Inhibition of 5alpha-reductase activity in SZ95 sebocytes and HaCaT keratinocytes in vitro.

Inhibition of 5alpha-reductase type 1 has been considered to be a promising target for treatment of androgen-dependent skin disorders, however, currently published clinical results on acne treatment are rather disappointing. In this study, the influence of selective inhibitors of 5alpha-reductase on testosterone metabolism within SZ95 sebocytes and HaCaT keratinocytes IN VITRO was investigated. In both cell types, the isotype 1 inhibitor MK386 completely inhibited the conversion of testosterone to 5alpha-dihydrotestosterone in concentrations higher than 10 (-9) M. Inhibitors of the isotype 2 such as finasteride, dihydrofinasteride, and turosteride, were >100-fold less active, while, as expected, androgen receptor inhibitors did not affect the 5alpha-reductase activity. MK386, but not finasteride, reduced testosterone-stimulated proliferation and slightly reduced the testosterone-induced increase in the amount of SZ95 sebocyte proteins. The androgen receptor inhibitor cyproterone acetate exhibited no effect on testosterone-induced proliferation, but inhibited the 5alpha-dihydrotestosterone-induced sebocyte proliferation. Our experimental findings and the existing clinical results indicate that the inhibition of 5alpha-reductase activity alone may be insufficient to reduce overall sebocyte activity and improve acne lesions.     

Inhibition of steroid 5 alpha-reductase and its effects on testosterone hydroxylation by rat liver microsomal cytochrome P-450

It has been shown previously that liver microsomal steroid 5 alpha-reductase activity increases with age in female but not male rats, which coincides with a female-specific, age-dependent decline in the cytochrome P-450-dependent oxidation of testosterone to 1 beta-, 2 alpha-, 2 beta-, 6 alpha-, 6 beta-, 7 alpha-, 15 beta-, 16 alpha-, 16 beta-, and 18-hydroxytestosterone and androstenedione. To determine whether the increase in steroid 5 alpha-reductase activity is responsible for the decrease in testosterone oxidation, we have examined the effects of the steroid 5 alpha-reductase inhibitor, 4-MA (17 beta-N,N-diethylcarbamoyl-4-methyl-4-aza-5 alpha-androstan-3-one), on the pathways of testosterone oxidation catalyzed by rat liver microsomes. We have also determined which hydroxytestosterone metabolites are substrates for steroid 5 alpha-reductase. At concentrations of 0.1 to 10 microM, 4-MA completely inhibited steroid 5 alpha-reductase activity without inhibiting the pathways of testosterone oxidation catalyzed by liver microsomes from rats of different age and sex, and from rats induced with phenobarbital or pregnenolone-16 alpha-carbonitrile. 4-MA (10 microM) had little or no effect on the oxidation of testosterone catalyzed by liver microsomes from mature male rats (which have low steroid 5 alpha-reductase activity). In contrast, the hydroxylated testosterone metabolites formed by liver microsomes from mature female rats (which have high steroid 5 alpha-reductase activity) accumulated to a much greater extent in the presence of 4-MA. Evidence is presented that 4-MA increases the accumulation of hydroxytestosterones by two mechanisms. First, 4-MA inhibited the 5 alpha-reduction of those metabolites (such as 6 beta-hydroxytestosterone) that were found to be excellent substrates for steroid 5 alpha-reductase. In the absence of 4-MA, these metabolites eventually disappeared from incubations containing liver microsomes from mature female rats. Second, 4-MA inhibited the formation of 5 alpha-dihydrotestosterone, which otherwise competed with testosterone for oxidation by cytochrome P-450. This second mechanism explains why 4-MA increased the accumulation of metabolites (such as 7 alpha-hydroxytestosterone) that were found to be poor substrates for steroid 5 alpha-reductase. Despite its marked effect on the accumulation of hydroxylated testosterone metabolites, 4-MA had no effect on their initial rate of formation by liver microsomes from either male or female rats.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of a novel steroid (PM-9) on the inhibition of 5alpha-reductase present in Penicillium crustosum broths

