Interaction between ethanol and contraceptive steroids on clotting activity in the rat

CONTRACE

INTERACTION BET

EN ET

NOL ACT IV I

D

NT CEPTIVE IN THE T

ION

STEROIDS ON CLOTTING

Charles D. Lox and M.Wayne Heine Department of Obstetrics and G y n e c o l o g y Texas Tech U n i v e r s i t y Health Sciences Center Lubbock, Texas 79430

ABST

CT

Female rats were given either ethinyl estradiol or n o r e t h i n d r o n e in conjunct ion with water or 5% ethanol. H e m a t o l o g i c a l parameters m e a s u r e d included the pro=hromb in time (PT), partial thro oplasr in time (APTT), fibrinogen (FIB), factors II, V, V!l, VIII, X, and XII activity; plus the platelet count (P ) and hematocrit (HCT). The data indicates that norethindrone together with water produces the greatest number of changes in clotting activity.

INTRODUCTION Many papers have appeared in the literature citing the r e l a t i o n s h i p between the use of oral contraceptives and the increased risk of thrombosis. O n e r e v i e w of t h i s s u b j e c t n o t e d the l a c k of ~ an a d e q u a t e test p r o c e d u r e to detect those women who when using oral contraceptives are at a higher risk to develop thromboe olytic disease or abnormalities of the h e m o s t a t i c s y s t e m (i). A n o v e r a l l t e n d e n c y towards h y p e r c o a g u l a b i l i t y including changes in specific clotting factors has been reported (2) This included cha,ges in platelet factor 3 activity, which showed an overall increase in the platelet factor 3-catalyzed activation of prothrombin (3). L i k e w i s e anti-thro in Ill levels have also been ~ shown to be altered in users of oral contraceptives (4). Overall m e a s u r e m e n t s of hemostasis has indicated hypercoagulabiiity to exist in some women taking combination type contraceptives. This" was primarily reflected in an accelerated partial thro oplastin ti as well as an increased activity of the fibr{nolytic. s y s t e m (5). It h a s b e e n r e p o r t e d that f a c t o r V I I , the f i r s t f a c t o r of the extrinsic pathway, was elevated in women using oral c o n t r a c e p t i v e s (6). A strong correlation has been reported between levels of factor VI~ activity and factor X!I activity, which links both the extrinsic and" istr~nsic pathways, suggesting an inherent residual ability to develop t h r o m b o e m b o lyric disease in women using contraceptives (7). Sub

tied for p u b l i c a t i o n Dece

Accepted

er 8, 1982

for p u b l i c a t i o n M a r c h 16, 1983

RIL 1983 VOL. 27 NO. 4

401

CONTRACE

ION

Although chronic ingestion of .alcohol has been k n o w n for some time to cause d i s r u p t i o n s in r e p r o d u c t i v e ftlnction, i n c l u d i n g e n d o c r i n e p h y s i ology, much less is known about the e f f e c t s of ethanol on the h e m o s t a t i c system. One recent report (8), has shown a d e c r e a s e in the p l a t e l e t count atld intrinsic p a t h w a y a c t i v i t y in male rats g i v e n ethanol. This was primarily reflected ~n a c t i v i t y changes for f a c t o r s V I I I , X, X l l , and the APTY. This s u g g e s t e d that intrinsic p a t h w a y a l t e r a t i o n s could result from a I coho i in,.ake. As many of the s t e r o i d h o r m o n e s as well as ethanol are m e t a b o l i z e d in the liver, the site of s y n t h e s i s of m a n y c l o t t i n g proteins, ~t w o u l d s e e m that an important area co i n v e s t i g a t e would be animals taking both

alcoho!

and steroidal

contraceptives.

Especially

looking for possible

s y n e r g i s t i c i n t e r a c t i o n b e t w e e n ethanol and c o n t r a c e p t i v e s , the r e s D o n s e of w h i c h might not be seen w i t h e i t h e r alone. This could be e x t r e m e l y important K i v e n the increased use of c o n t r a c e p t i v e s and social d r i n k i n g by women today. To this extent, the f o l l o w i n g study was u n d e r t a k e n .

