Surfactant alterations following acute bleomycin and hyperoxia-induced lung damage

Toxicology Letters, 32 (1986) 173-178

173

Elsevier

TOXLett.

I61 1

SURFACTANT ALTERATIONS FOLLOWING HYPEROXIA-INDUCED LUNG DAMAGE

(Bleomycin; hyperoxia;

M.E.

PECQUET

GOAD*,

surfactant;

A. FRANClNE

acute respiratory

failure)

TRYKA**

WITSCHI

Biology Division, Oak Ridge National Laboratory, (Received

March

(Accepted

June 20th,

23rd,

ACUTE BLEOMYCIN

and H.P.

AND

Oak Ridge, TN 37831 (U.S.A.)

1986) 1986)

SUMMARY Hamsters

treated

with 0.5 U/kg intratracheal

respiratory

failure

alterations

in pulmonary

choline

(DDPC)

treatment.

surfactant

lavage

treated

samples

with bleomycin

and inhibition

of surfactant

and exposed

indirectly Presence

(SM) were measured,

from treated

is the time of onset of respiratory

bleomycin

To examine

were measured.

and sphingomyelin

brochoalveolar Hamsters

72 h after

and control

function

The decreased

by the edema

and amount

animals

phosphatidyl

ratio was determined

24, 48, 72, and 96 h after

decreased

DPPC/SM

type II cell changes,

of dipalmitoyl

and the DPPC/SM

and 02 had a significantly failure.

for 24 h to 80% 02 develop acute

the lung epithelial

DPPC/SM

in

treatment.

ratio at 72 h, which

ratio may reflect type II cell damage

fluid.

INTRODUCTION

Pulmonary surfactant, produced by pulmonary type II epithelial cells, is a complex phospholipid mixture lining the alveoli and is responsible for decreasing surface tension to prevent alveolar collapse. Any gas, liquid, or small particulate agent entering through the airways first comes in contact with the surfactant lining of the * Guest nessee,

Investigator, Knoxville,

(U.S.A.). ** Current

address:

College

of Veterinary

TN. Current

address:

University

Medicine, M.I.T.,

of Arkansas

Department

Division

for the Medical

Abbreviations: DPPC, dipalmitoyl phosphatidylcholine; atography; MOP, morphometry measurement program;

of Pathobiology,

of Comparative Sciences,

Medicine,

Little Rock,

University

of Ten-

Cambridge,

MA

AR (U.S.A.).

HPTLC, high performance-thin-layer SM, sphingomyelin.

chrom-

174

alveoli. The layer may be physically disrupted. The phospholipids may react with the toxicants such that the qualitative or quantitative composition is altered. The surfactant layer may be removed by the toxic substance, or by a subsequent pulmonary reaction. Surfactant production may be altered by action of the substances

on the type II epithelial

cells,

which

produce

surfactant.

Therefore,

alterations of or damage to type II epithelial cells may be reflected in the surfactant produced [ 11. Bleomycin and hyperoxia in hamsters induce the onset of delayed pulmonary edema with clinical signs of acute respiratory failure and lesions of diffuse alveolar damage [2-51. Atelectasis is a component of the disease. Changes in surfactant or in the type II epithelial cells producing surfactant may play a part in this disease process. To determine if surfactant alterations occur in the bleomycin-hyperoxia model of acute respiratory failure in hamsters the quantity and quality of surfactant were measured. MATERIALS

AND METHODS

Animals and treatments male Syrian golden LVG/LAK hamsters, Mesocricetus auratus (loo-120 from Charles River Laboratories, Lakeview, NJ. Animals were screened for respiratory disease. Hamsters were divided into treatment groups of 24 animals each, housed 6 per cage, to a total of 96 animals. Treatment groups were: saline alone (S); saline + 24 h of 80% 02 exposure (SO); bleomycin alone (B), and bleomycin + 24 h of 80% 02 exposure (BO). Six animals from each group were killed after 24, 48, 72, and 96 h. Bleomycin sulfate (Blenoxane@ donated by Bristol Laboratories, Syracuse, NY), 5 U/kg bleomycin in saline or saline alone [3] as a control substance, was given intratracheally. Immediately after recovery, the hamsters were exposed to 24 h of 80% 02 or room air. Adult

g), were obtained

Surfactant sampling and measurement Hamsters were anesthetized with intraperitoneal sodium pentobarbital, placed on their backs and the diaphragm was incised. The trachea was exposed and a 22-gauge needle covered by a 3 cm sleeve of 19-gauge Teflon@ tubing was placed into the trachea and ligated. 4 ml of 0.9% saline was slowly instilled into the lungs, allowed to remain there 30 to 45 s, and then rapidly withdrawn. Animals were then killed by cutting the abdominal aorta. One lavage was done to keep blood contamination of bronchoalveolar lavage samples to a minimum. Lavage fluid was placed in 15ml plastic screw-capped test tubes. Phospholipids were extracted by a previously described method [6,7]. HPTLC was chosen as the method to qualitate and quantitate the phospholipids because it is sensitive and simple [6,7]. Nonfluorescent HPTLC plates with Silica Gel 60, F-254 for nano-TLC were used for the procedures (E. Merck, Darmstadt,

175

F.R.G.). 10 ~1 of each sample were applied to the plates, 6 per plate, with a Camag Linomat III (Muttenz, Switzerland). Charred HPTLC plates were scanned for reacted phospholipid bands in a densitometer, a Camag TLC-Scanner, at 525 nm with a tungsten visible light source. Densitometry tracings were analyzed for areasunder-the-curve with the MOP of a Zeiss Videoplanner. Standard curves for values of DPPC and SM were determined by use of a Zeiss Videoplanner. The DPPC/SM ratios were calculated from DPPC and SM values for each hamster and each group of hamsters. RESULTS

