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Volume 271, Number 22, Issue of May 31, 1996 pp. 12724-12736
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

27-Hydroxylated Low Density Lipoprotein (LDL) Cholesterol Can Be Converted to 7alpha ,27-Dihydroxy-4-cholesten-3-one (Cytosterone) before Suppressing Cholesterol Production in Normal Human Fibroblasts
EVIDENCE THAT AN ALTERED METABOLISM OF LDL CHOLESTEROL CAN UNDERLIE A DEFECTIVE FEEDBACK CONTROL IN MALIGNANT CELLS*

(Received for publication, December 22, 1995, and in revised form, March 4, 1996)

Magnus Axelson Dagger § and Olle Larsson

From the Dagger  Department of Clinical Chemistry and the  Department of Tumor Pathology, Karolinska Hospital, S-171 76 Stockholm, Sweden

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES


ABSTRACT

The formation of oxysterols in cultured human fibroblasts and their physiological roles as intracellular regulators of cholesterol production have been investigated. In the presence of low density lipoproteins (LDL), normal fibroblasts converted LDL cholesterol to 27hydroxycholesterol, which was further metabolized to 7alpha ,27-dihydroxycholesterol, 7alpha ,27-dihydroxy-4-cholesten-3-one, and 7alpha -hydroxy-3-oxo-4-cholestenoic acid. Autooxidation products of cholesterol contaminating the lipoproteins were also metabolized in the cells. 7alpha -Hydroxycholesterol was converted to 7alpha -hydroxy-4-cholesten-3-one prior to 27-hydroxylation and further oxidation to 7alpha -hydroxy-3-oxo-4-cholestenoic acid. 7beta -Hydroxycholesterol and 7-oxocholesterol were 27-hydroxylated and then oxidized to C27-acids. Oxidation of the 7beta -hydroxy group also occurred. 25-Hydroxycholesterol was 7alpha -hydroxylated and further oxidized to 7alpha ,25-dihydroxy-4-cholesten-3-one. 25-Hydroxylation of sterols was observed only under specific conditions. In contrast, only small amounts of oxysterols were formed in virus-transformed human fibroblasts when incubated with lipoproteins. This was due to very low activities of the 27- and 7alpha -hydroxylating enzymes. The rate of oxidation at C-3 was also decreased moderately.

A defective suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase by LDL and autooxidation products of cholesterol observed in the transformed fibroblasts could be caused by the deficiencies of the sterol-metabolizing enzymes, since these cells responded normally to the sterol metabolites 7alpha ,27-dihydroxy-4-cholesten-3-one, 7alpha ,25-dihydroxy-4-cholesten-3-one, and 27-hydroxy-7-oxo-cholesterol. These metabolites, which all possessed an oxo group with a conjugated double bond in the steroid nucleus and a hydroxyl group in the side chain, did not seem to require further metabolism in order to be active. An impaired response to LDL was also seen in other human tumor cells, including breast carcinoma, colonic carcinoma, and malignant melanoma cells. Common to all the malignant cells was an intracellular shortage of 7alpha ,27-dihydroxy-4-cholesten-3-one caused by a decreased formation or an increased metabolism.


INTRODUCTION

Low density lipoprotein (LDL)1 is the only regulator of cellular cholesterol production whose physiological role has been established (1, 2, 3, 4, 5). Internalized LDL suppresses 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme of cholesterol synthesis, but the underlying mechanisms are not fully understood. Recently, we reported that LDL cholesterol is converted to 27-hydroxycholesterol in human fibroblasts and that this oxysterol is an important intracellular mediator of the LDL-induced suppression of HMG-CoA reductase (6). The metabolism of LDL cholesterol and the biological effects of its metabolites in normal human fibroblasts have now been investigated further. Virus-transformed fibroblasts were included in the study, since a defective regulatory response to LDL has been observed previously in several different malignant cells (7, 8, 9, 10). This impaired cholesterol feedback system, the causes of which are unknown, seems to appear early in the development of cancer and has been considered to promote cell growth by increasing the cellular supply of cholesterol and intermediates in the mevalonate pathway (11). The two fibroblastic cell lines permitted us to make a direct comparison between the handling of LDL cholesterol and the suppression of HMG-CoA reductase in the normal and the corresponding tumor-transformed cell. Here we report that in normal human fibroblasts 27-hydroxylated LDL cholesterol is converted to 7alpha ,27-dihydroxy-4-cholesten-3-one, which does not seem to be metabolized further before suppressing HMG-CoA reductase. The study also shows that the metabolism of LDL cholesterol is markedly changed following transformation of fibroblasts and that these cells display a defective regulatory response to LDL. An altered metabolism of LDL cholesterol and an impaired suppression of HMG-CoA reductase were also noted in other human neoplastic cells.


MATERIALS AND METHODS

Steroids, Chemicals, and Sera

Diosgenin ((25R)-5-spirosten-3beta -ol) was from Sigma and was used as the starting material for the synthesis of 27-oxygenated steroids (12, 13, 14). In addition, 5-cholestene-3beta ,7alpha ,25-triol (7alpha ,25-dihydroxycholesterol) was prepared from the 3-acetate,25-trimethylsilyl ether derivative of 25-hydroxycholesterol, and, after hydrolysis, this steroid was further oxidized to 7alpha ,25-dihydroxy-4-cholesten-3-one as described for the corresponding 27-hydroxysteroids (14). 25-Hydroxycholesterol was oxidized in the same way to 25-hydroxy-4-cholesten-3-one. Other unlabeled steroids were those used in a previous study (15). [1alpha ,2alpha -3H]Cholesterol (44 Ci/mmol) and [1alpha ,2alpha -3H]cholesteryl oleate (49 Ci/mmol) were purchased from Amersham and 25-[26,27-3H]hydroxycholesterol (86 Ci/mmol) was from Du Pont de Nemours NV, NEN Products (Brussels, Belgium). Radioactivity was determined in an LKB/Wallac 1215 Rackbeta Scintillation Counter with OptiPhase ``HiSafe'' (Wallac) as the scintillation liquid. Cyclosporin A (CsA) and ketoconazole were kind gifts from Sandoz Pharma Ltd. (Basel, Switzerland) and Janssen Pharmaceutica (Beerse, Belgium), respectively. Fetal calf serum (FCS) having a total cholesterol concentration of 1.2 mM (12% free and 88% esterified cholesterol) was from Life Technologies, Inc. (Stockholm, Sweden). About 70% of the total cholesterol was found in the LDL fraction. The lipoprotein-deficient serum (LDS) was prepared by treating FCS with Cab-O-Sil (16), and the cholesterol concentration after this treatment was 0.1 mM. Cell growth was not affected negatively when this serum was used. LDL particles containing 3H-labeled cholesterol or cholesteryl oleate were obtained by incubating the radioactive steroid with FCS overnight at 20 °C (6).

