxide: superoxide oxidoreductase, EC 1.15.1.1) (SOD) and ferricytochrome c are used to check the effects on luminol chemiluminescence induced by a xanthine or hypoxanthine/xanthine oxidase/oxygen system. Luminol chemiluminescence has been attributed to superoxide anion radical (O2.-) in this system. From kinetic studies on the light intensity vs. time curves it is demonstrated that addition of SOD into the system does not affect the mechanism of O2.- generation, whilst ferricytochrome c dramatically alters the time-course of the reaction. This is interpreted as the effect of cytochrome c redox cycling by reaction with H2O2, modifying oxy-radical generation in the reaction medium. Also, an alternative mechanism for luminol chemiexcitation is proposed under certain experimental conditions.     

The mechanism of potentiation of horseradish peroxidase-catalysed oxidation of NADPH by porphyrins.

Several porphyrins, including HpD (haematoporphyrin derivative), potentiate the oxidation of NADPH by horseradish peroxidase/H2O2. To elucidate the mechanism of potentiation, the following observations are relevant. During peroxidase-catalysed NADPH oxidation, O2-.(superoxide radical) is generated, as judged from superoxide dismutase-inhibitable cytochrome c reduction. This generation of O2-. is suppressed by HpD. Peroxidase-catalysed NADPH oxidation is stimulated by superoxide dismutase and by anaerobic conditions. Under anaerobic conditions HpD has no influence on peroxide-catalysed NADPH oxidation. Previous studies have shown that horseradish peroxidase is inhibited by O2-.. Thus the experimental results indicate that the potentiating effect of HpD can be explained by its ability to inhibit O2-. generation in the horseradish peroxidase/H2O2/NADPH system.     

Mechanism of hydrogen peroxide formation catalyzed by NADPH oxidase in thyroid plasma membrane

The thyroid plasma membrane contains a Ca2(+)-regulated NADPH-dependent H2O2 generating system which provides H2O2 for the thyroid peroxidase-catalyzed biosynthesis of thyroid hormones. The plasma membrane fraction contains a Ca2(+)-independent cytochrome c reductase activity which is not inhibited by superoxide dismutase. But it is not known whether H2O2 is produced directly from molecular oxygen (O2) or formed via dismutation of super-oxide anion (O2-). Indirect evidence from electron scavenger studies indicate that the H2O2 generating system does not liberate O2-, but studies using the modified peroxidase, diacetyldeuteroheme horseradish peroxidase, to detect O2- indicate that H2O2 is provided via the dismutation of O2-. The present results provide indirect evidence that the cytochrome c reductase activity is not a component of the NADPH-dependent H2O2 generator, since it was removed by washing the plasma membranes with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid without affecting H2O2 generation. Spectral studies with diacetyldeuteroheme-substituted horseradish peroxidase showed that the thyroid NADPH-dependent H2O2 generator does not catalyze superoxide anion formation. The O2- adduct compound (compound III) was formed but was completely inhibited by catalase, indicating that the initial product was H2O2. The rate of NADPH oxidation also increased in the presence of diacetylheme peroxidase. This increase was blocked by catalase and was greatly enhanced by superoxide dismutase. The O2- adduct compound (compound III) was produced in the presence of NADPH when glucose-glucose oxidase (which does not produce O2-) was used as the H2O2 generator. NADPH oxidation occurred simultaneously and was enhanced by superoxide dismutase. We conclude that O2- formation occurs in the presence of an H2O2 generator, diacetylheme peroxidase and NADPH, but that it is not the primary product of the H2O2 generator. We suggest that O2- formation results from oxidation of NADPH, catalyzed by the diacetylheme peroxidase compound I, producing NADP degree, which in turn reacts with O2 to give O2-.     

DNA strand scission by enzymatically reduced mitomycin C: evidence for participation of the hydroxyl radical in the DNA damage

Phage DNA, as well as plasmid and mammalian DNA's, were exposed to a superoxide and hydroxyl radical-generating system containing NADPH-cytochrome P-450 reductase and mitomycin C, both with and without added Fe3+-ADP, in phosphate buffer at pH 7.5. The generation of superoxide (O2-.) and hydroxyl (.OH) radicals in the system was demonstrated by using ESR spectrometry with N-tert -butyl-alpha-phenylnitrone (PBN) as a spin trapping agent. Only the lambda DNA isolated after exposure to the O2-./.OH-generating system containing many lower molecular weight DNA fragments indicating DNA strand breaks. This breakage was completely inhibited by a .OH radical scavenger (sodium benzoate) and by catalase, but only slightly by superoxide dismutase. Thyroid and plasmid DNA's were both cleaved when exposed to the O2-./.OH-generating systems. It is suggested that the mechanism of DNA scission by mitomycin C described here closely resembles that induced by the anthracycline drugs.     

