THE SYNTHESIS AND METABOLISM OF [6-3H]-25-HYDROXYCHOLESTEROL IN RAT ADRENAL TUMOR CELLS
J. IAN MASON*, BARBARA A. MURRY* and D. JOHN ABERHARTt
*Cecil H. and Ida Green Center for Reproductive Biology Sciences, the Departments of Biochemistry and Obstetrics-Gynecology of the University of Texas Southwestern Medical School, 5323 Harry Hines Boulevard, Dallas, TX 75235, +Worcester Foundation for Experimental Biology, Shrewsbury, MA 01545, U.S.A.
(Received 10 January 1983)
SUMMARY
The synthesis of [6-3H]-25-hydroxycholesterol from 26-norcholest-5-en-3B-ol-25-one is described. The metabolism of the radiolabeled hydroxysterol in rat adrenocortical carcinoma cells and in mitochondria- enriched preparations of rat adrenal tissue was investigated. We found that [6-3H]-25-hydroxycholes- terol was metabolized efficiently to [3H]-pregnenolone in both preparations of rat adrenal tissue.
INTRODUCTION
Several investigations of the regulation of cholesterol metabolism have involved the use of 25-hydroxycho- lesterol, although the possible physiological import- ance of this sterol is not clear. The sterol has been used in two principal situations. First, 25-hydroxy- cholesterol is a potent inhibitor of cholesterol biosyn- thesis, apparently acting at the level of 3-hydroxy-3- methylglutaryl CoA reductase, the rate-limiting enzyme of cholesterol synthesis [1]. Second, the hy- droxysterol is a substrate of the cholesterol desmolase that is located in the mitochondria of steroidogenic tissue [2]. Importantly, 25-hydroxycholesterol appears to traverse both the plasma membrane and the mito- chondrial membrane rapidly without the need of transporting proteins such as apolipoproteins or the labile protein factor that is required in the mobiliza- tion of cholesterol for mitochondrial steroidogenesis [3-6]. Although 25-hydroxycholesterol radiolabeled in the octyl side chain (i.e. [26,27-3H]-25-hydroxy- cholesterol) is available commercially, in investiga- tions of the hormonal regulation of steroidogenesis there is a need for sterol that is radiolabeled in the steroid nucleus. The availability of such a radio- labeled 25-hydroxycholesterol also would permit the confirmation that pregnenolone is the initial product of 25-hydroxycholesterol metabolism in steroidogenic cells, since previously, investigators have relied pri-
# Please address correspondence to: Dr J. I. Mason, Cecil H. and Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical School, 5323 Harry Hines Boulevard, Dallas, TX 75235, U.S.A.
The following trivial names are used in the text: 25-hy- droxycholesterol, 5-cholesten-38,25-diol; pregnenolone, 3ß- hydroxy-5-pregnene-20-one; pregnenolone acetate, 38-ace- toxy-5-pregnene-20-one; cyanoketone 2-cyano-4,4,17x- trimethyl-17ß-hydroxy-5-androsten-3-one.
marily on the chromatographic properties and high cross-reactivity of the product with antisera to preg- nenolone-albumin conjugates [3, 6].
In this investigation, we developed a convenient method of synthesis of [6-3H]-25-hydroxycholesterol and the radiolabeled sterol was characterized. We have investigated the metabolism of the radiolabeled sterol in dispersed cells of a rat adrenocortical carci- noma as well as in mitochondria-enriched prep- arations of rat adrenal tissue.
