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 sug- gested 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 cor- ticosterone 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).