Gamma-glutamyl transferase deficiency results in lung oxidant stress in normoxia

gamma-Glutamyl transferase (GGT) is critical to glutathione homeostasis by providing substrates for glutathione synthesis. We hypothesized that loss of GGT would cause oxidant stress in the lung. We compared the lungs of GGT(enu1) mice, a genetic model of GGT deficiency, with normal mice in normoxia to study this hypothesis. We found GGT promoter 3 (P3) alone expressed in normal lung but GGT P3 plus P1, an oxidant-inducible GGT promoter, in GGT(enu1) lung. Glutathione content was barely decreased in GGT(enu1) lung homogenate and elevated nearly twofold in epithelial lining fluid, but the fraction of oxidized glutathione was increased three- and fourfold, respectively. Glutathione content in GGT(enu1) alveolar macrophages was decreased nearly sixfold, and the oxidized glutathione fraction was increased sevenfold. Immunohistochemical studies showed glutathione deficiency together with an intense signal for 3-nitrotyrosine in nonciliated bronchiolar epithelial (Clara) cells and expression of heme oxygenase-1 in the vasculature only in GGT(enu1) lung. When GGT(enu1) mice were exposed to hyperoxia, survival was decreased by 25% from control because of accelerated formation of vascular pulmonary edema, widespread oxidant stress in the epithelium, diffuse depletion of glutathione, and severe bronchiolar cellular injury. These data indicate a critical role for GGT in lung glutathione homeostasis and antioxidant defense in normoxia and hyperoxia.     

The function of gamma-glutamyl transpeptidase as a determinant in cell sensitivity to azaserine toxicity

The enzyme gamma-glutamyl transpeptidase (GGT) is characteristically present at high levels in mammalian cells that are vulnerable in vivo to the selectively toxic and carcinogenic effects of the naturally occurring diazo amino acid L-azaserine. The possible role of GGT as a determinant of cellular sensitivity to azaserine toxicity was investigated. No correlation was found between GGT activity and the abilities of different cell lines or GGT-deficient cell strains of TuWi, a human nephroblastoma-derived line high in GGT, to accumulate azaserine. However, the thiols glutathione and cysteine were found to inhibit the toxicity of azaserine in cultures of TuWi. In addition, maleate lowered both intracellular and extracellular glutathione levels and enhanced sensitivity of TuWi cells to azaserine, while serine-borate, a potent inhibitor of GGT, increased extracellular glutathione levels and inhibited azaserine toxicity. Since extracellular glutathione accumulation, which may reflect the rate of cellular glutathione turnover, is increased in cultures of azaserine-resistant, GGT-deficient strains of TuWi, we propose that GGT enhances cellular sensitivity to azaserine primarily by increasing the rate of glutathione turnover, thus removing the glutathione from detoxification pathways.     

Glutathione availability modulates alveolar macrophage function in the chronic ethanol-fed rat

We have previously demonstrated that chronic alcohol exposure decreases glutathione in the alveolar space. Although alcohol use is associated with decreased alveolar macrophage function, the mechanism by which alcohol impairs macrophage phagocytosis is unknown. In the current study, we examined the possibility that ethanol-induced alveolar macrophage dysfunction was secondary to decreased glutathione and subsequent chronic oxidative stress in the alveolar space. After 6 wk of ethanol ingestion, oxidant stress in the alveolar macrophages was evidenced by a 30-mV oxidation of the GSH/GSSG redox potential (P 相似文献   

Vitamin C is an essential antioxidant that enhances survival of oxidatively stressed human vascular endothelial cells in the presence of a vast molar excess of glutathione

