Effects of Anti-Androgen, Tzp-4238 and Chlormadinone Acetate on the Adrenal Cortex -Histo- Pathological and Immunocyto- Chemical Studies

ACTA HISTOCHEM. CYTOCHEM. Vol. 27, No. 4, pp. 365-372, 1994

EFFECTS OF ANTI-ANDROGEN, TZP-4238 AND CHLORMADINONE ACETATE ON THE ADRENAL CORTEX -HISTO- PATHOLOGICAL AND IMMUNOCYTO- CHEMICAL STUDIES-

MASANORI MURAKOSHI, RIE INADA, MASASHI TAGAWA, KAZUHIRO IIZUKA, SHUUJI MASUDA, MINORU SUZUKI, ATSUSHI MIZOKAMI* AND KEIICHI WATANABE **

Safety Research Department, Teikoku Hormone Mfg. Co. Ltd., 1604 Shimosakunobe, Takatsu-ku, Kawasaki 213, *Department of Molecular Cell Biology, University of Occupational and Environmental Health, Kitakyushu 806 and **Department of Pathology, Tokai University School of Medicine, Bohseidai, Isehara-city, Kanagawa 259-11

Received for publication May 12, 1994, in revised form July 22, 1994 and re-revised form August 24, 1994

The atrophic effects of two synthetic steroidal anti-androgens, chlormadinone acetate (CMA) and TZP-4238, on the adrenal gland of rats were investigated by histopathological and immunocytochemical procedures. Male Sprague-Dawley rats were divided into four experimental groups. Group 1 consisted of intact controls. Groups 2 and 3 received TZP-423810 mg/kg/day and CMA 50 mg/kg/day p.o., respective- ly, for 3 weeks. Group 4 received distilled water containing 2% Tween 80 instead of TZP-4238 or CMA. CMA caused marked atrophy of the adrenal gland. Histopathologically, the remarkable atrophy was observed in the adrenal cortical cells of zonae fasciculata and reticularis. Intracellular localization of glutathione-peroxidase (GSH-PO) which effectively reduces the lipid peroxides, was mainly observed in the cytoplasmic matrix (cytosol GSH-PO) near the mitochondria or lipid droplets. In ddition, immunoreactivi- ty of intramitochondrial GSH-PO (mitochondrial GSH-PO) was less than that in the controls. Androgen receptor (AR) was localized exclusively in the nuclei of the adrenal cortical cells. In CMA-treated rats, AR was localized also in the nuclei of the adrenal cortical cells of the atrophic cortical zones. In contrast, TZP- 4238 exerted no effect on the adrenal gland under the present experimental conditions. Therefore, it is suggested that TZP-4238 have a less inhibitory influence than CMA on the pituitary-adrenal axis. We further speculated that adrenal cortical cells of the atrophic zones induced by CMA treatment was decreased in corticosterone production and increased or remained in adrenal androgen production.

Both chlormadinone acetate (CMA) and 17a- prostatic glandular epithelial cells of rats (22). The acetoxy-6-chloro-2-oxa-4, 6-pregnadiene-3, 20-dione nuclear immunostaining for AR remarkably decreased (TZP-4238) are inhibitors of androgen action (18-21, after treatment with CMA or TZP-4238 (21, 22, 24). 24). TZP-4238 was synthesized in our laboratory by It is a well documented fact that AR was also localized introducing an oxygen atom at the C2 position of in the nuclei of the adrenal cortical cells (11). CMA. TZP-4238 was found to be a more potent anti- CMA and cyproterone acetate (CPA) have been androgen agent than CMA in the prostate (18-21, 24). used in the medical management of human benign pro- A rabbit polyclonal antibody referred as NH27 static hyperplasia or prostatic carcinoma (7, 10, 27, was rasied against human androgen receptor (AR) 32). The atrophic effects of CMA and CPA on the (16). Previous immunohistochemical study using this adrenal glands of rats have been reported by several antibody showed that AR is localized in nuclei of the authors (2, 3, 5, 26, 29). In the rat adrenal cortical cells, glutathione-peroxidase (GSH-PO), which effec- Correspondence to; Dr. Masanori Murakoshi, Safety tively reduces the lipid peroxides induced in some pro- Research Department, Teikoku Hormone Mfg, Co. Ltd., 1604 cesses of steroidogenesis has been observed both in the Shimosakunobe, Takatsu-ku, Kawasaki 213, Japan. cytoplasmic matrix (cytosol GSH-PO) and in the

