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.     

Regulation of testosterone hydroxylation by rat liver microsomal cytochrome P-450

The pathways of testosterone oxidation catalyzed by purified and membrane-bound forms of rat liver microsomal cytochrome P-450 were examined with an HPLC system capable of resolving 14 potential hydroxylated metabolites of testosterone and androstenedione. Seven pathways of testosterone oxidation, namely the 2 alpha-, 2 beta-, 6 beta-, 15 beta-, 16 alpha-, and 18-hydroxylation of testosterone and 17-oxidation to androstenedione, were sexually differentiated in mature rats (male/female = 7-200 fold) but not in immature rats. Developmental changes in two cytochrome P-450 isozymes largely accounted for this sexual differentiation. The selective expression of cytochrome P-450h in mature male rats largely accounted for the male-specific, postpubertal increase in the rate of testosterone 2 alpha-, 16 alpha, and 17-oxidation, whereas the selective repression of cytochrome P-450p in female rats accounted for the female-specific, postpubertal decline in testosterone 2 beta-, 6 beta-, 15 beta-, and 18-hydroxylase activity. A variety of cytochrome P-450p inducers, when administered to mature female rats, markedly increased (up to 130-fold) the rate of testosterone 2 beta-, 6 beta-, 15 beta-, and 18-hydroxylation. These four pathways of testosterone hydroxylation were catalyzed by partially purified cytochrome P-450p, and were selectively stimulated when liver microsomes from troleandomycin- or erythromycin estolate-induced rats were treated with potassium ferricyanide, which dissociates the complex between cytochrome P-450p and these macrolide antibiotics. Just as the testosterone 2 beta-, 6 beta-, 15 beta-, and 18-hydroxylase activity reflected the levels of cytochrome P-450p in rat liver microsomes, so testosterone 7 alpha-hydroxylase activity reflected the levels of cytochrome P-450a; 16 beta-hydroxylase activity the levels of cytochrome P-450b; and 2 alpha-hydroxylase activity the levels of cytochrome P-450h. It is concluded that the regio- and stereoselective hydroxylation of testosterone provides a functional basis to study simultaneously the regulation of several distinct isozymes of rat liver microsomal cytochrome P-450.     

Steroid metabolism by rat nasal mucosa: studies on progesterone and testosterone

The metabolism of [4-14C]progesterone and [4-14C]testosterone by slices of the nasal mucosa from rats was studied. As shown by gas chromatography-mass spectrometry there was a preferential formation of reduced progesterone-metabolites (5 alpha-pregnane-3,20-dione, 3 alpha- and 3 beta-hydroxy-5 alpha-pregnane-20-one, 20 alpha- and 20 beta-hydroxypregn-4-en-3-one, 2 alpha,3 alpha-dihydroxy-5 alpha-pregnane-20-one, 3 alpha,16 alpha-dihydroxy-5 alpha-pregnane-20-one) and reduced testosterone-metabolites (4-androstene-3,17-dione, 5 alpha-dihydrotestosterone, 3 alpha-hydroxy-5 alpha-androstane-17-one, and 5 alpha-androstane-3 alpha, 17 beta-diol, 2 alpha-hydroxy-5 alpha-dihydrotestosterone, 5 alpha-androstane-2 alpha,3 alpha, 17 beta-triol) indicating the presence of 5 alpha-reductase, 3 alpha-, 3 beta-, 17 beta-, 20 alpha- and 20 beta-hydroxysteroid oxidoreductase activities in this tissue. Progesterone-metabolites hydroxylated at positions 2 alpha, 6 alpha, 6 beta, 15 alpha and 16 alpha and testosterone-metabolites hydroxylated at positions 1 beta, 2 alpha, 6 beta, 15 beta and 16 alpha were also identified, indicating the presence of several steroid hydroxylases in the nasal mucosa. Autoradiography of the nasal region of rats injected with [4-14C]progesterone or [4-14C]testosterone showed a selective localization of radioactivity in the mucosa covering the olfactory region of the nasal cavity.     

