Supplementation with N-acetylcysteine and taurine failed to restore glutathione content in liver of streptozotocin-induced diabetics rats but protected from oxidative stress

Rats were rendered diabetic with streptozotocin and supplemented or not with N-acetylcysteine (NAC) and taurine (TAU). The liver was examined for the quantity of glutathione (GSH), both total and oxidised (GSSG), by HPLC assay. Moreover, the liver expression of gamma-glutamyl-cysteine synthetase, cysteine dioxygenase and heme oxygenase 1 was evaluated. Streptozotocin-diabetic rats showed decreased levels of liver glutathione (GSH); dietary supplementation with the antioxidants NAC and TAU failed to restore liver GSH to the level of control rats. Gamma-glutamyl-cysteine synthetase expression was not reduced in the diabetic rats, so the low hepatic GSH level in the supplemented diabetic rats cannot be ascribed to decreased expression of the biosynthetic key enzyme. Moreover, the diabetic rats showed no evidence of increased expression of cysteine dioxygenase, which could have indicated that NAC-derived cysteine was consumed in metabolic pathways different from GSH synthesis. However, NAC+TAU treatment provided partial protection from glutathione oxidation in the liver of diabetic rats; moreover, the antioxidant treatment reduced the hepatic overexpression of heme oxygenase 1 (HO-1) mRNA which was detected in the diabetic rats. In conclusion, although NAC was not able to restore liver GSH levels, the antioxidant treatment restrained GSH oxidation and HO-1 overexpression, which are markers of cellular oxidative stress: diabetic rats probably exploit NAC as an antioxidant itself rather than as a GSH precursor.     

蛋氨酸羟基类似物对肉鸡抗氧化能力及代谢激素的影响

以雄性罗斯肉鸡为动物模型,探讨蛋氨酸(DLM)及其液体羟基类似物(HMTBA)对机体抗氧化能力及代谢激素水平的影响差异。结果显示,与对照以及DLM相比,饲粮添加HMTBA显著提高了肉鸡体增重、腿肌重、腿肌率、胸肌重(p<0.05);饲粮添加HMTBA能显著升高肉鸡血浆、肝脏以及肌肉组织的总抗氧化能力(T-AOC),提高还原型谷胱甘肽/氧化型谷胱甘肽的比率(GSH/GSSG)及增强抗氧化氢酶(CAT)活性(p<0.05);可显著降低全血自由基(ROS)含量和肝脏、肌肉组织中丙二醛(MDA)含量(p<0.05);饲粮添加0.24%的HMTBA显著增加了血浆T3、T4、FT3含量(p<0.05)。结果表明,HTMBA较DLM更能有效地改善机体氧化还原状态,增强抗氧化能力,进而调控代谢激素,提高机体游离甲状腺激素的水平,促进肉鸡生长和肌肉沉积。     

Redox analysis of human plasma allows separation of pro-oxidant events of aging from decline in antioxidant defenses

Oxidative stress is a component of diseases and degenerative processes associated with aging. However, no means are available to assess causative oxidative events separately from decline in function of protective antioxidant systems. Previous studies show that ongoing oxidative processes maintain plasma cysteine/cystine redox at a value that is more oxidized than the antioxidant glutathione/glutathione disulfide (GSH/GSSG) system, suggesting that redox analysis of these plasma thiols could allow separate evaluation of an increase in oxidative events from a decline in antioxidant function. The present study uses measurement of cysteine/cystine and GSH/GSSG redox in plasma of 122 healthy individuals aged 19-85 years to determine whether thiol-disulfide redox changes occur with age. The results show a linear oxidation of cysteine/cystine redox state with age at a rate of 0.16 mV/year over the entire age span. In contrast, GSH/GSSG redox was not oxidized prior to 45 years and subsequently was oxidized at a nearly linear rate of 0.7 mV/year. These data suggest that there is a continuous, linear increase in oxidative events throughout adult life but that the capacity of the GSH antioxidant system is maintained until 45 years and then declines rapidly. The data further suggest that redox states of cysteine/cystine and GSH/GSSG provide an approach to clinically distinguish between increased causative oxidative events and decreased GSH antioxidant function. In principle, such analyses can be used to assess efficacy of intervention strategies against oxidative stress prior to or early after onset of clinical symptoms in aging and age-related disease.     

