JOURNAL OF PHARMACEUTICAL SCIENCES, Issue 7 2004
Verena M. Leitner
Abstract This study was aimed at investigating the potential of a new polycarbophil-cysteine (PCP-Cys)/glutathione (GSH) gel formulation to enhance the permeation of the model drug human growth hormone (hGH) across nasal mucosa in vitro and in vivo. The aqueous nasal gel contained PCP-Cys, GSH, and hGH in a final concentration of 0.3%, 0.5%, and 0.6% (m/v), respectively. In vitro permeation studies were performed in Ussing chambers on freshly excised bovine nasal mucosa using fluorescence-labeled dextran (molecular mass: 4.3 kDa; FD-4) and hGH (FITC-hGH). The release profile of FITC-hGH from the gel formulation and an unmodified PCP control formulation was determined. Furthermore, in vivo studies in rats were performed comparing the PCP-Cys/GSH/hGH gel with PCP/hGH control gel and physiological saline. The permeation of FD-4 and FITC-hGH across the nasal mucosa was improved two-fold and three-fold, respectively, in the presence of PCP-Cys/GSH. The PCP-Cys/GSH/hGH gel and the PCP/hGH control gel showed the same biphasic and matrix-controlled drug release. The nasal administration of the PCP-Cys/GSH/hGH gel formulation to rats resulted in a significantly increased and prolonged hGH plasma concentration,time profile versus unmodified PCP gel and physiological saline. According to these results, PCP-Cys gels might represent a promising new strategy for systemic nasal polypeptide delivery. © 2004 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 93:1682,1691, 2004 [source]

PRODUCTION OF PHYTOCHELATINS AND GLUTATHIONE BY MARINE PHYTOPLANKTON IN RESPONSE TO METAL STRESS,

JOURNAL OF PHYCOLOGY, Issue 5 2006
Silvia K. Kawakami
Phytoplankton deal with metal toxicity using a variety of biochemical strategies. One of the strategies involves glutathione (GSH) and phytochelatins (PCs), which are metal-binding thiol peptides produced by eukaryotes and these compounds have been related to several intracellular functions, including metal detoxification, homeostasis, metal resistance and protection against oxidative stress. This paper assesses our state of knowledge on the production of PCs and GSH by marine phytoplankton in laboratory and field conditions and the possible applications of PCs for environmental purposes. Good relationships have been observed between metal exposure and PC production in phytoplankton in the laboratory with Cd, Pb, and Zn showing the greatest efficacy, thereby indicating that PCs have a potential for application as a biomarker. Fewer studies on PC distributions in particulate material have been undertaken in the field. These studies show that free Cu has a strong relationship with the levels of PC in the particulate material. The reason for this could be because Cu is a common contaminant in coastal waters. However it could also be due to the lack of measurements of other metals and their speciation. GSH shows a more complex relationship to metal levels both in the laboratory and in the field. This is most likely due to its multifunctionality. However, there is evidence that phytoplankton act as an important source of dissolved GSH in marine waters, which may form part of the strong organic ligands that control metal speciation, and hence metal toxicity. [source]

Voltammetric Studies of the Interactions Between Ferrocene-Labeled Glutathione and Proteins in Solution or Immobilized onto Surface

ELECTROANALYSIS, Issue 16 2009
Yong Peng
Abstract Glutathione (GSH) tagged with a ferrocene (Fc) label at its C-terminal was synthesized via coupling ferrocenyl amine to glutathione using o -(benzotriazol-1-yl)- N,N,N,,N, -tetramethyluronium (HBTU)/1-hydroxybenzotrizole (HOBt). The presence of Fc yielded well defined voltammetric signals, rendering the Fc-tagged GSH (GSH-Fc) suitable for electrochemical studies of GSH binding to other biological species. The interaction of GSH-Fc with bovine serum albumin (BSA) was investigated, and a binding ratio of 1.41±0.06 (GSH-Fc/BSA) and an affinity constant Ka of 6.53±2.01×106,M,1 were determined. These results compare well with those measured by fluorescence using untagged GSH, suggesting that the attachment of Fc to GSH does not significantly perturb the GSH structure and binding behavior. By contrasting the binding behavior to several compounds that are known to conjugate to different domains of BSA, the voltammetric study confirmed that GSH-Fc binds at subdomain IIA of BSA with high affinity. The versatility of GSH-Fc for studying GSH binding to surface-confined proteins was also demonstrated with the GSH binding to electroinactive Zn-metallothionein (Zn7 -MT) through hydrogen binding at the region between the Zn7 -MT , and , domains. [source]

Voltammetric Investigation of Zinc Release from Metallothioneins Modulated by the Glutathione Redox Couple and Separated with a Porous Membrane

ELECTROANALYSIS, Issue 20 2008
Lin Liu
Abstract Glutathione (GSH), in addition to serving as a redox buffer in cellular environment, has been suggested as a modulator in metal regulation and homeostasis by metallothioneins (MTs). The interactions of MTs with both GSH and its oxidized form GSSG have been shown to govern the direction of metal transfer. Common methods for the determination of zinc release from MTs modulated by GSH/GSSG either involve radioactive species or enzymes or are labor-intensive. In this study, upon separation of Zn2+ from the reaction mixture of MTs and GSH with a centrifugal filter membrane, differential pulse voltammetry (DPV) was used for the Zn2+ quantification. The same approach is extended to the studies of metal transfer between Zn7MT with a GSH/GSSG mixture and that between Zn7MT with GSSG. The concomitant conversion between the free thiol and disulfide bonds was confirmed with UV-vis spectrophotometry. The results demonstrate that GSSG, GSH, and the GSH/GSSG mixture all modulate zinc release from Zn7MT. The percentage of zinc release increases in the order of GSH, GSSG, and the GSH/GSSG mixture. The new approach is demonstrated to be well suited for investigation of redox regulation of MT and its reaction with zinc-containing enzymes. [source]

