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5α-Reduction of norethisterone enhances its binding affinity for androgen receptors but diminishes its androgenic potency
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J. Steroid Biochem. Molec. Biol. Vol. 60, No. 1-2, pp. 121-129, 1997 Copyright ~) 1997 Elsevier Science Ltd. All rights reserved Printed in Great Britain
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5 -Reduction of Norethisterone Enhances its Binding Affinity for Androgen Receptors but D i m i n i s h e s its Androgenic Potency A n a E . L e m u s , 1. J u a n a E n r i q u e z , 2 G u s t a v o A . G a r c i a , 3 Ivonne Grillasca and Gregorio P rez=Palacios e ~Department of Reproductive Biology, UAM-Iztapalapa, Mexico City, Mexico; :Department of Reproductive Biology, National Institute of Nutrition S. Zubirdzn, Mexico City, Mexico and ~Faculty of Chemistry, National University of Mkxico, Mexico City, Mexico
Norethisterone (NET), a 19-nor synthetic progestin, undergoes enzyme-mediated 5~-reduction and e x e r t s p o t e n t a n d r o g e n i c e f f e c t s in t a r g e t o r g a n s . T o i n v e s t i g a t e its m o d e o f a n d r o g e n i c a c t i o n we e x a m i n e d , in a c o m p a r a t i v e m a n n e r , t h e in vitro m e t a b o l i s m o f N E T a n d t e s t o s t e r o n e (T), as well as t h e b i n d i n g a f f i n i t i e s to a n d r o g e n r e c e p t o r s (AR) a n d t h e a n d r o g e n i c p o t e n c y o f N E T , T , a n d t h e i r 5 u - r e d u c e d d e r i v a t i v e s . B i o c o n v e r s i o n o f [ 3 H ] - N E T a n d [ 3 H ] - T w a s s t u d i e d in r a t p r o s t a t e h o m o g e n a t e s , A R b i n d i n g a f f i n i t y w a s a s s e s s e d in r a t v e n t r a l p r o s t a t e s u s i n g [ 3 H ] - m i b o l e r o n e as t h e r a d i o l i g a n d , a n d t h e a n d r o g e n i c p o t e n c y w a s e v a l u a t e d b y t h e i n c r e a s e o f ~ - g l u c u r o n i d a s e a c t i v i t y in t h e m o u s e k i d n e y , a n d b y t h e g r o w t h o f a c c e s s o r y s e x o r g a n s in c a s t r a t e d m a l e r a t s . T h e r e s u l t s d e m o n s t r a t e d t h a t 5.z-NET d i s p l a y e d a h i g h e r A R b i n d i n g a f f i n i t y b u t a s i g n i f i c a n t l y l o w e r a n d r o genic potency than unchanged NET. The bioconversion studies indicated that the metabolism of N E T w a s s i m i l a r to t h a t o f T , a l t h o u g h to a l e s s e r e x t e n t , t h u s r u l i n g o u t t h e p o s s i b i l i t y t h a t t h e s y n t h e t i c p r o g e s t i n m e t a b o l i z e s r a p i d l y i n t o less a c t i v e d e r i v a t i v e s . T o i n v e s t i g a t e t h e n a t u r e o f t h e paradoxical effect of 5u-reductlon upon the NET molecule, the interaction with AR and the androgenic potency of T, 19-nortestosterone (19norT), 17~-ethynyl testosterone (ET) and their 5u-reduced derivatives were examined. The results of AR binding studies revealed that 5~-reduction of T and ET significantly enhanced their affinities, and that the 5~-derivative of 19norT displayed a similar b i n d i n g a f f i n i t y to t h a t e x h i b i t e d b y 1 9 n o r T . I n t e r m s o f b i o l o g i c a l a c t i v i t y , t h e r e s u l t s s h o w e d t h a t 5~-reduction of T and 19norT significantly increased their androgenic potency, whereas 5~-reduction o f E T r e s u l t e d in a s i g n i f i c a n t d i m i n u t i o n o f its a n d r o g e n i c i t y in a m a n n e r s i m i l a r to t h a t o b s e r v e d w i t h t h e 5 ~ - r e d u c t i o n o f N E T . W h e n N E T a n d 1 9 n o r T w e r e s i m u l t a n e o u s l y a d m i n i s t e r e d w i t h 5udihydrotestosterone they exhibited a potent synandrogenic activity, an effect that was cancelled by t h e i r 5 ~ - r e d u c t i o n . ]Interestingly, E T d i s p l a y e d a n a n t i a n d r o g e n i c a c t i v i t y , a n e f f e c t t h a t w a s also s u p p r e s s e d b y its 5 ~ - r e d u c t i o n . T h e o v e r a l l r e s u l t s d e m o n s t r a t e d a d i s t i n c t i v e , p a r a d o x i c a l e f f e c t o f 5 ~ - r e d u c t i o n u p o n t h e N E T m o l e c u l e , w h i c h w a s d i f f e r e n t f r o m t h a t s e e n in n a t u r a l l y o c c u r r i n g a n d r o g e n s , a n d w h i c h s u g g e s t s t h a t t h e p r e s e n c e o f t h e 1 7 ~ - e t h y n y l g r o u p p l a y s a k e y r o l e in t h i s phenomenon. The data provided further evidence that the metabolism of synthetic contraceptive p r o g e s t i n s m o d u l a t e s t h e e x p r e s s i o n o f t h e i r hot~tiione-like a c t i o n s . © 1997 E l s e v i e r S c i e n c e L t d .
J. Steroid Biochem. Mo,!ec. Biol., Vol. 60, No. 1-2, pp. 121-129, 1997
Trivial names and abbreviations: norethisterone (NET), 17~-ethynyl-17fl-hydroxy-19-nor-4-androstene-3-one;5~-dihydroNET(5a-NET), 17~-ethynyl-17fl-hydroxy-19-nor-5c~-androstane-3-one; 19-nortestosterone (19norT), 17fl-hydroxy-19-nor-4-androstene-3=one; 5c~dihydrol 9norT (Set-19norT),17fl-hydroxy-19-nor-5a-androstane-3-one;17a-ethynyltestosterone(ET),17a-ethynyl-17fl-hydroxy-4-androstene-3-one; ET acetate (ETAc), 17a,ethynyl-3-oxo-4-androstene-17fl-ylacetate; 5~-dihydro ET (5a-ET), 17~-ethynyl-17fl-hydroxy-5~androstane-3-one; 5~-dihydro ET acetate(5~-ETAc),17a-ethynyl-3-oxo-5~-androstane-17fl-yl-acetate;mibolerone,7~,17~-dimethyllTflhydroxy-4-androstene-3-one; testosterone (T), 3fl-hydroxy-4-androstene-3-one; 3cq5~-androstanediol (3cq5a-diol), 5c~-androstane3~,17fl-diol; 3fl,5ct-androstanediol (3fl,5~-diol), 5c~-androstane-3fl,17fl-diol; 3a,5ct-tetrahydroNET (3ct,5~-NET), 17c~-ethynyl-3a,17fldihydroxy-19-nor-Sa-androstane-3-one; 3fl,5a-tetrahydroNET(3fl,5~-NET), 17~-ethynyl-3fl,17fl-dihydroxy-19-norS~-androstane-3-one. *Correspondence to A. E. Lemus, Department of Reproductive Biology, National Institute of Nutrition S. Zubirfin, Vasco de Quiroga No. 15, Tlalpan, M6xico, D.F., 14000 Mexico. Tel: +(52 5) 573 1160; Fax: +(52 5) 655 9859. Received 17 Jan. 1996; zccepted 12 Aug. 1996. 121
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A.E. Lemus et al. INTRODUCTION
Norethisterone ( N E T ) , a synthetic 19-nor, 17~-ethynyl testosterone derivative, has been widely used as a contraceptive progestin in a n u m b e r of pharmaceutical formulations [1--4]. In addition to its well-known progestational activity, N E T displays potent androgen-like effects following its administration in a n u m ber of m a m m a l i a n species [5-8]. Previous studies from this and other laboratories have demonstrated that N E T , in a similar way to naturally occurring androgens, undergoes extensive in vivo and in vitro enzyme-mediated 5~-reduction [9-11], and that 5~dihydro N E T (5~-NET) exhibits a higher binding affinity for the androgen receptor (AR) c o m p a r e d to its parent c o m p o u n d [12]. T o assess whether the androgenic effects of N E T are mediated by its 5~-metabolic conversion product, we examined, in a comparative manner, the effects of N E T and 5 ~ - N E T u p o n fl-glucuronidase activity in the mouse kidney, and u p o n accessory sex organ growth in the castrated male rat. T h e results d e m o n strated that 5~-reduction of the N E T molecule induced a significant diminution of its androgenic potency, thus indicating that the androgenic effects of N E T are not mediated by its A-ring reduction. In addition, in vitro metabolic studies indicated that N E T and T exhibit similar metabolic patterns in a target tissue. T h e s e striking observations p r o m p t e d us to investigate whether the absence of the C-19 or the presence of the 17~-ethynyl group in the N E T molecule are involved in the paradoxical effect of 5~-reduction. Accordingly, the A R binding affinity and the androgenic potency of testosterone (T), 19-nortestosterone (19norT), 17c~-ethynyltestosterone (ET) and their corresponding 5~-derivatives were studied. AR binding affinities were assessed by displacement analysis in rat ventral prostate preparations using [3H]-mibolerone as the radioligand. Additional experiments were carried out to determine the effects of 5c~-reduction upon the synergistic/antagonistic activities of N E T , 19norT and E T . Further impetus to conduct this study was provided by the observation that 5~-reduction of N E T results in a dramatic diminution of its androgen-like behavioural potency in long-term castrated mate rats [13].
MATERIALS AND METHODS
Radiochemical purity of [3H]-steroids was established by paper or by thin-layer chromatographic behaviour, and by repeated crystallizations to constant specific activity. Radioinert T, 5~-dihydrotestosterone ( D H T ) , 3~,5c~ and 3fl,5c~, androstanediols, N E T , E T , and E T acetate (ETAc), N A D P H , and fl-glucuronidase assay reagents were supplied by Sigma Chemical Co. (St Louis, M O , U.S.A.). All solvents and reagents used were of analytical grade. Synthesis of steroid derivatives 5c~-NET and the 3~,5~ and 3fl,5~ tetrahydro N E T derivatives (3~,5~-NET; 3fi,5~-NET) were synthesized from N E T as previously described [14]. 5c~dihydro 19norT (5c~-19norT), 5~-dihydro E T (5~ET) and 5~-dihydro E T acetate (5c~-ETAc) were synthesized by lithium-ammonia reduction of their corresponding unsaturated compounds, crystallized from ethyl acetate-hexane and purified by flash chromatography [15]. Chemical purity of steroids was assessed by their melting points, high pressure liquid chromatography behaviour, infrared absorption, and 1Hnuclear magnetic resonance. T h e physical and spectroscopic constants of the A-ring reduced derivatives of 19norT, E T , and E T A c were as follows: 5~-19norT:
5~-ET:
5~-ETAc:
m.p. = 133°C; [~]~o (CH3-OH) = + 60°; i.r.: max 3300, 1712 cm-1; 1HNMR CDC13 (6) 0.76 (s, CH3), 1.5 (s, OH disappears with DzO), 3.6 (t, H-17) ppm. m.p. = 292°C; [e]~o (CH3-OH) = - 26°; i.r.: max 3380, 3280, 1710 cm 1; tHNMR CDC13 (6) 0.82 (s, CH3-18), 1.0 (s, CH3-19), 2.49 (s, C =- CH) ppm. m.p. = 189°C; [e]~o (CH3-OH) = - 13.8°; i.r.: max. 3280, 1740, 1710 cm x 1HNMR CDC13 (3) 0.84 (s, CH3-18), 1.0 (s, CH3-19), 2.01 (s, CH3C = O), 2.51 (s, C -= CH) pprn.
Animals and tissues Adult male Wistar rats (250-300 g) and adult male Balb/c mice (30-35 g) used throughout this study were kept under a 1 4 h light/10h dark cycle and maintained on food and water ad libitum. Gonadectomies were performed under light ether anaesthesia. Animals were killed by decapitation; the appropriate tissues were immediately removed, blotted and weighed and then submitted to experimental procedures.