Testosterone metabolism by cytochrome P-450 isozymes RLM3 and RLM5 and by microsomes. Metabolite identification

Testosterone metabolism by cytochrome P-450 isozymes RLM3 and RLM5 in a reconstituted system and by rat liver microsomes was examined. Eleven metabolites were detected. Two of these, found in spots 2 and 4 of a thin layer plate, were only formed by the rat liver microsomes and may represent reductive metabolites of testosterone. A number of monohydroxy metabolites were conclusively identified by gas chromatography-mass spectrometry. These include the 2-, 6 beta-, 7 alpha-, and 16 alpha-hydroxy isomers. Liver microsomes formed the 2 alpha- and 2 beta-epimers in a 1:2 ratio and both co-chromatographed with a third reduced metabolite in thin layer plate spot 4. In contrast with RLM5 about 90% of the 2-hydroxy isomer was the 2 alpha-epimer. RLM3 did not perform the 2-hydroxylation in detectable amounts. The 6 beta-isomer was a major metabolite of RLM3 and microsomes, but a minor product of metabolism by RLM5. In contrast, the 7 alpha-isomer was a minor metabolite of RLM3, was not formed by RLM5, and was a major microsomal metabolite. Hydroxylation at position 16 alpha was a major activity of RLM5 and the heterogeneous microsomal cytochromes, but with RLM3 it was a minor reaction. One new metabolite was found which appeared to be hydroxylated in the D-ring, had a mass spectrum different from both 16 alpha- and 16 beta-hydroxytestosterone, and was tentatively identified as a 15-hydroxy isomer. In agreement with the literature, androstene-3,17-dione was found to be an oxidative metabolite of testosterone by both microsomes and purified cytochrome P-450. It was a major metabolite of RLM5 but was not produced by RLM3. Studies with 18O2 and H218O conclusively show that oxidation of testosterone at C-17 does not involve transient incorporation of an oxygen atom in this position. A mechanism is suggested whereby cytochrome P-450 acts as a peroxidase in the formation of androstenedione.     

Simultaneous determination of testosterone metabolites in liver microsomes using column-switching semi-microcolumn high-performance liquid chromatography

A sensitive and selective column-switching semi-microcolumn high-performance liquid chromatographic (HPLC) method has been developed for the simultaneous determination of testosterone and eight of its metabolites (6alpha-, 6beta-, 16alpha-, 16beta-, 7alpha-, 2alpha-, and 2beta-hydroxytestosterone, and androstenedione) in liver microsomes. After incubation for 10 min, testosterone and its metabolites were extracted from the microsomes with ethyl acetate, and the extract was evaporated to dryness. The residue was dissolved in the mobile phase and loaded onto the HPLC system. The analytes were first concentrated in a precolumn and subsequently transferred to the analytical column, where they were separated using linear gradient elution. A UV detector set at 254 nm was used to detect the analytes. This newly developed method clearly separated TES and the metabolites with high resolution and was found to be reproducible with intra- and interday variability of <10.7%. This method has been subsequently used to determine the testosterone hydroxylation activities catalyzed by 15 different recombinant CYP isozymes. The results confirmed the formation of stereoselectively hydroxylated metabolites by each CYP isozyme.     

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.     

Catabolism of [4-14C]testosterone by subcellular fractions of human prostate

1. Incubation conditions were established in experiments with human-prostate homogenates for almost complete conversion of [4-(14)C]testosterone into at least ten transformation products. 2. Whole homogenates of tissue with benign hypertrophy were shown to contain 3alpha-, 3beta- and 17beta-hydroxy steroid dehydrogenases, Delta(4)-3-oxo steroid 5alpha- and 5beta- reductases and unidentified hydroxylases. 3. Most of the 17beta-hydroxy steroid-dehydrogenase activity was located in the mitochondria, which showed little other activity. 4. The 3alpha- and 3beta-hydroxy steroid dehydrogenases and the 5beta-reductase were located in the high-speed supernatant and required supplementation with NADPH for activity. 5. The 5alpha-reductase was located in both microsomal and high-speed supernatant fractions and also required supplementation with NADPH.     

