Intracellular antioxidants: from chemical to biochemical mechanisms.

Intracellular antioxidants include low molecular weight scavengers of oxidizing species, and enzymes which degrade superoxide and hydroperoxides. Such antioxidants systems prevent the uncontrolled formation of free radicals and activated oxygen species, or inhibit their reactions with biological structures. Hydrophilic scavengers are found in cytosolic, mitochondrial and nuclear compartments. Ascorbate and glutathione scavenge oxidizing free radicals in water by means of one-electron or hydrogen atom transfer. Similarly, ergothioneine scavenges hydroxyl radicals at very high rates, but it acts more specifically as a chemical scavenger of hypervalent ferryl complexes, halogenated oxidants and peroxynitrite-derived nitrating species, and as a physical quencher of singlet oxygen. Hydrophobic scavengers are found in cell membranes where they inhibit or interrupt chain reactions of lipid peroxidation. In animal cells, they include alpha-tocopherol (vitamin E) which is a primary scavenger of lipid peroxyl radicals, and carotenoids which are secondary scavengers of free radicals as well as physical quenchers of singlet oxygen. The main antioxidant enzymes include dismutases such as superoxide dismutases (SOD) and catalases, which do not consume cofactors, and peroxidases such as selenium-dependent glutathione peroxidases (GPx) in animals or ascorbate peroxidases (APx) in plants. The reducing coenzymes of peroxidases, and as a rule all reducing components of the antioxidant network, are regenerated at the expense of NAD(P)H produced in specific metabolic pathways. Synergistic and co-operative interactions of antioxidants rely on the sequential degradation of peroxides and free radicals as well as on mutual protections of enzymes. This antioxidant network can induce metabolic deviations and plays an important role in the regulation of protein expression and/or activity at the transcriptional or post-translational levels. Its biological significance is discussed in terms of environmental adaptations and functional regulations of aerobic cells.

Glutathione peroxidase mimics prevent TNFalpha- and neutrophil-induced endothelial alterations.

Based on the assumption that glutathione peroxidase (GPx) activity might be limiting in preventing peroxide-induced impairment of endothelial regulatory functions, we studied the effect of a series of new selenium-containing GPx mimics on endothelial cells exposed to an inflammatory stress. The two compounds that have the highest GPx activity, BXT-51072 and BXT-51077, were shown to be the most efficient inhibitors of leukocyte recruitment by human umbilical vein endothelial cells (HUVEC), upon incubation with neutrophils (10-fold excess over HUVEC) and with 1 ng/ml TNF-alpha for 1 or 3.5 h. When HUVEC were pre- and cotreated with 10 microM of either compound, neutrophil adhesion and endothelial alteration were markedly inhibited, as assessed by immunoassays of myeloperoxidase and von Willebrand factor, respectively. These two GPx mimics were also found to be the most efficient inhibitors of the TNFalpha-induced endothelial expression of P- and E-selectin and of the TNFalpha- or interleukin1-induced endothelial release of interleukin-8. Our results demonstrate that GPx mimics such as BXT-51072 behave as potent antagonists of TNF-alpha and interleukin-1 through the downregulation of endothelial proinflammatory responses.

ICAM-1 and VCAM-1 expression induced by TNF-alpha are inhibited by a glutathione peroxidase mimic.

Intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) are respectively involved in the endothelial recruitment of neutrophils, and in that of lymphocytes or tumor cells, in response to specific signals. We have used the glutathione peroxidase (GPx) mimic BXT-51072 to assess the possibility that endogenous hydroperoxides play a role in the tumor necrosis factor-alpha (TNFalpha)-induced expression of ICAM-1 and VCAM-1 by monolayers of human endothelial cells. The GPx mimic BXT-51072 strongly inhibits the TNFalpha-induced and cycloheximide-sensitive expression of ICAM-1 and VCAM-1. It also inhibits the TNFalpha-induced reorganization of the actin network and the associated formation of stress fibers. Actin reorganization induced by cytochalasin D treatment did not inhibit ICAM-1 expression. Our results are compatible with specific and synergistic effects of endogenous hydroperoxides on the biosynthesis and processing of cell adhesion molecules and cytoskeleton components.

