receiving MPA have failed to demonstrated an

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1 Direct effects of medroxyprogesterone acetate (MPA) and megestrol acetate (MGA) on rat testicular steroidogenesis Robert L. Barbieri and Kenneth J. Ryan Laboratory ofhuman Reproduction and Reproductive Biology, Harvard Medical School, Boston, Massachusetts 2115 Abstract. The effects of MPA and MGA on rat testicular steroidogenesis were examined by studying: 1) serum testosterone in hcg primed animals treated with MPA or MGA, 2) testosterone synthesis in rat Leydig cells cultured with MPA or MGA, 3) MPA and MGA binding to rat testis microsomal cytochrome P45 and 4) MPA and MGA inhibition of enzymes of rat testicular steroidogenesis. In immature rats receiving 1. IU of hcg per day 2 mg/kg of MPA or MGA reduced serum testosterone by 57 and 56%, respectively. In mature male rats receiving 5. IU of hcg per day 2 mg/kg of MPA or MGA reduced serum testosterone by 4 and 29%, respectively. In rat interstitial cells cultured with ng ofrat LH, 1 \g=m\m MPA or MGA inhibited testosterone production by 32 and 23%, respectively. Addition of MPA or MGA to microsomal preparations resulted in a type I cytochrome P45 difference spectrum. MPA and MGA inhibited rat testicular 17\g=a\hydroxylase,17,2lyase, and the 3\g=b\and 17\g=b\hydroxysteroiddehydrogenases. These findings suggest that MPA and MGA inhibit rat testicular steroidogenesis in vivo and in vitro. Medroxyprogesterone acetate (MPA) and megestrol acetate (MGA) are two clinically important synthetic 6methyl progestins. The 6methyl progestins have been given to boys and men for the treatment of a variety of disease states: 1) precoci ous puberty (Collip et al. 1964; Kaplan et al. 1968), 2) prostatic hypertrophy (Geller et al. 1976), and 3) Trivial names: medroxyprogesterone acetate (MPA) 4pregnen6a17aol3,2dione acetate; megestrol acetate (MGA) 4,6pregnadiene6methyl 17aol3,2dione acetate. sex offending behaviour (Money 197). In addi tion, MPA has been successfully employed as a male contraceptive agent (Frick et al. 1977; Meló & Coutinho 1977; AlvarezSanchez et al. 1977). The efficacy of the 6methyl progestins in the diverse clinical settings noted above might be due to their ability to suppress circulating testosterone. Investigators have proposed three mechanisms by which MPA might lower circulating testosterone: 1) by increasing the metabolic clearance rate of testosterone, 2) by decreasing circulating gonado trophins and 3) by directly interfering with Leydig cell steroidogenesis. Controversy continues as to the relative importance of each of these mecha nisms. Altman et al. (1972) have demonstrated that MPA induces hepatic testosterone A ring reductase activity in the rat liver. They postulated that MPA might thereby increase the metabolic clearance rate (MCR) of testosterone. In support of these findings Gordan et al. (197) reported that MPA increased the MCR of testosterone in nomal men and wo men. However, other experiments in boys and men receiving MPA have failed to demonstrated an altered MCR of testosterone (Rivarola et al. 1968; Clark et al. 1972; Nolten et al. 1976). Rivarola et al. (1968), Gordan et al. (197), Clark et al. (1972), and Nolten et al. (1976) have demon strated that MPA decreases the production rate of testosterone. MPA probably decreases the produc tion rate of testosterone by both inhibiting gonado trophin secretion and by directly inhibiting Leydig cell steroidogenesis. In boys and men MPA pro

