Antiandrogenic Effects of Spironolactone: Hormonal and Ultrastructural Studies in Dogs and Men

0022-534 7/78/ 1193-375$02. 00/0 Vol. 119, March

THE JOURNAL OF UROLOGY

Copyright © 1978 by The Williams & Wilkins Co.

Printed in U.SA.

ANTIANDROGENIC EFFECTS OF SPIRONOLACTONE: HORMONAL AND ULTRASTRUCTURAL STUDIES IN DOGS AND MEN SHIRO BABA, MASARU MURAI, SEIDO JITSUKAWA, MAKOTO HATA

AND

HIROSHI TAZAKI

From the Department of Urology, School of Medicine, Keio University, Tokyo, Japan

ABSTRACT

A decrease in the level of plasma testosterone and an increase in the level of plasma progesterone were noted after spironolactone had been administered for 20 days in 5 patients w~th prostatic carcinoma, as well as in 8 male dogs. Electron microscopic observation disclosed myelmlike bodies in the cytoplasm of Leydig and adrenocortical cells in dogs, contributing to a resolution of the mode of antiandrogenic action of spironolactone. Spironolactone, an aldosterone antagonist currently being used as a potassium-conserving diuretic, has an antiandrogenic effect by potentially inhibiting some enzymatic steps in the androgen synthesis in the testes and adrenals. To clarify the site and mode of the antiandrogenic action of spironolactone in vivo, we have studied spironolactone-induced alterations in the synthesis of sex steroids in normal mature male dogs and men with prostatic carcinoma, as well as the morphological changes in the adrenals, testes and prostate glands of dogs by conventional and electron microscopic observation. MATERIALS AND METHODS

The animal study was conducted using 10 mongrel mature male dogs, including 2 controls weighing 15.1 ± 4.2 kg. In the 8 dogs 40 mg./kg. oral spironolactone were administered daily for 20 days. Blood samples were obtained between 8 and 9 a.m. from the antecubital vein. The plasma was separated immediately by centrifugation at room temperature, and plasma progesterone and testosterone were determined by radioimmunoassay. Serum electrolyte determinations were available with the use of an autoanalyzer. On the morning of the 20th day all 10 dogs were sacrificed and the prostate, testes and adrenals were removed. The absolute weight of these organs and the weight of these organs per kilogram sacrificed body weight were recorded. All the specimens were prepared for conventional and electron microscopic observation. The specimens for electron microscopic study were fixed in 2.5 per cent glutaraldehyde, washed in 0.07M. borate buffer (pH 7.4), post-fixed in 2 per cent osmium tetroxide and_ then e:mbedded in Epon 812. Ultrathin sections were stamed with uranyl acetate and lead citrate. . Five patients, ranging in age from 50 to 76 years and havn~g histologically confirmed prostatic carcinoma, were included m the study. No patient had received any previous treatm~nt such as castration. Three of the patients were treated with full doses of 400 mg. per day spironolactone for more than 2 weeks. The remaining 2 were treated with reduced doses, 250 and 200 mg. per day, because of complications of angina pectoris and electrolyte imbalance caused by chronic r~nal failure, respectively (table 1). Blood samples were obtamed weekly to determine serum electrolytes, progesterone and testosterone. Plasma progesterone and testosterone were determined by radioimmunoassay using specific antibodies.* The 1,2-3H progesteronet and l,2- 3H testosteronet were diluted to a concenAccepted for publication May 6, 1977. Read at annual meeting of American Urological Association, Chicago, Illinois, April 24-28, 1977. * Clinical Research Laboratory, Central Institute for Experimental Animals, Tokyo, Japan. t New England Nuclear Corporation.

tration of 20 to 25 µ,Ci./ml. by a mixture of benzene and ethanol (9:1) and stored at 4C until use. RESULTS

