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Changes induced by treatment with aromatase inhibitors in testicular Leydig cells of rats and dogs
Exp Toxic Patholl994; 46: 211-213
Gustav Fischer Verlag Jena
Preclinical Safety, Pathology, Pharma Division, Ciba-Geigy AG, Basel, Switzerland
Changes induced by treatment with aromatase inhibitors in testicular Leydig cells of rats and dogs U. JUNKER WALKER and V. NOGUES With 1 figure Received: November 8, 1993; Accepted: December 21, 1993 Address for correspondence: Dr. U. JUNKER WALKER, Preclinical Safety, Pathology; Pharma Division, Ciba-Geigy AG, CH-4002 Basle, Switzerland. Key word: Aromatase inhibitors; Leydig cells, testicular; Testicular Leydig cells.
Summary Treatment of male rats and dogs with CGP 32 349 (formestane), a steroidal aromatase inhibitor, and CGS 20 267 (letrozo1e), a non-steroidal aromatase inhibitor, induced different alterations in testicular interstitial Leydig cells in the two species. Whereas in dogs Leydig cells were hypertrophic and hyperplastic, in rats either no effect (CGS 20 267) or atrophy of Leydig cells (CGP 32 349) was reported. The different response of the two species can be explained by different regulating mechanisms of gonadotropin secretion by the anterior pituitary.
Introduction Reduction of estrogen levels by inhibition of the enzyme aromatase, which converts androgens into estrogens, is a strategy in the treatment of estrogen-dependent mammary tumors in women. In toxicity studies in animals, effects resulting from the desired pharmacological properties of aromatase inhibitors are to be expected in the genital organs. We report the effects of two structurally different aromatase inhibitors on the male genital organs in toxicity studies with rats and dogs.
Material and Methods CGP 32349 [4-hydroxy-androst-4-ene-3,17-dione (4OHA), formestane]: Male rats [Tif:RAIf(SPF)] were treated intraperitoneally twice weekly for 6 months at doses of 0, 20, 100 and 500 mg/kg. Male pedigree beagle dogs were treated intramuscularly twice weekly for 6 months at doses of 0 and 30 mg/kg. CGS 20 267 [4,4' -(1H-l ,2,4-triazol-I-yl-methylene)bis-benzonitrile, letrozole]: Male rats Tif: RAIf(SPF)] received doses of 0, 0.3, 3 and 30 mg/kg orally each day for 3
months. Male pedigree beagle dogs received daily doses of 0, 0.03, 0.3 and 3 mg/kg orally for 3 months. At the end of the treatment periods the animals were killed by exsanguination under deep anesthesia induced by Halothane® (rats) or Pentothal® (dogs). The animals were examined macroscopically: organ weights, including testes, epididymides, seminal vesicles (rats), prostate and pituitary, were collected; and tissues, including testes, epididymides, seminal vesicles (rats), prostate and pituitary, were fixed in 4 % neutral phosphate buffered formalin. Paraffin sections of the genital organs and pituitary were stained with hematoxylin and eosin. Pituitary sections of rats and dogs treated with CGS 20 267 were stained immunohistochemically for luteinizing hormone (LH) and follicle-stimulating hormone (FSH) by alkaline phosphatase.
