Oxidative Stress In Human Erythrocytes Research Articles

Comparative study of antioxidant activity of dextran-coated iron oxide, gold, and silver nanoparticles against age-induced oxidative stress in erythrocytes.

Erythrocytes undergo several changes during human aging and age-related diseases and, thus, have been studied as biomarkers of the aging process. The present study aimed to explore the antioxidant ability of metal and metal oxide nanoparticles (NPs) such as iron oxide (Fe3O4), gold (Au), and silver (Ag) to mitigate age-related oxidative stress in human erythrocytes. Metal and metal oxide NPs behave like antioxidative enzymes, directly influencing redox pathways and thus have better efficiency. Additionally, biopolymer coatings such as dextran enhance the biocompatibility of these NPs. Therefore, dextran-coated Fe3O4, Au, and Ag NPs were synthesized using wet chemical methods and were characterized. Their hemocompatibility and ability to protect erythrocytes from age-induced oxidative stress were investigated. The Fe3O4 and Au NPs were observed to protect erythrocytes from hydrogen peroxide and age-induced oxidative damage, including decreased antioxidant levels, reduced activity of antioxidative enzymes, and increased amounts of oxidative species. Pre-treatment with NPs preserved the morphology and membrane integrity of the erythrocyte. However, Ag NPs induced oxidative stress in erythrocytes similar to hydrogen peroxide. Therefore, dextran-coated Fe3O4 and Au nanoparticles have the potential to be employed as antioxidant therapies against age-related oxidative stress.

Protective Effects of Mulberry (Morus atropurpurea Roxb.) Leaf Protein Hydrolysates and Their In Vitro Gastrointestinal Digests on AAPH-Induced Oxidative Stress in Human Erythrocytes.

Mulberry leaf protein hydrolysates (HMP), and their in vitro gastrointestinal digests (GHMP), have shown favorable chemical antioxidant activities. The aim of this study is to investigate the potential protective effects of HMP and GHMP against 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH)-induced oxidative stress in human erythrocytes. The inhibition rate of hemolysis, the reactive oxygen species (ROS) level, the concentration of malondialdehyde (MDA), the reduced glutathione (GSH) and oxidized glutathione (GSSH), and the enzymatic activities of total superoxide dismutase (SOD), catalase (CAT), and cellular glutathione peroxidase (GSH-Px) were evaluated as the biomarkers of oxidative status in human erythrocytes. The results showed that HMP and GHMP effectively inhibit the occurrence of erythrocyte hemolysis in the range of 0.025-1.0 mg/mL, and the inhibition rates of HMP and GHMP reached 92% and 90% at concentrations of 0.4 mg/mL and 1.0 mg/mL, respectively. HMP and GHMP reduced the AAPH-induced oxidative hemolysis damage via suppressing the generation of ROS by inhibiting the formation of MDA, maintaining the balance of GSH/GSSG, and preserving the activities of the antioxidant enzymes, including SOD, GSH-Px, and CAT. Our findings revealed that both HMP and GHMP could be used as natural antioxidants, and have the potential for further application in the development of functional foods.

