Prostate Microsomes Research Articles

New steroidal 17β-carboxy derivatives present anti-5α-reductase activity and anti-proliferative effects in a human androgen-responsive prostate cancer cell line

The androgens testosterone (T) and dihydrotestosterone (DHT), besides playing an important role in prostate development and growth, are also responsible for the development and progression of benign prostate hyperplasia (BPH) and prostate cancer. Therefore, the actions of these hormones can be antagonized by preventing the irreversible conversion of T into DHT by inhibiting 5α-reductase (5α-R). This has been a useful therapeutic approach for the referred diseases and can be achieved by using 5α-reductase inhibitors (RIs). Steroidal RIs, finasteride and dutasteride, are used in clinic for BPH treatment and were also proposed for chemoprevention of prostate cancer. Nevertheless, due to the increase in bone and muscle loss, impotency and occurrence of high-grade prostate tumours, it is important to seek for other potent and specific molecules with lower side effects. In the present work, we designed and synthesized steroids with the 3-keto-Δ(4) moiety in the A-ring, as in the 5α-R substrate T, and with carboxamide, carboxyester or carboxylic acid functions at the C-17β position. The inhibitory 5α-R activity, in human prostate microsomes, as well as the anti-proliferative effects of the most potent compounds, in a human androgen-responsive prostate cancer cell line (LNCaP cells), were investigated. Our results showed that steroids 3, 4 and 5 are good RIs, which suggest that C-17β lipophylic amides favour 5α-R inhibition. Moreover, these steroids induce a decrease in cell viability of stimulated LNCaP cells, in a 5α-R dependent-manner, similarly to finasteride.

Spectrophotometric Method for the Assay of Steroid 5α-Reductase Activity of Rat Liver and Prostate Microsomes

A simple spectrophotometric method for the assay of steroid 5α-reductase (5α-SR) was developed in which 5α-dihydrotestosterone (5α-DHT) and 5α-androstane-3α,17β-diol (5α-diol), metabolites formed in the NADPH-dependent reduction of testosterone with enzyme sources of 5α-SR, were measured by enzymatic cycling using 3α-hydroxysteroid dehydrogenase in the presence of excess thionicotinamide-adenine dinucleotide (thio-NAD) and NADH. It was found that 5α-SR activity was proportional to the accumulated thio-NADH having an absorption maximum at 400 nm. Because of the high cycling rate (> 600 cycle per min) and no interference from testosterone, enzymatic cycling can determine the sum of 5α-DHT and 5α-diol at the picomole level without separation from excess testosterone. The present method was readily applicable to the assay of 5α-SR activity of rat liver and prostate microsomes as well as to the assay of inhibitory activity of finasteride, a synthetic inhibitor of 5α-SR.

Development of a new gas chromatography–mass spectrometry (GC–MS) methodology for the evaluation of 5α-reductase activity

The androgens testosterone (T) and dihydrotestosterone (DHT) play a key role in the function and integrity of prostate tissue, but are also implicated in prostate cancer and benign prostatic hyperplasia (BPH). The reduction of androgen levels can be achieved by the inhibition of 3-oxo-5α-steroid-4-dehydrogenase (5α-reductase), which is responsible for the irreversible conversion of T into its more active metabolite DHT. In fact, the use of 5α-reductase inhibitors (RIs), like finasteride, can be a valuable strategy for the treatment of BPH and in chemoprevention of prostate tumors. In this work a new method based on a dispersive liquid–liquid microextraction (DLLME) procedure, followed by gas chromatography–mass spectrometry (GC–MS), to evaluate the 5α-reductase activity, by measuring the conversion percentage of T into DHT was optimized and validated. Enzymatic assays were carried out in human prostate microsomes, using T as substrate. T and DHT were extracted by the developed DLLME technique and quantified, after silylation, by GC–MS. Variables affecting the extraction efficiency and derivatization of T and DHT were evaluated. The optimized method showed good linearity (with correlation coefficients over 0.9994 for T and 0.9995 for DHT), good recoveries (higher than 80%), and good intra- and inter-day precision (below 13%, 3 levels, n=6). The detection limits for T and DHT were 0.5nM and the limits of quantification were 5nM. The new GC–MS method is a good alternative to the already described methods, to evaluate 5α-reductase activity, since it avoids the use of radioactive compounds and corresponds to a fast and sensitive methodology with a good extraction efficiency, accuracy and high recovery. As this method allows the evaluation of 5α-reductase activity, also permits the study of inhibitory efficacy of new molecules as potential RIs.