Results In-life Observations No overt signs of toxicity were seen with either compounds in both species. Macropathology CGP 32 349: Signs of local irritation caused by the test material were found in both species. Weights of testes, seminal vesicles and prostate were decreased in treated rats. In dogs testicular and prostatic weights were increased in treated animals. CGS 20 267: In rats a reduction in weights of prostate, epididymides and seminal vesicles was evident at 3 and 30 mglkg. Testes weights were decreased in dogs treated with 0.3 and 3 mglkg. Histopathology CGP 32 349: In rats atrophy of the interstitial cells of Leydig (fig. la) in the testes and atrophy of the seminal Exp Toxic Patho146 (1994) 3 211
Fig.!. a: CGP 32 349: Atrophy of Leydig cells in rats (hematoxylin & eosin, xlOO). b: CGS 20267: No effects on Leydig cells in rats (hematoxylin & eosin, xlOO). c: CGP 32 349: Hypertrophy/hyperplasia of Leydig cells in dogs (hematoxylin & eosin, xlOO). d: CGS 20267: Hypertrophy/hyperplasia of Leydig cells, disturbance of spermatogenesis in dogs (hematoxylin & eosin, xlOO). vesicles were observed at doses of 100 and 500 mg/kg. In dogs diffuse hypertrophy and hyperplasia of testicular Leydig cells (fig. lc) were present in all treated animals. No pituitary changes were observed in either species. CGS 20 267: No alterations were present in testes (fig. Ib) and pituitary of treated rats. Prostatic atrophy was observed at all doses. Testicular Leydig cells were hypertrophic and hyperplastic in all treated dogs and spermatogenesis was disturbed or arrested (fig. Id). In addition, 212
Exp Toxic Pathol46 (1994) 2/3
hypertrophic adenohypophysial cells which were positive for LH or FSH by immunohistochemistry were found at all doses.
Discussion Mechanism of action The function of Leydig cells is mediated by LH secretion from the pituitary. In rodents aromatization is not es-
sential for androgen-regulated feedback control of gonadotrophins (KREY et al. 1982). Gonadotropin secretion is reported to be suppressed in female rats treated with 4OHA (WING et al. 1988). The compound has androgenic activity (approx. 1 % of testosterone). Therefore, effects on sex steroid target tissues with GCP 32 349 appear to be related to the androgenic activity of the compound, either through the negative feedback mechanism on the anterior pituitary or a direct effect on LH secretion. Treatment of female rats with the non-steroidal compound CGS 20 267 results in an increase of serum LH levels (BHATNAGAR et al. in press). Leydig cells in males are not affected after treatment for 3 months, probably due to the lack of androgenic properties of CGS 20 267 and less pronounced effects on LH secretion. However, a variety of substances which influence the hypothalamic-pituitary-Leydig cell axis in the rat may cause Leydig cell hyperplasia in long term experiments (GREAVES 1990). In contrast to the rat, aromatization plays a significant role in the feedback regulation of gonadotrophins in dogs and inhibition of the enzyme invariably leads to an increase in LH (AWONIYI et al. 1986). In the study with CGS 20267 in dogs the hypertrophied gonadotrophs in the pituitary suggested elevated serum gonadotropin levels. Consequently Leydig cells were stimulated and became hypertrophic. Similar effects were seen with CGP 32 349 in the testes of dogs, although no histological alterations were observed in the pituitary.
Comparison with other species In nonhuman primates and humans aromatization also plays a significant role in the control of gonadotrophin secretion (ELLINWOOD et al. 1984; D' AGATA et al. 1981) and effects similar to those reported for dogs can be expected. Clinical studies with healthy male volunteers showed that CGS 20 267 induces an increase in LH levels (BHATNAGAR et al. 1992).
Comparison to related compounds Most data available from the literature about the effects of aromatase inhibitors on gonadotropin secretion are from female animals and few results for male animals. In male, estradiol-implanted, orchidectomized dogs the postcastration response of LH and FSH can be suppressed by treatment with the aromatase inhibitor aminoglutethimide in the presence of estradiol (AWONIYI et al. 1986). In male rhesus monkeys treated with the aromatase inhibitor 1,4,6-androstatriene-3,17-dione (ATD) a rise in both serum LH and testosterone concentrations
was reported (ELLINWOOD et al. 1984). ATD did not influence serum LH levels in intact male rats (KREy et al. 1982). In female rats the steroidal aromatase inhibitors exemestane and MDL 18 962 lowered serum LH levels (ZACCHEO et al. 1991) and treatment with CGS 16 949 A and CGS 18 320 B, two non-steroidal aromatase inhibitors, led to an increase in serum LH (BHATNAGAR et al. 1990). In male rats treatment with CGS 16949 A had no effect on serum LH levels (BHATNAGAR et al. 1992).