The conversion of testosterone (T) to 5alpha-dihydrotestosterone (DHT) has been demonstrated in Penicillium crustosum broth obtained from fermented pistachios, lemons and corn tortillas. Furthermore, the presence of 5alpha-reductase enzyme, which is responsible for this conversion, has been established by electrophoretical techniques in these cultures.5alpha-Reductase enzyme is also present in animal and human androgen-dependent tissues as well as in prostate and seminal vesicles. The increase of the conversion of T to DHT in prostate gland, has been related to some illnesses such as benign prostate hyperplasia and prostate cancer. Furthermore, treatment with 5alpha-reductase inhibitors such as finasteride reduces the prostate growth. These data have stimulated research for the synthesis of new molecules with antiandrogenic activity, whose biological effect needs to be demonstrated.The purpose of this study is to determine the inhibition pattern of 5alpha-reductase in P. crustosum by finasteride and the new steroidal compound PM-9. K(m) and V(max) values for T, were determined in the broths by Lineweaver-Burk plots using different testosterone concentrations. The inhibition pattern of finasteride and PM-9 was also determined by Lineweaver-Burk using different concentrations of T and inhibitors. Results show that finasteride and PM-9 inhibit 5alpha-reductase present in the broth in a competitive manner.     

Dutasteride affects progesterone metabolizing enzyme activity/expression in human breast cell lines resulting in suppression of cell proliferation and detachment

Recent evidence indicates that progesterone metabolites play important roles in regulating breast cancer. Previous studies have shown that breast carcinoma and tumorigenic breast cell lines have higher 5alpha-reductase and lower 3alpha-hydroxysteroid oxidoreductase (3alpha-HSO) and 20alpha-HSO activities and mRNA expression levels than normal tissue and non-tumorigenic cell lines. The 5alpha-reduced progesterone metabolites such as 5alpha-dihydroprogesterone (5alphaP) promote both mitogenic and metastatic activity in breast cell lines in culture, whereas the 4-pregnene metabolites, 4-pregnen-3alpha-ol-20-one (3alphaHP) and 4-pregnen-20alpha-ol-3-one (20alphaHP) have the opposite (anti-cancer-like) effects. The 5alpha-reductase inhibitor dutasteride has been shown to inhibit 5alpha-reduction of testosterone to 5alpha-dihydrotestosterone in prostate tissue, resulting in decreased prostate volume. The aim of this study was to determine if dutasteride is an effective inhibitor of progesterone 5alpha-reduction in human breast cell lines and if such inhibition reduces mammary cell proliferation and detachment. The effect of dutasteride on progesterone metabolizing enzyme activities and mRNA expression were examined in tumorigenic MCF-7 and non-tumorigenic MCF-10A human breast cell lines. Dutasteride (10(-6)M) inhibited progesterone conversion to 5alpha-pregnanes by >95% and increased 4-pregnene production. The results indicated that effects of dutasteride on the progesterone metabolizing enzymes are due to direct inhibition of 5alpha-reductase activity and to altered levels of expression of 5alpha-reductase and HSO mRNAs. Treatment of cells with progesterone without medium change for 72 h resulted in significant conversion to 5alpha-pregnanes and increases in cell proliferation and detachment. The increases in proliferation and detachment were blocked by dutasteride and were reinstated by concomitant treatment with 5alphaP, providing proof-of-principle that the effects were due not to progesterone but to the 5alpha-reduced metabolites. This study provides the first evidence that dutasteride is a potent progesterone 5alpha-reductase inhibitor and that such inhibition may be beneficial in breast cancer.     

Prolactin involvement in regulation of testicular 5 alpha-reductase activity in the immature rat

Treatment of intact immature (25-day-old) rats with bromoergocryptine (BR), which suppressed prolactin (Prl) secretion, decreased testicular 5 alpha-reductase activity, whereas treatment with Prl increased the enzyme activity in BR-treated animals. Serum luteinizing hormone (LH) concentrations were not reduced by BR treatment or elevated by Prl, suggesting that the BR and Prl effects on enzyme activity were not due to alterations in LH secretion. Hypophysectomy (at 21 days of age) caused a dramatic decrease in testicular 5 alpha-reductase activity, and treatment with LH partially reversed this effect. Treatment of hypophysectomized animals with Prl alone had no effect on the enzyme activity but enhanced the effect of LH. Testosterone propionate, given to hypophysectomized animals in a regimen that increased testicular testosterone to concentrations at least as high as those in intact (sham-hypophysectomized) controls, had no effect on enzyme activity, whether given alone or in combination with LH. These results indicate that Prl is involved, along with LH, in maintaining the high 5 alpha-reductase activity of the prepubertal rat testis; the action of Prl, apparently requiring the presence of LH, may be to decrease the rate of degradation of the enzyme. The data also suggest that the action of LH on testicular 5 alpha-reductase activity is not mediated by its stimulation of testosterone production.     

