Potential nitrite scavengers as inhibitors of the formation of N-nitrosamines in solution and tobacco matrix systems.

The ability of 20 compounds, all but one tobacco constituents, to inhibit the formation of tobacco-specific N-nitrosamines (TSNA) was investigated in buffer and detergent solution and in tobacco midrib and lamina systems. In solution at pH 5.5, ascorbic acid and the phenolic acids caffeic and ferulic acid were the most potent inhibitors of the reaction between nornicotine and nitrite, with nearly complete inhibition at molar ratios test compound/nitrite > 1:1. Also, cysteine > dihydrocaffeic acid > protocatechuic acid approximately catechin acted as strong inhibitors with >90% inhibition at a ratio of 3:1. Lower inhibitions were observed with chlorogenic acid > p-coumaric acid > sclareol > serine. Rutin showed an inhibition of 34% at a ratio of 0.1:1. Sclareol, alanine, proline, and serine did not significantly affect the N-nitrosonornicotine (NNN) formation. alpha-Tocopherol and glutathione enhanced NNN formation at pH 5.5 but were inhibitors at pH 3. Cinnamic acid, vanillic acid, eugenol, and esculin enhanced NNN formation. Increased NNN formation was also observed for dihydrocaffeic acid, chlorogenic acid, protocatechuic acid, and catechin at a less-than-equimolar ratio of test compound to nitrite. The tobacco matrix experiments were performed with air-cured, ground tobacco midrib and lamina. Caffeic acid, ferulic acid, dihydrocaffeic acid and catechin were potent inhibitors of the formation of TSNA in the midrib as well as in the lamina. Also protocatechuic acid, glutathione, ascorbic acid, p-coumaric acid, chlorogenic acid and cysteine were inhibitors, while alpha-tocopherol and rutin inhibited the reaction in the midrib but not in the lamina. Cinnamic acid, vanillic acid, eugenol, alanine, proline and serine showed small effects only. The molar ratio of secondary alkaloid(s)/nitrite in the test systems were 0.1:1 (solution), approximately 0.25:1 (midrib), and approximately 1:1 (lamina) and is most likely the major contributor to the observed order of inhibition potency (solution > midrib > lamina) of the test compounds. The vicinal phenolic hydroxyl groups of polyphenols and the simultaneous presence of a phenol group and an olefinic bond in hydroxycinnamic acids were the most characteristic structural elements of the potent inhibitors.

Influence of human plasma high density lipoproteins from septic patients on different functions of normal human neutrophils.

High Density Lipoproteins (HDL) from three patients with E. coli sepsis contained high, low and no Serum Amyloid Protein (Apo SAA), respectively. Preincubation of neutrophils from healthy persons for half an hour with sepsis HDL as well as normal HDL increased the phagocytosis, the stimulated nitroblue tetrazolium reduction, the chemotaxis and the random migrations of these cells. However, for all these functions, lower values were obtained after incubation with sepsis HDL containing high amounts of apo SAA than with normal HDL. A qualitative change of HDL might thus in part be responsible for the decreased function of neutrophils noted during the acute phase of bacterial infections.

Significance of oxygen availability for release of oxygen free radicals and lysozyme by neutrophils.

Neutrophilic granulocytes were exposed to an atmosphere of nearly 100% oxygen (hyperoxia) for one hour. The nitroblue tetrazolium (NBT) reduction, reflecting oxygen radical release, was decreased both in resting and stimulated cells, but lysozyme release was unchanged. Short time exposure of patients to oxygen hypertension might therefore be beneficial as therapy, in conditions where reduced production of oxygen radicals is required. The NBT reduction of resting and stimulated neutrophils in an atmosphere of purified argon (hypoxia) was also considerably decreased, and the lysozyme release unchanged. This reflects the anaerobic conditions in abscesses, where the contribution of neutrophil oxygen metabolites to the killing of microorganism might be reduced.

Alveolar macrophages in rabbits after combined exposure to nickel and trivalent chromium.

Rabbits were exposed to a combination of 0.7 mg/m3 Ni2+ as NiCl2 and 1.2 mg/m3 of Cr3+ as Cr(NO3)3, to 0.6 mg/m3 of Ni2+ as NiCl2, or to filtered air for about 4 months, 5 days/week and 6 hr/day. Alveolar macrophages were recovered by lung lavage and studied by light and electron microscopy. Metabolic activity, phagocytic capacity and lysozyme activity in the macrophages were studied. After the combined exposure, the effects on lung weight, number of macrophages, and appearance of surface and number of intracellular laminated inclusions in these cells were more than additive. These effects might be explained by a combination of increased production by Ni2+ and impaired catabolism of surfactant by Cr3+. Because the metal concentrations used were not far above occupational threshold limit values, combined exposures to nickel and trivalent chromium should be considered more seriously.

