Anticarcinogenic antioxidants as inhibitors against intracellular oxidative stress

Oxidative stress has been implicated in the pathogenesis of numerous diseases, including cancer. In the present study, the protective effect of natural antioxidants, such as quercetin and tea polyphenols, on intracellular oxidative stress was studied. Here we report a novel function of quercetin and tea polyphenols, as potential inhibitors of 4-hydroxy-2-nonenal (HNE)-induced intracellular oxidative stress and cytotoxicity. In rat liver epithelial RL34 cells, a potent electrophile HNE dramatically induced the productions of reactive oxygen species (ROS), which correlated well with the reduction in cell viability. We found that quercetin and tea polyphenols, such as epigallocatechin gallate and theaflavins and their gallate esters, significantly inhibited the HNE-induced ROS production and cytotoxicity. In addition, HNE induced a transient decrease in the mitochondrial membrane potential (Δψ), which was also retarded by the antioxidants. These data suggest that the antioxidants, such as quercetin and tea polyphenols, are inhibitors against mitochondrial ROS production.     

Reduced glutathione content in human sperm is decreased after cryopreservation: Effect of the addition of reduced glutathione to the freezing and thawing extenders

In this study, total glutathione content was determined in human spermatozoa before and after cryopreservation. Total GSH in fresh semen was 4.47 ± 0.46 nmol/10⁸ cells. Following semen cryopreservation, GSH decreased to 1.62 ± 0.13 nmol/10⁸ cells, a 64% reduction (p < 0.01). This decrease in GSH content was associated with a decrease in sperm progressive motility (68% of reduction, p < 0.01). Addition of 1 mM GSH to the freezing extender increased the percentage of total motility and sperm viability. It also modified the motility pattern measured by CASA with changes in the straight-line and average path velocities and wobble of the curvilinear trajectory. Addition of GSH to the freezing media reduced spermatozoa ROS levels and increased the level of sulfhydryl groups on membrane proteins. Nevertheless, no effect of GSH addition on lipid membrane disorder or chromatin condensation was detected. Addition of 1 or 5 mM GSH to the thawing media increased the percentage of motile and progressively motile spermatozoa, but no effect on viability was detected. In conclusion, the antioxidant defensive capacity of the GSH is severely altered by the freeze–thawing process. The addition of GSH to the freezing and thawing extender could be of partial and limited benefit in improving the function of frozen human spermatozoa.     

Enhanced sensitivity to oxidative stress in transgenic tobacco plants with decreased glutathione reductase activity leads to a decrease in ascorbate pool and ascorbate redox state

To investigate the possible mechanisms of glutathione reductase (GR) in protecting against oxidative stress, we obtained transgenic tobacco (Nicotiana tabacum) plants with 30–70% decreased GR activity by using a gene encoding tobacco chloroplastic GR for the RNAi construct. We investigated the responses of wild type and transgenic plants to oxidative stress induced by application of methyl viologen in vivo. Analyses of CO₂ assimilation, maximal efficiency of photosystem II photochemistry, leaf bleaching, and oxidative damage to lipids demonstrated that transgenic plants exhibited enhanced sensitivity to oxidative stress. Under oxidative stress, there was a greater decrease in reduced to oxidized glutathione ratio but a greater increase in reduced glutathione in transgenic plants than in wild type plants. In addition, transgenic plants showed a greater decrease in reduced ascorbate and reduced to oxidized ascorbate ratio than wild type plants. However, there were neither differences in the levels of NADP and NADPH and in the total foliar activities of monodehydroascorbate reductase and dehydroascorbate reductase between wild type and transgenic plant. MV treatment induced an increase in the activities of GR, ascorbate peroxidase, superoxide dismutase, and catalase. Furthermore, accumulation of H₂O₂ in chloroplasts was observed in transgenic plants but not in wild type plants. Our results suggest that capacity for regeneration of glutathione by GR plays an important role in protecting against oxidative stress by maintaining ascorbate pool and ascorbate redox state.     

