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Biochemical assessment of acute nitrogen dioxide toxicity in rat lung
rOXICOLOCY
AND
APPLIED
Biochemical
PHARMACOLOGY
Assessment DANIEL
81,
of Acute Nitrogen Dioxide Toxicity in Rat Lung J. GUTH’
Department of Radiation School of Medicine
Received
128-138 (1985)
AND RICHARD
Biology and Biophysics, and Dentistry, Rochester,
February
20. 1985; accepted
D. MAVIS* University of Rochester. New York 14642
June 3, 1985
Biochemical Assessment of Acute Nitrogen Dioxide Toxicity in Rat Lung. GUTH, D. J., AND R. D. (1985). Toxicol. Appl. Pharmacol. 81, 128-138. The early primary biochemical response of lung to NO, was studied separately from the later secondary responsesof inflammation and proliferation by measuring several biochemical parameters in lungs of rats immediately following a 4-hr exposure to nitrogen dioxide (NO*) at concentrations of 10, 20, 30, and 40 ppm. Cell-free lavage tluid contained elevated amounts of lactate dehydrogenase (LDH), malate dehydrogenase (MDH), isocitrate dehydrogenase (IDH), glucose-6-phosphate dehydrogenase (GDH), acid phosphatase (AP), and aryl sulfatase (AS) after 30 or 40 ppm NO*. Total protein and sialic acid were increased in cell-free lavage after 20, 30, or 40 ppm NOZ. The amounts of protein, siahc acid, and acid phosphatase recovered by airway lavage were equal to the amounts found in 0.7 ml of plasma, consistent with transudation of this volume of plasma into airways as a source of these parameters. The plasma activity of the other parameters measured was too low to account for their increase in lavage fluid by plasma leakage into airways. Decrease in the number and enzyme content of lavagable celfs indicated damage to free cells in the airways. The amount of the decrease in enzyme content of the lavagable cell fraction was similar to the increase in the cell-free lavage for all of the measured enzymes except acid phosphatase, suggesting the release of these enzymes into airways as a result of damage to free cells. However, the LDH isoenzyme profile in cell-free lavage after exposure is inconsistent with free cells as the source of this enzyme. No changes were observed in the whole-lung homogenate content of protein, DNA, lipid, LDH, MDH, IDH, GDH, AP, AS, glutathione reductase, NADPH cytochrome c, or succinate cytochrome c reductase immediately after NO* exposure. This study indicates that initial acute damage to lung by NO2 results in translocation of enzymes, proteins, and sialic acid into airways. Plasma is a likely source of translocated protein, sialic acid, and acid phosphatase. The sources of the other enzyme activities remain to be identified, with lung parenchyma and free cells as likely sources. MAWS,
0 1985 Academic PBS, he.
Acute or chronic exposure to nitrogen dioxide (NO*) results in a sequence of damage, inflammatory response, proliferation, and repair in the lung. Morphological damage to type I epithelial cells and ciliated bronchiolar cells is first seen after four hours of exposure to 17 ppm NO2 and, after 8 to 12 hr of exposure, cells are detached from the basement membrane (Stephens et al., 1972). Damage to the ’ Present address: California Primate Research Center, University of California, Davis, CA 95616. * To whom correspondence should be addressed.
0041-008X/85
$3.00
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epithelium results in increased permeability evidenced by transudation of serum protein (DeNicola et al., 1981; Selgrade et al., 1981) and penetration of high-molecular-weight tracers (Gordon et al., 1983). Exposure to NO2 is also followed by recruitment of inflammatory cells into the lung with increases in polymorphonuclear neutrophils (PMN) (DeNicola et al., 198 1; Gardner et al., 1969) and macrophages (DeNicola et al., 1981). Repair of lung epithelium after NO2 exposure involves proliferation of alveolar type II cells and nonciliated bronchiolar cells (Evans et al., 1972,
128
BIOCHEMICAL
ASSESSMENT
1973,1975,1976). The magnitude and the rate of onset and decline of the edema, inflammation, proliferation, and repair vary substantially with the concentration of NOz, the duration of the exposure, and the animal species under study. Measurement of biochemical changes in the NO*-exposed lung has provided information that is generally consistent with the morphological data describing the proliferative and inflammatory responses. Biochemical studies from this laboratory and others have defined parameters that increase in the lung after NOz exposure over a time course of days to weeks after initiation of exposure. These include protein, DNA, and various enzyme activities (Wright and Mavis, 198 1; DeNicola et al., 1981; Ospital et al., 1981; Sagai et al., 1982; Ichinose and Sagai, 1982). Isolation of alveolar epithelial type II cells from rat lung during NOz-induced proliferation has shown an increase in both the number of type II cells and their content of protein and enzymes, compared with type II cells isolated from control rats (Wright et al., 1982). This increase in cell number and size as a general response to lung damage could explain a number of biochemical changes in lung which occur a day or more after exposure to NO2 or other toxic agents. NOz exposure also results in recruitment of large numbers of inflammatory cells into the lung (DeNicola et al., 198 1) which could contribute significantly to biochemical changes in the whole lung. In order to identify early biochemical changes which reflect initial damage caused directly by interaction of NOz with lung tissue, such changes would ideally be studied in isolation from the secondary responses of proliferation and inflammation. In the work reported here we used an acute exposure protocol, in which rats were exposed to NOz for 4 hr and killed immediately after exposure in an attempt to characterize immediate biochemical changes resulting from primary damage before the complicating events of inflammation or proliferation.
OF NO* TOXICITY
129
METHODS Exposures. Male Long Evans rats from Charles River Breeding Labs (Portage, MA) were purchased as specific pathogen free, maintained in a barrier facility under a 12hr light cycle, 25°C temperature, and 30-35% relative humidity with free accessto rat chow (Agway) and water for at least 7 days prior to use. SurveilIance of specific pathogen status was performed periodically on selected animals. Animals weighed between 340 and 380 g when used. Six rats per group were exposed in a 29-liter glass chamber to NO2 (0.5% NO* in Nz, Air Products) diluted with compressed air at a final flow rate of 6 liters/min. Control groups were exposed to compressed air alone. Chamber concentrations of 10,20,30, or 40 ppm were continuously monitored with a Beckman Mode1 925A analyzer calibrated witb a standard 11.5 ppm NO* in N2 (Air Products). Concentration of calibration gas was established with the wet chemical method of Saltzman (I 954) and chamber concentration was checked three times during each exposure by this method. Chamber concentrations were maintained within 5% of stated concentration throughout exposure and NO concentration was less than 1% of NO2 concentration. Tissue preparation. After 4-hr exposure, rats were kihed immediately by ip injection of sodium pentobarbital. The lungs were perfused for 5 min through the right ventricle with 0.9% sodium chloride at a pressure of 30 cm of water and a temperature of 12°C. The dorsal aorta was severed prior to perfusion to decrease systemic resistance to perfusion. During perfusion, the lungs were gently ventilated through a trachea1 cannula. Following perfusion, the lungs were removed and immediately lavaged three times at room temperature with 6 ml per lavage of 0.9% sodium chloride buffered at pH 7.4 with 5 mM Tris-HCI. Each lavage was slowly injected and withdrawn four times through a tracheal cannula. Final recovery of injected lavage fluid was 85 to 90% both in control and exposed lungs. Lavaged cells were counted in a Coulter Counter (Hialeah, FL) and a hemocytometer. Differential cell counts were performed on Wright-stained cells prepared by cytocentrifugation. Lavage fluid was separated into cell-free lavage and cell pellet by centrifugation at 3OOgfor 15 min at 4°C. The cell pellet was resuspended in 10 ml of 5 mM Tris-HCl, pH 7.4. The cells were lysed for biochemical analysis in a Brinkman Polytron (Brinkman Instruments) at a setting of 6.5 for 15 sec. Our lavage procedure, adapted from Snyder et al., (1983) was designed to recover a major amount of airway material in a relatively small volume. We tested the efficiency of our procedure by more extensively lavaging the lungs of 12 rats (6 control, 6 exposed to 40 ppm NO2 for 4 hr) with an additional five lavages of 6 ml each beyond our normal procedure. We found that our normal procedure of three lavages recovered 74% of the total cells
