..- -,,_a,,‘,.~,"-‘__,“.--_-_ *-.----‘ _,~_,_, _.._._.. -.._' ..’-...;g.g.'....‘-- . STUDIES ON THE EFFECT OF PARAQUAT ON THE RAT LUNG Thesis for the Degree of M. S. MICHIGAN STATE UNIVERSITY GILBERT LANTEY BOYE, M. D. 1977 ,2 g‘W.SIS LIBRARY Michigan State University PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE 6/01 c:/CIRCIDateDue.p65-p. 15 ABSTRACT STUDIES ON THE EFFECT OF PARAQUAT ON THE RAT LUNG BY Gilbert Lantey Boye, M.D. Paraquat (Methyl Viologen) is the generic name of the compound, l,l'-dimethyl 4,4', dipyridylium. It is a broad Spectrum herbicide effective against broad leaf weeds, grasses and aquatic weeds, and it is rapidly deactivated on contact with the clay in soil. On account of its advantages over other herbicides, it has been increasingly used all over the world. Medical interest in paraquat has arisen as a result of the peculiar and often fatal pulmonary damage in the mammalian Species which may occur after ingestion of small quantities. It has caused over 200 deaths world-wide since its introduction in 1962. Death following paraquat poisoning is usually the result of progressive fibrosis and epithelial proliferation in the lungs. Thus, paraquat poisoning usually causes a progressive respiratory disease, unresponsive to any therapy, resulting in death. Rational therapeutic measures have not been developed because of lack of information on the precise mechanism of action and Optimal methods for removal of the poison. Thus, the overall purpose of this study was to determine the effect of other chemical agents on the toxic effects of paraquat and how these could be adepted for use in the management of paraquat poisoning. Gilbert Lantey Boye, M.D. The objectives of this project were three fold. The first was to study the effect of paraquat on the isolated rat lung preparation and how its effects may be modified by other chemical agents. The agents used were those that had been reported to offer some potential for protecting against or increasing paraquat toxicity and might provide insight into the mechanism of paraquat poisoning. The second objective was to study the modification of the effect of paraquat toxicity in_vivg_in the presence of other chemical agents in both acute and chronic experiments and how these may elucidate the mechanism of paraquat toxicity. The third objective was to investigate how the paraquat affected lung handled endogenous substances which are normally activated or metabolized by the lung and how their control may affect the outcome of paraquat poisoning. The experimental data presented in this dissertation show that perfusing the isolated rat lung with paraquat resulted in the rapid production of marked edema, measured by the percent change in the weight of the lung (mean 37.7 :_l7.7 SEM). It was also observed that perfusing the rat lung with paraquat in the presence of mannitol or propranolol significantly reduced the amount of edema formed. This observation suggested that propranolol and mannitol may afford some protection against paraquat poisoning, but this observa- tion was not confirmed in the in_!i!g experiments with prepranolol. The concentration of the cyclic nucleotide, cyclic AMP, in the lung was increased significantly after perfusing with paraquat and this increase was reduced in the presence of propranolol. Gilbert Lantey Boye, M.D. In both the acute and chronic in 3133 experiments, significant increases were observed in lung cyclic AMP concentrations in rats treated with paraquat. In the chronic experiments in which groups of rats received paraquat, paraquat and propranolol, or paraquat and theophylline, the highest concentrations of cyclic AMP and cyclic GMP were found in the group of rats which received paraquat and theophyl- line, and the percentage mortality was highest in this group but not significantly different from the other groups which received paraquat. The mean concentrations of paraquat in the lungs of the different groups were not significantly different. Cyclic AMP and cyclic GMP concentrations in other organs including the liver, spleen, kidney and thymus, which are known to be affected by paraquat, unlike the lung, were not significantly different from controls. It was also observed that paraquat induced cyclic nucleotide changes in the rat lung were not endogenous catecholamine dependent since levels in resperine treated rats were not significantly different from non-reserpinised paraquat treated rats. The isolated lung from the paraquat-treated rats perfused with SH-PGEZ showed significant inhibition of PGE2 metabolism. This was probably due to an impairment of the uptake mechanism for PGE2 since significantly more unchanged PGE2 was present in the venous effluent from the lungs of treated rats and the levels of the metabolites (lseketo-PGE and 13,l4-dihydro-15-keto-PGE2) in the lung 2 homogenates of the treated and control groups were not significantly different. Interference with the pulmonary mechanism for inactivating endogenous vasoactive hormones such as PGs by drugs, toxic chemicals, atmospheric pollutants or disease is probably more important than Gilbert Lantey Boye, M.D. hitherto appreciated. In paraquat poisoning, interference with excretor as a result of circulating abnormal levels of renal vaso- constrictor hormones such as PGFZa’ would accentuate toxic effects particularly on the target organ, the lung. STUDIES ON THE EFFECT OF PARAQUAT ON THE RAT LUNG BY Gilbert Lantey Boye, M.D. A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Pharmacology 1977 DEDICATION This work is dedicated to my wife Saah, and Koshie Odarley, Odarkor, Odartey and Odartei, our delightful children. ii ACKNOWLEDGMENTS I would like to express my sincere appreciation to the graduate committee members: Drs. Theodore M. Brody, Robert Echt, James E. Gibson, and Andrew M. Michelakis, for their assistance in the prepara- tion of this thesis. I would especially like to acknowledge Dr. Andrew M. Michelakis for his guidance, constructive criticism and encouragement during this investigation and throughout my graduate studies. I am indebted to Dr. Robert Echt for his constant interest and invaluable help. I gratefully acknowledge the technical assistance of Mr. Paul Susan and Ms. Ivy Mao, and the typing skills of Ms. Sharon Hallabrin. iii TABLE OF CONTENTS Page LIST OF TABLES . . . . . . . . . . . . . . . . . vi LIST OF FIGURES. . . . . . . . . . . . . . . . . Vii INTRODUCTION. . . . . . . . . . . . . 1 Human Toxicity of Paraquat . . . . . . . . . . . . 2 Signs and Symptoms . . . . . . . . . 3 Therapeutic Approach to Paraquat Poisoning. . . . . . . 5 Rapid Excretion of Absorbed Paraquat . . . . . . 6 Modification of Tissue Effects of Absorbed Paraquat . 6 Other Regimens . . . . . . . . . . . . . . 7 Animal Toxicity of Paraquat . . . . . . . . . . . 8 Mechanism of Paraquat Action. . . . . . . 9 Non-Respiratory Functions of the Lung. . . . . . . 12 The Handling of Paraquat and Other Amines by the Lung . 14 The Handling of Propranolol and Other B-Adrenergic Agents by the Lung. . . . . . . . . . 15 The B-Adrenergic System and Cyclic Nucleotides. . . . . . l6 PURPOSE . . . . . . . . . . . . . . . . . . 18 METHODS . . . . . . . . . . . . . . . . . . 19 Animals. . . . . . . . . . . . . 19 Perfusion . . . . . . . . . . . . . . 19 Surgery. . . . . . . . . . . . . . . . . . 24 Perfusate . . . . . . . . . . . . . 25 Assays . . . . . . . . . . . . . . . . 26 Glucose Studies . . . . . . . . . . . . . . . 28 Paraquat Studies. . . . . . . . . . . . . . 28 Effect of Paraquat on Rat Lung Cyclic AMP and Cyclic GMP 29 Rat Isolated Lung Perfusion Experiment . . . . . . 29 Rat Chronic In Vivo Experiments. . . . . . . 32 Rat Chronic In Vivo Experiment I . . . . . . . . . 33 Rat Chronic In Vivo Experiment II . . . . . . . . . 33 Rat Chronic In V1vo Experiment III. . . 34 Rat Chronic In Vivo Experiment IV . . . 35 iv Page Rat Acute In VLvo Experiment I . . . . . . . . . . . 35 Rat Acute In Vivo Experiment II. . . . . . . . . . . 36 Analysis of_Resu1ts. . . . . . . . . . . . . . . 37 Statistics. . . . . . . . . . . . . . . . . . 37 RESULTS . . . . . . . . . . . . . . . . . . . 39 Isolated Perfused Lung Experiments. . . . . . . . . . 39 Glucose Studies . . . . . . . . . . 40 Effect of Paraquat on Rat Lung Cyclic AMP . . . . . . . 40 Rat Isolated Lung Perfusion Experiment . . . . . . . . 40 Rat Chronic and Acute Ln Vivo Experiments . . . . . . . 41 Rat Acute Ln VLvo Experiment I . . . . . . . . . . . 45 Rat Acute Ln VLvo Experiment II. . . . . . . . . . 45 Effect of Paraquat on Cyclic Nucleotides of Rat Liver, Spleen, Kidney and Thymus . . . . . . . . . . . . 46 DISCUSSION . . . . . . . . . . . . . . . . . . 95 SUMMARY . . . . . . . . . . . . . . . . . . . 105 BIBLIOGRAPHY. . . . . . . . . . . . . . . . . . 