Methionine challenge paradoxically induces a greater activation of the antioxidant defence in subjects with hyper- vs. normohomocysteinemia

To determine whether hyperhomocysteinemia induced post-methionine loading (PML) is associated with different response in the aminothiol redox state and oxidative stress vs. normohomocysteinemia, we assessed PML plasma thiols, vitamins, free malondialdehyde (MDA), and blood reduced glutathione (GSH) in 120 consecutive subjects (50 [35-56] years, 83 males), divided into two groups according to PML plasma total Hcy < 35 microM (Group 1, n = 65) or > or = 35 microM (Group 2, n = 55).In the group as a whole, plasma reduced cysteine and cysteinylglycine, blood reduced GSH (all p for time = 0.0001) and plasma total GSH (p for time = 0.001) increased from baseline to PML. MDA values were unchanged. Group 1 and 2 differed in blood reduced GSH (p for group = 0.004, higher in Group 2), and MDA levels (p for group = 0.024, lower in Group 2).The oxidative stress induced by methionine challenge seems to be opposed by scavenger molecules activation, namely GSH, and lipid peroxidation does not increase. This mechanism paradoxically appears to be more efficient in hyperhomocysteinemic subjects.     

Pinealectomy increases oxidant damage in kidney and testis caused by hyperthyroidism in rats

Thyroid hormones regulate energy metabolism and act on mitochondria which are an important source of free radicals in the cell. The pineal gland activates antioxidant systems via melatonin secretion and thus has a protective function in body tissues. The present study was conducted to determine the oxidative damage caused by hyperthyroidism in kidney and testis tissues of pinealectomized rats. Experimental animals were allocated to three groups: 1, control group; 2, sham pinealectomy-hyperthyroidic group; and 3, pinealectomy-hyperthyroidic group. Hyperthyroidism was induced by A 3-week intraperitoneal administration of thyroxin after sham pinealectomy or pinealectomy. Malondialdehyde (MDA) and glutathione (GSH) levels were determined in kidney and testis tissues. MDA levels of the kidney and testis tissue in the pinealectomy and hyperthyroidic groups were significantly higher than those in the sham pinealectomy-hyperthyroidic group and the control group (p < 0.001). GSH levels of both kidney and testis tissues were significantly higher in the sham-pinealectomy-hyperthyroidic group when compared to the other two groups (p < 0.001). This increase in GSH levels was more evident in the pinealectomy-hyperthyroidic group than in the control group (p < 0.001). The results of our study demonstrate that MDA and GSH levels in kidney and testis tissues increased due to hyperthyroidism and that pinealectomy made the increase in MDA levels more apparent, while decreasing GSH levels.     

N‐nitrosodimethylamine changes the expression of glutathione S‐transferase in the liver of male mice: The role of antioxidants

The present study investigated the protective effect of gossypol, selenium, zinc, or glutathione (GSH) against dimethylnitrosamine (DMN)‐induced hepatotoxicity in the livers of male mice. The expression and the activity of glutathione S‐transferase (GST), levels of GSH, and free radicals (malondialdehyde (MDA)), as well as the activity of glutathione reductase were determined after the treatment of mice for seven consecutive days with low or high doses of gossypol, selenium, zinc, or GSH. In experimental groups, DMN was administered as a single dose for 2 h after the repeated dose treatments of mice for seven consecutive days with each antioxidant. DMN reduced the expression and inhibited the activity of GST. However, repeated treatments of mice with low‐dose gossypol or high dose of either selenium or GSH followed by a single dose of DMN induced the expression and the activity of GST. In contrast, low‐dose treatments of mice with zinc, selenium, or GSH followed by a single dose of DMN reduced the expression and the activity of GST compared to either control or DMN‐treated groups. In addition, high‐dose treatment with either gossypol or selenium markedly induced the levels of GSH compared to either control or DMN‐treated groups. Interestingly, pretreatment of mice with high dose of either gossypol or selenium for seven consecutive days followed by a single dose of DMN decreased the levels of MDA, whereas DMN induced such levels. It is concluded that high dose of either gossypol or selenium is a stronger protector than zinc and GSH in ameliorating the toxic effects of DMN. © 2008 Wiley Periodicals, Inc. J Biochem Mol Toxicol 22:389–395, 2008; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20255     

