Release Of Reactive Oxygen Intermediates Research Articles

Selective modulation of monocyte and neutrophil responses with activated protein C in preterm infants

Background Inflammation is associated with many disorders of preterm infants including periventricular leukomalacia, chronic lung disease, and necrotizing enterocolitis. Activated protein c (APC) has shown positive immunomodulatory effects. Objectives We aimed to study neutrophil and monocyte function in response to lipopolysaccharide (LPS) and APC stimulation ex vivo in preterm infants <32 weeks gestation over the first week of life compared to neonatal and adult controls. Methods Peripheral blood was taken on day 1, 3, and 7 and stimulated with LPS in the absence or presence of APC. Expression of toll-like receptor 4 (TLR4) and CD11b and reactive oxygen intermediate (ROI) release from neutrophils and monocytes was examined by flow cytometry. Results LPS induced neutrophil ROI in adults and preterm infants and was significantly reduced by APC. Baseline and LPS-induced monocyte ROI production in preterm neonates was increased compared to adult and term controls. Neutrophil TLR4 baseline expression was higher in term controls compared to preterm infants. Conclusion Increased systemic ROI release in preterm infants may mediate tissue damage, ROI was reduced by APC. However, due to the high risk of hemorrhage further examination of APC mutant forms with anti-inflammatory but decreased anticoagulant properties is merited.

Immunological Activities of Crude Polysaccharides Extracts of Climacodon pulcherrimus (Phanerochaetaceae) in Lipopolysaccharide-Induced Rat Macrophages and Neutrophils' Responses.

Mushroom contains a wide variety of components with high immune-modulating properties. Mushroom polysaccharides are known as immunocorrectors with wide areas of usage, which balance the immune system without resulting in the entire immune suppression or overreaction. To explore the potentially immunomodulatory role of Climacodon pulcherrimus, the present study was intended to evaluate the in vitro immunomodulatory activity of C. pulcherrimus polysaccharides (CPP) on rat neutrophils and macrophages. CPP was studied on lipopolysaccharide-induced neutrophils and macrophages' responses. The effect on phagocytosis, release of reactive oxygen intermediates (ROI), nitric oxide (NO), lysosomal enzyme release and neutrophil myeloperoxidase, and neutrophil adherence was assessed. The cytotoxicity of CPP against macrophages using 3-(4,5-dimethylthiazole-2-yl)-2, 5-diphenyltetrazolium bromide assay and neutral red inclusion assay was also assessed. ENPC (endopolysaccharides) markedly enhanced phagocytosis of macrophages and neutrophils, whereas the EXPC (exopolysaccharides) increased only that of neutrophils at 0.34 and 0.68 mg/mL. The production of NO evidently increased in ENPC- and EXPC-treated neutrophil and macrophage cultures. The production of ROI in a concentration-dependent manner increased in ENPC-treated (0.08-5.55 mg/mL) and EXPC-treated (0.17 and 0.34 mg/mL) macrophage cultures and ENPC-treated neutrophils, whereas it decreased in EXPC-treated neutrophils. The secretion of acid phosphatase increased in ENPC- and EXPC-treated macrophage and neutrophil cultures. Neutrophil myeloperoxidase release increased in EXPC-treated (5.55 mg/mL) cultures, whereas it decreased in ENPC-treated cultures. ENPC and EXPC increased the adhesion of neutrophils to microplate. These findings demonstrated that CPP displayed a dual mode of action, inhibiting or enhancing numerous phagocytic activities of macrophages and neutrophils.

