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Vol. 29, Issue 3, 242-251, March 2001
INSERM U128, IFR24, Campus Centre National de la Recherche Scientifique, Montpellier, France (S.G.-C., J.-M.P., L.P.-G., M.D., J.-M.F., P.M.); Syngenta AG, Basel, Switzerland (F.W.); Service de Chirurgie Digestive, Hopital Saint Eloi, Montpellier, France (J.-M.F.); and Service de Chirurgie Digestive, Hopital Purpan, Toulouse, France (N.C.)
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Abstract |
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 Top
 Abstract
 Introduction
Experimental Procedures
Results
Discussion
References
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The expression and inducibility of four CYP2C genes, including CYP2C8, -2C9, -2C18, and -2C19, was investigated in primary cultures of human hepatocytes. By the use of RNase protection assay and specific antibodies, each CYP2C mRNA and protein were quantified unequivocally. The four CYP2C mRNAs were expressed in human livers and cultured primary hepatocytes, but only the CYP2C18 protein was not detected. Compounds known to activate the pregnane X receptor (PXR) such as rifampicin, or the constitutively activated receptor (CAR) such as phenobarbital, induced CYP2C8, CYP2C9, and to a lesser extent CYP2C19 mRNAs and proteins. CYP2C18 mRNA was expressed but not inducible. The concentration dependence of CYP2C8 and CYP2C9 mRNAs in response to rifampicin and phenobarbital paralleled that of CYP3A4 and CYP2B6, the maximum accumulation being reached with 10 µM rifampicin and 100 µM phenobarbital. In contrast, dexamethasone produced maximum induction of CYP2C8 and CYP2C9 mRNAs at 0.1 µM while in these conditions neither CYP3A4 nor CYP2B6 was significantly induced. Moreover, the concentration dependence of CYP2C8 and CYP2C9 mRNAs in response to dexamethasone paralleled that of tyrosine aminotransferase. Furthermore, dexamethasone, which has been recently shown to up-regulate PXR and CAR expression through the glucocorticoid receptor, potentiated CYP2C8 and CYP2C9 mRNA induction in response to rifampicin and phenobarbital. Collectively, these results suggest the possible implication of at least three receptors in the regulation of CYP2C8 and CYP2C9 expression, i.e., glucocorticoid receptor, PXR, and/or CAR.
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Introduction |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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In
humans, the cytochrome P450 (CYP1) 2C subfamily
includes at least four functional genes: CYP2C8,
-2C9, -2C18, and -2C19 (Goldstein and
de Morais, 1994
). CYP2C8, -2C9, and -2C19 proteins are primarily
located in the liver where they account for approximately 20% of total
cytochrome P450 (Shimada et al., 1994). In contrast, CYP2C18 protein
seems to be primarily expressed in the skin (Zaphiropoulos, 1997). Low
levels of CYP2C mRNAs and proteins have also been detected in small
intestine and other extra-hepatic tissues (Klose et al., 1999). The
CYP2C proteins play a significant role in metabolizing currently
marketed drugs, including tolbutamide, phenytoin, tienilic acid,
warfarin (at position 7), diclofenac as substrates of CYP2C9 (Miners
and Birkett, 1998); and S-mephenytoin, warfarin (at
positions 6 and 8), omeprazole, diazepam, imipramine, pentamidine, and
moclobemide as substrates of CYP2C19 (Wrighton et al., 1993; Goldstein
and de Morais, 1994; Goldstein et al., 1994). CYP2C8 and CYP2C18
proteins exhibit a substrate specificity that is close to that of -2C9 or -2C19 but with either a lower Vmax or a
higher Km (Romkes et al., 1991; Goldstein
et al., 1994). CYP2C8 is also specifically involved in the oxidative
metabolism of Taxol, benzo[a]pyrene, arachidonic acid, and retinoids
(Nadin and Murray, 1999). The polymorphisms of CYP2C19 have
been well characterized using the 4-hydroxylation of the
S-enantiomer of mephenytoin. Several defective mutants
including CYP2C19*2 to CYP2C19*8 have been
described and shown to encode either a truncated, inactive, or
partially defective protein (De Morais et al., 1994; Ibeanu et al.,
1999). Several allelic variants of CYP2C9 including
CYP2C9*2 and 2C9*3 have also been described, the
latter being possibly responsible for the rare polymorphism of
tolbutamide (Stubbins et al., 1996; Bhasker et al., 1997; Gill et al.,
1999; Yasar et al., 1999).