THODS

D

ERIALS

E x p e r i m e n t a l Design. Seventy female S p r a g u e - D a w l e y rats b e t w e e n 175 and 210 grams were used in this experiment. The rats w e r e o b t a i n e d from SASCO Animal Fa~s, Omaha, N e b r a s k a . The animals were m a i n t a i n e d in w i r e - m e s h edges under constant t e m p e r a t u r e (23C) with 12 hours of light and 12 hours of d a r k daily t h r o u g h o u t the course of the experiment. P u r i n a rat c h o w and rig"inking s o l u t i o n s w e r e a v a i l a b l e ad l i b i d u m t h r o u g h o u t the experiment. At the init iat ion of the e x p e r i m e n t l-ani is were w e i g h e d and m a r k e d for identification. The 70 rats were s u b s e q u e n t l y s u b d i v i d e d into six groups30 rats ( I 5 each group) w e r e p l a c e d on an ad libid d r i n k i n g s o l u t i o n of w a t e r or five percent (V/V) ethanol in d r i n k i n g w a t e r for the d u r a t i o n of the e x p e r i m e n t , these animals serving as controls. The 40 other female r a t s w e r e d i v i d e d i n t o 4 g r o u p s of I0 r a t s e a c h and w e r e p l a c e d on the f o l l o w i n g treatment regimens. T w o groups of I0 rats each were placed on a solution of d r i n k i n g water for 45 d a y s , the last 15 d a y s of w h i c h the anima!s were given I00 m i c r o l i t e r s of e t h i n y l e s t r a d i o l or n o r e t h i n d r o n e p os for the d u r a t i o n of the e x p e r i m e n t a l period. The second two groups 0-f i 0 .......rats ... each were p l a c e d on a d r i n k i n g s o l u t i o n of 5% ethanol in w a t e r for 45 days, the last 15 days of w h i c h they were also given ethinyl e s t r a diol or n o r e t h i n d r o n e s o l u t i o n s p e r os. The steroids were o b t a i n e d from Sigma Chemical Company, St. L o u i s , M i s s o u r i and u s e d as s h i p p e d . Both s t e r o i d s were d i s s o l v e d in c h l o r o f o r m and s u s p e n d e d in v e g e t a b l e oil with the organic solvent then b e i n g e v a p o r a t e d off under a stream of nitrogen. The final i00 .~I steroid s o l u t i o n s which were given per os c o n t a i n e d either 0.2 m i c r o g r a m s of erh~nyl e s ~ r a d ~ o l or 2 m i c r o g r a m s o f ~ n o r e t h i n d r o n e , these s o l u t i o n s were given once d a i l y b e = w e t s 0800 and I000 hours. D u r i n g the last 5 days of the experiment, daily vaginal s ars were taken to dete ine the s t a t e of t h e e s t r o u s c y c l e . At the c o n c l u s i o n of t h e e x p e r l m e n t a l period the animals were s a c r i f i c e d with body w e i g h t s again being taken. The animals were s a c r i f i c e d under ether a n e s t h e s i a and a 4.5 ml sample of

402

RIL 1983 VOL. 27 NO. 4

CONTRACE

ION

sodium citrated (3.8%) whole blooa being collected via syringe from the dorsal aorta. P l a s m a w a s o b t a i n e d by c e n t r i f u g a t ion at 4C ( 2 5 0 0 r p m s ) after aliquots had been removed for hematocrit and platelet count determinations. The plasma was stored frozen until assayed for clotting activity, ich always occurred w i t h i n 7 days. Anal s. Hematocrit was d e t e r m i n e d on a Clay-Adams Autocrit I!. Platelet counts were d e t e r m i n e d manually using a hemocytoineter (American Optical). Clotting activity was determined on an E l e c t r a 750 C o a g u l a t i o n Timer, Medical Laboratories Automation, utilizing human factor deficient substrafes supplied by George King Biochemical Co., Wichita, Kansas. This i n c l u d e d the PT, A P T T , f i b r i n o g e n , and c o a g u l a t i o n factor activity for factors II, V, VII, VIII, X and XII. As human factor deficient substrate was used, percent activity was d e t e r m i n e d against a normal h u m a n control for c o m p a r a t i v e purposes. Statistics. the student's

All data was analyzed for statistical significance t-test or regression and c o r r e l a t i o n analysis.