2 duces a decrease in serum gonadotrophin concen trations in the range of 5% (Rifkind et al. 1969; Sadoff & Lusk 1974; Meyer et al. 1977). Evidence for a direct effect of MPA on Leydig cell steroidogenesis includes: 1) the administration of MPA to a boy with a hypothalamic hamartoma secreting LRH produced a large reduction in plasma T without altering plasma LH or FSH (Judge et al. 1977), and 2) MPA directly suppresses 17ßhydroxysteroid dehydrogenase activity in a dult rat testis (Satyaswaroop & Gurpide 1978). The purpose of this paper is to report further evidence of a direct effect of the 6methyl progestins on rat testicular steroidogenesis. Materials and Methods Unlabelled progesterone, 17ahydroxyprogesterone, and androstenedione were purchased from Steraloids and recrystallized before use. [l,2,6,7,3h]progesterone (SA, 9 Ci/mmol), 17ahydroxy[I,23H]progesterone (SA, 4 Ci/mmol), and [l,23h]androstenedione (SA, 58.6 Ci/mmol) were purchased from New England Nu clear and purified by thin layer chromatography (chloro form : methanol 98.5:1.5). Medroxyprogesterone acetate was a gift of the Upjohn Company. Megestrol acetate was a gift of MeadJohnson. Rat LH (rlh) was gift of the NIAMDD. All other reagents were purchased from Sigma. Administration ofmpa and MGA to immature weanling and mature male rats Fifteen day old weanling and 6 day old adult male SpragueDawley rats (CD strain, Charles River Labs) were kept under controlled lighting. The weanling rats were kept in litters of ten animals. In the experiments with the immature weanling rats the animals were divided into nine groups and were treated daily for five days according to the following protocol: Group 1 vehicle, Group 2, 3 MPA or MGA (2 mg/kg), Group 4 hcg 1. IU, Group 5, 6 MPA or MGA (2 mg/kg) and hcg 1. IU, Group 7 hcg. IU and Group 8, 9 MPA or MGA (2 mg/kg) and hcg. IU. In the experiments with the mature male rats the animals were divided into six groups and were treated daily for five days according to the following protocol: Group 1 vehicle. Group 2, 3 MPA or MGA (2 mg/kg), Group 4 hcg 5. IU and Group 5, 6 MPA or MGA (2 mg/kg) and hcg 5. IU. All injec tions were given sc. The steroids were administered as a crystalline suspension in saline. Twelve hours after the last injection blood was collected by cardiac puncture and assayed for testosterone (Challis et al. 1975). MPA and MGA had less than.19? crossreactivity with testo sterone for the testosteronespecific antibody. Isolated interstitial cell incubations Interstitial cells were isolated by the method of Dufau Be Catt (1974) and incubated as previously described (Barbieri et al. 1977). The final volume of the incubation was.4 ml. Preparation ofmicrosomal suspensions, spectral determinations and enzyme assays Microsomes were prepared from 6 dav old male rats as previously outlined (Barbieri et al. 1977). The microso mal fraction was used to perform all spectral determina tions. One ml cuvettes were employed. For the dehydrogenase assays the substrate concentration was. im. For all enzymatic determinations the final incubation volume was.4 ml. Effect of MPA and MGA on serum testosterone castrated in male rats implanted with testosterone silastic implants Sixty day old male rats were castrated and silastic cap sules (.1 cm OD,.62 cm ID. 4 cm in length) containing testosterone were implanted in each rat. After 4 weeks the rats were divided into three groups with eight rats in each group. One group received vehicle, the other two groups received 2 mg/kg of MPA or MGA. Sixteen hours after the injection the rats were bled by cardiac puncture and the serum was assayed for testo sterone. Table 1. Effect of medroxyprogesterone acetate (MPA) 2 mg/kg; and megestrol acetate (MGA) 2 mg/kg on serum testosterone concentration in immature male rats ± hcg. Treatment Serum testosterone (Xng/ml ± sn N = ) Vehicle.95 ±.23 MPA.51 ±.15* MGA.46 ±.16* Vehicle + hcg ( 1. IU) 8.71 ± 2.73 MPA 4 hcg ( 1. IU) 3.72 ± 1.6** MGA +hcg(l.oiu) 3.87 ± 1.39** Vehicle + hcg (. IU) ± 2.47 MPAHhCG(lO.OIU) 8.98±1.46*** MGAlhCG(lO.OIU) 8.83 ± 2.43*** * P <.1 compared to vehicle. ** P <.1 compared to vehicle + hcg (1. IU) ** P <.5 compared to vehicle 4 hcg (. IU)

