Toxicology, 4 (1975) 53--63 © Elsevier/North-Holland, Amsterdam -- Printed in The Netherlands
EFFECTS OF I N T R A T R A C H E A L INSTILLATION OF DACTINOMYCIN ON PULMONARY EDEMA AND PHOSPHATASE ACTIVITY OF THE LUNG LAVAGE FLUID IN RATS
SHRI N. GIRI, MANNFRED A. HOLLINGER and L. RICHARD KARTT Department of Physiological Sciences, School of Veterinary Medicine, and Department of Pharmacology, School of Medicine, University of California, Davis, Calif. 95616 (U.S.A.)
(Received August 8th, 1974)
SUMMARY Intratracheal (i.t.) administration of protein synthesis inhibitors produced pulmonary edema. Of those inhibitors studied, dactinomycin (act. D) was the most potent. Severity of lung damage due to act. D was dose- and almost age-related. Maximal intensity of pulmonary edema was reached on the 3rd day following administration and remained constant for 14 days. Histopathological studies revealed confluent edema of the entire lung. Pretreatm e n t with act. D induced tolerance to an LD100 edematogenic dose of thiourea. The effects of i.t. instillation of act. D appear to be localized in the pulmonary tissue. Lung lavage fluid collected from drug-treated rats had higher acid and alkaline phosphatase activities, higher protein c o n t e n t and more leukocyte infiltration than that of control.
INTRODUCTION The capacity of lung to synthesize protein actively in vivo as well as in vitro has been demonstrated by Massaro et al. [1,2]. Some protein synthesized by lung plays an important physiological role in the action of lung surfactant in maintaining its form and function [3,4]. As in most tissues, it is probable that the a m o u n t of protein in the lung at any given time is controlled in part by the protein-synthesizing and protein-degrading ability of the lung. An alteration in either of these processes induced by disease,
Abbreviation: act. D, dactinomycin; i.t., intratracheal; PMN, polymorphonuclear leukocytes~
53
chemicals or other means might therefore be expected to cause observable changes in lung structure and function. For example, enzymatic attack on structural proteins in the lung by papain administered i.t. is frequently employed in animal models to study the pathogenesis of emphysema [5,6]. A similar type of animal model utilizing i.t. administration of compounds which inhibit lung protein synthesis was recently reported in a preliminary study by Giri et al [7,8]. It was considered warranted to extend this previous study in order to understand the characteristic features and the mechanism of lung damage induced by the i.t. administration of act. D. METHODS AND MATERIALS Male Sprague--Dawley rats weighing 350 to 450 g and free of pulmonary pathogens were used t h r o u g h o u t the experiment unless the weight specified otherwise. They were housed two per cage in a room designed as the holding area. Rats were fed Purina rat chow and water ad libitum. The following four compounds which inhibit protein synthesis were used: ethionine, cycloheximide, act. D, and puromycin dihydrochloride. The first three compounds were purchased from the Sigma Chemical Company and the last from the National Biological Company. Drugs were dissolved in phosphate buffer (pH 7.4, 0.05 M) and instilled i.t. in a volume of 1 ml/kg body weight. This volume was kept constant for each drug regardless of the dosage injected. The procedure for i.t. administration has been described previously [8]. Briefly, rats were lightly anesthetized with sodium pentobarbital (25 to 30 mg/kg, i.p.), tracheae were exposed, and the drug was instilled via a 27-gauge needle. Rats were immediately held in an upright position to let the drug solution gravitate to the distal regions of the lung. Rats in the control group were anesthetized in the same way and received an equivalent volume of phosphate buffer by the same route. The incisions were sutured and an antiseptic was applied. Rats were allowed to recover and the extent of lung damage was examined under varying experimental conditions. At various times following drug administration rats were killed with sodium pentobarbital and the lungs immediately separated from the heart and extraneous tissues, blotted gently and weighed. The degree of pulmonary edema was quantitated as lung weight expressed as percent body weight [9]. For histopathological studies, the lungs and heart were removed en bloc, and fixed in Zenker-formalin solution. Lung lavage in a group of act. D-treated rats was carried out according to the m e t h o d of Brian et al. [10]. The lavage liquid resulting from repeated washings of lung was pooled and centrifuged at 12 000 × g for 10 min in a refrigerated centrifuge (Sorval RC2-B) at 4 °. The supernatant was aspirated and discarded. The packed cells from each rat lung lavage were suspended in isotonic saline to a total volume of 1.5 ml. The suspension was centrifuged at 300 rev./min for 10 min to sediment the heavier particles. The supernatant was carefully transferred into a tissue homogenizer (Tri-R) and homogenized in an equal volume of cold distilled deionized water, using 10 up-and-down
54
strokes. The h o m o g e n a t e was then assayed for acid and alkaline phosphatase activity [ 1 1 ] . The pr ot e i n c o n t e n t of t he h o m o g e n a t e was d e t e r m i n e d according to the m e t h o d of L ow r y et al. [ 1 2 ] . Plasma, liver and lung tissues were collected as follows: rats were etherized and b l o o d was collected in centrifuge tubes directly from the severed inferior vena cava. After being allowed to coagulate, the blood was spun at 3000 rev./min for 10 min, the serum being aspirated and diluted 1 : 20 for the d e t e r m i n a t i o n of acid and alkaline phosphatase activity. A 2--3-g lobe of the liver was removed and weighed in a tared beaker containing cold isotonic saline. The tissue was washed in saline a second time, b l o t t e d dry, cut up into smaller pieces and then hom ogeni zed in 0.25 M sucrose using 10 up-and-down strokes. The h o m o g e n a t e was made up to a final c o n c e n t r a t i o n of 10%. This was later diluted 1 : 100 with distilled deionized water just prior to e n z y m e assay. The lung and heart were removed en bloc, placed in a watch glass containing cold isotonic saline and perfused with saline through the trachea, in order to remove cellular c ont e nt s f r om air spaces, and through the right ventricle to