Radioactive material and chemicals
Androgen receptor binding studies
[6,7-3H]-Norethisterone ( 3 H - N E T ) specific activity: 55 Ci/mmol and [1,2,6,7- 3H]-testosterone (3H-T) specific activity: 102 Ci/mmol were obtained from N e w England Nuclear Research Products (Boston, MA, U.S.A.). [17~-methyl 3H]-dimethyl-19-nortestosterone (3H-mibolerone) specific activity: 85 Ci/ m m o l and unlabelled mibolerone were purchased from A m e r s h a m Intemational plc (Bucks., U.K.).
T h e presence of high affinity androgen binding sites in cytosol preparations of rat ventral prostates was assessed by a linear sucrose gradient labelling technique using [3H]-mibolerone as the radioligand, as previously described [16]. T h e equilibrium dissociation constant and the cytosol limited-capacity binding sites, for rat ventral prostate androgen receptor, were studied by a saturation
A Paradoxical Effect of 5~-Reduction analysis using [3H]-mibolerone as the radioligand [121. T o evaluate the interaction of N E T and its 5~reduced derivative with rat prostate AR, appropriate cytosol preparations obtained from animals castrated 48 h before the experiments, were incubated overnight with 2 n M [3H]-mibolerone at 4°C in the presence or absence of increasing concentrations of radioinert steroid competitors, including N E T , 5 a - N E T , mibolerone, T, and D H T . At the end of the incubation period, b o u n d and free fractions were separated by the addition of a dextran-coated charcoal suspension ( D C C ) and incubated for 1 0 m i n at 4°C. Incubations with D C C were terminated by centrifugation at 800 x g for 10 min, and aliquots were submitted to radioactive counting in a Packard T r i - C a r b liquid scintillation spectrometer using Instagel (Packard, Downers Grove, IL, U.S.A.) as the counting solution. Results we.re expressed as the percentage of [3H]-mibolerone specific binding. T h e relative binding affinities (RBA) for AR of 19norT, E T , and their 5~-reduced derivatives were also evaluated by their capability to displace b o u n d [3H]-mibolerone from rat ventral prostate androgen binding sites, as described above. T h e RBA and inhibition constants (Ki) of each steroid in displacement experiments were calculated according to the procedures described by Reel et al. [17] and C h e n g and Prusoff [18], respectively.
Assessment of androgenk: potency of unsaturated and 5~reduced steroids T o evaluate the androgenic potency of various natural and synthetic steroids and their 5~-reduced derivatives, two bioassays were used: the androgeninduced fl-glucuronidase activity in the mouse kidney and the androgen-induced growth of male rat accessory sex organs. T h e fl-glucuronidase bioassay was performed in adult mice castrated 5 days before the experiments. Animals were divided into eight groups (n = 7 - 9 each) and submitted to daily steroid treatments for five consecutive days. Steroids were dissolved in propyleneglycol (PG) and subcutaneously (s.c.) administered (40 #1); to enhance the solubility of E T and 5aE T , they were used in bioassays as their corresponding 17fi-acetate derivatives. T and D H T were given at a dose of 125 #g per day, whereas N E T , 5 a - N E T , 19norT, 5~-19norT, E T A c , and 5 a - E T A c were administered at 400 pg per day. T w o additional groups of intact (n = 5) and castrated (n = 5) mice, receiving vehicle alone, served as controls. At the end of the treatm e n t period the animals were killed and their kidneys removed immediately and homogenized in deionized water. Aliquots of kidney homogenates (200 #1; 16 m g protein) were submitted to fl-glucuronidase activity determination by the colorimetric m e t h o d of Fishman [19], with minor modifications, using the Sigma assay
123
kit reagents. Results were expressed as fl-glucuronidase units (U) per milligram of protein. T h e androgenic potency of unsaturated and 5~reduced steroids was also evaluated by their capability to induce ventral prostate and seminal vesicle growth in adult rats castrated eight days prior to the experiments. Animals were divided into eight groups (n = 7 - 9 each) and submitted to daily steroid treatments for 10 consecutive days. Steroids were dissolved in P G and administered s.c. (100 #1). T and D H T were given at the daily dose of 250 #g, whereas N E T , 5 ~ - N E T , 19norT, 5~-19norT, E T A c and 5aE T A c were administered at 2 m g per day. T w o additional groups of intact ( n = 9 ) and castrated (n = 12) rats, receiving vehicle alone, served as controls. At the end of the treatment period, animals were killed and their ventral prostates and seminal vesicles were immediately removed, blotted and weighed to the nearest 0.1 rag. Additional experiments were carried out to determine the effect of 5~-reduction u p o n the androgen agonistic, synergistic or antagonistic activities of N E T , 19norT, and E T using the mouse kidney fl-glucuronidase bioassay. Castrated male mice were divided into six groups (n = 8 each), and submitted to daily combined steroid treatments. Steroids were dissolved in P G and administered s.c. (40 #1). N E T , 5 ~ - N E T , 19norT, 5~-19norT, E T A c and 5 a - E T A c at a daily dose of 400 pg each were simultaneously administered with D H T (250/~g/day) for five consecutive days. T w o additional groups (n = 8 each) of castrated mice receiving either vehicle alone or D H T (250 pg/day) for five days served as controls. In vitro metabolic studies Ventral prostates from adult rats, castrated 48 h before the experiments, were homogenized in K r e b s Ringer phosphate buffer solution, p H 7.4. Tissue preparations (10 m g protein) were incubated in triplicate with a single concentration (2/~M) of either [3H]-labelled N E T or T (3 #Ci), in the presence or absence of 0.3 m M N A D P H in a final volume incubation of 1 ml. Incubations were carried out in a D u b n o f f metabolic incubator at 37.5°C for 2 h, using air as the gas phase. Tissue-less incubations were used as controls. At the end of the incubation period the steroids were extracted (4x) with three volumes of ethyl acetate. T h e organic extracts were partitioned between petroleum ether and 10% aqueous methanol. Methanolic extracts from the N E T incubations were added with 2.5 pg each of N E T , 5 a - N E T , 3~,5~N E T , and 3 f l , 5 a - N E T as steroid carriers, whereas 2.5 pg each of T , D H T , 3~,5a-androstanediol (3~,Sadiol) and 3fl,5~-androstanediol (3fi, Sa-diol) were added to T incubation extracts. Isolation and radiochemical purity of N E T and T metabolites were established by an identical m e t h o d to that of steroid carriers in both paper (petroleum ether: benzene:
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A.E. Lemus et al.
methanol: water; 5:1:5:1) and thin layer (benzene: absolute ethanol; 95:5) chromatographic systems and successive recrystallization to obtain a constant specific activity. T h e radioactive content in samples was determined in a Packard Tri-Carb liquid scintillation spectrometer, Model 2660 (Packard, Downers Grove, IL, U.S.A.). Counting efficiency of [3HI was 45% and quenching was corrected in all samples by external standardization. Formation of [3H]-NET and [3H]-T metabolites was calculated from the last crystal values and the results were expressed as pmol/mg protein/h.
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RESULTS -
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Competition of N E T and 5 ~ - N E T for androgen receptors T h e results of saturation analysis and linear sucrose gradient experiments, using [3H]-mibolerone as the radioligand, provided evidence of the presence of a specific 8.2 S cytosol-located androgen receptor in the rat ventral prostate, with an equilibrium dissociation constant of 1.71 n M and a saturation binding capacity of 2.3 nmol/mg of protein, confirming and extending our previous report using a different ligand [16]. T h e effects of increasing concentrations of nonradioactive N E T , 5~-NET, T and D H T upon the [3I-I]-mibolerone binding to androgen cytosol receptors from rat ventral prostate are shown in Fig. 1. T h e order to the affinity for the androgen receptor was: D H T > T >5or-NET>NET. T h e RBA and Ki values of each steroid are listed in Table 1. Competition of 19norT, E T and their 5~-reduced derivatives for androgen receptors T h e addition of increasing concentrations of radioinert 19norT, ET, and their 5~-reduced derivatives induced a varying degree of displacement of [3H]mibolerone from rat ventral prostate cytosol AR, as depicted in Fig. 2. T h e most potent steroid competitors for androgen binding sites were 5 ~ - l g n o r T and 19norT. 5~-ET also displayed a potent competitive effect, although to a lesser extent, whereas E T was effective only at high concentrations. T h e RBA and Ki values of each steroid are given in Table 1. Androgenic potency of N E T and 5 ~ - N E T T h e administration of N E T (400 pg/day for five days) to castrated mice induced an increase of kidney fl-glucuronidase activity to significantly higher levels ( P < 0.0001) than those observed in castrated control animals, whereas the administration of an identical
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S T E R O I D C O N C E N T R A T I O N (nM) Fig. 1. D i s p l a c e m e n t effect o f r a d i o i n e r t u n s a t u r a t e d a n d 5~r e d u c e d s t e r o i d s u p o n t h e specific b i n d i n g o f [ 3 H ] - m i b o l e r o n e to c y t o s o l a n d r o g e n r e c e p t o r s . A l i q u o t s o f v e n t r a l p r o s tate c y t o s o l (4.5 m g p r o t e i n / m l ) f r o m c a s t r a t e d a d u l t m a l e rats w e r e i n c u b a t e d o v e r n i g h t w i t h 2 n M [ 3 H ] - m i b o l e r o n e at 4°C (final i n c u b a t i o n v o l u m e 2 0 0 / d ) , in the a b s e n c e o r p r e sence of graded concentrations of non-labelled norethisterone (NET), 5~-dihydro-NET (5~-NET) (Panel A), and testostero n e (T), a n d 5 ~ - d i h y d r o t e s t o s t e r o n e ( D H T ) ( P a n e l B). T h e total b i n d i n g o f [ 3 H ] - m i b o l e r o n e in t h e a b s e n c e o f r a d i o i n e r t s t e r o i d s w a s set at 100%. E a c h p o i n t r e p r e s e n t s t h e m e a n o f three e x p e r i m e n t s i n t r i p l i c a t e . F o r details s e e t h e text.
dose of 5 ~ - N E T to castrated mice had no effect upon kidney enzyme activity, as shown in Fig. 3. T h e administration of T (125#g/day for five days) to castrated mice fully restored kidney fl-glucuronidase activity to levels observed in intact animals, whereas Table 1. Relative binding affinities (RBA) and inhibition constant (K~) of natural and synthetic steroidsfor androgen receptors Steroid competitors MB T DHT NET 5~-NET 19norT 5~-19norT ET 5~-ET
RBA* (%)
K i t (nM)
100.0 63.6 125.0 14.0 41.0 112.9 116.6 7.0 46.6
8.1 12.1 6.1 51.1 18.1 7.3 7.1 101.1 16.1
'~RBA was determined according to Reel et al. [17]. TKi values were calculated according to C h e n g and Prusoff [18].
A Paradoxical Effect of 5~-Reduction
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Fig. 2. C o m p e t i t i o n of radd[oinert u n s a t u r a t e d a n d 5ct-reduced s t e r o i d s for rat v e n t r a l p r o s t a t e a n d r o g e n b i n d i n g sites. C y t o s o l a n d r o g e n recepto:rs w e r e l a b e l l e d in v i t r o b y o v e r n i g h t i n c u b a t i o n s i d e n t i c a l to t h o s e d e s c r i b e d in Fig. 1. Increasing concentrations of the following radioinert synthetic s t e r o i d s w e r e a d d e d to t h e i n c u b a t i o n s : 1 9 - n o r t e s t o s t e r o n e ( 1 9 n o r T ) , 5ct-dihydro 1 9 - n o r T (5~t-19norT) ( P a n e l A), a n d 1 7 ~ - e t h y n y l t e s t o s t e r o n e ( E T ) , a n d 5 ~ - d i h y d r o E T (5~-ET) (Panel B). Each point represents the mean of three experi m e n t s in triplicate.
