Despite its hepatotoxic potential, cyclosporine A (CsA) has been
reported to positively influence compensatory liver growth. To probe
the physiological consequences of CsA on the recovery of liver
function, studies were initiated in the 2/3 partially hepatectomized
(PHx) rat, taking the recovery of cytochromes P-450-dependent drug
metabolism as primary outcome. CsA was administered at a dose of 3.33 mg/kg/day for 10 days. Drug metabolism was evaluated by the recovery of
14CO2 after administration of isotopically
labeled model drugs and by studying the expression of the P-450
transcripts involved in their biotransformation before and 24 to
96 h after PHx. Before PHx, neither the steady-state mRNA nor the
in vivo disposition of caffeine (CYP1A2), erythromycin (CYP3A2 and
3A1), or aminopyrine (CYP2B1 and 2C11) were influenced by CsA. Studies
24 h after PHx revealed a 29 to 39% reduction in the elimination
of [14C]aminopyrine and [14C]erythromycin,
which was unaffected by CsA. Their metabolism at 48 to 96 h after
PHx also remained unaffected by CsA. By contrast, postPHx,
[14C]caffeine elimination decreased to a level closely
proportional to the loss in liver mass. In addition, CsA accelerated
the recovery and/or prevented the decrease of caffeine elimination
24 h after PHx but not at later time points, indicating an early,
but unsustained, beneficial effect of CsA on the recovery of
CYP1A2-mediated activities. These data show that at the critical time
of greatest loss in liver mass, CsA has only a selective influence on
the biotransformation of cytochrome P-450 protein-dependent activities
and that its effect on the regeneration process does not translate into
an overall accelerated recovery of the hepatic drug-metabolizing function.
 |
Introduction |
Several investigators have reported that
cyclosporine A (CsA)1 can
positively influence the liver regeneration process after 2/3 partial
hepatectomy (PHx) in laboratory animals (Garcia-Alonso et al.,
1989
; Francavilla et al., 1990; Mazzaferro et al., 1990; Kahn et al.,
1990; Tanaka et al., 1993). However, despite numerous reports
indicating an increase in the hepatic regeneration phenomenon, several
studies have noticed that the liver weight restitution was
not influenced by CsA in the postPHx period (Makowka
et al., 1986; Kahn et al., 1990; Provencher et al., 1997).
Moreover, CsA has also been shown to induce oxidative stress in rat
hepatocytes (Wolf et al., 1997), to increase the growth of
carcinogen-induced liver foci (Yabu et al., 1991; Masuhara et al.,
1993), to inhibit both the hepatic uptake and synthesis of bile acids
(Kukonguiriyapan and Stacey, 1988; Levy et al., 1994), and to lead to
cholestasis (Stone et al., 1987; Kassianides et al., 1990). These
observations indicate that, in addition to its reported trophic effect
on the hepatic regeneration process, CsA also harbors the potential for inducing serious hepatic side effects, which, despite a CsA-mediated acceleration in the regeneration process, could dampen the recovery of
hepatic function after loss of liver mass.
The liver is the major site of drug metabolism and CsA administration
has been reported to compete with drugs metabolized by CYP3A2 (rat;
Zhang and Thomas, 1996) and CYP3A4 (human; Pichard et al., 1990), the
two cytochromes P-450 primarily involved in the biotransformation of
CsA (Combalbert et al., 1989; Prueksaritanont et al., 1993). By
contrast, CsA has been reported not to influence per se the in vivo
biotransformation of xenobiotics metabolized by other cytochromes P-450
as exemplified by studies on the N-demethylation of
aminopyrine and the level of CYP2B2, 2C6, 2C11, and 2C13 in the rat
(Gershbein, 1987; Isogai et al., 1993; Beckurts and Lauterburg, 1995).
