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Effect of silica and volcanic ash on the content of lung alveolar and tissue phospholipids
ENVIRONMENTAL
RESEARCH
35, 140-153 (1984)
Effect of Silica and Volcanic Ash on the Content of Lung Alveolar and Tissue Phospholipids’ DOUGLAS J. KORNBRUST* AND GARY E. HATCH~ *Research Triangle Institute, P.O. Box 12194, Research Triangle Park, North Carolina 27709-2194, and )‘Health Effects Research Laboratory, Inhalation Toxicology Branch, VS. Environmental Protection Agency, Research Triangle Park, North Car&u 27711 Received May 1, 1983 Silica or volcanic ash (VA) was administered to rats via intratracheal instillation and the changes in extracellular (i.e., lavage fluid) and tissue phospholipids, as well as various biochemical parameters, were monitored over a 6-month period. VA produced relatively minor (up to 2.8-fold) increases in lung tissue or lavage fluid phospholipids that were maximal at 1 month postinstillation. These increases were quantitatively similar to the increases in protein and DNA content of lung tissue and lavage fluid induced by VA and, thus, may be attributable to hypercellularity and accumulation of cellular breakdown products in the alveolar lumen. Instillation of silica produced a much greater (up to 1l-fold) increase than VA in total phospholipid over time, primarily due to a 1Cfold increase in phosphatidylcholine (PC). The accumulation of PC was more pronounced in the lavage fluid during the first month following silica instillation, but thereafter progressed more rapidly in the lung tissue. The relatively small increases (1.3- to 35fold) in other phospholipids induced by silica appeared to be nonspecific, since they did not differ greatly from the increases in lung weight, DNA, and protein. Collectively, these results indicate that intratracheal instillation of silica induces selective accumulation of lung PC, implying enhanced synthesis and secretion of pulmonary surfactant from alveolar epithelial Type II cells into the lumen.
INTRODUCTION Progressive lung fibrosis is generally recognized as a major biological response to silica inhalation. However, the condition which develops in humans exposed to silica is also characterized by a marked lipid accumulation (Ramirez-R. and Harlan, 1968). Experimental studies have provided a more precise evaluation of the changes in lung lipids induced in laboratory animals exposed by inhalation or intratracheal instillation of silica, usually in the form of quartz. Heppleston et al. (1970) described the response of rat lungs to inhaled silica as being histopathologically indistinguishable from a human disease state of obscure etiology referred to as “alveolar proteinosis.” Histochemical analysis revealed that the amorphous material occupying large areas of the lung parenchyma in silica-exposed rats consisted predominantly of lipid rather than protein. Early studies by Fallon (1937) had shown an increase in total lung lipid and crude phospholipid, and subsequent studies by various investigators confirmed that the major species of t This report has been reviewed by the Health Effects Research Laboratory, U.S. Environment Protection Agency, and approved for publication. Mention of trade names or commercial products does not constitute endorsement or recommendation for use. 140 0013-9351/84 $3.00 Copyright 0 1984 by Academic Press, Inc. Au r&h& of reproduction in any form reserved.
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lung phospholipid, phosphatidylcholine (PC), was present in much greater quantities in the silicotic lung (Marks and Marasas, 1960; Bailey e? al., 1963). The pattern of phospholipid response of the rat lung to an intratracheal instillation of quartz was further characterized by Gnmspan et al. (1973). As early as 2 days following the administration of silica, the quantity of total lung phospholipids was nearly doubled, primarily due to an increase in PC, and this accumulation continued over a 90-day period of study. A similar pattern of phospholipid accumulation in whole lung, characterized by a predominant excess of PC. was observed when rats were exposed to quartz dust via inhalation (Heppleston et al., 1974). Analysis of lung washings from human silicotic patients revealed an analogous phospholipidotic condition characterized by an abnormally high proportion of PC (Ramirez-R. et al., 1971). The studies of Heppleston et al. (1974) also demonstrated that the accumulation of PC in rat lung was primarily attributable to an increase in the dipalmitoyl species (DPPC). Since DPPC is the major surface-tension-lowering component of pulmonary surfactant (King, 1974), the specific accumulation of this lipid suggested that the lung was manufacturing increased amounts of surfactant phospholipids in response to silica. Consistent with this hypothesis, Heppleston et al. (1974) determined that the in vivo incorporation of labeled palmitic acid into DPPC was trebled while its rate of disappearance was doubled, resulting in a net overproduction of DPPC. Other investigators have recently proposed that the phospholipids which accumulate in silicotic lung may be recruited from the liver through a stimulation of phospholipogenesis in liver (Eskelson et al., in press). However, the relative contribution of liver as compared to lung de novo synthesis of lipids remains to be determined. In order to derive a better understanding of the mechanisms underlying silicainduced accumulation of lipids in the lung and the relationship of these changes to the overall pathogenesis of silicosis, it is important to more fully characterize the lipidotic response with respect to the question of whether the lungs are stimulated by silica exposure to produce surfactant lipids. Since pulmonary surfactant is normally secreted into alveolar spaces, overproduction of this material should result in a dramatic rise in extracellular lipids. This hypothesis is supported by the histopathological observation of an abundance of lipid-like material in the alveolar regions of silicotic lungs as well as a large number of “foamy” macrophages that have apparently phagocytized the secreted lipids (Heppleston et aI., 1970). However, previous determinations of the lipid composition of silicotic lungs were performed on a whole-lung basis and thus do not provide information about the relative quantities of lipids present in lung tissue and alveolar regions. Therefore, the present studies were undertaken in order to characterize the extent to which various phospholipids accumulate in alveolar spaces and lung tissue following intratracheal administration of silica to rats, The major phospholipids were monitored, including phosphatidylglycerol (PG), which, in addition to PC, is believed to be a marker for surfactant (Rooney et al., 1974), but has not previously been quantified in silcotic lungs. In addition, changes in lung tissue and lavage fluid protein, DNA, and nonprotein sulfhydryls (NPSH) were determined. For comparative purposes, the same analyses were performed on rats treated with Mt. St. Helens volcanic ash, since it has been reported to induce
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histopathological changes indicative (Sanders et al., 1982).
AND
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of a mild lipoproteinosis
condition
in lung
METHODS
grade silica with a nominal particle size of 5 pm was obtained from Pennsylvania Glass Sand Corporation (Pittsburgh, Pa.). Microspcopic analysis revealed that the material contained a large percentage of fine particles (i.e., less than 2 p,m). Recent reports have indicated that the majority of quartz particles present in the atmosphere are in the course fraction (i.e., greater than 2.5 p,rn aerodynamic diameter (Davis et al., 1982). Therefore, in order to obtain a silica sample with a particle size distribution similar to that which is present in the ambient atmosphere, the crude material was fractionated by a single pass through a Donaldson Accucut Classifier, adjusted to exclude particles with an aerodynamic diameter of less than 7 pm. The final product had a count median diameter of 4.3 p,rn (actual diameter) and a 83%/ 50% (6) ratio of 1.44, as determined by scanning electron microscopy. Volcanic ash was obtained from a site near Mt. St. Helens as previously described (Hatch et al., 1982). The material used had a mean particle diameter of 2.5 pm. Prior to injection, the silica and volcanic ash samples were suspended in isotonic saline and sterilized by boiling for 30 min. Administration of silica and volcanic ash. Male Charles River Sprague-Dawley (cesarean-derived) rats, weighing 280-330 g, were lightly anesthetized with halothane and injected intratracheally with 50 mg of silica or volcanic ash in a 0.5-ml volume of sterile saline. Control rats received sterile saline only. Prior to use, the animals were housed in humidity- and temperature-controlled rooms with 12-hr light/dark cycles and allowed access to food and water ad lib. Preparation of tissue and lavage jluid. Six control and six silica-treated rats were killed by an intraperitoneal injection of an overdose of sodium pentobarbital at 2, 7, 30, 90, and 180 days following instillation. Six volcanic ash-treated rats were killed at 30, 90, and 180 days postinstillation. The abdomen, chest, and neck were opened and the lungs were perfused as described in Kornbrust and Mavis (1980). The lungs plus trachea were excised, rinsed free of blood, and weighed. After insertion of a tracheal catheter, the lungs were lavaged with a total of 2535 ml of 0.9% saline at room temperature. Initially, a volume of 10 ml/g of lung was rinsed in and out of the lungs three times and delivered to a 50-ml collection tube. This procedure was repeated two more times with a volume of saline equal to that recovered from the previous washing. The three washings were pooled, 50 t.~lof 0.01% butylated hydroxytoluene in ethanol was added, and the samples were set aside temporarily in an ice bucket. The amount of phospholipid recovered by this method was found, in separate experiments, to be 62.5 4 7.9% (SD) of the amount which was obtained by 10 lung washes. The lung parenchyma was then separated from the trachea and visible bronchi by scraping with a scalpel. The weight of the residual bronchi and trachea was measured and subtracted from the total lung weight to yield a lung parenchymal tissue weight. The parenchyma was collected in a 15-ml tube and homogenized with a Polytron Pt. 10 (Brinkmann Instruments, Westbury, N.Y.) in 7 ml of icecold 0.15 M KCl/20 mM Tris-HCl/l.O mM EDTA (pH 7.4). The total volume of Preparation