wash o u t cellular contents f r o m the p u l m o n a r y capillary bed [ 1 3 ] . Lung lobes were cut away, c h o p p e d and homogenized (10 strokes) in 0.25 M sucrose and made up to 15 ml. The h o m o g e n a t e was filtered through two layers o f cold moist cheese cloth and the resulting filtrate was diluted with distilled deionized water (1 to 10) prior to acid and alkaline phosphatase assay. Urine f r o m rats housed individually in stainless steel metabolic cages was collected over a 24-h period in ice-cold test tubes. The total volume of urine voided was measured and centrifuged at 10 000 rev./min for 20 min in a refrigerated centrifuge. Urine was purified by gel filtration [14] at 4 ° prior to e n z y m e assay. The data expressed as the mean +- standard error. The S t u d e n t " t " test was e m p l o y e d to de t e r m i ne the degree of significance between the means. RESULTS The ef f ect of i.t. administration of protein synthesis inhibitors on the in d u ctio n o f p u l m o n a r y edema is summarized in Table I. When examined 48 h after drug administration, it was f ound that each c o m p o u n d p r o d u c e d a significant degree of p u l m o n a r y edema. Of the f o u r inhibitors studied, act. D at doses used was the most and ethionine the least pot ent . Purom yci n and c y c l o h e x i m i d e appeared to possess an intermediate p o t e n c y . As act. D produced the most severe lung damage, it alone was t herefore subsequently studied. The e x t e n t of lung damage induced by act. D was found to correlate with th e a m o u n t of drug administered (Fig. I). A small a m o u n t of pleural effusion varying in color from straw to bloodtinged was noticed in rats treated with 50 or 100 pg/kg act. D. Grossly, lungs o f these rats cont ai ned diffuse hemorrhagic spots involving one or m ore lobes. A f r o t h y m uc ous discharge was seen oozing from t he incised tracheae
55
TABLE I P U L M O N A R Y E D E M A I N D U C E D BY I N T R A T R A C H E A L A D M I N I S T R A T I O N O F PROTEIN SYNTHESIS INHIBITORS
Treatment
Lung wt
- - X
Body wt Mean ± SE
Sham-operated control A c t . D 50 p g / k g C y c l o h e x i m i d e 50 p g / k g C y c l o h e x i m i d e 500 ~g/kg P u r o m y c i n 50 p g / k g E t h i o n i n e 25 m g / k g
0.378 0.749 0.401 0.418 0.434 0.457
100
-+ 0.007b(5)a ± 0.053 (5) -+ 0.017 (6) + 0.013 c (5) + 0.009 d (6) + 0.015 d (5)
a The values in p a r e n t h e s e s are the n u m b e r o f animals used. b p value b e t w e e n c o n t r o l and a c t . D is significant at the 0.001 level. c p value b e t w e e n c o n t r o l and c y c l o h e x i m i d e is significant at the 0.05 level. d p values b e t w e e n c o n t r o l and p u r o m y c m and c o n t r o l and e t h i o n i n e are significant at the 0.01 level.
of these rats. Fig. 2 is a typical photomicrograph of histopathological changes in the lungs of rats treated with act. D (50 pg/kg i.t.). In contrast to the normal-appearing lungs in the buffer-inStilled control rats, the lungs of the drug-treated rats showed massive confluent edema and e r y t h r o c y t e aggregates in the alveolar, peribronchial and perivascular interstitial spaces of the lungs.
15 b0
, "
J
I0
X u~ l.I
d
o,L i
0
12 5
25 DOSE
50 OF
ACT
I0© ~g/~q
D
BW
IT
Fig. 1. E f f e c t s o f d i f f e r e n t doses o f act. D on p u l m o n a r y e d e m a . Rats were sacrificed and p u l m o n a r y e d e m a was d e t e r m i n e d 48 h a f t e r drug a d m i n i s t r a t i o n . Each p o i n t r e p r e s e n t s the m e a n o f six animals with s t a n d a r d e r r o r o f mean. Zero drug, c o n t r o l .
56
Fig. 2. Photomicrograph of a rat lung which received one injection of act. D (50 pg/kg) 48 h before killing. Note the generalized confluent edema with erythrocytes in the alveolar space (Zenker-formalin fixitive; H.E. stain; x 200). The time course for the onset and progression of p u l m o n a r y edem a following a single dose (50 pg/kg) of act. D is illustrated in Fig. 3. An increase in lung weight was f ound one day after drug administration, and the e x t e n t of lung damage progressed with time through the third day. From that poi nt in time no change in the severity of p u l m o n a r y edem a was observed for the entire duration (14 days) of the experiment. The ef f ect of act. D p r e t r e a t m e n t on acute lethal effect of an LD10o dose (10 mg/kg) of t hi our e a i.p. is indicated in Table II. P r e t r e a t m e n t with act. D was f o u n d to afford significant p r o t e c t i o n against acute t hi ourea lethality. The mo r talit y d r o p p e d f r o m 6 o u t of 7 in the cont rol group to 1 o u t o f 7 in the act. D-pretreated rats. The e f f e c t o f act. D (50 pg/kg i.t.) on the i nduct i on of p u l m o n a r y edem a in rats o f varying ages is indicated in Table III. These results d e m o n s t r a t e that the severity of lung damage in response to act. D is related t o the animal's
57
LL 0
qO
O8 ~0 a UJ ¢9 ¢0
06
~
o4
Q. X W
O2
Z _1
TIME AFTER ACT. D. INJECTION, 50.ug/kg IT (IN DAYS)
Fig. 3. Effects of a single dose of act. D (50 pg/kg) on the development of pulmonary edema for 14 days following drug administration. Each point represents the mean of 6 animals with standard error of mean. C, control.
age. T h u s , 2 3 - d a y - o l d r a t s w e r e l e a s t s u s c e p t i b l e w h i l e 7 3 - t o 9 8 - d a y - o l d r a t s w e r e m o s t s u s c e p t i b l e t o t h e e d e m a t o g e n i c e f f e c t o f a c t . D. T h e e f f e c t o f i.t. a d m i n i s t r a t i o n o f a c t . D ( 5 0 p g / k g ) o n a c i d a n d a l k a l i n e phosphatase activity and protein content of lung lavage fluid are summarized in T a b l e I V . T h e l u n g l a v a g e c o l l e c t e d f r o m d r u g - t r e a t e d r a t s c o n t a i n e d a higher amount of acid and alkaline phosphastase activity than that of cont r o l s . I n a d d i t i o n , a g r e a t e r a m o u n t o f p r o t e i n in t h e l u n g l a v a g e o f t h e s e r a t s was also obtained. On the other hand, act. D had no significant effect on the
TABLE II E F F E C T OF ACTINOMYCIN D PRETREATMENT TOLERANCE TO AN LD100 DOSE OF THIOUREA
ON THE DEVELOPMENT OF
Pretreatment
One LD100 dose of T hioureaa
Number of rats died Number of rats used
% Mortality
None Sham-operated control Act.D (50 pg/kg i.t.) Act.D (50 ttg/kg i.t.)
