Glutathione Reductase Promotes Fungal Clearance and Suppresses Inflammation during Systemic Candida albicans Infection in Mice.

Glutathione reductase (Gsr) catalyzes the reduction of glutathione disulfide to glutathione, which plays an important role in redox regulation. We have previously shown that Gsr facilitates neutrophil bactericidal activities and is pivotal for host defense against bacterial pathogens. However, it is unclear whether Gsr is required for immune defense against fungal pathogens. It is also unclear whether Gsr plays a role in immunological functions outside of neutrophils during immune defense. In this study, we report that Gsr-/- mice exhibited markedly increased susceptibility to Candida albicans challenge. Upon C. albicans infection, Gsr-/- mice exhibited dramatically increased fungal burden in the kidneys, cytokine and chemokine storm, striking neutrophil infiltration, histological abnormalities in both the kidneys and heart, and substantially elevated mortality. Large fungal foci surrounded by massive numbers of neutrophils were detected outside of the glomeruli in the kidneys of Gsr -/- mice but were not found in wild-type mice. Examination of the neutrophils and macrophages of Gsr-/- mice revealed several defects. Gsr -/- neutrophils exhibited compromised phagocytosis, attenuated respiratory burst, and impaired fungicidal activity in vitro. Moreover, upon C. albicans stimulation, Gsr -/- macrophages produced increased levels of inflammatory cytokines and exhibited elevated p38 and JNK activities, at least in part, because of lower MAPK phosphatase (Mkp)-1 activity and greater Syk activity. Thus, Gsr-mediated redox regulation is crucial for fungal clearance by neutrophils and the proper control of the inflammatory response by macrophages during host defense against fungal challenge.

Dynamic denitrosylation via S-nitrosoglutathione reductase regulates cardiovascular function.

Although protein S-nitrosylation is increasingly recognized as mediating nitric oxide (NO) signaling, roles for protein denitrosylation in physiology remain unknown. Here, we show that S-nitrosoglutathione reductase (GSNOR), an enzyme that governs levels of S-nitrosylation by promoting protein denitrosylation, regulates both peripheral vascular tone and ß-adrenergic agonist-stimulated cardiac contractility, previously ascribed exclusively to NO/cGMP. GSNOR-deficient mice exhibited reduced peripheral vascular tone and depressed ß-adrenergic inotropic responses that were associated with impaired ß-agonist-induced denitrosylation of cardiac ryanodine receptor 2 (RyR2), resulting in calcium leak. These results indicate that systemic hemodynamic responses (vascular tone and cardiac contractility), both under basal conditions and after adrenergic activation, are regulated through concerted actions of NO synthase/GSNOR and that aberrant denitrosylation impairs cardiovascular function. Our findings support the notion that dynamic S-nitrosylation/denitrosylation reactions are essential in cardiovascular regulation.

Glutathione reductase facilitates host defense by sustaining phagocytic oxidative burst and promoting the development of neutrophil extracellular traps.

Glutathione reductase (Gsr) catalyzes the reduction of glutathione disulfide to glutathione, which plays an important role in the bactericidal function of phagocytes. Because Gsr has been implicated in the oxidative burst in human neutrophils and is abundantly expressed in the lymphoid system, we hypothesized that Gsr-deficient mice would exhibit marked defects during the immune response against bacterial challenge. We report in this study that Gsr-null mice exhibited enhanced susceptibility to Escherichia coli challenge, indicated by dramatically increased bacterial burden, cytokine storm, striking histological abnormalities, and substantially elevated mortality. Additionally, Gsr-null mice exhibited elevated sensitivity to Staphylococcus aureus. Examination of the bactericidal functions of the neutrophils from Gsr-deficient mice in vitro revealed impaired phagocytosis and defective bacterial killing activities. Although Gsr catalyzes the regeneration of glutathione, a major cellular antioxidant, Gsr-deficient neutrophils paradoxically produced far less reactive oxygen species upon activation both ex vivo and in vivo. Unlike wild-type neutrophils that exhibited a sustained oxidative burst upon stimulation with phorbol ester and fMLP, Gsr-deficient neutrophils displayed a very transient oxidative burst that abruptly ceased shortly after stimulation. Likewise, Gsr-deficient neutrophils also exhibited an attenuated oxidative burst upon encountering E. coli. Biochemical analysis revealed that the hexose monophosphate shunt was compromised in Gsr-deficient neutrophils. Moreover, Gsr-deficient neutrophils displayed a marked impairment in the formation of neutrophil extracellular traps, a bactericidal mechanism that operates after neutrophil death. Thus, Gsr-mediated redox regulation is crucial for bacterial clearance during host defense against massive bacterial challenge.

