Altered turnover and synthesis rates of lung surfactant following thoracic irradiation

Im. J. Radiarm Oncology Bml. Phys., Vol. 13. pp. 233-237 Printed in the U.S.A. All rights reserved.

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0360-3016/87 0 1987 Pergamon

$3.00 + .OO Journals Ltd.

??Original Contribution

ALTERED TURNOVER AND SYNTHESIS RATES OF LUNG SURFACTANT FOLLOWING THORACIC IRRADIATION P.

G. COULTAS, PH.D.,’ R. G. AHIER, B.Sc.’ AND R. L. ANDERSON, PH.D.~

‘MRC Cyclotron Unit, Hammersmith Hospital, London W 12 OHS, U.K.; and ‘Department of Radiology, Stanford University Medical Center, California 94305, U.S.A. Between 2-6 weeks after thoracic irradiationwith 10 Gy X rays, when levels of surfactant in the alveoli show the greatest increase, there is a reduction in the rate of radioactivity loss from ‘H-choline labeled disaturated phosphatidylcholine from the lung. This indicates a reduced turnover of surfactant. Discrepancies between the halving times for specific activity and for total radioactivity of the disaturated phospholipids suggest that at between 2 and 3 weeks post-irradiation, removal and degradation of surfactant almost ceases, but that synthesis continues normally. However, by 3 weeks post-irradiation, choline-3H incorporation into disaturated phosphatidylcholine suggests that surfactant synthesis is increased about two-fold. The reduced number of macrophages recovered from alveolar lavage between about 2 and 6 weeks post-irradiation may indicate a reason for the lengthened turnover times of surfactant over this period. Nevertheless the stimulated surfactant production that takes place from about 3 weeks onward suggests an additional active response to radiation or to radiation damage by the type II pneumonocytes. These studies confirm that the maximum levels of alveolar surfactant seen at 3 weeks post-irradiation result from a different lung response than that responsible for the increase in surfactant, which occurs within hours of irradiation. Lung surfactant, Turnover, Synthesis, Irradiation.

tissue.2 This paper uses 3H-choline incorporation into disaturated PC to examine the changes responsible for the elevation of alveolar surfactant that occurs about 26 weeks post-irradiation.

INTRODUCIION Lung surfactant is produced by type II pneumocytes.3 These cells also have an important role as the stem cell for the renewal of the alveolar epithelium.’ Interference with the surfactant system and the ensuing modifications of alveolar surface tension could lead to the consequences, such as the lung edema, septal fibrosis, and atelectasis, that follow irradiation. I9Rubin et al. [e.g. I51have demonstrated that an increased level of alveolar surfactant is one of the earliest detectable changes following lung irradiation. However, the pattern of changes in amounts of alveolar surfactant after various doses of neutron or X-irradiation indicate an apparently biphasic increase in alveolar surfactant after radiation.2 The early phase, initiated within hours of radiation, probably results from a rapid loss of lamellar bodies from type II pneumonocytes by expulsion into the alveolar lumen.i6 The peak levels of surfactant in alveolar lavage fluid are, however, reached at about 3 weeks post-irradiation though without any apparent change in lipid composition.2 At this time amounts of disaturated phosphatidylcholine (disaturated PC is the characteristic and major component of surfactant12) are also increased in the lung

METHODS

AND

MATERIALS

CFLP female mice fourteen weeks old were irradiated with 10 Gy X rays (250 kVp at 1.7 Gy/min) on a rotating jig, which shielded all parts of the body except the thorax. Irradiation details are published elsewhere.5 At intervals of 2, 3, and 5 weeks after radiation, groups of animals were injected intraperitoneally with 10 uCi of 3H-choline* in 0.2 ml saline. Groups of 4 mice were killed at various intervals after injection by i.p. administration of 0.5 ml sodium pentobarbitone (60 mg/ml). The lungs were then lavaged (6 X 1 ml chilled saline) prior to removal. Phospholipids were extracted from aliquots of both lavage fluid and homogenized lung tissue and the amounts of disaturated phospholipid assayed using the methods of Mason et ~1.’ Details of these procedures have been published elsewhere.2 Total incorporated radioactivity and the specific activities of the disaturated

Reprint requests to: Dr. P. G. Coultas. Accepted for publication 4 September 1986.