Responses of tropical tree species to urban air pollutants: ROS/RNS formation and scavenging

Air pollution in an urban environment is the major stress factor for vegetation due to the direct generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS). To quantify urban air pollution-induced ROS/RNS formation, damage and detoxification, nine different biochemical parameters related to free radical formation, scavenging and membrane damage were estimated in twelve tropical tree species. The experiment was performed in three different seasons at four distinct urban environments in Varanasi city located in the Indo-Gangetic plain of India. Redundancy analysis was performed to statistically assess the relationship between air pollutants (PM2.5, NO2, SO2 and O3) and temperature with ROS/RNS generation and their detoxification. Significant effects of air pollution exposure and temperature on ROS/RNS formation, scavenging and membrane damage were recorded with increasing pollution load in the city for all the tree species. The extent of variability (47–87%) in responses of different tree species was due to their intrinsic ability to scavenge free radicals which minimized the membrane damage. PM2.5, NO2 and O3 were identified as major pollutants that influenced trees to different extents in regulating ROS/RNS. However, the response was maximum against NO2 (34–72%) followed by PM2.5 (16–64%) and O3 (3–31%), indicating that under urban environment, trees are considerably sensitive to the combined effects of both particulate and gaseous pollutants. Reactive oxygen intermediate release, total free radical scavenging activity, NO scavenging activity and membrane stability index were identified as major parameters which showed distinct responses with increasing pollution load. Caesalpinia sappan, Ficus religiosa and Albizia lebbeck were identified as most tolerant tree species having higher ROS/RNS scavenging potential resulted in lower membrane damage. Thus responses of urban trees to air pollution are governed by their intrinsic defence mechanisms to scavenge ROS/RNS by maintaining the membrane integrity through integrated cross-talk between different antioxidative pathways.

Trichinella spiralis Calreticulin Binds Human Complement C1q As an Immune Evasion Strategy.

As a multicellular parasitic nematode, Trichinella spiralis regulates host immune responses by producing a variety of immunomodulatory molecules to escape from host immune attack, but the mechanisms underlying the immune evasion are not well understood. Here, we identified that T. spiralis calreticulin (Ts-CRT), a Ca2+-binding protein, facilitated T. spiralis immune evasion by interacting with the first component of human classical complement pathway, C1q. In the present study, Ts-CRT was found to be expressed on the surface of different developmental stages of T. spiralis as well as in the secreted products of adult and muscle larval worms. Functional analysis identified that Ts-CRT was able to bind to human C1q, resulting in the inhibition of C1q-initiated complement classical activation pathway reflected by reduced C4/C3 generation and C1q-dependent lysis of antibody-sensitized sheep erythrocytes. Moreover, recombinant Ts-CRT (rTs-CRT) binding to C1q suppressed C1q-induced THP-1-derived macrophages chemotaxis and reduced monocyte–macrophages release of reactive oxygen intermediates (ROIs). Blocking Ts-CRT on the surface of newborn larvae (NBL) of T. spiralis with anti-Ts-CRT antibody increased the C1q-mediated adherence of monocyte–macrophages to larvae and impaired larval infectivity. All of these results suggest that T. spiralis-expressed Ts-CRT plays crucial roles in T. spiralis immune evasion and survival in host mostly by directly binding to host complement C1q, which not only reduces C1q-mediated activation of classical complement pathway but also inhibits the C1q-induced non-complement activation of macrophages.

Modulatory potential of resveratrol during lung inflammatory disease

Neutrophils are the first cells to achieve the sites of infection or inflammation in the lungs. The massive accumulation of these cells is associated with acute and chronic lung injury. Therefore, they have been implicated in the pathogenesis of many lung diseases through the release of reactive oxygen intermediates, proteolytic enzymes and Neutrophil Extracellular Traps (NETs). The excessive and continuous release of NETs, fibers composed by decondensed chromatin coated with neutrophil proteins, are associated to the impairment of lung function in different pathological settings.Flavonoids inhibit the respiratory burst of neutrophils in mammals. However, one of these flavonoids, resveratrol has a particular chemical property. It reduce Cu(II) to Cu(I) form with concomitant formation of reactive oxygen species, which can produce DNA breakage as reported in several in vitro models.We hypothesize that direct resveratrol administration in lungs can cleave DNA in NETs, improving lung function during acute airway infections or chronic inflammatory lung diseases.If the hypothesis is correct, the control of NET formation can be used to reduce the inflammatory environment in lung after neutrophil stimuli. Additionally, the production of proinflammatory cytokines by neutrophils could be also diminished by resveratrol administration. In this sense, this flavonoid provides a multifaceted opportunity for treatment of lung diseases with strong or chronic neutrophil activation.

