BBA 51417
IN VITRO STUDIES OF THE ADRENAL METABOLISM OF HALOGENATED SIDE-CHAIN ANALOGUES OF CHOLESTEROL
J. IAN MASON ª, THANGAVEL ARUNACHALAM b and ELIAHU CASPI b
” The Cecil H. and Ida Green Center for Reproductive Biology Sciences, Departments of Biochemistry and Obstetrics and Gynecology, The University of Texas Southwestern Medical School, 5323 Harry Hines Boulevard, Dallas, TX 75235 and + Worcester Foundation for Experimental Biology, 222 Maple Avenue, Shrewsbury, MA 01545 (U.S.A.)
(Received January 4th, 1983) (Revised manuscript received April 1st, 1983)
Key words: Sterol metabolism; Bromocholesterol; Iodocholesterol; (Rat adrenal mitochondria)
The cholesterol side-chain cleavage enzyme system of rat adrenal mitochondria and rat adrenocortical carcinoma cells was found to metabolize three halogenated side-chain cholesterol analogues to pregnenolone. The analogues were 26-bromocholesterol, 26-nor-25(RS)-bromocholesterol and 26-iodocholesterol. The addition of Ca2+ to rat adrenal mitochondria did not produce an increase in the rate of metabolism of the halogenated sterol to pregnenolone. The brominated sterols suppressed the de novo sterol biosynthesis of rat adrenocortical carcinoma cells. The experimental findings are supportive of the notion that a halogen atom at such a position in a sterol is analogous to a hydroxyl group but unlike a proton. ACTH, therefore, may not be a requirement for the uptake and utilization of such sterols. The halogenated sterols may have a use as probes in the study of sterol transfer into and within cells.
Introduction
The use of radioactively labeled sterols, i.e., iodinated or seleno-derivatives of cholesterol, as adrenal-imaging agents has been investigated in several studies [1-3]. The derivatives investigated so far are those that involve modification of the cholesterol molecule by either nuclear substitution of the steriod ring or substitution at an angular methyl group. It would appear that these deriva- tives are not metabolized to the corresponding bile acids or steroid hormone analogues, but rather are
metabolized to yet other products [4]. In previous studies, it was shown that in adrenocortical mitochondria pregnenolone was synthesized from a variety of cholesterol analogues in which the isooctyl side chain of cholesterol had been sub- stituted at various positions by hydroxyl groups [5].
The present study was undertaken to investigate the metabolic transformation of halogenated side- chain derivatives of cholesterol in rat adrenal cortical mitochondria and in rat adrenocortical tumor cells. In the rat adrenal cortex, the regula- tion of steroid hormone biosynthesis appears to involve at least two mechanisms. Cholesterol is stored within rat adrenocortical cells as cholesterol esters. There is evidence that, when the adrenal gland is stimulated by ACTH, the cholesterol ester hydrolase enzyme present in the cytosol of the cell is activated, and hence acts to release nonesterified
Common names have been used as follows: 26-iodocholesterol, 26-iodo-5-cholesten-33-ol; 26-bromocholesterol, 26-bromo-5- cholesten-38-ol; 26-nor-25-bromocholesterol, 26-nor-25( RS)- bromo-5-cholesten-38-ol; 25-hydroxycholesterol, 5-cholesten- 38,25-diol; cyanoketone, 2a-cyano-4,4,17a-trimethyl-178-hy- droxy-5-androsten-3-one.
cholesterol from the cholesterol ester stores [6]. The uptake of the nonesterified cholesterol by adrenocortical mitochondria is facilitated in an ACTH-mediated process. It would appear, how- ever, that rat adrenocortical carcinoma cells lack such a store of cholesterol esters and rely on de novo synthesis of cholesterol and on direct utiliza- tion of lipoprotein cholesterol as sources of steroidogenic cholesterol [7-9]. First, we evaluated the rate of metabolism of 26-nor-25-bromocho- lesterol, 26-bromocholesterol and 26-iodocho- lesterol to pregnenolone in rat adrenocortical mitochondria and compared these rates to that of the metabolism of cholesterol. Second, we have studied the ability of rat adrenocortical carcinoma cells to take up and utilize these halogenated sterols for pregnenolone biosynthesis. Third, the ability of these halogenated sterols to suppress the incorpo- ration of [1-14C]acetate into sterols has been de- termined.
Materials and Methods
Adrenal glands were obtained from male Spraque-Dawley rats (Charles River Breeding Laboratories, Wilmington, MA) that had been subjected to a diethyl ether anesthesia stress (10 min) immediately prior to killing. A mitochondria- enriched fraction from rat adrenal homogenate in sucrose (0.25 M) was prepared as previously de- scribed [10]. The mitochondrial fraction was washed once with sucrose (0.25 M) before final suspension in the same medium. Mitochondrial protein content was determined by the procedure of Lowry et al. [11], with bovine serum albumin as the protein standard.
