Localization of epoxide-metabolizing enzymes in rat testis.

Antibodies raised against rat hepatic epoxide hydrolase (EC 3.3.2.3) and glutathione S-transferases (EC 2.5.1.18) B, C and E were used to determine the presence and localizations of these epoxide-metabolizing enzymes in testes of sexually immature and mature Wistar and Holtzman rats. Unlabeled antibody peroxidase-antiperoxidase staining for each enzyme was readily detected in rat testes at the light microscopic level. Although significant strain-related differences were not apparent, staining intensity for certain enzymes differed markedly between Leydig cells and seminiferous tubules. Leydig cells of immature and mature rats were stained much more intensely for epoxide hydrolase and glutathione S-transferases B and E than were seminiferous tubules, whereas Sertoli cells, spermatogonia, spermatocytes and spermatids, as well as Leydig cells, were stained intensely by the anti-glutathione S-transferase C. Age-related differences in staining for glutathione S-transferase B were not obvious, while the anti-glutathione S-transferase C stained seminiferous tubules more intensely in immature rats, and antibodies to epoxide hydrolase and glutathione S-transferases C and E stained Leydig cells much more intensely in mature rats. These observations thus demonstrate that testes of both sexually immature and mature rats contain epoxide hydrolase and glutathione S-transferases. Except for glutathione S-transferase C in immature rats, Leydig cells appear to contain much higher levels of enzymes than do seminiferous tubules. During sexual maturation, the testicular level of glutathione S-transferase B appears to remain constant, while levels of epoxide hydrolase and glutathione S-transferases C and E increase within Leydig cells and the level of glutathione S-transferase C decreases within seminiferous tubules.

Contribution of cytochrome b5 to androgen synthesis in rat testicular microsomes.

Cytochrome b5 was purified from porcine testicular microsomes, and its amino acid composition was determined. Rabbit antibody against the purified cytochrome b5 was prepared in order to study the contribution of cytochrome b5 to testicular microsomal oxygenases related to androgen production. In the presence of NADPH alone as the electron donor, the antibody against cytochrome b5 inhibited the activities of steroid 17 alpha-hydroxylase and C-17-C-20 lyase of rat testicular microsomal fraction. Addition of NADH to the NADPH-supported oxygenase assay system enhanced both steroid oxygenase activities, and addition of the antibody against cytochrome b5 decreased the NADH-caused stimulation of steroid 17 alpha-hydroxylase and C-17-C-20 lyase activities. When dehydroepiandrosterone and NAD+ were added as substrates for 3 beta-hydroxy-delta 5-steroid dehydrogenase in order to synthesize NADH by enzymatic reaction, the NADPH-supported activities of steroid 17 alpha-hydroxylase and C-17-C-20 lyase were further stimulated as compared with the addition of NADH, and this stimulation was suppressed by the antibody against cytochrome b5. These results suggest that cytochrome b5, together with 3 beta-hydroxy-delta 5-steroid dehydrogenase, contributes to the activities of steroid 17 alpha-hydroxylase and C-17-C-20 lyase in the testicular microsomal fraction.

Steroid hormone production in testis, ovary, and adrenal gland of immature rats irradiated in utero with 60Co.

Pregnant rats received whole-body irradiation at 20 days of gestation with 2.6 Gy lambda rays from a 60Co source. Endocrinological effects before maturation were studied using testes and adrenal glands obtained from male offspring and ovaries from female offspring irradiated in utero. Seminiferous tubules of the irradiated male offspring were remarkably atrophied with free germinal epithelium and containing only Sertoli cells. Female offspring also had atrophied ovaries. Testicular tissue obtained from intact and 60Co-irradiated rats was incubated with 14C-labeled pregnenolone, progesterone, 17 alpha-hydroxyprogesterone, and androstenedione as a substrate. Intermediates for androgen production and catabolic metabolites were isolated after the incubation. The amounts of these metabolites produced by the irradiated testes were low in comparison with the control. The activities of delta 5-3 beta-hydroxysteroid dehydrogenase, 17 alpha-hydroxylase, C17,20-lyase, and delta 4-5 alpha-reductase in the irradiated testes were 30-40% of those in nonirradiated testes. Also, the activities of 17 beta- and 20 alpha-hydroxysteroid dehydrogenases were 72 and 52% of the control, respectively. In adrenal glands, the 21-hydroxylase activity of the irradiated animals was 38% of the control, but the delta 5-3 beta-hydroxysteroid dehydrogenase activity was comparable to that of the control. On the other hand, the activity of delta 5-3 beta-hydroxysteroid dehydrogenase of the irradiated ovary was only 19% of the control. These results suggest that 60Co irradiation of the fetus in utero markedly affects the production of steroid hormones in testes, ovaries, and adrenal glands after birth.

