EFFECT OF SEMICARBAZIDE ON THE SURFACTANT PHOSPHOLIPID PERCENTAGES DURING FETAL

AND POSTNATAL LUNG DEVELOPMENT IN RAT

MONICA IX LA FUENTE.* ANGEL HERNANZ and MARGARITA ALMA

J.C. Mutis Institute. C.S.I.C., Madrid, Spain

Abstract --1. The eFFect of 100 mg/kg of semicarba~ide on phosphatidylcholjne. phosp~~tidylcthano- lamine, sphingonlyei~ne. phosphatidylserine and lysophosphatidylcholine of the pulmonary surfactant was studied in offspring of treated rats on the 10th day of gestation.

2. The relative percentages of phosphatidylcholine were smaller in the offspring of treated rats than in controls, but the opposite was observed with the other phospholipids.

3. Significant statrstical ditrerences at almost all ages studied were observed in phosphatidylcholine and phosphatidylethnnolamine.

4. The ratio of phosphatidylcholine to sphingomyeline. an index of lung maturity. was smaller in the olrspring of treated rats. with statistically significant differences just before birth and on the first day of life

The fetal lung is an immature organ until the terminal period of gestation. At this time, pulmonary surfac- tant production is at its maximum. A deficiency in pulmonary surfactant is considered to be responsible for the idiopathic respiratory distress syndrome of the newborn (Avery & Mead, 1959; Farrell & Avery, 1975). The pulmonary surfactant is composed mostly of lipid, with 75”,,, of the phospholipids (Klaus et al., 1961: King, 1974) being responsible for the lowering of the surface tension; this is especially true of phos- phatidylcholine (King & Clements, 1972: King, 1974). In the terminal period of gestation the amount of this substance increases drastically in the lung, as has been shown for a variety of mammalian species (Brumley ct

rtl.. 1967; Soodsma ef (II., 1976: Okano & Akino, 1978). We found in previous unpublished research that

semicarbazide (NH,-CO--NIi--NH,) administered in different doses during the gestation of the rat pro- duced, besides reabsorptions and other fetal abnor- malities, a considerable increase in postnatal mor- tality during the first weeks of life. This mortality was

maximum, 68”,,, when 100 m&kg semicarbazide was administered. These newborn showed signs of asphyxia. This fact led us to believe that the cause of death could be poor development of the lung. The purpose of the present paper is to study the action of semicarbazide on the phosphoIipid levels of the pul- monary surfactant in order to try and find at least one of the possible reasons for this high postnatal mor- tality.

MATERIALS AND METHODS

The experiments were performed on Wistar rats, weigh- ing between 200 and 250 g. They were kept at 23 ‘C; food (Panlab) and water provided md libitum Female and male rats were placed together overnight and a vaginal smear

* Present address: Department of Biochemistry, Faculty of Medicine, University of C6rdoba. C6rdoba. Spain

was performed the next day. All the females with posittve smears were considered to be at day 1 of gestation.

The semicarbazide (Merck), dissolved in distilled water. was administered intraperitoneally in a single dose of lOOmg/kg on day 10 of gestation. Control rats only received the same volume of distilled water.

The pregnant rats were distributed into lots where: (a) two groups (control and treated) were sacrificed on day IX of gestation: (b) two groups (control and treated) were sac- rificed on day 21; and (c) two groups (control and treated) were allowed to deliver in order to study their otTspring. which was done so on days 1. 3, 7. 15. 22 and 30 of life.

The fetuses were delivered by Cesarian section. the mothers being anesthetized with 50 mg!kg sodium pento- barbital (Sigma, St. Louis, MO, USA). The lungs were im- mediately extracted and washed several times in 0.9”, NaCl (Merck Darmstadd, FRG) at a temperature between

0 and 5 C. The lungs from the offspring of the rats allowed to deliver received the same treatment. The surfac- tant was obtained by pressing the lungs into a container with I5 ml 0.9”,, NaCI.