Animal experiments. In dogs treated with spironolactone the plasma progesterone increased significantly to a maximum value of 1.9 ± 0.8 ng./ml. (mean ± standard deviation) from 0.27 ± 0.1 ng./ml. during a period of 2 weeks (p < 0.005), while plasma testosterone decreased significantly to 54.2 ± 25 ng./dl. from 125 ± 20 ng./dl. within 20 days (p < 0.025) (fig. 1). No significant changes were noted in serum electrolytes throughout the study. Spironolactone caused a significant decrease in the absolute and relative weights of the prostate (p < 0.025) (table 2). However, no significant difference was noted in the absolute and relative weights of the adrenals and testes. A conventional microscopic study revealed no significant changes in the adrenals and testes but remarkable atrophic epithelial changes were noted in the prostate of the animals treated with spironolactone, characterized by a change from tall to low columnar epithelium and loss of epithelial convolutions (fig. 2). Electron microscopic observation and light microscopic study showed that the normal prostates were lined by tall columnar cells with typical characteristics of secretory cells. The cytoplasm contained a large number of elongated cisternae of rough-surfaced endoplasmic reticulum and abundant mature secretory granules containing moderately dense material near the apex (fig. 3, A). In contrast, the prostates of the animals treated with spironolactone showed a marked decrease of these secretory granules and the appearance of many irregularly shaped vacuoles in the cytoplasm (fig. 3, B). Despite the negative findings by conventional microscopy an ultrastructural study revealed characteristic laminated membranous structures identified as so-called spironolactone bodies in the glomerular cells of the adrenal cortices of animals treated with spironolactone. Smooth endoplasmic reticulum was increased quantitatively and the mitochondria were larger and more pale than those of controls. No differences in nuclei or other cell membranes were observed between the animals treated with spironolactone and control animals (figs. 4 and 5). Also, seemingly similar myelin-like structures were found in the cytoplasm of Leydig cells (fig. 6). These structures were comprised of smooth-surfaced, doublelayered membranes, concentrically arranged about a homogeneous lipid-like core, that were morphologically identical to the so-called spironolactone body. No distinctive alterations in the cytoplasm of the spermatogonium and Sertoli cell were observed. Clinical study. While plasma progesterone increased from 0.25 ± 0.10 ng./ml. (mean ± standard deviation) to a maxi-

375

376

BABA AND ASSOCIATES TABLE

1. Patients treated with spironolactone

Pt.

Age (yrs.)

Serum Creatinine (mg./dl.)

Duration (days)

Dosage (mg./day)

Electrolyte Imbalance

1 2

76 69 65 50 70

1.1

1.0 1.2 0.9 2.2

27 20 24 20 22

400 400 250* 400 200t

None None None None Hyperkalemia (transient)

3 4

5

Other Complications None None Angina pectoris None Anorexia

* Lower dose owing to angina pectoris. t Lower dose owing to chronic renal failure. TABLE

2. Antiandrogenic effect of spironolactone on canine prostate and testis

Body Weight No. Arrimals Control Spironolactone*

2 8

Organ Weight

Organ Weight/Kg. Sacrificed Animal

Before Treatment (kg.)

After Treatment (kg.)

Adrenal

Testis

Prostate

(gm.)

(gm.)

(gm.)

Adrenal (mg./kg.)

Testis (mg./kg.)

Prostate (mg./kg.)

16.3 ± 6.2 14.7 ± 3.9

14.5 ± 4.9 11.8 ± 2.5

1.5 ± 0.8 1.2 ± 0.5

9.9 ± 4.1 5.9 ± l.2t

97 ± 20 94 ± 30

750 ± 32 731 ± 159

674 ± 53 506 ± 69t

10.9 ± 4.2 9.7 ± 2.5

* 40 mg./kg. orally daily for 20 days. t Significant decrease (p < 0.025).

mum value of 1.3 ± 0.31 ng./ml. by 20 days after the administration of spironolactone the plasma testosterone decreased to 200 ± 80.3 ng./dl. from 427 ± 74.3 ng./dl. during the same interval (fig. 7). These changes were statistically significant (p < 0.025). The serum sodium, chloride and potassium levels remained within normal range during the same period of spironolactone administration in all cases except 1. In this particular patient the serum potassium level increased to 5.3 mEq./1. transiently. The bilateral ureteral obstruction observed in this patient had resulted in an elevation of the serum level of blood urea nitrogen and creatinine to 28.3 and 1.4 mg./dl., respectively. Therefore, this patient was treated with a reduced dose of 200 mg. spironolactone daily. However, the patient experienced relief of pain from bony metastasis of prostatic carcinoma after 10 days. Of the 5 men receiving spironolactone 1 exhibited nodular induration of the breasts after 2 weeks. DISCUSSION

