Altered GSH Research Articles

Comparative toxicity of 4 commonly used intravitreal corticosteroids on rat retina

Objective: To investigate the effects of 4 commonly used steroids (dexamethasone, triamcinolone, betamethasone, and methylprednisolone) on 50 retinas of 25 adult pigmented rats. Study Design: Experimental animal study. Participants: Twenty-five pigmented Long-Evans male rats. Methods: Each steroid drug with 2 different doses (0.025 mL and 0.050 mL) was injected into the vitreous of each eye of 5 rats. The low drug dose was injected into the right eye and the high dose was injected into the left eye. Ten eyes of 5 randomly selected rats were used as a control group and intravitreal saline was injected into these eyes. Oxidative damage and intrinsic antioxidative capacity were determined by measuring retinal malondialdehyde (MDA) and glutathione (GSH) levels, respectively. Results: No statistically meaningful difference was observed in retinal GSH and MDA measurements in the lowand high-dose triamcinolone (1 and 2 mg), low-dose betamethasone (0.075 mg), and low-dose dexamethasone (0.1 mg) groups, compared with the control group. Both doses of methylprednisolone (1.6 mg and 3.2 mg), high-dose betamethasone (0.15 mg), and high-dose dexamethasone (0.2 mg) markedly altered retinal GSH and MDA levels. Conclusions: The results of our study show that the toxicity of triamcinolone is not evident even in high doses. It may be used safely. We also suggest that intravitreal use of low doses of betamethasone and dexamethasone is safer than higher doses of these drugs and both doses of methylprednisolone.

Pivotal role of glutathione depletion in plasma-induced endothelial oxidative stress during sepsis

Plasma from septic shock patients can induce production of reactive oxygen species (ROS) by human umbilical vein endothelial cells (HUVEC) in vitro. How endothelial cells defend themselves against ROS under increased oxidative stress has not yet been examined. This study investigates the antioxidant defenses of HUVEC exposed to plasma obtained from either septic shock patients or healthy volunteers. Prospective, observational study. Medical intensive care unit in a university hospital. Twenty-five patients with septic shock and 10 healthy volunteers. Blood samples were collected within the first 24 hrs of septic shock. In vitro HUVEC production of ROS was studied by spectrofluorimetry using 2',7'-dichlorodihydrofluorescein diacetate fluorescent dye. Reactive nitrogen species were also assessed. Intracellular reduced glutathione (GSH) levels were measured using monochlorobimane fluorescent dye. Activity of catalase and superoxide dismutase in HUVEC were also measured. Cell death was assessed using YOPRO fluorescent dye and the MTT assay. On admission, the septic shock population's mean age was 55 yrs old, the mean Sequential Organ Failure Assessment score was 12, mean simplified acute physiology score was 50, and intensive care unit mortality rate was 45%. Evaluation of HUVEC antioxidant defenses showed a significantly decreased GSH level, increased catalase activity, and unchanged superoxide dismutase activity. ROS levels and cell death were significantly reduced when cells were pretreated with N-acetylcysteine or GSH, but no changes in reactive nitrogen species were observed. This study demonstrates that plasma-induced ROS production by HUVEC is associated with an intracellular decrease in reduced GSH. Both ROS levels and cell death decreased when N-acetylcysteine or GSH were added before exposing the cells to plasma. These data suggest a pivotal role of alterations in GSH in damage caused by sepsis-generated ROS in endothelial cell.

Functional significance of the GAG trinucleotide-repeat polymorphism in the gene for the catalytic subunit of γ-glutamylcysteine ligase

γ-Glutamylcysteine ligase (GCL) is the rate-limiting enzyme in glutathione (GSH) synthesis. A GAG-repeat polymorphism in the 5′ UTR of the gene coding for the catalytic subunit of GCL ( GCLC) has been associated with altered GSH levels in vitro. Thus, we hypothesized that this polymorphism is associated with altered GCL activity and blood GSH levels in vivo. A total of 256 healthy United States black and white adults were genotyped for the GAG polymorphism and blood GSH levels were measured. In a subset of 107 individuals, blood GCL activity was determined. Five alleles with 4, 7, 8, 9, and 10 GAG repeats were observed. The most prevalent genotype was 7/9 (40%) followed by 7/7 (32%) and 9/9 (11%). GSH levels were 15% lower in 9/9 individuals than 7/9 individuals ( P = 0.05). GCL activity was 21% lower in 9/9 individuals than 7/7 individuals ( P = 0.04). A decreasing trend of GCL activity was observed in the order of 7/7 > 7/9 > 9/9 ( P = 0.04). These findings show that 9/9 individuals have lower blood GSH levels, which is likely due to a decrease in GCL activity. Such individuals might be more susceptible to oxidative stress-related diseases than individuals with other genotypes.