The sterol side-chain cleavage activity of the rat adrenal mitochondria was determined by use of a procedure described earlier which involves de- termination of pregnenolone by radioimmunoas- say [10]. Cyanoketone (5 u.M) was added to pre- vent further metabolism of pregnenolone. The various sterols were added in ethanol to achieve a final concentration of 100 uM. This sterol con- centration gave optimal enzyme activity using the described assay conditions. The final concentra- tion of ethanol did not exceed 1%. The mitochondrial assay media were maintained at 37°℃ for 10 min prior to initiation of the reaction
by the addition of isocitrate (final concentration, 10 mM). The mitochondrial protein content of the assay was approximately 0.5 mg/ml.
The rat adrenocortical carcinoma 494 was transplanted and maintained in male Sprague- Dawley rats (Charles River Breeding Laboratories Inc., Wilmington, MA) as described by Ney et al. [12]. A dispersed tumor cell preparation was ob- tained by a procedure similar to that described previously [9]. Briefly, viable tumor tissue from two rats was placed in a 125-ml Erlenmeyer flask that contained 50 ml Krebs-Ringer phosphate buffer, pH 7.4, glucose (2 mg/ml) and bovine serum albumin (fraction V, 5 mg/ml). The tissue was stirred for 15-20 min at 37℃ with a magnetic stirring bar. After the undissociated tissue settled out, the supernatant that contained dissociated cells was filtered through gauze into 50-ml centri- fuge tubes. The supernatant was centrifuged at 600 rpm for 20 min in a refrigerated centrifuge. The resulting pellet of packed cells was suspended in 50 ml buffer and allowed to settle by gravity for 60 min on ice. The resultant supernatant fraction, which contained most of the red blood cells and cellular debris, was discarded. The loosely packed cells were suspended in 50 ml buffer, and the gravity settling process was repeated at least twice. This procedure resulted in a preparation of nonag- gregated tumor cells virtually free of red blood cells and other small particles that did not settle with the relatively heavy tumor cells. The nucleated cells were counted in a hemocytometer after stain- ing with Nessler’s B stain.
Incubations of the isolated tumor cells were conducted in air at 37°℃ in the isolation buffer in a total volume of 0.5 ml that contained 1 . 106 cells. The incubations were terminated by rapid chilling. An inhibitor of 3ß-hydroxysteroid dehy- drogenase, cyanoketone (15 µM), was added to prevent further metabolism of pregnenolone. The sterols were added in ethanol and the final ethanol content did not exceed 1%.
Pregnenolone was determined by a RIA method reported in detail previously [9], based on the procedure described by Abraham et al. [13]. Anti- serum to pregnenolone-20-albumin conjugate was raised in New Zealand white rabbits and used at a dilution of 1: 3500. Briefly, the 0.5-ml chilled in- cubations were diluted with 2 ml of water and
aliquots (10-pl) were used directly in the radioim- munoassay. Protein from the lyzed cells did not interfere with the assay at the cell dilution em- ployed. The halogenated sterols cross-reacted less than 1% with equimolar amounts of pregnenolone. The zero time incubation blank was subtracted from the values determined in these assays.
Isolated tumor cells were incubated in a similar manner after the addition of 1 uCi [1-14C]acetate (sodium salt) to the medium to determine the incorporation of radioactive lable into lipids. The assays were terminated by freezing. After saponifi- cation with alcoholic potassium hydroxide, the lipids were extracted using chloroform/methanol (2: 1, v/v). The resulting organic phase was con- centrated and applied on to silica gel G thin-layer chromatoplates. The chromatograms were devel- oped twice with the solvent system diisopropyl ether / petroleum ether (60-80°℃ b.p.)/ acetic acid (35: 15: 1). The radioactive regions were located with a radiochromatogram scanner and the visuali- zation of authentic standards. Quantification of radioactivity was performed by liquid scintillation spectrometry [14].
The ACTH used in this study was the USP corticotropin reference standard. The 25-hydroxy- cholesterol was obtained from Steraloids Inc., Wil- ton, NH. Cyanoketone was kindly supplied by the Sterling-Winthrop Research Institute (Rensselaer, NY). Other standard steroids and bovine serum albumin (fraction V) were supplied by Sigma Chemical Company (St. Louis, MO). The [7a- 3H]pregnenolone (NET 039; 17 Ci/mmol) and [1-14C]acetate (sodium salt, 60 mCi/mmol) were supplied by New England Nuclear Chemicals Inc., Boston, MA. All other chemicals were of analyti- cal reagent grade.
