Quantitative study of natural antioxidant systems for cellular nitrofurantoin toxicity

The toxicity of nitrofurantoin was studied on human WI-38 fibroblasts: this chemical was lethal when added at concentrations higher than 5·10⁻⁵ M in the culture medium. The protection afforded by anitoxidants was then tested: α-tocopherol gave at 10⁻⁴ M a light protection in contrast to ascorbic acid which even became toxic at high concentrations. We also tested catalase, superoxide dismutase and glutathione peroxidase introduced intracellularly by the microinjection technique. On a molecular basis, glutathione peroxidase was 23-times more efficient than catalase and 3000-times more than superoxide dismutase. The results also showed that a similar range of enzyme concentrations was found for the protection against high oxygen pressure. This suggests that, in the case of both oxygen and nitrofurantoin toxicity, the peroxide derivatives are the most toxic intermediates of the free radical attacks.     

Oxidation of ascorbic acid with superoxide anion generated by the xanthine-xanthine oxidase system.

Ascorbic acid was found to be oxidized by O₂^$\text{?}$ which was generated by the xanthine-xanthine oxidase system. From a kinetic analysis of the inhibition of this reaction by superoxide dismutase, the second-order rate constant for the reaction between ascorbic acid and O₂^? at pH 7.4 was estimated to be 2.7 × 10⁵ M^?1 sec^?1. A function of ascorbic acid as a defense against O₂^? is presented.     

Formation of hydrogen peroxide by isolated cell walls from horseradish (Armoracia lapathifolia Gilib.)

Summary Isolated cell-wall suspensions from horseradish in the presence of 5×10^-4 M MnCl₂ catalyze the production of hydrogen peroxide at the expense of either NADPH or NADH. This reaction is inhibited by scavengers of the superoxide free radical ion such as ascorbate or dihydroxyphenols or by superoxide dismutase, and stimulated by monophenols such as p-coumaric acid. On comparison with isolated (commercial) horseradish peroxidase it becomes evident that (a) cell-wall-bound peroxidase(s) is (are) responsible for the production of hydrogenperoxide, involving the superoxide free radical ion as an intermediate of the complex reaction chain.Abbreviation SOD superoxide dismutase     

紫色红曲霉对沙棘青稞复合酵素性能的影响研究

【目的】为了对比分析紫色红曲霉是否对沙棘青稞酵素具有促进作用。【方法】以对照组、沙棘组、沙青组、沙青红曲组4种发酵液为研究对象,对其pH、总糖、总酸、可溶性固形物、总酚、总黄酮、抗坏血酸和洛伐他汀、超氧化物歧化酶、脂肪酶和蛋白酶含量、1,1-二苯基-2-苦苯肼自由基清除能力、2,2ʹ-联氮双(3-乙基苯并噻唑啉-6-磺酸)阳离子自由基清除能力、酵母菌活菌数和乳酸菌活菌数进行比较分析。【结果】沙青红曲组的总酚、总黄酮、抗坏血酸和洛伐他汀、超氧化物歧化酶、脂肪酶和蛋白酶含量、1,1-二苯基-2-苦苯肼自由基清除率、2,2ʹ-联氮双(3-乙基苯并噻唑啉-6-磺酸)阳离子自由基清除率、酵母菌活菌数和乳酸菌活菌数均显著高于沙棘组和沙青组(P<0.05)。【结论】该研究证明了紫色红曲霉的添加可提高酵素的性能,对酵素行业的多元化发展有潜在的促进作用。     

Pentoxifylline

Pentoxifylline (PTX), a tri-substituted purine and xanthine derivative, has been used for several years to improve microcirculation because of its hemorheological properties. PTX has also antifibrotic and anti-inflammatory effects. We studied the reaction of PTX with the hydroxyl radical and superoxide anion. Hydroxyl radical was generated by a mixture of ascorbic acid, H₂O₂ and Fe (III)-EDTA. We evaluated the iron-dependent degradation of deoxyribose, mediated by hydroxyl radical, in the presence of different concentrations of PTX (from 0.05 to 3 mM), measuring the degradation products of deoxyribose that react with 2-thiobarbituric acid (TBA). The reaction of PTX with hydroxyl radical occurred with a rate constant of (1.1±0.2)×10¹⁰ M ⁻¹/s. These results support the properties of PTX as a hydroxyl radical scavenger. Some authors verified that PTX decreases the release of superoxide anion from activated neutrophils. We studied the effect of PTX as a scavenger of superoxide generated in vitro by a hypoxanthine-xanthine oxidase system. PTX was not a superoxide anion scavenger in this system.     