365 366 Murakoshi et al.

mitochondria (mitochondrial GSH-PO). Both the phosphate-buffered 10% formalin, embedded in cytosolic and mitochondrial GSH-PO in these adrenal paraffin. Sections cut were mounted and stained with cortical cells are closely related to the cell function, hematoxylin and eosin (HE). mainly steroidogenesis (17). In the present study, an attempt to observe im- Immunocytochemicalstaining for adrenal gland munocytochemical localization of AR and GSH-PO 1) GSH-PO were made in order to clarify the atrophic effect of The adrenal glands were fixed in periodate-lysine- TZP-4238- or CMA-administration on rat adrenal 4% paraformaldehyde solution (13) for 4 to 6hr at 4°C glands. under constant agitation. The fixed tissues were then washed in 0.01 M phosphate-buffered saline (PBS) con- MATERIALSAND METHODS taining sucrose from 10 to eventually 20% overnight at 4°C. Subsequently, 6 pm-thick frozen sections Animals and tissue preparation were prepared from the washed tissues in a cryostat, Male Sprague-Dawley rats were purchased from and were placed on albumin-coated glass slides. The Charles River Japan Inc. (Atsugi, Japan) at the age of sections were washed in 0.01 M PBS and then were five weeks. The animals were kept in a barrier-main- stained by Nakane's direct peroxidase-labeled an- tained room, which were maintained at a temperature tibody method using rabbit anti-rat GSH-PO of 22 ± 2°C with a humidity of 55 ± 15%. The room polyclonal antibody IgG Fab fragment (25). was ventilated twenty one times per hr and provided For light microscopic observations of GSH-PO, with 12hr of light (from 8:00 to 20:00). The animals 6 lam-thick frozen sections were incubated with the an- were housed individually in plastic cages (CLEA tibody labeled with horseradish peroxidase (HRPO, Japan Inc.). Solid food (CE-2, CLEA Japan Inc.) Sigma Chemical Co., St. Louis, MO) for 1 hr. After and tap water were available to all animals ad libitum. the incubation was completed, the sections were Then one week was allowed for the adjustment of the treated in Graham-Karnovsky's reaction medium (4), animals to laboratory conditions. which contained 20 mg% 3, 3'-diaminobenzidine These animals were divided into four experimen- (DAB, Wako Pure Chemical Industries, Osaka) and tal groups. Group 1 consisted of untreated controls. 0.005% hydrogen peroxide in 0.05 M Tris-HC1 bu- In group 2, rats were administered 10 mg/kg of TZP- ffer, pH 7.6, for 5 to 10 min at room temperature. 4238 orally by gavage single shot daily for 3 weeks. In Then the sections were counterstained for nuclei with group 3, rats were administered 50 mg/kg of CMA 1% methyl green dissolved in veronal acetate buffer, orally by gavage single shot daily for 3 weeks. TZP- pH 4.2. 4238 and CMA were suspended in distilled water con- For electron microscopic observations of GSH- taining 2% Tween 80. The dosages of TZP-4238 and P0, 6 /Cm-thick frozen sections were incubated with CMA have been previously shown to cause prostatic HRPO-labeled antibody for 6hr. After the incuba- atrophy in the rat (19). The anti-androgenic potency tion was completed, the sections were incubated for of TZP-4238 relative to CMA has been 4 times in in- 30 min in Graham-Karnovsky's reaction medium (4), tact rats and 10 times in castrated rats supplemented from which the substrate hydrogen peroxide was omit- with testosterone propionate (14). Animals of the ted, and then they were incubated in the fully equip- group 4 were given only vehicle solution (2 ml/kg ped reaction medium for 5 min. The sections were each) consisting of distilled water and 2% Tween 80. post-fixed in 2% 0504 in 0.1 M phosphate buffer, Each group consisted of 5 animals, and the rats were pH 7.4, for 90 min, dehydrated in graded ethanol killed by decapitation at the end of the experimental series, and embedded in Quetol 812 by an inverted period and the ventral prostates and adrenal glands gelatin capsule method. Ultrathin sections were were removed. prepared with a LKB ultra-microtome and were observed under a JEOL 1200EX electron microscope. Organ weight As an immunologic negative control, normal rab- The weights of the ventral prostates and adrenal bit serum (NRS) IgG Fab fragment labeled with glands were recorded (absolute weight). Weights HRPO was applied on both light and electron relative to body weight (relative weight) were microscopic investigations instead of anti-GSH-PO calculated. IgG Fab fragment labeled with HRPO. 2) AR Histopathological examination The adrenal glands were frozen in dry-ice cooled The adrenal glands were fixed in 0.1 M ethanol. Frozen sections (6 pm in thickness) were Effects of TZP-4238 and CMA on Adrenals 367