1190 Activated Protein C Alters Neutrophil Reactive Oxygen Intermediates in Preterm Neonates

BackgroundInflammation and infection are important aetiological factors in development of preterm birth. Inflammation is associated with many disorders of preterm infants including periventricular leukomalacia, chronic lung disease and necrotising eneterocolitis.AimsTo...

Leishmania amazonensis fails to induce the release of reactive oxygen intermediates by CBA macrophages

SummaryCBA mouse macrophages effectively control Leishmania major infection, yet are permissive to Leishmania amazonensis. It has been established that some Leishmania species are destroyed by reactive oxygen species (ROS). However, other species of Leishmania exhibit resistance to ROS or even down-modulate ROS production. We hypothesized that L. amazonensis–infected macrophages reduce ROS production soon after parasite–cell interaction. Employing a highly sensitive analysis technique based on chemiluminescence, the production of superoxide () and hydrogen peroxide (H2O2) by L. major- or L. amazonensis-infected CBA macrophages were measured. L. major induces macrophages to release levels of 3·5 times higher than in uninfected cells. This production is partially dependent on NADPH oxidase (NOX) type 2. The level of accumulated H2O2 is 20 times higher in L. major-than in L. amazonensis-infected cells. Furthermore, macrophages stimulated with L. amazonensis release amounts of ROS similar to uninfected cells. These findings support previous studies showing that CBA macrophages are effective in controlling L. major infection by a mechanism dependent on both production and H2O2 generation. Furthermore, these data reinforce the notion that L. amazonensis survive inside CBA macrophages by reducing ROS production during the phagocytic process.

Coxiella burnetiiAcid Phosphatase Inhibits the Release of Reactive Oxygen Intermediates in Polymorphonuclear Leukocytes

Coxiella burnetii, the etiological agent of Q fever, is a small, Gram-negative, obligate intracellular bacterium. Replication of C. burnetii during infection has been shown to be increased by decreasing oxidative stress using p47(phox -/-) and iNOS(-/-) mice in vivo and by pharmacologic inhibitors in vitro. Building upon this model, we investigated the role polymorphonuclear leukocytes (PMN) play in the control of infection, since NADPH oxidase-mediated release of reactive oxygen intermediates (ROI) is a primary bactericidal mechanism for these cells that is critical for early innate clearance. Earlier studies suggested that C. burnetii actively inhibited release of ROI from PMN through expression of an unidentified acid phosphatase (ACP). Recent genomic annotations identified one open reading frame (CBU0335) which may encode a Sec- and type II-dependent secreted ACP. To test this model, viable C. burnetii propagated in tissue culture host cells or axenic media, C. burnetii extracts, or purified recombinant ACP (rACP) was combined with human PMN induced with 4-phorbol 12-myristate 13-acetate (PMA). The release of ROI was inhibited when PMN were challenged with viable C. burnetii, C. burnetii extracts, or rACP but not when PMN were challenged with electron beam-inactivated C. burnetii. C. burnetii extracts and rACP were also able to inhibit PMA-induced formation of NADPH oxidase complex on PMN membranes, suggesting a molecular mechanism responsible for this inhibition. These data support a model in which C. burnetii eludes the primary ROI killing mechanism of activated PMN by secreting at least one acid phosphatase.

