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Vol. 293, Issue 1, 24-32, April 2000
Novartis Pharma AG, Experimental Toxicology, Basel, Switzerland (S.G., E.P., A.W.)
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
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The immunosuppressive cyclosporine A derivative, O-hydroxyethyl-D(Ser)8-cyclosporine (SDZ IMM 125), was examined for its ability to induce apoptosis in rat hepatocytes cultured for 4 or 20 h. Four hours after SDZ IMM 125 treatment, chromatin condensation and fragmentation, and the number of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeled and Annexin V-positive cells increased dose dependently without any observable lactate dehydrogenase leakage. The activity of the cysteine protease, caspase-3, was increased, but not that of caspase-1 and -6. The specific caspase-3 inhibitor, Ac-Asp-Glu-Val-Asp-aldehyde, inhibited caspase-3 activation and attenuated SDZ IMM 125-induced apoptosis and lactate dehydrogenase leakage. After 20 h of SDZ IMM 125 incubation, the parameters of apoptosis were further increased. Decreased mitochondrial membrane potential (measured by rhodamine 123 uptake) and cytochrome c release went in parallel with ultrastructural mitochondrial changes, and might be regarded as early events that trigger the apoptotic cascade. Transmission electron microscopy showed cytoplasmic blebbing after 4 h of SDZ IMM 125 incubation. As observed by transmission electron microscopy, treatment with SDZ IMM 125 resulted in an increase in the number of necrotic cells after 20 h, but not after 4 h. Our findings suggest that in rat hepatocyte cultures, SDZ IMM 125 is a specific inducer of apoptosis after short-term incubation, and this overlaps with necrosis after longer treatment periods. It is very likely that the necrosis occurring later is the result of the early apoptotic events.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The
O-hydroxyethyl-D(Ser)8-cyclosporine
A derivative
O-hydroxyethyl-D(Ser)8-cyclosporine
(SDZ IMM 125), is almost equipotent to cyclosporine A (CsA) as concerns
its immunosuppressive properties, i.e., suppression of lymphokine
production; however, in animal studies, it possesses a wider
therapeutic window. The immunosuppressive effect of both compounds
derives from their inhibition of interleukin-2 (IL-2) synthesis in
T-helper cells at the mRNA level, thus inhibiting the maturation of
cytotoxic T cells. In the T cell, cyclosporines form complexes with
immunophilins and inhibit the original
peptidyl-prolyl-cis-trans isomerase activity. Because a
nonimmunosuppressive CsA analog also binds to and inhibits cyclophilin,
the inhibition of this enzyme activity alone does not explain the
immunosuppressive properties of CsA and SDZ IMM 125. The
immunosuppressant-immunophilin complexes also bind to and inhibit the
calcium-activated phosphatase, calcineurin. This could result in an
altered modification pattern of cytoplasmic components of transcription
factors, thereby disturbing the nuclear translocation, which is a
prerequisite for proper IL-2 transcription. As a sequel, the
biochemical cascade that transduces activation signals from the T-cell
receptor on the surface of the cell to its nucleus is interrupted
(Baumann et al., 1992
; Borel et al., 1996).
In preclinical studies, SDZ IMM 125 caused less renal dysfunction in
the rat, compared with CsA (Donatsch et al., 1992; Hiestand et al.,
1992). SDZ IMM 125 inhibited the uptake and secretion of bile acids in
rat hepatocytes, and, in the isolated perfused rat liver, also bile
flow (Wolf et al., 1998).
The drug was well tolerated in healthy volunteers, and in psoriatic
patients, in whom it had a dose-related beneficial effect in clearing
psoriasis (Witkamp et al., 1995). Clinical adverse effects were similar
to those reported for CsA, i.e., transient impairment of liver
function, which manifests itself by elevated, serum bile-acid levels,
together with hyperbilirubinemia. There was clear evidence for liver
intolerance of SDZ IMM 125, which resulted in significant
dose-dependent increases in the liver-specific serum transaminases.
Elevation of the aminotransferases was found more frequently than after
treatment with CsA (Witkamp et al., 1995).