Endogenous S-nitrosothiols protect against myocardial injury.

Despite substantial evidence that nitric oxide (NO) and/or endogenous S-nitrosothiols (SNOs) exert protective effects in a variety of cardiovascular diseases, the molecular details are largely unknown. Here we show that following left coronary artery ligation, mice with a targeted deletion of the S-nitrosoglutathione reductase gene (GSNOR(-/-)) have reduced myocardial infarct size, preserved ventricular systolic and diastolic function, and maintained tissue oxygenation. These profound physiological effects are associated with increases in myocardial capillary density and S-nitrosylation of the transcription factor hypoxia inducible factor-1alpha (HIF-1alpha) under normoxic conditions. We further show that S-nitrosylated HIF-1alpha binds to the vascular endothelial growth factor (VEGF) gene, thus identifying a role for GSNO in angiogenesis and myocardial protection. These results suggest innovative approaches to modulate angiogenesis and preserve cardiac function.

A genetic analysis of nitrosative stress.

Nitrosative stress is induced by pathophysiological levels of nitric oxide (NO) and S-nitrosothiols (e.g., S-nitrosoglutathione, GSNO) and arises, at least in significant part, from the nitrosylation of critical protein Cys thiols (S-nitrosylation) and metallocofactors. However, the mechanisms by which NO and GSNO mediate nitrosative stress are not well understood. Using yeast Saccharomyces cerevisiae strains lacking NO- and/or GSNO-consuming enzymes (flavohemoglobin and GSNO reductase, respectively), we measured the individual and combined effects of NO and GSNO on both cell growth and the formation of protein-bound NO species. Our results suggest an intracellular equilibrium between NO and GSNO, dependent in part on cell-catalyzed release of NO from GSNO (i.e., "SNO-lyase" activity). However, whereas NO induces multiple types of protein-based modifications, levels of which correlate with inhibition of cell growth, GSNO mainly affects protein S-nitrosylation, and the relationship between S-nitrosylation and nitrosative stress is more complex. These data support the idea of multiple classes of protein-SNO, likely reflected in divergent routes of synthesis and degradation. Indeed, a significant fraction of protein S-nitrosylation by NO occurs in the absence of O(2), which is commonly assumed to drive this reaction but instead is apparently dependent in substantial part upon protein-bound transition metals. Additionally, our findings suggest that nitrosative stress is mediated principally via the S-nitrosylation of a subset of protein targets, which include protein SNOs that are stable to cellular glutathione (and thus are not metabolized by GSNO reductase). Collectively, these results provide new evidence for the mechanisms through which NO and GSNO mediate nitrosative stress as well as the cellular pathways of protein S-nitrosylation and denitrosylation involving metalloproteins, SNO lyase(s) and GSNO reductase.

Nitrofurantoin cytotoxicity. In vitro assessment of risk based on glutathione metabolism.

