Glutathione metabolism at the blood-cerebrospinal fluid barrier

Glutathione metabolism and transport in the choroid plexus were probed by determining the effects of administration to rats of several compounds (buthionine sulfoximine, L-2-oxothiazolidine-4-carboxylate, L-(alpha 5,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazole acetic acid, gamma-glutamyl alanine, and glutathione monoethyl ester) on the levels of glutathione and cysteine in the cerebrospinal fluid. The findings indicate that glutathione is actively metabolized in the choroid plexus by pathways similar to those in kidney and other tissues. The level of glutathione in the cerebrospinal fluid can be decreased or increased by giving compounds that do not, under similar conditions, appreciably alter total brain levels of glutathione. Glutathione monoethyl ester is effectively transported into the cerebrospinal fluid.     

Protection against cisplatin toxicity by administration of glutathione ester

The role of cellular glutathione in the prevention of toxicity due to the anti-cancer drug cisplatin (cis-diamminedichloroplatinum) was explored in mice treated with buthionine sulfoximine (BSO), a selective inhibitor of gamma-glutamylcysteine synthetase (and therefore of glutathione synthesis), and with glutathione and glutathione monoisopropyl ester. Pretreatment of mice with BSO enhanced the lethal toxicity of cisplatin by about twofold. Administration of glutathione ester (dose, 2.5-7.5 mmol/kg) protected against lethal cisplatin toxicity; glutathione was also effective, but much less so. Glutathione ester, in contrast to glutathione, is effectively transported into cells and split to glutathione intracellularly. The previous findings that administered glutathione does not protect against lethal toxicity due to cadmium ions and mercuric ions, whereas glutathione ester does, suggest that intracellular glutathione is required for protection against these heavy metal ions. That administration of glutathione has a protective effect on cisplatin toxicity suggests that the toxic effects of cisplatin may be exerted both intracellularly and extracellularly, and that extracellular glutathione (or its degradation products) may form a complex with cisplatin extracellularly. The finding that glutathione ester is more effective than glutathione in protecting against the toxicity of cisplatin suggests that use of glutathione ester may be therapeutically advantageous.     

Glutathione monoethyl ester: high-performance liquid chromatographic analysis and direct preparation of the free base form

Glutathione monoethyl ester (L-gamma-glutamyl-L-cysteinylglycine ethyl ester) was shown by R. N. Puri and A. Meister (1983, Proc. Natl. Acad. Sci. USA 80, 5258-5260) to be taken up by several tissues and intracellularly hydrolyzed to GSH. Since GSH itself is not significantly taken up by tissues, glutathione monoesters provide the most direct and convenient means available for increasing the intracellular GSH concentration of many tissues and cell types. In previous studies glutathione esters were prepared by HCl- or H2SO4-catalyzed esterification, and the product esters were precipitated as acidic salts by addition of ether to the reaction mixtures. In the present studies, glutathione monoethyl ester was synthesized by H2SO4-catalyzed esterification in the presence of sodium sulfate as the dehydrating agent. When no GSH remained, alcohol-washed Dowex-1 resin (hydroxide form) was added to remove sulfate and neutralize the reaction mixture. After the resin was removed by filtration, glutathione monoethyl ester crystallized in the chilled filtrate. The product was free of sulfate, GSH, and glutathione diester; its solutions in water or saline were neutral. Preparations obtained to date are nontoxic when administered to mice in doses up to at least 10 mmol/kg. Progress of the esterification reaction and purity of the product were determined quantitatively by HPLC after derivatization of the thiols with monobromobimane. Elution times of GSH, glutathione diester, and glutathione monoesters involving either the glutamyl or the glycyl carboxylate groups are reported.     

Oxidative stress and inflammation contribute to lung toxicity after a common breast cancer chemotherapy regimen

Delayed pulmonary toxicity syndrome after high-dose chemotherapy (HDC) and autologous hematopoietic support occurs in up to 64% of women with advanced-stage breast cancer. Using a similar, but nonmyeloablative, HDC treatment regimen in mice, we found both immediate and persistent lung injury, coincident with marked decreases in lung tissue glutathione reductase activity and accompanied by increases in lung oxidized glutathione, bronchoalveolar lavage (BAL) lipid peroxidation, and BAL total cell counts. Most interestingly, at 6 wk, BAL total cell counts had increased fourfold, with lymphocyte cell counts increasing >11-fold. A single supplemental dose of glutathione prevented early lung injury at 48 h but showed no lung-protective effects at 6 wk, whereas single doses of other thiol-sparing agents (Ethyol and glutathione monoethyl ester) showed no benefit. These data suggest that this HDC regimen results in acute and persistent lung toxicity, induced in part by oxidative stress, that culminates with an acute lung cellular inflammatory response. Continuous glutathione supplementation and/or attenuation of the delayed pulmonary inflammatory response may prove beneficial in preventing lung toxicity after the use of these chemotherapeutic agents.     

