Anne P. Autor

Increase in manganese superoxide dismutase activity in the mouse heart after X-irradiation☆

Local X-irradiation of mouse heart caused a large increase in manganese superoxide dismutase activity (MnSOD) in this organ but not in copper and zinc containing superoxide dismutase (Cu-Zn SOD) activity. MnSOD induction was both dose and time dependent. Another mitochondrial enzyme, citrate synthase, was not induced by X-irradiation. The amount of immunoreactive MnSOD also increased after X-irradiation, showing that the amount of MnSOD protein increased after X-irradiation. The response to X-irradiation was found to be biphasic--with one large peak and one smaller peak of manganese superoxide dismutase activity. The effect of various inhibitors of cellular activities on these two peaks of MnSOD activity was examined. Cycloheximide, a cytosolic protein synthesis inhibitor, abolished both peaks of MnSOD activity, while chloramphenicol, a mitochondrial protein synthesis inhibitor, has no effect on either peak. Actinomycin D, a RNA-synthesis inhibitor, lowered both peaks, but had more of an effect on the second peak than on the first. In vivo protein synthesis studies using [3H]arginine showed that an increase in new protein synthesis occurred during the time period of the second peak, but did not occur during the first peak. These results are consistent with the hypothesis that MnSOD induction occurs in two peaks with the first peak due to a preformed MnSOD protein or mRNA for MnSOD and the second peak due to an increase in new protein synthesis.

Developmental Characteristics of Pulmonary Superoxide Dismutase: Relationship to Idiopathic Respiratory Distress Syndrome

Extract: Pulmonary superoxide dismutase (SOD) activity was determined for various groups of human fetuses, infants, and adults. Enzyme activity was found to increase with age from a low of 17 ± 1 units/nig DNA in fetal lung to 49 ± 6 units/nig DNA in infant lung and finally to 110.2 ± 14.8 units/mg DNA in adult lung (P < 0.05). No difference in lung SOD activity was demonstrated between normal infants and those with idiopathic respiratory distress/hyaline membrane disease (IRDS/HMD). No significant differences in SOD activity were found among all the samples of infant blood. Adult blood samples, however, contained significantly greater SOD activity both in terms of heme concentration and volume of whole blood (P < 0.05). SOD activity in lung tissue from both rats and rabbits was also found to increase with age from a low value in fetal animals to a maximum activity in adults (P < 0.05). Exposure of New Zealand White rabbits, prematurely delivered by caesarian section, to 80% oxygen for 24 hr resulted in a 42% increase in lung SOD activity. Similarly, 7-day-old Sprague-Dawley rats exposed to 85% oxygen for 24 hr showed a 43% increase in pulmonary SOD activity. No increase in pulmonary SOD was observed when adult rats were exposed to 85% oxygen for 24 hr. The effect of hyperoxia on SOD activity in excised lung was investigated. Rat lung, incubated in either heparinized whole blood or in plasma and exposed to 100% oxygen, showed a 30% increase in SOD activity after 2 hr. This capacity of lung tissue to respond to hyperoxia in vitro with increased SOD activity was age dependent. The maximum increase in SOD activity was seen with lungs from 10–12-day-old rats. The oxygen-stimulated increase in lung SOD activity disappeared at about 19–20 days of age.Speculation: Superoxide dismutase which catalyzes the dismutation of the oxygen free radical may well be a primary lung protectant against the depredations of environmental oxygen. SOD appears to be a maturationally important enzyme since the activity of this enzyme increases with development in both lung tissue and blood of animals and humans. The premature infant may be compromised when exposed to the relative hyperoxia of the extrauterine environment by a reduced complement of the enzyme or a reduced ability to increase pulmonary SOD activity in response to hyperoxia. Lung damage resulting from deficient endogenous protection may be a factor in the clinical picture of IRDS/HMD. Treatment of the immature lung with high concentrations of oxygen may further compromise a lung already deficient in SOD protection. Because prolonged expisure to hyperoxia precedes diagnosis of bronchopulmonary dysplasia, an SOD deficiency may also be important in the etiology of this condition.