Cell Culture Conditions

Normal human fibroblasts (line GM 08333) were obtained from NiGMS, Corriell Institute for Medical Research (Camden, NJ) and SV40 virus-transformed human fibroblasts (90-VA IV) were a kind gift from Dr. Stein (University of Colorado, Boulder, CO). Human colonic carcinoma (WiDr), breast carcinoma (MDA 231), and malignant melanoma (SK-MEL-2) cell lines were from American Type Culture Collection

All cell lines were grown in monolayers in tissue culture flasks maintained in a 95% air, 5% CO2 atmosphere at 37 °C in a humidified incubator and were cultured in either Dulbecco's modified Eagle's medium (MDA 231) or minimal Eagle's medium (the other cells) supplemented with essential and nonessential amino acids and 10% FCS (v/v). For experimental purposes, cells were cultured in dishes. Cells were seeded at a density of 5,000 cells per cm2. The experiments were started 48-72 h later, at which time a cell density of approximately 20,000 per cm2 had been reached. The cells were subconfluent also at the end of the incubations. When the metabolism of cholesterol or oxysterols was studied, normal or transformed fibroblasts (cell number 3-6 × 106 in 57-143-cm2 dishes) were first preincubated for 24 h in medium containing 10% LDS and were then incubated for 3-68 h with 7-10 ml of medium containing 4-10% FCS (with or without 3H-labeled cholesterol or cholesteryl oleate) or were incubated with the oxysterol in 10% LDS for 24-48 h. Control cells were incubated in the same way, but only for 15 min. Effects of CsA, ketoconazole, and oxysterols were tested on normal and transformed fibroblasts at concentrations of 10-30 µM, 30 µM, and 0.12 µM, respectively, in cell media containing 0-10% FCS and 10-0% LDS. The substances were added to the incubation media in freshly prepared ethanol solutions, and the ethanol concentrations of media became 0.1-0.5%. Control cells were incubated in the same way, but without CsA, ketoconazole, or oxysterols. The dish size and volume of media when HMG-CoA reductase activity was to be determined were 20 cm2 and 5 ml, respectively, and the incubations were carried out in duplicate for 3-24 h. Each oxysterol was tested in 2-5 separate experiments. Determination of HMG-CoA reductase activity was then carried out as described previously (6, 17, 18).

Analysis of Oxysterols and Steroid Acids

The procedure for extraction and purification of oxysterols present in incubation media and cells was essentially the same as described previously (6). Following the collection of a neutral oxysterol fraction from the lipophilic anion exchanger (6), a fraction containing steroids with a free carboxyl group was eluted with 0.15 M acetic acid in 95% aqueous methanol prior to elution of a fraction containing stronger acids (including steroid sulfates) with 0.5 M potassium acetate/potassium hydroxide, apparent pH 10.0, in 72% aqueous methanol (19).

Trimethylsilyl ethers of oxysterols and methyl ester trimethylsilyl ether derivatives of steroid acids were prepared (19) and were analyzed by gas chromatography-mass spectrometry (GC/MS) as described previously (6).

High Performance Liquid Chromatography (HPLC)

3H-Labeled cholesterol, cholesteryl oleate, and 25-hydroxycholesterol and/or their radioactive metabolites were analyzed by HPLC prior to or after group fractionation and purification as described above. 3H-Labeled cholesterol and cholesteryl esters were extracted from small aliquots of the incubation media with mixtures of isopropyl alcohol and hexane and from cells with mixtures of ethanol and water prior to separation by straight-phase HPLC using hexane/isopropyl alcohol, 98:2 (v/v), as the mobile phase (6). Appropriate fractions from the HPLC effluent were collected in vials, and the radioactivity was then determined by scintillation counting. Radioactive neutral and acidic metabolites of [3H]cholesterol or [3H]cholesteryl oleate were isolated from media and cells as described above and were then characterized by HPLC. For this purpose, three HPLC systems were used in the following order. Reversed-phase HPLC was carried out on a column of LiChrospher (250 × 4 mm, Hibar, 100RP-18, 5 µm, Merck, Darmstadt, Germany) using a pump (Constametric III) and a variable wavelength detector (Spectra Monitor D from LDC/Milton Roy, Riviera Beach, FL) set at 220 or 240 nm and a Rheodyne Model 7125 injector with a 100-µl loop. The mobile phase first used for neutral metabolites was a mixture of methanol/ethanol/water, 80:20:10 (by volume, flow rate 1 ml × min-1), and fractions were collected between 0 and 11 min (fraction 1; containing polar metabolites, e.g. 7alpha ,27-dihydroxy-4-cholesten-3-one, retention time about 4.5 min) and between 11 and 14 min (fraction 2; containing 7alpha -hydroxy-4-cholesten-3-one and 27-hydroxycholesterol having retention times 11.5 and 12.5 min, respectively). The mobile phase was then changed to 85% aqueous methanol (flow rate 1 ml × min-1) for separation of sterols in fraction 1 or for separation of steroid acids (as methyl ester derivatives). In the former case, a fraction of the effluent containing 7alpha ,27-dihydroxy-4-cholesten-3-one (retention time about 8.5 min) was collected between 8.0 and 9.0 min, and in the latter case a fraction of the effluent containing 7alpha -hydroxy-3-oxo-4-cholestenoic acid (retention time about 12 min) was collected between 11.0 and 13.0 min. These fractions and fraction 2 (containing 7alpha -hydroxy-4-cholesten-3-one and 27-hydroxycholesterol) were then reanalyzed by straight-phase HPLC. The latter was carried out with an instrument similar to that above, but with a column (250 × 4.5 mm) of LiChrospher (Hibar, Si 100, 5 µM, Merck). The mobile phase was hexane/isopropyl alcohol, 94:6 (v/v), when fraction 2 was analyzed (retention times of 7alpha -hydroxy-4-cholesten-3-one and 27-hydroxycholesterol were about 8 and 9 min, respectively), and 90:10 (v/v), when the fractions containing 7alpha ,27-dihydroxy-4-cholesten-3-one or 7alpha -hydroxy-3-oxo-4-cholestenoic acid methyl ester were analyzed. The flow rate was 1.0 ml × min-1 in all cases. The HPLC effluent during the latter analyses was collected in scintillation vials with 15-60-s intervals, and, after addition of scintillation fluid, the radioactivity was determined.

