Role of antioxidants in protecting cellular DNA from damage by oxidative stress

We have previously derived 2 V79 clones resistant to menadione (Md1 cells) and cadmium (Cd1 cells), respectively. They both were shown to be cross-resistant to hydrogen peroxide. There was a modification in the antioxidant repertoire in these cells as compared to the parental cells. Md1 presented an increase in catalase and glutathione peroxidase activities whereas Cd1 cells exhibited an increase in metallothionein and glutathione contents. The susceptibility of the DNA of these cells to the damaging effect of H₂O₂ was tested using the DNA precipitation assay. Both Md1 and Cd1 DNAs were more resistant to the peroxide action. In the case of Md1 cells it seems clear that the extra resistance is provided by the increase in the two H₂O₂ scavenger enzymes, catalase and glutathione peroxidase. In the case of Cd1 cells the activities of these enzymes as well as of superoxide dismutases (Cu/Zn and Mn) are unaltered as compared to the parental cells. The facts that parental cells exposed to 100 μM Zn²⁺ in the medium exhibit an increase in metallothionein but not in glutathione and that these cells become more resistant to the DNA-damaging effect of H₂O₂ suggest that this protein might play a protective role in vivo against the OH radical attack on DNA.     

Cellular protection of morin against the oxidative stress induced by hydrogen peroxide

Flavonoids are a class of secondary metabolites abundantly found in fruits and vegetables. In addition, flavonoids have been reported as potent antioxidants with beneficial effects against oxidative stress-related diseases such as cancer, aging, and diabetes. The present study was carried out to investigate the cytoprotective effects of morin (2′,3,4′,5,7-pentahydroxyflavone), a member of the flavonoid group, against hydrogen peroxide (H₂O₂)-induced DNA and lipid damage. Morin was found to prevent the cellular DNA damage induced by H₂O₂ treatment, which is shown by the inhibition of 8-hydroxy-2′-deoxyguanosine (8-OHdG) formation (a modified form of DNA base), inhibition of comet tail (a form of DNA strand breakage), and decrease of nuclear phospho histone H2A.X expression (a marker for DNA strand breakage). In addition, morin inhibited membrane lipid peroxidation, which is detected by inhibition of thiobarbituric acid reactive substance (TBARS) formation. Morin was found to scavenge the intracellular reactive oxygen species (ROS) generated by H₂O₂ treatment in cells, which is detected by a spectrofluorometer, flow cytometry, and confocal microscopy after staining of 2′,7′-dichlorodihydrofluorescein diacetate (DCF-DA). Morin also induces an increase in the activity of catalase and protein expression. The results of this study suggest that morin protects cells from H₂O₂-induced damage by inhibiting ROS generation and by inducing catalase activation.     

Effect of thiol redox state modulators on oxidative stress and sclerotial differentiation of the phytopathogenic fungus <Emphasis Type="Italic">Rhizoctonia solani</Emphasis>

This study showed that sclerotial differentiation in the filamentous phytopathogenic fungus Rhizoctonia solani is directly related to oxidative stress and thiol redox state (TRS). Sclerotial differentiation is modulated by the availability of non-cytotoxic −SH groups as was shown by the inhibition of sclerorial differentiation by the TRS modulator N-acetyl cysteine (AcCSH), and not necessarily with those of the TRS reduced components glutathione (GSH) and its precursor cysteine (CSH) as indicated by the GSH-biosynthesis inducer and inhibitor l-2–oxo-thiazolidine-4-carboxylate and l-buthionine-S,R-sulfoximine, respectively. Moreover, inhibition of sclerotial differentiation was accompanied by decrease of the high oxidative stress indicators, lipid peroxidation and DNA damage in the mycelial substrate where sclerotia initials are formed, which suggests that this phenomenon is related to oxidative stress as it is predicted by our theory on sclerotial differentiation.     