Reduction of paraquat toxicity by superoxide dismutase

Abstract The effect of intravenously administered superoxide dismutase on paraquat-treated rats kept either in air or an atmosphere of 90%–95% oxygen was investigated. Of those rats maintained in the oxygen-enriched atmosphere, 50% died within 30 hours whereas, 50 hours elapsed before 50% mortality was observed for the superoxide dismutase-treated rats. Those animals allowed to remain in air were more responsive to superoxide dismutase treatment. Of those animals for which paraquat was fatal, untreated rats showed 50% cumulative mortality within 35 hours after paraquat administration, whereas those rats treated with superoxide dismutase showed 50% mortality after 80 hours. Sections of lung tissue examined at low magnification indicated that the extensive alveolar and vascular damage caused by paraquat was ameliorated with the administration of superoxide dismutase. These findings may have particular relevance in the treatment of paraquat intoxication in humans.

Adaptation to hyperoxia in the neonatal rat: Kinetic parameters of the oxygen-mediated induction of lung superoxide dismutases, catalase and glutathione peroxidase

The activities of the enzymes superoxide dismutase, catalase and glutathione peroxidase increase in the lungs of neonatal rats exposed to normobaric hyperoxia. The oxygen-mediated increase in activity of these enzymes, known from previous studies to be an inductive response, was studied in 10- and 25-day-old rats as a function of both oxygen concentration and length of time of exposure to greater than 95% oxygen. In the lungs of 10-day-old rats the increase in superoxide dismutase, catalase, and glutathione peroxidase occurs only at 80% ambient oxygen or greater. In 25-day-old rats a similar pattern of response occurs with pulmonary catalase and glutathione peroxidase. However, unlike the response in 10-day-old rats, pulmonary superoxide dismutase does not increase in oxygen-exposed 25-day-old rats. The time course of enzyme induction was different for 10-day-old rats compared with 25-day-old rats. Exposure of 10-day-old rats to 95+% oxygen resulted in a significant increase in activity of superoxide dismutase after only 4 h when compared with air-exposed control animals. Catalasee and glutathione peroxidase in the same age group increased significantly after 6 h and 12 h of exposure to oxygen, respectively. Maximal levels of superoxide dismutase, catalase and glutathione peroxidase were reached after 6, 12 and 24 h of exposure to hyperoxia, respectively. This level of activity was then maintained throughout the subsequent exposure time up to 96 h. The activity of pulmonary catalase and glutathione peroxidase in 25-day-old rats did not increase significantly after 6 h of exposure to hyperoxia. An apparent plateau of increased activity was reached after 24 h of exposure. As observed with the 3 enzymes in 10-day-old rats, maximal enzyme activities were maintained throughout the subsequent period of oxygen exposure up to 96 h.

The effect of dietary fatty acids on the composition of adult rat lung lipids: relationship to oxygen toxicity.

Abstract Three groups of mature rats were placed on separate dietary regimens for a period of 33 days. One group was fed standard rat food, the second group was fed a diet high in saturated fatty acids provided by hydrogenated coconut oil, and the third group was fed a diet high in polyunsaturated fatty acids provided by cod liver oil. After 33 days of maintenance on these diets major changes in the fatty acid composition of lung triglycerides were observed. Fatty acids with two or more double bonds comprised 27.9% of the total lung triglyceride fatty acids in rats fed standard rat food, 24.7% in the rats fed cod liver oil, and 2.8% in the rats fed coconut oil. The fatty acid composition of lung phospholipids showed smaller changes which were confined to shifts in the relative content of arachidonic and docosahexaenoic acids in those rats fed cod liver oil. Exposure of all three groups of rats to 100% oxygen resulted in a greatly enhanced mortality in the group fed coconut oil compared with the other two groups. All of the rats fed coconut oil were dead within 68 hr, while, after 96 hr, only 54% of those fed either standard rat food or cod liver oil were dead. These data suggest that an increase in the saturated fatty acid content of lung triglycerides through dietery manipulation results in increased susceptibility to oxygen toxicity.