Cellular glutathione levels may exceed vitamin C levels by 10-fold, generating the question about the real antioxidant role that low intracellular concentrations of vitamin C can play in the presence of a vast molar excess of glutathione. We characterized the metabolism of vitamin C and its relationship with glutathione in primary cultures of human endothelial cells oxidatively challenged by treatment with hydrogen peroxide or with activated cells undergoing the respiratory burst, and analyzed the manner in which vitamin C interacts with glutathione to increase the antioxidant capacity of cells. Our data indicate that: (i) endothelial cells express transporters for reduced and oxidized vitamin C and accumulate ascorbic acid with participation of glutathione-dependent dehydroascorbic acid reductases, (ii) although increased intracellular levels of vitamin C or glutathione caused augmented resistance to oxidative stress, 10-times more glutathione than vitamin C was required, (iii) full antioxidant protection required the simultaneous presence of intracellular and extracellular vitamin C at concentrations normally found in vivo, and (iv) intracellular vitamin C cooperated in enhancing glutathione recovery after oxidative challenge thus providing cells with enhanced survival potential, while extracellular vitamin C was recycled through a mechanism involving the simultaneous neutralization of oxidant species. Therefore, in endothelial cells under oxidative challenge, vitamin C functions as an essential cellular antioxidant even in the presence of a vast molar excess of glutathione.     

Normal alveolar epithelial lining fluid contains high levels of glutathione

The epithelial cells on the alveolar surface of the human lower respiratory tract are vulnerable to toxic oxidants derived from inhaled pollutants or inflammatory cells. Although these lung cells have intracellular antioxidants, these defenses may be insufficient to protect the epithelial surface against oxidants present at the alveolar surface. This study demonstrates that the epithelial lining fluid (ELF) of the lower respiratory tract contains large amounts of the sulfhydryl-containing antioxidant glutathione (GSH). The total glutathione (the reduced form GSH and the disulfide GSSG) concentration of normal ELF was 140-fold higher than that in plasma of the same individuals, and 96% of the glutathione in ELF was in the reduced form. Compared with nonsmokers, cigarette smokers had 80% higher levels of ELF total glutathione, 98% of which was in the reduced form. Studies of cultured lung epithelial cells and fibroblasts demonstrated that these concentrations of reduced glutathione were sufficient to protect these cells against the burden of H2O2 in the range released by alveolar macrophages removed from the lower respiratory tract of nonsmokers and smokers, respectively, suggesting that the glutathione present in the alveolar ELF of normal individuals likely contributes to the protective screen against oxidants in the extracellular milieu of the lower respiratory tract.     

Expression of gamma-glutamyl transpeptidase protects ramos B cells from oxidation-induced cell death

The ectoenzyme, gamma-glutamyl transpeptidase (GGT, EC ) cleaves glutathione (GSH) to facilitate the recapture of cysteine for synthesis of intracellular GSH. The impact of GGT expression on cell survival during oxidative stress was investigated using the human B cell lymphoblastoid cell line, Ramos. Ramos cells did not express surface GGT and exhibited no GGT enzyme activity. In contrast, Ramos cells stably transfected with the human GGT cDNA expressed high levels of surface GGT and enzymatic activity. GGT-transfected Ramos cells were protected from apoptosis when cultured in cyst(e)ine-deficient medium. The GGT-expressing cells also had lower levels of intracellular reactive oxygen species (ROS). Homocysteic acid and alanine, inhibitors of cystine and cysteine uptake, respectively, caused increased ROS content and diminished viability of GGT expressing cells. Exogenous GSH increased the viability of the GGT-transfected cells more effectively than that of control cells, whereas the products of GSH metabolism prevented death of both the control and GGT-transfected cells comparably. These data indicate that GGT cleavage of GSH and the subsequent recapture of cysteine and cystine allow cells to maintain low levels of cellular ROS and thereby avoid apoptosis induced by oxidative stress.     

