Effect Of Glutathione Depletion Research Articles

2044 GLYOXAL METABOLISM: A NOVEL PATHWAY IN ENDOGENOUS OXALATE SYNTHESIS

You have accessJournal of UrologyStone Disease: Basic Research1 Apr 20112044 GLYOXAL METABOLISM: A NOVEL PATHWAY IN ENDOGENOUS OXALATE SYNTHESIS Kyle D. Wood, John Knight, Dean G. Assimos, and Ross P. Holmes Kyle D. WoodKyle D. Wood Winston-Salem, NC More articles by this author , John KnightJohn Knight Winston-Salem, NC More articles by this author , Dean G. AssimosDean G. Assimos Winston-Salem, NC More articles by this author , and Ross P. HolmesRoss P. Holmes Winston-Salem, NC More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2011.02.2275AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The immediate precursor of the endogenous synthesis of oxalate is glyoxylate. The sources of glyoxylate are not well defined. Glyoxal, a reactive di-aldehyde, may be a significant source. Plasma glyoxal levels are higher in diabetics; a cohort with higher levels of urinary oxalate excretion. Glyoxal is detoxified through the glutathione dependent glyoxalase pathway to glycolate. We propose an alternative metabolism of glyoxal via aldehyde dehydrogenase yielding glyoxylate which may be accelerated by oxidative stress. METHODS The following 3 in-vitro model systems were employed: human liver lysates, HepG2 cells (hepatoma cell line), and human red blood cells. Incubations over time with varying concentrations of glyoxal (0.01 mM to 5 mM) were performed with liver lysates and HepG2 cells. HepG2 cells and erythrocytes were also incubated with 5mM and 50mM 13C labeled glucose. The effect of glutathione depletion on erythrocyte conversion of glyoxal to oxalate and glycolate was also studied. Liver lysates were incubated with disulfiram, an inhibitor of aldehyde dehydrogenase, NAD+, a known cofactor of this enzyme, and NADP+. Glyoxylate, glycolate and oxalate were measured using ion chromatography, ion chromatography coupled with mass spectroscopy, and high pressure liquid chromatography. RESULTS In human liver lysates treated with increasing concentrations of glyoxal, there was a linear increase in glyoxylate production. Human liver lysates only converted glyoxal to glyoxylate in the presence of NAD+ and this conversion was inhibited by disulfiram. As glyoxal concentrations were increased from 0 to 2.5 mM in HepG2 cells, there was a linear increase in both glycolate and oxalate production; significantly more glycolate produced than oxalate. Incubating red blood cells with both glyoxal and menadione, an agent which depletes glutathione, resulted in significantly more oxalate and decreased glycolate production. Human erythrocytes cultured with 50 mM 13C labeled glucose yielded both 13C labeled glycolate and oxalate whereas 5mM 13C labeled glucose did not yield oxalate or glycolate. CONCLUSIONS Glyoxal appears to be converted to glyoxylate via aldehyde dehydrogenase resulting in endogenous oxalate synthesis. Under conditions of oxidative stress there is an increased flux of glyoxal to oxalate formation. The exposure of cells to high levels of glucose results in its conversion to both glycolate and oxalate, likely via glyoxal. This novel pathway may be the major source of endogenous oxalate synthesis. © 2011 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 185Issue 4SApril 2011Page: e818 Advertisement Copyright & Permissions© 2011 by American Urological Association Education and Research, Inc.MetricsAuthor Information Kyle D. Wood Winston-Salem, NC More articles by this author John Knight Winston-Salem, NC More articles by this author Dean G. Assimos Winston-Salem, NC More articles by this author Ross P. Holmes Winston-Salem, NC More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Effect of glutathione depletion, hyperthermia, and a 100‐mT static magnetic field on an hsp70/luc reporter system