of silica and volcanic ash. Industrial
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homogenate was measured; two 0.7-ml aliquots were removed for nonprotein sulfhydryl determination, and a 0.25-ml aliquot for DNA and protein determinations; and 4.0 ml of homogenate was used for lipid analysis. The pooled lavage fluid was centrifuged for 10 min at IOOg (a procedure which precipitates less than 5% of the total PC or PG, as determined in preliminary experiments). The resulting cell-free supernatant was concentrated to approximately 4-5 ml in a Speed-Vat (Savant Instruments, Hicksville, N.Y.) vacuum concentrator. The cellular pellet obtained from centrifugaton of the lavage fluid was resuspended in 2.0 ml of 0.9% saline. Lipid analyses. Lung homogenate (4.0-ml aliquot) and concentrated cell-free lavage fluid were extracted by the procedure of Bligh and Dyer (1959). The resulting chloroform layer was concentrated to 2-3 ml under a gentle stream of nitrogen, transferred to preweighed vials, and dried completely under N,. An estimate of recovery during extraction was determined to be 85 ~fr 4.1% (mean -+ SD, n = 6) in separate experiments using L-cY-dipalmitoylphosphatidylcholine (Sigma Chemical Co., St. Louis, MO.). All lipid data were corrected by this factor. Total lipid was determined by weighing the vials containing the lipid residue. The lipid residue was dissolved in 4.0 ml of CHCl, and stored in screw-cap glass vials under N, at - 20°C until further analyses were conducted. Total phospholipid (lipid phosphorus) was measured in appropriate size aliquots (5-100 ~1) of the lipid extracts by the procedures of Chen et al. (1956). The remaining lipid extract was divided into two equal portions and the CHCl, solvent was evaporated under N,. The lipid residue from each half of the extract was redissolved in 50 ~1 of CHCl, and spotted on Whatman Linear K (Whatman Inc., Clifton, N.J.) thin layer chromatography plates. One set of plates containing half of the lipid extract was developed in chloroform (65%)/methanol(30%)/acetic acid (5%) (System 1) while the other set of plates was developed in chloroform (65%) methanol (25%)aqueous ammonia (5%) (System 2). Reference plates containing phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylserine (PS), phosphatidylinositol (PI), sphingomyelin, phosphatidic acid, tripalmitin, and dipalmitin standards (all from Sigma Chemical Co.) were run simultaneously. After drying at room temperature in a separate tank under a gentle stream of N,, the locations of the phospholipids on the reference plates were visualized by exposure to iodine vapor. The locations of phospholipids on the sample plates were determined after brief exposure to I2 by comparison to the reference plates. In general, System 1 gave the best separation of PC from all other lipids, while System 2 gave a clear separation of PG and PE from all other lipids. PS and PI did not separate well from each other and were collected together from plates developed in System 1. Areas of silica corresponding to the phospholipids of interest were scraped from the glass plates onto wax paper and collected in glass tubes. The phospholipids were extracted from the silica by the method of Bligh and Dyer (1959), using either 20% acetic acid or 20% ammonia as the aqueous phase for samples derived from System 1 or System 2, respectively. The resulting chloroform layers were removed, evaporated under Nz, and the phospholipids redissolved in 1.0 ml of chloroform. Appropriate size aliquots (lo-400 ~1) were used for lipid phosphorus quantification (Chen ef al., 1956).
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Other biochemical analyses. The nonprotein sulfhydryl content of lung tissue was measured calorimetrically using Ellman’s reagent as described by Sedlak and Lindsay (I%@, with adjustments in volume to accomodate a 0.7-ml sample. Lung DNA content was determined by the method of Labarca and Paigen (1980). A 0.25-ml aliquot of lung homogenate was mixed with 1.75 of 2.0 M NaWO.05 M sodium phosphate/l.0 mM EDTA (pH 7.4) and sonicated briefly (20 set). Duplicate IOO-~1 aliquots were removed and added to 1.9 ml of the NaCl/NaPOd/EDTA buffer. Addition of 10 JL~of 0.2 mg/ml Hoechst Dye H33258 (Calbiochem-Behring Corp., San Diego, Calif.) yielded a fluorescent complex with DNA which was quantified spectrofluorometrically. Duplicate 50-pl aliquots of the eight-fold diluted tissue homogenate (from the DNA determination) were used for measurement of protein by the method of Lowry et al. (1951). Protein concentrations of the cell-free lavage supernatant and the cellular pellet were measured using 100and 50-p,l aliquots of the lavage supernatant and resuspended pellets, respectively. RESULTS
AND DISCUSSION
Effect on Lung Weight, Protein, DNA, and NPSH Lung weight was increased by silica administration in a manner similar to that previously described (Singh et al., 1977) (Fig. 1). A 20% increase was observed after 2 days which progressed to a nearly 2.5-fold difference by 180 days postinstillation. Tissue protein and DNA increased in parallel with lung weight in silicatreated rats. These changes may reflect the morphologic alterations which are characteristic of silicosis, such as Type II alveolar epithelial cell hyperplasia, interstitial pneumonitis, and increased production of collagen by fibroblasts (Heppleston et al., 1970; Dauber et al., 1980; Reiser et al., 1982). Further, at the 3-, and 6-month timepoints, approximately 10% of the increase in lung weight in silica-treated rats was accounted for by the accumulation of lipid in lung tissue (Table 1). In contrast to silica, treatment with volcanic ash produced smaller and, for the most part, non-statistically-significant increases in lung weight, DNA, and protein content, which were maximal at 1 month postinstillation. Neither treatment had an effect on the nonprotein sulfhydryl concentration of lung tissue when the data were expressed per milligram of DNA. It may be more relevant, however, to quantify the lung NPSH content relative to the amount of lipid present in view of the current conceptions about the role of NPSH (i.e., glutathione) in protecting against lipid peroxidation and the recent evidence for the occurence of this reaction in silicotic lung (Gupta and Kaw, 1982). The cellular pellet obtained by low speed centrifugation of the lavage fluid from silica-treated rats was found to contain a 1.4- to 2.5-fold greater amount of DNA than the pellet from saline-treated rats at all timepoints. The protein content of this fraction was increased to a slightly greater extent by silica administration. These changes are consistent with the previously observed increase in the bronchoalveolar free cell population resulting from silica instillation, which has been shown by Morgan et al. (1980) to consist of an influx of polymorphonuclear leukocytes (PMNs) into alveolar spaces and an elevation in the number of alveolar
FIG. 1. Effect of intratracheal instillation of silica (A) or volcanic ash (B) on lung weight, lung tissue DNA and protein, lavage-derived cellular DNA and protein, and cell-free lavage fluid protein. All values are derived from the mean from six treated rats expressed as a percentage of the mean from six control rats. The data were calculated on a whole lung basis except for NPSH which was quantified per milligram of DNA. Control values (mean 2 SD) for 2 and 180 days postinstillation were as follows: lung weight, 1.15 f 0.10 and 1.58 h 0.14 g; tissue DNA, 6.26 2 1.24 and 8.28 2 0.58 mgflung; tissue protein, 105 ? 9 and 140 ? 15 mgihrng; lavage pellet DNA, 18.0 & 9.0 and 27.7 2 6.8 t&lung; lavage pellet protein, 1.24 2 0.72 and 1.12 k 0.42 mg/lung; lavage fluid protein, 2.87 2 0.79 and 3.65 5 1.69 mg/lung; and tissue nonprotein sulfhydryls (NPSH), 0.50 2 0.08 pmole/mg DNA (2 days postinstillation control). Asterisks indicate statistically significant difference from controls, which was determined using Student’s t test and applying the Bonferroni correction factor for multiple I tests (Dayton and Schafer, 1973) to the a priori P value of 0.05, yielding P values of 0.01 and 0.0167 for the silica and volcanic ash data, respectively. 145
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c S VA
PS/PI (~mole/lung)
a Mean k SD from six rats. b It is suspected that these thereby artifactually increased ’ Not done-samples lost. * Statistically different from
c S VA
PG (pmolellung)
1.15 +- 0.09 2.81 k 0.39 (244)* -
C S VA
0.09 + 0.01 0.09 -1- 0.01 (100) -
0.08 4 0.01 0.22 2 0.01 (275)* -
0.05 ‘-c 0.01 0.11 ” 0.02 (220)* -
1.12 t 0.08 11.5 z!z 1.4 (1026)* -
1.59 + 0.25 13.3 ? 1.5 (836)* -
5.5 i 1.0 16.1 z!z 3.9 (292)* -
6.8 16.3 9.8 1.61 12.9 4.60 1.05 10.5 1.69 0.04 0.05 0.04 0.08 0.17 0.11
f ” +‘k ‘2 k +” k e 2 ” k
2.3 3.4 (241)* 2.2 (144) 0.29 2.5 (801)* 2.3 (286)* 0.15 0.9 (lOOO)* 0.14 (161)* 0.01 0.01 (125) 0.01 (100) 0.02 0.01 (213)* 0.02 (138) NDC ND ND
6.0 23.7 7.4 1.78 19.5 1.69 1.09 14.2 1.27 0.05 0.06 0.05 0.11 0.22 0.11 0.09 0.10 0.08
c 2.7 ‘- 5.0 (395-l* ‘- 2.1 (123) 2 0.22 k 5.1 (1095)* 2 0.24 (95) -+ 0.09 4 2.9 (1303)* ” 0.13 (117) 2 0.01 k 0.02 (120) 2 0.01 (100) * 0.03 ” 0.04 (200)* k 0.02 (100) “_ 0.03 % 0.01 (111) 2 0.01 (89)
2 1.6 2 3.9 (337)* zk 1.0 (96) 2 0.18 2 2.4 (636)* +- 0.25 (102)
2 2 2 2 2
0.02 0.02 0.04 0.06 0.03
(147)* (94)
(157) (114)
0.14 k 0.03 0.21 2 0.06 (150) 0.19 2 0.04 (136)
0.11 0.08 0.17 0.25 0.16
1.25 0.13(768)* 9.6 22 2.2 1.30 ? 0.09 (103) 0.07 2 0.03
8.2 27.6 8.0 1.89 11.9 1.92
control using the criteria defined in the legend to Fig. 1 (P < 0.05).
