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Oxygen Toxicity and Gas Mixtures: Morphology
honeycombing and interstitial scarring (Fig 3, 4). The severity was graded by a method previously applied to whole sections of human lungs.' Within three to ten days the majority of the adapted rats had a grade 10 to 20 emphysema, with a predominantly panlobular distribution. The lungs from the rats which survived 20-30 days had large bullous blebs, more marked at the bases of the lungs. In these longterm survivors the emphysema ranged from grade 30 to 60. Regions with thinned alveolar walls alternated with zones of cellular proliferation and interstitial collagen deposition. Large scars and recent, partly organized, thromboemboli were also present. The mild-to-moderate number of interstitial and intra-alveolar inflammatory cells included macrophages, lymphocytes and neutrophils. The intra-alveolar exudate and congestion in the adapted rats appeared less marked during the exposure to 100 percent 0, than in the acute phase controls, while the perivascular exudate was still very severe. A more exact comparison was not feasible, since the intratracheal perfusion fixation washed out
Dr. Pratt: I am bothered by the localized scarring we
saw in your rats; in humans the scars of chronic oxygen toxicity are more diffuse. Can you explain the difference? Remembering that your experimental animals were old discarded breeders, I am wondering if the scars may not have been there before the exposure to oxygen began. They looked old to me.
Dr. Paegle: I think the process is diffuse. The air spaces are enlarged and dilated throughout the entire lung. In the rat lung, however, you don't see the large blebs as
some of the intra-alveolar proteinaceous exudate and compressed the congested vessels. At least a portion of the large scars originated from confluence and organization of the large regions of perivascular exudate. After the initial, relatively acellular phase during the first two days of 100 percent O,, the numerical increase of fibroblasts in these regions was quite marked. In view of the quick induction of marked scarring and honeycombing, which would seem difficult to reverse, and because the general features of lung injury due to acute oxygen toxicity had seemed similar in man and rat,2 the clinical use of high 0, concentrations for long periods may have to be more carefully scrutinized in regard to production of similar chronic damage in man.
1 Spain DM, Siege1 H. Bradess VA: Emphysema in appar-
ently healthy adults. JAMA 224:322-325, 1973
2 Senior RM, Wessler S, Avioli LV: Pulmonary oxygen
toxicity. JAMA 217: 1373-1377
prominently as in some sections of the human lungs. The h e r scarring in the rat lungs is not well seen on the lower power sections I have shown. You could see this scarring with higher power. Regarding the large scars, one can hypothesize that while in some regions there is some regression and reabsorption of the perivascular exudate as the vessels recover a little bit, in other places for some reason or another, absorption hasn't taken place as fast and then organization progresses and leads to a large scar. Perhaps this process of organization is also helped along in areas where thromboemboli have occurred.
Oxygen Toxicity and Gas Mixtures: ~ o r ~ h o l o ~ ~ ' Philip C . Pratt, M . D., h n m P. Sanders, Ph.D., and W&m
D. Currie, Ph.D.
hi. project involves exposure of rats to progressive Tincrements of oxygen concentration in order to inves-
tigate the level at which oxygen becomes toxic to pulmonary cells and to examine associated morphologic and biochemical changes. Exposure concentrations ranged through 40, 70,90 and 100 percent. There was a slight consistent lag in weight gain among animals exposed to 40 and 70 percent. These animals began to lose weight during the Second day of exposure to percent oxygen. Increased lung weight and volume were demonstrable after 70, 90, and 100 percent whether singly or in sequence. Greater increases occurred in animals exposed only to 100 percent oxygen for seven days than in animals exposed over a period of 16 days to progressive increments of concentration including seven days of 100 percent oxygen. This excess weight is attributed primarily to alveolar and interstitial edema. The sequentially exposed animals showed less intense edema. These animals also showed alveolar septa1 thickening, with prominent alveolar epithelium and increased interstitial cells in alveolar walls and perivascular stroma. *From the Deparhnents of Pathology and Radiobiology, Duke Medical Center and the Durham Veterans Administration Hospital, Durham, N.C. Supported in part by Contract NIH-71-2145.