A flash-photometric method for determination of reactivity of superoxide: application to superoxide dismutase assay

Pulse-generation of O2- by a flash was used to determine the reactivity of O2-, O2- was produced within 10 ms by a flash of light through the excitation of FMN in the presence of N,N,N',N'-tetramethylethylenediamine and oxygen. Kinetic analysis of cytochrome c reduction by O2- generated by flash yielded the reaction rate constant between cytochrome c and O2- and the spontaneous disproportionation rate constant of O2-. We applied it for superoxide dismutase assay using a linear relation between superoxide dismutase concentration and the apparent rate constant of cytochrome c reduction by O2-. The catalytic rate constant and activation energy at pH 7.3 of bovine liver Cu,Zn-superoxide dismutase were found to be 1.75 x 10(9) M-1 . s-1 at 25 degrees C and 26.9 kJ . M-1, respectively. The kinetics of O2- decay can be also monitored at 240 nm in this flash-photometric system and gave the spontaneous disproportionation rate constant of O2- and the catalytic rate constant of superoxide dismutase.     

Superoxide dismutase-like activities of copper(II) complexes tested in serum

The superoxide dismutase-like activities of a series of coordination complexes of copper were evaluated and compared to the activities of bovine erythrocyte superoxide dismutase (superoxide: superoxide oxidoreductase, EC 1.15.1.1) in serum using the nitroblue tetrazolium chloride (NBT)-reduction assay and electron paramagnetic resonance (EPR) spectroscopy. A 40% inhibition was observed for the initial rate of the NBT reduction by superoxide dismutase in serum, but more than 40% inhibition was achieved with CuSO4, Cu(II)-dimethylglyoxime, Cu(II)-3,8-dimethyl-4,7-diazadeca-3,7-dienediamide, Cu2[N,N'-(2-(O-hydroxy-benzhydrylidene)amino)ethyl]2-1,2-ethane dia mine), Cu(II)-(diisopropylsalicylate)2, Cu(II)-(p-bromo-benzoate)2, Cu(II)-(nicotinate)2 and Cu(II)-(1,2-diamino-2-methylpropane)2. The electron paramagnetic resonance technique of spin trapping was used to detect the formation of superoxide (O2-.) and other free radicals in the xanthine-xanthine oxidase system under a variety of conditions. Addition of the spin trapping agent 5,5-dimethylpyrroline 1-oxide (DMPO) to the xanthine-xanthine oxidase system in fetal bovine serum produced the O2-.-spin adduct of DMPO (herein referred to as superoxide spin adduct, DMPO-OOH) as the well known short-lived nitroxyl whose characteristic EPR spectrum was recorded before its rapid decay to undetectable levels. The hydroxyl radical (HO.) adduct of the spin trap DMPO (herein referred to as DMPO-OH) was detected to a very small extent. When CuSO4, or the test complexes of copper, were added to the xanthine-xanthine oxidase system in serum containing the spin trap, the yield of DMPO-OOH was negligible. In addition to their superoxide dismutase-like activity, CuSO4 and the copper complexes also behaved as Fenton-type catalysts as seen by the accumulation of varying amounts of the hydroxyl spin adduct DMPO-OH. Both the Fenton-type catalysis and the superoxide dismutase-like action of these compounds were lost when a chelator such as EDTA was included in the xanthine-xanthine oxidase incubation mixture. Addition of superoxide dismutase instead of the copper compounds to this enzyme system abolished the formation of superoxide adduct DMPO-OOH, and no hydroxyl adduct DMPO-OH was detected. This effect of superoxide dismutase remained unaltered by EDTA.     