D H T given at an identical dose exhibited a significantly greater ( P < 0.001) potency than T to increase kidney e n z y m e activity in castrated mice, as depicted in Fig. 3. Similar results were obtained w h e n N E T , T, and their 5~-reduced derivatives were examined for their capability to induce growth of accessory sex organs in the castrated male rat. Indeed, N E T administered at the daily dose of 2 mg for 10 consecutive days induced a significant increase ( P < 0 . 0 0 0 1 ) in the weights of the ventral prostate and seminal vesicles compared with vehicle-treated castrated animals (Fig. 4). On the contrary, treatment with 5 ~ - N E T , administered at an identical dose, failed to induce ventral prostate and seminal vesicle growth in castrated rats, as s h o w n in Fig. 4. T h e administration of D H T (250 pg/day for 10 days) induced a significant increase in the weights of the ventral prostate (P<0.0001) and the seminal vesicles ( P < 0 . 0 0 1 ) compared with vehicle-treated castrated male rats. T h e androgenic effect of D H T was significantly more
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5etNET
Fig. 3. Effect o f n o r e t h i s t e r o n e ( N E T ) , t e s t o s t e r o n e (T) a n d their 5~-reduced derivatives (5~-NET and DHT) on mouse k i d n e y f l - g l u c u r o n i d a s e activity. A d u l t c a s t r a t e d m a l e Balb/c m i c e w e r e t r e a t e d w i t h s t e r o i d s ( N E T a n d 5 ~ - N E T : 400/~g! day; T a n d D H T : 125/zg/day) for five c o n s e c u t i v e d a y s . I n t a c t (I) a n d c a s t r a t e d (C) v e h i c l e - t r e a t e d m i c e s e r v e d as c o n t r o l groups. Each bar shows the mean and standard deviation ( S D ) for s e v e n to n i n e a n i m a l s . * P < 0.0001 c o m p a r e d w i t h t h e C v e h i c l e - t r e a t e d group; * * P < 0.001 c o m p a r e d w i t h t h e T - t r e a t e d g r o u p . F o r e x p e r i m e n t a l details s e e t h e text.
potent than T administered at an identical dose, particularly in terms of ventral prostate growth ( P < 0.001), as depicted in Fig. 4. T h e observation that 5~-reduction of the N E T molecule diminished and almost cancelled its androgenlike potency, as assessed by two different bioassays, prompted us to examine the nature of this most striking finding.
Androgenic potency of 19norT, ET, and their 5~-derivarives
T h e administration of 19norT (400 ttg/day for five days) induced an increase of kidney fl-glucuronidase activity in castrated mice, reaching significantly higher levels ( P < 0.0001) than those observed in vehicletreated control animals (Fig. 5). T h e 5~-reduced derivative of 19norT, given at an identical dose, exhibited a significantly greater potency than 19norT ( P < 0.01), in terms of kidney fi-glucuronidase activity increase in castrated mice, as depicted in Fig. 5.
A.E. Lemus et al.
126 250
_
VENTRAL PROSTATE
SEMINAL
T
200 -
50
/ / / / / / / / / / / / / /
i 'i 1 I
C
T
DHT NET 5c~NET
,
7I
i
150
100
60
VESICLES
C
/ /
/
/
/
¢
"/
/ / / / / / /
/ / / / / / / / /
50
//A I/,4 e~ 40 g~
T h e administration of E T A c (400 #g/day for five days) induced a significant increase ( P < 0.0001) of kidney fi-glucuronidase activity in castrated male mice, as s h o w n in Fig. 5. T h e 5~-reduced derivative of ETAc, given at an identical dose, also induced a rise of kidney /~-glucuronidase activity, although with a significantly lower ( P < 0.01) potency than E T A c (Fig. 5.) W h e n the potency of 19norT, ETAc, and their 5~reduced derivatives to induce accessory sex organ growth was assessed in castrated male rats, the results were similar but not identical to those obtained using the m o u s e kidney ~-glucuronidase assay. T h e administration of 19norT ( 2 m g / d a y for 10 days) to castrated rats resulted in a significant increase in the weights of the ventral prostate ( P < 0.0001) and the seminal vesicles ( P < 0.001). W h e n 5~-19norT was given at an identical dose, it induced an increase of both ventral prostate and seminal vesicle growth, with a significantly greater ( P < 0 . 0 0 1 ) potency than 19norT, as shown in Fig. 6. Interestingly, neither E T A c nor its 5~-reduced derivative, administered at the daily dose of 2 m g for 10 consecutive days to castrated male rats had any significant effect upon accessory sex organs.
Synandrogenic and antiandrogenic activities of NET, 19norT, ETAc and their 5~-derivatives T h e administration of N E T plus D H T and 19norT plus D H T revealed an androgenic synergistic effect of both synthetic steroids. Indeed, N E T and 19norT when given with D H T induced a significantly greater increase ( P < 0 . 0 1 and P < 0 . 0 0 0 1 , respectively) of
<
30
T
<
I/,I
T //A I/,4
k"
T DHT NET 5c~NET
Fig. 4. E f f e c t o f n o r e t h i s t e r o n e ( N E T ) , t e s t o s t e r o n e (T) a n d their 5~-reduced derivatives (5~-NET and DHT) on accessory s e x o r g a n g r o w t h i n t h e c a s t r a t e d m a l e rat. A d u l t c a s t r a t e d animals were treated with steroids (NET and 5~-NET: 2mgl day; T a n d D H T : 250 pg/day) f o r 10 c o n s e c u t i v e d a y s . I n t a c t (I) a n d c a s t r a t e d (C) v e h i c l e = t r e a t e d r a t s s e r v e d as c o n t r o l g r o u p s . E a c h b a r s h o w s t h e r e s u l t s ( m e a n + S D ) in t e r m s o f t h e w e i g h t (rag) o f t h e v e n t r a l p r o s t a t e a n d t h e s e m i n a l v e s icles for s e v e n to n i n e a n i m a l s . * P < 0.0001 c o m p a r e d w i t h t h e C v e h i c l e - t r e a t e d g r o u p ; * * P < 0.001 c o m p a r e d w i t h t h e C v e h i c l e = t r e a t e d g r o u p ; , P < 0.001 c o m p a r e d w i t h t h e T - t r e a ted group.
//A
//,4
© 20 C..)
//, I//
I/,,I I/,I //,4
/~/
I.ill
t- i :
I1.,I //2
10
//,1 //,i //,1 1
.
C
~
9"/
V,~
//,
NorT
5c~NorT
ETAc
Ii'/I
5~ETAc
Fig. 5. E f f e c t o f s y n t h e t i c u n s a t u r a t e d a n d 5 ~ = r e d u c e d s t e r oids on mouse kidney ~-glucuronidase activity. Adult c a s t r a t e d m a l e B a l b / c m i c e w e r e t r e a t e d w i t h 19=nortestos= terone (NorT), 17~=ethynyltestosterone acetate (ETAc) and t h e i r 5 ~ = r e d u c e d d e r i v a t i v e s (5mNorT a n d 5 ~ E T A c ) at t h e d a i l y d o s e o f 400 pg f o r five c o n s e c u t i v e d a y s . I n t a c t (I) a n d c a s t r a t e d (C) v e h i c l e = t r e a t e d m i c e s e r v e d as c o n t r o l g r o u p s . T h e r e s u l t s a r e e x p r e s s e d as t h e k i d n e y e n z y m e a c t i v i t y ( m e a n _+ S D ) f o r e a c h g r o u p (n = 7 - 9 ) o f a n i m a l s . * P < 0.0001 c o m p a r e d w i t h t h e C v e h i c l e = t r e a t e d g r o u p ; * * P < 0.01 corn= p a r e d w i t h t h e N o r T = t r e a t e d m i c e ; 0 P < 0.01 c o m p a r e d w i t h the ETAc=treated group.
kidney/%glucuronidase activity in castrated male mice compared with animals treated with D H T alone (Fig. 7). On the contrary, when 5 ~ - N E T and 5~19norT were given simultaneously with D H T a lack of synandrogenic effect of these reduced derivatives was observed in terms of m o u s e kidney enzyme activity (Fig. 7). T h e simultaneous administration of E T A c with DHT resulted in a significant diminution (P < 0.0001) of the D H T - i n d u c e d increase of kidney fl-glucuronidase activity in castrated mice. Indeed, the levels of kidney enzyme activity in animals treated with E T A c plus D H T resembled those found in vehicle-treated castrated mice. Interestingly, the c o m bined administration of 5~-ETAc plus D H T to castrated mice induced an increase o f kidney //-glucuronidase activity similar to that observed in animals treated with D H T alone (Fig. 7).
A Paradoxical Effect of 5~-Reduction 300 -
VENTRAL PROSTATE
SEMINAL VESICLES
250 -
~
~ T
~. 2 0 0 -
D
•
,]-
[3Hl-Substrate
/ / /
150
//
1oo
/ /
_~
Norethisterone (NET)
Testosterone(T)
/
~-
/
0 5~NorT 5~ETAc NorT ETAc
C
150 >
F100 _
T
< r~ z ©
50
N o cofactor
21.28 12.30 8.90 82.44 36.32 23.27
6.62 2.56 1.10 20.61 9.84 7.30
Metabolism of N E T and T in the rat prostate
T h e results from the in vitro incubations of rat vental prostate homogenates with radiolabelled N E T and T demonstrated that b o t h substrates undergo extensive bioconversion to A-ring reduced metabolites. As shown in Table 2, the enzyme-mediated A-ring reduction of both N E T and T was N A D P H - d e p e n d e n t , as has been previously described [20]. T h e in vitro metabolic fate of N E T and T in the prostate was similar, although T was a m o r e efficient substrate for the steroid metabolizing enzymes. T h e 5ct-dihydro derivatives of N E T and T were the major isolated incubation products followed by their corresponding 3~and 3/3-tetrahydro metabolites (Table 2).
DISCUSSION
200
E
U
5c~-NET 3~,5~-NET 3fl,5~-NET 5~-DHT 3a,5~-diol 3fl,5~-diol
pmol/mg protein/h NADPH
5c~NorT 5e~ETAc NorT ETAc
Fig. 6. A n d r o g e n i c p o t e n c y o f 1 9 - n o r t e s t o s t e r o n e ( N o r T ) , 17~-ethynyltestosterone acetate (ETAc) and their 5~-reduced m e t a b o l i t e s (5mNorT a n d 5.0dETAc) e v a l u a t e d b y t h e i r effects on t h e w e i g h t o f a c c e s s o r y s e x o r g a n s in t h e c a s t r a t e d m a l e rat. U n s a t u r a t e d a n d r e d u c e d s t e r o i d s w e r e a d m i n i s t e r e d ( 2 m g l d a y ) for 10 c o n s e c u t i v e d a y s to a d u l t m a l e rats c a s t r a t e d 8 d a y s b e f o r e t h e e x p e r i m e n t s . I n t a c t (I) a n d c a s t r a t e d (C) v e h i c l e - t r e a t e d a n i m a l s s e r v e d as c o n t r o l g r o u p s . T h e r e s u l t s a r e g i v e n as t h e w e i g h t o f t h e v e n t r a l p r o s t a t e a n d t h e s e m i n a l v e s i c l e s ( m e a n + S D ) for e a c h anim a l g r o u p (n = 7 - 9 ) . * P < 0.0001 c o m p a r e d w i t h the C v e h l c l e - t r e a t e d g r o u p ; **F' < 0.001 c o m p a r e d with the C v e h i c l e - t r e a t e d group; , P < 0.001 c o m p a r e d w i t h t h e N o r T treated group.
03
[3H]-isolated metabolites
F o r details see M a t e r i a l s a n d M e t h o d s .
/ C
Table 2. In vitro formation of A-ring reduced metabolites of f H]-norethisterone and testosterone in the rat ventral prostate
I
//
127
_
//l /A
D u D o C
DHT
NET +
5aNET +
NorT +
5c~NorT +
ETAc +
5aETAc +
DHT
DHT
DHT
DHT
DHT
DHT
Fig. 7. S y n a n d r o g e n l c a n d a n t i a n d r o g e n i c activities o f n o r ethisterone (NET), 19-nortestosterone (NorT), 17~-ethynyltestosterone acetate (ETAc) and their 5~-reduced metabolites in c a s t r a t e d B a l b l c m i c e . T h e effect o f u n s a t u r a t e d a n d r e d u c e d s t e r o i d s (400/z g/day) a d m i n i s t e r e d s i m u l t a n e o u s l y w i t h D H T (250/ag/day) for five c o n s e c u t i v e d a y s w a s a s s e s s e d b y t h e i r c a p a b i l i t y to m o d i f y t h e m o u s e k i d n e y /~-glucurould a s e activity. C a s t r a t e d a n i m a l s t r e a t e d w i t h D H T p l u s v e h i c l e ( D H T ) or v e h i c l e a l o n e (C) s e r v e d as c o n t r o l g r o u p s . T h e r e s u l t s a r e g i v e n as t h e k i d n e y e n z y m e a c t i v i t y ( m e a n + S D ) for e a c h a n i m a l g r o u p (n = 8), * P < 0.01, * * P < 0.0001, , P < 0.0001 c o m p a r e d w i t h t h e D H T + v e h i c l e - t r e a t e d g r o u p .
