Impaired glutathione metabolism in hemolytic anemia

During the delivery of oxygen by erythrocytes, highly reactive oxygen species such as superoxide anion arise. The presence of reactive species damages the cell constituents. Glutathione (GSH) functions to repair cells when they are attacked by oxidative stress. GSH is synthesized in erythrocytes and glutathione disulfide (GSSG) is transported outside the cells to maintain a high GSH/GSSG ratio. The redox cycle of GSH by glutathione reductase and glutathione peroxidase is closely related to G6PD. Hereditary enzyme deficiency related to GSH metabolism, with hemolytic anemia has been reported. G6PD deficiency causes hemolytic anemia due to insufficiency of the redox cycle of GSH. Deficiency of GSH synthesizing enzymes or glutathione reductase also causes hemolysis. Pyrimidine 5'-nucleotidase deficiency causes hemolytic anemia even when there is a high concentration of GSH. Accumulation of nucleotides in red cells causes inhibition of G6PD activity.     

The regulation and role of extracellular glutathione peroxidase

Reactive oxygen species and reactive nitrogen species are mediators of lung tissue damage. To minimize the effect of oxidative stress, the lung is well equipped with an integrated antioxidant system. In some circumstances, antioxidants increase in response to oxidants and reduce tissue injury. The lung is somewhat unique in that it has an extracellular surface, which is often directly exposed to oxidative stresses. In this context, the extracellular antioxidant system, comprised primarily of glutathione and glutathione peroxidase, is especially important in protecting against oxidant injury. Induction of extracellular glutathione peroxidase occurs in airway inflammation and undoubtedly plays an important defense against oxidative injury to the airway surface.     

Contrasting effects of SH-compounds on oxidative DNA damage: repair and increase of damage

The non-radical singlet oxygen (¹O₂) and the OH radical (^.OH) are the major damaging oxidative species that can be generated inside cells during normal aerobic metabolism and by processes such as photosensitization. Both reactive oxygen species fulfill essential prerequisites to be a genotoxic agent. Due to their continuous production the represent and ever-present threat to all vital cellular molecules, especially DNA. As might be anticipated from the difference in character between these reactive species (non-radical versus radical) the pattern of DNA modifications caused by singlet oxygen is different from that produced by OH radicals. All cells possess an elaborate defense system against oxidative damage. This paper focuses mainly on the effect of thiols such as glutathione, which are thought to play a role as antioxidants. Under certain conditions thiols can repair chemically, probably by H-donation, some of the DNA damage caused by ^.OH; for instance breaks can be rather easily prevented in this way. This process will complete with fixation of damage by oxygen. However, there is ample evidence that H-atom donation does not always lead to ‘correct’ repair. Moreover under aerobic conditions thiyl peroxy radicals might increase DNA damage. Although the repair/fixation process could be examined in the case of ¹O₂ yet, it could be demonstrated that reactive species can be formed out of the reaction of thios with ¹O₂ capable of enhancing the number of DNA modifications such as 8-oxoguanine and single-strand breaks, probably arising from different pathways. Although it si quite clear that thiols are to some extent excellent antioxidants they possess unexpected properties which, depending on the conditions, can have genotoxic consequences.     

Role of glutathione in macrophage control of mycobacteria

Reactive oxygen and nitrogen intermediates are important antimicrobial defense mechanisms of macrophages and other phagocytic cells. While reactive nitrogen intermediates have been shown to play an important role in tuberculosis control in the murine system, their role in human disease is not clearly established. Glutathione, a tripeptide and antioxidant, is synthesized at high levels by cells during reactive oxygen intermediate and nitrogen intermediate production. Glutathione has been recently shown to play an important role in apoptosis and to regulate antigen-presenting-cell functions. Glutathione also serves as a carrier molecule for nitric oxide, in the form of S-nitrosoglutathione. Previous work from this laboratory has shown that glutathione and S-nitrosoglutathione are directly toxic to mycobacteria. A mutant strain of Mycobacterium bovis BCG, defective in the transport of small peptides such as glutathione, is resistant to the toxic effect of glutathione and S-nitrosoglutathione. Using the peptide transport mutant as a tool, we investigated the role of glutathione and S-nitrosoglutathione in animal and human macrophages in controlling intracellular mycobacterial growth.     

