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In order to confirm the relationship between glutathione-peroxidase (GPx1) and biological significance on steroidogenesis, we have studied the immunocytochemical localization of GPx1 in the rat adrenal cortical cells. GPx1 was observed not only in cytoplasm (cytosol GPx1) but in mitochondria (mitochondrial GPx1). The staining intensity was altered by the functional state of the adrenal cortical cells. Furthermore, cytosol- and mitochondrial-GPx1 was modified by lipoperoxidative damage in the adrenal cortical cells. Therefore, we proposed that the pattern of GPx1 staining should be a more sensitive and specific indicator of oxidative damage in cells. Thus, the staining pattern of GPx1 is thought to be a useful marker for lipid peroxidation in the adrenal cortical cells.

The recurrence of estrus and fertility after removal of a subcutaneous chlormadinone acetate implant (CMA-I) administered to prevent estrus for 4 years, was investigated in 8 female dogs and the results compared with those for 4 untreated female dogs (control group). The sex hormones present during the estrous cycle were also investigated. There were no significant differences in the estrous cycle after removal of the implant between the CMA-I-treated group and the control group. However, although conception was achieved after mating and no uterine diseases developed in the control group, only 5 (4 dogs, 41.7%) of the 12 cases (6 dogs) in which mating took place at the second to fourth estrus after the removal of CMA-I resulted in pregnancy in the CMA-I-treated group. Furthermore, 6 (75.0%) of the 8 dogs in the CMA-I-treated group developed uterine diseases including pyometra or hydrometra. There were no significant differences in plasma progesterone, LH and prolactin levels between the non-pregnant and pregnant dogs in the CMA-I-treated group or control group. These results suggest that long-term implantation of CMA-I affects fertility after the implant is removed.

In order to confirm the relationship between glutathione-peroxidase (GSH-PO) and Bcl-2, we have studied the immunocytochemical localization of GSH-PO, Bcl-2, androgen receptor (AR) and apoptosis in the rat ventral prostatic cells in the presence and absence of testosterone. Male Crj:CD(SD) IGS rats were divided into four experimental groups. Group 1 consisted of untreated controls. In group 2, rats were sacrificed two days after castration. In groups 3 and 4, rats were subcutaneously administered 1 mg/animal of testosterone daily for 3 or 7 days after two days of castration, respectively. In group 1, GSH-PO was predominantly observed in the glandular epithelial cells. Bcl-2 was localized exclusively in the cytoplasm of the glandular epithelial cells. AR was localized in the nuclei of the glandular epithelial cells. In group 2, the intensity of the staining of GSH-PO, Bcl-2 and AR markedly decreased. However, the number of apoptotic cells (apoptotic index) markedly increased. In groups 3 and 4, the immunoreactivities of GSH-PO, Bcl-2 and AR clearly recovered. These findings strongly suggest that the expression of GSH-PO and Bcl-2 in the glandular epithelial cells of the rat ventral prostate is testosterone-dependent. Furthermore, co-expression of GSH-PO and Bcl-2 in the prostatic cells are considered to be normal or adaptive aspects of the cells.

The effect of testosterone and estradiol on the prostate of castrated rats (rat prostatic hyperplasia model after combined treatment with testosterone and estradiol) were investigated by histopathologically and immunohistochemical procedures. The castrated rats were treated for 6 weeks with 1) testosterone 1 mg/animal and 2) testosterone 1 mg/animal plus 17beta-estradiol (estradiol) 10 μg/animal. Histopathologically, glandular hyperplasia of the ventral prostate and glandular hyperplasia with fibromuscular stromal proliferation in the dorsolateral prostate, was clearly observed. Immunohistochemical localization of glutathione peroxidase (GSH-PO) which effectively reduces the lipid peroxides, was predominantly observed in the glandular epithelial cells of the ventral prostate. The intensity of GSH-PO staining in the glandular epithelial cells was remarkably decreased after castration, and that it was clearly recovered by testosterone or testosterone plus estradiol treatment to the castrated rats. In contrast, GSH-PO was clearly observed in the glandular epithelial cells of the dorsolateral prostate following testosterone alone or testos-terone plus estradiol. Androgen receptor (AR) is mainly localized in nuclei. In the dorsolateral prostate, AR was also detected in the nuclei of the proliferated stromal fibromuscular cells. Furthermore, immunodetectable AR rapidly declined after androgen withdrawal and returned to intact levels of staining intensity after androgen replacement. These findings strongly suggest that expressions of GSH-PO and AR in the prostate are testosterone-dependent. It is concluded that immunohistochemical analysis, using GSH-PO and AR, may be a useful method for prediction of the effects of androgen action on the rat prostate.

