Steroid biosynthesis by a sesterterpene pathway in the rat adrenal gland in vitro

Rat adrenal gland preparations were incubated with radioactive cholesterol and 23,24-dinor-5-cholen-3 beta-ol. Steroids were isolated, purified by thin-layer and high performance liquid chromatography and crystallised to constant specific activity. It was found that the rat adrenal gland can utilise 23,24-dinor-5-cholen-3 beta-ol to produce corticosterone. Also, in contrast to the conversion of cholesterol to corticosterone which occurs in the mitochondrial fraction, the conversion of 23,24-dinor-5-cholen-3 beta-ol to corticosterone occurs in the microsomal fraction. It was concluded that the sesterterpene pathway for steroid biosynthesis can function in the rat adrenal gland and that the intermediates are converted to steroid hormones in the microsomal fraction.     

Adrenal androgen biosynthesis by a sesterpene pathway

Rat and human adrenal gland preparations were incubated with radioactive cholesterol and 23,24-dinor-5-cholen-3 beta-ol, the latter being a proposed intermediate in the sesterterpene pathway for steroid biosynthesis. Steroids were isolated, purified by TLC and crystallised to constant specific activity. It was found that rat and human adrenal glands can utilise 23,24-dinor-5-cholen-3 beta-ol to produce androstenedione and dehydroepiandrosterone. Also, it was found that the conversion of 23,24-dinor-5-cholen-3 beta-ol to androgens occurs in the microsomal fraction. It was concluded that the sesterterpene pathway for steroid biosynthesis can function in the rat and human adrenal glands to produce androgens and that the intermediates are converted to androgens in the microsomal fraction.     

Inhibition of cholesterol side-chain cleavage by intermediates of an alternative steroid biosynthetic pathway

Mitochondrial preparations from endocrine tissues were incubated with radioactive cholesterol and the effect of hydroxylated metabolites of 23,24-dinor-5-cholen-3 beta-ol (23,24-dinor-5-cholene-3 beta,20-diol and 23,24-dinor-5-cholene-3 beta,21-diol) on the production of pregnenolone was measured. These compounds are intermediates in an alternative, sesterterpene pathway for steroid hormone biosynthesis. It was found that these materials, like the analogous side-chain-hydroxylated derivatives of cholesterol (20 alpha-hydroxycholesterol and 22S-hydroxycholesterol), inhibit cholesterol side-chain cleavage. The possibility that there could be a control mechanism whereby metabolites of 23,24-dinor-5-cholen-3 beta-ol inhibit steroidogenesis occurring by the cholesterol pathway is discussed.     

Biosynthesis of adrenal corticosteroids by the sesterterpene pathway via 23,24-dinor-4-cholen-3-one

Rat adrenal gland preparations were incubated with 23,24-dinor-5-cholen-3 beta-ol (Guneribol), a proposed intermediate in the sesterterpene pathway for steroid biosynthesis. Steroids were isolated, purified by thin layer and high-performance liquid chromatographies and crystallized to constant specific activity. These preparations converted the substrate to 23,24-dinor-4-cholen-3-one. Radioactive 23,24-dinor-4-cholen-3-one was synthesised and incubated with further tissue preparations and shown to be converted to corticosteroids. These findings suggest that 23,24-dinor-4-cholen-3-one is an intermediate on the sesterterpene pathway for steroidogenesis.     

Biosynthesis of androgens by the sesterterpene pathway via 23,24-dinor-4-cholen-3-one

Rat testicular and adrenal gland microsomal preparations were incubated with 23,24-dinor-5-cholen-3β-ol (Guneribol) a proposed intermediate in the sesterterpene pathway for steroid biosynthesis. Steroids were isolated, purified by thin layer and high-performance liquid chromatography and crystallized to constant specific radioactivity. These preparations converted the substrate to 23,24-dinor-4-cholen-3-one. Radioactive 23,24-dinor-4-cholen-3-one was synthesized and incubated with further tissue preparations and shown to be converted to steroid hormones. These findings suggest that 23,24-dinor-4-cholen-3-one is an intermediate on the sesterterpene pathway for steroidogenesis.     

