Assessment of DNA damage in sperm after repeated non‐invasive sampling in zebrafish Danio rerio

Repeated non‐invasive sampling of zebrafish Danio rerio sperm was conducted, sperm counts were obtained and a method for measurement of DNA damage in sperm was developed and validated (single‐cell gel electrophoresis, comet, assay). DNA damage in sperm increased with concentration of hydrogen peroxide (H₂O₂, 0–200 µM), and in vitro exposure of sperm to 200 µM H₂O₂ produced 88·7 ± 3·9% tail DNA compared to unexposed controls [12 ± 0·7% tail DNA (mean ± s.e ., n = 3)]. Frequency of sperm sampling (sampled every 2, 4 or 7 days) did not affect DNA damage in sperm, but sperm counts decreased 57 and 22% for fish sampled every 2 or 4 days, respectively.     

Effect of Tribulus terrestris saponins on proliferation of adipose-derived mesenchymal stem cells

Adipose-derived mesenchymal stem cells (ASCs) transplantation has shown great promise for treating various diseases; however, poor viability of transplanted ASCs because of oxidative stress has limited its therapeutic efficiency. Plant saponins are recently been reported to have antioxidant activity tested in various cancer cell lines. This study was designed to investigate the protective effects of Tribulus terrestris saponins (TTS) on the proliferation of ASCs. The cytotoxic activity of hydrogen peroxide (H₂O₂) was determined by treating ASCs with 100, 200, 300, 400, and 500 µM H₂O₂ for 2 hours. ASCs were treated with 6.25, 12.5, 25, 50, and 100 µg/mL concentrations of TTS for the proliferative experiment. To check the protective effect of TTS, experiments were designed in two ways. In one set, ASCs were pretreated with different concentrations of TTS for 2 hours and then apoptosis was induced by treating them with 400 µM H₂O₂ for next 2 hours, while in other set, ASCs were first treated with 400 µM H₂O₂ for 2 hours and subsequently with different concentrations of TTS for 24 hours. The vitality and proliferation potential of cells were detected by 3-(4,5-Dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The result of the current study shows that in response to stress-induced by H₂O₂ at concentration of 400 µM, ASCs underwent growth arrest and cell viability was reduced to half while treatment with TTS before and after H₂O₂ exposure significantly prevents premature apoptosis. The findings suggest that saponins may act as an effective protective agent against oxidative stress–induced ASCs apoptosis.     

Mercury-induced changes in growth variables and antioxidative enzyme activities in Indian mustard

Abstract

In a hydroponic system, experiments were conducted to study the effect of different levels of mercury treatments (0, 5, 10, 25 and 50 µM Hg) on Indian mustard (Brassica juncea L. Czern & Coss.) cv. Pusa Jai Kisan. Concentration-dependent inhibitory effects were observed on growth characteristics (plant dry mass, leaf area, shoot and root length). These were accompanied by an increase in shoot Hg content and in oxidative stress characteristics such as the MDA and H₂O₂ levels. The plant growth decreased maximally at 50 µM of Hg. Despite a reduction in growth, activities of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and glutathione reductase (GR) were enhanced with increase in Hg-treatments. The Hg-induced alterations in growth are linked with increase in lipid peroxidation (MDA and H₂O₂), whereas the enhancement in activities of antioxidant enzymes protects plants from Hg-induced oxidative stress.     

The role of insulin against hydrogen peroxide-induced oxidative damages in differentiated SH-SY5Y cells

Abstract

Exogenous hydrogen peroxide (H₂O₂) can easily penetrate into biological membranes and enhance the formation of other reactive oxygen species (ROS). In the present study, we have investigated the neuroprotective effects of insulin on H₂O₂-induced toxicity of retinoic acid (RA)-differentiated SH-SY5Y cells. To measure the changes in the cell viability of SH-SY5Y cells at different concentrations of H₂O₂ for 24?h, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT)-based assay was used and a 100?µM H₂O₂ was selected to establish a model of H₂O₂-induced oxidative stress. Further assays showed that 24?h of 100?µM H₂O₂-induced significant changes in the levels of lactate dehydrogenase (LDH), nitric oxide (NO), ROS, and calcium ion (Ca²⁺) in neuronal cells, but insulin can effectively diminish the H₂O₂-induced oxidative damages to these cells. Moreover, cells treated with insulin increased H₂O₂-induced suppression of glutathione levels and exerted an apparent suppressive effect on oxidative products. The results of insulin treatment with SH-SY5Y cells increased the Bcl-2 levels and decreased the Akt levels. The treatment of insulin had played a protective effect on H₂O₂-induced oxidative stress related to the Akt/Bcl-2 pathways.     

