Br. J. exp. Path. (1978) 59, 32
BIOLOGICAL EFFECTS OF CHRYSOLITE AFTER SO2 SORPTION. III. EFFECTS ON THE BIOCHEMICAL COMPONENTS OF ALVEOLAR WASHING A. OBLIN, J. M. WARNET, M. C. JAURAND, J. BIGNON AND J. R. CLAUDE From the Laboratoire de Toxicologie de l'uter des Mecanismes d'Action des Medicaments et des Toxiques de 1'Universitt Rene Descartes de Paris (Pr R. Truhaut); Departement de Recherches sur les Affections Respiratoires et l'Environnement de 1' UniversitW de Paris- Val de Marne; Laboratoire des Centres d'Examens Complementaires de la DGASS de la Prefecture de Paris (Directeur: Dr G. Bonnaud) Received for publication September 6, 1977
Summary.-The short-term effects of chrysotile asbestos before and after SO2 sorption are studied in the rabbit after intratracheal injection of low doses of these pollutants. Chrysotile, as well as S02-chrysotile, induces an increase in the unsaturated fatty acid content of lung surfactant, which is similar to that observed in the respiratory distress syndrome of the newborn, and an increase in the protein level of pulmonary washings which may be explained by an increase of the permeability of the blood-air barrier. These soluble proteins can interact with the surfacant and thus decrease its tensio-active properties. THE alveolar epithelium is covered by an alveolar lining material (ALM) consisting of phospholipids and proteins the biochemical composition of which has been extensively investigated (Scarpelli, 1968; Clements, 1970, 1973; King, 1974; Tierney, 1974). Studies of the effects of different inhaled or intratracheally injected pollutants have been carried out to investigate physical and/or biochemical changes in the ALM (Rosenberg, Alarie and Robillard, 1962; Rhoades, 1972; Griunspan, Antweiler and Demnen, 1973; Sherwin and Carlson, 1973; Heppleston et al., 1974, 1975). Sulphur dioxide has been shown to decrease surface tension of the lung (Kahana and Aronovitch, 1966, 1968), and it seems that SO2 has a hydrolytic action on pulmonary surfactant (Douste-Blazy and Prevost, 1976). On the other hand, inhaled asbestos has been found to increase the surface active material of the lung (McDermott et al., 1977). All these studies were concerned with uncombined pollutants. In order to study the possible synergistic effects of a fibrous particle chrysotile and a gaseous
pollutant-SO2 on the biochemistry of ALM, we have investigated the quantitative and qualitative composition of the major phospholipid fractions and the protein content of broncho-alveolar lavage fluid. In this study, only the primary harmful effects of these associated pollutants (chrysotile-802) have been considered. Short term intoxication was provoked by intratracheally introduced doses similar to those found in the environment. MATERIALS AND METHODS Four groups of rabbits (EC 601 Evic Ceba, France) were used; two served as control groups, one untreated (untreated controls) and the other receiving 1-2 ml of 0-9o w/v NaCl (NaCl controls). The two experimental groups received 10 irng of chrysotile fibres before and a,fter S02 sorption. SO2 was adsorbed on UICC A chrysotile as described elsewhere by Goni et al. (unpublished). The intratracheal injection was carried out as described by Jaurand et al., unpublished. Preparation of AL,l.-The rabbit lungs were washed as previously described (Jaurand et al., in press). The alveolar washings were centriftiged for 20 rnin at 300 g to remove alveolar
33
EFFECTS OF CHRYSOTILE ASBESTOS AFTER S02 SORPTION
free cells. Three supernatants (So) were pooled and centrifuged for 15 min at 850 g. The pellet (P1) was discarded and SI was recentrifuged for 90 min at 100,000 g. The pellet P2 was resuspended in 3 ml of 0-9% w/v NaCl and the supernatant S2 was concentrated against 20% (w/v) of polyvinylpyrrolidone at +4°. Protein analyis8.-Total proteins were estimated by the method of Lowry et al. (1951). Polyacrylamide gel electrophoresis of proteins was performed according to Weber and Osborn (1969) after dialysis of the samples against the running buffer (82.5 mM Tris, 400 mm borate, pH 7. 1). Bromophenol blue was used as a running control and bovine albumin (IV fraction SIGMA) as the internal control. The gels were stained after electrophoresis with 7% (w/v) in acetic Coomassie blue. Lipid analy8is.-Lipids were extracted from the washing fractions by the method of Folch, Lees and Sloane-Stanley (1957). Lipid phosphorus was determined according to Briggs (1924) after sulphonitroperchloric mineralization. The principal phospholipid components were separated by thin layer chromatography (TLC) on 0-25 mm silica gel plates (E. Merck, Darmstadt, Germany) in chloroform-methanolwater (65: 25: 4 by vol.) (Robinson and Philipps, 1963). Phospholipids were identified by cochromatography with standards (Sigma) and bands were revealed with 12 vapour. Each phospholipid fraction was eluted from TLC plates with methanol or with chloroformmethanol (2 : 1, v/v). Fatty acid components were analysed by gas liquid chromatography (GLC) after transmethvlation performed according to Rogozinski (1964) as modified by Fosbrooke and Tamir (1968). The fatty acid methyl esters were extracted by hexane, evaporated under reduced pressure and dissolved in carbon disulphide. Separations were achieved in 10% (w/v) diethylene glycol succinate on chromosorb W (80-100 mesh) in 10'x 1/8" columns by using temperature programming (+2°/min) in the range -1-165 to 1800 on a varian aerograph 1440 chromatograph. The identification of the methyl esters was performed by comparison of their retention time with that of known standards (Sigma). The percentage of fatty acids was calculated by the means of series 200 Disc integrator. RESULTS
PULMONARY LAVAGE
270 g, 20 min
PELLET Po (cells) kcei'sJ
SUPERNATANT So
Phospholipids - = 1.32 Proteins 850 g, 15 min
SUPERNATANT S
PELLET P
Phospholipids
Proteins
Phospholipids =0.87 Proteins =
8.90
100 000 g, 90 1 min
SUPERNATANT
Prntoteisos
PELLET P2
Phospholipids
S2
Phospholipidds
= =
n 14 l/.