Erdwion of the phospholiprd.\ in r/w pulmoncrr~~ srtrfirt~tanr

All the lipids were extracted according to Folch’s method (1Y51). The separation of the phospholipids was performed by hidimensional thin-layer chromatography on 20 x 2Ocm silica gel G plates. The solvents used in the first run were chloroform:methanol:~atcr (65:24:4v/‘v) and in the second were chloroform: methanol : acetic acid: water (YO:40: 17:2 v:v). The phospholipids were visualized by iodine vapor and each spot was identified by comparing it to the known phospholipid patterns of phosphatidylcho- line, phosphatidylethanolamine, sphingomyeline. phospha- tidylserine and lysophosphatidylcholine. The quantitative analysis of each phospholipid was carried out by the method of Rouser et al. (1966). The amount of each phos- pholipid, measured as icg lipid-bound phosphorus per g of lipids, was expressed as the percentage of the total amount of phospholipids in the plate. The statistical analysis was carried out according to Student’s “I” test. P < 0.05 being the minimum significant level.

Table I, Phosphatidylcholine and phosphalidyleth~~nolaminc percentages of the pulmonary surfactant in the perinntal development of the rat

Age Phosphatidylcholines (‘I,,) Phosphatidylcthanolamines (“,,)

Controls Treated P Controls Treated P

IX day 70.8 * 1.x 54.x 2 1.6 < 0.00 I 13.5 + I.6 30.6 t I. 1 10.01 fetuses (121 (12) (12) (13 ‘1 day 80.9 * 0.x 70,s & 1.0 < 0.00 1 10.3 * 1.3 15.3 * 1.5 < 0.05 fetuses (10) (101 (10) ( 10) 1 day 84.0 * 1.7 74.4 * 2.3 <ox 9.3 f I..? I’.:! * 0.5 < 0.05 offspring (1% (IW (IO) (10) 3 da> 86.1 & 1.1 77.2 * 2.3 <O.Ol 6.4 2 0.9 11.4 $: 0.7 < 0.00 1 offspring c 10) (l(l) (10) (10) 7 day x3.x t I.0 77.5 k I.0 < 0.0 I 7.7 & 0.5 12.8 rir 0.9 <O.Ol offspring (8) (I()) (8) (101 IS day 8X k 0.9 78.3 i. 0.7 < 0.05 7.6 + 0.8 Il.4 k 0.x < 0.05 offspring (8) (101 (81 (10) 22 day 79.5 f 0.8 72.4 ) I.6 CO.05 x.5 & I.1 14.3 i 1.6 < 0.0s offspring (8 (IO) (8) (101 30 day 77.2 + 0.x 74.0 * 7.0 NS’ X.9 + 0.6 13.3 i 1.3 < 0.05 offspring (16) (1’) (16) (12)

Table includes mean + SEM of the number of lots indicated between parentheses. Each lot contains hetween 8 and 12 fetusesor 4 and 6 offspring.

* NS. not significant,

RESULTS

The phosphatidylcholine and phosphatidylethano- lamine percentages obtained on the different fetal and postnatal days studied for the offspring of mothers (control and treated with semicarbazide during gesta- tion) are shown in Table 1. As can be seen in this table, the phosphatidylcholine. in the offspring of con- trols, increases before birth, continues to do so up to the 3rd day of life, and then begins to decrease. How- ever, in the descendants of treated rats these percent- ages not only increase at the end of gestation but continue to do so up to day 15 of postnatal life. We find that the treated animal phosphatidylcholine per-

centages are lower than in the offspring controls with statistically significant differences on all days studied, with only one exception-day 30. The phosphatidyl- ethanolamine percentages in the descendants of treated and control mothers decrease just before birth, being slowly restored thereafter. Comparing these percentages-higher in the offspring of treated animals-we find statistically significant differences on all days studied.