Increase in heart glutathione redox ratio and total antioxidant capacity and decrease in lipid peroxidation after vitamin e dietary supplementation in guinea pigs

Dietary treatment with three diets differing in vitamin E, Low E (15 mg of vitamin E/kg diet), Medium E (150 mg/kg), or High E (1,500 mg/kg), resulted in guinea pigs with low (but nondeficient), intermediate, or high heart a-tocopherol concentration. Neither the antioxidant enzymes superoxide dismutase, catalase, glutathione peroxidase, and reductase, nor the nonenzymatic antioxidants, GSH, ascorbate, and uric acid were homeostatically depressed by increases in heart a-tocopherol. Protection from both enzymatic (NADPH dependent) and nonenzymatic (ascorbate-Fe²⁺) lipid peroxidation was strongly increased by vitamin E supplementation from Low to Medium E Whereas no additional gain was obtained from the Medium E to the High E group. The GSH/GSSG and GSH/total glutathione ratios increased as a function of the vitamin E dietary concentration closely resembling the shape of the dependence of heart a-tocopherol on dietary vitamin E. The results show the capacity of dietary vitamin E to increase the global antioxidant capacity of the heart and to improve the heart redox status in both the lipid and water-soluble compartments. This capacity occurred at levels six times higher than the minimum daily requirement of vitamin E, even in the presence of optimum dietary vitamin C concentrations and basal unstressed conditions. The need for vitamin E dietary supplementation seems specially important in this tissue due to the low constitutive levels of endogenous enzymatic and nonenzymatic antioxidants present of the mammalian heart in comparison with those of other internal organs.     

The ketogenic diet increases mitochondrial glutathione levels

The ketogenic diet (KD) is a high-fat, low carbohydrate diet that is used as a therapy for intractable epilepsy. However, the mechanism(s) by which the KD achieves neuroprotection and/or seizure control are not yet known. We sought to determine whether the KD improves mitochondrial redox status. Adolescent Sprague–Dawley rats (P28) were fed a KD or control diet for 3 weeks and ketosis was confirmed by plasma levels of β-hydroxybutyrate (BHB). KD-fed rats showed a twofold increase in hippocampal mitochondrial GSH and GSH/GSSG ratios compared with control diet-fed rats. To determine whether elevated mitochondrial GSH was associated with increased de novo synthesis, the enzymatic activity of glutamate cysteine ligase (GCL) (the rate-limiting enzyme in GSH biosynthesis) and protein levels of the catalytic (GCLC) and modulatory (GCLM) subunits of GCL were analyzed. Increased GCL activity was observed in KD-fed rats, as well as up-regulated protein levels of GCL subunits. Reduced CoA (CoASH), an indicator of mitochondrial redox status, and lipoic acid, a thiol antioxidant, were also significantly increased in the hippocampus of KD-fed rats compared with controls. As GSH is a major mitochondrial antioxidant that protects mitochondrial DNA (mtDNA) against oxidative damage, we measured mitochondrial H₂O₂ production and H₂O₂-induced mtDNA damage. Isolated hippocampal mitochondria from KD-fed rats showed functional consequences consistent with the improvement of mitochondrial redox status i.e. decreased H₂O₂ production and mtDNA damage. Together, the results demonstrate that the KD up-regulates GSH biosynthesis, enhances mitochondrial antioxidant status, and protects mtDNA from oxidant-induced damage.     

(R)-alpha-lipoic acid reverses the age-related loss in GSH redox status in post-mitotic tissues: evidence for increased cysteine requirement for GSH synthesis

Age-related depletion of GSH levels and perturbations in its redox state may be especially deleterious to metabolically active tissues, such as the heart and brain. We examined the extent and the mechanisms underlying the potential age-related changes in cerebral and myocardial GSH status in young and old F344 rats and whether administration of (R)-alpha-lipoic acid (LA) can reverse these changes. Our results show that GSH/GSSG ratios in the aging heart and the brain declined by 58 and 66% relative to young controls, respectively (p < 0.001). Despite a consistent loss in GSH redox status in both tissues, only cerebral GSH levels declined with age (p < 0.001). To discern the potential mechanisms underlying this differential loss, the levels and the activities of gamma-glutamylcysteine ligase (GCL) and cysteine availability were determined. There were no significant age-related changes in substrate or enzyme levels, or GCL activity when saturating amounts of substrates were provided. However, kinetic analysis of GCL in brains of old rats displayed a significant increase (p < 0.05) in the apparent [Km] for cysteine (Km cys) vs. young rats (84.3+/-25.4 vs. 179.0+/-49.0; young and old, respectively), resulting in a 40% loss in apparent catalytic turnover of the enzyme. Thus, the age-related decline in total GSH appears to be mediated, in part, by a general decrement in GCL catalytic efficiency. Treating old rats with LA (40 mg/kg body wt; by i.p.) markedly increased tissue cysteine levels by 54% 12 h following treatment and subsequently restored the cerebral GSH levels. Moreover, LA improved the age-related changes in the tissue GSH/GSSG ratios in both heart and the brain. These results demonstrate that LA is an effective agent to restore both the age-associated decline in thiol redox ratio as well as increase cerebral GSH levels that otherwise decline with age.     