U.
= 12.27
Proteins
FIG. 1.-Preparation of pulmonary alveolar lining.
determined by electron microscopic observations (Bignon et al., 1976). In this pellet P2, the phospholipids/proteins (PL/ P) ratio is 12-27 so that there is a gain in phosphlipids in this fraction as compared to the supernatant So. This technique provided a 5-fold increase in PL in the pellet P2 as compared to the supernatant S2.
Lipid analysis Tables I and II show that there is no difference in the levels of lipids, phospholipids (about 92% of the lipid content), and phosphatidyleholine (about 80% of the phosphlipid content) of surfactant from the control and experimental groups. Analysis of the fatty acid content of ALM phosphatidyleholine (Table III) showed that chrysotile and S02-chrysotile seemed to provoke an increase in unsaturated fatty acids as compared to the untreated animals in which the separation of the 16: 0 and 16: 1 fatty acids cannot be made. However, a variation was also observed in NaCl controls as compared to untreated animals.
Preparation of ALM Fig. I gives the data concerning purification of the surfactant performed by succes- Protein analysis Table V shows the protein levels of the sive centrifugations. The pellet P1 contains cellular fragments and P2 consists pellet P2 and the supernatant S2 in the of lamellar bodies or myelin figures as four groups of rabbits. Chrysotile, as well
34
A. OBLIN, J. M. WARNET, M. C. JAURAND, J. BIGNON AND J. R. CLAUDE
TABLE I.-Lipid, Phospholipid and Phosphatidylcholine Content of Pulmonary Surfactant (P2) Untreated controls N=3
NaCl controls N=3 22-72±2- 10 21-62±1-92
Chrysotile N=3 Lipid (mg*) 19-83±0-54 26-46±4- 14 18-26±1-01 24-83±4-20 Phosp holipid (mg) 15-01+0-51 19-52±2-06 20-43±3-71 Phosp)hatidylcholine(mg) Results are expressed as mg per whole lavage. Means ±s.e. mean are given.
S02-chrys3otile N=3 24 13±0 *58 21-79±0-*20 18 04±0 *77
TABLE II.-Composition of Phospholipids from Rabbit Pulmonary Surfacant (P2) Untreated control % PL 4-6 5-0 78-9 5-2 1 6 1.9 3-0
Lysophosphatidy]choline Sphingomyelin Phosphatidylcholine Phosphatidylglycerol Diphosphatidylglycerol (?) Phosphatidylethanolamine Others
Chrysotile % PL 4-3 4-5 82-7 3-4 1*1 2-9 1*1
S02-chrysotile % PL 2-1 4-5 75-7 8-4 2-6 2-6 3-2
TABLE III.-Fatty Acid Composition of Phosphatidylcholine Purified from Pulmonary Surfacant Untreated control N=2 1 1 88-6*
Myristate (14 0) Palmitate (16 0) Palmitoleate (16 : 1) Stearate (18 : 0) 1-9 5-6 Oleate (18: 1) Linoleate (18 : 2) 2-5 * C16: 0+±16: 1. Results are expressed as % of the total fatty acid.
NaCl control N=I
Chrysotile N=I
1-4 71-4 7 0 1 -2 15-7 3-1
1 *1 75-7 6-8 1 -6 13 -8 1 -7
SO2-chrysotile N=i 10 71 -5 9-6 0 9 13-1 3 -8
TABLE IV.-Protein Content of Pellet (P2) and Supernatant S2 Isolated from Rabbit Pulmonary Lavage Pellet P2
Supernatant S2 -'
-
r
Protein mg 2-02±0 08
% total protein 4 9
1 * 67±0 18
1-93±0-20
r
A 20 9
Protein mg 39 36±3 40
% total protein 95-1
5-8
0
26 - 98±2 - 80
94 -2
0
4-6
15-6
40-27±2-54
95 4
49-2
2 - 21±0-05 4- 7 32 - 3 44-59±5 -24 95 3 N=3 A: Increase in protein expressed as a percentage of that obtained from NaCl controls. N: Number of groups studied (each group comprised three animals).
