Scientific Studies of THQueen

J Nutr Biochem 2007 Mar 13

A review of the interaction among dietary antioxidants and reactive oxygen species.

Seifried HE, et al

Division of Cancer Prevention, National Cancer Institute, Rockville, MD 20862, USA.

During normal cellular activities, various processes inside of cells produce reactive oxygen species (ROS). Some of the most common ROS are hydrogen peroxide (H(2)O(2)), superoxide ion (O(2)(-)), and hydroxide radical (OH(-)). These compounds, when present in a high enough concentration, can damage cellular proteins and lipids or form DNA adducts that may promote carcinogenic activity. The purpose of antioxidants in a physiological setting is to prevent ROS concentrations from reaching a high-enough level within a cell that damage may occur. Cellular antioxidants may be enzymatic (catalase, glutathione peroxidase, superoxide dismutase) or nonenzymatic (glutathione, thiols, some vitamins and metals, or phytochemicals such as isoflavones, polyphenols, and flavanoids). Reactive oxygen species are a potential double-edged sword in disease prevention and promotion. Whereas generation of ROS once was viewed as detrimental to the overall health of the organism, advances in research have shown that ROS play crucial roles in normal physiological processes including response to growth factors, the immune response, and apoptotic elimination of damaged cells. Notwithstanding these beneficial functions, aberrant production or regulation of ROS activity has been demonstrated to contribute to the development of some prevalent diseases and conditions, including cancer and cardiovascular disease (CVD). The topic of antioxidant usage and ROS is currently receiving much attention because of studies linking the use of some antioxidants with increased mortality in primarily higher-risk populations and the lack of strong efficacy data for protection against cancer and heart disease, at least in populations with adequate baseline dietary consumption. In normal physiological processes, antioxidants effect signal transduction and regulation of proliferation and the immune response. Reactive oxygen species have been linked to cancer and CVD, and antioxidants have been considered promising therapy for prevention and treatment of these diseases, especially given the tantalizing links observed between diets high in fruits and vegetables (and presumably antioxidants) and decreased risks for cancer.

Indian J Med Science 2003 Aug;57(8):335-7.

Low blood glutathione levels in acute myocardial infarction.

Kharb S.

Department of Biochemistry, Pt. B.D. Sharma PGIMS, Rohtak, Haryana. simmikh@rediffmail.com

BACKGROUND: Although experimental studies have demonstrated that reduced glutathione (GSH) is involved in cellular protection from deleterious effects of oxygen free radicals in ischaemia and reperfusion, there are controversial data on the correlation between levels of GSH and the ischaemic process. AIM: The present study was planned to evaluate erythrocyte GSH levels in patients with acute myocardial infarction (AMI). SETTING & DESIGN: Erythrocyte GSH levels were determined in 22 patients with AMI and 15 age matched healthy volunteers served as control. MATERIAL & METHODS: Erythrocyte GSH levels were measured by using Bentler in AMI and control patients. Also lipid profile was analyzed enzymatically in these subject. STATISTICS: The values were expressed as means +/- standard deviation (SD) and data from patients and controls was compared using student's 't'-test. Results and CONCLUSION: Gluthathione (GSH) levels were significantly decreased in patients with acute myocardial infarction (AMI) as compared to control (p<0.001). Also, total cholesterol and triglycerides were higher is AMI subjects (p<0.05). These finding suggest that depressed GSH levels may be associated with enhanced protective mechanism to oxidative stress in AMI.

PMID: 12944689

Med Sci Monitor 2001 May-Jun;7(3):415-20. =

Increased plasma glutathione peroxidase activity in patients with acute myocardial infarction.

Zachara BA, et al

Department of Biochemistry, The Ludwik Rydygier Medical University, 24 Karlowicza Str, 85-092 Bydgoszcz, Poland. bronz@aci.amb.bydgoszcz.pl

BACKGROUND: Aim of the study was to determine the concentration of selenium (Se) and the activity of glutathione peroxidase (GSH-Px) in patients with acute myocardial infarction (AMI) and to observe the behavior of these parameters during thrombolysis therapy. MATERIAL AND METHODS: The study comprised two groups of AMI patients and a control group. The first group consisted of 49 patients from whom blood samples were taken after admission to the intensive care unit and subsequently after 3, 7, 14 and 30 days of hospitalization. In the second group of patients (n = 18) blood was taken for measuring only the GSH-Px activity in plasma. In this group blood samples were collected after admission to the hospital, 6, 12, 24, 48 hours, 3, 7, 14 and 30 days later. Control group comprised of 58 healthy subjects. Se levels in whole blood and plasma were measured spectrofluorometrically with 2,3-diaminonaphthalene as a complexing reagent. GSH-Px activity in red cells and plasma was measured spectrofluorometrically with t-butyl hydroperoxide as substrate. RESULTS: In the first group of patients Se concentrations in whole blood and plasma as well as GSH-Px activities in red cells and plasma did not differ significantly from healthy subjects. Both Se levels and GSH-Px activities were stable during the entire period of the study. In the second group of patients, however, plasma GSH-Px activity increased after admission and reached the highest value after 48 hours. This activity was significantly higher compared to healthy subjects (p < 0.004) and to the mean initial activity of this group (p < 0.02). In the later period the activity decreased to the values of healthy subjects. CONCLUSION: We suggest that the increased activity of glutathione peroxidase (GSH-Px) in plasma of acute myocardial infarction (AMI) patients is the response of the organism to the increased levels of reactive oxygen species produced during reperfusion and thrombolysis.