Circadian rhythm of acid phosphatase activity in rat liver microsomes and dependence of NADH 5 alpha-reductase on phosphatase activity

The specific activity of acid phosphatase in male and female rats follows a circadian rhythm. Preincubation of liver microsomes with testosterone led to an increase of phosphatase activity and a loss of circadian rhythm. NADH 5 alpha-reductase was inactivated by several animal and bacterial acid and alkaline phosphatases while the acid phosphatase from potatoes was ineffective. The extent of inhibition depends on the course of circadian rhythm of NADH 5 alpha-reductase activity. Preincubation of microsomes in the presence of testosterone inhibited the NADH 5 alpha-reduction of testosterone. No such inhibition was observed after preincubation of microsomes with progesterone.     

New 5alpha-reductase inhibitors: in vitro and in vivo effects

The enzyme 5alpha-reductase is responsible for the conversion of testosterone (T) to its more potent androgen dihydrotestosterone (DHT). This steroid had been implicated in androgen-dependent diseases such as: benign prostatic hyperplasia, prostate cancer, acne and androgenic alopecia. The inhibition of 5alpha-reductase enzyme offers a potentially useful treatment for these diseases. In this study, we report the synthesis and pharmacological evaluation of several new 3-substituted pregna-4, 16-diene-6, 20-dione derivatives. These compounds were prepared from the commercially available 16-dehydropregnenolone acetate. The biological activity of the new steroidal derivatives was determined in vivo as well as in vitro experiments. In vivo experiments, the anti-androgenic effect of the steroids was demonstrated by the decrease of the weight of the prostate gland of gonadectomized hamster treated with T plus finasteride or the new steroids. The IC50 value of these steroids was determined by measuring the conversion of radio labeled T to DHT. The results of this study carried out with 5alpha-reductase enzyme from hamster and human prostate showed that four of the six steroidal derivatives (5, 7, 9, 10) exhibited much higher 5alpha-reductase inhibitory activity, as indicated by the IC50 values than the presently used Proscar 3 (finasteride). The comparison of the weight of the hamster's prostate gland indicated that compound 5 had a comparable weight decrease as finasteride. The overall data of this study showed very clearly those compounds 5, 7, 9, 10 are good inhibitors for the 5alpha-reductase enzyme.     

Androgen receptor and 5 alpha-reductase activity in the ductuli efferentes and epididymis of adult rhesus macaques

We measured androgen receptors (AR) and 5 alpha-reductase activity (5 alpha RA) in the ductuli efferentes and epididymides from adult rhesus macaques. Tissue samples were either assayed biochemically for AR or stained immunocytochemically (ICC) with a monoclonal antibody against AR. To estimate 5 alpha RA, tissue microsomes were incubated with [1 alpha,2 alpha-3H]testosterone, and the [3H]dihydrotestosterone formed was quantified. We found significant regional differences in the levels of both 5 alpha RA and AR in the excurrent ducts. In general, both enzyme activity and AR levels were higher in the caput and corpus epididymis than in ductuli efferentes and cauda epididymis. With ICC, positive nuclear AR staining was detected in all epithelial cell types, whereas variable numbers of stromal cells were positively stained. Our data demonstrate that there are segmental differences in the concentrations of 5 alpha RA and AR in epididymis and suggest that there may be regional differences in the regulation of epididymal functions by androgen.     