Heat shock proteins, in particular hsp70, are induced under conditions of cellular stress. It has been reported that environmental stimuli such as hyperthermia, oxidative stress, and exposure to magnetic fields increase levels of hsp70. It has also been reported that hyperthermia in combination with magnetic field exposure results in a synergistic increase in hsp70 production. We tested the hypothesis that oxidative stress induced by glutathione (GSH) depletion in combination with static magnetic field (SMF) exposure will produce a similar synergistic increase in hsp70 production. We exposed cells to heat, SMF, and diethylmaleate (DEM), which depletes GSH levels alone and in combination with each other, and measured hsp70 production using an hsp70/luciferase reporter and mRNA levels using PCR. We found that treatment with DEM significantly reduced the rate of luciferase bioluminescence production, particularly in the presence of heat. There was no significant effect of a 100-mT SMF exposure either alone or in combination with heat, DEM, or both on bioluminescence, however there was a significant interaction between SMF and DEM on hsp70 mRNA levels. Therefore, under our exposure conditions, GSH depletion reduced hsp70 levels but a synergistic effect of combining this stress with other external stimuli was only observed at the level of mRNA.

The Cystine/Glutamate Antiporter Regulates Dendritic Cell Differentiation and Antigen Presentation

The major cellular antioxidant glutathione is depleted during HIV infection and in obesity. Although the consequence of glutathione depletion on immune function is starting to emerge, it is currently not known whether glutathione dysregulation influences the differentiation and maturation of dendritic cells (DCs). Moreover, the effect of glutathione depletion on DC effector functions, such as Ag presentation, is poorly understood. Glutathione synthesis depends on the cystine/glutamate antiporter, which transports the rate-limiting precursor cystine into the cell in exchange for glutamate. In this paper, we present a detailed study of antiporter function in DCs and demonstrate a role for the antiporter in DC differentiation and cross-presentation. We show that the antiporter is the major mechanism for transport of cystine and glutamate and modulates the intracellular glutathione content and glutathione efflux from DCs. Blocking antiporter-dependent cystine transport decreases intracellular glutathione levels, and these effects correlate with reduced transcription of the functional subunit of the antiporter. We further demonstrate that blocking antiporter activity interferes with DC differentiation from monocyte precursors, but antiporter activity is not required for LPS-induced phenotypic maturation. Finally, we show that inhibiting antiporter uptake of cystine interferes with presentation of exogenous Ag to class II MHC-restricted T cells and blocks cross-presentation on MHC class I. We conclude that aberrant antiporter function disrupts glutathione homeostasis in DCs and may contribute to impaired immunity in the diseased host.

Effect of glutathione depletion on removal of copper from LEC rat livers by tetrathiomolybdate

Tetrathiomolybdate (TTM) is a powerful and selective copper (Cu) chelator that is used as a therapeutic agent for Wilson disease. TTM is the sole agent that can remove Cu bound to metallothionein (MT) in the livers of Long–Evans rats with a cinnamon-like coat color (LEC rats). However, the administration of excess TTM causes the deposition of Cu and molybdenum (Mo) in the liver. In the present study, the effect of hepatic glutathione (GSH) depletion on the removal of Cu from the livers of LEC rats was evaluated to establish an effective therapy by TTM. Pretreatment with l-buthionine sulfoximine (BSO), a depletor of GSH in vivo, reduced the amounts of Cu and Mo excreted into both the bile and the bloodstream, and increased the amounts of Cu and Mo deposited in the livers of LEC rats in the form of an insoluble complex 4 h after the TTM injection. The results suggest that GSH depletion creates an oxidative environment in the livers of LEC rats, and the oxidative environment facilitates the insolubilization of Cu and Mo in the livers of LEC rats after the TTM injection. Therefore, the effect of TTM on the removal of Cu from the liver was reduced in the oxidized condition. Wilson disease patients and LEC rats develop liver injury caused by oxidative damage. From a clinical viewpoint, increasing in the GSH concentration is expected to enhance the effect of TTM.

