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Biomedical subjects

B A Freeman

Publications and source records attributed to B A Freeman.

At least 127 records · Page 7Linked to original sources
PubMed1986

Augmentation of antioxidant enzymes in vascular endothelium.

The endothelium is a key site of injury from reactive oxygen species that can potentially be protected by the antioxidant enzymes superoxide dismutase and catalase. Large proteins, such as superoxide dismutase and catalase, do not readily penetrate cell membranes, which limits their efficacy in protecting cells from cellular reactions involving both intracellularly and extracellularly generated reactive oxygen species. Two methods are described that promote enzyme delivery to cultured endothelial cells and confer increased resistance to oxidative stress. The first method is to entrap the antioxidant enzymes within liposomes, which then become incorporated by endothelial cells and can increase enzyme specific activities by as much as 44-fold within 2 h. The second method involves covalent conjugation of polyethylene glycol (PEG) to superoxide dismutase and catalase, a technique that increases circulatory half-life and reduces protein immunogenicity. Conjugation of PEG to superoxide dismutase and catalase increased cellular-specific activities of these enzymes in cultured endothelial cells (but at a slower rate than for liposome entrapped enzymes) and rendered these cells more resistant to oxidative stress. Both liposome-mediated delivery and PEG conjugation offer an additional benefit over native superoxide dismutase and catalase because they can increase cellular antioxidant activities in a manner that can provide protection from both intracellular and extracellular superoxide and hydrogen peroxide.

Animals↗
PubMed1986

Antioxidant enzyme activity in alveolar type II cells after exposure of rats to hyperoxia.

The activity of antioxidant enzymes were measured in alveolar type II cells isolated from control and 85% oxygen-exposed rats to determine if type II cells, an oxygen-resistant lung cell type had constitutively high enzyme activities and to measure the effect of hyperoxia on these antioxidant enzyme. Type II cells were isolated from lungs of control rats and rats exposed to 85% O2 for 7 days. In whole lungs of rats exposed to 85% oxygen there is an increase in activity (per lung or per mg lung DNA) in the antioxidant enzymes CuZn superoxide dismutase, Mn superoxide dismutase, catalase, glutathione peroxidase and glucose-6-phosphate dehydrogenase. Oxygen exposure significantly increased (p less than 0.05) all type II cell antioxidant enzyme activities when expressed per mg DNA. The protein content of oxygen exposed type II cells increased 25% from (63.9 +/- 4.8 micrograms/10(6) cells to 79.6 +/- 4.2 micrograms/10(6) cells, p less than 0.05). When type II cell enzyme activities were expressed in U/mg cell protein, only CuZn superoxide dismutase and Mn superoxide dismutase increased in activity following oxygen exposure (by 43% and 28% relative to air exposed lung type II cells, respectively, p less than 0.05). This suggested that most lung cell antioxidant enzymes increased in activity following oxidant stress in proportion to increased cell mass. CuZn and Mn superoxide dismutase increased activity to an extent greater than the increase in type II cell protein content after oxygen exposure. Alveolar macrophages lavaged from control and oxygen-exposed rats were also evaluated, and they had no significant change in CuZn and Mn superoxide dismutase activities. Type II cells accounted for 10% and 17% of alveolar cells in control and oxygen treated rats. By knowing the antioxidant enzyme activities in type II cells, the total enzyme activity of whole lung and the number of type II cells in control and oxygen exposed rats from morphometric data, we calculated the percent of whole lung enzyme activity accounted for by type II cells. Type II cells accounted for a high percentage of lung glucose-6-phosphate dehydrogenase (58% in control rats, 65% in oxygen exposed rats) but a low percentage of Mn superoxide dismutase (4% in control rats, 6% in oxygen exposed rats).

Animals↗
PubMed1986

Intracellular localization of the copper-zinc and manganese superoxide dismutases in rat liver parenchymal cells.

