Microbial transformation of 17α-cyanomethyl-17-hydroxy-4,9-estradien-3-one (STS 557) and 17α-cyanomethyl-19-nortestosterone by Mycobacterium smegmatis

Microbial transformation of the new progestagen STS 557 (17α-cyanomethyl-17-hydroxy-4,9-estradien-3-one) by $\text{Mycobacterium smegmatis}$ yielded predominantly ring A-aromatized compounds: 17α-cyanomethyl-1,3,5(10),9(11)-estratetraene-3, 17-diol, 17α-cyanomethyl-1,3,5(10)-estratriene-3, 17-diol and the corresponding 3-methyl ethers. The analogous compound without the 9(10) double bond, 17α-cyanomethyl-19-nortestosterone, was transformed mainly to 5α-hydrogenated metabolites: 17α-cyanomethyl-17-hydroxy-5α-estran-3-one, 17α-cyanomethyl-17-hydroxy-5α-1-estren-3-one, 17α-cyanomethyl-5α-estrane-3α, 17-diol, and 17α-cyanomethyl-5α-estrane-3β, 17-diol. From these results, it is concluded that 4,9-dien-3-oxo compounds are not substrates for enzymatic 5α-hydrogenation.     

Metabolism of 17β-hydroxy-2-hydroxymethylene-5α-androstan-3-one in the rabbit

An acidic metabolite, 2α-carboxy-5α-androstane-3α, 16α, 17αtriol and two neutral metabolites, 2α-hydroxymethyl-5α-androstane-3α, 17α-diol, and 2α-hydroxymethyl-5α-androstane-3α, 16α, 17α-triol have been identified in the urine of rabbits orally dosed with 17β-hydroxy-2-hydroxymethylene-5α-androstan-3-one. 2α-Hydroxymethyl-5α-androstane-3α, 16α, 17α-triol was previously obtained from the urine of rabbits dosed with 17β-hydroxy-2α-methyl-5α-androstan-3-one. The acidic metabolite was the major urinary excretion product.     

In vitro metabolism of testosterone to 17ß-hydroxy-5α-androstane-3-one and 5α-androstane-3α, 17ß-diol by the rat intestine

The metabolism of testosterone to 17ß-hydroxy-5α-androstane-3-one and 5α-androstane-3α, 17ß-diol by the 800 g supernatant fraction by different parts of the gastrointestinal tract from male rats was investigated. This metabolism tended to be higher in immature than in mature animals. Administration of dexamethasone or long-acting ACTH to immature and mature rats increased testosterone metabolism to 17ß-hydroxy-5α-androstane-3-one and 5α-androstane-3α, 17ß-diol by ileum tissue. No such effect could be observed following administration of progesterone, estradiol, prolactin, LH or FSH in mature animals. Development of the gastrointestinal tract from the immature to themmature stage was associated with augmented metabolism of testosterone to 17ß-hydroxy-5α-androstane-3-one and 5α-androstane-3α,17ß-diol in the ileum.     

A direct radioiodination technique for the radioimmunoassay of 17α-ethynyl, 17β-hydroxy-4-estren-3-one

A new technique is described for the direct labelling of norethisterone with¹²⁵I for use in radioimmunoassay. This method allows the preparation of tracers possessing both high specific activity and unaltered immunological properties. The steroid is radioiodinated in the presence of H₂O₂ using acetic acid as the solvent, in a sealed vial heated at 100°C. The reaction mixture is then chromatographed on bidimensional t.l.c. and six major radioactive spots are separated; only one of these fractions possess immunoreactivity. When submitted to repeated t.l.c. it behaves as a single compound, possibly a monoiodinated derivative, substituted in Ring A.The new tracer was compared in RIA with both the tyrosine-iodinated and the tritiated tracers. The standard inhibition curve obtained with the new labelled ligand had the same slope as that obtained using the tritiated one; on the other hand the slope of the curve obtained with the tyrosine iodinated derivative was slightly higher than that of the directly iodinated one.The results obtained indicate that the possibility exists of radioiodinating steroids without losing their immunoreactivity.     

