Cytotechnology 1 : 275-283 (1988) 9 Kluwer Academic Publishers, Dordrecht - Printed in the Netherlands
In vitro test system for compounds affecting cholesterol pathway:
Studies in primary rat liver cell cultures* Johannes Pill, Johannes Aufenanger, Barbara Frey, Albrecht Frey, Gisela Johne and Kaflheinz Stegmeier Medizinische Forschung, Boehringer Mannheim GrnbH, D-6800 Mannheim, FRG Received 29 December 1987; accepted in revised form 8 April 1988
Key words: In vitro test system, liver cell cultures, sterol pathway, TLC separation Abstract
Rat liver cells derived from male and female animals in primary monolayer cultures were investigated for suitability as a test system for xenobiotics affecting the cholesterol pathway. An appropriate mode of extraction and separation of newly formed cholesterol and precursors is described. This system can be widely applied. Rat liver cells from females in oestrus cycle had a higher synthesis rate of cholesterol than those from males. The disadvantages related to the cycle phases make male rats more appropriate donor animals for the test system developed. The altered in vitro cholesterol synthesis is relevant to that in vivo. The extraction of newly synthesized cholesterol and its precursors by means of columns packed with large-pore kieselgur is precise and time saving. The modified separation by thin-layer chromatography on silica gel layers impregnated with silver nitrate enables direct separation from the extract and is sufficient to recognize cholesterol and its precursors. The method in this form is suitable for processing a large number of specimens e.g. for screening.
Introduction
Hypercholesterolemia has been found to be one of the factors responsible for the development of atherosclerosis in man (Assmann, 1982; Rifkind, 1984a and 1984b). One therapeutic approach is the lowering of elevated cholesterol by drugs (Thompson, 1981). Effective agents are therefore being sought to lower and/or to prevent a rise in plasma cholesterol levels by inhibiting cholesterol biosynthesis. The aim of this investigation was to set up a test system for the evaluation of
interference by xenobiotics in the cholesterol pathway. The system should be suitable for a wider application, e.g. screening. The results should have a strong relevance for in vivo conditions. The incorporation of radio-labelled precursors into cholesterol by various types of cells (David, 1983; Andersen, 1979; Krone, 1979) is a frequently used model for the investigation of biosynthesis. The liver is the central organ for cholesterol synthesis and catabolism (Dietschy, 1970). Rats are frequently used for studying
* Dedicated to Prof. F.H. Schmidt on the occasion o f his 60th birthday
276 drugs effect on lipid metabolism (Kritchevsky, 1975). Primary cultures of rat hepatocytes have been shown to be suitable for such investigations. Under normal conditions, cultured cells preferentially utilize exogenous cholesterol and endogenous cholesterol synthesis is suppressed. In a cholesterol-free medium the endogenous cholesterol synthesis is stimulated (Davis, 1979). From epidemiological studies in man and experiments in animals it is known that female sexual hormones influence lipid metabolism (Bencze, 1975). We therefore compared de novo cholesterol synthesis from 14C-acetate by rat hepatocytes in a cholesterol-free medium derived from male and female animals in different phases of oestrus cycle. The protein content of the cells and the glutamate pyruvate transaminase (GPT) in the medium were measured during a period up to 164 h to monitor the viability and integrity of the cells. The separation of newly synthesized lipids by shaking with CHC13/MeOH (Bligh, 1959), extraction of the neutral non-saponifiable lipids with n-heptane after treatment with KOH and, subsequently, the digitonin precipitation of sterols (Krone, 1979) is very time-consuming, not very sensitive and is not suitable for a large number of determinations. We used a simple and sensitive method which is based on the principle of liquid-liquid-extraction and carried out using prepacked kieselgur columns (Pill, 1985). Blocking a particular enzyme at a late stage of the cholesterol pathway generally leads to an accumulation of that enzyme's substrates, which might themselves be atherogenic (Holmes, 1962) or otherwise harmful. Drugs acting on these steps are not appropriate for a further development. The enhancement of cholesterol precursors by different drugs was investigated using a modified thin-layer chromatographic method based on silver ion absorption chromatography (Pill, 1987). Material and Methods Chemicals
BM 15766 was synthesized in the Chemical Research Department of Boehringer Mannheim,
FRG. Triparanol was kindly supplied by Merell Pharma GmbH, Riisselsheim, FRG, Clotrimazole by Bayer AG, Leverkusen, FRG, mevinolin by Merck Sharp and Dohme, Rahway, N.J., USA, and compactin by Prof. A. Endo, Tokyo, Japan. 2J4C-acetate, sodium salt, was obtained from Amersham Buchler, Braunschweig, FRG; cholesterol from Boehringer Mannheim; 7-dehydrocholesterol and lanosterol from Sigma Chemie GmbH, Munich, FRG; squalene from Roth, Kaflsruhe, FRG, and desmosterol from Serva, Heidelberg, FRG. The chemicals and media required for cell culture were obtained from Boehringer Mannheim, unless otherwise stated. All other chemicals were obtained in the necessary degrees of purity from the usual chemical suppliers.