Since spironolactone first became widely available in 195, there have been several reports of gynecomastia and loss of libido in patients being treated with it. The reports consist of individual case reports and an incidental single observation in a group of patients being treated with spironolactone. i-:i Since Greenblatt and Koch-Weser reported on 788 hospitalized patients who received spironolactone and found a relatively lower incidence of gynecomastia (1.2 per cent) it has been thought generally that gynecomastia is a rather uncommon occurrence associated with spironolactone. 4 However, Clark reported on gynecomastia occurring in 4 of 7 men during treatment with spironolactone and strongly recommended more careful examination of the breasts of all patients receiving spironolactone. 5 Castro and associates studied the clinical use of spironolactone for benign prostatic hyperplasia and found some temporary effects. 6 The mechanism of action of spironolactone causing these estrogenic effects remains unknown; however, it is thought that alteration of the normal gonadal-hypothalamic-pituitary interrelations may have an important role. Recent research indicates that spironolactone inhibits androgen formation in microsomes, characterized by a rapid loss of testicular microsomal p-450 with a concomitant decrease in the activity of 17 a-hydroxylase, which catalyzes the conversion of progesterone to 17 a-hydroxyprogesterone, a main precursor of the testosterone. 7 Menard and associates have suggested that the destruction of microsomal cytochrome p-450 by spironolactone may be limited to those tissues in which microsomal 17 ahydroxylase activity is high. 8 The adrenal glands that produce

cortisol in such species as dog and guinea pig have a high level of 17 a-hydroxy lase activity. Therefore, spironolactone administration in these species as well as in human beings may cause a substantial loss of testicular and adrenal microsomal cytochrome p-450 with a concomitant decrease in 17 ahydroxylase activity. If spironolactone significantly decreased the production of testosterone and adrenal androgens in dog and man the plasma steroid hormonal pattern also would have changed subsequently. Such drug-induced biochemical effects might account for the clinically observed endocrine effects of spironolactone therapy. Stripp and associates demonstrated the increase of plasma progesterone after treatment by spironolactone in man. However, they could not show any significant CAN I NE PLASMA PROGESTERONE

Ng/ml

2.0

1.0

CAN I NE PLASMA TESTOSTERONE

Ng/dl

150

lOJ

5J

Day

4

Day

9

SPIRONOLACTONE (40

Day

14

o,y 20

mg/Kg/dayl

Fm. 1. Effect of spironolactone on plasma levels of progesterone and testosterone in dogs.

ANTIANDROGENIC EFFECTS OF SPIRONOLACTONE

377

FIG. 2. A, cross-section of prostate taken from control dog that had received no spironolactone. B, cross-section of prostate taken from dogs given 40 mg./kg. spironolactone daily for 20 days. Note decreased number of convolutions and change to low columnar epithelial cells. Reduced from x 25.

Fm. 3. A, electron microscopic appearance of prostate from control dog. Much of cytoplasm is filled with elongated cistemae of roughsurfaced endoplasmic reticulum. Mature secretory granules are abundant and more common toward apex of cell. N, nucleus. RER, roughsurfaced endoplasmic reticulum. SG, secretory granules. B, electron microscopic appearance of prostate trom dog given 40 mg./kg. spironolactone daily for 20 days. Note decreased number of secretory granules and appearance of many irregularly shaped vacuoles ir1 cytoplasm. V, vacuole. Reduced from x3,000.

change of plasma testosterone, probably owing to the short duration and small dosage of spironolactone administered. 9 Walsh and Siiteri found suppression of plasma testosterone, androstenedione and dehydroepiandrosterone by high doses of spironolactone in 7 castrated men with prostatic carcinoma. 10 In our study plasma testosterone decreased and progesterone increased significantly after spironolactone had

been given for 20 days in 8 male dogs and in 5 men with prostatic carcinoma. These studies indicate that spironolactone suppresses testicular and adrenal androgen production by the inhibition of the enzymatic step involved in the conversion of progesterone to androgen. In addition, it has been shown that spironolactone also reduces aldosterone secretion in men. 11 Although the enzymes involved in the inhibition of

