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Immunocytochemical localization of glutathione-reductase (GSSG-RD) in rat adrenal glands was studied in 1) normal untreated, 2) hypophysectomized, and 3) ACTH administered groups in order to clarify the biological significance of GSSG-RD in the adrenal cortical lipid metabolism including steroidogenesis. In the normal untreated group, GSSG-RD was immunohistochemically localized in all three zones of the adrenal cortex. In immunoelectron microscopic investigations, GSSG-RD was observed mainly in mitochondria. The hypophysectomized group showed atrophy of the zonae fasciculata and reticularis. GSSG-RD staining of the atrophic cortical zones was weakened. In immunoelectron microscopic investigations, GSSG-RD was observed not only in mitochondria but also in cytoplasmic matrix. On ACTH administration, the adrenal cortex became markedly hypertrophic, especially in the outer fasciculata. Immunohistochemically, GSSG-RD was mainly localized in the cells of those hypertrophic cortical zones. In immunoelectron microscopic investigations, GSSG-RD was observed not only in mitochondria but also in cytoplasmic matrix. These staining patterns were similar to those of glutathione-peroxidase (GSH-PO). GSH-PO reduces the lipid peroxides with concomitant oxidation of glutathione. Oxidized glutathione is reduced by GSSG-RD under the presence of NADPH. These two enzymes are known to be key enzymes of the lipid peroxides scavenging system. Based on our findings, it is strongly suggested that a very close relationship lies between the adrenal cortical GSSG-RD and steroidogenesis.

The effects of testosterone and 17β-estradiol (E2) on the dorsolateral prostate of castrated rats were investigated by histopathological and immunohistochemical procedures. Male Sprage-Dawley rats were divided into four experimental groups. Group 1 consisted of intact controls. The other animals were castrated. In group 2, rats were sacrificed 2 days after castration. The castrated animals were treated for 6 weeks with 1) testosterone I mg/head (Group 3) and 2) testosterone I mg/head plus E2 10 μg/head (Group 4). A significant increase in the dorsolateral prostatic weight occurred after 6 weeks treatment with testosterone plus E2 (Group 4). Histopathologically, glandular hyperplasia with fibro-muscular stromal proliferation was clearly observed, and the number of bromo-deoxyuridine (BrdU)-positive cells showed a significant increase over that induced by testosterone alone. Immunohisto-chemical localization of glutathione peroxidase (GSH-PO) which effectively reduces the lipid peroxides, was clearly observed in the glandular epithelial cells of the dorsolateral prostate following testosterone alone or testosterone plus E2-treatment. Therefore, it appeared that GSH-PO protein synthesis in the glandular epithelium of the dorsolateral prostate can be enhanced (induced?) by sex hormones such as testosterone and E2. In the dorsolateral prostate of intact rats, positive staining for androgen receptor (AR) was observed in nuclei of the glandular epithelial cells. In addition, immunodetectable AR decreased within 2 days after castration, but returned to intact levels after administration of testosterone. These findings agree with previous work on the ventral prostate. Furthermore, in group 4, AR was also detected in the nuclei of the proliferated stromal fibro-muscular cells. It is concluded that immunohisto-chemical analysis, using GSH-PO and AR, may be a useful method for predication of the effects of androgen. action on the rat prostate.

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-4238 10mg/kg/day and CMA 50mg/kg/day p. o., respectively, 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, immunoreactivity 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.