Molecular mechanism of transforming growth factor (TGF)-beta1-induced glutathione depletion in alveolar epithelial cells. Involvement of AP-1/ARE and Fra-1

Glutathione (GSH) is a ubiquitous antioxidant in lung epithelial cells and lung lining fluid. Transforming growth factor beta1 (TGF-beta1) is a pleiotropic cytokine involved in cellular proliferation and differentiation. The level of TGF-beta1 is elevated in many chronic inflammatory lung disorders associated with oxidant/antioxidant imbalance. In this study, we show that TGF-beta1 depletes GSH by down-regulating expression of the enzyme responsible for its formation, gamma-glutamylcysteine synthetase (gamma-GCS) and induces reactive oxygen species production in type II alveolar epithelial cells (A549). To investigate the molecular mechanisms of inhibition of glutathione synthesis, we employed reporters containing fragments from the promoter region of the gamma-GCS heavy subunit (h), the gene that encodes the catalytic subunit of gamma-GCS. We found that TGF-beta1 reduced the expression of the long gamma-GCSh construct (-3802/GCSh-5'-Luc), suggesting that an antioxidant response element (ARE) may be responsible for mediating the TGF-beta1 effect. Interestingly, the electrophoretic mobility shift assay revealed that the DNA binding activity of both activator protein-1 (AP-1) and ARE was increased in TGF-beta1-treated epithelial cells. The gamma-GCSh ARE contains a perfect AP-1 site embedded within it, and mutation of this internal AP-1 sequence, but not the surrounding ARE, prevented DNA binding. Further studies revealed that c-Jun and Fra-1 dimers, members of the AP-1 family previously shown to exert a negative effect on phase II gene expression, bound to the ARE sequence. We propose a novel mechanism of gamma-GCSh down-regulation by TGF-beta1 that involves the binding of c-Jun and Fra-1 dimers to the distal promoter. The findings of this study provide important information, which may be used for the modulation of glutathione biosynthesis in inflammation.     

The gamma-glutamyltransferase activity and non-protein sulfhydryl compounds levels in rat kidney of different age groups

The present work was aimed to obtain information about age-dependent changes of gamma-glutamyltransferase (GGT) activity and the levels of non-protein sulfhydryl compounds (NPSH) in rat kidneys. In addition, protein-bound cysteine (PB-Cys), sulfane sulfur compounds and reactive oxygen species (ROS) were estimated. The results indicate that the activity of GGT and NPSH levels in the kidneys are reduced with age. At the same time, a significant increase in the level of protein-bound cysteine was observed. Simultaneously, the content of sulfane sulfur compounds was increased in the group of the oldest animals. These findings indicate that the capacity for extracellular glutathione degradation and, in consequence, the availability of cysteine for intracellular glutathione biosynthesis may be impaired. The increased PB-Cys level indicates potentiation of the thiolation reaction, i.e. development of protein-mixed disulfides. These results reveal age dependent disturbances in the thiol-disulfide equilibrium in the kidneys which leads to an imbalance between pro- and antioxidatory processes.     

Antioxidant imbalance in the lungs of cystic fibrosis transmembrane conductance regulator protein mutant mice

Recent studies suggest that the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) protein modulates epithelial reduced glutathione (GSH) transport and when defective creates an antioxidant imbalance. To test whether the CFTR protein modulates lung antioxidant defenses in vivo, epithelial lining fluid (ELF) and lung tissue from CFTR knockout (CFTR-KO) and wild-type (WT) mice were compared for GSH content and the activities of glutathione reductase, glutathione peroxidase, and gamma-glutamyltransferase. In the CFTR-KO mice, the ELF concentration of GSH was decreased (51%) compared with that in WT mice. The concentration of GSH in the lung tissue of CFTR-KO mice, however, was not significantly different from that in WT mice. The activities of glutathione reductase and glutathione peroxidase in the lung tissue of CFTR-KO mice were significantly increased compared with those in WT mice (48 and 28%, respectively). Tissue lipid and DNA oxidation were evaluated by measurement of thiobarbituric acid-reactive substances and 8-hydroxy-2'-deoxyguanosine, respectively. The levels of thiobarbituric acid-reactive substances and 8-hydroxy-2'-deoxyguanosine in the lung tissue of CFTR-KO mice were significantly increased compared with those in WT mice. These data support our hypothesis that a mutation in the CFTR gene can affect the antioxidant defenses in the lung and may contribute to the exaggerated inflammatory response observed in CF.     

