Enhanced lipid peroxidation in cancer tissue homogenates in non-small cell lung cancer.

The oxidative modification of nucleic acids by reactive oxygen species may lead to malignant conversion, but its exact role in lung cancer biology is still not clear. Lipid peroxidation, a well-known index of free radicals activity, is a process of oxidative polyunsaturated acids destruction. Our study was aimed to investigate the level of lipid peroxidation ex vivo in tumor tissue and lung parenchyma obtained from patients with non-small cell lung cancer. Thirty-two patients with lung cancer (including 19 with squamous cell lung cancer) were enrolled in the study. During a surgical resection, tumor tissue and lung parenchyma were obtained and the concentration of lipid peroxidation products, i.e. conjugated dienes and lipid hydroperoxides, measured. In the whole group of patients the concentrations of conjugated dienes and lipid hydroperoxides in the tumor tissue were higher than those in lung parenchyma (1.008 +/- 0.503 A233 nm vs. 0.717 +/- 0.283 A233 nm; p < 0.05 and 0.109 +/- 0.062 A532 nm vs. 0.102 +/- 0.087 A532 nm; p < 0.05, respectively). Similar results were obtained in squamous cell carcinoma patients (0.975 +/- 0.348 A233 nm vs. 0.708 +/- 0.300 A233 nm; p > 0.02 and 0.094 +/- 0.029 A532 nm vs. 0.080 +/- 0.071 A532 nm; p < 0.05, respectively). In both groups of patients, a positive correlation between concentration of conjugated dienes in tumor tissue and clinical stage (R = 0.45; R = 0.52; p < 0.05, respectively) was found. Our results confirm the enhanced lipid peroxidation in cancer tissue as compared with matched lung parenchyma. Additionally, a higher level of oxidative stress, expressed as the concentration of conjugated dienes in tumor tissue, was associated with clinical progression of the tumor.

Comparison of hydrogen peroxide generation and the content of lipid peroxidation products in lung cancer tissue and pulmonary parenchyma.

Lipid peroxidation, as a well-known index of reactive oxygen species activity, not only in lung biochemistry, is an oxidative process associated with membrane lipid destruction. Also, the oxidative modification of nucleic acids by reactive oxygen species is of remarkable biological importance as it may contribute to malignant conversion, but its exact role in lung cancer biology is still not clear. Our study aimed to investigate the level of lipid peroxidation ex vivo in tumour tissue and lung parenchyma obtained from patients with lung cancer. Forty-two patients with lung cancer were enrolled into the study. During a surgical resection, tumour tissue and lung parenchyma were obtained and concentration of lipid peroxidation products, thiobarbituric acid-reactive substances and Schiff bases, and spontaneous generation of hydrogen peroxide, were measured. The concentration of thiobarbituric acid-reactive substances (P<0.001) in the tumour tissue was higher than that in lung parenchyma. In small cell lung cancer as well as in squamous cell carcinoma patients, a positive correlation between spontaneous generation of hydrogen peroxide in tumour tissue and clinical stage (r = 0.43; r = 0.46; respectively) was found. Our results prove enhanced lipid peroxidation in cancer tissue as compared with matched-lung parenchyma. In small cell lung cancer and squamous cell carcinoma patients, the high level of oxidative stress, expressed as a spontaneous generation of hydrogen peroxide in tumour tissue, was associated with clinical progression of tumour's stage.

Cefodizime enhances phagocytosis and intracellular killing of Staphylococcus aureus but does not influence polymorphonuclear leukocytes response to fMLP stimulation.

The influence of Cefodizime (CDZ) on in vitro activity of polymorphonuclear leukocytes (PMNL) from healthy subjects was assessed. Preincubation with CDZ enhanced phagocytosis and intracellular killing of Staphylococcus aureus by PMNL. Contrary to numerous clinical reports, no significant effect of CDZ preincubation on PMNL response to n-formyl-methionyl-leucyl-phenylalanine was found with respect to intracellular calcium changes, degranulation, hydrogen peroxide production, and chemiluminescence. These results suggest that augmented microbicidal activity of PMNL is not related to the enhanced production of reactive oxygen species in healthy subjects.