The atrophic effect of a synthetic steroidal antiandrogen, chlormadinone acetate (CMA), on spontaneous benign prostatic hyperplasia (BPH) was investigated. Male beagle dogs (5-8 years old) were divided into four experimental groups. Group 1 consisted of untreated controls. Groups 2 to 4 received CMA 0.03, 0.1 and 0.3 mg/kg/day, p.o., respectively, for 6 months. In group 1, glandular hyperplasia of the prostate was clearly detected. The glandular epithelial cells showed uniformly intense nuclear staining for androgen receptor (AR). AR was also localized in the nuclei of the fibro-muscular stromal cells. Immunoreactively of 5 alpha-reductase type I was positive in most glandular epithelial cells. No fibro-muscular stromal cells were stained. Immunolocalization of 5 alpha-reductase type II was clearly detected in the interacinar fibro-muscular stromal cells, but not in the glandular epithelial cells. In groups 2 to 4, CMA produced marked atrophy of the glandular epithelium. The interacinar fibro-muscular stroma was prominent. The nuclear staining for AR in both epitheial and stromal cells was remarkably decreased. Furthermore, the immunoreaction for 5 alpha-reductase type I in most glandular epithelial cells was negative or very weak. The immunoreaction of 5 alpha-reductase type II in the interacinar fibro-muscular stromal cells was negative or very weak. The decreased AR-immunostaining may be explained by the decrease in the number of AR and it may play a key role in the down-regulation of 5 alpha-reductase gene expression by CMA. The atrophy after treatment with CMA may be due to shrinkage of both glandular and stromal compartments in the prostate tissue.

The histopathological changes related to chlormadinone acetate (CMA) implantation were examined using female beagle dogs given 10 mg/kg for four years. Estrus was completely inhibited in the CMA-implanted animals. In contrast, all control animals showed sign of estrus during the experiment, with periods of anestrus of normal duration. Histopathlogically, uterine sections from the CMA-implanted animals showed cystic glandular hyperplasia, but no histologic evidence of endometritis, myometritis, and pyometra was found. In the ovaries of the CMA-implanted animals, developing ovarian follicles were observed but no mature follicles were noted in addition to an absence of corpus luteum. No remarkable changes were observed in liver, adrenal, mammary gland, gallbladder, and implanted site. Furthermore, the intensity of staining and number of adrenocorticotropic hormone (ACTH)- and luteininzing hormone (LH)-positive cells in the pituitary sections of CMA-implanted animals were not different from the control animals. It was concluded, therefore, that subcutaneous implantation of CMA (10 mg/kg) is nearly equivalent to the minimal ovulation-suppressing dose.

In order to confirm the relationship between benign prostatic hyperplasia (BPH) regression and atrophic effect of chlormadinone acetate (CMA), the prostates of spontaneous BPH dogs administered CMA for 6 months were investigated histopathologically and immunohistochemically. Male beagle dogs (5-8 years old) were divided into four experimental groups; Group 1 consisted of untreated controls. Groups 2 to 4 received CMA 0.03, 0.1, and 0.3 mg/kg/day, p.o., respectively, for 6 months. In group 1, glandular hyperplasia of the prostate was clearly detected. In groups 2 to 4, CMA produced marked atrophy of the glandular epithelium. The interacinar fibro-muscular stroma was prominent. To evaluate the frequency of apoptosis, we counted the positive cells stained by the nick end labeling method. In group 1, the apoptotic index was 0.68 ± 0.03 %. In groups 2 to 4, apoptotic indices were 10.32 ± 0.96 %, 2.05 ± 0.88 %, and 1.15 ± 0.75 %, respectively. Apoptotic cell death was mainly observed in the glandular epithelial cells. Based on our data, regression of BPH after treatment with CMA may be apoptotic cell death.

