NADPH oxidase produces reactive oxygen species and maintains survival of rat astrocytes

Reactive oxygen species (ROS) produced by activated astrocytes have been considered to be involved in the pathogenesis of neurodegenerative diseases, while NADPH oxidase is an essential enzyme involved in ROS-mediated signal transduction. The goal of the present study was to determine whether NADPH oxidase plays a role in ROS generation and cell survival in rat astrocytes. We found that the release of ROS in rat astrocytes was significantly increased by stimulation with calcium ionophore or opsonized zymosan, which are known to trigger a respiration burst in phagocytes by the NADPH oxidase pathway. Further study indicated that diphenylene iodonium (DPI), an inhibitor of NADPH oxidase, significantly suppressed the increase of ROS release caused by the calcium ionophore or opsonized zymosan. Cell survival assay and fluorescence double dyeing with acridine orange and ethidium bromide showed that DPI dose- and time-dependently decreased the viability of normal astrocytes, whereas exogenous supplementation of H2O2 can reverse the survival of DPI-treated astrocytes. For the first time, our results suggest that NADPH oxidase is an important enzyme for the generation of ROS in astrocytes, and the ROS generated by NADPH oxidase play an essential role in astrocyte survival.     

Anti-inflammatory action of gentamycin through inhibitory effect on neutrophil NADPH oxidase activity

The effects of gentamycin on the NADPH oxidase (EC 1.6.99.6) from human neutrophils in both whole-cell and fully soluble (cell-free) systems were investigated. Gentamycin was found to inhibit, concentration-dependently, the superoxide generation of neutrophils exposed to phorbol myristate acetate in a whole-cell system and the activation of superoxide-generating NADPH oxidase by sodium dodecyl sulfate in a cell-free system. The concentrations of the drug required for 50% inhibition of the oxidase (IC₅₀) were 150 μM in the whole-cell system and 10 μM in the cell-free system. In addition, in the cell-free system, the drug did not change the K_m value for NADPH of the oxidase. However, gentamycin did not the superoxide generation of NADPH oxidase after its activation in the cell-free system, suggesting that the drug do not have superoxide-scavenger action. These results suggest that gentamycin, an aminoglycoside antibiotic, may exhibit an anti-inflammatory action due to inhibition of neutrophil NADPH oxidase activation.     

Endogenous production of reactive oxygen species by the NADPH oxidase complexes is a determinant of γ-glutamyltransferase expression

Abstract

γ-Glutamyltransferase (GGT) plays a significant role in antioxidant defence and participates in the metabolism of glutathione (GSH). The enzyme is up-regulated after acute oxidative stress and during pro-oxidant periods, but the underlying regulatory mechanisms are not well known. The present investigation studied whether the endogenous reactive oxygen species (ROS) level was a determinant for GGT expression. A substantial amount of ROS is produced through the NADPH oxidase (NOX) system and knockdown of p22phox, a sub-unit of NOX1-4, resulted not only in reduced ROS levels but also in reduced GGT expression in human endometrial carcinoma cells. Phorbol-12-myristate-13-acetate (PMA) is an activator of NOX and it was found that PMA treatment of human colon carcinoma cells both increased cellular ROS levels and subsequently up-regulated GGT expression. On the other hand, the NOX inhibitor apocynin reduced ROS levels as well as GGT expression. The GGT mRNA sub-type A was increased after PMA-induced NOX activation. These results demonstrate that ROS generated from NOX enzymes are a significant determinant for GGT expression and activity.     

Simultaneous use of electrochemistry and chemiluminescence to detect reactive oxygen species produced by human neutrophils

