Hiroshi Inano

Prevention of radiation-induced mammary tumors☆

The radiation-induced rat mammary tumor model is useful for studying tumor prevention by treatment in the initiation or promotion stage. In anti-initiation experiments, the administration of radical scavengers or spin-trapping agents before or immediately after irradiation reduced the incidence of mammary tumors, suggesting that free radicals produced by exposure are a potent initiator. To evaluate the role of nitric oxide (NO) in the initiation, NO-specific scavengers or NO synthase inhibitors were administered during the initiation. These agents partially prevented the tumorigenesis, suggesting that radiation-induced NO contributes to tumor initiation. The administration of curcumin during irradiation reduced the incidence of the tumors in the presence of tumor promotor. In anti-promotion experiments on preventing diethylstilbestrol (DES)-dependent tumor development from mammary primodial cells exposed to radiation, tamoxifen decreased the tumor incidence. From the results, estrogen itself or prolactin induced by estrogen may be a promoter for the tumorigenesis. Bezafibrate and simvastatin, agents inducing hypolipidemia and hypocholesterolemia respectively, cause a decrease in the DES-dependent promotion of radiation-induced tumorigenesis. The simultaneous administration of curcumin and DES significantly reduces the development of mammary tumors in irradiated rats. In this review, the endocrinologic and pharmacologic significance of the anti-initiation and anti-promotion is discussed.

Submicrosomal distribution of adrenal enzymes and cytochrome P-450 related to corticoidogenesis

By density-gradient centrifugation in the absence of CsCl, the microsomal fractions obtained from porcine and rat adrenal glands were separated into two subfractions: one fraction consisted of smooth-surfaced membrane structures which bore no ribosomes on their outer surface, and the other consisted of abundant free ribosomes with membrane structures, according to electron microscopic observation. The activities of three microsomal enzymes related to corticoidogenesis, namely Δ5-3β-hydroxysteroid dehydrogenase (EC 1.1.1.51) coupled with the Δ5-Δ4 isomerase (EC 5.3.3.1), 17α-hydroxylase (EC 1.14.1.7) and 21-hydroxylase (EC 1.14.1.8), were predominantly located in the smooth-surfaced subfraction. In accordance with the distribution of the hydroxylases, adrenal microsomal cytochrome P-450 was concentrated exclusively in the smooth-surfaced subfraction. As 21-hydroxylation was significantly inhibited in a CO-containing atmosphere, the cytochrome P-450 in the smooth-surfaced subfraction was participating as the direct activating site of O2 during 21-hydroxylation. The Km values of the submicrosomal Δ5-3β-hydroxysteroid dehydrogenase for pregnenolone and of 17α-hydroxylase for progesterone were estimated as 0.2 and 0.13 mM, respectively. The Km of the adrenal 21-hydroxylase was evaluated as 99 μM for progesterone and as 0.22 mM for 17α-hydroxyprogesterone. Substrate specificities of the adrenal submicrosomal enzymes, dynamic analysis of the corticoidogenesis throughout the incubation time, and the relationship between the amounts of enzyme preparations and the products, were examined.

Studies on enzyme reactions related to steroid biosynthesis: I. Presence of the cytochrome P-450 in testicular tissue and its role in the biogenesis of androgens

Abstract Among the subcellular fractions of testicular homogenatcs of rats, the cytochrome P-450 was localized exclusively in the microsomal fraction (10,000–105,000 g precipitate) of the cells such as the interstitial cells, which were radio-resistant, and also responsive to gonadotrophin. In the atmosphere of CO:O 2 (95:5) mixture, the activities of the testicular microsomal 17α-hydroxylase and C 17 -C 20 lyase which cleaves the side-chain of 17α-hydroxyprogesterone were seriously decreased, in comparison with those exhibited in an Ar: O 2 (95:5) atmosphere, after the preparation had previously been gassed with CO or Ar. Therefore, the above mentioned two enzymes related to androgen biosynthesis were concluded to involve the cytochrome P-450 as the site of molecular oxygen activation which was required by the two enzymes.

