Effects of intratracheal administration of xanthine plus xanthine oxidase on lung antioxidant enzymes, lipid peroxidation, and collagen in hamsters

EXPERIMENTAL

AND

MOLECULAR

PATHOLOGY

49, 395-409 (1988)

Effects of Intratracheal Administration of Xanthine plus Xanthine Oxidase on Lung Antioxidant Enzymes, Lipid Peroxidation, and Collagen in Hamsters’ SHRI N. GIRI, DALLAS M. HYDE,’ PETEREMAU, AND HARA P. MISIW~ Departments of Veterinary Pharmacology and Toxicology and 2Anatomy, School of Veterinary Medicine, University of California, Davis, California 95616, and 3College of Veterinary Medicine, University of Virginia, Blacksburg, Virginia 24061 Received

March

28, 1988, and in revised

form

July 20, 1988

Xanthine (X) and xanthine oxidase (X0) were injected intratracheally (IT) in hamsters at Day 0 (38 mg X, 100 pg X0) and Day 5 (38 mg X, 250 pg X0). Control hamsters received saline or X (38 mg) plus boiled X0 (100,250 kg). Cytoplasmic superoxide dismutase (SOD) activity increased from control of 286 to 337 and 335 unitsihmg at Days 12 and 19, respectively, but decreased to 228 units/lung at Day 33; mitochondrial SOD activity increased at Day 12 from control of 57 to 71 unitsflung and then decreased at Days 26 and 33 to 42 and 33 units/Iung, respectively. Glutathione peroxidase (GP) and glutathione reductase (GR) activities rose from their control values of 1161 and 1151 to 1561 and 2287 units/lung at Day 12, respectively; thereafter, GR activity decreased to 512 and 462 unitsflung at Days 19 and 26, respectively. Glutathione transferase declined at Day 12 but increased at Day 26 after initial treatment. Glucose&phosphate dehydrogenase activity declined from control of 1071 to 693 units/lung at Day 2 and returned to control thereafter. Catalase activity remained unaffected. Hydroxyproline was increased from 903 @lung in control to 1080, 1301, 1195, and 1148 CLg/lungat Days 12,19,26, and 33, respectively. Malonaldehyde increased from 40 nmole/lung in control to 70 and 113 nmole/Iung at Days 12 and 33, respectively. The ratio of right ventricle to left ventricle and septum increased significantly from control of 0.277 to 0.318 at Day 33. Histopathology at Days 2 and 4 revealed peribronchiolar and arteriolar infIammation, and diffuse alveolitis. By Day 12 there were thickened alveolar septa and foci of fibrotic consolidation. 8 1988Academic press, IX.

INTRODUCTION Formation of oxygen free radicals is considered to be an important mechanism for lung injury leading to fibrosis following exposure to air pollutants, herbicides, hyperoxia, and a variety of pulmonary toxicants (Menzel, 1976; Thomas, 1986; Clark et al., 1985; Fridovich, 1978). The free radicals interact with cellular components such as nucleic acids, proteins, and membranes to cause dysfunctions in biological processes (Proctor, 1984). However, in tissues and cells, injury can be prevented by a complex set of protective mechanisms, including superoxide dismutase, catalase, glutathione oxidation-reduction enzyme systems, vitamins E and C, p-carotene, and repair processes (Kimball et al., 1976). Despite current advances in understanding of the pathogenesis of oxidant-mediated tissue injury, little is known about the mechanism by which oxygen free radical formation leads to lung fibrosis. To study the role of free radicals in lung injury, animal models have been developed in which the effects of specific scavengers of free radicals on lung toxicity are evaluated (Johnson et al., 1981). Additionally, intratracheal adminisi Part of this work was presented at the 71st Annual Meeting at FASEB in Washington, DC, March 29-April5, 1987, and published as an abstract in Fed. Proc. 47564 (1987). This work was supported by the National Heart Lung and Blood Institute, under Grant 5Rol HL-23754-06. 395 0014-4800188 $3.00 Copyrieht 0 1988 by Academic Press, Inc. Au rights of reproduction in any form nserved.