The effect of a synthetic steroidal anti-androgen, TZP-4238, on steroid-induced rat dorsolateral prostatic hyperplasia was investigated. Male Sprague-Dawley rats were divided into five experimental groups. Group 1 consisted of intact controls. The other animals were castrated. The castrated animals were treated for seven weeks with 1) testosterone 1mg/animal plus 17β-estradiol (E2) 10μg/animal (Group 2), 2) testosterone plus E2 plus TZP-4238 2mg/kg (Group 3), 3) testosterone plus E2 plus TZP-4238 8mg/kg (Group 4) and 4) testosterone plus E2 plus Tween 80 (Group 5) instead of TZP-4238. TZP-4238 was administered orally for four weeks after three weeks of treatment with testosterone plus E2. In groups 2 and 5, glandular hyperplasia of the dorsolateral prostate was clearly observed, and the fibro-muscular stromal proliferation was also noted. The glandular epithelial cells showed uniformly intense nuclear immunostaining for androgen receptors (AR). Furthermore, AR was also localized in the nuclei of the proliferated fibromuscular cells. In contrast, combined treatment with TZP-4238 (Groups 3 and 4) produced inhibition of the glandular hyperplasia. Furthermore, nuclear immunostaining of AR of both epithelial and stromal cells was remarkably decreased. These results indicate that the uptake of testosterone and/or its androgenic effect on the dorsolateral prostate may be suppressed by TZP-4238. Furthermore, the decreased AR immunostaining may be explained by the decrease in number of AR and/or antibody binding site for AR.

The effects of testosterone and 17β-estradiol (E2) on the dorsolateral prostate of castrated rats were investigated by histopathological and immunohistochemical procedures. Male Sprage-Dawley rats were divided into four experimental groups. Group 1 consisted of intact controls. The other animals were castrated. In group 2, rats were sacrificed 2 days after castration. The castrated animals were treated for 6 weeks with 1) testosterone I mg/head (Group 3) and 2) testosterone I mg/head plus E2 10 μg/head (Group 4). A significant increase in the dorsolateral prostatic weight occurred after 6 weeks treatment with testosterone plus E2 (Group 4). Histopathologically, glandular hyperplasia with fibro-muscular stromal proliferation was clearly observed, and the number of bromo-deoxyuridine (BrdU)-positive cells showed a significant increase over that induced by testosterone alone. Immunohisto-chemical localization of glutathione peroxidase (GSH-PO) which effectively reduces the lipid peroxides, was clearly observed in the glandular epithelial cells of the dorsolateral prostate following testosterone alone or testosterone plus E2-treatment. Therefore, it appeared that GSH-PO protein synthesis in the glandular epithelium of the dorsolateral prostate can be enhanced (induced?) by sex hormones such as testosterone and E2. In the dorsolateral prostate of intact rats, positive staining for androgen receptor (AR) was observed in nuclei of the glandular epithelial cells. In addition, immunodetectable AR decreased within 2 days after castration, but returned to intact levels after administration of testosterone. These findings agree with previous work on the ventral prostate. Furthermore, in group 4, AR was also detected in the nuclei of the proliferated stromal fibro-muscular cells. It is concluded that immunohisto-chemical analysis, using GSH-PO and AR, may be a useful method for predication of the effects of androgen. action on the rat prostate.

The effect of a synthetic steroidal anti-androgen, TZP-4238, on steroid-induced rat prostatic hyperplasia was investigated. Male Wistar rats were divided into four experimental groups. Group 1 consisted of intact controls. The other animals were castrated. The castrated animals were treated for 7 weeks with 1) testosterone 1 mg/head plus 17 beta-estradiol (E2) 0.01 mg/head (Group 2), 2) testosterone plus E2 + TZP-4238 8 mg/kg (Group 3) and 3) testosterone plus E2 + chlormadinone acetate (CMA) 20 mg/kg (Group 4). TZP-4238 and CMA were administered orally for 4 weeks after 3 weeks treatment with testosterone plus E2. In group 2, glandular hyperplasia of the prostate was clearly observed, and the number of bromo-deoxyuridine (BrdU)-positive cells showed a significant increase. In contrast, combined treatment with TZP-4238 (Group 3) or CMA (Group 4) produced marked atrophy of the glandular epithelium, and the number of BrdU-positive cells were remarkably decreased compared with Group 2. In addition, the localization of glutathione-peroxidase (GSH-PO) which effectively reduces the lipid peroxides in the glandular epithelial cells was markedly decreased. Furthermore, nuclear immunostaining of androgen receptor was remarkably decreased after combined treatment with TZP-4238 or CMA. Our data indicate that TZP-4238 is a potent steroidal androgen receptor antagonist for the prevention of rat prostatic growth in the steroid-induced prostatic hyperplasia model.

