Cell-type-specific expression of rat steroid 5 alpha-reductase isozymes

The enzyme steroid 5 alpha-reductase (EC 1.3.99.5) is a component of an intercellular signaling pathway that determines cell fate in the primordium of the mammalian reproductive tract. During male phenotypic sexual differentiation, the dihydrotestosterone product of this enzyme binds to the androgen receptor and initiates development of the external genitalia and prostate. Genes encoding two isozymes of steroid 5 alpha-reductase with different biochemical properties and tissue distributions have recently been isolated. In the current study, we utilize in situ hybridization analysis to determine cell-type-specific expression patterns of the 5 alpha-reductase isozyme mRNAs in two androgen target tissues (regenerating ventral prostate and epididymis) and a peripheral tissue (liver). In regenerating ventral prostate, the type 1 mRNA is expressed in basal epithelial cells whereas expression of the type 2 mRNA is largely confined to stromal cells. These results were confirmed by immunohistochemical analysis and are consistent with distinct roles played by the isozymes in the prostate. In the epididymis, both 5 alpha-reductase isozyme mRNAs are expressed in epithelial cells. Only the type 1 mRNA is present in the liver. This mRNA is distributed in a striking spatial gradient extending from hepatocytes surrounding the portal triad (high expression) to those surrounding the central vein (low to absent expression). These findings demonstrate cell-type-specific expression of the steroid 5 alpha-reductase isozymes and underscore their distinct and overlapping functions in androgen physiology.     

Identification and partial characterization of two steroid 5 alpha-reductase isozymes in the canine prostate

BACKGROUND AND OBJECTIVE: Intensive induction and post-remission therapies have improved the prognosis in adult acute lymphoblastic leukemia (ALL). However, different from children, the impact of late intensification therapy in the overall results of treatment has not been consistently evaluated. The objective of this study was to analyze the results of a multicenter prospective protocol, PETHEMA ALL-89, in which, after intensive induction and consolidation therapy, randomization to receive delayed intensification treatment was performed. DESIGN AND METHODS: One hundred and eight adults (age > or = 15 years) diagnosed with ALL (ALL L3 excluded) in 22 Spanish hospitals from 1989 to 1994 were treated with a five-drug induction therapy, followed by four cycles of early post-remission treatment during four months, and maintenance therapy for two years. Patients in remission at the end of the first year were randomized to receive one six-week cycle of late intensification therapy. Uni- and multivariate analyses of early response to treatment, complete remission (CR), leukemia-free survival (LFS) and overall survival (OS) were performed. RESULTS: The median (range) age of the series was 28 (15-74) years and leukocyte count 26 x 10(9)/L (1-600). ALL L1/L2 was present in 38/70 patients, early pre-B in 13, common in 53, pre-B in 12 and T in 30 cases. The CR rate was 86%, and refractory disease 9%. Median LFS was 34 months, with a 5-yr probability of 41% (95% CI, 29-53), whereas median OS was 51 months and 5-year probability 47% (34-59%). There were no differences in either LFS and OS between patients who did or did not receive delayed intensification therapy. Prognostic factors for CR attainment were advanced age and slow response to therapy. These two features were, in addition to high leukocyte counts, the parameters with negative influence in both LFS and OS. INTERPRETATION AND CONCLUSIONS: The results of PETHEMA ALL-89 are similar to those referred in other chemotherapy-based protocols in adult ALL. Delayed intensification has not improved the length of remission and survival. Efforts to improve the prognosis of adult ALL patients must be mainly focused in early intensification treatment.     

Expression and regulation of steroid 5 alpha-reductase in the genital tubercle of the fetal rat

Nociceptin/orphanin FQ is a heptadecapeptide which was recently isolated from brains. It induces hyperalgesia, in contrast to the analgesic effects of opioid ligands, although it and its receptor structurally resemble opioid peptides and opioid receptors, respectively. To investigate the molecular mechanism underlying nociceptin/orphanin FQ actions, we performed Xenopus oocyte expression assays, in situ hybridization histochemistry and electrophysiological analyses of neurons. We found that the nociceptin/orphanin FQ receptor is functionally coupled with the G-protein-activated K+ (GIRK) channel in Xenopus oocytes, and that the receptor mRNA and GIRK1 mRNA co-exist in various neurons, including hippocampal pyramidal cells. Furthermore, we found that nociceptin/orphanin FQ induces hyperpolarizing currents via inward-rectifier K+ channels in hippocampal pyramidal cells, suggesting that the nociceptin/orphanin FQ receptor couples with the GIRK channel in this region. We conclude that the nociceptin/orphanin FQ receptor couples with the GIRK channel in various neurons, including hippocampal pyramidal cells, thereby modulating neuronal excitability.     

