Effect of the glutathione/glutathione disulfide redox couple on thiopurine methyltransferase

The susceptibility of recombinant human thiopurine methyltransferase (hTPMT) to thiol-disulfide exchange was investigated. The enzyme was incubated in buffers of the redox couple GSH and GSSG. The values of the chosen concentrations and concentration ratios of the redox couple equaled those expected to occur in vivo. Activity measurements of the enzyme over time in these buffers at 30 degrees C indicated that thiol-disulfide exchange may be a part of the posttranslational modulation of hTPMT activity. Activity varied between 5% and 100%, with the lowest activities in buffers of low [GSH]/[GSSG] concentration ratios and of low total concentration of the redox couple. A thiol-disulfide exchange mechanism involving a mixed disulfide was proposed. Titration of the protein thiol groups with Ellmann's reagent (5,5'-dithiobis[2-nitrobenzoic acid]) revealed that at least two protein thiols were readily accessible for conjugation with the reagent, while others were conjugated more slowly. The previous model of hTPMT constructed by our group was in accordance with the experimental results. Inspection of the model indicated that one of the protein thiols subject to slow thiol-disulfide exchange may be situated at the binding site of the co-substrate of the enzyme and thus be responsible for the glutathione/glutathione disulfide modulation of the activity of hTPMT.     

Acetaminophen-induced depletion of glutathione and cysteine in the aging mouse kidney.

Glutathione (GSH) plays an essential role in the detoxification of acetaminophen (APAP) and the prevention of APAP-induced toxicity in the kidney. Our previous results demonstrated that a GSH deficiency is a general property of aging tissues, including the kidney, suggesting a hypothesis that senescent organisms are at greater risk to APAP-induced renal damage. To test this, C57BL/6NIA mice of different ages through the life span were injected with various doses of APAP, and the extent of GSH and cysteine (Cys) depletion and recovery were determined. At time intervals up to 24 hr, kidney cortex samples were obtained, processed and analyzed for glutathione status, namely GSH, glutathione disulfide (GSSG), Cys and cystine, using an HPLC method with dual electrochemical detection. In the uninjected controls, GSH and Cys concentrations decreased about 30% in the aging mouse, but the GSSG and cystine levels were unchanged during the life span. APAP administration depleted the kidney GSH and Cys contents in a dose- and time-dependent manner. Four hours after APAP administration, GSH levels of the young, growing (3- to 6-month) and the mature (12-month) mice decreased 34 and 58%, respectively, and recovered to near control values by 24 hr (95 and 98%). In contrast, the extent of depletion in old (31-month) mice was greater (64%) and the 24-hr recovery was less, returning only to 56%. Likewise, Cys levels of the young and mature mice decreased 49 and 65%, respectively, 4 hr following APAP, and increased to 99 and 85% by 24 hr. In contrast, in old mice, there was a 78% depletion after 4 hr followed by a recovery of only 65% by 24 hr. These results demonstrated clearly that in the aging mouse kidney, a GSH and Cys deficiency occurs that is accompanied by an impaired APAP detoxification capacity.     

Antioxidant status of erythrocytes and their response to oxidative challenge in humans with argemone oil poisoning

Oxidative damage of biomolecules and antioxidant status in erythrocytes of humans from an outbreak of argemone oil (AO) poisoning in Kannauj (India) and AO intoxicated experimental animals was investigated. Erythrocytes of the dropsy patients and AO treated rats were found to be more susceptible to 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH) induced peroxidative stress. Significant decrease in RBC glutathione (GSH) levels (46, 63%) with concomitant enhancement in oxidized glutathione (172, 154%) levels was noticed in patients and AO intoxicated animals. Further, depletion of glutathione reductase (GR), glucose-6-phosphate dehydrogenase (G-6-PDH) and glutathione-S-transferase (GST) (42-52%) was observed in dropsy patients. Oxidation of erythrocyte membrane lipids and proteins was increased (120-144%) in patients and AO treated animals (112-137%) along with 8-OHdG levels in whole blood (180%) of dropsy patients. A significant reduction in alpha-tocopherol content (68%) was noticed in erythrocytes of dropsy patients and hepatic, plasma and RBCs of AO treated rats (59-70%) thereby indicating the diminished antioxidant potential to scavenge free radicals or the limited transport of alpha-tocopherol from liver to RBCs leading to enhanced oxidation of lipids and proteins in erythrocytes. These studies implicate an important role of erythrocyte degradation in production of anemia and breathlessness in epidemic dropsy.     