PPARγ Controls Dectin-1 Expression Required for Host Antifungal Defense against Candida albicans

We recently showed that IL-13 or peroxisome proliferator activated receptor γ (PPARγ) ligands attenuate Candida albicans colonization of the gastrointestinal tract. Here, using a macrophage-specific Dectin-1 deficient mice model, we demonstrate that Dectin-1 is essential to control fungal gastrointestinal infection by PPARγ ligands. We also show that the phagocytosis of yeast and the release of reactive oxygen intermediates in response to Candida albicans challenge are impaired in macrophages from Dectin-1 deficient mice treated with PPARγ ligands or IL-13. Although the Mannose Receptor is not sufficient to trigger antifungal functions during the alternative activation of macrophages, our data establish the involvement of the Mannose Receptor in the initial recognition of non-opsonized Candida albicans by macrophages. We also demonstrate for the first time that the modulation of Dectin-1 expression by IL-13 involves the PPARγ signaling pathway. These findings are consistent with a crucial role for PPARγ in the alternative activation of macrophages by Th2 cytokines. Altogether these data suggest that PPARγ ligands may be of therapeutic value in esophageal and gastrointestinal candidiasis in patients severely immunocompromised or with metabolic diseases in whom the prevalence of candidiasis is considerable.

The Glycosylphosphatidylinositol-Anchored Protease Sap9 Modulates the Interaction ofCandida albicanswith Human Neutrophils

Human polymorphonuclear neutrophils (PMNs) play a major role in the immune defense against invasive Candida albicans infection. This fungal pathogen produces a set of aspartic proteases that directly contributes to virulence properties such as adhesion, tissue invasion, and immune evasion. We show here that, in contrast to other secreted proteases, the cell surface-associated isoform Sap9 has a major impact on the recognition of C. albicans by PMNs. SAP9 is required for the induction of PMN chemotaxis toward C. albicans filaments, an essential prerequisite of effective PMN activation. Furthermore, deletion of SAP9 leads to a mitigated release of reactive oxygen intermediates (ROI) in human PMNs and decreases C. albicans-induced apoptosis triggered by ROI formation. In confrontation assays, killing of a SAP9 deletion mutant is reduced in comparison to wild-type C. albicans. These data clearly implicate Sap9 protease activity in the initiation of protective innate immunity and suggest novel molecular mechanisms in C. albicans-host interaction leading to neutrophil activation.

Rice isoflavone reductase‐like protein is associated with root development and ROS stress resistance

Isoflavone phytoalexins and isoflavone reductase gene in the rice has not been identified and characterized. We characterized OsIRL protein using Northern, Western, 2‐DE, immunohistochemical analysis, and transgenic plant in rice. The transcript and proteins were more abundant in stems and roots than in flower and leaves. Expression of OsIRL was more accumulated in the root than in the shoot and seed. To investigate the release of reactive oxygen intermediates (ROI) by seed during germination, we utilized the apoplastic oxidation of 2¡‐,7¡‐dichlorofluorescin to fluorescent 2¡‐7¡‐dichloroflurescein and DAB staining as an in vivo assay. Expression of OsIRL was increased in the root elongation and maturation position carrying accumulation of ROI. Expression of OsIRL was increased in the oxidants (H2O2, FeCl3, MV) and decreased in the antioxidants such as reduced glutathione in the root and rice suspension cell. OsIRL was accumulated in the root sclerenchyma, outside of cortex, endodermis, and metaphloem cell. A certain concentration of H2O2 was necessary for root elongation, suggesting that OsIRL protein may play a role as antioxidant. The OsIRL transcripts levels were highly increased in callus, leaves, stem, and roots by treatment with ABA. The transgenic OsIRL plants have methyl viologen resistance, ROS‐generator, indicating that OsIRL have involved in root growth and resistance to oxidative stress.