106 Table 10. LIST OF TABLES Rat Isolated Lung Perfusion Experiment-Change in Lung weights 0 O O O O O O O I O O O 0 O 0 Summary Rat Chronic lg_!ixg_£xperiment l. . . . Rat Chronic £n_!1!g_Experiment 1 . . . . . . . . Summary Rat-Chronic ln_yi!g_Experiment 2. . . . . Summary Rat Chronic lg Vi!g_Experiment 3. . . . . Rat Acute ln_yi!g_Bxperiment 2 . . . . . . . . The Effect of Paraquat on Cyclic Nucleotides in Thymus, Liver, Spleen, Kidney, and Lung . . . . . . The Rf Values of Prostaglandin PGEZ and Its Three Metabolites in TLC System . . . . . . . . . Effect of Paraquat on Prostaglandin E2 Metabolism by the Rat Isolated Lung (Using TLC). . . . . . . . . Effect of Paraquat on the Amount of Prostaglandin Ez Metabolized by the Rat Isolated Lung . . . . . vi Page 85 86 87 88 89 90 91 92 93 94 LIST OF FIGURES Figure Page 1. Lung Perfusion Apparatus--showing general outlay . . . 21 2. Lung Perfusion Apparatus--showing diagram of perfusate flow during perfusion . . . . . . . . . . . 23 3. Structure of paraquat . . . . . . . . . . . . 48 4. Rat isolated lung perfusion experiment showing percent weight change in the lung on perfusing with paraquat, propranolol, paraquat and propranolol, paraquat and mannitol, histamine, Bovine-serum albumin-Kreb's solution (BSA). . . . . . . . . . . . . . SO 5. Rat isolated lung perfusion experiment-~showing glucose utilization with time . . . . . . . . . . . 52 6. Rat isolated lung perfusion experiment--showing cyclic AMP changes after perfusing with isoproterenol, paraquat, paraquat and propranolol and BSA solution . 54 7. Effect of paraquat on body weight of rats compared to saline treated rats . . . . . . . . . . . . 56 8. Effect of paraquat on lung total proteins compared to saline treated rats . . . . . . . . . . . . 58 9. Effect of paraquat on lung c-AMP compared to saline treated rats . . . . . . . . . . . . . . 60 10. Effect of paraquat on lung c—GMP compared to saline treated rats . . . . . . . . . . . . . . 62 11. Effects of paraquat, paraquat and propranolol, and paraquat and theophylline on rat body weight compared to rats treated with saline, propranolol or theophylline . . . . . . . . . . . . . . 64 vii Figure Page 12. Effects of paraquat, propranolol, and paraquat and propranolol on rat lung c-AMP . . . . . . . . . 66 13. Effects of paraquat, theophylline, and paraquat and theophylline on rat lung c-AMP. . . . . . . . . 68 14. Effect of paraquat, prOpranolol, and paraquat and propranolol on lung c-GMP . . . . . . . . . . 70 15. Effect of paraquat, theophylline and paraquat and theophylline on rat lung c-GMP. . . . . . . . . 72 16. Long concentrations of paraquat and propranolol in groups of rats treated with paraquat (Group B), paraquat + propranolol (Group C), propranolol (Group D), or paraquat + theophylline (Group F). . . . . . . . 74 17. Lung concentrations of paraquat and theophylline in groups of rats treated with paraquat (Group B), theophylline (Group E) or paraquat and theophylline (Group F). . . 76 18. Effects of paraquat, propranolol, theophylline, paraquat and pr0pranolol, and paraquat and theophylline on rat lung total proteins, total c-AMP and total c-GMP after 9 days treatment . . . . . . . . . . . 78 19. Rat lung concentrations of paraquat, propranolol and theOphylline after treatment with paraquat, paraquat and propranolol, paraquat and theophylline, propranolol or theophylline. . . . . . . . . . . . . . 80 20. Effect of paraquat, paraquat and l-prOpranolol, paraquat and d—propranolol, l-propranolol, and d-propranolol on rat lung c-AMP and c-(NP. . . . . . . . . . . 82 21. Acute effect of paraquat on lung c-AMP and c-GMP . . . 84 viii INTRODUCTION Paraquat (methyl viologen) is the generic name for l,l'-dimethyl— 4,4'-dipyridylium. It is available either as the dichloride or dimethyl sulphate salt, both of which are water soluble. Paraquat is a broad spectrum herbicide effective against broad leaf weeks, grasses and aquatic weeds. Paraquat has been reported to be firmly bound when it contacts most types of soils and upon adsorption can be degraded by several micro-organisms present in the soil. Application to crops results in a low level of contamination. The following formulations are available in the United States: 1. ORTHO Paraquat Cl 29.1% solution (21.7 weight-% cation) 2. ORTHO Dual Paraquat 42% Solution (21.7 weight—% cation) 3. Chevron Industrial Weed 21.07 weight-% cation and Grass Killer 4. ORTHO Spot Weed and Grass 0.44% solution (0.2 weight-% Killer cation) ORTHO Spot Weed and Grass Killer is unlikely to be a problem in accidental poisoning because of its low paraquat content and the fact that it is only marketed in pressurized cans which dispense a foam. Outside the U.S. paraquat is marketed as 20% concentrate, Gramoxone, and in a 5% granular form, Weedol. Medical interest in paraquat has arisen as a result of the peculiar and often fatal pulmonary toxicity in mammals which may occur after ingestion of small quantities. It is estimated to have caused over two hundred human deaths world wide since its introduction in 1962. Most of these deaths have followed accidental oral ingestion. The fatal dose of paraquat in adult man is thought to be about 15ml of a 20% solution, or a "mouthful" as described in most case histories (Kimbrough, 1974). Death following paraquat poisoning is usually the result of progressive fibrosis and epithelial proliferation. This effect has been described in both humans and animals, although there is a marked difference in the oral acute lethal doses observed between species (Clark g£_al,, 1966; Murray and Gibson, 1972). Rational therapeutic measures have not been developed for paraquat poisoning because of the lack of information on the precise mechanisms of action, and optimal methods for the removal of the poison. Human Toxicity of Paraquat Serious paraquat poisoning in humans has occurred only after ingestion or parenteral administration (Almog and Tel, 1967). Exposure of the skin to a solution of paraquat produces erythema and reactive hyperkeratosis which may be associated with pustule formation. There is ulceration and necrosis two to three days after short periods of contact with the mucous membrane and conjunctiva of the eye (Canning 9131., 1969). §ign§ and Symptoms In cases of paraquat ingestion, there is usually an immediate burning discomfort of the mouth and pharynx, followed by ulceration due to the severe irritating effect of paraquat. This is generally followed by repeated vomiting. If the dose ingested was large (i.e., 6-8 02.), the lungs, kidneys, liver, and adrenals may be severely affected initially followed by possible fatal pulmonary edema within 24 to 72 hours. When smaller amounts are taken there may be oliguria, increase in blood urea nitrogen and albuminuria as a result of acute renal failure. Jaundice is also sometimes noted. The initial phase is followed by a latent period sometimes lasting as long as two weeks during which time the patient feels well and kidney function generally improves. These symptoms are superceded by predominantly pulmonary signs and symptoms including increasing dyspnoea, cyanosis, and pulmonary congestion. Pathologic changes include necrosis of alveolar epithelial cells and accumulation of alveolar macrophages (Witchi and Kacew,'l974). Pulmonary infiltrates are detected on chest roentgenograms, and physiologic studies reveal hypoxemia, decreased lung volumes, low lung compliance, and impaired diffusing capacity for carbon monoxide (DLCO) (Gardner, 1972; Matthew ggugl., 1968). Later stages are characterized by the development of pulmonary fibrosis and eventual death from respiratory failure. Lung pathology in paraquat poisoning has been studied exten- sively. Thurlbeck and Thurlbeck (1976), noted a considerable topo- graphic variation in the severity and nature of lung lesions. They described two distinct forms of fibrosis. In one form, there was marked intra—alveolar edema in much of the lung and active fibroblastic proliferation of the lung interstitium. Hyaline membranes lined some of the air spaces and honey-combing with disorganization of the alveolar pattern of the lung was evident. Also, distinctive areas of the lung structure were completely disorganized and replaced by many small (0.05mm-2.0mm) cysts lined by fibrous tissue. This type of lesion is similar to fibrosing alveolitis in man. A second type of fibrosis is characterized by preservation of the framework of the lining architecture with the formation of abundant loose fibrous tissue within the alveolar spaces. Thurlbeck and Thurlbeck (1976) suggested this may represent the organization of protein-rich edema fluid which pours into alveoli at an early stage of paraquat poisoning. 