The contribution of thioredoxin-2 reductase and glutathione peroxidase to H2O2 detoxification of rat brain mitochondria

Brain mitochondria are not only major producers of reactive oxygen species but they also considerably contribute to the removal of toxic hydrogen peroxide by the glutathione (GSH) and thioredoxin-2 (Trx2) antioxidant systems. In this work we estimated the relative contribution of both systems and catalase to the removal of intrinsically produced hydrogen peroxide (H₂O₂) by rat brain mitochondria. By using the specific inhibitors auranofin and 1-chloro-2,4-dinitrobenzene (DNCB), the contribution of Trx2- and GSH-systems to reactive oxygen species (ROS) detoxification in rat brain mitochondria was determined to be 60 ± 20% and 20 ± 15%, respectively. Catalase contributed to a non-significant extent only, as revealed by aminotriazole inhibition. In digitonin-treated rat hippocampal homogenates inhibition of Trx2- and GSH-systems affected mitochondrial hydrogen peroxide production rates to a much higher extent than the endogenous extramitochondrial hydrogen peroxide production, pointing to a strong compartmentation of ROS metabolism. Imaging experiments of hippocampal slice cultures showed on single cell level substantial heterogeneity of hydrogen peroxide detoxification reactions. The strongest effects of inhibition of hydrogen peroxide removal by auranofin or DNCB were detected in putative interneurons and microglial cells, while pyramidal cells and astrocytes showed lower effects. Thus, our data underline the important contribution of the Trx2-system to hydrogen peroxide detoxification in rat hippocampus. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).     

Relative contributions of heart mitochondria glutathione peroxidase and catalase to H(2)O(2) detoxification in in vivo conditions

This study was aimed at assessing the relative contributions to H(2)O(2) detoxification by glutathione peroxidase and catalase in the mitochondrial matrix of heart. For this purpose, mitoplasts from rat heart were used in order to minimize contamination with microperoxisomes, and the kinetic rate constants of both enzymatic activities were determined along with a simulation profile. Results show that the contribution of catalase to H(2)O(2) removal in heart mitochondria is not significant, even under strong oxidative conditions, such as those achieved in ischemia-reperfusion and involving extensive glutathione depletion and high H(2)O(2) concentrations. Conversely, maintenance of the steady state levels of H(2)O(2) in the heart mitochondrial matrix seems to be the domain of glutathione peroxidase. It is suggested that the physiological role of the low amounts of catalase found in heart mitochondria is related to its peroxidatic rather than catalatic activity.     

Developmental study of rat brain glutathione peroxidase and glutathione reductase

Glutathione peroxidase and glutathione reductase activities were measured in whole rat brains at selected ages from birth to adulthood. On a wet weight basis glutathione peroxidase activity increased 70% during development and glutathione reductase activity increased 160%. On a protein basis glutathione peroxidase declined slightly in activity during the first two weeks of life and then maintained the 14-day activity into adulthood while glutathione reductase showed a 30% increase in activity. While less than the developmental changes in many enzymes involved in aerobic glycolysis or catecholamine metabolism, these increases do suggest a role in CNS metabolism.     

Distribution of glutathione peroxidases glutathione reductase in rat brain mitochondria

The distribution of glutathione reductase (GR), glutathione peroxidase (GPx) and phospholipid hydroperoxide glutathione peroxidase (PHGPx) in isolated rat brain mitochondria was investigated. using a fractionation procedure for the separation of inner and outer membranes, contact sites between the two membranes and a soluble fraction mainly originating from the mitochondrial matrix. The data indicate that GR and GPx are concentrated in the soluble fraction, with a minor portion of the two enzymes being associated with the contact sites. PHGPx is localized largely in the inner membrane. The possible functional significance of these findings is discussed.     