Absence of linkage between radiosensitivity and the predisposing atp7b gene mutation for heritable hepatitis in the LEC rat.

The LEC rat is known to be a mutant strain that spontaneously develops heritable hepatitis due to copper accumulation, caused by mutation of the copper-transporting ATPase gene (Atp7b). Immunodeficiency and radiosensitivity have also been observed. Hayashi et al. extensively examined the radiosensitivity of the LEC rat and concluded that its hypersensitivity is controlled by a single autosomal gene. Furthermore, they suggested the possibility that it correlates to copper accumulation due to the Atp7b gene mutation, because ionizing radiation-induced hydroxyl radicals might act in concert with copper-induced hydroxyl radicals. In the present experiment, we analyzed linkage between radiosensitivity and the mutation responsible for hepatitis in F(1) animals of a cross with the F344 rat. Our results clearly demonstrated an absence of any significant association. In addition, partial dominance for radiosensitivity was observed, and radiosensitive (F(1) x LEC) backcross rats were twice as numerous as their radioresistant counterparts, suggesting the possibility of control by two or more recessive genes.

Chemoprevention by dietary dehydroepiandrosterone against promotion/progression phase of radiation-induced mammary tumorigenesis in rats.

When pregnant rats received whole body irradiations with 260 cGy gamma-ray at day 20 of pregnancy, and were then implanted with a diethylstilbestrol (DES) pellet for an experimental period of 1 year under feeding of a control diet, a high incidence (96.2%) of mammary tumors was observed. Administration of dietary 0.6% dehydroepiandrosterone (DHEA) together with DES implantation significantly decreased the incidence (35.0%) of mammary tumors. The first appearance of palpable tumors in the DHEA-fed group was 4.5 months later than that in the control group. For clarification of the mechanism of the chemopreventive action, we measured hormone levels in the serum of DHEA-fed rats. In the DHEA diet rats, the concentration of estradiol-17 beta exceeded, by approximately 6-fold, that in the control rats, while the levels of progesterone and prolactin were decreased by 30 and 45%, respectively. Interestingly, DHEA feeding prevented DES-induced hypertrophy of pituitary glands and DES-induced high level of prolactin in pituitary glands detected by immunohistochemical studies, but stimulated the development of mammary glands more than that in control rats treated with DES alone. These findings suggest that DHEA has a potent preventive activity against the promotion/progression phase of radiation-induced mammary tumorigenesis. The mechanism of chemoprevention by change of endocrinological environment is discussed.

Subcellular distribution of 3 beta-hydroxysteroid dehydrogenase-isomerase in bovine and murine adrenocortical tissue: species differences in the localization of activity and immunoreactivity.

Key to the production of biologically active steroids is the enzyme 3 beta-hydroxysteroid dehydrogenase-isomerase. Some controversy has arisen concerning the subcellular distribution of this enzyme within steroidogenic cells. The distribution of 3 beta-hydroxysteroid dehydrogenase-isomerase was assessed in subcellular fractions obtained from homogenates of rat, bovine, and mouse adrenal glands in two ways. The activity of 3 beta-hydroxysteroid dehydrogenase-isomerase was quantitated by measuring the conversion of radiolabeled pregnenolone to radiolabeled progesterone in an aliquot of each of the fractions obtained. The presence of the enzyme was assessed by performing Western analyses on aliquots of each of the fractions obtained with the use of a specific polyclonal antiserum against 3 beta-hydroxysteroid dehydrogenase-isomerase, the characterization of which is described. In control experiments, the degree of contamination of the fractions was determined by assessing the presence of known subcellular fraction markers with Western analysis. In the bovine and mouse adrenal glands, 3 beta-hydroxysteroid dehydrogenase-isomerase appears to be localized solely in the microsomal fraction, while in the rat, 3 beta-hydroxysteroid dehydrogenase-isomerase appears to have dual subcellular distribution: the microsomes and the inner mitochondrial membrane. We conclude that there is a species difference in the subcellular distribution of this important steroidogenic enzyme and that this species difference may be related to the steroidogenic pathway preferred in that species.