8S 16TH ASPEN LUNG CONFERENCE
The lesions in sequentially exposed rats are somewhat more comparable to hdings in lungs of patients who die after treatment with oxygen than those in animals exposed to 100 percent oxygen only. However, alveolar septa are not as thick, fibrosis is not as prominent and hyaline membranes are absent although usually seen in human lungs. The significance of these differences remains to be demonstrated. Lung tissue from animals exposed simultaneously with these has been examined for metabolic activity. Preliminary results indicate that there is depression of respiration with succinate and a-ketoglutarate as substrates in homogenized lung tissue of animals killed after as few as three days of exposure to 40 percent oxygen. For example, basal oxygen consumption with succinate, dropped from 3.5 to 3.0 p-liters of 02/mg of mitochondrial protein/min and a-ketoglutarate, from 1.75 to 1.25. The ADP stimulated rate was reduced to a somewhat greater degree indicating a loss of respiratory reserve capacity. Despite this reduction, the measured ATP level after three days of exposure to 40 percent oxygen was increased (from 0.3 to 0.35 pnol/mg of mitochondrial protein) A drop in ATP first appeared after the 3 days in 90 percent oxygen.
CHEST 66: 1, JULY, 1974 SUPPLEMENT
DISCUSSION Dr. Thurlbeck: A question of information. In your summary you said that 40 percent oxygen produced lesions, but when You showed Your $ides You said the 40 percent 0,lung looked normal. Dr. Pratt: After three days of exposure, the lungs looked normal, but by six days, early lesions were visible. This
was shown in the last slide of my series. Dr. Lauweryns: In your control animals as compared to your oxygen treated animals, was there a werence in the appearance of the lymphatics? Dr. P m : Lymphatic vessels were dilated and could be seen in the perivascular adventitia. However, most of the increase in width of this area was due to, interstitial accumulation of edema fluid.
Common Oxidant Lesion of Mitochondria1 Redox State Produced by Nitrogen Dioxide, Ozone, and High Oxygen In Alveolar ~ a c r o ~ h a ~ e s * JeffreyR. Simons, M.D.,Oe JamesTheodore, M.D., and Eugene D. Robin, M.D.
P
hysiologic and biochemical effects of inhaled oxidants on the lung are currently areas of major interest and concern. Two of the oxidizing agents present in the urban environment are ozone and the oxides of nitrogen, in particular, nitrogen dioxide. It has been conclusively shown that exposure to relatively high concentrations of NO, for a short period produces bronchiolitis obliteransl and severe, often fatal lung disease. Similar alterations were reported for ozone by other investigator^.^ Oxygen, clearly an oxidant agent in high concentrations, alters pulmonary structure and function after a relatively short period in various mammalian specie^.^ It would, therefore, seem appropriate to study the effects of oxidizing agents on isolated pulmonary cells to better understand mechanisms by which these agents produce derangements of pulmonary structure and function. One pulmonary cell available in relatively pure form is the alveolar macrophage, which by its location in the air-exchanging portion of the acinus is subjected to intimate contact with inhaled gases. Previous studies in our' laboratory showed that rabbit alveolar macrophages in vitro exhibited biochemical changes under hypoxic conditions. The redox (oxidationreduction) state of the cells was decreased in both the cytoplasmic and mitochondria1 compartments after exposure to hypoxia. The present studies were undertaken to observe the effects of 80 percent oxygen and low concentrations of nitrogen dioxide and ozone on this fundamental physicochemical variable. Oxidation-reduction reactions are vital to many fundamental biochemical processes, with important biologic and clinical implications. The functions that are sibserved by these reactions include the provision of energy, oxidative biosynthesis, biodegradation reactions and detoxification. The reactions usually involve certain common electron camers rather than involving electron flow directing from one substrate to another. Among the most important of these electron camers are the nicotinamide adenine dinucleotide pair (NAD+/NADH). Direct measurements of the biologically active NAD+ and its reduced form NADH are not possible due to their 'From the De artment of Medicine, Stanford University School of ~ d c i n e Stanford, , Ca. O°Currently Assistant Professor of Medicine, University of Utah and Staff Physician, Salt Lake City Veterans Adminktration Hospital.