Mechanisms of glutathione disulfide efflux from erythrocytes

Glutathione (GSH) plays numerous critical protective roles in the erythrocyte and GSH turnover is likely an important factor in regulating susceptibility to oxidative stress and toxins. Efflux of glutathione disulfide (GSSG) from erythrocytes is an important component in the regulation of GSH levels; however, little is known of the mechanisms involved. We hypothesize that multidrug resistance associated protein 1 (MRP1) is responsible, in part, for GSSG transport from erythrocytes. To test this, we determined the levels of MRP1 protein in erythrocyte membranes from healthy adults and compared them with intracellular levels of GSH. MRP1 levels varied substantially from person to person and were inversely correlated with levels of GSH (r = -0.39, P < 0.05). In contrast, activity levels of glutamyl cysteine ligase, the rate limiting GSH biosynthetic enzyme, were unrelated to GSH levels. To directly determine the role of MRP1 in GSSG transport, in vitro studies were conducted examining the effects of MRP1 inhibitors MK571 and verapamil on GSSG efflux. Both compounds resulted in significant but not complete inhibition (20-53%) of GSSG efflux from cells. Overall, these findings support a role for MPR1 in the regulation of erythrocyte GSH levels through the transport and elimination of GSSG from cells.     

Assessing the risk of epidemic dropsy from black salve use

Epidemic dropsy is a potentially life‐threatening condition resulting from the ingestion of argemone oil derived from the seeds of Argemone mexicana Linn. Exposure to argemone oil is usually inadvertent, arising from mustard cooking oil adulteration. Sanguinarine, an alkaloid present in argemone oil, has been postulated as a causative agent with the severity of epidemic dropsy correlating with plasma sanguinarine levels. Cases of epidemic dropsy have also been reported following the topical application of argemone containing massage oil. Black salve, a topical skin cancer therapy also contains sanguinarine, but at significantly higher concentrations than that reported for contaminated massage oil. Although not reported to date, a theoretical risk therefore exists of black salve inducing epidemic dropsy. This literature review explores the presentation and pathophysiology of epidemic dropsy and assesses the risk of it being induced by black salve.     

Identification and quantitation of glutathione in hepatic protein mixed disulfides and its relationship to glutathione disulfide

The amount of glutathione present in hepatic protein mixed disulfides was determined to be 20–30 nmole/g liver. This was established using two specific enzymatic methods: (a) the coupled assay with DTNB and glutathione (GSSG) reductase and (b) a newly developed test using GSH transferase and 1-chloro-2,4-dinitrobenzene for the estimation of GSH released from proteins after borohydride treatment; further, these results were confirmed by HPLC analysis. Thus, authentic glutathione makes up only 2–6% of the value for total protein mixed disulfides. The latter were determined with the generally employed o-phthalaldehyde assay, which is not necessarily specific for GSH. The amount of glutathione mixed disulfides depends linearly on the content of glutathione disulfide in the liver cell in the range studied. By increasing the GSSG levels from 20 to about 60 nmole/g liver with paraquat, nitrofurantoin or t-butyl hydroperoxide, glutathione protein mixed disulfides are increased by a similar amount.     