Cytochrome P450 3A5 is highly expressed in normal prostate cells but absent in prostate cancer

Testosterone is essential for the growth and function of the luminal prostate cells, but it is also critical for the development of prostate cancer, which in the majority of the cases derives from luminal cells. Cytochrome P450 3A (CYP3A) enzymes hydroxylate testosterone and dehydroepiandrosterone to less active metabolites, which might be the basis for the association between CYP3A polymorphisms and prostate cancer. However, it is unknown whether the CYP3A enzymes are expressed at relevant levels in the prostate and which polymorphisms could affect this tissue-specific CYP3A activity. Thus, we measured CYP3A4, CYP3A5, CYP3A7, and CYP3A43 mRNA in 14 benign prostatic hyperplasias and ten matched non-tumoral/tumoral prostate samples. We found that CYP3A5 mRNA in non-tumoral prostate tissue was 10% of the average amount of liver samples, whereas the expression of the other CYP3A genes was much lower. Similarly to liver, CYP3A5*3 polymorphism decreased CYP3A5 mRNA content 13-fold. CYP3A5 protein was detected in non-tumoral prostate microsomes by western blot, and immunohistochemistry (IHC) localized CYP3A5 exclusively in the basolateral prostate cells. In contrast to the normal tissue, IHC and RT-PCR showed that tumoral tissue lacked CYP3A5 expression. In conclusion, prostate basolateral cells express high levels of CYP3A5 which dramatically decrease in tumoral tissue. This finding supports an endogenous function of CYP3A5 related to the metabolism of intra-prostatic androgens and cell growth, and that polymorphisms affecting CYP3A5 activity may result in altered prostate cancer risk and aggressiveness.

FLUTAMIDE METABOLISM IN FOUR DIFFERENT SPECIES IN VITRO AND IDENTIFICATION OF FLUTAMIDE METABOLITES IN HUMAN PATIENT URINE BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY/TANDEM MASS SPECTROMETRY

A new metabolic scheme of flutamide is proposed in this article. Some patients treated with flutamide, a nonsteroidal antiandrogen, have developed severe hepatic dysfunction. Toxic metabolites have been proposed to be responsible for these negative effects. In this study, the qualitative aspects of the in vitro metabolism of flutamide in liver microsomes from human, dog, pig, and rat were evaluated. A direct comparison of the flutamide metabolism in liver and prostate microsomes from pig was made, and the in vivo metabolism of flutamide was investigated in urine from orally treated prostate cancer patients. Liquid chromatography/tandem mass spectrometry was used for analysis. The mass spectrometer was equipped with an electrospray interface and operated in the negative ion mode. In liver microsomes from pig, dog, and rat, extensive hydroxylation of flutamide occurred. One, two, or three hydroxy groups were attached, and isomeric forms were detected for both monohydroxylated and trihydroxylated drug. In pig liver microsomes, isomers of a third metabolite, hydroxylated 4-nitro-3-(trifluoromethyl)-aniline, were also found after incubation with either flutamide or 2-hydroxyflutamide. In human liver microsomes, the pharmacologically active 2-hydroxyflutamide was the only metabolite detected. Several phase I metabolites as well as four intact phase II metabolites could be recovered from the urine samples. For the first time in humans, glucuronic acid conjugates of hydroxylated 4-nitro-3-(trifluoromethyl)-aniline, and mono- and dihydroxylated flutamide were identified, together with hydroxylated 4-nitro-3-(trifluoromethyl)-aniline conjugated with sulfate. In addition, one mercapturic acid conjugate of hydroxylated flutamide, probably formed from flutamide via a reactive intermediate, was detected.

Anti-tumour effects and pharmacokinetic profile of 17-(5'-isoxazolyl)androsta-4,16-dien-3-one (L-39) in mice: an inhibitor of androgen synthesis.