Disposable Amperometric Sensors for Thiols with Special Reference to Glutathione

ELECTROANALYSIS, Issue 18 2008
Dipankar Bhattacharyay
Abstract The antioxidant ,reduced glutathione' tripeptide is conventionally called glutathione (GSH). The oxidized form is a sulfur-sulfur linked compound, known as glutathione disulfide (GSSG). Glutathione is an essential cofactor for antioxidant enzymes; it provides protection also for the mitochondria against endogenous oxygen radicals. The ratio of these two forms can act as a marker for oxidative stress. The majority of the methods available for estimation of both the forms of glutathione are based on colorimetric and electrochemical assays. In this study, electrochemical sensors were developed for the estimation of both GSH and GSSG. Two different types of transducers were used: i) screen-printed three-electrode disposable sensor (SPE) containing carbon working electrode, carbon counter electrode and silver/silver chloride reference electrode; ii) three-electrode disposable system (CDE) consisting of three copper electrodes. 5,5,-dithiobis(2-nitrobenzoic acid) (DTNB) was used as detector element for estimation of total reduced thiol content. The enzyme glutathione reductase along with a co-enzyme reduced nicotinamide adenine dinucleotide phosphate was used to estimate GSSG. By combining the two methods GSH can also be estimated. The detector elements were immobilized on the working electrodes of the sensors by bulk polymerization of acrylamide. The responses were observed amperometrically. The detection limit for thiol (GSH) was less than 0.6,ppm when DTNB was used, whereas for GSSG it was less than 0.1,ppm. [source]

Cadmium induced oxidative stress influence on glutathione metabolic genes of Camellia sinensis (L.) O. Kuntze

ENVIRONMENTAL TOXICOLOGY, Issue 4 2007
Prashant Mohanpuria
Abstract Glutathione, a tripeptide with sulfhydryl (-SH) group is a very crucial compound primarily involved in redox balance maintenance of the cellular environment. In this study, we monitored the influence of Cd exposure on the transcript levels of glutathione metabolic genes in bud tissues, the youngest leaf, of Camellia sinensis L. In addition, some physiochemical parameters were also studied. Cd exposure decreased chlorophyll and protein contents, while increase was observed in lipid peroxidation upon Cd treatments. These changes were found to be concentration and duration dependent, indicating the occurrence of oxidative stress upon Cd exposure. The transcript levels of glutathione biosynthetic genes viz. ,-glutamylcysteine synthetase (,-ECS) and glutathione synthetase (GSHS) increased upon Cd exposure. Furthermore, transcript levels of glutathione reductase (GR), an enzyme involved in reduction of oxidized glutathione (GSSG) to reduced glutathione (GSH), also showed upregulation on Cd exposure. However, the transcript levels of glutathione-S-transferase (GST), an enzyme involved in forming metal,GSH complex and help in sequestration of high levels of metal ions to vacuole, did not show any change on Cd treatment. This study document that Cd exposure induces oxidative stress in Camellia sinensis and the upregulation in transcript levels of glutathione metabolic genes except GST have suggested the role of these enzymes in the protection of plants from high level Cd exposure. © 2007 Wiley Periodicals, Inc. Environ Toxicol 22: 368,374, 2007. [source]

Analysis of glutathione endpoints for measuring copper stress in Chlamydomonas reinhardth

ENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 8 2007
Tasha L. Stoiber
Abstract Glutathione (GSH) is the most abundant nonprotein thiol in eukaryotic cells and it protects cells by functioning as an antioxidant and a metal-binding ligand. Because glutathione readily undergoes oxidation-reduction reactions to combat oxidative stress, intracellular ratios of the reduced (GSH) to the oxidized (GSSG) forms of glutathione may serve as an important biomarker of exposure and effect of trace metals in eukaryotic cells. We compared sensitivity of glutathione ratios in the freshwater alga Chlamydomonas reinhardtii to the traditional endpoints of cell growth rates and chlorophyll a following exposure to Cu for periods of 6 and 24 h. A response of the GSH:GSSG ratio to Cu concentration was observed at Cu levels of 40 and 80 nM after exposure for both 6 and 24 h. The concentration of total GSH at 24 h was roughly half the value at 6 h after exposure to either 40 or 80 nM Cu. A response for cell growth rate was observed only at 24 h, whereby the average specific growth rate decreased from about 1.1 to 0.4 d,1. The total Cu concentrations eliciting a cell response of 50%, effect concentrations (EC50s), after 24 h of exposure were similar (49.2, 49.8, and 38.2 nM Cu) and not significantly different for GSH:GSSG ratio, GSH levels, and specific growth, respectively. Total cell-associated Cu concentrations after exposure for 24 h were calculated from the EC50 endpoints and ranged from 13.3 to 17.0 fg/cell. Overall, thiol ratios were indicative of toxicity resulting from exposure to Cu, but precision may be greater for the cell growth rate endpoints. [source]

Glutathione, vitamin E and oxidative stress in coronary artery disease: relevance of age and gender

EUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 4 2009
V. Cavalca
Abstract Background, Observational studies suggest that low levels of antioxidants are associated with high risk for coronary artery disease (CAD). We investigated whether the biomarkers of oxidative balance undergo the same modifications in all CAD patient groups, regardless of gender and age. Materials and methods, One hundred sixty-eight CAD patients and 107 healthy controls were assayed for plasma levels of reduced glutathione (GSH), ,- and ,-tocopherol (,- and ,-T) as endogenous antioxidants. A damage score (DS), representative of oxidative stress status, was calculated. ancova models were used to test the association between antioxidants, DS and CAD and its modulation by age and gender. Results, The DS was higher in CAD than in controls. GSH levels, were lower in CAD patients (mean ± SEM: 57·61 ± 1·87 ,mol 10 g,1 haemoglobin vs. 68·55 ± 2·23 in controls, P < 0·0006) in males and in older subjects. Levels of other antioxidants exhibited a complex pattern. Overall, no difference was found in ,- and ,-T contents between CAD and controls, but lower ,-T values were observed in CAD females. A significant interaction between CAD status and gender was observed (P = 0·003). Conclusions, Our study shows that the involvement of antioxidants in CAD is related to patients' characteristics. These findings may be relevant in planning antioxidant therapies. [source]