Morphology and function of blood monocytes after incubation with lung surfactant.

Human blood monocytes were incubated for different periods of time with lung surfactant (phospholipid concentration 1-2.5 mg/ml). After short-term (30 min) incubation, there was an increase in the nitroblue tetrazolium (NBT) reduction of the monocytes both at rest and during stimulation with E. coli bacteria, and enhanced ingestion of fluorescein-labelled yeast particles. Electron microscopic examination of the same monocytes showed an active cell surface with numerous protrusions. Long-term (24 h) incubation with surfactant resulted in a reduced ability of the cells to adhere to plastic dishes. Although the NBT-reduction of resting monocytes was increased after long-term incubation with surfactant, the additional enhancement of NBT-reduction after stimulation with bacteria was decreased. These cells were rounded, usually devoid of surface structures, their nuclei were condensed, and their cytoplasm filled with surfactant material. Thus, monocytes are initially activated in the presence of surfactant, but if the cells become overfed with surfactant lipids their functional capacity decreases.

Rabbit alveolar macrophages after long-term inhalation of soluble cobalt.

Rabbits were exposed to 2 or 0.4 mg/m3 of cobalt as CoCl2 for 14-16 weeks (5 days/week and 6 hr/day). More macrophages were lavaged from the lungs of rabbits exposed to the higher Co2+ concentration, and the diameter and variation of the diameter of the macrophages were significantly larger than in controls. The activity of lysozyme in the lavage fluid and in the macrophages was increased in the two exposed groups. Some macrophages in the exposed animals were large and engorged with intracellular lamellar inclusions and lipid droplets. Most of these cells had a smooth surface. The oxidative metabolic activity measured by reduction of nitroblue tetrazolium was increased in the exposed groups. The number of yeast cell particles attached to the surface of the macrophages was increased in the group exposed to the high concentration, but the number of ingested particles was not affected by cobalt exposure. Apart from the fact cobalt increased lysozyme activity whereas nickel decreased it, cobalt produced the same type of effects on macrophages as nickel did in earlier studies. Cobalt affected only a minor proportion whereas nickel affected most macrophages. This can be explained by the fact nickel produced a general increase in the volume density of the type II cells while cobalt affected the type II cells only in some areas of the lungs.

High-density lipoproteins inhibit the bacterial lipopolysaccharide mediated increase in oxidative metabolism and lysozyme release by neutrophilic granulocytes in vitro.

The effects of purified Salmonella endotoxin (LPS) and of LPS combined with isolated human plasma high density lipoproteins (HDL) on oxidative metabolism, measured by nitroblue tetrazolium (NBT)-test, and on lysozyme release of human granulocytes have been studied in vitro. A considerable increase in the NBT-reduction and in lysozyme release was noted in granulocytes exposed to LPS. The stimulating effect of LPS on NBT-reduction and lysozyme release was significantly diminished when the cells were incubated with LPS together with HDL. These observations suggest that HDL in vivo may play an important part in inhibition of metabolic changes induced in granulocytes by LPS which leads to the production and secretion of tissue damaging mediators.

Rabbit alveolar macrophages after inhalation of hexa- and trivalent chromium.

Rabbits inhaled aerosols of hexavalent chromium (Na2CrO4) and trivalent chromium (Cr(NO3)3) at concentrations of 0.9 and 0.6 mg/m3 of the metal, respectively, for 4-6 weeks (5 days/week and 6 hr/day). Significantly more macrophages were obtained from the lungs of rabbits exposed to Cr(VI) but not from rabbits exposed to Cr(III) as compared with the controls. Macrophages from rabbits exposed to Cr(III) showed several conspicuous changes. About one-third of the macrophages contained round dark inclusions, 0.5-1.5 micron diameter, rich in chromium. Most cells had very large lysosomes which contained membranous fragments of different sizes surrounded by a more homogeneous matrix. Laminated inclusions similar to the lamellar bodies in the type II cells increased in number as did the percentage of cells with a smooth cell surface. Also macrophages from rabbits exposed to Cr(VI) showed morphological changes. The most pronounced one was enlarged lysosomes which contained short lamellae and electron-dense patchy inclusions. Only Cr(III) produced functional changes of the macrophages. The metabolic activity measured by reduction of nitroblue tetrazolium was increased and the phagocytic activity reduced.

Rabbit lung after inhalation of manganese chloride: a comparison with the effects of chlorides of nickel, cadmium, cobalt, and copper.