RP-HPLC method for quantitative determination of cystathionine, cysteine and glutathione: An application for the study of the metabolism of cysteine in human brain

The RP-HPLC method for a simultaneous separation and quantitation of the dinitrophenyl derivative of cystathionine (N,N'-di-DNP) in biological samples together with GSH, GSSG, cysteine and cystine, provides a very useful tool for investigation of the transsulfuration pathway in biological samples, at the same providing results which reflect the redox status (GSH/GSSG ratio) and the potential of the generation of H?S. An application of the method for the study of the process of transsulfuration in various human brain regions shows the presence of cystathionine in all the investigated regions; it also demonstrates that cystathionine levels vary greatly between particular regions. The highest level in the thalamus and the lowest in the cerebellum were associated with respectively a low or high γ-cystathionase activity, and at the same time, a high cysteine and GSH level in the thalamus and a low value in the cerebellum. Based on the above results, one may suggest a regulatory mechanism responsible for inhibition of the CGL activity at high concentration values of cysteine and/or GSH. Simultaneous determinations of GSH and GSSG levels allow for determining the GSH/GSSG ratio, which reflects tissue redox status. The method may be also employed in determining the activity of γ-cystathionase and cystathionine-β synthase.     

Thiol supplementation in aged animals alters antioxidant enzyme activity after heat stress.

Declines in oxidative and thermal stress tolerance are well documented in aging systems. It is thought that these alterations are due in part to reductions in antioxidant defenses. Although intracellular thiols are major redox buffers, their role in maintaining redox homeostasis is not completely understood, particularly during aging, where the reliance on antioxidant enzymes and proteins may be altered. To determine whether thiol supplementation improved the antioxidant enzyme profile of aged animals after heat stress, young and old Fischer 344 rats were treated with N-acetylcysteine (NAC; 4 mmol/kg ip) 2 h before heat stress. Liver tissue was collected before and 0, 30, and 60 min after heat stress. Aging was associated with a significant decline in tissue cysteine and glutathione (GSH) levels. There was also an age-related decrease in copper-zinc superoxide dismutase activity. Heat stress did not alter liver GSH, glutathione disulfide, or antioxidant enzyme activity. With NAC treatment, old animals took up more cysteine than young animals as reflected in an increase in liver GSH and a corresponding decrease in glutamate cysteine ligase activity. Catalase activity increased after NAC treatment in both age groups. Copper-zinc superoxide dismutase activity did not change with heat stress or drug treatment, whereas manganese superoxide dismutase activity was increased in old animals only. These data indicate that GSH synthesis is substrate limited in old animals. Furthermore, aged animals were characterized by large fluctuations in antioxidant enzyme balance after NAC treatment, suggesting a lack of fine control over these enzymes that may leave aged animals susceptible to subsequent stress.     

Burn-induced oxidative stress is altered by a low zinc status: kinetic study in burned rats fed a low zinc diet

As an initial subdeficient status of zinc, considered as an essential antioxidant trace element, is frequent in burned patients, we aim to assess the effects of low zinc dietary intakes on burn-induced oxidative stress, in an animal model. After 8 weeks of conditioning diets containing 80 ppm (control group) or 10 ppm of zinc (depleted group), Wistar rats were 20% TBSA burned and sampled 1-10 days after injury. Kinetic evolutions of zinc status, plasma oxidative stress parameters, and antioxidant enzymes were also studied in blood and organs. The zinc-depleted diet induced, before injury, a significant decrease in zinc bone level and the increase of oxidative stress markers without stimulation of antioxidant enzyme activity. After burn, more markedly in zinc depleted animals than in controls, zinc levels decreased in plasma and bone, while increasing in liver. The decrease of thiol groups and GSH/GSSG ratio and the depression of GPx activity in liver are also moderately emphasized. Nevertheless, depleted zinc status could not be considered as determining for oxidative damages after burn injury. Further investigations must also be done to enlighten the mechanism of beneficial effects of zinc supplementation reported in burned patients.     