Cat’s Claw Nutrients for Medical Treatments

Uncaria tomentosa (Willd. ex Schult.) DC is commonly known as cat’s claw that is derived from the Spanish word Uña de Gato that identifies the small, curved-back thorns on the stem at the leaf junction. It is a tropical medicinal vine of the Rubiaceae family that is widely distributed in the Amazon rainforest and other areas of South and Central America. Cat's claw, which is scientifically known as Uncaria tomentosa, has been historically used to treat and prevent inflammatory disorders, particularly osteoarthritis. Traditionally, it has been used to treat chronic viral infections, bacterial infections, numerous inflammatory and immunological disorders, asthma, dysentery, cancers, arthritis, and as a birth control agent. Nutraceuticals, including food or products containing cat’s claw, can be used to prevent and modify symptoms in osteoarthritis patients. So, cat’s claw is possibly effective in reducing the pain in patient’s joints. U. tomentosa is a potent complimentary herb for treating most parasites. Various chemical constituents are reported from the extracts of U. tomentosa along with their biological activities. It is worth noting that more than 50 phytochemical molecules have been identified and isolated from U. tomentosa, some of them are considered new to that species. It also was used for the treatment of various ailments including abscesses, urinary tract infections, contraception, rheumatism, and weakness. Additionally, it was used as a treatment option for mental disorders (anxiety). The preclinical assessment revealed that the cat’s claw defends towards various oxidative stresses, involving peroxynitrite that has been included in arthritis and other chronic inflammatory diseases along with inhibiting acute or chronic gastritis caused by high doses of nonsteroidal anti-inflammatory drugs (NSAIDs). U. tomentosa aqueous extract was used to protect against oxidative stress in human erythrocytes and relieve chronic intestinal inflammation. Cat’s claw has antitumor and immunostimulatory effects because of its oxindole alkaloids content. Anti proliferative effect of U. tomentosa against several cancer cell lines, namely cervical carcinoma, osteosarcoma, and breast cancer were documented. U. tomentosa hot water extract prevents inflammatory responses as well as tumor cell proliferation by inhibiting the transcriptional regulator nuclear factor kappa beta (NF-B) activation without interfering with interleukin-2 (IL-2) production or IL-2 receptor signaling. Medical technologists documented the ant proliferative effect of cat’s claw extracts against several cell lines, including glioma, premyelocytic leukemia, MCF7 breast cancer, acute lymphoblastic leukaemia, and neuroblastoma.

Mancozeb-induced cytotoxicity in human erythrocytes: enhanced generation of reactive species, hemoglobin oxidation, diminished antioxidant power, membrane damage and morphological changes

Mancozeb is an ethylene bis-dithiocarbamate fungicide extensively used in agriculture to safeguard crops from various fungal diseases. The general population is exposed to mancozeb through consumption of contaminated food or water. Here, we have investigated the effect of mancozeb on isolated human erythrocytes under in vitro conditions. Erythrocytes were treated with different concentrations of mancozeb (0, 5, 10, 25, 50, 100 μM) and incubated for 24 h at 37 °C. Analysis of biochemical parameters and cell morphology showed dose-dependent toxicity of mancozeb in human erythrocytes. Mancozeb treatment caused hemoglobin oxidation and heme degradation. Protein and lipid oxidation were enhanced, while a significant decrease was seen in reduced glutathione and total sulfhydryl content. A significant increase in the generation of reactive oxygen and nitrogen species was detected in mancozeb-treated erythrocytes. The antioxidant capacity and the activity of key antioxidant enzymes were greatly diminished, while crucial metabolic pathways were inhibited in erythrocytes. Damage to the erythrocyte membrane on mancozeb treatment was apparent from increased cell lysis and osmotic fragility, along with the impairment of the plasma membrane redox system. Mancozeb also caused morphological alterations and transformed the normal discoid-shaped erythrocytes into echinocytes and stomatocytes. Thus, mancozeb induces oxidative stress in human erythrocytes, impairs the antioxidant defense system, oxidizes cellular components, that will adversely affect erythrocyte structure and function.

Association of Different ABO and Rh Blood Groups with the Erythroprotective Effect of Extracts from Navicula incerta and Their Anti-Inflammatory and Antiproliferative Properties.

Previous studies have reported that different blood groups are associated with the risk of chronic degenerative diseases that mainly involve inflammation and neoplastic processes. We investigate the relationship between blood groups and the erythroprotective effect of extracts from Navicula incerta against oxidative damage as a proposal to develop drugs designed for people with a specific blood type related to chronic pathology. The study was carried out through the elucidation of the erythroprotective potential, anti-inflammatory and antiproliferative activity of Navicula incerta. Research suggests that the presence or absence of certain blood groups increases or decreases the abilities of certain phytochemicals to inhibit oxidative stress, which is related to the systemic inflammatory response involved in the development of different types of cancer. The pigment-rich extracts from Navicula incerta inhibit ROO•- induced oxidative stress in human erythrocytes on the A RhD+ve antigen without compromising the structure of the cell membrane. This result is very important, since the A antigen is related to the susceptibility of contracting prostate cancer. Similarly, it was possible to inhibit the proliferation of cervical (HeLa) and prostate (PC-3) carcinoma. The combinatorial analysis of different biological activities can help design phytochemicals as new candidates for preventive drugs treating the chronic degenerative diseases associated with a specific blood group.

