©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Cell Toxicity Induced by Inhibition of Acyl Coenzyme A:Cholesterol Acyltransferase and Accumulation of Unesterified Cholesterol (*)

(Received for publication, November 18, 1994; and in revised form, December 19, 1994)

Gregory J. Warner Genevieve Stoudt Mark Bamberger (1) William J. Johnson George H. Rothblat (§)

From the Medical College of Pennsylvania, Department of Biochemistry, Philadelphia, Pennsylvania 19129 and Pfizer Central Research, Groton, Connecticut 06340

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Considerable evidence supports the involvement of acyl-CoA:cholesterol acyltransferase (ACAT) in the maintenance of intracellular cholesterol homeostasis. A number of recently developed ACAT inhibitors may have potential use as pharmacological agents to reduce the development of atherosclerosis. Recently, however, reports arose describing cytotoxic effects following administration of a specific ACAT inhibitor to experimental animals. In order to address the specific intracellular mechanisms involved with the cytotoxic effect, we examined the consequences of ACAT inhibition in cholesterol-enriched mouse peritoneal macrophages. Mouse peritoneal macrophages were cholesterol-enriched by incubation with acetylated low density lipoprotein and free cholesterol:phospholipid dispersions prior to the addition of an ACAT inhibitor, either Sandoz 58-035 or Pfizer CP-113,818. The adenine pool of the macrophages was radiolabeled prior to addition of the ACAT inhibitors, in order to monitor the release of radiolabeled adenine, a technique shown to be a sensitive method to monitor drug-induced toxicity. The ACAT inhibitors were added for up to 48 h and at concentrations up to 2 µg/ml. These conditions resulted in an approximately 2-fold increase in adenine release. The increase in cell toxicity paralleled an increase in the cellular free cholesterol content. Reducing the cellular free cholesterol content, by the addition of extracellular acceptors, decreased the cytotoxic effects of the ACAT inhibitors. Addition of an intracellular cholesterol transport inhibitor, either progesterone or U18666A, together with CP-113,818 blocked the toxic effect of CP-113,818. These results suggest that ACAT inhibition of cholesterol-enriched macrophages increases cell toxicity due to the buildup of cellular free cholesterol. Removal of free cholesterol by the addition of extracellular cholesterol acceptors or by blocking intracellular sterol transport relieves the ACAT inhibitor-induced toxicity.


INTRODUCTION

Intracellular esterification of cholesterol is accomplished by the microsomal enzyme, acyl coenzyme A:cholesterol acyltransferase (ACAT)(^1)(1) . This reaction is believed to play a crucial role in the maintenance of cellular cholesterol homeostasis. Considerable evidence supports the involvement of ACAT in the intestinal absorption of cholesterol(1) , in the secretion of hepatic lipoproteins(2, 3) , and in the accumulation of cholesteryl esters in atherosclerotic lesions(4) . Inhibition of ACAT at any of these points may be beneficial in slowing the development of atherosclerosis.

Several ACAT inhibitors have been developed that show beneficial effects toward reducing atherosclerosis in experimental animals(5, 6) . The structural characteristics of these compounds vary considerably, although most inhibit ACAT activity with IC values less than 1 µM(7) . While the potential benefits of ACAT inhibitors have been widely reported, recent data suggests a potential toxic effect of these compounds(8, 9, 10) . These researchers noted an acute toxic effect of a specific ACAT inhibitor PD132301-2 in adrenal cells of both guinea pigs and beagle dogs. Vernetti et al.(10) concluded that this toxic effect may be linked to ATP depletion of mitochondria.

Maintenance of cellular free cholesterol concentrations within small ranges appears to be critical for preservation of cellular functions (11) . Inhibition of the enzyme catalyzing cholesteryl ester formation, and therefore controlling intracellular free cholesterol concentrations, may have consequences with regard to cell viability. The aim of this study was to more closely examine the intracellular mechanisms that may be responsible for the loss of cell viability as a consequence of ACAT inhibitor treatment. We hypothesize that free cholesterol accumulation, resulting from the blocking the ACAT arm of the cholesteryl ester cycle, is responsible for the observed toxic effects following ACAT inhibitor treatment.