Effect of glutathione depletion on mitochondrial‐mediated apoptotic signaling in rat skeletal muscle

Glutathione (GSH) is an abundant thiol that is critical in cellular redox status. Reduced cellular antioxidant capacity has been shown to increase cellular damage and apoptosis. In this study we investigated the effect of GSH depletion, by L‐buthionine‐[S,R]‐sulfoximine (BSO), on redox status and apoptotic signaling in rat quadriceps muscle. BSO treatment significantly (p<0.05) reduced whole muscle and mitochondrial GSH content by 95% and 84%, respectively. Whole muscle reactive oxygen species (ROS) generation was also found to be significantly (p<0.05) elevated in BSO‐treated animals compared to controls. In addition, CuZnSOD protein content was significantly (p<0.05) increased in BSO‐treated animals compared to controls. Flow cytometric analysis also revealed that membrane potential was significantly (p<0.05) reduced in isolated mitochondria of BSO‐treated animals. Interestingly, the activity of caspase‐3 and ‐9 in skeletal muscle was not significantly different between groups. This data suggests that glutathione depletion significantly alters muscle/mitochondrial redox status as well as mitochondrial membrane potential but not basal apoptotic signaling. It remains to be determined if these changes influence apoptotic signaling/susceptibility to additional cellular stressors. Research supported by NSERC of Canada.

Effect of Glutathione Depletion on Susceptibility of MA-10 Leydig Cell Steroidogenesis to Oxidative Stress.

Effect of Glutathione Depletion on Susceptibility of MA-10 Leydig Cell Steroidogenesis to Oxidative Stress.

Effect of Glutathione Depletion on Leydig Cell Steroidogenesis in Young and Old Brown Norway Rats

Changes in the oxidant/antioxidant environment of aging Leydig cells have been shown to be correlated with the reduced ability of these cells to produce testosterone. With this in mind, we hypothesized that the experimental depletion of glutathione (GSH), an abundant Leydig cell intracellular antioxidant, might result in reduced testosterone production. Incubation of Leydig cells isolated from the testes of adult Brown Norway rats with buthionine sulfoximine (BSO) reduced GSH content by more than 70% and testosterone production by about 40%. The antioxidants vitamin E, N-tert-butyl-alpha-phenylnitrone and Trolox countered BSO's effect on steroidogenesis but not on GSH depletion. Together, BSO and glutathione ethyl ester maintained intracellular GSH and also testosterone production, whereas 1,2-dithiole-3-thione, which increases intracellular GSH, increased testosterone production. In vivo studies also were conducted. Young (4 month old) and old (24 month old) rats were injected with BSO twice a day for 7 d, after which Leydig cells were isolated and analyzed in vitro. BSO treatment reduced Leydig cell GSH content by 70% and the ability of the Leydig cells to produce testosterone by more than 50%. As with aging, decreases were seen in LH-stimulated cAMP production, steroidogenic acute regulatory protein, cholesterol side-chain cleavage, 3beta-hydroxysteroid dehydrogenase, and 17alpha-hydroxylase/17,20-lyase. The results of these studies, taken together, are consistent with the hypothesis that alteration in the oxidant/antioxidant environment may play a significant, causative role in the age-related reduced ability of Leydig cells to produce testosterone.

Glutathione depletion inhibits dendritic cell maturation and delayed-type hypersensitivity: Implications for systemic disease and immunosenescence

Dendritic cells (DCs) play a key role as antigen-presenting cells in the immune system. There is growing evidence that the redox equilibrium of these cells influences their ability to induce T-cell activation and to regulate the polarity of the immune response. This could affect the outcome of the immune response during systemic diseases and aging. Our aim was to elucidate the mechanism by which the redox equilibrium of antigen-presenting DCs affects the delayed-type hypersensitivity (DTH) response during experimental modification of glutathione levels, as well as during aging. We looked at the effect of glutathione depletion by diethyl maleate in DCs as well as during systemic administration on the DTH response to the contact-sensitizing antigens, oxazolone, and 2,4-dinitro-1-fluorobenzene. We also determined whether glutathione repletion with N-acetyl cysteine could influence the decline of the DTH response in aged mice. Glutathione depletion in bone marrow-derived DCs interfered in their ability to mount a DTH response on adoptive transfer into recipient mice. Glutathione depletion interfered in IL-12 production and costimulatory receptor expression in DCs, leading to decreased IFN-gamma production in the skin of recipient mice. Systemic diethyl maleate treatment exerted similar effects on the DTH response and IFN-gamma production, whereas N-acetyl cysteine administration reversed the decline of the DTH response in aged animals. Glutathione depletion downregulates T(H)1 immunity through a perturbation of DC maturation and IL-12 production. These data show that the induction of oxidative stress in the immune system, under disease conditions and aging, interferes in T(H)1 immunity.