25-[3H]Hydroxycholesterol and its metabolites in media and cells were analyzed by HPLC following extraction. Medium was extracted with ethanol, and, after centrifugation and removal of the supernatant, the pellet was re-extracted with ethanol/isopropyl alcohol, 1:1 (v/v). The extracts were combined and the solvent was then evaporated. Nonpolar compounds were dissolved in hexane, and, after removal, the solid residue was dissolved in 60% aqueous methanol, which was passed through a column (1.5 × 0.8 cm) of octadecylsilane-bonded silica (Preparative C18; Waters Associates Inc., Milford, MA) and collected. The methanol in the eluate was then removed in vacuo, and the aqueous solution was re-extracted on the same column. After washing the column with water, sorbed steroids (polar metabolites) were eluted with methanol/chloroform, 1:1 (v/v), and were combined with the nonpolar metabolites present in the hexane fraction. This combined extract was evaporated to dryness and dissolved in methanol or hexane/isopropyl alcohol, 90:10 (v/v), prior to analysis by reversed-phase HPLC (mobile phase: methanol/ethanol/water, 80:20:10 (v/v) or straight-phase HPLC (mobile phase: hexane/isopropyl alcohol, 97:3 (v/v)).


RESULTS

Formation of Oxygenated Cholesterol Derivatives in Human Fibroblasts

The formation of oxygenated cholesterol derivatives in normal and virus-transformed human fibroblasts has been investigated in detail. A general method for the isolation of C27-steroids from media and cells were used, so that no major steroid was expected to escape detection. The final analysis was based on GC/MS.

When normal fibroblasts were incubated for 48 h in the absence of lipoproteins (media containing 10% LDS), only trace amounts (<1-10 pmol/mg of cell protein) of oxysterols could be detected in the medium and cells, with the exception of 7alpha -hydroxycholesterol, 7beta -hydroxycholesterol, and 7-oxocholesterol (amounts about 5-50 pmol/mg of cell protein). Most of these sterols were probably formed by autooxidation of cholesterol during the purification of the samples, since their amounts did not differ significantly from those of the controls (15-min incubations).

In contrast, when normal fibroblasts were incubated for 24-48 h with lipoproteins (media containing 10% FCS), 13 neutral and 5 acidic C27-steroids were found in the media. Steroids identified are listed in Table I. The identification was based on the GC retention indices of the derivatives and mass spectra, which were compared with those of the authentic steroids. Most of the steroids had additional oxygen groups both at C-7 and in the side chain. No additional steroids were identified in the cell extracts, and, with the exception of the autooxidation products of cholesterol (see above), the amounts of oxysteroids in the cell extracts were low and barely detectable (<10-20% of those in media). Because of this, oxysterols in the cells were usually not analyzed, unless otherwise stated. Fig. 1 shows a GC/MS analysis of neutral C27-steroids isolated from the medium after incubating normal fibroblasts with lipoproteins.

Table I.

Oxysterols in medium after incubating normal fibroblasts with lipoproteins

Oxygenated cholesterol derivatives identified in the neutral and acidic fractions from media (containing 10% FCS) after incubation with normal human fibroblasts and their gas chromatographic-mass spectrometric characteristics as trimethylsilyl ethers and methyl estertrimethylsilyl ether derivatives, respectively. Oxygenated cholesterol derivatives identified in the neutral and acidic fractions from media (containing 10% FCS) after incubation with normal human fibroblasts and their gas chromatographic-mass spectrometric characteristics as trimethylsilyl ethers and methyl estertrimethylsilyl ether derivatives, respectively.
No. Steroid name Structurea Retention indexb Molecular and significant ionsc

m/z
Neutral C27-steroids
1 7alpha -Hydroxycholesterol C5-3beta ,7alpha -ol 3115 546, 456
2 7alpha -Hydroxy-4-cholesten-3-one C4-7alpha -ol-3-one 3210 472, 457, 382, 269
3 7beta -Hydroxycholesterol C5-3beta ,7beta -ol 3235 546, 456
4 7-Oxocholesterol C5-3beta -ol-7-one 3375 472, 382, 367, 129
5 24-Hydroxycholesterol C5-3beta ,24 (R/S)-ol 3385 546, 413, 145, 129
6 25-Hydroxycholesterol C5-3beta ,25-ol 3405 546, 456, 131
7 7alpha ,25-Dihydroxycholesterol C5-3beta ,7alpha ,25-ol 3390 634, 544, 131
8 7alpha ,25-Dihydroxy-4-cholesten-3-one C4-7alpha ,25-ol-3-one 3490 560, 545, 412, 131
9 27-Hydroxycholesterol C5-3beta ,27-ol 3455 546, 456, 417, 129
10 7alpha ,27-Dihydroxycholesterol C5-3beta ,7alpha ,27-ol 3445 634, 544
11 7alpha ,27-Dihydroxy-4-cholesten-3-one C4-7alpha ,27-ol-3-one 3545 560, 545, 470, 269
12 7beta ,27-Dihydroxycholesterol C5-3beta ,7beta ,27-ol 3555 634, 544
13 27-Hydroxy-7-oxo-cholesterol C5-3beta ,27-ol-7-one 3710 560, 545, 470, 129
C27-steroid acids
14 3beta -Hydroxy-5-cholestenoate CA5-3beta -ol 3425 502, 412, 373, 129
15 3beta ,7alpha -Dihydroxy-5-cholestenoate CA5-3beta ,7alpha -ol 3415 590, 500
16 7alpha -Hydroxy-3-oxo-4-cholestenoate CA4-7alpha -ol-3-one 3515 516, 501, 426, 269
17 3beta ,7beta -Dihydroxy-5-cholestenoate CA5-3beta ,7beta -old 3530 590, 500
18 3beta -Hydroxy-7-oxo-5-cholestenoate CA5-3beta -ol-7-oned 3680 516, 426, 411, 129

a C, cholestane; CA, cholestanoate; superscript indicates position of double bond; greek letters denote configuration of hydroxyl groups.
b Kovats, on a fused silica capillary column coated with cross-linked methyl silicone.
c Intensities of fragment ions with m/z values above 200-300 were enhanced relative to those of lighter fragments; base peak is shown in italics; m/z, mass/charge.
d Tentative identification, reference compound not available.