Localization by in situ hybridization of steroid 5alpha-reductase isozyme gene expression in the human prostate and preputial skin

PURPOSE: The synthesis of dihydrotestosterone is catalyzed by steroid 5alpha-reductase isozymes, designated as types 1 and 2. Controversial results have been reported on the identification of the cell types expressing each isozyme in the human prostate and genital skin. The objective of the present study was to clearly identify at the cellular level the cells containing each type of 5alpha-reductase isozyme. MATERIALS AND METHODS: We used for the first time an in situ hybridization technique involving use of [35S]-labeled oligonucleotide probes to determine the cell type expression patterns of the two 5alpha-reductase isozymes in human prostate and preputial skin. RESULTS: In the prostate, hybridization with types 1 and 2 5alpha-reductase antisense probes led to a positive reaction in both epithelial and stromal cells, while the sense probes did not generate any signal. The silver grain counts for both isozymes revealed a higher labeling in the epithelial cells. In fact, the ratio epithelial cells/stroma was around 2 for both 5alpha-reductase types 1 and 2. In the preputial skin, 5alpha-reductase type 1 was found to be highly expressed in all the layers of the epidermis with the exception of the stratum corneum while a lower labeling was observed in some fibroblasts as well as in the secretory cells of sebaceous glands and excretory duct cells of sweat glands. Type 2 5alpha-reductase showed a very similar pattern of distribution. CONCLUSION: It appears that, in two human tissues, the two 5alpha-reductase isozymes mRNAs are expressed by the same cell types. This new observation should help clarify the physiological role of each type of 5alpha-reductase isozyme.     

5 alpha-reductase inhibitors, chemical and clinical models

An active site model of 5 alpha-reductase type 2 isoenzyme on an "active-analog approach" and based on 4-azasteroidal inhibitors has been constructed to evaluate the effects on the inhibitory potency of substituents on the steroid A ring. This model has proven able to predict the potential inhibitory activity of 19-nor-10-azasteroid and 6-azasteroid compounds. A model for the evaluation of clinical efficacy of an inhibitor, based on in vitro data, has also been developed and applied to finasteride. This inhibitory potency evaluation of finasteride in human scalp homogenates, plus pharmacokinetic data, allows the calculation of a theoretical in situ inhibition value for human scalp. From the IC50 curve of finasteride in scalp homogenates, it is possible to calculate that for an inhibition level similar to that obtained in prostate with 5 mg of finasteride, the necessary plasma concentration of the drug is 1 microM, a level obtained after the acute administration of 50 mg of finasteride.     

Inhibition of rat prostate carcinogenesis by a 5alpha-reductase inhibitor, FK143

We begin this article with the distinction between Deontology, Moral and Ethics. We also review the concept and the relevance of Bioethics, as the "science of survival", and as part of Ethics, a section of Philosophy. We tried to answer two further questions considering the role of Science in orienting Ethics, or the possible place of utilitarianism in controlling Ethics. The author discusses some new aspects of the doctor/patient relationship, and their evolution in the last 100 years, as well as the relations between patients and Health care institutions. Some ethical problems were also raised related to the beginning and the end of life. Finally the author reflects on the difficulties of defining ethical concepts in the near future.     

Influence of serum-free culture conditions on subcellular localization of steroid 5alpha-reductase in rat C6 glioma cells

Rat C6 glioma cells are considered to be well characterized, and therefore commonly used as a model system to investigate the function of glial cells. However, recent study has shown that an alteration in the expression of their phenotypic antigens is observed when the cells are maintained under the serum-free conditions, proposing the possibility that various properties of glioma cells can be altered by the growth conditions. To test this possibility, the effects of serum-free culture conditions on the expression of steroid 5alpha-reductase (5alpha-R) type 1 isozyme in glioma cells were examined using immunocytochemical technique. Immunoreactivity of 5alpha-R type 1 was confined to the perinuclear region of glioma cells cultured in serum-containing medium, and observed in the cytoplasmic space as well as the perinuclear region of the cells cultured in serum-free medium. In contrast, serum deprivation failed to affect the expression of phenotypic antigens, glial fibrillary acidic protein (GFAP) and 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase). Further studies showed that the expression of cytoplasmic 5alpha-R immunoreactivity induced by serum deprivation was reversible, and might be attributed to removal of serum proteins rather than biologically active small molecules from culture medium. This alteration in the expression of 5alpha-R immunoreactivity is therefore considered to reflect the translocation of the enzyme from the perinuclear region to the cell cytoplasm rather than the induction of cytoplasmic enzyme, and suggest that the culture conditions cause an alteration in the subcellular localization of 5alpha-R type 1 isozyme without phenotypic change of the glioma cell.     