In the canine prostate, glandular epithelial cells showed uniformly intense nuclear staining for androgen receptor (AR). AR was also localized in the nuclei of the fibro-muscular stromal cells. Immunoreactivity of 5 alpha-reductase type I was positive in most glandular epithelial cells. No fibro-muscular stromal cells were stained. Immunolocalization of 5 alpha-reductase type II was clearly detected in the interacinar fibro-muscular stromal cells, but not in the glandular epithelial cells. After treatment with antiandrogen, chlormadinone acetate (CMA) on canine spontaneous benign prostatic hyperplasia (BPH), glandular atrophy was evident. The interacinar fibro-muscular stroma was prominent. The nuclear staining for AR in both epithelial and stromal cells was remarkably decreased. Furthermore, the immunoreaction for 5 alpha-reductase type I in most glandular epithelial cells was negative or very weak. The immunoreaction of 5 alpha-reductase type II in the interacinar fibro-muscular stromal cells was negative or very weak. The decreased AR-immunostaining may be explained by the decrease in the number of AR and it may play a key role in the down-regulation of 5 alpha-reductase gene expression by CMA. The atrophy after treatment with CMA may be due to shrinkage of both glandular and stromal compartments in the prostate tissue.

The effect of chlormadinone acetate (CMA), a synthetic steroidal antiandrogen, on spontaneous benign prostatic hyperplasia (BPH) in dogs was investigated. Male beagle dogs (5-8 years old) were divided into four experimental groups. Group 1 consisted of untreated controls. Groups 2 and 3 received CMA 0.03 and 0.1 mg/kg/day, p.o., respectively, for 6 months. In group 1, glandular hyperplasia of the prostate was clearly detected. The glandular epithelial cells showed uniformly intense nuclear staining for androgen receptor (AR). AR was also localized in the nuclei of the fibro-muscular stromal cells. In groups 2 and 3, CMA produced marked atrophy of the glandular epithelium. The interacinar fibro-muscular stroma was prominent. The nuclear staining for AR in both epithelial and stromal cells was remarkably decreased. In addition, a histopathological study showed that CMA medication for 6 months exerted no effect on the testes and adrenal glands or on immunoreactive positive cells to LH- and ACTH-antibody (pituitary LH- and ACTH-cells). Therefore, it is concluded that CMA (0.03 and 0.1 mg/kg) causes regression of spontaneous canine BPH without any histopathological effects on the testes, adrenal glands or pituitary LH- and ACTH-cells.

The effect of a synthetic steroidal antiandrogen, chlormadinone acetate (CMA), on spontaneous benign prostatic hyperplasia (BPH) in dogs was investigated. Male beagle dogs (5-8 years old) were divided into four experimental groups. Group 1 consisted of untreated controls. Groups 2 to 4 received CMA 0.03, 0.1, and 0.3 mg/kg/day, p.o., respectively, for 6 months. In group 1, glandular hyperplasia of the prostate was clearly detected. In groups 2 to 4, CMA produced marked atrophy of the glandular epithelium. The interacinar fibro-muscular stroma was prominent. To evaluate the frequency of apoptosis, we counted the positive cells stained by the nick end labeling method. In group 1, the apoptotic index was 0.76±0.03%. In groups 2 to 4, apoptotic indices were 15.41±1.26%, 2.63±0.98% and 1.45±0.85%, respectively. Apoptotic cell death was mainly observed in the glandular epithelial cells. Based on our data, regression of BPH after treatment with CMA may be apoptotic cell death.

