Mechanisms of hormonal and antihormonal action of contraceptive progestins at the molecular level

J. Steroid Biochem. Molec. BioL Vol.41, No. 3-8, pp. 479-485, 1992

0960-0760/92 $5.00+ 0.00 Copyright© 1992PergamonPressplc

Printed in Great Britain. All rightsreserved

MECHANISMS ACTION

OF OF

HORMONAL

CONTRACEPTIVE

THE

MOLECULAR

AND

ANTIHORMONAL

PROGESTINS

AT

LEVEL

GREGORIOPI~REZ-PALACIOS,*MARCO A. CERB6N, ANA M. PASAPERA,J. IVONECASTRO, JUANAENRIQUEZ,FELIPEVILCHIS,GUSTAVOA. GARCiA,GABRIELAMORALtand ANA E. LEMUS Departments of Reproductive Biology,National Institute of Nutrition S. Zubri~.n and UAM-Iztapalapa, School of Chemistry, National University of M~xico, IMSS, and ENEP-Zaragoza, UNAM, M~xicoCity, M~xico Summary--19-Nor synthetic progestins undergo extensive metabolism at the target cells. The resulting metabolic conversion products interact with putative steroid receptors within the cells, and through those interactions, they may exert either agonistic, synergistic and antagonistic hormonal effects. Studies conducted in our laboratories have disclosed that norethisterone (NET) and D-(1) norgestrel (LNG), two widely used contraceptive progestins, are biotransformed to several A-ring reduced (dihydro and tetrahydro) derivatives. The resulting metabolites 5~t-dihydro NET (5~t-NET) and 5~t-dihydro LNG bind with relative high affinity to the progesterone and androgen receptors. To gain insight into the underlying molecular events mediating the mode of action of NET and its neutral metabolites, we have examined the expression of their biological effects at target organs by using the rabbit uterogiobin gone model and the fl-glucuronidase activity of the mouse kidney. The results of a series of experiments seem to indicate that the enzyme-mediated formation of the 5~ (trans A/B ring junction) NET derivative results in a significant diminution of its progestational and androgenic potencies. Furthermore, 5~t-NET acquire a potent anti-progestational/ contragestational effect as assessed in the female rabbit. These results demonstrated that 5or-reduction of 19-nor progestins exerts a paradoxical effect, at least in terms of their hormone-like effects. The overall data are in line with the concept that metabolism of synthetic progestins at hormone-sensitive organs modulates their mechanisms of action.

INTRODUCTION

considered to be under strict control of estrogens [5-10]. These findings provided a plausible The observation that norethisterone (170texplanation for the estrogen agonist effects of ethinyl- 17fl-hydroxy-4-estren-3-one) (NET), this 19-nor progestin which neither binds to ER but not progesterone, significantly suppresses nor undergoes aromatization in vivo. serum LH levels in postmenopausal This report summarizes the results of a varwomen [1, 2] and in non-estrogen primed casiety of studies aimed at assessing the molecular trated female rats [3, 4], indicated that this synthetic progestin could be recognized by events involved in the expression of hormonal and anti-hormonal effects of synthetic intracellular steroid binding sites other than the progestins. Particular focus is given to the interestrogen-dependent progesterone receptor (PR). action of N E T and its neutral metabolites with Further studies from our laboratories demonthe PR and androgen receptor (AR) in the strated that some hormone-like effects of N E T rabbit endometrium and the mouse kidney reare mediated by its A-ring reduced metabolic spectively, as well as to the biological activities conversion products. Thus, the 3fl,5~t-tetrawhich derive from those interactions. Progestahydro N E T derivative specifically binds with high affinity to the estrogen receptor (ER) and tional agonistic and antagonistic effects of N E T is able to initiate early cellular events that are and its derivatives were evaluated by using rabbit endometrial uteroglobin (Utg), a progesterone-regulated protein, and its specific Proceedings of the lOth International Symposium of the m R N A as molecular markers. Advantage was Journal of Steroid Biochemistry and Molecular Biology, Recent Advances in Steroid Biochemistry and Molecular taken from the fact that the Utg m R N A has Biology, Paris, France, 26-29 May 1991. been characterized and translated in vitro [11], *To whom all correspondence should be addressed at: National Institute of Nutrition S. Zubir~in, Calle Vasco and that its complementary and genomic D N A s de Quiroga No. 15, 14000 Mrxieo, D.F., Mrxico. have been cloned [12]. The androgen related 479