Although an important hepatic reserve exists for several biotransformation activities, a loss in liver mass secondary to either
the progression of hepatic diseases, or toxic or surgical aggression is
usually accompanied by a loss, albeit not always of the same magnitude
(Rikkers and Moody, 1974; Lauterburg and Bircher, 1976; Sendama et al.,
1985), in the liver-metabolizing capacity. Despite a steady stream of
studies reporting a positive effect of CsA on hepatic compensatory
hyperplasia, the physiological consequences of the CsA-mediated
increase on the regeneration process have not been evaluated. The
purpose of the present studies was, therefore, to investigate the
influence of CsA on the functional recovery of the liver after PHx,
taking the behavior of three cytochrome P-450 (CYP)-dependent
activities as the primary outcome of the studies. We now report that in
the postPHx period, CsA has no influence on the recovery of CYP2B1-,
2C11-, 3A1-, and 3A2-dependent activities and their corresponding
hepatic steady-state mRNA levels. By contrast, caffeine metabolism, a
CYP1A2-dependent substrate, was shown to be increased by CsA 24 h
after PHx but not at later time points.
| |
Materials and Methods |
Animal Treatment and Drug Regimen.
The studies were designed to evaluate the effect of CsA administration
on the recovery of the phase 1 drug-metabolizing function after PHx in
the rat, a well characterized model of liver regeneration (Michalopoulos and DeFrances, 1997). Male Sprague-Dawley rats weighing
200 to 250 g were used in all studies. All animals used in this
study were treated in accordance with the standards of ethics for
animal experimentation of the Canadian Council on Animal Care. All
protocols were approved by a local animal ethics committee.
A preliminary dose- and time-response study established that doses of
3.33 to 10 mg/kg/day CsA had similar effects on DNA synthesis, whereas
a 20 mg/kg dose inhibited DNA synthesis after PHx. Further studies for
periods of 3, 6, 9 (dose of 3.33 mg/kg/day), and 12 (doses of 3.33 and
10 mg/kg/day) days were found to have a similar effect of the
regeneration process. The lowest effective dose (3.33 mg/kg/day) was
chosen for all subsequent studies with a 10-day pretreatment period
before partial liver resection. Drug administration was continued at
the same dose during the postresection period. CsA was diluted in olive
oil and administered i.p. Placebo-treated controls received the vehicle
only and were subjected to the same experimental regimen as CsA-treated rats.
Partial hepatectomy was performed under light isoflurane anesthesia
after pretreatment with CsA or placebo. A 3-cm abdominal midline
incision was made and the median and left lobes of the liver
were extruded and excised (PHx; Higgins and Anderson, 1931), or
returned to the abdominal cavity (sham operation). The animals were
then returned to their cages and had access to food and water ad libitum.
Parameters Indicative of Drug-Metabolizing Enzyme Activities.
[14C]Aminopyrine, [14C]erythromycin,
and [14C]caffeine metabolism in vivo.
The influence of CsA administration on the in vivo disposition of
[14C]aminopyrine,
[14C]erythromycin, and
[14C]caffeine (all obtained from NEN-DuPont
Canada Inc., Markham, Ontario) was evaluated before PHx as well as at
several time points (24-96 h) after liver resection.
[Dimethylamine-14C] aminopyrine (0.25 µCi;
sp. act. 99 mCi/mmol),
[N-methyl-14C]erythromycin (0.25 µCi; sp. act. 55 mCi/mmol), or
[1-methyl-14C]caffeine (0.50 µCi; sp. act.
53.3 mCi/mmol) elimination was measured essentially as described by
Villeneuve et al. (Villeneuve et al., 1978). Briefly, the compounds
were injected i.v. under light isoflurane anesthesia. Rats were then
immediately allowed to awaken and were housed in individual air-tight
cages. Exhaled 14CO2 was
first drawn through a Dryrite dehumidifying cylinder (W.A. Hammond
Drierite Company, Xenia, Ohio) to remove water and then through
a scintillation vial to collect all exhaled CO2
during eight consecutive 15-min periods starting immediately after drug administration. Ten milliters of a 2:1 (v:v) methanol/ethanolamine mixture was used as a CO2-trapping agent. Trapped
radioactivity was determined after adding 10 ml Hionic-Fluor (Packard
Instrument Co., Meriden, CT) and evaluated by liquid scintillation spectroscopy.
Evaluation of cytochrome P-450 transcripts.
