Discriminatory inhibition of adrenocortical 17 alpha-hydroxylase activity by inhibitors of cholesterol side chain cleavage cytochrome P-450

Two inhibitors of the cholesterol side chain cleavage reaction were tested for their ability to inhibit bovine adrenocortical 17 alpha-hydroxylase and 21-hydroxylase activities. One inhibitor, 22-amino-23,24-bisnor-5-cholen-3 beta-ol (22-ABC), was found to be a potent inhibitor of 17 alpha-hydroxylation of either progesterone or pregnenolone but was inactive on 21-hydroxylase activity. 22-ABC was found to be a competitive inhibitor of 17 alpha-hydroxylase (cytochrome P-45017 alpha) activity, having an apparent inhibitor constant of 29 nM when using pregnenolone as the substrate. Spectral binding studies showed that 22-ABC produces a type II difference spectrum when added to a bovine adrenocortical microsomal preparation, due presumably to a coordination of its amine nitrogen atom to the heme-iron of cytochrome P-45017 alpha. The second cholesterol side chain cleavage inhibitor tested, (20R)-20-phenyl-5-pregnene-3 beta,20-diol (20-PPD), was found not to inhibit either the 21- or 17 alpha-hydroxylase activities. It is proposed that the phenyl group projecting from C-20 of 20-PPD prevents this steroid from binding to cytochrome P-45017 alpha. The discriminatory interaction of these two steroids with adrenocortical cytochromes P-450 provides some insight with respect to possible structural features of the active-site regions of these enzymes.     

Identification of P-glycoprotein mutations causing a loss of steroid recognition and transport.

P-glycoproteins transport a wide variety of hydrophobic compounds out of cells. While the diversity of transported molecules suggests a mechanism involving broad specificity, there is evidence of significant discrimination within given classes of molecules. One example of this behavior is transport of corticosteroids by the murine mdr1 P-glycoprotein. The presence of hydroxyl groups, associated with specific steroid carbon atoms, regulates the ability of corticosteroids to be transported. This specificity is demonstrated here by experiments measuring the ability of steroids to inhibit drug transport. The results indicate that a keto oxygen associated with the 3- and 20-carbon atoms, as well as a 17-carbon hydroxyl group, each acts to enhance steroidal P-glycoprotein inhibitory activity. Moreover, inhibitory steroids can be used for directed selection of variant cells, expressing mutated P-glycoproteins with a severely impaired ability to transport dexamethasone. The five mutations, reported here, are located within transmembrane domains 4-6, proximal to the cytoplasmic interface. The altered P-glycoproteins exhibit reduced capacity to be inhibited by specific steroids, suggesting decreased capacity to bind these molecules avidly. Studies comparing the relative inhibitory activity of a series of steroids indicate that these mutations alter recognition of the 17alpha-hydroxyl group and the 20-keto oxygen atom.     

Neuroactive steroids,their precursors and polar conjugates during parturition and postpartum in maternal and umbilical blood: 3.3beta-hydroxy-5-ene steroids

Five 3beta-hydroxy-5-ene steroids involved in the metabolic route from pregnenolone sulfate to dehydroepiandrosterone and its sulfate, of which three are known allosteric modulators of neurotransmitter receptors, were monitored in the serum of 20 women around parturition. In addition, their levels in maternal and umbilical serum were compared at delivery. On the basis of these data, a scheme of steroid biosynthesis in maternal organism during the critical stages around parturition is proposed.In maternal serum, all the steroids except dehydroepiandrosterone sulfate decreased during labor and even first day after delivery, although their changes were less distinct the more distant from pregnenolone sulfate (PregS) in the metabolic pathway. Calculation of product/immediate precursor ratios in maternal serum over all stages around parturition enabled identification of the respective changes in the activities of the relevant enzymes. The ratio of 17-hydroxypregnenolone/pregnenolone did not change significantly, while that of dehydroepiandrosterone/17-hydroxypregnenolone grew, indicating increased C17,20 side chain cleavage on the account of C17-hydroxylation both catalyzed by C17-hydroxylase-C17,20-lyase. As was shown by factor analysis, the changes in the maternal steroids were associated with a single common factor, which strongly correlated with all the steroids except dehydroepiandrosterone sulfate. The lack of change in the pregnenolone sulfate/pregnenolone ratio and a marked increase of the ratio dehydroepiandrosterone sulfate to unconjugated dehydroepiandrosterone indicate a different means of formation of both steroid sulfates. On the basis of these data, a scheme of steroid biosynthesis in maternal organism during the critical stages around parturition is proposed.     

