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.

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.

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.

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.

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.

Membrane fluidity and platelet fibrinogen receptor exposure by proteolytic enzymes.

Incubation of platelets with pronase or chymotrypsin results in the exposure of fibrinogen receptors. We determined that these enzymes did not affect the membrane fluidity as evaluated by the depolarization of the fluorescence of 1,6-diphenyl-1,3,5 hexatriene (DPH). There was no significant difference in either the depolarization or in its temperature dependence for control, pronase or chymotrypsin-treated platelets. Thus, it can be concluded that the exposure of fibrinogen receptors on the platelet surface by proteolytic enzymes does not depend on the changes of membrane fluidity. We also propose that the proteolytic enzymes do not cause a major alteration in the extent of protein chains embedded in the lipid layers of the platelet membranes.

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.

Dissociation between aggregation and superoxide production in human granulocytes.

Aggregation and the activation of the granulocyte (PMN) superoxide (O2-) generating system occur when certain stimuli are added to resting cells. It had previously been postulated that PMN aggregation is essential for maximal O2- production. This study was undertaken to test the hypothesis that PMN aggregation is required for full expression of PMN O2- production. We examined aggregation and O2- production induced by four stimuli; concanavalin A (Con A), phorbol myristate acetate (PMA), N-formylmethionyl-leucyl-phenylalanine (FMLP), and ionophore A23187. Cytochalasin B enhanced aggregation by all four stimuli but only enhanced the rate of O2- production by Con A; 2-deoxyglucose inhibited aggregation by all stimuli. Dissociation of PMN aggregation and O2- production was achieved by using NEM, TPCK, and divalent cations. NEM and TPCK prevent Con A-induced O2- production but have no effect on Con A-induced aggregation. PMA-stimulated PMN generate O2- in the presence or absence of Ca++ and Mg++. In contrast, PMA stimulated maximum PMN aggregation only in the presence of both Ca++ and Mg++. Thus PMN can generate O2- without aggregating, and PMN can aggregate without producing O2-. PMN from patients with chronic granulomatous disease do not generate O2- or undergo membrane potential depolarization in response to PMA. These PMN aggregated when stimulated with PMA, providing evidence that depolarization is not required for PMN aggregation. We conclude that aggregation and the activation of the O2- generating system, though temporally related, are not necessarily causally related.

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)

Graded responses of human neutrophils induced by serum-treated zymosan.

A modified zymosan preparation was used to probe the interaction of particulate stimuli with human neutrophils (PMNs). After extraction with alkali and detergent, the zymosan particles retained their ability to be opsonized in serum and to stimulate PMNs. Serum-treated zymosan (STZ) induced dose-dependent superoxide (O2-) production and membrane potential depolarization in the range of 1 to 10 mg/mL of STZ. The rate and extent of secretion of lysozyme and beta-glucuronidase were also dose-dependent in the range of 1 to 10 mg/mL of STZ. Cytochemical studies using nitroblue tetrazolium, however, showed that 92% of PMNs were stimulated to produce O2- at 0.1 mg/mL of STZ. The dose response of O2- production induced by STZ is therefore due to increasing O2- production by individual PMNs and not to the stimulation of more PMNs to produce O2-. Evidence for O2- production was found only in the area of PMN-zymosan contact, suggesting a mechanism for the graded responses of PMNs treated with particulate stimuli. In order to determine the nature of the dose dependence of depolarization (a measure of PMN activation), PMNs equilibrated with the fluorescent probe 3,3'-dipentyloxacarbocyanine were analyzed by flow cytometry. The results demonstrate that STZ induces a dose-dependent depolarization of the membrane potential of individual PMNs. These results also demonstrate that increasing concentrations of STZ can induce increasing PMN responses even when all of the PMNs have been activated. These results are consistent with the hypothesis that receptor-mediated particulate stimulation of PMNs is a phenomenon that results in graded PMN responses.

Impaired granulocyte superoxide production and prolongation of the respiratory burst due to a low-affinity NADPH-dependent oxidase.

A 7-yr-old girl with self-limited pulmonary aspergillosis was found to have a defect in granulocyte superoxide production. Her cells produced superoxide at 3% of control rates in response to phorbol myristate acetate (PMA) and opsonized zymosan. Lag times for O-2 production were normal with PMA, opsonized zymosan, and concanavalin-A stimulation. Her granulocyte membranes depolarized in response to all of these stimuli. Superoxide produced by podosomes and a particulate fraction demonstrated an enzyme activity with a normal maximal velocity but a decreased affinity for NADPH. NADH-dependent superoxide production by particles was similar with patient and control material. The duration of superoxide production was prolonged in the patient's intact granulocytes and in the particulate fractions from her cells. Bacterial killing by the patient's granulocytes was initially low, but approached control values after 90 min of incubation. These results are explained by an enzyme activity that has a decreased affinity for its substrate and a decreased rate of inactivation. Family studies indicate an autosomal recessive mode of inheritance.

The effect of vitamin E deficiency on some platelet membrane properties.