In hepatocyte primary cultures and in the isolated perfused liver, SDZ
IMM 125 caused the release of lactate dehydrogenase (LDH), which
correlated very well with that of serum transaminases (Wolf et al.,
1998). In addition, we have shown that CsA and SDZ IMM 125 caused
oxidative stress, and that cyclosporine cytotoxicity can be inhibited
by antioxidants (Wolf and Donatsch, 1990; Wolf and Broadhurst, 1992;
Wolf et al., 1994, 1997; Trendelenburg, 1995). SDZ IMM 125 enhanced the
uptake of Ca2+ into liposomal vesicles (Wolf et
al., 1998). In the current literature, oxidative stress and increased
intracellular Ca2+ concentrations have been
described as inducers of apoptosis (Buttke and Sandstrom, 1994), which
suggests the potential role of SDZ IMM 125 as an inducer of apoptosis.
However, neither inhibition nor induction of apoptosis by SDZ IMM 125 in the liver has been described after in vivo treatment. In contrast,
CsA has recently been shown to induce apoptosis in rat hepatocyte
cultures treated for 4 and 20 h (Grub et al., 2000).
This study was designed to investigate the mechanisms underlying the hepatic side effects of SDZ IMM 125 in relation to apoptosis and necrosis. Morphological and biochemical parameters were determined under in vitro conditions in which specific hepatocellular functions are preserved for up to 20 h.
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Materials and Methods |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Animals. Permission for animal studies was obtained from the Veterinäramt Basel-Landschaft, CH-4410 Liestal, and all study protocols were in compliance with institutional guidelines. Male Han Wistar rats were obtained from Biological Research Laboratories (CH-4414 Füllinsdorf, Switzerland). They were kept in Macrolon cages with wood shavings as bedding under optimal hygienic conditions, at a temperature of 22-23°C, a relative humidity of 50 to 74%, and fluorescent light for a 12-h light/dark cycle. They were given water and rodent pellets ad libitum.
Hepatocyte Isolation and Cell-Culture Conditions.
Rat
hepatocytes (rats 180-220 g) were isolated according to the two-step
liver perfusion method (Boelsterli et al., 1993). The cells were seeded
in 35-mm six-well Primaria culture dishes (Becton Dickinson, Basel,
Switzerland) at a density of 0.7 × 106 cells in 2 ml of William's medium E (WME),
or in 60-mm culture dishes (Primaria; Becton Dickinson) at a density of
2 × 106 cells in 5 ml of WME (Life
Technologies AG, Basel, Switzerland). The culture medium contained 10%
fetal calf serum, 100 U/ml penicillin, 0.1 mg/ml streptomycin,
10
7 M insulin, and 107
M dexamethasone. After an attachment period of 2 h at 37°C in a
5% CO2, 95% air atmosphere, the medium was
changed. The test compound was added together with the new medium. SDZ
IMM 125 (Novartis, Basel, Switzerland) was dissolved in dimethyl
sulfoxide (DMSO), and this solution was added to the culture medium,
which resulted in a final concentration of 1% DMSO in the culture
medium. Control plates received the DMSO-containing medium without SDZ
IMM 125.
Transmission Electron Microscopy (TEM).
The cell cultures
were fixed with 1% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, for 1 h or overnight at 4°C. Then, after fixing with 1%
OsO4 in 0.1 M cacodylate buffer, pH 7.4, for
1 h at 4°C, the cell cultures were dehydrated in graded ethanol solutions and embedded in Epon according to the method of Pease (1984).
Ultrathin sections of hepatocyte cultures from at least two
selected tissue blocks per well were counterstained with uranyl acetate
(Ac) and lead citrate, and were examined with a Philips CM10
transmission electron microscope.
Determination of Cytotoxicity.
LDH activity in the culture
media, as an index of plasma-membrane damage and loss of membrane
integrity, was measured spectrophotometrically (Wedler and Acosta,
1994). Enzyme activity was expressed as the percentage of extracellular
LDH activity of the total LDH activity on the plates.
Determination of Chromatin Condensation and Degradation.