MATERIALS AND METHODS
The scheme used for the synthesis of [6-3H]-25- hydroxycholesterol was analogous to the first steps in a recent synthesis of [6-3H]-1x-hydroxycholecalci- ferol [7]. 26-Norcholest-5-en-30-ol-25-one, 1 was con- verted by the method of Dauben and Bradlow[8] to 25-hydroxycholesterol diacetate, 2a. Hydrobora- tion [9] of 2a, followed by treatment with H2O2 in the presence of a phosphate buffer, pH 7 [10], gave a moderate yield of 5g-cholestane-38,6x,25-triol-3,25- diacetate, 3, which was oxidized with Jones re- agent [11] to 5x-cholestan-6-one-30,25-diol diacetate, 4. Treatment of 4 with NaBH4 or [3H] NaBH4 in glyme [12] led to the formation of 5x-cholestane- 38,60,25-triol-3,25-diacetate, 5a or 5b, respectively. Treatment of 5b with POC13 in pyridine [13] gave [6-3H]-25-hydroxycholesterol diacetate, 2b, which was converted to [6-3H]-25-hydroxycholesterol, 6, by treatment with LiAIH4. The structures of the sterols under discussion are given in Fig. 1.
A dispersed cell preparation of rat adrenocortical carcinoma cells was obtained from a rat adrenocorti- cal Snell 494 carcinoma grown in 4 week-old male Sprague-Dawley rats (Charles River Breeding Labor- atories, Wilmington, MA). These procedures have been described in detail previously [3]. Adrenocorti-
0
OAc
HO
AcO
R
1
2
& R=H
b R = 3H
OAc
OAc
ACO
H
#
AcO
OH
H
0
3
4
OAc
OH
AcO
H
HO
3H
5
HO
R
a R = H
6
b R = 3H
cal tumor cells (106 cells per 0.5 ml) were incubated in Krebs-Ringer phosphate buffer, pH 7.4, that con- tained 0.5% bovine serum albumin (Fraction V) and glucose (0.2%) in the presence of ACTH (20 nM) and cyanoketone (15 uM). The [6-3H]-25-hydroxycholes- terol (20 uM) was added in ethanol; the final concen- tration of ethanol in the incubation mixture was less than 1%. A maximal rate of pregnenolone formation was obtained with 20 uM 25-hydroxycholesterol. De- creased pregnenolone formation was seen at concen- trations of 25-hydroxycholesterol above 50 uM. This was likely due to the known membrane-disruptive action of the hydroxysterol. After incubation at 37℃ for varying times the cells and media were extracted with chloroform-methanol (2:1, v/v). The concen- trated organic layer was applied to thin-layer chroma- toplates (solvent; chloroform-ethyl acetate, 4:1 v/v). Radioactive regions on the chromatograms were located with a radiochromatographic scanner and visualization of authentic standards. Radioactivity was quantified by liquid scintillation spectrometry. After acetylation of the radiolabeled product in acetic anhydride in pyridine, repeated crystallization with nonradiolabeled pregnenolone 3ß-acetate was per- formed.
Male Sprague-Dawley rats (150-200 g), obtained from Charles River Breeding Laboratories (Wilming- ton, MA), were treated by ether anesthesia for 10 min, a process that increases blood ACTH levels [14]. A mitochondria-enriched fraction from the homogen- ates of adrenal glands was prepared as previously de- scribed [4] and the mitochondrial pellet was washed
once with sucrose (0.25 M). The resultant pellet was suspended in the same medium at a protein concen- tration of about 5 mg protein x ml-1. Protein con- tent was estimated using the method of Lowry with bovine serum albumin as the standard [15]. Sterol desmolase activity was assayed in a buffer, pH 7.4, that was composed of: sucrose (250 mM), KCI (20 mM), triethanolamine hydrochloride (15 mM), potassium phosphate (10 mM), MgCl2 (5 mM), EDTA (0.2 mM), NADP+ (0.2 mM) and 0.1% (w/v) bovine serum albumin (essentially fatty-acid-free) at a mito- chondrial protein concentration of about 1 mg pro- tein x ml-1 in a total volume of 1 ml. The 38-hy- droxysteroid dehydrogenase inhibitor, cyanoketone, was included in incubation mixtures (final concen- tration, 15 µM) to prevent further metabolism of preg- nenolone. The reaction was initiated by the addition of DL-isocitrate (10 mM) after the mitochondria were incubated at 37℃ for 10 min with [6-3H]-25-hy- droxycholesterol (50 µM) added in 10 ul ethanol. We found on the basis of initial experiments that the above concentrations of mitochondrial protein and sterol gave optimal sterol desmolase activity [4]. Ali- quots (0.2 ml) of the reaction medium were transferred into methanol (4 ml) to terminate the reaction after 2. 5 and 10 min incubation times after the addition of isocitrate. Chloroform (5 ml) and water (4 ml) were added to the extract and the concentrated organic layer was chromatographed as described before.