Redox biology of the intestine

The intestinal tract, known for its capability for self-renew, represents the first barrier of defence between the organism and its luminal environment. The thiol/disulfide redox systems comprising the glutathione/glutathione disulfide (GSH/GSSG), cysteine/cystine (Cys/CySS) and reduced and oxidized thioredoxin (Trx/TrxSS) redox couples play important roles in preserving tissue redox homeostasis, metabolic functions, and cellular integrity. Control of the thiol-disulfide status at the luminal surface is essential for maintaining mucus fluidity and absorption of nutrients, and protection against chemical-induced oxidant injury. Within intestinal cells, these redox couples preserve an environment that supports physiological processes and orchestrates networks of enzymatic reactions against oxidative stress. In this review, we focus on the intestinal redox and antioxidant systems, their subcellular compartmentation, redox signalling and epithelial turnover, and contribution of luminal microbiota, key aspects that are relevant to understanding redox-dependent processes in gut biology with implications for degenerative digestive disorders, such as inflammation and cancer.     

Redox biology of the intestine

Abstract

The intestinal tract, known for its capability for self-renew, represents the first barrier of defence between the organism and its luminal environment. The thiol/disulfide redox systems comprising the glutathione/glutathione disulfide (GSH/GSSG), cysteine/cystine (Cys/CySS) and reduced and oxidized thioredoxin (Trx/TrxSS) redox couples play important roles in preserving tissue redox homeostasis, metabolic functions, and cellular integrity. Control of the thiol-disulfide status at the luminal surface is essential for maintaining mucus fluidity and absorption of nutrients, and protection against chemical-induced oxidant injury. Within intestinal cells, these redox couples preserve an environment that supports physiological processes and orchestrates networks of enzymatic reactions against oxidative stress. In this review, we focus on the intestinal redox and antioxidant systems, their subcellular compartmentation, redox signalling and epithelial turnover, and contribution of luminal microbiota, key aspects that are relevant to understanding redox-dependent processes in gut biology with implications for degenerative digestive disorders, such as inflammation and cancer.     

Oxidation of extracellular cysteine/cystine redox state in bleomycin-induced lung fibrosis

Several lines of evidence indicate that depletion of glutathione (GSH), a critical thiol antioxidant, is associated with the pathogenesis of idiopathic pulmonary fibrosis (IPF). However, GSH synthesis depends on the amino acid cysteine (Cys), and relatively little is known about the regulation of Cys in fibrosis. Cys and its disulfide, cystine (CySS), constitute the most abundant low-molecular weight thiol/disulfide redox couple in the plasma, and the Cys/CySS redox state (E(h) Cys/CySS) is oxidized in association with age and smoking, known risk factors for IPF. Furthermore, oxidized E(h) Cys/CySS in the culture media of lung fibroblasts stimulates proliferation and expression of transitional matrix components. The present study was undertaken to determine whether bleomycin-induced lung fibrosis is associated with a decrease in Cys and/or an oxidation of the Cys/CySS redox state and to determine whether these changes were associated with changes in E(h) GSH/glutathione disulfide (GSSG). We observed distinct effects on plasma GSH and Cys redox systems during the progression of bleomycin-induced lung injury. Plasma E(h) GSH/GSSG was selectively oxidized during the proinflammatory phase, whereas oxidation of E(h) Cys/CySS occurred at the fibrotic phase. In the epithelial lining fluid, oxidation of E(h) Cys/CySS was due to decreased food intake. Thus the data show that decreased precursor availability and enhanced oxidation of Cys each contribute to the oxidation of extracellular Cys/CySS redox state in bleomycin-induced lung fibrosis.     

Cathepsin B, L, and S cleave and inactivate secretory leucoprotease inhibitor.