The numbers in parentheses are the data expressed as a percentage of control. samples contained a small amount of contaminating moisture which was not removed during the drying process and the apparent weight of the lipid (see Methods).
0.10 + 0.01 0.11 -t 0.01 (110) -
0.10 2 0.01 0.17 2 0.02 (170)* -
0.06 ” 0.01 0.07 2 0.01 (117) -
1.10 2 0.27 3.55 2 0.39 (209)* -
C S VA
C S VA
11.0 It I.9b 11.3 2 1.1 (103) -
C S VA
PE (pmole/lung)
Lavage fluid Total lipid (mg/lung) Total phospholipid (pmolkmg) PC (*mole/lung)
2 0 z
3 s g i; b z
5
F
[I) P
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macrophages. The influx of PMNs was reported by Morgan et al. to be very rapid during the first 24 hr following silica instillation, but by 48 hr, the number of PNMs had declined dramatically. Therefore, this transient wave of PNM infiltration would not be detected in the present studies since no measurements were performed prior to 2 days postinstillation. However, the increases in the DNA and protein content of the cellular lavage fraction were, in general, less than anticipated since Morgan et al. (1980) had observed a three- to five-fold increase in the total number of cells recovered by lavage. This lack of quantitative correspondence may be attributable to variations in the immunological response evoked by the different silica particle sizes or due to the difference in rat strains used. Adamson and Bowden (1981) have shown that increases in the alveolar macrophage population are more closely related to the number of particles instilled rather than the total dose by weight. Therefore, the relatively large particles employed in the present experiments would constitute a relatively small dose in terms of the number of particles administered, which may account for their weaker effect on macrophage proliferation. Further, recovery of free cells from the three lavages performed in the present study was estimated (assuming 20 pg of DNA/cell) (Bonney, 1974) to be approximately 10% of the total number of cells which reportedly is obtainable by exhaustive lavaging or mincing of lung tissue (Kikkawa and Yoneda, 1974). Therefore, the results probably lack quantitative precision, but may serve as qualitative indicators of changes in the alveolar free cell population. Increases in the protein content of the cell-free lavage fluid were observed at all timepoints following silica instillation. The effect was greatest at 2 days postinstillation and may indicate that considerable transudation of proteinaceous material occurs relatively rapidly following exposure to silica. These findings are consistent with those of Reiser et al. (1982), who observed granular eosinophilic material in the alveoli of the lungs of rats 1 week following exposure to silica. Accumulation of extracellular protein in the alveolar spaces, however, apparently does not occur to nearly the same extent as does lipid (see Table 1). No significant change in the cellular DNA content or cell-free protein content of lavage fluid was produced by exposure to volcanic ash, A significant increase in the amount of protein in the cellular pellet derived from lavage fluid was observed at 1 month postinstillation, which, considering the lack of an accompanying rise in DNA content, may indicate that some degree of hemorrhage had occurred in these animals. In a previous report by Sanders er al. (1982) concerning histopathological changes induced by vocanic ash, localized areas of inflammation and necrosis were noted. However, biochemical evidence for the occurrence of such lesions was not obtained in the present investigation, probably because the lesions were not sufficiently pronounced or widespread to significantly alter lavage fluid composition. Effect on Lipids
Instillation of volcanic ash had no significant effects on lung tissue total lipid or phospholipids, except for a small (30%) increase in total phospholipids ob-
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served at 30 days postinstillation (Table 1). In the lavage fluid, however, total phospholipid was increased almost threefold by 30 days, and the change was accompanied by a significant increase in PC. By 90 days postinstillation, the levels of phospholipid and PC had returned to normal. These changes are consistent with the histopathological findings of Sanders et al. (1982), who observed lipidlike material in the alveolar spaces beginning 37 days after the administration of volcanic ash. However, unlike the short-lived biochemical response demonstrated in the present study, the histopathological condition described by Sanders et al. appeared to persist over a 109-day period of observation, The reason for this discrepancy is not clear, but may be due to differences in ash samples or to differences in response patterns between the different strains of rats used. Exposure to silica produced a progressive increase in total lipid, total phospholipid, and the individual phospholipids (with the exception of the PSlPI fraction) of both lung tissue and lavage fluid (i.e., extracellular lipid). Relative to control levels, the increases were more pronounced for the lavage fluid lipids at the earlier timepoints. For example, total phospholipid and PC in lavage fluid had increased approximately S- and IO-fold, respectively, by 1 week postinstillation whereas only 1.9- and 2.4-fold increases in the tissue lipids were observed at this time. However, in terms of the absolute amount of phospholipid or PC which had accumulated (i.e., the difference between silica and control levels), the changes in the tissue and lavage fluid were roughly comparable at this time. Beyond I week postadministration, the magnitude of the increases in tissue phospholipid greatly exceeded the absolute increases in lavage fluid phospholipids. On a percentage of control basis, however, the accumulation of lipids in lung tissue did not surpass the accumulation in alveolar spaces until 6 months postinstillation. This was attributable not only to a substantial increase in tissue lipids between 3 and 6 months postinstillation, but also to an apparent reduction in the amount of lipid present in the alveoli during this period. These findings suggest that the structural derangement of lung architecture accompanying the progression of the silicotic condition may eventually impair the secretion of lipid into the alveoli, thereby augmenting the accumulation of lipid in the tissue. Alternatively, the development of fibrosis and regions of consolidation (Heppleston et al., 1970; Reiser et al., 1982) in the silicotic lung may reduce the efficiency of the lavage procedure and thereby artifactually decrease the amount of lipid recovered. Another possible explanation is that mechanisms responsible for the removal of alveolar lipid may be incapacitated or overwhelmed during the early stages of silicosis, but may recover or compensate in some manner by 6 months postadministration, resulting in a more efficient removal of alveolar lipid. A disproportionate increase in phospholipid, compared to total lipid, occurred in both lung tissue and lavage fluid. Whereas the phospholipids constituted 27 to 36% and 23 to 30% of the control tissue and lavage total lipids, respectively, administration of silica increased the proportion of phospholipids to 76% of the total tissue lipids at 180 days postinstillation and to 82% of the total lavage lipids at 90 days postinstillation (the timepoints at which maximal enrichment of phospholipids was observed). These figures compare well with the 75 to 80% values obtained by Heppleston et al. (1974) for the relative proportion of whole lung
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phospholipid from silica-treated rats. Similarly, the proportion of phospholipid represented by phosphatidylcholine increased to a variable extent such that, by 180 days postinstillation, the PC fraction was 67 and 80% of the total tissue and lavage phospholipid, respectively, compared to 42 and 67%, respectively, for the control rats at this timepoint. In contrast, the relative amounts of PE, PG, and PUPI as a proportion of the total tissue or lavage fluid phospholipid was decreased by the administration of silica. Thus, the accumulation of PC appears to be selective and is probably not a function of a general increase in lung phospholipid production. Other investigators have observed a qualitatively similar selective increase in whole lung PC (Grunspan et al., 1973; Heppleston et al., 1974), although the extent of the accumulation of PC in the present investigation was somewhat less than in previous studies (e.g., 13-fold vs. 20-fold for Grunspan et al., 1973). This difference in response may be due to the larger silica particles employed in the present investigations which have a lesser surface area per unit mass and may, therefore, have a less potent effect on lung lipid homeostasis. In addition, the larger particles used may not have penetrated as deeply into the respiratory areas of the lung (i.e., the alveoli) as did the smaller particles used in previous investigations. Increases in other major lipid classes have been reported to occur in silicosis. The total amount of lung triacylglycerols was shown to increase following intratracheal administration of silica (Heppleston et al., 1974; Eskelson et al., 1979a). This increase may be attributable in part to the increased cellularity of the silicotic lung since the concentration of triacylglycerols (per gram of lung) actually decreased (Eskelson et al., 1979a). A substantial increase in the cholesterol content of silicotic lungs has been observed (Heppleston et al., 1974; Eskelson et al., 1979b), which appears to constitute an enrichment relative to other neutral lipids (Heppleston et al., 1974). Evidence has been presented that the accumulated lung cholesterol is derived from liver (Eskelson et al., 1979b), but the physiological significance of this accumulation remains to be determined. Thus, cholesterol and phosphatidylcholine are the only species of lipid which have been shown to be progressively concentrated in silicotic lung, whereas the changes in other lipid species appear to parallel the increases in lung dry weight or DNA content and may, therefore, be merely a function of increased numbers of cells. The present studies have also demonstrated that a considerable fraction of the PC which accumulates is secreted into the alveolar lumen. The fact that the fraction of total lung PC residing in the alveolar compartment (i.e., lavage fluid) is increased from 14 to 16% in control rats to a maximum of 45% in silicatreated animals (7 days postinstillation) is indicative of this secretion process. The minor (1.5- to 2.0-fold) increases in PE, PG, and PWPI in the lavage fluid (Table 1) are not necessarily a consequence of secretion by the lung since these lipids may be derived from the cell debris that has been observed in the alveoli of silica-exposed animals (Heppleston et al., 1970; Reiser et al., 1982). Hence, the accumulation of PC in response to silica exposure appears to be exceptional not only in quantitative terms, but also because it is the only species for which there is evidence of an increased synthetic capacity (Heppleston et al., 1974) as well as active secretion from lung tissue induced by exposure to silica. Most of the PC produced in silicotic lung has been shown to be the dipalmitoyl
SILICA
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variety (DPPC) Heppleston et al., 1974). Since DPPC is the major suface-tensionlowering component of pulmonary surfactant (King, 1974), it may be postulated that silica exposure stimulates lung surfactant production. Phosphatidylglycerol also has been suggested to be an important component of pulmonary surfactant (Hallman and Gluck, 1976), yet this species did not accumulate in lung tissue or lavage fluid to nearly the same extent as PC. However, in recent investigation by Suzuki (1982), addition of PG to a reconstituted surfactant complex had a negligible effect on the surface activity of the complex. Thus, the role of PG in pulmonary surfactant is unclear and the present data for lavage PG content are, therefore, difficult to interpret with respect to the relationship between silica exposure and surfactant production. The overproduction of PC observed in the present and previous studies may be attributable in part to the hyperplasia of Type II alveolar epithelial cells induced by silica (Dauber et al., 1980; Reiser et al., 1982), since this cell type is believed to be the site of surfactant synthesis (King, 1974). In addition, Type II cell hyperplasia was shown to be produced by volcanic ash exposure (Sanders et al., 1982), and is probably a sufficient explanation for the moderate accumulation of extracellular phospholipid which occurred following administration of the ash. However, it is likely that additional mechanisms are involved in the silica-induced accumulation of PC since other agents which induce Type II cell hyperplasia, such as cadmium chloride (Hayes et al., 1976), oxidant gases (Mustafa and Tierney, 1978), butylated hydroxytoluene (Adamson et al., 1977), and volcanic ash (Sanders et al., 1982) do not produce lipid changes of the same magnitude and specificity as silica. Impaired removal has been suggested (Ramirez-R. and Harlan, 1968) as the cause of the accumulation of alveolar lipid in the human silicotic condition, although Heppleston et al. (1974) have shown that in rats exposed to silica, massive accumulation of DPPC occurs in spite of a two-fold increase in the turnover rate of this lipid, In an earlier investigation, Heppleston et al. (1970) noted that granular pneumocytes (Type II cells) appeared “hyperactive. ’ ’ Other investigators (Dauber et al., 1980) have described histopathological changes (i.e., an abundance of lamellar bodies) which are consistent with hypertrophy of alveolar Type II cells. Hence, stimulation of PC synthesis within existing or newly formed Type II cells may be the primarily mechanism for the accumulation of this lipid in silicotic lung. The impetus for the increased production of PC is unknown, but may relate to the impairment of the surfactant properties of the alveolar lining layer by accumulated deposits of cholesterol or products of cellular degradation (Heppleston et al., 1975) which, in turn, may serve as a stimulus for the synthesis of surfactant lipid (i.e., PC) as a compensatory mechanism. REFERENCES Adamson, I. Y. R., and Bowden, D. H. (1981). Dose response of the pulmonary macrophagic system to various particulates and its relationship to transepithelial passage of free particles. Exp. Lung Res. 2, 165-175. Adamson, I. Y. R., Bowden, D. H., Cote, M. G., and Witschi, H. P. (1977). Lung injury induced by butylated hydroxytoluene. Lab. Invest. 36, 26-32. Bailey, P., Kilroe-Smith, T. A., and Harington, J. S. (1963). Some lipid constituents of normal and quartz-dusted guinea-pig lungs. Nature (London) 198, 856-857.
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Bligh, E. G., and Dyer, W. J. (1959). A rapid method of total lipid extraction and purification. Cunad. J. Biochem. Physiol. 37, 911-917. Bonney, R. J. (1974). Adult liver parenchymal cells in primary culture: Characteristics and cell recognition standards. In Vitro 10, 103-142. Chen, P S., Toribara, T. Y., and Warner, H. (1956). Microdetermination of phosphorus. Anal. C/rem. 28, 1756-1758. Dauber, J. H., Rossman, M. D., Pietra, G. G., Jimenez, S. A., and Daniele, R. P. (1980). Experimental silicosis. Morphologic and biochemical abnormalities produced by intratracheal instillation of quartz into guinea pig lungs. Amer. .I. Pathol. 101, 595-612. Davis, B. L., Johnson, L. R., Stevens, R. K., Gatz, D. F., and Stensland, G. J. (1982). The observation of sulfate compounds on filter substrates by means of x-ray diffraction. Heterogenous Atmos. Chem. Geophys. Monogr. Ser. 26, 149-156. Dayton, C. M., and Schafer, W. D. (1973). Extended tables of t and chi square for Bonferroni tests with unequal error allocation. .I. Amer. Stat. Assoc. 68, 78-83. Eskelson, C. D., Stiffel, V., Owen, J. A., and Chvapil, M. (1979a). The importance of liver in normal and silicotic lung-lipid homeostasis: 3. Ifiacylglycerols. Physiol. Chem. Phys. 11, 135-141. Eskelson, C. D., Stiffel, V., Owen, J. A., and Chvapil, M. (1979b). The importance of the liver in normal and silicotic lung-lipid homeostasis. 2. Cholesterol, Environ. Res. 19, 432-441. Eskelson, C. D., Stiffel, V., Owen, J. A., and Chvapil, M. The importance of liver in normal and silicotic lung-lipid homeostasis. 1. Phospholipids. J. Environ. Puthol. Toxicol., in press. Grunspan, M., Antweiler, H., and Dehnen, W. (1973). Effect of silica on phospholipids in the rat lung. Brit. J. Ind. Med. 30, 74-77. Gupta, G. S., and Kaw, J. L. (1982). Formation of lipid peroxides in the subcellular fractions of silicotic lungs in rats, Eur. J. Respir. Dis. 63, 183- 187. Hallman, M., and Gluck, L. (1976). Phosphatidylglycerol in lung surfactant. III. Possible modifier of surfactant function. J. Lipid Res. 17, 257-262. Hatch, G. E., Boykin, E., Miller, E J., and Graham, J. A. (1982). Effects of fly ash and its constituents on sensory irritation in mice. Fundam. Appl. Toxicol. 2, 77-81. Hayes, J. A., Snider, G. L., and Palmer, K. C. (1976). The evolution of biochemical damage in the rat lung after acute cadmium exposure. Amer. Rev. Respir. Dis. 113, 121-130. Heppleston, A. G., Fletcher, K., and Wyatt, I. (1974). Changes in the composition of lung lipids and the turnover of dipalmitoyl lecithin in experimental alveolar lipo-proteinosis induced by inhaled quartz. Brit. J. Exp. Pathol. 55, 384-395. Heppleston, A. G., McDermott, M., and Collins, M. M. (1975). The surface properties of the lung in rats with alveolar lipoproteinosis. Brit. J. Exp. Pathol. 56, 444-453. Heppleston, A. G., Wright, N. A., and Stewart, J. A. (1970). Experimental alveolar lipo-proteinosis following the inhalation of silica. J. Puthol. 101, 293-307. Kikkawa, Y., and Yoneda, K. (1974). The type II epithelial cell of the lung. I. Method of isolation. Lab. Invest. 30, 76-84. King, R. J. (1974). The surfactant system of the lung. Fed. Proc. 33, 2238-2247. Kombrust, D. J., and Mavis, R. D. (1980). The effect of paraquat on microsomal lipid peroxidation in vitro and in vivo. Toxicol. Appl. Pharmacol. 53, 323-332. Labarca, C., and Paigen, K. (1980). A simple, rapid, and sensitive DNA assay procedure. Anal. Biochem. 102, 344-352. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. Marks, G. S., and Marasas, L. W. (1960). Changes in the lung lipids of rabbits and guinea pigs exposed to the inhalation of silica dust. Brit. J. Znd. Med. 17, 31-36. Morgan, A., Moores, S. R., Holmes, A., Evans, J. C., Evans, N. H., and Black A. (1980). The effect of quartz, administered by intratracheal instillation, on rat lung. I. The cellular response. Environ. Res. 22, 1-12. Mustafa, M. G., and Tiemey, D. E (1978) Biochemical and metabolic changes in the lungs with oxygen, ozone, and nitrogen dioxide toxicity. Amer. Rev. Respir. Dis. 118, 1061-1090. Ramirez-R., J., and Harlan, W. R. (1968). Pulmonary alveolar proteinosis. Nature and origin of alveolar lipid. Amer. J. Med. 45, 502-512.