Effect of antioxidant-enriched diets on glutathione redox status in tissue homogenates and mitochondria of the senescence-accelerated mouse

The main purpose of this study was to investigate whether consumption of diets enriched in antioxidants attenuates the level of oxidative stress in the senescence-accelerated mouse (SAM). In separate and independent studies, two different dietary mixtures, one enriched with vitamin E, vitamin C, L-carnitine, and lipoic acid (Diet I) and another diet including vitamins E and C and 13 additional ingredients containing micronutrients with bioflavonoids, polyphenols, and carotenoids (Diet II), were fed for 8 and 10 months, respectively. The amounts of glutathione (GSH) and glutathione disulfides (GSSG) and GSH:GSSG ratios were determined in plasma, tissue homogenates, and mitochondria isolated from five different tissues of SAM (P8) mice. Both diets had a reductive effect in plasma; however Diet I had relatively little effect on the glutathione redox status in tissue homogenates or mitochondria. Remarkably, Diet II caused a large increase in the amount of glutathione and a marked reductive shift in glutathione redox state in mitochondria. Overall, the effects of Diet II were tissue and gender specific. Results indicated that the glutathione redox state in mitochondria and tissues can be altered by supplemental intake of a relatively complex mixture of dietary antioxidants that contains substances known to induce phase 2 enzymes, glutathione, and antioxidant defenses. Whether corresponding attenuations occur in age-associated deleterious changes in physiological functions or life span remains unknown.     

Influence of organic selenium on hsp70 response of heat-stressed and enteropathogenic Escherichia coli-challenged broiler chickens (Gallus gallus)

The effect of dietary selenium yeast, a source of organic selenium, on heat shock protein 70 (hsp70) responses, redox status, growth and feed utilization were evaluated either in enteropathogenic Escherichia coli-challenged (EPEC) or in heat-stressed (HS) male broiler chickens grown to 42 days of age. One day-old chicks in experiment 1 were challenged orally with EPEC (10(6) cfu/chicken on day 1 and boosted by water application on days 2, 3, and 4) and fed diets with or without selenium yeast. Body weight (BW), feed conversion ratio (FCR), and total mortality were determined at 42 days of age, and this was followed by collection of ileal tissue for the quantification of total glutathione (TGSH), reduced glutathione (GSH), oxidized glutathione (GSSG), and hsp70 in randomly selected chickens from each treatment. In experiment 2, male broiler chickens were fed diets with or without selenium yeast under a thermoneutral rearing condition. At four weeks of age, blood and hepatic tissue were collected from chickens maintained in the thermoneutral environment and from chickens subjected to HS (40 degrees C for 1 h) and analyzed for TGSH, GSH, GSSG, and hsp70. Selenium yeast improved BW, FCR, and decreased mortality in both control and EPEC-challenged chicks. Selenium yeast significantly attenuated hsp70 expression in EPEC-challenged chickens and in those subjected to HS. The EPEC challenge increased TGSH and GSSG levels and decreased GSH/GSSG ratio. However, GSSG level accumulated in chickens fed diets without selenium supplementation resulting in a lower GSH/GSSG ratio in the selenium yeast-fed group. Heat stress increased GSSG level and decreased GSH/GSSG ratio. Selenium yeast-fed groups maintained higher levels of GSSG before and after HS with a resultant lower GSH/GSSG ratio. The hsp70 response was significantly less in those chickens fed selenium yeast and challenged with either EPEC or HS than in those chickens given no supplemental selenium. The results of this study suggest that selenium yeast supplementation had imparted resistance to oxidative stress associated with enteric bacteria infection and to high temperature exposure. It is believed that the resistance to the stressors was due to an improved redox status of the selenium yeast-fed chickens.     

