TNP-470 Inhibits Oxidative Stress,Nitric Oxide Production and Nuclear Factor Kappa B Activation in a Rat Model of Hepatocellular Carcinoma

The objective of this study is to determine if treatment with the angiogenesis inhibitor TNP-470 results in impairment of oxidative stress, inhibition of nuclear factor kappa B (NF-κB) activation and decrease of nitric oxide production in an experimental model of rat hepatocarcinogenesis. Tumour was induced by diethylnitrosamine and promoted by two-thirds hepatectomy plus acetaminofluorene administration. Experiments were carried out at 28 weeks after initiating the treatment. TNP-470 was administered at 30 mg/kg, three times per week from 20 to 28 weeks. Carcinomatous tissue growing outside dysplastic nodules and a marked expression of placental glutathione S-transferase were detected in rats with induced carcinogenesis. Liver concentrations of thiobarbituric acid reactive substances, reduced glutathione (GSH) and glutathione disulfide (GSSG) were significantly higher than those of controls and there was a significant increase in the GSSG/GSH ratio. Tumour growth was accompanied by augmented expression of inducible nitric oxide synthase, activation of (NF-κB) and proteolysis of IkappaB. All these effects were absent in animals receiving TNP-470. Our results indicate that TNP-470 inhibits oxidative stress, nitric oxide production and NF-κB activation induced by experimental hepatocarcinogenesis. These changes would contribute to the beneficial effects of TNP-470 in cancer treatment.     

TNP-470 inhibits oxidative stress, nitric oxide production and nuclear factor kappa B activation in a rat model of hepatocellular carcinoma

The objective of this study is to determine if treatment with the angiogenesis inhibitor TNP-470 results in impairment of oxidative stress, inhibition of nuclear factor kappa B (NF-κB) activation and decrease of nitric oxide production in an experimental model of rat hepatocarcinogenesis. Tumour was induced by diethylnitrosamine and promoted by two-thirds hepatectomy plus acetaminofluorene administration. Experiments were carried out at 28 weeks after initiating the treatment. TNP-470 was administered at 30 mg/kg, three times per week from 20 to 28 weeks. Carcinomatous tissue growing outside dysplastic nodules and a marked expression of placental glutathione S-transferase were detected in rats with induced carcinogenesis. Liver concentrations of thiobarbituric acid reactive substances, reduced glutathione (GSH) and glutathione disulfide (GSSG) were significantly higher than those of controls and there was a significant increase in the GSSG/GSH ratio. Tumour growth was accompanied by augmented expression of inducible nitric oxide synthase, activation of (NF-κB) and proteolysis of IkappaB. All these effects were absent in animals receiving TNP-470. Our results indicate that TNP-470 inhibits oxidative stress, nitric oxide production and NF-κB activation induced by experimental hepatocarcinogenesis. These changes would contribute to the beneficial effects of TNP-470 in cancer treatment.     

Hepatic response to the oxidative stress induced by E. coli endotoxin: Glutathione as an index of the acute phase during the endotoxic shock

Reactive oxygen species are important mediators of cellular damage during endotoxic shock. In order to investigate the hepatic response to the oxidative stress induced by endotoxin, hepatic and plasma glutathione (total, GSH and GSSG), GSSG/GSH ratio as well as Mn-superoxide dismutase and catalase activities were determined during the acute and recovery phases of reversible endotoxic shock in the rat. A significant increase in liver and plasma total glutathione content was observed 5 h after endotoxin treatment (acute phase), followed by a diminution of these parameters below control values at 48 h (recovery phase). The significant increases of GSSG levels and GSSG/GSH ratio are indicative of oxidative stress occurring during the acute phase. Liver Mn-SOD activity showed a similar time dependency as the GSSG/GSH ratio; however, a marked decrease in the liver catalase activity was observed during the process. These results indicate the participation of liver glutathione in the response to endotoxin and the possible use of plasma glutathione levels and GSSG/GSH ratio as indicators of the acute phase during the endotoxic process. (Mol Cell Biochem 159: 115-121, 1996)     