Synthesis of 5α-Androstane-3α,16α,17β-triol from 3β-hydroxy-5-androsten-17-one

5α-Androstane-3α, 16α 17β-triol was synthesized from 3β-hy-droxy-5-androsten-17-one. The procedure Involved catalytic hydrogenation of 3β-hydroxy-5-androsten-17-one to 3β-hydroxy-5α-androstan-17-one. This was followed by conversion of the 3β-hydroxy group to 3α-benzoyloxy group by the Mitsunobu reaction. Further treatment with isopropenyl acetate yielded 5α-androsten-16-ene-3α, 17-diol 3-benzoate 17-acetate. This was then converted to 3α, 17-dihydroxy-5α-androstan-16-one 3-benzoate 17-acetate via the unstable epoxide intermediate after treatment with $\text{m}$-cloroperoxybenzoic acid. LiAlH₄ reduction of this compound formed 5α-androstane-3α, 16α, 17β-trlol. ¹H and ¹³C NMR of various steroids are presented to confirm the structure of this compound.     

Metabolism of 17β-hydroxy-2α,3α-cyclopropano-5α-androstane in the rabbit

The neutral urinary excretion products of 17β-hydroxy-2α,3α-cyclopropano-5α-androstane from the rabbit, dosed orally, were investigated. Column chromatography yielded five crystalline metabolites which were identified by GLC and spectroscopic measurements. Three of these substances were hydroxylated in the 4α-position and one in the 6a-position with the cyclopropane ring intact. The fifth substance, 17β-hydroxy-3β-methyl-5α-androstan-2-one, can be derived from initial hydroxylation of the cyclopropane ring at C-2 followed by ring opening. The dosed substance and triol material was shown to be present by GLC and m.s. measurements. GLC determinations show that hydroxylation has occurred at C-4?C-6>C-2.     

Metabolism of 3β-hydroxy-5α- and 3β-hydroxy-5β-pregnan-20-one by leaf homogenates

Leaf homogenates of Cheiranthus cheiri, Nerium oleander, Strophanthus kombé, Digitalis purpurea, and Corchorus capsularis were ex     

5α-reduction of an anti-androgen TSAA-291, 16β-ethyl-17β-hydroxy-4-estren-3-one,by nuclear 5α-reductase in rat prostates

Inhibition of 5α-reduction of testosterone by an anti-androgen TSAA-291 (16β-ethyl-17β-hydroxy-4-estren-3-one) was studied in rat ventral prostates and the metabolic conversion of ³H-TSAA-291 was examined both $\text{in vitro}$ and $\text{in vivo}$. In the $\text{in vitro}$ experiment using nuclear 5α-reductase of the prostate, 5α-dihydrotestosterone formation from ³H-testosterone was inhibited in a competitive manner by the anti-androgen. In the $\text{in vitro}$ experiment using ³H-TSAA-291, 5α-reduction of the anti-androgen occurred. One, 2 and 4 hr after an intravenous administration of 140 μCi/rat of ³H-TSAA-291 to castrated rats, the unchanged TSAA-291 accumulated in higher amounts in the ventral prostate than in the plasma, skeletal muscle and levator ani muscle, thereby indicating the selective uptake of the anti-androgen by the androgen target organ. No appreciable amounts of the 5α-reduced metabolite of TSAA-291 were detected in the prostate, thus suggesting that TSAA-291 itself may be responsible for the anti-androgenic properties. The inhibitory potency on the 5α-reductase activity of several other 16β-substituted androstane and estrane analogues was also examined.     

The crystal and molecular structure of d1-17β-hydroxy-8α-androst-4-en-3-one (8-isotestosterone)

The molecular conformation of d1-8-isotestosterone has been determined crystallographically. Crystals of the title compound belong to the space group P2₁/c with a = 11.449(4), b = 10.962(4), c = 25.860(5) , β = 100.95(4)⁰, with two molecules in the asymmetric unit. The structure has been refined to a final R value of 0.052 for 2227 reflections. Unlike testosterone, which is a flat molecule, its 8-isomer has a folded conformation. The conformations of the ring-B in the two crystallographically independent molecules (A and B) correspond to the twist form and differ significantly from one another.     