Cobalt-protoporphyrin, a synthetic heme analogue, feminizes hepatic androgen metabolism in the rat

A single injection of cobalt-protoporphyrin (50 mumol/kg) produced marked changes in the metabolism of 14C-labeled testosterone and 4-androstenedione by male rat liver microsomes and this effect was maintained for at least 3 weeks. The rate of 3 beta- and 5 alpha-reduction was increased to levels observed in untreated adult female animals and cobalt-protoporphyrin altered the metabolic profile of testosterone towards that observed after infusion of growth hormone whereas hypophysectomy produced a more general inhibition of androgen metabolism. The reduction of testosterone or 4-androstenedione by liver microsomes was also increased when cobalt-protoporphyrin (10-30 microM) was added in vitro but a higher concentration (100 microM) led to inhibition of androgen metabolism. The identity of the main androgen metabolites was established by TLC, HPLC and mass spectrometry and the role of 5 alpha-reductase was demonstrated using a specific inhibitor of this enzyme. The possible sites of action of cobalt-protoporphyrin are discussed in relation to its in vivo effects on serum testosterone and LH concentrations.     

Rapid quantitation of testosterone hydroxyl metabolites by ultra-performance liquid chromatography and mass spectrometry

A rapid and sensitive ultra-performance liquid chromatography and mass spectrometry (UPLC/MS) method was developed to simultaneously quantify seven monohydroxyl testosterone metabolites (16alpha-, 2alpha-, 7alpha-, 6alpha-, 2beta-, 6beta-, and 16beta-hydroxyl testosterones) in rat liver microsomes. The UPLC system used a short 1.7-microm particle size column coupled to a Sciex 4000 Q trap in multiple reaction monitor (MRM) mode. All hydroxyl testosterones were resolved within 2.5 min. A 4-day validation was performed to determine the linearity, repeatability, reproducibility and accuracy of the method in rat liver microsomes. This method is applicable to the measurement of the testosterone hydroxylase activity in biological matrices such as the liver microsome incubates.     

Purification of two isozymes of rat liver microsomal cytochrome P450 with testosterone 7 alpha-hydroxylase activity

Cytochrome P450a was purified to electrophoretic homogeneity from liver microsomes from immature male Long-Evans rats treated with Aroclor 1254. Rabbit polyclonal antibody raised against cytochrome P450a cross-reacted with cytochromes P450b, P450e, and P450f (which are structurally related to cytochrome P450a). The cross-reacting antibodies were removed by passing anti-P450a over an N-octylamino-Sepharose column containing these heterologous antigens. The immunoabsorbed antibody recognized only a single protein (i.e., cytochrome P450a) in liver microsomes from immature male rats treated with Aroclor 1254 (i.e., the microsomes from which cytochrome P450a was purified). However, the immunoabsorbed antibody recognized three proteins in liver microsomes from mature male rats, as determined by Western immunoblot. As expected, one of these proteins (Mr 48,000) corresponded to cytochrome P450a. The other two proteins did not correspond to cytochromes P450b, P450e, or P450f (as might be expected if the antibody were incompletely immunoabsorbed), nor did they correspond to cytochromes P450c, P450d, P450g, P450h, P450i, P450j, P450k, or P450p. One of these proteins was designated cytochrome P450m (Mr approximately 49,000), the other cytochrome P450n (Mr approximately 50,000). Like cytochrome P450a, cytochrome P450n was present in liver microsomes from both male and female rats. However, whereas cytochrome P450a was detectable in liver microsomes from 1-week-old rats, cytochrome P450n was barely detectable until the rats were at least 3 weeks old. Furthermore, in contrast to cytochrome P450a, the levels of cytochrome P450n did not decline appreciably with age in postpubertal male rats. Cytochrome P450m was detectable only in liver microsomes from postpubertal (greater than 4 week-old) male rats. Cytochromes P450m and P450n were isolated from liver microsomes from mature male rats and purified to remove cytochrome P450a. When reconstituted with NADPH-cytochrome P450 reductase and lipid, cytochrome P450n exhibited little testosterone hydroxylase activity, whereas cytochrome P450m catalyzed the 15 alpha-, 18-, 6 beta-, and 7 alpha-hydroxylations of testosterone at 10.8, 4.6, 2.0, and 1.9 nmol/nmol P450/min, respectively. The ability of cytochrome P450m to catalyze the 7 alpha-hydroxylation of testosterone was not due to contamination with cytochrome P450a, which catalyzed this reaction at approximately 25 nmol/nmol P450a/min. Cytochrome P450m also converted testosterone to several minor metabolites, including androstenedione and 15 beta-, 14 alpha-, and 16 alpha-hydroxytestosterone.(ABSTRACT TRUNCATED AT 400 WORDS)     