Rabbits were exposed to aerosols of MnCl2 (mass median aerodynamic diameter 1 micron) in metal concentrations of 1.1 and 3.9 mg/m3 for 4-6 weeks, 5 days/week, 6 h/day. The effects of alveolar type II cells, phospholipids, alveolar macrophages, and lung structure in general were compared with earlier reported effects of Ni2+, Cd2+, Cu2+, and Co2+. Except for a significant increase in the diameter of the alveolar macrophages after exposure to the higher Mn2+ concentration, no abnormalities were seen. The results of this and earlier studies indicate that these five metal ions have different, specific effects on the alveolar part of the lung.

Toxicology of nickel.

Rabbits were exposed to low levels of airborne metals for 1-8 months, 5 days/week, 6 hours/day. After exposure, lung tissue was examined by light and electron microscopy. Macrophages lavaged from the left lung were examined morphologically and functionally. Phospholipids were analysed in lung tissue or lavage fluid. Metallic nickel dust, 0.1-1 mg/m3, affected alveolar macrophages, alveolar epithelial type II cells and phospholipids. In the lung tissue, nodular accumulation of macrophages was seen, and the volume density of alveolar type II cells was elevated. The amount of phospholipids was markedly increased, mainly due to an increase in disaturated phosphatidylcholines. After 1 month of exposure the macrophages appeared active. After 3 months they appeared 'overfed' and inactive. Metallic iron, chromium and cobalt did not produce the same effects as nickel. Exposure to 0.2 mg/m3 soluble nickel as nickel chloride produced almost identical effects to those of metallic nickel, indicating that the effect of the metallic nickel particles was caused by nickel ions. Exposure to cadmium chloride produced nearly all the effects produced by nickel chloride. However, cadmium chloride increased the level of lysozyme in the macrophages whereas nickel chloride decreased it. Cadmium chloride also produced interstitial alveolitis and cytoplasmic blebs on the surface of the macrophages. Cobalt chloride affected the growth of the type II cells, which formed nodules, but did not seem to affect the production of surfactant material by those cells. Copper chloride produced no effect apart from a slight increase in volume density of the type II cells. Thus, of four divalent metal ions, three (Ni2+, Cd2+ and Co2+) in similar concentrations in the inhaled air produced clear but different pathological effects in the lungs.

Opposite effects of different plasma lipoprotein fractions on granulocyte function.

Different plasma lipoprotein fractions in physiological concentrations have opposite effects on the phagocytosis and migration of human neutrophilic granulocytes. Preincubation for half an hour with very low density lipoproteins decreased the phagocytosis as well as the chemotactic and random migrations of the cells, while preincubation with high density lipoproteins increased the same functions. No effects were seen with low density lipoproteins. It is suggested that disturbances in plasma lipoprotein pattern may affect the function of phagocytes.

Rabbit alveolar macrophages after inhalation of soluble cadmium, cobalt, and copper: a comparison with the effects of soluble nickel.

Rabbits were exposed to aerosols of chlorides of cadmium, copper, and cobalt (0.4-0.6 mg/m3 as metal) for 1 month (5 days/weeks and 6 hr/day). The effects of alveolar macrophages were compared with earlier reported effects of nickel chloride (0.3 mg/m3 as Ni). Effects of Cd2+ exposure resembled those of Ni2+ exposure. The number of macrophages in lavage fluid and the variance of cell diameters were thus increased and many cells contained lamellated inclusions. Contrary to macrophages from Ni2+-exposed rabbits, the surface of about 50% of the cells had cytoplasmic blebs. However, such cells were rarely seen by scanning electron microscopy. There were significantly more polymorphonucleated neutrophils and small lymphocytes, suggesting lung parenchymal damage. Cells from Cd2+-exposed animals, like cells from Ni2+-exposed ones, showed an increased oxidative metabolic activity after stimulation with Escherichia coli bacteria. Bactericidal capacity, on the other hand, tended to be enhanced rather than decreased, as in the nickel experiment. After CO2+ exposure, the number of macrophages was slightly increased in the lavage fluid and the cells showed an increased metabolic activity both at rest and upon stimulation with bacteria. Cu2+ exposure gave a slight increase in lamellated inclusions in the macrophages.

The effect of intralipid on mononuclear and polymorphonuclear phagocytes.

Eight healthy subjects were given Intralipid, a soybean oil emulsion, 20% intravenously for 2 h. During the infusion a significant increase in the nitroblue tetrazolium-reduction of blood monocytes was noted. Preincubation of monocytes in vitro with Intralipid (20 to 100 mg/ml) for 30 min was found to increase the ability of the cells to migrate chemotactically and to phagocytize yeast particles. On the contrary, when neutrophilic granulocytes were preincubated with Intralipid in the same concentrations for 30 min. their nitroblue-tetrazolium-reduction, chemotactic and spontaneous locomotion, as well as their ingestion of yeast particles was depressed.

Rabbit lung after inhalation of soluble nickel. I. Effects on alveolar macrophages.

