Competitive inhibition by Danazol of oestradiol binding to rabbit and ovine uterine oestradiol receptor

J~mrnul of Sfemid Biochemisrry Vol. 13. pp. 1325 to 1329 Perpamon Press Ltd 1980 Prmted in Great Britain

COMPETITIVE INHIBITION BY DANAZOL OF OESTRADIOL BINDING TO RABBIT AND OVINE UTERINE OESTRADIOL RECEPTOR D. B. COOK and I. GIBB* Department of Clinical Biochemistry, University of Newcastle upon Tyne. Royal Victoria Infirmary, Newcastle upon Tyne, NEI 4LP, England (Recriwd

IO March

1980)

SUMMARY

The antigonadotrophin, Danazol, binding to soluble uterine receptor

was investigated macromolecules.

using rabbit and lamb uterine preparations

as a possible competitive inhibitor of oestradiol A simple in citro competitive protein binding assay

was employed to demonstrate the extent of this inhibition.

The drug was shown to be capable of competing with tritium-labelled oestradiol for receptor binding when present in excess of oestradiol to an extent which has been reported in patients on high oral Danazol dosage. These findings were used as a basis from which to consider target-tissue binding as a mode of action adjunctive to the gonadotrophin suppressing activity of the drug.

INTRODUCHON

U.K. (Code TRK 322) and is subsequently referred to as [3H]-oestradiol. Absolute ethanol, A.R. quality Danazol (17a-pregn-4-en-2O-yne-(2,3d)isoxasol-17-01) was obtained from James Burroughs Limited (Lonis a derivative of the first oral progestagen, 17or-ethidon) and A.R. benzene from B.D.H. Limited. /I-mernyl-testosterone. Various studies have reported that it captoethanol was purchased from Sigma. suppresses pituitary gonadotrophins in males [I] and females [2] without exhibiting progestational or oesTris-HCI buffer, 0.01 M, pH 8 trogenic activity [3,4]. Use of the drug is thus advo1.2551 g Trizma base (Sigma) was dissolved in apcated in the management of severe endometriosis [S], proximately 900 ml distilled water along with 0.3722 g the suppression of endometrial function being supposA.R. disodium-EDTA (BDH) and 85.58 g A.R. suedly secondary to the inhibition of gonadotrophin secrose (BDH). The pH was adjusted to 8.0 with 40”,, cretion [6]. Decreased levels of FSH and oestradiol HCI (BDH), IOg.bovine serum albumin (Fraction V) during chronic administration were reported [S] and (BDH) was added and the volume made to I litre the mid-cycle surges of LH and FSH have been generwith distilled water; 0.1% sodium azide (BDH) was ally prevented [S, 71. included as preservative. However, other studies have failed to demonstrate Dextran-churcoal suspension suppression of gonadotrophin levels in serum [8]. Indeed, it has been suggested by some workers that 0.005 g Dextran T70 (Pharmacia) was dissolved in endometrial atrophy in danazol-treated patients, in 2OOml Tris-HCI buffer at room temperature. To this the absence of suppression, indicates competition was added 0.5 g Norit GSX (Hopkin and Williams) between the drug and oestrogens at the target tissue and the suspension was stirred for at least 30 min to level [9. IO]. ensure effective coating of the charcoal. The dextranThe present work reports investigations of the effect charcoal was stored at 4’C and was restirred prior to of Danazol on binding of tritiated-oestradiol to rabbit use as well as while being dispensed. and lamb uterine cytosol oestrogen receptor, using Scintillator solution the Saturation Analysis Principle. 5 g PPO (Intertechnique Limited) and 100 mg POPOP (Intertechnique Limited) were dissolved in I EXPERIMENTAL litre of A.R. toluene (BDH). The solution was thorSteroids were obtained from Sigma (London) oughly mixed with 500ml Triton X-100 (Rohm and Chemical Company Limited with the exception of Hass Limited) and was both stored in and dispensed Danazol which was a gift from Sterling-Winthrop from an amber glass “Zipette” (Jencons Scientific Laboratories Limited. 2,4,6,7 (n) [3H]-oestradiol was Limited). obtained from the Radiochemical Centre. Amersham, Preparation of’ uterine cytosol receptor protein * Present address: Department of Clinical Biochemistry, Freeman Hospital, Newcastle upon Tyne. 1325