Sexual dimorphism of growth plate prehypertrophic and hypertrophic chondrocytes in response to testosterone requires metabolism to dihydrotestosterone (DHT) by steroid 5-alpha reductase type 1

Rat costochondral growth plate chondrocytes exhibit sex-specific and cell maturation dependent responses to testosterone. Only male cells respond to testosterone, although testosterone receptors are present in both male and female cells, suggesting other mechanisms are involved. We examined the hypothesis that the sex-specific response of rat costochondral cartilage cells to testosterone requires further metabolism of the hormone to dihydrotestosterone (DHT). Resting zone (RC) and growth zone (GC, prehypertrophic and upper hypertrophic zones) chondrocytes from male and female Sabra strain rats exhibited sex-specific responses to testosterone and DHT: only male cells were responsive. Testosterone and DHT treatment for 24 h caused a comparable dose-dependent increase in [3H]-thymidine incorporation in quiescent preconfluent cultures of male GC cells, and a comparable increase in alkaline phosphatase specific activity in confluent cultures. RC cells responded in a differential manner to testosterone and DHT. Testosterone decreased DNA synthesis in male RC cells but DHT had no effect and alkaline phosphatase specific activity of male RC cells was unaffected by either hormone. Inhibition of steroid 5alpha-reductase activity with finasteride (1, 5, or 10 microg/ml), reduced the response of male GC cells to testosterone in a dose-dependent manner, indicating that metabolism to DHT was required. RT-PCR showed that both male and female cells expressed mRNAs for steroid 5alpha-reductase type 1 but lacked mRNAs for the type 2 form of the enzyme. Male cells also exhibited 5alpha-reductase activity but activity of this enzyme was undetectable in female cells. These observations show that sex-specific responses of rat growth zone chondrocytes to testosterone requires the further metabolism of the hormone to DHT and that the effect of DHT in the male growth plate is maturation-state dependent. Failure of female chondrocytes to respond to testosterone may reflect differences in testosterone metabolism, since these cells possess greater ability to aromatize the hormone to estradiol.     

Modulation of epididymal delta 4-steroid 5 alpha-reductase activity in vitro by the phospholipid environment

Epididymal 5 alpha-reductase converts testosterone to 5 alpha-dihydrotestosterone. The enzyme is localized to the nuclear and microsomal membranes, and using two approaches, we investigated the relationship between 5 alpha-reductase activity and the membrane environment. In the first, nuclear and microsomal membrane fractions were treated with phospholipases to modify specifically the structure of the phospholipid component of the membranes, and the effects of these treatments on the kinetic parameters of 5 alpha-reductase were examined. The second approach was to observe the effects of phospholipids of known structure on solubilized 5 alpha-reductase activity. Treatment of the membrane fractions with phospholipase C increased the Km(app) of both the nuclear and microsomal 5 alpha-reductases for testosterone. Phospholipase A2 treatment also increased the Km(app) of the microsomal enzyme, but in contrast, the Km(app) of the nuclear 5 alpha-reductase for testosterone was unaffected. This demonstrated a fundamental difference in the role of the membrane environment in the expression of 5 alpha-reductase activity in these subcellular compartments. The ability of phospholipids to enhance the activity of solubilized 5 alpha-reductase was highly specific and structure related. Only phosphatidylcholines containing either unsaturated acyl chains or saturated acyl chains of 12 carbon atoms were found to activate 5 alpha-reductase. The most potent activator was dilauroyl phosphatidylcholine, which reduced the Km(app) values of both nuclear and microsomal 5 alpha-reductases for testosterone, without affecting the concentration of active 5 alpha-reductase (Vmax(app) ). This is the first time that an activator of 5 alpha-reductase has been found. These findings suggest that epididymal 5 alpha-reductase activity may be regulated by changes in the phospholipid environment.     

A kinetic analysis of the inhibition of human prostatic 5 alpha-reductase by 4-hydroxyandrostenedione and related steroids

The inhibition of human prostatic 5 alpha-reductase by androstenedione (A), 4-hydroxyandrostenedione (4-OH-A), and 4-methoxyandrostenedione (4-MeO-A) was studied. All three steroids inhibited 5 alpha-reductase in a concentration-dependent manner. The inhibition was competitive with respect to testosterone and non-competitive with respect to NADPH, indicating that these compounds inhibit 5 alpha-reductase by acting as alternative substrates. Ki values obtained were in the range 0.21-0.3 microM (A), 1.01-2.04 microM (4-OH-A), and 10.2-28.3 microM (4-MeO-A). Thus the two derivatives of androstenedione are poor inhibitors of 5 alpha-reductase and appear to have limited clinical potential.     