Synthesis of (4-C)-labeled and non-labeled 3β,15α-dihydroxy-5-pregnen-20-one and 3β,15α-dihydroxy-5-androsten-17-one

The synthesis of labeled and non-labeled 3β,15α-dihydroxy-5-pregnen-20-one (V) and 3β, 15α-dihydroxy-5-androsten-17-one (XI) is described. Treatment of 15α-hydroxy-4-pregnene-3,20-dione (I) with acetic anhydride and acetyl chloride gave 3,15α-diacetoxy-3,5-pregnadien-20-one (II). The enol acetate (II) was ketalized by a modification of the general procedure to yield 3,15α-diacetoxy-3,5-pregnadien-20-one cyclic ethylene ketal (III) which was then reduced with NaBH₄ and LiAlH₄ to give 3β, 15α-dihydroxy-5-pregnen-20-one cyclic ethylene ketal (IV). Cleavage of the ketal group of IV gave V. Similarly, XI was prepared by starting with 15α-hydroxy-4-androstene-3,17-dione (VII). The (4-¹⁴C)-3β,15α-dihydroxy-5-pregnen-20-one was prepared by a modification of the above procedure in that the enol acetate (II)was directly reduced with NaBH₄ and LiAlH₄ to yield 5-pregnene-3β,15α,20β-triol (XIII) which was then oxidized enzymatically with 20β-hydroxysteroid dehydrogenase to V.     

The fate of 2,4,6-tri-(3′,5′-di-tert.-butyl-4′- hydroxybenzyl)mesitylene (Ionox 330) in the dog and rat

1. Unchanged Ionox 330 is quantitatively eliminated in the faeces of dogs, rats and man after oral administration, and ¹⁴C is absent from the urine and expired gases of rats intubated with [¹⁴C]Ionox 330. Dogs and rats do not show a sex difference in this pattern of elimination. 2. Quantitative elimination of [¹⁴C]Ionox 330 and the absence of ¹⁴C in the carcass and viscera of rats 72hr. after dosage show that this substance does not accumulate in the body. 3. No metabolites are formed in consequence of the ingestion of Ionox 330. 4. Rats eliminate three-quarters or more of a dose (285·7mg./kg. body wt.) of Ionox 330 in 24hr. and the remainder during 24–48hr., and dogs eliminate the whole dose (90mg./kg. body wt.) within 48hr. and a variable proportion within 24hr. These rates of elimination are consistent with the passage of unabsorbed material through the alimentary canal. 5. After removal of the alimentary canal, radioactivity is absent from the carcass and remaining viscera of rats 8, 16 and 24hr. after ingestion of [¹⁴C]Ionox 330, and this strongly suggests the absence of alimentary absorption. 6. The absence of ¹⁴C in the 24hr. bile of animals with biliary fistulae establishes that [¹⁴C]Ionox 330 is not absorbed from the gastro-intestinal tract.     

The time-dependent inactivation of aromatase by 17β-hydroxy-10-methylthioestra-1,4-dien-3-one

17β-Hydroxy-10-methylthioestra-1,4-dien-3-one is an active-site irreversible inhibitor of aromatase, the cytochrome P-450 dependent enzyme responsible for the conversion of androst-4-ene-3,17-dione to estrone. Two time-dependent pathways to inactivation are observed, one of which requires NADPH activation.     

Purification and properties of a nad: 3α-hydroxy-5α-pregnan-20-one-oxidoreductase from rat liver microsomes

From rat liver microsorties a NAD: 3α-hydroxy-5α-pregnan-20-one oxidoreductase was isolated and purified up to a specific activity of 73 nmol/min.mg by affinity chromatography and DEAE-cellulose chromatography. Various Km-values have been determined. The enzyme exhibits highest affinity for 5α-pregnane-3,20-dione and NADH. The 3-oxo group of 5α-dihydrocortisone (17, 21-dihydroxy-5α-pregnane-3,11,20-trione) was not reduced by the purified enzyme preparation and NADH and no dehydrogenation with NAD was observed of 3α, 11β, 17, 21-tetrahydroxy-5α-pregnan-20-one. The optimal pH for the hydrogenation of the 3-oxo group was at pH 5.3 and for the dehydrogenation at pH 8.9. Disc gel electrophoresis in presence of 0.1% sodium dodecylsulfate yielded a homogeneous preparation.     

