Americat Journal of Pathology, Vol. 138, No. 1, January 1991 CopJyright © American Association of Pathologists

Effects of Smoke Inhalation on Surfactant Phospholipids and Phospholipase A2 Activity in the Mouse Lung M. Oulton,* H. K. Moores,II J. E. Scott, D. T. Janigan,t and R. Hajelat From the Departments of Physiology and Biophysics* and Pathology,t Dalbousie University, Halifax, Nova Scotia; the Department of Oral Biology,J University of Manitoba, Winnipeg, Manitoba, Canada; and the Webb-Waring Lung Institute, || University of Colorado, Denver, Colorado

The effects of smoke inhalation on the pulmonary surfactant system were examined in mice exposed for 30 minutes to smoke generatedfrom the burning of polyurethane foam. At 8 or 12 hours after exposure, surfactants were isolated separately from lung lavage (extracellular surfactant) and residual lung tissue (intracellular surfactant) for phospholipid analysis. Calcium-dependent phospholipase A2 (PLA2) was measured on a microsomal fraction prepared from the tissue homogenate. Smoke inhalation produced a twofold increase in extracellular surfactant total phospholipid. While there was no change in the total phospholipid or phosphatidylcholine (PC) content of the intracellular surfactant, smoke inhalation significantly decreased the disaturated species of PC (DSPC). The specific activity of PLA2 was reduced by more than 50% in both groups of exposed mice. Smoke inhalation appears to result in selective depletion of the DSPC of intracellular surfactant and PLA2 involved in its synthesis. This depletion may be compensated for by increased secretion or slower breakdown of the materialpresent in the extracellular compartment. (AmJPathol 1991, 138:195-202)

of the fire deaths in 1984 occurred in residential structures.4 In that country nearly all upholstered furniture manufactured in the mid 1980s contained flexible polyurethane foam and fires in upholstered furniture, usually initiated accidentally by cigarettes, are a major cause of residential fire deaths. As a result, fire victims frequently are exposed to the smoke generated by the burning of this material. Considering the complexity of foam-generated smoke,5 it is not surprising that little is known concerning its specific effects on the lung. Because there is some evidence that inhalation of smoke may, over a period of days, cause detrimental effects on lung parenchymal tissue,6 we have begun studies to examine some of these effects. Specifically we are studying the effects of smoke inhalation on the pulmonary surfactant system. There is considerable evidence to indicate that many noxious agents and toxic substances cause derangement of the pulmonary surfactant system by interruption of each or any of the processes of biosynthesis,9'10 secretion,1'112 or clearance of the used surfactant from the alveoli.'1314 However little is known about the effects of smoke inhalation on these dynamic processes.

Recently we developed methods for the quantitative and separate isolation of surfactant from alveolar lavage and the postlavaged lung tissue.15'16 In the present study we applied this methodology to study the effects of smoke inhalation on these two surfactant pools. We also examined the effects on the activity of phospholipase A2. This enzyme controls the rate-limiting step whereby the remodeling of 2-unsaturated phosphatidylcholine produces dipalmitoylphosphatidylcholine, the major constituent of the pulmonary surfactant.17

Methods Lung injury from fire smoke inhalation is a major prognostic factor in the survival of victims rescued from accidental fires.1 2 Residential fires comprise the largest single class of accidental fires in Canada, close to 50% of the annual total, and they were responsible for 70% of the nearly 500 fire deaths reported in 1988.3 In the United States, where there are about 5000 fire deaths each year, 80%

Random-bred, pathogen-free, Carworth Farms White (CFW; Swiss-Webster) male mice 8 to 9 weeks old were Supported by MRC and Nova Scotia Lung Association. Accepted for publication August 28, 1990. Address reprint requests to Dr. M. Oulton, Room 5G4, Tupper Medical Building, Dalhousie University, Halifax, Nova Scotia, B3H 4H7, Canada.