480

GREGORIOPI~REZ-PALACIOSet al.

effects were examined by the induction of fl-glucuronidase activity in the mouse kidney. The potency of these 19-nor steroids to restore copulatory behavior in long-term castrated male rats was used as an additional suitable procedure to screen their hormonal effects.

INTERACTION OF NORETHISTERONE AND ITS METABOLITES WITH PUTATIVE STEROID RECEPTORS IN TARGET ORGANS

It has been well established that most of the biological effects of natural and synthetic steroid hormones at the target organ level are exerted via intracellular steroid receptors [13, 14]. Accordingly, before evaluating the hormone- or antihormone-like effects of NET and its metabolites, we studied their relative binding affinities to the PR and AR using displacement analysis. To evaluate the interaction of NET and its derivatives with the rabbit uterine PR, appropriate cytosol preparations obtained from estradiol benzoate-primed (5 #g/day/4 days) immature New Zealand White female animals were incubated overnight with 1 nM [3H]ORG 2058 at 4°C in the presence or absence of increasing concentrations of radioinert steroid competitors including ORG 2058, progesterone, NET, 5~tdihydro NET (50t-NET), 3fl,5~t-tetrahydro NET (3fl,Sct-NET), levonorgestrel (LNG), RU 486 and estradiol. At the end of incubation period, bound and free fractions were separated by the addition of a dextran-coated charcoal suspenSTEREOSPECIFICITY

OF

sion. The results were expressed as the percentage of [3H]ORG 2058 specific binding. As depicted in Fig. 1, ORG 2058, progesterone, LNG, RU 486, NET and 50t-NET were efficient competitors for progesterone specific binding sites in the rabbit uterus, while 3fl,5~-NET and estradiol did not interact at all with the PR. The relative high binding affinity to the PR of unchanged NET and its 5g-reduced dihydro derivative founded in this study confirms and extends previous results in the rat uterus. Indeed, Chfivez et al. [6] demonstrated that 5~tNET is able to displace [3H]ORG 2058 from the rat uterine PR, though with a low relative binding affinity to that of unmodified NET. These results are interpreted as demonstrating that 5or-reduction of NET does not preclude its interaction with the uterine PR. To examine the interaction of NET and its A-ring reduced metabolites with the mouse kidney AR we also used the displacement analysis technique [15]. Using [3H]mibolerone as radioligand, the presence of a high affinity (Kd: 0.91 nM) androgen binding site was demonstrated in kidney cytosol preparations from castrated Balb/c mice, as shown in Fig. 2. The capability of NET and its derivatives to displace bound [3H]mibolerone from the mouse kidney AR is shown in Fig. 3. As can be noticed, 5or-NET was a competitor for AR with a potency higher than that exhibited by unmodified NET, while 3fl,5~-NET and its 3~-isomeric alcohol (3ct,5~t-NET) were ineffective. These findings were almost identical to those reported by Chav6z et al. [6] in the rat ventral prostate. PROGESTERONE

RECEPTOR

• x

~ o

40

x

20

0

0

10

COMPETITORS

100

(nM)

Fig. 1. Competition of radioinert natural and synthetic steroids for rabbit uterine cytosol progesterone binding sites. Progesterone receptors (PR) were labeled with l nM [3H]ORO 2058. Increasing concentrations of non-labeled ORG 2058 (O), progesterone ('k), northisterone (l'q), 5~t-NET ( x ), 3fl,5ct-NET (~)), levonorgestrel (~), 5e-LNG ( + ), RU 486 (*), and estradiol (E2) were added. [3H]ORG 2058 binding to PR in the absence of competitor was set at 100%. Results are expressed as the percentage of [3H]ORG 2058 specific binding. Each point represents the mean value of three experiments in triplicate.