17-(5′-Isoxazolyl)androsta-4,16-dien-3-one (L-39), a novel androstene derivative, was synthesized and evaluated in vitro and in vivo. L-39 showed potent and non-competitive inhibition of human testicular microsomal 17α-hydroxylase/C 17,20-lyase with an IC 50 value of 59 n M and Ki of 22 n M. L-39 also showed potent and competitive inhibition of 5α-reductase in human prostatic microsomes with IC 50 and Ki values of 33 and 28 n M respectively. L-39 (5 μM) has also been shown to manifest anti-androgenic activity in cultures of human prostate cancer cell lines (LNCaP) by preventing the labelled synthetic androgen R1881 (5 n M) from binding to the androgen receptors. Androgen-ependent human próstate cancer xenografts (PC-82) were grown in nude mice and the effects of L-39 (50 mg kg–1day–1) on tumour growth and prostate-specific antigen (PSA) levels were determined after 28 days. L-39 significantly (P< 0.01) diminished tumour growth and wet weights to a similar extent as castration or flutamide treatment. L-39 also significantly (P< 0.01) reduced serum PSA levels by more than 80% in the mice bearing human prostate cancer xenografts. Pharmacokinetic studies were also conducted in male Balb/c mice. After subcutaneous administration of a single bolus dose, L-39 was rapidly absorbed into the systemic circulation. Peak plasma levels occurred at 0.75 h and then declined with a t1/2 of 1.51 h. The bioavailability of L-39 after subcutaneous administration was 28.5%. These results demonstrate that L-39 is a potent inhibitor of androgen synthesis and is effective in reducing the growth of human prostate cancer xenografts in nude mice. Although improvements in the bioavailability are necessary, L-39 is a potential lead compound with this profile as an inhibitor of prostate cancer growth. © 2000 Cancer Research Campaign

Effects of novel 17-azolyl compounds on androgen synthesis in vitro and in vivo

17-Azolyl steroids were synthesized and evaluated as inhibitors of androgen synthesis in vitro and in vivo. Several of the novel compounds exhibit potent noncompetitive inhibition of human 17α-hydroxylase/C 17,20-lyase with IC 50 values ranging from 7 to 90 nM, and K i values from 1.2 to 41 nM. VN/85-1 and VN/108-1 were the most potent inhibitors against this enzyme with IC 50 value of 8 nM ( K i of 1.2 nM) and 7 nM ( K i of 1.9 nM), respectively. VN/107-1, VN/108-1 and VN/109-1 also showed moderate inhibition of 5α-reductase in human prostatic microsomes. Normal adult male rats were treated with these novel 17-azolyl steroidal compounds at a dose level of 50 mg/kg, s.c., for 14 consecutive days, sacrificed 1–2 h after the last administered dose and blood, prostate and other tissues were collected. The organs were weighed and tissue concentrations of testosterone (T) and dihydrotestosterone (DHT) were measured. Tissue T levels were significantly ( p<0.05) lower in rats treated with the novel 17-azolyl steroids by more than 50% compared to the control group. Similarly, the concentration of DHT in the serum and prostates was significantly ( p<0.05) diminished in rats treated with the 17-azolyl steroids by 39–80% compared to the control group. Furthermore, the wet weights of the prostates and seminal vesicles were significantly ( p<0.05) reduced by several of the novel steroids. Although only one dose was evaluated in these studies, VN/85-1 was the most effective compound and reduced prostatic androgen levels by more than 80% and the wet weights of the prostate and seminal vesicles in rats by about 50%. These findings suggest that these novel compounds may provide useful leads for the research and development of suitable agents for the treatment of androgen dependent prostate cancer.

Inhibition of Androgen Synthesis in Human Testicular and Prostatic Microsomes and in Male Rats by Novel Steroidal Compounds

Inhibition of Androgen Synthesis in Human Testicular and Prostatic Microsomes and in Male Rats by Novel Steroidal Compounds

Inhibition of androgen synthesis in human testicular and prostatic microsomes and in male rats by novel steroidal compounds.

The C(17,20)-lyase and 5alpha-reductase are key enzymes in the biosynthesis of androgens. The effects of novel steroidal compounds were evaluated as inhibitors against both human C(17,20)-lyase and 5alpha-reductase in vitro. The concentrations of testosterone (T) and dihydrotestosterone (DHT) in the prostate, testis and serum and changes in the tissue weights were also determined in rats treated with the novel inhibitors. L-12 and L-26 showed potent inhibition of human testicular C(17,20)-lyase with IC50 values of 50 and 25 nM, respectively. L-12, L-38, and I-47 showed moderate inhibition of human testicular C(17,20)-lyase with IC50 values of 75, 108, and 70 nM, respectively similar to ketoconazole (78 nM). Interestingly, L-6, L-26, and L-38 also showed some inhibitory activity against 5alpha-reductase with IC50 values of 75, 125, and 377 nM, respectively. Finasteride, an inhibitor of 5alpha-reductase had an IC50 value of 33 nM. However, ketoconazole did not inhibit 5alpha-reductase nor did finasteride inhibit C(17,20)-lyase. Treatment of normal male rats with several of these novel inhibitors (50 mg/kg x day, s.c., for 14 consecutive days) caused about 45-91% decrease in serum, testicular and prostatic T concentration. Similarly, serum and prostatic DHT concentration were significantly decreased in rats treated with these novel compounds by 50-90% compared with controls. Surgical castration caused almost complete elimination of circulating T and DHT concentration in rat tissues. L-6 and L-12 were the most effective and reduced the wet weight of the prostate by 50%. Although future improvements in their bioavailability are necessary, these novel steroidal compounds show promise as potential agents for reducing T and DHT levels in patients with androgen dependent diseases.