0n the contrary, Smith and Heath (1974), claimed that the formation of a fluid exudate during the early stages of paraquat poisoning is not necessary for the development of pulmonary fibrosis and that paraquat or its metabolites, directly stimulates an infiltra- tion of profibroblasts into the lung. Oxygen poisoning shares with paraquat the special sensitivity of type I epithelial-cells to damage but the early phase of paraquat poisoning is different from early oxygen poisoning (and ozone and radiation), where damage to endothelial cells plays a prominent role. The fibrotic and proliferative lesions described, as well as marked edema are very similar to the lesions described in oxygen poisoning. Although some of the patients studied were treated with oxygen terminally, Thurlbeck and Thurlbeck (1976), clearly identified the lung lesions as not caused by oxygen therapeutically administered since clinical pulmonary involvement occurred before oxygen was administered and also occurs in animals to which oxygen has not been given. However, some features of oxygen poisoning, notably severe edema may have been an added feature to the underlying paraquat lesion. The similarity to oxygen poisoning and the observation that hyperoxic environment increased the lethality of paraquat in rats (Fisher 33.21,, 1973) has led to the suggestion that paraquat may particularly affect the lung because of its high ambient oxygen. Although death due to paraquat poisoning is usually due to progressive respiratory failure (Malone g£_al,, 1971; Matthew gt_al,, 1968; Gardner, 1972), fatalities have occurred less frequently from uremia or from cardiac involvement (Oreopulous g£_§l,, 1968; Gardner, 1972). In most cases of paraquat poisoning in man, death occurred approximately one to four weeks after ingestion. Therapeutic Approach to Paraquat Poison£2g_ The therapeutic measures used thus far to manage paraquat poisoning have been unsuccessful. The therapeutic approach has been based on three general principles: a. Prevention of paraquat absorption b. Rapid excretion of absorbed paraquat c. Modification of the tissue effects of absorbed, non-excreted paraquat. Prevention of paraquat absorption--Gastric lavage and adminis- tration of cathartics are well established general measures for the treatment of some cases of poisoning including paraquat poisoning. Many adsorbents have been shown to be effective against absorption of I paraquat lg 112g, but only bentonite and Fuller's earth have been effective in_vivg_(Clark, 1975; Staiff, gt_gl,, 1973). The effectiveness of these agents is probably due to their prevention of gastro-intestinal absorption of paraquat. Smith §£_al, (1974), have demonstrated an active process for the uptake of paraquat into the lungs in both human and rat lung slices and it is, thus, important that specific measures to inhibit paraquat absorption should be considered an important part of the management of paraquat poisoning. Rapid Excretion of Absorbed Paraquat Increased urinary excretion of paraquat following forced diuresis with saline solution and mannitol has been documented (Fisher gt_al,, Kerr g£_al,, 1968). Forced diuresis is apparently a more effective means of removing absorbed paraquat than peritoneal dialysis (Fisher gt_§1,, 1971). Since paraquat has been detected in the urine for as long as 31 days after ingestion (Beebeejaum gt_gl,, 1971), con- tinued, as well as early efforts to eliminate absorbed paraquat may be lifesaving. Hemodialysis used early is effective in reducing the plasma levels of absorbed paraquat (Grundies g£_gl,, 1971). Modification of Tissue Effects of Absorbed Paraquat Paraquat toxicity in rats is greatly enhanced by oxygen. Hyperoxic environment (100% inspired oxygen) markedly accelerates the mortality of rats given lethal doses of paraquat (Fisher etugl., 1973), on the other hand, Smith and Rose (1977), have shown that paraquat poisoned rats placed immediately after dosing in atmosphere containing 10% oxygen, paradoxically died sooner than those left in air and there was no reduction in overall mortality. This is claimed to be due to an increased rate of accumulation of paraquat by the lung perhaps resulting from an increased perfusion of the lung by blood (Smith and Rose, 1977). Other Regimens Some reports have described treatment of patients who have ingested paraquat with corticosteroids (Malone g£_§l,, 1971; Duffy g£.al,, 1968; Lathwaite, 1975), immunosuppressive agents GMalone ggna£., 1971), or the antifibrotic agent, potassium aminobenzoate (Laithwaite, 1975). However, in the majority of cases, suppressive agents have not been effective. Other therapeutic measures available include the use of d-propranolol and superoxide dismutase (SOD). A regimen currently in use at the Royal Postgraduate School, Hammersmith, London, involves a stomach washout as soon as possible after ingestion followed by the administration of a 30% suspension of Fuller's earth (200—300 ml), along with a cathartic. This is continued for several days in order to prevent the absorption of paraquat. In cases of serious paraquat poisoning where there is impairment of renal function, hemodialysis is used. Oxygen administration is avoided as much as possible. Along with the above measures, SOD is administered both intravenously and by inhalation for a period of at least one week following the incident. In addition, d-propranolol is given intra- venously in an attempt to reduce the uptake of paraquat into the lung. Beclomethasone, a synthetic corticosteroid preparation, is administered from pressurized aerosol cannisters to limit the inflammatory process in the lung. The use of oxygen in paraquat poisoning has become a controversial issue. There are theoretical reasons and evidence from animal studies which contraindicate the use of oxygen. There is in addition, recent evidence which contraindicates the use of low oxygen therapy in cases of human poisoning unless it can be shown that the paraquat concentration in circulating blood is extremely low (below 0.05 nmoles/ml) such that enhanced lung accumulation will not occur (Smith and Rose, 1977). Superoxide dismutase is an enzyme found in aerobic cells which converts superoxide into hydrogen peroxide and molecular oxygen. SOD is found in two forms, one in the extramitochondrial cytosol and another in the mitochondria. The mitochondrial superoxide dismutase of eukaryotes is similar to the 500 of many bacteria with respect to 2+ its characteristic content of Mn and many homologies in amino acid sequence. The cytosol form of SOD has quite a different structure and 2+ 2+ contains Cu and Zn . These enzymes are present in high concentra- tion and are extraordinarily active. Animal Toxicity of Paraquat The toxicity of paraquat has been studied in many animal species including rats (Kimbrough and Gaines, 1970; Robertson gt_gl,, 1971; Short §£_31,, 1972), rabbits (Butler and Kleinerman, 1971), guinea pigs and monkeys (Murray and Gibson, 1972). Rats and monkeys exhibit lung damage similar to that observed in humans, while rabbits appeared to be resistant to paraquat and failed to develop the lung lesion described. The absorption of 14C-paraquat in rat after oral administration was poor, with elimination occurring in the urine and faeces (Daniel and Gage, 1966). No radioactivity appeared in the bile. Lichtfield g£_§l,, (1973) observed that an intravenous injection of paraquat was rapidly distributed in most tissues except the brain and spinal cord. After 24 hours, however, paraquat was selectively concentrated in the lung and skeletal muscle from where it was slowly excreted. The pathology of the lung lesion in rat has been extensively studied. The primary change found in the rat lung consists of vacuolization and degeneration of the membranous pneumocytes (type I), followed by increase of collagen and reticulin in the basement membrane and proliferation of granular pneumocytes (type II). Swelling of the endothelial cells, proliferation of fibroblasts, and increased numbers of endothelial cells follow. A well developed lung lesion shows areas where alveoli are completely obliterated or filled with an amorphous material which either formed a lattice or a large whorl of a very electrodense material similar to the material observed in the lamellar bodies (Kimbrough and Linder, 1973). Paraquat has also been found to cause centrilobular necrosis in the rat liver and proximal tubular necrosis in the kidney (Murray and Gibson, 1972). Atrophy of the thymus has been noted in rats and rabbits (Butler and Kleinerman, 1971), and rats. Mechanism of Paraquat Action The mechanism of paraquat's toxic effects on the main target organ, lung is not known. In 1967, Manktelow put forward the hypothesis that paraquat specifically interfered with the production of pulmonary surfactant. This conclusion was essentially based on histopathologic findings in paraquat poisoned animals. The tissue alterations resembled pathologic features observed in some cases of 10 human respiratory distress syndrome. The absence of stable bubble formation over thick tissue sections corroborated that surface active material was decreased. This view has been shared by Robertson §t_§l:, (1970) and Fisher §£_§1,, (1969 and 1972). On