References AWONIYI C, HASSON T, CHANDRASHEKAR V, et al.: Regulation of gonadrotropin secretion in the male: effect of an aromatization inhibitor in estradiol-implanted, orchidectomized dogs. 1 Andro11986; 7: 234-239. D' AGATA R, VICARI E, AUFFI A, et al.: Direct evidence in men for a role of endogenous oestrogens on gonadotropin release. Acta Endocrinol (Copenh) 1981; 97: 145-149. BHATNAGAR AS, HAUSLER A, SCHIEWECK K, et al.: Novel aromatase inhibitors. 1 Steroid Biochem Molec BioI 1990;37: 363-367. BHATHNAGAR AS, MULLER PH, SCHENKEL L, et al.: Inhibition of estrogen biosynthesis and its consequences on gonadotropin secretion in the male. 1 Steroid Biochem Molec Bioi 1992; 41: 437-443. BHATHNAGAR, AS, BATZL C, HAUSLER A, et al.: Role of estrogen in the feedback regulation of folliclestimulation hormone secretion in the female rat. 1 Steroid Biochem Molec BioI: In press. ELLINWOOD WE, HESS DL, ROSELLI CE, et al.: Inhibition of aromatization stimulates luteinizing hormone and testosterone secretion in adult male rhesus monkeys. 1 Clin Endocrinol Metab 1984; 59: 1088-1096. GREAVES P: Histopathology of preclinical toxicity studies, X. Male genital tract. Elsevier, Amsterdam 1990; pp 584-624. KREy LC, MACLUSKY Nl, DAVIS PG, et al.: Different intracellular mechanisms underlie testosterone's suppression of basal and stimulation of cyclic luteinizing hormone release in male and female rats. Endocrinology, 1982; 110: 2159-2167. WING L-YC, HAMMOND 10, BRODIE AMH: Differential responses of sex steroid target tissues of rats treated with 4-hydroxyandrostenedione. Endocrinology, 1988; 122: 2418-2427. ZACCHEO T, GIUDICE D, ORNATI G, et al.: Comparison of the effect of the irreversible aromatase inhibitor exemestane with atamestane and MDL 18962 in rats with DMBAinduced mammary tumours. Eur 1 Cancer 1991; 27: 1145-1150.
Exp Toxic Pathol46 (1994) 3 213
Gustav Fischer Verlag Jena
Preclinical Safety, Pathology, Pharma Division, Ciba-Geigy AG, Basel, Switzerland
Changes induced by treatment with aromatase inhibitors in testicular Leydig cells of rats and dogs U. JUNKER WALKER and V. NOGUES With 1 figure Received: November 8, 1993; Accepted: December 21, 1993 Address for correspondence: Dr. U. JUNKER WALKER, Preclinical Safety, Pathology; Pharma Division, Ciba-Geigy AG, CH-4002 Basle, Switzerland. Key word: Aromatase inhibitors; Leydig cells, testicular; Testicular Leydig cells.
Summary Treatment of male rats and dogs with CGP 32 349 (formestane), a steroidal aromatase inhibitor, and CGS 20 267 (letrozo1e), a non-steroidal aromatase inhibitor, induced different alterations in testicular interstitial Leydig cells in the two species. Whereas in dogs Leydig cells were hypertrophic and hyperplastic, in rats either no effect (CGS 20 267) or atrophy of Leydig cells (CGP 32 349) was reported. The different response of the two species can be explained by different regulating mechanisms of gonadotropin secretion by the anterior pituitary.
Introduction Reduction of estrogen levels by inhibition of the enzyme aromatase, which converts androgens into estrogens, is a strategy in the treatment of estrogen-dependent mammary tumors in women. In toxicity studies in animals, effects resulting from the desired pharmacological properties of aromatase inhibitors are to be expected in the genital organs. We report the effects of two structurally different aromatase inhibitors on the male genital organs in toxicity studies with rats and dogs.