Chromatin condensation and fragmentation were determined by Feulgen
staining and using light microscopy to count the percentage of cells
containing alterations in the nuclear structure (Lillie and Fullmer,
1976). Hepatocyte samples were treated as follows: after the
cell culture medium was removed, the cells were fixed overnight with
4% formaldehyde in PBS. After hydrolyzing in 5 N HCl for 90 min at
room temperature, the cells were rinsed in distilled water and stained
for 30 min in Schiff reagent (Merck, Dietikon, Switzerland). After
this, the hepatocytes were rinsed in 0.05 M
Na2S2O3
solution for 2 min, washed first in tap water, and then washed in
distilled water, and mounted by Crystal Mount (Biomeda, Foster City,
CA). After staining, hepatocyte nuclei were violet in color.
Determination of DNA Fragmentation.
Terminal
deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end
labeling (TUNEL) assay was performed using the DNA fragmentation kit
TdT-FragEL (Calbiochem, Lucerne, Switzerland). The principle of the
assay is based on the fact that TdT binds to exposed 3'-OH ends of DNA
fragments generated in response to apoptotic signals, and catalyzes the
addition of biotin-labeled and unlabeled deoxynucleotides (Gavrieli et
al., 1992). Biotinylated nucleotides were detected using a
streptavidin-horseradish peroxidase conjugate. Diaminobenzidine reacts
with the labeled sample to generate an insoluble, colored substrate at
the site of DNA fragmentation. Nonapoptotic cells do not incorporate
significant amounts of labeled nucleotide because they lack an excess
of 3'-OH ends. Nonapoptotic cells were counterstained with methyl
green. Positive TUNEL staining was indicated by a dark brown
diaminobenzidine signal, whereas shades of blue-green signified
a nonreactive cell.
Determination of Membrane Phosphatidylserine Distribution.
Phosphatidylserine distribution was detected by labeling the cells with
the biotin conjugate of Annexin V (Roche, Basel, Switzerland) according
to the method of Vermes et al. (1995).
Determination of Caspase-1, -3, and -6 Activity.
Caspase
activity was determined according to the method of Rodriguez et al.
(1996). After incubation, 2 × 106
cells were washed once in ice-cold PBS and lysed in 1 ml of buffer A
[10 mM HEPES, pH 7.4, 42 mM KCl, 5 mM MgCl2, 1 mM dithiothreitol (DTT), and protease inhibitors (Complete; Roche,
Basel, Switzerland)]. After three thaw-freeze cycles, the lysate was
centrifuged for 20 min at 13,000g at 4°C. The supernatant
(lysate) was removed and stored at 
80°C until the assay was performed.
Determination of Mitochondrial Membrane Potential.
Mitochondrial membrane potential was determined by the uptake of
rhodamine 123 according to the method of Wu et al. (1990). After
treatment, hepatocytes cultured on 96-well plates (Primaria; Becton
Dickinson), were washed in PBS and incubated with 10 µg/ml rhodamine 123 (Molecular Probes, Leiden, the Netherlands) for 30 min at
37°C. After additional washing, the hepatocytes were incubated with
WME for 30 min. Ethanol/water 1:1 was used to extract the amount of
dye retained by the cells. Fluorescence was measured with a Cytofluor
2300 from Millipore, with an excitation wavelength of 485 nm and an
emission wavelength of 530 nm.
Determination of Cytosolic and Mitochondrial Cytochrome
c Content.
Cytochrome c was determined
after subcellular fractionation by the Western blotting technique
according to the method of Tang et al. (1998). After incubation, 2 × 106 cells were washed once in ice-cold PBS and
lysed in 1 ml of buffer A (10 mM HEPES, pH 7.4, 42 mM KCl, 5 mM
MgCl2, 1 mM DTT and protease inhibitor). After
three thaw-freeze cycles, the lysate was centrifuged for 20 min at
13,000g at 4°C. The pellet fraction (mitochondria) was
first washed in buffer A containing sucrose and then solubilized in 50 µl of TNC buffer (10 mM Tris-Ac, pH 8.0, 0.5% NP-50, 5 mM CaCl2). The supernatant was recentrifuged at
100,000g (4°C, 1 h) to generate the cytosol, and then
removed and stored at 80°C.
.
After fractionation, the proteins were electroblotted onto a
polyvinylidene difluoride transfer membrane (0.2 µm) (Bio-Rad, Glattbrugg, Switzerland). Immunostaining of cytochrome c was
carried out with an anti-cytochrome c monoclonal antibody
(7H8.2C12; PharMingen, San Diego, CA). For detection, the membrane was
washed in PBS and incubated with Streptavidin peroxidase complex for 30 min. After additional washing, the ABC substrate was added for 5 to 15 min. Protein bands (15 kDa) were revealed using nitroblue
tetrazolium/5-bromo-4-chloro-3-indolylphosphate.