Nitrofurantoin, a commonly used urinary tract antiseptic, has been associated with idiosyncratic pulmonary and hepatic damage. We have used human lymphocytes in vitro to explore the biochemical basis of susceptibility to nitrofurantoin toxicity. The drug itself did not damage the cells as assessed by trypan blue dye exclusion. In the presence of a mouse hepatic microsomal drug-activating system, however, nitrofurantoin metabolites produced dose dependent toxicity to the lymphocytes. Inhibition of the enzyme epoxide hydrolase did not enhance toxicity; the metabolite thus does not appear to be a furan epoxide. Binding of reactive metabolites to cell macromolecules may lead directly to cell death, or in vivo, by acting as haptens to secondary immunologic responses. The metabolite caused a dose-dependent depletion of lymphocyte glutathione content. Cells from a patient with glutathione synthetase deficiency showed markedly enhanced nitrofurantoin toxicity. The findings suggest that glutathione plays a major role in protecting cells from nitrofurantoin-induced damage, and that studies of lymphocyte toxicity and glutathione metabolism in patients experiencing idiosyncratic reactions to nitrofurantoin may lead to elucidation of the biochemical and genetic basis of drug susceptibility.

Evidence for an additional disulfide reduction pathway in Escherichia coli.

An Escherichia coli cell-free protein synthesis cell extract has been created that lacks all known cytoplasmic disulfide reduction pathways but still retains significant reductase activity. Oxidized glutathione was partially stabilized by deleting the gene for glutathione reductase. To avoid previously reported AhpC mutations, thioredoxin reductase was only removed after extract preparation. The trxB gene was extended to encode a hemagglutinin tag so that TrxB could be removed by affinity adsorption. However, significant glutathione reductase activity remained. The unknown glutathione reductase pathway is disabled by iodoacetamide, is inhibited by NADH, and appears to use NADPH as an electron source.

GSR is not essential for the maintenance of antioxidant defenses in mouse cochlea: Possible role of the thioredoxin system as a functional backup for GSR.

Glutathione reductase (GSR), a key member of the glutathione antioxidant defense system, converts oxidized glutathione (GSSG) to reduced glutathione (GSH) and maintains the intracellular glutathione redox state to protect the cells from oxidative damage. Previous reports have shown that Gsr deficiency results in defects in host defense against bacterial infection, while diquat induces renal injury in Gsr hypomorphic mice. In flies, overexpression of GSR extended lifespan under hyperoxia. In the current study, we investigated the roles of GSR in cochlear antioxidant defense using Gsr homozygous knockout mice that were backcrossed onto the CBA/CaJ mouse strain, a normal-hearing strain that does not carry a specific Cdh23 mutation that causes progressive hair cell degeneration and early onset of hearing loss. Gsr-/- mice displayed a significant decrease in GSR activity and GSH/GSSG ratios in the cytosol of the inner ears. However, Gsr deficiency did not affect ABR (auditory brainstem response) hearing thresholds, wave I amplitudes or wave I latencies in young mice. No histological abnormalities were observed in the cochlea of Gsr-/- mice. Furthermore, there were no differences in the activities of cytosolic glutathione-related enzymes, including glutathione peroxidase and glutamate-cysteine ligase, or the levels of oxidative damage markers in the inner ears between WT and Gsr-/- mice. In contrast, Gsr deficiency resulted in increased activities of cytosolic thioredoxin and thioredoxin reductase in the inner ears. Therefore, under normal physiological conditions, GSR is not essential for the maintenance of antioxidant defenses in mouse cochlea. Given that the thioredoxin system is known to reduce GSSG to GSH in multiple species, our findings suggest that the thioredoxin system can support GSSG reduction in the mouse peripheral auditory system.

Extracellular superoxide provokes glutathione efflux from Escherichia coli cells.

The aim of the study was to elucidate a possible relationship between transmembrane cycling of glutathione and changes in levels of external superoxide. Exposure of growing Escherichia coli to exogenous reactive oxygen species (ROS) generated by xanthine and xanthine oxidase (XO) stimulates reversible glutathione (GSH) efflux from the cells that is considerably lowered under phosphate starvation. This GSH efflux is prevented by exogenous SOD, partially inhibited by catalase, and is not dependent on the GSH exporter CydDC. The γ-glutamyl transpeptidase (GGT) deficiency completely prevents a return of GSH to the cytoplasm. In contrast to wild-type E. coli, mutants devoid of GGT and glutathione reductase (GOR) show enhanced accumulation of oxidized glutathione in the medium after exposure to xanthine and XO. Under these conditions, sodC, ggt and especially gshA mutants reveal more intensive and prolonged inhibition of growth than wild-type cells. Treatment with XO does not influence E. coli viability, but somewhat increases the number of cells with lost membrane potential. In summary, data obtained here indicate that transmembrane cycling of GSH may be involved in E. coli protection against extracellular ROS and may promote rapid growth recovery.

