The action of chlormadinone acetate on the estradiol uptake by rat organ cultures

CURRENT INVESTIGATION

The action of chlormadinone acetate on the estradiol uptake by rat organ cultures JORGE

M.

GRETA

DECLERCQ

MARTHA

ROSNER*

DE

San Miguel,

DE

PEREZ

OLIVEIRA

BEDBS**

GUERRA

Argentina

Chlormadinone acetate was able to inhibit significantly (p < 0.01) the estradiol uptake by rat organ cultures of the hypothalamus and uterus when added simultaneously to estradiol-17B-6, 7-sH, suggesting that this drug can act as an antiestrogen by competing with estradiol for the binding sites. When chlormadinone acetate was added previously, it significantly (p < 0.01) increased the uptake of estradiol by both hypothalamic and uterine explants. These results could indicate estrogenic-like effect of chlormadinone acetate probably by increasing the binding sites.

E F FECT s of chlormadinone acetate on the in vivo estradiol uptake by the rat target organs have been studied previously.** 2 Chlormadinone acetate inhibited significantly the uptake of estradiol by the uterus and the hypophysis and modified its subcellular distribution. Nevertheless, the hy-

pothalmic uptake of estradiol was not inhibited, even when doses as high as 100 pg of chlormadinone acetate were utilized. The in vivo experiments1 were performed after a single injection of estradiol, and we thought that it was worthwhile to study the effects of exposing isolated rat target tissues to chlormadinone acetate for longer periods of time. For these reasons and because it also permits observation of the effects of drugs without the interaction of exogenous metabolic changes, the organotypic culture of rat hypothalami and uteri was selected.

THE

From

the Instituto Latinoamericano de la Reproduccidn (ILAFIR), Universidad de1 Salvador.

de

Fisiologia

This work was supported by Grant No. 099 from the Comisidn de Investigaciones Cientlficas, Provincia de Buenos Aires, Argentina. Reprint Director, Salvador, Miguel,

an

requests: Dr. Jorge M. Rosner, ILAFIR, Universidad de1 Casilla de Correo 10, San Prov. Bs. As. Argentina.

Material

and

methods

Diestrous Wistar rats weighing 200 grams were utilized and killed by decapitation; the hypothalami and uteri were obtained under aseptic conditions. Hypothalami were cut in halves and uteri were cut in 1 mm. thick pieces. Ten milliliters of TC 199 medium

*Member of the Carrera de1 Investigador, Consejo National de Investigaciones Cientificas y Tkcnicas,

Argentina.

**Member of the Carrera de1 Investigador, Comisidn National de Estudios Geo-Helioffsicos, Argentina. 430

Volume Number

Chlormadinone

112 3

kperiment NO.

1 I

action

2

on estradiol

uptake

431

5

I

24 hr

prelr

u

medium

change

0. “/lg chlormad,none

1 hr

acetate

450,000 tritiated

dpm 17p estradiol

450,000 tritiated

acetate

dpm 17 /, estradlol

450.000 trltiatvd

dpm 171 estradiol

+ 0. bpg chlormadinon acetate

Fig. 1. Experimental

(Difco Labs., Detroit, Michigan) was utilized in Petri dishes. Streptomycin, 100 pg per milliliter, and penicillin, 100 U. per milliliter, were added. Estradiol-I 7/.-6,7-$H, specific activity 40 c per millimole (New England Nuclear, Boston, Massachusetts), was used after purification by paper chromatography in a benzene/formamide system.3 Labeled estradiol was dissolved in isotonic saline solution (9 x lo6 disintegrations per minute [ d.p.m.1 per milliliter) and chlormadinone acetate in propylene glycol : ethanol (4: 1, v/v) (100 per milliliter). Fifty milligrams of tissue (5 hypothalami or 10 uterine fragments) was incubated in 10 ml. of TC 199 medium for 49 or 72 hr. at 37’ C. in a 95 per cent oxygen, 5 per cent carbon dioxide, moist atmosphere. Culture media were changed every 24 hr., and histologic controls were made at the end of each culture. Explants were washed 3 times with fresh culture medium, weighed, and digested with 1 ml. of hyamine hydroxide for 3 hr. at 60* C. in counting vials. Before the determination of radioactivity, 0.25 ml. of acetic acid and 10 ml. of scintillation fluid were added to the vials (4 Gm. of 2,5-diphenyloxazole and 40 mg. of p-bis 2[5 phenyloxazoyl] benzene/l toluene) . Radioactivity was determined in a Packard Tri-Carb liquid scintillation spectrometer, and quenching was corrected by the internal

design

for

uterine

studies.

