Effects of heavy metal ions on selected oxidative metabolic processes in rat alveolar macrophages.
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Animals↗
Biomedical subjects
V Castranova
Publications and source records attributed to V Castranova.
Toxicity of metal ions to alveolar macrophages.
Significant concentrations of metals are found in the respirable particulate effluents associated with metallurgical smelters. In this investigation the effects of the metallic ions lead, cadmium, iron, mercury, aluminum, chromium, and nickel on various aspects of alveolar macrophage function were studied. The production of antibacterial substances (ie, reactive forms of oxygen) by these cells and oxygen consumption are very sensitive to the metals. Particle uptake displays moderate sensitivity, while lysosomal enzyme activity and membrane integrity are fairly resistant to metals. In addition, the effects of the organic solvents carbon tetrachloride, toluene, and xylene on alveolar macrophage function were tested. These solvents were found to inhibit oxygen consumption and the release of antibacterial substances while not greatly affecting membrane integrity. The results of these experiments indicate that some metals and some organic substances are toxic to alveolar macrophage function.
Animals↗
Purification of human granulocytes by centrifugal elutriation and measurement of transmembrane potential.
Viable cell samples containing 93% pure granulocytes were obtained from human blood using the techniques of dextran sedimentation followed by centrifugal elutriation. The resting transmembrane potential (Em) of human granulocytes was estimated using the fluorescent lipophilic cation, Di-S-C3(5), from the null point for potassium--i.e., the external K concentration at which there is no change in Em in response to valinomycin (a K ionophore). The Em of human granulocytes, as calculated from the Nernst potential for K at the null point, is approximately -100 mV. Data indicate that this large transmembrane potential is due in part to the presence of an electrogenic Na-K pump in human granulocytes which is stimulated by external potassium and inhibited by ouabain.
Cell Separation↗
Transmembrane potential and ionic content of rat alveolar macrophages.
The cell volume, cell water, intracellular ionic concentrations, and transmembrane potential of rat alveolar macrophages were determined. The measurements were made on cells which had been separated from the medium by centrifugation through dibutyl phthalate in order to greatly reduce the trapped extracellular space. The mean cell volume of the alveolar macrophages is 1,525 cubic microns and 72% of this volume is water. The intracellular fluid is high in Na+ (97 mM) and lower in K+ (50 mM) and the intracellular Cl- concentration in 64 mM. The transmembrane potential, as measured from the equilibrium distribution of tritiated triphenylmethyl phosphonium and by using the fluorescent probe, Di-S-C3(5), is approximately -37 millivolts. Neither Na+, K+, nor Cl- is distributed at equilibrium. However, the K+ permeability of alveolar macrophage membranes appears to be greater than Na+ permeability.
Animals↗
Characteristics of anion transport in cat and dog red blood cells.
Self-exchange of chloride and sulfate in dog and cat red cells has been measured under equilibrium conditions. The rats of efflux for these anions are approximately twofold higher in dog compared to cat red blood cells. Although the rates differ, the anion exchange systems of these two red cell types exhibit many common properties. The dependence of 35SO4 efflux on the intracellular SO4 concentration, the pH dependence and the inhibition of 35SO4 efflux by Cl and SITS are almost identical in dog and cat red cells. Nystatin treatment was used to study the dependence of 36Cl efflux on internal Cl. Chloride efflux exhibits saturation in both cell types with dog red cells possessing a higher Vmax and K1/2 than cat red cells. The number of anion transport sites was estimated by extrapolation to the number of molecules of dihydro DIDS (H2DIDS, where DIDS is 4,4'-diisothiocyano-2,2' stilbene-disulfonic acid) which were bound at 100% inhibition of transport. The results indicate that either the turnover numbers for anion transport differ in dog, cat, and human red cells or that there is heterogeneity in the function of the membrane components which bind H2DIDS.
4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
Biochemical characteristics of rat alveolar macrophages with chlorphentermine-induced phospholipidosis: variations with increasing cell size.
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Animals↗
Heterogeneity in dog red blood cells: sodium and potassium transport.
After incubation in isotonic KCl, dog red blood cells can be separated by centrifugation into subgroups which assume different cell volumes and possess different transport characteristics. Those red cells which swell in isotonic KCl exhibit a higher permeability to K and possess a greater volume dependence for transport of K than those red cells which shrink. A high Na permeability characterizes cells which shrink in isotonic KCl and these cells exhibit a larger volume-dependent Na flux than those red cells which swell. These two subgroups of red cells do not seem to represent two cell populations of different age. The results indicate that the population of normal cells is evidently heterogeneous in that the volume-dependent changes in Na and K permeability are distributed between differnt cell types rather than representing a single cell type which reciprocally changes its selectivity to Na and K.