EPR kinetic studies of superoxide radicals generated during the autoxidation of 1-methyl-4-phenyl-2,3-dihydropyridinium, a bioactivated intermediate of parkinsonian-inducing neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.

1-Methyl-4-phenyl-2,3-dihydropyridinium (MPDP+), a metabolic product of the nigrostriatal toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), has been shown to generate superoxide radicals during its autoxidation process. The generation of superoxide radicals was detected as a 5,5-dimethyl-1-pyrroline-N-oxide (DMPO).O2- spin adduct by spin trapping in combination with EPR techniques. The rate of formation of spin adduct was dependent not only on the concentrations of MPDP+ and oxygen but also on the pH of the system. Superoxide dismutase inhibited the spin adduct formation in a dose-dependent manner. The ability of DMPO to trap superoxide radicals, generated during the autoxidation of MPDP+, and of superoxide dismutase to effectively compete with this reaction for the available O2-, has been used as a convenient competition reaction to quantitatively determine various kinetic parameters. Thus, using this technique the rate constant for scavenging of superoxide radical by superoxide dismutase was found to be 7.56 x 10(9) M-1 s-1. The maximum rate of superoxide generation at a fixed spin trap concentration using different amounts of MPDP+ was found to be 4.48 x 10(-10) M s-1. The rate constant (K1) for MPDP+ making superoxide radical was found to be 3.97 x 10(-6) s-1. The secondary order rate constant (KDMPO) for DMPO-trapping superoxide radicals was found to be 10.2 M-1 s-1. The lifetime of superoxide radical at pH 10.0 was calculated to be 1.25 s. These values are in close agreement to the published values obtained using different experimental techniques. These results indicate that superoxide radicals are produced during spontaneous oxidation of MPDP+ and that EPR spin trapping can be used to determine the rate constants and lifetime of free radicals generated in aqueous solutions. It appears likely that the nigrostriatal toxicity of MPTP/MPDP+ leading to Parkinson's disease may largely be due to the reactivity of these radicals.     

Formation of α-tocopherol radical and recycling of α-tocopherol by ascorbate during peroxidation of phosphatidylcholine liposomes: An electron paramagnetic resonance study

The events accompanying the inhibitory effect of α-tocopherol and/or ascorbate on the peroxidation of soybean L-α-phosphatidylcholine liposomes, which are an accepted model of biological membranes, were investigated by electron paramagnetic resonance, optical and polarograpic methods. The presence of α-tocopherol radical in the concentration range 10^?8–10^?7 M was detected from its EPR spectrum during the peroxidation of liposomes, catalysed by the Fe³⁺-triethylnetatramine complex. The α-tocopherol radical, generated in the phosphatidylcholine bilayer, is accessible to ascorbic acid, present in the aqueous phase at physiological concentrations. Ascorbic acid regenerates from it the α-tocopherol itself. A kinetic rate constant of about 2·10⁵ M^?·s^?1 was estimated from the reaction as it occurs under the adopted experimental conditions. The scavenging effect of α-tocopherol on lipid peroxidation is maintained as long a ascorbic acid is present.     