Effect of fresh red grape juice and grape fermentative product on oxidative-stress in human erythrocytes in vitro

Introduction: Oxidative stress is a phenomenon induced by an imbalance between production and the biological system's ability to readily detoxify oxygen reactive species (ROS) in cells. It has been shown that grape juice can reduce oxidative stress due to the presence of polyphenols. The aim of this study was to evaluate the effect of fresh red grape juice and grape fermentative product on oxidative stress in human erythrocytes. Methods: 5 ml of blood from 125 healthy individuals as control group collected in EDTA containing tubes. To perform biochemical assays, erythrocytes were incubated at 37 ºC for different times including 4, 24, 48, and 72 hours in the presence or absence of grape juice and grape red wine in amounts of 5 ml. Results: Grape juice and grape red wine reduced lipid peroxidation and increase of thiol groups, and total antioxidant capacity after 24 hours of treatment (p < 0.05). Also, the activity of catalase enzyme was increased 4 and 24 hours after treatment with red wine and grape juice, respectively. Conclusion: Grape juice and grape fermentative product may improve the antioxidant power of erythrocytes. This may lead to reducing the risk of free-radical damage and chronic diseases. However, more research with a higher number of samples is necessary to confirm the antioxidant effect of grape juice and red wine on human erythrocytes.

Piperine protects oxidative modifications in human erythrocytes.

Piperine (1-piperoyl piperidine), a major alkaloid constituent of Piper nigrum L. and Piper longum L. has pleiotropic biological effects, but the mechanism(s) involved remain to be elucidated. The current study was conducted to examine the efficacy of antioxidant ability of piperine on t-BHP induced markers of oxidative stress in human erythrocytes. Healthy human erythrocytes and erythrocytes membrane was stressed with free radical inducer chemical; t-BHP (10-5M), and the effects of piperine was measured against free radical mediated modification in lipid and protein content,-SH and GSH value with antioxidant potential. The results demonstrate that treatment of erythrocytes with piperine (10-5 to 10-7M) significantly (p<0.05) ameliorated the adverse consequences of oxidative stress as evidenced by prevention of oxidation of erythrocyte reduced glutathione, membrane thiols, proteins, and peroxidation of lipids; the effects were in correlation with ferric reducing and radical scavenging abilities of piperine. The study concludes that piperine possesses potent anti-oxidant potential which may explain many of its observed biological effects.

Antiviral, Antioxidant, and Antihemolytic Effect of Annona muricata L. Leaves Extracts.

Annona muricata L. is a tropical tree that is used in traditional medicine around the world. The high content of flavonoid, alkaloid, acetogenin, phenolic and lipophilic compounds of this tropical tree forms the basis of its traditional medical uses. Our objective was to study soursop leaf extracts to support their use as antiviral therapies and investigate their protective effects against oxidative damage. The aqueous extract (AE) and acidified ethanolic extract (AEE) of soursop leaves were characterized by ultra performance liquid chromatography (UPLC), and their effects on human erythrocytes and in vitro antioxidant capacity, as evaluated by 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS) assays, were investigated. The antiviral effects were evaluated using a bacteriophage surrogate. AEE showed the highest phenolic content, with rutin as the predominant compound. This extract showed higher values in the DPPH and ABTS assays, with 23.61 ± 0.42 and 24.91 ± 0.16 mmol of Trolox equivalent per gram, respectively. Inhibition of hemolysis was 34% and 51% for AE and AEE, respectively. AEE was selected for the antiviral study because of its higher antioxidant activity. The viral reduction ranged from 5–6 log10 plaque-forming units/volume (PFU) at contact times of 15–360 min. Soursop leaves have a positive effect on reducing oxidative stress in human erythrocytes and viral infections.

Microvesicle Formation Induced by Oxidative Stress in Human Erythrocytes.