In order to further clarify the biological significance of glutathione-peroxidase (GSH-PO) in the prostate, we have studied the immunocytochemical localization of GSH-PO in glandular epithelial cells of the ventral prostate in castrated rats treated with testosterone or testosterone plus 17β-estradiol (E2). In the control rat ventral prostate, GSH-PO was predominantly observed in the glandular epithelial cells. The intensity of GSH-PO staining in the glandular epithelial cells were remarkably depleted two days after castration, and it was clearly recovered by daily injection of testosterone, 1mg/head, or testosterone, 1mg/head, plus E2, 0.01mg/head. Furthermore, glandular hyperplasia of the prostate was definitely detected in the castrated rats with testosterone plus E2 for six weeks. In this case, the intensity of GSH-PO staining was markedly increased and the number of GSH-PO-positive granules increased remarkably, as compared with those of other experimental groups. These findings strongly suggest that castration causes marked suppression of GSH-PO protein synthesis, and lipid peroxides (or peroxidation), in the glandular epithelial cells, may be increased by testosterone-or testosterone plus E2-administration.

Rat prostate was stained immunohistochemically using polyclonal androgen receptor antibody. A rabbit polyclonal antibody referred as NH27 was raised against human androgen receptor. Male SpragueDawley rats were divided into four experimental groups. Group 1 consisted of intact controls. In group 2, rats were sacrified two days after castration. In group 3, rats were administered subcutaneously 1mg/ animal of testosterone daily for three days after two days of castration. In group 4, rats were given 10mg/ kg of TZP-4238, an anti-androgen agent, orally by gavage once a day for 1 week. Frozen sections of the prostates were fixed in Zamboni's fixative. The sections were incubated overnight at 4°C with NH27. In the control rat prostate, positive staining for the antibody appeared in nuclei of the glandular epithelium. In addition, immunodetectable androgen receptor remarkably decreased 2 days after castration and returned to intact levels after testosterone-administration in the castrated rats. These findings are in agreement with results of previous work on the prostate. Furthermore, nuclear immunostaining of the androgen receptor remarkably decreased after the treatment with TZP-4238. The precise mechanism which produces atrophy of the prostatic glandular epithelium is not yet clear from the present study. We further speculated that TZP-4238 was considered to be a steroidal androgen receptor antagonist. It is concluded that immunohistochemical analysis using NH27 may be a very useful method for predication of the effects of antiandrogen treatment for rats.

The effect of a synthetic steroidal anti-androgen, TZP-4238, on spontaneous benign prostatic hyperplasia (BPH) in dogs was investigated. Old male beagle dogs (5–9 years old) were divided into three experimental groups. Group 1 consisted of BPH controls. Groups 2 and 3 received TZP-4238 0.1 mg/kg/day and chlormadinone acetate (CMA) 0.3 mg/kg/day P.o., respectively, for 5 months. In group 1, glandular hyperplasia of the prostate was clearly detected. In contrast, TZP-4238 (Group 2) or CMA (Group 3) produced marked atrophy of the glandular epithelium. In addition, a histopathological study showed that TZP-4238 or CMA medication for 5 months exerted no effect on the testes and the pituitary luteinizing hormone (LH) cells. Therefore, it is suggested that TZP 4238 (0.1 mg/kg) or CMA (0.3 mg/kg) causes regression of spontaneous canine BPH without any histopathological effects on the testes and pituitary LH cells. However, slightly decreased serum testosterone levels were found in TZP 4238 treated animals, due apparently to a direct and/or indirect effect on the testes. Thus, it is suggested that a marginal antigonadotrophic effect cannot be excluded. It is concluded that TZP-4238 is a potent anti-androgen for the treatment of spontaneous canine BPH, without any negative influence on the function of the testes and the pituitary LH cells. Acta Pathol Jpn 42: 151–157, 1992.