[Assessment of nitric oxide level in aqueous humor under physiological conditions and after lens extraction with PMMA during experimental work in rabbits]. / Ocena stezenia tlenku azotu w cieczy wodnistej w warunkach fizjologicznych oraz po usunieciu soczewki i wszczepie sztucznej soczewki z PMMA w badaniach doswiadczalnych u królika.

PURPOSE: To evaluate the presence of nitric oxide and measure its level in the aqueous humor of the rabbit's eye, in physiological conditions and after extracapsular lens extraction and PMMA artificial lens implantation. We also investigated nitric oxide maintenance during early postoperative period (between 1-5 day after surgery). MATERIAL AND METHODS: We used 30 rabbits (weighing 3.0-3.5 kg) Just before surgery samples of aqueous humor were aspirated by anterior chamber puncture. Lens was extracted with extracapsular (envelope) technique. In 15 eyes PMMA IOL was implanted in the bag and 15 eyes were left aphakic. The aqueous samples were collected on 1st, 3rd, 5th days after surgery. Nitric oxide in each sample was measured with respect to fluorometric assay. RESULTS: In aqueous humor in physiological conditions we detected nitric oxide. Its level was estimated on the value of 26.52 nM/dl. After extracapsular lens extraction in both groups the level of nitric oxide was significantly higher than in control group. The day and value of NO level was different among examined groups. Nitric oxide level diminished significantly on 5th postoperative day. CONCLUSION: We came to conclusion that after ECCE and PMMA IOL implantation NO level was significantly higher as compared with control. This higher NO level after lens extraction can be responsible for the blood aqueous breakdown.

[Assessment of nitric oxide level in aqueous humor under physiological conditions and after lens extraction and artificial foldable acrylic lens implantation during experimental work in rabbits]. / Ocena stezenia tlenku azotu w cieczy wodnistej w warunkach fizjologicznych oraz po usunieciu soczewki i wszczepie sztucznej zwijalnej soczewki akrylowej w badaniach doswiadczalnych u królika.

PURPOSE: To evaluate the presence of nitric oxide (NO) and its level in the aqueous humor of rabbit's eye physiological conditions and after phacoemulsification and acrylic foldable artificial lens implantation. We also investigated nitric oxide maintenance during early postoperative period (between 1-5 days after surgery). MATERIAL AND METHODS: We examined 30 rabbits (weighing 3.0-3.5 kg) Just before surgery samples of aqueous humor were aspirated. Lens was extracted with phacoemulsification technique. In 15 eyes acrylic foldable IOL (group III) was implanted and 15 eyes were left aphakic (group IV). The aqueous samples were collected on 1, 3, 5 days after surgery. Nitric oxide in each sample was determined with fluorometric assay. RESULTS: The level of NO in aqueous humor in physiological conditions was estimated to 26.52 nM/dl. After phacoemulsification in both groups the level of nitric oxide was higher than in control group. The day and value of the highest NO level was different among examined groups. The highest level of NO was released during 1st day in group III and on the 3rd day in the group IV. CONCLUSION: We came to conclusion that the level of nitric oxide in aqueous humor after phacoemulsification in both groups is higher than in control group but significantly lower than in previously examined groups in which the surgery was made with extracapsular technique.

Increased serum concentrations of conjugated diens and malondialdehyde in patients with pulmonary tuberculosis.