Extracellular vesicles (EVs) released by different cell types play an important role in many physiological and pathophysiological processes. In physiological conditions, red blood cell (RBC)-derived EVs compose 4–8% of all circulating EVs, and oxidative stress (OS) as a consequence of different pathophysiological conditions significantly increases the amount of circulated RBC-derived EVs. However, the mechanisms of EV formation are not yet fully defined. To analyze OS-induced EV formation and RBC transformations, we used flow cytometry to evaluate cell esterase activity, caspase-3 activity, and band 3 clustering. Band 3 clustering was additionally analyzed by confocal microscopy. Two original laser diffraction-based approaches were used for the analysis of cell deformability and band 3 activity. Hemoglobin species were characterized spectrophotometrically. We showed that cell viability in tert-Butyl hydroperoxide-induced OS directly correlated with oxidant concentration to cell count ratio, and that RBC-derived EVs contained hemoglobin oxidized to hemichrome (HbChr). OS induced caspase-3 activation and band 3 clustering in cells and EVs. Importantly, we showed that OS-induced EV formation is independent of calcium. The presented data indicated that during OS, RBCs eliminated HbChr by vesiculation in order to sacrifice the cell itself, thereby prolonging lifespan and delaying the untimely clearance of in all other respects healthy RBCs.

Changes in Antioxidant Activity of Peptides Identified from Brown Rice Hydrolysates under Different Conditions and Their Protective Effects against AAPH-Induced Oxidative Stress in Human Erythrocytes.

Four antioxidant peptides (Ile-Tyr, Leu-Tyr, Val-Tyr, and Tyr-Leu-Ala), identified from brown rice protein hydrolysates, showed strong ROO· and ABTS·+ scavenging activities. Changes in the antioxidant activity of peptides and GSH (control) under different processing conditions, namely, NaCl, temperature, pH, and gastrointestinal proteases, were evaluated by the oxygen radical absorbance capacity assay and the Trolox equivalent antioxidant capacity assay. Results indicated that with the increase in NaCl concentration, temperature, and pH (beyond neutral), the antioxidant activity of the peptides decreased, while the decrease was lower than that of GSH. The antioxidant activity of the four antioxidant peptides changed slightly after in vitro digestion, indicating a relatively high digestion resistance. The protective effect on the oxidative damage model of 2,2-azobis (2-methylpropionamide)-dihydrochloride-induced human red blood cells was also studied. Leu-Tyr and Tyr-Leu-Ala could alleviate but not totally inhibit oxidative damage in red blood cells, and their protective effects were dependent on concentration.

D‐glucose and D‐galactose affect Band 3 protein function and oxidative stress in human erythrocytes

Metabolic dysfunctions continuously expose human erythrocytes to sugars present in plasma during their circulatory life span of 120 days. Hyperglycemia in diabetes mellitus causes glycation of proteins along with oxidative stress in erythrocytes. Moreover, acute systemic administration of D‐galactose was used to artificially induce blood senescence in human models as well to mimic galactosemia. The purpose of this study is to understand the effect of D‐glucose and D‐galactose in human erythrocytes and in particular on the function of Band 3 protein, which is an excellent tool for studying the oxidative events on the erythrocyte membrane. Human erythrocytes in vitro were exposed to increasing concentrations of D‐glucose (5‐15‐35 mM) for 3h or D‐galactose (0.1‐3‐5‐10 mM) for 1h. The rate constant for SO4= uptake, accounting for the efficiency of anion exchange through Band 3 protein along with levels of TBARS levels and membrane –SH groups have been measured. When erythrocytes were exposed to high D‐glucose (15–35 mM) for 3 h, TBARS levels and membrane –SH groups were unchanged while the rate constant for SO4= uptake was significantly accelerated with respect to control (5 mM). After exposure to high D‐galactose (5–10 mM) for 1 h, TBARS levels were higher and membrane –SH levels were lower than control, while the rate constant for SO4= uptake was significantly slower with respect to control (0.1 mM). The present findings show that the measurement of the rate constant for SO4= uptake is a suitable tool to monitor the effect of sugars on erythrocytes; hyperglycemia due to high glucose applied for 3 h affects anion exchange capability without producing lipid peroxidation and oxidative protein damage. High concentrations of D‐galactose (&gt; 5 mM) decreased the rate constant for SO4= uptake, induced lipid peroxidation and reduced total sulfhydryl content. Based on this evidence, future studies will evaluate if the acceleration of Band 3 protein in presence of high D‐glucose depends on altered Bp3 conformation, affecting crosslink with Hb, or on altered glycosylation or phosphorylation signaling underlying Band 3 protein function. In addition, alterations of parameters of oxidative stress caused by D‐galactose on the erythrocytes membrane open the view to consider a potential therapeutic strategy ameliorating the damage caused by D‐galactose.