The contribution of oxidative stress to drug-induced organ toxicity and its detection in vitro and in vivo

INTRODUCTION: Nowadays the 'redox hypothesis' is based on the fact that thiol/disulfide couples such as glutathione (GSH/GSSG), cysteine (Cys/CySS) and thioredoxin ((Trx-(SH)2/Trx-SS)) are functionally organized in redox circuits controlled by glutathione pools, thioredoxins and other control nodes, and they are not in equilibrium relative to each other. Although ROS can be important intermediates of cellular signaling pathways, disturbances in the normal cellular redox can result in widespread damage to several cell components. Moreover, oxidative stress has been linked to a variety of age-related diseases. In recent years, oxidative stress has also been identified to contribute to drug-induced liver, heart, renal and brain toxicity. AREAS COVERED: This review provides an overview of current in vitro and in vivo methods that can be deployed throughout the drug discovery process. In addition, animal models and noninvasive biomarkers are described. EXPERT OPINION: Reducing post-market drug withdrawals is essential for all pharmaceutical companies in a time of increased patient welfare and tight budgets. Predictive screens positioned early in the drug discovery process will help to reduce such liabilities. Although new and more efficient assays and models are being developed, the hunt for biomarkers and noninvasive techniques is still in progress.     

Status of glutathione and other thiols and disulfides in human plasma

While plasma thiols, including homocysteine (HCys), glutathione (GSH), and cysteine (Cys), are being investigated as potential indicators of disease risk and health status, low levels, poor stability, and the lack of comprehensive methodologies have hampered their accurate assessment. Using our previously described HPLC with electrochemical detection method, our goal was to assess levels, stability, and distribution of biologically relevant thiols and disulfides in human plasma. In fresh plasma, processed immediately after collection, low levels of Cys, cystine, Cys-Gly, and the mixed disulfide Cys-GSH (CSSG) were consistently observed, whereas the levels of GSH and Cys-Gly disulfide were often below the limits of detection. These profiles were a consequence of poor thiol stability, as thiol standards added to human plasma were lost rapidly due to autoxidation or formation of mixed disulfides. A 75% loss of added GSH observed after 30 min was accounted for completely by the formation of GSH disulfide (24%) and CSSG (74%). Similar changes were found with other thiols when added to plasma. Thiols lost to oxidation were recovered quantitatively by reducing samples with potassium borohydride (KBH(4)) prior to analysis. In a study of 106 healthy adults, mean total thiol levels in plasma were: Cys (201 microM) > Cys-Gly (101 microM) > HCys (7 microM) > gamma-Glu-Cys (5 microM) > GSH (4 microM). All together, these results account for the poor stability of thiols in plasma and provide a method for their comprehensive and accurate determination.     

A simultaneous liquid chromatography/mass spectrometric assay of glutathione,cysteine, homocysteine and their disulfides in biological samples

A liquid chromatography/mass spectrometric (LC/MS) method was developed for simultaneous detection and quantitation of glutathione (GSH), glutathione disulfide (GSSG), cysteine (CysSH), homocysteine (HCysSH) and homocystine in biological samples (rat brain, lung, liver, heart, kidneys, erythrocytes and plasma). Thiols were derivatized with a large excess of Ellman's reagent, a thiol-specific reagent, to ensure an instantaneous and complete derivatization. The derivatization blocked the oxidation of the thiols to disulfides, preventing errors caused by thiol oxidation. The samples were then analyzed by LC/MS. The method provides a highly selective and sensitive assay for these endogenous thiols and their corresponding disulfides. The detection limits for GSH, GSSG, CysSH, HCysSH and homocystine were 3.3, 3.3, 16.5, 29.6 and 14.9 pmol, respectively. An attempt for cystine analysis was unsuccessful due to earlier elution of the compound and strong interferences caused by other endogenous compounds. This method will be a useful tool in the investigation of the roles of these important thiol-containing compounds and their corresponding disulfides in physiological and pathological processes.     

