Teratogenic interaction of two substances: Effect of sodium salicylate and defibrase upon pregnant mice

THROMBOSIS

Vol.1, pp. 127-134, 1972 Pergamon Press, Inc.

RESEARCH

TERATOGENIC INTERACTIONOF TWO SUBSTANCES:EFFECT OF SODIUM SALICYLATEAND DEFIBRASE UPON PREGNANT MICE MARCELA GUTOVA'and K. SUNE LARSSON Laboratoryof Teratologyand Departmentof Blood CoagulationResearch, Karolinska Inntitutet,Stockholm

(Received

16.3.72.

Accepted

by

Editor

P.

Olsson)

ABSTRACT

The effect of the combined treatmentwith Defibrase,a thrombin-like enzyme preparationpurified from the venom of the snake Bothrops atrox, and sodium salicylatewas studied in NMRI mice during early (7, 8, 9 day) and late (15, 16, 17 day) gestation period. Increased embryolethalityand potentiationof the teratogenicand the foetal damaging effects of sodium salicylateby Defibrasewere observed, Blood coagulationtests performed on mothers revealed no significant change in either the number of platelets or the prothrombintime, except for animals on combined treatment in late gestation period, Persistingdefibrinogenationof the plasma was observed following Defibrase treatment. The possible risk of teratogenicsynergism in clinical therapy of thrombosiswith thrombin like enzymes in fertile women is indicated,

INTRODUCTION Various teratogeniceffects can be experimentallyinduced with different salicylate compounds (see review by Larsson, 1970). The incidenceof

'Researchfellow; permanent address: Department of Pharmacology,Faculty of Pediatrics,Charles University,Prague, Czechoslovakia

127

128

TEFUTOGENIC

INTERACTION

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foetal death, superficialand liver hemorrhagesis remarkablyhigh in mice after salicylate treatment during the last part of pregnancy (Eriksson,lg71), Furthermore,prolonged prothrombin time has been demonstratedin mouse foetuses with salicylate-Inducedhemorrhages (Eriksson,1971). Defibrase, a thrombinilikeenzyme preparationpurified from the venom of the snake Bothrops atrox, has so far not been proved to be teratogenlcin laboratoryanimals (Douchene-Marullaz,1963). A synergisticeffect of these two substanceson blood coagulationhas, however, been observed in adult humans treated with Xfibrase when an anti-inflammatorydrug was given simultaneously(Egberg, personal communication), The present study was undertaken in mice to investigatewhether Defibrase could also enhance the foetal damaging effect of sodium salicylate at two different gestationalperiods,

In addition, blood coagulationtests

were performed on some mothers in order to study the combined effect of these substances,both of which are known to interferewith the coagulationmechanism. MATERIAL and METHODS Pregnant primiparousmice of NMRI strain, weighing 20 g., were used. They were mated overnight and the day on which vaginal plug was found was denoted as day zero of pregnancy, The animals were injected intravenouslywith DefibraseR ’ for three consecutivedays, either on days 7, 8, and 9, or on days 15, 16, and 17 of gestation, The dose was 7.5 ml/kg per day which is approximatelyl/3 - l/6 of the acute LD 50, A sodlum salicylate injection intramuscularlywas superimposed one hour after the last injection of Defibrase (on day g or 17 of gestation) in a dose of 500 mg/kg, Furthermore,two groups of animals were injectedwith Defibrase or sodium sallcylatealone and the control group was given saline. On day 18 of gestation, the animals were sacrificedand laparotomised, The numbers of foetuses alive, late dead (after day 16) and resorbedwere recorded. Individualfoetuses were examined for external anomalies and

'DefibraseRcontaining4.4 NIH units/ml dissolved in chlorobutanolwas obtained from PentapharmAG, Basle, Ltd.