Glutathione and lipid peroxide levels in rat liver following administration of methapyrilene and analogs

The possibility was examined that the induction of tumors in rat liver by feeding methapyrilene, which is not mutagenic, is related to effects on glutathione levels and lipid peroxidation. Fischer 344 rats were given single-dose and multiple-dose treatments with the anti-histamine methapyrilene (MP), which is carcinogenic in rats, and with two non-carcinogenic analogs, methafurylene (MF) and thenyldiamine (TD) and the effects on malonaldehyde (MDA) formation and glutathione (GSH) levels in the liver were investigated. After a single dose, MDA levels were increased at 6 h by MF and TD and at 24 h by MP. MDA levels returned to normal after 30 h with MP and MF, but not with TD. Levels of MDA (and other TBA-reactive products) after four daily treatments were most elevated by TD, less elevated by MP, and were lowered by MF. Forty-two hours following treatment with both MP and MF, MDA levels had returned to normal, but in TD-treated animals MDA remained high. GSH levels were highest after MF and MP, and remained high at 42 h, but TD induced only a small increase. There appears to be increased lipid peroxidation in the liver as a result of treatment of rats with MP, MF and TD. The greater response induced by TD, as well as the increased liver GSH levels after repeated administration of all three drugs indicate that lipid peroxidation in rat liver is not a particular effect related to the liver carcinogen methapyrilene.     

Effect of metal ion catalyzed oxidation of rifamycin SV on cell viability and metabolic performance of isolated rat hepatocytes

The effect of rifamycin SV on metabolic performance and cell viability was studied using isolated hepatocytes from fed, starved and glutathione (GSH) depleted rats. The relationships between GSH depletion, nutritional status of the cells, glucose metabolism, lactate dehydrogenase (LDH) leakage and malondialdehyde (MDA) production in the presence of rifamycin SV and transition metal ions was investigated. Glucose metabolism was impaired in isolated hepatocytes from both fed and starved animals, the effect is dependent on the rifamycin SV concentration and is enhanced by copper (II). Oxygen consumption by isolated hepatocytes from starved rats was also increased by copper (II) and a partial inhibition due to catalase was observed. Cellular GSH levels which decrease with increasing the rifamycin SV concentration were almost depleted in the presence of copper (II). A correlation between GSH depletion and LDH leakage was observed in fed and starved cells. Catalase induced a slight inhibition of the impairment of gluconeogenesis, GSH depletion and LDH leakage in starved hepatocytes incubated with rifamycin SV, iron (II) and copper (II) salts. Lipid peroxidation measured as MDA production by isolated hepatocytes was also augmented by rifamycin SV and copper (II), especially in hepatic cells isolated from starved and GSH depleted rats. Higher cytotoxicity was observed in isolated hepatocytes from fasted animals when compared with fed or GSH depleted animals. It seems likely that in addition to GSH level, there are other factors which may have an influence on the susceptibility of hepatic cells towards xenobiotic induced cytotoxicity.     

Cocaine-induced biochemical changes and cytotoxicity in hepatocytes isolated from both mice and rats

The mechanism of cocaine-induced cytotoxicity was investigated in hepatocytes isolated from both male C3H mice and male Sprague-Dawley rats. Cocaine was more cytotoxic to mouse hepatocytes than rat and induced reduced glutathione (GSH) depletion prior to marked increases in cytotoxicity in both systems. In both mouse and rat cells, GSH depletion was accompanied by GSSG production, but in rat cells, quantitative measures suggested that other mechanisms contributed to GSH depletion. No cocaine-induced depletion of protein-thiol groups or generation of protein-glutathione mixed disulfides could be detected in rat cells. Cocaine induced lipid peroxidation, using malondialdehyde (MDA) production as an index of the peroxidation process, in both mouse and rat hepatocytes. Inhibition of MDA production to below control levels using the antioxidant N,N'-diphenyl-phenylene diamine (DPPD) however, had no inhibitory effect on cocaine-induced cytotoxicity in either mouse or rat cells. These data suggest that neither generalized protein thiol depletion nor lipid peroxidation are critical determinants of cocaine-induced cytotoxicity in cellular systems.     