A number of serine proteases, matrix metalloproteases, and cysteine proteases were evaluated for their ability to cleave and inactivate the antiprotease, secretory leucoprotease inhibitor (SLPI). None of the serine proteases or the matrix metalloproteases examined cleaved the SLPI protein. However, incubation with cathepsins B, L, and S resulted in the cleavage and inactivation of SLPI. All three cathepsins initially cleaved SLPI between residues Thr(67) and Tyr(68). The proteolytic cleavage of SLPI by all three cathepsins resulted in the loss of the active site of SLPI and the inactivation of SLPI anti-neutrophil elastase capacity. Cleavage and inactivation were catalytic with respect to the cathepsins, so that the majority of a 400-fold excess of SLPI was inactivated within 15 min by cathepsins L and S. Analysis of epithelial lining fluid samples from individuals with emphysema indicated the presence of cleaved SLPI in these samples whereas only intact SLPI was observed in control epithelial lining fluid samples. Active cathepsin L was shown to be present in emphysema epithelial lining fluid and inhibition of this protease prevented the cleavage of recombinant SLPI added to emphysema epithelial lining fluid. Taken together with previous data that demonstrates that cathepsin L inactivates alpha(1)-antitrypsin, these findings indicate the involvement of cathepsins in the diminution of the lung antiprotease screen possibly leading to lung destruction in emphysema.     

Expression of gamma-glutamyltransferase 1 in glioblastoma cells confers resistance to cystine deprivation–induced ferroptosis

Ferroptosis is an iron-dependent mode of cell death caused by excessive oxidative damage to lipids. Lipid peroxidation is normally suppressed by glutathione peroxidase 4, which requires reduced glutathione. Cystine is a major resource for glutathione synthesis, especially in cancer cells. Therefore, cystine deprivation or inhibition of cystine uptake promotes ferroptosis in cancer cells. However, the roles of other molecules involved in cysteine deprivation–induced ferroptosis are unexplored. We report here that the expression of gamma-glutamyltransferase 1 (GGT1), an enzyme that cleaves extracellular glutathione, determines the sensitivity of glioblastoma cells to cystine deprivation–induced ferroptosis at high cell density (HD). In glioblastoma cells expressing GGT1, pharmacological inhibition or deletion of GGT1 suppressed the cell density–induced increase in intracellular glutathione levels and cell viability under cystine deprivation, which were restored by the addition of cysteinylglycine, the GGT product of glutathione cleavage. On the other hand, cystine deprivation induced glutathione depletion and ferroptosis in GGT1-deficient glioblastoma cells even at an HD. Exogenous expression of GGT1 in GGT1-deficient glioblastoma cells inhibited cystine deprivation–induced glutathione depletion and ferroptosis at an HD. This suggests that GGT1 plays an important role in glioblastoma cell survival under cystine-limited and HD conditions. We conclude that combining GGT inhibitors with ferroptosis inducers may provide an effective therapeutic approach for treating glioblastoma.     

Glutathione S-transferase omega in the lung and sputum supernatants of COPD patients

Background

The major contribution to oxidant related lung damage in COPD is from the oxidant/antioxidant imbalance and possibly impaired antioxidant defence. Glutathione (GSH) is one of the most important antioxidants in human lung and lung secretions, but the mechanisms participating in its homeostasis are partly unclear. Glutathione-S-transferase omega (GSTO) is a recently characterized cysteine containing enzyme with the capability to bind and release GSH in vitro. GSTO has not been investigated in human lung or lung diseases.

Methods

GSTO1-1 was investigated by immunohistochemistry and Western blot analysis in 72 lung tissue specimens and 40 sputum specimens from non-smokers, smokers and COPD, in bronchoalveolar lavage fluid and in plasma from healthy non-smokers and smokers. It was also examined in human monocytes and bronchial epithelial cells and their culture mediums in vitro.

Results

GSTO1-1 was mainly expressed in alveolar macrophages, but it was also found in airway and alveolar epithelium and in extracellular fluids including sputum supernatants, bronchoalveolar lavage fluid, plasma and cell culture mediums. The levels of GSTO1-1 were significantly lower in the sputum supernatants (p = 0.023) and lung homogenates (p = 0.003) of COPD patients than in non-smokers.

