Ozone exposure and the production of reactive oxygen species by bronchoalveolar cells in humans.

Exposure to ozone injures respiratory epithelium, and the mechanisms may involve the generation of reactive oxygen species (ROS). This study tested the hypothesis that ozone exposure increases the airway burden of ROS to a greater degree in smokers than nonsmokers, and that this effect is independent of ozone-induced changes in spirometry. Healthy subjects were selected as either responders (decrement in FEV1 > 15%) or nonresponders (decrement in FEV1 < 5%) to ozone; each underwent 2 exposures to ozone and 1 to air, with bronchoalveolar lavage (BAL) performed 30 min (early) and 18 h (late) after exposure. Release of superoxide anion (O2(-)) was used as a measure of ROS release by all BAL cells, and flow cytometry was used to detect ROS production in alveolar macrophages (AM) only. Recovery of AM was approximately threefold greater in smokers than nonsmokers. Unstimulated, but not stimulated, cells obtained by BAL from smokers released approximately twofold greater amounts of O2(-) than cells from nonsmokers, both early and late after ozone exposure (p =.012 and p =.046, respectively). Stimulated, but not unstimulated, ROS generation by AM from smokers increased following ozone exposure, but the ozone effect was not significant. ROS production by AM decreased in nonsmokers (air vs. ozone late, p =.014). Total protein, albumin, and immunoglobulin M (IgM) increased in BAL fluid, consistent with an increase in epithelial permeability. In addition, the concentration of alpha2-macroglobulin increased approximately threefold 18 h after exposure in nonsmokers (p <.001). No relationship was found between measures of ROS production and lung function responsiveness to ozone. These studies suggest the airways of smokers experience a greater burden of ROS than those of nonsmokers following ozone exposure.

Intracellular reduction of selenite into glutathione peroxidase. Evidence for involvement of NADPH and not glutathione as the reductant.

Selenium (Se) in selenite is present in an oxidized state, and must be reduced for it to be incorporated as selenocysteine into selenoenzymes such as glutathione peroxidase (GPx). In vitro, Se, as in selenite, can be reduced utilizing glutathione (GSH) and glutathione reductase (GRed). We determined the effects of decreasing GSH levels, inhibiting GRed activity, and decreasing cellular NADPH on the selenite-dependent rate of GPx synthesis in cultured cells: PC3, CHO, and the E89 glucose-6-phosphate dehydrogenase (G-6-PD)-deficient cell line. A novel statistical analysis method was developed (using Box Cox transformed regression and a bootstrap method) in order to assess the effects of these manipulations singly and in combinations. Buthionine sulfoximine (BSO) was used to decrease GSH levels, 1,3 bis-(2 chloroethyl)-1 -nitrosourea (BCNU) was used to inhibit GRed activity and methylene blue (MB) was used to decrease cellular NADPH levels. This statistical method evaluates the effects of BSO, BCNU, MB and selenite alone and in combinations on GPx activity. Decreasing the GSH level (< 5% of control) did not have an effect on the selenite-dependent rate of GPx synthesis in PC3 or CHO cells, but did have a small inhibitory effect on the rate of GPx synthesis in E89 cells. Inhibiting GRed activity was also associated with either no effect (CHO, E89) or a small effect (PC3) on GPx activity. In contrast, decreasing NADPH levels in cells treated with MB was associated with a large decrease in the selenite-dependent rate of GPx synthesis to 36, 34 and 25% of control in PC3, CHO, and E89 cells, respectively. The effects of BSO plus BCNU were not synergistic in any of the cell lines. The effects of BSO plus MB were synergistic in G-6-PD-deficient E89 cells, but not in PC3 or CHO cells. We therefore conclude that under normal culture conditions, NADPH, and not glutathione, is the primary reductant of Se in selenite to forms that are eventually incorporated into GPx. For cells with abnormal ability to generate NADPH, lowering the GSH levels had a small effect on selenite-dependent GPx synthesis. GRed activity is not required for the selenite-dependent synthesis of GPx.

Increase in extracellular glutatione peroxidase in plasma and lungs of mice exposed to hyperoxia.