To elucidate the effects of synthetic salmon calcitonin (sCT) on the cells in the rat pituitary gland, we histopathologically and immunohistochemically examined the early changes after 4 or 13 weeks treatment with sCT 120 IU/kg. Focal proliferative lesions of the anterior pituitary glands were consistently found after treatment with sCT for 13 weeks. Histologically, the cells with the focal proliferative lesions were classified into the following three groups: 1) enlarged basophilic cell focus, 2) vacuolated cell focus and 3) chromophobe cell focus. These focal proliferative lesions had positive staining only for the alpha-subunit and failed to show Pit-1 protein immunoreactivity. The sCT treatment also increased the thickness of the pars intermedia. Hypertrophy of the pars intermediate cells was characteristically seen. Furthermore, Pit-1 protein immunoreactivity was clearly detected in the nuclei of the hyperplastic pars intermediate cells. All pars intermediate cells were equally stained by alpha- or beta-MSH and beta-endorphin in both vehicle- and sCT-treatment. No difference was seen. These findings strongly suggest a very close relationship between Pit-1 protein immunoreactivity and cellular proliferation induced by sCT.

The effect of a synthetic steroidal anti−androgen, chlormadinone acetate(CMA), on spontaneous benign prostatic hyperplasia(BPH)in dogs was investigated. Male beagle dogs(5−8 years old) were divided into four experimental groups. Group 1 consisted of untreated controls. Groups 2 to 4 received CMA 0.03, 0.1, and 0.3mg/kg/day, p.o., respectively, for 6 months. In group 1, glandular hyperplasia of the prostate was clearly detected. The glandular epithelial cells showed uniformly intense nuclear staining for androgen receptor(AR). AR was also localized in the nuclei of the fibro−muscular stromal cells. Immunolocalization of 5 alpha−reductase type II was clearly detected in the interacinar stromal cells, but not in the glandular epithelial cells. In groups 2 to 4, CMA produced marked atrophy of the glandular epithelium. The interacinar fibro−muscular stroma was prominent. The intensity of the nuclear staining for AR in both epithelial and stromal cells was negative or very weak. Furthermore, the immunoreaction for 5 alpha−reductase type II in the interacinar stromal cells was negative or very weak. The decreased AR−immunostaining may be explained by the decrease in the number of AR and it may play a key role in the down−regulation of 5 alpha−reductase type II gene expression by CMA. The atrophy after treatment with CMA may be due to shrinkage of both glandular and stromal compartments in the prostate tissue.

TZT-1027, a dolastatin 10 derivative, is an antimicrotubule agent with potent antitumor activity both in vitro and in vivo. In this study, we performed biochemical and histopathological examinations, and evaluated TZT-1027-induced tumoral vascular collapse and tumor cell death in an advanced tumor model, murine colon 26 adenocarcinoma. In addition, we studied the effects of TZT-1027 on cultured human umbilical vein endothelial cells (HUVEC). Tolerable doses of TZT-1027 induced tumor-selective hemorrhage within 1 h. This hemorrhage occurred mainly in the peripheral area of the tumor mass. Measurements of tumoral hemoglobin content and dye permeation revealed that the hemorrhage occurred firstly and tumor blood flow stopped secondarily. The vascular damage was followed by continuous induction of apoptosis of the tumor cells, tumor tissue necrosis, and tumor regression. In cultured HUVEC, TZT-1027 induced marked cell contraction with membrane blebbing in 30 min. These cell changes were completely inhibited by K252a, a broad-spectrum inhibitor of protein kinases. These effects of TZT-1027 on both tumor vasculature and HUVEC were greater than those of vincristine. In conclusion, TZT-1027 quickly attacked the well-developed vascular system of advanced tumors by a putative protein kinase-dependent mechanism, and then blocked tumor blood flow. Therefore, TZT-1027 has both a conventional antitumor activity and a unique anti-tumoral vascular activity, making it a potentially powerful tool for clinical cancer therapy.