+ + + b
10/10 6/7 1/7 0/7
100 85.5 14.5 0.0
a 10 mg/kg administered i.p. 7 days after pretreatment. b Challenged with saline i.p. 7 days after pretreatment.
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TABLE III PULMONARY EDEMA IN RATS OF VARYING AGES INDUCED BY THE INTRATRACHEAL ADMINISTRATION OF ACTINOMYCIN D (50 pg/kg)
Age in days
Lung wt - X 100 (Mean + SE) Body wt Control
23 47 73 98
0.726 -+ 0.039 0.564 + 0.041 0.378 -+ 0.007 0.351-+0.01
Act.D
(5) a (5) (5) (5)
0.807 -+ 0.021 b 0.742 -+ 0.043 c 0.749 -+ 0.053 d 0.639+0.033 d
% of control (5) (6) (4) (4)
111.0 131.5 197.5 181.5
a The values in parentheses are the number of animals used. b p value between control and act.D not significant. c p value between control and act.D significant at the 0.05 level. d p value between control and act.D significant at the 0.001 level.
a c t i v i t y o f t h e s e e n z y m e s i n liver, l u n g , s e r u m a n d u r i n e ( T a b l e V). S m e a r s m a d e f r o m t h e 1 2 0 0 0 X g s e d i m e n t s u s p e n s i o n o f t h e l u n g lavage f l u i d , a f t e r m o d i f i e d W r i g h t - G i e m s a s t a i n , r e v e a l e d m a s s i v e i n f i l t r a t i o n o f large v a c u o l a t e d m a c r o p h a g e s , n e u t r o p h i l s a n d m a s t cells i n act. D - t r e a t e d r a t s . N o a t t e m p t was m a d e , h o w e v e r , t o c o m p a r e q u a n t i t a t i v e l y t h e d i f f e r e n t i a l cell c o u n t s i n t h e l u n g lavage f l u i d b e t w e e n t h e d r u g - t r e a t e d a n d t h e c o n t r o l rats.
TABLE IV EFFECT OF i.t. ADMINISTRATION OF PHOSPHATE BUFFER OR ACT.D ON ACID AND ALKALINE PHOSPHATASE ACTIVITY AND PROTEIN CONTENT OF THE LUNG LAVAGE FLUID
Treatment
Control (PO4 buffer) Act.D (50 pg/kg) p value between control and Act.D
Enzyme activity expressed as pmoles of beta-naphthol released/h/lung lavage (Mean + SE)
Protein content expressed in mg/lung lavage (Mean -+ SE)
Acid phosphatase
Alkaline phosphatase
0.76 + 0.08 (10) a 1.28+0.21(9)
0.53 +- 0.07 (5) 1.63+0.20(5)
2.47 -+ 0.30 (5) 6.06-+0.65(5)
~0.05
~0.01
(0.01
a The values in parentheses are the number of animals used.
59
O BUFFER
O R ACT.
D ON ACID
AND
60]
Act.D (50 p g / k g )
67.3 -+ 24.9
108.2-+ 34.9 177.7 ± 23.7
231.9-+35 169.2 ± 24.7
178.2±25
Alkaline
a N o n e of the values b e t w e e n c o n t r o l a n d A c t . D are significant at t h e 0.05 level.
± 22.5
572.6-+ 50.1
Acid
Acid
Alkaline
Lung B-N/h/g
Liver B - N / h / g
+0.1
1.26 ± 0.2
1.4
Acid
6.8 + 0.6
7.7 ± 0 . 5
Alkaline
Serum B-N/h/ml
PHOSPHATASE
63.1 ± 8.4
49.3+7.8
Acid
10.4±1.9
7.9±1.1
Alkaline
Urine B-N/h/total urine v o l u m e (24 h)
ALKALINE
E n z y m e activity e x p r e s s e d in p m o l e s of b e t a - n a p h t h o l (B-N) released a ( M e a n ± SE o f 4 a n i m a l s )
Control (PO4 buffer)
Treatment
E F F E C T S OF i.t. A D M I N I S T R A T I O N O F P H O S P H A T E A C T I V I T Y OF L I V E R , L U N G , S E R U M A N D U R I N E
TABLE V
DISCUSSION I.t. administration of act. D, cycloheximide, puromycin and ethionine was found to induce the development of a significant degree of pulmonary edema. These c o m p o u n d s have in c o m m o n the ability to inhibit protein synthesis. Protein has been demonstrated to be an integral part of surfaceactive material and is synthesized within the lung [ 1 5 - - 1 7 ] . Scarpelli [18] suggested that a deficiency in the synthesis of the surfactant protein moiety could produce alveolar instability with increased fluid transudation from pulmonary capillaries resulting in pulmonary edema. Inhibition of lung protein synthesis may, therefore, be involved in the mediation of edematogenic effects produced by these compounds. Alternative mechanisms may be involved, however, since inhibition of protein synthesis is n o t the only effect produced b y these agents. Act. D, for example, inhibits phospholipid synthesis [19] as well as glycolysis and respiration [20]. The antibiotic can also produce development of acute lung inflammation secondary to an increase in histamine c o n t e n t [21]. Any of these effects may be involved in the formation of pulmonary edema. Histopathological studies of lungs performed 48 h after act. D instillation revealed extensive extravasation of erythrocytes from lung capillaries into lung interstitial areas, alveoli and airways. This suggests that act. D may have damaged not only the endothelial lining of the capillaries b u t the alveolar epithelial barrier as well. This, presumably, would have facilitated the development of massive confluent pulmonary edema as seen in the photomicrograph (Fig. 2). One of the properties of edematogenic c o m p o u n d s is their ability to produce tolerance to the edema-producing effects of other agents [ 2 2 , 2 3 ] . In this regard act. D is no exception. Tolerance provided by act. D pretreatment appears to be nonspecific inasmuch as act. D and thiourea possess varied structure and produce different effects. A property c o m m o n to both drugs, however, is their ability to induce pulmonary edema. It