prepared in a cryostat and mounted on albumin- spicuous changes were observed in the absolute or coated glass slides. The sections were fixed for 10 min relative weight of adrenal glands treated with TZP- at 4°C in Zamboni's fixative (33). After washing in 4238 10 mg/kg/day. 0.01 M PBS containing 20% sucrose, the sections were soaked in absolute methanol containing 0.3% 2. Histopathological and immunocytochemicalfindings hydrogen peroxide for 30 min at room temperature to Group1(Intact control) inactivate endogenous peroxidase. After washing in Representative light microscopic feature of the 0.01 M PBS, the sections were incubated overnight at adrenal gland is shown in Fig. la. Immuno- 4°C with NH 27, a rabbit polyclonal anti-androgen histochemical localization of GSH-PO was observed in receptor antibody (1:1000) (16, 21-24). After the cortical cells in all three zones of the adrenal cortex washing in 0.01 M PBS, the sections were covered (Fig. 2a). No reaction products were seen in the with biotin-conjugated goat anti-rabbit IgG for lhr, medulla. The control serum (NRS) was negative for washed and then treated with streptavidin-biotin- immunohistochemical localization of GSH-PO in the peroxidase complex (Histofine, SAB-PO (R) Kit, adrenal gland (data not shown). In immunoelectron Nichirei, Tokyo) for lhr. After washing in 0.01 M microscopic investigations, GSH-PO was observed PBS, the immuno-peroxidase staining was performed not only in cytoplasmic matrix (cytosol GSH-PO) but as described above. also in mitochondria (mitochondrial GSH-PO) in the adrenal cortical cells. Cytosol GSH-PO was observed Statistics mainly in lipid-laden outer fasciculata cells (Fig. 3a). The data was expressed as means ± S . D. . Mitochondrial GSH-PO which was present in the Homogeneity of variance was tested by Bartlett's mitochondrial cristae, was mostly observed in lipid- method, and when the assumption of homogeneity of depleted inner fasciculata cells (Fig. 3b). AR was variance was met, one-way layout analysis of variance localized exclusively in the nuclei of the adrenal cor- was performed. When significant difference was tical cells (Fig. 4a). No reaction products were seen in observed, Scheffe's or Dunnett's multiple comparative the medulla. No immunostaining was observed in the test was performed between the intact control group nuclei when the sections were incubated with preim- and the other experimental groups. mune serum (data not shown). Group2 (TZP-4238 administration) RESULTS In adrenal gland, no histopathological alterations were detected in rats treated with TZP-423810 mg/kg/ 1. Organ weights day (Fig. lb). Immunohistochemical localization of The organ weights of the controls and the animals GSH-PO (Fig. 2b) and AR (Fig. 4b) were similar to after treatment are shown in Table 1. Statistically those of the intact controls. significant decrease was noted in the absolute Group3 (CMA administration) (p < 0.01) and relative (p < 0.01) weight of ventral pro- The adrenal cortex became markedly atrophic state treated with TZP-423810 mg/kg/day or CMA 50 (Fig. lc). Immunohistochemically, GSH-PO was mg/kg/day. The administration of CMA 50 mg/kg/ observed in the cells of atrophic cortical zones (Fig. day to the rats marked a reduction in the absolute and 2c). The intensity of GSH-PO staining was weaker relative weight of adrenals. These values were than that of the intact control (Fig. 2c). In im- statistically significant (p < 0.01). In contrast, no con- munoelectron microscopic investigations, GSH-PO

TABLE i. Effect of TZP-4238 or chlormadinoneacetate (CMA) on prostatic weight and adrenal weight

Values are mean ±S.D. ** , p<0.01 vs intact control (Dunnett's multiple comparison test). 368 Murakoshi et al.