T h e results presented herein provide strong evidence that enzyme-mediated 5u-reduction of the synthetic progestin norethisterone enhances its relative binding affinity for the intracellular androgen receptor, but diminishes and almost cancels its androgenlike effects. Indeed, experiments carried out in rat ventral prostates using displacement analysis revealed a significant increase in the relative binding affinity of 5 u - N E T to the 8.2 S cytosol-located androgen receptor, with a concomitant significant decrease in the Ki value c o m p a r e d with unchanged N E T (Table 1), confirming and extending previous results from this laboratory [12]. W h e n the androgenic potency of N E T and 5 ~ - N E T was assessed by two different bioassays, it became evident that A-ring reduction of the N E T molecule results in a complete loss of its potent androgen-like effects as shown in Figs 3 and 4. These findings are in a sharp contrast with the well-established concept that formation of the 5~- (trans A/B ring junction) derivative of naturally occurring testosterone enhances both its RBA to the AR and its androgenic potency. These striking observations raise the important question as to why 5c~-reduction of N E T , a 19-nor, 17~-ethynyl testosterone derivative, induced a dra-
128
A.E. Lemus et al.
matic diminution of its androgenicity in spite of the fact that 5 ~ - N E T exhibits a high RBA for the androgen receptor, and p r o m p t e d us to delineate the nature of this paradoxical effect. This unexpected effect of 5~-reduction on N E T could be caused by a more rapid bioconversion of 5~N E T into less active molecules. T o rule out this possibility, a study aimed to compare the in vitro metabolism of radiolabelled N E T and T in the rat ventral prostate was conducted. T h e results disclosed that both N E T and T were efficiently bioconverted to several A-ring reduced c o m p o u n d s , including their corresponding 5~-dihydro and their 3~- and 3r-tetrahydro derivatives. T h e formation rate of A-ring reduced metabolites of N E T and T presented a similar pattern, it was N A D P H - d e p e n d e n t and it occurred to an almost identical extent, although T was a m o r e efficient substrate than N E T (Table 2). Indeed, the 5~-reduction of N E T seems to be slower than that of T, whereas the further 3~- or 3r-reduction of 5~N E T appears to be slightly faster than that of D H T . T o assess whether the lack of the C-19 methyl group or the presence of the 17~-ethynyl moiety of the N E T molecule may be involved in the paradoxical effect of 5a-reduction upon this synthetic progestin, a series of experiments was conducted, which aimed at evaluating the A R affinities and androgenic potency of 19norT, E T and their 5a-reduced derivatives. T h e results showed that 5~-reduction of 19-norT neither increased nor decreased its affinity for the androgen receptor, whereas 5~-reduction of E T significantly enhanced its relative binding to the androgen receptor (Fig. 2 and T a b l e 1). These combined results provided an explanation for the finding that 5~-reduction of N E T enhanced (two- to three-fold) the specific interaction with androgen binding sites in a m a n n e r similar to that observed with T, its parent c o m p o u n d (Fig. 1 and T a b l e 1). T h e androgenic potency of 19norT, E T A c and their corresponding 5~-derivatives was assessed by their effects upon the fl-glucuronidase activity in the mouse kidney, and upon the growth of rat accessory sex organs. T h e results showed that 5~-reduction of 19norT significantly increased the kidney enzyme activity (Fig. 5), in a fashion similar to that observed with T after its 5~-reduction (Figs 3 and 4). A similar increase in the androgenic potency of the 19norT molecule, induced by its 5~-reduction, was d e m o n strated in terms of the weights of the ventral prostate and seminal vesicles in castrated male rats (Fig. 6). These results were interpreted as indicating that removal of the C-19 methyl group from the T molecule does not interfere with the 5~-reductioninduced amplification of its androgenic potency. On the contrary, 5~-reduction of E T A c resulted in a significant diminution of its capability to increase flglucuronidase activity in the mouse kidney bioassay (Fig. 5). As shown in Fig. 6, E T A c was unable to re-
store the weights of the ventral prostate and seminal vesicles of castrated male rats, indicating that this c o m p o u n d was completely devoid of androgenic potency. Furthermore, 5~-reduction of E T A c had no effect at all on its biological potency (Fig. 7). T h e data indicate that the addition of a 17~-ethynyl group to the testosterone molecule precludes the androgen amplifying effect induced by 5~-reduction, and offers a plausible explanation for the paradoxical effect of Aring reduction u p o n norethisterone. These results confirm and extend the observations of Morali et al. [13] that N E T , when administered with estradiol-17fl to long-term castrated rats, was able to restore full copulatory behaviour, whereas 5~N E T failed to induce masculine behaviour when given with estradiol 17-fl, indicating that 5~-reduction of N E T results in a significant diminution of its behavioural androgenic potency. This paradoxical effect of 5~-reduction u p o n the androgen-like potency of N E T also seems to occur with its progestational activity. Guti6rrez-Sagal et al. [21] have demonstrated that Aring reduction of N E T significantly diminishes its progestational activity, as assessed by the endometrial content of the uteroglobin gene expression products in prepuberal rabbits; furthermore, the 5~-reduced N E T derivative exhibits potent anti-progestational activity. Reel et al. [17] have also demonstrated the capability of 5 ~ - N E T to inhibit progesterone-induced endometrial proliferation. M o r e recently Castro et al. [22] have reported that 5 ~ - N E T displays anti-implantation effects, when given to m a t e d adult female rabbits, with a potency similar to that observed with RU-486, the synthetic steroidal antiprogestin. Because the synandrogenic and antiandrogenic effects of several synthetic progestins have been well d o c u m e n t e d [23], we felt it would be of interest to assess the effect of 5~-reduction upon N E T , 19norT, and ETAc, in terms of their androgen synergistic and antagonistic activities. T h e simultaneous administration of D H T plus unchanged N E T revealed that this synthetic progestin exhibited a synandrogenic effect in the mouse kidney, as was expected, whereas the simultaneous administration of D H T plus the 5~-reduced N E T derivative had neither synandrogenic nor androgenic additive effects. This observation is consistent with the diminution of the androgenic activity of the N E T molecule induced by its 5~-reduction. T h e lack of antiandrogenic effects of 5~-NET, at the dose level employed, may be explained by the observations of Mowszowicz et al. [23], indicating that the synandrogenic and antiandrogenic effects of synthetic progestins vary in a dose-dependent manner. Similar results were observed following the administration of 19norT plus D H T and 5~-19norT plus D H T . Indeed, 19norT exhibited a synandrogenic potency identical to that of N E T , whereas 5~-19norT did not. Interestingly, E T A c displayed an antiandrogenic effect when admi-
A Paradoxical Effect of 5 ~ - R e d u c t i o n nistered
simultaneously
with
DHT,
as
shown
in
F i g . 7, w h e r e a s 5 c ~ - E T A c e x h i b i t e d n e i t h e r a n t i a n d r o genic nor synandrogenic effects in the mouse kidney. These data indicate that 5~-reduction modifies the expression of androgen synergistic and antagonistic activities of NET. T a k i n g all t h e s e r e s u l t s t o g e t h e r , it c a n b e c o n cluded that 5e-reduction of NET does not amplify, b u t d o e s s u p p r e s s its a n d r o g e n - l i k e a c t i v i t y i n t a r g e t o r g a n s , i n s p i t e o f its h i g h R B A t o t h e a n d r o g e n receptor. This paradoxical effect of 5e-reduction seems to be caused by the presence of a 17e-ethynyl group in the NET molecule. Whether similar findings can be obtained in isolated hormone-responsive systems remains to be ascertained, and deserves further experimental work in culture-maintained androgend e p e n d e n t a n d a n d r o g e n - s e n s i t i v e cells. T h e o v e r a l l data provide additional support for the concept that the metabolism of synthetic progestins at target o r g a n s m o d u l a t e s t h e i r h o r m o n e - l i k e effects. Acknowledgements--This study was supported in part by grants from the Rockefeller Foundation (RF 93029-allocation 5), New York and The Special Programme :in Human Reproduction of the World Health Organization, Geneva. The authors are indebted to Ms B. Alarc6n and Mr J. L. Ramirez for assistance with the preparation of the manuscript.
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129
9. Stillwell W. G., Homing E. C., Homing M. G., Stillwell R. N. Zlatkis A.: Characterization of metabolites of steroid contraceptives by gas chromatography and mass spectrometry. J. Steroid Biochem. 3 (1972) 699-706. 10. Braselton W.E. Jr, Lin T. J., Ellegood J. O., Mills T. M. and Mahesh V. B.: Accumulation of norethindrone and individual metabolites in human plasma during short- and long-term administration of a contraceptive dosage. Am. J. Obstet. Gynecol. 133 (1979) 154-160. 11. Larrea F., Vilchis F., Chfivez B., Prrez A. E., Garza-Flores J. P~rez-Palacios G.: The metabolism of 19-nor contraceptive progestins modulates their biological activity at the neuroendocrine level, ft. Steroid Biochem. 27 (1987) 657-663. 12. Chfivez B. A., Vilchis F., Pdrez A. E., Garcia G. A., Grillasca I. and Prrez-Palacios G.: Stereospecificity of the intraceUular binding of norethisterone and its A-ring reduced metabolites. J. Steroid Biochem. 22 (1985) 121-126. 13. Morali G., Lemus A. E., Oropeza V. M., Garcia G. A. and Prrez-Palacios G.: Induction of male sexual behavior by norethisterone: role of its A-ring reduced metabolites. Pharmacol. Biochem. Behav. 37 (1990) 477-484. 14. Vilchis F., Chfivez B., Pfirez A. E., Garcia G. A., Angeles A. P6rez-Palacios G.: Evidence that a non-aromatizable metabolite of norethisterone induces estrogen-dependent pituitary progestin receptors. J. Steroid Bioehem. 24 (1986) 525-531. 15. Still W. C., Kahan M. and Mitra A.: Rapid chromatographic technique for preparative separations with moderate resolution. J. Organ. Chem. 43 (1978) 2923-2925. 16. Vilchis F., Hernfindez A., P~rez A. E. and Prrez-Palacios G.: Hormone regulation of the rodent Harderian gland: binding properties of the androgen receptor in the male golden hamster. J. Endoer. 112 (1987) 3-8. 17. Reel J. R., Humphrey R. R., Shih Y.-H., Windsor B. L., Sakowski R., Creger P. L. and Edgren R. A.: Competitive progesterone antagonists: receptor binding and biologic activity of testosterone and 19-nortestosterone derivatives. Fertil. Steril. 31 (1979) 552-561. 18. Cheng Y.-C. and Prusoff W. H.: Relationship between the inhibition constant (Ki) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction. Biochem. Pharmacol. 22 (1973) 3099-3108. 19. Fishman W. H., Springer B. and Brunetti R.: Application of an improved glucuronidase assay method to the study of human blood ~-glucuronidase. J. Biol. Chem. 173 (1948) 449-456. 20. Frederiksen D. W. and Wilson J. D.: Partial characterization of the nuclear reduced nicotinamide adenine dinucleotide phosphate: delta 4-3-ketosteroid 5~-oxidoreductase of rat prostate. J. Biol. Chem. 246 (1971) 2584-2593. 21. Gutirrrez-Sagal R., Prrez-Palacios G., Langley E., Pasapera A. M., Castro I. and Cerbrn M. A.: Endometrial expression of progesterone receptor and uteroglobin genes during early pregnancy in the rabbit. Molec. Reprod. Dev. 34 (1993) 244-249. 22. Castro I., Cerb6n M. A., Pasapera A. M., Guti~rrez-Sagal R., Garcla G. A., Orozco C., Camacho-Arroyo I., Anzaldua R. Prrez-Palacios G.: Molecular mechanisms of the antihormonal and antiimplantation effects of norethisterone and its A-ring reduced metabolites. Molec. Reprod. Dev. 40 (1995) 157-163. 23. Mowszowicz I., Bieber D. E., Chung K. W., Bullock L. P. and Bardin C. W.: Synandrogenic and antiandrogenic effect of progestins: comparison with nonprogestational antiandrogens. Endocrinology 95 (1974) 1589-1599.