is possible therefore that the tolerance-producing ability of act. D might have a basic toxicological mechanism which is responsible for this action. The production of acute lung inflammation by act. D [21] is consistent with the observation made by Henschler and Laux [24] that a certain degree of inflammatory reaction of pulmonary tissues is a prerequisite for inducing tolerance to edematogenic agents. The innate resistance of younger animals to the edematogenic effect of various agents has been reported previously [25,26]. It was interesting to observe that the ability of act. D to produce pulmonary edema in rats of varying ages appears to parallel that of thiourea and NO2. The exact mechanism which determines the insensitivity of pulmonary tissue in y o u n g rats to edematogenic insult is n o t known, although several possibilities have been suggested [ 2 6 , 2 7 ] . The effects of i.t. instillation of act. D appear to be localized in pulmonary tissue since the c o m p o u n d produced edema of the lungs only and failed
61
to a f f e c t o t h e r organs such as liver and k i d n e y . In a d d i t i o n , t h e l e u k o c y t e infiltration and increased acid and alkaline p h o s p h a t a s e activities were obtained in t h e lung lavage fluid o f act. D - t r e a t e d rats. N o e f f e c t o n t h e activity o f these e n z y m e s was n o t e d in plasma, urine a n d liver a n d lung tissues. This w o u l d suggest t h a t act. D s o m e h o w increases the p e r m e a b i l i t y o f b o t h b r o n chial a n d p u l m o n a r y m i c r o c i r c u l a t i o n , allowing t h e m o b i l i z a t i o n o f leukoc y t e s into t h e lungs. L y s o s o m a l e n z y m e s p r e s e n t in PMN and m a c r o p h a g e s m a y be partially responsible f o r the p a t h o g e n e i t y o f act. D - i n d u c e d lung damage. This view is s u p p o r t e d b y o u r earlier findings [ 2 8 , 2 9 ] t h a t act. D p r o d u c e s p e r i t o n e a l e f f u s i o n w h e n instilled in t h a t l o c a t i o n . In a d d i t i o n , the e f f u s i o n was s h o w n to c o n t a i n i n f l a m m a t o r y cells (PMN, m a c r o p h a g e s , mast cells a n d eosinophils) as well as free l y s o s o m a l e n z y m e and histamine. ACKNOWLEDGEMENT This investigation was s u p p o r t e d in part b y a grant f r o m the California Research and Medical E d u c a t i o n F u n d o f the T u b e r c u l o s i s and R e s p i r a t o r y Disease A s s o c i a t i o n o f California and F a c u l t y Research G r a n t f r o m the University o f California at Davis. REFERENCES 1 D. Massaro, H. Weiss and M.R. Simon, Am. Resp. Dis., 101 (1970) 198. 2 D. Massaro, M.R. Simon and H. Steinkamp, J. Appl. Physiol., 30 (1971) 1. 3J.A. Clements and D.R. Tierney, W.O. Fenn and H. Rahn (Eds.),in Handbook of Physiology: A Critical, Comprehensive Presentation of Physiological Knowledge and Concepts, Vol. 2, Section 3, Respiration, American Physiological Society, Washington, D.C., 1965, p. 1565. 4 J.B. McClenahan, Ann. Intern. Med., 66 (1967) 1055. 5 P. Gross, M. Babyak, E. Tolker and M. Kaschak, J. Occup. Med., 6 (1964) 481. 6 R. Pushpakom, J.C. Hogg, A.J. Woolcock, A.E. Angus, P.T. Macklem and W.M. Thurlbeck, Am. Rev. Resp. Dis., 102 (1970) 778. 7 S.N. Giri, M.A. Hollinger, D.L. Dungworth' and C.E. Cross, Federation Proc., 32 (1973) 813. 8 S.N. Girl, M.A. Hollinger, C.E. Cross and D.L. Dungworth, Toxicology, 2 (1974) 211. 9 F.C. Courtice, Aust. N.Z.J. Surg., 22 (1953) 177. 10 J.D. Brian and N.R. Frank, J. Appl. Physiol., 25 (1968) 63. 11 )aM. Seligmann, H.H. Chauncey, M.M. Nichlas, L.H. Manheimer and H.A. Ravin, J. Biol. Chem., 190 (1951) 7. 12 O.H. Lowry, N.J. Rosebrough, A.L. Farr and R.J. Randall, J. Biol. Chem., 193 (1951) 265. 13 S.R. Cronin and S.N. Giri, Proc. Soc. Exptl. Biol. Med., 146 (1974) 120. 14 M. Werner, J. Chromatog., 40 (1969) 254. 15 R.J. King and J.A. Clements, Am. J. Physiol., 223 (1972) 707. 16 R.J. King and J.A. Clements, Am. J. Physiol., 223 (1972) 715. 17 K.J. Dickie, G.D. Massaro, V. Marshall and D. Massaro, J. Appl. Physiol., 34 (1973) 606. 18 E.M. Scarpelli, The Surfactant System of the Lung, Lea and Febiger, Philadelphia, 1968. 19 I. Pastan and R.M. Friedman, Science, 160 (1968) 316. 20J. Laszlo, D.S. Miller, K.S. McCarty and P. Hochstein, Science, 151 (1966) 1008.
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21 M.A. Reilly and R.W. Schayer, Brit. J. Pharmacol., 37 (1969) 489. 22 tCK. Carroll and R.L. Noble, J. Pharmacology, 97 (1949) 478. 23 H.E. Stokinger and W.D. Wagner, Arch. Industr. Health, 14 (1956) 158. 24 D. Henschler and W. Laux, Arch. Exptl. Pathol. Pharmacol., 239 (1960) 433. 25 S.H. Diekes and C.P. Richter, Proc. Soc. Exptl. Biol. Med., 62 (1946) 22. 26 A.R. Gregory and C.P. Hine, Proc. Soc. Exptl. Biol. Med., 128 (1968) 693. 27 E.J. Fairchild and S.L. Graham, J. Pharmacol. Exptl. Therap., 139 (1963) 177. 28 S.N. Giri and A. Burkhalter, Arch. Environ. Health, 18 (1969) 730. 29 S.N. Giri, L.R. Kartt, B. Joshi and S.R. Cronin, Federation Proc., 33 (1974) 558.