EXPERIMENTAL PROCEDURES

Materials

Heat-inactivated fetal bovine serum, bovine serum albumin (BSA, essentially fatty acid free), phosphatidylcholine (PC), unesterified cholesterol, progesterone, cholesteryl methyl ether, and gentamicin were purchased from Sigma. Organic solvents were purchased from Fisher. Tissue culture plates (12-well with 22-mm well diameter) were obtained from Corning Glass Works (Corning, NY). Culture media was from Life Technologies, Inc. Human LDL (1.019 g/ml < d < 1.063 g/ml) and HDL(3) (1.125 g/ml < d < 1.21 g/ml) were fractionated by sequential ultracentrifugation(12) . ApoE-containing particles in total HDL preparations were removed by heparin-Sepharose chromatography(13) . Human LDL was acetylated by the method of Basu et al.(14) . Free cholesterol and PC dispersions (2-3:1 mol of free cholesterol/mol of PC) were prepared according to Arbogast et al.(15) . Human apoAI was prepared as described previously (16) and stored at -20 °C. Apo-HDL/PC vesicles were prepared according to the method of Adelman et al.(17) . All lipoproteins, dispersions, and vesicles were sterilized by filtration (0.45 µM) (Millipore Corporation, Bedford, MA) prior to addition to cell culture media. Compound 58-035 (3-[decyldimethylsilyl]-N-[2-(4-methylphenyl)-1-phenylethyl]propanamide) was a gift from Dr. John Heider of Sandoz, Inc. (East Hanover, NJ). Compound 113,818 (-)-N-(2,4-bis(methylthio)-6-methylpyridin-3-yl)-2-(hexylthio)decanoic amide) was provided by Pfizer Central Research (Groton, CT). Compound U18666A (3beta-[2-(diethylamino)ethoxy]androst-5-en-17-one) was provided by the Upjohn Co. (Kalamazoo, MI).

Cell Culture and Isolation of Mouse Peritoneal Macrophages

B(6)C(3)F(1) mice (Taconic Farms, Germantown, NY) were injected intraperitoneally with 0.5 ml of sterile 10% thioglycolate solution. Five days after injection, the exudate cells were isolated by peritoneal lavage with 5-ml washes of sterile phosphate-buffered saline containing 10 IU/ml of heparin. The macrophages were pelleted by centrifugation at 1000 rpm for 15 min at 22 °C and resuspended in RPMI 1640 containing 50 mM HEPES buffer, 50 µg/ml gentamicin, and 2 g/liter sodium bicarbonate (Buffer A). Cells (1.5 times 10^6) were plated in 22-mm wells in Buffer A containing 7.5% heat-inactivated fetal bovine serum for 4 h. Following this time, cells were washed 1-3 times with 1 ml of HEPES-buffered minimum essential medium containing 2 g/liter sodium bicarbonate and 50 µg/ml gentamicin (Buffer B) to remove nonadherent cells. At this point, macrophages were incubated (unless otherwise indicated) with Buffer A containing 250 µg of free cholesterol/ml of free cholesterol:PC dispersions, 100 µg of protein/ml of acetylated LDL, and 1% heat-inactivated fetal bovine serum for 24 h. After this cholesterol-enrichment phase, cells were washed 3 times with 1 ml of Buffer B and then incubated for 18 h with Buffer A containing 0.2% BSA. At this point, cells were ready for the addition of ACAT inhibitors. All cells were incubated at 37 °C in a humid atmosphere containing 95% air and 5% CO(2).

Measurement of Oleic Acid Incorporation into Cholesteryl Oleate

A complex of bovine serum albumin with sodium [9,10-^3H]oleate (9.2 Ci/mmol, DuPont NEN) was prepared (18) and added to the macrophage incubations during the final 2 h of each experiment. Each well received 31 nmol of [^3H]oleate complex (molar ratio of oleate/BSA, 6:1) containing 23,000 dpm/nmol.

Cell monolayers were extracted with isopropyl alcohol containing cholesteryl [1-^14C]oleate (50,000 dpm, 59.5 mCi/mmol) (DuPont NEN) as an internal standard to correct for losses during extraction. After drying under N(2), the lipids were dissolved in 50 µl of chloroform/methanol (1:1) and plated onto Silica Gel G thin-layer chromatography plates (Whatman PE SIL G, Whatman, Ltd, Maidstone, Kent, United Kingdom). Cholesteryl esters were separated from other lipids using hexane/ethyl ether/acetic acid (80:20:1) as a developing solvent and visualized by co-migration with nonradioactive cholesteryl oleate. Areas were cut and analyzed for radioactivity by dual isotope liquid scintillation spectroscopy (Beckman LS 3801, Beckman Instruments, Inc., Irvine, CA).