Both the copper-zinc (CuZn) and the manganese (Mn) containing superoxide dismutases (SOD) have been immunolocalized in rat liver sections using protein A-gold labeling on ultrathin cryosections. The CuZnSOD was found to distribute uniformly throughout the nuclear and cytoplasmic matrix. The CuZn enzyme was excluded from membrane-bound compartments such as the nuclear envelope, endoplasmic reticulum, Golgi elements, secretory vesicles, and mitochondria. The primary exception was that lysosomes frequently labeled heavily for CuZnSOD. The lysosomal nature of these membrane-bound bodies was confirmed by double immunolabeling with a lysosomal enzyme, cathepsin-D. The MnSOD was located in mitochondria, particularly the matrix between the cristae. Light but distinct labeling for the MnSOD occurred in the cytoplasmic matrix.

Animals↗
PubMed1985

Free radical damage to cultured porcine aortic endothelial cells and lung fibroblasts: modulation by culture conditions.

Culture conditions modulating cell damage from xanthine plus xanthine oxidase-derived partially reduced oxygen species were studied. Porcine thoracic aorta endothelial cells and porcine lung fibroblasts were maintained in monolayer culture. Cells were prelabeled with 51Cr before xanthine plus xanthine oxidase exposure. Endothelial cells showed 30 to 100% more lysis than fibroblasts and thus seemed more sensitive to this oxidant stress. The effect of cell culture age, as indicated by population doubling level (PDL), was examined. Response of low PDL endothelial cells and fibroblasts subjected to oxidant stress was compared with the response of PDL 15 cells. Both low PDL endothelial cells and fibroblasts responded differently to the lytic effect of xanthine oxidase-derived free radicals than did higher PDL cells. Specific activities of the antioxidant enzymes catalase, manganese superoxide dismutase, copper-zinc superoxide dismutase, glutathione peroxidase, and glucose-6-phosphate dehydrogenase were measured in both low and high PDL fibroblasts and endothelial cells. Antioxidant enzyme specific activities could only partially explain the differences in response to oxidant stress between fibroblasts and endothelial cells and between low and high PDL cells. Cell culture medium composition modulated the rate of production, and relative proportions of xanthine plus xanthine oxidase-derived partially reduced species of oxygen, i.e. superoxide, hydrogen peroxide, and hydroxyl radical. Serum content of medium was important in modulating free radical generation; superoxide production rates decreased 32%, H2O2 became undetectable, and hydroxyl radical generation decreased 54% in the presence of 10% serum. The medium protein and iron content also modulated free radical generation. The data suggest that cell culture media constituents, cell type, and cell culture age greatly affect in vitro response of cells subjected to oxidant stress.

Animals↗
PubMed1985

Effect of superoxide dismutase encapsulated in liposomes or conjugated with polyethylene glycol on neutrophil bactericidal activity in vitro and bacterial clearance in vivo.

Encapsulation in liposomes (LIP) or conjugation with polyethylene glycol (PEG) are methods being used to increase circulating half-life periods and/or improve delivery of antioxidant enzymes that could decrease lung injury induced by O2 metabolites. We found that pretreatment with LIP, with or without the antioxidant enzyme, superoxide dismutase (SOD), decreased killing of Staphylococcus aureus 502A by neutrophils in vitro and clearance of intravenously injected S. aureus in rabbits in vivo. In contrast, pretreatment with PEG (with or without SOD) had no effect on neutrophil bactericidal activity in vitro or bacterial clearance in vivo. Our results suggest that conjugation with PEG may be a better way than encapsulation in liposomes for delivering antioxidant enzymes, especially if one is concerned about preserving host-defense mechanisms.

Animals↗
PubMed1985

Modulation of oxidant lung injury by using liposome-entrapped superoxide dismutase and catalase.