Glutathione depletion and cardiomyocyte apoptosis in viral myocarditis

EUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 3 2004
V. Kytö
Abstract Background, The course of viral myocarditis is highly variable. Oxidative stress and Bcl-2 family genes may play a role in its pathogenesis by regulating the amount of cardiomyocyte apoptosis. Apoptosis is difficult to detect and quantify in vivo. Therefore, we set to look for indicators of this potentially preventable form of cell death during various phases of experimental murine coxsackievirus B3 myocarditis. Methods, BALB/c mice were infected with the cardiotropic coxsackievirus B3 variant. Glutathione (HPLC), cardiomyocyte apoptosis (TUNEL and caspase-3 cleavage), Bax and Bcl-XL mRNA expression (real time RT-PCR), histopathology and viral replication (plaque assay and real time RT-PCR) were measured from day 3 to day 20 after infection. Results, Infection caused severe myocarditis and led to progressive decrease of plasma glutathione levels. Myocardial mRNA levels of pro-apoptotic Bax and antiapoptotic Bcl-XL were significantly increased from day 3 onwards. Bax mRNA and ratio of Bax to Bcl-XL correlated with cardiomyocyte apoptosis (r = 0·77, P = < 0·001 and r 0·51, P < 0·01, respectively). Cardiomyocyte apoptosis was highest on day 5, coinciding with a rapid decline in plasma glutathione (r = ,0·52, P = 0·003). Conclusions, Systemic oxidative stress as indicated by decreased plasma glutathione levels coincides with cardiomyocyte apoptosis in experimental coxsackievirus myocarditis. Decreased plasma glutathione levels and changes in cardiac Bax and Bcl-XL mRNA expression identify a phase of myocarditis in which the potentially preventable cardiomyocyte apoptosis is mostly observed. [source]

Sulfur assimilation and glutathione metabolism under cadmium stress in yeast, protists and plants

FEMS MICROBIOLOGY REVIEWS, Issue 4 2005
David Mendoza-Cózatl
Abstract Glutathione (,-glu-cys-gly; GSH) is usually present at high concentrations in most living cells, being the major reservoir of non-protein reduced sulfur. Because of its unique redox and nucleophilic properties, GSH serves in bio-reductive reactions as an important line of defense against reactive oxygen species, xenobiotics and heavy metals. GSH is synthesized from its constituent amino acids by two ATP-dependent reactions catalyzed by ,-glutamylcysteine synthetase and glutathione synthetase. In yeast, these enzymes are found in the cytosol, whereas in plants they are located in the cytosol and chloroplast. In protists, their location is not well established. In turn, the sulfur assimilation pathway, which leads to cysteine biosynthesis, involves high and low affinity sulfate transporters, and the enzymes ATP sulfurylase, APS kinase, PAPS reductase or APS reductase, sulfite reductase, serine acetyl transferase, O -acetylserine/O -acetylhomoserine sulfhydrylase and, in some organisms, also cystathionine ,-synthase and cystathionine ,-lyase. The biochemical and genetic regulation of these pathways is affected by oxidative stress, sulfur deficiency and heavy metal exposure. Cells cope with heavy metal stress using different mechanisms, such as complexation and compartmentation. One of these mechanisms in some yeast, plants and protists is the enhanced synthesis of the heavy metal-chelating molecules GSH and phytochelatins, which are formed from GSH by phytochelatin synthase (PCS) in a heavy metal-dependent reaction; Cd2+ is the most potent activator of PCS. In this work, we review the biochemical and genetic mechanisms involved in the regulation of sulfate assimilation-reduction and GSH metabolism when yeast, plants and protists are challenged by Cd2+. [source]

Glutathione depletion in hippocampal cells increases levels of H and L ferritin and glutathione S-transferase mRNAs

GENES TO CELLS, Issue 5 2007
Nadya Morozova
Glutathione plays an essential role in maintaining cellular redox balance, protecting cells from oxidative stress and detoxifying xenobiotic compounds. Glutathione depletion has been implicated in neurodegenerative disorders, including Alzheimer's and Parkinson's diseases. Cells of neuronal origin are acutely sensitive to glutathione depletion, providing an avenue for studying the mechanisms invoked for neuronal survival in response to oxidant challenge. We investigated the changes in mRNA profile in HT22 hippocampal cells following administration of homocysteic acid (HCA), a glutathione-depleting drug. We report that HCA treatment of HT22 murine hippocampal cells increases the levels of the mRNAs encoding at least three proteins involved in protection from oxidant injury, the mRNAs encoding heavy (H) and light (L) ferritin and glutathione S-transferase (GST). [source]

Inhibition of the decrease of volatile esters and terpenes during storage of a white wine and a model wine medium by glutathione and N -acetylcysteine

INTERNATIONAL JOURNAL OF FOOD SCIENCE & TECHNOLOGY, Issue 6 2008
Despima Papadopoulou
Summary Glutathione and N -acetylcysteine, each at 20 mg L,1, were tested as inhibitors of the decrease of volatile esters and terpenes during storage of Debina white wine. Moreover, the inhibition of the decrease of isoamyl acetate, ethyl hexanoate and linalool in a model wine medium by glutathione and N -acetylcysteine, each at 0,20 mg L,1, was also tested. Several volatiles, such as isoamyl acetate, ethyl hexanoate, ethyl octanoate, ethyl decanoate and linalool, decreased during wine storage. Glutathione or N -acetylcysteine significantly restricted the decrease of these volatiles. In the model medium, each thiol inhibited the decrease of the three volatiles in a dose-dependent manner. N -acetylcysteine inhibited the decrease of all three volatiles at 2.5 mg L,1 while glutathione at 2.5 or 5.0 mg L,1. The present results indicate that glutathione and N -acetylcysteine may be taken into account as potent inhibitors of the disappearance of aromatic esters and terpenes in wines. [source]