Structural specificity of steroids in stimulating DNA synthesis and protooncogene expression in primary rat hepatocyte cultures

Among the chemical compounds of varied structure which possess liver tumour-promoting are steroids, such as estrogens, pregnenolone derivatives and anabolic steroids. Although the mechanism(s) of tumour promotion in liver by these xenobiotics is not well understood, it is clear that growth stimulation is one important element in their action. As a basis for better defining whether steroids stimulate growth by a common mechanism or fall into sub-groups with differing actions, the effects of 46 steroids on DNA synthesis and the expression of protooncogenes c-fos and c-myc were examined in primary cultures of normal rat hepatocytes. Tentative groupings of steroids have been identified based on apparent structural requirements for stimulation of DNA synthesis, and effects of auxiliary factors in modulating this growth stimulus. For a "progestin" group, insulin appeared to be permissive for stimulation of DNA synthesis, and presence of an ester or hydroxyl group at 17alpha-position in combination with a non-polar group at C(6) appeared to be required for stimulation. For the pregnenes, dexamethasone was stimulatory. Structural requirements include a non-polar substitution at 16alpha-position and presence of a 6alpha-methyl group. Androgens were weak or ineffective stimulators of DNA synthesis. Anabolic steroids were weak to strong stimulators and alteration to A ring structure in combination with non-polar substitution at 17alpha-position appeared to be required for the activity. With the exception of the anabolic steroid, dianabol, there do not appear to be strong correlation between ability to stimulate DNA synthesis and ability to induce protooncogene expression among the steroids. This study provides a starting point for future more detailed examination of growth-stimulatory mechanism(s) of action of steroids in the liver.     

Sterol molecular modifications influencing membrane permeability

Various sterols and related steroids were tested for their ability to influence ethanol-induced electrolyte leakage from Hordeum vulgare roots. Cholesterol had the greatest influence and, depending on concentration, it stimulated or inhibited the loss of electrolyte. Cholesterol, however, was ineffective if the roots were pretreated with ethanol. These data suggest that sterols protect rather than restore membrane structure. First, modifications in the cholesterol perhydrocyclopentanophenanthrene ring system suggest that at least one double bond is required for membrane activity. Second, increasing the bulkiness of the C₁₇ side chain of cholesterol, as shown with campesterol, stigmasterol, and sitosterol, decreased its activity. Apparently for maximum effectiveness the sterol molecule should have a relatively flat configuration. Third, the C₃-hydroxyl group is required for membrane activity since cholesteryl methyl ether, cholest-5-ene-3β-thiol and cholesteryl halogens were without activity. Exception to the foregoing rule was cholestane which was slightly active but which has neither a C₃-hydroxyl group nor a double bond in the ring system.     

Steroid and sterol 7-hydroxylation: ancient pathways

B-ring hydroxylation is a major metabolic pathway for cholesterols and some steroids. In liver, 7 alpha-hydroxylation of cholesterols, mediated by CYP7A and CYP39A1, is the rate-limiting step of bile acid synthesis and metabolic elimination. In brain and other tissues, both sterols and some steroids including dehydroepiandrosterone (DHEA) are prominently 7 alpha-hydroxylated by CYP7B. The function of extra-hepatic steroid and sterol 7-hydroxylation is unknown. Nevertheless, 7-oxygenated cholesterols are potent regulators of cell proliferation and apoptosis; 7-oxygenated derivatives of DHEA, pregnenolone, and androstenediol can have major effects in the brain and in the immune system. The receptor targets involved remain obscure. It is argued that B-ring modification predated steroid evolution: non-enzymatic oxidation of membrane sterols primarily results in 7-oxygenation. Such molecules may have provided early growth and stress signals; a relic may be found in hydroxylation at the symmetrical 11-position of glucocorticoids. Early receptor targets probably included intracellular sterol sites, some modern steroids may continue to act at these targets. 7-Hydroxylation of DHEA may reflect conservation of an early signaling pathway.     