130
GUTH
AND MAVIS
and 90% of the total protein removed in the more extensive lavage. There was no difference in trypan blue exclusion in any lavagable cell pellet. The lungs were minced, weighed, and placed in ice-cold 300 mM sucrose, 1 mM EDTA, buffered with Tris-HC1 at pH 7.4. The minced lungs were homogenized in a Teflonglass homogenizer followed by a Brinkman-Polytron for I min at setting of 6.5. The homogenate was filtered through 160~pm Nytex nylon screen to remove connective tissue fragments. Filtering resulted in loss of I .6% of total protein and 1.1% of total acid phosphatase and NADPHcytochrome c reductase. The filtrate was designated whole homogenate. Biochemical assays. Biochemical measurements made in whole-lung homogenate and in lavage fluid were chosen to reflect general tissue composition (protein, DNA, and lipid extractable phosphate) and to assessthe effect of NO* exposures on soluble enzyme activities as well as on enzymes associated with membranous subcellular structures. Protein was measured by the method of Lowry et al. (195 1). NADPH-cytochrome c reductase was measured as described by Williams and Kamin (1962) except that we used 68 PM cytochrome c and pH 8.1. Succinate cytochrome c reductase was measured according to Sottocasa et al. (1967) except that we used 48 mM succinate and 25°C. 5’-Nucleotidase was measured in 0.2 ml final volume with 100 mM KCl, 10 mM MgClz, 50 mM Tris-HCI, pH 7.4, 10 mM NaK tartrate, 2 mM AMP. ABer 15 min incubation at 37”C, 0.8 ml of 1% ascorbate, 1% sodium dodecyl sulfate, 0.8 N H2S04, and 3.36% ammonium molybdate was added and absorbance measured at 820 nm after incubation for 30 min at 45°C. Acid phosphatase (AP) was measured with pnitrophenol phosphate as sub strate in 50 mM Na acetate, pH 5.0, with 0.1% Triton X100. AAer 15 min incubation at 37°C the reaction was terminated by bringing the solution to 0.1 N NaOH, and absorbance of pnitrophenol was measured at 410 nm. Aryl sulfatase (AS) was measured according to the procedure described by Wasserman and Austen (1976) for arylsultatase B. Lactate dehydrogenase (LDH) was assayed in 1.O ml of 50 mM K-phosphate, pH 7.5, with 0.16 mM NADH and 0.48 rnM pyruvate by measuring decrease in absorbance at 340 nm. Malate dehydrogenase (MDH) was measured in 50 mM K-phosphate, pH 7.4, with 0.2 mM NADH and 0.75 mM oxaloacetate also by the decrease in absorbance at 340 nm. Glucose-6-phosphate dehydrogenase (GDH) was assayed in 1.0 ml of 50 mM triethanolamine, pH 7.6, with 0.15 mM NADP, 2.0 mM glucose 6phosphate+ 2.0 mM 6-phosphoghrconate, and 10 mM MgCl, by measuring the increase in absorbance at 340 nm. These conditions measure the combined activities of GDH and 6-phosphogluconate dehydrogenase. Isocitrate dehydrogenase (IDH) was assayed in 1.O ml of 50 mM triethanolamine, pH 7.5, with 2.7 mM DL-iSOCitEite, 0.3 mM NADP, and 2.7 mM MnS04 by following the increase in absorbance at 340 nm. Total siahc acid was measured by the
thiobarbituric acid method described by Downs and Pigman (1976) after acid hydrolysis. Glutathione reductase was assayedasdescribed by Bergmeyer et al. (1974). Lipid extractable phosphorus was determined by the procedure for total phosphate described by Ames ( 1966) on extracted lipid (Bligh and Dyer, 1959). DNA was assayed thtorometrically using the product formed with DABA prepared as described by Wright and Mavis (198 1). For all enzyme activities, one unit is defined as 1 nmol product formed/min. All enzyme assayswere proportional to incubation time and added protein under the conditions used. Multiple assaysof single samples varied by no more than 5% from the mean. LDH isoenzymes. LDH isoenzymes were electrophoretically separated and stained on 5.5% polyacrylamide gels as described by Dietz et al. ( 1970) and Dietz and Lubrano (1967). &enzyme patterns were scanned by a Gilford 2410 scanning attachment on a Gilford 250 spectrophotometer. Peak areas were measured with a compensating polar planimeter (Lasico). Cell-free lavage and lavagable cell sample volumes were concentrated by lyophilization before electrophoresis. The various isoenzymes of LDH were found to be uniformly stable to lyophihzation. Whole-lung homogenate was centrifuged at 16OOg for 15 min prior to electrophoresis. Statistics. The Mann-Whitney test was used to compare parameters measured in exposed animals with those of controls.
RESULTS The results of measurement of the chosen parameters in whole-lung homogenate from rats exposed to 40 ppm NO;? for 4 hr are shown in Table 1. No significant changes from control values were observed in lipid extractable phosphate, protein, DNA, or in several enzymes assayed. In pooled cell-free lavage fluid, however, NO2 exposure produced increases in several of these parameters, as seen in Table 2. Protein and sialic acid amounts were elevated to 24 and 15 times control values, respectively. The activities of two lysosomal enzymes and several soluble enzymes were also significantly increased after exposure. No detectable activity of the microsomal enzyme NADPH cytochrome c reductase or the mitochondrial enzyme succinate cytochrome c reductase was present in the lavage fluid of control or exposed animals. The concentration dependence of the bio-
BIOCHEMICAL
ASSESSMENT
131
OF NO, TOXICITY
TABLE 1 COMPARISON
OF WHOLE LUNG CONTENT OF PROTEIN, DNA, LIPID PHOSPHATE, AND ENZYMES” IN CONTROL BATS AND BATS EXPOSED TO 40 ppm NO* FOR 4 HOURS*
Lipid extractable phosphate, pmol/lung Protein, mg/lung DNA, mg/lung S-Nucleotidase Acid phosphatase Aryl sulfatase NADPH-cytochrome c reductase Succinate-cytochrome c reductase Lactate dehydrogenase Malate dehydrogenase Isocitrate dehydrogenase Glucosed-phosphate dehydrogenase Glutathione reductase
Control
NO7 EXDOS&
19.9 f 2.7 127.7 + 16.7 12.4 f 1.8 3.25 + 0.35 7.08 +- 1.72 4.89 + 0.98 0.51 + 0.10 0.37 k 0.10 33.9 It 9.0 47.4 * 11.0 4.72 zk 1.31 2.71 + 0.32 1.84 + 0.22
21.0 + 3.2 139.3 + 27.7 12.9 r!~ 1.3 3.53 xk0.86 7.64 z!z0.92 5.77 f 1.16 0.55 + 0.09 0.32 + 0.05 31.0 + 5.5 51.0 + 20.0 4.55 + 0.58 2.77 + 0.46 1.97 + 0.30
a All enzyme activities are expressed as 10’ units/lung. * Values are expressed as X of six animals f SD.
chemical changes in cell free lavage was investigated as shown in Figs. l-3. Protein and sialic acid content were increased significantly after 20, 30, or 40 ppm NO2 for 4 hr but not
after 10 ppm (Fig. 1). LDH, MDH, GDH, IDH, and AP were increased significantly above controls after 30 and 40 ppm NO* but not after 10 or 20 ppm (Figs. 2 and 3).
TABLE 2 COMPARISON
OF
CELL-FREE LAVAGE CONTENT OF PROTEIN, SIALIC ACID, LIPID PHOSPHATE, IN CONTROL RATS AND RATS EXFQSED TO 40 ppm NO2 FOR4 HOURS*
Protein Sialic acid Lipid extractable phosphate Lactate dehydrogenase Malate dehydrogenase Glucose-6-phosphate dehydrogenase Isocitrate dehydrogenase Acid phosphatase Aryl sulfatase NADPH-cytochrome c reductase Succinatecytochrome c reductase
AND ENZYMES’
Control
NO2 Exposed
3.63 k 1.00 49.1 + 9.9 2.01 f 0.75 381 + 144 457 + 264 7.03 + 2.42 10.7 f 9.9 22.2 + 9.2 90.1 + 42.0 n.d. (~2.21)~ n.d. (~3.53)
85.3 * 41 .o* 747.3 + 386.8* 2.94 f 0.62* 647 f 334** 709 k 367** 20.9 + 5.8* 39.4 f 12.2* 55.9 f 18.8: 273.3 k 33.5. nd. (~2.38) n.d. (~3.61)
’ All enzyme activities are expressed as units/lung; protein as mgjlung; sialic acid as &lung; fimol/lung. * Values are expressed as X of six animals + SD. ’ nd., Activity was not detectable. Lower limit of detection is given in parentheses. * Different from control at p < 0.05 by Mann-Whitney test. ** Different from control at p < 0.10 by Mann-Whitney test.
lipid
phosphate
as
132
GUTH
AND MAVIS
NOI Concentration (ppm)
NO2Concentration (ppm)
FIG. 1. Effect of NO2 concentration on recovery of protein and sialic acid in cell-free lavage after 4-hr exposure. Data points represent the X 2 SD for six rats expressed as mg protein/lung or pg sialic acid/lung in the cell free lavage. *Significantly different from control at p < 0.05 by the Mann-Whitney test.
