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The effect of chlorcyclizine on cholesterol metabolism in mice
TOXICOLOGY
The
AND
Effect
APPLIED
PHARMACOLOGY,
of Chlorcyclizine
27,449-455 (1974)
on Cholesterol
Metabolism
in Mice
JAMESW. BARNHART The Dow Chemical Company, Development Evaluation Laboratories, Zionsville, Indiana 46077 Received July 19,1972; accepted August 7,1973
The Effect of Chlorcyclizine on Cholesterol Metabolism in Mice. BARNJ. W. (1974). Toxicol. Appl. Pharmacol. 27, 449455. The effect of chlorcyclizine, a known hypocholesterolemic agent, on cholesterol metabolism in mice was reexamined. The reduction of serum cholesterol and increase in liver weight and total cholesterol after chlorcyclizine treatment were confirmed. After a single ip injection of chlorcyclizine (50 mg/kg), a significant elevation of liver cholesterol was observed after 12 hr and a reduction of serum cholesterol was seen after 24 hr. The elevation of liver cholesterol was accounted for exclusively in the esterified fraction. Oral doses effective in reducing serum cholesterol were also effective in elevating liver esterified cholesterol. The incorporation of [%]acetate into cholesterol in vivo was not reduced after chlorcyclizine treatment, suggesting that cholesterol biosynthesis was not inhibited. A marked inhibition of steryl ester hydrolase was caused by chlorcyclizine in vitro. The large excessof cholesteryl esters in the liver was possibly a result of this effect.
HART,
Chlorcyclizine [ I-(p-chloro-cc-phenylbenzyl)-4-methylpiperazine] has been found to reduce serum cholesterol in various mammalian species (Schmidt and Martin, 1965; Barnhart and Sefranka, 1966). This effect was associated with an increase in liver cholesterol and may simply reflect a change in cholesterol distribution. Salvador et al. (1970) also found a hypocholesterolemic effect in mice after treatment with chlorcyclizine, but they concluded that this effect was not due to an accumulation of cholesterol in the liver. Schmidt and Martin (1965) investigated the possibility that chlorcyclizine affects cholesterol biosynthesis, and found a moderate but not significant reduction of [14C]acetate into cholesterol in vivo in mice. The effect of chlorcyclizine on serum and liver cholesterol and cholesterol biosynthesis was reexamined in the experiments reported here, and its effect on another area of cholesterol metabolism, steryl ester hydrolase, was observed. The results are discussed in relation to the hypocholesterolemic activity of this compound.
METHODS Experiments were carried out in Swiss-Webster male mice weighing 25 g.’ Chlorcyclizine hydrochloride2 was given ip in some experiments and incorporated into the ground rodent mash” at a level of 0.06 % in other experiments (as indicated in the tables). 1 Harlan Industries, Cumberland, Indiana. ZK and K Laboratories, Inc., Plainview,New York. 3 Wayne Lab Blox meal, Allied Mills, Inc., Chicago, Illinois. 449 Copyright 0 1974 by Academic Press, Inc. All rights of reproduction Printed in Great Britain
in any form reserved.
450
JAMES W. BARNHART
Serum cholesterol was determined by the method of Henly (1957). Liver cholesterol (free and total) was determined as the digitonide (Sperry and Webb, 1950) after homogenization in 1 : 1 acetone-absolute ethanol. Total protein of crude enzyme preparations was determined by the method of Lowry et al. (1951). Incorporation of sodium [l-14C]acetate into mouse liver lipids was determined 2 hr after iv injections of 0.1 ml of a solution containing4pCi of sodium [1-14C]acetate (4.0 ,&i/2.0 pmol). The animals were decapitated and their livers were homogenized in 20 ml of absolute ethanol as rapidly as possible. Twenty ml of 20 ‘A alcoholic KOH (w/v) were added, and the mixture was heated at 70°C for 1 hr. After cooling, 30 ml of water were added, and the nonsaponifiable lipids were extracted with 3 portions of petroleum ether. Portions of this solution were taken for radiochemical determination of nonsaponifiable material, digitoninprecipitable material, and cholesterol purified via the dibromide procedure (Schwenk and Werthessen, 1952). Digitonides were dissolved in 1.O ml of NCS reagent,4 and all samples were taken up in toluene (5 g PPO + 0.1 g dimethyl POPOP/liter) and counted in a liquid scintillation spectrometer.5 Steryl ester hydrolase (EC 3.1.1.13) activity was determined by using a defatted 104,000 g supernatant of mouse liver homogenate. The supernatant was diluted to a protein concentration of approximately 5 mg/ml. The isolation and assay were done as described by Deykin and Goodman (1962) except that free and total cholesterol were determined by the digitonide procedure (Sperry and Webb, 1950). The substrate was [4J4C]cholesteryl oleate4 with a total activity of 5.43 x lo4 dpm/1.22 nmol added in 50 ~1 of acetone. The in vitro biosynthesis of cholesteryl esters was determined using a mouse liver mitochondrialpreparation (Goodman et al., 1964). The substrate was [4-14C]cholesterol (0.10 &i/O.052 pmol) added in 100 ~1 of acetone. The amount of total protein in each incubation flask was about 8 mg. Free cholesterol and cholesteryl esters were separated on a 1 cm x 4 cm alumina column as described by Deykin and Goodman (1962). There was ~0.2% 14C contamination in the cholesteryl ester fraction from a sample inactivated by heat, and this was disregarded in the calculations. Statistical significance was determined using Student’s t test unless otherwise stated.