481

Hormonal actions of 19-norprogestins 0.50.

•

0.40

O.6.

~

o.,o

o.o\

"

. /

0

o.,o/ O.IO

I MIBOLERONE ( t i M )

B

-~"

0.3-

0.2-

O.I

i

i

0.2

0.4

i

0.6

B

Fig. 2. Representative Scatchard plot and saturation analysis of specific androgen binding in the mouse kidney (BALB/c) cytosol. Cytosol samples were incubated in triplicate with [3H]mibolerone at increasing concentrations (1.0-15.0 nM), in the presence or absence of a 100-fold excess of radioinert mibolerone. Incubations were carried out overnight at 4°C. Specificbinding was calculated by substracting non-specific binding from total binding. The saturation curve is shown in the inset. The apparent affinity of the androgen receptor in the castrated mouse kidney is 0.91 nM. A-RING REDUCTION OF NORETHISTERONE ABOLISHES ITS PROGESTATIONAL POTENCY

The o b s e r v a t i o n that 5~-reduction of the N E T molecule increases its affinity for the A R is in parallel with a n u m b e r of reports indicating that 50t-reduction of testosterone enhances its binding affinity for A R [16, 17].

T o ascertain whether enzyme-mediated structural modifications of N E T are able to modify

I00"

,3a-NET

3~-NET

80-

~e 60O U.I

~ 40ko _J O

i4- zo-

NET

I

5

i~

I

ao

t

I) DHT ~MO

~'o

i~o

STEROID CONCENTRATION (nM)

Fig. 3. Competition of noretlgsterone and its metabolites with [3H]miboleronefor binding to the cytosol androgen receptor of castrated mice kidney (BALB/c). Aliquots of kidney cytosol were incubated overnight with 1 nM [3H]miboleroneat 4°C, in the presence or absence of graded concentrations of the following non-labeled steroids: Mibolerone (MB), 5a-dihydrotestosterone (DHT), norethisterone (NET), 5~-dihydronorethisterone (5~-NET), 3fl,5~,-tetrahydronorethisterone(3fl-NET) and, 3~,5~-tetrahydronorethisterone (3e-NET). Bound and free fractions were separated by the addition of a dextrancoated charcoal suspension. The results are expressed as the percentage of [3H]miboleronespecificbinding.

482

GgEC, OR]O PI~REZ-PALACIO$ et al.

its progestational potency, a series of experiments assessing the Utg gene expression products after the administration of N E T and its A-ring reduced metabolites were conducted in prepubertal female rabbits. The rabbit Utg gene model was selected because data from a number of laboratories have demonstrated that progesterone and synthetic progestins specifically regulate the synthesis of this low molecular weight secretory protein at the endometrial level [18-20]. The s.c. administration of N E T to prepubertal female rabbits was followed by an increase of the Utg content in uterine flushings as analyzed in SDS-polyacrilamide gel electrophoresis. The augmentation of the uterine Utg content induced by N E T was similar to that observed in the progesterone-treated rabbits, whereas the A-ring reduction of N E T abolished the potency of Utg induction; in fact, 5or-NET 3fl,5ct-NET and estradiol were almost ineffective [21]. Furthermore, to quantitatively assess the effects of N E T and its metabolites upon the Utg content in uterine flushings, samples were submitted to a specific double antibody radioimmunoassay as described by Mayol and Longenecker[22], with minor modifications. As shown in Table 1, the results obtained confirmed that: (i) N E T exhibits a progestational activity similar to that of progesterone, (ii) reduction at the 5 position and later at the 3 position of the N E T molecule results in a significant diminution of its capability to induce uteroglobin synthesis, and (iii) estradiol exhibits very little, if any, progestational potency. To investigate the mechanisms involved in the NET-induced Utg rise in the rabbit uterine flushings, the uterine content of specific Utg m R N A was determined. Total uterine RNA from steroid-treated and control (oil-treated) animals was submitted to molecular hybridization with [ct-32P]d-ATP Utg cDNA. The identical quantity of total RNA used in the experiments was assessed by optical density, staining with ethidium bromide and methylen