195

196

Oulton et al

AJPJanuary 1991, Vol. 138, No. 1

housed (five per cage) in a controlled environment with food and water available ad libitum. The model used for studying smoke toxicity incorporated the guidelines of the National Bureau of Standards1819 and the National Research Council4 and was described elsewhere.' Briefly, the fuel was flexible polyurethane foam, synthesized from toluene diisocyanate and free of fire retardants and pigments. It was decomposed thermally, without flaming, in a tube furnace, preheated, and maintained at 400 ± 80C. Air was made to flow through the furnace and to carry the diluted smoke into a 20-I volume exposure chamber, where the smoke was dispersed uniformly by an internal fan and allowed to escape. Nine hundred milligrams of foam are decomposed, based on the ratio of 45 mg/I of chamber volume. The exposure duration was 30 minutes. Usually 12 or 13 mice and, rarely, up to 16 mice were exposed, and in all instances the body mass of the mice never exceeded 5% of the chamber volume; usually it was less than 3%. Throughout the exposures, the chamber temperature never exceeded 26°C and the chamber oxygen levels did not decrease to less than 18.5%, ie, there was neither heat stress nor significant hypoxic atmospheres. Although not consistently monitored, chamber humidity sometimes increased to more than 50%, depending on the ambient humidity. Following the exposure, mice were returned to their cages for either 8 or 12 hours, at which time they were anesthetized by an intraperitoneal injection of sodium pentobarbital for lung lavage with isotonic saline in 4 X 1.0 ml aliquots, as described previously.9 The lavage washings from two to five mice were pooled, centrifuged for 10 minutes at 700g to remove alveolar cells and debris, and the resultant cell-free supernatants, which are considered to be representative of the alveolar surfactant pool, were stored at -70°C for biochemical analysis. Following the lavage procedure, the lungs were perfused with 0.26 mol/l (molar) NaCI, 5 mmol/ (millimolar) ethylenedinitrilo tetraacetic acid (EDTA) via the right ventricle until the perfusate from a hole cut in the left ventricle was clear. The lungs were removed and pooled (as indicated above for the lavage) to provide sufficient material for phospholipid and enzymatic analysis. The lungs were weighed, homogenized in 0.010 mol/l TRIS/0. 145 mol/l Nacl buffer, pH 7.4, and subcellular fractions were isolated according to the technique of Oulton et al, 15,16 as previously described. Briefly this involves 1) centrifugation for 5 minutes at 140g yielding a crude nuclear pellet; 2) a further centrifugation for 30 minutes at 10,000g, the pellet of which is suspended in 2 ml TRIS buffer and applied to a discontinuous sucrose density gradient consisting of 5 ml each of 0.68 mol/l and 0.25 mol/l sucrose, which then was centrifuged at 65,000g for 60 minutes to yield a mitochondrial (density gradient pellet) and a lamellar body or tissue-stored surfactant (sucrose gradient interface) fraction; and 3) centrifugation of the

supernatant resulting from the 10,000g spin at 100,000g for 80 minutes to yield a microsomal pellet and soluble supernatant (cytosolic) fraction. Each lavage fraction and aliquots of the lamellar body fraction prepared from the lung tissue were extracted with chloroform/methanol (2:1, vol/vol) for phospholipid analysis, as described previously.15 Individual phospholipids were separated by two-dimensional thin-layer chromatography, and the spots were visualized and their phosphorus content determined as described.21 The disaturated species of phosphatidylcholine (DSPC) was isolated for analysis, as described by Mason et al.22 Subcellular marker enzyme analysis and ultrastructural characterization of the lamellar body fraction from both control and smoke-exposed mice, as well as preparation of other subcellular fractions, were performed as described in detail for the rabbit.15'16 Phospholipase A2 activity was assayed in all tissuederived fractions under optimal conditions (as determined previously) by measuring the enzymatic hydrolysis of [14C]oleic acid from 1 -palmitoyl-2[14C]-oleoyl phosphatidylcholine, as described previously.923 The assay mixture contained 0.10 mol/l TRIS (pH 8.0), 1 mmol/l ethylene glycol-bis (p3-aminoethyl ether) N,N,N',N', tetraacetic acid (EGTA), 15 mmol/l CaCI2, 0.024 mmol/l deoxycholic acid, and 0.26 mmol/l substrate, which included 1 -palmitoyl2[14C]-oleoyl phosphatidylcholine (specific activity, 40 to 60 mCi/mmole) (Amersham, Oakville, Ontario, Canada). The reaction was conducted at 37°C at a pH of 8.0 and was initiated by the addition of Ca++ and protein. The reaction was terminated with chloroform:methanol after 80 minutes. Previously we showed that the reaction is linear for at least 80 minutes. All incubations were conducted in triplicate. Unreacted substrate was separated from hydrolyzed fatty acid on LK6D silica gel plates in petroleum ether:diethyl ether:formic acid (80:20:1.5). Internal standards identified the position of the fatty acid. Control incubations included duplicate assays with no addition of protein to measure nonenzymatic fatty acid hydrolysis and a microsomal preparation from adult rabbit lung of known specific activity. Radioactivity in free fatty acid was determined on a Beckman LS5801 using H# that is based on the spectrum of 137Cs (Beckman Scientific, Palo Alto, CA) for quench correction. Disintegrations per minute were converted to total picomoles and nonenzymatic hydrolysis in the control incubations was subtracted from this value. Picomoles of products were standardized to milligrams of protein per minute. Protein was determined using the Bio-Rad Protein Assay kit (BioRad Laboratories, Mississauga, Ontario, Canada). Statistical comparison of the results was performed using Students' t-test24 or Duncan's new multiple range test (MRT).25 For the Duncan's test, an analysis of variance