Fig. 1. Gas chromatographic-mass spectrometric analysis of neutral oxysterols isolated from the medium after incubating normal fibroblasts with lipoproteins. Normal human fibroblasts (protein content: 1.1 mg, dish size: 143 cm2) were incubated with 10 ml of medium containing 10% FCS (cholesterol concentration: 1.2 mM) for 24 h, and the medium was then taken for analysis by GC/MS. Fragment ion current chromatograms characteristic of the trimethylsilyl ethers of oxysterols were constructed by the computer from mass spectra taken every 2 s during the analysis and for the purpose of illustration the intensities of the ions (m/z) were multiplied by appropriate factors. The principal sterols indicated by the numbers are listed in Table I. The equivalent of about 0.2 ml of medium was injected onto a Finnigan SSQ 710 instrument housing a 25-m fused-silica column coated with methyl silicone, and the oven temperature was programmed from 185 to 280 °C at a rate of 5 °C × min-1.

The quantitative results after incubating normal and transformed fibroblasts for 48 h with lipoproteins are shown in Table II. In addition to 27-hydroxycholesterol which is formed from LDL cholesterol (6), the amounts of three other 27-hydroxylated sterols also increased 10-20-fold when incubating normal fibroblasts. These sterols were 7alpha ,27-dihydroxy-4-cholesten-3-one, 27-hydroxy-7-oxocholesterol, and 7beta ,27-dihydroxycholesterol. In fact, the former of these was the major oxysterol formed. The amounts of their corresponding C27-acids also increased (about 5-10-fold). Also, 25-hydroxycholesterol and 7alpha ,25-dihydroxy-4-cholesten-3-one increased, but the amounts of the former varied considerably, possibly indicating that a part of this sterol had been produced by autooxidation of cholesterol. A decrease of the amount of 7alpha -hydroxycholesterol was also noted indicating a consumption of this sterol during the incubations. The amounts of the other steroids were similar to those of the controls, suggesting that they were present in FCS when added to the media.

Table II.

Production of oxysterols in normal and virus-transformed human fibroblasts when incubated with lipoproteins

The amounts of neutral and acidic oxygenated cholesterol derivatives were determined in the media (10 ml) containing 10% FCS (cholesterol concentration: 1.2 mM) after incubation with fibroblasts for 48 h. Cells incubated for 0.25 h served as controls. The amounts of neutral and acidic oxygenated cholesterol derivatives were determined in the media (10 ml) containing 10% FCS (cholesterol concentration: 1.2 mM) after incubation with fibroblasts for 48 h. Cells incubated for 0.25 h served as controls.
No. Steroid structurea Amountb of oxysteroid found in media
Normal fibroblastsc
Virus-transformed fibroblastsc
0.25 h; n = 2d 48 h; n = 4d 0.25 h; n = 2d 48 h; n = 4d

pmol
1 C5-3beta ,7alpha -ole 171: 117-225 54: 39-81 156: 81-228 60: 54-123
2 C4-7alpha -ol-3-one <6: <3-<6 <12: <6-<18 <9: <6-<9 69: 51-159
3 C5-3beta ,7beta -ole 138: 108-165 114: 99-156 144: 81-207 129: 75-216
4 C5-3beta -ol-7-onee 879: 840-915 816: 609-1140 993: 633-1350 867: 657-1881
5 C5-3beta ,24-ol 6: <6-6 6: <6-9 3: <3-6 9: <6-18
6 C5-3beta ,25-ole 6: <3-12 45: 25-150 9: <3-18 27: 15-30
7 C5-3beta ,7alpha ,25-ol <3: <3-3 <6: <3-<9 <3: <3-3 <3: <3-<3
8 C4-7alpha ,25-ol-3-one <6: <3-<6 21: <9-27 <3: <3-<3 <3: <3-<3
9 C5-3beta ,27-ol 12: 9-18 219: 153-267 12: 9-12 21: 15-33
10 C5-3beta ,7alpha ,27-ol <3: <3-<3 <3: <3-<3 <3: <3-<3 <3: <3-<3
11 C4-7alpha ,27-ol-3-one <12: <12-<12 270: 144-321 <9: <3-<12 <12: <9-<15
12 C5-3beta ,7beta ,27-ol <3: <3-<3 42: 33-48 <3: <3-<3 <3: <3-<3
13 C5-3beta ,27-ol-7-one <9: <6-<12 81: 33-108 <12: <3-<18 <9: <6-15
14 CA5-3beta -ol 21: 12-27 21: 18-27 15: 12-15 15: 12-18
15 CA5-3beta ,7alpha -ol 9: 6-9 6: 3-6 6: 6-6 6: 3-6
16 CA4-7alpha -ol-3-one 21: 18-21 75: 63-117 18: 15-18 9: 6-15
17 CA5-3beta ,7beta -ol 12: 9-12 51: 45-72 3: <3-3 <3: <3-<3
18 CA5-3beta -ol-7-one <3: <3-<3 27: 18-30 <3: <3-<3 <3: <3-<3

a For abbreviations and steroid names, see Table I.
b Expressed as median: range; < = an amount at or below the detection limit.
c Protein contents of normal and virus-transformed fibroblasts were 0.6 mg and 1.4 mg, respectively. The size of the incubation dishes was 57 cm2. All cells had been preincubated for 24 h in media containing 10% LDS.
d n = number of incubations.
e Can also be formed by autooxidation of cholesterol during the incubations or during purification of samples.

Much smaller amounts of oxysterols were formed by the transformed fibroblasts (Table II). For example, the amounts of 27-hydroxycholesterol only increased about 2-fold, and the other 27-hydroxylated sterols were barely detectable in the media. Only the amounts of 7alpha -hydroxy-4-cholesten-3-one increased significantly (this was not the case in normal fibroblasts) which may be related to a decrease of the amount of its potential precursor 7alpha -hydroxycholesterol. These results indicated that 27-hydroxylation of sterols may be obstructed in transformed fibroblasts (see below).

The oxysterols were also studied with regard to the time course of their cellular production. Incubation of normal human fibroblasts for different lengths of time showed that the formation of 27-hydroxylated sterols had started 3-8 h after exposure to lipoproteins (Table III). No production of 25-hydroxylated sterols was observed during the first 24 h. A decrease of the amounts of 7alpha -hydroxycholesterol was noted after 8 h of incubation. Since the oxysterols having oxy groups both in the 7- and 27-positions could be derived from either 27-hydroxylated LDL cholesterol or 7-oxygenated cholesterol derivatives (autooxidation products present in the medium), their origin was investigated.