Animals↗
Anion transport in dog, cat, and human red cells. Effects of varying cell volume and Donnan ratio.
Membrane potential and the rate constants for anion self-exchange in dog, cat, and human red blood cells have been shown to vary with cell volume. For dog and cat red cells, the outward rate constants for SO4 and Cl increase while the inward rate constant for SO4 decreases as cells swell or shrink. These changes coincide with the membrane potential becoming more negative as a result of changes in cell volume. Human red cells exhibit a similar change in the rate constants for SO4 and Cl efflux in response to cell swelling, but shrunken cells exhibit a decreased rate constant for SO4 efflux and a more positive membrane potential. Hyperpolarization of shrunken dog and cat red cells is due to a volume-dependent rate constant for SO4 efflux and a more positive membrane potential. Hyperpolarization of shrunken dog and cat red cells is due to a volume-dependent increase in PNa. If this increase in PNa is prevented by ATP depletion or if the outward Na gradient is removed, the response to shrinking is identical to human red cells. These results suggest that the volume dependence of anion permeability may be secondary to changes in the anion equilibrium ratio which in red cells is reflected by the membrane potential. When the membrane potential and cell volume of human red cells were varied independently by a method involving pretreatment with nystatin, it was found that the rate of anion transport (for SO4 and Cl) does not vary with cell volume but rather with membrane potential (anion equilibrium ratio); that is, the rate constant for anion efflux is decreased and that for influx is increased as the membrane potential becomes more positive (internal anion concentration increases) while the opposite is true with membrane hyperpolarization (a fall in internal anion concentration).
Animals↗
Reactive forms of oxygen and chemiluminescence in phagocytizing rabbit alveolar macrophages.
Chemiluminescence (CL), superoxide anion (O2-) production, and particle uptake were measured to determine the role of antibacterial substances in the chemiluminescent response associated with phagocytosis in rabbit alveolar macrophages (AM). Exposure of AM to zymosan particles induced both CL and the production of extracellular O2-. CL is inhibited by superoxide dismutase, an enzyme which catalyzes the conversion of O2- to hydrogen peroxide (H2O2), by catalase, an enzyme which destroys H2O2, and by the hydroxyl radical (.OH) scavengers, benzoate and ethanol. Superoxide dismutase and catalase probably exert their effects in the extracellular fluid. CL can also be produced by the addition of NaO2 or H2O2 to zymosan in a noncellular system. The chemiluminescent response occurs before particle uptake is complete, which also indicates that CL occurs in the extracellular fluid. These results suggest that CL induced by zymosan in AM is due to the extracellular reaction between various reactive forms of oxygen and zymosan.
Animals↗
The effect of inorganic phosphate on sodium fluxes in dog red blood cells.
The effect of extracellular inorganic phosphate on Na+ movements in dog red blood cells has been studied. As the phosphate concentration is increased from 0 to 30 mM, Na+ efflux increases by 2- to 3-fold and Na+ influx increases approximately 2-fold. This enhancement of Na+ fluxes by phosphate can be prevented by the addition of iodoacetate (1 mM), an inhibitor of glycolysis, or 4-acetamido-4'-iso-thiocyantostilbene-2,2'-disulfonic acid (0.01 mM), which blocks anion transport, to the medium. The increases in Na+ movements are not caused by changes in cell volumes. These results suggest that phosphate must enter the cell to enhance Na+ fluxes and that the mechanism of action may be via a stimulatory effect on glycolysis.
Animals↗
Study of amino and sulfhydryl sites in the sodium pathway in dog red blood cell membranes.
Amino reactive TNBS (2,4,6-trinitrobenzene sulfonic acid). SITS (4-acetamido-4'-isothiocyano-stilbene-2-2' -disulfonic acid), and Zn++, and SH reactive Hg++ were employed to study sodium channels in dog red blood cells. Simultaneous modification of the membrane with both a SH and an amino modifier results in an increase in Na+ permeability which is equal to the sum of their individual effects. This indicates that SH and amino sites are separate units. Three lines of evidence indicate that the amino sites are more superficial than the SH sites. (1) Pretreatment with an amino modifer decreases the effectiveness of subsequent SH modification. (2) SITS, a nonpenetrating amino reagent, enhances Na+ permeability while DTNB, a nonpenetrating SH modifier, is ineffective. (3) Pretreatment of amino sites decreases the apparent affinity of Hg++ for SH sites. In addition, three lines of evidence indicate that TNBS and Zn++ modify different amino sites. First, simultaneous modification with TNBS and Zn++ results in an increase in Na+ permeability equal to the sum of their individual effects. Secondly, Zn++ causes an increase in Na+ permeability in cells previously treated with TNBS. Finally, the pH dependence of Zn++ modification is oposite that for TNBS modification. These pH experiments suggest than Zn++ enhances Na+ permeability by reacting with unprotonated amino sites while TNBS modifies protonated amino sites. It is concluded that the sodium permeability of dog red blood cells is normally limited by superficial amino sites and deeper slfhydryl sites in the sodium channels.