Role of glutathione in neuroprotective effects of mood stabilizing drugs lithium and valproate

Mood stabilizing drugs lithium and valproate are the most commonly used treatments for bipolar disorder. Previous studies in our laboratory indicate that chronic treatment with lithium and valproate inhibits oxidative damage in primary cultured rat cerebral cortical cells. Glutathione, as the major antioxidant in the brain, plays a key role in defending against oxidative damage. The purpose of this study was to determine the role of glutathione in the neuroprotective effects of lithium and valproate against oxidative damage. We found that chronic treatment with lithium and valproate inhibited reactive oxygen metabolite H(2)O(2)-induced cell death in primary cultured rat cerebral cortical cells, while buthionine sulfoximine, an inhibitor of glutathione rate-limiting synthesis enzyme glutamate-cysteine ligase, reduced the neuroprotective effect of lithium and valproate against H(2)O(2)-induced cell death. Further, we found that chronic treatment with lithium and valproate increased glutathione levels in primary cultured rat cerebral cortical cells and that the effects of lithium and valproate on glutathione levels were dose-dependent in human neuroblastoma SH-SY5Y cells. Chronic treatment with lithium and valproate also increased the expression of glutamate-cysteine ligase in both rat cerebral cortical cells and SH-SY5Y cells. In addition, chronic treatment with other mood stabilizing drugs lamotrigine and carbamazepine, but not antidepressants desipramine and fluoxetine, increased both glutathione levels and the expression of glutamate-cysteine ligase in SH-SY5Y cells. These results suggest that glutathione plays an important role in the neuroprotective effects of lithium and valproate, and that glutathione may be a common target for mood stabilizing drugs.     

Mycobacterium bovis BCG recA deletion mutant shows increased susceptibility to DNA-damaging agents but wild-type survival in a mouse infection model

Pathogenic microorganisms possess antioxidant defense mechanisms for protection from reactive oxygen metabolites which are generated during the respiratory burst of phagocytic cells. These defense mechanisms include enzymes such as catalase, which detoxifies reactive oxygen species, and DNA repair systems, which repair damage resulting from oxidative stress. To (i) determine the relative importance of the DNA repair system when oxidative stress is encountered by the Mycobacterium tuberculosis complex during infection of the host and to (ii) provide improved mycobacterial hosts as live carriers to express foreign antigens, the recA locus was inactivated by allelic exchange in Mycobacterium bovis BCG. The recA mutants are sensitive to DNA-damaging agents and show increased susceptibility to metronidazole, the first lead compound active against the dormant M. tuberculosis complex. Surprisingly, the recA genotype does not affect the in vitro dormancy response, nor does the defect in the DNA repair system lead to attenuation as determined in a mouse infection model. The recA mutants will be a valuable tool for further development of BCG as an antigen delivery system to express foreign antigens and as a source of a genetically stable vaccine against tuberculosis.     

Oxidative and antioxidative potential of brain microglial cells

Microglial cells are the resident immune cells of the central nervous system. These cells defend the central nervous system against invading microorganisms and clear the debris from damaged cells. Upon activation, microglial cells produce a large number of neuroactive substances that include cytokines, proteases, and prostanoids. In addition, activated microglial cells release radicals, such as superoxide and nitric oxide, that are products of the enzymes NADPH oxidase and inducible nitric oxide synthase, respectively. Microglia-derived radicals, as well as their reactive reaction products hydrogen peroxide and peroxynitrite, have the potential to harm cells and have been implicated in contributing to oxidative damage and neuronal cell death in neurological diseases. For self-protection against oxidative damage, microglial cells are equipped with efficient antioxidative defense mechanisms. These cells contain glutathione in high concentrations, substantial activities of the antioxidative enzymes superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase, as well as NADPH-regenerating enzymes. Their good antioxidative potential protects microglial cells against oxidative damage that could impair important functions of these cells in defense and repair of the brain.     

Mitochondrial inhibition and oxidative stress: reciprocating players in neurodegeneration

Although the etiology for many neurodegenerative diseases is unknown, the common findings of mitochondrial defects and oxidative damage posit these events as contributing factors. The temporal conundrum of whether mitochondrial defects lead to enhanced reactive oxygen species generation, or conversely, if oxidative stress is the underlying cause of the mitochondrial defects remains enigmatic. This review focuses on evidence to show that either event can lead to the evolution of the other with subsequent neuronal cell loss. Glutathione is a major antioxidant system used by cells and mitochondria for protection and is altered in a number of neurodegenerative and neuropathological conditions. This review also addresses the multiple roles for glutathione during mitochondrial inhibition or oxidative stress. Protein aggregation and inclusions are hallmarks of a number of neurodegenerative diseases. Recent evidence that links protein aggregation to oxidative stress and mitochondrial dysfunction will also be examined. Lastly, current therapies that target mitochondrial dysfunction or oxidative stress are discussed.     