Dual proapoptotic and pronecrotic effect of hydrogen peroxide on human umbilical vein endothelial cells

Human umbilical vein endothelial cells were exposed in culture to hydrogen peroxide (H₂O₂), keeping them close to physiological conditions (high cell density, high serum content, H₂O₂ concentration not over 500 µM). Cell viability was assessed by flow cytometry using simultaneous staining with the fluorescent dye PO-PRO-1 to detect early apoptotic cells and DRAQ7 to detect late apoptotic and necrotic cells. The data obtained suggest that the primary mechanism of the cytotoxic response to H₂O₂ is apoptosis. The critical concentration of H₂O₂ causing death in a dense monolayer is 250 µM. Lower H₂O₂ concentrations (up to 200 µM) cause death of individual cells. The population of endothelial cell retains viability and response to calcium activating agonists does not change compared to control cells.     

Prooxidative effects of green tea polyphenol (−)-epigallocatethin-3-gallate on the HIT-T15 pancreatic beta cell line

Epigallocatechin-3-gallate (EGCG) is the main polyphenolic constituent in green tea and is believed to function as an antioxidant. However, increasing evidence indicates that EGCG produces reactive oxygen species (ROS) and subsequent cell death. In this study, we investigated the prooxidative effects of EGCG on the HIT-T15 pancreatic beta cell line. Dose-dependent cell viability was monitored with the cell counting kit-8 assay, while the induction of apoptosis was analyzed by a cell death ELISA kit and comet assay. Extracellular H₂O₂ was determined using the Amplex Red Hydrogen Peroxide Assay Kit. Intracellular oxidative stress was measured by fluorometric analysis of 2′,7′-dichlorofluorescin (DCFH) oxidation using DCFH diacetate (DA) as the probe. Treatment with EGCG (5–100 μM) decreased the viability of pancreatic beta cells, caused concomitant increases in apoptotic cell death, and increased the production of H₂O₂ and ROS. Catalase, the iron-chelating agent diethylenetriaminepentaacetic acid, and the Fe(II)-specific chelator o-phenanthroline all suppressed the effects of EGCG, indicating the involvement of both H₂O₂ and Fe(II) in the mechanism of action of EGCG. The antioxidant N-acetyl-cysteine and alpha-lipoic acid also suppressed the effects of EGCG. Furthermore, EGCG did not scavenge exogenous H₂O₂, but rather, it synergistically increased H₂O₂-induced oxidative cell damage in pancreatic beta cells. Together, these findings suggest that in the HIT-T15 pancreatic beta cell line, EGCG mediated the generation of H₂O₂, triggering Fe(II)-dependent formation of a highly toxic radical that in turn induced oxidative cell damage.     

Speciation dependant antioxidative response in roots and leaves of sorghum (Sorghum bicolor (L.) Moench cv CO 27) under Cr(III) and Cr(VI) stress

