Simultaneous radioimmunoassay of testosterone,dihydrotestosterone, 5α-androstane-3α, 17β-diol and 5α-androstane-3β, 17β-diol in the plasma of adult male rats

A single thin layer chromatography and three antibodies were used for the specific radioimmunoassay of four androgens in pooled rat plasma (Sprague-Dawley adult males). The following values were found (pg/ml ± SD). Testosterone : 3, 138 ± 173; dihydrotestosterone : 374 ± 20; 5α-androstane-3α 17β-diol : 284 ± 24; 5α-androstane-3β, 17β-diol : 223 ± 11.     

The identification and characterization of the c19o3 steroid metabolites of 5α-androstane-3β, 17β-diol produced by the canine prostate: 5α-androstane-3β, 6α, 17β-triol and 5α-androstane-3β, 7α, 17β-triol

This study has identified the polar metabolites of 5α-androstane-3β, 17β-diol(3β-diol) produced by the canine prostate. The major metabolite is 5α-androstane-3β, 7α, 17β-triol (7α-triol) accounting for approximately 80% of the total polar metabolites of 3β-diol. The remaining 20% is accounted for exclusively by another triol, 5α-androstane-3β, 6α, 17β-triol(6α-triol). This study has also characterized two enzymatic hydroxylases responsible for respective triol formation: 5α-androstane-3β, 17β-diol 6α-hydroxylase (6α-hydroxylase) and 5α-androstane-3β, 17β-diol 7α-hydroxylase (7α-hydroxylase). Both of these irreversible hydroxylases are located in the particulate fraction of the prostate and can utilize either NADH or NADPH as cofactor. Several $\text{in vitro}$ steroid inhibitors of these hydroxylases were identified including cholesterol, estradiol and diethylstilbestrol. Neither of the hydroxylases were found to be decreased by castration (3 months) when expressed as activity/DNA. Using a variety of C₁₉ androstane substrates, 6α- and 7α-triol were found to be major components of the total 3β-hydroxy-5α-androstane metabolites produced by the canine prostate.     

Measurement of 5α-androstane-3α,17β-diol in human spermatic venous plasma by mass-fragmentography

Five alpha-androstane-3α,17β-diol (3α-diol) an active metabolite of testosterone (T) was measured in the spermatic and peripheral venous blood of 6 normal males using mass-fragmentography. Using this method 3α-diol was clearly separated from the following isomers: 5α-androstane-3β,17β-diol, 5β-androstane-3α,17β-diol and 5β-androstane-3β,17β-diol. The mean concentrations (±SE) of 3α-diol in spermatic and peripheral venous blood were respectively 100 ± 38 ng/100 ml and 7.7 ± 1.9 ng/100 ml. The existence of a significant (P < 0.01) gradient between spermatic and peripheral vein clearly demonstrates that the human testis secretes 3α-diol.     

Synthesis of new steroid haptens for radioimmunoassay. Part III. 15β-carboxyethylmercaptosteroid-bovine serum albumin conjugates. Specific antisera for radioimmunoassay of 5α-dihydrotestosterone, 5α-androstane-3β, 17β-diol and 5α-androstane-3α, 17β-diol

The syntheses of 15β-carboxyethylmercapto-5α-dihydrotestosterone, 15β-carboxy-ethylmercapto-5α-androstane-3β, 17β-diol and 15β-carboxyethylmercapto-5α-androstane-3α, 17β-diol and the preparation of their bovine serum albumin (BSA) conjugates are described. These conjugates were employed for the generation of specific antisera suitable for radioimmunoassay (RIA) of 5α-dihydrotestosterone (5α-DHT), 5α-androstane-3β, 17β-diol (3β3-diol) and 5α-androstane-3α, 17β-diol (3α-diol).     