L-Xylulose reductase is involved in 9,10-phenanthrenequinone-induced apoptosis in human T lymphoma cells

9,10-Phenanthrenequinone (9,10-PQ), a major component in diesel exhaust particles, is suggested to generate reactive oxygen species (ROS) through its redox cycling, leading to cell toxicity. l-Xylulose reductase (XR), a NADPH-dependent enzyme in the uronate pathway, strongly reduces alpha-dicarbonyl compounds and was thought to act as a detoxification enzyme against reactive carbonyl compounds. Here, we have investigated the role of intracellular ROS generation in apoptotic signaling in human acute T-lymphoblastic leukemia MOLT-4 cells treated with 9,10-PQ and the role of XR in the generation of ROS. Treatment with 9,10-PQ elicited not only apoptotic signaling, including mitochondrial membrane dysfunction and activation of caspases and poly(ADP-ribose) polymerase, but also intracellular ROS generation and consequent glutathione depletion. The apoptotic effects of 9,10-PQ were drastically mitigated by pretreatment with intracellular ROS scavengers, such as N-acetyl-l-cysteine, glutathione monoethyl ester, and polyethylene glycol-conjugated catalase, indicating that intracellular ROS generation is responsible for the 9,10-PQ-evoked apoptosis. Surprisingly, the ROS generation and cytotoxicity by 9,10-PQ were augmented in an XR-transformed cell line. XR indeed reduced 9,10-PQ and produced superoxide anion through redox cycling. In addition, the expression levels of XR and its mRNA in the T lymphoma cells were markedly enhanced after the exposure to 9,10-PQ, and the induction was completely abolished by the ROS scavengers. Moreover, the 9,10-PQ-induced apoptosis was partially inhibited by the pretreatment with XR-specific inhibitors. These results suggest that initially produced ROS induce XR, which accelerates the generation of ROS.     

The role of oxidant injury in tumor cell sensitivity to recombinant human tumor necrosis factor in vivo. Implications for mechanisms of action

The intracellular glutathione levels of two human tumor lines and seven murine tumor lines were determined in order to investigate the role of oxidant injury in tumor cell sensitivity to human rTNF (rhTNF). Correlations were found between high intracellular glutathione levels and in vivo tumor resistance to rhTNF, and on the other hand, low glutathione levels and rhTNF sensitivity. The transplantable murine fibrosarcoma, Meth A, a TNF-sensitive line in vivo, was less sensitive to rhTNF and host toxicity was reduced when the hosts were pretreated with uric acid, a major reactive oxygen scavenger in humans and certain other primates. Conversely, pretreatment of the tumor-bearing hosts with DL-buthionine-(S,R)-sulfoximine, an inhibitor of GSH biosynthesis, resulted in an increased sensitivity of Meth A to rhTNF. This effect was not limited to tumor-bearing mice, as rats pretreated with diethyl maleate, a compound which irreversibly binds glutathione, were more sensitive to rhTNF toxicity than control rats. On the other hand, pretreatment with N-acetyl cysteine, an oxidant scavenger, reduced the toxicity of rhTNF treatment in rats. The data are consistent with the hypothesis that tumor cell sensitivity to rhTNF in vivo is dependent on its capacity to buffer oxidative attack. In addition, host toxicity is also related to the production of reactive oxygen species. Activated effector cells such as granulocytes and macrophages are hypothesized to produce most of this damage by their respiratory burst and oxidant release, although the direct action of rhTNF may also contribute to oxidative injury in vivo.     

Glutathione, a first line of defense against cadmium toxicity

Experimental modulation of cellular glutathione levels has been used to explore the role of glutathione in cadmium toxicity. Mice treated with buthionine sulfoximine [an effective irreversible inhibitor of gamma-glutamylcysteine synthetase (EC 6.3.2.2) that decreases cellular levels of glutathione markedly] were sensitized to the toxic effects of CdCl2. Mice pretreated with a sublethal dose of Cd2+ to induce metallothionein synthesis were not sensitized to Cd2+ by buthionine sulfoximine. Mice sensitized to Cd2+ by buthionine sulfoximine were protected against a lethal dose of Cd2+ by glutathione mono isopropyl ester (L-gamma-glutamyl-L-cysteinylglycylisopropyl ester), but not by glutathione. These results are in accord with studies that showed that glutathione mono esters (in contrast to glutathione) are efficiently transported into cells and converted intracellularly to glutathione. The findings indicate that intracellular glutathione functions in protection against Cd2+ toxicity, and that this tripeptide provides a first line of defense against Cd2+ before induction of metallothionein synthesis occurs. The experimental approach used here in which cellular levels of glutathione are decreased or increased seems applicable to investigation of other types of metal toxicity and of other glutathione-dependent biological phenomena.     