In order to assess the contribution of the transudation of plasma to the increase in activity seen in the lavage fluid, plasma amounts of proteins, sialic acid, and enzyme activities were measured in control animals. These results are shown in Table 3, together with the magnitude of the increases in these parameters in lavage fluid NOz-exposed animals. No change in the plasma amounts of these parameters was observed after NO* exposure. The increases in lavage fluid content of protein, sialic acid, and AP are equivalent to the amount of these components found in 0.72, 0.54, and 0.69 ml of plasma, respectively. These volumes are within the range of plasma which might be expected to enter the airways by transudation. Plasma amounts of all other components in Table 3 are such that transudation of at least 3 ml of plasma would be indicated if plasma were the sole source of these enzymes. The effect of the NOz exposure on the lavagable cell population is shown in Table 4. Total cells recovered in the lavage were decreased by 44% as measured in the hemocytometer. A similar decrease was found with a Coulter Counter in the size range characteristic
of macrophages as well as an increase in material of smaller size, possibly debris and nuclei from the disruption of intact cells. The retention of nuclear material in the cell pellet in spite of the decrease in countable cells is evidenced by the lack of change in the amount of DNA in the lavagable cell fraction. Lavaged cells were 98 and 96% macrophages in the control and exposed animals, respectively. Polymorphonuclear (PMN) cells increased slightly in exposed animals to 2.7% compared to 0.22% of recovered cells in the control animals. Although the DNA content of the cell pellet did not change significantly, there was a significant increase in DNA remaining in the cell free lavage, and the total lavagable DNA increased by 25%. The increase in lavagable DNA is equal to 0.05% of the total lung DNA content. Because of the apparent damage to the free cells, we examined changes in protein, sialic acid, lipid extractable phosphate, and enzyme content of the lavagable cells. In Table 5 are presented the total content of these parameters in the lavagable cells and cell-free lavage as well as the change in exposed animals. This presentation allows comparison of the in-
BIOCHEMICAL
ASSESSMENT
a
35’
133
OF NOz TOXICITY
b
’
’
’
’
NO2 Concentration
’
’
‘**----7
0 10 20 NO1 Concentration
(ppm)
30
40 (ppm)
600
100
o-
0 10 20 NOz Concentration
30
40 (ppm)
0
I
I
I
0 10 20 NOI Concentration
,
30
I
40 (ppm)
FIG. 2. Effect of NO2 concentration on recovery of soluble enzymes in cell free lavage after 4-hr exposure. Data points represent the X k SD for six rats expressed as units/lung in the cell-free lavage. (a) Isocitrate dehydrogenase; (b) malate dehydrogenase; (c) glucose-6-phosphate dehydrogenase; (d) lactate dehydrogenase. *Significantly different from control at p < 0.05 by the Mann-Whitney test.
creases in lavagable content with the decrease in cell content in order to evaluate the possibility that lavagable cells are a source of the increase in cell-free lavage contents. The cellfree lavage contents of protein and sialic acid are, respectively, 10 I- and 17 l-fold greater than the changes in cell content, eliminating free cells as the source of these parameters. The lipid-soluble phosphate content increased
slightly in the cell pellet and significantly in the cell-free lavage, indicating another source of phospholipid, probably lung tissue or plasma. In the case of the soluble and lysosomal enzymes, the increase in the content of cell-free lavage is the same, within experimental error, as the decrease in content in the lavagable cells, suggesting these cells as a source of the cell-free lavage activity. The exception
134
GUTH 70 j-
AND MAVIS TABLE 3
I
COMPARISONOFPLASMAAHOUNTSINCONTROLANIMALS WITH THE INCREASE IN CELL-FREE LAVAGE AMO~NTSOFPROTEIN, SIALICACID, ANDENZYMES" IN ANIMAL~EXPOSEDTO 40ppm NO2 ~0~4 Hou~s~ Plasma Plasma component
IL---J 0
10
20
30
NO2 Concentration
40
(ppm)
FIG. 3. Effect of NO2 concentration on recovery of acid phosphatase in cell-free lavage after 6hr exposure. Data points represent the 1+ SD for six rats expressed as units/ lung in the cell-free lavage. *Significantly different at Q < 0.05 by the Mann-Whitney test.
ProteinC Sialic acid’ Lactate dehydrogenas& Malate dehydrogenase” Isocitrate dehydrogemw? Glucose&phosphate dehydrogenas& Acid phosphatask
am0UM.S’
62.4
2.5
f
1.12 * 91.2 38.7
Increase in cell-free lavage b 44.9 0.61 276 345 13.86
0.12
f 51.3 21.2
+
n.d. (<1.41)’ 3.16 44.4
f k
0.72 10.7
16.88 30.8
a Plasma amounts are expressed as X + SD for six rats. b Data are the difference between the X of six rats exposed to 40 ppm NO* and the X of six control rats. Data are taken from Figs. I, 2 and 3. ’ For protein and sialic acid, plasma amounts are expressed as mg/ml and cell free lavage amounts are expressed as mg/lung. d For enzymes, plasma amounts are expressed as units/ml and cell-free lavage amounts are expressed as units/lung. ’ n.d., Not detectable. Lower limit of detectability is given in parentheses.
to this was AP, which was increased in an amount consistent with plasma as the source of increase, as described above. In a further attempt to identify the source of the LDH activity increase in lung lavage after NO2 exposure, we determined the iso-
L “I b
CONTROL
FREE CELL
TABLE 4 EFFETE OFEXPOSURETO 40ppmN02 FOR~HOURS ONLAVAGEABLECELLSANDDNA' Control Total cells X 10’ 1
2
3 LDH ISOZYME
4
5
#
FIG. 4. Effect of exposure to 40 ppm NO, for 4 hr on LDH isoenzyme pattern in rat lung lavage. Data are presented as the X k SD for five rats expressed as a percentage of total activity for each isoenzyme. (a) Control and exposed cell-free lavage; (b) control and exposed lavagable cells. *Significantly different than control at p < 0.05 by the Mann-Whitney test.
Percentage macrophages Percentage PMN DNA Lavageable cells (pg) Cell-free lavage (a) Total Iavagable Whw
NOzExposed
0.27 1.3 0.22
0.46 2 0.11; 96.0 + 3.5 2.66 + 2.51*
4.8 0.48
19.1 13.8
f
3~
f
5.6 2.7*
2
4.6
33.7
_+
6.0*
0.82 97.8 0.22
f
24.2 2.87
f
27.0
+ +
’ Values are expressed as X f SD for 6 control and 12 exposed rats. * Values are significantly different from controls at p < 0.05 by the Mann-Whitney test.
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ASSESSMENT
135
OF NO2 TOXICITY
TABLE 5 COMPARISON
OF THE EFFECTS OF 40 ppm NO2 ON PROTEIN, SIALIC ACID, LIPID EXTRACTABLE AND ENZYME CONTENT IN LAVAGABLE CELU AND CELL-FREE LAVAGE’
Cell-free lavage
Cells
Control Protein Sialic acid Lactate dehydrogenase Malate dehydrogenase Glucose-6-phosphate dehydrogenase Acid phosphatase Aryl sulfatase Lipid phosphate
PHOSPHATE,
Exposed
Difference
Control
Exposed
Difference
1.87 f 0.67 19.1 f 4.0
1.31 f 0.36 15.7 f 4.1
0.56 3.37
3.09 f 0.62 21.5 f 8.3
59.7 f 16.9* 599 + 174*
56.6 577
607 f 172
387 f 86*
219
421 + 164
679 + 118*
258
1190 f 405
800 + 234*
390
647 2 195
1052 + 210*
405
11.5 47.2 367 1.44
7.1 57.6 236 1.89
4.4 10.7 131 0.45
<2.8 15.4 rt 8.8 76.9 f 22.1 3.27 f 0.60
14.1 + 44.1 + 228 + 4.03 +
11.3 28.7 151 0.76
+ f f +
7.2 17.8 127 0.69
f + f f
2.7 24.4 86* 0.58**
5.0* 9.0; 47* 0.59*
a Values are expressed as X + SD for six control and six exposed rats. Units are mg/lung for protein, &lung sialic acid, ~mol/lung for lipid phosphate, and U/lung for all enzymes. * Values are significantly different from controls at p < 0.05 by the Mann-Whitney test. ** Values are significantly different from controls at p < 0.10 by the Mann-Whitney test.
enzyme composition of the LDH activity in the control and NOz-exposed lavage as shown in Fig. 4. The isoenzyme profiles of control lung homogenate, plasma, and red blood cells are shown in Fig. 5 as possible sources of the LDH released into the airways. In cell-free lavage from NOz-exposed animals, the proportions of isoenzymes 1, 2, and 3 are increased while isoenzymes 4 and 5 are decreased compared to the control lavage profile. No significant change in isoenzyme profile of lavagable free cells occurred in response to NO;!. The isoenzyme profiles of lung homogenate, red blood cells, or plasma did not correspond to the changes seen in the cell-free lavage.
for
CL 25 0 :
b
PLASMA
DISCUSSION The biochemical changes reported here as increases in lavagable parameters appear to result directly from lung damage. A minimal increase in PMNs suggests little inflammatory response at this early time after insult. A decrease in the number of lavagable intact mat-
LDH ISOZYME
#
FIG. 5. Isoenzyme patterns of LDH in (a) lung homogenate. (b) \ , .dasma. and (c) red blood cells.