Permeability of bilayer lipid membranes for superoxide (O2-.) radicals

Egg yolk phosphatidylcholine monolamellar liposomes (1000 A in diameter) loaded with cytochrome c were placed into an external solution, in which superoxide radicals, O2-., were generated by a xanthine-xanthine oxidase system. The penetration of the superoxide radicals across the liposomal membrane was detected by cytochrome c reduction in the inner liposome compartment. The effects of modifiers and temperature on this process were studied. The permeability of liposomal membrane for O2-. (P'O-2 = (7.6 +/- 0.3) X 10(-8) cm/s), or HO.2 (P'HO.2 = 4.9 X 10(-4) cm/s) were determined. The effect of the transmembrane electric potential (K+ concentration gradient, valinomycin) on the permeability of liposomal membranes for O2-. were investigated. It was found that O2-. can penetrate across liposomal membrane in an uncharged form. The feasibility of penetration of superoxide radicals through liposomal membrane, predominantly via anionic channels, was demonstrated by the use of an intramolecular cholesterol-amphotericin B complex.     

Enzymic formation of glycolate in Chromatium. Role of superoxide radical in a transketolase-type mechanism.

Chromatophores prepared from Chromatium exhibit a light-dependent O2 uptake in the presence of reduced 2,6-dichlorophenolindophenol, the maximum rate observed being 10.8 micronmol (mg of Bchl)-1 h-1 (air-saturated condition). As it was found that the uptake of O2 was markedly inhibited by superoxide dismutase, it is suggested that molecular oxygen is subject to light-dependent monovalent reduction, resulting in the formation of the superoxide anion radical (O2-). By coupling baker's yeast transketolase with illuminated chromatophore preparations, it was demonstrated that [U-14C]-fructose 6-phosphate (6-P) is oxidatively split to produce glycolate, and that the reaction was markedly inhibited by superoxide dismutase and less strongly by catalase. A coupled system containing yeast transketolase and xanthine plus xanthine oxidase showed a similar oxidative formation of glycolate from [U-14C] fructose 6-P. It is thus suggested that photogenerated O2- serves as an oxidant in the transketolase-catalyzed formation of glycolate from the alpha, beta-dihydroxyethyl (C2) thiamine pyrophosphate complex, whereas H2O2 is not an efficient oxidant. The rate of glycolate formation in vitro utilizing O2- does not account for the in vivo rate of glycolate photosynthesis in Chromatium cells exposed to an O2 atmosphere (10 micronmol (mg of Bchl)-1 h-1). However, the enhancement of glycolate formation by the autoxidizable electron acceptor methyl viologen in Chromatium cells in O2, as well as the strong suppression by 1,2-dihydroxybenzene-3,5-disulfonic acid (Tiron), an O2- scavenger, suggest that O2- is involved in the light-dependent formation of glycolate in vivo.     

Naloxone inhibits superoxide release from human neutrophils

Using the superoxide dismutase inhibitable reduction of cytochrome c assay, we studied, the effect of (-) naloxone on N-formyl-methionyl-leucyl-phenylalanine (FMLP) stimulated superoxide (O2-) release from human neutrophils. Neutrophils were pre-incubated with the range of concentrations of (-) naloxone that is administered in models of experimental sepsis (10(-6) - 10(-4.5) M). (-) Naloxone inhibited O2- release in a dose dependent manner. 02- produced by a cell-free xanthine-xanthine oxidase system was not inhibited by (-) naloxone, indicating that (-) naloxone was not scavanging O2-. There was no difference between the effect of (-) and (+) naloxone suggesting that the inhibition of O2- was not specific for an opiate receptor. Another opiate antagonist, nalorphine, as well as the opiate agonist, morphine, also inhibited O2- release in the same concentration range. There was no difference between the effect of naloxone and morphine.     

In vitro scavenging activity for reactive oxygen species by N-substituted indole-2-carboxylic acid esters.

The hydroxyl radical (HO*)- and superoxide anion radical (O* (2))-scavenging activity, as well as the singlet oxygen ((1)O(2))-quenching property of N-substituted indole-2-carboxylic acid esters (INDs) were investigated by deoxyribose degradation assay, a chemiluminescence method and the electron spin resonance (ESR) spin-trapping technique. This novel group of compounds was developed as a search for cyclooxygenase-2 (COX-2)-selective enzyme inhibitors. The results obtained demonstrated that of the 16 compounds examined, five inhibited light emission from the superoxide anion radical (O* (2))-DMSO system by at least 60% at a concentration of 1 mmol/L, nine prevented the degradation of deoxyribose induced by the Fenton reaction system (range 3-78%) or scavenged hydroxyl radicals (HO*) directly (range 8-93%) and 14 showed the (1)O(2)-quenching effect (range 10-74%). These results indicate that majority of the indole esters tested possess the ability to scavenge O(-) (2) and HO radicals and to quench (1)O(2) directly, and consequently may be considered effective antioxidative agents.     