Extracellular glutathione peroxidase (E-GPx) is a selenium-dependent enzyme that can reduce hydrogen peroxide and phospholipid hydroperoxides. E-GPx is found in plasma and extracellular fluids such as bronchoalveolar lavage fluid. Because lung is one of the tissues that is capable of synthesizing and secreting E-GPx, the effect of exposure to hyperoxia on E-GPx in plasma and lung were studied in an injury model of hyperoxia exposure in adult mice. Exposure to 100% oxygen for 72 h resulted in an increase of 55% in plasma GPx activity and an increase of 50% in the amount of E-GPx protein in the plasma. Exposure to hyperoxia was also associated with an increase in the amount of E-GPx protein in lungs. The 7-fold increase in the amount of E-GPx protein in lungs was not due to plasma contamination of lungs from mice exposed to hyperoxia. E-GPx in the lung is calculated to account for 10% of lung GPx activity in control mice. However, E-GPx is calculated to account for 45% of lung GPx activity in the lungs of mice exposed to hyperoxia for 72 h. Further studies are needed to determine whether the increase in lung E-GPx is due to changes in translation or stability of E-GPx. The role of E-GPx in protecting the lung from oxidative damage warrants further study.

Expression of extracellular glutathione peroxidase in human and mouse gastrointestinal tract.

Extracellular glutathione peroxidase (EGPx) is a glycosylated selenoprotein capable of reducing hydrogen peroxide, organic hydroperoxides, free fatty acid hydroperoxides, and phosphatidylcholine hydroperoxides. We found that human large intestinal explant cultures synthesize EGPx and cellular glutathione peroxidase (CGPx) and secrete EGPx. The level of EGPx mRNA expression relative to alpha-tubulin was similar throughout the mouse gastrointestinal tract. EGPx mRNA transcripts have been localized to mature absorptive epithelial cells in human and mouse large intestine. Western blot analysis of mouse intestinal protein has demonstrated the presence of EGPx protein in the small intestine, cecum, and large intestine, with the highest protein levels found in the cecum. Immunohistochemistry studies of human large intestine and mouse small and large intestine sections demonstrated the presence of EGPx protein within mature absorptive epithelial cells. In human large intestine and mouse small intestine, EGPx protein is also present in the extracellular milieu. These results suggest a role for EGPx in protection of the intestinal tract from peroxidative damage and/or in intercellular metabolism of peroxides.

Plasma glutathione peroxidase and its relationship to renal proximal tubule function.

Selenium-dependent extracellular glutathione peroxidase (E-GPx) is found in plasma and other extracellular fluids. Previous studies have indicated that patients with chronic renal failure on dialysis have low plasma GPx activity. In this study, dialysis patients had approximately 40% of control plasma GPx activity, while anephric individuals had lowest plasma GPx activities ranging from 2 to 22% of control. The residual plasma GPx activity in anephric individuals could be completely precipitated by anti-E-GPx antibodies, indicating that all plasma GPx activity can be attributed to E-GPx in both normal and anephric individuals. Plasma GPx activity rises rapidly following kidney transplantation, often reaching normal values within 10 days. The plasma GPx activity in some transplanted patients rises to levels higher than the normal range, followed by a return to the normal range. Since E-GPx in the kidney is primarily synthesized in the proximal tubules, we investigated whether nephrotoxic agents known to disrupt proximal tubule function also affected plasma GPx activity. The beta-lactam antibiotic cephaloglycin rapidly caused a decrease in plasma GPx activity in rabbits. In addition, the chemotherapeutic agent ifosfamide caused a decrease in plasma GPx activity in pediatric osteosarcoma patients. Fanconi syndrome associated with either ifosfamide therapy or valproic acid therapy also caused a decrease in plasma GPx activity. Thus plasma GPx activity is related to kidney function and is decreased in certain situations where nephrotoxic drugs are administered. Monitoring plasma GPx activity may have predictive value in evaluating the function of transplanted kidneys or in predicting those patients particularly at risk of nephrotoxic injury associated with certain medications.