136
GUTH
AND MAWS
rophages occurred which, together with an increase in lavagable cell debris, suggests direct destruction of these cells by NO;!. The absence of an increase in whole-lung content of protein, phospholipid, or DNA demonstrates a lack of significant proliferation, in agreement with previous studies at 5 or 6 hr after exposure in rats (Blank et al., 1978; Evans et al., 1971) and in hamsters (Creasia, 1978). No damage was detectable as changes in the parameters measured in whole-lung homogenate. The amount of enzyme activity increases detectable in lavage fluid represents 2% or less of the total lung activity, however (except for AS activity, which was 5%) and thus could be derived from lung parenchyma as a result of focal cell damage and release of enzymes without detectable loss of activity from whole-lung homogenate. The increase in lavagable DNA represents a very small fraction (0.05%) of the total lung DNA content and could similarly be a result of focal release of DNA from lung parenchyma. Alternatively, a modest increase in the number of free lavagable cells together with an increase in their fragility as a result of NOz exposure could be responsible for the increase in total lavagable DNA if the fragile cells released DNA into the cell-free supernatant fraction where the lavagable cell increase is localized. The similarity of the amount of enzyme activities lost from the lavagable cell fraction as a result of NO1 exposure to the increases observed in the cell-free lavage supernatant fraction suggests translocation from lavagable cells into the supernatant fraction as a result of cell damage. However, isoenzyme analysis of LDH activity in lavage and free cells clearly shows that free cells cannot be the source of released LDH. Isoenzymes numbers 4 and 5 comprise over 80% of the activity in free cells while the cell-free lavage after NOz shows increases in isoenzymes numbers 1, 2, and 3, and a decrease in the percentage of isoenzymes 4 and 5 compared to control. Thus the LDH activity increase in lavage cannot be from free cells. The profile of LDH isoenyzmes in cell-free la-
vage after NOa exposure most nearly resembles that of the whole-lung homogenate. The changes in the various isoenzymes compared to control, however, do not correspond to the profile of whole lung homogenate, which contains major percentages of isoenzymes 4 and 5. The major isoenzyme in plasma is isoenzyme 2, which is one of the isoenzymes which increases in lavage after NO*. The magnitude of LDH activity in plasma, however, is relatively low. The increase in lavagable LDH after NO2 exposure represents an amount of activity found in 3 ml of plasma. Transudation of this volume of plasma would cause an increase in lung weight of around 150%, which is unlikely. Maximum changes in lung weight reported by Blank et al. (1978) for rat lungs 6 hr after a 5hr exposure to 40 ppm NO2 was a 50% increase. We have observed no statistically significant changes in lung weight immediately after 4 hr of exposure to 40 ppm NOz (unpublished observation). Red blood cell LDH is composed predominantly of isoenzyme 5 and therefore red blood cells cannot be a significant source of the increase in lavage. Thus LDH isoenzyme analysis allows elimination of free cells as the sole source of the LDH activity increase in cell-free lavage after NO2, but does not yield an unambiguous identification of the source of the translocated LDH. None of the sources analyzed showed an isoenzyme profile which corresponded to the increases observed in lavage. An additional complicating factor in the interpretation of LDH profiles is the possibility of selective inactivation of isoenzymes by NOz. In particular, isoenzymes 4 and 5 are lost from lavagable cells and yet are not recovered in the lavage supernatant fraction. Beck et al. (1983) have analyzed LDH isoenzymes in hamster lung lavage after injury by a variety of agents. Instillation of a-quartz or iron oxide produced lavagable LDH isoenzymes which resembled the distribution in unexposed PMNs and macrophages. High oxygen yielded a pattern similar to unexposed serum, and lavage with a solution of Triton X-100 removed isoen-
BIOCHEMICAL
ASSESSMENT
zymes of LDH in a pattern typical of unexposed whole lung homogenate. In contrast to the increases in enzyme activities, the amount of protein in the lavage fluid after NOz increases in an absolute amount which corresponds to a major percentage of the whole lung protein. Since protein is not lost from the whole-lung homogenate in response to NOz, the relatively large protein increase in lavage must derive from a source outside of the lung, with plasma as a likely source. Comparison of plasma protein content with the amount of protein appearing in lavage after NO2 reveals that transudation of 0.7 ml of plasma into the airways would result in the observed increase in protein. This volume of plasma would also account for the amount of sialic acid and AP, appearing in lavage, but not the amounts of other enzymes studied, due to their relatively low activities in plasma as shown in Table 3. Another difference between the appearance of protein and sialic acid and the appearance of the enzymes is that the appearance of protein and sialic acid in the lavage fluid was statistically significant at an NOz concentration of 20 ppm while the enzyme appearance was significant at 30 ppm or above. This suggests that the release of enzymes results from a different and possibly less sensitive event than that which releases protein and sialic acid. An interpretation of this may be that permeability change in the alveolar septum may precede damage to cells and release of cellular components. Selgrade et al. (198 1) reported significant increases in lavage protein 12 hr after a 3-hr exposure of vitamin C-deficient guinea pigs to 5 ppm NOz. Comparing our results immediately following a 4-hr exposure to NO2 with the reported biochemical changes in hamster lungs after exposure to NO* for 48 hr (DeNicola et al., 198 1) yields some interesting parallelisms and contrasts. The longer exposure resulted in the several-fold elevation of a number of enzymes, sialic acid, and protein in lavage fluid, a result comparable to the present study. In contrast
OF NO2 TOXICITY
137
to our results, however, the lungs exposed for 48 hr showed marked inflammation as evidenced by a several-fold increase in lavagable cells, over 65% of which were PMNs. In addition, several enzyme activities and total protein were elevated in the homogenate of lung tissue as a result of the longer exposure, consistent with proliferation of lung cells to replace those damaged by NOz (Blank et al., 1978; Wright et al., 1982). Thus elevation of enzymes, protein, and sialic acid in lavage fluid appears to be an indicator of direct damage which begins during the first few hours of exposure and persists during 48 hr of continuous exposure. Elevation of lavagable cells appears to be an indicator of the later appearing process of inflammation, and elevation of enzymes and protein in the whole-lung homogenate is also a later event possibly reflecting proliferative repair. These results, together with a growing body of evidence (Henderson, 1984) lead to the conclusion that the appearance of various biochemical entities in the lavage is a useful indicator of early toxic damage to the lung, detectable prior to the secondary events of inflammation and proliferation, and that identifying the source of these entities may yield information on the location and nature of the damage. ACKNOWLEDGMENTS This paper is based on work performed under Contract DE-AC02-76EV03490 with the U.S. Department of Energy at the University of Rochester Department of Radiation Biology and Biophysics, and has been assigned Report No. DOE/EV/03490-2427. Daniel Guth was supported by NIH Training Grants 5T32-ES07026 and 5T32HLO7216-08. The expert technical assistance of Michael J. Vang is gratefully acknowledged.
REFERENCES B. N. (1966). Assay of inorganic phosphate, total phosphate and phosphatases. In Methods in Enzymology (E. F. Neufeld and V. Ginsburg, eds.). Vol. 8, pp. 115 118. Academic Press, New York.
AMES,
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AND MAWS
BECK, B. D., GERSON, B., FELDMAN, H. A., AND BRAIN, J. D. (1983). Lactate dehydrogenase isozymes in hamster lung lavage fluid after lung injury. Toxicol. Appl. Pharmacol. 71,59-l 1. BERGMEYER, H. U., GAWEHN, K., AND GROSS, I. M. (1974). Enzymes as biochemical reagents. In Methods of Enzymatic Analysis (H. U. Bergmeyer, ed.), Vol. 1, pp. 425-522. Academic Press, New York. BLANK, M. L., DALBEY, W., NE~ESHEIM, P., PRICE,J., CREASIA, D., AND SNYDER, F. (1978). Sequential changes in phospholipid composition and synthesis in lungs exposed to nitrogen dioxide. Amer. Rev. Resp. Dis. 117, 273-280. BLIGH, E. G., AND DYER, W. J. (1959). A rapid method of total lipid extraction and purification. Cunud. J. Biochem. Biophys. 37,9 1 l-9 17. CREASIA, D. A. ( 1978). Stimulation of DNA synthesis in lungs of hamsters tolerant to nitrogen dioxide. J. Tox. Environ. Health 4, 755-762. DENICOLA, D. B., REBAR, A. H., AND HENDERSON, R. F. (1981). Early damage indicators in the lung. V. Biochemical and cytological responses to NO, inhalation. Toxicol. Appl. Pharmacol. 60, 301-3 12. DIETZ, A. A., LUBRANO, T., AND RUBINSTEIN, H. M. (1970). Disc electrophoresis of lactate dehydrogenase isozymes. Clin. Chim. Acta 27, 225-232. DIETZ, A. A., AND LUBRANO, T. (1967). Separation and quantitation of lactic dehydrogenase isozymes by disc electrophoresis. Anal. Biochem. 20, 246-257. Downs, F., AND PIGMAN, W. (1976). Qualitative and quantitative determination of sialic acids. In Methods in Carbohydrate Chemistry (R. H. Whistler and J. N. BeMiller, eds.), Vol. 7, pp. 233-240. Academic Press, New York. EVANS, M. J., STEPHENS,R. J., AND FREEMAN, G. ( 197 1). Effects of nitrogen dioxide on cell renewal in the rat lung. Arch. Int. Med. 128, 57-60. EVANS, M. J., STEPHENS,R. J., CABRAL, L. J., ANDFREEMAN, G. (1972). CelI renewal in the lungs of rats exposed to low levels of NO*. Arch. Environ. Health 24, 180188. EVANS, M. J., CABRAL, L. J., STEPHENS,R. J., AND FREEMAN, G. (1973). Renewal of alveolar epithelium in the rat following exposure to NO,. Amer. J. Pathol. 70, 175198. EVANS, M. J., CABRAL, L. J., STEPHENS,R. J., ANDFREEMAN, G. (1975). Transformation ofalveolar type 2 cells to type 1 cells following exposure to NO,. Exp. Mol. Puthol. 22, 142-I 50. EVANS, M. J., JOHNSON, L. V., STEPHENS, R. J., AND FREEMAN, G. (1976). Renewal of the terminal bronchiolar epithelium in the rat following exposure to NO2 or 0,. Lab. Invest. 35, 246-257. GARDNER, D. E., HOLZMAN, R. S., AND CORN, D. L. (1969). Effects of nitrogen dioxide on pulmonary cell population. J. Bacterial. 98, 1041-1043.