Formation of 5-[17beta-2H]androstene-3beta,17alpha-diol from 3beta-hydroxy-5-[17,21,21,21-2H]pregnen-20-one by the microsomal fraction of boar testis

After incubation of 3beta-hydroxy-5-[17,21,21,21-2H]-pregnen-20-one with the microsomal fraction of boar testis, the metabolites were analyzed by gas chromatography and gas chromatography-mass spectrometry. The following metabolites were identified: 3beta,17alpha-dihydroxy-5-[21,21,21-3H]pregnen-20-one, 3beta-hydroxy-5-androsten-17-one, 5-androstene-3beta,17beta-diol, and 5-[17beta-2H]androstene-3beta,17alpha-diol. The presence of a 2H atom at the 17beta position of 5-androstene-3beta,17alpha-diol was confirmed by oxidizing the steroid with 3beta-hydroxy-steroid dehydrogenase of Pseudomonas testosteroni to obtain 17alpha-hydroxy-4-[2H]androsten-3-one and then by oxidizing the latter steroid with chromic acid to obtain nonlabeled 4-androstene-3,17-dione. Among these metabolites, the first three can be interpreted to be synthesized by a well documented pathway, including 17alpha-hydroxylation followed by side chain cleavage as follows: 3beta-hydroxy-5-[17,21,21,21-2H]pregnen-20-one leads to 3beta,17alpha-dihydroxy-2-[21,21,212H]-pregnen-20-one leads to 3beta-hydroxy-5-androsten-17-one leads to 5-androstene-3beta,17beta-diol. On the other hand, 5-androstene-3beta,17alpha-diol, which contained a 2H atom at the 17beta position, is not likely to be synthesized via above mentioned pathway in which nonlabeled 3beta-hydroxy-5-androsten-17-one is formed as the first C19-steroid. It seems that an alternate side chain cleavage mechanism leading from pregnenolone to 17alpha-hydroxy-C19-steroid exists in boar testis.     

Catabolism of cholesterol by bovine adrenal-cortex enzymes: in vitro formation of oxygenated sterols and side-chain cleavage products.

The identification of sterols and steroids formed by incubation of [7alph-3H, 26-14C]cholesterol with mitochondrial enzymes from bovine adrenal cortex is reported. Only 17% of the radioactivity associated with cholesterol metabolites was found in pregnenolone, whereas 15% was reliable to oxygenated sterols and 6% to steroid compounds. The significance of the formation of these compounds is discussed particularly as regards oxygenated cholesterol derivatives.     

Minor and trace sterols in marine invertebrates : VI. Occurrence and possible origins of sterols possessing unusually short hydrocarbon side chains

Sterols with biosynthetically unusually short side chains (fewer than eight carbon atoms expected for primary squalene cyclization products) have been identified in the extracts of numerous marine invertebrates. The structures of the short side chain and conventional side chain sterols have been determined for various species of Porifera and Coelenterata. Sterol structures were determined by comparison of their mass spectra and gas chromatographic retention times with those of authentic or synthetic samples. Evidence is presented supporting the natural occurrence of these compounds in the tissues of the marine invertebrates as opposed to formation by degradative processes during sample handling or laboratory work-up. The short side chain sterols were found to possess predominantly the androst-5-en-3β-ol nucleus with C-17 alkyl side chains ranging from zero to six carbon atoms. Concentrations of short side chain sterols range from trace levels to over 5% of the sterol mixture in various species. The possible origins of these short side chain sterols are evaluated in the light of current knowledge of sterol function, biosynthesis, dealkylation, microbial degradation, and autoxidation. Known sterol autoxidations are reviewed, and possible singlet oxygen and free radical mechanisms of sterol side chain autoxidation (at physiological temperatures) which may lead to sterols with shortened hydrocarbon side chain are suggested. The possible autoxidative generation of short side chain sterols from known marine sterols by the suggested mechanisms is evaluated through application of the REACT computer program. Predicted short side chains are tabulated for each parent marine sterol side chain and then compared with the compositions of the actual sterols found in the marine extracts examined. The possible natural environmental or in vivo autoxidative formation of the short side chain marine sterols is supported by these evaluations.     