Effects of Smoke on Lung Phospholipids

197

AJPJanuary 1991, Vol. 138, No. 1

performed before the test. The F value was significant to 5%. was

Results As described elsewhere,' exposed mice manifested labored respirations by the end of the exposures and this persisted for up to 12 hours. While histology revealed changes in proximal trachea and main bronchi, it proved an insensitive indicator of parenchymal injury as compared to the changes in lavage fluid.' No significant differences were observed in the mean lung weight (grams wet lung per mouse) in the individual groups (control: 0.258 ± 0.034, n = 10; 8 hours after exposure: 0.271 ± 0.047, n = 8; and 12 hours after exposure 0.292 ± 0.045, n = 7; P > 0.05 for each pair by Duncan's MRT). Therefore the data were expressed on the basis of wet lung weight. The purity of the lamellar body preparations in both control and smoke-exposed mice was assessed by marker enzyme analysis and electron microscopy. As indicated in Table 1, preparations obtained from both control and smoke-exposed mice contained minimal levels of succinate dehydrogenase (mitochondrial marker) and 5'-nucleotidase (plasma membrane marker). While substantially more nicotinamide adenine dinucleotide phosphate: cytochrome, reductase (microsomal marker) appeared to be present in the preparations, there was no difference between the control and smoke-exposed groups. Ultrastructurally both preparations appeared to consist of mainly intact lamellar body structures with minimal extraneous material. A representative micrograph is shown (Figure 1) for the untreated group. The effect of smoke exposure on the total phospholipid content in lavage fluids and lamellar body fractions is shown in Table 2. While there was nearly twice as much phospholipid in the lavage fluid at both 8 and 12 hours after smoke exposure, no change was observed in lamellar body fraction phospholipid content at either of these times. The cellular pellet obtained by low-speed centrifugation

of lavage fluid consistently constituted from 5% to 8% of total lavage phospholipid for both the untreated control and the smoke-exposed groups. In some experiments, the 700g lavage supernatant was centrifuged further to yield various surfactant sedimentable and nonsedimentable subfractions, but because it was found that the various subfractions were of identical phospholipid composition, which was distinctive from that of the cellular pellet (the cellular pellet containing less phosphatidylcholine [70.6% ± 3.2% versus more than 80%] and more phosphatidylinositol [4.3% ± 0.3% versus less than 2%] and lysophosphatidylcholine [5.4% ± 3.2% versus barely detectible levels] than the surfactant subfractions), the 700g supernatant was thought to be representative of the total extracellular surfactant pool. Exposure to smoke significantly increased the absolute content of the DSPC in the lavage fluids (Table 3). Like the total phospholipid content, the lavage DSPC almost doubled 12 hours after exposure. In contrast, the DSPC content of the lamellar body fraction, which constitutes the intracellular pool of stored surfactant, was decreased significantly by smoke exposure (Table 3). This decrease was reflected in the DSPC/PC ratio, which was significantly reduced in the lamellar body fraction at both 8 and 12 hours after smoke exposure (Table 4). The DSPC/PC ratio of the lavage fluid, on the other hand, was not affected by smoke exposure. In fact, as shown in Table 5, the relative distribution of all the phospholipids present in lavage fluid was unaffected by smoke inhalation. Thus the increases observed in the total phospholipid content of this extracellular surfactant pool appears to be distributed uniformly over each of the individual phospholipid constituents. For comparative purposes, blood was taken from several mice and the plasma prepared for phospholipid analysis and determination of the DSPC/PC ratio. Plasma contained much less phosphatidylcholine (64.1% ± 4.1 % of the total lipid phosphorus; n = 4) than any of the surfactant fractions and considerably less was present as the disaturated species (plasma DSPC/PC ratio: 0.187 ±0.014, n = 4).