Neutrophil-mediated oxidative burst and host defense are controlled by a Vav-PLCγ2 signaling axis in mice

Oxidative burst, a critical antimicrobial mechanism of neutrophils, involves the rapid generation and release of reactive oxygen intermediates (ROIs) by the NADPH oxidase complex. Genetic mutations in an NADPH oxidase subunit, gp91 (also referred to as NOX2), are associated with chronic granulomatous disease (CGD), which is characterized by recurrent and life-threatening microbial infections. To combat such infections, ROIs are produced by neutrophils after stimulation by integrin-dependent adhesion to the ECM in conjunction with stimulation from inflammatory mediators, or microbial components containing pathogen-associated molecular patterns. In this report, we provide genetic evidence that both the Vav family of Rho GTPase guanine nucleotide exchange factors (GEFs) and phospholipase C-gamma2 (PLC-gamma2) are critical mediators of adhesion-dependent ROI production by neutrophils in mice. We also demonstrated that Vav was critically required for neutrophil-dependent host defense against systemic infection by Staphylococcus aureus and Pseudomonas aeruginosa, 2 common pathogens associated with fatal cases of hospital-acquired pneumonia. We identified a molecular pathway in which Vav GEFs linked integrin-mediated signaling with PLC-gamma2 activation, release of intracellular Ca2+ cations, and generation of diacylglycerol to control assembly of the NADPH oxidase complex and ROI production by neutrophils. Taken together, our data indicate that integrin-dependent signals generated during neutrophil adhesion contribute to the activation of NADPH oxidase by a variety of distinct effector pathways, all of which require Vav.

Oxidative burst without phagocytes: the role of respiratory proteins

Respiratory proteins of vertebrates (hemoglobin) and invertebrates (hemocyanin) can generate highly reactive oxygen intermediates. New work shows that microbial compounds directly activate this release of reactive oxygen intermediates that kills the microbes.

Apis Mellifera Venom and Melittin Block neither NF-κB-p50-DNA Interactions nor the Activation of NF-κB, Instead They Activate the Transcription of Proinflammatory Genes and the Release of Reactive Oxygen Intermediates

Many alternative treatment approaches, originating from Asia, are becoming increasingly popular in the Western hemisphere. Recently, an article published in a renowned journal reported that venom of apis mellifera (bee venom (BV)) and melittin mediate immune-modulating effects by blocking the activation of the transcription factor NF-kappaB. Such a modus operandi would corroborate the many claims of beneficial effects of BV treatment and give immediate credit to this form of therapy. Fibroblast-like synoviocytes from rheumatoid arthritis patients and dermal fibroblast cells and white blood cells from healthy volunteers were used to study the effects of BV and melittin on the activation of NF-kappaB and a series of genes that are markers of inflammation. EMSAs demonstrate that neither BV nor melittin blocked IL-1beta-induced NF-kappaB activation; neither did they affect phosphorylation or degradation of IkappaB. Contrary to published data, even high concentrations of BV and melittin were without any effect on NF-kappaB-p50-DNA interactions. More importantly, in fibroblast-like synoviocytes, but also in dermal fibroblasts as well as in mononuclear cells exposed to BV or melittin, mRNA levels of several proinflammatory genes are significantly increased, and Western blot data show elevated cyclooxygenase-2 protein levels. Furthermore, exposure to BV higher than 10 mug/ml resulted in disintegration of all cell types tested. In addition, large quantities of oxygen radicals are produced in a dose-dependent manner in leukocytes exposed to BV. Taken together, data presented in this work do not corroborate an earlier report regarding the effectiveness of BV as an inhibitor of the transcription factor NF-kappaB.

Chemokines: Central Mediators of the Innate Response to Sepsis

Sepsis represents a perplexing challenge for the innate immune system. In response to microbial challenge, an immunocompetent host initiates an immediate robust response to contain and clear infection. However, if the infection is not controlled and spreads beyond the local site, this pervasive immune response, once systemic, often results in detrimental complications such as systemic inflammatory response syndrome, multiple organ failure, and immune paralysis, contributing to the high mortality observed in sepsis. Resident tissue macrophages, as sentinels of the host immune response to infection, are responsible for recognition of infection through several pattern recognition molecules and initiation of a complex cytokine/chemokine cascade. Chemokines are central mediators involved in recruitment of specific leukocyte populations to the site of infection, differentiation of classically- and alternatively-activated macrophages, and regulation of important processes such as pathogen phagocytosis and killing, release of reactive oxygen intermediates and proteases, and subsequent chemokine production. This review explores the role of the peritoneal macrophage and the importance of chemokines in macrophage activation during septic peritonitis. Continued investigation of chemokines and their mechanisms of action will provide valuable insight into the development of effective clinical therapeutics for the treatment of sepsis as well as other inflammatory disorders.