[Glutathione peroxidases: value of their determination in clinical biology]. / Les glutathion peroxydases: intérêt de leur dosage en biologie clinique.

The role of glutathione peroxidase in the oxidative metabolism and recent advances in the demonstration of the consequences of the desequilibrium in the proxidant/antioxidant balance on biological molecules oxidation, intracellular signals transduction, apoptosis and necrosis, have led to new approach in the knowledge of many pathological processes. Methods for determining antioxidant capacity have been developed. The measurement of glutathione peroxidase activity is a key step in the study of oxidative stress. Its determination in clinical biology needs optimal conditions for standardised assays which will be used for epidemiological studies aimed to evaluate the role of nutritional factors involved in the pathogeny of diseases caused or accompanied by oxidative stress.

[Metabolism and antioxidant function of glutathione]. / Métabolisme et fonction antioxydante du glutathion.

Glutathione (gamma-glutamyl-cysteinyl-glycine or GSH) is a cysteine-containing tripeptide with reducing and nucleophilic properties which play an important role in cellular protection from oxidative damage of lipids, proteins and nucleic acids. GSH regulates the metabolism of proteins and their activities by means of thiol-disulfide exchange. During oxidative stress, GSH plays a key role of protection and detoxification as a cofactor of glutathione peroxidases and glutathione-S-transferases. There are synergistic interactions between GSH and other components of the antioxidant defense system such as vitamin C, vitamin E and superoxide dismutases.

[Pharmacological modulation of alterations of endothelial cytoskeleton induced by hydrogen peroxyde and by TNF-alpha]. / Modulation pharmacologique des altérations du cytosquelette endothélial induites par le péroxyde d'hydrogène et par le TNF-alpha.

New selenium containing compounds which act as mimics of glutathione peroxidase (GPx) protect vascular endothelial cells (HUVEC) from the toxicity of 140 microM hydrogen peroxide. In the absence of GPx mimic, hydrogen peroxide destroys the tightness of the cellular monolayer and transforms the actin network into compact stress fibers. The pre-treatment of the cells by 4 microM of the lead-compound BXT-51072 for 1 hours inhibits the morphological modifications induced by hydrogen peroxide. This GPx mimic can also prevent the alterations of the endothelial cytoskeleton which are induced by Tumor Necrosis Factor-alpha (TNF-alpha) and which consist in a reorganization of actin filaments with the formation of stress fibers. Fluorescent labeling of polymerized actin has been performed by means of phalloidine coupled with rhodamine. The protective effect of this antioxidant catalyst against the toxicity of hydrogen peroxide and TNF-alpha includes the maintenance of a structural configuration of the cytoskeleton which is required for the function of endothelial barrier.

[Antioxidant and anti-inflammatory protection of vascular endothelial cells by new synthetic mimics of glutathione peroxidase]. / Protection antioxydante et anti-inflammatoire des cellules endothéliales vasculaires par de nouveaux mimes synthétiques de la glutathion peroxydase.

New selenium-containing compounds behave as GPx mimics and protect endothelial cells (HUVEC) from damage upon exposure to 55 microM linoleic acid hydroperoxide or to 200 microM hydrogen peroxide. The simultaneous presence of the GPx mimic and the hydroperoxyde is not necessary, since a pre-treatment of endothelial monolayers with 1 to 10 microM of such compounds, preserves their morphology, their cell density and their longer-term viability. The compounds which are most efficient in this model of oxidative stress also protect endothelial monolayers which have been incubated with an excess (10:1) of polymorphonuclear neutrophils (PMN) and with 1 ng/ml of TNF-alpha, if such monolayers are pre- and co-treated (10 microM). They inhibit the adhesion of activated neutrophils which show-up as polymorphous and very dense particles, in the vicinity of which endothelial alterations can be seen. The inhibition of leucocyte adhesion and that of endothelial activation/alteration have been quantified by means of immunoassays of myeloperoxidase and von Willebrand factor (vWf). The lead-compound BXT-51072 is not a direct inhibitor of the NADPH oxidase of PMN. TNF-alpha alone induces the endothelial release of Interleukin-8 (Il-8) as well as the expression of P- and E-selectin. The extent and the kinetics of inhibition of such processes by compound BXT-51072 would explain several of the effects observed in the presence of PMN. The GPx mimics also inhibit the endothelial production of Il-8 which is induced by Interleukin-1 alpha. Finally, compound BXT-51072 inhibits the endothelial expression of the adhesion factor VCAM-1 which is more slowly induced by TNF-alpha. Such antioxidant catalysts therefore protect endothelial cells from the toxic effects of TNF-alpha through mechanisms which include a down-regulation of cytokines and cell-adhesion factors.