Reactive oxygen species mediate central cardiorespiratory network responses to acute intermittent hypoxia

Although oxidative stress and reactive oxygen species generation is typically associated with localized neuronal injury, reactive oxygen species have also recently been shown to act as a physiological signal in neuronal plasticity. Here we define an essential role for reactive oxygen species as a critical stimulus for cardiorespiratory reflex responses to acute episodic hypoxia in the brain stem. To examine central cardiorespiratory responses to episodic hypoxia, we used an in vitro medullary slice that allows simultaneous examination of rhythmic respiratory-related activity and synaptic neurotransmission to cardioinhibitory vagal neurons. We show that whereas continuous hypoxia does not stimulate excitatory neurotransmission to cardioinhibitory vagal neurons, acute intermittent hypoxia of equivalent duration incrementally recruits an inspiratory-evoked excitatory neurotransmission to cardioinhibitory vagal neurons during intermittent hypoxia. This recruitment was dependent on the generation of reactive oxygen species. Further, we demonstrate that reactive oxygen species are incrementally generated in glutamatergic neurons in the ventrolateral medulla during intermittent hypoxia. These results suggest a neurochemical basis for the pronounced bradycardia that protects the heart against injury during intermittent hypoxia and demonstrates a novel role of reactive oxygen species in the brain stem.     

Acute exhaustive exercise does not alter lipid peroxidation levels and antioxidant enzyme activities in rat hippocampus, prefrontal cortex and striatum

Although regular physical exercise is beneficial to the body, it is well known that exhaustive exercise causes oxidative stress in muscle. Recent studies suggest that regular moderate physical exercise has the beneficial effects on brain. However, there is little information regarding whether or not exhaustive exercise could generate oxidative stress in brain and the findings are conflicting. The aim of this study was to investigate the effects of exhaustive exercise on thiobarbituric acid reactive substances, as an indicator of lipid peroxidation, in the hippocampus, prefrontal cortex and striatum. Additionally we examined antioxidant enzymes activities, superoxide dismutase and glutathione peroxidase, to assess the effects of reactive oxygen species. Exhaustive exercise did not change superoxide dismutase and glutathione peroxidase enzyme activities and thiobarbituric acid reactive substances levels neither immediately (0 min) nor at 3, 6, 12, 24 and 48 h after the cessation of exercise in the brain. These results indicate that acute exhaustive exercise may not cause significant lipid peroxidation in the hippocampus, prefrontal cortex and striatum during the post-exercise period.     

Lung protection by thiol-containing antioxidants

It is becoming increasingly clear that certain types of pulmonary injury may be closely related to oxidant-antioxidant imbalance in the lung, resulting from the production of reactive oxygen species within the lung during endogenous metabolism and xenobiotic insult. We have investigated the role of glutathione in pneumoprotection from such reactive species and, in particular, methods of manipulating the resident antioxidant capacity of lung glutathione. One such approach has been the use of the thiol-containing drug N-acetylcysteine. We have shown that N-acetylcysteine is able to both support intracellular glutathione biosynthesis and act as a 'scavenger' of reactive electrophilic species through the chemical reactivity of its thiol group. N-acetylcysteine reduces hydrogen peroxide to water, with the commensurate formation of N-acetylcysteine disulphide both when the peroxide was supplied directly or generated enzymatically. This basal reduction of hydrogen peroxide by N-acetylcysteine was greatly enhanced by the inclusion of catalytic amounts of the selenium-containing heterocycle, Ebselen, in the incubations. Thus, Ebselen mimics the activity of glutathione peroxidase but, unlike the enzyme, is able to use N-acetylcysteine as a co-substrate. Thus, the combination of N-acetylcysteine and Ebselen may provide a useful therapeutic tool in conditions of pulmonary toxicity associated with oxidant insult.     