(1-Benzylindole-3-yl)alkanoic acids; novel nonsteroidal inhibitors of steroid 5 alpha-reductase (I)

A novel series of indole-3-alkanoic acids with varied N-benzyl substituents were synthesized as nonsteroidal inhibitors of steroid 5 alpha-reductase. The structure-activity relationships in this series were studied and the optimum carboxylic acid side chain was butyric acid. Furthermore, compounds with a diaryl substituent at the 1-position of the indole ring displayed strong inhibitory activities in vitro. Amongst these derivatives, 4-[1-(6,6-dimethyl-6H-dibenzo[b,d]pyran-3-yl)methylindol-3-yl]b uty ric acid (FR119680) displayed very high inhibitory activity in vitro against rat prostatic 5 alpha-reductase (IC50 = 5.0 nM) and good in vivo activity in the castrated young rat model.     

Kinetic analysis of androstenedione 5 alpha-reductase in epithelium and stroma of human prostate

In the human prostate, various androgen-metabolizing enzymes are present. Among these enzymes, testosterone 5 alpha-reductase seems to be dominant. However, androstenedione is also a potential substrate of the prostatic 5 alpha-reductase. To address the question of to what extent the reduction of androstenedione to androstanedione occurs, the present study describes in detail the kinetic characteristics (Km and Vmax) and possible age-dependent alterations of this enzymatic step in epithelium and stroma of the human prostate. In normal prostate (NPR), the mean Km (nM) and Vmax (pmol/mg protein.h) were about twofold higher in stroma (Km, 211; Vmax, 130) than in epithelium (Km, 120; Vmax, 56), whereas in the benign prostatic hyperplasia (BPH), the mean Km (nM; mean +/- SEM) and Vmax (pmol/mg protein.h; mean +/- SEM) were about sixfold higher in stroma (Km, 668 +/- 121; Vmax, 415 +/- 73) than in epithelium (Km, 120 +/- 10; Vmax, 73 +/- 8). In BPH, those differences between epithelium and stroma were highly significant (p < 0.001). However, the efficiency ratios (Vmax/Km) of neither BPH nor NPR showed any significant differences between epithelium (NPR, 0.47; BPH, 0.62 +/- 0.06) and stroma (NPR, 0.70; BPH, 0.63 +/- 0.05). With respect to age-related changes, only stroma showed a significant increase of Km (p < 0.01) and Vmax (p < 0.05) with age. In summary, in both epithelium and stroma of the human prostate, a 5 alpha-reductase converts in measurable amounts androstenedione to androstanedione. The kinetic data were, in part, different between epithelium and stroma; the reason for this difference remains unclear. In comparison to other metabolic conversions, such as testosterone to dihydrotestosterone and androstenedione to testosterone, it is unlikely that, in the human prostate, the adrenal androgen androstenedione contributes significantly to the formation of testosterone and, further, of dihydrotestosterone.     

Synthesis and 5 alpha-reductase inhibitory activities of benzofuran derivatives with a carbamoyl group

Pentoxifylline, which has immunomodulatory effects in addition to its better known rheologic effects, might potentiate the effectiveness of traditional immunosuppressive drugs. We therefore studied the suppressive effect of pentoxifylline in combination with clinically relevant concentrations of prednisolone, methylprednisolone, cyclosporine, tacrolimus, rapamycin, and mycophenolic acid on mitogen-stimulated lymphocytes from 29 patients with glomerular diseases. Inhibition of lymphocyte proliferation obtained with 10(-7) and 10(-8) mol/L concentrations of the glucocorticoids and with 300 ng/mL cyclosporine was significantly increased when each was combined with 5, 25, or 50 microg/mL of pentoxifylline. The additive inhibitory effect of pentoxifylline in combination with 10(-7) mol/L glucocorticoids was inversely proportional to the inhibitory effect of the 10(-7) mol/L concentration of glucocorticoid alone, suggesting that the less sensitive the patient's cells, the greater the potentiation by pentoxifylline. The greatest degree of potentiation by pentoxifylline occurred when combined with the lower (10(-8) mol/L) concentration of glucocorticoids. Pentoxifylline also significantly increased lymphocyte suppression in combination with 150 and 300 ng/mL concentrations of cyclosporine, 5 ng/mL of tacrolimus, 2.5 x 10(-7) mol/L mycophenolic acid, and 10 ng/mL of rapamycin. These in vitro results suggest that pentoxifylline might have steroid-sparing effects and contribute to improved clinical outcomes from immunosuppressive treatment of renal diseases.     