Fluid replacement following dehydration reduces oxidative stress during recovery

To investigate the effects of hydration status on oxidative DNA damage and exercise performance, 10 subjects ran on a treadmill until exhaustion at 80% VO_2max during four different trials [control (C), 3% dehydration (D), 3% dehydration + water (W) or 3% dehydration + sports drink (S)]. Dehydration significantly decreased exercise time to exhaustion (D < C and S). Plasma MDA levels were significantly higher at pre-exercise in D than C. Plasma TAS was significantly lower at pre-exercise in C and S than in D, and was significantly lower in S than D at 60 min of recovery. Dehydration significantly increased oxidative DNA damage during exercise, but fluid replacement with water or sports drink alleviated it equally. These results suggest that (1) dehydration impairs exercise performance and increases DNA damage during exercise to exhaustion; and (2) fluid replacement prolongs exercise endurance and attenuates DNA damage.     

Effects of microcystins over short- and long-term memory and oxidative stress generation in hippocampus of rats

Microcystins produced by cyanobacteria are potent inhibitors of some protein phosphatases, but recent evidence also indicates its potential to generate oxidative stress. In the present study, the effects of microcystin raw extracts (Mic; 0.01 and 20microg/L) and purified okadaic acid (OA; 0.01 and 10microg/L) on short- and long-term memory alteration and antioxidant and oxidative damage were investigated in hippocampus of rats. The results showed an amnesic effect with 0.01 and 20microg/L Mic on retrieval and only with 0.01microg/L Mic on spatial learning. Parallel to these effects oxidative damage was observed as evidenced by augmented levels of lipid peroxides and DNA damage and the absence of antioxidant responses in terms of total oxyradical scavenging capacity. Phase II reactions catalyzed by glutathione-S-transferase were not modified after microcystins exposure. Overall this study showed physiological events (retrieval and spatial learning) that can be related to the classical toxic effects of microcystins (i.e., phosphatase inhibition). In addition, evidence of alternative toxicity mechanisms via oxidative stress generation was also obtained. The fact that organic anion transporter polypeptides (OATP) involved in microcystins uptake are expressed not only in liver but also in brain points to the environmental relevance of the observed effects.     

Persistent response of Fanconi anemia haematopoietic stem and progenitor cells to oxidative stress

Oxidative stress is considered as an important pathogenic factor in many human diseases including Fanconi anemia (FA), an inherited bone marrow failure syndrome with extremely high risk of leukemic transformation. Members of the FA protein family are involved in DNA damage and other cellular stress responses. Loss of FA proteins renders cells hypersensitive to oxidative stress and cancer transformation. However, how FA cells respond to oxidative DNA damage remains unclear. By using an in vivo stress-response mouse strain expressing the Gadd45β-luciferase transgene, we show here that haematopoietic stem and progenitor cells (HSPCs) from mice deficient for the FA gene Fanca or Fancc persistently responded to oxidative stress. Mechanistically, we demonstrated that accumulation of unrepaired DNA damage, particularly in oxidative damage-sensitive genes, was responsible for the long-lasting response in FA HSPCs. Furthermore, genetic correction of Fanca deficiency almost completely abolished the persistent oxidative stress-induced G₂/M arrest and DNA damage response in vivo. Our study suggests that FA pathway is an integral part of a versatile cellular mechanism by which HSPCs respond to oxidative stress.     

Diphenyl diselenide reduces temporarily hyperglycemia: possible relationship with oxidative stress

This study was designed to determine the effect of diphenyl diselenide and ebselen, synthetic organoselenium compounds with antioxidant properties, in diabetic rats. Diabetes was induced by the administration of streptozotocin (STZ) (45mg/kg, intravenous). In experimental trials, diphenyl diselenide, but not ebselen, caused a significant reduction in blood glucose levels of STZ-treated rats. This effect of diphenyl diselenide was accompanied by a reduction in the levels of glycated proteins. Diphenyl diselenide ameliorate superoxide dismutase activity (liver and erythrocytes) and Vitamin C levels (liver, kidney and blood), which were decreased in STZ-treated rats. In normal rats, diphenyl diselenide caused per se an increase in hepatic, renal and blood GSH levels. Similarly, treatment with diphenyl diselenide restored hepatic and renal GSH levels in STZ-treated rats. TBARS and protein carbonyl levels were not modified by STZ and/or diphenyl diselenide and ebselen treatments. Our findings suggest that diphenyl diselenide can be considered an anti-diabetogenic agent by exhibiting anti-hyperglycemic and antioxidant properties.     