Antagonistic Effects of Hydrogen Peroxide and Glutathione on Acclimation to Excess Excitation Energy in Arabidopsis

IUBMB LIFE, Issue 1 2000
Barbara Karpinska
Abstract The redox status of the quinone B (QB) and plastoquinone (PQ) pools plays a key role in the cellular and systemic signalling processes that control acclimatory responses in plants. In this study, we demonstrate the effects of hydrogen peroxide and glutathione on acclimatory responses controlled by redox events in the proximity of the QB-PQ pools. Our results suggest that the chloroplast is a sink for H2O2 and that, paradoxically, high concentrations of H2O2 in the chloroplast protect the photosynthetic apparatus and the plant cell from photoinhibition and photooxidative damage. Excess glutathione, however, caused an effect antagonistic to that observed for high H2O2. An explanation of this apparent paradox and a hypothetical redox-signalling model are suggested. [source]

Early detection of resistance to tebufenozide in field populations of Cydia pomonella L.: methods and mechanisms

JOURNAL OF APPLIED ENTOMOLOGY, Issue 7 2007
C. Ioriatti
Abstract:, Four populations of codling moth Cydia pomonella L. were collected as overwintering larvae from apple orchards with different pesticide pressure (S. Michele, Roncafort, Revò and Vervò) in the Trento province (northern Italy). Mortality rate caused by a predetermined discriminating concentration of tebufenozide topically applied on overwintering larvae was evaluated. Neonate F1 progeny of the same populations were assayed for susceptibility to tebufenozide by feeding them on thinning apples treated with an appropriate discriminating dose of the insecticide. The activities of the main enzyme systems involved in the detoxification of insecticides were also evaluated in each population and related to their susceptibility to tebufenozide. The topical test detected a significant loss in susceptibility to tebufenozide in two populations, S. Michele and Roncafort, while all the overwintering larvae collected in the orchards of Revò and Vervò died when treated topically with the discriminating concentration. The apple-dipping test performed on the neonate larvae showed a highly significant reduction in the susceptibility of the two populations of S. Michele and Roncafort. A less significant reduction in mortality rate was found in the Revò population; however, no statistical difference was found between the Vervò population and the susceptible reference. None of the four field populations significantly differed from the susceptible strain for Glutathione- S -transferase and esterase activity. A significantly higher frequency of individuals of the S. Michele and Roncafort populations exhibited a higher mixed function oxidase activity than the susceptible strain. The small resistance ratio values found for the two populations together with the low frequency of individuals exibiting enhanced enzymatic activity, reveals that the selection process was still at the early stage. Because of its efficiency in early detection of resistance to tebufenozide, topical application on diapausing larvae can thus be considered an appropriate, simple and robust tool for implementing resistance monitoring programmes for tebufenozide. [source]

Glutamine administration in patients undergoing cardiac surgery and the influence on blood glutathione levels

ACTA ANAESTHESIOLOGICA SCANDINAVICA, Issue 10 2009
J. M. ENGEL
Background: Cardiac surgery with an extracorporeal circulation cardiopulmonary bypass (CPB) is characterized by an oxidative stress response. Glutathione (GSH) belongs to the major antioxidative defense. In metabolic stress, glutamine (GLN) may be the rate-limiting factor of GSH synthesis. Decreased GLN plasma levels were observed after various critical states. We evaluated, in patients undergoing open heart surgery with CPB, the effects of a peri-operative GLN supplementation on GSH in whole blood and assessed their influence on the Sequential Organ Failure Assessment score and the intensive care unit length of stay. Methods: In this prospective, randomized, double-blinded study, we included 60 patients (age older than 70 years, ejection fraction <40% or mitral valve replacement) undergoing an elective cardiac surgery with CPB. We randomly assigned each subject to receive an infusion with either GLN (0.5 g/kg/day, group 1) or an isonitrogeneous, isocaloric, isovolemic amino acids solution (group 2) or saline (group 3). Results: From the first post-operative day GLN plasma levels in group 1 were significantly increased compared with the other groups. With saline GSH the levels decreased significantly post-operatively compared with GLN. We observed a significant correlation between GLN delivery and GSH levels. Conclusions: A peri-operative high-dose GLN infusion increased plasma GLN concentrations and maintained the GSH levels after cardiac surgery with CPB. [source]

Open-labeled pilot study of cysteine-rich whey protein isolate supplementation for nonalcoholic steatohepatitis patients

JOURNAL OF GASTROENTEROLOGY AND HEPATOLOGY, Issue 6 2009
Taned Chitapanarux
Abstract Background and Aims:, Glutathione (GSH) depletion contributes to liver injury and development of steatohepatitis. Undenatured cysteine-rich whey protein isolate has been clinically proven to raise GSH in several patient groups. The aim of this study was to evaluate the effect of oral supplementation with whey protein on patients with nonalcoholic steatohepatitis (NASH). Methods:, In an open-labeled clinical trial, 38 patients (18 male, 20 female; mean age 48 ± 14 years) with NASH confirmed by computed tomography measurements and liver biochemistries were given with a daily dose of 20 g whey protein isolate for 12 weeks. Results:, A significant reduction in alanine aminotransferase (ALT) (64 ± 72 vs 46 ± 36, P = 0.016) and aspartate aminotransferase (AST) (45 ± 49 vs 33 ± 18, P = 0.047) were observed. Plasma glutathione and total antioxidant capacity increased significantly at the end of study (53 ± 11 vs 68 ± 11, P < 0.05 and 1.26 ± 0.10 vs 2.03 ± 0.10, P < 0.05). Liver attenuation index improved from ,13.4 ± 11.1 to ,9.7 ± 13.1 (P = 0.048). Hepatic macrovesicular steatosis decreased significantly after 12 weeks of supplementation (33.82 ± 12.82 vs 30.66 ± 15.96, P = 0.046). Whey protein isolate was well tolerated. No serious adverse events were observed. Conclusions:, The results indicate that oral supplementation of cysteine-rich whey protein isolate leads to improvements in liver biochemistries, increased plasma GSH, total antioxidant capacity and reduced hepatic macrovesicular steatosis in NASH patients. The results support the role of oxidative stress in the pathogenesis of this disease. [source]