S-nitrosoglutathione reductase-dependent PPARγ denitrosylation participates in MSC-derived adipogenesis and osteogenesis.

Bone marrow-derived mesenchymal stem cells (MSCs) are a common precursor of both adipocytes and osteoblasts. While it is appreciated that PPARγ regulates the balance between adipogenesis and osteogenesis, the roles of additional regulators of this process remain controversial. Here, we show that MSCs isolated from mice lacking S-nitrosoglutathione reductase, a denitrosylase that regulates protein S-nitrosylation, exhibited decreased adipogenesis and increased osteoblastogenesis compared with WT MSCs. Consistent with this cellular phenotype, S-nitrosoglutathione reductase-deficient mice were smaller, with reduced fat mass and increased bone formation that was accompanied by elevated bone resorption. WT and S-nitrosoglutathione reductase-deficient MSCs exhibited equivalent PPARγ expression; however, S-nitrosylation of PPARγ was elevated in S-nitrosoglutathione reductase-deficient MSCs, diminishing binding to its downstream target fatty acid-binding protein 4 (FABP4). We further identified Cys 139 of PPARγ as an S-nitrosylation site and demonstrated that S-nitrosylation of PPARγ inhibits its transcriptional activity, suggesting a feedback regulation of PPARγ transcriptional activity by NO-mediated S-nitrosylation. Together, these results reveal that S-nitrosoglutathione reductase-dependent modification of PPARγ alters the balance between adipocyte and osteoblast differentiation and provides checkpoint regulation of the lineage bifurcation of these 2 lineages. Moreover, these findings provide pathophysiological and therapeutic insights regarding MSC participation in adipogenesis and osteogenesis.

Glutathione reductase-deficient erythrocytes as host cells of malarial parasites.

BCNU [1,3-bis(2-chloroethyl)-1-nitrosourea] and its less toxic derivative HeCNU [1-(2-chloroethyl)-3-(2-hydroxyethyl)-1-nitrosourea] are clinically-used antitumour drugs. In erythrocytes BCNU is a highly specific inhibitor of the enzyme glutathione reductase [H. Frischer and T. Ahmad, J. Lab. clin. Med. 89, 1080 (1977)]. When treating erythrocytes in vitro, 50% enzyme inhibition was obtained with 1 microM BCNU or 3 microM HeCNU within 2 hr. The two drugs were used for preparing red cell populations with various levels of glutathione reductase activity; complete inhibition (greater than or equal to 98%) was only achieved when the medium contained glucose as a source of reducing equivalents. The erythrocytes were then tested in drug-free media as host cells for the malaria parasite Plasmodium falciparum. In the range of 0-300 mU/ml cells, there was a correlation between glutathione reductase activity and parasite growth; erythrocytes with an activity of less than 20 mU/ml did not serve as host cells for P. falciparum at all although these erythrocytes were viable. When the culture medium was supplemented with 20 mM glutathione (GSH), parasite growth was normal irrespective of the glutathione reductase level in the erythrocytes. This is consistent with the finding that poisoning glutathione reductase led to a 10-fold decrease of the cytosolic GSH level. Our results corroborate the concept that intraerythrocytic inhibition of glutathione reductase mimicks the biochemistry of drug-sensitive glucose-6-phosphate dehydrogenase deficiency (favism), an inherited condition which confers protection from malaria.

Disorders of respiratory burst termination.