In contrast to the large amount of data on the biochemistry,
enzymology, pharmacology, toxicology, and genetics of CYP2C
genes, little is known on the inducibility of the different members of this subfamily in response to xenobiotics in humans. Yet, significant induction of these genes could further amplify the interindividual variability of CYP2C-related monooxygenase activities observed in human
populations. In this respect, a number of clinical studies (reviewed in
Jang and Maurel, 1999) have shown increased clearance and/or a
decreased half-life of drugs known to be CYP2C substrates when given in
association with known enzyme inducers, including rifampicin,
phenobarbital, and glucocorticoids. On the other hand, in vitro studies
in human hepatocyte cultures provided conflicting observations. For
example, Runge et al. (2000) reported recently that CYP2C9 to -2C19
were not inducible in response to rifampicin and phenobarbital, whereas
Morel et al. (1990) and Chang et al. (1997) reported that the levels of
CYP2C immunoreactive proteins and mRNAs were increased in human primary
hepatocytes treated with rifampicin, dexamethasone, and phenobarbital.
However, because of the high level of similarity in both nucleotide and
amino acid sequences, these last studies did not discriminate between
the inducibility of the various members of this subfamily at the mRNA and protein levels. Previous papers from different groups have shown
that rifampicin- and phenobarbital-mediated induction of CYP3A4 to -3A7
and CYP2B6 is controlled by two nuclear receptors: the pregnane X
receptor (PXR) (Bertilsson et al., 1998; Blumberg et al., 1998; Kliewer
et al., 1998; Lehmann et al., 1998) and the constitutively activated
receptor (CAR) (Baes et al., 1994; Honkakoski et al., 1998; Sueyoshi et
al., 1999), respectively. Whether these receptors are involved in the
control of CYP2C gene regulation is currently unknown.
In the present work, we used a RNase protection assay and specific antibodies to investigate the inducible expression of CYP2C8, CYP2C9, CYP2C18, and CYP2C19 genes in response to various xenobiotics, including rifampicin, dexamethasone, and phenobarbital, in cultured human hepatocytes. CYP2C8, CYP2C9, and CYP2C19 mRNAs and proteins were induced by previously characterized PXR and CAR activators, the rank order of inducibility being CYP2C8 > CYP2C9 > CYP2C19, while CYP2C18 was not inducible. The data suggest that several nuclear receptors, including the glucocorticoid receptor (GR), PXR, and/or CAR, might be involved in the control of basal and xenobiotic-inducible expression of CYP2C genes.
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Experimental Procedures |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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Human Liver Samples. Liver samples were lobectomies resected from adult patients for medically required purposes unrelated to our research program. The use of these human hepatic specimens for scientific purposes has been approved by the French National Ethics Committee. Clinical characteristics of liver donors are presented in Table 1.
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Materials.
Ham F-12 and Williams' E culture media, vitamins and hormones,
collagenase (type IV), dimethylsulfoxide (DMSO), actinomycin D,
dexamethasone, rifampicin, and mifepristone (RU486) were
purchased from Sigma (St. Louis, MO). Phenobarbital was purchased from
Specia (Paris, France). 2,3,7,8-Tetrachlorodibenzo-p-dioxin
(TCDD) was obtained from BCP instruments (Lyon, France);
1,4-bis[2-(3,5-dichloropyridyloxy)]-benzene (TCPOBOP) was a gift from
P. Lesca (INRA, Toulouse, France). Collagen-coated culture dishes were
obtained from Corning (Iwaki Glass, Tokyo, Japan).
-[32P]dCTP,
-[32P]UTP, and enhanced chemiluminescence
developing reagent were purchased from Amersham Pharmacia
Biotech (Amersham, England).
Primary Cultures of Human Hepatocytes.
Primary cultures of human hepatocytes were prepared and cultured as
described in previous papers (Diaz et al., 1990). Xenobiotic inducers
were diluted in DMSO and added to the culture medium at the indicated
concentrations. In all cases the concentration of DMSO was 0.1%, and
control cultures received only DMSO at the same concentration. The
treatments lasted for 8 to 96 h and were renewed every 24 h
as the culture medium was changed.
Preparation of Total RNA. Total RNA and protein were isolated using Trizol reagent (Life Technologies, Cergy-Pontoise, France), from 107 cultured hepatocytes according to the manufacturer's instructions. For quality control, 30 µg of total RNA were analyzed by Northern blot using a rat glyceraldehyde phosphate dehydrogenase cDNA probe (J.M. Blanchard, IGMM, Montpellier, France).
Preparation of Plasmids.
The CYP2C cDNA probes were synthesized by reverse transcriptase-PCR
from human liver total RNA by using specific oligonucleotides (Kimura
et al., 1987; Ged et al., 1988; Romkes et al., 1991; Ged and Beaune,
1992). These probes do not overlap with the regions in which the
mutations are found for the known variants of both CYP2C9
and CYP2C19 (Fig. 1A).