standard method. Statistics were performed by analysis of variance (Duncan’s4 test) . Six experiments, 10 organ cultures each, were done, with both hypothalamic and uterine explants. Experiments. Dose and time experiments were previously performed for establishing the optimal conditions for estradiol uptake. Dose experiments. Uterine explants were cultured for 48 hr. in separate Petri dishes; 300,000 to 600,000 d.p.m. of tritiated estradiol was added to the fresh culture medium, and organ cultures were stopped 1 hr. later. Four hundred and fifty thousand d.p.m. was selected as the optimum dose. Hypothalami explants were cultured for 72 hr. in separate Petri dishes; 150,000 to 160,000 d.p.m. of tritiated estradiol was added to the fresh culture medium, and organ cultures were stopped 1 hr. later. Three hundred thousand d.p.m. was selected as the optimum dose. Time exjeriments. Uterine explants were cultured for 48 hr. and stopped va, 1, and 2 hr. after the addition of the previously established optimal dose of estradiol. The best time was 1 hr. Hypothalami explants were cultured with the previously established optimal dose of labeled estradiol for 72 hr. and stopped f/2, 1, 24, 48, and 72 hr. after the addition of the hormone. The best time was 24 hr. Ten points were determined for each experiment.

432

Rower

et

February 1, 1972 Am. J. Obstet. Gynecol.

al.

Experiment NO.

b

24 hr

7

R

prPincuhation

medium

change

24 hr

24 hr

0.8/e chlormadinonr acetate

300,000 tritiated

dpm 17/3 estradiol

medium

change

300,000 tritiated

dpm 17 P Pstradial

300,000 tritiatrd

dpm 17 P estradiol

+ 0.8 pg chlormadinone acetate

Fig. 2. Experimental design for hypothalamus studies. Uterus (Fig. I). ExPERrMENr 1. Uterine explants were cultured for 48 hr., and 450,000 d.p.m. of tritiated estradiol-17/3 was added to the fresh culture medium. Organ cultures were stopped 1 hr. later. EXPERIMENT 2. Uterine explants were cultured for 48 hr. and transferred to another Petri dish containing 10 ml. of TC 199 medium, 0.6 JL~ of chlormadinone acetate, and 450,090 d.p.m. of estradiol-17P-6,7-3H. Organ cultures were stoppered 1 hr. later. EXPERIMENT 3. Uterine explants were cultured for 24 hr. ; after changing the medium, 0.6 pg of chlormadinone acetate was added for another 24 hr. period. The medium was changed again, and 450,000 d.p.m. of tritiated estradiol was added, with the cultures stopped 1 hr. later. In the following experiments the actions of estradiol and chlormadinone acetate were compared by adding 0.3 PLg of unlabeled estradiol. ExPERrMENr 4. Uterine explants were cultured for 48 hr. and transferred to another Petri dish containing 10 ml. of TC 199 and 0.3 pg of unlabeled estradiol-17P; 450,000 d.p.m. of tritiated estradiol-17/? was added. Organ cultures were stopped 1 hr. later. EXPERIMENT 5. Uterine explants were cultured for 24 hr. ; after changing the medium, 0.3 pg of estradiol was added for 24 hr. The

medium was changed again, and 450,000 d.p.m. of tritiated estradiol was added, with the culture stopped 1 hr. later. Hypothalamus (Fig. 2). EXPERIMENT 6. After 48 hr. of organ culture, hypothalami explants were transferred to fresh TC 199 medium containing 300,000 d.p.m. of tritiated estradiol. The experiment was stopped 24 hr. later. EXPERIMENT 7. After 48 hr. of culture, hypothalamic explants were transferred to fresh TC 199 medium containing 0.8 pg of chlormadinone acetate and 300,000 d.p.m. of tritiated estradiol-17/?. Organ cultures were stopped 24 hr. later. EXPERIMENT 8. After 24 hr. of culture, 0.8 pg of chlormadinone acetate was added to fresh TC 199. The medium was changed, and the explantes were cultured for 24 hr. after adding 300,000 d.p.m. of labeled estradiol- 17p. Results

Chlormadinone acetate added simultaneously to tritiated estradiol inhibited the estradiol uptake significantly (p < 0.01). When added 24 hr. previously, the uptake increased (p < 0.01) in both uterus and hypothalamus. Unlabeled estradiol also diminished significantly the estradiol uptake when added simultaneously and increased its uptake when added prior to the labeled hormone (Tables I to III).