Thiram-induced cytotoxicity and oxidative stress in human erythrocytes: an in vitro study

Tetramethylthiuram disulfide, commonly known as thiram, is an organosulfur compound which is used as a bactericide, fungicide and ectoparasiticide to prevent disease in seeds and crops. Being a fungicide there is a high probability of human occupational exposure to thiram and also via consumption of contaminated food. In this work, the cytotoxicity of thiram was studied under in vitro conditions using human erythrocytes as the cellular model. Erythrocytes were incubated with different concentrations of thiram (25–500 μM) for 4 h at 37 °C. Control cells (thiram untreated) were similarly incubated at 37 °C. Whole cells and hemolysates were analyzed for various biochemical parameters. Treatment of erythrocytes with thiram increased protein and lipid oxidation and hydrogen peroxide level in hemolysates but decreased glutathione and total sulfhydryl group content. This was accompanied by hemoglobin oxidation, heme degradation and release of free iron. Activities of all major antioxidant enzymes were inhibited. The antioxidant power of thiram treated erythrocytes was lowered resulting in decreased metal reducing and free radical quenching ability. These results suggest that thiram enhances the generation of reactive species that cause oxidative modification of cell components. This was confirmed by experiments that showed enhanced generation of reactive oxygen and nitrogen species in thiram treated erythrocytes. Activities of marker enzymes of glucose metabolism and erythrocyte membrane were also inhibited. All effects were seen in a thiram concentration-dependent manner. Electron microscopy further supported the damaging effect of thiram on erythrocytes. Thus thiram induces oxidative stress condition in human erythrocytes and causes oxidative modification of cell components.

Effects of L-carnitine on the erythrocytes of stored human blood.

This study aimed to assess the effects of L-carnitine on oxidative stress in human erythrocytes during storage. Using antioxidants as components of blood storage solutions may combat the effects of storage-induced oxidative stress on erythrocytes. Blood from male adults was stored at 4°C for 55 days in citrate phosphate dextrose adenine solution, without L-carnitine (Control) and with L-carnitine as an additive (at concentrations of 10, 30 and 60 mM-Experiments). Every fifth day, erythrocyte markers (morphology, count, haemoglobin, haemolysis and osmotic fragility), antioxidant defences (antioxidant enzymes and total antioxidant capacity) and oxidative stress markers (superoxides, lipid peroxidation and protein oxidation products) were analysed. Oxidative damage was observed in controls (day 25 onwards) and in experiments (day 35 onwards). L-carnitine (10 and 30 mM) protected erythrocytes from damage up to day 35 by maintaining haemoglobin and lipid peroxidation, assisting antioxidant enzymes and increasing antioxidant capacity by elevating sulfhydryls and ascorbic acid. L-carnitine was beneficial in prolonging storage up to 55 days but could not prevent oxidative damage completely in terms of haemolysis and osmotic fragility. L-carnitine ameliorated oxidative stress, but combinations with other antioxidants may provide comprehensive protection to erythrocytes during storage.

Pyridoxine Decreases Oxidative Stress on Human Erythrocyte Membrane Protein in vitro

Background: Pyridoxine has reduction and prevention against the levels of reactive oxygen species in in vitro studies. However, the biochemical mechanism that explains this behavior has not yet been fully clarified. Objective: To evaluate the effect of pyridoxine against oxidative damage on the membrane of human erythrocytes. Methods: Cumene hydroperoxide was used to induce oxidative stress in protein and lipid. Human erythrocytes were incubated with pyridoxine and cumene hydroperoxide, either alone or together for 8 h. Oxidative damage was determined by measuring lipid peroxidation and membrane protein carbonylation. Results: The results indicate that the malondialdehyde concentration decreased with increasing concentration of pyridoxine. The membrane protein content also decreased with increasing concentration of vitamin B6, which was confirmed by the decreased signal intensity in the western blot when compared to control without pyridoxine. Results demonstrate that pyridoxine can significantly decrease lipid peroxidation and protein carbonylation in red cell membrane exposed to high concentrations of oxidant agent. Conclusion: Pyridoxine showed a protective effect against the oxidative stress in human erythrocytes in vitro, inhibiting the carbonylation and the oxidative damage of erythrocyte membrane proteins. To date, such an effect has not yet been reported in terms of protein oxidation.