Effects of Reduced and Oxidized Glutathione on Sphingomyelinase Activity and Contents of Sphingomyelin and Lipid Peroxidation Products in Murine Liver

Like the phosphatidyl inositol cycle, the sphingomyelin cycle produces a series of the secondary messengers transmitting extracellular signals from the cytoplasmic membrane into the nucleus. Sphingomyelin, ceramide, sphingosine, sphingomyelinase, and ceramidase are the main components of the sphingomyelin cycle. In spite of numerous data on the functional properties of sphingomyelin cycle products, the activation mechanism for the key enzyme of the sphingomyelin cycle, sphingomyelinase (SMase), is not well understood. We have discovered effects of both reduced (GSH) and oxidized (GSSG) glutathione on the activity of neutral SMase in animals. GSH administration (18 mg per mouse) inhibits this enzymatic activity in liver for 2 h and increases the sphingomyelin level exactly as occurs in cell culture. The levels of diene conjugates and ketodienes decrease simultaneously during the experiment, thus indicating the ability of GSH to suppress oxidative processes in the cell. GSSG administration (18 mg per mouse) has no effect on the SMase activity during the first 15 min, but increases it twofold after 1 h. A short-term decrease in this activity after 30 min may depend on the conversion of excess GSSG into its reduced form by glutathione reductase. Unlike GSH, GSSG has no effect on the level of ketodienes after 1 h, but it induces the accumulation of diene conjugates. A strong correlation exists between the changes in SMase activity and in the level of oxidation products caused by either GSH or GSSG. These data indicate a relationship between SMase activity and the level of peroxidation products and possibly a relation between two signaling systems: the sphingomyelin cycle and the oxidative system.     

Effects of acute aerobic and anaerobic exercise on blood markers of oxidative stress

The purpose of this study was to compare oxidative modification of blood proteins, lipids, DNA, and glutathione in the 24 hours following aerobic and anaerobic exercise using similar muscle groups. Ten cross-trained men (24.3 +/- 3.8 years, [mean +/- SEM]) performed in random order 30 minutes of continuous cycling at 70% of Vo(2)max and intermittent dumbbell squatting at 70% of 1 repetition maximum (1RM), separated by 1-2 weeks, in a crossover design. Blood samples taken before, and immediately, 1, 6, and 24 hours postexercise were analyzed for plasma protein carbonyls (PC), plasma malondialdehyde (MDA), and whole-blood total (TGSH), oxidized (GSSG), and reduced (GSH) glutathione. Blood samples taken before and 24 hours postexercise were analyzed for serum 8-hydroxy-2'-deoxyguanosine (8-OHdG). PC values were greater at 6 and 24 hours postexercise compared with pre-exercise for squatting, with greater PC values at 24 hours postexercise for squatting compared with cycling (0.634 +/- 0.053 vs. 0.359 +/- 0.018 nM.mg protein(-1)). There was no significant interaction or main effects for MDA or 8-OHdG. GSSG experienced a short-lived increase and GSH a transient decrease immediately following both exercise modes. These data suggest that 30 minutes of aerobic and anaerobic exercise performed by young, cross-trained men (a) can increase certain biomarkers of oxidative stress in blood, (b) differentially affect oxidative stress biomarkers, and (c) result in a different magnitude of oxidation based on the macromolecule studied. Practical applications: While protein and glutathione oxidation was increased following acute exercise as performed in this study, future research may investigate methods of reducing macromolecule oxidation, possibly through the use of antioxidant therapy.     

Glutathione and antioxidant enzymes in skeletal muscle: effects of fiber type and exercise intensity.