65-3
Untreated controls N=3 NaCl controls N=4 Chrysotile
N=3 S02-chrysotile
as S02-chrysotile, provoked a significant increase in the soluble proteins of the supernatant S2 as compared to NaCl controls (P< 01). However, there is no variation as compared to untreated animals. The qualitative analysis of these soluble proteins by polyacrylamide gel
A 45 - 88
electrophoresis showed that the major component of these proteins was albumin (Fig. 2). DISCUSSION
The methods used in these experiments to purify ALM consisted of successive
EFFECTS OF CHRYSOTILE ASBESTOS AFTER S02 SORPTION
centrifugations of the alveolvar washing. The PL/P ratio obtained in the untreated rabbits was similar to that reported by King and Clements (1972a, b), King (1974), and Harwood et al. (1975). In the present study, 92% of the lipid content of the lung surfactant was represented by phospholipids. The major component of these polar lipids was phosphatidylcholine,
35
Experiments bv other authors were carried out to study the delayed changes induced by inhaled particles in the rat. (Rhoades (1972) observed a significant increase in total phospholipid and lecithin content of lung washings due to inhaled carbon in the rat. Grunspan et al. (1973) found that intratracheal injection of quartz provokes an increase in the total phospholipids of the rat lung. Tetley et al. (1976) observed that rats exposed to high doses of chrysotile or silica have a higher level of surfactant production. The data obtained in this present study did not show any increase in total phospholipid and phosphatidylcholine content of ALM in the rabbits treated with chrysotile and 802-chrysotile. These discrepancies may be explained by the low dose of these pollutants which have been used to simulate the environmental pollution and by the experimental conditions designed to study the early changes of the alveolar biochemistry. Indeed, after a longer delay most of the mineral particles have penetrated the alveolar interstitium (Susuki, Churg and Ono, 1972) and have induced a hyperplastic reaction of the type II pneumocytes (Bruch. 1974). By contrast in our experiments histological studies did not show any interstitial reaction, and there was only a very slight hyperplasia of Fi(e. Polyacrylamidle gel electrophoresis of type II pneumocytes. supernatant of rabbit ptulmonary lavage compared to bovine serum albumin The study of the fatty acid profile of (BSA) migrationi. a-Albumin (BSA). b= showed that chrysophosphatidylcholine S2 NaCl control. =S2 chrysotile. =82 chrysotile + BSA tile and S02-chrysotile, at low doses, induced an increase in the unsaturated fatty acid content of lung surfactant. Such with a percentage of 80% whlich is very modification has been observed in the close to the 83% found by Harwood et al. respiratory distress syndrome of the new(1975) in the rabbit, and to 7400 and 7850/ born (Brumley, Hodson and Avery, 1967). found respectively by Pfleger and Thomas The action of chrysotile and SO2(1971) and King and Clements (1972b) in chrysotile on the rabbit ALM consisted the dog. primarily of an increase in the protein level The data obtained in the two groups of of the pulmonary washings as compared controls were conflicting, in as much as to the NaCl controls. The major protein the protein level was higher in untreated component was albumin. These data are in animals than in NaCl controls and as the accord with immunocytochemical and unsaturated fatty acid content of the immunochemical studies (Bignon et al., phosphatidylcholine was lower in the NaCl 1976) which showed the presence of serum controls than in the untreated animals. proteins in the alveolar lining material of 1
S2
c
d
36
A. OBLIN, J. M. WARNET, M. C. JAURAID, J. BIGNON AND J. R. CLAUDE
the rat lung. The increase of the lung protein content observed in the experimental animals (both chrysotile and SO2chrysotile) might be explained by an increase in permeability of the blood-air barrier to macromolecules with transfer of various plasma proteins into the alveolus. These proteins could interact with the surfactant and thus decrease the tensioactive properties of this film (McDermott etal., 1977). Finally, our study did not find any significant differences in phospholipid and protein content of ALM in either experimental group. This work received financial support from the European Community Commission (ECC Contract No. 089 75 1 Env F), to whom we are grateful. REFERENCES BIGNON, J., JAURAND, M. C., PINCHON, M. C., SAPIN, C. & WARNET, J. M. (1976) Immunoelectron Microscopic and Immunochemical Demonstration of Serum Proteins in the Alveolar Lining Material of the Rat Lung. Amer. rev. resp. Dis., 113, 109. BRIGGS, A. P. (1924) Some Applications of the Colorimetric Phosphate Method. J. Biol. Chem., 59, 225. BRUCH, J. (1974) Comportement des Macrophages Alv6olaires et des Autres Cellules Alveolaires apres Inhalation de Poussieres d'Amiante. Rev. Franc. Mal. Resp., 2, Suppl. 1, 143. BRUMLEY, G. W., HODSON, W. A. & AVERY, M. E. (1967) Lung Phospholipids and Surface Tension Correlations in Infants with and without Hyaline Membrane Disease and in Adults. Pediatrics, 40,
13. CLEMENTS, J. A. (1970) Pulmonary Surfactant. Amer. Rev. Resp. Dis., 101, 984. CLEMENTS, J. A. (1973) Composition and Properties of Pulmonary Surfactant in Respiratory Distress Syndrome. Eds. C. A. Villee, D. B. Villee anid J. Zuckerman, New York: Academic Press, p. 77. DOUSTE-BLAZY, L. & PREVOST, M. C. (1976) Modifications Biochimiques du Film Tensio-actif Alv6olaire sous Effet de Certains Polluants. Rev. Fran,. Mal. Resp., 4, Suppl. 3, 77. FOLCH, J., LEES, M. & SLOANE-STANLEY, G. M. (1957) A Simple Method for the Isolation and Purification of Total Lipids from Animal Tissues. J. Biol. Chem., 226, 497. FOSBROOKE, A. S. & TAMIR, I. (1968) A Modified Method for the Preparation of Methyl Esters of a Mixture of Medium Chain and Long Chain Fatty Acids. Clin. chim. Acta, 20, 517.