Effect of endotoxin pretreatment on hepatic stellate cell response to ethanol and acetaldehyde

JOURNAL OF GASTROENTEROLOGY AND HEPATOLOGY, Issue 11 2001
Silvia C Quiroz
Abstract Background and Aim: The role of endotoxin in alcohol-induced liver damage is well recognized. How pre-exposure to endotoxin might affect alcohol injury is not known. We herein studied the effect of endotoxin pretreatment on hepatic stellate cell (HSC) response to ethanol and acetaldehyde. Methods: Rat HSC (CFSC-2G) were exposed to media supplemented with 1 ,g/mL lipopolysaccharide (LPS). This was followed by a 24 h exposure to media containing LPS plus 50 mmol/L ethanol or 175 ,mol/L acetaldehyde. Lipid peroxidation, collagen, and tumor necrosis factor (TNF)-,, interleukin (IL)-1,, IL-6 and transforming growth factor (TGF)-,1 secretion were determined at the end of both periods of exposure. Results: Lipopolysaccharide pretreatment did not modify lipid peroxidation induced by ethanol or acetaldehyde alone. Glutathione (GSH) content decreased to 4.2 ± 0.5 and 16.3 ± 0.8 nmol protein after exposure to ethanol or acetaldehyde alone, and decreased further with LPS pretreatment (2.4 ± 0.2 and 2.7 ± 0.3 nmol/mg protein, respectively). Oxidized GSH (GSSG) content increased in ethanol and acetaldehyde LPS-pretreated cells only. Collagen secretion increased to 988 ± 82 and 1169 ± 91 ,g/106 cells after exposure to acetaldehyde or LPS alone. Lipopolysaccharide pretreatment enhanced collagen secretion significantly in both ethanol- and acetaldehyde-treated cells (969 ± 56 and 1360 ± 72 ,g/106 cells, respectively). Interleukin-6 production increased to 288 ± 48, 1195 ± 86 and 247 ± 35 pg/mL per 106 cells after ethanol, acetaldehyde and LPS exposure, and increased further with LPS pretreatment in ethanol-exposed cells (680 ± 23 pg/mL 106 cells). Conclusion: Lipopolysaccharide pretreatment of HSC adds to the damage produced by ethanol and acetaldehyde by diminishing GSH content and increasing GSSG content, collagen and IL-6 secretion. [source]

p -quinone mediates 6-hydroxydopamine-induced dopaminergic neuronal death and ferrous iron accelerates the conversion of p -quinone into melanin extracellularly

JOURNAL OF NEUROSCIENCE RESEARCH, Issue 6 2005
Yasuhiko Izumi
Abstract Parkinson's disease (PD) is characterized by the selective loss of dopaminergic neurons in the substantia nigra (SN). 6-Hydroxydopamine (6-OHDA), a dopaminergic neurotoxin, is detected in human brains and the urine of PD patients. Using SH-SY5Y, a human neuroblastoma cell line, we demonstrated that 6-OHDA toxicity was determined by the amount of p -quinone produced in 6-OHDA auto-oxidation rather than by reactive oxygen species (ROS). Glutathione (GSH), which conjugated with p -quinone, provided significant protection whereas catalase, which detoxified hydrogen peroxide and superoxide anions, failed to block cell death caused by 6-OHDA. Although iron accumulated in the SN of patients with PD can cause dopaminergic neuronal degeneration by enhancing oxidative stress, we found that extracellular ferrous iron promoted the formation of melanin and reduced the amount of p -quinone. The addition of ferrous iron to the culture medium inhibited caspase-3 activation and apoptotic nuclear morphologic changes and blocked 6-OHDA-induced cytotoxicity in SH-SY5Y cells and primary cultured mesencephalic dopaminergic neurons. These data suggested that generation of p -quinone played a pivotal role in 6-OHDA-induced toxicity and extracellular iron in contrast to intracellular iron was protective rather than harmful because it accelerated the conversion of p -quinone into melanin. © 2005 Wiley-Liss, Inc. [source]

,-glutamylcysteine ethyl ester-induced up-regulation of glutathione protects neurons against A,(1,42)-mediated oxidative stress and neurotoxicity: Implications for Alzheimer's disease

JOURNAL OF NEUROSCIENCE RESEARCH, Issue 5 2005
Debra Boyd-Kimball
Abstract Glutathione (GSH) is an important endogenous antioxidant found in millimolar concentrations in the brain. GSH levels have been shown to decrease with aging. Alzheimer's disease (AD) is a neurodegenerative disorder associated with aging and oxidative stress. A,(1,42) has been shown to induce oxidative stress and has been proposed to play a central role in the oxidative damage detected in AD brain. It has been shown that administration of ,-glutamylcysteine ethyl ester (GCEE) increases cellular levels of GSH, circumventing the regulation of GSH biosynthesis by providing the limiting substrate. In this study, we evaluated the protective role of up-regulation of GSH by GCEE against the oxidative and neurotoxic effects of A,(1,42) in primary neuronal culture. Addition of GCEE to neurons led to an elevated mean cellular GSH level compared with untreated control. Inhibition of ,-glutamylcysteine synthetase by buthionine sulfoximine (BSO) led to a 98% decrease in total cellular GSH compared with control, which was returned to control levels by addition of GCEE. Taken together, these results suggest that GCEE up-regulates cellular GSH levels which, in turn, protects neurons against protein oxidation, loss of mitochondrial function, and DNA fragmentation induced by A,(1,42). These results are consistent with the notion that up-regulation of GSH by GCEE may play a viable protective role in the oxidative and neurotoxicity induced by A,(1,42) in AD brain. © 2005 Wiley-Liss, Inc. [source]