the other hand, Fletcher and Wyatt (1970 and 1972), found that the phospholipid compo- sition of rat lungs was unchanged after treatment with paraquat and that paraquat neither affected the amount of dipalmitoyl lecithin, the major lecithin constituent present, nor its rate of destruction. Thus, the effect of paraquat on surfactant material in the lung is unclear. Lipid Peroxidation has been defined as the oxidative deteriora— tion of polyunsaturated lipids. Paraquat and other related bipyridy- lium compounds have been investigated in the past and shown to be capable of being reduced in solution to give intensely colored, relatively stable free radicals (Michaelis and Hill, 1933). This property was shown to be a preprequisite for herbicidal activity (Calderbank, 1964). In the presence of oxygen, these radicals are rapidly reoxidized to the parent cations with production of super- oxide radical ions (02-), and hydrogen peroxide (Calderbank, 1964; Farrington g£_§l,, 1973). Paraquat is reduced by photosystems present in green leaves of plants, and cyclic redox reactions then lead to production of reactive oxygen Species which are thought to be the molecules responsible for herbicidal activities. Bus gt_a1, (1974), present in_vi££9 and in_vivg_evidence that mammalian toxicity of paraquat may be the consequence of cellular lipid peroxidation. The peroxidation being mediated through the single electron reduction of paraquat catalysed by NADPH-Cytochrome c 11 reductase with the subsequent transfer of the electron from reduced paraquat to molecular oxygen to form superoxide anion. The super- oxide may non-enzymatically dismutate to form singlet oxygen which reacts with unsaturated fatty acids to form fatty acid hydroperoxides. Lipid free radicals which form spontaneously from lipid hydroperoxides, react with membrane polyunsaturated lipids with the formation of more lipid free radicals, thus continuing the chain reaction process of lipid peroxidation. The consequence of such free radical catalysed peroxidation is extensive damage to cell membranes with resultant loss of functional integrity. Paraquat and other bipyridylium compounds, like Diquat, can be reduced to free radicals by homogenates of liver, kidney, or lung (Gage, 1968; Baldwin ggfla£., 1975). Incubation of paraquat with liver microsomes or a system containing NADPH-cytochrome c reductase, NADPH, and microsomal lipid greatly increase the formation of malonaldehyde which is dependent upon the concentrations of paraquat in the incubation mixture. However, Illet gg_al, (1974), found that paraquat inhibited in 31559 lipid peroxidation. It has been demonstrated that paraquat toxicity is significantly enhanced in selenium or vitamin E deficient mice or mice pretreated with dimethylmaleate and there is partial protection against paraquat toxicity by pretreatment with the enzyme, superoxide dismutase (Bus gt_ _a_1_., 1974). During the last few years, considerable attention has been focused on the pathological and biochemical changes brought about in lung tissue by oxygen and the oxidant gases, nitrogen dioxide and ozone. There are several recent reviews on the mechanism of the anatomic-pathologic and biochemical consequences of oxygen toxicity in 12 in the lung (Pfister and Nogues, 1974; Clark and Lambertsen, 1971; Haugaard, 1968). It is generally thought that lipid peroxidation may be an important consequence of exposure to normobaric or hyperbaric oxygen. It is interesting to note that the pathological findings in the paraquat lung have been likened to changes in the lung following oxygen toxicity. It_wou1d seem that, at normal oxygen tension, only small amounts of superoxide are formed and the endogenous enzyme, superoxide dismutase (SOD) is sufficient to degrade it to peroxide. But, when breathing pure oxygen, the amount of superoxide formed exceeds the capacity of endogenous superoxide dismutase to inactivate it, and then the superoxide is free to damage cellular components leading to cell death. The corrollary to paraquat poisoning is that the lung is exposed to higher concentrations of paraquat than any other tissue, and also achieves higher levels of paraquat. Thus, it is possible that even at normal oxygen tensions this may lead to the formation of more superoxide in the lung than the available superoxide dismutase can metabolise. Non-Respiratory Functions of the Lung_ The long held traditional view that the lung is solely and passively involved in gaseous exchange has been profoundly altered by contemporary research which has established its capacity to perform various metabolic functions. The location of the lung in the body is strategic for modifi— cation of drug action. The lungs are the first visceral organs to receive parenterally administered drugs, thus, the uptake or metabolism of a drug by the lung may greatly affect the action of the drug. 13 The ability of the lung to modify the biological activity of substances passing through the pulmonary circulation has been referred to as the pharmacokinetic function of the lung. Thus, the lung is capable of altering the biological activity of many substances, endogenous or exogenous, brought to it via the blood. The lung has been shown to affect many vasoactive substances by degrading them or converting an inactive product to its active form. Thus, angiotensin I is converted into its active form, angiotensin II. The degree of inactivation seems to be specific for a given substance, ranging from almost complete inactivation of bradykinin (Farreira and Vane, 1967), S-hydroxytryptamine (Thomas and Vane, 1967), prostaglandins E1, E2, and an (Ferreira and Vane, 1967), and minor inactivation of more- pinephrine (Ginn and Vane, 1968), to relatively free passage of com- pounds like epinephrine and angiotensin II (Hodge g£_al,, 1967). These phenomena are often quantitatively sufficient to markedly modify the concentration of some circulatory substances and to create large arterio-venous differences. These pharmacokinetic functions of the lung seem to chiefly reflect the metabolic activities of the endothelial cells of the pulmonary vasculature, and have been known to be affected by age, pregnancy, gaseous anesthetics and heart-lung by-pass (Junod, 1975). The lung is also capable of acting as an endocrine organ, releasing into the circulation a variety of active substances in- cluding histamine, SRS-A and prostaglandins in response to stimuli such as anaphylaxis, peptides, and physical deformation. The signifi- cance of such release has not been fully investigated, but it is clear 14 that the lung provides an essential control of the blood levels of many biologically active substances. The Handlinngf Paraquat and Other Amines by the Lung Several classes of drugs seem to be concentrated preferentially in lung tissue. Localized high concentrations may result in selective or even generalized toxicity. It has been suggested that in some cases, the lung acts as a depot which buffers the remainder of the body from high concentrations of a compound. An example are the phenothiazines, which almost always appear in pulmonary tissue in high concentrations. The depot in the lung could maintain blood levels for several days. In 1968, Vane reviewed the role of the lung in clearing cir- culating endogenous and exogenous substances. He listed basic, lipophilic amines as the compounds most likely to be concentrated in the lung. S—hydroxytryptamine and imipramine are examples of amines whose handling by the lung have been extensively studied. The uptake of these compounds occurs by different saturable mechanisms and the Km and Vmax values are thirty and one hundred times greater for imipramine suggesting basic differences in terms of the affinity and the number of the sites for uptake (Junod, 1975). The administration of another basic amine with similar physicochemical properties can result in release of previously bound imipramine and this may result in unwanted side effects. The lungs appear to have various means of processing and of eliminating drugs. As already discussed, amines are taken up by the lung via what appears to be an active transport mechanism of endothelial cells. Similarly, prostaglandins E and F 15 series are taken up and degraded but the site of the metabolic reactions are not yet known. The lungs also process steroid hormones (Hartiala, 1974), apparently for their own use, but it may be signi- ficant that the lungs can convert cortisone to its more potent analog, cortisol. Some of the cortisol thus formed leaves the lung and enters the arterial circulation. The Handling of Pr0pranolol and Other B-Adreneggic Agents by the Lung Two main types of B—adrenergic receptors have been identified. These are 8-1 receptors in the heart and 8-2 receptors in the trachea, bronchi, and blood vessels. Isoproterenol seems to stimulate both 8-1 and 8-2 receptors. Salbutamol, carbuterol, and fenoterol stimulate 8-2 receptors (Cullum et al., 1969; Giles EE“§1°' 1973; Wardell ggnal., 1974). The B-blocking agents include pr0pranolol, bunolol, sotalol, and K5592. Propranolol is the most commonly used B-adrenergic blocking agent. The aliphatic hydroxyl group on the propranolol molecule appears to be essential for activity and it gives the molecule its optical activity. The l-form is more potent than the d-form, and this difference is used to distinguish B-blockade from those other pharma- cological actions of the molecule, such as, local anesthetic effect. Propranolol is well absorbed after oral administration. It is con— centrated in the lung, and to a lesser extent, in the brain, liver, kidney and heart and excreted in urine after being almost completely metabolized. Two main metabolities, naphthoxy lactic acid and 4- hydroxypropranolol are found. 