A novel approach for the simultaneous optical and electrochemical detection of biologically produced reactive oxygen species has been developed and applied. The set-up consists of a luminol-dependent chemiluminescence assay combined with two amperometric biosensors sensitive to superoxide anion radicals (O(2)(-)) and hydrogen peroxide (H(2)O(2)), respectively. The method permits direct, real-time in vitro determination of both extra- and intracellular O(2)(-) and H(2)O(2) produced by human neutrophil granulocytes. The rate of O(2)(-) production by stimulated neutrophils was calculated to about 10(-17)mol s(-1) per single cell. With inhibited NADPH oxidase, a distinct extracellular release of H(2)O(2) instead of O(2)(-) was obtained from stimulated neutrophils with the rate of about 3 x 10(-18)mol s(-1) per single cell. When the H(2)O(2) release was discontinued, fast H(2)O(2) utilisation was observed. Direct interaction with and possibly attachment of neutrophils to redox protein-modified gold electrodes, resulted in a spontaneous respiratory burst in the population of cells closely associated to the electrode surface. Hence, further stimulation of human neutrophils with a potent receptor agonist (fMLF) did not significantly increase the O(2)(-) sensitive amperometric response. By contrast, the H(2)O(2) sensitive biosensor, based on an HRP-modified graphite electrode, was able to reflect the bulk concentration of H(2)O(2), produced by stimulated neutrophils and would be very useful in modestly equipped biomedical research laboratories. In summary, the system would also be appropriate for assessment of several other metabolites in different cell types, and tissues of varying complexity, with only minor electrode modifications.     

Induction of NADPH oxidase activity and reactive oxygen species production by a single Trypanosoma cruzi antigen

Trypanosoma cruzi is an intracellular protozoan parasite that predominantly invades mononuclear phagocytes and is able to establish a persistent infection. The production of reactive oxygen species (ROS) by phagocytes is an innate defence mechanism against microorganisms. It has been postulated that ROS such as superoxide anion (O₂), hydrogen peroxide and peroxynitrite, may play a crucial role in the control of pathogen growth. However, information on parasite molecules able to trigger ROS production is scarce. In this work, we investigated whether cruzipain, an immunogenic glycoprotein from T. cruzi, was able to trigger the oxidative burst by murine cells. By employing chemiluminiscense and flow-cytometric analysis, we demonstrated that cruzipain induced ROS production in splenocytes from non-immune and cruzipain immune C57BL/6 mice and in a Raw 264.7 macrophage cell line. We also identified an O₂⁻ molecule as one of the ROS produced after antigen stimulation. Cruzipain stimulation induced NOX2 (gp91^phox) and p47^phox expression, as well as the co-localisation of both NADPH oxidase enzyme subunits. In the current study, we provide evidence that cruzipain not only increased ROS production but also promoted IL-6 and IL-1β cytokine production. Taken together, we believe these results demonstrate for the first time that cruzipain, a single parasite molecule, in the absence of infection, favors oxidative burst in murine cells. This represents an important advance in the knowledge of parasite molecules that interact with the phagocyte defence mechanism.     

Reactive oxygen species produced by NADPH oxidase are involved in pollen tube growth

Tip-localized reactive oxygen species (ROS) were detected in growing pollen tubes by chloromethyl dichlorodihydrofluorescein diacetate oxidation, while tip-localized extracellular superoxide production was detected by nitroblue tetrazolium (NBT) reduction. To investigate the origin of the ROS we cloned a fragment of pollen specific tobacco NADPH oxidase (NOX) closely related to a pollen specific NOX from Arabidopsis. Transfection of tobacco pollen tubes with NOX-specific antisense oligodeoxynucleotides (ODNs) resulted in decreased amount of NtNOX mRNA, lower NOX activity and pollen tube growth inhibition. The ROS scavengers and the NOX inhibitor diphenylene iodonium chloride (DPI) inhibited growth and ROS formation in tobacco pollen tube cultures. Exogenous hydrogen peroxide (H2O2) rescued the growth inhibition caused by NOX antisense ODNs. Exogenous CaCl2 increased NBT reduction at the pollen tube tip, suggesting that Ca2+ increases the activity of pollen NOX in vivo. The results show that tip-localized ROS produced by a NOX enzyme is needed to sustain the normal rate of pollen tube growth and that this is likely to be a general mechanism in the control of tip growth of polarized plant cells.     

Cisplatin induces production of reactive oxygen species via NADPH oxidase activation in human prostate cancer cells

Abstract

This study aimed to examine the roles of reactive oxygen species (ROS) in cisplatin treatment of human prostate cancer cells; hormone-sensitive LNCaP and hormone-refractory PC3 and DU145 cells. Intracellular levels of ROS and H₂O₂ were measured and visualized using specific fluorescent probes. NADPH oxidase (NOX) activity was detected by lucigenin chemiluminescence assay. Expression levels of NOX isoforms were determined by semi-quantitative RT-PCR. Cisplatin treatment increased the intracellular levels of ROS and H₂O₂ in three prostate cancer cell lines. The increase was transient and robust in hormone-sensitive LNCaP cells compared with hormone-refractory PC3 and DU145 cells. Consistent with these findings, the NOX activity induced by cisplatin was higher in LNCaP cells than in PC3 and DU145 cells. Expression pattern of NOX isoforms varied among three cell lines and the NOX activity was independent of NOX expression. Taken together, we have shown that cisplatin induces production of ROS and H₂O₂ via NOX activation in human prostate cancer cell lines, which is most prominent in hormone-sensitive LNCaP cells.     

