Generation of reactive oxygen species, lipid peroxidation, and human sperm function

Recent studies have demonstrated that human spermatozoa are capable of generating reactive oxygen species and that this activity is significantly accelerated in cases of defective sperm function. In view of the pivotal role played by lipid peroxidation in mediating free radical damage to cells, we have examined the relationships between reactive oxygen species production, lipid peroxidation, and the functional competence of human spermatozoa. Using malondialdehyde production in the presence of ferrous ion promoter as an index of lipid peroxidation, we have shown that lipid peroxidation is significantly accelerated in populations of defective spermatozoa exhibiting high levels of reactive oxygen species production or in normal cells stimulated to produce oxygen radicals by the ionophore, A23187. The functional consequences of lipid peroxidation included a dose-dependent reduction in the ability of human spermatozoa to exhibit sperm oocyte-fusion, which could be reversed by the inclusion of a chain-breaking antioxidant, alpha-tocopherol. Low levels of lipid peroxidation also had a slight enhancing effect on the generation of reactive oxygen species in response to ionophore, without influencing the steady-state activity. At higher levels of lipid peroxidation, both the basal level of reactive oxygen species production and the response to A23187 were significantly diminished. In contrast, lipid peroxidation had a highly significant, enhancing effect on the ability of human spermatozoa to bind to both homologous and heterologous zonae pellucidae via mechanisms that could again be reversed by alpha-tocopherol. These results are consistent with a causative role for lipid peroxidation in the etiology of defective sperm function and also suggest a possible physiological role for the reactive oxygen species generated by human spermatozoa in mediating sperm-zona interaction.     

Radicaux libres et spermatozoïdes humains: physiologie et physiopathologie

The role of reactive oxygen species in the physiopathology of human sperm function has been emphasized in recent years. Their production in semen has been associated with loss of motility, decreased capacity for spermoocyte fusion and loss of fertility. In semen preparations, there are two major sources of reactive oxygen species: leucocytes and spermatozoa themselve. It has been proposed that reactive oxygen species production by human spermatozoa was dependent upon a membrane-bound NADPH oxidase or a mitochondrial diaphorase. Hydrogen peroxide produced by the dismutation of superoxide anion has been recognized as the most toxic oxidizing species for human spermatozoa. Owing to their high content of polyunsaturated fatty acids, it has been proposed that lipid peroxidation of the sperm plasma membrane is largely responsible for defective sperm function. Reactive oxygen species also affect the sperm axoneme as a result of ATP depletion, inhibit mitochondrial functions, and synthesis of DNA, RNA and proteins, produce cytoskeletal modifications and inhibit sperm-oocyte fusion. Human spermatozoa possess enzymatic defence systems such as superoxide dismutase, glutathion peroxidas/reductase and catalase to counteract the toxic effects induced by reactive oxygen species. Correlations have been reported between their effectiveness and the duration of sperm motility. If the excessive production of reactive oxygen species is detrimental for human spermatozoa, they could also participate in the physiological function of the spermatozoa when present at low concentrations. Indeed, reactive oxygen species have been shown to be involved in the activation of several enzymes. Furthermore, sperm capacitation, acrosome reaction and sperm-zona interaction would be enhanced by reactive oxygen species.     

Reactive oxygen species generation and human spermatozoa: The balance of benefit and risk

Although the generation of reactive oxygen species is an activity normally associated with phagocytic leucocytes, mammalian spermatozoa were, in fact, the first cell type in which this activity was described. In recent years it has become apparent that spermatozoa are not the only nonphagocytic cells to exhibit a capacity for reactive oxygen species production, because this activity has been detected in a wide variety of different cells including fibroblasts, mesangial cells, oocytes, Leyding cells endothelial cells, thryroid cells, adipocytes, tumour cell and platelets. Since the capacity to generate reactive oxygen species is apparently so widespread, the risk-benefit equation for these potentially pernicious molecules becomes a matter of intese interest. In the case of human spermatozoa, the risk of manufacturing reactive oxygen metabolites is considerable because these cells are particularly vulnerable to lipid peroxidation. Indeed, there is now good evidence to indicate that oxygen radicals are involved in the initiation of peroxidative damage to the sperm plasma membrane, seen in many cases of male infertility. This risk is off-set by recent data suggesting that superoxide anions and hydrogen peroxide also participate in the induction of key biological events such as hyperactiavated motility and the acrosome reaction. Thus, human spermatozoa appear to use reactive oxygen species for a physiological purpose and have the difficult task of ensuring the balanced generation of these potentially harmful, but biologically important, modulators of cellular function.     