Developmental expression of extracellular glutathione peroxidase suggests antioxidant roles in deciduum, visceral yolk sac, and skin.

Extracellular glutathione peroxidase (EGPx) is a secreted selenium-dependent enzyme that reduces hydroperoxides and organic hydroperoxides. Selenium deficiency in females is associated with infertility and spontaneous abortion, suggesting a role for selenium-requiring proteins during embryonic development. To gain insight into functions of EGPx in vivo, we determined sites of murine EGPx synthesis by in situ hybridization during embryogenesis and in adult tissues. At E7.5 of development, high EGPx expression was found in the maternally derived deciduum, with lower levels of accumulation in the embryonic visceral endoderm. At E9.5, the major sites of expression were the yolk sac endoderm and heart musculature. By E16.5, EGPx mRNA expression persisted in yolk sac endoderm but also accumulated significantly in atrially derived myocytes, ossification centers, adipose tissue, intestinal epithelium, and in a ventral-to-dorsal gradient in developing skin. Glutathione peroxidase activity due to EGPx protein was identified in the fluids surrounding the developing mouse embryo at midgestation. The expression of EGPx in tissues at the maternal-fetal interface--deciduum, visceral yolk sac, and skin--suggests that EGPx may serve to protect the embryo from oxidant damage. In adult mice, we identified the S1 segment of the kidney proximal tubules as the primary site of EGPx mRNA accumulation, with lower EGPx levels in atrial cardiac muscle, intestine, skin, and adipose tissue. These findings suggest that EGPx may serve a wider antioxidant role than previously recognized in the interstitium of multiple localized tissues, particularly those associated with the active transport of lipids.

Airway inflammation in smokers and nonsmokers with varying responsiveness to ozone.

Exposure to ozone causes symptoms, changes in lung function, and airway inflammation. We studied whether individuals who differ in lung-function responsiveness to ozone, or in smoking status, also differ in susceptibility to airway inflammation. Healthy subjects were selected on the basis of responsiveness to a classifying exposure to 0.22 ppm ozone for 4 h with exercise (responders, with a decrease in FEV1 > 15%; and non-responders, with a decrease in FEV1 < 5%). Three groups were studied: nonsmoker-nonresponders (n = 12), nonsmoker-responders (n = 13), and smokers (n = 13, 11 nonresponders and two responders). Each subject underwent two exposures to ozone and one to air, separated by at least 3 wk; bronchoalveolar and nasal lavages were performed on three occasions: immediately (early) and 18 h (late) after ozone exposure, and either early or late after air exposure. Recovery of polymorphonuclear leukocytes (PMN) increased progressively in all groups, and by up to 6-fold late after ozone exposure. Interleukin-6 (IL-6) and IL-8 increased early (by up to 10-fold and up to 2-fold, respectively), and correlated with the late increase in PMN. Lymphocytes, mast cells, and eosinophils also increased late after exposure. We conclude that ozone-induced airway inflammation is independent of smoking status or airway responsiveness to ozone.

Standardization of an opsonophagocytic assay for the measurement of functional antibody activity against Streptococcus pneumoniae using differentiated HL-60 cells.

Host protection against pneumococcal disease i primarily mediated by phagocytosis. We developed and standardized an opsonophagocytic assay using HL-60 cells (human promyelocytic leukemia cells). Fifty-five serum samples were analyzed for the presence of functional antibody against seven pneumococcal serogroups or serotypes (4, 6B, 9V, 14, 18C, 19F, and 23F) by using differentiated HL-60 cells (granulocytes) and peripheral blood leukocytes (PBLs). Six of the 55 serum samples were from unvaccinated adult volunteers, 31 serum samples were from adults who received one dose of the 14-valent or the 23-valent polysaccharide vaccine, and 18 serum samples were from 16-month-old infants who received four doses of an investigational 7-valent polysaccharide-protein conjugate vaccine. The results of an opsonophagocytic assay with HL-60 cells correlated highly with those of an assay with PBLs as effector cells (median r for seven serotypes = 0.87: P < 0.01). Opsonophagocytic titers were compared with the immunoglobulin G antibody concentrations determined by enzyme-linked immunosorbent assay (ELISA). The r values for serogroups or serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F were 0.61, 0.60, 0.67 0.90, 0.61, 0.39, and 0.57, respectively, when HL-60 cells were used as effector cells and 0.56, 0.47, 0.61, 0.90, 0.71, 0.31, and 0.62, respectively, when PBLs were used. The assay requires small amounts of serum (40 microliters per serotype), making this test suitable for assaying infant sera. Culturable cells aid in assay standardization and likely reduce donor-to-donor variability. This standardized assay, in combination with the standardized ELISA, can be used to evaluate current and developing pneumococcal vaccines, in which functional opsonophagocytic antibody activity may correlate with protection against pneumococcal disease.