Amines↗
Dog red blood cells: Na and K diffusion potentials with extracellular ATP.
External ATP causes a prompt increase in the Na and K permeability of dog red blood cells. By manipulating intra- and extracellular ion composition it is possible to observe ATP-induced net fluxes which can be explained in terms of the contribution of Na or K diffusion potentials to the membrane potential. Measurements of membrane voltage by a fluorescent dye technique confirm the existence of such potentials. A rough calculation of chloride permeability gives a value of the order of 10(-8) cm/s, which agrees with results in other species. The cells appear to be somewhat more permeable to bromide than to chloride.
Adenosine Triphosphate↗
Sodium permeability of dog red blood cell membranes. I. Identification of regulatory sites.
Divalent cations and group-specific chemical modifiers were used to modify sodium efflux in order to probe the molecular structure of sodium channels in dog red blood cells. Hg++, Ni++, Co++, and PCMBS (parachloromercuribenzene sulfonic acid), a sulfhydryl reactive reagent, induce large increases in Na+ permeability and their effects can be described by a curve which assumes 2:1 binding with the sodium channel. The sequence of affinities, as measured by the dissociation constants, reflects the reactivity of these divalent cations with sulfhydryl groups. In addition, the effects of Hg++ and PCMBS can be reversed by the addition of dithiothreitol, an SH-containing compound, to the medium. Much smaller increases in Na+ permeability are produced by Zn++ and the amino-specific reagents, TNBS (2,4,6-trinitrobenzene sulfonic acid) and SITS (4-acetamido-4'-isothiocyano-stilbene-2-2'-disulfonic acid). The Zn++ effect can be described by a curve which assumes bimolecular binding with the channel, and its effect on Na+ permeability can be reversed by the addition of glycine to the medium. The effects of Ni++ and SITS can be completely reversed by washing the cells in 0.16 M NaCl while TNBS binding is partially irreversible. Measurements of mean cell volumes (MCV) indicate that the modifier-induced increases in Na+ permeability are not caused by shrinkage of the cells. It is concluded that the movement of sodium ions through ionic channels in dog red blood cells can be enhanced by modification of amino and sulfhydryl groups. Zn++, TNBS, and SITS increase Na+ permeability by modifying amino groups in the channel while Hg++, Ni++, Co++, and PCMBS act on sulfhydryl groups.
Animals↗
Pharmacological study of oyster mushroom (Pleurotus ostreatus) extract on isolated guinea pig trachea smooth muscle.
Mushroom farm workers suffer from respiratory symptoms during the farming of mushrooms. The objective of this study was to analyze the effects of oyster mushroom (Pleurotus ostreatus) extract (OME) on isolated guinea pig tracheal smooth muscle in vitro. Isolated guinea pig tracheal tissue from 27 nonsensitized guinea pigs were studied. The OME was obtained from indoor mushroom growing fields and prepared as a 1:10 w/v aqueous solution. Dose-related contractions of nonsensitized guinea pig trachea were demonstrated using these extracts. The OME contained significant quantities of bacterial components (eg., endotoxin: 43,072.92 EU/mg). Parallel, pharmacological studies were performed by pre-treating the tissues with mediator-modifying agents including atropine, indomethacin, pyrilamine, BPB, acivicin, NDGA, captopril, TMB8 and capsaicin. Atropine consistently and strikingly reduced the contractile effects of this extract. These observations suggest an interaction of the OME with parasympathetic nerves or more directly with muscarinic receptors. Pretreatment with TMB8 (inhibitor of intracellular calcium mobilization) also significantly blocked the constrictor effect of OME, indicating a role of calcium mobilization in the constricting effect of OME. Inhibition of contraction by blocking of other mediators was less effective and varied depending on the drug. We conclude that OME causes a dose-related airway smooth muscle constriction by nonimmunological mechanisms involving a variety of airway mediators and possibly cholinergic receptors. This effect is not dependent on pre-sensitization of the guinea pigs.
Agricultural Workers' Diseases↗
Effect of silica inhalation on the pulmonary clearance of a bacterial pathogen in Fischer 344 rats.