GORDON, R. E., CASE,B. W., ANDKLE~NERMAN, J. ( 1983). Acute NO* effects on penetration and transport of horseradish peroxidase in hamster respiratory epithelium. Amer. Rev. Resp. Dis. 128, 528-533. HENDERSON,R. F. (1984). Use of bronchoalveolar Iavage to detect lung damage. Environ. Health Persp. 56, 115129.
ICHINOSE,T., AND SAGAI, M. (1982). Studies on the biochemical effectsof nitrogen dioxide. III. Changes of the antioxidative protective systemsin rat lungs and of lipid peroxidation by chronic exposure. Toxicol. Appl. Pharmacol. 66, l-8. LOWRY, 0. H., ROSEBROUGH,N. J., FARR, A. L. AND RANDALL, R. J. (195 1). Protein measurement with the Folin phenol reagent 1. Bioi. Chem. 193,265-275. OSPITAL, J. J., HACKER, A. D., AND MUSTAFA, M. G. (198 1). Biochemical changes in rat lungs after exposure to nitrogen dioxide. J. Tox. Environ. Health 8, 47-58. SAGAI, M., ICHINOSE, T., ODA, H., AND KUBOTA, K. (1982). Studies on the biochemical effects of nitrogen dioxide. II. Changes of the protective systemsin rat lungs and of lipid peroxidation by acute exposure. J Tax. Environ. Health 9, 153-164. SALTZMAN, B. E. (1954). Calorimetric microdetermination of NO2 in the atmosphere. Anal. Chem. 26, 1949- 1955. SELGRADE, M. K., MOLE, M. L., MILLER, F. J., HATCH, G. E., GARDNER, D. E., AND Hu, P. C. (1981). Effect of NO* inhalation on protein and lipid accumulation in the lung. Environ. Res. 26,422-437. SNYDER,A., SKOZA, L., ANDKIKKAWA, Y. (1983). Comparative removal of ascorbic acid and other airway substancesby sequential bronchoalveolar lavages.Lung 161, 111-121. SOITOCASA, G. L., KUYLENSTIERNA, B., ERNSTER, L., AND BERGSTRAND, A. (1967). An electron transport system associated with the outer membrane of liver mitochondria. J. Cell Biol. 32,4 1S-438. STEPHENS,R. J., FREEMAN, G., AND EVANS, M. J. (I 972). Early response of lungs to low levels of nitrogen dioxide. Arch. Environ. Health 24, 160- 179. WASSERMAN, S. I., AND AUSTEN, K. F. (1976). Arylsulfatase B of human lungs-isolation, characterization, and interaction with slow reacting substance of anaphylaxis. J. Clin. Invest. 57, 738-744. WILLIAMS, C. H., AND KAMIN, H. (1962). Microsomal triphosphopyridine nucleotide cytochrome c reductase of liver. J. Biol. Chem. 237, 587-595. WRIGHT, E. S., ANLI MAILS, R. D. (198 I). Changes in pulmonary phospholipid biosynthetic enzymes after nitrogen dioxide exposure. Toxicol. Appl. Pharmacol. 58, 262-268.
WRIGHT, E. S., VANG, M. J., FINKELSTEIN, J. N., AND MAVIS, R. D. (1982). Changes in phospholipid biosynthetic enzymes in type II cells and alveolar macrophages isolated from rat lungs after NOr exposure. Toxicol. Appl. Pharmacol. 66,305-3 11.
AND
APPLIED
Biochemical
PHARMACOLOGY
Assessment DANIEL
81,
of Acute Nitrogen Dioxide Toxicity in Rat Lung J. GUTH’
Department of Radiation School of Medicine
Received
128-138 (1985)
AND RICHARD
Biology and Biophysics, and Dentistry, Rochester,
February
20. 1985; accepted
D. MAVIS* University of Rochester. New York 14642
June 3, 1985
Biochemical Assessment of Acute Nitrogen Dioxide Toxicity in Rat Lung. GUTH, D. J., AND R. D. (1985). Toxicol. Appl. Pharmacol. 81, 128-138. The early primary biochemical response of lung to NO, was studied separately from the later secondary responsesof inflammation and proliferation by measuring several biochemical parameters in lungs of rats immediately following a 4-hr exposure to nitrogen dioxide (NO*) at concentrations of 10, 20, 30, and 40 ppm. Cell-free lavage tluid contained elevated amounts of lactate dehydrogenase (LDH), malate dehydrogenase (MDH), isocitrate dehydrogenase (IDH), glucose-6-phosphate dehydrogenase (GDH), acid phosphatase (AP), and aryl sulfatase (AS) after 30 or 40 ppm NO*. Total protein and sialic acid were increased in cell-free lavage after 20, 30, or 40 ppm NOZ. The amounts of protein, siahc acid, and acid phosphatase recovered by airway lavage were equal to the amounts found in 0.7 ml of plasma, consistent with transudation of this volume of plasma into airways as a source of these parameters. The plasma activity of the other parameters measured was too low to account for their increase in lavage fluid by plasma leakage into airways. Decrease in the number and enzyme content of lavagable celfs indicated damage to free cells in the airways. The amount of the decrease in enzyme content of the lavagable cell fraction was similar to the increase in the cell-free lavage for all of the measured enzymes except acid phosphatase, suggesting the release of these enzymes into airways as a result of damage to free cells. However, the LDH isoenzyme profile in cell-free lavage after exposure is inconsistent with free cells as the source of this enzyme. No changes were observed in the whole-lung homogenate content of protein, DNA, lipid, LDH, MDH, IDH, GDH, AP, AS, glutathione reductase, NADPH cytochrome c, or succinate cytochrome c reductase immediately after NO* exposure. This study indicates that initial acute damage to lung by NO2 results in translocation of enzymes, proteins, and sialic acid into airways. Plasma is a likely source of translocated protein, sialic acid, and acid phosphatase. The sources of the other enzyme activities remain to be identified, with lung parenchyma and free cells as likely sources. MAWS,
0 1985 Academic PBS, he.
Acute or chronic exposure to nitrogen dioxide (NO*) results in a sequence of damage, inflammatory response, proliferation, and repair in the lung. Morphological damage to type I epithelial cells and ciliated bronchiolar cells is first seen after four hours of exposure to 17 ppm NO2 and, after 8 to 12 hr of exposure, cells are detached from the basement membrane (Stephens et al., 1972). Damage to the ’ Present address: California Primate Research Center, University of California, Davis, CA 95616. * To whom correspondence should be addressed.
0041-008X/85
$3.00
Copyright 0 1985 by Academic PBS, Inc. AU rights of reproduction in any form rserved.
epithelium results in increased permeability evidenced by transudation of serum protein (DeNicola et al., 1981; Selgrade et al., 1981) and penetration of high-molecular-weight tracers (Gordon et al., 1983). Exposure to NO2 is also followed by recruitment of inflammatory cells into the lung with increases in polymorphonuclear neutrophils (PMN) (DeNicola et al., 198 1; Gardner et al., 1969) and macrophages (DeNicola et al., 1981). Repair of lung epithelium after NO2 exposure involves proliferation of alveolar type II cells and nonciliated bronchiolar cells (Evans et al., 1972,
128
BIOCHEMICAL
ASSESSMENT
1973,1975,1976). The magnitude and the rate of onset and decline of the edema, inflammation, proliferation, and repair vary substantially with the concentration of NOz, the duration of the exposure, and the animal species under study. Measurement of biochemical changes in the NO*-exposed lung has provided information that is generally consistent with the morphological data describing the proliferative and inflammatory responses. Biochemical studies from this laboratory and others have defined parameters that increase in the lung after NOz exposure over a time course of days to weeks after initiation of exposure. These include protein, DNA, and various enzyme activities (Wright and Mavis, 198 1; DeNicola et al., 1981; Ospital et al., 1981; Sagai et al., 1982; Ichinose and Sagai, 1982). Isolation of alveolar epithelial type II cells from rat lung during NOz-induced proliferation has shown an increase in both the number of type II cells and their content of protein and enzymes, compared with type II cells isolated from control rats (Wright et al., 1982). This increase in cell number and size as a general response to lung damage could explain a number of biochemical changes in lung which occur a day or more after exposure to NO2 or other toxic agents. NOz exposure also results in recruitment of large numbers of inflammatory cells into the lung (DeNicola et al., 198 1) which could contribute significantly to biochemical changes in the whole lung. In order to identify early biochemical changes which reflect initial damage caused directly by interaction of NOz with lung tissue, such changes would ideally be studied in isolation from the secondary responses of proliferation and inflammation. In the work reported here we used an acute exposure protocol, in which rats were exposed to NOz for 4 hr and killed immediately after exposure in an attempt to characterize immediate biochemical changes resulting from primary damage before the complicating events of inflammation or proliferation.