RESU

ut

il izing

S

en compared to water controls, the female rats who were d r i n k i n g the 5% ethanol solution for 45 days showed a slight decrease in body weight which was not statistically significant (60.5 grams versus 51.7 grams). The o n l y animals who showed a significant decrease in body weight over the 4 5 - d a y treatment period were the rats who were taking the combinaton of 5% ethanol and ethinyl estradiol (38.9 + 16.5 grams, X ~ S.D.). This r e p r e s e n t ed a significant decrease from both controls and from the rats who were taking ethinyl estradiol and w a t e r a l o n e (61 8 ~ 27 g r a m s ) . T h e r a t s+ who were receiving the n o r e t h i n d r o n e treatment either with w a t e r (51.1 16.1 g r a m s ) or n o r e t h i n d r o n e in c o m b i n a t i o n with ethanol (47.2 ÷ 14.8 grams) were not s t a t i s t i c a l l y different from either control in terms of body weight changes.

M i c r o s c o p i c e x a m i n a t i o n of the v a g i n a l s m e a r s i n d i c a t e d that b o t h steriodal contraceptives altered the normal vaginal cytology of the rat with many of the animals being arrested in the diestrous phase of the cycle. R a n d o m samplings of the control rats d r i n k i n g the ethanol solutions indicated that there was no d i s r u p t i o n of estrus in these animals. Comparison of c l o t t i n g activity in the w a t e r c o n t r o l s versus the etl,anol controls indicated that only one h e m a t o l o g i c a l parameter measured was s t a t i s t i c a l l y different. This occurred in the platelet count (Table I). All other clot~ing ~ctivities m e a s u r e d were not statistically different when comparing water controls versus ethanol controls. Therefore, with the e x c e p t i o n of the platelet count, all control data was pooled for statistical purposes.

L 1 9 8 3 VOL. 27 NO. 4

403

NTRACE

TABLE

ION

I. C h a n g e s

in p l a t e l e t ........

.......

~_

Controls

counts - ~ = ~ , , , ~ _

:

~

_

in c o n t r o l s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Est radiol

and ............

treated - - -

rats

........................................................................

_ ~

~

o

_

Norethindrone .

889 + 72

+_.

812"* +

44

83

7~4"

78+1

782

147

103

62

H20

768 ***

ETOH

~ S.D. compared

The water or and I I I .

x 103/ to its

effects ethanol

3 , p < 0.01, between controI, *** P < 0.001

of ethinyl drinking

controls, compared

** P < 0.01 to its control.

estradiol or norethindrone on c l o t t i n g a c t i v i t y can

be

in combination with seen in Tables II

DISCUSSION It is interesting to note that a significant body weight reduction occurred only in the female rats o w e r e on the c o m b i n a t i o n of e t h i n y l e s t r a d i o l and e t h a n o l ; a r e d u c t i o n ~ h i c h was not seen in the rats w h o w e r e taking either ethinyl esCradiol and water or ethanol alone. This suggests that perhaps the combination of these two drugs taken jointly has an adverse effect on body weight gain. However, this particular regimen did not cause any notable changes in clotting activity, which suggests that in terms of the clotting activity measurements in this study, the observed decrease in body weight was not particularly significant. Reported changes in platelet counts following alcohol consumption are widespread in the literature and were also noted to occur in this study. It is i n t e r e s t i n g to note that both e t h i n y l estradiol and n o r e t h i n d r o n e in c o n j u n c t i o n w i t h r a t e r c a u s e d the p l a t e l e t count to be s i g n i f i c a n t l y decreased fr the controls, yet when these steroids sere coupled with ethanol there was no significant reduction in plate!st count. This suggests that perhaps somehow the tendency towards thrombocytopenia often =ten with ethanol consumption is either modulated by contraceptive steriods or perhaps that a maximal response had already been reached. Notelovttz et al. (5) observed that women taking combittation type steroidal contraceptives contai~ling both e t h i n y l e s t r a d i o ! and" n o r e t h i n drone had i n c r e a s e d levels of f i b r i n o g e n a n t i g e n and activity. We did not