The susceptibility to autoxidation of erythrocytes in diabetic mice: Effects of melatonin and pentoxifylline

Oxidative stress had a great importance in development of complications in diabetes. We investigated effects of melatonin and pentoxifylline in diabetic mice. Swiss albino mice (n = 40) were divided into four groups: alloxan‐induced diabetes mellitus (DM), alloxan‐induced diabetes with melatonin supplementation (DM + MLT), alloxan‐induced diabetes with pentoxifylline supplementation (DM + PTX), and control. Glutathione‐peroxidase (GSH‐Px) activity, malondialdehyde (MDA) and reduced glutathione (GSH) levels, and susceptibility to oxidation of erythrocytes were measured. MDA levels were higher than control in the DM and DM + MLT. The DM had more MDA level than the DM + MLT and DM + PTX (P < 0.001). After in vitro oxidation, MDA levels of all groups were found higher than the control. However, they were significantly lower than the DM in DM + PTX and DM + MLT (P < 0.001). Although GSH levels of the DM and DM + PTX were less than the control, GSH‐Px activity of the DM was lower than the control and DM + PTX (P < 0.05). We suggest that there is increased oxidative stress and compromised antioxidant status of erythrocytes in diabetes; however, it can be effectively prevented by melatonin or pentoxifylline supplementation.     

Paracetamol-stimulated lipid peroxidation in isolated rat and mouse hepatocytes

Treatment of isolated hepatocytes from 3-methylcholanthrene induced rats with 1 mM paracetamol has been found to greatly decrease cellular reduced glutathione (GSH) content and to promote lipid peroxidation, evaluated as malonaldehyde (MDA) production and conjugated diene absorbance. A similar dosing of hepatocytes from phenobarbital-induced or normal rats is ineffective in that respect. On the other hand, the aspecific stimulation of the cytochrome P-450-mediated paracetamol activation due to acetone addition further increases GSH depletion as well as MDA production.Isolated hepatocytes with basal low GSH content are also more susceptible to paracetamol-induced lipid peroxidation, indicating that the rate of the drug metabolism and the cellular GSH content are critical factors in the determination of such peroxidative attack.In isolated mouse liver cells paracetamol does not require preliminary cytochrome P-450 induction to stimulate MDA formation, even at concentrations ineffective in rat cells.However, 5 mM paracetamol, despite a great depletion of cellular GSH content, does not promote MDA formation either in the rat or in the mouse hepatocytes. This effect may be due to the ability of paracetamol to scavenge lipid peroxides under defined conditions, as tested in various lipid peroxidizing systems.Membrane leakage of lactate dehydrogenase (LDH) is evident in paracetamol treated cells undergoing lipid peroxidation, but not when MDA formation is inhibited by high doses of the drug or by addition of antioxidants such as α-tocopherol and diphenylphenylenediamine (DPPD).Nevertheless in these conditions the covalent binding of activated paracetamol metabolites is not affected, suggesting that lipid peroxidation might play a role in the pathogenesis of liver damage following paracetamol overdose.     

Transport of glutathione across the mitochondrial membranes

Transport of glutathione (GSH) into mitochondria was observed when mitochondria in state 4 respiration were incubated with high concentrations of GSH. This transport was suppressed by antimycin A or dicyclohexyl-carbodiimide, or in state 3 respiration. Upon dissipation of the proton gradient by a proton ionophore, mitochondrial GSH was released into the medium. GSH moved freely across the proton-permeated mitochondrial membrane, its movement depending only on the GSH gradient across the inner membrane. These results indicate that there is a transport system for GSH in the mitochondrial membrane, and that a proton gradient is necessary to maintain GSH in the matrix, and to transport GSH into mitochondria.     