Effect of magnesium pretreatment on reduced glutathione levels in tissues of mice exposed to acute and subacute cadmium intoxication: a time course study.

The study was designed to investigate the role of magnesium (Mg) pretreatment on reduced glutathione (GSH) levels in kidney, liver and testis of mice intoxicated with cadmium (Cd). Animals were divided into four groups: I--controls, II--Cd group: mice intoxicated with Cd, III--Mg+Cd group: mice given Mg 1 h before Cd, and IV--Mg group: mice given only Mg. In acute time--response studies, the single oral dose of Cd was 20 mg Cd/kg b.w. and 40 mg Mg/kg b.w. GSH levels were determined after 4, 6, 12, 24, and 48 h. In subacute experiments, mice were given 10 mg Cd/kg b.w. orally every day and 20 mg Mg/kg b.w., and GSH content was determined in investigated organs after 1 and 2 weeks. Acute cadmium intoxication significantly decreased the GSH content in liver 4, 6 and 12 h after Cd administration and increased GSH in kidney after 12, 24 and 48 h, but did not cause significant GSH alterations in testis. Mg pretreatment reduced the observed changes of GSH content in kidney and liver. Subacute Cd intoxication induced diminished renal GSH levels compared with the controls while the increased GSH levels were observed in liver and testes after 2 weeks Cd treatment. Mg pretreatment was efficient in restoring renal and testis GSH levels towards the control group, but had no effect on hepatic GSH.

Local glutathione redox status does not regulate ileal mucosal growth after massive small bowel resection in rats.

Glutathione (GSH) concentration affects cell proliferation and apoptosis in intestinal and other cell lines in vitro. However, in vivo data on gut mucosal GSH redox status and cell turnover are limited. We investigated the effect of altered GSH redox status on the ileal mucosa in a rat model of short bowel syndrome following massive small bowel resection (SBR). Rats underwent 80% mid-jejunoileal resection (RX) or small bowel transection (TX; as operative controls), with administration of either saline or D, L-buthionine-sulfoximine (BSO), a specific inhibitor of cellular GSH synthesis. Ileal mucosal redox, morphology, and indices of cell proliferation and apoptosis were determined at different days after surgery. Ileal GSH redox status was assessed by GSH and GSH disulfide (GSSG) concentrations and the redox potential of GSH/GSSG (Eh). Ileal lipid peroxidation [free malondialdehyde (MDA)] was measured as an index of lipid peroxidation. BSO markedly decreased ileal mucosal GSH, oxidized GSH/GSSG Eh, and increased MDA content without inducing morphological damage as assessed by light or electron microscopy. As expected, SBR stimulated adaptive growth of ileal villus height and total mucosal height at 7 d after surgery, but this response was unaffected by BSO treatment despite a modest increase in crypt cell apoptosis. Ileal cell proliferation (crypt cell bromodeoxyuridine incorporation) increased at 2 d after SBR but was unaffected by BSO. Collectively, our in vivo data show that marked depletion of ileal GSH and oxidation of the GSH redox pool does not alter indices of ileal epithelial proliferation or SBR-induced ileal mucosal adaptive growth.

Depletion of reduced glutathione enhances motor neuron degeneration in vitro and in vivo

The mechanism of selective and age-dependent motor neuron degeneration in human amyotrophic lateral sclerosis (ALS) has not been defined and the role of glutathione (GSH) in association with motor neuron death remains largely unknown. A motor neuron-like cell culture system and a transgenic mouse model were used to study the effect of cellular GSH alteration on motor neuron cell death. Exposure of NSC34 motor neuron-like cells to ethacrynic acid (EA) or l-buthionine sulfoximine (BSO) dramatically reduced the cellular GSH levels, and was accompanied by increased production of reactive oxygen species (ROS) measured by the dichlorofluorescin (DCF) fluorescent oxidation assay. In addition, GSH depletion enhanced oxidative stress markers, AP-1 transcriptional activation, c-Jun, c-Fos and heme oxygenase-1 (HO-1) expression in NSC34 cells analyzed by a luciferase reporter, Western blotting and quantitative PCR assays respectively. Furthermore, depletion of GSH decreased mitochondrial function, facilitated apoptosis inducing factor (AIF) translocation, cytochrome c release, and caspase 3 activation, and consequently led to motor neuron-like cell apoptosis. In an ALS-like transgenic mouse model overexpressing mutant G93A-Cu, Zn-superoxide dismutase (SOD1) gene, we showed that the reduction of GSH in the spinal cord and motor neuron cells is correlated with AIF translocation, caspase 3 activation, and motor neuron degeneration during ALS-like disease onset and progression. Taken together, the in vitro and in vivo data presented in the current report demonstrated that decreased GSH promotes multiple apoptotic pathways contributing, at least partially, to motor neuron degeneration in ALS.

