Maintain GSH Levels Research Articles

Control of Redox State and Redox Signaling by Neural Antioxidant Systems

The glutathione/glutathione disulfide (GSH/GSSG) redox pair forms the major redox couple in cells and as such plays a critical role in regulating redox-dependent cellular functions. Not only does GSH act as an antioxidant but it can also modulate the activity of a variety of different proteins. An impairment in GSH status is thought to be the precipitating event in a wide range of neurological disorders. Therefore, understanding how to maintain GSH in the CNS could provide a valuable therapeutic approach. Intracellular GSH levels are regulated by a complex series of pathways that include substrate transport and availability, rates of synthesis and regeneration, GSH utilization, and GSH efflux. To date, the most effective approaches for maintaining GSH levels in the CNS include enhancing cyst(e)ine uptake both directly and indirectly via transcriptional upregulation of system x(c)(-), increasing GSH synthesis via transcriptional upregulation of the rate limiting enzyme in GSH biosynthesis, and decreasing GSH utilization. Among the transcription factors that play critical roles in GSH metabolism are NF-E2-related factor 2 (Nrf2) and activating transcription factor 4 (ATF4). Thus, compounds that can upregulate these transcription factors may be particularly useful in promoting the functional maintenance of the CNS through their effects on GSH metabolism.

N,N-Bis-(8-hydroxyquinoline-5-yl methyl)-benzyl substituted amines (HQNBA): Peroxisome proliferator-activated receptor (PPAR-γ) agonists with neuroprotective properties

We report on the neuroprotective effects of N,N-bis-(8-hydroxyquinoline-5-yl methyl)-benzyl substituted amines (HQNBA) in a model of oxidative stress-induced nerve cell death using mouse hippocampal-derived HT22 cells. The four derivatives (JLK1472, JLK1486, JLK1522 and JLK1535) protected the HT22 cells from death at concentrations ranging from 0.1 to 1 μM. Their action is partially dependent on their ability to act as PPARγ agonists. These analogues also maintain GSH levels suggesting that they have indirect anti-oxidant effects.

Aging Adversely Affects the Cigarette Smoke–induced Glutathione Adaptive Response in the Lung

Cigarette smoke (CS) is the leading cause of chronic obstructive pulmonary disease, accounting for more than 90% of cases. The prevalence of chronic obstructive pulmonary disease is much higher in the elderly, suggesting an age dependency. A prominent defense against the oxidant burden caused by CS is the glutathione (GSH) adaptive response in the lung epithelial lining fluid (ELF) and tissue. However, as one ages the ability to maintain GSH levels declines. Examine the effect of aging on the GSH adaptive response to CS and resulting lung sensitization to inflammation and oxidation. Both young (2 mo old) and aged (8, 13, 19, and 26 mo old) mice were used to study the effects of age on the GSH adaptive response after an acute exposure to CS. Young mice had a robust sixfold increase in ELF GSH after a single exposure to CS. The GSH response to CS decreased as a function of age and diminishes in the older mice to only a twofold increase over air controls. As a consequence, levels of CS-induced tumor necrosis factor-α and nitric oxide synthase, markers of inflammation, and 8-hydroxy-2-deoxyguanosine, a marker of DNA oxidation, were elevated in the aged mice compared with the young mice. Additionally, depletion of ELF GSH with buthionine sulfoximine in young mice recapitulated changes in ELF tumor necrosis factor-α as seen in old mice. These data suggest that the age-related maladaptive response to CS sensitizes the lung to both inflammation and oxidation potentially contributing to the development of CS-induced chronic obstructive pulmonary disease.

Aging Adversely Affects the Cigarette Smoke Induced Glutathione Adaptive Response in the Lung

Rationale: Cigarette smoke (CS) is the leading cause of chronic obstructive pulmonary disease, accounting for more than 90% of cases. The prevalence of chronic obstructive pulmonary disease is much higher in the elderly, suggesting an age dependency. A prominent defense against the oxidant burden caused by CS is the glutathione (GSH) adaptive response in the lung epithelial lining fluid (ELF) and tissue. However, as one ages the ability to maintain GSH levels declines. Objectives:ExaminetheeffectofagingontheGSHadaptiveresponse toCSandresultinglungsensitizationtoinflammationandoxidation. Methods: Both young (2 mo old) and aged (8, 13, 19, and 26 mo old) mice were used to study the effects of age on the GSH adaptive response after an acute exposure to CS. Measurements and Main Results: Young mice had a robust sixfold