GRUNSPAN, M., ANTWEILER, M. & DEMNEN, W. (1973) Effect of Silica on Phospholipids in the Rat Lung. Br. J. industr. Med., 30, 74. HARWOOD, J. L., DESAI, R., HEXT, P., TETLEY, T. & RICHARDS, R. (1975) Characterization of Pulmonary Surfactant from Ox, Rabbit, Rat and Sheep. Biochem. J., 151, 707. HEPPLESTON, A. G., FLETCIHER, K. & WYATT, I. (1974) Changes in the Composition of Lung Lipids and the "Turnover" of Dipolmitoyl Lecithin in Experimental Alveolar Lipo-proteinosis Induced by Inhaled Quartz. Br. J. exp. Path., 55, 384.
HEPPLESTON, A. G., McDERMOTT, M. & COLLINS, M. M. (1975) The Surface Properties of the Lung in Rats with Alveolar Lipo-proteinosis. Br. J. exp. Path., 56, 444. KAHANA, L. & ARONOVITCH, M. (1966) Effects of Sulfur Dioxide on Surface Properties of the Lung. Amer. Rev. resp. Di8., 94, 201. KAHANA, L. & ARONOVITCH, M. (1968) Pulmonary Surface Tension after Sulfur Dioxide Exposure. Amer. Rev. resp. Dis., 98, 34. KING, R. J. & CLEMENTS, J. A. (1972a) Surface Active Materials from Dog Lung. I. Method of Isolation. Am. J. Physiol., 223, 707. KING, R. J. & CLEMENTS, J. A. (1972b) Surface Active Materials from Dog Lung. II. Composition and Physiological Correlations. Am. J. Physiol., 223, 715. KING, R. J. (1974) The Surfactant System of the Lung. Fed. Proc., 33, 2238. LowRy, 0. H., ROSEBROUGH, A., FARR, A. L. & RANDALL, R. J. (1951) Protein Measurement with the Folin Phenol Reagent. J. Biol. Chem., 193, 265. MCDERMOTT, M., WAGNER, J. C., TETLEY, T., HARWOOD, J. & RICHARDS, R. J. (1977) The Effects of Inhaled Silica and Chrysotile on the Elastic Properties of Rat Lungs. Physiological, Physical and Biochemical Studies of Lung Surfactant. IV International Symposium on Inhaled Particles and Vapours, Edinburgh. PFLEGER, R. C. & THOMAS, H. G. (1971) Beagle Dog Pulmonary Surfactant Lipids. Lipid Composition of Pulmonary Tissue Exfoliated Lining Cells, and Surfactant. Arch. intern. Med., 127, 863. RHOADES, R. A. (1972) Effect of Inhaled Carbon on Surface Properties of Rat Lung. Life Sci., 11, 33.
ROBINSON, N. & PHILLIPS, B. M. (1963) Quantitative Thin Layer Chromatography of Serum Phospholipids. Clin. chim. Acta, 8, 385. RoGOZINSKI, M. (1964) The Methanol Sulphuric Acid Esterification Methods. II. An Improved Extraction Procedure. J. Gas Chromatography, 2, 328.
RoSENBERG, E., ALARIE, Y. & ROBILLARD, E. (1962) Effect of Aluminium Dust Inhalation on the Surface Tension of the Alveolar Lining of Rat Lungs. Fed. Proc., 21, 447. SCARPELLI, E. M. (1968) The Surfactant System of the Lung. Philadelphia: Lea and Febiger, p. 31. SHERWIN, R. P. & CARLSON, D. A. (1973) Protein Content of Lung Lavage Fluid of Guinea-pigs Exposed to 0 * 4 ppm Nitrogen Dioxide. Arch. Environ. Health, 27, 90. SUZUKI, Y., CHURG, J. & ONO, T. (1972) Phagocytic Activity of the Alveolar Epithelial Cells in Pulmonary Asbestosis. Am. J. Pathol., 69, 373.
EFFECTS OF CHRYSOTILE ASBESTOS AFTER S02 SORPTION TETLEY, T. D., HEXT, P. M., RICHARDS, R. J. & McDERMOTT, M. (1976) Chrysotile-induced Asbestosis: Charnges in the Free Cell Population, Pulmonary Surfactant and Whole Lung Tissue of Rats.
Br. J. exp. Pathol., 5, 505.
37
TIERNEY, D. F. (1974) Lung Metabolism and Biochemistry. Ann. Rev. Physiol., 36, 209. WEBER, K. & OSBORN, M. (1969) The Reliability of Molecular Weight Determination by Dodecyl Sulfate Polyacrylamide Gel Electrophoresis. J. Biol. Chem., 244, 4406.