Effect of hyperbaric oxygen on experimental acute distal colitis

It has been demonstrated that hyperbaric oxygen (HBO) is useful as an adjunctive therapy for Crohn's disease. However, its effects on ulcerative colitis have not been investigated. In the present study, HBO was tested for acetic acid-induced colitis, and antioxidant systems were evaluated to clarify its possible mode of action. Thirty-six Sprague-Dawley rats were randomly divided into three groups: sham control (Group I), colitis induced by acetic acid without any therapy (Group II), colitis induced by acetic acid and treated with HBO (Group III). HBO was given for 5 days, 2 sessions per day at 2.5-fold absolute atmosphere pressure (ATA) for a period of 90 min in rats in which colitis had been induced (Group III). Rats were sacrificed on the 5th day after the procedure. Superoxide dismutase (SOD), malondialdehyde (MDA) and glutathione peroxidase (GSH Px) activity were measured in the intestinal tissue and erythrocyte lysate. MDA and GSH Px were also determined in the plasma. Whereas MDA levels in erythrocyte, plasma and intestinal tissue were decreased, the levels of GSH Px and SOD were significantly increased in Group III as compared to those of Group II. The results of our study suggest that hyperbaric oxygen therapy has beneficial effects on the course of experimental distal colitis and that antioxidant systems may be involved in its mode of action.     

Oxidative stress and DNA damage induced by a drinking-water chlorination disinfection byproduct 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) in mice

3-Chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), a water chlorine disinfection byproduct, can induce DNA damage (e.g., modification of nucleotides and DNA strand breaks) and subsequent DNA repair in vitro. However, the underlying mechanism(s) how DNA damage is induced by MX is unknown. We hypothesized that MX may cause oxidative stress that leads to DNA damage in vivo. In the present study, we exposed groups of mice to MX at concentrations of 0 (solvent control), 11 (low), 33 (medium) and 99 (high) mg/kg b.w. by single intraperitoneal injection. After treating the mice for 3h, we detected cellular levels of malondialdehyde (MDA) and glutathione (GSH) to assess oxidative stress in the target cells. In addition, we also evaluated DNA damage using single cell gel electrophoresis (SCGE or Comet assay). We found that the levels of DNA damage in all cell types were correlated positively with levels of MDA but negatively with levels of GSH (P<0.05 for all). Also, there were negative correlations between levels of MDA and GSH (r=-0.995 for liver cells, -0.916 for kidney cells, -0.975 for intestine cells, respectively; P<0.05 for all but kidney cells). Our findings suggest that MX may induce DNA damage by the mechanism of causing cellular oxidative stress as measured by increased MDA and decreased GSH, at least in mice.     

Effects of hyperthyroidism induced by L-thyroxin administration on lipid peroxidation in various rat tissues

Thyroid dysfunctions are associated with many pathological signs in the body. One of these is lipid peroxidation that develops due to over- or under-secretion of thyroid hormones. The present study was conducted to determine lipid peroxidation that develops in different tissues including the brain, liver and heart of rats in experimental hyperthyroidism induced by L-thyroxin. The study was carried out on 30 male Sprague-Dawley rats. They were divided into three groups as control, sham hyperthyroidism and hyperthyroidism. Malondialdehyde (MDA) and glutathione (GSH) levels in rat tissues were determined at the end of a 3-weeks period of L-thyroxin administration. It was observed that MDA levels in the hyperthyroidism group were significantly higher in the cerebral cortex, liver and ventriculer tissue of heart (p < 0.001) than in the control and in sham hyperthyroidism groups. GSH levels were higher in the hyperthyroidism group than in control and sham hyperthyroidism groups in all tissues (p < 0.001). Results demonstrate that hyperthyroidism induced by L-thyroxin activates both oxidant and antioxidant systems in cerebral, hepatic and cardiac tissues. However, the increase in antioxidant activity cannot adequately prevent oxidative damage.     

Effects of Bacillus thuringiensis toxin on hepatic lipid peroxidation and free-radical scavengers in rats given alpha-tocopherol or acetylsalicylate

The effect of Dipel (D), a Bacillus thuringiensis-based bioinsecticide, on hepatic antioxidant enzyme activities and lipid peroxidation in rat liver was investigated. Administration of D in a dose of 1 mg/100 g body mass for 4 successive days increased the activities of glutathione peroxidase (GPx), glutathione reductase (GR) and the level of malondialdehyde (MDA) in rat hepatocytes. The activity of superoxide dismutase (SOD) and glutathione (GSH) level were decreased. Administration of D in rats pretreated with alpha-tocopherol (alphaT) or acetylsalicylic acid (ASA) decreased the activities of GPx, GR and MDA levels, while the GSH level was increased compared with rats treated with D alone. The SOD activity was increased in rats pretreated with alphaT before D, but decreased on pretreatment with ASA, compared with rats treated with D alone. The results indicated that D induced oxidative stress in rat liver that has been protected by prior administration of alphaT or ASA.     