Effect of glutathione depletion on apoptosis induced by thiram in Chinese hamster fibroblasts

Fungicide thiram, which is also known as an inducer of allergic contact dermatitis (ACD), was used as a model compound of thiuram chemicals, and its cellular effects were investigated in cultured Chinese hamster V79 cells. The level of intracellular reduced glutathione (GSH), protein sulfhydryl (PSH) groups, protein carbonyls (PC), membrane lipid peroxidation reflected by enhanced thiobarbituric acid reactive substrates (TBARS) production, as well as apoptotic effect were determined. The apoptosis induction was determined by assessing DNA fragmentation by TUNEL, annexin V binding, and caspases activation assays, using fluorescent microscope or flow cytometry, respectively. The concentrations of thiram required to induce cellular GSH depletion (by 40–50%), protein, and membrane lipid peroxidation (2-fold, and 1.7-fold, respectively), as well as to induce apoptosis in V79 Chinese hamster fibroblasts without causing necrosis through cytotoxic effects were between 50–100 μM. To investigate the role of decreased GSH content in the toxicity of thiram, GSH level was modified prior to exposure. Pretreatment of V79 cells with N-acetyl- l-cysteine (NAC), a GSH biosynthesis precursor, prevented GSH decrease, PC and TBARS production, as well as caspases activation induced by thiram exposure. On the other hand, thiram effects were enhanced by the previous depletion of cellular GSH by l-buthionine-(S,R)-sulfoximine (BSO).

Effect of glutathione depletion on antioxidant enzymes in the epididymis, seminal vesicles, and liver and on spermatozoa motility in the aging brown Norway rat.

Reactive oxygen species (ROS) play a role in male infertility, where excessive amounts impair spermatozoal motility. Epididymal antioxidant enzymes protect spermatozoa from oxidative damage in the epididymal lumen. Antioxidant secretions from the seminal vesicle protect spermatozoa after ejaculation. As it is known that with age there is increased generation of ROS, the goals of this study were to determine how aging affects the response of antioxidant enzymes in the epididymis, seminal vesicles, and liver to l-buthionine-S,R-sulfoximine (BSO) mediated glutathione (GSH) depletion, and to examine the impact of GSH depletion on motility parameters of spermatozoa from the cauda epididymidis in young (4-mo-old) and old (21-mo-old) rats. Levels of GSH and glutathione disulfide (GSSG), as well as activities of glutathione peroxidase, glutathione reductase, catalase, and superoxide dismutase, were measured in the caput, corpus and cauda epididymidis, seminal vesicles, and liver. Spermatozoal motility was assessed by computer-assisted sperm analysis. Significant age-related changes in antioxidant enzyme activities were found in the liver and cauda epididymidis. Glutathione depletion clearly affected tissues in both young and old. The compounding effect of age was most evident in the cauda epididymidis, seminal vesicles, and liver, where antioxidant enzyme activities changed significantly. Additionally, spermatozoa motility was adversely affected after BSO treatment in both age groups, but significantly more so in older animals. In summary, the male reproductive tissues and liver undergo age-related changes in antioxidant enzyme activities and in their response to GSH depletion.

Effect of glutathione depletion on sites and topology of superoxide and hydrogen peroxide production in mitochondria.