Substrate and nucleotide specificity of placental microsomal 3 beta-hydroxysteroid dehydrogenase

Recent kinetic studies on the placental microsomal 3 beta-hydroxysteroid dehydrogenase have shown that apparent Km values for 3 beta-hydroxy-5-androsten-17-one (dehydroepiandrosterone) and 3 beta-hydroxy-5-pregnen-20-one (pregnenolone) are 15nM and 40nM respectively, which are orders of magnitude lower than found in earlier studies. The purpose of this study was to investigate the substrate and nucleotide specificity of the 3 beta-hydroxysteroid dehydrogenase, and the ability of various steroids to inhibit the reaction at these lower steroid concentrations. Each steroid inhibited the metabolism of the other competitively, and the Ki values obtained were not significantly different from their respective Km values. The ability of various steroids to inhibit the reaction at concentrations of 100nM was usually less than that found at micromolar concentrations. However, certain steroids showed marked inhibition. For example, estrone and estradiol-17 beta inhibit the oxidation of both substrates competitively with Ki values of between 15 and 24nM. The Km values of dehydroepiandrosterone and pregnenolone with NADP+ as cofactor are higher than those with NAD+ as cofactor and the V values are much lower. These data indicate that in human placental microsomes a single 3 beta-hydroxysteroid dehydrogenase, essentially NAD+ specific, metabolizes dehydroepiandrosterone and pregnenolone.     

Oxidation of cholesterol and cholesterol analogues by mitochondrial preparations of steroid-hormone-producing tissue.

The rate of oxidation of cholesterol and its analogues to pregnenolone (3beta-hydroxypregn-5-en-20-one) by various mitochondrial preparations was measured. Sterols with the cholest-5-en-3beta-ol ring system and saturated side chains of different lengths were converted into pregnenolone rat rates similar to that of cholesterol. This marked lack of mitochondrial specificity towards the steroid side chains is in direct contrast with the rat liver microsomal cholesterol 7alpha-hydroxylase, which has a high specificity for the side chain. Steroids that retain the ring system, but contain hydroxyl groups at various points in the side chain, are converted into pregnenolone at rates three to eight times higher than in cholesterol. The results are discussed with reference to current ideas on the mechanism of the side-chain cleavage of cholesterol. The results are discussed with reference to current ideas on the mechanism of the side-chain cleavage of cholesterol.     

Pregnenolone sulphate-independent inhibition of TRPM3 channels by progesterone

Transient Receptor Potential Melastatin 3 (TRPM3) is a widely expressed calcium-permeable non-selective cation channel that is stimulated by high concentrations of nifedipine or by physiological steroids that include pregnenolone sulphate. Here we sought to identify steroids that inhibit TRPM3. Channel activity was studied using calcium-measurement and patch-clamp techniques. Progesterone (0.01-10μM) suppressed TRPM3 activity evoked by pregnenolone sulphate. Progesterone metabolites and 17β-oestradiol were also inhibitory but the effects were relatively small. Dihydrotestosterone was an inhibitor at concentrations higher than 1μM. Corticosteroids lacked effect. Overlay assays indicated that pregnenolone sulphate, progesterone and dihydrotestosterone bound to TRPM3. In contrast to dihydrotestosterone, progesterone inhibited nifedipine-evoked TRPM3 activity or activity in the absence of an exogenous activator, suggesting a pregnenolone sulphate-independent mechanism of action. Dihydrotestosterone, like a non-steroid look-alike compound, acted as a competitive antagonist at the pregnenolone sulphate binding site. Progesterone inhibited endogenous TRPM3 in vascular smooth muscle cells. Relevance of TRPM3 or the progesterone effect to ovarian cells, which have been suggested to express TRPM3, was not identified. The data further define a chemical framework for competition with pregnenolone sulphate at TRPM3 and expand knowledge of steroid interactions with TRPM3, suggesting direct steroid binding and pregnenolone sulphate-independent inhibition by progesterone.     

Minor and trace sterols in marine invertebrates. 1. General methods of analysis.