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5 -Reduction of Norethisterone Enhances its Binding Affinity for Androgen Receptors but D i m i n i s h e s its Androgenic Potency A n a E . L e m u s , 1. J u a n a E n r i q u e z , 2 G u s t a v o A . G a r c i a , 3 Ivonne Grillasca and Gregorio P rez=Palacios e ~Department of Reproductive Biology, UAM-Iztapalapa, Mexico City, Mexico; :Department of Reproductive Biology, National Institute of Nutrition S. Zubirdzn, Mexico City, Mexico and ~Faculty of Chemistry, National University of Mkxico, Mexico City, Mexico
Norethisterone (NET), a 19-nor synthetic progestin, undergoes enzyme-mediated 5~-reduction and e x e r t s p o t e n t a n d r o g e n i c e f f e c t s in t a r g e t o r g a n s . T o i n v e s t i g a t e its m o d e o f a n d r o g e n i c a c t i o n we e x a m i n e d , in a c o m p a r a t i v e m a n n e r , t h e in vitro m e t a b o l i s m o f N E T a n d t e s t o s t e r o n e (T), as well as t h e b i n d i n g a f f i n i t i e s to a n d r o g e n r e c e p t o r s (AR) a n d t h e a n d r o g e n i c p o t e n c y o f N E T , T , a n d t h e i r 5 u - r e d u c e d d e r i v a t i v e s . B i o c o n v e r s i o n o f [ 3 H ] - N E T a n d [ 3 H ] - T w a s s t u d i e d in r a t p r o s t a t e h o m o g e n a t e s , A R b i n d i n g a f f i n i t y w a s a s s e s s e d in r a t v e n t r a l p r o s t a t e s u s i n g [ 3 H ] - m i b o l e r o n e as t h e r a d i o l i g a n d , a n d t h e a n d r o g e n i c p o t e n c y w a s e v a l u a t e d b y t h e i n c r e a s e o f ~ - g l u c u r o n i d a s e a c t i v i t y in t h e m o u s e k i d n e y , a n d b y t h e g r o w t h o f a c c e s s o r y s e x o r g a n s in c a s t r a t e d m a l e r a t s . T h e r e s u l t s d e m o n s t r a t e d t h a t 5.z-NET d i s p l a y e d a h i g h e r A R b i n d i n g a f f i n i t y b u t a s i g n i f i c a n t l y l o w e r a n d r o genic potency than unchanged NET. The bioconversion studies indicated that the metabolism of N E T w a s s i m i l a r to t h a t o f T , a l t h o u g h to a l e s s e r e x t e n t , t h u s r u l i n g o u t t h e p o s s i b i l i t y t h a t t h e s y n t h e t i c p r o g e s t i n m e t a b o l i z e s r a p i d l y i n t o less a c t i v e d e r i v a t i v e s . T o i n v e s t i g a t e t h e n a t u r e o f t h e paradoxical effect of 5u-reductlon upon the NET molecule, the interaction with AR and the androgenic potency of T, 19-nortestosterone (19norT), 17~-ethynyl testosterone (ET) and their 5u-reduced derivatives were examined. The results of AR binding studies revealed that 5~-reduction of T and ET significantly enhanced their affinities, and that the 5~-derivative of 19norT displayed a similar b i n d i n g a f f i n i t y to t h a t e x h i b i t e d b y 1 9 n o r T . I n t e r m s o f b i o l o g i c a l a c t i v i t y , t h e r e s u l t s s h o w e d t h a t 5~-reduction of T and 19norT significantly increased their androgenic potency, whereas 5~-reduction o f E T r e s u l t e d in a s i g n i f i c a n t d i m i n u t i o n o f its a n d r o g e n i c i t y in a m a n n e r s i m i l a r to t h a t o b s e r v e d w i t h t h e 5 ~ - r e d u c t i o n o f N E T . W h e n N E T a n d 1 9 n o r T w e r e s i m u l t a n e o u s l y a d m i n i s t e r e d w i t h 5udihydrotestosterone they exhibited a potent synandrogenic activity, an effect that was cancelled by t h e i r 5 ~ - r e d u c t i o n . ]Interestingly, E T d i s p l a y e d a n a n t i a n d r o g e n i c a c t i v i t y , a n e f f e c t t h a t w a s also s u p p r e s s e d b y its 5 ~ - r e d u c t i o n . T h e o v e r a l l r e s u l t s d e m o n s t r a t e d a d i s t i n c t i v e , p a r a d o x i c a l e f f e c t o f 5 ~ - r e d u c t i o n u p o n t h e N E T m o l e c u l e , w h i c h w a s d i f f e r e n t f r o m t h a t s e e n in n a t u r a l l y o c c u r r i n g a n d r o g e n s , a n d w h i c h s u g g e s t s t h a t t h e p r e s e n c e o f t h e 1 7 ~ - e t h y n y l g r o u p p l a y s a k e y r o l e in t h i s phenomenon. The data provided further evidence that the metabolism of synthetic contraceptive p r o g e s t i n s m o d u l a t e s t h e e x p r e s s i o n o f t h e i r hot~tiione-like a c t i o n s . © 1997 E l s e v i e r S c i e n c e L t d .
J. Steroid Biochem. Mo,!ec. Biol., Vol. 60, No. 1-2, pp. 121-129, 1997
Trivial names and abbreviations: norethisterone (NET), 17~-ethynyl-17fl-hydroxy-19-nor-4-androstene-3-one;5~-dihydroNET(5a-NET), 17~-ethynyl-17fl-hydroxy-19-nor-5c~-androstane-3-one; 19-nortestosterone (19norT), 17fl-hydroxy-19-nor-4-androstene-3=one; 5c~dihydrol 9norT (Set-19norT),17fl-hydroxy-19-nor-5a-androstane-3-one;17a-ethynyltestosterone(ET),17a-ethynyl-17fl-hydroxy-4-androstene-3-one; ET acetate (ETAc), 17a,ethynyl-3-oxo-4-androstene-17fl-ylacetate; 5~-dihydro ET (5a-ET), 17~-ethynyl-17fl-hydroxy-5~androstane-3-one; 5~-dihydro ET acetate(5~-ETAc),17a-ethynyl-3-oxo-5~-androstane-17fl-yl-acetate;mibolerone,7~,17~-dimethyllTflhydroxy-4-androstene-3-one; testosterone (T), 3fl-hydroxy-4-androstene-3-one; 3cq5~-androstanediol (3cq5a-diol), 5c~-androstane3~,17fl-diol; 3fl,5ct-androstanediol (3fl,5~-diol), 5c~-androstane-3fl,17fl-diol; 3a,5ct-tetrahydroNET (3ct,5~-NET), 17c~-ethynyl-3a,17fldihydroxy-19-nor-Sa-androstane-3-one; 3fl,5a-tetrahydroNET(3fl,5~-NET), 17~-ethynyl-3fl,17fl-dihydroxy-19-norS~-androstane-3-one. *Correspondence to A. E. Lemus, Department of Reproductive Biology, National Institute of Nutrition S. Zubirfin, Vasco de Quiroga No. 15, Tlalpan, M6xico, D.F., 14000 Mexico. Tel: +(52 5) 573 1160; Fax: +(52 5) 655 9859. Received 17 Jan. 1996; zccepted 12 Aug. 1996. 121
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A.E. Lemus et al. INTRODUCTION
Norethisterone ( N E T ) , a synthetic 19-nor, 17~-ethynyl testosterone derivative, has been widely used as a contraceptive progestin in a n u m b e r of pharmaceutical formulations [1--4]. In addition to its well-known progestational activity, N E T displays potent androgen-like effects following its administration in a n u m ber of m a m m a l i a n species [5-8]. Previous studies from this and other laboratories have demonstrated that N E T , in a similar way to naturally occurring androgens, undergoes extensive in vivo and in vitro enzyme-mediated 5~-reduction [9-11], and that 5~dihydro N E T (5~-NET) exhibits a higher binding affinity for the androgen receptor (AR) c o m p a r e d to its parent c o m p o u n d [12]. T o assess whether the androgenic effects of N E T are mediated by its 5~-metabolic conversion product, we examined, in a comparative manner, the effects of N E T and 5 ~ - N E T u p o n fl-glucuronidase activity in the mouse kidney, and u p o n accessory sex organ growth in the castrated male rat. T h e results d e m o n strated that 5~-reduction of the N E T molecule induced a significant diminution of its androgenic potency, thus indicating that the androgenic effects of N E T are not mediated by its A-ring reduction. In addition, in vitro metabolic studies indicated that N E T and T exhibit similar metabolic patterns in a target tissue. T h e s e striking observations p r o m p t e d us to investigate whether the absence of the C-19 or the presence of the 17~-ethynyl group in the N E T molecule are involved in the paradoxical effect of 5~-reduction. Accordingly, the A R binding affinity and the androgenic potency of testosterone (T), 19-nortestosterone (19norT), 17c~-ethynyltestosterone (ET) and their corresponding 5~-derivatives were studied. AR binding affinities were assessed by displacement analysis in rat ventral prostate preparations using [3H]-mibolerone as the radioligand. Additional experiments were carried out to determine the effects of 5c~-reduction upon the synergistic/antagonistic activities of N E T , 19norT and E T . Further impetus to conduct this study was provided by the observation that 5~-reduction of N E T results in a dramatic diminution of its androgen-like behavioural potency in long-term castrated mate rats [13].
MATERIALS AND METHODS
Radiochemical purity of [3H]-steroids was established by paper or by thin-layer chromatographic behaviour, and by repeated crystallizations to constant specific activity. Radioinert T, 5~-dihydrotestosterone ( D H T ) , 3~,5c~ and 3fl,5c~, androstanediols, N E T , E T , and E T acetate (ETAc), N A D P H , and fl-glucuronidase assay reagents were supplied by Sigma Chemical Co. (St Louis, M O , U.S.A.). All solvents and reagents used were of analytical grade. Synthesis of steroid derivatives 5c~-NET and the 3~,5~ and 3fl,5~ tetrahydro N E T derivatives (3~,5~-NET; 3fi,5~-NET) were synthesized from N E T as previously described [14]. 5c~dihydro 19norT (5c~-19norT), 5~-dihydro E T (5~ET) and 5~-dihydro E T acetate (5c~-ETAc) were synthesized by lithium-ammonia reduction of their corresponding unsaturated compounds, crystallized from ethyl acetate-hexane and purified by flash chromatography [15]. Chemical purity of steroids was assessed by their melting points, high pressure liquid chromatography behaviour, infrared absorption, and 1Hnuclear magnetic resonance. T h e physical and spectroscopic constants of the A-ring reduced derivatives of 19norT, E T , and E T A c were as follows: 5~-19norT:
5~-ET:
5~-ETAc:
m.p. = 133°C; [~]~o (CH3-OH) = + 60°; i.r.: max 3300, 1712 cm-1; 1HNMR CDC13 (6) 0.76 (s, CH3), 1.5 (s, OH disappears with DzO), 3.6 (t, H-17) ppm. m.p. = 292°C; [e]~o (CH3-OH) = - 26°; i.r.: max 3380, 3280, 1710 cm 1; tHNMR CDC13 (6) 0.82 (s, CH3-18), 1.0 (s, CH3-19), 2.49 (s, C =- CH) ppm. m.p. = 189°C; [e]~o (CH3-OH) = - 13.8°; i.r.: max. 3280, 1740, 1710 cm x 1HNMR CDC13 (3) 0.84 (s, CH3-18), 1.0 (s, CH3-19), 2.01 (s, CH3C = O), 2.51 (s, C -= CH) pprn.
Animals and tissues Adult male Wistar rats (250-300 g) and adult male Balb/c mice (30-35 g) used throughout this study were kept under a 1 4 h light/10h dark cycle and maintained on food and water ad libitum. Gonadectomies were performed under light ether anaesthesia. Animals were killed by decapitation; the appropriate tissues were immediately removed, blotted and weighed and then submitted to experimental procedures.