In order to confirm the relationship between glutathione-peroxidase (GSH-PO) localization and biological testosterone action in the rat ventral prostate, immunocytochemical localization of GSH-PO in glandular epithelial cells of the rat ventral prostate was investigated. In the untreated group, GSH-PO was predominantly demonstrated in glandular epithelial cells of the ventral prostate. Intracellular localization of GSH-PO in the glandular epithelial cells was mainly observed in cytoplasmic matrix near the rough endoplasmic reticulum and was occasionally noted as a small granular structure (GSH-PO-positive granule) at the supranuclear region. In a castrated animal, the intensity of GSH-PO staining in the glandular epithelial cells was remarkably decreased. By testosterone administration to the castrated animal, GSH-PO was clearly detected in the glandular epithelial cells. Intracellular localization of GSH-PO was mainly observed in cytoplasmic matrix and the number of GSH-PO-positive granules increased remarkably. These findings suggest that immunostainable GSH-PO in the glandular epithelial cells of the rat ventral prostate is testosterone-dependent, and that its staining pattern is a useful marker for biological testosterone action.

The effect of a synthetic steroidal antiandrogen, chlormadinone acetate (CMA), on spontaneous benign prostatic hyperplasia (BPH) in dogs was investigated. Male beagle dogs (5-8 years old) were divided into four experimental groups. Group 1 consisted of untreated controls. Groups 2 to 4 received CMA 0.03, 0.1, and 0.3 mg/kg/day, p.o., respectively, for 6 months. In group 1, glandular hyperplasia of the prostate was clearly detected. In groups 2 to 4, CMA produced marked atrophy of the glandular epithelium. In addition, a histopathological study showed that CMA medication for 6 months exerted no effect on the testes except group 4 or on immunoreactive positive cells to LH antibody (pituitary LH cells). Therefore, it is suggested that CMA (0.03 and 0.1 mg/kg) causes regression of spontaneous canine BPH without any histopathological effects on the testes or pituitary LH cells. However, atrophy of seminiferous tubules in testes was found in CMA 0.3 mg/kg/day-treated animals, due apparently to a direct and/or indirect effect on the testes. It is suggested that a marginal antigonadotrophic effect can not be excluded.

In order to confirm the relationship between testosterone- administration and glutathione-peroxidase (GSH-PO) in the rat ventral prostate, we have studied the levels of GSH-PO mRNA, GSH-PO activity, and lipid peroxide value in the ventral prostate under the presence or absence of androgen. Male Sprague-Dawley rats were divided into four experimental groups. Group 1 consisted of intact controls. In group 2, rats were sacrificed two days after castration. In groups 3 and 4, rats were administered subcutaneously I mg/animal of testosterone-propionate daily for three or seven days after two days of castration. The prostatic GSH-PO mRNA levels were diminished in the castrated rat ventral prostate but greatly increased by testosterone (Groups 3 and 4). Furthermore, GSH-PO activity was increased and the TBA value was decreased by testosterone-administration (Groups 4). These findings strongly suggest that expression of GSH-PO in the glandular epithelial cells of the rat ventral prostate is considered to be testosterone-dependent.

Immunolocalization of glutathione-peroxidase (GSH-PO), apoptosis and bcl-2 protein in the rat ventral prostate was investigated under the presence or absence of androgen. Male Sprague-Dawley rats were divided into four experimental groups. Group 1 consisted of intact controls. In group 2, rats were sacrificed two days after castration. In groups 3 and 4, rats were subcutaneously administered 1 mg/animal of testosterone-propionate daily for three or seven days after two days of castration. The intensity of GSH-PO staining in the glandular epithelial cells of the ventral prostate was remarkably decreased after castration (Group 2), and it was clearly recovered by testosterone-administration (Groups 3 and 4) to the castrated rats. The prostatic GSH-PO mRNA levels were diminished in the castrated rat ventral prostate but greatly increased by testosterone (Groups 3 and 4). Furthermore, castration (Group 2) induced apoptosis in the prostatic glandular epithelial cells and apoptosis was reduced by testosterone-administration (Groups 3 and 4) to the castrated rats. In groups 3 and 4, expression of bcl-2 protein was clearly detected in the glandular epithelial cells of the ventral prostate. These findings strongly suggest that expression of GSH-PO and bcl-2 protein in the glandular epithelial cells of the rat ventral prostate is considered to be testosterone-dependent.