Dehydroascorbate reduction

Dehydroascorbic acid is generated in plants and animal cells by oxidation of ascorbic acid. The reaction is believed to occur by the one-electron oxidation of ascorbic acid to semidehydroascorbate radical followed by disproportionation to dehydroascorbic acid and ascorbic acid. Semidehydroascorbic acid may recycle to ascorbic acid catalyzed by membrane-bound NADH-semidehydroscorbate reductase. However, disproportionation of the free radical occurs at a rapid rate, 10⁵ M^–1 s^–1, accounting for measurable cellular levels of dehydroascorbate. Dehydroascorbate reductase, studied earlier and more extensively in plants, is now recognized as the intrinsic activity of thioltransferases (glutaredoxins) and protein disulfide isomerase in animal cells. These enzymes catalyze the glutathione-dependent two-electron regeneration of ascorbic acid. The importance of the latter route of ascorbic acid renewal was seen in studies of GSH-deficient rodents (Meister, A. (1992)Biochem. Pharmacol. 44 1905–1915). GSH deficiency in newborn animals resulted in decreased tissue ascorbic acid and increased dehydroascorbate-to-ascorbate ratios. Administration of ascorbic acid daily to GSH-deficient animals decreased animal mortality and cell damage from oxygen stress. A cellular role is proposed for dehydroascorbate in the oxidation of nascent protein dithiols to disulfides catalyzed in the endoplasmic reticulum compartment by protein disulfide isomerase.     

Adriamycin-induced leakage of lysosomal enzymes in vitro

Chronic treatment of rats with adriamycin has been shown to affect myocardial lysosomes as well as enzyme activities in the serum fraction. In this study, we examined in vitro effects of adriamycin (10^–6 to 10^–3 M) on the lysosomal fraction isolated from rat ventricular tissue. Morphological examination revealed that the isolated fraction was mainly vesicular in nature. Higher concentrations of adriamycin (10^–3 M) caused a significant loss of acid phosphatase and N-acetyl-B-d-glucosaminidase activity from the lyosomal vesicles. The enzyme leakage was not accompanied by any intravesicular localization of lanthanum, an extravesicular electron dense tracer. Preincubation of lysosomal vesicles with 10 g/ml superoxide dismutase did not protect against adriamycin-induced loss of lysosomal enzymes. The study shows that adriamycin induces loss of lysosomal enzymes in vitro and the superoxide radical may not be involved in this change.     

Lucigenin chemiluminescence in human seminal plasma

Seminal plasma protects spermatozoa from the detrimental effects of reactive oxygen species such as hydrogen peroxide. We investigated the lucigenin-dependent chemiluminescence in cell-free seminal plasma from andrological patients. The seminal plasma was separated from cells by centrifugation. In all seminal plasmas studied lucigenin-dependent chemiluminescence (LCL) was detected. The LCL showed a strong pH-dependence. The signal was stable if samples were stored at +4°C for up to 4 days or up to 8 days at -80°C. Filtration of the samples (0.45 and 0.22 μm pore size) did not lower their luminescence. The addition of superoxide dismutase (SOD) and ascorbic acid oxidase (AAO) lowered LCL nearly to baseline values while trolox and desferal showed moderate effect, whereas allopurinol had no effect. Electron paramagnetic resonance spectroscopy demonstrated ascorbyl radicals in seminal plasma. Physiological concentrations of ascorbic acid yielded SOD-inhibitable lucigenin-chemiluminescence. The nitroblue-tetrazolium assay showed that ascorbic acid in buffer solution produced formazan. Superoxide-anion radicals were not detected in seminal plasma by the spin-trap DEPMPO due to their low steady state concentration. It is concluded that in seminal plasma ascorbate reacts with molecular oxygen yielding ascorbyl radicals and superoxide anion. If lucigenin is added to seminal plasma, reducing substances present, such as ascorbate, reduce lucigenin to the corresponding radical; this radical reacts with molecular oxygen and also forms O₂^-2. So LCL in human seminal plasma results from the autoxidation of ascorbate and the oxidation of the reduced lucigenin. While the physiological relevance of the former mechanism is unknown, the latter is an artifact.     