Primary rat liver cell cultures
Male and female rats weighing between 190 and 240 g were used. The cycle phase was determined according to Preissecker (1958). Liver cells were obtained under sterile conditions using the collagenase recirculation technique modified from Berry and Friend (1969) (cellular yield: 2 - 3 x l0 g cells per liver, trypan blue exclusion: _>_95%) and cultivated as a monolayer in Dulbeccos's modified Eagle's medium (Morton, 1970) with the following additives: 16.5% fetal calf serum, L-glutamine (4 mM), omithine (0.4 raM), insulin CS (0.25 IU/ml, Hoechst, Frankfurt, FRG), streptomycin (100 lxg/ml) and penicillin (100 IU/ml). 3 ml of the cell suspension (10 6 cells/ml) were plated on a petri dish (6 cm in diameter) with a semipermeable membrane (petriperm| Heraeus, Hanau, FRG). The cells were incubated at 37~ in 10% CO 2 and 90% air and the medium was exchanged after 4 h and at the intervals indicated for one without fetal calf serum, but containing all the above-mentioned ingredients plus 2-14Cacetate (37 kBq/ml) in 100 pM acetate. Test compounds dissolved in DMSO (final concentration 0.1%) were added after 4 h and incubated for 48 h without exchange of medium.
277
Determination of the radioactivity incorporated into non-saponifiable neutral lipids After incubation of the cells with radioactive substrate, the monolayer was removed from the membrane surface with a rubber spatula, washed twice in isotonic sodium chloride solution and centrifuged at 400 x g. The resuspended cell pellet or culture medium was saponified at 75~ in twice its volume of 0.5 M potassium hydroxide in absolute ethanol. The mixture was then applied to a column packed with large pore kieselgur (Extrelut | Merck, Darmstadt, FRG). The neutral lipophilic, non-saponifiable substances were eluted with n-heptane directly into the scintillation vessels. The radioactivity was measured in Aqualuma | (Baker) in a fluid scintillation spectrometer (Packard Tricarb 460 C).
Autoradiography of the thin-layer chromatogram An X-ray film coated on one side (Kodak SB) was laid on the TLC plate and, depending on the radioactivity, each plate was exposed at - 2 0 ~ for up to one day and then developed.
Protein determination The protein determination was conducted in the cell lysate of parallel incubations. After removal of incubation medium the monolayer was rinsed three times with 3 ml saline (0.9% NaC1) scraped off with a rubber spatula and sonicated (50 Watt, 30 sec; Sonifier B-12, Branson Sonic Power Company, Danbury, Connecticut) in 10 ml demineralized water under cooling in ice water. The total protein content of the lysate was measured according to Lowry (1951).
Thin-layer chromatography (TLC) of the radioactive lipid Glutamate pyruvate transanimase (GPT) activity Silica gel plates (silica gel 60 F 254, 20 x 20 cm, Merck, Darmstadt, FRG) were immersed for approximately 4 min in a solution comprising 3% silver nitrate in 97% methanol, left to dry briefly in the air and activated in a drying oven at 120~ for 15 min. The eluate containing the neutral, non-saponiflable lipids obtained with Extrelut | was evaporated to dryness in a vacuum concentrator (Savant Instruments, Inc., Hicksville, N.Y.), taken up in 50 p.1 mobile phase (n-heptane/ ethylacetate 2:1, v/v) and applied with an Autospotter (Desaga, Heidelberg, FRG) to the TLCplates. The development time for a plate was about 40-45 min for a path length of 16 cm. After the first development, the plate was left to dry for approximately 30 min in the air before it was developed for a second time. The path length was reproduced exactly. After the second development, the mobile phase was once again allowed to evaporate. The compounds separated by TLC were identified by comparison with authentic samples and mass spectrometry (Pill, 1987).