C-19-steroid 7α-hydroxylation by rat testes. isolation and identification of: 7α, 17β-dihydroxy-5α-androstan-3-one, 5α-androstan-3α,7α,17β-triol and 5α-androstane-3β,7α, 17β-triol

From incubations of testosterone with rat testicular homogenates in the presence of a NADPH-generating system, the following 7α-hydroxylated metabolites could be isolated and identified: 7α,17β-dihydroxy-4-androsten-3-one (7α-hydroxy-testosterone), 7α-17β-dihydroxy-5α-androstan-3-one (7α-hydroxy-Dht), 5α-androstan-3α,7α,17β-triol (7α-hydroxy-3α-A'DIOL) and 5α-androstane-3β,7α,l7β-triol (7α-hydroxy-3β-A'DIOL). To our knowledge this is the first demonstration of the formation of 5α-reduced-7α-hydroxylated metabolites of testosterone in the male gonad. These 5α-reduced-7α-hydroxylated metabolites could also be isolated after incubations of 5α-androstane-3α,17β-diol (3α-A'D10L) with testicular homogenates in the presence of a NADPH-generating system.Measured as the sum of 7α-hydroxy-testosterone, 7α-hydroxy-Dht. 7α-hydroxy-3α-A'DIOL and 7α-hydroxy-3β-A'DIOL formed using testosterone as substrate, total 7α-hydroxylase activity was six times higher in testes of mature rats than in testes from animals 23 days old. With 3α-A'DIOL as substrate total 7α-hydroxylase in the mature testis was about three times greater than in the sexually immature testis.     

5α-androstane-3β,17β-diol in feedback between gonads and hypothalamo-pituitary system of male rats

Serum level of LH and levels of LH-RH in the arcuate nucleus and in the median eminence of hypothalamus were measured by radioimmunoassay in castrated male rats 1, 3, 6, 12, 24 h after single administration of 25 μg/100 g b.w. testosterone, 5α-androstane-3α,17β-diol (3α-diol) or 5α-androstane-3β, 17β-diol (3β-diol).The levels were then measured 6 h after single administration of 3α-diol and 3β-diol in different doses: 5 μg; 25 μg; 100 μg; 250 μg; 2500 μg/100 g b.w.The results suggest basic differences between the influence of testosterone and of its 5α-reduced metabolites upon the hypothalamo-pituitary system as to speed and strength of effect. Both 3α-diol and 3β-diol are active in the negative feedback between the gonads and the hypothalamo-pituitary system, but their effect depends on the dose. 3β-Diol is active in the low doses we have studied as opposed to its 3α-epimer.     

5α-androstane-3α,17β-diol selectively activates the canonical PI3K/AKT pathway: a bioinformatics-based evidence for androgen-activated cytoplasmic signaling

5α-Androstane-3α,17β-diol (3α-diol) is reduced from the potent androgen, 5α-dihydrotestosterone (5α-DHT), by reductive 3α-hydroxysteroid dehydrogenases (3α-HSDs) in the prostate. 3α-diol is recognized as a weak androgen with low affinity toward the androgen receptor (AR), but can be oxidized back to 5α-DHT. However, 3α-diol may have potent effects by activating cytoplasmic signaling pathways, stimulating AR-independent prostate cell growth, and, more importantly, providing a key signal for androgen-independent prostate cancer progression. A cancer-specific, cDNA-based membrane array was used to determine 3α-diol-activated pathways in regulating prostate cancer cell survival and/or proliferation. Several canonical pathways appeared to be affected by 3α-diol-regulated responses in LNCaP cells; among them are apoptosis signaling, PI3K/AKT signaling, and death receptor signaling pathways. Biological analysis confirmed that 3α-diol stimulates AKT activation; and the AKT pathway can be activated independent of the classical AR signaling. These observations sustained our previous observations that 3α-diol continues to support prostate cell survival and proliferation regardless the status of the AR. We provided the first systems biology approach to demonstrate that 3α-diol-activated cytoplasmic signaling pathways are important components of androgen-activated biological functions in human prostate cells. Based on the observations that levels of reductive 3α-HSD expression are significantly elevated in localized and advanced prostate cancer, 3α-diol may, therefore, play a critical role for the transition from androgen-dependent to androgen-independent prostate cancer in the presence of androgen deprivation.     

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.     

Antagonism of the actions of estrogens,androgens and progesterone by anordrin (2α,17α-diethynyl-A-nor-5α-androstane-2β, 17β-diol dipropionate)

Anordrin, an antifertility agent that is an antiestrogen with weak estrogenic activity, has been studied to further characterize its hormonal activities. A dose of 2.0 μg/mouse·day for 7 days did not increase the uterine content of protein, but it did inhibit to a small extent the effect of administered estradiol-17β on uterine protein content and more significantly the effect of estradiol-17β on the uterine content of progesterone receptors. Anordrin also decreased serum corticosteroid-binding globulin levels. Administration of an average daily dose of 160 μg/day of anordrin to intact male mice had no effect on weights of kidney, testis, or seminal vesicle after 10 days, but seminal vesicle weight was significantly decreased after 30 days at a slightly lower dose. Similarly, anordrin inhibited the increase in seminal vesicle weight induced by testosterone propionate treatment of castrated mice. In female mice anordrin failed to maintain deciduomata and blocked the ability of progesterone (2.0 mg/mouse·day) to do so. However, anordrin did not compete with the androgen [³H]R1881 for binding in kidney cytosol or with the progestin [³H]R5020 for uterine receptor sites. Anordrin also did not compete with [³H]corticosterone for binding to serum proteins.     