26-Norcholest-5-en-3B-ol-25-one was obtained from Steraloids, Inc. [3H]-NaBH4, specific activity, 5.0 Ci/mmol, was obtained from Amersham Corp ..
Arlington Heights, IL. Glyme (1,2-dimethoxyethane) and tetrahydrofuran (THF) were distilled from LiAIH4. Pyridine was distilled from BaO. Thin layer chromatography (t.l.c.) was performed with plates pre- pared with E. Merck silica gel HF 254 + 366. Radiochromatogram scanning was performed with a Nuclear Chicago Actigraph III instrument. Liquid scintillation spectrometry was performed with a Nuclear Chicago Mark II instrument. Samples were dissolved in 10 ml New England Nuclear Aquasol. NMR spectra were conducted in CDC13 solutions with internal TMS as reference, on a Varian EM-360 instrument. Melting points were taken on a hot stage apparatus, and are corrected. Microanalyses were per- formed by Galbraith Laboratories, Knoxville, TN. The mass spectrum was obtained by Shrader Analyti- cal and Consulting Laboratories, Detroit, Michigan, by use of an AEI-MS-30 mass-spectrometer at 70 eV.
RESULTS
Characterization of steroids in the synthetic scheme for the preparation of [6-3 H]-25-hydroxycholesterol
5a-Cholestane-30,60,25-triol-3,25-diacetate, 3. 25-Hy- droxycholesterol diacetate, 2a (595 mg, 1.18 mmol) in 11 ml dry THF under N2 was treated with 1.0 M bor- ane-THF solution (1.5 ml) at 25℃ for 5 h. Thereafter, phosphate buffer (2 M, pH 7.0, 2 ml) was added fol- lowed by 30% H2O2 (0.55 ml) and 95% EtOH (4.5 ml), and the mixture was stirred at 25℃ for 16h. Ad- ditional H2O (25 ml) was added, and the mixture was extracted with ether. The ether extract was washed with saturated NaCI, dried (Na2SO4), and evaporated to yield crude 3 as a glass (593 mg) that was purified by preparative t.l.c. (solvent: hexane-ethyl acetate, 3:1, v/v, RF 0.4) to yield pure 3 as a noncrystalline glass (260 mg); nmr 8 0.78 (3H, s, 18 Me), 0.85 (3H, s, 19 Me), 1.44 (6H, s, 26,27 Me), 1.98 (3H, s, OAc), 2.03 (3H, s, OAc), 3.40 (1H, br s, W1 = 16 Hz, 68 H), 4.67 (1H, br s, W] = 20 Hz, 3x H).
In the mass spectrum of 2a a molecular ion was not indicated, but peaks at m/z 444 (25%; M-CH3CO2H), 429 (22%; M-CH3CO2H-CH3), 426 (11%; M- CH3CO2H-H2O), 284 (24%; M-2 × CH3CO2H), 369 (16%; M-2× CH3CO2H-CH3), 366 (28%; M-2 x CH3CO2H-H2O). 332 (21%). 331 (60%). 313 (25%), 231 (32%). 213 (37%), 147 (20%), 133 (24%), 123 (52%), 122 (31%), 121 (25%), 109 (46%), 107 (35%), 105 (28%), 95 (100%), 94 (23%), 93 (35%), 82 (23%), 81 (55%) were found.
5x-Cholestan-6-one-30,25-diol diacetate, 4. The above product, 3 (244 mg, 0.48 mmol) in 40 ml ace- tone was treated with Jones reagent (2 ml) at 25℃ for 5 min. The mixture was diluted with H2O (100 ml) and extracted with ether. The extract was washed with H2O and thence with a solution that was satu- rated with NaCl, dried (Na2SO4) and evaporated to yield 4, 225 mg, prisms from MeOH, mp 140-141°C; NMR 8 0.67 (3H, s, 18 Me), 0.77 (3H, s, 19 Me). 1.42
(6H, s), 1.97 (3H, s, OAc), 2.02 (3H, s, OAc), 4.65 (1H, br s, W1 = 20 Hz, 3x H).