Effects on Skeletal Muscle Glutathione Status of Ischemia and Reperfusion Following Abdominal Aortic Aneurysm Surgery

Glutathione (GSH) is an important endogenous scavenger against reactive oxygen species. Elective abdominal surgery without ischemia and reperfusion leads to decreased muscle GSH concentrations 4-72 hr postoperatively without altering GSH redox status. In the present study, we investigated to what extent muscle GSH status was affected during and following elective abdominal aortic aneurysm repair. From patients (n = 10) undergoing abdominal aortic repair, thigh muscle specimens were taken preoperatively, at maximal ischemia, and at 10 min and 4, 24, and 48 hr of reperfusion. Specimens were analyzed for GSH, amino acids, and energy-rich compounds. At maximal ischemia, phosphocreatine decreased by 37% (p < 0.05) and lactate and creatine increased by 274% and 57% (p < 0.001 and 0.05), respectively, indicating ischemia during the clamping of aorta. Adenosine triphosphate, on the other hand, remained unaltered during the entire study period. Total GSH (tGSH) decreased by 46% at 24 hr and by 43% at 48 hr of reperfusion (p < 0.001), while reduced GSH decreased by 48% at 24 hr and by 44% at 48 hr (p < 0.001). The redox status (GSH/tGSH) of GSH and oxidized GSH remained unaltered. Among the constituent amino acids of GSH, glycine and cysteine remained unaltered while glutamine and glutamate decreased by 55% and 55%, respectively (p < 0.001). Abdominal aortic aneurysm repair induces metabolic alterations characteristic for ischemia. The antioxidative capacity in terms of muscle levels of GSH was decreased. However, the oxidative stress during reperfusion did not change GSH status more than what has been reported following abdominal surgery without ischemia and reperfusion. The results indicate that the oxidative stress elicited by elective abdominal aortic aneurysm repair is outbalanced by a compensated GSH metabolism not giving rise to an increased amount of oxidized GSH or an altered GSH redox status.

Chronopharmacology of Morphine in Mice

Dosing‐time–dependent changes in the effect and toxicity of morphine were examined in mice housed under alternating 12 h light (07:00 to 19:00 h) and dark (19:00 to 07:00 h) cycles. Morphine (0.5 mg/kg) was injected intraperitoneally (i.p.) in animals to assess its beneficial effect (i.e., protection against the kaolin‐induced, bradykinin‐mediated, writhing reaction) and its toxicity (i.e., alteration of the hepatic enzymes of aspartate aminotransferase [AST] alanine aminotransferase [ALT], and glutathione [GSH] in separate experiments). The magnitude of the analgesic effect of morphine depended on dosing time, with minimum effect at 02:00 h and maximum effect at 14:00 h. The serum hepatic enzyme levels of AST and ALT increased after dosing morphine (100 mg/kg) at 02:00 and 14:00 h. Time courses of these enzymes did not differ between the two trials. However, hepatic GSH, which is involved in the detoxification of chemical compounds, significantly decreased after i.p. morphine injection at 02:00 but not at 14:00 h. Overall, the results suggest that the analgesic effect of morphine is greater after dosing during the resting than during the activity phase of mice that have been induced with bradykinin‐mediated pain. Drug‐induced hepatic damage as inferred by GSH alteration, however, may be greater after dosing during the active phase.