Influence of glutathione- S -transferase theta (GSTT1) and mu (GSTM1) gene polymorphisms on the susceptibility of hepatocellular carcinoma in Taiwan,

JOURNAL OF SURGICAL ONCOLOGY, Issue 4 2010
Chia-Chun Kao MD
Abstract Background and Objectives Hepatocellular carcinoma (HCC) is one of the most frequent malignant neoplasms worldwide and is the second leading cause of cancer death in Taiwan. Genetic polymorphism has been reported as a factor for increased susceptibility of HCC. Glutathione- S -transferases theta (GSTT1) and mu (GSTM1) play essential roles in detoxification of ingested xenobiotics and modulation of the susceptibility of gene-related cancer. The aim of this study was to estimate the relationships between these two gene polymorphisms and HCC risk and clinicopathological status in Taiwanese. Methods Polymerase chain reaction (PCR) was used to determine gene polymorphisms of 102 patients with HCC and 386 healthy controls. Results Both gene polymorphisms were not associated with the clinical pathological status of HCC and serum levels of liver-related clinical pathological markers. While no relationship between GSTM1 gene polymorphism and HCC susceptibility was found, individuals of age <56 years old with GSTT1 present genotype have a risk of 2.77-fold (95% CI: 1.09,7.09) for HCC compared to that with null variant, after adjustment for other confounders. Conclusions GSTT1 and GSTM1 null genotypes do not associate with increased risk of HCC. J. Surg. Oncol. 2010;102:301,307. © 2010 Wiley-Liss, Inc. [source]

Effect of Chronic Ethanol Ingestion on Alveolar Type II Cell: Glutathione and Inflammatory Mediator-Induced Apoptosis

ALCOHOLISM, Issue 7 2001
Lou Ann S. Brown
Background : In septic patients, chronic alcohol abuse increases the incidence of the acute respiratory distress syndrome, a syndrome that requires alveolar type II cell proliferation and differentiation for repair of the damaged alveolar epithelium. We previously showed in a rat model that chronic ethanol ingestion decreased the antioxidant glutathione (GSH) in type II cells and exacerbated endotoxin-mediated acute lung injury. We hypothesized that this GSH depletion by ethanol, particularly mitochondrial GSH, predisposed type II cells to inflammatory mediator-induced apoptosis. Methods: Adult male rats were fed the Lieber-DeCarli diet for 2, 6, or 16 weeks. Alveolar type II cells were then isolated and treated with hydrogen peroxide or TNF-,. The effect on glutathione (cytosolic and mitochondrial), apoptotic events, and necrosis were determined. In other studies, rats were fed ethanol for 6 weeks and were treated with endotoxin and apoptosis of type II cells determined by the TUNEL method. Results: Chronic ethanol ingestion alone resulted in a progressive decrease in mitochondrial GSH and a progressive increase in the basal apoptosis and necrosis rate (p, 0.05). Furthermore, there was a progressive increase in the sensitivity of the cells to H2O2 or TNF-, induced cytochrome c release, caspase 3 activation, apoptosis, and necrosis (p, 0.05). Finally, there was a 2-fold increase in apoptotic type II cells in vivo when chronic ethanol ingestion was superimposed on endotoxemia. Conclusions: These results suggested that chronic ethanol ingestion resulted in a progressive depletion of mitochondrial GSH and sensitization of type II cells to inflammatory mediator-induced apoptosis and necrosis. These effects may be particularly relevant during acute stress when proliferation and differentiation of these cells are critical to repair of the damaged alveolar epithelium and may have important ramifications for the treatment of acute respiratory distress syndrome in patients with a history of alcohol abuse. [source]

Chromatin changes on the GSTP1 promoter associated with its inactivation in prostate cancer

MOLECULAR CARCINOGENESIS, Issue 10 2007
Steven T. Okino
Abstract Glutathione- S -transferases (GSTs) are metabolic enzymes that help detoxify and eliminate harmful chemicals. In prostate tumors, expression of GST , (encoded by GSTP1) is frequently lost because of promoter hypermethylation. Here we analyze the native GSTP1 promoter in cancerous and noncancerous human prostate cells to identify structural features associated with its cancer-related transcriptional silencing. We find that in noncancerous prostate cells (RWPE-1 and PWR-1E) GSTP1 is constitutively expressed, not methylated, highly accessible, bound by transcription factors and associated with histones with activating modifications (histone H3 methylated at lysine 4 and acetylated histones H3 and H4). In contrast, in cancerous prostate cells (LNCaP) GSTP1 is not expressed, extensively methylated, inaccessible, lacks bound transcription factors and is not associated with histones with activating modifications. We do not detect significant levels of histones with repressive modifications (histone H3 methylated at lysine 9 or 27) on GSTP1 in any cell line indicating that they are not associated with cancer-related GSTP1 silencing. Treatment of LNCaP cells with 5-azacytidine restores activating histone modifications on GSTP1 and reactivates transcription. We conclude that, in the process of prostate carcinogenesis, activating histone modifications on GSTP1 are lost and the DNA becomes methylated and inaccessible resulting in transcriptional silencing. © 2007 Wiley-Liss, Inc. [source]

Aberrant protein expression is associated with decreased developmental potential in porcine cumulus,oocyte complexes