OF NO* TOXICITY
129
METHODS Exposures. Male Long Evans rats from Charles River Breeding Labs (Portage, MA) were purchased as specific pathogen free, maintained in a barrier facility under a 12hr light cycle, 25°C temperature, and 30-35% relative humidity with free accessto rat chow (Agway) and water for at least 7 days prior to use. SurveilIance of specific pathogen status was performed periodically on selected animals. Animals weighed between 340 and 380 g when used. Six rats per group were exposed in a 29-liter glass chamber to NO2 (0.5% NO* in Nz, Air Products) diluted with compressed air at a final flow rate of 6 liters/min. Control groups were exposed to compressed air alone. Chamber concentrations of 10,20,30, or 40 ppm were continuously monitored with a Beckman Mode1 925A analyzer calibrated witb a standard 11.5 ppm NO* in N2 (Air Products). Concentration of calibration gas was established with the wet chemical method of Saltzman (I 954) and chamber concentration was checked three times during each exposure by this method. Chamber concentrations were maintained within 5% of stated concentration throughout exposure and NO concentration was less than 1% of NO2 concentration. Tissue preparation. After 4-hr exposure, rats were kihed immediately by ip injection of sodium pentobarbital. The lungs were perfused for 5 min through the right ventricle with 0.9% sodium chloride at a pressure of 30 cm of water and a temperature of 12°C. The dorsal aorta was severed prior to perfusion to decrease systemic resistance to perfusion. During perfusion, the lungs were gently ventilated through a trachea1 cannula. Following perfusion, the lungs were removed and immediately lavaged three times at room temperature with 6 ml per lavage of 0.9% sodium chloride buffered at pH 7.4 with 5 mM Tris-HCI. Each lavage was slowly injected and withdrawn four times through a tracheal cannula. Final recovery of injected lavage fluid was 85 to 90% both in control and exposed lungs. Lavaged cells were counted in a Coulter Counter (Hialeah, FL) and a hemocytometer. Differential cell counts were performed on Wright-stained cells prepared by cytocentrifugation. Lavage fluid was separated into cell-free lavage and cell pellet by centrifugation at 3OOgfor 15 min at 4°C. The cell pellet was resuspended in 10 ml of 5 mM Tris-HCl, pH 7.4. The cells were lysed for biochemical analysis in a Brinkman Polytron (Brinkman Instruments) at a setting of 6.5 for 15 sec. Our lavage procedure, adapted from Snyder et al., (1983) was designed to recover a major amount of airway material in a relatively small volume. We tested the efficiency of our procedure by more extensively lavaging the lungs of 12 rats (6 control, 6 exposed to 40 ppm NO2 for 4 hr) with an additional five lavages of 6 ml each beyond our normal procedure. We found that our normal procedure of three lavages recovered 74% of the total cells
130
GUTH
AND MAVIS
and 90% of the total protein removed in the more extensive lavage. There was no difference in trypan blue exclusion in any lavagable cell pellet. The lungs were minced, weighed, and placed in ice-cold 300 mM sucrose, 1 mM EDTA, buffered with Tris-HC1 at pH 7.4. The minced lungs were homogenized in a Teflonglass homogenizer followed by a Brinkman-Polytron for I min at setting of 6.5. The homogenate was filtered through 160~pm Nytex nylon screen to remove connective tissue fragments. Filtering resulted in loss of I .6% of total protein and 1.1% of total acid phosphatase and NADPHcytochrome c reductase. The filtrate was designated whole homogenate. Biochemical assays. Biochemical measurements made in whole-lung homogenate and in lavage fluid were chosen to reflect general tissue composition (protein, DNA, and lipid extractable phosphate) and to assessthe effect of NO* exposures on soluble enzyme activities as well as on enzymes associated with membranous subcellular structures. Protein was measured by the method of Lowry et al. (195 1). NADPH-cytochrome c reductase was measured as described by Williams and Kamin (1962) except that we used 68 PM cytochrome c and pH 8.1. Succinate cytochrome c reductase was measured according to Sottocasa et al. (1967) except that we used 48 mM succinate and 25°C. 5’-Nucleotidase was measured in 0.2 ml final volume with 100 mM KCl, 10 mM MgClz, 50 mM Tris-HCI, pH 7.4, 10 mM NaK tartrate, 2 mM AMP. ABer 15 min incubation at 37”C, 0.8 ml of 1% ascorbate, 1% sodium dodecyl sulfate, 0.8 N H2S04, and 3.36% ammonium molybdate was added and absorbance measured at 820 nm after incubation for 30 min at 45°C. Acid phosphatase (AP) was measured with pnitrophenol phosphate as sub strate in 50 mM Na acetate, pH 5.0, with 0.1% Triton X100. AAer 15 min incubation at 37°C the reaction was terminated by bringing the solution to 0.1 N NaOH, and absorbance of pnitrophenol was measured at 410 nm. Aryl sulfatase (AS) was measured according to the procedure described by Wasserman and Austen (1976) for arylsultatase B. Lactate dehydrogenase (LDH) was assayed in 1.O ml of 50 mM K-phosphate, pH 7.5, with 0.16 mM NADH and 0.48 rnM pyruvate by measuring decrease in absorbance at 340 nm. Malate dehydrogenase (MDH) was measured in 50 mM K-phosphate, pH 7.4, with 0.2 mM NADH and 0.75 mM oxaloacetate also by the decrease in absorbance at 340 nm. Glucose-6-phosphate dehydrogenase (GDH) was assayed in 1.0 ml of 50 mM triethanolamine, pH 7.6, with 0.15 mM NADP, 2.0 mM glucose 6phosphate+ 2.0 mM 6-phosphoghrconate, and 10 mM MgCl, by measuring the increase in absorbance at 340 nm. These conditions measure the combined activities of GDH and 6-phosphogluconate dehydrogenase. Isocitrate dehydrogenase (IDH) was assayed in 1.O ml of 50 mM triethanolamine, pH 7.5, with 2.7 mM DL-iSOCitEite, 0.3 mM NADP, and 2.7 mM MnS04 by following the increase in absorbance at 340 nm. Total siahc acid was measured by the
thiobarbituric acid method described by Downs and Pigman (1976) after acid hydrolysis. Glutathione reductase was assayedasdescribed by Bergmeyer et al. (1974). Lipid extractable phosphorus was determined by the procedure for total phosphate described by Ames ( 1966) on extracted lipid (Bligh and Dyer, 1959). DNA was assayed thtorometrically using the product formed with DABA prepared as described by Wright and Mavis (198 1). For all enzyme activities, one unit is defined as 1 nmol product formed/min. All enzyme assayswere proportional to incubation time and added protein under the conditions used. Multiple assaysof single samples varied by no more than 5% from the mean. LDH isoenzymes. LDH isoenzymes were electrophoretically separated and stained on 5.5% polyacrylamide gels as described by Dietz et al. ( 1970) and Dietz and Lubrano (1967). &enzyme patterns were scanned by a Gilford 2410 scanning attachment on a Gilford 250 spectrophotometer. Peak areas were measured with a compensating polar planimeter (Lasico). Cell-free lavage and lavagable cell sample volumes were concentrated by lyophilization before electrophoresis. The various isoenzymes of LDH were found to be uniformly stable to lyophihzation. Whole-lung homogenate was centrifuged at 16OOg for 15 min prior to electrophoresis. Statistics. The Mann-Whitney test was used to compare parameters measured in exposed animals with those of controls.
RESULTS The results of measurement of the chosen parameters in whole-lung homogenate from rats exposed to 40 ppm NO;? for 4 hr are shown in Table 1. No significant changes from control values were observed in lipid extractable phosphate, protein, DNA, or in several enzymes assayed. In pooled cell-free lavage fluid, however, NO2 exposure produced increases in several of these parameters, as seen in Table 2. Protein and sialic acid amounts were elevated to 24 and 15 times control values, respectively. The activities of two lysosomal enzymes and several soluble enzymes were also significantly increased after exposure. No detectable activity of the microsomal enzyme NADPH cytochrome c reductase or the mitochondrial enzyme succinate cytochrome c reductase was present in the lavage fluid of control or exposed animals. The concentration dependence of the bio-
BIOCHEMICAL
ASSESSMENT
131
OF NO, TOXICITY
TABLE 1 COMPARISON
OF WHOLE LUNG CONTENT OF PROTEIN, DNA, LIPID PHOSPHATE, AND ENZYMES” IN CONTROL BATS AND BATS EXPOSED TO 40 ppm NO* FOR 4 HOURS*
Lipid extractable phosphate, pmol/lung Protein, mg/lung DNA, mg/lung S-Nucleotidase Acid phosphatase Aryl sulfatase NADPH-cytochrome c reductase Succinate-cytochrome c reductase Lactate dehydrogenase Malate dehydrogenase Isocitrate dehydrogenase Glucosed-phosphate dehydrogenase Glutathione reductase
Control
NO7 EXDOS&
19.9 f 2.7 127.7 + 16.7 12.4 f 1.8 3.25 + 0.35 7.08 +- 1.72 4.89 + 0.98 0.51 + 0.10 0.37 k 0.10 33.9 It 9.0 47.4 * 11.0 4.72 zk 1.31 2.71 + 0.32 1.84 + 0.22
21.0 + 3.2 139.3 + 27.7 12.9 r!~ 1.3 3.53 xk0.86 7.64 z!z0.92 5.77 f 1.16 0.55 + 0.09 0.32 + 0.05 31.0 + 5.5 51.0 + 20.0 4.55 + 0.58 2.77 + 0.46 1.97 + 0.30
a All enzyme activities are expressed as 10’ units/lung. * Values are expressed as X of six animals f SD.
chemical changes in cell free lavage was investigated as shown in Figs. l-3. Protein and sialic acid content were increased significantly after 20, 30, or 40 ppm NO2 for 4 hr but not
after 10 ppm (Fig. 1). LDH, MDH, GDH, IDH, and AP were increased significantly above controls after 30 and 40 ppm NO* but not after 10 or 20 ppm (Figs. 2 and 3).