Table III.

Time-response for the production of oxysterols in normal fibroblasts when incubated with lipoproteins

The amounts of neutral oxygenated cholesterol derivatives were determined in the media (15 ml) containing 10% FCS (cholesterol concentration: 1.2 mM) after incubation for 0.25-24 h with normal human fibroblasts (protein content 1.7 mg, size of dishes 143 cm2). All cells had been preincubated for 24 in media containing 10% LDS. The amounts of neutral oxygenated cholesterol derivatives were determined in the media (15 ml) containing 10% FCS (cholesterol concentration: 1.2 mM) after incubation for 0.25-24 h with normal human fibroblasts (protein content 1.7 mg, size of dishes 143 cm2). All cells had been preincubated for 24 in media containing 10% LDS.
No. Steroid structurea Amount of oxysterol found in media after incubation for
0.25 h 3 h 8 h 24 h

pmol
1 C5-3beta ,7alpha -olb 92 92 31 15
2 C4-7alpha -ol-3-one 55 65 58 45
3 C5-3beta ,7beta -olb 92 92 58 66
4 C5-3beta -ol-7-oneb 508 500 369 412
5 C5-3beta ,24-ol 15 15 10 10
6 C5-3beta ,25-olb 28 28 10 15
7 C5-3beta ,7alpha ,25-ol 25 20 5 5
8 C4-7alpha ,25-ol-3-one 33 28 25 30
9 C5-3beta ,27-ol 23 30 70 240
10 C5-3beta ,7alpha ,27-ol 2 2 1 1
11 C4-7alpha ,27-ol-3-one 65 58 138 308
12 C5-3beta ,7beta ,27-ol 3 3 15 13
13 C5-3beta ,27-ol-7-one 8 5 43 108

a For abbreviations and steroid names, see Table I.
b Can also be formed by autooxidation of cholesterol during the incubations or during purification of samples.

Metabolism of LDL Cholesterol and 7-Oxygenated Sterols in Normal Fibroblasts

The metabolism of 7alpha -hydroxycholesterol, 7beta -hydroxycholesterol, and 7-oxocholesterol was studied by incubating these sterols (5 nmol) with normal human fibroblasts (protein content about 0.5 mg, dish size 57 cm2) for 24 or 48 h in media (10 ml) containing 10% LDS. The values given in percent represent the distribution of observed metabolites.

When 7alpha -hydroxycholesterol was incubated (24 h), the major metabolites found were 7alpha -hydroxy-4-cholesten-3-one (57%) and 7alpha ,27-hydroxy-4-cholesten-3-one (43%) (acids were not analyzed). Only a small amount of 7alpha ,27-dihydroxycholesterol (0.5%) was noted suggesting that oxidation/isomerization of 7alpha -hydroxycholesterol to 7alpha -hydroxy-4-cholesten-3-one precedes 27-hydroxylation. The corresponding enzyme activities have been found previously in fibroblasts (20). When 7alpha -hydroxy-4-cholesten-3-one was incubated with the fibroblasts for 48 h, the steroid was extensively converted to 7alpha ,27-dihydroxy-4-cholesten-3-one (37%) and 7alpha -hydroxy-3-oxo-4-cholestenoic acid (63%). A small amount of 25-hydroxylated 7alpha -hydroxy-4-cholesten-3-one (0.5%) was also found. These results show that 7alpha -hydroxycholesterol can be converted to 7alpha -hydroxy-4-cholesten-3-one, 7alpha ,27-dihydroxy-4-cholesten-3-one, and 7alpha -hydroxy-3-oxo-4-cholestenoic acid by normal fibroblasts and this could explain the appearance of these metabolites and the disappearance of 7alpha -hydroxycholesterol in media during the incubations with FCS (Table II).

The metabolism of 7beta -hydroxycholesterol differed from that of 7alpha -hydroxycholesterol. When this sterol was incubated for 48 h with normal fibroblasts, the major metabolites were 7beta ,27-dihydroxycholesterol (1%) and 3beta ,7beta -dihydroxy-5-cholestenoic acid (62%). In addition, a large portion (about one-third) of 7beta -hydroxycholesterol was converted to 7-oxocholesterol (20%), 27-hydroxy-7-oxocholesterol (1%), and 3beta -hydroxy-7-oxo-5-cholestenoic acid (14%). Oxidation of the 7beta -hydroxy group also occurred when 7beta ,27-dihydroxycholesterol was incubated. No conversion of 7beta -hydroxycholesterol to the corresponding 3-oxo-Delta 4 derivative was observed, which was consistent with the absence of 7beta -hydroxylated 3-oxo-Delta 4 steroids in media after incubating fibroblasts with FCS (Table I).

Incubation of 7-oxocholesterol with normal fibroblasts resulted in the formation of 27-hydroxy-7-oxocholesterol (55%) and the corresponding C27-acid (35%). A small amount was converted to 7beta -hydroxycholesterol (10%), but a conversion to 7alpha -hydroxylated products was not observed. Thus, the formation of C27-steroids having oxygen groups in both the 7- and 27-positions by normal fibroblasts could be due to the presence of autooxidation products of cholesterol in the medium. However, this did not exclude the possibility that 7alpha ,27-dihydroxy-4-cholesten-3-one and 7alpha -hydroxy-3-oxo-4-cholestenoic acid could also be derived from 27-hydroxycholesterol. 7alpha -Hydroxylation of 27-hydroxycholesterol in human fibroblasts was first noted by us (15) and was later found to be occurring generally in these cells (21). The product 7alpha ,27-dihydroxycholesterol is extensively converted to 7alpha ,27-dihydroxy-4-cholesten-3-one and the corresponding acid in the cells (15, 21).