MOLECULAR REPRODUCTION & DEVELOPMENT, Issue 1 2010
Melissa Paczkowski
Oocyte developmental competence is progressively obtained during pubertal development in females. Poor developmental potential in oocytes derived from prepubertal females suggests that essential processes required for oocyte development have not been fulfilled. The objective of this experiment was to analyze the protein profiles of porcine cumulus,oocyte complexes (COC) derived from cyclic and prepubertal females to identify alterations in protein abundance that correlate with developmental potential. COC complexes, aspirated from prepubertal and cyclic ovaries, were pooled into three replicates of 400 COCs each per treatment in ,100,µl SOF-HEPES medium. Protein samples were extracted and analyzed by two-dimensional differential in gel electrophoresis (2D-DIGE). Over 1,600 proteins were resolved on each of the three replicate gels. Sixteen protein spots were identified by mass spectrometry, representing 14 unique, differentially expressed proteins (volume ratio greater than 1.3). Glutathione- S -transferase and pyruvate kinase 3 were more abundant in COCs derived from cyclic females, whereas soluble epoxide hydrolase and transferrin were more abundant in prepubertal derived COCs. Abundance of several glycolytic enzymes (enolase 1, pyruvate kinase 3, and phosphoglycerate kinase) was increased in COCs derived from cyclic females, suggesting glucose metabolism is decreased in prepubertal derived COCs. We conclude that the abundance of proteins involved in metabolism and oxidative stress regulation is significantly altered in prepubertal derived COCs and may play a role in the mechanisms resulting in developmental competence. Mol. Reprod. Dev. 77: 51,58, 2010. © 2009 Wiley-Liss, Inc. [source]

Glutathione and adenosine triphosphate content of in vivo and in vitro matured porcine oocytes

MOLECULAR REPRODUCTION & DEVELOPMENT, Issue 4 2003
A.M. Brad
Abstract Glutathione (GSH) content in mature porcine oocytes is correlated with subsequent fertilization and developmental success. Adenosine triphosphate (ATP) is an important energy source for maintaining cellular activities and protein synthesis. The objective of this study was to compare GSH and ATP concentrations of in vivo and in vitro matured porcine oocytes. Ovulated, in vivo matured oocytes were frozen at ,80°C in groups of 10,20 (GSH) or 5,10 (ATP). In vitro oocytes were matured in either tissue culture medium-199 (TCM199) supplemented with polyvinyl alcohol (PVA) or hyaluronic acid (MAP5), or North Carolina State University-23 (NCSU23) supplemented with porcine follicular fluid (pFF) and frozen as described, or fertilized and cultured. GSH content was determined by the dithionitrobenzoic acid,glutathione disulfide (DTNB,GSSG) reductase recycling assay. ATP content was determined by using the Bioluminescent Somatic Cell Assay Kit. Oocytes matured in vitro in defined TCM199 with PVA or hyaluronic acid, or NCSU23 with pFF had significantly lower concentrations (P,<,0.05) of GSH (n,=,207, 9.82,±,0.71 pmol/oocyte; n,=,104, 9.73,±, 0.81 pmol/oocyte; n,=,108, 7.89,±,0.66 pmol/oocyte, respectively) compared to in vivo matured oocytes (n,=,217, 36.26,±,11.00 pmol/oocyte). Concentrations of ATP were not different between treatments (in vivo, n,=,70, 0.97,±,0.07 pmol/oocyte; TCM,PVA, n,=,117, 0.81,±,0.13 pmol/oocyte; TCM,MAP, n,=,107, 1.02,±,0.18 pmol/oocyte; NCSU,pFF, n,=,134, 0.71,±,0.08 pmol/oocyte). Intracellular ATP content does not appear to be related to developmental potential in porcine oocytes. Low intracellular GSH may be responsible, in part, for lower developmental competence observed in in vitro matured porcine oocytes. Mol. Reprod. Dev. 64: 492,498, 2003. © 2003 Wiley-Liss, Inc. [source]

Glutathione- S -transferase-1 and interleukin-1, gene polymorphisms in Japanese patients with Parkinson's disease

MOVEMENT DISORDERS, Issue 7 2005
Masataka Nishimura MD
Abstract We studied genetic polymorphisms in the glutathione- S -transferase-1 (GST-1) gene region and the interleukin-1, (IL-1,) promoter region in patients with Parkinson's disease (PD, n = 361), as well as controls (n = 257). Although we have confirmed the previous results, in a larger sample, that the IL-1, genotype has affected the age at onset of PD patients, no contribution of the GST-1 gene polymorphism was observed in the allele frequency or the onset age of the disease in Japanese persons. © 2005 Movement Disorder Society [source]

Topically Applied Eicosapentaenoic Acid Protects Against Local Immunosuppression Induced by UVB Irradiation, cis -Urocanic Acid and Thymidine Dinucleotides,

PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 1 2001
Ralf M. W. Moison
ABSTRACT UVB-induced immunosuppression, a promoter of photocarcinogenesis, involves the formation of pyrimidine dimers and cis -urocanic acid (cis -UCA), but reactive oxygen species (ROS) also plays an important role. Eicosapentaenoic acid (EPA) can inhibit photocarcinogenesis, but due to its polyunsaturated nature it is susceptible to oxidative damage by ROS. The antioxidant defense system may therefore be challenged upon ultraviolet-B (UVB) irradiation in the presence of EPA. We investigated whether topically applied EPA in mice could protect against local immunosuppression (contact hypersensitivity response to dinitrofluorobenzene) induced by UVB radiation (1.5 J/cm2), or topically applied cis -UCA (150 nmol/cm2) or thymidine dinucleotides (pTpT) (5 nmol/cm2). The influence of EPA on epidermal lipid peroxidation and antioxidant status was also measured. UVB irradiation, cis -UCA and pTpT all caused 70% immunosuppression. Topical pretreatment of mice with EPA partially protected against immunosuppression; the EPA dose needed to accomplish this was 10 nmol/cm2 for UVB irradiation, 100 nmol/cm2 for cis -UCA and 1000 nmol/cm2 for pTpT. Higher EPA doses caused higher UVB-induced lipid peroxidation and lower vitamin C levels. Glutathione only decreased with the highest EPA dose whereas vitamin E was not decreased after UVB irradiation. In conclusion, topically applied EPA protects against UVB-, cis -UCA- and pTpT-induced immunosuppression and maintenance of an adequate antioxidant defense seems to be an important prerequisite for the protective action by EPA. [source]