Protective effect of Calendula officinalis extract against UVB-induced oxidative stress in skin: Evaluation of reduced glutathione levels and matrix metalloproteinase secretion

Background and purpose Calendula officinalis flowers have long been employed time in folk therapy, and more than 35 properties have been attributed to decoctions and tinctures from the flowers. The main uses are as remedies for burns (including sunburns), bruises and cutaneous and internal inflammatory diseases of several origins. The recommended doses are a function both of the type and severity of the condition to be treated and the individual condition of each patient. Therefore, the present study investigated the potential use of Calendula officinalis extract to prevent UV irradiation-induced oxidative stress in skin. Methods Firstly, the physico-chemical composition of marigold extract (ME) (hydroalcoholic extract) was assessed and the in vitro antioxidant efficacy was determined using different methodologies. Secondly, the cytotoxicity was evaluated in L929 and HepG2 cells with the MTT assay. Finally, the in vivo protective effect of ME against UVB-induced oxidative stress in the skin of hairless mice was evaluated by determining reduced glutathione (GSH) levels and monitoring the secretion/activity of metalloproteinases. Results and conclusions The polyphenol, flavonoid, rutin and narcissin contents found in ME were 28.6 mg/g, 18.8 mg/g, 1.6 mg/g and 12.2 mg/g, respectively and evaluation of the in vitro antioxidant activity demonstrated a dose-dependent effect of ME against different radicals. Cytoxicity experiments demonstrated that ME was not cytotoxic for L929 and HepG2 cells at concentrations less than or equal to of 15 mg/mL. However, concentrations greater than or equal to 30 mg/mL, toxic effects were observed. Finally, oral treatment of hairless mice with 150 and 300 mg/kg of ME maintained GSH levels close to non-irradiated control mice. In addition, this extract affects the activity/secretion of matrix metalloproteinases 2 and 9 (MMP-2 and -9) stimulated by exposure to UVB irradiation. However, additional studies are required to have a complete understanding of the protective effects of ME for skin.

Effects of Glutamine Supplementation on Patients Undergoing Abdominal Surgery

To evaluate the effects of supplementation of glutamine (GLN) on maintaining glutathione (GSH) level, immune system function, liver function, and clinical outcome of patients receiving abdominal operation. Forty patients undergoing elective abdominal surgical treatment were randomly divided into 2 groups: study group (n = 20) and control group (n = 20). All patients received total parenteral nutrition (TPN) for up to 7 days during perioperative period. The study group received TPN supplemented with GLN dipeptide while the control group received TPN without GLN dipeptide. Patients in both groups received equivalent nitrogen and caloric intake. Blood sample was taken on preoperative day, and the 1st, 3rd, 6th postoperative day to measure GSH level, immune indexes, and liver function indexes. The decrease of GSH level in plasma and red blood cell (RBC) in study group was less than that in control group during postoperative period. Ratio of GSH/glutathione disulfide (GSSG) in plasma in study group was higher than that in control group on the 3rd postoperative day (52.53 +/- 11.46 vs. 31.43 +/- 7.27, P = 0.001). Albumin level in study group was higher than that in control group on the 3rd postoperative day (37.7 +/- 3.8 g/L vs. 33.8 +/- 4.2 g/L, P = 0.02). There was no significant difference in the levels of immunoglobin (IgG, IgM, IgA) or T lymphocyte subgroup (CD4, CD8, CD4/CD8) in both groups during postoperative period. There was one case with infectious complication in control group, while none in study group. A trend of shortened hospital stay was observed in study group compared with control group (22.3 +/- 2.1 d vs. 24.9 +/- 1.7 d, P = 0.32). Supplementation of GLN-enriched TPN has beneficial effects on maintaining GSH levels in plasma and RBC, sustaining GSH/GSSG ratio and albumin level, and keeping antioxidant abilities during postoperative period in patients with abdominal operation, with the trends of decreasing incidence of infectious complication and shortening hospital stay.