Radioactive material and chemicals
Androgen receptor binding studies
[6,7-3H]-Norethisterone ( 3 H - N E T ) specific activity: 55 Ci/mmol and [1,2,6,7- 3H]-testosterone (3H-T) specific activity: 102 Ci/mmol were obtained from N e w England Nuclear Research Products (Boston, MA, U.S.A.). [17~-methyl 3H]-dimethyl-19-nortestosterone (3H-mibolerone) specific activity: 85 Ci/ m m o l and unlabelled mibolerone were purchased from A m e r s h a m Intemational plc (Bucks., U.K.).
T h e presence of high affinity androgen binding sites in cytosol preparations of rat ventral prostates was assessed by a linear sucrose gradient labelling technique using [3H]-mibolerone as the radioligand, as previously described [16]. T h e equilibrium dissociation constant and the cytosol limited-capacity binding sites, for rat ventral prostate androgen receptor, were studied by a saturation
A Paradoxical Effect of 5~-Reduction analysis using [3H]-mibolerone as the radioligand [121. T o evaluate the interaction of N E T and its 5~reduced derivative with rat prostate AR, appropriate cytosol preparations obtained from animals castrated 48 h before the experiments, were incubated overnight with 2 n M [3H]-mibolerone at 4°C in the presence or absence of increasing concentrations of radioinert steroid competitors, including N E T , 5 a - N E T , mibolerone, T, and D H T . At the end of the incubation period, b o u n d and free fractions were separated by the addition of a dextran-coated charcoal suspension ( D C C ) and incubated for 1 0 m i n at 4°C. Incubations with D C C were terminated by centrifugation at 800 x g for 10 min, and aliquots were submitted to radioactive counting in a Packard T r i - C a r b liquid scintillation spectrometer using Instagel (Packard, Downers Grove, IL, U.S.A.) as the counting solution. Results we.re expressed as the percentage of [3H]-mibolerone specific binding. T h e relative binding affinities (RBA) for AR of 19norT, E T , and their 5~-reduced derivatives were also evaluated by their capability to displace b o u n d [3H]-mibolerone from rat ventral prostate androgen binding sites, as described above. T h e RBA and inhibition constants (Ki) of each steroid in displacement experiments were calculated according to the procedures described by Reel et al. [17] and C h e n g and Prusoff [18], respectively.
Assessment of androgenk: potency of unsaturated and 5~reduced steroids T o evaluate the androgenic potency of various natural and synthetic steroids and their 5~-reduced derivatives, two bioassays were used: the androgeninduced fl-glucuronidase activity in the mouse kidney and the androgen-induced growth of male rat accessory sex organs. T h e fl-glucuronidase bioassay was performed in adult mice castrated 5 days before the experiments. Animals were divided into eight groups (n = 7 - 9 each) and submitted to daily steroid treatments for five consecutive days. Steroids were dissolved in propyleneglycol (PG) and subcutaneously (s.c.) administered (40 #1); to enhance the solubility of E T and 5aE T , they were used in bioassays as their corresponding 17fi-acetate derivatives. T and D H T were given at a dose of 125 #g per day, whereas N E T , 5 a - N E T , 19norT, 5~-19norT, E T A c , and 5 a - E T A c were administered at 400 pg per day. T w o additional groups of intact (n = 5) and castrated (n = 5) mice, receiving vehicle alone, served as controls. At the end of the treatm e n t period the animals were killed and their kidneys removed immediately and homogenized in deionized water. Aliquots of kidney homogenates (200 #1; 16 m g protein) were submitted to fl-glucuronidase activity determination by the colorimetric m e t h o d of Fishman [19], with minor modifications, using the Sigma assay
123
kit reagents. Results were expressed as fl-glucuronidase units (U) per milligram of protein. T h e androgenic potency of unsaturated and 5~reduced steroids was also evaluated by their capability to induce ventral prostate and seminal vesicle growth in adult rats castrated eight days prior to the experiments. Animals were divided into eight groups (n = 7 - 9 each) and submitted to daily steroid treatments for 10 consecutive days. Steroids were dissolved in P G and administered s.c. (100 #1). T and D H T were given at the daily dose of 250 #g, whereas N E T , 5 ~ - N E T , 19norT, 5~-19norT, E T A c and 5aE T A c were administered at 2 m g per day. T w o additional groups of intact ( n = 9 ) and castrated (n = 12) rats, receiving vehicle alone, served as controls. At the end of the treatment period, animals were killed and their ventral prostates and seminal vesicles were immediately removed, blotted and weighed to the nearest 0.1 rag. Additional experiments were carried out to determine the effect of 5~-reduction u p o n the androgen agonistic, synergistic or antagonistic activities of N E T , 19norT, and E T using the mouse kidney fl-glucuronidase bioassay. Castrated male mice were divided into six groups (n = 8 each), and submitted to daily combined steroid treatments. Steroids were dissolved in P G and administered s.c. (40 #1). N E T , 5 ~ - N E T , 19norT, 5~-19norT, E T A c and 5 a - E T A c at a daily dose of 400 pg each were simultaneously administered with D H T (250/~g/day) for five consecutive days. T w o additional groups (n = 8 each) of castrated mice receiving either vehicle alone or D H T (250 pg/day) for five days served as controls. In vitro metabolic studies Ventral prostates from adult rats, castrated 48 h before the experiments, were homogenized in K r e b s Ringer phosphate buffer solution, p H 7.4. Tissue preparations (10 m g protein) were incubated in triplicate with a single concentration (2/~M) of either [3H]-labelled N E T or T (3 #Ci), in the presence or absence of 0.3 m M N A D P H in a final volume incubation of 1 ml. Incubations were carried out in a D u b n o f f metabolic incubator at 37.5°C for 2 h, using air as the gas phase. Tissue-less incubations were used as controls. At the end of the incubation period the steroids were extracted (4x) with three volumes of ethyl acetate. T h e organic extracts were partitioned between petroleum ether and 10% aqueous methanol. Methanolic extracts from the N E T incubations were added with 2.5 pg each of N E T , 5 a - N E T , 3~,5~N E T , and 3 f l , 5 a - N E T as steroid carriers, whereas 2.5 pg each of T , D H T , 3~,5a-androstanediol (3~,Sadiol) and 3fl,5~-androstanediol (3fi, Sa-diol) were added to T incubation extracts. Isolation and radiochemical purity of N E T and T metabolites were established by an identical m e t h o d to that of steroid carriers in both paper (petroleum ether: benzene:
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A.E. Lemus et al.
methanol: water; 5:1:5:1) and thin layer (benzene: absolute ethanol; 95:5) chromatographic systems and successive recrystallization to obtain a constant specific activity. T h e radioactive content in samples was determined in a Packard Tri-Carb liquid scintillation spectrometer, Model 2660 (Packard, Downers Grove, IL, U.S.A.). Counting efficiency of [3HI was 45% and quenching was corrected in all samples by external standardization. Formation of [3H]-NET and [3H]-T metabolites was calculated from the last crystal values and the results were expressed as pmol/mg protein/h.
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Competition of N E T and 5 ~ - N E T for androgen receptors T h e results of saturation analysis and linear sucrose gradient experiments, using [3H]-mibolerone as the radioligand, provided evidence of the presence of a specific 8.2 S cytosol-located androgen receptor in the rat ventral prostate, with an equilibrium dissociation constant of 1.71 n M and a saturation binding capacity of 2.3 nmol/mg of protein, confirming and extending our previous report using a different ligand [16]. T h e effects of increasing concentrations of nonradioactive N E T , 5~-NET, T and D H T upon the [3I-I]-mibolerone binding to androgen cytosol receptors from rat ventral prostate are shown in Fig. 1. T h e order to the affinity for the androgen receptor was: D H T > T >5or-NET>NET. T h e RBA and Ki values of each steroid are listed in Table 1. Competition of 19norT, E T and their 5~-reduced derivatives for androgen receptors T h e addition of increasing concentrations of radioinert 19norT, ET, and their 5~-reduced derivatives induced a varying degree of displacement of [3H]mibolerone from rat ventral prostate cytosol AR, as depicted in Fig. 2. T h e most potent steroid competitors for androgen binding sites were 5 ~ - l g n o r T and 19norT. 5~-ET also displayed a potent competitive effect, although to a lesser extent, whereas E T was effective only at high concentrations. T h e RBA and Ki values of each steroid are given in Table 1. Androgenic potency of N E T and 5 ~ - N E T T h e administration of N E T (400 pg/day for five days) to castrated mice induced an increase of kidney fl-glucuronidase activity to significantly higher levels ( P < 0.0001) than those observed in castrated control animals, whereas the administration of an identical
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S T E R O I D C O N C E N T R A T I O N (nM) Fig. 1. D i s p l a c e m e n t effect o f r a d i o i n e r t u n s a t u r a t e d a n d 5~r e d u c e d s t e r o i d s u p o n t h e specific b i n d i n g o f [ 3 H ] - m i b o l e r o n e to c y t o s o l a n d r o g e n r e c e p t o r s . A l i q u o t s o f v e n t r a l p r o s tate c y t o s o l (4.5 m g p r o t e i n / m l ) f r o m c a s t r a t e d a d u l t m a l e rats w e r e i n c u b a t e d o v e r n i g h t w i t h 2 n M [ 3 H ] - m i b o l e r o n e at 4°C (final i n c u b a t i o n v o l u m e 2 0 0 / d ) , in the a b s e n c e o r p r e sence of graded concentrations of non-labelled norethisterone (NET), 5~-dihydro-NET (5~-NET) (Panel A), and testostero n e (T), a n d 5 ~ - d i h y d r o t e s t o s t e r o n e ( D H T ) ( P a n e l B). T h e total b i n d i n g o f [ 3 H ] - m i b o l e r o n e in t h e a b s e n c e o f r a d i o i n e r t s t e r o i d s w a s set at 100%. E a c h p o i n t r e p r e s e n t s t h e m e a n o f three e x p e r i m e n t s i n t r i p l i c a t e . F o r details s e e t h e text.
dose of 5 ~ - N E T to castrated mice had no effect upon kidney enzyme activity, as shown in Fig. 3. T h e administration of T (125#g/day for five days) to castrated mice fully restored kidney fl-glucuronidase activity to levels observed in intact animals, whereas Table 1. Relative binding affinities (RBA) and inhibition constant (K~) of natural and synthetic steroidsfor androgen receptors Steroid competitors MB T DHT NET 5~-NET 19norT 5~-19norT ET 5~-ET
RBA* (%)
K i t (nM)
100.0 63.6 125.0 14.0 41.0 112.9 116.6 7.0 46.6
8.1 12.1 6.1 51.1 18.1 7.3 7.1 101.1 16.1
'~RBA was determined according to Reel et al. [17]. TKi values were calculated according to C h e n g and Prusoff [18].
A Paradoxical Effect of 5~-Reduction
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Fig. 2. C o m p e t i t i o n of radd[oinert u n s a t u r a t e d a n d 5ct-reduced s t e r o i d s for rat v e n t r a l p r o s t a t e a n d r o g e n b i n d i n g sites. C y t o s o l a n d r o g e n recepto:rs w e r e l a b e l l e d in v i t r o b y o v e r n i g h t i n c u b a t i o n s i d e n t i c a l to t h o s e d e s c r i b e d in Fig. 1. Increasing concentrations of the following radioinert synthetic s t e r o i d s w e r e a d d e d to t h e i n c u b a t i o n s : 1 9 - n o r t e s t o s t e r o n e ( 1 9 n o r T ) , 5ct-dihydro 1 9 - n o r T (5~t-19norT) ( P a n e l A), a n d 1 7 ~ - e t h y n y l t e s t o s t e r o n e ( E T ) , a n d 5 ~ - d i h y d r o E T (5~-ET) (Panel B). Each point represents the mean of three experi m e n t s in triplicate.