The previously synthesized 26-bromocholesterol and 26-iodocholesterol [15] was used.
26-nor-25(RS)-bromocholesterol
The required starting material, 25-keto-26-nor- cholesteryl acetate, was prepared from pregnen- olone (16). The 25-ketone was reduced with sodium borohydride in dioxane water (10: 1) at 10°C. The resulting 26-nor-25( RS)-hydroxycholestery1-3- acetate was treated with N-bromosuccinimide and triphenylphosphine to yield 25(RS)-bromide 3- acetate. After removal of the acetate (5% aqueous
H2SO4 in dioxane; 80°C, 1 h), 26-nor-25(RS)- bromocholesterol was obtained (m.p., 124-125℃). NMR(C2HCI3) 8 0.68 (3H,s,C18-H), 0.92 (3H,d, J = 6 Hz,C27-H), 3.51 (1H,m,C3-H), 4.13 (1H,m,C25-H) and 5.37 (1H,b,d,C,-H). MS s m/e 452 (81Br), 450 (79Br) (M+).
Results
Pregnenolone formation in rat adrenal mitochondria in the presence of cholesterol and halogenated cholesterol derivatives
The rate of synthesis of pregnenolone in rat adrenal mitochondria in the presence of added halogenated sterols and cholesterol (100 µM) was
(a) Cholesterol
(b)26-Bromocholesterol
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(c) 26-nor-25-Bromocho- lesterol
(d) 26-lodocholesterol
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determined. The results are given in Fig. 1. The rate of pregnenolone formation from endogenous precursors, in the absence of an exogenous sterol, is also illustrated. The rates of pregnenolone synthesis for the initial 2 min of the assays in the presence of 26-bromocholesterol (100 µM), 26- nor-25-bromocholesterol (50 MM) and 26-iodo- cholesterol (100 p.M) were 12, 8.5 and 12.5 nmol/ min per mg protein, respectively, compared with the rates of 3.0 and 8.0 nmol/ min per mg protein observed utilizing endogenous precursors and cholesterol (100 p.M), respectively.
Effect of Ca2+ on pregnenolone formation in rat adrenal mitochondria after the addition of cholesterol and halogenated cholesterol derivatives
In Fig. 2, we illustrate the effect of calcium
50
nmol PREGNENOLONExmg-1 protein
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Time (min)
chloride (1 mM) on pregnenolone synthesis in rat adrenal mitochondria in the presence of cholesterol (100 µ M) and 26-nor-25(RS)-bromocholesterol (50 uM). We found that after the addition of Ca2+ the rate of pregnenolone synthesis from cholesterol was increased but Ca2+ did not affect significantly the rate of pregnenolone synthesis found in the presence of the halogenated sterol. Similar results were obtained by use of 26-bromocholesterol and 26-iodocholesterol (data not shown).
Pregnenolone biosynthesis in rat adrenal tumor cells in the presence of halogenated sterols
When the halogenated sterols were added to isolated adrenocortical cells of the rat Snell adrenal carcinoma 494, a marked increased in the pregnen- olone secretion by these tumor cells was found; this observation is indicative of the uptake and metabolism of these sterols to pregnenolone. After the addition of cholesterol in the similar vehicle (ethanol; final concentration, 1%), however, the rate of pregnenolone biosynthesis was not differ- ent from that of the endogenous rate. The halogenated sterols, however, were not utilized for
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pregnenolone biosynthesis as efficiently as 25-hy- droxycholesterol. The results are presented in Fig. 3.
Effect of halogenated sterols on the incorporation of [14C]acetate into non-saponifiable lipids of rat adrenal tumor cells
We evaluated the effect of 26-bromocholesterol, 26-nor-25-bromocholesterol, 26-iodocholesterol, cholesterol and 25-hydroxycholesterol (50 uM of each sterol) on the incorporation of radioactively labeled [1-14C]acetate into pregnenolone, cholesterol and esterified cholesterol of isolated Snell adrenocortical carcinoma cells. All assays were conducted at 37ºC for 2 h in the presence of ACTH (20 nM) and cyanoketone (15 µM). The 26-nor-25-bromocholesterol and 26-bromocho- lesterol, in addition to 25-hydroxycholesterol, were more effective inhibitors of cholesterol and preg- nenolone de novo biosynthesis than was cholesterol. The 26-iodocholesterol did not affect significantly the rate of cholesterol biosynthesis, but inhibited de novo pregnenolone formation. It is pertinent to note that the greater reduction of labeling of pregnenolone in the presence of sterol analogues is due to the competition of the sterol analogues with labeled sterol for the side-chain
| Sterol added (50 µ M) | Percentage incorporation into: | |
|---|---|---|
| Pregnenolone | Total cholesterol | |
| None | 100±5 | 100±6 |
| Cholesterol | 97±2 | 119±5 |
| 26-nor-25-Bromocholesterol | 40±4 | 55±4 |
| 26-Iodocholesterol | 34±3 | 119±2 |
| 26-Bromocholesterol | 49±2 | 68±4 |
| 25-Hydroxycholesterol | 22±3 | 59±2 |
cleavage enzyme as well as the inhibition of sterol synthesis. The results are presented in Table I.