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tration of substances such as xanthine plus xanthine oxidase which generate oxygen free radicals has been utilized to cause acute lung injury (Johnson et al., 1981). The present studies were carried out to investigate further the timedependent changes in lung injury, antioxidant enzymes activities, lipid peroxidation, and lung collagen following intratracheal instillation of two doses of a mixture of xanthine plus xanthine oxidase at an interval of 5 days. The results of the study showed than an initial dose of xanthine plus xanthine oxidase mixture produced acute transient lung injury without any marked effects on antioxidant enzymes. However, the second dose of the mixture induced severe lung injury, lipid peroxidation, and changes in antioxidant enzyme activities, lung fibrosis, and right ventricular hypertrophy. MATERIALS

AND METHODS

Animals

Hamsters weighing W-100 g were purchased from Simonsen, Inc. (Gilroy, CA) and housed in a group of three to four in a large plastic cage and had access to water and laboratory chow ad libitum. Treatment

of Animals

Xanthine oxidase was purified from cream of cow’s milk. Xanthine was purchased from Sigma Chemical Co. (St. Louis, MO). The hamsters were allowed 7 days to acclimatize to the housing conditions and then given two intratracheal injections of xanthine plus xanthine oxidase solutions as follows: At Day 0, 38 mg xanthine in 0.25 ml sterile phosphate-buffered saline (PBS, pH 7.4) plus 100 kg xanthine oxidase in 0.25 ml ice-cold PBS were injected intratracheally into hamsters under sodium pentobarbital anesthesia (70-80 mg/kg ip). Xanthine and xanthine oxidase remained separated by an airspace in the syringe. Five days later, the same hamsters received intratracheal injections of 38 mg xanthine in 0.25 ml PBS plus 250 pg xanthine oxidase in 0.25 ml ice-cold PBS. Control animals were treated as follows: One group of control hamsters received intratracheal injections of 0.5 ml PBS at days 0 and 5. Another group of control hamsters were given intratracheal injection of 38 mg xanthine in 0.25 ml PBS plus 100 pg heatinactivated xanthine oxidase in 0.25 ml PBS at Day 0 followed by 38 mg xanthine in 0.25 ml PBS and 250 kg heat-inactivated xanthine oxidase at Day 5. Sacrifice

of Animals

and Tissue Processing

Hamsters in treated and control groups were sacrificed under anesthesia induced by sodium pentobarbital (gO-90 mg) at 2,4, 12, 19,26, and 33 days following treatment with the initial dose of xanthine plus xanthine oxidase, or xanthine plus heat-inactivated xanthine oxidase. Blood was collected from the inferior vena cava into heparinized syringes. Blood was centrifuged at 1 lOOg, at 4°C for 20 min. Plasma was harvested and stored at - 80°C. Histopathology

Hamsters designated for histopathology studies were sampled by time and treatment (four in each group) and anesthetized as described above. After bronchoalveolar lavage and thoracotomy, the heart was ligated at the base for isolation of the pulmonary vasculature. The trachea was cannulated and lungs and heart