Effects of ozone on normal and potentially sensitive human subjects. Part II: Airway inflammation and responsiveness to ozone in nonsmokers and smokers.

Exposure to ozone at levels near the National Ambient Air Quality Standard causes respiratory symptoms, changes in lung function, and airway inflammation. Although ozone-induced changes in lung function have been well characterized in healthy individuals, the relationship between airway inflammation and changes in pulmonary function have not been prospectively examined. The purpose of this study was to determine whether individuals who differ in, lung function responsiveness to ozone also differ in susceptibility to airway inflammation and injury. A secondary goal was to determine whether ozone exposure induces airway inflammation in smokers, a population known to have airway inflammation and an increased burden of toxic oxygen species. Healthy nonsmokers (n = 56) and smokers (n = 34) were exposed to 0.22 parts per million (ppm)* ozone for 4 hours, with intermittent exercise, for the purpose of selecting ozone "responders" (decrement in forced expiratory volume in 1 second [FEV1] > 15%) and "nonresponders" (decrement in FEV1 < 5%). Selected subjects then were exposed twice to ozone (0.22 ppm for 4 hours with exercise) and once to air (with the same exposure protocol), each pair of exposures separated by at least 3 weeks, in a randomized, double-blind fashion. Nasal lavage (NL) and bronchoalveolar lavage (BAL) were performed immediately after one ozone exposure and 18 hours after the other, and either immediately or 18 hours after the air exposure. Indicators of airway effects in lavage fluid included changes in inflammatory cells, proinflammatory cytokines, protein markers of epithelial injury and repair, and generation of toxic oxygen species. In the classification exposure, fewer smokers than nonsmokers were responsive to ozone (11.8% vs. 28.6%, respectively); an insufficient number of smoker-responders were identified to study as a separate group. In the BAL study, all groups developed a similar degree of airway inflammation, consisting of increases in interleukins 6 and 8 (maximal immediately after exposure), and increases in polymorphonuclear leukocytes (PMNs), lymphocytes, and mast cells (maximal 18 hours after exposure). The increase in PMNs was inversely correlated with age (p = 0.013), but gender, nonspecific airway responsiveness, and allergy history were not predictive of inflammation. Alveolar macrophage production of toxic oxygen species decreased after ozone exposure in nonsmokers; however, not in smokers. Findings from nasal lavage did not mirror lower airway inflammatory responses in these studies. We conclude that, in response to ozone exposure, smokers experienced smaller decrements in lung function and fewer symptoms than nonsmokers; however, the intensity of the airway inflammatory response was independent of smoking status or airway responsiveness to ozone. Furthermore, the burden of toxic oxygen species following ozone exposure was greater for smokers than for nonsmokers. Subjects were young, healthy, and able to sustain exercise; the results may not be representative of nonsmokers or smokers in general. Nevertheless, the findings indicate that measuring symptoms and spirometric changes is not sufficient to assess the potential risks associated with ozone exposure.

Comparative pharmacokinetic studies of three asparaginase preparations.