CHEST 66: 1, JULY, 1974 SUPPLEMENT
localization within specific cellular compartments and the binding of NAD+ and especially NADH to proteins. This ratio, however, can be determined by using suitable substrate pairs involved in NO+-NADH-dependent reactions. Two such reactions are the oxidation of lactate of pyruvate, occurring primarily in the cytosol; and the oxidation of 8-hydroxybutyrate to acetoacetate, occurring in the cristae of the mitochondria. Determination of the ratios of the B-hydroxybutyrate to acetoacetate and the ratio of lactate-to-pyruvate would enable one to calculate the free NAD+/NADH ratio for the respective compartment. Alveolar macrophages from several rabbits were obtained by the method of Myrviks and after washing with Krebs-Ringer's solution, were divided into aliquots for exposure to the gas in question: 15 ppm of NO, in ambient air, 1.0 ppm of 0, in ambient air or to 80 percent oxygen, while the other paired aliquot was exposed to ambient air and served as a control. At the end of the exposure time, the cellular suspensions were rapidly centrifuged and the sediment deproteinized with perchloric acid. The deproteinized sediment was centrifuged, and the supematent was neutralized with KOH. The neutralized supematent was used for analysis. Lactate, p-hydroxybutyrate and pyruvate levels were determined by fluorometic modifications of well established spectrophotometic techniques.' The acetoacetate level was determined by a modification of the Lowry technique.7 The free NAD+/NADH ratios for each compartment were calculated using an expression derived from the mass action law.
Mitochondria1 Compartment &OH butyric acid + NAP acetoacetic acid + NADH + H+ NAD+/NADH= acetoacetic acid x IH'I Keq = 4.93 p-OH butyric acid x K, Cytoplasmic Compartment lactic acid + N A P pyruvic acid + NADH + H+ pyruvate x [H'] Keq = 1.11x 10.11 NAD"NADH = lactic acid x K,, An assumed pH of 7.0 was used and the K, used was the value found in the literature. Variance between matched control and experimental aliquots was tested by the paired t test.
=
=
l8TH ASPEN LUNG CONFERENCE 95
Dr. Pratt: I am bothered by the localized scarring we
saw in your rats; in humans the scars of chronic oxygen toxicity are more diffuse. Can you explain the difference? Remembering that your experimental animals were old discarded breeders, I am wondering if the scars may not have been there before the exposure to oxygen began. They looked old to me.
Dr. Paegle: I think the process is diffuse. The air spaces are enlarged and dilated throughout the entire lung. In the rat lung, however, you don't see the large blebs as
some of the intra-alveolar proteinaceous exudate and compressed the congested vessels. At least a portion of the large scars originated from confluence and organization of the large regions of perivascular exudate. After the initial, relatively acellular phase during the first two days of 100 percent O,, the numerical increase of fibroblasts in these regions was quite marked. In view of the quick induction of marked scarring and honeycombing, which would seem difficult to reverse, and because the general features of lung injury due to acute oxygen toxicity had seemed similar in man and rat,2 the clinical use of high 0, concentrations for long periods may have to be more carefully scrutinized in regard to production of similar chronic damage in man.
1 Spain DM, Siege1 H. Bradess VA: Emphysema in appar-
ently healthy adults. JAMA 224:322-325, 1973
2 Senior RM, Wessler S, Avioli LV: Pulmonary oxygen
toxicity. JAMA 217: 1373-1377
prominently as in some sections of the human lungs. The h e r scarring in the rat lungs is not well seen on the lower power sections I have shown. You could see this scarring with higher power. Regarding the large scars, one can hypothesize that while in some regions there is some regression and reabsorption of the perivascular exudate as the vessels recover a little bit, in other places for some reason or another, absorption hasn't taken place as fast and then organization progresses and leads to a large scar. Perhaps this process of organization is also helped along in areas where thromboemboli have occurred.