378

BABA AND ASSOCIATES

FIG. 4. A, portion of cells in zona glomerulosa of adrenal cortex from control dog. Mitochondria are round, oval or elongated with lamellar cristae. M, mitochondria. Reduced from x7,000. B, portion of cells in zona glomerulosa of dog given 40 mg./kg. spironolactone daily for 20 days, demonstrating laminated membranous structure (arrow). Smooth endoplasmic reticulum is abundant. Mitochondria are large and pallid. SER, smooth endoplasmic reticulum. Reduced from x7,200.

FIG. 5. Cytoplasm of another zona glomerulosa cell of spironolactone-treated dog exhibiting characteristic laminated membranous inclusion identified as so-called spironolactone body (arrows). Reduced from x 26,700.

FIG. 6. Electron microscopic appearance of testis from spironolactone-treated dog. Laminated myelin-like structures (arrows) in cytoplasm of Leydig cell look seemingly similar to so-called spironolactone bodies. Reduced from x9,500.

ANTIANDROGENIC EFFECTS OF SPIRONOLACTONE Ng/ml

PLASMA PROGESTERONE

1.50

1.00

0.50

0.00

Ng/dl

500

PLASMA TESTOSTERONE

100

o..,__________________ Day

10

Day

15

Day

20

Fm. 7. Effect of spironolactone on plasma levels of progesterone and testosterone in patients with prostatic carcinoma.

379

spironolactone bodies might appear in the process of inhibitory action of spironolactone on the enzymatic step involved in the progesterone metabolic pathway, because the site of conversion is predominantly located in the smooth endoplasmic reticulum of the adrenocortical cells and Leydig cells as well. From a hormonal standpoint this observation corresponds to the morphological observations by electron microscopy obtained from our current study. Our conclusion, based upon this animal experiment, strongly indicates that spironolactone causes remarkable atrophy of the canine prostate, confirmed by light and electron microscopy, and this antiandrogenic effect of spironolactone is, so to speak, an indirect action of decreasing the plasma testosterone level, although we are aware of direct antiandrogenic action of spironolactone reported by Steelman and associates. 17 The principal goal of hormonal therapy in the treatment of prostatic cancer is the suppression of androgenic stimuli to the prostate. This effect can be achieved by 1 of 4 mechanisms: 1) suppression of pituitary luteinizing hormone release, 2) removal of the androgen-producing organ, 3) direct inhibition of steroidogenesis in the testes and adrenals and 4) the inhibition of androgenic action at target tissues. Even though our speculation stemmed from our current animal experiment of non-malignant canine prostates, it is our belief that this speculation should be extended further to malignant disease of the prostate and that spironolactone and its related agents could be used as antiandrogenis drugs, leading to their clinical application to the treatment of patients with cancer of the prostate. Drs. N. Osawa and S. Yamamoto assisted in the preparation of the hormonal assay and Mr. N. Komatsu provided technical assistance for electron microscopy.