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pGEMT-2C8. A 311-bp fragment (nucleotides 982-1293) was amplified by PCR with oligonucleotides sense 5'-GATCATGTAATTGGCAGACACA and reverse 5'-CCTGCTGAGAAAGGCATGAAG and cloned in pGEMT vector (Promega, Madison, WI). The Ribonuclease Protection Assay probe was synthesized with SP6 RNA polymerase after linearization of the plasmid with ApalI. The native probe contains 365 bp, and the digested probe is 239 bp long.
pGEMT-2C9. A 437-bp fragment (nucleotides 856-1293) was amplified by PCR with oligonucleotides sense 5'-AGCTTGGAAAACACTGCAGT and reverse 5'-CCTGCTGAGAAAGGCATGAAG and cloned in pGEMT vector (Promega). The Ribonuclease Protection Assay probe was synthesized with T7 RNA polymerase after linearization of the plasmid with ApalI. The native probe contains 284 bp, and the digested probe is 239 bp long.
pGEMT-2C18. A 431-bp fragment (nucleotides 869-1293) was amplified by PCR with oligonucleotides sense 5'-CTGTAACTGATATGTTTGGG and reverse 5'-CCTGCTGAGAAAGGCATGAAG and cloned in pGEMT vector (Promega). The Ribonuclease Protection Assay probe was synthesized with SP6 RNA polymerase after linearization of the plasmid with ApalI. The native probe contains 365 bp, and the digested probe is 239 bp long.
pGEMT-2C19. A 431-bp fragment (nucleotides 862-1293) was amplified by PCR with oligonucleotides sense 5'-GTAATCACTGCAGCTGACTTAC and reverse 5'-CCTGCTGAGAAAGGCATGAAG and cloned in pGEMT vector (Promega). The Ribonuclease Protection Assay probe was synthesized with SP6 RNA polymerase after linearization of the plasmid with ApalI. The native probe contains 365 bp, and the digested probe is 239 bp long.
For in vitro sense RNA synthesis, pGEMT-2C plasmids were linearized using NdeI/NcoI/NdeI/NdeI, and synthetic RNA was generated using T7/SP6/T7/T7 RNA polymerase for CYP2C8/9/18/19, respectively.pBS-IIK-CYP2B6.
A 263-bp fragment of CYP2B6 (nucleotides 25-288) was amplified by PCR
with oligonucleotides sense 5'-AGCCTCCCACCAGGGCCCCGCCC and reverse
5'-TGGCAAAGATCACACCATATCCCC, digested by Pst-1 and Sal-1, and cloned in
Pst-1/Sal-1 digested pBluescript II-KS+ vector (Stratagene, La Jolla,
CA). The Ribonuclease Protection Assay probe was synthesized
with T3 RNA polymerase after linearization of the plasmid with Sma-1.
The native CYP2B6 probe contains 192 bp, and the digested probe is 180 bp long. CYP3A4 plasmid was prepared as previously described (Greuet et
al., 1996). All CYP inserts were verified by nucleotide sequencing.
Ribonuclease Protection Assay.
Total RNA (30 µg) was analyzed by the RNase protection assay using
specific riboprobes as previously described (Greuet et al., 1996) with
minor modification. Total RNA was hybridized with the radiolabeled
antisense RNA probe (100,000-150,000 cpm) overnight at 37°C after
incubation for 10 min at 95°C. For Northern blot experiments, 30 µg
of total RNA were analyzed using
-[32P]dCTP-labeled human CYP3A4, rat
glyceraldehyde phosphate dehydrogenase, or mouse tyrosine
aminotransferase (TAT, kindly provided by Dr. T Grange, Institut J. Monod, Paris, France). cDNA probes and autoradiography were carried out
by exposing the dried gel to Kodak X-AR film; the signals were
quantified by analyzing the radioactivity with a PhosphoImager
apparatus and ImageQuant software (Molecular Dynamics, Sunnyvale, CA).
Hepatocyte Microsome Preparation.
Microsomes were prepared from liver samples and hepatocyte cultures by
differential centrifugation and stored as described previously (Diaz et
al., 1990). Protein concentration was determined by the bicinchoninic
acid method, according to the protocol provided by the manufacturer
(Pierce Chemical Co., Rockford, IL). Bovine serum albumin (Pierce
Chemical Co.) was used as the standard.