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EFFECTS OF I N T R A T R A C H E A L INSTILLATION OF DACTINOMYCIN ON PULMONARY EDEMA AND PHOSPHATASE ACTIVITY OF THE LUNG LAVAGE FLUID IN RATS
SHRI N. GIRI, MANNFRED A. HOLLINGER and L. RICHARD KARTT Department of Physiological Sciences, School of Veterinary Medicine, and Department of Pharmacology, School of Medicine, University of California, Davis, Calif. 95616 (U.S.A.)
(Received August 8th, 1974)
SUMMARY Intratracheal (i.t.) administration of protein synthesis inhibitors produced pulmonary edema. Of those inhibitors studied, dactinomycin (act. D) was the most potent. Severity of lung damage due to act. D was dose- and almost age-related. Maximal intensity of pulmonary edema was reached on the 3rd day following administration and remained constant for 14 days. Histopathological studies revealed confluent edema of the entire lung. Pretreatm e n t with act. D induced tolerance to an LD100 edematogenic dose of thiourea. The effects of i.t. instillation of act. D appear to be localized in the pulmonary tissue. Lung lavage fluid collected from drug-treated rats had higher acid and alkaline phosphatase activities, higher protein c o n t e n t and more leukocyte infiltration than that of control.
INTRODUCTION The capacity of lung to synthesize protein actively in vivo as well as in vitro has been demonstrated by Massaro et al. [1,2]. Some protein synthesized by lung plays an important physiological role in the action of lung surfactant in maintaining its form and function [3,4]. As in most tissues, it is probable that the a m o u n t of protein in the lung at any given time is controlled in part by the protein-synthesizing and protein-degrading ability of the lung. An alteration in either of these processes induced by disease,
Abbreviation: act. D, dactinomycin; i.t., intratracheal; PMN, polymorphonuclear leukocytes~
53
chemicals or other means might therefore be expected to cause observable changes in lung structure and function. For example, enzymatic attack on structural proteins in the lung by papain administered i.t. is frequently employed in animal models to study the pathogenesis of emphysema [5,6]. A similar type of animal model utilizing i.t. administration of compounds which inhibit lung protein synthesis was recently reported in a preliminary study by Giri et al [7,8]. It was considered warranted to extend this previous study in order to understand the characteristic features and the mechanism of lung damage induced by the i.t. administration of act. D. METHODS AND MATERIALS Male Sprague--Dawley rats weighing 350 to 450 g and free of pulmonary pathogens were used t h r o u g h o u t the experiment unless the weight specified otherwise. They were housed two per cage in a room designed as the holding area. Rats were fed Purina rat chow and water ad libitum. The following four compounds which inhibit protein synthesis were used: ethionine, cycloheximide, act. D, and puromycin dihydrochloride. The first three compounds were purchased from the Sigma Chemical Company and the last from the National Biological Company. Drugs were dissolved in phosphate buffer (pH 7.4, 0.05 M) and instilled i.t. in a volume of 1 ml/kg body weight. This volume was kept constant for each drug regardless of the dosage injected. The procedure for i.t. administration has been described previously [8]. Briefly, rats were lightly anesthetized with sodium pentobarbital (25 to 30 mg/kg, i.p.), tracheae were exposed, and the drug was instilled via a 27-gauge needle. Rats were immediately held in an upright position to let the drug solution gravitate to the distal regions of the lung. Rats in the control group were anesthetized in the same way and received an equivalent volume of phosphate buffer by the same route. The incisions were sutured and an antiseptic was applied. Rats were allowed to recover and the extent of lung damage was examined under varying experimental conditions. At various times following drug administration rats were killed with sodium pentobarbital and the lungs immediately separated from the heart and extraneous tissues, blotted gently and weighed. The degree of pulmonary edema was quantitated as lung weight expressed as percent body weight [9]. For histopathological studies, the lungs and heart were removed en bloc, and fixed in Zenker-formalin solution. Lung lavage in a group of act. D-treated rats was carried out according to the m e t h o d of Brian et al. [10]. The lavage liquid resulting from repeated washings of lung was pooled and centrifuged at 12 000 × g for 10 min in a refrigerated centrifuge (Sorval RC2-B) at 4 °. The supernatant was aspirated and discarded. The packed cells from each rat lung lavage were suspended in isotonic saline to a total volume of 1.5 ml. The suspension was centrifuged at 300 rev./min for 10 min to sediment the heavier particles. The supernatant was carefully transferred into a tissue homogenizer (Tri-R) and homogenized in an equal volume of cold distilled deionized water, using 10 up-and-down
54
strokes. The h o m o g e n a t e was then assayed for acid and alkaline phosphatase activity [ 1 1 ] . The pr ot e i n c o n t e n t of t he h o m o g e n a t e was d e t e r m i n e d according to the m e t h o d of L ow r y et al. [ 1 2 ] . Plasma, liver and lung tissues were collected as follows: rats were etherized and b l o o d was collected in centrifuge tubes directly from the severed inferior vena cava. After being allowed to coagulate, the blood was spun at 3000 rev./min for 10 min, the serum being aspirated and diluted 1 : 20 for the d e t e r m i n a t i o n of acid and alkaline phosphatase activity. A 2--3-g lobe of the liver was removed and weighed in a tared beaker containing cold isotonic saline. The tissue was washed in saline a second time, b l o t t e d dry, cut up into smaller pieces and then hom ogeni zed in 0.25 M sucrose using 10 up-and-down strokes. The h o m o g e n a t e was made up to a final c o n c e n t r a t i o n of 10%. This was later diluted 1 : 100 with distilled deionized water just prior to e n z y m e assay. The lung and heart were removed en bloc, placed in a watch glass containing cold isotonic saline and perfused with saline through the trachea, in order to remove cellular c ont e nt s f r om air spaces, and through the right ventricle to wash o u t cellular