Effect of oltipraz on the susceptibility of adultSchistosoma mansoni to killing by mouse peritoneal exudate cells

Incubation of the adultSchistosoma mansoni with the anti-schistosomal compound oltipraz (OPZ) (40 nM) resulted in a significant decrease in schistosome-reduced glutathione (GSH), a thiol compound which may have a role in protection against oxidant-mediated damage. A significant proportion (20–47%) of worms treated with OPZ became susceptible to in vitro killing by zymosan-stimulated peritoneal exudate cells from mice infected withS. mansoni or inoculated with Bacillus Calmette Guérin (BCG). Killing of the worms was partially inhibited by the addition to the assay system of exogenous glutathione peroxidase with GSH but not by superoxide dismutase. These results suggested that killing of parasites exposed to the drug was partly mediated by cell-generated hydrogen peroxide. They indicate also that depletion of schistosome GSH levels could render the parasites susceptible to killing by oxidative mechanisms, and suggest that there is potential in exploiting schistosome oxidant defense systems and reactive oxygen byproducts in the treatment of schistosomiasis. Inhibition of schistosome oxidant defense systems with drugs may render the parasites susceptible to killing by reactive oxygen byproducts of effector cells.     

Oxidative stress in autism

Autism is a severe developmental disorder with poorly understood etiology. Oxidative stress in autism has been studied at the membrane level and also by measuring products of lipid peroxidation, detoxifying agents (such as glutathione), and antioxidants involved in the defense system against reactive oxygen species (ROS). Lipid peroxidation markers are elevated in autism, indicating that oxidative stress is increased in this disease. Levels of major antioxidant serum proteins, namely transferrin (iron-binding protein) and ceruloplasmin (copper-binding protein), are decreased in children with autism. There is a positive correlation between reduced levels of these proteins and loss of previously acquired language skills in children with autism. The alterations in ceruloplasmin and transferrin levels may lead to abnormal iron and copper metabolism in autism. The membrane phospholipids, the prime target of ROS, are also altered in autism. The levels of phosphatidylethanolamine (PE) are decreased, and phosphatidylserine (PS) levels are increased in the erythrocyte membrane of children with autism as compared to their unaffected siblings. Several studies have suggested alterations in the activities of antioxidant enzymes such as superoxide dismutase, glutathione peroxidase, and catalase in autism. Additionally, altered glutathione levels and homocysteine/methionine metabolism, increased inflammation, excitotoxicity, as well as mitochondrial and immune dysfunction have been suggested in autism. Furthermore, environmental and genetic factors may increase vulnerability to oxidative stress in autism. Taken together, these studies suggest increased oxidative stress in autism that may contribute to the development of this disease. A mechanism linking oxidative stress with membrane lipid abnormalities, inflammation, aberrant immune response, impaired energy metabolism and excitotoxicity, leading to clinical symptoms and pathogenesis of autism is proposed.     

Analysis of the impact of intracellular reactive oxygen species generation on the structural and functional integrity of human spermatozoa: lipid peroxidation, DNA fragmentation and effectiveness of antioxidants

Exposure of human spermatozoa to nicotinamide adenine dinucleotide phosphate (NADPH) resulted in the dose dependent generation of reactive oxygen species (ROS) which, at a critical level of intensity, induced lipid peroxidation, DNA damage and a dramatic decline of sperm motility. This system was then used as a model for screening the ability of different antioxidants to combat oxidative stress created through the excessive intracellular generation of toxic oxygen products of metabolism. A variety of antioxidants that has previously been shown to be protective against extracellularly derived oxidants (e.g. superoxide dismutase, catalase, vitamin E, hypotaurine) were ineffective in this system. Albumin, however, could provide complete protection against NADPH induced oxidative stress via mechanisms that did not involve the suppression of the lipid peroxidation cascade but rather the inactivation of lipid peroxides generated during this process. Albumin did not protect against DNA damage induced by NADPH but was extremely effective at preventing DNA fragmentation arising from the suppression of glutathione peroxidase activity with mercaptosuccinate. These studies emphasize that the design of clinically effective antioxidant treatments will depend, critically, upon the source of the oxidative stress. For cases involving excessive intracellular ROS generation, albumin appears to be an important means of neutralizing lipid peroxide-mediated damage to the sperm plasma membrane and DNA.      

Hypoxia and HIF1alpha repress the differentiative effects of BMPs in high-grade glioma

Hypoxia commonly occurs in solid tumors of the central nervous system (CNS) and often interferes with therapies designed to stop their growth. We found that pediatric high-grade glioma (HGG)-derived precursors showed greater expansion under lower oxygen tension, typical of solid tumors, than normal CNS precursors. Hypoxia inhibited p53 activation and subsequent astroglial differentiation of HGG precursors. Surprisingly, although HGG precursors generated endogenous bone morphogenetic protein (BMP) signaling that promoted mitotic arrest under high oxygen tension, this signaling was actively repressed by hypoxia. An acute increase in oxygen tension led to Smad activation within 30 minutes, even in the absence of exogenous BMP treatment. Treatment with BMPs further promoted astroglial differentiation or death of HGG precursors under high oxygen tension, but this effect was inhibited under hypoxic conditions. Silencing of hypoxia-inducible factor 1alpha (HIF1alpha) led to Smad activation even under hypoxic conditions, indicating that HIF1alpha is required for BMP repression. Conversely, BMP activation at high oxygen tension led to reciprocal degradation of HIF1alpha; this BMP-induced degradation was inhibited in low oxygen. These results show a novel, mutually antagonistic interaction of hypoxia-response and neural differentiation signals in HGG proliferation, and suggest differences between normal and HGG precursors that may be exploited for pediatric brain cancer therapy.     