A short review on the role of glutathione in the response of yeasts to nutritional, environmental, and oxidative stresses

Glutathione (L-γ-Glutamyl-L-Cysteinylglycine) appears as the major nonprotein thiol compound in yeasts. Recent advances have shown that glutathione (GSH) seems to be involved in the response of yeasts to different nutritional and oxidative stresses. When the yeast Saccharomyces cerevisiae is starved for sulfur or nitrogen nutrients, GSH may be mobilized to ensure cellular maintenance. Glutathione S-transferases may be involved in the detoxification of electrophilic xenobiotics. Vacuolar transport of metal derivatives of GSH ensure resistance to metal stress. Growth of methylotrophic yeasts on methanol results in the formation of an excess formaldehyde that is detoxified by a GSH-dependent formaldehyde dehydrogenase. Growth of yeasts on glycerol results in the accumulation of methylglyoxal detoxified by the glyoxalase pathway. Glutathione per se can react with oxidative agents or is involved in the oxidative stress response through glutathione peroxidase.     

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.     

Variable regulation of glutamate cysteine ligase subunit proteins affects glutathione biosynthesis in response to oxidative stress

Glutamate cysteine ligase (GCL), composed of a catalytic (GCLC) and modulatory (GCLM) subunit, catalyzes the first step of glutathione (GSH) biosynthesis. Using 4-hydroxy-2-nonenal (4HNE), 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), and tertiary-butylhydroquinone (tBHQ) as models of oxidative stress which are known to work through different mechanisms, we measured changes in cellular GSH, GCL mRNA, and GCL protein. 4HNE and tBHQ treatments increased cellular GSH levels, while DMNQ exposure depleted GSH. Furthermore, changes in the two GCL mRNAs largely paralleled changes in the GCL proteins; however, the magnitudes differed, suggesting some form of translational control. The molar ratio of GCLC:GCLM ranged from 3:1 to 17:1 in control human bronchial epithelial (HBE1) cells and all treatments further increased this ratio. Data from several mouse tissues show molar ratios of GCLC:GCLM that range from 1:1 to 10:1 in support of these findings. These data demonstrate that alterations in cellular GSH are clearly correlated with GCLC to a greater extent than GCLM. Surprisingly, both control HBE1 cells and some mouse tissues have more GCLC than GCLM and GCLM increases to a much lesser extent than GCLC, suggesting that the regulatory role of GCLM is minimal under physiologically relevant conditions of oxidative stress.     

Monitoring the in vivo redox state of plant mitochondria: Effect of respiratory inhibitors, abiotic stress and assessment of recovery from oxidative challenge

In animals, the impact of ROS production by mitochondria on cell physiology, death, disease and ageing is well recognised. In photosynthetic organisms such as higher plants, however, the chloroplast and peroxisomes are the major sources of ROS during normal metabolism and the importance of mitochondria in oxidative stress and redox signalling is less well established. To address this, the in vivo oxidation state of a mitochondrially-targeted redox-sensitive GFP (mt-roGFP2) was investigated in Arabidopsis leaves. Classical ROS-generating inhibitors of mitochondrial electron transport (rotenone, antimycin A and SHAM) had no effect on mt-roGFP oxidation when used singly, but combined inhibition of complex III and alternative oxidase by antimycin A and SHAM did cause significant oxidation. Inhibitors of complex IV and aconitase also caused oxidation of mt-roGFP2. This oxidation was not apparent in the cytosol whereas antimycin A + SHAM also caused oxidation of cytosolic roGFP2. Menadione had a much greater effect than the inhibitors, causing nearly complete oxidation of roGFP2 in both mitochondria and cytosol. A range of severe abiotic stress treatments (heat, salt, and heavy metal stress) led to oxidation of mt-roGFP2 while hyperosmotic stress had no effect and low temperature caused a slight but significant decrease in oxidation. Similar changes were observed for cytosolic roGFP2. Finally, the recovery of oxidation state of roGFP in mitochondria after oxidation by H₂O₂ treatment was dramatically slower than that of either the cytosol or chloroplast. Together, the results highlight the sensitivity of the mitochondrion to redox perturbation and suggest a potential role in sensing and signalling cellular redox challenge.     