Immunoquantitation of CYP Proteins. Thirty micrograms of microsomal proteins or 80 µg of total proteins were separated by SDS-polyacrylamide gel electrophoresis (10%), then electroblotted onto Immobilon P (Millipore, Bedford, MA). Membranes were incubated with specific antibodies, AK1 (a monoclonal antibody directed against rat CYP2C6 cross reacting with CYP2C9/18/19), or a rabbit polyclonal antibody that recognizes CYP2C8/18 (kindly provided by Drs. P. Beaune and I. de Waziers, INSERM, Paris, France), and the blots were developed with the enhanced chemiluminescence detection system (Amersham Pharmacia Biotech). The relative amount of CYP protein was estimated from densitometric analysis (NIH Image Software, by Dr. W. Rasbond). Authentic standards for immunoblots were microsomes from human lymphoblastoid cells transfected with the human CYP1A1, -1A2, -2A6, -2B6, -2C9, -2C19, -2D6, -2E1, and -3A4 cDNA (GENTEST Corp., Woburn, MA), or microsomes from yeast transfected with the human CYP2C8 or -2C18 cDNA.
Measurement of Tolbutamide 4-Hydroxylation Activity. The rate of tolbutamide 4-hydroxylation was measured directly in cultured hepatocytes. After 96 h of induction, the culture medium was renewed in the absence of inducers and in the presence of 26.4 µM tolbutamide and [3H]tolbutamide (1 µCi, Amersham Pharmacia Biotech). Extracellular medium samples collected after 8 or 24 h were analyzed for tolbutamide and 4-hydroxytolbutamide by high-performance liquid chromatography using a Zorbax Bond C18 (150- × 4.6-mm; Interchim, Montluçon, France) column, protected with a column of the same phase. Elution was performed at room temperature with a 1.3-ml/min flow of isocratic mobile phase consisting of 40% acetonitrile (Carlo Erba, Val de Rueil, France), 59.96% water, and 0.04% orthophosphoric acid (Carlo Erba). The elution times of tolbutamide and 4-hydroxytolbutamide were 5 min 36 s and 2 min 30 s, respectively. The radioactivity of the effluent from the high-performance liquid chromatography column was analyzed in a Radioactivity Monitor LB 506-CI (Berthold, Wildbad, Germany). Radioactivity peaks were integrated with a computer and converted to molar concentrations of tolbutamide and 4-hydroxytolbutamide.
Statistical Analysis. Statistical analysis of data was performed using the Macintosh Stat View program (Abacus Concepts, Inc., Berkeley, CA). For mRNA analysis (Table 2), induction ratios (mean ± S.D.) relative to each inducer (mRNA level in inducer-treated cells to mRNA level in untreated cells) were calculated for all different cultures, and the statistical significance of the induction was assessed using the paired t test. For protein analysis (Table 3), relative levels were standardized with respect to levels measured in rifampicin-treated cells (arbitrarily taken as 100 for each culture), because in some cultures no CYP2C protein was detectable in untreated cells. The statistical significance of these relative level variations (mean ± S.D.) were then analyzed using the paired t test.
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Results |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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RNase Protection Assay.
CYP2C mRNAs display a high similarity in their nucleotide sequences
(82-94%) (Goldstein and de Morais, 1994). We therefore used RNase
protection to quantify these mRNAs individually (Fig. 1A). The
specificity of the assay was verified by using synthetic sense mRNA
fragments prepared from the CYP2C plasmids. The results presented in
Fig. 1B for CYP2C9 show that no interference was detected with CYP2C8,
-2C18, or -2C19 mRNAs. The intensity of the band relative to the
protected probe increased with the amount of synthetic sense CYP2C9
mRNA within the range 0.1 to 50 ng. This assay enabled us to detect 0.1 ng of the CYP2C9 mRNA riboprobe, i.e., 1 fmol of CYP2C mRNA. Similar
results were obtained with the other CYP2C riboprobes in terms of
specificity and sensitivity (not shown).
Expression of CYP2C mRNAs and Proteins in Human Livers.
This assay was used to evaluate the levels of CYP2C mRNAs in a bank of
human livers (n = 12). The results are shown in Fig. 2. First, we verified that this assay was
linear on a range of total liver RNA amount between 10 and 100 µg.
The four different CYP2C mRNAs were expressed at detectable levels in
all the human liver samples analyzed. The levels of CYP2C8, -2C9, and
-2C19 mRNA exhibited a large interindividual variability (by factors of
11, 10, and 14, respectively), while CYP2C18 mRNA was more stable
(3.4-fold variations). CYP2C9 mRNA accounted for approximately 50% of
CYP2C mRNAs, while the relative amount of the other mRNAs was 26% for
CYP2C8, 9% for CYP2C18, and 16% for CYP2C19, in reasonable agreement
with previous evaluations by other methods (Furuya et al., 1991; Romkes
et al., 1991). Statistical analysis of the data indicated that CYP2C
mRNA levels are not correlated.