Glutathionylation of proteins by glutathione disulfide S-oxide

Aqueous solution of S-nitrosoglutathione (GSNO) underwent spontaneous chemical transformation that generated several glutathione derivatives including glutathione sulfonic acid (GSO3H), glutathione disulfide S-oxide (GS(O)SG), glutathione disulfide S-dioxide, and glutathione disulfide. Surprisingly, GS(O)SG (also called glutathione thiosulfinate), which was not identified as a metabolite of GSNO previously, was one of the major products derived from GSNO. This compound was very reactive toward any thiol and the reaction product was a mixed disulfide. The rate of reaction of GS(O)SG with 5-mercapto-2-nitro-benzoate was nearly 20-fold faster than that of GSNO. The mechanism for the formation of GS(O)SG was believed to involve the sulfenic acid (GSOH) and thiosulfinamide (GS(O)NH2) intermediates; the former underwent self-condensation and the latter reacted with GSH to form GS(O)SG. Many reactive oxygen and nitrogen species were also capable of oxidizing GSH or GSSG to form GS(O)SG, which likely played a central role in integrating both the oxidative and nitrosative cellular responses through thionylation of thiols. Treatments of rat brain tissue slices with oxidants resulted in an enhanced thionylation of proteins with a concomitant increase in cellular level of GS(O)SG, suggesting that this compound might play a second messenger role for stimuli that produced a variety of oxidative species.     

Ameliorative effect of vitamins (alpha-tocopherol and ascorbic acid) on PCB (Aroclor 1254) induced oxidative stress in rat epididymal sperm

The aim of this study was to investigate the possible protective role of vitamins on PCB (Aroclor 1254)-induced spermiotoxicity using qualitative, quantitative and biochemical approaches. Adult male albino rats of Wistar strain were randomly divided into four groups, each group consists of six animals. The control group received corn oil, the second group of rats were administered Aroclor 1254 at a dose of 2 mg/kg bw/day intraperitoneally for 30 days. The third group of rats were treated with Aroclor 1254 along with alpha-tocopherol (50 mg/kg of bw/day) for 30 days, while the fourth group of rats were treated with Aroclor 1254 along with ascorbic acid (100 mg/kg bw/day) orally for 30 days. Twenty-four hours after the last treatment, control and experimental animals were killed by decapitation. Sperm was collected from the cauda epididymal region and its count and motility were detected. Sperm was sonicated and used for the estimation of reactive oxygen species (ROS) [hydroxyl radical (HO(*)) and hydrogen peroxide (H(2)O(2))], non-enzymic antioxidants [alpha-tocopherol, ascorbic acid and reduced glutathione (GSH)], activity of enzymic antioxidants [superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) glutathione reductase (GR) and glutathione-S-transferase (GST)] and lipid peroxidation (LPO). The result of this experiment shows that PCB significantly decreases the level of alpha-tocopherol, ascorbic acid and GSH and the activities of SOD, CAT, GPx, GR and GST with elevated levels of ROS and LPO. In addition, decreased epididymal sperm motility and count were observed. Simultaneous supplementation with alpha-tocopherol and ascorbic acid restored these parameters to that of normal range. In conclusion, alpha-tocopherol and ascorbic acid exhibited protective effect on sperm by inhibiting PCB-induced ROS generation.     

Antioxidant defense disruption by polycyclic aromatic hydrocarbons-coated onto Fe(2)O(3) particles in human lung cells (A549).