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were removed and weighed. The lungs were fixed by airway instillation of cacodylate-buffered glutaraldehyde-paraformaldehyde fixative (400 mOsm) at a pressure of 30 cm Hz0 for a minimum of 2 hr (Giri and Hyde, 1987). The cannula was removed, the trachea was tied off, and the lung and heart were stored in fixative. Blocks of tissue were cut from at least two sagittal slabs (2-3 mm thick) from the right cranial, right caudal, and left lung lobes of each lung. Each block was cut with about a l-cm* face. These blocks were dehydrated in a graded series of ethanol and embedded in paraffin. Five-micrometer-thick sections were cut from the paraffin blocks and stained with hematoxylin and eosin. From selected blocks, adjacent sections were cut and stained with Sirius Red for specific staining of collagenous fibers (James et al., 1986). Electron Microscopy One slab from each lobe was used to select a random block of about a OS-cm’ face for embedding in plastic. One-micrometer-thick sections were cut and stained with Toluidine Blue. Representative lesions were selected from these sections for further observation by electron microscopy. One-square-millimeter regions were cut from the larger plastic blocks, mounted on BEEM capsules, sectioned at 50-80 nm thick, and stained with uranyl acetate and lead citrate. These sections were examined on a Zeiss EM10 electron microscope. Biochemical Study The lungs of hamsters for biochemical studies were perfused in situ with 35 ml ice-cold isotonic saline through the right side of the heart. These lungs were then dissected free of extraneous tissues, immediately frozen in liquid nitrogen, and stored frozen at - 80°C until used for enzyme and collagen assays. The lungs were prepared for the assays as follows: The lungs were homogenized in 10 ml potassium phosphate-buffered saline (0.05 M, pH 7.8) using a Brinkmann Polytron. The resulting homogenate was distributed in l-ml volumes to ice-cold tubes for determination of collagen and lipid peroxidation. The rest of the homogenate was centrifuged at 102,OOOg, at 4°C for 60 min. The supernatants were distributed in l-ml volumes to ice-cold tubes and stored at -80°C until they were used for antioxidant defense enzyme assays. Collagen Analysis The l-ml homogenate for collagen determination was precipitated with 0.25 ml 50% trichloroacetic acid and the precipitate was hydrolyzed in 6 N HCl for 18-20 hr at 110°C. The amount of lung collagen expressed as micrograms of hydroxyproline per lung was measured by a method described by Woessner (1961). Catalase The catalase activity in the 102,OOOg supernatant of the lung homogenate was determined according to the procedure described by Holmes and Master (1970). The rate at which catalase causes decomposition of H202 was followed through the measurement of decreases in absorbances of H,O, at 240 nm in a Varian Cary 219 spectrophotometer. The rate constant of a first-order reaction (K) was used as a measure of catalase activity per lung where K (see-‘) = (l/At) x (Absorbancer/Absorbance& Absorbance, equals absorbance of H,02 at time 1 and absorbance, equals absorbance of H202 at time 2.

398 Glutathione

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Reductase

The glutathione reductase activity in the 102,OOOg supernatant of the lung homogenate was determined using a procedure described by Racker (1955). The oxidation of NADPH to NADP+ with oxidized glutathione as a substrate was followed. The catalytic activity of glutathione reductase was measured by following the decrease in absorbance of NADPH at 340 nm in a Varian Cary 219 spectrophotometer. One unit of enzyme activity was considered to be 1 kmole of NADPH oxidized per lung per hour. Glutathione

Peroxidase

The glutathione peroxidase activity in the hydroperoxide as a substrate as described by zyme was coupled to NADPH by glutathione oxidation was measured spectrophotometrically activity was considered equivalent to 1 pmole hour. Glutathione

lungs was assayed using cumene Chow and Tappel (1974). The enreductase and the rate of NADPH at 340 nm. One unit of enzyme of NADPH oxidized per lung per

Transferase

The glutathione transferase activity in the 102,OOOg supernatant of lung homogenate was determined using l-chloro-2, 4-dinitrobenzene as a substrate as described previously (Habig et al., 1974). Glucose-6-phosphate

Dehydrogenase

The activity of glucose-6-phosphate dehydrogenase in the lungs was measured with glucosed-phosphate as a substrate according to a method described by Deutsch (1986). Glucose-6-phosphate dehydrogenase utilizes NADP to oxidize glucose-6-phosphate. The rate of formation of NADPH is determined at 340 nm using a Varian Cary 219 spectrophotometer. One unit of enzyme activity was considered to be 1 p,mole of NADPH formed per lung per hour. Superoxide

Dismutase

Superoxide dismutase activity in the lungs was determined from the rate at which it inhibits the autoxidation of epinephrine to adrenochrome, as described by Misra and Fridovich (1972). The reaction mixture consisted of NaHCO,-CO3 buffer (pH 10.2), EDTA, and epinephrine. The rate of formation of adrenochrome was 0.025 absorbance units/min at 480 nm in a Varian Cary 219 spectrophotometer. Under these defined conditions, the amount of superoxide dismutase required to inhibit the rate of formation of adrenochrome by 50% (i.e., rate of 0.0125 absorbance units/min) is defined as 1 unit of activity. Cyanide (CN-) inhibits cytoplasmic, but not mitochondrial superoxide dismutase activity (Misra and Fridovich, 1972). Therefore, total activity of the enzyme represents the activity of superoxide dismutase in the sample in absence of CN- , while mitochondrial SOD activity represents the activity of the sample in the presence of CN- . Cytoplasmic SOD activity is the difference between total and mitochondrial SOD activity. Lipid Peroxidation