BIOLOGICAL EFFECTS OF CHRYSOLITE AFTER SO2 SORPTION. III. EFFECTS ON THE BIOCHEMICAL COMPONENTS OF ALVEOLAR WASHING A. OBLIN, J. M. WARNET, M. C. JAURAND, J. BIGNON AND J. R. CLAUDE From the Laboratoire de Toxicologie de l'uter des Mecanismes d'Action des Medicaments et des Toxiques de 1'Universitt Rene Descartes de Paris (Pr R. Truhaut); Departement de Recherches sur les Affections Respiratoires et l'Environnement de 1' UniversitW de Paris- Val de Marne; Laboratoire des Centres d'Examens Complementaires de la DGASS de la Prefecture de Paris (Directeur: Dr G. Bonnaud) Received for publication September 6, 1977
Summary.-The short-term effects of chrysotile asbestos before and after SO2 sorption are studied in the rabbit after intratracheal injection of low doses of these pollutants. Chrysotile, as well as S02-chrysotile, induces an increase in the unsaturated fatty acid content of lung surfactant, which is similar to that observed in the respiratory distress syndrome of the newborn, and an increase in the protein level of pulmonary washings which may be explained by an increase of the permeability of the blood-air barrier. These soluble proteins can interact with the surfacant and thus decrease its tensio-active properties. THE alveolar epithelium is covered by an alveolar lining material (ALM) consisting of phospholipids and proteins the biochemical composition of which has been extensively investigated (Scarpelli, 1968; Clements, 1970, 1973; King, 1974; Tierney, 1974). Studies of the effects of different inhaled or intratracheally injected pollutants have been carried out to investigate physical and/or biochemical changes in the ALM (Rosenberg, Alarie and Robillard, 1962; Rhoades, 1972; Griunspan, Antweiler and Demnen, 1973; Sherwin and Carlson, 1973; Heppleston et al., 1974, 1975). Sulphur dioxide has been shown to decrease surface tension of the lung (Kahana and Aronovitch, 1966, 1968), and it seems that SO2 has a hydrolytic action on pulmonary surfactant (Douste-Blazy and Prevost, 1976). On the other hand, inhaled asbestos has been found to increase the surface active material of the lung (McDermott et al., 1977). All these studies were concerned with uncombined pollutants. In order to study the possible synergistic effects of a fibrous particle chrysotile and a gaseous
pollutant-SO2 on the biochemistry of ALM, we have investigated the quantitative and qualitative composition of the major phospholipid fractions and the protein content of broncho-alveolar lavage fluid. In this study, only the primary harmful effects of these associated pollutants (chrysotile-802) have been considered. Short term intoxication was provoked by intratracheally introduced doses similar to those found in the environment. MATERIALS AND METHODS Four groups of rabbits (EC 601 Evic Ceba, France) were used; two served as control groups, one untreated (untreated controls) and the other receiving 1-2 ml of 0-9o w/v NaCl (NaCl controls). The two experimental groups received 10 irng of chrysotile fibres before and a,fter S02 sorption. SO2 was adsorbed on UICC A chrysotile as described elsewhere by Goni et al. (unpublished). The intratracheal injection was carried out as described by Jaurand et al., unpublished. Preparation of AL,l.-The rabbit lungs were washed as previously described (Jaurand et al., in press). The alveolar washings were centriftiged for 20 rnin at 300 g to remove alveolar
33
EFFECTS OF CHRYSOTILE ASBESTOS AFTER S02 SORPTION
free cells. Three supernatants (So) were pooled and centrifuged for 15 min at 850 g. The pellet (P1) was discarded and SI was recentrifuged for 90 min at 100,000 g. The pellet P2 was resuspended in 3 ml of 0-9% w/v NaCl and the supernatant S2 was concentrated against 20% (w/v) of polyvinylpyrrolidone at +4°. Protein analyis8.-Total proteins were estimated by the method of Lowry et al. (1951). Polyacrylamide gel electrophoresis of proteins was performed according to Weber and Osborn (1969) after dialysis of the samples against the running buffer (82.5 mM Tris, 400 mm borate, pH 7. 1). Bromophenol blue was used as a running control and bovine albumin (IV fraction SIGMA) as the internal control. The gels were stained after electrophoresis with 7% (w/v) in acetic Coomassie blue. Lipid analy8is.-Lipids were extracted from the washing fractions by the method of Folch, Lees and Sloane-Stanley (1957). Lipid phosphorus was determined according to Briggs (1924) after sulphonitroperchloric mineralization. The principal phospholipid components were separated by thin layer chromatography (TLC) on 0-25 mm silica gel plates (E. Merck, Darmstadt, Germany) in chloroform-methanolwater (65: 25: 4 by vol.) (Robinson and Philipps, 1963). Phospholipids were identified by cochromatography with standards (Sigma) and bands were revealed with 12 vapour. Each phospholipid fraction was eluted from TLC plates with methanol or with chloroformmethanol (2 : 1, v/v). Fatty acid components were analysed by gas liquid chromatography (GLC) after transmethvlation performed according to Rogozinski (1964) as modified by Fosbrooke and Tamir (1968). The fatty acid methyl esters were extracted by hexane, evaporated under reduced pressure and dissolved in carbon disulphide. Separations were achieved in 10% (w/v) diethylene glycol succinate on chromosorb W (80-100 mesh) in 10'x 1/8" columns by using temperature programming (+2°/min) in the range -1-165 to 1800 on a varian aerograph 1440 chromatograph. The identification of the methyl esters was performed by comparison of their retention time with that of known standards (Sigma). The percentage of fatty acids was calculated by the means of series 200 Disc integrator. RESULTS
PULMONARY LAVAGE
270 g, 20 min
PELLET Po (cells) kcei'sJ
SUPERNATANT So
Phospholipids - = 1.32 Proteins 850 g, 15 min
SUPERNATANT S
PELLET P
Phospholipids
Proteins
Phospholipids =0.87 Proteins =
8.90
100 000 g, 90 1 min
SUPERNATANT
Prntoteisos
PELLET P2
Phospholipids
S2
Phospholipidds
= =
n 14 l/.