Material and Methods CGP 32349 [4-hydroxy-androst-4-ene-3,17-dione (4OHA), formestane]: Male rats [Tif:RAIf(SPF)] were treated intraperitoneally twice weekly for 6 months at doses of 0, 20, 100 and 500 mg/kg. Male pedigree beagle dogs were treated intramuscularly twice weekly for 6 months at doses of 0 and 30 mg/kg. CGS 20 267 [4,4' -(1H-l ,2,4-triazol-I-yl-methylene)bis-benzonitrile, letrozole]: Male rats Tif: RAIf(SPF)] received doses of 0, 0.3, 3 and 30 mg/kg orally each day for 3
months. Male pedigree beagle dogs received daily doses of 0, 0.03, 0.3 and 3 mg/kg orally for 3 months. At the end of the treatment periods the animals were killed by exsanguination under deep anesthesia induced by Halothane® (rats) or Pentothal® (dogs). The animals were examined macroscopically: organ weights, including testes, epididymides, seminal vesicles (rats), prostate and pituitary, were collected; and tissues, including testes, epididymides, seminal vesicles (rats), prostate and pituitary, were fixed in 4 % neutral phosphate buffered formalin. Paraffin sections of the genital organs and pituitary were stained with hematoxylin and eosin. Pituitary sections of rats and dogs treated with CGS 20 267 were stained immunohistochemically for luteinizing hormone (LH) and follicle-stimulating hormone (FSH) by alkaline phosphatase.
Results In-life Observations No overt signs of toxicity were seen with either compounds in both species. Macropathology CGP 32 349: Signs of local irritation caused by the test material were found in both species. Weights of testes, seminal vesicles and prostate were decreased in treated rats. In dogs testicular and prostatic weights were increased in treated animals. CGS 20 267: In rats a reduction in weights of prostate, epididymides and seminal vesicles was evident at 3 and 30 mglkg. Testes weights were decreased in dogs treated with 0.3 and 3 mglkg. Histopathology CGP 32 349: In rats atrophy of the interstitial cells of Leydig (fig. la) in the testes and atrophy of the seminal Exp Toxic Patho146 (1994) 3 211
Fig.!. a: CGP 32 349: Atrophy of Leydig cells in rats (hematoxylin & eosin, xlOO). b: CGS 20267: No effects on Leydig cells in rats (hematoxylin & eosin, xlOO). c: CGP 32 349: Hypertrophy/hyperplasia of Leydig cells in dogs (hematoxylin & eosin, xlOO). d: CGS 20267: Hypertrophy/hyperplasia of Leydig cells, disturbance of spermatogenesis in dogs (hematoxylin & eosin, xlOO). vesicles were observed at doses of 100 and 500 mg/kg. In dogs diffuse hypertrophy and hyperplasia of testicular Leydig cells (fig. lc) were present in all treated animals. No pituitary changes were observed in either species. CGS 20 267: No alterations were present in testes (fig. Ib) and pituitary of treated rats. Prostatic atrophy was observed at all doses. Testicular Leydig cells were hypertrophic and hyperplastic in all treated dogs and spermatogenesis was disturbed or arrested (fig. Id). In addition, 212
Exp Toxic Pathol46 (1994) 2/3
hypertrophic adenohypophysial cells which were positive for LH or FSH by immunohistochemistry were found at all doses.
Discussion Mechanism of action The function of Leydig cells is mediated by LH secretion from the pituitary. In rodents aromatization is not es-
sential for androgen-regulated feedback control of gonadotrophins (KREY et al. 1982). Gonadotropin secretion is reported to be suppressed in female rats treated with 4OHA (WING et al. 1988). The compound has androgenic activity (approx. 1 % of testosterone). Therefore, effects on sex steroid target tissues with GCP 32 349 appear to be related to the androgenic activity of the compound, either through the negative feedback mechanism on the anterior pituitary or a direct effect on LH secretion. Treatment of female rats with the non-steroidal compound CGS 20 267 results in an increase of serum LH levels (BHATNAGAR et al. in press). Leydig cells in males are not affected after treatment for 3 months, probably due to the lack of androgenic properties of CGS 20 267 and less pronounced effects on LH secretion. However, a variety of substances which influence the hypothalamic-pituitary-Leydig cell axis in the rat may cause Leydig cell hyperplasia in long term experiments (GREAVES 1990). In contrast to the rat, aromatization plays a significant role in the feedback regulation of gonadotrophins in dogs and inhibition of the enzyme invariably leads to an increase in LH (AWONIYI et al. 1986). In the study with CGS 20267 in dogs the hypertrophied gonadotrophs in the pituitary suggested elevated serum gonadotropin levels. Consequently Leydig cells were stimulated and became hypertrophic. Similar effects were seen with CGP 32 349 in the testes of dogs, although no histological alterations were observed in the pituitary.