Hemoglobin autoxidation and regulation of endogenous H2O2 levels in erythrocytes

Red cells from mice deficient in glutathione peroxidase-1 were used to estimate the hemoglobin autoxidation rate and the endogenous level of H2O2 and superoxide. Methemoglobin and the rate of catalase inactivation by 3-amino-2,4,5-triazole (3-AT) were determined. In contrast with iodoacetamide-treated red cells, catalase was not inactivated by 3-AT in glutathione peroxidase-deficient erythrocytes. Kinetic models incorporating reactions known to involve H2O2 and superoxide in the erythrocyte were used to estimate H2O2, superoxide, and methemoglobin levels. The experimental data could not be modeled unless the intraerythrocytic concentration of Compound I is very low. Two additional models were tested. In one, it was assumed that a rearranged Compound I, termed Compound II*, does not react with 3-AT. However, experiments with an NADPH-generating system provided evidence that this mechanism does not occur. A second model that explicitly includes peroxiredoxin II can fit the experimental findings. Insertion of the data into the model predicted a hemoglobin autoxidation rate constant of 4.5 x 10(-7) s(-1) and an endogenous H2O2 and superoxide concentrations of 5 x 10(-11) and 5 x 10(-13) M, respectively, lower than previous estimates.     

臭氧浓度升高对油松抗氧化系统活性的影响

以生长在开顶箱内的油松为试材,对高浓度臭氧(80 nmol·mol^-1)条件下油松(Pinus tabulaeformis)针叶中超氧阴离子自由基(O₂^·)产生速率、过氧化氢(H₂O₂)含量、超氧化物歧化酶(SOD)、抗坏血酸过氧化物酶、脱氢抗坏血酸还原酶、单脱氢抗坏血酸还原酶、谷胱甘肽还原酶活性与抗坏血酸(ASA)含量进行测定.结果表明:高浓度臭氧使O₂^·产生速率提高,H₂O₂ 和MDA含量增加.ASA含量与SOD、抗坏血酸过氧化物酶、脱氢抗坏血酸还原酶、单脱氢抗坏血酸还原酶、谷胱甘肽还原酶活性在高浓度臭氧熏蒸的前期升高,随后下降并低于对照.说明生长季前期,油松抗氧化系统对高浓度臭氧存在适应性反应,但不能抵抗长期臭氧胁迫带来的氧化伤害.     

Permeability of bilayer phospholipid membranes to superoxide oxygen radicals

Lecithin monolayer liposomes (1000 A in diameter) loaded with cytochrome c were placed into the external solution, in which O2 superoxide radicals were regenerated by the xanthine-xanthine oxidase system. The penetration of superoxide radicals across the liposomal membranes was followed by cytochrome c reduction in the interval volume of the liposomes. The effects of lipid membrane modifiers and temperature on this process were investigated. The results obtained were used for calculation of the permeability coefficients of bilayer lipid membranes for O(2) (P'O(2) = (7.6 +/- 0.3) . 10(-8) cm . s-1) or HO . 2(P'HO(2) = 4.9 x 10(-4) cm . s-1). The effect of the transmembrane electric potential (concentration gradient of H+, valinomycin) on the permeability of liposomal membranes for the superoxide radical was studied. The superoxide radical was down to penetrate across the bilayer lipid membranes in an unloaded state. Using an intramolecular cholesterol-amphotericin B-complex, the superoxide radicals were shown to penetrate across the bilayer lipid membranes, predominantly via the anionic channels.     

Carboxymethylcellulose–gelatin–superoxidase dismutase electrode for amperometric superoxide radical sensing

A novel, highly sensitive superoxide dismutase biosensor for the direct and simultaneous determination of superoxide radicals was developed by immobilization of superoxide dismutase within carboxymethylcellulose-gelatin on a Pt electrode surface. The parameters affecting the performance of the biosensor were investigated. The response of the CMC-G-SOD biosensor was proportional to O (2) (·-) concentration and the detection limit was 1.25 × 10(-3) mM with a correlation coefficient of 0.9994. The developed biosensor exhibited high analytical performance with wider linear range, high sensitivity and low response time. The biosensor retained 89.8% of its sensitivity after use for 80 days. The support system enhanced the immobilization of superoxide dismutase and promoted the electron transfer of superoxide dismutase minimizing its fouling effect. The biosensor was quite effective not only in detecting O (2) (·-) , but also in determining the antioxidant properties of acetylsalicylic acid-based drugs and the anti-radical activity of healthy and cancerous human brain tissues.     