Regioselectivity and stereoselectivity of androgen hydroxylations catalyzed by cytochrome P-450 isozymes purified from phenobarbital-induced rat liver

The regioselectivity and stereoselectivity of androgen hydroxylations catalyzed by five isozymes of cytochrome P-450 purified from phenobarbital-induced rat liver were studied in a reconstituted monooxygenase system using testosterone (T) and androst-4-ene-3,17-dione (delta 4-A) as substrates. P-450 PB-3, an isozyme exhibiting low catalytic activity with many xenobiotic substrates, catalyzed efficient (turnover = 15.7 to 18.5 min-1 P-450-1 at 25 microM substrate) and highly stereoselective B-ring hydroxylations of both steroid substrates, with the corresponding 7 alpha- and 6 alpha-hydroxy alcohols formed in ratios of approximately 20 to 30:1, respectively. P-450 PB-2c metabolized testosterone to a mixture of 16 alpha OH-T, 2 alpha OH-T, and delta 4-A (product ratio = 1.0/0.78/0.33; turnover = 10.2 min-1 P-450-1). PB-2c is present in significantly larger amounts in mature male rats as compared to immature males, and probably catalyzes the male-specific testosterone 16 alpha-hydroxylase activity known to be induced at puberty and subject to endocrine control. P-450 PB-4, the major phenobarbital-induced isozyme in rat liver, catalyzed efficient D-ring hydroxylations, yielding 16 beta OH- delta 4-A as the predominant product with delta 4-A as substrate (turnover = 12.0 min-1 P-450-1) and a mixture of 16 beta OH-T, 16 alpha OH-T, and delta 4-A (the latter compound presumably formed via 17 alpha hydroxylation) with testosterone as substrate (turnover = 5.2 min-1 P-450-1). P-450 isozymes PB-1 and PB-5 hydroxylated both steroids with essentially the same regioselectivity as PB-4 but at only 5 to 10% the catalytic rate. Cytochrome b5 stimulated most of these steroid hydroxylations up to 2-fold with no change in regio- or stereoselectivity. The identification of specific steroid metabolites as diagnostic of particular P-450 isozymes should be useful for the assessment of isozymic contributions to microsomal activities and, in addition, facilitate comparisons of P-450 isozymes isolated in different laboratories.     

Studies on rat liver microsomal steroid metabolism using 18O-labelled testosterone and progesterone

In order to investigate the possible involvement of oxygen functions in the rat liver microsomal metabolism of progesterone and testosterone these steroids were specifically labelled with 18O in their oxo-functions and incubated with NADPH supplemented 105,000 g sediments. Gas chromatography-mass spectrometry was used to identify the metabolites formed as well as to quantitate the losses of 18O-label. With 18O-labelled testosterone as substrate two of the major monohydroxylated metabolites, i.e. 2 beta- and 6 beta-hydroxytestosterone were shown to have lost about 25 and 50% of their 18O respectively. A complete retention of label was found in 7 alpha- and 16 alpha-hydroxytestosterone. None of the monohydroxylated progesterone metabolites, i.e. the 2 alpha-, 6 beta- and 16 alpha-hydroxyprogesterone had lost any 18O following incubation with 3,20-18O-labelled progesterone. Control incubation (30', 37 degrees C) with buffer and 18O-labelled progesterone and testosterone revealed no exchange of 18O. Thus the partial loss of 3-18O-label during 2 beta- and 6 beta-hydroxylation of testosterone may indicate a covalent interaction between the steroid 3-oxo-group and one or more cytochrome P-450 species in the rat liver microsomes. In view of the potentiating effect of a 3-imine group in spontaneous 6 beta-hydroxylation the present in vitro data suggest that a steroid protein-interaction may occur via a 3-imine group during 6 beta-hydroxylation of testosterone in rat liver microsomes. Analysis of 5 alpha-reduced metabolites of both progesterone and testosterone showed significant losses of 3-18O, but due to the ease with which 3-oxo-5 alpha-steroids exchange their 3-18O with aqueous media an enzymatically induced loss of 3-18O could not be safely established. The 20-oxido-reductase which converted progesterone did not induce a loss of 20- or 3-18O thus indicating that the oxofunctions were not covalently engaged in the enzymatic binding of the steroid.     