In this work, the topology of mitochondrial O2(-)(radical) and H2O2 generation and their interplay with matrix GSH in isolated heart mitochondria were examined. We observed that complex I releases O2(-)(radical) into the matrix (where it is converted to H2O2 by Mn-SOD) but not into the intermembrane space. No free radical generation was observed from complex II, but succinate treatment caused H2O2 generation from the matrix through a reverse electron flow to complex I. Complex III was found to release O2(-)(radical) into the matrix and into the intermembrane space. Antimycin, which increases steady-state levels of UQO>- (ubisemiquinone at the Qo site) in complex III, enhanced both H2O2 generation from the matrix and O2(-)(radical) production from the intermembrane space. On the other hand, myxothiazol, which inhibits UQO>- formation, completely inhibited antimycin induced O2(-)(radical) toward the intermembrane space and inhibited H2O2 generation from the matrix by 70%. However, myxothiazol alone enhanced H2O2 production from complex III, suggesting that other components of complex III besides the UQO- can cause O2(-)(radical) generation toward the matrix. As expected, mitochondrial GSH was found to modulate H2O2 production from the matrix but not O2- generation from the intermembrane space. Low levels of GSH depletion (from 0-40%, depending on the rate of H2O2 production) had no effect on H2O2 diffusion from mitochondria. Once this GSH depletion threshold was reached, GSH loss corresponded to a linear increase in H2O2 production by mitochondria. The impact of 50% mitochondrial GSH depletion, as seen in certain pathological conditions in vivo, on H2O2 production by mitochondria depends on the metabolic state of mitochondria, which governs its rate of H2O2 production. The greater the rate of H2O2 generation the greater the effect 50% GSH depletion had on enhancing H2O2 production.

Effect of Glutathione Depletion and Hydrophilic Bile Acids on Hepatic Acute Phase Reaction in Rats with Extrahepatic Cholestasis

Background: Extrahepatic cholestasis by biliary obstruction induces an acute phase reaction in the liver. It is a complex process involving cytokines, hormones and growth factors. To determine whether the regulation of acute phase proteins (APP) in cholestasis depends on glutathione (GSH), the effect of buthionine sulfoximine-induced (BSO-induced) GSH depletion on the expression of various APP was studied. In addition, we determined the influence of hepatoprotective bile acids on hepatic APP and underlying cytokine events. Methods: Liver samples of bile-duct-ligated or sham-operated rats were examined. mRNA expression was quantified by densitometric analysis of Northern blots. Results: Expression of APP increased 2-5-fold in bile-duct-ligated rats as compared to sham-operated controls. This acute phase reaction remained similar independently of whether cholestasis occurred for 5 days or 3 weeks. In contrast to ! 2-macroglobulin and tissue inhibitor of metalloproteinases-1 (TIMP-1), mRNA levels of both # -fibrinogen and haptoglobin were significantly up-regulated after GSH depletion by BSO in cholestasis. Feeding of ursodeoxycholic and iso-ursodeoxycholic acid markedly down-regulated ! 2-macroglobulin and TIMP-1 expression in cholestasis but did not affect overexpression of # -fibrinogen and haptoglobin. Cholestasis leads to an increased APP expression accompanied by an increased expression of inflammatory cytokines (IL-6, TNF- ! ). After feeding of hydrophilic bile acids, increases in inflammatory cytokines were abrogated. Conclusions: We show that GSH is involved in the acute phase reaction during obstructive cholestasis. In addition, bile acids might selectively ameliorate the acute phase response by reducing expression of the APP not affected by GSH depletion ( ! 2-macroglobulin and TIMP-1).

Effect of glutathione depletion on caspase-3 independent apoptosis pathway induced by curcumin in Jurkat cells