Mercury chloride toxicity in human erythrocytes: enhanced generation of ROS and RNS, hemoglobin oxidation, impaired antioxidant power, and inhibition of plasma membrane redox system.

Mercury is among the most toxic heavy metals and a widespread environmental pollutant. Mercury chloride (HgCl2) is an inorganic compound of mercury which is easily absorbed in the gastrointestinal tract and then enters the blood where it can interact with erythrocytes. In this study, the effect of HgCl2 on human erythrocytes was studied under in vitro conditions. Erythrocytes were treated with different concentrations of HgCl2 (1-100μM) for 1h at 37°C. Cell lysates were prepared and assayed for several biochemical parameters. HgCl2 treatment resulted in oxidation of ferrous iron of hemoglobin to ferric form giving methemoglobin which is inactive as an oxygen transporter. However, the activity of methemoglobin reductase was increased. Hemoglobin oxidation was accompanied by heme degradation and the release of free iron. Protein oxidation was greatly increased with a simultaneous decrease in free amino and sulfhydryl groups and glutathione content. The antioxidant power of HgCl2-treated erythrocytes was impaired resulting in lowered metal reducing and free radical quenching ability of these cells. This suggests that HgCl2 induces oxidative stress in human erythrocytes. This was confirmed when superoxide anion, hydrogen peroxide, peroxynitrite, and nitric oxide generation were found to be dose-dependently increased in HgCl2-treated erythrocytes. Glycolysis and pentose phosphate pathway, the two major pathways of glucose metabolism in erythrocytes, were also inhibited. HgCl2 treatment also inhibited the plasma membrane redox system while the activities of AMP deaminase and glyoxalase-I were increased. These results show that HgCl2 induces oxidative and nitrosative stress, oxidizes hemoglobin, impairs the antioxidant defense mechanism, and alters metabolic pathways in human erythrocytes.

Correcting glucose-6-phosphate dehydrogenase deficiency with a small-molecule activator

Glucose-6-phosphate dehydrogenase (G6PD) deficiency, one of the most common human genetic enzymopathies, is caused by over 160 different point mutations and contributes to the severity of many acute and chronic diseases associated with oxidative stress, including hemolytic anemia and bilirubin-induced neurological damage particularly in newborns. As no medications are available to treat G6PD deficiency, here we seek to identify a small molecule that corrects it. Crystallographic study and mutagenesis analysis identify the structural and functional defect of one common mutant (Canton, R459L). Using high-throughput screening, we subsequently identify AG1, a small molecule that increases the activity of the wild-type, the Canton mutant and several other common G6PD mutants. AG1 reduces oxidative stress in cells and zebrafish. Furthermore, AG1 decreases chloroquine- or diamide-induced oxidative stress in human erythrocytes. Our study suggests that a pharmacological agent, of which AG1 may be a lead, will likely alleviate the challenges associated with G6PD deficiency.

Ameliorative effects of quercetin against bisphenol A-caused oxidative stress in human erythrocytes: an in vitro and in silico study.

Bisphenol A (BPA) is an endocrine disruptor of xenobiotic type, mainly used for the production of polycarbonate plastic, epoxy resins and non-polymer additives. Because of its wide usages in the environment, the toxic effects of BPA have proved to be harmful to human health. However, its effects on human haemoglobin remain unclear. The affinity between BPA and haemoglobin, as well as erythrocytes, is an important factor in understanding the mechanism of the toxicity of BPA. Flavonoids are strong antioxidants that prevent oxidative stress and Quercetin is a flavonoid found in numerous vegetables and fruits. Therefore, the present investigation was undertaken to investigate whether Quercetin can be used to alleviate the toxic effects of BPA in vitro in human red blood cells (RBC). Venous blood samples were collected from healthy, well-nourished adult volunteers (25-30 years old) by phlebotomy. In a RBC suspension with a cell density of 2 × 104 cell per mL, the concentration of BPA (25-150 µg mL-1) was found to cause an increase in the lipid peroxidation (LPO) and a decrease in the activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX) in human RBC. However, the concurrent addition of BPA (150 µg mL-1) and quercetin (10-50 µg mL-1) lead to significant amelioration. In silico studies gave structural insight into BPA and quercetin to decipher the plausible binding mechanism and molecular level recognition.