The in vivo and in vitro influence of methyl jasmonate on oxidative processes in Arabidopsis thaliana leaves

In Arabidopsis thaliana leaves a strong increase of H₂O₂ content was induced by application of methyl jasmonate (JAMe) through the root system, but the induction only slightly depended on JAMe concentration. The activity of superoxide dismutase and ascorbic acid peroxidase increased at lower JAMe concentrations and decreased at higher ones. Catalase activity decreased proportionally to JAMe concentration (in comparison with control plants). The sum of ascorbic acid and dehydroascorbate content at 10⁻⁶ M JAMe was similar to the control, but at higher concentrations it increased, especially due to a higher ascorbate accumulation. Methyl jasmonate applied directly to the extract of leaves (in vitro experiment) also induced a strong increase in H₂O₂ level, even at a low concentration (10⁻⁸ M). Since lower JAMe concentrations induced weak superoxide dismutase and did not change catalase and peroxidase activity, it is suggested that in this case a high level of hydrogen peroxide was not the result of the activity of the mentioned enzymes. JAMe-induction of H₂O₂ increase at the highest JAMe concentration resulted from SOD activity. Our in vivo and in vitro experiments suggest that jasmonate can influence oxidative stress not only through gene expression but also by its direct effect on enzyme activity.     

Suicide Inactivation of Catechol 2,3-Dioxygenase from Pseudomonas putida mt-2 by 3-Halocatechols

The inactivation of catechol 2,3-dioxygenase from Pseudomonas putida mt-2 by 3-chloro- and 3-fluorocatechol and the iron-chelating agent Tiron (catechol-3,5-disulfonate) was studied. Whereas inactivation by Tiron is an oxygen-independent and mostly reversible process, inactivation by the 3-halocatechols was only observed in the presence of oxygen and was largely irreversible. The rate constants for inactivation (K₂) were 1.62 × 10⁻³ sec⁻¹ for 3-chlorocatechol and 2.38 × 10⁻³ sec⁻¹ for 3-fluorocatechol. The inhibitor constants (K_i) were 23 μM for 3-chlorocatechol and 17 μM for 3-fluorocatechol. The kinetic data for 3-fluorocatechol could only be obtained in the presence of 2-mercaptoethanol. Besides inactivated enzyme, some 2-hydroxyhexa-2,4-diendioic acid was formed from 3-chlorocatechol, suggesting 5-chloroformyl-2-hydroxypenta-2,4-dienoic acid as the actual suicide product of meta-cleavage. A side product of 3-fluorocatechol cleavage is a yellow compound with the spectral characteristics of a 2-hydroxy-6-oxohexa-2,4-dienoic acid indicating 1,6-cleavage. Rates of inactivation by 3-fluorocatechol were reduced in the presence of superoxide dismutase, catalase, formate, and mannitol, which implies that superoxide anion, hydrogen peroxide, and hydroxyl radical exhibit additional inactivation.     

Stimulation of transepithelial sodium and chloride transport by ascorbic acid. Induction of Na+ channels is inhibited by amiloride

Ascorbic acid increases the short circuit current (I_sc) across the amphibian cornea when it is present at either surface of this epithelium. These effects were additive. The effect was greater when it was on the tear side. The response returned to baseline levels when the ascorbic acid was washed from the bathing media. The effect of ascorbic acid on I_sc when it was on the aqueous humor side of the cornea could be blocked by bumetanide but that due to the vitamin's presence on the tear side was unchanged. The ascorbic acid could enter the tissue and crossed the cornea at similar rates in either direction. When the cornea was bathed by a Cl^?-free solution or exposed to bumetanide, the rise in I_sc observed with ascorbic acid on the tear side was equivalent to an increased Na⁺ flux from the tear to the aqueous humor side. In normal (Cl^? present) Conway solution the rise in the I_sc seen with ascorbic acid on the aqueous humor side was equal to an increased flux of Cl^? from the aqueous to the tear surface. However, when ascorbic acid was present on the opposite, tear, side the increased I_sc reflected a rise in both Cl^? and Na⁺ transport, aqueous-to-tear side, and tear-to-aqueous side, respectively. Thiol reagents (tear side), including reduced glutathione (10^?5 M), blocked the effect of ascorbic acid (10^?3 M) providing they were added to the bathing solution prior to the vitamin. However, they had no effect once the response had been established. The effect of the reduced glutathione appeared to be of a non-competitive nature. Oxidized glutatione (10^?4 M) (and cystamine) blocked the effect of ascorbic acid (10^?3 M) when present on the tear side prior to the vitamin. However, they also increased the rate of decline of the response when added subsequently to the ascorbic acid. Amiloride (as low as 5·10^?9 M), on the tear side but not the aqueous humor side, prevented the response to ascorbic acid but could not reverse it, once it was established. The possible nature of the effect of ascorbic acid is discussed in relation to its pharmacological interactions with thiol and disulfide reagents and amiloride.     