H(2)O(2) disposal in cardiolipin-enriched brain mitochondria is due to increased cytochrome c peroxidase activity

The mitochondrial electron transport chain is a source of oxygen superoxide anion (O(2)(-)) that is dismutated to H(2)O(2). Although low levels of ROS are physiologically synthesized during respiration, their increase contributes to cell injury. Therefore, an efficient machinery for H(2)O(2) disposal is essential in mitochondria. In this study, the ability of brain mitochondria to acquire cardiolipin (CL), phosphatidylglycerol (PG), and phosphatidylserine (PS) in vitro through a fusion process was exploited to investigate lipid effects on ROS. MTT assay, oxygen consumption, and respiratory ratio indicated that the acquired phospholipids did not alter mitochondrial respiration and O(2)(-) production from succinate. However, in CL-enriched mitochondria, H(2)O(2) levels where 27% and 47% of control in the absence and in the presence of antimycin A, respectively, suggesting an increase in H(2)O(2) elimination. Concomitantly, cytochrome c (cyt c) was released outside mitochondria. Since free oxidized cyt c acquired peroxidase activity towards H(2)O(2) upon interaction with CL in vitro, a contribution of cyt c to H(2)O(2) disposal in mitochondria through CL conferred peroxidase activity is plausible. In this model, the accompanying CL peroxidation should weaken cyt c-CL interactions, favouring the detachment and release of the protein. Neither cyt c peroxidase activity was elicited by PS in vitro, nor cyt c release was observed in PS-enriched mitochondria, although H(2)O(2) levels were significantly decreased, suggesting a cyt c-independent role of PS in ROS metabolism in mitochondria.     

Expression of ascorbate peroxidase and glutathione reductase in roots of rice seedlings in response to NaCl and H2O2

The accumulation of H2O2 by NaCl was observed in the roots of rice seedlings. Treatment with NaCl caused an increase in the activities of ascorbate peroxidase (APX) and glutathione reductase (GR) and the expression of OsAPX and OsGR in rice roots. Exogenously applied H2O2 also enhanced the activities of APX and GR and the expression of OsAPX and OsGR in rice roots. The accumulation of H2O2 in rice roots in response to NaCl was inhibited by the NADPH oxidase inhibitors, diphenyleneiodonium chloride (DPI) and imidazole (IMD). However, DPI, IMD, and dimethylthiourea, a H2O2 trap, did not reduce NaCl-enhanced activities of APX and GR and expression of OsAPX and OsGR. It appears that H2O2 is not involved in the regulation of NaCl-induced APX and GR activities and OsAPX and OsGR expression in rice roots.     

Selenium-dependent glutathione peroxidase gene expression during gonad development and its response to LPS and H(2)O(2) challenge in Scylla paramamosain

A selenium-dependent glutathione peroxidase cDNA was obtained from green mud crab Scylla paramamosain (SpGPx) by homology PCR technique and rapid amplification of cDNA ends (RACE) methods. The 1135?bp full-length cDNA contains a 9?bp 5'-untranslated region (UTR), an open reading frame (ORF) of 564 bp encoded a deduced protein of 187 amino acids (aa), and a 562?bp 3'-UTR with a 100 bp conserved eukaryotic selenocysteine insertion sequence (SECIS). It involves a putative selenocysteine (Sec(40), or U(40)) residue which is encoded by an opal codon, (127)TGA(129), and forms an active site with residues Q(74) and W(142). Sequence characterization revealed that SpGPx contain a characteristic GPx signature motif 2 ((64)LAFPCNQF(71)), an active site motif ((152)WNFEKF(157)), a potential N-glycosylation site ((76)NTT(78)), and two residues (R(90) and R(168)) which contribute to the electrostatic architecture by directing the glutathione donor substrate. Multiple sequence alignment and phylogenetic analysis showed that SpGPx share a high level of identities and closer relationship with other selected invertebrate GPxs and vertebrate GPx1 and GPx2. Molecular modelling analysis results also supported these observations. Real time quantitative PCR analysis revealed that SpGPx was constitutively expressed in 10 selected tissues, and its expression level in gill and testis was higher than that in the other tissues (p?相似文献   