Table 2 shows the phosphatidy~serine, sphingomye- line and lysophosphatidylcholine percentages of the offspring of control and treated rats. These percent- ages decrease just before birth, increasing afterwards. In general, these values are higher in the descendants

Table 2. Sphingomyeline, phosphatldylserine and lysophosphatidylcholine percentages of the pulmon- ary surfactant in the perinatal development of the rat

Age

Sphingomyelines (“,,) Phosphlltidylserincs (‘I,,) Controls Treated Controls Treated

Lysophosphatidylcholmes (‘I,,) Controls Treated

IX day fetuses 21 day fetuses 1 day offspring 3 day offspring 7 day otlspring I5 day offspring 22 day offspring 30 day offspring

6.7 t 0.9 (12)

4.9 * 0.9

(10) 3.2 * 0.R

(10) 4.4 * 0.7

(10) 4. I * 0.7

(8) 5.5 * 0.4

(8) 6.4 + I.1

(8) 7.2 * 0.9

(16)

I I.4 & I.21 (12)

X.1 f 1,7* (10,

7.1 i_ l,fH (10)

5.5 _i 0.6 18)

5.x & I.2 (IO)

5.6 & 0.5

(10) 6.2 & I.1

(IO) 6.4 f 0.5

(12)

3.6 * 0.3 ii’1

2.5 + 0.4 (10)

I.6 * 0.2 ( IO)

I.5 * 0.2

(10) 2.4 & 0.6

(8) 2.5 ri: 0.3

(8) 2.6 + 0.5

(8) 3.5 rf: 0.7

(16)

4.x * 0.6 (12)

2.2 +- 0.8

(10) 2.3 & 0.5

(1(X 2.3 + 0.5

18) I .o * 0.2

(10) 2.3 * 0.6

(10) 3.2 + 0.7

(10) 3.1 & 0.x

(12)

x.7 i 0.9~ (Ill

4.1 * 1.0* (10)

3.8 + 0.3* (2))

3.8 * 0.3:

(8) 3.0 * 0.x

(10) 2.9 + 0.7

(I()) 3.1 i I.3

(101 3.2 i 04

(12)

Table includes mean f SEM of the number of lots indicated between parentheses. Each lot contains between X :tnd 12 fetuses or 4 and 6 offspring.

* P < 0.05. t P < 0.01. $ P < 0.001.

Effect of semicarbazide on surfactant phospholipid 117

Table 3. Ratio of phosphatidylcholines to sphingomyelines of the pulmonary surfactant in perinatal development of the rat

Age Controls Treated P

18 day fetuses 21 day fetuses I day offspring 3 day offspring 7 day offspring

15 day offspring 22 day offspring 30 day offspring

11.1 * 1.3(12) 4.X f 0.4(12) < 0.00 I 16.5 f 2.9 (10) 8.7 f 1.2(10) < 0.00 I 26.3 i 4.5 (10) 10.5 f 1.2(10) <O.OOl 19.6 f 2.6 (10) 14.0 + I.1 (IO) NS* 20.4 + 3.3 (8) 13.3 i 2.7(10) NS 15.0 i 0.9 (8) 13.9 k 1.1 (IO) NS 12.4 i 2.1 (8, 11.7 + 1.9(10) NS 10.7 i 1.2(16) 11.5 i 0.6(12) NS

Table includes mean f SEM of the number of lots indicated between oarentheses. Each lot contains between 8 and 12 fetuses or 4 and 6 offspring.

* NS. not significant.

of treated mothers. The sphingomyeline percentages shows statistically significant differences during the fetal period and the first day of postnatal life. These statistical differences were also found in the lysophos- phatidylcholine percentages on these days as well as on the third day of life.