Thiobarbituric

acid reacting product (TBA) as an index of lipid peroxidation

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was measured in the whole lung by the method of Ohkawa et al. (1979), and the method of Yagi (1984) was used for plasma. Estimation

of Right Heart Hypertrophy

After the heart was isolated both left and right auricles were removed. The right ventricle was carefully separated from the septum and left ventricle and each ventricle was weighed separately. The ratio of weights of right ventricle to that of left ventricle plus septum was used to estimate right ventricular hypertrophy (Turner et al., 1986). Statistical

Analysis

The data for all biochemical measurements were expressed per total lung in order to avoid the artificial lowering of the values in xanthine plus xanthine oxidase treated animals. This was necessary to minimize the effects of lung perfusion, residual plasma proteins, and infiltration of leukocytes in the lung alveoli of hamsters treated with this mixture since these factors would lower the values if the data were expressed either per gram tissue or per milligram protein (Karlinsky and Goldstein, 1980). The values are reported as the means + standard error of mean. Means of treated and control groups were compared using unpaired Student’s t test. The P values ~0.05 were considered significant. RESULTS As shown in Table I, xanthine plus xanthine oxidase had no effect on the catalase activities in the lung. However, the activity of glucosed-phosphate dehydrogenase decreased at 2 days following treatment and thereafter returned to the basal level. At 12 days after initial treatment the activities of glutathione reductase and glutathione peroxidase were significantly elevated; thereafter, glutathione peroxidase activity returned to control value while glutathione reductase activity declined at 19 and 26 days. Table II summarizes the effects of xanthine plus xanthine oxidase on superoxide dismutase activity in the lung. Cytoplasmic superoxide dismutase activity in the lungs was increased gradually to significant levels at 12 and 19 days after initial treatment of hamsters, but decreased at 33 days. Mitochondrial superoxide dismutase activity was significantly elevated 12 days after initial treatment but decreased at 26 and 33 days. These changes resulted in increases in total lung SOD activity at 12 and 19 days, but a decrease at 33 days after initial treatment. Figure 1 summarizes the effects of xanthine plus xanthine oxidase on lung glutathione transferase activity in hamsters. There was a significant decline in the activity of glutathione transferase from the control values of 5.1 2 0.2 to 4.2 + 0.3 units/lung at 12 days followed by a significant increase to 6.0 + 0.3 units/lung at 26 days after initial treatment. The accumulation of hydroxyproline in the lungs following treatment of hamsters with xanthine plus xanthine oxidase is shown in Fig. 2. Significant increases in total lung hydroxyproline as a measure of collagen relative to control were observed in animals treated with xanthine plus xanthine oxidase at 12, 19,26, and 33 days after initial treatment. The TBA reacting products as a measure of lipid peroxidation increased signif-

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TABLE I Effects of Intratracheal (IT) Administration of Xanthine plus Xanthine Oxidase on Lung Glutathione Reductase (GR), Glutathione Peroxidase (GP), Glucose-6-phosphate Dehydrogenase (G-6-PDH), and Catalase Activities in Hamsters” Time after IT administration of first dose (days) Control First dose 2 4 Second dose (5) 12 19 26 33

Enzyme activity G-6-PDH’ (unitskrng)

Catalased K (set-‘)/lung

GRb activity

Gp activity

11.51 k 106 (10)

1161 k 72 (10)

1071 2 84 (10)

0.88 k 0.06 (10)

1299 2 102 (5)

1219 * 150 (5)

0.95 f 0.05 (5)

926 f 82 (6s

1167 _’ 110 (6)

693 f 48 (5) P < 0.01’ 976 2 10 (6)