PURPOSE: As part of pharmacologic studies of asparaginase (ASNase), we determined the half-life of ASNase activity and protein, and the effect of dose, repeated doses, different drug preparations, and hypersensitivity reactions on the half-life (t1/2) of serum ASNase activity. PATIENTS AND METHODS: We measured ASNase activity (spectrophotometric assay) in serum samples obtained from patients with acute lymphoblastic leukemia (ALL) at various times during their therapy with intramuscular ASNase. ASNase protein was measured by enzyme-linked immunoadsorbent assay (ELISA). RESULTS: Studies following the initial dose of Escherichia coli-derived ASNase demonstrated no difference in apparent t1/2 following 25,000 IU/m2 versus 2,500 IU/m2 (1.24 v 1.35 days, P = .2). The apparent t1/2s following maintenance doses of E coli ASNase (middle dose t1/2, 1.28 days, or last dose t1/2, 1.14 days) showed no difference when compared with the initial dose of ASNase (P = .3 to .9). There was no significant difference between the apparent t1/2s of ASNase activity and ASNase protein (n = 8, P = .2 to .6). The serum t1/2 was 0.65 and 5.73 days for patients receiving Erwinia or polyethylene glycol (PEG)-modified E coli ASNase, respectively, as the induction dose. ASNase activity was undetectable in sera of four patients studied in the week following an anaphylactic reaction to E coli ASNase and the t1/2 was significantly shorter in five patients with a history of allergic reaction to E coli ASNase who were studied following a dose of PEG ASNase, (t1/2, 1.80 days). CONCLUSION: We conclude that (1) the apparent t1/2 of ASNase is dependent on enzyme preparation used, but is not affected by dose or by repeated use; (2) the apparent t1/2 of E coli ASNase as a protein is the same as the apparent t1/2 of enzymatic activity; and (3) patients who have had a hypersensitivity reaction to E coli ASNase have a decreased apparent t1/2 with both E coli and PEG ASNase.

Role of antioxidant enzymes in the induction of increased experimental metastasis by hydroxyurea.

BACKGROUND: Treatment of tumor cells with hydroxyurea and other DNA-damaging agents has been shown to increase the experimental metastatic potential of these cells. PURPOSE: We sought to elucidate some of the biochemical and genetic changes that promote tumor cell metastasis in hydroxyurea-treated cells. We hypothesized that drug treatment induces resistance to oxidative damage and that elimination of this resistance reverses the drug-induced experimental metastatic capabilities of tumor cells. METHODS: We examined the effect of hydroxyurea treatment on B16 melanoma cells with respect to experimental metastatic potential, resistance to hydrogen peroxide (H2O2), glutathione peroxidase activity and messenger RNA (mRNA) level, glutathione reductase activity, glutathione levels, glutathione-S-transferase activity, and catalase activity and mRNA level. RESULTS: Hydroxyurea-treated cells were transiently more metastatic following intravenous injection in syngeneic mice and transiently more resistant than untreated cells to exogenous H2O2. Hydroxyurea-induced experimental metastases and H2O2 resistance were eliminated by depletion of intracellular glutathione with buthionine sulfoximine. Glutathione peroxidase activity and mRNA level, glutathione reductase activity, and reduced glutathione levels were all transiently increased in hydroxyurea-treated cells, whereas the increase in glutathione-S-transferase activity was sustained. Catalase activity was modestly increased with no increase in its mRNA levels. CONCLUSIONS: In B16 melanoma cells, experimental metastasis induced by hydroxyurea appears to depend on a process that requires glutathione. Hydroxyurea treatment also induces resistance to exogenous H2O2, which may be due to induction of glutathione and antioxidant enzyme activity. IMPLICATIONS: The role of antioxidants in B16 melanoma cells offers new insights into the metastatic process and the cellular response to chemotherapy.

Modulation of the respiratory burst in human neutrophils by isoproterenol and dibutyryl cyclic AMP.

The effects of a beta-adrenergic agonist and a cyclic AMP analogue on activation, activity, and termination of FMLP-stimulated superoxide anion production were investigated. Incubation with isoproterenol resulted in a 50% reduction in the maximal rate of superoxide production and a 3-4-fold increase in the rate of termination of superoxide production. Exposure to 1 mM dibutyryl cyclic AMP resulted in a 40% decrease in the maximal rate and a 3-fold increase in the rate of termination of FMLP-induced superoxide production. Neither agent had a significant effect on the lag time prior to superoxide anion generation.