PMID: 11386018

Exp Neurol 2007 Feb;203(2):512-20. Epub 2006 Oct 17.

Characterization of intracellular elevation of glutathione (GSH) with glutathione monoethyl ester and GSH in brain and neuronal cultures: relevance to Parkinson's disease.

Zeevalk GD, et al

Department of Neurology, UMDNJ-Robert Wood Johnson Medical School, Building UBHC, Rm. 405D, 675 Hoes Lane, Piscataway, NJ 08854, USA. zeevalgd@umdnj.edu

Parkinson's disease (PD) is associated with loss of total glutathione (GSH) which may contribute to progressive cell death. Peripheral GSH administration has been used clinically with reported benefits. Despite this, there is little specific information to characterize its cellular uptake or clearance, brain elevation with peripheral delivery or neuroprotective efficacy in PD models. The current study was carried out to provide this information using in vitro and in vivo approaches. In rat mesencephalic culture, the monoethyl ester of GSH (GEE), but not GSH (1-10 mM, 24 h) produced a dose-dependent elevation in GSH. The half-life for clearance was 10.14 h and was not different in cells depleted of GSH prior to loading. Elevation of GSH with GEE protected neurons from oxidative stress with H2O2 or metabolic stress with the complex I and II inhibitors MPP+ and malonate, respectively. To determine if peripheral administration of GEE could elevate brain GSH levels, rats were administered 0.1-50 mg/kg/day GEE via osmotic minipump either subcutaneously (sc) or via a cannula placed into the left cerebral ventricle (icv) for 28 days. Only central delivery of GEE resulted in significant elevations of brain GSH. Elevation of brain GSH by icv infusion of GEE was examined for its neuroprotective effects against chronic central delivery of MPP+. Infusion of 0.142 mg/kg/day MPP+ for 28 days caused a selective ipsilateral loss of striatal dopamine. Co-infusion of MPP+ with 10 mg/kg/day GEE significantly protected against striatal dopamine loss. These findings show that the ethyl ester of GSH but not GSH per se can elevate intracellular GSH, that brain elevation of GSH requires central delivery of the ethyl ester and that this elevation provides neuroprotection against oxidative stress or chronic mitochondrial impairment.

PMID: 17049515

AIDS Res Ther 2006 Feb 20;3:5.

Glutathione and growth inhibition of Mycobacterium tuberculosis in healthy and HIV infected subjects.

Venketaraman V, et al

Division of Infectious Diseases, UMDNJ-New Jersey Medical School, Newark, NJ 07103, USA. venketvi@umdnj.edu.

ABSTRACT : Intracellular levels of glutathione are depleted in patients with acquired immunodeficiency syndrome in whom the risk of tuberculosis, particularly disseminated disease is many times that of healthy individuals. In this study, we examined the role of glutathione in immunity against tuberculosis infection in samples derived from healthy and human immunodeficiency virus infected subjects. Our studies confirm that glutathione levels are reduced in peripheral blood mononuclear cells and in red blood cells isolated from human immunodeficiency virus-infected subjects (CD4>400/cumm). Furthermore, treatment of blood cultures from human immunodeficiency virus infected subjects with N-acetyl cysteine, a glutathione precursor, caused improved control of intracellular M. tuberculosis infection. N-acetyl cysteine treatment decreased the levels of IL-1, TNF-alpha, and IL-6, and increased the levels of IFN-gamma in blood cultures derived from human immunodeficiency virus-infected subjects, promoting the host immune responses to contain M. tuberculosis infection successfully.

PMID: 16504020

J Biochem Mol Toxicology 2006;20(1):39-47.

Glutathione-enhancing agents protect against steatohepatitis in a dietary model.

Oz HS, et al

Department of Internal Medicine, Digestive Diseases and Nutrition, University of Kentucky Medical Center, Lexington, KY 40536, USA. helieh.oz@uky.edu

Nonalcoholic fatty liver (NAFL) and steatohepatitis (NASH) may accompany obesity, diabetes, parenteral nutrition, jejeuno-ileal bypass, and chronic inflammatory bowel disease. Currently there is no FDA approved and effective therapy available. We investigated the potential efficacy of those agents that stimulate glutathione (GSH) biosynthesis on the development of experimental steatohepatitis. Rats fed (ad libitum) amino acid based methionine-choline deficient (MCD) diet were further gavaged with (1) vehicle (MCD), (2) S-adenosylmethionine (SAMe), or (3) 2(RS)-n-propylthiazolidine-4(R)-carboxylic acid (PTCA). RESULTS: MCD diet significantly reduced hematocrit, and this abnormality improved in the treated groups (p < 0.01). Serum transaminases were considerably elevated (AST: 5.8-fold; ALT: 3.22-fold) in MCD rats. However, administration of GSH-enhancing agents significantly suppressed these abnormal enzyme activities. MCD rats developed severe liver pathology manifested by fatty degeneration, inflammation, and necrosis, which significantly improved with therapy. Blood levels of GSH were significantly depleted in MCD rats but normalized in the treated groups. Finally, RT-PCR measurements showed a significant upregulation of genes involved in tissue remodeling and fibrosis (matrix metalloproteinases, collagen-alpha1), suppressor of cytokines signaling1, and the inflammatory cytokines (IL-1beta, IL-6, TNF-alpha, and TGF-beta) in the livers of rats fed MCD. GSH-enhancing therapies significantly attenuated the expression of deleterious proinflammatory and fibrogenic genes in this dietary model. This is the first report that oral administration of SAMe and PTCA provide protection against liver injury in this model and suggests therapeutic applications of these compounds in NASH patients. 2006 Wiley Periodicals, Inc.