RESULTS The degree of incorporation of sodium [i4C]acetate in 2 hr into cholesterol was determined in vivo in mice pretreated with chlorcyclizine for 8 days (Table 1). The quantity of labeled cholesterol in liver (dpm/g liver) in the treated group was increased by about 80 %, but the specific activity (dpm/mg cholesterol) was not increased significantly because of the high total cholesterol in liver. The lowering of serum cholesterol, and increased liver weight and liver total cholesterol after chlorcyclizine treatment in mice (Table 2) confirmed previous reports (Schmidt and Martin, 1965; Barnhart and Sefranka, 1966). However, it was also demonstrated that the increase in liver total cholesterol of over 70 % was accounted for entirely in the esterified fraction. 4 Amersham/Searle Corp., Arlington Heights, Illinois. 5 Packard Instrument Co., Inc., La Grange, Illinois.
CHLORCYCLIZINEAND
451
CHOLESTEROL
TABLE 1 EFFECTOFCHLORCYCLIZINETREATMENTONINCORPORATIONOFSODIUM [ 1J4C] ACETATEINTO MOUSELIVERLIPIDS"
Activity of lipid fractions Control (x103)
Fraction Nonsaponifiable fraction (dpm/g liver) Digitonin-precipitable material (dpm/mg sterol) (dpm/g liver) Cholesterol (dpm/mg cholesterol) (dpm/g liver)
Chlorcyclizine, 0.06 % (x103)
1.81 + 0.62 0.50 f. 0.19
2.26 f 0.86 0.52 + 0.16
1.10 + 0.36 0.39 + 0.25 0.96 + 0.41
1.78 5 0.47b 0.51 + 0.19 1.74 + 0.55b
a After 8 daysof 0.06 % dietary chlorcyclizineHCl4.0 &i of sodium acetatewere injectediv and the mice werekilled 2 hr later. Analysisof total cholesterolindicated a 27‘A reduction of serumcholesterol and a 60% elevation (per gram) of liver total cholesterol in the chlorcyclizine means+ SD from 6 animals. b Significantly higher than control, p < 0.02.
group. The results are
Changes in liver and serum cholesterol in mice were also measured after a single ip injection of chlorcyclizine hydrochloride (Table 3). Groups of animals were sacrificed at 12-hr intervals. Serum cholesterol was unchanged after 12 hr but was lowered by 16 % 24 hr after dosing and remained at about the same level for the next 24 hr. Liver total cholesterol was elevated (15 %) as early as 12 hr after dosing and remained elevated throughout the 48 hr of the experiment. The effect of different dietary levels of chlorcyclizine on serum and liver cholesterol was also studied (Table 4). Drug levels of 0.03 % and 0.06 % were both effective in reducing serum cholesterol about 35 % and elevating total liver cholesterol from a control value of 2.99 mg/g to values over 4 mg/g. Lower
drug levels of 0.0075 % and 0.015 % did not significantly affect cholesterol levels, but liver size was increased at the level of 0.015 %. TABLE 2 EFFECTOFCHLORCYCLIZINETREATMENTONMOUSESERUMAND LIVER CHOLESTEROL"
Control Serum cholesterol (mg/lOO ml) Liver weight (g/100 g body wt) Liver cholesterolb (mg/g liver) : Free Total
137 * 5.5 k 2.41 ? 2.87 f
38 0.8 0.29 0.31
Chlorcyclizine 78 * 11” 9.5 k 1.4d 2.37 + 0.09 5.00 & 0.70d
n Results are means f SD from groups of 5 mice. The treated animals were maintained dietary chlorcyclizine for 8 days. b Digitonin-precipitable material. c Differs significantly from control, p < 0.02. d Differs significantly from control, p i 0.01.
on 0.06 %
452
JAMES
W.
BARNHART
TABLE ACUTE
CHOLESTEROL
CHANGES
IN MICE
3 AFTER
Serum cholesterol, total (mg/lOO ml)
Time (W 0 12 24 36 48 0 12 24 36 48
CHLORCYCLIZINE
TREATMENT”
Liver cholesterolYb free bw/s>
Liver cholesterol,b total (mdd
Control
124 t 122 * 124+ 134 & 128 &
15 11 15 13 21
2.36 2.21 2.24 2.14 2.27
f rt + + f
0.16 0.16 0.20 0.18 0.16
2.58 2.41 2.50 2.30 2.51
k f + k f
0.21 0.18 0.24 0.21 0.19
Chlorcyclizine
114+ 117* 104 * llO? 105 &
15 10 19” 29” 18”
2.35 2.35 2.38 2.14 2.15
rt k f + +
0.21 0.19 0.11 0.22 0.10
2.57 2.76 2.93 2.73 2.83
k + + -t -t
0.21 0.30’ 0.22’ 0.48’ 0.45’
’ Groups of 8 male mice were given ip injections of chlorcyclizine HCl(50 mg/kg) and killed after various intervals. Results are expressed as means k SD. b Digitonin-precipitable material. c Significantly different from corresponding control, p < 0.05. TABLE CHOLESTEROL
Dietary
CHANGES
drug level (%I
IN MICE
AFTER
6.92 7.74 9.09 9.15 8.77
+ + f + f
DOSAGE
LEVELS
Serum cholesterol (mg/lC@ ml)
Liver weight (g/100 g body wt)
0.00 0.0075 0.015 0.03 0.06
4
DIFFERENT
127 f 23 127 + 17 118 k 23 83k27‘ 78 + 25”
0.86 0.50 1.14’ 0.91’ 0.93’
OF CHLORCYCLIZINF?