Measurement of ACAT Inhibitor-induced Cell Toxicity

Macrophages were isolated and cultured as described under ``Experimental Procedures.'' ACAT inhibitor-induced cell toxicity was quantitated essentially as described by Shirhatti and Krishna (19) except as modified by Reid et al.(20) . Briefly, following the 18-h incubation period of macrophages with 0.2% BSA, either 1 µCi of [8-^3H]adenine (23 Ci/mmol) (Amersham Corp.) or 0.5 µCi of [8-^14C]adenine (54 mCi/mmol) (Amersham Corp.) was added to each well for 2 h unless otherwise indicated. Following this time, wells were washed 3 times with 1 ml of Buffer B containing 0.2% BSA and then incubated an additional 10 min with 1 ml of Buffer B containing 0.2% BSA. Media were removed, and treatment media were added for 24 h unless otherwise indicated. After the indicated times, triplicate 200-µl aliquots of media were removed and filtered (MultiScreen filtration system, Millipore Corp., Bedford, MA) to remove any cellular debris. Aliquots were analyzed for the release of cellular ^3H or ^14C by liquid scintillation spectroscopy (Beckman LS 3801, Beckman Instruments Inc., Irvine, CA).

Cellular Cholesterol Quantification

Following ACAT inhibitor incubations, cell media were removed, and the macrophage monolayers were washed 3 times with 1 ml of phosphate-buffered saline. Lipids were extracted with isopropyl alcohol containing 10 µg of cholesteryl methyl ether as an internal standard. Free and total cholesterol contents were quantified by gas-liquid chromatography (21) using the method of Ishikawa et al.(22) except for the omission of methyl butyrate from the extraction procedure following saponification. (^2)Phospholipid phosphorus was determined by the method of Sokoloff and Rothblat(23) . Cellular protein content was measured according to the method of Lowry et al.(24) as modified by Markwell and colleagues(25) .

Data Analysis

[^3H]Adenine release from macrophages was expressed as a percentage of release compared with the control treatment (0.2% BSA): (cpm in medium of treatment - cpm in medium of control)/([^3H]adenine cpm at time zero) times 100, or as a percent release of total cell [^3H]adenine content at time zero. [^3H]Oleic acid incorporation into cholesteryl [^3H]oleate was expressed as the percent inhibition compared with the control treatment (0.2% BSA): ((cpm control - cpm treatment)/cpm control) times 100. Values are expressed as the mean ± S.D. for triplicate determinations unless otherwise indicated. GraphPad Prism software package (version 1.0, GraphPad Software Inc., San Diego, CA) was used to analyze ACAT inhibitor concentration-dependence curves. Data were fitted to a four-parameter logistics equation that generated the best line to the experimental data and calculated the ACAT inhibitor concentrations (EC) that gave half-maximal [^3H]adenine release, ACAT inhibition, and cellular free cholesterol concentrations. The unpaired Student's t test was used to analyze for statistical differences between treatment and controls. Differences were determined to be significantly different at p < 0.05.


RESULTS

The goal of this investigation was to establish if the intracellular accumulation of free cholesterol in macrophagederived foam cells could lead to cell toxicity. For this purpose, we used cholesterol-enriched peritoneal macrophages as a model for foam cells and induced free cholesterol accumulation by inhibiting the ACAT arm of the cholesteryl ester cycle. This approach has been used in a number of investigations and results in the cellular deposition of substantial stores of free cholesterol(26, 27, 28, 29) . Our measure of toxicity was based on the established method of monitoring the release of radiolabeled adenine from control and drug-treated cells(19) , a technique that has been demonstrated to accurately quantify cell integrity in cell lines including the cholesterol-loaded macrophage(20) . In preliminary studies, we observed that the release of radiolabeled adenine correlated with the release of lactate dehydrogenase, a well accepted marker of cell toxicity. Therefore, this assay appears to be a reliable marker for ACAT inhibitor-induced toxicity in the cholesterol-loaded macrophage.

Time Course of ACAT Inhibitor Toxicity

Fig. 1illustrates the time course of [^3H]adenine release from cholesterolenriched macrophages incubated in the absence and presence of the ACAT inhibitor CP-113,818 (2.0 µg/ml). Adenine leakage increased over time and was significantly greater (p < 0.001) than the control after 12 h of exposure to the drug. By 36 h, the release of [^3H]adenine from ACAT inhibitor-treated macrophages was 2-fold greater than the background release of the adenine observed with control macrophages.