At the time of euthanasia (pre- and 48 h postPHx), the livers of
animals receiving CsA or vehicle were excised, trimmed of adherent
tissue, and flushed with ice-cold saline and flash frozen in liquid
nitrogen. Samples were kept at 
80°C until RNA extraction for the
determination of the expression of the genes encoding CYP1A2,
CYP2B1, CYP2C11, CYP3A1, CYP3A2, cyclophilin, GAPDH, and 18S ribosomal RNA. The latter two genes were used as
controls to monitor RNA loading on membranes. Additional animals were
treated with dexamethasone (DEX; one single i.p. injection: 400 mg/kg b.wt.), 3-methylcholanthrene (3-MC; one single i.p. injection: 30 mg/kg b.wt.), phenobarbital (PB; 350 mg/l in drinking water for 10 days), 
-naphthoflavone (NF; three daily i.p. injections: 80 mg/kg
b.wt.), and acetone (1% v:v in drinking water for 10 days).
These xenobiotics were used as selective inducers of the following
cytochrome P-450 subfamilies: CYP3A1 and 3A2
(Dex; Gonzalez et al., 1986), CYP1A1 and 1A2
(3-MC; Kim et al., 1995), CYP2B1 and 2B2 (PB;
Canivenc-Lavier et al., 1996), CYP1A1 and 1A2
(NF; Canivenc-Lavier et al., 1996), and CYP2C11 (AC).
They served to control for the in vivo modulation of the gene transcripts.
The probes used in this study were: for CYP1A2, a synthetic
20-mer oligonucleotide complementary to bases 1580 to 1599 of the P-450 1A2 cDNA sequence (Kim et al., 1995); for CYP2B1,
a synthetic 18-mer oligonucleotide complementary to bases 1000 to 1017 of the P-450 2B1 cDNA sequence (Canivenc-Lavier et al., 1996); for
CYP2C11, a synthetic 30-mer oligonucleotide corresponding to
the complement of nucleotides 945 to 974 of the coding sequence of
P-450 2C11 (Chen et al., 1995); for CYP3A1, a synthetic
32-mer oligonucleotide complementary to bases 1593 to 1624 of the P-450 PCN1 nucleotide sequence (Gonzalez et al., 1986); for
CYP3A2, a synthetic 24-mer oligonucleotide complementary to
bases 1652 to 1675 of the 6-A nucleotide sequence (Miyata et al.,
1994); for rat cyclophilin, a 0.8 kbp cDNA fragment inserted
in pCD vector in BamHI site (provided by Dr. G.N. Hendy,
McGill University, Montreal, Canada); for GAPDH, a 1.2 kb
rat cDNA fragment inserted in the PstI site of pBR322 (ACTT
#57090); and for 18S ribosomal RNA, a 1.5 kb human cDNA
insert from the EcoRI site of the pBluescript SK-vector
(ATCC # 77242). Nucleotides were synthesized by the Sheldon
Biotechnology Center, McGill University.
RNA extraction and Northern blot analyses were made as already
described (Demers et al., 1997). Poly(A)+ RNA
were purified by oligo(dT)-cellulose chromatography. Five micrograms of
poly(A)+ RNA were used for each Northern blot of
liver RNA, except for the induction study, where 15 µg of total RNA
were used. The 18S ribosomal RNA was analyzed using a
radiolabeled DNA probe. Other probes were labeled using
[
-32P]dCTP (3,000 Ci/mmol) and Klenow
according to the random oligo-priming method; hybridization and washing
were performed as described earlier (Demers et al., 1997).
CYP2B1, 2C11, 3A1, and 3A2
oligonucleotides were end-labeled by phosphorylation with T4
polynucleotide kinase and [
-32P]ATP (3,000 Ci/mmol). Hybridization was performed for 16 h at 52°C in 0.5 M
sodium phosphate buffer (pH 7.2) containing 7% SDS, 1% BSA, 1 mM
EDTA, and 240 µg salmon sperm DNA in the presence of 1 × 106 dpm/ml probe. After hybridization, the
filters were washed three times with 0.2 M sodium phosphate buffer pH
7.2, containing 1% SDS and 1 mM EDTA at hybridization temperature for
15 min. Exposure and densitometry were performed as described earlier
(Demers et al., 1997).
Biochemical Analyses.
CsA was measured in whole blood by fluorescence polarization (Abbott
TDX, Chicago, IL) taking care that serial dilutions of normal rat blood
were linearly correlated with respect to expected CsA concentrations.