Solubilization of human prostatic 5 alpha-reductase

A sensitive assay for 5 alpha-reductase was introduced which is capable of detecting at least 0.2 U of activity per sample. The assay was used in developing a method for the solubilization of human prostatic 5 alpha-reductase. Homogenisation conditions were devised under which 95% of the total prostatic 5 alpha-reductase was released into the microsomal fraction. A combination of 0.1 M sodium citrate, 0.1 M KCl, 20% (v/v) glycerol, 0.5 mM NADPH and 1 microM testosterone was found to stabilise 5 alpha-reductase in the presence of detergents. The effect of the presence of low concentrations of detergents in the assay on the activity of 5 alpha-reductase was studied. Triton X-100, Lubrol PX and Nonidet P-40, caused a concentration-dependent inhibition of activity. The ability of several detergents (Triton X-100 MEGA-9, Tween 20, Tween 80, digitonin, Lubrol PX and Nonidet P-40) to solubilise 5 alpha-reductase was studied. All detergents caused a concentration-dependent solubilization of 5 alpha-reductase. Significant amounts of active solubilized enzyme were recovered only with Lubrol PX at concentrations less than 1.1 mg/ml. Seventy percent of the 5 alpha-reductase was solubilized in an active form by extracting the membranes 3 times with 0.8 mg/ml Lubrol PX.     

Subcellular distribution and properties of progesterone (delta4-steroid) 5alpha-reductase in rat medial basal hypothalamus.

The subcellular distribution and properties of rat hypothalamic progesterone 5 alpha-reductase, which accelerates the conversion of progesterone to 5 alpha-pregnane-3,20-dione, have been investigated by utilizing 3H-labeled substrate and a reverse isotopic dilution assay system. The enxymic activity was associated primarily with a cell debris-membranes fraction deribed from the 100 x g pellet. This fraction contained mainly membrane-like particulates and was free of nuclei. Little or no activity was associated with the purified nuclei. The hypothalamic 5 alpha-reductase was stimulated by NADPH but not by NADH. The reaction proceeded optimally over a pH range of 6.0 to 7.2 and at a temperaturhe substrate specificity of the enzyme for other delta 4-3-ketosteroids and the ability of these steroids to inhibit the 5 alpha reduction of [1,2-3H]progesterone as well as the effect of 17 beta-estradiol were also studied. 20 alpha-hydroxypregn-4-en-3-one was more reactive that progesterone, while testosterone was the least reactive. The estimated Km for 20 alpha-hydroxypregn-4-en-3-one was 8.6 +/- 1.9 x 10(-7) M, and for testosterone, 1.6 +/- 1.4 x 10(-5) M. The inhibition studies indicate that 20 alpha-hydroxypregn-4-en-3-one and 17 beta-estradiol are competitive and noncompetitive inhibitors, respectively, of the 5 alpha reduction of progesterone with Ki of 6.0 +/- 3.0 x 10(-8) M for 20 alpha-hydroxypregn-4-en-3-one and Kii (intercept inhibition constant) of 2.6 +/- 0.7 x 10(-5) M and Kis (slope inhibition constant) of 3.6 +/- 0.6 x 10(-5) M for 17 beta-estradiol. Testosterone is a poor competitive inhibitor of the reaction.     

New progesterone derivatives as inhibitors of 5alpha-reductase enzyme and prostate cancer cell growth

In this study we report the synthesis and pharmacological evaluation, in vivo as well as in vitro, of four new progesterone derivatives 4-7. The evaluation in vivo was carried out on gonadectomized male hamsters that were injected subcutaneously daily with 1 mg/Kg of testosterone (T) and/or 1 mg/Kg of finasteride, or with 2 mg/Kg of the novel compounds. It was observed that when testosterone (T) and finasteride or compound 4 were injected together, the weight of the prostate decreased significantly as compared to that oftestosterone-treated animals. Compounds 5-7 did not show any in vivo activity. The 5alpha-reductase inhibitory activity of the novel compounds was determined in vitro using human prostate homogenates; the steroids 4-7 inhibited the 5alpha-reductase activity with IC50 values lower than that for the reference compound finasteride. 3. The effect of compounds 4-7 on the growth of lymphocytes and prostate cancer culture cells line was that steroid 4 inhibited the growth of both cells lines at a concentration of 50 microM and showed a cytotoxic effect whereas compounds 5-7 showed a much lower inhibition. Nevertheless steroids 4-7 didn't exhibit any toxic effects in vivo since the animals remained alive during the six days of treatment.     