Determination of Protein Concentration.
Protein content was
determined according to Bradford (1976). BSA served as standard.
Statistics. A two-way ANOVA was performed (group and animal; each of them were regarded as qualitative). If the effect of the animal number was not significant, it was omitted. A quantile plot was used to judge visually the normality of the residuals. If the residuals were not normally distributed, we tried to achieve (approximate) normality by transforming the response or omitting outliners.
A multiple comparison method was applied using the three methods of Turkey (Hayter, 1989), Sidak (1967), and Dunnett (1964). The Dunnett
test compares every treated group with the control group, whereas the
other two methods can be used to compare each group with each other group.
The multiple comparison method delivers an estimation of the difference
in the response expected between the two groups compared, the S.E. of
the response, and a lower and an upper confidence limit for the
difference. If the two limits do not include zero, the difference is
significantly different from zero, on the level of 5%. By repeating
the method for 1 and 0.1%, we could analyze how big the significance was.
S-Plus software (version 5) was used for the computations; the three
methods of Turkey (Hayter, 1989), Sidak (1967), and Dunnett (1964) were
used adaptively, i.e., in every case, the most sensitive method was used.
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Results |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Cytotoxicity.
The cytotoxicity of SDZ IMM 125 was determined
after 4 and 20 h by measuring the release of LDH in the cell
culture medium. After 4 h, SDZ IMM 125 did not induce an increase
in LDH at any concentration, whereas after 20 h at concentrations
of 25 and 50 µM, LDH values were significantly increased (Fig.
1).
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Morphology.
Chromatin condensation and fragmentation, as
determined by Feulgen staining, and DNA fragmentation measured by TUNEL
assay, showed a time- and dose-dependent increase after SDZ IMM 125 treatment. In control hepatocytes, the percentage of condensed
chromatin was 1 to 2%. Incubation with 50 µM SDZ IMM 125 increased
chromatin condensation by a factor of 9.3 after 4 h, and by a
factor of 30.5 after 20 h (Fig. 2).
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Ultrastructural Alterations.
For electron microscopy
investigations, hepatocytes were incubated for 4 and 22 h with 0, 10, and 50 µM SDZ IMM 125. The main ultrastructural changes seen in
all treated cultures (4 and 22 h) were the occurrence of cells
with aggregated chromatin in compact masses, condensed cytoplasm with
marked crowding of organelles, which was frequently associated with the
development of translucent cytoplasmic vacuoles, and apoptotic bodies
or blebs. The number of mitochondria was increased, their matrix often
darker, and several were larger in size (Fig. 5,
A-D). In addition, the induction of
dilated endoplasmic reticulum and vacuoles was observed. At 22 h, all SDZ IMM 125-treated cultures and control cultures
displayed necrotic changes, including swelling of all cytoplasmic
compartments, swelling and disappearance of mitochondrial cristae,
disintegration of membranes, and accumulation of large, dense granules
in the mitochondrial matrix. The necrotic changes were more pronounced in cultures treated with 50 µM SDZ IMM. There was no
significant difference in necrosis between control preparations and SDZ
IMM 125-treated preparations after 4 h of incubation.
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Biochemical Markers of Apoptosis.
After SDZ IMM 125 treatment,
Annexin V-positive cells were dose and time dependently increased. Four
hours after treatment, Annexin V reaction was increased from 8% in
control preparations to 23.15% in hepatocytes treated with SDZ IMM 125 at 50 µM; a 2.8-fold increase compared with controls. After 20 h, Annexin V-positive cells increased from 7% in controls to 37.6% in
SDZ IMM 125-treated hepatocytes, an increase of 5.5 times (Fig.
6).
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Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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In preclinical toxicological studies, the CsA derivative, SDZ IMM
125, was found to cause fewer renal side effects than CsA (Donatsch et
al., 1992). In humans, there was clear evidence of liver intolerance of
SDZ IMM 125, which results in significant increases in the
liver-specific serum transaminases ALAT and ASAT (Witkamp et al, 1995).