Increased cellular generation of partially reduced species of oxygen mediates the toxicity of hyperoxia to cultured endothelial cells and rats exposed to 95-100% oxygen. Liposomal entrapment and intracellular delivery of superoxide dismutase (SOD) to cultured porcine aortic endothelial cells increased the specific activity of cellular SOD up to 15-fold. The liposome-mediated augmentation of SOD activity persisted in cell monolayers and rendered these cells resistant to oxygen-induced injury in a cell SOD activity-dependent manner. Addition of free SOD to culture medium had no effect on cell SOD activity or resistance to oxygen toxicity. SOD and catalase-containing liposomes injected i.v. into rats increased lung-associated enzyme specific activities two- to fourfold. Liposome entrapment of both SOD and catalase significantly increased the circulating half-lives of these enzymes and was critical for prevention of in vivo oxygen toxicity. Free SOD and catalase injected i.v. in the absence or presence of control liposomes did not increase corresponding lung enzyme activities or survival time in 100% oxygen. These studies show that O2- and H2O2 are important mediators of oxygen toxicity and that intracellular delivery of oxygen protective enzymes can reduce tissue injury owing to overproduction of partially reduced oxygen species.

Animals↗
PubMed1984

Hyperoxia enhances lung and liver nuclear superoxide generation.

Porcine lung and liver nuclei generated superoxide (O-2) at a rate which increased with increasing oxygen concentration. NADH-dependent O-2 generation increased from 0 to 2.21 +/- 0.11 nmol/min per mg protein for lung nuclei and from 0.16 +/- 0.09 to 1.34 +/- 0.14 nmol/min per mg protein for liver nuclei, when oxygen concentration increased from 0 to 100%. NADPH-dependent O-2 generation increased similarly in liver nuclei (from 0.20 +/- 0.09 to 1.20 +/- 0.12 nmol/min per mg protein), while lung nuclei produced only 0.45 +/- 0.09 nmol/min per mg protein at 100% oxygen. NADH and NADPH had an additive effect on O-2 generation by liver nuclei, yielding 2.58 +/- 0.21 nmol/min per mg protein at 100% oxygen. Very little or no superoxide dismutase activity was present in washed nuclear preparations. The oxygen-dependence of nuclear O-2 generation shows that nuclear-derived partially reduced species of oxygen may affect nuclear function during hyperoxia or other metabolic situations where overproduction of oxygen radicals is problematic.

Aerobiosis↗
PubMed1984

Detection of superoxide generated by endothelial cells.

Superoxide and lipid free-radical generation in cultured endothelial cells treated with menadione or nitrazepam were measured using electron paramagnetic resonance spectroscopy. Superoxide was detected both intracellularly and extracellularly. Extracellular generation of superoxide and hydrogen peroxide was also measured, either by spectrophotometric measurement of succinoylated cytochrome c reduction or by polarography. Extracellular superoxide was generated due to reduced menadione diffusing across the plasma membrane and reacting with oxygen to generate superoxide in the medium. Increased intracellular oxygen tension favored intracellular oxidation of reduced menadione, thus decreasing diffusion of reduced menadione from the cells and, hence, decreasing extracellular superoxide production. The nitro anion free radical of reduced nitrazepam, which cannot cross the plasma membrane, did not generate detectable extracellular superoxide. Our results show that intracellular superoxide can be spin-trapped using 5,5-dimethyl-1-pyrroline-1-oxide and that secondary free-radical injury to membrane lipids, due to excess production of partially reduced species of oxygen by intact cells, can be detected by spin-trapping lipid free radicals with phenyl N-tert-butylnitrone.

Animals↗
PubMed1984

Liposome-mediated augmentation of brain SOD and catalase inhibits CNS O2 toxicity.

Enzymes specific for O-2 and H2O2 metabolism [superoxide dismutase (SOD) and catalase] can be delivered to the rat brain following entrapment in liposomes and intravenous injection and will protect against hyperbaric O2-induced convulsions in rats. Liposome-mediated superoxide dismutase and catalase augmentation of brain enzyme activity was 2.7-fold and 1.9-fold, respectively, 15 min after intravenous injection of superoxide dismutase plus catalase-entrapped liposomes. Rats treated with liposomes containing superoxide dismutase plus catalase 2 h before 6 ATA 100% O2 exposure had the time to convulsion extended three times that of controls. This protective effect was dose-dependent and was primarily due to augmentation of catalase activity. These findings show O-2 and H2O2 are important mediators of hyperbaric O2-induced central nervous system toxicity and that liposome-mediated augmentation of brain antioxidant enzymes has a biological effect.

Animals↗
PubMed1984

Protection against oxygen toxicity by intravenous injection of liposome-entrapped catalase and superoxide dismutase.