Grape Seed and Skin Extracts Inhibit Platelet Function and Release of Reactive Oxygen Intermediates

Red wine and purple grape juice contain polymeric flavonoids with antioxidant properties believed to be protective against cardiovascular events but the alcohol and sugar content of these beverages has curtailed their medicinal use. Acute cardiac events are also associated with enhanced inflammation and thrombosis. In this study, the extracts from grape skins or seeds were examined for their anti-inflammatory properties and effect on platelet release of reactive oxygen intermediates. Incubation of platelets with seed or skin extract led to a decrease in platelet aggregation from 68.8+/-19.8% to 45+/-3.6% for seeds and to 27+/-7.2% for skin, respectively (P<0.05). Platelet incubation with grape skin or seed extracts led to a marked decrease in superoxide release from 73+/-6.2 to 2+/-3.4 for grape seeds and to 0.33+/-0.57 for grape skin (chemilum. units; P<0.05) as well as a significant increase in radical-scavenging activity, decrease in reactive oxygen species release by confocal microscopy, and enhanced platelet NO was measured using an NO-sensitive microelectrode. These effects were dose dependent for both grape extracts. Coincubation with seeds and skins led to additive inhibition of platelet aggregation, enhanced NO release, and prevented superoxide production. Incubation with seed or skin extracts led to an immediate attenuation of release of the inflammatory mediator, soluble CD40 ligand. Thus, the extracts from purple grape skins and seeds inhibit platelet function and platelet-dependent inflammatory responses at pharmacologically relevant concentrations. These findings suggest potentially beneficial platelet-dependent antithrombotic and anti-inflammatory properties of purple grape-derived flavonoids.

Reactive molecules and microorganisms and copper intrauterine devices

Copper in the intrauterine device (IUD) has been shown to have bactericidal activity in addition to enhancing contraceptive activity. However the exact mechanism of antimicrobial action of copper IUD is not known. The increase in the number of polymorphonuclear leukocytes in the endometrium following IUD insertion has been documented. These inflammatory cells get activated and release reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI). However metals like iron and copper act as catalyst in augmenting the toxicity of reactive oxygen intermediates. Gupta et al. observed that the copper levels in the uterine flushings were significantly higher one-week post insertion along with increased ROI levels. For control of infection interaction of nitric oxide with microbial enzymes containing iron—sulphydryl proteins and a balance between ROI and RNI could account for antimicrobial action. (excerpt)

Engineered Saccharomycescerevisiae Strain BioS‐OS1/2, for the Detection of Oxidative Stress

One of the major stress factors during space and high-altitude flight is the oxidative damage caused by the release of reactive oxygen intermediates (ROIs) in human tissues. ROIs are released in response to several stress factors including radiation in space. Since ROIs contribute to several pathological conditions, there has been a great interest in developing a biosensor that can monitor the impact of ROIs on biological systems. Toward this goal, we sought to engineer a yeast stain that can monitor oxidative stress and be easily integrated into a biosensor platform. Saccharomyces cerevisiae respond to hyperoxidative stress by activating the expression of many proteins including the transcription factor, Yap1. Activated Yap1 primarily binds to the Yap-1 response elements in the promoters of genes that combat oxidative stress. Based on these observations, we genetically altered the Yap-1 pathway in the YCR094W BY4742 strain of S. cerevisiae by fusing the YREs in the promoter region of TRX2 gene to a cDNA-insert encoding green fluorescent protein (GFP). Exposure of this engineered yeast strain BioS-OS1 to varying levels of oxidative stress, as generated by different concentrations of H(2)O(2) or diamide, elicits robust expression of GFP that can be monitored by the fluorescence of GFP by as early as 1 h. BioS-OS1 can detect a H(2)O(2) concentration from 300 microM onward. We also show that the signaling strength of the strain can be increased by engineering multiple YREs in the upstream of the cDNA-insert encoding GFP. Thus, the results presented here demonstrate that the engineered BioS-OS yeast strain can detect ROI-generating oxidative stress and validate the use of this prototypic strain for the development of a biosensor to detect and monitor oxidative stress factors during space and high altitude flights.