Antioxidant Small Molecules Confer Variable Protection against Oxidative Damage in Yeast Mutants

To assess the capacity of small molecules to function as antioxidants in pathologic conditions, a set of yeast assays utilizing strains deficient in the antioxidant machinery was applied with measurements of reactive oxygen species (ROS), glutathione (GSH/GSSG), and induction of the stress responsive proteins oye2 and oye3. Yeast strains deficient in superoxide dismutase (Delta sod1), catalase A (Delta cta1), and double-deficient in Old Yellow enzyme 2 and glutathione reductase 1 (Delta oye2 glr1) were supplemented with ascorbic acid, beta-carotene, caffeic acid, or quercetin, subjected to pro-oxidant insult, and monitored for growth recovery. Ascorbic acid and caffeic acid protected cells under most circumstances, whereas beta-carotene and quercetin protection was highly context dependent, exhibiting protection in some cases and inhibition in others. Beta-carotene and quercetin elevated substantially endogenous levels of ROS in some yeast mutants. Quercetin supplementation increased significantly GSH and GSSG levels but could not maintain GSH levels in H(2)O(2)-exposed cells. Induction of the stress response machinery was manifested by the strong up-regulation of a chromosomally encoded OYE2-GFP fusion. In the case of quercetin, there was simultaneous induction of OYE3-GFP, which was previously shown to sensitize cells to H(2)O(2)-induced programmed cell death (PCD). Taken together, the results show that mutations in the antioxidant machinery affect significantly the capacity of dietary antioxidants to protect cells.

A novel approach to enhancing cellular glutathione levels

GSH and GSH-associated metabolism provide the major line of defense for the protection of cells from oxidative and other forms of toxic stress. Of the three amino acids that comprise GSH, cysteine is limiting for GSH synthesis. As extracellularly cysteine is readily oxidized to form cystine, cystine transport mechanisms are essential to provide cells with cysteine. Cystine uptake is mediated by system x(c)(-), a Na(+)-independent cystine/glutamate antiporter. Inhibition of system x(c)(-) by millimolar concentrations of glutamate, a pathway termed oxidative glutamate toxicity, results in GSH depletion and nerve cell death. Recently, we described a series of compounds derived from the conjugation of epicatechin (EC) with cysteine and cysteine derivatives that protected nerve cells in culture from oxidative glutamate toxicity by maintaining GSH levels. In this study, we characterize an additional EC conjugate, cysteamine-EC, that is 5- to 10-fold more potent than the earlier conjugates. In addition, we show that these EC conjugates maintain GSH levels by enhancing the uptake of cystine into cells through induction of a disulfide exchange reaction, thereby uncoupling the uptake from system x(c)(-). Thus, these novel EC conjugates have the potential to enhance GSH synthesis under a wide variety of forms of toxic stress.

Oxidative stress on EAAC1 is involved in MPTP‐induced glutathione depletion and motor dysfunction

Excitatory amino acid carrier 1 (EAAC1) is a glutamate transporter expressed on mature neurons in the CNS, and is the primary route for uptake of the neuronal cysteine needed to produce glutathione (GSH). Parkinson's disease (PD) is a neurodegenerative disorder pathogenically related to oxidative stress and shows GSH depletion in the substantia nigra (SN). Herein, we report that 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice, an experimental model of PD, showed reduced motor activity, reduced GSH contents, EAAC1 translocation to the membrane and increased levels of nitrated EAAC1. These changes were reversed by pre-administration of n-acetylcysteine (NAC), a membrane-permeable cysteine precursor. Pretreatment with 7-nitroindazole, a specific neuronal nitric oxide synthase inhibitor, also prevented both GSH depletion and nitrotyrosine formation induced by MPTP. Pretreatment with hydrogen peroxide, L-aspartic acid beta-hydroxamate or 1-methyl-4-phenylpyridinium reduced the subsequent cysteine increase in midbrain slice cultures. Studies with chloromethylfluorescein diacetate, a GSH marker, demonstrated dopaminergic neurons in the SN to have increased GSH levels after NAC treatment. These findings suggest that oxidative stress induced by MPTP may reduce neuronal cysteine uptake, via EAAC1 dysfunction, leading to impaired GSH synthesis, and that NAC would exert a protective effect against MPTP neurotoxicity by maintaining GSH levels in dopaminergic neurons.