4-hydroxypropranolol is found only after oral or intraperitoneal administration and has blocking 16 activity similar to that of propranolol but with a shorter duration of action.. Junod (1975) reported on studies of dl-propranolol accumula- tion in the rat lung. It is a saturable process and compounds with similar physicochemical properties can inhibit the binding or accelerate the release of bound propranolol. There is, however, partial Na+ dependence and marked temperature dependence at low substrate con- centrations. The B-Adrenergic System and Cyclic Nucleotides The key compound that is involved in the mediation of most of the metabolic effects of the B-adrenergic system (as well as a great number of hormones) is cyclic 3',5', adenosine monophosphate (c-AMP). This compound was first described in 1957 by Sutherland as a co—factor for the conversion of liver phosphorylase from its inactive to its active form, allowing liver glycolysis to proceed (Cleveland 33 31" 1972). c-AMP is formed from adenosine triphosphate (ATP) by the action of adenyl cyclase, an enzyme located within the cell membrane of the target cell, and it is inactivated (hydrolysed to 5' AMP) by phospho- diesterase (PDE), a soluble cytoplasmic enzyme. ATP——A-C——) c-AMP .315.) 5' AMP The equilibrium concentration of c-AMP, therefore, depends on the relative activities of these two enzymes. The activity of adenyl cyclase is increased by a number of factors including adrenergic stimulation while PDE is inhibited by xanthine derivatives, 17 particularly the0phylline, so that either may result in an increase in c—AMP levels. Cyclic AMP acts not only as a second messenger for the effects of nearly all hormones, but also, together with the other known natural cyclic nucleotide, cyclic guanosine 3',5'mon0phosphate (c-GMP), appears to be intimately involved in the control of almost all facets of cellular activity (Robison, et_al,, 1971; Greengard et_al,, 1972; Hardman gt_gl,, 1971). PURPOSE The overall purpose of this study was to determine the effect of other chemical agents on the toxicity of paraquat and how these could be adapted for use in the management of paraquat poisoning. The objectives were three fold. The first was to study the effect of paraquat on the isolated perfused rat lung preparation, and how its effects may be modified by other chemical agents; and second, to conduct acute and chronic in_vivg_experiments to determine the effect of other agents on paraquat toxicity. The third objective was to study how the paraquat affected lung handles endogenous substances such as the prostaglandins, peptides and biogenic amines which are effectively regulated by the normal lung. 18 METHODS Animals Adult male Sprague—Dawley rats (from Spartan Research Animals, Inc., Haslett, Michigan), weighing 150 - 250g were used in all experi- ments. The animals were housed in plastic cages in groups of three and were allowed food and water $9.119: The room temperature was maintained at 21 - 24°C and the light - dark cycle was twenty-four hours. Perfusion The perfusion apparatus is shown in Figures 1 and 2. In this set up for the isolated perfused lung experiment, the conditions under which the lung is normally maintained in the chest of the animal were simulated. This involved placing the lungs in an "artificial thorax" and connecting the trachea in such a way that gases can pass in and out of the lungs. The "artificial thorax" is a sealed chamber main- tained at normal body temperature (37°C). The chamber was arranged in such a manner that the isolated lung can be suspended in it and can be subjected to alterations that are somewhat similar to the changes in pressure that the lungs undergo while in the thorax of the intact animal. The changes in pressure in the "artificial thorax" were produced by connecting the sealed chamber to a pump (Rodent respirator pump by Harvard Apparatus Co.), that will alternate the pressure 19 20 .xefiuoo “anocom mcwzocmsumspewmmm< nowmsmhom mesa .H chewed 21 mar—Ragga? zo_m3n_mmm 023.. \v s: mwhuioz<2 » a mu x455 m o z 44.9....54 / . '.-III \. 2‘ {’Hw E as.» /- mucosa rm \& iv \ @ its-4501 « V . o ‘EI- [HI =1 s. _ m K 0 © 0 o M“ .53... mol m 3: @ ass“. gbfimEmLII «0.21....3". .22m3k2. :2: 20.351 .538. 4551”! rd 7...: I llllllll IIIIIIIIIIIIIIIIIIIIIIIIIIJ 562398 , as: T . .I All .52 T 333 ..I 532. him: 040u2<2 20:02.8. 293...:— ¢.O>¢mmu¢ 02¢ x5325. I I I I I I I ¢Ohxo 45032.3( zuucom j - . . FIWIII PII IIIII I‘IIIIIIIUIIII'U' .523 ZO_mDumwa ._ 0.05), but the deaths occurred earlier in the paraquat and theophylline 43 treated group than in the other groups referred to above (1 on the 7th day, 1 on the 8th day, 2 on the 9th day, 2 on the 10th day, and 3 on the 12th day) (Table 4). Body weight changes were also followed in the different groups (Figure 11). Rats in the groups receiving paraquat showed slower weight gain up to the 6th day and then a progressive decrease in weight with a significantly higher rate of weight loss in the group which received paraquat and theophylline compared to rats which received paraquat only or paraquat and propranolol. The three control groups showed a steady increase in weight. The concentration of paraquat in the lungs of the different groups of rats which received paraquat was determined. The highest level of paraquat in the lungs was found on the 10th day in the groups which received paraquat. The group which received paraquat and theophylline had significantly (p < 0.05) higher level of paraquat in the lungs compared to rats which received paraquat only or paraquat and propranolol (Figure 12). Lung propranolol concentration was highest on the 3rd day in both the group which received only propranolol and that which received paraquat and propranolol (Figure 12). There was significantly higher concentration of propranolol in the lungs of rats which received paraquat and propranolol. Lung theophylline con- centrations peaked on the 3rd day in rats which were treated with paraquat and theophylline, and on the 6th day in rats which received theophylline only. The theophylline concentrations at the two peaks in the two groups were not significantly different (Figure 13). Mean total lung cyclic AMP concentrations in all rats increased with time and the increases were highest in rats which received paraquat or paraquat and theophylline. 44 Rat Chronic In Vivo Experiment III: In this experiment, two rats from the group treated with paraquat and theophylline died (Table 5). There was significant increase in total lung proteins in the groups of rats which were treated with paraquat, paraquat and propranolol, or paraquat and theophylline compared to their respective controls. Lung cyclic AMP concentrations were significantly increased in rats which received paraquat (four-fold increase), or paraquat and theophylline (ten-fold increase) (Figure 18). Lung cyclic GMP concentrations were significantly increased in the rats which received paraquat (eight-fold increase), paraquat and propranolol (eight-fold increase), and paraquat and theophylline (three-fold increase) compared to controls (Figure 18). Lung concen- trations of paraquat in the rats which received paraquat, paraquat and propranolol, or paraquat and theophylline were not significantly different but lung propranolol concentrations in rats which received paraquat and propranolol were significantly higher while theophylline concentrations were significantly lower in rats treated with paraquat and theophylline compared to controls (Figure 19). Rat Chronic In Vivo Experiment IV: The d- and 1- forms of propranolol were given with paraquat in this experiment. Two rats died from the group treated with paraquat and l-propranolol. Lung cyclic AMP levels in rats treated with paraquat and d-propranolol showed about three-fold increase while the paraquat and l-propranolol treated rats showed a significant reduction. The cyclic AMP levels in the paraquat and d-propranolol treated rats were also significantly higher compared to the paraquat treated group (Figure 20). 