During pulmonary inflammation increased amounts of reactive oxygen species (ROS) and reactive nitrogen intermediates (RNI) are produced as a consequence of phagocyte respiratory burst. One of the manifestation of these free radical-mediated processes is lipid peroxidation (LP). The aim of our study was to assess the concentration of lipid peroxidation products (LPPs), conjugated diens (CD) and malondialdehyde (MDA), in patients with active TB. Forty-two patients were enrolled into the study. Half (group I) had advanced TB and were sputum smear-positive. The remainder (group II) had only small radiographical changes and were sputum smear-negative. Serum concentrations of CD and MDA were measured at days 0, 7, 14 and 28 in group I and day 0 in group II. We found that in all patients with active TB CCD (1.0 +/- 0.05A233) and CMDA (2.01 +/- 0.16 nmol dl-1) were significantly elevated compared to healthy controls (0.67 +/- 0.03A233 and 1.36 +/- 0.08 nmol dl-1, respectively) (P < 0.001). The highest levels of LPPs were in patients with advanced TB. These concentrations were stable during the first month of anti-tuberculous therapy. Our data indicated that, as in bacterial pneumonia, LPPs were enhanced in active TB. The levels of LPPs depended on the form of the disease as they were higher in subjects with advanced disease than in those with only small radiographical changes. Further studies are needed to assess the role of antioxidants as adjuvant therapy in patients with pulmonary TB.

Increased hydrogen peroxide and thiobarbituric acid-reactive products in expired breath condensate of asthmatic patients.

Symptoms of bronchial asthma are a manifestation of airway inflammation. Circulatory leucocytes (predominantly eosinophils, mast cells and neutrophils), release inflammatory mediators, including reactive oxygen species, i.e. superoxide anion which is dismutated to hydrogen peroxide (H2O2). Neutrophils from asthmatics generate greater amounts of these species than those of healthy subjects. Some of the H2O2 and thiobarbituric acid-reactive products (TBARs) can evaporate from alveolar lining fluid, and could be expired from the airways of asthmatics. In this study, therefore, we determined whether asthmatic patients exhale more H2O2 and TBARs than healthy subjects. We examined 10 healthy subjects as a control group and 21 asthmatic subjects. In asthmatic subjects, forced expiratory volume in one second (FEV1), was 68+/-9% of predicted value, peak expiratory flow rate (PEFR) was 65+/-8% pred, and bronchial reversibility was 34+/-5% of prebronchodilated FEV1. The mean H2O2 level measured spectrofluorimetrically in the expired breath condensate of asthmatic subjects was 26 fold higher than that in healthy controls (0.26+/-0.29 vs 0.01+/-0.03 nM; p<0.05). The concentration of TBARs in breath condensate was also higher in asthmatic patients compared with nonasthmatics (0.073+/-0.071 vs 0.004+/-0.009 nM; p<0.05). There was a significant correlation between H2O2 level and concentration of TBARs in asthmatic patients (r=0.74; p<0.01). There was also a strong inverse correlation between H2O2 content of all asthmatics and FEV1% pred (r=-0.63; p<0.005) and PEFR% pred (r=-0.52; p<0.05). We conclude that there are elevated levels of hydrogen peroxide and thiobarbituric acid-reactive products in expired breath condensate of asthmatic patients, and that measurement of these substances in the expired breath condensate could be a simple, noninvasive method that could be used as a biochemical marker of airway inflammation.

Polymorphonuclear leukocytes from asthmatics release more calcium from intracellular stores and have enhanced calcium increase after stimulation with N-formyl-methionyl-leucyl-phenylalanine.

Polymorphonuclear leukocytes isolated from peripheral blood of asthmatics appear to be primed to release more reactive oxygen species than cells of healthy subjects. The enhanced agonist-induced rise in the intracellular free calcium concentration may be responsible for this increased respiratory burst. To test this hypothesis we studied the N-formyl-methionyl-leucyl-phenylalanine- and cyclopiazonic acid--(an inhibitor of Ca(2+)-ATPase of intracellular calcium stores) induced calcium increase in the polymorphonuclear leukocytes of 28 subjects (16 with moderate asthma, 69.6% +/- 8.3% predicted normal peak expiratory flow and 12 normal controls) using a fluorescent probe Fura-2AM at 100 nM and 1 mM extracellular calcium concentrations. In 1 mN calcium, the N-formyl-methionyl-leucyl-phenylalanine-induced calcium increase was 1.7-fold higher in asthmatics than in healthy subjects. Similarly, the contribution of calcium from intracellular stores to the calcium response to N-formyl-methionyl-leucyl-phenylalanine was higher in asthmatics (55% +/- 14% vs. 39% +/- 14%, P < 0.01). The pool of calcium released from intracellular stores by N-formyl-methinoyl-leucyl-phenylalanine and cyclopiazonic acid was 2.3- and 2.2-fold larger than in control cells. There was a correlation between maximal intracellular calcium concentration related to N-formyl-methionyl-leucyl-phenylalanine-induced calcium release from intracellular stores and forced expiratory volume in 1 s expressed as percentage predicted and reversibility in asthmatics (r = 0.63, r = -0.53, P < 0.05). In conclusion, polymorphonuclear leukocytes of asthmatics exhibit an altered calcium response that is mainly dependent on increased calcium release from intracellular stores.