Differential Modulation of Growth and Glutathione Metabolism in Cultured Rat Astrocytes by 4-Hydroxynonenal and Green Tea Polyphenol, Epigallocatechin-3-Gallate

Oxidative stress has been implicated in the pathogenesis of cancer and prominent neurodegenerative disorders such as Alzheimer’s and Parkinson’s diseases. Apoptosis and cell cycle deregulation appear to be the mode of cell death in these disorders. Green tea polyphenol, epigallocatechin-3-gallate (EGCG) has been shown to be a potent anti-inflammatory, apoptotic and cancer chemopreventive agent. 4-Hydroxynonenal (HNE), a by-product of lipid peroxidation (LPO), has been reported to induce apoptosis and inhibit growth in many cell systems including neuroglial cultures. We have studied both the dose and time dependent effects of HNE and EGCG on the viability of primary astrocyte cell cultures prepared from neonatal rats. HNE was found to be cytotoxic at a higher dose (0.1 mM) and markedly reduced (up to 80%) the astrocyte viability while EGCG did not appear to be cytotoxic under similar conditions. In addition, we have also studied the alterations in glutathione (GSH) and LPO levels and the activities of GSH metabolizing enzymes after treatment with HNE and EGCG. A 40% decrease in GSH level and a moderate increase in LPO were observed in HNE treated cells suggesting an increase in oxidative stress. HNE treatment caused a 50% decrease in GSH reductase and a 35% increase in GSH peroxidase activities. Although HNE treatment did not lead to any significant alterations in GSH- S-transferase (GST) activity, an increased expression of GST isoenzymes was seen following the exposure to HNE. EGCG treatment caused a significant increase in LPO even in the presence of elevated GSH content. In contrast to HNE, EGCG treatment resulted in a significant decrease (50%) in the activity and expression of GSTs. Treatment of astrocyte cultures with HNE, resulted in a severe impairment in mitochondrial respiration as measured by MTT exclusion assay, while treatment with EGCG had no effect on mitochondrial respiratory activity. Both HNE and EGCG were found to initiate apoptosis in astrocytes as measured by DNA fragmentation assay. However, HNE seems to be a stronger apoptotic and cytotoxic agent than EGCG. These results suggest that HNE and EGCG differentially modulate oxidative stress and regulate the growth and survival of astrocytes.

Up-regulation of K(+) channels in diabetic rat ventricular myocytes by insulin and glutathione.

The cardiac pathogenesis of diabetes mellitus involves oxidative stress that elicits profound changes in myocardial glutathione, an endogenous regulator of cell function. This study examined the role of glutathione in regulating K(+) channel activity in isolated ventricular myocytes from diabetic rats and its relationship to insulin signaling. Colorimetric analysis of extracts of ventricular tissue from Sprague-Dawley rats showed that the basal level of reduced glutathione (GSH) was significantly less in rats with experimental diabetes compared with sham controls, consistent with oxidative stress conditions. This change in GSH status paralleled a significant decrease in the activity of gamma-glutamylcysteine synthetase, a major pathway involved in GSH homeostasis. Voltage-clamp studies confirmed that, compared with control myocytes, K(+) channels carrying the transient outward current (I(to)) are down-regulated in the diabetic state and that this electrophysiological change is reversed by in vitro treatment with insulin for 2-3 h. Incubation of diabetic rat myocytes with GSH also normalized I(to) density compared with untreated myocytes, but with a longer time course than insulin. To determine if up-regulation of I(to) by insulin was mediated by alterations in myocyte GSH, insulin-responsiveness of diabetic rat myocytes was tested in the presence of 1,3-bis-chloroethyl-nitrosourea, an inhibitor of glutathione reductase, or buthionine sulfoximine, a blocker of gamma-glutamylcysteine synthetase. Neither blocker alone altered I(to) density in diabetic rat myocytes when compared with untreated cells, but each blocked the effect of insulin to up-regulate I(to). These data suggest that oxidative stress-induced alteration in GSH redox state plays an important role in regulating I(to) channel function and that GSH homeostasis in ventricular myocytes is functionally coupled to insulin signaling.

