Oxidative Stress In Human Lymphocytes Research Articles

Hydroquinone triggers pyroptosis and endoplasmic reticulum stress via AhR-regulated oxidative stress in human lymphocytes

Benzene is a frequent component of environmental pollution and is abundant in petrochemicals, decorative materials, motor vehicle exhaust and cigarette smoke. Benzene is a well-known carcinogen in humans and animals, but the molecular mechanism has not yet been elucidated. Our earlier research indicated that hydroquinone (HQ), one of the main reactive metabolites of benzene, could activate aryl hydrocarbon receptor (AhR), which is essential for HQ-induced toxicity, including apoptosis and DNA damage. Since AhR is an important regulator of the immune system that integrates the environmental stimulus and immune response, we examined whether and how HQ-induced AhR activity could lead to NLRP3 inflammasome-dependent pyroptosis in JHP cells. Our results showed that HQ could cause inflammation process and resultant pyroptosis. In JHP cells, HQ also induced endoplasmic reticulum stress (ERS) by releasing excessive reactive oxygen species (ROS). The activation of pyroptosis induced by HQ treatment was reversed by an antioxidant (NAC) and an ERS inhibitor (4-PBA). Interestingly, the treatment of CH223191, an AhR inhibitor, reversed HQ-induced oxidative stress, ERS and pyroptosis. These data suggested that AhR-mediated HQ-induced ERS, ROS and inflammasome activation may play vital roles in the toxic effects of benzene. This work provides insights and prospective strategies into potential mechanisms for reducing benzene-induced hematotoxicity.

Effects of different mobile phone UMTS signals on DNA, apoptosis and oxidative stress in human lymphocytes

Different scientific reports suggested link between exposure to radiofrequency radiation (RF) from mobile communications and induction of reactive oxygen species (ROS) and DNA damage while other studies have not found such a link. However, the available studies are not directly comparable because they were performed at different parameters of exposure, including carrier frequency of RF signal, which was shown to be a critical for appearance of the RF effects. For the first time, we comparatively analyzed genotoxic effects of UMTS signals at different frequency channels used by 3G mobile phones (1923, 1947.47, and 1977 MHz). Genotoxicity was examined in human lymphocytes exposed to RF for 1 h and 3 h using complimentary endpoints such as induction of ROS by imaging flow cytometry, DNA damage by alkaline comet assay, mutations in TP53 gene by RSM assay, preleukemic fusion genes (PFG) by RT-qPCR, and apoptosis by flow cytometry. No effects of RF exposure on ROS, apoptosis, PFG, and mutations in TP53 gene were revealed regardless the UMTS frequency while inhibition of a bulk RNA expression was found. On the other hand, we found relatively small but statistically significant induction of DNA damage in dependence on UMTS frequency channel with maximal effect at 1977.0 MHz. Our data support a notion that each specific signal used in mobile communication should be tested in specially designed experiments to rule out that prolonged exposure to RF from mobile communication would induce genotoxic effects and affect the health of human population.

Oxidative stress-mediated genotoxicity of malathion in human lymphocytes

Applying the single-cell gel electrophoresis (comet) assay, we show that the widely used organophosphorus pesticide malathion is cytotoxic, genotoxic, and induces oxidative stress in human lymphocytes.

Toxicity of Fe2 O3 nanoparticles on human blood lymphocytes.

Magnetic nanoparticles (NPs) are used to a large extent in the targeted delivery of therapeutic agents. In this study, we aimed to investigate the possible toxicity of Fe2 O 3 NPs on human cells, including blood lymphocytes. We isolated blood lymphocytes from healthy humans using Ficoll polysaccharide and subsequently by gradient centrifugation. Then, the toxicity parameters, including cell viability, reactive oxygen species (ROS) formation, lipid peroxidation, cellular glutathione (GSH) level, mitochondrial and lysosomal damage, were measured in blood lymphocytes after exposure to Fe 2 O 3 NPs. Our results indicated that Fe 2 O 3 NPs significantly (dependent on concentration) reduced the cell viability, and the IC 50 was determined to be 1 mM. With increasing concentrations, we found that Fe 2 O 3 NPs-induced cell toxicity was associated with a significant increase in intracellular ROS and loss of mitochondrial membrane potential and lysosomal membrane leakiness. Consequently, these NPs at different concentrations affect GSH level and cause oxidative stress in human lymphocytes.