Growth, lipid peroxidation, H₂O₂ produciton and the response of the antioxidant enzymes and metabolites of the ascorbate glutathione pathway to oxidative stress caused by two concentrations (50 and 100 µM) of Cr(III) and Cr(VI) was studied in 15 day old seedlings of sorghum (Sorghum bicolor (L.) Moench cv CO 27) after 10 days of treatment. Cr accumulation in sorghum plants was concentration and organ dependant. There was no significant growth retardation of plants under 50 µM Cr(III) stress. 100 µM Cr(VI) was most toxic of all the treatments in terms of root and leaf growth and oxidative stress. 50 µM Cr(VI) treated roots exhibited high significant increase in superoxide dismutase (SOD), dehydroascorbate reductase (DHAR) and glutathione reductase (GR) (p < 0.01) and significant increases in catalse (CAT), ascorbate peroxidase (APX) and monodehydroascorbate reductase (MDHAR) (p < 0.05). A high increase in ascorbic acid (AA) level was seen in roots of 50 µM Cr(VI) treated plants in comparison with control. Levels of reduced glutathione (GSH) showed a varied and complex response in all the treatments in both plant parts. GSH/GSSG ratio was not affected by Cr(III) treatment in leaves, in contrast, roots exhibited significant reduction in the ratio. Results indicate that GSH depletion increased sensitivity to oxidative stress (Cr(VI) roots and leaves and Cr(III) 100 µM roots) and AA in tandem with APX compensated for GSH depletion by acting directly on H₂O₂ and the mechanism of defensive response in roots as well as leaves varied in its degree and effectiveness due to the concentration dependant differences observed in translocation of the element itself, reactive oxygen species (ROS) generation and enzyme inhibition based on the oxidation state supplied to the plants.     

Nitric oxide interferes with plant photo-oxidative stress by detoxifying reactive oxygen species

Oxidative stress within chloroplasts is originated due to light‐dependent O₂ reduction. This may be exacerbated by bipyridinium herbicides, which act at photosystem I as artificial electron acceptors. Their oxidation produces a superoxide anion that further dismutates to H₂O₂ and then, by the Fenton reaction, H₂O₂ may be reduced to the hydroxyl radical (OH^?). Reactive oxygen species (ROS), when produced in high amounts, provoke severe damage to the plant cell. Herein it is reported that two nitric oxide (NO) donors, sodium nitroprusside (100 µm ) and S‐nitroso‐N‐acetylpenicillamine (200 µm ), greatly reduced lipid peroxidation and the protein loss caused by the application of a high dose of the bipyridinium herbicide diquat to potato leaf pieces or isolated chloroplasts. Nitric oxide donors also protected the RNA against oxidative damage. Photo‐oxidative toxicity was correlated with an increase in photosynthetic electron transport and ROS production, but the rate of electron transport was restored and the ROS free amount was markedly reduced in the presence of NO. The specific activity of superoxide dismutase was not affected by diquat or NO donors, whereas just a small increase in catalase activity was observed after 24 h of treatment. These results provide strong evidence that NO is a potent antioxidant in plants and that its action may, at least in part, be explained by its ability to directly scavenge ROS.     

Electrochemical detection of extracellular hydrogen peroxide in Arabidopsis thaliana: a real‐time marker of oxidative stress

An electrochemical approach to directly measure the dynamic process of H₂O₂ release from cultures of Arabidopsis thaliana cells is reported. This approach is based on H₂O₂ oxidation on a Pt electrode in conjunction with continuous measurement of sample pH. For [H₂O₂] <1 mm , calibration plots were linear and the amperometric response of the electrode was maximum at pH 6. At higher concentrations ([H₂O₂] >1 mm ), the amperometric response can be described by Michaelian‐type kinetics and a mathematical expression relating current intensity and pH was obtained to quantitatively determine H₂O₂ concentration. At pH 5.5, the detection limit of the sensor was 3.1 µm (S/N = 3), with a response sensitivity of 0.16 Am ^?1 cm^?2 and reproducibility was within 6.1% in the range 1–5 × 10^?3 m (n = 5). Cell suspensions under normal physiological conditions had a pH between 5.5–5.7 and H₂O₂ concentrations in the range 7.0–20.5 µm (n = 5). The addition of exogenous H₂O₂, as well as other potential stress stimuli, was made to the cells and the change in H₂O₂ concentration was monitored. This real‐time quantitative H₂O₂ analysis is a potential marker for the evaluation of oxidative stress in plant cell cultures.     