Inhibitory effects of Serenoa repens on the kinetic of pig prostatic microsomal 5alpha-reductase activity.

The pathogenesis of benign prostatic hyperplasia is linked to the accumulation of dihydrotestosterone (DHT), the active form of testosterone (T), in prostatic tissue. We have defined characteristics of 5alpha-reductase enzyme which catalyzes the conversion of T into DHT in prostatic microsomes of growing pigs. Peaks for the 5alpha-reductase activity were found at pH 5.5 and 8.0, which indicates the presence of both type 1 and type 2 isozymes. Kinetic parameters of porcine 5alpha-reductase in the presence of Serenoa repens extracts revealed uncompetitive, noncompetitive, and mixed types of inhibitions. Our results show the inhibitory action of S. repens on prostate porcine microsomal 5alpha-reductase activity.

Inhibition of androgen synthesis by 22‐hydroximino‐23,24‐bisnor‐4‐cholen‐3‐one

The 22-hydroximino-23,24-bisnor-4-cholen-3-one (22-oxime) was synthesized and evaluated as an inhibitor of 17 alpha-hydroxylase/C17,20-lyase in rat testicular microsomes and the 5 alpha-reducatase of human prostatic microsomes from patients with benign prostatic hypertrophy. The 22-oxime demonstrated moderate inhibition for the 17 alpha-hydroxylase (Ki 74 nM vs. Km 29 nM) with progesterone as substrate and potent inhibition (Ki 18 nM vs. Km 76 nM) for the C17,20-lyase activity with 17 alpha-hydroxyprogesterone as substrate. Further investigation of this enzyme with progesterone as substrate demonstrated the inhibition occurred mainly at the 17 alpha-hydroxylation step of the progesterone substrate. The 22-oxime also demonstrated potent and competitive inhibition of 5 alpha-reductase in human prostatic microsomes (Ki 1.4 nM vs. Km 14 nM). When adult male rats were injected subcutaneously (sc) daily with 22-oxime (50 mg/kg/day) for 21 days, the concentrations of serum and testicular testosterone were significantly reduced by 65% and 59%, respectively, in comparison to vehicle-treated controls. Furthermore, both testosterone and DHT concentrations in rat prostatic tissue were significantly decreased by 60% and 44% compared to control tissue. Serum LH concentrations were unchanged in the 22-oxime-treated group compared to the control group. This indicates that the reduction in androgen concentrations in animals treated with this compound is not due to its influence on pituitary feedback mechanisms which result in reduced LH secretion. These results suggest that 22-oxime is effective in reducing androgen synthesis through the inhibition of 17 alpha-hydroxylase, C17,20-lyase, and 5 alpha-reductase both in vitro and in vivo.

Species differences in 5α-androstane-3β,17β-diol hydroxylation by rat, monkey, and human prostate microsomes