The enzyme activity was determined in the incubation medium (Dulbeccos's MEM without pH indicator) in the supematant after centrifugation (Empfehlungen, 1972).
Results
The 14C-acetate incorporation in cholesterol, GPT activity in the medium and the protein content in rat liver cell monolayer cultures from male and female donor animals (oestrus phase) over a period up to 164 h is described (Figs. 1 - 4). In cultures from female donors the amount of newly synthesized 14C-cholesterol is about 3 times higher than that in cultures from males, both in the medium and in the cells. Independent of the sex, the main fraction of cholesterol is found within the cells. The content of newly synthesized cholesterol in liver cells from female donors increased during the entire duration of the experi-
278 14C_CH P-
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1
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20
28
44
52
68
76
92
100
116
12LI
1LI0 1Li8
164 time (h)
Fig. 1. Release of newly synthesized 14C--cholesterol(I'hU-CH) from 2-14C-acetate into the medium in liver cell monolayer cultures in serum-free MEM Dulbeeeo. Cells derived from male (-oestrus phase) Sprague-Dawley rats (R 5: SEM).
-- 14 dishes, 5 preparations) and female (o
o 8 dishes, 4 preparations,
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~
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92 100
116 12tt
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Fig. 2. 14C-cholesterol (14C-CH) in liver ceils synthesized from 2-14C-acetate in monolayer cultures in serum-free MEM Dulbecco. Cells derived from male (-Dawley rats (R 5: SEM).
-- 10 dishes, 5 preparations) and female (o
ment of 164 h, whereas there was no further increase in the cells from the males between the l l 6 t h and 164th h. The release of newly synthesized cholesterol into the medium decreased in liver cell cultures from both sexes between the 20th and 44th h of incubation. Thereafter, an increase was observed in cultures from females up to 148 h and in males up to 116 h, which was followed by a decrease in the cholesterol release. The GPT-activity in the medium was low in both sexes until the 92nd h and increased drastically thereafter in cultures derived from male animals only (Fig. 3).
o 8 dishes, 4 preparations, oestrus phase) Sprague-
The protein content of the liver cells from male rats was approximately 40% higher at the beginning of the experiment than that from females, but it decreased drastically with the duration of the experiment (Fig. 4). Five per cent of the initial amount was found for male and 25% for female cells at the end of the experiment. The 14C-acetate incorporation into cholesterol in rat liver cell monolayer cultures from donor animals in different phases of the oestrus cycle was determined. The cholesterol content of the liver cells (Fig. 5) formed from 14C-acetate increased almost linearly during the 44 h incuba-
279 6PT e-
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Fig. 3. Glutamate-pyruvate-transaminase (GPT) activity in medium of liver cells in monolayer cultures in serum-free MEM Dulbecco. Cells derived from male (--~ 12 dishes, 4 preparations) and female (o Sprague-Dawley rats (it + SEM).
o 12 dishes, 4 preparations, oeslxus phase)
Protein
2
tl
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28
~
52
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76
92
100
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Fig. 4. Protein content of liver cells in monolayer cultures in serum-free MEM Dulbecco. Cells derived from male (-preparations) and female (o
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o 8 dishes, 4 preparations, oestrus phase) Sprague-Dawley rats (it + SEM).