404

APRIL 19

V O L 27 NO. 4

CONTRACEPTION

T A B L E If. Effects of e t h i n y l e s t r a d i o l (EE 2) on c l o t t i n g rats d r i n k i n g w a t e r o r 5 % e t h a n o l ( ~ ~ S . D ~

PT

SEC APTT

--mg/dl-FIB

II

activity

% Activity VII VIII

V

X

in female

XII

HCT %

Control

13,7

31,6

168

110

1~7

129

1t4

I~0

14+0

39+

N ffi 30

0.8

4.7

35

9

13

13

20

22

I0

3

104 . 7

141 . 6

1t3

36

.

N.S.

N.S.

0.01

N.S.

172÷

II0 ÷

EE 2 and

Water P < EE 2

30,2 . 4.6

N.S.

N.S.

13.6 ÷

and Ethanol P < N.S.

13¢3 . 0.3

.

.

31.4 ÷

0.6

- Not

156+ .

. 4

.

3.9

.

.

55

N.S. N.S. statistically

.

149 ÷

4

N.S. N.S. slgnificant

APTT

FIB

II

139 ÷

.

~1 .

.

N.S.

N.S.

0.001

0.01

IIO ÷

124 ÷

37÷

8

10

9

.

.

7

9

I19 ÷

.

13

activity

,% A c t i v i t y VII VIII

V

7

N.S. N.S. N.S. 0.001 ..........................

on c l o t t i n g

--mg/dl--

.... SEC

1t4 .

6

13 N.S.

T A B L E III. Effects of n o r e t h i n d r o n e (NED) d r i n k i n g w a t e r or 5% e t h a n o l ( ~ ~ S.D

PT

l~l

in female

2

2 0.05

rats

X

XII

HCT

Control

13,7

31,6

168

1~0

1~7

119

114

I00 +

140 +

39+

N - 30

0.8

4.7

35

9

13

13

20

22

I0

3

13,4 .

25j.7 . 2.3 0.01

122 .

151 .

125 .

1+53

36

NED and Water

0.5

P <

N.S.

NED

13,7

and

.

Ethanol

1.2

P < N.S.

N.S. "

Not

II0 .

31,5

.

.

~I .

.

46

9

19

14

12

I0

19

0.001

0.001

N.S.

0.02

N.S.

N.S.

0.01

0.02

106 ÷

150 ÷

97

141 ÷

3 8÷

150

.

.

5.6

31

N.S.

N.S.

statistically

127 .

.

.

.

139 ÷

.

1 ÷1 7

.

÷

.

2

6

7

5

12

5

16

2

N.S.

N.S.

N.S.

N.S.

N.S.

N.S.

N.S.

significant

APRIL 1983 VOL 27 NO. 4

405

CONTR ACEPTION

see this response when these steriods were used s e p a r a t e l y in rats. In fact, when n o r e t h i n d r o n e was used in c o n j u n c t i o n with water, there was a signif[cant decrease in fibr[nogen activity (Table I[I). The accelerated partial t h r o m b o p l a s t i n t£nle which they noted with thls c o m b i n a t l o n s t e r o l dal regimen was also seen in our study in those rats treated with n o r e t h i n drone and water. This o b s e r v a t i o n on the APTT supports a suggestion that the intrinsic pathway is being i n f l u e n c e d by c o n t r a c e p t i v e steroids; as the TT is a m e a s u r e of intrinsic p a t h w a y activity. Gordon e al. (7) noted a strong association between factor Vll and factor XII activity in women o were using combination contraceptives. Likewise, a highly significant correlation (P < 0.01) between factor VII and factor XI[ activities was also seen in this study for both ethinyl e s t r a d [ o ~ and n o r e t h i n d r o n e - i n g e s t ing r a t s w h o w e r e a l s o d r i n k i n g water. Those animals who were taking e i t h e r of these two steroids in c o n n e c t i o n with ethanol did not show this correlation If one looks at the overall c l o t t i n g data, it appears t h a t norethindrone is re potent ~n terms of inducing changes in c l o t t i n g activity than is ethinyl estradiol, e s p e c i a l l y en taken in c o n j u n c t i o n with water. It is interesting to note that aside from the plate!at count and the h e m a t o crit reduction, only one clotting a c t i v i t y was a b n o r m a l in t h o s e r a t s ingesting c o n t r a c e p t i v e steroids and ethanol, th~s being factor XII in the echinyl e s t r a d ~ o l - T r e a t e d rac. C o u p l e d together w ~ h the fact that those rats drinking water and receiving either ethinyl estradiol or n o r e t h i n d r o n e likewise had significant changes in factor XII activity, this again supports the suggestion that intrinsic pathway alterations were occurring.