Effect of training on H(2)O(2) release by mitochondria from rat skeletal muscle

In this study, oxygen consumption and H(2)O(2) release rate by succinate or pyruvate/malate supplemented mitochondria isolated from skeletal muscle of trained and untrained rats were investigated. The overall mitochondrial antioxidant capacity and the effect of preincubation of mitochondria with GDP, an inhibitor of uncoupling proteins UCP1 and UCP2, on both succinate-supported H(2)O(2) release and membrane potential were also determined. The results indicate that training does not affect mitochondrial oxygen consumption with both complex-I- and complex II-linked substrates. Succinate-supported H(2)O(2) release was lower in trained than in untrained rats both in State 4 and State 3. Even the antimycin A-stimulated release was lower in trained rats. When pyruvate/malate were used as substrates, H(2)O(2) release rate was lower in trained rats only in the presence of antimycin A. The increase of mitochondrial protein content (determined by the ratio between cytochrome oxidase activities in homogenates and mitochondria) in trained muscle was such that the succinate-supported H(2)O(2) release per g of tissue was not significantly different in trained and untrained rats, while that supported by pyruvate/malate was higher in trained than in untrained animals. The lack of training-induced changes in overall antioxidant capacity of mitochondria indicates that the decrease in mitochondrial H(2)O(2) release cannot be attributed to a greater capacity of mitochondria to scavenge the reactive oxygen intermediates derived from univalent O(2) reduction by respiratory chain components. In contrast, the above decrease seems to depend on the drop induced by training in mitochondrial membrane potential. These training effects are not due to an increased level of mitochondrial uncoupling protein, because in the presence of GDP the increase in both membrane potential and H(2)O(2) release was greater in untrained than in trained rats.     

Characteristics of alpha-glycerophosphate-evoked H2O2 generation in brain mitochondria

Characteristics of reactive oxygen species (ROS) production in isolated guinea-pig brain mitochondria respiring on alpha-glycerophosphate (alpha-GP) were investigated and compared with those supported by succinate. Mitochondria established a membrane potential (DeltaPsi(m)) and released H(2)O(2) in parallel with an increase in NAD(P)H fluorescence in the presence of alpha-GP (5-40 mm). H(2)O(2) formation and the increase in NAD(P)H level were inhibited by rotenone, ADP or FCCP, respectively, being consistent with a reverse electron transfer (RET). The residual H(2)O(2) formation in the presence of FCCP was stimulated by myxothiazol in mitochondria supported by alpha-GP, but not by succinate. ROS under these conditions are most likely to be derived from alpha-GP-dehydrogenase. In addition, huge ROS formation could be provoked by antimycin in alpha-GP-supported mitochondria, which was prevented by myxothiazol, pointing to the generation of ROS at the quinol-oxidizing center (Q(o)) site of complex III. FCCP further stimulated the production of ROS to the highest rate that we observed in this study. We suggest that the metabolism of alpha-GP leads to ROS generation primarily by complex I in RET, and in addition a significant ROS formation could be ascribed to alpha-GP-dehydrogenase in mammalian brain mitochondria. ROS generation by alpha-GP at complex III is evident only when this complex is inhibited by antimycin.     

A study on the inhibition of rat myocardium glutathione peroxidase and glutathione reductase by moniliformin

The yeasts of patients with oral cancer has been studied before and during Xr-therapy. Gram and PAS smears revealed an increase of yeast-like structures, during treatment, from 56% to 66% of the cases. Before radiotherapy oral yeasts were isolated from 56% of the patients with cancer represented by Candida albicans (30%); C. tropicalis (12%); C. glabrata and C. krusei (4%), besides six other different species (2%). During radiotherapy yeasts were isolated in 72% of the cases, as follow: C. albicans (36%); C. tropicalis (16%); Rhodotorula rubra (6%); C. kefyr; C. krusei and Pichia farinosa (4%), besides other nine species (2%). C. albicans serotype A represented 93% of the isolated samples, before treatment and 88,8% during Xr-therapy.     