Beneficial effect of succinic acid monoethyl ester on erythrocyte membrane bound enzymes and antioxidant status in streptozotocin-nicotinamide induced type 2 diabetes

Succinic acid monoethyl ester (EMS) was recently proposed as an insulinotropic agent for the treatment of non-insulin dependent diabetes mellitus. In the present study the effect of EMS and metformin on erythrocyte membrane bound enzymes and antioxidants activity in plasma and erythrocytes of streptozotocin-nicotinamide induced type 2 diabeteic model was investigated. Succinic acid monoethyl ester was administered intraperitonially for 30 days to control and diabetic rats. The effect of EMS on glucose, insulin, hemoglobin, glycosylated hemoglobin, TBARS, hydroperoxide, superoxide dismutase (SOD), catalase (CAT), glutathione peroxide (Gpx), glutathione-S-transferase (GST), vitamins C and E, reduced glutathione (GSH) and membrane bound enzymes were studied. The effect of EMS was compared with metformin, a reference drug. The levels of glucose, glycosylated hemoglobin, TBARS, hyderoperoxide, and vitamin E were increased significantly whereas the level of insulin and hemoglobin, as well as antioxidants (SOD, CAT, Gpx, GST, vitamin C and GSH) membrane bound total ATPase, Na(+)/K(+)-ATPase, Ca(2+)-ATPase and Mg(2+)-ATPase were decreased significantly in streptozotocin-nicotinamide diabetic rats. Administration of EMS to diabetic rats showed a decrease in the levels of glucose, glycosylated hemoglobin, lipid peroxidation markers and vitamin E. In addition the levels of insulin, hemoglobin, enzymic antioxidants, vitamin C, and GSH and the activities of membrane bound enzymes also were increased in EMS and metformin treated diabetic rats. The present study indicates that the EMS possesses a significant beneficial effect on erythrocyte membrane bound enzymes and antioxidants defense system in addition to its antidiabetic effect.     

The involvement of intracellular glutathione in methyl jasmonate signaling in Arabidopsis guard cells

We examined the involvement of intracellular glutathione (GSH) in methyl jasmonate (MeJA) signaling. The chlorina1-1 (ch1-1) mutation decreased GSH in guard cells and narrowed the stomatal aperture. GSH monoethyl ester increased intracellular GSH, diminishing this phenotype. GSH did not affect MeJA-induced reactive oxygen species production or cytosolic Ca(2+) oscillation, suggesting that GSH modulates MeJA signaling downstream of production and oscillation.     

Effect of certain dicarboxylic acid monoesters on growth,chlorophyll content,chlorophyllase and peroxidase activities,and gas-exchange of young maize plants

The effect of some dicarboxylic acid monoesters on growth, chlorophyll content, chlorophyllase (EC 3.1.1.14), and total peroxidase (EC 1.11.1.7.) activities was examined in detached and intact leaves of maize (Zea mays) plants grown in a greenhouse. The -monomethyl ester of itaconic acid (MEIA) at 1250 ppm had no effect on growth. However, application of the monoethyl ester of succinic (MESA) and monoethyl ester of adipic (MEAdA) acids (1250 ppm) resulted in an increased leaf area, fresh and dry weight of leaves and stems. These compounds retarded chlorophyll degradation in both detached and intact leaves. Chlorophyllase activity of the control and treated leaves was measured and related to chlorophyll content. Delaying of senescence by treatment with monoesters resulted in greater chlorophyll and protein content, compared with the control. However, the chlorophyllase activity/chlorophylla ratio in the treated plants decreased. Total peroxidase activity was higher in senescent leaves, but all treatments inhibited the increase of this enzyme activity. Prolonged carbon assimilative activity and enhanced leaf water use efficiency in treated plants was noted.     