外源GSH对盐胁迫下番茄幼苗生长及抗逆生理指标的影响

采用营养液栽培法,研究外源谷胱甘肽(GSH)对NaCl胁迫下番茄幼苗生长、根系活力、电解质渗透率和丙二醛(MDA)、脯氨酸(Pro)、可溶性糖含量以及超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)活性的影响,为利用外源物质减轻盐胁迫伤害提供理论依据。结果显示:(1)NaCl胁迫显著抑制了番茄幼苗的生长、根系活力和SOD、POD、CAT活性,提高了电解质渗透率及MDA、Pro、可溶性糖含量;(2)外源喷施GSH能够诱导NaCl胁迫下番茄幼苗叶片抗氧化酶SOD、POD、CAT活性上调,电解质渗透率及MDA含量下降,Pro和可溶性糖含量恢复至对照水平;(3)外源喷施还原型谷胱甘肽抑制剂(BSO)使NaCl胁迫下番茄幼苗的根系活力以及抗氧化酶SOD、POD、CAT活性下降,脯氨酸含量提高;(4)喷施GSH可诱导BSO和NaCl共处理番茄植株的根系活力、SOD、POD、CAT活性提高,MDA和Pro含量降低。研究表明,外源GSH可通过提高促进盐胁迫下番茄幼苗植株渗透调节能力及清除活性氧的酶促系统的防御能力、降低细胞膜脂过氧化程度、保护膜结构的完整性,从而有效缓解NaCl胁迫对番茄幼苗生长的抑制,提高其耐盐性。     

Acute glutathione depletion restricts mitochondrial ATP export in cerebellar granule neurons

Decreases in GSH pools detected during ischemia sensitize neurons to excitotoxic damage. Thermodynamic analysis predicts that partial GSH depletion will cause an oxidative shift in the thiol redox potential. To investigate the acute bioenergetic consequences, neurons were exposed to monochlorobimane (mBCl), which depletes GSH by forming a fluorescent conjugate. Neurons transfected with redox-sensitive green fluorescent protein showed a positive shift in thiol redox potential synchronous with the formation of the conjugate. Mitochondria within neurons treated with mBCl for 1 h failed to hyperpolarize upon addition of oligomycin to inhibit their ATP synthesis. A decreased ATP turnover was confirmed by monitoring neuronal oxygen consumption in parallel with mitochondrial membrane potential (Deltapsi(m)) and GSH-mBCl formation. mBCl progressively decreased cell respiration, with no effect on mitochondrial proton leak or maximal respiratory capacity, suggesting adequate glycolysis and a functional electron transport chain. This approach to "state 4" could be mimicked by the adenine nucleotide translocator inhibitor bongkrekic acid, which did not further decrease respiration when administered after mBCl. The cellular ATP/ADP ratio was decreased by mBCl, and consistent with mitochondrial ATP export failure, respiration could not respond to an increased cytoplasmic ATP demand by plasma membrane Na(+) cycling; instead, mitochondria depolarized. More prolonged mBCl exposure induced mitochondrial failure, with Deltapsi(m) collapse followed by cytoplasmic Ca(2+) deregulation. The initial bioenergetic consequence of neuronal GSH depletion in this model is thus an inhibition of ATP export, which precedes other forms of mitochondrial dysfunction.     

The effect of chronic diazepam administration on lipid peroxidation and Ca2+ -ATPase activity in rat liver

Chronic administration of diazepam (DZP) caused an increase in malondialdehyde (MDA) levels and a decrease in glutathione (GSH) content. DZP also markedly lowered Ca2+ATPase activity. Treatment with Se plus vitamin E reduced MDA levels and increased GSH content. Our results suggest that, increased lipid peroxidation together with alteration in Ca2+ -ATPase activity may play a role in DZP induced hepatic injury and Se plus vitamin E treatment may contribute to the attenuation of DZP induced hepatotoxicity.     