4S Lung cyclic GMP concentrations were significantly increased in rats treated with paraquat and d-propranolol compared to their controls but were not different from rats treated with only paraquat. Lung cyclic GMP levels in paraquat and l-propranolol treated rats were not significantly different compared to l-propranolol treated rats (Figure 20). Rat Acute In Vivo Experiment I In this experiment, two groups of rats received either saline or paraquat 20 mg cation (27.6 mg) per Kg body weight. Lung cyclic GMP was elevated with a peak at 30 min after paraquat injection, the level dropping to the control level, followed by a slight rise at about 8 hr and then a gradual fall to the control value (Figure 21). The lung cyclic AMP also showed a rise, the peak level occurring at 1 1/2 hr followed by a fall to the control value which was maintained over the 24 hr period of the experiment. There was a rapid uptake of paraquat into the lungs with concentration peak (10 nm/mg protein) at 2 hr, then a gradual fall to about 6 ng/mg protein over the rest of the period of the experiment (Figure 21). Rat Acute In Vivo Experiment 11 In this experiment, the effect of the adrenergic and cho- linergic systems on lung cyclic nucleotides was tested. Lung cyclic AMP values in the rats pretreated with reserpine were not significantly different from those of saline pretreated rats (Table 6). Lung cyclic GMP results were inconsistent in the reserpine pretreated rats compared to controls. The lung cyclic AMP values in the rats pretreated with atr0pine were all significantly lower compared 46 to saline pretreated controls (Table 6). Lung cyclic GMP levels in atrOpine pretreated rats were only significantly different in rats killed at 2 hr after paraquat injection (Table 6). Effect of Paraquat on Cyclic Nucleotides of Rat Liver, Spleen, Kidney and Thymus In this experiment it was observed that the cyclic nucleotides, cyclic AMP and cyclic GMP were significantly higher in lungs of rats treated with paraquat for 8 days while levels in the organs studied-- namely; liver, spleen, thymus, and kidney were not significantly differ- ent from their controls (Table 7). Figure 3. Structure of paraquat. 47 PARAGUAT + —— / \ + (oxidized) —— + (reduced) Figure 3 49 Figure 4. Rat isolated lung perfusion experiment showing percent weight change in the lung on perfusing with paraquat, propranolol, paraquat and propranolol, paraquat and mannitol, histamine, Bovine-serum albumin-Kreb's solution (BSA). Values represent mean :_SEM. LUNG PERCENT WEIGHT CHANGE 35 30 20 IO 50 RAT: ISOLATED PERFUSED LUNG- PERCENT WEIGHT CHANGE PERFUSING DIFFERENT DRUGS In)= no. lungs perfused (I .___J I O) V2 (IO) I .. m. % Prop (3) v — a: E ('9) (7) E E (3) 'lil _— . ' Pq+Prop P+q Histamine BSA Mannitol 51 Figure 5. Rat isolated lung perfusion experiment--showing glucose utilization with time. Values represent mean :_SEM. The slopes of the glucose disappearance curves were significantly different (p < 0.05). GLUCOSE OONCENTRATION m PERFUSATE (mass) 52 RAT: ISOLATED LUNG PERFUSUII EXPERIMENT ' GLUCOSE UTILIZATION] .1 L "Mint w a f 1: N 1 3} é With Lung Values - mean 3- sern 0 With the Luna (6) a Without the Luna (2) l 1 I5 55 45 so TIME OF PERFUSION (MINUTES) S3 .zmm H :85 acomowmow mosfim> .5333 4.2 use Hofiocmwmowm v.8 Hangman Susannah .Hocowououmomfi spa: wcfimsmaom poumm momcmcu mz< owauxo mcfizonmnuucosflwomxo :OMmsmwom mesa managemfl Ham .0 cannot 54 mmmmx-fim .momou unaccommonnou a“ aoaocmumoua pee Hmsvmnem no flow wx\ms my Hofiocmhmoum .mmfl mx\ms my umscmnmm .aon\Ha mo.ov Huez wm.o no“: xafimp panacea who: mama .d:<-o aqua any :9 Hofiocwumoua com umscmwem can .aoflocmhmoum .Amscmwmm mo muoowmm .NH 8A39Aa 66 2mm H Z .9399... .3 25051 .9359... doi .20... page... UdIIIII .9399... doi + calf! .dOmm .w 0n. .0”: .0; 7.4m cow _. 38F .ooom . ocmw . occm .oomm i .ooov I._._>> 09—3.me 2.41 2. 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'9’ “\ 'l'; [SK ~ ’ /.-' axe I 3 6 8 IO I3 DAY OF EXPERIMENT 01 O N U'I N O LUNG THEOPHYLLINE CONCENTRATION (pg) 8 IO Ul 77 .amo.o v my mHouucou scum pooHDMMMv xHuomuflmwcmwm mosfim> moumuwecw xmfinoum< .mumu m Rom 2mm.H came Hammonmon mosfim> .ucosumonu mxmp a “coma mzuiu Hmpou cam dz.> z. 0.20.5.0 ram (selowd) 9Nn‘l use dWV-O 'lVlOl 79 .nMo.o v my afiouucou scam uconommflc xfiucmofimwcwwm one coax: m99~m> moumuflvcw xmwhoum< .zmm + name acomonmou mosfim> .ocfififixzmoocu no Hofiocehmowm .ocfifiaxgmoonp pom «mscmhmm .HoHocmHmon can umscmhmm .pmsumnwm no“: unoEuwowu Houmm mafiaflxzdoonu new HoHocmHmonm .Hmsvmnmm mo mcowumeueoucou w:9~ Hem .mH unamflm 80 no.0 Va * 25 ER. 5. l m av . o. * 8; . m. . 8 a: E .9d 8 mucomocd of E 9.: Lo 9:90 8.9 vat. 89$ flea E UnTQLQ gem 5.7.8 90.78 On. 0 H... 0 ON ”u o. 09 m. 8 0m Om 01 00. E: 99 E “Os 8: v .6. 95.995 Lo 8585 5 9a to .980 ON. dd *9 99:32.”. 95 5 Ed *9 .890 any kzmgmwaxw 0).) Z. 0.20110941 Figure 20. 81 Effect of paraquat, paraquat and l-propranolol, paraquat and d-propranolol, l-propranolol, and d-propranolol on rat lung c-AMP and c-GMP. Rats were treated daily with paraquat (5 mg/kg ip). l-pr0pranolol (5 mg/kg sc 3x), d-propranolol (5 mg/kg sc 3x). 0n the 10th day all the rats were killed and the lung c-AMP and c-GMP determined. Values represent mean :_SEM. Asterisk indicates values significantly different from controls (p < 0.05). TOTAL C-AMP PER LUNG- pmoles 82 RAT: CHRONIC IN VIVO EXPERIMENT-(4l- LUNG CHANGES AFTER TREATMENT WITH Pq, Pq+L-Pro, Pq+D-Pro, L- Pro AND D-Pro alt I60 I50 l30 NO 90 70 o 0 50 PqL-Progo'fioliq PaL-PmfiqD-PI'DPE bPlo D-Fto L-Pro D-Pro C-AMP C-GMP * Significantly different from controls (P<0.05) TOTAL C-GMP PER LUNG-pmoles 83 .zmm.H :fiouonm me you coflumnucoocoo umscmnwm Ho QZUID .mzflm .ocfiamm Ho acoflumo we omv «escaped spa: Gunman» one: mpmm .mzuuu pom mz .xfifiac mx\wam uazvmham no“: aw voumunu onus mama Dame pasuw> 9H owaougu ummii.m odnmh Table 4.--Summary Rat-Chronic In Vivo Experiment 2. 88 Group 1 2 3 4 5 6 Treatment Saline Pq Pq + Prop Prop Th Pq + Th No. in Group 25 30 30 25 25 30 No. Dead Nil 4 6 Nil Nil 8 Percent Dead 0 13 20 O 0 27 Group 2 Deaths Group 3 Deaths Group 6 Deaths Day 9 - 1 Died Day 9 — l Died Day 7 - 1 Died 10 - 2 10 - 2 8 - l 11 - 1 ll - 0 9 - 2 12 - Q_ 12 - §_ 10 - 2 Total 4 Total 6 ll - 0 12 -.3 Total 8 89 Table 5.--Summary Rat Chronic In Vivo Experiment 3. Group 1 2 3 4 5 Treatment Pq Pq + Prop Prop Th Pq + Th No. in Group 10 10 10 10 10 No. Dead Nil Nil Nil Nil 2 Percent Dead 0 0 0 0 20 NB. 3 injections of Propranolol given daily. 90 mIHQ< mIHQm "mIHnm I H: N mIHm< ”mIHmm "miamm I :w5 on m-Hu< m-Aum "m-Hum - A; A m-A<< ”m-s<¢ um-H OH.9u oA.9u 00.9“ NH.QH. ow.en. o~.eu. ”H.0u. ov.9u om.9u om.qu om.9n AH.9H anouona .mn.~ mw.~ om.~ mm.~ oo.n om.m .wo.e .H~.e He.» e~.m oo.m an.~ \aze-o ewe: mm.qu mm.qu om.9fl em.qu om.qu vm.qu oe.qu om.mu oo.mu mH.mu 4w.qu oo.mw :Aosoua .me.m .ow.m .om.o .ew.m me.w mm.a w~.w ee.m we.HH MH.~H mo.o~ 99.x \az<-o cam: 92.... SEM To.“ c.2H mg...“ 9.3.“ Tau 52H 9.5..“ «an 9:.“ 93H 339 3 $6-0 m.smm o.omm ~.mHe o.mov «.mme o.mme N.oee w.am¢ 9.4N4 w.mme G.Hem «.mmm Aesop can: e.mnu 4.4wu o.Anu m.emu 4.0m“ v.99“ ~.~eu o.mfiwu o.owu. «.meu w.mwu e.m9fl Aofioa av az<-u m.s~m m.emw m.mow «.mem c.5aw o.HoHA s.cwm s.coofi m.eam~ «.mwmfi o.o~m~ o.mmoA Aesop can: m.mu m.~u a.wu w.mu ”.4“ m.nu. v.4“ m.wu m.wu N.nu ~.wu m.mu “may compona o.mes o.wms «.mefi e.HeH 4.NNH N.mHH o.on N.on «.mmfi o.NmH e.wHH 8.4NH Aesop cam: m-Ha< m-Hu< m-Am< m-A<< m-Hoa m-zuz m-Hma m-sfi> 9H ouso< Hemii.o Danae 91 Table 7.--The Effect of Paraquat on Cyclic Nucleotides in Thymus, Liver, Spleen, Kidney, and Lung. Mean c-AMP (p mole) Mean c-GMP (p mole) Protein (mg) Protein (mg) Thymus control 10.78 :_0.74 1.83 :_0.04 treated 11.30 :_0.71 1.88 :_0.23 Kidney control 5.91 :_0.35 1.15 :_0.10 treated 6.28 :_0.21 l 31 :_0.90 Liver control 1.98 I 0.20 0.33 :_0.03 treated 2.49 :_0.17 0.44 :_ 16 Spleen control 6.71 :_0.32 1.15 :_0.05 treated 6.30 :_0.37 1.45 :_0.11 Lung control 8.46 I 1.00 0.38 :_0.16 treated 20.38 :_l.22* 2.23 :_0.69* Values represent Mean :_SEM for 3 rats. *Values significantly different from control (p < 0.05). Male Sprague-Dawley rats were treated with paraquat (5mg/kg) ip daily for eight days. Control rats received saline ip daily for the same period. Rats were anesthetized and the thymus, kidney, liver, spleen, and lung were removed and quickly frozen in liquid nitrogen. These Specimens were homogenized in 10% TCA and assayed for c—AMP, c-GMP, and total proteins. 92 Table 8.--The Rf Values of Prostaglandin PGEZ and Its Three Metabolites in TLC System. Compound Rf Value PGE2 0.186 :_0.011 lS-Keto PGE2 0.567 :_0.026 13,14-dihydr0-15-ket0 PGE 0.747 :_0.025 2 13,14—dihydro PGE 0.260 1 0.016 2 Values represent mean :_SEM for 5 determinations. Twenty pl of each compound was applied to the silica gel G thin-plate chromatogram (Analtech, Newark, Del.). The plates were developed in chloroform:tetrahydrofuran:acetic acid (100210:5 by vol) for 80 min. The solvent front travelled 15 cm from the origin. The plates were sprayed with 5% phosmolybdic acid and dried at 120°C for 15 min. 93 .Amo.o v my Hoppcou scum ucuuommwp xfipcmufimMcmflm moaaa>w .moamemm m Rom 2mm.H :99: pcomoamou m99Hm> 4N.HHdN.a es.euom.me No.Nu.om.oA ON.