Glutathione Induces Neuronal Differentiation in Rat Bone Marrow Stromal Cells

It has been reported that rat bone marrow stromal cells (BMSCs) are differentiated into neuronal cells by administration of 2-mercaptoethanol [Woodbury et al (2000) J Neurosci Res 61:364–370]. In this study, we examined the effects of various sulfhydryl (SH) compounds on the differentiation of BMSCs obtained from rat femurs. Neuronal differentiation was detected morphologically and immunocytochemically. It was found that the cells treated with reduced glutathione (GSH) apparently differentiated into neurons, showing extensive processes, and expressing neuron-specific enolase and microtubule-associated protein 2. Glutathione monoethyl ester (GEE), which increased the cellular GSH content, showed no effect on the expression of neuronal markers. It is concluded that the neural differentiation of BMSCs occurs by the administration of GSH. It was suggested that extracellular and not intracellular GSH have effects on the induction of the neuronal differentiation of BMSCs.     

Activation of the antioxidant response element in primary cortical neuronal cultures derived from transgenic reporter mice

Many phase II protective genes contain a cis -acting enhancer region known as the antioxidant response element (ARE). Increased expression of these genes contributes to the protection of cells from oxidative stress. Transgenic reporter mice were created that carry in their genome the core ARE coupled to the human placental alkaline phosphatase (hPAP) reporter gene. Primary cortical cultures derived from these mice were treated with tBHQ resulting in a dose-dependent increase in hPAP activity. Histochemical staining for hPAP activity was observed in both glia and neurons from tBHQ-treated cultures. The tBHQ-mediated increase in hPAP was not affected by the antioxidant glutathione monoethyl ester (GSHEE), whereas the increase in hPAP following DEM treatment was completely blocked by GSHEE. Pre-treatment of cultures with the PI3-kinase inhibitor LY 294002 demonstrated a dose-dependent decrease in tBHQ-induced hPAP activity. In addition, the tBHQ-mediated expression of ARE-driven genes in primary cortical cultures was blocked by LY 294002. Interestingly, basal expression of Nrf2 was also inhibited by LY 294002. We theorize that increased levels of genes controlled by the ARE are important for cellular protection against oxidative stress. These ARE-hPAP transgenic mice will be an important in vivo model for testing our hypothesis.     

Hematotoxicity and blood glutathione levels after cisplatin treatment of tumor-bearing mice

The involvement of glutathione, a major cellular antioxidant, in cisplatin-mediated development of various hematological changes in mice bearing ascites Dalton lymphoma tumor was investigated. With tumor growth, glutathione levels decreased in blood but increased in tumor cells. Cisplatin treatment of tumor-bearing mice caused a decrease in glutathione levels in blood, ascites supernatant, and tumor cells. Blood hemoglobin, erythrocytes, packed cell volume and leukocytes (eosinophils, basophils, and lymphocytes) were also decreased along with the development of various morphological abnormalities in erythrocytes (microcytes, macrocytes, echinocytes, acanthocytes, etc.) after cisplatin treatment. All these hematotoxic features were noted to be increased more when buthionine sulfoximine (a specific glutathione-depleting agent) was also given prior to cisplatin treatment. However, combination treatment of cysteine (precursor for glutathione synthesis) plus cisplatin resulted in an improvement in the glutathione levels and decrease in hematological toxicities. It is noted that the glutathione levels in blood and abnormalities in erythrocytes and other hematological parameters are inversely related in cisplatin-mediated cancer chemotherapy. It is suggested that blood glutathione may play an important role in the development of cisplatin-mediated hematological toxicity in the host. This revised version was published online in July 2006 with corrections to the Cover Date.     

The Involvement of Intracellular Glutathione in Methyl Jasmonate Signaling in Arabidopsis Guard Cells

We examined the involvement of intracellular glutathione (GSH) in methyl jasmonate (MeJA) signaling. The chlorina1-1 (ch1-1) mutation decreased GSH in guard cells and narrowed the stomatal aperture. GSH monoethyl ester increased intracellular GSH, diminishing this phenotype. GSH did not affect MeJA-induced reactive oxygen species production or cytosolic Ca²⁺ oscillation, suggesting that GSH modulates MeJA signaling downstream of production and oscillation.     