The effects of alpha-tocopherol (vitamin E) deficiency on membrane properties of platelets were studied to determine if vitamin E has a measureable stabilizing role in biological membranes. Three groups of rats and three of mice were studied: two groups consisted of Fisher strain rats and one of Sprague-Dawley rats fed a Draper corn oil diet with and without high levels of supplementary vitamin E. The mice were two groups of BALB/c animals maintained on an 8% hydrogenated coconut oil diet, and one group of CBA/J mice on an 8% lard diet, in each case either deficient in or supplemented with vitamin E. The relative content of fatty acids obtained from both rat platelets and erythrocytes was unchanged by vitamin E deficiency. Depletion of vitamin E had no effect on the degree of fluorescence polarization of 1,6-diphenyl-1,3,5,-hexatriene-labeled rat platelets. No changes in hematocrit values were seen in any of the studies. The platelet count of only the vitamin E-deficient Sprague-Dawley rats was elevated with respect to vitamin E-supplemented counterparts; the others remained constant. Platelet reactivities, as measured by ADP-and thrombin-induced platelet aggregation and by the thrombin-induced changes in platelet transmembrane potential, were unaffected by vitamin E deficiency in all three groups of rats. Our results indicate that a membrane stabilizing effect of vitamin E on rat platelet or erythrocyte membrane fatty acids or on platelet response to external stimuli could not be demonstrated, nor was elevation in platelet count a general phenomenon associated with vitamin E deficiency.-Whitin, J. C., R. K. Gordon, L. M. Corwin, and E. R. Simons. The effect of vitamin E deficiency on some platelet membrane properties.

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.

Activation of human granulocytes by arachidonic acid: its use and limitations for investigating granulocyte functions.

The effects of arachidonic acid on human granulocytes (polymorphonuclear neutrophil leukocytes, PMN) were examined with respect to early events associated with activation of the superoxide (O2-)-generating system and its reactivation with other stimuli after the addition of fatty acid-free bovine serum albumin. As with other stimuli, O2- production occurred after a lag that was dependent on the amount of arachidonic acid used. Although the lag, rate, and extent of O2- production were dose dependent, at all concentrations used, the duration of O2- production was four to six minutes. As previously described, when fatty acid-free albumin was added one minute after stimulation of PMN by arachidonic acid, O2- production ceased immediately. The O2(-)-generating system could then be reactivated by the addition of either phorbol myristate acetate (PMA), concanavalin A, or serum-treated zymosan. In previously activated cells, the lag time for reactivation was shorter and the rate of O2- production greater with PMA. PMN membrane potential was depolarized by arachidonic acid. This depolarization was not reversed with the addition of albumin. PMN cytoplasts were also capable of reversible activation by arachidonic acid in a manner identical to whole cells. Divalent cations were found to be necessary for the activation of PMN by arachidonic acid. In the absence of calcium, arachidonic acid caused rapid lysis of the cells, as manifested by the release of lactic acid dehydrogenase. We were unable to measure later events in PMN stimulated by arachidonic acid since even in the presence of divalent cations lactic acid dehydrogenase was released from the cells after five minutes of incubation. We conclude that, in addition to its ability to activate PMN, arachidonic acid is toxic to the cells and cannot be used to study late events in granulocyte activity, and, in the absence of calcium, it may be difficult to interpret its action as a stimulant of the oxidase.

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.

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.

Effects of the myeloperoxidase system on fluorescent probes of granulocyte membrane potential.

Activation of normal or myeloperoxidase-deficient human granulocytes by phorbol myristate acetate resulted in an initial membrane depolarization as indicated by an increase in fluorescence of the lipophilic cation probe of membrane potential, 3,3'-dipropylthiodicarbocyanine. A subsequent apparent hyperpolarization (decrease in fluorescence) was observed in normal but not myeloperoxidase-deficient cells. Addition of purified myeloperoxidase restored a normal pattern of fluorescence changes to the enzyme-deficient granulocytes. The secondary decrease in fluorescence in normal cells was markedly blunted by addition of azide, cyanide, or catalase. In a cell-free system, the fluorescence of 3,3'-dipropylthiodicarbocyanine, but not that of 3,3'-dipentyloxadicarbocyanine, was rapidly eliminated by myeloperoxidase in the presence of hydrogen peroxide and a halide; this loss of fluorescence was inhibited by azide, cyanide, or catalase. These findings indicate that secretion of myeloperoxidase and hydrogen peroxide by activated granulocytes results in decreased fluorescence of 3,3'-dipropylthiodicarbocyanine, probably by thioether oxidation. While the determination of initial rates of depolarization using this probe is unaffected by the myeloperoxidase system, measurement of extent of depolarization and any subsequent membrane potential changes requires the addition of inhibitors. In the absence of inhibitors, the secondary decrease in fluorescence can be used as an indicator of secretion of myeloperoxidase and hydrogen peroxide.

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.

Opsonized zymosan-stimulated granulocytes-activation and activity of the superoxide-generating system and membrane potential changes.

Phagocytic cells generate superoxide in response to stimulation by opsonized particles. A continuous assay for opsonized zymosan-stimulated granulocyte superoxide production shows that there is a lag time between the addition of particles and the onset of detectable superoxide production. Superoxide production is preceded by membrane potential depolarization. Neither superoxide production nor membrane depolarization occurs in granulocytes from patients with chronic granulomatous disease. The extent of activation by opsonized zymosan is affected by the dose of zymosan from 0.5 to 4.5 mg/ml, but the time necessary for activation (lag time) is not. Similarly, the extent of depolarization but not the time necessary for attaining maximum depolarization is concentration-dependent. Effects of temperature, divalent cations, 2-deoxyglucose, cyanide, and N-ethyl maleimide on superoxide production are similar for granulocytes treated with soluble stimuli and with opsonized zymosan. Thus, zymosan stimulates granulocytes to generate superoxide and undergo membrane depolarization in a manner similar to that elected by soluble stimuli.