Partial prevention of glutathione depletion in rats following acute intoxication with diethylmaleate.

To assess the ability of L-2-oxothiazolidine-4-carboxylate (OTC) to stimulate the biosynthesis of glutathione (GSH) in non-fasted male rats, the time-courses of GSH and cysteine contents were studied in liver, kidney, heart and brain, following a single intraperitoneal injection of OTC (5 mmol/kg), with or without co-administration of the GSH depletor diethylmaleate (3 mmol/kg). In the absence of diethylmaleate, OTC did not change the GSH or cysteine content of heart and kidney. The liver was the only organ where systemic administration of OTC resulted in a fast and quasi-linear increase in GSH as a function of time, with no appreciable lag-time. A maximal, i.e. 2.1-fold increase in liver GSH was induced by OTC at the times corresponding to the low GSH values of the diurnal cycle observed in control rats. A smaller, i.e. 1.4-fold increase in brain GSH was observed after 6 hours. A marked increase in cysteine always preceded that of GSH in liver and brain. In the liver, the OTC-mediated stimulation of GSH biosynthesis was optimal when cysteine delivery was achieved at the onset of the cysteine decrease that was observed in the diurnal cycle of control rats. These results support the view that cysteine is a limiting factor in the biosynthesis of GSH. Following an acute dose of diethylmaleate (3 mmol/kg), OTC afforded a general and significant protection of rat tissues against GSH depletion.(ABSTRACT TRUNCATED AT 250 WORDS)

Glutathione oxidase activity of selenocystamine: a mechanistic study.

Selenocystamine (RSe-SeR) was shown to catalyze the oxygen-mediated oxidation of excess GSH to glutathione disulfide, at neutral pH and ambient PO2. This glutathione oxidase activity required the heterolytic reduction of the diselenide bond, which produced two equivalents of the selenolate derivative selenocysteamine (RSe-), via the transient formation of a selenenylsulfide intermediate (RSe-SG). Formation of RSe- was the only reaction observed in anaerobic conditions. At ambient PO2, the kinetics and stoichiometry of GSSG production as well as that of GSH and oxygen consumptions demonstrated that RSe- performed a three-step reduction of oxygen to water. The first step was a one-electron transfer from RSe- to dioxygen, yielding superoxide and a putative selenyl radical RSe., which decayed very rapidly to RSe-SeR. In the second step, RSe- reduced superoxide to hydrogen peroxide through a much faster one-electron transfer, also associated with the decay of RSe. to RSe-SeR. The third step was a two-electron transfer from RSe- to hydrogen peroxide, again much faster than oxygen reduction, which resulted in the production of RSe-SG, presumably via a selenenic acid intermediate (RSeOH) which was trapped by excess GSH. This third step was studied on exogenous hydroperoxide in anaerobic conditions, and it could be eliminated from the glutathione oxidase cycle in the presence of excess catalase. The role of RSe- as a one- and two-electron reductant was confirmed by competitive carboxymethylation with iodoacetate. RSe- was able to rapidly reduce ferric cytochrome c to its ferrous derivative. The overall rate of catalytic glutathione oxidation was GSH concentration dependent and oxygen concentration independent. Excess glutathione reductase and NADPH increased the catalytic oxidation of GSH, probably by switching the rate-limiting step from selenylsulfide to diselenide cleavage. When GSH was substituted for dithiothreitol, it was shown to reduce RSe-SeR to RSe- in a fast and quantitative reaction, and selenocystamine behaved as a dithiothreitol oxidase, whose catalytic cycle was dependent on oxygen concentration. The oxidase cycle of glutathione was inhibited by mercaptosuccinate, while that of dithiothreitol was not affected. When mercaptosuccinate was substituted for GSH, a stable selenenylsulfide was formed. These observations suggest that electrostatic interactions affect the reductive cleavage of diselenide and selenenylsulfide linkages. This study illustrates the ease of one-electron transfers from RSe- to a variety of reducible substrates. Such free radical mechanisms may explain much of the cytotoxicity of alkylselenols, and they demonstrate that selenocystamine is a poor catalytic model of the enzyme glutathione peroxidase.