Studies on the effect of 5α-androstane-3α, 17α-diol in the canine prostate in vivo

Adult beagle dogs, castrated one month previously, were treated with 5α-androstane-3α, 17α-diol (total dose 300 mg) over a period of one month. Examination of the prostate after treatment showed no significant change in size, weight or histological appearance from the castrate dog prostate. Subsequent administration of 5α-androstane-3α, 17β-diol (300 mg) over the same period of time resulted in restoration of the prostate size, weight and histological appearance to that of the normal intact dog prostate. It is concluded that exogenously administered 5α-androstane-3α, 17α-diol does not promote prostatic growth in the castrate beagle dog.     

Synthesis of 3α,7α-dihydroxy-5β-androstan-17-one

A short and efficient method for the stereospecific synthesis of 3α,7α-dihydroxy-5β-androstan-17-one was accomplished from the readily available 4-androstene-3,17-dione. Key steps are the stereospecific and selective epoxidation of 4,6-androstadiene-3,17-dione, followed by hydrogenations with carefully selected reagents, solvents and reaction conditions.     

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.     

5α,6α- AND 5β,6β-dichloromethylene adducts of 3β-acetoxy-5-androsten-17-one

Both the 5α, 6α- and 5β, 6β-dichloromethylene adducts ($\text{2a}$ and $\text{2b}$) of 3β-acetoxy-5-androsten-17-one ($\text{1}$) are produced when the latter is exposed to dichlorocarbene generated from chloroform and base by Phase Transfer Catalysis using ultrasound as a means of agitation. The ¹H NMR substituent effects of 5α, 6α- and 5β, 6β-dichloromethylene on the angular methyl groups (Zürcher values) are given. The ¹³C NMR spectra for both compounds are presented and discussed.     

Alteration in the normal pattern of serum testosterone and 5α-androstane-3α,17β-diol in the immature male rat following chronic treatment with luteinizing hormone releasing hormone or luteinizing hormone

A major component of sexual maturation in the male rat is a progressive decline in serum concentrations of 5α-androstane-3α,17β-diol (3α-diol) and a concomitant increase in testicular testosterone biosynthesis and secretion. Chronic administration of synthetic luteinizing hormone releasing hormone (LHRH) or luteinizing hormone (LH)/human chorionic gonadotropin (hCG) to immature male rats has been shown to result in a delay in sexual maturation as evidenced by decreased sex accessory gland weights and altered testicular testosterone production. We have examined the postulate that such treatments may either reverse or retard the normal developmental pattern of serum testosterone and 3α-diol concentrations. Chronic $\text{in vivo}$ treatment of 28 day old immature male rats for 2 weeks with daily injections of either 0.5 μg of LHRH, 1.0 μg of LHRH, or 30 μg of LH was found to result in significant reductions in weights of the seminal vesicles and ventral prostate glands and diminutions in serum testosterone concentrations. Serum content of 3α-diol was either unchanged or slightly elevated in the LHRH treated animals and increased significantly in the LH treated animals. These data suggest that either a reversal of or retardation in the normal developmental pattern of serum testosterone and 3α-diol content has been achieved in the immature male rat by chronic LHRH or LH treatment.     

Metabolism of 17α-methyl-5α-dihydrotestosterone in the rabbit

17α-Methyl-5α-dihydrotestosterone and the reduced metabolites, 17α-methyl-5α-androstane-3α, 17β-diol and -3β, 17β-diol together with two hydroxylated metabolites, 17α-methyl-5α-androstane-3β, 15α, 17β-triol and 17α-methyl-5α-androstane-3α, 6α, 17β-triol were isolated and identified in the urine of rabbits orally dosed with 17α-methyl-5α-dihydrotestosterone. Formation of the C-6 hydroxylated derivative demonstrates that the 4,6-enolization of a 4-en-3-one is not a necessary requirement for hydroxylation at C-6 of the androstane nucleus in the rabbit. No evidence was obtained for the presence of 17α-methyl/17β-hydroxyl epimerization.     