(1) Rabbit. Four groups each of three New Zealand White’ prepubertal rabbits were used. The animals

1325

D. 8.

COOK

were killed by cervical disiocation and their uteri excised as quickly as possible. The organs were dissected free of fat, weighed, minced and homogenised in 10 times V/W Tris-WC1 buffer at ice bath temperature. The homogenates were centrifuged at 4’C and lOO,ooOg for I I-I.The supernatant cytosols containing the receptor macromolecules were preserved with mercaptoethanol (final concentration 0. I M), divided into aliquots and stored in polystyrene tubes at - 7r)“C. (2) Ocitre. The uterus of a freshly killed Iamb was dissected to expose the endometriai surfaces which were scraped off, weighed and homogenised in ice cold Tris-HCl buffer (three times V/W) as described above. Preparation of steroids for assessment of competitive bi~d~i~~e&cts Stock solutions of danazol, oestradial, oestriol, oestrone, 17~OH progesterone, cortisol, 1I-deoxycorticosterone and cholesterol were prepared in A.R. ethanol and stored at 4°C. A stock solution of oestrone-3methyl ether was prepared in A.R. benzene and similarly stored. Less concentrated solutions were prepared by diluting the stocks in buffer such that the required amount of steroid to be added to each tube was contained in 0.1 ml.

and I. QBB

unlabelled oestradioi and (b) increasing amounts of [3H]-oestradiol alone, to establish that the labelling of the oestrogen had not significantly altered its binding affinity. Scatchard analyses of subsequent batches of cytosot preparations were also carried out (labelled and unlabetled oestradiol present) to establish the concentration of binding sites. Each batch was then diluted in buffer for use in such a manner as to maintain the constancy of the binding material per tube.

incubation 5f steroids with uterine cytosoi was carried out in a reaction mixture consisting of 20pg [3HJ-oestradiol in 0.1 ml buffer, unlabelled steroid in a similar buffer volume and 0.1 ml af appropriately diluted cytosol preparation. The incubation, phase separation and scintillation measurements were performed as described above. Results were plotted as percentage bound oestradioi against the logarithm of the steroid concentration. With each batch of tubes, non-specific binding tubes containing buffer in place of cytosol were assayed. Total activity tubes contained 20pg [3Hf-oestradiol in 0.8 mt buffer and these were not charcoal treated. All tubes were assayed in triplicate.

RESULTS

Optimisutiorr of the binding system The optimum receptor protein content for use in the competitive binding system was established for the initial batch of cytosol as described by Mester[ I I], Two series of glass tubes (12 x 75 Gallenkamp) were prepared, one containing 20 pg [3H]-oestradiol in buffer and the second 150 pg unlabelled oestradiol plus 2Opg [3H]-oestradiol in buffer. Varying volumes of uterine cytosol were added to each series of tubes in a total volume of0.3 ml. After 30 min incubation at 30°C. the tubes were chilled in an ice bath for 5 min. 0.5 ml Dextran-charcoal suspension was added and the contents vortex mixed and allowed to stand in the ice bath for 5 min to remove the free oestradiot. The tubes were then centrifuged at 15OOg for 5 min in a Mistral 6L centrifuge at 4°C The supernatants containing the bound oestradiol were transferred to 2.5 ml scintillator solution, gelled with 1.25 ml distilled water (to improve stability) and counted for 5 min in polythene minivials in an ICN/ Tracerlab Corumat 2700 Liquid Scintillation Counter. Results uere plotted as percentage bound [3HJ-oestradiol against volume of cytosol. The optimal volume of cytosol was selected as that gibing the greatest displacement between the binding curves in the presence and absence of unlabelled oestradiol. Using this optimal volume of cytosol, the concentration of the binding sites in the original cytosol preparation and the oestrogen receptor binding atTinity was established by Scatchard Analysis [ 121. This was performed on assays which contained (a) a fixed amount of C3HJ-oestradiol and increasing amounts of