U.
= 12.27
Proteins
FIG. 1.-Preparation of pulmonary alveolar lining.
determined by electron microscopic observations (Bignon et al., 1976). In this pellet P2, the phospholipids/proteins (PL/ P) ratio is 12-27 so that there is a gain in phosphlipids in this fraction as compared to the supernatant So. This technique provided a 5-fold increase in PL in the pellet P2 as compared to the supernatant S2.
Lipid analysis Tables I and II show that there is no difference in the levels of lipids, phospholipids (about 92% of the lipid content), and phosphatidyleholine (about 80% of the phosphlipid content) of surfactant from the control and experimental groups. Analysis of the fatty acid content of ALM phosphatidyleholine (Table III) showed that chrysotile and S02-chrysotile seemed to provoke an increase in unsaturated fatty acids as compared to the untreated animals in which the separation of the 16: 0 and 16: 1 fatty acids cannot be made. However, a variation was also observed in NaCl controls as compared to untreated animals.
Preparation of ALM Fig. I gives the data concerning purification of the surfactant performed by succes- Protein analysis Table V shows the protein levels of the sive centrifugations. The pellet P1 contains cellular fragments and P2 consists pellet P2 and the supernatant S2 in the of lamellar bodies or myelin figures as four groups of rabbits. Chrysotile, as well
34
A. OBLIN, J. M. WARNET, M. C. JAURAND, J. BIGNON AND J. R. CLAUDE
TABLE I.-Lipid, Phospholipid and Phosphatidylcholine Content of Pulmonary Surfactant (P2) Untreated controls N=3
NaCl controls N=3 22-72±2- 10 21-62±1-92
Chrysotile N=3 Lipid (mg*) 19-83±0-54 26-46±4- 14 18-26±1-01 24-83±4-20 Phosp holipid (mg) 15-01+0-51 19-52±2-06 20-43±3-71 Phosp)hatidylcholine(mg) Results are expressed as mg per whole lavage. Means ±s.e. mean are given.
S02-chrys3otile N=3 24 13±0 *58 21-79±0-*20 18 04±0 *77
TABLE II.-Composition of Phospholipids from Rabbit Pulmonary Surfacant (P2) Untreated control % PL 4-6 5-0 78-9 5-2 1 6 1.9 3-0
Lysophosphatidy]choline Sphingomyelin Phosphatidylcholine Phosphatidylglycerol Diphosphatidylglycerol (?) Phosphatidylethanolamine Others
Chrysotile % PL 4-3 4-5 82-7 3-4 1*1 2-9 1*1
S02-chrysotile % PL 2-1 4-5 75-7 8-4 2-6 2-6 3-2
TABLE III.-Fatty Acid Composition of Phosphatidylcholine Purified from Pulmonary Surfacant Untreated control N=2 1 1 88-6*
Myristate (14 0) Palmitate (16 0) Palmitoleate (16 : 1) Stearate (18 : 0) 1-9 5-6 Oleate (18: 1) Linoleate (18 : 2) 2-5 * C16: 0+±16: 1. Results are expressed as % of the total fatty acid.
NaCl control N=I
Chrysotile N=I
1-4 71-4 7 0 1 -2 15-7 3-1
1 *1 75-7 6-8 1 -6 13 -8 1 -7
SO2-chrysotile N=i 10 71 -5 9-6 0 9 13-1 3 -8
TABLE IV.-Protein Content of Pellet (P2) and Supernatant S2 Isolated from Rabbit Pulmonary Lavage Pellet P2
Supernatant S2 -'
-
r
Protein mg 2-02±0 08
% total protein 4 9
1 * 67±0 18
1-93±0-20
r
A 20 9
Protein mg 39 36±3 40
% total protein 95-1
5-8
0
26 - 98±2 - 80
94 -2
0
4-6
15-6
40-27±2-54
95 4
49-2
2 - 21±0-05 4- 7 32 - 3 44-59±5 -24 95 3 N=3 A: Increase in protein expressed as a percentage of that obtained from NaCl controls. N: Number of groups studied (each group comprised three animals).