¢HNV.N mm.nntqm.me GH.¢H.Nm.NN amputee NN.QHNH.A oe.nHwN.ee NN.QHNN.4N wfi.wuso.w mN.muNo.Am NN.muoe.mA Honpcou N - N N N - N - N a 99a 0989-mH 999-0009-mfl mom moa ouox-mH mua-ouo9 NH mom e990 900 oueane 4A.NH oumcowoso: was; ouexefie 4H.NA ucoaammm msoco> .muqe wcfimsv mesa pauNHomH wax 0:9 »9 Emflaonmuuz mm :Nccmfiweumoum :0 pmsceamd mo «00mmmii.m Danae 94 Table 10.--Effect of Paraquat on the Amount of Prostaglandin E2 Metabolized by the Rat Isolated Lung. PGEZ lS-keto-PGE 13,14-dihydro-1S Metabolized (a) Formed (%) 2 Keto P0132 Formed (9.) Control 72.30 1 1.22 56.30 i 1.23 4.59 + 1.19 Treated 59.18*:_4.16 44.57 :_3.55 5.10 + 1.20 Values represent Mean :_SEM. *Values significantly different from control (p < 0.05). Data drived from Table 9 by substracting correspdong values obtained from perfusion experiment without rat lung. DISCUSSION The isolated perfused lung is a useful model for studying a chemical compound like paraquat which has the lung as its main target organ, On perfusing the rat isolated lung with bovine serum albumin- Kreb's Ringer bicarbonate and measuring the glucose content of the perfusate over the period of study, it was observed that the glucose concentration steadily decreased with time, a change that was not observed when the lung was not included (Figure 4). This has been used as a criterion of viability of the lung in conjunction with other gross anatomical criteria. The isolated rat lung perfused with paraquat, propranolol, histamine, paraquat and propranolol, or paraquat and mannitol, showed different degrees of edema, as measured by the percentage change in lung weight. Paraquat produced a rapid and marked edema during the 90 minute period of perfusion and in the presence of propranolol and mannitol, the edema produced on perfusing the lung with paraquat was significantly reduced (Figure 3). This observation suggested a protection of the lung from the toxic effect of paraquat although this was not borne out by the rat ip_yiyg_studies. Although the isolated lung is a useful model for studying the effect of a chemical agent with a specific effect on the lung, in the 95 96 final analysis the only means of establishing an effect unequivocally is by its effect in the intact animal. It is possible to explain the effect of propranolol in reducing the edema observed on perfusing paraquat, on the basis of the fact that both paraquat and propranolol are basic lipophilic amines with similar physico—chemical properties and known to be extensively accumulated by the lung and to competitively inhibit the uptake of each other. Other amines including biogenic amines may behave in a similar manner. The rate of accumulation of paraquat by rat lung from plasma ip_yiyg, following oral dosing was about one seventh of that predicted from ip_yip£g_(Rose pp 21,, 1976). This observation led to the suggestion that inhibitors of paraquat accumulation may be present in the circulation. A number of endogenous amines including norepinephrine, S-hydroxytryptamine, and histamine, have been reported to reduce concentrations of paraquat accumulation into lung slices, as have several other drugs including imipramine, d-propranolol, betazole, diquat, and burinamide (Lock g£_§l:, 1976). No precise structural requirement has emerged for compounds which inhibit the accumulation of paraquat, although the substitution of a carboxyl group on the o—carbon carrying the amino group to an amino acid (e.g., tyramine to tyrosine, histamine to histidine), abolishes the ability of the amine to inhibit paraquat (Lock g£_al,, 1976). Lock g£_§l, (1976), have suggested two types of inhibition, namely the inhibition produced by norepinephrine, imipramine, diquat and lysine which is linear with time and may be explained by simple competition between paraquat and inhibitor at the uptake site, and a non-linear inhibition produced by betazole and histamine. 97 An understanding of the mechanism of inhibition of paraquat uptake is important in the search for agents that reduce the uptake of paraquat into the human lung. Compounds which will be of therapeutic value will be those which can be given in quantities sufficient to inhibit paraquat accumulation into the lung, until such time that the paraquat is excreted from the body or removed by other therapeutic measures. The lung accumulates paraquat very much more effectively than any other organ examined. This selectivity must be a primary factor in the deve10pment of lung damage and partially explains why this organ is the most severely effected. The main lung cell types that accumulate paraquat are the alveolar epithelial type I and 11 cells (Ilett §£_§l,, 1974). Mannitol, a polyhydric alcohol used mainly as an osmotic diuretic is an effective scavenger of hydruxyl radicals as it has a protective effect against hydroxyl (OH') depolymerizing species generated secondarily by a reaction between the enzymatically gen- erated superoxide radical from peroxidation pathway and hydrogen peroxide (The Haber and Weiss reaction) in hyaluronidase deploymeriza- tion (McCord, 1974). On the basis of the hypothesis that lipid peroxidation is the underlying mechanism in paraquat toxicity, mannitol was tried out and it was interesting to observe that it significantly reduced the edema from paraquat perfusion. Perfusing the isolated rat lung with isoproterenol or paraquat resulted in an increase in the lung cyclic AMP and perfusing paraquat and propranolol resulted in significantly less cyclic AMP in the lung (Figure 6). This finding was also observed in the ip_vivo experiments. 98 Kuo and Kuo (1973), studied the effects of adrenergic and cholinergic agents on the levels of cyclic AMP and cyclic GMP in slices of rat lung. They reported that isoproterenol significantly increased pulmonary cyclic AMP levels (about three fold) and this increase was abolished by pr0pranolol but not by phenoxybenzamine. Stoner §p_al, (1973), reported that pulmonary cyclic AMP was markedly increased by exposure to isoproterenol, epinephrine, or prostaglandin E1. The rat ip_yiyg experiments carried out in this project involved acute and chronic exposure to paraquat. Weight loss and lethality were the most useful indices of paraquat toxicity in these experiments. In all the chronic experimemtns, weight loss was observed in rats which received paraquat by itself or together with propranolol or theophylline. There was in the chronically treated rats a slower weight gain compared to control rats but after about the sixth day of chronic treatment progressive weight loss was observed and this was more marked in rats which received paraquat and theophylline than those which received paraquat alone or paraquat and propranolol (Figure 11). Mortality in the paraquat and theophylline treated rats was higher but not signifi- cant at the 5% level compared to the controls (Figure 6). Weight loss associated with paraquat poisoning is due to failure of many paraquat treated animals to accept food. Lung total proteins in paraquat treated rats showed a steady and progressive increase in all the chronic experiments (Figure 8). This increase in lung protein is due to increase in protein synthesis and Van Osten and Gibson (1975), correlated it with the proliferation of endoplasmic reticulum and the enlargement of "ribosomal" granules. They further suggested that the synthesis of cellular protein and 99 nucleic acid components in paraquat poisoned animals may be secondary to paraquat initiated free radical toxicity rather than the direct result of paraquat action on cellular control mechanisms. The exact mechanism of the lung protein increase observed on chronic paraquat treatment is not clear. This observed increase in lung protein concentration makes it unacceptable to express specific values such as cyclic AMP in terms of mg. of protein. Thus, in the chronic experiments in this project, it seemed that to express data per total lung was the most satisfactory approach. Figures arrived at this way will not be affected secondarily by changes in chemical composition of the lung and therefore they allowed one to compare the entire biochemical capacity of the damaged lung with the values found in the appropriate controls. Maling g£_§l, (1975), reported what appeared to be an encouraging result from the use of the B-adrenergic blocking drug, propranolol, in rats. Since ingestion of paraquat in humans produced respiratory distress, it was considered reasonable to treat paraquat poisoned rats with the B-adrenergic agonist, l-isoproterenol to dilate the bronchi and improve gaseous exchange. However, it was found that treatment with l-isoproterenol (Maling gp_gl,, 