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;
Lasker et al., 1998). A significant correlation was found between the
levels of CYP2C19 mRNA and protein (r = 0.895;
P < 0.0001) and the levels of CYP2C8 and CYP2C9
proteins (r = 0.863; P < 0.001).
However, neither the levels of CYP2C8 and CYP2C9 mRNA and protein nor
the levels of CYP2C19 protein with other CYP2C proteins appeared to be
correlated. As expected, the levels of CYP2C9 and the rates of
4-hydroxylation of tolbutamide exhibited a significant correlation
(r = 0.7 and P = 0.009, not shown).
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Induction of CYP2C mRNAs and Proteins by Various Compounds in Cultured Primary Human Hepatocytes. Hepatocytes prepared from different patients were cultured for 96 h in the absence or presence of several compounds previously characterized as CYP inducers. Then, total RNA and microsomes were extracted and analyzed by RNase protection assay and immunoblot, respectively.
The four CYP2C mRNAs were expressed constitutively in our cultures (Fig. 4). Compounds previously characterized as CYP2B/3A inducers (Maurel, 1996a) including
rifampicin, dexamethasone, and phenobarbital were also inducers of
CYP2C8 and CYP2C9 mRNAs. However, the fold induction was on average 2 to 6, that is, significantly smaller than that classically observed
with CYP2B6 mRNA in response to phenobarbital or with CYP3A4 mRNA in
response to rifampicin (see Fig. 6 for example). In one culture
(FT137), several other compounds were tested, including TCDD, TCPOBOP,
bosentane, omeprazole, prednisone, sulfadimidine, and phenylbutazone.
TCDD (a prototypical CYP1A inducer) and TCPOBOP (a
CYP2B inducer in rodents) were not significant inducers,
whereas the other compounds (which were all CYP3A4 inducers) gave
induction ratios close to those found with rifampicin, dexamethasone,
and phenobarbital (not shown). Interestingly, the levels of CYP2C8 and
CYP2C9 mRNAs exhibited a significant correlation after induction (with
r = 0.66; P = 0.03), suggesting that
the expression of these genes may be coregulated, at least in part, in
response to inducers. CYP2C19 mRNA was also inducible, notably by
rifampicin and phenobarbital but to a lesser extent as compared with
CYP2C8 and -2C9 (Table 2). In contrast, the levels of CYP2C18 mRNAs
were either not affected or decreased in response to the various
treatments.
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; Pichard-Garcia et al.,
2000), was not related to the sex, age, or pathology of the patient
from whom hepatocytes were isolated, nor to the quality of the cell monolayer as assessed by phase contrast microscopy.
Comparative Analysis of CYP2C8, CYP2C9, CYP3A4, and CYP2B6 mRNA Induction. Since these observations suggest that CYP2C8 and CYP2C9 share the same inducers as CYP3A4 and CYP2B6, we sought to determine whether these different CYP families respond identically to inducers in terms of time and concentration dependence. Three compounds were selected for this detailed investigation, i.e., dexamethasone, rifampicin, and phenobarbital.
Effect of dexamethasone.
The effect of this glucocorticoid must be considered separately from
the other compounds as it is not only an inducer of CYP genes, but it
has also an important effect on the phenotype of hepatocytes
(up-regulation of PXR and CAR, see next section). In a first series of
experiments, hepatocytes were cultured for 36 h in our standard
culture medium but in the absence of dexamethasone (our standard
culture medium contains 0.1 µM dexamethasone and no other
glucocorticoid). Control experiments (cellular morphology, CYP3A4
inducibility in response to rifampicin; see Fig. 8) demonstrated that
no significant loss of differentiation of cells occurred under these
conditions. Then, culture medium was supplemented with 1 µM
dexamethasone, and cells were harvested 8, 16, 24 and 48 h later.
As reported in Fig. 6A, CYP2C8 and CYP2C9
mRNAs were induced with close if not identical time dependence. In
contrast, CYP3A4 was only detectable after 48 h of treatment, and
CYP2B6 mRNA remained undetectable for the duration of experiments
(Pascussi et al., 2000a; data not shown). The same experiments were
carried out on TAT mRNA, known to be controlled by the GR.
Interestingly, TAT mRNA levels displayed a time dependence identical to
that of CYP2C8 and CYP2C9 mRNAs in response to dexamethasone (Fig. 6B).
To determine whether induction of CYP2C mRNAs was of transcriptional origin or due to mRNA stabilization, hepatocytes were cultured for
48 h in the standard culture medium (0.1 µM dexamethasone) and
exposed to actinomycin D for 32 h, in the absence or presence of
0.1 µM dexamethasone. Data reported in Fig.