Follicle-stimulating hormone inhibits granulosa cell 5 alpha-reductase activity. Possible role of 5 alpha-reductase as a steroidogenic pubertal switch.

In order to elucidate the role of 5 alpha-reductase in the ovarian pubertal transition from 5 alpha-reduced to non-5 alpha-reduced steroids, we examined the characteristics and regulation of granulosa cell (GC) 5 alpha-reductase activity. Maximum activity was observed at 37 degrees C and at a pH of 6.5-8.0. Synthetic 4-aza-3-oxosteroids proved to be potent inhibitors (76% inhibition at 0.1 microM) of ovarian 5 alpha-reductase activity, and 20 alpha-DHP was a better substrate than either progesterone or testosterone (4- or 7-fold higher affinity constants, respectively). The Km (20 alpha-DHP) of the enzyme was 0.50 +/- 0.03 microM and 0.75 +/- 0.20 microM in homogenates of whole ovaries and GC, respectively. 17 beta-Estradiol was a non-competitive inhibitor (KI = 6.97 microM). 5 alpha-Reductase activity was 22-fold (immature) to 68-fold (mature) higher in liver than ovary and 4-fold higher in theca-interstitial shells than in isolated GC. Ovarian 5 alpha-reductase activity decreased markedly with age (greater than 60% inhibition in mature, randomly cycling rats as compared to immature rats). In vivo administration of follicle-stimulating hormone (FSH) to immature rats produced a dose-dependent decrease in GC 5 alpha-reductase activity (36 +/- 1.1% and 46 +/- 5.9% inhibition following 12 micrograms and 24 micrograms FSH, respectively). Similarly, the in vitro provision of FSH (100 ng/ml) to cultured GC from immature rats resulted in (36-59%) inhibition in 5 alpha-reduced steroids. Inasmuch as FSH promotes GC development and the advancement of puberty, its ability to "switch-off" ovarian 5 alpha-reductase activity may enhance the formation of biologically potent (i.e. non-5 alpha-reduced) progestins as well as the availability of aromatizable androgens, in the best interests of pubertal steroidogenesis.     

Functional characteristics of nuclear 5 alpha-reductase from rat ventral prostate

The subcellular distribution and functional characteristics of 5 alpha-reductase (3-oxo-5 alpha-steroid: NADP+ 4-ene-oxidoreductase, EC 1.3.1.22) from rat ventral prostate were studied and compared to the 5 alpha-reductase from female rat liver. Tissue fractionation retained main enzymic activity in the microsomal fraction of rat liver, while 5 alpha-reductase from rat prostate was localized in the nuclear membrane with a specific activity 160 times that of the initial homogenate. The purity of nuclear envelopes was checked by electron microscopy. Solubilization experiments indicated that the hepatic 5 alpha-reductase is attached to the endoplasmic reticulum as a peripheral protein, while the nuclear prostatic enzyme is an integral membrane protein. Incubation experiments with phospholipases suggested a decisive role of the surrounding phospholipids for the prostatic enzyme activity. To elucidate the characteristics of hydrogen transfer of the enzyme, the effect of flavins and different other cofactors on 5 alpha-reductase activity in isolated prostatic nuclei were studied. Our findings indicate that in rat ventral prostate the conversion of testosterone to 5 alpha-dihydrotestosterone proceeds by a direct hydrogen transfer from NADPH to testosterone. Concerning these parameters the behaviour of hepatic 5 alpha-reductase is absolutely different from the prostatic enzyme. The localization of 5 alpha-reductase within the nuclear envelope of rat ventral prostate as an integral membrane protein seems to be of physiological significance with regard to the action of androgens.     