Amer J Physiol Heart Circ Physiol 2005 Aug;289(2):H768-76. Epub 2005 Apr 1.

Cardiomyocyte apoptosis induced by short-term diabetes requires mitochondrial GSH depletion.

Ghosh S, et al

Division of Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences, Univ. of British Columbia, 2146 East Mall, Vancouver, BC, Canada V6T 1Z3.

Oxidative stress due to excessive reactive oxygen species (ROS) and depleted antioxidants such as glutathione (GSH) can give rise to apoptotic cell death in acutely diabetic hearts and lead to heart disease. At present, the source of these cardiac ROS or the subcellular site of cardiac GSH loss [i.e., cytosolic (cGSH) or mitochondrial (mGSH) GSH] has not been completely elucidated. With the use of rotenone (an inhibitor of the electron transport chain) to decrease the excessive ROS in acute streptozotocin (STZ)-induced diabetic rat heart, the mitochondrial origin of ROS was established. Furthermore, mitochondrial damage, as evidenced by loss of membrane potential, increases in oxidative stress, and reduction in mGSH was associated with increased apoptosis via increases in caspase-9 and -3 activities in acutely diabetic hearts. To validate the role of mGSH in regulating cardiac apoptosis, L-buthionine-sulfoximine (BSO; 10 mmol/kg ip), which blocks GSH synthesis, or diethyl maleate (DEM; 4 mmol/kg ip), which inactivates preformed GSH, was administered in diabetic rats for 4 days after STZ administration. Although both BSO and DEM lowered cGSH, they were ineffective in reducing mGSH or augmenting cardiomyocyte apoptosis. To circumvent the lack of mGSH depletion, BSO and DEM were coadministered in diabetic rats. In this setting, mGSH was undetectable and cardiac apoptosis was further aggravated compared with the untreated diabetic group. In a separate group, GSH supplementation induced a robust amplification of mGSH in diabetic rat hearts and prevented apoptosis. Our data suggest for the first time that mGSH is crucial for modulating the cell suicide program in short-term diabetic rat hearts.

PMID: 15805231

Intensive Care Medicine 2005 Aug;31(8):1072-8. Epub 2005 Jul 6.

Temporal changes in whole-blood and plasma glutathione in ICU patients with multiple organ failure.

Flaring UB, et al

Department of Anesthesia and Intensive Care, Astrid Lindgren Children's Hospital, Karolinska University Hospital, 17176, Stockholm, Sweden. urban.flaring@karolinska.se

OBJECTIVE: This study investigated the temporal changes in whole-blood and plasma glutathione in ICU patients with multiple organ failure. DESIGN AND SETTING: Prospective and descriptive pilot study performed in an ICU with eight beds at a university hospital. PATIENTS: Critically ill patients (n=11) with multiple organ failure and ICU stay of at least 6 days were consecutively included. Patients with chronic obstructive pulmonary disease (n=21) and healthy volunteers (n=10) were used as reference groups. MEASUREMENTS AND RESULTS: Whole-blood and plasma glutathione were measured every 72 h. Total glutathione and the reduced fraction were determined in whole blood. The oxidized fraction and the redox status were calculated from these values. In plasma only the total concentration was determined. Patients were studied for 6-15 days. Nutrition was supplied according to routines supplying basal needs including glutamine. Both total and reduced glutathione was found to be depleted in whole blood compared to the reference groups. Redox status indicated continuing oxidative stress. Plasma glutathione showed higher values in total concentrations than the reference groups. CONCLUSIONS: This study demonstrates that glutathione remains depleted in whole blood. This contrasts to what has previously been shown in skeletal muscle where a restitution of glutathione concentration is seen.

PMID: 15999254

Antioxid Redox Signal 2005 Jul-Aug;7(7-8):900-10.

Glutathione depletion in a midbrain-derived immortalized dopaminergic cell line results in limited tyrosine nitration of mitochondrial complex I subunits: implications for Parkinson's disease.

Bharath S, Andersen JK

Buck Institute for Age Research, Novato, CA 94945, USA.

Oxidative stress and mitochondrial dysfunction signify two important biochemical events associated with the loss of dopaminergic neurons in Parkinson's disease (PD). Studies using in vitro and in vivo PD models and in affected tissues from the disease itself have demonstrated a selective inhibition of mitochondrial complex I activity that appears to affect normal mitochondrial physiology leading to neuronal cell death. Earlier experiments from our laboratory have demonstrated that induced depletion of glutathione (GSH + GSSG) in cultured dopaminergic cells resulted in increased oxidative stress and a decrease in mitochondrial function. Furthermore, this dysfunction was linked to a selective decrease in mitochondrial complex I activity that appears to be due to oxidation of this complex. Glutathione depletion is the earliest detectable biochemical event during PD progression and occurs prior to complex I inhibition. Recent observations have also indicated that oxidative damage to complex I via naturally occurring free radicals such as peroxynitrite leads to modification of tyrosine and/or cysteine residues resulting in complex I inhibition. Using the sucrose gradient method, we detected in complex I-enriched fractions from a glutathione-depleted dopaminergic cell line two bands corresponding to approximately 25-kDa and approximately 30-kDa polypeptides that demonstrate anti-nitrotyrosine immunoreactivity, suggesting the possible involvement of protein nitration by peroxynitrite in glutathione depletion-mediated complex I inhibition.