Toxicity of 1,2-dibromoethane in isolated hepatocytes: Role of lipid peroxidation

Treatment of isolated hepatocytes with 1,2-dibromoethane (DBE) caused a concentration dependent depletion of cellular glutathione (GSH) content and a parallel increase in the covalent binding of reactive intermediates to cell proteins, as a consequence of the haloalkane activation. The reduction of the hepatocyte GSH content, induced by DBE, stimulated the onset of lipid peroxidation, as measured by malondialdehyde (MDA) accumulation. N-Acetylcysteine (1 mM) was found to partially prevent GSH loss and to inhibit MDA formation, whereas equal concentrations of cysteine and methionine were ineffective on these respects. The stimulation of the peroxidative reactions appeared to be also associated with an increase in the leakage of lactate dehydrogenase (LDH) from the cells, indicative of a severe hepatocyte injury. Antioxidants such as -tocopherol, N,N′-phenyl-phenylenediamine (DPPD) and promethazine, as well as N-acetylcysteine reduced MDA formation to various extents and also protect against LDH release, yet without interfering with the covalent binding of DBE reactive intermediates to hepatocyte proteins. These results suggest the involvement of lipid peroxidation, consequent to GSH depletion, in the pathogenesis of liver cell necrosis due to DBE.     

Protective effect of melatonin on contractile activity and oxidative injury induced by ischemia and reperfusion of rat ileum

Free radicals derived from molecular oxygen have been reported to be responsible for changes in motility and mucosal damage observed in intestinal ischemia-reperfusion injury. Melatonin has been considered as an antioxidant that prevents injuries resulted from I/R in various tissues. The present study was designed to determine the effect of melatonin on the contractile responses of acetylcholine (Ach) and KCl, on malondialdehyde (MDA), a product of lipid peroxidation, and reduced glutathione (GSH) levels and to assess histopathological changes in the smooth muscle of terminal ileum subjected to ischemia-reperfusion. The intestinal ischemia-reperfusion was induced by occlusion of superior mesenteric artery of rat for 30 min, followed by a period of reperfusion for 3 h. Melatonin at doses of 10 or 50 mg/kg was administered via the tail vein in 5 min prior to reperfusion. Following reperfusion, segments of terminal ileum were rapidly taken and transferred into isolated organ bath and responses to Ach and KCl were recorded. Samples of terminal ileum were also taken for measuring the MDA and GSH levels. EC50 values of these contracting substances were seriously reduced in the ischemia-reperfusion group compared to that of the sham-operated control group. The decreased contraction response to Ach and KCl was significantly ameliorated by a dosage of 50 mg/kg of melatonin, while not by a dosage of 10 mg/kg. Similar pattern of the effect was observed in the tissue levels of MDA and GSH as well as in histological improvement. Melatonin appeared to be restoring the amounts of tissue MDA and GSH back to about control levels. These results suggest that the high dose of melatonin not only physiologically but also biochemically and morphologically could be useful to normalize contractility injured by oxidative stress in intestinal ischemia-reperfusion.     

Administration of glutathione and lipid peroxidation induced during fasting

It is well known that lipid peroxidation may be initiated or exaggerated by conditions leading to hepatic GSH depletion or altered GSH/GSSG ratio. In our study we evaluated the effects of GSH administration on hepatic, bile and plasma GSH, GSSG and MDA in rats depleted of the tripeptide by a prolonged. fasting. An exteriorized biliary-duodenal fistula was established and GSH or saline solution was administered i.p. for a period of 6h. Rats treated with GSH exhibited an increased GSH and decreased GSSG biliary excretion. Whereas in control rats an opposite pattern was observed, namely enhanced GSSG and decreased GSH biliary excretion. While hepatic GSH and GSSG concentrations were comparable in the two groups, a significant increase in liver and plasma MDA production was found in controls compared to GSH treated rats. Our data suggest a protective role of GSH against the production of lipoperoxidation as evidenced by the decrease of hepatic, biliary and plasma MDA levels and by a decreased percentage of biliary GSSG. In addition, the significant increase of biliary GSH excretion, observed in rats treated with GSH compared to controls, may be due to an increased supply of the tripeptide which is known to be preferentially excreted into bile in the reduced form.