Superoxide and hydrogen peroxide formation during enzymatic oxidation of DOPA by phenoloxidase

Generation of superoxide anion and hydrogen peroxide during enzymatic oxidation of 3-(3,4-dihydroxyphenyl)-DL-alanine (DOPA) has been studied. The ability of DOPA to react with O2*- has been revealed. EPR spectrum of DOPA-semiquinone formed upon oxidation of DOPA by O2*- was observed using spin stabilization technique of ortho-semiquinones by Zn2+ ions. Simultaneously, the oxidation of DOPA by O2*- was found to produce hydrogen peroxide (H2O2). The analysis of H2O2 formation upon oxidation of DOPA by O2*- using 1-hydroxy-3-carboxy-pyrrolidine (CP-H), and SOD as competitive reagents for superoxide provides consistent values of the rate constant for the reaction between DOPA and O2*- being equal to (3.4+/-0.6)x10(5) M(-1) s(-1).The formation of H2O2 during enzymatic oxidation of DOPA by phenoloxidase (PO) has been shown. The H2O2 production was found to be SOD-sensitive. The inhibition of H2O2 production by SOD was about 25% indicating that H2O2 is produced both from superoxide anion and via two-electron reduction of oxygen at the enzyme. The attempts to detect superoxide production during enzymatic oxidation of DOPA using a number of spin traps failed apparently due to high value of the rate constant for DOPA interaction with O2*-.     

Effect of free-radical scavengers on enumeration of thermally stressed cells of Staphylococcus aureus MF-31.

Exposure of crude cell lysates of Staphylococcus aureus MF-31 to 5 or 10 mM hydrogen peroxide resulted in a linear decrease in superoxide dismutase activity. Approximately 13% of the superoxide dismutase activity was lost after 16 min. Thermally stressed and nonstressed cells were exposed to a photochemically generated exogenous flux of superoxide radicals (O2.-). The death of thermally stressed cells was linear with time. Addition of superoxide dismutase or catalase to the O2.- generating system resulted in protection of thermally stressed and nonstressed cells, with the protective effect being greater for thermally stressed cells. Incorporation of O2-, hydroxyl radical, or singlet oxygen scavengers or antioxidants to tryptic soy agar containing 7.5% NaCl did not increase the enumeration of thermally stressed cells.     

1,2-Di-O-α-linolenoyl-3-O-β-galactosyl-sn-glycerol as a superoxide generation inhibitor from Perilla frutescens var. crispa

Using a superoxide (O(2)(-)) generation assay system with differentiated HL-60 cells, 1,2-di-O-α-linolenoyl-3-O-β-galactosyl-sn-glycerol (DLGG) was identified as an O(2)(-) generation inhibitor from Perilla frutescens var. crispa (a local variety, kida-chirimen shiso). DLGG suppressed the O(2)(-) level in a dose-dependent manner with an IC(50) value of 21 μM, comparable to those of rosmarinic acid (RoA, IC(50) = 29 μM) and caffeic acid (CA, IC(50) = 30 μM). While RoA and CA also dose-dependently inhibited O(2)(-) generation in a xanthine-xanthine oxidase system, DLGG had no effect in the same system. Thus DLGG appeared to decrease the O(2)(-) level in the HL-60 assay system by mechanisms different from those of RoA and CA, which appeared to act as O(2)(-) scavengers.     

Sustained Photoproduction of Ammonia from Nitrate by Anacystis nidulans

Exposure of crude cell lysates of Staphylococcus aureus MF-31 to 5 or 10 mM hydrogen peroxide resulted in a linear decrease in superoxide dismutase activity. Approximately 13% of the superoxide dismutase activity was lost after 16 min. Thermally stressed and nonstressed cells were exposed to a photochemically generated exogenous flux of superoxide radicals (O2.-). The death of thermally stressed cells was linear with time. Addition of superoxide dismutase or catalase to the O2.- generating system resulted in protection of thermally stressed and nonstressed cells, with the protective effect being greater for thermally stressed cells. Incorporation of O2-, hydroxyl radical, or singlet oxygen scavengers or antioxidants to tryptic soy agar containing 7.5% NaCl did not increase the enumeration of thermally stressed cells.