7 show that CYP2C8 and CYP2C9 mRNAs were
rather stable as they exhibited minor decay for the duration of
exposure to actinomycin D (i.e., 32 h), irrespective of the
presence of dexamethasone. These results suggest that the increased
accumulation of CYP2C8 and CYP2C9 mRNAs in response to dexamethasone is
of transcriptional origin.
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; data not shown).
Control experiments showed that accumulation of TAT mRNA reached at
least 60% of its maximum level at 0.1 µM dexamethasone and only
slightly increased at greater concentrations (Fig. 8B). In aggregate,
these experiments show that induction of CYP2C8, CYP2C9, and TAT mRNAs
in response to dexamethasone display close time and concentration
dependence.
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Effect of rifampicin and phenobarbital. Next, we investigated similarly both the time and concentration dependence of CYP2C8 and CYP2C9 mRNAs' increase in response to rifampicin and phenobarbital under the same conditions as those described above for dexamethasone, except that dexamethasone was not withdrawn from the culture medium (concentration: 0.1 µM). Here again, expression of CYP3A4 and CYP2B6 mRNAs in response to rifampicin and phenobarbital, respectively, was analyzed in parallel. The time dependence of CYP2C8 and CYP2C9 mRNAs was not drastically different from that of CYP3A4 or CYP2B6 mRNAs, irrespective of the inducers (Fig. 6, C-F). However, in contrast to what was observed with dexamethasone, greater concentrations of rifampicin and phenobarbital were necessary to induce CYP2C8 and CYP2C9 mRNAs, the maximum levels being reached with approximately 10 and 100 µM, respectively (Fig. 8, C-F). Interestingly, the changes in CYP2C8 and CYP2C9 mRNAs roughly paralleled those of CYP3A4 and CYP2B6 mRNAs in response to rifampicin and phenobarbital. Thus, CYP2C8, CYP2C9, CYP3A4, and CYP2B6 mRNAs' inductions display similar time and concentration dependence in response to rifampicin and phenobarbital, but not in response to dexamethasone.
Dexamethasone Enhances Induction of CYP2C8 and CYP2C9 mRNAs in
Response to Rifampicin and Phenobarbital.
We observed recently that both PXR (Pascussi et al., 2000a) and CAR
(Pascussi et al., 2000b) are up-regulated by dexamethasone in human
primary hepatocytes most likely through the glucocorticoid receptor
pathway. Consequently, the levels of both PXR and CAR decrease in cells
cultured in the absence of dexamethasone (or other glucocorticoid).
This accounts for the previous observations that induction of CYP3A4 or
CYP2B6 mRNAs in response to compounds known to activate PXR and/or CAR
(i.e., rifampicin and/or phenobarbital, for example) is potentiated by
pretreatment of hepatocytes with dexamethasone. We report a similar
observation here with respect to CYP2C8 and CYP2C9 mRNA induction.
,b),
this was also the case for CYP3A4 and CYP2B6 mRNA induction, in the
same cultures (Table 4). Control experiments clearly confirmed that, in
the presence of dexamethasone, GR, PXR, and CAR were expressed in our
cultures at a level similar to that observed in the tissue (not shown).
In aggregate, these observations suggest that CYP2C8 and CYP2C9 mRNAs
are coregulated in response to rifampicin and phenobarbital and that
this response is likely to be mediated, at least in part, by PXR and/or
CAR, both receptors being up-regulated by glucocorticoids.
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Discussion |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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Primary cultures of human hepatocytes represent the most reliable
in vitro model for evaluating the inducibility of CYP genes in response
to xenobiotics in humans. Indeed, we and others have reported on the
reasonably good in vivo-in vitro correlation concerning the
inducibility of CYP1A1/1A2 and CYP3A4
by several drugs (Maurel, 1996b; Li et al., 1997). In this work we show
that CYP2C8, CYP2C9, and CYP2C19
respond to the same inducers as CYP3A4 and
CYP2B6, suggesting that PXR and/or CAR might be involved in
this process.