Figure 1: Time course of mouse peritoneal macrophage cell toxicity following incubation with compound CP-113,818. Mouse peritoneal macrophages were enriched with cholesterol for 24 h with acetylated LDL, free cholesterol/phospholipid dispersion, and fetal bovine serum as described under ``Experimental Procedures.'' The cells were then incubated with 1 µCi of [^3H]adenine for 2 h before the addition of the ACAT inhibitor. Macrophages were incubated with CP-113,818 (2.0 µg/ml) dissolved in Me(2)SO or Me(2)SO alone as control. The final concentration of Me(2)SO did not exceed 0.1%. The release of [^3H]adenine from the macrophages was monitored for up to 36 h. Values are presented as the percent of total cellular ^3H cpm released to the culture medium for control (box) and CP-113,818-treated (bullet) macrophages. Values are averages from triplicate wells. *, p < 0.001 versus control at same time point.



Relationships between Free Cholesterol Accumulation and Cell Toxicity

To establish if a direct correlation existed between the level of free cholesterol that accumulated in macrophages and the leakage of [^3H]adenine, it was necessary to design experiments in which the level of accumulated free cholesterol could be varied in a controlled manner. This was achieved in two ways: 1) by varying the extent of cholesteryl ester loading (since the concentration of free cholesterol that results from the inhibition of ACAT is a function of the amount of esterified cholesterol undergoing hydrolysis by the neutral cholesteryl ester hydrolase(26, 30) ) and 2) by varying the concentration of extracellular acceptors present during the ACAT inhibition phase of the experiment. The presence of increasing concentrations of extracellular acceptors stimulates free cholesterol efflux (31) and thus modulates cell free cholesterol content.

Fig. 2shows the results of an experiment in which monolayers of macrophages were loaded with increasing levels of cholesteryl esters and then exposed to the ACAT inhibitor CP-113,818 for 24 h in the absence of any extracellular cholesterol acceptors. The different degrees of cholesteryl ester loading were achieved by prior exposure of the cells to acetylated LDL (100 µg of protein/ml) together with increasing concentrations of a 2:1 (mol/mol) free cholesterol/phospholipid dispersion. As expected(32) , this treatment resulted in more than a 4-fold increase in cell total cholesterol above unloaded, control macrophages, with the major accumulation occurring in the cholesteryl ester pool (Fig. 2A). As shown in Fig. 2B, the treatment of each group of macrophages with the ACAT inhibitor for 24 h led to a significantly higher (p < 0.001) release of [^3H]adenine than observed in parallel cholesterol-loaded cells incubated under identical conditions but in the absence of the inhibitor. In unloaded control macrophages the effect of CP-113,818 (2.0 µg/ml) on cell toxicity was small but statistically significant (p < 0.001). In cells cholesterol-enriched by prior incubation with the highest concentration of dispersion, the ACAT inhibitor CP-113,818 (2.0 µg/ml) increased cell toxicity nearly 5-fold over controls. Fig. 2C illustrates that upon incubation of cholesteryl ester-enriched macrophages with CP-113,818, these cells accumulate free cholesterol. At all levels of added cholesterol/phospholipid dispersion tested, the free cholesterol content of the ACAT inhibitor-treated macrophages was significantly higher than untreated controls (p < 0.05). Maximal [^3H]adenine release and cell free cholesterol content (time 24 h) was reached with a concentration of 250 µg of free cholesterol added via free cholesterol/phospholipid dispersion.


Figure 2: Effect of varying levels of macrophage cholesterol enrichment on the accumulation of free cholesterol and the release of adenine after treatment with ACAT inhibitors. Macrophages were cholesterol-loaded for 24 h with fetal bovine serum, acetylated LDL, and the specified amounts of free cholesterol/phospholipid dispersion. Following an 18-h equilibration period in 0.2% BSA media, some wells were analyzed for free and esterified cholesterol as described under ``Experimental Procedures.'' The remaining monolayers were then incubated with 1 µCi of [^3H]adenine for 2 h before the addition of the ACAT inhibitor. Macrophages were incubated in the absence (control) and presence of CP-113,818 (2.0 µg/ml) for 24 h. A, cholesterol content of macrophages at time 0 following cholesterol enrichment with increasing amounts of free cholesterol via a free cholesterol/phospholipid dispersion. The values are presented as the average free () and esterified (&cjs2106;) cholesterol (µg/well) from triplicate dishes. B, influence of increasing cholesterol-loading on cell toxicity. The release of [^3H]adenine from the macrophages was monitored for 24 h. Values are presented as the mean ± S.D. release of [^3H]adenine (% of control) from triplicate wells. C, free cholesterol content following a 24-h incubation with ACAT inhibitor. Values are presented as the mean ± S.D. (from triplicate wells) for cellular free cholesterol content (µg/well) in the absence (&cjs2106;) and presence () of CP-113,818 (2.0 µg/ml). a, p < 0.05 versus control.