Circulating levels of apoptotic markers and oxidative stress parameters in women with polycystic ovary syndrome: a case-controlled descriptive study

Context: Apoptotic dysregulation plays a role in the pathogenesis of polycystic ovary syndrome (PCOS).

Objective: To evaluate circulatory apoptotic markers and oxidative stress in patients with PCOS.

Materials and methods: Forty-four women with PCOS, and 44 healthy women as controls were enrolled in the study. Oxidative stress parameters and caspases levels were measured in serum.

Results: The caspase 9 level was significantly lower and related with oxidant status in patients with PCOS, while the circulating levels of caspases 3 and 7 were statistically similar in both groups.

Discussion: This study is the first report demonstrating the circulating levels of apoptotic markers and their relationship with oxidant status in PCOS.

Conclusion: The circulating caspase 9 and oxidant status might contribute to apoptotic dysregulation in PCOS.     

Inhibition of catalase activity by oxidative stress and its relationship to salicylic acid accumulation in plants

The decrease in catalase activity and its relationship to change in salicylic acid content were investigated in rice, wheat, and cucumber seedlings exposed to oxidative stresses. A decrease in chlorophyll fluorescence (F/Fm), measured as an indicator of the oxidative stress, and a drop in catalase activity were observed following treatment with NaCl in all plant seedlings tested . Furthermore, such decreases in F/Fm and catalase activity were also observed under low temperature conditions in both rice cultivars, whereas the degrees of decrease were dependent on their low temperature tolerance . Although the content of salicylic acid increased in rice seedlings stressed by NaCl treatment, it was inversely correlated with the decrease in the catalase activity . Such a relationship between the decrease in catalase activity and increase in salicylic acid content was confirmed with paraquat treatment of the rice seedlings . These results suggested that the fall in catalase activity is a phenomenon occurring in many plant species under oxidative stress and is related to the accumulation of salicylic acid in oxidatively-stressed plants.     

Oxidative balance score and oxidative stress biomarkers in a study of Whites,African Americans,and African immigrants

Abstract

Context: Oxidative balance score (OBS) is a composite measure of multiple pro- and antioxidant exposures.

Objective: To investigate associations of OBS with F2-isoprostanes (FIP), mitochondrial DNA copy number (mtDNA), and fluorescent oxidative products (FOP), and assess inter-relationships among the biomarkers.

Methods: In a cross-sectional study, associations of a thirteen-component OBS with biomarker levels were assessed using multivariable regression models.

Results: Association of OBS with FIP, but not with FOP, was in the hypothesized direction. The results for mtDNA were unstable and analysis-dependent. The three biomarkers were not inter-correlated.

Conclusions: Different biomarkers of oxidative stress may reflect different biological processes.     

The oxidative induction of DNA lesions in cancer cells by 5-thio-d-glucose and 6-thio-d-fructopyranose and their genotoxic effects. Part 3

Thio-sugars have been described as potent inhibitors of cancer cell growth but the detailed mechanism of action remains unknown. Herein we investigated the mechanism of their anticancer action in the HeLa cell line. We investigated two thio-sugars: 5-thio-d-glucose (FCP1) and 6-thio-β-d-fructopyranose (FCP2). We have observed that FCP1 as well as FCP2 clearly induced oxidative DNA lesions in cancer cells and increased the level of cellular ROS. A spin trap and antioxidants have decreased the level of DNA lesions induced by FCPs. FCPs also induced significant changes in the oxidative-stress gene expression. Therefore, we assume that ROS generation is correlated with the increased NOX5 expression by FCPs. Higher cyto- and genotoxicity of FCPs for HeLa cells in a low glucose environment suggested their role in the glucose metabolism. The data indicates that thio-sugars may become drug alternatives for the cancer treatment but such undertaking needs further studies.     