Enhanced Mitochondrial Radical Production in Patients with Rheumatoid Arthritis Correlates with Elevated Levels of Tumor Necrosis Factor alpha in Plasma

Mitochondrial dysfunction contributes to cell damage in a number of human diseases. One significant mechanism by which mitochondria damage cells is by producing reactive oxygen species from the respiratory chain. In this study we measured the production of reactive oxygen species by leukocyte mitochondria in blood from rheumatoid arthritis patients. To do this we used the chemiluminescence of lucigenin, which is accumulated by mitochondria within cells and reacts with superoxide to form a chemiluminescent product. By using specific inhibitors we could distinguish between the production of reactive oxygen species by mitochondria and by NADPH oxidase. There was a five-fold increase in mitochondrial reactive oxygen species production in whole blood and monocytes from patients with rheumatoid arthritis, when compared to healthy subjects or patients with non-rheumatic diseases. There was no increase in mitochondrial reactive oxygen species production by neutrophils from rheumatoid arthritis patients. The enhanced mitochondrial radical production in rheumatoid arthritis patients correlated significantly with increased levels of tumor necrosis factor alpha in plasma (p<0.0001). As tumor necrosis factor alpha is known to increase mitochondrial reactive oxygen species production the elevated mitochondrial radical formation seen in rheumatoid arthritis patients may be due to activation of the mitochondrial radical production. These data suggest that elevated mitochondrial oxidative stress contributes to the pathology of rheumatoid arthritis.     

Mass Spectrometry Reveals New Insights into the Production of Superoxide Anions and 4‐Hydroxynonenal Adducted Proteins in Human Sperm

The free‐radical theory of male infertility suggests that reactive oxygen species produced by the spermatozoa themselves are a leading cause of sperm dysfunction, including loss of sperm motility. However, the field is overshadowed on several fronts, primarily because: i) the probes used to measure reactive oxygen species (ROS) are imprecise; and ii) many reports suggesting that oxygen radicals are detrimental to sperm function add an exogenous source of ROS. Herein, a more reliable approach to measure superoxide anion production by human spermatozoa based on MS analysis is used. Furthermore, the formation of the lipid‐peroxidation product 4‐hydroxynonenal (4‐HNE) during in vitro incubation using proteomics is also investigated. The data demonstrate that neither superoxide anion nor other free radicals that cause 4‐HNE production are related to the loss of sperm motility during incubation. Interestingly, it appears that many of the 4‐HNE adducted proteins, found within spermatozoa, originate from the prostate. A quantitative SWATH analysis demonstrate that these proteins transiently bind to sperm and are then shed during in vitro incubation. These proteomics‐based findings propose a revised understanding of oxidative stress within the male reproductive tract.     

Cyclic nucleotide metabolism and reactive oxygen production by macrophages

The production of reactive oxygen species by elicited rat peritoneal macrophages was assessed by $\text{in vitro}$ measurement of chemiluminescence in the presence of luminol. The divalent ion ionophore A23187 stimulated the production of reactive oxygen species. This action was inhibited by monobutyryl and dibutyryl derivatives of cyclic AMP but was not affected by derivates of cyclic GMP. Cyclic AMP and cyclic GMP concentrations increased rapidly in macrophages exposed to A23187 or zymosan. Indomethacin (20 μmol/1) inhibited the increase in cyclic AMP concentration but not the increase in cyclic GMP concentration. Neither A23187 nor zymosan stimulated adenylate cyclase activity in broken cell preparations of macrophages. The observations are consistent with the hypothesis that PGE produced by macrophages after phagocytotic stimuli may inhibit certain macrophage functions and perform a regulatory role in these cells. This action of PGE may be mediated by cyclic AMP.     