The biochemical mechanisms for the termination of the respiratory burst are likely to be multifactorial. Alterations in the endogenous oxidant-scavenging mechanisms can modulate the kinetics of the termination phase of neutrophil superoxide production. These are most apparent when those alterations are found in the neutrophils of certain patients. The use of inhibitors of the oxidant scavenging systems in normal neutrophils has not yielded results that exactly mimic the studies with neutrophils from the patients. For example, the glutathione reductase-inhibited neutrophils (from the action of BCNU) do not show the same degree of abnormality as do the neutrophils from the patient with glutathione reductase deficiency. Further investigations on the mechanisms of inactivation of the NADPH oxidase are warranted in order to gain a greater understanding of this important regulating mechanism.

Lens capsule and epithelium in age-related cataract.

On the basis of preoperative assessment of patient characteristics, intraoperative obtainment of a lens-capsule and epithelium specimen, histopathologic investigation of lens capsule and epithelium, and biochemical analysis of glutathione reductase in lens epithelium, age-related cataract was studied in 50 adult patients who underwent consecutive extracapsular cataract-posterior chamber lens implant surgery. Patients (25 men and 25 women; age range, 41 to 91 years; mean age, 75 years) had a wide range of systemic and ocular disease; 17 of 50 (34%) patients had a history of severe vision-impairing cataract in a first-degree relative. Anterior lens-capsule thickness ranged from 10 to 22 microns, with a mean of 17 microns. Statistical analysis of lens-epithelium ultrastructure in 41 of 50 specimens documented mixing of normal and abnormal cells, verified a gradation in the degree of abnormal ultrastructural features, and demonstrated a statistically significant decrease in epithelial cytologic activity with advancing age (P = .038). Biochemical analysis documented a severe glutathione reductase deficiency in nine of 39 (23%) lens-epithelium specimens, possibly reflecting a dietary deficiency of riboflavin.

Enzyme deficiencies in neonates with jaundice.

Numerous findings have shown that enzyme deficiencies, especially those involved in the protection of red cells from oxidation may lead to hemolysis and hyperbilirubinemia. It is established that G6PD deficiency may be the cause of neonatal hyperbilirubinemia, as has been found in several countries and among widely different ethnic groups. We try to establish the incidence of G6PD, PK and GSSG-R deficiencies in neonates with jaundice for a better assessment of the population at risk. The present investigation was carried out in the attempt to be certain whether these enzymes could play a part in the development of neonatal jaundice. A total of 341 neonates of both sexes with jaundice were analyzed: 47 with G6PD deficiency; 9 with PK deficiency and 2 with GSSG-R deficiency.

Erythrocyte enzyme disorders in children.

Erythrocyte metabolic abnormalities should be considered as a possible cause of hemolysis when there is no evidence of an immune-mediated hemolytic anemia, no consumptive red blood cell disorder, no morophologic or laboratory data to suggest a problem of the red cell membrane, and no evidence of a quantitative or qualitative defect in hemoglobin synthesis. Glucose-6-phosphate dehydrogenase deficiency is clearly the most common enzyme deficiency causing clinical problems.

Glutathione reductase in the red blood cells.

Glutathione reductase plays an important role in protecting hemoglobin, red cell enzymes, and biological cell membranes against oxidative damage by increasing the level of reduced glutathone (GSSGR) in the process of aerobic glycolysis. The enzyme deficiency may result in mild to moderately severe hemolytic anemia upon exposure to certain drugs or chemicals. However, hereditary deficiency of the enzyme is extremely rare. Recent studies on glutathione reductase in the red cell have shown more insight in the understanding of red cell metabolism and interactions with other enzymes, especially glucose-6-phosphate dehydrogenase (G-6-PD). Glutathione reducatase in serum may be a source of error in any clinical laboratory test in which an enzyme activity is determined indirectly by measuring the change in reduced nicotinamide-adenine dinucleotide (NADH) or reduced nicotinamide adenine dinucleotide phosphate (NADPH) absorbance. Glutathione reductase levels are reduced in banked blood when citrate-phosphate-dextrose (CPD) is used as a preservative. Reviewed is the role of glutathione reductase in the metabolism of the red cell and its clinical implication and usefulness.