Transfer of cholesterol and a fluorescent cholesterol analog, 3'-pyrenylmethyl-23,24-dinor-5-cholen-22-oate-3 beta-ol, between human plasma high density lipoproteins

A fluorescent cholesterol analog, 3'-pyrenylmethyl-23,24-dinor-5-cholen-22-oate-3 beta-ol (PMCA), has been synthesized as a spectroscopic probe of cholesterol function. The substrate activity of PMCA, about two-thirds that of cholesterol, with lecithin:cholesterol acyltransferase indicates that PMCA is a reasonable cholesterol analog and that the orientation of the substituted sterol in the phospholipid interface is similar to that of cholesterol. The fluorescence properties of PMCA are similar to those of other pyrene-containing compounds that exhibit concentration-dependent excimer fluorescence. The rate of transfer of [3H]PMCA between HDL is about six times faster than cholesterol. These results indicate that the analog will be useful in studies of cholesterol function.     

Amino-steroids as inhibitors and probes of the active site of cytochrome P-450scc. Effects on the enzyme from different sources

A series of analogues of cholesterol, each having a primary amine attached to a shortened side chain, were tested for their effects on cytochrome P-450scc from several different sources. Reconstituted enzyme systems using disrupted mitochondria from bovine adrenal and placenta, adult human adrenal and placenta, neonatal human adrenal, and rat adrenal and testis were used to assay for inhibitory effects on the side chain cleavage of cholesterol to pregnenolone. Two of the derivatives tested, 22-amino-23,24-bisnor-5-cholen-3 beta-ol and 23-amino-24-nor-5-cholen-3 beta-ol, were found to be potent inhibitors of this reaction; the derivatives in which the amine was attached closer to or further from the steroid ring, (20 R and S)-20-amino-5-pregnen-3 beta-ol and 24-amino-5-cholen-3 beta-ol, were much weaker inhibitors. In addition, spectral studies with rat adrenal mitochondria and a soluble preparation of human placental cytochrome P-450scc showed that binding of the 22-amine derivative to the enzyme produces difference spectra characteristic of nitrogen bonding to the heme; this indicates that the heme is positioned close to C-22 in the steroid-enzyme complex. These findings on the relative effectiveness of the amino-steroid inhibitors and the type of complex formed are similar to results obtained with purified bovine adrenocortical cytochrome P-450scc. This establishes that the proximity of the substrate binding site and the heme-iron catalytic site is a feature common to the enzyme from several sources and is therefore likely to be a necessary property of the active site structure.     

Competitive inhibition of cytochrome P-450scc by (22R)- and (22S)-22-aminocholesterol. Side-chain stereochemical requirements for C-22 amine coordination to the active-site heme

Two diastereomeric aminocholesterols, (22R)-22-aminocholesterol and (22S)-22-aminocholesterol, are both found to be potent inhibitors of the biosynthesis of pregnenolone from cholesterol by purified bovine mitochondrial P-450scc. Both steroids are competitive versus cholesterol, but the stereochemically correct analog (22R)-22-aminocholesterol is bound approximately 1000 times more tightly than (22S)-22-aminocholesterol. The dissociation constants are 25 nM and 13 microM, respectively. Direct comparisons between spectroscopic and enzymatic properties of the two enzymesterol complexes and the 22-amino-23,24-bisnor-5-cholen-3 beta-ol complex are made, underlining the importance of the stereochemistry at the C-22 position.     

The conversion of 23,24-dinor-5-cholen-3β-ol to cortisol by the canine adrenal

An alternative pathway for steroidogenesis, via a sesterterpene, has been proposed. This communication presents evidence that the canine adrenal can utilise 23,24-dinor-5-cholen-3β-ol to biosynthesise cortisol.It has been proposed that steroid hormones could be biosynthesised by a pathway other than that through cholesterol, possibly by a sesterterpene pathway (1,2).The previous studies were carried out using bovine adrenal tissue. This communication extends these studies to include the canine adrenal gland.     