Purification and properties of testicular 3 beta-hydroxy-5-ene-steroid dehydrogenase and 5-ene-4-ene isomerase.

Through the treatment of rat testicular microsomes with sodium cholate, 3 beta-hydroxy-5-ene-steroid dehydrogenase and 5-ene-4-ene isomerase (abbreviated as the 3 beta-hydroxysteroid dehydrogenase and isomerase, respectively) were solubilized, and then purified by DEAE and hydroxylapatite column chromatographies. The findings were as follows: With this purification procedure, the 3 beta-hydroxysteroid dehydrogenase activity could not be separated from the isomerase. For 3-oxo-4-ene-steroid formation from 3 beta-hydroxy-5-ene-steroids, NAD+ was required as a cofactor. While the 3 beta-hydroxysteroid dehydrogenase required NAD+, the isomerase also required NAD+ or its reduced form, in contrast to the microbial enzyme. On treatment of the purified enzyme with 5'-p-fluorosulfonyl-benzoyladenosine (FSBA), both enzyme activities were markedly reduced. The enzyme, affinity labeled with [adenine-8-14C]FSBA, showed a mol. wt of 46.8 K. During 4-androstenedione production from DHA, 5-androstenedione was detected as an intermediate.

Influence of the estrous cycle at gamma-ray exposure on radiation-induced mammary tumorigenesis in virgin rats.

Wistar rats received whole body irradiation with 260 cGy gamma-rays at 10 a.m. of individual phases of their estrous cycle and then had diethylstilbestrol pellets implanted for 1 year. When the radiation was given during di-estrus II, the highest incidence (73.3%) of mammary tumorigenesis was observed, and mean latency until the first tumor appearance was 8.5 +/- 0.7 months. Also, rats irradiated on estrus had significantly lower incidence (35.3%) of mammary tumors than those irradiated on pro-estrus and di-estrus II, but there was no significant difference in latency period. Iball's indices of total mammary tumors, fibroadenoma plus adenocarcinoma, were 14.3 +/- 0.9, 28.8 +/- 2.1, 23.3 +/- 0.8 and 12.2 +/- 0.2 in rats irradiated during di-estrus I, di-estrus II, pro-estrus and estrus respectively. The percentage of adenocarcinomas was comparatively uniform (25.0 to 34.8%) throughout the various phases of the estrous cycles. Also, Iball's indices calculated from adenocarcinoma indicated no significant differences between all groups. From our results, the highest incidence of mammary tumors arose in rats after irradiation at di-estrus II with minimum level of prolactin in serum. We discuss different mechanisms of radiation-induced and chemical-carcinogen-induced tumorigenesis of mammary glands.

Testicular and adrenal 3 beta-hydroxy-5-ene-steroid dehydrogenase and 5-ene-4-ene isomerase.

The purified multifunctional enzyme, 3 beta-hydroxysteroid dehydrogenase with steroid 5-ene-4-ene isomerase from rat testes and adrenals showed similar catalytic properties. They exhibited the same molecular weight of 46,500. Either NAD+ or NADH was required for steroid isomerizing activity, probably as an allosteric effector. It was clearly demonstrated by using the purified enzyme that without NAD(H) no isomerizing activity was detected. In the presence of NADH, or its analogue, 3 beta-hydroxysteroid dehydrogenase obtained from both tissues was inhibited; however, steroid isomerizing activity remained due to the allosteric effect. The results suggest that in these endocrine organs, both enzyme activities reside within the same protein.

Reasons for reduced activities of 17 alpha-hydroxylase and C17-C20 lyase in spite of increased contents of cytochrome P-450 in mature rat testis fetally irradiated with 60Co.