PXR (Bertilsson et al., 1998; Blumberg et al., 1998; Kliewer et al.,
1998; Lehmann et al., 1998) and CAR (Baes et al., 1994; Honkakoski et
al., 1998; Sueyoshi et al., 1999) are members of the
steroid/retinoid/thyroid hormone receptor superfamily (NR1) of
ligand-activated transcription factors. These receptors share a common
heterodimerization partner, retinoid X receptor (RXR). Heterodimers
PXR/RXR and CAR/RXR bind their target CYP genes by common nuclear
receptor AGGTCA-based DNA response element motifs, including everted
repeats spaced by 6 bp (ER-6) or direct repeats spaced by 4 bp (DR-4),
respectively. PXR and CAR have been implicated in the inducible
expression of CYP3A4 through -3A7 and CYP2B6, respectively, in response
to xenobiotics. The present findings that 1) CYP2C8 and CYP2C9 mRNAs
are inducible in response to compounds previously characterized as
CYP3A4 inducers and PXR activators (such as rifampicin); 2) the
concentration and time dependence of CYP2C8 and CYP2C9 mRNA induction
in response to rifampicin is identical to that of CYP3A4 mRNA; and 3)
dexamethasone enhances the induction of CYP2C8 and CYP2C9 mRNAs in
response to rifampicin, as previously observed with CYP3A4 mRNA
(Pascussi et al., 2000a), suggest collectively that PXR is involved at
least partly in the control of the inducible expression of
CYP2C8 and CYP2C9 in response to rifampicin. Data
obtained on CYP2C19 are more difficult to interpret because
of the moderate induction of the mRNA and the large interindividual
variability. However, the finding that rifampicin is the strongest
inducer of this gene is again in favor of a possible implication of
PXR.
Phenobarbital is not a potent inducer of CYP2C8 and
CYP2C9 genes. Indeed large concentrations of this compound
(at least 100 µM) are required to produce significant induction. This
would appear to be consistent with recent data from Lehmann et al.
(1998) showing that this barbiturate is able to activate the human PXR at concentrations greater than 100 µM. However, recent work
(Honkakoski et al., 1998; Sueyoshi et al., 1999) has shown that
induction of CYP2B6 and CYP3A4 by phenobarbital
could be mediated through CAR. Although this receptor binds to and
activates NR1 cis-element in the CYP2B6 upstream
region, it can bind to and activate as well the PXR-responsive ER-6
element present in the CYP3A4 regulatory region. It is
therefore not known whether PXR and/or CAR is/are responsible for
phenobarbital-mediated induction of CYP2C8 and CYP2C9 mRNAs. To attempt
to answer this question, we decided to evaluate the actual contribution
of CAR by investigating the effect of androstenol, a potent CAR
deactivator, on this process (Moore et al., 2000). Conflicting
observations were made: this compound inhibited the induction of CYP2C8
and CYP2C9 mRNAs in response to phenobarbital in some cultures but not
in others. A possible reason for this is that androstenol per se is
able to activate PXR as revealed by its ability to induce CYP3A4 mRNA
in our cultures (not shown). Therefore, although androstenol might
inhibit the phenobarbital-mediated induction of CYP2C8 and CYP2C9 mRNA
by deactivating CAR, its ability to activate PXR might reverse this effect. The relative amplitude of these two pathways probably varies
from one culture to another with the respective levels of CAR
and PXR proteins, thus explaining our conflicting observations.
The finding that induction of CYP2C8 and CYP2C9 mRNAs is obtained
within 24 h in response to submicromolar concentrations of
dexamethasone is interesting (Figs. 6 and 8). Lehmann et al. (1998)
have shown that significant activation of PXR by dexamethasone requires
concentrations on the order of or greater than 10 µM, consistent with
the finding that this compound is not a CYP3A4 inducer below
this concentration. Thus, PXR cannot be responsible for the CYP2C8 and
CYP2C9 induction observed here at 0.1 and 1 µM dexamethasone, and we
therefore suspected GR to be responsible. Our observations that
induction of TAT, CYP2C8, and CYP2C9 mRNAs displays similar time and
concentration dependence is consistent with this possibility (Figs. 6,
A and B and 8, A and B). Especially interesting in this respect is the
finding that neither CYP3A4 nor CYP2B6 mRNAs were inducible in parallel
to CYP2C8 and CYP2C9 mRNAs in response to dexamethasone. For
supramicromolar concentration of dexamethasone (10 µM) and for much
longer duration of treatment (96 h), it is possible that PXR becomes
partly activated, which would account for the further induction of
CYP2C8 and CYP2C9 mRNAs as observed in experiments shown in Fig. 5 and
Table 2. As discussed above for androstenol and CAR contribution, we
attempted to use RU486 (a prototypical GR antagonist) to evaluate the
contribution of GR in dexamethasone-mediated CYP2C8 and CYP2C9 mRNAs'
induction. However, these experiments were not conclusive either
(presence or absence of inhibition from one culture to another, not
shown). Indeed, the inhibitory effect of RU486 on dexamethasone
induction mediated by GR might or might not be masked by the activation of PXR by this compound.