PMID: 15998245

Parkinsonism Relat Disorders 2002 Sep;8(6):385-7.

Glutathione depletion and oxidative stress.

Mytilineou C, et al

Department of Neurology, Mount Sinai School of Medicine, Box 1137, New York, NY 10029, USA. catherine.mytilneou@mssm.edu

Oxidative stress is believed to contribute to the pathogenesis of Parkinson's disease. One of the indices of oxidative stress is the depletion of the antioxidant glutathione (GSH), which may occur early in the development of Parkinson's disease. To study the role of GSH depletion in the survival of dopamine neurons we treated mesencephalic cultures with the GSH synthesis inhibitor L-buthionine sulfoximine. Our studies have shown that the depletion of GSH causes a cascade of events, which ultimately may result in cell death. An early event following GSH depletion is a phospholipase A(2)-dependent release of arachidonic acid. Arachidonic acid can cause damage to the GSH-depleted cells through its metabolism by lipoxygenase. The generation of superoxide radicals during the metabolism of arachidonic acid is likely to play an important role in the toxic events that follow GSH depletion.

PMID: 12217624

Alcohol 2002 Jul;27(3):179-83.

S-Adenosyl-L-methionine and mitochondrial reduced glutathione depletion in alcoholic liver disease.

Fernandez-Checa JC, et al

Liver Unit, Hospital Clinic I Provincial, 08036 Barcelona, Spain. checa@medicina.ub.es

The pathogenesis of alcohol-induced liver disease is not well understood, and many factors have been described to contribute to the progressive loss of liver functions, including the overgeneration of reactive oxygen species. Mitochondria are specific targets of the toxic effects of ethanol, reflected in the loss of phosphorylative oxidation and defective ATP generation, which underlie one of the hallmarks of the hepatic alterations induced by chronic alcohol intake. Mitochondrial reduced glutathione (GSH), whose primary function is to maintain a competitive functional organelle, becomes depleted by alcohol intake. Furthermore, GSH depletion in hepatocyte mitochondria has been revealed as an important mechanism in the sensitization of liver to alcohol-induced injury. This depletion of the mitochondrial GSH level is determined by an impaired transport of GSH from the cytosol into the mitochondrial matrix owing to a partial inactivation of mitochondrial GSH carrier. The loss of function of this specific mitochondrial transporter is due to the alterations in the physicochemical properties of the inner mitochondrial membrane caused by alcohol. Because of the primary defect in the transport of cytosolic GSH into mitochondria, GSH precursors are inefficient in replenishing the levels of mitochondrial GSH despite significant increase in cytosolic GSH. Supplementation of S-adenosyl-L-methionine (SAM) to rats fed alcohol chronically has been shown to replete the mitochondrial GSH levels because of normalization of the microviscosity of the mitochondrial inner membrane. Because of the instrumental role of GSH in mitochondria in hepatocyte survival against inflammatory cytokines, its repletion by SAM feeding may underlie the potential therapeutic use of this hepatoprotective agent in the treatment of alcohol-induced liver injury.

PMID: 12163147

Pancreatology 2002;2(1):34-9.

Effect of chronic hypoxia on glutathione status and membrane integrity in the pancreas.

Ip SP, et al

School of Chinese Medicine, Chinese University of Hong Kong, Shatin, NT, Hong Kong SAR.

BACKGROUND: Our recent study has shown that chronic hypoxia could upregulate significantly a local renin-angiotensin system in the pancreas. The activation of such a local renin-angiotensin system may provide an alternate mechanism that leads to the generation of reactive radical species in the pancreas during chronically hypoxic exposure. The present study aims at elucidating the antioxidant status in the pancreas during varying degrees of chronic hypoxia. METHODS: Sprague-Dawley rats were exposed to an isobaric hypoxic (10% oxygen) chamber for a period up to 28 days. The glutathione status and membrane integrity of the pancreas were studied with a time course of chronic hypoxia (3, 7, 14, 21 and 28 days). The effect of chronic hypoxia on changes of oxidative states in the pancreas was assessed based on the measurements of glutathione, malondialdehyde, alpha-amylase and DNA fragmentation using biochemical assays. RESULTS: Pancreatic glutathione was decreased drastically after 3-day hypoxia and its level was almost completely recovered after 7-day hypoxia. Malondialdehyde was not affected while DNA fragmentation was increased significantly in a time-dependent manner during the course of chronic hypoxia. Membrane integrity of the pancreatic cells was improved, as evidenced by the decrease of plasma alpha-amylase during the time-course study of chronic hypoxia. CONCLUSION: Pancreatic glutathione was depleted only in the early period of chronic hypoxia followed by a rapid recovery, suggesting that adaptive response of the pancreas may occur during chronic hypoxia. The enhancement of glutathione-dependent antioxidant capacity during chronic hypoxia prevented oxidative damage to the membrane of the pancreatic cells.