Glutathione status and antioxidant enzymes in various types of rat skeletal muscle were studied after an acute bout of exercise (Ex) at different intensities. Glutathione (GSH) and glutathione disulfide (GSSG) concentrations were the highest in soleus (SO) muscle, followed by those in deep (DVL) and then superficial (SVL) portions of vastus lateralis. In DVL, but not in SO or SVL, muscle GSH increased proportionally with Ex intensity and reached 1.8 +/- 0.08 mumol/g wet wt compared with 1.5 +/- 0.03 (P < 0.05) in resting controls (R). GSSG in DVL was increased from 0.10 +/- 0.01 mumol/g wet wt in R to 0.14 +/- 0.01 (P < 0.05) after Ex. Total glutathione (GSH + GSSG) contents in DVL were also significantly elevated with Ex, whereas GSH/GSSG ratio was unchanged. Activities of GSH peroxidase (GPX), GSSG reductase (GR), and catalase (CAT) were significantly higher in SO than in DVL and SVL, but there was no difference in superoxide dismutase activity between the three muscle types. Furthermore, Ex at moderate intensities elicited significant increases in GPX, GR, and CAT activities in DVL muscle. None of the antioxidant enzymes was affected by exercise in SO. It is concluded that rat DVL muscle is particularly vulnerable to exercise-induced free radical damage and that a disturbance of muscle GSH status is indicative of an oxidative stress.     

Increased lymphocyte antioxidant defences in response to exhaustive exercise do not prevent oxidative damage

It has been reported that exercise induces oxidative stress and causes adaptations in antioxidant defences. The aim of this study was to determine the adaptations of lymphocytes to the oxidative stress induced by an exhaustive exercise. Nine voluntary male subjects participated in the study. The exercise was a cycling mountain stage (171.8 km), and the cyclists took a mean of 283 min to complete it. Blood samples were taken the morning of the cycling stage day, after overnight fasting, and 3 h after finishing the stage. We determined the blood glutathione redox status (GSSG/GSH), lymphocyte antioxidant enzyme activities and superoxide dismutase (SOD) levels; the plasma and lymphocyte vitamin E levels; the serum lactate dehydrogenase (LDH) and creatine kinase (CK) activities and urate levels; the plasma carotene and malonaldehyde (MDA) levels; and the lymphocyte carbonyl index. The cycling stage induced significant increases in blood-oxidized (glutathione/GSSG), plasma MDA and serum urate levels. The exercise also produced increases in CK and LDH serum activities. The mountain cycling stage induced significant increases in lymphocyte vitamin E levels, glutathione peroxidase and glutathione reductase activities as well as increased SOD activity and protein levels. The protein carbonyl levels increased significantly in lymphocytes after the stage. In conclusion, in spite of increasing antioxidant defences in response to the oxidative stress induced by the exhaustive exercise, lymphocyte oxidative damage was produced after the stage as demonstrated by the increased carbonyl index even in very well trained athletes.     

Accumulation of glutathione disulfide mediates NF-kappaB activation during immune stimulation with CpG DNA

Innate immune cells recognize pathogens by detecting molecular patterns that are distinct from those of the host. One such pattern is unmethylated CpG dinucleotides, which are common in bacterial DNA but not in vertebrate genomes. Macrophages respond to such CpG motifs in bacterial DNA or synthetic oligodeoxynucleotides (ODN) by inducing NF-kappaB and secreting proinflammatory cytokines, such as interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha), but the mechanisms regulating this have been unclear. CpG ODN-stimulated cells produce reactive oxygen species (ROS) and have a decreased ratio of intracellular glutathione/glutathione disulfide (GSH/GSSG), indicating a shift to a more oxidized intracellular redox state. To determine whether this may play a role in mediating the CpG-induced macrophage activation, the GSH/GSSG redox state was manipulated in the murine macrophagelike cell line RAW264.7. Treatment of cells with BCNU to inhibit glutathione reductase (GR) enhanced the CpG-induced intracellular oxidation and decreased the GSH/GSSG, with increased activation of NF-kappaB and a doubling in the CpG-induced production of IL-6 and TNF-alpha. Experimental manipulation of the intracellular GSSG concentration during inhibition of cellular prooxidant production demonstrated that increased intracellular GSSG is a primary signal that is directly or indirectly required for CpG-induced NF-kappaB activation but is not in itself sufficient to trigger this in the absence of CpG ODN. These data suggest the existence of a second CpG-induced intracellular signal, independent of GSSG, mediating the activation of innate immunity by bacterial DNA.     

