Effects of norethynodrel on the tissue distribution of H3-estradiol- 17β in ovariectomized rats

463

EFFECTS

OF NORETHYNODREL

ON THE TISSUE

DISTRIBUTION

H3-ESTRADIOL - 178 IN OVARIECTOMIZED

R. Saucier,

R.C.

D~partement

Banerjee,

P. Brazeau,

de Biologie,

Universit~

Sherbrooke,

Received:

Jr.

Qua.

OF

RATS

and S.M.

Husain

de Sherbrooke,

Canada.

May 19, 1970 ABSTRACT

Injection of tritiated estradiol to ovariectomized mature female rats in a dose of 0.i ~ g per i00 g body weight showed a selective uptake of this steroid by certain tissues in 1 hour. Injection of i00 ~ g norethynodrel per i00 g body weight 15 mts before and 15 mts after the estradiol injection reduced the radioactivity in the pituitary, hypothalamus, uterus, vagina and other tissues. It is proposed that there exists a competitive inhibition of estradiol by norethynodrel in these tissues in the rat.

INTRODUCTION

In order the

to elucidate

incorporation

target

cluding been works

of tritiated

and non-target

Selective

uptake

tissues

vagina,

shown by many workers to the mode

of action

estradiol

of estrogens

in the ~o-called

had been extensively

of H3-estradiol

the uterus,

related

the mode

in various

pituitary (1-4).

of action

studied.

tissues

in-

and hypothalamus

In connection

with

of contraceptive

has other

steroids

464

that

ST ER O I D S

are now in progress

necessary lished

two somewhat

(5),

thynodrel),

of estradiol (6),

lective

which titive

Enovid,

component

acts

of labelled in rats.

suggest

that norethynodrel

inhibitor

(nore-

inhibitor

rat tissues.

norethynodrel

study was undertaken

and

of one of the popular

were unable

In view

pub-

suggested

as a competitive

in certain

on the other hand,

by any tissue

Eisenfeld

findings,

felt

reports

-hydroxyestr-5(10)-en-3-one

for binding

uptake

the present

of their

the progestin

contraceptives,

it was

contradictory

laboratories.

on the basis

17~ -ethynyl-17B

et al

in our laboratories

from two different

Axelrod that

to verify

16:4

of these

to find

any se-

or other progestins conflicting

and evidences does

Watanabe

indeed

reports

are presented

act as a compe-

of H3-estradiol.

MATERIALS

AND METHODS

Estradiol-17B -6,7-H3, chromatographically pure, had a specific activity of 40 curies per millimole and was supplied in benzene-ethanol solution by the New England Nuclear Corporation. Six mature female Sprague Dawley rats, ovariectomized at least 2 weeks previously, were injected i.v. with chromatographically pure tritiated estradiol in 10% ethanolic saline (0.I ~ g / 1 0 0 g body weight) through the jugular vein. The animals were sacrificed 1 hour later and the tissues were processed according to the method of Watanabe et al (6). Accurately weighed segments of liver, kidney, uterus, vagina, adrenal and cerebellum were placed in separate scintillation vials to which 0.5 ml of hyamine hydroxide was added. After standing at room temperature for 16 hours, the vials were heated for 3 hrs at 60oc. We added 0.5 ml of ethanol and 15 ml of scintillation mixture (con-

Oct. 1970

ST ER O I D S

TABLE

Effect

of n o r e t h y n o d r e l

on

the

465

1

tissue

dpm/mg

distribution

of w e t

of H 3 - e s t r a d i o l .

weight

H3-estradiol

Norethynodrel g i v e n 15 mts before H3-estradiol injection

Norethynodrel g i v e n 15 mts after H3-estradiol injection

Mean

Mean

Mean

Control:

± S.E.M.

+ S.E.M.

± S.E.M.

Pituitary

1599

± 88

293

± 25*

694

± 77*

Uterus

1273

± 115

422

± 52*

371

± 40*

Liver

647

± 33

576

± 30

374

± 42*

Vagina

490

± 52

164

± 22*

268

± 54*

Kidney

217

± 18

97

± 7 *

106

± 19"

Adrenal

194

± 16

107

± 4 *

138

± 17"

Hypothalamus

ii0

± 5

36

± 2 *

67

± 5 *

Serum

41 ± 1

38

± 3

28

± 1 *

Cerebellum

30

25 ± 2

* P<0.05

compared

± 1

to c o n t r o l .

21 ± 2 *

466

S T ER O I D S

16:4

taining 200 mg of dimethyl POPOP and 6 g of PPO per liter of toluene) to each vial. Radioactivity was determined in a Packard spectrometer model no. 3380 with automatic dpm calculator. In case of the hypothalamus and pituitary, entire tissues from each rat were used. Radioactivity in serum was determined by adding 0.3 ml of serum to 3 ml of hyamine hydroxide and 1.5 ml of ethanol, standing at room temperature for 16 hrs and counting as described above. Chromatographically pure norethynodrel in 50% ethanolic saline (I00 ~g/100 g body weight) was injected 15 mts before and 15 mts after the H3-estradiol injection. Six animals were sacrificed and the tissues processed as described above.

RESULTS

The distribution shown

in Table

i.

of H3-estradiol

There was

an unequal

radioactivity

in the H3-estradiol

In descending

order

liver,

vagina,

these

kidney,

organs

adrenal,

cerebellum.