Liver cholesterol
(mg/db
Free 2.32 2.30 2.10 2.34 2.52
+ + f f +
Total
0.28 0.33 0.05 0.38 0.28
2.99 3.53 3.02 4.36 4.02
f f + f +
1.00 0.92 0.47 0.65’ 0.67d
’ Results are expressed as means f SD from groups of 8 mice (16 in control group). The treated animals were maintained on specified levels of dietary chlorcyclizine HCl for 8 days. A multiple comparison test was used for the statistical evaluation (Dunnett, 1964). b Digitonin-precipitable material. c Differs significantly from control, p < 0.01. d Differs significantly from control, p < 0.05. TABLE EFFECT OF CHLORCYCLIZINE [4-14C]C~~~~~~~~~
Concentration chlorcyclizine 5
X
0 lo-’
lXlo4M
2X104M
M
of
5 ON HYDROLYSIS OLEATE IN VITRO’
% Hydrolysis 47.8 26.7 16.4
7.2
OF
% Inhibition 0 44.1 65.7
84.9
’ Cholesteryl oleate, 1.22 nmol, was present in a total volume of 2.0 ml of phosphate buffer, pH 7.3. Each flask contained about 9 mg of total protein. Results are the average of duplicate determinations. The results were corrected for the small amount of hydrolysis (2.2 %) that occurred nonenzymatitally.
CHLORCYCLIZINE
AND
4.53
CHOLESTEROL
TABLE 6 EFFECT
OF CHLORCYCLIZINE CHOLESTERYL
[4J4C] Concentration chlorcyclizine
of
0
5 X 1o-5 M 1X104M
2X
1o-4
M
ON SYNTHESIS OF ESTERS IN VITRO’
Cholesteryl esters (dpm/flask x 104)
Free cholesterol (dpm/flask x 104) --
3.86 3.82 3.79 3.43
16.2 16.2 16.4 16.7
a The substrate was 0.052 pmol of [4-‘4C]cholesterol in a total volume of 3.0 ml of phosphate buffer, pH 7.3. Each flask contained 25 pmol ATP, 2 pmol of CoA and 8 mg of total protein. Results are the average of duplicate determinations.
The hydrolysis of [4-14C]cholesteryl oleate by a soluble liver fraction was determined after incubation with 5 x 1OJ M, 1 x 1O-4M and 2 x 1O-4M concentrations of chlorcyclizine (Table 5). Chlorcyclizine was an effective inhibitor in this system, causing a 44% inhibition at the lowest concentration. At the same concentrations, chlorcyclizine exerted a very minor effect on a mitochondrial cholesterol esterifying system (Table 6). DISCUSSION
The hypocholesterolemic response and apparent shift of cholesterol from serum to liver caused by chlorcyclizine in mice was reported by Schmidt and Martin (1965). They also studied the effect of chlorcyclizine on the incorporation of [14C]acetate into nonsaponifiable material in vivo in mice. A moderate decrease was observed in the treated group, but the change was not significant. Our results indicate that chlorcyclizine does not inhibit the biosynthesis of cholesterol. There was no significant change in the incorporation of [14C]acetate into cholesterol when the results were expressed in terms of specific activity. This was due to the large increase in liver cholesterol, since there was a significant increase in incorporation of the labeled substrate when the results were expressed in terms of tissue weight. The results were not markedly different when expressed in terms of total sterols or simply as cholesterol, intimating that chlorcyclizine exerts no major effect on the later steps of the biosynthetic pathway. The serum and liver cholesterol changes reported by Schmidt and Martin (1965) were confirmed in the present experiment. Chlorcyclizine is an effective hypocholesterolemit agent in mice, and this effect is accompanied by an increase in liver cholesterol and liver weight. Although it was shown that an increase in liver cholesterol resulted from chlorcyclizine treatment, there was no prior evidence that the serum and liver changes were associated during the acute period of change immediately after dosing. We found that the hepatic hypercholesterolemia was coincident with the reduction of serum cholesterol, and in this instance preceded the serum changes by a few hours. Schmidt and Martin (1965) suggested that dosage levels effective in reducing serum cholesterol were also effective in raising liver cholesterol. We confirmed this observation, substantiating the conclusion that one effect accompanies the other. Salvador
454
JAMESW.BARNHART
et al. ( 1970) found that liver cholesterol was increased in female, but not in male, mice after chlorcyclizine treatment, and inferred that this deposition mechanism was not responsible for the hypocholesterolemic effect in serum, which was detected in both sexes. The possibility that chlorcyclizine has a direct effect on cholesteryl ester metabolism is suggested by the discovery of a marked in vitro inhibition of steryl ester hydrolase. However, there is as yet no direct evidence to substantiate this suggestion. The liver is a major site for cholesterol synthesis in the body and also contains synthetic and hydrolytic systems involving cholesteryl esters. The introduction of an inhibitor of steryl ester hydrolase such as chlorcyclizine into this system could very well upset the normal equilibrium which favors a high proportion of free cholesterol. Chlorcyclizine had no effect on in vitro synthesis of cholesteryl esters. It was, therefore, not a nonspecific enzyme inhibitor. Schweppe and Jungmann (1971) investigated the effect of several hypocholesterolemic agents on cholesteryl ester synthesis in vitro and