In Vitro Study Towards Role of Acrylamide-Induced Genotoxicity in Human Lymphocytes and the Protective Effect of L- Carnitine

ABSTRACT Acrylamide (AA), is an important industrial compound, formed during food processing under high temperature. Due to its potential carcinogenic effect, it has been recognized as a human health concern. In this study, we investigated the role of oxidative stress in acrylamide’s genotoxicity and protective role of L- carnitine (LCA) in human lymphocytes. The micronucleus test (MNT) was used to monitor AA genotoxicity after 20h exposure to concentrations of 0.25,50 and 100 mM. Furthermore we evaluated oxidative stress markers, such as reactive oxygen species (ROS), lipid peroxidation (LPO) and glutathione (GSH) content. In MNT, It has been found that AA at high concentrations (50 and 100 µM) significantly increased the micronuclei (MN) frequencies. On the one hand, AA exposure induced ROS formation, LPO and also GSH oxidation in treated lymphocytes. On the other hand, pretreatment with LCA (100 and 200µM) inhibited AA-induced oxidative stress which subsequently led to decreasing of the micronuclei (MN) frequencies in human lymphocytes. In overall, the clastugenic effect of acrylamide was confirmed. Also, it has been observed that LCA prevented AA genotoxicity via amelioration of oxidative stress in human lymphocytes. Therefore, oxidative stress suggested as an important factor involved in acrylamide-induced genotoxicity.

Changes in lymphocyte oxidant/antioxidant parameters after carbonyl and antioxidant exposure

During normal B- and T-cell life, processes including activation, proliferation, signaling pathways and apoptosis are markedly dependent on ROS generation. However, these cells can also suffer the effect of oxidant overproduction. Thus, the purpose of the present study was to examine the possible pro-oxidant effects of MGO/high glucose and antioxidant effects of astaxanthin associated with vitamin C on some oxidative and antioxidant parameters of human lymphocytes in vitro. Lymphocytes from healthy subjects were treated with 20mM of glucose and 30μM MGO followed or not by the addition of the antioxidants astaxanthin (2μM) and vitamin C (100μM) for up to 24h. We examined superoxide dismutase (SOD), catalase, glutathione peroxidase, glutathione reductase, and glucose-6-phosphate dehydrogenase (G6PDH) activities, GSH/GSSG ratio and total thiol and carbonyl content. Oxidative parameters included superoxide anion, hydrogen peroxide and nitric oxide production. The association of astaxanthin and vitamin C proved to be a powerful antioxidant in human lymphocytes as showed by the marked reduction in superoxide anion, and hydrogen peroxide production as well as increased GSH content, GSH/GSSG ratio, GPx and GR activities. The antioxidant association showed to be more potent than their individual application. High glucose and methylglyoxal did not promote oxidative stress in human lymphocytes, since neither the oxidative parameters nor the antioxidant defense system was altered. According to these results, new therapies with the association of astaxanthin and vitamin C may be helpful to improve the immune function of patients with exacerbated production of ROS.

Oxidative stress in human lymphocytes treated with fatty acid mixture: Role of carotenoid astaxanthin

Fatty acids (FA) have been shown to alter leukocyte function, and depending on concentration and type, they can modulate both inflammatory and immune responses. Astaxanthin (ASTA) is a carotenoid that shows notable antioxidant properties. In the present study we propose to evaluate the oxidative stress in human lymphocytes induced by a FA mixture and the possible protective role of ASTA. The present study showed that the FA mixture at 0.3mM caused a marked increase in the production of superoxide anion, hydrogen peroxide and nitric oxide, which was accompanied by an increase in total-SOD activity, in TBARS levels and a reduction of catalase activity and content of GSH and free thiol groups. The FA mixture also promoted an increase in intracellular Ca2+ mobilization and in the proliferative capacity of B-lymphocytes. The addition of ASTA (2μM) partially decreased the ROS production and TBARS levels and increased the levels of free thiol groups. ASTA decreased the proliferative capacity of cells treated with FA but not by reducing intracellular calcium concentration. Based on these results we can conclude that ASTA can partially prevent oxidative stress in human lymphocytes induced by a fatty acid mixture, probably by blenching/quenching free radical production.