Pregnant rats received whole body irradiation with 2.6 Gy gamma-ray from a 60Co source at Day 20 of gestation. When pups were 4 months old, activities of electron transport system and steroid monooxygenase in tests were assayed. The content of total cytochrome P-450 in the irradiated testes had increased to 170% of that in non-irradiated rats, but NADPH-cytochrome P-450 reductase activity had reduced to 36% of the control. Also, amounts of cytochrome b5 in testicular microsomal fraction were decreased markedly after irradiation, but no significant change of NADH-cytochrome b5 reductase activity was observed in the treated pups. Because both 17 alpha-hydroxylase and C17-C20 lyase activities tended to be decreased by fetal irradiation, testosterone production from progesterone and 17 alpha-hydroxyprogesterone was reduced to about 30% of the control. From these results, it has been suggested that the testicular cytochrome P-450 is radioresistant but steroid monooxygenase activities are reduced after the fetal irradiation. We propose that the discrepancy arises from the marked decrement of NADPH-cytochrome P-450 reductase activity.

Promotive effects of diethylstilbestrol, its metabolite (Z,Z-dienestrol) and a stereoisomer of the metabolite (E,E-dienestrol) in tumorigenesis of rat mammary glands pregnancy-dependently initiated with radiation.

Wistar-MS rats received whole body irradiation with 260 cGy gamma-rays at day 20 of pregnancy and then were treated with diethylstilbestrol (DES), E,E-dienestrol (E,E-DIES) or Z,Z-dienestrol (Z,Z-DIES) for 1 year. DES administration caused the highest incidence of mammary tumors with a concomitant reduction of gain in body weight. When E,E-DIES or Z,Z-DIES in pellet form was implanted, the incidence of tumors was significantly lower than that observed in rats treated with DES. To clarify the increased susceptibility to mammary tumorigenesis after DES administration we measured hormone levels in the serum of rats implanted with pellets containing derivatives of the synthetic estrogens. The serum prolactin concentration was significantly increased by DES administration. When E,E-DIES or Z,Z-DIES pellets were implanted the prolactin level was markedly reduced to 4.5% and 0.7% of that observed in DES-treated rats, respectively. In addition, the serum concentrations of estradiol-17 beta and progesterone in rats with Z,Z-DIES pellets were higher than those of rats with DES or E,E-DIES pellets. A large number of DES-induced mammary tumors were positive for both estrogen and progesterone receptors, but no tumors negative for both receptors were obtained. The findings suggest that DES acts directly on radiation-initiated mammary cells via binding with estrogen receptors and/or stimulates the secretion of prolactin from the pituitary glands.

Pregnancy-dependent initiation in tumorigenesis of Wistar rat mammary glands by 60Co-irradiation.

Pregnant Wistar rats received whole body irradiation with 260 cGy gamma-rays at days 7, 14 and 20 of pregnancy and then were treated with diethylstilbestrol (DES) for 1 year. The highest incidence (92.9%) for tumorigenesis of mammary glands was observed in the rats irradiated in late pregnancy. Histological examination showed that tumors were classified as fibroadenoma and adenocarcinoma. To determine the reasons for specific induction of mammary tumors by irradiation in late pregnancy, hormone concentrations in serum and estrogen receptors in mammary glands during pregnancy were measured. Concentrations of estradiol, progesterone, 11-deoxycorticosterone and placental lactogen at day 20 were higher than at days 7 and/or 14, but no difference was observed in the concentrations of prolactin and thyroid-stimulating hormone during pregnancy. The estrogen receptor in mammary glands at day 20 was indicated to have the highest affinity and the highest binding capacity during pregnancy. Normal mammary glands at day 20 were suggested to have more abundant epithelial cells in the mammary lobes than those at days 7 and 14. The data suggest that the critical requirements for the initiation of tumorigenesis by gamma-rays are dependent upon the differentiated state of mammary glands exposed to various hormones, and that the concentration and persistence of the synthetic estrogen (DES) are necessary for the promotion of tumorigenesis of the irradiated mammary glands.

Purification and characterization of rat adrenal 3 beta-hydroxysteroid dehydrogenase with steroid 5-ene-4-ene-isomerase.

After solubilization of rat adrenal microsomes with sodium cholate, 3 beta-hydroxysteroid dehydrogenase with steroid 5-ene-4-ene isomerase (abbreviated as steroid isomerase) activity was purified to a homogeneous state. The following characteristics of the enzyme were obtained: 3 beta-Hydroxysteroid dehydrogenase together with steroid isomerase was detected as a single protein band in SDS-polyacrylamide gel electrophoresis, where its mol. wt was estimated as 46,500. Either NAD+ or NADH was required for demonstration of steroid isomerase activity. Treatment of the enzyme with 5'-p-fluorosulfonylbenzoyladenosine, an affinity labeling reagent for NAD+-dependent enzyme, diminished both the enzyme activities.