The graph derived by plotting percentage bound [~~I-oestradiol against cytosol volume in the initial optimisation procedure is presented in Fig. 1. The volume of cytosol selected as containing a satisfactory amount of the receptor protein is seen to be 50~1. This volume of supernatant was found by Scatchard Analysis to contain 88 fmol of binding sites. Figure 2 shows that the binding sites were of a singie high atEnity type (tinear Scatchard ptotsf and that tritiation of the oestradiol had not materially altered that affinity. Similar evaluations were performed on subsequent batches of rabbit and ovine cytosols in order 80 r 60-

Fig+ I. Effect on binding of increasing rabbit eytosol votume in the aresence of (A) 20oa lWl-oestradior only per tube, and iB) 20pg E3’~j-oestradioi.-and 15Opg u& labelled oestradiol per tube. Maximal curve separation is at 50 ~1 of cytosol per assay tube. Each point is the mean of triplicate determinations.

Danazol

and oestradiol

receptor

1327

binding

B/F B/B,

P9 I

1

0

0.10

0.05

0.15

0.20

pmol

20

B oestradiolltube

Fig. 2. Scatchard plots using the original rabbit cytosol preparation (O---O) with labelled oestradiol only binding sites = 0.195 = 1.66 x 10g I mol-t, W ~ISOEiP,i0” pmol/tube), and M with both labelled and unlabelled oestradiol present (K.I.OCi,,,iOn= 1.85 x lo9 I mol-‘, binding sites = 0.165 pmol/tube).

to standard& the amount of binding material in the assays and the characteristics of those batches are shown in Table 1. Competitive

binding

t

00’

Fig. 3. Binding competition between labelled oestradiol and unlabelled steroids in rabbit uterine cytosol preparations. (A-A, oestradiol; -0, oestrone: 04. oestriol; M, Danazol: A-A. oestrone-3-methyl ether; A-----A, cholesterol; O-----O 1710H progesterone; m---1. cortisol; O-----O, I I-deoycorticosterone), The data points are means of replicate assays using four separate batches of rabbit cytosol preparations.

I. Scatchard

data of uterine

80 -

BIB,

60 -

40 -

20 -

000

8 log pg steroid added

Fig. 4. Binding competition between labelled oestradiol and unlabelled steroids in an ovine uterine cytosol preparation. Symbols as in Fig. 3. The data points arc the means of replicate assays using a single batch of ovine cytosol preparation.

oestradiol

receptor

preparations

Binding

Ovine

I 2 3 4

8

loo-

Table

Rabbit Rabbit Rabbit Rabbit

6

log pg steroid added

assays

Binding inhibition curves for the various steroids are shown in Fig. 3 (rabbit cytosol) and Fig. 4 (ovine cytosol) and are seen to be virtually identical. The curve for oestradiol itself represents a standard calibration curve in a conventional saturation assay system for oestradiol using uterine receptor. The sensitivity of the assay was 4.7 pg and a paired difference estimate of precision [ 131 gave a coefficient of variation of 4.8% with a mean of 58.2pg oestradiol per tube. Recoveries of added oestradiol were 9(rlOS% over the (r150 pg/tube calibration range. The curves for the other steroids represent cross-reaction in such a system. The parallel natures of the binding curves for unlabelled oestradiol, oestriol and oestrone in the presence of 20 pg [3H]-oestradiol/0.3 ml incubation volume and a standard amount of binding protein permit the calculation of overall cross-reactivities for these compounds (Table 2). No significant cross-reactions were demonstrable with either receptor species when cholesterol, l7rOH progesterone, I I -deoxycorticosterone or cortisol were included in the assay