Table 1. Distribution of Marker Enzymes in Subcellular Fractions from Untreated and Smoke-exposed Mouse Lung

Fraction

Tissue homogenate Crude mitochondria Crude microsomes Crude plasma membranes Lamellar bodies

Succinate dehydrogenase Smoke exposed Untreated

11.8 ± 1.0 22.0 ± 4.2

11.2 ± 4.2 25.7 ± 7.5

NADPH: Cytochrome, reductase Untreated Smoke exposed nmol/min. per mg protein 11.9 ± 1.0 12.3 ± 3.3

84.3 ± 11.7

5'-Nucleotidase Smoke exposed

Untreated

6.8 ± 3.0

5.2 ± 0.6

82.4 ± 9.3 22.1 ± 4.2 5.9 ± 2.1

ND 6.3 ± 3.5 Each value represents the mean ± 1 SD of from three to five determinations. In the smoke-exposed group the mice were examined 8-hour after a 30-

5.0 ± 1.5

8.4 ± 2.1

37.3 ± 16.1

40.6 ± 18.9

minute exposure. No significant differences were found between the groups (P > 0.05 by Student's t-test). NADPH, nicotinamide adenine dinucleotide phosphate; ND, not determined.

198

Oulton et al

AJPJanuary 1991, Vol. 138, No.

1

*Y.2S;it .s .Sw,. g. S. [&.8^;sT.s^;

d*': W.;

a

t..'

s -# sa # ,z,,.e .....

D .Ss l.

E

j

#/ ali. X s e

Figure 1. Representative electron micrograph of mouse lamellar body preparation indicating the presence of mainly intact structures with minimal contamination by other membranous components (34,0003).

Smoke exposure resulted in small but significant changes in the overall phospholipid composition of the lamellar body fractions (Table 5). In addition to phosphatidylcholine, significant increases were observed in the relative proportion of phosphatidylserine with concomitant decreases in phosphatidylinositol, phosphatidylglycerol,

Table 2. Effect of Smoke Exposure on Total Phospholipid Content in Lavage Fluids and Lamellar Body Fractions in Mouse Lung

Phospholipid content (jsg/g wet lung weight) Treatment

n*

Control 8 hours after

7

exposure

7

2617.7

6

2706.9 ± 477.7t

Lavage fluids 1473.5

±

243.1

Lamellar body fractions 979.0

±

Table 3. Effect ofSmoke Exposure on the Disaturated Phosphatidicholine Content in Lavage Fluids and Lamellar Body Fractions in Mouse Lung DSPC content (,ug/g wet lung) Lamellar Lavage fluids body fractions Treatment (n) (n) Control 561.6 ± 130.2 412.8 ± 57.9

148.3

8 hours after

±

511.5t

861.0

±

202.1

12 hours after exposure

and the unidentified phospholipid. Thus, while smoke inhalation appeared to affect several of the individual phospholipids present in the intracellular surfactant pool, the major effect appeared to be in the almost twofold decrease in the DSPC content of this fraction.

950.2 ± 90.2

Mice were killed either 8 or 12 hours after a 30-minute exposure. Each value represents the mean ± 1 SD for the number of determinations shown. * Each determination was obtained by separately pooling lavage fluids and corresponding lung tissue from two to three mice. t Significantly different from control values (P < 0.01) as determined by Duncan's Multiple Range Test.

(10)

(5)

942.3 ± 153.5t 273.2 ± 97.8* exposure (7) (5) 12 hours after 1054.2 ± 212.4t 304.9 ± 49.6* exposure (6) (5) DSPC, disaturated species of phosphatidylcholine (PC). Treatment of mice, presentation of data and statistical analysis were as described for Table 2. DSPC content was determined according to the method of Mason et al.17 * Significantly different from control values (P < 0.05). t Significantly different from control values (P < 0.01).

Effects of Smoke on Lung Phospholipids

199

AJPJanuary 1991, Vol. 138, No. 1

Table 4. Effect of Smoke Exposure on the DSPC/total Phosphatidylcholine (PC) Ratio in Lavage Fluids and Lamellar Body Fractions in Mouse Lung DSPC/PC ratio Lamellar body fractions Lavage fluids (n) (n) Treatment 0.54 ± 0.02 0.53 ± 0.08 Control 8 hours after exposure 12 hours after exposure

(10)

0.56 ± 0.08 (8) 0.52 ± 0.03 (7)

(5)

0.38 ± 0.06*

(5) 0.30 ± 0.17t (5)

DSPC, disaturated species of phosphatidylcholine (PC). Treatment of mice, presentation of data and statistical analysis were as described for Table 2. * Significantly different from control values (P < 0.05).

t Significantly different from control values (P < 0.01).

Consistent with this decrease in the DSPC content, we observed that the specific activity of the microsomal phospholipase A2 (Table 6) also was significantly reduced (P < 0.05) 8 hours and again 12 hours after exposure to the smoke. Other subcellular fractions did not show any significant alteration in enzymatic activity after smoke exposure.