PMID: 12120004

Eur J Gastroenterol Hepatol 2001 Nov;13(11):1309-13.

Low levels of gastric mucosal glutathione during upper gastric bleeding associated with the use of nonsteroidal anti-inflammatory drugs.

Savoye G, et al

The Digestive Tract Research Group, EA 3234, IFR MP 23, Rouen University Hospital, Rouen, France. g_savoye@hotmail.com

OBJECTIVES: To investigate the glutathione concentrations in gastric mucosa from patients with acute gastric bleeding related to nonsteroidal anti-inflammatory drugs (NSAIDs), and to test the influence of nutritional status on mucosal glutathione. Glutathione protects the gastrointestinal mucosa against reactive oxygen species, and glutathione content in various tissues may be depleted during malnutrition. METHODS: Endoscopic biopsies were obtained from 39 patients. Eighteen of these (9 well-nourished, 9 malnourished) presented with gastric bleeding ulcers related to NSAIDs. Twenty-one other patients (12 well-nourished, 9 malnourished) underwent normal routine diagnostic endoscopy and served as controls. Malnutrition was defined as a loss of over 10% of normal body weight and/or plasma albumin levels below 30 g/l. Gastric biopsies were taken from the fundus and antrum (controls) and from the region of the ulcer (patients with acute bleeding) and frozen quickly until glutathione analysis by high-performance liquid chromatography (HPLC) coulometric detection. Results were expressed as nmol/mg wet tissue. RESULTS: Gastric mucosal glutathione levels were significantly (P < 0.05) lower in both the antrum (0.81 +/- 0.34 v. 1.41 +/- 0.88 nmol/mg tissue) and the fundus (1.04 +/- 0.54 v. 1.43 +/- 0.92 nmol/mg tissue, P < 0.05) in malnourished than in well-nourished control patients. Glutathione mucosal concentrations were decreased significantly in patients with NSAID-induced gastric bleeding compared with control patients (0.38 +/- 0.36 v. 1.12 +/- 0.56 nmol/mg tissue, P < 0.001), and the lowest glutathione levels were observed in malnourished patients (0.28 +/- 0.20 v. 0.48 +/- 0.15 nmol/mg tissue in well-nourished patients, not significant). CONCLUSION: Malnutrition is associated with low levels of gastric glutathione. This may contribute to the severity and the onset of haemorrhage in NSAID-induced gastric ulcers.

PMID: 11692056

Mech Ageing Dev 2000 Dec 20;121(1-3):217-30.

Antioxidants may contribute in the fight against ageing: an in vitro model.

Hu HL, et al

Molecular Physiology, Unilever Research Laboratory Colworth, Sharnbrook, Bedford MK44 1LQ, UK.

Elderly humans have altered cellular redox levels and dysregulated immune responses, both of which are key events underlying the progression of chronic degenerative diseases of ageing, such as atherosclerosis and Alzeimer's disease. Poorly maintained cellular redox levels lead to elevated activation of nuclear transcription factors such as NFkB and AP-1. These factors are co-ordinately responsible for a huge range of extracellular signaling molecules responsible for inflammation, tissue remodeling, oncogenesis and apoptosis, processes that orchestrate many of the degenerative processes associated with ageing. It is now clear that levels of endogenous anti-oxidants such as GSH decrease with age. This study aimed to investigate the potential of exogenous anti-oxidants to influence inflammatory responses and the ageing process itself. We investigated the potential of the dietary antioxidant, quercetin, to reverse the age related influences of GSH depletion and oxidative stress using in vitro human umbilical vein endothelial cells (HUVEC) and human skin fibroblast (HSF) cell models. Oxidative stress-induced inflammatory responses were investigated in a GSH depletion and a Phorbol 12-myristate 13-acetate (PMA)-induced stress model. As measured with a sensitive HPLC fluorescence method, GSH in HUVEC was depleted by the addition of L-buthionine-[S,R]-sulfoxiniine (BSO), a gamma-glutamylcysteine synthetase inhibitor, to the culture medium at a concentration of 0.25 mM. Time course studies revealed that the GSH half-life was 4.6 h in HUVEC. GSH depletion by BSO for 24 h led to a slight increase in intracellular adhesion molecule - 1 (ICAM1) expression and prostaglandin E2 (PGE2) secretion in both types of cells. However, GSH depletion markedly enhanced PMA-induced ICAM and PGE2 production in HUVEC. Responses were progressively elevated following prolonged BSO treatment. Inhibition studies showed that 1-(5-Isoquinolinylsulfonyl)-2-methylpiperazine (H7), a protein kinase C (PKC) inhibitor, not only abolished most of PMA-induced ICAM-1 expression and PGE2, production, but also eliminated GSH depletion-enhanced PMA stimulation. This enhancement was also inhibited by supplementation with quercetin. The results clearly demonstrate that GSH depletion increased the susceptibility of vascular endothelial cells and fibroblasts to oxidative stress associated inflammatory stimuli. This increased in vitro susceptibility may be extrapolated to the in vivo situation of ageing, providing a useful model to study the influence of micronutrients on the ageing process. In conclusion, these data suggest that dietary antioxidants could play a significant role in the reduction of inflammatory responses.