tion period independently of the cycle phase of the donor animals. In liver cells from female rats in oestrus, however, a synthesis rate that was about double that in animals in other cycle phases was observed, t4C-cholesterol formation was at its lowest in dioestrus. The release of newly formed cholesterol into the medium (Fig. 5) was at its greatest in liver cells obtained in the oestrus phase. The release ofl4C-cholesterol decreased independently of the cycle the longer the experiment lasted. This reduction was most marked in cell cultures obtained during oestrus, so that after an incubation period of 44 h, the 14C-cholesterol activity in the medium was comparable with that in prooestrus. The effect of various inhibitory substances on
t4C-acetate incorporation into the non-saponifiable lipid faction is summarized in Table 1. Figures 6 and 7 are autoradiograms of the thinlayer chromatographic separation of the nonsaponifiable lipid fraction after extraction with wide pore kieselgur. In the solvent treated controis nearly all the radioactivity is detectable at the position of cholesterol. BM 15.766, (4-(2[ 1- (4-chlorocinnamyl)-4-piperazinyl]-ethyl)-benzoic acid), a AS'7-sterol-A7-reductase inhibitor (Aufenanger, 1986), produced a reduction of the incorporation rate and an accumulation of 7-dehydrocholesterol. The inhibition of AS,24-sterolA24-reductase by triparanol (Steinberg, 1960) led to a clear increase in desmosterol. Accumulation of lanosterol and dihydrolanosterol was seen
280
1tiC_OH
ltlc_cH
2rio
~dl~
Cells
210 2
T 150 e~D
x
90
x
60
x
1i
lncubotlon t i m (~)
Incubation lime (h)
Fig. 5. De novo synthesized 14C-cholesterol from 2-14C-acetate in liver cell monolayer cultures in serum-free MEM Dulbecco; content
in cells (A) and release into medium (B). The ceils were obtained from female Sprague-Dawley rats in various phases of their oestrus cycle (o o oestrus, -~ -- prooestrus, II-------ll metoestrus, 9 9 dioestrus, X + SEM, 6 culture dishes from 2 preparations).
Table 1. Effect of various inhibitory substances on 2-1'k2-acetate
incorporation into cholesterol and its precursors in primary monolayer cultures of liver cells derived from male SpragueDawley rats during an incubation time of 48 h in serum-fw.e MEM Dulbeceo. The differences from solvent-treated controls (DMSO 0.1%, v/v) in per cent are given (X + SEM, 6 culture dishes from 3 preparations) Compound
Concentration [M]
14C-acetate incorporation [%]
BM 15.766 Triparanol Clotrimazole Mevinolin
1~ 5 1~ 5 1~ 5 1~ 5 1~ 6 1~ 7 1~ $ 1~ 9
~ • 1• 3• 81• ~ • 35• 14• 7•
1~ 6
56•
Compactin
w i t h t h e a n t i m y c o t i c c l o t r i m a z o l e ( B e r g , 1981). N e i t h e r c o m p o u n d a f f e c t e d t h e total i n c o r p o r a t i o n in c h o l e s t e r o l a n d p r e c u r s o r s . B e s i d e s t h e a c c u m u l a t i o n o f t h e e x p e c t e d sterols, a s l i g h t a d d i t i o n a l c h a n g e in s t e r o l p a t t e r n o c c u r r e d . C o m p a c t i n a n d m e v i n o l i n ( E n d o , 1976), i n h i b i tors o f t h e 3 - h y d r o x y - 3 - m e t h y l g l u t a r y l - C o A reductase, led only to a reduction of newly formed 14C-cholesterol. T h e e f f e c t o f m e v i n o l i n o n t h e i n c o r p o r a t i o n rate is c o n c e n t r a t i o n d e p e n d e n t .
Discussion
T h e c o n t i n u o u s i n c r e a s e in n e w l y s y n t h e s i z e d c h o l e s t e r o l in l i v e r c e l l s f r o m f e m a l e a n i m a l s s h o w s t h a t t h e y are a b l e to p r o d u c e c h o l e s t e r o l throughout the entire duration of the experiment, w h i l e n o c h o l e s t e r o l s y n t h e s i s is o b s e r v e d in the l i v e r c e i l s o f m a l e a n i m a l s a f t e r t h e 116th h. T h e
281
Fig. 6. Autoradiogrmn of the separation of non-saponifiable lipids by thin-layer chromatography. Effect of various inhibitory substances on sterol biosynthesis from 2-14C-acetate in primary monolayer cultures of liver cells derived from male Sprague-Dawley rats after an incubation period of 48 h in serum-flee MEM Dulbecco. The corresponding controls were treated with the same amount of solvent (DMSO 0.1%, v/v).