We addressed the hypothesis that in vitro short-term exposure to hematite (Fe(2)O(3)) and polycyclic aromatic hydrocarbons (PAHs) is more deleterious by virtue of their combinations being able to cause higher oxidative stress conditions in human lung cells (A549), than either chemical alone. Lipid peroxidation (malondialdehyde; MDA), antioxidant enzyme activities (superoxide dismutase; SOD, glutathione peroxidase; GPx, glutathione reductase; GR), glutathione status (reduced glutathione; GSH, oxidized glutathione; GSSG) and alpha-tocopherol (alpha-Toc) consumption were studied in cells exposed to Fe(2)O(3), benzo(a)pyrene (B(a)P) or pyrene, alone or in association. We found that increases in GSSG/GSH (P<0.01) and in alpha-Toc consumption (P<0.01) counteracted Fe(2)O(3)-induced lipid peroxidation. Exposure to B(a)P did not induce oxidative injury because of the involvement of non-enzymatic antioxidants in cell homeostasis. Pyrene did not induce free radicals (FR)-induced injury. Exposure to PAHs-coated onto Fe(2)O(3) particles damaged both the enzymatic (i.e. increases in SOD and GR activities; P<0.01) and the non-enzymatic (i.e. increases in GSSG/GSH; P<0.001, alpha-Toc consumption; P<0.01) antioxidant defenses, thereby allowing lipid peroxidation (i.e. MDA production; P<0.05). Exposure to PAHs-coated onto Fe(2)O(3) particles induced not only higher lipid peroxidation (i.e. MDA production; P<0.05) but also higher antioxidant alterations (i.e. SOD and GR activities; P<0.05, GSSH/GSH; P<0.01 or P<0.05) than either chemical alone. Several mechanisms could account for this result, enhanced uptake of Fe(2)O(3) and/or greater availability of PAHs. Hence, our results indicate that exposure to PAHs-coated onto Fe(2)O(3) particles is more deleterious in lungs than either chemical alone.     

Influence of cysteine upon the glutathione status of isolated rat hepatocytes

Contrary to a previous report [J. Viña, R. Hems and H. A. Krebs, Biochem. J.170, 627 (1978)], we have observed that incubation of isolated rat hepatocytes in a medium containing high concentrations of cysteine did not deplete intracellular glutathione (GSH), but instead caused a net increase of GSH. Hepatocyte-dependent accumulation of the mixed disulfide of cysteine and GSH (CYSSG) in incubation media within a 2-hr period was increased several-fold in medium containing cysteine or cystine compared to methionine-containing medium. Hepatocytes (10⁶ cells/ml) placed in medium containing normal levels of cysteine, cystine, or methionine established about a 1:1 ratio of extracellular GSH to GSSG at a concentration of about 6μM each. These results indicate a possible intracellular-extracellular relationship for glutathione and an intracellular response to the extracellular status of glutathione and cyst(e)ine.     

The toxicity of N-methyl-alpha-methyldopamine to freshly isolated rat hepatocytes is prevented by ascorbic acid and N-acetylcysteine

In the past decade, clinical evidence has increasingly shown that the liver is a target organ for 3,4-methylenedioxymethamphetamine (MDMA, "ecstasy") toxicity. The aims of the present in vitro study were: (1) to evaluate and compare the hepatotoxic effects of MDMA and one of its main metabolites, N-methyl-alpha-methyldopamine (N-Me-alpha-MeDA) and (2) to investigate the ability of antioxidants, namely ascorbic acid and N-acetyl-L-cysteine (NAC), to prevent N-Me-alpha-MeDA-induced toxic injury, using freshly isolated rat hepatocytes. Cell suspensions were incubated with MDMA or N-Me-alpha-MeDA in the final concentrations of 0.1, 0.2, 0.4, 0.8, and 1.6 mM for 3 h. To evaluate the potential protective effects of antioxidants, cells were preincubated with ascorbic acid in the final concentrations of 0.1 and 0.5 mM, or NAC in the final concentrations of 0.1 and 1 mM for 15 min before treatment with 1.6 mM N-Me-alpha-MeDA for 3 h (throughout this incubation period the cells were exposed to both compounds). The toxic effects were evaluated by measuring the cell viability, glutathione (GSH) and glutathione disulfide (GSSG), ATP, and the cellular activities of GSH peroxidase (GPX), GSSG reductase (GR), and GSH S-transferase (GST). MDMA induced a concentration- and time-dependent GSH depletion, but had a negligible effect on cell viability, ATP levels, or on the activities of GR, GPX, and GST. In contrast, N-Me-alpha-MeDA was shown to induce not only a concentration- and time-dependent depletion of GSH, but also a depletion of ATP levels accompanied by a loss in cell viability, and decreases in the antioxidant enzyme activities. For both compounds, GSH depletion was not accompanied by increases in GSSG levels, which seems to indicate GSH depletion by adduct formation. Importantly, the presence of ascorbic acid (0.5 mM) or NAC (1 mM) prevented cell death and GSH depletion induced by N-Me-alpha-MeDA. The results provide evidence that MDMA and its metabolite N-Me-alpha-MeDA induce toxicity to freshly isolated rat hepatocytes. Oxidative stress may play a major role in N-Me-alpha-MeDA-induced hepatic toxicity since antioxidant defense systems are impaired and administration of antioxidants prevented N-Me-alpha-MeDA toxicity.     