Glutamate induces H2O2 synthesis in nonsynaptic brain mitochondria

Mitochondrial reactive oxygen species regulate many important biological processes. We studied H₂O₂ formation by nonsynaptic brain mitochondria in response to the addition of low concentrations of glutamate, an excitatory neurotransmitter. We demonstrated that glutamate at concentrations from 10 to 50 μM stimulated the H₂O₂ generation in mitochondria up to 4-fold, in a dose-dependent manner. The effect of glutamate was observed only in the presence of Ca²⁺ (20 μM) in the incubation medium, and the rate of calcium uptake by the brain mitochondria was increased by up to 50% by glutamate. Glutamate-dependent effects were sensitive to the NMDA receptor inhibitors MK-801 (10 μM) and D-AP5 (20 μM) and the inhibitory neurotransmitter glycine (5 mM). We have shown that the H₂O₂ formation caused by glutamate is associated with complex II and is dependent on the mitochondrial potential. We have found that nonsynaptic brain mitochondria are a target of direct glutamate signaling, which can specifically activate H₂O₂ formation through mitochondrial respiratory chain complex II. The H₂O₂ formation induced by glutamate can be blocked by glycine, an inhibitory neurotransmitter that prevents the deleterious effects of glutamate in brain mitochondria.     

The effect of age and sex on glutathione reductase and glutathione peroxidase activities and on aerobic glutathione oxidation in rat liver homogenates

1. Changes in liver glutathione reductase and glutathione peroxidase activities in relation to age and sex of rats were measured. Oxidation of GSH was correlated with glutathione peroxidase activity. 2. Glutathione reductase activity in foetal rat liver was about 65% of the adult value. It increased to a value slightly higher than the adult one at about 2-3 days, decreased until about 16 days and then rose after weaning to a maximum at about 31 days, finally reaching adult values at about 45 days old. 3. Weaning rats on to an artificial rat-milk diet prevented the rise in glutathione reductase activity associated with weaning on to the usual diet high in carbohydrate. 4. In male rats glutathione peroxidase activity in the liver increased steadily up to adult values. There were no differences between male and female rats until sexual maturity, when, in females, the activity increased abruptly to an adult value that was about 80% higher than that in males. 5. The rate of GSH oxidation in rat liver homogenates increased steadily from 3 days until maturity, when the rate of oxidation was about 50% higher in female than in male liver. 6. In the liver a positive correlation between glutathione peroxidase activity and GSH oxidation was found. 7. It is suggested that the coupled oxidation-reduction through glutathione reductase and glutathione peroxidase is important for determining the redox state of glutathione and of NADP, and also for controlling the degradation of hydroperoxides. 8. Changes in glutathione reductase and glutathione peroxidase activities are discussed in relation to the redox state of glutathione and NADP and to their effects on the concentration of free CoA in rat liver and its possible action on ketogenesis and lipogenesis.     

Micromethods in single muscle fibers. 2. Determination of glutathione reductase and glutathione peroxidase

This paper extends the previous study for systems which control intracellular oxidative events in muscle and describes procedures suitable to assay glutathione peroxidase (GSHPx), glutathione reductase (GR), and total glutathione (GSH + GSSG) after fiber typing of individual muscle fibers. In human skeletal muscle, both GR and GSHPx activities were relatively low when compared to those of other tissue. No difference was found among fiber types (I, IIA, and IIB) with regard to GR activity, but in contrast GSHPx activity was significantly lower in type IIB fibers than in the other types. These results suggest that type IIB fibers may have a reduced ability to cope with hydroperoxides generated during oxidative stress, which, in turn, could lead to increased damage to membrane structures by lipid peroxidation or oxidation of sensitive intracellular thiol (-SH) enzymes by hydrogen peroxide. The Km of skeletal muscle GR for GSSG was 27 microM and for NADPH was 22 microM. If one assumes approximately 95% of total glutathione is present in the reduced state, then GSSG concentration would be of the order of 0.3 mmol/kg and under these conditions skeletal muscle GR would be efficient in all muscle fiber types.     

Spatial and temporal ontogenies of glutathione peroxidase and glutathione disulfide reductase during development of the prenatal rat.