Collectively, our results suggest the possible implication of at least
three receptors in the xenobiotic-inducible expression of CYP2C8
and CYP2C9, i.e., GR, PXR, and/or CAR. We propose that GR is
responsible, at least in part, for the basal expression of these genes
under physiological conditions in our standard cultures (presence of
0.1 µM dexamethasone). Note that GR is expressed constitutively in
our cultures (Pascussi et al., 2000a,b), as are CYP2C8 and CYP2C9
mRNAs. In the presence of xenobiotics (including glucocorticoids at
supramicromolar concentrations) able to activate PXR and/or CAR,
CYP2C8 and CYP2C9 gene expression is further
enhanced. The finding that the time course of induction of CYP2C8 and
CYP2C9 mRNAs is similar, whatever the inducer (see Fig. 6), suggests that this process is only dependent on the activation of latent receptor molecules (GR, PXR, and/or CAR) present in the cells and that
CYP2C gene transactivation by these receptors is not rate
limiting. Analysis of cis-elements responsible for
CYP2C8 and CYP2C9 gene induction in response to
glucocorticoids and xenobiotics is being performed.
In contrast to the other CYP2C messengers, CYP2C18 mRNA was not
inducible in any of the cultures tested. Furthermore, we were unable to
detect the CYP2C18 protein in microsomes prepared either from human
liver tissue or cultured hepatocytes. These data suggest that
CYP2C18 is not significantly expressed at the protein level in the liver and is regulated differently from the other members of
this subfamily. Indeed, Zaphiropoulos (1997) reported recently that
CYP2C18 is the member of the CYP2C subfamily most
abundantly expressed in the human epidermis.
Our observations on the induction of proteins CYP2C9 and CYP2C19 and
related monooxygenase activities (tolbutamide and
S-mephenytoin 4-hydroxylations) in our cultures are
consistent with previous observations in vivo. Several clinical reports
have focused on the changed pharmacokinetic parameters of drugs known
as CYP2C substrates, in patients receiving rifampicin, dexamethasone,
phenobarbital, or a high concentration of prednisone (Jang and Maurel,
1999). For example, the systemic clearance of phenytoin, tolbutamide, and S-warfarin exhibited a 2- to 3-fold increase in patients
receiving rifampicin, suggesting clinically significant CYP2C9
induction. Zhou et al. (1990) observed an increase in the urinary
excretion of 4'-hydroxy-S-mephenytoin (CYP2C19) in extensive
(but not in poor) metabolizers of this anticonvulsant treated with
rifampicin. Lackner (1991) reported an increase in i.v. phenytoin
clearance with concomitant administration of dexamethasone, requiring
an increased dose of the drug for therapeutic efficacy, again
suggesting a clinically significant induction of CYP2C9. Finally,
phenobarbital and prednisone were found to decrease the half-life of
elimination of cyclophosphamide, a drug recently shown to be a low
Km substrate of CYP2C9 and CYP2C19 (Jao et
al., 1972; Faber et al., 1974), whereas dexamethasone produced an
increase in the body clearance of this molecule (Yule et al., 1996).
The present in vitro data are consistent with these clinical reports
and provide clear evidence in favor of a significant influence of
xenobiotics, in addition to genetic determinants, to the wide
interindividual variability of the CYP2C-related biotransformations in humans.
| |
Acknowledgments |
|---|
We are grateful to Drs. Philippe Beaune and Isabelle de Waziers (INSERM, Paris, France) for providing authentic CYP2C8 and -2C18 proteins and anti-CYP2C18 antibodies, and to Dr. Colin Young for careful reading of the manuscript.
| |
Footnotes |
|---|
Received September 13, 2000; accepted November 16, 2000.
This work was supported in part by Laboratoires Fournier Dijon, France (S.G.-C.) and la Ligue Nationale contre le Cancer (J.M.P.), and by Hoffman-La Roche (Basel, Switzerland).
Send reprint requests to: P. Maurel, INSERM U128, IFR24, Campus CNRS, 1919 Route de Mende, 34293 Montpellier, France. E-mail: maurel{at}u128.crbm.cnrs-mop.fr
| |
Abbreviations |
|---|
Abbreviations used are: CYP, cytochrome P450; DMSO, dimethylsulfoxide; TAT, tyrosine aminotransferase; GR, glucocorticoid receptor; PXR, pregnane X receptor; RXR, retinoid X receptor; CAR, constitutively activated receptor; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; TCPOBOP, 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene; PCR, polymerase chain reaction; bp, base pair.
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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) or presence of different inducers. Total RNA was extracted
and 30 µg were analyzed by RNase protection assay with specific CYP2C
cDNA probes. The signals relative to protected probes were quantified
by analyzing the radioactivity with a PhosphoImager apparatus and
ImageQuant software and normalized with respect to the levels in
untreated cells taken as 1. Inducers: RIF, rifampicin; DEX,
dexamethasone; PB, phenobarbital. Concentrations are indicated in
micromolar. MW, size markers; NP, native probe.