3 Results Effect of MPA and MGA on serum testosterone in immature and adult male rats Table 1 shows that immature male rats have low basal levels of testosterone which can be increased by treatment with hcg. MPA and MGA signifi cantly decreased serum testosterone in rats treated with vehicle or hcg. In the rats receiving hcg approximately 9% of the serum T could be attri buted to the exogenously administered gonadotro phin. In the rats receiving 1. IU of hcg 2 mg/kg of MPA or MGA decreased the serum testosterone by 57 and 56%, respectively. In animals receiving. IU of hcg MPA and MGA decreased serum testosterone by 22 and 23%, respectively. Table 2 shows that in adult male rats MPA and MGA significantly decreased serum testosterone in vehicle or hcg treated animals. In the rats receiv ing hcg, approximately 8% of the serum testo sterone could be attributed to the exogenously administered gonadotrophin. In rats receiving 5 IU hcg, PMA and MGA decreased serum testo sterone by 4 and 29%, respectively. Effect of MPA and MGA on testosterone production in isolated interstitial cells In interstitial cells stimulated with ng of rat LH, 1 XM MPA and MGA inhibited testosterone pro duction by 32 and 23%, respectively (P<.1) (Fig. 1). Ten (IM MPA or MGA inhibited the production of testosterone by 54 and 36%, respec tively (P<.1). In cells not stimulated with rat Table 2. Effect of medroxyprogesterone acetate (MPA) 2 mg/kg; and megestrol acetate (MGA) 2 mg/kg on the serum testosterone concentration in adult male rats ±hcg 5 IU. Treatment Serum testosterone (X ng/ml ± sn N = 8) Vehicle 3.63 ±.61 MPA.84 ±.21* MGA.75 ±.16* Vehicle + hcg ± 2.99 MPA + hcg 11.8 ±3.62** MGA + hcg ± 1.46** * P <.1 compared to vehicle. ** P <.1 compared to vehicle 4 hcg Table 3 a and b. Interaction of MPA and MGA with rat testicular cytochrome P45. a) Binding of MPA and MGA to rat testicular cytochrome P45. Steroid MPA MGA Apparent Ks* (um) Spectrum type b) Inhibition of rat testicular microsomal 17ahydroxylase and 17,2lvase by MPA and MGA. Steroid MPA MGA K ** (um) 17ahydroxylase * Ks spectral dissociation constant.!* K[ enzymatic inhibition constant. Ki**(um) 17,2lyase LH both MPA and MGA produced smaller decre ments in testosterone production at each dose tested. Inhibition ofmicrosomal enzymes ofsteroidogenesis by MPA and MGA Addition of MPA or MGA to microsomal prepara tions resulted in a type I cytochrome P45 differ ence spectrum. A representative spectral titration is shown in Fig.2. The apparent spectral dissociation constants (Ks) are listed in Table 3a. Ks values varied less than 5% between experiments. MPA and MGA competitively inhibited the mi crosomal 17ahydroxylase and the 17,2lyase. The enzymatic inhibition constants (Ki) are shown in Table 3 b. A LineweaverBurk plot for the inhibition of the 17ahydroxylase by MPA is shown in Fig. 3. MPA and MGA inhibited the microsomal 3ß and the 17ßhydroxysteroid dehydrogenases (Table 4). Both MPA and MGA were better inhibi tors of the 3ßhydroxysteroid dehydrogenase than of the 17ßhydroxysteroid dehydrogenase. Effect ofmpa and MGA on serum testosterone in castrate male rats implanted with testosterone silastic implants In eight male castrated rats implanted sterone silastic implants with testo for four weeks the mean

4 i 3H 2H 3 2 OH o> i oh /h // I~7 I"6 I"5 I~4 "'!"b I"5 I"4 Medroxyprogesterone Acetate (M) Megestrol Acetate (M) Figs, la and b. a. Medroxyprogesterone acetate and b. Megestrol acetate inhibition of testosterone production in rat interstitial cell incubations. ng rlh per incubation; 2 ng rlh per incubation and A ng rlh per incubation. All points are the average of four incubations ± SD...5 < < Wavelength (nm) Fig. 2. Medroxyprogesterone acetate binding to rat testis microsomal cytochrome P45. Concentrations medroxyprogesterone acetate: 1) 2 (AM; 2) 4 \m; 3) 6 (ím; 4) 11 um; 5) 26 ím. Inset: Hanes plot of medroxyprogesterone acetate binding to cytochrome P45.