The 6α-, 7α-, and 7β-hydroxylation of 5α-androstane-3β,17β-diol by rat prostate microsomes appears to be catalyzed by a single, high-affinity cytochrome P450 enzyme. In the present study we have examined the hydroxylation of 5α-androstane-3β,17β-diol by prostate microsomes from cynomolgus monkeys and from normal subjects and patients with benign prostatic hyperplasia. Our results suggest that although rat, monkey, and human prostate microsomes catalyze the 6α-, 7α-, and 7β-hydroxylation of 5α-androstane-3β,17β-diol, these pathways of oxidation in monkeys and humans are not catalyzed by a single cytochrome P450 enzyme. The ratio of the three metabolites was not uniform among prostate microsomal samples from individual humans or monkeys. The 6α-hydroxylation of 5α-androstane-3β,17β-diol varied independently of both the 7α- and 7β-hydroxylation, which varied in unison. The 6α-, 7α-, and 7β-hydroxylation of 5α-androstane-3β,17β-diol by monkey prostate microsomes appeared to be differentially affected by in vivo treatment of monkeys with β-naphthoflavone or dexamethasone. Treatment of a monkey with dexamethasone appeared to cause a 2.5-fold increase in both the 7α- and the 7β-hydroxylation of 5α-androstane-3β,17β-diol without increasing the 6α-hydroxylation. The 7α- and 7β-hydroxylation of 5α-androstane-3β,17β-diol by human and monkey prostate microsomes, but not the 6α-hydroxylation, was inhibited by antibody against rat liver NADPH-cytochrome P450 reductase. Similarly, the 7α- and 7β-hydroxylation of 5α-androstane-3β,17β-diol by human prostate microsomes, but not the 6α-hydroxylation, was markedly inhibited (>85%) by equimolar concentrations of the imidazole-containing antimycotic drugs ketoconazole, clotrimazole, and miconazole. These results suggest that the 7α- and 7β-hydroxylation of 5α-androstane-3β,17β-diol by monkey and human prostate microsomes is catalyzed by a cytochrome P450 enzyme, whereas the 6α-hydroxylation is catalyzed by a different enzyme which may or may not be a cytochrome P450 monooxygenase. The hydroxylation of 5α-androstane-3β,17β-diol by prostate microsomes from normal human subjects was quantitatively and qualitatively similar to its hydroxylation by prostate microsomes from patients with benign prostatic hyperplasia. This suggests that an impairment of androgen inactivation is not the mechanism for the proposed accumulation of 5α-dihydrotestosterone in the hyperplastic prostate. The rate of 5α-androstane-3β,17β-diol hydroxylation by human prostate microsomes ranged from 1 to 10 pmol/mg protein/min, which is considerably less than the rate catalyzed by rat prostate microsomes (~600 pmol/mg protein/min). This marked species difference in the rate of inactivation of androgens by prostate microsomes could potentially explain why humans, in contrast to rats, are prone to develop benign prostatic hyperplasia.

Hydroxylation of 5α-androstane-3β,17β-diol by rat prostate microsomes: Effects of antibodies and chemical inhibitors of cytochrome P450 enzymes

The purpose of the present study was to test the hypothesis that rat prostate microsomes contain a single cytochrome P450 enzyme responsible for the conversion of 5α-androstane-3β,17β-diol to a series of trihydroxylated products. The three major metabolites formed by in vitro incubation of 5α-[ 3H]androstane-3β,17β-diol with rat prostate microsomes were apparently 5α-androstane-3β,6α,17β-triol, 5α-androstane-3β,7α,17β-triol, and 5α-androstane-3β,7β,17β-triol, which were resolved and quantified by reverse-phase HPLC with a flow through radioactivity detector. The ratio of the three metabolites remained constant as a function of incubation time, microsomal protein concentration, ionic strength, and substrate concentration. The ratio of the three metabolites was dependent on pH, apparently because the hydroxylation of 5α-androstane-3β,17β-diol shifted from the 6α-to the 7α-position with increasing pH (6.8–8.0). The V max values were 380, 160, and 60 pmol/mg microsomal protein/min for the rate of 6α-, 7α-, and 7β-hydroxylation, respectively. Similar K m values (0.5–0.7 μ m) were measured for enzymatic formation of all three metabolites, which suggests that formation of all three metabolites was catalyzed by a single, high-affinity enzyme. Testosterone, 5α-dihydrotestosterone, and 5α-androstane-3α,17β-diol did not appreciably inhibit the hydroxylation of 5α-androstane-3β,17β-diol, suggesting that this enzyme exhibits a high degree of substrate specificity. Formation of all three metabolites was inhibited by antibody against rat liver NADPH-cytochrome P450 reductase (85%) and by a 9:1 mixture of carbon monoxide and oxygen (60%). Several chemical inhibitors of cytochrome P450 enzymes, especially the antimycotic drug clotrimazole, also inhibited the formation of all three metabolites. Polyclonal antibodies that recognize liver cytochrome P450 1A, 2A, 2B, 2C, and 3A enzymes did not inhibit 5α-androstane-3β,17β-diol hydroxylase activity. Overall, these results are consistent with the hypothesis that the 6α-, 7α-, and 7β-hydroxylation of 5α-androstane-3β,17β-diol by rat prostate microsomes is catalyzed by a single, high-affinity P450 enzyme. This cytochrome P450 enzyme appears to be structurally distinct from those in the 1A, 2A, 2B, 2C, and 3A gene families.