found a potentiation in some cases. Steryl ester hydrolase was not examined. The increase in liver total cholesterol caused by chlorcyclizine was wholly accounted for in the esterified fraction. The high proportion of cholesteryl esters in the liver is a distinct departure from normality, but is known to be induced by various dietary regimens (Goodman, 1965). The mechanism responsible for this change is unknown. A familial disorder in humans involving high levels of hepatic cholesteryl esters has been described by Schiff et al. (1968); liver cholesteryl esterase activity was found to be normal, and serum total cholesterol levels were moderately elevated. The metabolic defect was thought by these authors to be a block in the biliary excretion of cholesterol; however, no reason for the high proportion of cholesteryl esters was suggested. It is not known whether the high proportion of cholesteryl esters in the liver could be responsible for a decreased cholesterol flux from the liver and the ultimate hypocholesterolemic effect of chlorcyclizine. Differences in the transport of cholesterol and cholesteryl esters are known. Goodman (1965) reported that cholesteryl esters must be hydrolyzed to free cholesterol before intestinal absorption. It was suggested that cholesteryl esters may be taken up intact by the liver. A small amount of cholesteryl esters may be incorporated into lipoprotein and released by the liver, but the major portion of the esters in plasma probably are formed by the plasma lecithin-cholesterol acyltransferase reaction (Glomset, 1970). ACKNOWLEDGMENTS The author is grateful to Dr. John N. Eble for many valuable comments during the preparation of the manuscript. REFERENCES BARNHART, J. W. AND SEFRANKA, J. A. (1966). Hypocholesterolemic activity of antihista-
minics. Life Sci. 5, 871-874. DEYKIN, D. AND GOODMAN, DE W. S. (1962). The hydrolysis of long-chain fatty acid esters
of cholesterol with rat liver enzymes.J. Biol. Chem. 231,3649-3656. DUNNETT, C. W. (1964). New tables for multiple comparisons with a control. Biometrics
20,482-491. GLOMSET, J. A. (1970). Physiological role of lecithin-cholesterol acyltransferase. Amer. J.
Clin. Nutr. 23, 1129-1136.
CHLORCYCLIZINE
AND
CHOLESTEROL
455
GOODMAN, DE W. S. (1965). Cholesterol ester metabolism. Physiol. Rev. 45,747-839. GOODMAN, DE W. S., DEYKIN, D. AND SHIRATORI, L. (1964). The formation of cholesterol esters with rat liver enzymes. J. Biol. Chem. 239, 1335-1345.
A. A. (1957). The determination of serum cholesterol. Analyst 82,286-287. 0. H., ROSEBROUGH, N. J., FARR, A. L. AND RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193,265-275. SALVADOR, R. A., ATKINS, C., HABER, S. AND CONNEY, A. H. (1970). Changes in the serum concentration of cholesterol, triglycerides and phospholipids in the mouse and rat after administration of either chlorcyclizine or phenobarbital. Biochem. Pharmacol. 19, 14631469. SCHIFF, L., SCHUBERT, W. K., MCADAMS, J., SPIEGEL, E. L. AND O’DONNELL, J. F. (1968). Hepatic cholesterol ester storage disease,a familial disorder. Amer. J. Med. 44,538-546. SCHMIDT, J. L. AND MARTIN, D. L. (1965). The effect of diarylalkylpiperazines and related compounds on plasma and liver lipids and liver size. Toxicol. Appl. Pharmacol. 7,257-267. SCHWENK, E. AND WERTHESSEN, N. T. (1952). Studies on the biosynthesis of cholesterol. III. Purification of 14C-cholesterol from perfusions of livers and other organs. Arch. Biochem. Biophys. 40, 334-341. SCHWEPPE, J. S. AND JUNGMANN, R. A. (1971). The effects of hypocholesterolemic agents on cholesterol esterification in vitro. Proc. Sot. Exp. Biol. Med. 136,449-451. SPERRY, W. M. AND WEBB, M. (1950). A revision of the Schoenheimer-Sperry method for cholesterol determination. J. Biol. Chem. 187,97-106. HENLY, LOWRY,
The
AND
Effect
APPLIED
PHARMACOLOGY,
of Chlorcyclizine
27,449-455 (1974)
on Cholesterol
Metabolism
in Mice
JAMESW. BARNHART The Dow Chemical Company, Development Evaluation Laboratories, Zionsville, Indiana 46077 Received July 19,1972; accepted August 7,1973
The Effect of Chlorcyclizine on Cholesterol Metabolism in Mice. BARNJ. W. (1974). Toxicol. Appl. Pharmacol. 27, 449455. The effect of chlorcyclizine, a known hypocholesterolemic agent, on cholesterol metabolism in mice was reexamined. The reduction of serum cholesterol and increase in liver weight and total cholesterol after chlorcyclizine treatment were confirmed. After a single ip injection of chlorcyclizine (50 mg/kg), a significant elevation of liver cholesterol was observed after 12 hr and a reduction of serum cholesterol was seen after 24 hr. The elevation of liver cholesterol was accounted for exclusively in the esterified fraction. Oral doses effective in reducing serum cholesterol were also effective in elevating liver esterified cholesterol. The incorporation of [%]acetate into cholesterol in vivo was not reduced after chlorcyclizine treatment, suggesting that cholesterol biosynthesis was not inhibited. A marked inhibition of steryl ester hydrolase was caused by chlorcyclizine in vitro. The large excessof cholesteryl esters in the liver was possibly a result of this effect.