Glucose-6-phosphate dehydrogenase plays a central role in modulating reduced glutathione levels in reed callus under salt stress

In the present study, we investigated the role of glucose-6-phosphate dehydrogenase (G6PDH) in regulating the levels of reduced form of glutathione (GSH) to the tolerance of calli from two reed ecotypes, Phragmites communis Trin. dune reed (DR) and swamp reed (SR), in a long-term salt stress. G6PDH activity was higher in SR callus than that of DR callus under 50-150 mM NaCl treatments. In contrast, at higher NaCl concentrations (300-600 mM), G6PDH activity was lower in SR callus. A similar profile was observed in GSH contents, glutathione reductase (GR) and glutathione peroxidase (GPX) activities in both salt-stressed calli. After G6PDH activity and expression were reduced in glycerol treatments, GSH contents and GR and GPX activity decreased strongly in both calli. Simultaneously, NaCl-induced hydrogen peroxide (H2O2) accumulation was also abolished. Exogenous application of H2O2 increased G6PDH, GR, and GPX activities and GSH contents in the control conditions and glycerol treatment. Diphenylene iodonium (DPI), a plasma membrane (PM) NADPH oxidase inhibitor, which counteracted NaCl-induced H(2)O(2) accumulation, decreased these enzymes activities and GSH contents. Furthermore, exogenous application of H2O2 abolished the N-acetyl-L: -cysteine (NAC)-induced decrease in G6PDH activity, and DPI suppressed the effect of buthionine sulfoximine (BSO) on induction of G6PDH activity. Western-blot analyses showed that G6PDH expression was stimulated by NaCl and H2O2, and blocked by DPI in DR callus. Taken together, G6PDH activity involved in GSH maintenance and H2O2 accumulation under salt stress. And H2O2 regulated G6PDH, GR, and GPX activities to maintain GSH levels. In the process, G6PDH plays a central role.

Expression and function of cystine/glutamate transporter in neutrophils

Reactive oxygen species (ROS) produced by neutrophils are essential in the host defense against infections but may be harmful to neutrophils themselves. Glutathione (GSH) plays a pivotal role in protecting cells against ROS-mediated oxidant injury. Cystine/glutamate transporter, designated as system xc- and consisting of two proteins, xCT and 4F2hc, is important to maintain GSH levels in mammalian-cultured cells. In the present paper, we have investigated system xc- in neutrophils. In human peripheral blood neutrophils, neither the activity of system xc- nor xCT mRNA was detected. The activity was induced, and xCT mRNA was expressed when they were cultured in vitro. The mRNA expression was much enhanced in the presence of opsonized zymosan or PMA. In contrast, mouse peritoneal exudate neutrophils, immediately after preparation, exhibited system xc- activity and expressed xCT mRNA. The activity and the expression were heightened further when they were cultured. Peritoneal exudate cells (mostly neutrophils) from xCT-deficient (xCT-/-) mice had lower cysteine content than those from the wild-type mice. GSH levels in the xCT-/-cells decreased rapidly when they were cultured, whereas those in the wild-type cells were maintained during the culture. Apoptosis induced in culture was enhanced in the xCT-/-cells compared with the wild-type cells. These results suggest that system xc- plays an important role in neutrophils when they are activated, and their GSH consumption is accelerated.

Increased Myocardial Dysfunction After Ischemia-Reperfusion in Mice Lacking Glucose-6-Phosphate Dehydrogenase

Free radical injury contributes to cardiac dysfunction during ischemia-reperfusion. Detoxification of free radicals requires maintenance of reduced glutathione (GSH) by NADPH. The principal mechanism responsible for generating NADPH and maintaining GSH during periods of myocardial ischemia-reperfusion remains unknown. Glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme in the pentose phosphate pathway, generates NADPH in a reaction linked to the de novo production of ribose. We therefore hypothesized that G6PD is essential for maintaining GSH levels and protecting the heart during ischemia-reperfusion injury. Susceptibility to myocardial ischemia-reperfusion injury was determined in Langendorff-perfused hearts isolated from wild-type mice (WT) and mice lacking G6PD (G6PD(def)) (20% of WT myocardial G6PD activity). During global zero-flow ischemia, cardiac function was similar between WT and G6PD(def) hearts. On reperfusion, however, cardiac relaxation and contractile performance were greatly impaired in G6PD(def) myocardium, as demonstrated by elevated end-diastolic pressures and decreased percent recovery of developed pressure relative to WT hearts. Contractile dysfunction in G6PD(def) hearts was associated with depletion of total glutathione stores and impaired generation of GSH from its oxidized form. Increased ischemia-reperfusion injury in G6PD(def) hearts was reversed by treatment with the antioxidant MnTMPyP but unaffected by supplementation of ribose stores. These results demonstrate that G6PD is an essential myocardial antioxidant enzyme, required for maintaining cellular glutathione levels and protecting against oxidative stress-induced cardiac dysfunction during ischemia-reperfusion.