Comparison of PAF- and fMLP-induced [Ca2+]i transients in human polymorphonuclear leukocytes.

Changes of [Ca2+]i in human polymorphonuclear leukocytes (PMNL) were studied. PMNL suspension was activated three times every 5 min with 10(-7) M PAF and fMLP. Both PAF and fMLP, induced three consecutive [Ca2+]i transients in PMNL suspended in medium with 1 mM Ca2+. The first Ca2+ response was a result of Ca2+ release from internal stores and the extracellular Ca2+ influx, while the second and third responses were completely dependent on Ca2+ influx from extracellular space. The contribution of Ca2+ from intracellular stores to the first PAF-induced Ca2+ response was about 1.4-fold lower in comparison with the first fMLP induced Ca2+ response (27 +/- 1 vs 37 +/- 6% (p < 0.05). Previous addition of PAF enhanced 3-fold (p < 0.001) the PMNL response to fMLP while cells pretreated with fMLP failed to increase their [Ca2+]i after challenge with PAF. PMNL from 40% of donors did not respond to PAF in the presence of 100 nM Ca2+. However, the cells responding to PAF as the cells treated with fMLP or cyclopiazonic acid released almost the entire Ca2+ from intracellular stores after challenge. Subtraction of mean [Ca2+]i transients in the presence of 100 nM Ca2+ from that obtained in medium with 1 mM Ca2+ showed that, in PMNL stimulated with PAF in contrast to the cells treated with fMLP, the onset of Ca2+ influx from extracellular space precedes Ca2+ release from intracellular stores. These results suggest that PAF-induced Ca2+ influx from extracellular space is at least partly independent of Ca2+ release from intracellular stores.

Effect of substance P and its precursor alpha-protachykinin on intracellular free calcium concentration in human polymorphonuclear leukocytes.

The increase in intracellular free calcium concentration is an important step in signal transduction leading to phagocyte activation. The undecapeptide substance P can influence various functions of human polymorphonuclear leukocytes, including chemotaxis, phagocytosis, and respiratory burst. In this study we investigated the ability of low-concentration (that can occur in vivo) substance P (10(-7) M) and its precursor alpha-protachykinin (3 x 10(-7) M) to increase the intracellular free calcium concentration in human polymorphonuclear leukocytes. Cells isolated from ten healthy donors were incubated with substance P or alpha-protachykinin in 1 mM calcium medium for 5 min and the intracellular free calcium concentration was monitored using the fluorescent calcium indicator Fura-2am. Polymorphonuclear leukocytes from 40% of donors responded to both agonists. The substance P- and alpha-protachykinin-induced increase in intracellular free calcium concentration was 59 +/- 13 nM and 58 +/- 12 nM and the extracellular calcium influx contributed to 87 +/- 8% and 54 +/- 8% of the calcium response, respectively. alpha-Protachykinin released almost all the calcium from intracellular stores, while substance P mobilized only 24 +/- 5% of this calcium pool. Finally, cells that responded to a single challenge with substance P and alpha-protachykinin were able to increase their intracellular free calcium concentration in response to each of three consecutive stimulations with these agonists. This may be an additional mechanism by which substance P and its precursor modify the function of human polymorphonuclear leukocytes.

Changes of intracellular free calcium concentration in human polymorphonuclear leukocytes after repeated stimulations with N-formyl-methionyl-leucyl-phenylalanine.