The magnitude and the incidence of such effects have never been
observed after administration of CsA, either in healthy volunteers or
in transplant patients. However, CsA was found to induce apoptosis in
primary rat hepatocytes (Roman et al., 1998; Grub et al., 2000),
whereas the apoptotic potential of SDZ IMM 125 was then unknown.
In this study, by applying morphological and biochemical methods, and
determining apoptosis at different subcellular levels, we have shown,
for the first time, that SDZ IMM 125 specifically induced apoptosis in
primary rat hepatocyte cultures after 4 and 20 h of treatment.
With TEM, it was possible to demonstrate that SDZ IMM 125 induced
apoptosis according to the classical criteria, and to distinguish the
effects observed from necrosis. Apoptosis appeared as typical cell
shrinkage accompanied by condensed cytoplasm with focally dilated
endoplasmic reticulum, structurally intact mitochondria and other
organelles compacted together and maintaining their integrity,
formation of cytoplasmic protuberances on the cell surface (blebbing)
and cytoplasmic bodies, nuclear shrinkage with invagination of nuclear
membrane, and massive deposits of chromatin in compact masses adjoining
the nuclear envelope. It was also possible to clearly distinguish
necrosis, characterized by swelling of all cytoplasmic compartments,
swelling and disappearance of mitochondrial cristae, disintegration of
membranes and accumulation of large dense granules in the mitochondrial
matrix, detachment of ribosomes from rough endoplasmic reticulum
membranes, and nuclear swelling with clumping of loosely textured
nuclear chromatin (Wyllie et al., 1980).
LDH release might be a component of apoptosis in cell culture. Although LDH release in cell culture does not necessarily imply necrosis, plasma membrane damage is one feature of necrotic cells, which could serve among other parameters to determine necrosis. Because under in vitro cell culture conditions the apoptotic cells can not undergo rapid phagocytosis as in vivo in the intact tissue, it might also be that LDH release is a result of late apoptotic cells.
The fact that SDZ IMM 125 can induce necrosis was clearly shown by TEM
examinations using morphological criteria 20 h after incubation.
There were also clear indications for transition figures, showing cells
with both apoptotic and necrotic properties. From these results, it
seems possible that the necrosis observed in the cultures in this study
could partly be "secondary necrosis" of apoptotic cells (Eanshaw,
1995).
Annexin V staining, chromatin condensation and fragmentation, and the
TUNEL assay have a certain potential to give positive results also with
necrotic cells (Gold et al., 1994; Vermes et al., 1995). By means of
ultrastructural investigation with TEM, we could exclude the
possibility of necrosis at 4 h after SDZ IMM 125 treatment at all
concentrations. Exclusion of necrosis can also be supported by the
determination of LDH activity in the cell culture supernatant, which is
proof of the integrity of the outer cell membrane, a typical criterion
of necrotic cell death. The results obtained from the parameters of
apoptosis applied after 4 h can therefore be considered to be
highly specific for the induction of apoptosis.
Our results indicate that caspase-3 might be involved in the early
apoptotic steps. After 20 h, caspase-3 activity was maximally increased by incubation with 25 µM SDZ IMM 125, whereas its activity increased less by incubation with 50 µM SDZ IMM 125. SDZ IMM 125 is
cytotoxic after 20 h of treatment, and the cells, therefore, contain less ATP. Because caspase activation is only observed in
ATP-containing cells, it could be explained that caspase-3 is less
activated by incubation with the highest dose of SDZ IMM 125 than with 25 µM SDZ IMM 125. It is described that inhibition of
caspase activity induces a switch from apoptosis to necrosis (Lemaire
et al., 1998). Caspase-6 activity was also found to be increased by SDZ
IMM 125, but not statistically significantly. A possible explanation
for the minor effects of the caspase-6 substrate Ac-VEID-AMC and its
inhibitor Ac-VEID-CHO might be that both compounds have overlapping
activity with caspase-3 and that the observed caspase-6-related changes
are not specific for caspase-6.
