Increased acridine orange uptake and lysosomal enzyme levels in rat liver after steroid administration

A steroid which stabilizes lysosomes $\text{in vitro}$ and a pyrogenic steroid which labilizes lysosomes $\text{in vitro}$ were compared with respect to their ability to modify lysosomal uptake and lysosomal enzyme levels $\text{in vivo}$. Cortisone acetate increased the uptake of acridine orange by rat liver lysosomes when the dye was administered by intrathoracic injection. The steroid increased and accelerated the uptake of acridine orange so that, in liver lysosomes from treated rats, the maximum uptake was double that of controls and was reached at 2h, whereas in controls the maximum uptake was at 4h after the injection of the dye. This large elevation of uptake is specific to the lysosomal fraction and is not seen in other subcellular fractions of rat liver. The specific activities of a lysosomal enzyme β-N-acetylglucosaminidase were increased in lysosomal fractions from cortisone acetate-treated rats. Etiocholanolone, a steroid which labilizes lysosome $\text{in vitro}$, similarly accelerated and increased acridine orange uptake by lysosomes but had little effect on lysosomal β-N-acetylglucosaminidase levels. Thus the ability of steroids to stabilize or labilize lysosomes $\text{in vitro}$ does not correlate with their effect on lysosomal uptake of injected substances $\text{in vivo}$, or with their ability to induce increased specific activities of lysosomal enzymes.     

Formation of neutral steroids from endogenous precursors in minced human fetal adrenals in vitro

The role of endogenous precursors in steroid biosynthesis in the human fetal adrenals was studied in $\text{in vitro}$ incubations with no added exogenous substrate. The identification and quantitative determination of the steroids was carried out by gas-liquid chromatography and gas chromatography - mass spectrometry. During the incubation a 10–60-fold increase in the concentration of dehydroepiandrosterone sulfate was observed. An increase was also seen in the concentrations of the other two steroid sulfates detected, pregnenolone and 17-hydroxypregnenolone sulfate. The concentrations of the corresponding free steroids were seen to decrease during the incubations. Only traces of free dehydroepiandrosterone and progesterone were detected endogenously or at any stage during the incubations. No corticoids could be found. Endogenous cholesterol was found in high concentrations (1.5 – 3.0 mg/g wet tissue) in the tissue samples studied. A small proportion of it was present as a sulfate conjugate.It is concluded that fetal adrenals can form neutral steroid sulfates of the 3β-hydroxy-5-ene series from endogenous precursors $\text{in vitro.}$ Cholesterol which was detected in high concentrations in the adrenal tissue is a possible precursor of these metabolites. The results obtained would suggest that this endogenous metabolism involves sulfated intermediates principally.     

Binding of steroids to P.testosteroniΔ5-3-ketosteroid isomerase: Measurement of the number of binding sites by equilibrium dialysis

The binding of progesterone, 17β-estradiol and 19-nortestosterone acetate to the Δ⁵-3-ketosteroid isomerase from $\text{Pseudomonas}$$\text{testosteroni}$ has been investigated by the technique of equilibrium dialysis. Under the conditions used, all three steroids formed 2:1 complexes with each molecule of enzyme dimer (M.W. = 26,788). No evidence of any cooperative binding phenomena was obtained. The dissociation constants of the enzyme steroid complexes at 25°C were: progesterone, 2.2 μ$\text{M}$; estradiol, 2.5 μ$\text{M}$; 19-nortestosterone acetate, 9.2 μ$\text{M}$.     

Interaction of prostaglandins with adrenal microsomal cytochrome P-450 in the guinea pig

Studies were carried out to investigate the effects of prostaglandins (PG) $\text{in vitro}$ on adrenal microsomal steroid and drug metabolism in the guinea pig. The addition of PGE₁, PGE₂, PGA₁, PGF_1α or PGF_2α to isolated adrenal microsomes produced typical type I difference spectra. The sizes of the spectra (ΔA_385–420) produced by prostaglandins were smaller than those produced by various steroids including progesterone, 17-hydroxyprogesterone and 11β-hydroxyprogesterone. However, the affinities of prostaglandins and steroids for adrenal microsomal cytochrome P-450, as estimated by the spectral dissociation constants, were similar. Prior addition of prostaglandins to isolated adrenal microsomes did not affect steroid binding to cytochrome P-450 or the rate of steroid 21-hydroxylation. In contrast, prostaglandins inhibited adrenal metabolism of ethylmorphine and diminished the magnitude of the ethylmorphine-induced spectral change in adrenal microsomes. The results indicate that prostaglandins inhibit adrenal drug metabolism by interfering with substrate binding to cytochrome P-450. Since 21-hydroxylation was unaffected by PG, different cytochrome P-450 moieties are probably involved in adrenal drug and steroid metabolism.     