The removal of cell cholesterol from mouse peritoneal macrophage-derived foam cells can be accomplished by adding to the incubation medium either native HDL (26) or reconstituted apo-HDLbulletphospholipid complexes (rHDL)(33) . In a preliminary experiment, cholesterol-loaded macrophages were incubated with CP-113,818, either alone or together with the apoprotein-phospholipid complex or with human HDL(3). In addition, a second, extensively used ACAT inhibitor, Sandoz 58-035(34) , was added to parallel cultures alone or together with the cholesterol acceptors. Incubation with Sandoz 58-035 (2.0 µg/ml) resulted in [^3H]adenine release comparable with that observed with 2.0 µg/ml of CP-113,818 (176 and 205% of control, respectively). Co-incubation of either ACAT inhibitor with HDL(3) or rHDL blocked this toxic response. The data for [^3H]adenine release closely paralleled the free cholesterol content of the cells, with free cholesterol accumulating in cells exposed to either of the ACAT inhibitors and with the deposition of cell free cholesterol prevented by the diversion of the cholesterol to extracellular cholesterol acceptors (data not shown). These data, together with those of Fig. 2, indicate that ACAT inhibitor-induced cell toxicity is the result of free cholesterol buildup.

The ability to modulate the extent of free cholesterol accumulation in the drug-treated cells by exposure to graded levels of extracellular cholesterol acceptors provided us the opportunity to more precisely examine the relationship between cell free cholesterol content and cell toxicity as measured by labeled adenine leakage. The data presented in Fig. 3were obtained from a study in which the cholesteryl ester-enriched macrophages were incubated for 24 h in medium supplemented with CP-113,818 (2.0 µg/ml) together with increasing concentrations of rHDL complexes. In this protocol, the cells had been previously labeled with [^3H]cholesterol during the loading phase of the experiment so that the efflux of cell cholesterol could also be measured. Fig. 3A presents the free cholesterol mass data for the cells after a 24-h exposure to rHDL. As the concentration of rHDL increased, cell free cholesterol decreased until free cholesterol contents reached 40-45 µg/well; levels that were equivalent to untreated control cells. Fig. 3B illustrates that the rHDL particles acted as efficient acceptors of the cell cholesterol and stimulated cholesterol efflux in a dose-dependent manner. The release of [^14C]adenine from the treated cells was inversely related to the fractional efflux of cell cholesterol (r^2 = 0.95, p < 0.001) and very highly correlated with the actual mass of cell free cholesterol (r^2 = 0.92, p < 0.001, Fig. 3C).


Figure 3: Effects of increasing apo-HDL/PC complexes on cellular free cholesterol content, cell toxicity, and cholesterol efflux from cholesterol-loaded macrophages. Mouse peritoneal macrophages were cholesterol-loaded for 24 h with acetylated LDL, free cholesterol/phospholipid dispersion, and fetal bovine serum as described under ``Experimental Procedures.'' In wells where [^3H]cholesterol efflux was measured, 4 µCi/well of [1,2-^3H]cholesterol was present in the loading medium during the period of cholesterol enrichment. In this experiment, macrophages were labeled for 18 h with 1 µCi/well of [^14C]adenine during the equilibration period. The appearance of [1,2-^3H]cholesterol and [^14C]adenine in the media were measured after 24 h of incubation with compound CP-113,818 (2.0 µg/well) alone or in combination with apo-HDL/PC at amounts ranging from 0 to 200 µg of phospholipid/ml. A, influence of increasing the media cholesterol acceptor concentration on cell free cholesterol content. Values are presented as the mean ± S.D. for cellular free cholesterol (µg/well) from triplicate wells after a 24-h incubation with CP-113,818 (2.0 µg/well) and various levels of cholesterol acceptor. B, correlation between relative cholesterol efflux and [^14C]adenine release. Values for the fractional efflux of [1,2-^3H]cholesterol are presented as the average of triplicate wells. [^14C]Adenine release is expressed as the mean ± S.D. (% of control) from triplicate wells. The r^2 value was 0.95 (p < 0.001). C, correlation between [^14C]adenine release and cellular free cholesterol content. Values for cell free cholesterol are the average from triplicate wells after a 24-h treatment and are the same as those in B. [^14C]Adenine release is expressed as the mean ± S.D. (% of control) from triplicate wells. The r^2 value was 0.92 (p < 0.001).