Reduced expression of GSTM2 and increased oxidative stress in spontaneously hypertensive rat

Human essential hypertension is a complex polygenic trait with underlying genetic components that remain unknown. The spontaneously hypertensive rat (SHR) is a well-characterized experimental model for essential hypertension. By comparative proteomics, we previously identified glutathione S-transferase, mu 2 (GSTM2), a protein involved in detoxification of reactive oxygen species, which had a significant reduction in left ventricles of 16-week-old SHR compared with WKY rats. In parallel, Western blotting and RT-PCR showed a similar reduction of GSTM2 in left ventricles and aortas of 4-, 8-, and 16-week-old SHR, which is before the onset of hypertension. This suggests that differential expression is not attributable to long-term changes in blood pressure. Meanwhile, the activities of GSTM2 were significantly decreased in different ages old SHR. Conversely, there was an enhanced generation of superoxide anion and activation of NADPH oxidase in SHR, which was accompanied by an increase in the protein expression of p47phox, a subunit of NADPH oxidase. These data suggest that it maybe a reduction in antioxidant defenses, evident by a reduced expression and activity of GSTM2, in the left ventricles and aortas of SHR that leads to increased levels of superoxide anion and activation of NADPH oxidase.     

Role of respiration and glutathione in cadmium-induced oxidative stress in Escherichia coli K-12

Cadmium is a widespread pollutant that has been associated with oxidative stress, but the mechanism behind this effect in prokaryotes is still unclear. In this work, we exposed two glutathione deficient mutants (ΔgshA and ΔgshB) and one respiration deficient mutant (ΔubiE) to a sublethal concentration of cadmium. The glutathione mutants show a similar increase in reactive oxygen species as the wild type. Experiments performed using the ΔubiE strain showed that this mutant is more resistant to cadmium ions and that Cd-induced reactive oxygen species levels were not altered. In the light of these facts, we conclude that the interference of cadmium with the respiratory chain is the cause of the oxidative stress induced by this metal and that, contrary to previously proposed models, the reactive oxygen species increase is not due to glutathione depletion, although this peptide is crucial for cadmium detoxification.     

Cu/Zn-superoxide dismutase and glutathione are involved in response to oxidative stress induced by protein denaturing effect of alachlor in Saccharomyces cerevisiae

Alachlor is a widely used pre-emergent chloroacetanilide herbicide which has been shown to have many harmful ecological and environmental effects. However, the mechanism of alachlor-induced oxidative stress is poorly understood. We found that, in Saccharomyces cerevisiae, the intracellular levels of reactive oxygen species (ROS) including superoxide anions were increased only after long-term exposure to alachlor, suggesting that alachlor is not a pro-oxidant. It is likely that alachlor-induced oxidative stress may result from protein denaturation because alachlor rapidly induced an increased protein aggregation, leading to upregulation of SSA4 and HSP82 genes encoding heat shock proteins (Hsp) of Hsp70 and Hsp90 family, respectively. Although only SOD1 encoding Cu/Zn-superoxide dismutase (SOD), but not SOD2 encoding Mn-SOD, is essential for alachlor tolerance, both SODs play a crucial role in reducing alachlor-induced ROS. We found that, after alachlor exposure, glutathione production was inhibited while its utilization was increased, suggesting the role of glutathione in protecting cells against alachlor, which becomes more important when lacking Cu/Zn-SOD. Based on our results, it seems that alachlor primarily causes damages to cellular macromolecules such as proteins, leading to an induction of endogenous oxidative stress, of which intracellular antioxidant defense systems are required for elimination.     

Adaptation to oxidative stress and impact of chronic oxidative stress on immunity in heat-stressed broilers

1. Glutathione peroxidase activity and serum malondialdehyde of heat-stressed broilers were increased in the early period of heat exposure, and then these parameters decreased.

2. The lesion scores of bursa of Fabricius in heat-stressed broilers were increased and decreased in accordance with the activity of glutathione peroxidase and serum malondialdehyde.

3. High environmental temperature had not affected relative bursa of Fabricius weight and NDV-HI titer of heat-stressed broilers.

4. We concluded that heat-stressed broilers could adapt to oxidative stress, and environmental temperature set at 38±2 °C had not affected humoral immunity.