Properties and biochemical characterization of NADH 5 alpha-reductase from rat liver microsomes

NADH 5 alpha-reductase is present in microsomes of various rat organs: heart and skeletal muscle, liver, adrenal glands, kidney, testes and prostate. The enzyme from rat liver microsomes utilizes B-hydrogen from the coenzyme NADH for steroid reduction. After solubilization of the enzyme with the nonionic detergent lubrol, phosphatidylcholine is necessary to restore the activity. This reactivation of the enzyme activity is paralleled by a corresponding increase of Vmax for testosterone (17 beta-hydroxy-4-androsten-3-one). Km and Vmax for testosterone change, Km and Vmax for the coenzyme NADH remain constant with an alteration of phosphate concentration in the incubation medium. The NADH 5 alpha-reductase is inhibited by numerous substances: amytal, phenobarbital, mepacrin, thenoyltrifluoracetone, gallic acid propyl ester, dicoumarol, pentachlorophenol, NADP and antibodies against rat liver NADPH ferrihemoprotein reductase. Antibodies against rat liver cytochrome-b5 reductase cause an activation of NADH 5 alpha-reductase.     

Pronounced and differential effects of ionic strength and pH on testosterone oxidation by membrane-bound and purified forms of rat liver microsomal cytochrome P-450

The aim of this study was to determine the effects of ionic strength and pH on the different pathways of testosterone oxidation catalyzed by rat liver microsomes. The catalytic activity of cytochromes P-450a (IIA1), P-450b (IIB1), P-450h (IIC11) and P-450p (IIIA1) was measured in liver microsomes from mature male rats and phenobarbital-treated rats as testosterone 7 alpha-, 16 beta-, 2 alpha- and 6 beta-hydroxylase activity, respectively. An increase in the concentration of potassium phosphate (from 25 to 250 mM) caused a marked decrease in the catalytic activity of cytochromes P-450a (to 8%), P-450b (to 22%) and P-450h (to 23%), but caused a pronounced increase in the catalytic activity of cytochrome P-450p (up to 4.2-fold). These effects were attributed to changes in ionic strength, because similar but less pronounced effects were observed with Tris-HCl (which has approximately 1/3 the ionic strength of phosphate buffer at pH 7.4). Testosterone oxidation by microsomal cytochromes P-450a, P-450b, P-450h and P-450p was also differentially affected by pH (over the range 6.8-8.0). The pH optima ranged from 7.1 (for P-450a and P-450h) to 8.0 (for P-450p), with an intermediate value of 7.4 for cytochrome P-450b. Increasing the pH from 6.8 to 8.0 unexpectedly altered the relative amounts of the 3 major metabolites produced by cytochrome P-450h. The decline in testosterone oxidation by cytochromes P-450a, P-450b and P-450h that accompanied an increase in ionic strength or pH could be duplicated in reconstitution systems containing purified P-450a, P-450b or P-450h, equimolar amounts of NADPH-cytochrome P-450 reductase and optimal amounts of dilauroylphosphatidylcholine. This result indicated that the decline in testosterone oxidation by cytochromes P-450a, P-450b and P-450h was a direct effect of ionic strength and pH on these enzymes, rather than a secondary effect related to the increase in testosterone oxidation by cytochrome P-450p. Similar studies with purified cytochrome P-450p were complicated by the atypical conditions needed to reconstitute this enzyme. However, studies on the conversion of digitoxin to digitoxigenin bisdigitoxoside by liver microsomes, which is catalyzed specifically by cytochrome P-450p, provided indirect evidence that the increase in catalytic activity of cytochrome P-450p was also a direct effect of ionic strength and pH on this enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)     