Hydroxylation of 5α-androstane-3β,17β-diol by rat prostate microsomes: Potent inhibition by imidazole-type antimycotic drugs and lack of inhibition by steroid 5α-reductase inhibitors

5α-Dihydrotestosterone, the principal androgen mediating prostate growth and function in the rat, is formed from testosterone by steroid 5α-reductase. The inactivation of 5α-dihydrotestosterone involves reversible reduction to 5α-androstane-3β,17β-diol by 3β-hydroxysteroid oxidoreductase followed by 6α-, 7α-, or 7β-hydroxylation. 5α-Androstane-3β,17β-diol hydroxylation represents the ultimate inactivation step of dihydrotestosterone in rat prostate and is apparently catalyzed by a single, high-affinity ( K m ~ 0.5 μM) microsomal cytochrome P450 enzyme. The present studies were designed to determine if 5α-androstane-3β,17β-diol hydroxylation by rat prostate microsomes is inhibited by agents that are known inhibitors of androgen-metabolizing enzymes. Inhibitors of steroid 5α-reductase (4-azasteroid analogs; 10 μ m) or inhibitors of 3β-hydroxy-steroid oxidoreductase (trilostane, azastene, and cyanoketone; 10 μ m) had no appreciable effect on the 6α-, 7α-, or 7β-hydroxylation of 5α-androstane-3β,17β-diol (10 μ m) by rat prostate microsomes. Imidazole-type antimycotic drugs (ketoconazole, clotrimazole, and miconazole; 0.1–10 μ m) all markedly inhibited 5α-androstane-3β,17β-diol hydroxylation in a concentration-dependent manner, whereas triazole-type antimycotic drugs (fluconazole and itraconazole; 0.1–10 μ m) had no inhibitory effect. The rank order of inhibitory potency of the imidazole-type antimycotic drugs was miconazole > clotrimazole > ketoconazole. In the case of clotrimazole, the inhibition was shown to be competitive in nature, with a K i of 0.03 μ m. The imidazole-type antimycotic drugs inhibited all three pathways of 5α-androstane-3β,17β-diol hydroxylation to the same extent, which provides further evidence that, in rat prostate microsomes, a single cytochrome P450 enzyme catalyzes the 6α-, 7α-, and 7β-hydroxylation of 5α-androstane-3β,17β-diol. These studies demonstrate that certain imidazole-type compounds are potent, competitive inhibitors of 5α-androstane-3β,17β-diol hydroxylation by rat prostate microsomes, which is consistent with the effect of these antimycotic drugs on cytochrome P450 enzymes involved in the metabolism of other androgens and steroids.

Distribution and regulation of 5 alpha-androstane-3 beta,17 beta-diol hydroxylase in the rat central nervous system.

5 alpha-Androstane-3 beta,17 beta-diol hydroxylase (3 beta-diol OHase), a form of cytochrome P-450 whose main function is thought to be the elimination of dihydrotestosterone (DHT) from its target tissues, was found in the central nervous system (CNS) of both male and female rats at a level which is at least 300-fold higher than any other P-450 catalyzed steroid hydroxylase in the brain. In order to gain some insight into the physiological function of this enzyme we have studied its distribution throughout the CNS as well as the effects of age and hormonal manipulations of rats. We have also partially purified the enzyme from the brain and compared it with the 3 beta-diol OHase in the prostate. 3 beta-Diol OHase activity was distributed throughout the CNS of adult male and female rats with levels between 70-153 nmol products formed/g tissue.h. The products were identified as 5 alpha-androstane-3 beta,7 beta,17 beta-triol,5 alpha-androstane-3 beta,6 alpha,17 beta-triol and 5 alpha-androstane-3 beta,7 alpha,17 beta-triol. The ratio of these three triols was 1:9:3. Catalytic activity was not affected by castration or adrenalectomy of male rats, or during late pregnancy or lactation in females. In the brains of 14-day-old fetuses and 2-day-old rats, the level of 3 beta-diol OHase was 30% of that of adults and it increased to adult levels by day 28. Cytochrome P-450 was partially purified from microsomes of whole brain, hypothalamus, and prostate and 3 beta-diol OHase activity reconstituted. On the basis of the ratios of the three triols formed from 3 beta-diol, it can be concluded that the 3 beta-diol OHase in the brain is similar to that in the prostate. From a comparison of the turnover numbers in reconstituted systems with P-450 prepared from the prostate, brain, and hypothalamus, it can be estimated that up to 50% of the P-450 in the hypothalamus and 10% in whole brain is 3 beta-diol OHase. Supportive evidence for this relative distribution of 3 beta-diol OHase in the hypothalamus was obtained by comparing the sodium dodecyl sulfate electrophoretic profiles of P-450 obtained from the hypothalamus and prostate microsomes.