TABLE 2 COMPARISON
OF
CELL-FREE LAVAGE CONTENT OF PROTEIN, SIALIC ACID, LIPID PHOSPHATE, IN CONTROL RATS AND RATS EXFQSED TO 40 ppm NO2 FOR4 HOURS*
Protein Sialic acid Lipid extractable phosphate Lactate dehydrogenase Malate dehydrogenase Glucose-6-phosphate dehydrogenase Isocitrate dehydrogenase Acid phosphatase Aryl sulfatase NADPH-cytochrome c reductase Succinatecytochrome c reductase
AND ENZYMES’
Control
NO2 Exposed
3.63 k 1.00 49.1 + 9.9 2.01 f 0.75 381 + 144 457 + 264 7.03 + 2.42 10.7 f 9.9 22.2 + 9.2 90.1 + 42.0 n.d. (~2.21)~ n.d. (~3.53)
85.3 * 41 .o* 747.3 + 386.8* 2.94 f 0.62* 647 f 334** 709 k 367** 20.9 + 5.8* 39.4 f 12.2* 55.9 f 18.8: 273.3 k 33.5. nd. (~2.38) n.d. (~3.61)
’ All enzyme activities are expressed as units/lung; protein as mgjlung; sialic acid as &lung; fimol/lung. * Values are expressed as X of six animals + SD. ’ nd., Activity was not detectable. Lower limit of detection is given in parentheses. * Different from control at p < 0.05 by Mann-Whitney test. ** Different from control at p < 0.10 by Mann-Whitney test.
lipid
phosphate
as
132
GUTH
AND MAVIS
NOI Concentration (ppm)
NO2Concentration (ppm)
FIG. 1. Effect of NO2 concentration on recovery of protein and sialic acid in cell-free lavage after 4-hr exposure. Data points represent the X 2 SD for six rats expressed as mg protein/lung or pg sialic acid/lung in the cell free lavage. *Significantly different from control at p < 0.05 by the Mann-Whitney test.
In order to assess the contribution of the transudation of plasma to the increase in activity seen in the lavage fluid, plasma amounts of proteins, sialic acid, and enzyme activities were measured in control animals. These results are shown in Table 3, together with the magnitude of the increases in these parameters in lavage fluid NOz-exposed animals. No change in the plasma amounts of these parameters was observed after NO* exposure. The increases in lavage fluid content of protein, sialic acid, and AP are equivalent to the amount of these components found in 0.72, 0.54, and 0.69 ml of plasma, respectively. These volumes are within the range of plasma which might be expected to enter the airways by transudation. Plasma amounts of all other components in Table 3 are such that transudation of at least 3 ml of plasma would be indicated if plasma were the sole source of these enzymes. The effect of the NOz exposure on the lavagable cell population is shown in Table 4. Total cells recovered in the lavage were decreased by 44% as measured in the hemocytometer. A similar decrease was found with a Coulter Counter in the size range characteristic
of macrophages as well as an increase in material of smaller size, possibly debris and nuclei from the disruption of intact cells. The retention of nuclear material in the cell pellet in spite of the decrease in countable cells is evidenced by the lack of change in the amount of DNA in the lavagable cell fraction. Lavaged cells were 98 and 96% macrophages in the control and exposed animals, respectively. Polymorphonuclear (PMN) cells increased slightly in exposed animals to 2.7% compared to 0.22% of recovered cells in the control animals. Although the DNA content of the cell pellet did not change significantly, there was a significant increase in DNA remaining in the cell free lavage, and the total lavagable DNA increased by 25%. The increase in lavagable DNA is equal to 0.05% of the total lung DNA content. Because of the apparent damage to the free cells, we examined changes in protein, sialic acid, lipid extractable phosphate, and enzyme content of the lavagable cells. In Table 5 are presented the total content of these parameters in the lavagable cells and cell-free lavage as well as the change in exposed animals. This presentation allows comparison of the in-
BIOCHEMICAL
ASSESSMENT
a
35’
133
OF NOz TOXICITY
b
’
’
’
’
NO2 Concentration
’
’
‘**----7
0 10 20 NO1 Concentration
(ppm)
30
40 (ppm)
600
100
o-
0 10 20 NOz Concentration
30
40 (ppm)
0
I
I
I
0 10 20 NOI Concentration
,
30
I
40 (ppm)
FIG. 2. Effect of NO2 concentration on recovery of soluble enzymes in cell free lavage after 4-hr exposure. Data points represent the X k SD for six rats expressed as units/lung in the cell-free lavage. (a) Isocitrate dehydrogenase; (b) malate dehydrogenase; (c) glucose-6-phosphate dehydrogenase; (d) lactate dehydrogenase. *Significantly different from control at p < 0.05 by the Mann-Whitney test.
creases in lavagable content with the decrease in cell content in order to evaluate the possibility that lavagable cells are a source of the increase in cell-free lavage contents. The cellfree lavage contents of protein and sialic acid are, respectively, 10 I- and 17 l-fold greater than the changes in cell content, eliminating free cells as the source of these parameters. The lipid-soluble phosphate content increased
slightly in the cell pellet and significantly in the cell-free lavage, indicating another source of phospholipid, probably lung tissue or plasma. In the case of the soluble and lysosomal enzymes, the increase in the content of cell-free lavage is the same, within experimental error, as the decrease in content in the lavagable cells, suggesting these cells as a source of the cell-free lavage activity. The exception
134
GUTH 70 j-
AND MAVIS TABLE 3
I
COMPARISONOFPLASMAAHOUNTSINCONTROLANIMALS WITH THE INCREASE IN CELL-FREE LAVAGE AMO~NTSOFPROTEIN, SIALICACID, ANDENZYMES" IN ANIMAL~EXPOSEDTO 40ppm NO2 ~0~4 Hou~s~ Plasma Plasma component
IL---J 0
10
20
30
NO2 Concentration
40
(ppm)
FIG. 3. Effect of NO2 concentration on recovery of acid phosphatase in cell-free lavage after 6hr exposure. Data points represent the 1+ SD for six rats expressed as units/ lung in the cell-free lavage. *Significantly different at Q < 0.05 by the Mann-Whitney test.
ProteinC Sialic acid’ Lactate dehydrogenas& Malate dehydrogenase” Isocitrate dehydrogemw? Glucose&phosphate dehydrogenas& Acid phosphatask
am0UM.S’
62.4
2.5
f
1.12 * 91.2 38.7
Increase in cell-free lavage b 44.9 0.61 276 345 13.86
0.12
f 51.3 21.2
+
n.d. (<1.41)’ 3.16 44.4
f k
0.72 10.7
16.88 30.8
a Plasma amounts are expressed as X + SD for six rats. b Data are the difference between the X of six rats exposed to 40 ppm NO* and the X of six control rats. Data are taken from Figs. I, 2 and 3. ’ For protein and sialic acid, plasma amounts are expressed as mg/ml and cell free lavage amounts are expressed as mg/lung. d For enzymes, plasma amounts are expressed as units/ml and cell-free lavage amounts are expressed as units/lung. ’ n.d., Not detectable. Lower limit of detectability is given in parentheses.
to this was AP, which was increased in an amount consistent with plasma as the source of increase, as described above. In a further attempt to identify the source of the LDH activity increase in lung lavage after NO2 exposure, we determined the iso-
L “I b
CONTROL
FREE CELL
TABLE 4 EFFETE OFEXPOSURETO 40ppmN02 FOR~HOURS ONLAVAGEABLECELLSANDDNA' Control Total cells X 10’ 1
2
3 LDH ISOZYME
4
5
#
FIG. 4. Effect of exposure to 40 ppm NO, for 4 hr on LDH isoenzyme pattern in rat lung lavage. Data are presented as the X k SD for five rats expressed as a percentage of total activity for each isoenzyme. (a) Control and exposed cell-free lavage; (b) control and exposed lavagable cells. *Significantly different than control at p < 0.05 by the Mann-Whitney test.
Percentage macrophages Percentage PMN DNA Lavageable cells (pg) Cell-free lavage (a) Total Iavagable Whw
NOzExposed
0.27 1.3 0.22
0.46 2 0.11; 96.0 + 3.5 2.66 + 2.51*
4.8 0.48
19.1 13.8
f
3~
f
5.6 2.7*
2
4.6
33.7
_+
6.0*
0.82 97.8 0.22
f
24.2 2.87
f
27.0
+ +
’ Values are expressed as X f SD for 6 control and 12 exposed rats. * Values are significantly different from controls at p < 0.05 by the Mann-Whitney test.