Possible regulation mechanism of microsomal glutathione S-transferase activity in rat liver

After rats were injected with the reduced glutathione (GSH) depletor phorone (diisopropylidene acetone, 250 mg/kg, i.p.), there was a significant increase in microsomal glutathione S-transferase activity in the liver. The maximum activity was observed 24 hr after injection and was about 2-fold that of the control activity. Diethylmaleate (500 mg/kg, i.p.) had the same effect. Twenty-four hours after phorone injection (250 mg/kg, i.p.), the concentrations of GSH and oxidized glutathione (GSSG) in the liver were increased about 2-fold. Under the same conditions, the level of mixed disulfides with microsomal proteins (GSS-protein) was also increased. Further, the activity of microsomal glutathione S-transferases was increased by the in vitro addition of disulfide compounds such as GSSG, cystine and homocystine, and the activity increased by GSSG was reduced to control levels by incubating with the corresponding sulfhydryl compounds such as GSH, cysteine and homocysteine respectively. Thus, microsomal glutathione S-transferase activity appears to be regulated by the formation and/or cleavage of a mixed disulfide bond between the sulfhydryl group present in the enzyme and GSSG. Therefore, the increase of microsomal glutathione S-transferase activity after phorone injection may be due to the formation of a mixed disulfide bond between the sulfhydryl group in the enzyme and GSSG.     

Prospective type 1 and type 2 disulfides of Keap1 protein

Experiments were carried out to detect cysteine residues on human Keap1 protein that may be sensors of oxidative stress that gives rise to changes in the GSH/GSSG redox couple. Human Keap1 protein, at a final concentration of 6 microM, was incubated for two hours in aqueous buffer containing 0.010 M GSH, pH 8, in an argon atmosphere. Subsequently, excess iodoacetamide and trypsin were added to generate a peptide map effected by LCMS analysis. Peptides containing all 27 carboxamidomethylated cysteines were identified. Replacement of GSH by 0.010 M GSSG yielded a map in which 13 of the original carboxamidomethylated peptides were unperturbed, while other caboxamidomethylated cysteine-containing peptides were undetected, and a number of new cysteine-containing peptide peaks were observed. By mass analysis, and in some cases, by isolation, reduction, carboxamidomethylation, and reanalysis, these were identified as S-glutathionylated (Type 1) or Cys-Cys (Type 2) disulfides. Such peptides derived from the N-terminal, dimerization, central linker, Kelch repeat and C-terminal domains of Keap1. Experiments were carried out in which Keap1 was incubated similarly but in the presence of various GSH/GSSG ratios between 100 and 1 ([GSH + GSSG] = 0.010 M), with subsequent caraboxamidomethylation and trypsinolysis to determine differences in sensitivities of the different cysteines to the type 1 and type 2 modifications. Cysteines most sensitive to S-glutathionylation include Cys77, Cys297, Cys319, Cys368, and Cys434, while cysteine disulfides most readily formed are Cys23-Cys38 and Cys257-Cys297. The most reducing conditions at which these modifications are at GSH/GSSG = 10, which computes to an oxidation potential of E h = -268.5 mV, a physiologically relevant value. Under somewhat more oxidizing, but still physiologically relevant, conditions, GSH/GSSG = 1 ( E h = -231.1 mV), a Cys319-Cys319 disulfide is detected far from the dimerization domain of the Keap1 homodimer. The potential impact on protein structure of the glutathionylation of Cys434 and Cys368, the two modified residues in the Kelch repeat domain, was analyzed by docking and energy minimizations of glutathione residues attached to the Kelch repeat domain, whose coordinates are known. The energy minimizations indicated marked alterations in structure with a substantial constriction of Neh2 binding domain of the Keap1 Kelch repeat domain. This alteration appears to be enforced by an extended hydrogen-bonding network between residues on the glutathione moiety attached to Cys434 and amino acid side chains that have been shown to be essential for repression of Nrf2 by Keap1. The modifications of Keap1 detected in the present study are discussed in the context of previous work of others who have examined the sensitivity of cysteines on Keap1 to electrophile assault.     