Bone-marrow derived hematopoietic stem/progenitor cells express multiple isoforms of NADPH oxidase and produce constitutively reactive oxygen species

Consolidated evidence highlights the importance of redox signalling in poising the balance between self-renewal and differentiation in adult stem cells. The present study shows that human hematopoietic stem/progenitor cells (HSCs) constitutively generate low levels of hydrogen peroxide whose production is inhibited by DPI, apocynin, catalase, and LY294002 and scarcely stimulated by PMA. Moreover, it is shown that HSCs express at the mRNA and protein levels the catalytic subunits of NOX1, NOX2, and NOX4 isoforms of the NADPH oxidase family along with the complete battery of the regulatory subunits p22, p40, p47, p67, rac1, rac2, NOXO1, and NOXA1 as well as the splicing variant NOX2s and that the three NOX isoforms are largely co-expressed in the same HSC. These findings are interpreted in terms of a positive feed-back mechanism of NOXs activation enabling a fine tuning of the ROS level to be possibly used in redox-mediated signalling for growth and differentiation of HSCs.     

Pigment epithelium-derived factor stimulates skeletal muscle glycolytic activity through NADPH oxidase-dependent reactive oxygen species production

Pigment epithelium-derived factor is a multifunctional serpin implicated in insulin resistance in metabolic disorders. Recent evidence suggests that exposure of peripheral tissues such as skeletal muscle to PEDF has profound metabolic consequences with predisposition towards chronic conditions such as obesity, type 2 diabetes, metabolic syndrome and polycystic ovarian syndrome. Chronic inflammation shifts muscle metabolism towards increased glycolysis and decreased oxidative metabolism. In the present study, we demonstrate a novel effect of PEDF on cellular metabolism in mouse cell line (C2C12) and human primary skeletal muscle cells. PEDF addition to skeletal muscle cells induced enhanced phospholipase A2 activity. This was accompanied with increased production of reactive oxygen species in a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-dependent manner that triggered a shift towards a more glycolytic phenotype. Extracellular flux analysis and glucose consumption assays demonstrated that PEDF treatment resulted in enhanced glycolysis but did not change mitochondrial respiration. Our results demonstrate that skeletal muscle cells express a PEDF-inducible oxidant generating system that enhances glycolysis but is sensitive to antioxidants and NADPH oxidase inhibition.     

Endotoxin priming of neutrophils requires endocytosis and NADPH oxidase-dependent endosomal reactive oxygen species

NADPH oxidase 2 (Nox2)-generated reactive oxygen species (ROS) are critical for neutrophil (polymorphonuclear leukocyte (PMN)) microbicidal function. Nox2 also plays a role in intracellular signaling, but the site of oxidase assembly is unknown. It has been proposed to occur on secondary granules. We previously demonstrated that intracellular NADPH oxidase-derived ROS production is required for endotoxin priming. We hypothesized that endotoxin drives Nox2 assembly on endosomes. Endotoxin induced ROS generation within an endosomal compartment as quantified by flow cytometry (dihydrorhodamine 123 and Oxyburst Green). Inhibition of endocytosis by the dynamin-II inhibitor Dynasore blocked endocytosis of dextran, intracellular generation of ROS, and priming of PMN by endotoxin. Confocal microscopy demonstrated a ROS-containing endosomal compartment that co-labeled with gp91(phox), p40(phox), p67(phox), and Rab5, but not with the secondary granule marker CD66b. To further characterize this compartment, PMNs were fractionated by nitrogen cavitation and differential centrifugation, followed by free flow electrophoresis. Specific subfractions made superoxide in the presence of NADPH by cell-free assay (cytochrome c). Subfraction content of membrane and cytosolic subunits of Nox2 correlated with ROS production. Following priming, there was a shift in the light membrane subfractions where ROS production was highest. CD66b was not mobilized from the secondary granule compartment. These data demonstrate a novel, nonphagosomal intracellular site for Nox2 assembly. This compartment is endocytic in origin and is required for PMN priming by endotoxin.     