Vol.l.No.2

TERATOGEXCC

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INTERACTION

superficialhemorrhagesaccording to Eriksson, 1970,

*

Sune of the animals were subjected to blood sampling, Sodium citrate was used as anticoagulant(1 part 3.8 per cent sodium citrate (x 2H20) + 9 parts of blood), Blood samples (30~1) for determinationof prothrombin' and platelet number were taken from the tail vein, For the determinationof fibrinogen concentrationin plasma (0.2 ml) blood samples taken from the orbital plexus were used, Blood samples were taken before initiatingtherapy, one hour after the last Defibrase injection (prior to sodium salicylateadministration),and 10 hours after the sodium salfcylateinjection or 10 hours after Defibrase given alone, The numbers of platelets were estimated according to Kristenson (1922), the prothrombin times according to Nor&

(1970), and fibrinogen concentra-

tions according to BergstrUm et al (1960). Statisticalevaluation was performed using "Student's"t-test, RESULTS As can be seen from Table I, an Increasednumber of resorbed embryos was found only after combined Defibrase and sodium salicylatetreatatent during the early part of gestation, In this group of animals a slight increase in the percentage of malformed foetuses was also found, After treatment in the late gestation period an increase in the frequency of late dead foetuses was demonstratedafter sodium salicylate fnjections alone and after its use in combinationwith Defibrase, In these two groups a few foetuses with hemorrhageswere found, The influence of Defibrase and sodium salicylatetreatment in animals which had in addition been subjected to blood sampling is seen in Table II, Many mothers died after combined therapy, Furthermore,following the treatment in the 1ate gestation period premature deliveriesoccurred, In the group of animals treated during early gestation period and subjected to blood sampling an increased resorption rate was observed in those treated with Defibrase. The embryolethalitywas significantlyincreased in the group given sodium salicylate in addition to Defibrase injectionsin comparisonwith other groups.

'All reagents were provided by the Department of Blood CoagulationResearch, Karolfnska Institute,Stockholm

I

6 4 4

15.16.17

15.16.17

15,16,17

Na Salicylate + Defibrase

Defibrase

Controls

50

35

70

59

17

Na Salicylate

3.5

-

1.6

-

1

48 96.0

-

-

12 17.1

11 18.8

2

-

1

-

Dead No. %

32 94.3

54 77.1

48 81.2

52 91.2

57

7.88

Controls

52 91.2

75 92.5

57

7.8.9

Defibrase

5

81

Living No. %

43 69.3

7.w

Na Salicylate + Defibrase

7

sites

Implantation

62

9

Mothers

Treatment

Substance

Na Salicylate

No. of

Day of

Administered

Not Subjected to Blood Sampling

7.5

2

3

4

-

3

5

4.0

6.7

5.8

-

3.6

8.8

18 29.1

6

Resorbed No. %

Foetuses

-

-

-

-

-

1 1.4

-

2 3.5

1 1.7

5 8.0

1 1.2

Malformed No, %

-

-

2

3

-

-

2

-

-

-

2.8

5.0

-

-

3.2

-

Hemorrhage No. %

The Foetal Damaging Effect of Sodium Salicylate and Defibrase Treatment in NMRI Mice,

TABLE

II

No. of

7.8.9

17

15.16.17

15.16.17

15.16.17

Controls

Na Salicylate

Na Salicylate + Defibrase

Defibrase

Controls 0.05

7.8.9

Defibrase

PC

7.8.9

Na Salicylate + Defibrase

1

9

Treatment Mothers

Day of

Na Salicylate

Substance

Administered

2

1 dead, abortion

62

36

62

67

65

98

17

75

sites

Per 0.01

2 delivered 2 dead

1 partly delivered

4 dead

Remarks

Implantation

3

P<

6; 98.3

30 83.3

23 3.2

39l58.2

61 93.8

86 85.7

14382.3

74 97.3

Living NQ. %

Subjected to Blood Sampling

38.8

-

0.0015

-

-

_

_

572 91.9

26

_

Dead No, % 2.7

4.9

3.0

6.2

1

1.7

6 16.7

3

2

4

12 14.3

3 17.3

1

Resorbed No. %

Foetuses

_ -

_ -

_

_

_

_

_

_

-

2 2.7

Malformed No. '16

The Foetal Damaging Effect of Sodium Salicylate and Defibrase Treatment in NMRI Mice,