1975) or salbutamol (Fletcher, 1973), increased the lethal effect of paraquat. Theophylline, a xanthine derivative, also produced a potentiating effect with paraquat. Maling gp_gl, (1975), reported further that pretreatment with d1- propranolol or l-propranolol was relatively ineffective. These findings suggested the B-adrenergic receptor may be involved in the toxicity of paraquat since it is known that the B-adrenergic receptor blocking activity is associated mainly with the L-isomer (Barret, 1969). Other 100 B-adrenergic blocking agents including bunolol, sotalol, pronethalol, and K0592 also reduced the mortality from paraquat although bunolol and propranolol proved more effective than the others. In the light of the above findings, a part of this project was designed to find out whether cyclic AMP, which is involved in the mediation of most of the metabolic effects of the B-adrenergic system, and the other naturally occurring cyclic nucleotide, cyclic GMP, have a role in paraquat toxicity. It was thus interesting to observe increases in pulmonary cyclic AMP and cyclic GMP on treatment with paraquat. The rat chronic ip_yiyg_experiment 1 showed that paraquat significantly increased intracellular cyclic AMP (by two fold), (Figure 9) and cyclic GMP (by ten fold), (Figure 10). This observation was found in subsequent ip_yiyg_experiments and theophylline was observed to potentiate the increases in these cyclic nucleotides. Thus, rats treated with paraquat and theophylline showed a rapid and greater weight loss compared to controls (Figure 11), and significantly higher pulmonary paraquat concentrations (Figure 16), although the lung paraquat concentrations were not consistently statistically different from the other groups in some of the experiments. The mortality in the paraquat and theophylline treated rats (27%), was higher but not significantly different from rats treated with paraquat only (13%), or with paraquat and propranolol (20%), (Table 6). Rats treated with paraquat and the0phylline had signifi- cantly less pulmonary theophylline compared to rats which only received the0phylline (Figure 17). The protection afforded by propranolol in reducing paraquat induced pulmonary edema in the isolated lung 101 experiment was not reproduced in the ip_yiyg_experiments. This observation may be due to a difference in rate of uptake of pr0pranolol ip_yiyg_compared with that ip_yi£rg, This may be the result of propranolol uptake inhibitory substances in plasma, binding of propranolol by components of plasma or increased metabolism of propranolol to less active metabolites thus reducing the concentration of free propranolol. Differences in the behavior of lung ip_yiyg_ compared with the isolated perfused lung cannot be ignored. The pulmonary paraquat concentration was not significantly different from rats treated with only paraquat but the propranolol concentration was significantly higher (by half as much) in rats which received paraquat and propranolol compared to rats which received only propranolol (Figure 16). Gardiner and Shanker (1976), reported that the per- meability of the lung is markedly increased in the presence of paraquat induced damage and pointed out that relatively lipid insoluble com- pounds primarily absorbed by diffusion through aqueous membrane pores showing marked increase in absorption rate by paraquat damaged lungs, is consistent with the idea of an increased porosity for the absorbing membrane. This explanation applies to propranolol which is a relatively lipid insoluble compound. In the case of theophylline, a lipid soluble drug, increased membrane porosity would be expected to have a lesser effect on the absorption rate, since although diffusing through pores, the drug is absorbed predominately by crossing lipid regions of the pulmonary membrane (Enna and Shanker, 1972). Other factors which could contri— bute to changes in membrane permeability of paraquat damaged lungs to drugs include reduced pulmonary surfactant activity (Manktelow, 1967), 102 and a change in pulmonary blood flow. The presence of edema fluid itself could slow absorption rates owing to the decreased absorbing surface/volume ratio. In the chronic ip_yiyg_experiments other organs including the liver, spleen, thymus, and kidney, which are known to be affected by paraquat were assayed for cylic AMP and cyclic GMP and compared to controls. It was found that the cyclic nucleotide content of these organs was not significantly different from controls, while it was significant in the lungs (Table 7). This finding suggested the pulmonary cyclic nucleotide changes observed were probably related to the peculiar toxic effect of paraquat on the target organ, the lung. Acute iplyiyg_experiment II was designed to determine whether increased endogenous catecholamines were responsible for the increased cyclic nucleotides observed in the lung. Rats pretreated with reserpine to deplete catecholamine stores, then given paraquat had lung cyclic AMP values not significantly different from controls (Table 6). This observation suggested that endogenous catecholamine was not a factor in the cyclic AMP elevation in the lung discussed above. On the other hand, rats pretreated with atropine followed by paraquat had pulmonary cyclic AMP values signifi- cantly lower compared to controls (Table 6). Kuo and Kuo (1973), reported that increases in cyclic GMP levels in the lung tissue are closely regulated by muscarinic cholinergic receptor activation, elevated by acetycholine and abolished by atropine. Acetylcholine a1so increased the cyclic AMP content. Thus, in the 103 present study it was observed that atropine abolished an increase in cyclic GMP after paraquat treatment and cyclic AMP level was signifi- cantly reduced compared to controls (Table 6). It is rather difficult to assign specific sites or functions to the changes in cyclic nucleotide concentrations in a tissue as heterogenous as the lung. Stoner §£_al: (1973), suggested that increased cyclic GMP concentrations produced by acetycholine occurred in cells innervated by the parasympathetic nervous system, that is, cyclic GMP serves as a "second messenger" for acetylcholine. The position with cyclic AMP is much more complicated since the lung is a site for synthesis or storage of a number of agents that can increase cyclic AMP concentration, for example, norepinephrine, prostaglandins, and histamine. Alternatively, the accumulation of cyclic GMP might itself increase cyclic AMP concentration as has been shown to occur in fat cells, kidney slices, and avian erythrocytes (Murad gg_al,, 1970), presumably by inhibiting cyclic AMP phosphodiesterase (Ferrendelli £3 a_l_., 1970). The Acute 1211.22 Experiment I showed an increase in cyclic GMP with a peak at 30 minutes followed by a cyclic AMP peak at 1.5 hours after paraquat treatment. Recently there has been increased awareness of the role played by the lung in regulating the systemic arterial blood levels of impor- tant vasoactive hormones including prostaglandins, peptides, and bio- genic amines. Such a regulation may be achieved by uptake and metabolism of substances reaching the lung via the pulmonary artery, for example, prostaglandins of the E and F series, serotonin and bradykinin, or by synthesis within the lung e.g., angiotensin. 104 The isolated lung from paraquat treated rats perfused with SH-PGEZ showed significant inhibition of PGE2 due either to an impairment of the uptake mechanism of PGE2 to the site metabolism. This may be of metabolism or to interference with the enzymes involved in prosta- glandin metabolism. Bito and Baroody (1975) suggested that the capacity of the lung to metabolize PGs required the transmembrane transport as an initial step. Thus, any interference with that mechanism would reduce the amount of PG metabolized. There is also evidence that the capacity of the lung to metabolize PCs can be modified by various conditions and changes which affect the enzymes involved in the metabolic processes. Parkes and Eling (1975) reported that the activity of the enzyme, prostaglandin dehydrogenase (PG-DH) in the lung is reduced on exposure to 100% oxygen or endotoxin shock. Interference with the pulmonary mechanism for inactivating endogenous vasoactive hormones such as the PCs by drugs, toxic chemicals, atmospheric pollutants or disease may be more important than hitherto appreciated. In paraquat poisoning, interference with excretion as a result of circulating abnormal levels of vasoconstrictor hormones would accentuate toxic effects particularly on the target organ, the lung. SUMMARY Paraquat, is a bipyridylium compounds used as a broadspectrum herbicide, which is very toxic to the mammalian Species. The lung is the main organ affected by paraquat although other organs such as the kidney, liver, spleen and thymus may be affected to a lesser extent. 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