contents f r o m the p u l m o n a r y capillary bed [ 1 3 ] . Lung lobes were cut away, c h o p p e d and homogenized (10 strokes) in 0.25 M sucrose and made up to 15 ml. The h o m o g e n a t e was filtered through two layers o f cold moist cheese cloth and the resulting filtrate was diluted with distilled deionized water (1 to 10) prior to acid and alkaline phosphatase assay. Urine f r o m rats housed individually in stainless steel metabolic cages was collected over a 24-h period in ice-cold test tubes. The total volume of urine voided was measured and centrifuged at 10 000 rev./min for 20 min in a refrigerated centrifuge. Urine was purified by gel filtration [14] at 4 ° prior to e n z y m e assay. The data expressed as the mean +- standard error. The S t u d e n t " t " test was e m p l o y e d to de t e r m i ne the degree of significance between the means. RESULTS The ef f ect of i.t. administration of protein synthesis inhibitors on the in d u ctio n o f p u l m o n a r y edema is summarized in Table I. When examined 48 h after drug administration, it was f ound that each c o m p o u n d p r o d u c e d a significant degree of p u l m o n a r y edema. Of the f o u r inhibitors studied, act. D at doses used was the most and ethionine the least pot ent . Purom yci n and c y c l o h e x i m i d e appeared to possess an intermediate p o t e n c y . As act. D produced the most severe lung damage, it alone was t herefore subsequently studied. The e x t e n t of lung damage induced by act. D was found to correlate with th e a m o u n t of drug administered (Fig. I). A small a m o u n t of pleural effusion varying in color from straw to bloodtinged was noticed in rats treated with 50 or 100 pg/kg act. D. Grossly, lungs o f these rats cont ai ned diffuse hemorrhagic spots involving one or m ore lobes. A f r o t h y m uc ous discharge was seen oozing from t he incised tracheae
55
TABLE I P U L M O N A R Y E D E M A I N D U C E D BY I N T R A T R A C H E A L A D M I N I S T R A T I O N O F PROTEIN SYNTHESIS INHIBITORS
Treatment
Lung wt
- - X
Body wt Mean ± SE
Sham-operated control A c t . D 50 p g / k g C y c l o h e x i m i d e 50 p g / k g C y c l o h e x i m i d e 500 ~g/kg P u r o m y c i n 50 p g / k g E t h i o n i n e 25 m g / k g
0.378 0.749 0.401 0.418 0.434 0.457
100
-+ 0.007b(5)a ± 0.053 (5) -+ 0.017 (6) + 0.013 c (5) + 0.009 d (6) + 0.015 d (5)
a The values in p a r e n t h e s e s are the n u m b e r o f animals used. b p value b e t w e e n c o n t r o l and a c t . D is significant at the 0.001 level. c p value b e t w e e n c o n t r o l and c y c l o h e x i m i d e is significant at the 0.05 level. d p values b e t w e e n c o n t r o l and p u r o m y c m and c o n t r o l and e t h i o n i n e are significant at the 0.01 level.
of these rats. Fig. 2 is a typical photomicrograph of histopathological changes in the lungs of rats treated with act. D (50 pg/kg i.t.). In contrast to the normal-appearing lungs in the buffer-inStilled control rats, the lungs of the drug-treated rats showed massive confluent edema and e r y t h r o c y t e aggregates in the alveolar, peribronchial and perivascular interstitial spaces of the lungs.
15 b0
, "
J
I0
X u~ l.I
d
o,L i
0
12 5
25 DOSE
50 OF
ACT
I0© ~g/~q
D
BW
IT
Fig. 1. E f f e c t s o f d i f f e r e n t doses o f act. D on p u l m o n a r y e d e m a . Rats were sacrificed and p u l m o n a r y e d e m a was d e t e r m i n e d 48 h a f t e r drug a d m i n i s t r a t i o n . Each p o i n t r e p r e s e n t s the m e a n o f six animals with s t a n d a r d e r r o r o f mean. Zero drug, c o n t r o l .
56
Fig. 2. Photomicrograph of a rat lung which received one injection of act. D (50 pg/kg) 48 h before killing. Note the generalized confluent edema with erythrocytes in the alveolar space (Zenker-formalin fixitive; H.E. stain; x 200). The time course for the onset and progression of p u l m o n a r y edem a following a single dose (50 pg/kg) of act. D is illustrated in Fig. 3. An increase in lung weight was f ound one day after drug administration, and the e x t e n t of lung damage progressed with time through the third day. From that poi nt in time no change in the severity of p u l m o n a r y edem a was observed for the entire duration (14 days) of the experiment. The ef f ect of act. D p r e t r e a t m e n t on acute lethal effect of an LD10o dose (10 mg/kg) of t hi our e a i.p. is indicated in Table II. P r e t r e a t m e n t with act. D was f o u n d to afford significant p r o t e c t i o n against acute t hi ourea lethality. The mo r talit y d r o p p e d f r o m 6 o u t of 7 in the cont rol group to 1 o u t o f 7 in the act. D-pretreated rats. The e f f e c t o f act. D (50 pg/kg i.t.) on the i nduct i on of p u l m o n a r y edem a in rats o f varying ages is indicated in Table III. These results d e m o n s t r a t e that the severity of lung damage in response to act. D is related t o the animal's
57
LL 0
qO
O8 ~0 a UJ ¢9 ¢0
06
~
o4
Q. X W
O2
Z _1
TIME AFTER ACT. D. INJECTION, 50.ug/kg IT (IN DAYS)
Fig. 3. Effects of a single dose of act. D (50 pg/kg) on the development of pulmonary edema for 14 days following drug administration. Each point represents the mean of 6 animals with standard error of mean. C, control.
age. T h u s , 2 3 - d a y - o l d r a t s w e r e l e a s t s u s c e p t i b l e w h i l e 7 3 - t o 9 8 - d a y - o l d r a t s w e r e m o s t s u s c e p t i b l e t o t h e e d e m a t o g e n i c e f f e c t o f a c t . D. T h e e f f e c t o f i.t. a d m i n i s t r a t i o n o f a c t . D ( 5 0 p g / k g ) o n a c i d a n d a l k a l i n e phosphatase activity and protein content of lung lavage fluid are summarized in T a b l e I V . T h e l u n g l a v a g e c o l l e c t e d f r o m d r u g - t r e a t e d r a t s c o n t a i n e d a higher amount of acid and alkaline phosphastase activity than that of cont r o l s . I n a d d i t i o n , a g r e a t e r a m o u n t o f p r o t e i n in t h e l u n g l a v a g e o f t h e s e r a t s was also obtained. On the other hand, act. D had no significant effect on the
TABLE II E F F E C T OF ACTINOMYCIN D PRETREATMENT TOLERANCE TO AN LD100 DOSE OF THIOUREA
ON THE DEVELOPMENT OF
Pretreatment
One LD100 dose of T hioureaa
Number of rats died Number of rats used
% Mortality
None Sham-operated control Act.D (50 pg/kg i.t.) Act.D (50 ttg/kg i.t.)