Oxygen therapy and hyaline membrane disease: the effect of hyperoxia on pulmonary superoxide dismutase activity and the mediating role of plasma or serum.

In vitro and in vivo hyperoxic exposure of the lungs of normal immature animals resulted in a rapid increase of pulmonary superoxide dismutase activity. The increase of pulmonary SOD activity with in vitro hyperoxic exposure requires the presence of plasma or serum in the incubation medium. Twenty-three out of 26 plasma samples from premature infants without hyaline membrane disease were found to support the hyperoxic increaase of pulmonary SOD activity, whereas only five of 15 plasma samples from infants with HMD were effective. A defective plasma-lung interaction in infants with HMD may result in an inability to increase pulmonary levels of this presumed protective enzyme during hyperoxic therapy.

Changes in the fatty acid composition of rat lung lipids during development and following age-dependent lipid peroxidation

Analyses of the fatty acid content and composition of various lung lipids were conducted in rats 1 day, 5 days, and 12 days after birth and in adult animals in order to define more clearly the specific lipid peroxidizing system found in neonatal rat lungs. Lipid peroxidation occurs in the 900×g supernatant fraction of rat lung homogenates in an age-dependent manner independent of the addition of any factor and is maximal at 5 days of age. No lipid peroxidation is evident in similar preparations of either newborn or adult lung tissue. As the animals develop, arachidonic and docosahexaenoic acids, fatty acids which are both highly susceptible to lipid peroxidation in the presence of a suitable catalyst, decrease gradually when measured as the percentage of the total fatty acids in the triglyceride fraction of the lung. The total quantity of triglycerides, however, is significantly lower in lungs from 1-day-old rats than at any other age. The fatty acid composition and total quantity of both lung phospholipids and lung free fatty acids do not show similar changes. Following in vitro incubation of the 900×g supernatant fraction of peroxidizing lung homogenates, an appreciable decrease in the amount of arachidonic and docosa-hexaenoic acid could be detected in lung triglycerides. Less extensive decreases were observed in the phospholipid fraction. No changes in these components were observed in newborn or adult animals. The addition of triarachidonin to the 900×g supernatant fraction of lung homogenates resulted in increased malondialdehyde release at all ages tested while added arachidonic acid increased the formation of malondialdehyde only in 5- and 12-day-old rat lung preparations. The addition of triolein, cholesterol arachidonate, and diarachidonyl phosphatidylcholine had no effect on malondialdehyde formation at any age. The age-dependent lipid peroxidation observed after in vitro incubation of rat lung homogenate preparations, therefore, may result from the relatively high concentration of triglycerides containing polyunsaturated fatty acids present in the neonatal tissue. As the susceptible polyunsaturated fatty acids of lung triglycerides are replaced by less unsaturated species, this activity may diminish concomitantly.

Age-dependent lipid peroxidation in neonatal rat lung tissue.

Abstract A unique, age-specific pulmonary lipid peroxidation has been found to occur after incubation of neonatal rat lung homogenates in the absence of any added factors. As measured by the formation of malondialdehyde, lipid peroxidation was not detectable in rat lung homogenates prepared from animals immediately after birth but appeared by the second day and reached a maximum at 5 days of age. The effect gradually disappeared by 20 to 21 days after birth. The addition of NADPH did not enhance lipid peroxidation in the sensitive age group nor did it initiate lipid peroxidation when added to lung homogenates from either 1-day-old or adult rats. The activities and concentrations of various endogenous antioxidants were measured in neonatal lung tissue. When measured in lung tissue obtained from rats during the sensitive age period, no concomitant deficiencies of glutathione peroxidase, glutathione reductase, glucose 6-phosphate dehydrogenase, reduced glutathione, or a-tocopherol were observed. With the exception of α-tocopherol, none of these factors inhibited malondialdehyde formation when added to homogenized lung tissue prepared from 5-day-old rats. α-Tocopherol did inhibit malondialdehyde formation in 5-day-old rat lung homogenates but at a concentration much greater than the endogenous concentration found in adult rat lungs. The 21-day neonatal age period during which malondialdehyde is produced following incubation of lung tissue is similar to the 3-week period immediately after birth reported to be the time of maximum proliferation of rat lung fibroblasts, type 1 pneumocytes and type II pneumocytes.