The generation of hydroxyl radicals following superoxide production by neutrophil NADPH oxidase

Stimulated neutrophils generate superoxide and hydroxyl radicals. A membrane-bound NADPH oxidase, inactive in the resting state, is responsible for superoxide production. The production of hydroxyl radicals is through a secondary reaction. A Fenton-catalysed Haber—Weiss reaction is proposed. Transferrin was used as the catalyst in this investigation.     

Relationship between p38 mitogen-activated protein kinase and small GTPase Rac for the activation of NADPH oxidase in bovine neutrophils

Superoxide production by NADPH oxidase is essential for bactericidal properties of neutrophils. However, molecular mechanisms underlying the activation of this enzyme remain largely unknown. Here, using bovine neutrophils we examined the role of p38 mitogen-activated protein kinase (p38 MAPK) in the signaling pathways of the NADPH oxidase activation. Superoxide production was induced by stimulation with serum-opsonized zymosan (OZ) and attenuated by p38 MAPK inhibitor, SB203580. OZ stimulation induced the translocation of p47(phox) and Rac to the plasma membrane and SB203580 completely blocked the translocation of Rac, but only partially blocked that of p47(phox). Furthermore, SB203580 abolished the OZ-elicited activation of Rac, which was assessed by detecting the GTP-bound form of this protein. Phosphatidylinositol 3-kinase (PI3K) inhibitors, wortmannin and LY294002, blocked not only p38 MAPK activation but also Rac activation. However, SB203580 showed no effect on the PI3K activity. These results suggested that PI3K/p38 MAPK/Rac pathway was present in the activation of NADPH oxidase in bovine neutrophils.     

Caveolin-1 is a negative regulator of NADPH oxidase-derived reactive oxygen species

Changes in the expression and function of caveolin-1 (Cav-1) have been proposed as a pathogenic mechanism underlying many cardiovascular diseases. Cav-1 binds to and regulates the activity of numerous signaling proteins via interactions with its scaffolding domain. In endothelial cells, Cav-1 has been shown to reduce reactive oxygen species (ROS) production, but whether Cav-1 regulates the activity of NADPH oxidases (Noxes), a major source of cellular ROS, has not yet been shown. Herein, we show that Cav-1 is primarily expressed in the endothelium and adventitia of pulmonary arteries (PAs) and that Cav-1 expression is reduced in isolated PAs from multiple models of pulmonary artery hypertension (PH). Reduced Cav-1 expression correlates with increased ROS production in the adventitia of hypertensive PA. In vitro experiments revealed a significant ability of Cav-1 and its scaffolding domain to inhibit Nox1–5 activity and it was also found that Cav-1 binds to Nox5 and Nox2 but not Nox4. In addition to posttranslational actions, in primary cells, Cav-1 represses the mRNA and protein expression of Nox2 and Nox4 through inhibition of the NF-κB pathway. Last, in a mouse hypoxia model, the genetic ablation of Cav-1 increased the expression of Nox2 and Nox4 and exacerbated PH. Together, these results suggest that Cav-1 is a negative regulator of Nox function via two distinct mechanisms, acutely through direct binding and chronically through alteration of expression levels. Accordingly, the loss of Cav-1 expression in cardiovascular diseases such as PH may account for the increased Nox activity and greater production of ROS.     

Origin of cadmium-induced reactive oxygen species production: mitochondrial electron transfer versus plasma membrane NADPH oxidase

* Cadmium (Cd(2+)) is an environmental pollutant that causes increased reactive oxygen species (ROS) production. To determine the site of ROS production, the effect of Cd(2+) on ROS production was studied in isolated soybean (Glycine max) plasma membranes, potato (Solanum tuberosum) tuber mitochondria and roots of intact seedlings of soybean or cucumber (Cucumis sativus). * The effects of Cd(2+) on the kinetics of superoxide (O2*-), hydrogen peroxide (H(2)O(2)) and hydroxyl radical ((*OH) generation were followed using absorption, fluorescence and spin-trapping electron paramagnetic resonance spectroscopy. * In isolated plasma membranes, Cd(2+) inhibited O2*- production. This inhibition was reversed by calcium (Ca(2+)) and magnesium (Mg(2+)). In isolated mitochondria, Cd(2+) increased and H(2)O(2) production. In intact roots, Cd(2+) stimulated H(2)O(2) production whereas it inhibited O2*- and (*)OH production in a Ca(2+)-reversible manner. * Cd(2+) can be used to distinguish between ROS originating from mitochondria and from the plasma membrane. This is achieved by measuring different ROS individually. The immediate (相似文献   