Active site-directed inhibitors of cytochrome P-450scc. Structural and mechanistic implications of a side chain-substituted series of amino-steroids

A series of analogues of cholesterol, each having a shortened side chain and a primary amine group, were prepared and tested for their effects on bovine adrenocortical cholesterol side chain cleavage cytochrome P-450 (P-450scc). A previous study had shown that one derivative, 22-amino-23,24-bisnor-5-cholen-3 beta-ol, is a potent competitive inhibitor of the enzyme and forms a complex in which the steroid ring binds to the cholesterol site and the side chain amine forms a bond with the heme iron (Sheets, J. J., and Vickery, L. E. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 5773-5777). In the studies reported here, the 23-amine derivative, 23-amino-24-nor-5-cholen-3 beta-ol, was found to be an equally potent inhibitor and to be competitive with respect to cholesterol (Ki = 38 nM). Binding of the 23-amine to P-450scc also caused formation of a low spin complex with an absorption maximum at 422 nm, indicative of a nitrogen-donor ligand. Other derivatives in which the side chain amine was linked closer to the steroid, 17 beta-amino-5-androsten-3 beta-ol and (20 R + S)-20-amino-5-pregnen-3 beta-ol, were found to be only very weak inhibitors (I50 greater than 100 microM) and did not produce the 422 nm spectral form when bound. Derivatives in which the amine was attached a greater distance from the steroid ring, 24-amino-5-cholen-3 beta-ol and 25-amino-26,27-bisnor-5-cholesten-3 beta-ol, caused a progressive decrease in inhibitory potency and a failure to produce the 422 nm form on binding. The dependence of the type of interaction of these amino-steroids with P-450scc upon the amine position establishes that the steroid binding site and the heme catalytic site of the enzyme are fixed within a specific distance of one another. The heme appears to be located sufficiently close to the position that the side chain of cholesterol would occupy to allow for direct attack of an iron-bound oxidant to occur during hydroxylation and side chain cleavage.     

The formation and reduction of the 14,15-double bond in cholesterol biosynthesis

It was shown that 100mug quantities of 4,4'-dimethyl[2-(3)H(2)]cholesta-8,14-dien-3beta-ol (IIIa), tritiated cholesta-8,14-dien-3beta-ol, 4,4'-dimethyl[2-(3)H(2)]cholesta-7,14-dien-3beta-ol, dihydro[2-(3)H(2)]lanosterol and [24-(3)H]lanosterol were converted by a 10000g supernatant of rat liver homogenate into cholesterol in 17%, 54%, 6%, 9.5% and 24% yields respectively. From an incubation of dihydro[3alpha-(3)H]lanosterol with a rat liver homogenate in the presence of a trap up to 38% of the radioactivity was found to be associated with a fraction that was unambiguously shown to be 4,4'-dimethylcholesta-8,14-dien-3beta-ol. Another related compound, 4,4'-dimethylcholesta-7,14-dien-3beta-ol was also shown to be equally effective in its ability to trap compound (IIIa) from an incubation of dihydro[3alpha-(3)H]lanosterol. The mechanism of the further conversion of the compound (IIIa) into cholesterol occurred by the reduction of the 14,15-double bond and involved the addition of a hydrogen atom from the medium to C-15 and another from the 4-position of NADPH to C-14. Two possible mechanisms for the removal of the 14alpha-methyl group in sterol biosynthesis are discussed.     