A rapid transient rise in the intracellular free calcium concentration ( Ca2+]i) is an important step in human polymorphonuclear leukocytes (PMNL) activation. This can be caused by many inflammatory mediators and has been implicated in the regulation of various cellular reactions. In this study we investigated the changes of [Ca2+]i in human PMNL activated three times with 10(-7)M n-formyl-methionyl-leucyl-phenylalanine (FMLP). PMNL in the presence of 1 mM Ca2+ were able to respond to three consecutive stimulations with FMLP. The first Ca2+ response was the highest one and was a result of Ca2+ release from internal stores (which was responsible for about 30% of maximal increment in [Ca2+]i) and the extracellular Ca2+ influx. Experiments with PMNL suspended in a medium containing 100 nM Ca2+ and pretreated with 1 nM Ni2+ (an inorganic calcium channel blocker) revealed that the second and third response is completely dependent on the extracellular Ca2+ influx. Changes of the time interval between stimulations had no influence on the occurrence of extracellular Ca2+ influx related to second addition of FMLP. Elongation of the time interval up to 30 min did not restore the release of Ca2+ from internal stores. It indicates the occurrence of dissociation of Ca2+ release from intracellular stores and extracellular Ca2+ influx during the second and third PMNL response to FMLP.

Effect of cytochalasin B on intracellular free calcium concentration in human polymorphonuclear leukocytes after repeated stimulation with n-formyl-methionyl-leucyl-phenylalanine.

Cytochalasin B can influence various functions of human polymorphonuclear leukocytes, including chemotaxis, lysosomal enzyme release, and reactive oxygen species generation. In this study we investigated the effect of cytochalasin B on the increase in intracellular free calcium concentration after three consecutive additions of 10(-7) M N-formyl-methionyl-leucyl-phenylalanine. The interval between stimulations was 5 min. Intracellular free calcium concentration was monitored using the fluorescent calcium indicator FURA-2AM. Cytochalasin B (3.3 micrograms/ml) added 60 s before the cell stimulation enhanced all three polymorphonuclear leukocyte calcium responses by increasing the N-formyl-methionyl-leucyl-phenylalanine-induced calcium influx from the extracellular space. Cytochalasin B increased the peak intracellular free calcium concentration and elevated the plateau phase level, but had no influence on its shape. In addition, pretreatment with cytochalasin B of polymorphonuclear leukocytes suspended in low calcium medium restored their capacity to respond to a third stimulation with N-formyl-methionyl-leucyl-phenylalanine. Finally, in resting cells cytochalasin B caused a moderate increase in intracellular free calcium concentration which was independent of extracellular calcium.

Ambroxol inhibits endotoxin-induced lipid peroxidation in mice.

Administration of ambroxol (70 mg/kg ip) once a day for 3 days protected lung and heart lipids from lipopolysaccharide (LPS, 17 mg/kg)-induced oxidative stress in mice. Ambroxol as a lipid peroxidation inhibitor was almost as active as an equivalent dose of N-acetylcysteine (27.6 mg/kg), a well known antioxidant. The lung and heart levels of conjugated dienes in animals pretreated with ambroxol were 3.3- and 1.7-times lower (p < 0.05 and p < 0.01) than those observed in the control group which received only buffer and subsequently LPS. These results indicate that ambroxol can sufficiently inhibit the harmful process of lipid peroxidation in vivo.

Effect of bacterial lipopolysaccharide on the content of lipid peroxidation products in lungs and other organs of mice.