Production of reactive oxygen species by spermatozoa undergoing cooling, freezing, and thawing

In the present study, we provide evidence for the production of reactive oxygen species (ROS) during cryopreservation of bovine spermatozoa. Cooling and thawing of spermatozoa cause an increase in the generation of superoxide radicals. Although nitric oxide production remains unaltered during sperm cooling from 22-4 degrees C, a sudden burst of nitric oxide radicals is observed during thawing. Increase in lipid peroxidation levels have been observed in frozen/thawed spermatozoa and appears to be associated with a reduction in sperm membrane fluidity as detected by spin labeling studies. The data presented provide strong evidence that oxygen free radicals are produced during freezing and thawing of bovine spermatozoa and suggest that these reactive oxygen species may be a cause for the decrease in sperm function following cryopreservation. Mol. Reprod. Dev. 59: 451-458, 2001.     

The use of excised roots from in vitro culture for the determination of superoxide production in plants

The production of reactive oxygen species (ROS) in plants is a common event in metabolic and physiological processes as well as in the response to biotic and abiotic stress. In this paper we will report that root tissue from axenically grown tomato cultivars and Lycopersicon wild species can be used for the determination of superoxide production. Superoxide generation was evaluated following the treatment of root tissues with two general elicitors of the defence response: laminarin and calcium ionophore A23187. Results demonstrated that elicitor reactivity in terms of superoxide generation of the tomato cultivars and the wild species used was different. This suggested varying levels of competence for non-specific active defence. The proposed technique merges the advantages of in vitro cultures and of whole tissues and also demonstrates that root tissue is a suitable material for evaluating free radical release.     

TLR-Mediated Production of Reactive Oxygen Species and Tumor Necrosis Factor Alpha by Human Peripheral Blood Neutrophils

This paper presents the study on TLR-mediated production of reactive oxygen species and tumor necrosis factor alpha by peripheral blood neutrophils in healthy donors stimulated with zymosan (TLR2/6 ligand), peptidoglycan (TLR2/1 ligand), and lipopolysaccharide (TLR4 ligand). Luminol- and lucigen-independent chemiluminescence was used to detect the production of reactive oxygen species. The concentration of tumor necrosis factor alpha was measured by enzyme immunoassay. The plots of dependence of the light sums of luminol- and lucigenin-dependent chemiluminescence on the concentration of each ligand were shaped as saturation curves. The comparison of the light sums of lucigenin-dependent chemiluminescence (the production of superoxide anion radical) and luminol-dependent chemiluminescence (the total production of reactive oxygen species) showed that the contribution of NADPH oxidase to the total TLR-mediated production of oxidants can reach 40–50%. Stimulation indices were calculated to compare the ability of TLR ligands to stimulate the production of reactive oxygen species and tumor necrosis factor alpha by neutrophils. It has been established that the activation of neutrophils with zymosan leads to higher (more than 8-fold) production of reactive oxygen species rather than production of tumor necrosis factor alpha. Unlike zymosan, lipopolysaccharide stimulated the production of tumor necrosis factor alpha to a greater extent (by more than 2 times) than the production of reactive oxygen species. Peptidoglycan takes an intermediate position between these ligands. Thus, the production of effector molecules (reactive oxygen species and tumor necrosis factor alpha) by human peripheral blood neutrophils depends on the nature of the TRL ligand.     

Inhibition of Mitochondrial Complex I Leads to Decreased Motility and Membrane Integrity Related to Increased Hydrogen Peroxide and Reduced ATP Production,while the Inhibition of Glycolysis Has Less Impact on Sperm Motility

Mitochondria have been proposed as the major source of reactive oxygen species in somatic cells and human spermatozoa. However, no data regarding the role of mitochondrial ROS production in stallion spermatozoa are available. To shed light on the role of the mitochondrial electron transport chain in the origin of oxidative stress in stallion spermatozoa, specific inhibitors of complex I (rotenone) and III (antimycin-A) were used. Ejaculates from seven Andalusian stallions were collected and incubated in BWW media at 37°C in the presence of rotenone, antimycin-A or control vehicle. Incubation in the presence of these inhibitors reduced sperm motility and velocity (CASA analysis) (p<0.01), but the effect was more evident in the presence of rotenone (a complex I inhibitor). These inhibitors also decreased ATP content. The inhibition of complexes I and III decreased the production of reactive oxygen species (p<0.01) as assessed by flow cytometry after staining with CellRox deep red. This observation suggests that the CellRox probe mainly identifies superoxide and that superoxide production may reflect intense mitochondrial activity rather than oxidative stress. The inhibition of complex I resulted in increased hydrogen peroxide production (p<0.01). The inhibition of glycolysis resulted in reduced sperm velocities (p<0.01) without an effect on the percentage of total motile sperm. Weak and moderate (but statistically significant) positive correlations were observed between sperm motility, velocity and membrane integrity and the production of reactive oxygen species. These results indicate that stallion sperm rely heavily on oxidative phosphorylation (OXPHOS) for the production of ATP for motility but also require glycolysis to maintain high velocities. These data also indicate that increased hydrogen peroxide originating in the mitochondria is a mechanism involved in stallion sperm senescence.     