Suppression of oxidative stress in aging NZB/NZW mice: effect of fish oil feeding on hepatic antioxidant status and guanidino compounds

Oxidative stress caused by excessive reactive species (RS) and lipid peroxidation is known to be casually linked to age-related inflammation. To test the hypothesis that fish oil (FO) intake has a beneficial effect on nephritis due to its suppressive action of oxidative stress and the enhancement of antioxidant defenses, we examined the effect of dietary FO on various oxidative stress-related parameters and guanidino compound (GC) levels using (NZB × NZW) F₁ (B/W) mice. These mice were fed diets supplemented with either 5% corn oil (control) or 5% FO. At 4 and 9 months of age, the hepatic oxidative status was estimated by assessing RS generation produced from xanthine oxidase, the prostaglandin pathway and lipid peroxidation. To evaluate the effect of FO on redox status, including antioxidant defenses, GSH and GSSG levels and antioxidant enzyme activities were measured. To correlate the extent of oxidative status with the nephritic condition, creatinine, guanidino acetic acid and arginine levels were measured. Results indicated that increased levels of lipid peroxidation, RS generation and xanthine oxidase activity with age were all significantly suppressed by FO feeding. Furthermore, reduced GSH levels, GSH/GSSG ratio and antioxidant enzyme activities in the FO-fed mice were effectively enhanced compared to the corn oil-fed mice. Among several GCs, the age-related increase of creatinine level was blunted by FO. Based on these results, we propose that dietary FO exerts beneficial effects in aged, nephritic mice by suppressing RS, superoxide and lipid peroxidation, and by maintaining a higher GSH/GSSG ratio and antioxidant enzyme activities.     

Impact of a dietary challenge with peroxidized oil on the glutathione redox status and integrity of the small intestine in weaned piglets

Glutathione (GSH) is considered to play an important role in maintaining the integrity of the small intestine. In piglets, altered mucosal GSH levels might therefore be involved in weaning-induced changes of the small intestinal morphology and barrier function. To test this hypothesis, we aimed to challenge the mucosal GSH redox status during the first 28 days after weaning, by feeding diets containing 5% fresh linseed oil (CON), or 2.5% (OF1) or 5% (OF2) peroxidized linseed oil (peroxide value 225 mEq O₂/kg oil) and exploring the effects on gut integrity. Piglets were pair-fed and had a total daily feed allowance of 32 g/kg BW. A fourth treatment included animals that were fed the control diet ad libitum (ACON). Animals were sampled at days 5 and 28 post-weaning. The malondialdehyde (MDA) concentration and GSH redox status (GSH/GSSG E_h) were determined in blood, liver and small intestinal mucosa. Histomorphology of the duodenal and jejunal mucosa was determined, and Ussing chambers were used to assess fluorescein isothiocyanate dextran (FD4) and horseradish peroxidase (HRP) fluxes across the mucosa. Results show that peroxidized linseed oil imposed an oxidative challenge at day 28, but not at day 5 post-weaning. At day 28, increasing levels of dietary peroxides to pair-fed pigs linearly increased MDA levels in duodenal and jejunal mucosa. Moreover, FD4 fluxes were significantly increased in OF1 (+75%) and OF2 (+64%) in the duodenum, and HRP fluxes tended (P=0.099) to show similar differences, as compared to CON. This co-occurred with a significant 11 mV increase of the hepatic GSH/GSSG E_h, potentiated by a significantly increased GSH peroxidase activity for treatments OF1 (+47%) and OF2 (+63%) in liver as compared to CON. Furthermore; duodenal HRP flux significantly correlated with the hepatic glutathione disulphide (GSSG) level (r=0.650), as also observed in the jejunum for hepatic GSSG (r=0.627) and GSH/GSSG E_h (r=0.547). The jejunal permeability was not affected, but FD4 and HRP fluxes significantly correlated with the local GSH (r=0.619; r=0.733) and GSSG (r=0.635; r=0586) levels. Small intestinal histomorphology was not affected by dietary lipid peroxides, nor were there any correlations found with the GSH redox system. To conclude, under oxidative stress conditions, jejunal barrier function is related to the local and hepatic GSH redox system. It is suggested that the hepatic GSH system participates in the elimination of luminal peroxides, and thereby impacts on duodenal barrier function.     