Alpha-tocopherol and alpha-lipoic acid enhance the erythrocyte antioxidant defence in cyclosporine A-treated rats

The aim of this study was to determine the effects of dietary antioxidant supplementation with alpha-tocopherol and alpha-lipoic acid on cyclosporine A (cyclosporine)-induced alterations to erythrocyte and plasma redox balance. Rats were randomly assigned to either control, antioxidant (alpha-tocopherol 1000 IU/kg diet and alpha-lipoic acid 1.6 g/kg diet), cyclosporine (25 mg/kg/day), or cyclosporine + antioxidant treatments. Cyclosporine was administered for 7 days after an 8 week feeding period. Plasma was analysed for alpha-tocopherol, total antioxidant capacity, malondialdehyde, and creatinine. Erythrocytes were analysed for glutathione, methaemoglobin, superoxide dismutase, catalase, glutathione peroxidase, glucose-6-phosphate dehydrogenase, alpha-tocopherol and malondialdehye. Cyclosporine administration caused a significant decrease in superoxide dismutase activity (P<0.05 control versus cyclosporine) and this was improved by antioxidant supplementation (P<0.05 cyclosporine versus cyclosporine + antioxidant; P<0.05 control versus cyclosporine + antioxidant). Animals receiving cyclosporine and antioxidants showed significantly increased (P<0.05) catalase activity compared to both groups not receiving cyclosporine. Cyclosporine administration induced significant increases in plasma malondialdehyde and creatinine concentration (P<0.05 control versus cyclosporine). Antioxidant supplementation prevented the cyclosporine induced increase in plasma creatinine (P<0.05 cyclosporine versus cyclosporine + antioxidant; P>0.05 control versus cyclosporine + antioxidant), however, supplementation did not alter the cyclosporine induced increase in plasma malondialdehyde concentration (P>0.05 cyclosporine versus cyclosporine + antioxidant). Antioxidant supplementation resulted in significant increases (P<0.05) in plasma and erythrocyte alpha-tocopherol in both of the supplemented groups compared to non-supplemented groups. In conclusion, dietary supplementation with alpha-tocopherol and alpha-lipoic acid enhanced the erythrocyte antioxidant defence and reduced nephrotoxicity in cyclosporine treated animals.     