Comparison with other species In nonhuman primates and humans aromatization also plays a significant role in the control of gonadotrophin secretion (ELLINWOOD et al. 1984; D' AGATA et al. 1981) and effects similar to those reported for dogs can be expected. Clinical studies with healthy male volunteers showed that CGS 20 267 induces an increase in LH levels (BHATNAGAR et al. 1992).
Comparison to related compounds Most data available from the literature about the effects of aromatase inhibitors on gonadotropin secretion are from female animals and few results for male animals. In male, estradiol-implanted, orchidectomized dogs the postcastration response of LH and FSH can be suppressed by treatment with the aromatase inhibitor aminoglutethimide in the presence of estradiol (AWONIYI et al. 1986). In male rhesus monkeys treated with the aromatase inhibitor 1,4,6-androstatriene-3,17-dione (ATD) a rise in both serum LH and testosterone concentrations
was reported (ELLINWOOD et al. 1984). ATD did not influence serum LH levels in intact male rats (KREy et al. 1982). In female rats the steroidal aromatase inhibitors exemestane and MDL 18 962 lowered serum LH levels (ZACCHEO et al. 1991) and treatment with CGS 16 949 A and CGS 18 320 B, two non-steroidal aromatase inhibitors, led to an increase in serum LH (BHATNAGAR et al. 1990). In male rats treatment with CGS 16949 A had no effect on serum LH levels (BHATNAGAR et al. 1992).
References AWONIYI C, HASSON T, CHANDRASHEKAR V, et al.: Regulation of gonadrotropin secretion in the male: effect of an aromatization inhibitor in estradiol-implanted, orchidectomized dogs. 1 Andro11986; 7: 234-239. D' AGATA R, VICARI E, AUFFI A, et al.: Direct evidence in men for a role of endogenous oestrogens on gonadotropin release. Acta Endocrinol (Copenh) 1981; 97: 145-149. BHATNAGAR AS, HAUSLER A, SCHIEWECK K, et al.: Novel aromatase inhibitors. 1 Steroid Biochem Molec BioI 1990;37: 363-367. BHATHNAGAR AS, MULLER PH, SCHENKEL L, et al.: Inhibition of estrogen biosynthesis and its consequences on gonadotropin secretion in the male. 1 Steroid Biochem Molec Bioi 1992; 41: 437-443. BHATHNAGAR, AS, BATZL C, HAUSLER A, et al.: Role of estrogen in the feedback regulation of folliclestimulation hormone secretion in the female rat. 1 Steroid Biochem Molec BioI: In press. ELLINWOOD WE, HESS DL, ROSELLI CE, et al.: Inhibition of aromatization stimulates luteinizing hormone and testosterone secretion in adult male rhesus monkeys. 1 Clin Endocrinol Metab 1984; 59: 1088-1096. GREAVES P: Histopathology of preclinical toxicity studies, X. Male genital tract. Elsevier, Amsterdam 1990; pp 584-624. KREy LC, MACLUSKY Nl, DAVIS PG, et al.: Different intracellular mechanisms underlie testosterone's suppression of basal and stimulation of cyclic luteinizing hormone release in male and female rats. Endocrinology, 1982; 110: 2159-2167. WING L-YC, HAMMOND 10, BRODIE AMH: Differential responses of sex steroid target tissues of rats treated with 4-hydroxyandrostenedione. Endocrinology, 1988; 122: 2418-2427. ZACCHEO T, GIUDICE D, ORNATI G, et al.: Comparison of the effect of the irreversible aromatase inhibitor exemestane with atamestane and MDL 18962 in rats with DMBAinduced mammary tumours. Eur 1 Cancer 1991; 27: 1145-1150.
Exp Toxic Pathol46 (1994) 3 213