Energy restriction but not protein source affects antioxidant capacity in athletes

The primary purpose of this study was to examine the effect of energy restriction on antioxidant capacity in trained athletes. Secondly, our study determined whether dietary protein source influenced the antioxidant response, performance, and immunity. Twenty male cyclists consumed either whey or casein supplement (40 g/day) in addition to their diet for 17 days. All subjects subsequently underwent 4 days of energy restriction using a formula diet (20 kcal/kg) while continuing protein supplementation. Energy restriction caused 2.7 +/- 0.3 kg weight loss, increased lymphocyte total glutathione (tGSH) 37%, red blood cell glutathione peroxidase 48%, plasma cysteine 12%, and decreased whole blood reduced to oxidized GSH (rGSH/GSSG) ratio by 52%. The only immunity factor altered by energy restriction was an increase in stimulated phagocytosis (65%). Acute submaximal exercise reduced blood tGSH but increased glutathione peroxidase. Performance of a high intensity cycle test following 45 min of moderate exercise tended to be reduced by energy restriction (P = 0.06) but was unaffected by protein source. Energy restriction caused a negative nitrogen balance with no difference from dietary protein source. In conclusion, acute energy restriction increased plasma cysteine and several markers of the glutathione antioxidant system in trained athletes. A high cysteine dietary protein source did not influence these responses.     

Comparison between the effects of normoxia and hypoxia on antioxidant enzymes and glutathione redox state in ex vivo culture of CD34(+) cells

Hypoxia maintained biological characteristics of CD34(+) cells through keeping lower intracellular reactive oxygen specials (ROS) levels. The effects of normoxia and hypoxia on antioxidant enzymes and glutathione redox state were compared in this study. Hypoxia decreased the mRNA expression of both catalase (CAT) and glutathione peroxidase (GPX), but not affected mRNAs expression of superoxide dismutase (SOD). While the cellular GPX activities under hypoxia were apparently less than those under normoxia, neither SOD activities nor CAT activities were affected by hypoxia. The analysis of glutathione redox status and ROS products showed the lower oxidized glutathione (GSSG) levels, the higher reduced glutathione (GSH) levels, the higher GSH/GSSG ratios, and the less O(2)- and H(2)O(2) generation under hypoxia (versus normoxia). Meanwhile more primary CD34(+)CD38(-) cells were obtained when cultivation was performed under hypoxia or with N-acetyl cysteine (the precursor of GSH) under normoxia. These results demonstrated the different responses of anti-oxidative mechanism between normoxia and hypoxia. Additionally, the present study suggested that the GSH-GPX antioxidant system played an important role in HSPCs preservation by reducing peroxidation.     

Effects of Dietary Glutathione on Plasma and Liver Lipid Levels in Rats Fed on a High Cholesterol Diet

The effects of dietary glutathione (GSH) on plasma and liver lipid concentrations were investigated with rats fed on a high cholesterol diet. When graded levels of GSH, 0.75 to 5.0%, were added to the 25% casein basal diet, the plasma total cholesterol level was significantly decreased and the HDL-cholesterol level was inversely increased in all addition levels without influence on the growth of animals except for the 5% addition level; the dietary addition of 5% GSH markedly depressed the growth and food consumption of rats and caused a slight diarrhea. Plasma triglyceride and phospholipid levels were decreased by the dietary addition of GSH. The contents of cholesterol and triglyceride in the liver were decreased as the dietary addition level of GSH was increased. The dietary addition of a mixture of glutamic acid, cysteine and glycine, or cysteine alone corresponding to 2.5% GSH resulted in a cholesterol-lowering effect which could not be distinguished from the effect of GSH in rats fed on the 25% casein diet. When 1.5% GSH was added to a low (10%) casein diet, the plasma cholesterol-lowering effect of GSH was also observed and the effect was comparable to that of cysteine. These results indicate that dietary-added GSH has a plasma and liver cholesterol-lowering efficacy and that this effect is largely attributable to the cysteine residue of GSH rather than to the tripeptide itself or the other amino acid residues.     

Opposite effect of methionine-supplemented diet, a model of hyperhomocysteinemia, on plasma and liver antioxidant status in normotensive and spontaneously hypertensive rats

Hyperhomocysteinemia is often associated with an increase in blood pressure. However our previous study has shown that methionine supplementation induced an increase in blood pressure in Wistar-Kyoto (WKY) rats and a decrease in blood pressure in spontaneously hypertensive rats (SHR) with significant differences in plasma homocysteine (Hcy) metabolites levels. Previously liver antioxidant status has been shown to be decreased in SHR compared to WKY rats. It has been suggested that oxidative stress may predispose to a decrease in NO bioavailability and induce the flux of Hcy through the liver transsulfuration pathway. Thus the aim of this study was 1) to investigate the effect of methionine supplementation on NO-derived metabolites in plasma and urine 2) to investigate whether abnormalities in Hcy metabolism may be responsible for the discrepancies observed between WKY rats and SHR concerning blood pressure and 3) to investigate whether a methionine-enriched diet, differently modified plasma and liver antioxidant status in WKY rats an SHR. We conclude that the increase in blood pressure in WKY rats is related to high plasma cysteine levels and is not due to a decrease in NO bioavailability and that the decrease in blood pressure in SHR is associated with high plasma GSH levels after methionine supplementation. So GSH synthesis appears to be stimulated by liver oxidative stress and GSH is redistributed into blood in SHR. So the great GSH synthesis can be rationalized as an autocorrective response that leads to a decreased blood pressure in SHR.     