Localization of Nitric Oxide Synthases and Nitric Oxide Production in the Rat Mammary Gland

We investigated nitric oxide (NO) production and the presence of nitric oxide synthase (NOS) in the mammary gland by use of an organ culture system of rat mammary glands. Mammary glands were excised from the inguinal parts of female Wistar-MS rats primed by implantation with pellets of 17β-estradiol and progesterone and were diced into approximately 3-mm cubes. Three of these cubes were cultured with 2 ml of 10% FCS/ DMEM plus carboxy-PTIO (an NO scavenger, 100μM) in the presence or absence of LPS (0.5μg/ml) for 2 days. The amount of NO produced spontaneously by the cultured mammary glands was relatively minute at the end of the 2-day culture period, and the NO production was significantly enhanced by the presence of LPS. This enhancement of NO production was completely eliminated by addition of hydrocortisone (3μM), an inhibitor of inducible NOS (iNOS), to the incubation medium. Immunoblot analyses with specific antisera against NOS isoforms such as iNOS, endothelial NOS (eNOS), and brain NOS (bNOS) showed immunoreactive bands of iNOS (122 ± 2 kD) and eNOS (152 ± 3 kD) in extracts prepared from the mammary glands in the culture without LPS. The immunoreactive band of iNOS was highly intense after the treatment of mammary glands with LPS, whereas the corresponding eNOS immunoreactive band was faded. The immunohistochemical study of anti-iNOS antiserum on frozen sections of the cultured mammary glands showed that an immunoreactive substance with the antiserum was localized to the basal layer (composed of myoepithelial cells of alveoli and lactiferous ducts) of the mammary epithelia and to the endothelium of blood vessels that penetrated into the interstitium of the mammary glands. Histochemical staining for NADPH-diaphorase activity, which is identical to NOS, showed localization similar to that of iNOS in the mammary glands. Similar observations were noted in the immunohistochemistry of eNOS. In contrast, the immunoreactive signal with the bNOS antiserum was barely detected in the epithelial parts of alveoli and lactiferous ducts of the mammary glands. These observations demonstrate that three isoforms of NOS are present not only in the endothelium of blood vessels but also in the parenchymal cells (the glandular epithelium) of the rat mammary gland, such as epithelial cells and myoepithelial cells, and suggest that NO may have functional roles in the physiology of the mammary glands.

Susceptibility of fetal, Virgin, pregnant and lactating rats for the induction of mammary tumors by gamma rays

Pregnant Wistar-MS rats received a whole-body irradiation of 0-2.6 Gy gamma rays at day 20 of pregnancy. The mother rats were implanted with a diethylstilbestrol (DES) pellet 30 days after weaning, and the female pups delivered by the irradiated mother were treated with DES after maturation. Lactating rats were irradiated with gamma rays 21 days after parturition and then treated with DES. Virgin rats 70 days of age were also irradiated and then administered DES. The rats which received intrauterine irradiation did not develop mammary tumors at doses less than 2.1 Gy and showed a low incidence of tumors at 2.6 Gy. In virgin rats, the maximum tumor incidence was obtained with 1 Gy. The incidence of total mammary tumors in the mother rats and lactating rats increased in a dose-dependent manner with increasing doses of gamma rays up to 2.1 Gy. With 0.1-1 Gy, the incidence of adenocarcinoma in the mother rats was significantly lower than that observed in the lactating rats. However, the incidence in the mother rats irradiated with 1.0-1.5 Gy was significantly higher than that of virgin rats treated with the corresponding gamma-ray doses. These findings suggest that the susceptibility of the mammary glands to radiation depends upon the differentiation at the time of exposure.

Side-chain cleavage as related to steroid hormone synthesis☆

The mechanism of side-chain cleavage during the course of steroid hormone biosynthesis by rat testicular and adrenal enzyme preparations has been investigated. 17α-Hydroxypregnene-C-17-C-20 lyase in testicular tissue required molecular oxygen in addition to NADPH. Progesterone and 17α-hydroxyprogesterone, under an 18O2-enriched atmosphere, and 17α-18O-hydroxylated progesterone under an 16O2 atmosphere were incubated with a rat testicular enzyme preparation, and the products were analyzed by high-resolution mass spectrometry. Progesterone-17α-hydroxylase required molecular oxygen, which was incorporated in the 17α-hydroxyl group of 17α-hydroxyprogesterone. However, in the case of the side-chain cleavage of 17α-hydroxyprogesterone, the molecular oxygen required was not incorporated into the direct steroid product, androstenedione, nor into the further product testosterone. It was thus established that the oxygen atoms of the 17-ketone and 17β-hydroxyl groups of androstenedione and testosterone, respectively, which were derived by testicular enzymes from progesterone, originated from the 17α-hydroxyl group of 17α-hydroxyprogesterone. The side-chain cleavage of cholesterol, in the synthesis of pregnenolone, was also studied, by incubation of 20α, 22R-dihydroxycholesterol with rat adrenal preparation under an 18O2 atmosphere. The immediate product pregnenolone and its further metabolite progesterone were isolated, and analysed by mass-spectrometry. No incorporation of molecular oxygen into the metabolites was observed, suggesting that the oxygen in the 20-ketone group of pregnenolone and progesterone is derived from the oxygen of the 20α-hydroxyl group introduced into cholesterol prior to the side-chain cleavage.