Effect of cyclooxygenase inhibitors and protease inhibitors on phorbol-induced stimulation of oxygen consumption and superoxide production by rat pulmonary macrophages☆

Oxygen consumption and superoxide anion production by pulmonary macrophages are both increased by phorbol myristate acetate (PMA) but the two processes have been separated using protease inhibitors and cyclooxygenase inhibitors. Pretreatment with the protease inhibitors (L-1-tosylamido-2-phenylethylchloromethyl ketone (TPCK) and N-alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK), as well as with the cyclooxygenase inhibitors acetylsalicylic acid (ASA) and ibuprofen (IBU), inhibited the stimulation of superoxide production and oxygen consumption by phorbol myristate acetate. However, whereas the order of potency for inhibition of stimulation of superoxide production was TPCK greater than TLCK greater than IBU greater than ASA, the order of potency for inhibition of stimulation of oxygen consumption was ASA greater than IBU greater than TPCK = TLCK. Although all four agents were effective inhibitors of PMA-stimulated superoxide production and oxygen consumption when added before PMA, in contrast to the cyclooxygenase inhibitors. TPCK was unable to inhibit oxygen consumption by more than 70-80% regardless of the concentration used, although superoxide generation could be inhibited completely. When added after PMA, ASA did not suppress either oxygen consumption or superoxide production and ibuprofen was only one-half as effective as an inhibitor. TPCK and TLCK, when added after PMA, accelerated the return to basal rates of both oxygen consumption and superoxide production. None of the four agents had any effect on basal superoxide production or oxygen consumption at the concentrations used. The data support the interpretation that both prostaglandin biosynthesis and protease activity may be associated with the activation of the superoxide-generating system of pulmonary macrophages. The consumption of molecular oxygen following stimulation of the cells with phorbol myristate acetate is not due solely to the generation of superoxide, however, since each process is inhibited with different potency by the same group of inhibitors. There appears to be a component of oxygen consumption which results from the activation of cyclooxygenase and, unlike superoxide production, cannot be completely inhibited by treatment with protease inhibitors.

Production of superoxide anion by an nadph-oxidase from rat pulmonary macrophages

Superoxide anion (0;) is produced by polymorphonuclear leukocytes coincident to the respiratory burst which occurs in response to certain soluble and particulate stimuli [ 1,2]. This univalently reduced form of oxygen plays a role directly or indirectly in the microbicidal function of leukocytes ]3,4]. The production of 0; by pulmonary macrophages following activation of the cells with soluble or particulate stimuli has also been demonstrated [5]. A membrane-bound flavoprotein dormant in resting leukocytes but activated by specific stimuli catalyzes the reduction of molecular oxygen with NADPH as the source of electrons. Enzymes of this type have been demonstrated to reside in the subcetlular granules and plasma membrane of activated leukocytes [6-91. Cytochrome b involvement in this process has also been suggested [ 101. A superoxide-generating enzyme has also been reported to be present in the endoplasmic reticulum and mitochond~al membrane, but not the plasma membrane, of resting, non-activated pulmonary macrophages [ 111. Here, we describe a pyridine nucleotide-dependent superoxide generating activity with a high affinity for NADPH which may be solubilized from a mixed membrane preparation from resting rat pulmonary macrophages and ma~rophages stimulated with opsonized, killed yeast particles.