* Radiochemical Centre, Amersham U.K.; S.A. 10 Ci/m mole. 233

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I. J. Radiation Oncology 0 Biology 0 Physics

lipid from both lavage and tissue were determined scintillation count.?

by

February 1987, Volume 13, Number 2 d.p.m 6.104

in

DPL

1 “--mm,.

?? ??

RESULTS

4.103

per

pg

Disat.

PC

T

T

1

5.1021 4 0

100

I 160

??

495 f

Figure 1 shows changes in the specific activity of the disaturated phospholipid (>95% DPC) from both lavage fluid (open symbols) and from tissue (solid symbols) as a function of time after ‘H-choline injection. Data for control mice and for mice injected at 2, 3, or 5 weeks after 10 Gy X rays are shown separately. Tissue and lavage fluid specific activities did not differ significantly in any series of measurements and best fit straight line regressions have therefore been fitted using the combined data from the lavage fluid and tissue measurements. The slope of these regressions would provide a measure of the biological half-life of the disaturated PC if total amounts of disaturated PC were constant and its output and removal equal. Figure 2 illustrates the changes in the total activity (d.p.m.) of disaturated phospholipid as a function of time after 3H choline injection with data for control mice

d.p.m.

?? ?? ‘------ll_,

hours

4 0

after 100

‘H-choline , 160

Fig. 1. The specific activities (d.p.m. per ug disaturated lipid P) for disaturated PC from both lavage fluid and tissue are shown for the four panels for the control mice and for mice irradiated with 10 Gy X rays at 2,3, and 5 weeks before 3H-choline injection. Equal proportions of the extracts from 4 mice were pooled and the specific activity measurements shown are the mean of duplicate determinations made on these bulked extracts. Because there were no significant differences between the specific activities measured for material from lavage fluid (0) and from tissue (O), the best fit linear regressions shown have been fitted to combined sets of data. Linear regressions are an adequate fit for all sets of data except the 2 week post-irradiation (P.I.) series.

7 Beckman LS 6800; High Wycombe, U.K.

16.104

2 weeks

PI

Sweehs

PI

T 3 weeks

i

-

GO

PI

-

I

--_ 100

0

60

-

ii0

Fig. 2. The total radioactivity present in disaturated phosphatidyl choline (total d.p.m.) is shown as a function of time after injection with ‘H-choline. Estimations were made on pooled material from 4 mice and the total activity shown represents one quarter of the sum of the disaturated PC activity in lavage fluid plus that in tissue. The different series of measurements were made in control mice and in mice injected 2, 3, and 5 weeks after 10 Gy thoracic X-irradiation. The best fit linear regressions are shown for the control data and for data at the three different times P.I. (post-irradiation).

and for mice injected at 2, 3, or 5 weeks after 10 Gy X rays, again illustrated separately. For this figure the counts from tissue and lavage have been aggregated and the totals shown are on a per mouse basis. Straight line regressions have been fitted to the data and extrapolated back to the time of injection. The intercept indicates the relative incorporations of choline-3H during the period of precursor availability and the slope is again a measure of the biological half life of the disaturated PC. Table 1 summarizes various data for each series of animals. The total amounts of disaturated lipid per mouse (lavage plus tissue) are given (column I) together with the numbers of macrophages recovered from the alveolar lavage fluid (column II). (More detailed information on disaturated lipid levels is given in reference 2.) Also shown are the half-life values calculated from the slopes of best fit regressions for both specific activity (column III) and for total activity (column IV). DISCUSSION Lung surfactant phospholipids differ from those extracted from other tissues in possessing two saturated acid moieties (predominantly palmitic acid).13 This highly saturated nature of the surfactant phospholipids is apparently essential for their surface tension reducing

235

Post-radiationsurfactantturnover 0 P. G. COULTAS et al.