Mechanistic studies with solubilized rat liver steroid 5 alpha-reductase: elucidation of the kinetic mechanism

A solubilized preparation of steroid 5 alpha-reductase (EC 1.3.1.30) from rat liver has been used in studies focused toward an understanding of the kinetic mechanism associated with enzyme catalysis. From the results of analyses with product and dead-end inhibitors, a preferentially ordered binding of substrates and release of products from the surface of the enzyme is proposed. The observations from these experiments were identical with those using the steroid 5 alpha-reductase activity associated with rat liver microsomes. The primary isotope effects on steady-state kinetic parameters when [4S-2H]NADPH was used also were consistent with an ordered kinetic mechanism. Normal isotope effects were observed for all three kinetic parameters (Vm/Km for both testosterone and NADPH and Vm) at all substrate concentrations used experimentally. Upon extrapolation to infinite concentration of testosterone, the isotope effect on Vm/Km for NADPH approached unity, indicating that the nicotinamide dinucleotide phosphate is the first substrate binding to and the second product released from the enzyme. The isotope effects on Vm/Km for testosterone at infinite concentration of cofactor and on Vm were 3.8 +/- 0.5 and 3.3 +/- 0.4, respectively. Data from the pH profiles of these three steady-state parameters and the inhibition constants (1/Ki) of competitive inhibitors versus both substrates indicate that the binding of nicotinamide dinucleotide phosphate involves coordination of its anionic 2'-phosphate to a protonated enzyme-associated base with an apparent pK near 8.0. From these results, relative limits have been placed on several of the internal rate constants used to describe the ordered mechanism of the rat liver steroid 5 alpha-reductase.     

5 Alpha-reductase inhibitory and anti-androgenic activities of some 4-azasteroids in the rat

Inhibition of 5 alpha-reductase and anti-androgenicity were studied in rats treated with various 4-azasteroids. The known inhibitor, N,N-diethyl-4-methyl-3-oxo-4-aza-5 alpha-androstane-17 beta-carboxamide (4-MA) served as a reference compound, and analogs of this basic molecule were assayed. Enhancement of enzyme inhibitory potency was usually seen with delta 1 analogs, whereas reduction in activity was noted with substitutuents such as delta 5, a spirotetrahydrofuran ring at C-17 or 4-deaza groups. Many of the 4-azasteroids had a much greater oral anti-androgenic effect against testosterone propionate (TP) than dihydrotestosterone propionate (DHTP). This difference in activity versus the two androgens is believed to reflect the necessity for TP to undergo reduction to DHT before becoming capable of stimulating prostatic growth. Inhibition of 5 alpha-reductase by active compounds prevented the conversion, thereby producing an anti-androgenic effect. In this regard, certain delta 1 analogs of 4-MA, particularly those bearing a 17 beta-(N-tert butylcarbamoyl) group, proved very effective against TP but were relatively inactive versus DHTP.     

Human liver microsomal steroid metabolism: identification of the major microsomal steroid hormone 6 beta-hydroxylase cytochrome P-450 enzyme