In the current literature, CsA was often used as a specific inhibitor
of mitochondrial Ca2+ release and of mitochondrial membrane
potential, and as a blocker of the mitochondrial permeability
transition in rat hepatocytes when incubated for 1 h at
concentrations in the nanomolar range (Qian et al., 1997; Lemasters et
al., 1998). Our data strongly suggest that CsA (Grub et
al., 2000) and SDZ IMM 125 induce apoptosis by decreasing mitochondrial
membrane potential, which is followed by mitochondrial permeability
transition as determined by cytochrome c release from the
mitochondria. The difference of our data to those of other
investigators might be explained by the different CsA concentrations
used and differences in the treatment time. CsA can serve as a specific
inhibitor at low concentrations and after short-term expo-sure, but not
after longer treatment with high concentrations.
Our results suggest that the mechanism of SDZ IMM 125-induced apoptosis
proceeds from disruption of mitochondrial membrane potential to
destruction of the mitochondrial membrane, which results in cytochrome
c release into the cytosol. This results in an activation of
caspases, especially caspase-3. As a consequence, DNA is attacked,
leading to condensation and fragmentation of chromatin. We cannot
exclude that there exist other mechanisms, parallel to the activation
of caspases, which result in SDZ IMM 125-induced apoptosis. It is also
possible that intracellular calcium and reactive oxygen play a role in
this complex mechanism. This would be consistent with the hypothesis of
Jewell et al. (1982; Orrenius, 1993) who postulated that morphological
changes in the plasma membrane are associated with disturbances in
intracellular calcium homeostasis. It is known that an increase in
intracellular calcium concentration may directly cause toxicity.
In comparison with CsA, SDZ IMM 125 is more cytotoxic at equimolar
doses. Chromatin condensation and fragmentation, DNA fragmentation, as
well as Annexin V-positive cells were, on average, 1.5 to 2 times
higher. Whereas SDZ IMM 125 already induced caspase-3 activity after
4 h, it takes 20 h for CsA to elicit a similar effect (Grub et al., 2000). These results indicate that apoptosis induced by SDZ IMM
125 occurs earlier, and to a greater extent than after CsA treatment.
These data suggest that SDZ IMM 125 is more cytotoxic, and causes
stronger apoptotic effects than CsA. The results imply that, for
therapeutic reasons and to circumvent liver adverse effects of SDZ IMM
125, other administration schedules than the oral route should be
envisaged, for instance, the inhalative route.
In summary, our results showed that SDZ IMM 125 specifically induced apoptosis in rat hepatocyte primary cultures after short-term incubation, and this overlapped with necrosis after longer-term treatment. It is likely that the necrosis that occurs after long-term treatment is, partially, a secondary result of apoptosis.
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Acknowledgments |
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We thank B. Greiner and M. Gianella for their excellent technical assistance, and Dr. W. Seewald from Aicos, Basel, Switzerland, for performing the various statistical analyses.
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Footnotes |
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Accepted for publication December 6, 1999.
Received for publication July 29, 1999.
1 Current address: University of Kaiserslautern, Department of Chemistry, Kaiserslautern, Germany.
Send reprint requests to: Dr. A. Wolf, Novartis Pharma AG, WSH-2881.3.27, CH-4002 Basel, Switzerland. E-mail: armin.wolf{at}pharma.novartis.com
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Abbreviations |
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CsA, cyclosporine A; TdT, terminal deoxynucleotidyl transferase; Ac, acetate; AMC, 7-amino-4-methyl coumarin; CHO, aldehyde; DEVD, Asp-Glu-Val-Asp; DMSO, dimethyl sulfoxide; DTT, dithiothreitol; LDH, lactate dehydrogenase; SDZ IMM 125, O-hydroxyethyl-D(Ser)8-cyclosporine; TEM, transmission electron microscopy; TUNEL, TdT-mediated dUTP-biotin nick end labeling; VEID, Val-Glu-Ile-Asp; WME, William's medium E; YVAD, Tyr-Val-Ala-Asp.
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References |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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)
and 20 h (
). Data are expressed as mean ± S.D.
(n = 3). Statistically significant differences
versus the control group are expressed as ***P < .001.
) and 20 h (
) of incubation in
hepatocyte primary cultures. Data are expressed as mean ± S.D.
(n = 3). Statistically significant differences
versus the control group are expressed as *P < .05 and
***P < .001.
, 0.5 µM;
, 1 µM; 
, 2.5 µM; 
, 10 µM; 
, 25 µM;
, 50 µM.