Role of mitochondria in exercise-induced oxidative stress in skeletal muscle from hyperthyroid rats

Previous study showed that exercise induces higher oxidative damage and respiratory capacity reduction in hyperthyroid than in euthyroid skeletal muscle. Because impaired cell function can result from mitochondrial dysfunction, we evaluated the changes induced by exercise in oxygen consumption of skeletal muscle mitochondria from euthyroid and hyperthyroid rats. The mitochondrial function was related with indices of oxidative damage and nitric oxide production, scavenger levels and mitochondrial ROS production rates. Our results show that exercise increased state 4 and decreased state 3 respiration, and the highest changes happened in hyperthyroid preparations. This was consistent with the observation that oxidative damage and NO(*) derivative content were increased by T(3) administration and exercise, reaching the highest levels in hyperthyroid exercised rats. Our results also indicate that the high mitochondrial oxidative damage induced by T(3) and exercise is due to enhanced ROS production, which is dependent on increases in mitochondrial content and reduction degree, respectively, of autoxidizable electron carriers.     

Phorate triggers oxidative stress and mitochondrial dysfunction to enhance micronuclei generation and DNA damage in human lymphocytes

Herein, we studied phorate for its toxicological effects in human lymphocytes. Phorate treatment for 3 h has induced significant increase in the lymphocytic DNA damage. Compared to control, comet data from highest concentration of phorate (1000 µM) showed 8.03-fold increase in the Olive tail moment (OTM). Cytokinesis blocked micronucleus (CBMN) assay revealed 6.4-fold increase in binucleated micronucleated (BNMN) cells following the exposure with phorate (200 µM) for 24 h. The nuclear division index (NDI) in phorate (200 µM) treated cells reduced to 1.8 vis-à-vis control cells showed NDI of 1.94. Comparative to untreated control, 60.43% greater DCF fluorescence was quantitated in lymphocytes treated with phorate (500 µM), affirming reactive oxygen species (ROS) generation and oxidative stress. Flow cytometric data of phorate (200 µM) treated lymphocytes showed 81.77% decline in the fluorescence of rhodamine 123 (Rh123) dye, confirming the perturbation of mitochondrial membrane potential (ΔΨm). Calf thymus DNA (ct-DNA) treated with phorate (1000 µM) exhibited 2.3-fold higher 8-Hydroxy-2′-deoxyguanosine (8-oxodG) DNA adduct formation, signified the oxidative DNA damage. The alkaline unwinding assay revealed 4.0 and 6.5 ct-DNA strand breaks when treated to phorate and phorate-Cu (II) complex. Overall, the data unequivocally suggests the cyto- and genotoxic potential of phorate in human lymphocytes, which may induce comparable toxicological consequences in persons occupationally or non-occupationally exposed to insecticide phorate.     

Evaluation of oxidative DNA damage and antioxidant defense in patients with nasal polyps

Abstract

Objectives

The presence of inflammatory cells indicates the development of epithelial cell injury in nasal polyposis (NP) and the potential for production of high levels of reactive oxygen and nitrogen species. The aim of our study was to clarify the role of oxidative stress and antioxidant status in the deterioration accompanying NP.

Methods

Twenty patients (11 men) aged 47.2 ± 17.0 years with nasal polyps were included in the study. Twenty healthy subjects (7 men) aged 48.2 ± 15.3 years formed the control group. The erythrocyte activities of antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx), and plasma nitric oxide (NO) concentrations were measured. An alkaline comet assay was used to determine the extent of blood lymphocyte DNA damage of oxidized purines as glicosylo-formamidoglicosylase (Fpg) sites, and oxidized pyrimidines as endonuclease III (Nth) sites.

