Pulmonary NO2 toxicity: Phosphatidylcholine levels and incorporation of [3H]thymidine into DNA

ENVIRONMENTAL

RESEARCH

27, 352-360 (1982)

Pulmonary NO, Toxicity: Phosphatidylcholine Levels and Incorporation of [3H]Thymidine into DNA MERLE L. BLANK,* WALDEN DALBEY,? EDGAR A. CRESS,* SUSAN GARFINKEL, t AND FRED SNYDER*,’ *Medical tBiology

and Health Sciences Division, Oak Ridge

Division, National

Oak Ridge Laboratory.

Associated Oak Ridge,

Universities, Tennessee

and 37830

Received March 16, 1981 Lungs from rats exposed to 30 ppm NO, (PI/liter) for either 1 or 5 hr had higher incorporation rates of [3H]thymidine into DNA and greater amounts of phosphatidylcholine than the lungs from control animals. Although there was no difference between the pulmonary phosphatidylcholine levels in rats exposed to NO, for 1 or 5 hr, the incorporation of [3H]thymidine into DNA was considerably higher in the lungs of animals exposed to NO, for 5 hr than in those exposed for 1 hr. Analyses of autoradiograms of lung tissue showed essentially the same labeling index for the alveoli from animals exposed to NO, for 1 or 5 hr; however, the animals exposed to NO, for 5 hr had a greater number of labeled cells in the lower airways and alveolar ducts than in the lungs from rats exposed for only 1 hr. These results suggest that the increased levels of phosphatidylcholine after exposure to NO, are directly related to damage of the alveolar cells, whereas the incorporation of [3H]thymidine into lung DNA represents more widespread damage to areas involving the lower airways. Second and third exposures (24 hr apart) of rats to 30 ppm NO, for either 1 or 5 hr caused little increase in the incorporation of rH]thymidine into pulmonary DNA and no further increase in lung phosphatidylcholine levels. Decreased incorporation of thymidine into the DNA of pulmonary cells after second and third exposures to NO, is referred to as “adaptation” in this report. When NO,-adapted animals were again exposed to NO, 7 days after the previous NO, treatment, the lungs again exhibited increases in both rH]thymidine incorporation and levels of phosphatidylcholine.

INTRODUCTION

Exposure to high concentrations of nitrogen dioxide (NO,), a common air pollutant, causes necrosis of certain epithelial cells in the respiratory tract. The major sites of damage by NO, are the terminal bronchioles, alveolar duct, and nearby alveoli. In the bronchiole, it is primarily the ciliated cells that are damaged by NO,; the nonciliated cells divide and eventually differentiate into new ciliated cells (Evans et al., 1976; Hackett, 1979). An analogous sequence occurs in lung tissue where type II cells divide and replace damaged type I cells (Evans et al., 1973). Incorporation of [3H]thymidine into DNA during the repair phase was found to be related to the initial damage caused by NO, (Creasia et al., 1977: Evans et al., 1971). During this period thymidine incorporation and epithelial cell hyperplasia reach a peak at 24 to 48 hr after initial exposure to NO, and then return to normal levels even with continued exposure; this phenomenon has been ’ The U.S. Government’s right to retain a nonexclusive royalty-free license in and to the copyright covering this paper, for governmental purposes, is acknowledged. 352 0013-9351/82/020352-09$02.00/O

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termed adaptation (Creasia et al., 1977; Evans et al., 1971). Possible defense mechanisms that may be operating in lungs of animals exposed repeatedly to oxidant gases have been recently reviewed (Mustafa and Tierney, 1978). We have previously shown with rat lungs that the cellular response to a single exposure to NO, is accompanied by increases in the levels of pulmonary surfactant (disaturated phosphatidylcholine) and total phosphatidylcholine as early as 6 hr after a 5-hr exposure to 15 to 40 ppm NOz @l/liter). The peak level of phosphatidylcholine was dependent on NO, concentration and always occurred at 48 hr after exposure (Blank et al., 1978). Moreover, the phosphatidylcholine fraction contained a higher proportion of dipalmitoylglycerophosphocholine (surfactant lipid) after exposure to the NO,. Similar results have recently been reported for lungs of rats exposed to high concentrations of oxygen (Brumley et al., 1979). Our objectives in the present study were threefold, all dealing with acute exposures. The first was to determine if the NO,-induced increase in pulmonary [3H]thymidine incorporation and phosphatidylcholine levels was dependent on the duration of a short-term exposure to NO,. Since adaptation apparently occurs in a matter of several hours during exposure, we wanted to know if a significant portion of pulmonary damage occurs during the first few hours after exposure. The second objective was to determine the time required for adaptation to develop with repeated exposures. If a significant portion of pulmonary damage is initiated in the first hours of exposure, is adaptation induced during the same time? Our third objective was to establish how long the adaptation persists in the lung after the last exposure to NO,. MATERIALS