O n l y one h e p a t i c synthesized clotting factor activity was a b n o ~ a l in t h o s e r a t s t a k i n g e t h i n y l e s t r a d i o l , this w a s f a c t o r V in the w a t e r d r i n k i n g group. However, those animals who were receiving n o r e t h i n d r o n e and water had several abno al c l o t t i n g activities of hepatic derivation, fibrinogen, factor I[ and factor VII In the case of f i b r i n o g e n and factor VII, these activities were d e c r e a s e d ile the factor II a c t i v i t y was increased. If indeed clotting activity is more disturbed in rats taking ethinyl estradiol or n o r e t h i n d r o n e in c o n j u n c t i o n with water than when coupled with ingestion of alcohol, this would suggest thaC i n t e ~ s of altering hemostasis, s~ehow joint usage of contraceptive steroids and e t h a n o l are inhibitory on o n e a n o t h e r . W%ether this means that ingesting both steroid contracept ires and ethanol s y n e r g i s t i c a l l y decreases the potent ial for abnormal h e m o s t a s i s is unclear at this time, but c e r t a i n l y would be an interesting finding. If th~s is indeed the case, perhaps en e~hanol and c o n t r a c e p t i v e s are taken in combination, there i{ a selective inhib[tion of the hepatic enz es that are involved in c l o t t i n g factor activity or synthesis. Several other q u e s t i o n s from this study arose which stil! need answering. Such as, how do these two steroids be ve en c o m b i n e d and given to the animal with d r i n k i n g water and/or ethanol? Or, a t effect on clotting a c t i v i t y would be seen when the animal was primed f[rst with c o n t r a c e p t i v e s then dr~nks alcohol? These further questions are now under study in our laboratory.

4

RIL 1983 V O L 27 NO. 4

CONTRACEPTION

FE

Q

Q

Q

Q

O

•

NCES

M en, E.F. Oral contraceptives and blood coagulation" a critical review . . . . d. Obstet. Gyneeol. 142,781-790, 1982. HoOt,ten, E.G. Hormonal steroid contraceptives." a further review of adverse reactions. Drugs 16,223-357, 1978. Leff, B., Henriksen, R.A., and Owen, W.G. Effect of oral contraceptive use on platelet prothro in converting (platelet factor 3) activity. Thromb Res. 15,631-638, 1979. Conard, J. Ant ithro in III and the oestrogen content of co ined oesto-progestogen contraceptives. Lancet (2) 1148-1149, 15 Nov 1972. telovitz, M., Kitchens, C.S., Coons, L., McKenzie, L. and Carter, R. Low-dose oral contracetive usage and coagulation. . J. Obstet. Gynecol. 141,71-75, 1981. Meade, T.W. Oral contraceptives, clotting factors, and thrombosis. . J .... Obstet. Gynecol., 142,758-761, 1982. Gordon, E.M., Ratnoff, O.D., and Jones, P.K. The role of au eared Hageman factor (factor Xll) titers in the cold-promoted activation of factor VII and spontaneous shortening of the prothrombin time in women using oral contraceptives. J. Lab. Clin. Med. 99,363-369, 1982. Lox, C.D., and Heine, M.W. Evidence for an ethanol mediated disruption of hemostasis. Gen Pha acol, in press.

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407