In vitro metabolism of testosterone in hepatic tissue of a hagfish, Eptatretus burgeri

The metabolism of testosterone in hepatic tissue (combined microsomal and cytosol fractions) of a hagfish, Eptatretus burgeri, was examined in the presence of NADPH and in aerobic or CO-saturated atmosphere. The metabolites formed in CO atmosphere were identified as 4-androstene-3,17-dione, 5α-dihydrotestosterone, 3β-hydroxy-5α-androstan-17-one, and 5α-androstane-3β,17β-diol. From formation of these metabolites of testosterone, the activities of 5α-reductase, 3β- and 17β-hydroxysteroid dehydrogenases were indicated. Under aerobic condition, 7α-hydroxytestosterone, 7α-hydroxy-5α-dihydrotestosterone, 3β,7α-dihydroxy-5α-androstan-17-one and 5α-androstane-3β,7α,17β-triol were formed, in addition to 4-androstene-3,17-dione. These results demonstrate the presence of 5α-reductase, 3β- and 17β-hydroxysteroid dehydrogenases and 7α-hydroxylase in the hagfish liver. Since the hagfish is regarded as a very primitive vertebrate, it may be suggested that these enzymes are phylogenetically old and that the 7α-hydroxylase represents one of the first forms of hepatic cytochrome P-450 appearing during development.

Prostate 3α-hydroxysteroid dehydrogenase: Its partial purification and properties

Abstract 3α-Hydroxysteroid dehydrogenase (E.C. 1.1.1.50) has been purified from the cytosol fraction (superna-tant fluid at 105,000 g ) of rat prostate by precipitation with ammonium sulphate (40–100% saturation), followed by chromatography on DEAE-cellulose and Sephadex G-100. Electrophoretic analysis of the enzyme preparation at the final stage revealed two major protein bands which were closely located on the gel, and a few minor bands. The enzyme activity was exclusively localized in the major bands. The final preparation was purified about 190 fold in S.A., compared with the cytosol fraction precipi-tated at 40–100% of ammonium sulphate saturation. Molecular weight of the 3α-hydroxysteroid de-hydrogenase was estimated as 34,000 daltons from the elution pattern of the activity through the Sephadex G-100 gel filtration. The final enzyme preparation was devoid of receptor component with high affinity for 5α-dihydrotestosterone. Optimal temperature of the final 3α-hydroxysteroid dehydro-genase preparation at pH 7.4 was about 45–47.5°C. The enzyme preferred NADP(H) to NAD(H), and reduction of 5α-dihydrotestosterone was more efficiently catalyzed by the enzyme than oxidation of 5α-androstane-3α,17β-diol.

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.

Sexual differences of steroidogenic enzymes in embryonic gonads of the chicken (Gallus domesticus).

The left ovary and testis of 15-day-old embryos of the chicken were compared in the enzyme activities related to steroidogenesis. The activity of delta 5-3 beta-hydroxysteroid dehydrogenase coupled with delta 5-delta 4 isomerase in the ovary was similar to that of the testis. Activities of 17 alpha-hydroxylase and C-17-C-20 lyase in the ovary were 2.5 and 2.6 times those in the testis. From the CO-induced difference spectrum, the content of cytochrome P-450 in the ovarian microsomes was estimated as 27.6 pmol/mg protein. However, no detectable amount of cytochrome P-450 was observed in the testicular microsomal fraction. The substrate (progesterone)-induced difference spectrum was appreciable only in the ovarian microsomes. The activity of microsomal NADPH-cytochrome c reductase in the ovary was significantly higher than that in the testis. The activities of 17 beta-hydroxysteroid dehydrogenase in both gonads were similar to each other, when androstenedione was used as substrate. However, its activity in the ovary was 1.4 and 3.1 times that in the testis, when dehydroepiandrosterone and estrone were used as substrate, respectively. Aromatase activity in the ovary was over 100 times that in the testis, as assessed by release of [3H]water from [1-3H]testosterone. Appreciable amounts of radioactive estradiol-17 beta and estrone were formed from [4-14C]testosterone and [7-3H]androstenedione, respectively, only by the ovarian tissue. 5 beta-Reductase activity in the ovary was 1.4 times that in the testis.