AND METHODS

Male F344/Bd specific-pathogen-free, lo-week-old rats were used in all experiments. Animals were obtained from a breeding colony within Oak Ridge National Laboratory. Rats were exposed to 30 ppm NO, (pi/liter) in chambers made of stainless steel and Plexiglass (Nettesheim et al., 1970). Chambers were operated under dynamic conditions at 21.1 t 1. 1°C and 45 to 50% humidity, with an airflow of 15 ft3/min. Concentrations of NO, were monitored with a Bendix chemiluminescence NOz detector (Bendix Process Instruments Division, Ronceverte, W.V.). The level of NO, was higher than that normally found in the environment in order to amplify biochemical changes that might have been undetected at lower exposure levels. At daily intervals after exposure to NOz, rats were injected intraperitoneally with [3H-methyl]thymidine (Schwarz/Mann, Orangeburg, N.Y; 6 Ci/mmole, 2 mCi/kg body wt). Forty-five minutes after injection, animals were anesthetized with methoxyflurane, killed by aortic bleeding, and the lungs were removed. The left bronchus was tied off, and the left lobe was removed, weighed, and frozen in liquid nitrogen for subsequent analyses. The right lobes were fixed by tracheal instillations of buffered formalin for subsequent autoradiography. Thymidine incorporation into pulmonary DNA was used as an index of cellular proliferation resulting from injury during exposure to NO, (Evans et al., 1971). The specific radioactivity of DNA in the left lobe was determined after homogenizing the lobe in 5 ml water with a Polytron (Brinkman Instruments, Westbury, N.Y.). Aliquots of the homogenate were spotted onto glass-fiber fil-

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ET AL.

ters, dried, washed with cold 5% trichloroacetic acid (TCA), and heated in 5% TCA at 80°C for 20 min. Aliquots were then removed for determination of tritium activity in a liquid scintillation counter and for calorimetric assay of DNA (Giles and Myers, 1965). Lipids were extracted from the remaining homogenate of the left lobe by the procedure of Bligh and Dyer (1959), except the methanol contained 2% (by volume) glacial acetic acid. The extracted lipids were dissolved in 2 ml chloroform and stored at -23°C until analyzed. Extracts of total lipids were separated into phospholipid classes by thin-layer chromatography on silica gel HR layers (250 pm) in a solvent system of chloroform:methanol:glacial acetic acid:water (50:25:8:2, v/v). The developed chromatoplates were sprayed with sulfuric acid, charred at 180°C for 1 hr, and the amount of phosphorus in the phosphatidylcholine band (resolved by thin-layer chromatography) was determined by the method of Rouser et al. (1966). The amounts of phosphatidylcholine phosphorus were expressed on a per lung basis. Since we had previously shown that the phosphatidylcholine in lung after exposure to NO, contained a higher content of the disaturated class (surfactant type) than control lung (Blank et al., 1978), no effort was made to repeat the analysis of this specific molecular species in these experiments. Autoradiograms were prepared from paraffin-embedded sections of the right inferior lobe cut along the bronchus. After being stained with hematoxylin and eosin, the sections were examined under 450x for the incidence of labeled cells (at least five grains per nucleus) at various sites in the respiratory tract. In the bronchi and bronchioles, at least 1000 epithelial cells were counted per slide. Labeled cells in all alveolar ducts and associated alveoli in each section were counted. The number of labeled cells in 1000 randomly selected alveoli was determined for each animal. Means and standard deviations (SD) were calculated and all data were analyzed for significance by the method of analysis of variance with hypothesis testing of particular contrasts. RESULTS

