Effects of chronic exposure to ozone on pulmonary lipids in rats

Toxicology, 64 (1990) 313--324 Elsevier Scientific Publishers Ireland Ltd.

Effects of chronic exposure to ozone on pulmonary lipids in rats Elaine S. Wright, David M. White* and Kathleen L. Smiler Biomedical Science Department, General Motors Research Laboratories, Warren, All 48009 (U. S.A.) (Received Octoberl2th, 1990 ; accepted February 23th, 1990)

Summary Ozone is the most toxic component of photochemical oxidant air pollution. Exposure to high concentrations of ozone produces a variety of toxic effects in the lung, but it is not known to what extent prolonged exposure to low concentrations of ozone may contribute to the development of chronic lung disease. Phospholipids, important components of cellular membranes and surfactant, are necessary for the maintenance of normal lung structure and function. In order to test the effects of chronic exposure to environmentally relevant concentrations of ozone on phospholipid metabolism in the lung, rats were exposed to clean air or to 0.12, 0.25 or 0.50 ppm ozone for up to 18 months. The content and biosynthesis of phospholipids in both lung tissue and bronchopulmonary lavage fluid (surfactant) were measured. Incorporation of [J'C]acetate into lung tissue phospholipids, an estimate of overall biosynthesis, decreased significantly at some time points in the study, while tissue phospholipid content tended to increase with both ozone concentration and with age. No changes were detected in phospholipid content of bronchopulmonary lavage fluid. These findings did not support the hypothesis that prolonged exposure of rats to environmentally relevant concentrations of ozone results in either qualitative or quantitative deficits in the pulmonary surfactant system.

Key words: Ozone; Lung; Phospholipids; Surfactant

Introduction O z o n e , a h i g h l y r e a c t i v e o x y g e n s p e c i e s , is a t o x i c c o m p o n e n t o f p h o t o c h e m i cal o x i d a n t air p o l l u t i o n . T h e r e is c o n c e r n t h a t p r o l o n g e d e x p o s u r e o f h u m a n populations to low concentrations of ozone might contribute to the development of chronic pulmonary

d i s e a s e . P o t e n t i a l sites f o r o z o n e i n t e r a c t i o n in t h e l u n g

include the lipid c o m p o n e n t s

o f cell m e m b r a n e s ,

subcellular organelles and sur-

*Present address: Department of Biochemistry, University of Chicago, Chicago, U.S.A. Address all correspondence to: Elaine S. Wright, PhD, Biomedical Science Department, GM Research Laboratories, Warren, MI 48090, U.S.A. The Research Biomedical Laboratory of GM Research Laboratories is accredited by the American Association for the Accreditation of Laboratory Animal Care (AAALAC). The rationale and experimental protocol for the use of an animal model in this program have been reviewed by the Research Laboratories' Animal Research Committee. 0300-483X/90/$03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

313

factant, a mixture of phospholipids which lines the alveolar surface and which is necessary for maintaining structural and functional integrity of the deep lung [1]. Loss of functional surfactant from the alveolar surface could result from direct interaction of the ozone molecule with surfactant phospholipids, or from loss of biosynthetic or secretory activity o f alveolar epithelial type II cells [2--4]. Altered content and metabolism o f lung lipids have been reported after short term exposure to high ( > 1 ppm) ozone concentrations in vivo [5--9]. Exposure in vitro of type II cells to ozone has been shown to inhibit lipid synthesis [10], although short term exposures to ozone in vivo have produced evidence of both stimulation and inhibition of phospholipid synthesis or secretion in the lung [3,7,8]. Intermittant exposures to 0.15 or 0.30 ppm ozone for 3--7 months caused shifts in fatty acid and phospholipid composition in lung or lavage fluids [11,12], but the effects of prolonged exposures on phospholipid metabolism and total content of important surfactant lipids have not been investigated. The early pulmonary response to ozone is a dynamic one and includes proliferation o f type II cells, influx and proliferation of alveolar macrophages and transudation of proteins and serum into the alveolar space. These responses subside after a few days whether or not the exposure continues [1,13]. All of these changes could have an effect on the results of measurements of lipid content and metabolism in the lung, and the lack of agreement among various studies is likely due, at least in part, to the different times after the initiation of exposure at which measurements were made [2,4]. Thus, the results of studies using acute exposures to ozone, while important for gaining insight into possible mechanisms of ozone toxicity, are of limited utility for predicting effects from long term low level exposures. These studies were designed to determine whether continuous long-term exposure to ozone, at concentrations which do not produce severe overt toxicity, could effect subtle changes in phospholipid or fatty acid metabolism in the lung. Rats were exposed to clean air or 0.12, 0.25 or 0.50 ppm ozone for 3, 6, 12 or 18 months. The appearance of 14C in specific phospholipids in lung tissue and bronchopulmonary lavage fluid was used to estimate lipid biosynthesis, and total content of major lipid classes was measured in lavage fluid and lung tissue. Materials and methods