Effects of exogenous nitric oxide on photosynthesis,antioxidative ability,and mineral element contents of perennial ryegrass under copper stress

A hydroponics experiment was conducted to test the effects of sodium nitroprusside (SNP, a donor of NO) supplied with different concentrations on copper (Cu) toxicity in ryegrass seedlings (Lolium perenne L.). Excess Cu (200 µM) reduced chlorophyll content, resulting a decrease in photosynthesis. Cu stress induced the production of hydrogen peroxide (H₂O₂) and superoxide anion (O₂^{? ?}), leading to malondialdehyde (MDA) accumulation. Furthermore, activities of antioxidant enzymes in Cu-treated seedlings such as superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) were decreased. In addition, Cu stress inhibited the uptake of K, Mg, Fe, and Zn and increased Ca content in roots. Moreover, in leaves of Cu-stressed seedlings, K, Fe, and Zn contents were decreased and the contents of Ca and Mg were not affected significantly. In Cu-treated seedlings, Cu concentration in roots was higher than in leaves. Addition of 50, 100, 200 µM SNP in Cu-mediated solutions increased chlorophyll content and photosynthesis, improved antioxidant enzyme activities, reduced Cu-induced oxidative damages, kept intracellular ion equilibrium under Cu stress, increased Cu concentration in roots and inhibited Cu accumulation in leaves. In particular, addition of 100 µM SNP had the best effect on promoting growth of ryegrass seedlings under Cu stress. However, the application of 400 µM SNP had no obvious alleviating effect on Cu toxicity in ryegrass seedlings.     

Glutamate receptor-mediated taurine release from the hippocampus during oxidative stress

Background

Hippocampal slices swell and release taurine during oxidative stress. The influence of cellular signalling pathways on this process is unclear. Glutamate signalling can facilitate volume regulation in other CNS preparations. Therefore, we hypothesize activation of taurine release by oxidative stress results from tissue swelling and is coupled to activation of glutamate receptors.

Methods

Rat hippocampi were incubated at room temperature for 2 hr in artificial cerebrospinal fluid (aCSF) equilibrated with 95% O₂ plus 5% CO₂. For some slices, 1 mM taurine was added to the aCSF to maintain normal tissue taurine content. Slices then were perfused with aCSF at 35° C and baseline data recorded before 2 mM H₂O₂ was added. For some studies, mannitol or inhibitors of glutamate receptors or the volume-regulated anion channel (VRAC) were added before and during H₂O₂ treatment. The intensity of light transmitted through the slice (the intrinsic optical signal, IOS) was determined at 1-min intervals. Samples of perfusate were collected at 2-min intervals and amino acid contents determined by HPLC. Data were analyzed by repeated measures ANOVA and post hoc Dunnett’s test with significance indicated for p<0.05.

Results

IOS of slices prepared without taurine treatment increased significantly by 3.3±1.3% (mean±SEM) during oxidative stress. Little taurine was detected in the perfusate of these slices and the rate of taurine efflux did not change during H₂O₂ exposure. The α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate antagonist, 25 µM CNQX, but not the N-methyl-D-aspartate (NMDA) receptor antagonist, 10 µM MK-801, inhibited the increase in IOS during H₂O₂ treatment. Taurine-treated slices exposed to H₂O₂ showed no change in IOS; however, taurine efflux increased by 335±178%. When these slices were perfused with hypertonic aCSF (350 mOsm) or exposed to the VRAC inhibitor, 20 µM DCPIB, no increase in the taurine efflux rate was observed during H₂O₂ exposure. Taurine-treated slices perfused with 10 µM MK-801 during H₂O₂ exposure showed a 4.6±1.9% increase in IOS but no increase in the taurine efflux rate.

Conclusions

Taurine efflux via VRAC is critical for volume regulation of hippocampal slices exposed to oxidative stress. This increased taurine efflux does not result from direct activation of the taurine release pathway by H₂O₂. NMDA receptor activation plays an important role in taurine release during oxidative stress.

     

Oxidant Injury in PC12 Cells—A Possible Model of Calcium "Dysregulation" in Aging: I. Selectivity of Protection Against Oxidative Stress