Single l- and 5-hr Exposures Both total pulmonary phosphatidylcholine and incorporation of thymidine into DNA were increased after a single exposure to 30 ppm NOa for either 1 or 5 hr (Fig. 1). Control values were 297 ? 23 pg P (mean + SD) in phosphatidylcholine and 31 2 10 dpm/pg DNA (mean t SD) for thymidine incorporation. The increase in thymidine incorporation was transient, peaking at about 24 hr after exposure to NO, before returning to control levels. The rise in phosphatidylcholine content was slower; it peaked at 48 hr and remained above the control levels for at least 3 days after exposure. Thymidine incorporation 1 day after exposure was significantly greater with a 5-hr than with a 1-hr exposure (P < 0.001). However, the response was not proportional to exposure duration (419% of control for 1 hr and 755% for 5 hr). Although phosphatidylcholine levels were also significantly elevated above control values, no significant difference was observed in response to the l- and 5-hr exposures to NO,. An increased labeling index was observed at all five sites within the lungs, with peak values occurring 1 day after exposure (Table 1). The labeling index for the four sites located in the airways was higher for the 5-hr than for the 1-hr exposures

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300

200

I

100

0 0

1

2 Days

After

3

Exposure

FIG. 1. Effects of single I-hr (solid lines) or Shr-(broken lines) exposures to 30 ppm NO, on [3H]thymidine incorporation into lung DNA (solid circles) and levels of lung phosphatidylcholine (open circles). Data points represent the means 2 SD of at least 13 animals.

1 day after the exposure to NO, (P < 0.003). However, a 5-hr exposure did not increase the labeling index in the peripheral alveoli above the response observed for the 1-hr exposure. The response in the peripheral alveoli, although significantly higher than controls, was not as dramatic as in the airways. Multiple Exposures

The number of daily exposures to NO, required to produce adaptation was investigated for both the l- and 5-hr exposures (Tables 2 and 3). Groups of rats received either 1, 2, or 3 successive daily exposures and both thymidine incorporation and phosphatidylcholine levels were measured at daily intervals after the last exposure to NOz. Adaptation, based on the [3H]thymidine results, was nearly complete within a day after the initial exposure to NO, (Table 2), and daily exposures subsequent to the first exposure had little effect on the return to control values. Again, single l- and 5-hr exposures produced a dose response in the thymidine uptake similar to that observed in the experiment shown in Fig. 1. Second and third exposures to NO, did not cause the pulmonary level of phosphatidylcholine to increase beyond that observed after the initial exposure (Table 3).

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TABLE INCIDENCE

OF RADIOLABELED AT VARIOUS

Site in respiratory tract

1

CELLS AFTER LABELING WITH [3H]T~~~~~~~~ DAYS AFTER EXPOSURE TO NO?

Days after exposure to NO,

Exposure (hr)

0

1

2

3

3H-Labeled cells Main bronchF’

Secondary bronchi*

Terminal bronchioles*

Alveolar duct and alveoli’

Peripheral alveohd

0 1 5

0.18 t 0.10

0 1 5

0.13 k 0.2

0 1 5

0.16 + 0.2

0 1 5

0.19 2 0.19

0 1 5

4.3 k 3.1

0.26 k 0.3” 1.30 f 1.3’

0.21 k 0.2 0.40 + 0.6

0.35 + 0.2 0.20 + 0.1

0.75 + 0.5” 3.36 k 1.1’

0.25 + 0.2 0.51 + 0.5

0.43 2 0.3 0.43 k 0.3

5.50 2 3.9” 12.73 zk 2.9“

0.60 k 0.5 0.83 k 0.6

0.70 + 0.3 0.90 k 0.16

4.25 2 3.28’ 10.83 2 5.11’

1.20 k 1.68 2.34 k 1.85

2.82 t 1.66 4.76 + 2.52

23.4 + 15.5 22.9 k 11.6

2.9 k 4.1 4.3 2 2.6

N.D.’ N.D.’

u Date expressed as mean values t SD; see Materials and Methods for labeling protocol. b Percentage of epithelial cells that were radiolabeled. CNumber of radiolabeled cells per alveolar duct and associated alveoli. d Number of radiolabeled cells per 1000 peripheral alveoli. I’ Values for I- and 5-hr exposures are significantly different from each other (P < 0.003). f Not determined as values for second day were at control levels.