Digital imaging fluorescence microscopy: spatial heterogeneity of photobleaching rate constants in individual cells

Photobleaching and related photochemical processes are recognized experimental barriers to quantification of fluorescence by microscopy. We have measured the kinetics of photobleaching of fluorophores in living and fixed cells and in microemulsions, and have demonstrated the spatial variability of these processes within individual cells. An inverted fluorescence microscope and a high-sensitivity camera, together with high-speed data acquisition by a computer-controlled image processor, have been used to control precisely exposure time to excitation light and to record images. To improve the signal-to-noise ratio, 32 digital images were integrated. After correction for spatial variations in camera sensitivity and background fluorescence, the images of the relative fluorescence intensities for 0.065 micron2 areas in the object plane were obtained. To evaluate photobleaching objectively, an algorithm was developed to fit a three-parameter exponential equation to 20 images recorded from the same microscope field as a function of illumination time. The results of this analysis demonstrated that the photobleaching process followed first-order reaction kinetics with rate constants that were spatially heterogeneous and varied, within the same cell, between 2- and 65-fold, depending on the fluorophore. The photobleaching rate constants increased proportionally with increasing excitation intensity and, for benzo(a)pyrene, were independent of probe concentration over three orders of magnitude (1.25 microM to 1.25 mM). The propensity to photobleach was different with each fluorophore. Under the cellular conditions used in these studies, the average rates of photobleaching decreased in this order: N-(7-nitrobenz-2-oxa-1,3-diazole)-23,24-dinor-5-cholen-22-amine-3 beta-ol greater than acridine orange greater than rhodamine-123 greater than benzo(a)pyrene greater than fluorescein greater than tetramethylrhodamine greater than 1,1'dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine. The photobleaching appears to be an oxidation reaction, in that the addition of saturated solutions of Na2S2O5 to mineral oil microemulsions eliminated photobleaching of N-(7-nitrobenz-2-oxa-1,3-diazole)-23,24-dinor-5-cholen-22-amine-3 beta-ol or benzo(a)pyrene. We identified experimental conditions to observe, without detectable photobleaching, fluorophores in living cells, which can not be studied anaerobically. Useful images were obtained when excitation light was reduced to eliminate photobleaching, as determined from zero-time images calculated from the exponential fit routine.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cellular uptake and intracellular localization of benzo(a)pyrene by digital fluorescence imaging microscopy

Uptake of benzo(a)pyrene by living cultured cells has been visualized in real time using digital fluorescence-imaging microscopy. Benzo(a)pyrene was noncovalently associated with lipoproteins, as a physiologic mode of presentation of the carcinogen to cells. When incubated with either human fibroblasts or murine P388D1 macrophages, benzo(a)pyrene uptake occurred in the absence of endocytosis, with a halftime of approximately 2 min, irrespective of the identity of the delivery vehicles, which were high density lipoproteins, low density lipoproteins, very low density lipoproteins, and 1-palmitoyl-2-oleoylphosphatidylcholine single-walled vesicles. Thus, cellular uptake of benzo(a)pyrene from these hydrophobic donors occurs by spontaneous transfer through the aqueous phase. Moreover, the rate constant for uptake, the extent of uptake, and the intracellular localization of benzo(a)pyrene were identical for both living and fixed cells. Similar rate constants for benzo(a)pyrene efflux from cells to extracellular lipoproteins suggests the involvement of the plasma membrane in the rate-limiting step. The intracellular location of benzo(a)pyrene at equilibrium was coincident with a fluorescent cholesterol analog, N-(7-nitrobenz-2-oxa-1,3-diazole)-23,24-dinor-5-cholen-22-amine-3 beta-ol. Benzo(a)pyrene did not accumulate in acidic compartments, based on acridine orange fluorescence, or in mitochondria, based on rhodamine-123 fluorescence. When the intracellular lipid volume of isolated mouse peritoneal macrophages was increased by prior incubation of these cells with either acetylated low density lipoproteins or with very low density lipoproteins from a hypertriglyceridemic individual, cellular accumulation of benzo(a)pyrene increased proportionately with increased [1-14C]oleate incorporation into cellular triglycerides and cholesteryl esters. Thus, benzo(a)pyrene uptake by cells is a simple partitioning phenomenon, controlled by the relative lipid volumes of extracellular donor lipoproteins and of cells, and does not involve lipoprotein endocytosis as an obligatory step.     