The uptake

was

when norethynodrel

affected

norethynodrel jection, uterus,

was

~njected

reduced.

Reduction

same tissues

and,

injection. H3-estradiol

These

was data

levels

rats

hypothalamus,

was

kidney

as,

administered.

and tissues When

H3-estradiol

in-

in the pituitary,

and adrenal

was markedly

also noted

in the

the liver,

cerebellum

and serum

15 mts

after H3-estradiol

are also presented I.

serum

was

injected

in Fig.

of

uterus,

by various

of radioactivity

is

(control).

are the pituitary,

in radioactivity

as well

when norethynodrel

treated

15 mts before

hypothalamus,

tissues

distribution

of radioactivity

the accumulation vagina,

in various

as percent

of control

Oct. 1970

S T E R O I D S

467

I00

BO

_1

w

60

..

•

!

j~.bI

"1n.Z 0

//

't4~l

4o

/ /

/ /

t~ /

IA. 0 I-Z b.l

~J

20

/ / /

/ /

!~

W

/

o

~ ~

iiiiiil 7L lii~!ii

i

/

Fi B, |. Effect of norechynodrel on the tissue concentrations o[ :.3_ estradio]. Norethynodre] was given 15 mrs before (dotted bsrs~ and 15 mrs after (diagonal bars) the 113-estradlol injection. "P ( 0.05, • -p
468

ST ER O I D S

16:4

DISCUSSION

Norethynodrel,

as a contraceptive

likely to act on the following organs effect,

e.g.,

the hypothalamus,

fallopian tube and possibly

steroid,

is most

for its antifertility

pituitary,

the vagina.

uterus,

ovary,

The discussion

of

the results will be limited to data with regard to the hypothalamus, the present

pituitary,

uterus

and vagina obtained during

investigation.

The results obtained by us and by Eisenfeld (5) indicate activity

that there is an unequal

in the various

tissues

uterus.

distribution

and the highest

was accumulated by the pituitary

reduced H3-estradiol

However,

estradiol

injection reduced radioactivity Eisenfeld and Axelrod

than it is in displacing

tissues

estradiol

injected

injection

compared

that

in all target

following H 3-

only in the uterus

therefore,

in preventing

inferred the

into the cell of estradiol once it is already bound.

show no such difference:

norethynodrel was estradiol

(5),

is more effective

intracellular binding or entrance

Our results

in stating

they found that treatment

that norethynodrel

radioactivity

concentration

tissues.

and vagina.

of radio-

and to a lesser degree by the

We also agree with these workers

norethynodrel

and Axelrod

irrespective

of whether

15 mts before or 15 mts after

the radioactivity was lowered

to the control.

This appears

in all target

to suggest

that

Oct. 1970

S T ER O I D S

they act as competitive no specific sites ference

inhibitors to

each other and probably

in the target tissues

are involved with pre-

for the binding of these two steroids.

Our findings strongly

suggest

H3-estradiol

and those of Eisenfeld that there

from estrogenic

of Eisenfeld impurities

by the latter workers.

to find any indication

and Axelrod

contaminants

present

et al (6) injected

Reportedly, (5) contained

undetectable

crystallisation

and were unable

less than

used

.05% ar-

et al (6) was

In order to avoid the we purified nore-

and checked

the purity

before using.

the effect of any possible by our procedure,

0.i ~ g

the norethynodrel

(and criticisms)

by thin layer and gas chromatography

used

uptake of radioactivity

and that used by Watanabe

thynodrel by repeated

to minimize

(5) as resulting

/I00 g body weight

of such contaminants

of

(6) criticized

in the norethynodrel

purified by thin layer chromatography. presence

(5)

inhibition

et al

and Axelrod

of selective

by any tissue of the rat. by Eisenfeld

Watanabe

Watanabe

of tritiated norethynodrel

and Axelrod

is a competitive

by norethynodrel.

the observations

omatic

q~

estrogenic

Moreover, impurities,

the dose of norethynodrel

used was kept 50% lower than that used by Eisenfeld

that we

and Axelrod

(5).

47o

ST ER O I D S

16:4

Acknowledgments This investigation was supported by grants from the National Research Council of Canada and from the Minist~re de l'Education, Gouvernement du Qu@bec. Norethynodrel was generously supplied by the G.D. Searle & Co.

REFERENCES .

Jensen, E.V., Jacobson, H.E., Flesher, J.W., Saha, N.N., Gupta, G.N., Smith, S., Colucci, V., Shiplacoffe, D., Newmann, H.G., De Sombre, E.R. and Jungblut, P.W. STEROID DYNAMICS, Editors G. Pincus, T. Nakao and J.F. Tait. Academic Press, New York, 1966, p. 133-156.

.

Stone, G.M., Baggett R. and Donnelly R.B., J. ENDOCRINOL. 27, 271 (1963).

3.

Stumpf, W.E., ENDOCRINOLOGY, 83, 777 (1968).

4.

Stumpf, W.E., ENDOCRINOLOGY, 85, 31 (1969).

5.

Eisenfeld, A.J. and Axelrod J., J. Pharmacol. Exp. Therapeut., 150, 469 (1965).

6.

Watanabe, H., Saha, N.N. and Layne, D,S., STEROIDS, I_!, 97 (1968).

and