Glutathione and thioredoxin redox during differentiation in human colon epithelial (Caco-2) cells

Cellular redox, maintained by the glutathione (GSH)- and thioredoxin (Trx)-dependent systems, has been implicated in the regulation of a variety of biological processes. The redox state of the GSH system becomes oxidized when cells are induced to differentiate by chemical agents. The aim of this study was to determine the redox state of cellular GSH/glutathione disulfide (GSH/GSSG) and Trx as a consequence of progression from proliferation to contact inhibition and spontaneous differentiation in colon carcinoma (Caco-2) cells. Results showed a significant decrease in GSH concentration, accompanied by a 40-mV oxidation of the cellular GSH/GSSG redox state and a 28-mV oxidation of the extracellular cysteine/cystine redox state in association with confluency and increase in differentiation markers. The redox state of Trx did not change. Thus the two central cellular antioxidant and redox-regulating systems (GSH and Trx) were independently controlled. According to the Nernst equation, a 30-mV oxidation is associated with a 10-fold change in the reduced/oxidized ratio of a redox-sensitive dithiol motif. Therefore, the measured 40-mV oxidation of the cellular GSH/GSSG couple or the 28-mV oxidation of the extracellular cysteine/cystine couple should be sufficient to function in signaling or regulation of differentiation in Caco-2 cells.     

Effect of Dietary Vitamin C and Catalase Inhibition on Antioxidants and Molecular Markers of Oxidative Damage in Guinea Pigs

Guinea pigs were fed for five weeks with two diets with different levels of vitamin C, low (33 mg of Vit C/Kg diet) and high (13,200 mg of Vit C/Kg of diet). Catalase was inhibited with 3-amino-1,2,4-triazole (AT) in half of the animals from each dietary group. AT caused an almost complete depletion of liver catalase activity (90%) in both dietary groups. Vitamin C supplementation increased total glutathione peroxidase activity and tissue vitamin C level and decreased levels of protein carbonyls and malondialdehyde (MDA) in both treated and non-treated animals. This vitamin C supplementation did not change any of the other antioxidant defences studied. Our results show that dietary vitamin C supplementation increases global antioxidant capacity and decreases endogenous oxidative damage in the guinea pig liver under normal non-stressful conditions. This supports the protective value of dietary antioxidant supplementation.     

Effect of Procysteine on aging-associated changes in hepatic GSH and SMase: evidence for transcriptional regulation of smpd3

In hepatocytes, aging-associated decline in GSH has been linked to activation of neutral SMase (nSMase), accumulation of bioactive ceramide, and inflammation. In this study, we seek to test whether dietary supplementation with the cysteine precursor, L-2-oxothiazolidine-4-carboxylic acid (OTC), would correct the aging-associated differences in hepatic GSH, nSMase, and ceramide. Young and aged mice were placed on a diet that either lacked sulfur-containing amino acids (SAAs) or had 0.5% OTC for 4 weeks. Mice fed standard chow were used as an additional control. SAA-deficient mice exhibited significant aging-associated differences in hepatic GSH, GSH/GSSG, ceramide, and nSMase. C24:1 ceramide, the major ceramide species in liver, was affected the most by aging, followed by the less abundant C16:0 ceramide. OTC supplementation eliminated the aging-associated differences in hepatic GSH and GSH/GSSG ratio. Surprisingly, however, instead of decreasing, the nSMase activity and ceramide increased in the OTC-fed mice irrespective of their age. These effects were due to elevated nSMase-2 mRNA and protein and appeared to be direct. Similar increases were seen in HepG2 cells following treatment with OTC. The OTC-fed aged mice also exhibited hepatic steatosis and triacylglyceride accumulation. These results suggest that OTC is a potent stimulant of nSMase-2 expression and that there may be unanticipated complications of OTC supplementation.