Exposures and in-life quality control This experiment represents one portion of a larger study of the effects of chronic exposure to ozone on rat lung. Details of animal shipment, housing, quality control methods and mortality and morbidity data for the overall exposure study have been published [14]. Male Fischer 344 rats (CDF [F-344/CrlBR]), 28 days old, were obtained from Charles River Breeding Laboratories, Kingston NY and Portage, MI. Animals were exposed to ozone (0.12, 0.25 or 0.50 ppm) or clean air for 20 h/day, 7 days/week, in 12.6-m 3 stainless steel and glass Rochester-style chambers. The total air flow rate was maintained at 14 chamber volumes per hour. Ozone was generated from extra dry oxygen using a Welsbach model T-408 ozone generator. Chamber concentrations were measured continuously

314

using Dasibi (model 1003AH and 1003PC) ozone monitors. Animals were maintained on a 12 h light/dark cycle, temperature 21 _.+ 1°C, relative humidity 50 -.+ 10070. Control animals were exposed under identical conditions to purified air. Certified laboratory rodent chow (Purina 5002, Ralston Purina Co., St. Louis, MO) and untreated tap water were provided ad libitum. Fatty acid analysis of samples from each of a total of 12 lots of chow showed no significant differences between lots, thereby assuring that results were not confounded by effects due to changes in dietary lipid content over the course of the 18 month experiment. Chow in cage feeders within the chambers was replenished daily and discarded weekly to insure against excessive accumulation of oxidation products in the food due to exposure to ozone.

Experimental Immediately upon removal from inhalation chambers, rats were anesthetized with Na-pentobarbital and injected via the tail vein with 2-['4C]acetate (1--3 mCi/mmol DuPont NEN; 10 laCi/lO0 g body weight in 0.1 /aCi/ml solution in 0.9°70 NaCI). There were 12 rats in each of the four exposure groups at the 3 months exposure time point and 6 animals in each group at 6, 12 and 18 months. One half the animals from each exposure group were sacrificed 1 h after injection of the isotope; the remaining animals were sacrificed 18 h later. Rats were sacrificed by exsanguination under deep Na-pentobarbital anesthesia. Lungs were perfused with 0.9070 NaCI to remove blood, and excised. A catheter was tied into the trachea and 7 ml of 0.9070 NaCI was infused and withdrawn with a syringe three times. This procedure was repeated twice and all recovered fluid was pooled, centrifuged at 300 g for 10 rain to remove free cells and stored at - 7 0 ° C for later analysis. Lung tissue was blotted, weighed, frozen in liquid nitrogen and stored at - 7 0 ° C for later analysis. Lung parenchymal tissue was dissected away from the trachea and major airways. The lung tissue was minced and homogenized (Tekmar Tissuemizer model SDT-1810, SDT-100EN probe). All tissue processing steps were carried out on ice. Lipids were extracted from tissue homogenates and from bronchoalveolar lavage fluid [15]. Neutral and polar lipid fractions were separated from total lung tissue lipid extracts by silicic acid column chromatography (100--200 mesh, Biorad Biosil A) [161. Phospholipid classes in lung tissue lipids and lavage fluid were separated by two-dimensional thin layer chromatography (Baker Si250-PA plates) using modifications of the methods of Jobe et al. [17] and Gilfillan et al. [18]. Samples were dissolved in 200/al chloroform and two separate 40-/al aliquots were applied to the preabsorbant area of each TLC plate. Duplicate plates were run for each sample. Plates were developed in the first dimension with chloroform/methanol/ acetic acid/water (65:25:8:4). After drying, plates were developed in the same direction with chloroform/methanol/petroleum ether/acetic acid (40:20:30:10). Sequential development in these two solvent systems was necessary in order to obtain reliable separation of sphingomyelin (SP) from phosphatidylcholine (PC) and phosphatidylethanolamine (PE) from phosphatidylglycerol (PG). After development, spots were visualized in iodine vapor and SP, phosphatidylserine and 315