Cytochrome P-450-dependent steroid hormone metabolism was studied in isolated human liver microsomal fractions. 6 beta hydroxylation was shown to be the major route of NADPH-dependent oxidative metabolism (greater than or equal to 75% of total hydroxylated metabolites) with each of three steroid substrates, testosterone, androstenedione, and progesterone. With testosterone, 2 beta and 15 beta hydroxylation also occurred, proceeding at approximately 10% and 3-4% the rate of microsomal 6 beta hydroxylation, respectively, in each of the liver samples examined. Rates for the three steroid 6 beta-hydroxylase activities were highly correlated with each other (r = 0.95-0.97 for 25 individual microsomal preparations), suggesting that a single human liver P-450 enzyme is the principal microsomal 6 beta-hydroxylase catalyst with all three steroid substrates. Steroid 6 beta-hydroxylase rates correlated well with the specific content of human P-450NF (r = 0.69-0.83) and with its associated nifedipine oxidase activity (r = 0.80), but not with the rates for debrisoquine 4-hydroxylase, phenacetin O-deethylase, or S-mephenytoin 4-hydroxylase activities or the specific contents of their respective associated P-450 forms in these same liver microsomes (r less than 0.2). These correlative observations were supported by the selective inhibition of human liver microsomal 6 beta hydroxylation by antibody raised to either human P-450NF or a rat homolog, P-450 PB-2a. Anti-P-450NF also inhibited human microsomal testosterone 2 beta and 15 beta hydroxylation in parallel to the 6 beta-hydroxylation reaction. This antibody also inhibited rat P-450 2a-dependent steroid hormone 6 beta hydroxylation in uninduced adult male rat liver microsomes but not the steroid 2 alpha, 16 alpha, or 7 alpha hydroxylation reactions catalyzed by other rat P-450 forms. Finally, steroid 6 beta hydroxylation catalyzed by either human or rat liver microsomes was selectively inhibited by NADPH-dependent complexation of the macrolide antibiotic triacetyloleandomycin, a reaction that is characteristic of members of the P-450NF gene subfamily (P-450 IIIA subfamily). These observations establish that P-450NF or a closely related enzyme is the major catalyst of steroid hormone 6 beta hydroxylation in human liver microsomes, and furthermore suggest that steroid 6 beta hydroxylation may provide a useful, noninvasive monitor for the monooxygenase activity of this hepatic P-450 form.     

Neonatal hormone manipulations and the maintenance of perineal muscles and their motor neurones in Albino Swiss rats

Newborn female Albino Swiss rats received testosterone propionate, dihydrotestosterone benzoate or oestradiol benzoate for 4 days after birth. The neonatal administration of all three hormones maintained neurones of the spinal nucleus of bulbocavernosus (SNB) complex in adulthood at levels intermediate between those found in normal females (approximately 40 neurones) and those found in normal males (approximately 220 neurones). Dihydrotestosterone benzoate was the most effective treatment. Oestradiol benzoate, while as potent as testosterone propionate in maintaining SNB neurone numbers, could not maintain the perineal muscles which are their normal target. Dihydrotestosterone benzoate and testosterone propionate maintained both neurones and muscles. Newborn male Albino Swiss rats received either the aromatase inhibitor 4-OH-androstenedione, or the 5 alpha-reductase inhibitor aza-steroid 17 beta-N,N-diethylcarbamoyl-4-methyl-4-aza-5 alpha-androstan-3-one(4-MA). Only neonatal treatment with 4-MA led to reduced SNB neurone numbers in adulthood, but the reduction was modest (-16%). The results of the two experiments suggest that several hormones can maintain SNB neurone numbers in Albino Swiss rats, but that 5 alpha-reduced metabolites of testosterone may be particularly effective.     

Regulation of two electrophoretically distinct proteins recognized by antibody against rat liver cytochrome P450 3A1.

We recently reported that antibody against purified P450 3A1 (P450p) recognizes two electrophoretically distinct proteins (50 and 51 kDa) in liver microsomes from male and female rats, as determined by Western immunoblotting. Depending on the source of the liver microsomes, the 51-kDa protein corresponded to 3A1 and/or 3A2 which could not be resolved by sodium dodecyl sulfate (SDS)polyacrylamide gel electrophoresis. The other protein (50 kDa) appears to be another member of the P450 IIIA gene family. Both proteins were markedly intensified in liver microsomes from male or female rats treated with pregnenolone-16 alpha-carbonitrile, dexamethasone, troleandomycin, or chlordane. In contrast, treatment of male or female rats with phenobarbital intensified only the 51-kDa protein. Treatment of male rats with Aroclor 1254 induced the 51-kDa protein, but suppressed the 50-kDa form. In addition to their changes in response to inducers, the 50- and 51-kDa proteins also differed in their developmental expression. For example, the 50-kDa protein was not expressed until weaning (3 weeks), whereas the 51-kDa protein was expressed even in 1-week-old rats. At puberty (between weeks 5 and 6), the levels of the 50-kDa and 51-kDa proteins markedly declined in female but not in male rats, which introduced a large sex difference (male greater than female) in the levels of both proteins. Changes in the level of the 51-kDa protein were paralleled by changes in the rate of testosterone 2 beta-, 6 beta-, and 15 beta-hydroxylation. In male rats, the marked increase in the levels of the 50-kDa protein between weeks 2 and 3 coincided with a three- to four fold increase in the rate of testosterone 2 beta-, 6 beta-, and 15 beta-hydroxylation, which suggests that the 50-kDa protein catalyzes the same pathways of testosterone oxidation as the 51-kDa protein. However, this developmental increase in testosterone oxidation may have resulted from an activation of the 51-kDa 3A protein. These results indicate that the two electrophoretically distinct proteins recognized by antibody against P450 3A1 are regulated in a similar but not identical manner, and suggest that the 51-kDa 3A protein is the major microsomal enzyme responsible for catalyzing the 2 beta-, 6 beta-, and 15 beta-hydroxylation of testosterone.     