The role of external and matrix pH in mitochondrial reactive oxygen species generation

Reactive oxygen species (ROS) generation in mitochondria as a side product of electron and proton transport through the inner membrane is important for normal cell operation as well as development of pathology. Matrix and cytosol alkalization stabilizes semiquinone radical, a potential superoxide producer, and we hypothesized that proton deficiency under the excess of electron donors enhances reactive oxygen species generation. We tested this hypothesis by measuring pH dependence of reactive oxygen species released by mitochondria. The experiments were performed in the media with pH varying from 6 to 8 in the presence of complex II substrate succinate or under more physiological conditions with complex I substrates glutamate and malate. Matrix pH was manipulated by inorganic phosphate, nigericine, and low concentrations of uncoupler or valinomycin. We found that high pH strongly increased the rate of free radical generation in all of the conditions studied, even when DeltapH=0 in the presence of nigericin. In the absence of inorganic phosphate, when the matrix was the most alkaline, pH shift in the medium above 7 induced permeability transition accompanied by the decrease of ROS production. ROS production increase induced by the alkalization of medium was observed with intact respiring mitochondria as well as in the presence of complex I inhibitor rotenone, which enhanced reactive oxygen species release. The phenomena revealed in this report are important for understanding mechanisms governing mitochondrial production of reactive oxygen species, in particular that related with uncoupling proteins.     

Reduction of clofazimine by mycobacterial type 2 NADH:quinone oxidoreductase: a pathway for the generation of bactericidal levels of reactive oxygen species

The mechanism of action of clofazimine (CFZ), an antimycobacterial drug with a long history, is not well understood. The present study describes a redox cycling pathway that involves the enzymatic reduction of CFZ by NDH-2, the primary respiratory chain NADH:quinone oxidoreductase of mycobacteria and nonenzymatic oxidation of reduced CFZ by O(2) yielding CFZ and reactive oxygen species (ROS). This pathway was demonstrated using isolated membranes and purified recombinant NDH-2. The reduction and oxidation of CFZ was measured spectrally, and the production of ROS was measured using a coupled assay system with Amplex Red. Supporting the ROS-based killing mechanism, bacteria grown in the presence of antioxidants are more resistant to CFZ. CFZ-mediated increase in NADH oxidation and ROS production were not observed in membranes from three different Gram-negative bacteria but was observed in Staphylococcus aureus and Saccharomyces cerevisiae, which is consistent with the known antimicrobial specificity of CFZ. A more soluble analog of CFZ, KS6, was synthesized and was shown to have the same activities as CFZ. These studies describe a pathway for a continuous and high rate of reactive oxygen species production in Mycobacterium smegmatis treated with CFZ and a CFZ analog as well as evidence that cell death produced by these agents are related to the production of these radical species.     

NOX5 in human spermatozoa: expression, function, and regulation

Physiological and pathological processes in spermatozoa involve the production of reactive oxygen species (ROS), but the identity of the ROS-producing enzyme system(s) remains a matter of speculation. We provide the first evidence that NOX5 NADPH oxidase is expressed and functions in human spermatozoa. Immunofluorescence microscopy detected NOX5 protein in both the flagella/neck region and the acrosome. Functionally, spermatozoa exposed to calcium ionophore, phorbol ester, or H(2)O(2) exhibited superoxide anion production, which was blocked by addition of superoxide dismutase, a Ca(2+) chelator, or inhibitors of either flavoprotein oxidases (diphenylene iododonium) or NOX enzymes (GKT136901). Consistent with our previous overexpression studies, we found that H(2)O(2)-induced superoxide production by primary sperm cells was mediated by the non-receptor tyrosine kinase c-Abl. Moreover, the H(V)1 proton channel, which was recently implicated in spermatozoa motility, was required for optimal superoxide production by spermatozoa. Immunoprecipitation experiments suggested an interaction among NOX5, c-Abl, and H(V)1. H(2)O(2) treatment increased the proportion of motile sperm in a NOX5-dependent manner. Statistical analyses showed a pH-dependent correlation between superoxide production and enhanced sperm motility. Collectively, our findings show that NOX5 is a major source of ROS in human spermatozoa and indicate a role for NOX5-dependent ROS generation in human spermatozoa motility.     