American Ginseng Modifies 137Cs-Induced DNA Damage and Oxidative Stress in Human Lymphocytes

The multifold bioactive medicinal properties of ginseng have been closely linked to its antioxidative ability, which is related to its ginsenoside content. Since the key mechanism of radiation-induced cell death and tissue damage is the generation of reactive oxygen species (ROS) that attack cellular DNA, this study focuses on the impact of a standardized North American ginseng extract (NAGE) on (137)Cs-induced oxidative stress in human peripheral lymphocytes (PBL) obtained from 10 healthy individuals (6M/4F), 42.7 +/- 4.6 years of age. At two different time points (0 h and 24 h before irradiation), we applied NAGE (250 - 1000 microg ml(-1)) to mononuclear cell cultures for cytokinesis-block micronuclei (MN) assay and determination of the state of oxidative stress in PBL. We found that at both time points, NAGE significantly reduced the MN yields in PBL after irradiation (1 and 2 Gy) in a concentration-dependent manner (P<0.001). Compared with radiation alone, the maximum reduction rate of MN yield were 51.1% and 49.1% after 1 Gy and 2 Gy exposures, respectively. We also found that before irradiation the presence of NAGE in the culture medium resulted in a significant increased intracellular total antioxidant capacity (TAC) in PBL. At both time points, the increment of (137)Cs-induced MN yields in PBL was positively correlated with the increment of intracellular ROS production (R = 0.6 - 0.7, P = 0.002), but negatively correlated with the reduction of TAC levels (R = -0.4 -0.5, P = 0.02 - 0.004). However, the presence of NAGE in the culture medium significantly increased the TAC levels, while concomitantly decreasing both ROS production and MN yields in PBL (P<0.001). Our findings that NAGE is effective in protecting human PBL against radiation-induced oxidative stress should encourage further in vivo study of dietary supplementation with NAGE as an effective natural radiation countermeasure.

The antioxidant effects of vitamin A, C, and E on aflatoxin B1-induced oxidative stress in human lymphocytes

The aim of this study was to investigate the possible protective role of vitamin A, C, and E on aflatoxin B(1)-induced in human lymphocytes using biochemical approaches. The control group received dimethyl sulfoxide, the second group of cultures were administered aflatoxin B(1) (AFB(1)) at a dose of 5 muM. The other group of cultures were treated with AFB(1)+vitamin A (0.5 and 1.0 and 1.5 microM) and AFB(1)+vitamin C (25, 50, and 100 microM) and AFB(1)+vitamin E (40, 100, and 200 microM). The results of this experiment show that AFB(1) significantly decreased the level of GSH and the activities of superoxide dismutase and GPx and increased level of malondialdehyde. Simultaneous supplementation with vitamin A, C, and E restored these parameters to that of normal range. In conclusion, vitamin A, C, and E exhibited protective effects in human lymphocytes by inhibiting AFB(1)-induced ROS generation.

Ferulic acid inhibits UV-B–induced oxidative stress in human lymphocytes

UV-B induces lipid peroxidation and oxidative stress in mammalian cells by producing reactive oxygen species. In this present study, we report the protective effect of ferulic acid (FA; 3-methoxy-4-hydroxycinnamic acid), a dietary antioxidant, on UV-B–induced oxidative stress and lipid peroxidation using normal human lymphocytes. Treatment of the human peripheral lymphocytes with UV-B irradiation caused a significant (P b .05) depletion of reduced glutathione (GSH) levels, superoxide dismutase, catalase, and GSH peroxidase activities, and increased the levels of thiobarbituric acid–reactive substances. Treatment of human lymphocytes with FA before 30 minutes of irradiation inhibited UV-B–induced lipid peroxidation. Ferulic acid also inhibited UV-B–induced depletion of antioxidant defense components, such as GSH peroxidase, catalase, superoxide dismutase, and GSH. The maximum dose of FA (10 μg/mL) normalized the UV-B–induced oxidative stress, indicating the protective effect of FA in human peripheral lymphocytes under in vitro conditions against UV-B exposure. Our observations suggest that FA reduces UV-B–induced lipid peroxidation and enhances antioxidant status in human lymphocytes exposed to UV-B radiation, and therefore, FA could serve as a protector against the UV-B–induced pathologic changes. © 2007 Elsevier Inc. All rights reserved.