Survival of rats exposed to 100% oxygen was increased from 69.5 +/- 1.5 to 118.1 +/- 9.9 h (mean +/- SEM, P less than 0.05) when liposomes containing catalase and superoxide dismutase were injected intravenously before and during exposure. The increased survival time in 100% oxygen was also associated with significantly less fluid in the pleural cavity. Rats injected with catalase- and superoxide dismutase-containing liposomes, which had increased survival in 100% oxygen, had increased lung wet weight upon autopsy compared with saline-injected controls (2.9 +/- 0.2 g/lung vs. 4.8 +/- 0.4 g/lung, mean +/- SE, P less than 0.05). Intravenous injection of control liposomes along with catalase and superoxide dismutase in the suspending buffer decreased the mean pleural effusion volume 89% and had no significant effect on survival time. Lung catalase and superoxide dismutase activities were increased 3.1- and 1.7-fold, respectively, 2 h after a single intravenous injection of liposomes containing catalase or superoxide dismutase. Superoxide dismutase activity was also significantly greater than controls in both air- and 100% oxygen-exposed rat lungs, when enzyme activity was assayed 24 h after cessation of injection of control and oxygen-exposed rats with enzyme-containing liposomes every 12 h for 36 h. Free superoxide dismutase and catalase injected intravenously in the absence of liposomes did not increase corresponding lung enzyme activities, affect pleural effusion volume, lung wet weight, or extend the mean survival time of rats exposed to 100% oxygen. The clearance of liposome-augmented 125I-labeled catalase from lung and plasma obeyed first order kinetics according to a one-compartment model. When clearance of liposome-augmented catalase activity or radioactivity were the parameters used for pharmacokinetic studies, the half-life of augmented lung catalase was 1.9 and 2.6 h, respectively. The half-life of liposome-entrapped catalase and superoxide dismutase activity in the circulation was 2.5 and 4 h, respectively, while intravenously injected catalase and superoxide dismutase had a circulation half-life of 23 and 6 min, respectively.

Animals↗
PubMed1984

Pulmonary antioxidant enzyme maturation in the fetal and neonatal rat. I. Developmental profiles.

Neonatal, adult, and fetal rat lungs of 18, 20, and 22 d gestation from four to six litters were examined for cytochrome oxidase, glucose-6-phosphate dehydrogenase, catalase, glutathione peroxidase, copper-zinc and manganese superoxide dismutase activities. All results were corrected for the contribution of enzymes in blood that contaminate homogenates. Because lung protein/DNA ratios and body water change significantly with gestational age, enzyme activities were expressed as U/mg DNA. All activities were low in d 18 lung and increased with advancing gestational age. Only catalase and copper-zinc superoxide dismutase increased activity in response to air breathing, suggesting that maturation of the antioxidant enzyme system is virtually complete before delivery. Activities of glucose-6-phosphate dehydrogenase, catalase, glutathione peroxidase, and manganese superoxide dismutase were higher in neonatal than in adult lung.

Animals↗
PubMed1984

Pulmonary antioxidant enzyme maturation in the fetal and neonatal rat. II. The influence of maternal iron supplements upon fetal lung catalase activity.

It was observed that the lung catalase activity of premature (day 21 of gestation; term = 22 days) rat pups is affected by maternal iron intake. Pups from control dams receiving Purina Lab Chow and water ad libitum have only 50% of the lung catalase activity of pups from dams who received 1 mg/kg parenteral iron dextran daily from day 7 to day 20 of gestation. Other oxygen-protective enzymes, copper-zinc and manganese superoxide dismutase, glutathione peroxidase, and glucose-6-phosphate dehydrogenase, were unaffected by maternal iron supplements.

Animals↗
PubMed1984

Tissue slices in the study of lung metabolism and toxicology.