The correlative data in Fig. 3C indicate that the ability of extracellular cholesterol acceptors to reduce or eliminate the toxicity of ACAT inhibitors is related to their ability to prevent cell free cholesterol accumulation. However, another mechanism that could explain the protective effect of the extracellular cholesterol acceptors could be related to a possible ability to bind the ACAT inhibitors and prevent uptake into cells. Both CP-113,818 and Sandoz 58-035 are hydrophobic compounds that could associate with the cholesterol acceptors and thus have a reduced effective concentration in medium containing HDL or rHDL. Cholesterol esterification was reduced more than 97% compared with controls when cells were exposed to CP-113,818 (2.0 µg/ml) alone or together with rHDL complexes, whereas rHDL alone reduced esterification only 80%. This result indicates that the ACAT inhibitor, at the concentrations used in this study, was equally effective in blocking ACAT activity in both the absence and presence of the cholesterol acceptor.

Dose Curve of ACAT Inhibition, Cholesterol Accumulation, and Adenine Release

In all of the studies described above, the amount of ACAT inhibitor used was more than sufficient to inhibit greater than 97% of cellular cholesterol esterification activity. To further examine the relationships between ACAT inhibition, cell free cholesterol accumulation and cell toxicity, a series of determinations was conducted examining the dose response of these parameters to increasing concentrations of either CP-113,818 or Sandoz 58-035. Fig. 4shows the relationship between the concentrations of the two ACAT inhibitors and 1) the degree of ACAT inhibition as measured by [^3H]oleate incorporation into cholesteryl esters, 2) cellular free cholesterol content, and 3) the release of [^3H]adenine. At the highest levels tested, both ACAT inhibitors reduced the incorporation of [^3H]oleate into cholesteryl [^3H]oleate by more than 95%, with EC values being 10.4 ng/ml for Sandoz 58-035 and 2.5 ng/ml for CP-113,818. The relationships among ACAT inhibition, free cholesterol buildup, and cell toxicity for both inhibitors are similar. The accumulation of free cholesterol is saturable and occurs at concentrations of CP-113,818 and Sandoz 58-035, which do not fully inhibit ACAT after 24 h of incubation. The EC values for the cholesterol accumulation process are approximately 40% lower than the EC for ACAT inhibition. For CP-113,818 and Sandoz 58-035, the EC values for free cholesterol accumulation were 5.5 and 17.5 ng/ml, and for adenine release were 0.7 and 5.5 ng/ml, respectively. The relationships between free cholesterol accumulation, ACAT inhibition, and adenine release are very similar for both inhibitors, with the CP-113,818 compound being approximately 3-5-fold more potent.


Figure 4: Relationships between ACAT inhibition, cellular free cholesterol concentration, and cell toxicity with increasing concentrations of CP-113,818 and Sandoz 58-035. Mouse peritoneal macrophages were cholesterol-loaded for 24 h with acetylated LDL, free cholesterol/phospholipid dispersion, and fetal bovine serum as described under ``Experimental Procedures.'' Macrophages were incubated with compounds CP-113,818 or Sandoz 58-035 at concentrations up to 50 ng/ml. These concentrations shut down virtually all cholesterol esterification activity after a 24-h incubation. Values for [^3H]adenine release for CP-113,818 (box) and Sandoz 58-035 (), ACAT inhibition for CP-113,818 (up triangle) and Sandoz 58-035 (), and cellular free cholesterol concentrations for CP-113,818 (circle) and Sandoz 58-035 (bullet) are presented as the percent of maximum change measured and represent means from triplicate wells.