Purification and properties of a recombinant sulfur analog of murine selenium-glutathione peroxidase.

We previously constructed plasmids for synthesis of glutathione-peroxidase (GPx) mutants in an Escherichia coli expression system. In these recombinant proteins either cysteine ([Cys]GPx mutant) or serine ([Ser]GPx mutant) were present in place of the active-site selenocysteine (SeCys) of the natural enzyme. We have now investigated GPx activity of [Cys]GPx and [Ser]GPx mutants. Enzyme assays performed on preparations of these partially purified proteins demonstrated that the [Cys]GPx mutant exhibited a significant GPx activity, unlike the [Ser]GPx mutant. Purification of [Cys]GPx was performed in two steps of ion-exchange chromatography giving a 98% homogenous protein in 50% yield. The purified [Cys]GPx protein was shown to be a symmetrical tetramer by the means of gel-filtration HPLC and SDS/PAGE. Two isoelectric points were found (6.8 and 7.2) which may reflect two different oxidation states of the mutant protein. The GPx activity of the [Cys]GPx mutant was optimal at pH 8.5. The [Cys]GPx mutant had a specific activity approximately 1000-fold smaller than that of the natural enzyme, and was very easily inactivated by hydroperoxides. Inhibition of the activity with iodoacetate determined a pKa of 8.3, presumably that of the active-site cysteine. Unlike that of SeGPx, the GPx activity of [Cys]GPx was only slightly inhibited by mercaptosuccinate. We discuss hypothetical mechanistic constraints of either catalytic cycle, which may explain such results.

Glutathione and cysteine depletion in rats and mice following acute intoxication with diethylmaleate.

We examined the dose-dependent glutathione (GSH) depletion in liver, kidney, heart and brain of rats and mice, and cysteine depletion in rat kidney, following i.p. administration of diethylmaleate (DEM). In either rodent, the fall in total GSH concentration in liver and heart reached an upper value of 90 and 80% with 3 and 4 mmol DEM/kg respectively, which did not increase with higher doses. This study suggests that the residual level of GSH corresponds to the mitochondrial pool, in which case DEM might serve as a tool for the measurement of mitochondrial GSH ex vivo. In further experiments, we studied the time course of GSH and cysteine after administration of 3 mmol DEM/kg in rat tissues. Maximal depletion was reached approximately 1 hr after the i.p. injection. Subsequent GSH repletion was fast in liver and kidney, whereas it was slow in heart and brain, with a return to control values by 8-12 and by 48 hr after intoxication, respectively. This study provides new data for cardiac GSH and renal cysteine decrease after intoxication with DEM and should help to optimize GSH depletion models for further pharmacological investigations, especially when the use of inhibitors of glutathione metabolic turnover is undesirable and when side-effects other than GSH depletion must be avoided.

[Possible role of glutathione peroxidase in the regulation of collagenase activity]. / Rôle possible de la glutathion peroxydase dans la régulation de l'activité collagénase.