Factors influencing prostatic 5 alpha-reductase activity in possum (Trichosurus vulpecula)

1. There were marked differences in prostatic wts among individual possums, but no evidence of a seasonally related change in wt could be established. It was concluded that the wt differences are mainly due to the changes in secretory activities. After castration the prostate wts fell while after administration of testosterone or oestradiol partially reversed this process. 2. Seven steroid conversion products were isolated from prostatic homogenates incubated with [3H] testosterone; 5 alpha-androstane-3 beta,17 beta-diol forming the highest yield. 3. While the 5 alpha-reductase activity of prostates from intact possums was very low (approx. 8% of the total yield), it increased to over 50% after castration. 4. Administration of testosterone or oestradiol partially reversed the post-castration rise in 5 alpha-reductase, while 17 beta-hydroxy-5 alpha-androstane-3-one (DHT) was ineffective. Administration of porcine FSH-NIH-P2 to both intact or castrated possums caused a marked rise in prostatic 5 alpha-reductase activity. 5. It was concluded that in possum, FSH may have a direct stimulatory effect on prostatic 5 alpha-reductase activity. The results are discussed in relation to placental mammals.     

Establishment of type II 5alpha-reductase over-expressing cell line as an inhibitor screening model

Dihydrotestosterone (DHT) is the most potent male hormone that causes androgenetic alopecia. The type II 5alpha-reductase is an enzyme that catalyzes the conversion of testosterone (T) to DHT, therefore it can be expected that specific inhibitors for type II 5alpha-reductase may improve the pathophysiologic status of androgenetic alopecia. In this study, we attempted to establish the reliable and convenient screening model for type II 5alpha-reductase inhibitors. After transfection of human cDNA for type II 5alpha-reductase into HEK293 cells, the type II 5alpha-reductase over-expressing stable cells were selected by G418 treatment. RT-PCR and Western blot analyses confirmed that type II 5alpha-reductase gene was expressed in the stable cells. In in vitro enzymatic assay, 10 microg of stable cell extract completely converted 1 microCi (approximately 0.015 nmol) of T into DHT. The type II 5alpha-reductase activity was inhibited by finasteride in a dose-dependent manner, confirming the reliability of screening system. In cell culture condition, 2 x 10(5) of stable cells completely converted all the input T (approximately 0.03 nmol) into DHT by 4h incubation, demonstrating that the stable cell line can be used as a cell-based assay system. Using this system, we selected the extracts of Curcumae longae rhizoma and Mori ramulus as the potential inhibitors for type II 5alpha-reductase. These results demonstrate that the type II 5alpha-reductase over-expressing stable cell line is a convenient and reliable model for screening and evaluation of inhibitors.     

Sexual difference in properties of 5 alpha-reductase in microsome of rat submandibular glands

The properties of 5 alpha-reductase activity in the submandibular glands of rats were investigated using microsomal fraction in the presence of NADPH, and we found a sexual difference in these properties. The formation of 5 alpha-dihydrotestosterone and 5 alpha-androstane-3 alpha, 17 beta-diol from testosterone demonstrated 5 alpha-reductase in the rat microsomal fraction of this tissue sample. Apparent michaelis constants (Km) of the 5 alpha-reductase activity for testosterone was 1.02 x 10(-6) M for the female tissue. Microsomal fraction of submandibular glands of male rats had two Kms and were determined as 1.12 x 10(-6) M and 1.01 x 10(-5) M. The lower Km of 5 alpha-reductase for male rats was similar to for the females.     

Comparative study of human steroid 5alpha-reductase isoforms in prostate and female breast skin tissues: sensitivity to inhibition by finasteride and epristeride

Steroid 5alpha-reductase (5-AR) catalyses the reduction of testosterone (T) to dihydrotestosterone (DHT). The 5alpha-reductase found in human benign prostatic hyperplasia (BPH) has been compared with that found in human breast skin tissue in respect of sensitivity to inhibition by Finasteride and Epristeride. Kinetic studies showed the presence of two isoforms of 5alpha-reductase in benign prostatic hyperplasia indicated by low and high Km isoforms for testosterone, while female breast skin tissue contained only one isoform. The isoforms differ in their affinity for the inhibitors Finasteride and Epristeride, both compounds being more effective for the low Km 5alpha-reductase isoform than the high Km 5alpha-reductase of prostatic tissue, with Finasteride displaying competitive inhibition and Epristeride uncompetitive. Finasteride and Epristeride are also inhibitors of skin 5alpha-reductase, which possesses a comparable Ki for Finasteride to that of the low Km prostatic enzyme, but Epristeride was a less potent inhibitor of the skin enzyme relative to the prostate isoform. These results suggest that the inhibitors have therapeutic potential, other than for treatment of benign prostatic hyperplasia, for treating skin disorders influenced by the action of dihydrotestosterone and warrant further investigation.