Effect of polyphenolic compounds from Coriandrum sativum on H 2O 2-induced oxidative stress in human lymphocytes

Polyphenolic compounds are widely distributed in plants and known to be excellent antioxidants in vitro. They have the capacity to reduce free-radical formation by scavenging free radicals and protecting antioxidant defences. The present study evaluated the antioxidant potencies of polyphenolic compounds from a spice, Coriandrum sativum against hydrogen peroxide-induced oxidative damage in human lymphocytes. Pretreatment with polyphenolic rich fractions protected human lymphocytes against H 2O 2-induced oxidative damage. H 2O 2 treatment significantly decreased the activities of antioxidant enzymes, such as superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, glutathione- S-transferase and caused decreased glutathione content and increased thiobarbituric acid-reacting substances (TBARS). Treatment with polyphenolic fractions (50 μg/ml) increased the activities of antioxidant enzymes and glutathione content and reduced the levels of TBARS significantly. Observed reduction in the level of lipid peroxides showed a decreased tendency of peroxidative damage. We conclude that, under these experimental conditions, polyphenolic compounds effectively suppress hydrogen peroxide-induced oxidative stress.

Ex-vivo and in vitro protective effects of kolaviron against oxygen-derived radical-induced DNA damage and oxidative stress in human lymphocytes and rat liver cells.

The present study reports the protective effects of kolaviron, a Garcinia biflavonoid from the seeds of Garcinia kola widely consumed in some West African countries against oxidative damage to molecular targets ex-vivo and in vitro. Treatment with hydrogen peroxide (H2O2) at a concentration of 100 micromol/L increased the levels of DNA strand breaks and oxidized purine (formamidopyrimidine glycosylase (FPG) and pyrimidine (endonuclease III (ENDO III) sites) bases in both human lymphocytes and rat liver cells using alkaline single cell gel electrophoresis (the comet assay). Kolaviron was protective at concentrations between 30-90 micromol/L and decreased H2O2-induced DNA strand breaks and oxidized bases. Neither alpha-tocopherol nor curcumin decreased H2O2-induced DNA damage in this assay. In lymphocytes incubated with Fe3+/GSH, Fe3+ was reduced to Fe2+ by GSH initiating a free radical generating reaction which induced 11.7, 6.3, and 4.9 fold increase respectively in strand breaks, ENDO III and FPG sensitive sites compared with control levels. Deferoxamine (2 mmol/L), an established iron chelator significantly inhibited GSH/Fe3+-induced strand breaks and oxidized base damage. Similarly, kolaviron at 30 and 90 micromol/L significantly attenuated GSH/Fe3+-induced strand breaks as well as base oxidation. Kolaviron (100 mg/kg bw) administered to rats for one week protected rat liver cells against H2O2-induced formation of strand breaks, ENDO III, and FPG sensitive sites, Fe3+/EDTA/ascorbate-induced malondialdehyde formation and protein oxidation using gamma-glutamyl semialdehyde (GGS) and 2-amino-adipic semialdehyde (AAS) as biomarkers of oxidative damage to proteins. We suggest that kolaviron exhibits protective effects against oxidative damage to molecular targets via scavenging of free radicals and iron binding. Kolaviron may therefore be relevant in the chemoprevention of oxidant-induced genotoxicity and possibly human carcinogenesis.

Protective effect of docosahexaenoic acid against hydrogen peroxide-induced oxidative stress in human lymphocytes