Glutaredoxin 2 catalyzes the reversible oxidation and glutathionylation of mitochondrial membrane thiol proteins: implications for mitochondrial redox regulation and antioxidant DEFENSE

The redox poise of the mitochondrial glutathione pool is central in the response of mitochondria to oxidative damage and redox signaling, but the mechanisms are uncertain. One possibility is that the oxidation of glutathione (GSH) to glutathione disulfide (GSSG) and the consequent change in the GSH/GSSG ratio causes protein thiols to change their redox state, enabling protein function to respond reversibly to redox signals and oxidative damage. However, little is known about the interplay between the mitochondrial glutathione pool and protein thiols. Therefore we investigated how physiological GSH/GSSG ratios affected the redox state of mitochondrial membrane protein thiols. Exposure to oxidized GSH/GSSG ratios led to the reversible oxidation of reactive protein thiols by thiol-disulfide exchange, the extent of which was dependent on the GSH/GSSG ratio. There was an initial rapid phase of protein thiol oxidation, followed by gradual oxidation over 30 min. A large number of mitochondrial proteins contain reactive thiols and most of these formed intraprotein disulfides upon oxidation by GSSG; however, a small number formed persistent mixed disulfides with glutathione. Both protein disulfide formation and glutathionylation were catalyzed by the mitochondrial thiol transferase glutaredoxin 2 (Grx2), as were protein deglutathionylation and the reduction of protein disulfides by GSH. Complex I was the most prominent protein that was persistently glutathionylated by GSSG in the presence of Grx2. Maintenance of complex I with an oxidized GSH/GSSG ratio led to a dramatic loss of activity, suggesting that oxidation of the mitochondrial glutathione pool may contribute to the selective complex I inactivation seen in Parkinson's disease. Most significantly, Grx2 catalyzed reversible protein glutathionylation/deglutathionylation over a wide range of GSH/GSSG ratios, from the reduced levels accessible under redox signaling to oxidized ratios only found under severe oxidative stress. Our findings indicate that Grx2 plays a central role in the response of mitochondria to both redox signals and oxidative stress by facilitating the interplay between the mitochondrial glutathione pool and protein thiols.     

Effect of acute exercise on glutathione deficient heart

The role of glutathione (GSH) in myocardial antioxidant defense was investigated in Swiss-Webster mice either performing swim exercise to exhaustion or rested in both the GSH adequate (GSH-A) and GSH deficient (GSH-D) states. GSH deficiency was accomplished by injecting mice with L-buthionine [S,R]sulfoximine (BSO; 2 nmol/kg body wt, i.p.) and providing BSO (20 mM) in drinking water for 12 days. GSH and glutathione disulfide (GSSG) contents in the GSH-D hearts were decreased to 10 and 8%, respectively, of those in the GSH-A mice. This decrease was associated with a significant decline of the total glutathione level in the liver, skeletal muscle and plasma. Myocardial GSH peroxidase and GSH sulfur-transferase activities decreased significantly following GSH deficiency, whereas superoxide dismutase activity was significantly elevated. GSH deficiency did not affect exercise endurance performance. However, exhaustive exercise decreased GSH content in the myocardium of the GSH-A and GSH-D mice by 22 and 44% (p < 0.05), respectively. The GSH:GSSG ratio was not altered significantly following exercise because of a concomitant decrease in GSSG (p < 0.05). -Glutamyltranspeptidase activity was significantly increased after exercise, especially in the GSH-D hearts (72%; p < 0.05). GSH content after exercise correlated negatively with exercise time in both GSH-A and GSH-D mice (p < 0.05). These data indicate that GSH is actively used in the myocardium during prolonged exercise at moderate intensity and that GSH deficiency is tolerated by the heart, possibly compensated for by an increased GSH uptake from the plasma.     