1561 k 95 (6) P < 0.01 1349 f 69 (6) NS 1166 k 65 (7) NS 1322 f 130 (4) NS

1246 + 154 (6) NS* 1050 k 82 (6) NS 1079 f 58 (7) NS 917 -1- 34 NS

0.97 f 0.09 (6)

2287 P 512 P 462 P 937

f 397 (6) < 0.01 k 76 (6) < 0.001 _’ 63 (7) < 0.01 +- 104 (4) NS

0.97 -+ 0.05 (6)

0.87 f 0.02 (6) 0.77 4 0.05 (7) 0.96 k 0.05 (4)

a See Materials and Methods for treatment schedule. b One unit of enzyme activity was considered to be equivalent to 1 umole of NADPH oxidized/ hmg/hr. ’ One unit of enzyme activity was considered to be equivalent to 1 umole of NADPH formed/ lungihr. d K (see-i) = (1lAr) x (AbsorbanceJAbsorbance,). c P values are between treated and control. f Number in parentheses is the number of animals used. B NS, not significant.

icantly in the lung at 12 and 33 days and in plasma at 12 days following the initial administration of xanthine plus xanthine oxidase in hamsters (Fig. 3). The effects of xanthine plus xanthine oxidase on the hypertrophy of the right heart are summarized in Fig. 4. The ratio of the weights of right ventricle to left ventricle plus septum increased significantly over the controls at 33 days after initial treatment. Pulmonary parenchyma and intraalveolar septa from control hamsters were thin and had normal appearing cells (Fig. 5). At 2 days following treatment with xanthine plus xanthine oxidase there was diffuse alveolitis and epithelial necrosis involving mostly intraalveolar macrophages and some neutrophils (Fig. 6). There was also infiltration of the interstitium with neutrophils. At 4 days following treatment with xanthine plus xanthine oxidase (Fig. 7), the perivasculature was infiltrated with fibroblasts and neutrophils and there was thickening of alveolar septa. The adventitia of arteries and small veins were also thickened and alveolar macrophages and fibroblasts accumulated in some alveolar ducts. At 12 days after initial treatment the lungs showed a diffuse alveolitis and thickening of interstitial septa (Fig. 8). The thickened alveolar septa were lined with cuboidal epithelial cells and contained neutrophils and tibroblasts. There was some intraalveolar organization and hemorrhage. In some animals there was edema fluid in the alveoli.

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TABLE II Effects of Intratracheal (IT) Administration of Xanthine plus Xanthine Oxidase on Mitochondrial and Cytoplasmic Superoxide Dismutase (SOD) Activities of the Lung in Hamsters Time after IT administration of fust dose (days) Control First dose 2 4 Second dose 12 19 26 33

SOD activity (units/lung)a Mitochondrial’

Cytoplasmi&

Totalb

57 + 3 (10)

286 f I.5 (10)

333 + 11 (10)

49 2 3 (5) 53 2 5 (6)

291 2 8 (5) 308 f 25 (6)

340 ” 7 (5) 362 k 29 (6)

71 +- 6 (6)

337*

408 k 15 (6) P < 0.001 387 f 20 (6) P < 0.01 341 k 11 (7) NS 261 + 7 (4) NS

ll(6)

P < 0.05’

P < 0.05

51 ? 4 (6y NSg 42 5 3 (7) P < 0.01 33 +- 2 (4) P < 0.001

335 2 19 (6) P < 0.05

299 f 11 (7) NS 228 + 6 (4) NS

LIOne unit is equivalent to the volume of the sample that gives 50% inhibition of conversion of epinephrine to adrenochrome. b Total activity represents the activity of SOD in the sample in the absence of CN’ Mitochondrial SOD activity represents the activity of sample in presence of CNd Cytoplasmic SOD activity is the difference between total and mitochondrial SOD e P values are between treated and control. f Number in parentheses is the number of animals used. 8 NS, not significant.

control rate of (10e3 M). (lo-’ M). activity.