PMID: 11164475

Amer j Pathol 2000 Jun;156(6):2045-56.

Depletion of hepatic glutathione prevents death receptor-dependent apoptotic and necrotic liver injury in mice.

Hentze H, et al.

Department of Biochemical Pharmacology, University of Konstanz, Germany.

The activation of the death receptors, tumor necrosis factor-receptor-1 (TNF-R1) or CD95, is a hallmark of inflammatory or viral liver disease. In different murine in vivo models, we found that livers depleted of gamma-glutamyl-cysteinyl-glycine (GSH) by endogenous enzymatic conjugation after phorone treatment were resistant against death receptor-elicited injury as assessed by transaminase release and histopathology. In apoptotic models initiated by engagement of CD95, or by injection of TNF or lipopolysaccharide into galactosamine-sensitized mice, hepatic caspase-3-like proteases were not activated in the GSH-depleted state. Under GSH depletion, also caspase-independent, TNF-R1-mediated injury (high-dose actinomycin D or alpha-amanitin), as well as necrotic hepatotoxicity (high-dose lipopolysaccharide) were entirely blocked. In the T-cell-dependent model of concanavalin A-induced hepatotoxicity, GSH depletion resulted in a suppression of interferon-gamma release, delay of systemic TNF release, hepatic nuclear factor-kappaB activation, and an abrogation of sinusoidal endothelial cell detachment as assessed by electron microscopy. When GSH depletion was initiated 3 hours after concanavalin A injection, ie, after the peak of early pro-inflammatory cytokines, livers were still protected. We conclude that sufficient hepatic GSH levels are a prerequisite for the execution of death receptor-mediated hepatocyte demise.

PMID: 10854226

Amer J. Physiol 1999 Dec;277(6 Pt 1):L1067-88.

Lung glutathione and oxidative stress: implications in cigarette smoke-induced airway disease.

Rahman I, MacNee W.

Department of Respiratory Medicine, Medical School, University of Edinburgh, Edinburgh EH8 9AG, United Kingdom. ir@srv1.med.ed.ac.uk

Glutathione (GSH), a ubiquitous tripeptide thiol, is a vital intra- and extracellular protective antioxidant in the lungs. The rate-limiting enzyme in GSH synthesis is gamma-glutamylcysteine synthetase (gamma-GCS). The promoter (5'-flanking) region of the human gamma-GCS heavy and light subunits are regulated by activator protein-1 and antioxidant response elements. Both GSH and gamma-GCS expression are modulated by oxidants, phenolic antioxidants, and inflammatory and anti-inflammatory agents in lung cells. gamma-GCS is regulated at both the transcriptional and posttranscriptional levels. GSH plays a key role in maintaining oxidant-induced lung epithelial cell function and also in the control of proinflammatory processes. Alterations in alveolar and lung GSH metabolism are widely recognized as a central feature of many inflammatory lung diseases including chronic obstructive pulmonary disease (COPD). Cigarette smoking, the major factor in the pathogenesis of COPD, increases GSH in the lung epithelial lining fluid of chronic smokers, whereas in acute smoking, the levels are depleted. These changes in GSH may result from altered gene expression of gamma-GCS in the lungs. The mechanism of regulation of GSH in the epithelial lining fluid in the lungs of smokers and patients with COPD is not known. Knowledge of the mechanisms of GSH regulation in the lungs could lead to the development of novel therapies based on the pharmacological or genetic manipulation of the production of this important antioxidant in lung inflammation and injury. This review outlines 1) the regulation of cellular GSH levels and gamma-GCS expression under oxidative stress and 2) the evidence for lung oxidant stress and the potential role of GSH in the pathogenesis of COPD.

PMID: 10600876

Neurology 1998 Dec;51(6):1562-6.

Decreased glutathione transferase activity in brain and ventricular fluid in Alzheimer's disease.

Lovell MA, et al

Department of Chemistry, Sanders-Brown Center on Aging, University of Kentucky, Lexington 40536-0230, USA.

OBJECTIVE: To investigate the levels of glutathione transferase (GST), a protective enzyme against aldehydes, and especially 4-hydroxynonenal (HNE) in the brain and ventricular CSF of autopsied AD and normal control subjects. BACKGROUND: Studies have implicated increased levels of oxidative stress in the brain in the pathogenesis of AD. Decreased levels of polyunsaturated fatty acids and increased levels of markers of lipid peroxidation have been reported in the brain in AD, particularly in areas severely affected in the disease. HNE, one marker of lipid peroxidation, is neurotoxic in neuronal culture and in vivo and is elevated in AD brain and CSF. METHODS: We measured levels of GST activity and protein in multiple brain regions and ventricular CSF in short-postmortem-interval AD patients and age-matched prospectively evaluated control subjects. RESULTS: A decrease in GST activity in all brain areas was observed in AD compared with controls with significant decreases in the amygdala, hippocampus and parahippocampal gyrus, inferior parietal lobule, and nucleus basalis of Meynert. Levels of GST protein also were depleted in most brain regions in AD. A significant decrease in GST activity and protein levels was also found in ventricular CSF in AD. CONCLUSION: Reduced levels of GST, a protective mechanism against HNE, may have a role in the pathogenesis of neuron degeneration in AD.

PMID: 9855502

Brain Res Mol Brain Res 1998 Jan;53(1-2):196-205.