BIOCHEMICAL
ASSESSMENT
135
OF NO2 TOXICITY
TABLE 5 COMPARISON
OF THE EFFECTS OF 40 ppm NO2 ON PROTEIN, SIALIC ACID, LIPID EXTRACTABLE AND ENZYME CONTENT IN LAVAGABLE CELU AND CELL-FREE LAVAGE’
Cell-free lavage
Cells
Control Protein Sialic acid Lactate dehydrogenase Malate dehydrogenase Glucose-6-phosphate dehydrogenase Acid phosphatase Aryl sulfatase Lipid phosphate
PHOSPHATE,
Exposed
Difference
Control
Exposed
Difference
1.87 f 0.67 19.1 f 4.0
1.31 f 0.36 15.7 f 4.1
0.56 3.37
3.09 f 0.62 21.5 f 8.3
59.7 f 16.9* 599 + 174*
56.6 577
607 f 172
387 f 86*
219
421 + 164
679 + 118*
258
1190 f 405
800 + 234*
390
647 2 195
1052 + 210*
405
11.5 47.2 367 1.44
7.1 57.6 236 1.89
4.4 10.7 131 0.45
<2.8 15.4 rt 8.8 76.9 f 22.1 3.27 f 0.60
14.1 + 44.1 + 228 + 4.03 +
11.3 28.7 151 0.76
+ f f +
7.2 17.8 127 0.69
f + f f
2.7 24.4 86* 0.58**
5.0* 9.0; 47* 0.59*
a Values are expressed as X + SD for six control and six exposed rats. Units are mg/lung for protein, &lung sialic acid, ~mol/lung for lipid phosphate, and U/lung for all enzymes. * Values are significantly different from controls at p < 0.05 by the Mann-Whitney test. ** Values are significantly different from controls at p < 0.10 by the Mann-Whitney test.
enzyme composition of the LDH activity in the control and NOz-exposed lavage as shown in Fig. 4. The isoenzyme profiles of control lung homogenate, plasma, and red blood cells are shown in Fig. 5 as possible sources of the LDH released into the airways. In cell-free lavage from NOz-exposed animals, the proportions of isoenzymes 1, 2, and 3 are increased while isoenzymes 4 and 5 are decreased compared to the control lavage profile. No significant change in isoenzyme profile of lavagable free cells occurred in response to NO;!. The isoenzyme profiles of lung homogenate, red blood cells, or plasma did not correspond to the changes seen in the cell-free lavage.
for
CL 25 0 :
b
PLASMA
DISCUSSION The biochemical changes reported here as increases in lavagable parameters appear to result directly from lung damage. A minimal increase in PMNs suggests little inflammatory response at this early time after insult. A decrease in the number of lavagable intact mat-
LDH ISOZYME
#
FIG. 5. Isoenzyme patterns of LDH in (a) lung homogenate. (b) \ , .dasma. and (c) red blood cells.
136
GUTH
AND MAWS
rophages occurred which, together with an increase in lavagable cell debris, suggests direct destruction of these cells by NO;!. The absence of an increase in whole-lung content of protein, phospholipid, or DNA demonstrates a lack of significant proliferation, in agreement with previous studies at 5 or 6 hr after exposure in rats (Blank et al., 1978; Evans et al., 1971) and in hamsters (Creasia, 1978). No damage was detectable as changes in the parameters measured in whole-lung homogenate. The amount of enzyme activity increases detectable in lavage fluid represents 2% or less of the total lung activity, however (except for AS activity, which was 5%) and thus could be derived from lung parenchyma as a result of focal cell damage and release of enzymes without detectable loss of activity from whole-lung homogenate. The increase in lavagable DNA represents a very small fraction (0.05%) of the total lung DNA content and could similarly be a result of focal release of DNA from lung parenchyma. Alternatively, a modest increase in the number of free lavagable cells together with an increase in their fragility as a result of NOz exposure could be responsible for the increase in total lavagable DNA if the fragile cells released DNA into the cell-free supernatant fraction where the lavagable cell increase is localized. The similarity of the amount of enzyme activities lost from the lavagable cell fraction as a result of NO1 exposure to the increases observed in the cell-free lavage supernatant fraction suggests translocation from lavagable cells into the supernatant fraction as a result of cell damage. However, isoenzyme analysis of LDH activity in lavage and free cells clearly shows that free cells cannot be the source of released LDH. Isoenzymes numbers 4 and 5 comprise over 80% of the activity in free cells while the cell-free lavage after NOz shows increases in isoenzymes numbers 1, 2, and 3, and a decrease in the percentage of isoenzymes 4 and 5 compared to control. Thus the LDH activity increase in lavage cannot be from free cells. The profile of LDH isoenyzmes in cell-free la-
vage after NOa exposure most nearly resembles that of the whole-lung homogenate. The changes in the various isoenzymes compared to control, however, do not correspond to the profile of whole lung homogenate, which contains major percentages of isoenzymes 4 and 5. The major isoenzyme in plasma is isoenzyme 2, which is one of the isoenzymes which increases in lavage after NO*. The magnitude of LDH activity in plasma, however, is relatively low. The increase in lavagable LDH after NO2 exposure represents an amount of activity found in 3 ml of plasma. Transudation of this volume of plasma would cause an increase in lung weight of around 150%, which is unlikely. Maximum changes in lung weight reported by Blank et al. (1978) for rat lungs 6 hr after a 5hr exposure to 40 ppm NO2 was a 50% increase. We have observed no statistically significant changes in lung weight immediately after 4 hr of exposure to 40 ppm NOz (unpublished observation). Red blood cell LDH is composed predominantly of isoenzyme 5 and therefore red blood cells cannot be a significant source of the increase in lavage. Thus LDH isoenzyme analysis allows elimination of free cells as the sole source of the LDH activity increase in cell-free lavage after NO2, but does not yield an unambiguous identification of the source of the translocated LDH. None of the sources analyzed showed an isoenzyme profile which corresponded to the increases observed in lavage. An additional complicating factor in the interpretation of LDH profiles is the possibility of selective inactivation of isoenzymes by NOz. In particular, isoenzymes 4 and 5 are lost from lavagable cells and yet are not recovered in the lavage supernatant fraction. Beck et al. (1983) have analyzed LDH isoenzymes in hamster lung lavage after injury by a variety of agents. Instillation of a-quartz or iron oxide produced lavagable LDH isoenzymes which resembled the distribution in unexposed PMNs and macrophages. High oxygen yielded a pattern similar to unexposed serum, and lavage with a solution of Triton X-100 removed isoen-
BIOCHEMICAL
ASSESSMENT
zymes of LDH in a pattern typical of unexposed whole lung homogenate. In contrast to the increases in enzyme activities, the amount of protein in the lavage fluid after NOz increases in an absolute amount which corresponds to a major percentage of the whole lung protein. Since protein is not lost from the whole-lung homogenate in response to NOz, the relatively large protein increase in lavage must derive from a source outside of the lung, with plasma as a likely source. Comparison of plasma protein content with the amount of protein appearing in lavage after NO2 reveals that transudation of 0.7 ml of plasma into the airways would result in the observed increase in protein. This volume of plasma would also account for the amount of sialic acid and AP, appearing in lavage, but not the amounts of other enzymes studied, due to their relatively low activities in plasma as shown in Table 3. Another difference between the appearance of protein and sialic acid and the appearance of the enzymes is that the appearance of protein and sialic acid in the lavage fluid was statistically significant at an NOz concentration of 20 ppm while the enzyme appearance was significant at 30 ppm or above. This suggests that the release of enzymes results from a different and possibly less sensitive event than that which releases protein and sialic acid. An interpretation of this may be that permeability change in the alveolar septum may precede damage to cells and release of cellular components. Selgrade et al. (198 1) reported significant increases in lavage protein 12 hr after a 3-hr exposure of vitamin C-deficient guinea pigs to 5 ppm NOz. Comparing our results immediately following a 4-hr exposure to NO2 with the reported biochemical changes in hamster lungs after exposure to NO* for 48 hr (DeNicola et al., 198 1) yields some interesting parallelisms and contrasts. The longer exposure resulted in the several-fold elevation of a number of enzymes, sialic acid, and protein in lavage fluid, a result comparable to the present study. In contrast
OF NO2 TOXICITY
137
to our results, however, the lungs exposed for 48 hr showed marked inflammation as evidenced by a several-fold increase in lavagable cells, over 65% of which were PMNs. In addition, several enzyme activities and total protein were elevated in the homogenate of lung tissue as a result of the longer exposure, consistent with proliferation of lung cells to replace those damaged by NOz (Blank et al., 1978; Wright et al., 1982). Thus elevation of enzymes, protein, and sialic acid in lavage fluid appears to be an indicator of direct damage which begins during the first few hours of exposure and persists during 48 hr of continuous exposure. Elevation of lavagable cells appears to be an indicator of the later appearing process of inflammation, and elevation of enzymes and protein in the whole-lung homogenate is also a later event possibly reflecting proliferative repair. These results, together with a growing body of evidence (Henderson, 1984) lead to the conclusion that the appearance of various biochemical entities in the lavage is a useful indicator of early toxic damage to the lung, detectable prior to the secondary events of inflammation and proliferation, and that identifying the source of these entities may yield information on the location and nature of the damage. ACKNOWLEDGMENTS This paper is based on work performed under Contract DE-AC02-76EV03490 with the U.S. Department of Energy at the University of Rochester Department of Radiation Biology and Biophysics, and has been assigned Report No. DOE/EV/03490-2427. Daniel Guth was supported by NIH Training Grants 5T32-ES07026 and 5T32HLO7216-08. The expert technical assistance of Michael J. Vang is gratefully acknowledged.
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