Susceptibility of lactating rat mammary glands to gamma-ray-irradiation-induced tumorigenesis.

Lactating rats of the Wistar-MS strain were irradiated with 260 cGy of gamma rays 21 days after parturition (day 21). Diethylstilbestrol (DES) pellets were implanted one month after termination of nursing and were allowed to remain for one year. A significantly higher incidence (96.4%) of mammary tumors was observed in these rats irradiated during late lactation than in virgin irradiated animals (30.4%). A control group of lactating animals irradiated during late lactation but not treated with DES was also observed for one year; the final incidence of mammary tumors in this group was 35.3%. The latency period was shortest in the DES-treated group irradiated during late lactation. Histological examination showed that the mammary glands of lactating rats were highly developed, with alveoli filled with milk. Five days after weaning, there was degeneration of alveolar tissue, concomitant with a marked decrease in the concentration of estrogen and prolactin receptors. A considerable amount of epithelial tissue remained in the mammary glands during the process of atrophy. When the rats were irradiated 5 days after weaning, and then were treated with DES for one year, the incidence of mammary tumors was 73.3%, significantly higher than that in virgin irradiated rats. However, this incidence was not significantly different from that in animals irradiated during late lactation. These results suggested that the induction of mammary tumors by gamma irradiation before or after weaning was more dependent upon the stage of differentiation in mammary glands than upon the proliferative activity of epithelial cells, and that DES is essential as a promoter for radiation-induced mammary tumorigenesis.

Relationship between stages of mammary development and sensitivity to gamma-ray irradiation in mammary tumorigenesis in rats.

Mature Wistar-MS rats were ovariectomized and treated with estradiol benzoate and/or progesterone. Control animals were treated with olive oil. The rats were then exposed to gamma-rays and implanted with a pellet of diethylstilbestrol. The incidence of mammary tumors in rats treated with estradiol benzoate or with progesterone was significantly higher than in rats in the non-treated control group, whereas, in rats treated with both estradiol benzoate and progesterone, the incidence was not significantly different from that in the controls. Histological examination of the mammary tumors showed 2 types of neoplasm: adenocarcinoma and fibroadenoma. Interestingly, over half of all the tumors in the rats treated with estradiol benzoate were adenocarcinomas, while fibroadenomas were mainly induced in the rats treated with progesterone or with both estradiol benzoate and progesterone. The expression of estrogen and progesterone receptors in the tumor tissues showed some differences according to whether the groups were treated with estradiol benzoate or with progesterone. Morphologically, mammary glands at irradiation showed well-developed lobuloalveoli in both the estradiol-benzoate-treated rats and in those rats treated with both estradiol benzoate and progesterone. This was consistent with the higher incorporation of [3H]thymidine into the DNA in the mammary glands of rats in both of these groups. Our findings suggest that a more advanced developmental stage of the mammary glands, dependent upon ovarian hormones, is related to a higher incidence of mammary tumors induced by irradiation.

Immunocytochemical localization of 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD), and its relation to the ultrastructure of steroidogenic cells in immature and mature rat ovaries.

Immunocytochemical localization of 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) and its relation to the ultrastructure of steroidogenic cells were examined in mature and immature rat ovaries. In mature (8-10 weeks old) rat ovaries, the theca interna cells of secondary as well as Graafian follicles, and the interstitial gland cells were all strongly stained with anti-17 beta-HSD antibody. However, granulosa cells, corpus luteum cells, oocytes and peritoneal epithelial cells were negative against this staining. In the ovaries of 1-week-old rats, all these cells were negative to immunostaining for 17 beta-HSD. In the ovaries of 2-week-old rats, the theca interna cells of secondary follicles and the interstitial gland cells showed a positive reaction for the 17 beta-HSD activity. Electron microscopic examination demonstrated the presence of characteristic structures for steroid secretory cells such as many lipid droplets, well developed smooth endoplasmic reticulum, and oval mitochondria with tubular cristae in the theca interna cell of secondary as well as Graafian follicles and in the interstitial gland cell of mature rat ovaries. In the ovaries of 1-week-old rats, all the theca cells of the primary and secondary follicles were fibroblast-like in their shape and fine structure, and typical interstitial cells were not recognized. In the 2-week-old rats, some of the theca interna cells and interstitial cells were well differentiated in ultrastructure, showing characteristic features for steroid secretory cells. These findings indicate that by 2 weeks after birth, theca interna cells and interstitial gland cells acquire the ability for testosterone production as seen in mature rat ovaries.