Abstract: Previous research has suggested that the initial effects of cellular free radical neurotoxic insult involve large increases in intracellular Ca²⁺. However, the exact role of oxidative stress on the various parameters involved in these increases has not been specified. The present experiments were performed to examine these parameters in PC12 cells exposed to 5, 25, or 300 µM H₂O₂ for 30 min in growth medium alone or containing either nifedipine (L-type Ca²⁺ antagonist), conotoxin (N-type antagonist), Trolox (vitamin E analogue), or α-phenyl-n-tert-butylnitrone (nitrone trapping agent; PBN). The concentrations of H₂O₂ were chosen by examining the degree of cell killing induced by exposure to graded concentrations of H₂O₂. The 5 and 25 µM concentrations of H₂O₂ produced no significant cell killing at either 30 min or 24 h after treatment, whereas the 300 µM concentration produced a moderate degree of cell killing that did not increase between the two times. Fluorescent imaging was used to visualize intracellular Ca²⁺ changes in fura-2-loaded cells. Baseline (pre-30 mM KCI) Ca²⁺ levels were increased significantly by H₂O₂ treatment (e.g., 300 µM, 200%), but the rise in the level of free intracellular Ca²⁺ after KCI stimulation (i.e., peak) was decreased (e.g., 300 µM, 50%) and the cell's ability to sequester or extrude the excess Ca²⁺ (i.e., Ca²⁺ recovery time) after depolarization was decreased significantly. All compounds prevented baseline Ca²⁺ increases and, with the exception of conotoxin, antagonized the peak decreases in Ca²⁺. It is interesting that after 300 µM H₂O₂ exposure, only Trolox was partially effective in preventing these deficits in recovery. Conotoxin increased the decrement recovery in the absence of H₂O₂. However, in cells exposed to 5 or 25 µM H₂O₂, conotoxin as well as the other agents were effective in preventing the deficits in recovery.     

Curcumin inhibits apoptosis by regulating intracellular calcium release,reactive oxygen species and mitochondrial depolarization levels in SH-SY5Y neuronal cells

Neurological diseases such as Alzheimer’s and Parkinson’s diseases are incurable progressive neurological disorders caused by the degeneration of neuronal cells and characterized by motor and non-motor symptoms. Curcumin, a turmeric product, is an anti-inflammatory agent and an effective reactive oxygen and nitrogen species scavenging molecule. Hydrogen peroxide (H₂O₂) is the main source of oxidative stress, which is claimed to be the major source of neurological disorders. Hence, in this study we aimed to investigate the effect of curcumin on Ca²⁺ signaling, oxidative stress parameters, mitochondrial depolarization levels and caspase-3 and -9 activities that are induced by the H₂O₂ model of oxidative stress in SH-SY5Y neuronal cells. SH-SY5Y neuronal cells were divided into four groups namely, the control, curcumin, H₂O₂, and curcumin?+?H₂O₂ groups. The dose and duration of curcumin and H₂O₂ were determined from published data. The cells in the curcumin, H₂O₂, and curcumin?+?H₂O₂ groups were incubated for 24?h with 5?µM curcumin and 100?µM H₂O₂. Lipid peroxidation and cytosolic free Ca²⁺ concentrations were higher in the H₂O₂ group than in the control group; however, their levels were lower in the curcumin and curcumin?+?H₂O₂ groups than in the H₂O₂ group alone. Reduced glutathione (GSH) and glutathione peroxidase (GSH-Px) values were lower in the H₂O₂ group although they were higher in the curcumin and curcumin?+?H₂O₂ groups than in the H₂O₂ group. Caspase-3 activity was lower in the curcumin group than in the H₂O₂ group. In conclusion, curcumin strongly induced modulator effects on oxidative stress, intracellular Ca²⁺ levels, and the caspase-3 and -9 values in an experimental oxidative stress model in SH-SY5Y cells.     

Effect of lycopene on As2O3 induced oxidative stress in SH-SY5Y cells

It is known that oxidative stress may cause neuronal injury and several experimental models showed that As₂O₃ exposure causes oxidative stress. Lycopene, a carotenoid, has been shown to have protective effect in neurological disease models due to antioxidant activity, but its effect on As₂O₃-induced neurotoxicity is not identified yet. The aim of this study is to investigate the effects of lycopene on As₂O₃-induced neuronal damage and the related mechanisms. Cell viability was determined by the MTT assay. Lycopene was administrated with different concentrations (2, 4, 6 and 8 µM) one hour before 2 µM As₂O₃ exposure in SH-SY5Y human neuroblastoma cells. The anti-oxidant effect of lycopene was determined by measuring superoxide dismutase (SOD), catalase (CAT) hydrogen peroxide (H₂O₂), malondialdehyde (MDA), total antioxidant status (TAS) and total oxidant status (TOS). MTT results and LDH cytotoxicity analyses showed that pretreatment with 8 µM lycopene significantly improved the toxicity due to As₂O₃ exposure in SH?SY5Y neuroblastoma cells. Pretreatment with lycopene significantly increased the activities of anti?oxidative enzymes as well as total antioxidant status and decreased total oxidative status in As₂O₃ exposed cells. The results of this study indicate that lycopene may be a potent neuroprotective against oxidative stress and could be used to prevent neuronal injury or death in several neurological diseases.