Short-term exercise worsens cardiac oxidative stress and fibrosis in 8-month-old db/db mice by depleting cardiac glutathione

Abstract

Moderate exercise improves cardiac antioxidant status in young humans and animals with Type-2 diabetes (T2D). Given that both diabetes and advancing age synergistically decrease antioxidant expression in most tissues, it is unclear whether exercise can upregulate cardiac antioxidants in chronic animal models of T2D. To this end, 8-month-old T2D and normoglycemic mice were exercised for 3 weeks, and cardiac redox status was evaluated. As expected, moderate exercise increased cardiac antioxidants and attenuated oxidative damage in normoglycemic mice. In contrast, similar exercise protocol in 8-month-old db/db mice worsened cardiac oxidative damage, which was associated with a specific dysregulation of glutathione (GSH) homeostasis. Expression of enzymes for GSH biosynthesis [γ-glutamylcysteine synthase, glutathione reductase] as well as for GSH-mediated detoxification (glutathione peroxidase, glutathione-S-transferase) was lower, while toxic metabolites dependent on GSH for clearance (4-hydroxynonenal) were increased in exercised diabetic mice hearts. To validate GSH loss as an important factor for such aggravated damage, daily administration of GSH restored cardiac GSH levels in exercised diabetic mice. Such supplementation attenuated both oxidative damage and fibrotic changes in the myocardium. Expression of transforming growth factor beta (TGF-β) and its regulated genes which are responsible for such profibrotic changes were also attenuated with GSH supplementation. These novel findings in a long-term T2D animal model demonstrate that short-term exercise by itself can deplete cardiac GSH and aggravate cardiac oxidative stress. As GSH administration conferred protection in 8-month-old diabetic mice undergoing exercise, supplementation with GSH-enhancing agents may be beneficial in elderly diabetic patients undergoing exercise.     

Chronic iron overload enhances inducible nitric oxide synthase expression in rat liver.

Iron is an essential micronutrient promoting oxidative stress in the liver of overloaded animals and human, which may trigger the expression of redox-sensitive genes. We have tested the hypothesis that chronic iron overload (CIO) enhances inducible nitric oxide synthase (iNOS) expression in rat liver by extracellular signal-regulated kinase (ERK1/2) and NF-kappaB activation. CIO (diet enriched with 3%(wt/wt) carbonyl-iron for 12 weeks) increased liver protein carbonylation and decreased reduced glutathione (GSH) content and the GSH/GSSG ratio after 6 weeks, parameters that are normalized after 8-12 weeks of treatment. These changes are paralleled by higher phosphorylated-ERK1/2 to non-phosphorylated-ERK1/2 ratios at 6 and 8 weeks, increased NF-kappaB DNA binding to the iNOS gene promoter at 8-12 weeks, and higher iNOS mRNA expression and activity at 8 and 12 weeks. It is concluded that CIO triggers liver oxidative stress at early times, with upregulation of iNOS expression involving the ERK/NF-kappaB pathway at later times, a finding that may represent a hepatoprotective mechanism against CIO toxicity in addition to the recovery of GSH homeostasis.     

Seizure-induced changes in mitochondrial redox status

The aim of this study was to determine seizure-induced oxidative stress by measuring hippocampal glutathione (GSH) and glutathione disulfide (GSSG) levels in tissue and mitochondria. Kainate-induced status epilepticus (SE) in rats resulted in a time-dependent decrease of GSH/GSSG ratios in both hippocampal tissue and mitochondria. However, changes in GSH/GSSG ratios were more dramatic in the mitochondrial fractions compared to hippocampal tissue. This was accompanied by a mild increase in glutathione peroxidase activity and a decrease in glutathione reductase activity in hippocampal tissue and mitochondria, respectively. Since coenzyme A (CoASH) and its disulfide with GSH (CoASSG) are primarily compartmentalized within mitochondria, their measurement in tissue was undertaken to overcome problems associated with GSH/GSSG measurement following subcellular fractionation. Hippocampal tissue CoASH/CoASSG ratios were decreased following kainate-induced SE, the time course and magnitude of change paralleling mitochondrial GSH/GSSG levels. Cysteine, a rate-limiting precursor of glutathione was decreased following kainate administration in both hippocampal tissue and mitochondrial fractions. Together these changes in altered redox status provide further evidence for seizure-induced mitochondrial oxidative stress.     