The circulating concentrations of aspartate aminotransferase (AST),
alanine aminotransferase (ALT), and alkaline phosphatase (AP) were
measured with a multianalyzer system (Paramax, Dade County, Brea, CA)
in the Clinical Biochemistry department of our hospital.
Statistical Analysis.
Results are expressed as means ± S.E.M. Statistically significant
differences between group means were evaluated by analysis of variance
or by the Student's t test as indicated in the table and
figure legends.
| |
Results |
As indicated in Table 1, CsA
administration did not significantly affect the circulating
concentrations of AST, ALT, and AP. Body weight and liver weight were
also unaffected by CsA pretreatment. Whole blood CsA concentration was
found to be 727 ± 77 ng/ml at the end of the pretreatment period.
Figure 1 presents Northern blot analyses
of CYP1A2, 2B1, 2C11, 3A1, and 3A2 mRNAs in
control and CsA-treated rats and densitometric evaluation of the
CYP content. As illustrated, all transcripts were similarly
expressed in livers of both sham-operated groups (Fig. 1, A and B,
lanes 1 and 2). Furthermore, the level of steady-state expression of
CYP1A2, 2B1, 2C11, 3A1, and 3A2 was significantly reduced after 2/3 partial liver resection without, however, any effect
of CsA on the transcript levels (Fig. 1, A and B, lanes 3 and 4). By
contrast, studies on the expression of indicators of the in vivo
modulation of the CYP gene transcripts (Fig.
2) revealed that the administration of
dexamethasone increased mRNA levels of CYP3A1 and
3A2, 3-MC increased mRNA levels of CYP1A2, PB
increased mRNA levels of CYP2B1, whereas NF increased
those of CYP1A2. Cyclophilin (the CsA-binding protein) mRNA
levels were unaffected by CsA administration (data not shown).
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Fig. 1.
Influence of CsA administration and PHx on
the level of the hepatic CYP1A2, 2B1, 2C11, 3A1, and 3A2 mRNAs.
A representative Northern blot analyses of the hepatic
CYPs. All measurements were done in individual animals
after placebo (control rats receiving olive oil; lanes 1 and 3)
administration, or CsA (lanes 2 and 4). At day 10 after the beginning
of CsA administration, animals were subjected to sham operation (lanes
1 and 2) or to a 2/3 partial liver resection (lanes 3 and 4) and
CYP mRNA levels were measured 48 h after PHx.
Poly(A)+-enriched RNA (5 µg/lane) of individual rats were
used for all samples. GAPDH mRNA levels were used to
control for unequal loading of poly(A)+ RNA on the gels.
Results are representative of at least three separate experiments per
group in sham-operated rats and at least seven experiments per group in
PHx rats. B, densitometric evaluation of mRNA levels obtained in the
Northern analyses. Placebo  ; CsA-treated rats  . N.D. indicates
not detectable; PHs indicates sham-operated animals; PHx indicates
partially hepatectomized animals. Statistically significant differences
between group means were evaluated by analysis of variance. Effect of
PHx, p < .001; intragroup test post hoc
Bonferroni-Dunn: significantly different from PHs,
*p < .03, **p < .009, ***p < .0001.
|
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Fig. 2.
Influence of DEX, 3-MC, acetone, PB, and
NF administration (lines 1 to 6 respectively) on the levels of mRNA
of each CYP transcript.
RNA from normal untreated rat was used as control (C). RNA from female
rats was used as negative control for the male-specific
CYP2C11 ([female]; line 7).
18S ribosomal RNA levels were used to control for
unequal loading of total RNA on the gels. A, representative Northern
analyses. B, densitometric evaluation of the CYP/18S
ratios. n = 3 animals for each inducer except for
3-MC where n = 2, and CYP3A1 where
n = 1. N.D. indicates nondetectable, whereas N.A.
indicates not done. Stastistically significant differences between
group means were evaluated by analysis of variance with the
Bonferroni/Dunn post hoc test. Significantly different from control,
***p < .0001; **p < .002.
|
|
The effect of CsA treatment on the disposition of
[14C]aminopyrine,
[14C]erythromycin, and
[14C]caffeine is presented in Figs.
3 to 5 and in Tables 2 to 4. CsA
administration did not significantly influence
[14C]aminopyrine,
[14C]erythromycin, or
[14C]caffeine elimination before surgery.