Discussion Our data indicate that a 30-minute exposure to smoke generated by burning polyurethane foam, as described in this report, results in a nearly twofold increase in the phospholipid content of alveolar surfactant, with no alteration in its overall composition. On the other hand, there was no change in the quantity of the intracellular surfactant pool but dramatic changes were observed in the composition of this fraction. Our marker enzyme and ultrastructural analyses suggest that the preparations obtained from the untreated and smoke-exposed mice are reasonably devoid of contamination by other subcellular fractions. Also contamination by plasma phospholipids would be doubfful because the phospholipids, being soluble, would not coisolate with the lamellar bodies but remain in the soluble supernatant fraction that is routinely discarded. Based on the overall distribution of phospholipids in our lavage samples, we conclude that the phospholipids that accumulate in this fraction are indeed of a surfactant origin. The enrichment of phosphatidylcholine, particularly the disaturated species, the presence of phosphatidylglycerol as the second most abundant phospholipid, as well as the relatively low proportions of each of the other phospholipids that we found in both the untreated and smoke-exposed lavage samples, are characteristic of surfactants isolated by ourselves151'6 as well as by several

other investigators from a wide variety of species.26 While it might be argued that plasma phospholipids may leak across the alveolar membrane and contribute to the elevated phospholipid level observed in the smoke-exposed mice, our data, however, suggest that there is minimal contribution of phospholipids from this source. Phosphatidylcholine accounts for less than 70% of mouse plasma phospholipids and the plasma DSPC/PC ratio is less than 0.200. If plasma phospholipid were present to any appreciable extent, it would be reflected in the phospholipid analysis. This was not the case in either the untreated or the smoke-exposed mice. Centrifugation at 700g for 10 minutes appeared to be adequate to remove the cellular material from the lavage returns. While alveolar macrophages are known to increase after exposure to smoke27 as well as other noxious agents, this was not reflected in the present study by a disproportionate distribution of total lavage phospholipids following smoke exposure. Rather the cellular pellet consistently represented from 5% to 10% of the total phospholipid in both the treated and untreated mice. While routinely we did not further fractionate the lavage (700g) supernatant into sedimentable and nonsedimentable subfractions, in the few experiments we did perform we found such a remarkable consistency in the overall phospholipid composition of these subfractions in both the treated and untreated groups as to provide further support for the notion of a surfactant origin for the phospholipids that accumulate in this pool after exposure to smoke. It was interesting to find that on high-speed centrifugation (10,000g for 30 minutes) of the lavage samples that the bulk of the phospholipid was recovered in the soluble supernatant fraction. This is in contrast to reported findings in the rabbit in which the bulk of lavage phospholipids are recovered in the high-speed pellet152829 but correlates with studies with rats.30 Because the sedimentable surfactant is believed to represent newly secreted material and the nonsedimentable fraction to represent the already-used material,283' this suggests that in the mouse and rat, but not the rabbit, the bulk of lavage surfactant is in the latter form. Whether exposure to smoke or other noxious or toxic substances plays any role in the distribution of phospholipids over the subfractions is not known. Further studies are being conducted to clarify this issue. While it is well recognized that the lung surfactant system is seriously impaired by most agents that damage the parenchyma,31 the nature of the impairment has never been clarified fully. In some situations, for example exposure to cigarette smoke (either chronic or acute)32 or administration of certain lung damaging endotoxins,'127 alveolar surfactant pools appear to be significantly decreased, in some cases with altered composition" while in other situations, eg, following exposure to silica dust,14

200 Oulton et al AJPJanuary 1991, Vol. 138,

No. 1

Table 5. Effect of Smoke Exposure on Phospholipid Composition in Lavage Fluids and Lamellar Body Fractions in Mouse Lung Phospholipid (% of total) Treatment Lavage Fluids Control 8 hr post-exposure 12 hr post-exposure Lamellar Body Fractions Control 8 hr post-exposure 12 hr post-exposure