Curcumin, a yellow pigment from Curcuma longa, exhibits anti-inflammatory, antitumor, and antioxidative properties. Although its precise mode of action has not been elucidated so far, numerous studies have shown that curcumin may induce apoptosis in normal and cancer cells. Previously, we showed that in Jurkat cells curcumin induced nontypical apoptosis-like pathway, which was independent of mitochondria and caspase-3. Now we show that the inhibition of caspase-3 by curcumin, which is accompanied by attenuation of internucleosomal DNA fragmentation, may be due to elevation of glutathione, which increased in curcumin-treated cells to 130% of control. We have demonstrated that glutathione depletion does not itself induce apoptosis in Jurkat cells; though, it can release cytochrome c from mitochondria and caspase-3 from inhibition by curcumin, as shown by Western blot. The level of Bcl-2 protein was not affected by glutathione depletion even upon curcumin treatment. Altogether, our results show that in Jurkat cells curcumin prevents glutathione decrease, thus protecting cells against caspase-3 activation and oligonucleosomal DNA fragmentation. On the other hand, it induces nonclassical apoptosis via a still-unrecognized mechanism, which leads to chromatin degradation and high-molecular-weight DNA fragmentation.

Endothelium-dependent vasorelaxation is inhibited by in vivo depletion of vascular thiol levels: Role of endothelial nitric oxide synthase

Thiols like glutathione may serve as reducing co-factors in the production of nitric oxide (NO) and protect NO from inactivation by radical oxygen species. Depletion of thiol compounds reduces NO-mediated vascular effects in vitro and in vivo. The mechanisms underlying these actions are not clear, but may involve decreased synthesis of NO and/or increased degradation of NO. This study investigates the effect of glutathione depletion on the response to NO-mediated vasodilation induced by acetylcholine (Ach, 10 μg/kg), endothelial NO synthase (eNOS) activity and potential markers of vascular superoxide anion (O·-2) production in conscious chronically catheterized rats. Thiol depletion induced by buthionine sulfoximine (BSO, 1 g ip within 24 h) decreased the hypotensive effect of Ach by 30% (MAP reduction before BSO 27 ± 3 mmHg, 19 ± 3 mmHg after BSO, (mean ± SEM), p < .05n = 8). The impaired effect of Ach was associated with a significant reduction in eNOS activity (control: 7.7 ± 0.8, BSO: 3.9 ± 0.4 pmol/min/mg protein (p < .05), n = 6). In contrast, neither NADH/NADPH driven membrane-associated oxidases nor lucigenin reductase activity were significantly (p < .05) affected by BSO (BSO: 4415 ± 123, control: 4105 ± 455 counts/mg, n = 6) in rat aorta. It is concluded that in vivo thiol depletion results in endothelial dysfunction and a reduced receptor-mediated vascular relaxation. This effect is caused by reduced endothelial NO formation.

Protective role of intracellular glutathione against nitric oxide-induced necrosis in rat gastric mucosal cells.

Nitric oxide synthase activity is increased in the stomach in association with Helicobacter pylori infection and portal hypertension, but the mechanism by which nitric oxide contributes to mucosal damage remains unclear. To examine whether nitric oxide injures gastric mucosal cells and whether cellular glutathione affects nitric oxide-induced cytotoxicity. A confluent monolayer of RGM-1 gastric mucosal cells was exposed to nitric oxide donors (NOC5 or NOC12). Cell viability was determined by trypan blue dye exclusion, lactate dehydrogenase release and supravital staining with Hoechst 33342 and propidium iodide. The kinetics of the reduced/oxidized forms of glutathione were also measured, as well as the effect of glutathione-depletion or glutathione-precursor treatment on nitric oxide-induced cytotoxicity. Excess exogenous nitric oxide produced by NOC5 or NOC12 induced necrosis in RGM-1 cells in a time- and concentration-dependent manner. The level of reduced glutathione drastically decreased prior to the loss of cell viability and remained low, but oxidized glutathione was not affected. Glutathione depletion increased necrosis of both NOCs in an NOC-concentration-related fashion, while pre-treatment with gamma-glutamylcysteine ethyl ester reduced their necrotic susceptibility. Exogenous nitric oxide induced necrosis in gastric mucosal cells, and intracellular reduced glutathione protects gastric mucosal cells from damage by nitric oxide.