Light-dependent spin trapping of hydroxyl radical from human erythrocytes.

The generation of reactive oxygen species from human erythrocytes has previously been demonstrated. Furthermore, erythrocytic protoporphyrin IX has been shown to generate superoxide and singlet oxygen when exposed to light. These findings suggest that a component of erythrocytic reactive oxygen species production may be light-dependent. By inhibiting erythrocyte superoxide dismutase, catalase, and glutathione peroxidase with N,N-diethyldithiocarbamate or sodium cyanide, we demonstrate the light-dependent generation of hydroxyl radical in human erythrocytes using spin trapping/Electron Spin Resonance spectroscopy. This finding may be significant in tissues where blood is exposed to light, such as in the eye.     

Reactive oxygen species and human spermatozoa: analysis of the cellular mechanisms involved in luminol- and lucigenin-dependent chemiluminescence.

We have shown that human spermatozoa generate and release reactive oxygen species that can be detected by chemiluminescence techniques. Analysis of the cellular mechanisms responsible for this activity suggests that the probe, luminol, undergoes an intracellular dioxygenation reaction mediated by hydrogen peroxide and a sperm peroxidase located within the acrosome. Support for this model included the following observations: (1) the luminol-dependent signal could be suppressed with peroxidase inhibitors, phenylhydrazine and sodium azide; (2) this suppression could be reversed by the addition of an azide-insensitive peroxidase, horse radish peroxidase (HRP); (3) inhibition of intracellular superoxide dismutase (SOD) with potassium cyanide (KCN) suppressed the luminol signal; (4) peroxidase activity could be detected in purified populations of human spermatozoa with 3,3',5,5' tetramethylbenzidine (TMB); (5) this peroxidase was active at the pH prevailing within the acrosomal vesicle; and (6) peroxidase activity and luminol-dependent chemiluminescence were minimal in spermatozoa exhibiting a congenital absence of acrosomes. Human spermatozoa could also generate lucigenin-dependent chemiluminescent signals that could neither be suppressed with peroxidase inhibitors nor enhanced by the addition of peroxidase. However, these signals could be enhanced by suppression of intracellular SOD with KCN or inhibited by exogenous SOD, suggesting that lucigenin was responding to superoxide anion released into the extracellular space. The ability of chemiluminescent techniques to detect and discriminate the production of superoxide and hydrogen peroxide by spermatozoa should facilitate the further analysis of reactive oxygen species as mediators of normal and abnormal human sperm function.     

Intracellular free Ca2+ concentration in fowl spermatozoa and its relationship to motility and respiration in spermatozoa.

The ability of fowl spermatozoa to accumulate and de-esterify the intracellular fluorescent Ca2+ indicator fura-2 was established. The cytosolic Ca2+ concentrations, measured by this technique, did not change after the addition of 1 mmol EGTA l-1. Subsequently, addition of the calcium ionophore A23187 caused a reduction in cytosolic Ca2+ concentrations, presumably by efflux of Ca2+ from the spermatozoa. Intracellular free Ca2+ concentrations were then significantly increased by the addition of 1 mmol CaCl2 l-1. The motility of demembranated spermatozoa gradually decreased after the addition of EGTA alone or EGTA with A23187, but was instantly restored by the addition of CaCl2 in the presence of both EGTA and A23187. Unlike demembranated spermatozoa, intact spermatozoa maintained their motility, even after the addition of EGTA, but their motility was reduced by the addition of A23187 in the presence of EGTA. The addition of A23187 also reduced the rate of oxygen consumption, but not the ATP concentrations in intact spermatozoa. These results demonstrate that the motility and respiration of fowl spermatozoa are strongly influenced by their intracellular Ca2+ concentrations.     