Measurement of serum L-asparagine in the presence of L-asparaginase requires the presence of an L-asparaginase inhibitor.

The antileukemic activity of L-asparaginase (ASNase), an important component of therapy for acute lymphoblastic leukemia, is thought to result from depletion of serum L-asparagine (Asn). In studies of the pharmacological effects of ASNase, investigators have reported prolonged reduction in the serum concentration of Asn after the administration of ASNase. Such measurements may not be valid because ASNase present in the blood sample may hydrolyze Asn before its determination. We examined recovery of [U-14C]Asn from blood samples with and without various concentrations of added ASNase. In the presence of greater than or equal to 0.01 IU/ml of ASNase, the amount of [U-14C]Asn recovered was less than 15% of that without ASNase. Utilizing this assay, we studied the effect of 2 known inhibitors of ASNase in an attempt to improve Asn recovery. In the presence of aspartic beta semialdehyde (ASA), or 5-diazo-4-oxo-L-norvaline (DONV), and up to 1.0 IU/ml ASNase, Asn levels remained at greater than 90% of control. ASA prevented the hydrolysis of exogenous Asn in blood samples drawn from patients after ASNase injection. We also developed a method to determine Asn in serum utilizing high pressure liquid chromatography. Using this method, we found that the Asn level was greater than 90% of a normal level in the presence of 40 mM DONV and 1.0 IU/ml ASNase. Examination of serum from 4 patients treated with ASNase showed that Asn is detectable 7-19 days sooner when DONV is present in the blood collection system than in its absence. We conclude that: (a) as little as 0.01 IU/ml ASNase can hydrolyze Asn added to blood; (b) continued hydrolysis of Asn by ASNase ex vivo can result in falsely low serum Asn measurements; (c) ASA or DONV present in the collection tubes obviates the problem of continued ASNase activity; and (d) the degree and duration of Asn depletion after ASNase therapy is much less than previously believed. Thus, for accurate measurements of the duration and degree of Asn depletion by ASNase, an ASNase inhibitor such as ASA or DONV should be present in the blood collection system.

Signal transduction in N-formyl-methionyl-leucyl-phenylalanine and concanavalin A stimulated human neutrophils: superoxide production without a rise in intracellular free calcium.

Changes in intracellular ionized free calcium ([Ca]i), inositol triphosphate (IP3), and sn-1,2-diacylglycerol (DAG) were determined in relation to agonist-induced human neutrophil superoxide (O2-) production. With 0.1 microM N-formyl-methionyl-leucyl-phenylalanine (fMLP) stimulation, generation of IP3 and a peak rise in [Cai] occurred at 30 sec, preceding maximal O2- production (1.5 min) and the maximal rise in DAG mass (4 min). FMLP-induced O2- production was inhibited by pertussis toxin. In cytochalasin B-primed, concanavalin A (Con A) stimulated neutrophils, a peak rise in [Ca]i but not IP3 proceeded O2- production, and pertussis toxin did not inhibit O2- production. EGTA inhibited the cytochalasin B/fMLP-induced increment in [Ca]i and O2- production by 75% and 50%, respectively, and completely ablated the response to cytochalasin B/Con A, suggesting a role for extracellular as well as intracellular calcium in the respiratory burst. However, three types of experiments indicate that an increase in [Ca]i is neither sufficient nor always required for O2- production. First, treatment with ionomycin resulted in a marked increase in [Ca]i but did not cause O2- production. Second, pertussis toxin inhibited both fMLP-induced IP3 generation and O2- production but did not inhibit the rise in [Ca]i. Third, following neutrophil priming with dioctanoylglycerol (diC8), maximal O2- production occurred in response to 0.015 microM fMLP or Con A without a rise in [Ca]i, and diC8/fMLP-induced O2- production was not inhibited by EGTA. Taken together, these data suggest that 1) an increment in [Ca]i is not strictly essential for neutrophil O2- production, 2) unlike fMLP, Con A-induced O2- production does not proceed through a pathway involving the pertussis toxin-sensitive G protein, and 3) regulation of neutrophil [Ca]i involves mechanisms independent of IP3 concentration.