In order to confirm the relationship between glutathione-peroxidase (GSH-PO) and testosterone action in rat ventral prostate, we have studied the immunocytochemical localization of GSH-PO in glandular epithelial cells of rat ventral prostate after treatment of TZP-4238 or chlormadinone acetate (CMA) as antiandrogens. In the control rat ventral prostate, GSH-PO was predominantly observed in the glandular epithelial cells and intracellular localization of GSH-PO was mainly observed in the cytoplasmic matrix near the rough endoplasmic reticulum and it was occasionally noted as a small granular structure at the supranuclear region. In TZP-4238-or CMA-administered rats, the glandular epithelial cells of the ventral prostate were markedly atrophic. The intensity of GSH-PO staining in the glandular epithelial cells was markedly decreased. Immunoelectron microscopically, GSH-PO-positive granules were hardly seen in the atrophic glandular epithelial cells. These findings strongly suggest that loss of GSH-PO staining in the glandular epithelial cells of the rat ventral prostate treated with TZP-4238 or CMA as anti-androgens is thought to be caused by inhibition of testosterone action, and that its staining pattern is a useful for the effect of antiandrogens on prostate.

The atrophic effects of a synthetic steroidal anti-androgen, TZP-4238, on the pituitary, prostate and adrenal gland of rats were investigated. Male Sprague-Dawley rats were divided into three experimental groups. Group 1 consisted of controls. Groups 2 and 3 received chlormadinone acetate (CMA) 50 mg/kg/day and TZP-4238 10 mg/kg/day p.o., respectively, for 3 weeks. CMA (Group 2) produced marked atrophy of the prostate. Furthermore, CMA caused marked atrophy of the adrenal gland. Histopathologically, the remarkable atrophy was observed in the adrenal cortical cells of zonae fasciculata and reticularis. The most striking ultrastructural alterations were noted in the mitochondria. In addition, intramitochondrial localization of glutathione-peroxidase (GSH-PO) which effectively reduces the lipid peroxides, was less than that in the controls. In the anterior pituitary gland, CMA induced a reduction in the size of ACTH cells. TZP-4238 (Group 3) produced marked atrophy of the prostate. However, TZP-4238 exerted no effect on the adrenal gland or anterior pituitary ACTH cells. In addition, the present histopathological study showed that TZP-4238 or CMA exerted no effect on the testes or anterior pituitary LH cells. Therefore, it is suggested that TZP-4238 causes atrophy of the prostate without any significant histopathological changes in the adrenal glands or anterior pituitary ACTH cells under the present experimental conditions. We further speculated that TZP-4238 had a more potent anti-prostatic effect than CMA and TZP-4238 had a less inhibitory influence than CMA on the pituitary-adrenal axis.

The effect of a synthetic steroidal anti-androgen, TZP-4238, on steroid-induced canine prostatic hyperplasia was studied by light and electron microscopy. Male beagle dogs (1–2 years old) were divided into four experimental groups. Group 1 consisted of intact controls. The other animals were castrated. The castrated animals were treated for 25 weeks with 1) 5α-androstane-3α, 17β-diol (A-diol) plus 17β-estradiol (E2)(Group2), 2) A-diol plus E2+ TZP 4238 0.5mg/kg (Group 3) and 3) A-diol plus E2+ chlormadinone acetate (CMA) 2.5 mg/kg (Group 4). TZP-4238 and CMA were administered orally for 21 weeks after 4 weeks treatment with A-diol plus E., In group 2, glandular hyperplasia of the prostate was clearly noted. In contrast, combined treatment with TZP-4238 (Group 3) or CMA (Group 4) produced marked atrophy of the glandular epithelium. Loss of secretory and metabolic activities was confirmed by ultrastructural investigations. Our data indicate that TZP-4238 is a potent anti-androgen for the prevention of canine prostatic hyperplasia in the steroid-induced benign prostatic hyperplasia (BPH) model. Acta Pathol Jpn 40: 871–879, 1990.