3beta-Hydroxy sterols occurring at a concentration of at least 0.001% of the sterol mixtures of Pseudoplexaura porosa and Plexaura homomalla have been fractionated using a series of refined techniques and subsequently analyzed using combined gas chromatography-mass spectrometry (GC-MS) in the development of a procedure for examining the minor and trace components of marine sterol mixtures. A total of 49 sterols were found which spanned a molecular weight range of 274 to 440. In addition delta4-3-keto analogs of cholesterol, 24-methylcholesterol and gorgosterol were found in the extracts of P. homomalla. Initial separation of various natural sterol-containing conjugates and free sterols was found to have a number of advantages. Fractional digitonin precipitation and alumina column chromatography were found to possess greater sterol separation abilities than previously recognized. Many of the minor sterols were found to possess novel structures including a series of short side chain sterols, 19-nor sterols, 5beta-stanols and 4-monomethyl sterols for which structure elucidation work is continuing.     

The identification and simultaneous quantification of 7-hydroxylated metabolites of pregnenolone, dehydroepiandrosterone, 3beta,17beta-androstenediol, and testosterone in human serum using gas chromatography-mass spectrometry

7-Hydroxy-metabolites of dehydroepiandrosterone (DHEA) and 3beta,17beta-androstenediol (AD) possess immunomodulatory and neuroprotective properties; therefore, the measurement of these steroids in patients with autoimmune diseases or disturbances in the CNS may be of interest. A gas chromatography-mass spectrometry (GC-MS) method for the determination of 7-hydroxy-metabolites of pregnenolone, DHEA, AD, and testosterone including the parent steroids was applied to serum samples from 12 adult men (27-66 years), 13 male adolescents (13-20 years), 5 boys (6-10 years), 15 women in the follicular phase of the menstrual cycle (22-45 years), 17 women in the luteal phase (22-45 years), and 4 girls (6-10 years). The steroids were age and sex dependent, but independent of the menstrual cycle. The ratio of the 7alpha-hydroxy-metabolites to their parent steroids were age dependent, exhibiting an increasing trend (p < 0.0001, ANOVA) from pregnenolone (5%) to AD (20%). The ratio of 7beta- to 7alpha-metabolites ranged from 0.6 to 1. These results are consistent with models suggesting 7alpha-hydroxylation of the parent steroid, conversion to a 7-oxo-steroid and finally to the 7beta-hydroxylated-metabolite. Partial correlations suggested that 7-hydroxylation might reduce the concentration of circulating androgens. Despite the three times lower concentration of AD-metabolites, their antiglucocorticoid, immunomodulatory, and neuroprotective effects may be comparable to that of DHEA based on their reported greater biological activity.     

Hydration of amino acid side chains: dependence on secondary structure.

Energy calculations have been used to study the hydration sites around the polar groups of serine, threonine and tyrosine side chains. These hydration sites depend not only on the hybridization of the polar group but also on the local secondary structure, the chi 1 side chain torsion angle and the position of the hydroxyl hydrogen atom. For tyrosine side chains, two solvent sites are found approximately in the plane of the ring. Even for serine and threonine side chains only two minimum energy sites are found in general of which one is in an expected position within hydrogen bonding of the hydroxyl hydrogen atom (unless this is blocked from interaction with solvent molecules by, for example, Oi-4 or Oi-3. The position of the second of these sites depends not only on the position of the hydroxyl oxygen but also on neighbouring main chain atoms to which it can also hydrogen bond. There is good agreement with the solvent distributions obtained from crystallographic data.     

Synthesis and evaluation of some steroidal oximes as cytotoxic agents: structure/activity studies (I)

The side chain of a compound plays an important role in its biological function. In our studies, we have found that hydroximinosteroid derivatives with different side chains and position of hydroximino on ring A and B displayed remarkable distinct cytotoxicities against a diversity of cancer cell types. Presence of an oxime group on ring B and a hydroxy on ring A or B resulted in a higher cytotoxicity than other structural motifs. In addition, a cholesterol-type side chain at position 17 was required for the biological activity. Our findings provide new evidence showing the relationship between the chemical structure and biological function. The information obtained from the studies may be useful for the design of novel chemotherapeutic drugs.     