Plasma selenium-dependent glutathione peroxidase. Cell of origin and secretion.

Human plasma glutathione peroxidase (GSHPx) has been shown to be a glycosylated selenoprotein distinct enzymatically, structurally, and antigenically from known cellular glutathione peroxidases. The extracellular location of the enzyme and the fact that it is glycosylated suggested that it is a secreted protein. Utilizing mutually non-cross-reactive antibodies to human cellular and plasma GSHPx, we conducted a search to determine the tissue of origin for plasma GSHPx. The cells screened were endothelial cells because they are the main source of extracellular superoxide dismutase, HL-60 cells (myeloid cell line) because they are the main source of extracellular H2O2, and Hep G2 cells (hepatic cell line) because they are the source of many plasma proteins. Human umbilical vein endothelial cells were metabolically labeled with either [35S]methionine or [75Se]selenious acid, and HL-60 cells and Hep G2 cells were metabolically labeled with [75Se]selenious acid. Proteins were immunopurified from the labeled cells and their media with either anti-red blood cell (RBC) GSHPx IgG or with anti-plasma GSHPx IgG. Utilizing anti-RBC GSHPx IgG, only the cellular form of the enzyme was precipitated from all the cells tested but not from their media. When anti-plasma GSHPx IgG was applied to the cells and their media, a selenoprotein was precipitated only from the media of Hep G2 cells. When Hep G2 cells were incubated in the presence of the carboxylic ionophore monensin, an intracellular selenoprotein could be detected using anti-plasma GSHPx IgG. The precipitation of the cellular form from all three cell types was partially inhibited by preincubation of the anti-RBC GSHPx IgG with purified RBC GSHPx while the precipitation of the selenoprotein from the medium of Hep G2 cells by anti-plasma GSHPx IgG was prevented by preincubation of the antibody with purified plasma GSHPx. We suggest that plasma GSHPx is synthesized by and secreted from hepatic cells. This is, to the best of our knowledge, the only known selenoprotein with a defined function that has been shown to be synthesized for secretion by mammalian cells.

Antihuman plasma glutathione peroxidase antibodies: immunologic investigations to determine plasma glutathione peroxidase protein and selenium content in plasma.

Plasma glutathione peroxidase (GSHPx) (glutathione: H2O2 oxidoreductase) is a unique selenoglycoprotein. Treatment of this enzyme with glycopeptidase F partially deglycosylates it and establishes the presence of N-linked sugar moieties. Antibodies raised in a rabbit against the purified enzyme from plasma were found to be specific, noninhibitory, and capable of precipitating the enzymatic activity. The antibodies precipitated greater than 90% of the GSHPx activity of normal plasma, thus indicating that the selenoenzyme is the main if not the sole GSHPx activity of plasma. The antibodies did not precipitate RBC GSHPx. A slight cross-reactivity of the antibodies was found with rat plasma GSHPx. A GSHPx activity precipitation assay of normal plasma in the presence of selenium (Se)-deficient plasma indicates that no cross-reactive protein in the Se-deficient plasma interferes with the precipitation of the GSHPx activity from normal plasma. Thus, GSHPx protein as well as activity is deficient in plasma in the absence of Se. Antibodies against GSHPx either from RBCs or from plasma were used to specifically immunoprecipitate most of the GSHPx activity from RBCs or plasma, respectively, in healthy individuals to determine the amount of Se associated with the protein. GSHPx accounts for approximately 15% of the Se in RBCs and 12% of the Se in plasma. Thus, in normal individuals, these proteins account for only a fraction of plasma and RBC Se.

Disorders of respiratory burst termination.

The biochemical mechanisms for the termination of the respiratory burst are likely to be multifactorial. Alterations in the endogenous oxidant-scavenging mechanisms can modulate the kinetics of the termination phase of neutrophil superoxide production. These are most apparent when those alterations are found in the neutrophils of certain patients. The use of inhibitors of the oxidant scavenging systems in normal neutrophils has not yielded results that exactly mimic the studies with neutrophils from the patients. For example, the glutathione reductase-inhibited neutrophils (from the action of BCNU) do not show the same degree of abnormality as do the neutrophils from the patient with glutathione reductase deficiency. Further investigations on the mechanisms of inactivation of the NADPH oxidase are warranted in order to gain a greater understanding of this important regulating mechanism.