The ratios of phosphatidylcholine to sphingomye- line are illustrated in Table 3. There is a continuous increase in the control offspring up to the final day of fetal life and first day of postnatal life. followed by a continuous decrease. The offspring of treated mothers showed a similar behaviour, although this increase does not stop until the third day of life. The ratios are also somewhat lower. with statistically significant dif- ferences, on day 18 and 21 of fetal life and on the first day of postnatal life.

DISCUSSION

The results show that semicarbazide acts on differ- ent phospholipid levels of the pulmonary surfactant and accordingly also on the phosphatidylcholine, the principal source of surface active properties in the lung (King & Clements, 1972; King, 1974). The data found for the descendants of control rats agree with other data already established by different authors. These studies in rat have indicated the fact that there is a significant increase in the in riro pulmonary phos- pholipids and in the phosphatidylcholine content between day 21 and 22 of gestation (Weinhold & Villee. 1965: Fujiwara c’t (II.. 1968) This increase also occurs during the first few days after birth (Kikkawa c’r (II.. 1965; Cluck rf LI/.. 1967). Fujiwjara rr ctl. (1968) indicate that there is an increase of the phosphatidyl- choline during fetal life in sheep which reaches a con- tent of 86.0’1” at full-term. whereas the phosphatidyl- ethanolamine decreases until it reaches 4.4:,,, at birth. It is important to mention here that the percentages found by these authors in immature fetuses are 73.0”,, for the phosphatidylcholine and 9.0”,, for the phos- phatidylethanolamine. These are approximately the same values which we found in our research. This seems to suggest that the fetuses from treated rats have pulmonary phospholipid values corresponding to immature fetuses which do not even mature after birth.

The ratio of phosphatidylcholine to sphingomye- line, index of lung maturity (Gluck & Kulovich, 1973).

shows an increase in control animals which lasted up to the first day of life, decreasing afterwards. This agrees with the data found by other authors for the fetuses and offspring of rabbits (Rooney et al., 1975).

Our results suggest that semicarbazide possibly acts on the synthesis of phospholipids in lung and never reaches optimum phosphatidylcholine levels. It has been shown that the methylation pathway in the dr nom phosphatidylcholine synthesis is predominant in the fetal lung of rats up to day 19. However, after day 22 and during postnatal life the CDP-choline path- way becomes more predominant in rats (Weinhold. 1968; Rooney & Motoyama. 1976) and other animals (Farrel, 1973: Epstein & Farrel. 1975). Semicarbazide may inhibit the CDP-choline pathway because of its capacity to react specifically with the cytosine group (Hayatsu & Ukita. 1964, 1966). Nevertheless, this reaction is strongly pH-dependent and at physiologi- cal pH of 7.4 practically no reaction should occur. As was indicated above, this route is predominant just before birth and in postnatal life when the differences in the phosphatidylcholine percentages between the fetuses of treated mothers and control rats are signifi- cant. However, further research is necessary before it is possible to state whether the semicarbazide acts primarily on pulmonary dr ncm phospholipid syn- thesis or if its effect is advanced by other means.

A(,~/~o~~/~,d~/c,,~~rnr We are very grateful to Mrs C. Mendez for her help in the preparation of the manuscript.

REFERENCES

AV~KY M. E. & MEAD J. (1959) Surface properties in re- lation to atelectasis and hyaline membrane disease. Am. J. Dis. Child. 97, 517-523.

BKUMLEY G. W.. CHERNICK W. A., HODSON W. A., NOR- MAND C.. FENKER A. & AVERY M. E. (1967) Correlations of mechanical stability. morphology, pulmonary surfac- tant and phospholipid content of the developing lamb lung. J. c/i~ I~~Ku. 45. 863-873.

EPSTEIN M. F. & FARREL P. M. (1975) The choline incor- poration pathway: primary mechanism for de moo lecithin synthesis in fetal primate lung. fed&r. Res. 9, 658-665.

FARR~L P. M. (1973) Re.~pirtrtor~ Distress Syndrome, pp. 311~ 349. Academic Press, New York.