Table 1. Amounts of surfactant (pg lipid P in disat PC), alveolar macrophage numbers and half lives of total and specific DPL activity III

I

Total pg

II

disat PC per mouse (tiss. + lav.)

Number of macrophages x 10-5

Control 10 Gy Irradiated (i) 2 weeks

29.7 ? 7.1

1.75t0.15

48.6 f 5.0*

0.59 * 0.09*

(ii) 3 weeks

60.0 ? 3.4*

0.52 + O.ll*

(iii) 5 weeks

48.9 ? 3.0*

1.93 + 0.26

Half-life disat PC total activ. (hr) 81+5 495 + 112+ 131 + -

IV Half-life (hrs) disat PC specific activ. 71+2

524 168 25 17 29 20

89 f 3*t 110 I!r 5* 100 + 4*

* Significantly different (p < 0.0 1) from controls. t Significantly different (p < 0.0 1) from total activity half life.

properties. About 70% of total surfactant phospholipid is PC (phosphatidylcholine), and thus disaturated PC can be considered as both the most characteristic and the important surfactant component.12 This study has used the disaturated PC extracted from lavage fluid and from lung tissue as a measure of surfactant levels. Choline has a high specificity for PC’* and is incorporated into disaturated PC via its main biosynthetic pathway, but is not significantly incorporated into other choline-containing surfactant phospholipids. “,‘7 Since the increases in disaturated PC that are seen 2-6 weeks after 10 Gy X rays (ref. 2, but see also Table 1) could result either from increased synthesis or from altered degradation and removal, changes in total activity and in specific activity of the desaturated PC have been followed after 3H-choline. After 3H-choline injection, autoradiographs show radioactivity initially localized over the type II pneumonocytes and by about 2 hours, predominantly within the lamellar bodies.4 From this time on, the radioactivity of the disaturated PC within the alveolar spaces increases.496 However, active incorporation of labeled choline continues over a period of about 6 hours.’ In preliminary studies (data not shown), the specific activity of lavage phospholipids reached a maximum at about 24 hours after ‘H choline injection irrespective of whether the mice had been irradiated ( 10 Gy X rays) 3 weeks previously or not. This compares with data in studies on the premature rabbit in which peak specific activity was seen at 16-20 hours post-injection.6 Since our previous data had also indicated a fairly long turnover time, samples were restricted to after the time at which peak activity was seen and were taken 30 hours after 3H-choline injection, and subsequently. The specific activity data for both control and all three irradiated series (Fig. 1) show no significant differences between the activities of the disaturated PC in the tissue and in the lavage fluid. This indicates that, although partitioned between apparently anatomically distinct and

spatially separated tissue and lavage fluid compartments, disaturated PC nevertheless acts as if forming a single pool. In this respect our conclusions agree with those of Young and Tiemey2’ rather than with the data of Jobe and Gluck6 though the latter study involved premature animals and pool sizes were changing appreciably. This ‘single pool’ behavior demonstrates that there is very substantial recycling of secreted material. Such recycling will clearly mean that measured half-life values of both specific and total activity are lengthened so that they do not reflect the chemical stability of the surfactant material. As can be seen from the data for the control mice shown in the top left panels of Figures 1 and 2, both total and specific activities of the disaturated PC fell exponentially from 30 hours post-injection, with a half-life of between 70 and 80 hours. The agreement between the halflife estimates from total activity and those from specific activity (see Table 1) is to be expected in control animals because production, removal, reutilization, pool sizes, etc., are all in equilibrium and constant.7 Relative to the values calculated for control mice, the biological half-lives calculated from specific activity measurements in all 3 series of irradiated animals indicate significantly lengthened surfactant turnover-times (Table 1, column III). These calculated half-lives must, however, be treated with some caution as the total quantities of disaturated PC are changing during the period of observation (see Table 1, column I). In this context a comparison with the half-life values calculated from the total disaturated PC activity (Figure 2 and Table 1, col. IV) is of particular interest. In all cases the half-lives based on total radioactivity confirm the extended tumover of the disaturated PC in the various groups of irradiated mice. However, although there is reasonable agreement between the two half-life estimates for controls (see above) and for the 3 and 5 week post-irradiation series, there is a significant and marked discrepancy between