The dark side of a ‘detoxification’ mechanism

Drug metabolism is not always a detoxification; sometimes products are generated that are chemically reactive and can damage cells. In the case of acetaminophen [paracetamol (APAP)], although ‘safe’ metabolites are generated at low doses, once enough drug is ingested the reactive metabolite N-acetyl-p-benzoquinoneimine (NAPQI) can be formed. Fortunately, the cell has an abundant nucleophile, reduced glutathione (GSH), which can act as an antioxidant (forming GSSG in the process) or directly scavenge chemicals such as NAPQI, facilitating their excretion as inactivate conjugates. In addition, glutathione S-transferases (GSTs) accelerate GSH conjugation and thereby usually serve to protect the cell. Unfortunately, in APAP overdose this system is overwhelmed, GSH is depleted, crucial targets are apparently damaged by reactive metabolites 1xIdentification of the hepatic protein targets of reactive metabolites of acetaminophen in vivo in mice using two-dimensional gel electrophoresis and mass spectrometry. Qui, Y et al. J. Biol. Chem. 1998; 273: 17940–17953Crossref | PubMed | Scopus (185)See all References1 and hepatotoxicity ensues. Although it has been recognized for some time that in certain cases even GS-conjugates can be reactive, Henderson et al. 2xIncreased resistance to acetaminophen toxicity in mice lacking glutathione S-transferase Pi. Henderson, C.J et al. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12741–12745Crossref | PubMed | Scopus (156)See all References2 have recently proposed another way in which having a GST is not necessarily a good thing.Henderson et al. used mice in which both of the GST Pi family enzymes, GST P1 and GST P2, had been knocked out, and showed that this gene deletion did not aggravate APAP toxicity as had been expected; instead, the GstP1/P2−/− mice showed little or no signs of hepatoxicity, whereas the wild-type animals were clearly sensitive to APAP. GSH levels dropped rapidly in both groups but recovered faster in null animals. No significant difference was observed in overall drug–protein adduct formation.So, why was the absence of GST Pi enzymes protective? The enhanced recovery of GSH levels suggested that null mice were better able to maintain GSH levels under oxidative challenge. GST Pi enzymes might preferentially make a reversible ipso GS-adduct, and if the ipso adduct reacted with protein thiols (in addition to GSH), particularly those on crucial targets, three effects could ensue: APAP could be regenerated, GSH could be consumed and specific proteins could be inactivated by the formation of mixed disulfides, all of which could be potentiated by the presence of a catalytic GST. Although there was no evidence of GST Pi mediation of GSH conjugation in this report, other studies have suggested a role for rat and human Pi enzymes in the conjugation of NAPQI. GST Pi has also been identified as a target of adduct formation.Henderson et al. propose an alternative theory, namely that GST Pi, acting as a monomer, blocks activation of JUN by inhibiting a JUN N-terminal kinase (JNK). JUN has been implicated in responses to stress, and APAP has been shown to induce this activity. This highlights a hitherto unexpected role for xenoprotective enzymes: they might be involved in modulating cell signalling. It has been proposed that, under conditions of stress, GST Pi forms dimers and multimers that cannot bind to JNK, thus relieving the inhibition on signalling. It is therefore consistent that a complete absence of GST Pi should potentiate the stress response even further.The challenge now is to determine which, if either, mechanism is responsible for this unexpected effect of the GstP1/P2−/− knockout animal. A key point will be whether this effect is common to other cases of chemical-induced toxicity. Whatever the outcome, it is clear that even the best detoxification mechanisms can show a dark side under the right conditions.

Use of in vitro methaemoglobin generation to study antioxidant status in the diabetic erythrocyte