Lung tissue slices are model systems for the study of pulmonary metabolism. Because of the speed and simplicity of slice preparation, lung slices have been used in studies of oxygen, amino acid, carbohydrate and lipid utilization and adenine nucleotide metabolism. Dose-response characteristics for toxicants are readily described because multiple lung samples can be studied from the same animal or a population of animals. Lung slices prepared from animals exposed to oxidant air pollutants exhibit alterations in respiration, glucose consumption and lipid metabolism. These studies have indicated both direct toxic effects of air pollutants on enzyme systems and also air pollutant-induced changes in the cellularity of lungs.

Air Pollutants↗
PubMed1984

Differentiation-arrested rat fetal lung in primary monolayer cell culture. III. Antioxidant enzyme activity.

Differentiation-arrested monolayer lung cell cultures were developed from day 18, 20, and 22 rat fetuses and 3-day-old neonatal rats. These cultures were examined for antioxidant enzyme activity, and the values obtained were compared with previously reported in vivo activity. All cultures were catalase deficient, and activity could be restored by the addition of 0.25 microM Fe(NO3)3 X 9H2O to the culture medium. The other measured antioxidant enzymes--copper-zinc and manganese superoxide dismutase, glutathione peroxidase, and glucose 6-phosphate dehydrogenase-demonstrate gestation-dependent increases of activity in vivo that were not evident in vitro, supporting the concept of a circulating "maturation factor" during fetal life. When cultures from fetal days 20 and 22 and from neonatal day 3 lungs were challenged with 50% oxygen in the presence of serum, antioxidant enzyme activities were unchanged, and there was no evidence of cell damage as assessed by release of lactate dehydrogenase. In the absence of serum, however, fetal day 20 (but not fetal day 22 or neonatal day 3) lung cells showed evidence of cell damage and increased antioxidant enzyme activities. It is concluded that cultured immature fetal cells are more susceptible to oxygen toxicity than those derived from mature fetal or neonatal animals. This increased susceptibility cannot be explained on the basis of the reduced antioxidant enzyme activity observed in vivo.

Animals↗
PubMed1984

Scanning electron microscopy of vascular casts in experimental ocular vasoproliferation.

Scanning electron microscopy of vascular casts was used to investigate three experimental models of neovascularization. In each experimental situation, the casts provided a valuable three dimensional representation of the newly formed blood vessels and permitted subclassification of the vessels within normal and proliferating vascular networks. They defined also the predominant origin of new vessels from venules and capillaries, and enabled the evolution of proliferating vessels into arterioles and venules to be documented. Although vascular casts must be interpreted with caution in light of the possibility of incomplete filling and other artifacts, they are a valuable tool in the study of ocular vasoproliferation.

Animals↗
PubMed1983

Liposome-mediated augmentation of superoxide dismutase in endothelial cells prevents oxygen injury.

Increased intracellular production of oxygen radicals is a major etiology of cell damage from many quinoid antibiotics, environmental toxicants, and hyperoxia. Enhancing the intracellular content of protective enzymes can provide a means of limiting biological damage caused by free radicals. Liposomal entrapment and intracellular delivery of superoxide dismutase to cultured porcine aortic endothelial cells increased the specific activity of cellular superoxide dismutase 6 to 12-fold. This augmented superoxide dismutase activity persisted in cultured endothelial cell monolayers and rendered these cells resistant to oxygen-induced injury. Culture of confluent endothelial cells in hyperoxia increased 51Cr and lactate dehydrogenase release in an oxygen concentration-dependent manner. Superoxide dismutase-augmented endothelial cells were resistant to oxygen damage compared to untreated controls, in a superoxide dismutase concentration-dependent manner. Free superoxide dismutase in the absence or presence of liposomes containing no enzyme had no effect on cellular enzyme activity and did not protect from oxygen damage. Liposomes made from saturated fatty acid-containing phospholipids had a small but significant protective effect on oxygen-induced cell damage. These liposomes probably increased endothelial cell membrane saturated lipid content and thereby decreased peroxidative damage when the cells were exposed to hyperoxia. Conversely, preincubation of cells with arachidonic acid increased cell arachidonic acid content, sensitivity to hyperoxia, and hyperoxia-induced production of thiobarbituric acid material. These data suggest that intracellular delivery of superoxide dismutase prevents oxygen-induced cell damage and that superoxide is an important mediator of cellular oxygen toxicity.

Animals↗