Cytosol

4

2

batch

Association constant (x IO9 litre mol-‘)

(pmol/tube)

capacity (pmol/mg wet tissue)

I .82 I .47 1.85 2.34

0.176 0.183 0.145 0.194

0.086 0.064 0.084 0.045

0.57

0.486

0.049

D. B. COOKand 1. GI~B

1328 Table 2. Oestrogen

Oestrogen Oestradiol Oestrone Oestriol

cross reactivities with uterine receptor preparations Receptor Rabbit uterus (““J 100 71 45

oestradiol

source Ovine uterus (“0)

I00 45 3’

It has been shown that on 200 mg Danazol per day (100 mg b.d.) and 400 mg per day (200mg b.d.) mean plasma levels of 341 and 921 pg/l respectively were achieved within 14 days [ 171. However, considerably higher dose schedules have been used. Greenblatt et ~/.[6] found that 600-8OOmg per day were required to achieve an antigonadotrophic effect. Sherins et u/.[ I] showed that testosterone and androstenedione suppression in males was dose-related and used 600 mg per day over 11 weeks. Dosages of 800 mg per day have also been given [Z] while Lind and Cook[8] reported a patient on IOOOmg per day for 1 year and another on 600mg per day for 3 months. Though symptomatic relief was achieved, in neither of these two patients were gonadotrophins suppressed. Indeed. they were actually elevated but gonadotrophins were not estimated serially throughout the cycle to deterDISCUSSION mine whether or not the mid-cycle peaks were present. Such high dose schedules as described can Korenman [ 14. 151 first demonstrated binding of thus apparently give rise to equilibrium plasma levels oestradiol to uterine cytosol receptors and also of danazol of the order of 1000 pg/l. If one considers a showed binding of the synthetic oestrogen, diethylstilplasma oestradiol level of lOOng/l which is well boestrol, to be greater than that of oestradiol itself. within even the mid-cycle peak range at the point of Shutt[ 161 also described cross-reactivity of oestrone time most relevant to danazol’s therapeutic action, and oestriol similar to that shown here. The present work shows no binding of non-oestrogenic steroids as and a plasma danazol concentration of at least 1000 pg/l, the 104-fold oestradiol/drug relationship is control substances. but danazol and oestrone-3seen to be achievable in vitv. methyl ether both showed some degree of competition Oestradiol and danazol both diffuse passively into with [3H]-oestradiol. the cell where they come in contact with the cytosolic Wood YI u/.[5] suggested that the mode of action of oestradiol receptor. Since no active transport process Danazol was most probably not mediated via compeis involved, the rates of diffusion are largely contition with the oestradiol receptor present in the cytotrolled by concentration gradients. Thus the extrasol of uterine tissue. These authors, however, only cellular oestradiol/danazol concentration relationinvestigated amounts of danazol up to 500ng per ships are pertinent even though the receptors being tube. In our investigations, we have increased the conconsidered are located within the cells. centrations of the drug present in the assay. The It thus seems possible that at least part of the mode paper of Wood et u/.[S] demonstrates approximately 7.5”” decrease of the Bound [3H]-oestradiol in the of action of danazol may be to competitively inhibit oestradiol binding at target tissue receptor level. This presence of 5OOng Danazol using a similar rabbit cytosol preparation. This report shows about ZOO,, is in addition to and not exclusive of other actions at competition at this level increasing to 507; at 1O’pg other parts of the hypothalamic-gonadal axis. per tube. Since each assay contained labelled oestradiol at a level of < 10’ pg/tube it is evident that sigAck,lowledye,~lrrlt.s~~We wish to thank Dr T. Lind for his encouragement with this work and the Newcastle upon nificant competition for receptor binding arises when Tyne Municipal Abattoir for assistance in collection of danazol is present at 104-fold excess over the oestrolamb uteri. gen. Although we must exercise caution in extrapolating i/l vitro findings to irk riro situations, the quesREFERENCES tion which must be asked is. can such a quantitative oestradiol/Danazol relationship exist in patients being 1. Sherins R. J., Candy H. M., Thorslund T. W. and Paulsen C. A.: Pituitary and testicular function studies. 1. treated with the drug’! system even up to IO’-fold excess over [-‘H]-oestradie). Both oestrone-3-methyl ether and, more importantly danazol were shown to cross-react with the labelled oestradiol. The dose-response relationships were shallower for the drug than for unlabelled oestradiol with both species of receptor. Since there is a lack of absolute parallelism with the unlabelled oestradiol curves it is not possible to assign an overall potency to the drug’s competitive effect [ 141. There is nonetheless a quantitatively significant competition in that. with [3H]-oestradiol present at 20pg per tube an approximately 104-fold excess of danazol causes a 20”~ reduction in oestrogen binding while a 106-fold excess results in SO”/, reduction.