Steroid-lipid interactions in sonicated dipalmitoyl phosphatidyl choline vesicles: A steady-state and time-resolved fluorescence anisotropy study with all trans-1,6-diphenyl-1,3,5-hexatriene as probe

The effect of steroid chemical structure on steroid-lipid interaction is studied in sonicated dipalmitoyl phosphatidylcholine vesicles by steady-state and time-resolved fluorescence anisotropy techniques using diphenylhexatriene as probe. A large number of cholesterol derivatives and steroids, some of them being precursors involved in corticosteroids biosynthesis, has been studied. Addition of a polar group on the side chain in carbon-20 or 22 position evokes considerable difference for the interaction with the bilayer. The carbon-22 position appears to be of critical importance. This observation may be relevant for the functioning of mitochondria from steroid producing tissues, where a stereospecific hydroxylation of cholesterol at the carbon-22R position is required for pregnenolone synthesis.     

Pharmacological properties of new 1,2-benzisothiazolyloxypropanolamines on cardiac and tracheal beta-receptors.

This paper reports the pharmacological assessment of beta-blocking properties of new benzisothiazole and benzisoxazole derivatives, substituted in position 3-, 5- or 7- with the oxypropanolaminic side chain (I-VI), to of which contain the -OCH3 group in position 3- (III, V) in comparison with propranolol. The results, obtained on isoprenaline-induced chronotropic response of rat isolated atria and on isoprenaline-induced relaxation of guinea-pig tracheal strips precontracted by carbachol, suggest that the compounds (I, II, IV, VI), at variance with the methoxy-substituted (III, V), possess a beta 1-blocking activity 4-300 times lower than propranolol. pA2 values drop from 8.36 to 7.56 and 7.04 from the relative compounds substituted in position 7- (IV), 3- (I) and 5- (II), thus indicating that the position of the oxypropanolaminic chain in the benzisothiazole ring affects the ability of the molecules to interact with the beta 1-adrenoceptor. Furthermore, benzisothiazole rather than benzisoxazole ring seems to facilitate the drug-beta 1 adrenoceptor interaction, the compound (I) displaying a 10-fold higher affinity than compound (VI).     

Neurosteroid levels are increased in vivo after LPS treatment and negatively regulate LPS-induced TNF production

Neuroactive steroids such as dehydroepiandrosterone sulfate and pregnenolone inhibit lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF) production. Corticosteroids not only inhibit TNF production but their levels are increased in vivo after endotoxin injection, thus representing a feedback system that limits TNF production. We wondered whether the same could be true for neuroactive steroids. Thus, the possibility that neuroactive steroids might be increased concomitantly to TNF induction in vivo in mice treated with LPS was investigated. Increased plasma and hippocampal levels of allopregnanolone (but not of dehydroepiandrosterone or pregnenolone) were found 90 min after LPS injection. Allopregnanolone and progesterone (IC50 10- 7 and 10- 9 M, respectively) also inhibited TNF production by mouse peritoneal macrophages in vitro at concentrations in the range of those detected in vivo. These findings suggest that neuroactive steroids may act as endogenous inhibitors of cerebral and systemic TNF production.     

The Influence of Exogenous Steroids on the Growth of Aspergillus niger and Torula utilis

The yield of Aspergillus niger mycelium from a synthetic medium can be increased by the addition of microgram quantities of cholesterol, ergosterol, cholestanol, 7-dehydrocholesterol, stigmasterol, sitosterol, pregnenolone, and the vitamins D. The stimulation is not due to degradation to the acetate level. It is obtained only in highly aerated cultures. The rate of growth of Torula utilis was not increased. Both organisms were inhibited by desoxycorticosterone, testosterone, androstenedione, cortisone acetate, progesterone, and diethylstilbestrol. T. utilis was also inhibited by estradiol. A small decrease in progesterone inhibition of T. utilis was obtained by adding ergosterol, cholesterol, or pregnenolone. Of the compounds which have been adequately tested the order of stimulatory activity for A. niger is: ergosterol > cholesterol > stigmasterol > 7-dehydrocholesterol > cholestanol > pregnenolone. Progesterone was inhibitory at low concentrations but stimulatory at higher ones, while 17-hydroxyprogesterone was neither inhibitory nor stimulatory. Desoxycorticosterone and testosterone were inhibitory at all concentrations. Complete inhibition of the growth of the fungus was not obtained with any of the steroids. It is concluded that A. niger has a metabolic requirement for a steroid with a hydroxy group on carbon 3, a double bond in the 5–6 position, and a side chain similar to that in ergosterol or cholesterol and that this material is growth-limiting in the early stages of the cultures described.