Table I. Radioimmunoassayable uteroglobin content in uterine flushings of steroid-treated prepubertal rabbits Treatments Progesterone Norethisterone 5ct Dihydro-NET 3fl,5ct-NET Estradiol Oil (control)

Dose*' (mg/kg b.w./d)

Animals (n)

Uteroglobin (/~g/mg protein)

1.0 1.0 1.0 0.5 0.01 --

6 6 6 6 6 6

397.5 + 25.0 456.3 + 55.5 104.0 -I-_8.9 39.2 + 1.2 60.1 + 5.0 Non-detectable

aSteroids were s.c. administered at the dose indicated for five consecutive days (modified after Cerb6n et al., 1990 [21]).

125

2

P4

NET

6 ~ N E T 3B6 o~NET

E2

OiL

STEROID TREATMENTS

Fig. 4. Effects o f norethisterone ( N E T ) and its A-ring reduced metabolites (5et-NET, 3fl,5~t-NET) u p o n the

uteroglobin mRNA content in the uterus of prepubertal rabbits. The figure shows the densitometric analysis of a typical Northern blot autoradiography. Animals treated with progesterone,estradiol, and oil were used as controls.

blue throughout Northern blot analysis. The results as assessed by densitometric analysis of a typical Northern blot autoradiography are shown in Fig. 4. NET administration induced a significant increase in the uterine Utg m R N A content as compared with the oil-treated animals. This increase was similar to that observed in progesterone-treated rabbits. A similar increase of uterine Utg m R N A was noticed although to a higher extent in the pregnant rabbit uterus[21]. On the contrary, administration of 5~-NET, 3fl,5ct-NET, and estradiol resulted in a very limited increase of uterine Utg m R N A as compared with the oil-treated animals. Whether the increased endometrial content of Utg m R N A observed following N E T administration is due to an increase of gene transcription rate or to a messenger stabilization effect can not yet be drawn; however, since hormonal action of progesterone is exerted at the transcriptional level [23], it seems plausible that N E T may act through a similar mechanism. The striking observation that 50t-reduction of NET resulted in a significant diminution of its capability to induce progesterone-regulated uterine Utg and its mRNA was not in line with the previous demonstration that 5or-NET binds to the PR with an adequate relative affinity, and prompted us to investigate whether the 5or-reduced metabolite of NET may exert a progesterone synergistic or even an antagonistic effect. When 5~-NET was simultaneously administered with progesterone at equimolar doses to prepubertal rabbits, it abolished the progesterone-induced increase of uterine Utg[24] Although antiprogestational activity of 5~t-NET was noticed at all doses

Hormonal actions of 19-norprogestins employed, doses above I mg/kg of body weight were sufficient to completely abolish Utg content in uterine flushings. From these studies it was evident that 5or-NET possesses a potent antiprogestational activity. When 5~-NET was compared with the synthetic antiprogestin RU 486, the capability to suppress Utg production was similar at least at the dose of 1 mg/kg b.w. [25]. These data strongly suggest that 5orreduction of the NET molecule not only results in a dramatic diminution of its progestational intrinsic potency but also in the acquisition of an anti-hormonal effect. The precise mechanisms of the antiprogestational activity of 50t-NET still requires additional experimental work, though it can be advanced that its mode of action is different from that observed with RU 486, at least in the prepubertal rabbit uterus (data not shown). Interestingly, the findings correlate well with the anti-implantation activity of 5or-NET in the rat as reported by Reel et al. [26]. The correlation between the effects of 5~-NET upon the rabbit Utg gene expression and its anti-implantation activity is now under study at our laboratories. BIOLOGICAL ACTIONS OF NORETHISTERONE AND ITS METABOLITES MEDIATED VIA THE ANDROGEN RECEPTOR: PARADOXICAL EFFECTS OF 5~,-REDUCTION