Arjunolic acid ameliorates reactive oxygen species via inhibition of p47phox-serine phosphorylation and mitochondrial dysfunction

Impaired cardiovascular function during acute myocardial infarction (MI) is partly associated with recruitment of activated polymorphonuclear neutrophils. The protective role of arjunolic acid (AA; 2,3,23-trihydroxy olean-12-en-28-oic acid) is studied in the modulation of neutrophil functions in vitro by measuring the reactive oxygen species (ROS) generation. Neutrophils were isolated from normal and acute MI mice to find out the efficacy of AA in reducing oxidative stress. Stimulation of neutrophils with phorbol-12-myristate-13-acetate (PMA) resulted in an oxidative burst of superoxide anion (O₂⁻) and enhanced release of lysosomal enzymes. The treatment of neutrophils with PMA induced phosphorylation of Ser345 on p47^phox, a cytosolic component of NADPH oxidase. Furthermore, we observed activated ERK induced phosphorylation of Ser345 in MI neutrophils. Treatment with AA significantly inhibited the phosphorylation of P47^phox and ERK in the stimulated controls and MI neutrophils. Oxidative phosphorylation activities in MI cells were lower than in control, while the glycolysis rates were elevated in MI cells compared to the control. In addition, we observed AA decreased intracellular oxidative stress and reduced the levels of O₂⁻ in neutrophils. This study therefore identifies targets for AA in activated neutrophils mediated by the MAPK pathway on p47^phox involved in ROS generation.     

Glutathione transferase Omega 1 is required for the lipopolysaccharide-stimulated induction of NADPH oxidase 1 and the production of reactive oxygen species in macrophages

Bacterial lipopolysaccharide (LPS) stimulation of macrophages and inflammation via the Toll-like receptor 4 (TLR4) signaling pathway through NF-κΒ generates reactive oxygen species (ROS) and proinflammatory cytokines such as IL-1β, IL-6, and TNFα. Because glutathione transferase Omega 1-1 (GSTO1-1) can catalyze redox reactions such as the deglutathionylation of proteins and has also been implicated in the release of IL-1β we investigated its role in the development of LPS-mediated inflammation. Our data show that shRNA knockdown of GSTO1-1 in macrophage-like J774.1A cells blocks the expression of NADPH oxidase 1 and the generation of ROS after LPS stimulation. Similar results were obtained with a GSTO1-1 inhibitor. To maintain high ROS levels during an inflammatory response, LPS stimulation causes the suppression of enzymes such as catalase and glutathione peroxidase that protect against oxidative stress. The knockdown of GSTO1-1 also attenuates this response. Our data indicate that GSTO1-1 needs to be catalytically active and mediates its effects on the LPS/TLR4 inflammatory pathway upstream of NF-κΒ. These data suggest that GSTO1-1 is a novel target for anti-inflammatory intervention.     

NADPH oxidase-mediated generation of reactive oxygen species: A new mechanism for X-ray-induced HeLa cell death

Oxidative damage is an important mechanism in X-ray-induced cell death. Radiolysis of water molecules is a source of reactive oxygen species (ROS) that contribute to X-ray-induced cell death. In this study, we showed by ROS detection and a cell survival assay that NADPH oxidase has a very important role in X-ray-induced cell death. Under X-ray irradiation, the upregulation of the expression of NADPH oxidase membrane subunit gp91^phox was dose-dependent. Meanwhile, the cytoplasmic subunit p47^phox was translocated to the cell membrane and localized with p22^phox and gp91^phox to form reactive NADPH oxidase. Our data suggest, for the first time, that NADPH oxidase-mediated generation of ROS is an important contributor to X-ray-induced cell death. This suggests a new target for combined gene transfer and radiotherapy.     