In order to determine whether LDL cholesterol (via 27-hydroxycholesterol) could be converted to 7alpha ,27-dihydroxy-4-cholesten-3-one and the acid, the contribution from 7alpha -hydroxycholesterol (which is always present when the medium contains lipoproteins) had to be accounted for. This was made possible by the following experiment. LDL and other lipoproteins in FCS were first labeled with [3H]cholesteryl oleate and were then incubated with normal fibroblasts in the presence and absence of cyclosporin A (CsA), a selective inhibitor of the sterol 27-hydroxylase (6, 22, 23). When lipoproteins are labeled in this way, the cellular uptake of [3H]cholesteryl oleate is due solely to a LDL receptor-dependent process (i.e. a physiological uptake of LDL) (6). After the incubations, radioactive 27-hydroxycholesterol, 7alpha ,27-dihydroxy-4-cholesten-3-one, and 7alpha -hydroxy-3-oxo-4-cholestenoic acid were analyzed by HPLC. 3H-Labeled 7alpha -hydroxy-4-cholesten-3-one, the direct metabolite of 7alpha -hydroxycholesterol, was also determined. If 3H-labeled 7alpha -hydroxycholesterol (free or esterified) was present in the medium, its 3-oxidized metabolite was expected to accumulate in the presence of CsA, since the drug prevented further metabolism (see above). The amount of the metabolite would then reflect the contribution to 7alpha ,27-dihydroxy-4-cholesten-3-one and the acid from 7alpha -hydroxycholesterol in the absence of CsA. Obviously, essentially no formation of 27-hydroxylated compounds was expected in the presence of CsA (see also Fig. 5). For comparison, fibroblasts were also incubated with lipoproteins labeled with [3H]cholesterol, whose cellular uptake is not entirely dependent on LDL receptors.


Fig. 5. A simplified scheme of the metabolism of LDL cholesterol and major autooxidation products of cholesterol in human fibroblasts and the formation of potent HMG-CoA reductase suppressors. The names of the steroids are listed in Table I. In addition to hydrolysis and esterification (not shown), major reactions of sterols were: I, 27-hydroxylation; II, 7alpha -hydroxylation; III, 3-oxidation with isomerization of the 5-double bond; and IV, oxidation to a 27-carboxy group. Hydrolyzed LDL cholesterol was shown to be metabolized via these reactions (filled arrows). Oxidation of a 7beta -hydroxy group (V) to a ketone was also observed. Minor reactions noted are shown by broken arrows. The formation of 7alpha ,25-dihydroxy-4-cholesten-3-one by 25-hydroxylation of sterols (not shown) was observed under specific conditions. Reactions I, II, and III were obstructed in virus-transformed fibroblasts displaying a defective suppression of HMG-CoA reductase by LDL cholesterol and autooxidation products of cholesterol. Sterol metabolites with an apparently normal suppressive effect also in transformed cells are indicated in-frame.

The results of these incubations are summarized in Table IV. In addition to 27-hydroxycholesterol, both 7alpha ,27-dihydroxy-4-cholesten-3-one and 7alpha -hydroxy-3-oxo-4-chole-stenoic acid were found as 3H-labeled compounds after incubation with [3H]cholesteryl oleate. The HPLC analyses of these metabolites are shown in Fig. 2. Since no accumulation of radioactive 7alpha -hydroxy-4-cholesten-3-one was observed in the corresponding incubation with CsA (Table IV), autooxidation (7alpha -hydroxylation) of [3H]cholesteryl oleate had not occurred during the incubations. In the incubations with [3H]cholesterol, much larger amounts of radioactive 7alpha ,27-dihydroxy-4-cholesten-3-one and the corresponding acid were found, although the amount of 27-hydroxycholesterol was less than with [3H]cholesteryl oleate. However, the incubation with [3H]cholesterol and CsA resulted in a significant accumulation of 3H-labeled 7alpha -hydroxy-4-cholesten-3-one suggesting that most of the 27-oxygenated metabolites had been produced from autooxidized [3H]cholesterol (7alpha -hydroxycholesterol). The difference in chemical stability toward oxygen between [3H]cholesteryl oleate and [3H]cholesterol was surprising, but was confirmed by exposing them to air and heat in an aqueous/methanolic environment for 24 h. No autooxidation products (<0.1%) of [3H]cholesteryl oleate could be detected, whereas about 2% of [3H]cholesterol were autooxidized. Thus, the results demonstrate that LDL cholesteryl esters are hydrolyzed and are converted to 27-hydroxycholesterol, which is then 7alpha -hydroxylated and oxidized to 7alpha ,27-dihydroxy-4-cholesten-3-one and 7alpha -hydroxy-3-oxo-4-cholestenoic acid in normal human fibroblasts. The latter seems to be the major metabolic end product of this extended LDL pathway in fibroblasts.

Table IV.

Formation of radioactive metabolites from LDL [3H]cholesteryl oleate in normal fibroblasts

The amounts of 3H-labeled 27-hydroxycholesterol, 7alpha ,27-dihydroxy-4-cholesten-3-one and 7alpha -hydroxy-3-oxo-4-cholestenoic acid were determined in media and cells after incubating normal human fibroblasts (protein content, 0.7 mg; size of dishes, 57 cm2) for 68 h with media (10 ml) containing LDL (4% FCS; cholesterol concentration, 1.2 mM) labeled with [3H]cholesteryl oleate or [3H]cholesterol. When 27-hydroxylation of sterols was obstructed by cyclosporin A (CsA, 10 µM), the accumulation of 3H-labeled 7alpha -hydroxy-4-cholesten-3-one indicated the presence of autooxidized [3H]cholesterol or [3H]cholesteryl oleate (i.e. free or esterified 7alpha -hydroxycholesterol) in the media during the incubation. The amounts of 3H-labeled 27-hydroxycholesterol, 7alpha ,27-dihydroxy-4-cholesten-3-one and 7alpha -hydroxy-3-oxo-4-cholestenoic acid were determined in media and cells after incubating normal human fibroblasts (protein content, 0.7 mg; size of dishes, 57 cm2) for 68 h with media (10 ml) containing LDL (4% FCS; cholesterol concentration, 1.2 mM) labeled with [3H]cholesteryl oleate or [3H]cholesterol. When 27-hydroxylation of sterols was obstructed by cyclosporin A (CsA, 10 µM), the accumulation of 3H-labeled 7alpha -hydroxy-4-cholesten-3-one indicated the presence of autooxidized [3H]cholesterol or [3H]cholesteryl oleate (i.e. free or esterified 7alpha -hydroxycholesterol) in the media during the incubation.
Steroid structurea Amount of 3H-labeled oxysterol foundb
FCS + [3H]cholesteryl oleatec
FCS + [3H]cholesterolc
Control +CsA Control +CsA

dpm
C5-3beta ,27-ol 12,000 <1,970 7,090 <120
C4-7alpha ,27-ol-3-one 5,140 <1,140 88,800 12,800
CA4-7alpha -ol-3-one 10,800 <120 26,300 1,690
C4-7alpha -ol-3-one <410 <410 1,450 25,300

a For abbreviations and steroid names, see Table I.
b Values represent the sum of the amounts found in medium and cells; < = an amount at or below the detection limit.
c FCS was preincubated for 16 h at 20 °C with [3H]cholesteryl oleate (48 × 106 dpm) or [3H]cholesterol (49 × 106 dpm). All cells had been preincubated for 24 h in media containing 10% LDS.