Conclusion

GSTO1-1 is abundant in the alveolar macrophages, but it is also present in extracellular fluids and in airway secretions, the levels being decreased in COPD. The clinical significance of GSTO1-1 and its role in regulating GSH homeostasis in airway secretions, however, needs further investigations.     

nSMase2 activation and trafficking are modulated by oxidative stress to induce apoptosis

We have previously shown that accumulation of ceramide, triggered by hydrogen peroxide (H(2)O(2)), induces apoptosis of human airway epithelial (HAE) cells. Under oxidant exposure, a lung sphingomyelinase (SMase) is activated and displays continued ceramide generation and pro-apoptotic signaling, thus leading to the pathological apoptosis that causes lung injury. In a search for a specific SMase that is modulated by oxidative stress, we recently cloned nSMase2 from monkey lung tissue and HAE cells. Here, we show that this nSMase2 is up-regulated by an oxidant (H(2)O(2)) and is inhibited by an antioxidant (glutathione (GSH)). Moreover, nSMase2 subcellular localization is governed by oxidant exposure, which leads to its preferential trafficking to the plasma membrane, where it generates ceramide and induces apoptosis. On the other hand, exposure to GSH results in nSMase2 trafficking to the nucleus, where it neither generates ceramide nor induces apoptosis.     

Role of transferrin and ceruloplasmin in antioxidant activity of lung epithelial lining fluid

Lung epithelial lining fluid (ELF) is a thin layer of plasma ultrafiltrate and locally secreted substances that may provide antioxidant protection and serve as a "front-line" defense for the lower respiratory tract epithelium. To characterize the antioxidant properties of ELF, young, healthy, nonsmoking volunteers underwent bronchoalveolar lavage with determination of ELF volumes and ELF proteins. ELF (greater than 0.4 ml) is a potent inhibitor of lipid peroxidation as measured by malondialdehyde (MDA) production in an in vitro iron-dependent assay system. Two serum proteins, transferrin and ceruloplasmin, were quantitated in ELF and found to be potent inhibitors of lipid peroxidation. Other ELF components, including vitamin E, vitamin C, and albumin, did not function as antioxidants in this system. Several experimental observations suggest that ELF transferrin was more important than ceruloplasmin in inhibiting lipid peroxidation: 1) ELF concentrations of transferrin were 20-fold higher than those for ceruloplasmin; 2) ELF antioxidant activity was abolished by preincubation with Fe3+; 3) ELF antioxidant activity was minimally affected by sodium azide, which is known to inhibit ceruloplasmin ferroxidase activity; and 4) ELF ceruloplasmin ferroxidase activity was virtually nondetectable. ELF possesses a significant antioxidant activity that may be important in vivo in protecting the lung from oxidant injury.     

Synthetic chloride channel restores glutathione secretion in cystic fibrosis airway epithelia

Cystic fibrosis (CF), an inherited disease characterized by defective epithelial Cl- transport, damages lungs via chronic inflammation and oxidative stress. Glutathione, a major antioxidant in the epithelial lung lining fluid, is decreased in the apical fluid of CF airway epithelia due to reduced glutathione efflux (Gao L, Kim KJ, Yankaskas JR, and Forman HJ. Am J Physiol Lung Cell Mol Physiol 277: L113-L118, 1999). The present study examined the question of whether restoration of chloride transport would also restore glutathione secretion. We found that a Cl- channel-forming peptide (N-K4-M2GlyR) and a K+ channel activator (chlorzoxazone) increased Cl- secretion, measured as bumetanide-sensitive short-circuit current, and glutathione efflux, measured by high-performance liquid chromatography, in a human CF airway epithelial cell line (CFT1). Addition of the peptide alone increased glutathione secretion (181 +/- 8% of the control value), whereas chlorzoxazone alone did not significantly affect glutathione efflux; however, chlorzoxazone potentiated the effect of the peptide on glutathione release (359 +/- 16% of the control value). These studies demonstrate that glutathione efflux is associated with apical chloride secretion, not with the CF transmembrane conductance regulator per se, and the defect of glutathione efflux in CF can be overcome pharmacologically.