Meanings: L S: CH: D: 7DHC: I - IV:
lanosterol and dihydrolanosterol squalene cholesterol desmosterol 7-dehydrocholesterol unidentified sterols
Lanes: 1: 2: 3: 4: C:
BM 15.766 (10-SM) Triparanol(10-SM) Clotfimazole (10-SM) Compactin (10-6M) control
r e m a r k a b l e increase in G P T activity in the m e dium o f hepatocytes f r o m m a l e s during the s a m e period indicates leakage f r o m the cells. T h e de-
Fig. 7. Effect of different concentrations of mevinolin (10-5 10~s M) on sterol biosynthesis from 2-1'tC-aeetatein primary cultures of liver cells (for details see legend Fig. 6).
crease in delivery o f cholesterol to the m e d i u m during the s a m e period o f the experiment can be regarded as an indication o f secretion and not as a non-specific loss o f cholesterol f r o m the cells (Davis, 1979). T h e l o w loss o f protein, the low G P T activity in the m e d i u m and the higher cholesterol synthesis rate in the liver cells from female animals throughout the entire experimentation show that these cells possess a longer survival t i m e than those f r o m m a l e s under these conditions o f culture. L i v e r cells f r o m female rats in oestrus show a considerably greater accumulation and secretion o f n e w l y f o r m e d cholesterol, i.e. an increased synthesis rate, than those f r o m donors in other cycle phases. This observation correlates with the oestradiol levels in the p l a s m a (Taubert, 1981; T h o m e y c r o f t , 1974). T h e rate o f cholesterol synthesis from cells obtained in dioestrus is only slightly higher than that f r o m m a l e animals. This
282 cycle phase, which lasts 50-60 h, is the longest; oestrus lasts only 10-18 h (Preissecker, 1958). Observation of several cycles is advisable to determine the oestrus phase, because of the frequently considerable individual variations of the given times. This has to be borne in mind, along with the higher synthesis performance in oestrus, when choosing the sex of the donor animals. For reasons of practicability, further investigations with drugs were carried out using liver cell cultures derived from male donor rats. Because of the increase of the GPT activity in the medium, the incubation time should not exceed 92 h. The eluate, obtained by using Extrelut ~), contains the newly synthesized non-saponifiable lipids and can be used for liquid scintillation counting without any further purification process. Cholesterol and its precursors are extracted only under these conditions of culture and analysis, as shown by TLC separation. In contrast to extraction by shaking (Bligh, 1959), this procedure is simple, time saving and suitable for use on a large number of samples. Thin-layer chromatography on silica gel cartiers impregnated with silver nitrate enables cholesterol and its precursors, including squalene, to be separated in a single procedure. This means that changing the mobile phase or acetylating the substances to be separated is not necessary in contrast to Klein (1961). The resolution capacity of the TLC is sufficient to be able to recognize possible accumulation of intermediate products resulting from the inhibition of sterol biosynthesis. Not only the most important intermediates, such as squalene, lanosterol, desmosterol, and 7dehydrocholesterol can be clearly discriminated, but also others which have hitherto not been conclusively identified. Blocking a late step of the cholesterol pathway leads to accumulation of that enzyme's substrate and therefore provides an indication of the site of action of inhibitor substances within the sterol synthesis pathway. Sterols other than cholesterol might themselves be atherogenic or otherwise harmful (Holmes, 1962). Besides, the reduced 14C-acetate incorporation, the information from
TLC separation can be used as a further selection criterion for compounds for development. The expected changes by inhibitors of different steps in the cholesterol pathway from investigations in animals and men were found under culture conditions and demonstrate the relevance of the in vitro results for in vivo (Aufenanger, 1986; Steinberg, 1960; Berg, 1981; Endo, 1976). The 14C-acetate incorporation into cholesterol and its precursors in rat liver cell cultures and the modified extraction procedure and TLC separation is an appropriate method for investigating the influence of xenobiotics on the cholesterol pathway. A large number of compounds can be conveniently screened.
Acknowledgements We would like to thank Dr. Hooper for translation, and Mrs. I. Ebbe for secretarial help in preparation of this manuscript.
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