Volume Number

112 3

Chlormadinone

Table I. Estradiol

uptake by uterine in 10 ml. of TC 199

cultured

Experiment

Table III. Estradiol

explants

explants medium

Table II. Estradiol cultured

650 287 prior

1,106.8

t +

77 8

+ 117

uptake by uterine with unlabeled estradiol

No.

4-E2-sH 2 Ez simultaneously 5-E? prior to E$H

estradiol

uptake

433

uptake by hypothalamic in 10 ml. of TC 199 .-

(0.3 P.9) Experiment

cultured

on

Tissue

In all groups estradiol-17PH (450,000 d.p.m.) was added for 60 min. Experiment 1: Labeled &radio1 was added after 48 hr. organ culture. Experiment 2: Labeled e&radio1 and 0.6 pg of chlormadinone acetate was added after 48 hr. organ culture. Experiment 3: Chlormadinone acetate (0.6 fig) was added after 24 hr. organ culture, and 24 hr. later the tissue was washed and labeled estradiol was added. Mean + S.E. No. = 10 in each experiment. By the analysis of variance (Duncan’s test), all differences were significant (p < 0.01).

explants

action

Tissue (d.p.m./mg.)

No.

l-Control E*-sH 2--Simultaneous Es-sH + chlormadinone acetate 3-Chlormadinone acetate to E:-sH

acetate

Tissue (d.p.m./mg.) 239.42 8 783 t63

Experiment 1: Labeled estradiol (450,ooO d.p.m.) and unlabeled estradiol were added. After a 48 hr. organ culture, unlabeled estradiol was added, and, 24 hr. later, the tissue was washed, and labeled &radio1 (450,000 d.p.m.) was added. Mean f S.E. No. = 10 in each experiment. Student’s t test: The diierence was significant (p < 0.01).

Comment It was previously demonstrated that chlormadinone acetate inhibited significantly the in vivo uptake by the uterus and the anterior hypophysis but not by the hypothalamus. In this work, the in vitro effect of chlormadinone acetate was studied in organ culture. In the uterine experiments, chlormadinone acetate, both in vivo and in vitro, inhibited the estradiol uptake significantly when added simultaneously, suggesting that this drug can act as an antiestrogen by competing with estradiol for the binding sites.

Experiment

(d.&m./mg.)

No.

6-Control Ez-sH ‘I-Simultaneous E?-sH + chlormadinone acetate 8-Chlormadinone acetate to Ez-sH

53.3 t 13.1 + prior

427.7

3.8 3.6

f 18

In all groups, labeled estradiol-178 (300,ooO d.p.m.) was added for 24 hr. Experiment 6: Labeled estradiol was added after a 48 hr. organ culture. Experiment 7: After a 48 hr. organ culture, chlormadinone acetate (0.8 pg) and labeled estradiol were added for 24 hr. Experiment 8: After 24 hr. organ culture, chlormadinone acetate (0.8 &g) was added, and, 24 hr. later, the tissue was washed and cultured for 24 hr. in fresh medium containing labeled estradiol. Mean i: S.E. No. = 10 in each group. By the analysis of variance (Duncan’s test), all differences were significant (p < 0.01).

The disagreement between the in vivo and the in vitro effects of chlormadinone acetate on the hypothalamic estradiol uptake is not clearly understood. It is possible that the in vivo results reflect the short action of a single injection; and those of the in vitro experiment, the effect of a prolonged exposure to chlormadinone acetate. When chlormadinone acetate was added prior to estradiol, it increased the uptake significantly (p < 0.01) by both hypothalamic and uterine explants. These results could indicate an estrogenic-like effect of chlormadinone acetate, probably by increasing the binding sites. When unlabeled estradiol was added prior to labeled estradiol, it increased the uptake, similarly to the action of chlormadinone acetate, and when added simultaneously, it inhibited the uptake. The similar behavior on the estradiol uptake of chlormadinone acetate and unlabeled estradiol is a further hint to the estrogen-like action of chlormadinone acetate.

REFERENCES

1. 2.

Rosner, and De Maqueo, Martinez

J. M., Macome, J. C., Denari, J. H., Carli, N.: Acta Endocrinol. In press. M., Perez Vega, E., Goldzieher, J., Manautou, J., and Rude], H. W.:

3. 4.

AK J. OBSTET. GYNECOL. 85: 427, 1963. Rosner, J. M., Ho&a, S., and Forsham, P. H.: Endocrinology 75: 299, 1964. Duncan, D. B.: Biometrics 2: 1, 1955.