Discussion
We found that rat adrenal mitochondria metabolize derivatives of cholesterol that contain iodine or bromine functions in the sterol side chain to produce pregnenolone. The sterol de- smolase activity of rat adrenal mitochondria with these substrate analogues was at least twice that found with the presumed physiological substrate, cholesterol. This observation is further illustrative of the relative lack of specificity of the mitochondrial cholesterol desmolase for substrates with modifications of the sterol side chain [5]. In a recent report, Morisaki et al. [17] have shown, by use of a reconstituted cholesterol desmolase, that 25-fluorocholesterol is converted to pregnenolone at double the rate at which cholesterol is converted to pregnenolone. Interestingly, we found that the side-chain cleavage of the halogenated sterols was not stimulated by Ca2+. This finding was in con- trast to the Ca2+-stimulated side-chain cleavage of cholesterol by the rat adrenocortical mitochondria, a phenomenon that has been implicated in the tropic hormonal control of cholesterol metabolism in steroidogenic tissues. Thus, the facility of the uptake and transport of the halogenated sterols through the mitochondrion to the inner mitochondrial membrane location of the sterol desmolase is similar to that observed with the side-chain-hydroxy derivatives of cholesterol [10]. This observation is supportive of the notion that a halogen atom at such a position in a sterol is analogous to a hydroxyl group but unlike a proton [18]. ACTH, therefore, may not be a requirement for the uptake and utilization of such sterols.
We foresee the potential usefulness of the side- chain halogenated analogues of cholesterol as adrenal-imaging agents (with the appropriate y- emitting isotope of bromine or iodine) for aid in the diagnosis of function and conditions such as adrenal carcinoma or Cushing’s syndrome. By use of a dispersed cell preparation of rat Snell adrenocortical carcinoma 494 cells, we found that with the halogenated sterols there was an increase in pregnenolone formation. This result was indica- tive that such sterols were taken up and subse-
quently metabolized by the adrenal carcinoma cells.
In rat adrenocortical carcinoma 494 cells, a significant proportion of steroid production arises from cholesterol synthesized de novo [8]. It was of interest, therefore, that 26-nor-25-bromocho- lesterol and 26-bromocholesterol sterols were more potent inhibitors of the incorporation of radioac- tively labeled acetate into cholesterol and pregnen- olone than was cholesterol and produced a similar inhibitory profile to a well-recognized inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase activ- ity, viz., 25-hydroxycholesterol [19]. The inhibition of de novo pregnenolone biosynthesis observed in the presence of 26-iodocholesterol is suggestive of a cholesterol-sparing effect. Since the results pre- sented in Fig. 3 are suggestive that the halogenated sterols are taken up into the intracellular sites of sterol metabolism more efficiently than is cholesterol, it may well be that the weak inhibition produced by cholesterol is due to a lower intracell- ular amount of this sterol compared with the halogenated sterols. Since these sterols are also metabolized within the cell (see Fig. 3), a further degree of complexity must be considered in an interpretation of these data.
It would appear, therefore, that side-chain halogenated derivatives of cholesterol are readily taken up and metabilized by adrenocortical carcinoma cells to a characteristic product of the adrenal cortex, viz., pregnenolone. Since these tumor cells contain little cholesterol reserve, it is likely that exogenous sterol is readily incorporated into the cells. Thus, the use of a suitably radioac- tively labeled sterol should facilitate the imaging of tumors of steroidogenic tissue. Given the rapid metabolism, it would appear that concomitant ad- ministration of an inhibitor of cholesterol de- smolase might prove to be useful in the retention of the halogenated sterol in the adrenal cell. In this regard, aminoglutethimide, a known inhibitor of the cholesterol desmolase, also is strongly inhibi- tory of the metabolism of the halogenated sterols in adrenal carcinoma cells (Mason, J.I., unpub- lished data). Subsequent removal of the drug would facilitate the metabolism of the (radioactively labeled) cholesterol analogue. In combination with ion probe spectroscopic techniques, the use of
these or similar halogenated sterols may further our understanding of the transfer of sterols into and within cells.
Acknowledgements
This work was supported, in part, by grant Nos. CA-30253 and CA-16464, awarded by the Na- tional Cancer Institute, DHHS. We thank Debbie Staley and Sylvia Williams for expert editorial assistance.
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