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I. J. Radiation Oncology 0 Biology 0 Physics

total and specific activity half-lives calculated for the 2 week series (see Table 1). Here, total activity falls very slowly relative to specific activity. Similar results were seen in studies of PC labeling in premature rabbit6 and, as in that case, the discrepancy in the present results could be explained by a continued synthesis that substantially exceeds removal. Such a situation would result in an increase in the total amount of disaturated PC present, which does indeed take place (see Table 1, also ref. 2). Measurements of the relative 3H-choline incorporation into disaturated PC can be used to provide an indication of the relative rates of synthesis of disaturated PC (and thus, also, of surfactant). For such an approach it is important that choline incorporation into disaturated PC in lung should be small relative to total choline incorporation into the other tissues of the animal. In these experiments, this condition is met since less than 1% of the injected ‘H-choline was incorporated into lung disaturated PC. Further problems could arise if either pool sizes or fluxes in the biosynthetic pathway were substantially altered.’ It would be difficult, however, to envisage both the elevated level of product and the increased precursor incorporation which occurs (see below), without invoking an increase in disaturated PC synthesis. Backextrapolation of the regression lines shown in Figures 1 and 2 to the time of ‘H-choline injection enables estimates to be made of the initial incorporation of 3H-choline. For the total count data (Fig. 2), these to extrapolates will directly give a measure of the relative 3H-choline incorporations into disaturated PC. Similar b extrapolates for specific activity (Fig. 1) will, if multiplied by the appropriate total amounts of disaturated PC for the various series, also indicate the relative initial amounts of radioactivity incorporated. Table 2 shows the relative 3H-choline incorporations in controls and at 2, 3, and 5 weeks after 10 Gy X rays, calculated in the two ways outlined above. Both methods of calculation indicate that surfactant synthesis is increased by a factor of about two at 3 weeks post-irradiation; at 5 weeks post-irradiation, though closer to control level, it is still significantly elevated. However, at 2 weeks the two calculations give significantly different results. The estimate indicating synthesis rate comparable with controls and based on total counts (Table 2, column II) is probably more reliable, since the Q,extrapolate for specific activity may be spuriously high because the changes in specific activity at this time are caused not by the degradation and removal of desaturated PC, but by label dilution resulting from continued disaturated PC synthesis (see earlier discussion). Elevated surfactant recoveries from alveolar spaces have been reported within hours of irradiation.6Y7 At these early times electron micrographs show reduced numbers of lamellar bodies within the type II pneumonocytes. In viva studiesI of type II pneumonocytes also

February 1987, Volume 13, Number 2

Table 2. Calculation of total initial incorporation of radioactivity into surfactant (disat PC) after tritiated choline injection: an indication of relative surfactant synthesis rates in control mice and in mice at 2,3, and 5 weeks after 10 Gy X rays I

Total ~.cgdisat PC X II to extrapolate, to extrapolate, (specific activity) Total d.p.m. in disat. PC dpm X low4 dpm X lop4

Control 10 Gy - irradiated: (i) Inj. 2 wks. PI (ii) Inj. 3 wks. PI (iii) In. 5 wks. PI

6.5 -+ 0.5

5.8 + 0.3

10.1 f 1.3*t 11.3*0.7* 8.7 f 0.7*

5.2 + 0.4 10.7 f 0.6* 7.3 f 0.7*

* Significantly different from control (p < 0.05). t Significantly different from equivalent figure col. 2 (p < 0.05). PI = Post-irradiation.