D H T given at an identical dose exhibited a significantly greater ( P < 0.001) potency than T to increase kidney e n z y m e activity in castrated mice, as depicted in Fig. 3. Similar results were obtained w h e n N E T , T, and their 5~-reduced derivatives were examined for their capability to induce growth of accessory sex organs in the castrated male rat. Indeed, N E T administered at the daily dose of 2 mg for 10 consecutive days induced a significant increase ( P < 0 . 0 0 0 1 ) in the weights of the ventral prostate and seminal vesicles compared with vehicle-treated castrated animals (Fig. 4). On the contrary, treatment with 5 ~ - N E T , administered at an identical dose, failed to induce ventral prostate and seminal vesicle growth in castrated rats, as s h o w n in Fig. 4. T h e administration of D H T (250 pg/day for 10 days) induced a significant increase in the weights of the ventral prostate (P<0.0001) and the seminal vesicles ( P < 0 . 0 0 1 ) compared with vehicle-treated castrated male rats. T h e androgenic effect of D H T was significantly more
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Fig. 3. Effect o f n o r e t h i s t e r o n e ( N E T ) , t e s t o s t e r o n e (T) a n d their 5~-reduced derivatives (5~-NET and DHT) on mouse k i d n e y f l - g l u c u r o n i d a s e activity. A d u l t c a s t r a t e d m a l e Balb/c m i c e w e r e t r e a t e d w i t h s t e r o i d s ( N E T a n d 5 ~ - N E T : 400/~g! day; T a n d D H T : 125/zg/day) for five c o n s e c u t i v e d a y s . I n t a c t (I) a n d c a s t r a t e d (C) v e h i c l e - t r e a t e d m i c e s e r v e d as c o n t r o l groups. Each bar shows the mean and standard deviation ( S D ) for s e v e n to n i n e a n i m a l s . * P < 0.0001 c o m p a r e d w i t h t h e C v e h i c l e - t r e a t e d group; * * P < 0.001 c o m p a r e d w i t h t h e T - t r e a t e d g r o u p . F o r e x p e r i m e n t a l details s e e t h e text.
potent than T administered at an identical dose, particularly in terms of ventral prostate growth ( P < 0.001), as depicted in Fig. 4. T h e observation that 5~-reduction of the N E T molecule diminished and almost cancelled its androgenlike potency, as assessed by two different bioassays, prompted us to examine the nature of this most striking finding.
Androgenic potency of 19norT, ET, and their 5~-derivarives
T h e administration of 19norT (400 ttg/day for five days) induced an increase of kidney fl-glucuronidase activity in castrated mice, reaching significantly higher levels ( P < 0.0001) than those observed in vehicletreated control animals (Fig. 5). T h e 5~-reduced derivative of 19norT, given at an identical dose, exhibited a significantly greater potency than 19norT ( P < 0.01), in terms of kidney fi-glucuronidase activity increase in castrated mice, as depicted in Fig. 5.
A.E. Lemus et al.
126 250
_
VENTRAL PROSTATE
SEMINAL
T
200 -
50
/ / / / / / / / / / / / / /
i 'i 1 I
C
T
DHT NET 5c~NET
,
7I
i
150
100
60
VESICLES
C
/ /
/
/
/
¢
"/
/ / / / / / /
/ / / / / / / / /
50
//A I/,4 e~ 40 g~
T h e administration of E T A c (400 #g/day for five days) induced a significant increase ( P < 0.0001) of kidney fi-glucuronidase activity in castrated male mice, as s h o w n in Fig. 5. T h e 5~-reduced derivative of ETAc, given at an identical dose, also induced a rise of kidney /~-glucuronidase activity, although with a significantly lower ( P < 0.01) potency than E T A c (Fig. 5.) W h e n the potency of 19norT, ETAc, and their 5~reduced derivatives to induce accessory sex organ growth was assessed in castrated male rats, the results were similar but not identical to those obtained using the m o u s e kidney ~-glucuronidase assay. T h e administration of 19norT ( 2 m g / d a y for 10 days) to castrated rats resulted in a significant increase in the weights of the ventral prostate ( P < 0.0001) and the seminal vesicles ( P < 0.001). W h e n 5~-19norT was given at an identical dose, it induced an increase of both ventral prostate and seminal vesicle growth, with a significantly greater ( P < 0 . 0 0 1 ) potency than 19norT, as shown in Fig. 6. Interestingly, neither E T A c nor its 5~-reduced derivative, administered at the daily dose of 2 m g for 10 consecutive days to castrated male rats had any significant effect upon accessory sex organs.
Synandrogenic and antiandrogenic activities of NET, 19norT, ETAc and their 5~-derivatives T h e administration of N E T plus D H T and 19norT plus D H T revealed an androgenic synergistic effect of both synthetic steroids. Indeed, N E T and 19norT when given with D H T induced a significantly greater increase ( P < 0 . 0 1 and P < 0 . 0 0 0 1 , respectively) of
<
30
T
<
I/,I
T //A I/,4
k"
T DHT NET 5c~NET
Fig. 4. E f f e c t o f n o r e t h i s t e r o n e ( N E T ) , t e s t o s t e r o n e (T) a n d their 5~-reduced derivatives (5~-NET and DHT) on accessory s e x o r g a n g r o w t h i n t h e c a s t r a t e d m a l e rat. A d u l t c a s t r a t e d animals were treated with steroids (NET and 5~-NET: 2mgl day; T a n d D H T : 250 pg/day) f o r 10 c o n s e c u t i v e d a y s . I n t a c t (I) a n d c a s t r a t e d (C) v e h i c l e = t r e a t e d r a t s s e r v e d as c o n t r o l g r o u p s . E a c h b a r s h o w s t h e r e s u l t s ( m e a n + S D ) in t e r m s o f t h e w e i g h t (rag) o f t h e v e n t r a l p r o s t a t e a n d t h e s e m i n a l v e s icles for s e v e n to n i n e a n i m a l s . * P < 0.0001 c o m p a r e d w i t h t h e C v e h i c l e - t r e a t e d g r o u p ; * * P < 0.001 c o m p a r e d w i t h t h e C v e h i c l e = t r e a t e d g r o u p ; , P < 0.001 c o m p a r e d w i t h t h e T - t r e a ted group.
//A
//,4
© 20 C..)
//, I//
I/,,I I/,I //,4
/~/
I.ill
t- i :
I1.,I //2
10
//,1 //,i //,1 1
.
C
~
9"/
V,~
//,
NorT
5c~NorT
ETAc
Ii'/I
5~ETAc
Fig. 5. E f f e c t o f s y n t h e t i c u n s a t u r a t e d a n d 5 ~ = r e d u c e d s t e r oids on mouse kidney ~-glucuronidase activity. Adult c a s t r a t e d m a l e B a l b / c m i c e w e r e t r e a t e d w i t h 19=nortestos= terone (NorT), 17~=ethynyltestosterone acetate (ETAc) and t h e i r 5 ~ = r e d u c e d d e r i v a t i v e s (5mNorT a n d 5 ~ E T A c ) at t h e d a i l y d o s e o f 400 pg f o r five c o n s e c u t i v e d a y s . I n t a c t (I) a n d c a s t r a t e d (C) v e h i c l e = t r e a t e d m i c e s e r v e d as c o n t r o l g r o u p s . T h e r e s u l t s a r e e x p r e s s e d as t h e k i d n e y e n z y m e a c t i v i t y ( m e a n _+ S D ) f o r e a c h g r o u p (n = 7 - 9 ) o f a n i m a l s . * P < 0.0001 c o m p a r e d w i t h t h e C v e h i c l e = t r e a t e d g r o u p ; * * P < 0.01 corn= p a r e d w i t h t h e N o r T = t r e a t e d m i c e ; 0 P < 0.01 c o m p a r e d w i t h the ETAc=treated group.
kidney/%glucuronidase activity in castrated male mice compared with animals treated with D H T alone (Fig. 7). On the contrary, when 5 ~ - N E T and 5~19norT were given simultaneously with D H T a lack of synandrogenic effect of these reduced derivatives was observed in terms of m o u s e kidney enzyme activity (Fig. 7). T h e simultaneous administration of E T A c with DHT resulted in a significant diminution (P < 0.0001) of the D H T - i n d u c e d increase of kidney fl-glucuronidase activity in castrated mice. Indeed, the levels of kidney enzyme activity in animals treated with E T A c plus D H T resembled those found in vehicle-treated castrated mice. Interestingly, the c o m bined administration of 5~-ETAc plus D H T to castrated mice induced an increase o f kidney //-glucuronidase activity similar to that observed in animals treated with D H T alone (Fig. 7).
A Paradoxical Effect of 5~-Reduction 300 -
VENTRAL PROSTATE
SEMINAL VESICLES
250 -
~
~ T
~. 2 0 0 -
D
•
,]-
[3Hl-Substrate
/ / /
150
//
1oo
/ /
_~
Norethisterone (NET)
Testosterone(T)
/
~-
/
0 5~NorT 5~ETAc NorT ETAc
C
150 >
F100 _
T
< r~ z ©
50
N o cofactor
21.28 12.30 8.90 82.44 36.32 23.27
6.62 2.56 1.10 20.61 9.84 7.30
Metabolism of N E T and T in the rat prostate
T h e results from the in vitro incubations of rat vental prostate homogenates with radiolabelled N E T and T demonstrated that b o t h substrates undergo extensive bioconversion to A-ring reduced metabolites. As shown in Table 2, the enzyme-mediated A-ring reduction of both N E T and T was N A D P H - d e p e n d e n t , as has been previously described [20]. T h e in vitro metabolic fate of N E T and T in the prostate was similar, although T was a m o r e efficient substrate for the steroid metabolizing enzymes. T h e 5ct-dihydro derivatives of N E T and T were the major isolated incubation products followed by their corresponding 3~and 3/3-tetrahydro metabolites (Table 2).
DISCUSSION
200
E
U
5c~-NET 3~,5~-NET 3fl,5~-NET 5~-DHT 3a,5~-diol 3fl,5~-diol
pmol/mg protein/h NADPH
5c~NorT 5e~ETAc NorT ETAc
Fig. 6. A n d r o g e n i c p o t e n c y o f 1 9 - n o r t e s t o s t e r o n e ( N o r T ) , 17~-ethynyltestosterone acetate (ETAc) and their 5~-reduced m e t a b o l i t e s (5mNorT a n d 5.0dETAc) e v a l u a t e d b y t h e i r effects on t h e w e i g h t o f a c c e s s o r y s e x o r g a n s in t h e c a s t r a t e d m a l e rat. U n s a t u r a t e d a n d r e d u c e d s t e r o i d s w e r e a d m i n i s t e r e d ( 2 m g l d a y ) for 10 c o n s e c u t i v e d a y s to a d u l t m a l e rats c a s t r a t e d 8 d a y s b e f o r e t h e e x p e r i m e n t s . I n t a c t (I) a n d c a s t r a t e d (C) v e h i c l e - t r e a t e d a n i m a l s s e r v e d as c o n t r o l g r o u p s . T h e r e s u l t s a r e g i v e n as t h e w e i g h t o f t h e v e n t r a l p r o s t a t e a n d t h e s e m i n a l v e s i c l e s ( m e a n + S D ) for e a c h anim a l g r o u p (n = 7 - 9 ) . * P < 0.0001 c o m p a r e d w i t h the C v e h l c l e - t r e a t e d g r o u p ; **F' < 0.001 c o m p a r e d with the C v e h i c l e - t r e a t e d group; , P < 0.001 c o m p a r e d w i t h t h e N o r T treated group.
03
[3H]-isolated metabolites
F o r details see M a t e r i a l s a n d M e t h o d s .
/ C
Table 2. In vitro formation of A-ring reduced metabolites of f H]-norethisterone and testosterone in the rat ventral prostate
I
//
127
_
//l /A
D u D o C
DHT
NET +
5aNET +
NorT +
5c~NorT +
ETAc +
5aETAc +
DHT
DHT
DHT
DHT
DHT
DHT
Fig. 7. S y n a n d r o g e n l c a n d a n t i a n d r o g e n i c activities o f n o r ethisterone (NET), 19-nortestosterone (NorT), 17~-ethynyltestosterone acetate (ETAc) and their 5~-reduced metabolites in c a s t r a t e d B a l b l c m i c e . T h e effect o f u n s a t u r a t e d a n d r e d u c e d s t e r o i d s (400/z g/day) a d m i n i s t e r e d s i m u l t a n e o u s l y w i t h D H T (250/ag/day) for five c o n s e c u t i v e d a y s w a s a s s e s s e d b y t h e i r c a p a b i l i t y to m o d i f y t h e m o u s e k i d n e y /~-glucurould a s e activity. C a s t r a t e d a n i m a l s t r e a t e d w i t h D H T p l u s v e h i c l e ( D H T ) or v e h i c l e a l o n e (C) s e r v e d as c o n t r o l g r o u p s . T h e r e s u l t s a r e g i v e n as t h e k i d n e y e n z y m e a c t i v i t y ( m e a n + S D ) for e a c h a n i m a l g r o u p (n = 8), * P < 0.01, * * P < 0.0001, , P < 0.0001 c o m p a r e d w i t h t h e D H T + v e h i c l e - t r e a t e d g r o u p .