Silica inhalation predisposes workers to bacterial infection and impairments in pulmonary defense function. In this study, we evaluated the effect of pre-exposure to silica on lung defense mechanisms by use of a rat pulmonary Listeria monocytogenes infection model. Male Fischer 344 rats were exposed by inhalation to filtered air or silica (15 mg/m3 x 6 h/day x 5 days/wk). After 21 or 59 days of silica exposure, the rats were inoculated intratracheally with 5 x 10(3) L. monocytogenes. At 0 (noninfected controls), 3, and 7 days after infection, the left lungs were removed, homogenized, and the number of viable L. monocytogenes was counted after an overnight culture at 37 degrees C. Bronchoalveolar lavage (BAL) was performed on the right lungs. Alveolar macrophages (AM) were collected, and the AM production of chemiluminescence (CL), an index of reactive oxygen species generation, was measured. The number of lavagable neutrophils (PMNs) and acellular BAL lactate dehydrogenase (LDH) activity were determined as indices of inflammation and injury, respectively. Pre-exposure to silica for 59 days caused substantial increases in PMN number and LDH activity compared with the air controls, whereas silica inhalation for both 21 and 59 days significantly enhanced the pulmonary clearance of L. monocytogenes compared with air controls. Dramatic elevations were also observed in zymosan- and phorbol myristate acetate (PMA)-stimulated CL production by lung phagocytes recovered from rats pre-exposed to silica for 59 days. These results demonstrate that short-term exposure to inhaled silica particles activates lung phagocytes, as evidenced by increases in reactive oxygen species. This up-regulation in the production of antimicrobial oxidants is likely responsible for the enhancement in pulmonary clearance of L. monocytogenes observed with short-term silica inhalation.
Administration, Inhalation↗
Reactive oxygen species and silica-induced carcinogenesis.
Although silica has recently been designated as a carcinogen, its mechanism of carcinogenesis is not fully understood. Recent studies suggest that free-radical reactions may play an important role in the initiation and progression of cancer. This article summarizes literature on the generation of reactive oxygen species (ROS) directly from silica and from silica-stimulated cells. It also summarizes information concerning the role of ROS in silica-induced DNA damage as well as in silica-induced cell proliferation, including the effects of silica on the activation of nuclear transcription factors, induction of growth factors and oncogene expression, redox regulation of the p53 tumor suppressor gene, induction of apoptosis, and division of damaged cells. Understanding the role of ROS in silica-mediated reactions may help develop therapeutic agents to block silica-induced free radical reactions and thus prevent or attenuate silica-induced carcinogenesis.
Animals↗
Reduction of chromium(VI) and its relationship to carcinogenesis.
Although Cr(VI)-containing compounds are well-documented carcinogens, their mechanism of action is still not well understood. Recent studies have suggested that reduction of Cr(VI) to its lower oxidation states and related free-radical reactions play an important role in carcinogenesis. This article summarizes recent studies on (1) the reduction of Cr(VI) by ascorbate, diol- and thiol-containing molecules, certain flavoenzymes, cell organelles, intact cells, and whole animals; (2) free-radical production with emphasis on hydroxy radical generation via Fenton or Haber-Weiss type reactions; and (3) free-radical-induced cellular damage, such as DNA strand breaks, hydroxylation of 2'-deoxyguanosine, and activation of nuclear transcription factor kappa B.
Animals↗
Mechanistically identified suitable biomarkers of exposure, effect, and susceptibility for silicosis and coal-worker's pneumoconiosis: a comprehensive review.
Clinical detection of silicosis is currently dependent on radiological and lung function abnormalities, both late manifestations of disease. Markers of prediction and early detection of pneumoconiosis are imperative for the implementation of timely intervention strategies. Understanding the underlying mechanisms of the etiology of coal workers pneumoconiosis (CWP) and silicosis was essential in proposing numerous biomarkers that have been evaluated to assess effects following exposure to crystalline silica and/or coal mine dust. Human validation studies have substantiated some of these proposed biomarkers and argued in favor of their use as biomarkers for crystalline silica- and CWP-induced pneumoconiosis. A number of "ideal" biological markers of effect were identified, namely, Clara cell protein-16 (CC16) (serum), tumor necrosis factor-alpha (TNF-alpha) (monocyte release), interleukin-8 (IL-8) (monocyte release), reactive oxygen species (ROS) measurement by chemiluminescence (neutrophil release), 8-isoprostanes (serum), total antioxidant levels measured by total equivalent antioxidant capacity (TEAC), glutathione, glutathione peroxidase activity, glutathione S-transferase activity, and platelet-derived growth factor (PDGF) (serum). TNF-alpha polymorphism (blood cellular DNA) was identified as a biomarker of susceptibility. Further studies are planned to test the validity and feasibility of these biomarkers to detect either high exposure to crystalline silica and early silicosis or susceptibility to silicosis in gold miners in South Africa.
Air Pollutants, Occupational↗