phosphatidylinositol (PS + PI), PE and PG were scraped, extracted from the gel and analyzed. One PC spot was scraped, extracted and analyzed to give a total PC value. The preabsorbant area was removed and the plates were developed in 100070 acetone to remove iodine. After drying, 100 ~l of 5070 osmium tetroxide in chloroform was applied over the remaining PC spot. The plates were developed in the second dimension in chloroform/methanol/ammonia (85:35:5) and the separated disaturated PC (DSPC) was scraped, eluted from the gel and quantirated. Separated phospholipids were extracted from gel by the method of Bligh and Dyer [15]. Recovery from gel was greater than 95070. Phosphorus content of lipid fractions was measured by a modification of the method of Ames [19]. '4C incorporated into phospholipids was measured by scintillation counting and specific activity in each phospholipid class was calculated as dpm/pg lipid phosphorus. For the measurement of lung tissue fatty acid content, fatty acid methyl esters were formed by reaction of lipid extract with BFJmethanol and separated by capillary column GC (J&W DB 225 30 m x 0.25/~m). L-a-dinonadecanoyl phosphatidylcholine was added to lipid samples as an internal standard.

Data analysis Data showing significant differences between group means by ANOVA were further analysed using Dunnett's or Duncan's multiple range tests. Dunnett's multiple range test was used to test for the effects of ozone at each time point; Duncan's multiple range test was used to test for the effects of exposure duration within each experimental group. All comparisons were two-sided tests and the null hypothesis (that the means of groups being compared were not different) was rejected at values of P < 0.05. Results

The mean body weight of the group exposed to 0.50 ppm ozone was significantly decreased relative to controls and the other ozone-exposed groups (P < 0.05) after 3 months of exposure (Fig. 1). After one year this group showed a mean body weight depression of approximately 10070. No significant differences in lung wet weights were detected. Lung wet weight to body weight ratios in the group exposed to 0.50 ppm were about 2007o and 40070 greater than controls after 3 and 6 months of exposure, respectively, although only the 6-month change showed statistical significance (P < 0.05) (data not shown). There were no exposure-related increases in lung weight to body weight ratios after 12 or 18 months of exposure. No evidence of infectious disease in controls or in any of the ozoneexposed groups was detected during the study. A description of spontaneous morbidity and mortality in controls and ozone-exposed animals has been previously reported [14]. The results in Fig. 2 show increases in total phospholipid content in lung tissue related to both exposure duration and concentration. In both control and ozoneexposed groups, total lung phospholipid increased significantly after 12 and 18 months when compared to the two earlier time points. Lung phospholipid con-

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Fig. 2. Effect o f exposure to ozone for 3, 6, 12 or 18 months on rat lung content of lipid-extractable phosphorus, a measure of total lung phospholipid. Error bars represent standard error of the mean. N -- 12 rats per group. °Mean of exposed group is different from control group at same time point. •Mean is different from mean of same dose group at 3 months, bMean is different from mean of same dose group at 6 months. All tests P • 0.05.

317

tent t e n d e d to increase with o z o n e c o n c e n t r a t i o n at all time p o i n t s in the study, b u t changes were statistically significant o n l y after 6 a n d 12 m o n t h s o f e x p o s u r e to 0.50 p p m . W h e n lung p h o s p h o l i p i d s were s e p a r a t e d b y class, a similar t r e n d was a p p a r e n t in t o t a l lung c o n t e n t o f P C a n d D S P C ( d a t a n o t shown). T h e f a t t y acid c o n t e n t o f lung tissue a f t e r e x p o s u r e to 0.50 p p m o z o n e for 18 m o n t h s s h o w e d no d i f f e r e n c e s c o m p a r e d to clean air c o n t r o l s (Table I). In b r o n c h o p u l m o n a r y lavage fluid, t o t a l p h o s p h o l i p i d c o n t e n t t e n d e d to increase with e x p o s u r e d u r a t i o n in c o n t r o l s a n d in the two g r o u p s e x p o s e d to higher o z o n e c o n c e n t r a t i o n s (Fig. 3). T h e r e were no significant c o n c e n t r a t i o n related changes in t o t a l lavage p h o s p h o l i p i d at a n y time p o i n t in the study. Significant increases were d e t e c t e d in m i n o r p h o s p h o l i p i d classes ( P S - P I a n d P E ) after 3 a n d 12 m o n t h s o f e x p o s u r e to 0.50 p p m , b u t no o z o n e - r e l a t e d trends were a p p a r e n t in P C , D S P C or P G ( d a t a n o t shown). M e a s u r e m e n t o f m4C i n c o r p o r a t i o n into lung tissue lipids 1 h a f t e r injection gave a n e s t i m a t e o f t o t a l lung lipid biosynthesis, while the a m o u n t o f ~4C in lava g e a b l e lipids 18 h a f t e r label i n j e c t i o n r e p r e s e n t e d the i n t e g r a t e d result o f overall tissue biosynthesis, secretion o f s u r f a c t a n t lipids into the extracellular space, degr a d a t i o n a n d clearance. In all e x p e r i m e n t a l a n d c o n t r o l g r o u p s , specific r a d i o a c tivity o f '4C m e a s u r e d in lung tissue p h o s p h o l i p i d s 1 h after i s o t o p e a d m i n i s t r a t i o n was 5-fold g r e a t e r t h a n specific activity in the tissue lipids 18 h later ( d a t a n o t shown). E i g h t e e n h o u r s a f t e r i s o t o p e a d m i n i s t r a t i o n , '4C a p p e a r e d in lavage fluid lipids with a specific r a d i o a c t i v i t y 5-fold greater t h a n that m e a s u r e d in lavage lipids l h a f t e r a d m i n i s t r a t i o n o f the label. T h e specific r a d i o a c t i v i t y o f '4C m e a s u r e d in lung tissue 1 h after a d m i n i s t r a tion o f p ' C ] a c e t a t e is s h o w n in Figs. 4 a w d . A t all f o u r time p o i n t s in the study,