65-3
Untreated controls N=3 NaCl controls N=4 Chrysotile
N=3 S02-chrysotile
as S02-chrysotile, provoked a significant increase in the soluble proteins of the supernatant S2 as compared to NaCl controls (P< 01). However, there is no variation as compared to untreated animals. The qualitative analysis of these soluble proteins by polyacrylamide gel
A 45 - 88
electrophoresis showed that the major component of these proteins was albumin (Fig. 2). DISCUSSION
The methods used in these experiments to purify ALM consisted of successive
EFFECTS OF CHRYSOTILE ASBESTOS AFTER S02 SORPTION
centrifugations of the alveolvar washing. The PL/P ratio obtained in the untreated rabbits was similar to that reported by King and Clements (1972a, b), King (1974), and Harwood et al. (1975). In the present study, 92% of the lipid content of the lung surfactant was represented by phospholipids. The major component of these polar lipids was phosphatidylcholine,
35
Experiments bv other authors were carried out to study the delayed changes induced by inhaled particles in the rat. (Rhoades (1972) observed a significant increase in total phospholipid and lecithin content of lung washings due to inhaled carbon in the rat. Grunspan et al. (1973) found that intratracheal injection of quartz provokes an increase in the total phospholipids of the rat lung. Tetley et al. (1976) observed that rats exposed to high doses of chrysotile or silica have a higher level of surfactant production. The data obtained in this present study did not show any increase in total phospholipid and phosphatidylcholine content of ALM in the rabbits treated with chrysotile and 802-chrysotile. These discrepancies may be explained by the low dose of these pollutants which have been used to simulate the environmental pollution and by the experimental conditions designed to study the early changes of the alveolar biochemistry. Indeed, after a longer delay most of the mineral particles have penetrated the alveolar interstitium (Susuki, Churg and Ono, 1972) and have induced a hyperplastic reaction of the type II pneumocytes (Bruch. 1974). By contrast in our experiments histological studies did not show any interstitial reaction, and there was only a very slight hyperplasia of Fi(e. Polyacrylamidle gel electrophoresis of type II pneumocytes. supernatant of rabbit ptulmonary lavage compared to bovine serum albumin The study of the fatty acid profile of (BSA) migrationi. a-Albumin (BSA). b= showed that chrysophosphatidylcholine S2 NaCl control. =S2 chrysotile. =82 chrysotile + BSA tile and S02-chrysotile, at low doses, induced an increase in the unsaturated fatty acid content of lung surfactant. Such with a percentage of 80% whlich is very modification has been observed in the close to the 83% found by Harwood et al. respiratory distress syndrome of the new(1975) in the rabbit, and to 7400 and 7850/ born (Brumley, Hodson and Avery, 1967). found respectively by Pfleger and Thomas The action of chrysotile and SO2(1971) and King and Clements (1972b) in chrysotile on the rabbit ALM consisted the dog. primarily of an increase in the protein level The data obtained in the two groups of of the pulmonary washings as compared controls were conflicting, in as much as to the NaCl controls. The major protein the protein level was higher in untreated component was albumin. These data are in animals than in NaCl controls and as the accord with immunocytochemical and unsaturated fatty acid content of the immunochemical studies (Bignon et al., phosphatidylcholine was lower in the NaCl 1976) which showed the presence of serum controls than in the untreated animals. proteins in the alveolar lining material of 1
S2
c
d
36
A. OBLIN, J. M. WARNET, M. C. JAURAID, J. BIGNON AND J. R. CLAUDE
the rat lung. The increase of the lung protein content observed in the experimental animals (both chrysotile and SO2chrysotile) might be explained by an increase in permeability of the blood-air barrier to macromolecules with transfer of various plasma proteins into the alveolus. These proteins could interact with the surfactant and thus decrease the tensioactive properties of this film (McDermott etal., 1977). Finally, our study did not find any significant differences in phospholipid and protein content of ALM in either experimental group. This work received financial support from the European Community Commission (ECC Contract No. 089 75 1 Env F), to whom we are grateful. REFERENCES BIGNON, J., JAURAND, M. C., PINCHON, M. C., SAPIN, C. & WARNET, J. M. (1976) Immunoelectron Microscopic and Immunochemical Demonstration of Serum Proteins in the Alveolar Lining Material of the Rat Lung. Amer. rev. resp. Dis., 113, 109. BRIGGS, A. P. (1924) Some Applications of the Colorimetric Phosphate Method. J. Biol. Chem., 59, 225. BRUCH, J. (1974) Comportement des Macrophages Alv6olaires et des Autres Cellules Alveolaires apres Inhalation de Poussieres d'Amiante. Rev. Franc. Mal. Resp., 2, Suppl. 1, 143. BRUMLEY, G. W., HODSON, W. A. & AVERY, M. E. (1967) Lung Phospholipids and Surface Tension Correlations in Infants with and without Hyaline Membrane Disease and in Adults. Pediatrics, 40,
13. CLEMENTS, J. A. (1970) Pulmonary Surfactant. Amer. Rev. Resp. Dis., 101, 984. CLEMENTS, J. A. (1973) Composition and Properties of Pulmonary Surfactant in Respiratory Distress Syndrome. Eds. C. A. Villee, D. B. Villee anid J. Zuckerman, New York: Academic Press, p. 77. DOUSTE-BLAZY, L. & PREVOST, M. C. (1976) Modifications Biochimiques du Film Tensio-actif Alv6olaire sous Effet de Certains Polluants. Rev. Fran,. Mal. Resp., 4, Suppl. 3, 77. FOLCH, J., LEES, M. & SLOANE-STANLEY, G. M. (1957) A Simple Method for the Isolation and Purification of Total Lipids from Animal Tissues. J. Biol. Chem., 226, 497. FOSBROOKE, A. S. & TAMIR, I. (1968) A Modified Method for the Preparation of Methyl Esters of a Mixture of Medium Chain and Long Chain Fatty Acids. Clin. chim. Acta, 20, 517.