n

PS

Pi

SM

PC

PG

PE

X

6 7 6

0.8 ± 0.4 0.6 ± 0.2 0.7 + 0.4

1.6 ± 0.3 1.6 ± 0.4 1.6 ± 0.3

0.6 ± 0.2 0.8 ± 0.4 0.6 ± 0.2

81.1 ± 1.3 81.5 ± 2.0 82.5 + 1.7

11.4 ± 0.6 11.7 ± 1.3 10.6 ± 1.1

2.6 ± 0.5 2.4 ± 0.8 2.1 ± 0.7

1.7 ± 0.4 1.5 ± 0.8 2.0 ± 0.7

3.2 ± 0.7 1.7 ± 0.2 75.3 ± 1.5 10.7 ± 1.0 6.7 ± 0.6 1.4 ± 0.6 1.8 ± 0.4t 1.5 ± 0.7 78.4 ± 2.0* 9.4 ± 1.0* 6.0 ± 0.6 0.7 ± 0.5* 9.5 ± 1.1* 6.0 ± 1.2 0.7 ± 0.6* 2.2 ± 0.9* 1.6 ± 0.8 78.0 ± 3.4 Treatment of mice and statistical analysis as for Table 2. PS, phosphatidylserine; Pi, phosphatidylinositol; SM, sphingomyelin; PC, phosphatidylcholine; PG, phosphatidylglycerol; PE, phosphatidylethanolamine; 7 7 6

1.0 ± 0.8 2.1 ± 0.7* 1.9 ± 0.5*

X, unidentified phospholipid. * Significantly different from control values (P < 0.05). t Significantly different from control values (P < 0.01).

diesel fuel particles,`3 or ozone,34 there is an increase in the level not only of alveolar surfactant but also in the total lung surfactant pool. In a recent study in our laboratory in which we examined the effects of trichloroethylene on mouse lung surfactant,9 we found no change in the alveolar pool but a drastic reduction (close to 85%) in the phospholipid content of the intracellular pool. A different response was found in the present study in that the quantity of phospholipids in the intracellular pool remained unchanged, but drastic alterations were found in the overall composition. It is thus apparent that while several agents may lead to destruction of the pulmonary surfactant, there seems to be no generalized response to a toxic insult. Some studies32 35suggest that impairment of the surfactant system can result not necessarily from a decrease in the absolute content of lavage phospholipid but from an inactivation resulting from the exudation of plasma proteins due to a damaged alveolar membrane.3 While we did not measure the surface properties of our samples, we did find a somewhat elevated protein level in the lavage content from the smoke-exposed mice. It is possible that the significant increase in the phospholipid content of these same samples may constitute a defense mechanism whereby an increased release of surfactant is triggered

Table 6. Activity of Phospholipase A2 in the Microsomal Fraction (100, OOOg pellet) of Mouse Lung Exposed to Polyurethane Smoke n Mean Treatment 9.22 5 Control 3 4.29* 8 hours post exposure 2.21* 3 12 hours post exposure

SEM

0.95 0.84 0.57

Mice were treated as described for Table 2. Results are expressed as picomoles of fatty acid hydrolyzed/minute/mg protein. Background

nonenzymatic hydrolysis of the fatty acid has been subtracted from the values. Pooling of tissue and statistical analysis were as described for

Table 2. *

Significantly different from control values (P < 0.01).

to compensate for the diminishment of surface activity. Another possible benefit that could be brought about by an increased release of surfactant involves its recently described role in transporting foreign particles toward the ciliary escalator for removal from the lung.337 It is possible that the accumulation of alveolar surfactant phospholipid, which we observed after smoke exposure, results not from an increased release of surfactant from the type 11 cell but rather from a decrease in its rate of removal from the alveoli. This could result from an inhibition of either or both of the clearance or recycling processes. While we cannot make the distinction as to which of these two processes are in operation, our data nevertheless suggest that the increased accumulation of lavage phospholipid may indeed be due to a slowing of the removal of this material from the airways. If, for example, the increase in alveolar surfactant resulted from an increased secretion of newly or recently produced lamellar bodies, then not only would the overall phospholipid composition but more importantly the DSPC/PC ratio would be expected to be similar for the surfactants isolated from the two lung compartments. This is true for normal lung tissue as reported by ourselves15'6 and others143338 for a number of species and in the present study in the untreated mice. It is not so for the smoke-exposed mice, however, in which case not only were there differences in phospholipid composition but the greatly decreased DSPC/PC ratio found in the lamellar body fraction was not observed in the alveolar pool. Thus, while the alveolar pool in the smokeexposed mice does not appear to represent newly secreted material, because our studies were performed at either 8 or 12 hours following exposure, we cannot exclude the possibility that an earlier response to the smoke would be a transient stimulation of the secretory process and that the material present in the lavage at 8 or 12 hours is representative of this earlier-secreted material. Further study is necessary to clarify this point.