Oxidatively stressed lymphocytes exhibit decreased proliferative response to mitogenic stimulation. Although several sensitive targets involved in lymphocyte suppression have already been identified, little is known about the influence of oxidative stress on cyclic nucleotide phosphodiesterases (PDE) (EC 3.1.4.17), thought to play a major role in the control of cyclic AMP (cAMP) level, a well-recognized negative effector of lymphoproliferation. Although the polyunsaturated fatty acid content of membrane phospholipids is thought to be directly related to the extent of oxidant-induced lipid peroxidation, some n-3 fatty acids also seem to have antioxidant effects, depending on the concentration used and the overall redox status of the cells in question. Results of the present study showed that human peripheral blood mononuclear cells (PBMC) as well as rat thymocytes were relatively resistant to a short-term exposure (10 min) to hydrogen peroxide (H 2O 2). Indeed, H 2O 2-induced lipid peroxidation, estimated by malondialdehyde (MDA) production, was only 2-fold increased by H 2O 2 concentrations lower than 2 mM, whereas a larger increase (10-fold) could be observed in PBMC at the highest dose (5 mM). Previous enrichment of PBMC with 5 μM docosahexaenoic acid (22:6n-3), brought to the cells as a fatty acid–albumin complex (ratio 1), significantly reduced MDA production induced by low doses of H 2O 2, the protective effect no longer being observed at the highest doses. In contrast, eicosapentaenoic acid (20:5n-3) did not have any protective effect. Cytosolic PDE activities of both human PBMC and rat thymocytes were significantly inhibited (40–50%) after H 2O 2 treatment of the cells, whereas particulate PDE activities were not modified. Different responses of PDE activities to H 2O 2 treatment were observed when PBMC were first enriched with 22:6n-3 prior to H 2O 2 addition. In 22:6n-3-treated cells, the H 2O 2-induced inhibition of both cAMP- and cGMP-PDE cytosolic activities was abolished, whereas the particulate activities were increased by the highest H 2O 2 concentration used (5 mM). At the same time, the glutathione peroxidase (glutathione: oxidoreductase, EC 1.11.1.9) (GSH-Px) activity of PBMC and thymocytes was only marginally inhibited by H 2O 2 addition (20%), and pretreatment of the cells with 22:6n-3 did not modify the slight inhibitory effect of H 2O 2. Collectively, these results suggest that lymphocytes are relatively resistant to H 2O 2-induced lipid peroxidation due to their high GSH-Px content, and that low doses of 22:6n-3 are able to prevent some of the H 2O 2-induced alterations such as lipid peroxidation and PDE inhibition. Docosahexaenoic acid might thus offer some protection against oxidant-induced lymphocyte suppression.

Oxygen radicals induce stress proteins and tolerance to oxidative stress in human lymphocytes.

A set of eight proteins is induced in peripheral blood lymphocytes from normal donors by exposure to hydrogen peroxide or to xanthine oxidase plus hypoxanthine. Four of them (hsp90, hsp72 and proteins 65 and 50 kDa) are also expressed after heat shock, together with proteins 110, 100 and 38 kDa. Among proteins induced after oxidative stress is a 32 kDa protein-probably corresponding to heme oxygenase-1 (HO-1)- and a 27 kDa protein, both known to be induced by reactive oxygen species. Although ionizing radiation is known to generate a number of pro-oxidant intermediates, using our one-dimensional electrophoresis system we can detect no differences in the proteins synthesized after exposure to gamma-ray doses between 5 and 20 Gy as compared with control cells. Pre-exposure to a mild hyperthermia or to moderate oxidative stress significantly increases survival of lymphocytes challenged with high doses of reactive oxygen species, in conditions compatible with a protective rôle exerted by stress proteins. The increase in survival is accompanied by the maintenance of the proliferative capacity of the cells. The physiological rôle played by stress proteins in prevention and repair of damage and the relationships between stress protein induction, oxidative state, proliferation and mode of cell death are discussed.

The effect of various antioxidants and other modifying agents on oxygen-radical-generated DNA damage in human lymphocytes in the COMET assay

The effects of antioxidants and various other modifying agents on oxygen-radical-generated DNA damage in human lymphocytes have been investigated using the COMET assay. Hydrogen peroxide (H 2O 2) and bleomycin (BLM) have produced clear dose-related responses. In 38 independent experiments, there was consistency between the two donors used in the study for the negative and positive control data. The endogenous antioxidant catalase abolished effects with H 2O 2, but only slightly affected the response with BLM. Superoxide dismutase did not alter the response with H 2O 2 and only slightly affected BLM. The exogenous antioxidant vitamin C produced a clear dose-related response on its own. In combination with H 2O 2, there were small protective effects at low doses and exacerbating effects at high doses, but these were within the inter-experimental variability range. Vitamin E (trolox) produced no effects with either H 2O 2 or BLM, or on its own. Silymarin protected against the effect due to H 2O 2. Other modifying agents such as apo-transferrin and deferoxamine mesylate produced a clear dose-related protection of effects due to BLM. This protection was less due to H 2O 2. In the presence of ferrous chloride, the effect due to BLM was exacerbated. In a small sample of 6 smokers and 6 non-smokers, responses from smokers approached borderline significance ( P = 0.054) by comparison with non-smokers. These observations would suggest that the COMET assay is a useful tool for examining issues related to oxidative stress in human lymphocytes.