TABLE I FATTY ACID CONTENT OF RAT LUNG TISSUE AFTER 18 MONTHS EXPOSURE TO CLEAN AIR OR 0.50 ppm OZONE Fatty acid

0.50 ppmozone

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1.97 1.54 0.05 1.53 0.54 0.29 0.30

Total

10.98 ± 0.93

318

± ± ± ± ± ± ±

0.32 0.22 0 0.30 0.12 0.05 0.07

0.51 2.05 27.60 1.36 12.15 17.84 14.00 0.41 14.03 4.95 2.67 2.73

± ± ± ± ± ± ± ± ± ± ± ±

0.67 0.60 3.18 0.20 0.62 2.14 1.15 0.03 2.94 1.19 0.51 0.72

mg/lung

Per cent

0.05 0.22 3.21 0.16 1.38 1.89 1.52 0.05 1.71 0.50 0.33 0.32

0.49 1.92 28.46 1.38 12.21 16.75 13.39 0.42 15.12 4.41 2.88 2.81

± ± :t: ± ± ± ± ± ± ± ± ±

0.01 0.04 0.31 0.04 0.08 0.16 0.19 0 0.25 0.08 0.05 0.08

11.29 ± 0.61

± ± ± ± ± ± ± ± ± ± ± ±

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incorporation of t4C into DSPC, PG, PC and total phospholipid tended to decrease with increasing ozone concentration, however changes were statistically significant only after 3 and 12 months exposure. In lavage fluid lipids, significant concentration-related decreases in ~4C incorporation into phospholipids was detected after 6 months of exposure to 0.12, 0.25 and 0.50 ppm ozone, but the effect was not apparent at any other time in the study (data not shown). Discussion This study was designed to measure overall effects of chronic ozone exposure on fatty acid and phospholipid metabolism, rather than to distinguish changes in a single pathway. Incorporation of acetate carbon into complex lipids in the present study represents the sum o f de novo fatty acid synthesis, plus acetylation of glycerolphosphate to yield newly synthesized phospholipids and remodelling of phospholipids into disaturated species characteristic o f surfactant [20,21]. Jobe and co-workers showed that intravenously injected [J4C]acetate was incorporated into lung tissue lipids of rabbits within 10 rain and was thus virtually a pulse label [22]. In the same study, incorporation of []4C]acetate into lavageable surfactant lipids was shown to be maximal between 10 and 28 h after injection. In the study reported here, measurement o f ]4C incorporation into lung tissue lipids one

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c Fig. 4 (a,b,c,d) Effect of exposure to ozone on appearance of ["C]acetate in phospholipids in rat lung tissue. Rats were sacrificed 1 h after intravenous administration of ["C]acetate. Data are expresseed as specific radioactivity (dpm/microgram lipid extractable phosphorus) in each phospholipid class, or in total phospholipid PL(T). SP = sphingomyelin, PC(T) = total phosphatidylcholine, DSPC = disaturated phosphatidylcholine, PS + Pl = combined phosphatidylserine and phosphatidylinositol, PE = phosphatidylethanolamine, PG = phosphatidylglycerol. N = 6 rats/group. Error bars represent standard error of the mean. *Mean is different from control group, P < .05. b~