Influence of Progesterone and U18666A on Cell Toxicity and Blockage of ACAT Inhibitor-induced Cell Toxicity

Progesterone and U18666A have been reported to act as an inhibitors of intracellular cholesterol transport (28, 35, 36) and as inhibitors of cholesterol esterification in the endoplasmic reticulum(26, 35, 36) . This ACAT inhibition is thought to arise from inhibition of intracellular cholesterol transport rather than a direct effect on ACAT(28, 36, 37) . To determine whether transport of free cholesterol from the site of hydrolysis to another cellular pool was required to induce toxicity, experiments were carried out in which either progesterone or U18666A was added in combination with CP-113,818 to cholesterol-enriched macrophages. As seen in Fig. 5, 1 µg/ml of CP-113,818 caused an approximately 2-fold increase in [^3H]adenine release. The addition of 5 µg/ml progesterone or 1 µg/ml U18666A in combination with 1 µg/ml of CP-113,818 reduced the ACAT inhibitor-induced toxicity by 70% (p < 0.01).


Figure 5: Addition of progesterone or U18666A to relieve ACAT inhibitor-induced cell toxicity. Mouse peritoneal macrophages were cholesterol-loaded for 48 h with acetylated LDL, free cholesterol/phospholipid dispersion, and fetal bovine serum as described under ``Experimental Procedures.'' The cells were incubated with 1 µCi of [^3H]adenine for 2 h before the addition of the treatments. Macrophages were incubated for 24 h with 1 µg/ml CP-113,818 alone or in combination with 5 µg/ml progesterone or 1 µg/ml U18666A. Values for [^3H]adenine release are presented as the mean ± S.D. (% of control) from triplicate wells. a, p < 0.01 versus CP-113,818 alone.



When increasing concentrations of progesterone were added to medium containing a constant amount of CP-113,818 (1 µg/ml), we observed that the addition of progesterone, together with CP-113,818, decreased cell toxicity in a manner that was dose-dependent on the concentration of progesterone (Fig. 6). The addition of 5 µg/ml of progesterone in combination with 1 µg/ml of CP-113,818 showed no increase in toxicity compared with 5 µg/ml of progesterone alone, or control incubations with no additions.


Figure 6: Dose curve of the effect of progesterone on ACAT inhibitorinduced cell toxicity. Mouse peritoneal macrophages were cholesterol-loaded for 24 h with acetylated LDL, free cholesterol/phospholipid dispersion, and fetal bovine serum as described under ``Experimental Procedures.'' The cells were incubated with 1 µCi of [^3H]adenine for 2 h before the addition of the treatments. Macrophages were incubated for 24 h with up to 5 µg/ml progesterone alone or in combination with 1 µg/ml CP-113,818. Values for [^3H]adenine release are presented as the mean ± S.D. (% of control) from triplicate wells for progesterone alone () or progesterone plus CP-113,818 (up triangle).



Free cholesterol concentrations in macrophages treated with CP-113,818 alone or in combination with increasing concentrations of progesterone showed similar increases (data not shown).


DISCUSSION

Inhibition of cholesterol esterification may provide a mechanism by which progression of atherosclerosis is reduced(7) . However, inhibition of ACAT in intact cells, in vitro or in vivo, may result in a redistribution of cellular cholesterol by increasing the free cholesterol and decreasing the cellular cholesteryl ester contents. In this study, using cholesterol-loaded macrophages, we show that such a change in the cellular cholesterol homeostasis resulted in an increased cell toxicity.

Subacute adrenal toxicity caused by a specific inhibitor of ACAT has recently been reported(8, 9, 10) . These researchers attributed the toxic effect to a direct inhibition of mitochondrial respiration in adrenocortical cells. Short term incubation of the ACAT inhibitor with adrenocortical cells resulted in a rapid loss of cell viability. Further examination into the mechanism of cell toxicity showed that cellular ATP levels were severely compromised prior to loss of cell viability. The present cell toxicity data are consistent with these findings.

In the present study, we have shown that two specific ACAT inhibitors, Sandoz 58-035 and CP-113,818, induce a free cholesterol accumulation in macrophage-derived foam cells. In each case, the free cholesterol buildup coincides with an increase in cell toxicity. Several lines of evidence suggest that free cholesterol is responsible for this induced toxic effect. First, increasing the cellular content of cholesteryl esters, before ACAT inhibitor treatment, resulted in higher free cholesterol buildup as well as higher [^3H]adenine release (Fig. 2). Second, cellular free cholesterol content and the release of ^14C-labeled nucleotides were highly correlated, while the latter showed a highly negative correlation with efflux of [^3H]cholesterol from macrophages (Fig. 3). Third, the EC for cholesterol buildup is lower than that for ACAT inhibition, indicating that only partial inhibition of ACAT will produce cell free cholesterol accumulation in this closed cell system (Fig. 4). Finally, CP-113,818-induced cell toxicity could be blocked by the combined addition of progesterone or U18666A ( Fig. 5and Fig. 6), compounds which inhibit intracellular sterol transport(35, 36, 38) .