Glutathione depletion exacerbates impairment by oxidative stress of phosphoinositide hydrolysis, AP-1, and NF-kappaB activation by cholinergic stimulation.

Li , et al

Department of Psychiatry and Behavioral Neurobiology University of Alabama at Birmingham, Birmingham, AL 35294-0017, USA.

Oxidative stress appears to contribute to neuronal dysfunction associated with Alzheimer's disease and other CNS neurodegenerative disorders. This investigation examined if oxidative stress might contribute to impairments in cholinergic receptor-linked signaling systems and if intracellular glutathione levels modulated responses to oxidative stress. To do this the activation of the AP-1 and NF-kappaB transcription factors and of the phosphoinositide second-messenger system was measured in human neuroblastoma SH-SY5Y cells after exposure to the oxidants H2O2 or diamide, with or without prior depletion of cellular glutathione. H2O2 concentration-dependently inhibited carbachol-stimulated AP-1 activation and this inhibition was potentiated in glutathione-depleted cells. Carbachol-stimulated NF-kappaB activation was unaffected by H2O2 unless glutathione was depleted, in which case there was a H2O2 concentration-dependent inhibition. Glutathione depletion also potentiated the inhibition by H2O2 of carbachol- or G-protein (NaF)-stimulated phosphoinositide hydrolysis, whereas phospholipase C activated by the calcium ionophore ionomycin was not inhibited. The thiol-oxidizing agent diamide also inhibited phosphoinositide hydrolysis stimulated by carbachol or NaF, and glutathione depletion potentiated the diamide concentration-dependent inhibition. Unlike H2O2, diamide also inhibited ionomycin-stimulated phosphoinositide hydrolysis. Activation of both AP-1 and NF-kappaB stimulated by carbachol was inhibited by diamide, and glutathione depletion potentiated the inhibitory effects of diamide. Thus, diamide inhibited a wider range of signaling processes than did H2O2, but glutathione depletion increased the susceptibility of phosphoinositide hydrolysis and of transcription factor activation to inhibition by both H2O2 and diamide. These results demonstrate that the vulnerability of signaling systems to oxidative stress is influenced by intracellular glutathione levels, indicating that cell-selective susceptibility to inhibition of signal transduction systems by oxidative stress can arise from cellular variations in antioxidant capacity.

PMID: 9473671

J Viral Hepat 1997 Mar;4(2):139-41.

Reduced glutathione concentration in erythrocytes of patients with acute and chronic viral hepatitis.

Swietek K, Juszczyk J.

Department of Infectious Diseases, Karol Marcinkowski University of Medical Sciences, Poznań, Poland.

Reduced glutathione (GSH), the main intracellular mechanism that protects against oxidative stress, is the subject of considerable interest in viral hepatitis. In patients with chronic hepatitis C, results reported from different centres are controversial, demonstrating either a reduction or an elevation of GSH concentration. The aim of this study was to evaluate the glutathione concentration in erythrocytes (normal range 2.45 +/- 0.15 mmol l-1) in patients with acute and chronic viral hepatitis. In 52 patients with acute viral hepatitis (hepatitis A virus (HAV), hepatitis B virus (HBV) and hepatitis C virus (HCV) infection) there was marked reduction of GSH at the beginning of the disease (0.79 +/- 0.43 mmol l-1, P < 0.001) with high alanine aminotransferase (ALT) activity (1549 +/- 772.9 IU l-1). In 37 patients with chronic HCV infection the mean value of GSH was below the normal range (1.92 +/- 0.62 mmol l-1, P < 0.001). In 60% of patients (n = 22), depletion of GSH was observed and 40% (n = 15) presented with a normal concentration of GSH. In 10 patients with chronic HBV infection the mean value of GSH was also below the normal range (1.93 +/- 0.32 mmol l-1, P < 0.001); in 80% of cases (n = 8) depletion of GSH was observed and 20% of patients (n = 2) had normal GSH concentrations. The ALT activity was not significantly different in patients with depleted and normal GSH concentrations (P > 0.05) in groups with chronic HBV and HCV infection.

PMID: 9097271

Brain Res 1996 Apr 15;716(1-2):118-22.

Depletion of brain glutathione results in a decrease of glutathione reductase activity; an enzyme susceptible to oxidative damage.

Barker JE, et al

Department of Neurochemistry, Institute of Neurology, London, UK.

Loss of the intracellular antioxidant glutathione (GSH) from the substantia nigra is considered to be an early event in the pathogenesis of Parkinson's disease (PD). While the cause of the loss is unclear, an imbalance in the enzymes associated with the synthesis, utilisation, degradation and translocation of GSH has been implicated. The enzyme glutathione reductase is also important in GSH homeostasis: it regenerates GSH from the oxidised from (GSSG). However, to date the activity and regulation of glutathione reductase in conditions such as PD have not been explored. In view of this we have measured the effects of GSH depletion on glutathione reductase activity of the rat brain. Other glutathione related enzymes were also measured. Using pre-weanling rats, brain GSH was depleted by up to 60% by subcutaneous administration of L-buthionine sulfoximine. The only enzyme affected by GSH depletion was glutathione reductase; its activity being reduced by approximately 40%. As GSH inactivates a number of oxidising species including peroxynitrite (ONOO-), we additionally investigated the susceptibility of glutathione reductase to ONOO- in vitro, using purified enzyme. ONOO- decreased glutathione reductase activity in a concentration dependent manner with an apparent 50% inhibition occurring at an initial concentration of 0.09 mM. These data suggest that GSH is important in the maintenance glutathione reductase activity. This may arise in part from its ability to inactivate oxidising agents such as ONOO-.