Cyclosporine A-induced changes to erythrocyte redox balance is time course-dependent

Cyclosporine A-treated transplant recipients develop pronounced cardiovascular disease and have increased oxidative stress and altered antioxidant capacity in erythrocytes and plasma. These experiments investigated the time-course of cyclosporine A-induced changes to redox balance in plasma and erythrocytes. Rats were randomly assigned to either a control or cyclosporine A-treated group. Treatment animals received 25 mg/kg of cyclosporine A via intraperitoneal injection for either 7 days or a single dose. Control rats were injected with the same volume of the vehicle. Three hours after the final injections, plasma was analysed for total antioxidant status, alpha-tocopherol, malondialdehyde, and creatinine. Erythrocytes were analysed for reduced glutathione (GSH), alpha-tocopherol, methaemoglobin, malondialdehyde, and the activities of superoxide dismutase, catalase, GSH peroxidase, and glucose-6-phosphate dehydrogenase (G6PD). Cyclosporine A administration for 7 days resulted in a significant increase (P<0.05) in plasma malondialdehyde, methaemoglobin, and superoxide dismutase and catalase activities. There was a significant decrease (P<0.05) in erythrocyte GSH concentration and G6PD activity in cyclosporine A animals. There were no significant differences (P>0.05) between groups following a single dose of cyclosporine A in any of the measures. In summary, cyclosporine A alters erythrocyte redox balance after 7 days administration, but not after a single dose.     

Evolution of oxidative stress parameters and response to oral vitamins E and C in streptozotocin-induced diabetic rats

Type I diabetes in humans and streptozotocin (STZ)-induced diabetes in rats has been associated with oxidative stress, but antioxidant therapy has given contradictory results, in part related to the absence of common conditions used to evaluate in-vivo antioxidant properties. This prompted the study of an experimental model of antioxidant therapy in STZ-treated rats. Adult female rats received STZ (50 mgkg(-1)) and were studied 7 or 14 days later. Adipose tissue weight progressively decreased with the time of treatment, whereas plasma triglycerides increased at 7 days, before returning to control values at 14 days after STZ treatment. STZ diabetic rats had increased plasma thiobarbituric acid reacting substances and alpha-tocopherol levels, but the latter variable was decreased when corrected for total lipids. STZ diabetic rats showed a higher GSSG/GSH ratio at Day 14 and lower GSH + GSSG at Day 7 in liver. To evaluate the effect of short-term antioxidant therapy, rats received 5 doses of vitamins C and E over 3 days before being killed on Day 14. Treatment with antioxidants decreased plasma lactic acid and thiobarbituric acid reacting substances, as well as urine 8-isoprostane, and decreased plasma uric acid in controls. Vitamins increased the plasma alpha-tocopherol/lipids ratio only in control rats, although the plasma and liver alpha-tocopherol concentration increased in both groups. STZ diabetic rats showed moderate oxidative stress and treatment with antioxidant vitamins caused a significant change in a selected group of oxidative stress markers, which reflected an improvement in some of the complications associated with this disease. The present experimental conditions can be used as a sensitive experimental model to study the responsiveness of diabetes to other antioxidant interventions.     

冠心病患者血浆谷胱甘肽水平变化的临床分析

目的 研究冠心病(CHD)患者血浆氧化还原态抗氧化系统的改变及临床意义.方法 对根据有无致动脉粥样硬化危险因素及冠状动脉造影结果筛选出的60例冠脉造影异常患者(CHD组)、28例有不同程度的动脉粥样硬化危险因素但冠脉造影正常患者(危险组)及30例正常患者(对照组)测定血浆还原型谷胱甘肽(GSH)和氧化型谷胱甘肽(GSSG)浓度,并根据Nernst方程计算出氧化还原电位(EhGSH/GSSG).其中CHD组又分为不稳定型心绞痛(UAP)组32例和急性心肌梗死(AMI)组28例.结果 GSH浓度CHD组与对照组比较差异有统计学意义(P＜0.05).GSSG浓度和EhGSH/GSSG值CHD组及危险组与对照组比较差异均有统计学意义(P＜0.05).UAP组与AMI组之间各指标差异均无统计学意义(P＞0.05).结论 动脉粥样硬化时机体的抗氧化作用减弱,存在氧化应激,提示改善机体的氧化还原状态有可能减慢或阻止动脉粥样硬化的发生与发展.