The metabolism of 2-chloro-1-(2′,4′-dichlorophenyl)vinyl diethyl phosphate (Chlorfenvinphos) in the dog and rat

1. A single oral dose of [(14)C]Chlorfenvinphos to rats is quantitatively eliminated in 4 days. Rats do not show a sex difference in the elimination pattern and show only a small degree of biological variation in the total excretion data. Of the label 87.2% is excreted in the urine (67.5% in the first day after dosage), 11.2% in the faeces and 1.4% in the expired gases; less than 0.9% of (14)C is present in the gut and contents after 4 days. 2. After oral administration of [(14)C]Chlorfenvinphos to dogs, 94.0% (91.8-97.6%) of the (14)C is excreted in the urine and faeces during 4 days. Dogs do not show a sex difference in the pattern of elimination, and excretion of radioactivity in the urine is very rapid: 86.0% of (14)C during 0-24hr. 3. Chlorfenvinphos is completely metabolized in rats and dogs: unchanged Chlorfenvinphos is absent from the urine and from the carcass, when elimination is complete. In rats, 2-chloro-1-(2',4'-dichlorophenyl)vinyl ethyl hydrogen phosphate accounts for 32.3% of a dose of Chlorfenvinphos, [1-(2',4'-dichlorophenyl)ethyl beta-d-glucopyranosid]uronic acid for 41.0%, 2,4-dichloromandelic acid for 7.0%, 2,4-dichlorophenylethanediol glucuronide for 2.6% and 2,4-dichlorohippuric acid for 4.3%; in dogs, 2-chloro-1-(2',4'-dichlorophenyl)vinyl ethyl hydrogen phosphate accounts for 69.6%, [1-(2',4'-dichlorophenyl)ethyl beta-d-glucopyranosid] uronic acid for 3.6%, 2,4-dichloromandelic acid for 13.4% and 2,4-dichlorophenylethanediol glucuronide for 2.7%. 4. Dogs and rats show a species difference in the rate of excretion of (14)C in the urine, and in the proportions of the metabolites, with the exception of 2,4-dichlorophenylethanediol glucuronide, that are excreted in the urine. Alternative explanations for the latter species difference are suggested. 5. 2-Chloro-1-(2',4'-dichlorophenyl)vinyl ethyl hydrogen phosphate and 2,4-dichlorophenacyl chloride probably lie on the main metabolic pathway of Chlorfenvinphos, since, in common with that insecticide, they give rise to [1-(2',4'-dichlorophenyl)ethyl beta-d-glucopyranosid]uronic acid and 2,4-dichloromandelic acid as major metabolites in the urine. 6. The proposed scheme for the metabolism of Chlorfenvinphos represents a detoxication mechanism.     

Radioimmunoassay of 17β-hydroxy-5α-androstan-3-one, 4-androstene-3,17-dione,dehydroepiandrosterone, 17-hydroxyprogesterone and progesterone and its application to human male plasma

RIA methods for the measurement of 5α-dihydrotestosterone (DHT), androstenedione (Δ4), dehydro-epiandrosterone (DHEA), 17-hydroxyprogesterone (17-OHP) and progesterone (P) have been developed and applied to the study of factors determining the levels of these steroids in males.It has been shown that DHT, 17-OHP and DHEA levels are lower in elderly males than in young (<50 years) subjects, Δ4 and P levels remaining constant. All steroids, with the exception of DHT, show significant nyctohemeral variations: upon ACTH stimulation T levels decrease, whereas Δ4 and DHEA, 17-OHP and P increase significantly. Dexamethasone has the reverse effect, although T levels do not decrease. HCG stimulation results in a significant increase of T, DHT, 17-OHP and Δ4 levels, whereas DHEA and P levels are hardly influenced. In a small group of orchidectomized males, all steroids studied, with the exception of P were significantly lower than in normal males of similar age.It is concluded that in males T, DHT and 17-OHP have an almost exclusive testicular origin; Δ4 has a mixed testicular and adrenal origin, whereas DHEA has a predominant and P an exclusive adrenal origin in males.     

Development of hepatic microsomal activity of 3-hydroxy-3-methylglutaryl-coenzyme a reductase and cholesterol 7α-hydroxylase in the young chick

• 1.1. The 3-hydroxy-3-methylglutaryl-CoA reductase activity increased from 1 to 4 weeks of age, but decreased from 4 to 8 weeks of age.
• 2.2. Cholesterol 7α-hydroxylase activity increased from 1 to 4 weeks, decreased from 4 to 6 weeks, and increased again from 6 to 8 weeks of age.
• 3.3. Serum total and free cholesterol concentrations decreased from 1 to 6 weeks of age, but increased from 6 to 8 weeks of age.