Indomethacin attenuation of hepatic perfusion and plasma 6-ketoPGF1 alpha elevations following glutathione depletion in rabbits

Glutathione (GSH) is important in detoxification and regulating cyclooxygenase activity. Since the liver has high levels of GSH, xenobiotic-induced changes in hepatic GSH could affect hepatic tissue blood perfusion (HP) via alterations in prostaglandin synthesis. In anesthetized male New Zealand rabbits, elevating GSH with GSH monoethyl ester had no affect on HP. Treatment of rabbits with diethyl maleate to deplete GSH also had no affect on HP in animals previously given GSH monoethyl ester. However, HP increased within 20 min in rabbits treated with diethyl maleate prior to GSH monoethyl ester. In another experiment, a similar rise in HP following GSH depletion was accompanied by arterial plasma 6-ketoPGF1 alpha (the stable metabolite of prostacyclin) levels that were 4-times higher than in the controls. Plasma TxB2 (the stable metabolite of thromboxane) also increased following diethyl maleate, but only to levels that were 25-times lower than for 6-ketoPGF1 alpha. Since indomethacin blocked the rise in HP, as well as the increases in 6-ketoPGF1 alpha and TxB2, these results indicate changes in HP may occur following GSH depletion as a result of increased synthesis of one or more arachidonic acid metabolites and implicate prostacyclin as a possible mediator of this phenomenon.     

Dopamine induces proteasome inhibition in neural PC12 cell line

The autoxidation and enzymatic catabolism of dopamine results in the generation of reactive oxygen species (ROS), which may possibly contribute to oxidative stress in multiple neurodegenerative disorders. Recent studies indicate that proteasome inhibition occurs in numerous neurodegenerative conditions, possibly as the result of oxidative stress, although the effects of dopamine on proteasome activity have not been determined. In the present study we examined the effects of dopamine on proteasome activity in the neural PC12 cell line. Application of dopamine induced a dose- and time-dependent decrease in proteasome activity, which occurred prior to cell death. Application of an antioxidant (gluthathione monoethyl ester), monoamine oxidase inhibitors (deprenyl, clogyline, paragyline), or an inhibitor of dopamine uptake (nomifensine) attenuated dopamine toxicity and dopamine-induced proteasome impairment. Application of the proteasome inhibitor lactacystin increased the toxicity of dopamine and the levels of protein oxidation following administration of dopamine. Together, these data indicate that dopamine induces proteasome inhibition that is dependent, in part, on ROS and dopamine uptake, and suggest a possible role for proteasome inhibition in dopamine toxicity.     

LPS-induced ROS generation and changes in glutathione level and their relation to the maturation of human monocyte-derived dendritic cells

Lipopolysaccharide (LPS)-induced reactive oxygen species (ROS) generation and the concomitant decline in the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) were demonstrated in human monocyte-derived dendritic cells (DC). Further, their relation to the maturation of DC, characterized by the production of cytokines, up-regulation of cell surface molecules and allo-stimulatory capacity, was examined. The LPS-induced ROS generation was demonstrated using electron paramagnetic resonance spectroscopy in intact cells, and was also confirmed using laser scanning confocal microscopy. The GSH/GSSG was assesed using a glutathione assay kit. When the DC were treated with alpha-phenyl-tert-butylnitrone, the ROS generation was attenuated, but the declined GSH/GSSG was not attenuated, and only cytokine production was suppressed among the above-mentioned maturation characteristics. When the DC were treated with glutathione monoethyl ester, both the ROS generation and the declined GSH/GSSG were attenuated, and the maturation characteristics were all suppressed. These findings suggest that the LPS-induced ROS generation and the concomitant decline in GSH/GSSG occur in human monocyte-derived DC and that the former is involved in cytokine production, while the latter is involved in the up-regulation of cell surface molecules and allo-stimulatory capacity. Since the cytokine production and the allo-stimulatory capacity of DC play an important role in inflammatory and immune responses, differential regulation of the ROS generation and the declined GSH/GSSG may be useful as therapeutic tools in diseases where both responses become entangled, such as sepsis and graft-versus-host disease.     

Diet, DDT, and the toxicity of drugs and chemicals.

The toxic and carcinogenic effects of many compounds depend on their activation to reactive molecules in the cytochrome P-450 system of the endoplasmic reticulum of cells. Changes in dietary input alter P-450 levels in different tissues and so alter toxicity. In this way, low protein diets protect against carbon tetrachloride poisoning. Fats, proteins, and nonnutrients such as flavones, antioxidants, and contaminants like DDT all affect P-450 levels. The activated molecules may be diverted from their target sites by inactivation processes, such as epoxide hydratase or glutathione trappin. This is also under nutritional control. Low protein diets render animals sensitive to acetaminophen by reducing glutathione levels. In the sequence of events leading from initial contact of toxin with organism to eventual cell injury or neoplasm, nutritional factors are of import at every stage. Assessments of the toxicity of chemicals that do not take into account the nutritional variable in man are likely to be incorrect.