GRUNSPAN, M., ANTWEILER, M. & DEMNEN, W. (1973) Effect of Silica on Phospholipids in the Rat Lung. Br. J. industr. Med., 30, 74. HARWOOD, J. L., DESAI, R., HEXT, P., TETLEY, T. & RICHARDS, R. (1975) Characterization of Pulmonary Surfactant from Ox, Rabbit, Rat and Sheep. Biochem. J., 151, 707. HEPPLESTON, A. G., FLETCIHER, K. & WYATT, I. (1974) Changes in the Composition of Lung Lipids and the "Turnover" of Dipolmitoyl Lecithin in Experimental Alveolar Lipo-proteinosis Induced by Inhaled Quartz. Br. J. exp. Path., 55, 384.
HEPPLESTON, A. G., McDERMOTT, M. & COLLINS, M. M. (1975) The Surface Properties of the Lung in Rats with Alveolar Lipo-proteinosis. Br. J. exp. Path., 56, 444. KAHANA, L. & ARONOVITCH, M. (1966) Effects of Sulfur Dioxide on Surface Properties of the Lung. Amer. Rev. resp. Di8., 94, 201. KAHANA, L. & ARONOVITCH, M. (1968) Pulmonary Surface Tension after Sulfur Dioxide Exposure. Amer. Rev. resp. Dis., 98, 34. KING, R. J. & CLEMENTS, J. A. (1972a) Surface Active Materials from Dog Lung. I. Method of Isolation. Am. J. Physiol., 223, 707. KING, R. J. & CLEMENTS, J. A. (1972b) Surface Active Materials from Dog Lung. II. Composition and Physiological Correlations. Am. J. Physiol., 223, 715. KING, R. J. (1974) The Surfactant System of the Lung. Fed. Proc., 33, 2238. LowRy, 0. H., ROSEBROUGH, A., FARR, A. L. & RANDALL, R. J. (1951) Protein Measurement with the Folin Phenol Reagent. J. Biol. Chem., 193, 265. MCDERMOTT, M., WAGNER, J. C., TETLEY, T., HARWOOD, J. & RICHARDS, R. J. (1977) The Effects of Inhaled Silica and Chrysotile on the Elastic Properties of Rat Lungs. Physiological, Physical and Biochemical Studies of Lung Surfactant. IV International Symposium on Inhaled Particles and Vapours, Edinburgh. PFLEGER, R. C. & THOMAS, H. G. (1971) Beagle Dog Pulmonary Surfactant Lipids. Lipid Composition of Pulmonary Tissue Exfoliated Lining Cells, and Surfactant. Arch. intern. Med., 127, 863. RHOADES, R. A. (1972) Effect of Inhaled Carbon on Surface Properties of Rat Lung. Life Sci., 11, 33.
ROBINSON, N. & PHILLIPS, B. M. (1963) Quantitative Thin Layer Chromatography of Serum Phospholipids. Clin. chim. Acta, 8, 385. RoGOZINSKI, M. (1964) The Methanol Sulphuric Acid Esterification Methods. II. An Improved Extraction Procedure. J. Gas Chromatography, 2, 328.
RoSENBERG, E., ALARIE, Y. & ROBILLARD, E. (1962) Effect of Aluminium Dust Inhalation on the Surface Tension of the Alveolar Lining of Rat Lungs. Fed. Proc., 21, 447. SCARPELLI, E. M. (1968) The Surfactant System of the Lung. Philadelphia: Lea and Febiger, p. 31. SHERWIN, R. P. & CARLSON, D. A. (1973) Protein Content of Lung Lavage Fluid of Guinea-pigs Exposed to 0 * 4 ppm Nitrogen Dioxide. Arch. Environ. Health, 27, 90. SUZUKI, Y., CHURG, J. & ONO, T. (1972) Phagocytic Activity of the Alveolar Epithelial Cells in Pulmonary Asbestosis. Am. J. Pathol., 69, 373.
EFFECTS OF CHRYSOTILE ASBESTOS AFTER S02 SORPTION TETLEY, T. D., HEXT, P. M., RICHARDS, R. J. & McDERMOTT, M. (1976) Chrysotile-induced Asbestosis: Charnges in the Free Cell Population, Pulmonary Surfactant and Whole Lung Tissue of Rats.
Br. J. exp. Pathol., 5, 505.
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TIERNEY, D. F. (1974) Lung Metabolism and Biochemistry. Ann. Rev. Physiol., 36, 209. WEBER, K. & OSBORN, M. (1969) The Reliability of Molecular Weight Determination by Dodecyl Sulfate Polyacrylamide Gel Electrophoresis. J. Biol. Chem., 244, 4406.