To assess the androgen-like effects of NET and its derivatives, the fl-glucuronidase activity of the mouse kidney was used as a biological marker. Although this enzyme comprises less than 1% of the total kidney proteins when fully induced, its assay in vitro has been well characterized and it may serve as a suitable tool for the study of androgen action [27]. As described above, we have demonstrated the presence of a specific, high affinity androgen binding protein in BALB/c mice kidney cytosols (Fig. 2), as well as the relative binding affinities of NET and its metabolites to the mice kidney androgen receptors (Fig. 4). To evaluate if the increased affinity of 5orNET for the AR correlated with the expression of its androgen-like effect, we have examined in a comparative manner the effects of NET and 5~-NET upon the growth of ventral prostate and seminal vesicles, as well as upon the fl-glucuronidase activity of the mouse kidney. The results demonstrated that NET possesses an intrinsic androgenic potency and that 5or-NET induced a significantly diminished androgen-

483

like effect, an observation that markedly differs from that found with testosterone and its natural occurring 5~-dihydro derivative (data not shown). Interestingly, depending upon the dose employed, 5or-NET may exert a synergist or antagonist androgenic effect when administered simultaneously with dihydrotestosterone. These results raise the question as to why 5~t-reduction of NET induced a significant diminution of its androgenic potency, even that 50t-NET exhibits a relatively high affinity for the AR. Whether the lack of the C-19 methyl group or the presence of the 17ct-ethynyl moiety of NET may explain this paradoxical effect observed after its A-ring reduction, still remains to be ascertained. Studies employing 19-norethisterone and 17ct-ethyniltestosterone are ongoing at our laboratories.

BEHAVIORAL EFFECTS OF NORETHISTERONE AND ITS DERIVATIVES IN RODENTS

In an attempt to correlate the hormone-like effects of NET and its A-ring reduced derivatives exerted at peripheral target organs with those occurring at the central nervous system, we decided to investigate their effectiveness in the activation of the neural substrate responsible for the expression of masculine sexual behavior in the castrated male rat. Advantage was taken from the fact that male sexual behavior in rodents in under strict steroid hormone regulation [28, 29]. Full restoration of ejaculation in the castrated animals was achieved when NET was simultaneously administered with estradiol, indicating the androgen-like intrinsic potency of this progestin [30]. The most potent compound to induce male sexual behavior when given simultaneously with dihydrotestosterone was 3fl,5~-NET, demonstrating its estrogen agonist effect. When administered alone, NET exerted the most potent effect on male behavior, followed by 5or-NET, while the tetrahydro NET derivatives were ineffective. The observation that NET alone restored male sexual activity at a level identical to that induced by testosterone, demonstrated an androgenic-estrogenic activity of NET exerted through its intrinsic androgenic effect and the estrogenic effect of its tetrahydro derivatives. These data are consistent with the notion that pharmacological activation of mating requires a potent androgenic effect with a concomitant estrogen-like action, and NET fulfills both hormonal requirements.

484

GREGORIO PI~REZ-PALACIOSet al. CONCLUSIONS

The data presented herein indicate that the metabolism of synthetic progestins and the further interactions of their bioconversion products with putative steroid receptors, within the target cells, modulate and direct the expression of their biological activities. The enzyme-mediated structural modifications of norethisterone, presented as a model, induce profound changes in the hormone-like effects of this 19-nor synthetic molecule. The use of appropriate molecular models will contribute to a better understanding of the mode of action of these compounds, widely used in a number of contraceptive formulations. Additional strategies are required to disclose the nature of the underlying mechanisms involved in the expression of the hormonal and antihormonal effects of these steroids and their derivatives. Acknowledgements--This work was supported by the Special Programme in Human Reproduction, W.H.O. (Geneva), The Rockefeller Foundation (New York) and the University Program on Health Research (PUIS) of the National Autonomous University of Mexico. The authors wish to thank Professor E. Milgrrm (Bicetre, France) for the provision of bio-reagents. The assistance of Ms B. Alarc6n in preparing the manuscript is acknowledged.

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