Acrolein causes inhibitor kappaB-independent decreases in nuclear factor kappaB activation in human lung adenocarcinoma (A549) cells

Acrolein is a highly electrophilic alpha,beta-unsaturated aldehyde to which humans are exposed in various situations. In the present study, the effects of sublethal doses of acrolein on nuclear factor kappaB (NF-kappaB) activation in A549 human lung adenocarcinoma cells were investigated. Immediately following a 30-min exposure to 45 fmol of acrolein/cell, glutathione (GSH) and DNA synthesis and NF-kappaB binding were reduced by more than 80%. All parameters returned to normal or supranormal levels by 8 h post-treatment. Pretreatment with acrolein completely blocked 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced activation of NF-kappaB. Cells treated for 1 h with 1 mM diethyl maleate (DEM) showed a 34 and 53% decrease in GSH and DNA synthesis, respectively. DEM also reduced NF-kappaB activation by 64% at 2 h post-treatment, with recovery to within 22% of control at 8 h. Both acrolein and DEM decreased NF-kappaB function approximately 50% at 2 h after treatment with TPA, as shown by a secreted alkaline phosphatase reporter assay. GSH returned to control levels by 8 h after DEM treatment, but proliferation remained significantly depressed for 24 h. Interestingly, DEM caused a profound decrease in NF-kappaB binding, even at doses as low as 0.125 mM that had little effect on GSH. Neither acrolein nor DEM had any effect on the levels of phosphorylated or nonphosphorylated inhibitor kappaB-alpha (IkappaB-alpha). Furthermore, acrolein decreased NF-kappaB activation in cells depleted of IkappaB-alpha by TPA stimulation in the presence of cycloheximide, demonstrating that the decrease in NF-kappaB activation was not the result of increased binding by the inhibitory protein. This conclusion was further supported by the finding that acrolein modified NF-kappaB in the cytosol prior to chemical dissociation from IkappaB with detergent. Together, these data support the conclusion that the inhibition of NF-kappaB activation by acrolein and DEM is IkappaB-independent. The mechanism appears to be related to direct modification of thiol groups in the NF-kappaB subunits.     

Protective effect of silymarin on oxidative stress in rat brain

Brain is susceptible to oxidative stress and it is associated with age-related brain dysfunction. Previously, we have pointed out a dramatic decrease of glutathione levels in the rat brain after acetaminophen (APAP) oral administration overdose. Silymarin (SM) is a mixture of bioactive flavonolignans isolated from Silybum marianum (L.) Gaertn., employed usually in the treatment of alcoholic liver disease and as anti-hepatotoxic agent in humans. In this study, we have evaluated the effect of SM on enzymatic and non enzymatic antioxidant defensive systems in rat brain after APAP-induced damage. Male albino Wistar rats were treated with SM (200 mg/kg/die orally) for three days, or with APAP single oral administration (3 g/kg) or with SM (200 mg/kg/die orally) for 3 days and APAP single oral administration (3 g/kg) at third day. Successively the following parameters were measured: reduced and oxidized glutathione (GSH and GSSG), ascorbic acid (AA), enzymatic activity variations of superoxide dismutase (SOD) and malondialdehyde levels (MDA). Our results showed a significant decrease of GSH levels, AA levels and SOD activity and an increase of MDA and GSSG levels after APAP administration. After SM administration GSH and AA significantly increase and SOD activity was significantly enhanced. In the SM+APAP group, GSH values significantly increase and the others parameters remained unchanged respect to control values. These results suggest that SM may to protect the SNC by oxidative damage for its ability to prevent lipid peroxidation and replenishing the GSH levels.