Estrogen and progestin receptors and aromatase activity in rhesus monkey prostate.

We examined nuclear estrogen receptors (ER) and progestin receptors (PR) in the rhesus monkey prostate. Tissues were obtained from six intact males, five untreated castrates, six castrates treated with testosterone (T) for 6 weeks, and four castrates treated with estradiol (E2) for 6 weeks. Samples of the caudal lobe were either assayed biochemically for ER or stained immunocytochemically (ICC) with monoclonal antibodies against the ER or PR. Prostates from untreated castrates had significantly more ER than tissues from intact or T-treated castrates. In E2-treated castrates, ER number increased compared to that in intact and T-treated castrates. With ICC, ER was found only in the nuclei of the fibroblasts and smooth muscle cells of the stroma, not the glandular, ductal, or urethral epithelial cells. Intact and T-treated castrates had a very small number of positive cells, while untreated and E2-treated castrates had a significantly increased number of positive ER cells in the fibromuscular stroma. With ICC, PR was absent in intact or T-treated animals and barely evident in untreated castrates, but was significantly increased in the fibromuscular stroma of E2-treated castrates. The histological preparations indicated there was no stromal hypertrophy in the E2-treated castrates, but the E2 treatment did cause dilation of the glandular acini. Aromatase activity was measured in prostatic microsomes with a radiometric assay. Levels were low (3-30 fmol/h.mg protein) compared to those in brain and placenta, and no differences in activity were seen between castrates and T-treated castrates. Our data demonstrate that androgens can suppress the level of nuclear ER in the rhesus prostate, and that E2 treatment of castrates can induce PR in the same cells as those that contain ER. Thus, under appropriate conditions, estrogens could affect the rhesus prostate through a receptor-mediated pathway.

Nicotine and cotinine effects on 3 alpha hydroxysteroid dehydrogenase in canine prostate

We have recently observed that cigarette smoking affects plasma androgen concentrations. The effects of nicotine and cotinine, two products of cigarette smoking, on testosterone metabolism were determined. The activity of delta 4 steroid 5 alpha-reductase, which converts testosterone to 5 alpha-dihydrotestosterone (DHT) was measured in isolated dog prostate nuclei using testosterone (0-200 nM) as substrate and NADPH as cofactor. Activity of 3 alpha-hydroxysteroid dehydrogenase (HSD), which converts DHT to 3 alpha-androstanediol (3 alpha-diol) and is a reversible enzyme, was measured in isolated dog prostate microsomes with DHT (0-20 microM) as substrate and NADPH as cofactor. When microsomal fractions were incubated for 1 hour with and without nicotine (0-50 microM) and cotinine (0-100 microM), enzyme activity of HSD was significantly suppressed (p less than 0.001). The Vmax was not affected significantly (p greater than 0.60) and Km increased with increasing concentrations of nicotine and cotinine (p less than 0.05). Both nicotine and cotinine are competitive inhibitors of HSD in dog prostate microsomes with Ki's of 61 and 89 microM, respectively. The apparent 5 alpha-reductase activity was unaffected by nicotine and cotinine. The inhibitors produced a marked effect on activity of HSD when used in concentrations achieved in humans who smoke cigarettes. The results suggest that nicotine and cotinine are competitive inhibitors of the HSD, an important enzyme involved in the metabolism of DHT and produce an accumulation of DHT. These products of cigarette smoking could alter androgen action in tissue such as skin and prostate.

Isolation and catalytic activity of cytochrome P-450 from ventral prostate of control rats.