     

Role of 11β-OH-C(19) and C(21) steroids in the coupling of 11β-HSD1 and 17β-HSD3 in regulation of testosterone biosynthesis in rat Leydig cells

Here we describe further experiments to support our hypothesis that bidirectional 11β-HSD1-dehydrogenase in Leydig cells is a NADP(H) regenerating system. In the absence of androstenedione (AD), substrate for 17β-HSD3, incubation of Leydig cells with corticosterone (B) or several C₁₉- and C₂₁-11β-OH-steroids, in the presence of [³H]-11-dehydro-corticosterone (A), stimulated 11β-HSD1-reductase activity. However, in presence of 30 μM AD, testosterone (Teso) synthesis is stimulated from 4 to 197 picomole/25,000 cells/30 min and concomitantly inhibited 11β-HSD1-reductase activity, due to competition for the common cofactor NADPH needed for both reactions. Testo production was further significantly increased (p < 0.05) to 224-267 picomole/25,000 cells/30 min when 10 μM 11β-OH-steroids (in addition to 30 μM AD) were also included. Similar results were obtained in experiments conducted with lower concentrations of AD (5 μM), and B or A (500 nM).Incubations of 0.3-6.0 μM of corticosterone (plus or minus 30 μM AD) were then performed to test the effectiveness of 17β-HSD3 as a possible NADP⁺ regenerating system. In the absence of AD, increasing amounts (3-44 pmol/25,000 cells/30 min) of 11-dehydro-corticosterone were produced with increasing concentrations of corticosterone in the medium. When 30 μM AD was included, the rate of 11-dehydro-corticosterone formation dramatically increased 1.3-5-fold producing 4-210 pmol/25,000 cells/30 min of 11-dehydro-corticosterone. We conclude that 11β-HSD1 is enzymatically coupled to 17β-HSD3, utilizing NADPH and NADP in intermeshed regeneration systems.     

Opposing actions of the progesterone metabolites, 5α-dihydroprogesterone (5αP) and 3α-dihydroprogesterone (3αHP) on mitosis,apoptosis, and expression of Bcl-2, Bax and p21 in human breast cell lines

Previous studies have shown that breast tissues and breast cell lines convert progesterone (P) to 5α-dihydroprogesterone (5αP) and 3α-dihydroprogesterone (3αHP) and that 3αHP suppresses, whereas 5αP promotes, cell proliferation and detachment. The objectives of the current studies were to determine if the 5αP- and 3αHP-induced changes in cell numbers are due to altered rates of mitosis and/or apoptosis, and if 3αHP and 5αP act on tumorigenic and non-tumorigenic cells, regardless of estrogen (E) and P receptor status. The studies were conducted on tumorigenic (MCF-7, MDA-MB-231, T47D) and non-tumorigenic (MCF-10A) human breast cell lines, employing several methods to assess the effects of the hormones on cell proliferation, mitosis, apoptosis and expression of Bcl-2, Bax and p21. In all four cell lines, 5αP increased, whereas 3αHP decreased cell numbers, [³H]thymidine uptake and mitotic index. Apoptosis was stimulated by 3αHP and suppressed by 5αP. 5αP resulted in increases in Bcl-2/Bax ratio, indicating decreased apoptosis; 3αHP resulted in decreases in Bcl-2/Bax ratio, indicating increased apoptosis. The effects of either 3αHP or 5αP on cell numbers, [³H]thymidine uptake, mitosis, apoptosis, and Bcl-2/Bax ratio, were abrogated when cells were treated simultaneously with both hormones. The expression of p21 was increased by 3αHP, and was unaffected by 5αP. The results provide the first evidence that 5αP stimulates mitosis and suppresses apoptosis, whereas 3αHP inhibits mitosis and stimulates apoptosis. The opposing effects of 5αP and 3αHP were observed in all four breast cell lines examined and the data suggest that all breast cancers (estrogen-responsive and unresponsive) might be suppressed by blocking 5αP formation and/or increasing 3αHP. The findings further support the hypothesis that progesterone metabolites are key regulatory hormones and that changes in their relative concentrations in the breast microenvironment determine whether breast tissues remain normal or become cancerous.