+ + + b
10/10 6/7 1/7 0/7
100 85.5 14.5 0.0
a 10 mg/kg administered i.p. 7 days after pretreatment. b Challenged with saline i.p. 7 days after pretreatment.
58
TABLE III PULMONARY EDEMA IN RATS OF VARYING AGES INDUCED BY THE INTRATRACHEAL ADMINISTRATION OF ACTINOMYCIN D (50 pg/kg)
Age in days
Lung wt - X 100 (Mean + SE) Body wt Control
23 47 73 98
0.726 -+ 0.039 0.564 + 0.041 0.378 -+ 0.007 0.351-+0.01
Act.D
(5) a (5) (5) (5)
0.807 -+ 0.021 b 0.742 -+ 0.043 c 0.749 -+ 0.053 d 0.639+0.033 d
% of control (5) (6) (4) (4)
111.0 131.5 197.5 181.5
a The values in parentheses are the number of animals used. b p value between control and act.D not significant. c p value between control and act.D significant at the 0.05 level. d p value between control and act.D significant at the 0.001 level.
a c t i v i t y o f t h e s e e n z y m e s i n liver, l u n g , s e r u m a n d u r i n e ( T a b l e V). S m e a r s m a d e f r o m t h e 1 2 0 0 0 X g s e d i m e n t s u s p e n s i o n o f t h e l u n g lavage f l u i d , a f t e r m o d i f i e d W r i g h t - G i e m s a s t a i n , r e v e a l e d m a s s i v e i n f i l t r a t i o n o f large v a c u o l a t e d m a c r o p h a g e s , n e u t r o p h i l s a n d m a s t cells i n act. D - t r e a t e d r a t s . N o a t t e m p t was m a d e , h o w e v e r , t o c o m p a r e q u a n t i t a t i v e l y t h e d i f f e r e n t i a l cell c o u n t s i n t h e l u n g lavage f l u i d b e t w e e n t h e d r u g - t r e a t e d a n d t h e c o n t r o l rats.
TABLE IV EFFECT OF i.t. ADMINISTRATION OF PHOSPHATE BUFFER OR ACT.D ON ACID AND ALKALINE PHOSPHATASE ACTIVITY AND PROTEIN CONTENT OF THE LUNG LAVAGE FLUID
Treatment
Control (PO4 buffer) Act.D (50 pg/kg) p value between control and Act.D
Enzyme activity expressed as pmoles of beta-naphthol released/h/lung lavage (Mean + SE)
Protein content expressed in mg/lung lavage (Mean -+ SE)
Acid phosphatase
Alkaline phosphatase
0.76 + 0.08 (10) a 1.28+0.21(9)
0.53 +- 0.07 (5) 1.63+0.20(5)
2.47 -+ 0.30 (5) 6.06-+0.65(5)
~0.05
~0.01
(0.01
a The values in parentheses are the number of animals used.
59
O BUFFER
O R ACT.
D ON ACID
AND
60]
Act.D (50 p g / k g )
67.3 -+ 24.9
108.2-+ 34.9 177.7 ± 23.7
231.9-+35 169.2 ± 24.7
178.2±25
Alkaline
a N o n e of the values b e t w e e n c o n t r o l a n d A c t . D are significant at t h e 0.05 level.
± 22.5
572.6-+ 50.1
Acid
Acid
Alkaline
Lung B-N/h/g
Liver B - N / h / g
+0.1
1.26 ± 0.2
1.4
Acid
6.8 + 0.6
7.7 ± 0 . 5
Alkaline
Serum B-N/h/ml
PHOSPHATASE
63.1 ± 8.4
49.3+7.8
Acid
10.4±1.9
7.9±1.1
Alkaline
Urine B-N/h/total urine v o l u m e (24 h)
ALKALINE
E n z y m e activity e x p r e s s e d in p m o l e s of b e t a - n a p h t h o l (B-N) released a ( M e a n ± SE o f 4 a n i m a l s )
Control (PO4 buffer)
Treatment
E F F E C T S OF i.t. A D M I N I S T R A T I O N O F P H O S P H A T E A C T I V I T Y OF L I V E R , L U N G , S E R U M A N D U R I N E
TABLE V
DISCUSSION I.t. administration of act. D, cycloheximide, puromycin and ethionine was found to induce the development of a significant degree of pulmonary edema. These c o m p o u n d s have in c o m m o n the ability to inhibit protein synthesis. Protein has been demonstrated to be an integral part of surfaceactive material and is synthesized within the lung [ 1 5 - - 1 7 ] . Scarpelli [18] suggested that a deficiency in the synthesis of the surfactant protein moiety could produce alveolar instability with increased fluid transudation from pulmonary capillaries resulting in pulmonary edema. Inhibition of lung protein synthesis may, therefore, be involved in the mediation of edematogenic effects produced by these compounds. Alternative mechanisms may be involved, however, since inhibition of protein synthesis is n o t the only effect produced b y these agents. Act. D, for example, inhibits phospholipid synthesis [19] as well as glycolysis and respiration [20]. The antibiotic can also produce development of acute lung inflammation secondary to an increase in histamine c o n t e n t [21]. Any of these effects may be involved in the formation of pulmonary edema. Histopathological studies of lungs performed 48 h after act. D instillation revealed extensive extravasation of erythrocytes from lung capillaries into lung interstitial areas, alveoli and airways. This suggests that act. D may have damaged not only the endothelial lining of the capillaries b u t the alveolar epithelial barrier as well. This, presumably, would have facilitated the development of massive confluent pulmonary edema as seen in the photomicrograph (Fig. 2). One of the properties of edematogenic c o m p o u n d s is their ability to produce tolerance to the edema-producing effects of other agents [ 2 2 , 2 3 ] . In this regard act. D is no exception. Tolerance provided by act. D pretreatment appears to be nonspecific inasmuch as act. D and thiourea possess varied structure and produce different effects. A property c o m m o n to both drugs, however, is their ability to induce pulmonary edema. It is possible therefore that the tolerance-producing