Do invading leucocytes contribute to the decrease in glutathione concentrations indicating oxidative stress in exercised muscle, or are they important for its recovery?

Mice were subjected to one session of strenuous running exercise and their soleus muscles were examined in respect of changes in ultrastructure and to their concentration of reduced glutathione [GSH] which are indicators of oxidative stress. It was hypothesized that invading leucocytes contributed to oxidative stress and they were functionally inhibited in one experimental group by the administration of colchicine. Exercise led to an immediate decrease in [GSH] of about 60%, which slowly recovered during 96 h after exercise. With the administration of colchicine after exercise, [GSH] was higher than in the untreated exercise group 48 h after exercise, indicating an inhibition of the ability of leucocytes to produce oxidative stress. However, at 96 h after exercise, [GSH] was lower in the treated exercise group than in the untreated group. The morphological evaluation of the percentage of affected fibres showed that the invasion of leucocytes increased muscle fibre damage. The results suggested that invading leucocytes enhanced production of reactive species of oxygen that may have participated in inducing muscle damage. However, inhibition of leucocyte invasion did not permit their scavenger action of removing cell debris, which appeared to produce even more oxidative stress in the muscle.     

Evaluation of a dithiocarbamate derivative as a model of thiol oxidative stress in H9c2 rat cardiomyocytes

Thiol redox state (TRS) refers to the balance between reduced thiols and their corresponding disulfides and is mainly reflected by the ratio of reduced and oxidized glutathione (GSH/GSSG). A decrease in GSH/GSSG, which reflects a state of thiol oxidative stress, as well as thiol modifications such as S-glutathionylation, has been shown to have important implications in a variety of cardiovascular diseases. Therefore, research models for inducing thiol oxidative stress are important tools for studying the pathophysiology of these disease states as well as examining the impact of pharmacological interventions on thiol pathways. The purpose of this study was to evaluate the use of a dithiocarbamate derivative, 2-acetylamino-3-[4-(2-acetylamino-2-carboxyethylsulfanylthiocarbonylamino)phenylthiocarbamoylsulfanyl]propionic acid (2-AAPA), as a pharmacological model of thiol oxidative stress by examining the extent of thiol modifications induced in H9c2 rat cardiomyocytes and its impact on cellular functions. The extent of thiol oxidative stress produced by 2-AAPA was also compared to other models of oxidative stress including hydrogen peroxide (H₂O₂), diamide, buthionine sulfoximine, and N,N׳-bis(2-chloroethyl)-N-nitroso-urea. Results indicated that 2-AAPA effectively inhibited glutathione reductase and thioredoxin reductase activities and decreased the GSH/GSSG ratio by causing a significant accumulation of GSSG. 2-AAPA also increased the formation of protein disulfides as well as S-glutathionylation. The alteration in TRS led to a loss of mitochondrial membrane potential, release of cytochrome c, and increase in reactive oxygen species production. Compared to other models, 2-AAPA is more potent at creating a state of thiol oxidative stress with lower cytotoxicity, higher specificity, and more pharmacological relevance, and could be utilized as a research tool to study TRS-related normal and abnormal biochemical processes in cardiovascular diseases.     

Ameliorating effects of troxerutin on nickel-induced oxidative stress in rats

Abstract

Objective

This study investigates the effects of troxerutin on nickel (Ni)-induced oxidative stress in rats.

Methods

Nickel as nickel sulfate (20 mg/kg body weight (b.w.)) was administered intraperitoneally for 20 days to induce toxicity in the subject rats. The levels of stress markers AST, ALT, ALP, LDH, and GGT in the hepatic tissue were significantly increased while a decrease in the levels of enzymic and non-enzymic antioxidants was observed in Ni intoxicated rats.

Results

Oral administration of troxerutin along with Ni for 20 days in a dose-dependent manner significantly reverted the stress markers in the liver tissue to near normal level. Troxerutin exhibited significant protection at 100 mg/kg b.w. Histopathological studies also supported the above findings.

Conclusions

Thus, we conclude that troxerutin preserved the histo-architecture and ameliorated stress markers in the liver tissue of Ni-intoxicated rats.