Total synthesis of heterocyclic steroids,part IV: Mass spectrometry in stereochemical determination of the C/D ring fusion geometry in some heteroaromatic steroids

The mass spectral fragmentation pattern and the relative intensities of the fragment peaks of the D ring in some 8-dehydroheteroaromatic steroids were studied. Characteristic differences for the $\text{cis and trans}$ isomers were ascertained and were used for the determination of the stereochemistry of thiasteroids of which only one isomer was obtained.     

Isolation and preliminary characterization of corticosterone-receptor complexes in mouse placental tissue

The $\text{in vitro}$ binding of several radioactive steroids was examined in mouse placental tissue, using Sephadex chromatography to separate the labelled steroid complex from free steroids. Binding was exhibited by cortisol, corticosterone and progesterone, but not cortisone. Cortexolone, an antiglucocorticoid, was tested in the system and found to reduce the binding of the active steroids. The displacement of labelled corticosterone by addition of unlabelled steroids was also examined. Preliminary characterization of the corticosterone receptor using hydrolytic enzymes suggested a protein nature on the basis of degradation by pronase but not by nucleases.     

Marine natural products: 5alpha-cholestane-3beta,5,6beta,9-tetrol: a polyhydroxylated steroid from the gorgonian Pseudopterogorgia elisabethae.

A tetrahydroxylated steroid isolated from a gorgonian, $\text{Pseudopterogorgia elisabethae}$, has been shown by spectral analyses and degradative studies to be 5α-cholestane-3β,5,6β,9-tetrol.     

Irreversible active-site-directed inhibition of Δ5-3-ketosteroid isomerase by steroidal 17-β-oxiranes. Evidence for two modes of binding in steroid-enzyme complexes

The spiro-17β-oxiranyl derivatives of d-equilenin, epiandrosterone, dehydroepiandrosterone, and Δ⁴-androsten-3,17-dione are active-site-directed irreversible inhibitors of Δ⁵-3-ketosteroid isomerase of $\text{P.}\text{testosteroni}$. The 17β-oxiranyl steroids rapidly inhibit the isomerase in a time-dependent manner which exhibits saturation kinetics. The enzyme is protected against inactivation by the competitive inhibitor 19-nortestosterone. In addition, prolonged dialysis against neutral buffer leads to no regeneration of enzyme activity. Fluorescent spectral changes associated with the incubation of the enzyme with the 17β-oxirane derived from d-equilenin indicate that there are two modes of binding for steroids to the isomerase. These results suggest that 17β-oxiranes may inhibit the enzyme by a mechanism similar to that for the previously studied spiro-3β-oxiranyl steroids.     

Steroid t-butyldimethylsilyl ethers as derivatives for mass fragmentography.

The rates of formation, chromatographic properties and mass spectral characteristics of various steroid t-butyldimethylsilyl ($\text{t}$-BDMS) ethers are examined and their application to mass fragmentography discussed.     

Steroid induction of nerve growth factor synthesis in cell culture

Nerve growth factor (NGF) is a peptide hormone which is necessary for the development of sympathetic neurons. Exposing a rat central nervous system glioma cell line (C-6) to the steroid hormone 17β-estradiol increases the amount of NGF secreted by these cells into the surrounding medium. This induction is highly specific to 17β-estradiol in that similar steroids do not increase NGF levels. Both NGF activity and protein levels increase upon estradiol stimulation and there is a parallel increase in NGF $\text{de}$$\text{novo}$ synthesis. The estradiol effect can be blocked with actinomycin D but not with puromycin or cycloheximide. This is the first report demonstrating regulation of NGF synthesis by a steroid hormone in a clonal cell line of glial origin. We propose this system as a model system for the study of the regulation of NGF synthesis and the isolation and analysis of putative precursors to the NGF molecule.     

Steroid hormone receptors in human malignancy.

Steroid hormones induce responses in target tissues by a mechanism involving the specific initial interaction of hormone with cytoplasmic receptor molecules. These receptors, usually localized in target tissues have high binding affinities and limited binding specificities for biologically active steroids. Examination of human leukemic lymphoblasts has revealed these receptors in some tumor samples. Their presence is well correlated with hormone responsiveness of the tumor $\text{in vitro}$. Similar studies on human breast cancer tumor homogenates has indicated that about $\text{2}\text{3}$ of primary tumors contain estrogen receptor. The absence of receptor predicts a lack of response to hormone therapy almost invariably, while the presence of receptor increases but does not assure that the tumor will be hormone responsive. Recently $\text{in vitro}$ tissue culture systems which mimic the hormone responses observed $\text{in vivo}$ have been developed which should significantly aid in the clarification of the mechanisms whereby steroid hormones stimulate and inhibit growth in target tissues.     