Steroid 5alpha-reductase type 1 immunolocalized in the rat peripheral nervous system and paraganglia

Steroid 5alpha-reductase is an enzyme that converts a number of steroids with a C-4, 5 double bond and C-3 ketone to 5alpha-reduced metabolites. This enzyme has been suggested to play a role in brain development and myelination in the rat nervous system. In the present study, we examined the cellular and subcellular localization of the enzyme immunocytochemically in the rat peripheral nervous system and paraganglia using a polyclonal antibody against rat 5alpha-reductase type 1. Light and electron microscopical studies localized 5alpha-reductase in the Schwann cells of myelinated and unmyelinated nerve fibres, the satellite cells of the ganglia, the enteric glial cells and the supporting/sustentacular cells of the paraganglia. In the myelinated nerve fibres, immunoreactivity was observed in the outer loops, the nodes of Ranvier and the Schmidt-Lanterman incisures. Subcellularly, the immunoreactivity was localized in the cytoplasm of various glial cells. No immunoreactivity was observed in the myelin membrane, the axon or the neuronal perikaryon. These findings suggest that 5alpha-reductase is widely distributed in glial cells, and that, in addition to myelination, 5alpha-reduced steroids play a role in some glial functions in the peripheral nervous system.     

Synthesis and in vitro activity of some epimeric 20 alpha-hydroxy, 20-oxime and aziridine pregnene derivatives as inhibitors of human 17 alpha-hydroxylase/C17,20-lyase and 5 alpha-reductase

Some epimeric 20-hydroxy, 20-oxime, 16 alpha, 17 alpha-, 17,20- and 20,21-aziridine derivatives of progesterone were synthesized and evaluated as inhibitors of human 17 alpha-hydroxylase/C17,20-lyase (P450(17) alpha) and 5 alpha-reductase (5 alpha-R). The reduction of 16-dehydropregenolone acetate (3a) was reinvestigated. NaBH4 in the presence of CeCl3 gave better stereo-selectivity for 20 beta-ol [20 alpha/20 beta-OH (4 alpha/4 beta) = 1/2.7] than LTBAH or the Meerwein-Pondroff method reported; reduction with Zn in HOAc formed exclusively 20 alpha-ol (4 alpha b). The 20 alpha- and 20 beta-hydroxy-4,16-pregnadien-3-one (9 alpha) and (9 beta) were synthesized from the alcohols 4 alpha b and 4 beta b. Several 20-oxime pregnadienes and 16 alpha, 17 alpha-, 17,20- and 20,21-aziridinyl-5-pregnene derivatives were also synthesized. LiAlH4 reduction of the 16-en-20-oxime (12b) yielded 20 (R)-(13a) and 20(S)-17 alpha,20-aziridine (13b) and 20(R)-17 beta,20-aziridine (14a). Several compounds inhibited the human P450(17) alpha with greater potency than ketoconzole. The 5 alpha-R enzyme assay showed that while (9 alpha) did not have any activity, (9 beta) and (3b) were potent 5 alpha-reductase (IC50 = 21 and 31 nM) inhibitors with activities similar to finasteride. The 20-oximes (17a) and (17b) were potent dual inhibitors for both 5 alpha-R (IC50 = 63 and 115 nM, compared to 33 nM for finasteride) and P450(17) alpha (IC50 = 43 and 25 nM, compared to 78 nM for ketoconazole).     

Pharmacokinetics and pharmacodynamics of FK143, a nonsteroidal inhibitor of steroid 5 alpha-reductase, in healthy volunteers

Results from a comparative analysis of the efficiency of countermeasures in agriculture in a long term after the ChNPP accident are presented. Based on criteria such as reduction factor for 137Cs transfer to plants, averted dose and cost of 1 manSv relative ratings of countermeasures are given. Using one of the farms, located in the contaminated area as an example radiological justification of the optimal systems of countermeasures application is provided.     