HART,
Chlorcyclizine [ I-(p-chloro-cc-phenylbenzyl)-4-methylpiperazine] has been found to reduce serum cholesterol in various mammalian species (Schmidt and Martin, 1965; Barnhart and Sefranka, 1966). This effect was associated with an increase in liver cholesterol and may simply reflect a change in cholesterol distribution. Salvador et al. (1970) also found a hypocholesterolemic effect in mice after treatment with chlorcyclizine, but they concluded that this effect was not due to an accumulation of cholesterol in the liver. Schmidt and Martin (1965) investigated the possibility that chlorcyclizine affects cholesterol biosynthesis, and found a moderate but not significant reduction of [14C]acetate into cholesterol in vivo in mice. The effect of chlorcyclizine on serum and liver cholesterol and cholesterol biosynthesis was reexamined in the experiments reported here, and its effect on another area of cholesterol metabolism, steryl ester hydrolase, was observed. The results are discussed in relation to the hypocholesterolemic activity of this compound.
METHODS Experiments were carried out in Swiss-Webster male mice weighing 25 g.’ Chlorcyclizine hydrochloride2 was given ip in some experiments and incorporated into the ground rodent mash” at a level of 0.06 % in other experiments (as indicated in the tables). 1 Harlan Industries, Cumberland, Indiana. ZK and K Laboratories, Inc., Plainview,New York. 3 Wayne Lab Blox meal, Allied Mills, Inc., Chicago, Illinois. 449 Copyright 0 1974 by Academic Press, Inc. All rights of reproduction Printed in Great Britain
in any form reserved.
450
JAMES W. BARNHART
Serum cholesterol was determined by the method of Henly (1957). Liver cholesterol (free and total) was determined as the digitonide (Sperry and Webb, 1950) after homogenization in 1 : 1 acetone-absolute ethanol. Total protein of crude enzyme preparations was determined by the method of Lowry et al. (1951). Incorporation of sodium [l-14C]acetate into mouse liver lipids was determined 2 hr after iv injections of 0.1 ml of a solution containing4pCi of sodium [1-14C]acetate (4.0 ,&i/2.0 pmol). The animals were decapitated and their livers were homogenized in 20 ml of absolute ethanol as rapidly as possible. Twenty ml of 20 ‘A alcoholic KOH (w/v) were added, and the mixture was heated at 70°C for 1 hr. After cooling, 30 ml of water were added, and the nonsaponifiable lipids were extracted with 3 portions of petroleum ether. Portions of this solution were taken for radiochemical determination of nonsaponifiable material, digitoninprecipitable material, and cholesterol purified via the dibromide procedure (Schwenk and Werthessen, 1952). Digitonides were dissolved in 1.O ml of NCS reagent,4 and all samples were taken up in toluene (5 g PPO + 0.1 g dimethyl POPOP/liter) and counted in a liquid scintillation spectrometer.5 Steryl ester hydrolase (EC 3.1.1.13) activity was determined by using a defatted 104,000 g supernatant of mouse liver homogenate. The supernatant was diluted to a protein concentration of approximately 5 mg/ml. The isolation and assay were done as described by Deykin and Goodman (1962) except that free and total cholesterol were determined by the digitonide procedure (Sperry and Webb, 1950). The substrate was [4J4C]cholesteryl oleate4 with a total activity of 5.43 x lo4 dpm/1.22 nmol added in 50 ~1 of acetone. The in vitro biosynthesis of cholesteryl esters was determined using a mouse liver mitochondrialpreparation (Goodman et al., 1964). The substrate was [4-14C]cholesterol (0.10 &i/O.052 pmol) added in 100 ~1 of acetone. The amount of total protein in each incubation flask was about 8 mg. Free cholesterol and cholesteryl esters were separated on a 1 cm x 4 cm alumina column as described by Deykin and Goodman (1962). There was ~0.2% 14C contamination in the cholesteryl ester fraction from a sample inactivated by heat, and this was disregarded in the calculations. Statistical significance was determined using Student’s t test unless otherwise stated.