Anal. Calcd for C31H50O5: C, 74.06; H, 10.03. Found: C, 74.32; H. 10.33.
5x-Cholestane-3,60,25-triol-3,25-diacetate, 5a. The above product, 4 (60 mg, 0.119 mmol) in dry glyme (2 ml) was treated with NaBH4 (50 mg) with stirring at 25℃ under N2 for 24 h, then at 50℃ for 7 h. The mixture was diluted with H2O (50 ml) and treated with 3 drops conc HCI. The mixture was then extracted with ether and the extract was washed with H2O and a solution that was saturated with NaCl, dried (Na2SO4) and evaporated to yield a crude product (48 mg). Pure 5a, 31 mg, was then isolated by preparative t.l.c. (solvent: hexane-ethyl acetate 3:1, v/v, RF 0.5) and crystallized from MeOH, plates, m.p. 153-154°C; n.m.r. 8 0.74 (3H, s, 18 Me), 1.06 (3H, s, 19 Me), 1.44 (6H, s, 26, 27 Me), 1.99 (3H, s, OAc), 2.06 (3H, s, OAc), 3.76 (1H, br s, WA = 7 Hz, 6% H), 4.70 (1H, br s, WA = 20 Hz, 3x H).
Anal. Calcd for C31H5205: C, 73.77; H. 10.38. Found: C, 73.99; H, 10.13.
[6-3H]-25-Hydroxycholesterol, 6. 5x-Cholestan-6- one-3,25-diol diacetate, 4 (50 mg) in dry glyme (2 ml) was treated under N2 with [3H]NaBH4 (100 mCi, 0.8 mg) with stirring at 25°℃ for 24 h, then at 50℃ for 24 h. Then additional NaBH4 (28 mg) was added and the mixture was stirred at 50℃ for an additional 24 h. A crude product (45 mg, 5 x 1010 d.p.m.) was obtained that consisted mainly (>90%) of [6-3H]-5x- cholestane-30,60,25-triol-3,25-diacetate, 5b, as shown by radiochromatogram scanning. All of the product was dissolved in dry pyridine (1.5 ml) and treated with POC13 (0.45 ml) at 28℃ for 18 h. The resultant black solution was added to 10 ml of ice-cold H2O and the mixture was extracted with ether. The extract was washed with a solution that was saturated with NaCl, dried (Na2SO4), and evaporated to a semi-crystalline residue (45 mg, 3.9 × 101º d.p.m.) from which chromatographically pure [6-3H]-25-hydroxycholes- terol diacetate, 2b, (27 mg, 2.5 × 101º d.p.m.) was iso- lated by preparative t.l.c. (solvent: hexane-ethyl acet- ate, 9:1, v/v). This product in absolute ether (10 ml), was treated with LiAIH4 (100 mg) at reflux for 3.5 h. After cooling to 0°C, saturated Na2SO4 was added dropwise to yield a flocculent precipitate that was fil- tered and washed with ether. The filtrate was evapor- ated to yield radiochromatographically pure [6-3H]- 25-hydroxycholesterol, 6 (14.7 mg, 1.6 x 101º d.p.m.), (RF 0.27, solvent: hexane-ethyl acetate 3:1, v/v), which was rendered crystalline by dissolving in a small volume of MeOH and then by evaporation under reduced pressure.
Metabolism of [6-3 H]-25-hydroxycholesterol in rat adrenocortical carcinoma cells
The incubation of radiolabeled 25-hydroxycholes- terol (50 µM) with dispersed cells of Snell 494 adreno- cortical carcinoma cells resulted in the production of a single principal metabolite with a mobility on t.l.c.