Danazol

2.

3.

4.

5.

6.

and oestradiol

Experience with a new gonadal inhibitor, Danazol. J c/in. E&ocr. Metah. 32 (197 I ) 527-53 I, Andrews M. C. and Wentz A. C.: The effects of Danazol on gonadotrophins and steroid blood levels in normal and anovulatory women. Atn. J. Ohsret. Gyrtecol. 121 (1975) 817-823. Dmowski W. P., Scholer H., Mahesh V. B. and Greenblatt R. B.: Danazol-a synthetic steroid derivative with interesting physiological properties. Frrt. Strril. 22 (1971) 9-18. Potts B. 0.. Beyler A. L. and Schane H. P.: Pituitary gonadotrophin -inhlhltory activity of Danazoi. Frrr Steril. 22 (1974) 367-371. Wood G. P.. Wu C. H.. Flickinger G. L. and Mikhail G.: Hormonal changes associated with Danazol therapy. Ohstrt. Gynecol. 45 (1975) 302-304. Greenblatt R. B.. Dmowski W. P., Mahesh V. and Scholer H. F.: Clinical studies with an antigonadotrophin-Danazol. Fert. Sleril. 22 (1971) 102-l 12. Colle M. 0. and Greenblatt R. B.: Contraceptive properties of Danazol. J. Reprod. Med. 17 (1976) 98%lOl. Lind T. and Cook D. B.: How does Danazol work? Lu,,cut I(l976) 1401-1401. Ingerslev M.: Experience with Danazol in severe and extensive endometriosis. J. ht. Med. Res. 5 (Suppl. 3) (1977) 81-85.

receptor

binding

1329

J.. Fraser I. S.. Thorburn G. D. and Jenkin IO. Guillebaud G.: Endocrine effects of Danazol in menstruating women. J. inr. Med. Res. 5 (Suppl. 3) (1977) 57 ~66. D. M. and Kellie A. E.: Assay of I I. Mester J.. Robertson plasma oestradiol by competitive protein binding to uterine supernatant protein. J. sreroid Riochem. 2 (1971) l-10. G.: The attractions of proteins for small 12. Scatchard molecules and ions. Ann. N. Y. Acud. Sci. 51 (1949) 66664. comment. Bionlrrric> 13. Snedecor G.: Query 92-editorial 8 (1952) 85-86. S. G.: Comparative binding affinity of oes14. Korenmann trogens and its relation to oestrogenic potency. Steroids 13 (1969) 163-177. S. G.: Radioligand binding assay of specific IS. Korenman oestrogens using a soluble uterine macromolecule. J. c/in. Endocr. Merab. 28 (1968) 127-130. of oestradiol in plasma by 16. Shutt D. A.: Measurement competitive protein binding radioassay. Sleroidt 13 (1969) 69-82. J. G., Labross A.. Williams-Ross T.. Ross I7 Lloyd-Jones R. W., Edelson J. and Davison G.: Danazol plasma concentrations in man. J. int. Med. Res. 5 (Suppl. 3) (1977) 18-24.