Comparative Hepatocarcinogenicity of Hexachlorobenzene, Pentachlorobenzene, 1,2,4,5-Tetrachlorobenzene, and 1,4-Dichlorobenzene: Application of a Medium-Term Liver Focus Bioassay and Molecular and Cellular Indices

Of the twelve different chlorobenzene isomers, a thorough evaluation of carcinogenicity has only been assessed on monochlorobenzene, 1,2-, and 1,4-dichlorobenzene, and hexachlorobenzene. In the studies presented here, we measured the ability of 1,4-dichlorobenzene (DCB), 1,2,4,5-tetrachlorobenzene (TeCB), pentachlorobenzene (PeCB), and hexachlorobenzene (HCB) to promote glutathione S-transferase pi (GSTP1-1) positive preneoplastic foci formation in rat liver, following diethylnitrosamine (DEN) initiation. The results from these studies show that TeCB, PeCB, and HCB all promote the formation of GSTP1-1 positive foci and that DCB does not. The numbers and area of foci were greatest following HCB promotion, and TeCB and PeCB were approximately equal in their promoting ability. Levels of hepatic CYP1A2, CYP2B1/2, non-focal GSTP1-1, and c-fos were measured in response to treatment with the 4 chlorobenzene isomers, as were reduced glutathione (GSH) and oxidized glutathione (GSSG) levels. Results from these studies show that induction of CYP1A2 and CYP2B1/2 have correlation with both the presence and degree of GSTP1-1 foci promotion by the 4 chlorobenzenes. Alterations in GSH and GSSG levels were similar in PeCB- and TeCB-treated animals in that GSSG levels were significantly decreased, whereas HCB and DCB did not have this effect, although HCB treatment led to a significant increase in GSH levels. We conclude that induction of CYP1A2 or CYP2B1/2 by chlorobenzene isomers may indicate promotional ability, and that this property might be exploited to predict the hepatocarcinogenicity of other chlorobenzene isomers.

Gluthatione level is altered in lymphoblasts from patients with familial Alzheimer's disease

Intracellular levels of glutathione (GSH), glutathione disulphide (GSSG), glutamic acid and γ-glutamyl cysteine synthetase (γ-GCS) were measured in lymphoblast lines from patients with familial and sporadic Alzheimer's disease (AD) and from age-matched controls. Lymphoblasts carrying presenilins (PS) and amyloid precursor protein (APP) genes mutations showed significantly decreased GSH content with respect to controls. Levels of GSSG and glutamic acid, as well as the activity of γ-GCS were not significantly different in lymphoblasts carrying genes mutations as compared with control cells. These results indicate that even peripheral cells not involved in the neurodegenerative process of AD show altered GSH content when carrying PS and APP genes mutations. The provided data appear to be in accordance with the known alteration of GSH levels in central nervous system and strengthen the hypothesis of oxidative stress as an important, possibly crucial mechanism in the pathogenesis of AD.

Protection from 1‐cyano‐3,4‐epithiobutane nephrotoxicity by aminooxyacetic acid and effect on xenobiotic‐metabolizing enzymes in male fischer 344 rats

1-Cyano-3,4-epithiobutane (CEB), a naturally occurring nitrile derived from cruciferous plants, causes nephrotoxicity and increased renal glutathione (GSH) concentration in male F-344 rats. This CEB-induced nephrotoxicity is dependent on GSH conjugation and bioactivation. The objectives of the present study were to investigate the effect of CEB on several xenobiotic-metabolizing enzymes and to evaluate the effect of modulators of GSH transport and metabolism on CEB-induced nephrotoxicity and GSH concentration. Animals received 125 mg kg−1 CEB alone or following pretreatment with one of three selective inhibitors of GSH metabolism: acivicin, probenecid or aminooxyacetic acid. There were no significant alterations in epoxide hydrolase (EH), P-450, ethoxyresorufin O-deethylase (EROD) or pentoxyresorufin O-depentylase (PROD) enzyme activity, but renal glutamyl cysteine synthetase (GCS) activity was decreased at 12 and 24 h, as was renal glutathione S-transferase 4 h after CEB administration. Renal ECOD activity was also diminished at 24 h and at 12 and 24 h in liver. Aminooxyacetic acid (AOAA) abrogated the nephrotoxicity, the renal GSH-enhancing effect, and decreased GCS of CEB alone. These findings provide further evidence for the importance of GSH conjugation as a significant pathway in CEB metabolism and the role of a reactive thiol in nephrotoxicity and altered renal GSH. Copyright © 1999 John Wiley & Sons, Ltd.

Protection from 1-cyano-3,4-epithiobutane nephrotoxicity by aminooxyacetic acid and effect on xenobiotic-metabolizing enzymes in male Fischer 344 rats.