FIGS. la-c. Adrenal glands of control (a), TZP-4238 (b)- and CMA (c)-treated rats. The zonae fasciculata (F) and reticularis (R) are markedly atrophic in C. In contrast, no remarkable changes are detectable in b. G: Zona glomerulosa. H-E stain. a-c. X 120

FIGS. 2a-c. Immunohistochemical localization of GSH-PO in control (a), TZP-4238 (b)- and CMA (c)-treated rat adrenal glands. GSH-PO is localized in cortical cells of all zones of the adrenal cortex (a, b). In atrophic cortical zones of CMA-treated rats, GSH-PO staining is weaker than that of a orb. G: Zona glomerulosa, F: Zona fasciculata, R: Zona reticularis. Perox- idase labeled antibody method. a-c. X 150 Effects of TZP-4238 and CMA on Adrenals 369

FIGS. 3a-d. Immunoelectron microscopic localization of GSH-PO in control (a, b) and CMA (c, d)-treated rat adrenal cortical cells. GSH-PO is observed not only in cytoplasmic matrix (a, cytosol GSH-PO) but in mitochondria (b, mitochondrial GSH-PO) in the rat adrenal cortical cells of the control. In CMA-treated animals, GSH-PO is mainly observed in cytoplasmic matrix (c, d). Peroxidase labeled antibody method. a, b. X 12,000 c, d. X 15,000 370 Murakoshi et al.

Fits. 4a-c. Immunohistochemical localization of AR in control (a), TZP-4238 (b)- and CMA (c)-treated rat adrenal gland. AR is observed in the nuclei of the adrenal cortical cells (a, b). In CMA-treated rats, AR is observed also in the nuclei of the adrenal cortical cells of the atrophic cortical zones (c). G: Zona glomerulosa, F: Zona fasciculata, R: Zona reticularis. ABC method. a-c. x 150

was predominantly noted in cytoplasmic matrix generally accepted that CMA has some glucocorticoid- (cytosol GSH-PO) (Figs. 3c, d). The number of like activities and suppressive effect on adrenal func- GSH-PO-positive mitochondria was less than that in tion in rodents (2, 6). On the basis of our observa- the intact control. AR was localized also in the nuclei tions and these data, it is suggested that at least in of the atrophic cortical zones (Fig. 4c). part, the influence of CMA on the adrenal function is Group4 (Tween80 administration) not a direct one, but rather indirect acts by inhibiting This appearance was nearly the same as that of ACTH secretion of the pituitary glands. group 1. It is well documented fact that zona fasciculata cells synthesize corticosterone as a final product in the DISCUSSION rat adrenal cortex and cytochrome P-450 (P-450) 11A activity is mainly restricted to the zona fasciculata cells In the present study, CMA caused a marked (5, 8, 9, 15). Mitochondrial GSH-PO has been main- atrophy and loss of weight of the adrenal gland. ly observed in the adrenal cortical cells of the zona Histopathologically, the remarkable atrophy was fasciculata. Upon NADPH-dependent lipid peroxida- observed in the adrenal cortical cells of the zonae tion reaction, adrenal cortex mitochondrial P-450 has fasciculata and reticularis. Previously we been destroyed in a parallel manner as the formation demonstrated that the number of GSH-PO-positive of malondialdehyde (12). The cytochrome responsi- mitochondria markedly increased when ACTH was ad- ble for 1lp-hydroxylation has been more susceptible to ministered to normal or hypophysectomized rats degradation than for cholesterol side-chain cleavage (17). We therefore postulated that the mitochondrial reaction (12). Timcenko-Youssef et al. (31) GSH-PO in the adrenal cortical cells may be ACTH demonstrated that the adrenal cortical mitochondrial dependent (17). In the CMA-treated group, GSH- GSH-PO protects the degradation of P-450 by lipid PO was mostly observed in the cytoplasmic matrix peroxidation. Therefore, it is strongly suggested that (cytosol GSH-PO) near the mitochondria or lipid mitochondrial GSH-PO may be a very important en- droplets. The number of GSH-PO-positive mitochon- zyme for steroidogenesis, especially, corticosterone dria was less than that in the intact controls. It is synthesis. Further work along this line is now pro- Effects of TZP-4238 and CMA on Adrenals 371