Extract of Ginkgo biloba induces glutamate cysteine ligase catalytic subunit (GCLC)

PHYTOTHERAPY RESEARCH, Issue 3 2008
Xiao-Ping Liu
Abstract The extract of Ginkgo biloba (EGb), containing 24% flavone glycosides and 6% terpenoids, is widely used to treat early-stage Alzheimer's disease, vascular dementia, peripheral claudication and vascular tinnitus. Its marked antioxidant activity has recently been demonstrated in both cell lines and animals. Glutathione (GSH) plays an important role in the antioxidant system by conjugating to xenobiotics to facilitate their export from cells. Glutamate cysteine ligase (GCL) is the rate-limiting enzyme for GSH synthesis and its catalytic subunit (GCLC) determines this de novo synthesis. Thus, induction of GCLC is a strategy to enhance the antioxidant capability in cells. The present study aimed to investigate the induction effect of EGb on GCLC in HepG2 and Hep1c1c7 cell lines. Real-time PCR, Western blot and enzyme activity assay were used to detect induction and it was found that GCLC was induced by EGb in these two cell lines. It is suggested that the antioxidant activity of EGb is (or is partly) through the induction of GCLC. Copyright © 2007 John Wiley & Sons, Ltd. [source]

Functional characterization and expression analysis of a glutathione transporter, BjGT1, from Brassica juncea: evidence for regulation by heavy metal exposure

PLANT CELL & ENVIRONMENT, Issue 10 2003
J. BOGS
ABSTRACT Glutathione and its derivatives play an important role in the tolerance of plants against heavy metals. A glutathione transporter, BjGT1 (AJ561120), was cloned and functionally characterized from Brassica juncea, a plant which may be used for phytoremediation. The full-length BjGT1 cDNA showed homology with the high affinity glutathione transporter HGT1 from Saccharomyces cerevisiae and shares 92% identity with a putative glutathione transporter from A. thaliana (At4g16370). When expressed in the S. cerevisiae hgt1, strain, BjGT1 complemented the mutant on medium with glutathione as the only sulphur source and mediated the uptake of [3H]GSH. Immunoblot analysis with a peptide-specific antiserum directed against a C-terminal sequence revealed high BjGT1 expression in leaf tissue and relatively low expression in stem tissue, whereas BjGT1 protein was not detectable in root tissue. The amounts of BjGT1 mRNA and protein were analysed during a 6 d exposure of B. juncea to 25 µm Cd(NO3)2. BjGT1 mRNA was strongly induced by cadmium in stems and leaves. Unexpectedly, the amount of BjGT1 protein in leaves showed a pronounced decrease with a minimum after 96 h of Cd exposure, followed by partial recovery. The strong regulation of BjGT1 by cadmium suggests a role of this glutathione transporter during heavy metal exposure. [source]

Glutathione- S -transferase pi as a model protein for the characterisation of chemically reactive metabolites

PROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 2 2008
Rosalind E. Jenkins Dr.
Abstract Chemically reactive metabolites (CRMs) are thought to be responsible for a number of adverse drug reactions through modification of critical proteins. Methods that defined the chemistry of protein modification at an early stage would provide invaluable tools for drug safety assessment. Here, human GST pi (GSTP) was exploited as a model target protein to determine the chemical, biochemical and functional consequences of exposure to the hepatotoxic CRM of paracetamol (APAP), N -acetyl- p -benzoquinoneimine (NAPQI). Site-specific, dose-dependent modification of Cys47 in native and His-tagged GSTP was revealed by MS, and correlated with inhibition of glutathione (GSH) conjugating activity. In addition, the adaptation of iTRAQ labelling technology to define precisely the quantitative relationship between covalent modification and protein function is described. Multiple reaction monitoring (MRM)-MS of GSTP allowed high sensitivity detection of modified peptides at physiological levels of exposure. Finally, a bioengineered mutant cytochrome P450 with a broad spectrum of substrate specificities was used in an in vitro reaction system to bioactivate APAP: in this model, GSTP trapped the CRM and exhibited both reduced enzyme activity and site-specific modification of the protein. These studies provide the foundation for the development of novel test systems to predict the toxicological potential of CRMs produced by new therapeutic agents. [source]

SHORT COMMUNICATION: Increased Expression of Glutathione by Estradiol, Tumor Necrosis Factor-Alpha, and Interleukin 1-Beta in Endometrial Stromal Cells

AMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY, Issue 6 2009
Sa Ra Lee
Problem, The intracellular antioxidant system, based on glutathione (GSH), plays a key role in endometrial detoxification reactions and has been proposed to be involved in the pathogenesis endometriosis. This study was designed to evaluate whether estradiol (E2) and proinflammatory cytokines have any effects on expression of glutathione in endometrial stromal cells (ESCs). Method of study, Glutathione levels were measured utilizing high-performance liquid chromatography following in vitro culture and treatment of ESCs with estradiol, tumor necrosis factor-alpha (TNF-,) and interleukin 1-beta (IL-1,). Results, The GSH level in E2 (10,8 m) treatment group was significantly higher than in the control group at 48 h (P < 0.05). In vitro treatment of ESCs with TNF-, 10 ng/mL as well as E2 (10,8 m) plus TNF-, 10 ng/mL for 48 hr also led to a significant increase in GSH level (P < 0.05; P < 0.05, respectively). Both IL-1, 10 ng/mL and E2 (10,8 m) plus IL-1, 10 ng/mL for 48 hr increased GSH level significantly (P < 0.05; P < 0.05, respectively) as well. Conclusions, These findings might suggest that increased production of estradiol and proinflammatory cytokines in the peritoneal cavity possibly leads to the establishment of endometriosis through increased level of GSH. [source]