Effects of Smoke on Lung Phospholipids

201

AJP January 1991, Vol. 138, No. 1

Our results also suggest that, in contrast to reports for several other noxious agents,39 exposure to polyurethanegenerated smoke does not appear to stimulate the surfactant biosynthetic process. This is evidenced by the drastic reduction in the activity of phospholipase A2, which, although it was assayed in whole lung fractions and therefore does not specifically reflect changes in only the surfactant-producing type 11 cells, no doubt contributes to the depletion of DSPC observed in the lamellar body fraction. It is not known whether enzymes involved in the synthesis of other surfactant phospholipid also are affected, but in view of the relatively small changes observed in these phospholipids it seems that any effects that do occur would be minimal. It is also not known if other enzymes involved in the synthesis of phosphatidylcholine, or more importantly, the disaturated species, are affected by smoke inhalation. We are currently investigating this possibility using our mouse lung model. Our results show that exposure of mice to polyurethane-generated smoke results in an increased accumulation of alveolar surfactant phospholipid without any alteration in composition and no change in the content but significant alterations in the composition of the intracellular pool. This suggests that the major effects of smoke inhalation are inhibitory in nature, acting on both the removal of surfactant from the alveoli and its biosynthesis in the type 11 cell. While the former response appears to be a compensatory mechanism and therefore is defensive in nature, the latter is clearly destructive. Further study is necessary, however, at both shorter and longer time intervals following exposure to elucidate more clearly the underlying mechanisms involved in these responses.

Acknowledgments The authors thank 1. Winter and J. M. MacDonald for technical assistance.

References 1. Demling RH: Burns. N Engl J Med 1985, 313:1389-1398 2. Heimbach DM: Inhalation injuries. Ann Emerg Med 1988,17: 1316-1320 3. Fire Losses in Canada. Annual Report, 1988, Minister of Labour, Govemment of Canada, Ottawa, Ont. Cat. No. W51, 1988, p 7 4. Committee on Fire Toxicology. Fire and Smoke, Understanding The Hazards. National Research Council. Washington, DC, National Academy Press, 1986, p 18 5. Levin BC, Paabo M, Fultz ML, Bailey C: Generation of hydrogen cyanide from flexible polyurethane foam decomposed under different combustion conditions. Fire Mat 1985, 9:125134

6. Paabo M, Levin M: A review of the literature on the gaseous products and toxicity generated from the pyrolysis combustion of rigid polyurethane foams. Fire Mat 1987, 11:1-29 7. Woolley WD: Smoke and toxic gas production from burning polymers. J Macromol Sci. Chem 1982, A17:1-33 8. Cohen MA, Guzzardi LJ: Inhalation of products of combustion. Ann Emerg Med 1983, 12:628-632 9. Scott JE, Forkert PG, Oulton M, Rasmusson MG, Temple S, Fraser MO, Whitefield S: Pulmonary toxicity of trichloroethyline: Induction of changes in surfactant phospholipids and phospholipase A2 activity in the mouse lung. Exp Mol Pathol 1988, 49:141-150 10. Giri SN: Effects of intratracheal instillation of bleomycin on phospholipid synthesis in hamster lung tissue slices. Proc Soc Exp Biol Med 1987, 186:327-332 11. Tahvanainen J, Hallman M: Surfactant abnormality after endotoxin-induced lung injury in guinea pigs. Eur J Respir Dis 1987, 71:250-258 12. LeMesurier SM, Lykle WJ, Stewart BW: Reduced yields of pulmonary surfactant: Patterns of response following administration of chemicals to rats by inhalation. Toxicol Lett 1980, 5:89-93 13. Amanuma K, Suzuki KT: Effects of intratracheal instillation of cadmium chloride on phospholipids in alveolar wash fluid. Toxicol 1987, 44:321-328 14. Dethloff LA, Gilmore LB, Hook GER: The relationship between intra- and extra-cellular surfactant phospholipids in the lungs of rabbits and the effects of silica-induced lung injury. Biochem J 1986, 239:59-67 15. Oulton M, Fraser M, Dolphin M, Yoon R, Faulkner G: Quantification of surfactant pool sizes in rabbit lung during perinatal development. J Lipid Res 1986, 27:602-614 16. Oulton M, Dolphin MA: Subcellular distribution of disaturated phosphatidylcholine in developing rabbit lung. Lipids 1988, 23:55-61 17. Batenburg JJ: Biosynthesis and secretion of pulmonary surfactant. In Robertson B, Van Golde LMG, Batenburg JJ, eds. Pulmonary Surfactant. Elsevier, Amsterdam, 1984, p 237 18. Levin BC, Fowell AJ, Birky MM, Paabo M, Stolte A, Malek D: Further development of a test method for the assessment of the acute inhalation toxicity of combustion products. Nat Bur Stand Washington, DC, 1982 19. Levin BC, Paabo M, Fultz ML: An acute inhalation toxicological evaluation of combustion products from fire-retarded and non fire-retarded flexible polyurethane foam and polyester. Nat Bur Stand Washington, DC, 1983 20. Jannigan DT, Moores H, Hajela R: Polyurethane foam smoke inhalation in mice: Early decreases in lavagable lung macrophages. Chest 1989, 96(Suppl): 2925 21. Oulton M, Martin TR, Faulkner GT, Stinson D, Johnson JP: Developmental study of a lamellar body fraction isolated from human amniotic fluid. Pediatr Res 1980, 14:722-728 22. Mason RJ, Nellenbogen J, Clements JA: Isolation of disaturated phosphatidylcholine with osmium tetroxide. J Lipid Res 1976, 17:281-284 23. Scott JE, Oulton M, Boylan M, Dolphin MA, Temple S: Profile of phospholipase A2 activity in subcellular fractions and la-