DISCUSSION Previous studies have shown that a single intratracheal administration of xanthine plus xanthine oxidase causes an acute lung injury followed by resolution of the lesions and return to a normal appearance (Johnson et al., 1981). This finding is consistent with the present study which showed that the initial dose of 38 mg xanthine plus 100 pg xanthine oxidase produced transient respiratory distress in hamsters followed by recovery at 4 days after treatment. Lung injury consisted of focal alveolitis characterized by thickened alveolar septa, epithelial necrosis, and intraalveolar infiltration of neutrophils and macrophages. The adventitia around arterioles and small veins were thickened with numerous fibroblasts. No change in the antioxidant enzyme activities was observed. However, the significant findings of the present study were that a second intratracheal administration of 38 mg xanthine plus 250 pg xanthine oxidase caused severe lung injury associated with changes in antioxidant enzyme activities, elevated plasma and lung lipid peroxidation, lung fibrosis, and right ventricular hypertrophy. The lungs had peribronchiolar and arteriolar inflammation, thickening of veins, and diffuse alveolitis with intraalveolar accumulation of macrophages, lymphocytes, and neutrophils and some fibrotic consolidation. In some animals, there were intraalveolar edema and hemorrhages. These inflammatory reactions of the lungs were associated with increased activities of glutathione peroxidase, glutathione reductase, glutathione transferase, and both cytoplasmic and mitochondrial superoxide dismutase. The development of lung fibrosis was demonstrated by increased lung collagen content which remained elevated over a 3-week period. Right ventricular hypertrophy was

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6 5 4 3 2 1 0 12 dose

1

‘f

‘t

dose

TIME AFTER

16

20

24

28

32

36

2

x + x0

TREATMENT(DAYS)

FIG. 1. Effects of intratracheal administration of xanthine plus xanthine oxidase on lung glutathione transferase activity in hamsters. One unit of enzyme activity was considered equivalent to 1 kmole of I-chloro-2, 4-dinitro-benzene conjugated/lung/min. The data are expressed as means * SEM of the number of hamsters shown in parentheses. P values indicate significant differences between treated animals and pooled controls (C).

evidence of myocardial adaptation to the rise in pulmonary vascular resistance during lung fibrosis induced by xanthine plus xanthine oxidase. The increased inflammatory response to the second dose of xanthine and xanthine oxidase at 12 days was associated with increased numbers of libroblasts in lesions, hydroxyproline, and malonaldehyde levels. Neutrophils which were prevalent in lesions at 12 days could inflict severe lung injury by several mechanisms. Reactive oxygen species produced by PMN and proteolytic enzymes contained in the azurophilic granules of these inflammatory cells would cause deterioration of the normal tissue after being exocytosed by PMN (Henson, 1980; Fantone and Ward, 1983). In addition, the lactoferrin contained in the specific granules of PMN 1500

p(O.01

p(0.02 1300

I2

pco.05,i\J<;.a

1 1100-l

i'

1

(6)

i

(7)

co dose

1 f

4

a f

dose

TIME AFTER

12

16

20

24

(4)

28

32

I 36

2

x + x0

TREATMENT(DAYS)

2. Effects of intratracheal administration of xanthine plus xanthine oxidase on lung hydroxyproline. The data are expressed as means f SEM of the number of hamsters shown in parentheses. P values indicate significant difference between treated animals and pooled controls (C). FIG.

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m--m

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p( 0.001

120-

T i

loo-

(4)

p( 0.01

80-

/

T

,446)

(5)

/

60 1

P E i

8 dose

1

12

16

20

24

26

32

‘dose2

TIME AFTER

X + X0

TREATMENT

(DAYS)

FIG. 3. Effects of intratracheal administration of xanthine plus xanthine oxidase on lung and plasma thiobatbituric acid reacting products measured as malonaldehyde. The data are expressed as means f SEM of the number of hamsters shown in parentheses. P values indicate significant difference between treated animals and pooled controls. CP, control plasma; CL, control lung.

may further promote the generation of reactive oxygen species in the lung of xanthine and xanthine oxidase treated animals (Ambruso and Johnston, 1981). The reactive oxygen species are shown to damage lung parenchymal cells (Martin et al., 1981) and endothelial cells (Sacks et al., 1978) and oxidize polyunsaturated lipids (Ford-Hutchinson et al., 1980), leading to synthesis and release of a wide spectrum of arachidonate derived products (Ford-Hutchinson et al., 1980). Some p(O.02

0.35 T

0.30 I

ln + 3

0.25 0.20 I

CONTROL 0 DAYS

FIG.