Results

A significant increase of NO (P < 0.05) and non-significant decreases of SOD (P > 0.05), CAT (P > 0.05), and GPx (P > 0.05) were seen in NP patients compared to healthy controls. The level of blood lymphocyte oxidative DNA damage in NP patients was significantly higher compared to the control group (P = 0.01).

Discussion

The blood lymphocyte DNA damage level increased in patients with NP. Elevated DNA damage may be related to overproduction of reactive oxygen and nitrogen species and/or decreased antioxidant protection.     

Calcium microdomains and oxidative stress

The phenomenon of calcium microdomains is firmly established in the field of subcellular physiology. These regions of localized, transient calcium increase are exemplified by the spontaneous 'sparks' released through the ryanodine receptor in myocytes, but include subplasmalemmal microdomains, focal calcium oscillations and microdomains enclosed within organelles, such as the endoplasmic reticulum, golgi and mitochondria. Increasing evidence suggests that oxidative stress regulates both the formation and disappearance of microdomains. Calcium release channels and transporters are all modulated by redox state, while several mechanisms that generate oxidative or nitrosative stress are regulated by calcium. Here, we discuss the evidence for the regulation of calcium microdomains by redox state, and, by way of example, demonstrate that the frequency of calcium sparks in cardiomyocytes is increased in response to oxidative stress. We consider the evidence for the existence of analogous microdomains of reactive oxygen and nitrogen species and suggest that the refinement of imaging techniques for these species might lead to similar concepts. The interaction between Ca(2+) microdomains and proteins that modulate their formation results in a complex and dynamic, spatial signaling mechanism, which is likely to be broadly applicable to different cell types, adding new dimensions to the calcium signaling 'toolkit'.     

Changes in the ascorbic acid levels of peritoneal lymphocytes and macrophages of mice with endotoxin-induced oxidative stress

Ascorbic acid (AA) is an important cytoplasmic antioxidant that mice synthesize in the liver, the intracellular levels of which decrease in an oxidative stress situation such as endotoxic shock. The present work deals with the changes in AA levels, that modulate the immune function, in the two main immune cells, namely macrophages and lymphocytes, from female BALB/c mice suffering endotoxic shock caused by intraperitoneal injection of Escherichia coli lipopolysaccharide (LPS) (100 mg/kg). The intake by cells of this antioxidant present in vitro at different concentrations was also studied. The animals show an oxidative stress, standardized in previous studies, that causes mortality at 30h after LPS injection. The cells were obtained from the peritoneum at 2, 4, 12 and 24h after LPS or PBS (control) injections and were incubated without or with AA at 0.01, 0.1 and 1 mM for 10, 30, 60, 120 or 180 min. The hepatic AA levels were also studied at 0, 2, 4, 12 and 24h after LPS injection. The peritoneal cells obtained from animals injected with LPS showed increased AA levels in relation to the control cells at all times after LPS injection, with maximal effect at 12h. The AA levels decreased after this time, in agreement with changes in the AA hepatic levels. The increase was due to the AA of lymphocytes since macrophages showed a decrease in AA at different times after LPS injection. Both cells showed an increase in the intracellular levels of AA when this antioxidant was added in vitro. This takes place mainly at 30–60 min of incubation in cells from controls and at 10 min in cells from treated mice 12–24 h after LPS injection. The incorporation decreased at these times of endotoxic shock, a few hours before death. In all cases AA levels were higher in lymphocytes than in macrophages, and 1 mM was the most effective concentration. These results suggest that the immune cells need appropriate levels of antioxidants, such as AA, under oxidative stress conditions, and that while lymphocytes take and accumulate AA, macrophages use it.     