Cross-talk between calcium and reactive oxygen species originated from NADPH oxidase in abscisic acid-induced antioxidant defence in leaves of maize seedlings

The signal interactions between calcium (Ca2+) and reactive oxygen species (ROS) originated from plasma membrane NADPH oxidase in abscisic acid (ABA)-induced antioxidant defence were investigated in leaves of maize (Zea mays L.) seedlings. Treatment with ABA led to significant increases in the activity of plasma membrane NADPH oxidase, the production of leaf O2-, and the activities of several antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR). However, such increases were blocked by the pretreatment with Ca2+ chelator EGTA or Ca2+ channel blockers La3+ and verapamil, and NADPH oxidase inhibitors such as diphenylene iodonium (DPI), imidazole and pyridine. Treatment with Ca2+ also significantly induced the increases in NADPH oxidase activity, O2- production and the activities of antioxidant enzymes, and the increases were arrested by pretreatment with the NADPH oxidase inhibitors. Treatment with oxidative stress induced by paraquat, which generates O2-, led to the induction of antioxidant defence enzymes, and the up-regulation was suppressed by the pretreatment of Ca2+ chelator and Ca2+ channel blockers. Our data suggest that a cross-talk between Ca2+ and ROS originated from plasma membrane-bound NADPH oxidase is involved in the ABA signal transduction pathway leading to the induction of antioxidant enzyme activity, and Ca2+ functions upstream as well as downstream of ROS production in the signal transduction event in plants.     

Cytochrome P450 oxidase activity and its role in NADPH dependent lipid peroxidation

A comparison is made between microsomal NADPH-dependent H₂O₂ production and malondialdehyde (MDA) formation in rat liver microsomes, obtained from phenobarbital pretreated rats. An increase in H₂O₂ formation was observed during NADPH-dependent disposition (10 min) of 100 μM diazepam (33%) and 2 mM hexobarbital (69%). In contrast orphenadrine (100 μM) and its mono-N-demethylated metabolite tofenacine (100 μM) decreased the H₂O₂ formation (35% and 55%, respectively). However, all these substrates were found to inhibit NADPH-dependent lipid peroxidation (60 min), estimated by measuring MDA formation, to various extents. These data strongly suggest that the oxidase activity of cytochrome P450 (H₂O₂ production) is not involved in a rate-limiting step in NADPH-dependent lipid peroxidation.     

Generation of reactive oxygen species by equine neutrophils and their effect on motility of equine spermatozoa

Contaminating leukocytes in the ejaculate are an important source of reactive oxygen species (ROS) in human semen. When present in sufficient numbers, they can have a detrimental influence on sperm function in humans. Unfortunately, there is little published information regarding the importance of leukocytes in stallion semen. The objectives of this study were to determine the production of hydrogen peroxide (H2O2) by activated equine neutrophils and to examine the effect of this ROS production on equine sperm motility in vitro. Motile equine spermatozoa (two ejaculates each from four stallions) and peripheral blood neutrophils were isolated on discontinuous Percoll gradients, washed and resuspended in a modified Tyrode's medium. Spermatozoa (25 x 10(6)/ml) were incubated for 30 min at 38 C with neutrophils (0,0.5 x 10(6),1 x 10(6), 5 x 10(6) and 10 x 10(6)/ml) activated by either the protein kinase C agonist, 12-myristate, 13-acetate phorbol ester (PMA; 100 nM) or the leukocyte chemotactic peptide, formyl-methionyl-leucyl-phenylalanine (FMLP; 0.1 mM). Sperm motility was determined by computer-assisted semen analysis (CASA) at time 0 min (T0) and time 30 min (T30), and H2O2 was measured at T30 with the Amplex Red assay kit. At T30, there was a significant (P < 0.01) increase in H2O2 with the addition of 5 x 10 and 10 x 10(6) neutrophils/ml activated by FMLP (0.76 +/- 0.3 and 0.99 +/- 0.4 microM, respectively, versus 0.0024 +/- 0.002 microM in sperm alone), and this increase was associated with a significant (P < 0.001) decrease in total motility (52 +/- 5.1 and 48 +/- 6.0%, respectively, versus 80 +/- 4.7% in sperm alone). At T30, there was also a significant (P < 0.001) increase in H2O2 with the addition of 5 x 10(6) and 10 x 10(6) neutrophils/ml activated by PMA (1.88 +/- 0.2 and 2.07 +/- 0.3 microM, respectively, versus 0.0009 +/- 0.0006 microM in sperm alone). The results of this study demonstrate that 5 x 10(6) activated neutrophils/ml are sufficient to impair equine sperm motility in vitro.