Partial hepatectomy similarly decreased the elimination of
[14C]aminopyrine and
[14C]erythromycin in placebo-control and
CsA-treated animals but CsA administration did not significantly
accelerate the biotransformation recovery of either substrate as
illustrated in Figs. 3 and 4 and Tables
2 and 3.
In contrast, CsA significantly enhanced the biotransformation of
[14C]caffeine 24 h after PHx (CsA versus
control: p < .04; Fig.
5, Table 4)
but its elimination progressively increased in placebo-treated rats
between 48 and 96 h after surgery where no differences between the
two groups were observed. A significant interaction between drug
administration and time in relation to partial hepatectomy was observed
(p < .02) due to the CsA-mediated early
beneficial effects (early recovery and/or protection against a
decrease) on [14C]caffeine elimination, which
was followed by a gradual tapering off of its biotransformation
activity at times 48, 72, and 96 h after partial hepatectomy.
[14C]caffeine elimination was also shown to
exhibit an overshoot in its recovery 72 and 96 h after surgery
most particularly in placebo-controls. Sham-operated animals showed
biotransformation activities similar to those observed before surgery
(data not shown).
| |
Discussion |
The metabolizing capacity related to several cytochromes
P-450-mediated biotransformation activities [CYP 2B1 and CYP2C11 (aminopyrine; Kotake et al., 1982; Ching et al., 1996) and CYP1A2 (caffeine; Butler et al., 1989)] revealed that neither their
steady-state mRNA levels nor their in vivo disposition were influenced
by the chronic administration of CsA in sham-operated animals. The
N-demethylation of erythromycin (CYP3A2, 3A1; Craig et al.,
1993) was shown to be slightly decreased, albeit in a nonsignificant
manner (p < .055), by CsA administration, an
observation concurring with that of others where the elimination of
erythromycin was shown to be unaffected by CsA treatment in vivo
(Watkins, 1996). In addition, we reported here that CYP3A1
and 3A2 mRNAs were not influenced by CsA in both the pre-
and posthepatectomy (48 h) periods. In vitro studies, however, have
reported competition between CsA and many other substrates metabolized
by CYP3As including erythromycin (Pichard et al., 1990). In these
studies, however, CsA concentrations of 5 µM were used compared to a
mean CsA circulation concentration of less than 1 nM observed in the
present studies, indicating that at clinically relevant concentration,
CsA has little effect on CYP3A2 and 3A1-mediated
N-demethylation activities and their respective mRNAs.
Studies of the hepatic drug-metabolizing capacity after PHx revealed,
however, a significant reduction in the elimination of both aminopyrine
(29 to 32%) and erythromycin (29 to 39%), a reduction that was also
shown not to be influenced by chronic CsA administration. The
relatively small loss in the biotransformation capacity of the liver
observed after 2/3 hepatic resection illustrates well the adaptive
capacity of the organ as extrahepatic metabolism of aminopyrine or of
erythromycin has been shown to be negligible in the rat and in humans
(Sendama et al., 1985; Watkins et al., 1992). It also concurs with
observations on the biotransformation of aminopyrine (Lauterburg and
Bircher, 1976; Sendama et al., 1985) and on the circulating
concentration of liver-specific proteins (Rikkers and Moody, 1974)
where no concordance between hepatic function and liver mass were
observed. Our data thus clearly show that CsA does not perturb nor
accelerate the recovery of drug-metabolizing enzyme activity associated
with three major cytochrome P-450 enzymes (CYP2B1, CYP2C11, and CYP3A2)
after partial liver resection. This observation might be somewhat
surprising in view of the apparent increase in the hepatic regeneration
process reported by several investigators (Mazzaferro et al., 1990;
Kahn et al., 1990; Francavilla et al., 1991) or of the acceleration in
DNA synthesis reported by Garcia-Alonso et al. (Garcia-Alonso et al.,
1990) as well as by our own laboratory (Provencher et al., 1997),
observations certainly indicative of a CsA-mediated increased
efficiency in the liver recovery process.