Poor glycaemic control in diabetes and a combination of oxidative, metabolic, and carbonyl stresses are thought to lead to widespread non-enzymatic glycation and eventually to diabetic complications. Diabetic tissues can suffer both restriction in their supply of reducing power and excessive demand for reducing power. This contributes to compromised antioxidant status, particularly in the essential glutathione maintenance system. To study and ultimately correct deficiencies in diabetic glutathione maintenance, an experimental model would be desirable, which would provide in vitro a rapid, convenient, and dynamic reflection of the performance of diabetic GSH antioxidant capacity compared with that of non-diabetics. Xenobiotic-mediated in vitro methaemoglobin formation in erythrocytes drawn from diabetic volunteers is significantly lower than that in erythrocytes of non-diabetics. Aromatic hydroxylamine-mediated methaemoglobin formation is GSH-dependent and is indicative of the ability of an erythrocyte to maintain GSH levels during rapid thiol consumption. Although nitrite forms methaemoglobin through a complex GSH-independent pathway, it also reveals deficiencies in diabetic detoxification and antioxidant performance compared with non-diabetics. Together with efficient glycaemic monitoring, future therapy of diabetes may include trials of different antiglycation agents and antioxidant combinations. Equalization in vitro of diabetic methaemoglobin generation with that of age/sex-matched non-diabetic subjects might provide an early indication of diabetic antioxidant status improvement in these studies.

Mg2+Antagonism on Ni2+-Induced Changes in Microtubule Assembly and Cellular Thiol Homeostasis

As an essential metal for cell metabolism, Mg2+is known to exert antagonism on Ni2+genotoxic and other effects. This study examined the influence of Mg2+on Ni2+-induced changes in microtubule (MT) assemblyin vitro,cytoplasmic MT organization, cellular glutathione (GSH), and cytoskeletal and cytosolic protein sulfhydryls (PSH). As determined by a turbidity assay at 27°C, Ni2+enhanced thein vitroMT assembly in a Pipes buffer by shortening the initial lag (nucleation phase) and increasing the rate of polymerization with a higher final plateau. However, presence of 1 mMexogenous MgCl2abolished the Ni2+enhancing effect. Exposure of 3T3 cells to 2 mMNiCl2for 20 hr resulted in perinuclear bundling of MTs and decreases in cytoskeletal and cytosolic PSH and cellular GSH levels. However, coincubation of cells with MgCl2(1.25–20 mM) added to the culture medium markedly diminished the Ni2+injury to MT organization. Under these conditions the Ni2+interference with PSH was blocked such that both the cytoskeletal and cytosolic PSH levels returned to the range of control cells without metal treatment. Treatment of cells with Mg2+(1.25–5 mM) for 20 hr slightly increased, while with higher Mg2+doses (>10 mM) decreased, cellular GSH content. Importantly, in Ni2+-treated cultures, addition of Mg2+(1.25–10 mM) elevated GSH levels to ≥200% of that in cells treated with Ni2+alone. Furthermore, these Ni2+and Mg2+(1.25–10 mM) treated cells actually maintained GSH levels which were essentially unchanged from the basal level of control cells with no metal treatment. Although Mg2+replacement of Ni2+bound to MT proteins could be an important mechanism, cellular GSH may also be a critical factor in Mg2+antagonism on Ni2+-enhanced MT assembly in view of the essential role of tubulin PSH in modulating MT assembly and, in turn, the GSH modulation of PSH.

Styrene Inhalation Toxicity Studies in Mice: II. Sex Differences in Susceptibility of B6C3F1 Mice

Styrene Inhalation Toxicity Studies in Mice. II. Sex Differences in Susceptibility of B6C3F1 Mice. Morgan, D. L., Mahler, J. F., Dill, J. A., Price, H. C., Jr., and Adkins, B., Jr. (1993). Fundam. Appl. Toxicol. 21, 317-325.Styrene is a commercially important chemical used in the production of plastics and resins. In initial short-term styrene inhalation studies, toxicity was significantly greater in male B6C3F1 mice than in females, suggesting that males may metabolize styrene more extensively and/or may be less able to detoxify reactive metabolites. In addition, a nonlinear dose-response was observed where toxicity and mortality were greater in mice exposed to 250 ppm than in those exposed to 500 ppm. These studies were conducted to investigate potential mechanism(s) for sex differences and the nonlinear dose-response in styrene toxicity by evaluating the effects of repeated styrene exposure on styrene oxide production, hepatic GSH availability, and hepatotoxicity in male and female B6C3F1 mice. Mice (36/sex/dose) were exposed to 0, 125, 250, or 500 ppm styrene 6 hr/day for up to 3 days. Styrene exposure caused increased mortality and hepatotoxicity (centrilobular necrosis, increased serum liver enzymes) in males and females after one or two exposures to 250 and 500 ppm. Hepatic GSH levels were decreased in a dose-dependent manner in males and females. After one exposure, GSH levels in males rebounded above controls in all dose groups. After three exposures to 125 or 250 ppm males appeared to maintain GSH levels; GSH was still decreased in the 500 ppm group. GSH levels in females were decreased after each exposure in all dose groups to lower levels than in males, and did not rebound above controls. Male mice had significantly greater blood styrene levels than females after one exposure to 500 ppm; however, there were no significant sex differences in blood styrene after subsequent exposures. Levels of SO in blood were not significantly greater in male mice than females within a dose group, and did not change significantly with repeated styrene exposures for 3 days. Blood styrene and SO levels were significantly higher at 500 ppm than at 250 ppm indicating that styrene uptake and metabolism are greater at 500 ppm than at 250 ppm. The higher incidence of mortality in male mice and the nonlinear dose-response to styrene cannot be explained by gender- or dose-related differences in hepatotoxicity, GSH depletion, or blood styrene or SO levels.