1-Cyano-3,4-epithiobutane (CEB), a naturally occurring nitrile derived from cruciferous plants, causes nephrotoxicity and increased renal glutathione (GSH) concentration in male F-344 rats. This CEB-induced nephrotoxicity is dependent on GSH conjugation and bioactivation. The objectives of the present study were to investigate the effect of CEB on several xenobiotic-metabolizing enzymes and to evaluate the effect of modulators of GSH transport and metabolism on CEB-induced nephrotoxicity and GSH concentration. Animals received 125 mg kg-1 CEB alone or following pretreatment with one of three selective inhibitors of GSH metabolism: acivicin, probenecid or aminooxyacetic acid. There were no significant alterations in epoxide hydrolase (EH), P-450, ethoxyresorufin O-deethylase (EROD) or pentoxyresorufin O-depentylase (PROD) enzyme activity, but renal glutamyl cysteine synthetase (GCS) activity was decreased at 12 and 24 h, as was renal glutathione S-transferase 4 h after CEB administration. Renal ECOD activity was also diminished at 24 h and at 12 and 24 h in liver. Aminooxyacetic acid (AOAA) abrogated the nephrotoxicity, the renal GSH-enhancing effect, and decreased GCS of CEB alone. These findings provide further evidence for the importance of GSH conjugation as a significant pathway in CEB metabolism and the role of a reactive thiol in nephrotoxicity and altered renal GSH.

Cytoprotective effects of trimetazidine in carmustine cholestasis

Carmustine [1 ,3-bis(2-chloroethyl)-1-nitrosurea (BCNU)] is an antitumour agent, however, its usefulness has been limited by a side effect; which involves pericholangitis and intrahepatic cholestasis. The primary effects of cholestasis is well known; bile flow retention, intracellular Ca++ accumulation and acidosis although it may lead to hepatotoxicity by dose-dependent manner. Recent studies provide evidence that lipoperoxidation (LPO) and alterations in the antioxidant system may significantly contribute to BCNU induced hepatotoxicity. Trimetazidine, (1-[2,3,4-Trimethoxy-benzyl] piperazine HCl; TMZ) introduced as an antianginal compound, is found to exhibit various cytoprotective features by preserving cellular ATP levels, limiting intracellular acidosis and inorganic phosphate as well as Na+ and Ca++ accumulation in ischemic cardiac injury. No study was undertaken to investigate the cytoprotective role of TMZ in cholestatic injury till today; therefore we initiated this study to investigate if its cytoprotective features also exhibit in the liver and to characterize further the cholestatic response to BCNU administration. Male rats were randomly seperated to control (CONT) (n = 15), BCNU administered (BCNU) (n = 16) and BCNU+TMZ administered (BCNU+TMZ) (n = 12) groups. The control rats received a single dosage of 2 ml/kg of corn oil (i.p.) while the BCNU group received a single dosage of BCNU (20 mg/kg, i.p.) in corn oil. In the BCNU + TMZ group 2,5 mg/kg/day (i.p.) of TMZ was administered for three days. This group also received BCNU (20 mg/kg, i.p.) in corn oil, 12 hours after the initial dose of TMZ. The cholestatic effect of BCNU was monitored by stasis markers such as ALP, GGT and total bilirubin levels. Hepatic TBARS analysis was determined with the modified method of OKHAWA et al. based on the reaction of lipid peroxides with thiobarbituric acid. Oxidized (GSSG) and reduced (GSH) glutathione levels were measured by the modified enzymatic recycling method of TEARE et al. Statistical tests were performed using Kruskal Wallis one-way Anova test and posthoc analysis by Newman-Keuls test. The BCNU group and the BCNU + TMZ group showed significant increases (p = 0.029) in hepatic TBARS levels compared to the CONT group; however the difference between the BCNU and BCNU + TMZ groups in regard to TBARS was not significant. BCNU and BCNU + TMZ groups manifested a significant decrease (p = 0.0005) in GSH levels as compared to controls. GSH/GSSG ratios in the BCNU and BCNU + TMZ group also manifested a significant decrease (p = 0.0013) as compared to the CONT group. TMZ administration caused a significant increase in total GSH levels (p = 0.0026) in BCNU + TMZ group when compared to the BCNU group. Our results support the hypothesis that BCNU induced cholestasis partly involves LPO revealed by the distinct increase in the content of TBARS in the liver after BCNU administration. BCNU is a potent inhibitor of GSSG reductase altering the preservation of the thiol redox balance in the system. As a result, supranormal concentrations of intracellular GSSG would accumulate in the hepatocyte and the extrusion of this oxidized compound would require active transport leading to ATP hydrolysis. This would deplete the energy stores of the cell which would accelerate further the possible prooxidant status. Although administration of TMZ did not provoke any significant alterations in LPO, it preserved the total GSH levels of the cell probably by improving the energy status of the cell by protection of ATP-producing processes at the mitochondrial level and provision of the necessary substrates for GSH synthesis. This protective role in the antioxidant system normalizes the altered GSH levels by BCNU and hence proposes TMZ to be a promising agent in the cholestatic injury.