PMID: 8738227

Basic Res Cardiology 1989 Jul-Aug;84(4):388-95.

Free radical mediated injuries after coronary artery occlusion.

Rith E, et al

Department of Experimental Surgery, University Medical School of Pé, Hungary.

The left descending coronary artery (LAD) of mongrel dogs was ligated for one month to evaluate the effect of a dihydroquinoline-type antioxidant (MTDQ-DA) during the healing process of myocardial infarction. Thirty animals were divided into three groups: I) control animals (10 dogs) were treated with saline infusion during operation and postoperative days; II) 10 dogs were treated with intravenous MTDQ-DA during the operation followed by postoperative oral treatment, and III) 10 animals were treated with preoperative and postoperative oral treatment plus intraoperative MTDQ-DA infusion. The antioxidant effects were evaluated by measurements of the lipid peroxidation and product malondialdehyde (MDA) and of endogenous scavengers: reduced glutathione (GSH) and superoxide dismutase (SOD) in the ischemic and in intact areas. Ultrathin sections were investigated by JEOL 100 C electron microscope. Elevation of lipid peroxidation (LPO) was present in each group. The most remarkable changes were seen in the control ischemic areas (152 +/- 6.7%). GSH was depleted in the infarcted areas of the control hearts (60 +/- 5.6%), in the antioxidant treated animals this thiol compound was only slightly decreased (81 +/- 5.6%). SOD activity showed a sharp decline (group I) in both ischemic (52 +/- 8.3 U/g) and non-ischemic parts of the left ventricle (78 +/- 7 U/g). After MTDQ-DA treatment (groups II, III) SOD activity diminished only in the ischemic area (83 +/- 8.5 U/g); other parts showed normal (123 +/- 7.2 U/g) activity. According to ultrastructural examinations, MTDQ-DA diminished the degree of structural injury.

PMID: 2818438

Amer Rev Respir Dis 1989 Feb;139(2):370-2.

Glutathione deficiency in the epithelial lining fluid of the lower respiratory tract in idiopathic pulmonary fibrosis.

Cantin AM, et al

Pulmonary Branch, National Heart, Lung and Blood Institute, Bethesda, Maryland 20892.

Glutathione (L-gamma-glutamyl-L-cysteinyl-glycine, GSH), a sulfhydryl-containing tripeptide produced by most mammalian cells, is an efficient scavenger of toxic oxidants, including hydrogen peroxide, an oxidant that plays a major role in the oxidant burden placed on the epithelial surface of the lower respiratory tract in chronic inflammatory states. GSH is present in the epithelial lining fluid of the normal lower respiratory tract, where it is thought to play a major role in providing antioxidant protection to the epithelial cells. In this regard, we hypothesized that the lower respiratory tract of patients with IPF may be chronically depleted of this antioxidant, thus leading to an increased susceptibility of lung epithelial cells to oxidant injury. To evaluate this concept, the concentration of glutathione was determined in the epithelial lining fluid of the lower respiratory tract of 15 patients with IPF and compared to that of 19 normal subjects. Strikingly, whereas ELF glutathione concentrations were high in normal subjects (429 +/- 34 microM), a fourfold decrease was found in patients with IPF (97 +/- 18 microM, p less than 0.001). In the context of the known oxidant burden present in the lower respiratory tract of patients with IPF, these observations of a "GSH deficiency" in IPF ELF suggest that there is a marked oxidant-antioxidant imbalance at the alveolar surface of these persons, thus increasing the susceptibility to the severe epithelial cell damage characteristic of this disease.

PMID: 2913886

Cardiovascu Res 1988 Nov;22(11):833-9.

Depletion of myocardial glutathione: its effects on heart function and metabolism during ischaemia and reperfusion.

Chatam JC, et al

Department of Biochemistry, University of Oxford, England.

To determine the importance of intracellular reactive oxygen metabolites in ischaemia and reperfusion, rat hearts were depleted of glutathione, a major intracellular antioxidant, prior to total global or low flow ischaemia. Chronic pretreatment of rats with up to 7 mmol.kg-1 L-buthionine-S,R-sulphoximine over a period of 96 hours resulted in 85% depletion of total myocardial glutathione. Subsequent recovery of glutathione after treatment with the sulphoximine was slow, reaching only 54% of control levels, 96 hours after the final dose. Depletion of myocardial glutathione by 70% did not increase the metabolic consequences of either total global or low flow ischaemia in the isolated perfused heart, as determined by 31P NMR spectroscopy and high performance liquid chromatographic analysis of purine release. During low flow ischaemia glutathione depletion caused a significant reduction in purine release. On reperfusion, functional recovery was depressed compared to controls. Hearts depleted of glutathione also showed a decrease in developed tension during normoxic perfusion prior to low flow ischaemia. There was no difference between the two groups before or after total global ischaemia. These results do not support the hypothesis of free radical mediated reperfusion injury; however, the method described here for the depletion of glutathione should prove a useful tool for further investigation into the role of glutathione in cardiac metabolism and function.

PMID: 3256425