indicate that pre-labeled surfactant phospholipids are released into the culture medium after radiation doses above lo- 15 Gy X rays and that p-adrenergic stimulants, which normally result in surfactant release, are then unable to produce an additional response. These observations suggest that irradiation elicits an early release of the pre-existing surfactant stored in the lamellar bodies of the type II pneumonocytes, thus augmenting surfactant in alveolar spaces and depleting it within lung tissue. In a detailed examination of the time course and extent of changes in surfactant levels after irradiation, Ahier et ~1.~ found that although surfactant recoveries from alveolar lavage fluid were elevated 2 days after irradiation, they had returned to nearer control levels by 9 days before peaking again at 3 weeks post-irradiation. At this later time a dose of 5 Gy X rays produced a significant increase in alveolar surfactant level. These various observations suggested a biphasic response to irradiation by the surfactant system with an initial phase resulting from direct radiation action on the type II pneumonocytes and a second more prolonged phase between 2 and 6 weeks after irradiation. The present studies of surfactant turnover during this later phase confirm this suggestion, but indicate that the nature of the response is complex. Both synthesis and removal of surfactant are affected. Slowed turnover is seen by 2 weeks post-irradiation and occurs earlier than the onset of increased synthesis. The well documented decrease in alveolar macrophage number after irradiation [for example, see ref. 1 l] may be involved in extending the half-life of the labeled disaturated PC in view of the possible role of these cells in surfactant turnover.” Our data on macrophage numbers (Table 1, column II) show a similar depression to that found by other workers, but the correlation with the DPL half-life estimates (Table 1, columns 3 and 4) is not exact.

Post-radiation surfactant turnover 0 P. G. COULTAS et al.

However the later onset of increased surfactant synthesis also suggests a separate active response by the type II pneumonocytes, although the cause of this response is as

231

yet unclear. Thus the overall changes in surfactant level reported earlier by us2 probably arise from more than one cause.

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12. Perelman, R.H., Engle, M., Farrell, P.: Perspectives on fetal lung development. Lung 159: 53-80, 198 1. 13. Pfleger, F.C., Thomas, H.G.: Beagle dog pulmonary surfactant lipids. Arch. Intern. Med. 127: 863-872, 1971. 14. Rooney, S., Motoyama, E.: Studies on the biosynthesis of pulmonary surfactant. The role of the methylation pathway of phosphatidylcholine biosynthesis in primate and non-primate lung. Clin. Chim. Acta. 69: 525-531, 1976. 15. Rubin, P., Shapiro, D.L., Finkelstein, J.N., Penney, D.P.: The early release of surfactant following lung irradiation by alveolar type II cells. Znt. J. Radiat. Oncol. Biol. Phys. 6: 75-77, 1980. 16. Shapiro, D., Finkelstein, J., Rubin, P., Penney, D., Siemann, D.: Radiation induced secretion of surfactant from cell cultures of type II pneumonocytes: an in vitro model of radiation toxicity. Znt. J. Radiat. Oncol. Biol. Phys. 10: 375-378,1984. 17. Spitzar, H., Morrison, K., Norman, J.: Incorporation of LMe-14C methionine and Me-14C choline into lung phospholipids. Biochim. Biophys. Acta. 152: 552-558, 1968. 18. Stein, O., Stein, Y.: Lecithin synthesis, intracellular transport and secretion in rat liver. IV. An radioautographic and biochemical study of choline deficient rat injected with 3H-choline. J. Cell Biol. 40: 46 l-483, 1969. 19. Van den Brenk, H.A.: Radiation effects on the pulmonary system. In Pathology of Radiation Berdjis, C. (Ed.). Baltimore, Williams and Wilkins, 1969, pp. 569-59 1. 20. Young, S., Tiemey, D.: Dipalmitoyl lecithin secretion and metabolism by the rat lung. Am. J. Physiol. 222: 15391544, 1972.