A fluorescent cholesterol analog traces cholesterol absorption in hamsters and is esterified in vivo and in vitro.

The fluorescent cholesterol analog 22-(N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3beta-ol (fluoresterol) was characterized as a tool for exploring the biochemistry and cell biology of intestinal cholesterol absorption. Hamsters absorbed fluoresterol in a concentration- and time-dependent manner, with an efficiency of about 15-30% that of cholesterol. Fluoresterol absorption was blocked by compounds known to inhibit cholesterol absorption, implying that fluoresterol interacts with those elements of the normal pathway for cholesterol absorption on which the inhibitors act. Confocal microscopy of small intestinal tissue demonstrated that fluoresterol was taken up by absorptive epithelial cells and packaged into lipoprotein particles, suggesting a normal route of intracellular trafficking. Uptake of fluoresterol was confirmed by biochemical analysis of intestinal tissue, and a comparison of [(3)H] cholesterol and fluoresterol content in the mucosa suggested that fluoresterol moved through the enterocytes more rapidly than did cholesterol. This interpretation was supported by measurements of fluoresterol esterification in the mucosa. Four hours after hamsters were given fluoresterol and [(3)H]cholesterol orally, 44% of the fluoresterol in the intestinal mucosa was esterified, compared to 8% of the [(3)H]cholesterol. Caco-2 cells took up 2- to 5-fold more [(3)H]cholesterol than fluoresterol from bile acid micelles, and esterified 21-24% of the fluoresterol but only 1-4% of the [(3)H]cholesterol. Thus fluoresterol apparently interacts with the proteins required for cholesterol uptake, trafficking, and processing in the small intestine.     

The biosynthesis of some androst-16-enes from C21 and C19 steroids in boar testicular and adrenal tissue

1. The formation of androst-16-enes from [4-(14)C]progesterone has been investigated with long-term incubations and short-term kinetic studies. After 4hr., 1.7 and 10.3% respectively of 3alpha- and 3beta-hydroxy-5alpha-androst-16-enes were formed in boar testis minces, but much smaller yields were obtained in boar adrenal. Both tissues formed small quantities of androsta-4,16-dien-3-one. 2. The amounts of androst-4-ene-3,17-dione and testosterone isolated were small, suggesting that androst-16-ene formation may occur preferentially in the boar testis. 3. In the absence of tissue no radioactive androst-16-enes were formed. 4. Incubation of both [4-(14)C]pregnenolone and [7alpha-(3)H]progesterone resulted in 3alpha- and 3beta-hydroxy-5alpha-androst-16-enes containing (3)H/(14)C ratios of near unity and confirmed that both C(21) steroids were precursors. A similar incubation with 17alpha-hydroxy[4-(14)C]-progesterone and [7alpha-(3)H]progesterone gave the same Delta(16)-alcohols, but they contained only (3)H, indicating that side-chain cleavage of pregnenolone and progesterone occurred before 17alpha-hydroxylation. 5. Dehydroepiandrosterone, testosterone, testosterone acetate and 16-dehydroprogesterone were not found to be precursors of Delta(16)-steroids. 6. A pathway is proposed for the biosynthesis of 3alpha- and 3beta-hydroxy-5alpha-androst-16-enes from pregnenolone and progesterone; this may involve androsta-4,16-dien-3-one as an intermediate, but excludes 17alpha-hydroxyprogesterone, testosterone and dehydroepiandrosterone.     