202

Oulton et al

AJPJanuary 1991, Vol. 138, No. 1

24.

25. 26.

27.

mellar bodies of developing and newborn rabbit lung. Correlation with intracellular levels of disaturated phosphatidylcholine. Biochim Biophys Acta 1987, 921:75-81 Mendenhall W: Introduction to Probability and Statistics, 4th Edition. Belmont, CA, Duxbury Press, 1975 Ott L: An Introduction to Statistical Methods and Data Analysis. North Scituate, CA, Duxbury Press, 1977, pp 392,1977 Possmayer F: Biochemistry of pulmonary surfactant during fetal development and in the prenatal period. In Robertson B, VanGolde LMG, Batenburg JJ, eds. Pulmonary Surfactant. Amsterdam, Elsevier, 1984, pp 296 Miller K, Cottrell RC: Adverse effects of toxins and drugs on the surfactant systems. Eur J Respir Dis 1987, 71(Suppl

153):237-241 28. Baritussio A, Bellina L, Carraro R, Rossi A, Euzi G, Magoon MW, Mussini I: Heterogeneity of alveolar surfactant in the rabbit: Composition, morphology and labelling of subfractions isolated by centrifugation of lung lavage. Eur J Clin Invest 1984,14:24-29 29. Bruni R, Baritussio A, Quaglino D, Gabelli C, Benevento M, Ronchetti IP: Postnatal transformations of alveolar surfactant in the rabbit. Changes in pool size, pool morphology and isoforms of the 32-38 kDa apolipo protein. Biochim Biophys Acta 1988, 958:255-267 30. Spain CL, Silbajoris R, Young SL: Alterations of surfactant pools in fetal and newborn rat lung. Pediatr Res 1987, 21: 5-9

31. Magoon MW, Wright JR, Baritussio A, Williams MC, Goerke J, Benson BJ, Hamilton RL, Clements JA: Subfractions of lung surfactant: Implications for metabolism and surface activity. Biochim Biophys Acta 1983, 750:18-31 32. Higenbottam T: Pulmonary surfactant and chronic lung disease. Eur J Respir Dis 1987, 71(Suppl) 153:222-228 33. Nieman GF, Clank WR, Wax SD, Webb WR: The effect of smoke inhalation on pulmonary surfactant. Ann Surg 1980, 191:171-181 34. Balis JU, Paterson JF, Haller EM, Shelley SA, Montgomery MR: Ozone-induced lamellar body responses in a rat model for alveolar injury and repair. Am J Pathol 1988, 132:330344 35. Eskelson CD, Chvapil M, Strom KA, Vostal JJ: Pulmonary phospholipidosis in rats respiring air containing diesel particulates. Environ Res 1987, 44:260-271 36. Notter RH, Finkelstein JN: Pulmonary surfactant: An interdisciplinary approach. J Appl Physiol 1984, 57:613-624 37. Gebhardt KF, Rensch H, Seefeld H von: Model study on transport properties of lung surfactant. Prog Resp Res 1984,

18:40-43 38. Weiss JM, Gebhardt KF, Ziegler H, Rensch H: Role of surfactant in peripheral transport mechanisms. Eur J Respir Dis 1987, 71 (Suppl 153):205-208 39. Foster JR, Cottrell RC, Herod IA, Atkinson HAC, Miller K: A comparative study of the pulmonary effects of NO2 in the rat and hamster. Br J Exp Path 1985, 66:193-204

Effects of smoke inhalation on surfactant phospholipids and phospholipase A2 activity in the mouse lung.

The effects of smoke inhalation on the pulmonary surfactant system were examined in mice exposed for 30 minutes to smoke generated from the burning of...
2MB Sizes 0 Downloads 0 Views