4.

26 DAYS

33 DAYS

TIME AFTER X + X0 TREATMENT Effects of intratracheal administration of xanthine plus xanthine oxidase on heart in ham-

sters: -= RV LV + S

weight of right ventricle weight of left ventricle plus septum

The data are expressed as means f SEM of the number of hamsters shown. P value indicates significant different between treated animals and pooled controls.

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FIG. 5. Interalveolar septa from a hamster given two doses of xanthine plus heat-inactivated xanthine oxidase intratracheally and killed at 12 days of the experiment. Note the thin septa with sparse collagen fibers (arrows), capillaries (C), and a type II epithelial cell. Bar = 5 km.

of these products are known to participate in the inflammatory and edemagenic process of the lung (Dahlen et al., 1981; Williams and Piper, 1980). Why a second dose of xanthine and xanthine oxidase should produce a greater inflammatory reaction than the initial dose is unknown. However, it is clear from our results that epithelial necrosis and inflammation were not resolved when the second dose was given. We suggest that our fibrotic lesion represents an inappropriate repair process. Fibrosis could occur from irreparable damage to epithelial cells which normally keep fibroblastic responses in check (Terzhogi et al., 1978). Denuded epithelium from tracheal rings that were implanted subcutaneously resulted in massive accumulation of granulation tissue, unless isolated tracheal cells were implanted at the same time, in which case fibrosis was prevented and epithelialization occurred (Terzhogi et al., 1978). The implications are that denuded and/or altered basement membranes can initiate fibroblast responses. The lung injury due to intratracheal administration of xanthine plus xanthine oxidase is linked to the generation of oxygen free radicals (Johnson et al., 1981). In the presence of oxygen, xanthine oxidase metabolizes xanthine to uric acid with the formation of superoxide radicals (O-,). The superoxide radical has a

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FIG. 6. Evidence of hemorrhage (erythrocytes, E, and fibrin, arrows) present in an alveolus of a hamster given a single intratracheal dose of xanthine plus xanthine oxidase followed by 2 days of recovery. Note the neutrophil (N) and other mononuclear phagocytes (M) in the alveolus. Bar = 5 pm.

potential to react with a variety of biological substrates in the lung (Proctor, 1984). Also, the superoxide anions can decompose to yield hydroxyl radicals, peroxy radicals, and hydrogen peroxide which in turn are known to initiate free radical mediated tissue damage (Martin et al., 1981). For example, the oxidants convert membrane unsaturated fatty acids to lipid peroxides (Freeman and Crapo, 1982). Malonaldehyde, the breakdown product of lipid peroxides, is commonly used as an indicator of lipid peroxidation (Pryor and Stanley, 1975). In the present study the lung and plasma malonaldehyde levels were unchanged following initial treatment of hamsters with 38 mg xanthine plus 100 pg xanthine oxidase. The most likely explanation for this observation is that the basal antioxidant and repair processes in the lung were capable of preventing any rise in lipid peroxidation. However, both lung and plasma malonaldehyde levels were significantly elevated at 1 week after the second treatment with xanthine plus xanthine oxidase. In this case, it is likely that excessive oxygen free radicals were generated in the lungs following the second intratracheal administration of 38 mg xanthine plus 250 kg xanthine oxidase. These free radicals may have initiated peroxidative changes of lung lipids, induced severe lung damage directly, caused increased activities of glutathione peroxidase, glutathione reductase, and both cytoplasmic and mitochondrial superoxide dismutase (Martin et al., 1981; Freeman and Crapo, 1982; Pryor and Stanley, 1975; Faiman and Januszkiewicz, 1986; Kellog, 1975). The origin of plasma lipid peroxides is unclear, but they may arise from the injured lung tissue. Elevation of lipid peroxides in plasma and lung suggests that these substances could mediate pulmonary toxicity induced by xanthine plus xanthine oxidase. The interaction of free radicals with biological membranes as a primary mech-