spironolactone on aldosterone production are not understood clearly it is reasonable to suggest that spironolactone acts 1. directly on the enzyme system contributing to the progesterone metabolic pathway in the adrenal and testis. 2. As shown in the animal experiments in our study electron microscopic observation revealed numerous enlarged mito3. chondria and smooth endoplasmic reticulum, as well as socalled spironolactone bodies, in the cytoplasm of the adreno4. cortical cell in the animals treated with spironolactone. Tlie 5. appearance of the laminated bodies has been reported previously in the glomerular zone of the adrenal glands of patients 6. treated with spironolactone. 12 The histochemical study by Jenis and Hertzog has shown that the core of the spironolactone bodies consists of neutral lipids whereas the rings are 7. probably a phospholipid-protein complex. 13 Electron microscopy revealed that the bodies are composed of concentric agranular membranes surrounding a lipid-like core. 14 These results strongly suggested that the bodies are derived from 8. the smooth endoplasmic reticulum. Mitochondria may have an important role in the formation of spironolactone bodies but the mechanisms involved in the course of the formation of 9. these bodies were not understood clearly. 15 In our study similar laminated structures also were found in the cytoplasm of Leydig cells. Dietert and Scallen have suggested the mech- 10. anisms of formation of similar myelin-like structures appearing in the process of inhibition of cholesterol biosynthesis in murine adrenal gland and testis. 16 It is now generally agreed 11. that the enzymes involved in the cholesterol biosynthesis and metabolic pathway of progesterone into testosterone are found mainly in the microsomal fraction derived from the endoplasmic reticulum. The predominant cytological feature in the 12. adrenals and testes observed in our series by electron micro- 13. scopic study is the occurrence of myelin-like, cytoplasmic inclusions that are considered to be implicated in the same morphogenesis. 14. From these observations it would seem that the so-called

REFERENCES

Smith, W. G.: Spironolactone and gynecomastia. Lancet, 2: 886, 1962. Williams, E.: Spironolactone and gynecomastia. Lancet, 2: 1113, 1962. Mann, N. M.: Gynecomastia during therapy with spironolactone. J.A.M.A., 184: 778, 1963. Greenblatt, D. J. and Koch-Weser, J.: Adverse reactions to spironolactone. J.A.M.A., 225: 40, 1973. Clark, E.: Spironolactone therapy and gynecomastia. J.A.M.A., 193: 157, 1965. Castro, J. E., Griffiths, H. J. L. and Edwards, D. E.: A doubleblind, controlled, clinical trial of spironolactone for benign prostatic hypertrophy. Brit. J. Surg., 58: 485, 1971. Menard, R. H., Stripp, B. and Gillette, J. R.: Spironolactone and testicular cytochrome p-450: decreased testosterone formation in several species and changes in hepatic drug metabolism. Endocrinology, 94: 1628, 1974. Menard, R. H., Martin, H. F., Stripp, B., Gillette, J. R. and Bartter, F. C.: Spirnolactone and cytochrome p-450: impairment of steroid hydroxylation in the adrenal cortex. Life Sci., 15: 1639, 1974. Stripp, B., Taylor, A. A., Bartter, F. C., Gillette, J. R., Loriaux, D. L., Easley, R. and Menard, R.H.: Effect of spironolactone on sex hormones in man. J. Clin. Endocr., 41: 777, 1975. Walsh, P. C. and Siiteri, P. K.: Suppression of plasma androgens by spironolactone in castrated men with carcinoma of the prostate. J. Urol., 114: 254, 1975. Sundsfjord, J. A., Marton, P., J0rgensen, H. and Aakvaag, A.: Reduced aldosterone secretion during spironolactone treatment in primary aldosteronism: report of a case. J. Clin. Endocr., 39: 734, 1974. Janigan, D. T.: Cytoplasmic bodies in the adrenal cortex of patients treated with spironolactone, Lancet, 1: 850, 1963. Jenis, E. H. and Hertzog, R. W.: Effect of spironolactone on the zona glomerulosa of the adrenal gland: light and electron microscopy. Arch. Path., 88: 530, 1969. Davis, D. A. and Medline, N. M.: Spironolactone (aldactone) bodies, concentric lamellar formations in the adrenal cortices

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BABA AND ASSOCIATES

of patients treated with spironolactone. Amer. J. Clin. Path., 54: 22, 1970.

15. Fisher, E. R. and Horvat, B.: Experimental production of socalled spironolactone bodies. Arch. Path., 91: 471, 1971. 16. Dietert, S. E. and Scallen, T. J.: An ultrastructural and biochemical study of the effects of three inhibitors of cholesterol

biosynthesis upon murine adrenal gland and testis. Histochemical evidence for a lysosome response. J. Cell Biol., 40: 44, 1969. 17. Steelman, S. L., Brooks, J. R., Morgan, E. R. and Patanelli, D. J.: Anti-androgenic activity of spironolactone. Steroids, 14: 449, 1969.