Reactive oxygen species in spermatozoa: methods for monitoring and significance for the origins of genetic disease and infertility

Human spermatozoa generate low levels of reactive oxygen species in order to stimulate key events, such as tyrosine phosphorylation, associated with sperm capacitation. However, if the generation of these potentially pernicious oxygen metabolites becomes elevated for any reason, spermatozoa possess a limited capacity to protect themselves from oxidative stress. As a consequence, exposure of human spermatozoa to intrinsically- or extrinsically- generated reactive oxygen intermediates can result in a state of oxidative stress characterized by peroxidative damage to the sperm plasma membrane and DNA damage to the mitochondrial and nuclear genomes. Oxidative stress in the male germ line is associated with poor fertilization rates, impaired embryonic development, high levels of abortion and increased morbidity in the offspring, including childhood cancer. In this review, we consider the possible origins of oxidative damage to human spermatozoa and reflect on the important contribution such stress might make to the origins of genetic disease in our species.     

Metabolic activation of chlorpromazine by stimulated human polymorphonuclear leukocytes. Induction of covalent binding of chlorpromazine to nucleic acids and proteins.

Human polymorphonuclear leukocytes (PMNs) have been stimulated with either phorbol 12-myristate 13-acetate (PMA), calcium ionophore A23187 or a combination of both to induce the respiratory burst and myeloperoxidase (MPO) release. Chlorpromazine (CPZ) but not chlorpromazine sulfoxide (CPZSO) inhibited the respiratory burst as measured with lucigenin chemiluminescence. The inhibition was due to interference with processes in the cell leading to the respiratory burst and not to scavenging of produced oxygen radicals that provoke the luminescence. CPZ was metabolized by stimulated PMNs. HPLC analysis revealed formation of CPZSO and an unidentified product. Both products result from decay of chlorpromazine radical cation (CPZ+.), indicating formation of this radical intermediate in CPZSO oxidation by stimulated PMNs. CPZ conversion correlated with H2O2 production and MPO release. The largest CPZ conversion was observed with phorbol ester plus A23187 stimulation. The conversion was reduced by catalase and sodium azide, an inhibitor of MPO, with 70% and 40%, respectively. This indicates only partial involvement of extracellularly released MPO in CPZ metabolism by PMNs. Considerable covalent binding of [3H]CPZ to nucleic acids and proteins of intact stimulated PMNs was observed. This binding was larger upon co-stimulation with phorbol ester and A23187. Azide did not reduce covalent binding. This indicates that covalent binding is not mediated by extracellularly released MPO and that CPZ is probably activated intracellularly. Activation of PMNs and production of H2O2 is a prerequisite for both CPZ conversion and covalent binding. This study demonstrates that phagocytic cells might contribute to drug metabolism and drug-induced toxicity.     

Evidence for a nonoxidative mechanism of human natural killer (NK) cell cytotoxicity by using mononuclear effector cells from healthy donors and from patients with chronic granulomatous disease

In vitro natural killer (NK) activity expressed by blood mononuclear cells from patients with chronic granulomatous disease of childhood (CGD) was equivalent to that expressed by cells from normal, healthy volunteers. Because neutrophils and monocytes from these same donors exhibited extremely depressed oxidative functions, our data could be interpreted to show that a) NK cells derived from a unique and separate cellular lineage unaffected by the disease-related oxidative defect, or b) the in vitro cytolytic mechanism(s) of NK cells were not dependent on oxygen metabolites. These hypotheses were examined by using as NK effector cells large granular lymphocytes (LGL) from healthy donors whose monocytes and neutrophils had normal oxidative functions. Such functions were measured in the nitroblue tetrazolium dye reduction assay, which is a qualitative measurement of superoxide anion production; by reduction of ferric cytochrome c, a more specific and quantitative measurement of superoxide anion production; and in the luminol-enhanced chemiluminescence assay, an extremely sensitive measure of several reactive oxygen radicals, including superoxide anion, hydroxyl radical, and singlet oxygen. Whereas monocytes and neutrophils from healthy donors were readily stimulated with zymosan or phorbol myristate acetate (PMA) in each of these assays. LGL produced no detectable amounts of oxygen metabolites when co-incubated either with K562 erythroleukemia cells, PMA, E. coli endotoxin, or the calcium ionophore A23187. Thus, because NK cell activity is normal in CGD patients with major oxidative defects, and because no reactive oxygen metabolites could be detected in LGL that simultaneously exhibited potent NK activity, we conclude that in vitro NK activity by human mononuclear cells involves a lytic mechanism(s) independent of oxygen metabolites.