Inhibition of Lipid Peroxidation by Dihydroquinoline-Type Antioxidant (CH 402)

The in vitro effect of a non-toxic, water soluble, low molecular weight, stable dihydroquinoline-type antioxidant, CH 402 (Na (2,2-dimethyl-1,2-dihydroquinoline-4-yl) –- methane sulphonic acid) was studied on free radical reactions in brain subcellular fractions. Experiments were performed using rat and mouse brain homogenate and microsomal fractions. Non … enzymatically induced lipid peroxidation by ascorbic acid was studied in correlation with ascorbic acid and CH 402 concentrations and incubation time. Malondialdehyde production during lipid peroxidation was measured by the thiobarbituric acid test. In a concentration range of 10^?2–10^?5 M CH 402 dose - dependently inhibited the ascorbic acid induced in vitro lipid peroxidation in mouse and rat brain subcellular fractions.     

Pro-oxidant and antioxidant processes in gas gland and other tissues of cod (Gadus morhua)

Partial reduction of molecular oxygen produces reactive oxyradicals, including the superoxide anion radical (O _- ² ) and hydroxyl radical (·OH). The gas gland functions under hyperoxic and acidic conditions and therefore is likely to be subjected to enhanced oxidative stress. Aspects of pro- and antioxidant processes in gas gland were compared with other tissues likely to be subject to differing degrees of oxyradical production, viz. liver (site of chemically-mediated oxyradical production), gills and skeletal muscle. Antioxidant enzyme activities (superoxide dismutase, catalase, selenium-dependent and total glutathione peroxidase) per g wet weight were highest in liver and lowest in muscle. Catalase and glutathione peroxidase activies per g wet weight were higher in gills than in gas gland, whereas the reverse was seen for superoxide dismutase. Cytosolic superoxide dismutase activities per mg protein were two- and nine-fold higher in gas gland than in liver and gills. The pH characteristics of the antioxidant enzymes were generally similar in all the tissues. Glutathione, vitamin E and unsaturated (peroxidizable) lipid levels were generally highest in liver followed by gas gland. Lipid peroxidation (malonaldehyde equivalents) was evident in all tissues except gas gland. Hydrogen peroxide and O _- ² were involved in the NAD(P)H-dependent ferric/EDTA-mediated formation of ·OH (as measured by 2-keto-4-methiolbutyrate oxidation) by mitochondrial and postmitochondrial fractions of gas gland. Tissue maximal potentials for ·OH production paralled superoxide dismutase but not catalase or glutathione peroxidase activities. Overall, the results confirm the presence of effective antioxidant defences in gas gland and support previous workers' contentions of a central role for superoxide dismutase in this process.Abbreviations EDTA di-sodium ethylenediaminetetra-acetic acid - G-6-P glucose-6-phosphate - GPX total glutathione peroxidase - GSH reduced glutathione - GSSG oxidised glutathione - GST glutathion-S-transferase - HPLC high performance liquid chromatography - KMBA 2-keto-4-methiolbutyric acid - MOPS 3-[N-morpholino] propane-sulphonic acid - PMS postmitochondrial supernatant - Se-GPX selenium-dependent glutathion peroxidase - SOD superoxide dismutase - TCA trichloroacetic acid     

Chilling Induced Oxidative Stress in Germinating Wheat Grains as Affected by Water Stress and Calcium

Wheat (Triticum aestivum L.) plants were subjected to mild water stress during grain filling at milk (early, medium, and late) and dough (early, soft, hard) stages. The grains harvested from stressed plants were subjected to low temperature stress of 10 °C for 24 h in presence or absence of 1 mM CaCl₂, and embryos were examined for oxidative injury. The embryos of grains water stressed at milk and soft dough stages showed lowest contents of H₂O₂ and malondialdehyde and highest membrane stability index, ascorbic acid content, and activities of catalase, ascorbate peroxidase, and superoxide dismutase as compared to control embryos or water-stressed at other stages. Presence of Ca²⁺ in the medium reduced H₂O₂ and malondialdehyde content and increased ascorbic acid content, and catalase, ascorbate peroxidase and superoxide dismutase activities.     