RESULTS The degree of incorporation of sodium [i4C]acetate in 2 hr into cholesterol was determined in vivo in mice pretreated with chlorcyclizine for 8 days (Table 1). The quantity of labeled cholesterol in liver (dpm/g liver) in the treated group was increased by about 80 %, but the specific activity (dpm/mg cholesterol) was not increased significantly because of the high total cholesterol in liver. The lowering of serum cholesterol, and increased liver weight and liver total cholesterol after chlorcyclizine treatment in mice (Table 2) confirmed previous reports (Schmidt and Martin, 1965; Barnhart and Sefranka, 1966). However, it was also demonstrated that the increase in liver total cholesterol of over 70 % was accounted for entirely in the esterified fraction. 4 Amersham/Searle Corp., Arlington Heights, Illinois. 5 Packard Instrument Co., Inc., La Grange, Illinois.
CHLORCYCLIZINEAND
451
CHOLESTEROL
TABLE 1 EFFECTOFCHLORCYCLIZINETREATMENTONINCORPORATIONOFSODIUM [ 1J4C] ACETATEINTO MOUSELIVERLIPIDS"
Activity of lipid fractions Control (x103)
Fraction Nonsaponifiable fraction (dpm/g liver) Digitonin-precipitable material (dpm/mg sterol) (dpm/g liver) Cholesterol (dpm/mg cholesterol) (dpm/g liver)
Chlorcyclizine, 0.06 % (x103)
1.81 + 0.62 0.50 f. 0.19
2.26 f 0.86 0.52 + 0.16
1.10 + 0.36 0.39 + 0.25 0.96 + 0.41
1.78 5 0.47b 0.51 + 0.19 1.74 + 0.55b
a After 8 daysof 0.06 % dietary chlorcyclizineHCl4.0 &i of sodium acetatewere injectediv and the mice werekilled 2 hr later. Analysisof total cholesterolindicated a 27‘A reduction of serumcholesterol and a 60% elevation (per gram) of liver total cholesterol in the chlorcyclizine means+ SD from 6 animals. b Significantly higher than control, p < 0.02.
group. The results are
Changes in liver and serum cholesterol in mice were also measured after a single ip injection of chlorcyclizine hydrochloride (Table 3). Groups of animals were sacrificed at 12-hr intervals. Serum cholesterol was unchanged after 12 hr but was lowered by 16 % 24 hr after dosing and remained at about the same level for the next 24 hr. Liver total cholesterol was elevated (15 %) as early as 12 hr after dosing and remained elevated throughout the 48 hr of the experiment. The effect of different dietary levels of chlorcyclizine on serum and liver cholesterol was also studied (Table 4). Drug levels of 0.03 % and 0.06 % were both effective in reducing serum cholesterol about 35 % and elevating total liver cholesterol from a control value of 2.99 mg/g to values over 4 mg/g. Lower
drug levels of 0.0075 % and 0.015 % did not significantly affect cholesterol levels, but liver size was increased at the level of 0.015 %. TABLE 2 EFFECTOFCHLORCYCLIZINETREATMENTONMOUSESERUMAND LIVER CHOLESTEROL"
Control Serum cholesterol (mg/lOO ml) Liver weight (g/100 g body wt) Liver cholesterolb (mg/g liver) : Free Total
137 * 5.5 k 2.41 ? 2.87 f
38 0.8 0.29 0.31
Chlorcyclizine 78 * 11” 9.5 k 1.4d 2.37 + 0.09 5.00 & 0.70d
n Results are means f SD from groups of 5 mice. The treated animals were maintained dietary chlorcyclizine for 8 days. b Digitonin-precipitable material. c Differs significantly from control, p < 0.02. d Differs significantly from control, p i 0.01.
on 0.06 %
452
JAMES
W.
BARNHART
TABLE ACUTE
CHOLESTEROL
CHANGES
IN MICE
3 AFTER
Serum cholesterol, total (mg/lOO ml)
Time (W 0 12 24 36 48 0 12 24 36 48
CHLORCYCLIZINE
TREATMENT”
Liver cholesterolYb free bw/s>
Liver cholesterol,b total (mdd
Control
124 t 122 * 124+ 134 & 128 &
15 11 15 13 21
2.36 2.21 2.24 2.14 2.27
f rt + + f
0.16 0.16 0.20 0.18 0.16
2.58 2.41 2.50 2.30 2.51
k f + k f
0.21 0.18 0.24 0.21 0.19
Chlorcyclizine
114+ 117* 104 * llO? 105 &
15 10 19” 29” 18”
2.35 2.35 2.38 2.14 2.15
rt k f + +
0.21 0.19 0.11 0.22 0.10
2.57 2.76 2.93 2.73 2.83
k + + -t -t
0.21 0.30’ 0.22’ 0.48’ 0.45’
’ Groups of 8 male mice were given ip injections of chlorcyclizine HCl(50 mg/kg) and killed after various intervals. Results are expressed as means k SD. b Digitonin-precipitable material. c Significantly different from corresponding control, p < 0.05. TABLE CHOLESTEROL
Dietary
CHANGES
drug level (%I
IN MICE
AFTER
6.92 7.74 9.09 9.15 8.77
+ + f + f
DOSAGE
LEVELS
Serum cholesterol (mg/lC@ ml)
Liver weight (g/100 g body wt)
0.00 0.0075 0.015 0.03 0.06
4
DIFFERENT
127 f 23 127 + 17 118 k 23 83k27‘ 78 + 25”
0.86 0.50 1.14’ 0.91’ 0.93’
OF CHLORCYCLIZINF?