The inhibition of ACAT by itself is not sufficient to produce the toxic accumulation of free cholesterol as evidenced by the observation that ACAT inhibition by progesterone does not produce toxicity equivalent to that observed with Sandoz 58-035 or CP-113,818. In addition, treatment with progesterone or U18666A, together with the ACAT inhibitor, CP-113,818, eliminates the toxicity. As shown in Fig. 7, we propose that interruption of the cholesteryl ester cycle results in the accumulation of unesterified cholesterol. Excess free cholesterol is transported from the site of hydrolysis, and its incorporation into membranes results in the eventual destabilization of the plasma membrane (Fig. 7B). We further propose that progesterone or U18666A interferes with the transport of unesterified cholesterol from intracellular sites of cholesteryl ester hydrolysis to the plasma membrane (Fig. 7C). In support of this hypothesis is the observation that the accumulation of free cholesterol in progesterone-treated cells resulted in the formation of membrane-bound vacuoles containing myelin figures(27, 28) , whereas inhibition of ACAT with pharmacological inhibitors such as Sandoz 58-035 does not induce intracellular storage of cholesterol in intracellular organelles(28) . Thus, our data would be consistent with the free cholesterol-induced toxicity resulting from a destabilization of the plasma membrane upon cholesterol enrichment and the protection produced by progesterone and U18666A being a reflection of the retention of the free cholesterol in intracellular pools.


Figure 7: Diagram of possible mechanism for ACAT inhibitor-induced cell toxicity. A, mouse peritoneal macrophages incubated for 24 h with acetylated LDL, free cholesterol/phospholipid dispersion, and fetal bovine serum have an enlarged cholesteryl ester pool compared with the free cholesterol pool. The cholesteryl ester cycle proceeds uninterrupted. B, the addition of an ACAT inhibitor blocks the conversion of free cholesterol (FC) to cholesteryl ester (CE) resulting in the intracellular buildup of free cholesterol, some of which is transferred to a membrane pool. Enrichment of this membrane pool ultimately results in a loss of cell viability. C, as above, the addition of an ACAT inhibitor causes an intracellular buildup of free cholesterol. However, co-incubation of either progesterone or U18666A, together with the ACAT inhibitor, blocks the toxic effect by inhibiting the intracellular transport of the free cholesterol to the membrane pool.



Our present observations are relevant to considerations of the development of ACAT inhibitors that function at the level of the vessel wall. Studies have shown that unstable plaques are associated with lipid accumulation and foam cells (39, 40) and suggested that a reduction in the extent of lipid deposition will result in the stabilization of the pathologic processes (for a review, see (41) ). Based on our observations, it can be predicted that if ACAT inhibitors are employed to reduce the intracellular levels of cholesteryl esters, cell death could result unless sufficient cholesterol acceptors were present to prevent free cholesterol accumulation.


FOOTNOTES

*
This work was supported by Program Project Grant HL22633, Training Grant HL07443 from the National Institutes of Health, and support from Pfizer Central Research. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Medical College of Pennsylvania, Dept. of Biochemistry, 2900 Queen Ln., Philadelphia, PA 19129.

(^1)
The abbreviations used are: ACAT, acyl coenzyme A:cholesterol acyltransferase; BSA, bovine serum albumin; PC, phosphatidylcholine; Sandoz 58-035, (3-[decyldimethylsilyl]-N-[2-(4-methylphenyl)-1-phenylethyl]propanamide); CP-113,818, (-)-N-(2,4-bis(methylthio)-6methylpyridin-3-yl)-2-(hexylthio) decanoic amide); U18666A, (3beta-[2-(diethylamino)ethoxy]androst-5-en-17-one); LDL, low density lipoprotein; HDL, high density lipoprotein; rHDL, reconstituted high density lipoprotein; apo-HDL/PC, apolipoproteins (from high density lipoproteins) and phosphatidylcholine-reconstituted particles.

(^2)
J. Klansek, personal communication.


ACKNOWLEDGEMENTS

We thank Vinh Nguyen, Dr. Patricia Yancey, and Faye Baldwin for contributions. We also thank Dr. Michael Phillips for helpful discussion.


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