5 I7aHYDRXYLASE. I I r. /A. 9 / y t> / jt O / jf a/ / /.7 / / s' œ.5 //J*^^^ /. I 3 5 1/(progesterone) I/íM"') Fig. 3. LineweaverBurk plot of medroxyprogesterone acetate inhibition of the rat testis 17ahydroxylase. Medroxy progesterone acetate concentrations: [im; A A 1.5 XM; 5. (CM; A A. (J.M. Cytochrome P45 concentration 1. nm. Ki = 3. i.m. serum testosterone was 5.98 ± 1.7 ng/ml (X ± SD). In the animals which received 2 mg/kg of MPA or MGA 16 h before sacrifice, the serum testosterone was 5.84 ± 1.6 and 5.63 ± 1.5 ng/ml, respectively. The differences between the control and the MPA or MGA treated groups was not statistically signi ficant. Discussion The ability of MPA and MGA to lower testosterone in males is possibly related to three pharmacologie actions of these drugs: 1) decreased LH secretion, 2) altered MCR of testosterone, 3) direct inhibition of rat testicular steroidogenesis. The ability of MPA to suppress circulating LH is well established and no new evidence on this point is presented here. In the male rat it has been shown that within 16 h of a large parenteral dose of MPA hepatic testo sterone A ring reductase activity increases signifi cantly (Altman et al. 1972). Based on this observa tion it has been postulated that MPA increases the MCR of testosterone. Since circulating levels of a steroid are determined by production rate (PR) and MCR we decided to examine the effects of MPA and MGA on serum testosterone in a model system where PR of testosterone is constant, i.e., castrated male rats with testosterone silastic im plants. In this model system the administration of a Table 4. Inhibition of rat testis microsomal steroid dehydrogenases by MPA and MGA. Enzyme Inhibitor ((AM) MPA Relative rate of product formation Inhibitor ((ím) MGA Relative rate of product formation 3ßHydroxvsteroid dehydrogenase 17ßHydroxvsteroid dehydrogenase I

6 large parenteral dose of MPA or MGA did not produce a change in serum testosterone. This would suggest that in this experiment MPA and MGA did not affect the MCR of testosterone. The hypothesis that MPA or MGA can directly suppress testicular steroidogenesis was supported by the in vivo observation that MPA and MGA decrease serum testosterone in the hcg treated immature and mature male rat. In this model system the majority of testosterone synthesis is supported by the exogenous gonadotrophin. Un less MPA and MGA can alter the clearance of hcg the above observation would suggest that MPA and MGA can directly decrease testosterone synthesis without decreasing circulating gonadotrophins. The observation that MPA and MGA decreased testosterone production in rat interstitial cell incu bations provides in vitro evidence that MPA and MGA can directly inhibit testicular steroidogenesis The results presented here indicate that MPA and MGA inhibit multiple enzymes or rat testicular steroidogenesis including the 17ahydroxylase, the 17,2lyase, and the 3ß and 17ßhydroxysteroid dehvdrogenases. Inhibition of rat testis 17ßhydroxysteroid dehydrogenase by MPA has been pre viously reported (Satyaswaroop & Gurpide 1978). In addition, the inhibition of human corpus luteum 3ßhydroxysteroid dehydrogenase by medroxy progesterone and megestrol has been reported by Saure et al. (1977). Aakvaag (197) has observed that chlormadinone acetate, a compound closely related to MPA and MGA, is a potent inhibitor of porcine ovarian 3ßhydroxysteroid dehydrogen ase. The inhibition of steroid dehvdrogenases by synthetic progestins is probably due to direct bind ing of the synthetic progestin to the active site of the dehydrogenase. However, there is no available evidence to support this hypothesis. Cytochrome P45 has been implicated as the terminal oxidase of the testicular 17ahydroxylase (Menard & Purvis 1973) and the 17,2iyase (Betz & Michels 1973). Since MPA and MGA can elicit a type I cytochrome P45 binding spectrum when added to microsomal suspensions and can competi tively inhibit the 17ahydroxylase and the 17,2 lyase, it can be inferred that MPA and MGA bind to the active site of these two enzymes. This inference is further supported by the similarity of the spec tral dissociation constants and the enzymatic inhibi tion constants for the binding of MPA and MGA to the 17ahydroxylase and the 17,2lyase. The search for potent inhibitors of steroid hydroxvlases requires an efficient method for screen ing promising compounds in order to select a few for detailed analysis. The data presented in this paper suggest that the binding of MPA and MGA to the active site of the 17ahydroxylase and the 17,2lvase can be quantitatively described with equal precision by spectral or enzymatic techni ques. Other investigators have previously reported that when assaying for inhibitors of steroid hydroxylases in the testis (Barbieri et al. 1977), the adrenal (Cheng et al. 1976) and the placenta (Uzgiris et al. 