Spatial and temporal expression and regulation of the antioxidant enzymes, glutathione peroxidase (GSH-Px), glutathione disulfide reductase (GSSG-Rd) may be important in determining cell-specific susceptibility to embryotoxicants. Creation of tissue-specific ontogenies for antioxidant enzyme activities during development is an important first step in understanding regulatory relationships. Early organogenesis-stage embryos were grouped according to the somite number (GD 9-13), and fetuses were evaluated by gestational day (GD 14-21). GSH-Px activities in the visceral yolk sac (VYS) increased on consecutive days from GD 9 to GD 13, representing a 5.7-fold increase during this period of development. GSH-Px activities in VYS decreased after GD 13, ultimately constituting a 37% decrease at GD 21. Head, heart, and trunk specific activities generally increased from GD 9 to GD 13 albeit not to the same magnitude as detected in the VYS. GSSG-Rd activities showed substantial increases in the VYS from GD 9 to GD 13, 6.3-fold and decreased thereafter to 50% by GD 21. The greatest changes in enzyme activities were noted in the period between GD 10 and GD 11, where the embryo establishes an active cardiovascular system and begins to convert to aerobic metabolism. Generally, from GD 14-21, embryonic organ GSH-Px and GSSG-Rd activities either remained constant or increased as gestation progressed. These studies suggest the importance of the VYS in dealing with ROS and protecting the embryo. Furthermore, understanding the consequences of lower antioxidant activities during organogenesis may help to pinpoint periods of teratogenic susceptibility to xenobiotics and increased oxygen.     

Differential oxidation of thioredoxin-1, thioredoxin-2, and glutathione by metal ions

Metal toxicity often includes the generation of reactive oxygen species (ROS) and subsequent oxidative stress, but whether metals have different effects on the major thiol antioxidant systems is unknown. Here, we examine the effects of arsenic, cadmium, cesium, copper, iron, mercury, nickel, and zinc on glutathione (GSH), cytoplasmic thioredoxin-1 (Trx1), and mitochondrial thioredoxin-2 (Trx2) redox states. GSH/GSSG redox states were determined by HPLC, and Trx1 and Trx2 redox states were determined by Redox Western blot methods. Copper, iron, and nickel showed significant oxidation of GSH but relatively little oxidation of either Trx1 or Trx2. Arsenic, cadmium, and mercury showed little oxidation of GSH but significantly oxidized both Trx1 and Trx2. The magnitude of effects of arsenic, cadmium, and mercury was greater for the mitochondrial Trx2 (>60 mV) compared to the cytoplasmic Trx1 (20 to 40 mV). Apoptosis signal-regulating kinase 1 (ASK1) may be activated by two different pathways, one dependent upon GSH and glutaredoxin and the other independent of GSH and dependent upon thioredoxin. ASK1 activation and cell death were observed with metals that oxidized thioredoxins but not with metals that oxidized GSH. These findings show that metals have differential oxidative effects on the major thiol antioxidant systems and that activation of apoptosis may be associated with metal ions that oxidize thioredoxin and activate ASK1. The differential oxidation of the major thiol antioxidant systems by metal ions suggest that the distinct thiol/disulfide redox couples represented by GSH/GSSG and the thioredoxins may convey different levels of control in apoptotic and toxic signaling pathways.     

Succinate modulation of H2O2 release at NADH:ubiquinone oxidoreductase (Complex I) in brain mitochondria

Complex I (NADH:ubiquinone oxidoreductase) is responsible for most of the mitochondrial H2O2 release, both during the oxidation of NAD-linked substrates and during succinate oxidation. The much faster succinate-dependent H2O2 production is ascribed to Complex I, being rotenone-sensitive. In the present paper, we report high-affinity succinate-supported H2O2 generation in the absence as well as in the presence of GM (glutamate/malate) (1 or 2 mM of each). In brain mitochondria, their only effect was to increase from 0.35 to 0.5 or to 0.65 mM the succinate concentration evoking the semi-maximal H2O2 release. GM are still oxidized in the presence of succinate, as indicated by the oxygen-consumption rates, which are intermediate between those of GM and of succinate alone when all substrates are present together. This effect is removed by rotenone, showing that it is not due to inhibition of succinate influx. Moreover, alpha-oxoglutarate production from GM, a measure of the activity of Complex I, is decreased, but not stopped, by succinate. It is concluded that succinate-induced H2O2 production occurs under conditions of regular downward electron flow in Complex I. Succinate concentration appears to modulate the rate of H2O2 release, probably by controlling the hydroquinone/quinone ratio.