De novo synthesis of steroids (from acetate) by isolated rat Sertoli cells.

Sertoli cells from 17 day old rats were shown to convert [¹⁴C]acetate to [¹⁴C]-labelled cholesterol, pregnenolone and 17α-hydroxypregnenolone$\text{in}$$\text{vitro}$. Identification was by several systems of thin layer and gas chromatography of the extracted steroids and their sylil and acetyl derivatives and by recrystallizations with authentic and acetylated unlabelled steroids. Several other steroids formed from acetate were tentatively identified. No androstenedione or testosterone were formed. That the Sertoli cell cultures were free of Leydig cells was established by the absence of histochemically detectable 3β-hydroxysteroid dehydrogenase activity and the inability of the cultures to oxidize the 3β-hydroxyl group of [¹⁴C]pregnenolone. This is the first direct evidence that Sertoli cells have the capacity to synthesize steroids $\text{de}$$\text{novo}$ from acetate.     

Rapid and intensive conversion of 5α-androstane-3α,17β-diol into 5α-dihyorotestosterone in the male rat anterior pituitary: in vivo and in vitro studies

The $\text{in vivo}$ and $\text{in vitro}$ metabolism of $\text{(}{}^3\text{H}\text{)-5α-}\text{androstane}\text{-α, 17β-}\text{diol}$ by the male rat anterior pituitary was studied. A rapid and intensive conversion of 5α-androstane-3α,17β-diol into 5α-dihydrotestosterone was demonstrated, since following a 30 min. incubation time, 73 % of the recovered radioactivity were constituted by 5α-dihydrotestosterone. Studies on the subcellular distribution of steroids showed that 5α-dihydrotestosterone was the main steroid recovered except from the 105,000 × g pellet. From $\text{in vivo}$ and $\text{in vitro}$ experiments it was concluded that the transformation of 5α-dihydrotestosterone into 5α-androstane-3α,17β-diol was a reversible process, and that this last steroid could exert its biological action mainly $\text{via}$ 5α-dihydrotestosterone.     

Pregnancy hormone concentrations in marijuana users

The maternal serum concentrations of human chorionic gonadotropin, pregnancy-specific beta-l-glycoprotein, placental lactogen, progesterone, 17-hydroxyprogesterone, estradiol and estriol were measured in 13 women who smoked marijuana regularly throughout pregnancy. Cannabinoid use in these women was confirmed by RIA measurements of their sum Δ ⁹- tetrahydrocannabinol (THC) concentrations. These THC using women were matched within $\text{2}\text{1}\text{2}$ weeks of gestational age with 13 pregnant non-THC using controls drawn from the same population. Placental protein and steroid hormone concentrations were within established normal ranges for gestational age and there were no significant differences between the groups in the concentrations of any of the protein and steroids measured. In addition, no significant differences between THC users were found following linear regression analysis of placental hormone concentrations as a function of gestational age. Thus, this study suggests that marijuana use during pregnancy does not significantly alter the circulating maternal concentrations of trophoblastic protein hormones or major fetoplacental steroid hormones.     

A radioimmunoassay for 11-oxotestosterone: Its application in the measurement of levels in blood sebum of rainbow trout (S. Gairdneri).

17β-Hyd.roxyandrost-4-ene-3,11-dione was linked via its 3-(O-carboxymethyl) oxime to bovine serum albumin to give a conjugate which was used to generate antiserum in rabbits. The antiserum, at an overall dilution of 1 in 16,000, together with [1,2-³H] 17β-hydroxyandrost-4-ene-3,11-dione synthesized from [1,2-³H] cortisone have been used to develop a radioimmunoassay for the parent steroid. The assay incorporates a purification step in which serum or plasma extracts are chromatographed on silica gel layers bound to plastic or aluminium sheets and the steroid, containing zones cut out and eluted directly with assay buffer. The cross-reactivities of several steroids with the antiserum and the specificity, sensitivity, accuracy and precision of the assay are described. Blood sera from Immature male rainbow trout contain $\text{ca}$ 0.2–0.4 μg/100 ml of 17β-hydroxyandrost-4-ene-3,11-dione. As male fish mature, serum levels rise sharply to reach values of 2 to >9 μg/100 ml. Levels in immature females rarely exceeded the assay sensitivity but serum from three ripe females showed low but detectable levels ($\text{ca}$ 0.2 μg/100 ml) of steroid. The assay has found application in sexing live fish for experimental purposes.     

An A-nor-3-thia-5beta-pregnane.