Activation of neutrophil superoxide production by concanavalin A can occur at low levels of intracellular ionized calcium.

The effect of concanavalin A (Con A) on the concentration of ionized intracellular calcium [( Cai++]) in human granulocytes (PMN) was monitored using the fluorescent calcium indicator and chelator, Quin2. The addition of Con A to PMN resulted in a rise in [Cai++] that was markedly enhanced by the presence of Ca++ in the external buffer. The onset of the increment in [Cai++] preceded the onset of O2- production. The rise in [Cai++] induced by Con A is not transient, with a new, higher steady-state level of [Cai++] being attained within five minutes. The addition of alpha-methylmannoside (alpha-MM) one minute after Con A resulted in the return of [Cai++] to the original baseline level and the cessation of O2- production. The addition of a second stimulus (such as arachidonic acid) to these cells resulted in a second increment in [Cai++] and the return of O2- production. Thus the rise in [Cai++] induced by Con A is tightly coupled to the activation, inactivation, and reactivation of the O2- generating system by Con A. Further experiments were undertaken to assess the possible requirement for the rise in [Cai++] in the activation of PMN by Con A. PMN could be depleted of Cai++ by loading with Quin2 in the absence of extracellular Ca++. These Ca++- depleted PMN can be induced to produce O2- after treatment with PMA but not with Con A. The addition of Ca++ to Ca++ -depleted PMN results in a return of [Cai++] to the normal resting level of Ca++ -replete PMN. The time required to return to baseline is a function of the concentration of intracellular Quin2. The addition of Ca++ to Ca++ -depleted, Con A-treated PMN results in the elevation of [Cai++] and the production of O2-. Over a tenfold range of intracellular Quin2 concentration, the onset of O2- production always occurred at [Cai++] that were less than the normal resting level. Thus, activation of the O2- generating system by Con A can occur at [Cai++] which are much lower than the incremental level induced by Con A in Ca++ -replete PMN. Supporting this is the observation that only a very small increment in [Cai++] is induced by Con A in PMN cytoplasts, even though Con A could induce O2- production in the Quin2-loaded cytoplasts.(ABSTRACT TRUNCATED AT 400 WORDS)

The role of glutathione reductase in maintaining human granulocyte function and sensitivity to exogenous H2O2.

Human granulocytes (polymorphonuclear leukocytes, PMN) produce H2O2 and other reactive oxygen species while undergoing phagocytosis. To examine the role of the glutathione cycle in metabolizing H2O2, we incubated PMN with 1,3-bis (2-chloroethyl) nitrosourea (BCNU). Incubation of PMN with BCNU results in a dose-dependent inhibition of PMN glutathione reductase (GRED), with 50% inhibition occurring at approximately 2 micrograms/mL BCNU. PMN hexose monophosphate shunt activity stimulated with an exogenous H2O2-generating system was inhibited only when the GRED activity was reduced to less than 30% of control. BCNU-treated cells contained lower levels of reduced sulfhydryls and reduced glutathione, which decreased even more in the presence of an exogenous H2O2-generating system. The effect of BCNU and exogenous H2O2 on various aspects of phagocytosis were examined. Exposure of BCNU-treated PMN to an H2O2-generating system resulted in an inhibition of chemotactic peptide-induced shape changes and degranulation. The ability of BCNU-treated cells to produce O2- was diminished only when the PMN were incubated with an H2O2-generating system in the presence of cyanide. Ingestion of opsonized bacteria by BCNU-treated PMN was unaffected by incubation in an H2O2-generating system even in the presence of cyanide. We conclude that PMN GRED is inhibited by BCNU, the ability of PMN to metabolize H2O2 is affected only when GRED is reduced more than 70%, this inhibition affects the glutathione content of these cells, and some, but not all of the phagocytic functions of GRED-inhibited PMN are inhibited after exposure to an H2O2-generating system.