Erythrocyte glutathione deficiency in symptom-free HIV infection is associated with decreased synthesis rate.

Although several studies have documented intra- and extracellular glutathione (GSH) deficiency in asymptomatic human immunodeficiency virus (HIV) infection, the mechanisms responsible for the altered GSH homeostasis remain unknown. To determine whether decreased synthesis contributes to this alteration of GSH homeostasis, a primed-constant infusion of [2H2]glycine was used to measure the fractional and absolute rates of synthesis of GSH in five healthy and five symptom-free HIV-infected subjects before and after supplementation for 1 wk with N-acetylcysteine. The erythrocyte GSH concentration of the HIV-infected group was lower (P < 0.01) than that of the control group (1.4 +/- 0.16 vs. 2.4 +/- 0.08 mmol/l). The smaller erythrocyte GSH pool of the HIV-infected group was associated with a significantly slower (P < 0.01) absolute synthesis rate of GSH (1.15 +/- 0.14 vs. 1.71 +/- 0.15 mmol. l-1. day-1) compared with controls. Cysteine supplementation elicited significant increases in both the absolute rate of synthesis and the concentration of erythrocyte GSH. These results suggest that the GSH deficiency of HIV infection is due in part to a reduced synthesis rate secondary to a shortage in cysteine availability.

Ferric nitrilotriacetate promotes N-diethylnitrosamine-induced renal tumorigenesis in the rat: implications for the involvement of oxidative stress.

Ferric nitrilotriacetate (Fe-NTA) is a known complete renal carcinogen. In this study we show that Fe-NTA is a potent inducer of renal ornithine decarboxylase (ODC) activity and DNA synthesis and promoter of N-diethylnitrosamine (DEN)-induced renal tumorigenesis in rat. Fe-NTA induced renal ODC activity several fold as compared with saline-treated rats. Renal DNA synthesis, measured as [3H]thymidine incorporation into DNA, was increased after Fe-NTA treatment. Similar to other known tumor promoters, Fe-NTA also depleted the antioxidant armory of the tissue. It depleted glutathione (GSH) levels to approximately 55% of saline-treated controls. It also led to a dose-dependent decrease in the activities of glutathione reductase and glutathione S-transferase. Similarly, activities of catalase, glutathione peroxidase and glucose 6-phosphate dehydrogenase decreased significantly (45-65%). In contrast, gamma-glutamyl transpeptidase activity showed an increase. The maximum changes in activities of these enzymes could be observed at 12 h following Fe-NTA treatment. In addition, Fe-NTA augmented renal microsomal lipid peroxidation >150% over saline-treated controls, which was concomitant with the alterations in GSH metabolizing enzymes and depletion of the antioxidant armory. These effects were alleviated in rats which received a pretreatment with an antioxidant, BHA or BHT. Fe-NTA promoted DEN-induced renal tumorigenesis. In saline alone- and DEN alone-treated animals no tumors could be recorded, whereas in Fe-NTA alone-treated animals 17% tumor incidence was observed. However, in DEN-initiated and Fe-NTA-promoted animals tumor incidence increased to 71%. Our results show that Fe-NTA induces oxidative stress in the kidney and decreases antioxidant defenses, as indicated by the fall in GSH level and in the activities of glutathione peroxidase and catalase. Concomitantly, Fe-NTA increases ODC activity and DNA synthesis, which may be compensatory changes following oxidative injury to renal cells in addition to providing a strong stimulus for renal tumor promotion. Thus oxidative stress and impaired antioxidant defenses induced by Fe-NTA in the kidney may contribute to the observed nephrotoxicity and carcinogenicity.

CD28 co-stimulation is intact and contributes to prolonged ex vivo survival of hyporesponsive synovial fluid T cells in rheumatoid arthritis.