Intracellular antioxidant activities of selected cereal phenolic extracts and mechanisms underlying the protective effects of adlay phenolic extracts on H2O2-induced oxidative stress in human erythrocytes

The intracellular antioxidant activities of millet, rice, wheat, brown rice, oat, buckwheat, corn and adlay phenolic extracts were evaluated in an ex vivo erythrocytes system. Buckwheat, corn, and brown adlay free phenolic extracts could effectively attenuate H2O2-induced erythrocyte hemolysis. The protective effects and involved mechanisms of polished adlay, brown adlay and adlay bran phenolic extracts were studied further. The intracellular antioxidant activities of adlay phenolic extracts decreased in the following order: adlay bran>brown adlay>polished adlay. Adlay bran free phenolic extract possessed stronger intracellular antioxidant activity than chlorogenic acid, p-coumaric acid and ferulic acid. The free phenolic extracts were more effective than bound phenolic extracts to suppress hemolysis and plasma oxidation through inhibition of MDA equivalents, MetHb and ROS generation and to restore the activities of Na+K+-ATPase, Ca2+Mg2+-ATPase and intracellular antioxidant enzyme to normal levels. Adlay-rich diets could reduce the incidence of cancer, cardiovascular and neurodegenerative diseases.

SO4= uptake and catalase role in preconditioning after H2O2-induced oxidative stress in human erythrocytes.

Preconditioning (PC) is an adaptive response to a mild and transient oxidative stress, shown for the first time in myocardial cells and not described in erythrocytes so far. The possible adaptation of human erythrocytes to hydrogen peroxide (H2O2)-induced oxidative stress has been here verified by monitoring one of band 3 protein functions, i.e., Cl-/HCO3- exchange, through rate constant for SO4= uptake measurement. With this aim, erythrocytes were exposed to a mild and transient oxidative stress (30min to either 10 or 100μM H2O2), followed by a stronger oxidant condition (300- or, alternatively, 600-μM H2O2 treatment). SO4= uptake was measured by a turbidimetric method, and the possible role of catalase (CAT, significantly contributing to the anti-oxidant system in erythrocytes) in PC response has been verified by measuring the rate of H2O2 degradation. The preventive exposure of erythrocytes to 10μM H2O2, and then to 300μM H2O2, significantly ameliorated the rate constant for SO4= uptake with respect to 300μM H2O2 alone, showing thus an adaptive response to oxidative stress. Our results show that (i) SO4= uptake measurement is a suitable model to monitor the effects of a mild and transient oxidative stress in human erythrocytes, (ii) band 3 protein anion exchange capability is retained after 10 μM H2O2 treatment, (iii) PC response induced by the 10 μM H2O2 pretreatment is clearly detected, and (iv) PC response, elicited by low-concentrated H2O2, is mediated by CAT enzyme and does not involve band 3 protein tyrosine phosphorylation pathways. Erythrocyte adaptation to a short-term oxidative stress may serve as a basis for future studies about the impact of more prolonged oxidative events, often associated to aging, drug consumption, chronic alcoholism, hyperglycemia, or neurodegenerative diseases.

The role of intracellular zinc in H2O2-induced oxidative stress in human erythrocytes

In vitro exposure of human erythrocytes to H2O2 at concentrations of 30–1000 μM resulted in a dose-dependent increase of the intracellular levels of Zn2+ and inhibition of the cytosolic esterase activity, which is a major marker of erythrocyte viability. The observed effect depended on the concentration of H2O2 and the duration of exposure of the cells to this compound. An inverse relationship between the changes in the intracellular level of labile zinc ions and esterase activity in the cells exposed to hydrogen peroxide was detected; this was indicative of the role of Zn2+ in the programmed death of red blood cells. The combined action of hydrogen peroxide and N',N'-tetrakis-(2-pyridyl-methyl)-ethylenediamine, an intracellular zinc ion chelator, has been found to eliminate the cytotoxic effect of H2O2, whereas the addition of Zn2+ to the erythrocyte incubation medium enhanced the effects of hydrogen peroxide. The reduction of the concentration of non-protein thiol groups due to a decrease of the level of reduced glutathione was shown to contribute to the release of Zn2+ from the intracellular binding sites during oxidative stress induced by H2O2 in human erythrocytes.