Cross talk of nitric oxide,oxygen radicals,and superoxide dismutase regulates the energy metabolism and cell death and determines the fates of aerobic life

Although oxygen is required for the energy metabolism in aerobic organisms, it generates reactive oxygen and nitrogen species that impair a wide variety of biological molecules, including lipids, proteins, and DNA, thereby causing various diseases. Because mitochondria are the major site of free radical generation, they are highly enriched with enzymes, such as Mn-type superoxide dismutase in matrix, and antioxidants including GSH on both sides of inner membranes, thus minimizing oxidative stress in and around this organelle. We recently showed that a cross talk of nitric oxide and oxygen radicals regulates the circulation, energy metabolism, reproduction, and remodeling of cells during embryonic development, and functions as a major defense system against pathogens. The present work shows that Cu/Zn-type superoxide dismutase, which has been postulated for a long time to be a cytosolic enzyme, also localizes bound to inner membranes of mitochondria, thereby minimizing oxidative stress in and around this organelle, while mitochondrial association decreases markedly with the variant types of the enzyme found in patients with familial amyotrophic lateral sclerosis. We also report that a cross talk of nitric oxide, superoxide, and molecular oxygen cooperatively regulates the fates of pathogens and their hosts and that oxidative stress in and around mitochondria also determines cell death in the development of animals and tissue injury caused by anticancer agents by some carnitine-inhibitable mechanism.     

Characterization of neural cell types expressing peroxiredoxins in mouse brain

The differential expression patterns of antioxidant enzymes observed in the brains of patients with neurodegenerative diseases suggest an important role for reactive oxygen species and antioxidant enzymes in neurodegeneration. The six mammalian peroxiredoxins (Prxs) comprise a novel family of anti-oxidative proteins that are widely distributed in most tissues, but few studies of Prx in brain tissue have been reported. The specific histology of the neural cell types in which Prxs are expressed is an important issue related to biological function and defense against oxidative stress in the brain. This study analyzed mouse brain neural cell types expressing Prx isoforms using single- or double-label immunohistochemical techniques. In neurons, immunoreactivity for Prx II-V was observed in the cytoplasm. In particular, Prx II was found in the habenular nuclei, and Prx III and V were found in the stratum lucidum of the hippocampus. Astrocytes and microglia were immunoreactive only for Prx VI and Prx I, respectively. Prx I and IV immunoreactivity was apparent in oligodendrocytes, where it was principally localized in the nuclei. The observed distribution of Prx isoforms in the mammalian brain may be indicative of their specific roles in their preferred neural cell types and subcellular locales. The results of this study will help in unraveling the physiological and pathophysiological roles of the different Prx isoforms in neural function.     

The role of oxidative stress in the in vitro induction of micronuclei by pesticides in mouse lung fibroblasts

The involvement of the antioxidant enzymes catalase and glutathione peroxidase (both at 0.1 mg/ml) in defence against the genotoxicity of phosphamidon (80 microg/ml) and dieldrin (25 microM) was investigated in order to demonstrate that the two pesticides damage DNA through the generation of reactive oxygen species and therefore of oxidative stress. The pesticide genotoxicity was determined by the cytokinesis-block micronucleus test performed on primary mouse lung fibroblast cultures. Also, 3-aminotriazole (40 mM) and mercaptosuccinate (0.5 mM), inhibitors of catalase and glutathione peroxidase, respectively, were added to the cultures. Data indicate that catalase causes a decrease only in the damage induced by phosphamidon, while glutathione peroxidase protects against damage induced by both phosphamidon and dieldrin. Simultaneous treatment with antioxidant inhibitors and pesticides results in a decrease in micronucleus frequency and cell number, due to apoptotic death. Our results indicate that clastogenic DNA damage produced by the two pesticides is modulated by antioxidant enzymes and their inhibitors and thus could be due to oxidative stress induction.