T h e results presented herein provide strong evidence that enzyme-mediated 5u-reduction of the synthetic progestin norethisterone enhances its relative binding affinity for the intracellular androgen receptor, but diminishes and almost cancels its androgenlike effects. Indeed, experiments carried out in rat ventral prostates using displacement analysis revealed a significant increase in the relative binding affinity of 5 u - N E T to the 8.2 S cytosol-located androgen receptor, with a concomitant significant decrease in the Ki value c o m p a r e d with unchanged N E T (Table 1), confirming and extending previous results from this laboratory [12]. W h e n the androgenic potency of N E T and 5 ~ - N E T was assessed by two different bioassays, it became evident that A-ring reduction of the N E T molecule results in a complete loss of its potent androgen-like effects as shown in Figs 3 and 4. These findings are in a sharp contrast with the well-established concept that formation of the 5~- (trans A/B ring junction) derivative of naturally occurring testosterone enhances both its RBA to the AR and its androgenic potency. These striking observations raise the important question as to why 5c~-reduction of N E T , a 19-nor, 17~-ethynyl testosterone derivative, induced a dra-
128
A.E. Lemus et al.
matic diminution of its androgenicity in spite of the fact that 5 ~ - N E T exhibits a high RBA for the androgen receptor, and p r o m p t e d us to delineate the nature of this paradoxical effect. This unexpected effect of 5~-reduction on N E T could be caused by a more rapid bioconversion of 5~N E T into less active molecules. T o rule out this possibility, a study aimed to compare the in vitro metabolism of radiolabelled N E T and T in the rat ventral prostate was conducted. T h e results disclosed that both N E T and T were efficiently bioconverted to several A-ring reduced c o m p o u n d s , including their corresponding 5~-dihydro and their 3~- and 3r-tetrahydro derivatives. T h e formation rate of A-ring reduced metabolites of N E T and T presented a similar pattern, it was N A D P H - d e p e n d e n t and it occurred to an almost identical extent, although T was a m o r e efficient substrate than N E T (Table 2). Indeed, the 5~-reduction of N E T seems to be slower than that of T, whereas the further 3~- or 3r-reduction of 5~N E T appears to be slightly faster than that of D H T . T o assess whether the lack of the C-19 methyl group or the presence of the 17~-ethynyl moiety of the N E T molecule may be involved in the paradoxical effect of 5a-reduction upon this synthetic progestin, a series of experiments was conducted, which aimed at evaluating the A R affinities and androgenic potency of 19norT, E T and their 5a-reduced derivatives. T h e results showed that 5~-reduction of 19-norT neither increased nor decreased its affinity for the androgen receptor, whereas 5~-reduction of E T significantly enhanced its relative binding to the androgen receptor (Fig. 2 and T a b l e 1). These combined results provided an explanation for the finding that 5~-reduction of N E T enhanced (two- to three-fold) the specific interaction with androgen binding sites in a m a n n e r similar to that observed with T, its parent c o m p o u n d (Fig. 1 and T a b l e 1). T h e androgenic potency of 19norT, E T A c and their corresponding 5~-derivatives was assessed by their effects upon the fl-glucuronidase activity in the mouse kidney, and upon the growth of rat accessory sex organs. T h e results showed that 5~-reduction of 19norT significantly increased the kidney enzyme activity (Fig. 5), in a fashion similar to that observed with T after its 5~-reduction (Figs 3 and 4). A similar increase in the androgenic potency of the 19norT molecule, induced by its 5~-reduction, was d e m o n strated in terms of the weights of the ventral prostate and seminal vesicles in castrated male rats (Fig. 6). These results were interpreted as indicating that removal of the C-19 methyl group from the T molecule does not interfere with the 5~-reductioninduced amplification of its androgenic potency. On the contrary, 5~-reduction of E T A c resulted in a significant diminution of its capability to increase flglucuronidase activity in the mouse kidney bioassay (Fig. 5). As shown in Fig. 6, E T A c was unable to re-
store the weights of the ventral prostate and seminal vesicles of castrated male rats, indicating that this c o m p o u n d was completely devoid of androgenic potency. Furthermore, 5~-reduction of E T A c had no effect at all on its biological potency (Fig. 7). T h e data indicate that the addition of a 17~-ethynyl group to the testosterone molecule precludes the androgen amplifying effect induced by 5~-reduction, and offers a plausible explanation for the paradoxical effect of Aring reduction u p o n norethisterone. These results confirm and extend the observations of Morali et al. [13] that N E T , when administered with estradiol-17fl to long-term castrated rats, was able to restore full copulatory behaviour, whereas 5~N E T failed to induce masculine behaviour when given with estradiol 17-fl, indicating that 5~-reduction of N E T results in a significant diminution of its behavioural androgenic potency. This paradoxical effect of 5~-reduction u p o n the androgen-like potency of N E T also seems to occur with its progestational activity. Guti6rrez-Sagal et al. [21] have demonstrated that Aring reduction of N E T significantly diminishes its progestational activity, as assessed by the endometrial content of the uteroglobin gene expression products in prepuberal rabbits; furthermore, the 5~-reduced N E T derivative exhibits potent anti-progestational activity. Reel et al. [17] have also demonstrated the capability of 5 ~ - N E T to inhibit progesterone-induced endometrial proliferation. M o r e recently Castro et al. [22] have reported that 5 ~ - N E T displays anti-implantation effects, when given to m a t e d adult female rabbits, with a potency similar to that observed with RU-486, the synthetic steroidal antiprogestin. Because the synandrogenic and antiandrogenic effects of several synthetic progestins have been well d o c u m e n t e d [23], we felt it would be of interest to assess the effect of 5~-reduction upon N E T , 19norT, and ETAc, in terms of their androgen synergistic and antagonistic activities. T h e simultaneous administration of D H T plus unchanged N E T revealed that this synthetic progestin exhibited a synandrogenic effect in the mouse kidney, as was expected, whereas the simultaneous administration of D H T plus the 5~-reduced N E T derivative had neither synandrogenic nor androgenic additive effects. This observation is consistent with the diminution of the androgenic activity of the N E T molecule induced by its 5~-reduction. T h e lack of antiandrogenic effects of 5~-NET, at the dose level employed, may be explained by the observations of Mowszowicz et al. [23], indicating that the synandrogenic and antiandrogenic effects of synthetic progestins vary in a dose-dependent manner. Similar results were observed following the administration of 19norT plus D H T and 5~-19norT plus D H T . Indeed, 19norT exhibited a synandrogenic potency identical to that of N E T , whereas 5~-19norT did not. Interestingly, E T A c displayed an antiandrogenic effect when admi-
A Paradoxical Effect of 5 ~ - R e d u c t i o n nistered
simultaneously
with
DHT,
as
shown
in
F i g . 7, w h e r e a s 5 c ~ - E T A c e x h i b i t e d n e i t h e r a n t i a n d r o genic nor synandrogenic effects in the mouse kidney. These data indicate that 5~-reduction modifies the expression of androgen synergistic and antagonistic activities of NET. T a k i n g all t h e s e r e s u l t s t o g e t h e r , it c a n b e c o n cluded that 5e-reduction of NET does not amplify, b u t d o e s s u p p r e s s its a n d r o g e n - l i k e a c t i v i t y i n t a r g e t o r g a n s , i n s p i t e o f its h i g h R B A t o t h e a n d r o g e n receptor. This paradoxical effect of 5e-reduction seems to be caused by the presence of a 17e-ethynyl group in the NET molecule. Whether similar findings can be obtained in isolated hormone-responsive systems remains to be ascertained, and deserves further experimental work in culture-maintained androgend e p e n d e n t a n d a n d r o g e n - s e n s i t i v e cells. T h e o v e r a l l data provide additional support for the concept that the metabolism of synthetic progestins at target o r g a n s m o d u l a t e s t h e i r h o r m o n e - l i k e effects. Acknowledgements--This study was supported in part by grants from the Rockefeller Foundation (RF 93029-allocation 5), New York and The Special Programme :in Human Reproduction of the World Health Organization, Geneva. The authors are indebted to Ms B. Alarc6n and Mr J. L. Ramirez for assistance with the preparation of the manuscript.
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9. Stillwell W. G., Homing E. C., Homing M. G., Stillwell R. N. Zlatkis A.: Characterization of metabolites of steroid contraceptives by gas chromatography and mass spectrometry. J. Steroid Biochem. 3 (1972) 699-706. 10. Braselton W.E. Jr, Lin T. J., Ellegood J. O., Mills T. M. and Mahesh V. B.: Accumulation of norethindrone and individual metabolites in human plasma during short- and long-term administration of a contraceptive dosage. Am. J. Obstet. Gynecol. 133 (1979) 154-160. 11. Larrea F., Vilchis F., Chfivez B., Prrez A. E., Garza-Flores J. P~rez-Palacios G.: The metabolism of 19-nor contraceptive progestins modulates their biological activity at the neuroendocrine level, ft. Steroid Biochem. 27 (1987) 657-663. 12. Chfivez B. A., Vilchis F., Pdrez A. E., Garcia G. A., Grillasca I. and Prrez-Palacios G.: Stereospecificity of the intraceUular binding of norethisterone and its A-ring reduced metabolites. J. Steroid Biochem. 22 (1985) 121-126. 13. Morali G., Lemus A. E., Oropeza V. M., Garcia G. A. and Prrez-Palacios G.: Induction of male sexual behavior by norethisterone: role of its A-ring reduced metabolites. Pharmacol. Biochem. Behav. 37 (1990) 477-484. 14. Vilchis F., Chfivez B., Pfirez A. E., Garcia G. A., Angeles A. P6rez-Palacios G.: Evidence that a non-aromatizable metabolite of norethisterone induces estrogen-dependent pituitary progestin receptors. J. Steroid Bioehem. 24 (1986) 525-531. 15. Still W. C., Kahan M. and Mitra A.: Rapid chromatographic technique for preparative separations with moderate resolution. J. Organ. Chem. 43 (1978) 2923-2925. 16. Vilchis F., Hernfindez A., P~rez A. E. and Prrez-Palacios G.: Hormone regulation of the rodent Harderian gland: binding properties of the androgen receptor in the male golden hamster. J. Endoer. 112 (1987) 3-8. 17. Reel J. R., Humphrey R. R., Shih Y.-H., Windsor B. L., Sakowski R., Creger P. L. and Edgren R. A.: Competitive progesterone antagonists: receptor binding and biologic activity of testosterone and 19-nortestosterone derivatives. Fertil. Steril. 31 (1979) 552-561. 18. Cheng Y.-C. and Prusoff W. H.: Relationship between the inhibition constant (Ki) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction. Biochem. Pharmacol. 22 (1973) 3099-3108. 19. Fishman W. H., Springer B. and Brunetti R.: Application of an improved glucuronidase assay method to the study of human blood ~-glucuronidase. J. Biol. Chem. 173 (1948) 449-456. 20. Frederiksen D. W. and Wilson J. D.: Partial characterization of the nuclear reduced nicotinamide adenine dinucleotide phosphate: delta 4-3-ketosteroid 5~-oxidoreductase of rat prostate. J. Biol. Chem. 246 (1971) 2584-2593. 21. Gutirrrez-Sagal R., Prrez-Palacios G., Langley E., Pasapera A. M., Castro I. and Cerbrn M. A.: Endometrial expression of progesterone receptor and uteroglobin genes during early pregnancy in the rabbit. Molec. Reprod. Dev. 34 (1993) 244-249. 22. Castro I., Cerb6n M. A., Pasapera A. M., Guti~rrez-Sagal R., Garcla G. A., Orozco C., Camacho-Arroyo I., Anzaldua R. Prrez-Palacios G.: Molecular mechanisms of the antihormonal and antiimplantation effects of norethisterone and its A-ring reduced metabolites. Molec. Reprod. Dev. 40 (1995) 157-163. 23. Mowszowicz I., Bieber D. E., Chung K. W., Bullock L. P. and Bardin C. W.: Synandrogenic and antiandrogenic effect of progestins: comparison with nonprogestational antiandrogens. Endocrinology 95 (1974) 1589-1599.