The influence of lipopolysaccharide from Escherichia coli (LPS, 17 mg/kg body weight) on the lipid peroxidation process in organs of mice was studied. The content of conjugated dienes (CD), lipid peroxides (LP), malondialdehyde (MDA) (all three lipid peroxidation by-products), peroxidase (PO) activity and wet-to-dry weight ratio in lungs, heart, spleen, kidneys and liver were determined 1.5 h after intravenous injection of LPS. Animals observed at this time-point had reduced activity and decreased body temperature by about 2 degrees C, however, all analysed organs did not reveal any changes of wet-to-dry weight ratio comparing to organs from mice injected with sterile, pyrogen free 0.9% NaCl. Only extracts from heart and lungs showed significant increase in the tissue level of at least two lipid peroxidation products. The heart content of CD, MDA, and LP was about 1.5-, 1.3-, and 2.4-fold higher than in control group. In lungs CD and MDA increased 3.3- and 1.3-times but in spleen only content of LP was elevated. In these organs the suppression of PO activity was also observed. Liver and kidneys did not reveal any convincing enhancement of lipid peroxidation process and alterations of PO activity. Since free radical reactions are involved in lipid peroxidation process and inactivation of PO these results suggest that heart, lungs and spleen are the organs mostly exposed to oxidative stress during the first 1.5 h after single injection of LPS in mice.

Ascorbic acid enhances the decrease in peroxidase activity in inflamed tissues of mice.

The aim of this study was to explore whether intraperitoneal administration of ascorbic acid (AA) at a dose of 500 mg/kg, once a day for 3 following days, affected the peroxidase (PO) activity in inflamed feet of mice. The foot inflammatory reaction induced by the carrageenan (CAR), n-formyl-methionyl-leucyl-phenylalanine (FMLP) and xanthine-xanthine oxidase was accompanied by suppression of PO activity. Administration of AA, having no effect on the degree of foot oedema, skin temperature and microscopic picture of tissue specimens significantly enhanced the decline in PO activity provoked by inflammatory agents. This activity decreased 2.0-, 1.6- and 1.9-fold (p < 0.001, p < 0.01, p < 0.05) when inflammatory response was induced with FMLP, CAR and X-XO, respectively. Also in vitro AA (50-100 micrograms/ml) inhibited PO activity of leukocyte lysate and foot extract obtained from untreated animals. In conclusion we found that AA, having no effect on inflammatory response, significantly enhanced inhibition of PO activity in inflamed tissues in mice which could be a result of direct action of AA on the enzyme molecule.

Ascorbic acid did not alter the content of conjugated dienes and malondialdehyde in organs of mice.

The aim of this study was to explore whether ip administration of ascorbic acid (AA) in a dose of 500 mg/kg, once a day for 3 following days, affected the content of lipid peroxidation products: malondialdehyde (MDA) and conjugated dienes (CD) in organs of mice. Injection of AA caused 2.1-, 1.3- and 1.8-fold increase in the concentration of this vitamin in liver, spleen and lungs, respectively, while the content of MDA and CD in these organs did not differ from values found in animals treated with 0.9% NaCl. It suggests that in our animal model AA did not act as a prooxidant enhancing the lipid peroxidation in various tissues.

Erythrocytes protect alpha-1-proteinase inhibitor from oxidative inactivation induced by chemicals, the myeloperoxidase-H2O2-halide system and stimulated polymorphonuclear leukocytes.

The oxidative inactivation of the alpha-1-proteinase inhibitor (alpha 1PI) is one of the mechanisms responsible for creating the elastase/antielastase imbalance during inflammation in the lower respiratory tract. Chronic supremacy of elastase may cause degradation of elastin fibers leading to the pulmonary emphysema. In this study we have investigated the effect of erythrocytes (RBC, concentrations 0.125 to 1.5%) on the oxidative inactivation of alpha 1PI by the phorbol myristate acetate-stimulated polymorphonuclear leukocytes (PMNL) and PMNL myeloperoxidase-H2O2-halide system. During exposure of alpha 1PI to both systems in the presence of RBC the significant protection (p less than 0.001) of alpha 1PI was found for all RBC concentrations, e.g. at RBC concentration of 1% the elastase inhibitory capacity (EIC) of alpha 1PI increased from 0 to 60 +/- 6 and 88 +/- 9% of the control value (untreated alpha 1PI), n = 5, respectively. The preincubation of RBC with chloramine-T (1 mM), inhibition of RBC catalase or depletion of RBC reduced glutathione alone did not diminish the capacity of RBC to protect alpha 1PI. However, these treatments together completely deprived RBC of their protective properties. Moreover, we have compared the decrease in the EIC of human blood and its plasma after incubation with H2O2 (0.1 mM to 0.1 M) or chloramine-T (1 microM to 1 mM). For the incubation with H2O2 no decrease in blood EIC was found whereas in plasma the loss of EIC was already visible at a H2O2 concentration of 0.1 mM. Also for the incubation with chloramine-T the EIC of blood was more resistant to oxidant damage than EIC of plasma. It is suggested that RBC contaminations present in the phagocyte inflammatory infiltration in the lower airways may protect alpha 1PI from oxidative inactivation and thus indirectly diminish proteolytic lung injury related to inflammation.