Effect of glutathione depletion on inhibition of cell cycle progression and induction of apoptosis by melphalan (L-phenylalanine mustard) in human colorectal cancer cells

Intracellular levels of glutathione have been shown to affect the sensitivity of cells to cell death-inducing stimuli, as well as the mode of cell death. We found in five human colorectal cancer cell lines (HT-29, LS-180, LOVO, SW837, and SW1116) that GSH depletion by L-buthionine-[ S, R]-sulfoximine (BSO) below 20% of control values increased L-phenylalanine mustard (L-PAM; Melphalan) cytotoxicity 2- to 3-fold. Effects on kinetics of both cell cycle progression and cell death were further investigated in the HT-29 cell line. BSO treatment alone had no effect on cell cycle kinetics, but did enhance the inhibition of S phase progression as induced by L-PAM; at high concentration of of L-PAM, BSO pretreatment resulted in blockage in all phases of the cell cycle. Yet, BSO pretreatment did not affect the intracellular L-PAM content. L-PAM induced apoptosis in both normal and GSH-depleted cells. A combination of annexin V labeling and propidium iodide staining revealed that even the higher concentration of L-PAM (420 μM) did not induce apoptosis until 48 hr after treatment, but that induction of cell death was markedly accelerated as a result of GSH depletion: 48 hours after L-PAM (420 μM) treatment, GSH-depleted cells showed a 4-fold increase in DNA fragmentation and a 7-fold increase in the fraction of apoptotic (annexin V-positive) cells as compared to cells with normal GSH levels. Various antioxidant treatment modalities could not prevent this potentiating effect of GSH depletion on L-PAM cytotoxicity, suggesting that reactive oxygen species do not play a role. These data show that after BSO treatment the mode of L-PAM-induced cell death does not necessarily switch from apoptosis to necrosis.

Astrocyte-Enhanced Neuronal Survival is Mediated by Scavenging of Extracellular Reactive Oxygen Species

The survival of cultured neurons is promoted by the presence of antioxidants or astrocytes. This indicates that extracellular reactive oxygen species (ROS) impair neuronal survival and suggests that astrocytes exert their survival-enhancing effect through inactivation of these toxicants. However, to our knowledge, data supporting this hypothesis are lacking. Previously, we showed that loss of the antioxidant glutathione abolishes the neuronal survival-stimulating action of astrocytes in cocultures, consisting of rat striatal astrocytes and mesencephalic, dopaminergic neurons. Using uptake of [ 3H]dopamine as marker of neuronal survival, we presently investigated whether this effect of glutathione depletion is mediated by extracellular ROS. For this purpose, we incubated glutathione-depleted cocultures with superoxide dismutase, catalase or both. Whereas superoxide dismutase had no effect and catalase only partially protected, addition of the enzymes together completely prevented the impairment of neuronal survival caused by glutathione loss. No change in neuronal survival occurred upon exposure of control cocultures to superoxide dismutase and/or catalase. These data strongly implicate scavenging of extracellular ROS in astrocyte-stimulated neuronal survival and moreover suggest a crucial role for glutathione in this process.

Oxidative Damage in Tissues of Rats Exposed to Cigarette Smoke

Cigarette smoke is known to contain high concentrations of free radicals and oxidants. To examine the oxidative effect of cigarette smoking, we subjected rats to inhalation of cigarette smoke, and measured cellular free glutathione, the degree of protein S-thiolation, and 8-oxo-2′-deoxyguanosine (oxo 8dG) in DNA. Inhalation of the cigarette smoke for 30 days, three times a day, resulted in a significant decrease of the total free glutathione contents in tissues, especially in the lung. Elevated levels of oxidized glutathione and protein S-thiolation were observed in the lung but not in other tissues. Increased contents of oxo 8dG in DNA were found in all tissues analyzed. When rats were treated with buthionine sulfoximine (BSO, 80 mg/kg/day) to deplete glutathione, the oxidative effect of cigarette smoking was greatly potentiated. The effect of glutathione depletion was most evident in the lung. Cigarette smoking for only 7 days resulted in extreme depletion of the glutathione both in the lungs and in the liver of BSO-treated rats. Furthermore, oxo 8dG in DNA increased markedly, especially in lung. The results verified that the lung is a primary target of cigarette smoke-induced oxidative damage, and cigarette smoke exerts its oxidative effects on the rest of the entire organs eventually. Our results indicate that glutathione plays crucial roles in protecting proteins and DNA from oxidation caused by cigarette smoking.