The results of the surfactant analysis of lavage samples are summarized in Table I. Hamsters treated with saline and air served as controls. Values measured were

TABLE

I

SURFACTANT

PHOSPHOLIPID

AND HAMSTERS

WITH

CONTENT

BLEOMYCIN

AND

AND

RATlONS

FROM

HYPEROXIA-INDUCED

CONTROL LUNG

HAMSTERS

DAMAGE

Treatment

Hours

DPPC

SM

DPPC/SM

gTOUD=

treatment

(Pg)

&g)

ratio

Sd

24 48

19.0

f

3.9b

1.1 f O.lb

18.4

+- 4.2b

37.2

i

5.5

1.7 + 0.3

22.6

5 2.6

SO’

after

12

22.1

+ 3.2

1.0 * 0.1

24.0

4 3.5

96

13.7

f

1.2 f

0.2

12.3

+ 1.5

24

10.4

+ 0.3

1.1 ?z 0.3

11.1

* 1.4

48

19.8“ + 0.8

1.1 * 0.1

18.1

+ 1.9

+ 0.6

0.6 + 0.1

12.5’ t

1.4

1.1 * 0.1

10.5

f

1.4

12.6

+ 1.0

72

B’

BO’

7.16’

96

11.5

t

24

12.0

* 0.9

1.0 f

48

13.1C

+ 1.5

1.3 f 0.2

10.6’ f

72

21.2’

k 1.6

1.5 * 0.1

15.3’ + 1.8

96

12.8

+ 1.2

1.5 * 0.4

10.5

24

18.5

f

1.9

2.5 + 1.1

11.2 + 2.4

48

12.6

* 2.0

1.7 k 0.2

7.3’

f 0.50

* 0.7

1.8 f

4.5’

k 0.8

* 2.4

1.7 + 0.5

72

7.5c

96 a Six animals b Mean

d lntratracheal

16.6

per treatment

k standard

’ Significant

group

1.2

0.1

0.2

11.5 f

0.9

* 1.7

1.4

per time point.

error.

at PcO.05,

compared

to control

values,

saline and air, at the same time points.

saline and air.

’ lntratracheal

saline and exposure

f lntratracheal

5 U/kg 5 U/kg

g Intratracheal

0.9

bleomycin bleomycin

to 24 h of 80% OZ. and air. and 24 h 80% OZ.

176

DPPC and SM, and DPPCSM ratios were calculated. The values from groups SO, B, and BO were compared to values from group S values at the same time points which were defined as the control values. Measurements were compared as pg amounts and as deviations from control values. Group SO had decreased DPPC at 48 and 72 h. The DPPC/SM ratio for SO hamsters was significantly decreased at 72 h. Group B hamsters had decreased DPPC at 24 and 48 h. Values for SM in group B were not increased or decreased at any time point. Group B DPPWSM ratios were decreased at 48 and 72 h. Hamsters in group BO had significant differences in DPPC, SM, and DPPC/SM ratio values. At 48 h DPPC was significantly decreased, as it was at 72 h. The SM values were significantly increased at 24 h. The DPPC/SM ratio of group BO was significantly decreased at 48 and 72 h. The major change measured in pulmonary surfactant in hamsters treated with bleomycin and 24 h 80% 0~ was a marked decrease in DPPC/SM ratio at 48 and 72 h, mainly due to a marked decrease in DPPC. DISCUSSION

DPPC is a phospholipid unique to pulmonary surfactant and is considered the active component. SM is a component of surfactant and is also found in cell mem-

Fig.

1. Electron

micrograph

of surfactant

aggregates

(arrow)

in lungs

from

hamsters

tratracheal bleomycin and 24 h of 80% 02 (uranyl acetate and lead citrate; magnification is a 20000x view of surfactant; tubular myelin structures are typical of phospholipids

treated

with in-

4000 x). Inset in surfactant.

177

branes and serum [l]. The DPPC/SM ratio reflects changes in lung surfactant by comparing the amounts of pulmonary phospholipids [7]. Decreases in DPPC and DPPC/SM ratios in group B and BO hamsters may reflect a change in activity of pulmonary type II cells [ 1,7]. Bleomycin is known to inhibit normal type II cell division and DNA synthesis in acute phase damage and to cause hyperplasia and metaplasia in subacute phases [8]. Lungs injured by butylated hydroxytoluene also have altered type II cell metabolism and acute decreases in surfactant [9]. The additional insult of 24 h of exposure to 80% 02 increases the functional damage to type II cells. Type II cells are then less able to divide to repair damage and thus to synthesize surfactant. Ultrastructural studies, performed in this laboratory, show that surfactant aggregates are prominent in groups B and BO (Fig. 1). Prominent surfactant aggregates may indicate protein-surfactant interactions or deactivation of surfactant [ 11. Therefore, decreased surfactant in group BO hamsters may be due to ‘wash-out’ by alveolar edema fluid at 72 h as well as aggregation and deactivation by protein interactions. The 96-h DPPC/SM ratio in group BO samples were normal, probably because those animals would survive the insult by repair of the damaged tissues. Surfactant alterations in bleomycin and hyperoxia-induced acute respiratory failure may be caused by alterations of type II cell metabolism or may be secondary to surfactant interactions with high-protein edema fluid. Regardless of the cause, surfactant amounts and composition, reflected in DPPC content and DPPC/SM ratios, are altered in the bleomycin and hyperoxia-induced acute respiratory failure of hamsters. ACKNOWLEDGEMENT

Research sponsored by the Office of Health and Environmental Research, U.S. Department of Energy, under contract DE-AC05840R21400 with the Martin Marietta Energy Systems, Inc. REFERENCES 1 Y. Kikkawa disease,

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