hour after injection gave an estimate of total lung lipid biosynthesis, while the amount of ~4C in lavageable lipids 18 h after label injection represented the integrated result of overall tissue biosynthesis, secretion of surfactant lipids into the extracellular space, degradation and clearance. Tissue and alveolar pool sizes were measured by quantitating total phospholipids and fatty acids and of separated lipid classes and species in lung tissue and lavage fluid. The increase in tissue phospholipid content measured after 6 and 12 months of ozone exposure (Fig. 2) was accompanied by a general trend towards concentration-related decreases in the incorporation of [J'Clacetate into lung phospholipids (Fig. 4). However, similar increases in phospholipid content were not found in the lavage fluid, which was assumed to be representative of the in situ surfactant lipid pool. An increase in lung tissue phospholipid content is consistent with an increase in overall phospholipid biosynthesis due to increased synthetic activity within type II cells, or to an increase in the total number of type II cells in the lung [4,13]. Lung tissue phospholipid content could also increase if the rate of secretion of surfactant phospholipid into the extracellular pool decreased, resulting in an accumulation of 'stored' phospholipid in type II cells. Our results showing an increase in tissue phospholipid content in the presence of a decrease in apparent phospholipid biosynthesis are consistent with a decrease in turnover of surfactant phospholipid in lung tissue due to a decreased rate of secretion by type II cells. The decrease in 14C incorporation into phospholipids measured in the present study could also have resulted from changes in endogenous substrate levels in lung tissue, rather than actual inhibition of phospholipid biosynthesis. For example, an increase in the endogenous free acetate pool would result in an apparent decrease in utilization of 14C-labeled acetate. Similarly, a shift from de novo fatty acid synthesis to endogenous fatty acid pools as a source of fatty acid substrate for phospholipid synthesis would also have been measured as a decrease in utilization o f administered ~4C-acetate. While lung tissue content of free fatty acids was neither qualitatively nor quantitatively different from controls after 18 months of exposure to 0.50 ppm ozone, this group failed to gain weight at the same rate as controls and other ozone-exposed groups. Decreased food intake due to ozone exposure could have resulted in shifts in availability of endogenous substrates for lipid biosynthesis in the lung. Thus, while prolonged exposure to 0.50 ppm ozone affected phospholipid metabolism in the lung, the mechanisms which account for the changes are not clear. The present study showed an age-related increase in total phospholipid content of lung and lavage fluid that was independent of ozone exposure. Other studies of chronic exposure of rats to the same range of concentrations of ozone have shown similar age-related changes in lung structure and function. When collagen synthesis and content were measured in rat lungs after exposure to 0.12, 0.25 or 0.50 ppm of ozone for 3, 6, 12 or 18 months, lung content of hydroxyproline increased with exposure duration in all groups, including clean air controls, whether data were normalized to lung dry weight or expressed as total lung content [14]. An apparent concentration-related increase in total lung collagen content was observed after 18 months, but did not show statistical significance.

322

Qualitative histologic examination o f chronically exposed rat lungs showed a concentration-related deposition of excess connective tissue in the bronchiolar walls associated with the ozone lesion. However, systematic evaluation of the entire parenchyma revealed a diffuse accumulation of connective tissue protein in the alveolar septal walls over time in both control and ozone-exposed groups [29]. In studies of lung ventilatory function, there were both age-related and ozonerelated changes. Exposure to 0.50 ppm ozone for 1 year produced a decrease in CO diffusion capacity and increases in both functional residual capacity and residual volume that were restored to control values when animals were allowed to breathe clean air for 3 months post-exposure [24]. Most humans are exposed to environmental toxicants at low concentrations which do not produce overt toxicity and exposures frequently occur over significant fractions o f a lifetime. Experiments in which animals are chronically exposed to low concentrations o f toxicants contribute to an understanding o f the implications for human health o f subtle alterations in structure or function due to toxic exposures superimposed on the normal progression of change over time. It is important to recognize that when experimental animals are exposed for long periods of time to low concentrations of ozone (or other toxicants), subtle effects due to chemical exposure may be difficult to detect against a background of agerelated changes. However, because of the difficulties inherent in attempting to predict chronic effects from the results of acute, high level exposures, chronic animal studies represent an important means for investigating the potential for effects of prolonged exposure to low concentrations o f pollutants on human populations.

Acknowledgements The authors gratefully acknowledge the expert technical assistance of J.B. D'Arcy, Y. Sanford, K. Suni, R.G. Wooley, and of the veterinary technicians of the Bioresources and Histopathology Section of the Biomedical Science Department, GM Research Laboratories.

References 1 2 3 4 5 6

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11

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22 23

24

324

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