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Ramirez-R., .I., Schwartz, B., Dowell. A. R., and Lee, S. D. (1971). Biochemical composition of human pulmonary washings. Arch. Intern. Med. 127, 395-400. Reiser, K. M., Hesterberg, T. W., Haschek, W. M., and Last, J. M. (1982). Experimental silicosis. I. Acute effects of intratracheally instilled quartz on collagen metabolism and morphologic characteristics of rat lungs. Amer. J. Pathol 107, 176-185. Rooney, S. A.. Canavan, P. M., and Motoyama, E. K. (1974). The identification of phosphatidylglycerol in the rat, rabbit, monkey, and human lung. Biochim. Biophys. Acta 360, 56-67. Sanders, C. L., Conklin, A. W., Gelman, R. A., Adee, R. R., and Rhoads, K. (1982). Pulmonary toxicity of Mount St. Helens volcanic ash. Environ. Res. 27, 118-135. Sedlak, J., and Lindsay, R. H. (1968). Estimation of total, protein bound and nonprotein sulfhydryl groups in tissue with Ellman’s reagent. Anal. Biochem. 25, 192-205. Singh, J. Kaw, J. L., Pandey, S. D., Viswanathan, P. N., and Zaidi, S. H. (1977). Amino acid changes and pulmonary response of rats to silica dust. Environ. Res. 14, 452-462. Suzuki, Y. (1982). Effect of protein, cholesterol, and phosphatidylglycerol on the surface activity of the lipid-protein complex reconstituted from pig pulmonary surfactant. J. Lipid Res. 23, 62-69.
RESEARCH
35, 140-153 (1984)
Effect of Silica and Volcanic Ash on the Content of Lung Alveolar and Tissue Phospholipids’ DOUGLAS J. KORNBRUST* AND GARY E. HATCH~ *Research Triangle Institute, P.O. Box 12194, Research Triangle Park, North Carolina 27709-2194, and )‘Health Effects Research Laboratory, Inhalation Toxicology Branch, VS. Environmental Protection Agency, Research Triangle Park, North Car&u 27711 Received May 1, 1983 Silica or volcanic ash (VA) was administered to rats via intratracheal instillation and the changes in extracellular (i.e., lavage fluid) and tissue phospholipids, as well as various biochemical parameters, were monitored over a 6-month period. VA produced relatively minor (up to 2.8-fold) increases in lung tissue or lavage fluid phospholipids that were maximal at 1 month postinstillation. These increases were quantitatively similar to the increases in protein and DNA content of lung tissue and lavage fluid induced by VA and, thus, may be attributable to hypercellularity and accumulation of cellular breakdown products in the alveolar lumen. Instillation of silica produced a much greater (up to 1l-fold) increase than VA in total phospholipid over time, primarily due to a 1Cfold increase in phosphatidylcholine (PC). The accumulation of PC was more pronounced in the lavage fluid during the first month following silica instillation, but thereafter progressed more rapidly in the lung tissue. The relatively small increases (1.3- to 35fold) in other phospholipids induced by silica appeared to be nonspecific, since they did not differ greatly from the increases in lung weight, DNA, and protein. Collectively, these results indicate that intratracheal instillation of silica induces selective accumulation of lung PC, implying enhanced synthesis and secretion of pulmonary surfactant from alveolar epithelial Type II cells into the lumen.
INTRODUCTION Progressive lung fibrosis is generally recognized as a major biological response to silica inhalation. However, the condition which develops in humans exposed to silica is also characterized by a marked lipid accumulation (Ramirez-R. and Harlan, 1968). Experimental studies have provided a more precise evaluation of the changes in lung lipids induced in laboratory animals exposed by inhalation or intratracheal instillation of silica, usually in the form of quartz. Heppleston et al. (1970) described the response of rat lungs to inhaled silica as being histopathologically indistinguishable from a human disease state of obscure etiology referred to as “alveolar proteinosis.” Histochemical analysis revealed that the amorphous material occupying large areas of the lung parenchyma in silica-exposed rats consisted predominantly of lipid rather than protein. Early studies by Fallon (1937) had shown an increase in total lung lipid and crude phospholipid, and subsequent studies by various investigators confirmed that the major species of t This report has been reviewed by the Health Effects Research Laboratory, U.S. Environment Protection Agency, and approved for publication. Mention of trade names or commercial products does not constitute endorsement or recommendation for use. 140 0013-9351/84 $3.00 Copyright 0 1984 by Academic Press, Inc. Au r&h& of reproduction in any form reserved.
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lung phospholipid, phosphatidylcholine (PC), was present in much greater quantities in the silicotic lung (Marks and Marasas, 1960; Bailey e? al., 1963). The pattern of phospholipid response of the rat lung to an intratracheal instillation of quartz was further characterized by Gnmspan et al. (1973). As early as 2 days following the administration of silica, the quantity of total lung phospholipids was nearly doubled, primarily due to an increase in PC, and this accumulation continued over a 90-day period of study. A similar pattern of phospholipid accumulation in whole lung, characterized by a predominant excess of PC. was observed when rats were exposed to quartz dust via inhalation (Heppleston et al., 1974). Analysis of lung washings from human silicotic patients revealed an analogous phospholipidotic condition characterized by an abnormally high proportion of PC (Ramirez-R. et al., 1971). The studies of Heppleston et al. (1974) also demonstrated that the accumulation of PC in rat lung was primarily attributable to an increase in the dipalmitoyl species (DPPC). Since DPPC is the major surface-tension-lowering component of pulmonary surfactant (King, 1974), the specific accumulation of this lipid suggested that the lung was manufacturing increased amounts of surfactant phospholipids in response to silica. Consistent with this hypothesis, Heppleston et al. (1974) determined that the in vivo incorporation of labeled palmitic acid into DPPC was trebled while its rate of disappearance was doubled, resulting in a net overproduction of DPPC. Other investigators have recently proposed that the phospholipids which accumulate in silicotic lung may be recruited from the liver through a stimulation of phospholipogenesis in liver (Eskelson et al., in press). However, the relative contribution of liver as compared to lung de novo synthesis of lipids remains to be determined. In order to derive a better understanding of the mechanisms underlying silicainduced accumulation of lipids in the lung and the relationship of these changes to the overall pathogenesis of silicosis, it is important to more fully characterize the lipidotic response with respect to the question of whether the lungs are stimulated by silica exposure to produce surfactant lipids. Since pulmonary surfactant is normally secreted into alveolar spaces, overproduction of this material should result in a dramatic rise in extracellular lipids. This hypothesis is supported by the histopathological observation of an abundance of lipid-like material in the alveolar regions of silicotic lungs as well as a large number of “foamy” macrophages that have apparently phagocytized the secreted lipids (Heppleston et aI., 1970). However, previous determinations of the lipid composition of silicotic lungs were performed on a whole-lung basis and thus do not provide information about the relative quantities of lipids present in lung tissue and alveolar regions. Therefore, the present studies were undertaken in order to characterize the extent to which various phospholipids accumulate in alveolar spaces and lung tissue following intratracheal administration of silica to rats, The major phospholipids were monitored, including phosphatidylglycerol (PG), which, in addition to PC, is believed to be a marker for surfactant (Rooney et al., 1974), but has not previously been quantified in silcotic lungs. In addition, changes in lung tissue and lavage fluid protein, DNA, and nonprotein sulfhydryls (NPSH) were determined. For comparative purposes, the same analyses were performed on rats treated with Mt. St. Helens volcanic ash, since it has been reported to induce
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histopathological changes indicative (Sanders et al., 1982).