Data obtained during the study on caffeine elimination suggest that CsA
might have mediated the early recovery of CYP1A2-linked drug-metabolizing activity although the breath test approach does not
allow the exact metabolic pattern resulting from the biotransformation of the drug to be measured. However, as already shown by others, the
caffeine breath test is a valuable method to measure CYP1A2-mediated N-demethylation activities as it has been shown to exhibit a
high correlation with the in vivo metabolic clearance rate of caffeine in humans as well as in the rat (Willson and Hart, 1981; Renner et al.,
1984). Moreover, using the breath test as a probe, data obtained in our
laboratory (G. Raymond and J.P.V., unpublished data) have also
shown that NF (a known inducer of CYP1A2) administration increased
the elimination of caffeine whereas treatment with PB, DEX, or
4-methylpyrasole had no influence on its biotransformation, indicating
that the caffeine breath test is a good indicator of CYP1A2-dependent
activity. Interestingly, the decrease in caffeine elimination (a 60%
decrease) after PHx was also closely related to the loss of liver mass
contrary to the smaller decrease observed in the elimination of
aminopyrine and erythromycin. This observation indicates that the
hepatic metabolic reserve related to CYP1A2 seems to be smaller (and
closely related to liver mass) than that observed for the other
cytochromes P-450 probed in this study, an observation also made by
others in rats as well as humans (Renner et al., 1984; Schaad et al.,
1995) and where extrahepatic metabolism has also been reported to be
negligible (De Waziers et al., 1989). It is tempting to suggest a link
between the accelerated first wave of DNA synthesis (which takes place
in the PHx model 22 to 24 h after the 2/3 resection; Mazzaferro et
al., 1990; Kahn et al., 1990; Francavilla et al., 1991) and in the
regeneration process, mediated by CsA with the early recovery of the
caffeine biotransformation function. With time, however, the effect of
CsA would be expected to diminish as liver regeneration will progress
in placebo-treated rats to catch up with the CsA-treated animals. In
fact, the early beneficial effect of CsA on the recovery of
CYP1A2-mediated biotransformation was, indeed, short-lived, its effect
gradually diminishing over the following 72 h. It is not excluded
that the deleterious hepatic effect(s) of CsA might have played a role
in counterbalancing the positive influence of the drug on the
hyperplastic process dampening the expected accelerated recovery in
liver mass and the hepatic drug-metabolizing function. The data
obtained do not preclude, however, an accelerated CsA-mediated recovery
(and/or protection from an initial decrease) on other substrates but
they show that its positive effect is limited. Indeed, at the critical time of greatest loss in liver parenchyma, CsA was only successful in
improving the in vivo biotransformation of CYP1A2-linked drug metabolizing activity, whereas it did not increase the
biotransformation of CYP2B1-, 2C11-, and 3A2-dependent substrates. It
illustrates, however, the lack of impact of the drug on the whole organ
recovery function associated with these drug-metabolizing activities.
Our data, thus, lead us to conclude that the reported effect of CsA on
the regeneration process does not translate into an overall accelerated
recovery of the drug-metabolizing function of the liver despite an
apparent early acceleration of the regeneration process. The effect of
CsA may, then, mostly affect those drug-metabolizing activities
exhibiting the lowest reserve that would be expected, therefore, to be
the most sensitive to losses in liver mass or occur when hepatic mass
is inadequate for body size, such as after transplantation of livers
that are small for recipients.
We thank Dr. G.N. Hendy from the Departments of Medicine and
Physiology, McGill University for providing the rat cyclophilin probe,
Dr. Pierre Banon from the Clinical Biochemistry Department, Centre
Hospitalier de l'Université de Montréal, Campus St-Luc for
measuring the CsA, and Manon Livernois for her excellent secretarial assistance.
These studies were supported by the Medical Research Council of
Canada (Grant No. MT-6511). S.P. is the recipient of a Studentship award from the Canadian Liver Foundation.
Abbreviations used are:
CsA, cyclosporine A;
3-MC, 3-methylcholanthrene;
ALT, alanine aminotransferase;
NF, -naphthoflavone;
CYP, cytochrome P-450 gene;
CYP, cytochrome P-450 protein;
DEX, dexamethasone;
PB, phenobarbital;
PHs, sham-operated animals;
PHx, 2/3 partial hepatectomy/hepatectomized.
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Abstract
Introduction
... ... ... 