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FIG. 7. Extensive areas of epithelial necrosis (left side of septum) were observed in hamsters given a single intratracheal dose of xanthine plus xanthine oxidase followed by 4 days of recovery. Note the mononuclear phagocytes (M) in alveoli and the interstitium open to an alveolus (A) Bar = 5 pm.

anism for pulmonary oxygen toxicity has focused on the ability of oxygen free radicals to destroy cells that form the lung air-blood interface (Clark ef al., 1985; Johnson et al., 1981; Martin et al., 1981). The lung damage caused by oxygen free radical may also occur due to free radical induced inflammatory reactions mediated by the inflammatory cells such as neutrophils, macrophages, and lymphocytes as well as chemical mediators (Giri and Hyde, 1987; Faiman and Januszkiewicz, 1986). The pathologic changes induced by oxidant injury included tissue necrosis, edema, and inflammation. It was demonstrated in the present study that excessive oxygen free radical produced by xanthine plus xanthine oxidase led to the development of lung fibrosis. This suggests that there is a critical quantity of oxygen free radicals and/or extent of lung injury which is essential for initiation and perpetuation of the fibrotic processes. Many tissues, including the lung, have antioxidant and repair systems to protect against oxygen free radical damage. The cytosolic enzyme, glutathione peroxidase uses reduced glutathione to metabolize lipid hydroperoxides to relatively unreactive hydroxy fatty acids (Chance et al., 1979). The antioxidant activity of this enzyme is tightly coupled to the intracellular levels of glutathione, glutathione reductase, and NADPH. Reduced glutathione is resupplied from the oxidized glutathione by glutathione reductase in the presence of NADPH. Superoxide dismutase, both cytoplasmic and mitochondrial, plays an important role in detoxifying the superoxide radical and thus limiting the reactivity of O-, (McCord er al., 1969). In the present study, the increases in the activities of lung glutathione peroxidase and glutathione reductase following the intratracheal administration of a second dose of xanthine plus xanthine oxidase may represent a protective re-

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FIG. 8. A portion of a thickened interalveolar septum from a hamster given two intratracheal doses of xanthine and xanthine oxidase and killed at 12 days of the experiment. Neutrophils (N) and mononuclear phagocytes (M) were commonly observed in alveoli and septa. Spindle-shaped fibroblasts (F) were observed throughout the septum. Bar = 5 Fm.

sponse to increased lipid peroxidation in the lungs. Similarly, elevation of both lung cytoplasmic and mitochondrial superoxide dismutase activities suggests an adaptive response to the generation of an excessive amount of superoxide radicals during xanthine plus xanthine oxidase reaction. The increases in lung glutathione peroxidase, glutathione reductase, and superoxide dismutase but not catalase activities suggest that superoxide dismutase and glutathione shuttle enzymes are the important lung antioxidant enzymes during xanthine plus xanthine oxidase induced injury. Previous studies with the lung where the potential for lipid peroxidation is increased in response to oxidant induced injury have demonstrated elevated activities of antioxidant enzymes (Kimball et al., 1976; Giri et al., 1983; Reasor and Koshut, 1980). Stimulation of these antioxidant enzyme activities is necessary to minimize cellular toxicity of oxygen free radicals. Increases in the antioxidant enzyme activities may also be part of the repair process for the damaged tissue. It was observed in the present study that initial increases in the activities of superoxide dismutase and glutathione reductase were followed by decreases, in the later part of the study. The most likely explanation for the initial rise followed by decreases in the activities of these antioxidant enzymes is that they might have been inactivated by the free radicals. The present study shows that the oxygen free radicals, released from intratracheal administration of a single dose of xanthine plus xanthine oxidase induced acute lung injury which resolved quickly. However, increased antioxidant enzyme activity, lipid peroxidation, and severe lung damage leading to fibrosis was produced by a second intratracheal administration of xanthine plus xanthine ox-

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