TABLE

6

24

3

-

16.7

39.01

4.4

Hemorrhage No. %

d

3 g z

E

ti

w

.2

w

c1

.r

132

TERATOGENIC

INTERACTION

Vol.1,No.P

The frequency of late-dead foetuseswas higher in the groups treated with sodium salicylate alone or in combinationwith Defibrase than in other groups, The percentage of living foetuses in the group of animals with combined treatmentwas as low as 3.2%. Furthermore,a very high frequency of superficialhemorrhageswas found in foetuses whose mothers were treated with Oefibrase and sodium salicylate in late pregnancy, The blood coagulationtests (Table III)

revealed no significantdif-

ferences in the number of platelets and prothrombin time, except for animals on combined treatment in late gestation, Prolonged prothrombin time occurred in this group of animals in which the harmful effect on foetuseswas most evident. Oefibrinogenationof plasma was found after Oefibrase treatment and the low concentrationof fibrinogenpersisted for 10 hours after the last Defibrase injection; the difference is statisticallysignificant, DISCUSSION The results of the present investigationsconfirm our earlier observations that salicylate given late in pregnancy causes a higher incidence of foetal death than when given in early gestation (Larsson and Eriksson, 1966) and that salicylate injections in late pregnancy induce premature birth (Erikssonand Larsson, 1968). Potentiationof the teratogeniceffect of one agent by another agent, even when non-teratogenicitself or given in subthresholddose, has been described (Wilson, 1964). In the present study the synergisticeffect of Defibrase and sodium salicylatehas been observed as an increase in the frequency of resorptionsand ma.lformations in the early stage of the gestation period, Furthermare,it is known (Olson et al., 1971) that a combination of Defibrase and salicylate severely impairs the blood coagulationmechanisms, In our experiment a much higher incidence of maternal mortality (due to massive bleeding) occurred in a group injectedwith both Uefibrase and sodium salicylate. These conditionshad a marked effect on the foetuses as well, causing their bleeding and death. An increasedfrequency of superficial hemorrhageswas also found. Thus, it is evident that risks in Defibrase and sodium salicylate therapy is not only limited to the early stages of pregnancy,but could also be harmful in late pregnancy,

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TERATOGENIC

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Vol.1,No.Z

ACKNOWLEDGEMENTS This study was supported by grants from the Swedish Medical Research Council, nos. 872014P-993-07,872-14X-993-07and 872-19X-520-08, REFERENCES BERGSTRUM,K., BLOMMCK, B, and KLEEN, G, Studies on the plasma fibrinolytic activity in a case of liver cirrhosis, Acta Med. Stand, 168, 291, 1960, DOUCHENE-MARULLAZ,P, Effect of reptilase on the gestation in the rat and mouse, Teratologicalstudy on request of LaboratoriesStago, 1963, Unpublished, EGBERG, N, Personal Communication, ERIKSSON,M, Effect of salicylatetreatment on foetal and maternal prothrombin time in the mouse, Acta Physiol, Stand, 78, 39, 1970, ERIKSSON,M, Salicylate-inducedfoetal damage late in pregnancy, An experimental study in mice, Acta Paediat. Scan& Suppl, 211, 1971, ERIKSSON,M, and LARSSON, K.S, Premature birth induced in mice by salicylate. Nature 220, 385, 1968. KRISTENSON,A. A new method for the direct counting of the so-called blood platelets in man, Acta Med. Stand, 57, 301, 1922, LARSSON, K,S, Action of salicylateson prenatal development, In: Congenital malformationof mammalia, Ed, H, Tuchmann-Duplessis, Masson & Cie, Paris, 1970, p. 171, LARSSON, K,S, and ERIKSSON,M, Salicylate-inducedfoetal death and malformations in two mouse strains, Acta Paediat, Stand, 55, 569, 1966, NOREN, I, Specific assay of prothrombin, A method using a freeze-dried reagent of intrinsic coagulationfactors. Stand, J. Clin. Lab. Invest. 25, 47, 1970, OLSSON, P,, BLOM~CK, M,, EGBERG, N,, EKESTRUM, S., GURANSSON,L. and JOHNSSON, H, Studies on the bleeding tendency and on the possibilityOf surgery in states of Reptilase induced defibrinogenation, Thromb. Diath. Haemorrhag,Suppl, 47, 389, 1971, SMITH, M.J.H. and SMITH, P,K, The Salicylates;a critical bibliographic review, IntersciencePublishers;London, 1966, WILSON, J,G, Teratogenicinteractionof chemical agents in the rats, J, Pharmacol, Exp, Ther, 144, 429, 1964,