gress in our laboratory. 14; 291-302, 1966. On the other hand, the present histopathological 5. Griffiths, K., Grant, J. K, and Symington, T.: A bioche- and immunocytochemical studies showed that TZP- mical investigation of the functional zonation of the 4238 medication for 3 weeks exerted no effect on the adrenal cortex in man. J. Clin. Endocrinol. Metab. 23; adrenal gland. Therefore, it is suggested that TZP- 776-785, 1963. 4238 causes atrophy of the prostate without any signifi- 6. Grvner, M. L. and Rochefort, J. G.: Effect of synthetic cant histopathological changes in the adrenal gland progestogens and estradiol- 1 73 on adrenal function in the rat. Endocrinology 90; 1238-1244, 1972.

and pituitary ACTH cells under the present ex- 7. Habenicht, U. F. and Eletreby, M. F.: Synergistic in- perimental conditions. In fact, Mieda et al. (14) hibitory effects of the aromatase inhibitor 1-methyl-an- demonstrated that the binding affinities of TZP-4238 drosta-1, 4-diene-3, 17-dione and the antiandrogen and CMA for glucocorticoid receptor was about cyproterone acetate on androstenedione-induced hyper- 1/20,000 and 1/1 , ,000 that of dexamethasone, respec- plastic effects in the prostates of castrated dog. Prostate tively. 11; 133-143, 1987. AR was localized exclusively in the nuclei of the 8. Hamerg, M., Samuelsson, S., Bjorkhem, I. and adrenal cortical cells. The male and female adrenal Danielsson, H.: Oxygenesis in fatty acid and steroid metabolism. In "Molecular Mechanisms of Oxygen Ac-

showed the same pattern of AR staining. It is believ- tivation", ed. by 0. Hayaishi, Academic Press, New ed that adrenal androgens are secreted by zona York, 1974, pp 447 reticularis. Thus, AR in the adrenal cortical cells 9. Hayano, N., Saba, N., Dorfman, R. I. and Hechter, may be involved in an autocrine function. In TZP- .: Some aspects of the biosynthesis of adrenal O steroid 4238-treated rats, staining pattern of AR was similar hormones. Recent Prog. Horm. Res. 12; 79-123, 1956. to that of the intact control. Interestingly, AR was 10. Ito, Y., Kurosawa, I., Yamanaka, H., Koya, A., Imai, also localized in the nuclei of the adrenal cortical cells K., Kasaku, N. and Shida, K.: The mechanism of an- of the atrophic cortical zones induced by CMA treat- tiandrogenic action of chlormadinone acetate. Hinyokika Kiyo 68; 537-552, 1977 (In Japanese with English ment. These results indicated that TZP-4238 or abstract).

CMA exerted no effect on the adrenal AR. Pham- 11. Kimura, N., Mizokami, A., Oonuma, T., Sasano, H.

Huu-Trung et al. (38) reported that CPA, an anti-an- and Nagura, H.: Immunocytochemical localization of drogen agent, decreased cortisol production and in- androgen receptor with polyclonal antibody in paraffin- creased adrenal androgen levels. Androgens have embedded human tissues. J. Histochem. Cytochem. 41; been shown to increase both synthesis (1, 30) and half- 671-678, 1993. life of AR (30), which result in elevated receptor pro- 12. Klimek, J., Schaap, A. P. and Kimura, T.: The relation- tein levels within the tissues. Based on our data and ship between NADPH-dependent lipid peroxidation and degradation of cytochrome P-450 in adrenal cortex these facts, it is suggested that adrenal cortical cells of mitochondria. Biochem. Biophys. Res. Commun. 110; 559- the atrophic cortical zones induced by CMA treatment 566, 1983. was decreased in corticosterone production and in- 13. McLean, I. W, and Nakane, P. K.: Periodate-lysine- creased or remained in adrenal androgen production. paraformaldehyde fixative. A new fixative for im- The biological significance of AR in the adrenal cor- munoelectron microscopy. J Histochem. Cytochem. 22; tical cells is to be further clarified. 1077-1083,1974. 14. Mieda, M., Oota, Y., Saito, T., Takahashi, H.,

Shimazawa, E. and Miyasaka, K.: Anti-androgenic ac-

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