     

Cytoprotective propensity of Bacopa monniera against hydrogen peroxide induced oxidative damage in neuronal and lung epithelial cells

Hydrogen peroxide (H₂O₂), a major reactive oxygen species (ROS) produced during oxidative stress, is toxic to the cells. Hence, H₂O₂ has been extensively used to study the effects of antioxidant and cytoprotective role of phytochemicals. In the present investigation H₂O₂ was used to induce oxidative stress via ROS production within PC12 and L132 cells. Cytoprotective propensity of Bacopa monniera extract (BME) was confirmed by cell viability assays, ROS estimation, lipid peroxidation, mitochondria membrane potential assay, comet assay followed by gene expression studies of antioxidant enzymes in PC12 and L132 cells treated with H₂O₂ for 24 h with or without BME pre-treatment. Our results elucidate that BME possesses radical scavenging activity by scavenging 2,2-diphenyl-1-picrylhydrazyl, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid), superoxide radical, and nitric oxide radicals. The IC₅₀ value of BME against these radicals was found to be 226.19, 15.17, 30.07, and 34.55 µg/ml, respectively). The IC₅₀ of BME against ROS, lipid peroxidation and protein carbonylation was found to be 1296.53, 753.22, and 589.04 µg/ml in brain and 1137.08, 1079.65, and 11101.25 µg/ml in lung tissues, respectively. Further cytoprotective potency of the BME ameliorated the mitochondrial and plasma membrane damage induced by H₂O₂ as evidenced by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase leakage assays in both PC12 and L132 cells. H₂O₂ induced cellular, nuclear and mitochondrial membrane damage was restored by BME pre-treatment. H₂O₂ induced depleted antioxidant status was also replenished by BME pre-treatment. This was confirmed by spectrophotometric analysis, semi-quantitative RT-PCR and western blot studies. These results justify the traditional usage of BME based on its promising antioxidant and cytoprotective property.     

Heat-shock response protects peripheral blood mononuclear cells (PBMCs) from hydrogen peroxide-induced mitochondrial disturbance

The present study was designed to investigate ex vivo the protective mechanisms of heat-shock response against H₂O₂-induced oxidative stress in peripheral blood mononuclear cells (PBMCs) of rats. Twenty-four hours later, heat-shock treatment was executed in vivo; rat PBMCs were collected and treated with H₂O₂. The accumulation of reactive oxygen species and the mitochondrial membrane potential were evaluated by intracellular fluorescent dHE and JC-1 dye staining, respectively, and expression of HSP72 and cytochrome c was detected by Western blot analysis. Cellular apoptosis was assayed by TUNEL staining and double staining of Annexin V and PI. The results showed that H₂O₂-induced oxidative stress leads to intracellular superoxide accumulation and collapse of the mitochondrial membrane potential in rat PBMCs. Moreover, cellular apoptosis was detected after H₂O₂ treatment, and the release of mitochondrial cytochrome c from mitochondria to cytosol was significantly enhanced. Heat-shock pretreatment decreases the accumulation of intracellular superoxide in PBMCs during H₂O₂-induced oxidative stress. Moreover, heat-shock treatment prevents the collapse of the mitochondrial membrane potential and cytochrome c release from mitochondria during H₂O₂-induced oxidative stress. In conclusion, mitochondria are critical organelles of the protective effects of heat-shock treatment. Cellular apoptosis during H₂O₂-induced oxidative stress is decreased by heat-shock treatment through a decrease in superoxide induction and preservation of the mitochondrial membrane potential.     