Effect of Glutathione Depletion and Metallothionein Gene Expression on Arsenic-Induced Cytotoxicity and c-myc Expression in Vitro

Arsenic exposure is clearly linked to human cancer. In rodent cells, arsenic has been reported to induce aberrant gene expression, including activation of the proto-oncogene c-myc. Abnormal or altered expression of such oncogenes can be involved in the acquisition of a malignant phenotype. Although its mechanism of action is unclear, arsenic is known to exert at least some of its toxic effects through interaction with sulfhydryl groups, and the nonprotein sulfhydryl glutathione (GSH) appears to play an important role in detoxication of arsenic. Similarly, metallothionein (MT), a metal-binding protein with high sulfhydryl content, often functions in defense against metal-induced or oxidative cellular injury. Therefore, we examined the relationship among GSH, MT gene expression, and arsenic-induced toxicity or c-myc expression in cultured rat myoblast (L6) cells. In initial toxicity studies, arsenic was used in both the trivalent (arsenite) and pentavalent (arsenate) forms. The role of GSH was studied by pretreating cells with L-buthionine sulfoximine (BSO), which induces a marked depletion of GSH. In vitro exposure of L6 cells to BSO (1 to 25 μM) resulted in dose-dependent decreases in GSH. GSH depletion sensitized cells to both arsenite and arsenate. Zinc pretreatment, at levels which highly activated MT expression, had no effect on arsenite-induced cytotoxicity. Arsenite (1 μM) alone modestly increased c-myc expression from 1 to 4 h after treatment (maximum of 2.0-fold over control). After GSH depletion cells responded to arsenite exposure with much larger increases in c-myc transcription (3.2-fold over control). Zinc pretreatment had no reductive effect on arsenite-induced c-myc expression despite markedly activating the MT gene. Thus, it appears that the cellular levels of GSH, but not MT gene expression, play an important role in resistance to arsenic toxicity and aberrant gene activation. Moreover, depletion of GSH enhances arsenic-induced proto-on-cogene activation, which might contribute to subsequent transformation.

Effect of glutathione depletion on exocyclic adduct levels in the liver DNA of F344 rats.

The effects of glutathione (GSH) depletion on the in vivo formation of cyclic 1,N2- propanodexoxyguanosine adducts (AdG and CdG) as background lesions in the liver DNA of F344 rats were investigated. A group of 5 male F344 rats were given drinking water containing 30 mM L-buthionine (S,R)-sulfoximine (BSO) for 21 days, and another group of 8 rats were given only drinking water as controls. The BSO-treated rats had significantly lower weight gain than control rats. The hepatic GSH levels in the BSO-treated group were reduced by 84% as compared with the control group, from 4.43 to 0.72 mumol/g of tissue. The isomeric AdG3, CdG1, and CdG2 were detected by the 32P-postlabeling/HPLC method in the liver DNA of rats without carcinogen treatment, as we reported previously [Nath, R. G., and Chung, F.-L. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 7491-7495. Nath, R. G., et al. (1996) Cancer Res. 56, 452-456]. The mean levels (mumol/mol of guanine) for AdG3, CdG1, and CdG2 were 0.57 +/- 0.25, 0.15 +/- 0.18, and 0.16 +/- 0.22 for the control group and 1.18 +/- 1.03, 3.16 +/- 3.26, and 2.50 +/- 2.59 for the BSO group, respectively. These increases correspond to approximately 2-fold for AdG and 15-21-fold for CdG adducts. The dramatic increase in the cyclic adduct levels in rat liver DNA could have resulted mainly from GSH depletion as a result of the BSO treatment, even though other unknown effects due to the toxicity of BSO cannot be ruled out. These results suggest that GSH plays an important role in protecting the liver against cyclic propano DNA adduction and provide further support for the endogenous origin of these adducts.