The conversion of a 5β-pregnan-3-one into a 3-thia-A-nor-pregnane is described. The single epimer, characterized as the 5β-isomer by nmr spectroscopy, was obtained.A previous report [1] from this laboratory described the synthesis of the epimeric 3-oxa-A-norpregnan-20-ones $\text{1}$ and $\text{2}$ from A-norprogesterone. Recently, the total synthesis of the related A-nor-3-thiaestra-1,-5(10)-diene derivative $\text{3}$ has been described [2]. Continuing interest [3] in A-ring-modified steroids as probes of drug-receptor interactions prompts this report of the preparation of the 3-thia-5β-A-norpregnan-20-one $\text{4}$.     

Mass spectra of sterically crowded trialkylsilyl ether derivatives of steroids

The electron-impact mass spectra of the title compounds have some important features which give these derivatives certain advantages over the widely studied trimethylsilyl analogues. There is significantly less extensive fragmentation, and abundant ions at (M — $\text{t}$.Bu)⁺ or (M — $\text{i}$.Pr)⁺ serve as indicators of molecular weight and should be useful for selected ion monitoring. From various precursors, the ease of elimination of HX₂SiOH, via a proposed multi-centre transition state, appears to depend upon conformational and stereochemical factors, as well as the position of the parent silyloxy group, RX₂SiO, on the steroid skeleton. This particular fragmentation appears to be a powerful diagnostic method for distinguishing between stereoisomers, being especially useful for differentiation between epimers. In addition, the presence of a 17-silyloxy function promotes a characteristic cleavage of ring B in a skeletally saturated steroid. Elimination of silanol, RX₂SiOH, at various stages in the fragmentations of bis-silylated steroids is also an important process, but other familiar features of the spectra of steroid trimethylsilyl ethers, though usually present, are very much suppressed.     

Androgen metabolism by rat epididymis. 2. Metabolic conversion of 3H-testosterone in vitro

The epididymis of adult rats metabolizes ³H-testosterone by experiments $\text{in}$$\text{vitro}$. After incubation of slices from epididymal tissue for 2 hrs at 37°C, 8% of the total radioactivity was found in the water-soluble fraction, whereas 92% in the ether soluble fraction (free steroids). The free steroids were examined further and the following metabolites identified: $\text{testosterone}$ (17β-hydroxy-4-androsten-3-one) 10,4%, $\text{androstendione}$ (4-androstene-3,17-dione) 6,2%, $\text{5α-A-dione}$ (5α-androstane-3,17-dione) 7,3%, $\text{DHT}$ (17β-hydroxy-5α-androstane-3-one) 39,3%, $\text{3α-diol}$ (5α-androstane-3α,17β-diol) 22,7%, $\text{3β-diol}$ (5α-androstane-3β,17β-diol) 4,6% and androsterone(3α-hydroxy-5α-androstan-17-one) 8,9%. The relative amount of each metabolite is given in per cent of the total radioactivity in the ether soluble fraction. When segments (caput, corpus, cauda) of epididymis were incubated in the same way, differences in steroid metabolism were demonstrated. Characteristic for caput epididymidis was high formation of DHT (58,4%) and 3α-diol (23,5%). Corpus epididymidis showed lower formation of DHT (50,6%) and 3α-diol (12,7%), but an approximately 3 times higher formation of 5α-A-dione (12,0%) than caput (3,4%) and cauda (3,5%). Cauda epididymis showed the lowest formation of DHT (38,3%), whereas 3α-diol (29,1%) and androsterone (11,4%) formation were relatively high. The ratio between 17β-hydroxy metabolites (DHT and androstanediols) and 17-keto metabolites were much higher in the caput (8,8) than in the corpus (3,2) and cauda (3,6), indicating a higher 5α-reductase activity in this segment.     

The pharmacokinetics of tritiated ethinylestradiol in the rabbit

The disappearance of ethinylestradiol from the blood of rabbits has been studied, following the intravenous administration of this steroid. The disappearance followed two exponentials, the first having a half life ($\text{t}\text{1}\text{2}$) of 5.5 min and the second, apparently terminal exponential was also rapid ($\text{t}\text{1}\text{2}\text{= 69}\text{min}$). The plasma clearance was 150 ml/min which suggests almost total clearance of this steroid during a single passage through the liver. Bile contained a significant concentration of EE conjugates and thus this steroid could undergo enterohepatic recirculations. A large oral dose of unlabelled EE, given prior to intravenous administra tion of tritiated EE, considerably altered the pharmacokinetics of the latter by saturating both phase one metabolism (changes of the steroid nucleus) and the secretion of conjugates into bile. It was not clear whether phase two metabolism (conjugation) was also saturated.     

3-Epi-20-hydroxyecdysone from meconium of the tobacco hornworm

A new ecdysteroid, 3-epi-20-hydroxyecdysone, with biological activity $\text{1}\text{10}$ to $\text{1}\text{15}$ that of α-ecdysone has been isolated and identified from the meconium of the tobacco hornworm. The possible role or function of this steroid as an inactivation product and/or regulator of molting hormone titer is discussed.