AND
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of a mild lipoproteinosis
condition
in lung
METHODS
grade silica with a nominal particle size of 5 pm was obtained from Pennsylvania Glass Sand Corporation (Pittsburgh, Pa.). Microspcopic analysis revealed that the material contained a large percentage of fine particles (i.e., less than 2 p,m). Recent reports have indicated that the majority of quartz particles present in the atmosphere are in the course fraction (i.e., greater than 2.5 p,rn aerodynamic diameter (Davis et al., 1982). Therefore, in order to obtain a silica sample with a particle size distribution similar to that which is present in the ambient atmosphere, the crude material was fractionated by a single pass through a Donaldson Accucut Classifier, adjusted to exclude particles with an aerodynamic diameter of less than 7 pm. The final product had a count median diameter of 4.3 p,rn (actual diameter) and a 83%/ 50% (6) ratio of 1.44, as determined by scanning electron microscopy. Volcanic ash was obtained from a site near Mt. St. Helens as previously described (Hatch et al., 1982). The material used had a mean particle diameter of 2.5 pm. Prior to injection, the silica and volcanic ash samples were suspended in isotonic saline and sterilized by boiling for 30 min. Administration of silica and volcanic ash. Male Charles River Sprague-Dawley (cesarean-derived) rats, weighing 280-330 g, were lightly anesthetized with halothane and injected intratracheally with 50 mg of silica or volcanic ash in a 0.5-ml volume of sterile saline. Control rats received sterile saline only. Prior to use, the animals were housed in humidity- and temperature-controlled rooms with 12-hr light/dark cycles and allowed access to food and water ad lib. Preparation of tissue and lavage jluid. Six control and six silica-treated rats were killed by an intraperitoneal injection of an overdose of sodium pentobarbital at 2, 7, 30, 90, and 180 days following instillation. Six volcanic ash-treated rats were killed at 30, 90, and 180 days postinstillation. The abdomen, chest, and neck were opened and the lungs were perfused as described in Kornbrust and Mavis (1980). The lungs plus trachea were excised, rinsed free of blood, and weighed. After insertion of a tracheal catheter, the lungs were lavaged with a total of 2535 ml of 0.9% saline at room temperature. Initially, a volume of 10 ml/g of lung was rinsed in and out of the lungs three times and delivered to a 50-ml collection tube. This procedure was repeated two more times with a volume of saline equal to that recovered from the previous washing. The three washings were pooled, 50 t.~lof 0.01% butylated hydroxytoluene in ethanol was added, and the samples were set aside temporarily in an ice bucket. The amount of phospholipid recovered by this method was found, in separate experiments, to be 62.5 4 7.9% (SD) of the amount which was obtained by 10 lung washes. The lung parenchyma was then separated from the trachea and visible bronchi by scraping with a scalpel. The weight of the residual bronchi and trachea was measured and subtracted from the total lung weight to yield a lung parenchymal tissue weight. The parenchyma was collected in a 15-ml tube and homogenized with a Polytron Pt. 10 (Brinkmann Instruments, Westbury, N.Y.) in 7 ml of icecold 0.15 M KCl/20 mM Tris-HCl/l.O mM EDTA (pH 7.4). The total volume of Preparation
of silica and volcanic ash. Industrial
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homogenate was measured; two 0.7-ml aliquots were removed for nonprotein sulfhydryl determination, and a 0.25-ml aliquot for DNA and protein determinations; and 4.0 ml of homogenate was used for lipid analysis. The pooled lavage fluid was centrifuged for 10 min at IOOg (a procedure which precipitates less than 5% of the total PC or PG, as determined in preliminary experiments). The resulting cell-free supernatant was concentrated to approximately 4-5 ml in a Speed-Vat (Savant Instruments, Hicksville, N.Y.) vacuum concentrator. The cellular pellet obtained from centrifugaton of the lavage fluid was resuspended in 2.0 ml of 0.9% saline. Lipid analyses. Lung homogenate (4.0-ml aliquot) and concentrated cell-free lavage fluid were extracted by the procedure of Bligh and Dyer (1959). The resulting chloroform layer was concentrated to 2-3 ml under a gentle stream of nitrogen, transferred to preweighed vials, and dried completely under N,. An estimate of recovery during extraction was determined to be 85 ~fr 4.1% (mean -+ SD, n = 6) in separate experiments using L-cY-dipalmitoylphosphatidylcholine (Sigma Chemical Co., St. Louis, MO.). All lipid data were corrected by this factor. Total lipid was determined by weighing the vials containing the lipid residue. The lipid residue was dissolved in 4.0 ml of CHCl, and stored in screw-cap glass vials under N, at - 20°C until further analyses were conducted. Total phospholipid (lipid phosphorus) was measured in appropriate size aliquots (5-100 ~1) of the lipid extracts by the procedures of Chen et al. (1956). The remaining lipid extract was divided into two equal portions and the CHCl, solvent was evaporated under N,. The lipid residue from each half of the extract was redissolved in 50 ~1 of CHCl, and spotted on Whatman Linear K (Whatman Inc., Clifton, N.J.) thin layer chromatography plates. One set of plates containing half of the lipid extract was developed in chloroform (65%)/methanol(30%)/acetic acid (5%) (System 1) while the other set of plates was developed in chloroform (65%) methanol (25%)aqueous ammonia (5%) (System 2). Reference plates containing phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylserine (PS), phosphatidylinositol (PI), sphingomyelin, phosphatidic acid, tripalmitin, and dipalmitin standards (all from Sigma Chemical Co.) were run simultaneously. After drying at room temperature in a separate tank under a gentle stream of N,, the locations of the phospholipids on the reference plates were visualized by exposure to iodine vapor. The locations of phospholipids on the sample plates were determined after brief exposure to I2 by comparison to the reference plates. In general, System 1 gave the best separation of PC from all other lipids, while System 2 gave a clear separation of PG and PE from all other lipids. PS and PI did not separate well from each other and were collected together from plates developed in System 1. Areas of silica corresponding to the phospholipids of interest were scraped from the glass plates onto wax paper and collected in glass tubes. The phospholipids were extracted from the silica by the method of Bligh and Dyer (1959), using either 20% acetic acid or 20% ammonia as the aqueous phase for samples derived from System 1 or System 2, respectively. The resulting chloroform layers were removed, evaporated under Nz, and the phospholipids redissolved in 1.0 ml of chloroform. Appropriate size aliquots (lo-400 ~1) were used for lipid phosphorus quantification (Chen ef al., 1956).
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Other biochemical analyses. The nonprotein sulfhydryl content of lung tissue was measured calorimetrically using Ellman’s reagent as described by Sedlak and Lindsay (I%@, with adjustments in volume to accomodate a 0.7-ml sample. Lung DNA content was determined by the method of Labarca and Paigen (1980). A 0.25-ml aliquot of lung homogenate was mixed with 1.75 of 2.0 M NaWO.05 M sodium phosphate/l.0 mM EDTA (pH 7.4) and sonicated briefly (20 set). Duplicate IOO-~1 aliquots were removed and added to 1.9 ml of the NaCl/NaPOd/EDTA buffer. Addition of 10 JL~of 0.2 mg/ml Hoechst Dye H33258 (Calbiochem-Behring Corp., San Diego, Calif.) yielded a fluorescent complex with DNA which was quantified spectrofluorometrically. Duplicate 50-pl aliquots of the eight-fold diluted tissue homogenate (from the DNA determination) were used for measurement of protein by the method of Lowry et al. (1951). Protein concentrations of the cell-free lavage supernatant and the cellular pellet were measured using 100and 50-p,l aliquots of the lavage supernatant and resuspended pellets, respectively. RESULTS
AND DISCUSSION
Effect on Lung Weight, Protein, DNA, and NPSH Lung weight was increased by silica administration in a manner similar to that previously described (Singh et al., 1977) (Fig. 1). A 20% increase was observed after 2 days which progressed to a nearly 2.5-fold difference by 180 days postinstillation. Tissue protein and DNA increased in parallel with lung weight in silicatreated rats. These changes may reflect the morphologic alterations which are characteristic of silicosis, such as Type II alveolar epithelial cell hyperplasia, interstitial pneumonitis, and increased production of collagen by fibroblasts (Heppleston et al., 1970; Dauber et al., 1980; Reiser et al., 1982). Further, at the 3-, and 6-month timepoints, approximately 10% of the increase in lung weight in silica-treated rats was accounted for by the accumulation of lipid in lung tissue (Table 1). In contrast to silica, treatment with volcanic ash produced smaller and, for the most part, non-statistically-significant increases in lung weight, DNA, and protein content, which were maximal at 1 month postinstillation. Neither treatment had an effect on the nonprotein sulfhydryl concentration of lung tissue when the data were expressed per milligram of DNA. It may be more relevant, however, to quantify the lung NPSH content relative to the amount of lipid present in view of the current conceptions about the role of NPSH (i.e., glutathione) in protecting against lipid peroxidation and the recent evidence for the occurence of this reaction in silicotic lung (Gupta and Kaw, 1982). The cellular pellet obtained by low speed centrifugation of the lavage fluid from silica-treated rats was found to contain a 1.4- to 2.5-fold greater amount of DNA than the pellet from saline-treated rats at all timepoints. The protein content of this fraction was increased to a slightly greater extent by silica administration. These changes are consistent with the previously observed increase in the bronchoalveolar free cell population resulting from silica instillation, which has been shown by Morgan et al. (1980) to consist of an influx of polymorphonuclear leukocytes (PMNs) into alveolar spaces and an elevation in the number of alveolar