The efficacy and clinical safety of chlormadinone acetate (CMA) in preventing estrus were assessed in queens on condition that CMA was subcutaneously implanted in silastic silicon rubber. Thirteen queens were divided into the following four groups according to dose of CMA-administered: group 1 (n=3), control; group 2 (n=3), 2.5 mg/kg; group 3 (n=3), 5 mg/kg; group 4 (n=4), 20 mg/kg. The implants were left in these queens for 12 months after implantation. All control animals showed signs of estrus during the experiment, with periods of anestrus of normal duration. In contrast, estrus was completely inhibited in the CMA-treated groups. Plasma concentrations of CMA had started to decrease by l month after implantation and continued to decrease gradually thereafter. Histopathologically, the uterus from group 4 had coiled branched glands with little secretion in the endometrium. Mammary glands from the CMA-treated groups showed mild lobular development with acinar proliferation and secretion. Sections through the other organs (pituitary gland, adrenal gland, ovary, and implant site) had no distinct or consistent changes that could be related to the CMA-treated. It was concluded, therefore, that low, stable levels of CMA maintained in plasma by subcutaneous implantation could be the main reason for few changes due to the antigonadotropic and glucocorticoid-like activities and less serious condition in the uterine and mammary glands due to progestagenic activity.

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.

Rat pituitary tumors induced by synthetic salmon calcitonin (TZ-CT) were studied by the indirect peroxidase-labeled antibody method, together with ultrastructure and serum hormone measurement. Immunohistochemically, TZ-CT-induced pituitary tumors showed staining for only rLH alpha subunit, and were negative for other peptide hormones including GH, PRL, alpha MSH and ACTH, and the beta subunit of glycoprotein hormones. Electron microscopic examination showed that the majority of tumor cells possessed numerous small secretory granules, 100 to 200 nm in diameter. The serum PRL concentrations of rats with TZ-CT-induced pituitary tumors were markedly elevated, but not beyond 130 ng/ml. From our data, TZ-CT-induced pituitary tumors are considered to be endocrinologically inactive and to produce alpha subunit. Furthermore, these tumors are thought to be potentially useful models of alpha subunit-producing pituitary tumors in humans. This is the first report to document the tumorigenesis of alpha subunit-producing pituitary tumors in rats after long-term treatment with calcitonin.

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.

In order to confirm the relationship between sex hormone administration and glutathione-peroxidase (GSH-PO) in the rat ventral prostate, the levels of GSH-PO mRNA, GSH-PO activity, and lipid peroxide (Thiobarbituric acid: TBA) value in the ventral prostate were investigated. Male Crj: CD (SD) IGS rats were divided into six experimental groups. Group 1 consisted of intact controls. In group 2, rats were sacrificed two days after castration. In groups 3 and 4, rats were subcutaneously administered 1 mg/animal of testosterone daily for three- or seven-day administration two days after castration, respectively. In groups 5 and 6, rats were subcutaneously administered 1 mg/animal of testosterone plus 0.01 mg/animal of 17beta-estradiol (E2) daily for three- or seven-day administration two days after castration, respectively. GSH-PO activity of the ventral prostate homogenate for testosterone or testosterone plus E2 administration to the castrated rat was increased and the TBA value was remarkably decreased. The prostatic GSH-PO mRNA level was diminished in the castrated rat ventral prostate, but was increased by testosterone or testosterone plus E2 administration. In particular, the GSH-PO mRNA level of testosterone plus E2-treated animals was higher than that of testosterone-treated animals. These findings strongly suggest that expression of GSH-PO in the rat ventral prostate is testosterone- or E2-dependent. We speculate that the transcription of prostatic GSH-PO mRNA was regulated by testosterone or E2 and de novo synthesis of GSH-PO would thus be regulated at transcription level by testosterone or E2.