TABLE ~HITHYMIDINE INCORPORATION AFTER MULTIPLE DAILY

Exposure to NO,

DAYS

Days after first exposure to NO,

Duration

Number

1

1 hr

1 2 3

363 k 33

1 2 3

544 k 18 -

5 hr

2 IN LUNGS AT VARIOUS EXPOSURES TO NO?

2

3

% of control 128 f 43 118 f 43 118 + 29 121 z!z 32 196 2 57 1442 2 282 2 146 -

113 t 22 81 ? 2 104 + 16

4 115 k 27 135 k 10 97k 16 82 2 5

a Values are means of three rats + SD; control values were 33 + 6 dpm/pg DNA for rH]thymidine incorporation.

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TABLE PHOSPHATIDYLCHOLINE CONTENT AFTER MULTIPLE DAILY

Exposure to NO,

3 OP LUNGS EXPOSURES

AT VARIOUS TO NO,”

DAYS

Days after first exposure to NO,

Duration

Number

1

1 hr

1 2 3

168 f 14

1 2 3

155 2 26 -

5 hr

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DNA AND SURFACTANT

2

-

3

4

% of control 174 k 10 165 2 4 166k 3 168 c 8 165 f 10

122 k 16 123 k 12

165 2 3 147 r 17 -

137 ? 20 122 t_ 12

164 ? 22 126 + 8 133 i 11

” Values are means of three rats k SD: control values are based on the analysis of six rats (292 k 40 pg P in phosphatidylcholine).

On the basis of these results, we predicted that two short-term exposures (1 hr) would protect against a subsequent 5-hr exposure to 30 ppm NO,. This possibility was tested in three separate experiments and because all gave similar results, only data from one typical experiment are shown (Table 4). A single 5-hr exposure to NO, increased [3H]thymidine incorporation into DNA to about 600% of the control values. When rats were initially exposed to NO2 for two I-hr periods, a subsequent 5-hr exposure merely delayed the rate of return of [3H]thymidine incorporation to control values, instead of causing a six- to sevenfold increase. However, the incorporation of [3H]thymidine into DNA 1 day after this subsequent 5-hr exposure was greater than in animals exposed to NO, for only two TABLE PULMONARY AFTER

4

THYMIDINE INCORPORATION AND PHOSPHATIDYLCHOLINE Two I-hr EXPOSURES TO NO, FOLLOWED BY A SUBSEQUENT

Paran eter

Conditions for exposure to NO,

LEVELS AT VARIOUS 5-hr EXPOSURE TO

DAYS

NO;

Days after first exposure to NO, 1

2

3

4

5

% of control THjThymidine incorporation

Phosphatidylcholine levels

A B C

590 k 94

A B C

187 t 3 -

165 + 19 233 + 89

195 k 108 122 t 18 72 rf 10 104 _f 12

198 k 30 208 -c 21

206 + 49 200 k 14 160 t 0

159% 5 141t11

-

155 + 7 14357

” Values are means of three rats ? SD; control values were 30 IT 5 dpm/pg DNA for [3H]thymidine incorporation and 263 ? 8 pg P in phosphatidylcholine. Rats in Group A received a single 5-hr exposure to NO,; Group B received two daily I-hr exposures followed by a 5-hr exposure to NO,; and Group C received two daily 1-hr exposures to NO,.

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TABLE 5 PULMONARY THYMIDINE INCORPORATION AND PHOSPHATIDYLCHOLINE LEVELSDURINGADAPTATION" Thymidine

Exposure to NO,

Phosphatidylcholine % of control

None Single exposure Adapted, l-day Adapted, 7-day Adapted, 7-day

of control rats period, reexposed period, reexposed period, not reexposed

100 k 493 + 330 k 527 +

9.1 410 25b 39O

10027 227 2 35’ 137 c 8’ 106 + 5

(1Values are means of five rats + SD; control values were 44 + 4 dpm/wg DNA for thymidine incorporation and 387 t 26 pg P in phosphatidylcholine. b Measured 24 hr after exposure to NO,. CMeasured 48 hr after exposure to NO,.