50
PERCENT CONVERSION
25
0
0
1
2
TIME (hours)
similar to pregnenolone. The time course of formation of this metabolite is illustrated in Fig. 2. The initial rate of formation of this metabolite was 3 nmol x 30 min-1 x (106 cells)-1. It should be noted that cya- noketone was present in these assays to prevent further metabolism of pregnenolone to progesterone. We confirmed the identity of the metabolite after acetylation of the radiolabeled product with acetic an- hydride in a solution of pyridine and subsequent repeated crystallizations with authentic pregnenolone 36-acetate. After addition of 32,800 c.p.m. of acety- lated radiolabeled metabolite to 57.1 mg of pregneno- lone 3B-acetate, the respective specific activities of crystals and mother liquor of sequential crystal- lizations were 477.5, 454.2, 477.5 and 471.1 c.p.m. x mg -1.
A second but very minor metabolite was found (RF 0.76) on the chromatogram that was developed in chloroform-ethyl acetate (4:1, v/v). The rate of forma- tion of this metabolite was less than 5% that of the principal metabolite. This minor metabolite was less “polar” than pregnenolone (RF, 0.52) but more “polar” than cholesterol ester (RF = 1.0). This product possibly is an acyl ester of 25-hydroxycholes- terol, since the mobility of 25-hydroxycholesterol-3฿- acetate was similar. Further characterization of this minor metabolite was not attempted with the small amount of radiolabeled material available.
Metabolism of [6-3H]-25-hydroxycholesterol in mito- chondria-enriched preparations of rat adrenal
As illustrated in Fig. 3, adrenal mitochondria metabolized the radiolabeled 25-hydroxycholesterol to a single product with a t.l.c. mobility similar to that of pregnenolone. The initial rate of product formation was 3.6 nmol x min-1 x mg-1 protein. We con- firmed the identity of the product as follows: the radiolabeled product was extracted from the silica gel with chloroform-methanol (2:1, v/v) and subsequently acetylated with acetic anhydride in pyridine. In repeated crystallizations with authentic pregnenolone acetate there was little change in the specific activity of the pregnenolone acetate crystals. Thus, in the
nmol steroid x mg-1 protein
50
25
0
0
5
10
Time (min)
presence of cyanoketone (an inhibitor of 3ß-hydroxy- steroid dehydrogenase-isomerase activity) pregneno- lone was the single product of 25-hydroxycholesterol metabolism in rat adrenal mitochondria detected.
DISCUSSION
In the present investigation, we synthesized [6-3H]-25-hydroxycholesterol starting from 26-nor- cholest-5-en-30-ol-25-one. Appropriate chemical authentication of the identity of intermediates and the final product are described.
Based on the data presented in Figs 2 and 3 as well as the recrystallization experiments, we conclude that [6-3H]-25-hydroxycholesterol is converted very effi- ciently to [3H]-pregnenolone in both rat adrenocor- tical carcinoma cells and rat adrenal mitochondria when compared to the metabolism of cholesterol in similar preparations [3,4].
In the rat adrenocortical carcinoma cells, the for- mation of a second minor metabolite of radiolabeled 25-hydroxycholesterol was observed. This metabolite possibly could be an acyl ester of 25-hydroxycholes- terol, but further characterization of this metabolite was not undertaken. It is not surprising that very little esterification of 25-hydroxycholesterol is observed in the Snell 494 adrenocortical carcinoma cells since it has been reported [16] that in this tumor there is low activity of microsomal acyl CoA:cholesterol acyl transferase (ACAT), the enzyme required to catalyze the esterification of 25-hydroxycholesterol. Further studies of 25-hydroxycholesterol metabolism to its acyl esters in cells with an active ACAT remain to be undertaken. Such studies will be facilitated by the use of [6-3H]-25-hydroxycholesterol. In particular, the relative flux of the sterol into pregnenolone and sterol ester in steroidogenic cells should provide insight into the fate of free sterol as it is generated in the cell.
Furthermore, since 25-hydroxycholesterol is metab- olized rapidly to pregnenolone in steroidogenic cells and subsequently metabolism of pregnenolone occurs, the use of [6-3H]-25-hydroxycholesterol will facilitate the identification of sterol metabolites in such cells.