1977), the spectral and enzymatic techni ques provide quantitatively equivalent results. Since a spectral assay is at least an order of magni tude less time consuming than an enzymatic assay, spectral analysis of drug binding to cytochrome P45 in steroidogenic tissues is probably the pre ferred method for screening for inhibitors of ste roid hydroxylases. Acknowledgments This work was supported bv grants from the Rockefeller Foundation (RF 654) and the USPHS (HD 79234). References Aakvaag A (197): Steroid formation in porcine ovarian tissue in vitro. Acta Endocrinol (Kbh) 65: Altman K, Gordan G G, Southren A L, Vittek J & Wilker S (1972): Induction of hepatic testosterone Aring reductase by medroxyprogesterone acetate. Endocri nology 9: AlverezSanchez F, Faundes A, Brache V & Leon P (1977): Attainment and maintenance of azoospermia with combined monthly injections of depot medroxy progesterone acetate and testosterone enanthate. Con traception 15: Barbieri R L. Canick J A & Ryan K J (1977): Danazol inhibits steroidogenesis in the rat testis in vitro. Endo crinology 1: Betz G 8c Michels D (1973): The effects of metyrapone on the 17,2 lvase from rat testis microsomes. Biochem Biophys Res Commun 5: Challis J R, Davies I J, Benirschke K, Hendricks A G & Ryan K J (1975): The effects of dexamethasone on the peripheral plasma concentration of androstenedione, testosterone and cortisol in the pregnant Rhesus mon key. Endocrinology 96:

7 Cheng S C, Suzuki K, Wolfgang S & Harding B S (1976): Effects of spironolactone. canrenone and canrenoate K on cytochrome P45, and 1 Iß and 18hydroxylation in bovine and human adrenal microsomes. Endocrino logy 99: Clark A F, Calandra R D & Bird C E (1972): Kinetics of testosterone metabolism in normal young men : effects of medroxyprogesterone acetate administration. J Steroid Biochem 3: Collip P J, Kaplan S A, Boyle D C, Plachte F 8c Kugut M D (1964): Constitutional isosexual precocious puberty. Am J Dis Child 8: Dufau M Be Catt K J (1974): A highly sensitive in vitro bioassay for luteinizing hormone and chorionic gonadotropin: testosterone production by dispersed Leydig cells. J Clin Endocrinol Metab 39: Frick J, Bartsch G 8c Weiske W H (1977): The effect of monthly depot medroxyprogesterone acetate and tes tosterone on human spermatogenesis. Contraception 15: Geller J, Albert J, Geller S, Lopez D, Cantor T & Yen S ( 1976) : Effect of megestrol acetate on steroid metabo lism and steroidprotein binding in the human pros tate. J Clin Endocrinol Metab43: 8. Gordan G G, Southren A L, Tochimoto S, Olivo J, Altman K, Rand J Be Lemberger I. (197): Effect of medroxyprogesterone acetate on the metabolism and biological activity of testosterone. J Clin Endocrinol Metab 3: Judge D M, Kulin H E, Page R, Santen R & Trapukdi A (1977): Hypothalamic hamartoma: a source of I.HRH in precocious pubertv. N Engl J Med 296: 7. Kaplan S A, Ling S M & Irani N G (1968): Idiopathic isosexual precocity: therapy with medroxvprogesterone. Am J Dis Child 116: Melo J F 8c Coutinho E M (1977): Inhibition of spermato genesis in men with monthly injections of medroxy progesterone acetate and testosterone enanthate. Con traception 15: Menard R H & Purvis J L (1973): Studies on cytochrome P45 in testis microsomes. Arch Biochem Biophys 154:818. Mever W J, Walker P A, Weideking C, Money J, Kowarski A A, Migeon CJ Be Borgonkav D S (1977): Pituitaryfunction in adult males receiving medroxyprogester one acetate. Fértil Steril 28: Money [ (197): Use of androgendepleting hormone in the treatment of male sex offenders. J Sex Res 6: Nolten W E, Sholiton L J, Srivastava I. S, Knowles H C Be Werk E E (1976): The effects of diethylstilbestrol and medroxyprogesterone acetate on kinetics and produc tion of testosterone and dihydrotestosterone in patients with prostatic carcinoma. J Clin Endocrinol Metab 43 : Rifkind A B. Kulin H E. Cargille C M, Rayford P C Be Ross G T (1969): Suppression of urinary excretion of LH and FSH by medroxyprogesterone acetate. J Clin Endocrinol Metab 29: Rivarola M A, Camacho A M & Migeon C J (1968): Effect of treatment with medroxyprogesterone acetate on testicular function. J Clin Endocrinol Metab 28: Sadoff I. Be I.usk W (1974): The effect of large doses of medroxyprogesterone acetate on urinary estrogen levels and serum levels of cortisol, Tt, LH and testo sterone in patients with advanced cancer. Obstet Gynecol 43: Satvaswaroop P G Be Gurpide E (1978): A direct effect of medroxyprogesterone on 17ßhvdroxysteroid dehy drogenase in adult rat testis. Endocrinology 2: Saure A. Karjalainen O & Teravainen T (1977): The formation in effect of synthetic gestagens on estrogen vitro bv human corpus lutetim. Acta Endocrinol (Kbh), Suppl212:89. Uzgiris V I. Graves P F. & Salhanick H A (1977): Ligand modification of corpus lutetim mitochondrial mono 6. oxvgenation. Biochemistry 16: 593 Received on July 28th, 1979.