1-hr periods (P < 0.025); these results indicated that complete adaptation to the 5-hr exposure to NOz had not occurred. Rats exposed to NO2 for 5 hr after they had previously inhaled NO, for two I-hr periods (24 hr apart) failed to exhibit any additional increase in the level of phosphatidylcholine. Duration of Adaptation

It was of interest to determine how long adaptation persisted once it was induced. Either 2 or 7 days were allowed to elapse between an initial 5-hr exposure to 30 ppm NOz and a subsequent 5-hr exposure to NO,. Thymidine incorporation was measured 24 hr and phosphatidylcholine levels 48 hr after the second exposure to NOz (Table 5). Phosphatidylcholine levels were not measured at 2 days after the initial exposures to NOz, as the elevated response had already been characterized (Blank et al., 1978). Adaptation, as measured by [3H]thymidine incorporation, disappeared completely at 7 days and partially after 2 days if there was no subsequent exposure to NO,. If rats previously subjected to NO, at two separate periods were not exposed to NOz again for 2 days, the incorporation of thymidine into DNA was intermediate between the value observed for controls and those that had received a single exposure to NO, (i.e., exhibited a partial loss of adaptation). In contrast, the phosphatidylcholine levels of lungs 48 hr after the second exposure, although above the control levels, were not as high as after a single exposure to NOP. The results also indicated a partial adaptation in the phosphatidylcholine response in rat lung after 7 days without a second exposure to 30 ppm NOz. DISCUSSION

Effects of both concentration and duration of exposure to NO, on the lung have been studied extensively. However, it appears that no information is available on [3H]thymidine incorporation into DNA or on phosphatidylcholine levels in lung after exposures to NO* for short periods, e.g., 1 hr. Our previous studies have shown that an increase in phosphatidylcholine was proportional to the concentration of NO, (Blank et al., 1978), and a 2.4-fold increase in NO, concentration (12.5

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to 30 ppm) resulted in a 3.5-fold increase in rH]thymidine incorporation (unpublished observations). Results presented here show that increasing the exposure from 1 to 5 hr did not produce the magnitude of change in the parameters measured that had been seen by increasing the levels of NO,. This is consistent with the observation that duration of exposure is relatively less significant than the concentration of NOz (Larsen et al., 1979), as assessed by susceptibility of mice to infective microorganisms. In the cell proliferative response to 5-hr exposures to NO* (relative to 1-hr exposures), autoradiography indicated the main site of increased cell proliferation was in the epithelium of the lower airways and alveolar duct. A single 1-hr exposure to NO, caused the maximum increase in both the peripheral alveoli labeling index and levels of phosphatidylcholine. Since alveolar type II cells, which are responsible for synthesis of pulmonary surfactant lipid (Frosolono, 1977; Van Golde, 1976), proliferate after exposure to NO, (Evans et al., 1973), it appears that the increased levels of phosphatidylcholine found after exposure to NO, are related to the relative number and the metabolic activity of the alveolar type II cells. Increased pulmonary levels of phosphatidylcholine after exposure to NOz could also be explained by a lower clearance rate of surfactant from the lung. [3H]Thymidine incorporation into DNA and the level of surfactant can yield valuable information regarding total pulmonary damage and damage confined to the alveolar cells, respectively, in animals exposed to NO,. There was a dichotomy in the duration of adaptation as measured by the thymidine and phosphatidylcholine endpoints. Adaptation in terms of [3H]thymidine incorporation was lost after 7 days without subsequent exposure to NOz, but a partial adaptive response was still evident in the phosphatidylcholine levels. This could be explained if the replacement type I cells formed from type II cells during recovery from NO, damage (Evans et al., 1973) are more resistant to subsequent exposures to NO*, as proposed by Stephens et al. (1978). We conclude, on the basis of [3H]thymidine labeling of pulmonary DNA, the rat lungs apparently “adapted” after two exposures (24 hr apart) to 30 ppm NO,. A similar adaptation was reflected by the response in pulmonary surfactant lipids; i.e., second and third exposures of rats to NOz did not result in increased levels of phosphatidylcholine beyond that caused by the first exposure to NO,. These results indicate that an adaptation of the pulmonary phosphatidylcholine response to exposure to NO, occurs in lung surfactant production as well as in the cell proliferative response. ACKNOWLEDGMENTS We thank Dr. Richard L. Schmoyer, Jr., a biostatistician at the Oak Ridge National Laboratory, for the statistical analysis of the data. Work performed at Oak Ridge Associated Universities was supported by the U.S. Department of Energy (Contract DE-AC05-760R00033). Work performed at Oak Ridge National Laboratory (operated by Union Carbide Corp.) was supported by the U.S. Department of Energy (Contract WR-7405-eng-26).

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