Alveolar macrophages from eight rabbits, exposed for about 1 month (5 days/week, 6 hr/day) to an aerosol of nickel chloride, 0.3 mg/m3 (as Ni), were studied. The number of macrophages in the lavage fluid and the variance of the cell diameter increased. The macrophages contained laminated structures and most cells had an active cell surface. A few macrophages had a large number of laminated structures and a smooth cell surface. The capacity of the macrophages to reduce nitroblue tetrazolium (NBT) tended to be increased at rest and was significantly increased after stimulation with Escherichia coli. The bactericidal capacity of the macrophages was decreased. The effects were similar to those earlier described after exposure of rabbits for 1 month to about 1 mg/m3 of metallic nickel dust. After exposure both to metallic and soluble nickel the effects are probably caused by an increased amount of surfactant produced by the type II cells in response to nickel ions.

Rabbit lungs after long-term exposure to low nickel dust concentration. II. Effects on morphology and function.

For 4 and 8 months (5 days/week, 6 hr/day) rabbits were exposed to 0.13 +/- 0.05 (mean +/- SD) mg/m3 of metallic nickel dust. Volume density of alveolar type II cells was estimated with electron microscopy. Lavaged alveolar macrophages were studied with light and electron microscopy and their abilities to reduce nitroblue tetrazolium (NBT) and to phagocytize particles were tested. The effects seemed to be similar after 4 and 8 months of exposure and when the exposed animals were combined, volume density of type II cells was increased and also significantly correlated with concentration of disaturated phosphatidylcholines in the lung. The macrophages had an active surface. Their NBT activity at rest was increased but a further increase during stimulation with E. coli was low, suggesting an impaired function. Phagocytic activity, however, was not significantly changed.

The effect of phospholipid-containing surfactant from nickel exposed rabbits on pulmonary macrophages in vitro.

Alveolar macrophages from 9 normal rabbits were incubated in vitro for 3 h with and without phospholipid-containing surfactant from nickel-treated ones. The macrophages treated with surfactant showed morphological and functional criteria of increased activity. The cell surface had many protrusions and the cytoplasma contained several lamellated structures. The oxidative metabolism, measured by the nitroblue tetrazolium (NBT)-test, at rest and after E. coli stimulation was increased, as was the attachment and ingestion of yeast particles. The NBT-values were about the same as corresponding values of macrophages lavaged from the lungs of nickel-treated rabbit. Macrophages incubated with surfactant from untreated animals, had NBT values and phagocytic activity similar to cells incubated without surfactant. As this substance was administered in excess, the difference in macrophage response would probably be due to a qualitative alteration of the surfactant after nickel exposure.

Decreased chemotactic and random migration of leukocytes during Intralipid infusion.

The soybean oil emulsion Intralipid was given intravenously to 12 healthy subjects for 2 hr. During the infusion an impairment of the chemotactic and random migration of leukocytes was noted. It was correlated to the dose given and to the degree of hypertriglyceridemia induced. Migration was fully restituted 22 hr after the infusion. Also when added in vitro Intralipid caused an impairment of leukocyte motility that followed a dose response pattern.

Alveolar macrophage function in nickel dust exposed rabbits.

8 rabbits were exposed to metallic nickel dust (2 mg/m3, of which about half was respirable) for 4 weeks. The lungs were lavaged and the macrophages were collected. In comparison with 8 control rabbits, a significant increase was noted in the nickel exposed rabbits as concerned the weight and density of the lungs, the size variation of the lung cells, the phagocytosis of silver coated particles, and the metabolic activity as measured by NBT reduction. The last mentioned increase was recorded during basal conditions as well as during phagocytosis. The NBT reduction during phagocytosis was significantly correlated with the degree of phagocytosis of silver coated particles in both control and exposed rabbits. It is suggested that the exposure to nickel dust has unspecifically activated the macrophages perhaps by increased production of phospholipids.

The value of immunoelectroosmophoresis (IEOP) for etiological diagnosis of acute respiratory tract infections due to pneumococci and Mycoplasma pneumoniae.

Immunoelectroosmophoresis (IEOP) has been used to detect pneumococcal and Mycoplasma pneumoniae antigen in 324 sputum samples from 224 patients. Pneumococcal antigen was found in 30/37 samples from which pneumococci had earlier been isolated and in 72/243 specimens where they had not been found. Of these 72 samples 69 were from patients treated with antibiotics. Mycoplasma antigen was found in 9/57 sputum samples from which Mycoplasma had been isolated and in 2/32 other samples from patients with a serologically verified diagnosis of mycoplasmal infection. As to pneumococci, the IEOP is of value because of its rapidity and especially because antigen findings can be made in patients treated with antibiotics. In spite of sonication and concentration, mycoplasma antigen was too rarely found for the method, as now carried out, to be useful in diagnostic work.