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 chlormadinone acetate CMA) as antiandrogen. In the control rat ventral prostate, GSH-PO was predominantly observed in the glandular epithelial cells and intracellular localization of GSH-PO was exclusively observed in the cytoplasmic matrix near the rough endoplasmic reticulum and it was occasionally noted as small granular structure. In 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 CMA a s antiandrogen is thought to be caused by inhibition of testosterone action, and that its staining pattern is useful for the effect of antiandrogens or antiprostatic agents on prostate.

Four-aminopyrazolopyrimidine (4-APP)-induced fatty livers were studied immunohistochemically and biochemically. Immunohistochemical localization of glutathione-peroxidase (GSH-PO) in control rat liver was predominantly observed in the hepatocytes of portal zones of the hepatic lobules. After 3 days of 4-APP administration, the intensity of GSH-PO staining was weaker than that of control. Especially, this tendency was predominantly observed in portal zones of the hepatic lobules. Biochemcally, lipid peroxide levels measured by thiobarbituric acid method in the liver homogenates were markedly increased following 4-APP administration. However, glutathione-peroxidase (GSH-PO) activity in the same homogenates was decreased. Based on our data, we strongly suggested that pathogenesis of 4-APP induced-fatty liver may be due to uncontrolled free radical formation, peroxidation of lipids, and an associated lipid accumulation in the liver.

Pathologic changes related to chlormadinone acetate (CAP) implantation were examined using 14 bitches given doses 2.5 to 25 mg/kg for 2 years. Absence of corpus luteum in bitches given 5 mg/kg or more supported long-term preventive effect of CAP on estrus. The uteri were dose-dependently enlarged and mucometra was occasionally found. Endometrial epithelium hyperplasia was observed but less in smaller doses. Changes in the mammary gland were only growth and lactation at normal degree. No remarkable changes were observed in ACTH and LH cells in the pituitary gland. Low, stable levels of CAP maintained in plasma by subcutaneous implantation seemed to be the main reason for absence or slight CAP-related pathologic changes.

One of the major dose-limiting toxicities induced by antimicrotubule antitumor agents such as vinca alkaloids and taxanes is peripheral neuropathy. The neurotoxicity of TZT-1027 (a dolastatin 10 derivative antimicrotubule agent) was thus assessed using the animal models for antimicrotubule agent-induced neurotoxicity. Rabbits were intravenously given TZT-1027 or vincristine weekly for 5 weeks. In the mouse study, TZT-1027, vincristine or paclitaxel was intravenously given every 2 days and/or weekly. Despite the neuropathologic evidence such as myelinated axonal and fiber degeneration in the peripheral nerves and in the sensory tracts of the spinal cord following the treatment with vincristine or paclitaxel, no drug-induced alteration was observed in the TZT-1027 groups. Although there are reports that some other dolastatin derivatives with antimicrotubule activity showed no neurotoxic potential in humans, the present study represents the first demonstration in experimental animals that a dolastatin derivative has no, or at least a lower, neurotoxic potential compared to other antimicrotubule agents.