Liver cholesterol
(mg/db
Free 2.32 2.30 2.10 2.34 2.52
+ + f f +
Total
0.28 0.33 0.05 0.38 0.28
2.99 3.53 3.02 4.36 4.02
f f + f +
1.00 0.92 0.47 0.65’ 0.67d
’ Results are expressed as means f SD from groups of 8 mice (16 in control group). The treated animals were maintained on specified levels of dietary chlorcyclizine HCl for 8 days. A multiple comparison test was used for the statistical evaluation (Dunnett, 1964). b Digitonin-precipitable material. c Differs significantly from control, p < 0.01. d Differs significantly from control, p < 0.05. TABLE EFFECT OF CHLORCYCLIZINE [4-14C]C~~~~~~~~~
Concentration chlorcyclizine 5
X
0 lo-’
lXlo4M
2X104M
M
of
5 ON HYDROLYSIS OLEATE IN VITRO’
% Hydrolysis 47.8 26.7 16.4
7.2
OF
% Inhibition 0 44.1 65.7
84.9
’ Cholesteryl oleate, 1.22 nmol, was present in a total volume of 2.0 ml of phosphate buffer, pH 7.3. Each flask contained about 9 mg of total protein. Results are the average of duplicate determinations. The results were corrected for the small amount of hydrolysis (2.2 %) that occurred nonenzymatitally.
CHLORCYCLIZINE
AND
4.53
CHOLESTEROL
TABLE 6 EFFECT
OF CHLORCYCLIZINE CHOLESTERYL
[4J4C] Concentration chlorcyclizine
of
0
5 X 1o-5 M 1X104M
2X
1o-4
M
ON SYNTHESIS OF ESTERS IN VITRO’
Cholesteryl esters (dpm/flask x 104)
Free cholesterol (dpm/flask x 104) --
3.86 3.82 3.79 3.43
16.2 16.2 16.4 16.7
a The substrate was 0.052 pmol of [4-‘4C]cholesterol in a total volume of 3.0 ml of phosphate buffer, pH 7.3. Each flask contained 25 pmol ATP, 2 pmol of CoA and 8 mg of total protein. Results are the average of duplicate determinations.
The hydrolysis of [4-14C]cholesteryl oleate by a soluble liver fraction was determined after incubation with 5 x 1OJ M, 1 x 1O-4M and 2 x 1O-4M concentrations of chlorcyclizine (Table 5). Chlorcyclizine was an effective inhibitor in this system, causing a 44% inhibition at the lowest concentration. At the same concentrations, chlorcyclizine exerted a very minor effect on a mitochondrial cholesterol esterifying system (Table 6). DISCUSSION
The hypocholesterolemic response and apparent shift of cholesterol from serum to liver caused by chlorcyclizine in mice was reported by Schmidt and Martin (1965). They also studied the effect of chlorcyclizine on the incorporation of [14C]acetate into nonsaponifiable material in vivo in mice. A moderate decrease was observed in the treated group, but the change was not significant. Our results indicate that chlorcyclizine does not inhibit the biosynthesis of cholesterol. There was no significant change in the incorporation of [14C]acetate into cholesterol when the results were expressed in terms of specific activity. This was due to the large increase in liver cholesterol, since there was a significant increase in incorporation of the labeled substrate when the results were expressed in terms of tissue weight. The results were not markedly different when expressed in terms of total sterols or simply as cholesterol, intimating that chlorcyclizine exerts no major effect on the later steps of the biosynthetic pathway. The serum and liver cholesterol changes reported by Schmidt and Martin (1965) were confirmed in the present experiment. Chlorcyclizine is an effective hypocholesterolemit agent in mice, and this effect is accompanied by an increase in liver cholesterol and liver weight. Although it was shown that an increase in liver cholesterol resulted from chlorcyclizine treatment, there was no prior evidence that the serum and liver changes were associated during the acute period of change immediately after dosing. We found that the hepatic hypercholesterolemia was coincident with the reduction of serum cholesterol, and in this instance preceded the serum changes by a few hours. Schmidt and Martin (1965) suggested that dosage levels effective in reducing serum cholesterol were also effective in raising liver cholesterol. We confirmed this observation, substantiating the conclusion that one effect accompanies the other. Salvador
454
JAMESW.BARNHART