Evaluation of antigenotoxicity effects of umbelliprenin on human peripheral lymphocytes exposed to oxidative stress

The protective properties of a prenylated coumarin, umbelliprenin (UMB), on the human lymphocytes DNA lesions were tested. Lymphocytes were isolated from blood samples taken from healthy volunteers. DNA breaks and resistance to H₂O₂-induced damage were measured using a single-cell microgel electrophoresis technique under alkaline conditions (comet assay). Human lymphocytes were incubated in UMB (10, 25, 50, 100, 200, and 400 μM) alone or a combination of different concentrations of UMB (10, 25, 50, 100, 200, and 400 μM) and 25 μM H₂O₂. Untreated cells, ascorbic acid (AA; 25, 50, 100, 200, and 400 μM) and H₂O₂ (25 μM) were considered as negative control, positive control, and the standard antioxidant agent for our study, respectively. Single cells were analyzed with “TriTek Cometscore version 1.5” software. The DNA damage was expressed as percent tail DNA. UMB exhibited a concentration-dependent increase in protection activity against DNA damage induced by 25 μM H₂O₂ (from 67.28% to 39.17%). The antigenotoxic activity of AA, in the range 0–50 μM, was greater than that of UMB. However, no significant difference (p > 0.05) in the protective activity was found between UMB and AA at concentrations of approximately higher than 50 μM.     

Madecassoside,a triterpenoid saponin isolated from Centella asiatica herbs,protects endothelial cells against oxidative stress

This study aimed to investigate the effect of madecassoside against oxidative stress‐induced injury of endothelial cells. Hydrogen peroxide (H₂O₂, 500 µmol/L) was employed as an inducer of oxidative stress in human umbilical vein endothelial cells (HUVECs). Cell apoptosis was detected by Hoechst 33258 staining and flow cytometry. Caspase‐3 activity and mitochondria membrane potential were further examined. As a result, madecassoside (10, 30, 100 µmol/L) could reverse morphological changes, elevate cell viability, increase glutathione levels, and decrease lactate dehydrogenase and malondialdehyde levels caused by H₂O₂ in a concentration‐dependent manner. It attenuated apoptosis, preventing the activation of caspase‐3 and the loss of mitochondria membrane potential, as well as the phosphorylation of p38 mitogen‐activated protein kinase (MAPK) in HUVECs. These data suggested that madecassoside could protect HUVECs from oxidative injury, which was probably achieved by inhibiting cell apoptosis via protection of mitochondria membranes and downregulation of the activation of caspase‐3 and p38 MAPK. © 2012 Wiley Periodicals, Inc. J Biochem Mol Toxicol 26:399–406, 2012; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.21434     

Cadmium-induced subcellular accumulation of O2·− and H2O2 in pea leaves

Cadmium is a toxic metal that produces disturbances in plant antioxidant defences giving rise to oxidative stress. The effect of this metal on H₂O₂ and O₂^·? production was studied in leaves from pea plants growth for 2 weeks with 50 µm Cd, by histochemistry with diaminobenzidine (DAB) and nitroblue tetrazolium (NBT), respectively. The subcellular localization of these reactive oxygen species (ROS) was studied by cytochemistry with CeCl₃ and Mn/DAB staining for H₂O₂ and O₂^·?, respectively, followed by electron microscopy observation. In leaves from pea plants grown with 50 µm CdCl₂ a rise of six times in the H₂O₂ content took place in comparison with control plants, and the accumulation of H₂O₂ was observed mainly in the plasma membrane of transfer, mesophyll and epidermal cells, as well as in the tonoplast of bundle sheath cells. In mesophyll cells a small accumulation of H₂O₂ was observed in mitochondria and peroxisomes. Experiments with inhibitors suggested that the main source of H₂O₂ could be a NADPH oxidase. The subcellular localization of O₂^·? production was demonstrated in the tonoplast of bundle sheath cells, and plasma membrane from mesophyll cells. The Cd‐induced production of the ROS, H₂O₂ and O₂^·?, could be attributed to the phytotoxic effect of Cd, but lower levels of ROS could function as signal molecules in the induction of defence genes against Cd toxicity. Treatment of leaves from Cd‐grown plants with different effectors and inhibitors showed that ROS production was regulated by different processes involving protein phosphatases, Ca²⁺ channels, and cGMP.