Oxygen Toxicity and Gas Mixtures: ~ o r ~ h o l o ~ ~ ' Philip C . Pratt, M . D., h n m P. Sanders, Ph.D., and W&m
D. Currie, Ph.D.
hi. project involves exposure of rats to progressive Tincrements of oxygen concentration in order to inves-
tigate the level at which oxygen becomes toxic to pulmonary cells and to examine associated morphologic and biochemical changes. Exposure concentrations ranged through 40, 70,90 and 100 percent. There was a slight consistent lag in weight gain among animals exposed to 40 and 70 percent. These animals began to lose weight during the Second day of exposure to percent oxygen. Increased lung weight and volume were demonstrable after 70, 90, and 100 percent whether singly or in sequence. Greater increases occurred in animals exposed only to 100 percent oxygen for seven days than in animals exposed over a period of 16 days to progressive increments of concentration including seven days of 100 percent oxygen. This excess weight is attributed primarily to alveolar and interstitial edema. The sequentially exposed animals showed less intense edema. These animals also showed alveolar septa1 thickening, with prominent alveolar epithelium and increased interstitial cells in alveolar walls and perivascular stroma. *From the Deparhnents of Pathology and Radiobiology, Duke Medical Center and the Durham Veterans Administration Hospital, Durham, N.C. Supported in part by Contract NIH-71-2145.
8S 16TH ASPEN LUNG CONFERENCE
The lesions in sequentially exposed rats are somewhat more comparable to hdings in lungs of patients who die after treatment with oxygen than those in animals exposed to 100 percent oxygen only. However, alveolar septa are not as thick, fibrosis is not as prominent and hyaline membranes are absent although usually seen in human lungs. The significance of these differences remains to be demonstrated. Lung tissue from animals exposed simultaneously with these has been examined for metabolic activity. Preliminary results indicate that there is depression of respiration with succinate and a-ketoglutarate as substrates in homogenized lung tissue of animals killed after as few as three days of exposure to 40 percent oxygen. For example, basal oxygen consumption with succinate, dropped from 3.5 to 3.0 p-liters of 02/mg of mitochondrial protein/min and a-ketoglutarate, from 1.75 to 1.25. The ADP stimulated rate was reduced to a somewhat greater degree indicating a loss of respiratory reserve capacity. Despite this reduction, the measured ATP level after three days of exposure to 40 percent oxygen was increased (from 0.3 to 0.35 pnol/mg of mitochondrial protein) A drop in ATP first appeared after the 3 days in 90 percent oxygen.
CHEST 66: 1, JULY, 1974 SUPPLEMENT
DISCUSSION Dr. Thurlbeck: A question of information. In your summary you said that 40 percent oxygen produced lesions, but when You showed Your $ides You said the 40 percent 0,lung looked normal. Dr. Pratt: After three days of exposure, the lungs looked normal, but by six days, early lesions were visible. This
was shown in the last slide of my series. Dr. Lauweryns: In your control animals as compared to your oxygen treated animals, was there a werence in the appearance of the lymphatics? Dr. P m : Lymphatic vessels were dilated and could be seen in the perivascular adventitia. However, most of the increase in width of this area was due to, interstitial accumulation of edema fluid.
Common Oxidant Lesion of Mitochondria1 Redox State Produced by Nitrogen Dioxide, Ozone, and High Oxygen In Alveolar ~ a c r o ~ h a ~ e s * JeffreyR. Simons, M.D.,Oe JamesTheodore, M.D., and Eugene D. Robin, M.D.