Mitochondrial Signal Transduction Abnormalities in Systemic Lupus Erythematosus

Engagement of T cell receptors by antigen-presenting cells or stimulation by cytokines determine whether the cell will become activated, anergic or die via apoptosis or necrosis. Ca2+ is a key second messenger that delivers signal from the cell surface, reactive oxygen intermediates (ROI) and nitric oxide (NO) are recently recognized as important mediators of T-cell activation. NO as a multifunctional intracellular and intercellular messenger induces mitochondrial biogenesis in many cell types such as lymphocytes. Mitochondria produce ROI and store and release Ca2+ in response to activation and death signals. Rapid Ca2+ fluxing is increased while sustained Ca2+ signaling is decreased in lupus T cells. Lupus T cells contain increased numbers and mass of mitochondria. Serum NO levels and production of NO by monocytes is increased in patients with systemic lupus erythematosus (SLE). Lupus T cells exhibit mitochondrial hyperpolarization and increased mitochondrial mass which confer predisposition to necrosis rather than apoptosis in response to repetitive activation and death signals. Exposure of normal T cells to NO dose-dependently increase the mitochondrial mass and mimic rapid and sustained Ca2+ signal abnormalities observed in lupus T cells. Thus, increased mitochondrial biogenesis may account for altered Ca2+ handling and represents novel targets for pharmacological intervention in SLE. Keywords: Mitochondrial Signal Transduction, Systemic Lupus, necrosis, T cells, mitochondrial biogenesis

The Effects of Tamoxifen and Its Metabolites on Platelet Function and Release of Reactive Oxygen Intermediates

Tamoxifen is effective in the prevention and treatment of breast cancer, but its use is associated with an increased risk of thrombosis. The mechanism for this effect is unknown. Reactive oxygen intermediates enhance platelet-dependent thrombosis, and in oncological studies, tamoxifen has been shown to increase production of reactive oxygen species. Therefore, the effects of tamoxifen and its bioactive metabolites on platelet activity and platelet reactive oxygen species were determined. Platelets were incubated with tamoxifen or the metabolites 4-hydroxy-tamoxifen (4-OH), N-desmethyl tamoxifen, or 4-hydroxy-N-desmethyl tamoxifen (endoxifen). Tamoxifen metabolites have been previously shown to possess enhanced bioactivity, and consistent with this observation, tamoxifen metabolites but not tamoxifen modestly increased platelet aggregation. These effects were similar with platelets isolated from male or female subjects. Platelet nitric oxide release or cGMP levels were not altered by incubation with tamoxifen or any of its metabolites. Incubation with tamoxifen metabolites increased stimulation-dependent platelet superoxide release [8.1 +/- 1.6 arbitrary units (a.u.) for control versus 15.2 +/- 3.5 a.u. for 4-OH; P < 0.01]. Coincubation with a superoxide dismutase mimetic eliminated the tamoxifen metabolite-induced enhancement of platelet aggregation. Corresponding to increased superoxide release, incubation with tamoxifen metabolites enhanced the functional activation of NADPH oxidase as determined by phosphorylation of its subunits p47(phox) and p67(phox). In summary, incubation of platelets with the active metabolites of tamoxifen increases stimulation-dependent superoxide release through a NADPH oxidase-dependent mechanism. This results in modest changes in platelet function and seems to be consistent with previous oncological studies demonstrating tamoxifen-dependent increase in reactive oxygen species generation.