Glutathione uptake and protection against oxidative injury in isolated kidney cells

Analysis with radiotracer and high performance liquid chromatography techniques showed that glutathione (GSH) is transported intact into cells primarily of proximal tubule origin. Characteristics of GSH uptake were the same as previously reported for basal-lateral membrane vesicles, namely, uptake was Na+-dependent, inhibited by gamma-glutamylglutamate and/or probenecid, and not inhibited by cysteinylglycine or the constituent amino acids. Studies with inhibitors of gamma-glutamyltransferase (acivicin) and gamma-glutamylcysteine synthetase (buthionine sulfoximine) showed that GSH uptake, degradation and resynthesis are independent processes. The GSH uptake rate with 1 mM GSH was approximately three-fold greater than the GSH synthetic rate with 1 mM amino acids. To examine whether uptake of GSH can supplement synthesis to protect against injury, we incubated cells with a toxic concentration of t-butylhydroperoxide with or without GSH or its constituent amino acids. Although amino acids provided significant protection, GSH provided greater protection (cells with t-butylhydroperoxide plus GSH were not significantly different from cells alone). This protection by GSH was eliminated by gamma-glutamylglutamate or probenecid, indicating that GSH uptake was required for the protection seen. Protection was also eliminated when the GSSG reductase/GSH peroxidase system was inhibited by bischloronitrosourea (BCNU), indicating that GSH transport affords protection by maintaining GSH levels in the cell. Thus, intact GSH is transported into isolated proximal tubule cells by a Na+-dependent system, and this transported GSH can be used to supplement endogenous synthesis and GSSG reduction to protect cells against oxidative injury.

Renal and hepatic glutathione pool modifications in response to depletion treatments.

In this study we examined the response of the renal and hepatic glutathione (GSH) pool in rats to drastic GSH depletion treatments. For this purpose, we used a protein-free diet, starvation, and the injection of varying doses of diethyl maleate as depleting agents. We analysed GSH levels in both kidney and liver tissue homogenates after rats were fed a protein-free diet for 2 or 7 days or starved for 1, 2, or 3 days, as well as after diethyl maleate administration in a single maximal dose or in varying doses. The results indicated that the liver GSH pool was always more labile than the kidney GSH pool. Moreover, kidney GSH levels were almost unchanged after 7 days on a protein-free diet or after 2 days of starvation, while liver showed significant changes in GSH levels. When we analysed the repletion rate, kidney had higher kinetic parameters (k = 0.148 h-1) than liver (0.097 h-1). We conclude that efficient mechanisms of maintaining GSH levels exist in the kidney and these may serve to avoid GSH diminution and hence preserve renal function during states of GSH depletion.

The effect of bcnu (carmustine) on tissue glutathione reductase activity

A single, 50 mg/kg i.p. dose of 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU) to mice significantly inhibited glutathione reductase activity in liver within 10 min, and in lung and heart within 30 min. Maximal inhibition was reached at 4 h in all tissues and glutathione reductase activity remained significantly depressed for 48 h in liver and for 96 h in lung and heart. BCNU doses of less than 25 mg/kg had no effect on glutathione reductase activity. Increasing the BCNU dose to 100 mg/kg caused greater inhibition of glutathione reductase activity in all tissues. In spite of significantly decreased glutathione reductase activity, reduced glutathione (GSH) levels were unaffected in all tissues both 1 and 24 h after 50 mg/kg BNCU. These results suggest that lower than normal glutathione reductase activity is sufficient to maintain GSH levels. These results also demonstrate that BCNU inhibits tissue glutathione reductase activity for several days, but only at doses significantly greater than previously reported.