FIG. 1. Effect of intratracheal instillation of silica (A) or volcanic ash (B) on lung weight, lung tissue DNA and protein, lavage-derived cellular DNA and protein, and cell-free lavage fluid protein. All values are derived from the mean from six treated rats expressed as a percentage of the mean from six control rats. The data were calculated on a whole lung basis except for NPSH which was quantified per milligram of DNA. Control values (mean 2 SD) for 2 and 180 days postinstillation were as follows: lung weight, 1.15 f 0.10 and 1.58 h 0.14 g; tissue DNA, 6.26 2 1.24 and 8.28 2 0.58 mgflung; tissue protein, 105 ? 9 and 140 ? 15 mgihrng; lavage pellet DNA, 18.0 & 9.0 and 27.7 2 6.8 t&lung; lavage pellet protein, 1.24 2 0.72 and 1.12 k 0.42 mg/lung; lavage fluid protein, 2.87 2 0.79 and 3.65 5 1.69 mg/lung; and tissue nonprotein sulfhydryls (NPSH), 0.50 2 0.08 pmole/mg DNA (2 days postinstillation control). Asterisks indicate statistically significant difference from controls, which was determined using Student’s t test and applying the Bonferroni correction factor for multiple I tests (Dayton and Schafer, 1973) to the a priori P value of 0.05, yielding P values of 0.01 and 0.0167 for the silica and volcanic ash data, respectively. 145
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c S VA
PS/PI (~mole/lung)
a Mean k SD from six rats. b It is suspected that these thereby artifactually increased ’ Not done-samples lost. * Statistically different from
c S VA
PG (pmolellung)
1.15 +- 0.09 2.81 k 0.39 (244)* -
C S VA
0.09 + 0.01 0.09 -1- 0.01 (100) -
0.08 4 0.01 0.22 2 0.01 (275)* -
0.05 ‘-c 0.01 0.11 ” 0.02 (220)* -
1.12 t 0.08 11.5 z!z 1.4 (1026)* -
1.59 + 0.25 13.3 ? 1.5 (836)* -
5.5 i 1.0 16.1 z!z 3.9 (292)* -
6.8 16.3 9.8 1.61 12.9 4.60 1.05 10.5 1.69 0.04 0.05 0.04 0.08 0.17 0.11
f ” +‘k ‘2 k +” k e 2 ” k
2.3 3.4 (241)* 2.2 (144) 0.29 2.5 (801)* 2.3 (286)* 0.15 0.9 (lOOO)* 0.14 (161)* 0.01 0.01 (125) 0.01 (100) 0.02 0.01 (213)* 0.02 (138) NDC ND ND
6.0 23.7 7.4 1.78 19.5 1.69 1.09 14.2 1.27 0.05 0.06 0.05 0.11 0.22 0.11 0.09 0.10 0.08
c 2.7 ‘- 5.0 (395-l* ‘- 2.1 (123) 2 0.22 k 5.1 (1095)* 2 0.24 (95) -+ 0.09 4 2.9 (1303)* ” 0.13 (117) 2 0.01 k 0.02 (120) 2 0.01 (100) * 0.03 ” 0.04 (200)* k 0.02 (100) “_ 0.03 % 0.01 (111) 2 0.01 (89)
2 1.6 2 3.9 (337)* zk 1.0 (96) 2 0.18 2 2.4 (636)* +- 0.25 (102)
2 2 2 2 2
0.02 0.02 0.04 0.06 0.03
(147)* (94)
(157) (114)
0.14 k 0.03 0.21 2 0.06 (150) 0.19 2 0.04 (136)
0.11 0.08 0.17 0.25 0.16
1.25 0.13(768)* 9.6 22 2.2 1.30 ? 0.09 (103) 0.07 2 0.03
8.2 27.6 8.0 1.89 11.9 1.92
control using the criteria defined in the legend to Fig. 1 (P < 0.05).
The numbers in parentheses are the data expressed as a percentage of control. samples contained a small amount of contaminating moisture which was not removed during the drying process and the apparent weight of the lipid (see Methods).
0.10 + 0.01 0.11 -t 0.01 (110) -
0.10 2 0.01 0.17 2 0.02 (170)* -
0.06 ” 0.01 0.07 2 0.01 (117) -
1.10 2 0.27 3.55 2 0.39 (209)* -
C S VA
C S VA
11.0 It I.9b 11.3 2 1.1 (103) -
C S VA
PE (pmole/lung)
Lavage fluid Total lipid (mg/lung) Total phospholipid (pmolkmg) PC (*mole/lung)
2 0 z
3 s g i; b z
5
F
[I) P
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macrophages. The influx of PMNs was reported by Morgan et al. to be very rapid during the first 24 hr following silica instillation, but by 48 hr, the number of PNMs had declined dramatically. Therefore, this transient wave of PNM infiltration would not be detected in the present studies since no measurements were performed prior to 2 days postinstillation. However, the increases in the DNA and protein content of the cellular lavage fraction were, in general, less than anticipated since Morgan et al. (1980) had observed a three- to five-fold increase in the total number of cells recovered by lavage. This lack of quantitative correspondence may be attributable to variations in the immunological response evoked by the different silica particle sizes or due to the difference in rat strains used. Adamson and Bowden (1981) have shown that increases in the alveolar macrophage population are more closely related to the number of particles instilled rather than the total dose by weight. Therefore, the relatively large particles employed in the present experiments would constitute a relatively small dose in terms of the number of particles administered, which may account for their weaker effect on macrophage proliferation. Further, recovery of free cells from the three lavages performed in the present study was estimated (assuming 20 pg of DNA/cell) (Bonney, 1974) to be approximately 10% of the total number of cells which reportedly is obtainable by exhaustive lavaging or mincing of lung tissue (Kikkawa and Yoneda, 1974). Therefore, the results probably lack quantitative precision, but may serve as qualitative indicators of changes in the alveolar free cell population. Increases in the protein content of the cell-free lavage fluid were observed at all timepoints following silica instillation. The effect was greatest at 2 days postinstillation and may indicate that considerable transudation of proteinaceous material occurs relatively rapidly following exposure to silica. These findings are consistent with those of Reiser et al. (1982), who observed granular eosinophilic material in the alveoli of the lungs of rats 1 week following exposure to silica. Accumulation of extracellular protein in the alveolar spaces, however, apparently does not occur to nearly the same extent as does lipid (see Table 1). No significant change in the cellular DNA content or cell-free protein content of lavage fluid was produced by exposure to volcanic ash, A significant increase in the amount of protein in the cellular pellet derived from lavage fluid was observed at 1 month postinstillation, which, considering the lack of an accompanying rise in DNA content, may indicate that some degree of hemorrhage had occurred in these animals. In a previous report by Sanders er al. (1982) concerning histopathological changes induced by vocanic ash, localized areas of inflammation and necrosis were noted. However, biochemical evidence for the occurrence of such lesions was not obtained in the present investigation, probably because the lesions were not sufficiently pronounced or widespread to significantly alter lavage fluid composition. Effect on Lipids
Instillation of volcanic ash had no significant effects on lung tissue total lipid or phospholipids, except for a small (30%) increase in total phospholipids ob-
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served at 30 days postinstillation (Table 1). In the lavage fluid, however, total phospholipid was increased almost threefold by 30 days, and the change was accompanied by a significant increase in PC. By 90 days postinstillation, the levels of phospholipid and PC had returned to normal. These changes are consistent with the histopathological findings of Sanders et al. (1982), who observed lipidlike material in the alveolar spaces beginning 37 days after the administration of volcanic ash. However, unlike the short-lived biochemical response demonstrated in the present study, the histopathological condition described by Sanders et al. appeared to persist over a 109-day period of observation, The reason for this discrepancy is not clear, but may be due to differences in ash samples or to differences in response patterns between the different strains of rats used. Exposure to silica produced a progressive increase in total lipid, total phospholipid, and the individual phospholipids (with the exception of the PSlPI fraction) of both lung tissue and lavage fluid (i.e., extracellular lipid). Relative to control levels, the increases were more pronounced for the lavage fluid lipids at the earlier timepoints. For example, total phospholipid and PC in lavage fluid had increased approximately S- and IO-fold, respectively, by 1 week postinstillation whereas only 1.9- and 2.4-fold increases in the tissue lipids were observed at this time. However, in terms of the absolute amount of phospholipid or PC which had accumulated (i.e., the difference between silica and control levels), the changes in the tissue and lavage fluid were roughly comparable at this time. Beyond I week postadministration, the magnitude of the increases in tissue phospholipid greatly exceeded the absolute increases in lavage fluid phospholipids. On a percentage of control basis, however, the accumulation of lipids in lung tissue did not surpass the accumulation in alveolar spaces until 6 months postinstillation. This was attributable not only to a substantial increase in tissue lipids between 3 and 6 months postinstillation, but also to an apparent reduction in the amount of lipid present in the alveoli during this period. These findings suggest that the structural derangement of lung architecture accompanying the progression of the silicotic condition may eventually impair the secretion of lipid into the alveoli, thereby augmenting the accumulation of lipid in the tissue. Alternatively, the development of fibrosis and regions of consolidation (Heppleston et al., 1970; Reiser et al., 1982) in the silicotic lung may reduce the efficiency of the lavage procedure and thereby artifactually decrease the amount of lipid recovered. Another possible explanation is that mechanisms responsible for the removal of alveolar lipid may be incapacitated or overwhelmed during the early stages of silicosis, but may recover or compensate in some manner by 6 months postadministration, resulting in a more efficient removal of alveolar lipid. A disproportionate increase in phospholipid, compared to total lipid, occurred in both lung tissue and lavage fluid. Whereas the phospholipids constituted 27 to 36% and 23 to 30% of the control tissue and lavage total lipids, respectively, administration of silica increased the proportion of phospholipids to 76% of the total tissue lipids at 180 days postinstillation and to 82% of the total lavage lipids at 90 days postinstillation (the timepoints at which maximal enrichment of phospholipids was observed). These figures compare well with the 75 to 80% values obtained by Heppleston et al. (1974) for the relative proportion of whole lung