Quantitative study of natural antioxidant systems for cellular nitrofurantoin toxicity

The toxicity of nitrofurantoin was studied on human WI-38 fibroblasts: this chemical was lethal when added at concentrations higher than 5.10(-5) M in the culture medium. The protection afforded by antioxidants was then tested: alpha-tocopherol gave at 10(-4) M a light protection in contrast to ascorbic acid which even became toxic at high concentrations. We also tested catalase, superoxide dismutase and glutathione peroxidase introduced intracellularly by the microinjection technique. On a molecular basis, glutathione peroxidase was 23-times more efficient than catalase and 3000-times more than superoxide dismutase. The results also showed that a similar range of enzyme concentrations was found for the protection against high oxygen pressure. This suggests that, in the case of both oxygen and nitrofurantoin toxicity, the peroxide derivatives are the most toxic intermediates of the free radical attacks.     

Electron paramagnetic resonance spin trapping investigation into the kinetics of glutathione oxidation by the superoxide radical: re-evaluation of the rate constant

The ability of glutathione to scavenge the superoxide radical is a matter of serious contention in the literature: reported values for the second-order rate constant range from 10(2) to greater than 10(5) M(-1) s(-1). The physiological implications of this discrepancy will determine, for example, whether or not glutathione can compete with Mn-superoxide dismutase for reaction with the radical in the mitochondrial matrix, leading to formation of the potentially harmful glutathionyl radical. Several authors have investigated the kinetics of glutathione oxidation by superoxide using spectrophotometric assays, based on competition between either ferricytochrome c or epinephrine for reaction with the radical. However, these approaches have received criticism because the contributions of various secondary reactions to the overall kinetics have been largely overlooked (e.g., the reduction of ferricytochrome c by glutathione). In the present investigation, we have used electron paramagnetic resonance spectroscopy to monitor competition between GSH and the spin trap 5,5-dimethyl-1-pyrroline N-oxide for reaction with superoxide. This method has been used previously and a rate constant of 1.8 x 10(5) M(-1) s(-1) obtained (Dikalov, S.; Khramtsov, V.; Zimmer, G. Arch. Biochem. Biophys. 326:207-218; 1996). However, we demonstrate that this value is a gross overestimation because the spectrum of the hydroxyl radical adduct of the spin trap was incorrectly assigned to the glutathionyl radical adduct. The relatively high yield of the DMPO hydroxyl radical adduct is shown to be due to the two-electron reduction of the corresponding superoxide radical adduct by glutathione. Taking these factors into consideration, we estimate the second order rate constant for the oxidation of glutathione by superoxide to be approximately 200 M(-1) s(-1).     

Ascorbic acid uptake in guinea pig intestinal mucosa

Cellular accumulation of ascorbic acid was investigated in vitro in distal intestinal mucosa of guinea pig. With ¹⁴C-ascorbic acid present at 8 μM/L in the bathing media, tissue/media (T/M) concentration ratios of at least 5 were routinely achieved. Recently absorbed ascorbic acid appeared to be free in solution in the cellular fluid in that it diffused from tissue exposed to poisons with a disappearance half-time of approximately 10 minutes. Ascorbic acid uptake was highly dependent on the presence of sodium in the bathing media; total Tris substitution resulted in a 97% decrease in uptake. Also, metabolically depleted tissue did not accumulate ascorbic acid against a concentration gradient. Uptake of ¹⁴C-ascorbic acid from a bathing solution concentration of 8 μM/L was reduced 67% in the presence of 0.8 mM/L nonlabeled ascorbic acid. Recently absorbed ¹⁴C-ascorbic acid moved more rapidly back into the lumen when the luminal solution contained nonlabeled ascorbic acid (5 mM) than when it contained mannitol (5mM). This demonstration of counter transport substantiates a carrier mechanism in the brush border.