A novel olefinic rearrangement. The enzymic conversion of cholesta-7,9-dien-3β-ol into cholesta-8,14-dien-3β-ol

1. [3alpha-(3)H]Cholesta-7,9-dien-3beta-ol is converted in high yield into cholesterol by a 10000g(av.) supernatant fraction of rat liver homogenate. 2. Incubation of cholesta-7,9-dien-3beta-ol with [4-(3)H]NADPH and rat liver microsomal fractions under anaerobic conditions resulted in (3)H being incorporated into the 14alpha-position of cholest-7-en-3beta-ol. 3. Under anaerobic conditions in the absence of NADPH cholesta-7,9-dien-3beta-ol was isomerized into cholesta-8,14-dien-3beta-ol by rat liver microsomal fractions.     

Sterol synthesis: studies of the metabolism of 14 alpha-methyl-5 alpha-cholest-7-en-3 beta-ol

[3 alpha-3H]14 alpha-Methyl-5 alpha-cholest-7-en-3 beta-ol has been prepared by chemical synthesis. The metabolism of this compound has been studied in the 10,000 g supernatant fraction of liver homogenates of female rats. Efficient conversion to cholesterol was observed. Other labeled compounds recovered after incubation of [3 alpha-3H]14 alpha-methyl-5 alpha-cholest-7-en-3 beta-ol with the enzyme preparations include the unreacted substrate, 5 alpha-cholesta-7,14-dien-3 beta-ol, 5 alpha-cholesta-8,14-dien-3 beta-ol, cholesta-5,7-dien-3 beta-ol, 5 alpha-cholest-8(14)-en-3 beta-ol, 5 alpha-cholest-8-en-3 beta-ol, and 5 alpha-cholest-7-en-3 beta-ol. In addition, significant amounts of incubated radioactivity were recovered in steryl esters. The steroidal components of these esters were found to contain labeled 14 alpha-methyl-5 alpha-cholest-7-en-3 beta-ol, 5 alpha-cholesta-8,14-dien-3 beta-ol, 5 alpha-cholesta-7,14-dien-3 beta-ol, 5 alpha-cholest-8-en-3 beta-ol, 5 alpha-cholest-7-en-3 beta-ol, and cholesterol.     

The biosynthesis of 23,24-dinor-5-cholene-3beta,20-diol and 23,24-dinor-5-cholene-3beta,21-diol

An alternative pathway for steroidogenesis, via a sesterterpene, has been proposed. This communication presents evidence that two of the proposed compounds with the 23,24-dinorcholane carbocyclic system, 23,24-dinor-5-cholene-3β,20-diol and 23,24-dinor-5-cholene3β,21-diol, can be biosynthesised from sodium [³H]acetate in a bovine adrenal preparation.     

Studies on the biosynthesis of 16-dehydro steroids: The metabolism of [4-14C]pregnenolone by boar adrenal and testis tissue in vitro

1. The metabolism of [4-(14)C]pregnenolone in vitro by boar adrenocortical and testis tissue has been studied. 2. Boar testis tissue formed three labelled Delta(16)-steroids, 5alpha-androst-16-en-3alpha-ol, 5alpha-androst-16-en-3beta-ol and androsta-4,16-dien-3-one. In adrenal tissue very much smaller yields of the same metabolites were obtained. 3. Both tissues produced labelled progesterone, androst-4-ene-3,17-dione and testosterone in varying quantities. The amount of progesterone was about 120 times greater in the adrenal tissue. In testis tissue dehydroepiandrosterone was found only in small quantity. 4. A pathway is suggested for the biosynthesis of Delta(16)-steroids from pregnenolone in boar testis tissue. The possibility that progesterone may be an intermediate is discussed.     

Deoxycholic acid degradation by a Pseudomonas species. Acidic intermediates from the initial part of the catabolic pathway.

The microbial catabolism of deoxycholic acid by a Pseudomonas species was studied, and three acidic products were isolated as their methyl esters. Evidence is presented that the compounds are methyl 3 alpha,12 alpha-dihydroxy-23,24-dinor-5 beta-cholan-22-oate, methyl 12 alpha-hydroxy-3-oxo-5 beta-cholan-24-oate and methyl 12 alpha-hydroxy-3-oxo-23,24-dinor-5 beta-cholan-22-oate.