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phospholipid from silica-treated rats. Similarly, the proportion of phospholipid represented by phosphatidylcholine increased to a variable extent such that, by 180 days postinstillation, the PC fraction was 67 and 80% of the total tissue and lavage phospholipid, respectively, compared to 42 and 67%, respectively, for the control rats at this timepoint. In contrast, the relative amounts of PE, PG, and PUPI as a proportion of the total tissue or lavage fluid phospholipid was decreased by the administration of silica. Thus, the accumulation of PC appears to be selective and is probably not a function of a general increase in lung phospholipid production. Other investigators have observed a qualitatively similar selective increase in whole lung PC (Grunspan et al., 1973; Heppleston et al., 1974), although the extent of the accumulation of PC in the present investigation was somewhat less than in previous studies (e.g., 13-fold vs. 20-fold for Grunspan et al., 1973). This difference in response may be due to the larger silica particles employed in the present investigations which have a lesser surface area per unit mass and may, therefore, have a less potent effect on lung lipid homeostasis. In addition, the larger particles used may not have penetrated as deeply into the respiratory areas of the lung (i.e., the alveoli) as did the smaller particles used in previous investigations. Increases in other major lipid classes have been reported to occur in silicosis. The total amount of lung triacylglycerols was shown to increase following intratracheal administration of silica (Heppleston et al., 1974; Eskelson et al., 1979a). This increase may be attributable in part to the increased cellularity of the silicotic lung since the concentration of triacylglycerols (per gram of lung) actually decreased (Eskelson et al., 1979a). A substantial increase in the cholesterol content of silicotic lungs has been observed (Heppleston et al., 1974; Eskelson et al., 1979b), which appears to constitute an enrichment relative to other neutral lipids (Heppleston et al., 1974). Evidence has been presented that the accumulated lung cholesterol is derived from liver (Eskelson et al., 1979b), but the physiological significance of this accumulation remains to be determined. Thus, cholesterol and phosphatidylcholine are the only species of lipid which have been shown to be progressively concentrated in silicotic lung, whereas the changes in other lipid species appear to parallel the increases in lung dry weight or DNA content and may, therefore, be merely a function of increased numbers of cells. The present studies have also demonstrated that a considerable fraction of the PC which accumulates is secreted into the alveolar lumen. The fact that the fraction of total lung PC residing in the alveolar compartment (i.e., lavage fluid) is increased from 14 to 16% in control rats to a maximum of 45% in silicatreated animals (7 days postinstillation) is indicative of this secretion process. The minor (1.5- to 2.0-fold) increases in PE, PG, and PWPI in the lavage fluid (Table 1) are not necessarily a consequence of secretion by the lung since these lipids may be derived from the cell debris that has been observed in the alveoli of silica-exposed animals (Heppleston et al., 1970; Reiser et al., 1982). Hence, the accumulation of PC in response to silica exposure appears to be exceptional not only in quantitative terms, but also because it is the only species for which there is evidence of an increased synthetic capacity (Heppleston et al., 1974) as well as active secretion from lung tissue induced by exposure to silica. Most of the PC produced in silicotic lung has been shown to be the dipalmitoyl
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variety (DPPC) Heppleston et al., 1974). Since DPPC is the major suface-tensionlowering component of pulmonary surfactant (King, 1974), it may be postulated that silica exposure stimulates lung surfactant production. Phosphatidylglycerol also has been suggested to be an important component of pulmonary surfactant (Hallman and Gluck, 1976), yet this species did not accumulate in lung tissue or lavage fluid to nearly the same extent as PC. However, in recent investigation by Suzuki (1982), addition of PG to a reconstituted surfactant complex had a negligible effect on the surface activity of the complex. Thus, the role of PG in pulmonary surfactant is unclear and the present data for lavage PG content are, therefore, difficult to interpret with respect to the relationship between silica exposure and surfactant production. The overproduction of PC observed in the present and previous studies may be attributable in part to the hyperplasia of Type II alveolar epithelial cells induced by silica (Dauber et al., 1980; Reiser et al., 1982), since this cell type is believed to be the site of surfactant synthesis (King, 1974). In addition, Type II cell hyperplasia was shown to be produced by volcanic ash exposure (Sanders et al., 1982), and is probably a sufficient explanation for the moderate accumulation of extracellular phospholipid which occurred following administration of the ash. However, it is likely that additional mechanisms are involved in the silica-induced accumulation of PC since other agents which induce Type II cell hyperplasia, such as cadmium chloride (Hayes et al., 1976), oxidant gases (Mustafa and Tierney, 1978), butylated hydroxytoluene (Adamson et al., 1977), and volcanic ash (Sanders et al., 1982) do not produce lipid changes of the same magnitude and specificity as silica. Impaired removal has been suggested (Ramirez-R. and Harlan, 1968) as the cause of the accumulation of alveolar lipid in the human silicotic condition, although Heppleston et al. (1974) have shown that in rats exposed to silica, massive accumulation of DPPC occurs in spite of a two-fold increase in the turnover rate of this lipid, In an earlier investigation, Heppleston et al. (1970) noted that granular pneumocytes (Type II cells) appeared “hyperactive. ’ ’ Other investigators (Dauber et al., 1980) have described histopathological changes (i.e., an abundance of lamellar bodies) which are consistent with hypertrophy of alveolar Type II cells. Hence, stimulation of PC synthesis within existing or newly formed Type II cells may be the primarily mechanism for the accumulation of this lipid in silicotic lung. The impetus for the increased production of PC is unknown, but may relate to the impairment of the surfactant properties of the alveolar lining layer by accumulated deposits of cholesterol or products of cellular degradation (Heppleston et al., 1975) which, in turn, may serve as a stimulus for the synthesis of surfactant lipid (i.e., PC) as a compensatory mechanism. REFERENCES Adamson, I. Y. R., and Bowden, D. H. (1981). Dose response of the pulmonary macrophagic system to various particulates and its relationship to transepithelial passage of free particles. Exp. Lung Res. 2, 165-175. Adamson, I. Y. R., Bowden, D. H., Cote, M. G., and Witschi, H. P. (1977). Lung injury induced by butylated hydroxytoluene. Lab. Invest. 36, 26-32. Bailey, P., Kilroe-Smith, T. A., and Harington, J. S. (1963). Some lipid constituents of normal and quartz-dusted guinea-pig lungs. Nature (London) 198, 856-857.
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Ramirez-R., .I., Schwartz, B., Dowell. A. R., and Lee, S. D. (1971). Biochemical composition of human pulmonary washings. Arch. Intern. Med. 127, 395-400. Reiser, K. M., Hesterberg, T. W., Haschek, W. M., and Last, J. M. (1982). Experimental silicosis. I. Acute effects of intratracheally instilled quartz on collagen metabolism and morphologic characteristics of rat lungs. Amer. J. Pathol 107, 176-185. Rooney, S. A.. Canavan, P. M., and Motoyama, E. K. (1974). The identification of phosphatidylglycerol in the rat, rabbit, monkey, and human lung. Biochim. Biophys. Acta 360, 56-67. Sanders, C. L., Conklin, A. W., Gelman, R. A., Adee, R. R., and Rhoads, K. (1982). Pulmonary toxicity of Mount St. Helens volcanic ash. Environ. Res. 27, 118-135. Sedlak, J., and Lindsay, R. H. (1968). Estimation of total, protein bound and nonprotein sulfhydryl groups in tissue with Ellman’s reagent. Anal. Biochem. 25, 192-205. Singh, J. Kaw, J. L., Pandey, S. D., Viswanathan, P. N., and Zaidi, S. H. (1977). Amino acid changes and pulmonary response of rats to silica dust. Environ. Res. 14, 452-462. Suzuki, Y. (1982). Effect of protein, cholesterol, and phosphatidylglycerol on the surface activity of the lipid-protein complex reconstituted from pig pulmonary surfactant. J. Lipid Res. 23, 62-69.