Acknowledgements-This study was supported, in part, by Grant CA-30253, NCI. DHHS (J.I.M.) and Grant RR-05528, Division of Research Resources, DHHS (DJA). We are very grateful to Debbie Staley and Sylvia Williams for excellent editorial assistance.
REFERENCES
1. Kandutsch A. A. and Chen H. W .: Regulation of sterol synthesis in cultured cells by oxygenated derivatives of cholesterol. J. cell Physiol. 85 (1975) 415-424.
2. Jefcoate C. R., Simpson E. R. and Boyd G. S .: Spectral properties of rat adrenal mitochondrial cytochrome P450. Eur. J. Biochem. 42 (1974) 539-551.
3. Mason J. I. and Robidoux W. F .: Steroidogenesis in isolated cells and mitochondria of rat Snell adrenocor- tical carcinoma 494. Endocrinology 105 (1979) 1230-1236.
4. Mason J. I., Arthur J. R. and Boyd G. S .: Control of sterol metabolism in rat adrenal mitochondria. Bio- chem. J. 173 (1978) 1045-1051.
5. Falke H. E., Degenhart H. J., Abeln G. H. A. and Visser H. K. A .: Studies on isolated rat adrenal cells: metabolism of hydroxylated sterols. Mol. cell. Endocr. 3 (1975) 375-383.
6. Toaff M. E., Strauss III J. F., Flickinger G. I. and Shattil S. J .: Relationship of cholesterol supply to luteal mitochondrial steroid synthesis. J. biol. Chem. 254 (1979) 3977-3982.
7. Holick M. F., Holick S. A., Tavela T., Gallagher B., Schnoes H. K. and DeLuca H. F .: Synthesis of [6-3H]-1x-hydroxyvitamin D3 and its metabolism in vivo to [3H]-1x,25-Dihydroxyvitamin D3. Science 190 (1975). 576-578.
8. Dauben W. G. and Bradlow H. L .: The preparation of
cholesterol from 45-norcholestene-38-ol-25-one. J. Am. chem. Soc. 72 (1950) 4248-4250.
9. Brown H. C. and Sharp R. L. Hydroboration XXVII .: The hydroboration of 1-butenyl and related vinyl de- rivatives containing representative substituents. An un- usually powerful directive influence of the ethoxy sub- stituent. J. Am. chem. Soc. 90 (1968) 2915-2927.
10. Brown H. C. and Coleman R. A .: Reaction of B-alkyl-9-borabicyclo [3.3.1] nonanes with carbon monoxide in the presence of lithium tri-t-butoxy- aluminohydride. The conversion of functionally substi- tuted olefins into aldehydes via hydroboration. J. Am. chem. Soc. 91 (1969) 4606-4607.
11. Bowden K., Heilbron I. M., Jones E. R. H. and Wee- don B. C. L .: Researches on acetylenic compounds. I. The preparation of acetylenic ketones by oxidation of acetylenic carbinols and glycols. J. chem. Soc. (1946) 39-45.
12. Akhtar M. and Marsh S .: The stereochemistry of the hydrogen elimination in the biological conversion of cholest-7-en-38-ol into cholesterol. Biochem. J. 102, (1967) 462-467.
13. Aberhart D. J., Lloyd-Jones J. G. and Caspi E .: Bio- synthesis of cardenolides in digitalis lanata. Phyto- chemistry 12 (1973) 1065-1071.
14. Matsuyama H., Ruhmann-Wennhold A. and Nelson D. H .: Radioimmunoassay of plasma ACTH in intact rats. Endocrinology 88 (1971) 692-695.
15. Lowry O. H., Rosebrough N. J., Farr A. L. and Ran- dall R. J .: Protein measurement with the Folin phenol reagent. J. biol. Chem. 193 (1951) 267-275.
16. Lossow W. J., Shyamala G., Shah, S. and Chaikoff I. L .: Uptake, hydrolysis and synthesis of cholesterol esters by a transplantable adrenal cortical tumor. Proc. Soc. exp. Biol. Med. 119 (1965) 126-131.