et al. ( 1970) found that liver cholesterol was increased in female, but not in male, mice after chlorcyclizine treatment, and inferred that this deposition mechanism was not responsible for the hypocholesterolemic effect in serum, which was detected in both sexes. The possibility that chlorcyclizine has a direct effect on cholesteryl ester metabolism is suggested by the discovery of a marked in vitro inhibition of steryl ester hydrolase. However, there is as yet no direct evidence to substantiate this suggestion. The liver is a major site for cholesterol synthesis in the body and also contains synthetic and hydrolytic systems involving cholesteryl esters. The introduction of an inhibitor of steryl ester hydrolase such as chlorcyclizine into this system could very well upset the normal equilibrium which favors a high proportion of free cholesterol. Chlorcyclizine had no effect on in vitro synthesis of cholesteryl esters. It was, therefore, not a nonspecific enzyme inhibitor. Schweppe and Jungmann (1971) investigated the effect of several hypocholesterolemic agents on cholesteryl ester synthesis in vitro and found a potentiation in some cases. Steryl ester hydrolase was not examined. The increase in liver total cholesterol caused by chlorcyclizine was wholly accounted for in the esterified fraction. The high proportion of cholesteryl esters in the liver is a distinct departure from normality, but is known to be induced by various dietary regimens (Goodman, 1965). The mechanism responsible for this change is unknown. A familial disorder in humans involving high levels of hepatic cholesteryl esters has been described by Schiff et al. (1968); liver cholesteryl esterase activity was found to be normal, and serum total cholesterol levels were moderately elevated. The metabolic defect was thought by these authors to be a block in the biliary excretion of cholesterol; however, no reason for the high proportion of cholesteryl esters was suggested. It is not known whether the high proportion of cholesteryl esters in the liver could be responsible for a decreased cholesterol flux from the liver and the ultimate hypocholesterolemic effect of chlorcyclizine. Differences in the transport of cholesterol and cholesteryl esters are known. Goodman (1965) reported that cholesteryl esters must be hydrolyzed to free cholesterol before intestinal absorption. It was suggested that cholesteryl esters may be taken up intact by the liver. A small amount of cholesteryl esters may be incorporated into lipoprotein and released by the liver, but the major portion of the esters in plasma probably are formed by the plasma lecithin-cholesterol acyltransferase reaction (Glomset, 1970). ACKNOWLEDGMENTS The author is grateful to Dr. John N. Eble for many valuable comments during the preparation of the manuscript. REFERENCES BARNHART, J. W. AND SEFRANKA, J. A. (1966). Hypocholesterolemic activity of antihista-
minics. Life Sci. 5, 871-874. DEYKIN, D. AND GOODMAN, DE W. S. (1962). The hydrolysis of long-chain fatty acid esters
of cholesterol with rat liver enzymes.J. Biol. Chem. 231,3649-3656. DUNNETT, C. W. (1964). New tables for multiple comparisons with a control. Biometrics
20,482-491. GLOMSET, J. A. (1970). Physiological role of lecithin-cholesterol acyltransferase. Amer. J.
Clin. Nutr. 23, 1129-1136.
CHLORCYCLIZINE
AND
CHOLESTEROL
455
GOODMAN, DE W. S. (1965). Cholesterol ester metabolism. Physiol. Rev. 45,747-839. GOODMAN, DE W. S., DEYKIN, D. AND SHIRATORI, L. (1964). The formation of cholesterol esters with rat liver enzymes. J. Biol. Chem. 239, 1335-1345.
A. A. (1957). The determination of serum cholesterol. Analyst 82,286-287. 0. H., ROSEBROUGH, N. J., FARR, A. L. AND RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193,265-275. SALVADOR, R. A., ATKINS, C., HABER, S. AND CONNEY, A. H. (1970). Changes in the serum concentration of cholesterol, triglycerides and phospholipids in the mouse and rat after administration of either chlorcyclizine or phenobarbital. Biochem. Pharmacol. 19, 14631469. SCHIFF, L., SCHUBERT, W. K., MCADAMS, J., SPIEGEL, E. L. AND O’DONNELL, J. F. (1968). Hepatic cholesterol ester storage disease,a familial disorder. Amer. J. Med. 44,538-546. SCHMIDT, J. L. AND MARTIN, D. L. (1965). The effect of diarylalkylpiperazines and related compounds on plasma and liver lipids and liver size. Toxicol. Appl. Pharmacol. 7,257-267. SCHWENK, E. AND WERTHESSEN, N. T. (1952). Studies on the biosynthesis of cholesterol. III. Purification of 14C-cholesterol from perfusions of livers and other organs. Arch. Biochem. Biophys. 40, 334-341. SCHWEPPE, J. S. AND JUNGMANN, R. A. (1971). The effects of hypocholesterolemic agents on cholesterol esterification in vitro. Proc. Sot. Exp. Biol. Med. 136,449-451. SPERRY, W. M. AND WEBB, M. (1950). A revision of the Schoenheimer-Sperry method for cholesterol determination. J. Biol. Chem. 187,97-106. HENLY, LOWRY,