Fig. 2. HPLC analyses of 3H-labeled 7alpha ,27-dihydroxy-4-cholesten-3-one and 7alpha -hydroxy-3-oxo-4-cholestenoic acid isolated from the medium of normal human fibroblasts. The cells (0.7 mg of protein) were incubated for 68 h with LDL (4% FCS) labeled with [3H]cholesteryl oleate, and the medium was then taken for analysis by HPLC. For experimental details, see ``Materials and Methods'' and Table IV. After the injections, fractions of the HPLC column effluent were collected in scintillation vials with 15-60-s intervals, and the radioactivity was then determined. For purpose of illustration, unlabeled 7alpha ,27-dihydroxy-4-cholesten-3-one (results shown in top chromatograms) or 7alpha -hydroxy-3-oxo-4-cholestenoic acid (as methyl ester derivative, bottom chromatograms) were injected together with the samples, and the peaks of these compounds are seen in the UV chromatograms. A column of silica (LiChrospher) connected to a UV detector was used with hexane/isopropyl alcohol (90:10) as mobile phase, and the flow rate was 1.0 ml × min-1.

Metabolism of LDL Cholesterol and Side-chain Hydroxylated Sterols in Transformed Fibroblasts

In contrast to normal fibroblasts, only small amounts of 27-oxygenated sterols were detected in media after incubating transformed human fibroblasts with lipoproteins (Table II). Although the increased amounts of 7alpha -hydroxy-4-cholesten-3-one could indicate that 27-hydroxylation of sterols was obstructed in these cells (see above), the lack of 27-hydroxylated sterols in the media could also be due to a decreased cellular uptake of LDL and oxysterols, or to an increased formation of conjugates (e.g. fatty acid esters or sulfate esters).

Table V shows the distribution of 3H-labeled cholesterol and cholesteryl esters after incubating normal and transformed fibroblasts with lipoproteins labeled with radioactive cholesterol or cholesteryl oleate for 48 h. Results on the oxysterol production from the same incubations are those shown in Table II. As seen in Table V, the cellular uptake and retention (cell content) of [3H]cholesterol in normal and transformed cells were about 16 and 22%, respectively. About 4% had been esterified by both cell types. After the incubations with [3H]cholesteryl oleate, the retention of the compound was about 11% in the normal cells and 15% in the transformed cells, although a major portion had been hydrolyzed in the cells. About 9% and 35% of hydrolyzed [3H]cholesterol were also present in media of the normal and transformed fibroblasts, respectively, due to an efflux of hydrolyzed LDL cholesterol from the cells (6, 24). When the cellular content and the efflux of cholesterol in the incubations with [3H]cholesteryl oleate were added together, the total uptake of [3H]cholesteryl oleate in the normal and the transformed cells was 19% (32%/mg of protein) and 50% (36%/mg of protein), respectively. These results show that the uptake of LDL (reflected by that of [3H]cholesteryl oleate) was not decreased but possibly increased in the transformed cells also when the protein content of the cells was taken into account. Thus, a possible reduced formation of 27-hydroxylated sterols by transformed fibroblasts (Table II) was not due to a decreased uptake of LDL.

Table V.

Uptake and handling of free and esterified [3H]cholesterol in normal and transformed fibroblasts

Distribution of radioactivity after incubating normal and virus-transformed human fibroblasts for 48 h with media containing lipoproteins (10% FCS) labeled with [3H]cholesterol or [3H]cholesteryl oleate. The total concentration of unlabeled cholesterol in FCS was 1.2 mM. Incubations for 0.25 h served as controls. Distribution of radioactivity after incubating normal and virus-transformed human fibroblasts for 48 h with media containing lipoproteins (10% FCS) labeled with [3H]cholesterol or [3H]cholesteryl oleate. The total concentration of unlabeled cholesterol in FCS was 1.2 mM. Incubations for 0.25 h served as controls.
Incubation conditionsa
Distribution of free and esterified [3H]cholesterol
Time Additions to incubation mediumb Medium
Cells
Free Esters Total in medium Free Esters Total in cells

h % recovered radioactivityc
Normal cells
0.25 [3H]Cholesterol in FCS 95 4 99 1 <1 1
48 [3H]Cholesterol in FCS 82 3 85 11 4 15
48 [3H]Cholesterol in FCS 80 4 84 11 4 16
0.25 [3H]Cholesteryl oleate in FCS 2 98 100 <1 <1 <1
48 [3H]Cholesteryl oleate in FCS 10 81 91 6 4 9
48 [3H]Cholesteryl oleate in FCS 11 77 88 9 3 12
Transformed cells
0.25 [3H]Cholesterol in FCS 95 4 99 1 <1 1
48 [3H]Cholesterol in FCS 66 12 79 17 4 21
48 [3H]Cholesterol in FCS 73 5 78 18 4 22
0.25 [3H]Cholesteryl oleate in FCS 1 99 100 <1 <1 <1
48 [3H]Cholesteryl oleate in FCS 38 49 86 11 3 14
48 [3H]Cholesteryl oleate in FCS 34 51 84 12 3 16

a Protein contents of normal and virus-transformed fibroblasts were 0.6 mg and 1.4 mg, respectively. The size of the incubation dishes was 57 cm2. All cells had been preincubated for 24 h in media containing 10% LDS. Results from these incubations are also shown in Table II.
b The amounts of [3H]cholesterol and [3H]cholesteryl oleate added to the medium were 22.8 × 106 dpm and 18.9 × 106 dpm, respectively. The sterols were preincubated with FCS for 16 h at 20 °C.
c The total recovery was >90% of the radioactivity added.