Benzisothiazole-1,1-dioxide alkanoic acid derivatives as inhibitors of rat lens aldose reductase

Derivatives of 4-substituted 1,2-benzisothiazole-1,1-dioxide alkanoic acids were prepared and their in vitro aldose reductase inhibitory activity was tested in rat lens enzyme. The acetic derivatives 10, 12, and 16a-d proved to be much more potent inhibitors than the propionic derivatives 11, 13, and 17a-d. The presence of an acyl moiety on the amino group in position 4 of the acetic derivatives 16a-d led to a significant increase in activity with respect to the parent compound 14. One of the most active compounds in vitro, 10, was also evaluated in vivo as an inhibitor of glutathione lens depletion in galactosemic rats, but it did not show any activity in maintaining the rat lens glutathione level, probably due to problems of ocular bioavailability or metabolism.     

5 alpha-reductase isozymes in the central nervous system

The enzyme 5 alpha-reductase (5 alpha-R) activates several delta 4-3keto steroids to more potent derivatives which may also acquire new biological actions. Testosterone gives rise to the most potent natural androgen dihydrotestosterone (DHT), and progesterone to dihydroprogesterone (DHP), a precursor of the endogenous anxiolytic/anesthetic steroid tetrahydroprogesterone (THP). Two isoforms of 5 alpha-R, with a limited degree of homology, different biochemical properties and distinct tissue distribution have been cloned: 5 alpha-R type 1 and type 2. In androgen-dependent structures DHT is almost exclusively formed by 5 alpha-R type 2; 5 alpha-R type 1 is widely distributed in the body, with the highest levels in the liver, and may be involved in steroid catabolism. In the brain, the roles of the two isozymes are still largely unknown. This brief review will summarize recent experimental data from our laboratory which try to assign possible functional roles to the process of 5 alpha-reduction, and to the two 5 alpha-R isoforms in the CNS.     

Steroidal and nonsteroidal sulfamates as potent inhibitors of steroid sulfatase

Synthetic routes to potent steroidal and nonsteroidal sulfamate-based active site-directed inhibitors of the enzyme steroid sulfatase, a topical target in the treatment of postmenopausal women with hormone-dependent breast cancer, are described. Novel compounds were examined for estrone sulfatase (E1-STS) inhibition in intact MCF-7 breast cancer cells and placental microsomes. Reaction of the sodium salt of estrone with sulfamoyl chloride gave estrone 3-O-sulfamate (EMATE, 2) which inhibits E1-STS activity potently (> 99% at 0.1 microM in intact MCF-7 cells, IC50 = 65 pM) in a time- and concentration-dependent manner, suggesting that EMATE is an active site-directed inhibitor. EMATE is also active in vivo orally. 5,6,7,8-Tetrahydronaphthalene 2-O-sulfamate (7) and its N-methylated derivatives (8 and 9) were synthesized, and 7 inhibits the E1-STS activity in intact MCF-7 cells by 79% at 10 microM. 4-Methylcoumarin 7-O-sulfamate (COUMATE) and its derivatives (14, 16, and 18) were prepared to extend this series of nonsteroidal inhibitors, and COUMATE reduces the E1-STS activity in placental microsomes by > 90% at 10 microM. Although the orally active COUMATE is less potent than EMATE as an active site-directed inhibitor, it has the important advantage of being nonestrogenic. Analogues (20, 22, 24, 26, 27, 31, 33, 39, and 44) of COUMATE were synthesized to study its structure-activity relationships, and sulfamates of tetralones (46 and 48) and indanones (49, 51, and 53) were also prepared. While most of these compounds were found to inhibit E1-STS activity less effectively than COUMATE, one analogue, 3,4-dimethylcoumarin 3-O-sulfamate (24), was found to be some 12-fold more potent than COUMATE as an E1-STS inhibitor in intact MCF-7 cells (IC50 = 30 nM for 24, cf. 380 nM for COUMATE). Hence, highly potent sulfamate-based inhibitors of steroid sulfatase, such as EMATE, COUMATE, and 24, possess therapeutic potential and will allow the importance of estrogen formation in breast tumors via the E1-STS pathway to be assessed. A pharmacophore for active site-directed sulfatase inhibition is proposed.     

Combined use of 5 alpha-reductase inhibitors and alpha-l adrenergic receptor blockers in patients with benign prostatic hyperplasia

Historically, the ethics of a professional were the ethics of a gentleman. The social changes in the 1960s, where citizens asked for a greater voice in all affairs that affected them gave rise to formal approaches to ethics in the health fields. Principle-based and case-based reasoning have been dominant. Neither codes nor approaches based on virtue (the character of the professional) are perfect solutions in all cases, but professions are strengthened through the development and discussion of statements about ethical conduct.