Transfusion requirements of patients with enzyme deficient red blood cells.

Red cell enzyme deficiency disease states are reviewed to outline the requirements for transfusion therapy in the neonatal period, during spontaneous crises, drug or infection-induced crises and chronic anemia.

[Relationship between vitamin status (A, B1, B2, B6, and C), clinical features and nutritional habits in a population of old people (author's transl)]. / Beziehungen zwischen dem Vitaminstatus (Vitamin A, B1, B2, B6 und C), klinischen Befunden und den Ernährungsgewohnheiten in einer Gruppe von alten Leuten in Wien.

112 patients in a medical ward were examined clinically and biochemically with regard to their vitamin status (plasma vitamin A, plasma vitamin C, blood glutathione reductase, vitamin B1, vitamin B2, N-methyl nicotinic acid amide and pyridoxic acid excretion). The nutritional habits were assessed by means of a questionnaire. The results of these two examinations were correlated with the clinical findings. The assessment of the vitamin B2 status showed a deficiency in 8 cases and a marginal vitamin B2 supply in a further 4 cases. The thiamine intake was insufficient in 43 cases and marginal in 42 cases. The biochemical assessment of vitamins C, B1 and PP indicated a deficiency of these vitamins. Dermatological signs pointed to a deficiency of vitamins A and B2. There was a significant correlation between the plasma vitamin A level and the serum iron level. The importance of milk and vegetables in the diet is stressed and also the association between milk consumption and the serum cholesterol level.

[Glucose-6-phosphate dehydrogenase deficiency and other erythrocyte enzyme abnormalities]. / Glukose-6-Phosphatdehydrogenasemangel und andere Enzymanomalien in Erythrozyten.

Normal values of glucose-6-phosphate dehydrogenase (G6PD), 6-phosphogluconate dehydrogenase (6PGD), glutathione reductase (GR), glucosephosphate isomerase (GPI), pyruvate kinase (PK) and pyrimidine 5'-nucleotidase (P5N) have been determined in normocytes, reticulocytes, newborn cord erythrocytes, and leucocytes. Metabolic and clinical aspects of G6PD and the classification of its genetic variants are reviewed. Enzyme determinations and their variation in drug-induced haemolysis are critically presented. Extensive tables are published listing the drugs and compounds that can cause haemolysis in G6PD-deficient patients, as well as those preparations which may, probably, be administered safely. Clinical and biochemical data in patients with the inherited enzyme defects GR, GPI, PK, and P5N, as well as acquired deficiency of the last-mentioned in chronic lead intoxication, are reviewed in the light of our personal experience in this field.

Glutathione reductase deficiency in Saudi Arabia.

Glutathione reductase (GR) is a ubiquitous enzyme required for the conversion of oxidized glutathione (GSSG) to reduced glutathione (GSH) concomitantly oxidizing reduced nicotinamide adenine dinucleotide phosphate (NADPH) in a reaction essential for the stability and integrity of red cells. Mutations in the GR gene and nutritional deficiency of riboflavin, a co-factor required for the normal functioning of GR, can cause GR deficiency. We conducted a study on 1691 Saudi individuals to determine the overall frequency of GR deficiency and to identify whether the deficiency results from genetic or acquired causes or both. The activity of GR was measured in freshly prepared red cell haemolysate in the presence and absence of flavin adenine dinucleotide (FAD) and the activity coefficient (AC) was determined. Samples with low GR activity (> 2.0 IU/g haemoglobin) both in the presence and absence of FAD and an AC between 0.9 and 1.2 were considered GR-deficient. Samples with AC > or = 1.3 were considered riboflavin-deficient. The overall frequency of partial GR deficiency was 24.5% and 20.3% in males and females respectively. In addition, 17.8% of males and 22.4% of females suffered from GR deficiency due to riboflavin deficiency. This could be easily corrected by dietary supplementation with riboflavin. No cases of severe GR deficiency were identified.