In order to determine whether the shortage of 27-hydroxycholesterol and other 27-hydroxylated sterols in media of transformed fibroblasts could be due to an increased metabolism (other than formation of C27-acids) or conjugation, the metabolism of 25-hydroxycholesterol was studied. The major reason for selecting this sterol instead of 27-hydroxycholesterol was that 25-hydroxycholesterol was available in a 3H-labeled form, so that major metabolites or conjugates could be traced and would not escape detection. Because of their similarities in structure (both having a 3beta -hydroxy-Delta 5 structure and one hydroxyl group in the side chain), the cellular handling of the two sterols was expected to be similar (except that the 25-hydroxy group could not be oxidized to a carboxyl group). Table VI shows the distribution of radioactivity when 3H-labeled 25-hydroxycholesterol (plus unlabeled, 1.2 nmol) had been incubated with normal and transformed fibroblasts for 48 h in 10% LDS. In addition to 25-[3H]hydroxycholesterol, two major radioactive metabolites, one polar and one nonpolar, were found by HPLC. Other metabolites constituted less than 1% each of the recovered radioactivity. No radioactivity (<0.1%) corresponding to oxidized 25-[3H]hydroxycholesterol without a 7alpha -hydroxy group (i.e. 25-hydroxy-4-cholesten-3-one, see below) was found. Neither did we find any radioactivity (<1%) in fractions containing weak acids (e.g. having a free carboxyl group) or stronger acids (e.g. glucuronides or mono- or disulfates) which were isolated from the extracts by anion exchange chromatography. After collecting a fraction of the HPLC effluent containing the polar metabolite and derivatization, it was identified by GC/MS as 7alpha ,25-dihydroxy-4-cholesten-3-one (21). The nonpolar metabolite(s) had a retention time (3.7 min), which was similar to that of the 3-acetate derivative of 25-hydroxycholesterol (retention time 4.9 min) by straight-phase HPLC (retention time of free 25-hydroxycholesterol was 11.8 min). It was therefore tentatively characterized as being fatty acid esters of 25-[3H]hydroxycholesterol. This was supported further by the recovery of free 25-[3H]hydroxycholesterol after treating the nonpolar metabolite(s) with mild alkali in a methanolic solution. Table VI clearly reveals large differences in the handling of 25-[3H]hydroxycholesterol between the two cell lines. Intact 25-[3H]hydroxycholesterol was found mainly in the cells (32% in the normal and 50% in the transformed cells). A large portion (about 43%; 71%/mg of protein) of 25-[3H]hydroxycholesterol had been converted to 7alpha ,25-dihydroxy-4-cholesten-3-one by normal cells (21) but much less so (about 3%; 2%/mg of protein) by the transformed cells. This sterol was recovered mainly in the media. On the other hand, esterification of 25-[3H]hydroxycholesterol was noted only in the transformed cells, although the amount of esters was relatively small (about 7%). These results show that 25-hydroxycholesterol is readily taken up by both normal and transformed cells, but whereas the sterol is extensively 7alpha -hydroxylated in normal cells, this reaction is obstructed in transformed cells. The results also suggested that the apparent shortage of 27-hydroxycholesterol in transformed cells was due to a decreased formation rather than an increased conjugation, since only a minor amount of the analogous sterol 25-hydroxycholesterol was esterified and since no other conjugates were found.

Table VI.

Metabolism of 25-[3H]hydroxycholesterol in normal and transformed fibroblasts

Distribution of recovered radioactivity after incubating normal and virus-transformed human fibroblasts for 48 h with media containing 3H-labeled and unlabeled 25-hydroxycholesterol and 10% LDS (cholesterol concentration, 0.1 mM). Incubations for 0.25 h served as controls. Distribution of recovered radioactivity after incubating normal and virus-transformed human fibroblasts for 48 h with media containing 3H-labeled and unlabeled 25-hydroxycholesterol and 10% LDS (cholesterol concentration, 0.1 mM). Incubations for 0.25 h served as controls.
Radioactive compound Relative amount founda
Normal fibroblasts
Transformed fibroblasts
0.25 h 48 h 48 h 0.25 h 48 h 48 h

% recovered radioactivityb
In medium
25-[3H]Hydroxycholesterol 47 5 4 73 21 21
Nonpolar metabolite(s)c <1 <1 <1 <1 2 2
Polar metabolite(s)d 2 43 37 2 2 2
Total radioactivity in mediume 57 57 51 79 30 29
In cells
25-[3H]Hydroxycholesterol 35 30 34 18 49 50
Nonpolar metabolite(s)c <1 <1 <1 <1 5 5
Polar metabolite(s)d <1 4 5 <1 2 3
Total radioactivity in cellse 43 43 49 22 70 71

a The amounts of 3H-labeled and unlabeled 25-hydroxycholesterol added to the medium (10 ml, containing 10% LDS) were 1.4 × 106 dpm and 1.2 nmol, respectively. Protein contents of normal and virus-transformed fibroblasts were 0.6 mg and 1.4 mg, respectively. The size of the incubation dishes was 57 cm2. All cells had been preincubated for 24 h in media containing 10% LDS.
b The total recovery was about 90% of the radioactivity added.
c Retention time on straight-phase HPLC was 3.5-4.0 min using hexane/isopropyl alcohol (97:3) as the mobile phase and a flow-rate of 1 ml × min-1. It was tentatively identified as 25-hydroxycholesterol esterified with a fatty acid.
d Retention time on reversed-phase HPLC was 3.0-4.5 min using methanol/ethanol/water (80:20:10) as the mobile phase and a flow rate of 1 ml × min-1. In this fraction, 7alpha ,25-dihydroxy-4-cholesten-3-one was identified by gas chromatography-mass spectrometry, and the amounts were similar to those calculated from the radioactivity.
e Also includes other radioactive compounds, each accounting for less than 1% of the total radioactivity.

After these observations, the rates of oxidation/isomerization of 3beta ,7alpha -dihydroxy-Delta 5 steroids to 3-oxo-Delta 4 steroids in normal and transformed cells were also investigated. 7alpha ,27-Dihydroxycholesterol (1.2 nmol) was therefore incubated with normal and transformed fibroblasts (protein contents 0.4 and 1.1 mg, respectively, size of dishes 57 cm2) for 48 h in media (10 ml) containing 10% LDS, and the metabolites were then analyzed by GC/MS. Incubations for 15 min served as controls. The analyses showed that this sterol was readily taken up by the cells, since only about 1% remained in the media. In media from normal cells, about 26% and 39% were recovered as 7alpha ,27-dihydroxy-4-cholesten-3-one and 7alpha -hydroxy-3-oxo-4-cholestenoic acid, respectively. The corresponding values for the transformed cells were 28% and 19%. Trace amounts (about 1%) were converted to 3beta ,7alpha -dihydroxy-5-cholestenoic acid in the transformed cells. No 7-oxo-, 7beta -hydroxy-, or other metabolites were found. Thus, almost the same amounts of 7alpha ,27-dihydroxycholesterol were oxidized by the normal and transformed fibroblasts. However, when the number of incubated cells (cell protein content) were taken into account, the oxidation rate in transformed cells was calculated to be about 25% of that of normal cells. These studies show that the apparent activities of 27- and 7