FARREL P. M. & AV~:RY M. E. (1975) Hyaline membrane disease. Am. Rrr. rrsp. Dis. I II, 657-688.

118 M~~NI~A UE LA F~JENTE zf ui.

F’OLCH J. (1951) J. hid. Chew. 191, 807. FUJIWAKA T.. ADAMS F. H.. SIPOS S. & EL-SALAWY A.

(196X) Alveolar and whole lung phospholipids of newborn lambs. Proc Sm. exp. Bid. Med. 127, 967. 969.

Glum L. & KULOVI~H M. V. (1973) Lecithin~sphingom~e- lin ratios in amniotic fluid in normal and abnormal pregnancy. AHI. J. Ohsret. G~ncc,o/. 115, 539.

GLIJC.K L., SKIBNEY M. & KULOVICJ-I M. V. (1967) The biochemical development of surface activity in mam- malian lung. It. The biosynthesis of phospholipids in the lung of the developing rabbit fetus and newborn. Prdirtr. Res. 1, 247-265.

H~YATS~J H. & (JKIT.~ T. (1964) Selective modification of cytidine nucteosides in ribonucteic acid by semicarba- zide. Biockm biophys. Rrs. Cornmum 14, 198~203.

HAYATS~! H. & Unlr~ T. (1966) The modification of nu- cleosides and nucleotides. IV. The reaction of semicarha- ride with nucleic acid. Biocliiitt. hiopi)rx .dctli 123, 45X470.

KIKKAWA Y.. MOT~YAMA E. K. & GI.~(.K L. (1965) Study of the lung of fetal and newborn rabbits. Rex Dis. 52, 177 208.

KIN<; R. J. (1974) The surfxtant system of the lung. Feri. Proc. 33, 223X- 2248.

KING R. J. & CL~MENTS J. A. (1972) Surface active materials from dog lung. II. Composition and physio- logical correlations. Am. d. Physiof. 223, 715 726.

KLAUS M. I-I., CL~MNTS J. A. & Hnvtt. R. (1961) Compo- sition of surface-active material isolated from beef lung. Proc. tmfn. Acnd. Sci. USA. 47, 1X5X 1859.

OKANO G. & Atxxo T. (1978) Changes in the structural and metabolic heterogeneity of lecithins in the develop- ing rat lung. Bi~~~~~i~~i. hiophys. Ac,rcr 528, 373- 384.

ROON~:Y S. A. & MOTOYAMA E. K. (1976) Studies on the biosynthesis of pulmonary surfxtant. The role of the methylation pathway of phosphatidylcholinc biosyn- thesis in primate and non-primate lung. Cliti. c,hitrr. .Icrtr 69, 525553 I.

Roo~ity S. A., PAGE-R• BEKTS B. A. & MOTOYA~~A E.. K. (1975) Role of lamellur inclusions in surfactant produc- tion: studies on phospholipid composition and biosyn- thesis in rat and rabbit lung subccllular fractions. J. Lipid Rrs. 16, 418-425.

RO~JS~:R G.. SIAI(ATOS A. N. & FLEISC.HI,R S. (1966) Regtt- lation of pulmonary lecithin synthesis. Lipids I, 85.

SOODSMA J. F.. Mtrxs L. C. & HARLOW R. D. (1976) The analysis of the molecular species of fetal rabbit lung phosphatid~icholinc by consecutive chromatog~phic techniques. Bictc~hinz. hioplt~s. .4crrt 424, 159-t 67.

W~INHOI.I) P. A. Cyr VnLt-t- C. ( 1965) Phospholipid metab- olism in the liver and lung of rats during development. Bioc~/ii/,r. hi”p/i.,..s. ;l<~r~r 106, 540 550.

W~:INHOLI> P. A. ( IOhX) Biosynthests of phosphatidqlcho- line during prenatal development of the rat lung. .1. Lipkl Rcs. 9, 262 266.