5 alpha-Androstane-3 beta, 17 beta-diol hydroxylase (3 beta-diol hydroxylase), a form of cytochrome P-450, was purified from rat ventral prostate, and its regulation as a function of age and 5 alpha-dihydrotestosterone (DHT) treatment was examined. Cytochrome P-450 could be quantitated by its CO difference spectrum only after partial purification from the microsomal membrane, and this was achieved by chromatography on p-chloroamphetamine-coupled Sepharose. Further purification of prostate microsomal P-450 by anion exchange chromatography yielded a preparation with a P-450 content of 8-10 nmol/mg of protein, which upon sodium dodecyl sulfate electrophoresis showed, in the molecular weight region between 50,000 and 60,000 where P-450 is expected to migrate, a single protein band of Mr 54,000. This preparation upon reconstitution with cytochrome P-450 reductase and microsomal lipid catalyzed the formation of three triols, 5 alpha-androstane-3 beta, 7 beta, 17 beta-triol, 5 alpha-androstane-3 beta, 6 alpha, 17 beta-triol, and 5 alpha-androstane-3 beta, 7 alpha, 17 beta-triol from 3 beta-diol in the ratio 1:7:3. Both turnover number and the ratio of the three products in the reconstituted system were similar to that found in prostate microsomes. These data indicate that a single form of P-450 catalyzes the formation of all three triols and that 3 beta-diol hydroxylase is the major, if not the only, form of P-450 in the prostate microsomes of untreated rats. The yield of P-450 from prostate microsomes varied as a function of age from a high level of 0.05 nmol/mg of microsomal protein in 6-week-old rats to 0.002 nmol/mg of microsomal protein in rats 11 weeks or older. 3 beta-Diol hydroxylase activity followed a similar age-related pattern varying between 2,000 and 4,000 nmol of triols formed/g of tissue/h in 6-week-old rats to 100 nmol of triols formed/g of tissue/h in 11-week-old rats. Treatment of 6-week-old rats with DHT did not prevent the age-related decrease in 3 beta-diol hydroxylase activity. However, DHT does play a role in the regulation of this enzyme since castration resulted in a loss of catalytic activity from the prostate and treatment of castrated rats with DHT caused an induction of the enzyme.

Aromatase activity in microsomes from rat ventral prostate and dunning R3327H rat prostatic adenocarcinoma

We have measured aromatase activity in microsomes obtained from rat ventral prostate, using the 3H 2O release method as described by Weisz[1]. Production of 3H 2O from 1β-[ 3H]androstenedione correlated with estrogen production measured by RIA and by TLC. The assay was optimized for incubation time and protein concentration, and used to determine the aromatase activity of ventral prostate microsomes from rats of varying age. Aromatase activity per mg microsomal protein increased from an average of 4 pmol/mg protein × h in 3-month old rats to 68 pmol/mg protein × h in 8-month old rats. Aromatase activity was also measured in microsomes from the Dunning R3327H rat prostatic adenocarcinoma, and was increased in tumors removed 225 days after implantation compared to tumors removed 141 days after implantation. Tumors removed 225 days after implantation from rats which had been treated with DES for 14 days displayed increased aromatase activity compared to untreated tumors. The presence of aromatase activity in the rat ventral prostate and rat prostatic adenocarcinoma would allow regulation of estrogen levels independent of circulating estrogen. Thus, in situ changes in estrogen production with age may contribute to the development of prostatic disease.

Photoaffinity labeling of steroid 5 alpha-reductase of rat liver and prostate microsomes.

21-Diazo-4-methyl-4-aza-5 alpha-pregnane-3,20-dione (Diazo-MAPD) inhibits steroid 5 alpha-reductase in liver microsomes of female rats with a Ki value of 8.7 +/- 1.7 nM, and the inhibition is competitive with testosterone. It also inhibits the binding of a 5 alpha-reductase inhibitor, [3H] 17 beta-N,N-diethylcarbamoyl-4-methyl-4-aza-5 alpha-androstan-3-one ([3H]4-MA), to the enzyme in liver microsomes. The inhibition of 5 alpha-reductase activity and of inhibitor binding activity by diazo-MAPD becomes irreversible upon UV irradiation. [1,2-3H]Diazo-MAPD binds to a single high affinity site (Kd 8 nM, 125 pmol binding sites/mg of protein) in liver microsomes of female rats, and this binding requires NADPH. Without UV irradiation, this binding is reversible, and it becomes irreversible upon UV irradiation. Both the initial reversible binding and the subsequent irreversible conjugation after UV irradiation are inhibited by inhibitors (diazo-MAPD and 4-MA) and substrates (progesterone and testosterone) of 5 alpha-reductase, but they are not inhibited by 5 alpha-reduced steroids (5 alpha-dihydrotestosterone and 5 alpha-androstan-3 alpha, 17 beta-diol). NADPH stimulates the binding of [3H] diazo-MAPD to microsomes of male rat liver and prostate. UV irradiation also induces conjugation of [3H] diazo-MAPD to these microsomes. Photoaffinity labeled liver microsomes of female rats were solubilized and fractionated by high performance gel filtration. The radioactive conjugate eluted in one major peak at Mr 50,000.