Glutathione peroxidase (Se-GPx) is a selenoenzyme which catalyzes the reduction of hydroperoxides by glutathione (GSH), in most mammalian cells. Several Slow-acting drugs that are used in the treatment of rheumatoid arthritis, including D-Penicillamine, alpha-mercaptopropionylglycine and gold salts, are specific inhibitors of Se-GPx. In situation of oxidant stress, Se-GPx activity is a major source of glutathione disulfide (GSSG), an essential activator of leucocyte collagenase. Hence the possibility that the enzymatic reduction of hydroperoxides produced during chronic inflammation would play an important role in the destruction of joint tissue of arthritic patients. Inhibition of a protective system such as Se-GPx may therefore be involved in the mechanism of action of D-Penicillamine and gold salts, but it could also explain some of their undesirable or toxic effects. Confirmation of this hypothesis would open the way to new pharmacological strategies.

Mechanism of selenium-glutathione peroxidase and its inhibition by mercaptocarboxylic acids and other mercaptans.

In a systematic search for effectors of glutathione peroxidase, a number of mercaptocarboxylic acids and tertiary mercaptans were found to be strong and specific inhibitors of the enzyme glutathione peroxidase. Assessment of various models was made by linear and nonlinear least squares fitting techniques. The results support the formation of reversible enzyme-inhibitor complexes. The active site selenium is trapped by the rapid binding of the inhibitor in competition with GSH. Data are consistent with the formation of thioselenenate adducts of the active site. The kinetic model which best describes the observed inhibition by the very strong inhibitor mercaptosuccinate implies that a selenenic acid with a kinetically significant lifetime is not formed when hydroperoxide is reduced. A noncovalent binding site for GSH or the presence of a cysteine residue at the active site of the enzyme provides a mechanistic rationale for the observed kinetics. Three of the most potent inhibitors found in this study, mercaptosuccinate, penicillamine, and alpha-mercaptopropionylglycine, are currently used as slow-acting drugs in the treatment of rheumatoid arthritis. Overall, the evidence suggests that glutathione peroxidase may be involved in the etiology of this disease.

Purification and characterization of selenium-glutathione peroxidase from hamster liver.

Hamster liver glutathione peroxidase was purified to homogeneity in three chromatographic steps and with 30% yield. The purified enzyme had a specific activity of approximately 500 mumol cumene hydroperoxide reduced/min/mg of protein at 37 degrees C, pH 7.6, and 0.25 mM GSH. The enzyme was shown to be a tetramer of indistinguishable subunits, the molecular weight of which was approximately 23,000 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A single isoelectric point of 5.0 was attributed to the active enzyme. Amino acid analysis determined that selenocysteine, identified as its carboxymethyl derivative, was the only form of selenium. One residue of cysteine was found to be present in each glutathione peroxidase subunit. The presence of tryptophan was colorimetrically determined. Pseudo-first-order kinetics of inactivation of the enzyme by iodoacetate was observed at neutral pH with GSH as the only reducing agent. An optimal pH of 8.0 at 37 degrees C and an activation energy of 3 kcal/mol at pH 7.6 were found. A ter-uni-ping-pong mechanism was shown by the use of an integrated-rate equation. At pH 7.6, the apparent second-order rate constants for reaction of glutathione peroxidase with hydroperoxides were as follows: k1 (t-butyl hydroperoxide), 7.06 X 10(5) mM-1 min-1; k1 (cumene hydroperoxide), 1.04 X 10(6) mM-1 min-1; k1 (p-menthane hydroperoxide), 1.2 X 10(6) mM-1 min-1; k1 (diisopropylbenzene hydroperoxide), 1.7 X 10(6) mM-1 min-1; k1 (linoleic acid hydroperoxide), 2.36 X 10(6) mM-1 min-1; k1 (ethyl hydroperoxide), 2.5 X 10(6) mM-1 min-1; and k1 (hydrogen peroxide), 2.98 X 10(6) mM-1 min-1. It is concluded that for bulky hydroperoxides, the more hydrophobic the substrate, the faster its reduction by glutathione peroxidase.