5α-Estrane-3β,17β-diol and 5β-estrane-3α,17β-diol: definitive screening biomarkers to sign nandrolone abuse in cattle?

17β-Nandrolone (17β-NT) is one of the most frequently misused anabolic steroids in meat producing animals. As a result of its extensive metabolism combined with the possibility of interferences with other endogenous compounds, detection of its illegal use often turns out to be a difficult issue. In recent years, proving the illegal administration of 17β-NT became even more challenging since the presence of endogenous presence of 17β-NT or some of its metabolite in different species was demonstrated. In bovines, 17α-NT can occur naturally in the urine of pregnant cows and recent findings reported that both forms can be detected in injured animals. Because efficient control must both take into account metabolic patterns and associated kinetics of elimination, the purpose of the present study was to investigate further some estranediols (5α-estrane-3β,17β-diol (abb), 5β-estrane-3α,17β-diol (bab), 5α-estrane-3β,17α-diol (aba), 5α-estrane-3α,17β-diol (aab) and 5β-estrane-3α,17α-diol (baa)) as particular metabolites of 17β-NT on a large number of injured (n=65) or pregnant (n=40) bovines. Whereas the metabolites abb, bab, aba and baa have previously been detected in urine up to several days after 17β-NT administration, the present study showed that some of the isomers abb (5α-estrane-3β,17β-diol) and bab (5β-estrane-3α,17β-diol) could not be detected in injured or pregnant animals, even at very low levels. This result may open a new way for the screening of anabolic steroid administration considering these 2 estranediols as biomarkers to indicate nandrolone abuse in cattle.     

Synthesis of 3β-hydroxy-androsta-5,7-dien-17-one from 3β-hydroxyandrost-5-en-17-one via microbial 7α-hydroxylation

The synthesis of 3β-hydroxy-androsta-5,7-dien-17-one from 3β-hydroxy-androst-5-en-17-one (dehydroepiandrosterone, DHEA) via microbial 7α-hydroxylation has been accomplished. At the first stage, 3β,7α-dihydroxy-androst-5-en-17-one was obtained in high yield (71.2%) using a strain of Gibberella zeae VKM F-2600, which was first applied for DHEA conversion. The further route included the substitution of 7α-hydroxyl group with chlorine followed by a dehydrochlorination stage, and required minimal purifications of the intermediate products. The steroids obtained at every step were characterized by TLC_,¹H NMR, MS, UV- and IR-spectrometry.The combination of microbial and chemical steps ensured 54.6% yield of the target 3β-hydroxy-androsta-5,7-dien-17-one from DHEA and can be applied for obtaining novel vitamin D derivatives.     

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.     

Androgen metabolism by rat epididymis: 5. Metabolic conversion and nuclear binding after injection of 5α-androstane-3α,17β-diol, in vivo

The pattern of androgenic metabolites in blood, muscle, caput and cauda epididymidis has been investigated in functionally hepatectomized 24 hours castrated rats, 3 hours after the intra-muscular injection of 200 μCi of ³H -3α-diol. Identification of the radioactive metabolites showed only negligible differences between the epididymal regions. In both caput and cauda the main metabolite was DHT (17β-hydroxy-5α-androstane-3-one); 3α- and 3β-diol, androsterone (3α-hydroxy-5α-androstane-17-one), 5-A-dione (5α-androstane-3,17-dione), Δ¹⁶-3α-ol (5α-androst-l6-en-3α-ol), Δ¹⁶-3β-ol (5α-androst-l6-en-3α-ol) and Δ¹⁶-3-one (5α-androst-l6-en-3-one) were also present.$\text{Androsterone}$ and $\text{3α-diol}$ were the predominant metabolites in blood and muscle. No Δ¹⁶ compounds could be detected and in constrast to epididymis, more than 50% of the radioactivity was associated with polar compounds. From determination of total radioactivity, it was seen that retention by epididymis varied from two to four times that of muscle. Purification and identification of the radioactivity associated with the nuclear fraction demonstrated that DHT was the only nuclear bound androgen.It is suggested from these results that at least one effect of 3α-diol on the rat epididymis is exerted through its conversion to DHT.