The effects of diethyl-4-methyl-3-oxo-4-aza-5 alpha-androstane-17 beta-carboxamide (4-MA) and (4R)-5,10-SECO-19-norpregna-4,5-diene-3,10,20-trione (SECO) on androgen biosynthesis in the rat testis and epididymis

We have investigated the effects of two 4-ene-steroid 5 alpha-reductase inhibitors, diethyl-4-methyl-3-oxo-4-aza-5 alpha-androstane-17 beta-carboxamide (4-MA) and (4R)-5,10-seco-19-norpregna-4, 5-diene-3,10,20-trione (SECO), on testicular and epididymal androgen biosynthesis. Kinetic analyses revealed that both compounds inhibited epididymal DHT biosynthesis. 4-MA was a competitive inhibitor of epididymal nuclear and microsomal 4-ene-steroid 5 alpha-reductases (3-oxo-5 alpha-steroid: NADP 4-ene-oxidoreductase EC 1.3.1.22) with Kiapp values of 12.8 and 15.1 nmol/l compared to the respective Kmapp values of 185 and 240 nmol/l. Values for the Vmaxapp were always within 70-130% of the control. SECO at 1.0 mumol/l, also inhibited epididymal nuclear and microsomal 4-ene-steroid-5 alpha-reductases, causing respectively 2.9 and 5.2-fold increases in Kmapp. The Vmaxapp values were unchanged. However, SECO concentrations of 5 and 25 mumol/l abolished 4-ene-steroid 5 alpha-reductase activity at all testosterone concentrations. To examine the specificity of these compounds, we investigated their effects on the enzymes that convert pregnenolone to testosterone. Rat testis microsomes converted pregnenolone to testosterone via the 4-ene-3-oxo pathway, with the major metabolites being progesterone, 17-hydroxyprogesterone, 4-androstenedione and testosterone; some 17-hydroxypregnenolone was also formed. Very small amounts of dehydroepiandrosterone (DHA) and 5-androstenediol were detected. SECO, at a concentration that completely inhibited epididymal 4-ene-steroid 5 alpha-reductase activity, did not alter the metabolic profile of pregnenolone metabolism. However, 4-MA prevented the appearance of 4-ene steroids, and large quantities of 17-hydroxypregnenolone and DHA accumulated, suggesting that inhibition of the 3 beta-hydroxysteroid: NAD(P)+ oxidoreductase (EC 1.1.1.51) and 3-oxosteroid 5-ene-4-ene-isomerase (EC 5.3.3.1) [3 beta-hydroxysteroid dehydrogenase-isomerase] was occurring. Optimal conditions for the microsomal conversion of DHA to 4-androstenedione were determined; kinetic analyses of the 3 beta-hydroxysteroid dehydrogenase-isomerase activity revealed that 4-MA inhibited this reaction non-competitively, reducing Vmaxapp values to 25% of the control. The Kiapp determined from the intercept replot, was 121 nmol/l, and the Kmapp was always between 90 and 130% of the control value. It is concluded that SECO is more specific than 4-MA in its effects on androgen biosynthesis in the testis and epididymis and that both these drugs should provide useful tools in assessments of the relative contributions of 5 alpha-reduced androgens to androgen dependent processes.