Light and electron microscopic immunocytochemistry on the localization of 3 beta-hydroxysteroid dehydrogenase/isomerase in the bovine adrenal cortical cells.

The localization of 3 beta-hydroxysteroid dehydrogenase/isomerase (3 beta-HSD) was studied in bovine adrenal glands by light as well as electron microscopic immunocytochemistry, using anti-bovine adrenal 3 beta-HSD antibody. With light microscopy the cytoplasm of the glomerulosa cells was weakly immunostained, while that of the fasciculata-reticularis cells was intensely immunostained though both the capsular connective tissue cells and the medullary cells were entirely negative for this reaction. Electron microscopic immunocytochemistry revealed that the positive reaction products for 3 beta-HSD were present on the membrane of smooth endoplasmic reticulum of the cortical cells, especially that of the fasciculata and reticularis cells. Other cell organelles such as mitochondria and Golgi apparatus were entirely negative. The present results indicate that 3 beta-HSD is present in the membrane of smooth endoplasmic reticulum of bovine adrenal cortical cells.

Steroidogenesis in the testes and the adrenals of adult male rats after gamma-irradiation in utero at late pregnancy.

Pregnant rats were irradiated with 2.1 Gy gamma-ray of 60Co at day 20 of gestation. Seventy days after birth, the body weight of the fetally irradiated male pups was significantly lower than the control. The testes, ventral prostates and seminal vesicles were atrophied by irradiation, whereas no decreased weight of the adrenals was observed. Histological examination of the testes of the irradiated rats revealed a complete disappearance of germinal cells. Sertoli cells and Leydig cells appeared normal, and no apparent histological difference was observed in the adrenals between the control and the irradiated rats. Activities of microsomal delta 5-3 beta-hydroxysteroid dehydrogenase (HSD) + isomerase, 17 alpha-hydroxylase/C17,20-lyase, 17 beta-HSD and 7 alpha-hydroxylase per pair of testes were decreased in the irradiated rats (36-86% of the control). In contrast, no decreased activity of 20 alpha-HSD in the cytosol fraction was observed by irradiation. No decreased activity of adrenocortical enzymes, such as delta 5-3 beta-HSD + isomerase, 21-hydroxylase, 11 beta-/18-hydroxylase and 5 alpha-reductase, was also observed in the irradiated group. Concentrations of LH, FSH, TSH, prolactin, testosterone, progesterone and aldosterone in serum were measured by radioimmunoassay. Only the FSH concentration was significantly increased by the irradiation, while no difference was found in the concentration of other hormones. It was concluded that irreversible damage was induced in spermatogenesis and androgen production by the fetal irradiation, whereas corticoidogenesis was not affected.

Leydig cell tumor of the ovary associated with endometrial carcinoma and containing 17 beta-hydroxysteroid dehydrogenase.

A case of ovarian Leydig cell tumor associated with adenocarcinoma of the endometrium in a 66-year-old woman is described herein, the eighth such case published. Clinically, both masculinizing and feminizing symptoms were observed: an increase in facial hair growth, slight baldness, clitoromegaly, and postmenopausal genital bleeding. Three biopsies of the endometrium during a 5-month preoperative period showed atypical hyperplasia. Surgically resected material contained a Leydig cell tumor of the left ovary and focal adenocarcinoma in atypical hyperplasia of the endometrium. Serum levels of androgens and estrogens measured by radioimmunoassay decreased after removal of the tumor. Immunohistochemical studies revealed that the Leydig cell tumor contained testosterone, estrogens, and 17 beta-hydroxysteroid dehydrogenase (HSD) in the cytoplasm. This is the first report of a Leydig cell tumor in which the localization of 17 beta-HSD was demonstrated immunohistochemically.