In rheumatoid arthritis (RA), T cells in the inflamed joint are considered to play a crucial role in the pathogenesis. However, despite the fact that synovial T cells have an activated memory phenotype, they are functionally suppressed upon combined CD3 and CD28 stimulation. Here, we analyzed the contribution of both CD3 and CD28 to the hyporesponsiveness of synovial T cells in RA. In contrast to the low CD3 responsiveness of synovial fluid (SF) T cells compared to peripheral blood (PB) T cells, the CD28 co-stimulatory response was observed to be unaffected. Hyporesponsiveness of SF T cells has previously been associated with decreased levels of intracellular glutathione (GSH), an antioxidant and regulator of the intracellular redox state. Treatment of SF T cells with N-acetylcysteine, an antioxidant and replenisher of GSH, selectively improved CD3-induced responses, while leaving CD28 responsiveness unaffected. These data show that the CD3 pathway is highly sensitive to intracellular GSH alterations, whereas CD28 responsiveness is relatively refractory. Furthermore, in support for a functional role of CD28 co-stimulation, it was demonstrated that CD28 ligation acted in synergy with the IL-2 receptor gamma chain signaling cytokine IL-15 in the enhancement of the ex vivo survival of SF T cells. These data indicate that CD28 co-stimulatory capacity of SF T cells, in contrast to CD3 stimulation, remains intact despite an altered intracellular redox state. Thereby, CD28 stimulation may contribute to the persistence of T cells at the site of inflammation, which might be of relevance in the pathogenesis of RA.

Role of exogenous glutathione in teleost fish and its effects on antioxidant defense responses in rainbow trout exposed to 3,3',4,4'-tetrachlorobiphenyl

Intraperitoneal injection of glutathione (GSH) is shown to effectively increase tissue glutathione content in two teleost species, the rainbow trout and the American eel. GSH uptake into teleost tissues is a distinct piscine feature of GSH biochemistry compared with mammalian species where little or not uptake occurs of exogenously added GSH. Enhancement and depletion of tissue GSH status was used as a tool to examine the effects of altered GSH content on other cellular antioxidants in 3,3’,4,4’-tetrachlorobiphenyl (TCB) exposed rainbow trout. α-Tocopherol content was unchanged and thiobarbituric acid reactive substances (TBARS), an indicator of lipid peroxidation, changed little in liver and muscle of trout exposed to TCB and the various GSH treatments. However, γ-tocopherol levels in muscle and superoxide dismutase activities in the various tissues were significantly higher in controls and TCB-saline treated fish than in GSH deficient and lipoate supplemented TCB exposed trout. GSH status thus appears to affect some cellular antioxidant defenses in TCB exposed trout.

Glutathione S-transferase 8-8 is localized in smooth muscle cells of rat aorta and is induced in an experimental model of atherosclerosis.

Allylamine (AA) is an electrophilic amine with a long history of experimental usage because of its extremely potent and relatively specific cardiovascular toxicity; it has been utilized in a variety of experimental models attempting to mimic human atherosclerotic lesions, myocardial infarction, and vascular injury. Even though the exact mechanisms by which AA causes vascular lesions remain unresolved, recent studies on the acute effects of AA exposure in rats strongly suggest that deamination to the aldehyde acrolein, oxidative stress, and the resultant increase in lipid peroxidation, generation of .OH radicals, and acute depletion of glutathione (GSH) may be some of the causative factors in AA-induced vascular lesions. Since glutathione S-transferase 8-8 (GST8-8) of rat belongs to a distinct subgroup of GST isozymes involved in the detoxification of products of lipid peroxidation, we designed studies to examine the effects of AA exposure on this GST isoform in rat aorta using Western blotting and immunohistochemical techniques. The results of these studies demonstrate that GST8-8 is expressed in rat aorta and is dramatically induced upon AA exposure. By immunohistochemistry, GST8-8 was localized in the smooth muscle cells of the vascular media which is believed to be the site of metabolism of AA. A significant increase in gamma-glutamylcysteine synthetase activity and GST activity toward 4-hydroxynonenal and acrolein, which are preferred substrates of GST8-8, was seen as early as 3 days following AA treatment. Alterations in GSH and other GSH-related enzymes at 3 and 10 days support the concept that--upon AA exposure--aortic defense mechanisms respond early and induction of GSH biosynthesis and rat GST8-8 occur to alleviate the toxic effects of acrolein, a major, genotoxic product of AA metabolism. The presence of GST8-8 in the vasculature, which is constantly exposed to products of lipid peroxidation, and its induction by AA, suggest that GST8-8 plays a key role in protecting blood vessels against oxidative stress and hence, may be involved in the atherogenic process.