P
hysiologic and biochemical effects of inhaled oxidants on the lung are currently areas of major interest and concern. Two of the oxidizing agents present in the urban environment are ozone and the oxides of nitrogen, in particular, nitrogen dioxide. It has been conclusively shown that exposure to relatively high concentrations of NO, for a short period produces bronchiolitis obliteransl and severe, often fatal lung disease. Similar alterations were reported for ozone by other investigator^.^ Oxygen, clearly an oxidant agent in high concentrations, alters pulmonary structure and function after a relatively short period in various mammalian specie^.^ It would, therefore, seem appropriate to study the effects of oxidizing agents on isolated pulmonary cells to better understand mechanisms by which these agents produce derangements of pulmonary structure and function. One pulmonary cell available in relatively pure form is the alveolar macrophage, which by its location in the air-exchanging portion of the acinus is subjected to intimate contact with inhaled gases. Previous studies in our' laboratory showed that rabbit alveolar macrophages in vitro exhibited biochemical changes under hypoxic conditions. The redox (oxidationreduction) state of the cells was decreased in both the cytoplasmic and mitochondria1 compartments after exposure to hypoxia. The present studies were undertaken to observe the effects of 80 percent oxygen and low concentrations of nitrogen dioxide and ozone on this fundamental physicochemical variable. Oxidation-reduction reactions are vital to many fundamental biochemical processes, with important biologic and clinical implications. The functions that are sibserved by these reactions include the provision of energy, oxidative biosynthesis, biodegradation reactions and detoxification. The reactions usually involve certain common electron camers rather than involving electron flow directing from one substrate to another. Among the most important of these electron camers are the nicotinamide adenine dinucleotide pair (NAD+/NADH). Direct measurements of the biologically active NAD+ and its reduced form NADH are not possible due to their 'From the De artment of Medicine, Stanford University School of ~ d c i n e Stanford, , Ca. O°Currently Assistant Professor of Medicine, University of Utah and Staff Physician, Salt Lake City Veterans Adminktration Hospital.
CHEST 66: 1, JULY, 1974 SUPPLEMENT
localization within specific cellular compartments and the binding of NAD+ and especially NADH to proteins. This ratio, however, can be determined by using suitable substrate pairs involved in NO+-NADH-dependent reactions. Two such reactions are the oxidation of lactate of pyruvate, occurring primarily in the cytosol; and the oxidation of 8-hydroxybutyrate to acetoacetate, occurring in the cristae of the mitochondria. Determination of the ratios of the B-hydroxybutyrate to acetoacetate and the ratio of lactate-to-pyruvate would enable one to calculate the free NAD+/NADH ratio for the respective compartment. Alveolar macrophages from several rabbits were obtained by the method of Myrviks and after washing with Krebs-Ringer's solution, were divided into aliquots for exposure to the gas in question: 15 ppm of NO, in ambient air, 1.0 ppm of 0, in ambient air or to 80 percent oxygen, while the other paired aliquot was exposed to ambient air and served as a control. At the end of the exposure time, the cellular suspensions were rapidly centrifuged and the sediment deproteinized with perchloric acid. The deproteinized sediment was centrifuged, and the supematent was neutralized with KOH. The neutralized supematent was used for analysis. Lactate, p-hydroxybutyrate and pyruvate levels were determined by fluorometic modifications of well established spectrophotometic techniques.' The acetoacetate level was determined by a modification of the Lowry technique.7 The free NAD+/NADH ratios for each compartment were calculated using an expression derived from the mass action law.
Mitochondria1 Compartment &OH butyric acid + NAP acetoacetic acid + NADH + H+ NAD+/NADH= acetoacetic acid x IH'I Keq = 4.93 p-OH butyric acid x K, Cytoplasmic Compartment lactic acid + N A P pyruvic acid + NADH + H+ pyruvate x [H'] Keq = 1.11x 10.11 NAD"NADH = lactic acid x K,, An assumed pH of 7.0 was used and the K, used was the value found in the literature. Variance between matched control and experimental aliquots was tested by the paired t test.
=
=
l8TH ASPEN LUNG CONFERENCE 95