ability of act. D might have a basic toxicological mechanism which is responsible for this action. The production of acute lung inflammation by act. D [21] is consistent with the observation made by Henschler and Laux [24] that a certain degree of inflammatory reaction of pulmonary tissues is a prerequisite for inducing tolerance to edematogenic agents. The innate resistance of younger animals to the edematogenic effect of various agents has been reported previously [25,26]. It was interesting to observe that the ability of act. D to produce pulmonary edema in rats of varying ages appears to parallel that of thiourea and NO2. The exact mechanism which determines the insensitivity of pulmonary tissue in y o u n g rats to edematogenic insult is n o t known, although several possibilities have been suggested [ 2 6 , 2 7 ] . The effects of i.t. instillation of act. D appear to be localized in pulmonary tissue since the c o m p o u n d produced edema of the lungs only and failed
61
to a f f e c t o t h e r organs such as liver and k i d n e y . In a d d i t i o n , t h e l e u k o c y t e infiltration and increased acid and alkaline p h o s p h a t a s e activities were obtained in t h e lung lavage fluid o f act. D - t r e a t e d rats. N o e f f e c t o n t h e activity o f these e n z y m e s was n o t e d in plasma, urine a n d liver a n d lung tissues. This w o u l d suggest t h a t act. D s o m e h o w increases the p e r m e a b i l i t y o f b o t h b r o n chial a n d p u l m o n a r y m i c r o c i r c u l a t i o n , allowing t h e m o b i l i z a t i o n o f leukoc y t e s into t h e lungs. L y s o s o m a l e n z y m e s p r e s e n t in PMN and m a c r o p h a g e s m a y be partially responsible f o r the p a t h o g e n e i t y o f act. D - i n d u c e d lung damage. This view is s u p p o r t e d b y o u r earlier findings [ 2 8 , 2 9 ] t h a t act. D p r o d u c e s p e r i t o n e a l e f f u s i o n w h e n instilled in t h a t l o c a t i o n . In a d d i t i o n , the e f f u s i o n was s h o w n to c o n t a i n i n f l a m m a t o r y cells (PMN, m a c r o p h a g e s , mast cells a n d eosinophils) as well as free l y s o s o m a l e n z y m e and histamine. ACKNOWLEDGEMENT This investigation was s u p p o r t e d in part b y a grant f r o m the California Research and Medical E d u c a t i o n F u n d o f the T u b e r c u l o s i s and R e s p i r a t o r y Disease A s s o c i a t i o n o f California and F a c u l t y Research G r a n t f r o m the University o f California at Davis. REFERENCES 1 D. Massaro, H. Weiss and M.R. Simon, Am. Resp. Dis., 101 (1970) 198. 2 D. Massaro, M.R. Simon and H. Steinkamp, J. Appl. Physiol., 30 (1971) 1. 3J.A. Clements and D.R. Tierney, W.O. Fenn and H. Rahn (Eds.),in Handbook of Physiology: A Critical, Comprehensive Presentation of Physiological Knowledge and Concepts, Vol. 2, Section 3, Respiration, American Physiological Society, Washington, D.C., 1965, p. 1565. 4 J.B. McClenahan, Ann. Intern. Med., 66 (1967) 1055. 5 P. Gross, M. Babyak, E. Tolker and M. Kaschak, J. Occup. Med., 6 (1964) 481. 6 R. Pushpakom, J.C. Hogg, A.J. Woolcock, A.E. Angus, P.T. Macklem and W.M. Thurlbeck, Am. Rev. Resp. Dis., 102 (1970) 778. 7 S.N. Giri, M.A. Hollinger, D.L. Dungworth' and C.E. Cross, Federation Proc., 32 (1973) 813. 8 S.N. Girl, M.A. Hollinger, C.E. Cross and D.L. Dungworth, Toxicology, 2 (1974) 211. 9 F.C. Courtice, Aust. N.Z.J. Surg., 22 (1953) 177. 10 J.D. Brian and N.R. Frank, J. Appl. Physiol., 25 (1968) 63. 11 )aM. Seligmann, H.H. Chauncey, M.M. Nichlas, L.H. Manheimer and H.A. Ravin, J. Biol. Chem., 190 (1951) 7. 12 O.H. Lowry, N.J. Rosebrough, A.L. Farr and R.J. Randall, J. Biol. Chem., 193 (1951) 265. 13 S.R. Cronin and S.N. Giri, Proc. Soc. Exptl. Biol. Med., 146 (1974) 120. 14 M. Werner, J. Chromatog., 40 (1969) 254. 15 R.J. King and J.A. Clements, Am. J. Physiol., 223 (1972) 707. 16 R.J. King and J.A. Clements, Am. J. Physiol., 223 (1972) 715. 17 K.J. Dickie, G.D. Massaro, V. Marshall and D. Massaro, J. Appl. Physiol., 34 (1973) 606. 18 E.M. Scarpelli, The Surfactant System of the Lung, Lea and Febiger, Philadelphia, 1968. 19 I. Pastan and R.M. Friedman, Science, 160 (1968) 316. 20J. Laszlo, D.S. Miller, K.S. McCarty and P. Hochstein, Science, 151 (1966) 1008.
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21 M.A. Reilly and R.W. Schayer, Brit. J. Pharmacol., 37 (1969) 489. 22 tCK. Carroll and R.L. Noble, J. Pharmacology, 97 (1949) 478. 23 H.E. Stokinger and W.D. Wagner, Arch. Industr. Health, 14 (1956) 158. 24 D. Henschler and W. Laux, Arch. Exptl. Pathol. Pharmacol., 239 (1960) 433. 25 S.H. Diekes and C.P. Richter, Proc. Soc. Exptl. Biol. Med., 62 (1946) 22. 26 A.R. Gregory and C.P. Hine, Proc. Soc. Exptl. Biol. Med., 128 (1968) 693. 27 E.J. Fairchild and S.L. Graham, J. Pharmacol. Exptl. Therap., 139 (1963) 177. 28 S.N. Giri and A. Burkhalter, Arch. Environ. Health, 18 (1969) 730. 29 S.N. Giri, L.R. Kartt, B. Joshi and S.R. Cronin, Federation Proc., 33 (1974) 558.
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