Effect of ascorbic acid on hydroxyl radical generation by chemical, enzymatic and cellular systems. Importance for antioxidant prevention of pulmonary emphysema.

The ability of ascorbic acid (AA) (25 to 500 microM) to increase OH production by a chemical (Fe(2+)-EDTA-H2O2), an enzymatic (xanthine-xanthine oxidase-Fe(2+)-EDTA) and a cellular system (3.10(6) human polymorphonuclear leukocytes (PMNL) or murine peritoneal macrophages (PM) activated with 7.2 ng PMA/ml) was studied. At all concentrations used AA strongly enhanced OH generation by the chemical and the enzymatic systems. However, the maximal increase of about 14-fold was found for incomplete chemical system (10 microM Fe(2+)-20 microM EDTA) and 500 microM AA. In the case of phorbol-myristate-acetate-activated-PMNL and macrophages, the moderate increase in OH formation was only caused by low AA concentrations. At 50 microM AA, the OH formation was 112 +/- 3 and 117 +/- 4% of control, respectively. Higher AA concentrations had no influence or even decreased OH formation by phagocytes. It is suggested that administration of AA will not significantly enhance OH generation from pulmonary phagocytes and could be useful for prevention of the oxidant-mediated lung injury related to inflammation.

Effect of ascorbic acid on killing of lymphocytes and macrophages by hydrogen peroxide.

The ability of ascorbic acid (AA) to modify the H2O2 (0.6 to 9 mM) toxic activity for human lymphocytes (PBMC) and murine peritoneal macrophages (PM) was studied in vitro. 100 microM AA added simultaneously with H2O2 to the cell medium enhanced the killing of PBMC but not of PM independently of the presence of Fe(2+)-EDTA. Similarly, preincubation of PBMC with 500 microM AA that resulted in a 2.5-fold rise in the intracellular level of AA increased their susceptibility to both H2O2 and H2O2-Fe(2+)-EDTA. On the contrary, PM preincubated with AA were more resistant to the toxic action of the H2O2-Fe(2+)-EDTA system and did not reveal any changes of the susceptibility to H2O2 alone. It is suggested that AA under certain conditions may have opposite effects on the H2O2-induced cell damage related to inflammation.

The main serum protease inhibitors; alpha-1-proteinase inhibitor and alpha-2-macroglobulin inhibit H2O2 release from human polymorphonuclear leukocytes stimulated with phorbol myristate acetate.

Since various functions of phagocytes can be affected by protease inhibitors, the ability of alpha-1-proteinase inhibitor (alpha 1PI) and alpha-2-macroglobulin (alpha 2M) to modulate the H2O2 production by human polymorphonuclear leukocytes (PMNL) was studied. The preincubation of PMNL for 30 min with these protease inhibitors at concentrations which may occur in human blood diminished in a dose dependent manner their H2O2 generation induced with phorbol myristate acetate. At alpha 1PI 600 mg/dl and alpha 2M 800 mg/dl the H2O2 response decreased to 61 +/- 2 and 58 + 3% (p less than 0.001, n = 4) of the control value obtained with cells preincubated in phosphate buffered saline with glucose, respectively. Autologous serum alone and with addition of pure alpha 1 PI or alpha 2M also suppressed H2O2 release from PMNL. It is suggested that inhibition of H2O2 generation from PMNL may be another additional way by which these serum protease inhibitors may protect tissues (especially lungs) from acute injury related to inflammation.