Cellular accumulation of Cys326-OGG1 protein complexes under conditions of oxidative stress

The common Ser326Cys polymorphism in the base excision repair protein 8-oxoguanine glycosylase 1 is associated with a reduced capacity to repair oxidative DNA damage particularly under conditions of intracellular oxidative stress and there is evidence that Cys326-OGG1 homozygous individuals have increased susceptibility to specific cancer types. Indirect biochemical studies have shown that reduced repair capacity is related to OGG1 redox modification and also possibly OGG1 dimer formation. In the current study we have used bimolecular fluorescence complementation to study for the first time a component of the base excision repair pathway and applied it to visualise accumulation of Cys326-OGG1 protein complexes in the native cellular environment. Fluorescence was observed both within and around the cell nucleus, was shown to be specific to cells expressing Cys326-OGG1 and only occurred in cells under conditions of cellular oxidative stress following depletion of intracellular glutathione levels by treatment with buthionine sulphoximine. Furthermore, OGG1 complex formation was inhibited by incubation of cells with the thiol reducing agents β-mercaptoethanol and dithiothreitol and the antioxidant dimethylsulfoxide indicating a causative role for oxidative stress in the formation of OGG1 cellular complexes.     

Mechanisms involved in the development of diabetic retinopathy induced by oxidative stress

Background: Diabetic retinopathy (DR) is one of the main complications in patients with diabetes and has been the leading cause of visual loss since 1990. Oxidative stress is a biological process resulting from excessive production of reactive oxygen species (ROS). This process contributes to the development of many diseases and disease complications. ROS interact with various cellular components to induce cell injury. Fortunately, there is an antioxidan t system that protects organisms against ROS. Indeed, when ROS exceed antioxidant capacity, the resulting cell injury can cause diverse physiological and pathological changes that could lead to a disease like DR.

Objective: This paper reviews the possible mechanisms of common and novel biomarkers involved in the development of DR and explores how these biomarkers could be used to monitor the damage induced by oxidative stress in DR, which is a significant complication in people with diabetes.

Conclusion: The poor control of glucemy in pacients with DB has been shown contribute to the development of complications in eyes as DR.     

Quercetin protects HCT116 cells from Dichlorvos-induced oxidative stress and apoptosis

The present study was designed to assess the possible protective effects of Quercetin (QUER), a flavonoid with well-known pharmacological effects, against Dichlorvos (DDVP)-induced toxicity in vitro using HCT116 cells. The cytotoxicity was monitored by cell viability, reactive oxygen species (ROS) generation, anti-oxidant enzyme activities, malondialdehyde (MDA) production, and DNA fragmentation. The apoptosis was assessed through the measurement of the mitochondrial transmembrane potential (ΔΨm) and caspase activation. The results indicated that pretreatment of HCT116 cells with QUER, 2 h prior to DDVP exposure, significantly decreased the DDVP-induced cell death, inhibited the ROS generation, modulated the activities of catalase (CAT) and superoxide dismutase (SOD), and reduced the MDA level. The reductions in mitochondrial membrane potential, DNA fragmentation, and caspase activation were also attenuated by QUER. These findings suggest that dietary QUER can protect HCT116 cells against DDVP-induced oxidative stress and apoptosis.     

Proteomic profiling and identification of cofilin responding to oxidative stress in vascular smooth muscle

We used 2-DE and MALDI-TOF/TOF to identify proteins of vascular smooth muscle cells whose expression was or was not altered by exposure to 500 microM H2O2 for 30 min. We detected more than 800 proteins on silver-stained gels of whole protein extracts from rat aortic smooth muscle strips. Of these proteins, 135 clearly unaffected and 19 having levels altered by exposure to H2O2 were identified. Protein characterization revealed that the most prominent vascular smooth muscle proteins were those with antioxidant, cytoskeletal structure, or muscle contraction. In addition, cofilin, an isoform of the actin depolymerizing factor family, shifted to its basic site on the 2-DE gel as a result of H2O2 treatment. In Western blot analysis of proteins from A7r5 aortic smooth muscle cells, the phosphorylation, but not the expression, of cofilin was decreased by H2O2 in a dose-dependent manner. The H2O2-induced dephosphorylation of cofilin and apoptosis was inhibited by Na3VO4, an inhibitor of protein tyrosine phosphatase (PTP). These results suggest that cofilin is one of the proteins regulated by H2O2 treatment in vascular smooth muscle, and has an important role in the induction of vascular apoptosis through PTP-dependent mechanisms.