In order to confirm the relationship between sex hormone administration and glutathione-peroxidase (GSH-PO) in the rat ventral prostate, the levels of GSH-PO mRNA, GSH-PO activity, and lipid peroxide (TBA) value in the ventral prostate were investigated. Male Crj:CD(SD)IGS rats were divided into six experimental groups. Group 1 consisted of intact controls. In group 2, rats were sacrificed two days after castration. In groups 3 and 4, rats were subcutaneously administered 1 mg/animal of testosterone daily for three or seven days after two days of castration, respectively. In groups 5 and 6, rats were subcutaneously administered 1 mg/animal of testosterone plus 0.01 mg/animal of 17 beta-estradiol (E2) daily for three or seven days after two days of castration, respectively. GSH-PO activity of the ventral prostate homogenate for testosterone or testosterone plus E2 administration to the castrated rat was increased and the TBA value was remarkably decreased. The prostatic GSH-PO mRNA level was diminished in the castrated rat ventral prostate, but was increased by testosterone or testosterone plus E2 administration. In particular, the GSH-PO mRNA level of testosterone plus E2-treated animals was higher than that of testosterone-treated animals. These findings strongly suggest that expression of GSH-PO in the rat ventral prostate is considered to be testosterone- or E2-dependent. Furthermore, it is suggested that the transcription of prostatic GSH-PO mRNA was regulated by testosterone or E2 and de novo synthesis of GSH-PO would thus be regulated at transcription level by testosterone or E2.

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.

Testosterone-secreting adrenal adenoma is rare. We recently experienced a 17-year-old pubertal girl who showed signs of virilization and a high serum level of testosterone. The excised adrenal gland showed a 3.5 x 3 x 3-cm cortical adenoma. Light and electron microscopic findings together with the high serum level and high tumor tissue contents of testosterone and dehydroepiandrosterone (DHA) indicated that the tumor was a testosterone-secreting adrenal cortical adenoma. This appears to be a rather rare tumor from a review of the literature. Interestingly, in this case, the cytoplasm of the tumor cells contained structures resembling spironolactone bodies. From the results of enzyme histochemistry, the steroidogenetic pathways in this tumor were speculated.

Immunohisto-cytochemical localization of glutathione-peroxidase (GSH-PO) in adrenocortical cells was studied on rats treated with aminoglutethimide (Elipten), which blocks the conversion of cholesterol into pregnenolone, and 4-aminopyrazolopyrimidine (4-APP), which increases the adrenocortical cholesterol synthesis. In a normal untreated group, GSH-PO was observed immunohistochemically in zonae fasciculata and reticularis and intracellular localization of GSH-PO was observed not only in cytosol but in mitochondria. In Elipten treated group, immunohistochemically, GSH-PO was mainly observed in lipid accumulated zona fasciculata. And intracellular localization of GSH-PO was mainly observed in cytosol near the electron dense lipid droplets. Intensity of immunocytochemical staining of GSH-PO was stronger than that in the normal untreated group. However, number of GSH-PO positive mitochondria was less than that in the normal untreated group. In 4-APP treated group, immunohistochemical localization of GSH-PO was mainly observed in inner fasciculata-reticularis and intracellular localization of GSH-PO was mostly observed in cytosol near the smooth endoplasmic reticulum.These findings may suggest that GSH-PO plays an important role in prevention of damage to mitochondrial and microsomal components with lipid peroxidation.

Immunocytochemical localization of glutathione-peroxidase (GSH-PO) in the ventral prostate was studied in rats under the following experimental conditions; 1) normal untreated 2) 4 weeks after castration 3) testosterone-administration to castrated, in order to clarify the role of GSH-PO. In normal untreated group, GSH-PO was predominantly observed in glandular epithelial cells of the ventral prostate. Intracellular localization of GSH-PO in the glandular epithelial cells was mainly observed in cytoplasmic matrix near the rough endoplasmic reticulum, and it was occasionally noted as a small granular structure at the supranuclear region. In the castrated animal, the intensity of GSH-PO staining in the glandular epithelial cells of atrophic prostate was markedly decreased. By testosterone-administration to the castrated animal, GSH-PO was clearly detected in the glandular epithelial cells of stimulated prostate. Intracellular localization was mainly observed in cytoplasmic matrix and the number of GSH-PO-positive granules increased remarkably as compared with those of other experimental groups.These findings strongly suggest that GSH-PO in the glandular epithelial cells of the rat ventral prostate is testosterone-dependent, and that its staining pattern is a useful marker for testosterone action.